Piperine analogs as potent Staphylococcus aureus NorA efflux pump inhibitors

Article abstract of DOI:10.1016/j.bmc.2008.09.042

Based on our recent findings that piperine is a potent Staphylococcus aureus NorA efflux pump inhibitor (EPI), 38 piperine analogs were synthesized and bioevaluated for their EPI activity. Twenty-five of them were found active with potentiating activity equivalent or more than known EPIs like reserpine, carsonic acid and verapamil. The inhibitory mechanism of the compounds was confirmed by efflux inhibition assay using ethidium bromide as NorA substrate. The present communication describes the synthesis, bioevaluation and structure related activity of these efflux pump inhibitors.

Full text of DOI:10.1016/j.bmc.2008.09.042

Bioorganic & Medicinal Chemistry 16 (2008) 9847–9857
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Piperine analogs as potent Staphylococcus aureus NorA efflux pump inhibitors
a
a
Payare L. Sangwan a, Jawahir L. Koul a, Surrinder Koul a, , Mallepally V. Reddy , Niranjan Thota ,
*
Inshad A. Khan b, Ashwani Kumar b, Nitin P. Kalia b, Ghulam N. Qazi b
a Bioorganic Chemistry Division, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu Tawi 180001, India
b Biotechnology Division, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu Tawi 180001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on our recent findings that piperine is a potent Staphylococcus aureus NorA efflux pump inhibitor
(EPI), 38 piperine analogs were synthesized and bioevaluated for their EPI activity. Twenty-five of them
were found active with potentiating activity equivalent or more than known EPIs like reserpine, carsonic
acid and verapamil. The inhibitory mechanism of the compounds was confirmed by efflux inhibition
assay using ethidium bromide as NorA substrate. The present communication describes the synthesis,
bioevaluation and structure related activity of these efflux pump inhibitors.
Received 5 August 2008
Revised 15 September 2008
Accepted 16 September 2008
Available online 19 September 2008
Keywords:
Ó 2008 Elsevier Ltd. All rights reserved.
Piperine analogs
NorA efflux pumps inhibitor
Potentiator
Ciprofloxacin
1. Introduction
used in combination with anti-infective can restore the activity
of the drug molecule/s.19 Combination of anti-infective amoxicillin
Emergence of multi drug resistant (MDR) pathogenic bacteria is
proving a menace for the management of the bacterial infections
and this MDR phenomenon1,2 that occur (independently or syner-
gistically) through several mechanisms3–6 is responsible for reduc-
ing or preventing the accessibility of the drug to the target by
decreasing the transport of the antibiotic into the cell or by
enhancing the efflux of the drug from the cell to the outside med-
ium resulting in a low or ineffective concentration of the drug
found in many species of bacteria, fungi, and tumor cell.7–10 This
MDR mechanism also recognizes many structurally unrelated com-
pounds.11–15 Molecular properties of bacterial multidrug trans-
porters of unlike specific drug resistance transporters and their
presence may be there as a part of the detoxifying mechanisms
in xenobiotics.16 Among several MDR transporters encompassing
Gram-positive and Gram-negative bacteria, MDR pumps such as
NorA transporter17 (member of the major facilitator family-MF
family), considered to be one of the major contributors towards
drug effluxing, contributes to the resistance of Staphylococcus aur-
eus to wide range of structurally unrelated compounds such as
ethidium bromide, acriflavin, quaternary amine compounds, fluo-
roquinolones, rhodamine-6-G, puromycin, and chloramphenicol
by promoting their active extrusion from the cell.18 However, at-
tempts are being made to evolve an alternative approach wherein
natural and synthetic molecules are being identified which when
with anti-infective resistant inhibitor clavulanic acid is a classical
example of that sort.20 Therefore, development of clinically useful
inhibitors that decrease the effectiveness of efflux pumps would
represent a significant advance to provide successful treatment
of multi drug resistant conditions.
In continuation of our research interest21–29 towards drug
development, we have earlier demonstrated several applications
of piperine and piperine analogs such as inhibitors of cytochrome
P450 when co-administered with different drugs,28 besides their
property as potent pungent/thermogenic agents,27 and have, re-
cently, reported the application of piperine as the inhibitor of bac-
terial NorA efflux pump,21,29 capable of reducing the minimum
inhibitory concentration (MIC) of ciprofloxacin resistant when
tested against the strain of S. aureus or Methicillin resistant S. aur-
eus [MRSA].21 Based on piperine molecule, further studies towards
the development of more potent EPIs have been carried out and in
this communication, we report the preparation and identification
of piperine analogs as potent EPIs and also discuss the structure–
activity relationship of these analogs.
2. Results and discussion
One of the natural products that forms major constituent of Pi-
per nigrum and Piper longum is 5-(3,4-methylenedioxyphenyl)-
2E,4E-pentadienoic acid piperidine amide27 commonly known as
piperine and like many other amides both from natural and syn-
thetic sources which possess many biological activities,30–34 this
* Corresponding author. Tel.: +91 191 2569000; fax: +91 191 2569333.
E-mail address: surrinderkoul@gmail.com (S. Koul).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.09.042

9848
P. L. Sangwan et al. / Bioorg. Med. Chem. 16 (2008) 9847–9857
amide also exhibits several biological activities such as P-gp inhib-
itor activity,35 as melanocyte replication stimulant36 and bioavail-
ability enhancer activity (reported from our institute).37 Of late, an
important property that has been recognized with aromatic
amides is that of they being efflux pump inhibitors of bacteria to
check the emergence of multi drug resistance offered by the micro-
organisms,38,39 which have also been demonstrated for piperine
molecule in the recent studies carried out by us.21,23 The piperine
molecule reduced the MIC of antibiotic ciprofloxacin by twofold
when used in combination against S. aureus 1199. In order to de-
velop more potent efflux pump inhibitor/s than piperine, we have
been exploring different strategies cum modifications of this mol-
ecule.23,24,26,29 Piperine, is made up of a 1,3-benzodioxol group
(represented as compartment A), a pentadienoic acid side chain
(represented as compartment B), and piperidine as the amine moi-
ety (represented as compartment C) as shown in Figure 1.
Modification of compartment B of piperine molecule (1) by way
of introduction of an alkyl substituent at C4 position, or replace-
ment of 1,3-benzodioxol by other substituted phenyl moiety has
been recently carried out by us. The modification incurred gener-
ated a new series of compounds29 (Fig. 2) and on bioevaluation,
several of these molecules exhibited potent efflux pump inhibitory
activity showing fourfold reduction in MIC of ciprofloxacin against
S. aureus 1199 compared to piperine which showed only twofold
reduction.23,29 The QSAR studies of these molecules have also been
documented by us.25
In the present study, we have carried out the modifications in
the compartment C in the form of replacement of the piperidine
moiety by aliphatic and aromatic amines and have studied the
influence of these substituent’s on the overall efflux pump inhibi-
tory activities of the synthesized molecules co-administered with
ciprofloxacin and tested against S. aureus 1199 and NorA over-
expressing S. aureus 1199B.
Piperic acid (2) used as the starting material for the preparation
of piperine analogs was obtained by the saponification of naturally
occurring piperine (1) isolated from the methanolic extract of P. ni-
grum. Conversion of piperic acid (2) into its acid chloride followed
by the condensation of the latter with an appropriate amine
(Scheme 1) afforded piperine analogs (3–32) in overall yields of
60–93%.
O
NR2R3
R
R
1
Figure 2. R = substituents; R1 = C1–C10 aliphatic chain; NR2R3 = alkyl, aryl, alky aryl
or aryl alkyl amine, etc.
of the molecules failed to potentiate the antibacterial activity of
the drug.
However, the combination when tested against NorA over-
expressing S. aureus 1199B showed encouraging results with
molecules 22, 26–28, and 31 showing the reduction of MIC of
ciprofloxacin by fourfold while twofold reduction was observed
for molecules 4, 6, 7, 8, 10, 12, 13, 17–21, 23, 25, 29, 30, 32,
33, 35, and 36, respectively. In the present studies, the concen-
tration of the synthesized molecules was evaluated at dose
dependent concentration that ranged from 0.8 to 50 lg/mL and
used in combination with the anti-infective against S. aureus
1199B (Table 2). Since a large number of molecules (25 out of
38) exhibited efflux pump inhibitory activity against NorA over-
expressing S. aureus 1199B, compared to wild type strain S. aur-
eus 1199 where only a few were found active, this could be
attributed due to the over expression of the target protein
(NorA) by S. aureus 1199B strain.
In SAR studies, combination of drug with EPIs showed that
the nature of the basic moiety is an important factor for its re-
sponse towards drug potentiation, that is, exhibition of high, low
or no potentiation of the drug activity when tested against S.
aureus 1199B (NorA overexpressing). In comparison to piperine
bearing piperidinyl as the basic moiety, the replacement of it
by small alkyl chain substituents like isopropyl (4), isobutyl (6)
or diisopropyl (8) proved slightly better potentiators than
straight chain like n-propyl (3) or n-butyl substituents (5) as
shown in Table 1. Replacement of piperidinyl moiety by anilinyl
moiety proved to be a good substituent (13), but introduction of
electron releasing group in the anilinyl moiety annihilated the
effect and no potentiation of the drug could be observed for
the molecules such as toluidinyl (14), anisidinyl (15) or 3,4-
methylenedioxyanilinyl moieties (16). However, introduction of
substituent such as 2-hydroxymethyl group in the anilinyl moi-
ety (19- 21) (irrespective of the positional isomers ortho, meta,
and para) potentiated ciprofloxacin activity. Introduction of elec-
tron withdrawing substituent (nitrile group) in the anilinyl moi-
ety (22 and 23) showed potentiation of ciprofloxacin (with m-
isomer proving to be better potentiator than p-isomer). Among
o- and p-cyanomethyl anilinyl compounds 24 and 25, p-isomer
exhibited better potentiating activity than o-isomer. Ortho, meta,
and para phenylacetamide moieties as replacement for piperidi-
nyl moiety exhibited the best potentiating effect for the drug
and fourfold reduction in the MIC of ciprofloxacin was observed
for all the three isomers (26–28). Benzamidyl or 2-thiazolyl or 6-
thiozolinyl moiety did not prove to be good potentiator of cipro-
floxacin (18, 29, and 30). Three known NorA efflux pump inhib-
itors namely verapamil, carsonic acid, and reserpine were taken
for comparative studies and from the results obtained, several
molecules were identified which exhibited better or equivalent
potentiating activity to those of known inhibitors as shown in
Table 1.
The MIC of the synthesized molecules was determined so as to
use these molecules at concentration devoid of antibacterial
activity, a prerequisite of any compound to be used as safe EPIs.
The amide molecules (including the saturated molecules 33–38)
were used in combination with antibiotic ciprofloxacin and bio-
evaluated against S. aureus 1199 and NorA overexpressing S. aur-
eus 1199B.40–42 Along with these synthetic molecules, piperine
and three known efflux pump inhibitors namely carsonic acid,43
reserpine,44 and verapamil45 were also used for the comparative
studies. Ciprofloxacin alone showed MIC at 0.25
lg/mL and
8
lg/mL against S. aureus 1199 and NorA overexpressing S. aureus
1199B, respectively. Among the library of 38 molecules used in
combination with ciprofloxacin and tested against S. aureus
1199, only compounds 8, 13, 22, 26–28, 31, and 36 could reduce
the MIC of the drug by twofold (as shown in Table 1) and the rest
O
O
Compounds 1, 6, 7, 22, 26, and 31 were subjected to hydrogena-
tion (Scheme 2) and saturated molecules 33–38 thus obtained
were bioevaluated. The saturation of the double bonds resulted
in lowering of potentiation of ciprofloxacin, for example, lowering
or loss of EPI activity was observed for compound 33, and 36–38.
These results are in agreement with our earlier results obtained
for other different set of compounds.22–24 The above studies
N
Compartment B
O
Compartment A
Compartment C
Piperine (1)
Figure 1.

P. L. Sangwan et al. / Bioorg. Med. Chem. 16 (2008) 9847–9857
9849
O
O
O
O
O
Ethylene glycol
KOH/Reflux, H3O+
O
O
O
O
a
N
NR1R2
OH
1
2
3-32
Scheme 1. Reagents: (a) SOCl2/C6H6; NHR1R2. NR1R2 = n-propyl amine (3), isopropyl amine (4), n-butyl amine (5), isobutyl amine (6), N,N-diethyl amine (7), diisopropyl
amine (8), -2-amino butanol (9), -2-amino butanol (10), morpholine (11), N-methyl piperazine (12), aniline (13), o-toluidine (14), p-anisidine (15), 3,4-methylenedi-
D
L
oxyphenyl amine (16), 2-amino-1-phenyl ethanol (17), 4-benzamide amine (18), o-hydroxymethylphenyl amine (19), m-hydroxymethylphenyl amine (20), p-
hydroxymethylphenyl amine (21), m-cyanophenyl amine (22), p-cyanophenyl amine (23), o-cyanomethylphenyl amine (24), p-cyanomethylphenyl amine (25),
o-acetylaminophenyl amine (26), m-acetylaminophenyl amine (27), p-acetylaminophenyl amine (28), benzothiazol-2-yl amine (29), benzothiazol-6-yl amine (30), 4-
benzanilide amine (31), and 40-benzanilide (32).
Table 1
Piperine analogs as potent inhibitor of multi drug efflux pump in S. aureus 1199B and S. aureus 1199 and potentiation of ciprofloxacin MIC
Entry
MECa of EPI
S. aureus 1199B MICb of cipro. (
l
g/mL)
MICb of cipro. (
With EPI
lg/mL)
Without EPI
With EPI
Fold reduction
Without EPI
Fold reduction
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
>100
25
100
25
25
25
>100
25
>100
25
12.5
>100
>100
>100
12.5
25
25
25
6.25
6.25
1.5
25
>100
25
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
4
8
4
4
4
8
4
8
4
4
8
8
8
4
4
4
4
4
2
4
4
8
4
2
4
2
4
2
4
4
4
2
4
4
4
8
4
8
4
8
4
2
4
4
0
2
0
2
2
2
0
2
0
2
2
0
0
0
2
2
2
2
2
4
2
2
0
2
4
2
4
2
4
2
2
2
4
2
2
2
0
2
0
2
0
2
4
2
2
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.12
0.25
0.25
0.25
0.25
0.12
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.12
0.25
0.25
0.25
0.25
0.12
0.25
0.12
0.25
0.12
0.25
0.25
0.25
0.12
0.25
0.25
0.25
0.25
0.25
0.25
0.12
0.25
0.12
0.12
0.12
0.12
0
0
0
0
0
2
0
0
0
0
2
0
0
0
0
0
0
0
0
0
0
0
0
0
2
0
2
0
2
0
2
0
0
0
0
0
0
0
0
2
0
2
2
2
2
23
24
25
26
25
6.25
12.5
6.25
25
6.25
6.25
3.12
25
3.12
3.12
50
>100
50
>100
50
>100
50
25
25
27
28
29
30
31
32
33
34
35
36
37
38
Piperine
Reserpine
Carsonic acid
Verapamil
50
No antibacterial activity of EPIs was observed at 100
lg/mL that was the highest concentration tested.
For determining potentiation of ciprofloxacin, EPI were tested at concentration range of 50–0.8
lg/mL.
a
MEC, minimum effective concentration.
b
MIC, minimum inhibitory concentration.
showed that unsaturation apparently does seem to be an impor-
The inhibitory mechanism of the compounds was confirmed by
tant factor responsible for drug potentiation.
efflux inhibition assay using ethidium bromide as substrate of
NorA and compounds 22, and 26 which emerged as the most po-
tent inhibitors among the piperine analogs bioevaluated along
with the inactive EPI 5 and reserpine (known EPI). The ethidium
bromide fluorescences only when it is bound to nucleic acids inside
the cells. The NorA mediated ethidium bromide efflux from the
The MIC of ciprofloxacin was determined against S. aureus 1199
and NorA overexpressing S. aureus 1199B in Muller–Hinton Broth
in the presence of increasing amounts of efflux pump inhibitors
by broth checkerboard synergy method in a microtitre plates using
twofold serial dilutions.46

9850
P. L. Sangwan et al. / Bioorg. Med. Chem. 16 (2008) 9847–9857
Table 2
Dose dependent MIC of ciprofloxacin in presence of EPI’s against S. aureus 1199B
Entry
Cipro. alone
MIC (lg/mL) of ciprofloxacin
In presence of EPI’s (lg/mL)
+50
+25
+12.5
+6.25
+3.12
+1.56
+0.8
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
4
8
4
4
4
8
4
8
4
2
8
8
8
2
4
4
4
4
1
4
8
4
2
2
2
4
4
2
4
4
8
4
8
4
8
4
2
4
4
8
4
8
4
4
4
8
4
8
4
4
8
8
8
4
4
4
4
4
2
4
8
4
2
2
2
4
4
2
4
8
8
8
8
8
8
4
2
4
8
8
8
8
8
8
8
8
8
8
8
4
8
8
8
4
8
8
8
4
2
8
8
8
4
2
4
4
4
4
4
8
8
8
8
8
8
8
4
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
4
2
8
8
8
4
4
4
4
4
4
4
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
4
8
8
8
8
8
8
8
4
4
4
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
4
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
35
36
37
38
Piperine
Reserpine
Carsonic acid
Verapamil
O
O
O
O
O
NR1R2
NR1R2
H2 , Pd / C
4 hrs
O
1, 6, 7, 22, 26, 31
33-38
Scheme 2. Reagents: NR1R2 = piperidine (33), isobutyl amine (34), N,N-diethyl amine (35), m-cyanophenyl amine (36), o-acetylaminophenyl amine (37), and 4-benzanilide
amine (38).
cells resulted in a rapid decrease in fluorescence. Results presented
in Figure 3 are the averages from triplicate measurements. As
shown inFigure 3, only the control cells without EPIs or in presence
of inactive EPI 5 extruded ethidium bromide, resulting in a signif-
icant decrease in fluorescence over the time of the assay. Potent
inhibitors such as compound 22 and 26 effectively blocked the
extrusion of ethidium bromide, which resulted in the decreased
fluorescence.
of the two set of molecules. In case of other known inhibitors
namely carsonic acid, and verapamil, twofold reduction of the
MIC is observed. The mechanism of action of these compounds
has been established by ethidium bromide fluorescence experi-
ment. In SAR studies, the nature of basic moiety and its effect on
the inhibitory property of efflux pump NorA besides the role of
unsaturation has also been described. The potential clinical utility
of these potent EPIs molecules warrants further investigations.
3. Conclusion
4. Experimental
Preparation of piperine analogs and their bioevaluation as po-
tent EPIs is described. Five out of 38 synthesized molecules are
shown responsible for fourfold lowering of MIC of ciprofloxacin
against overexpressing NorA S. aureus 1199B which is also ob-
served for known EPI reserpine showing thereby the equipotency
4.1. General methods
All reagents for chemical synthesis were purchased from Sig-
ma–Aldrich and used as received. Piperine and carsonic acid were
isolated from the methanol extract of P. nigrum and Rosmarinus

P. L. Sangwan et al. / Bioorg. Med. Chem. 16 (2008) 9847–9857
9851
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
5
10
15
20
25
30
Time (min)
Control
Piperine (50 ug/ml)
26 (12.5 ug/ml)
22 (6.25 ug/ml)
5 (50 ug/ml)
Reserpine (25 ug/ml)
Figure 3. Ethidium bromide efflux inhibition assay from S. aureus 1199B cells. The cells were loaded with ethidium bromide and the efflux was allowed to occur in the
absence of EPI (control) or in the presence of EPIs. Each time point represents the mean log10 SD of three readings.
officinalis plants, respectively, in our lab and characterized by spec-
troscopic techniques. All the solvents used in reactions were dis-
tilled and dried before use. All reactions were monitored by TLC
on 0.25 mm silica gel 60 F254 plates coated on aluminum sheet
(E. Merck). 1H NMR and 13C NMR spectra were recorded on Brucker
Avance DPX-200 instrument at 200 MHz and 50 MHz, respectively,
using CDCl3 as solvent with TMS as internal standard. Chemical
shift is expressed in d (ppm) and coupling constant in Hertz. Mass
spectra were recorded on Jeol MSD-300 instrument and IR spectra
on Perkin-Elmer FT-IR spectrometer as KBr Pellet or neat sample.
removed on rotavapour under reduced pressure and thereafter
the reconstituted acid chloride in dichloromethane (20 mL) was al-
lowed to condense with n-propyl amine (0.59 g, 10 mmol) dis-
solved in dichloromethane. The contents stirred for 1hr, diluted
with water and the organic layer was separated out and washed
with water (2ꢀ 25 mL), dried over anhydrous sodium sulfate and
concentrated to give crude product which on crystallization from
ethyl acetate/petroleum ether (1:4) gave a colorless crystalline
compound 3 (2.38 g, yield 91%), mp 151–153 °C. Anal. Calcd for
C15H17NO3: C, 69.48; H, 6.607; N, 5.40%. Found: C, 69.81; H,
6.603; N, 5.46%. MS (%) M+ at m/z 259 (20), 201 (100), 172 (32),
143 (53), 115 (45). IR: 3268, 2917, 2849, 1642, 1605, 1542, 1255,
4.2. Preparation of substituted aryl pentadienoic acid amides
1041, 991, 755 cmꢁ1 1H NMR: d 0.98 (3H, t, J = 6.8, –CH2CH3),
.
The amides were prepared from piperic acid (2) through acid
chloride formation and subsequent condensation with appropriate
amines. The structure analyses were carried out mainly through IR,
NMR, and MS studies.
1.58 (2H, m, –CH2CH3), 3.38 (2H, m, –NHCH2), 5.96 (2H, s,
–OCH2O–), 6.01 (1H, d, J = 15.02 Hz, –CH@CHCO), 6.67–7.01 (5H,
m, olefinic and Ar-H), 7.38 (2H, dd, J = 15.02, 9.2 Hz, –CH@CHCO).
13C NMR: d 12.1, 25.2, 46.6, 99.5, 107.2, 108.2, 120.2, 124.3,
126.5, 129.5, 129.7, 143.5, 147.1, 147.3, and 167.9.
4.2.1. Preparation of 5-(3,4-methylenedioxyphenyl)-2E,4E-
pentadienoic acid (piperic acid) (2)
4.2.3. Preparation of 5-(3,4-methylenedioxyphenyl)-2E,4E-
pentadienoic acid isopropyl amide (4)
A mixture of piperine 1 (28.5 g, 100 mmol, mp 132 °C) dis-
solved in ethylene glycol (200 mL) and potassium hydroxide
(25 g) was refluxed at 180 °C. Completion of reaction was mon-
itored on TLC and the reaction worked up by diluting with ex-
cess of ice-water (500 mL) followed by acidification with 2 N
HCl. The resulting precipitate filtered, washed with water, and
dried to give the crude product, which on crystallization from
ethanol gave 2 as pale yellow crystals (17.4 g, 80%) mp 217 °C
(lit. mp 217 °C).28
The isopropyl amide 4 was prepared from piperic acid 2 (2.2 g,
10 mmol) and isopropyl amine (0.59 g, 10 mmol) by the method as
described for 3 to give crude product which on crystallization from
ethyl acetate furnished a crystalline colorless solid 4 (2.38 g, yield
92%) mp 172–173 °C [lit.47,48 mp 171–173 °C]. Anal. Calcd for
C15H17NO3: C, 69.48; H, 6.607; N, 5.40%. Found: C, 69.58; H,
6.613; N, 5.71%. MS (%) M+ at m/z 259 (14), 244 (12), 216 (100),
201 (75), 172 (15), 143 (30), 116 (25). IR: 3278, 2972, 2917,
2849, 1644, 1604, 1503, 1252, 1039, 993 cmꢁ1 1H NMR: d 1.19
.
4.2.2. Preparation of 5-(3,4-methylenedioxyphenyl)-2E,4E-
pentadienoic acid n-propyl amide (3)
To piperic acid 2 (2.18 g, 10 mmol) in dry dichloromethane
(50 mL) was added freshly distilled thionyl chloride (1.0 mL) and
the contents were refluxed for 1 h, excess of thionyl chloride was
(6H, d, J = 6.54 Hz, –CH(CH3)2), 4.17–4.21 (1H, m, –NHCH–), 5.89
(1H, d, J = 14.85 Hz, –CH@CHCO), 5.98 (2H, s, –OCH2O–), 6.67–
6.98 (5H, m, olefinic and Ar-H), 7.34 (1H, dd, J = 14.85, 9.80 Hz,
–CH@CHCO). 13C NMR: d 19.5, 21.1, 37.4, 101.7, 107.7, 108.5,
118.6, 119.3, 120.5, 126.9, 134.8, 136.8, 146.9, 147.1, and 163.1.

9852
P. L. Sangwan et al. / Bioorg. Med. Chem. 16 (2008) 9847–9857
4.2.4. Preparation of 5-(3,4-methylenedioxyphenyl)-2E,4E-
pentadienoic acid n-butyl amide (5)
4.2.8. Preparation of 5-(3,4-methylenedioxyphenyl)-2E,4E-
pentadienoic acid -2-amino-butanol amide (9)
Compound 9 was prepared from piperic acid 2 (1.1 g, 10 mmol)
and -2-amino butanol (0.45 g, 5 mmol) by the method as de-
D
Compound 5 was prepared from piperic acid 2 (1.1 g, 5 mmol)
and n-butyl amine (0.37 g, 5 mmol) by the method as described
for 3 to give crude product as gummy mass which on crystalliza-
tion in ethyl acetate compound 5 (1.26 g, yield 92%) obtained as
colorless solid mp 142–143 °C [lit.28,48 mp 144–145 °C]. Anal. Calcd
for C16H19NO3: C, 70.30; H, 7.006; N, 5.12%. Found: C, 70.78; H,
7.014; N, 5.32%. MS (%) M+ at m/z 273 (24), 258 (22), 201 (100),
172 (23), 143 (52), 116 (35). IR: 3284, 2958, 2916, 1640, 1615,
D
scribed for 3 to give crude product which on crystallization from
ethyl acetate furnished a colorless crystalline solid 9 (1.10 g, yield
76%), mp 206–207 °C. Anal. Calcd for C16H19NO4: C, 66.42; H,
6.618; N, 4.84%. Found: C, 66.92; H, 6.621; N, 5.02%. MS (%) M+
at m/z 289 (45), 271 (24), 242 (100), 201 (53), 172 (12), 143 (30),
116 (25). IR: 3410, 2925, 2854, 1725, 1665, 1458, 1378, 1050,
1445, 1255, 990 cmꢁ1
.
1H NMR: d 0.93 (3H, t, J = 6.5 Hz, –CH2CH3),
678 cmꢁ1 1H NMR: d 0.90 (3H, t, J = 8.5 Hz, –CH2CH3), 1.17–1.58
.
1.34 (4H, m, (CH2)2CH3), 3.75 (2H, m, –NHCH2), 5.90 (1H, d,
J = 14.90 Hz, –CH@CHCO), 5.98 (2H, s, –OCH2O–), 6.65–6.98 (5H,
m, olefinic and Ar-H), 7.37 (1H, dd, J = 14.90, 9.80 Hz, –CH@CHCO).
13C NMR: d 20.2, 27.3, 28.4, 46.8, 101.6, 107.8, 108.9, 118.7, 120.5,
121.6, 126.8, 136.9, 137.8, 147.7, 148.0, and 167.4.
(2H, m, –CH2CH3), 3.68 (2H, br s, CH2OH), 3.95 (1H, br s, –NHCH),
5.91 (1H, d, J = 14.78 Hz, –CH@CHCO), 5.96 (2H, s, –OCH2O–),
6.72–6.97 (5H, m, olefinic and Ar-H), 7.35 (1H, dd, J = 14.78,
9.81 Hz, –CH@CHCO). 13C NMR: d 10.5, 27.3, 52.5, 67.7, 100.5,
107.9, 108.7, 120.8, 124.7, 126.3, 129.3, 130.5, 143.4, 147.8,
147.9, and 165.3.
4.2.5. Preparation of 5-(3,4-methylenedioxyphenyl)-2E,4E-
pentadienoic acid isobutyl amide (6)
4.2.9. Preparation of 5-(3,4-methylenedioxyphenyl)-2E,4E-
Compound
6
was prepared from piperic acid
2
(2.2 g,
pentadienoic acid
Compound 10 was prepared from piperic acid 2 (1.1 g, 5 mmol)
and -2-amino butanol (0.45 g, 5 mmol) by the method as described
L-2-amino-butanol amide (10)
10 mmol) and isobutyl amine (0.73 g, 10 mmol) by the method
as described for 3 to give crude product which on crystallization
L
from ethyl acetate furnished
a
colorless crystalline solid
6
for 3 to give crude product which on crystallization from ethyl ace-
tate furnished a white crystalline solid 10 (1.13 g, yield 78%), mp
210–211 °C. Anal. Calcd for C16H19NO4: C, 66.42; H, 6.618; N,
4.84%. Found: C, 66.73; H, 6.625; N, 4.99%. MS (%) M+ at m/z 289
(68), 271 (56), 242 (75), 201 (53), 172 (12), 143 (30), 116 (25). IR:
(2.53 g, yield 93%), mp 165–167 °C [lit.48 mp 167–169 °C]. Anal.
Calcd for C16H19NO3: C, 70.30; H, 7.006; N, 5.12%. Found: C,
70.81; H, 7.011; N, 5.32%. MS (%) M+ at m/z 273 (28), 258
(12), 243 (15), 201 (100), 172 (18), 143 (38), 116 (25). IR:
3283, 2959, 2917, 1643, 1613, 1446, 1256, 989 cmꢁ1
.
1H NMR:
3410, 2925, 2854, 1725, 1665, 1458, 1378, 1050, 678 cmꢁ1 1H
.
d 0.98 (6H, d, J = 6.5 Hz, –CH(CH3)2), 2.35 (1H, m, CH(CH3)2),
3.74 (2H, m, –NHCH2), 5.91 (1H, d, J = 14.95 Hz, –CH@CHCO),
5.99 (2H, s, –OCH2O–), 6.64–6.97 (5H, m, olefinic and Ar-H),
NMR: d 0.93 (3H, t, J = 8.5 Hz, CH2CH3), 1.18–1.52 (2H, m, –CH2CH3),
3.71 (2H, br s, CH2OH), 3.98 (1H, br s, –NHCH), 5.89 (1H, d,
J = 14.81 Hz, –CH@CHCO), 5.94 (2H, s, –OCH2O–), 6.76–6.96 (5H,
m, olefinic and Ar-H), 7.39 (1H, dd, J = 14.81, 9.85 Hz, –CH@CHCO).
13C NMR: d 11.5, 26.4, 54.4, 66.8, 100.1, 107.8, 108.9, 121.3, 123.5,
126.8, 128.9, 131.1, 142.5, 146.9, 147.1, and 164. 7.
7.36 (1H, dd, J = 14.95, 9.82 Hz, –CH@CHCO). 13C NMR:
d
20.5 ꢀ 2, 28.3, 47.4, 101.9, 107.6, 108.7, 118.9, 120.3, 121.7,
126.7, 136.6, 137.3, 147.9, 148.2, and 167.2.
4.2.6. Preparation of 5-(3,4-methylenedioxyphenyl)-2E,4E-
pentadienoic acid N,N-diethyl amide (7)
4.2.10. Preparation of 5-(3,4-methylenedioxyphenyl)-2E,4E-
pentadienoic acid morpholide (11)
Compound 7 was prepared from piperic acid 2 (1.1 g, 5 mmol)
and N,N-diethyl amine (0.37 g, 5 mmol) by the method as de-
scribed for 3 to give a solid 7 (1.25 g, yield 92%) mp 86 °C [lit.47
mp 85–87 °C]. Anal. Calcd for C16H19NO3: C, 70.30; H, 7.006; N,
5.12%. Found: C, 70.81; H, 7.016; N, 5.41%. MS (%) M+1 at m/z 274
(72), 259 (8), 244 (12), 201 (100), 173 (23), 135 (35), 105 (5). IR:
Compound 11 was prepared from piperic acid 2 (1.1 g, 5 mmol)
and morpholine (0.4 g, 5 mmol) by the method as described for 3
to give white crystalline compound 11 (1.29 g, yield 93%) mp
162–163 °C [lit.48,49 mp 167–168 °C]. Anal. Calcd for C16H17NO4:
C, 66.88; H, 5.963; N, 4.87%. Found: C, 67.04; H, 5.969; N, 5.13%.
MS (%) M+1 at m/z 287 (25), 269 (14), 201 (100), 156 (11), 135
(28), 105 (10). IR: 3362, 2942, 2855, 1636, 1489, 1445, 1403,
3164, 2977, 2929, 1737, 1641, 1444, 1404, 1250, 1038, 805 cmꢁ1
.
1H NMR: d 1.08 and 1.15 (3H each, t, J = 6.8 Hz, 2ꢀ CH2CH3), 3.36
(4H, m, 2ꢀ –CH2CH3), 5.89 (2H, s, –OCH2O–), 6.28 (1H, d,
J = 14.50 Hz, –CH@CHCO), 6.67–6.91 (5H, m, olefinic and Ar-H),
7.37 (1H, dd, J = 14.50, 9.82 Hz, –CH@CHCO). 13C NMR: d 12.3,
14.0, 40.3, 41.2, 100.3, 104.7, 107.5, 119.3, 121.5, 124.4, 130.0,
137.4, 141.5, 147.2, 147.3, and 164.9.
1251, 1114, 1038, 759 cmꢁ1
.
1H NMR: d 3.71 (8H, br s, 2ꢀ –
CH2CH2O–), 5.98 (2H, s, –OCH2O–), 6.38 (1H, d, J = 14.60 Hz,
–CH@CHCO), 6.68–6.99 (5H, m, olefinic and Ar-H), 7.41 (1H, dd,
J = 14.60, 9.40 Hz, –CH@CHCO). 13C NMR: d 67.0, 101.6, 105.9,
108.7, 118.6, 123.1, 125.1, 131.0, 139.9, 144.2, 148.5, 148.7, and
166.6.
4.2.7. Preparation of 5-(3,4-methylenedioxyphenyl)-2E,4E-
pentadienoic acid N,N-diisopropyl amide (8)
4.2.11. Preparation of 5-(3,4-methylenedioxyphenyl)-2E,4E-
pentadienoic acid N-methyl piperazine amide (12)
Compound 8 was prepared from piperic acid 2 (1.1 g, 5 mmol)
and diisopropyl amine (0.51 g, 5 mmol) by the method as de-
scribed for 3 to give a colorless solid 8 (1.32 g, yield 88%) mp
83 °C [lit.47 mp 81–83 °C]. Anal. Calcd for C18H23NO3: C, 71.73; H,
7.691; N, 4.64%. Found: C, 72.04; H, 6.699; N, 4.92%. MS (%) M+1
at m/z 302 (86), 286 (15), 271 (12), 201 (100), 173 (13), 135 (10),
Compound 12 was prepared from piperic acid 2 (1.1 g, 5 mmol)
and N-methyl piperazine (0.5 g, 5 mmol) by the same method as
described for 3 to give a white crystalline compound 12 (1.36 g,
yield 88%) mp 185–186 °C [lit.49 mp 167–168 °C]. Anal. Calcd for
C17H20N2O3: C, 67.98; H, 6.711; N, 9.32%. Found: C, 68.37; H,
6.717; N, 9.63%. MS (%) M+1 at m/z 301 (58), 285 (12), 201 (100),
173 (41), 135 (18), 105 (13). IR: 3357, 2956, 2927, 1732, 1641,
105 (15). IR: 3194, 2960, 2927, 1738, 1651, 1404, 1041, 761 cm
ꢁ1
.
1H NMR: d 1.33 (12H, br s, 4ꢀ CH3), 4.02 (2H, m, 2ꢀ –NCH),
1455, 1405, 1040, 748 cmꢁ1 1H NMR: d 2.94 (3H, s, N–CH3), 3.19
.
5.98 (2H, s, –OCH2O–), 6.35 (1H, d, J = 14.61 Hz, –CH@CHCO),
6.72–6.99 (5H, m, olefinic and Ar-H), 7.33 (1H, dd, J = 14.61,
9.52 Hz, –CH@CHCO). 13C NMR: d 19.8, 19.9, 20.6, 20.7, 44.8,
46.4, 100.3, 104.7, 107.5, 121.4, 122.1, 124.6, 130.2, 136.8, 140.3,
147.1, 147.2, and 165.7.
(4H, m, –N(CH2)2), 3.51 (4H, m, –N(CH2)2), 5.97 (2H, s, –OCH2O–),
6.38 (1H, d, J = 14.62 Hz, –CH@CHCO), 6.74–7.02 (5H, m, olefinic
and Ar-H), 7.35 (1H, dd, J = 14.62, 9.45 Hz, –CH@CHCO). 13C NMR:
d 39.2, 50.2, 55.4, 101.3, 104.9, 107.8, 120.6, 122.8, 125.8, 131.7,
136.8, 141.3, 147.3, 147.4, and 165.8.

P. L. Sangwan et al. / Bioorg. Med. Chem. 16 (2008) 9847–9857
9853
4.2.12. Preparation of 5-(3,4-methylenedioxyphenyl)-2E,4E-
pentadienoic acid anilide (13)
4.2.16. Preparation of 5-(3,4-methylenedioxyphenyl)-2E,4E-
pentadienoic acid 2-amino-1-phenyl ethanol amide (17)
Compound 17 was prepared from piperic acid 2 (2.2 g,
The amide 13 was prepared from piperic acid 2 (1.1 g, 5 mmol)
and aniline (0.47 g, 5 mmol) by the method as described for 3 to
give crude product which on crystallization from ethyl acetate fur-
nished a colorless crystalline solid 13 (1.2 g, yield 82%), mp 174–
175 °C. Anal. Calcd for C18H15NO3: C, 73.70; H, 5.154; N, 4.77%.
Found: C, 73.91; H, 5.152; N, 4.98%. MS (%) M+ at m/z 293 (74),
215 (28), 201 (14), 172 (100), 143 (15), 116 (12). IR: 3259, 2917,
10 mmol) and 2-amino-1-phenyl ethanol (1.37 g, 10 mmol) by
the method as described for 3 to give crude product which on col-
umn chromatography on silica eluted in pet ether/ethyl acetate
(3:2) furnished colorless crystalline solid 17 (2.76 g, yield 82%),
mp 202–203 °C. Anal. Calcd for C20H19NO4; C, 71.20; H, 5.676; N,
4.15%. Found: C, 71.66; H, 5.672; N, 4.27%. MS (%) M+ at m/z 337
(14), 319 (25), 241 (70), 201 (100), 172 (35), 144 (35), 115 (25).
IR: 3313, 2923, 2852, 2360, 1649, 1502, 1447, 1252, 1038, 758,
700 cmꢁ1. 1H NMR: d 4.02 (2H, m, –NHCH2–), 4.80 (1H, m, –CHOH),
5.96 (2H, s, –OCH2O–), 6.05 (1H, d, J = 14.95 Hz, –CH@CHCO), 6.68–
6.96 (5H, m, olefinic and Ar-H), 7.23–7.32 (5H, m, Ar-H0), 7.35 (1H,
dd, J = 14.95, 9.25 Hz, –CH@CHCO). 13C NMR: d 53.5, 77.2, 101.6,
107.8, 108.7, 119.9, 124.1, 125.6, 125.7, 125.71, 125.8, 128.4,
128.45, 128.49, 139.4, 141.2, 142.3, 147.2, 147.3, and 167.8.
2849, 1599, 1489, 1442, 1252, 1038, 753 cmꢁ1 1H NMR: d 5.99
.
(2H, s, –OCH2O–), 6.01 (1H, d, J = 14.15 Hz, –CH@CHCO), 6.66–
6.98 (5H, m, olefinic and Ar-H), 7.01–7.26 (5H, m, Ar-H0), 7.32
(1H, dd, J = 14.15, 9.31 Hz, –CH@CHCO). 13C NMR: d 101.4, 107.5,
108.4, 120.5, 121.23, 121.24, 124.1, 124.6, 126.4, 128.7, 129.4,
130.1 ꢀ 2, 139.4, 142.8, 147.1, 147.2, and 165.2.
4.2.13. Preparation of 5-(3,4-methylenedioxyphenyl)-2E,4E-
pentadienoic acid o-toluidide (14)
Compound 14 was prepared from piperic acid 2 (1.1 g, 5 mmol)
and o-toluidine (0.54 g, 5 mmol) by the method as described for 3 to
give crude product which on column chromatography over silica gel
and eluted with petroleum ether and ethyl acetate (4:1) furnished a
colorless crystalline compound 14 (1.41 g, yield 91%), mp 194–
195 °C. Anal. Calcd for C19H17NO3: C, 74.25; H, 5.574; N, 4.55%. Found:
C, 74.70; H, 5.579; N, 4.76%. MS (%) M+ at m/z 307 (18), 292 (100), 201
(35), 172 (41), 143 (63) 16 (25). IR: 3359, 2933, 2853, 2360, 1742,
4.2.17. Preparation of 5-(3,4-methylenedioxyphenyl)-2E,4E-
pentadienoic acid 4-benzamide (18)
This compound was prepared by condensing piperic acid 2
(2.2 g, 10 mmol) with 4-amino benzamide (1.36 g, 10 mmol) as
per the method described for 3 to furnish a crude product which
on CC over SiO2 and elution with ethyl acetate/pet ether gave a
brown crystalline compound 18 (2.06 g, yield 60%), mp 190–
192 °C. Anal. Calcd for C19H16N2O4: C, 67.84; H, 4.794; N, 8.32%.
Found: C, 68.02; H, 4.793; N, 8.49%. MS (%) M+ at m/z 336 (25),
292 (100), 201 (10), 173 (14), 143 (5), 115 (24). IR: 3395, 2916,
1654, 1403, 1250, 1094, 1042, 760 cmꢁ1 1H NMR: d 2.24 (3H, s,
.
–CH3), 5.98 (1H, d, J = 14.78 Hz, –CH@CHCO), 6.02 (2H, s, –OCH2O),
6.60–6.67 (2H, m, olefinic-H), 6.98–7.18 (7H, m, Ar-H, and Ar-H0),
7.36 (1H, d, J = 14.78, 9.23 Hz, –CH@CHCO). 13C NMR: d 21.4, 101.8,
107.8, 108.8, 120.4, 121.4, 124.6, 124.9, 125.9, 126.7, 128.9, 129.7,
129.9, 130.5, 139.7, 143.2, 147.4, 147.5, and 165.0.
2848, 1673, 1603, 1446, 1251, 1037 cmꢁ1 1H NMR: d 5.94 (1H, d,
.
J = 15.0 Hz, –CH@CHCO), 5.99 (2H, s, –OCH2O–), 6.72–7.10 (5H,
m, olefinic, Ar-H), 7.36 (1H, dd, J = 15.0 and 9.5 Hz, –CH@CHCO),
7.66–7.97 (4H, m, Ar-H0). 13C NMR: d 101.1, 107.8, 108.9, 119.5,
121.9, 121.91, 123.9, 125.9, 127.9, 128.0, 128.01, 128.6, 130.1,
141.1, 142.6, 147.1, 147.5, 164.1, and 168.5.
4.2.14. Preparation of 5-(3,4-methylenedioxyphenyl)-2E,4E-
pentadienoic acid p-anisidide (15)
The compound 15 was prepared from piperic acid 2 (1.1 g,
5 mmol) and p-anisidine (0.62 g, 5 mmol) by the method as de-
scribed for 3 to give crude product which on column chromatogra-
phy over silica gel and eluted with 15% EtOAc in pet ether afforded
a colorless crystalline solid 15 (1.17 g, yield 75%), mp 167–168 °C.
Anal. Calcd for C19H17NO4: C, 70.57; H, 5.298; N, 4.33%. Found: C,
70.97; H, 5.302; N, 4.52%. MS (%) M+ at m/z 323 (5), 293 (12),
201 (35), 172 (100), 144 (43), 116 (52). IR: 3239, 2917, 2849,
4.2.18. Preparation of 5-(3,4-methylenedioxyphenyl)-2E,4E-
pentadienoic acid 2-hydroxymethylphenyl amide (19)
Compound 19 was prepared from piperic acid
2 (2.18 g,
10 mmol) using thionyl chloride (1.0 mL) and 2-amino benzyl alco-
hol (1.23 g, 10 mmol) by the procedure as described for 3 to give
crude product which on crystallization from methanol furnished
white crystalline solid 19 (2.32 g, yield 72%), mp 162–163 °C. Anal.
Calcd for C19H17NO4: C, 70.57; H, 5.299; N 4.33%. Found: C, 70.85;
H, 5.294; N, 4.41%. MS (%) M+ at m/z 323 (49), 291 (100), 202 (51),
173 (36) 142 (9). IR: 3347, 2917, 2849, 2360, 1605, 1449, 1253,
1604, 1509, 1248, 1034, 757 cmꢁ1 1H NMR: d 3.89 (3H, s,
.
–OCH3), 5.99 (2H, s, –OCH2O–), 6.09 (1H, d, J = 15.0 Hz,
–CH@CHCO), 6.72 (2H, d, J = 8.2 Hz, Ar-H-30 and 50), 6.82–7.35
(6H, m, olefinic and Ar-H), 7.53 (2H, d, J = 8.2 Hz, Ar-H-20 and 60).
13C NMR: d 58.3, 99.8, 107.8, 108.9, 115.1, 120.1, 123.3, 124.2,
126.6, 128.8, 129.1, 131.2, 143.5, 148.1, 148.2, 158.2, and 165.2.
1038, 755 cmꢁ1 1H NMR: d 4.74 (2H, s, –CH2OH), 5.99 (2H, s,
.
–OCH2O–), 6.11 (1H, d, J = 15.0 Hz, –CH@CHCO), 6.72–6.98 (5H,
m, olefinic and Ar-H), 7.01–7.19 (2H, m, Ar- H-40 and 50), 7.26–
7.56 (2H, m, –CH@CHCO and Ar-H-30), 7.67 (1H, d, J = 8.4 Hz, Ar-
H-60). 13C NMR: d 57.6, 100.1, 107.4, 108.6, 119.4, 121.6, 122.4,
125.1, 126.5, 127.9, 128.1, 128.6, 128.7, 134.0, 141.4, 142.6,
147.1, 147.6, and 164.1.
4.2.15. Preparation of 5-(3,4-methylenedioxyphenyl)-2E,4E-
pentadienoic acid 3,4-methylenedioxyphenyl amide (16)
The compound 16 was prepared from piperic acid 2 (2.2 g,
10 mmol) and 3,4-methylenedioxy phenyl amine (1.37 g,
10 mmol) by the method as described for 3 to give crude product
which on crystallization from ethyl acetate and hexane yielded a
colorless crystalline solid 16 (2.90 g, yield 86%), mp 202–203 °C.
Anal. Calcd for C19H15NO5: C, 67.65; H, 4.481; N, 4.15%. Found: C,
68.11; H, 4.484; N, 4.33%. MS (%) M+ at m/z 337 (12), 201 (100),
172 (22), 141 (38), 115 (45). IR: 3346, 2924, 2853, 1641, 1403,
4.2.19. Preparation of 5-(3,4-methylenedioxyphenyl)-2E,4E-
pentadienoic acid 3-hydroxymethylphenyl amide (20)
This compound was prepared by condensing piperic acid 2
(2.20 g, 10 mmol) with 3-amino benzyl alcohol (1.23 g, 10 mmol)
as per the method described for 3 to furnish a crude product which
on CC over SiO2 and elution with ethyl acetate/pet ether (1:4) gave
a pale yellow crystalline compound 20 (1.93 g, yield 60%), mp 189–
190 °C. Anal. Calcd for C19H17NO4: C, 70.57; H, 5.298; N, 4.33%.
Found: C, 70.01; H, 5.293; N, 4.50%. MS (%) M+ at m/z 323 (15),
305 (70), 290 (100), 270 (80), 201 (15), 173 (8), 121 (7). IR: 3406,
1259, 1125, 1039, 761 cmꢁ1 1H NMR: d 5.94 and 5.97 (2H each,
.
s, 2ꢀ –OCH2O–), 6.13 (1H, d, J = 14.84 Hz, –CH@CHCO), 6.73–7.01
(8H, m, olefinic, Ar-H, and Ar-H0), 7.43 (1H, dd, J = 14.84, 9.23 Hz,
CH@CHCO). 13C NMR: d 101.4, 101.5, 106.7, 107.9, 108.7, 108.8,
109.5, 120.5, 124.3, 126.4, 128.9, 129.3, 132.3, 143.1, 144.2,
146.6, 147.2, 147.4, and 165.5.
2916, 2848, 1598, 1442, 1250, 1036 cmꢁ1 1H NMR: d 4.66 (2H,
.
br s, –CH2OH), 5.98 (2H, s, –OCH2O–), 6.14 (1H, d, J = 15.0 Hz,
–CH@CHCO), 6.76–7.15 (7H, m, olefinic, Ar-H and Ar-H-40 and 50),

9854
P. L. Sangwan et al. / Bioorg. Med. Chem. 16 (2008) 9847–9857
7.35 (1H, m, –CH@CHCO), 7.65 (2H, m, Ar-H-20 and 60). 13C NMR: d
61.0, 99.4, 107.6, 108.8, 119.6, 122.0, 122.1, 122.9, 125.3, 126.8,
128.1, 128.6, 128.9, 138.6, 141.5, 142.8, 147.1, 147.5, and 164.4.
3373, 3230, 2916, 2849, 1645, 1607, 1501, 1490, 1455, 1290,
1035, 997, 761 cmꢁ1 1H NMR: d 3.85 (2H, s, –CH2CN), 5.98 (2H,
.
s, –OCH2O–), 6.08 (1H, d, J = 15.0 Hz, –CH@CHCO), 6.74–6.98 (5H,
m, olefinic and Ar-H), 7.05 (1H, d, J = 8.2 Hz, Ar-H-30), 7.14–7.50
(4H, m, –CH@CHCO and Ar-H0). 13C NMR: d 20.7, 101.1, 107.9,
108.5, 115.0, 119.3, 121.9, 122.6, 124.1, 124.2, 125.9, 128.0,
128.1, 128.6, 129.2, 141.6, 142.6, 147.2, 147.6, and 164.3.
4.2.20. Preparation of 5-(3,4-methylenedioxyphenyl)-2E,4E-
pentadienoic acid 4-hydroxymethylphenyl amide (21)
This compound was prepared by condensing piperic acid 2
(1.1 g, 5 mmol) with 4-amino benzyl alcohol (0.62 g, 5 mmol) as
per the method described for 3 to furnish a crude product which
on crystallization with ethyl acetate: pet ether gave a white crys-
talline compound 21 (1.3 g, yield 80%), mp 140–142 °C. Anal. Calcd
for C19H17NO4: C, 70.57; H, 5.298; N, 4.33%. Found: C, 70.93; H,
5.295; N, 4.47%. MS (%) M+ at m/z 323 (50), 306 (35), 292 (90),
271 (100), 201 (10), 166 (48), 143 (24), 115 (12). IR: 3395, 2917,
4.2.24. Preparation of 5-(3,4-methylenedioxyphenyl)-2E,4E-
pentadienoic acid 4-cynomethylphenyl amide (25)
The compound 25 was prepared by condensing piperic acid 2
(2.18 g, 10 mmol) with 4-amino benzyl cyanide (1.32 g, 10 mmol)
by the method as described for 3 to give a crude product which
on crystallization from ethyl acetate/petroleum ether (1:4) gave a
light brown crystalline compound 25 (2.65 g, yield 80%), mp
186 °C. Anal. Calcd for C20H16N2O3: C, 72.27; H, 4.850; N 8.42%.
Found: C, 72.49; H, 4.849; N, 8.47%. MS (%) M+ at m/z 332 (40),
294 (100), 201 (59), 172 (66), 131 (20). IR: 3346, 2917, 2849,
2849, 1653, 1602, 1502, 1488, 1448, 1252, 1038, 756 cmꢁ1 1H
.
NMR: d 4.67 (2H, br s, –CH2OH), 6.00 (2H, s, –OCH2O–), 6.25 (1H,
d, J = 15.0 Hz, –CH@CHCO), 6.76–6.98 (5H, m, olefinic and Ar-H),
7.24 (2H, d, J = 8.4 Hz, Ar-H-30 and 50), 7.40 (1H, m, –CH@CHCO),
7.65 (2H, d, J = 8.4 Hz, Ar-H-20 and 60). 13C NMR: d 65.5, 99.3,
108.4, 109.6, 119.4, 122.5, 122.51, 125.1, 126.6, 128.19, 128.2,
128.4, 128.6, 137.0, 141.7, 142.6, 147.9, 148.0, and 164.3.
1605, 1516, 1502, 1252, 1038, 755 cmꢁ1 1H NMR: d 3.73 (2H, s,
.
–CH2CN), 5.98 (2H, s, –OCH2O–), 6.05 (1H, d, J = 15.0 Hz,
CH@CHCO), 6.72–7.01 (5H, m, olefinic and Ar-H), 7.19 (2H, d,
J = 8.6 Hz, Ar-H-30 and 50), 7.31–7.56 (1H, m, CH@CHCO), 7.62
(2H, d, J = 8.6 Hz, Ar-H-20 and 60). 13C NMR: d 24.9, 101.5, 106.1,
108.9, 115.2, 119.6, 121.0, 121.1, 123.0, 124.1, 126.1, 128.1,
128.9, 131.0, 131.0, 141.2, 142.8, 147.9, 148.0, and 164.2.
4.2.21. Preparation of 5-(3,4-methylenedioxyphenyl)-2E,4E-
pentadienoic acid 3-cyanophenyl amide (22)
Compound 22 was prepared by condensing piperic acid 2 (2.2 g,
10 mmol) with 3-amino benzonitrile (1.20 g, 10 mmol) by the
method as described for 3 to give a light brown crystalline com-
pound 22 (2.54 g, yield 80%) after crystallization from ethyl ace-
tate/pet ether (1:4), mp 175–176 °C. Anal. Calcd for C19H14N2O3:
C, 71.68; H, 4.432; N, 8.80%. Found: C, 71.98: H, 4.431; N, 8.93%.
MS (%) M+ at m/z 318 (25), 294 (100), 201 (19), 175 (26) 143
(15), 117 (40). IR: 3376, 2917, 2849, 2230, 1602, 1543, 1486,
4.2.25. Preparation of 5-(3,4-methylenedioxyphenyl)-2E,4E-
pentadienoic acid 2-acetylaminophenyl amide (26)
Compound 26 was prepared by condensing piperic acid 2 (2.2 g,
10 mmol) with 20-amino acetanilide (1.50 g, 10 mmol) by the
method as described for 3 to give a brown crystalline compound
26 (2.81 g, yield 80%) after crystallization from ethyl acetate/pet
ether (1:4), mp 189–190 °C. Anal. Calcd for C20H18N2O4: C, 68.56;
H, 5.173; N, 7.99%. Found: C, 68.85; H, 5.171; N, 8.03%. MS (%)
M+ at m/z 350 (25), 305 (12), 291 (100), 201 (23), 173 (16), 149
(15), 115 (20). IR: 3282, 2917, 2850, 1653, 1602, 1505, 1486,
1252, 1038, 756 cmꢁ1 1H NMR: d 5.90 (2H, s, –OCH2O–), 6.02
.
(1H, d, J = 15.0 Hz, –CH@CHCO), 6.70–7.00 (5H, m, olefinic and
Ar-H), 7.26 (1H, dd, J = 15.0 and 9.2 Hz, –CH@CHCO), 7.31–7.40
(2H, m, Ar-H-40 and 50), 7.85 (1H, s, Ar-H-20), 7.91 (1H, d,
J = 8.5 Hz, Ar-H-60). 13C NMR: d 101.2, 107.7, 108.8, 116.1, 117.5,
121.2, 123.0, 123.6, 124.7, 125.8, 127.6, 128.0, 128.5, 129.4,
138.9, 142.7, 146.8, 147.5, and 163.8.
1252, 1038, 754 cmꢁ1 1H NMR: d 2.14 (3H, s, –COCH3), 5.99 (2H,
.
s, –OCH2O–), 6.09 (1H, d, J = 14.70 Hz, –CH@CHCO), 6.75–7.00
(5H, m, olefinic and Ar-H), 7.10–7.49 (5H, m, –CH@CHCO and Ar-
H0). 13C NMR: d 18.1, 101.5, 107.3, 108.6, 120.0, 120.1, 121.5,
123.5, 123.8, 124.3, 126.1, 128.3, 128.8, 135.1, 136.5, 142.6,
147.6, 147.7, 163.9, and 168.6.
4.2.22. Preparation of 5-(3,4-methylenedioxyphenyl)-2E,4E-
pentadienoic acid 4-cyanophenyl amide (23)
Compound 23 was prepared by condensing piperic acid 2 (2.2 g,
10 mmol) with 4-amino benzonitrile (1.20 g, 10 mmol) by the
method as described for 3 to furnish a gummy mass 23 (1.90 g,
yield 60%). Anal. Calcd for C19H14N2O3: C, 71.68; H, 4.432; N,
8.80%. Found: C, 72.04; H, 4.439; N, 8.94%. MS (%) M+ at m/z 318
(15), 294 (100), 201 (9), 175 (76) 143 (75), 117 (30). IR: 3370,
2916, 2848, 2214, 1627, 1604, 1515, 1316, 1252, 1170, 1037,
4.2.26. Preparation of 5-(3,4-methylenedioxyphenyl)-2E,4E-
pentadienoic acid 3-acetylaminophenyl amide (27)
Compound 27 was prepared by condensing piperic acid 2 (2.2 g,
10 mmol) with 30-amino acetanilide (1.50 g, 10 mmol) by the
method as described for 3 to give a light brown crystalline com-
pound 27 (2.99 g, yield 85%) after crystallization from ethyl ace-
tate/pet ether (1:4), mp 186–187 °C. Anal. Calcd for C20H18N2O4:
C, 68.56; H, 5.173; N, 7.99%. Found: C, 68.79; H, 5.172; N, 8.13.
MS (%) M+ at m/z 350 (15), 305 (10), 291 (100), 201 (13), 173
(25), 149 (35), 115 (25). IR: 3327, 2916, 2849, 1655, 1605, 1545,
830 cmꢁ1 1H NMR:
. d 5.94 (2H, s, –OCH2O–), 6.20 (1H, d,
J = 15.0 Hz, –CH@CHCO), 6.72–6.98 (5H, m, olefinic and Ar-H),
7.52–7.60 (3H, m, –CH@CHCO and Ar-H-30 and 50), 7.81 (2H, d,
J = 8.5 Hz, Ar-H-20 and 60). 13C NMR: d 101.3, 107.8, 108.2, 110.3,
117.8, 120.4, 121.2, 123.7, 125.1, 125.9, 127.4, 127.6, 133.8,
135.0, 139.0, 141.2, 147.2, 147.6, and 163.5.
1502, 1253, 1038, 802 cmꢁ1 1H NMR: d 2.12 (3H, s, COCH3), 5.90
.
(2H, s, –OCH2O–), 6.03 (1H, d, J = 15.0 Hz, –CH@CHCO), 6.72–7.01
(5H, m, olefinic and Ar-H), 7.05–7.48 (4H, m, –CH@CHCO and Ar-
H0), 7.87 (1H, s, Ar-H-20). 13C NMR: d 17.6, 101.3, 107.1, 108.2,
115.2, 117.0, 117.2, 119.8, 123.6, 125.9, 127.2, 128.2, 128.5,
140.5, 142.1, 142.4, 147.2, 147.5, 163.5, and 168.2.
4.2.23. Preparation of 5-(3,4-methylenedioxyphenyl)-2E,4E-
pentadienoic acid 2-cyanomethylphenyl amide (24)
The compound 24 was prepared by condensing piperic acid 2
(2.2 g, 10 mmol) with 2-amino benzyl cyanide (1.30 g, 10 mmol)
by the method as described for 3 to give a crude gummy mass
which was purified on column chromatography over silica and elu-
tion with pet ether/ethyl acetate to give a semi solid compound 24
(2.65 g, yield 80%). Anal. Calcd for C20H16N2O3: C, 72.27; H, 4.852;
N, 8.42%. Found: C, 72.63; H, 4.858; N, 8.60%. MS (%) M+ at m/z 332
(100), 294 (10), 201 (15), 173 (75) 143 (5), 131 (12), 115 (8). IR:
4.2.27. Preparation of 5-(3,4-methylenedioxyphenyl)-2E,4E-
pentadienoic acid 4-acetylaminophenyl amide (28)
Compound 28 was prepared by condensing piperic acid 2 (2.2 g,
10 mmol) with 40-amino acetanilide (1.50 g, 10 mmol) by the
method as described for 3 to give a brown crystalline compound
28 (2.91 g, yield 83%) after crystallization from ethyl acetate/pet

P. L. Sangwan et al. / Bioorg. Med. Chem. 16 (2008) 9847–9857
9855
ether (1:4), mp 169–170 °C. Anal. Calcd for C20H18N2O4: C, 68.56;
H, 5.173; N, 7.99%. Found: C, 68.92; H, 5.175; N, 8.08%. MS (%)
M+ at m/z 350 (5), 305 (15), 291 (100), 201 (28), 171 (43), 149
(30), 117 (20). IR: 3327, 2917, 2849, 1670, 1607, 1514, 1448,
13C NMR: d 100.3, 107.8, 108.0, 119.5, 120.4, 120.41, 120.5,
120.51, 123.8, 124.1, 125.9, 127.5, 127.51, 128.5, 128.7, 128.71,
128.8, 129.0, 138.0, 141.0, 142.7, 146.8, 147.5, 163.8, and 165.5.
1253, 1039, 756 cmꢁ1
.
1H NMR: d 2.15 (3H, s, COCH3), 5.95 (2H,
4.2.31. Preparation of 5-(3,4-methylenedioxyphenyl)-2E,4E-
s, –OCH2O–), 6.04 (1H, d, J = 15.0 Hz, –CH@CHCO), 6.76–6.98 (5H,
m, olefinic and Ar-H), 7.47–7.62 (5H, m, –CH@CHCO and Ar-H0).
13C NMR: d 17.9, 101.5, 107.5, 108.6, 119.8, 119.81, 120.1, 120.4,
120.41, 123.8, 126.1, 128.4, 128.8, 140.1, 141.3, 142.6, 147.4,
147.8, 163.8, and 168.5.
pentadienoic acid 40-benzanilide amide (32)
Compound 32 was prepared from piperic acid
2 (2.2 g,
10 mmol) and 40-amino benzanilide (2.12 g, 10 mmol) by the
method as described for 3 to give crude product which on crystal-
lization from ethyl acetate furnished a light brown crystalline com-
pound 32 (3.66 g, yield 90%), mp 210–211 °C. Anal. Calcd for
C25H20N2O4: C, 73.80; H, 4.887; N, 6.79%. Found: C, 73.11; H,
4.884; N, 6.97%. MS (%) M+ at m/z 412 (45), 382 (24), 292 (100),
201 (53), 172 (12), 143 (30), 116 (25). IR: 3390, 2918, 2850,
4.2.28. Preparation of 5-(3,4-methylenedioxyphenyl)-2E,4E-
pentadienoic acid benzothiazol-2yl amide (29)
To piperic acid 2 (1.1 g, 5 mmol) dissolved in dry dichlorometh-
ane (30 mL) was added thionyl chloride (0.5 mL) and the resulting
acid chloride was condensed with 2-amino benzothiazole (0.75 g,
5 mmol) and worked up as described for 3 to furnish a crude prod-
uct which on crystallization with ethyl acetate/pet ether gave a
brown crystalline compound 29 (1.23 g, yield 70%), mp 179–
180 °C. Anal. Calcd for C19H14N2O3S: C, 65.12; H, 4.026; N, 7.99%.
Found: C, 65.64; H, 4.023; N, 8.04%. MS (%) M+ at m/z 350 (28),
279 (39), 262 (100), 201 (15), 173 (5), 143 (10), 116 (20). IR:
3374, 2917, 2849, 1670, 1602, 1502, 1447, 1254, 1130, 1038,
1651, 1608, 1512, 1252, 1038 cmꢁ1 1H NMR: d 5.80 (2H, s,
.
–OCH2O–), 6.03 (1H, d, J = 15.0 Hz, –CH@CHCO), 6.73–7.02 (5H,
m, olefinic and Ar-H), 7.10–7.35 (3H, m, –CH@CHCO and Ar-H-300
and 500), 7.50–7.67 (5H, m, Ar-H-20, 30, 50, 60 and Ar-H-400), 7.88
(2H, d, J = 8.2 Hz, Ar-H-200 and 600). 13C NMR: d 101.6, 107.1,
108.3, 119.8, 120.5, 120.51, 120.6, 120.61, 124.1, 126.1, 128.7,
129.5, 129.15, 130.0, 130.9, 131.5, 131.5, 131.6, 135.8, 135.9,
142.9, 147.7, 147.8, 164.0, and 165.5.
754 cmꢁ1
.
1H NMR:
d
5.99 (2H, s, –OCH2O–), 6.37 (1H, d,
4.2.32. Preparation of 5-(3,4-methylenedioxyphenyl) pentanoic
acid piperidine amide (tetra hydro piperine) (33)
J = 15.0 Hz, –CH@CHCO), 6.77–6.97 (5H, m, olefinic and Ar-H),
7.28–7.46 (3H, m, –CH@CHCO and Ar-H0), 7.79 (1H, d, J = 8.3 Hz,
Ar-H0), 7.88 (1H, d, J = 8.3 Hz, Ar-H0). 13C NMR: d 101.6, 107.9,
108.2, 120.7, 122.0, 122.1, 122.3, 122.5, 123.3, 123.9, 125.6,
128.3, 128.6, 142.8, 146.1, 147.9, 148.1, 164.1, and 176.2.
A mixture of methanolic solution of Piperine 1 (142 mg) and 5%
Pd/C (30 mg) was hydrogenated at 40 psi for 4 h and the contents
filtered, washed with methanol (5ꢀ 15 mL), and concentrated on a
rotavapour to give crude product that on column chromatography
over silica gel and elution with pet ether/ethyl acetate (4:1) affor-
ded compound 33 (141.8 mg, yield 98.5%) as a gummy mass [the
spectral data was in agreement with data reported in the
lit.28,50]. Anal. Calcd for C17H23NO3: C, 70.56; H, 8.010; N, 4.84%.
Found: C, 70.75; H, 8.017; N, 4.98%. MS (%) M+1 at m/z 290 (42),
205 (28), 177 (41), 156 (100), 119 (32), 85 (18). IR 3162, 2929,
4.2.29. Preparation of 5-(3,4-methylenedioxyphenyl)-2E,4E-
pentadienoic acid benzothiazol-6yl amide (30)
To piperic acid 2 (1.1 g, 5 mmol) dissolved in dry dichlorometh-
ane (30 mL) was added thionyl chloride (0.5 mL) and the resulting
acid chloride was condensed with 6-amino benzothiazole (0.75 g,
5 mmol) and worked up as described for 3 to furnish a crude prod-
uct which on crystallization with ethyl acetate: pet ether gave a
brown crystalline compound 30 (1.4 g, yield 80%), mp 174–
176 °C. Anal. Calcd for C19H14N2O3S: C, 65.12; H, 4.026; N, 7.99%.
Found: C, 65.81; H, 4.023; N, 8.06%. MS (%) M+ at m/z 350 (24),
279 (29), 262 (100), 201 (4), 143 (12), 116 (12). IR: 3296, 2916,
1734, 1637, 1481, 1405, 1241, 1037, 932 cmꢁ1 1H NMR: d 1.22–
.
1.58 (10H, m, –N–CH2(CH2)3 and –C–CH2)2), 2.28 (2H, t, J = 7.0 Hz,
COCH2), 2.50 (2H, t, J = 7.0 Hz, Ar-CH2), 3.38 (4H, m, –N–(CH2)2),
5.90 (2H, s, –OCH2O–), 6.62 (3H, m, Ar-H). 13C NMR: d 24.1, 24.2,
24.6, 30.8, 31.1, 32.9, 34.9, 42.3, 46.4, 100.4, 107.7, 108.5, 120.7,
134.9, 144.5, 146.5, and 170.3.
2849, 1655, 1603, 1575, 1488, 1252, 1140, 1038, 755 cmꢁ1
.
1H
NMR: 5.99 (2H, s, –OCH2O–), 6.10 (1H, d, J = 15.0 Hz,
d
4.2.33. Preparation of 5-(3,4-methylenedioxyphenyl) pentanoic
acid isobutyl amide (34)
–CH@CHCO), 6.71–6.83 (4H, m, olefinic and Ar-H), 6.93 (1H, d,
J = 8.2 Hz, Ar-H), 7.29–7.35 (2H, m, –CH@CHCO and Ar-H-60), 8.01
(1H, d, J = 8.4 Hz, Ar-H-50), 8.05 (1H, s, Ar-H-20), 8.92 (1H, s,
N@CH–S). 13C NMR: d 101.3, 107.0, 108.1, 116.3, 119.2, 120.5,
123.5, 123.8, 125.9, 128.5, 128.8, 135.2, 139.6, 142.7, 146.2,
146.8, 147.0, 157.3, and 163.8.
The compound 34 was prepared in 96% yield as a colorless solid
from compound 6 by the same procedure as described for com-
pound 33 mp 62 °C. [Lit.50,51 mp 61–63 °C]. Anal. Calcd for
C16H23NO3: C, 69.29; H, 8.357; N, 5.05%. Found: C, 69.82; H,
8.365; N, 5.31%. MS (%) M+ at m/z 278 (100), 205 (35), 177 (58),
144 (32), 119 (13). IR: 3292, 2932, 1642, 1551, 1489, 1251, 1039,
4.2.30. Preparation of 5-(3,4-methylenedioxyphenyl)-2E,4E-
pentadienoic acid 4-benzanilide amide (31)
856 cmꢁ1 1H NMR: d 0.97 (6H, d, J = 6.5 Hz, –CH(CH3)2), 1.65
.
(4H, m, (CH2)2), 2.16 (2H, m, CO–CH2), 2.36 (1H, m, –CH(CH3)2),
2.53 (2H, t, J = 6.5 Hz, Ar-CH2), 3.72 (2H, m, –NHCH2), 5.89 (2H, s,
–OCH2O–), 6.66 (3H, m, Ar-H). 13C NMR: d 12.8, 19.1, 24.3, 30.4,
30.7, 34.4, 35.7, 38.2, 99.7, 107.1, 107.8, 120.1, 135.1, 144.5,
146.5, and 171.9.
The compound 31 was prepared from piperic acid 2 (1.1 g,
5 mmol) and 4-amino benzanilide (1.06 g, 5 mmol) by the proce-
dure as described for 3 to give crude product which on column
chromatography over silica gel and elution with pet ether/ethyl
acetate furnished a light brown crystalline compound 31 (1.23 g,
yield 60%), mp 232–233 °C. Anal. Calcd for C25H20N2O4: C, 72.80;
H, 4.887; N 6.79%. Found: C, 72.99; H, 4.883; N, 6.88%. MS (%) M+
at m/z 412 (15), 381 (30), 264 (25), 202 (100), 173 (24), 143 (5),
116 (11), 92 (24). IR: 3376, 2917, 2849, 2230, 1602, 1543, 1486,
4.2.34. Preparation of 5-(3,4-methylenedioxyphenyl) pentanoic
acid N,N-diethyl amide (35)
Compound 35 was prepared in 98% yield as a gummy mass from
compound 7 by the procedure as described for compound 33 [the
spectral data was in agreement with data reported in the lit.28].
Anal. Calcd for C16H23NO3: C, 69.28; H, 8.357; N, 5.05%. Found: C,
69.98; H, 8.366; N, 5.29%. MS (%) M+1 at m/z 278 (75), 205 (13),
175 (23), 134 (35), 127 (100), 114 (45), 99 (23), 71 (5). IR: 3381,
1252, 1038, 756 cmꢁ1 1H NMR: d 5.97 (2H, s, –OCH2O–), 6.45
.
(1H, d, J = 15.0 Hz, –CH@CHCO), (6.79–7.15 (5H, m, olefinic and
Ar-H), 7.18–7.27 (3H, m, Ar-H00), 7.38 (1H, dd, J = 15.0, 9.2 Hz,
–CH@CHCO), 7.52 (2H, d, J = 8.4 Hz, Ar-H-200 and 600), 7.63 (2H, d,
J = 8.4 Hz, Ar-H-20 and 60), 7.81 (2H, d, J = 8.4 Hz, Ar-H-30 and 50).
2928, 1638, 1485, 1403, 1244, 1039, 932 cmꢁ1 1 H NMR: d 1.10
.

9856
P. L. Sangwan et al. / Bioorg. Med. Chem. 16 (2008) 9847–9857
and 1.15 (3H each, t, J = 7.2 Hz, 2ꢀ –CH3), 1.64 (4H, m, (CH2)2), 2.30
(2H, t, J = 7.0 Hz, –COCH2), 2.55 (2H, t, J = 6.7 Hz, Ar-CH2), 3.30 (4H,
q, J = 7.2 Hz, –NH(CH2)2), 5.91 (2H, s, –OCH2O–), 6.59–6.74 (3H, m,
Ar-H). 13C NMR: d 13.5, 14.7, 25.4, 31.9, 33.3, 35.9, 40.4, 42.3, 101.1,
108.4, 109.2, 121.4, 136.6, 145.9, 147.9, and 172.4.
4.4. Efflux studies
Ethidium bromide accumulation and its efflux were determined
from the active cells by reported method.53 S. aureus 1199B was
grown overnight on Trypticase Soya Agar. Bacterial suspension
(0.2 OD550) was prepared in uptake buffer (NaCl, 110 mM, KCl,
7 mM; NH4Cl, 50 mM; Na2HPO4, 0.4 mM; Tris base, 52 mM; glu-
cose, 0.2%, adjusted the solution to pH 7.5 with HCl). The suspen-
4.2.35. Preparation of 5-(3,4-methylenedioxyphenyl) pentanoic
acid 3-cyano-phenyl amide (36)
This was prepared in 84% yield as a gummy mass from com-
pound 22 by the procedure as described for compound 33. Anal.
Calcd for C19H18N2O3: C, 70.79; H, 5.627; N, 8.69%. Found: C,
71.13; H, 5.633; N, 8.93%. MS (%) M+1 at m/z 321(85), 296 (18),
220 (39), 205 (100), 143 (12), 117 (24). IR: 3326, 2926, 2855,
sions were exposed to 2
the most active EPIs (22 and 26 of 6.25 and 12.5
tion, respectively), inactive EPI 5 (100 g/mL), piperine (50
and standard EPI reserpine (25 g/mL) for 30 min at 37 °C. The
cells were pellet down by centrifugation and re-suspended in fresh
buffer. The loss of fluorescence was recorded for 30 min at 5 min
interval at excitation and emission wavelengths of 530 and
600 nm in a spectrophotometer (Perkin-Elmer model LS50).
l
g/mL ethidium bromide in presence of
g/mL concentra-
g/mL)
l
l
l
l
2231, 1669, 1604, 1585, 1487, 1252, 1039, 795 cmꢁ1 1H NMR: d
.
1.67 (4H, m, (CH2)2), 2.33 (2H, t, J = 7.0 Hz, –COCH2), 2.55 (2H, t,
J = 6.7 Hz, Ar-CH2), 5.96 (2H, s, –OCH2O–), 6.67–6.93 (3H, m, Ar-
H), 7.21–7.43 (2H, m, Ar-H0), 7.82 (1H, s, Ar-H-20), 8.01 (1H, d,
J = 8.2 Hz, Ar-H-60). 13C NMR: d 24.4, 30.8, 34.1, 35.2, 100.3,
107.3, 108.1, 113.7, 118.5, 120.4, 122.1, 123.4, 126.6, 129.3,
134.7, 144.7, 146.6, 149.5, and 172.1.
Acknowledgment
The authors are highly thankful to Prof. G. W. Kaatz of Wayne
State University, School of Medicine, Detroit, Michigan, USA for
providing S. aureus 1199 and S. aureus 1199B as gift samples.
4.2.36. Preparation of 5-(3,4-methylenedioxyphenyl)-
pentanoic acid 2-acetylamino-phenyl amide (37)
This was prepared in 48% yield as a gummy mass from compound
26 by the procedure as described for compound 33. Anal. Calcd for
C20H22N2O4: C, 67.71; H, 6.256; N, 7.90%. Found: C, 68.01; H,
6.265; N, 8.04%. MS (%) M+ at m/z 354 (21), 309 (72), 295 (30), 205
(100), 173 (18), 149 (34), 115 (12). IR: 3162, 2926, 1667, 1601,
References and notes
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(4H, m, (CH2)2), 2.32 (2H, t, J = 7.0 Hz, –COCH2), 2.54 (2H, t,
J = 6.7 Hz, Ar-CH2), 5.86 (2H, s, –OCH2O–), 6.62–6.84 (3H, m, Ar-H),
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7.69 (2H, d, J = 8.2 Hz, Ar-H-20 and 60), 7.86 (2H, d, J = 8.4 Hz, Ar-H-
30 and 50). 13C NMR: d 23.9, 30.5, 32.3, 33.9, 99.6, 107.2, 108.4,
111.3, 118.6, 120.3, 120.4, 120.6, 123.5, 127.9, 128.0, 128.3, 128.7,
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of the EPIs
The MIC of ciprofloxacin was determined against S. aureus 1199
and S. aureus 1199B in Muller–Hinton Broth in the presence of
increasing amounts of efflux pump inhibitors by broth checker-
board synergy method in a microtitre plates using twofold serial
dilutions.46 Each candidate EPI was tested at seven concentrations
(100–1.56
lg/mL) and ciprofloxacin was tested at 10 concentra-
tions (16–0.03
lg/mL). The plates were incubated for 18 h at
37 °C and the wells were assessed visually for growth. The minimal
effective concentration (MEC) was determined to be the minimal
concentration of EPI that produced the maximal reduction in sub-
strate MIC. No further decrease in substrate MIC was observed at
EPI concentrations greater than the MEC.52
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