GB2370989A

GB2370989A - Piperine analogues for the treatment of skin conditions

Info

Publication number: GB2370989A
Application number: GB0101146A
Authority: GB
Inventors: Amala Raman; Robert Charles Hider; Radhakrishnan Venkatasamy
Original assignee: BTG International Ltd
Current assignee: BTG International Ltd
Priority date: 2001-01-16
Filing date: 2001-01-16
Publication date: 2002-07-17
Also published as: US7361685B2; ZA200305432B; GB0101146D0; US20040254165A1; MXPA03006324A; WO2002057260A1; CN1486313A; JP2004520366A; EP1377571A1; KR20040028711A; CA2434775C; CA2434775A1

Abstract

Compounds of formula (I) wherein m is 1,2 or 3, R<SP>1</SP> being an alkoxy or hydroxy group when m is 1, each R<SP>1</SP> being independently an alkoxy group or together being a 3',4'-methylenedioxy group when m is 2 and each R<SP>1</SP> being independently an alkoxy group or two R<SP>1</SP>s being a 3',4'-methylenedioxy group and the other R<SP>1</SP> being an alkoxy group when m is 3, n is 0, 1 or 2, R<SP>2</SP> and R<SP>3</SP> are both H or together represent a double bond, R<SP>4</SP> and R<SP>5</SP> are both H or together represent a double bond and R<SP>6</SP> is a pyrrolidino, piperidino, 4-methylpiperidino, morpholino, N-monoalkylamino, N-monocycloalkylamino, 3',4'-methylenedioxybenzylamino, 2-phenethylamino or alkoxy group for use in the treatment of skin conditions. The skin conditions are either melanomas or those which are treatable by stimulation of melanocyte proliferation, such as vitiligo. The compounds are analogues of piperine and may be applied topically. <EMI ID=1.1 HE=38 WI=103 LX=494 LY=1216 TI=CF>

Description

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 Wild. (=Centratherum anthelminticum Kuntze) are well known for their use in this disease. Psoralens, which are employed in the modem PUVA and khellin in KUVA therapy were originally derived from plant sources (Psoralea corylifolia L and Ammi visnaga respectively) used in traditional remedies for vitiligo. However these therapies rely on the use ofUV irradiation for their efficacy, which is associated with the aetiology of skin cancer.

The fruit of black pepper (Piper nigrum L.) and long pepper (Piper longum 1.) 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 (Indiam Medicinal Plants, 2nd Edition, Vol. 3, (1935) pages 2128-2135), including its use in the treatment ofleucoderma. Black pepper has also been implicated as a possible adjunct to Vernonia anthelmintica in the treatment of leucoderma (Indian Medicinal Journal, Vol. 1, 3rd Edition, (1982) 1267-1270).

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.

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

As reported in prior PCT Application Publication NO WO 00/02544, 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 (E, E)-l- [5- (l, 3-benzodioxol-5-yI)-l-oxo-2, 4-pentadienyI] piperidine and should not be confused with piperidine. Certain named analogues and derivatives of piperine are also mentioned in the said prior application to have such effects.

In that application, piperine is also disclosed as being able to inhibit the proliferation of melanoma cells, thus making it useful in the treatment of skin cancer.

Summary of the invention The invention provides a method of treating a subject (human or animal) having a skin condition selected from the group consisting of (a) those requiring stimulation of melanocyte proliferation and (b) melanomas, which comprises administering to the subject, preferably to the site of the condition, an effective amount of a piperine analogue or derivative thereof, as hereinafter defined. 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. Where patent law permits, the invention includes such a piperine analogue or derivative thereof for the use mentioned where such use is medical, i. e. the skin condition is a disorder.

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 bum 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 piperine derivative or analogue thereof may be administered by oral, topical, intravenous or subcutaneous (intra-muscular) routes but is preferably applied topically (to the area of the skin where treatment is desired).

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

The method of the invention 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.

Piperine 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 the piperine analogue or derivative thereof, as hereinafter defined.

The piperine analogue or derivative thereof may be administered by oral or topical routes. Suitable dosage forms may be any of those discussed above.

The formula of piperine derivatives and analogues thereof usable in this invention is given below.

wherein n = 0,1 or 2; when n = 0, R2 and R3 represent hydrogen atoms or together represent a carbon to carbon double bond;

when n = 1, or 2 R2 and R3 together and R4 and Rs together represent carbon to carbon double bonds, R2 and R3 together represent a carbon to carbon double bond and R4 and Rs represent hydrogen atoms, or R2, R3, R4 and Rs all represent hydrogen atoms ; m= 1, 2 or 3 ;

when m = 1, Ru represents an alkoxy group having from 1 to 3 carbon atoms or a hydroxy group ; when m = 2, each R'independently represents an alkoxy group having from 1 to 3 carbon atoms or the two Rls together represent a 3', 4'-methylenedioxy group; when m = 3, two Rls together represent a 3'-4'-methylenedioxy group and the other R represents an alkoxy group having from 1 to 3 carbon atoms or a hydroxy group; R6 represents a pyrrolidino, piperidino, 4-methylpiperidino or morpholino group, a Nmonoalkylamino group of 4 to 6 carbon atoms, a N-monocyclooalkylamino group of 4 to 7 carbon atoms, a 3'-4'-methylenedioxy-substituted benzylamino or 2-phenethylamino group or R6 represents an alkoxy group of 1 to 6 carbon atoms ; in any of its E, Z geometrically isomeric forms, but excluding the named analogues and derivatives in our prior application.

The excluded analogues and derivatives of formula (1) named in our prior application are as follows: (a) n is 0,1, or 2, m is 2, the R's together represent a 3', 4'-methylenedioxy group, R2 and R3, together with the carbon atoms to which they are attached form a carbon to carbon double bond and, when n is 1 or 2, R4 and R5, together with the carbon atoms to which they are attached, form a carbon to carbon double bond and R6 is piperidino, or (b) n is 1 and (i) m is 3, the RIS being 3', 4'-methylenedioxy and 6'-methoxy or (ii) m is 2, the R's being 3'-hydroxy-4'-methoxy; or (iii) m is 1 and the RI is 4'-hydroxy; and R2 to R6 are as defined in case (a) above, or (c) n is 1, R6 is piperidino, pyrrolidino, isobutylamino or methoxy and all other symbols are as defined in case (a) above, or (d) n is 1, R4 and R5 represent hydrogen atoms and either R2 and R3 also do or R2 and R3 together with the carbon atoms to which they are attached form a carbon to carbon double bond; and m, Rl and R6 are as defined in case (a) above; and in all of which cases (a) to (d) the molecule is in the E, E or all E geometric configuration or in case (a) when n is 1 may be in the Z, Z, Z, E or E, Z geometric configuration.

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

Brief description of the drawings Figure 1 : Plots of growth of melan-a-cells cultured in different media. Each point represents the mean and standard deviation (SD) of 6 replicates. FBS = fetal bovine serum.

TPA = tetradecanoyl phorbol acetate.

Figure 2: Plots of growth melan-a-cells cultured from different initial plating densities of cells. Medium was supplemented with 20 nM TPA. On day 4 the medium in the remaining plates was replaced. Each point shows mean and SD of 6 replicates.

Figure 3: Effect of P. nigrum extract on the growth of melan-a cells. Culture was maintained for 8 days. Medium and extract were replaced with fresh ones on day 4. Each point designates mean and SD of 6 replicates, except that 12 replicates were done for cells only.

Figure 4: Effects of P. nigrum extract and TPA on the proliferation of melan-a cell line. Each point shows mean and SD of 6 replicates, except that 12 replicates were done for cells only.

Figure 5: Effects of piperine and TPA on the growth of melan-a cells in the presence of RO-31-8220. n = 6 for piperine and TPA treated wells, whereas n = 12 for RO-31-8220 alone.

Figure 6: Effects of piperine and TPA on the growth of human melanoblasts in the presence ofET3. *P < 0.05 when compared to vehicle control (One way Anova, followed by Dunnett's t-test).

Figure 7: Effects of piperine on the growth of human melanocytes in the presence of ET1. *P < 0.05 when compared to ETI InM treatment (One way Anova, followed by Dunnett's t-test).

Figure 8: Dose response curve showing the growth of melan-a cells in presence of a compound of formula (1), RV-A01, as % of control plotted against concentration.

Description of the preferred embodiments The following are preferred features of the compounds of formula (1) used in the invention considered alone or in any possible combination of two or more: n is 1, R2 and R3 together and R4 and Rs together represent double bonds or R2, R3, R4 and Rs all represent hydrogen atoms

m is 2 or 3, two RIS represent 3', 4'-methylenedioxy and optionally a third R', representing 6'-methoxy, is also present R 6 represents a piperidino, 4-methylpiperidino, pyrrolidino or morpholino group or an alkylamino group having 4,5 or 6 atoms, preferably branched chain and especially an isobutylamino (2-methylpropylamino) group, a cycloalkylamino group having from 4 to 7 carbon atoms, especially a

cyclohexylamino group, or a 3, 4-methylenedioxy-substituted benzylamino or 2-phenethylamino group alternatively ruz is an alkoxy group having from 1 to 6 carbon atoms, preferably 3 to 6 the geometric configuration at the double bonds is as in piperine (all E, E) While the preferred meaning of RI is a 3', 4'-methylenedioxy group, RI may alternatively be provided by one, two or even three groups selected from hydroxy and alkoxy having from 1 to 3 carbon atoms, preferably methoxy, e. g. as in 3'-methoxy, 4'-methoxy, 6'methoxy and 3', 4'-dimethoxy substitution of the left-hand benzene ring.

Most preferably m is 2, two RIS represent 3', 4'-methylenedioxy and R6 represents an alkylamino group having 5 or 6 carbon atoms or a cycloalkylamino having from 4 to 7 carbon

atoms, especially a cyclohexylamino group, or R6 represents a 3, 4-methylenedioxy-substituted benzylamino or 2-phenethylamino group. The symbols n, R2, R3, R4, and R5 may have any of the permitted meanings, but preferably n = 1. Preferably the geometric configuration is all-E.

Specific preferred compounds of formula (1) for use in the invention are shown below. Compounds 1-14 fall within the exclusion clause above, but the substituents and variations to which they refer are illustrative: one or more such substituents or variations can be present in analogues and derivatives of piperine usable in this invention. Variations and alterations (illustratively. all other structural features of the molecule are as in piperine unless otherwise indicated. but see above) Variation in stereochemistry at double bonds and in extent of conjugation in chain

Variation in separation of rings (conjugated)

Alterations to nitrogen substituent

Alterations to the phenyl substituent

Alterations to connecting chain and amide group

Compounds of formula (1) and trivial names 1 (E, E)-Piperine 2 (Z, Z)-Chavicine 3 (Z, E)-Isopiperine 4 (E, Z)-Isochavicine 5 3, 4-dihydropiperine-Piperanine 6 1,2, 3,4-tetrahydropiperine Structures (all E) 7 n = 0 - Ilepcimide 1 n = I-Piperine 8 n = 2-Piperettine

Structures (all E, E) R= 1 piperidino-Piperine 9 pyrrolidino-Trichostachine 10 isobutylamino-Piperlonguminine 11 methoxy-Despiperidylmethoxypiperine 17 morpholino 18 hexylamino 19 3', 4'-methylenedioxybenzylamino Structures (all E, E)

1 3', 4'-methylenedioxyphenyl ; n = I-Piperine 12 As 1 + 6'-methoxy ; n = 1 ; -Wisanine 13 3'-hydroxy, 4'-methoxyphenyl ; n = 1 4'-Methoxyisocoumaperine 14 4'-hydroxyphenyl ; n = 1-Coumaperine

20 4'-methoxyphenyl ; n = 0 R'= 21 cyclohexylamino ; n = 1 22 cyclohexylamino ; n = 0

The piperine analogues or derivatives can be synthesised using methods known to a skilled person (see, for example, Chapman and Hall, Combined Chemical Dictionary on CD Rom, Release 1 : 1 (1997) and The Merck Index (1983), 10th edition. Publ. Merck and Co, Rahway, USA. PP. 1077-1078). Thus, they 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 or equivalent amount of plant extract.

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

Examples Plant samples and preparation of extracts Piper nigrum L. fruit (black pepper, Piperaceae), originally from India, was purchased from the Food Centre, 70 Turnpike Lane, London N8, UK. The rest of the herbs were either supplied by East-West Herbs, Kingham, Oxon, UK or by Cipla Ltd, Mumbai, India.

For the preliminary screening programme, the powdered dry herb (10 g) was heated to boiling in distilled water (100 ml) and allowed to boil for 10 min, using a hot plate as heat

source. The plant material was filtered off under vacuum through filter paper (Whatman), and the filtrate freeze-dried.

Cell culture experiments 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 C for 5-10 min) and inoculated with a repeater-pipettor (Finn pipette, Labsystems, Finland) into 96-well microtiter plates (Costar, Cambridge, MA, USA). The plates were incubated at 37 C in a 10% CO2, 90% air humidified atmosphere for the stated length of time.

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% w/v). The plate was placed at 4 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 RI 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 ul 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) Example 1 Optimisation of incubation conditions-FBS concentration and cell seeding density Prior to testing the herbal extracts, optimal culture conditions were established. The variable factors regarding incubation conditions include foetal bovine serum (FBS) concentration, initial cell seeding density and incubation period. To determine optimum FBS concentration, 1,2, and 5% FBS were used to culture the melan-a cell line, the growth pattern with each concentration of FBS was monitored by SRB assay. For the determination of

optimum cell seeding density, a series of initial seeding density of 0. 15 to 1. 2 x 104 cell per well of melan-a cells were plated into 96-well plates with 5% FBS and 20 nM tetradecanoyl phorbol acetate (TPA) supplemented growth medium. The growth pattern was monitored with SRB assay at daily intervals. The culture was extended to 8 days; on day 4, the medium in the remaining plates was replaced.

Results The effect of FBS concentrations on melan-a growth The optimal condition for the negative experimental control, is that cells neither grow too fast nor decline dramatically. Rapid growth might mask any subtle stimulatory effect brought about by the herbal extracts, whereas a dramatic decline in cell numbers indicates unfavourable culture conditions for cell survival, which could lead to cell damage. Figure 1 shows the growth curves of melan-a cell line at three different concentrations of FBS. Neither 1% nor 2% FBS supplemented medium was able to maintain cell survival; cell numbers declined significantly in 4 days of culture. However, 5% FBS was capable of keeping melan-a cell line alive with only a small increase in cell numbers observed over 4 days. TPA (20 nM) was able to cause further proliferation in the presence of 5% FBS indicating that cells were capable of responding to mitogenic stimuli at 5% FBS. Morphological observations under a microscope revealed that with 1% and 2% FBS supplemented medium, cell bodies were round, lightly pigmented with few dendritic processes and the culture displayed an ageing growth pattern. However in 5% FBS, cells possessed more melanosomes and some short dendrites without an ageing appearance. Therefore 5% FBS was used throughout in the herbal screening experiments.

Growth curves of melan-a cell line with various seeding densities In Figure 2, growth curves over 8 days with different initial cell numbers were plotted to elucidate the melan-a cell line's growth pattern in 96-well plates in the presence of 5% FBS and 20 nM TPA. The optimal initial plating density together with proper harvesting time was determined. All of the initial plating number of cells showed a net growth in the presence of TPA and 5% FBS supplemented medium, although the higher plating density of 1.2 x 104 cells/well depleted the growth medium on day 3 of culture and the cells ceased to grow until

the medium was replaced. With the lower plating densities (2-4 x 103 cells/well) the SRB assay OD readings remained relatively low after 8 days'culture. The initial plating density of 6 x 103 cells/well exhibited exponential growth, and after 4 days of culture, the OD reading increased to a value of about 0.4. Since the higher OD values are associated with greater precision and accuracy, it was determined that the initial inoculation of 6 x 103 cells/well was the optimum density for the herbal test experiment. For the simplicity of the experiment, harvesting time was day 4 since the cells at this stage was not confluent and after 4 days, growth medium tended to become depleted and replacement was necessary for the further growth.

Example 2 Preliminary herbal screening experiments Melan-a cells were seeded at a density of 6 x 103/1001/well in standard medium supplemented with 0 nM TPA and 5% FBS. After 4 hours of incubation, herbal extracts, which were reconstituted in growth medium and sterilised by filtration (pore size 0. 2 urn), of different concentrations was added into each well. Final concentrations of plant extract were 0 (negative control), 10,100 and 1000 Jlg dry extract per ml. 6 replicate wells were used for each concentration tested. The negative control (12 wells), positive control (20 nM TPA, 6 wells), and test wells were all in the same 96-well plate. The culture was terminated after 4 days and SRB assay performed according to the methods given above.

Results The effect of 30 herbal extracts on the proliferation of melan-a cell line Table 1 shows the results of the preliminary screening of 30 aqueous herbal extracts on the proliferation of melan-a cell line. Crude extracts of Astragales membranaceous (Fisch. ) Bunge, unripe Citrus reticulate Blanco, Dictamnus dasycarpus Turcz., Ophiopogon japonicus (Thunb.) Kergawe, Piper nigrum L. , Poria cocos (Schw. ) Wolf and Tribulus terestris L. were observed to stimulate melanocyte proliferation, sometimes even at the lowest dose level of 10 ug/ml. Other extracts either had no significant effect or were cytotoxic. Among these positive responses, that of Piper nigrum L. extract at 0. 01 and 0.1 mg/ml was the most pronounced.

Piper nigrum extract at these two concentrations not only strikingly enhanced cell growth, but

this extract also altered the cell morphology. In the presence of Piper nigrum extract, the cellular bodies were smaller, with more and longer bipolar or polydendritic processes, an effect similar to that observed with TPA.

Example 3

Repeats of the tests on Pi Repeats of the tests on Piper nigrum extract on the melan-a cells A newly prepared Piper nigrum fruit extract was tested on a new batch of melan-a cell line with the culture in microplates extended to 8 days. The effects of Piper nigrum extract on the growth of melan-a cell line were evaluated by SRB assay.

Results

Repeats of the tests of Piper nigrum extract on melan-a cells Rgpeats of the tests of Pi In the light of the positive results from the preliminary experiment, further investigations on Piper nigrum extract were carried out. Figure 3 shows that the result of the significant proliferant effect brought about by the Piper nigrum extract was even more marked on the extension of the incubation period to 8 days of culture, the growth was 272% of the control (cells only). Microscopically, the morphology of the cells was altered as those seen in the preliminary experiments.

Example 4 Confirmation of the proliferant effect of Piper nigrum by haemocytometer counting Melan-a cells were plated in petri dishes (035 mm, Nunclon, Denmark) with a plating density of 2 x 104/ml and Piper nigrum extract at concentrations of 0.01 and 0.1 mg/ml. A negative control (cells in medium only) and positive TPA (20 nM) control were also set up.

After 4 days the cells in each dish were harvested and counted with haemocytometer.

Results

Confirmation of the proliferant effect of Piver nigrum by haemocytometer counting ] per nizmm by haemoczometer countin SRB assay indirectly estimates cell number through protein staining and spectrophotometric measurement. To confirm if Piper nigrum extract stimulates melan-a cell proliferation, a direct cell counting with haemocytometer method was employed. Table 2

shows the cell numbers in the presence of Piper nigrum extract and 20 nM TPA. Cell number under the influence of Piper nigrum extract at 0. 01 and 0. 1 mg/ml were increased significantly compared to control, but less than that with 20 nM TPA. This result is consistent with the finding in 96-well microplate SRB assay.

Example 5 Effect of Piperine on the growth of melan-a cell line Piperine (Sigma-Aldrich Co. Ltd, Irvine, UK) was dissolved in MeOH, sterilised by filtration through a membrane (pore size 0.2 Jim) and diluted with standard growth medium. The final concentrations in culture were 0.1 and 1 JIM. A separate experiment (data not shown) showed that the concentration of MeOH present in these experiments was not toxic or proliferant to the cells.

Results The effect of piperine on the proliferation of melan-a cell line The effect of this compound on melan-a cell line is shown in Figure 4. Piperine at the two concentrations tested significantly stimulated melan-a proliferation. This compound brought about morphologic changes to melan-a cells, with smaller cell bodies, more and longer cellular dendrites, resembling those alterations induced by Piper nigrum extract and TPA. This indicates that piperine is an active principle responsible for the observed proliferant effect of Piper nigrum.

Example 6 Test of piperine on different cell types to determine its specificity In order to determine the specificity of piperine, a panel of different cell types were employed to facilitate this investigation. These included melan-a, melan-c, SVK14, CSM, XB2, SC1, B16F10, IM9, CACO2, Swiss 3T3 cell lines and normal human lymphocytes. TPA (20 nM) was also tested on these cells. Table 3 shows the biological origin of the cells and an outline of the cell culture protocols.

Results The effects of piperine and TPA on the growth of a panel of cell types.

From Table 4, it can be seen that piperine has a highly selective effect on the growth of a panel of cell types, since it only stimulates the mouse melanocytes (melan-a, melan-c), human melanoblasts (FM21E), human foetal melanocytes (FM 2 1 E) and the mouse fibroblast SC1 cell lines at the concentration tested. The SC1 cell line may have a particular sensitivity to TPA due to the way in which it has been derived, i. e. it has been cultured in the presence of TPA. However, piperine has either no effect or a cytotoxic effect on other cells. This result implies that piperine may have desirable specificity index for the proliferation of melanocytes in culture and is not a general mitogen. In our experimental system, TPA, a well known PKC activator and a tumour promoting agent, had similar effects to piperine on all cell types tested, except that TPA strikingly stimulated human lymphocyte and 3T3 fibroblast proliferation whereas piperine obviously lacked such an activity. Piperine seems to be a less potent stimulant than TPA.

Example 7 Mode of Action: effect of RO-31-8220 on the growth of melan-a cells with piperine and TPA Melan-a cell line cultured with piperine 1 uM and TPA 20 nM separately was set up in a 96-well plate, 1 J. of different concentrations of RO-31-8220 (Calbiochem-Novabiochem) in DMSO was introduced with a micro-syringe into the wells to make up the final RO-31-8220 concentrations of 0 (control), 0.1, 1,5, 10,100 nM, with final DMSO concentrations smaller than 0.01% v/v, at which the DMSO showed neither toxic nor proliferant effect to the cells in a separate experiment (data not shown). 6 replicate wells were used for each concentration. The culture was incubated for 4 days before it was terminated and processed with SRB assay to evaluate the growth of melan-a cells.

Results Mode of Action: effect of RO-31-8220 on the growth of melan-a cells with piperine and TPA Figure 5 shows the effect of RO-31-8220 on the survival and growth of melan-a cell line in the presence or absence of piperine and TPA. RO-31-8220 alone did not have significant cytotoxic effect to the cells at the concentrations up to 100 nM. However, the

proliferant effects of piperine, and TPA (as indicated by the Y axis values) on melan-a cells were effectively inhibited by the presence of RO-31-8220 at the concentrations of 0. 1-100 nM. It thus appears that piperine and TPA exert their proliferant effects through the activation of PKC cell signalling pathway.

The selectivity of piperine on the growth of a panel of cell types has also been tested. It was found that piperine possessed a fairly high specificity and selectivity towards melanocytes, since it significantly stimulated the growth of melan-a, melan-c and FM21E melanoblasts and FM21E melanocytes in culture, whereas it did not stimulate all other cells apart from a TPA-sensitive fibroblast cell line. Piperine was observed to have inhibitory effects on B16 mouse melanoma cell line which is syngeneic with melan-a cells. Thus piperine may be a specific stimulant for the proliferation of melanocytes in vitiliginous skin without the risk of stimulating melanoma cells.

Example 8 Experiments on human melanoblasts in culture Human melanoblasts in culture in this experiment were established from human foetal skin. Subconfluent melanoblasts maintained in MCDB 153 medium supplemented with 10% FBS, 10 ng/ml stem cell factor (SCF) and 1 nM endothelin 3 were subculture and inoculated into 96-well microplate with 6 x 103 cells/l00ul/well. After incubation in the 10% CO2, humidified atmosphere, at 370C for 3-4 hours to allow the attachment of the cells on the plate, piperine dissolved in MeOH and water was added into the wells. The final concentrations of piperine were 1,5, 10,100 uM, with TPA (20 nM) as positive control. Six replicates were used in each group of treatment, with 12 wells used for vehicle control. The incubation was conducted for 5 days before cells were harvested by fixing with cold trichloroacetic acid (TCA, at 4 C, final concentration 20% v/v), and evaluated for cell number using an SRB assay. One way ANOVA and Dunnett's t-test was employed to test the significance of any differences between treatment groups and vehicle control. Growth in the presence of piperine and TPA was expressed as % of control incubations containing no piperine or TPA. The experiments were repeated using melanoblasts from 3 different donors.

Results Figure 6 shows the effect of piperine on the growth of human melanoblasts in vitro.

Piperine at the concentrations of 1,10, 100 AM was found to cause significant stimulation to human melanoblasts in a dose response manner, with 34% more cell yield compared to vehicle control when the culture was exposed to 100 AM piperine in culture for 5 days. TPA, a wellknown melanocytic growth-stimulating agent, was also able to cause significant cell growth at tested concentrations, with over 50% of more cell yield observed when the culture was exposed to 20 nM for 5 days. In the other repeated experiments, piperine was consistently observed to induce significant cell growth at the concentrations ranging from 5-100 uM ; these stimulatory effects were generally less than that of TPA. Morphologically, in the presence of piperine, melanoblasts appeared to be more dendritic and the cell bodies were flatter and smaller.

Example 9 Experiments on human melanocytes in culture Human melanocytes used in this experiment were derived from induced differentiation of human foetal melanoblasts. The key character of human melanocytes that is different from its precursor melanoblasts is their ability to synthesise melanin. Melanin is a valid marker for melanocytes. The cell pellet of human melanocytes exhibits a characteristic brown to black colour, whereas human melanoblasts cannot produce melanin thus devoid of brown or black colour in the cell pellet.

Two protocols were employed for the experiments on human melanocytes in culture.

The first employed 24-well plates and evaluated cell number with SRB assay. The second employed petri dishes and cell number was counted with a haemocytometer chamber.

For the first protocol, subconfluent human melanocytes maintained in a 0100 mm petri dish were subculture into two 24-well plates (Falcon) using basic culture medium of RPMI 1640 supplemented with FBS (10%), bFGF (100 pM), CT (1 nM) and endothelin 1 (1 nM). The initial plating density was 20,000 cells/cm2 (38,200 cells/well) with each well containing 1000 l medium. After incubation in a 10% CO2, humidified atmosphere, at 370C for 2-3 hours to allow the attachment of the cells, piperine in 500 Al medium was added into wells to made up final concentrations of 0, 1,5, 10 and 100 AM. Cells only in the medium

with above supplement lacking of endothelin 1 were also set up as negative control. Six replicates were used in each group of treatment, and culture was incubated for 5 days before the cells were harvested by fixing with cold TCA (final concentration 20%) and processed with SRB assay. The solubilized SRB dye solution was transferred to a 96-well plate for optical density reading.

For the second protocol, subconfluent human melanocytes were subcultured in a 060 mm petri dishes (28 cm2, Falcon) with RPMI 1640 basic medium supplemented with FBS (10%), CT (1 nM), bFGF (100 pM) and endothelin 1 (1 nM). The initial plating density was 10, 000 cells/cm2, with 5 ml medium per dish. Cells were incubated for 2-3 hours in 10% CO2, humidified atmosphere, at 37oC, followed by addition of piperine solution in to the dishes, making the final concentrations of 0,1, 5,10 and 100 p. M. Cells in the above supplemented medium lacking endothelin 1 were also set up as a negative control. Three dishes were used for each treatment and the culture was maintained for 5 days before cells were harvested with trypsinisation and counted with a haemocytometer chamber. For melanin production experiment, the harvested cells were centrifuged and pelleted. After carefully removing the medium, NaOH (1 M) was used to solubilized the cell pellets and optical density read at 475 nm in a Perkin-Elmer UV spectrophotometer (model OVNIS Lambda 2). The melanin content was calculated by using a regression equation y = 0.005 + 0.005x corresponding to the calibration curve for synthetic melanin.

Results Figure 7 delineates the effects of piperine on the growth of human melanocytes cultured in 24-well plate. Piperine at the concentrations of 5 and 10 uM markedly stimulates the growth of these pigmented cells, with 36% more cells yielded when the culture was under

the influence of 10 uM piperine for 5 days. However, at 100 uM, piperine exerted inhibitory effect on the growth of these cells. In addition, in the presence of 1 nM endothelin 1, TPA at 20 nM was not able to stimulate cell growth in our culture system, a result that is of great difference with that observed in human melanoblasts.

Table 5 shows the effects of piperine on the growth of human melanocytes cultured in petri dishes. It is conspicuous that in the presence of ET1 (1nom), piperine at the concentrations of 5 and 10 u. M significantly stimulated the growth of human melanocytes,

with cell number over twice as many as that of ETl (lnM) control when this melanocyte cell type was exposed to 5 uM piperine for 5 days. This result was consistent with that obtained from the 24-well plate experiments, and it served to confirm that the stimulatory effects observed by SRB assay were indeed due to increased cell number rather than augmentation of protein production alone.

TABLES Table 1. Preliminary screening of 30 herbal aqueous extracts on the proliferation of melan-a cell line detected with SRB assay after 4 days culture.

Names of herbs Plant part Cell number (% of control) after 4 days incubation when grown in the presence of extract at : 1 mg ml''0. 1 mg ml''0. 01 mg ml'' plants with a significant stimulatory effect Astragalus membranaceous (Fisch.) Bunge Root 163. 2* 123. 6* 105. 6 Citrus reticulata Blanco (Qing Pi-unripe) Peel 16. 0 138. 5* 127. 6* Dictamnus dasycarpus Turcz. root bark 105. 0 159. 4* 98. 0 Ophiopogonjaponicus (Thunb.) Kergawe Root 127. 8* 126. 5* 108. 4 Piper nigrumZ. Fruit 11. 5 215. 4* 151. 3* Porta cocos (Schw.) Wolf (fungus) Sclerotium 79. 0 134. 6* 128. 8* Tribulus terrestris L. Fruit 80. 7 136. 1* 142. 2* plants with no significant stimulatory effect Angelica dahurica (Fisch.) Benth. & Hook. Root 50. 4 118. 3 107. 0 Chaenomeles lagenaria (Loisel.) Koldz. fruit 57. 1 74. 5 99. 0 Citrus reticulata Blanco (Chen Pi-ripe) Peel 34. 6 101. 1 81. 1 Corydalis bulbosa D. C. Root 91. 8 101. 2 92. 9 Curcuma longa L. Root 84. 1 104. 8 108. 3

Cyperus rotundus L. Rhizome 27. 5 52. 8 55. 8 Cornus ojftcinalis Sieb. et Zucc. Fruit 30. 4 92. 1 101. 6 Gentiana scabra Bunge Root 42. 2 107. 4 108. 6 Ligustrum lucidum Ait. Fruit 97. 6 58. 1 98. 4 Lithospermum erythrorhizon Sieb. et Zucco Root 43. 8 103. 8 111. 3 Notopterygium incisium Ting root/rhizome 18. 1 97. 4 94. 8 Paeonia lactiflora Pall. Root 31. 8 62. 2 100. 7 Paeonia suffruticosa Andr. Root 53. 2 72. 0 132. 8 Picrorhiza kurroa Royle ex. Benth Rhizome 42. 5 77. 5 90. 0 Platycodon grandiflorum (Jacq.) A. DC. Root 35. 1 94. 1 96. 8 Plumbago zeylanica L. Root 30. 2 103. 9 114. 1 Polygala tenuifolia Willd. Root 12. 7 43. 7 79. 6 Ramulus mari (insect) Whole 41. 1 87. 1 89. 4 Siesgesbeckia pubescens Makino Herb 17. 0 40. 8 51. 7 Spirodela polyrrhiza (L.) Scheid Herb 100 79. 3 96. 6 Trichosanthes kirilowii Maxim Root 112. 9 108. 1 116. 1 Tripterygium wilfordii Hook. Root 89. 8 36. 7 63. 3 Zingiber officinale Roscoe Rhizome 7. 9 105. 8 90. 6 *P < 0. 01 compared with vehicle treatment (one-way ANOVA, followed by Dunnett's t-test).

Table 2. Effects of Piper nigrum extract on the proliferation of melan-a cells counted with haemocytometer Treatment to cells cell number (x 104/mil) Control 2. 02 20 nM TPA 5. 0* Piper nigrum at 0. 01 mg/ml 3. 06* Piper nigrum at 0. 1 mg/ml 3. 13* *P < 0. 01 compared with vehicle treatment (one-way ANOVA, allowed by Dunnett's t-test).

Table 3. Biological origin and the culture conditions of a panel of different cell types used in selectivity experiment.

Cell name biological origin optimum culture conditions FBS Medium incubation (4days) Melan-a normal epidermal melanoblasts from embryos of inbred 5% RPMI1640 3 7oC, 10% CO2 C57BL mice Melan-c albino embryos of outbred LAC-MF strain mice 10% RPMI1640 37oC, 10% C02 FM21E human foetal melanoblasts from epidermis (strain 21) 10% MCDB153 37oC, 10% CO2 melanoblast FM21E Human melanocytes derived from FM21E melanoblasts RPMI1640 37OC, 10% CO2 melanocyte SVK14 human keratinocytes 10% DMEM 37oC, 10% CO2 CSM14. 1. 4 neuronal cells from mesencephalin of rat 10% DMEM 34oC, 5% CO2 SCI Fibroblastoids from neonatal murine skin 10% DMEM 3 7oC, 10% CO2 XB2 murine keratinocytes 10% DMEM 37oC, 10% CO2

B6F10 mouse melanoma 5% RPMI1640 370C, 10% CO2 CACAO2 human colon cancer 10% RPMI1640 37oC, 10% CO2 IM9 human lymphoblastoid B cells 10% RPMI1640 37oC, 5% CO2 Swiss 3T3 mouse fibroblasts 10% DMDM 370C, 5% CO2 Human healthy human blood samples 10% DMEM 37oC, 5% CO2 lymphocytes

Table 4 Effects of piperine and TPA on the growth of a panel of cell types. (see Table 3 for details of cells) cell number as a % of control Cell type piperine at the concentration of (uM) TPA at the concentration of 0. 01 0. 1 1 10 100 20nM 200nM Melan-a ND 130* 169* 153* ND 295* ND Melan-c 109 208* 198* 119* 137* 186* 222* FM21E Melanoblast ND 101 101 119* 134* 153* ND FM21E human melanocytes ND ND 98 143* 75* 98 102 SVK14 97 101 92 84* 23* 71* 66* CSM14. 1. 4 93 94 95 89 64* 85* 76* SCI 191* 178* 175* 204* 190* 178* 199* XB2 80 90 86 90 42* 96 99 B16F10 71* 64* 47* 33* 0* 35* 55* CACAO2 103 99 102 81* 34* 95 90 IM9 ND 101 103 69*'ND ND ND Swiss 3T3 113 104 106 102 51* 185* 207* Human Lymphocytes ND 93 93 ND ND 282* ND * P < 0. 05 compared to treatment with vehicle alone (one-way ANOVA followed by Dunnett's t-test) ; ND = not done.

Relative standard deviations of all values were less than 10% of the mean

Table 5 : Effects of piperine on the proliferation and melanin production of human melanocytes cultured in petri dishes Treatments Cell no. (x 10-4) SD % of control OD reading : t SD Melanin after cultured for 5 (ETIlnM) at 475nm content/10 cells days Cells only 17. 71i6. 16* 49. 1 0. 02610. 0049*0. 23 : 0. 001 g ET1 (lNm) 36. 04 6. 16 100. 0 0. 08710. 044 0. 46 : 0. 01 zig ET (lnM) + 60. 83il6. 78 168. 8 0. 134 0. 014' 0. 42 : 0. 03 g piperine (I M) ET1 (lnM) + 78. 96 5. 63* 219. 1 0. 137 0. 0085* 0. 334 0. 01 g piperine (5 uM) ET1 (lnM) + 64. 79i13. 47* 179. 8 0. 139 0. 028* 0. 41 0. 07 fg Piperine (10 jM) ET1 (lnM) + 61. 04il0. 04 169. 4 0. 144 0. 00464 0. 46 0. 001 ig piperine (100 M) *, * P < 0. 05 when compared to ET1 (1 nM) control (one way ANOVA, followed by Dunnett's t-test).

Example 10 Derivatives of Piperine 1.0 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 of melanocytes which is often familial with lack of established aetiology.

Various piperine derivatives of formula (1) were synthesised and tested for melanocyte (mouse melan-a) proliferant activity in-vitro. Cells were incubated with the test compound for 4 days, as previously for mouse cells but with 10% FBS, after which the sulphorhodamine-B (SRB) assay was performed to determine cell number SRB uptake was measured as optical density at 550nm. The 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.

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 1.1). 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 piperine analogues Analogues of piperine were synthesised using methods described in the literature, adapted from the literature or devised in the inventors'laboratory. Structures of 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.

1.5 Results Table 6 presents an overall summary of the results appearing in detail in other Tables which follow. Tables 7-12 relate to results at a single concentration of test compound (lOuM). They are 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 lOuM but more active at 50uM. This is illustrated for one compound (RV-A01) in Figure 8 of the Drawings.

Table 6 : Overall Summary of Results

Test Change with Respect to Active at Active at More active CPD. Piperine lOM ? higher cone. than piperine at (gm) ? higher conc.

(uM) ? Vary amide (amino group listed below) RV-A01 Pyrrolidino Yes 25, 50 50 RV-A02 Morpholino Yes 25, 50 50 RV-A04 3, 4-methylenedioxy- 50 0 benzylamino RV-A05 Hexylamino Yes 25, 50 No RV-A06 Isobutylamino Yes 25 No RV-A07 Methylamino Yes No No RV-A08 Ethylamino Yes No No RV-A09 Isopropylamino Yes No No RV-A10 Cyclohexylamino Yes 50 50 RV-A11 Butylamino Yes 50 50 Shorten connecting chain by 2 C-atoms (1 double bond) RV-B01 I Piperidino I Yes 25, 50 50 Shorten connecting chain by 2 C-atoms (1 double bond) and vary amino part of amide group RV-B02 Pyrrolidino Nob Not doned Not done RV-B03 Morpholino No Not doned Not done Replace amide by ester (alkyl group listed below) RV-AB1 Methyl Yes 50, 100 50, 100 RV-AB2 Ethyl No"Not done Not done RV-AB4 Isopropyl Yes 50 50 RV-AB5 Propyl Yes 50 50, 1 oye RV-AB6 Butyl Yes 50, 100 50, 100 Shorten connecting chain as above and replace amide by ester (alkyl group listed below) RV-BB1 I Methyl I Nob I Not done I Not done Reduce double bonds in connecting chain, making it saturated RV-C02 Yes 25, 50 No Reduce double bonds in connecting chain and shorten it by 2 C-atoms RV-C03 I I Nob I Not done I Not done Replace 3', 4'-methylenedioxy by methoxy and shorten connecting chain by 2 C-atoms (1 double bond)

Test Change with Respect to Active at Active at More active CPD. Piperine lOM ? higher cone. than piperine at (p. M) ? higher cone.

(UM) ? RV-G01 6'-MeO No lODe No RV-G02 3'-MeO No love No RV-G03 4'-MeO No 100 No RV-G04 3', 4'-Di-MeO No 100 No

Footnotes a. In dose response test. b. But the cells displayed weak dendricity suggestive of activity. c. This result is considered anomalous. It is intended to re-run the experiment. d. Expected to show activity at 50aM. e. Not corroborated by the t-test at I OOPLM.

Table 7 Variation on Nitrogen Substituent of Piperine Effect on melan-a cells at M concentration Code No Structure Percentage cell growth Stimulant Relative Dendricity (Repeated experiments) activity activity to Test cpd. Piperine piperine 0 RV-A01 OA 18334** 18050 1. 03 RV-AO1 Positive +++ 202 ""f) 20284* 19163* osmve +++ 0 &num;.

0 ! t 156d : 58 21065** 0. 5 RV-A02 (Nî 18740** 17022 Positive 1. 02 +++ zu o 15319** 15519** 0. 9 Non0 significant P 14947 17039** here, but 0. 7 RV-A04 -ifY-Nro. 11927 16929** positive in 0. 27 + b-''0'14722 17328** dose 0. 66 response test 0 RV-A05 o - Positive 0. +++ KVAU (JV"14017* 16929** smve +++ 0, 9

J) 14766 17039** 1. 69 RV-A06 OrT 15824** 16929** Positive 0. 83 +++ \ JH J ! 15640** 15518** 1. 0 0 0 RV-A07 y"NH 17024* 21633* Positive 0. 6 ++ 0 0 RV-A08 ---fNpr 20014 23617 Positive 0. 73 +++ < 0 RV-A09 YNH 22419 26316** Positive 0. 76 +++ < os RV-A10 0 ; 30829** 30217** Positive 1. 02 +++ o-'L' ODD

0 RV-All oYN) 26421** 34714** Positive 0. 66 +++ 0 &num; *P < O. 05, **P < 0. 01 compared to vehicle treatment (Dunnett's t test) +++ highly dendritic, ++ moderately dendritic, + weakly dendritic,-no effect

Table 8 Variation in connecting chain length and amide Effect on melan-a cells at 11M concentration group Code NO Structure Percentage cell growth Stimulant Relative Dendricity (Repeated experiments) activity activity to e Test Piperine piperine 0 J) 17133** 18050** 0. 88 RV-BOI --f {N. 14820 19163** Positive 0. 52 ++ \ Ji J LJ 15222** 15518** 0. 97 0 ! t 14014 18050** 0. 2 RV-B02/, "N\ 15433 19163** , 0. 59 + R 0 &num; 0 ocm 0. . '\., \. 10312 210=L65** 0. 02 RV-B03/Yl 7 1 117 17022- 0. 22 \) 0 OD **P < 0. 01 compared to vehicle treatment (Dunnett's t test) ++ moderately dendritic, + weakly dendritic,-no effect

Table 9 Repl cement of amide by ester group Effect on melan-a cells at tgt concentration Code Structure Percentage cell growth Stimulant Relative Dendricity (Repeated experiments) activity activity to Test Piperine piperine 0 j ! 16338 21065** 0. 57 RV-ABI 0 0 14118* 17022** Positive 0. 59 ++ \ J J 1517* 15518** 0. 93 0 0 RV-AB2 0 299 171+39** Positive-I Toxic < 22 : : 1 : : 0. 4 171 : : 1 : : 39**-1. 09 0 &num; RV-AB4 . "0" 22412** 25515** Positive 0. 8 ++ 0 0 RV-AB5 16635** 16929** Positive 0. 95 ++ 0 &num;

0 RV-AB6 o 14818** 18111** Positive 0. 59 + 0 &num; *P < 0. 05, **P < 0. 01 compared to vehicle treatment (Dunnett's t test) +++ highly dendritic, ++ moderately dendritic, + weakly dendritic,-no effect

Table 10 Replacement of amide by ester group and Effect on melan-a cells at uM concentration variation in connecting chain length Code N'Structure Percentage cell growth Stimulant Relative Dendricity (Repeated experiments) activity activity to Test Piperine piperine 0 J ! 14927 21065** 0. 44 RV-BB1/j""0 12915 17022** 0. 41 + < J. j 12112 15518-gmiicant **P < 0. 01 compared to vehicle treatment (Dunnett's t test), ++moderately dendriticy

Table 11 Reduction of double bonds in connecting chain Effect on melan-a cells at uM concentration and variation in chain length Code NO Structure Percentage cell growth Stimulant Relative Dendricity (Repeated experiments) activity activity to Test Piperine piperine 0 RV-C02 0 No 169 : L29** 18050** Positive 0. 8...

19589** l9l : L63** 1. 04 0 0 Dvrm 0. , 1045 1717** 0. 056.

Yl ?) 12 1717 '0. 18 \) '' **P < 0. 01 compared to vehicle treatment (Dunnett's t test) +++ highly dendritic, + weakly dendritic,-no effect

Table 12 Variation in the phenyl substituent and Effect on melan-a cells at uM concentration connecting chain length Code NO Structure Percentage cell growth Stimulant Relative Dendricity (Repeated experiments) activity activity to Test Piperine piperine 0 RV-G01 ff 1058 20229** None 0. 04 0 RV-G02 119 18 17139** Negative 0. 26 RV-G02 YY g , Negative 0 0 rf 17139** Non-0. 29 H ) j 8* 171d=7** significant 0. 30 I I

I 0 RV-G04 J) J 1009 22411** None 0 I *P < 0. 05, **P < O. Ol compared to vehicle treatment (Dunnett's t test)-no effect I I I I *P < 0. 05, **P < 0. 01 compared to vehicle treatment (Dunnett's t test)-no effect

Dose response experiments

Code ? Structure 0 RV-A01 -. Y 0 &num;

Compounds 111M 10 M 25 M 50 M Tested Piperine 151~7**# 202~12**# 171~15**# 142~9** RV-A01 109~7 122~7 142~21** 186~14**#

** P0. 01 Compared to vehicle treatment (Dunnet's t test) * P < 0. 05 Compared to vehicle treatment (Dunnet's t test) + Piperine is significantly more active than test compound P < 0. 05 D Test compound is significantly more active than Piperine P < 0. 05

Code NO Structure 0 RV-A02/-'---N" \- kO < Ili 0 1

Compounds 1 M 10 M 25 M 50 M Tested Piperine 147~11**# 192~13**# 167~19** 142~15** RV-A02 125~10 167~17** 171~8** 168~12**#

** P < 0. 01 Compared to vehicle treatment (Dunnet's t test) + Piperine is significantly more active than test compound P < 0. 05 0 Test compound is significantly more active than Piperine P < 0. 05

Code ? Structure 0 RV-A04. o-YYNH 0 &num;'&num;'0 0

Compounds 1 M 10 M 50 M 100 M Tested Piperine 12011 178~11**# 116~13 92~9 RV-A04 10112 138~10** 150~15**# 71~9 Dentricity-+ + RV-A04

** P < 0. 01 Compared to vehicle treatment (Dunnet's t test) * P < 0. 05 Compared to vehicle treatment (Dunnet's t test) + Piperine is significantly more active than test compound P < 0. 05 D Test compound is significantly more active than Piperine P < 0. 05 - no effect, + weakly dendritic, ++ moderately dendritic

Code NO Structure 0 0 N NH RV-A05 yNH0 : 1 ( : 11) 0

Compounds 1 M 10 M 25 50 M Tested Piperine 173~6**# 230~13**# 188~19** 182~15** RV-A05 155~9** 18~13** 178~18** 174~8**

** P0. 01 Compared to vehicle treatment (Dunnet's t test) . Piperine is significantly more active than test compound P < 0. 05

Code ? Structure 0 RV-A06 --YY-0 &num;

Compounds 1 M 10 M 25 M 50 M Tested Piperine 147~8# 195~22** 173~17* 159~14 RV-A06 1347 188~14* 172~15* 135~24 ** P < 0. 01 Compared to vehicle treatment (Dunnet's t test) * P < 0. 05 Compared to vehicle treatment (Dunnet's t test) + Piperine is significantly more active than test compound P < 0.05

Code NO Structure 0 RV-A07 -YyNt-r 00

Compounds 1 M 10 M 50 M 100 M Tested Piperine 211~16**# 216~33** 52~15 16~3 RV-A07 140~12** 170~24** 71~5 46~2 Dentricity ++ ++ + + ofRV-A07 ** P0. 01 Compared to vehicle treatment (Dunnet's t test) + Piperine is significantly more active than test compound P < 0.05 ++ moderately dendritic, + weakly dendritic

Code ? Structure 0 RV-A08 "Nhf . 11 D0

Compounds 1 M 10 M 50 M 100 M Tested Piperine 216~14**# 236~17** 61~11 32~5 RV-A08 139~27** 200~14** 81~12 62~13 Dendricity ++ +++i + + of RV-A08 ** 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

Code ? Structure 0 RV-A09/---NH < I ODC)

Compounds 1 M 10 M 50 M 100 M Tested Piperine 221~17**# 263~16** 77~12 24~2 RV-A09 187~15** 224~19** 85~5 42~6 Dendricity +++ +++ + + of RV-A09

** P0. 01 Compared to vehicle treatment (Dunnet's t test) . Piperine is significantly more active than test compound P < 0. 05 +++ highly dendritic, + weakly dendritic

Code ? Structure rT RV-A10 o, 0..-7

Compounds 1 M 10 M 50 M 100 M Tested Piperine 236~30** 302~17** 78~11 21~4 RV-A10 301~20**# 308~29** 155~22**# 100~13 Dendricity +++ +++ ++ + of RV-A10 ** P < 0. 01 Compared to vehicle treatment (Dunnet's t test) 0 Compound is significantly more active than piperine P < 0. 05 +++ highly dendritic, ++ moderately dendritic, + weakly dendritic

Code ? Structure 0 0 14, Zz 14ZZZ RV-AL I < I- NH \)

Compounds 1 M 10 M 50 M 100 M Tested Piperine 251~19** 347~14**# 61~7 25~2 RV-A11 189~6** 264~21** 158~20**# 84~6 Dendricity +++ +++ ++ + of RV-A11 ** P < 0. 01 Compared to vehicle treatment (Dunnet's t test) . Piperine is significantly more active than test compound P < 0. 05 0 Compound is significantly more active than Piperine P < 0. 05

Code NO Structure 0 RV-B01 0 *-) -1 4 4 NC) ODC"

Compounds I 10 M 25 M 50 M Tested Piperine 144~27**# 190~7** 172~11** 153~10** RV-B01 1116 147~7** 187~18** 187~8**# ** P < 0. 01 Compared to vehicle treatment (Dunnet's t test) + Piperine is significantly more active than test compound P < 0.05 0 Compound is significantly more active than Piperine P < 0. 05

Code ? Structure 0 RV-ABI < < 0

Compounds 1 M 10 M 50 M 100 M Tested Piperine 133~31** 177~14**# 139~16* 95~24 RV-AB1 125~13 147~16** 187~12**# 171~8**# Dendricity-+ ++ ++ of RV-AB1 ** P < 0. 01 Compared to vehicle treatment (Dunnet's t test) * P < 0. 05 compared to vehicle treatment (Dunnett's t test) + Piperine is significantly more active than test compound P0. 05 0 Compound is significantly more active than Piperine P < 0. 05 - no effect, + weakly dendritic, ++ moderately dendritic

Code ? Structure 0 RV-AB4 < OO 0-L

Compounds Igm 10 M 50 M 100 M Tested Piperine 223~18**# 255~15** 60~16 24~6 RV-AB4 175~6** 224~12** 148~19**# 90~7 Dendricity ++ ++ ++ + of RV-AB4 ** P < 0. 01 Compared to vehicle treatment (Dunnet's t test) # Piperine is significantly more active than test compound P < 0.05 # Compound is significantly more active than Piperine P < 0. 05 ++ moderately dendritic, + weakly dendritic

Code NO Structure 0 RV-AB5 < 0 o'' I I I

Compounds 1 M 10 M 50 M 100 M Tested Piperine 141~26**# 220~29**# 45~12 23~4 RV-AB5 120~21 151~19** 163~8**# 123~8 Dendricity ++ + ofRV-AB5

** P < 0. 01 Compared to vehicle treatment (Dunnet's t test) + Piperine is significantly more active than test compound P < 0. 05 0 Compound is significantly more active than Piperine P < 0. 05 ++ moderately dendritic, + weakly dendritic

Code NO Structure 0 RV-AB6 to 'o-'

Compounds 1 M 10 M 50 M 100 M Tested Piperine l13 181~11** 43~6 23~6 RV-AB6 1035 148~18** 190~11**# 128~17**# Dendricity-+ ++ + of RV-AB6 ** P < 0. 01 Compared to vehicle treatment (Dunnet's t test) 0 Compound is significantly more active than Piperine P < 0. 05 ++ moderately dendritic, + weakly dendritic, -no effect

Code Structure 0 RV-C02 No < I 0

Compounds 1 M 10 M 25 M 50 M Tested Piperine 158~10**# 203~11** 188~12** 164~6** RV-C02 134~15** 183~33** 199~31** 175~12**

** P < 0. 01 Compared to vehicle treatment (Dunnet's t test) . Piperine is significantly more active than test compound P0. 05

Code ? Structure 0 RV-G01 fT < "N I cnl-l -1 o

Compounds 1 M 10 M 50 M 100 M Tested Piperine 161~23** 202~29** 61~5 40~7 RV-G01@ 99~8 105~8 103~6 119~9 Dendricity of RV-G01

** P < 0. 01 Compared to vehicle treatment (Dunnet's t test) - no effect

Code NO Structure 0 RV-G02 YN 10

Compounds 1 M 10 M 50 M 100 M Tested Piperine 151~17** 201~15** 57~15 39~11 RV-G02 99 95~18 110~11 127~9 Dendricity of RV-G02

** P < 0. 01 Compared to vehicle treatment (Dunnet's t test) - no effect

Code ? Structure 0 RV-G03 1 NC) oio I t

Compounds 1 M 10 M 50 M 100 M Tested Piperine 163~9** 181~23** 59~11 40~12 RV-G03 90~10 108~20 111~10 133~15** Dendricity of RV-G03

** P < 0. 01 Compared to vehicle treatment (Dunnet's t test) - no effect

Code ? Structure I 0 Nit RV-G04 jj J) J I I

Compounds 1 M 10 M 50 M 100 M Tested Piperine 179~12** 224~11** 92~19 35~4 RV-G04 95~11 100~9 114~8 123~7* Dendricity ofRV-G04 ** P < 0. 01 Compared to vehicle treatment (Dunnet's t test) - no effect

2. Synthesis of amide derivatives of piperinic acid piperinic aci 2. 1 Preparation of piperinic acid (RV-AOO) To piperine (1) (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, 60% yield). m. p. 206o-208oC (Lit m. p. 217 -218C)'

Piperine (1) Piperinic acid 2.2 Synthesis of piperlonguminine (RV-A06)

A mixture of piperinic acid (350mg, 0. 0016mole, leq) and triethylamine (0. 4ml, 0. 0032mole, 2eq) in dichloromethane (50ml) was stirred for 15min at OOC. To this mixture methanesulfonyl chloride (0. 18ml, 0. 0024mole, 1.5eq) was added and stirred for further 30 min at DoC. Isobutylamine (0. 23ml, 0. 0024mole, 1.5eq) was added to the mixture and stirred for Ih at OOC and 2h at room temperature. Dichloromethane (50ml) was added to the mixture which was then washed with 5% HCl (3xlOOml), saturated aqueous NaHCO3 (3xlOOml) and water (3xlOOml). The organic fraction was dried over anhydrous sodium sulphate, filtered and rotary evaporated to yield a yellowish solid residue. Recrystallisation from methanol yielded colourless needles of piperlonguminine (120mg, 32% yield) The reaction is presumed to proceed through a mesylate ester intermediate.

Piperlonsuminine (RV-AO6)

'H-NMR (CDCIs) : 5. 96 (d, 1H, J=14. 8, CH=CH-CH=CH), 7. 36 (d, d, 1H, J=10. 5, 14. 8, CH=CH-CH=CH), 6. 66 (d, d, 1H, J=15. 4, 10. 5, CH=CH-CH=CH), 6. 76 (d, 1H, J=15. 4 CH=CH-CH=CH), 6.96 (d, lH J=1.6, Ar-7H), 6.76 (d, lH J=8.0, Ar-lOH), 6.87 (d, d, 1H J=1.6, 8.0 Ar-llH), 5.97 (s, 2H, O-CHrO), 3.18 (t, 2H, J=6.5 CH2-CH), 1.83 (m, 1H, J=6. 5 CH2-CH), 0.94 (d, 6H, J=6.5, (CH3) 2), 5.82 (t, 1H, NH J=5.3) 13C-NMR (CDCl3): 20.4 (CH3), 29.4 (CH), 47.3 (CH2), 102.2 (CH2), 106.2 (CH), 109.1 (CH), 123.3 (CH), 125.5 (CH), 126.0 (CH), 132.0 (C), 138.0 (CH), 140.4 (CH), 148.9 (C), 149.2 (C), 166.2 (C) MS m/z (%): 273 #(M+ 98), 216 (20), 201 (100), 174 (25), 173 (65), 172 (23), 171 (17) 143 (20), 115 (40), 96 (11).

IR (KBr) : Vmax (carbonyl group) 1644 m. p. 161. 2 -161. 7 C (Lit m. p. 156 -1600C)' 2.3 Synthesis of other amide derivatives of nioerinic acid The general method was as for piperlonguminine (section 1.2), using the same proportions of reactive amine, triethylamine and methanesulfonyl chloride relative to piperinic acid (200 or 300 mg., leq). Recrystallisation from ethyl acetate/petroleum spirit yielded the other amide derivatives of piperinic acid.

5-E, E-oiperinoylpyrrolidine (RV-A01)

IH-NMR (CDC13) 8 : 6. 26 (d, 1H, J=14. 7, CH=CH-CH=CH), 7. 43 (d, d, 1H, J=9. 5, 14. 7, CH=CH-CH=CH), 6. 73 (d, d, 1H, J=15. 3, 9. 5, CH=CH-CH=CH), 6. 78 (d, IH, J=15. 3 CH=CH-CH=CH), 6. 98 (d, IH J=1. 6, Ar-7-H), 6. 77 (d, lH J=8. 0, Ar-10-H), 6. 89 (d, d, IH J=1. 6,8. 0 Ar-11-H), 5.97 (s, 2H, O-CH2-O), 3.57 (t, 2H, J=4. 0 N-CH2 (pyrrolidine)) 3.54 (t, 2H, J=4. 0 N-CH2 (pyrrolidine) 1.90 (m, 2H, CH2- CH2(pyrrolidine)) 1.87 (m, 2H, CH2-CH2(pyrrolidine)) 13C-NMR (CDCI3) : 24.3 (CH2), 26.1 (CH2), 45.9 (CH2), 46.4 (CH2), 101.2 (CH2), 105.7 (CH), 108.4 (CH), 121.4 (CH), 122.5 (CH), 125.2 (CH), 130.9 (C), 138.7 (CH), 141.7 (CH), 148.1 (C), 148.2 (C), 164.9 (C) MS m/z (%): 271 (M'78), 201 (100), 173 (30), 172 (15), 171 (13) 143 (13), 115 (27) IR (KBr): Via,, (carbonyl group) 1637

m. p. 142. 9'- 1430C (Lit m. p. 142"-143''C) yield 49. 2% 5-E, E-piperinoyl morpholine (RV-A02)

IH-NMR (CDCI3) : 6. 37 (d, 1H, J=14. 6, CH=CH-CH=CH), 7. 45 (d, d, 1H, J=10. 2, 14. 6, CH=CH-CH=CH), 6. 72 (d, d, 1H, J=15. 5, 10. 2, CH=CH-CH=CH), 6. 79 (d, 1H, J=15. 5 CH=CH-CH=CH), 6. 98 (d, 1H J=1. 5, Ar-7-H), 6. 80 (d, 1H J=8. 0, Ar-10-H), 6. 89 (d, d, 1H J=1.5, 8.0 Ar-11-H), 5.98 (s, 2H, O-CH2-0), 3.70 (t, 2H, J=4. 0 CH2-N-CH2 (morpholine)) 3.60 (t, 2H, J=4. 0 CH2-O-CH2 (morpholine))

13C-NMR (CDC13) : 42. 3 (CH2), 46. 1 (CH2), 66 (CH2), 66 (CH2), 101. 3 (CH2), 106. 5 (CH), 108. 5 (CH), 118. 7 (CH), 122. 7 (CH), 124. 9 (CH), 130. 8 (C), 139. 1 (CH), 143. 4 (CH), 148.2 (C), 148.3 (C), 165.6 (C) MS m/z (%): 287 (Ms 57), 201 (100), 173 (25), 171 (10) 143 (10), 115 (30) IR (KBr): vmax (carbonyl group) 1641

m. p. 161. 8o-162. 50C (Lit m. p. 167-168"C) 3, yield 44. 1% 5-E. E-pinerinoylpiperino ! vmine (RV-A04)

'H-NMR (CDC13) 5 : 5.98 (d, 1H, J=14. 9, CH=CH-CH=CH), 7.34 (d, d, 1H, J=10. 7,14. 9, CH=CH-CH=CH), 6.73 (d, d, 1H, J=15. 5,10. 7, CH=CH-CH=CH), 6.79 (d, 1H, J=15. 5 CH=CH-CH=CH), 6.98 (d, 2H J=1. 5, Ar-7,3'-H), 6.78 (d, 2H J=8.0, Ar-10, 6'-H), 6.89 (d, d, 2H J=1. 6,8. 0 Ar-ll, 7'-H), 5.98 (s, 2H, O-CH2-O), 5.93 (s, 2H, O-CHz-O), 4.40 (d, 2H, CH2) 3.57 (br, 1H, NH) 'C-NMR (CDC13) : 43.4 (CH2), 101.1 (CH2), 101.4 (CH2), 105.8 (CH), 108. 3 (CH) 108.5 (CH), 108.6 (CH), 121.2 (CH), 122.8 (CH), 124.7 (CH), 130.9 (C), 132.2 (C) 139.9 (CH), 141.6 (CH), 147.0 (C) 147.9 (C) 148.3 (C), 148.4 (C), 166.9 (C) MS m/z (%): 351 (mu81), 216 (15), 203 (12), 202 (53) 201 (29), 174 (31), 173 (22), 150 (23) 144 (11), 143 (10), 135 (100), 116 (12) 115 (29) m. p. 190. 5 -191. 7 C, yield 50. 1% 5-E,E-piperinoylhexylamine RV-A05

'H-NMR (CDCl3) : 5. 90 (d, IH, J=14. 8, CH=CH-CH=CH), 7. 35 (d, d, 1H, J-10. 6, 14. 8, CH=CH-CH=CH), 6. 66 (d, d, 1H, J=15. 4, 10. 6, CH=CH-CH=CH), 6. 76 (d, IH, J=15. 4 CH=CH-CH=CH), 6. 97 (d, lH J=1. 4, Ar-7H), 6. 77 (d, IH J=8. 0, Ar-lOH), 6. 88 (d, d, IH J=1. 5, 8. 0 Ar-llH), 5. 97 (s, 2H, O-CHrO), 3. 34 (q, 2H, CH2-CH2-CH2-CH2-CH2) 1. 54 (m, 2H, CH2-CH2-CH2-CH2-CH2) 1. 32 (m, 6H, CH2-CH2-CH2-CH2-CH2) 0.88 (t, 3H, CH3), 5.54 (br, NH) 13C-NMR (CDCl3) : 14.3 (CH3), 22.5 (CH2), 26.6 (CH2), 29.6 (CH2), 31.5 (CH2), 39.7 (CH2), 101. 3 (CH2), 105. 7 (CH), 108. 5 (CH), 122.5 (CH), 123.2 (CH), 124.6 (CH), 130.8 (C), 138.7 (CH), 140.9 (CH) 148.2 (C), 148.2 (C), 166.0 (C) MS m/z (%): 301 (M+94), 202 (18) 201 (73), 174 (40), 173 (100), 172 (31), 171 (15) 143 (24), 115 (63) IR (KBr): Vmax (carbonyl group) 1641 m. p. 149. So-149. 8 C (Lit m. p. 139'-141 OC) 4, yield 50. 1% 5-E, E-piperinovlmethylamine (RV-A07)

'H-NMR (CDC13) 8 : 5. 91 (d, 1H, J=14. 8, CH=CH-CH=CH), 7. 36 (d, d, 1H, J=10. 7, 14. 8, CH=CH-CH=CH), 6. 66 (d, d, 1H, J=15. 4, 10. 6, CH=CH-CH=CH), 6. 77 (d, 1H, J=15. 4 CH=CH-CH=CH), 6. 97 (d, IH J=1. 5, Ar-7H), 6. 77 (d, IH J=8. 0, Ar-lOH), 6. 88 (d, d, IH J=1. 6, 8. 0 Ar-llH), 5. 97 (s, 2H, O-CH2-O), 2. 91 (t, 3H, CH3), 5. 61 (br, NH) 13C-NMR (CDC13) : 26.9 (CH3), 101.7 (CH2), 106.1 (CH), 108.9 (CH), 123.0 (CH), 123.3 (CH), 125.0 (CH), 131.2 (C), 139.2 (CH), 141.4 (CH), 148.6 (C), 148.6 (C), 167.2 (C) MS m/z (%) : 231(M+89), 201 (42), 173 (67), 172 (32), 171 (17), 143 (27), 116 (21) 115 (100), 89 (12) m. p. 181. 10-182. 4 C (Lit m. p. 186oc, yield 48.2%

5-E. E-piperinovlethv ! aminefRV-A08)

IH-NMR (CD30D) : 6. 14 (d, IH, J=15. 0, CH=CH-CH=CH), 7. 37 (d, d, IH, J=10. 2, 15. 0, CH=CH-CH=CH), 6. 93 (d, d, 1H, J=15. 7, 10. 6, CH=CH-CH=CH), 6. 87 (d, IH, J=15. 7 CH=CH-CH=CH), 6. 97 (d, IH J=1. 5, Ar-7H), 6. 77 (d, lH J=8. 0, Ar-lOH), 6. 88 (d, d, 1H J=1. 6, 8. 0 Ar-llH), 5. 97 (s, 2H, O-CHz-O), 3. 39 (m, 2H, J= 6. 2, CH2), 1. 22 (t, 3H, J= 6. 1, CH3), "C-NMR (CDCI3) : 14.7 (CH3), 36.9 (CH2), 103.2 (CH2), 107.2 (CH), 109.8 (CH), 121.2 (CH), 124.9 (CH), 125.9 (CH), 132.4 (C), 142.9 (CH), 145.2 (CH), 150.2 (C), 150.6 (C), 170 (C) MS m/z (%): 245 (M+78), 218 (34), 201 (71), 200 (49), 174 (64), 173 (80), 172 (76), 171 (65), 143 (75), 116 (68), 115 (100) m. p. 158. 5"-159. 9 C (Litm. p. 162 -164 C)4, yield 45. 6%

5-E. E-pipennoy ! ! soprooy ! amine (RV-A09)

'H-NMR (CDC13) 5 : 5. 87 (d, IH, J=14. 8, CH=CH-CH=CH), 7. 36 (d, d, 1H, J=10. 7, 14. 8, CH=CH-CH=CH), 6.66 (d, d, 1H, J=15.4, 10.6, CH=CH-CH=CH), 6.76 (d, 1H, J=15.2 CH=CH-CH=CH), 6.97 (d, lH J=1.6, Ar-7H), 6.77 (d, lH J=8. 0, Ar-lOH), 6.88 (d, d, IH J=1.6, 8.0 Ar-llH), 5.97 (s, 2H, O-CH2-O), 4.15 (m, IH, J=6.6, CH), 5.36 (d, IH, J=7. 3 NH), 1.19 (d, 6H, J=6.6, (CH3) 2) 'C-NMR (CDC13) : 23.2 (CH3) 2, 41.9 (CH), 101. 9 (CH2), 106.4 (CH), 108.9 (CH), 123.0 (CH), 123.8 (CH), 124.1 (CH), 131.3 (C), 140.2 (CH), 141.2 (CH), 148.8 (C), 148.6 (C) 165.6 (C) MS m/z (%): 259 (M+80), 201 (62), 174 (34), 173 (74), 172 (31), 171 (15), 143 (30), 116 (16), 115 (100) m. p. 169 -169. 4 C (Lit m. p. 171 -173 C)4, yield 52% 5-E, E-piperinoy] cyclohexylamin (RV-A10)

IH-NMR (CDC13) 6 : 5. 93 (d, IH, J=14. 8, CH=CH-CH=CH), 7. 35 (d, d, IH, J=10. 6, 14. 8, CH=CH-CH=CH), 6.66 (d, d, 1H, J=15.3, 10.6, CH=CH-CH=CH), 6.76 (d, IH, J=15.4 CH=CH-CH=CH), 6.96 (d, IH J=1.6, Ar-7H), 6.76 (d, IH J=8.0, Ar-lOH), 6.87 (d, d, 1H J=1. 6,8. 0 Ar-llH), 5.97 (s, 2H, O-CH2-O), 3.87 (m, 1H, CH (cyclohexyl) ) 1.99 (m, 2H, CH2 (cyclohexyl)) 1.65 (m, 4H, CH2-CH2 (cyclohexyl) 1.39 (m, 2H, CH2 (cyclohexyl)) 1.18 (m, 2H, CH2 (cyclohexcyl)) 5.48 (d, J=8.0 NH)

"C-NMR (CDC13) : 25. 3 ( (CH2) 2), 25. 9 (CH2), 33. 6 ( (CHz) z), 48. 6 (CH), 101. 3 (CHz), 101. 7 (CH), 106. 1 (CH), 108. 9 (CH), 123. 0 (CH), 124. 0 (CH), 125. 1 (CH), 131. 3 (C), 139.0 (CH), 141.2 (CH) 148.5 (C), 148.5 (C), 165.5 (C) MS m/z (%): 299 (M+56), 259 (48) 216 (33), 201 (60), 174 (33), 173 (61), 172 (18), 171 (16) 143 (17), 115 (100) m. p. 196. 4 -197.3 C (Lit m. p. 199 -200 C) 4, yield 57. 4% 5-E, E-piperinoylbutylamino (RV-A11)

'H-NMR (CDC13) : 5.97 (d, IH, J=14.8, CH=CH-CH=CH), 7.35 (d, d, 1H, J=10. 7,14. 8,

CH=CH-CH=CH), 6. 66 (d, d, IH, J=15. 4, 10. 6, CH=CH-CH=CH), 6. 76 (d, IH, J=15. 4 CH=CH-CH=CH), 6. 97 (d, IH J=1. 6, Ar-7H), 6. 77 (d, lH J=8. 0, Ar-lOH), 6. 89 (d, d, 1H J=1.5, 8.0 Ar-llH), 5.97 (s, 2H, O-CH2-O), 3.36 (q, 2H, CH2-CH2-CH2-) 1.54 (m, 2H, CH2- CH2- CH2) 1.39 (m, 6H, CH2-CH2-CH2) 0.93 (t, 3H, CH3), 5.47 (br, NH) 13C-NMR (CDCl3) : 14.2 (CH3), 20.5 (CH2), 32.2 (CH2), 39.8 (CH2), 101.7 (CH2), 106.1 (CH), 108.9 (CH), 123.0 (CH), 123.6 (CH), 125.0 (CH), 131.3 (C), 139.2 (CH), 141.3 (CH) 148.6 (C), 148.6 (C), 166.4 (C) m. p. 144. 2 -145. 6 C (Lit m. p. 144o-145oCt, yield 38.4% References: 'Chatteqee, A. , and Dutta, C. P. (1967). Alkaloids of Piper longum Linn-I Structure and synthesis of piperlongumine and piperlonguminine, Tetrahedron, 23, 1769-1781.

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

3H. Oediger and A. Schulze (Bayer AG), (1979), Deutsche Auslegeschrift 2757 483

4Paula, Vanderlucia F. de ; A Barbosa, Luiz C. de ; Demuner, Antonio J. ; Pilo-Veloso, Dorila ; Picanco, Marcelo C. (2000) Pest Management Science 56, 2, 168-174.

5Gokale et al., (1948) Journal of University Bombay Science 16/5A 32-35 3. Synthesis of ester derivatives of piperinic acid 3.1 Preparation of piperinic acid (RV-AOO) As described above.

3. 2 Synthesis of 5-E,E-piperinic acid methyl ester (RV-AB1) A mixture of piperinic acid (300mg, 0. 0014mole, leq) and triethylamine (0.39ml, 0.0028mole, 2eq) in dichloromethane (50ml) was stirred for 15min at OOC. To this mixture methanesulfonyl chloride (0.16ml, 0. 0021mole, 1.5eq) was added and stirred for further 30 min at OOC. Methanol in excess (10ml) was added to the mixture and stirred for lh at OOC and 1h at room temperature. Dichloromethane (50ml) was added to the mixture which was then washed with water (3xlOOml), 5% NaHCO3 (3xlOOml) and water (3xlOOml). The organic fraction was dried over anhydrous sodium sulphate, filtered and rotary evaporated to yield a yellowish solid residue. Recrystallisation from ethyl acetate/petroleum spirit yielded ester (180mg, 56.2% yield). m. p. 142. 9-143 C (Lit m. p.

140oC) 6 3.3 Synthesis of other esters of piperinic acid.

They were synthesised as described in section 3.2, replacing methanol (10ml) ethanol (10mol), isopropanol, butanol or propanol (15ml).

5-E, E-pipernic acid methyl ester (RV-AB1)

'H-NMR (CDCh) 6 : 5. 94 (d, 1H, J=15. 2, CH=CH-CH=CH), 7. 41 (d, d, 1H, J=10. 8, 15. 2, CH=CH-CH=CH), 6. 70 (d, d, 1H, J=15. 4, 10. 8, CH=CH-CH=CH), 6. 81 (d, 1H, J=15. 7 CH=CH-CH=CH), 6. 99 (d, lH J=1. 6, Ar-7H), 6. 79 (d, lH J=8. 1, Ar-lOH), 6. 91 (d, d, 1H J=1. 5, 8. 1 Ar-llH), 5. 98 (s, 2H, O-CHrO), 3. 57 (t, 3H, br, OCH3J=4. 7) 'C-NMR (CDC13) 8 : 51. 5 (CH3), 101. 8 (CH2), 106. 2 (CH) 108. 9 (CH), 120. 0 (CH), 123. 4 (CH) 124. 7 (CH), 130. 8 (CH), 140. 9 (C), 145. 5 (CH), 148. 6 (C), 148. 9 (C), 168. 9 (C) MS m/z (%) : 232 (M"69), 201 (19), 174 (12), 173 (100), 172 (39), 171 (12) 143 (33), 116 (11), 115 (53) 101 (15), 100 (12) rl 5-E. E-piperinic acid ethyl ester (RV-AB2)

IH-NMR (CDC13) 8 : 5. 94 (d, 1H, J=15. 2, CH=CH-CH=CH), 7. 41 (d, d, 1H, J=10. 8, 15. 3, CH=CH-CH=CH), 6. 70 (d, d, 1H, J=15. 4, 10. 8, CH=CH-CH=CH), 6. 81 (d, 1H, J=15. 5 CH=CH-CH=CH), 6. 99 (d, lH J=1. 6, Ar-7H), 6. 78 (d, lH J=8. 1, Ar-lOH), 6. 91 (d, d, 1H J=1. 6, 8. 1 Ar-llH), 5. 98 (s, 2H, O-CH2-O), 4. 22 (q, 2H, OCH2 J=7. 2), 1. 31 (t, 3H, CH3 J=7. 2) C-NMR (CDC13) : 14. 7 (CH3), 60. 7 (CH2), 101. 6 (CH2), 106. 3 (CH) 108. 9 (CH), 120. 8 (CH), 123. 3 (CH) 124. 9 (CH), 131. 0 (CH), 140. 5 (CH), 145. 1 (CH), 148. 7 (C), 148. 9 (C), 167. 6 (C)

5-E, E-piperinic acid isopropyl ester (RV-AB4) Physical data are not available for this compound.

5-E. E-piperinic acid propyl ester (RV-AB5)

'H-NMR (CDCl3) 5 : 5.94 (d, IH, J=15. 2, CH=CH-CH=CH), 7.41 (d, d, 1H, J=10. 7,15. 2, CH=CH-CH=CH), 6.70 (d, d, IH, J=15. 4,10. 8, CH=CH-CH=CH), 6.76 (d, IH, J=15.4 CH=CH-CH=CH), 6.99 (d, IH J=1.6, Ar-7H), 6.78 (d, IH J=8.1, Ar-lOH), 6.91 (d, d, IH J=1. 5,8. 0 Ar-llH), 5.98 (s, 2H, O-CH2-O), 4.12 (t, 2H, OCHzJ=6. 7) 1.69 (m, 2H, CH2 J=7.3) 0.97 (t, 3H, CH3 J=7.4) 13C-NMR (CDCl3) : 10.9 (CH3), 22.5 (CH2), 66.3 (CH2), 101. 8 (CH2), 106.2 (CH) 108.9 (CH), 120.9 (CH), 123.3 (CH) 124.9 (CH), 131. 0 (CH), 140.5 (CH), 145.1 (CH), 148.7 (C), 148.9 (C), 167.7 (C)

MS m/z (%) : 260 (mu 59), 201 (26), 174 (18), 173 (100), 172 (39), 171 (14) 143 (34), 116 (16), 115 (73), 100 (12) m. p. 119 -120 C References 6Avijit Banerjee, Tapasree Ghosal, and Aditi Kacharya. (1984). Indian Journal of Chemistry, 23B, 546-549.

5-E. E-piperinic acid butyl ester (RV-AB6)

IH-NMR (CDC13) 6 : 5. 94 (d, IH, J=15. 2, CH=CH-CH=CH), 7. 40 (d, d, 1H, J=10. 7, 15. 3, CH=CH-CH=CH), 6.70 (d, d, IH, J=15.4, 10.8, CH=CH-CH=CH), 6.76 (d, IH, J=15. 4 CH=CH-CH=CH), 6.99 (d, IH J=1.6, Ar-7H), 6.78 (d, IH J=8.0, Ar-lOH), 6.91 (d, d, IH

J=1. 5, 8. 0 Ar-llH), 5. 98 (s, 2H, O-CHz-O), 4. 12 (t, 2H, OCH2 J=6. 7) 1. 69 (m, 2H, CH2 J=7. 3) 1. 69 (m, 2H, CH2J=7. 6), 0. 95 (t, 3H, CH3 J=7. 5) MS m/z (%) : 274 (M''50), 201 (15), 174 (14), 173 (100), 172 (30), 171 (14) 143 (21), 115 (55) Obtained as an oil.

4. Synthesis of amide derivatives of 3. 4-methvlenedioxvcinnamic acid These 3, 4-methylenedioxycinnamide derivatives were synthesised as described in Section 2. 2, but using 3, 4-methylenedioxycinnamic acid (500mg) as the starting acid and reducing the proportion of triethylamine to 1. 5 equivalent with respect to the starting acid.

Also, in the first stage, the reaction mixture was stirred for 2 hours, instead of 30 minutes, again at 0 C.

1- (3-trans-benzo-l, 3-dioxol-5-vlacrvlovl) piperidine (RV-B01)

'H-NMR (CDC13) 6 : 7. 56 (d, IH, J=15. 3, CH=CH), 6. 73 (d, 1H, J=15. 3, CH=CH-), 7. 03 (d, IH J=1. 5, Ar-7H), 6. 79 (d, lH J=8. 0, Ar-8H), 6. 99 (d, d, 1H J=1. 6, 8. 0 Ar-9H), 5. 98 (s, 2H, O-CHrO), 3. 57 (br, 2H, CH2-N-CH2), 3. 65 (br, 2H, CH2-N CH2 (piperidine)), 1. 65 (m, 6H, CH2-CH2-CH2- (piperidine)) 13C-NMR (CDC13) : 24. 8 (CH2), 25. 6 (CH2), 26. 7 (CH2), 43. 3 (CH2), 46. 9 (CH2), 101. 3 (CH2), 106. 7 (CH), 108. 4 (CH), 115. 6 (CH), 123. 5 (CH), 129. 9 (C), 141. 9 (CH) 148. 1 (CH), 148. 8 (C), 165. 4 (C) m. p. 80. 1o-82oC (Lit m. p. 80o-82oC) 7, yield 49. 2%

1- (3-trans-benzo-l, 3-dioxol-5-ylacryloyl) pyrrolidine (RV-B02)

'H-NMR (CDCI3) 8 : 7. 60 (d, IH, J=15. 2, CH=CH), 6. 73 (d, IH, J=15. 3, CH=CH-), 7. 04 (d, IH J=1. 5, Ar-7H), 6. 80 (d, lH J=8. 0, Ar-8H), 7. 01 (d, d, IH J=1. 5, 8. 0 Ar-9H), 5.99 (s, 2H, O-CH2-O), 3.61 (br, 2H, CH2-N-CH2 (pyrrolidine) ), 3.57 (br, 2H, CH2-N- CH2(pyrrolidine)), 1.99 (4H, CH2-CH2 (pyrrolidine)), 13C-NMR (CDCl3) : 24.3 (CH2), 26.1 (CH2) 46.0 (CH2), 46.5 (CH2), 101.4 (CH2), 106.4 (CH), 108.5 (CH), 116.8 (CH), 123.8 (CH), 129.7 (C), 141.0 (CH) 148.1 (C), 148.9 (C), 164.8 (C) MS m/z (%): 245 (W 62), 176 (41) 175 (100) 145 (36), 117 (11), 89 (14). m. p. 152. 50-1530C, yield 44. 1%

1- (3-trans-benzo-l, 3-dioxol-5-vlacrylovl) morpholine (RV-B03)

IH-NMR (CDCIs) 6 : 7. 61 (d, 1H, J=15. 3, CH=CH), 6. 73 (d, IH, J=15. 3, CH=CH-), 7. 03 (d, lH J=1. 4, Ar-7H), 6. 80 (d, IH J=8. 0, Ar-8H), 7. 01 (d, d, IH J=1. 4, 8. 0 Ar-9H), 5.99 (s, 2H, O-CH2-0), 3.72 (br, 4H, CH2-N-CH2 (morpholine) ), 3.67 (br, 4H, CH2-O CH2(morpholine)), 'C-NMR (CDCi3) : 42.6 (CH2), 46.2 (CH2), 66.8 (CH2), 46.5 (CH2), 101.4 (CH2), 106.3 (CH), 108.5 (CH), 114.4 (CH), 123.9 (CH), 129.5 (CH), 143.0 (CH) 148.2 (C), 148.9 (C), 149.1 (C), 165.6 (C) MS m/z (%): 261 (M+ 60), 176 (24) 175 (100) 145 (30), 117 (10), 89 (11). m. p. 160 -160. 3 C, yield 50. 1%

5. Synthesis of 3-trans-benzo-l. 3-dioxol-5-ylacrylic acid methyl ester (RV-BBl) To 3, 4-methylenedioxycinnamic acid (2g, O. Olmol, leq) methanol (4ml, lOeq) was added. Sulphuric acid (0. 2ml) was added and refluxed overnight. The solvent was rotary evaporated to yield solid residue. This residue was dissolved in ether and washed

with water (2xlOOml) and 5% NaHCO3 (3xlOOml) and with water (2xlOOml). The organic fraction was dried over anhydrous sodium sulphate and rotary evaporated to yield white solid. Recrystallisation from ethyl acetate/petroleum spirit yielded crystals (69.4% yield) m. p. 133. 7 -134. 2 C (Lit m. p. 134oC) 8 3-trans-benzo-1,3-dioxol-5-ylacrylic acid methyl ester (RV-BBl)

1H-NMR (CDCh) 5 : 7.59 (d, 1H, J=15.9, CH=CH), 6.26 (d, 1H, J=15.9, CH=CH-), 7.03 (d, lH J=1.5, Ar-7H), 6.81 (d, lH J=8.0, Ar-8H), 7.01 (d, d, 1H J=1. 5,8. 0 Ar-9H), 6.00 (s, 2H, O-CHrO), 3.79 (s, 3H, OCH3) 13C-NMR (CDCl3) : 51.6 (CH3), 101.5 (t CH2), 106.5 (CH), 108.5 (CH), 115.7 (CH), 124.4 (CH), 128.8 (CH), 144.5 (CH) 148.3 (C), 148.6 (C), 148.2 (C), 167.6 (C) MS m/z (%): 206 (M+ 100), 175 (68) 175 (100) 145 (27), 117 (10), 89 (11).

References: 7H. Staudinger and H. Schneider. (1923). Chem. Ber. 56,699.

8Takemoto et al. (1985). Chemical and Pharmaceutical Bulletin 23, 1161.

6. Synthesis of tetrahydropiperine (RV-C02) Piperine (2g, 7mmol) was hydrogenated in ethanol (50ml) over 5% Pd-C under a pressure of hydrogen at 10 psi for 30mins to give tetrahydropiperine (1.59g, 78% yield) as an oil2.

Piperine (1) 1- (5-benzo-1, 3-dioxol-5-yl-pentanoyl)-piperidme Tetrahvdropiperine (RV-C02)

IH-NMR (CDC13) 8 : 2.55 (t, 4H, J=7. 0 CH2-CH2- CH2-CH2), 2.32 (t, 4H, J=7. 0 CH2 CH2- CH2-CH2) 6.66 (d, lH J=1. 3, Ar-7H), 6.70 (d, lH J=8.0, Ar-lOH), 6.61 (d, d, 1H J=1.2, 8.0 Ar-llH), 5.89 (s, 2H, O-CHz-O), 3.53 (t, 2H, N-CHEZ (piperidine) ) 3.35 (t, 2H, N-CH2 (piperidine) ) 1.63 (m, 2H, CH2-CH2-CH2 (piperidine) ) 1.54 (m, 2H, CH2 CH2-CH2(piperidine)) 13C-NMR (CDC13) : 24.5 (CH2), 24.9 (CH2), 25.5 (CH2), 26.5 (CH2), 31.4 (CH2), 33.2

(CH2), 35. 4 (CH2), 42. 5 (CH2), 46. 6 (CH2), 100. 7 (CH2), 108. 0 (CH), 108. 8 (CH), 109. 0 (CH), 121. 0 (C), 145. 4 (C) 147. 4 (C), 171. 1 (C) MS mlz (%) : 289 (mu 71), 204 (31), 154 (23), 148 (22), 141 (23), 140 (38), 135 (28) 127 (100), 112 (23), 86 (12), 84 (24), 70 (10), 36 (11)

7. Synthesis of 3-benzo-l < 3-dioxol-5-ylpropionic acid piperidide 7. 1 Synthesis of 3-benzo-L3-dioxol-5-ylpropionic acid 3-benz-1, 3-dioxol-5-ylacrylic acid (2g) was hydrogenated in ethanol (50ml) over 5% Pd-C under a pressure of hydrogen at 10 psi for 40mins to give 3-benzo1, 3-dioxol-5-ylpropionic acid (1.67g, 80% yield) as a solid, m. p. 86. 1 -88. 3 C (Lit m. p.

87-880qIO

7. 2 Synthesis of 3-benzo-L3-dioxol-5-vlpropionic acid piperidide (RV-C03) The method was adapted from that reported for piperlonguminine (section 2.2) but utilising 3-benzo-1, 3-dioxol-5-ylpropionic acid and piperidine as the acid and amine components respectively. A mixture of 3-benzo-1, 3-dioxol-5-ylpropionic acid (200mg, 0. 0026mole, leq) and triethylamine (0.27ml, 0. 002mole, 2eq) in dichloromethane (50ml)

was stirred for 15min at OOC. To this mixture methanesulfonyl chloride (O. llml, 0. 0015mole, 1. 5eq) was added and stirred for further 30 min at OOC. Piperidine (0. 15ml, 0. 0015mole, 1.5eq) was added to the mixture and stirred for Ih at 0 C and Ih at room temperature. Dichloromethane (50ml) was added to the mixture which was then washed with 5% HCI (3xlOOml), saturated aqueous NaHCO3 (3xlOOml) and water (3x100ml). The organic fraction was dried over anhydrous sodium sulphate, filtered and rotary evaporated to yield brown oil (65% yield).

3-benzo-l, 3-dioxol-5-ylpropionic acid pioeridide (RV-C03)

IH-NMR (CDCI3) : 2. 87 (t, 2H, J=7. 3 Cl2), 2. 57 (t, 2H, J=7. 0 CHCHz) 6. 70 (d, lH J=1. 5, Ar-7H), 6. 72 (d, IH J=8. 0, Ar-lOH), 6. 66 (d, d, 1H J=1. 2, 8. 0 Ar-llH), 5. 90 (s, 2H, O-CHz-O), 3. 55 (t, 2H, N-CHEZ (piperidine)) 3. 34 (t, 2H, N-CHEZ (piperidine)) 1. 62 (m, 2H, CH2-CH2-CH2 (piperidine)) 1. 49 (m, 2H, CH2-CH2-CH2 (piperidine)) 13C-NMR (CDCI3) : 25.7 (CH2), 25.9 (CH2), 26.6 (CH2), 31.7 (CH2), 35.8 (CH2), 43.1 (CH2), 47.1 (CH2), 101.2 (CH2), 109.2 (CH), 109.3 (CH), 121.5 (CH), 135. 6 (C), 146.2 (C) 148. 0 (C), 170. 8 (C) References 9Biswanath Das. , A. Kasinatham. , and P. Madhusudhan. (1998). Regioselective reduction of ap-double bond of some naturally occuring dienamides using NABH system.

Tetrahedron Letters 39,677-678.

'"pekin, Robinson, (1907) Journal of Chemical Society 91,1084 8 Synthesis of amide derivatives of methoxy-substituted cinnamic acid A mixture of monomethoxycinnamic acid (200mg, 0.89mmol, leq) and triethylamine (2. 4ml, 1.78mmol, 2eq) in dichloromethane (50ml) was stirred for 15min at OOC. To this mixture methanesulfonyl chloride (1.02ml, 1. 33mmol, 1.5eq) was added and stirred for further 30 min at OOC. Piperidine (0.23ml, 1.33mmol, 1.5eq) was added to the mixture and stirred for Ih at 0 C and 1h at room temperature. Then dichloromethane (50ml) was added to the mixture, which was then washed with 5% HCI (3xlOOml), saturated aqueous NaHCO3 (3xlOOml) and water (3xlOOml). The organic fraction was dried over anhydrous sodium sulphate, filtered and rotary evaporated to yield an oil. This oil was purified by chromatography on silica gel using ethyl acetate/petroleum spirit (2: 8) as an eluant.

The piperidine amide of 3,4 dimethoxycinnamic acid was prepared in the same way utilising 200mg of the acid.

1- (2-methoxy-cinnamoyl)-piperidine (RV-GO1)

'H-NMR (CDC13) 8 : 7. 56 (d, IH, CH=CH), 7. 29 (d, 1H, J=7. 8 ArH), 7. 12 (d, 1H, J=7. 6 ArH) 7. 0 (d, d 1H, J=1. 8 ArH) 6. 86-6. 90 (m, ArH), 6. 88 (d, 1H, J=15. 4 CH=CH), 3. 583. 66 (br, 4H, CHz-N-CH2 (piperidine)) 1. 56-1. 71 (m, 6H, CHz. CHz CH2 (piperidine)) 3. 83 (s, 3H, OCH3) 13C-NMR (CDCl3) : 25.7 (CH2), 26.0 (CH2), 27.1 (CH2), 43.7 (CH2), 47.4 (CH2), 55.7 (CH3), 113.4 (CH), 115.3 (CH), 118.5 (CH), 120.6 (CH), 130.1 (CH), 142.4 (CH), 137.3 (C), 160.2 (C), 165.6 (C) MS m/z (%): 245 (M+ 28), 162 (22), 161 (100), 133 (20), 118 (24), 113 (14), 84 (51) yield 25.5%

1- (3-methoxy-cinnamovl) oiteridine (RV-G02)

'H-NMR (CDC13) 8 : 7. 60 (d, 1H, J=15. 4, CH=CH), 7. 29 (d, 1H, J=7. 8 ArH), 7. 12 (d, 1H, J=7. 6 ArH) 7. 0 (d, d 1H, J=1. 8 ArH) 6. 86-6. 90 (m, ArH), 6. 88 (d, 1H, J=15. 4 CH=CH), 3.58-3. 66 (br, 4H, CH2-N-CH2 (piperidine) ) 1.56-1. 71 (m, 6H, CH2-CH2 CH2 (piperidine) ) 3.83 (s, 3H, OCH3) 13C-NMR (CDCI3) : 25.7 (CH2), 26.0 (CH2), 27.1 (CH2), 43.7 (CH2), 47.4 (CH2), 55.7 (CH3), 113.4 (CH), 115.3 (CH), 118.5 (CH), 120.6 (CH), 130.1 (CH), 142.4 (CH), 137.3 (C), 160.2 (C), 165.6 (C) MS m/z (%): 245 (M+ 77), 162 (65), 161 (100), 133 (20), 118 (24), 113 (14), 84 (51)

m. p. 680-700C, yield 31. 4% 1- (4-methoxv-cinnamov piperidine (RV-G03)

IH-NMR (CDC13) : 7.61 (d, 1H, J=15. 4, CH=CH), 7.47 (d, 2H, J=7.8 ArH), 6.87-6. 90 (m, 2H, ArH), 6.77 (d, IH, J=15. 4 CH=CH), 3.58-3. 65 (br, 4H, CH2-N-CH2 (piperidine)) 1.52-1. 69 (m, 6H, CH2-CH2 CH2 (piperidine)) 3.82 (s, 3H, OCH3) 13C-NMR (CDCl3) : 25.6 (CH2), 26.0 (CH2), 26.4 (CH2), 43.7 (CH2), 47.4 (CH2), 55.7 (CH3), 114.5 (CH), 115.6 (CH), 118.5 (CH), 121.9 (CH), 129.6 (CH), 142.2 (CH), 132.8 (C), 161. 0 (C), 166.0 (C) MS m/z (%): 245 (M+ 71), 162 (17), 161 (100), 133 (26), 118 (12), 113 (14), 84 (24), 77 (36) 1-(3,4-dimethoxycinnamoyl)piperidine (RV-G04)

IH-NMR (CDCI3) 60MHz 8 : 7.61 (IH, CH=CH), 7.23 ( 1H, ArH), 6.98 ( 1H, ArH) 6.82 (IH, J=1.8 ArH) 6.68 (IH, CH=CH), 3.58-3. 65 (br, 4H, CH2-N-CH2 (piperidine) ) 1.51.8 (6H, CH2-CH2 CH2 (piperidine) ) 3.91 (s, 6H, OCH3) 2) MS m/z (%): 275 (M+ 62), 192 (48), 191 (100), 161 (18), 118 (11), 84 (26), 77 (12), yield 42.3%

Claims

CLAIMS 1. A method of treating a subject having a skin condition selected from the group of conditions (a) those treatable by stimulation of me ! anocyte proliferation and (b) melanomas, which comprises administering to the subject an effective amount of a compound of formula (1)

wherein n = 0, 1 or 2; when n = 0, R ? and R3 represent hydrogen atoms or together represent a carbon to carbon double bond; when n = 1, or 2 R2 and R3 together and R4 and Rs together represent carbon to carbon double bonds, R2 and R3 together represent a carbon to carbon double bond and R4 and Rs represent hydrogen atoms, or R2, R3, R4 and R5 all represent hydrogen atoms; m= 1, 2 or 3 ; when m = 1, R1 represents an alkoxy group having from 1 to 3 carbon atoms or a hydroxy group; when m = 2, each R'independently represents an alkoxy group having from 1 to 3 carbon

atoms or the two Rls together represent a 3', 4'-methylenedioxy group ; when m = 3, two RIS together represent a 3'-4'-methylenedioxy group and the other R represents an alkoxy group having from 1 to 3 carbon atoms or a hydroxy group; R6 represents a pyrrolidino, piperidino, 4-methylpiperidino or morpholino group, a Nmonoalkylamino group of 4 to 6 carbon atoms, a N-monocyclooalkylamino group of 4 to 7 carbon atoms, a 3'-4'-methylenedioxy-substituted benzylamino or 2-phenethylamino group or R6 represents an alkoxy group of 1 to 6 carbon atoms;

in any of its E, Z geometrically isomeric forms, but excluding compounds of formula (1) in which : (a) n is 0, 1, or 2, m is 2, the R's together represent a 3', 4'-methylenedioxy 3 group, R2 and R3, together with the carbon atoms to which they are attached form a carbon to carbon double bond and, when n is 1 or 2, R4 and R5, together with the carbon atoms to which they are attached, form a carbon to carbon double bond and R6 is piperidino, or (b) n is 1 and (i) m is 3, the R's being 3', 4'-methylenedioxy and 6'-methoxy or (ii) m is 2, the R's being 3'-hydroxy-4'-methoxy; or (iii) m is 1 and the RI is 4' hydroxy; and R2 to R6 are as defined in case (a) above, or (c) n is 1, R6 is piperidino, pyrrolidino, isobutylamin or methoxy and all other symbols are defined as in case (a) above, or (d) n is 1, R4 and R5 represent hydrogen atoms and either R2 and R3 also represent hydrogen atoms or R2 and R3 together with the carbon atoms to which they are attached form a carbon to carbon double bond ; and m, R1 and R6 are as defined in case (a) above; and in all of which cases (a) to (d) the molecule is in the E, E or all E geometric configuration or in case (a) when n is I may be in the Z, Z, Z, E or E, Z geometric configuration.
2. The method of Claim 1, wherein the subject is a patient suffering from a melanoma.
3. The method of Claim 1, wherein the subject is a patient suffering from a skin disorder treatable by stimulation of melanocyte proliferation.
4. The method of Claim 1, wherein the skin disorder is vitiligo.
5. The method of Claim 1, wherein the compound is administered topically to the area of the skin to be treated.
6. The method of any preceding claim, wherein the compound of formula (1) is one in which: m =2, the R'groups together represent 3', 4'-methylenedioxy and R6 represents an alkylamino group having 5 or 6 carbon atoms or a cycloalkylamino group aving from 4 to 7 carbon atoms, especially a cyclohexylamino group, or R6 represents a 3,4methylenedioxy-substitued benzylamino or 2-phenethylamino group.
7. The method of Claim 6, wherein the compound is of the E, E geometric configuration.
8. Compounds having the definition given within Claim 1, for use in treating a skin condition defined within Claim 1 which is a disorder.
9. Compounds according to Claim 8, wherein the skin condition is defined in Claim 2,3 or 4.
10. Compounds according to Claim 8 or 9, for topical application to the area of the skin to be treated.
11. Compounds according to Claim 8, as further defined by Claim 6 or 7.
12. Each of the compounds specifically hereinbefore listed in Example 10, other than those excluded by the exclusion clause of Claim 1, for use in treating a skin condition defined within Claim I which is a disorder.