Structure-activity relationship of piperine and its synthetic analogues for their inhibitory potentials of rat hepatic microsomal constitutive and inducible cytochrome P450 activities

Article abstract of DOI:10.1016/S0968-0896(99)00273-4

Inhibitors of drug metabolism have important implications in pharmaco- toxicology and agriculture. We have reported earlier that piperine, a major alkaloid of black and long peppers inhibits both constitutive and inducible cytochrome P450 (CYP)-dependent drug metabolising enzymes. In the present study, an attempt has been made to prepare several novel synthetic analogues so as to relate various modifications in the parent molecule to the inhibition of CYP activities. Two types of mono-oxygenase reactions arylhydrocarbon hydroxylase (AHH) and 7-methoxycoumarin-O-demethylase (MOCD) have been studied. Inhibition studies were investigated in rat microsomal fraction prepared from untreated, 3MC- and PB- treated rat liver in vitro. Modifications were introduced into the piperine molecule: (i) in the phenyl nucleus, (ii) in the side chain and (iii) in the basic moiety. Thus, 38 compounds have been subjected to such studies, and simultaneously an attempt has also been made to arrive at the structure-activity relationship of synthetic analogues. In general, most of the inhibitory potential of the parent molecule is lost with modification in either of the three components of piperine. Saturation of the side chain resulted in significantly enhanced inhibition of CYP while modifications in the phenyl and basic moieties in few analogues offered maximal selectivity in inhibiting either constitutive or inducible CYP activities. Thus few novel analogues as CYP inactivators have been synthesized which may have important consequences in pharmacokinetics and bioavailability of drugs. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(99)00273-4

Bioorganic & Medicinal Chemistry 8 (2000) 251±268
Structure±Activity Relationship of Piperine and its Synthetic
Analogues for their Inhibitory Potentials of Rat Hepatic
Microsomal Constitutive and Inducible Cytochrome P450 Activities
Surrinder Koul, a Jawahir L. Koul, a Subhash C. Taneja, a Kanaya L. Dhar, a
Deshvir S. Jamwal, b Kuldeep Singh, b Rashmeet K. Reen b and Jaswant Singh b,*
aNatural Products Chemistry Division, Regional Research Laboratory (CSIR), Jammu-tawi 180001, India
bBiochemistry Laboratory, Division of Pharmacology, Regional Research Laboratory (CSIR), Jammu-tawi 180001, India
Received 16 July 1999; accepted 25 September 1999
AbstractÐInhibitors of drug metabolism have important implications in pharmaco-toxicology and agriculture. We have reported
earlier that piperine, a major alkaloid of black and long peppers inhibits both constitutive and inducible cytochrome P450 (CYP)-
dependent drug metabolising enzymes. In the present study, an attempt has been made to prepare several novel synthetic analogues
so as to relate various modi®cations in the parent molecule to the inhibition of CYP activities. Two types of mono-oxygenase
reactions arylhydrocarbon hydroxylase (AHH) and 7-methoxycoumarin-O-demethylase (MOCD) have been studied. Inhibition
studies were investigated in rat microsomal fraction prepared from untreated, 3MC- and PB- treated rat liver in vitro. Modi®ca-
tions were introduced into the piperine molecule: (i) in the phenyl nucleus, (ii) in the side chain and (iii) in the basic moiety. Thus, 38
compounds have been subjected to such studies, and simultaneously an attempt has also been made to arrive at the structure±
activity relationship of synthetic analogues. In general, most of the inhibitory potential of the parent molecule is lost with mod-
i®cation in either of the three components of piperine. Saturation of the side chain resulted in signi®cantly enhanced inhibition of
CYP while modi®cations in the phenyl and basic moieties in few analogues o€ered maximal selectivity in inhibiting either con-
stitutive or inducible CYP activities. Thus few novel analogues as CYP inactivators have been synthesized which may have
important consequences in pharmacokinetics and bioavailability of drugs. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
drug bioavailability consequent to modulation of drug
metabolism.2 Subsequently, it was shown to enhance the
bioavailabilty of phenytoin in healthy volunteers,8
reduce a¯atoxin B1 binding of DNA9 and protected
hepatoma cells4 and V79 constructs of rat CYP2B110
in cultures from cytotoxicity and genotoxicity of AFB1
by impairing CYP mediated activation of the myco-
toxin. Piperine also produced di€erential inhibition of
glucuronidation in guinea pig enterocytes and rat liver,
while conjugated double bonds appeared essential for in
vitro inhibition of hepatic UDP-glucose dehydrogenase6
irrespective of the oxidation state of piperidine or
methylenedioxyphenyl (MDP) rings. Piperine, thus,
appears pharmacologically an important molecule
despite the fact that it is a natural compound of very
low toxicological consequences11 and has been in use
the world over for the palatability of food in the form of
black pepper. At least four major metabolites of piper-
ine from human urine have been reported, viz. 5-(3,4-
dihydroxy phenyl)-2,4-pentadienoic acid piperidide and
its 4-hydroxy-piperidine analogues and their respective
Piperine (trans-trans-isomer of 1-piperoyl piperidine) is
a major ingredient of piper species, Piper nigrum Linn
and Piper longum Linn, which are commonly used as
spices and in various traditional systems of medicines.1
Earlier studies from this laboratory have demonstrated
that piperine inhibits several constitutive and inducible
cytochrome P450 (CYP) activities in vitro and in vivo.2±7
We postulated that the use of piperine in the form of
piper species in several traditional herbal formulations
might have been responsible for the enhancement of
Keywords: piperine analogues; rat microsomes; cytochrome P450
inhibition; structure±activity relationship.
Abbreviations: AHH, arylhydrocarbon hydroxylase; BP, benzo(a)-
pyrene; CYP, cytochrome P450; MOC, 7-methoxycoumarin; MOCD,
7-methoxycoumarin-O-demethylase; 3MC, 3-methylcholanthrene;
MDP, methylenedioxyphenyl; PB, phenobarbital.
*Corresponding author. Tel.: +191-572002/579117/549084; fax:
+191-548607/546383; e-mail: rrlj@nde.vsnl.net.in
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00273-4

252
S. Koul et al. / Bioorg. Med. Chem. 8 (2000) 251±268
tetrahydro-analogues12 without any studies of their
e€ect on drug metabolism. We became primarily inter-
ested to know whether or not any structural modi®ca-
tions in the parent molecule piperine would result in
di€erential inhibition of rat hepatic constitutive and
inducible CYP activities. In addition, such studies might
also prove useful in developing speci®c inactivators of
certain CYPs from natural lead bioactive molecules
such as piperine.
appeared to be the consequence of direct interaction of
piperine with CYP1A1 gene product.7 Thus apparent
shortcomings, if any, in piperine molecule could per-
haps be overcome by introducing various structural
modi®cations in the molecule.
In this study we, therefore, have introduced several
modi®cations in piperine so as to correlate its structure
to the inhibition of constitutive and inducible rat hepa-
tic CYP activities. For this purpose microsomes from
untreated-, 3MC and PB -treated rat liver and two types
of diagnostic substrates benzo(a)pyrene and 7-methoxy-
coumarin have been used. Piperine earlier was reported
to inhibit atleast two CYP-dependent marker reac-
tions, viz. arylhydrocarbon (benzo(a)pyrene) hydro-
xylase (AHH) and 7-methoxycoumarin O-demethylase
(MOCD) in H4IIEC3 cells.4 The former reaction pri-
marily is mediated by members of CYP family which
are inducible by polycyclic aromatic hydrocarbons, i.e.
CYP1A.20 The latter is catalyzed by constitutive, phe-
nobarbital or dexamethasone inducible CYP forms.21
Further, MOCD activity was observed in preparations
of SD1 cells containing only CYP2B1, the major PB-
inducible CYP of rat liver and not in XEM1 cells
containing only CYP1A1.21 It may be mentioned that
dexamethasone and PB- inducible CYPs involve several
members of CYP2 and CYP3 families. Measurement of
the activities of AHH and MOCD assayed in the pre-
sent study o€er a simple system of choice for in vitro
screening of a large number of synthetic analogues of
piperine as CYP inhibitors in contrast to the measure-
ment of hexobarbital induced sleeping time in intact
animals.22 In this study we, therefore, report the
synthesis of several piperine analogues (Table 1) and
attempted to correlate their structures with the inhibi-
tion of constitutive and inducible CYP activities. The
study may be found very useful in developing new ana-
logues as selective inactivators of CYPs.
A number of distinct CYPs are present in normal
hepatic and extrahepatic tissues whose relative popula-
tion may be altered due to the in vivo exposure to drugs
and chemicals. For instance, 3-methylcholanthrene
(3MC) induces members of CYP1A family and pheno-
barbital (PB) induces several members of CYP2 and
CYP3 families.13 Accordingly, the disposition of drugs
and CYP substrates would proceed along pathways dif-
ferent from normal tissues. Therefore, the inhibition or
induction of drug metabolising enzymes has important
consequences on the pharmacokinetics of drugs and
hence their bioavailability. For that matter, a large
number of inhibitors of CYP have reportedly been
evaluated for the management of breast or uterine can-
cer, in¯ammation and other diseases.13±15 In this con-
cern use of aromatase inhibitors16 and development of
synergists as inhibitors of CYPs have already shown
their due bene®ts. Moreover, in clinical therapeutics, the
treatment regimens ought to be changed because of the
inducibility of particular CYPs towards a drug substrate
(s). It is, therefore, desirable to search for potential CYP
inhibitors with maximal isoenzymic selectivity which
indeed appears extremly dicult in view of the diversity
and overlapping substrate speci®city of various CYP
isoforms.
Piperine structure (Fig. 1) consists of three important
components, viz. methylenedioxyphenyl (MDP) ring,
side chain with conjugated double bonds and a basic
piperidine moiety attached through a carbonylamide
linkage to side chain. Each of these moieties might
in¯uence the constitutive and inducibility characteristics
of various CYPs. Furthermore, an MDP ring appears to
be a common functional group of several naturally
occuring compounds of pharmacological importance17
contributing signi®cantly to modulate drug meta-
bolism.18 The natural alkaloid consumed world-wide
produces di€erential inhibition of CYPs.2,19 When
compared to another MDP-containing insecticide
synergist piperonyl butoxide, the latter produced a
strong biphasic e€ect, an initial inhibition followed by
induction.19 Though piperine also induced the CYP1A1
activity by transcription activation, the overall inhibi-
tion of benzo(a)pyrene metabolism and AHH activity
Results
Structure±activity relationship of substituted
phenylpentadienoic acid derivatives with the inhibition of
hepatic microsomal monooxygenase activitities in vitro
The speci®c activities of AHH and MOCD in micro-
somes from untreated, 3MC- and PB-treated rat liver,
and the e€ect of piperine thereon are given in Table 2.
These values are taken as controls for comparing the
CYP activities of corresponding microsomes under the
in¯uence of various piperine analogues. Piperine as such
produced concentration-dependent and equipotent
inhibition of both constitutive and inducible AHH and
MOCD activities. Similarly, the in¯uence of various
piperine analogues on the CYP activities of liver micro-
somes from untreated, 3MC- and PB- treated rats was
investigated in vitro at three di€erent concentrations of
10, 30 and 100 mM (Tables 3 and 4). The mono-
oxygenases inhibitory potential of each compound was
compared with the parent molecule piperine by evalu-
ating the IC50 values, the concentration which brings
about 50% of enzymatic inhibition.
Figure 1. Structure of piperine.

S. Koul et al. / Bioorg. Med. Chem. 8 (2000) 251±268
253
Table 1.
Compound
Structure
MF
Mp ( ꢀC)
135
Compound
Structure
MF
Mp (ꢀC)
8
C17H21NO2
23
C17H19NO3
113
116
24
25
26
27
28
C24H27NO3
C23H25NO3
C23H27NO3
C19H25NO4
C18H23NO4
9
C16H19NO2
C17H22N2O2
C17H26N2O2
137
201
183
147
10
11
155
183
214
190
12
13
14
15
16
17
18
C16H17NO3
C16H19NO3
C17H23NO3
C16H21NO3
C16H23NO3
C16H23NO3
C18H23NO3
144
a
a
a
a
29
30
C21H27NO2
C20H25NO2
164
117
31
32
33
34
35
36
37
C21H29NO2
C15H17NO3
C14H15NO3
C19H25NO2
C17H23NO2
C18H25NO2
C20H29NO2
137
89
118
148
114
148
162
146
122
89
19
20
21
22
C17H21NO3
C17H23NO3
C19H27NO3
C18H21NO3
86
71
aSemi-solid.

254
S. Koul et al. / Bioorg. Med. Chem. 8 (2000) 251±268
Table 2. In¯uence of piperine on hepatic microsomal AHH and
MOCD activities of untreated and inducers Ð pretreated ratsa
(a) In¯uence of modi®cations of the basic moiety on
AHH activity. Presence of piperidine moiety in piperine
o€ered an appropriate combination for inhibiting both
the constitutive and the 3MC- inducible AHH activities.
Replacement of piperidine moiety by pyrrolidine or n-
butyl amines (12 and 13) rendered the inducible AHH
activitity insensitive to these analogues while the con-
stitutive one was inhibited poorly.
Monooxygenase activities (pmol/min/mg protein)
AHH
MOCD
Compound
Control
Untreated 3MC-inducible Untreated PB-inducible
57‹7
530‹47
388‹25
1048‹81
Piperine (mM):
10
30
100
(b) In¯uence of modi®cations in aromatic ring on AHH
activity. Replacement of the MDP ring by either ethyl-
enedioxyphenyl, 3-methoxy-4-benzyloxyphenyl, 4-meth-
oxyphenyl, 3,4-dimethoxyphenyl, 3,4,5-trimethoxyphenyl
and 2,2-dimethyl-3,4-dihydrobenzopyran produced
compounds (8, 18, 22, 24, 27 and 29) which in general
did not in¯uence the inducible AHH while the con-
stitutive one exhibited poor sensitivity compared to
strong inhibition elicited by piperine. The inhibitory
e€ect in general was remarkably reduced from null
e€ect in the inducible form to a moderate inhibition in
the constitutive enzyme. The studies suggested that var-
ious compounds synthesised by modifying the phenyl
ring without disturbing the side chain and the basic
moiety again had no in¯uence on the inducibile AHH
activity. Furthermore, this e€ect again was found to be
comparable with compounds having modi®cations in
both the phenyl and the basic moieties (9, 19, 20, 23, 26,
28, 30 and 31). An exception was found with analogue
46.7‹5.1
29.1‹3.2
17‹2.5
439‹50
312‹40
138‹14
310‹23
241‹27
128‹11
880‹65
618‹46
419‹44
aThe data are mean values‹SD (n=8) from rat liver microsomal
preparations. These enzyme activities were used as controls for com-
parison of inhibitory ecacy of the above monooxygenases by various
synthetic analogues shown in Tables 3 and 4. Conditions for prepara-
tion of microsomes and enzyme assays are given in Materials and
Methods. P>0.01 (Student's `t' test).
(i) In¯uence of substituted derivatives of phenylpentadie-
noic acid on microsomal constitutive and 3MC-inducible
AHH activity of rat liver in vitro. Piperine elicited a
strong concentration related inhibition of both con-
stitutive and inducible AHH activities (Tables 3). All
the three structural components in the piperine molecule
appeared obligatory for inhibition of both constitutive
and inducible AHH activities (Table 3).
Table 3. Comparative in¯uence of piperine and its synthetic analogues on the constitutive and 3MC-inducible rat hepatic microsomal AHH
activity in vitroa
AHH activity (pmol 3-OH-BP formed per min per mg protein)
Untreated
30 mM
3MC-inducible
30 mM
Compounds
10 mM
100 mM
IC50
35
10 mM
100 mM
IC50
45
Piperine
46.7‹5.1
29.1‹3.2
17.1‹1.5
439‹50
312‹40
138‹14
Analogues
12
13
22
23
24
25
26
8
53.5‹4.8
59.8‹6.0
51.9‹6.8
54.1‹5.6
52.5‹4.5
49.0‹3.7
55.9‹4.7
53.6‹3.9
55‹5.0
37.6‹3.5
40.5‹3.9
50.2‹4.9
53.0‹6.0
30.3‹3.5
28.8‹4.5
48.5‹5.9
46.3‹3.5
49.0‹3.9
51.9‹4.4
48.5‹5.1
57.0‹4.1
49.1‹6.7
54.2‹5.1
43.9‹5.6
45.0‹3.4
41.6‹2.9
34.2‹2.9
42.8‹4.1
50.7‹5.9
48.5‹4.2
18.2‹2.4
34.7‹3.1
34.2‹2.7
29.1‹3.0
34.5‹3.5
33.1‹4.1
43.9‹5.1
47.3‹4.1
28.6‹7.5
21.7‹1.9
34.8‹4.2
32.4‹2.5
43.8‹4.8
38.2‹4.1
39.9‹2.1
53.0‹2.5
41.1‹3.1
41.1‹5.9
37.1‹4.2
30.8‹4.6
35.9‹4.7
28.5‹3.0
30.2‹4.2
31.9‹4.1
35.9‹4.0
7.41‹0.4
25.9‹3.4
18.8‹1.1
10.3‹0.9
80
105
>100
NI
100
525‹50
581‹32
464‹37
510‹37
500‹30
505‹67
495‹61
521‹56
503‹47
525‹71
490‹39
459‹51
495‹46
454‹61
484‹51
444‹29
485‹34
459‹45
525‹45
459‹36
490‹51
492‹54
469‹45
474‹40
505‹39
549‹34
653‹47
418‹55
490‹40
513‹35
551‹59
576‹53
510‹42
508‹42
540‹47
474‹27
444‹38
510‹35
510‹47
459‹60
479‹50
479‹37
408‹39
291‹21
270‹19
464‹49
540‹45
464‹51
449‹50
439‹39
548‹25
617‹51
444‹48
474‹44
520‹36
525‹61
525‹48
490‹30
511‹50
500‹37
413‹43
464‹25
511‹21
505‹48
459‹49
490‹45
515‹42
357‹31
133‹11
127‹16
479‹45
490‹39
479‹48
469‹47
428‹27
NIb
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
>100
40
35
>100
>100
NI
>100
NI
NI
9
10
18
19
20
27
28
29
30
31
32
33
34
14
15
16
17
57‹4.8
57.6‹3.0
53.0‹4.5
54.2‹6.9
53‹3.7
NI
>100
>100
105
>100
100
>100
>100
>100
23
70
50
48.5‹6.8
54.7‹4.6
51.3‹3.1
47.3‹4.0
55.9‹4.0
53.6‹6.8
53.6‹4.9
49.6‹3.4
46.2‹3.9
46.7‹3.9
48.4‹4.9
35
NI
NI
NI
NI
NI
32
aTest compounds were dissolved in 50% methanol and introduced in 10 mL of the vehicle in 1 mL of the assay system before initiation of the reaction
with the substrate. Controls received the vehicle only. Assays were performed in duplicate and data are mean‹ SD of three experiments. Speci®c
activities of untreated controls are given in Table 2.
bNI=no inhibition.

S. Koul et al. / Bioorg. Med. Chem. 8 (2000) 251±268
255
Table 4. Comparative in¯uence of piperine and its synthetic analogues on the constitutive and PB-inducible rat hepatic microsomal 7-methoxy-
coumarin O-demethylase activity in vitroa
MOCD activity (pmol 7-hydroxycoumarin formed /min/mg protein)
Untreated
30 mM
PB-inducible
30 mM
Compounds
10 mM
100 mM
IC50
45
10 mM
100 mM
IC50
47
Piperine
310‹23
241‹27
128‹11
880‹65
618‹46
419‹44
Analogues
12
13
22
23
24
25
26
8
9
18
19
20
27
28
29
30
31
32
33
34
14
15
16
17
335‹30
314‹24
415‹49
314‹23
367‹42
400‹34
334‹34
380‹37
388‹31
322‹27
361‹23
376‹40
353‹37
361‹26
361‹48
376‹26
369‹41
369‹18
330‹31
450‹40
303‹21
357‹49
341‹31
392‹40
339‹26
353‹31
376‹30
303‹32
326‹50
349‹30
353‹25
320‹31
331‹52
291‹23
272‹22
323‹29
314‹29
337‹27
295‹25
334‹30
349‹33
334‹34
302‹19
318‹27
132‹12
326‹29
124‹16
204‹21
312‹22
427‹37
144‹15
229‹23
240‹35
244‹19
244‹18
278‹25
321‹37
213‹20
233‹19
241‹24
225‹20
229‹21
140‹17
198‹14
353‹29
256‹26
244‹12
202‹18
109‹11
198‹20
97‹11
100
NI
80
1032‹72
1100‹98
954‹79
713‹62
938‹80
891‹70
985‹89
991‹68
NDc
1017‹97
1048‹79
765‹67
775‹63
911‹99
796‹67
859‹81
943‹78
1037‹51
1079‹90
870‹43
796‹68
964‹90
1048‹89
954‹81
922‹80
1048‹87
828‹80
482‹45
838‹76
796‹68
681‹68
680‹62
ND
744‹62
880‹71
514‹45
681‹59
796‹59
597‹53
451‹36
954‹69
890‹46
943‹72
702‹50
503‹41
796‹77
681‹43
618‹56
728‹46
964‹100
545‹45
367‹41
645‹56
660‹56
639‹56
550‹44
ND
440‹26
629‹80
356‹32
513‹48
629‹42
338‹21
335‹27
838‹81
713‹37
744‹45
325‹39
367‹33
492‹50
352‹37
272‹29
90
NI
100
25
>100
>100
>100
90
>100
>100
>100
>100
>100
NIb
>100
>100
>100
>100
>100
65
>100
NI
>100
>100
100
70
>100
28
90
>100
40
27
NI
>100
>100
50
27
85
25
100
23
39
50
45
115‹16
aSpeci®c activities of untreated controls are given in Table 2. Other conditions were the same as described in Table 3.
bNI, no inhibition.
cND, not determined.
25 where replacement of MDP by 3-methoxy-4-benzyl-
oxyphenyl and piperidine by pyrrolidine resulted in
inhibition of only constitutive activity which was com-
parable to piperine.
pronounced inhibition of constitutive AHH activity.
The potentiation of this inhibition of constitutive activity
was almost higher by 2-fold compared to piperine.
Further, it is important to note that modi®cation of basic
moiety in 14 resulted into compounds (15, 16, 17) having
similar or less inhibitory e€ect as that of piperine on the
constitutive AHH while such modi®cations in the parent
molecule have rendered them almost ine€ective (12, 13).
(c) In¯uence of modi®cations of the side chain on AHH
activity. Having modi®ed the two terminals of the
piperine molecule, we focused our attention on the cen-
tral ole®nic part of the molecule. Two types of major
modi®cations were introduced in this case, e.g. removal
of one double bond, (32) and saturation of the con-
jugated double bonds (14). These modi®cations, without
any change in other parts of the parent molecule,
o€ered interesting features. Removal of a double bond
(32) resulted into marked loss of inhibitory e€ect on
constitutive AHH while it retained its inhibitory e€ect
on the inducible AHH almost equipotent to piperine. A
similar e€ect was observed when the basic moiety of this
molecule was replaced by pyrrolidine moiety (33). On
the contrary, modi®cation of MDP to benzopyranyl in
32, i.e. 34 resulted in complete abolition of both the
constitutive and inducible AHH.
Besides the above mentioned compounds, we also syn-
thesised some other analogues of piperine, viz. 11, 21
and 37, where modi®cations were introduced in all the
three parts of the molecules. Such compounds did not
show any di€erential or speci®c inhibitory e€ects
against enzyme activities assayed (not shown).
(ii) In¯uence of substituted derivatives of phenylpentadi-
enoic acid on microsomal constitutive and PB-inducible
MOCD activity of rat liver in vitro. Like AHH activity,
modi®cations introduced in the piperine molecule also
a€ected both the constitutive and PB-inducible MOCD
activities (Table 4).
Therefore, presence of conjugated double bonds in
piperine appeared essential for overall inhibition of
CYP activities and the level of saturation may tilt the
balance of inhibition singularly either to constitutive or
inducible forms of CYPs. For instance, saturation of
double bonds of piperine (14) rendered it ine€ective
towards 3MC-inducible form of CYP while it elicited
(a) In¯uence of modi®cations of the basic moiety on
MOCD activity. Replacement of piperidine in piperine
by pyrrolidine and n-butylamine (12 and 13) had no
in¯uence on the constitutive with inducible MOCD
activities, unlike the strong inhibition produced by
piperine. We also observed a similar e€ect on AHH
activity discussed above.

256
S. Koul et al. / Bioorg. Med. Chem. 8 (2000) 251±268
(b) In¯uence of the modi®cations in the aromatic ring on
MOCD activity. Modi®cation of the MDP ring almost
abolished the inhibitory in¯uence on both the con-
stitutive and inducible MOCD activities. For instance,
replacement of MDP by either ethylenedioxyphenyl, 3-
methoxy-4-benzyloxyphenyl, 4-methoxyphenyl, 3,4-di-
methoxyphenyl, 3,4,5-trimethoxyphenyl, and 2,2-di-
methyl-3,4-dihydrobenzopyran produced compounds
(8, 18, 22, 24, 27 and 29) which in general did not
in¯uence the PB-inducible MOCD activity while the
constitutive one exhibited poor sensitivity with an
exception of 29 which was sensitive to induced activity
as much as piperine. Further, when both the MDP and
piperidine moieties were modi®ed (9, 19, 20, 23, 25, 26,
28, 30 and 31), most of the compounds lost their inhi-
bitory potentials towards both constitutive and induci-
ble MOCD with the exception of 20, 23 and 30 which
exhibited pronounced inhibitory e€ect only on inducible
MOCD where the IC50 values were half of piperine.
Table 5. In¯uence of piperine analogues on monooxygenase activities
of hepatoma H4IIEC3/G cells in culturea,c
Enzyme activity (pmol/min/mg protien)
Arylhydrocarbon
hydroxylase
7-Methoxycoumarindemethylase
Compound Control BA-treated
Control
PB-treated
DMSO
Piperine
14
16
17
68.4‹2.3 75‹4
6.4‹2.3 60‹5
2.8‹1.0 63‹6
5.3‹0.8 70‹10
16.5‹1.5
13.0‹1.7
7.0‹0.1
10.0‹1.5
8.0‹0.5
13.5‹3.0
8.5‹1.0
7.5‹1.5
12‹1.0
9.5‹2.0
3.6‹0.1
NDb
aCells were grown in 90 mm culture dishes near con¯uency. Cultures
were incubated with medium containing 50 mM of piperine analogues
for 4 h. In case of pretreatment of cultures to BA (20 mM, 18 h) or PB
(2 mM, 3 days), the experimental protocols were staggered so that the
treatment with the above compounds started at the same time. Other
conditions were the same as described in Materials and Methods.
bND, not determined.
cData are mean‹SD from three culture plates.
(c) In¯uence of modi®cations of the side chain on
MOCD activity. Decrease in length of the side chain of
piperine (removal of one double bond) without altering
the MDP or the basic moiety formed compound (32)
which had no in¯uence on both the constitutive and
inducible MOCD activities. This compound otherwise
inhibited speci®cally the inducible AHH activity (see
Table 3). A modi®cation of the basic moiety in 32 lead
to 33 having similar e€ects as 32. However, a modi®ca-
tion in the MDP of 32 resulted in 34 which is capable of
inhibiting PB-inducible MOCD as much as piperine but
has no e€ect on the constitutive one. Such modi®cations
are likely to yield more compounds which might impart
di€erential speci®city to the PB-inducible CYPs.
was not a€ected signi®cantly. These compounds, never-
theless appeared equipotent in the inhibition of MOCD
activity from untreated and PB-treated cultures. These
results correlated well with the in vitro microsomal
inhibition of mono-oxygenases by piperine. Further,
long-term in¯uence of these analogues on the inhibition
or induction of AHH and MOCD has been investigated
in cell cultures. Cell cultures were exposed to the med-
ium containing 60 mM analogues for 27 h (Fig. 2). Both
piperine and 14 induced AHH activity by about 70%
while the inducibility was of low magnitude with 16 and
17. However, MOCD continued exhibiting marginal
impairment even after 27 h of exposure with these ana-
logues while the magnitude of inhibition remained rela-
tively higher with 16 and 17 compared to piperine.
It is again interesting to note that saturation of double
bonds of piperine, i.e. 14 leads to potentiation of the
inhibition of both the constitutive and inducible MOCD
activities as observed with AHH activity. However,
replacement of basic moiety in 14 by pyrrolidine (15)
resulted into a complete loss of inhibitory potential
while its replacement by n-butylamide (16) elicited rela-
tively stronger inhibition of only constitutive activity
while replacement by N,N-diethylamide (17) exhibited
sensitivity almost comparable to piperine. Other analo-
gues of piperine, where modi®cations were introduced
in all the three parts of the molecules (11, 21 and 37) had
no in¯uence on MOCD activity as has been observed
earlier at least against AHH activity (not shown).
In¯uence of piperine analogues on hexobarbital induced
sleeping time in mice
Intraperitoneal administration of the above selected
compounds viz. 14, 16 and 17 potentiated hexobarbital-
induced sleeping time over piperine (Fig. 3).
Comparative e€ect of piperine analogues on the kinetics
of MOC demethylation by rat liver microsomes in vitro
Microsomes from untreated rat liver were used. Kinetics
of enzyme inhibition in the presence and absence of
piperine and two selected analogues using Lineweaver±
Burk double reciprocal plot and Dixon plot of analysis
has been determined. The values of the kinetic data
are given in Table 6. All the three analogues caused
non-competitive inhibition. The Vmax decreased with
increasing inhibitor concentrations while the apparent
Km of 151 mM was almost similar when dealkylation of
MOC was studied in the presence and absence of
piperine and its analogues. The values of half-maximal
Ki of enzyme inactivation obtained for 14 and 17 were
much lower than piperine, and correlated with the
intenstity of inhibition of MOC dealkylation in hepa-
toma cell cultures (Table 5).
In¯uence of piperine analogues 14, 16 and 17 on the
constitutive and inducible AHH and MOCD activities of
the H4IIEC3/G hepatoma cells in culture
To investigate the initial interaction of these analogues
with the mono-oxygenase activities, the cell cultures
were exposed to the above compounds for 4 h. Piperine
mediated inhibition intensity of the monooxygenase
activities in the hepatoma cells in culture was compared
with three tetrahydropiperine derivatives (Table 5). Like
in vitro inhibition of constitutive AHH, these com-
pounds elicited inhibition stronger than piperine in cell
cultures while the BA-inducible activity in comparison

S. Koul et al. / Bioorg. Med. Chem. 8 (2000) 251±268
257
Figure 2. In¯uence of piperine and its synthetic analogues on monooxygenase activities in rat hepatoma cells. Rat Reuber hepatoma H4IIEC3/G
cells (1Â106) were seeded in 90 mm tissue culture plates and allowed to grow for 48 h. The medium was changed with fresh medium and cultures
were treated with 60 mM piperine or anologues in 15 mL DMSO/plate as vehicle. After 27 h of exposure, medium was removed, cultures washed with
PBSÂ2. Cells were scrapped and collected in PBS and centrifuged. The cell pellet was stored in liquid nitrogen. Before assay of monooxygenases the
pellet was suspended in 0.5 mL of 50 mM Tris±HCl, pH 7.4. Other conditions were the same as described in Materials and Methods. The data are
mean ‹SD from four culture plates. P<0.05 (Student's `t' test).
Table 6. Comparative e€ect of piperine analogues on the kinetics of
MOC demethylation by rat liver microsomes in vitroa
Vmax (pmol/min/mg protein)
Km (mM) Ki (mM)
Compound 0 mM 15 mM 30 mM 60 mM
Piperine
14
17
278
283
284
242
193
NDb
194
133
143
125
96
100
151
150
152
50
35
42
aValues derived from Lineweaver±Burk double reciprocal and Dixon
plots of MOC dealkylation by microsomal MOCD from untreated rat
liver in the presence and absence of indicated inhibitors.
bND, not determined.
activities and that (ii) the modi®cation of any one moi-
ety in the piperine molecule may not only alter the
status of inhibition but also could elicit di€erential
inhibition of the two types of monooxygenase activities
examined.
Piperine was shown earlier to inhibit the oxidation of
benzo(a)pyrene, 7-ethoxy-O-coumarin and ethylmor-
phine in untreated, 3MC- and PB-treated rat liver.2 In
this study we examined the oxidation of two types of
CYP substrates B(a)P and MOC by using microsomes
from untreated, 3MC- and PB- pre-treated rat liver.
Because we have earlier reported that piperine inhibits
at least two CYP-dependent marker reactions, viz. aryl-
hydrocarbon B(a)P hydroxylase (AHH) and 7-methoxy-
coumarin O-demethylase (MOCD) in H4IIEC3 cells.39
The former reaction primarily is mediated by members
of CYP family which are inducible by polycyclic aro-
matic hydrocarbons, i.e. CYP1A.20 The later is cata-
lyzed by constitutive, phenobarbital or dexamethasone
inducible CYP forms comprising members of CYP2 and
CYP3 families.13,21 Further, MOCD activity was observed
in preparations of genetically constructed V79 cells (SD1)
containing only CYP2B1, the major PB-inducible CYP
Figure 3. In¯uence of piperine analogues on the hexobarbital-induced
sleeping time in mice. Compounds were administered (5 mg/kg body
wt, ip) 30 min before hexobarbital (60 mg/kg body wt, ip). Data are
mean‹SD (n=4). P values<0.01 (Student's `t' test).
Discussion
The present studies have been undertaken to relate the
role of various functional groups in piperine molecule to
the inhibition of constitutive and inducible CYP activ-
ities of rat liver by employing the oxidation of marker
CYP substrates of CYP1A and CYP2 gene families. The
results suggested that (i) all the three components of
piperine, viz. MDP ring, side chain and the piperidine
moiety together, are essential for maximal inhibition of
both the constitutive and inducible AHH and MOCD

258
S. Koul et al. / Bioorg. Med. Chem. 8 (2000) 251±268
of rat liver and not in XEM1 cells containing only
CYP1A1.21
in the induction of CYP450 has also been demonstrated
earlier43 despite the fact that these compounds are
found potent inhibitors of mixed function oxidases both
in vitro and in vivo.44,45 The noncompetitive inhibition
of mixed function oxidases by MDP compounds essen-
tially appears due to the binding of MDP metabolite to
reduced CYP.46 We observed earlier that piperine is not
a suicidal inhibitor of monooxygenases. It was found
that despite its ability to activate moderately CYP1A1
gene transcription, the alkaloid regulate CYP1A1 gene
expression posttranslationally where it inhibits its cata-
lytic activity by binding with the enzyme without
destroying the AHH.7 However, it is not known that
which CYP form (s) is involved in the metabolism of
piperine.
From the two CYP marker reactions studied against
several analogues of piperine, some interesting features
emerged about the structure±activity relationship on the
inhibition of mono-oxygenase activities examined. For
instance all the three structural components in piperine
molecule occuring in nature are essential for overall
inhibition. In the case of AHH various compounds with
modi®ed MDP ring in general were insensitive to both
constitutive and inducible AHH with only the exception
of 25. However, the inhibition becomes selective in
favour of the constitutive AHH only when the con-
jugated side chain is saturated with or without any
alteration in the basic moiety (14, 15, 16, 17). This sug-
gested that di€erences in the protein domain in active
site architecture exist between the constitutive and
inducible CYP1A1 enzymes. Saturation of the side
chain of piperine induces ¯exibility in the molecule
which may facilitate interaction of the inactivator with
protein domain and may thereby enhance constraints
for orientation of the CYP substrates to the active site.
On the other hand, compounds 32 and 33 are short by
one double bond which not only confers rigidity in the
molecule but also renders them selective inhibitors of
only inducible AHH activity. Saturation of the con-
jugated double bonds to tetrahydro-derivatives of
MDP ring appear to result in higher ¯exibility of the
side chain which perhaps acts as a handle to orient
MDP group to the active site of the CYP450 anchored
in a strong hydrophobic environment. Thus, altering the
functional groups in piperine would determine its inter-
action with the hydrophobic environment of the active
site and hence its potential to determine the speci®city
and extent of inhibition. In addition, the lipophillic
nature of the synthetic analogues also in¯uences
strongly the inhibition of AHH and MOCD. This is
evidenced from the facts that compounds 14 and 16
have displayed higher inhibition than their unsaturated
counterpart 11. The latter although, it is a tetrahydro
derivative, has displayed null inhibition due to qua-
ternary ammonium salt which imparts higher water
solubility to the compound. This also suggests the sig-
ni®cance of 5-carbon ring or piperidine molecule in
supplementing the hydrophobicity of piperine. Synthesis
of such compounds thus appear useful in undestanding
the environment of active site of di€erent CYP enzymes.
In contrast to the B(a)P oxidation by CYP1A family,
MOC is a preferred substrate for liver constitutive and
PB-inducible CYPs.21 PB induces several CYP members
of CYP2 (A1, B1, B2, B4, C5, C6) and CYP3 (A2, A4)
families.13 We do not know the relative preference of
each isoform to MOC dealkylation. Under such cir-
cumstances it is not easy to design inhibitors of su-
cient selectivity to target individual CYP isoforms
because of several forms of CYP and wide range of
overlapping substrates speci®cty. However, after intro-
ducing structural alterations in piperine a number of
compounds synthesised exhibited preferential selectivity
either towards constitutive or inducible CYPs (25, 29,
30). These compounds relatively exhibited maximal
selectivity and higher CYP inactivating potency than
the parent molecule as evidenced by their e€ect on
microsomal MOCD activities. However, unlike insensi-
tivity to the MC-inducible AHH activity, the tetra-
hydroderivatives (14, 15, 16, 17) were equipotent to
both constitutive and inducible forms while analogues
14 and 17 exhibited higher sensitivity than piperine. It
appears that the presence of the side chain with satu-
rated double bonds linked through amide linkage
appeared to impart speci®city for inhibiting di€erent
forms of CYP450s. This in turn would again depend
upon the amino acids located in the putative substrate
recognition sites of CYP which regulate the accessibility
of the substrates to generate MI which would sequester
CYP to modulate drug biotransformation. In fact, it
would require further studies to test these selected ana-
logues for their speci®city and selectivity with individual
CYP members.
Certain substituted methylenedioxy benzenes are also
known as synergists for a number of classes of pesticides
of di€erent structure types40 which act by inhibition of
drug biotransformation. MDP ring is generally con-
sidered to require hepatic metabolism for inhibition of
microsomal oxidation through an active metabolite
carbene.41,42 Part of the inhibitory action of MDP-rela-
ted compounds is due to the metabolite intermediate
(MI) complexation of CYP. Food ¯avouring agent iso-
safrol forms MI complex selectively with PB-inducible
CYP2B1 and MC-inducible 1A2.13 This inhibition in
general was not observed with various compounds we
synthesised with change in substitutions in phenyl
nucleus. The importance of the methylendioxy carbon
Besides studying the relationship of functional groups
of piperine with the inhibition of CYP activities, we
attempted to compare the potency of some analogues
such as 14, 16 and 17 with piperine in di€erent systems
in vitro and in vivo. These compounds were insensitive
to 3MC-inducible but expressed higher sensitivity to
constitutive AHH and as well as to constitutive and
inducible MOCD. This type of response was also evi-
dent from experiments on sleeping time in mice, and
monooxygenase inhibition in the hepatoma cells pre-
treated with inducers. Poor inducibility of AHH by 16
and 17 compared to piperine in this regard was inter-
esting when cells were exposed to these anologues for
longer period while they inhibited the enzymes strongly

S. Koul et al. / Bioorg. Med. Chem. 8 (2000) 251±268
259
during initial period. Like piperine,2 the noncompetitive
nature of inhibition was not altered by tetra-
hydroderivatives, the Ki values however, were further
lowered compared to piperine although Km value was
similar in each case. Similarly, other selective com-
pounds such as 20, 23, 25 and 35 could be tested for
selectivity and speci®city against certain pesticides as
synergists or for enhancing the bioavailability of some
therapeutically important drugs.
simultaneously increasing the potential drug inhibitory
property of the molecule. Modi®cations of piperine
molecule may thus prove useful in the development of
selective CYP inhibitors.
Experimental
Chemistry
General methods. Reagents for chemical synthesis were
of AR grade and obtained commercially. All reactions
were monitored by TLC carried out on 0.25 mm E.
Merck silica gel plates using UV light. Silica gel of mesh
Conclusion
In conclusion, the structure of piperine is ideally suited
to a€ect the microsomal oxidation of large number of
compounds. Piperine has a number of inherent advan-
tages in that it is a simple molecule, can be used with
intact cells in culture and in vivo, easily available com-
mercially, and a highly indispensable ingredient of spi-
ces in platability of food used for ages. Presence of
piperidine function at the terminal end of conjugated
double bond in the side chain and MDP ring o€ered
di€erential sensitivity in inhibiting the CYP450 activ-
ities examined in the present study. By introducing
modi®cations at di€erent positions desired inhibitors of
drug metabolism with strong implications in agriculture
and pharmaco-toxicology could be developed We have
earlier reported that presence of unsaturated double
bonds in the side chain of piperine molecule are
responsible for inhibition of UDP-glucose dehydro-
genases6 and certain dehydrogenase complex associated
with electron transport chain47 in vitro. This drawback
is obviously removed by saturating the double bond and
1
size 60±120 was used for column chromatography. H
NMR spectra were determined at either 60 MHz or
90 MHz using Varian F-60 or Jeol Fx-90 spectrometers,
respectively. Mass spectra were determined on Jeol
MSD-300 mass spectrometer while IR spectra were
recorded on Perkin±Elmer FT-IR spectrometer.
Preparation of substituted aryl pentadienoic acid amides
and other derivatives. The structure of piperine (Fig. 1)
may be divided into three main components, i.e.
methylenedioxyphenyl part, a conjugated side chain and
piperidine moiety. For the preparation of its synthetic
analogues modi®cations were envisaged in all the three
components. The key intermediate, the substituted
phenyl-2E,4E-pentadienoic acid was constructed start-
ing from corresponding benzaldehyde in a ®ve-step
reaction sequence via cinnamaldehyde intermediate as
depicted in Scheme 1. The amides were readily obtained
from the carboxylic acids through acyl chloride formation
Scheme 1.

260
S. Koul et al. / Bioorg. Med. Chem. 8 (2000) 251±268
and condensation with appropriate amines. For the
synthesis of pyrano analogues, the required benzopyran
moiety was prepared from the corresponding phenols
by condensing with isoprene in presence of an acid
(Scheme 3) which is followed by the same reaction
sequence as described in Scheme 1. The synthesis of
substituted phenyl-2E-propenoic acid is shown in
Schemes 2 and 3. The tetrahydro-analogues were
obtained by catalytic reduction of the phenylpentadie-
noic acid or the phenylpentadienoamides in presence of
palladium-charcoal (10%). The structure analysis was
carried out mainly through elemental analysis, IR,
PMR and MS studies. Various compounds synthesized
through Schemes 1±3 are given in Table 1.
Scheme 3. R1R2NH: piperidine, pyrrolidine, diethylamine, n-butyl-
amine, n-pentylamine, isopropylamine, isobutylamin, n-methylpiper-
azine and n-hexylamine. S. No. R (a) 4-methoxy; (b) 3,4-dimethoxy;
(c) 3,4-ethylendedioxy; (d) 4-benzyloxy-3-methoxy; (e) 3,4,5,-tri-
methoxy; (f) 4-hydroxy; (g) [2H]-2,2-dimethyl-3,4-dihydro pyranyl; (h)
3,4-methylendioxy.
Preparation of ꢀ-methyl-4-methoxy benzyl alcohol (2a).
To an ethereal solution of Mg metal (2.6 g, 110 mmol)
and methyl iodide (9.8 mL, 130 mmol) added an ethereal
solution of p-anisaldehyde (1a) (15 mL, 110 mmol) and
the contents stirred for 2 h at 0±5 ꢀC. The reaction mix-
ture worked up by adding saturated aqueous solution of
ammonium chloride (10 mL), followed by dilution with
water (100 mL), separation of organic layer followed by
extraction of aqueous layer with solvent ether
(2Â100 mL). The combined organic layer washed with
water (2Â20 mL), dried over anhydrous sodium sulfate
and concentrated in vacuo to give a gummy mass (2a)
(16.0 g, 95%) analysed for C9H12O2, MS (%) M+ at
m/z 152 (36), 137 (57), 134 (100), 109 (62), 106 (14), 103
1
C10H10O2, MS (%) M+ at m/z 162. n cm (KBr) 1694,
1650, 1600, 1582, 1500, 1462, 1334, 1246, 1124, 996 and
818. 1H NMR (CDCl3) d: 3.90 (3H, s, OCH3), 6.57 (1H,
d d, J=16.0 Hz and 7.0 Hz, -CH=CH CO), 6.78
(2H, d, J=8.5 Hz, 2ÂAr-H), 7.43 (1H, d, J=16.0 Hz,
-CH=CH CO), 7.46 (2H, d, J=8.5 Hz, 2ÂAr H) and
9.73 (1H, d, J=7.0 Hz,=CH CHO).
Preparation of 5-(4-methoxy phenyl)-2E,4E-pentadienoic
acid (4a). To a stirring mixture of (3a) (6.5 g, 40 mmol)
and the ylide, prepared from ethyl bromoacetate
(4.8 mL, 44 mmol) and triphenyl phosphine (11.7 g,
44 mmol), in dry dimethoxy ethane (100 mL) was added
sodium hydride (2.0 g) in small proportions. The pro-
gress of the reaction monitored by TLC; after the com-
pletion of the reaction, the contents poured carefully in
ethyl acetate to quench the excess of sodium hydride,
followed by addition of water, the organic layer sepa-
rated and the aqueous layer extracted with ethyl acetate
(3Â125 mL). The combined organic layer washed with
water (3Â40 mL), dried over anhydrous sodium sulfate
and concentrated in vacuo. The crude solid product
taken up in 10% methanolic KOH solution (140 mL)
and the contents re¯uxed on water bath for 6 h. On
cooling, the contents were diluted with water (300 mL)
and extracted with ethyl acetate (3Â25 mL). The aqu-
eous phase washed with petroleum ether (30 mL) and
then acidi®ed with 2N HCl solution. The resulting
precipitate ®ltered, washed with ice cold water and
dried to give acid (4a) (6.7 g, 82%) crystallised from
ethyl acetate:petroleum ether (9:1) as colourless com-
pound, mp 183 ꢀC (lit. mp 182±183 ꢀC),23,25,26 analysed
1
(10), 94 (50), 91 (62) and 78 (19). n cm (KBr) 3352,
2968, 1612, 1588, 1510, 1450, 1422, 1300, 1242, 1174,
1068, 1030, 1016, 888 and 810. 1H NMR (CDCl3) d 1.45
(3H, d, J=6.5 Hz, CH3 C OH), 3.80 (3H, s, OCH3),
4.80 (1H, q, J=6.5 Hz CH3 CHOH), 6.90 (2H, d,
J=8.5 Hz 2ÂAr H) and 7.28 (2H, d, J=8.5 Hz,
2ÂAr H).
Preparation of 3-(4-methoxy phenyl)-2E-propenal (3a).
To a chilled solution of (2a) (12.2 g, 80 mmol) in DMF
(15 mL) added phosphoryl chloride (7 mL) slowly at
0±5 ꢀC for 1 h.23 The contents were stirred further for 2 h
and then allowed to attain room temperature followed
by heating on a water bath for 3 h. The reaction mixture
cooled and a saturated solution of sodium acetate
(15 mL) added, followed by dilution with water
(150 mL). The contents of the reaction mixture were
extracted with ethylacetate (5Â100 mL), the organic
layer washed with water (3Â30 mL) and dried over
anhydrous sodium sulfate to give crude product which
on CC over SiO2 and elution with pet.ether:ethyl acetate
(9:1) gave yelꢀlow crystalline compound (3a) (8.4 g,
65%), mp 58 C (lit. mp. 56±57 ꢀC),24 analysed for
Scheme 2.

S. Koul et al. / Bioorg. Med. Chem. 8 (2000) 251±268
261
1
for C12H12O3, MS (%) M+ at m/z 204. ncm (KBr)
2960, 1688, 1668, 1612, 1550, 1440, 1320, 1040, 960 and
and Ar H) and 7.30 (1H, dd, J=15.0 Hz and 7.0 Hz, -
CH CHˆCH-CO).
1
810. H NMR (acetone-d6) d 3.90 (3H, s, OCH3), 6.10
(1H, d, J=15.0 Hz, -CHˆCH CO), 6.60±7.13 (3H, m,
ole®nic and Ar H) and 7.40±8.20 (4H, m, ole®nic and
Ar H).
Preparation of 5-(4-methoxy phenyl) pentanoic acid N-
methyl piperazine amide (11). To the compound (10)
(0.57 g, 2 mmol) in ethylacetate (30 mL) added Pd/C
(5%, 30 mg) and hydrogenated the contents at 30 psi.
Work up of the reaction mixture a€orded 11 (0.54 g,
93%), mp 183 ꢀC (analysed for C17H26N2O2; found C
70.3172, H 9.0280, N 9.6507; calcd C 70.3112, H 9.0237,
N 9.6465). MS (%) M+ at m/z 290 (32), 191 (100) 163
Preparation of 5-(4-methoxy phenyl)-2E,4E-pentadienoic
acid piperidine amide (8). To the acid (4a) (2.0 g,
10 mmol) in dry methylene chloride (50 mL) added
freshly distilled thionyl chloride (0.8 mL) and re¯uxed
for 1 h, excess of thionyl chloride removed in vacuo and
thereafter condensed with methylenechloride solution of
piperidine (1 mL) and stirred for 30 min. The organic
layer washed with water (2Â25 mL), dried over anhy-
drous sodium sulfate and concentrated to give crude
product which on CC over SiO2 and elution with pet.
ether:ethyl acetate (4:1), yielded colourless crystalline
compound 8 (2.56 g, 94%), mp 135 ꢀC (lit. mp 97 ꢀC),25
(analysed for C17H21NO2; found C 75.2481, H 7.8004,
N 5.1637; calcd C 75.2463, H 7.7999, N 5.1618) MS (%)
M+ at m/z 271 (48), 187 (100), 159 (39), 129 (40), 115
(60) and 84 (10). ncm 1(KBr) 2930, 1640, 1600, 1560,
1450, 1320, 1250, 1110, 1050 and 970. 1H NMR
(CDCl3) d: 1.50 (6H, bs, -N CH2 (CH2)3), 3.36 (4H,
m, N (CH2)2, 3.82 (3H, s, OCH3), 6.36 (1H, d,
J=15.0 Hz, CHˆCH CO) 6.80±7.00 (6H, m, ole®nic
and Ar H) and 7.25 (1H, dd. J=15.0 Hz and 7.0 Hz,
-CH CH=CH CO).
1
(46) 148 (10) 120 (48) and 99(15). ncm (KBr) 2972,
1640, 1560, 1450, 1350, 1310, 1250, 1128, 1010, 930 and
1
827. H NMR (CDCl3) d 1.60 (4H, bs, (CH2)2), 2.50
(4H, m, Ar CH2 CH2 CO), 3.10 (3H, bs, -N CH3),
3.40±3.70 (8H, m, 2Â N(CH2)2 3.86 (3H, s, OCH3) and
6.60±7.00 (4H, m, Ar H).
Preparation of 5-(3,4-methylenedioxy phenyl)-2E,4E-
pentadienoic acid (piperic acid) 4h. Piperine (28.0 g,
98 mmol), mp 132 ꢀC, dissolved in ethylene glycol
(200 mL) and re¯uxed at 180 ꢀC after adding potassium
hydroxide (25 g) and after the completion of the reac-
tion the contents diluted with sucient amount of water
and acidi®ed with 2N HCl. The resulting precipitate ®l-
tered and dried to give crude product which on crystal-
lisation from ethanol gave 4h as pale yellow solid
(13.8 g, 65%) mp 217 ꢀC (lit. mp 217 ꢀC).27
Preparation of 5-(3,4-methylenedioxyphenyl)-2E,4E-pen-
tadienoic acid pyrrolidine amide (12). Compound (12)
was prepared from 4 h (2.2 g, 10 mmol) using thionyl-
chloride (0.9mL) and pyrrolidine (0.95mL, 11mmol) by
the same method as described for 8 to give a crude pro-
duct which on crystallisation with pet.ether:ethylacetate
(4:1) furnished a pale yellow crystalline compound (2.46g,
90%) mp 147 ꢀC (lit. mp. 144±146 ꢀC)28 (analysed for
C16H17NO3; found C 70.8444, H 6.3169, N 5.1666; calcd
C 70.8315, H 6.3152, N 5.1626) ;MS (%) M+ at m/z 271
(11) 201 (66) 173 (100) 143 (10) and 70 (9). ncm 1 (KBr)
1642, 1616, 1598, 1505, 1490, 1450, 1418, 1364, 1252,
1194, 1148, 1142, 1038, 994, 932 and 844. 1H NMR
(CDCl3) d 1.90 (4H, m, (CH2)2), 3.52 (4H, m, -N(CH2)2),
5.93 (2H, s, O CH2 O), 6.20 (1H, d, J=15 Hz,
CO CHˆCH), 6.62±7.50 (6H, m, ole®nic and Ar H).
Preparation of 5-(4-methoxyphenyl)-2E,4E-pentadienoic
acid pyrrolidine amide (9). Compound (9) was prepared
from acid (4a) (1.0 g, 5 mmol) using thionyl chloride
(0.4 mL) and pyrrolidine (0.5 mL, 6 mmol) by the same
method as described for 8 to give a crude product
which on CC over SiO2 and elution with pet.ether: ethyl
acetate (4:1) furnished white crystalline compound
(1.18 g, 91.8%), mp. 137 ꢀC (analysed for C16H19NO2;
found C 74.6824, H 7.4421, N 5.4451; calcd C 74.6804,
H 7.4418, N 5.4431). MS (%) M+ at m/z 257 (35) 187
1
(100) 159 (12) 129 (16) 116 (32) and 70 (38). ncm
(KBr) 2955, 1630, 1600, 1520, 1485, 1436, 1310, 1250,
1
1184, 1136, 1022, 838 and 732. H NMR (CDCl3) d:
1.93 (4H, m, (CH2)2), 3.56 (4H, m, -N(CH2)2) 3.85 (3H,
s, OCH3), 6.23 (1H, d, J=15 Hz, CO CHˆCH), 6.66±
7.00 and 7.20±7.75 (7H, m, ole®nic and Ar-H).
Preparation of 5-(3,4-methylenedioxy phenyl)-2E,4E-
pentadienoic acid n-butyl amide (13). It was prepared
from (4h) (4.4g, 20mmol) using thionyl chloride and n-
butyl amine (2 mL, 20mmol) employing the process as
described for the preparation of 8, to give the amide (13)
(4.6 g, 84%), mp 144 ꢀC (lit. mp 151±152 ꢀC)28 (analysed
for C16H19NO3; found C 70.3120, H 7.0071, N 5.1296;
calcd C 70.3019, H 7.0062, N 5.1245); MS (%) M+ at m/z
273 (100), 216 (14), 201 (17), 173 (87), 152 (11), 143 (30),
135 (12), 115 (71) and 96 (7). ncm 1(KBr) 3328, 2936,
1640, 1550, 1506, 1490, 1466, 1442, 1398, 1364, 1316,
Preparation of 5-(4-methoxy phenyl)-2E,4E-pentadienoic
acid N-methyl piperazine amide (10). Compound (10)
was prepared from acid (4a) (2.0 g, 10 mmol) using
thionyl chloride (0.8 mL) and N-methyl piperazine
(1.0 mL, 11 mmol) by the method as described for 8 to
give a crude product which on CC over SiO2 and elution
with pet.ether:ethyl acetete furnished white crystalline
compound, (2.45 g, 86%), mp 201±202 ꢀC (analysed for
C17H22N2O2; found C 71.3139, H 7.7420, N 9.7852;
calcd C 71.3011, H 7.7429, N 9.7823). MS (%) M+ at
m/z 286 (46), 187 (100), 159 (31), 144 (28), 116 (14) and
99 (15). ncm 1(KBr) 3250, 2930, 1640, 600, 1560, 1420,
1
1248, 1208, 1192, 1100, 1042, 942 and 812. H NMR
(CDCl3) d 0.97 (3H, d, J=6.5Hz CH2-CH3), 1.48 (4H, m,
-C (CH2)2), 3.36 (2H, m, -NH CH2 CH2), 5.92 (1H, d,
J=15.0 Hz CHˆCH-CO), 5.96 (2H, s, -O CH2 O-),
6.64±7.00 (5H, m, ole®nic and Ar-H) and 7.24±7.52 (1H,
m, CHˆCH CO).
1
1310, 1250, 1020 and 970. H NMR (CDCl3) d 2.95
(3H, s, -N-CH3), 3.20 (4H, m, -N (CH2)2), 3.50 (4H, m,
-N (CH2)2), 3.92 (3H, s, OCH3), 6.30 (1H, d,
J=15.0 Hz CHˆCH CO), 6.80±7.00 (6H, m, ole®nic

262
S. Koul et al. / Bioorg. Med. Chem. 8 (2000) 251±268
Preparation of 5-(3,4-methylenedioxy phenyl) pentanoic
acid (tetrahydro piperic acid) 5h. Piperic acid (4h)
(6.70 g, 31 mmol) was dissolved in methanol (100 mL)
and to it was added Pd/C (5%, 200 mg) and subjected to
hydrogenation at 40ꢀpsi to yield tetrahydꢀro piperic acid
(5h) (6.85 g), mp 95 C (lit. mp 100±101 C)29 analysed
for C12H14O4.
6.5 Hz, CH2 CH2 CO), 2.69 (2H, t, J=6.5 Hz,
Ar CH2-CH2), 3.29 (4H, q, J=6.5 Hz, -N (CH2)2), 5.86
(2H, s, -O CH2 O-) and 6.59 (3H, bs, Ar H).
Preparation of ꢀ-methyl-3,4-dimethoxy benzyl alcohol
(2b). It was prepared from 3,4-dimethoxy benzaldehyde
(1b) (20 g, 100 mmol) and Grignard reagent [Mg metal,
3.0 g, 120 mmol) and methyl iodide (9.0 mL) as per the
procedure described for compound 2a to give a
gummy mass (2b) (18.8 g, 90%), analysed for C10H14O3,
MS (%) M+ at m/z 182 (60), 166 (86), 164 (5), 138
(100), 123 (29), 107 (20) and 77 (23). ncm 1(KBr) 3476,
2932, 1596, 1508, 1454, 1418, 1366, 1312, 1262, 1234,
Preparation of 5-(3,4-methylenedioxy phenyl) pentanoic
acid piperidine amide (14). Compound 5h (2.0 g, 9 mmol)
was condensed with piperidine (0.9 mL) as described for
8 to yield a gummy mass (2.4 g, 92%) (analysed for
C17H23NO3; found C 70.5700, H 8.0118, N 4.8434; calcd
C 70.5619, H 8.0110, N 4.8404). MS (%) M+ at m/z 289
(50), 205 (100), 177 (47), 147 (57), 119 (32) and 84 (16).
ncm 1(KBr) 2935, 1640, 1565, 1440, 1250, 1020 and 870.
1H NMR(CDCl3) d: 1.56 (10H, m, -NCH2 (CH2)3 and
-C (CH2)2), 2.30 (2H, t, J=7.0 Hz, COCH2), 2.54 (2H, t,
J=7.0Hz, Ar CH2), 3.46 (4H, m, -N (CH2)2), 5.82 (2H,
s, -O CH2 O-) and 6.62 (3H, m, Ar H).
1
136, 1018, 838, 756, and 720. H NMR (CDCl3) d 1.42
(3H, d, J=7.0 Hz, -CH3 CHOH), 3.84 (6H, s,
2ÂOCH3), 4.74 (1H, q, J=7.0 Hz, CHOH CH3) and
6.88 (3H, s, Ar H).
Preparation of 3-(3,4-dimethoxyphenyl)-2E-propenal (3b).
It was prepared from 2b (15.0 g, 80 mmol) and POCl3
(14 mL) and DMF (40 mL) as described for 3a to give
crude product which on crystallisation from ethyl acet-
ate:pet.ether (1:9) gave 3b (9.8 g, 63%), mp 85 ꢀC (lit.
mp 81 ꢀC)30 analysed for C11H12O3, MS (%) M+ at m/z
Preparation of 5-(3,4-methylenedioxy phenyl) pentanoic
acid pyrrolidine amide (15). To 5h (2.0 g, 9 mmol) dis-
solved in dry methylene chloride (40 mL) was added
thionyl chloride (0.8 mL) and the resulting acid chloride
condensed with pyrrolidine (0.9 mL) and worked up as
described for compound 8 to furnish a gummy mass
(15) (2.40 g, 94%) (analysed for C16H21NO3; found C
69.7801, H 7.6874, N 5.0893; calcd C 69.7943, H 7.6870,
N 5.0870). MS (%) M+ at m/z 275 (66), 148 (27), 126
1
192. ncm (KBr) 1705, 1638, 1595, 1350, 1135, 1010
1
and 830. H NMR (CDCl3) d 3.86 (6H, s, 2ÂOCH3)
6.56 (1H, dd, J=16.0 Hz and 7.0 Hz, -CHˆCH CHO),
6.88±7.14 (3H, m, 3ÂAr H), 7.36 (1H, d, J=16.0 Hz,
-CHˆCH CHO) and 9.65 (1H, d, J=7.0 Hz, -CHˆ
CH CHO).
1
(98), 113 (100), 105 (16), 98 (57) and 70 (75). ncm
(KBr) 2960, 1640, 1550, 1455, 1350, 1305, 1268, 1120,
Preparation of 5-(3,4-dimethoxy phenyl)-2E,4E-pentadi-
enoic acid (4b). This compond was prepared from 3b
(9.0 g, 46 mmol) by Wittig reaction as described for 4a
to give ꢀ4b (9.0 g, 83%), crꢀystallised from ethylacetate,
mp 166 C (lit. mp 203±205 C)30 analysed for C13H14O4,
MS (%) M+ at m/z 234 (87), 220 (19), 189 (100), 174
(56), 158 (38), 145 (19), 131 (32), 115 (66), 103 (52), 91
1
930 and 840. H NMR (CDCl3) d 1.38±1.94 (8H, m,
-(CH2)4) 2.23 (2H, t, J=7.0 Hz, -COCH2), 2.58 (2H, t,
J=7.0 Hz, Ar CH2), 3.33 (4H, m, -N(CH2)2), 5.90 (2H,
s, -O CH2 O-) and 6.65 (3H, bs, 3ÂAr H).
Preparation of 5-(3,4-methylenedioxy phenyl)-pentanoic
acid n-butyl amide (16). (5h) (2.0 g, 9 mmol) was con-
densed with n-butyl amine (1 mL) by the method as
described for 8 to yield 16 as a gummy mass (2.0 g,
98%) (analysed for C16H23NO3; found C 69.2940, H
8.3588 N 5.0522; calc. C 69.2871, H 8.3579, N 5.0500). MS
(%) M+ at m/z 277 (49), 205 (100), 177 (33), 144 (36) and
119 (28). ncm 1 (K Br) 3260, 2932, 1638, 1557, 1448, 1351,
1300, 1252, 1119, 1056, 930 and 835. 1H NMR (CDCl3) d
0.96 (3H, d, J=6.5 Hz, CH3), 1.16±1.84 (8H, bm,
4ÂCH2), 2.16 (2H, m, -CH2 CO), 2.53 (2H, t, J=6.5Hz,
Ar CH2), 3.30 (2H, q, J=6.5Hz, -NH CH2), 5.88 (2H,
s, -O CH2 O-) and 6.66 (3H, m, Ar H).
1
(74) and 77 (41). ncm (KBr) 2936, 1682, 1628, 1518,
1444, 1320, 1212, 1168, 1140, 1024, 886 and 827. H
1
NMR (CDCl3) d 3.86 (6H, s, 2ÂOCH3), 5.92 (1H, d,
J=15.0 Hz, -CHˆCH CO), 6.84±7.16 (5H, m, ole®nic
and Ar H) and 7.24±7.56 (1H, m, -CH CHˆCH-CO).
Preparation of 5-(3,4-dimethoxy phenyl)-2E,4E-pentadi-
enoic acid piperidine amide (18). Compound 4b (2.0 g,
8.5 mmol) was condensed with piperidine (0.9 mL) by
the method described for compound 8 to yield 18 (2.40 g,
91%), a crystalline solid, mp 118 ꢀC (lit. mp 110 ꢀC)25
(analysed for C18C23NO3; found C 71.7441, H 7.6922,
N 4.6503; calcd C 71.7351, H 7.6917, N 4.6475). MS (%)
Preparation of 5-(3,4-methylenedioxy phenyl)pentanoic
acid diethyl amide (17). The compound was prepared
from tetrahydro piperic acid 5h (2.0 g, 9 mmol) and
diethylamine(1 mL) by the method as described for 8,
as a gummy mass (2.30 g, 92%) (analysed for C16H23
NO3; found C 69.2879, H 8.3590, N 5.0510; calcd C
69.2871, H 8.3579, N 5.0500). MS (%) M+ at m/z 277
(61), 205 (18), 175 (8), 134 (78), 127 (39), 114 (100), 99
M
+ at m/z 301 (49), 217 (100), 159 (57), 114 (17) and 84
1
(16). ncm (KBr) 2930, 1635, 1605, 1565, 1513, 1452,
1440, 1312, 1250, 1131 1025, 870 and 808. H NMR
1
(CDCl3) d 1.66 (6H, bs, N (CH2)2 (CH2)3), 3.59 (4H,
bs, -N (CH2)2, 3.92 and 3.94 (6H, 2Âs, 2ÂOCH3), 6.43
(1H, d, J=15.0 Hz, -CHˆCH CO), 6.70±7.06 (5H, m,
ole®nic and Ar-H) and 7.24±7.75 (1H, m, CHˆCH
CHˆCH CO).
1
(78) and 71(47). ncm (KBr) 2957, 1642, 1553, 1457,
1361, 1241, 1128, 1053, 927 and 843. 1H NMR (CCl4) d:
1.13 (3H, t, J=6.5Hz, -CH2-CH3), 1.18 (3H, t, J=6.5Hz,
-CH2 CH3), 1.79 (4H, m, -(CH2)2-), 2.39 (2H, t, J=
Preparation of 5-(3,4-dimethoxy phenyl)-2E,4E-pentadi-
enoic acid pyrrolidine amide (19). Compound 4b (2.0 g,
8.5 mmol) was condensed with pyrrolidine (0.9 mL) as

S. Koul et al. / Bioorg. Med. Chem. 8 (2000) 251±268
263
per the procedure described for 8 to furnish a crystalline
solid (2.25 g, 92%), mp 148 ꢀC (analysed for C17C21NO3;
found C 71.0803, H 7.3666, N 4.8767; calcd C 71.0569,
H 7.3657, N 4.8744) MS (%). M+ at m/z 287 (41), 217
(4 mL) in solvent ether (80 mL) by the procedure
described for 2a to give a semisolid (5.1 g, 94%), ana-
lysed for C10H12O3, MS (%) M+ at m/z 180 (10), 164
(100), 136 (90), 106 (66) and 92 (90). ucm 1 (KBr) 3400,
2968, 1584, 1500, 1432, 1284, 1260, 1198, 1154, 1054,
1
(100), 189 (66), 174 (22), 98 (37) and 70 (35). ucm
(KBr) 2965, 1640, 1605, 1560, 1514, 1450, 1322, 1250,
1
and 868. H NMR (CDCl3) d 1.44 (3H, d, J=7.0 Hz,
CH3), 4.21 (4H, s, -OCH2 CH2O-), 4.76 (1H, q,
1
1020, and 870. H NMR (CDCl3) d 1.85 (4H, m, N-
CH2-(CH2)2), 3.46 and 3.49 (4H, 2Ât, J=6.0 Hz,
-N(CH2)2), 3.82 and 3.86 (6H, 2Âs, 2ÂOCH3), 6.18
(1H, d, J=15.0 Hz, -CHˆCH CO), 6.62±7.10 (5H, m,
ole®nic and Ar-H) and 7.20±7.79 (1H, m, CHˆCH
CH=CH CO).
J=7.0 Hz, CHOH) and 6.96 (3H, m, 3Â Ar H).
Preparation of 3-(3,4-ethylenedioxy phenyl)-2E-propenal
(3c). It was prepared from 2c (4.0 g, 22 mmol), POCl3
(4 mL) and DMF (8 mL) by the procedure described for
3a to give a yellow solid (2.8 g, 67%), mp 63±64 ꢀC,
analysed for C11H10O3, MS (%) M+ at m/z 190 (100),
Preparation of 5-(3,4-dimethoxy phenyl)-2E,4E-pentadi-
enoic acid n-butyl amide (20). Compound 4b (1.5 g,
6.4 mmol) was made to react with n-butylamine (0.7 mL)
as per the procedure described for 8 to give 20 (1.6 g,
86%), a crystalline solid, mp 114 ꢀC (analysed for
C17H23NO3; found C 70.5704, H 8.0119, N 4.8434;
calcd C 70.5619, H 8.0110, N 4.8404). MS (%) M+ at
m/z 289 (44), 217 (9), 189 (23), 188 (100), 157 (16), 151
(13), 99 (10) and 71 (38). ucm 1 (KBr) 3240, 2950, 1650,
1
162 (16), 134 (22), 106 (42) and 78 (53). ucm (KBr)
2928,1668, 1612, 1576, 1502, 1452, 1436, 1394, 1288,
1202, 1112, 1036, 968, 912 and 876. 1H NMR (CDCl3) d
4.28 (4H, s, -OCH2CH2O-), 6.56 (1H, dd, J=15.0 and
7.0 Hz, CH=CH CHO), 6.88±7.12 (3H, m, 3Â Ar H),
7.36 (1H, d, J=15.0 Hz, -CHˆCH CHO) and 9.60
(1H, d, J=7.0 Hz,ˆCH CHO).
1
1510, 1450, 1380, 1250, 1170, 1025, 1005 and 870. H
Preparation of 5-(3,4-ethylenedioxy phenyl)-2E,4E-pen-
tadienoic acid (4c). This compound was prepared from
3c (4.0g, 21 mmol) by the procedure described for 4a to
furnish 4c (4.0 g, 82%), mp 178±80 ꢀC, analysed for
NMR (CDCl3) d 0.92 (3H, t, J=6.5 Hz, -CH2 CH3),
1.46 (4H, m, -NCH2 (CH2)2), 3.31 (2H, m, -NCH2
CH2), 3.85 (6H, s, 2ÂOCH3), 5.94 (1H, d, J=14.0 Hz,
-CHˆCH CO), 6.62±7.00 (5H, m, ole®nic and Ar-H)
and 7.17±7.62 (1H, m, =CH CHˆCH-CO).
1
C13H12O4, MS (%) M+ at m/z 232. ucm (KBr) 2968,
1704, 1608, 1582, 1502, 1462, 1432, 1374, 1288, 1256, 1158,
1054, 860 and 810. 1H NMR (CDCl3 and DMSO-d6) d 4.28
(4H, s, -OCH2CH2O-), 6.10 (1H, d, J=15.0 Hz, -CHˆCH-
CHO) and 6.72±7.60 (6H, m, ole®nic and Ar H).
Preparation of 5-(3,4-dimethoxy phenyl)-2E,4E-penta-
dienoic acid-n-hexylamine amide (21). Compound 4b
(1.5 g, 6.4 mmol) was condensed with n-hexylamine
(0.8 mL) by the procedure described for 8 to give 21
(1.8 g, 94%), a crystalline solid, mp 148 ꢀC (analysed for
C19H27NO3; found C 71.9014, H 8.5740, N 4.4182;
calcd C 71.8935, H 8.5731, N 4.4126). MS (%) M+ at
m/z 317 (40.0), 217 (92.0), 189 (100), 159 (14.0), 114
(10.0), 89 (15.0) and 63 (13.0). ucm 1 (KBr) 3250, 2950,
2450, 1650, 1610, 1510, 1440, 1260, 1140, 1020, 990
Preparation of 5-(3,4-ethylenedioxy phenyl)-2E,4E-pen-
tadienoic acid piperidine amide (22). Compound 4c
(1.4 g, 6 mmol) was condensed with piperidine (0.6 mL)
by the procedure described for 8 to yield 22 (1.4 g,
78%), mp 162 ꢀC (analysed for C18H21NO3; found C
72.2207, H 7.0710, N 4.6813; calcd C 72.2181, H 7.0701,
N 4.6788). MS (%) M+ at m/z 299 (62), 215 (90), 189
(100), 162 (80), 114 (31) and 84 (80). ucm 1 (KBr) 2990,
1630, 1590, 1500, 1420, 1300, 1256, 1150, 990, 926 and
880. 1H NMR (CDCl3) d: 1.66 (6H, m, -N C (CH2)3),
3.60 (4H, m, -N (CH2)2), 4.26 (4H, s, -OCH2CH2O-),
6.17 (1H, d, J=14.0 Hz, -CHˆCH CO), 6.62±7.10
(5H, m, ole®nic and Ar H) and 7.29±7.79 (1H, m,
-CHˆCH-CO).
1
and 850. H NMR (CDCl3) d 0.90 (3H, t, J=6.5 Hz,
-CH2 CH3), 1.34±1.71 (8H, m, (CH2)4), 3.33 (2H, m,
-NH CH2), 3.94 (6H, s, 2ÂOCH3), 5.97 (1H, d,
J=14.0 Hz, -CHˆCH CO), 6.73±7.10 (5H, m, ole®nic
and Ar H) and 7.26 (1H, m, CH CHˆCH-CO).
Preparation of 3,4-ethylenedioxy benzaldehyde (1c). To
a
solution of 3,4-dihydroxy benzaldehyde (6.9 g,
50 mmol), in dry acetone (110 mL) was added 1,2-
dibromoethane (6 mL) and anhydrous potassium car-
bonate (5g) and the contents re¯uxed for 48 h to give
after usual work up, 3,4-ethylenedioxy benzaldehyde
(1c) (7.5 g, 90%), crystallised from hexane:acetone, mp
54±55 ꢀC, analysed for C9H8O3, MS (%) M+ at m/z 164
Preparation of 5-(3,4-ethylenedioxy phenyl)- 2E,4E-pen-
tadienoic acid pyrrolidine amide (23). Compound 4c
(1.6 g, 7 mmol) was reacted with pyrrolidine (0.8 mL) by
the procedure described for 8 to yield 23 (1.90 g, 95%),
mp 113 ꢀC (analysed for C17H19NO3 found C 71.5612,
H 6.7121, N 4.9101 calc. C 71.5589, H 6.7113, N 4.9088)
MS (%) M+ at m/z 285 (40), 215 (18), 188 (100), 98 (14)
1
(100) 135 (10) 119 (4) 91 (3) and 79 (22). ucm (KBr)
1680, 1608, 1576, 1500, 1458, 1394, 1288, 1206, 1108,
1
and 70 (23). ucm (KBr) 2960, 1645, 1600, 1577, 1510,
1400, 1285, 1248, 1190, 1114, 1052 and 775. H NMR
1
1
1040, 944, 910 and 862. H NMR (CDCl3) d: 4.24 (4H,
s, -O CH2 CH2 O-), 6.89 (1H, d, J=8.5 Hz, Ar H),
7.35 (2H, m, Ar H) and 9.71 (1H, s, CHO).
(CDCl3) d: 1.94 (4H, m, -N-C-(CH2)2), 3.57 (4H, t, J=
7.0 Hz, N-(CH2)2), 4.30 (4H, s, -OCH2CH2O-), 6.25 (1H,
d, J=14.0 Hz, -CH=CH-CO), 6.73±7.16 (5H, m, Ole-
®nic and Ar-H) and 7.30±7.70 (1H, m, -CH=CH-CO).
Preparation of ꢀ-methyl-3,4-ethylenedioxy benzyl alco-
hol (2c). This compound was prepared from 3,4-ethyle-
nedioxy benzaldehyde (5.0 g, 30 mmol) and Grignard
reagent (mg metal, 0.86 g, 36 mmol) and methyl iodide
Preparation of 4-benzyloxy-3-methoxy benzaldehyde (1d).
To a solution of 4-hydroxy-3-methoxy benzaldehyde

264
S. Koul et al. / Bioorg. Med. Chem. 8 (2000) 251±268
(13.0 g, 85 mmol) in acetone (250 mL) was added benz-
ylbromide (12.0 mL, 88 mmol) and anhydrous potas-
sium carbonate (10 g) and re¯uxed for 24 h. The usual
work up a€orded 1d (19.0 g, 92%), mp 67 ꢀC, analysed
for C15H14O3 MS (%) M+ at m/z 242 (100), 227 (18),
CO CHˆCH), 6.84±7.00 (6H, m, ole®nic and Ar H)
and 7.28±7.78 (5H, bs, ole®nic and Ar H).
Preparation of 5-(4-benzyloxy-3-methox phenyl)-2E,4E-
pentadienoic acid pyrrolidine amide (25). Compound 4d
(1.55 g, 5 mmol) was condensed with pyrrolidine
(0.6 mL) by the method described for 8 to yield 25
(1.7 g, 94%), mp 155 ꢀC (analysed for C23H25NO3; found
C 76.0120, H 6.9336, N 3.8551; calcd C 76.0075, H 6.9327,
N 3.8538). MS (%) M+ at m/z 363 (19), 273 (44), 244 (5),
201 (17), 155 (3), 145 (4), 131 (6), 115 (14) and 91 (100).
1
213 (21) 181 (29), and 91 (50). ucm (KBr) 2928, 1674,
1598, 1586, 1500, 1460, 1428, 1400, 1386, 1236, 1184,
1
1128, 916 and 818. H NMR (CDCl3) d: 4.12 (3H, s,
OCH3), 5.44 (2H, s, -OCH2 Ar), 7.20 (1H, d,
J=8.5 Hz, Ar H), 7.48±7.84 (7H, m, Ar H) and 9.92
(1H, s, Ar CHO).
1
ucm (KBr) 2914, 1636, 1610, 1589, 1504, 1420, 1384,
Peparation of ꢀ-methyl-4-benzyloxy-3-methoxy benzyl
alcohol (2d). It was prepared from 1d (14.5 g, 60 mmol)
and magnesium iodide (62 mmol) as described for 2a
to furnish a semisolid (14.0 g, 90%), analysed for
C16H18O3, MS (%) M+ at m/z 259 (10), 241 (4), 151
1354, 1260, 1230 1138 and 980. 1H NMR (CDCl3) d: 1.90
(4H, bs, -N C (CH2)2), 3.52 (4H, bs, -N (CH2)2-), 3.88
(3H, s, OCH3), 5.14 (2H, s, Ar CH2O), 6.24 (1H, d,
J=15.0 Hz, -CHˆCH CO), 6.68±7.08 (5H, m, ole®nic
and Ar H) and 7.16±7.80 (6H, m, ole®nic and Ar H).
1
(16), 121 (14) and 91 (100). ucm (KBr) 3410, 2972,
1594, 1500, 1454, 1414, 1382, 1258, 1222, 1150, 1115
Preparation of 5-(4-benzyloxy-3-methoxy phenyl)-2E,4E-
pentadienoic acid isobutyl amide (26). Compound 4d
(1.55 g, 5 mmol) was condensed with isobutyl amine
(0.6 mL) by the method described for 8 to furnish a solid
(1.65 g, 90%), mp 183 ꢀC (analysed for C23H27NO3;
found C 75.5910, H 7.4472, N 3.8347; calcd C 75.5883,
H 7.4461, N 3.8325). MS (%) M+ at m/z 365 (14), 293
(3), 274 (34), 201 (18), 175 (17), 143 (12), 131 (6), 115
1
and 1072. H NMR (CDCl3) d 1.38 (3H, d, J=7.0 Hz,
CHOH CH3), 3.83 (3H, s, OCH3), 4.72 (1H, q, J=
7.0 Hz, CHOH-CH3), 5.04 (2H, s, Ar CH2O), 6.76
(1H, s, Ar H), 6.82 (2H, d, J=8.5 Hz, 2Â Ar H) and
7.16±7.52 (5H, m, 5Â Ar H).
Preparation of 3-(4-benzyloxy-3-methoxy phenyl)-2E-
propenal (3d). This compound was prepared from 2d
(11 g, 43 mmol) and Vilsmeier reagent as described for 3a
to yield a crystalline solid 3d (7.0g, 64%), mp 91 ꢀC (lit.
mp. 90 ꢀC)31 analysed for C17H16O3, MS (%) M+ at m/z
268 (51), 242 (23), 178 (41), 162 (9), 147 (20), 124 (10) and
91 (100). (KBr) 2940, 1666, 1620, 1598, 1502, 1480, 1460,
1426, 1384, 1260, 1220, 1168, 1120, 1028 and 972. 1H NMR
(CDCl3) d 3.88 (3H, s, OCH3), 5.20 (2H, s, Ar CH2O),
6.56 (1H, dd, J=15.0 and 7.0 Hz, CHˆCH CHO), 6.84±
7.14 (3H, m, Ar-H), 7.22±7.54 (6H, m, ole®nic and Ar H)
and 9.60 (1H, d, J=7.0 Hz, -CH CHO).
1
(17) and 91 (100). ucm (KBr) 2956, 1642, 1610, 1594,
1512, 1458, 1414, 1332, 1248, 1234 and 1122. H NMR
1
(CDCl3): 0.92 (6H, d, J=6.5 Hz, -CH (CH3)2), 1.80
(1H, m, CH (CH3)2), 3.20 (2H, t, J=6.5 Hz, -NH CH2),
3.90 (3H, s, OCH3), 5.20 (2H, s, Ar CH2O), 5.94 (1H,
d, J=14.0 Hz, -CHˆCH CO), 6.70±7.08 (5H, m, ole®nic
and Ar-H). and 7.24±7.80 (6H, m, ole®nic and Ar H.)
Preparation of ꢀ-methyl-3,4,5-trimethoxy benzyl alcohol
(2e). This compound was prepared from 3,4,5-tri-
methoxy benzaldehyde 1e (10.0 g, 51 mmol) by react-
ing with methyl magnesium iodide (53 mmol) as
described for 2a to yield a semisolid 2e (9.80 g, 90%)
analysed for C11H16 O4, MS (%) M+ at m/z 212 (100),
196 (52), 168 (80), 153 (31), 137 (32), 122 (11), 108 (13),
Preparation of 5-(4-benzyloxy-3-methoxyphenyl)-2E,4E-
pentadienoic acid (4d). This compound was prepared
from 3d (5.0 g, 19 mmol) by Wittig reaction as descri-
bed for 4a to yield 4d (4.8 g, 81%), a crystalline solid,
mp 196 ꢀC, analysed for C19H18O4, MS (%) M+ at m/z
310 (20), 297 (18), 266 (24), 219 (16), 175 (30) 115 (11)
and 91 (100). ucm 1 (KBr) 2932, 1666, 1618, 1592, 1504,
1
94 (13) and 77 (13). ucm (KBr) 3480, 2972, 1594,
1502, 1456, 1420, 1328, 1234, 1118, 1000 and 832. H
1
NMR (CDCl3) d 1.44 (3H, d, J=7.0 Hz, CH3 CHOH),
3.76 (3H, s, OCH3), 3.82 (6H, s, 2ÂOCH3), 4.76 (1H, q,
J=7.0 Hz, CHOH CH3) and 6.56 (2H, s, 2Â Ar H).
1
1454, 1422, 1352, 1308 and 1266. H NMR (CDCl3) d
3.90 (3H, s, OCH3), 5.14 (2H, s, Ar CH2O), 5.92 (1H,
d, J=14.0 Hz, CHˆCH CO), 6.73±7.10 (5H, m,
Ar H), 7.21±7.70 (6H, m, ole®nic and Ar H).
Preparation of 3-(3,4,5-trimethoxy phenyl-2E-propenal
(3e). This compound was prepared from 2e (8.5 g,
40 mmol) by reacting with Vilsmeier reagent as descri-
bed for 3a to give a yellow crystalline solid (5.9 g,
66%), mp 110±111 ꢀC32 analysed for C12H14O4. MS (%)
M+ at m/z 222, 207, 179 and 151. ucm 1(KBr) 1695,
Preparation of 5-(4-benzyloxy-3-methoxyphenyl)-2E,4E-
pentadienoic acid piperidine amide (24). Compound 4d
(1.55 g, 5 mmol) was condensed with piperidine (0.6 mL,
6 mmol) by the method described for 8 to furnish a
solid (1.8 g, 95%) crystallised from ethylacetate:pet.ether
(9:1) to give a crystalline solid mp 116±17 ꢀC (lit. mp.
118 ꢀC)31 (analysed for C24H27NO3; found C 76.3667, H
7.2103, N 3.7142; calcd C 76.3651, H 7.2091, N 3.7106);
MS (%) M+ at m/z 377 (42) 286 (100) 258 (12) 201 (13)
1
1638, 1595, 1570, 1350, 1135, 1010 and 830 H NMR
(CDCl3) d 3.88 (9H, s, 3xOCH3), 6.46 (1H, dd,
J=16.0 Hz and 7.0 Hz, -CHˆCH CHO), 6.80 (2H, bs,
2 Ar H), 7.40 (1H, d, J=16.0 Hz, CHˆCH CHO)
and 9.70 (1H, d, J= 7.0 Hz,ˆCH CHO).
1
and 91 (11). ucm (KBr) 2928, 2856, 1620, 1510, 1450,
Preparation of 5-(3,4,5-trimethoxy phenyl)-2E,4E-pent-
adienoic acid (4e). This compound was prepared from
3e (5.1 g, 23 mmol) through Wittig reaction as descri-
bed for 4a to give (4.6 g, 76%), mp 190 ꢀC33 analysed
1
1390, 1368, 1266 and 1196. H NMR (CDCl3) d: 1.50
(6H, bs, 3Â CH2), 3.44 (4H, bs, -N(CH2)2), 3.98 (3H, s,
OCH3) 5.04 (2H, s, Ar CH2), 6.34 (1H, d, J=15 Hz,

S. Koul et al. / Bioorg. Med. Chem. 8 (2000) 251±268
265
for C14H16O5 MS (%) M+ at m/z 264 ucm 1(KBr)
2932, 1700, 1626, 1605, 1506, 1424, 1338, 1232, 1160,
1120, 1010, 974 and 846. 1H NMR (CDCl3) d: 3.81 (9H,
s, 3ÂOCH3), 6.20 (1H, d, J=15.0 Hz, -CHˆCH CO),
6.70±7.10 (4H, m, ole®nic and Ar H), and 7.30±7.84
(1H, m, CH CH CO).
MS (%) M++1 at m/z 207 (48), 188 (100),121 (80) and 96
1
(61). ucm (KBr) 3352, 2932, 1618, 1588, 1494, 1450,
1370, 1348, 1252, 1208, 1150, 1120, 1070, 926 and 878. 1H
NMR (CDCl3) d 1.28 (6H, s, (CH3)2), 1.44 (3H, d,
J=6.5Hz, CH3-CHOH), 1.76 (2H, t, J=7.0Hz,
Ar CH2 CH2), 2.76 (2H, t, J=7.0 Hz, Ar CH2 CH2),
4.76 (1H, q, J=6.5 Hz, CHOH CH3) 6.72 (1H, d,
J=8.5Hz, Ar H) and 7.04 (2H, bs, Ar H).
Preparation of 5-(3,4,5-trimethoxy phenyl)-2E,4E-penta-
dienoic acid piperidine amide (27). Compound 4e (1.6 g,
6 mmol) was condensed with piperidine (0.5 mL) as
described for 6 to furnish 27 (1.86 g, 94%), mp 214 ꢀC
(analysed for C19H25NO4; found C 68.8644, H 7.6040, N
4.2296; calcd C 68.8601, H 7.6031, N 4.2264). MS (%) M+
at m/z 331 (37), 277 (70), 247 (14), 219 (23), 189 (12), 112
Preparation of 3-[(2H)-2,2-dimethyl-3,4-dihydro-benzo-
pyran-6yl]-2-propenal (3g). This compound was prepared
from 2g (6.20 g, 30mmol) and Vilsmeier reagent by the
method as described for 3a to give 3g (4.2 g, 65%), a
semi solid, analysed for C14H16O2, MS (%) M+ at m/z
216 (33), 188 (30), 174 (28), 160 (48), 146 (31), 103 (40) and
69 (100). ucm 1 (KBr) 2968, 2916, 1664, 1606, 1574, 1492,
1426, 1386, 1365, 1336, 1244, 1106, 944 and 800. 1H NMR
(CDCl3) d 1.24 (6H, s, C(CH3)2), 1.76 (2H, t, J=7.0Hz,
Ar CH2 CH2), 2.72 (2H, t, J=7.0 Hz, Ar CH2 CH2),
6.48 (1H, dd, J=14.0 and 7.0 Hz, CHˆCH CHO), 6.74
(1H, d, J=8.5 Hz, Ar-H), 7.20 (2H, bs, 2ÂAr-H), 7.32
(1H, d, J=15.0 Hz, Ar-CHˆCH CHO) and 9.56 (1H, d,
J=7.0Hz, Ar CHO).
1
(11) and 84 (100). ucm (KBr) 2930, 1640, 1605, 1560,
1440, 1350, 1250, 1140, 1010, 960 and 870. H NMR
1
(CDCl3) d: 1.60 (6H, bs, -N C (CH2)3), 3.62 (4H, m, N
(CH2)2), 3.89 (3H, s, OCH3), 3.94 (6H, s, 2ÂOCH3), 6.47
(1H, d, J=15.0 Hz, CHˆCH CO), 6.66±7.00 (4H, m, ole-
®nic and Ar H) and 7.23±7.72 (1H, m, -CHˆCH CO).
Preparation of 5-(3,4,5-trimethoxy phenyl)-2E,4E-penta-
dienoic acid pyrrolidine amide (28). Compound 4e (1.6 g,
6 mmol) was condensed with pyrrolidine (0.6mL) as
described for 8 to yield 28 (1.80 g, 95%), a crystalline solid,
mp 190 ꢀC (analysed for C18H23NO4; found C 68.1212, H
7.3047, N 4.4150; calcd C 68.1190, H 7.3040, N 4.4132).
MS (%) M+ at m/z 317 (57), 219 (100), 189 (63), 159 (12),
98 (35), 70 (16). ucm 1 (KBr) 2960, 1640, 1605, 1560, 1450,
1330, 1250, 1135, 1015, 970 and 860. 1H NMR (CDCl3) d
1.95 (4H, m, N C (CH2)2), 3.64 (4H, t, J=6.5Hz,
-N(CH2)2), 3.85 (3H, s, OCH3), 3.92 (6H, s, 2ÂOCH3), 6.26
(1H, d, J=15.0 Hz, CHˆCH CO), 6.65±6.93 (4H, m, ole-
®nic and Ar H) and 7.23±7.60 (1H, m, -CHˆCH CO).
Preparation of 5-[(2H)-2,2-dimethyl-3,4-dihydro-benzo-
pyran-6yl]-2E,4E-pentadienoic acid (4g). This compound
was prepared from 3g (3.90 g, 18mmol) by Wittig reac-
tion as described for 4a to give [4.1g, 89%], a crystalline
solid, mp 217 ꢀC, analysed for C16H18O3, MS (%) M+ at
m/z 258 (57), 213 (19), 203 (26), 171 (10), 157 (100), 145
(26), 129 (82), 115 (54), 102 (13) and 91 (33). ucm 1(KBr)
2944, 1644, 1598, 1494, 1408, 1388, 1362, 1214, 1120, 970
and 866. 1H NMR (CD3OD) d 1.46 (6H, s, C(CH3)2), 1.90
(2H, t, J=7.0 Hz, Ar CH2 CH2), 2.87 (2H, t, J=7.0Hz,
Ar CH2 CH2), 5.95 (1H, d, J= 15.0 Hz, CHˆCH
CO), 6.53±7.00 (5H, m, ole®nic and Ar-H) and 7.43±7.80
(1H, m, CHˆCH CHO).
Preparation of (2H)-2,2-dimethyl-3,4-dihydro-6-formyl
benzopyran (1g). To a stirring solution of 4-hydroxy
benzaldehyde 1f (25 g, 205 mmol) and orthophos-
phoric acid (20 mL) in hexane (200 mL), a slow addition
of freshly distilled isoprene (25 mL) in n-hexane
(40 mL) was made in 9 h at room temperature and the
reaction mixture stirred further for 24 h. The reaction
mixture was worked up by dilution with water, followed
by extraction of the aqueous layer with solvent ether
(2Â100 mL). The combined organic layer washed with
water, dried over anhydrous sodium sulfate and con-
centrated to give crude resinous product which on CC
over SiO2 and elution with hexane:benzene (4:1) a€or-
ded 1g (11.5 g, 30%), as a gummy mass34 analysed for
C12H14O2, MS (%) M+ at m/z 190 (36), 189 (73), 160
(33), 146 (38), 134 (100) and 106 (13). ucm 1(KBr) 2940,
1682, 1606, 1576, 1488, 1432, 1384, 1362, 1352, 1324,
1270, 1236, 1114, 1108, 874 and 818. 1H NMR (CDCl3)
d: 1.35 (6H, bs, -C(CH3)2), 1.85 (2H, t, J=6.0 Hz,
-CH2 CH2 Ar), 2.85 (2H, t, J=6.0 Hz, Ar CH2
CH2), 6.87 (1H, d, J=8.5 Hz, Ar H), 7.64 (2H, bs,
Ar H) and 9.80 (1H, s, Ar CHO).
Preparation of 5-(2H)-2,2-dimethyl-3,4-dihydro-benzo-
pyran-6yl-2E,4E-pentadienoic acid piperidine amide (29).
Compound 4g (1.0g, 4 mmol) was condensed with
piperidine (0.6mL) as described for 8 to yield 29 (1.16 g,
89%), mp 164 ꢀC (analysed for C21H27NO2; found C
77.5093, H 8.3624, N 4.3055; calcd C 77.5023, H 8.3617, N
4.3038) MS (%) M+ at m/z 325 (85), 297 (37), 241 (100),
213 (48), 189 (39), 111 (92) and 84 (92). ucm 1(KBr) 2950,
1650, 1610, 1548, 1447, 1383, 1314, 1010, 972 and 865. 1H
NMR (CDCl3) d 1.34 (6H, bs, -C (CH3)2), 1.59±2.16
(6H, m, N (CH2)3), 1.80 (2H, t, J=6Hz, Ar C CH2),
2.72 (2H, t, J=6.0 Hz, Ar-CH2), 3.52 (4H, m,
-N (CH2)2), 6.16 (1H, d, J=14.0 Hz, -CHˆCH CO),
6.59±6.82 and 6.92±7.69 (6H, m, ole®nic and Ar H).
Preparation of 5-[(2H)-2,2-dimethyl-3,4-dihydrobenzo-
pyran-6yl]-2E,4E-pentadienoic acid pyrrolidine amide
(30). Compound 4g (1.0 g, 4 mmol) was condensed with
pyrrolidine (0.5 mL) as described for the 8 to give 30
(0.98 g, 85%), mp 117 ꢀC (analysed for C20H25NO2,
found C 77.1400, H 8.0917, N 4.5004, calcd C 77.1363,
H 8.0910, N 4.4977) MS (%) M+ at m/z 311 (73), 241
(100), 184 (50), 158 (40), 127 (31), 97 (31) and 70 (46).
Preparation of 1-[-(2H)-2,2-dimethyl-3,4-dihydro-benzo-
pyran-6yl]-ethanol (2g). This compound was prepared
from 1g (7.0 g, 37mmol) and methyl magnesium iodide
(39 mmol) reagent by the method as described for 2a to
give 2g (7.3 g, 94%) a semisolid, analysed for C13H18O2,
1
ucm (KBr) 2972, 1653, 1610, 1540, 1450, 1385, 1364,
1320, 1250, 1040, 972 and 865. 1H NMR (CDCl3) d 1.32

266
S. Koul et al. / Bioorg. Med. Chem. 8 (2000) 251±268
(6H, s, -C (CH3)2), 1.62 (4H, m, 2ÂCH2), 1.80 (2H, t,
J=6.5 Hz, Ar CH2 CH2), 2.75 (2H, t, J=6.5 Hz, Ar-
CH2 CH2), 3.58 (2H, m, -N (CH2)2), 6.40 (1H, d,
J=14.0 Hz, -CHˆCH CO), 6.60±6.92 (3H, m, ole®nic
and Ar H) and 7.04±7.68 (3H, m, ole®nic and Ar H)
Preparation of 3-[(2H)-2,2-dimethyl-3,4-dihydrobenzo-
pyran-6yl]-2E-propenoic acid (7). Compound 7 was pre-
pared from 1g (6.0 g, 32 mmol) and malonic acid (4.2 g,
40 mmol) as described for 6 to give crystalline com-
pound 736 (6.7 g, 90%), mp 188 ꢀC analysed for
C14H16O3, MS (%) M+ at m/z 232 (93), 215 (13), 188
1
Preparation of 5-[(2H)-2,2-dimethyl-3,4-dihydrobenzo-
pyran-6yl]-2E,4E-pentadienoic acid n-pentyl amide (31).
Compound 4g (1.0 g, 4 mmol) was condensed with n-
pentyl amine (0.6 mL) as described for 8 to yield 31, a solid
(1.20 g, 92%), mp 137 ꢀC (analysed for C21H29NO2; found
C 77.0317, H 8.9267, N 4.2793; calcd C 77.0253, H 8.9258,
N 4.2774). MS (%) M+ at m/z 327 (22), 241 (23), 185 (22),
(6), 176 (100) and 131 (20). ucm (KBr) 2964, 1674,
1609, 1574, 1494, 1430, 1386, 1370, 1304, 1286, 1260,
1234, 1150, 932 and 820. 1H NMR (CDCl3) d 1.32 (6Hs,
-C(CH3)2), 1.82 (2H, t, J=7.0 Hz, Ar CH2 CH2), 2.74
(2H, t, J=7.0 Hz, Ar CH2 CH2), 6.72 (1H, d,
J=15.0 Hz, CHˆCH CO), 6.80 (1H, d, J=8.5 Hz,
Ar H), 7.24 (1H, s, Ar H), 7.28 (1H, d, J=8.5 Hz,
Ar H) and 7.68 (1H, d, J=15.0 Hz, -CHˆCH CO).
1
175 (13), 157 (77), 128 (49), 96 (28) and 69 (100). ucm
(KBr) 3270, 2910, 1640, 1600, 1490, 1380, 1260, 1125, 1000,
920 and 805. 1H NMR (CDCl3) d 0.90 (3H, t, J=6.0Hz,
-CH2 CH3), 1.16±1.56 [12H, bs, -NH CH2 (CH2)3 and
C (CH3)2], 1.80 (2H, t, J=6.0 Hz, Ar CH2 CH2), 2.72
(2H, t, J=6.0 Hz, Ar CH2 CH2), 3.34 (2H, m,
-N (CH2)), 5.94 (1H, d, J=15.0 Hz, -CHˆCH CO),
6.52±6.82 and 6.90±7.66 (6H, m, ole®nic and Ar H).
Preparation of 3-[(2H)-2,2-dimethyl-3,4-dihydro-benzo-
pyran-6yl]-2E-propenoic acid piperidine amide (34).
Compound 7 (0.93 g, 4 mmol) was condensed with
piperidine (0.6 mL) as described for 8 to give 34 (1.10 g,
92%), mp 122 ꢀC (analysed for C19H25NO2; found C
76.2221, H 8.4160, N 4.6802; calcd C 76.2191, H 8.4156,
N 4.6781) MS (%) M+at m/z 299 (100), 214 (93), 187
1
Preparation of 3-(3,4-methylenedioxy phenyl)-2E-prope-
noic acid (6). To 1h (4.5 g, 30 mmol) in pyridine
(25 mL) and piperidine (1 mL) was added malonic acid
(3.7 g, 36 mmol) and contents stirred for 24 h, followed
by heating on water bath for 6 h. The contents were
cooled, poured in ice-cold water, acidi®ed with 2 N
HCl. The resulting precipitate ®ltered, washed with
water and air dried to give 6 (6.2 g, 95%), crystallised
from ethyl acetate:n-hexane (9:1), mp. 244±646 ꢀC (lit.
mp 247 ꢀC).35
(31), 158 (60) 84 (61) and 69 (30). ucm (KBr) 2932,
1640, 1598, 1578, 1494, 1434, 1382, 1368, 1270, 1244,
1232, 1214, 1110, 1010, 982 and 830. 1H NMR (CDCl3)
d: 1.23 (6H, s, -C(CH3)2), 1.56 (6H, bs, N C (CH2)3),
1.72 (2H, t, J=6.5 Hz, Ar CH2 CH2), 2.72 (2H, t,
J=6.5 Hz, Ar CH2 CH2), 3.50 (4H, m, NH (CH2)2),
6.66 (1H, d, J=15.0 Hz, CHˆCH CO), 6.62 (1H, d,
J=8.5 Hz, Ar H), 7.06±7.24 (2H, m, 2ÂAr-H) and
7.43 (1H, d, J= 15.0 Hz, -CHˆCH CO).
Preparation of 3-[(2H)-2,2-dimethyl-3,4-dihydrobenzo-
pyran-6yl]-2E-propenoic acid isopropyl amide (35).
Compound 7 (1.2 g, 5 mmol) was condensed with iso-
propyl amine (0.7 mL) by the method as described for 8
to give 35 (1.24 g, 91%), mp 89 ꢀC (analysed for C17
H23NO2; found C 74.6898, H 8.4823, N 5.1255; calcd C
74.6900, H 8.4816, N 5.1236); MS (%) M+ at m/z 273
(100), 215 (61), 188 (58), 159 (51) and 58 (48). ucm 1(KBr)
3260, 2968, 1654, 1612, 1580, 1496, 1420, 1384, 1370,
Preparation of 3-(3,4-methylenedioxy phenyl)-2E-prope-
noic acid piperidine amide (32). Compound 6 (2.1 g,
11 mmol) was condensed with piperidine (1.2 mL) as
described for 8 to yield 32 (2.80 g, 91%), mp 89 ꢀC
(analysed for C15H17NO3; found C 69.4901, H 6.6089,
N 5.4071; calcd C 69.4804, H 6.6078, N 5.4017) MS (%)
M+ at m/z 259 (4), 147 (21), 111 (100) and 84 (29).
1
ucm (KBr) 2940, 1642, 1588, 1494, 1436, 1350, 1298,
1250, 1216, 1100, 1018, 972 and 808. 1H NMR (CDCl3)
1304, 1236, 1200, 1152, 1112, 1000, 920 and 874. H
1
d
1.64 (6H, bs, N C (CH2)3), 3.60 (4H, bs,
NMR (CDCl3) d 1.20 (6H, d, J=6.5 Hz, -C(CH3)2),
1.33 (6H, s, (CH3)2), 1.76 (2H, t, J=6.50 Hz, Ar CH2
CH2), 2.70 (2H, t, J=6.5 Hz, Ar CH2), 4.13 (1H, m,
CH N), 6.13 (1H, d, J=15.0 Hz, CHˆCH CO), 6.63
(1H, d, J=8.5 Hz, Ar H), 6.95±7.23 (2H, m, Ar H)
and 7.43 (1H, d, J=15.0 Hz, -CHˆCH CO).
-N (CH2)2), 5.94 (2H, s, -OCH2O-), 6.70 (1H, d,
J=15.0 Hz, CHˆCH CO), 6.76 (1H, d, J=8.5 Hz,
Ar H), 6.98 (1H, d, J=8.5 Hz, Ar H), 7.02 (1H, s,
Ar H) and 7.52 (1H, d, J=15.0 Hz, CH=CH CO).
Preparation of 3-(3,4-methylenedioxy phenyl)-2E-prope-
noic acid pyrrolidine amide (33). Compound 6 (2.0 g,
10 mmol) was condensed with pyrrolidine (1.0 mL) as
described for 8 to yield 33 (2.4 g, 90%), mp 146 ꢀC
(analysed for C14H15NO3; found C 68.5603, H 6.1644,
N 5.7133; calcd C 68.5569, H 6.1638, N 5.7106). MS
(%) M+ at m/z 245 (15) 174 (32) 147 (9) 98 (30) and
70 (100). ucm 1(KBr) 2960, 1646, 1594, 1496, 1454,
1414, 1354, 1320, 1278, 1242, 1198, 1018, 992, 928 and
Preparation of 3-[(2H)-2,2-dimethyl-3,4-dihydrobenzo-
pyran-6yl-2E- propenoic acid n-butylamide (36). Com-
pound 7 (1.2g, 5 mmol) was condensed with n-butylamine
(0.6 mL) by the method as described for 8 to give 36
(1.29 g, 90%), mp 86ꢀC (analysed for C18H25NO2; found
C 75.2301, H 8.7677, N 4.8771; calcd C 75.2253, H 8.7673,
N 4.8736). MS (%) M+ at m/z 287 (100), 242 (79), 214
(79), 187 (21), 170 (12), 158 (60) 130 (30) and 72 (14).
1
1
826. H NMR (CDCl3) d 1.92 (4H, bs, N C (CH2)2),
ucm (KBr) 3200, 2932, 1648, 1616, 1580, 1544, 1450,
3.56 (4H, m, -N (CH2)2), 5.96 (2H, s, -OCH2O-),
6.50 (1H, d, J=15.0 Hz, CHˆCH CO), 6.78 (1H, d,
J=8.5Hz, Ar-H), 6.97 (1H, d, J=8.5Hz, Ar-H), 7.00
(1H, s, Ar-H) and 7.56 (1H, d, J=15.0 Hz, CHˆ
CH CO).
1424, 1384, 1370, 1308, 1264, 1236, 1152, 968 and 852. 1H
NMR (CDCl3) d 0.92 (3H, t, J= 6.5 Hz, -CH2 CH3),
1.33 (6H, s, -C(CH3)2), 1.49 (4H, m, -C (CH2)2), 1.79
(2H, t, J=6.5 Hz, Ar CH2 CH2), 2.74 (2H, t, J=6.5 Hz,
Ar CH2 CH2), 3.34 (2H, m, NH-CH2), 6.38 (1H, d,

S. Koul et al. / Bioorg. Med. Chem. 8 (2000) 251±268
267
J=15.0 Hz, CHˆCH CO), 6.66 (1H, d, J=8.5 Hz, Ar-
H), 7.09±7.42 (2H, m, Ar H) and 7.67 (1H, d,
J=15.0 Hz, -CHˆCH CO).
terminated with 75 mL of 15% cold TCA (w/v). The
product 7-OH-coumarin was extracted and measured
¯uorometrically at 396 nm excitation and 520 nm emission.
Preparation of 3-[(2H)-2,2-dimethyl-3,4-dihydrobenzo-
pyran-6yl]-2E-propenoic acid n-hexylamide (37). Com-
pound 7 (0.93 g, 4 mmol) was condensed with hexylamine
(0.5 mL) as described for 8 to give 37 (1.15 g, 91%), mp
71 ꢀC (analysed for C20H29NO2; found C 76.1600, H
9.2666, N 4.4443; calcd C 76.1505, H 9.2657, N 4.4402).
MS (%) M+ at m/z 315 (82), 258 (16), 215 (92), 187 (22),
173 (100) and 99 (19). ucm 1(KBr) 3190, 2900, 1654, 1608,
1573, 1540, 1494, 1283, 1381, 1367, 1306, 1238, 1214,
1154, 1120, 928 and 818. 1H NMR (CDCl3) d: 0.89 (3H, d,
J=6.5Hz, -CH3), 1.30 (6H, s, -C(CH3)2), 1.33 (8H, m,
-C(CH2)4 CH3), 1.76 (2H, t, J=6.0Hz, Ar CH2 CH2),
2.73 (2H, t, J=6.0 Hz, Ar CH2), 3.33 (2H, m, NH CH2)
6.23 (1H, d, J=15.0 Hz, CHˆCH CO), 6.70 (1H, d,
J=8.5Hz, Ar H), 7.10±7.33 (2H, m, Ar H), and 7.54
(1H, d, J=15.0 Hz,-CHˆCH CO).
The activity of arylhydrocarbonhydroxylase (AHH) was
measured according to Wiebel et al.38 Brie¯y, the reac-
tion mixture in a total volume of 1 mL contained 50 mM
Tris±HCl bu€er, pH 7.6, 3 mM MgCl2, 0.6 mM
NADPH and 0.5 to 2.5 mg microsomal protein. Piper-
ine or test compound was added in 10 mL of 50%
methanol. The reaction was started under subdued light
with 0.1 mM benzo (a) pyrene in 20 mL of 50% metha-
nol and incubated at 37 ꢀC for 30 min in a shaking water
bath. The reaction was terminated and the relative
¯uorescence of the aqueous phase was measured at
396 nm excitation and 520 nm emission using 3-OH-
benzo(a)pyrene as reference standard.
Sleeping time. The hexobarbital induced sleeping time
was determined as the time required for the mice to
regain their rightening re¯ex.2 The control animals
received only the vehicle.
Biology
Chemicals. Chemicals and cell culture medium were
purchased from the following sources: NADPH from
Sigma Chemie, Munchen, FRg; 7-methoxycoumarin
(MOC) from Aldrich Chemie, Steinheim, FRg and pur-
i®ed as described earlier.21 The sources of piperine and
other chemicals are described elsewhere.2,6 All other
chemicals used were of analytical grade and available
locally.
Cell culture and treatment. H4IIEC3/G cells used in
the present study are the descendants of rat Reuber
hepatoma. Their source, growth characteristics and
incubation conditions are described earlier.4,39 Cells
were seeded at a density of 1Â106 cells per 90 mm plastic
dishes in DMEM containing fetal calf serum and anti-
biotics and allowed to grow for 48 h. Cultures were
exposed for 24 h to fresh medium containing test com-
pound delivered in 15 mL DMSO. Control plates
received only the vehicle. Cells were washed with phos-
phate bu€er saline (PBS) and harvested in 1 mL PBS
and centrifuged. The pellet was stored in liquid nitrogen
and used for assay of monooxygenase activities after
suspension in 50 mM Tris±HCl, pH 7.4.
Animals and treatment. Adult male albino Charles Fos-
ter rats (200‹20 g, body wt) and male Swiss albino mice
(25‹2 g, body wt) used were bred in the animal house
of this institute. The animals were maintained on stan-
dard commercial pellet food (Hindustan Lever Ltd.,
Bombay) and water ad libitum. Rats were treated with
3MC and PB for inducibility of CYP activities as
described earlier.2
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tion.37 The pellet was resuspended in 0.25 M sucrose/
1 mM phosphate bu€er, pH 7.6 so as to obtain protein
30±40 mg/ mL. The preparation was stored in small
portions at 70 ꢀC.
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