DIARYLALKANES AS POTENT INHIBITORS OF BINUCLEAR ENZYMES

Abstract

The present invention implements a strategy that combines an enzyme inhibition assay with a chemical dereplication process to identify active plant extracts and the particular compoundsdiarylalkanes and/or diarylalkanols within those extracts that specifically inhibit binuclear enzyme function. Included in the present invention are compositions of matter comprised of one or more of diarylalkanes and/or diarylalkanols, which inhibit the activity of binuclear enzymes, particularly tyrosinase and which prevent melanin overproduction. The present invention also provides a method for inhibiting the activity of a binuclear enzyme, particularly tyrosinase and a method for preventing and treating diseases and conditions related to binuclear enzyme function. The present invention further includes a method for preventing and treating melanin overproduction and diseases and conditions of the skin related thereto. The method for preventing and treating diseases and conditions related to binuclear enzyme function and melanin overproduction is comprised of administering to a host in need thereof an effective amount of a composition comprising one or more diarylalkanes and/or diarylalkanols synthesized and/or isolated from one or more plants together with a pharmaceutically acceptable carrier.

Claims

1-40. (canceled)

41. A method for treating a disease or condition associated with overproduction or uneven distribution of melanin, the method comprising administering an effective amount of a composition comprising a compound having the following structure: ##STR00016## wherein: R.sub.1, R.sub.2, R.sub.4, R.sub.5, R.sub.1, R.sub.2, R.sub.4 and R.sub.5 are each independently H, OH, SH, CN, SR, NH.sub.2, NHR, NR.sub.2 or a glycoside of a monosaccharide or oligosaccharide comprised of 2-6 monosaccharides, wherein said monosaccharide is an aldopentose, methyl-aldopentose, aldohexose, ketohexose or derivative thereof; R.sub.3 is SH, CN, SR, NH.sub.2, NHR, NR.sub.2, X or a glycoside of a monosaccharide or oligosaccharide comprised of 2-6 monosaccharides, wherein said monosaccharide is an aldopentose, methyl-aldopentose, aldohexose, ketohexose or derivative thereof; R.sub.3 is H, SH, CN, SR, NH.sub.2, NHR, NR.sub.2, X or a glycoside of a monosaccharide or oligosaccharide comprised of 2-6 monosaccharides, wherein said monosaccharide is an aldopentose, methyl-aldopentose, aldohexose, ketohexose or derivative thereof; R.sub.6 and R.sub.7 are, at each occurrence, each independently H, OH, OR, CN, NHR, NH.sub.2 or X; R is an alkyl group having from 1-20 carbon atoms; X is Cl, Br, F or I; and n is 3, wherein: i) at least 2 of R.sub.1-R.sub.5 are not H, or at least 2 of R.sub.1R.sub.5 are not H; and ii) at least one occurrence of R.sub.6 or R.sub.7 is OH; and iii) when R.sub.1, R.sub.3, and R.sub.5 are OH, R.sub.4 is OH, OR, SH, CN, SR, NH.sub.2, NHR, NR.sub.2 or a glycoside of a monosaccharide or oligosaccharide comprised of 2-6 monosaccharides, wherein said monosaccharide is an aldopentose, methyl-aldopentose, aldohexose, ketohexose or derivative thereof.

42. The method of claim 41, wherein the compound is selected from the group consisting of 1-(2,4-dihydroxyphenyl)-3-(3,4-dihydroxyphenyl)-1-propanol, 1-(2,4-dihydroxyphenyl)-3-(3,4-dimethoxyphenyl)-1-propanol, 1-(2,4-dihydroxyphenyl)-3-(2-hydroxyphenyl)-1-propanol, 1-(2,4-dihydroxyphenyl)-3-(2-methoxyphenyl)-1-propanol, 1-(2,4-dihydroxyphenyl)-3-(4-methoxyphenyl)-1-propanol, 1-(2,4,6-trihydroxyphenyl)-3-(4-aminophenyl)-1-propanol, dihydroxyphenyl)-3-phenyl-1-propanol, 1-(2,4-dihydroxyphenyl)-3-(3-methoxy-4-hydroxyphenyl)-1-propanol, 1-(2-hydroxy-4-methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-1-propanol, 1-(2-hydroxy-4-methoxyphenyl)-3-(2,4-dimethoxyphenyl)-1-propanol, 1-(2-hydroxy-5-methoxyphenyl)-3-(3,4-dimethoxyphenyl)-1-propanol, 1-(2,4-dihydroxyphenyl)-2-(4-methoxyphenyl)-1-ethanol, 1-(2-hydroxy-4-methoxyphenyl)-3-(2,3,4,5-tetrahydro-benzo(b)dioxocin-8-yl)-1-propanol, 3-(5-chloro-1 -methyl-1-hydro-imidazol-2-yl)-1-(2-hydroxy-4-m ethoxyphenyl)-1-propanol, 1-(2-hydroxy-4,6-dimethoxyphenyl)-3-(3-methoxy-4-hydroxyphenyl)-1-propanol, 1-(2,4-dihydroxyphenyl)-2-(3,4-dimethoxyphenyl)-1-ethanol, 1-(2-hydroxy-5-methoxyphenyl)-3-(2,4-dimethoxyphenyl)-1-propanol, 1-(2,4-dihydroxyphenyl)-2-(2,4-dichlorophenyl)-1-ethanol, 1-(2,4,6-trihydroxyphenyl)-3-(3-hydroxy-4-methoxyphenyl)-1-propanol, 1,3-bis(2,4-dimethoxyphenyl)-propan-1,3-diol, 1-(2,4,6-trihydroxyphenyl)-3-(3-hydroxy-4-methoxyphenyl)-1-propanol and 1-(2,4,6-trimethoxyphenyl)-3-(3,4-dimethoxyphenyl)-1-propanol.

43. The method of claim 41, wherein the compound is 1-(2,4-dihydroxyphenyl)-3-(2-hydroxyphenyl)-1-propanol.

44. The method of claim 41, wherein the administration lightens dark skin pigmentation, reduces skin dullness, evens skin tone or corrects skin discoloration.

45. The method of claim 41, wherein the administration reduces signs of aging on the skin and reduces wrinkles associated with aging by neutralization of active oxygen species.

46. The method of claim 41, wherein the administration inhibits synthesis of melanin.

47. The method of claim 41, wherein the disease or condition is: skin darkening or damage due to exposure to ultra violet light; sun tan; hyper pigmentation spots; thermal burns; topical wounds; skin pigmentation due to inflammatory conditions caused by fungal, microbial or viral infections; vitiligo; or a carcinoma.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0044] FIG. 1 illustrates the process of melanogenesis, together with various potential mechanisms for regulating this process.

[0045] FIG. 2 illustrates the structure of the active site of tyrosinase. As can be seen in this figure, two copper ions are closely spaced and each is coordinated to three histidines through the N-E nitrogen atom of its side chain.

[0046] FIGS. 3A and 3B depict the structure, name, mechanism of action, and other effects of known tyrosinase inhibitors.

[0047] FIG. 4 illustrates the HPLC/UV chromatogram of a HTP fraction that contains the UP288 (1-(2-methoxy-4-hydroxyphenyl)-3-(2-hydroxy-5-methoxyphenyl)-propane) (1) as highlighted.

[0048] FIG. 5 depicts the chemical structure and .sup.13C-NMR spectrum of UP288 (1).

[0049] FIG. 6 illustrates tyrosinase inhibitory dose response curves and IC.sub.50 values for UP288 and kojic acid.

[0050] FIG. 7 depicts the bioassay-guided isolation of two active compounds (UP302a and UP302b) from Dianella ensifolia (P0389) (whole plant).

[0051] FIG. 8 depicts the HPLC/UV chromatogram of the enriched UP302 fraction after multiple column separations.

[0052] FIG. 9 depicts a gHSQC spectrum of UP302a (2), which illustrates the links between protons and carbons.

[0053] FIG. 10 illustrates graphically inhibition of the activity of tyrosinase at various concentrations of inhibitor UP302a and substrate L-DOPA. This kinetic study revealed that UP302a is a competitive inhibitor of the tyrosinase enzyme.

[0054] FIG. 11 illustrates the inhibition of endogeneous melanin production from mouse B16 F1 cells by kojic acid and UP302a (2). Each sample was tested in triplicate at 10 different concentrations.

[0055] FIG. 12 depicts three-dimensional conformation of UP302a after MM2 energy minimization.

[0056] FIG. 13 illustrates the three-dimensional conformation of UP302a, when coordinated to two copper ions in the Cu.sup.II-O.sub.2-Cu.sup.II oxidation state.

[0057] FIG. 14 depicts the distances between adjacent atoms of UP302a when chelated with copper ions in the peroxide form (Cu.sup.II-O.sub.2-Cu.sup.II) as calculated in Example 17.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0058] The present invention relates generally to the prevention and treatment of diseases and conditions mediated by binuclear enzymes. Specifically, the present invention includes a method for inhibiting the activity of an enzyme having a binuclear active site. Included in the present invention are novel compositions comprised of one or more diarylalkane(s). The diarylalkanes of the present invention can be isolated from one or more plant sources or can be obtained by organic synthesis. Further included in the present invention are methods for isolating these compounds from a natural source and methods for synthesizing these compounds. In one embodiment, the diarylalkanes are obtained by synthetic modification of a naturally occurring compound isolated from a plant source.

[0059] Various terms are used herein to refer to aspects of the present invention. To aid in the clarification of the description of the components of this invention, the following definitions are provided. Unless defined otherwise all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

[0060] It is to be noted that as used herein the term a or an entity refers to one or more of that entity; for example, a diarylalkane refers to one or more diarylalkanes. As such, the terms a or an, one or more and at least one are used interchangeably herein.

[0061] Diarylalkanes as used herein are a specific class of aromatic compounds having the following general structure: The present invention also includes a novel composition of matter comprised of one or more diarylalkanes, wherein said diarylalkanes are selected from the group of compounds represented by the following general structure:

##STR00005##

wherein

[0062] Ar.sub.1 and Ar.sub.2 are independently selected from the group consisting of a substituted 5- or 6-membered aromatic or heteroaromatic ring, wherein each 6-membered aromatic or heteroaromatic ring is independently substituted with 1-5 R groups (R.sub.1R.sub.5), and each 5-membered aromatic or heteroaromatic ring is substituted with 1-4 R groups (R.sub.1R.sub.4), except when Ar.sub.1 and Ar.sub.2 are both a 6-membered aromatic ring, i.e. a phenyl group at least one of Ar.sub.1 and Ar.sub.2 are substituted with 1-5 R groups (R.sub.1R.sub.5), wherein at least 2 of said of R.sub.1R.sub.5 are not H wherein

[0063] R is independently selected from the group consisting of H, OH, SH, OR, CN, SR, NH.sub.2, NHR, NR.sub.2 and X, and a glycoside of a monosaccharide or oligosaccharide comprised of 2-6 monosaccharides, wherein said monosaccharide(s) are independently selected from the group consisting of an aldopentose, methyl-aldopentose, aldohexose, ketohexose and chemical derivatives thereof; wherein R is an alkyl group having between 1-20 carbon atoms and X is a halogen, selected from the group consisting of Cl, Br, F and I;

[0064] R.sub.6, and R.sub.7 are independently selected from the group consisting of H, OH, OR, CN, NHR, NH.sub.2, and X, wherein R is an alkyl group having between 1-20 carbon atoms and wherein X is a halogen, selected from the group consisting of Cl, Br, F, I; and n=1 to 10. In a preferred embodiment n=2-4.

[0065] In one embodiment, said diarylalkanes and diarylalkanols are selected from the group of compounds represented by the following general structure:

##STR00006##

wherein

[0066] R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5 R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 are independently selected from the group consisting of H, OH, SH, OR, CN, SR, NH.sub.2, NHR, NR.sub.2, X, and a glycoside of a monosaccharide or oligosaccharide comprised of 2-6 monosaccharides, wherein said monosaccharide(s) are independently selected from the group consisting of an aldopentose, methyl-aldopentose, aldohexose, ketohexose and chemical derivatives thereof; wherein R is an alkyl group having between 1-20 carbon atoms and X is a halogen, selected from the group consisting of Cl, Br, F, I, and wherein at least 2 of R.sub.1-R.sub.5 or at least 2 of R.sub.1R.sub.5 are not H;

[0067] R.sub.6, and R.sub.7 are independently selected from the group consisting of H, OH, OR, CN, NHR, NH.sub.2, and X, wherein R is an alkyl group having between 1-20 carbon atoms and wherein X is a halogen, selected from the group consisting of Cl, Br, F and I; and

[0068] n=1 to 10. In a preferred embodiment n=2-4.

[0069] Diarylalkanols as used herein are a specific type of diarylalkanes having at least one hydroxyl group (R.sub.6 and/or R.sub.7=OH) attached to the alkyl carbons between the two aromatic rings.

[0070] Binuclear enzyme as used herein refers to an enzyme which has a binuclear active site, an example of which is tyrosinase which has two copper ions at its active site as discussed above. Binuclear enzymes include, but are not limited to tyrosinase, arginase, urease, cytochrome c oxidase, proton pumping heme-copper oxidase, bifunctional carbon monoxide dehydrogenase/acetyl-coenzyme A synthase, ribonucleotide reductase, metalo-beta-lactamase, H(+)-ATPase and alternative oxidase, and bacterial phosphotriesterase.

[0071] Therapeutic as used herein, includes prevention, treatment and/or prophylaxis. When used, therapeutic refers to humans as well as other animals.

[0072] Pharmaceutically or therapeutically effective dose or amount refers to a dosage level sufficient to induce a desired biological result. That result may be the alleviation of the signs, symptoms or causes of a disease or any other alteration of a biological system that is desired. The precise dosage will vary according to a variety of factors, including but not limited to the age and size of the subject, the disease and the treatment being effected.

[0073] Placebo refers to the substitution of the pharmaceutically or therapeutically effective dose or amount dose sufficient to induce a desired biological that may alleviate the signs, symptoms or causes of a disease with a non-active substance.

[0074] A host or patient or subject is a living mammal, human or animal, for whom therapy is desired. The host, patient or subject generally refers to the recipient of the therapy to be practiced according to the method of the invention. It should be noted that the invention described herein may be used for veterinary as well as human applications and that the term host should not be construed in a limiting manner. In the case of veterinary applications, the dosage ranges can be determined as described below, taking into account the body weight of the animal.

[0075] As used herein a pharmaceutically acceptable carrier refers to any carrier, which does not interfere with effectiveness of the biological activity of the active ingredient and which is not toxic to the host to which it is administered. Examples of pharmaceutically acceptable carriers include, but are not limited to, any of the standard pharmaceutical carriers such as a saline solution, i.e. Ringer's solution, a buffered saline solution, water, a dextrose solution, serum albumin, and other excipients and preservatives for tableting and capsulating formulations.

[0076] The present invention includes a method for inhibiting the activity of an enzyme with a binuclear active site, referred to herein as a binuclear enzyme, said method comprising administering to a host in need thereof an effective amount of one or more diarylalkane(s), wherein said diarylalkanes are synthesized and/or isolated from a one or more plants. Examples of binuclear enzymes included within the scope of the present invention include, but are not limited to tyrosinase, arginase, urease, cytochrome c oxidase, proton pumping heme-copper oxidase, bifunctional carbon monoxide dehydrogenase/acetyl-coenzyme A synthase, ribonucleotide reductase, metalo-beta-lactamase, H(+)-ATPase and alternative oxidase, and bacterial phosphotriesterase. In one embodiment, the binuclear enzyme is tyrosinase.

[0077] The present invention also includes a method for the prevention and treatment of diseases and conditions related to the activity of binuclear enzymes. The method of prevention and treatment according to this invention comprises administering internally or topically to a host in need thereof a therapeutically effective amount of one or more diarylalkane(s). Depending on the binuclear enzyme being inhibited the diarylalkane may be used as an anti-microbial, anti-fungal, anti-malaria, or anti-viral agent, a regulator for the production of nitric oxide as a means of controlling male and female sexual arousal, an anti-inflammatory drug, an antioxidant, a regulator of drug metabolism, for treatment and prevention of periodontal diseases, oral pre-cancerous conditions, oral cancers, and other oral malignancies, sensitive gums and teeth, sequelae, pulpitis, irritation, pain and inflammation caused by the physical implantation of oral dentures, trauma, injuries, bruxism and other minor wounds in mouth, on the gums or on the tongue, dental plague and calculus, tooth decalcification, proteolysis and caries (decay). and an inhibitor of the growth of cancers and solid tumors.

[0078] The present invention further includes methods for the prevention and treatment of diseases and conditions related to the overproduction or uneven distribution of melanin, said method comprising administering internally or topically to a host in need thereof a therapeutically effective amount of one or more diarylalkane(s). Diseases and conditions related to the overproduction or uneven distribution of melanin include, but not limited to suntan, hyper pigmentation spots caused by skin aging, liver diseases, thermal burns and topical wounds, skin pigmentation due to inflammatory conditions caused by fungal, microbial and viral infections, vitilago, carcinoma, melanoma, as well as other mammalian skin conditions.

[0079] The method can also be used for preventing and treating skin darkening and damage resulting from exposure to ultraviolet (UV) radiation, chemicals, heat, wind and dry environments. Finally, the method can be used for preventing and treating wrinkles, saggy skin, lines and dark circles around the eyes, soothing sensitive skin and preventing and treating dermatitis and other allergy related conditions of the skin. In addition to their use for the prevention and treatment of the above described diseases and conditions of the skin, the therapeutic compositions described herein provide an efficacious composition that yields the benefit of smooth and youthful skin appearance with improved skin color, enhanced elasticity, reduced and delayed aging, enhanced youthful appearance and texture, and increased flexibility, firmness, smoothness and suppleness.

[0080] By chelating with metal ions diarylalkanes also can be used to deliver essential metal ions into the blood stream of the host, and/or carry metal ions through the skin or blood/brain barrier, as well as, other membranes. In this embodiment, the method comprises administering to a host in need thereof a therapeutically effective amount of one or more diarylalkane(s), together with the metal ion(s) to be delivered. In this capacity the diarylalkanes can be used to treat diseases and conditions including, but not limited to anemia and other iron deficiencies, inflammation; obesity and diabetes, periodontal diseases, oral pre-cancerous conditions, oral cancers, and other oral malignancies, sensitive gums and teeth, sequelae, pulpitis, irritation, pain and inflammation caused by the physical implantation of oral dentures, trauma, injuries, bruxism and other minor wounds in mouth, on the gums or on the tongue, dental plague and calculus, tooth decalcification, proteolysis and caries (decay), and viral infections. The metal ions are selected from the group including, but not limited to copper, iron, zinc, selenium, magnesium and other metal ions.

[0081] In yet another embodiment, the dialkylalkanes can be used in the food industry to prevent browning and color changes in fruits, mushrooms and other food products.

[0082] The diarylalkanes that can be used in accordance with the following include compounds illustrated by the general structure set forth above. The diarylalkanes of this invention may be obtained by synthetic methods or may be isolated from one or more families of plants selected from the group including, but not limited to Compositae, Fabaceae, Lauraceae, Leguminosae, Liliaceae, Loranthaceae, Moracea, and Myristicaceae. The diarylalkanes of this invention can be extracted, concentrated, and purified from the genera of high plants, including but not limited to Acacia, Broussonetia, Dianella, Helichrysum, Iryanthera, Knema, Lindera, Pterocarpus, Viscum, and Xanthocercis. The diarylalkanes can be found in different parts of the plant, including but not limited to stems, stem barks, heart woods, trunks, trunk barks, twigs, tubers, roots, root barks, young shoots, seeds, rhizomes, flowers and other reproductive organs, leaves and other aerial parts. In a one embodiment, the diarylalkanes are isolated from a plant or plants in the Broussonetia, Dianella, and Iryanthera genera.

[0083] In another embodiment, the diarylalkanes of this invention are obtained by synthetic methods. Included in this invention is a method of synthesizing diarylalkanes and diarylalkanols said method comprising reducing a compound having the following general structure:

##STR00007##

wherein

[0084] R.sub.1-R.sub.5 and R.sub.1R.sub.5 and n are as defined above and wherein R.sub.6 and R.sub.7 together form one or more carbonyl group(s). The reducing agent can be selected from any known reducing agent for the reduction of ketones to alcohols including, but not limited to borohydrides, H.sub.2 in the presence of a catalyst, NaH and LiAlH.sub.4. In one embodiment the reducing agent is NaBH.sub.4.

[0085] In yet another embodiment, the diarylalkanes are obtained by synthetic modification of a naturally occurring compound isolated from a plant source. For example, the naturally occurring compound butein is isolated from a plant source, dehydrated and reduced to yield the corresponding diarylalkanol.

[0086] In yet another embodiment, the diarylalkanes are obtained by the reaction of two appropriately substituted aromatic compounds. Feasible chemical reactions for synthesizing these compounds from two substituted aromatic compounds include, but are not limited to Aldol condensation between a substituted benzaldehyde and a substituted acetophenone; Claisen-Schmidt reaction or crossed aldol condensation between an aldehyde and a ketone; Grignard reaction using an organomagnesium halide of one substituted aromatic ring to link the second substituted aromatic ring through addition reaction to the carbonyl group on the molecule; Claisen rearrangement by an intra-molecular isomerization, in which an esterified phenol with appropriate substitution groups will be isomerized to link the second aromatic rings at the ortho-position of the phenol followed by a reducing reaction; and a Suzuki coupling reaction, in which two substituted aromatic rings are converted to arylboronic acids and then linked by an alkyl halide by using a carefully selected palladium catalyst. These reactions are well known to those of skill in the art and the conditions for such reactions can be determined using the information disclosed herein for the synthesis of these compounds.

[0087] Note that throughout this application various citations are provided. Each of these citations is specifically incorporated herein by reference in its entirety.

[0088] The present invention implements a strategy that combines a tyrosinase inhibition assay with a chemical dereplication process to identify active plant extracts and the particular compounds within those extracts that specifically inhibit the binuclear enzyme tyrosinase. As noted above, enzymes that inhibit tyrosinase may lead to a reduction in the production of melanin thereby effectively lightening the skin. A library of plant extracts was generated by extracting dry plant powders with an organic solvent, as described in Example 1. The tyrosinase inhibition assay was developed following a method reported by Jones et al. (2002) Pigment. Cell Res. 15:335, as described in Example 2. Using this assay, a total of 1144 plant extracts were screened for their ability to inhibit the activity of mushroom tyrosinase. This primary screen identified 20 plant extracts (1.75% hit rate) with potent tyrosinase inhibitory activity. Table 1 delineates percent inhibition of tyrosinase by four of these extracts isolated from four different genera.

[0089] In order to efficiently identify active compounds from the active plant extracts, a high throughput fractionation process was used, as described in Example 3. Briefly, the active extracts were fractionated using a high throughput purification (HTP) system. Each of the fractions was then tested for its ability to inhibit tyrosinase activity as per the primary assay described in Example 2. After dereplication, using a combination of HPLC with PDA and MS detectors coupled with a structure database search and elimination of fractions that contained known tyrosinase inhibitors, such as polyphenols and chromones, a total of seven active extracts were chosen for bioassay guided large-scale isolation and purification as described in Examples 4-6, using the extracts of Broussonetia kazinoki Sieb. Et Zucc (Moraceae), and Dianella ensifolia (L.) DC. (Liliaceae) for purposes of illustration.

[0090] Example 4 describes the extraction, separation and purification of the novel diarylpropane: 1-(2-methoxy-4-hydroxyphenyl)-3-(2-hydroxy-5-methoxyphenyl)-propane (UP288) (1) from Broussonetia kazinoki Sieb. Et Zucc (Moraceae) (whole plant) using the general method set forth in Examples 1-3. FIG. 4 illustrates the HPLC/UV chromatogram of a HTP fraction that contains the UP288. The structure of the active compound UP288 was elucidated using a combination of spectroscopic methods as set forth in Example 4. FIG. 5 depicts the chemical structure and .sup.13C-NMR spectrum of UP288. FIG. 6 illustrates tyrosinase inhibitory dose response curves and IC.sub.50 values for UP288 relative to kojic acid. The figure illustrates that UP288 (1) is as potent a tyrosinase inhibitor as kojic acid, having an IC.sub.50=24 M.

[0091] Surprisingly, two similar diarylalkanes were isolated and identified from a totally different family of plantDianella ensifolia (L.) DC. (Liliaceae), as described in Example 5. FIG. 7 depicts schematically the bioassay-guided isolation of these two active compounds (UP302a (2) and UP302b (3)) from Dianella ensifolia (P0389) (whole plant). With reference to FIG. 7, it can be seen that only fifteen column fractions from a total of 264 collected samples exhibited potent inhibition of tyrosinase. A HPLC analysis (FIG. 8) of the combined active fractions showed that active compounds were minor components in the best pool, which has already been heavily enriched. Laborious separation and purification efforts yielded two novel active compounds that have been fully characterized by NMR and other spectroscopic methods as illustrated in Example 5 and FIG. 9 as 1-(3-methyl-2,4-dimethoxyphenyl)-3-(2,4-dihydroxyphenyl)-propane (UP302a, IC.sub.50=0.24 M) (2) and 1-(3-methyl-2,4-dimethoxyphenyl)-3-(2,5-dihydroxyphenyl)-propane (IP302b, IC.sub.50=1.2 M) (3).

[0092] Example 6 describes the large-scale isolation of UP302a (2), the most potent tyrosinase inhibitor, isolated from Dianella ensifolia (DE) (whole plant). With reference to Example 6, from 4.3 kg of dried biomass, a total of 30 mg of pure UP302a (2) was obtained after multiple column fractionations on silica gel, CG-161, and C-18 resins. The structure and biological function of the isolated compound were confirmed.

[0093] Due to the low natural abundance of diarylalkanes/diarylalkanols methods to synthesize these biologically active compounds as an alternative commercial source of this class of compounds was developed. Example 7 describes a general method for the synthesis of diarylalkanes via the reduction of substituted chalcones. For purposes of illustration the reduction of 2,4-dihydroxyphenyl)-3,4-dimethoxyphenylchalcone (4) to 1-(2,4-dihydroxyphenyl)-3-(3,4-dimethoxyphenyl)-1-propanol (5) using sodium borohydride is described. However, as set forth in Example 7, a number of other diarylalkanes have been synthesized using this general method. All of the compounds synthesized showed high to moderate tyrosinase inhibitory activity. With respect to the general method described in Example 7, any other known reducing agents, can be used to effect this reduction, including, but are not limited to other borohydrides, H.sub.2 in the presence of a catalyst, NaH and LiAlH.sub.4.

[0094] Using the general reaction described in Example 7, several other substituted diarylpropanones have been converted to diarylpropanes and/or diarylpropanols as demonstrated in Examples 8, 9 and 10. Example 11 demonstrates the synthesis of a diarylpropanol using a flavonoid glycoside isolated from a natural source as the starting material.

[0095] In another embodiment, the present invention includes methods for synthesizing this class of compounds by reaction of two appropriately substituted aromatic compounds. This embodiment is illustrated in Example 12, using the reaction of resorcinol with 3-methoxy-4-hydroxycinnamic acid for purposes of illustration. Feasible chemical reactions for synthesizing these compounds from two substituted aromatic compounds include, but are not limited to Aldol condensation between a substituted benzaldehyde and a substituted acetophenone; Claisen-Schmidt reaction or crossed aldol condensation between an aldehyde and a ketone; Grignard reaction using an organomagnesium halide of one substituted aromatic ring to link the second substituted aromatic ring through addition reaction to the carbonyl group on the molecule; Claisen rearrangement by an intra-molecular isomerization, in which an esterified phenol with appropriate substitution groups will be isomerized to link the second aromatic rings at the ortho-position of the phenol followed by a reducing reaction; and a Suzuki coupling reaction, in which two substituted aromatic rings are converted to arylboronic acids and then linked by an alkyl halide by using a carefully selected palladium catalyst. These reactions are well known to those of skill in the art and the conditions for such reactions can be determined using the information disclosed herein for the synthesis of these compounds.

[0096] Example 13 sets forth the IC.sub.50 values for a number of diarylalkanes and diarylalkanols synthesized according the methods of this invention. The compounds were evaluated using the general method described in Example 2. The IC.sub.50 value of each sample was calculated using kinetics software to verify that the reaction was linear at a specified time and concentration. Using the methods described in Examples 7-12a total of 24 compounds were synthesized and evaluated for their ability to inhibit tyrosinase. The results are set forth in Table 2. With reference to Table 2, it can be seen that the IC.sub.50's of the synthetic diarylalkanols were comparable to the naturally occurring diarylpropanes. Thus, these two classes of compounds are capable of inhibiting tyrosinase to approximately the same extent. The most active diarylalkanes and/or diarylalkanols had three carbons between the two aromatic rings. Using the calculations described in Example 17, this structural feature was demonstrated to be critical in order to generate a parallel and superimposed intra-molecular conformations. However, diarylalkanols, which contained two and four carbons between the two aromatic rings, such as 1-(2,4-dihydroxyphenyl)-2-(4-methoxyphenyl)-1-ethanol (IC.sub.50=77 M) and 1,4-bis-(3,4-dihydroxyphenyl)-2,3-dimethyl-buthane (IC.sub.50=700 M) also were able to significantly inhibit tyrosinase activity.

[0097] Using the method described in Example 2, the inhibition of tryosinase by UP302a (2) was evaluated using L-DOPA as the substrate as set forth in Example 14. The results are set forth in FIG. 10. This study revealed that UP302a (2) is a powerful competitive inhibitor having a long lasting effect. Interestingly, tyrosinase activity was not resumed for several days after incubation with UP302a. In contrast, tyrosinase activity was totally restored after only 1 hour following incubation with kojic acid. Since two of the substituents on the aromatic rings of UP302a were methoxy groups, the inhibitor cannot be easily hydroxylated and/or oxidized. This may explain both the effectiveness and duration of the inhibitory activity of UP302a. Thus, it can be concluded that these compounds will have a long lasting effect.

[0098] The efficacy of the claimed composition was also demonstrated by measuring the inhibition of melanin produced in an in vitro test on a B-16 cell line as described in Example 15. The results are set forth in FIG. 11. The reduction of endogenous melanin by UP302a (2) was almost six fold greater than that of kojic acid. Additionally, inhibition of MSH induced melanin production by UP302a was also significantly greater than kojic acid. As expected, UP288 (1) was comparable to kojic acid in the B-16 cell line model.

[0099] Example 16 describes an assay to assess the cytotoxicity of two diarylpropanes UP288 (1) and UP302a (2) relative to kojic acid. At a concentration of 250 M, which was above IC.sub.50 of all three tested compounds, the diarylpropanes demonstrated similar safety profiles to that of kojic acid.

[0100] Example 17 describes the molecular modeling analyses performed to determine the most stable 3-D conformation of the active diarylalkanes and diarylalkanols. Molecular mechanics calculations were performed using Chem3D software. These calculations revealed that the most potent tyrosinase inhibitor1-(3-methyl-2,4-dimethoxyphenyl)-3-(2,4-dihydroxyphenyl)-propane (UP302a (2), IC.sub.50=0.24 M) has a very unique 3-dimensional conformation with two the aromatic rings superimposed on each other as illustrated in FIG. 12. The minimized total energy for the conformation is 4.7034 KJ/Mol and the distance between the two aromatic rings is 3.28 . The phenolic hydroxyl groups on the first aromatic ring are right above the two methoxy groups on the second aromatic ring with the distance between two oxygen atoms being as 2.99 and 3.16 , respectively as illustrated in FIG. 14. The active site of the binuclear enzyme tyrosinase has two copper ions complexed to an oxygen molecule in a peroxide oxidation state [Cu.sup.II-O.sub.2-Cu.sup.II], which is key to the mechanism by which tyrosinase catalyzes the introduction of a hydroxyl group in the ortho position of the aromatic ring of a mono-phenol (such as tyrosine). (Decker et al. (2000) Angew. Chem. Int. Ed. 39:1591). The interactomic distances were reported as 3.56 for Cu-Cu. 1.90 for Cu-O and 1.41 for 0-0. (Kitajima et al. (1989) J. Am. Chem. Soc. 111:8975). The parallel conformation of 1-(3-methyl-2,4-dimethoxyphenyl)-3-(2,4-dihydroxyphenyl)-propane (UP302a, IC.sub.50=0.24 M) will perfectly chelate with both copper ions of the [Cu.sup.II-O.sub.2-Cu.sup.II] complex from both the top and the bottom as illustrated in FIGS. 13 and 14. This dual chelation by the inhibitor to both copper ions at the active site will totally block the access of the substrate, such as L-Dopa to the enzyme, thus effectively inhibiting the function of the protein. Using the same approach, the isolated and synthetic diarylalkanes and diarylalkanols listed in Table 2 were analyzed. The results of this analysis indicated that the compounds with twisted or non-parallel conformations possessed either no ability or only a weak ability to inhibit the activity of tyrosinase.

[0101] From these studies it has been determined that the most effective diarylalkane inhibitors have two to three substituents on one aromatic ring and zero to multiple substituents on the second aromatic ring. The most favorable structures are those in which at least one aromatic ring is substituted in the 2 and 4-positions. Preferably the rings are 6-membered aromatic and/or heteroaromatic as demonstrated by two of the compounds isolated 1-(2-hydroxy-4-methoxyphenyl)-3-(2,3,4,5-tetrahydro-bezo(b)dioxocin-8-yl)-1-propanolIC.sub.50=72 M and 3-(5 -chloro-1-methyl-1-hydro-imidazol-2-yl)-1-(2-hydroxy-4-methoxyphenyl)-1-propanolIC.sub.50=225 M.

[0102] The compositions of this invention can be administered by any method known to one of ordinary skill in the art. The modes of administration include, but are not limited to, enteral (oral) administration, parenteral (intravenous, subcutaneous, and intramuscular) administration and topical application. The method of treatment according to this invention comprises administering internally or topically to a patient in need thereof a therapeutically effective amount of a diarylalkane or a mixture comprised of two or more diarylalkanes.

[0103] The compositions of the present invention can be formulated as pharmaceutical compositions, which include other components such as a pharmaceutically and/or cosmetically acceptable excipient, an adjuvant, and/or a carrier. For example, compositions of the present invention can be formulated in an excipient that the host to be treated can tolerate. An excipient is an inert substance used as a diluent or vehicle for a therapeutic agent such as a diarylalkane or a mixture of diarylalkanes. Examples of such excipients include, but are not limited to water, buffers, saline, Ringer's solution, dextrose solution, mannitol, Hank's solution, preservatives and other aqueous physiologically balanced salt solutions. Nonaqueous vehicles, such as fixed oils, sesame oil, ethyl oleate, or triglycerides may also be used. Other useful formulations include suspensions containing viscosity-enhancing agents, such as sodium carboxymethylcellulose, sorbitol or dextran. Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability. Examples of buffers include phosphate buffer, bicarbonate buffer, tris buffer, histidine, citrate, and glycine, or mixtures thereof, while examples of preservatives include, but are not limited to EDTA, disodium EDTA, BHA, BHT, vitamin C, vitamin E, sodium bisulfite, SnCl.sub.2, thimerosal, m- or o-cresol, formalin and benzyl alcohol. Standard formulations can be either liquid or solids, which can be taken up in a suitable liquid as a suspension or solution for administration. Thus, in a non-liquid formulation, the excipient can comprise dextrose, human serum albumin, preservatives, etc., to which sterile water or saline can be added prior to administration.

[0104] In one embodiment of the present invention, the composition can also include an adjuvant or a carrier. Adjuvants are typically substances that generally enhance the biological response of a host to a specific bioactive agent. Suitable adjuvants include, but are not limited to, Freund's adjuvant, other bacterial cell wall components, aluminum, magnesium, copper, zinc, iron, calcium, and other metal ion based salts, silica, polynucleotides, toxoids, serum proteins, viral coat proteins, other bacterial-derived preparations, gamma interferon; block copolymer adjuvants; such as Hunter's Titermax adjuvant (Vaxcel, Inc. Norcross, Ga.), Ribi adjuvants (available from Ribi ImmunoChem Research, Inc., Hamilton, Mont.); and saponins and their derivatives, such as Quil A (available from Superfos Biosector A/S, Denmark). Carriers are typically compounds that increase the half-life of a therapeutic composition in the treated host. Suitable carriers include, but are not limited to, polymeric controlled release formulations, biodegradable implants, liposomes, bacteria, viruses, oils, esters, and glycols.

[0105] In one embodiment, the composition is prepared as a controlled release formulation, which slowly releases the composition of the present invention into the host. As used herein, a controlled release formulation comprises a composition of the present invention in a controlled release vehicle. Suitable controlled release vehicles will be known to those skilled in the art. Preferred controlled release formulations are biodegradable (i.e., bioerodible).

[0106] The therapeutic agents of the instant invention are preferably administered topically by any suitable means, known to those of skill in the art for topically administering therapeutic compositions including, but not limited to as an ointment, gel, lotion, or cream base, or as a toothpaste, mouth-wash, or coated on dental flossing materials or as an emulsion, as a patch, dressing or mask, a nonsticking gauze, a bandage, a swab or a cloth wipe. Example 18 describes the preparation of two cream formulations with an active content at 0.01% and 0.1% of a pure and/or mixture of diarylalkane(s) in the total weight of the formula. Such topical application can be locally administered to any affected area, using any standard means known for topical administration. A therapeutic composition can be administered in a variety of unit dosage forms depending upon the method of administration. For particular modes of delivery, a therapeutic composition of the present invention can be formulated in an excipient of the present invention. A therapeutic reagent of the present invention can be administered to any host, preferably to mammals, and more preferably to humans. The particular mode of administration will depend on the condition to be treated.

[0107] In one embodiment, a suitable ointment is comprised of the desired concentration of a single diarylalkane or a mixture of two or more diarylalkanes, that is an efficacious, nontoxic quantity generally selected from the range of 0.001% to 100% based on the total weight of the topical formulation, from 65 to 100% (preferably 75 to 96%) of white soft paraffin, from 0 to 15% of liquid paraffin, and from 0 to 7% (preferably 3 to 7%) of lanolin or a derivative or synthetic equivalent thereof. In another embodiment the ointment may comprise a polyethylene-liquid paraffin matrix.

[0108] In one embodiment, a suitable cream is comprised of an emulsifying system together with the desired concentration of a single diarylalkane or a mixture of two or more diarylalkanes as provided above. The emulsifying system is preferably comprised of from 2 to 10% of polyoxyethylene alcohols (e.g. the mixture available under the trademark Cetomacrogol 1000), from 10 to 25% of stearyl alcohol, from 20 to 60% of liquid paraffin, and from 10 to 65% of water; together with one or more preservatives, for example from 0.1 to 1% of N,N-methylenebis[N-[3-(hydroxymethyl)-2,5-dioxo-4-imidazolidinyl]urea] (available under the name Imidurea USNF), from 0.1 to 1% of alkyl 4-hydroxybenzoates (for example the mixture available from Nipa Laboratories under the trade mark Nipastat), from 0.01 to 0.1% of sodium butyl 4-hydroxybenzoate (available from Nipa Laboratories under the trade mark Nipabutyl sodium), and from 0.1 to 2% of phenoxyethanol.

[0109] In one embodiment, a suitable gel is comprised of a semi-solid system in which a liquid phase is constrained within a three dimensional polymeric matrix with a high degree of cross-linking The liquid phase may be comprised of water, together with the desired amount of a single diarylalkane or a mixture of two or more diarylalkanes, from 0 to 20% of water-miscible additives, for example glycerol, polyethylene glycol, or propylene glycol, and from 0.1 to 10%, preferably from 0.5 to 2%, of a thickening agent, which may be a natural product, selected from the group including, but not limited to tragacanth, pectin, carrageen, agar and alginic acid, or a synthetic or semi-synthetic compound, selected from the group including, but not limited to methylcellulose and carboxypolymethylene (carbopol); together with one or more preservatives, selected from the group including, but not limited to for example from 0.1 to 2% of methyl 4-hydroxybenzoate (methyl paraben) or phenoxyethanol-differential. Another suitable base, is comprised of the desired amount of a single diarylalkane or a mixture of diarylalkanes, together with from 70 to 90% of polyethylene glycol (for example, polyethylene glycol ointment containing 40% of polyethylene glycol 3350 and 60% of polyethylene glycol 400, prepared in accordance with the U.S. National Formulary (USNF)), from 5 to 20% of water, from 0.02 to 0.25% of an anti-oxidant (for example butylated hydroxytoluene), and from 0.005 to 0.1% of a chelating agent (for example ethylenediamine tetraacetic acid (EDTA)).

[0110] The term soft paraffin as used above encompasses the cream or ointment bases white soft paraffin and yellow soft paraffin. The term lanolin encompasses native wool fat and purified wool fat. Derivatives of lanolin include in particular lanolins which have been chemically modified in order to alter their physical or chemical properties and synthetic equivalents of lanolin include in particular synthetic or semisynthetic compounds and mixtures which are known and used in the pharmaceutical and cosmetic arts as alternatives to lanolin and may, for example, be referred to as lanolin substitutes.

[0111] One suitable synthetic equivalent of lanolin that may be used is the material available under the trademark Softisan known as Softisan 649. Softisan 649, available from Dynamit Nobel Aktiengesellschaft, is a glycerine ester of natural vegetable fatty acids, of isostearic acid and of adipic acid; its properties are discussed by H. Hermsdorf in Fette, Seifen, Anstrichmittel, Issue No. 84, No. 3 (1982), pp. 3-6.

[0112] The other substances mentioned hereinabove as constituents of suitable ointment or cream bases and their properties are discussed in standard reference works, for example pharmacopoeia. Cetomacrogol 1000 has the formula CH.sub.3(CH.sub.2).sub.m(OCH.sub.2CH.sub.2)OH, wherein m may be 15 or 17 and n may be 20 to 24. Butylated hydroxytoluene is 2,6-di-tert-butyl-p-cresol. Nipastat is a mixture of methyl, ethyl, propyl and butyl 4-hydroxybenzoates.

[0113] The compositions of the invention may be produced by conventional pharmaceutical techniques. Thus the aforementioned compositions, for example, may conveniently be prepared by mixing together at an elevated temperature, preferably 60-70 C., the soft paraffin, liquid paraffin if present, and lanolin or derivative or synthetic equivalent thereof. The mixture may then be cooled to room temperature, and, after addition of the hydrated crystalline calcium salt of mupirocin, together with the corticosteroid and any other ingredients, stirred to ensure adequate dispersion.

[0114] Regardless of the manner of administration, the specific dose is calculated according to the approximate body weight of the host. Further refinement of the calculations necessary to determine the appropriate dosage for treatment involving each of the above mentioned formulations is routinely made by those of ordinary skill in the art and is within the scope of tasks routinely performed by them without undue experimentation, especially in light of the dosage information and assays disclosed herein. These dosages may be ascertained through use of the established assays for determining dosages utilized in conjunction with appropriate dose-response data.

[0115] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention.

EXAMPLES

Example 1

Preparation of Organic Extracts from Dry Plants

[0116] Dried plant material was ground to a particle size of no larger than 2 mm and a portion (60 g) was transferred to an Erlenmeyer flask and extracted with 600 ml of methanol:dichloromethane (1:1). The mixture was shaken for one hour, filtered and the biomass was extracted again with methanol:dichloromethane (1:1) (600 ml). The organic extracts were combined and evaporated under vacuum to provide an organic extract from each plant material. Each extract (approximately 75 mg) was then dissolved in 1.5 ml DMSO to a concentration of 50 mg/ml, which was then stored in a 70 C. freezer. An aliquot of the extract solution was used for tyrosinase assay as described in Example 2.

Example 2

Tyrosinase Inhibition Assay

[0117] A tyrosinase inhibition assay was carried out using the method reported by Jones et al. (2002) Pigment. Cell Res. 15:335. Using this method, the conversion of L-Dopa, a substrate of tyrosinase, into dopachrome is followed by monitoring absorption at 450 nm. Tyrosinase was prepared in 50 mM potassium phosphate buffer, pH 6.8 (assay buffer) at 2000 U/ml and stored at 20 C. in 1 ml aliquots prior to use. For use in assays, stock enzyme solutions were thawed and diluted to 200 U/ml with assay buffer. A 2 mM working solution of substrate, L-DOPA, was prepared in assay buffer for each assay. Samples were dissolved in 10% DMSO (0.5 ml) and diluted to 5 ml with assay buffer. The reaction mixture consisted of 0.050 ml 2 mM L-DOPA, 0.050 ml 200 U/ml mushroom tyrosinase and 0.050 ml inhibitor. Reaction volume was adjusted to 200 l with assay buffer. Assays were performed in 96 well Falcon 3097 flat-bottom microtiter plates (Beckton Dickinson, N.J.). Appearance of dopachrome was measured with a WALLAC 1420 Multilable Counter (Turku, Finland). Average velocity was determined from linear enzyme rate as measured by change in absorbance (A.sub.450) at 450 nm per minute. Percent inhibition of tyrosinase by test samples was determined by comparison of absorbance of samples versus control using formula (I):

(Negative control absorptionsample absorption)/Negative control absorption100(1)

[0118] The results are set forth in Table 1.

TABLE-US-00001 TABLE 1 Tyrosinase inhibitory activity of four plant extracts Plant Latin Weight of Percent Inhibition Name and the Organic of Tyrosinase Parts Amount Extract (concentration mg/ml) Broussonetica 20 g 1.1 g 68% kazinoki whole (at 0.125 mg/ml) plant Rhus chinensis 20 g 12.8 g 31% cecidiums (at 0.125 mg/ml) Polygonum 20 g 2.4 g 43% multiflorum (at 0.125 mg/ml) tubers Dianella 20 g 1.7 g 57% ensifolia whole (at 0.125 mg/ml) plant

Example 3

HTP Fractionation of Active Plant Extracts

[0119] Active organic extract (400 mg) was loaded onto a prepacked, normal phase, flash column. (2 cm ID8.2 cm, 10 g silica gel). The column was eluted using a Hitachi high throughput purification (HTP) system with a gradient mobile phase of (A) 50:50 EtOAc:hexane and (B) methanol from 100% A to 100% B in 30 minutes at a flow rate of 5 mL/min. The separation was monitored using a broadband wavelength UV detector and the fractions were collected in a 96-deep-well plate at 1.9 mL/well using a Gilson fraction collector. The sample plate was dried under low vacuum and centrifugation. DMSO (1.5 mL) was used to dissolve the samples in each cell and a portion (100 L) was taken for the tyrosinase inhibition assay in duplicate.

Example 4

Extraction, Separation and Purification of 1-(2-Methoxy-4-Hydroxyphenyl)-3-(2-Hydroxy-5-Methoxyphenyl)-Propane (1) from Broussonetia kazinoki (BK) (Whole Plant)

[0120] ##STR00008##

[0121] Broussonetia kazinoki (100 g whole plant) was ground and extracted three times with 800 ml of MeOH:DCM (1:2). Dried extract (6 g) was fractionated using a silica gel column with gradient solvent elution of hexane/ethyl acetate (50/50) to MeOH. Fractions were collected in 2 sets of 88 test tubes. LC/MS/PDA was utilized to check each of the fractions, which were then combined based on the similarity of their composition. The combined fractions were evaporated to remove solvent, dried and their tyrosinase inhibition activity measured as described in Example 2. It was found that fractions (P0346-HTP-F2-P0346-HTP-F4) were the most active and these fractions were combined and labeled as BK-F2-4. After solvent evaporation, BK-F2-4 was further separated on a pre-packed reverse phase column (C-18 column) using a water/MeOH gradient. Eighteen compound peaks were observed following separation. Fourteen reverse phase columns were performed and the similar fractions from each run were combined. One compound peak referred to as UP288 in the combined and enriched fraction showed strong tyrosinase inhibition activity (FIG. 4). After separation and purification with preparative HPLC, 6 mg of 1-(2-methoxy-4-hydroxyphenyl)-3-(2-hydroxy-5-methoxyphenyl)-propane (UP288) (1) was obtained. The structure of this compound was elucidated using MS and NMR spectroscopy (1H, .sup.13C, HMQC and HMBC). FIG. 5 depicts the chemical structure and .sup.13C-NMR spectrum of UP288. UP288 is an inhibitor of tyrosinase having activity comparable with that of kojic acid with IC.sub.50 value of 24 M. FIG. 6 illustrates tyrosinase inhibitory dose response curves and IC.sub.50 values for UP288 and kojic acid.

[0122] 1-(2-Methoxy-4-hydroxyphenyl)-3-(2-hydroxy-5-methoxyphenyl)-propane (UP288). Yield 0.006% (purity >96%, HPLC); UV .sub.max: 281.0 nm; MS (Super Sound Ionization, Positive ion detection): m/z 289 (M+1, 100%); .sup.1H-NMR (400 MHz, (CD.sub.3).sub.2SO): 1.70 (2H, m, CH.sub.2), 2.46 (4H, m, 2 CH.sub.2), 3.68 (3H, s, OCH.sub.3), 3.73 (3H, s, OCH.sub.3), 6.26 (1H, q, H-5), 6.35 (1H, d, H-3), 6.55 (1H, q, H-14), 6.65 (1H, d, H-13), 6.72 (1H, d, H-16), 6.86 (1H, d, H-6), 8.69 (1H, s, OH), 9.20 (1H, s, OH); .sup.13C-NMR (100 MHz, (CD.sub.3).sub.2S0): A 28.5 (C-8), 31.6 (C-9), 34.5 (C-10), 55.0 (C-7), 55.6 (C-17), 98.9 (C-3), 106.4 (C-5), 112.4 (C-16), 115.2 (C-13), 119.7 (C-1), 119.8 (C-14), 120.3 (C-11), 120.4 (C-6), 132.9 (C-12), 144.6 (C-4), 147.2 (C-17) & 158.3 (C-7).

Example 5

Extraction, Separation and Purification of 1-(3-Methyl-2,4-Dimethoxyphenyl)-3-(2,4-Dihydroxyphenyl)-Propane (UP302a) (2) and 1-(3-Methyl-2,4-Dimethoxyphenyl)-3-(2,5-Dihydroxyphenyl)-Propane (UP302b) (3) from Dianella ensifolia (P0389) (Whole Plant)

[0123] ##STR00009##

[0124] Dianella ensifolia (P0389, 300 g whole plant) was ground and extracted three times 800 ml of MeOH:DCM (1:2). Dried extract (5 g) was fractionated using a silica gel column with gradient solvent elution of hexane/ethyl acetate (50/50) to MeOH. Fractions were collected in 2 sets of 264 test tubes. LC/MS/PDA was utilized to check each of the fractions, which were then combined into 22 fractions based on the similarity of their composition. (FIG. 7). The combined fractions were evaporated to remove solvent, dried and their tyrosinase inhibition activity measured as described in Example 2. It was found that fractions P0389-HTP-F12, P0389-HTP-F13 and P0389-HTP-F14 were the most active and these fractions were combined and relabeled as DE-F12-14. After solvent evaporation, DE-F12-14 was further separated on a pre-packed reverse phase column (RP-column) using a water/MeOH gradient. Two major and eleven minor compound peaks were observed following separation. The compounds corresponding to each of these peaks were isolated following 7 additional separations on RP-columns. All of the compounds collected were dried and tested for tyrosinase inhibitory activity. Two of the eleven minor peaks referred to as UP302a and UP302b, respectively, exhibited strong tyrosinase inhibitory activity. (FIG. 8). After separation and purification, two active compounds were obtained: 1-(3-methyl-2,4-dimethoxyphenyl)-3-(2,4-dihydroxyphenyl)-propane (UP302a, 10 mg) (2) and 1-(3-methyl-2,4-dimethoxyphenyl)-3-(2,5-dihydroxyphenyl)-propane (UP302b, 6 mg) (3). The structures of these two compounds were elucidated using MS and NMR spectroscopy (1H, .sup.13C, gHSQC and HMBC). FIG. 9 depicts the gHSQC spectrum of UP302a. Tyrosinase inhibition assays showed that UP302a was the most potent inhibitor with an IC.sub.50 of 0.24 M, while UP302b has an IC.sub.50 of 12 M.

[0125] 1-(3-methyl-2,4-dimethoxyphenyl)-3-(2,4-dihydroxyphenyl)-propane (UP302a) (2). Yield 0.02% (purity >98%, HPLC); UV .sub.max: 279.8 nm; MS (Super Sound Ionization, Positive ion detection): m/z 303 (M+1, 100%); .sup.1H-NMR (400 MHz, (CD.sub.3).sub.2SO): 1.70 (2H, m, CH.sub.2), 2.03 (3H, s, CH.sub.3), 2.43 (2H, m, CH.sub.2), 2.49 (2H, m, CH.sub.2), 3.58 (3H, s, OCH.sub.3), 3.73 (3H, s, OCH.sub.3), 6.11 (1H, q, H-16), 6.25 (1H, d, H-14), 6.65 (.sup.1H, d, H-5), 6.76 (1H, d, H-17), 6.97 (1H, d, H-6), 8.93 (1H, s, OH), 9.03 (1H, s, OH); .sup.13C-NMR (100 MHz, (CD.sub.3).sub.2SO): 28.8 (C-9), 29.3 (C-11), 31.1 (C-10), 55.3 (C-7), 55.9 (C-8), 102.4 (C-14), 105.8 (C-16), 106.1 (C-5), 118.4 (C-1), 118.6 (C-12), 126.9 (C-3), 127.0 (C-6), 130.1 (C-17), 155.7 (C-13), 156.2 (C-15), 156.3 (C-4) and 156.8 (C-2).

[0126] 1-(3-methyl-2,4-dimethoxyphenyl)-3-(2,5-dihydroxyphenyl)-propane (UP302b) (3). Yield 0.01% (purity >95%, HPLC); UV .sub.max: 279.8 nm; MS (Super Sound Ionization, Positive ion detection): m/z 303 (M+1, 100%); .sup.1H-NMR (400 MHz, (CD.sub.3COCD.sub.3): 1.82 (2H, m, CH.sub.2), 2.07 (3H, s, CH.sub.3), 2.52 (2H, m, CH.sub.2), 2.56 (2H, m, CH.sub.2), 3.63 (3H, s, OCH.sub.3), 3.77 (3H, s, OCH.sub.3), 6.64 (1H, q, H-15), 6.72 (1H, d, H-14), 6.64 (1H, d, H-5), 6.70 (1H, d, H-17), 7.00 (1H, d, H-6), 7.65 (1H, s, OH), and 7.69 (1H, s, OH).

Example 6

Large-Scale Isolation of 1-(3-Methyl-2,4-Dimethoxyphenyl)-3-(2,4-Dihydroxyphenyl)-Propane (UP302a) (2) from Dianella ensifolia (DE) (Whole Plant)

[0127] Dianella ensifolia (4.3 kg whole plant) was collected, ground and extracted three times using a percolation extractor with methanol as the solvent. The extracts were combined and evaporated to remove the methanol. The crude extract was then suspended in water and partitioned with DCM. The layers were separated and the DCM layer was evaporated to provide 60 g of material. LC-MS/PDA analysis of both layers revealed that the majority of the UP302a was present in the DCM layer with only a minor amount present in the water layer. The DCM extract was fractionated on three separate silica gel columns eluting with a gradient of hexane-ETOAC. A total of 15 sub-fractions were obtained and analyzed by HPLC-MS/PDA. Targeted compound (UP302a) was found in fractions 6 to 9, which were combined to yield a total of 3 g of enriched UP302a. The enriched UP302a was further separated on an open column packed with CG-161 resin eluting with a water-MeOH gradient. A total of 23 fractions were collected with the UP302a eluting in fractions 15 to 21. Fractions 15-21 were then combined and the solvent was evaporated to yield 700 mg of a solid, which was further purified by preparative HPLC on a C-18 column to generate 30 mg of UP302a. The structure, tyrosinase inhibitory activity and purity of the purified product was confirmed by NMR, enzyme inhibition assay and LC-MS/PDA.

Example 7

Synthesis of Diarylalkanes by Sodium Borohydride Reduction of Substituted Chalcones

[0128] A general method for the synthesis of diarylalkanes by sodium borohydride reduction of substituted chalcones is described below using the reduction of 2,4-dihydroxy)-3,4-dimethoxychalcone (4) for purposes of illustration.

##STR00010##

[0129] 2,4-Dihydroxy-3,4-dimethoxychalcone (4) (40 mg) was dissolved in 1-propanol (5 ml), followed by the addition of sodium borohydride (15 mg) and the mixture was allowed to react at room temperature for 2 hours. Upon completion of the reaction, 20% acetic acid (0.2 ml) was added and the mixture was heated at 80 C. for 5 minutes and cooled down. The mixture was then separated on a pre-packed C.sub.18 column eluting with a MeOH/H.sub.2O gradient to provide 1-(2,4-dihydroxyphenyl)-3-(3,4-dimethoxyphenyl)-1-propanol (5). The structure of compound (5) was confirmed by MS, UV spectroscopy, 1D and 2D .sup.1H-NMR.

[0130] 1-(2,4-dihydroxyphenyl)-3-(3,4-dimethoxyphenyl)-1-propanol (5). Yield 60% (purity >98%, HPLC); UV .sub.max: 278.5 nm; MS (Super Sound Ionization, Positive ion detection): m/z 305 (M+1, 100%); .sup.1H-NMR (400 MHz, (CD.sub.3).sub.2SO): 1.93 (2H, m, CH.sub.2), 2.60 (2H, m, CH.sub.2), 4.49 (1H, m, CHOH), 3.78 (3H, s, OCH.sub.3), 3.80 (3H, s, OCH.sub.3), 6.28 (1H, q, H-5), 6.31 (1H, d, H-3), 6.98 (1H, d, H-6), 6.71 (1H, q, H-5), 6.77 (1H, d, H-2), 6.83 (1H, d, H-6).

[0131] Using the above-described general method the following compounds were reduced to their corresponding alcohols: 2,4-dihydroxy-2-hydroxychalcone, 2-hydroxy-4-methoxy-2,4-dimethoxy-chalcone, 4-hydroxy-4-hydroxy-chalcone, 2,4-dihydroxy-2-hydroxy-chalcone, 2,4-dihydroxy-3,4-dimethoxy-chalcone, 2,4,6-trimethoxy-3,4-dimethoxy-chalcone and 2-hydroxy-4-methoxy-3,4,5-trimethoxy-chalcone to provide 1-(2,4-dihydroxyphenyl)-3-(2-hydroxyphenyl)-1-propanol, 1-(2-hydroxy-4-methoxyphenyl)-3-(2,4-dimethoxyphenyl)-1-propanol, 1-(4-hydroxyphenyl)-3-(4-hydroxyphenyl)-1-propanol, 1-(2,4-dihydroxyphenyl)-3-(2-hydroxyphenyl)-1-propanol, 1-(2,4-dihydroxyphenyl)-3-(3,4-di-methoxyphenyl)-1-propanol, 1-(2,4,6-trimethoxyphenyl)-3-(3,4-dimethoxyphenyl)-1-propanol and 1-(2-hydroxy-4-methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-1-propanol.

Example 8

Synthesis of Substituted Diphenylpropanols by Sodium Borohydride Reduction of Substituted Diarylpropanones

[0132] A general method for the synthesis of substituted diphenylpropanols by sodium borohydride reduction of substituted diarylpropanones is described below using the reduction of 1-(2-hydroxy-5-methoxyphenyl)-3-(2,4-dimethoxyphenyl)-1-propanone (6) for purposes of illustration.

##STR00011##

[0133] 1-(2-hydroxy-5-methoxyphenyl)-3-(2,4-dimethoxyphenyl)-1-propanone (6) (5 mg) was dissolved in 1-propanol (1 ml), followed by the addition of sodium borohydride (2 mg) and the mixture was allowed to react at room temperature for 5 hours. Upon completion of the reaction, 20% acetic acid (0.2 ml) was added to neutralize the excess sodium borohydride. The reaction mixture was then separated on a pre-packed C.sub.18 column eluting with a MeOH/H.sub.2O gradient to provide 1-(2-hydroxy-5-methoxyphenyl)-3-(2,4-dimethoxyphenyl)-1-propanol (7).

[0134] Following the above-described general synthetic procedure the following diarylalkane compounds were reduced: 1-(2-hydroxy-4,6-dimethoxyphenyl)-3-(3-methoxy-4-hydroxyphenyl)-1-propanone, 3-(5-benzyloxy-4-methoxy-2-methylphenyl)-1-(2-hydroxy-4,5-dimethoxyphenyl)-1-propanone, 1-(2-hydroxy-4-methoxyphenyl)-3-(2,3,4,5-tetrahydro-bezo(b)dioxocin-8-yl)-1-propanone and 3-(5-chloro-1-methyl-1-hydro-imidazol-2-yl)-1-(2-hydroxy-4-methoxyphenyl)-1-propenone to provide 1-(2-hydroxy-4,6-dimethoxyphenyl)-3-(3-methoxy-4-hydroxyphenyl)-1-propanol, 3-(5 -benzyloxy-4-methoxy-2 -methylphenyl)-1-(2-hydroxy-4,5-dimethoxyphenyl)-1-propanol, 1-(2-hydroxy-4-methoxy-phenyl)-3-(2,3,4,5-tetrahydro-bezo(b)dioxocin-8-yl)-1-propenol and 3-(5-chloro-1-methyl-1-hydro-imidazol-2-yl)-1-(2-hydroxy-4-methoxy-phenyl)-1-propenol, respectively.

Example 9

Synthesis of 1,3-Bis(2,4-Dimethoxyphenyl)-Propan-1,3-Diol (9)

[0135] ##STR00012##

[0136] 1,3-Bis(2,4-dimethoxyphenyl)-propan-1,3-dione (8) (5 mg) was dissolved in 1-propanol (1 ml), followed by the addition of sodium borohydride (3 mg) and the mixture was allowed to react at room temperature for 3 hours. Upon completion of the reaction, 20% acetic acid (0.2 ml) was added to neutralize the excess sodium borohydride. The mixture was then separated on a pre-packed C.sub.18 column eluting with a MeOH/H.sub.2O gradient to provide 1,3-bis(2,4-dimethoxyphenyl)-propan-1,3-diol (9).

Example 10

Synthesis of 1-(2,4,6-Trihydroxyphenyl)-3-(3 -Hydroxy-4-Methoxyphenyl)-1-Propanol (11) from Neohesperidin

[0137] ##STR00013##

[0138] Neohesperidin is a glycoside of dihydrochalcone. A total weight of 100 mg of neohesperidin was suspended in 10 ml of 1 N HCl and heated at 80 C. for 2 hours. The hydrolyzed product (10) was cooled down and extracted with ethyl acetate (310 ml). The ethyl acetate layers were combined, evaporated to remove ethyl acetate and dissolved in 1-propanol (5 ml). Sodium borohydride (25 mg) was added to the propanol solution and stirred at room temperature for 2 hours. After the completion of the reaction, the mixture was separated on a pre-packed C.sub.18 column eluting with a MeOH/H.sub.2O gradient to provide 1-(2,4,6-trihydroxyphenyl)-3-(3 -hydroxy-4-methoxyphenyl)-1-propanol (11).

Example 11

Extraction, Purification and Structure Modification of Butrin to Synthesize 142,4-Dihydroxyphenyl)-3-(3 ,4-Dihydroxyphenyl)-1-Propanol (14)

[0139] ##STR00014##

[0140] Butrin is a high content flavanone-glycoside that has been extracted with methanol from the dried flowers of Butea frondosa and purified by multiple reverse phase column chromatographic separations. After removing sugars by hydrolysis with HCl, butin (12) was produced and purified by RP-HTP (1.5% yield from the whole plant). Butin was then treated with 10% sodium hydroxide at 80 C. to obtain butein (13), which was reduced with sodium borohydride to obtain 1-(2,4-dihydroxyphenyl)-3-(3,4-dihydroxyphenyl)-1-propanol (14) (IC.sub.50=250 nM).

Example 12

Synthesis of 1-(2,4-Dihydroxyphenyl)-3-(3 -Methoxy-4-Hydroxyphenyl)-1-Propanol (19)

[0141] ##STR00015##

[0142] Resorcinol (15), 3-methoxy-4-hydroxy-cinnamic acid (16) and H.sub.2SO.sub.4 (5%) were refluxed in THF for 4 hours to provide 7,4-dihydroxy-3-methoxy flavanone (17) (90% yield). The product, 7,4-dihydroxy-3-methoxy flavanone (17) was then treated with 10% sodium hydroxide at 80 C. for 1 hour, followed by reduction with sodium borohydride in propanol to provide, as confirmed LC-MS/PDA detection, 1-(2,4-dihydroxyphenyl)-3-(3-methoxy-4-hydroxyphenyl)-1-propanol (19). The crude product exhibits quite strong tyrosinase inhibitory activity. The mixture was further purified by HTP.

Example 13

IC.SUB.50 .Measurements of Tyrosinase Inhibition by Synthetic Diarylalkanes

[0143] Inhibition of tyrosinase by synthetic diarylalkanes was measured using the method described in Example 2. The IC.sub.50 value of each sample was calculated using kinetics software to verify that the reaction was linear at a specified time and concentration. Using the methods described in Examples 7-12a total of 24 compounds were synthesized and evaluated for their ability to inhibit tyrosinase. The results are set forth in Table 2.

TABLE-US-00002 TABLE 2 IC.sub.50 values of synthetic diarylalkanes and/or diarylalkanols Tyrosinase Inhibition Compound Name (IC.sub.50) 1-(2,4-dihydroxyphenyl)-3-(3,4-dihydroxyphenyl)-1-propanol 0.5 m 1-(2,4-dihydroxyphenyl)-3-(3,4-dimethoxyphenyl)-1-propanol 0.85 m 1-(2,4-dihydroxyphenyl)-3-(2-hydroxyphenyl)-1-propanol 0.7 m 1-(2,4-dihydroxyphenyl)-3-(2-methoxyphenyl)-1-propanol 3 m 1-(2,4-dihydroxyphenyl)-3-(4-methoxyphenyl)-1-propanol 6 m 1-(2,4,6-trihydroxyphenyl)-3-(4-aminophenyl)-1-propanol 8 m 1-(2,4-dihydroxyphenyl)-3-phenyl-1-propanol 8 m 1-(2,4-dihydroxyphenyl)-3-(3-methoxy-4-hydroxyphenyl)-1-propanol 8.5 m 1-(2-hydroxy-4-methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-1-propanol 11 m 1-(2-hydroxy-4-methoxyphenyl)-3-(2,4-dimethoxyphenyl)-1-propanol 25 m 1-(2-hydroxy-5-methoxyphenyl)-3-(3,4-dimethoxyphenyl)-1-propanol 30 m 1-(2,4-dihydroxyphenyl)-2-(4-methoxyphenyl)-1-ethanol 77 m 1-(2-hydroxy-4-methoxyphenyl)-3-(2,3,4,5-tetrahydro-benzo(b)dioxocin-8-yl)- 72 m 1-propanol 3-(5-chloro-1-methyl-1-hydro-imidazol-2-yl)-1-(2-hydroxy-4-methoxyphenyl)- 225 m 1-propanol 1-(4-hydroxyphenyl)-3-(4-hydroxyphenyl)-1-propanol 305 m 1-(2-hydroxy-4,6-dimethoxyphenyl)-3-(3-methoxy-4-hydroxyphenyl)-1-propanol 375 m 1-(2,4-dihydroxyphenyl)-2-(3,4-dimethoxyphenyl)-1-ethanol 431 m 1,4-bis-(3,4-dihydroxyphenyl)-2,3-dimethylbutane 700 m 1-(2-hydroxy-5-methoxyphenyl)-3-(2,4-dimethoxyphenyl)-1-propanol 1000 m 1-(2,4-dihydroxyphenyl)-2-(2,4-dichlorophenyl)-1-ethanol 1000 m 1-(2,4,6-trihydroxyphenyl)-3-(3-hydroxy-4-methoxyphenyl)-1-propanol 1200 m 1,3-bis(2,4-dimethoxyphenyl)-propan-1,3-diol 1200 m 1-(2,4,6-trihydroxyphenyl)-3-(3-hydroxy-4-methoxyphenyl)-1-propanol 1200 m 1-(2,4,6-trimethoxyphenyl)-3-(3,4-dimethoxyphenyl)-1-propanol 1500 m

Example 14

Enzyme Inhibition Kinetics

[0144] Using the method described in Example 2, the inhibition of tryosinase was evaluated at different concentrations (0, 261, 522, 1044 nM) of an inhibitor (UP302a) using L-DOPA at concentrations of 0.75, 1.25, and 2.5 mM as the substrate. As shown in FIG. 10, it was found that UP302a is a competitive inhibitor with potent and long lasting inhibitory effect. Tyrosinase activity was not resumed for several days after incubation with UP302a. In contrast, tyrosinase activity was totally restored after only 1 hour following incubation with kojic acid.

Example 15

Inhibition of Melanin Production from B-16 Cell Line

[0145] The inhibition of melanin production was evaluated using two different assays. In the first assay, the inhibition of melanin production was evaluated without induction by -MSH; whereas in the second assay the inhibition of melanin production was measured with -MSH induction in cell culture medium. Briefly, B16 F1 cells (ATCC #CRL-622) were grown to confluency and then seeded at 40,000 cells per well. The cells were allowed to attach overnight at 37 C. in a humidified environment containing 5% CO.sub.2. On day 2, inhibitors were added to the cells at concentrations ranging from 0-1000 M in triplicate and allowed to incubate for four days. The amount of -MSH required to induce melanin formation was determined by the addition of hormone at concentrations ranging from 0-1000 nM in ten-fold increments. Once the proper -MSH concentration was determined, cells were seeded as above, allowed to attach overnight and then co-incubated with tyrosinase inhibitors at concentrations ranging from 0-1000 M. Color development was visually monitored each morning. After the development of color, 200 l of cell supernatant was removed from each well and absorbance was measured at 450 nm. The resulting readings were used to determine the IC.sub.50 for melanin formation in the cell assay with and without -MSH induction. For an initial comparison of cell toxicity, the 250 M treated wells were used to perform a lactate dehydrogenase assay (LDH). LDH is a metabolic enzyme that leaks out of damaged or dead cells. The enzyme converts a chromophore in the presence of NAD.sup.+ to yield a color change that can be monitored spectrophotometrically.

[0146] The results of this assay revealed that all of the natural inhibitors tested (UP288, UP302a, and UP302b) are at least as good, if not better inhibitors than kojic acid. There were some differences in the IC.sub.50 values under the two sets of conditions. Inhibition by kojic acid improved from an IC.sub.50 of 170 M for the endogenous experiment to an IC.sub.50 of 67 M in the induced experiment. Of the inhibitors tested relative to kojic acid, compound UP302b was only one that that showed an increase in IC.sub.50 under the two sets of conditions increasing from an IC.sub.50 of 5.2 M to an IC.sub.50 of 34 M. The IC.sub.50's measured for inhibition of tyrosinase were relatively the same for all of the compounds tested with the exception of the two compounds UP302 and UP302b, which had low IC.sub.50's of 0.2 M and 0.3 M, respectively, compared to 28 M and 5.2 M in the endogenous assay and 40 M and 34 M in the induced assay. These differences may be due to decreased cell penetration by UP302a (2) and UP302b (3), as compared to the other inhibitors. This is overcome, however by the strength of their inhibition of the enzyme.

[0147] Table 3 provides the results of these two assays for inhibitors UP288 and UP302a relative to kojic acid.

Example 16

Cell Toxicity Assay

[0148] The compound treated wells were used to perform a lactate dehydrogenase assay (LDH). LDH is a metabolic enzyme that leaks out of damaged or dead cells. The enzyme converts a chromophore in the presence of NAD to yield a color change that can be monitored spectrophotometrically. The cytotoxicity was examined at a concentration of 250 M. At this concentration none of these compounds are significantly more cytotoxic than kojic acid. It should be noted however, that cytotoxicity at only one concentration (250 M) was tested. As shown in the Table 3, UP288 (1) and UP302a (2) showed moderate cytotoxicity, which were comparable with kojic acid.

TABLE-US-00003 TABLE 3 Inhibition of mushroom tyrosinase and melanin formation in mouse B16 F1 cells by isolated compounds and comparison of cell toxicity Endogenous MSH Induced Tyrosinase Melanin Melanin Cell Inhibition Inhibition Inhibition Toxicity Compound IC.sub.50 (M) IC.sub.50 (M) IC.sub.50 (M) (LDH) UP288 24.0 108 105 0.315 UP302a 0.24 28 40 0.265 Kojic acid 29 170 67 0.260

Example 17

Molecular Mechanics (MM2) Calculation

[0149] Molecular mechanics calculations were performed using Chem3D software for purposes of energy minimization and determination of the most stable 3-D conformation. The following parameters were used: Step interval=2.0 fs, frame interval=10 fs, terminate after 10,000 steps, heating/cooling rate=1.000 Kcal/atom/PS, target temperature=3000K. Properties: pi bond orders and steric energy summary. All natural and synthetic compounds and other diarylalkane and diarylalkanol structures were analyzed. It was found that the most potent tyrosinase inhibitor1-(3-methyl-2,4-dimethoxyphenyl)-3-(2,4-dihydroxyphenyl)-propane (UP302a (2), IC.sub.50=0.24 M)isolated from whole plants of Dianella ensifolia (L.) DC. has a very unique 3-dimensional conformation in which the two aromatic rings were superimposed on each other. The minimized total energy for the conformation is 4.7034 KJ/Mol. The distance between the two aromatic rings was 3.28 . The phenolic hydroxyl groups on the first aromatic ring were right above the two methoxyl groups on the second aromatic ring with the distance between two oxygen atoms being 2.99 and 3.16 , respectively as illustrated in FIGS. 12-14. This intramolecular parallel conformation allows this compound to perfectly chelate both copper ions of the binuclear enzyme when it is in the peroxide form [Cu.sup.II-O.sub.2-Cu.sup.II] from both the top and the bottom.

Example 18

Formulation of the Diarylalkane Composition into a Cream

[0150] UP302a is comprised of a substituted diarylpropane as the major active component. These compounds are soluble in high polarity solvents including, but not limited to ethanol, propylene glycol and ethylene glycol. They can be formulated with a pharmaceutically and/or cosmetically acceptable excipient, an adjuvant, and/or a carrier. Examples of such excipients include, but are not limited to water, buffers, saline, Ringer's solution, dextrose solution, mannitol, Hank's solution, preservatives and other aqueous physiologically balanced salt solutions. Nonaqueous vehicles including, but not limited to fixed oils, sesame oil, ethyl oleate, or triglycerides may also be used. Other useful formulations include, but are not limited to suspensions containing viscosity enhancing agents, including, but not limited to sodium carboxymethylcellulose, sorbitol, or dextran. Excipients can also contain minor amounts of additives or preservatives, such as antioxidants that enhance color and chemical stability. UP302 also can be prepared in a liposome formulation to increase its skin penetration or as a controlled release formulation, which slowly releases the composition of the active ingredient into the host.

[0151] UP302a is preferably administered topically as an ointment, gel, lotion, or cream base or as an emulsion, a patch, dressing or mask, a nonsticking gauze, a bandage, a swab or a cloth wipe. Such topical application can be locally administered to any affected area, using any standard means known for topical administration. UP302 can be administered to both humans and animals.

[0152] A therapeutic composition of UP302a can be administered in a variety of unit dosage forms depending upon the method of administration and targeted indications. An efficacious, nontoxic quantity is generally recommended in the range of 0.01% to 5% based on total weight of the topical formulation. Two different concentrations of UP302a (0.01% and 0.5% by weight) were formulated in creams as illustrated in Tables 4 and 5. To prepare these creams the diarylalkane was dissolved in water at room temperature and homogenized in a blender until it was fully dispersed in solution (approximately 5 minutes) to yield a composition A. At room temperature and without stifling or agitating, Ultrez-21 carbomer was added to the homogenized solution by sprinkling it onto the surface and allowing it to fully wet (no white areas visible) and fall into the solution. With gentle stifling, the solution was then heated to 40 C. and glycerin was added and the composition was mixed for an additional 5 minutes to provide Composition B. At 40 C., Composition A is added to Composition B and the composition is mixed well until homogenous (approximately 5 minutes). The resulting emulsion is cooled to 30 C. and the pH adjusted to approximately 5.5 (5.3 to 5.7) by titrating with neutralizer while stifling with a stir bar and/or spatula. The emulsion became highly viscous due to the neutralization-induced conformational change of the carbomer. Upon stifling the emulsion will achieved a suitable viscosity for an emulsion cream. The composition was mixed until uniform, poured into clean storage vessels and stored at 2 C. to 8 C.

TABLE-US-00004 TABLE 4 Composition of 0.01% Diarylalkane Cream Phase Ingredient % (w/w) Weight (g) Aqueous Water, Purified 85.00 12 Diarylalkane (UP302a) 0.01 0.0015 Ultrez 21 Carbomer 0.50 0.075 Glycerin 8.00 1.2 Oil PEG-7 Glyceryl Cocoate 3.00 0.45 Caprylic/Capric Triglyceride 2.67 0.4 PH Neutralizer Sodium Hydroxide (18% w/v), 0.00 0.0 Molecular Biology Grade SUM 7 Ingredients 100 15

TABLE-US-00005 TABLE 5 Composition of 0.1% UP302 Cream Phase Ingredient % (w/w) Weight (g) Aqueous Water, Purified 84.00 12.6 Diarylalkane (UP302a) 0.1 0.0015 Ultrez 21 Carbomer 0.50 0.075 Glycerin 8.00 1.2 Oil PEG-7 Glyceryl Cocoate 3.00 0.45 Caprylic/Capric Triglyceride 2.67 0.4 PH Neutralizer Sodium Hydroxide (18% w/v), Molecular Biology Grade SUM 7 Ingredients 99.7 15