Effects of piperine analogues on stimulation of melanocyte proliferation and melanocyte differentiation

Bioorganic & Medicinal Chemistry 12 (2004) 1905–1920

Eﬀects of piperine analogues on stimulation of melanocyte

proliferation and melanocyte diﬀerentiation

Radhakrishnan Venkatasamy,^aLaura Faas,^aAntony R. Young,^bAmala Raman^a

and Robert C. Hider^a,*

^aDepartment of Pharmacy, King’s College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NN, UK

^bSt John’s Institute of Dermatology, King’s College London, Lambeth Palace Road, London SE1 7EH. UK

Received 11 November 2003; accepted 23 January 2004

Abstract—A wide range of piperine analogues has been synthesised in order to undertake a structure–activity study of their ability

to stimulate melanocyte proliferation. Results demonstrate that an aromatic ring containing at least one ether function and a car-

bonyl group containing side chain is essential for this activity. A number of highly active piperine analogues have been identiﬁed,

for instance 1-(3,4-methylenedioxyphenyl)-penta-2E,4E-dienoic acid methyl ester (5a), 1-E,E-piperinoyl-isobutylamine (4f) and 1-

(3,4-methylenedioxyphenyl)-pentanoic acid cyclohexyl amide (20). A selection of analogues has also been evaluated for their eﬀect

on melanocyte morphology and melanogenesis. The piperine analogues altered cell morphology by increasing dendrite formation

leading to bi-, tri- and quadripolar cells. These same analogues were found to increase total melanin in cell cultures, although

melanin content per cell was not signiﬁcantly altered from control in the presence of these compounds.

1. Introduction

medicine to treat vitiligo. Most of the formulations had

psoralen as a main constituent but a few did not.²

Vitiligo is a common skin pigment disorder aﬀecting

1% of the world population, the incidence varying

between 0.1–9% in diﬀerent countries.¹Vitiligo is

deﬁned as a ‘circumscribed, acquired, idiopathic, pro-

gressive hypomelanotic skin disorder, which is char-

acterised by the development of patchy depigmented

macules due to progressive loss of melanocytes’. From

this deﬁnition vitiligo can be diﬀerentiated from other

hypomelanotic skin disorders such as chemically

induced depigmentation, depigmentaton associated with

melanoma, and depigmentation secondary to various

other dermatoses.¹In ancient times various herbal

remedies were used in Ayurvedic, Unani and Chinese

In 1999, twenty-eight herbs were screened for potential

stimulants of proliferation using a microtitre colori-

metric assay.³In this study aqueous extracts of seven

herbs were found to be active melanocyte proliferants,

black pepper (Piper nigrum L.) being the most potent

herb screened,^3,4Piperine, the main pepper alkaloid,

was also found very eﬀective at micromolar concentra-

tions.⁴This melanocyte proliferant activity of piperine

had not previously been reported. Since Piper nigrum

extract demonstrated high melanocyte proliferant activ-

ity it was assumed that not only piperine possesses this

activity but also possibly other structural analogues

may share the eﬀect. In order to investigate this possi-

bility, we have synthesized various piperine analogues in

order to monitor their melanocyte proliferation stimu-

lation activity.

Keywords: Piperine analogues; Melanocytes; Dendricity; Melanogen-

esis; Vitiligo.

Abbreviations: TPA, 12-O-Tetradecanoyl phorbol-13-acetate; CT,

Cholera toxin; SRB, Sulphorhodamine B; DCM, Dicholormethane;

DMSO, Dimethyl sulfoxide; D_7.4, Distribution coeﬃcient at pH 7.4;

TCA, Trichloroacetic acid; OD, Optical density; PBS, Phosphate

buﬀer saline.

A number of technical problems persist with culturing

human melanocytes, for instance human melanocytes

can not be passaged for more than three cycles and will

not survive during routine frozen storage,⁵hence mela-

nocyte cell lines from mouse skin were adopted for

* Corresponding author. Tel.: +44-20-7848-4882; fax: +44-20-7848-

4800; e-mail: robert.hider@kcl.ac.uk

doi:10.1016/j.bmc.2004.01.036

1906

R. Venkatasamy et al. / Bioorg. Med. Chem. 12 (2004) 1905–1920

bioassay. One such cell line is melan-a which is an

immortal line of pigmented mouse cells cultured from

epidermal melanoblasts obtained from embryos of

inbred C57BL mice.⁵This cell line is non-tumorigenic and

12-O-tetradecanoyl phorbol-13-acetate (TPA) is essen-

tial for the proliferation of these cells. Cells can be readily

cultured and survive well in routine frozen storage.⁵

verted to an ester intermediate 3 by reacting with methane

sulfonyl chloride and triethylamine. By reacting this

intermediate in situ with diﬀerent amines, the correspond-

ing amides 4a–k were synthesised (Scheme 1).⁸The amide

function of piperine was also replaced by a range of

ester groups, yielding the corresponding analogues 5a–d.

The methylenedioxy phenyl group was substituted with

either methyl or phenyl groups, the corresponding

amide derivatives 7a–b were also obtained. The dime-

thoxyphenyl containing analogue 9 was also investi-

gated. In order to achieve the synthesis of this latter

molecule, Piperine 1 was sequentially treated with BBr₃

and methyl iodide⁹(Scheme 1). In addition to these

structural modiﬁcations, the eﬀect of length of aliphatic

chain and degree of saturation on proliferation activity

was investigated. The chain length was reduced to two

carbons and the amide groups were systematically var-

ied and also substituted by an ester group The methyle-

nedioxy phenyl function was also substituted and these

analogues 10a–d, 11a–c, and 12 were synthesised from

the corresponding acids. Piperettine 14, which contains

a six carbon aliphatic chain was synthesised by modify-

ing the method of Dehmlow and Shamout¹⁰(Scheme 2).

2. Chemistry

Other workers have previously prepared a wide range of

piperine analogues, for instance Koul et al.⁶investigat-

ing inducible cytochrome P450 activities and Paula et

al.⁷investigating insecticidal activity. In order to inves-

tigate the chemical structure responsible for the pro-

liferant activity, four diﬀerent series of compounds were

prepared by using standard synthetic routes. All modi-

ﬁcations were structural analogues of piperine. In order

to determine the role of the amide function in the pro-

liferation activity, a series of aliphatic amines (C₁–C₆),

aromatic amines and heterocyclic amines were used to

synthesise a series of amide analogues. Piperinic acid 2,

obtained by alkaline hydrolysis of piperine 1, was con-

Scheme 1. (a) KOH (b) MeSO₂Cl, Et₃N , CHCl₂, 0 ^ꢀC 45 min (c) R¹R²NH, 0 ^ꢀC 1 h, rt 2 h (d) BBr₃in CH₂Cl₂rt 48 h (e) DMSO/KOH, CH₃I,

2

nitrogen atomosphere, 30 min (f) NaBH₄/I₂, THF, 0 ^ꢀC nitrogen atomosphere, 48 h.

Scheme 2. (a) Aliquat 336, K₂CO₃/NaH, toluene, 90 ^ꢀC 6 h (b) H₂, Pd-C (c) MeSO₂Cl, Et₃N , CHCl₂, 0 ^ꢀC 45 min, RNH, 0 ^ꢀC 1 h, rt 1 h.

2

R. Venkatasamy et al. / Bioorg. Med. Chem. 12 (2004) 1905–1920

Table 1. Structures of piperine analogues

1907

Code

n

R

Code

n

R

1

2

piperidinyl

pyrrolidinyl

morpholinyl

aminobenzyl

amino-5-methyl

aminohexyl

aminoisobutyl

aminomethyl

aminoethyl

aminoisopropyl

aminocyclohexyl

4k

5a

5b

5c

2

1

3

aminobutyl

methoxy

ethoxy

propyloxy

butyloxy

piperidinyl

pyrrollidinyl

morpholinyl

methoxy

4a

4b

4c

4d

4e

4f

4g

4h

4i

5d

11a

11b

11c

12

Figure 1. Comparison of practical LogP value and calculated logP

value of piperine and its analogues.

14

piperidinyl

of ionisation is diminishing small at pH 7.4, their dis-

tribution coeﬃcients at pH 7.4 are expected to be iden-

tical to their partition coeﬃcients. When designing a

dosage form for transdermal application, compounds

possessing Log P values around 3–4 are considered to

be optimum.¹²CLog P values were calculated for the

entire series¹³and Log P values were also measured by

the shake-ﬂask method for a small selection of com-

pounds. These determined log P values were compared

with the corresponding CLog P values (Fig. 1). The

dotted line is indicative of the theoretically perfect

correlation between ClogP and the experimentally

determined log P. There was broad agreement between

these two values for 1, 5a, 4j, 19 and 20 but the com-

pound 11c deviated to an appreciable degree; this is

likely to be associated with the presence of the mor-

pholine group.

4j

Table 2. Structures of piperine analogues

Code

n

R

Code

n

R¹

7a

7b

9

10a

10b

10c

10d

2

1

phenyl

methyl

3,4-dimethoxyphenyl

2methoxyphenyl

3-methoxyphenyl

4-methoxyphenyl

3,4-dimethoxyphenyl

18

19

20

21

1

2

3

piperidinyl

cyclohexylaminyl

piperidinyl

4. Results and discussion

4.1. Melanocyte proliferation activity

Due to non-availability of a suitable medium capable of

maintaining culture of normal melanocytes, melanoma

cells have frequently been used to investigate the eﬀect

of growth factors on melanocyte growth in vitro.¹⁴

However normal melanocytes are essential for the study

of the eﬀect of biological agents on melanocyte pro-

liferation, as normal melanocytes possess critical prop-

erties¹⁴. Eisinger and Mako (1982)¹⁴reported that 12-O-

tetradecanoyl phorbol-13-acetate (TPA) and cholera

toxin (CT) are eﬀective at supporting normal human

melanocyte proliferation. These two compounds there-

fore provide a useful medium for the support of mouse

melanocyte growth. The microplate assay is an eﬃcient

way of screening compounds for cytotoxic and cell pro-

The degree of saturation of the aliphatic chain was also

modiﬁed. 5-(3,4-methylenedioxyphenyl)-penta-2E,4E-

dienyl piperidine 15 (Scheme 2) was isolated during an

attempt to reduce a single double bond of piperine

according to the method of Das et al.¹¹No trace of

double bond reduction was observed despite systematic

variation of reaction conditions.¹¹3,4-Methylenedioxy-

cinnamic acid was hydrogenated in ethanol over 5%

Pd-C under hydrogen to yield 5-(3,4-methlyenedi-

oxyphenyl)-propanoic acid and this saturated acid

was converted to the amide derivative 18 by condensa-

tion with piperidine (Scheme 2). Catalytic hydrogena-

tion was used to reduce amides 1, 4j, and 14 to the

corresponding saturated analogues 19, 20, and 21

(Scheme 2). Systematic presentation of the complete

series of molecules synthesised in this work is given in

Tables 1 and 2.

liferation activity and the sulphorhodamine B (SRB)

3,4

staining procedure

was adopted to evaluate these

activities. Melan-a cell proliferation activity was also

established using the microplate assay.

(i) Eﬀect of amide moiety

The amide containing compounds 4a–k were found to

possess proliferation activity with the exception of

4c (Table 3). Compound 4a stimulated melanocyte

proliferation at 1–50 mM having weak activity at 1 mM

3. Partition coeﬃcient measurement

Since the majority of the molecules investigated in this

study are neutral over the pH range 4–9 and their degree

1908

R. Venkatasamy et al. / Bioorg. Med. Chem. 12 (2004) 1905–1920

and peak activity at 10 mM. 4b was found to be active in

the range of 1–100mM with peak activity at 10 mM.

Compound 4d has peak activity at 50 mM and 4e at 100

mM. Compound 4f showed similar response at 1–100

mM. Compound 4g–i showed melanocyte proliferation

activity at 1–10 mM while being cytotoxic at 50 and 100

mM. Replacement of piperidine in piperine by cyclo-

hexylamine and n-butylamine (4j and 4k) showed most

promising melanocyte proliferation eﬀect at 1–50 mM.

but no eﬀect at 100 mM.

(iii) Eﬀect of aromatic ring

The aromatic-substituted compounds 7a and b were not

found to be active at 1–100 mM concentrations. Com-

pound 9 was found be moderately active over the range

10–100 mM, however this compound was ineﬀective at

1mM. This indicates that an etheriﬁed phenyl group is

essential for activity (Table 5).

(iv) Eﬀect of aliphatic chain length

(ii) Eﬀect of ester moiety

None of the short chain piperine analogues 10a–d, 11a–

c, and 12 possessed activity in the range of 1–100 mM,

with the single exception of 11a. The compound 11a was

not eﬀective at 1 mM but it showed melanocyte pro-

liferative activity at the other concentration studied.

However, introduction of one more double bond in the

piperine molecule, i.e., 14 showed melanocyte prolifera-

tion activity at 1–10 mM and 100 mM. From this obser-

vation it is possible to deduce that both the

All piperine ester analogues 5a–d were found to be

eﬀective at both 10 and 50 mM (Table 4). Compound 5a

showed melanocyte proliferation stimulatory activity at

1–100 mM. 5b was cytotoxic at 100 mM. Compound 5c

showed greatest melanocyte proliferative stimulatory

activity at 10 mM but no eﬀect at 100 mM. 5d possesses

activity at 10–100 mM with no eﬀect at 1 mM.

Table 3. Piperine amide analogues

Code

n

R

ClogP

Biological activity

Melanocyte proliferation stimulatory activity

of piperine amide analogues

Stimulant

activity

Dendricity

Proliferation stimulatory activity^yat

1 mM

10 mM

50 mM

100 mM

1

2

piperidinyl

pyrrolidinyl

morpholinyl

aminobenzyl

piperinolaminyl

aminohexyl

aminoisobutyl

aminomethyl

aminoethyl

aminoisopropyl

aminocyclohexyl

aminobutyl

2.963

2.354

1.783

3.883

3.448

4.760

3.572

1.824

1.597

2.662

3.855

3.441

Strong activity

No eﬀect

+++

–

192Æ19**

126Æ9**

129Æ9**

93Æ6

211Æ18**

185Æ3**

171Æ9**

99Æ9

181Æ14**

158Æ11**

165Æ7**

ND

99Æ10

92Æ13

4a

4b

4c

4d

4e

4f

4g

4h

4i

130Æ14**

104Æ12

71Æ10

Positive

+

101Æ12

138Æ10**

152Æ11**

172Æ6**

170Æ24**

200Æ14**

224Æ19**

308Æ29**

264Æ21**

150Æ16**

181Æ18**

160Æ14**

71Æ5

Strong activity

+++

++

+++

93Æ6

197Æ18**

156Æ11**

46Æ2

145Æ9**

140Æ12**

139Æ27**

187Æ15**

301Æ20**

189Æ6**

81Æ12

62Æ13

85Æ5

42Æ6

4j

4k

155Æ22**

100Æ13

158Æ20**

84Æ6

*p<0.05, **p<0.01 when compared to vehicle treatment (One way Anova followed by Dunnett’s t-test) ^yExpressed as percentage cell growthÆSEM

compared to control (medium only) – no eﬀect, +weakly dendritic, ++ moderately dendritic, +++ highly dendritic.

Table 4. Piperine ester analogues

Compd

n

R

ClogP

Biological activity

Melanocyte proliferation stimulatory activity

of ester analogues

Stimulant

activity

Dendricity

Proliferation stimulatory activity^yat

1 mM

10 mM

50 mM

100 mM

5a

5b

5c

5d

2

methoxy

ethoxy

propyloxy

butyloxy

2.484

2.783

3.542

4.071

Strong activity

+

125Æ14**

149Æ7**

175Æ6**

103Æ5

147Æ17**

207Æ10**

224Æ12**

148Æ18**

187Æ13**

169Æ7**

148Æ19**

190Æ11**

171Æ8**

52Æ13

++

90Æ7

128Æ17**

*p<0.05, **p<0.01 when compared to vehicle treatment (One way Anova followed by Dunnett’s t-test) ^yExpressed as percentage cell growthÆSEM

compared to control (medium only) – no eﬀect,+weakly dendritic, ++ moderately dendritic, +++ highly dendritic.

R. Venkatasamy et al. / Bioorg. Med. Chem. 12 (2004) 1905–1920

1909

methylenedioxyphenyl group and aliphatic chain length

are important for activity (Table 6).

piperine analogues. Thus with the introduction of ben-

zylamine 4c results in total loss of activity, whereas a

wide range of acylic and cyclic aliphatic amines, were

found to all possess activity (Table 3). Interestingly the

compounds with smaller residues (4g and i) lose potency

at high concentration (100 mM), in contrast to the more

lipophilic analogue 4e which maintains activity over a

wide range (1–100 mM) (Fig. 2). When converted to the

free acid 2 of the corresponding amine 1 activity is

completely lost. In contrast simple aliphatic esters pos-

sess activity, albeit somewhat weaker than the corres-

ponding amides (Table 4). The ﬁnding that a simple

alkyl analogue 22 of identical length to 4k totally lacks

activity (data not shown) conﬁrms that the concept the

carbonyl function of the amide and ester is essential for

receptor binding.

Saturated analogues 19, 20, and 21 all possessed strong

activity except short chain saturated analogue 18.

Compound 19 showed the peak activity at 10 mM. It

also showed very weak activity at 1 mM and was highly

toxic to the cells at 100 mM. 20 showed consistent

activity throughout the concentration range of 10–100

mM having weaker activity at 1 mM. 21 showed greatest

melanocyte proliferative stimulatory activity at 50 mM.

This compound was also found to be active at 10 mM

(Table 7). However 15, where the carbonyl group is

reduced, did not show melanocyte proliferative stimu-

latory activity over the range 1–100 mM (Table 6), indi-

cating that the carbonyl group is essential for maximum

activity. Thus the length of connection chain between

the two pharmacophores is apparently more important

than the hybridization state of this carbon chain.

From these results it is clear that the nature of the amide

residue has an important role for the activity of the

Table 5. Eﬀect of aromatic moiety

Code

R

ClogP

Biological activity

Melanocyte proliferation stimulatory activity

of aromatic substituted analogues

Stimulant

activity

Dendricity

Proliferation stimulatory activity^yat

1 mM

10 mM

50 mM

100 mM

7a

7b

9

methyl

phenyl

3,4-dimethoxyphenyl

2.484

2.783

3.542

No eﬀect

Positive

—

83Æ10

96Æ8

95Æ6

N

ND

165Æ23**

DÆ10

98Æ13

72

92Æ10

104Æ7

127Æ11**

161Æ5**

*p<0.05, **p<0.01 when compared to vehicle treatment (One way Anova followed by Dunnett’s t-test) ND:Not done yexpressed as percentage cell

growthÆSEM compared to control (medium only) — no eﬀect.

Table 6. Eﬀect of aliphatic chain length

Code

R

R¹

x

n

ClogP

Biological activity

Melanocyte proliferation stimulatory

activity of piperine analogues with modiﬁed

connecting chain and connecting chain length

Stimulant

activity

Dendricity

Proliferation stimulatory activity^yat

1 mM

10 mM

50 mM

100 mM

10a

10b

10c

10d

11a

11b

11c

12

2-methoxy

3-methoxy

4-methoxy

piperidinyl

O

1

3

2

2.813

2.552

No eﬀect

Weak

—

++

—

99Æ8

99Æ5

105Æ8

103Æ6

110Æ11

111Æ10

114Æ8

197Æ12**

ND

119Æ9

95Æ18

127Æ9

90Æ10

95Æ11

119Æ10

106Æ11

85Æ8

108Æ20

100Æ9

133Æ15*

123Æ7

3,4-dimethoxy

Weak

3,4-methylenedioxy piperidinyl

3,4-methylenedioxy pyrrolidinyl

3,4-methylenedioxy morpholinyl

3,4-methylenedioxy methoxy

3,4-methylenedioxy piperidinyl

2.459 Strong Activity

180Æ8**

140Æ14

103Æ12

129Æ15

217Æ15**

95Æ18

195Æ13**

98Æ9

1.900

1.329

2.030

Positive

No eﬀect

ND

134Æ12

110Æ14

122Æ11*

127Æ9

100Æ10

199Æ13**

99Æ5

14

15

4.072 Strong Activity

2.813 No eﬀect

+++

—

59Æ6

3,4-methylenedioxy piperidinyl H₂

110Æ11

*p<0.05, **p<0.01 when compared to vehicle treatment (One way Anova followed by Dunnett’s t-test) ND: Not done ^yExpressed as percentage cell

growthÆSEM compared to control (medium only) - no eﬀect, ++ moderately dendritic.

1910

R. Venkatasamy et al. / Bioorg. Med. Chem. 12 (2004) 1905–1920

Table 7. Eﬀect of aliphatic chain-saturated analogues

Code

n

R

ClogP

Biological activity

Melanocyte proliferation stimulatory activity

of saturated piperine analogues

Stimulant

activity

Dendricity

Proliferation stimulatory activity^yat

1 mM

10 mM

50 mM

100 mM

18

19

20

21

1

2

3

piperidinyl

aminocyclohexyl

piperidinyl

1.643

2.551

3.046

3.459

No eﬀect

—

90Æ10

118Æ12

129Æ6**

118Æ9

108Æ20

111Æ10

133Æ15**

80Æ2

Strong Activity

+++

++

189Æ16**

193Æ6**

144Æ6**

160Æ15**

192Æ10**

160Æ16**

191Æ13**

111Æ20

*p<0.05, **p<0.01 when compared to vehicle treatment (One way Anova followed by Dunnett’s t-test) ND:Not done ^yExpressed as percentage cell

growthÆSEM compared to control (medium only) – no eﬀect, ++ moderately dendritic.

When the linking chain length between the aromatic

nucleus and the amide function is reduced to 2 carbons

the activity remains virtually unchanged for the piperine

analogue 11a whereas compounds 11b, c, and 12 (Table

6) are weaker than their 4 carbon chain analogues 4a, b,

and 5a respectively (Tables 4 and 5).

maintained at 100mM (195%) but not at 1 mM. In con-

trast with piperine (1) (n=2) peak activity was observed

at 10 mM and the compound being ineﬀective at 100 mM.

14 (n=3) showed peak activity at 10 mM but was toxic

at 50mM. This phenomenon could be due to diﬀerent

modes of action at lower and higher concentrations and

reveal that the number of carbons might inﬂuence the

cells in diﬀerent ways Thus although the structure of

both the aromatic residue and the amide portion of the

molecules are essential for activity, the distance and

type of linkage between these two functionalities is

apparently less critical.

One analogue series was used to investigate the inﬂu-

ence of changes on the aromatic nucleus. Conversion to

the dimethyl analogue 10d and to various monomethyl

analogues 10a, b, and c leads to a reduced potency but

all were found to possess some activity. In contrast

when no methoxy or methylenedioxy group is present in

the ring, the activity is totally abolished, for example

with 7b. In contrast, substitution of the methylenedioxy

function in the parent compound (1) by two methoxy

functions 9 failed to have a major inﬂuence on the bio-

logical activity compared to piperine (Table 5). Com-

pound 9 did not alter the morphology of cells. Clearly at

least one ether function is required for the melanocyte

proliferation inducing-activity. Surprisingly when the

aromatic and amide residues are conserved but the

length of linking unsaturated chain is varied, activity is

maintained. The length of the chain can range between

4.3 A (11a) and 9.9 A (14) and yet virtually full activity

is conserved. Signiﬁcantly when the linking chain is

saturated, the activity is also conserved in many of the

analogues (Tables 6). Interestingly the peak activity for

11a (n=1) was at 50 mM (197%) and this eﬀect was

5. Dendricity

Vitiliginous skin melanocytes lose their dendrites before

disappearing from the epidermis.¹⁵In order to achieve

pigmentation in vitiligo patients, it is important that

treatments should aim not only to increase melanocyte

proliferation but also increase melanocyte dendricity

and melanogenesis.

Eﬀect on dendricity of melan-a cells by the test com-

pounds was given a rating based on observation under

microscope. Very long dendrites and more number of

dendrites were designed as highly dendritic. Medium

length and bipolar dendrites were considered as moder-

ate and bipolar dendrites with smaller length were

designed as weakly dendritic. The results showed in

Table 3–7 indicated that piperine amide analogues sti-

mulate more dendricity when compared to piperine

ester analogues. The inactive compounds did not show

any morphological changes in melanocytes.

The detailed quantitative analysis of dendricity has been

carried out. Melanocyte (melan-a) cultures were main-

tained for 72 h in the medium containing piperine or

other piperine analogues (10 mM). More than 90% of

these melanocytes displayed lengthy dendritic processes,

varying from a bipolar to a quadruple polar nature.

Figure 2. Inﬂuence of 4e, g, i on the growth of melan-a cells. Cells

were incubated at 37 ^ꢀC for 4 days (n=6, meanÆSEM). Percentage

growth is expressed as percentage cell number compared to control

(medium only).

Dendrites were analysed with the aid of a computer-

aided image analyzer. Each compound was found to

increase both the average dendrite length and dendrite

R. Venkatasamy et al. / Bioorg. Med. Chem. 12 (2004) 1905–1920

1911

Figure 3. Eﬀect of piperine and its analogues on dendricity.

numbers per cell. Compounds 1, 4b, e, j, 14, 19, and 20

showed a marked increase in dendricity of melanocytes

(Fig. 3A) when compared to the control. This group of

compounds increased the dendricity mainly to a bipolar

state with a few cells possessing a tripolar and quad-

ruple polar nature. In contrast, in the presence of com-

pound 5a, b and 11a, melanocytes were limited to a

bipolar nature (Fig. 3D). Having increased the number

of dendrites per melanocyte all these compounds 1, 4b,

e, j, 11a, 14, 19, and 20 were also found to increase the

total length of the dendrites whereas 5a, b did not

increase the total length of dendrites (Fig. 3B and E).

The typical highly dendritic appearance of melanocytes

treated with 1 and 4j and 19 and 20 is shown in Figure 4.

The phenomenon of increasing dendricity appears to be

close to an ‘all or nothing’ eﬀect. With the possible

exception of 5a, b and 11a, the total dendritic length of

the cells and the length of individual dendrites were

found to be remarkably constant.

Piperine analogues (10 mM) were added to the melano-

cyte cell culture (n=4). The cells were incubated at

37 ^ꢀC for 3 days. The piperine amide analogues 4b, 4e,

Figure 4. Eﬀect of piperine (1) and its analogues 4j, 19 and 20 on

dendricity.

1912

R. Venkatasamy et al. / Bioorg. Med. Chem. 12 (2004) 1905–1920

Table 8. Eﬀect of piperine and its analogues on melanogenesis

of two major domains, a membrane-bound regulatory

domain and a cytoplasmic catalytic domain.¹⁶Both

domains possess multiple ligand binding sites and it is

tempting to propose that because the distance between the

two parts of the pharmacophore in active molecules, namely

the aromatic centre and the aliphatic amide function,

can be separated by a wide range of distances and yet

retain full activity, that they interact simultaneously with

two binding sites, one on each of the two receptor domains.

Work is in progress to investigate this possibility.

Compd

Total melanin content

per dish (mg/mL)

Melanin content/

cell (ng/mL)

Control

1

19

4j

21

16.4Æ0.7

24.5Æ1.7**

19.6Æ3.5

22.3Æ4.1*

21.6Æ2.2

0.29Æ. 0.03

0.28Æ0.04

0.24Æ0.03

0.30Æ0.06

0.28Æ0.02

*p<0.05, **p<0.01 when compared to vehicle treatment saturated

(One way Anova followed by Dunnett’s t-test).

The major observations resulting from these structure–

activity studies are that the logP values of the molecules

are not critical, but probably should exceed unity; the

aliphatic chain can be either unsaturated or fully satu-

rated, but must include a carbonyl function for optimal

activity and that the aromatic function must include at

least one ether group. Some of these analogues, for

instance, 20 and 21, by virtue of their saturated nature,

are more stable towards autooxidation than the parent

molecule piperine and hence will have advantages as

pharmaceutical agents.

4j and its saturated analogues 19, 20 showed increase in

number of dendrites (A) increase in dendrite length (B)

and increase in average dendrite length (C). Short chain

piperine amide analogue 11a, piperine ester analogues

5a, b showed similar in dendrite numbers (D) but

decrease in dendrite length (E) and average dendrite

length (F) when compared to piperine whereas piper-

ettine 14 showed the similar response to piperine.

*p<0.05 when compared to vehicle treatment (One way

Anova followed by Dunnett’s t-test).

All the active compounds investigated in this study

appear to induce identical changes to melanocytes,

namely transformation to bipolar cells each possessing

dendrites of similar length. The virtually identical

response produced by these piperine analogues indicates

that at a certain threshold level they activate a switching

mechanism. Piperine and some of the analogues identi-

ﬁed in this manuscript are under preclinical develop-

ment for the treatment of vitiligo.

6. Melanogenesis

Pigmentation in the skin depends on the amount, size

and type of melanins produced by melanocytes. Mela-

nin quantiﬁcation is necessary to investigate the ability

of the compounds to inﬂuence melanogenesis. After 3

days incubation piperine and three analogues 4j, 19, and

21 showed evidence of increased total melanin although

melanin content per cell was the same. This indicates

that the compounds have neither a stimulatory nor sig-

niﬁcant inhibitory action on melanogenesis (Table 8).

8. Experimental

8.1. General

7. Conclusion

Melting points were recorded using an Electrothermal

IA9100 digital melting point apparatus and are cor-

The work reported in this communication conﬁrms the

previous observation that piperine induces melanocyte

proliferation and enhanced dendricity (Lin et al.).⁴

Furthermore, it has now been demonstrated that a

range of closely related analogues also possess this

property. Most of the compounds showed maximum

activity at 10 mM. Variation in peak activity was

observed with 4d, 5a, d, 11a, 20, 9 (50 mM), and 4e (100

mM), Toxic eﬀects were observed with 4d, g, h, i, 5b, and

19 at 100 mM.

1

rected. H and ¹³C NMR spectra were recorded using a

Bruker (360 MHz) NMR spectrophotometer unless

speciﬁed. Mass spectra (EI) were recorded on a Joel

AX505W. Elemental analysis was performed by Micro-

Analytical Laboratories, Department of Chemistry, The

University of Manchester, Manchester M13 9PL UK.

8.2. (2E,4E)-Piperinic acid (2)

Methanolic KOH (20%, 100 mL) was added to piperine

(1) (2 g, 0.7 mmol, 1 equiv), and reﬂuxed for 2 days.

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 (50 mL) and acidiﬁed with 6NHCl to pH <1

yielding a yellowish precipitate of (2E,4E)-piperinic acid

(2). Recrystallization from methanol gave yellow nee-

dles (0.9 g, 60% yield). mp 206–208 ^ꢀC.

Many of the active compounds retain their activity over

a relatively wide concentration range (1–100 mM) for

instance 4b, e, 5a, and 20. In contrast others including 1,

4a, g, h, i, 5b, c, and 19 were found to possess reduced

potency at higher concentrations, indicating a satura-

tion of the stimulus. The melanocyte proliferation eﬀect

is inhibited by RO-31-8220, a selective protein kinase C

inhibitor strongly suggests that protein kinase C signal-

ing is central to this activity (Lin et al.).⁴With the

piperine analogues 19, 4j melanocyte proliferation

activity was also abolished by protein kinase C inhibitors

(unpublished data). Protein kinase C is a Ca²⁺-activated

phospholipid-dependent protein kinase, which consists

8.3. Compounds (4a–k)

A mixture of (2E,4E)-piperinic acid (2) (200 mg, 0.9

mmol, 1 equiv) and triethylamine (0.25 mL, 1.8 mmol, 2

R. Venkatasamy et al. / Bioorg. Med. Chem. 12 (2004) 1905–1920

1913

equiv) in dichloromethane (50 mL) was stirred for 15

min at 0 ^ꢀC. To this mixture methane sulfonylchloride

(0.1 mL, 1.3 mmol, 1.5 equiv) was added and stirred for

further 30 min at 0 ^ꢀC. The amine (1.5 equiv) was added

to the mixture and stirred for 1 h at 0 ^ꢀC and 2 h at

room temperature. Dichloromethane (50 mL) was

added to the mixture, which was then washed with 5%

HCl (3Â100 mL), saturated aqueous NaHCO₃(3Â100

mL) and water (3Â100 mL). The organic fraction was

dried over anhydrous sodium sulphate, ﬁltered and

rotary evaporated to yield a solid residue. Recrystalli-

sation from ethylacetate and petroleum spirit gave

crystals of amide (4a–k).

J=5.96, Ph-CH₂), 7.2–7.3 (m, 5H, Ar), 8.55 (t, 1H,

J=5.96 NH); ¹³C NMR (CDCl₃): d 43.8 (CH₂), 101.8

(CH₂), 106.0 (CH), 108.8 (CH), 123.1 (CH), 123.2 (CH),

125.1 (CH), 127.6 (CH), 128.0 (CH), 128.9 (CH), 139.6

(CH), 141.7 (CH), 148.7 (C), 148.8 (C), 167.6 (C); MS

m/z (%): 307 (M⁺100), 216 (13), 202 (28), 201 (26), 174

(36), 173 (49), 172 (16), 144 (10), 143 (13), 115 (26), 91

(25); IR (KBr): n_max(carbonyl group) cm^ꢁ11637. Calcd

for C₁₉H₁₇NO₃: C 74.23%, H 5.58%, N4.56%; found:

C 74.18%, H 5.35%, N4.53%. Yield: 214 mg (50.1%);

mp 177–178 ^ꢀC.

8.7. 1-E,E-piperinoyl-3,4-methylenedioxyphenyl methyl-

amine (4d)

8.4. 1-E,E-piperinoyl-pyrrolidine (4a)

¹H NMR (CDCl₃): d 5.98 (d, 1H, J=14.9, CH¼CH–

CH¼CH), 7.34 (dd, 1H, J=10.7, 14.9, CH¼CH–CH¼

CH), 6.73 (dd, 1H, J=15.5, 10.7, CH¼CH–CH¼CH),

6.79 (d, 1H, J=15.5 CH¼CH–CH¼CH), 6.98 (d, 2H

J=1.5, Ar-H), 6.78 (d, 2H J=8.0, Ar-H), 6.89 (dd, 2H

J=1.6, 8.0 Ar-H), 5.98 (s, 2H, O–CH₂–O), 5.93 (s, 2H,

O–CH₂–O), 4.40 (d, 2H, CH₂N), 3.57 (br, 1H, NH);

¹³C NMR (CDCl₃): d 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 (CH), 132.2

(CH), 139.9 (CH), 141.6 (C), 147.0 (C), 147.9 (C)148.3

(C), 148.4 (C), 166.9 (C); MS m/z (%): 351 (M⁺81), 216

(15), 203 (12), 202 (53), 201 (29), 174 (31), 173 (22), 150

(23), 144 (11), 143 (10), 135 (100), 116 (12), 115 (29).

Calcd for C₂₀H₁₇NO₅: C 68.37%, H 4.88%, N3.99%;

found: C 68.27%, H 4.66%, N3.86%. Yield: 157 mg

(50.1%); mp 190.5–191.7 ^ꢀC.

¹H NMR (CDCl₃): d 6.26 (d, 1H, J=14.7, CH¼CH–

CH¼CH), 7.43 (dd, 1H, J=9.5, 14.7, CH¼CH–CH=

CH), 6.73 (dd, 1H, J=15.3, 9.5, CH¼CH–CH¼CH),

6.78 (d, 1H, J=15.3, CH¼CH–CH¼CH), 6.98 (d, 1H

J=1.6, Ar-H), 6.77 (d, 1H J=8.0, Ar-H), 6.89 (dd, 1H

J=1.6, 8.0, Ar-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(pyrroli-

dine)), 1.87 (m, 2H, CH₂–CH₂(pyrrolidine)); ¹³C N MR

(CDCl₃): d 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): n_max(carbonyl group)

cm^ꢁ11637. Yield: 186 mg (49.2%). Calcd for

C₁₆H₁₇NO₃: C 70.83%, H 6.31%, N5.16%; found: C

70.90%, H 6.99%, N4.58%

8.8. 1-E,E-piperinoyl-hexylamine (4e)

¹H NMR (CDCl₃): d 5.90 (d, 1H, J=14.8, CH¼CH–

CH¼CH), 7.35 (dd, 1H, J=10.6, 14.8, CH¼CH–CH¼

CH), 6.66 (dd, 1H, J=15.4, 10.6, CH¼CH–CH¼CH),

6.76 (d, 1H, J=15.4 CH¼CH–CH¼CH), 6.97 (d, 1H

J=1.4, Ar-H), 6.77 (d, 1H J=8.0, Ar-H), 6.88 (dd, 1H

J=1.5, 8.0 Ar-H), 5.97 (s, 2H, O–CH₂–O), 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): d 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): n_max(carbonyl group) cm^ꢁ1

1641. Yield: 168 mg, (40.1%); mp 149.5–149.8 ^ꢀC (lit.

mp 139–141 ^ꢀC). Calcd for C₁₈H₂₃NO₃: C 71.72, H

7.69%, N4.64%; found: C 71.09%, H 7.81%, N

4.56%.

8.5. 1-E,E-piperinoyl-morpholine (4b)

¹H NMR (CDCl₃): d 6.37 (d, 1H, J=14.6, CH¼CH–

CH¼CH), 7.45 (dd, 1H, J=10.2, 14.6, CH¼CH–CH¼

CH), 6.72 (dd, 1H, J=15.5, 10.2, CH¼CH–CH¼CH),

6.79 (d, 1H, J=15.5 CH¼CH–CH¼CH), 6.98 (d, 1H

J=1.5, Ar-H), 6.80 (d, 1H J=8.0, Ar-H), 6.89 (dd, 1H

J=1.5, 8.0 Ar-H), 5.98 (s, 2H, O–CH₂–O), 3.70 (t, 4H,

J=4.0 CH₂–N–CH₂(morpholine)), 3.60 (t, 4H, J=4.0

CH₂–O–CH₂(morpholine)); ¹³C NMR (CDCl₃): d 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 (M⁺57), 201

(100), 173 (25), 171 (10), 143 (10), 115 (30); IR (KBr):

n_max(carbonyl group) cm^ꢁ11641; Yield: 113 mg

(44.1%); mp 161.8–162.5 ^ꢀC (lit. mp 167–168 ^ꢀC). Calcd

for C₁₆H₁₇NO₄: C 66.88%, H 5.96%, N4.84%; found:

C 66.47%, H 5.78%, N4.79%.

8.6. 1-E,E-piperinoyl-benzylamine (4c)

8.9. 1-E,E-piperinoyl-isobutylamine (4f)

¹H NMR (CDCl₃): d 6.15 (d, 1H, J=15.0, CH¼CH–

CH¼CH), 7.19 (dd, 1H, J=10.2, 15.0, CH¼CH–CH¼

CH), 6.92 (dd, 1H, J=15.5, 10.2, CH¼CH–CH¼CH),

6.85 (d, 1H, J=15.5 CH¼CH–CH¼CH), 7.22 (d, 1H

J=1.4, Ar-H), 6.89 (d, 1H J=8.0, Ar-H), 6.98 (dd, 1H

J=1.4, 8.0 Ar-H), 6.03 (s, 2H, O–CH₂–O), 4.36 (d, 2H,

¹H NMR (CDCl₃): d 5.96 (d, 1H, J=14.8, CH¼CH–

CH¼CH), 7.36 (dd, 1H, J=10.5, 14.8, CH¼CH–CH¼

CH), 6.66 (dd, 1H, J=15.4, 10.5, CH¼CH–CH¼CH),

6.76 (d, 1H, J=15.4 CH¼CH–CH¼CH), 6.96 (d, 1H

J=1.6, Ar-H), 6.76 (d, 1H J=8.0, Ar-H), 6.87 (dd, 1H

1914

R. Venkatasamy et al. / Bioorg. Med. Chem. 12 (2004) 1905–1920

J=1.6, 8.0 Ar-H), 5.97 (s, 2H, O–CH₂–O), 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): d 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): n_max(car-

bonyl group) cm^ꢁ11644. Yield: 120 mg, (32%); mp

161.2–161.7 ^ꢀC (lit. mp 156–160 ^ꢀC). Calcd for

C₁₆H₁₉NO₃: C 70.29%, H 7.01%, N5.12%; found: C

69.79%, H 7.02%, N5.01%.

143 (30), 116 (16), 115 (100). Yield: 188 mg (52%); mp

169–169.4 ^ꢀC (lit. mp 171–173 ^ꢀC). Calcd for

C₁₅H₁₇NO₃: C 69.46%, H 6.61%, N5.40%; found: C

69.13%, H 6.34%, N5.24%.

8.13. 1-E,E-piperinoyl-cyclohexylamine (4j)

¹H NMR (CDCl₃): d 5.93 (d, 1H, J=14.8, CH¼CH–

CH¼CH), 7.35 (dd, 1H, J=10.6, 14.8, CH¼CH–CH¼

CH), 6.66 (dd, 1H, J=15.3, 10.6, CH¼CH–CH¼CH),

6.76 (d, 1H, J=15.4 CH¼CH–CH¼CH), 6.96 (d, 1H

J=1.6, Ar-H), 6.76 (d, 1H J=8.0, Ar-H), 6.87 (dd, 1H

J=1.6, 8.0 Ar-H), 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(cyclohexyl)), 5.48 (d,

J=8.0 NH); ¹³C NMR (CDCl₃): d 25.3 ((CH₂)₂), 25.9

(CH₂), 33.6 ((CH₂)₂), 48.6 (CH), 101.7 (CH₂), 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). Yield: 239 mg (57.4%); mp

196.4–197.3 ^ꢀC (lit. mp 199–200 ^ꢀC). Calcd for

C₁₈H₂₁NO₃: C 72.20%, H 7.07%, N4.68%; found: C

72.14%, H 6.90%, N4.61%.

8.10. 1-E,E-piperinoyl-methylamine (4g)

¹H NMR (CDCl₃): d 5.91 (d, 1H, J=14.8, CH¼CH–

CH¼CH), 7.36 (dd, 1H, J=10.7, 14.8, CH¼CH–CH¼

CH), 6.66 (dd, 1H, J=15.4, 10.6, CH¼CH–CH¼CH),

6.77 (d, 1H, J=15.4 CH¼CH–CH¼CH), 6.97 (d, 1H

J=1.5, Ar-H), 6.77 (d, 1H J=8.0, Ar-H), 6.88 (dd, 1H

J=1.6, 8.0 Ar-H), 5.97 (s, 2H, O–CH₂–O), 2.91 (t, 3H,

CH₃), 5.61 (br, NH); ¹³C NMR (CDCl₃): d 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). Yield: 155 mg (48.2%); mp

181.1–182.4 ^ꢀC (lit. mp 186 ^ꢀC). Calcd for C₁₃H₁₃NO₃:

C 67.50%, H 5.66%, N6.01%; found: C 66.96%, H

5.61%, N5.90%.

8.14. 1-E,E-piperinoyl-butylamine (4k)

¹H NMR (CDCl₃): d 5.97 (d, 1H, J=14.8, CH¼CH–

CH¼CH), 7.35 (dd, 1H, J=10.7, 14.8, CH¼CH–CH¼

CH), 6.66 (dd, 1H, J=15.4, 10.6, CH¼CH–CH¼CH),

6.76 (d, 1H, J=15.4 CH¼CH–CH¼CH), 6.97 (d, 1H

J=1.6, Ar-H), 6.77 (d, 1H J=8.0, Ar-H), 6.89 (dd, 1H

J=1.5, 8.0 Ar-H), 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): d 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). Yield: 146 mg (38.4%); mp 144.2–

145.6 ^ꢀC (lit. mp 144–145 ^ꢀC). Calcd for C₁₆H₁₉NO₃: C

70.29%, H 7.01%, N5.12%; found: C 70.16%, H

6.56%, N4.78%.

8.11. 1-E,E-piperinoyl-ethylamine (4h)

¹H NMR (CD₃OD): d 6.14 (d, 1H, J=15.0, CH¼CH–

CH¼CH), 7.37 (dd, 1H, J=10.2, 15.0, CH¼CH–CH¼

CH), 6.93 (dd, 1H, J=15.7, 10.6, CH¼CH–CH¼CH),

6.87 (d, 1H, J=15.7 CH¼CH–CH¼CH), 6.97 (d, 1H

J=1.5, Ar-H), 6.77 (d, 1H J=8.0, Ar-H), 6.88 (dd, 1H

J=1.6, 8.0 Ar-H), 5.97 (s, 2H, O–CH₂–O), 3.39 (m, 2H,

J=6.2, CH₂), 1.22 (t, 3H, J=6.1, CH₃), ¹³C N MR

(CDCl₃): d 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). Yield: 156 mg (45.6%); mp 158.5–

159.9 ^ꢀC (lit. mp 162–164 ^ꢀC).

8.15. Compounds (5a–d)

A mixture of (2E,4E)-piperinic acid (2) (200 mg, 0.9

mmol, 1 eq) and triethylamine (0.25 mL, 1.8 mmol, 2

eq) in dichloromethane (50 mL) was stirred for 15 min

at 0 ^ꢀC. To this mixture methane sulfonylcholride (0.1

mL, 1.3 mmol, 1.5 equiv) was added and stirred for

further 30 min at 0 ^ꢀC. The alcohol (10 mL) was added

to the mixture and stirred for 1 h at 0 ^ꢀC and 2 h at

room temperature. Dichloromethane (50 mL) was

added to the mixture, which was then washed with

water (3Â100 mL), 5% NaHCO₃(3Â100 mL) and

water (3Â100 mL). The organic fraction was dried over

anhydrous sodium sulphate, ﬁltered and rotary evapo-

rated to yield a brownish solid residue. Recrystallisation

from ethylacetate and petroleum spirit gave piperinic

acid esters (5a–d).

8.12. 1-E,E-piperinoyl-isopropylamine (4i)

¹H NMR (CDCl₃): d 5.87 (d, 1H, J=14.8, CH¼CH–

CH¼CH), 7.36 (dd, 1H, J=10.7, 14.8, CH¼CH–CH¼

CH), 6.66 (dd, 1H, J=15.4, 10.6, CH¼CH–CH¼CH),

6.76 (d, 1H, J=15.2 CH¼CH–CH¼CH), 6.97 (d, 1H

J=1.6, Ar-H), 6.77 (d, 1H J=8.0, Ar-H), 6.88 (dd, 1H

J=1.6, 8.0 Ar-H), 5.97 (s, 2H, O–CH₂–O), 4.15 (m, 1H,

J=6.6, CH), 5.36 (d, 1H, J=7.3 NH), 1.19 (d, 6H,

J=6.6, (CH₃)₂); ¹³C NMR (CDCl₃): d 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),

R. Venkatasamy et al. / Bioorg. Med. Chem. 12 (2004) 1905–1920

1915

8.16. 5-(3,4-Methylenedioxyphenyl)-penta-2E,4E-dienoic

acid methyl ester (5a)

J=1.6, Ar-H), 6.78 (d, 1H J=8.0, Ar-H), 6.91 (dd, 1H

J=1.5, 8.0 Ar-H), 5.98 (s, 2H, O–CH₂–O), 4.12 (t, 2H,

OCH₂J=6.7), 1.69 (m, 2H, CH₂J=7.3), 1.69 (m, 2H,

CH₂J=7.6), 0.95 (t, 3H, CH₃J=7.5); MS m/z (%): 274

(M⁺50), 201 (15), 174 (14), 173 (100), 172 (30), 171

(14), 143 (21), 115 (55). Yield: 102 mg 41.4%. Calcd for

C₁₆H₁₈O₄: C 70.04%, H 6.61%; found: C 69.56%, H

6.27%.

¹H NMR (CDCl₃): d 5.94 (d, 1H, J=15.2, CH¼CH–

CH¼CH), 7.41 (dd, 1H, J=10.8, 15.2, CH¼CH–CH¼

CH), 6.70 (dd, 1H, J=15.4, 10.8, CH¼CH–CH¼CH),

6.81 (d, 1H, J=15.7 CH¼CH–CH¼CH), 6.99 (d, 1H

J=1.6, Ar-H), 6.79 (d, 1H J=8.1, Ar-H), 6.91 (dd, 1H

J=1.5, 8.1 Ar-H), 5.98 (s, 2H, O–CH₂–O), 3.57 (t, 3H,

br, OCH₃J=4.7); ¹³C NMR (CDCl₃): d 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). Yield: 117 mg

(56.2%); mp 142.9–143 ^ꢀC (lit. mp 140 ^ꢀC). Calcd for

C₁₃H₁₂O₄: C 67.22%, H 5.21%; found: C 66.91%, H

5.21%.

8.19. 5-(Phenyl)-penta-2E,4E-dienoic acid (6a)

Cinnamaldehyde (1 mL, 0.0075 mol, 1 equiv) and

malonic acid (1.56 g, 0.015 mol, 2 eq) was stirred in

pyridine (15 mL) for 10 min and reﬂuxed for overnight.

Mixture is boiled for 15 min. Mixture was cooled and

acidiﬁed with 1NHCl yielding a yellowish precipitate of

mixture of 1-(phenyl)-penta-2,4-dienoic acid. Recrys-

tallisation from chloroform gave 5-(phenyl)-penta-

2E,4E-dienoic acid (6a) (56.2% yield). mp 164.5–165 ^ꢀC.

(lit. mp 164–165 ^ꢀC).

8.16.1.

5-(3,4-Methylenedioxyphenyl)-penta-2E,4E-di-

1

enoic acid ethyl ester (5b). H NMR (CDCl₃): d 5.94 (d,

1H, J=15.2, CH¼CH–CH¼CH), 7.41 (dd, 1H, J=10.8,

15.3, CH¼CH–CH¼CH), 6.70 (dd, 1H, J=15.4, 10.8,

CH¼CH–CH¼CH), 6.81 (d, 1H, J=15.5 CH¼CH–

CH¼CH), 6.99 (d, 1H J=1.6, Ar-H), 6.78 (d, 1H

J=8.1, Ar-H), 6.91 (dd, 1H J=1.6, 8.1 Ar-H), 5.98 (s,

2H, O–CH₂–O), 4.22 (q, 2H, OCH₂J=7.2), 1.31 (t, 3H,

CH₃J=7.2); ¹³C NMR (CDCl₃): d 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 (C), 140.5 (CH),

145.1 (CH), 148.7 (C), 148.9 (C), 167.6 (C); MS m/z

(%): 246 (M⁺83), 201 (26), 174 (14), 173 (100), 143

(15), 115 (53). Calcd for C₁₄H₁₄O₄: C 68.27%, H 5.73%;

found C 68.61%, H 5.54%. Yield: 120 mg (55%) mp

43.1–44 ^ꢀC (lit. mp 45 ^ꢀC).

8.20. 5-(Phenyl)-penta-2E,4E-dienoyl piperidine (7a)

A mixture of 5-(phenyl)-penta-2E,4E-dienoic acid (200

mg, 0.0011 mol, 1 equiv) and triethylamine (0.3 mL,

0.0022 mol, 2 eq) in dichloromethane (50 mL) was stir-

red for 15 min at 0 ^ꢀC. To this mixture methane sulfonyl

chloride (0.12 mL, 0.0016 mol, 1.5 equiv) was added

and stirred for further 30 min at 0 ^ꢀC. Piperidine (0.16

mL, 0.0016 mol, 1.5 equiv) was added to the mixture

and stirred for 1 h at 0 ^ꢀC and 1 h at room temperature.

Dichloromethane (50 mL) was added to the mixture

which was then washed with 5% HCl (3Â100 mL),

saturated aqueous NaHCO₃(3Â100 mL) and water

(3Â100 mL). The organic fraction was dried over anhy-

drous sodium sulphate, ﬁltered and rotary evaporated

to yield a yellowish solid residue. Recrystallisation from

ethylacetate/ether yielded white crystals of 5-(phenyl)-

8.17. 5-(3,4-Methylenedioxyphenyl)-penta-2E,4E-dienoic

acid propyl ester (5c)

1

penta-2E,4E-dienoyl piperidine. H NMR (CDCl₃) (60

¹H NMR (CDCl₃): d 5.94 (d, 1H, J=15.2, CH¼CH–

CH¼CH), 7.41 (dd, 1H, J=10.7, 15.2, CH¼CH–

CH¼CH), 6.70 (dd, 1H, J=15.4, 10.8, CH¼CH–

CH¼CH), 6.76 (d, 1H, J=15.4 CH¼CH–CH¼CH),

6.99 (d, 1H J=1.6, Ar-H), 6.78 (d, 1H J=8.1, Ar-H),

6.91 (dd, 1H J=1.5, 8.0 Ar-H), 5.98 (s, 2H, O–CH₂–O),

4.12 (t, 2H, OCH₂J=6.7), 1.69 (m, 2H, CH₂J=7.3),

0.97 (t, 3H, CH₃J=7.4); ¹³C NMR (CDCl₃): d 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 (C), 140.5 (CH), 145.1 (CH), 148.7 (C), 148.9 (C),

167.7 (C); MS m/z (%): 260 (M⁺59), 201 (26), 174 (18),

173 (100), 172 (39), 171 (14), 143 (34), 116 (16), 115 (73),

100 (12). Yield: 84 mg 34% mp 119–120 ^ꢀC. Calcd for

C₁₅H₁₆O₄: C 69.20%, H 6.19%; found: C 69.04%, H

6.49%.

mHZ): d 7.1–7.4 (1H ArH), 7.25 (1H ArH), 6.78 (2H

ArH), 6.45 (1H, ArH), 3.4–3.6 (br, 4H, CH₂–N–CH₂

(piperidine)), 1.5–1.8 (m, 6H, CH₂–CH₂CH₂(piper-

idine)) (36% yield). mp 73–75 ^ꢀC (lit. mp 77 ^ꢀC).

8.21. 1-[(2E,4E)-Hexadienoyl]-piperidine (7b)

A mixture of 2,4-hexadienoic acid (300 mg, 0.0026 mol,

1 eq) and triethylamine (0.7 mL, 0.0052 mol, 2 equiv)

in dichloromethane (50 mL) was stirred for 15 min at

0 ^ꢀC. To this mixture methane sulfonyl chloride (0.3

mL, 0.0039 mol, 1.5 equiv) was added and stirred for a

further 30 min at 0 ^ꢀC. Piperidine (0.23 mL, 0.0039

mol, 1.5 equiv) was added to the mixture and stirred for 1

h at 0 ^ꢀC and 1 h at room temperature. Dichloro-

methane (50 mL) was added to the mixture which was

then washed with 5% HCl (3Â100 mL), saturated aqu-

eous NaHCO₃(3Â100 mL) and water (3Â100 mL). The

organic fraction was dried over anhydrous sodium sul-

phate, ﬁltered and rotary evaporated to yield a yellow-

ish solid residue. Recrystallisation from ethylacetate/

ether yielding colourless crystals of 2,4-hexadienoyl

piperidine (58.3% yield). mp 79.2–81.2 ^ꢀC (lit. mp 83–

84 ^ꢀC).

8.18. 5-(3,4-Methylenedioxyphenyl)-penta-2E,4E-dienoic

acid butyl ester (5d)

¹H NMR (CDCl₃): d 5.94 (d, 1H, J=15.2, CH¼CH–

CH¼CH), 7.40 (dd, 1H, J=10.7, 15.3, CH¼CH–CH¼

CH), 6.70 (dd, 1H, J=15.4, 10.8, CH¼CH–CH¼CH),

6.76 (d, 1H, J=15.4 CH¼CH–CH¼CH), 6.99 (d, 1H

1916

R. Venkatasamy et al. / Bioorg. Med. Chem. 12 (2004) 1905–1920

¹H NMR (CDCl₃): d 6.23 (d, 1H, J=15.7, CH¼CH–

CH¼CH), 7.25 (dd, 1H, J=10.7, 15.7, CH¼CH–CH¼

CH), 6.73 (dd, 1H, J=15.3, 9.5, CH¼CH–CH¼CH),

6.78 (d, 1H, J=15.3 CH¼CH–CH¼CH), 3.61 (br, 2H,

N–CH₂(piperidine)), 3.48 (br, 2H, N–CH₂(piper-

idine)), 1.65 (m, 2H, CH_2-CH₂CH₂(piperidine)), 1.57

(m, 4H, CH₂–CH₂CH₂(piperidine)), 1.83 (d, 3H,

J=6.6, CH₃); ¹³C NMR (CDCl₃): d 18.5 (CH₃), 24.6

(CH₂), 25.6 (CH₂), 26.7 (CH₂), 43.1 (CH₂), 46.8 (CH₂),

118.3 (CH), 130.0 (CH), 137.0 (CH), 142.6 (CH), 165.7

(C); MS m/z (%): 179 (M⁺5), 168 (100), 167 (14), 138

(12), 112 (11), 85 (18), 83 (31), 69 (10), 47 (11). Calcd for

C₁₁H₁₇ON: C 73.70%, H 9.55%, N 7.81%; found: C

72.95%, H 9.34%, N7.81%.

141.2 (CH), 147.6 (C), 148.2 (C), 148.2 (C), 164.0 (C);

MS m/z (%): 301 (M⁺71), 218 (27), 217 (81), 188 (52),

182 (34), 148 (17), 140 (52), 127 (100), 115 (22), 84 (40).

8.24. Compounds (10a–d)

A mixture of monomethoxycinnamic acid (200 mg, 0.89

mmol, 1 equiv) and triethylamine (2.4 mL, 1.78 mmol, 2

eq) in dichloromethane (50 mL) was stirred for 15 min

at 0 ^ꢀC. To this mixture methane sulfonyl chloride (1.02

mL, 1.33 mmol, 1.5 equiv) was added and stirred for

further 30 min at 0 ^ꢀC. Piperidine (0.23 mL, 1.33 mmol,

1.5 eq) was added to the mixture and stirred for 1 h at

0 ^ꢀC and 1 h at room temperature. Then dichloro-

methane (50 mL) was added to the mixture, which was

then washed with 5% HCl (3Â100 mL), saturated aqu-

eous NaHCO₃(3Â100 mL) and water (3Â100 mL). The

organic fraction was dried over anhydrous sodium sul-

phate, ﬁltered and rotary evaporated to yield an oil.

This oil is puriﬁed by chromatography on silica gel

using ethylacetate/petroleum spirit (2:8) as an eluent

(10a–c). The piperidine amide of 3,4-dimethoxy-

cinnamic acid was prepared in the same way utilising

200 mg of the acid (10d).

8.22. Piperidine-5-(3,4-dihydroxyphenyl)penta-2E,4E-

dienoic acid amide (8)

Piperine (1) (500 mg, 1.75 mmol, 1 eq) was dissolved in

dichloromethane (DCM, 10 mL) and BBr3 in dichloro-

methane (0.7 mL, 8.7 mmol, 5 equiv) was added and

stirred at room temperature for 2 days. After the com-

pletion of reaction, DCM was removed under reduced

pressure and a yellow solid was obtained. The yellow

solid was washed with DCM and ﬁltered to give a pro-

duct (354 mg, 76.2%). ¹H NMR (MeOD): d 6.54 (d, 1H,

J=14.6, CH¼CH–CH¼CH), 7.32 (dd, 1H, J=9.5,

14.6, CH¼ CH–CH¼CH), 6.78 (dd, 1H, J=13.2, 9.5,

CH¼CH–CH¼CH), 6.75 (d, 1H, J=13.2 CH¼CH–

CH¼CH), 6.98 (d, 1H J=1.9, Ar-H), 6.74 (d, 1H J=8.2,

Ar-H), 6.85 (dd, 1H J=1.9, 8.2 Ar-H), 3.59 (br, 4H,

CH₂–N–CH₂(piperidine)), 1.59 (m, 6H, CH₂–CH₂–CH₂

(piperidine)); ¹³C NMR (MeOD): 25.9 (CH₂), 27.3 (CH₂),

28.2 (CH₂), 45.1 (CH₂), 48.6 (CH₂), 114.8 (CH), 116.9

(CH), 119.7 (CH), 121.7 (CH), 125.5 (CH), 141.7 (CH),

145.7 (CH), 147.0 (C), 148.3 (C), 148.3 (C), 168.3 (C).

8.25. 1-(2-methoxycinnamoyl)-piperidine (10a)

¹H NMR (CDCl₃): d 7.56 (d, 1H, CH¼CH), 7.29 (d,

1H, J=7.8 Ar-H), 7.12 (d, 1H, J=7.6 Ar-H), 7.0 (dd

1H, J=1.8 Ar-H), 6.86–6.90 (m, Ar-H), 6.88 (d, 1H,

J=15.4 CH¼CH), 3.58–3.66 (br, 4H, CH₂–N–CH₂

(piperidine)), 1.56–1.71 (m, 6H, CH₂–CH₂CH₂(piper-

idine)), 3.83 (s, 3H, OCH₃); ¹³C NMR (CDCl₃): d 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: 55 mg

(25.5%), mp 68–70 ^ꢀC.

8.23. Piperidine-5-(3,4-dimethoxyphenyl)penta-2E,4E-

dienoic acid amide (9)

Potassium hydroxide (328 mg) was added to dimethyl-

sulfoxide (5 mL, DMSO) and stirred under nitrogen

atmosphere for 5mins at room temperature. The com-

pound 8 (200 mg) was added to the mixture and fol-

lowed by iodomethane (0.3 mL) and stirred for another

30 min at same temperature. The mixture was poured

into water (50 mL) and extracted with dichoromethane

(3Â30 mL). The combined extracts were washed with

water (50 mL) and dried over sodium sulphate, ﬁltered

and rotary evaporated to yield a solid. Recrystallisation

using petroleum spirit and ethylacetate yielded pure

8.26. 1-(3-methoxycinnamoyl)-piperidine (10b)

¹H NMR (CDCl₃): d 7.60 (d, 1H, J=15.4, CH¼CH),

7.29 (d, 1H, J=7.8 ArH), 7.12 (d, 1H, J=7.6 Ar-H), 7.0

(dd 1H, J=1.8 Ar-H), 6.86–6.90 (m, Ar-H), 6.88 (d, 1H,

J=15.4 CH¼CH), 3.58–3.66 (br, 4H, CH₂–N–CH₂

(piperidine)), 1.56–1.71 (m, 6H, CH₂–CH₂–CH₂(piper-

idine)), 3.83 (s, 3H, OCH₃); ¹³C NMR (CDCl₃): d 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); mp 68–70 ^ꢀC.

Yield: 68 mg, (31.4%). Calcd for C₁₅H₁₉NO₂: C

73.42%, H 7.81%, N5.71%; found: C 73.37%, H

7.96%, N5.86%.

1

greenish crystals (122 mg, 58%). H NMR (CDCl₃): d

6.46 (d, 1H, J=14.6, CH¼CH–CH¼CH), 7.25–7.46 (m,

1H, CH¼CH–CH¼CH), 6.78 (1H, CH¼CH–CH¼CH),

6.80 (d, 1H, J=14.6 CH¼CH–CH¼CH), 6.98 (d, 1H

J=1.8, Ar-H), 6.83 (d, 1H J=8.1, Ar-H), 7.0 (dd, 1H

J=1.6, 8.1 Ar-H), 3.89 and 3.91 (2s, 6H, (O–CH₃–)₂),

3.63 (br, 2H, N–CH₂(piperidine)), 3.52 (b, 2H, N–CH₂

(piperidine), 1.64 (m, 2H, CH_2-CH₂(piperidine)), 1.87

8.27. 1-(4-methoxycinnamoyl)-piperidine (10c)

(m, 4H, CH₂–CH₂–CH₂(piperidine)); ¹³C

N

MR ¹H NMR (CDCl₃): d 7.61 (d, 1H, J=15.4, CH¼CH),

7.47 (d, 2H, J=7.8 Ar-H), 6.87–6.90 (m, 2H, Ar-H),

6.77 (d, 1H, J=15.4 CH¼CH), 3.58–3.65 (br, 4H, CH₂–

N–CH₂(piperidine)), 1.52–1.69 (m, 6H, CH₂–CH₂–CH₂

(CDCl₃): d 23.3 (CH₂), 24.2 (CH₂), 25.3 (CH₂), 41.8

(CH₂), 45.5 (CH₂), 54.4 (OCH₃), 54.5 (OCH₃), 107.5

(CH), 109.7 (CH), 118.4 (CH), 123.8 (CH), 137.0 (CH),

R. Venkatasamy et al. / Bioorg. Med. Chem. 12 (2004) 1905–1920

1917

(piperidine)), 3.82 (s, 3H, OCH₃); ¹³C NMR (CDCl₃): d

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). Yield:

79.1 mg (36.3%). Calcd for C₁₅H₁₉NO₂: C 73.44%, H

7.80%, N5.71%; found: C 73.49%, H 7.63%, N

5.89%.

(CH), 123.8 (CH), 129.7 (C), 141.0 (CH), 148.1 (C),

148.9 (C), 164.8 (C); MS m/z (%): 245 (M⁺62), 176

(41), 175 (100), 145 (36), 117 (11), 89 (14). Yield: 280 mg

(44.1%); mp 152.5–153 ^ꢀC. Calcd for C₁₄H₁₅NO₃: C

68.54%, H 6.16%, N5.71%; found: C 68.02%, H

6.26%, N5.74%.

8.32. 1-(3,4-Methylenedioxy-cinnamoyl)-morpholine (11c)

¹H NMR (CDCl₃): d 7.61 (d, 1H, J=15.3, CH¼CH),

6.73 (d, 1H, J=15.3, CH¼CH), 7.03 (d, 1H J=1.4, Ar-

H), 6.80 (d, 1H J=8.0, Ar-H), 7.01 (dd, 1H J=1.4, 8.0

Ar-H), 5.99 (s, 2H, O–CH₂–O), 3.72 (br, 4H, CH₂–N–

CH₂(morpholine)), 3.67 (br, 4H, CH₂-O-CH₂(mor-

pholine)), ¹³C NMR (CDCl₃): d 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). Yield: 339 mg (50.1%); mp 160–160.3 ^ꢀC. Calcd

for C₁₄H₁₅NO₄: C 64.34%, H 5.78%, N5.36%; found:

C 64.20%, H 5.88%, N5.38%.

8.28. 1-(3,4-dimethoxycinnamoyl)-piperidine (10d)

¹H NMR (CDCl₃), 60 MHz: d: 7.61 (1H, CH¼CH),

7.23 (1H, Ar-H), 6.98 (1H, Ar-H), 6.82 (1H, J=1.8 Ar-

H), 6.68 (1H, CH¼CH), 3.58–3.65 (br, 4H, CH₂–N–

CH₂(piperidine)), 1.5–1.8 (6H, CH₂–CH₂CH₂(piper-

idine)), 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: 111 mg (42.3%).

8.29. Compounds (11a–c)

A mixture of 3,4-(methylenedioxy)-cinnamic acid (500 mg,

0.0026 mol, 1 equiv) and triethylamine (0.54 mL, 3.9

mmol, 1.5 eq) in dichloromethane (50 mL) was stirred

for 15 min at 0 ^ꢀC. To this mixture methane sulfo-

nylcholride (0.3 mL, 3.9 mmol, 1.5 equiv) was added

and stirred for further 30 min at 0 ^ꢀC. The amine (1.5

equiv) was added to the mixture and stirred for 2 h at

0 ^ꢀC and 1 h at room temperature. Dichloromethane (50

mL) was added to the mixture, which was then washed

with water (3Â100 mL), 5% NaHCO₃(3Â100 mL) and

water (3Â100 mL). The organic fraction was dried over

anhydrous sodium sulphate, ﬁltered and rotary evapo-

rated to yield a brownish solid residue. Recrystallisation

from ethylacetate and petroleum spirit gave 3,4-

(methylenedioxy)-cinnamic acid amides (11a–c).

8.33. 3,4-(Methylenedioxy)-cinnamic acid methyl ester

(12)

Methanol (4 mL, 10 equiv) was added to 3,4-(methylene-

dioxy)-cinnamic acid (2 g, 0.01 mol, 1 equiv). 0.2 mL of

sulphuric acid was added to the methanolic solution and

reﬂuxed overnight. The solvent was rotary evaporated

to yield solid residue. This residue was dissolved in ether

and washed with water (2Â100 mL) and 5% NaHCO₃

(3Â100 mL) and with water (2Â100 mL). The organic

fraction was dried over anhydrous sodium sulphate and

rotary evaporated to yield white solid. Recrystallisation

from ethylacetate/petroleum spirit yielded crystals (1.42

g, 69.4% yield); mp 133.7–134.2 ^ꢀC (lit. mp 134 ^ꢀC). H

1

NMR (CDCl₃): d 7.59 (d, 1H, J=15.9, CH¼CH), 6.26

(d, 1H, J=15.9, CH¼CH), 7.03 (d, 1H J=1.5, Ar-H),

6.81 (d, 1H J=8.0, Ar-H), 7.01 (dd, 1H J=1.5, 8.0 Ar-

H), 6.00 (s, 2H, O–CH₂–O), 3.79 (s, 3H, OCH₃); ¹³C

NMR (CDCl₃): d 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).

8.30. 1-(3,4-Methylenedioxy-cinnamoyl)-piperidine (11a)

¹H NMR (CDCl₃): d 7.56 (d, 1H, J=15.3, CH¼CH),

6.73 (d, 1H, J=15.3, CH¼CH), 7.03 (d, 1H J=1.5, Ar-

H), 6.79 (d, 1H J=8.0, Ar-H), 6.99 (dd, 1H J=1.6, 8.0

Ar-H), 5.98 (s, 2H, O–CH₂–O), 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 (CDCl₃):

d 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 (C),

148.8 (C), 165.4 (C). Yield :328 mg (58.2%); mp 80.1–82 ^ꢀC

(lit. mp 80–82 ^ꢀC). Calcd for C₁₅H₁₇O₃N: C 69.47%, H

6.60%, N5.40%; found: C 69.56%, H 6.58%, N5.39%.

8.34. 7-(3,4-methylenedioxyphenyl)-2E,4E,6E-heptatri-

enoic acid piperidine amide (14)

1-[(2E,4E)-Hexadienoyl]-piperidine (7b) (500 mg, 2.79

mmol) piperonal (13) (419 mg, 2.69 mmol) and aliquat

336 (50 mg) were dissolved in toluene and anhydrous

potassium carbonate (100 mg) and sodium hydride (20

mg) was added and reﬂuxed under nitroger for 6 h at

90 ^ꢀC. The reaction mixture was poured into water and

extracted with dichloromethane (3Â100 mL). The

organic layer was dried over anhydrous sulphate, ﬁl-

tered and rotary evaporated to yield a solid residue. The

mixture was puriﬁed by column chromatography using

hexane:ethylacetate (1:9) as an eluent to give yellow

8.31. 1-(3,4-Methylenedioxy-cinnamoyl)-pyrrolidine (11b)

¹H NMR (CDCl₃): d 7.60 (d, 1H, J=15.2, CH¼CH),

6.73 (d, 1H, J=15.3, CH¼CH), 7.04 (d, 1H J=1.5, Ar-

H), 6.80 (d, 1H J=8.0, Ar-H), 7.01 (dd, 1H J=1.5, 8.0

Ar-H), 5.99 (s, 2H, O–CH₂–O), 3.61 (br, 2H, CH₂–N–

CH₂(pyrrolidine)), 3.57 (br, 2H, CH₂–N–CH₂(pyrroli-

dine)), 1.99 (4H, CH₂–CH₂(pyrrolidine)), ¹³C N MR

(CDCl₃): d 24.3 (CH₂), 26.1 (CH₂), 46.0 (CH₂), 46.5

(CH₂), 101.4 (CH₂), 106.4 (CH), 108.5 (CH), 116.8

1

solid H NMR (CDCl₃): d 6.95 (d, 1H J=1.6, Ar-H),

6.76 (d, 1H J=8.0, Ar-H), 6.85 (dd, 1H J=1.6, 8.0 Ar-H),

1918

R. Venkatasamy et al. / Bioorg. Med. Chem. 12 (2004) 1905–1920

6.37 (d, 1H J=14.6, CH¼CH–CH¼CH–CH¼CH),

6.42 (dd, 1H J=11.4, 14.0, CH¼CH–CH¼CH–

CH¼CH), 7.35 (dd, 1H J=11.4, 14.6, CH¼CH–

CH¼CH–CH¼CH), 6.59 (d, 1H J=15.0, CH¼CH–

CH¼CH–CH¼CH), 6.66 (dd, 1H J=15.0, 10.0,

CH¼CH–CH¼CH–CH¼CH), 6.63 (dt, 1H J=10.0,

14.0, CH¼CH–CH¼CH–CH¼CH), 5.96 (s, 2H, O–

CH₂–O), 3.51 (br s, 2H, CH_2-N–CH₂), 3.62 (br s, 2H,

J=5.7, (CH₂–N–CH₂), 1.54-1.59 (m, 4H, CH₂–CH₂–

CH₂(piperidine)), 1.60–1.68 (m, 2H, CH₂–CH₂–CH₂

(piperidine)); ¹³C NMR (CDCl₃): d 25.0 (CH ), 26.0

(CH), 27.1 (CH), 43.6 (CH), 47.3 (CH), 101.6 (CH₂),

105.8 (CH), 108.8 (CH), 120.4 (CH), 122.4 (CH), 127.0

(CH), 130.8 (CH), 135.8 (CH), 139.5 (CH), 142.7 (CH),

135.9 (C), 148.1 (C), 148.5 (C), 165.8 (C); MS m/z (%):

311 (M⁺99), 301 (18), 227 (27), 217 (21), 199 (42), 182

(30), 169 (59), 149 (29), 141 (100), 127 (86), 115 (43), 84

(81), 69 (43). Yield 45.6%; mp 144.3–145.6 ^ꢀC.

propanoic acid and piperidine as the acid and amine

components respectively. A mixture of 3-benzo[1,3]-

dioxol-5-yl-propionic acid (200 mg, 0.0026 mole, 1

equiv) and triethylamine (0.27 mL, 0.002 mol, 2 eq) in

dichloromethane (50 mL) was stirred for 15 min at 0 ^ꢀC.

To this mixture methane sulfonyl chloride (0.11 mL,

0.0015 mol, 1.5 equiv) was added and stirred for further

30 min at 0 ^ꢀC. Piperidine (0.15 mL, 0.0015 mole, 1.5 eq)

was added to the mixture and stirred for 1 h at 0 ^ꢀC and

1 h at room temperature. Dichloromethane (50 mL) was

added to the mixture which was then washed with 5%

HCl (3Â100 mL), saturated aqueous NaHCO₃(3Â100

mL) and water (3Â100 mL). The organic fraction was

dried over anhydrous sodium sulphate, ﬁltered and

rotary evaporated to yield a brown oil (203 mg, 65%

1

yield). H NMR (CDCl₃): d 2.87 (t, 2H, J=7.3 CH₂),

2.57 (t, 2H, J=7.0 CH₂–CH₂), 6.70 (d, 1H J=1.5, Ar-

7H), 6.72 (d, 1H J=8.0, Ar-10H), 6.66 (dd, 1H J=1.2,

8.0 Ar-11H), 5.90 (s, 2H, O–CH₂–O), 3.55 (t, 2H, N–

CH₂(piperidine)), 3.34 (t, 2H, N–CH₂(piperidine)),

1.62 (m, 2H, CH₂–CH_2-CH₂(piperidine)), 1.49 (m, 2H,

CH₂–CH₂–CH₂(piperidine)); ¹³C NMR (CDCl₃): d

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).

8.35. 5-(3,4-methylenedioxyphenyl)-penta-2E,4E-dienyl

piperidine (15)

Piperine (1) (1 g, 3.5 mmol) and NaBH₄(0.35 g, 8.75

mmol) were stirred in THF (20 mL) at 0 ^ꢀC under

nitrogen atmosphere. Iodine (0.65 g, 2.62 mmol) was

added dropwise to the mixture and left for 2 days. The

reaction was quenched with methanol until the eﬀer-

vescence ceased. The solvent was rotary evaporated to

yield a residue (0.9 g). This residue was puriﬁed by col-

umn chromatography on silicagel (31 cm/10 mm) using

petroleum spirit/ethylacetate as an eluent. Recrystalli-

sation of the fraction from methanol gave white crystals

(16) (120 mg, 13%). mp 126.7–127.3 ^ꢀC. ¹H N MR

(CDCl₃): d 3.48 (d, 2H, J=7.4 CH¼CH–CH¼CH–

CH₂), 6.1 (m, 1H, J=7.5, 15.1, CH¼CH–CH¼CH),

6.35 (dd, 1H, J=15.1, 10.2, CH¼CH–CH¼CH), 6.67

(dd, 1H, J=10.2, 15.4, CH¼CH–CH¼CH), 6.48 (d,

1H, J=15.6 CH¼CH–CH¼CH), 6.94 (d, 1H J=1.6,

Ar-H), 6.76 (d, 1H J=8.0, Ar-H), 6.82 (dd, 1H J=1.5,

8.0 Ar-H), 5.97 (s, 2H, O–CH₂–O), 3.57 (br, 2H, CH₂–

N–CH₂), 3.65 (br, 2H, CH₂–N–CH₂(piperidine)), 1.65

(m, 6H, CH₂–CH₂–CH₂– (piperidine)); ¹³C N MR

(CDCl₃): d 20.5 (CH₂), 20.5 (CH₂), 22.7 (CH₂), 53.0

(CH₂), 57.8 (CH₂), 101.2 (CH₂), 124.1 (CH), 108.4

(CH), 147.6 (C), 148.1 (C), 105.5 (CH), 131.3 (C), 133.5

(CH), 126.2 (CH), 137.1 (CH), 121.6 (CH), 57.78 (CH₂);

MS (m/z) 271 (M⁺) 187, 157, 136, 110, 98, 84, 55. Calcd

for C₁₇H₂₁NO₂.H₂O: C 70.23%, H 8.01%, N4.84%;

found: C 69.79%, H 7.72%, N4.47%.

8.38. Tetrahydropiperine (19)

Piperine (1) (2 g, 7 mmol) was hydrogenated in ethanol

(50 mL) over 5% Pd-C under a pressure of hydrogen at

10 PSI for 30 min to give tetrahydropiperine (1.59g,

78%) as an oil. ¹H NMR (CDCl₃): d 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, 1H J=1.3, Ar-H), 6.70 (d, 1H

J=8.0, Ar-H), 6.61 (dd, 1H J=1.2, 8.0 Ar-H), 5.89 (s,

2H, O–CH₂–O), 3.53 (t, 2H, N–CH₂(piperidine)), 3.35

(t, 2H, N–CH₂(piperidine)), 1.63 (m, 2H, CH₂–CH₂–

CH₂(piperidine)), 1.54 (m, 2H, CH₂–CH₂–CH₂(piper-

idine)); ¹³C NMR (CDCl₃): d 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 m/z (%): 289 (M⁺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). Calcd

for C₁₇H₂₃NO₃: C 70.54%, H 8.01%, N4.84%; found:

C 70.70%, H 8.38%, N4.85%.

8.39. 5-(3,4-Methylenedioxyphenyl)-pentanoicacid cyclo-

hexyl amide (20)

8.36. 5-(3,4-methylenedioxyphenyl)-propanoic acid (17)

3,4-methylenedioxycinnamic acid (2g) was hydro-

genated in ethanol (50 mL) over 5% Pd-C under a

pressure of hydrogen at 10 PSI for 40 min to give 5-(3,4-

methlyenedioxyphenyl)-propanoic acid (1.67 g, 80%

yield) as a solid. mp 86.1–88.3 ^ꢀC (lit. mp 87–88 ^ꢀC).

5% Pd/C (30 mg) was added to 5-(3,4-methylenedioxy

phenyl)-2E,4E-pentadienoic acid cyclohexyl amide 4j

(300 mg) and hydrogenated at 30 psi for 1 h. The solu-

tion was ﬁltered and rotary evaporated to yield a white

solid. Recrystallisation from ethylacetate and petroleum

spirit yielded pu_ꢀre white crystals (255 mg, yield 84%).

1

mp 145.4–146.3 C. H NMR (CDCl₃): d 6.65 (d, 1H

8.37. 5-(3,4-methylenedioxyphenyl)-propanoyl piperidine

(18)

J=1.6, Ar-H), 6.71 (d, 1H J=7.8, Ar-H), 6.60 (dd, 1H

J=1.6, 8.0 Ar-H), 5.90 (s, 2H, O–CH₂–O), 5.43 (s, 1H,

NH), 3.87 (m, 1H, CH (cyclohexyl amide)), 2.53 (t, 2H,

J=7.7 (CH₂–CH₂–CH₂CH₂)), 2.14 (t, 2H, J=7.7

The method was adapted from that reported for piper-

longuminine⁸but utilising 1-(3,4-methlyenedioxyphenyl)-

R. Venkatasamy et al. / Bioorg. Med. Chem. 12 (2004) 1905–1920

1919

((CH₂–CH₂–CH₂–CH₂)), 1.62–1.91 (m, 10H, CH₂–

8.42. Cell proliferation

CH₂–CH₂–CH₂, CH₂–CH₂–CH₂(cyclohexyl amide),

1.07–1.30 (m, 4H, CH₂–CH–CH₂(cyclohexylamide));

¹³C NMR (CDCl₃): d 25.3 ((CH₂)₂), 25.7 (CH₂), 25.9

(CH₂), 31.3 (CH₂), 31.7 (CH₂), 33.6 (CH₂), 35.8 (CH₂),

37.3 (CH₂), 48.4 (CH), 101.1 (CH₂), 108.4 (CH), 109.2

(CH), 121.4 (CH), 136.4 (C), 145.8 (C), 147.8 (C), 172.2

(C); MS m/z (%): 303 (M⁺98), 204 (72), 176 (13), 168

(16), 162 (12), 161 (14), 154 (27), 148 (66), 141 (61), 135

(100), 74 (24), 60 (60). Calcd for C₂₀H₁₇NO₅: C 71.24%,

H 8.31%, N4.62%; found C 71.22%, H 8.66%, N

4.61%.

Melan-a cells were diluted with RPMI 1640 medium.

Aliquots (100 mL) containing 0.6Â10⁴cells were inocu-

lated into 96 well microlitre plates using a Finn Pipette.

A 50 mL volume of each compound solution was added

into 6 replicates of 100 mL of cells in 96-well microtitre

plates. The ﬁnal concentration of each compound in the

well was 1–100 mM. After adding compounds into

the melan-a cells (96 well plate), the plates were incu-

bated at 37 ^ꢀC in a 10% CO₂, 90% air humidiﬁed

atmosphere incubator for 4 days.

After the stated length time of incubation, cells were

attached to the substratum of the plate. These cells

were ﬁxed by addition of 50% of cold trichloroacetic

acid (TCA, 40 mL) solution (20% v/v) on top of the cells

and growth medium already present in each well. The

plate was incubated at 4ÂC for 1 h. After the incubation

period, each well was gently washed with tap water to

remove TCA, growth medium and dead cells and

allowed to dry in air.

8.40. 7-(3,4-Methylenedioxyphenyl)-heptanoic acid piper-

idine amide (21)

7-(3,4-Methylenedioxyphenyl)-hepta-2E,4E,6E-trienoyl

piperidine 14 (150 mg) was dissolved in 30 mL of etha-

nol. 5% Pd/C (15 mg) was added and the contents

hydrogenated at 30 psi. The solution was ﬁltered and

rotary evaporated to yield oil (83 mg, 43.6%). ¹H N MR

(CDCl₃): d 6.66 (d, 1H J=1.5, Ar-H), 6.71 (d, 1H

J=7.8, Ar-H), 6.60 (d, d, 1H J=1.6, 8.0 Ar-H), 5.90 (s,

2H, O–CH₂–O), 3.53 (t, 2H, J=5.4 CH_2-N–CH₂), 3.37

(t, 2H, J=5.7, (CH₂–N–CH₂), 2.51 (t, 2H, J=7.7

(CH₂–CH₂–CH₂–CH₂–CH₂–CH₂)), 2.33 (t, 2H, J=7.7

((CH₂–CH₂–CH₂–CH₂–CH₂–CH₂)), 1.52-1.65 (m, 10H,

To the dried cells, 50 mL volume of SRB (Sulphorho-

damine B) dissolved in 1% acetic acid in water was

added to each well and left for 30 min. At the end of the

staining period, unbound SRB was removed by washing

5 times with 1% acetic acid. The plate was air-dried and

150 mL of Tris base tris (hydroxymethyl) aminomethane

was added into each well to solubilize the cell bound

dye. The plate was shaken for 15–30 min on a gyratory

shaker followed by reading the optical density (OD) at

550nm in a microplate spectrophotometer.

CH₂–CH₂–CH₂–CH₂–CH₂–CH₂,

CH₂–CH₂–CH₂

(piperidine)), 1.34 (m, 4H, CH₂–CH₂–CH₂–CH₂–CH₂–

CH₂); ¹³C NMR (CDCl₃): d 24.9 (CH₂), 25.8 (CH₂),

25.9 (CH₂), 26.9 (CH₂), 29.3 (CH₂), 29.7 (CH₂), 31.3

(CH₂), 31.9 (CH₂), 33.8 (CH₂), 42.9 (CH₂), 47.1 (CH₂),

101.8 (CH₂), 108.4 (CH), 109.2 (CH), 121.4 (CH), 137.0

(C), 145.7 (C), 147.8 (C), 171.8 (C); MS m/z (%): 317

(M⁺78), 232 (11), 204 (10), 183 (30), 182 (15), 154 (21),

148 (43), 141 (41), 127 (100), 112 (43), 85 (49).

8.43. Measurement of dendricity

Three sterile, round cover slips were placed in 60-mm

culture dishes. Aliquots (2.9 mL) containing 1.5Â10⁴/

mL cells were plated in the 60-mm culture dishes. A 100

mL volume of each compound solution was added into 3

replicates of 2.9 mL of cells in the culture dishes. The

ﬁnal concentration of each compound in the culture

dish was 10 mM. After adding compounds and media

into the melan-a cells (culture dish), the dishes were

incubated at 37 ^ꢀC in a 10% CO₂, 90% air humidiﬁed

atmosphere incubator for 3 days.

8.41. Determination of distribution coeﬃcients

Distribution coeﬃcients between n-octanol and phos-

phate buﬀer (pH 7.4) were determined using the shake

ﬂask method. The aqueous and organic phases were

presaturated with respect to each other before use.

Compounds were dissolved in octanol (at 0.25 mM to

0.5 mM) and UV absorbance was measured before

mixing with buﬀer. A 10 mL aliquot of octanol was

taken and 100 mL of phosphate buﬀer was added to the

solution and shaken well for 4 h. The solution was left

overnight at 25 ^ꢀC. After equilibrium, the octanol phase

was collected very carefully and UV absorbance

was obtained. The distribution coeﬃcient (D_7.4) was

calculated using the following equation. This experi-

ment was repeated for four times and the average value

taken.

After incubation, viable cells were found to be attached

to the cover slips. The growth medium and dead cells

were removed by aspiration and the attached cells

gently washed with sterile phosphate buﬀer saline (PBS)

twice. These cells were ﬁxed by addition of 2 mL of

methanol: acetone (1:1) to the culture dish. The dish

was incubated at ꢁ20 ^ꢀC for 10 min. Each dish was then

gently washed with PBS to remove methanol: acetone

and followed by addition of 2 mL of PBS and culture

dishes were stored at 4 ^ꢀC.

A₁V_w

D_7;4

¼

A₀ꢀ A₁V_o

Staining procedures were carried out in order to view

the cells and dendrites eﬀectively. Eosin 1% (Raymond

Alamb Ltd) was added to the culture dish and left for 1

min. After washing with tap water, 0.1% haematoxylin

(Sigma Aldrich) was added to the dish and left for 5

A₀— Absorbance of n-octanol before addition of buﬀer

A₁— Absorbance of n-octanol layer after equilibrium

V_w— Volume of aqueous layer

V_o— Volume of octanol

1920

R. Venkatasamy et al. / Bioorg. Med. Chem. 12 (2004) 1905–1920

min. The cover slips in the dish were then washed with

tap water. After staining the cells within the dish, cover

slips were mounted in D.P.X mounting medium (Ray-

mond Alamb Ltd) on a glass slide and representative

ﬁelds were taken through a phase contrast microscope

(Nikon). These were used to assess melanocyte mor-

phology and dendrite formation. At least a total of 30

cells from 3 cover slips were evaluated for each com-

pound. Cell diameter and dendrite length was measured

with a computer aided image analyzer (PC_IMAGE for

Windows version 5.3).

texed for 10 min. The melanin content was calculated

on the basis of absorbance at 475 nm and compared

with standard curve of synthetic melanin. Synthetic

melanin was analysed spectrophotometrically over the

range of 3.25–100 mg/mL.

Acknowledgements

Mr. R. Venkatasamy was funded by Overseas Research

Students Award Scheme (ORS) and the project was

funded by British Technology Group (BTG), UK. The

authors also wish to thank Prof. D. C. Bennett of St.

George Medical School, UK for the gift of the melan-a

cell line.

Number of dendrites per cell was calculated as: total

number of dendrites / number of cells

Total average dendrite length per cell was calculated on

the basis of:

References and notes

total dendrite length / number of cells

1. Ortonne, J. P., Mosher, D. B., Fitzpatrick, D. B., Eds.

Vitiligo and Other Hypomelanosis of Hair and Skin;

Plenum: New York/London, 1983; pp 129–310.

2. Donata, K.; Austin, M.; Rajagopalan, K. Ancient Science

of Life 1990, 9, 202.

Average dendrite length was calculated by using:

total dendrite length / number of dendrites

3. Lin, Z.; Hoult, R.; Raman, A. J. Ethnopharmacol. 1999,

66, 141.

4. Lin, Z.; Hoult, R.; Bennet, D.; Raman, A. Planta Med.

1999, 65, 600.

5. Bennett, D. C.; Cooper, P. J.; Hart, I. R. Int. J. Cancer

1987, 39, 414.

6. Koul, S.; Koul, J. L.; Taneja, S. C.; Dhar, K. L.; Jamwal,

D. S.; Singh, K.; Reen, R. K.; Singh, J. Bioorg. Med.

Chem. 2000, 8, 251.

7. Paula, V. F.de.; Barbosa, A.; Luiz, C.de.; Demuner, A. J.;

Pilo-Velosa, D.; Picanco, M. C. Pest. Manag. Sci. 2000,

56 (2), 168.

8. Chatterjee, A.; Dutta, C. P. Tetrahedron 1967, 23, 1769.

9. Johnstone, R. A. W.; Rose, M. E. Tetrahedron 1979, 35,

2169.

8.44. Melanin assay

Melan-a cells (2.9 mL) were plated in a 60 mm culture

dish at a density of 2Â10⁴cells/mL. Piperine and its

analogues were prepared by dissolving in methanol and

diluting in medium. A 100 mL aliquot of piperine or

piperine analogues were added to 4 replicates of each

dish to get a ﬁnal concentration of 10 mM. After adding

the compounds the dishes were incubated at 37 ^ꢀC in a

10% CO₂, 90% air humidiﬁed atmosphere incubator

for 3 days. The concentration of methanol in the ﬁnal

volume of culture was 0.33% and this concentration

was found to be non-toxic to the cells (data not shown).

10. Dehmlow, E. V.; Shamout, A. R. J. Chem. Res.-S. 1981,

106.

11. Das, B.; Kasinathan, A.; Madhusudhan, P. Tetrahedron

Lett. 1998, 39, 677.

12. http://www.raell.demon.co.uk/chem./logp/logppka.html.

13. http://www.daylight.com.

14. Eisenger, M.; Marko, O. P. Natl. Acad. Bio-Sci. 1982, 79,

2018.

After incubation, the medium was removed and the cells

were washed with sterile PBSA and trypsinized using

250 mg/mL trypsin/EDTA at 37 ^ꢀC for 5–7 min. Cells

were collected and centrifuged at 1100 rpm for 7 min. A

pellet was formed and the media was removed.

Fresh medium (2 mL) was added to the pellet. After

dispersing the cells, 1 mL of melan-a cells were cen-

trifuged again and the medium was removed. The

remaining pellet was dissolved in 1.0M NaOH and vor-

15. Cui, J.; Shen, L.; Wang, G. J. Invest. Dermatol. 1991,

97 (3), 410.

16. Burgoyone, R. D.; Peterson, O. H.; Portland Press: London,

1997; pp 139–151.

Article Doi

Effects of piperine analogues on stimulation of melanocyte proliferation and melanocyte differentiation

DOI: 10.1016/j.bmc.2004.01.036

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Full text of DOI:10.1016/j.bmc.2004.01.036

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