Ultrasound-assisted bismuth nitrate-induced green synthesis of novel pyrrole derivatives and their biological evaluation as anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2012.01.055

A series of novel N-substituted pyrrole derivatives have been designed and synthesized following ultrasound-assisted and bismuth nitrate-catalyzed eco-friendly route. This reaction has also provided a general method to prepare diverse varieties of N-substituted pyrroles with less nucleophilic polyaromatic amines. Based on ¹H NMR spectroscopy, a plausible mechanistic pathway has been advanced. Cytotoxicity of some selected N-substituted pyrrole derivatives has been evaluated in vitro in a panel of mammalian cancer cell lines which includes liver cancer cell lines (HepG2 and Hepa1-6), colon cancer cell lines (HT-29 and Caco-2), a cervical cancer cell line (HeLa) and NIH3T3 cells. Two compounds, 5-(1H-pyrrol-1-yl)-1,10-phenanthroline (9) and 1-(phenanthren-2-yl)-1H-pyrrole (10) have shown good cytotoxicity against some cancer cell lines. Furthermore, these compounds have exhibited cytotoxic specificity against liver cancer cell lines in vitro when compared with normal cultured primary hepatocytes.

Full text of DOI:10.1016/j.ejmech.2012.01.055

European Journal of Medicinal Chemistry 50 (2012) 209e215
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Ultrasound-assisted bismuth nitrate-induced green synthesis of novel pyrrole
derivatives and their biological evaluation as anticancer agents
a
a
b
b,c,
Debasish Bandyopadhyay , Sanghamitra Mukherjee , Jose C. Granados , John D. Short **,
Bimal K. Banik
a
a,
*
Department of Chemistry, The University of Texas-Pan American, 1201 West University Drive, Edinburg, TX 78539, USA
University of Texas Health Science Center at San Antonio-Regional Academic Health Center, Medical Research Division, 1214 West Schunior Street, Edinburg, TX 78541, USA
Department of Pharmacology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 October 2011
Received in revised form
A series of novel N-substituted pyrrole derivatives have been designed and synthesized following
ultrasound-assisted and bismuth nitrate-catalyzed eco-friendly route. This reaction has also provided
a general method to prepare diverse varieties of N-substituted pyrroles with less nucleophilic poly-
aromatic amines. Based on 1H NMR spectroscopy, a plausible mechanistic pathway has been advanced.
Cytotoxicity of some selected N-substituted pyrrole derivatives has been evaluated in vitro in a panel of
mammalian cancer cell lines which includes liver cancer cell lines (HepG2 and Hepa1-6), colon cancer cell
lines (HT-29 and Caco-2), a cervical cancer cell line (HeLa) and NIH3T3 cells. Two compounds,
25 January 2012
Accepted 27 January 2012
Available online 3 February 2012
Keywords:
Pyrrole
5-(1H-pyrrol-1-yl)-1,10-phenanthroline (9) and 1-(phenanthren-2-yl)-1H-pyrrole (10) have shown good
cytotoxicity against some cancer cell lines. Furthermore, these compounds have exhibited cytotoxic
specificity against liver cancer cell lines in vitro when compared with normal cultured primary
hepatocytes.
Ultrasound
Bismuth nitrate
Polyaromatic
Anticancer
Cytotoxicity
Ó 2012 Published by Elsevier Masson SAS.
1. Introduction
for the synthesis of pyrroles is the PaaleKnorr reaction [25,26].
Previous studies from our group have focused on the synthesis of
According to the World Health Organization, cancer is a leading
cause of death worldwide that accounted for 7.6 million deaths in
polyaromatic compounds and their biological activities as anti-
cancer agents [27e32]. On this basis, we have become interested in
the synthesis of pyrroles bound to polyaromatic skeletons and their
potential use as anticancer agents.
2
008 [1]. In addition to the extensive use of irradiation and surgical
treatment, chemotherapy still plays an important role for the
treatment of cancer. In this context, the search for novel chemo-
therapeutic agents is an interesting and continually evolving field
of cancer research.
Pyrroles are found to be as the fundamental structural motifs in
various classes of natural and biologically important molecules
such as porphyrins, bile pigments, coenzymes, and alkaloids [2e7].
This moiety is also present in several synthetic pharmaceuticals as
well as electrically conducting polymers [8e10]. Consequently,
many methods for the synthesis of diversely substituted pyrroles
have been developed [11e24]. However, the most reliable method
Herein we report a simple ultrasound-assisted eco-friendly
practical method for the synthesis of N-substituted pyrroles by
reacting 2,5-dimethoxytetrahydrofuran (1) with various amines in
the presence of catalytic amounts (5 mol%) of bismuth nitrate
pentahydrate under solvent-free conditions. This reaction produces
pyrroles in excellent yield and within a short period of time. Two of
these pyrrole conjugated derivates have caused cytotoxicity in
multiple cancer cell lines in vitro, and have demonstrated cytotoxic
specificity against liver cancer cell lines in vitro when compared
with normal cultured primary hepatocytes.
2
. Results and discussion
*
Corresponding author. Tel.: þ1 956 6658741; fax: þ1 956 3845006.
** Corresponding author. University of Texas Health Science Center at San
2.1. Chemistry
Antonio-Regional Academic Health Center, Medical Research Division, 1214
West Schunior Street, Edinburg, TX 78541, USA. Tel.: þ1 956 6658741;
fax: þ1 956 3845006.
In recent years organic reactions in solvent-free condition have
E-mail addresses: shortj@uthscsa.edu (J.D. Short), banik@utpa.edu (B.K. Banik).
received tremendous priority in view of green methodology
0223-5234/$ e see front matter Ó 2012 Published by Elsevier Masson SAS.
doi:10.1016/j.ejmech.2012.01.055

210
D. Bandyopadhyay et al. / European Journal of Medicinal Chemistry 50 (2012) 209e215
[
33,34]. In view of the fact that PaaleKnorr procedure requires
irradiation. Demethylation should take place in compound 1 only
because electrophilic reagent bismuth nitrate should attack
compound 1. There is sufficient scope to use this reaction for the
synthesis of several biologically active compounds as described
herein.
Our method has been tested with a wide range of amines and no
side products are identified in any of these examples. In the case of
adamantanyl amine (Entry 6, Table 2), there was a possibility of
molecular rearrangement in the presence of Lewis acid but inter-
estingly no rearranged product was observed. The corresponding
product viz. 1-(adamantan-1-yl)-1H-pyrrole was isolated in good
(79%) yield. The spectral and m.p. data were identical with our
previous reports [15e17]. The structure is confirmed by X-ray
crystallographic analysis [37]. The ORTEP projection is shown in
Fig. 1.
a 1,4-dicarbonyl compound and Lewis acid as catalyst, we predict
that our work on bismuth nitrate-catalyzed [35,36] reactions on
acetal and glycosylation may prove useful for the success of the
reaction if 2,5-dimethoxytetrahydrofuran (1) is selected as one of
the reactants. This concept has been tested and we disclose
herein the preparation of a number of new pyrroles using ultra-
sound irradiation. Ultrasonic exposure of amine (2) with
2
,5-dimethoxytetrahydrofuran (1) in the presence of catalytic
amount (5 mol%) of bismuth nitrate pentahydrate produced
excellent yield of the corresponding pyrroles (3) (Scheme 1).
The methodology has also been successfully examined with
a number of amines. First of all, the amount of the catalyst viz.
Bi(NO
2
3 3 2
) $5H O was optimized by reacting aniline (1 mmol) with
,5-dimethoxytetrahydrofuran (1.2 mmol) under ultrasound irra-
diation at room temperature for 5 min. The results are presented in
Table 1. This data clearly shows that the presence of 5 mol% of
The reaction between 1 and 2 can give pyrrole (about 55%) in the
absence of bismuth nitrate by irradiating the reaction mixture for
a long time (approximately 5 h). Multicyclic aromatic amines failed
to yield the products in the absence of catalyst. However, the
reaction gives products when ultrasound was used (Table 2). The
presence of small amounts of bismuth nitrate (5 mol%) is necessary
for the success of the reaction. This method has also been applied
with other bismuth-based reagents like bismuth chloride, bismuth
subnitrate, bismuth oxide and bismuth bromide in catalytic
proportion (5 mol%). Stannic chloride, zinc chloride and boron
trifluoride etherate failed to improve the yield of the products. The
best results have been observed with bismuth nitrate as the
catalyst.
Bi(NO
99%) yield.
Diverse
3
)
3
$5H
2
O is necessary for a successful reaction in excellent
(
amines (for example, aromatic, aliphatic,
polyaromatic and heteropolyaromatic amines) react with 2,5-
dimethoxytetrahydrofuran (1) in presence of catalytic amount
(
5 mol%) of bismuth nitrate pentahydrate under ultrasound irra-
diation to give excellent yield of the corresponding pyrroles. The
results are depicted in Table 2.
Notably, our current method is better than the existing methods
3 3 2
because we have not used any solvent. Our catalyst Bi(NO ) $5H O
is eco-friendly and non-toxic. According to our scientific knowl-
edge, this solvent-free method is superior to the method where
acetic acid was used as solvent. Most importantly, we could not
prepare pyrroles by reacting polyaromatic amines and 1 in the
presence of acetic acid. These amines are not nucleophilic enough
to undergo condensation with a carbonyl group unless there is
appropriate activation. Acetic acid was used for the preparation of
pyrroles under drastic conditions with amines that have much
higher basicity and nucleophilicity. In contrast, our method with
non-toxic bismuth nitrate worked very well with different types of
amines, even with amines that are very weakly nucleophilic in
nature.
Based on the nature of the substrate, catalyst and condition of
the experiment, a most probable mechanism is advanced. The
methoxy groups in 1 can undergo a deprotection reaction under
mild acidic conditions and this process can be highly facilitated by
ultrasound irradiation. The intermediate can easily form the reac-
tive dialdehyde 5. The reactive dialdehyde 5 on reaction with
amines can lead to pyrroles 3 following a nucleophilic addition and
subsequent dehydrationearomatization route. This reaction
suggests the capability of bismuth nitrate to serve as a Lewis acti-
vator. Proton NMR spectroscopy has been used to strengthen the
2.2. Biology
As a part of our research to synthesize novel polyaromatic
anticancer agents, biological assays for the four pyrrole containing
polyaromatic compounds viz. 1-(chrysen-6-yl)-1H-pyrrole (7)
(Entry 7, Table 2), 1-(pyren-1-yl)-1H-pyrrole (8) (Entry 8, Table 2),
5-(1H-pyrrol-1-yl)-1,10-phenanthroline (9) (Entry 9, Table 2) and
1-(phenanthren-2-yl)-1H-pyrrole 10 (Entry 10, Table 2) have been
carried out. Due to its fused ring structure as well as puckered
configuration 1-(adamantan-1-yl)-1H-pyrrole (6) (Entry 6, Table 2)
was also included in that series.
The effect of pyrrole conjugated compounds (6e10) on spectral
properties has been studied. Previous reports [38e40] have iden-
tified that the peak emission wavelength of both pyrene and
chrysene is less than 400 nM. Therefore, we sought to determine
the effects of the addition of a pyrrole to these PAH molecules
would affect the absorption and spectral emission patterns of these
molecules. In addition, we sought to determine the absorbance and
spectral emission patterns after conjugation of a pyrrole moiety to
either polyaromatic or adamantyl scaffold. No significant absor-
bance could be detected for any of the pyrrole conjugated
compounds, and no spectral emission could be detected for the
proposed mechanism as in Scheme 2.
1
Upon irradiating a CDCl
3
solution of 1 for 5 min, H NMR has
been taken. A downfield signal due to the eCHO group is observed.
The intensity of the eCHO group becomes more predominant in the
Table 1
1
H NMR when 1 was irradiated in CDCl
3
in the presence of catalytic
Bi(NO
mmol) and 2,5-dimethoxytetrahydrofuran (1.2 mmol) for
temperature: optimization of the amount of catalyst.
3 3 2
) $5H O catalyzed ultrasound-assisted synthesis of pyrrole using aniline
(1
5
min at room
amounts of bismuth nitrate. This suggests the formation of 5 in the
reaction media in the presence of bismuth nitrate under ultrasonic
Entry
3 3 2
Bi (NO ) $5H O (mol %)
Yield (%)a
1
2
3
4
5
6
7
8
30
25
20
15
10
5
89
91
91
94
97
99
57
33
2
1
3 3 2
Scheme 1. Bi(NO ) .5H O catalyzed ultrasound-induced synthesis of N-substituted
pyrroles.
a
Isolated yield.

D. Bandyopadhyay et al. / European Journal of Medicinal Chemistry 50 (2012) 209e215
211
Table 2
Ultrasound-induced Bi(NO
Scheme 1.
3
)
3
$5H
2
O
catalyzed synthesis of pyrroles following
Entry
Amine
Product
Time (min)
5
Yield(%)a
1
99
Scheme 2. Ultrasound-assisted bismuth nitrate-catalyzed synthesis of pyrroles:
plausible mechanism of the reaction.
2
5
95
3
4
5
5
92
95
5
6
7
30
35
10
87
79
92
Fig. 1. X-ray crystallographic structure (ORTEP projection) of 1-(adamantan-1-yl)-1H-
pyrrole (6).
pyrrole conjugated adamantyl derivative (6), the pyrrole conju-
gated heteropolyaromatic compound (9), or most of the pyrrole
conjugated PAH derivatives including chrysene (7) and phenan-
threne (10) in this study.
However, we were able to detect a fluorescence emission
spectrum for the pyrrole conjugated pyrene compound (8), with
a peak emission occurring between 460 nM and 470 nM (Fig.1). The
shoulders for this emission peak were at 420 nM and 550 nM
(Fig. 2). This data suggests that the addition of pyrrole to pyrene
8
9
15
87
60
76
86
1
0
5
a
Isolated yield.
Fig. 2. Emission spectrum of the pyrene derivative 8: The compound 8 (50 mM) was
diluted in PBS and excited at a wavelength of 350 nM.

212
D. Bandyopadhyay et al. / European Journal of Medicinal Chemistry 50 (2012) 209e215
Table 3
Estimated IC50 values (mM) for the compounds (6e10) in a small panel of mammalian cell lines.
Compounds
HepG2
Hepa1-6
Caco-2
HT-29
HeLa
NIH3T3
6
7
8
9
38.6 ꢀ 11.5
>50
19.9 ꢀ 6.1
>50
>50
>50
ND
11.9 ꢀ 1.0
>50
24.3 ꢀ 0.7
4.2 ꢀ 0.5
>50
12.9 ꢀ 5.9
>50
17.7 ꢀ 9.9
27.9 ꢀ 20.7
>50
24.0 ꢀ 18.5
>50
ND
1.9 ꢀ 1.5
2.1 ꢀ 1.3
8.5
a
0.7 ꢀ 0.8
b
>50
10.7 ꢀ 0.4
3.4 ꢀ 0.4
3.9 ꢀ 0.3
4.0
a
3.0 ꢀ 1.6
10
13.4 ꢀ 4.8
>50
Cisplatin
7.0
10.8a
16.8
11.7
a
Only reduced cell viability to a minimum of w45e50%.
Not determined.
b
causes a red-shift in its fluorescent emission spectrum but ablates
the fluorescent emission when conjugated to a chrysene molecule.
It has been found [30,41,42] that certain derivatives of poly-
aromatic hydrocarbons (PAHs), including pyrene and chrysene
derivatives, reduce the viability of transformed cell lines, and some
of these PAH derivatives have been reported to reduce cell viability
by induction of apoptosis [42,43]. Therefore, we tested the effects of
compounds 6e10 on the viability of a small panel of human and
mouse cell lines, which included liver cancer cell lines (HepG2 and
Hepa1-6), colon cancer cell lines (HT-29 and Caco-2), a cervical
cancer cell line (HeLa), and NIH3T3 cells. Although Caco-2 cells
were resistant to the effects of all of these pyrrole conjugated
compounds, each pyrrole conjugated compound was capable of
reducing viability of the other cell types (Table 3).
value of 13 mM or lower (Table 3). Furthermore, compound 9 and
compound 10 mediated reduction in viability occurred between 24
and 48 h after treatment, since compounds 9 and 10 (when used at
twice the calculated IC50 concentrations) caused a >50% decrease in
viable HepG2 cells after 48 and 72 h treatments but only about a 20%
decrease after a 24 h treatment (Fig. 3).
Since compounds 9 and 10 were the most potent compounds for
reducing cell viability in vitro, we sought to monitor their effects on
anchorage-independent growth of HepG2 cells. Both of these
compounds inhibited anchorage-independent growth greater than
60% when cells were treated with 25
mM of compound and greater
than 80% when used at a lower concentration of 12.5
mM (Fig. 4).
We also sought to determine whether compounds 9 and 10
reduced cell viability by inducing apoptosis. We examined whether
these two compounds would lead to increased Caspase 3/7 activity
or increased DNA fragmentation (as measured by a TUNEL assay) in
HepG2 cells. However, we did not detect an increase in either of
these features of apoptosis (data not shown). Taken together, these
data suggest that the ability of compounds 9 and 10 to reduce cell
viability occurs through an apoptosis-independent mechanism.
Since compounds 9 and 10 were the most potent pyrrole
conjugated compounds for reducing cell viability in vitro and in
anchorage-independent growth assays, we next asked whether
these compounds would be less effective at reducing the viability of
non-cancerous cells in vitro (i.e., exhibit tumor cell specificity). We
examined the effects of increasing dosages of compounds 9 and 10
on the viability of Hepa1-6 cells and normal primary hepatocytes.
As stated in Table 3, compounds 9 and 10 were both capable of
The addition of pyrrole to a chrysene or pyrene molecule, 7 and
8
, respectively, were the least effective in reducing cell viability in
this small panel of cell lines as the majority of cell lines had an
estimated IC50 value greater than 50 M in most cell lines when
treated with these compounds. Although compound had
a calculated IC50 value of less than 1 M when used to treat Hepa1-
cells, the maximum reduction in Hepa1-6 cell viability for this
compound was only w50%, even when used at a dosage of 50 M,
m
7
m
6
m
suggesting that this compound may be inducing cytostasis but not
cytotoxicity in this cell line.
In contrast, other pyrrole derived compounds did cause cyto-
toxicity in multiple cell lines in vitro. Compound 8 effectively
decreased the viability in Hepa1-6, HT-29, and HeLa cells with an
estimated IC50 values of 24 mM or lower, and the pyrrole conjugated
adamantane molecule, 6, reduced the viability of all cell lines tested,
reducing Hepa1-6 cell viability at doses 2.5 mM and 5 mM (Fig. 4).
other than Caco-2 and HepG2, with an estimated IC50 value less than
However, neither compound 9 nor compound 10 was capable of
2
4
m
M (Table 3). Most importantly, compounds with addition of
a pyrrole to heteropolyaromatic and phenanthrene molecules (9 and
0, respectively) showed the most significant potency for reduction
reducing viability of normal primary hepatocytes, even when used
at dosages of 10
mM as shown in Fig. 5 (A & B). Therefore,
1
compounds 9 and 10 showed distinct inhibitory effects on viability
of HepG2, Hepa1-6, and NIH3T3 cell viability, with an estimated IC50
of cancer cells when compared with normal cells.
Fig. 3. The effects of pyrrole conjugated compounds (9 and 10) on the viability of
HepG2 cells in a time-dependent manner. HepG2 cells were treated with compounds 9
and 10 at 2ꢁ IC50 concentrations for 24, 48, and 72 h, and then cell viability was
analyzed using SRB assay. A representative experiment of three independent experi-
ments performed in duplicates is shown. Each data point represents the
average ꢀ standard deviation value.
Fig. 4. Effect of pyrrole conjugated compounds (9 and 10) on anchorage-independent
growth of HepG2 cells. HepG2 cells, grown in soft agar, were treated with the indicated
doses of pyrrole conjugated compounds for 7 day as described in Materials and
Methods. A representative experiment is shown and each dose of compound was
administered in duplicate e the standard deviation bars are shown.

D. Bandyopadhyay et al. / European Journal of Medicinal Chemistry 50 (2012) 209e215
213
DNA fragmentation [42,43]; however, features of apoptosis were
not observed upon treatment of liver cancer cell lines with either
compound 9 or 10 as part of this study. These data suggest that
N-substituted pyrroles may have a different mechanism of cyto-
toxicity when compared with other polyaromatic derivatives. In
summary, some promising anticancer pyrrole derivatives have
been identified, and the method as reported herein may find
applications in other areas of research. The mechanism of action
of these new compounds is being evaluated and will be reported
in due course.
4
4
4
. Experimental protocols
.1. Chemistry
.1.1. General
Melting points were determined in a Fisher Scientific electro-
chemical Mel-Temp* manual melting point apparatus (Model 1001)
ꢂ
equipped with a 300 C thermometer. Elemental analyses (C, H, N)
were conducted using the PerkineElmer 2400 series II elemental
analyzer, their results were found to be in good agreement (ꢀ0.2%)
with the calculated values for C, H, N. Sonication was performed in
B5510-DTH, Branson ultrasonic cleaner (Model-5510, frequency of
42 kHz and an output power of 135 Watts) with digital timer,
heater, temperature monitor and degas. Inside tank dimensions are
0
0
00
00
1
2
1.5 ꢁ 9.5 ꢁ 6 (length ꢁ width ꢁ height) with a fluid capacity of
Fig. 5. Inhibitory effects of pyrrole conjugated compounds (9 and 10) on the viability
of normal mouse hepatocytes (Mouse CD-1 cells) and mouse hepatoma cells (Hepa1-
.5 gallons. FT-IR spectra were registered on a Bruker IFS 55
Equinox FT-IR spectrophotometer as KBr discs. H NMR (300 MHz)
and C NMR (75.4 MHz) spectra were obtained at room tempera-
ture with JEOL Eclipse-300 equipment using TMS as internal
1
6
). Normal (Mouse CD-1) and cancer (Hepa1-6) cells were treated with the indicated
concentrations of compounds 9 (A) and 10 (B) for 48 h, and then cell viability
normalized to non-treated cells) was measured using MTS assay. Date denotes the
1
3
(
average ꢀ standard deviation value of a representative experiment of two independent
standard and CDCl
3
as solvent. Bismuth nitrate pentahydrate
experiments performed in duplicates.
(reagent grade) 98% (Cat # 248592-500G, Batch # MKBC6772)
purchased from SigmaeAldrich was used. All other chemicals were
purchased from SigmaeAldrich Corporation (analytical grade).
Throughout the project solvents were purchased from Fisher
Scientific. Deionized water was used for the preparation of all
aqueous solutions.
Although many of the pyrrole conjugated polycyclic compounds
did not demonstrate low IC50 values in this study, we clearly show
that two compounds (compounds 9 and 10 in this paper) were
cytotoxic to some cell lines at doses less than 5 mM, a dosage range
that is similar to that of drugs such as Cisplatin and 5-FU in certain
cell lines. It is very crucial to note that these compounds are new
and no synthesis and biological studies of these molecules have
been investigated. Our data has confirmed selective cytotoxic
activity in important cancer cell lines without destroying the
normal cell. Despite significant progress in the development of
anticancer agents, selective and non-toxic new cytotoxic molecules
are in demand.
4.1.2. General procedure for the synthesis of pyrroles (3)
Amine 2 (1.0 mmol), 2,5-dimethoxytetrahydrofuran (1,1.2 mmol)
and bismuth nitrate pentahydrate (24 mg, 5 mol%) was irradiated in
a B5510-DTH (Branson ultrasonic cleaner; Model-5510, frequency
4
2 kHz with an output power 135 Watts), as specified in Table 2.
After completion of the reaction (monitored by TLC) diethyl ether
10 mL) was added to the reaction mixture and filtered. Pure product
was isolated from the reaction mixture after evaporation of ether.
(
3
. Conclusion
4.1.3. Spectral data
In conclusion, the present synthetic protocol allows the
Spectroscopic data for representative compounds, e.g. mono-
preparation of a variety of N-substituted pyrroles in high yields
without the use of expensive or sensitive reagents/instruments.
Due to simplicity of the procedure, products can be isolated very
easily. The novelty of the reaction is that it gives good yield (76%,
Compound 9) even if the substrate is a potential metal chelator.
A plausible mechanistic route has been suggested and cytotox-
icity of some selected compounds was evaluated in vitro in
a panel of mammalian cancer cell lines which included liver
cancer cell lines (HepG2 and Hepa1-6), colon cancer cell lines
aromatic (Entries 1 and 2, Table 2), benzylic (Entry 5, Table 2),
alicyclic (Entry 6, Table 2), polyaromatic (Entries 7 and 8, Table 2) as
well as heteropolyaromatic (Entry 9, Table 2) are as follows:
4.1.3.1. 1-Phenyl-1H-pyrrole. (Entry 1, Table 2): Brown sticky oil. IR:
ꢃ1
1
2923, 1312, 1106, 782, 611 cm
; H NMR d (ppm): 6.39 (m, 2H,
pyrrole), 7.24 (d, 2H, J ¼ 3.12 Hz, pyrrole), 7.42e7.53 (m, 5H, AreH);
1
3
C NMR
d
(ppm): 109.07 (2C), 115.51 (2C), 122.96 (2C), 124.57,
N: C, 83.88; H, 6.34; N,
128.62 (2C), 137.08. Anal. Calcd. for C10
H
9
(
HT-29 and Caco-2), a cervical cancer cell line (HeLa) and NIH3T3
9.78. Experimental: C, 83.79; H, 6.31; N, 8.72.
cells. Compounds 9 and 10 showed good cytotoxicity against
some cancer cell lines. Furthermore, these compounds exhibited
selective cytotoxicity of hepatic cancer cell lines when compared
with normal hepatocytes in vitro. Interestingly, previous studies
have shown that polyaromatic compounds cause apoptosis in
certain cell types in vitro, as indicated by Caspase 3 activation and
4.1.3.2. 1-(4-Methoxyphenyl)-1H-pyrrole. (Entry 2, Table 2): Black
amorphous solid. Solidified from dichloromethane/hexane
ꢂ
ꢃ1 1
mixture; M.p.: 89 C; IR: 2954, 1301, 1191, 850, 748 cm ; H NMR
(ppm): 3.87 (s, 3H, OCH ), 6.33 (m, 2H, pyrrole), 6.67 (d, 2H,
J ¼ 3.30 Hz), 6.96e7.73(m, 4H, AreH); C NMR
d
3
13
d
(ppm): 55.66

214
D. Bandyopadhyay et al. / European Journal of Medicinal Chemistry 50 (2012) 209e215
(
1
OCH
58.21. Anal. Calcd. for C11
Experimental: C, 76.20; H, 6.27; N, 7.97.
3
), 109.93 (2C), 114.71 (2C), 120.15 (2C), 126.07 (2C), 132.56,
analyzed between the wavelengths of 350 nMe750 nM using
a SpectraMax M5 plate reader.
H11NO: C, 76.28; H, 6.40; N, 8.09.
4.2.2. Cell culture
4
.1.3.3. 1-Trityl-1H-pyrrole. (Entry 5, Table 2): This compound was
HepG2, Hepa1-6, NIH3T3 and HeLa cells were cultured in Dul-
becco’s Modified Eagle’s Medium (DMEM) (Invitrogen, Carlsbad,
CA) containing 10% Fetal Bovine Serum (FBS, Invitrogen); Caco-2
cells were cultured in DMEM containing 20% FBS, and HT-29 cells
were cultured in McCoy’s media (Invitrogen) containing 10% FBS.
Mouse (CD-1) fresh hepatocytes were purchased and maintained in
Williams’ E medium according to manufactures instructions (Invi-
trogen). HepG2, Hepa1-6, NIH3T3, HeLa, Caco-2, and HT-29 cell
lines were purchased from American Type Culture Collection
obtained according to above general procedure; yellowish brown
amorphous solid. M.p.: 238e240 C; IR (KBr disc.
ꢂ
ꢃ1
n
cm ): 2599,
2
8
6
337, 1594, 1476, 1423, 1322, 1261, 1222, 1182, 1073, 1033, 1001, 971,
1
61, 738, 718, 622; H NMR
d
(ppm): 6.16 (t, 2H, J ¼ 2.19 Hz, pyrrole),
.63 (t, 2H, J ¼ 2.19 Hz, pyrrole), 7.17e7.20 (m, 6H, AreH), 7.29e7.32
m, 9H, AreH); 1 C NMR
3
d
(ppm): 92.82, 107.45 (2C), 123.60 (2C),
(
127.58 (3C), 127.79 (6C), 130.13 (6C), 143.86 (3C). Anal. Calcd. for
C
23
H19N: C, 89.28; H, 6.19; N, 4.53. Experimental: C, 89.03; H, 6.08;
ꢂ
N, 4.41.
(ATCC, Manassas, VA), and all cells were incubated at 37 C with 5%
2
CO .
4.1.3.4. 1-(Adamntan-1-yl)-1H-pyrrole. (Entry 6, Table 2): Yellow
crystals. Crystallized from diethyl ether/hexane mixture; M.p.:
4.2.3. Cell viability assays
Cells were plated (5000 cells/well) onto a 96-well dish and
incubated overnight at 37 C. The following day, cells were treated
with increasing dosages (0.1 mMe100 mM) of each pyrrole conju-
gated compound, which had been dissolved in DMSO. The DMSO
concentration of treatments was limited to 0.25%, and cells were
ꢂ
ꢃ1 1
;
71
C; IR: 2923, 2855, 1479, 1450, 1219, 713, 619 cm
H NMR
ꢂ
d
(ppm): 1.77 (m, 3H), 2.12 (d, 6H, J ¼ 3.00), 2.23 (m, 6H), 6.19 (t, 2H,
13
J ¼ 2.19 Hz, pyrrole), 6.91(m, 2H, pyrrole); C NMR
d
(ppm): 29.79
(
3C), 36.35 (3C), 44.04 (3C), 55.00, 107.24 (2C), 116.55 (2C). Anal.
Calcd. for C14H19N: C, 83.53; H, 9.51; N, 6.96. Experimental: C,
8
3.41; H, 9.50; N, 6.89.
treated with DMSO alone (0.25%) or 10 mM Doxorubicin as negative
and positive controls for cytotoxicity, respectively. After 48-h, cells
were fixed and cell viability was analyzed using the Sulforhod-
amine B colorimetric assay [44]. Absorbance of SRB was measured
utilizing a SpectraMax M5 plate reader and absorbance values
were normalized to non-treated cells. Normalized cell viabilities,
with increasing drug doses, were plotted on a 4-parameter logis-
tical curve, and the IC50 of each compound in each cell line was
calculated using SigmaPlot software (Systat Software, Inc.). Each
compound was used in two independent cell viability assays to
generated doseeresponse curves. The mean IC50, with the corre-
sponding standard deviation, of the two independent treatments
were then calculated. Reduction of cell viability in a time-
dependent manner was assessed by treating HepG2 cells
(5000 cells/well) with compounds at twice the calculated IC50
value (concentrations) of each compound for 24, 48, and 72 h. After
cells were treated at the indicated times and doses, cells were fixed
and cell viability was analyzed using the SRB assay as described
above.
4
.1.3.5. 1-(Chrysen-6-yl)-1H-pyrrole. (Entry 7, Table 2): Brown
crystals. Crystallized from ethyl acetate/hexane mixture; M.p.:
ꢂ
ꢃ1 1
1
39 C; IR: 2947, 2924, 1513, 1461, 1071, 812 cm ; H NMR
.49 (t, 2H, J ¼ 2.21 Hz, pyrrole), 7.12 (m, 2H, pyrrole), 7.70e8.72 (m,
1H, AreH); 13C NMR
(ppm): 109.27 (2C), 120.98, 121.29 (2C),
23.23, 123.49 (2C), 123.64 (2C), 124.05, 126.45 (2C), 126.75 (2C),
26.88 (2C), 127.42, 128.64 (2C), 132.26, 137.94. Anal. Calcd. for
15N: C, 90.08; H, 5.15; N, 4.77. Experimental: C, 89.91; H, 5.04;
N, 4.76.
d (ppm):
6
1
1
1
d
22
C H
4.1.3.6. 1-(Pyren-1-yl)-1H-pyrrole. (Entry 8, Table 2): Pale yellow
crystals. Crystallized from diethyl ether/hexane mixture; M.p.:
ꢂ
ꢃ1 1
;
7
8
C; IR: 2956, 2927,1598, 1511, 1304, 1072, 848 cm
(ppm): 6.53 (m, 2H, pyrrole), 7.15 (m, 2H, pyrrole), 8.01e8.19
(ppm): 109.45 (2C), 122.36, 123.83
2C), 124.89, 125.42, 126.51, 126.72, 127.19, 127.89 (2C), 128.63 (2C),
28.92, 130.62, 131.01, 131.39, 133.43, 135.97. Anal. Calcd. for
13N: C, 89.86; H, 4.90; N, 5.24. Experimental: C, 89.77; H, 4.81;
N, 5.17.
H NMR
d
(
(
m, 9H, AreH); 13C NMR
d
1
C
20
H
The comparison of the effects of compounds on the viability of
normal mouse hepatocytes (Mouse CD-1 cells) and Hepa1-6 cells
Ò
was determined by the colorimetric CellTiter 96 AQueous One
4.1.3.7. 5-(1H-Pyrrol-1-yl)-1,10-phenanthroline. (Entry 9, Table 2):
Solution Cell Proliferation Assay (MTS) (Promega, Madison, WI)
Yellowish brown amorphous solid. Solidified from methanol/
according to the manufacturer’s instructions. Briefly, cells were
ꢂ
hexane mixture; M.p.: 192 C; IR: 3186, 2362, 2336, 1591, 1539,
plated onto a 96-well dish (200
overnight at 37 C. After 24 h wells were replaced with fresh media
m
L media/well) and incubated
ꢃ1
1
ꢂ
1
(
1
074, 739 cm ; H NMR
broad s, 2H, pyrrole), 7.62e9.20 (m, 7H, AreH); C NMR
10.14 (2C), 122.79, 123.24 (2C), 123.55 (2C), 123.74 (2C), 129.04,
d
(ppm): 6.44 (broad s, 2H, pyrrole), 7.02
13
d
(ppm):
and cells were treated with compounds 9 and 10 (0.5, 2.5, 5, and
10 mM) for 48 hrs, then MTS solution (20 mL per 100 mL of culture
1
C
32.26, 136.17, 143.91, 144.09, 150.80, 150.91. Anal. Calcd. for
media) was added to each well and each plate was incubated at
ꢂ
16 11
H N
3
: C, 78.35; H, 4.52; N,17.13. Experimental: C, 78.23; H, 4.43;
37 C with 5% CO
2
for 4 h. The absorbance was then read at 490 nm
N, 17.02.
using a SpectraMax M5 plate reader, and absorbance values were
normalized to non-treated cells.
4
4
.2. Biology
4
.2.4. Anchorage-independent cell growth assays
Anchorage-independent cell growth assays were performed
.2.1. Spectral analyses of pyrrole conjugated compounds (6e10)
Compounds (6e10) were dissolved in DMSO at a concentration
using the CytoSelectÔ 96-well Cell Transformation (Soft Agar
Colony Formation) Assay according to the manufacturer’s instruc-
tions. Briefly, HepG2 cells were plated at 5000 cells/well in top agar.
Cells were then exposed to the indicated dose of each compound
for seven days e the compound and fresh media was replaced once,
on day 4 of the 7-day assay. On day 7, the agar was solubilized and
cells were quantified using a SpectraMax M5 plate reader with
filters set to capture light between 485 nM and 520 nM.
of 20 mM. For spectral analyses, compounds were then diluted to
M in either DMSO or phosphate-buffered saline (PBS). The
5
0 m
absorbance of each compound, dissolved in either DMSO or PBS,
was then analyzed between the wavelengths of 350 nMe750 nM
using a SpectraMax M5 plate reader (Molecular Devices, Sunny-
vale, CA). In addition, diluted compounds were excited with light at
a wavelength of 350 nM, and the subsequent light emission was

D. Bandyopadhyay et al. / European Journal of Medicinal Chemistry 50 (2012) 209e215
215
Authors’ contributions
[22] S.Z. Yuan, J. Liu, L. Xu, A convenient synthesis of pyrroles catalyzed by acidic
resin under solvent-free condition, Chin. Chem. Lett. 21 (2010) 664e668.
[
23] E. Li, X. Xu, H. Li, H. Zhang, X. Xu, X. Yuan, Y. Li, Copper-catalyzed synthesis of
five-membered heterocycles via double CeN bond formation: an efficient
synthesis of pyrroles, dihydropyrroles, and carbazoles, Tetrahedron 65 (2009)
DB and SM conducted the sonochemical reactions, isolated and
characterized the compounds by spectral analyses. JCG, DB and JDS
carried the biological evaluation of the compounds on cancer cell
lines.
8961e8968.
[
[
24] B. Zuo, J.X. Chen, M.C. Liu, H.Y. Wu, W.K. Su, Yb(OTf)
3
-catalyzed synthesis of
pyrroles under solvent-free conditions, Chin. Chem. Lett. 20 (2009) 423e426.
25] P. Ruault, J.-F. Pilard, B. Touaux, F.T. Boullet, J. Hamelin, Rapid generation of
amines by microwave irradiation of ureas dispersed on clay, Synlett (1994)
Acknowledgment
935e936.
[
[
26] T.N. Danks, Microwave assisted synthesis of pyrroles, Tetrahedron Lett. 40
(1999) 3957e3960.
27] F.F. Becker, B.K. Banik, Polycyclic aromatic compounds as anticancer agents:
synthesis and biological evaluation of some chrysene derivatives, Bioorg. Med.
Chem. Lett. 8 (1998) 2877e2880.
We gratefully acknowledge the funding support from National
Cancer Institute (NIH/NCI-P20, Grant# 5P20CA138022-02).
References
[
28] F.F. Becker, C. Mukhopadhyay, L. Hackfeld, I. Banik, B.K. Banik, Polycyclic
aromatic compounds as anticancer agents: synthesis and biological evaluation
of dibenzofluorene derivatives, Bioorg. Med. Chem. 8 (2000) 2693e2699.
[
[
[
[
[
1] Please see website: http://www.who.int/mediacentre/factsheets/fs297/en/
11.04.11).
2] R.A. Jones, G.P. Bean, The Chemistry of Pyrroles, Academic Press, London,
977, p 1.
3] R.J. Sundberg, in: A.R. Katritzky, C.W. Rees (Eds.), Comprehensive Heterocyclic
Chemistry, vol. 4, Pergamon Press, Oxford, 1984, p. 370.
4] R.J. Sundberg, in: A.R. Katritzky, C.W. Rees, E.F.V. Scriven (Eds.), Comprehen-
sive Heterocyclic Chemistry II, vol. 2, Pergamon Press, Oxford, 1996, p. 149.
5] D.L. Boger, C.W. Boyce, M.A. Labroli, C.A. Sehon, Q. Jin, Total syntheses of
Ningalin A, Lamellarin O, Lukianol A, and Permethyl Storniamide A utilizing
heterocyclic azadiene DielseAlder reactions, J. Am. Chem. Soc. 121 (1999)
(
[29] B.K. Banik, F.F. Becker, Polycyclic aromatic compounds as anticancer agents. 4.
Structure-activity relationships of chrysene and pyrene derivatives, Bioorg.
Med. Chem. 9 (2001) 593e605.
[30] B.K. Banik, F.F. Becker, Synthesis, electrophilic substitution and structur-
eeactivity relationship studies of polycyclic aromatic compounds towards the
development of anticancer agents, Curr. Med. Chem. 8 (2001) 1513e1533.
1
[31] I. Banik, F.F. Becker, B.K. Banik, Stereoselective synthesis of
b-lactams with
polyaromatic imines: entry to new and novel anticancer agents, J. Med. Chem.
46 (2003) 12e15.
[32] D. Bandyopadhyay, J.C. Ggranados, J.D. Short, B.K. Banik, Polycyclic aromatic
compounds as anticancer agents: evaluation of synthesis and in vitro cyto-
toxicity, Oncol. Lett. 3 (2012) 45e49.
[33] W.C. Shearouse, D.C. Waddell, J. Mack, Alternative solvent-free methodologies
in the synthesis of pharmaceutical drugs, Curr. Opin. Drug Discov. Devel. 12
(2009) 772e783.
[34] N.R. Candeias, L.C. Branco, P.M.P. Gois, C.A.M. Afonso, A.F. Trindade, More
sustainable approaches for the synthesis of N-based heterocycles, Chem. Rev.
109 (2009) 2703e2802.
54e62.
[
[
[
[
6] J.T. Gupton, Pyrrole natural products with antitumor properties, Top. Heter-
ocycl. Chem. 2 (2006) 53e92.
7] H. Fan, J. Peng, M.T. Hamann, J.-F. Hu, Lamellarins and related pyrrole-derived
alkaloids from marine organisms, Chem. Rev. 108 (2008) 264e287.
8] A. Fuerstner, Venturing into catalysis based natural product synthesis, Synlett
(
1999) 1523e1533.
9] S.J. Higgins, Conjugated polymers incorporating pendant functional groups-
synthesis and characterization, Chem. Soc. Rev. 26 (1997) 247e258.
10] R.D. McCullough, P.C. Ewbank, in: T.A. Skotheim, R.L. Elsenbaumer,
[35] B.K. Banik, D. Adler, P. Nguyen, N. Srivastava, A new bismuth nitrate-induced
stereospecific glycosylation of alcohols, Heterocycles 61 (2003) 101e104.
[
J.R. Reynolds, M. Dekker (Eds.), Handbook of Conducting Polymers, second ed.
[36] N. Srivastava, S.K. Dasgupta, B.K. Banik,
A remarkable bismuth nitrate-
(
1998) New York, ch. 9.
11] T.L. Gilchrist, Synthesis of aromatic heterocycles, J. Chem. Soc. Perkin Trans. 1
1998) 615e628.
catalyzed protection of carbonyl compounds, Tetrahedron Lett. 44 (2003)
1191e1193.
[37] X-ray crystallographic data were recorded with an automatic single crystal
X-ray diffractometer consists of a 3 kw X-ray generator, Saturn 724 CCD
detector, AFC12K goniometer, and X-stream 2000 (for nitrogen generation).
[
[
(
12] K.K.A.D.S. Kathriarachchi, A.I. Siriwardana, I. Nakamura, Y. Yamamoto,
Synthesis of 1,2,3,4-tetrasubstituted pyrrole derivatives via the palladium-
catalyzed reaction of 1,3-diketones with methyleneaziridines, Tetrahedron
Lett. 48 (2007) 2267e2270.
13] H. Hamaguchi, S. Kosaka, H. Ohno, N. Fujii, T. Tanaka, Bromoallenes as allyl
dication equivalents in the presence or absence of palladium(0): direct
construction of bicyclic sulfamides containing five- to eight-membered rings
by tandem cyclization of bromoallenes, Chem. Eur. J. 13 (2007) 1692e1708.
14] M.L. Crawley, I. Goljer, D.J. Jenkins, J.F. Mehlmann, L. Nogle, R. Dooley,
P.E. Mahaney, Regioselective synthesis of substituted pyrroles: efficient
palladium-catalyzed cyclization of internal alkynes and 2-amino-3-
iodoacrylate derivatives, Org. Lett. 8 (2006) 5837e5840.
The compound crystallizes in the P2
1
/n space group with only one molecule in
the asymmetric unit, situated on a general position. All non-hydrogen atoms
were refined anisotropically. Hydrogen atoms were located in a difference
Fourier map and were refined with Uiso(H) ¼ 1.2Ueq (C). Crystals were selected
from the original sample. An X-ray quality colorless chip-shaped crystal of
dimensions 0.22 ꢁ 0.10 ꢁ 0.17 mm was selected for structural analysis. The
ORTEP projection is shown in Fig. 1. Crystallographic data for 1-(Adamntan-1-
yl)-1H-pyrrole have been deposited as DBSMPy-5 with Cambridge Crystallo-
graphic Data Centre as CCDC 787708. Copies of the data can be obtained, free
of charge, on application to CCDC, 12 Union Road, Cambridge CB2 IEZ, UK.
(fax: þ44 1223 336033 or e-mail: deposit@ccdc.cam.ac.uk).
[
[
[
[
[
15] D. Bandyopadhyay, S. Mukherjee, B.K. Banik, An expeditious synthesis of
N-substituted pyrroles by microwave-induced iodine-catalyzed reactions
under solventless conditions, Molecules 15 (2010) 2520e2525.
[38] L. Búcsiová, P. Hrdlovi ꢀc , S. Chmela, Spectral characteristics of fluorescence
probes based on pyrene in solution and in polymer matrices, J. Photochem.
Photobiol. A Chem. 143 (2001) 59e68.
[39] P. Hrdlovi ꢀc , S. Chmela, Spectral characteristics of probes based on ionic
derivatives of pyrene in polar polymer matrices, J. Photochem. Photobiol. A
Chem. 118 (1998) 137e142.
[40] M. Spiro, P. Vigny, R.M. Hodgson, Fluorescence spectral studies on the
metabolic activation of chrysene by hamster embryo cells, Carcinogenesis 3
(1982) 1491e1493.
16] D. Bandyopadhyay, G. Rivera, I. Salinas, H. Aguilar, B.K. Banik, Remarkable
iodine-catalyzed synthesis of novel pyrrole-bearing N-polyaromatic
tams, Molecules 15 (2010) 1082e1088.
17] S. Rivera, D. Bandyopadhyay, B.K. Banik, Facile synthesis of N-substituted
pyrroles via microwave-induced bismuth nitrate-catalyzed reaction, Tetra-
hedron Lett. 50 (2009) 5445e5448.
b
-lac-
[
[
[
18] M. Rueping, A. Parra, Fast, efficient, mild, and metal-free synthesis of pyrroles
by domino reactions in water, Org. Lett. 12 (2010) 5281e5283.
19] V. Estevez, M. Villacampa, C.J. Menendez, Multicomponent reactions for the
synthesis of pyrroles, Chem. Soc. Rev. 39 (2010) 4402e4421.
20] B.M. Trost, M. Osipov, G. Dong, Palladium-catalyzed dynamic kinetic asym-
metric transformations of vinyl aziridines with nitrogen heterocycles: rapid
access to biologically active pyrroles and indoles, J. Am. Chem. Soc. 132 (2010)
[41] B.K. Banik, F.F. Becker, Novel 6,12-disubstituted chrysene as potent anticancer
agent: synthesis, in vitro and in vivo study, Eur. J. Med. Chem. 45 (2010)
4687e4691.
[42] K. Ohara, M. Smietana, A. Restouin, S. Mollard, J. Borg, Y. Collette, J. Vasseur,
Amine-guanidine switch: a promising approach to improve DNA binding and
antiproliferative activities, J. Med. Chem. 50 (2007) 6465e6475.
[43] K.R. Landis-Piwowar, D. Chen, Q.C. Cui, V. Minic, F.F. Becker, B.K. Banik,
Q.P. Dou, Apoptotic-inducing activity of novel polyaromatic compounds in
human leukemic cells, Int. J. Mol. Med. 17 (2006) 931e935.
[44] V. Vichai, K. Kirtikara, Sulforhodamine B colorimetric assay for cytotoxicity
screening, Nat. Protoc. 1 (2006) 1112e1116.
1
5800e15807.
21] S. Lamande-Langle, M. Abarbri, J. Thibonnet, A. Duchene, J.-L. Parrain, Domino
allylic amination/Sonogashira/heterocyclisation reactions: palladium-
catalyzed three-component synthesis of pyrroles, Chem. Commun. 46
2010) 5157e5159.
[
(