AU2002219386B2 - Treatment of skin disorders - Google Patents

Description

WO 02/057260 PCT/GB02/00158 COMPOUNDS FOR USE IN THE TREATMENT OF SKIN CONDITIONS Field of the invention This invention relates to the treatment of skin conditions, comprising those conditions requiring stimulation of melanocyte proliferation and to the inhibition of melanomas. The invention is of especial application to the treatment of vitiligo and skin cancer.

Vitiligo is a common skin pigment disorder characterised by the development of patchy de-pigmented lesions. Current treatments which include the use of photosensitisers (eg psoralens) with UVA radiation (PUVA), corticosteroids or skin grafting have low success rates and are generally accompanied by unpleasant side effects. Vitiligo has a highly detrimental impact on the emotional well-being of the sufferer, the disfiguring effects of the disease being compounded by the absence of a suitable treatment. Although vitiligo patches are not believed to contain melanocytes (pigment producing cells), a reservoir exists in hair follicles in vitiliginous skin. Thus activation of hair follicular melanocytes is a crucial process in the repigmentation of vitiliginous skin.

Certain plant remedies, usually administered as mixtures of herbs or extracts, particularly those used in traditional Chinese medicine and Indian Ayurvedic medicine, have been employed for the treatment of vitiligo for a long time and in many cases have given positive results in small scale studies. Herbs such as Psoralea corylifolia L. and Vernonia anthelmintica Willd. (=Centratherum anthelminticum Kuntze) are well known for their use in this disease. Psoralens, which are employed in the moder PUVA and khellin in KUVA therapy were originally derived from plant sources (Psoralea colylifolia L and Ammi visnaga respectively) used in traditional remedies for vitiligo. However these therapies rely on the use of UV irradiation for their efficacy, which is associated with the aetiology of skin cancer.

The fruit of black pepper (Piper nigrum and long pepper (Piper longum are both important medicinal herbs in Ayurvedic and Unani (traditional Indian) medicine systems, in which remedies generally consist of mixtures of herbs. A wide range of the medicinal uses of black pepper have been documented by Kirtikar and Basu (Indian Medicinal Plants, 2 nd Edition, Vol. 3, (1935) pages 2128 2135), including its use in the treatment of leucoderma.

Black pepper has also been implicated as a possible adjunct to Vernonia anthelmintica in the treatment of leucoderma (Indian Medicinal Journal, Vol. 1, 3 rd Edition, (1982) 1267 1270).

-1- SUBSTITUTE SHEET (RULE 26) WO 02/057260 PCT/GB02/00158 These two herbs are employed as a constituent in many traditional herbal preparations for a variety of uses, including gastro-intestinal and skin ailments. Compositions comprising black pepper, ginger and pipali have been used in the treatment of vitiligo (Ancient Science of Life, Vol. IX, No. 4 (1990) 202 206); however, the specific therapeutic action of black pepper in this orally administered composition has not been established.

It has been found (WO 001/02544) that, piperine, which is present in the fruit of Piper nigrum, stimulates the replication of melanocytes. The action of piperine is to increase the number of cells which confer pigmentation. Piperine is the compound benzodioxol-5-yl)-l-oxo-2,4-pentadienyl]piperidine and should not be confused with piperidine.

Pharmaceutical compositions containing piperine have been used in the treatment of tuberculosis and leprosy (EP 0 650 728). It has also been suggested that piperine is able to enhance the bioavailability of the other constituents of a pharmaceutical composition (WO 96/25939).

There is, therefore, a need for further compounds and compositions, which are able to stimulate the proliferation of melanocytes.

Summary of the invention The invention provides a compound or formula for use in the treatment of a skin condition requiring stimulation ofmelanocyte proliferation and melanomas, in which

R

2

R

4

R

6

R

8

(RR

(R

R

3 R 5 7 0

(I)

mis 2 n is 0 or 1 p is 0 or 1 q is 0 or 1 WO 02/057260 PCT/GB02/00158 the two R' groups together represent a 3',4'-methylenedioxy group

R

2 is hydrogen

R

3 and R 4 represent hydrogen atoms or together represent a carbon to carbon double bond;

R

5 and R 6 represent hydrogen atoms or together represent a carbon to carbon double bond;

R

7 and R 8 represent hydrogen atoms or together represent a carbon to carbon double bond; and

R

9 represents piperidino, morpholino, cyclohexylamino, methylamino, ethylamino and isopropylamino in any of its E, Z geometrically isomeric forms or an active analogue or derivative thereof, as hereinafter defined or optionally when n is 1 R 2 and R 3 together represent a carbon to carbon double bond and one or more of R 4 and R 5 together, R 5 and R 6 together,

R

6 and R 7 together or R 7 and R 8 together represent a carbon to carbon double bond the other of

R

4 to Ri representing hydrogen with the proviso that the compound is not piperine, 3,4dihydropiperine; 1,2,3,4-tetrahydropyridine, Ilepeimide or piperettine.

The invention also provides the use of a compound of formula in the preparation of a medicament for use in the treatment of a skin condition requiring stimulation of melanocyte proliferation and melanomas. Pharmaceutical compositions comprising a compound of formula and a pharmaceutically acceptable carrier are also provided.

The active ingredient may be used on its own, but is more suitably used in combination with a carrier or excipient and optionally one or more further active ingredients.

Stimulation of melanocyte proliferation greatly facilitates the re-pigmentation of depigmented skin, e.g. post traumatised de-pigmented skin. The term "post traumatised depigmented skin" means the skin formed during the healing process that occurs after a skin trauma. De-pigmentation may arise, for example, from scar tissue formed as a result of a skin trauma such as burn or other skin lesion or may be due to vitiligo. The present invention can be used to treat any of these skin disorders in a patient.

Generally in this invention, the compounds of formula or active derivatives or analogues thereof may be administered by oral, topical, intravenous or subcutaneous (intramuscular) routes but are preferably applied topically (to the area of the skin where treatment is desired). Indeed, the twice-daily topical application of compounds of formula has been -3-

\O

found to induce significant pigmentation in mice. Skin coloration in the mouse Spopulation under study was first observed at approximately four weeks after the Streatment was started. This coloration was enhanced further as a result of subsequent 00 0topical applications.

The active ingredient may be formulated as a solid powder; a paste, ointment or IND cream; a tablet or capsule or a solution.

00 Mc, The compounds of formula may also be used to treat a person having a skin _condition which would benefit from coloration, e.g. to enhance or promote the natural colouring of the skin. The treatment may be used for prophylactic, therapeutic or cosmetic purposes.

I The compounds of formula and their analogues or derivatives as hereinafter defined inhibit the proliferation of melanoma cells. Thus, they may also be used in the treatment of skin cancer. Another aspect of the invention therefore provides a method of treating skin cancer in a human or animal patient comprising the administration to said patient of a therapeutically effective amount of a compound of formula or an active analogue or derivative thereof, as hereinafter defined.

The compounds of formula or active analogues or derivatives thereof may be administered by oral or topical routes. Suitable dosage forms may be any of those discussed above.

Certain of the active analogues or derivatives of the compound of formula (1) are new. The present invention therefore includes such compounds, and pharmaceutical compositions containing them together with a carrier or excipient.

The present invention also provides a compound for use in the treatment of a skin condition, said compound comprising the formula

R

2 R' R 6

R

8 IIr Ii I -R 9

O

O

c in which Sm is 2; 00 n is 0 or 1; p is 0 or 1; q is 0 or 1; 0 0 the two R' groups together represent a 3',4'-methylenedioxy group;

R

2 to R 8 are hydrogen atoms; and

R

9 represents cyclohexylamino or an active analogue thereof.

O

0 The present invention also provides use of a compound in the preparation of a medicament for use in the treatment of a skin condition selected from the group of conditions consisting of those treatable by stimulation of melanocyte proliferation and a melanoma, said compound comprising the formula

R

2

R

4

R

6

R

8

R'

(R m I)

R

3

R

5

R

7

O

in which m is 2; n is 0 or 1; p is 0 or 1; q is 0 or 1; the two R' groups together represent a 3',4'-methylenedioxy group;

R

2 is hydrogen;

R

3 and R 4 represent hydrogen atoms or together represent a carbon to carbon double bond; IN 4b 6 C- R 5 and R represent hydrogen atoms or together represent a carbon to carbon double bond; R 7 and R 8 represent hydrogen atoms or together represent a carbon to carbon double bond; and

R

9 represents morpholino, cyclohexylamino, methylamino, ethylamino, IN isopropylamino, isopropoxy, propoxy and butoxy in any of its E,Z geometrically 00 2 3 M^ isomeric forms or an active analogue thereof or optionally when n is I R and R together represent a carbon to carbon double bond and one or more of R 4 and R c together, R 5 and R 6 together, R 6 and R 7 together or R 7 and R 8 together represent a carbon to carbon double bond the other of R4 to R8 representing hydrogen atoms.

C-K Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Description of the preferred embodiments Preferred compounds of formula are those in which n is 0, one ofp or q is other than 0, R 3 and R 4 together form the second bond of a carbon to carbon double bond and either R 5 and R 6 together or R 7 and R 8 together form the second bond of a carbon to carbon double bond; and n is 0, one of p or q is other than 0, R 3 and R 4 are each hydrogen and either

R

5 and R 6 or R 7 and R 8 are also hydrogen and R 9 is cyclohexylamino or piperidino.

Particularly preferred compounds are those in which WO 02/057260 PCT/GB02/00158 n is 0, one of p or q is other than 0, R 3 and R 4 together form the second bond of a carbon to carbon double bond and either R 5 and R 6 together or R 7 and R 8 together form the second bond of a carbon to carbon double bond and R 9 is selected from morpholino, cyclohexylamino, methylamino, ethylamino and isopropylamino; n is 0, p is 0, q is 1, R 3

R

4

R

7 and R 8 represent hydrogen and R is cyclohexylamino; and n is 0, p is 1, q is 1, R 3 R R R 6

R

7 and R 8 represent hydrogen and R 9 is piperidino.

The compounds of formula can be prepared from the appropriate acid with the appropriate connecting chain between the carboxylic acid function and the benzene ring and having the appropriate stereochemistry. Where necessary, this may be preceded or followed by reduction to reduce the double bond or bonds in the connecting chain. Methods of preparing amides and esters from these acids are illustrated by the Examples below. They may also be adapted from the references cited herein, the disclosure of which is herein incorporated by reference.

The active compounds may be formulated for topical use in the form of creams, soft paraffin or lotions. Aqueous cream BP or Yellow Soft Paraffin BP may suitably contain the active at 0.03-3.0 mg w/w or an equivalent amount of plant extract. A suitable lotion is typically prepared from 20% glycerol and 80% ethanol in purified water and contains 0.03-3.0 mg w/w of the active material. These topical formulations may also contain penetration enhancers such as oleic acid, propylene glycol, ethanol, urea, lauric diethanolamide or azone, dimethyl sulphoxide, decylmethyl sulphoxide, or pyrrolidone derivatives. Liposomal delivery systems may also be used.

Compositions for oral formulation include tablets or capsules containing 1.5-150 mg active for daily administration.

The invention will now be described with reference to the following non-limiting examples, with reference to the accompanying tables and drawings.

Examples Introduction Cell culture experiments WO 02/057260 PCT/GB02/00158 Microplate culture and sulforhodamine B (SRB) assay Cells of mouse melan-a cell line (passage number 18-24), a first known line of nontumorigenic pigmented mouse melanocytes were maintained in a flask (Costar, Cambridge, MA, USA) using RPMI 1640 (ICN, Costa, Mesa, CA, USA) as a basic medium. For microplate proliferation assays, subconfluent melan-a cultures were trypsinized (0.25% trypsin at 37 0 C for 5-10 min) and inoculated with a repeater-pipettor (Finn pipette, Labsystems, Finland) into 96-well microtiter plates (Costar, Cambridge, MA, USA) at a seeding concentration of 6 x 10 cells per well. A supplemental growth medium of 10% foetal bovine serum (FBS) was added to the 36-well microtiter plates. The plates were incubated at 37 0 C in a 10% CO2, 90% air humidified atmosphere for 4 days. At the end of the incubation, an SRB assay was performed. Briefly, cells attached to the bottom of the plate were fixed by addition of cold trichloroacetic acid (TCA, 4°C, Aldrich, Dorset, UK) on the top of the growth medium (final TCA 20% The plate was placed at 4 0 C for 1 hour before being gently washed five times with tap water. It was allowed to dry in air, or aided with a hair dryer to speed up the drying process, then 50 il of 4% w/v SRB dissolved in 1% acetic acid in water was added to each well for 30 min. At the end of the staining period, unbound SRB was removed by washing 4 times with 1% acetic acid. The plate was air dried again, and 150 pl of 10 mM aqueous Tris base (Sigma-Aldrich Co. Ltd, Irvine, UK) was added into each well to solubilize the cell-bound dye. The plate was shaken for 15 min on a gyratory shaker followed by reading the optical density (OD) at 550 nm in a microplate spectrophotometer (Anthos Labtec HT3, version 1.06). A control assay was carried out on cells incubated without test compound.

There were 2 or 3 series of experiments, each of which consisted of six replicate experiments.

The results are tabulated below.

Example 1 Compounds of formula (1) Introduction Vitiligo is defined as a circumscribed, acquired, idiopathic, progressive hypomelanotic skin disorder which is characterised by the development of patchy depigmented macules due to progressive loss ofmelanocytes which is often familial with lack of established aetiology.

WO 02/057260 PCT/GB02/00158 Various compounds of formula were synthesised and tested for melanocyte (mouse melan-a) proliferant activity in-vitro. Cells were incubated with the test compound for 4 days, as described above.

1.1 Percentage cell growth (A) Percentage cell growth was obtained with a given compound calculated as (optical density in the presence of the compound/ control optical density) x 100.

1.2 Relative activity to piperine Melan-a cell proliferant activity for tested compounds was compared with that obtained with piperine. Percentage stimulant activity is (A-100) where A stands for piperine or a test compound's percentage cell growth (see All figures are given with Standard Error of the Mean.

Relative activity to piperine was calculated as (A-100) compound (A-100) piperine).

Interpretation of the relative active value is as follows 0 Inhibition of cell growth 0 No effect (equal to control) 0-1 Stimulant but weaker effect than piperine 1 Equal stimulant effect to piperine 1 Stimulant and stronger effect than piperine 1.3 Dendricity Effect on dendricity of melan-a cells by the test compounds was by observation under microscope. Dendricity is relevant to vitiligo since normal skin melanocytes have dendrites, but in vitiligo the melanocytes seem to lose these before they disappear from the patches.

1.4 Synthesis of Compounds of formula (1) The compounds of formula were synthesised using methods described in the literature, adapted from the literature or devised in the inventors' laboratory. Structures of WO 02/057260 PCT/GB02/00158 compounds were verified using NMR, MS, IR spectroscopy and melting point. Unless a synthetic method is given, reagents and reactants were purchased from Sigma Aldrich.

Results The activity of compounds of formula at a single concentration of test compound (10 OM) is shown in Table 1. This is followed by data showing results at other concentrations. Many compounds showed a "cross-over" effect in which the test compound was less active than piperine at 10 OM but more active at -8- Table 1 Variation on Nitrogen Substituent of Piperine Effect on melan-a cells at ~IMA concentration Code N' Structure Percentage cell growth Stimulant Relative Dendricity (Repeated experiments) activity activity to Test cpd. IPiperine piperine 0 0156=L58 2 1 0±65* RY-A02 K N N187±40** 170±22 Positive 1.02 153±19** 155±1I9** 0.9 00 RV-A07 <0 OD a N NH 170±24* 216±33* Positive 0.6 0 RV-AO8 0 N NH'" 200±14 236±-17 Positive 0.73 RV-A09 <0 NH' 224±:19 263±1L6** Positive 0.76 0 RY-lO 0 0 N 3M&-29** 302id7** Positive 1.24H **P<0.0l compared to vehicle treatment (Dunnett's t test) highly dendritic, moderately dendritic, wealy denciritic, no effect WO 02/057260 WO 02/57260PCT/GB02/00158 Compounds 1IM IlOPLM 25iiM -Tested Pipeline 147±1 1**0 192+13*"** 167±19** 142115"* RV-A02 125:dd0 167dd17** 171+8** 168Adl2**O **P<O.O0 1 Compared to vehicle treatment (Dunnet' s t test) *Piperine, is significantly more active than test compound P<0.05 I] Test compound is significantly more active than Piperine P<0.05 11 WO 02/057260 WO 02/57260PCT/GB02/00158 Code N' Structure 0 RV-A07 0 j NH Compounds 1gM 10gM 50gM 100gM Tested Igm 10 ,M 50pM IOOPM Piperine 211:116**+ 216+33** 52=1=15 16d=3 RV-A07 140±12** 170±24** 71:15 46±2 Dentricity of RV-A07 P<0.0 1 Compared to vehicle treatment (Dunnet' st test) Piperine, is significantly more active than test compound P<0.05 moderately dendritic, weakly dendritic 12 WO 02/057260 WO 02/57260PCT/GB02/00158 Code N' Structure 0 RV-A08 0 N NH- Compounds ijiM 1O0.iM 5O iM 1OO[iM Tested Piperine 216±14**+ 236:07"* 61±11 32:L5 RV-A08 139±27** 200±1I4** 81±12 62+13 Dendricity -of RV-AO8 **P<0.01 Compared to vehicle treatment (Dunnet's t test) *Piperine is significantly more active than test compound P<0.05 highly dendritic, moderately dendritic, weakly dendritic 13 WO 02/057260 WO 02/57260PCT/GB02/00158 Code NO Structure 0 0 RV-A0 NH

OD

Compounds Iji pl jiM 50[IM 100P~M Tested IPiperine 221±17**+ 263+:16** 77112 24±2 RV-A09 187115** 224L=19** 854 5 42d=6 Dendricity of RV-A09__ .01 Compared to vehicle treatment (Dunnet's t test) 4Piperine is significantly more active than test compound P<0.05 highly denchitic, weakly dendritic -14- WO 02/057260 WO 02/57260PCT/GB02/00158 Code N' Structure RV-A1O 0 NH0 Compounds lRO10 M 5OjiM 1OO[tM Tested Piperine 236±30** 302±17** 78±11 21±4 RV-A1O 301±20**El 308±29** 155±22**O 100:L13 Dendricity of RY-AlO P<0. 01 Compared to vehicle treatment (Dunnet's t test) I] Compound is significantly more active than piperine P<0.05 highly dendritic, moderately dendritic, weakly dendritic 15 WO 02/057260 WO 02/57260PCT/GB02/00158 RV-C04 Code No Structure RV-C04 0 0 N <I

H

0D Compound 1pLm 10ptm 50gtM 100[pM Piperine 191±12**, 216±18** 184±6** 96±6 RV-C04 129±6** 192±6** 192±10** 191±12** Dendricity of RV-C04 16- WO 02/057260 WO 02/57260PCT/GB02/00158 Code No Structure 0 oDi 4

NC

Compound 1I M 1QtM 50VM1 lO0ptM IPiperine 161±13** 192±2** 189±15** 87±13 118±1 160±5** 158±19** 113±15 Dendricity of -17- WO 02/057260 PCT/GB02/00158 2. Synthesis of amide derivatives of piperinic acid 2.1 Preparation of piperinic acid (RV-AOO) To piperine (2g, 0.7mmol, leq), 20% ofmethanolic KOH (100ml) was added and refluxed for 2days. After completion of the hydrolysis, methanol was removed under reduced pressure and a yellow coloured oily solid was obtained. This residue was dissolved in water (50ml) and acidified with 6N HCI to pH <1 yielding a yellowish precipitate of piperinic acid. Recrystallization from methanol gave yellow needles (0.9g, yield). m.p. 206 0 C-208 0 C (Lit m.p. 217 0 C-218 0

C)

1 0 0 S N 20% KOH H Piperine Piperinic acid 2.2 Synthesis of amide derivatives of piperinic acid The following amines were reacted with piperinic acid in accordance with the following procedure: morpholine; methylamine; ethylamine; isopropylamine and cyclohexylamine. A mixture of piperinic acid (leq) and triethylamine (2eq) in dichloromethane (50ml) was stirred for 15min at 0 0 C. To this mixture methanesulfonyl chloride (1.5eq) was added and stirred for further 30 min at 0°C. The amine (1.5eq) was added to the mixture and stirred for lh at 0°C and 2h at room temperature.

Dichloromethane (50ml) was added to the mixture which was then washed with 5% HCI (3x100ml), saturated aqueous NaHCO 3 (3xl00ml) and water (3xl00ml). The organic fraction was dried over anhydrous sodium sulphate, filtered and rotary evaporated to yield a yellowish solid residue. Recrystallisation from ethylacetate/petroleum spirit yielded colourless needles of piperlonguminine (120mg, 32% yield) 2 The reaction is presumed to proceed through a mesylate ester intermediate.

-18- WO 02/057260 WO 02/57260PCT/GB02/00158 morpholine (IRV-A02) 0 0

N

'11-NMR (CDC1 3 6: 6.37 11-1, J=14.6, CI-P"CH-CII=CIL), 7.45 U-1, J=10.2, 14.6, CH=CH-CH=CH), 6.72 111, J=15.5, 10.2, CH=CH-CH=CH), 6.79 111, J=15.5 CH=CH-CH=CH), 6.98 (d,1H J=1.5, Ar-7-H1), 6.80 (d,1H J=8.0, Ar-10-H), 6.89 d, III J=1.5, 8.0 Ar-li-H), 5.98 211, O-C11 2 3.70 2H, J=4.0 CH 2

CR

2 (morpholine)) 3.60 2H, J=4.0 C11 2

CH

2 (morpholine)) 13 C-NMR (CDC1 3 :42.3 (CHA) 46.1(C11 2 66(C][1 2 66(CI- 2 101.3 (CH 2 106.5 108.5 (CHI), 118.7 122.7 (CII), 124.9 130.8 139.1 143.4 (CHI), 148.2 148.3 165.6 (C) MS m/z :287 (Mi 57), 201 (100), 173 171 (10) 143 115 IR (KBr): (carbonyl group) 1641 m.p. 161.8 0 -162.5()C (Lit m.p. 167-168 0

C)

3 yield 44.1 (RV-A07) 0 31 o 8 N 1

H

o

NH

'1T-NMR (crDCI 3 8: 5.91 1H, J=14.8, C~HCH-CH=CMf, 7.36 111, J 1O.7, 14.8, CH=CHf-CIFCH), 6.66 M1, J=15.4, 10.6, CH=CLI-CH=CH), 6.77 1II, J=15.4 CUI=CH-CH=CH), 6.97 (d,lH Ar-71), 6.77 (d,IH J=8.0, Ar-l1ll), 6.88 d, 111 J=1.6, 8.0 Ar-lull), 5.97 2H, O-CH 2 2.91(t, 3H1, CR 3 5.61 (hr, NH) 123.3 125.0 (CHI), 131.2 139.2 141.4 (CHI), 148.6 148.6 167.2

(C)

-19- WO 02/057260 PCT/GB02/00158 MS m/z 231(M+89), 201 173 172 171 143 116 (21) 115 (100), 89 (12) m.p. 181.1 0 -182.4 0 C (Lit m.p. 186C) 5 yield 48.2% 5-EE-piperinoylethVIamine (RV-A8) 0 3 1' oYi 64~ 2 lNHf> 0 I Z oI

NH

'H-NMR (CD 3 OD) 8: 6.14 1H, J=15.0, CH=CH-CH=CEi, 7.37 111, J=10.2, 15.0, CH=CH-CH=CH), 6.93 1I, J=15.7, 10.6, CH=CI-CH=CH), 6.87 1I, J 15.7 CH CH-CH=CH), 6.97 (d1H J 1.5, Ar-7M), 6.77 (d,IH J 8.O, Ar-lOH), 6.88 (d, d, 1H J=1.6, 8.0 Ar-tIll), 5.97 2H, O-Cl 2 3.39 2H, J= 6.2, CH 2 31, J= 6.1, C1 3 3 C-NMR (CDC1 3 14.7 (CU 3 36.9 (CU 2 103.2 (CU 2 107.2 109.8 (CII), 121.2 124.9 125.9 132.4 142.9 145.2 150.2 150.6 (C),170 (C) MS m/z 245(M+78), 218 201 200 174 173 172 171 143 116 (68),115 (100) m.p. 158.5 0 -159.9 0 C (Lit m.p. 162 0 -164 0

C)

4 yield 45.6% WO 02/057260 WO 02/57260PCT/GB02/00158 (RV-A09) 1 0 9 11l 'H-NMLR (CDC1 3 8: 5.87 1H, J=14.8, CH=CH-CH=ClI), 7.36 IR, J=10.7, 14.8, CJ-I=CH-CII=CH), 6.66 III, J=15.4, 10.6, CH=CHI-CH=CH), 6.76 1H1, J=15.2 CH'CH-CH=CH), 6.97 (d,1H J=1.6, Ar-711), 6.77 (d,1H J=8.0, Ar-lOll), 6.88 1H J=1.6, 8.0 Ar-till), 5.97 2H, O-C11 2 4.15(m-4 LH, CH), 5.36 1H, J=7.3 NHl), 1. 19 6H, J=6.6, (CH 3 2 1 3 C-NMR (CDC1 3 23.2 (CR 3 2, 41.9 (CII), 101.9 (CR 2 106.4 108.9 (CHI), 123.0 123.8 (CHi), 124.1 131.3 140.2 141.2 148.8 148.6 165.6 (C) MS m/z 259(M 4 201 174 173 172 171 143 (3 116 115 (100) m.p. 169 0 169.4 0 C (Lit m.p. 171 0 -173 OC) 4 yield 52% 5-E,F-Piperinoyl cycdobexylamine (RV-A1 0) 3, 0 4' 0 6> NH 6',15 0 9 'H-NMR (CDC1 3 5: 5.93 1H1, f=14.8, CH=CII-CH=C), 7.35 LHf, J=10.6, 14.8, CH=CH-CH=CH), 6.66 JH, J=15.3, 10.6, CH=CH-CH=CH), 6.76 1H, J'=15.4 CH=CH-CH=CH), 6.96 (d,1H J=1.6, Ar-7H1), 6.76 (d,1H J=8.0, Ar-l1ll), 6.87 d, III J=1.6, 8.0 Ar-liR), 5.97 2H1, O-CH 2 3.87 (in, 11H, CH (cyclohexyl)) 1.99 (mn, 2H, C11 2 (cyclohexyl)) 1.65 (in, 4H, CH 2

-C

2 (cyclohexyl) 1.39 (in, 211, CR 2 (cyclohexyl)) 1. 18 (in, 211, CH1 2 (cyclohexyl)) 5.48 (d,J=8 .0 NIlI) -21- WO 02/057260 WO 02/57260PCT/GB02/00158 13 C-NIVR (CDCl 3 ):25.3 ((Cl 2 25.9 (CH 2 33.6 ((CH 2 48.6 101.3 (CH 2 101.7 (CII), 106.1 (CHI), 108.9 123.0 (CII), 124.0 (CHI), 125.1 (CII),131.3 139.0 (CMI, 141.2 (CH) 148.5 148.5 165.5 (C) MS m/z :299(M+56), 259 (48) 216 201 (60),174 173 172 171 (16) 143 115 (100) m.p. 196.4 0 -197.3 0 C (Lit m.p. 199 0 _200 0

C)

4 yield 57.4% References: 'Ghatterjee, and Dutta, C.P. (1967). Alkaloids of Piper longum Linn-I Structure and synthesis of piperlongumine and piperlonguminine, Tetrahedron, 23,1769-17 81.

2 Nokio Nakumara, Fumiyuki Kiuchi, and Yoshisuke Tsuda (1988). Infrared spectra of conjugated amides: Reassignment of the C=O and C=C absorptions: Chiemical and Pharmaceutical Bulletin, 36, 2647-2651.

3 1.Oediger and A. Schulze (Bayer AG), (1979), Deutsche Auslegeschrift 2757 483 4 Paula, Vanderlucia F. de; A Barbosa, Luiz C. de; Deniuner, Antonio Pilo-Veloso, Dorila; Picanco, Marcelo C. (2000) Pest Management Science 56, 2, 168 174.

sGokale et al., (1948) Journal of University Bombay Science 16/5A 32-3 4. Preparation of 5-(3,4-miethylenedioxy Ihey-pentanoic acid cyclohexylamide (RV-C04) To 5-(3,4-methylenedioxy phenyl)-2E,4E-peutadienoic acid cyclohexyl amide (300mg) was added 5% PcI/C (30mg) and hydrogenated the contents at 30 psi for ihr. The solution was filtered and rotary evaporated to yield a white solid. Recrystallisation from ethylacetate and petroleum spirit yielded pure white crystals (255mg, yield m.p.

145.4'C -146.30 C.

0 0 5% Pd/C o

H

0 NH

H

2 0 5-(3,4-methylenedioxy 5-(3,4-methylenedioxy phenyl)-2E,4E-pentadienoic phenyl)-2,4-pentatioic acid cyclohexyl amide acid cyclohexyl amide -22- WO 02/057260 WO 02/57260PCT/GB02/00158 Preparation of 7-(3,4-methylenedioxy phenyl)-heptanoic acid piperidineamlide To 7-(3,4-methylenedioxy phenyl)-2E,4E,6E-heptatrienoic acid piperidine amide (150mg, 0.O6mmole) was added 5% Pd/C (15mg) and hydrogenated the contents at 30 psi for 30min to give 7-(3,4-methylenedioxy phenyl)-heptanoic acid piperidine amide as an oil.

0 0,- 7-(3,4-methylenedioxy phenyl)-2E,4E,6E-heptatrienoic acid piperidine amide 0 Pd/C 7-(3 ,4-methylenedioxy phenyl)-2,4,6-heptanoic acid piperidine amide RV-C04 'H-NMR (CDCl 3 8: 6.65 (d,1H J=1.6, Ar-7-11), 6.71 (d,1H J=7.8, Ar-tO-H), 6.60 d, I1H J=1.6, 8.0 Ar-il-H), 5.90 2H, O-CI~z-O), 5.43 1H1, NH), 2.53 2H, J=7.7 (C11f 2

-CH

2

-CI

2

CH

2 2.14 2H, J=7,7 ((Cf 2

-CH

2

CH

2

-CH

2 1.62-1.9 1 (in, 1011,

CH

2 -C1 2 -C1 2 -C1 2

CH

2

.CH

2 .CH2 (cyclohexyl amnide) 1.07-1.30 (in, 4H, C11 2

.CH-CH

2 (cyclohexylamide) 13 C-NMR (CDC1 3 :25.3 ((CH 2 25.7 (CH 2 ),25.9 (C 2 31.3 (C 2 31.7

(CH

2 33.6 (CH 2 35.8 (CH 2 37.3 (C 2 48.4 101.1 (CH 2 108.4 (CHI), 109.2 121.4 (CII), 136.4 145.8 147.8 172.2 23 WO 02/057260 WO 02/57260PCT/GB02/00158 MS mn/z :303 98), 204 176 168(16), 162 (12) 161 154 148 141 (61) 135 (100) 74 (24) 60 9 7 5 3 0 8 0i 21N 2' Oil 13 510 31 12 4' 'H-NMR (CDC1 3 6: 6.66 (d,1H J 1.5, Ar-7-H), 6.71 (d,lH J=7.8, Ar-lO-il), 6.60 d, I114 J 1.6, 8.0 Ar-Il-Il), 5.90 2H, O-CHR 2 3.53 211, J=5.4 CH 2

.N-

CU

2 3.37 2H, J=5.7, (CH 2 Cl- 2 2.51 2H, J=7.7 (CH 2 -C11 2

-CH

2 -C1-1 2 -CF1 2

CH

2 2.33(t, 211, J='7.7 ((C11 2

-CH

2

CH

2

GCH

2

.CH

2

-CH

2 1.52-1.65 (in, 1011, hydrocarbon CE 2

CH

2 CHz-C11 2

-CH

2 (Piperidine)) 1.34 (Mi, 4H, CH 2 CHz) 13 C-NMR (CDC1 3 24.9 (CU- 2 25.8 (CH 2 ),25.9 (CU 2 26.9 (CH 2 29.3(C11 2 29.7 (C14 2 31.3 (Gil 2 31.9 (CH 2 33.8 (Gil 2 42.9 (C 2 47.1 (CU 2 10 1.89 (CU 2 108.4 (CII), 109.2 121.4 137.0 145.7 147.8 171.8 MS in/z :317 (W 4 78), 232 204 183 182 154 (21) 148 141 127 (100), 112 85 (49) Yield 5 1.2% -24-

Claims (10)

1. 2. A compound according to claim 1, wherein said compound is 5-(3,4- methylenedioxyphenyl)- pentanoic acid cyclohexylamide or 7-(3,4- methylenedioxyphenyl)-heptanoic acid cyclohexylamide.
2. 3. A pharmaceutical composition comprising a therapeutically effective amount of a compound of claim I or claim 2 and a pharmaceutically acceptable carrier.
3. 4. A pharmaceutical composition according to claim 3, which includes a penetration enhancer. Use of a compound in the preparation of a medicament for use in the Z treatment of a skin condition selected from the group of conditions consisting of(a) _those treatable by stimulation of melanocyte proliferation and a melanoma, said compound comprising the formula OO 00 SR2 R4 R 6 R 8 g R 9 (R')m (Rm R 3 R 5 R 7 O in which m is 2; n is 0 or 1; pis 0 or 1; q is 0 or 1; the two R' groups together represent a 3',4'-methylenedioxy group; R 2 is hydrogen; R 3 and R 4 represent hydrogen atoms or together represent a carbon to carbon double bond; R 5 and R 6 represent hydrogen atoms or together represent a carbon to carbon double bond; R 7 and R 8 represent hydrogen atoms or together represent a carbon to carbon double bond; and O 2/ O R 9 represents morpholino, cyclohexylamino, methylamino, ethylamino, isopropylamino, isopropoxy, propoxy and butoxy in any of its E,Z geometrically Z isomeric forms or an active analogue thereof or optionally when n is I R 2 and R 3 together represent a carbon to carbon double bond and one or more of R 4 and R together, R 5 and R 6 together, R 6 and R 7 together or R 7 and R 8 together represent a I carbon to carbon double bond the other of R 4 to R 8 representing hydrogen atoms. 00
4. 6. Use according to claim 5, wherein said compound is selected from the group CN 5-E,E piperinoyl morpholine, 5-E,E-piperinoylmethylamine, Spiperinoylethylamine, 5-E,E-piperinoylisopropylamine and C 10 piperinoylcyclohexylamine.
5. 7. Use according to claim 5 or claim 6, wherein in said formula n is 0.
6. 8. Use according to any one of claims 5 to 7, wherein in said formula p is 1, q is 0 and R 3 and R 4 together and R 5 and R 6 together are a carbon double bond.
7. 9. Use according to any one of claims 5 to 8, wherein in said formula R 2 to R 8 are hydrogen atoms. Use according to any one of claims 5 to 9, wherein said compound is 5-(3,4- methylenedioxyphenyl)-pentanoic acid cyclohexylamide or 7-(3,4-methylened ioxyphenyl)-heptanoic acid cyclohexylamide.
8. 11. Use according to any one of claims 5 to 10, in which the skin condition is vitiligo.
9. 12. A compound according to claim 1 or claim 2 for use in the treatment of a skin condition substantially as hereinbefore described.
10. 13. A pharmaceutical composition according to claim 3 or claim 4 substantially as hereinbefore described with reference to the examples and/or the preferred embodiments excluding, if any comparative examples. IO 28 O O S14. Use of a compound in the preparation of a medicament for use in the 0 treatment of a skin condition substantially as hereinbefore described with reference to the examples and/or the preferred embodiments excluding, if any comparative examples. 00 Dated this sixteenth day of November 2006 C BTG International Limited Patent Attorneys for the Applicant: F B RICE CO