WO2009126700A1 - Hydroxylated tolans and related compounds as cosmetics or therapeutics for skin conditions - Google Patents

Abstract

Compounds of the hydroxytolan family induce SIRT1 enzymatic activity or inhibit tyrosinase activity, and act to inhibit, counteract or prevent skin conditions associated with aging such as wrinkling, undesirable pigmentation and treat or prevent various skin conditions or diseases, including inhibition of cutaneous inflammation. Topical administration of cosmetic or pharmaceutical compositions comprising these compounds are used to treat the above skin conditions. The most preferred hydroxytolan compounds include 4,4'-dihydroxytolan. (HT-1), 4 hydroxy 4' trifluoromethyltolan or 4' hydroxy 4 trifluoromethyltolan (HT-2), 3,4',5-trihydroxytolan or 3',4,5'-trihydroxytolan (HT-3), and 3,3',5,5'-tetrahydroxytolan (HT-4).

Description

HYDROXYLATED TOLANS AND RELATED COMPOUNDS AS COSMETICS OR THERAPEUTICS FOR SKIN CONDITIONS

BACKGROUND OF THE INVENTION

Field of the Invention The invention in the field of biochemistry and medicine relates to methods of treating skin conditions, including cosmetic and therapeutic uses, with compounds of the tolan family, preferably hydroxytolans.

Description of the Background Art

In industrialized countries, the average human life expectancy has approximately doubled over the last 100 years due to improvements in nutrition, medicine and living and working conditions. Aging, once thought to be an active continuation of a genetically programmed development of organisms, is now perceived as a loss of equilibrium between the repair potential of an organism versus the frequency and intensity of the damages to which it is exposed. Skin is the organ most exposed to environmental insults. Free radicals generated by ultraviolet (UV) radiation or by internal metabolism are the most deleterious aging agents, acting on cellular proteins, lipids, glycans, and DNA (Rocquet and Bonte, 2002). Consequently, anti-aging skin care products were developed that contained natural free-radical scavengers or active ingredients which reinforced natural cellular proteins that detoxify these free radicals. Subsequent complementary strategies were based on activation of:

(a) chaperone proteins (Nizard, et al., 2004) to protect cellular proteins structure;

(b) detoxifying proteins such as proteasome peptidases (Bulteau, et al., 2006) to recycle damaged proteins or

(c) master genes found to affect organism or cellular life span. In more recent developments, anti-aging strategies were designed to improve stress resistance by controlling key metabolic and signalling pathways as part of a fundamental mechanism for surviving adversity (Moreau, et al., 2007).

The protein Sir2 (whose homologue in mammals is known as SIRTl, SIR2L1 or Sir2α) is the namesake of a family of closely related enzymes, the sirtuins (named from "Sir-two-ns"). Sirtuins are found in human skin, in keratinocytes and fibroblasts in the "aging-responsible interface" (ARI), (Dal Farra and Domloge, 2006). The ARI includes the lower epidermis, upper deraiis, and epidermal-dermal junction. Twenty "youth markers" have been localized to the ARI; these markers change with aging and are implicated in wrinkle formation, loss of firmness or tautness of the skin, irregular pigmentation, dryness, and other signs of aging. (Bosset, et al., 2002, 2003a,b; Dumas, et al, 2005; Juan, et al, 2005; Le Varlet, et al, 1998). Int'l Patent Pub. WO 2006/103110 also disclosed the involvement of the SIRTl protein in the mechanisms of skin cells and its role in the aging process and cell protection. This document discloses expression of SIRT 1 in skin cells and the relation of that expression to different stresses on cutaneous cells. Induction of SIRTl expression using a peptide that includes a 7 residue sequence of SIRTl protected the cells and improved the manifestations of stress and intrinsic aging. Increased expression of the sirtuin SIRTl in normal human dermal skin fibroblasts in vitro and in epidermal cells of healthy human skin ex vivo decreased cell senescence and DNA fragmentation induced by ultraviolet-B (UVB) exposure. Furthermore, improvements were seen in facial fine lines and wrinkles, hydration, pigmented spot color intensity, complexion radiance, firmness, complexion homogeneity, and texture. Therefore, it appears that compounds which increase SIRTl expression may be useful in treating multiple manifestations of skin aging (Moreau, et al., 2001).

Sirtuins are phylogenetically conserved from bacteria to humans, and regulate cell functions beyond gene silencing (Guarente, 2000; Gasser and Cockell, 2001; Blander and Guarente, 2004; Buck, Gallo and Smith, 2004; Lamming, Wood and Sinclair, 2004). Indeed, recent research indicates that sirtuins have pathophysiological relevance to cancer, obesity, muscle differentiation, inflammation, diabetes and neurodegeneration. In addition, experimental evidence shows that sirtuin activity extends the lifespan of several organisms (Guarente, 2000; Blander and Guarente, 2004; Buck, Gallo and Smith, 2004; Lamming, Wood and Sinclair, 2004). SIRTl is the human orthologue of yeast Sir2 and is the best-characterized member of the mammalian sirtuins. SIRTl is a nuclear protein endowed with deacetylase activity (Bitterman, et al., 2002; Imai, et al., 2000; Fulco, et al., 2003) that interacts with several transcription regulating factors. Mammalian SIRT2 is a cytoplasmic and nuclear deacetylase that is involved in mitosis and can target histones in vitro and tubulin in vivo (North, et al., 2003). Mammalian SIRT3 which can also deacetylate histones is localized in the mitochondrial matrix (Schwer, et al., 2002; Onyango, et al., 2002), a cell compartment that typically lacks histones. Much less is known about mammalian SIRT4-7, although they are expressed widely in human cells (Blander and Guarente, 2004; Frye, 1999).

The ability of SIRTl to modulate a wide variety of cell processes is related to its ability to remove acetyl-groups from transcription factors such as p53, FOXO family molecules, and nuclear histones (Luo, et al, 2001; Motta, et al, 2004; Yeung, et al, 2004). More recently, it has been shown that SIRTl modulates NFκB-dependent transcription by interacting with, and deacetylating, the active subunit of NFKB (RelA/p65) in a site-specific manner, leading to its deactivation (Yeung, et al, supra). Since NFKB plays a fundamental role as a proinflammatory transcription factor by enhancing the expression of various cytokine genes, which are implicated in a variety of human diseases, its deactivation by SIRTl may lead to the successful management of many diseases (Nayagam, et al., 2006).

Two of the enzymes downstream from NFKB are cyclooxygenase I (COX-I) and II (COX-2). Cyclooxygenases are a family of enzymes that are responsible for formation of prostanoids including prostaglandins, prostacyclin and thromboxane. Pharmacological inhibition of COX can relieve symptoms of inflammation and pain. Because the promoters of the genes encoding COX-I and COX-2 they have a NFKB binding site, expression of these enzymes is downregulated as a result of NFKB downregulation.

Because of the importance of sirtuin (or Sir2) enzymes in many cellular processes, there is a need in the art for small molecules capable of modulating Sir2 enzyme activity. Nicotinamide was found to be the most potent inhibitor of Sir2 to date (Sauve and Schramm, 2003; Jackson, et al., 2003; Borra, et al., 2004). Nicotinamide inhibits Sir2-dependent lifespan extension (Bitterman, et al., 2002). Through phenotypic screening, small molecules such as sirtinol (Grozinger, et al., 2001), splitomicin (Bedalov, et al., 2001) and splitomicin analogues (Posakony, et al., 2004; Hirao, et al., 2003), were identified as inhibitors of Sir2. Approximately, 500,000 compounds have been evaluated for their SIRT-I modulating activity (Stipp, D. (2007). In particular, fifteen plant phenols, including quercetin, piceatannol, and resveratrol, were shown to have SIRTl-activating properties (Howitz, KT et al., 2003, Nature 425:191-6; see, also, Howitz et al, US 2006/0014705 Al (based on Appl. No. 11/166,892, filed 6/24/2005) and Howitz et al. US 2006/0084135 Al (based on Appl. No. 10/884,062, filed 7/1/2004). Of the small molecules identified and tested, resveratrol, (3,4', 5- trihydroxy-^rαwi'-stilbene, also, 3,4',5-trihydroxystilbene), was found to be the most potent SIRTl activator (Howitz et al., supra). Resveratrol

Resveratrol, the chemical formula of which is shown below,

belongs to a class of polyphenolic compounds called stilbenes (Soleas GJ et al., 2002; 2001(a); 2001(b), 2001(c), 1997(a,) 1997(b); Goldberg DM et al, 2003) and is produced by some plants in response to stress, injury, fungal infection, and UV radiation (Aggarwal, et al, 2004). Resveratrol is fat-soluble and occurs in a trans and a cis configuration and can also occur as a glucoside (Romero-Perez, et al, 1999). Resveratrol has a diverse bioactivities (Aggarwal, et al, 2004; Fremont, 2000) including: antioxidant activity, modulation of lipid and lipoprotein metabolism, inhibition of platelet aggregation, vasorelaxing activity, anticancer activity and estrogenic activity. More recently, reports on the potential for resveratrol to inhibit the development of cancer (Jang, et al, 1997) and extend lifespan (Howitz, et al, 2003, supra ) in cell culture and animal models have continued to generate scientific interest. A variety of mechanisms have been reported for resveratrol including: increased levels of cell death, activation of phase II detoxification and decreased levels of cell division, DNA synthesis and inflammation (Aggarwal, et al, 2004; Aziz, Kumar and Ahmad, 2003; Dong, 2003; Fremont, 2003; Gusman, Malonne, and Atassi, 2001; Jang, et al, 1997; Mitchell, Zhu and Young, 1999; Savouret and Quesne, 2002; Signorelli and Ghidoni, 2005).

Resveratrol was reported to inhibit cell proliferation and cause apoptotic cell death by modulating numerous mediators of cell cycle and survival signaling. Depending on concentrations, resveratrol "switched" cells between reversible cell cycle arrest and irreversible apoptosis. Specifically, resveratrol treatment blocked the cell cycle in the GoZG₁, G₁ZS transition, S phase or G₂ZM phases by (a) suppressing cyclins and their corresponding kinases, (b) increasing p53 levels or (c) inhibiting DNA synthesis. Resveratrol also up-regulated pro- apoptotic members of the Bcl-2 family and down-regulated anti-apoptotic members of this family. Finally, resveratrol inhibits NFKB and AP-I signaling pathways, their upstream kinases and their downstream targets (including inducible COX-2, inducible nitric oxide synthase (iNOS) and matrix metalloprotease-9 (MMP-9). Thus, resveratrol can inhibit proliferation and induce cell death (Aggarwal, et ah, 2004; Aziz, Kumar and Ahmad, 2003; Dong, 2003; Fremont, 2003; Gusman, Malonne, and Atassi, 2001; Jang, et al, 1997; Mitchell, Zhu and Young, 1999; Savouret and Quesne, 2002; Signorelli and Ghidoni, 2005).

SUMMARY OF THE INVENTION The present invention relates to a method of using a composition comprising an effective amount of a compound of the tolan family, preferably a hydroxytolan, to modulate (preferably stimulate or induce) sirtuin activity, inhibit tyrosinase activity, inhibit NFKB binding to DNA, inhibit COXl and/or COX2 enzymatic activity. One or more of these activities endow the compound with "skin activity" (defined below) which includes, upon appropriate administratoin such as by the topical route, (i) inhibiting or reversing the effects of aging, (ii) whitening or inhibiting pigmentation, (ii) firming, tightening or tautening (which terms are interchangeable) skin, and thereby inhibiting or reversing skin wrinkling, in a mammal, preferably a human. The compound also has cutaneous antiinflammatory activity. A compound of this class may be used alone, in a combination (two or more tolan compounds) or in conjunction with other agents that include (a) cosmetic agents (b) skin-active pharmaceutical agents to treat a skin disease, condition or affliction, or agents that treat other pathological phenomena that also have undesirable effects on the condition of the skin.

Tolan, whose IUPAC name is diphenylacetylene has the formula CeHsC≡CC_όHs and consists of two phenyl groups attached to both ends of a -C≡C- (ethynyl) linker. The present invention focuses particularly on hydroxylated tolans also referred to as hydroxytolans, in which at least one of the two phenyl groups is substituted with at least one -OH group. As described in more detail below, the compounds useful in the present invention include additional derivatives of a tolan or hydroxytolan compound.

Also disclosed herein are methods for evaluating a tolan compound suspected of having "skin-activity" as defined herein for its sirtuin-inducing activity and thereby predicting its skin activity in a human which results in anti-aging, whitening and anti- wrinkle effects in the skin and an ability to treat, which includes (i) preventing, (ii) inhibiting progression of, or (iii) reversing, other undesirable skin conditions or diseases.

The present invention is directed to a method for stimulating SIRTl activity in the skin of a subject, and for mediating a desirable skin activity in a subject, the composition comprising a compound of the following Formula I: Formula

wherein:

L represents a linkage between the two phenyl rings that is preferably-C=C- (although in some embodiments, L can be -C=C- or -C-C-); R¹ and R² are independently substituents at any available position of the phenyl rings; m and n are independently, 0, 1, 2, 3, 4 or 5 representing the number of R¹ and R² substituents of the rings, respectively, and at least one of m or n must be >1 ; wherein R¹ and/or R² is:

-OH, a halogen, a haloalkyl group with one C atom substituted with from 1 to 3 halogen atoms, a C₁-C_O alkyl, C₂-C_O alkenyl, or C₂-C₆ alkynyl group, an acetyl group,

-OR³, wherein R³ is a C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl group, an acetyl group, or β-D-glucoside, a carboxyl group, an α hydroxyl carboxylic acid, or a nitro group, and wherein, optionally, one phenyl ring is replaced with a cyclohexyl group. Preferably, at least one occurrence of R¹ or R² is -OH,

Most preferably in the above composition, L is -C≡C-.

When the compound can occur as a trans or a cis stereoisomer, the trans stereoisomer is preferred.

Another preferred composition used in the above method comprises one or more hydroxylated tolans, in combination with one or more hydroxylated stilbenes and/or one or more hydroxylated diphenylethanes in accordance with Formula I and the above structural specifications.

Also provide herein is a cosmetic or pharmaceutical composition that comprises a composition as described above comprising one or more of the active compounds described and a cosmetically or a pharmaceutically acceptable carrier or excipient. This includes all cosmetic and dermatological forms of carriers. Cosmetically acceptable is defined as including "dermatologically acceptable, so that the composition is compatible with skin, mucous membranes, nails and hair and permits and promotes the action of the active compound on all cutaneous cells, including keratinocytes, fibroblasts and melanocytes. The pharmaceutical composition may be formulated so that one or more of the components, is formulated topical, parenteral, or oral administration.

The present invention provides a method of inhibiting the development of, the reversal, partial or complete, of skin wrinkling, skin pigmentation or other age-related skin condition, in a subject, administering to a subject in need thereof, an effective amount of one or more of a first compound of Formula I. (By "first compound" is intended one of a possible series of compounds that may be given with "a second compound" which generally refers to compounds of a different class).

The abbreviation "HT" as used herein to refer to a hydroxylated tolan such as any Formula 1 compound that comprises at least one -OH substituent on a phenol ring is useful in the present cosmetic or pharmaceutical compositions and methods.

Thus, one embodiment provides a method of treatment of skin with a combination that comprises

(a) an effective amount of a first compound, that is a compound of Formula 1 as described above in which L is -C≡C-, more preferably a compound selected from HT- 1 , HT-2, HT-3 and HT-4, as described.

(b) an amount of a second compound which is effective in combination with the first compound in promoting or enhancing or complementing the skin activity of the first compound in achieving an anti-aging effect or disease prevention or treatment effect. In a preferred embodiment of the above method, L is -C≡C- in Formula I. Preferred among these compounds are hydroxytolans. Particular preferred hydroxytolans are:

(1) HT-I, also referred to herein as KST 201, the structural formula of which is

Formula II;

(2) HT-2, also referred to herein as KST 213 the structural formula of which is

Formula III;

(3) HT-3, also referred to as KST 301, the structural formula of which is:

Formula IV; or

(4) HT-4, also referred to as KST 401, the structural formula of which is

Formula V

(5) HT-5 (3,4,3'4'-tetrahydroxytolan; formula not shown), a variant of HT-4, in which the - OH groups are at the 3, 4, 3' and 4' positions .

In the above method, the composition is preferably administered topically. The present invention also provides a method of inhibiting pigmentation or whitening skin in a subject, comprising administering to the subject in need thereof a compound of formula I, above, preferably a compound selected from HT-I, HT-2, HT-3, HT-4 or HT-5. Also included is such treatment with a combination that comprises

(a) an effective amount of a first compound, that is a compound of Formula 1 as described above in which L is -C≡C-, more preferably a compound selected from HT-I, HT-2, HT-3, HT-4 or HT-5, as described.

(b) an amount of a second compound which is effective in combination with the first compound in inhibiting pigmentation or whitening the skin.

The present active compounds (a) induce, stimulate and increase sirtuin, e.g., SIRTl, enzymatic or catalytic activity, and/or inhibit tyrosinase activity, and/or inhibit NFKB binding to DNA and/or inhibit COX-l/COX-2 activity by at least 10% compared to an appropriate control value. More preferably, they increase this activity by at least about 20%, preferably at least about 30%, more preferably at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or at least about 100%. All intermediate values in the above ranges, as well as increases of greater than 100%, are also contemplated. The above noted values, when expressed as "fold increases" in activity or as ratios of activity to a control, are, respectively, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 and 2.0.

In the foregoing method of administering the compound, the HT compound and any additional agent may be administered by the same or by different routes. The preferred route for each agent is topical, although those of skill in the art will know, depending on the nature of the additional agent, which route other than topical may be used - including, for example, oral, intravenous, intramuscular, subcutaneous, intranasal, etc.

The present methods may result in partial or complete responses, as there are recognized in the art of treating skin conditions or diseases. See, e.g., Rook's Textbook of Dermatology, D.A. Burns et al, eds., 7^th ed (or later), Blackwell Publishing Ltd., 2004.

In an embodiment of the above methods, the subject is administered, as a first compound, one or more hydroxytolan compounds of Formula I or another of the related tolan skin-active compounds described herein, together with one or more of a "second" compound that is skin reactive. Such a second compound may also be a /raws-stilbene (in which L is a - C=C- linkage in Formula I) or a diphenylethane (in which L is a -C-C-linkage in Formula I) that is known in the art (see, for example, references cited herein) to stimulate SIRTl enzymatic activity. In one embodiment wherein the second compound is a stilbene, resveratrol is excluded.

A most preferred embodiment of the present invention is a topical pharmaceutical composition comprising as the active compound, a compound described above, or a pharmacologically acceptable salt, ester, amide, prodrug or analogue of the disclosed compound,. More preferably, the compound is a hydroxylated tolan, such as one or more of HT-I, HT-2, HT-3 or HT-4, or said salt, ester, amide, prodrug or analogue thereof.

The present compounds act like resveratrol in their ability to increase sirtuin activity as while possessing a longer half-life and a greater selectivity index. Preferably, the active compound is more potent that resveratrol in achieving the same biochemical and biological (skin) effects. An effective "skin-active amount," or a therapeutically effective amount of a compound as described herein is used in a method to prevent and/or treat (including alleviation of symptoms) a skin condition, disorder or disease. This method comprises administering to a susceptible or affected subject, preferably by application to the skin, the above topical cosmetic or pharmaceutical composition. The skin conditions, disorders or diseases that are preventable or treatable in accordance with this invention include, without limitation, undesired skin changes resulting from natural aging and the like, skin damage caused by exposure to solar radiation or radiation from other light sources. Skin diseases or disorders include conditions that are limited to the skin, as well as cutaneous symptoms or cutaneous manifestations of other diseases or disorders, including of systemic diseases that have other effects elsewhere in the body. Such skin diseases or conditions include contact dermatitis (irritant-induced or allergic), atopic dermatitis (allergic eczema), actinic keratosis, keratinization disorders (including eczema), epidermolysis bullosa diseases (including pemphigus), exfoliative dermatitis, seborrheic dermatitis, erythemas (including erythema multiforme and erythema nodosum), discoid lupus erythematosus, or dermatomyositis.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph showing results of NFKB p65 DNA binding which quantitates the NFKB inhibitory activity of resveratrol and KST 201 in DU 145 human andro gen-independent prostate cancer cells (DU145) as a function of test agent dose (5, 25, 50 and 100 μM) for a fixed incubation time of 24 hours. DNA-binding activity of active p65 was determined by the intensity of chemiluminescence produced by DNA-p65-horse radish peroxidase (HRP) complex fixed on the well surface. The results represent mean ± SD of triplicate determinations. An inhibitor for p65-DNA binding was added to confirm specificity.

Figure 2 is a graph showing results of NFκB p65 DNA binding as above using the same compounds and cells, the assay being performed as indicated for Fig. 1. This was a time course study in which a single concentration of the test agent (100 μM) was incubated with the cells for 6, 12, and 24 hours. DNA-binding activity of active p65 was determined as above. Figure 3 is a graph showing results of an assay measuring COX-I enzyme activity to investigate inhibitory activity of resveratrol and KST 201 on prostaglandin (PG) biosynthesis. Varying concentrations of the test agents resveratrol and KST 201 (10, 25, 50, 100 and 200 μM) were examined in the presence of COX- 1. COX activity was quantified by measuring the amount of final product PGF_2α which competed with AChE- linked PGs for binding to its antibody fixed on the well surface. The color developed by adding substrate for AChE and was inversely proportional to the amount of PGF_2α and COX activity. The results represent mean ± SD of triplicate determinations.

Figure 4 is a graph showing results of a COX-2 enzyme activity assay performed as above (for Fig. 3) to investigate inhibitory activity of resveratrol and KST 201 on prostaglandin (PG) biosynthesis. Varying concentrations of the test agents resveratrol and KST 201 (10, 25, 50, 100 and 200 μM) were examined the presence of COX-2. COX activity was quantified as above. The results represent mean ± SD of triplicate determinations.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is directed to compositions and methods that modulate human sirtuin activity, inhibit tyrosinase as well as other enzymes or signaling pathways, and are "skin- active" as defined below. The present inventors have discovered, surprisingly, that modifying polyphenolic compounds by changing the chemical structure of the linkage between the aromatic rings and altering the number and/or position of the hydroxyl groups on the rings, leads to active, stable, relatively non-toxic compounds that, among other actions, cause activation of SIRTl to a level closely approximating that induced by resveratrol, and permit the use of these compounds inhibit aging-associated changes in skin, along with other age-related cosmetic changes, and to treat certain skin diseases that are noted elsewhere herein. It should be understood, that while one mechanism of action of the compounds of the present invention on cells, and, in particular, on skin and cutaneous tissue, is by SIRTl activation, this is by no means the only target or cellular pathway that is affected. Others may be as or more important. Therefore, while the modes of action for the compounds are described primarily as SIRTl activation or induction, NFKB binding to DNA, COX-inhibition, or tyrosinase inhibition, the inventor does not wish to be bound by these mechanisms. Other tolan compounds of generally similar chemical structure as the compounds disclosed herein may also function in accordance with the invention, e.g., without being potent activators of SIRTl catalytic activity or potent tyrosinase inhibitors.

A compound according to this invention "skin active" or posseses "skin activity" if the compound effects changes in skin, preferably in human skin, that include promoting skin tightness or tautness which constitute, collectively "anti-wrinkle" activty, promoting skin whitening or whiteness, or inhibition or reversal of pigmentation, presumably by inhibition of tyrosinase activity, preventing or diminishing damage from exposure to certain radiation, primarily solar or other UVA or UVB radiation. "Skin activity" includes preventing, diminishing or alleviating symptoms of any of a number of known skin diseaes or afflictions, including cutaneous inflammation, or diseases not considered skin disease per se but that involve skin pathology. Also included within the scope of "skin" as used herein are nails and hair as well as mucous membranes, all of which are also referred to collectively as "keratinous substrates." The present methods overcome or counteract and reverse at least partially the effects of various external "insults" to the skin and cutaneous environment which commonly have visible consequences, such as skin aging, depigmentation/hyperpigmentation, and inflammatory reactions. Treatment of skin or a keratinous substrates with a compound of the present invention includes all actions of the compound that preserves or restores the healthy functioning of skin (and/or hair and/or nails) or acts on any process that permits preservation or improvement of the appearance and/or texture of skin, hair or nails. These include hydration, protection against any type of external, environmental insults such as protection_? from effects of sunlight and counteracting or preventing any visible signs of aging. Such signs of aging include all of the changes in external appearance of skin due to aging, and include wrinkles, fine lines, limp skin, slackened skin, thin looking skin, the loss of elasticity and/or skin tone, dull skin, and skin which lacks radiance. Signs of aging also include internal alterations that are not directly visible, for example, the internal degradation resulting from ongoing exposure to UV radiation. "Enhanced skin appearance" refers to any phenomenon which is manifest as visual improvement in the skin's appearance - such as a more attractive appearance, the firmness and smoothness of youthful skin. Small visible imperfections are diminished or made to disappear.

The papery appearance of aged skin is attenuated. The active compounds of the present invention are intended to protect keratinous substrates and, particularly, the skin, hair, and nails from all types of external, environmental insults, so that use of these compositions allow protection of the keratinous substrates and increased resistance to stress inflicted upon them by the environment. External insults can be chemical, physical, biological, or thermal in origin.

The present methods achieve these effects by administration of the tolan compounds described herein, alone, in combination, and/or when applied or administered in conjunction with other cosmetic agents, skin-active agents, such as the related group of /raws-stilbene compounds that are know in the art, or other drugs or compounds that contribute to beneficial effects on skin. Many of the biological effects of the present compounds on skin can be viewed as "anti-aging" effects. They include improvements in wound healing of aging skin. As noted, resveratrol has been considered to be one of the most potent SIRTl activators (Howitz, et al., supra). However, significant toxicity to normal cells has also been reported (Aggarwal, et al. supra). As shown above, the chemical structure of resveratrol consists of two aromatic rings linked by a styrene double bond with two hydroxyl groups at the 3 and 5 positions of one ring and one hydroxyl group at the 4' position of the other ring.

Structure-activity relationship studies of resveratrol and its analogs revealed the functional groups and chemical structures essential to their bioactivities. The present inventor engaged in an intelligent design approach by modifying the structure of resveratrol (or resveratrol homologues or derivatives) in one or more of the following ways: (1) most importantly, changing the chemical structure of the linkage between the two aromatic (phenolic) rings;

(2) altering the number and/or position of the hydroxyl groups on either ring,

(3) adding to, or replacing the hydroxyl groups on either ring with other substituents. This led to the discovery of a novel series of hydroxytolan compounds not heretofore known to have the skin-activities discussed herein that are comparable or better SIRTl- activators than resveratrol or other resveratrol homologues or derivatives known in the art (see, for example, US2006/0276393 (referred to as the '393 application) and other related patent applications and patents by Sirtris), which may have longer half- lives (include UV breakdown and hplc data here) than does resveratrol and fewer side effects, based on their lower non- specific toxicity. Members of the "KST series" of molecules disclosed herein were discovered to modulate sirtuin activity and to exert anti-aging, whitening, anti-wrinkle activity on skin (see Examples), and to be active against other skin afflictions when used alone or in conjunction with other cosmetic or skin-active agents.

Other utilities of some of these hydroxytolan compounds were disclosed earlier by the present inventor (alone or with colleagues) in U.S. Patents 6,599,945 and 7,094,809, and co- pending U.S. provisional patent applications 60/945,276 and 60/945,263.

The present inventor made the surprising discovery that modifying various polyphenolic compounds by changing the chemical structure of the linkage between the aromatic rings, in particular to a -C≡C- acetylene linkage (i.e., tolans) and by altering the number and/or position of the hydroxyl groups on the rings, and/or derivatizing the rings with other groups in addition to, or in place, of the, hycroxyl groups, results in compounds which are more effective for the above uses while maintaining low toxicity to normal cells and tissue. Modulating SIRTl activity (or other signaling pathways) allows members of the KST series of compounds to coordinately regulate genes, transcription factors and other cellular processes that control skin aging, whiteness, wrinkling and are exploited in the treatment or amelioration of other skin afflictions. The present invention provides novel cosmetic and pharmaceutical compositions which induce SIRTl, inhibit NFκB-dependent transcription by deacetylating, the active subunit (RelA/p65) in a site-specific manner, inhibit tyrosinase activity, reduce or limit the expression of various cytokine genes, and thereby serve to inhibit skin- associated aging and various afflictions. The present compounds, acting via an NFKB binding site on their promoters also inhibit expression of genes encoding COX enzymes, and are therefore possess useful antiinflammatory activity.

As discussed in more detail below, the present invention provides a method of affecting skin in a desirable manner and thereby inhibiting or counteractiung aging effects including wrinkling and pigmentation using one or more tolan compounds, preferably a hydroxytolan member, as described herein.

As noted, the compounds are preferably aministered topically, although they may be administered by other routes, either alternatively, contemporaneously or sequentially. These routes include subcutaneously, intracutaneously, orally, intravenously, or intranasally to act on skin cells and skin tissue to achieve the desired effects. While the inventor does not wish to be bound by any mechanistic explanation of these effects or outcomes, nor must they be, it is believed that their advantageous activity on manifestations of skin aging or skin disorders is related to redox cycling and the possible generation of peroxides and other reactive oxygen species (ROS) as well as the inhibtion of tyrosine kinases and subsequent membrane lipid alterations, and also to the action of tyrosinase enzymes involved in formation of undesirable skin pigmentation.

Similarly, the mechanism by which the tolan compounds of the present invention activates SIRTl is not clear, and, the invention is not bound by proposed mechanisms. Milne, J. C. et al, 2007, Nature 450:712-716, studying other multi-ring (albeit non-tolan) small organic molecule activators of SIRTl examined the mechanism of activation. The effect of these compounds on the classic enzymatic parameters V_max (velocity of enzyme-catalyzed reaction at infinite concentration of substrate) and the K_m (Michaelis constant) of SIRTl for its two substrates, NADl and acetylated peptide were determined. None of the activating compounds tested affected Km for NADl or the V_max but they all decreased the K_m of SIRTl for acetylated peptide substrate. The magnitude of the Km effect positively correlated with the EC_{1 5} values for the compounds. A similar Km-type activation mechanism had been proposed for small molecule activators that bind to an allosteric site of another enzyme. The energetics of binding of one SIRT activator to purified SIRTl enzyme was studied by isothermal titration calorimetry. Titration of the compound against the purified human SIRTl-C did not result in detectable binding. In the presence of acetylated peptide substrate, the activating compound exhibited signs of protein-binding-site saturation (with best fit to a one site binding model). The dissociation constant (Kd) for this compound was 16.2 mM and the enthalpy was -6.1 kcal/mol. These data suggested that these SIRTl activators_? bind to a SIRTl-peptide substrate complex and promote a more productive conformation that enhances catalytic activity. The compounds in this study, like their tolan "homologues" disclosed herein (compounds X, Y and Z, below), were 1, 000-fold more potent activators than, and structurally unrelated to, resveratrol. Similar to resveratrol, these compounds bind directly to the SIRTl -acetylated peptide complex at the same site and lower the Km for peptide substrate resulting in a more productive catalytic complex. Also shown in this publication was that amino acids 183-225 of human SIRTl were critical for maintaining activation by these compounds and therefore defined the allosteric binding site. Acetylated peptide binding to SIRTl may induce a conformational change that exposes an allosteric site in this region of the enzyme. The authors speculated that an endogenous activator of SIRTl (not yet identified) exists and may be increased after various mild physiological stresses. These activating compounds seemed to mimic the beneficial effects of caloric restriction (among which are known to exist anti-aging effects, presumably on skin as well) on mitochondrial and metabolic function in mammals in vivo, supporting the present inventors conception that such SIRTl activators can be used to treat conditions and diseases of ageing include the skin conditions described herein.

Hydroxylated Tolans

The structural skeleton of the preferred compounds of the present invention, the hydroxylated tolans, comprises two aromatic rings joined by an acetylene bridge. The compounds preferred for the methods and uses of the present invention are described by Formula I, Formula

wherein:

L represents a linkage between the two phenyl rings and is preferably a -C≡C- linkage (tolans); R¹ and R² are substituents at any available ring position; m and n is the integer 0, 1, 2, 3, 4 or 5 representing the number of aromatic ring R¹ and R² substituents, respectively.

Preferred, though non-limiting examples of phenyl ring substituents R¹ and R² are: (i) OH, (ii) a halogen,

(iii) a haloalkyl group wherein one C atom is substituted with from 1 to 3 halogens; the halogen is preferably F, Cl and Br, most preferably F, so that most preferred haloalkyl substituents are CH₂F, CHF₂ and CF₃;

(iv) a C₁-C₆ alkyl (referred to also as "lower alkyl"), C₂-C₆ alkenyl ("lower alkenyl"), or C₂-C₆ alkynyl ("lower alkynyl")

(v) an acetyl group, (vi) OR³, wherein R³ is a lower alkyl, lower alkenyl, or lower alkynyl; R³ may also be an acetyl group or β-D-glucoside, (vii) a carboxyl group, (viii) an α hydroxyl carboxylic acid, or

(ix) a nitro group.

In embodiment, one of the phenyl rings is replaced with a cyclohexyl ring, preferably unsubstituted.

When m or n is 2 or greater, the R¹ and R² substituents may be the same or different. For example, if m=2, the ring may be disubstituted with one -OH group and one haloalkyl group, etc.

When L is -C≡C-, at least one of R¹ or R² is OH (and m or n is 1). The present invention includes methods of using, in addition to the above tolan compound, at least one second compound of Formula 1 which is a similar ring substituent of a stilbene (where L is -C=C- in Formula I) or a diphenylethane (where L is -C-C- in Formula I). In these embodiments, at least one of R¹ or R² is OH (and m or n is 1) so that the class of intended second compounds are hydroxystilbenes and hydroxyphenyl ethanes.

The most preferred compounds for use in the present methods, the chemical moieties of which were disclosed in U.S. Patents 6,599,945 and 7,094,809, are shown below, and written in terms of the features of Formula I. Each is a hydroxylated tolan in which L is a -C≡C- linkage: (A) "Hydroxytolan-1" ("HT-I"; also, KST 201)) shown below in Formula II: m=l and n=l (in Formula I); R¹ is an OH group at ring position 4;

R² is an OH group at ring position 4' ;

Formula Il

A chemical name for this compound is 4,4'-dihydroxytolan.

(B) "Hydroxytolan-2" ("HT-2"; also KST 213) shown below in Formula III: m=l and n=l (in Formula I);

R¹ is an OH group at position 4 and R² is a CF₃ at position 4', or alternatively R¹ is a CF₃ at position 4 and R² is an OH at position 4' .

Formula

Chemical names for this compound are 4-hydroxy-4'-trifluoromethyltolan and 4' -hydroxy-4-trifluoromethyltolan.

© "Hydroxytolan-3" ("HT-3"; also KST 301)) shown below in Formula IV: m=2 and n=l (in Formula I); R¹ is an OH group at positions 3 and 5 and R² is an OH group at position 4' or, alternatively, m=l and n=2, and

R¹ is an OH group at position 4 and R² represents OH groups at positions 3' and 5'. Formula IV

Chemical names for this compound include 3,4',5-trihydroxytolan (or alternatively, 3,5,4'-trihydroxytolan, 3',4,5'-trihydroxytolan or 3',5',4-trihydroxytolan).

(D) "Hydroxytolan-4" ("HT-4"; also KST 401) shown below in Formula V: m=2 and n=2 (in Formula I);

R¹ represents OH groups at positions 3 and 5 and R² represents OH groups at positions 3' and 5'

Formula V

A chemical name for this compound is 3,3',5,5'-tetrahydroxytolan (or, alternatively, 3,5,3'5'-tetrahydroxytolan)

(E) "Hydroxytolan-5" ("HT-5"): m=2 and n=2 (in Formula I);

R¹ represents OH groups at positions 3 and 4 and R² represents OH groups at positions 3' and 4' A chemical name for this compound is 3,3',4,5'-tetrahydroxytolan (or, alternatively,

3,4,3'4'-tetrahydroxytolan)

In other embodiments, the active compound is a sirtuin-inducing compound from the following list. (Some of these are specified by the above generic formulas or are the same as a specific compounds listed above). These are listed below in the approximate order of their expected reactivity in inducing Sirtl catalytic activity: 3,5,4' -trihydroxytolan (same as HT- 3 above); 3,5-dihydroxy-4-methoxytolan 3-O-β-glucosidep;3,3',5-trihydroxy-4'-methoxytolan 3- O-β-D-glucoside; 3,5-dihydroxy-4'-thiomethyltolan; 3,5-dihydroxy-4'-chlorotolan; 3, 5- dihydroxytolan; 3,5-dihydroxy-4'-ethyltolan; 3,5-dihydroxy-4'-fluorotolan; 3,5,3\4'-tetrahydroxytolan; 3_^5. dihydroxy-4'-methyltolan; 3,5-dihydroxy-4'-azidotolan; 3,5-dihydroxy-4'-nitrotolan; 3.5- dihydroxy-4-isopropyltolan; 3.5-dihydroxy-4'-methoxytolan; 3,5,3' -trihydroxy-4'- methoxytolan; 3,4'-dihydroxy-5-acetoxytolan; 3,5-dihydroxy-4'-acetoxytolan; (E)-l-3,5- dihydroxyphenyl)-2-(2-napthyl)ethyne ; 3-hydroxytolan; 3,5-dimethoxymethoxy-4'- thiomethyltolan; 3,5-dihidyroxy-4-acetamidetolan; 3,4-dihydroxytolan; (E)-l-3,5- dihydroxyphenyl)-2-(cyclohexyl)ethyne; and 3,4-dimethoxytolan.

Yet additional compounds are 3,4,3'-trihydroxy-4'-acetoxytolan; 3,4,4'-trihydroxy-3'- acetoxytolan; 3,5,4'-trihydroxy-3'-acetoxytolan; 3,5,3'-trihydroxy-4'-acetoxytolan; 3,4,3'- trihydroxy-4'-acetamidetolan; 3,4,4'-trihydroxy-3'-acetamidetolan; 3,4,3'-trihydroxy-3'- acetamidetolan; 3,4,4'-trihydroxy-4'-acetamidetolan; 3,4'-dihydroxy-4-acetoxytolan; 4,4'- dihydroxy-3-acetoxytolan; 3,4-dihydroxy-4'-acetoxytolan; 3,4'-dihydroxy-4'-acetamidetolan; 4,4'-dihydroxy-3-acetamidetolan; 3,4-dihydroxy-4'-acetamidetolan; 3,5,4' -trihydroxy-4- acetamidetolan; 3,4'-dihydroxy-4-acetamidetolan; 4,4'-dihydroxy-3-acetamidetolan; and 3,4- dihydroxy-4'-acetamidetolan.

In other embodiments, exemplary sirtuin-activating compounds are tolan homologues of sirtuin-activating stilbenes described in Howitz, KT et ah. (2003) Nature 425:191-6, and particularly in Milne, J. C. et ah, 2007, Nature 450:712-716 (for treatment of diabetes in the latter). In the preferred tolan compounds, an ethynyl (-C=C-) group bridges the two phenolic rings rather than the ethenyl -C=C- group or the amide { -NH-C (O)- } groups in the active compounds disclosed in the above-cited references. Preferred examples of such tolan compounds have the following structures:

Compound X Compound Y

Compound Z

These SIRTl activating compounds are structurally divergent from HT-I, HT-2, HT-3, or HT-4 in terms of the substituents of the phenyl rings of Formula 1, and are expected to be more potent than, HT-I in activating SIRTl and in their skin activity in vivo. These compounds are believed to bind to the SIRTl enzyme-peptide substrate complex at an allosteric site N-terminal to the catalytic domain and lower the Km for acetylated substrates.

For the sake of convenience, the tolan and hydroxytolan skin-active compounds used in the present invention are also referred to herein collectively as "active compounds," though it is to be understood that other compounds with which they may be combined, admixed, etc., in various embodiments disclosed herein are also "active" in a biological or pharmacological sense. As is disclosed in more detail below, the active compounds of this invention are typically admixed with one or more cosmetically or pharmaceutically acceptable carriers and/or excipients that are well known in the art for human (or veterinary) uses to produce cosmetic compositions, or pharmaceutical/therapeutic compositions. U.S. Patents 6,599,945 and 7,094,809 described above disclose HT-I, HT-2I and HT-3 and describe their use in some very specific methods - inhibiting the formation of infectious herpes virus particles or for treating gonorrhea caused by Neisseria gonorrhoeae. Synthesis of HT-4 was shown, although no biological activity for this compound was identified. None of these documents (nor the other documents focused on resveratrol) disclose or suggest the specific methods and uses of the compounds that are claimed herein.

To the extent that any specific disclosure in these publications or other publications may be considered to anticipate any generic aspect of the present invention, the disclosure of the present invention should be understood to include a proviso or provisos that exclude or disclaim any such species that were previously disclosed. The aspects of the present invention which are not anticipated by the disclosure of said publications are also unobvious from the disclosure of these publications, due at least in part to the unexpectedly superior results disclosed or alleged herein.

Synthesis of Hydroxylated and Poly-Hydroxylated Tolans

The synthetic schemes described below are those used by the present inventor and colleagues in producing the indicated compounds. They are not intended here as exclusive approaches or schemes, but rather are illustrative of preferred methods. A general scheme for preparing polyhydroxylated tolans is shown below.

(MeO)_n Pd(PPh₃J₂CI₂ Cu! (MeO)_n

Q-. + H-_^ -SiMe, *~ A V = SiMe₃ i Pr₂NH, r t

1 4,4'-(OH)₂

2 3,4XOH)₃

3 3,3',5,5'-(OH)₄

A. Synthesis of 3,5-dimethoxyiodobenzene from 3,5-dimethoxyaniline

In a 500 ml, 3-necked, round-bottomed flask equipped with a thermometer, a mechanical stirrer and an addition funnel was placed HCl (12 M, 100 ml, 1.2 mol) and crushed ice (100 g). The flask was immersed in a dry ice-Me₂CO cooling bath, and 3,5-dimethoxyaniline (15.3 g, 100 mmol) was added with stirring. To this cold mixture NaNO₂ (8.4 g, 120 mmol) in 40 ml H₂O was added dropwise at such a rate to maintain the temperature of the reaction mixture between -10⁰C and -5°C throughout the addition. The reaction mixture was stirred for 1 hour between 0°C and 5°C. The red dark solution of the diazonium salt was added to a well-stirred solution of KI (83 g, 500 mmol) in 200 ml H₂O at room temperature. The mixture was stirred for 2 hours, and then allowed to stand overnight. The resulting solution was extracted with ether (200 ml x 4). The pooled organic extracts were washed with brine (200 ml x 2) and an aqueous saturated Na₂S₂O₃ solution (200 ml x 2), dried over MgSO₄, filtered and concentrated to a small volume. Silica gel was added, and the mixture evaporated to dryness. This preloaded silica gel was placed on a pad of silica gel and eluted with petroleum to give 17.5 g (66%) of a colorless solid, 3,5-dimethoxyiodobenzene. ¹H-NMR (CDCl₃, 300 Mz): δ ppm: 6.85 (2H, d, J=2.3, Ar- H), 6.40 (IH, t, J=2.3, Ar-H), 3.76 (s, 6H, 2CH₃O).

B. Synthesis of Arylethynyltrimethylsilanes from Ethylnyltrimethylsilane and Aryl Iodides General Procedure:

To a solution of aryl methoxy substituted aryl iodide (40 mmol) in isopropylamine (250 ml) were added Pd(PPh₃)₂Cl₂ (0.4 mmol) and CuI (0.8 mmol), then trimethylsilylacetylene (44 mmol). The reaction mixture was stirred at ambient temperature for 2 to 4 hours under a slow stream of nitrogen. The reaction mixture was filtered and the residues were washed with ethyl acetate, and the solvent evaporated from the combined filtrates. The crude product was purified by column chromatography on silica gel using petroleum/ethyl acetate as an eluent to give the methoxy substituted arylethylyl trimethylsilanes.

( 1 ) 2-(4-methoxyphenyl)- 1 -trimethylsilyl-ethyne

Purified by column chromatography on silica gel using petroleum ether as an eluent to give 2-(4-methoxyphenyl)-l-trimethylsilyl-ethyne (96% yield) as a light yellow oil.

(2) 2-(3,5-dimethoxyphenyl)-l-trimethylsilyl-ethyne

Purified by column chromatography on silica gel using petroleum ether as an eluent to give 2.2 g (94%) light yellow needles.

T_GC=5.39 (T_init=50°C). ¹H-NMR(CDCl₃, 300 Mz): δ ppm: 6.6(s, 2H, Ar-H), 6.43(s, IH, Ar-H), 3.77(s, 6H, 2CH₃), 0.24(s, 9H, SiMe₃).

C. Synthesis of Methoxy Substituted Arylacetylenes

To a solution of arylethynyltrimethylsilanes (30 mmol) in methanol (30 ml) was added potassium fluoride (3.5 g, 60 mmol). The reaction mixture was stirred at room temperature for 2 hours. After removal of methanol, the product was extracted with ether (100 ml x 3) and purified by chromatography on silica gel using petroleum ether as eluent to afford pure products. (1) p-Methoxyethylnylbenzene Pale yellow oil was obtained in 92% yield. ¹H-NMR (CDCl₃, 300 Mz): δ ppm: 7.94(d, 2H, J=8.98, Ar-H), 6.83(d, 2H, J=8.55, Ar-H), 3.80(s, 3H, Ch₃O), 3.00 (s, IH- H).

(2) 3,5-Methoxyrthylnylbenzene

Pale yellow needle was obtained in 91% yield. ¹H-NMR (CDCl₃, 300 Mz): δ ppm: 7.94(d, 2H, J=2.4, Ar-H), 6.83(d, 2H, J=2.3, Ar-H), 3.78(s, 6H, 2Ch₃O), 3.94 (s,

IH- H).

D. Synthesis of Methoxytolans

General Procedure:

To a solution of methoxyethynylbenzenes (20 mmol) and methoxy substituted aryl iodide (22 mmol) in isopropylamine (120 ml) were added Pd(PPHj)₂Cl₂ (0.2 mmol) and CuI (0.4 mmol). The reaction mixture was stirred at ambient temperature for 6 hours under a slow stream of nitrogen. The reaction mixture was filtered and the residues were washed with ethyl acetate and the solvent evaporated from the combined filtrates. The crude product was purified by column chromatography on silica gel using petroleum ether/ethyl acetate (9:1) as an eluent to give methoxytolans.

(1) 3,4',5-Trimethoxyltolan:

A pale yellow oil was obtained in 93% yield. ¹H-NMR (CDCl₃, 300 Mz): δ ppm: 7.46(d,

2H, J=8.6, Ar-H), 6.88(d, 2H, J=8.8, Ar-H), 6.66(d, 2H, J=2.3, Ar-H), 6.44(t, 2H,

J=2.3, Ar-H), 3.83(s, 3H, CH₃O), 3.80(s, 6H, 2CH₃O). (2) 3,3',5,5'-Tetramethoxytolan:

A colorless needle crystal was obtained in 85% yield. ¹H-NMR (CDCl₃, 300 Mz): δ ppm: 6.69(d, 4H, J=2.3, Ar-H), 6.46(d, 2H, J=2.3, Ar-H), 6.66(d, 2H, J=2.3, Ar-H),

3.80(s, 12H, 4CH₃O).

(3) 4,4'-Dimethoxytolan: A colorless needle crystal was obtained in 91% yield. ¹H-NMR (CDCl₃, 300 Mz): δ ppm: 7.46(d, 4H, J=8.7, Ar-H), 6.87(d, 2H, J=8.7, Ar-H), 3.82(s, 6H, 2CH₃O).

E. Synthesis of Hydroxytolans

General Procedure:

In a dry 250 ml, 3-necked, round-bottomed flask was placed a solution of methoxytolans (10 mmol) in anhydrous methylene chloride under N₂. The reaction mixture was cooled to below -20°C, and BBr₃ (20 mmol x the number of methoxy groups) was added by syringe. Then the reaction mixture was permitted to warm up to room temperature and stirred for over 24 hours The reaction mixture (a reddish clear solution) was then poured into ice- water and stirred After sufficient stirring, an aqueous NaHCO₃ solution was added to adjust the pH of the mixture to between 7 and 8 Then the mixture was extracted with ethyl acetate 3-4 times The organic layer was washed with brine and dried over MgSO₄ Solvent was removed under reduced pressure The red brown color crude product was purified by column chromatography on silica gel using petroleum/ethyl acetate (1:1) as an eluent to give hydroxytolans

(1) 3,4',5-Tπhydroxytolan:

A pale yellow solid was obtained in 82% yield ¹H-NMR (CDCl₃, 300 Mz): δ ppm: 9 89(s, IH, OH), 9 45(s, 2h, 2-OH), 7 33(d, 2H, J=8 65, Ar-H), 6 78 (d, 2H, J=8 63,

Ar-H), 6 31(d, 2H, J=2 2, Ar-H), 6 23(d, 2H, J=2 2, Ar-H)

(2) 3,3',5,5'-Tetrahydroxytolan:

A pale red solid was obtained in 92% yield ¹H-NMR (CDCl₃, 300 Mz): δ ppm: 9 49(s, 4H, 4-OH), 6 33(d, 4H, J=2 2, Ar-H), 6 25(t, 2H, J=2 2, Ar-H) (3) 4,4'-Dihydroxytolan:

A white solid was obtained in 66% yield ¹H-NMR (CDCl₃, 300 Mz): δ ppm: 9 82(s, 2H, 2-OH), 7 31(d, 4H, J=8 7, Ar-H), 6 77(d, 4H, J=8 7, Ar-H)

References 1 Ah, MA et al , Chem Pharm Bull, 1992, 40 1130-6, 2 Pavia, MR et al , Bioorg Med Chem, 1996, 4 659-66 3 Jeffery, T, Tetrahedron Lett, 1994, 35 3051-4 4 Jeffery, T et al , Tetrahedron Lett, 1994, 35 4103-6 5 Schmidt-Radde, RH et al , JAm Chem Soc, 1992, 114 9713-15, 6 Schumm, JS et al , Angew Chem, lnt Ed Eng , 1994, 33 1360-3, 7 Pal, M et al , J Chem Soc Perkin Trans, 1996, 1 449-51, 8 Bumagin, NA et al , Russ J Org Chem, 1996, 32 996-1000, 9 Bumagin, NA et al , Tetrahedron Lett, 1996, 37 897-900, 10 Meier H et al , J Org Chem , 1997, 62 4821-6

Approaches to Synthesis of 4-Hvdroxy-4'-trifluoromethyltolan (HT-2) As above, the synthetic schemes described below are those used by the present inventor and colleagues in producing the indicated compounds They are not intended here as exclusive approaches or schemes, but rather are illustrative

A synthetic scheme for the preparation of 4-hydroxy-4'-trifluoromethyltolan (HT-2) is shown in the diagram below Synthetic details of the specific reaction steps are described below Most of these reactions are readily accomplished with high yields (over 90%) All products are preferably purified by column chromatography and characterized by GC and ¹H-

NMR spectrometry

1. l-Iodo-4-tetrahvdropyranyloxybenzene 1

To a stirred solution of 4-iodophenol (11.0 g, 50 mmol) in CH₂Cl₂ (50 ml) cooled with an ice bath, dihydropyran (5.0 g, 60 mmol) was added dropwise over 10 min at 0°C to 5°C. After the solution became clear, toluenesulfonic acid, TsOH (10 mg) was added. The solution was stirred at 20°C for 15 min. Then it was quenched by addition of NaHCO₃ (1 g) and 3 drops of water, and after stirring for 5 min at 20°C, the solvent was removed in vacuo and the residue was purified by column chromatography on silica gel with petroleum ether as eluent to give 14.0 g (92%) of 1 as colorless crystal; mp 66°C; δ _H(CDC1₃, 300 MHz): 7.55(d, J=8.3, 2H, Ar-H), 6.83(d, J=8.4, 2H, Ar-H), 5.37(t, J=3.1, IH, OCHO), 3.86(m, IH, THP), 3.59(m, IH, THP), 1.87~1.58(m, 6H, THP).

2. 4-Tetrahvdropyranyloxy-l-(trirnethylsilylethvnyl) benzene 2

To a degassed solution of compound 1 (9.12 g, 30 mmol) in diisopropylamine (180 ml) under N₂, Pd(PPh₃)₂Cl₂ (140 mg, 0.2 mmol) and CuI (78 mg, 0.4 mmol) were added. Then trimethylsilyl acetylene (3.3 g, 33 mmol) was added dropwise to this clear solution. The reaction mixture was stirred for 2 hours at room temperature. The salt formed during the reaction procedure was filtered off and washed well with ethyl acetate. The filtrate was evaporated to dryness and hydrolyzed with concentrated hydrochloric acid (5 ml), water (25 ml) and crushed ice (10 g), then extracted with ethyl acetate. The combined organic paste was washed with brine and dried with MgSO₄. The solvent was removed in vacuo and the residue was purified by column chromatography (petroleum ether-ethyl acetate=9:l) to give a yellow oil of 2. Yield 7.9 g (96%); δ _H(CDC1₃, 300 MHz): 7.39(d, J=8.7, 2H, Ar-H), 6.97 (d, J=8.6, 2H, Ar- H), 5.41(t, J=3.1, IH, OCHO), 3.84(m, IH, THP), 3.59(m, IH, THP), 1.86~1.61(m, 6H, THP), 0.23(s, 9H, 3 CH₃).

3. 4-Tetrahydropyranyloxyphenylacetylene 3 KF (9.3 g, 160 mmol) was added to a stirred solution of 2 (22.6 g, 80 mmol) in MeOH

(150 ml). The reaction mixture was stirred at room temperature for about 4 hours. After the reaction finished (GC shows no starting material remaining), the solvent was removed under reduced pressure on a rotary evaporator. The residue was purified by column chromatography on silica gel (petroleum ether-ethyl acetate=9:l) to give a pale yellow crystals of 3. Yield 15.7 g (97%); mp 65°C, δ _H(CDC1₃, 300 MHz): 7.42(d, J=8.7, 2H, Ar-H), 7.00(d, J=8.7, 2H, Ar-H), 5.43(t, J=3.2, IH, OCHO), 3.87(m, IH, THP), 3.60(m, IH, THP), 2.99(s, IH, C=C-H), 1.96~1.56(m, 6H, THP).

4. 4-Tetrahydropyranyloxy-4' -trifluormethyltolan 4

A solution of 3 (12.1 g, 60 mmol) and 4-bromobenzotriflouride (14.85 g, 66 mmol) in diisopropylamine (250 ml) was heated to 30°C under N₂, and the solution was degassed. Then Pd(PPh₃)₂Cl₂ (210 mg, 0.3 mmol) and copper(I) iodide (114 mg, 0.6 mmol) were added to this clear solution. The reaction mixture was stirred for 2 hours at 80°C, and then cooled to room temperature. The salt formed during the reaction procedure was filtered off and washed well with ethyl acetate. The filtrate was evaporated to dryness and hydrolyzed with concentrated hydrochloric acid (10 ml), water (100 ml) and crushed ice (50 g), then extracted with ethyl acetate. The combined organic paste was washed with brine and dried over MgSO₄. The solvent was removed in vacuo and the residue was purified by column chromatography (petroleum ether-ethyl acetate=9:l) to give a pale yellow crystals of 4. Yield 16.6 g (80%); mp 112~113°C; δ _H(CDC1₃, 300 MHz): 7.59(s, 4H, Ar-H), 7.48(d, J=8.7, 2H, Ar-H), 7.04(d, J=8.7, 2H, Ar- H), 5.46(t, J=3.1, IH, OCHO), 3.89(m, IH, THP), 3.62(m, IH, THP), 1.86~1.62(m, 6H, THP).

5. 5-Hydroxy-4' -trifluoromethyltolan 5

Compound 4 (13.84 g, 40 mmol), CH₂Cl₂ (75 ml) and MeOH (125 ml) were placed in a 250 ml round-bottomed flask, then TsOH (0.4 g, 0.4 mmol) was added. The reaction mixture was stirred at 30°C for 1 hour. When the reaction was finished (TLC showed no starting material remaining), the solvent was removed by rotary evaporation and the residue was dissolved in

EtOAc and filtered through silica gel. The solvent was removed and the solid was recrystallized from solvents of ethyl acetate and hexane (1:5) to give a pale yellow crystal 9.5 g (90%), mp 131-132°C, δ H(CDCl₃, 300 MHz): 7 59(s, 4H, Ar-H), 7 44(d, J=8 7, 2H, Ar-H), 6 82(d, J- 8 7, 2H, Ar-H), 5 16(s, IH, OH)

References Shen, D et al , J Matter Chem , 1999, 9 661 2 Praef eke, K et al , Angew Chem Int Ed Engl , 1990, 29 111, 3 Bouchta, A et al , Liq Crystals, 1992, 12 575, 4 Hsieh, CJ et al , Liq Crystals, 1994, 16469

Synthesis for other tolans utilize methods that are known in the art, and are thus not reiterated here

Conditions and Diseases of the Skin

The cosmetic and pharmaceutical compositions of this invention are useful for treating humans and animals suffering from, or prone to, certain skin conditions, disorders or diseases associated with natural aging, environmental exposure (such as solar), or caused by inflammation or by similar processes such as those that occur in inflammatory cells For example, the compositions disclosed herein maybe used in a method to prevent or treating skin wrinkles, unwanted pigmentation, damage caused by exposure to UVA or UVB radiation, or diseases or conditions such as psoriasis, contact dermatitis (irritant-induced or allergic), atopic dermatitis (e g , allergic eczema), actinic keratosis, keratinization disorders (including eczema), epidermolysis bullosa diseases (including pemphigus), exfoliative dermatitis, seborrheic dermatitis, erythemas (including erythema multiforme and erythema nodosum), discoid lupus erythematosus, dermatomyositis, and undesired skin changes resulting from natural aging For detailed descriptions of such skin disorders, see, for example, Rook's Textbook of Dermatology, supra The present compounds and method are also useful to treat melanoma (The anti-tumor and anti-cancer activities of these compounds are discussed more fully in co-pending PCT application of Tsai, PCT/US08/67733 published as WO2008/1577872 (which is hereby incorporated by reference in its entirety) Pharmaceutical/Cosmetic Formulations and Modes of Administration

Pharmaceutical or cosmetic compositions for use in accordance with the present methods may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients These compositions comprise the compounds of the invention described above, and their physiologically acceptable salts and solvates Pharmaceutically acceptable acid addition salts of the compounds of the invention containing a basic group are formed where appropriate with strong or moderately strong, non-toxic, organic or inorganic acids by methods known to the art Exemplary of the acid addition salts that are included in this invention are maleate, fumarate, lactate, oxalate, methanesulfonate, ethanesulfonate, benzenesulfonate, tartrate, citrate, hydrochloride, hydrobromide, sulfate, phosphate and nitrate salts.

Pharmaceutically acceptable base addition salts of compounds of the invention containing an acidic group are prepared by known methods from organic and inorganic bases and include, for example, nontoxic alkali metal and alkaline earth bases, such as calcium, magnesium, sodium, potassium and ammonium hydroxide; and nontoxic organic bases such as triethylamine, butylamine, piperazine, and tri(hydroxymethyl)methylamine.

A composition of this invention may be active per se, or may act as a "pro-drug" that is converted in vivo to the active form. The compounds of the invention, as well as the pharmaceutically acceptable salts thereof, may be incorporated into convenient dosage forms for the appropriate routes and modes of administration. These include, primarily forms for topical administration, but also may include capsules, impregnated wafers, tablets or injectable preparations. Solid, semi-solid or liquid pharmaceutically acceptable carriers or excipients may be employed. Preparations which can be administered orally or which can be used for other modes of administration, including suitable topical formulations or solutions for administration by injection or infusion, preferably contain from about 0.01% to 15%, preferably from about 0.1% to 10% by weight or by volume of active compound(s), together with the carrier or excipient. Preferred routes for injection are subcutaneous (sc), intracutaneous, intramuscular (im), intravenous (iv) or intraperitoneal (ip), or for inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral, sublingual or rectal administration. In one embodiment, the compound is administered locally, at the site of the target cells or tissue.

Techniques and formulations useful herein are well-known in the art and may be found, for example, in Gennaro, AE, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins Publishers; 2003 (or a later edition). Other useful references include,

Rowe, R.C. et ah, Handbook of Pharmaceutical Excipient, APhA Publications, 5^th ed, 2005 (or latest edition); Ansel, HC & Stoklosa, MJ, Pharmaceutical Calculations, Lippincott Williams & Wilkins Publishers, 12^th Ed, 2005 (or latest edition); and Watson, D (ed), Pharmaceutical Analysis: A Textbook for Pharmacy Students and Pharmaceutical Chemists, Churchill Livingstone; 2^nd ed, 2005 (or latest edition). For human administration, it will be understood that the preparations meet the sterility, pyrogenicity, general safety and purity standards required by FDA Office of Biological Standards and of other relevant regulatory bodies. The cosmetic or pharmaceutical preparations are made using conventional techniques of cosmetic or pharmaceutical chemistry and formulation involving such steps as mixing, granulating and compressing, when necessary for tablet forms, or mixing, filling and dissolving the ingredients, as appropriate, to give the desired products for the various routes of administration described herein. The pharmaceutical compositions may contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and so forth.

Although intended primarily for humans, the present invention may be used in the treatment of any of a number of animal genera and species, and are applicable in the practice of veterinary medicine. Thus, the pharmaceutical compositions can be used to treat domestic and commercial animals, preferably mammals.

In a preferred embodiment, a skin-active compound described herein, is incorporated into a topical formulation containing a topical carrier that is generally suited to topical drug administration and comprising any such material known in the art. The topical carrier may be selected so as to provide the composition in the desired form, e.g., as a solution or suspension, an ointment, a lotion, a cream, a salve, an emulsion or microemulsion, a gel, an oil, a powder, or the like. It may be comprised of naturally occurring or synthetic materials, or both. The carrier for the active ingredient may also be in a sprayable form. It is preferable that the selected carrier not adversely affect the active agent or other components of the topical formulation. Examples of suitable topical carriers for use herein can be soluble, semi-solid or solid and include water, alcohols and other nontoxic organic solvents, glycerin, mineral oil, silicone, petroleum jelly, lanolin, fatty acids, vegetable oils, parabens, waxes, and the like. Semisolid carriers preferably have a dynamic viscosity greater than that of water. More preferred vehicles include ointment bases, conventional creams such as HEB cream; gels; as well as petroleum jelly and the like. If desired, and depending on the carrier, the compositions may be sterilized or mixed with auxiliary agents, e.g., preservatives, stabilizers, wetting agents, buffers, or salts for influencing osmotic pressure and the like. Formulations may be colorless, odorless ointments, lotions, creams, microemulsions and gels.

Compounds incorporated into ointments are generally semisolid preparations which are typically based on petrolatum or other petroleum derivatives. The specific ointment base to be used, as will be appreciated by those skilled in the art, is one that will provide for optimum delivery of the active compound, and, preferably, will provide for other desired characteristics as well, e.g., emolliency or the like. As with other carriers or vehicles, an ointment base should be inert, stable, nonirritating and nonsensitizing. As explained in Remington's (supra) ointment bases may be grouped in four classes: oleaginous bases; emulsifiable bases; emulsion bases; and water-soluble bases. Oleaginous ointment bases include, for example, vegetable oils, fats obtained from animals, and semisolid hydrocarbons obtained from petroleum. Emulsifiable ointment bases, also known as absorbent ointment bases, contain little or no water and include, for example, hydroxystearin sulfate, anhydrous lanolin and hydrophilic petrolatum. Emulsion ointment bases are either water-in-oil (W/O) emulsions or oil-in- water (OAV) emulsions, and include, for example, cetyl alcohol, glyceryl monostearate, lanolin and stearic acid. Exemplary water-soluble ointment bases are prepared from polyethylene glycols (PEGs) of varying molecular weight, e.g., polyethylene glycol-1000 (PEG-1000). Oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, corn oil, or synthetic oils may be added.

Compounds may be incorporated into lotions, which generally are preparations to be applied to the skin surface without friction, and are typically liquid or semiliquid preparations in which solid particles, including the active agent, are present in a water or alcohol base. Lotions are usually suspensions of solids, and may comprise a liquid oily emulsion of the oil-in-water type. Lotions are preferred formulations for treating large body areas, because of the ease of applying a more fluid composition. It is generally necessary that the insoluble matter in a lotion be finely divided. Lotions will typically contain suspending agents to produce better dispersions as well as compounds useful for localizing and holding the active agent in contact with the skin, e.g., methylcellulose, sodium carboxymethylcellulose, or the like. An exemplary lotion formulation for use in conjunction with the present method contains propylene glycol mixed with a hydrophilic petrolatum such as that which may be obtained under the trademark Aquaphor® from Beiersdorf, Inc. (Norwalk, Conn.).

Compounds may be incorporated into creams, which generally are viscous liquid or semisolid emulsions, either oil-in-water or water-in-oil. Cream bases are water- washable, and contain an oil phase, an emulsifier and an aqueous phase. The oil phase is generally comprised of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol; the aqueous phase usually, although not necessarily, exceeds the oil phase in volume, and generally contains a humectant. The emulsifier in a cream formulation, as explained in Remington's, supra, is generally a nonionic, anionic, cationic or amphoteric surfactant. Compounds may be incorporated into microemulsions, which generally are thermodynamically stable, isotropically clear dispersions of two immiscible liquids, such as oil and water, stabilized by an interfacial film of surfactant molecules (Encyclopedia of Pharmaceutical Technology (New York: Marcel Dekker, 1992), volume 9). For the preparation of microemulsions, surfactant (emulsifier), co-surfactant (co-emulsifier), an oil phase and a water phase are necessary. Suitable surfactants include any surfactants that are useful in the preparation of emulsions, e.g., emulsifiers that are typically used in the preparation of creams. The co-surfactant (or "co-emulsifier") is generally selected from the group of polyglycerol derivatives, glycerol derivatives and fatty alcohols. Preferred emulsifier/co-emulsifier combinations are generally although not necessarily selected from the group consisting of: glyceryl monostearate and polyoxyethylene stearate; polyethylene glycol and ethylene glycol palmitostearate; and caprilic and capric triglycerides and oleoyl macrogolglycerides. The water phase includes not only water but also, typically, buffers, glucose, propylene glycol, polyethylene glycols, preferably lower molecular weight polyethylene glycols (e.g., PEG 300 and PEG 400), and/or glycerol, and the like, while the oil phase will generally comprise, for example, fatty acid esters, modified vegetable oils, silicone oils, mixtures of mono- di- and triglycerides, mono- and di-esters of PEG (e.g., oleoyl macrogol glycerides), etc.

Pharmaceutical compositions (including cosmetic preparations) may comprise from about 0.00001 to 100% such as from 0.001 to 10% or from 0.1% to 5% by weight of one or more compounds described herein. In topical formulations, the active agent is present in an amount in the range of approximately 0.25 wt. % to 75 wt. % of the formulation, preferably in the range of approximately 0.25 wt. % to 30 wt. % of the formulation, more preferably in the range of approximately 0.5 wt. % to 15 wt. % of the formulation, and most preferably in the range of approximately 1.0 wt. % to 10 wt. % of the formulation. Other active agents may also be included in formulations, e.g., anti-inflammatory agents, analgesics, antimicrobial agents, antifungal agents, antibiotics, vitamins, antioxidants, and sunblock agents commonly found in sunscreen formulations including, but not limited to, anthranilates, benzophenones (particularly benzophenone-3), camphor derivatives, cinnamates (e.g., octyl methoxycinnamate), dibenzoyl methanes (e.g., butyl methoxydibenzoyl methane), p- aminobenzoic acid (PABA) and derivatives thereof, and salicylates (e.g., octyl salicylate).

Compounds may be incorporated into gel formulations, which generally are semisolid systems consisting of either suspensions made up of small inorganic particles (two-phase systems) or large organic molecules distributed substantially uniformly throughout a carrier liquid (single phase gels). Single phase gels can be made, for example, by combining the active agent, a carrier liquid and a suitable gelling agent such as tragacanth (at 2 to 5%), sodium alginate (at 2-10%), gelatin (at 2-15%), methylcellulose (at 3-5%), sodium carboxymethylcellulose (at 2-5%), carbomer (at 0.3-5%) or polyvinyl alcohol (at 10-20%) together and mixing until a characteristic semisolid product is produced. Other suitable gelling agents include methylhydroxycellulose, polyoxyethylene-polyoxypropylene, hydroxyethylcellulose and gelatin. Although gels commonly employ aqueous carrier liquid, alcohols and oils can be used as the carrier liquid as well. Various additives, known to those skilled in the art, may be included in formulations, e.g., topical formulations. Examples of additives include, but are not limited to, solubilizers, skin permeation enhancers, opacifiers, preservatives (e.g., anti- oxidants), gelling agents, buffering agents, surfactants (particularly nonionic and amphoteric surfactants), emulsifiers, emollients, thickening agents, stabilizers, humectants, colorants, fragrance, and the like. Inclusion of solubilizers and/or skin permeation enhancers is particularly preferred, along with emulsifiers, emollients and preservatives. An optimum topical formulation comprises approximately: 2 wt. % to 60 wt. %, preferably 2 wt. % to 50 wt. %, solubilizer and/or skin permeation enhancer; 2 wt. % to 50 wt. %, preferably 2 wt. % to 20 wt. %, emulsifiers; 2 wt. % to 20 wt. % emollient; and 0.01 to 0.2 wt. % preservative, with the active agent and carrier (e.g., water) making of the remainder of the formulation.

A skin permeation enhancer serves to facilitate passage of therapeutic levels of active agent to pass through a reasonably sized area of unbroken skin. Suitable enhancers are well known in the art and include, for example: lower alkanols such as methanol ethanol and 2- propanol; alkyl methyl sulfoxides such as dimethyl sulfoxide (DMSO), decylmethylsulfoxide (CioMSO) and tetradecylmethyl sulfoxide; pyrrolidones such as 2-pyrrolidone, N-methyl-2- pyrrolidone and N-(-hydroxyethyl)pyrrolidone; urea; N,N-diethyl-m-toluamide; C₂-C₆ alkanediols; miscellaneous solvents such as dimethyl formamide (DMF), N,N- dimethylacetamide (DMA) and tetrahydrofurfuryl alcohol; and the 1 -substituted azacycloheptan- 2-ones, particularly l-n-dodecylcyclazacycloheptan-2-one (laurocapram; available as Azone^®). Examples of solubilizers include, but are not limited to, the following: hydrophilic ethers such as diethylene glycol monoethyl ether (ethoxydiglycol, available commercially as Transcutol^®) and diethylene glycol monoethyl ether oleate (available commercially as Softcutol"); polyethylene castor oil derivatives such as polyoxy 35 castor oil, polyoxy 40 hydrogenated castor oil, etc.; polyethylene glycol, particularly lower molecular weight polyethylene glycols such as PEG 300 and PEG 400, and polyethylene glycol derivatives such as PEG- 8 caprylic/capric glycerides (available commercially as Labrasoi^®); alkyl methyl sulfoxides such as DMSO; pyrrolidones such as 2-pyrrolidone and N-methyl-2-pyrrolidone; and DMA. Many solubilizers can also act as absorption enhancers. A single solubilizer may be incorporated into the formulation, or a mixture of solubilizers may be incorporated therein.

Suitable emulsifiers and co-emulsifiers include, without limitation, those emulsifiers and co-emulsifiers described with respect to microemulsion formulations. Emollients include, for example, propylene glycol, glycerol, isopropyl myristate, polypropylene glycol-2 (PPG-2) myristyl ether propionate, and the like.

Topical skin treatment compositions can be packaged in a suitable container to suit its viscosity and intended use by the consumer. For example, a lotion or cream can be packaged in a bottle or a roll-ball applicator, or a propellant-driven aerosol device or a container fitted with a pump suitable for finger operation. When the composition is a cream, it can simply be stored in a non-deformable bottle or squeeze container, such as a tube or a lidded jar. The composition may also be included in capsules such as those described in U.S. Pat. No. 5,063,507. Accordingly, also provided are closed containers containing a cosmetically acceptable composition as herein defined. In one embodiment, a topical pharmaceutical composition containing the active compound, preferably a hydroxytolan compound as disclosed herein, is prepared in the form of a cream as follows (Table 1):

Table 1

The topical composition may be prepared as follows. The xanthan gum is dispersed in water, and allowed to stand. Phase 1 (the oil phase) is heated to 75°C. Phase 2 is then heated to 75°C. Under high speed agitation, phase 1 is mixed into phase 2. The temperature is maintained at 75°C, and rapid stirring is continued for 10 min. The mixture is cooled slowly while stirring is continued at low speed. At 40°C, Phase is 3 is added. The active compound is then dispersed into diethylene glycol monoethyl ether, heated to 40°C, and then cooled to 30°C. while stirring slowly (Phase 4). At 30°C, Phase 4 is added to the cream, mixed well, and cooled to room temperature with slow mixing. A stable cream is obtained.

An example of a microemulsion of the active compound of the invention, e.g., HT-I, HT-2, HT-3 or HT-4, is prepared with the following components (Table 2):

Table 2

The active compound is dispersed into diethylene glycol monoethyl ether. PEG-8 caprylic/capric glycerides and oleoyl macrogolglycerides are added, with agitation. PEG 400 is then slowly added, again, with agitation, followed by addition of water. A stable microemulsion is thus obtained.

For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, the compounds can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution. In addition, the compounds may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.

For oral administration, the pharmaceutical compositions may take the form of, for example, tablets, lozenges, or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); nonaqueous vehicles (e.g., oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl -p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated to give controlled release of the active compound.

To prevent oxidation and preserve the sirtuin- stimulatory activity of the present compounds, the compounds may be stored in a nitrogen atmosphere or sealed in a type of capsule and/or foil package that excludes oxygen (e.g., Capsugel™).

For administration by inhalation, the compounds may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin, for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. Controlled release formula also includes patches.

In certain embodiments, pharmaceutical compositions can be administered with medical devices known in the art. For example, a pharmaceutical composition described herein can be administered with a needle-less hypodermic injection device (e.g., U.S. Pat. Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556). Examples of well-known implants and modules useful in the invention are described in U.S. Pat. No. 4,487,603 (an implantable micro-infusion pump for dispensing medication at a controlled rate); U.S. Pat. No. 4,486,194 (a therapeutic device for administering medicaments through the skin); U.S. Pat. No. 4,447,233 (medication infusion pump for delivering medication at a precise infusion rate; U.S. Pat. No. 4,447,224 (a variable flow implantable infusion apparatus for continuous delivery); U.S. Pats. No. 4,439,196 and 4,475,196 (osmotic drug delivery system).

One way to achieve sustained release kinetics is embedding or encapsulating the active compound into nanoparticles. Nanoparticles can be administrated as powder, as a powder mixture with added excipients or as suspensions. Colloidal suspensions of nanoparticles can easily be administrated through a cannula with small diameter. Nanoparticles are particles with a diameter from about 5 nm to up to about 2000 nm. The term "nanoparticles" as it is used hereinafter refers to particles formed by a polymeric matrix in which the active compound is dispersed, also known as "nanospheres", and also refers to nanoparticles which are composed of a core containing the active compound which is surrounded by a polymeric membrane, also known as "nanocapsules". In certain embodiments, nanoparticles are preferred having a diameter from about 50 nm to about 500 nm, in particular from about 100 nm to about 800 nm. Nanoparticles can be prepared by in situ polymerization of dispersed monomers or by using preformed polymers. Since polymers prepared in situ are often not biodegradable and/or contain toxicological serious byproducts, nanoparticles from preformed polymers are preferred. Nanoparticles from preformed polymers can be prepared by different techniques, e.g., by emulsion evaporation, solvent displacement, salting-out, mechanical grinding, microprecipitation, and by emulsification diffusion. With the methods described above, nanoparticles can be formed with various types of polymers. For use in the method of the present invention, nanoparticles made from biocompatible polymers are preferred. The term "biocompatible" refers to material that after introduction into a biological environment has no serious effects to the biological environment. From biocompatible polymers those polymers are especially preferred which are also biodegradable. The term "biodegradable" refers to material that after introduction into a biological environment is enzymatically or chemically degraded into smaller molecules, which can be eliminated subsequently. Examples are polyesters from hydroxycarboxylic acids such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), polycaprolactone (PCL), copolymers of lactic acid and glycolic acid (PLGA), copolymers of lactic acid and caprolactone, poly-ε- caprolactone, polyhydroxybutyric acid and poly(ortho)esters, polyurethanes, polyanhydrides, polyacetals, polydihydropyrans, polycyanoacrylates, natural polymers such as alginate and other polysaccharides including dextran and cellulose, collagen and albumin. Further description on preparing nanoparticles can be found in US Pat. No. 6,264,922, the contents of which are incorporated herein by reference.

Suitable surface modifiers can preferably be selected from known organic and inorganic pharmaceutical excipients. Such excipients include various polymers, low molecular weight oligomers, natural products and surfactants. Preferred surface modifiers include nonionic and ionic surfactants. Representative examples of surface modifiers include gelatin, casein, lecithin (phosphatides), gum acacia, cholesterol, tragacanth, stearic acid, benzalkonium chloride, calcium stearate, glycerol monostearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters, polyoxyethylene alkyl ethers, e.g., macrogol ethers such as cetomacrogol 1000, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, e.g., the commercially available Tweens™, polyethylene glycols, polyoxyethylene stearates, colloidal silicon dioxide, phosphates, sodium dodecylsulfate, carboxymethylcellulose calcium, carboxymethylcellulose sodium, methylcellulose, hydroxyethylcellulose, hydroxy propylcellulose, hydroxypropylmethylcellulose phthalate, noncrystalline cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol, and polyvinylpyrrolidone (PVP). Most of these surface modifiers are known pharmaceutical excipients. See, for example, Handbook of Pharmaceutical Excipients (supra) published by the American Pharmaceutical Association and The Pharmaceutical Society of Great Britain, Pharmaceutical Press, 1986. Liposomes, well-known in the art, are another delivery system which can be injectable or applied topically. Accordingly, the active compounds can also be administered in the form of a liposome delivery system. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine of phosphatidylcholines. Liposomes usable herein encompass, but are not limited to small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes are used for a variety of therapeutic purposes, and in particular, for carrying therapeutic agents to target cells. Advantageously, liposome-drug formulations offer the potential of improved delivery properties, which include, for example, controlled release. An extended circulation time may be needed for liposomes to reach a target region, cell or site. In particular, this is necessary where the target region, cell or site is not located near the site of administration. For example, when liposomes are administered systemically, it is desirable to coat the liposomes with a hydrophilic agent, for example, a coating of hydrophilic polymer chains such as polyethylene glycol (PEG) to extend the blood circulation lifetime of the liposomes. Such surface-modified liposomes are commonly referred to as "long circulating" or "sterically stabilized" liposomes. One surface modification is the attachment of PEG chains, typically having a molecular weight from about 1-5 kDa, and to about 5 mole % of the lipids making up the liposomes (see, for example, Stealth Liposomes, Lasic, D et ah, eds. CRC Press, Boca Raton, FIa., (1995)), and the cited references therein. The pharmacokinetics of such liposomes are characterized by a dose-independent reduction in uptake of liposomes by the liver and spleen via the mononuclear phagocyte system (MPS), and significantly prolonged blood circulation time, as compared to non-surface-modified liposomes, which tend to be rapidly removed from the blood and accumulated in the liver and spleen.

Another formulation, particularly a solution, of the present active agents is through the use of cyclodextrin. By cyclodextrin is meant α-, β-, or γ-cyclodextrin. Cyclodextrins are described in detail in U.S. Pat. No. 4,727,064, which is incorporated herein by reference.

Cyclodextrins are cyclic oligomers of glucose; these compounds form inclusion complexes with any drug whose molecule can fit into the lipophilic cavities of the cyclodextrin molecule. The cyclodextrin of the compositions according to the invention may be α-, β-, or γ-cyclodextrin. Most preferred cyclodextrins are amorphous cyclodextrin compounds. By amorphous cyclodextrin is meant non-crystalline mixtures of cyclodextrins wherein the mixture is prepared from α-, β-, or γ-cyclodextrin. Exemplary Kits

Also provided herein are kits, e.g., kits for therapeutic purposes, including kits for treating or preventing skin conditions or disorders or secondary conditions thereof. A kit may comprise one or more agent that modulates sirtuin protein activity or level, e.g., sirtuin activating or inhibitory compounds, such as those described herein, and optionally devices for contacting cells with the agents. Devices include syringes, stents and other devices for introducing a compound into a subject or applying it to the skin of a subject.

Further, a kit may also contain components for measuring a factor, e.g., described above, such as the activity of sirtuin proteins, e.g., in tissue samples. Other kits include kits for diagnosing the likelihood of having or developing skin condition or disorder or a secondary condition thereof. A kit may comprise an agent for measuring the activity and or expression level of a sirtuin.

The therapeutic dosage administered is an amount which is therapeutically effective in treating the target condition is known or readily ascertainable by those skilled in the art. The dose is also dependent upon the age, health, and weight of the recipient, state or stage of the condition, nature of concurrent treatment if any, the frequency of treatment, and the nature of the effect desired. Effective doses or amounts can be determined in view of this disclosure by one of ordinary skill in the art by carrying out routine trials with appropriate controls. Comparison of the appropriate treatment groups to the controls will indicate whether a particular dosage is effective. An effective amount of the compound is an amount sufficient to treat, heal, prevent, ameliorate, or reduce symptoms.

The preferred dose of active agent, preferably a hydroxytolan such as HT-I, HT-2, HT-3 or HT-4 is in the range of about 0.5 to about 500 μg/kg body weight /day, preferably about 10 to about 200 μg/kg/ day, more preferably about 20 to about 150 μg/kg/day. For topical administration, dosages of about 0.1-15% concentration (by weight) of the compound, preferably 1-10%, are suggested. The foregoing ranges are, however, suggestive, as the number of variables in an individual treatment regime is large, and considerable excursions from these preferred values are expected. Although a single application or administration of the present compounds may be sufficient to ameliorate some measurable symptoms or the pathologies, it is expected that multiple topical doses or concurrent or subsequent doses by one or more other routes, possibly for periods as long as weeks, months and longer, will be required for the desired therapeutic outcomes. Identifying Compounds that Modulate SIRTl Activity and are Skin-Reactive

To identify compounds that modulate SIRTl activity (or to verify that a compound has this activity), a group of compounds or even a chemical library is screened using a high throughput fluorescent deacetylation assay in 96-well plates (Bitterman et al. (2002) J Biol Chem 277:45099-45107). (The assay of the present examples may also be used.) The substrate used in the assay was a fluorogenic peptide based on the sequence encompassing the p53-K382 acetylation site, a known target of SIRTl in vivo (Vaziri et al. (2001) Cell 107:149-59; Luo et al. (2001) Cell 107:137-48; Langley et al. (2002) EMBO J 21:2383-96). This substrate has certain advantages over other fluorogenic peptide substrates that are based on other known HDAC targets.

The following is an example of a high-throughput screening protocol that may be used. The following wells are designated for control reactions: a) with enzyme; DMSO blank, b) with enzyme; with resveratrol (50 μM or 100 μM) positive control. The reaction mixture contains (final): 0.5 units/reaction SIRTl deacetylase (BIOMOL); 200 μM NAD⁺; 5 μM Fluor de Lys- SIRTl substrate (BIOMOL); buffer (25 mM Tris/Cl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl₂, and 1 mg/ml BSA). In addition, control reaction mixtures containing no enzyme are prepared so that each well receiving compound has a corresponding "no enzyme control" well. Reactions are performed in black 384 well plates {e.g., from NUNC) in a final volume of 25 μl/well. The reactions are initiated by combining enzyme and substrate in a reaction mixture immediately prior to aliquoting in plates (or substrate only for "no enzyme control" plates). Mixtures are aliquoted to plates using, e.g., Biotek Microfill™ (Biotek Instruments). Control mixtures are manually added to designated wells. A test or "library" compound is added at a desired concentration by pin transfer to both "+ enzyme" and "no enzyme" plates. Compounds are added in at least triplicate (with enzyme reaction in duplicate and "no enzyme" controls) at a final concentration of about 50 μM. The plates are incubated at 37°C for 30-60 minutes. Then 25 μl of Ix. Developer II™ (BIOMOL) plus 2 mM nicotinamide are added to all wells to stop the reactions. The reactions are incubated for at least 30 minutes at 37°C for the signal to develop. The plates are read in a micro fluorometer capable of excitation at a wavelength in the range of 350-380 nm and detection of emitted light in the range of 440-460 nm. A read time of 0.1 sec per well may be used. US Pat App2006/0276393 disclosed a structure activity relationship (SAR) analysis of SIRTl stimulating activity among a large number of natural polyphenol products (none of which were tolans or included any of the present compounds. This was based on identifying SIRTl activators as above. Inspection of two activating compounds suggested a possible SAR. Piceatannol (3,5,3\4'-tetrahydroxy-trans-stilbene) comprises two phenyl groups trans to one another across a linking ethylene moiety. The /raws-stilbene ring structures of piceatannol are superimposable on the flavonoid A and B rings of quercetin (a 3-ringed flavonoid compound with SIRTl activating activity) with the ether oxygen and carbon-2 of the C ring aligning with the ethylene carbons in piceatannol. Further, the 5, 7, 3' and 4' hydroxyl group positions in quercetin can be aligned, respectively, with the 3, 5, 3' and 4' hydroxyls of piceatannol. Both polyphenols, quercetin and piceatannol, and members of a large and diverse group of plant secondary metabolites that includes flavones, stilbenes, flavanones, isoflavones, catechins (flavan-3-ols), chalcones, tannins and anthocyanidins were examined. A secondary screen encompassing resveratrol was performed with various additional representatives from a number of the above polyphenol classes. The screen emphasized flavones due to the greater number of hydroxyl position variants available in this group. Additional potent SIRTl activators were found among the stilbenes, chalcones and flavones in this screen. The six most active flavones had 3' and 4' hydroxyls although the most active compound overall, resveratrol was more active than piceatannol, which differs only by its additional 3 '-hydroxyl. The document noted that the importance of the 4'-hydroxyl to activity was evidenced by the fact that each of the 12 most stimulatory flavones share this feature. Many, but not all of the most active compounds include hydroxyls in the two meta positions of the flavone A ring trans to that with the 4' or 3 ',4' pattern (in the B ring). A potentially coplanar orientation of the trans phenyl rings was suggested as possibly being important for activity since catechins and flavanones, which lack the 2,3 double- bond, had weak activity despite having equivalent hydroxylation patterns to various stimulatory flavones. The absence of activity in the isoflavone genistein, although hydroxylated in an equivalent way to stimulatory compounds (e.g., resveratrol) was consistent with the idea that the trans positioning and spacing of the hydroxylated rings contributes strongly to activity. It was noted that the biological effects of polyphenols are frequently attributed to antioxidant, metal ion chelating and/or free-radical scavenging activity, leading to the suggestion that the apparent polyphenol stimulation of SIRTl might simply represent repair of oxidative and/or metal-ion induced damage incurred during preparation of the recombinant SIRTl protein for testing. However, this was countered by the fact that (1) a variety of free-radical protective compounds, including antioxidants, chelators and radical scavengers, failed to stimulate SIRTl; and (2) diverse SIRTl stimulating activity was observed among various polyphenols of equivalent antioxidant capacity (comparing resveratrol, quercetin and the epicatechins). Using the above (or the exemplified) screening in combination with the SAR information developed by the present inventor together with that discussed above, it is within the skill of the art, in view of the present disclosure, to design and synthesize additional tolan compounds in accordance with this invention, that are SIRTl activators and thereby, expected to have the skin activity that would make them useful in the methods of the present invention. Compounds designed in this way are further tested using methods exemplified below for their effects on NFKB and COXl and COX2 activity, inhibition of tyrosinase, as well as in appropriate in vivo models of skin activity (based on anti- wrinkle activity, increasing skin tautness, etc., as are known in the art In vivo and Cellular Testing of the Active Compounds Sir2 and its closest eukaryotic homologs have a role in conserved pathways of stress- response and longevity regulation and this effect can be evaluated using yeast or the nematode worm C. elegans (Kenyon, C. Cell 705.-165-168 (2001); Guarente, L et al, Nature 408:255-62 (2000); Lin, SJ. et al., Science 289:2126-8 (2000); Anderson, R. M. et al., Nature 423:181-5 (2003) Tissenbaum, HA et al., Nature 410:221-30 (2001)). C. elegans Sir2.1 functions in the insulin/IGF- 1 signaling pathway (Kenyon, supra, Guarente et al., supra) which has also been shown to regulate lifespan in rodents (Holzenberger, M. et al., Nature 427:182-7 (2003); Bluher, M. et al., Science 299:489-90 (2003). This model can also be used to evaluate compounds.. Effects of activating SIRTl are evaluated by immuno staining, Western blotting, and cytometry of normal human skin cells in culture and on healthy skin samples ex vivo by comparing control samples with compound-treated samples Cellular integrity and aging is followed by comet assays measuring DNA fragmentation and β galactosidase activity (a marker of senescence) in test and control samples.

The effectiveness of the tolan test compounds in human subjects are evaluated by applying a formulation that includes 1% (w/w) of the test compound once daily to the face and neck for 4 weeks. Control subjects receive only the carrier formulation. Dermatologists use a graded scale (1-9) to score fine lines and wrinkles, hydration, pigment color intensity, complexion radiance, skin density, firmness, complexion homogeneity, and texture of the skin before and after the first application and again after 4 weeks of use. A Pixel Skin method, based on an analysis of the gray-level variance and surface of imperfections (age-related parameters) from numerical pictures of the faces, is used to objectively measure the skin care efficacy. See, for example Moreau, M et al, 2007). Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified.

EXAMPLE 1 Hydroxytolans and Resveratrol Stimulate SIRTl Enzymatic Activity The ability of a compound of the hydroxytolan series to modulate SIRTl activity was assayed using a SIRTl Fluorescent Activity Assay/Drug Discovery Kit which is designed to measure the lysyl deacetylase activity of recombinant human SIRTl included in the kit. The SIRTl Fluorescent Activity Assay is based on the "Fluor de Lys-SIRTl Substrate'VDeveloper II combination in which Fluor de Lys-SIRTl Substrate is a peptide comprising amino acids 379- 382 of human p53 (Arg-His-Lys-Lys(Ac)) (SEQ ID NO:1. The assay's fluorescence signal is generated in proportion to the amount of deacetylation of the Lys corresponding to Lys-382, a known in vivo target of SIRTl activity.

The assay procedure included two steps. First, the Fluor deLys-SIRTl substrate, indicated above was incubated with human recombinant SIRTl together with the co-substrate NAD+. Deacetylation of Fluor de Lys-SIRTl sensitizes it so that, in the second step, treatment with the Fluor de Lys™ Developer II produces a fluorophore. Resveratrol, a SIRTl activator, and the sodium salt of suramin, an inhibitor, were included as positive controls for these two types of activity modulation.

Results are shown in Table 3, below. Four HT compounds of the present invention and resveratrol at concentrations of 20 μM were assayed for their abilities to modulate SIRTl activity. A 15 min. incubation period was employed. Resveratrol deacetylated 20.1 ± 0.2 μM of the target peptide standard. KST 201, 213, 301 and 401 deacetylated 12.2 + 0.9 μM, 12.4 + 0.6 μM, 15.8 ± 0.6 μM, and 14.7 ± 1.5 μM of the peptide standard, respectively. These values represent 60 to 80% of the values of resveratrol. Suramin abrogated the modulating effect of all of the compounds. Table 3: Modulation of Human SIRTl by Members of the KST Series

Test Agent Deacetylated Fluor

(20μM) deLys Std. (μM)

Resveratrol 20.1 + 0.2

KST 201 12.2 + 0.9

KST 213 12.4 + 0.6

KST 301 15.8 + 0.6

KST 401 14.7 + 1.5

EXAMPLE 2 Hvdroxytolan Compound KST 201 Inhibits NFKB Binding Activity The NFKB family members are transcription factors playing multiple roles in the regulation of immune and inflammatory responses, developmental processes and diseases such as cancer. NFKB and its inhibitor IKB form an inactive complex in cytoplasm. Activation occurs by phosphorylation, ubiquitination and degradation of IKB, which then releases active NFKB into the nucleus where it regulates the expression of target genes leading to a variety of cellular behaviors. More recently, it has been shown that SIRTl modulates NFκB-dependent transcription by interacting with and deacetylating the active subunit of NFKB (RelA/p65) in a site-specific manner, leading to its deactivation (Yeung, et ah, 2004).

Since both resveratrol and KST 201 activated SIRTl which led to NFKB deactivation, the effects of both compounds on NFKB activity were evaluated. An enzyme-linked immunosorbent assay (ELISA) kit was used to detect the active forms of NFKB p65 by measuring its binding to a consensus DNA sequence. Cell lysates for DNA binding study of NFKB p65 were prepared from prostate cancer DU 145 cells treated with resveratrol or KST 201 at different concentrations or for different intervals. DNA sequences containing p65-binding site were immobilized to the surfaces of 96-well plates allowing activated p65 to interact. After washing, the amount of bound p65 was detected and quantified by the chemiluminescent sandwich ELISA. NFKB was constitutively activated in DU145 cells, and the basal level was measured for normalization of data obtained from treated cells. Results are shown in Figures 1 and 2.

The results revealed that the DNA binding activity decreased to 75% of its basal activity following 24-hour treatments with 5, 25 and 50 μM resveratrol and to 55% of its basal activity following a 100 μM treatment. In comparison, DNA binding activity decreased to 75% of its basal activity following 24-hour exposure to 5 or 25 μM KST 201, to 50% following exposure to 50 μM, and to 30% following exposure to 100 μM (Fig. 1).

Following exposure to 100 μM resveratrol (Fig. 2), the DNA binding activity of p65 was suppressed to 40.2% of its basal activity by 12 h. However, the DNA binding activity of p65 increased slightly to 55.3% of basal activity after 24-hour exposure, which may reflect the lability of resveratrol.

By comparison, following exposure to KST 201, the DNA binding activity of p65 was suppressed to 75% of its basal activity by 6 h and remained at this level until it dropped to 55% of basal activity after 24 h.

EXAMPLE 3

Hvdroxytolan Compound KST 201 Inhibits COXl and COX2 Activity

Cyclooxygenase enzymes (COX) enzymes catalyze the biosynthesis of PGH₂ from arachidonic acid employing their cyclooxygenase and peroxidase activities located at distinct active sites in the protein. The COX assay used here examined the inhibitory effect of the compound on both COXl and COX2 isomers by comparing the amount of the final stable product, PGF_2α, generated from PGH₂ by SnCl₂, produced in the presence or absence of either resveratrol or KST 201. This assay is described, for example, in Seaver, B et ah, (2004) . J. Herbal Pharmacother. 4:11-18.

The reaction for PGF_2α synthesis were prepared by mixing solutions of resveratrol or KST 201), COXl or COX2, heme, arachidonic acid and saturated SnCl₂ solution and then transferring to a 96- well plate followed by colorimetric development using Ellman's reagent. The intensity of color was inversely proportional to the amount of free prostaglandins, quantitatively representing the COX enzymatic activity under the influence of resveratrol or KST 201. The results shown in Figs, 3 and 4 indicated that resveratrol and KST 201 decreased the ability of both COXl and COX2 to catalyze the conversion of the prostaglandin precursor.

Treatment with resveratrol at concentrations from 10 to 200 μM, significantly inhibited enzymatic activity of both COX isomers. COXl inhibition ranged from 17% to 100% inhibition of initial activity (Fig. 3). COX2 inhibition ranged from 18% to 94% (Fig. 4. KST 201 was not as potent as resveratrol in this assay, although both COX isomers were inhibited by at least about 50%. EXAMPLE 4 Hydroxytolan Compounds Inhibit Tyrosinase Activity

Resveratrol is known to be an excellent tyrosinase inhibitor (for example, of mushroom tyrosinase) and a skin whitening agent. See, inter alia, Fremont, 2000, supra; Bernard et al. (2000). Resveratrol inhibits mushroom tyrosinase with an IC₅₀ value of about 150 μM. The ability of one of the compounds of the present invention, KST 201, was examined for its inhibition of tyrosinase.

This method was based on publications of Bernard et al. {supra ) and Lee, SH et al. , Biol Pharm Bull, 2002, 25:1045-48. The assay is based on the following reactions:

ryrosiπe L-DOPA

H

L-DOPA LX}PAquιn£,Φe

Materials

1. Sodium L-tyrosine (MW=225.2). 10 mM of working solution in 50 mM phosphate buffer (PB), pH 6.5

2. L-DOPA [3,4-Dihydroxy-l-phenylalanine]: ((MW=197.2). 5 mM stock reagent was dissolved in 50 mM PB, pH 5.0. Dilution of 1:10 in 50 mM PB, pH 6.5, was made for the working reagent.

3. Phosphate Buffer (PB): 50 mM, pH 6.5, made from 1 M Na₂HPO₄ in dH₂O and 1 M Na H₂PO₄ in dH₂0. For 50 mM PB, 4.07 ml IM Na₂HPO₄ and 5.97 ml Na H₂PO₄ were diluted in about 140 ml dH₂0, and the pH brought to 6.5; 200 ml dH₂0 were added.

4. 60 U mushroom tyrosinase (Sigma T3824- 25,000 U): stock vial was diluted in 1.5625 ml 50 mM PB yielding 16 U/μl. Aliquots of ~ lOOμl were frozen. Before use, the enzyme solution was diluted 10-fold in 50 mM PB, pH 6.5. 5. Kojic acid: (MW=142.11 ; 70 niM stock solution was diluted 10Ox in 10% ethanol to yield a 0.7 niM solution. Procedure

1. A 0.1M solution of a test compound was prepared and 100 fold dilutions were made with 150 μl 50 mM PB and 10 μl ImM test compound + 990 μl 50 mM PB, respectively. This yields 250 μM and 100 μM solutions.

2. 24 well plates were set up in a conformation as shown below (all numbers refer to concentrations μM). "Cpdl refers to a first test compound, e.g., KST-201 and RES to resveratrol. Cpd2 refers to another test compound that could be included in the plate.

"Blank" "Total" "KA 700" Res 1000 Cpd 1 1000 Cpd1 1000

Cpd1 250 Cpd1 250 Cpd1 100 Cpd1 100 Cpd2 1000 Cpd2 1000

Cpd2 250 Cpd2 250 Cpd2 100 Cpd2 100

3. 25 μl of a dilution of test compound delivered to wells of a 96 well microplate except for

"total" and "blank" wells. 25 μl of buffer went into the "total" well.

4. The tyrosinase was prepared by diluting 100 μl 10-fold in 50 mM PB, pH 6, mixed an kept on ice. 50 μl of this working mushroom tyrosinase (1600 U/ml) was added to each well (using a fresh pipet tip for in each well). The wells' contents were mixed and allow to incubate for 20 minutes

6. 75 μl 50 mM PB, pH 6.5 was added to the blank wells of the 24 well plate.

7. A master mix of the following reagents was prepared: 25 μl L-DOPA (0.5 mM)

25 μl Na L-tyrosine (10 mM). 875 μl mM PB, pH 6.5

8. About 3 minutes before the end of the incubation in step 4, the entire contents of each well was transferred to the respectively labeled well of the 24 well plate.

10. At the end of the incubation, 925 μl of the master mix was added to each of the wells as quickly as possible. 1 l.The plates were placed on the plate reader and read at 30 second intervals for 10 minutes at wavelength of 475 nm to obtain the results.

Enzyme Inhibition was shown as % Inhibition = ([A-B]/A) x 100 where A = Absorbance at 475 nm ("Total"); and B = Absorbance at 475 nm with test compound. Results of assays of several of the hydroxytolans of the present invention are shown in

Table 4, below. Table 4: Tyrosinase Inhibition by Hydroxytolan Compounds

The results (and others not shown) indicate that all three hydroxytolan compounds are dose-dependent inhibitors of tyrosinase. KST-201 is as active as resveratrol, whereas KST-301 has somewhat reduced activity and KST-401 is less active under these conditions. The improved toxicity profile of these compounds vs. resveratrol make them better as skin-reactive agents (for all physiologic or pathologic events that are tyrosinase-mediated or tyrosinase- dependent. Important among these activities is skin whitening.

Moreover, the structure of these tolan molecules, particularly KST 201, affords greater stability compared to resveratrol, and thereby provide better efficacy than resveratrol in vivo in achieving skin whitening. As shown in the earlier examples, KST-201 stimulated SIRTl and inhibited NFKB binding, activities, both of which are known to be correlated with "skin activity" as defined herein.

EXAMPLE 5 KST-201 Is Not A Skin Irritant in Assays of Cell Viability, IL-lα and PGE₇ Release MatTek EpiDerm™ MTT viability assay was performed on full thickness human skin sections. This test provides an estimate of dermal irritation potential using an alternative to the Draize methodology. The MatTek EpiDerm™ MTT Viability Assay has been demonstrated to be a quantitative method for assessing potential skin hazards.

EpiDermFT™ Full Thickness skin samples were received from MatTek and refrigerated until used (about 24 hrs). Before use, tissues were incubated (37°C in 5% CO₂) with assay medium for an equilibration period of 16-18 hours. The medium was replaced with fresh medium before dosing. Tissues were incubated in a 6-well microplates with 2 ml of medium.

Reduction of MIT: 100 mg of the test compound was mixed with 1 ml of the MTT (methylthiazole tetrazolium) solution. A negative control, 100 μl of tissue culture water, was tested concurrently. The solutions were incubated at room temperature in the dark for 60 minutes and then visually inspected for purple coloration, which indicates that the test article converted (reduced) MTT to a formazan product. Since tissue viability is based on MTT conversion, direct conversion of MTT by a test compound (i.e., not acting on the tissue or cells) can exaggerate the measured viability, making a test compound appear less irritating than it really is. KST-201 did not itself reduce MTT.

KST-201 was dosed neat. 100 mg of KST-201 solution were applied to the top of each tissue sample and remained in contact with the tissue for the indicated times. Each treatment was conducted in triplicate. A negative control, undosed tissues, was tested in duplicate at the 8 hour time point. A positive control (the detergent 1% Triton X-IOO; 200 μl/plate) was tested in duplicate for 6, 8 and 12 hour exposure times. The 8- and 12-hour time points were used to calculate the ET50.

Following this exposure period, the viability of the tissues was determined using MTT uptake and conversion. Each tissue sample was rinsed with phosphate buffered saline (PBS) and transferred to a 6-well plate containing 2 ml of MTT (Methyl thiazole tetrazolium ) solution (1 mg/ml MTT diluted in Dulbecco's Modified Eagle's Medium (DMEM)). The tissues were then returned to the incubator for a three-hour MTT incubation period. Following this, each sample was rinsed and then treated overnight with 3.0 ml of extractant solution in the well of a 6-well plate and 1.0 ml of extractant in an EpiDermFT™ millicell. The absorbency of an aliquot of the extracted MTT formazan product was measured at 540 nm using a microplate reader, subtracting the absorbance at a reference wavelength of 690 nm.

The viability was then expressed as a percent of control values. The mean percent viability for each time point was used to calculate an ET₅₀, which represents the time at which the tissue viability was reduced 50% compared to control tissues.

The mean absorbance value for each time point was calculated from the optical density (OD) of duplicate or triplicate samples and expressed as % viability for each sample using the following formula:

% Viability = 100 X (OD_sample/OD_neg_atlve control)

The ET₅₀, the time at which tissue viability was reduced 50% compared to control tissues, was determined using the equation: V=a + b x log t Where V= percentage viability, t = time in minutes, and a and b are constants that can be determined by using the viability data for two different exposure times of the test compound that must yield viabilities that flank 50%.

The results are shown below. The other hydroxytolan compounds described herein are also tested in this and the other assays described below and yield similar results indicating that they are non-irritating.

Table 5: Full Thickness Skin Viability Assay and Irritancy Determination

The calculated ET₅₀ and conclusions are summarized below:

Test Agent Exposure (hr) ET,n (hr) Irritancy Classification

KST-201 1, 4, 24 >24.0 Non-irritating

Pos. Control 8, 12 10.4 Within Range

5.50-10.50

Correlation of In vitro and In vivo

According to these assays (per MatTek) the following groups can be used to assign expected in vivo irritancy responses based on the ET₅₀ results of this assay

Expected In Vivo Irritancy Example ET₅₀ (hrs) Severe, Probably Corrosive Concentrated Nitric acid < 0.5 Moderate 1% Sodium Dodecyl Sulfate 0.5 - 4 Moderate to Mild 1% Triton X-100 4 - 12 Very Mild Baby shampoo 12 - 24 Non-irritating 10% Tween 20 >24 Based on the foregoing, KST-201 is not an irritant and is used in the cosmetic and therapeutic methods described herein at the indicated doses without concern for skin irritation. It is less irritating than baby shampoo. The other hydroxytolan compounds disclosed herein also expected to have this property of not being irritants. Measurement of the release of cytokines, such as interleukin lα (IL- lα) or prostaglandin

E₂ (PGE2), from the full thickness skin during incubation with a test compound is also considered a valuable predictor of the agent's in vivo irritation potential. The observations that the ET₅₀ values (based on viability, or survival, of cells) in the tissue sample in presence of KST-201 is an independent measure of the low irritancy and toxicity of this compound. The decreases in IL- lα and PGE2 release from cells in the same tissue samples when exposed to KST-201 (below) is an additional strong predictor that this compound has low toxicity and irritancy in vivo.

Enzyme-linked immunosorbent assay (ELISA) kits were obtained from Cayman Chemical (Ann Arbor, MI) to test for the presence of the cytokine Interleukin- 1 α (IL- lα) The kits used were Cayman Chemical Interleukin- lα (human) EIA Kit, Lot No. 0407089 and the

Prostaglandin E2 Express EIA Kit, Lot No. 0405151, in the assay medium. Approximately 1 ml of the media from beneath the tissue millicells were sampled at the end of the dosing and incubation and stored at approximately -8O⁰C until used. The conventional ELISA was conducted according to the manufacturer's directions on both undiluted samples and samples diluted to 1 : 10 in order to achieve the detectable protein concentration range of the ELISA kits. Protein standards were run with both ELISAs in order to derive standard curves.

Optical density readings of colorimetric indications of protein concentration for each well were determined using a spectrophotometer. Unknown protein concentrations (IL- lα and PGE₂) were determined by comparison of OD readings to the respective standard curve. In the IL-lα ELISA, results from the 100% (neat) concentration fell within the detectable protein concentration range of the kit, while in the PGE2 ELISA, results from the 10% concentration fell within range.

Assay results of IL-lα (in pg/ml) or PGE2 in ng/ml are shown in Tables 6 and 7 below. Table 6: Interleukin-lα (IL-lα) Release * from KST-201-treated Tissue

* pg/ml

Table 7: Prostaglandin E₇ (PGE2) Release * from KST-201-treated Tissue

* ng/ml

While interleukin-lα (IL-lα) and prostaglandin E₂ (PGE₂) are known primarily as proinflammatory cytokines, they also control a wide variety of other processes. IL-lα is a protein that plays a critical role in the regulation of the body's response to inflammation, microbial invasion, tissue injury, and immunological response (Dinarello, CA (1998). Int. Rev. Immunol. 16:457-99; Madinov, L et al, (2003) Thromb. Haemost. 90: 367-71). Recent studies suggest that IL-lα also has a role in wound healing, rheumatoid arthritis, Alzheimer's disease and tumor growth (Rajalingam, D et al. (2007) Biochem. Biophys. Res. Commun. 360:604-$; Infante, J et al. (2007) Dement. Geriatr. Cogn. Disord. 23 215-18).

Likewise, PGE₂ is an important mediator of numerous (patho)physiological processes, in addition to inflammation, including female reproduction, cell proliferation and repair, tumorigenesis, gastrointestinal ulceration, kidney development and function, cardiovascular development and function, bone formation and neurodegenerative disorders (Murakami, M et al, (2004) Prog. Lipid Res. 43: 3-35). Therefore, the diminished levels of IL-lα and PGE₂ observed following KST-201 treatment are indicative of the ability of this compound, and the other related hydroxytolan compounds of the present invention to modulate not only inflammation but a variety of processes. EXAMPLE 6

KST-201 Kills Tumor Cells and is Potent Against Melanoma

For a description of well established rodent models which are considered to be highly representative of a broad spectrum of human tumors, see, Geran, R.I. et al , "Protocols for Screening Chemical Agents and Natural Products Against Animal Tumors and Other Biological Systems (Third Edition)", Cane. Chemother. Reports, Pt 3, 3: 1-112,

Schepartz SA, Jpn J Antibiot. 30 Suppl:35-40 (1977) describes antitumor screening procedures of the National Cancer Institute. The screens (with respect to included tumors) have changed over the years and have included P388 tumor (initially for testing of anti-cancer natural products) and B 16 melanoma and Lewis lung carcinoma for special studies. P388 has also served as a pre-screen that is followed by a panel of transplanted tumors and human tumor xenografts representing the major tumor sites.

P388 is a murine lymphocytic leukemia P388 that has been a long established model to screen and study antitumor/anticancer agents. This line was established from a tumor induced in 1955 in a DBA/2 mouse by painting with MCA Scientific Proceedings, Pathologists and Bacteriologists 33:603, 1957.

Another commonly used tumor model is the 3LL Lewis Lung Carcinoma (Malave, I. et al., J. Nat'l. Cane. Inst. «52:83-88 (1979) which originated from a spontaneously carcinoma of the lung in a C57BL/6 mouse (Cancer Res 15:39, 1955). This model has been utilized by a number of investigators a lung carcinoma metastasis model. See, for example, Gorelik, E. et al., J. Nat'l. Cane. Inst. 65: 1257-1264 (1980); Gorelik, E. et al., Rec. Results Cane. Res. 75:20-28 (1980); Isakov, N. et al., Invasion Metas. 2: 12-32 (1982) Talmadge JE. et al, J. Nat'l. Cane. Inst. 69:975- 980 (1982); Hilgard, P. et al, Br. J. Cane 35:78-86 (1977)).

The B 16 murine melanoma is a rapidly growing, metastatic tumor of spontaneous origin that does not regress spontaneously (Greene. EL. Handbook on Genetically Standardized JAX Mice. Second Ed., Bar Harbor, Maine: Bar Harbor Times Publishing Co., 1977). It has been used in a wide variety of studies on tumor metastasis and has generally been characterized as poorly or nonimmuno genie (Leveson, SH et al, Cancer Res. 39:582-86 (1979). Highly metastatic sublines such as B16-F10 (Fidler, IJ, Nat. New Biol., 242:148-9, 1973) have been developed. This tumor is also used to study experimental metastasis by i.v. injection resulting in distinct, melanotic lung metastases.

The M5076 tumor, a transplantable murine reticulum cell sarcoma that arose spontaneously in the ovary of a C57BL/6 mouse (Talmadge JE et al , Cancer Res. 41 : 1271-80 (1981). This tumor displays functional and ultrastructural characteristics indicating that it is of macrophage origin. Karyotype analysis revealed that M5076 tumor cells are hypodiploid with another abnormality that serves as marker for tumor cell identification. M5076/ovarian carcinoma spontaneously metastasizes to abdominal organs (liver, spleen, kidney, ovary, uterus), but not to the lung in C57BL/6 mice (Mantovani A et al, Int J Cancer. 25:617-20 (1980).

DU- 145 is a human prostate cancer cell line derived from a brain metastasis (Stone KR et al. (1978) Int. J. Cancer 21: 274-81; Alimirah F et al. (2006) FEBS Lett. 580:2294-300). These cells have moderate metastatic potential compared. The DU145 cell line was derived from brain metastasis. DU145 are not hormone sensitive and do not express Prostate Specific Antigen. It grows as subcutaneous nodules in immune-deficient mice. Sublines with variable metastatic properties have been derived {e.g., Kozlowski, JM. et al., In: Tumor Progression and Metastasis, Fidler, IJ et al, eds, Alan R. Liss, Inc., New York, 1988, p. 189-231; Bex A et al, Cancer Genet Cytogenet. 724:98-104 (2001)).

KST-201 (Tolecine™) was tested against four murine tumors and one human tumor (growing in immunodeficient mice. Dose-response studies were done and an IC₅₀ value was calculated. Results appear in Table 8, below. KST-201 was effective against all these tumors, and it's highest potency was against the murine melanoma B 16 which, as noted, is a traditional and accepted melanoma tumor growth (and metastasis) model. Table 8: Killing of Tumor Cells by KST-201 (Tolecine™)

All references cited herein, including journal articles or abstracts, published or corresponding

U.S. or foreign patent applications, issued U.S. or foreign patents, or any other references, are entirely incorporated by reference herein, including all data, tables, figures, and text presented in the cited references. Additionally, the entire contents of the references cited within the references cited herein are also entirely incorporated by references.

Reference to known method steps, conventional methods steps, known methods or conventional methods is not in any way an admission that any aspect, description or embodiment of the present invention is disclosed, taught or suggested in the relevant art.

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art (including the contents of the references cited herein), readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art.

References

In addition to the more complete reference citations above, the following list provides full citations to those minimal citations above that indicated author(s) and publication year.

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Claims

y : sai- en - -WHAT IS CLAIMED IS:

1. A method of activating SIRTl protein enzymatic activity in skin cells in a subject, comprising administering to skin a subject in need thereof an effective amount of a composition that comprises:

(a) a cosmetically, dermatologically or pharmaceutically acceptable carrier or excipient and

(b) one or more of a first tolan compound, which is a compound of Formula I:

Formula I

L represents a -C≡C- acetylene linkage,

R¹ and R² are, independently substituents at any available position of the phenyl rings; m and n are, independently, 0, 1, 2, 3, 4 or 5 representing the number of R¹ and R² substituents of the rings, respectively, and at least one of m or n must be >1; wherein R¹ and/or R² is:

-OH, a halogen, a haloalkyl group with one C atom substituted with from 1 to 3 halogen atoms, a C₁-C_O alkyl, C₂-C_O alkenyl, or C₂-C_O alkynyl group, an acetyl group, -OR³, wherein R³ is a C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl group, an acetyl group, or β-D-glucoside, a carboxyl group, an α hydroxyl carboxylic acid, or a nitro group, and wherein, optionally one of the two phenyl rings is replaced with a cyclohexyl ring, which compound activates said SIRTl enzymatic activity, resulting in protection of the skin from, or reversal at least in part of, an effect of skin aging or environmental insult. y : sai- en - -

2. The method of claim 1 wherein the effect of aging or environmental insult being protected against or reversed comprises wrinkling, loss of tightness, facial fine lines, dehydration, color intensity of pigmented regions, or damage from exposure to solar radiation of radiation from another li ¹gOh¹ t source.

3. A method of inhibiting tyrosinase activity in skins cells and thereby promoting whitening of the skin, comprising administering to the skin of a subject in need thereof an effective amount of a composition that comprises

(a) a cosmetically, dermatologically or pharmaceutically acceptable carrier or excipient, and

(b) one or more of a first tolan compound, which is a compound of Formula I:

Formula I

wherein:

L represents a -C≡C- acetylene linkage,

R¹ and R² are, independently substituents at any available position of the phenyl rings; m and n are, independently, O, 1, 2, 3, 4 or 5 representing the number of R¹ and R² substituents of the rings, respectively, and at least one of m or n must be >1; wherein R¹ and/or R² is: -OH, a halogen, a haloalkyl group with one C atom substituted with from 1 to 3 halogen atoms, a C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl group, an acetyl group, -OR³, wherein R³ is a C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl group, an acetyl group, or β-D-glucoside, a carboxyl group, an α hydroxyl carboxylic acid, or y : sai- en - -

a nitro group, and wherein, optionally one of the two phenyl rings is replaced with a cyclohexyl ring, which compound inhibits said tyrosinase activity and promotes said whitening.

4. A method of treating or alleviating the symptoms of a skin condition or disease in a subject, comprising administering to a subject in need thereof an effective amount of a composition that comprises

(a) a dermatologically or pharmaceutically acceptable carrier or excipient, and

(b) one or more of a first tolan compound, which is a compound of Formula I:

Formula I

L represents a -C=C- acetylene linkage,

R¹ and R² are, independently substituents at any available position of the phenyl rings; m and n are, independently, 0, 1, 2, 3, 4 or 5 representing the number of R¹ and R² substituents of the rings, respectively, and at least one of m or n must be >1; wherein R¹ and/or R² is:

-OH, a halogen, a haloalkyl group with one C atom substituted with from 1 to 3 halogen atoms, a C₁-C_O alkyl, C₂-C_O alkenyl, or C₂-C_O alkynyl group, an acetyl group, -OR³, wherein R³ is a C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl group, an acetyl group, or β-D-glucoside, a carboxyl group, an α hydroxyl carboxylic acid, or a nitro group, and wherein, optionally one of the two phenyl rings is replaced with a cyclohexyl ring, y : sai- en - -

which compound activates a SIRTl enzymatic activity in skin cells, thereby treating said disease or condition or alleviating said symptoms.

5. The method of claim 4 wherein said skin condition or disease is psoriasis, contact dermatitis, atopic dermatitis, actinic keratosis, a keratinization disorder epidermolysis bullosa diseases, pemphigus, exfoliative dermatitis, seborrheic dermatitis, an erythemas, discoid lupus erythematosus, or dermatomyositis.

6. A method of inhibiting cutaneous inflammation in a subject, comprising administering to a subject in need thereof an antiinflammatory effective amount of a composition that comprises:

(a) a dermatologically or pharmaceutically acceptable carrier or excipient, and

(b) one or more of a first tolan compound, which is a compound of Formula I:

Formula I

L represents a -C=C- acetylene linkage,

R¹ and R² are, independently substituents at any available position of the phenyl rings; m and n are, independently, 0, 1, 2, 3, 4 or 5 representing the number of R¹ and R² substituents of the rings, respectively, and at least one of m or n must be >1; wherein R¹ and/or R² is:

-OH, a halogen, a haloalkyl group with one C atom substituted with from 1 to 3 halogen atoms, a C₁-C_O alkyl, C₂-C_O alkenyl, or C₂-C_O alkynyl group, an acetyl group, -OR³, wherein R³ is a C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl group, an acetyl group, or β-D-glucoside, a carboxyl group, y : sai- en - -

an α hydroxyl carboxylic acid, or a nitro group, and wherein, optionally one of the two phenyl rings is replaced with a cyclohexyl ring, which compound inhibits NFKB binding to DNA or activity of cyclooxygenase COXl and/or COX2 enzyme in skin cells, thereby inhibiting said inflammation.

7. A method of cosmetic treatment of a keratinous substrate in a subject, comprising applying to the keratinous substrate of a subject in need thereof a composition that comprises:

(a) a cosmetically or dermatologically acceptable carrier or excipient, and

(b) one or more of a first tolan compound, which is a compound of Formula I:

Formula I

wherein:

L represents a -C≡C- acetylene linkage,

R¹ and R² are, independently substituents at any available position of the phenyl rings; m and n are, independently, 0, 1, 2, 3, 4 or 5 representing the number of R¹ and R² substituents of the rings, respectively, and at least one of m or n must be >1; wherein R¹ and/or R² is:

-OH, a halogen, a haloalkyl group with one C atom substituted with from 1 to 3 halogen atoms, a C₁-C_O alkyl, C₂-C_O alkenyl, or C₂-C_O alkynyl group, an acetyl group, -OR³, wherein R³ is a C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl group, an acetyl group, or β-D-glucoside, a carboxyl group, an α hydroxyl carboxylic acid, or a nitro group, and wherein, optionally one of the two phenyl rings is replaced with a cyclohexyl ring, y : sai- en - -

which compound activates a SIRTl enzymatic activity in skin cells, thereby cosmetically treating said keratinous substrate.

8. The method of any of claims 1-7 wherein at least one occurrence of R¹ or R² is -OH.

9. The method of claim 8 wherein the compound is selected from the group consisting (a) HT-I, the structural formula of which is:

Formula Il

(b) HT-2, the structural formula of which is:

Formula

HT-3, the structural formula of which is:

Formula IV

(d) HT-4, the structural formula of which is:

Formula V

and (e) 3,4,3'4'-tetrahydroxytolan (HT-5). y : sai- en - -

10. The method of any of claims 1-7 wherein the compound is selected from the group consisting of: 3,5-dihydroxy-4-methoxytolan 3-O-β-glucoside; 3,3\5-trihydroxy-4'-methoxytolan 3-O-β-D-glucoside; 3,5-dihydroxy-4'-thiomethyltolan; 3,5-dihydroxy-4'-chlorotolan; 3, 5- dihydroxytolan; 3,5-dihydroxy-4'-ethyltolan; 3,5-dihydroxy-4'-fluorotolan; 3,5,3',4'- tetrahydroxytolan ; 3,5-dihydroxy-4'-methyltolan; 3,5-dihydroxy-4'-azidotolan; 3,5-dihydroxy-4'- nitrotolan; 3.5-dihydroxy-4-isopropyltolan; 3.5-dihydroxy-4'-methoxytolan; 3,5,3'-trihydroxy-4'- methoxytolan; 3,4'-dihydroxy-5-acetoxytolan; 3,5-dihydroxy-4'-acetoxytolan; (E)-l-3,5-dihyd- roxyphenyl)-2-(2-napthyl)ethyne ; 3-hydroxytolan ; 3,5-dimethoxymethoxy-4'-thiomethyltolan; 3,5-dihidyroxy-4-acetamidetolan; 3,4-dihydroxytolan; (E)- l-3,5-dihydroxyphenyl)-2- (cyclohexyl)ethyne ; 3,4-dimethoxytolan; 3,4,3'-trihydroxy-4'-acetoxytolan; 3,4,4'-trihydroxy-3'- acetoxytolan; 3,5,4'-trihydroxy-3'-acetoxytolan; 3,5,3'-trihydroxy-4'-acetoxytolan; 3,4,3'-tri- hydroxy-4'-acetamidetolan; 3,4,4' -trihydroxy-3'-acetamidetolan; 3,4,3' -trihydroxy-3'-acet- amidetolan; 3,4,4' -trihydroxy-4'-acetamidetolan; 3,4'-dihydroxy-4-acetoxytolan; 4,4'-dihydroxy-3- acetoxytolan ; 3 ,4-dihydroxy-4 ' - acetoxytolan ; 3 ,4 ' -dihydroxy-4 ' - acetamidetolan ; 4,4 ' -dihydroxy- 3 - acetamidetolan; 3,4-dihydroxy-4'-acetamidetolan; 3,5,4' -trihydroxy-4-acetamidetolan; 3,4'- dihydroxy-4-acetamidetolan; 4,4'-dihydroxy-3-acetamidetolan; and 3,4-dihydroxy-4'- acetamidetolan.

11. The method of any of claims 1 - 10 wherein the compound is administered topically to the subject.

12. The method of any of claims 1 - 11 which further comprises administering topically to said subject one or more additional compounds of Formula I wherein L is a -C=C- linkage or a - C-C-linkage, which additional compound activates SIRTl enzymatic activity and/or inhibits tyrosinase activity and/or inhibits NFKB binding to DNA and/or inhibits COXl and COX2 enzymatic activity in skin cells.

13. The method of claim 11 or 12 wherein the additional compound is administered topically to the subject. y : sai- en - -

14. The method of claim 11 or 12 wherein the additional compound is administered by a different route than the first compound or compounds, which route is selected from the group consisting of: oral, intravenous, intraperitoneal, intrathecal, intramuscular, subcutaneous, transdermal, rectal, and intranasal.

15. The method of any of claims 11-14, wherein the additional compound is not resveratrol.

16. A topical pharmaceutical or cosmetic composition formulated for topical administration, and useful for a keratinous substrate in a subject with a skin condition or disease, which composition comprises

(a) a topical, cosmetically or pharmaceutically acceptable carrier or excipient, and

(b) one or more of a first tolan compound, which is a compound of Formula I:

Formula I

L represents a -C=C- acetylene linkage,

R¹ and R² are, independently substituents at any available position of the phenyl rings; m and n are, independently, 0, 1, 2, 3, 4 or 5 representing the number of R¹ and R² substituents of the rings, respectively, and at least one of m or n must be >1; wherein R¹ and/or R² is:

-OH, a halogen, a haloalkyl group with one C atom substituted with from 1 to 3 halogen atoms, a C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl group, an acetyl group, -OR³, wherein R³ is a C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl group, an acetyl group, or β-D-glucoside, y : sai- en - -

a carboxyl group, an α hydroxyl carboxylic acid, or a nitro group, and wherein, optionally one of the two phenyl rings is replaced with a cyclohexyl ring, wherein said compound activates SIRTl enzymatic activity and protects the skin from, or reverses at least in part an effect of skin aging or environmental insult, inhibits tyrosinase activity and promotes skin whitening, treats condition or disease or alleviates said symptoms, or cosmetically treats said keratinous substrate.

17. The composition of claim method of claim 15 wherein at least one occurrence of R¹ or R² is -OH.

18. The composition of claim 17 wherein the compound is selected from the group consisting

(a) HT-I, the structural formula of which is:

Formula Il

(b) HT-2, the structural formula of which is:

Formula

© HT-3, the structural formula of which is:

Formula IV

y : sai- en - -

(d) HT-4, the structural formula of which is:

Formula V

and (e) 3,4,3'4'-tetrahydroxytolan (HT-5).

19. The composition of claims 16 wherein the compound is selected from the group consisting of: 3,5-dihydroxy-4-methoxytolan 3-0-β-glucoside; 3,3\5-trihydroxy-4'-methoxytolan 3-O-β-D-glucoside; 3,5-dihydroxy-4'-thiomethyltolan; 3,5-dihydroxy-4'-chlorotolan; 3, 5-dihyd- roxytolan; 3,5-dihydroxy-4'-ethyltolan; 3,5-dihydroxy-4'-fluorotolan; 3,5,3',4'-tetrahydroxytolan ; 3,5-dihydroxy-4'-methyltolan; 3,5-dihydroxy-4'-azidotolan; 3,5-dihydroxy-4'-nitrotolan; 3.5- dihydroxy-4-isopropyltolan; 3.5-dihydroxy-4'-methoxytolan; 3,5,3' -trihydroxy-4'-methoxytolan; 3,4'-dihydroxy-5-acetoxytolan; 3,5-dihydroxy-4'-acetoxytolan; (E)-l-3,5-dihydroxyphenyl)-2-(2- napthyl)ethyne ; 3-hydroxytolan ; 3,5-dimethoxymethoxy-4'-thiomethyltolan; 3,5-dihidyroxy-4- acetamidetolan; 3,4-dihydroxytolan; (E)-l-3,5-dihydroxyphenyl)-2-(cyclohexyl)ethyne ; 3,4- dimethoxytolan; 3,4,3'-trihydroxy-4'-acetoxytolan; 3,4,4'-trihydroxy-3'-acetoxytolan; 3,5,4'- trihydroxy-3'-acetoxytolan; 3,5,3'-trihydroxy-4'-acetoxytolan; 3,4,3'-trihydroxy-4'-acetamidetolan; 3,4,4'-trihydroxy-3'-acetamidetolan; 3,4,3' -trihydroxy-3'-acetamidetolan; 3,4,4'-trihydroxy-4'- acetamidetolan; 3,4'-dihydroxy-4-acetoxytolan; 4,4'-dihydroxy-3-acetoxytolan; 3,4-dihydroxy-4'- acetoxytolan; 3,4'-dihydroxy-4'-acetamidetolan; 4,4'-dihydroxy-3-acetamidetolan; 3,4-dihydroxy- 4'-acetamidetolan; 3,5,4'-trihydroxy-4-acetamidetolan; 3,4'-dihydroxy-4-acetamidetolan; 4,4'- dihydroxy-3-acetamidetolan; and 3,4-dihydroxy-4'-acetamidetolan.