Synthesis of piperine-amino acid ester conjugates and study of their cytotoxic activities against human cancer cell lines

Article abstract of DOI:10.1007/s00044-010-9500-5

A series of piperine-amino acid ester conjugates (4a-4r) were synthesized under mild conditions and screened for their cytotoxic activities against a panel of human cancer cell lines (IMR-32, MCF-7, PC-3, DU-145, Colo-205, and Hep-2). The parent compound

Full text of DOI:10.1007/s00044-010-9500-5

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:38–46
DOI 10.1007/s00044-010-9500-5
ORIGINAL RESEARCH
Synthesis of piperine–amino acid ester conjugates and study
of their cytotoxic activities against human cancer cell lines
•
V. Rama Subba Rao G. Suresh R. Ranga Rao
•
•
•
•
•
K. Suresh Babu G. Chashoo A. K. Saxena
J. Madhusudana Rao
Received: 9 September 2010 / Accepted: 2 November 2010 / Published online: 21 November 2010
Ó Springer Science+Business Media, LLC 2010
Abstract A series of piperine–amino acid ester conju-
gates (4a–4r) were synthesized under mild conditions and
screened for their cytotoxic activities against a panel of
human cancer cell lines (IMR-32, MCF-7, PC-3, DU-145,
Colo-205, and Hep-2). The parent compound piperine
lacked significant activity but the analogues were effective
to in all tested human cancer cell lines. The introduction of
D- and L-amino acid side chain to piperine through peptide
linkage significantly increased cytotoxic activity. Among
the tested conjugates, 4p showed significant cytotoxic
activity against DU-145 cell lines with IC50 of 21 lM. The
synthetic protocol is suitable for generating piperine
derivatives with various structural motifs for exploring the
desired activity.
engulf approx eight million people around the world for the
year 2007 (Shewach, 2009). Over the past few decades,
extensive research has led to the development of a plethora
of chemotherapeutic agents; however, none of these agents
are capable of completely eliminating cancer (Cragg and
Newman, 2005; Cragg et al., 2009). The limitations of
current anticancer drugs and rapid development of drug
resistance (Sreedhar and Csermely, 2004; Pechan, 1991;
McCubrey et al., 2006) have highlighted the need for the
discovery of new anticancer agents, preferably with novel
mechanisms of action. To identify new chemical entities
for a more effective treatment of cancer, drug designers can
follow many strategies, but the crucial decision is always
the selection of a suitable starting point from the vast
chemical space (Lloyd et al., 2006).
Keywords Piper nigrum Á Piperine Á Piperoic acid Á
Amino acid conjugates Á Amide linkage Á Peptide linkage Á
Boric acid Á Cytotoxic activity
In this respect, natural products can be viewed as
evolved privileged structures (Koehn and Carter, 2005) and
biologically prevalidated leads, in other words, as mole-
cules that have probably evolved evolutionarily to exert
highly specialized functions. Recent review pointed out
that, about 74% of anticancer compounds being either
natural or natural product-derived products, indicating
potency of these scaffolds (Newman et al., 2003). The
unprecedented structures of these molecules make them
excellent synthetic targets, and their potent activity against
a broad number of therapeutic indications makes these
natural products excellent drug lead candidates for new
therapeutics (Newman, 2008).
Introduction
Cancer is one of the most serious threats against human
health in the world. Recent statistical data showed that it
V. Rama Subba Rao Á G. Suresh Á R. Ranga Rao Á
K. Suresh Babu Á J. Madhusudana Rao (&)
Division of Organic Chemistry-I, Natural Product Laboratory,
Indian Institute of Chemical Technology, Hyderabad 500 607,
India
In connection with recent our investigations of piper
species for value-added products (Sumathykutty and Rao,
1991; Reddy et al., 2004; Srinivas and Rao, 1999; Srinivas
et al., 2006; Rao et al., 2009; Jyothi et al., 2009), we have
isolated large quantities of piperine (1) (2%) from Piper
nigrum, which prompted us to synthesize derivatives and
screen for the anticancer activity. Traditionally, pepper
e-mail: janaswamy@iict.res.in
G. Chashoo Á A. K. Saxena
Department of Pharmacology, Indian Institute of Integrative
Medicine (CSIR), Canal Road, Jammu Tawi 180001,
Jammu & Kashmir, India
123

Med Chem Res (2012) 21:38–46
39
has been used for many ailments particularly as anti
inflammatory agent and piperine has been identified as the
active principle of pepper (Parmar et al., 1997). Piperine
(1), a hydrophobic amide derived from the fruit of Piper
nigrum has been shown to exhibit a wide spectrum of
biological and pharmacological activities (Mujumdar et al.,
1990; Birkmayer et al., 1985; Kong et al., 2004; Pradeep
and Kuttan, 2002; Sunila and Kuttan, 2004; Bezerra et al.,
2006; Selvendran et al., 2005). Despite the prominent
potential of piperine, very little effort has been devoted into
the synthesis of piperine derivatives (Sangwan et al., 2008;
Nargotra et al., 2009; Mishra et al., 2005).
functionality in all amino acids (D and L) was protected as
its methyl ester by following literature method (Medina
and EI-Sayed, 2009). However, these approaches of
combining different active molecules present in nature,
resulting in enhancement of their bioavailability, are cer-
tainly encouraging. These molecules contain the unique
properties like chirality, hydrophilicity/hydrophobicity, and
optical properties (Frank-Furt and Krishnan, 2003; Thian-
tanwat et al., 2000). The distinctive internal composition
created by the amino acid building blocks offers stereo-
selective sites for noncovalent interactions with guest/drug
molecule. All target compounds (4a–4r) were character-
1
Recently, development of hybrid or conjugate molecules
between two different types of moieties emerged as a new
approach in the discovery of new cytotoxic agents, as they
consists of high potency and also different alkylation sites
and these properties are essential to play vital role in tumor
treatment (Kamal et al., 2005; Noh et al., 2009). Thus, we
wish to utilize amino acids as the diversity surrogates to
couple with the piperine. Such piperine–amino acid
derivatives may have the advantages of structural diversity,
improved solubility, and tunable anticancer activity. In
addition, the chemical processes and reactions involved are
concise and straightforward, which make it feasible for the
scale-up production of piperine derivatives of interest.
Based on the consideration of simple processes and ample
diversity, we derived a concise strategy for synthesizing
piperine–amino acid ester conjugates through the peptide
linkage. Herein, we report the synthesis and cytotoxic
activity of this new series of derivatives.
ized by H, 13C NMR, IR, and HREIMS analyses.
Biological activity
Discussion
The in vitro growth inhibition and cytotoxicity assay was
performed by NCI according to well-established proce-
dures (Skehan et al., 1990). Initially, all the synthesized
derivatives (4a–4r) along with the piperine were screened
in a one dose primary anticancer screen for their cytotoxic
activity against neuroblastoma (IMR-32), breast (MCF-7),
prostate (PC-3, DU-145), colon (Colo-205), liver (Hep-2)
cell lines using 5-flurouracil, adriamycin, and mitomycin C
as positive controls. As expected, most of the derivatives
showed good cytotoxic activity better than piperine itself.
Table 2 summarizes their IC50 values against panel of
human cancer cell lines. From these data, we found that
activity was enhanced when the L-amino acids were con-
jugated to piperine instead of D-amino acids. The amino
acids which possess isopropyl, isobutyl, and methyl
side chains, such as D-valine, L-leucine, L-isoleucin, and
L-alanine, were conjugated to piperine; its cytotoxic
activity was dramatically increased to over 10–90%.
Especially, the inhibitory activity of 4p, which possess the
heterocyclic side chain such as histidine showed highest
growth inhibition (91%) toward the prostate cell line and
its IC50 value was 21 0.09. However, it should also note
that the parent compound piperine itself is inactive against
the prostate cancer cells. It is important to note that com-
pound 4a displayed moderate activity toward neuro-
blastma, breast, and prostate cancer cell lines. It is
important to note that cell specificity was observed in
compound 4p against prostate cell lines, which showed
selective cytotoxicity against DU-145, but inactive toward
PC-3, suggesting the imidazole ring structure may con-
tribute to the binding. Similarly, compounds 4g and 4h
displayed moderate activity on breast cancer and colon
cancer cell lines. It is noteworthy to mention that among
the tested monobasic aminoacid esters (3a–3h), com-
pounds 4a, 4h showed moderate activity on PC-3 and
Chemistry
Results and discussion
The basic skeleton of piperine consists of methylenedi-
oxyphenyl (MDP) moiety, side chain which consists of
conjugated trans-double bonds and basic six-membered
piperidine moiety attached to side chain via amide linkage.
This study aimed to explain SAR studies on basic amide
moiety (–CO–N–) in piperine, by replacing with peptide
bond (–CO–NH–) through the conjugation of amino acid
ester. Thus, the target compounds 4a–4r were prepared
through one step condensation of piperic acid (2) and
amino acid esters (D and L) (3a–3r) using ecofriendly boric
acid in catalytic amount at reflux temperature in toluene
(Table 1) (Tang et al., 2005). In this reaction, as shown in
Scheme 1, the acid moiety of 2 gets activated with boric
acid, which in turn provides an electron-deficient acid
carbonyl center that reacts with amine (3a–3r) to afford
amide (4a–4r) in 70–88% yield. Piperic acid (2) was
obtained through basic hydrolysis of piperine. The acid
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40
Med Chem Res (2012) 21:38–46
Table 1 Structure, reaction time, and yield of conjugates (4a–4r)
Table 1 continued
(4a-4r)a
(4a-4r)a
S. no
R
Time (h)
S. no
R
Time (h)
Yield (%)
Yield (%)
COOMe
COOMe
COOMe
COOMe HN
N
1
a
6
75
4
4
4
85
80
88
16
p
N
N
H
H
COOMe
2
3
b
c
4
4
80
82
N
H
N
17
18
q
r
NH
COOMe
N
H
N
H
COOMe
COOMe
Yield refers to pure products after chromatography
a
4
5
d
e
6
6
85
86
N
All products were characterized by 1H, 13C NMR, IR and HRE-
SIMS spectroscopy
H
N
H
Colo-205 with IC50 (33 0.14, 36 0.11) cells. More-
over, the compound 4q which possesses amide linkage is
showed inactive against all cell lines. Overall, these results
imply that the mode of perception as well as the structure–
activity relationship of the piperine analogues differs con-
siderably between cell lines examined in this study. To the
best of our knowledge this is the first report on the anti-
cancer activity of the piperine–amino acid ester conjugates.
COOMe
6
7
f
4
4
88
87
N
H
COOMe
g
N
H
COOMe
OH
8
9
h
i
6
6
80
80
N
Conclusion
H
COOMe
COOMe
COOMe
COOMe
In conclusion, we have observed that the introduction of an
amino acid side chain to the piperine moiety causes a
significant increase in the cytotoxic activity. Esters of ali-
phatic L-amino acids were found to be more effective than
those of aromatic amino acids. Analogues with hetero
aromatic side chain were also good cytotoxic agents. In this
study compound 4p was found to be potent among the
tested compounds. This initial library represents our early
stage effort in diversifying piperine derivatives for
exploring useful structure–activity relationships. On the
basis of the results observed histidine and glycine could be
identified as the best possible side chain among the amino
acids screened against the cancer cell lines. By conjugating
with amino acids, the reported piperine derivatives mani-
fest improved solubility in various solvents including
water.
N
H
10
11
j
5
6
75
75
N
H
COOMe
k
N
H
COOMe
COOMe
12
13
l
6
6
80
80
N
H
m
N
H
OH
COOMe
6
6
75
70
14
15
n
o
Experimental
N
H
OH
General
COOMe
N
H
Optical rotations were recorded on a JASCO DIP 300
OH
digital polarimeter at 25°C. IR spectra were recorded on
123

Med Chem Res (2012) 21:38–46
41
Scheme 1 Synthesis of
piperine–amino acid ester
conjugates
O
O
KOH, methanol
reflux, 6 h
O
O
O
O
N
OH
Piperine (1)
Piperic acid (2)
O
O
O
R
B(OH)3, (3a-r), toluene
reflux, 7-12h
Conjugates (4a-r), R= aminoacid ester
3a = Methyl ester of glycine
3j = Methyl ester of (D)-aspartic acid
3k = Methyl ester of (D)-phenyl glycine
3l = Methyl ester of (L)-phenyl alanine
3m = Methyl ester of (D)-phenyl alanine
3n = Methyl ester of (L)-tyrosine
3b = Methyl ester of (L)-alanine
3c = Methyl ester of (D)-alanine
3d = Methyl ester of (L)-valine
3e = Methyl ester of (D)-valine
3f = Methyl ester of (L)-leucine
3g = Methyl ester of (L)-isoleucine
3h = Methyl ester of (L)-serine
3i = Methyl ester of (L)-aspartic acid
3o = Methyl ester of (L)-2,4-Dihydroxy phenyl alanine
3p = Methyl ester of (L)-histidine
3q = Methyl ester of (L)-proline
3r = Methyl ester of (L)-tryptopan
Table 2 IC50 (lM) for selected conjugates with piperine against human cell lines
Compound
IMR-32
Neuroblastoma
MCF-7
Breast
PC-3
Prostate
DU-145
COLO-205
Colon
HEP-2
Liver
IC-50 (lM)
4a
50 0.35
44 0.33
33 0.14
49 0.21
–
–
4b
–
–
–
51 0.19
–
–
4d
–
51 0.33
49 0.32
44 0.17
–
–
4e
–
–
–
–
51 0.23
–
4f
–
48 0.42
–
–
55 0.12
43 0.11
4g
–
48 0.35
–
–
–
–
4h
–
–
–
–
36 0.11
–
4i
–
–
–
46 0.13
–
–
4p
–
–
–
21 0.09
–
–
Piperine
5-FU
89 0.39
nd
99 0.41
2.9 0.3
0.8 0.01
nd
–
–
46 0.13
2.9 0.09
nd
70 0.1
nd
nd
2
0.3
Adriamycin
Mitomycin C
0.02 0.12
nd
nd
nd
nd
Nd
1.4 0.11
1.8 0.3
nd
The values are represented as mean SE of three experiments for each carried out in triplicate
– [50 lM
nd not determined
Nicolet-740 spectrometer with KBr Pellets. The 1H and 13
C
9100 and are uncorrected. Most of the reaction solvents
were purified by distillation under nitrogen from the indi-
cating drying agent and used fresh, dichloromethane (cal-
cium hydride), acetone (potassium permanganate). Column
chromatography was carried out using silica gel 100–200
mesh (Acme Silica gel) and precoated silicagel plates
(Merck, 60 F254) were used for preparative TLC. B(OH)3,
L and D-amino acids were purchased from Sigma Chemical
Co., St Louis, MO, USA. Other chemicals of analytical
grade were procured from indigenous manufacturers.
NMR spectra were recorded on a Bruker FT-300 MHz
1
spectrometer at 300 MHz for H and 75 MHz for 13C,
respectively, using TMS as internal standard. The chemical
shifts are expressed as (d) values in parts per million (ppm)
and the coupling constant (J) is given in hertz (Hz). Spin
multiplicities are described as s (singlet), br s (broad sin-
glet), d (doublet), t (triplet), q (quartet), and m (multiplet).
Mass spectra were measured on LC-MSD-Trap-SL instru-
ment. Melting points were recorded on Electrothermal
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Med Chem Res (2012) 21:38–46
Extraction and isolation of piperine from Piper nigrum
solid. Stirring is continued for an additional 15 min and
then the precipitate is filtered off with suction through a
sintered glass filter funnel. The collected solid, which was
purified by column chromatography (40% ethyl acetate–
hexane) to give target compounds (4a–4r). Reaction time
and yields of products (4a–4r) are given in Table 1.
The fruits of Piper nigrum also known as black pepper
were purchased from the local super market, Secunderabad
(Andhra Pradesh, India) in the month of December 2007
and identification was made by Department of Botany,
Osmania University, Hyderabad. A voucher specimen was
deposited at the herbarium of Indian Institute of Chemical
Technology, Hyderabad, India. The shade dried fruits of
Piper nigrum were powdered in a pulvarizer (5 kg) and
extracted with hexane in a soxhlet apparatus for 72 h. The
resulting extract was filtered to get 10 g of piperine.
Synthesis of piperic acid (2) from piperine (1)
Piperine (5 g, 17.5 mmol) was refluxed with methanolic
KOH (2 N, 20 ml) for 6 h and methanol was evaporated
under reduced pressure, solution cooled in ice bath, the
gummy potassium salt of piperic acid was suspended in
water and gradually acidified with dilute hydrochloric acid,
dark yellow precipitate was collected and stirred for 3–4 h
at cold ice bath, filtered through filter paper, and washed
with cold water (2 9 10 ml) and recrystallised from
methanol to afford yellow crystalline compound with yield
of 80%, final compound was submitted to spectral analysis
and the spectral values and mp were matched with reported
data.
Cell lines and cell cultures
The human cancer cell lines were obtained either from
National Center for Cell Science, Pune, India or National
Cancer Institute, Frederick, USA. The human neuroblas-
toma (IMR-32), breast (MCF-7), prostate (PC-3, DU-145),
colon (colo-205), liver (Hep-2) cell lines were grown and
maintained in RPMI-1640medium, pH 7.4, whereas
DMEM was used for neuroblastoma (IMR-32) and liver
(Hep-2). The media were supplemented with FCS (10%),
penicillin (100 U/ml), streptomycin(100 g/ml), glutamine
(2 mM) and cells were grown in CO2 incubator (Heraeus,
GmbH, Germany) at 37°C with 90% humidity and 5%
CO2. Cells were treated with A001 and F002 dissolved in
DMSO, while the untreated control cultures received only
the vehicle (DMSO, \0.2%).
Spectroscopic data of conjugates (4a–4r)
Methyl-2-((2E,4E)-5-(benzo[d][1,3]dioxol-6-yl)penta-2,4-
dienamido)acetate (4a)
It is a pale white solid, mp. 152–154°C; IR (KBr) tmax
:
663, 808, 993, 1036, 1289, 1489, 1605, 1742, 2853, 2922,
3066, and 3272 cm-1; 1H NMR (300 MHz, CDCl3): d ppm
3.78 (3H, s), 5.13–5.17 (2H, d, J = 5.0 Hz), 5.95–6.02
(1H, d, J = 14.7 Hz), 5.98 (2H, s), 6.09–6.18 (1H, br t,
J = 4.9 Hz), 6.62–6.73 (1H, dd, J = 15.4, 10.5 Hz),
6.74–6.83 (2H, m), 6.86–6.91 (1H, dd, J = 8.1, 1.7 Hz),
6.95–6.9 (1H, d, J = 1.5 Hz), 7.33–7.42 (1H, dd, J = 14.9,
10.5 Hz), 13C NMR (75 MHz, CDCl3): d 29.6, 41.4, 52.3,
101.4, 105.8, 108.5, 121.9, 122.7, 124.4, 130.6, 139.5,
142.0, 148.1, 166.2, 170.4; HRESIMS m/z 290.1019
[M? ? H], calcd for C15H15NO5 290.09502.
Determination of in vitro cytotoxicity against human
cancer cell lines
In vitro cytotoxicity against human cancer cell lines was
determined using sulphorhodamine B assay (Skehan et al.,
1990). Stock solutions of all the compounds were prepared
in DMSO and serially diluted with growth medium to
obtain desired concentrations.
Experimental procedure for synthesis of 4a–4r
(S)-Methyl-2-((2E,4E)-5-(benzo[d][1,3]dioxol-6-yl)penta-
2,4-dienamido)propanoate (4b)
The reaction vessel is charged with piperic acid 2 (0.050 g,
1 mmol), boric acid (0.020 g, 0.30 mmol), and 50 ml of
toluene, Dean-Stark trap topped with a reflux condenser.
To the stirred colorless reaction mixture is added amino-
acid ester (3a–3r) (1 mmol) in one portion. The reaction
mixture is heated at reflux for 12–15 h. The progress of the
reaction was monitored by TLC using 40% ethyl acetate–
hexane system. The mixture is allowed to cool to ambient
temperature and then is poured with stirring into 100 ml of
hexane leading to the immediate precipitation of a white
It is a yellow solid, mp. 158–160°C; [a]2D5 = ?9 (c 0.50,
CHCl3); IR (KBr) tmax: 770, 1039, 1250, 1495, 1649, 1740,
2851, 2919, and 3455 cm-1; 1H NMR (300 MHz, CDCl3):
d ppm 1.43–1.48 (3H, d, J = 7.1 Hz), 3.77 (3H, s),
4.66–4.77 (1H, m), 5.92–5.97 (1H, d, J = 13.0 Hz), 5.98
(2H, s), 6.10–6.17 (1H, br d, J = 6.98 Hz), 6.63–6.73 (1H,
dd, J = 15.4, 10.3 Hz), 6.75–6.82 (2H, m), 6.88–6.92 (1H,
dd, J = 8.1, 1.5 Hz), 6.99 (1H, d, J = 1.3 Hz), 7.31–7.42
(1H, dd, J = 14.9, 10.3 Hz). 13C NMR (75 MHz, CDCl3):
123

Med Chem Res (2012) 21:38–46
43
d 18.6, 48.1, 52.3, 101.2, 105.7, 108.4, 122.3, 122.7, 124.4,
130.8, 139.1, 141.7, 148.2, 148.3, 165.4, 173.6; HRESIMS
m/z 304.1180 [M? ? H], calcd for C16H17NO5 346.1179.
139.3, 141.8, 148.2, 148.3, 165.8, 172.6; HRESIMS m/
z 332.1492 [M? ? H], calcd for C18H21NO5 332.1492.
(R)-Methyl-2-((2E,4E)-5-(benzo[d][1,3]dioxol-6-yl)penta-
2,4-dienamido)-4-methylpentanoate (4f)
(R)-Methyl-2-((2E,4E)-5-(benzo[d][1,3]dioxol-6-yl)penta-
2,4-dienamido)propanoate (4c)
It is a pale yellow solid, mp. 111–115°C; [a]2D5 = ?20
(c 0.10, CHCl3); IR (KBr) tmax: 866, 1037, 1200, 1444,
It is a yellow solid, mp. 150–154°C; [a]2D5 = -15 (c 0.50,
CHCl3); IR (KBr) tmax: 927, 1030, 1249, 1450, 1538, 1652,
1
1536, 1653, 1740, 2857, 2921, and 3414 cm-1; H NMR
(300 MHz, CDCl3): d ppm 0.95 (6H, t, J = 5.2 Hz),
1.53–1.65 (2H, m), 1.67–1.76 (1H, m), 3.75 (3H, s),
4.72–4.82 (1H, m), 5.92–6.00 (4H, m), 6.62–6.72 (1H, dd,
J = 15.8, 10.5 Hz), 6.74–6.81 (2H, m), 6.87–6.92 (1H, dd,
J = 8.3, 1.5 Hz), 6.97–6.99 (1H, d, J = 1.5 Hz), 7.33–7.42
(1H, dd, J = 15.1, 10.5 Hz). 13C NMR (75 MHz, CDCl3):
d 22.0, 22.7, 24.9, 29.6, 41.9, 50.7, 52.3, 101.3, 105.7,
108.4, 122.3, 122.8, 124.6, 130.7, 139.4, 141.9, 148.2,
165.7, 173.7; HRESIMS m/z 346.1637 [M? ? H], calcd
for C19H23NO5 346.1649.
1
1740, 2852, 2920, and 3457 cm-1; H NMR (300 MHz,
CDCl3): d ppm 1.42–1.49 (3H, d, J = 7.1 Hz), 3.77 (3H,
s), 4.66–4.78 (1H, m), 5.92–5.94 (1H, d, J = 13.0 Hz),
5.95–6.00 (2H, br s), 6.11–6.18 (1H, br d, J = 6.98 Hz),
6.62–6.73 (1H, dd, J = 15.4, 10.3 Hz), 6.74 –6.82 (2H, m),
6.87–6.92 (1H, dd, J = 8.1, 1.5 Hz), 6.97–6.99 (1H, d,
J = 1.3 Hz), 7.32–7.42 (1H, dd, J = 14.9, 10.3 Hz). 13C
NMR (75 MHz, CDCl3): d 18.5, 47.1, 51.7, 100.1, 107.9,
109.4, 123.3, 123.6, 125.5, 131.8, 139.1, 141.6, 148.3,
148.4, 165.5, 173.7; HRESIMS m/z 304.1181 [M? ? H],
calcd for C16H17NO5 346.1179.
(2R,3R)-Methyl-2-((2E,4E)-5-(benzo[d][1,3]dioxol-6-
yl)penta-2,4-dienamido)-3-methylpentanoate (4g)
(R)-Methyl-2-((2E,4E)-5-(benzo[d][1,3]dioxol-6-yl)penta-
2,4-dienamido)-3-methylbutanoate (4d)
It is a pale yellow solid, mp. 112–115°C; [a]2D5 = ?68
(c 0.50, CHCl3); IR (KBr) tmax: 805, 929, 1037, 1255,
1
1444, 1534, 1644, 1741, 2964, and 3303 cm-1; H NMR
It is a white solid, mp. 140–142°C; [a]2D5 = ?39 (c 0.50,
CHCl3);IR (KBr) tmax: 670, 746, 807, 989, 1039, 1194,
(300 MHz, CDCl3): d ppm 0.91–0.98 (6H, t, J = 7.1 Hz),
1.40–1.54 (1H, m), 1.88–1.98 (1H, m), 3.76 (3H, s),
4.70–4.76 (1H, dd, J = 8.6, 5.0 Hz), 5.99 (2H, s),
5.95–6.01 (1H, d, J = 14.7 Hz), 6.02–6.07 (1H, br d,
J = 9.0 Hz), 6.63–6.73 (1H, dd, J = 15.4, 10.3 Hz),
6.76–6.82 (2H, m), 6.88–6.93 (1H, dd, J = 8.1, 1.7 Hz),
6.99 (1H, d, J = 1.5 Hz), 7.32–7.42 (1H, dd, J = 14.9,
10.3 Hz). 13C NMR (75 MHz, CDCl3): d 11.5, 15.3, 25.2,
38.1, 53.0, 56.3, 101.2, 105.6, 108.4, 122.5, 122.6, 124.4,
130.6, 139.2, 141.7, 148.1, 165.9, 172.7; HRESIMS m/
z 346.1643 [M? ? H], calcd for C19H23NO5 346.1649.
1
1504, 1645, 1741, 2875, 2961, and 3292 cm-1; H NMR
(300 MHz, CDCl3): d ppm 0.89–1.00 (6H, m), 2.12–2.26
(1H, m), 3.75 (3H, s), 4.62–4.74 (1H, m), 5.90–6.08 (4H,
m), 6.63–6.74 (1H, dd, J = 15.4, 10.3 Hz), 6.75–6.85 (2H,
m), 6.87–6.93 (1H, dd, J = 8.1, 1.3 Hz), 6.97–7.00 (1H, br
s), 7.32–7.43 (1H, dd J = 15.0, 10.3 Hz). 13C NMR (75
MHz, CDCl3): d 17.9, 18.9, 31.5, 52.1, 57.1, 101.4, 105.7,
108.5, 122.6, 124.5, 130.8, 139.2, 140.9, 148.2, 165.9,
172.7; HRESIMS m/z 332.1490 [M? ? H], calcd for
C18H21NO5 332.1492.
(R)-Methyl-2-((2E,4E)-5-(benzo[d][1,3]dioxol-6-yl)penta-
2,4-dienamido)-3-hydroxypropanoate (4h)
(S)-Methyl-2-((2E,4E)-5-(benzo[d][1,3]dioxol-6-yl)penta-
2,4-dienamido)-3-methylbutanoate (4e)
It is a yellow solid, mp. 148–150°C; [a]2D5 = ?60 (c 0.50,
CHCl3); IR (KBr) tmax: 663, 1263, 1644, 1731, 2361, 2854,
2925, and 3450 cm-1; 1H NMR (300 MHz, CDCl3): d ppm
2.70 (1H, br s), 3.80 (3H, s), 4.05 (2H, m), 4.80 (1H, m),
5.95 (2H, s), 6.55 (1H, br d, J = 7.5 Hz), 6.75 (1H, d,
J = 15.0 Hz), 6.80 (2H, m), 6.85 (1H, dd, J = 8.5, 1.3 Hz),
6.95 (1H, d, J = 1.3 Hz), 7.35–7.45 (1H, dd, J = 14.9,
10.5 Hz). 13C NMR (75 MHz, CDCl3): d 52.7, 54.9, 63.9,
101.6, 105.5, 108.2, 121.9, 122.5, 124.5, 128.6, 130.8,
139.8, 142.3, 166.3, 170.8; HRESIMS m/z 320.1113
[M? ? H], calcd for C16H17NO6 320.1129.
It is a white solid, mp. 139–141°C; [a]2D5 = ?45 (c 0.50,
CHCl3); IR (KBr) tmax: 758, 930, 1037, 1251, 1497, 1653,
1
1738, 2358, 2963, and 3304 cm-1; H NMR (300 MHz,
CDCl3): d ppm 0.91–0.95 (3H, d, J = 6.7 Hz), 0.95–1.00
(3H, d, J = 6.9 Hz), 2.13–2.27 (1H, m), 3.76 (3H, s),
4.65–4.73 (1H, m), 5.96–5.99 (3H, m), 5.95–6.01 (1H, d,
J = 14.9 Hz), 6.62–6.72 (1H, dd, J = 15.4, 10.5 Hz),
6.74–6.81 (2H, m), 6.81–6.91 (1H, dd, J = 8.1, 1.3 Hz),
6.97 (1H, br d, J = 1.3 Hz), 7.31–7.41 (1H, dd J = 14.9,
10.5 Hz). 13C NMR (75 MHz, CDCl3): d 17.8, 18.9, 31.5,
52.1, 57.0, 101.3, 105.7, 108.5, 122.4, 122.6, 124.4, 130.7,
123

44
Med Chem Res (2012) 21:38–46
(R)-Dimethyl-2-((2E,4E)-5-(benzo[d][1,3]dioxol-6-
yl)penta-2,4-dienamido)succinate (4i)
(R)-Methyl-2-((2E,4E)-5-(benzo[d][1,3]dioxol-6-yl)penta-
2,4-dienamido)-3-phenylpropanoate (4l)
It is a yellow solid, mp.154–156°C; [a]2D5 = ?26 (c 0.50,
CHCl3); IR (KBr) tmax: 999, 1248, 1345, 1495, 1649, 1738,
2851, 2920, and 3450 cm-1; 1H NMR (300 MHz, CDCl3):
d ppm 2.88–2.98 (1H, dd, J = 17.3, 4.5 Hz), 3.06–3.15
(1H, dd, J = 17.3, 4.1 Hz), 3.70 (3H, s), 3.78 (3H, s),
4.94–5.01 (1H, m), 5.95 (NH, br s), 5.98–6.00 (2H, s),
6.54–6.59 (1H, d, J = 7.9 Hz), 6.3–6.74 (1H, dd, J = 15.4,
10.5 Hz), 6.76–6.83 (2H, m), 6.88–6.93 (1H, dd, J = 8.1,
1.5 Hz), 6.98–7.00 (1H, d, J = 1.5 Hz), 7.34–7.43 (1H, dd,
J = 14.9, 10.3 Hz). 13C NMR (75 MHz, CDCl3): d 36.1,
48.6, 51.9, 52.8, 101.3, 105.7, 108.5, 122.1, 122.7, 124.5,
130.7, 139.6, 142.0, 148.2, 148.3, 165.6, 171.2, 171.6;
HRESIMS m/z 362.1234 [M? ? H], calcd for C18H19NO7
362.1234.
It is a yellow solid, mp. 118–120°C; [a]2D5 = ?148 (c 0.50,
CHCl3); IR (KBr) tmax: 807, 927, 1036, 1196, 1348, 1441,
1533, 1646, 1735, 2338, 2946, 3027, 3305, and 3733 cm-1
;
1H NMR (300 MHz, CDCl3): d ppm 3.16–3.23 (2H, t,
J = 5.3 Hz), 3.75 (3H, s), 4.98–5.05 (1H, m), 5.88–5.95
(1H, d, J = 14.9 Hz), 5.96–6.01 (3H, m) 6.62–6.74 (1H,
dd, J = 15.4, 10.7 Hz), 6.76–6.86 (2H, m), 6.87–6.93 (1H,
dd, J = 8.3, 1.8 Hz), 6.97–7.03 (1H, dd, J = 9.6, 1.7 Hz),
7.27–7.31 (5H, m), 7.32–7.42 (1H, dd, J = 14.9, 10.5 Hz).
13C NMR (75 MHz, CDCl3): d 37.9, 52.3, 53.2, 101.3,
105.7, 108.5, 122.2, 122.6, 124.4, 126.9, 128.5, 129.2,
130.6, 135.8, 139.4, 141.9, 148.2, 165.5, 172.1; HRESIMS
m/z 380.1482 [M? ? H], calcd for C22H21NO5 380.1492.
(S)-Methyl-2-((2E,4E)-5-(benzo[d][1,3]dioxol-6-yl)penta-
2,4-dienamido)-3-phenylpropanoate (4m)
(S)-Dimethyl-2-((2E,4E)-5-(benzo[d][1,3]dioxol-6-
yl)penta-2,4-dienamido)succinate (4j)
It is a yellow solid, mp. 123–125°C; [a]2D5 = -28 (c 0.25,
CHCl3); IR (KBr) tmax: 811, 997, 1036, 1149, 1251, 1369,
It is a white solid, mp.150–152°C; [a]2D5 = -20 (c 0.50,
CHCl3); IR (KBr) tmax: 850, 929, 1036, 1153, 1246, 1367,
1493, 1597, 1648, 1734, 2336, 2920, 3044, 3288, and
3729 cm-1; 1H NMR (300 MHz, CDCl3): d ppm 2.88–2.97
(1H, dd, J = 17.3, 4.5 Hz), 3.06–3.15 (1H, dd, J = 17.3,
4.1 Hz), 3.70 (3H, s), 3.78 (3H, s), 4.92–5.01 (1H, m),
5.94–6.01 (3H, m), 6.54–6.59 (1H, br d, J = 7.9 Hz),
6.63–6.74 (1H, dd, J = 15.4, 10.5 Hz), 6.77–6.85 (2H, m),
6.88–6.93 (1H, d, J = 8.1, 1.7 Hz), 6.99 (1H, d, J = 1.7
Hz), 7.33–7.43 (1H, dd, J = 14.9, 10.5 Hz). 13C NMR (75
MHz, CDCl3): d 36.1, 48.6, 51.9, 52.8, 101.3, 105.7, 108.5,
122.1, 122.7, 124.5, 130.7, 139.6, 142.0, 148.2, 148.3,
165.6, 171.2, 171.6; HRESIMS m/z 362.1236 [M? ? H],
calcd for C18H19NO7 362.1234.
1494, 1532, 1652, 1741, 2853, 2921, 3029, and 3299 cm-1
;
1H NMR (300 MHz, CDCl3): d ppm 3.16–3.22 (2H, t,
J = 5.4 Hz), 3.75 (3H, s), 4.96–5.05 (1H, m), 5.88–5.94
(1H, d, J = 14.7 Hz), 5.91–5.96 (NH, br d, J = 7.5 Hz),
5.99 (2H, br s), 6.62–6.72 (1H, dd, J = 15.4, 10.5 Hz),
6.77–6.80 (1H, d, J = 8.3 Hz), 6.76–6.82 (1H, d, J = 15.4
Hz), 6.88–6.92 (1H, dd, J = 8.1, 1.7 Hz), 6.98 (1H, d,
J = 1.5 Hz), 7.08–7.13 (2H, dd, J = 7.9, 1.8 Hz),
7.24–7.34 (3H, m), 7.32–7.42 (1H, dd, J = 14.7, 10.5 Hz).
13C NMR (75 MHz, CDCl3): d 37.9, 52.3, 53.2, 101.3,
105.7, 108.5, 122.1, 122.7, 124.3, 127.0, 128.6 (2), 128.7,
129.2 (2), 130.9, 135.8, 139.5, 141.8, 165.4, 172.0; HRE-
SIMS m/z 380.1485 [M? ? H], calcd for C22H21NO5
380.1492.
(S)-Methyl-2-((2E,4E)-5-(benzo[d][1,3]dioxol-6-yl)penta-
2,4-dienamido)-2-phenylacetate (4k)
(R)-Methyl-2-((2E,4E)-5-(benzo[d][1,3]dioxol-6-yl)penta-
2,4-dienamido)-3-(4-hydroxyphenyl)propanoate (4n)
It is a pale yellow solid, mp. 165–167°C; [a]2D5 = -41
(c 0.50, CHCl3); IR (KBr) tmax: 809, 929, 1040, 1255,
1369, 1496, 1529, 1644, 1736, 2782, 2890, 3026, and 3311
It is a yellow solid, mp. 157–160°C; [a]2D5 = ?54 (c 0.50,
CHCl3); IR (KBr) tmax: 804, 924, 1035, 1148, 1348, 1443,
1
1
cm-1; H NMR (300 MHz, CDCl3): d ppm 3.75 (3H, s),
1599, 1646, 1720, 2895, 2949, 3026, and 3374 cm-1; H
5.67–5.72 (1H, d, J = 7.3 Hz), 5.94–6.03 (1H, d, J = 14.6
Hz), 5.98 (2H, s), 6.49–6.66 (1H, dd, J = 15.4, 10.2 Hz),
6.72–6.76 (2H, m), 6.80 (1H, br s), 6.87–6.92 (1H, dd,
J = 8.0, 2.2 Hz), 6.96–6.99 (1H, d, J = 1.4 Hz), 7.30–7.44
(6H, m). 13C NMR (75 MHz, CDCl3): d 52.8, 56.4, 101.4,
105.6, 108.6, 122.0, 122.7, 124.5, 127.2, 128.5, 129.0,
130.6, 136.7, 139.7, 142.1, 148.0, 148.3, 165.3, 171.4;
HRESIMS m/z 366.1340 [M? ? H], calcd for C21H19NO5
366.1336.
NMR (300 MHz, CDCl3): d ppm 3.03–3.19 (2H, m), 3.75
(3H, s), 4.93–5.00 (1H, m), 5.88–5.95 (1H, d, J = 14.9
Hz), 5.98–5.99 (2H, br s), 5.99–6.02 (1H, br d, J = 7.5
Hz), 6.61–6.71 (1H, dd, J = 15.4, 10.5 Hz), 6.72 –6.74
(1H, br s), 6.85–6.78 (2H, m), 6.79–6.82 (1H, m),
6.88–6.92 (1H, dd, J = 8.1, 1.5 Hz), 6.93–6.99 (3H, m),
7.32–7.41 (1H, dd, J = 14.9, 10.5 Hz); 13C NMR (75
MHz, CDCl3): d 29.7, 52.2, 53.2, 96.2, 101.4, 105.6, 108.6,
115.4, 122.3, 122.7, 124.5, 130.0, 139.5, 142.0, 148.3,
123

Med Chem Res (2012) 21:38–46
45
1
151.6, 154.8, 165.5, 172.1; HRESIMS m/z 396.1451
[M? ? H], calcd for C22H21NO6 396.1442.
1606, 1652, 1738, 2361, 2950, and 3289 cm-1; H NMR
(300 MHz, CDCl3): d ppm 3.34–3.41 (2H, m), 3.68–3.70
(3H, s), 5.02–5.10 (1H, m), 5.80–5.88 (1H, d, J = 15.1 Hz),
5.96–5.98 (2H, br s), 6.09–6.17 (1H, d, J = 7.5 Hz),
6.56–6.67 (1H, dd, J = 15.1, 10.5 Hz), 6.71–6.84 (2H, m),
6.85–6.91 (1H, d, J = 8.3 Hz), 6.95–6.99 (1H, d, J = 1.5
Hz), 7.04–7.23 (3H, m), 7.25–7.40 (2H, m), 7.51–7.57 (1H,
d, J = 7.5 Hz), 8.26–8.31 (1H, br s). 13C NMR (75 MHz,
CDCl3): d 27.7, 52.4, 53.1, 101.3, 105.7, 108.5, 110.5, 111.2,
118.6, 119.6, 122.1, 122.3, 122.7, 122.9, 124.4, 127.6, 130.7,
136.1, 139.4, 141.7, 148.2, 165.6, 172.3; HRESIMS m/z
419.1607 [M? ? H], calcd for C24H22N2O5 419.1601.
(R)-Methyl-2-((2E,4E)-5-(benzo[d][1,3]dioxol-6-yl)penta-
2,4-dienamido)-3-(2,4-dihydroxyphenyl)propanoate (4o)
It is a yellow solid, mp. 140–142°C; IR (KBr) tmax: 809,
928, 1037, 1116, 1250, 1444, 1604, 1732, 2852, 2922, and
3361 cm-1; 1H NMR (300 MHz, CDCl3): d ppm 3.08–3.18
(2H, m), 3.73 (3H, s), 4.89–5.03 (1H, m), 5.89–6.03 (4H,
m), 6.06–6.20 (1H, m), 6.62–7.06 (7H, m), 7.28–7.41 (1H,
dd, J = 14.5, 10.7 Hz). 13C NMR (75 MHz, CDCl3): d
32.5, 52.1, 56.3, 100.8, 101.4, 105.2, 105.8, 108.9, 118.2,
120.9, 121.9, 126.1, 131.4, 131.9, 136.5, 140.4, 147.4,
147.9, 154.8, 156.8, 165.2, 171.9; HRESIMS m/z 412.1375
[M? ? H], calcd for C22H21NO7 412.1391.
Acknowledgments Authors gratefully acknowledge keen interest of
Dr. J. S. Yadav, Director, IICT, Hyderabad, India. RSR, GSK and
RRR thank CSIR, New Delhi for financial support.
(R)-Methyl-2-((2E,4E)-5-(benzo[d][1,3]dioxol-6-yl)penta-
2,4-dienamido)-3-(1H-imidazol-5-yl)propanoate (4p)
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1H NMR (300 MHz, CDCl3): d ppm 3.05–3.24 (2H, m),
3.75 (3H, s), 5.00 (1H, m), 5.90–6.08 (4H, m), 6.69–6.93
(4H, m), 6.96–7.09 (3H, m), 7.32–7.40 (1H, dd, J = 14.5,
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(R)-Methyl-1-((2E,4E)-5-(benzo[d][1,3]dioxol-6-yl)penta-
2,4-dienoyl)pyrrolidine-2-carboxylate (4q)
It is a pale yellow solid, mp. 115–117°C; [a]2D5 = ?48
(c 1.0, CHCl3); IR (KBr) tmax: 929, 1038, 1250, 1444,
1
1643, 1740, 2852, and 2921 cm-1; H NMR (200 MHz,
CDCl3): d ppm 1.86–2.31 (4H, m), 3.55–3.72 (2H, m), 3.74
(3H, s), 4.50–4.64 (1H, m), 5.97 (2H, s), 6.20–6.35 (1H, d,
J = 14.6 Hz), 6.71–6.84 (3H, m), 6.87–6.95 (1H, dd,
J = 8.0, 1.4 Hz), 6.98–7.01 (1H, br s), 7.38–7.53 (1H, dd,
J = 14.6, 10.2 Hz). 13C NMR (75 MHz, CDCl3): d 24.8,
29.2, 46.8, 52.1, 59.0, 101.3, 105.6, 108.4, 120.3, 122.7,
125.0, 130.8, 139.3, 143.0, 148.2, 165.2, 172.8; HRE-
SIMS m/z 330.1335 [M? ? H], calcd for C18H19NO5
330.1336.
(R)-Methyl-2-((2E,4E)-5-(benzo[d][1,3]dioxol-6-yl)penta-
2,4-dienamido)-3-(1H-indol-3-yl)propanoate (4r)
It is a yellow solid, mp. 118–120°C; [a]2D5 = ?111 (c 0.50,
CHCl3); IR (KBr) tmax: 805, 927, 1036, 1149, 1250, 1490,
123

46
Med Chem Res (2012) 21:38–46
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