INHIBITION OF MELANOGENESIS BY CHEMICALLY MODIFIED CURCUMINS
The present invention provides a method of treating a subject afflicted with hyperpigmentation or of lightening the skin tone of a subject comprising administering to the subject an amount of a compound having the structure: or a salt or ester thereof, so as to thereby treat the subject or lighten the skin tone of the subject.
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This application claims priority of U.S. Provisional Application No. 62/393,429, filed Sep. 12, 2016, the contents of which are hereby incorporated by reference.
Throughout this application, certain publications are referenced in parentheses. Full citations for these publications may be found immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to describe more fully the state of the art to which this invention relates.
BACKGROUND OF THE INVENTIONCurcumin is a naturally occurring compound of the curcuminoid family, isolated originally from the plant Curcuma longa. The rhizome of this plant, specifically, is used to create the spice known as turmeric, and is a major component of the daily diet in many Asian countries. Even before the modern characterization of curcumin's molecular structure and functionality, it has long been used in traditional eastern medicines.
With its natural medicinal history in mind, curcumin has been studied extensively over the past few decades in a wide variety of systems, and has been found to exhibit significant pleiotropic effects. These effects may be attributed to the chemistry of curcumin, consisting of two polyphenolic rings joined by a conjugated, flexible linker region with a R-diketone moiety at its center. The R-diketone moiety is capable of undergoing keto-enol tautomerization, though the enol form is more stable in both the solid phase and in solution (Gupta, S. C. et al. 2011) and is the dominant species at physiological pH (Gupta, S. C. et al. 2011; Zhang, Y. et al. 2012). The biological activities of curcumin are wide ranging: beyond having intrinsic antioxidant properties, it has been found to bind a wide spectrum of cellular constituents in vitro and in vivo, including inflammatory molecules, protein kinases, carrier proteins, cell survival proteins, structural proteins, the prion protein, antioxidant response elements, metal ions, and more (Gupta, S. C. et al. 2011). In addition, curcumin shows virtually no toxicity in humans (Gupta, S. C. et al. 2011; Ammon, H. P. T. et al. 1991).
While curcumin has been shown to have multiple beneficial effects, its poor oral absorption and lack of solubility in physiological fluid has all but precluded its use as a medicinal substance. Therefore, novel chemically-modified curcumins with enhanced pharmacokinetic and pharmacodynamic properties are needed.
Melanocytes are specialized cells which originate from the neural crest and have a key role in synthesis of melanin, a biopolymeric pigment inside organelles called melanosomes which are secreted and transferred to keratinocytes in the epidermis. Melanosomes progress through four stages of maturation [Cichorek, M. et al. 2013]. Melanocytes are present in basal layer of the epidermis and connect to neighboring keratinocytes via dendrites and one melanocyte contacts up to 30-40 keratinocytes to transfer melanin [Fitzpatrick, T. B. et al. 1963]. The process involves synthesis, packaging, transfer and uptake of melanin by keratinocytes [Ando, H. et al. 2012] which is ultimately responsible for skin coloration. Melanin provides UV photo-protection and scavenges free radicals; however, an excessive production of melanin in the skin can lead to hyperpigmentation, also called as hypermelanosis, and is associated with medical skin disorders such as melasma, post-inflammatory hyperpigmentation (PIH) and lentigosenilis (LS). It also causes significant psychosocial burden.
Tyrosinase (EC 1.14.18.1) is the rate-limiting enzyme in melanin synthesis pathway which catalyzes the conversion of L-tyrosine to L-Dopa and subsequent conversion to L-Dopaquinone. Tyrosinase is a membrane-bound glycoprotein consisting of two copper atoms in its active site [Chang, T. S. 2009]. Hence, compounds which can chelate copper can inhibit tyrosinase activity. The most popular commercial skin whitening agents, such as kojic acid, hydroquinone and arbutin (glycosylated hydroquinone) are tyrosinase inhibitors. However, all these exhibit serious side-effects; kojic acid causes pigmented contact dermatitis [Garcia-Gavin, J. et al. 2010], hydroquinone is carcinogenic [Kooyers, T. et al. 2006] and arbutin has potent genotoxicity [Cheng, S. L. et al. 2007]. These limitations have prompted an interest in identifying novel and natural plant-derived compounds without adverse effects, for treatment of hyperpigmentation both in cosmetic and clinical settings.
SUMMARY OF THE INVENTIONThe present invention provides a method of treating a subject afflicted with hyperpigmentation or of lightening the skin tone of a subject comprising administering to the subject an amount of a compound having the structure:
wherein bond α and β are each, independently, present or absent;
X is CR5 or N; Y is CR10 or N;
R1 is —SR12, —SO2R13, —COR14, —CSR14, —C(—NR12) R4, —C(—NH) R14, —SOR12, —POR12, —P(═O)(OR12)(OR13), or —P(OR12).(OR13),
-
- wherein R12 is C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
- R13 is H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
- R14 is C2-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, heteroaryl, heterocyclyl, methoxy, —OR15, or —NR16R17,
- wherein
- R15 is H, C3-10 alkyl, C2-10 alkenyl, C2-10 alkynyl;
- R16 and R17 are each, independently, H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 are each independently, H, halogen, —NO2, —CN, —NR28R29, —NHR28R29+, —SR28, —SO2R28, —OR28, —CO2R28, CF3, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
- wherein R28 and R29 are each, H, CF3, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, or —C(═O)-heterocyclyl; and
wherein each occurrence of alkyl, alkenyl, or alkynyl is branched or unbranched, unsubstituted or substituted,
or a salt or ester thereof, so as to thereby treat the subject or lighten the skin tone of the subject.
The present invention provides a method of treating a subject afflicted with hyperpigmentation comprising administering to the subject an amount of a compound having the structure:
wherein bond α and β are each, independently, present or absent;
X is CR5 or N; Y is CR10 or N;
R1 is —SR12, —SO2R13, —COR14, —CSR14, —C(—NR12)R14, —C(—NH)R14, —SOR12, —POR12, —P(═O)(OR12)(OR13), or —P(OR12).(OR13),
-
- wherein R12 is C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
- R13 is H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
- R14 is C2-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, heteroaryl, heterocyclyl, methoxy, —OR15, or —NR16R17,
- wherein
- R15 is H, C3-10 alkyl, C2-10 alkenyl, C2-10 alkynyl;
- R16 and R17 are each, independently, H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 are each independently, H, halogen, —NO2, —CN, —NR28R29, —NHR28R29+, —SR28, —SO2R28, —OR28, —CO2R28, CF3, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
- wherein R28 and R29 are each, H, CF3, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, or —C(═O)-heterocyclyl; and
wherein each occurrence of alkyl, alkenyl, or alkynyl is branched or unbranched, unsubstituted or substituted,
or a salt or ester thereof, so as to thereby treat the subject.
In some embodiments, wherein the compound reduces melanin synthesis in the subject.
In some embodiments, wherein the compound inhibits melanogenesis in the subject.
In some embodiments, wherein the compound inhibits tyrosinase activity in the subject.
In some embodiments, wherein the compound lightens the skin tone of the subject relative to the subject's natural skin tone.
The present invention further provides a method of lightening the skin tone of a subject comprising administering to the subject an amount of a compound having the structure:
wherein bond α and β are each, independently, present or absent;
X is CR5 or N; Y is CR10 or N; R1 is —SR12, —SO2R13, —COR14, —CSR14, —C(—NR12)R14, —C(—NH)R14, —SOR12, —POR12, —P(═O)(OR12)(OR13), or —P(OR12).(OR13),
-
- wherein R12 is C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
- R13 is H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
- R14 is C2-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, heteroaryl, heterocyclyl, methoxy, —OR15, or —NR16R17 wherein
- R15 is H, C3-10 alkyl, C2-10 alkenyl, C2-10 alkynyl;
- R16 and R17 are each, independently, H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 are each independently, H, halogen, —NO2, —CN, —NR28R29, —NHR28R29+, —SR28, —SO2R28, —OR28, —CO2R28, CF3, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl; - wherein R28 and R29 are each, H, CF3, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, or —C(═O)-heterocyclyl; and
wherein each occurrence of alkyl, alkenyl, or alkynyl is branched or unbranched, unsubstituted or substituted, or a salt or ester thereof, so as to thereby lighten the skin tone of the subject.
In some embodiments, wherein the compound reduces melanin synthesis in the subject.
In some embodiments, wherein the compound inhibits melanogenesis in the subject.
In some embodiments, wherein the compound inhibits tyrosinase activity in the subject.
In some embodiments, wherein the compound lightens the skin tone of the subject relative to the subject's natural skin tone.
The present invention yet further provides a method of inhibiting melanogenesis for reducing skin melanin levels in a subject comprising administering to the subject an amount of a compound having the structure:
wherein bond α and β are each, independently, present or absent;
X is CR5 or N; Y is CR10 or N;
R1 is —SR12, —SO2R13, —COR14, —CSR14, —C(═NR12)R14, —C(═NH) R14, —SOR12, —POR12, —P(═O)(OR12)(OR13), or —P(OR12).(OR13),
-
- wherein R17 is C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
- R13 is H, C1-10 alkyl, C1-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
- R14 is C2-10 alkyl, C1-10 alkenyl, C2-10 alkynyl, heteroaryl, heterocyclyl, methoxy, —OR15, or —NR16R17 wherein
- R15 is H, C3-10 alkyl, C2-10 alkenyl, C2-10 alkynyl;
- R16 and R17 are each, independently, H, C1-10 alkyl, C1-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 are each independently, H, halogen, —NO2, —CN, —NR28R29, —NHR28R29+, —SR28, —SO2R28, —OR28, —CO2R28, CF3, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl; - wherein R28 and R29 are each, H, CF3, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, or —C(═O)-heterocyclyl; and
wherein each occurrence of alkyl, alkenyl, or alkynyl is branched or unbranched, unsubstituted or substituted, or a salt or ester thereof, so as to thereby inhibit melanogenesis for reducing skin melanin levels in the subject.
In some embodiments, wherein in the compound a and 1 are each present.
In some embodiments, wherein in the compound a and 1 are each absent.
In some embodiments, wherein the compound has the structure:
R3, R4, R8 and R9 are each independently, H, halogen, —NO2, —CN, —NR28R29, —NHR28R29+, —SR28, —SO2R28, —OR28, —CO2R28, CF3, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
-
- wherein R28 and R29 are each, H, CF3, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, or —C(═O)-heterocyclyl; and
R14 is methoxy, —OR15, or —NR16R17, - wherein R15 is H, C3-10 alkyl, C2-10 alkenyl, C2-10 alkynyl; and R16 and R17 are each, independently, H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
wherein each occurrence of alkyl, alkenyl, or alkynyl is branched or unbranched, unsubstituted or substituted, or a salt or ester thereof.
- wherein R28 and R29 are each, H, CF3, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, or —C(═O)-heterocyclyl; and
In some embodiments, wherein the compound has the structure:
R3, R4, R8 and R9 are each independently, H, halogen, —NO2, —CN, —NR28R29, —NHR28R29+, —SR28, —SO2R28, —OR28, —CO2R28, CF3, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
-
- wherein R28 and R29 are each, H, CF3, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, or —C(═O)-heterocyclyl; and
R14 is methoxy, —OR15, or —NR16R17, - wherein R15 is H, C3-10 alkyl, C2-10 alkenyl, C2-10 alkynyl; and R16 and R17 are each, independently, H, C1-10 alkyl, C2-10 alkenyl, C2-10alkynyl, aryl, heteroaryl, or heterocyclyl;
wherein each occurrence of alkyl, alkenyl, or alkynyl is branched or unbranched, unsubstituted or substituted, or a salt or ester thereof.
- wherein R28 and R29 are each, H, CF3, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, or —C(═O)-heterocyclyl; and
In some embodiments, wherein R14 is methoxy or —NR16R17, wherein R16 and R17 are each, independently, aryl or heteroaryl.
In some embodiments, wherein R3, R4, R8 and R9 are each independently, H or —OR28, wherein R28 is H or C1-10 alkyl.
In some embodiments, wherein R3, R4, R8 and R9 are each —OR28, wherein each R28 is, independently, H or C1-10 alkyl.
In some embodiments, wherein R3, R4, R8, and R9 are each, independently, H, —OCH, or —OH; and R14 is methoxy or —N(CH3)2.
In some embodiments, wherein the compound has the structure:
or a salt thereof.
In some embodiments, wherein the compound has the structure:
or a salt thereof.
In some embodiments, wherein the compound contains an amide group. In some embodiments, wherein the compound contains an ester group.
In some embodiments, wherein the compound is administered topically to the subject.
In some embodiments, the subject is afflicted with a skin pigmentation disorder.
In some embodiments, a method of decreasing production of melanin in a subject in need thereof comprising administering to the subject an amount of the compound of the present invention or a salt or ester thereof, so as to thereby decrease production of the melanin in the subject.
In some embodiments, a method of inhibiting melanogensis in a subject in need thereof comprising administering to the subject an amount of the compound of the present invention or a salt or ester thereof, so as to thereby inhibit melanogensis in the subject.
In some embodiments, the melanin is extracellular melanin.
In some embodiments, the melanin is intracellular melanin.
In some embodiments, the method inhibits keratinocyte uptake of extracellular melanin.
In some embodiments, the method reduces synthesis of extracellular melanin and intracellular melanin.
The present invention also provides a method of treating a subject afflicted with hyperpigmentation or of lightening the skin tone of a subject comprising administering to the subject an amount of a compound having the structure:
wherein
bond α and β are each, independently, present or absent;
X is CR5 or N; Y is CR10 or N;
R1 is H, CF3, halogen, —NO2, —OCF3, —OR12, —NHCOR12, —CONR12R13, —CSNR12R13, —C(═NH) NR12R13 —SR12, —SO2R13, —COR14, —CSR14, —C(═NR12) R14, —C(═NR12) NR13R14, —SOR2, —SONR12R13, —SO2NR12R13, —P(O)R12, —PH(═O)OR12 —P(═O)(OR12)(OR13), or —P(OR12)(OR13),
-
- wherein R12 and R13 are each, independently, H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
- R14 is C2-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, heteroaryl, heterocyclyl, methoxy, —OR15, —NR16R17, or
-
-
- wherein R15 is H, C3-10 alkyl, C2-10 alkenyl, C2-10 alkynyl;
- R16 and R17 are each, independently, H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
- R18, R19, R21, and R22 are each independently H, halogen, —NO2, —CN, —NR23R24, —SR23, —SO2R23, —CO2R23, —OR25, CF3, —SOR23, —POR23, —C(═S) R23, —C(═NH) R23, —C(═N) R23, —P(═O)(OR23)(OR24), —P(OR23)(OR24), —C(═S)R23, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
- wherein R23, R24, and R25 are each, independently, H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
- R20 is halogen, —NO2, —CN, —NR26R27, CF3, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
- wherein R26 and R27 are each, independently, H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
R2, R3, R8, R5, R6, R7, R8, R9, R10, and R11 are each independently, H, halogen, —NO2, —CN, —NR2SR29, —NHR28R29, —SR28, —SO2R28, —OR28, —CO2R28, CF3, C1-10 alkyl, C2-10 alkenyl, C2-o alkynyl, aryl, heteroaryl, or heterocyclyl;
- wherein R26 and R27 are each, independently, H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
- wherein R28 and R29 are each, H, CF3, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, or —C(═O)-heterocyclyl; and
wherein when R1 is H, then R3, R4, R5, R8, R9, or R10, is halogen, —NO2, —CN, —NR28R29, —NHR28R29+, —SR28, —SO2R28, —CO2R28, CF3, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl; - wherein R28 and R29 are each, H, CF3, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, or —C(═O)-heterocyclyl; and
wherein each occurrence of alkyl, alkenyl, or alkynyl is branched or unbranched, unsubstituted or substituted;
or a salt or ester thereof, so as to thereby treat the subject or lighten the skin tone of the subject.
-
In some embodiments, the method wherein the in the compound, R1 is other than H.
In some embodiments, the method wherein the compound has the structure:
wherein R14 is C2-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, heteroaryl,
heterocyclyl, methoxy, —OR15, —NR16R17, or
-
- wherein R15 is H, C3-10 alkyl, C2-10 alkenyl, C2-10 alkynyl; R16 and R17 are each, independently, H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl; R18, R19, R21, and R22 are each independently H, halogen, —NO2, —CN, —NR23R24, —SR23, —SO2R23, —CO2R23, —OR25, CF3, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
- wherein R23, R24, and R25 are each, independently, H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
- R20 is halogen, —NO2, —CN, —NR26R27, CF3, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
- wherein R26 and R27 are each, independently, H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 are each independently, H, halogen, —NO2, —CN, —NR28R29, —SR28, —SO2R28, —OR28, —C02R28, CF3, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
wherein R28 and R29 are each, H, CF3, C1-10 alkyl, C2-10 alkenyl, or C2-10 alkynyl; and
wherein each occurrence of alkyl, alkenyl, or alkynyl is branched or unbranched, unsubstituted or substituted; and
or a salt thereof.
- wherein R26 and R27 are each, independently, H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
- wherein R15 is H, C3-10 alkyl, C2-10 alkenyl, C2-10 alkynyl; R16 and R17 are each, independently, H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl; R18, R19, R21, and R22 are each independently H, halogen, —NO2, —CN, —NR23R24, —SR23, —SO2R23, —CO2R23, —OR25, CF3, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
In some embodiments, the method wherein the compound has the structure:
wherein R14 is C2-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, heteroaryl,
heterocyclyl, —OR15, —NR16R17, or
-
- wherein R15 is H, C3-10 alkyl, C2-10 alkenyl, C2-10 alkynyl; R16 and R17 are each, independently, H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl; R18, R19, R21, and R22 are each independently H, halogen, —NO2, —CN, —NR23R24, —SR23, —SO2R23, —CO2R23, —OR25, CF3, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
- wherein R23, R24, and R25 are each, independently, H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
- R20 is halogen, —NO2, —CN, —NR26R27, CF3, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
- wherein R26 and R27 are each, independently, H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 are each independently, H, halogen, —NO2, —CN, —NR28R29, —SR28, —SO2R28, —OR28, —CO2R28, CF3, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
- wherein R26 and R27 are each, independently, H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
- wherein R28 and R29 are each, H, CF3, C1-10 alkyl, C2-10 alkenyl, or C2-10 alkynyl; and
wherein each occurrence of alkyl, alkenyl, or alkynyl is branched or unbranched, unsubstituted or substituted; and
or a salt thereof.
- wherein R15 is H, C3-10 alkyl, C2-10 alkenyl, C2-10 alkynyl; R16 and R17 are each, independently, H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl; R18, R19, R21, and R22 are each independently H, halogen, —NO2, —CN, —NR23R24, —SR23, —SO2R23, —CO2R23, —OR25, CF3, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
In some embodiments, the method wherein the compound has the structure:
wherein R14 is C2-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, heteroaryl,
heterocyclyl, —OR15, —NR16R17, or
-
- wherein R15 is H, C4-10 alkyl, C2-10 alkenyl, C2-10 alkynyl; R16 and R17 are each, independently, H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl; R18, R19, R21, and R22 are each independently H, halogen, —NO2, —CN, —NR23R24, —SR23, —SO2R23, —CO2R23, —OR25, CF3, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
- wherein R23, R24, and R25 are each, independently, H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
- R20 is halogen, —NO2, —CN, —NR26R27, CF3, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
- wherein R26 and R27 are each, independently, H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 are each independently, H, halogen, —NO2, —CN, —NR28R29, —SR28, —SO2R28, —OR28, —CO2R28, CF3, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
- wherein R26 and R27 are each, independently, H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
- wherein R28 and R29 are each, H, CF3, C1-10 alkyl, C2-10 alkenyl, or C2-10 alkynyl; and
wherein each occurrence of alkyl, alkenyl, or alkynyl is branched or unbranched, unsubstituted or substituted; and
or a salt thereof.
- wherein R15 is H, C4-10 alkyl, C2-10 alkenyl, C2-10 alkynyl; R16 and R17 are each, independently, H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl; R18, R19, R21, and R22 are each independently H, halogen, —NO2, —CN, —NR23R24, —SR23, —SO2R23, —CO2R23, —OR25, CF3, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
In some embodiments, the method wherein at least one of R2, R3, R4, R5, and R6 and at least one of R7, R8, R9, R10, and R11, are each, independently, —OR28.
In some embodiments, the method wherein
-
- R14 is methoxy, —OR15 or —NR16R17;
- R15 is H, C3-10 alkyl, C2-10 alkenyl, or C2-10 alkynyl;
- R16 and R17 are each, independently, H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
- or a salt thereof.
In some embodiments, the method wherein
-
- R14 is methoxy or —NR16R17;
- R16 and R17 are each, independently, H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
- or a salt thereof.
In some embodiments, the method wherein
-
- R14 is —OR15,
- R15 is H, C3-10 alkyl, C2-10 alkenyl, or C2-10 alkynyl;
or a salt thereof.
In some embodiments, the method wherein
-
- R14 is —NR16R17,
- wherein R16 and R17 are each, independently, H or aryl; R2, R3, R4, R5, R6, R7, R8, R9, R10, and Ru are each independently, H, —NR28R29, or —OR28,
- wherein R28 and R29 are each, H or C1-10 alkyl;
or a salt thereof.
- wherein R28 and R29 are each, H or C1-10 alkyl;
- wherein R16 and R17 are each, independently, H or aryl; R2, R3, R4, R5, R6, R7, R8, R9, R10, and Ru are each independently, H, —NR28R29, or —OR28,
- R14 is —NR16R17,
In some embodiments, the method wherein
-
- R14 is —NH-phenyl;
- R2, R5, R6, R7, R10, and R11 are each H;
- R3, R4, R8, and R9 are each, independently, H, —OH, or —OCH3;
or a salt thereof.
In some embodiments of the method, a compound having the structure:
or a salt thereof.
In some embodiments of the method, a compound having the structure:
or a salt thereof.
In some embodiments of the method, a compound having the structure:
or a salt thereof.
In some embodiments, melanin synthesis in the subject is reduced by at least 10%.
In some embodiments, melanin synthesis in the subject is reduced by at least 25%.
In some embodiments, melanin synthesis in the subject is reduced by at least 50%.
In some embodiments, melanin synthesis in the subject is reduced by 1 to 50%.
In some embodiments, melanin synthesis in the subject is reduced by 10 to 50%.
In some embodiments, melanin synthesis in the subject is reduced by 25 to 50%.
Melanogenesis herein relates to the production of melanin in melanocytes and/or transfer of melanin from melanocytes to keratinocytes.
The CMC's disclosed herein have improved solubility and greater metal binding capability and enhanced therapeutic anti-inflammatory effects and efficacy in vivo relative to curcumin or tetrahydrocurcumin.
In some embodiments, the compound is solubilized in a non-toxic organic solubilizing agent. A non-limiting example of a non-toxic organic solubilizing agent is N-methylglucamine, which is also known as “meglumine”. In some embodiments, the compound is solubilized in dimethylsulfoxide. In some embodiments, the compound is solubilized in dimethylsulfoxide (less than 0.5% concentration).
This invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and of the above compounds.
Variations on the following general synthetic methods (Pabon, H. 1964) will be readily apparent to those skilled in the art and are used to prepare the compounds of the method of the present invention.
The synthesis of the curcumin analogues of the present invention can be carried out according to general Scheme 1. The R groups designate any number of generic substituents.
The starting material is provided by 2,4-pentanedione, which is substituted at the 3-carbon (see compound a). The desired substituted 2,4-pentanedione may be purchased from commercial sources or it may be synthesized using conventional functional group transformations well-known in the chemical arts, for example, those set forth in Organic Synthesis, Michael B. Smith, (McGraw-Hill) Second ed. (2001) and March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Michael B. Smith and Jerry March, (Wiley) Sixth ed. (2007), and specifically by Bingham and Tyman (45) and in the case of 3-aryl-aminocarbonyl compounds by Dieckman, Hoppe and Stein (46), the contents of which are hereby incorporated by reference. 2,4-pentanedione a is reacted with boron trioxide to form boron enolate complex b.
Boron enolate complex b is a complex formed by coordination of the enolate of compound a with boron. It is understood by those having ordinary skill in the art that the number of compound a enolates that may coordinate to boron as well as the coordination mode, i.e. monodentate versus bidentate, are variable so long as reaction, such as Knoevenagel condensation, at the C-3 carbon of the 2,4-pentanedione is suppressed.
Boron enolate complex b is then exposed to a benzaldehyde compound in the presence of a base catalyst and a water scavenger to form curcumin analogue c via aldol condensation. The ordinarily skilled artisan will appreciate that the benzaldehyde may possess various substituents on the phenyl ring so long as reactivity at the aldehyde position is not hindered. Substituted benzaldehyde compounds may be purchased from commercial sources or readily synthesized using aryl substitution chemistry that is well-known in the art. Suitable base catalysts for the aldol step include, but are not limited to, secondary amines, such as n-butylamine and n-butylamine acetate, and tertiary amines. Suitable water scavengers include, but are not limited to, alkyl borates, such as trimethyl borate, alkyl phosphates, and mixtures thereof. Other suitable reaction parameters have also been described by Krackov and Bellis in U.S. Pat. No. 5,679,864, the content of which is hereby incorporated by reference.
The compounds of the present invention include all hydrates, solvates, and complexes of the compounds used by this invention. If a chiral center or another form of an isomeric center is present in a compound of the present invention, all forms of such isomer or isomers, including enantiomers and diastereomers, are intended to be covered herein. Compounds containing a chiral center may be used as a racemic mixture, an enantiomerically enriched mixture, or the racemic mixture may be separated using well-known techniques and an individual enantiomer may be used alone. The compounds described in the present invention are in racemic form or as individual enantiomers. The enantiomers can be separated using known techniques, such as those described in Pure and Applied Chemistry 69, 1469-1474, (1997) IUPAC. In cases in which compounds have unsaturated carbon-carbon double bonds, both the cis (Z) and trans (E) isomers are within the scope of this invention.
The compounds of the subject invention may have spontaneous tautomeric forms. In cases wherein compounds may exist in tautomeric forms, such as keto-enol tautomers, each tautomeric form is contemplated as being included within this invention whether existing in equilibrium or predominantly in one form.
In the compound structures depicted herein, hydrogen atoms are not shown for carbon atoms having less than four bonds to non-hydrogen atoms. However, it is understood that enough hydrogen atoms exist on said carbon atoms to satisfy the octet rule.
This invention also provides isotopic variants of the compounds disclosed herein, including wherein the isotopic atom is 2H and/or wherein the isotopic atom 13C. Accordingly, in the compounds provided herein hydrogen can be enriched in the deuterium isotope. It is to be understood that the invention encompasses all such isotopic forms.
It is understood that where a numerical range is recited herein, the present invention contemplates each integer between, and including, the upper and lower limits, unless otherwise stated.
Except where otherwise specified, if the structure of a compound of this invention includes an asymmetric carbon atom, it is understood that the compound occurs as a racemate, racemic mixture, and isolated single enantiomer. All such isomeric forms of these compounds are expressly included in this invention.
Except where otherwise specified, each stereogenic carbon may be of the R or S configuration. It is to be understood accordingly that the isomers arising from such asymmetry (e.g., all enantiomers and diastereomers) are included within the scope of this invention, unless indicated otherwise. Such isomers can be obtained in substantially pure form by classical separation techniques and by stereochemically controlled synthesis, such as those described in “Enantiomers, Racemates and Resolutions” by J. Jacques, A. Collet and S. Wilen, Pub. John Wiley & Sons, N Y, 1981. For example, the resolution may be carried out by preparative chromatography on a chiral column.
The subject invention is also intended to include all isotopes of atoms occurring on the compounds disclosed herein. Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium. Isotopes of carbon include C-13 and C-14.
It will be noted that any notation of a carbon in structures throughout this application, when used without further notation, are intended to represent all isotopes of carbon, such as 12C, 13C, or 14C. Furthermore, any compounds containing 13C or 14C may specifically have the structure of any of the compounds disclosed herein.
It will also be noted that any notation of a hydrogen in structures throughout this application, when used without further notation, are intended to represent all isotopes of hydrogen, such as 1H, 2H, or 3H. Furthermore, any compounds containing 2H or 3H may specifically have the structure of any of the compounds disclosed herein.
Isotopically-labeled compounds can generally be prepared by conventional techniques known to those skilled in the art using appropriate isotopically-labeled reagents in place of the non-labeled reagents employed.
In the compounds used in the method of the present invention, the substituents may be substituted or unsubstituted, unless specifically defined otherwise.
In the compounds used in the method of the present invention, alkyl, heteroalkyl, monocycle, bicycle, aryl, heteroaryl and heterocycle groups can be further substituted by replacing one or more hydrogen atoms with alternative non-hydrogen groups. These include, but are not limited to, halo, hydroxy, mercapto, amino, carboxy, cyano, carbamoyl and aminocarbonyl and aminothiocarbonyl.
It is understood that substituents and substitution patterns on the compounds used in the method of the present invention can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure results.
In choosing the compounds used in the method of the present invention, one of ordinary skill in the art will recognize that the various substituents, i.e. R1, R2, etc. are to be chosen in conformity with well-known principles of chemical structure connectivity.
As used herein, “alkyl” includes both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms and may be unsubstituted or substituted. Thus, C1-Cn as in “C1-Cn alkyl” is defined to include groups having 1, 2, . . . , n−1 or n carbons in a linear or branched arrangement. For example, C1-C6, as in “C1-C6 alkyl” is defined to include groups having 1, 2, 3, 4, 5, or 6 carbons in a linear or branched arrangement, and specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, pentyl, hexyl, and octyl.
As used herein, “alkenyl” refers to a non-aromatic hydrocarbon radical, straight or branched, containing at least 1 carbon to carbon double bond, and up to the maximum possible number of non-aromatic carbon-carbon double bonds may be present, and may be unsubstituted or substituted. For example, “C2-C6 alkenyl” means an alkenyl radical having 2, 3, 4, 5, or 6 carbon atoms, and up to 1, 2, 3, 4, or 5 carbon-carbon double bonds respectively. Alkenyl groups include ethenyl, propenyl, butenyl and cyclohexenyl.
The term “alkynyl” refers to a hydrocarbon radical straight or branched, containing at least 1 carbon to carbon triple bond, and up to the maximum possible number of non-aromatic carbon-carbon triple bonds may be present, and may be unsubstituted or substituted. Thus, “C2-C6 alkynyl” means an alkynyl radical having 2 or 3 carbon atoms and 1 carbon-carbon triple bond, or having 4 or 5 carbon atoms and up to 2 carbon-carbon triple bonds, or having 6 carbon atoms and up to 3 carbon-carbon triple bonds. Alkynyl groups include ethynyl, propynyl and butynyl.
“Alkylene”, “alkenylene” and “alkynylene” shall mean, respectively, a divalent alkane, alkene and alkyne radical, respectively. It is understood that an alkylene, alkenylene, and alkynylene may be straight or branched. An alkylene, alkenylene, and alkynylene may be unsubstituted or substituted.
As used herein, “heteroalkyl” includes both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms and at least 1 heteroatom within the chain or branch.
As herein, “cycloalkyl” shall mean cyclic rings of alkanes of three to eight total carbon atoms, or any number within this range (i.e., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl).
As used herein, “monocycle” includes any stable polyatomic carbon ring of up to 10 atoms and may be unsubstituted or substituted. Examples of such non-aromatic monocycle elements include but are not limited to: cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. Examples of such aromatic monocycle elements include but are not limited to: phenyl.
As used herein, “bicycle” includes any stable polyatomic carbon ring of up to 10 atoms that is fused to a polyatomic carbon ring of up to 10 atoms with each ring being independently unsubstituted or substituted. Examples of such non-aromatic bicycle elements include but are not limited to: decahydronaphthalene. Examples of such aromatic bicycle elements include but are not limited to: naphthalene.
As used herein, “aryl” is intended to mean any stable monocyclic, bicyclic or polycyclic carbon ring of up to 10 atoms in each ring, wherein at least one ring is aromatic, and may be unsubstituted or substituted. Examples of such aryl elements include phenyl, p-toluenyl (4-methylphenyl), naphthyl, tetrahydro-naphthyl, indanyl, biphenyl, phenanthryl, anthryl or acenaphthyl. In cases where the aryl substituent is bicyclic and one ring is non-aromatic, it is understood that attachment is via the aromatic ring.
As used herein, the term “polycyclic” refers to unsaturated or partially unsaturated multiple fused ring structures, which may be unsubstituted or substituted.
The term “arylalkyl” refers to alkyl groups as described above wherein one or more bonds to hydrogen contained therein are replaced by a bond to an aryl group as described above. It is understood that an “arylalkyl” group is connected to a core molecule through a bond from the alkyl group and that the aryl group acts as a substituent on the alkyl group. Examples of arylalkyl moieties include, but are not limited to, benzyl (phenylmethyl), p-trifluoromethylbenzyl (4-trifluoromethylphenylmethyl), 1-phenylethyl, 2-phenylethyl, 3-phenylpropyl, 2-phenylpropyl and the like.
The term “heteroaryl”, as used herein, represents a stable monocyclic, bicyclic or polycyclic ring of up to 10 atoms in each ring, wherein at least one ring is aromatic and contains from 1 to 4 heteroatoms selected from the group consisting of 0, N and S. Bicyclic aromatic heteroaryl groups include phenyl, pyridine, pyrimidine or pyridizine rings that are (a) fused to a 6-membered aromatic (unsaturated) heterocyclic ring having one nitrogen atom; (b) fused to a 5- or 6-membered aromatic (unsaturated) heterocyclic ring having two nitrogen atoms; (c) fused to a 5-membered aromatic (unsaturated) heterocyclic ring having one nitrogen atom together with either one oxygen or one sulfur atom; or (d) fused to a 5-membered aromatic (unsaturated) heterocyclic ring having one heteroatom selected from O, N or S. Heteroaryl groups within the scope of this definition include but are not limited to: benzoimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl, oxazolyl, oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyrimidyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, azetidinyl, aziridinyl, 1,4-dioxanyl, hexahydroazepinyl, dihydrobenzoimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl, dihydroisooxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, methylenedioxybenzoyl, tetrahydrofuranyl, tetrahydrothienyl, acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrrazolyl, indolyl, benzotriazolyl, benzothiazolyl, benzoxazolyl, isoxazolyl, isothiazolyl, furanyl, thienyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, tetra-hydroquinoline. In cases where the heteroaryl substituent is bicyclic and one ring is non-aromatic or contains no heteroatoms, it is understood that attachment is via the aromatic ring or via the heteroatom containing ring, respectively. If the heteroaryl contains nitrogen atoms, it is understood that the corresponding N-oxides thereof are also encompassed by this definition.
The term “alkylheteroaryl” refers to alkyl groups as described above wherein one or more bonds to hydrogen contained therein are replaced by a bond to an heteroaryl group as described above. It is understood that an “alkylheteroaryl” group is connected to a core molecule through a bond from the alkyl group and that the heteroaryl group acts as a substituent on the alkyl group. Examples of alkylheteroaryl moieties include, but are not limited to, —CH2—(C5H4N), —CH2—CH2—(C5H4N) and the like.
The term “heterocycle” or “heterocyclyl” refers to a mono- or poly-cyclic ring system which can be saturated or contains one or more degrees of unsaturation and contains one or more heteroatoms. Preferred heteroatoms include N, O, and/or S, including N-oxides, sulfur oxides, and dioxides. Preferably the ring is three to ten-membered and is either saturated or has one or more degrees of unsaturation. The heterocycle may be unsubstituted or substituted, with multiple degrees of substitution being allowed. Such rings may be optionally fused to one or more of another “heterocyclic” ring(s), heteroaryl ring(s), aryl ring(s), or cycloalkyl ring(s). Examples of heterocycles include, but are not limited to, tetrahydrofuran, pyran, 1,4-dioxane, 1,3-dioxane, piperidine, piperazine, pyrrolidine, morpholine, thiomorpholine, tetrahydrothiopyran, tetrahydrothiophene, 1,3-oxathiolane, and the like.
The alkyl, alkenyl, alkynyl, aryl, heteroaryl and heterocyclyl substituents may be substituted or unsubstituted, unless specifically defined otherwise. In the compounds of the present invention, alkyl, alkenyl, alkynyl, aryl, heterocyclyl and heteroaryl groups can be further substituted by replacing one or more hydrogen atoms with alternative non-hydrogen groups. These include, but are not limited to, halo, hydroxy, mercapto, amino, carboxy, cyano and carbamoyl.
As used herein, the term “halogen” refers to F, Cl, Br, and I.
The terms “substitution”, “substituted” and “substituent” refer to a functional group as described above in which one or more bonds to a hydrogen atom contained therein are replaced by a bond to non-hydrogen or non-carbon atoms, provided that normal valencies are maintained and that the substitution results in a stable compound. Substituted groups also include groups in which one or more bonds to a carbon(s) or hydrogen(s) atom are replaced by one or more bonds, including double or triple bonds, to a heteroatom. Examples of substituent groups include the functional groups described above, and halogens (i.e., F, Cl, Br, and I); alkyl groups, such as methyl, ethyl, n-propyl, isopropryl, n-butyl, tert-butyl, and trifluoromethyl; hydroxyl; alkoxy groups, such as methoxy, ethoxy, n-propoxy, and isopropoxy; aryloxy groups, such as phenoxy; arylalkyloxy, such as benzyloxy (phenylmethoxy) and p-trifluoromethylbenzyloxy (4-trifluoromethylphenylmethoxy); heteroaryloxy groups; sulfonyl groups, such as trifluoromethanesulfonyl, methanesulfonyl, and p-toluenesulfonyl; nitro, nitrosyl; mercapto; sulfanyl groups, such as methylsulfanyl, ethylsulfanyl and propylsulfanyl; cyano; amino groups, such as amino, methylamino, dimethylamino, ethylamino, and diethylamino; and carboxyl. Where multiple substituent moieties are disclosed or claimed, the substituted compound can be independently substituted by one or more of the disclosed or claimed substituent moieties, singly or pluraly. By independently substituted, it is meant that the (two or more) substituents can be the same or different.
As used herein, the term “electron-withdrawing group” refers to a substituent or functional group that has the property of increasing electron density around itself relative to groups in its proximity. Electron withdrawing property is a combination of induction and resonance. Electron withdrawal by induction refers to electron cloud displacement towards the more electronegative of two atoms in a a-bond. Therefore, the electron cloud between two atoms of differing electronegativity is not uniform and a permanent state of bond polarization occurs such that the more electronegative atom has a slight negative charge and the other atom has a slight positive charge. Electron withdrawal by resonance refers to the ability of substituents or functional groups to withdraw electron density on the basis of relevant resonance structures arising from p-orbital overlap. Suitable electron-withdrawing groups include, but are not limited to, —CN, —CF3, halogen, —NO2, —OCF3, —OR12, —NHCOR12, —SR12, —SO2R13, —COR14, —CSR14, —CNR14, —C(═NR12) R14, —C(═NH) R14, —SOR12, —POR12, —P(═O)(OR2)(OR13), or —P(OR12)(OR13),
-
- wherein R12 and R13 are each, independently, H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
- R14 is C2-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, heteroaryl,
-
- heterocyclyl, methoxy, —OR15, —NR16R17, or
- wherein R15 is H, C3-10 alkyl, C2-10 alkenyl, C2-10 alkynyl;
- R16 and R17 are each, independently, H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
- R18, R19, R21, and R22 are each independently H, halogen, —NO2, —CN, —NR23R24, —SR23, —SO2R23, —CO2R23, —OR25, CF3, —SOR23, —POR23, —C(═S) R23, —C(═NH) R23, C(═NR24) R23, —C(═N)R23, —P(═O)(OR23)(OR24), —P(OR23)(OR24), —C(═S)R23, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
- wherein R23, R24, and R25 are each, independently, H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
- R20 is halogen, —NO2, —CN, —NR26R27, CF3, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
- wherein R26 and R27 are each, independently, H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl.
- heterocyclyl, methoxy, —OR15, —NR16R17, or
In the subject invention, the biological activity of curcumin analogues is attributed in part to their ability to access and bind copper.
Without wishing to be bound by theory, it is believed that metal binding affinity arises from increased stability of the curcumin enolate formed by removal of hydrogen from the C-4 carbon, which then proceeds to form a complex with the metal. The stability of a carbanion, including an enolate, is directly related to the acidity of the ionizable hydrogen, such as an enolic hydrogen. In general, the stability of an enolate increases with increasing acidity of the enolic hydrogen. Herein, the enolic hydrogen refers to the hydrogen atom connected to the C-4 carbon of the curcumin skeleton.
The acidity of the enolic hydrogen of curcumin and its analogues can be enhanced by incorporation of an electron-withdrawing group at the C-4 carbon. Substituents which delocalize negative charge will enhance acidity and stability of the resulting carbanion, such as an enolate. Again, without wishing to be bound by theory, it is believed that the electron-withdrawing group allows the negative charge of the enolate to be delocalized into the electron-withdrawing group, thereby stabilizing the enolate, enhancing its stability, and increasing its metal binding affinity.
The choice of electron-withdrawing groups on the C-4 carbon and the choice of electron-donating groups on the aryl rings may be chosen using techniques well known by the ordinarily skilled artisan. In general, the electron donating ability of common substituents suitable for use on the aryl rings can be estimated by their Hammett σ values. The Hammett σpara value is a relative measurement comparing the electronic influence of the substituent in the para position of a phenyl ring to the electronic influence of a hydrogen substituted at the para position. Typically for aromatic substituents in general, a negative Hammett ara value is indicative of a group or substituent having an electron-donating influence on a pi electron system (i.e., an electron-donating group) and a positive Hammett σpara value is indicative of a group or substituent having an electron-withdrawing influence on a pi electron system (i.e., an electron-withdrawing group). Similarly, Hammett σmeta value is a relative measurement comparing the electronic influence of the substituent in the meta position of a phenyl ring to the electronic influence of a hydrogen substituted at the meta position. A list of Hammett σpara and σmeta values for common substituents can be found in Lowry and Richardson, “Mechanism and Theory in Organic Chemistry”, 3rd ed, p. 144. The effect of some substituents, including some electron-withdrawing groups, on C—H acidity can also be found on page 518 in Lowry and Richardson, “Mechanism and Theory in Organic Chemistry”, 3rd ed, the content of which is hereby incorporated by reference.
It is understood that substituents and substitution patterns on the compounds of the instant invention can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art, as well as those methods set forth below, from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure results.
In choosing the compounds of the present invention, one of ordinary skill in the art will recognize that the various substituents, i.e. R1, R2, etc. are to be chosen in conformity with well-known principles of chemical structure connectivity.
The various R groups attached to the aromatic rings of the compounds disclosed herein may be added to the rings by standard procedures, for example those set forth in Advanced Organic Chemistry: Part B: Reaction and Synthesis, Francis Carey and Richard Sundberg, (Springer) 5th ed. Edition. (2007), the content of which is hereby incorporated by reference.
The compounds used in the method of the present invention may be prepared by techniques well known in organic synthesis and familiar to a practitioner ordinarily skilled in the art. However, these may not be the only means by which to synthesize or obtain the desired compounds.
The compounds used in the method of the present invention may be prepared by techniques described in Vogel's Textbook of Practical Organic Chemistry, A. I. Vogel, A. R. Tatchell, B. S. Furnis, A. J. Hannaford, P. W. G. Smith, (Prentice Hall)5th Edition (1996), March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Michael B. Smith, Jerry March, (Wiley-Interscience) 5th Edition (2007), and references therein, which are incorporated by reference herein. However, these may not be the only means by which to synthesize or obtain the desired compounds.
Another aspect of the invention comprises a compound used in the method of the present invention as a pharmaceutically acceptable salt.
Another aspect of the invention comprises a compound used in the method of the present invention as a pharmaceutical composition.
In some embodiments, a pharmaceutical composition comprising the compound of the present invention and a pharmaceutically acceptable carrier.
As used herein, the term “pharmaceutically active agent” means any substance or compound suitable for administration to a subject and furnishes biological activity or other direct effect in the treatment, cure, mitigation, diagnosis, or prevention of disease, or affects the structure or any function of the subject. Pharmaceutically active agents include, but are not limited to, substances and compounds described in the Physicians' Desk Reference (PDR Network, LLC; 64th edition; Nov. 15, 2009) and “Approved Drug Products with Therapeutic Equivalence Evaluations” (U.S. Department Of Health And Human Services, 30th edition, 2010), which are hereby incorporated by reference. Pharmaceutically active agents which have pendant carboxylic acid groups may be modified in accordance with the present invention using standard esterification reactions and methods readily available and known to those having ordinary skill in the art of chemical synthesis. Where a pharmaceutically active agent does not possess a carboxylic acid group, the ordinarily skilled artisan will be able to design and incorporate a carboxylic acid group into the pharmaceutically active agent where esterification may subsequently be carried out so long as the modification does not interfere with the pharmaceutically active agent's biological activity or effect.
The compounds used in the method of the present invention may be in a salt form. As used herein, a “salt” is a salt of the instant compounds which has been modified by making acid or base salts of the compounds. In the case of compounds used to treat an infection or disease caused by a pathogen, the salt is pharmaceutically acceptable. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as phenols. The salts can be made using an organic or inorganic acid. Such acid salts are chlorides, bromides, sulfates, nitrates, phosphates, sulfonates, formates, tartrates, maleates, malates, citrates, benzoates, salicylates, ascorbates, and the like. Phenolate salts are the alkaline earth metal salts, sodium, potassium or lithium. The term “pharmaceutically acceptable salt” in this respect, refers to the relatively non-toxic, inorganic and organic acid or base addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or by separately reacting a purified compound of the invention in its free base or free acid form with a suitable organic or inorganic acid or base, and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like. (See, e.g., Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19).
The compounds of the present invention may also form salts with basic amino acids such a lysine, arginine, etc. and with basic sugars such as N-methylglucamine, 2-amino-2-deoxyglucose, etc. and any other physiologically non-toxic basic substance.
The compounds used in the method of the present invention may be administered in various forms, including those detailed herein.
The treatment with the compound may be a component of a combination therapy or an adjunct therapy, i.e. the subject or patient in need of the drug is treated or given another drug for the disease in conjunction with one or more of the instant compounds. This combination therapy can be sequential therapy where the patient is treated first with one drug and then the other or the two drugs are given simultaneously. These can be administered independently by the same route or by two or more different routes of administration depending on the dosage forms employed.
As used herein, a “pharmaceutically acceptable carrier” is a pharmaceutically acceptable solvent, suspending agent or vehicle, for delivering the instant compounds to the animal or human. The carrier may be liquid or solid and is selected with the planned manner of administration in mind. Liposomes are also a pharmaceutically acceptable carrier as are slow-release vehicles. Liposomes are also a pharmaceutically acceptable carrier as are slow-release vehicles, especially those intended for use in delivery of the active compounds to the epidermis.
The dosage of the compounds administered in treatment will vary depending upon factors such as the pharmacodynamic characteristics of a specific chemotherapeutic agent and its mode and route of administration; the age, sex, metabolic rate, absorptive efficiency, health and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment being administered; the frequency of treatment with; and the desired therapeutic effect.
A dosage unit of the compounds used in the method of the present invention may comprise a single compound or mixtures thereof with additional agents. The compounds can be introduced directly, e.g. by topical application, or other methods, onto a site of the skin condition, all using dosage forms well known to those of ordinary skill in the pharmaceutical arts.
The compounds used in the method of the present invention can be administered in admixture with suitable pharmaceutical diluents, extenders, excipients, or in carriers such as the novel programmable sustained-release multi-compartmental nanospheres (collectively referred to herein as a pharmaceutically acceptable carrier) suitably selected with respect to the intended form of administration and as consistent with conventional pharmaceutical practices. The unit will be in a form suitable for topical administration. The compounds can be administered alone or mixed with a pharmaceutically acceptable carrier. This carrier can be a solid or liquid, and the type of carrier is generally chosen based on the type of administration being used. The active agent can be co-administered in the form of a tablet or capsule, liposome, as an agglomerated powder or in a liquid form. Examples of suitable solid carriers include lactose, sucrose, gelatin and agar. Capsule or tablets can be easily formulated and can be made easy to swallow or chew; other solid forms include granules, and bulk powders. Tablets may contain suitable binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents. Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents. Oral dosage forms optionally contain flavorants and coloring agents. Parenteral and intravenous forms may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.
In some embodiments, the topical application restricts delivery solely to the epidermis and/or avoids, or at least fails to facilitate, systemic delivery.
Techniques and compositions for making dosage forms useful in the present invention are described in the following references: 7 Modern Pharmaceutics, Chapters 9 and 10 (Banker & Rhodes, Editors, 1979); Pharmaceutical Dosage Forms: Tablets (Lieberman et al., 1981); Ansel, Introduction to Pharmaceutical Dosage Forms 2nd Edition (1976); Remington's Pharmaceutical Sciences, 17th ed. (Mack Publishing Company, Easton, Pa., 1985); Advances in Pharmaceutical Sciences (David Ganderton, Trevor Jones, Eds., 1992); Advances in Pharmaceutical Sciences Vol. 7. (David Ganderton, Trevor Jones, James McGinity, Eds., 1995); Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms (Drugs and the Pharmaceutical Sciences, Series 36 (James McGinity, Ed., 1989); Pharmaceutical Particulate Carriers: Therapeutic Applications: Drugs and the Pharmaceutical Sciences, Vol 61 (Alain Rolland, Ed., 1993); Drug Delivery to the Gastrointestinal Tract (Ellis Horwood Books in the Biological Sciences. Series in Pharmaceutical Technology; J. G. Hardy, S. S. Davis, Clive G. Wilson, Eds.); Modem Pharmaceutics Drugs and the Pharmaceutical Sciences, Vol 40 (Gilbert S. Banker, Christopher T. Rhodes, Eds.). All of the aforementioned publications are incorporated by reference herein.
Tablets may contain suitable binders, lubricants, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents. For instance, for oral administration in the dosage unit form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, gelatin, agar, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.
The compounds used in the method of the present invention may also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids such as lecithin, sphingomyelin, proteolipids, protein-encapsulated vesicles or from cholesterol, stearylamine, or phosphatidylcholines. The compounds may be administered as components of tissue-targeted emulsions.
The compounds used in the method of the present invention may also be coupled to soluble polymers as targetable drug carriers or as a prodrug. Such polymers include polyvinylpyrrolidone, pyran copolymer, polyhydroxylpropylmethacrylamide-phenol, polyhydroxyethylasparta-midephenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues. Furthermore, the compounds may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, and crosslinked or amphipathic block copolymers of hydrogels.
The compounds used in the method of the present invention may also be administered via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will generally be continuous rather than intermittent throughout the dosage regimen.
The compounds and compositions of the present invention can be introduced directly, e.g. by topical administration, to the afflicted area, all using dosage forms well known to those of ordinary skill in the pharmaceutical arts.
Specific examples of pharmaceutically acceptable carriers and excipients that may be used to formulate oral dosage forms of the present invention are described in U.S. Pat. No. 3,903,297 to Robert, issued Sep. 2, 1975. Techniques and compositions for making dosage forms useful in the present invention are described-in the following references: 7 Modern Pharmaceutics, Chapters 9 and 10 (Banker & Rhodes, Editors, 1979); Pharmaceutical Dosage Forms: Tablets (Lieberman et al., 1981); Ansel, Introduction to Pharmaceutical Dosage Forms 2nd Edition (1976); Remington's Pharmaceutical Sciences, 17th ed. (Mack Publishing Company, Easton, Pa., 1985); Advances in Pharmaceutical Sciences (David Ganderton, Trevor Jones, Eds., 1992); Advances in Pharmaceutical Sciences Vol 7. (David Ganderton, Trevor Jones, James McGinity, Eds., 1995); Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms (Drugs and the Pharmaceutical Sciences, Series 36 (James McGinity, Ed., 1989); Pharmaceutical Particulate Carriers: Therapeutic Applications: Drugs and the Pharmaceutical Sciences, Vol 61 (Alain Rolland, Ed., 1993); Drug Delivery to the Gastrointestinal Tract (Ellis Horwood Books in the Biological Sciences. Series in Pharmaceutical Technology; J. G. Hardy, S. S. Davis, Clive G. Wilson, Eds.); Modem Pharmaceutics Drugs and the Pharmaceutical Sciences, Vol 40 (Gilbert S. Banker, Christopher T. Rhodes, Eds.). All of the aforementioned publications are incorporated by reference herein.
The term “prodrug” as used herein refers to any compound that when administered to a biological system generates the compound of the invention, as a result of spontaneous chemical reaction(s), enzyme catalyzed chemical reaction(s), photolysis, and/or metabolic chemical reaction(s). A prodrug is thus a covalently modified analog or latent form of a compound of the invention.
It is understood that where a parameter range is provided, all integers within that range, and tenths thereof, are also provided by the invention. For example, “0.2-5 mg/kg/day” is a disclosure of 0.2 mg/kg/day, 0.3 mg/kg/day, 0.4 mg/kg/day, 0.5 mg/kg/day, 0.6 mg/kg/day etc. up to 5.0 mg/kg/day.
As used herein, “treating” means preventing, slowing, halting, or reversing the progression of a disease or condition. Treating may also mean improving one or more symptoms of a disease or condition.
As used herein, “about” in the context of a numerical value or range means±10% of the numerical value or range recited or claimed, unless the context requires a more limited range.
In choosing the compounds of the present invention, one of ordinary skill in the art will recognize that the various substituents, i.e. R1, R2, etc. are to be chosen in conformity with well-known principles of chemical structure connectivity.
The various R groups attached to the aromatic rings of the compounds disclosed herein may be added to the rings by standard procedures, for example those set forth in Advanced Organic Chemistry: Part B: Reaction and Synthesis, Francis Carey and Richard Sundberg, (Springer) 5th ed. Edition. (2007), the content of which is hereby incorporated by reference.
Chemically-modified curcumins may be relatively insoluble in water. Such compounds may be solubilized in a safe organic solubilizing agent, such as meglumine (ie., N-methyl glucamine which is a deoxy(methylamino) glucitol, a derivative of glucose) or dimethylsulfoxide (below 0.5%). Dimethylsulfoxide (below 0.5%) is generally recognized as safe for veterinary applications and have not been associated with toxicity to humans in topical applications.
The compounds of the present invention and derivatives thereof are described in and can be synthesized according to methods described in PCT International Publication Nos. WO 2010/132815 A9 and WO 2013/059203 A1, each of which are hereby incorporated by reference.
The National Institutes of Health (NIH) provides a table of Equivalent Surface Area Dosage Conversion Factors below (Table A) which provides conversion factors that account for surface area to weight ratios between species.
Each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiments. Thus, all combinations of the various elements described herein are within the scope of the invention.
This invention will be better understood by reference to the Experimental Details which follow, but those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention as described more fully in the claims which follow thereafter.
Materials and Methods MaterialsMTS cytotoxicity assay (CellTiter Aqueous one) was purchased from Promega. Pure Curcumin (PC, 99% purity) was purchased from Selleck Chemicals. Tetrahydrocurcumin (THC, 96%, Sabiwhite™) was obtained via Biocogent LLC. The chemically synthesized curcumin derivatives—CMC2.24, CMC2.5, CMC2.14 and CMC2.23 (all 97% purity) were obtained from ChemMaster International through Dr. Francis Johnson. Pyrocatechol Violet (PV), copper sulfate, mushroom tyrosinase, L-DOPA, and kojic acid (KA) were purchased from Sigma. 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) dye were purchased from Molecular Probes.
Cell CultureB16F10 mouse melanoma cells (CRL-6475™) were obtained from ATCC (Manassas, Va.) and cultured in DMEM with 10% heat-inactivated fetal bovine serum (HI-FBS) and 1% antibiotics (penicillin-streptomycin). Human keratinocytes (HaCaT) cells were obtained from AddexBio (San Diego, Calif.) and primary human dermal fibroblasts (NHDF, purchased originally from Lonza) were obtained from Michael Ingrassia at Biocogent LLC (Stony Brook). Both these cells were cultured in DMEM with 10% HI-FBS and 1% antibiotics. Human primary epidermal melanocytes from darkly pigmented donor (HEM-DP) were obtained from Cascade Biologics. The cells were cultured using Medium 254 supplemented with 1% Human melanocyte growth supplement (HMGS, Cascade Biologics) and 1% antibiotics. Cells were detached using TrypLE Express (0.0.5%) and cultured in T-25 flasks.
MTS Cytotoxicity AssayIn order to test the four CMCs (CMC2.14, CMC2.24, CMC2.5 and CMC2.23) alongside PC and THC, for their effects on melanin content, we first screened their nontoxic concentrations using MTS cytotoxicity (Promega CellTiter Aqueous One) assay. MTS is a tetrazolium salt which is reduced to purple colored formazan upon reaction with mitochondrial dehydrogenases. Briefly, B16F10 cells were seeded at 4×103 cells/well in 96-well plates for 24 hrs. The test compounds were prepared from stocks (20 mM in DMSO) and further diluted using culture media (1:625 dilution) and added to the cells after 24 hrs, such that final DMSO concentrations in all groups were 0.16% which did not affect cell viability. Controls were treated with 0.16% DMSO. At the end of 48 hours, medium was aspirated and replaced by 100 μl of fresh media. MTS (20 μl) was added and incubated for 40 minutes and absorbance read at 490 nm using a Versamax® microplate reader. Cell viability was calculated from the absorbance values relative to control groups and expressed in %.
Melanin Content Quantification in B16F10 Cells
B16F10 cells were seeded at 1×105 cells/well in 1.5 ml media in 12-well plates and incubated for 24 hours. The media was then replaced with test compounds (PC, THC and 4 CMCs) at nontoxic doses (selected from MTS assay) and incubated for another 48 hours. For quantification of melanin content, cells were trypsinized and cell pellets were washed in PBS. Cell pellets were also visually observed for lightening of the intracellular pigment. After aspiration, 250 μL of 1N NaOH was added and heated to 70° C. to solubilize melanin. The aliquots were then transferred to a 96-well plate and absorbance was read at 475 nm using a microplate reader. A portion of the lysate was used to evaluate total protein content using BCA assay. The melanin absorbance was normalized by total protein content and expressed as % of control.
Mushroom Tyrosinase ActivityThe direct effects of the compounds on tyrosinase enzyme activity were tested using mushroom tyrosinase enzyme with L-DOPA substrate. Briefly, 80 μl of all the 6 test compounds prepared at different concentrations in 50 mM sodium phosphate (pH 6.5) buffer was added to 96 wells followed by 100 μl of freshly prepared substrate solution (3 mM L-DOPA in buffer). The reaction was initiated by adding 20 μl of mushroom tyrosinase enzyme (final concentration of enzyme in wells was 3.5 μg/ml). The production of DOPAchrome was monitored by measuring the kinetics of absorbance increase at 475 nm (for 30 min every 30 seconds) at 30° C. using microplate reader (Versamax®). The slopes of the kinetic readings were calculated to determine and compare tyrosinase activity from control.
Copper Chelating Activity AssayMetal ion chelation activity can be detected by pyrocatechol violet indicator assay which is similar to that reported in other studies with some modifications [Baek, S-H. et al. 2015]. Briefly, 100 μl of sample containing different concentrations of solutes were mixed with 50 mM sodium acetate buffer (pH 6.0) in a 96 well plate. Control group consisted of buffer only; KA at 500 μM was used as a positive control. Copper sulfate (2 mM; 10 μl) was added to the samples and incubated for 10 minutes. 10 μl of 2 mM pyrocatechol violet was then added and the plate was further incubated for 20 minutes. The absorbance was read at 632 nm using a microplate reader (Versamax). Lower absorbance compared to control was indicative of copper chelation.
Intracellular Tyrosinasa ActivityWe quantitated the cellular tyrosinase activity to delineate mechanism of melanogenesis inhibition by the compounds. Briefly, R16F10 cells were cultured in 24-well tissue culture plates at a density of 4×104 cells/well. After 24 hrs, media was changed and test compounds were added, and further incubated for 48 hrs. At the end of treatments, cells were trypsinized and cell pellets were washed in PBS and lysed with lysis buffer (containing 1% NP-40 to solubilize the normally membrane-bound enzyme and protease inhibitors) under ice for 20 minutes and then centrifuged to remove cellular debris. 50 μl of lysates were then aliquoted in 96-well microplate and 150 μl of 3 mM solution of freshly prepared L-DOPA in phosphate buffer, pH 6.8 was added. The absorbance was then measured kinetically at 475 nm every 30 seconds for 40 minutes at 300° C. using microplate reader (Versamax®). The % inhibition of tyrosinase activity was calculated by determining melanin formation rates from measurements of the linear regions of the slopes of melanin content as a function of time and comparing computed rates of melanin formation in the presence of the test compounds to those of the controls.
DPPH Antioxidant AssayMelanogenesis is often associated with higher oxidative stress and reactive oxygen species (ROS) generation. Hence, compounds which possess antioxidative activity can also act in inhibiting melanin production. DPPH is a stable free radical which changes color from purple to yellow upon reduction by antioxidant compounds by electron donation; the color change can be measured spectrophotometrically. PC, THC and CMCs were evaluated for antioxidant activity. Briefly, 2,2-Diphenyl-1-picrylhydrazyl (DPPH) was freshly prepared in methanol and mixed with different concentrations of the compounds in triplicates in a 96 well plate. The Negative Control group consisted of DPPH only and Ascorbic Acid (AA) at 100 μg/ml was used as a positive control. The final DPPH concentration was 100 μM and final volume was 200 μl in the plate (20 μl samples with 180 μl of DPPH). The plate was covered and incubated for 30 minutes. Absorbance was read at 517 nm. DPPH scavenging activity was reported as % relative to control.
Intracellular ROS AssayIntracellular ROS generation was quantified using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA), a non-fluorescent dye which is trapped intracellularly by cleavage by intracellular esterases after reaction with reactive oxygen species (ROS); only the product, DCF, is fluorescent. For measuring intracellular ROS, B16F10 cells were seeded in 24 well plates at 4×104 cells/well. After 24 hours, media was aspirated and PC, THC and CMCs were added to the wells at different concentrations and further incubated for 48 hours. At the end of the incubations, the media was aspirated and cells were washed with PBS. DCFDA (Molecular Probes) was dissolved in DMSO to make 10 mM stock. DCFDA dye working stock (50 μM) was prepared by diluting the stock in DMEM (serum-free, phenol-red free and sodium pyruvate free) media and added to cells and further incubated for 45 minutes at 37° C. The cells were then washed with DPBS and fluorescence was read in bottom read mode, using a fluorescence microplate reader (Gemini EM Spectramax, Molecular Devices) set to excitation and emission wavelengths of 485 nm and 535 nm, respectively. Data was expressed as relative fluorescence % of treated vs. control.
Ultrastructural Study of Melanosome Maturation Stages Using TEMIn order to study the effects of CMCs on melanosome maturation, TEM was conducted to study ultrastructure and the effects were evaluated qualitatively. Briefly, B16F10 cells were seeded in six-well plates at 2×105 cells/well and treated with compounds (CMC2.5 at 20 μM, CMC2.14 at 10 μM, CMC2.23 at 25 μM and CMC2.24 at 20 μM) for 48 hours. The cells were detached as described herein, the pellets were washed in PBS and fixed in glutaraldehyde fixative overnight, and then processed for resin embedding. The images were observed using Tecnai BioTwin G TEM (FEI, OR, US), at 80 kV.
Recovery Study of Intracellular Tyrosinase Activity in B16F10 CellsIn order to establish reversibility of tyrosinase inhibition by the CMCs, we conducted a “recovery” study over a 2 day interval using the highest concentrations of the compounds which produced maximum tyrosinase activity inhibition without toxicity. B16F10 were plated in six-well plates at 3.5×104 cells/well and compounds were added (PC—10 μM, CMC2.14—10 μM, CMC2.5—20 μM, CMC2.23—25 μM and CMC2.24 at 20 μM) next day. Tyrosinase activity in one set of cultures after detachment and lysis as described herein was estimated after 2 day exposure while another set of cultures were continued with fresh media without the compounds to study reversibility of tyrosinase activity inhibition for another 2 days. Results are expressed as % tyrosinase activity for both 2 day exposure and 2 day recovery post 2 day exposure.
MITF Protein Levels Measurement in B16F10 CellsMITF is the master regulator of melanogenesis and a known transcription factor for tyrosinase gene. The effects of PC and CMCs on micropthalmia transcription factor (MITF) proteins levels were assayed using a cell-based ELISA (LifeSpan Biosciences, Seattle). Briefly, B16F10 cells were cultured in a 96-well plate at 1×104 cells/well for 24 hours and then media was replaced with fresh media containing compounds in 0.1% DMSO for further 48 hours. The cells were then fixed and subsequent steps were conducted based on manufacturer instructions. The absorbance of MITF expression levels were normalized by absorbance of crystal violet stain (0.05%) and data was reported as % of control.
Mlanin Content Measurement in αMSH-Stimulated B16F10 Cells.UV irradiation is known to produce α-MSH, a pro-melanogenic stimulus and in order to test if CMCs exhibited antimelanogenic activity in presence of hormone stimulation; we tested the compounds in presence of MSH. B16F10 cells were seeded at 1×105 cells/well in 12-well plates and incubated for 24 hours. The test compounds were then added in presence or absence of αMSH (100 nM), and further incubated for another 48 hours. For evaluation of melanin content, cells were trypsinized, washed in PBS and then 250 μL of 1N NaOH was added and heated to 70° C. to solubilize melanin. The aliquots were then transferred to a 96-well plate and absorbance was read at 475 nm using microplate reader. The absorbance of melanin was normalized by total protein contents and reported as % of control.
MTS Cytotoxicity Test with Human Keratinocytes and Human Dermal fibroblasts
For testing toxicity of PC, THC, and CMCs during 48 hours incubation with HaCaT cells, we conducted MTS cytotoxicity assay. Briefly, 2×104 cells/well were seeded in 96-well plates and after 24 hours, the compounds at concentrations of 5-25 μM were added to the wells and incubated for 48 hours. Absorbance was read after 1 hour of incubation of plates and results were expressed as % of control.
For testing cytotoxicity of the curcumin compounds exposed to normal human dermal fibroblasts, a protocol similar to that described immediately above was employed. Briefly 5×103 cells/well were plated in 96 wells and compounds added for 48 hours. MTS assay was then conducted and plate incubated for 1 hour and then 100 μl of supernatants were aliquoted into 96 well plates and absorbance read at 490 nm using Versamax microplate reader.
Phagocytosis Assay Using Fluosphere Beads in KeratinocytesThe final steps of melanogenesis in the skin involve transfer of melanin contained within mature melanosomes from melanocytes to keratinocytes. In order to model melanosome uptake by keratinocytes in the presence of CMCs, we used uptake of fluosphere latex beads (0.5 μm, carboxylate-modified, Molecular Probes, Eugene, Oreg.) by a human keratinocyte cell line (HaCaT) as a model for melanosome uptake study; this model has been previously established [Cardinali G, et al. 2005; Cardinali, G. et al. 2008] to study phagocytosis by keratinocytes. HaCaT cells were cultured in 24 well plates (3.5×104 cells/well) for 48 hours and then media was replaced with fresh media containing CMCs (CMC2.14 at 10 μM, CMC2.5 at 20 μM, CMC2.23 at 20 μM and CMC2.24 at 20 μM). Control wells were treated with 0.1% DMSO. After 48 hours exposure, media was removed and wells were washed with PBS and Fluosphere bead solution was suspended in complete medium by sonication for 15 minutes was then diluted in complete medium before addition to the wells (180×107 beads/well) and further incubation for 24 hours. After the treatment, wells were washed in PBS and extracellular fluorescence was quenched using trypan blue (0.1% in PBS); Fluorescence intensity over the entire bottom surface of each well was measured at excitation/emission of 580/605 using the “well-scan” mode with average of 21 points/well for triplicate wells in a Gemini EM fluorescence plate reader. Controls without any beads were used as blank for background subtraction and results are reported as RFU values expressed as % relative to control.
MTS Cytotoxicity Test with Human Epidermal Melanocytes
For screening cytotoxicity of PC, THC and CMCs during 48 hours incubation on HEM-DP cells, we conducted MTS cytotoxicity assay. Briefly, 3×104 cells/well were seeded in 96 well plates and after 24 hours, compounds at various concentrations were added to the wells and incubated for 48 hours. MTS reagent was added and incubated for 90 minutes and absorbance was read at 490 nm. The results are expressed as % relative to control.
Melanin Content Quantification in Human Epidermal Melanocytes Melanin Content.Primary cultures of human epidermal melanocytes from a darkly pigmented donor were seeded at 2.2×105 cells/well in 1.5 ml media in 12-well plates and incubated for 48 hours. The test compounds were added to cultures and controls were treated with 0.16% DMSO, and further incubated for another 48 hours. For evaluation of melanin content, cells in the wells were trypsinized and cell pellets were washed in PBS. After aspiration, 250 μL of 1N NaOH was added and heated to 70° C. to solubilize melanin. The aliquots were then transferred to a 96-well plate and absorbance was read at 475 nm using microplate reader. A portion of the lysate was used to evaluate total protein content using 239 bincinchonic acid (BCA) assay (Pierce BCA kit, Thermo Scientific). The absorbance of melanin was normalized to the total protein content for each lysate and reported as % of control.
Intracellular Tyrosinase ActivityWe quantitated the cellular tyrosinase activity of human melanocytes based on established methods. Briefly, human epidermal melanocytes from a darkly pigmented donor (HEM-DP) were cultured in 12-well tissue culture plates at density of 1.5×105 cells/well. After 48 hrs. media was changed and test compounds were added, and further incubated for 48 hrs. At the end of treatments, cells were trypsinized and cell pellets were washed in PBS and lysed with lysis buffer under ice for 30 minutes and then centrifuged to remove cellular debris. 50 μl of lysates were then aliquoted in 96-well microplate and 150 μl of freshly prepared 3 mM L-DOPA was added. The absorbance was then measured kinetically at 475 nm every 30 seconds for 30 minutes at 30° C. using microplate reader (Versamax®). The % inhibition of tyrosinase activity was calculated by determining melanin formation rates from the linear range of the slopes of melanin formation over time for the different samples and calculating % inhibition from the measured velocities of melanin formation.
Recovery Study of Intracellular Tyrosinase Activity in Human MelanocytesIn order to study if the melanocytes could recover from inhibited tyrosinase activity after exposure to the CMCs, we employed a model for reversibility of inhibition of tyrosinase activity inhibition. This can establish safety of the compounds since irreversible inhibition of tyrosinase activity could damage melanocyte synthetic machinery with potential loss of the protective function of melanin. CMCs were used at highest concentration which gave potent inhibition of melanogenesis without toxicity (CMC2.24-10 μM, CMC2.23 at 20 μM and CMC2.5 at 20 μM). Briefly, HEM-DP were plated in 6-well plates at 2.3×105 cells/well and grown for 3 days and then CMCs were added. Tyrosinase activity was estimated after 2 day exposure in cellular lysates and another set of cultures were continued with fresh media without the compounds for further 5 days (with media change at day 3), in order to study reversibility of melanin inhibition.
Statistical AnalysisOne-way analysis of variance (ANOVA) with Tukey's post-hoc test was run in experiments where all compounds were tested together while Dunnett's post-hoc test was used when compounds were compared individually vs. control. All the analyses were conducted using GraphPad Prism software (Version 4.0) and differences were considered statistically significant at p<0.05. All data are reported as Mean±SD.
Example 1. Effect of Compounds on Cytotoxicity in B16F10 CellsMTS assay was conducted to screen the compounds (PC, THC and CMCs) for cytotoxicity and only select nontoxic ones to be further used for melanogenesis study. PC and CMC2.14 caused significant cytotoxicity at 20 and 25 μM. The mean value of cell viability was 24.44% and 16.55% for PC at 20 μM and 25 uM, respectively (
Melanin content was estimated in cell pellets to study effects of compounds on inhibition of melanogenesis.
As tyrosinase is the primary rate-limiting enzyme in melanogenesis pathway, we studied if the compounds inhibited melanin in part due to inhibition of tyrosinase. Mushroom tyrosinase activity was tested to screen the compounds to identify if the compounds had any direct inhibitory effect of the tyrosinase enzyme using L-DOPA as substrate.
Since tyrosinase is a binuclear copper enzyme, we further studied if the compounds inhibited tyrosinase via copper chelation, for which the PV dye method was used and results are summarized in
To identify the mechanism of depigmentation, we evaluated tyrosinase activity levels in B16F10 cellular lysates after treatment with the test compounds.
Antioxidant activity is desirable for compounds intend for use in diminishing pigmentation since excessive melanogenesis causes increased oxidative stress and generation of superoxide and hydrogen peroxide [Simon, J. D. et al. 2009]. The DPPH scavenging assay results for PC, THC and CMCs are summarized in
CMC2.24 showed the greatest antioxidant activity, reducing intracellular ROS in B16F10 cells by 40% at 20 μM (
We studied melanosome ultrastructure in B16F10 cells to qualitatively evaluate if the inhibitory mode of action of CMCs might encompass effect on melanosome maturation: previous studies have documented effects on maturation of melanosomes by antimelanogenic compounds [Bellei, B. et al. 2014; Liang, Y-R. et al. 2014; Wu, S-Y. et al. 2014]. Immature melanosomes are referred to as stage I+II while mature melanosomes are referred to as stage III+Stage IV. Control cells (
Normal melanin production is necessary for photo-protection and in immune regulation, and since tyrosinase is a key enzyme in melanogenesis pathway, skin lighteners which cause excessive or irreversible inhibition of tyrosinase activity could pose safety concerns. There have been reports in which recovery of tyrosinase activity in melanocytes has been studied by melanogenesis inhibitors [Charalambous, A. et al. 2015, Chawla, S. et al. 2008]. Hence, we evaluated CMCs for reversibility of tyrosinase activity inhibition using the highest concentrations for each of the CMCs. CMC2.14 at 10 μM caused loss of up to 75% of tyrosinase activity which was similar to CMC2.24 at 20 μM (
MITF is the master regulator of melanogenesis and a known transcription factor for tyrosinase gene. Downregulation of MITF is an attractive target for skin-whitening compounds.
In order to test if CMCs exhibited antimelanogenic activity in presence of hormone stimulation; we tested the compounds in presence of MSH and results are shown in
To establish safety for topical use in cosmetics, we evaluated cytotoxicity of the compounds in human keratinocyte and primary dermal human fibroblasts using MTS assay. PC was found to be significantly toxic in both HaCaT cells and NHDF cells. HaCaT cell viability was 90.12% at 10 μM (p<0.05) and reduced drastically at higher doses (
Taken together, the results show that PC was the most toxic in both skin cells from 10 μM onwards, while both CMC2.23 and CMC2.24 were nontoxic in the concentration range 5-25 μM in both cells. CMC2.14 was a significant stimulator of cell proliferation in both cells, which may implicate a therapeutic effect of this compound in terms of providing benefits in wound healing and CMC2.5 at 25 μM showed some cytotoxicity to keratinocytes.
Example 13. Effect of Compounds on Phagocytosis of Fluosphere Beads by KeratinocytesCompounds which can inhibit uptake of melanin by keratinocytes can offer attractive target for skin pigmentation inhibitors at later stages in melanin pathway. We selected concentrations for the 4 CMCs which was nontoxic and potent in activity and the results of the phagocytosis assay are summarized in
PC, THC and CMCs (2.14, 2.24, 2.23 and 2.5) were screened for cytotoxicity in HEM-DP cells using MTS assay for duration of 48 hours. PC (5, 10 μM), CMC2.24 (5, 10 μM) and CMC2.23 (5, 10, 20 μM) were screened as nontoxic (
Taken together, the results of CMCs on melanin production levels in HEM-DP cells suggest that PC and THC were inactive in concentration ranges tested for duration of 48 hours, while CMC2.24 was most potent by inhibiting 20% melanin production at 10 μM, while a similar inhibition rate could be achieved by both CMC2.23 and CMC2.5 at 20 μM. These results are in agreement with melanin inhibition activity obtained using B1610 mouse melanoma cells where CMC2.24 was most potent. Hence, in HEM-DP cells, the order of melanogenesis inhibitory activity is CMC2.24>CMC2.23=CMC2.5
Example 16. Effects of Compounds on Intracellular Tyrosinase Activity in HEM-DP CellsTo explore mechanisms of melanogenesis inhibition in HEM-DP cells, intracellular tyrosinase activity was quantitated in cellular lysates after treatment with compounds for 48 hours.
Overall, CMC2.5 and CMC2.23 both inhibited similar melanin levels at 20 μM as obtained before and the cellular tyrosinase data indicates that other mechanisms in melanogenesis pathway apart from tyrosinase enzyme pathway, could be at play in case of CMC2.5 since it only inhibited tyrosinase activity by 16% at 20 μM while CMC2.23 at similar concentration inhibited tyrosinase activity potently by 3-folds (49%).
Example 17. Recovery Study of Tyrosinase Activity in HEM-DP CellsAn amount of compound CMC2.5, 2.23 or 2.24 is topically administered to a subject afflicted with hyperpigmentation. The amount of the compound is effective to treat the hyperpigmentation.
An amount of compound CMC2.5, 2.23 and 2.24 is topically administered to a subject in need of skin lightening. The amount of the compound is effective to lighten the skin of the subject.
DiscussionTurmeric extract is comprised of three major curcuminoids (curcumin I, curcumin II and curcumin III), of which curcumin (curcumin I) is most abundant (77%), along with curcumin II (demethoxycurcumin, 17%) and curcumin III (bis-demethoxycurcumin, 3%) [Aggarwal, B. B. et al. 2007]. The commercial grade curcumin used in research studies is thus not chromatographically pure and usually consists of a mixture of curcumin I, II and III. Recently there have been several reports of melanogenesis inhibition by curcumin [Hosoya, T. et al. 2012; Jang, J. Y. et al. 2009; Lee, J. H. et al. 2010; Mustarichie, R. et al. 2013; Wolnicka-Glubisz, A. et al. 2015; Tu, C. X. et al. 2012]. However, the previous reports on curcumin as a melanogenesis inhibitor document use of curcumin preparations the source of whichis inconsistent and variable. For example, studies which tested analogs of curcumins used curcumin from Sigma which has >80% curcumin that contains contaminating curcuminoids (II and III) while some studies did not even report the source of the curcumin preparation. Hence, a true comparison of structure-activity relation (SAR) cannot be made. Additionally, some studies have used alpha-melanocyte stimulating hormone (αMSH) or IBMX as an exogenous stimulator in cell culture studies to induce melanogenesis.
Tetrahydrocurcumin (THC), a colorless hydrogenated metabolite of yellow curcumin has been recently commercialized for treatment of cosmetic hyperpigmentation and is marketed as Sabiwhite™. THC has appeal for cosmetic sector as it does not have an intrinsic color unlike yellow curcumin [Arct, J. et al. 2014]. However, to date, no cell culture studies have been conducted with THC to assess its effects on melanogenesis and no comparisons with curcumin or its derivatives have been reported. CMC2.24, a triketonic N-phenylaminocarbonyl derivative of bis-45 demethoxycurcumin, exhibits enhanced stability and solubility compared to curcumin and also demonstrated diverse potential pharmacological activities such as treatment of anthrax by inhibiting lethal factor enzyme [Antonelli, A. C. et al. 2014], treatment of prostate cancer [Botchkina, et al. 2013], normalizing wound healing in diabetes in rats [Zhang, Y. et al. 2016] and reduction of periodontitis in rats [Elburki, M. S. et al. 2014]. CMC2.5 (4-methoxycarbonyl curcumin), another derivative, has also demonstrated performance superior to that of the parent compound (curcumin) in treatment of inflammation in periodontitis [Gu, Y. et al. 2013]. The synthesis scheme for other CMCs which are based on the Pabon reaction has been summarized in previous report [Zhang, Y. et al. 2012].
In the present work, four CMCs (referred as CMC2.14, CMC2.5, CMC2.24 and CMC2.23) can be grouped into two categories which differ in the i) type of substituent on the P-diketone (—COOCH3 and —CONHPh), and ii) presence or absence of the methoxy group on the aryl rings All four CMCs were tested for inhibition of melanogenesis. Sabiwhite™ (tetrahydrocurcumin, referred as THC hereafter) was also used to compare alongside curcumin and CMCs to evaluate whether CMCs at low doses could have greater potential as antimelanogenic agents than white THC for cosmetic purposes. Moreover, to date, there is no study which has explored if the double bond conjugation in curcumin is unique in terms of melanogenesis inhibitory activity Hence testing both PC (curcumin) and THC (tetrahydrocurcumin) can help in dissecting the role of the double-bond conjugation in modulating melanin inhibition. The SAR study focused on evaluating the three regions of the parent curcumin structural skeleton—i) THC as a model for effects of hydrogenation of the two double bonds bridging the phenyl rings to the central β-diketone moiety; ii) CMC2.5 and CMC2.14 as models for effects of substituents on the two phenyl rings (—OCH3 and —H) and; iii) CMC2.23 and CMC2.24 as models for modification of the central R-diketone moiety (—COOCH3 and —CONHPh).
The results provide for the novel use of the CMCs (CMC2.5, CMC2.24 and CMC2.23) for skin depigmentation, which are far superior to the colored parent compound (curcumin) and the white curcumin (THC, Sabiwhite™). At low micro-molar ranges tested in 5-25 μM, the synthetic CMCs (CMC2.24, CMC2.23 and CMC2.5) were superior inhibitors of melanogenesis compared to both PC and THC, both of which were inactive as inhibitors of melanogenesis at similar doses when tested in primary human melanocytes (in addition, PC is highly toxic to human dermal fibroblasts and the HaCaT human keratinocyte line). Also, the color of CMCs at such low doses should not hinder their use in cosmetic formulations.
The mechanisms of action of the CMCs include direct inhibitory effects on tyrosinase enzyme activity (and at the cellular levels of enzyme protein), which is mostly or completely reversible upon removal of the compounds, antioxidant activity, suppression of MITF protein levels, inhibition of melanosome maturation and melanosome transport to keratinocytes. Additionally, since CMC2.24 is being pursued as a pharmacological candidate for treatment interventions for canine periodontitis, it holds potential for use in clinical settings to treat hyperpigmentation disorders in dark skinned individuals as its preclinical safety and toxicology studies have already been undertaken in preparation for future clinical trials. Hence, CMC2.24, as well as CMC2.5 and CMC2.23 and derivatives thereof, could be repurposed as drugs for treating hyperpigmentation disorders in dermatology.
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Claims
1. A method of treating a subject afflicted with hyperpigmentation comprising administering to the subject an amount of a compound having the structure: wherein bond α and β are each, independently, present or absent; wherein each occurrence of alkyl, alkenyl, or alkynyl is branched or unbranched, unsubstituted or substituted, or a salt or ester thereof, so as to thereby treat the subject.
- X is CR5 or N; Y is CR10 or N;
- R1 is —SR12, —SO2R13, —COR14, —CSR14, —C(—NR12) R4, —C(—NH)R14, —SOR12, —POR12, —P(═O)(OR12)(OR13), or —P(OR12).(OR13), wherein R12 is C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl; R13 is H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl; R14 is C2-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, heteroaryl, heterocyclyl, methoxy, —OR15, or —NR16R17, wherein R15 is H, C3-10 alkyl, C2-10 alkenyl, C2-10 alkynyl; R16 and R17 are each, independently, H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
- R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 are each independently, H, halogen, —NO2, —CN, —NR28R29, —NHR28R29+, —SR28, —SO2R28, —OR28, —CO2R28, CF3, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl; wherein R28 and R29 are each, H, CF3, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, or —C(═O)-heterocyclyl; and
2. The method of claim 1, wherein the compound reduces melanin synthesis in the subject.
3. The method of claim 1, wherein the compound inhibits melanogenesis in the subject.
4. The method of claim 1, wherein the compound inhibits tyrosinase activity in the subject.
5. The method of claim 1, wherein the compound lightens the skin tone of the subject relative to the subject's natural skin tone.
6. A method of lightening the skin tone of a subject comprising administering to the subject an amount of a compound having the structure: wherein bond α and β are each, independently, present or absent; wherein each occurrence of alkyl, alkenyl, or alkynyl is branched or unbranched, unsubstituted or substituted, or a salt or ester thereof, so as to thereby lighten the skin tone of the subject.
- X is CR5 or N; Y is CR10 or N;
- R1 is —SR12, —SO2R13, —COR14, —CSR14, —C(—NR12) R14, —C(NH) R14, —SOR12, —POR12, —P(═O)(OR12)(OR13), or —P(OR12).(OR13), wherein R12 is C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl; R13 is H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl; R14 is C2-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, heteroaryl, heterocyclyl, methoxy, —OR15, or —NR16R17 wherein R15 is H, C3-10 alkyl, C2-10 alkenyl, C2-10 alkynyl; R16 and R17 are each, independently, H, C1-10 alkyl, C2-10 alkenyl, C2-10alkynyl, aryl, heteroaryl, or heterocyclyl;
- R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 are each independently, H, halogen, —NO2, —CN, —NR28R29+, —NHR28R29+, —SR28, —SO2R28, —OR28, —CO2R28, CF3, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl; wherein R28 and R29 are each, H, CF3, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, or —C(—)-heterocyclyl; and
7. The method of claim 6, wherein the compound reduces melanin synthesis in the subject.
8. The method of claim 6, wherein the compound inhibits melanogenesis in the subject.
9. The method of claim 6, wherein the compound inhibits tyrosinase activity in the subject.
10. The method of claim 6, wherein the compound lightens the skin tone of the subject relative to the subject's natural skin tone.
11. The compound of claim 1 or 6, wherein a and 3 are each present.
12. The compound of claim 1 or 6, wherein a and R are each absent.
13. The method of any one of claims 1-11, wherein the compound has the structure: wherein each occurrence of alkyl, alkenyl, or alkynyl is branched or unbranched, unsubstituted or substituted, or a salt or ester thereof.
- R3, R4, R8 and R9 are each independently, H, halogen, —NO2, —CN, —NR28R29, —NHR28R29+, —SR28, —SO2R28, —OR28, —CO2R28, CF3, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl; wherein R28 and R29 are each, H, CF3, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, or —C(═O)-heterocyclyl; and
- R14 is methoxy, —OR15, or —NR16R17, wherein R15 is H, C3-10 alkyl, C2-10 alkenyl, C2-10 alkynyl; and R16 and R17 are each, independently, H, C1-10 alkyl, C2-10 alkenyl, C2-10alkynyl, aryl, heteroaryl, or heterocyclyl;
14. The method of any one of claims 1-10 or 12, wherein the compound has the structure: wherein each occurrence of alkyl, alkenyl, or alkynyl is branched or unbranched, unsubstituted or substituted, or a salt or ester thereof.
- R3, R4, R8 and R9 are each independently, H, halogen, —NO2, —CN, —NR28R29, —NHR28R29+, —SR28, —SO2R28, —OR28, —CO2R2, CF3, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl; wherein R28 and R29 are each, H, CF3, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, or —C(═O)-heterocyclyl; and
- R14 is methoxy, —OR15, or —NR16R17, wherein R15 is H, C3-10 alkyl, C2-10 alkenyl, C2-10 alkynyl; and R16 and R17 are each, independently, H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
15. The method of claim 13 or 14, wherein R14 is methoxy or —NR16R17, wherein R16 and R17 are each, independently, aryl or heteroaryl.
16. The method of claim 13 or 14, wherein R3, R4, R8 and R9 are each independently, H or —OR28, wherein R28 is H or C1-10 alkyl.
17. The method of claim 13 or 14, wherein R3, R4, R8 and R9 are each —OR28, wherein each R28 is, independently, H or C1-10 alkyl.
18. The method of claim 13 or 14, wherein R3, R4, R8, and R9 are each, independently, H, —OCH3, or —OH; and R14 is methoxy or —N(CH3)2.
19. The method of any one of claims 1-11, wherein the compound has the structure: or a salt thereof.
20. The method of any one of claims 1-10 or 12, wherein the compound has the structure: or a salt thereof.
21. The method of any one of claims 1-20, wherein the compound is administered topically to the subject.
22. A method of inhibiting melanogenesis for reducing skin melanin levels in a subject comprising administering to the subject an amount of a compound having the structure: wherein bond α and β are each, independently, present or absent; wherein each occurrence of alkyl, alkenyl, or alkynyl is branched or unbranched, unsubstituted or substituted, or a salt or ester thereof, so as to thereby inhibit melanogenesis for reducing skin melanin levels in the subject.
- X is CR5 or N; Y is CR10 or N;
- R1 is —SR12, —SO2R13, —COR14, —CSR14, —C(—NR12)R14, —C(—NH)R14, —SOR12, —POR12, —P(═O)(OR12)(OR13), or —P(OR12).(OR13), wherein R12 is C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl; R13 is H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl; R14 is C2-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, heteroaryl, heterocyclyl, methoxy, —OR15, or —NR16R17 wherein R15 is H, C3-10 alkyl, C2-10 alkenyl, C2-10 alkynyl; R16 and R17 are each, independently, H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl;
- R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 are each independently, H, halogen, —NO2, —CN, —NR28R29, —NHR28R29+, —SR28, —SO2R28, —OR28, —CO2R28, CF3, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, aryl, heteroaryl, or heterocyclyl; wherein R28 and R29 are each, H, CF3, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, or —C(═O)-heterocyclyl; and
Type: Application
Filed: May 3, 2019
Publication Date: Aug 22, 2019
Applicant: The Research Foundation for The State University of New York (Albany, NY)
Inventors: Shilpi GOENKA (East Setauket, NY), Sanford SIMON (Stony Brook, NY)
Application Number: 16/402,982
