A clean synthesis of 2,5-dihydro-1H-pyrrole-2-carboxylates under catalyst-free and solvent-free conditions: cytotoxicity and molecular docking studies

Article abstract of DOI:10.1007/s13738-018-1359-2

Abstract: A concise, clean synthetic approach to access of 2,5-dihydro-1H-pyrrole-2-carboxylates by two-component condensation reaction of ethyl pyruvate and aniline derivatives under catalyst-free and solvent-free conditions is described. The advantages of this method are practical simplicity, catalyst-free and solvent-free conditions, high atom economy, short reaction times, and good yields of the products. The anticancer potential of synthesized compounds was evaluated against MCF7, MOLT-4 and HL-60 cells by using MTT assay. Among the tested synthesized 2,5-dihydro-1H-pyrrole-2-carboxylates (3a-h, 4a-d), compound 4d having sulfonamide moiety exhibited good cytotoxic activity against all tested cell lines and molecular docking studies also demonstrated that the results of the docking study are in reasonable agreement with cytotoxicity activities. Graphical Abstract: A series of 2,5-dihydro-1H-pyrrole-2-carboxylate derivatives were synthesized by two-component condensation reaction of ethyl pyruvate and aniline derivatives under catalyst-free and solvent-free conditions.[Figure not available: see fulltext.].

Full text of DOI:10.1007/s13738-018-1359-2

Journal of the Iranian Chemical Society
https://doi.org/10.1007/s13738-018-1359-2
ORIGINAL PAPER
A clean synthesis of 2,5‑dihydro‑1H‑pyrrole‑2‑carboxylates
under catalyst‑free and solvent‑free conditions: cytotoxicity
and molecular docking studies
Khodabakhsh Niknam1 · Masoumeh Bavadi1 · Sanaz Mojikhalifeh1 · Omolbanin Shahraki2
Received: 6 October 2017 / Accepted: 4 April 2018
© Iranian Chemical Society 2018
Abstract
A concise, clean synthetic approach to access of 2,5-dihydro-1H-pyrrole-2-carboxylates by two-component condensation
reaction of ethyl pyruvate and aniline derivatives under catalyst-free and solvent-free conditions is described. The advantages
of this method are practical simplicity, catalyst-free and solvent-free conditions, high atom economy, short reaction times,
and good yields of the products. The anticancer potential of synthesized compounds was evaluated against MCF7, MOLT-4
and HL-60 cells by using MTT assay. Among the tested synthesized 2,5-dihydro-1H-pyrrole-2-carboxylates (3a-h, 4a-d),
compound 4d having sulfonamide moiety exhibited good cytotoxic activity against all tested cell lines and molecular dock-
ing studies also demonstrated that the results of the docking study are in reasonable agreement with cytotoxicity activities.
Graphical Abstract
A series of 2,5-dihydro-1H-pyrrole-2-carboxylate derivatives were synthesized by two-component condensation reaction of
ethyl pyruvate and aniline derivatives under catalyst-free and solvent-free conditions.
Keywords Clean synthesis · 2,5-Dihydro-1H-pyrrole-2-carboxylates · Ethyl pyruvate · Catalyst-free · Two-component
reactions · Cytotoxicity · Molecular docking
Introduction
(MCRs) have received a considerable attention in academic
research laboratories, synthetic, and pharmaceutical chem-
istry because of simple experimentation, mild conditions,
simplifed workup procedures, reduced pollution, and high
yields of the products [3–6].
Green chemistry and development of practical, economi-
cal, environmentally friendly methods for the synthesis of
bioactive medicinal motifs are major challenges in synthetic
chemistry [1, 2]. Accordingly, multi-component reactions
Dihydro-pyrrole-2-ones are an important class of nitro-
gen heterocycles that are frequently found in pharmaceuti-
cally active compounds and numerous natural products such
as alkaloids, pheromone, steroids, chlorophyll, and heme.
Dihydro-pyrrole-2-ones exhibit a wide spectrum of phar-
macological activities such as anticancer [7], herbicidal [8],
HIV integrase inhibition [9], and antibacterial activity [10].
For example, pyrrocidine A (I) is a potential antibiotic iso-
lated from the fermentation broth of a fungus (LL-Cyan426)
* Khodabakhsh Niknam
niknam@pgu.ac.ir
1
Department of Chemistry, Faculty of Sciences, Persian Gulf
University, Bushehr 75169, Iran
2
Department of Medicinal Chemistry, Faculty of Pharmacy,
Zahedan University of Medical Sciences, Zahedan, Iran
Vol.:(0123456789)
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Journal of the Iranian Chemical Society
(Fig. 1) [11]. Pyrrolidone derivatives (II) were used as
inhibitor of HCV polymerase [12]; violacein (III), a natural
product, possesses an antibiotic and antitumor activity which
was extracted from the gram-negative bacterium Chromo-
bacterium violaceum [13].
development of facile and greener methods for the prepa-
ration of these important heterocycles is still an attractive
option.
Sulfonamides have been reported to possess anticancer
[23], anti-infammatory [24], antihypertensive [25], antima-
larial [26], and carbonic anhydrase (CA) inhibitory [27].
Carbonic anhydrases (CAs, EC 4.2.1.1) are a group of met-
alloenzymes that catalyze the hydratation of carbon dioxide
to bicarbonate and protons. They play an important role in
many physiological processes such as pH and CO2 homeo-
stasis, electrolyte secretion, regulation of respiration and gas
exchange, gluconeogenesis, boneresorption, lipogenesis, cal-
cifcation, and tumorigenicity. Sixteen diferent human (h)
CA isoforms belonging to α class have been detected so far.
Among them, the human cytosolic isoforms hCA I and hCA
II are wide spread throughout the body and are targets for
antiglaucoma, diuretic, and anticonvulsant drugs [28–32].
According to the above facts and in continuation of our
studies on synthesis of dihydro-pyrrole-2-one compounds
[16, 33], herein we report a green and efcient protocol
for the synthesis of 2,5-dihydro-1H-pyrrole-2-carboxylate
derivatives 3 or 4 via two-component condensation reaction
aniline or aniline-bearing sulfonamide derivatives (1) and
ethyl pyruvate (2) under catalyst- and solvent-free condi-
tions (Scheme 1). In addition, their cytotoxicity against three
cancer cell lines and molecular docking study was evaluated.
1,5-Dihydro-2H-pyrrol-2-ones are found as substructures
in many natural products with interesting pharmacological
properties [14–16]. A class of dihydro-pyrrole-2-one skel-
etons is 5-oxo-1-aryl-2,5-dihydro-1H-pyrrole-2-carboxy-
lates, which are an appealing family of lactams. In addition,
pyrrole-2-carboxylates are the precursor for the synthesis
of other heterocyclic and spiro compounds [17, 18]. How-
ever, the synthetic methods to the 5-oxo-1-aryl-2,5-dihydro-
1H-pyrrole-2-carboxylate compounds are very rare. Mandal
and Achar reported the synthesis of 2-oxo-3-pyrrolines by
the reaction between amines with methyl pyruvate in the
presence of ultrasonic irradiation in CH2Cl2 [19]. Gein and
co-workers reported the synthesis and reaction with oxa-
lyl chloride of ethyl 1-aryl-4,5-dioxo-2-pyrrolidinecarbox-
ylates and their 3-arylamino derivatives [20]. Akkurt and
co-workers reported the synthesis of ethyl 4-anilino-2-me-
thyl-5-oxo-1-phenyl-2,5-dihydro-1H-pyrrole-2-carboxylate
via two-component reaction of ethyl pyruvate and aniline
in the presence of Fe3O4 nanoparticles [21, 22]. However,
this method sufers from certain drawbacks such as the use
of metal catalyst and harsh reaction conditions. Hence, the
Fig. 1 The biologically active compounds containing the pyrrol-2-one moiety
Scheme 1 Synthesis of
2,5-dihydro-1H-pyrrole-2-car-
boxylate derivatives
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Journal of the Iranian Chemical Society
Experimental
128.2, 128.5, 134.4, 134.5, 154.6, 158.7, 167.8, 171.6. MS
(EI, 70 eV), m/z (%): 396 (M+, 10.6), 323 (base peak), 148
(9.8), 91 (13.1). Anal. Calcd for C22H24N2O5: C, 66.65; H,
6.10; N, 7.07; found: C, 66.33; H, 6.08, N, 6.79.
Chemicals and reagents
Chemicals were purchased from Merck and Aldrich Chemi-
cal Companies. The products were characterized by compar-
ison of their spectral and physical data with those reported in
the literature. For recorded 1HNMR spectra, we use Bruker
(400 MHZ) Avance Ultrashield in pure deuterated DMSO-d6
and CDCl3 solvents with tetramethylsilane (TMS) as internal
standards. Mass spectra were recorded on a FINNIGAN-
MAT 8430 mass spectrometer operating at 70 eV. FTIR
spectroscopy (Shimadzu FTIR 8300 spectrophotometer)
was employed for characterization of the products. Melting
points determined in open capillary tubes in a Barnstead
Electrothermal 9100 BZ circulating oil melting point appa-
ratus. The reaction monitoring was accomplished by TLC
on silica gel PolyGram SILG/UV254 plates. MCF7 (human
breast adenocarcinoma), HL-60 (promyelocytic leukemia)
and MOLT-4 (human acute lymphoblastic leukemia) cells
were obtained from the National Cell Bank of Iran, Pas-
teur Institute, Tehran, Iran. The cell culture medium (RPMI
1640), fetal bovine serum (FBS), and penicillin–strepto-
mycin were obtained from Gibco BRL (Life technology,
Paisley, Scotland). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT) powder was purchased from
Sigma Chemicals Co. (Germany). The culture plates were
purchased from Nunc (Denmark).
Ethyl 2‑methyl‑5‑oxo‑1‑(p‑tolyl)‑4‑(p‑
tolylamino)‑2,5‑dihydro‑1H‑pyrrole‑2‑carboxylate (3b)
White solid, recrystallized from ethanol, mp 193–195 °C. IR
(KBr) (cm−1): 3307 (N–H), 3115 (=C–H), 1729 (C=O). 1H-
NMR (400 MHz, CDCl3): δ (ppm) 1.27 (t, 3H, J=7.0 Hz,
CH3), 1.66 (s, 3H, CH3), 2.35 (s, 3H, CH3), 2.40 (s, 3H,
CH3), 4.22 (q, 2H, CH2), 5.94 (s, 1H, = CH), 6.61 (s, 1H,
NH), 7.01 (d, 2H, J=8.0 Hz, Ar), 7.16 (d, 2H, J=7.6 Hz,
Ar), 7.24–7.27 (m, 4H, Ar). 13C NMR (100 MHz, CDCl3):
δ (ppm) 14.1, 20.7, 21.1, 21.3, 62.1, 68.7, 106.8, 117.1,
125.8, 129.8, 129.9, 131.0, 133.3, 133.9, 136.9, 138.6,
167.6, 171.7. MS (EI, 70 eV), m/z (%): 364 (M+, 8.0), 291
(base peak), 263 (4.5), 132 (13.4), 91 (17.8). Anal. Calcd for
C22H24N2O3: C, 72.50; H, 6.64; N, 7.69; found: C, 72.26;
H, 6.56, N, 7.29.
Ethyl‑2‑methyl‑5‑oxo‑1‑(m‑tolyl)‑4‑
(m‑tolylamino)‑2,5‑dihydro‑1H‑pyrrole‑2‑carboxylate (3c)
White solid, recrystallized from ethanol, mp 197–199 °C,
IR (KBr) ν (cm−1): 3306 (N–H), 3115 (=C–H), 1729
(C=O). 1H-NMR (400 MHz, CDCl3): δ (ppm) 1.27 (t, 3H,
J=7.0 Hz, CH3), 1.66 (s, 3H, CH3), 2.35 (s, 3H, CH3), 2.40
(s, 3H, CH3), 4.22 (q, 2H, CH2), 5.94 (s, 1H, = CH), 6.61
(s, 1H, NH), 7.00–7.11(m, 2H, Ar), 7.18 (d, 2H, J=8.6 Hz,
Ar), 7.21–7.26 (m, 3H, Ar), 7.46–7.53 (m, 1H, Ar). 13C
NMR (100 MHz, CDCl3): δ (ppm) 14.1, 20.6, 21.1, 21.2,
62.1, 68.7, 106.7, 117.1, 125.9, 129.8, 129.9, 131.0, 133.3,
133.9, 136.8, 138.6, 167.6, and 171.7. MS (EI, 70 eV): m/z
(%)=364 (M+, 7.9), 291 (base peak), 263 (4.2), 214 (2.1),
132 (12.9), 91(17.3). Anal. Calcd for C22H24N2O3: C, 72.51;
H, 6.64; N, 7.69; found: C, 72.18; H, 6.47; N, 7.43.
General procedure for the synthesis
of 2,5‑dihydro‑1H‑pyrrole‑2‑carboxylates
A mixture of aromatic amine (2 mmol) and ethyl pyruvate
(2 mmol) was heated with stirring in an oil bath at 80 °C
under catalyst-free and solvent-free conditions. The progress
of the reaction was monitored by TLC. After completion,
the crude products were purifed by recrystallization from
ethanol.
Ethyl 1‑(3,4‑dimethylphenyl)‑4‑((3,4‑dimethylphenyl)
amino)‑2‑methyl‑5‑oxo‑2,5‑dihydro‑
1H‑pyrrole‑2‑carboxylate (3d)
Ethyl‑1‑(4‑methoxyphenyl)
‑4((4‑methoxyphenyl)amino)‑2‑methyl‑5‑
oxo‑2,5‑dihydro‑1H‑pyrrole‑2‑carboxylate (3a)
White solid, recrystallized from ethanol, mp 174–176 °C.
IR (KBr) (cm−1): 3303 (N–H), 3120 (=C–H), 1725 (C=O).
1H-NMR (400 MHz, CDCl3): δ (ppm) 1.22–1.38 (m, 3H,
CH3), 1.66 (s, 3H, CH3), 2.20–2.45 (m, 12H, 4 × CH3),
4.13–4.38 (m, 2H, OCH2), 5.93(s, 1H, =CH), 6.56 (s, 1H,
NH), 6.80–7.25 (m, 6H, Ar). 13C NMR (100 MHz, CDCl3):
δ (ppm) 14.1, 19.0, 19.4, 20.0, 20.1, 21.3, 62.0, 68.7, 106.7,
114.4, 118.5, 123.4, 127.3, 129.8, 130.2, 130.4, 133.4,
133.9, 135.8, 137.5, 137.7, 138.9, 167.6, 171.8. MS (EI,
White solid, recrystallized from ethanol, mp 224–226 °C. IR
(KBr) (cm−1): 3309 (N–H), 3120 (=C–H), 1729 (C=O). 1H-
NMR (400 MHz, CDCl3): δ (ppm) 1.29 (t, 3H, J=7.0 Hz,
CH3), 1.64 (s, 3H, CH3), 3.83 (s, 3H, CH3), 3.85 (s, 3H,
CH3), 4.21 (q, 2H, J = 7.0 Hz, CH2), 5.86 (s, 1H, = CH),
6.50 (s, 1H, NH), 6.92 (d, 2H, J=8.8 Hz, Ar), 6.97 (d, 2H,
J = 8.8 Hz, Ar), 7.06 (d, 2H, J = 8.8 Hz, Ar), 7.30 (d, 2H,
J=8.8 Hz, Ar). 13C NMR (100 MHz, CDCl3): δ (ppm) 14.1,
21.5, 55.5, 55.6, 62.1, 68.9, 105.8, 114.4, 114.7, 118.7,
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Journal of the Iranian Chemical Society
70 eV), m/z (%): 392 (M+, 24.5), 319 (base peak), 291 (4.9),
146 (15.7). Anal. Calcd for C24H28N2O3: C, 73.44; H, 7.19;
N, 7.14; found: C, 73.11; H, 7.16, N, 6.85.
421 (base peak), 342 (6.9), 198 (11.7), 91 (7.8). Anal. Calcd
for C20H18Br2N2O3: C, 48.61; H, 3.67; Br, 32.34; N, 5.67;
found: C, 48.27; H, 3.61, N, 5.34.
Ethyl 1‑(4‑chlorophenyl)‑4‑((4‑chlorophenyl)amino)
Ethyl 2‑methyl‑1‑(3‑nitrophenyl)‑4‑((3‑nitrophenyl)
‑2‑methyl‑5‑oxo‑2,5‑dihdro‑1H‑pyrrole‑2‑carboxylate (3e)
amino)‑5‑oxo‑2,5‑dihydro‑1H‑pyrrole‑2‑carboxylate (3h)
Pale yellow solid, recrystallized from ethanol, mp
218–220 °C. IR (KBr) (cm−1): 3309 (N–H), 3115 (=C–H),
Yellow solid, recrystallized from ethanol, mp 196–198 °C.
IR (KBr) (cm−1): 3316 (N–H), 3121 (=C–H), 1730 (C=O).
1H-NMR (400 MHz, CDCl3): δ (ppm) 1.15–1.45 (m, 3H,
CH3), 1.79 (s, 3H, CH3), 4.15–4.45 (m, 2H, OCH2), 6.22
(s, 1H, = CH), 7.03 (s, 1H, NH), 7.29–7.74 (m, 3H, Ar),
7.80–8.05 (m, 3H, Ar), 8.17–8.47 (m, 2H, Ar). 13C NMR
(100 MHz, CDCl3): δ (ppm) 14.1, 21.2, 63.0, 69.0, 110.2,
110.7, 116.6, 119.7, 121.4, 122.9, 130.1, 130.3, 130.6,
132.5, 136.9, 141.7, 148.7, 149.2, 166.9, 170.4. MS (EI,
70 eV), m/z (%): 426 (M+, 5.9), 353 (base peak), 307 (9.8),
163 (6.8). Anal. Calcd for C20H18N4O7: C, 56.34; H, 4.26;
N, 13.14; found: C, 56.02; H, 4.28, N, 12.83.
1
1731 (C=O). H-NMR (400 MHz, CDCl3): δ (ppm) 1.26
(t, 3H, J=7.2 Hz, CH3), 1.67 (s, 3H, CH3), 4.15–4.30 (m,
2H, OCH2), 5.97 (s, 1H, =CH), 6.74 (s, 1H, NH), 7.02 (d,
2H, J = 8.8 Hz, Ar), 7.30 (d, 2H, J = 8.6 Hz, Ar), 7.36 (d,
2H, J=8.8 Hz, Ar), 7.42 (d, 2H, J=8.8 Hz, Ar). 13C NMR
(100 MHz, CDCl3): δ (ppm) 14.1, 21.2, 62.4, 68.8, 108.0,
118.1, 126.6, 126.7, 129.3, 129.4, 132.6, 133.3, 134.4,
139.4, 167.2, 171.1. MS (EI, 70 eV), m/z (%): 408 (M+4,
2.0), 406 (M+2, 6.9), 404 (M+, 9.8), 331 (base peak), 152
(21.6), 91 (2.9). Anal. Calcd for C20H18Cl2N2O3: C, 59.27;
H, 4.48; Cl, 17.50; N, 6.91; found: C, 58.94; H, 4.51, N,
6.59.
Ethyl 1‑(4‑methoxy‑3‑(N‑phenylsulfamoyl)
phenyl)‑4‑((4‑methoxy‑3‑(N‑phenyl‑sulfamoyl)
phenyl)amino)‑2‑methyl‑5‑oxo‑2,5‑dihdro‑1H‑
pyrrole‑2‑carboxylate (4a)
Ethyl‑1‑(3‑chlorophenyl)‑4‑((3‑chlorophenyl)amino)‑2
‑methyl‑5‑oxo‑2,5‑dihdro‑1H‑pyrrole‑2‑carboxylate (3f)
White Solid, recrystallized from ethanol, mp 213–215 °C,
IR (KBr) ν (cm−1): 3308 (N–H), 3115 (=C–H), 1731
(C=O). 1H-NMR (400 MHz, CDCl3): δ (ppm) 1.26 (t, 3H,
J=7.2 Hz, CH3), 1.66 (s, 3H, CH3), 4.23 (q, 2H, CH2), 5.97
(s, 1H, = CH), 6.75 (s, 1H, NH), 7.11–7.22 (m, 3H, Ar),
7.32 (d, 1H, J = 8.8 Hz, Ar), 7.40 (d, 2H, J = 8.6 Hz, Ar),
7.51–7.55 (m, 1H, Ar), 7.82–7.85 (m, 1H, Ar). 13C NMR
(100 MHz, CDCl3): δ (ppm) 14.1, 21.3, 62.4, 68.8, 108.5,
118.6, 126.6, 126.7, 129.1, 130.2, 132.6, 134.3, 135.4, 136.0,
139.4, 167.2, 171.2. MS (EI, 70 eV): m/z (%)=408 (M+4,
2.1), 406 (M+2, 6.9), 404 (M+, 9.7), 331(base peak), 303
(8.1), 152 (21.7), 91(3.2). Anal. Calcd for C20H18Cl2N2O3:
C, 59.27; H, 4.48; Cl, 17.49; N, 6.91; found: C, 58.94; H,
4.35; N, 6.59.
White solid, recrystallized from ethanol, mp > 250 °C.
1
IR (KBr) (cm−1): 3299 (N–H), 1736 (C=O). H-NMR
(400 MHz, DMSO-d6): δ (ppm) 1.20 (t, 3H, J = 7.2 Hz,
CH3), 1.44 (s, 3H, CH3), 3.85 (s, 3H, OCH3), 3.92 (s,
3H, OCH3), 4.08–4.18 (m, 2H, CH2), 5.98 (s, 1H, = CH),
6.97–7.01 (m, 2H, Ar), 7.08–7.13 (m, 5H, Ar), 7.18–7.23
(m, 4H, Ar), 7.28 (d, 1H, J = 9.2 Hz, Ar), 7.46 (dd, 1H,
J1 =8.8, J2 =2.8 Hz, Ar), 7.54 (dd, 1H, J1 =8.8, J2 =2.8 Hz,
Ar), 7.70 (d, 2H, J=2.8 Hz, Ar), 8.45 (s, 1H, NH), 10.06 (s,
1H, SO2NH), 10.16 (s, 1H, SO2NH). 13C NMR (100 MHz,
DMSO-d6): δ (ppm) 14.3, 21.2, 56.8, 57.0, 62.4, 68.5, 108.0,
114.2, 114.3, 119.7, 119.8, 124.0, 124.1, 124.2, 127.0,
127.3, 128.1, 128.5, 129.4, 129.5, 133.5, 134.6, 135.0,
138.1, 138.4, 151.1, 155.5, 167.2, 171.3. MS (EI, 70 eV),
m/z (%): 706 (M+), 530 (0.1), 438 (0.2), 300 (0.9), 207 (2.9),
93 (base peak), 43 (73). Anal. Calcd for C34H34N4O9S2: C,
57.78; H, 4.85; N, 7.93; S, 9.07; found: C, 57.45; H, 4.81,
N, 7.61.
Ethyl 1‑(4‑bromophenyl)‑4‑((4‑bromophenyl)amino)‑2
‑methyl‑5‑oxo‑2,5‑dihydro‑1H‑pyrrole‑2‑carboxylate (3g)
White solid, recrystallized from ethanol, mp 233–235 °C. IR
(KBr) (cm−1): 3313 (N–H), 3115 (=C–H), 1730 (C=O). 1H-
NMR (400 MHz, CDCl3): δ (ppm) 1.25 (t, 3H, J=7.1 Hz,
CH3), 1.67 (s, 3H, CH3), 4.14–4.30 (m, 2H, OCH2), 5.97 (s,
1H, = CH), 6.74 (s 1H, NH), 6.97 (d, 2H, J= 8.0 Hz, Ar),
7.30 (d, 2H, J = 8.3 Hz, Ar), 7.44 (d, 2H, J = 8.0 Hz, Ar),
7.56 (d, 2H, J=8.3 Hz, Ar). 13C NMR (100 MHz, CDCl3):
δ (ppm) 14.1, 21.2, 62.5, 68.7, 108.2, 113.9, 118.6, 120.5,
126.9, 132.3, 134.9, 139.9, 167.2, 171.1. MS (EI, 70 eV),
m/z (%): 496 (M+4, 4.9), 494 (M+2, 11.8), 492 (M+, 3.9),
Ethyl 1‑(3‑(N‑(4‑chlorophenyl)sulfamoyl)‑4‑
methoxyphenyl)‑4‑((3‑(N‑(4‑chlorophenyl)
sulfamoyl)‑4‑methoxyphenyl)‑amino)
‑2‑methyl‑5‑oxo‑2,5‑dihdro‑1H‑pyrrole‑2‑carboxylate (4b)
White solid, recrystallized from ethanol, mp > 250 °C.
IR (KBr) (cm−1): 3296 (N–H), 1738 (C=O). H-NMR
1
(400 MHz, DMSO-d6): δ (ppm) 1.19 (t, 3H, J = 7.2 Hz,
CH3), 1.46 (s, 3H, CH3), 3.84 (s, 3H, OCH3), 3.92 (s,
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Journal of the Iranian Chemical Society
3H, OCH3), 4.08–4.18 (m, 2H, CH2), 6.03 (s, 1H, = CH),
7.09–7.14 (m, 5H, Ar), 7.25–7.30 (m, 5H, Ar), 7.48 (dd,
1H, J1 = 8.8, J2 = 2.8 Hz, Ar), 7.56 (dd, 1H, J1 = 8.8,
J2 =2.8 Hz, Ar), 7.71–7.73 (m, 2H, Ar), 8.48 (s, 1H, NH),
10.25 (s, 1H, SO2NH), 10.34 (s, 1H, SO2NH). 13C NMR
(100 MHz, DMSO-d6): δ (ppm) 14.3, 21.1, 56.8, 57.0, 62.5,
68.6, 108.1, 114.2, 114.4, 119.8, 121.3, 121.5, 124.1, 126.7,
127.0, 128.1, 128.2, 128.3, 128.6, 129.3, 129.4, 133.7,
134.5, 135.0, 137.2, 137.4, 151.0, 155.4, 167.2, 171.2. MS
(EI, 70 eV), m/z (%):776 (M+2, 0.002), 774 (M+, 0.003), 438
(9.1), 300 (18.6), 207 (25.8), 149 (7.1), 93 (base peak). Anal.
Calcd for C34H32Cl2N4O9S2: C, 52.65; H, 4.16; Cl, 9.14; N,
7.22; S, 8.27; found: C, 52.32; H, 4.19, N, 6.90.
NH), 9.81 (s, 1H, SO2NH), 9.92 (s, 1H, SO2NH). 13C NMR
(100 MHz, DMSO-d6): δ (ppm) 14.3, 19.1, 20.0, 20.1, 21.0,
56.8, 56.9, 62.4, 68.5, 107.8, 114.1, 114.3, 117.6, 117.7,
119.7, 121.3, 121.5, 124.0, 127.1, 127.3, 128.1, 128.4,
130.2, 130.3, 132.1, 133.4, 134.6, 134.9, 135.7, 135.9,
137.1, 137.2, 151.1, 155.4, 167.1, 171.3. MS (EI, 70 eV),
m/z (%): 762 (M+, 0.4), 689 (7.7), 504 (18.8), 306 (33.5),
211 (23.9), 120 (base peak), 65.1 (18.1). Anal. Calcd for
C38H42N4O9S2: C, 59.83; H, 5.55; N, 7.34; S, 8.41; found:
C, 59.49; H, 5.51, N, 7.01.
Cell culture
All cell lines were cultured in RPMI 1640 supplemented
with 10% FBS, 100 units/mL penicillin-G, and 100 µg/mL
streptomycin. Cells were grown in monolayer (MCF7) or in
suspension (HL-60 and MOLT-4) cultures.
Ethyl 1‑(4‑methoxy‑3‑(N‑(p‑tolyl)
sulfamoyl)phenyl)‑4‑((4‑methoxy‑3‑
(N‑(p‑tolyl)sulfamoyl)phenyl)amino)‑2
‑methyl‑5‑oxo‑2,5‑dihdro‑1H‑pyrrole‑2‑carboxylate (4c)
Cell viability assay
White solid, recrystallized from ethanol, mp > 250 °C.
1
IR (KBr) (cm−1): 3296 (N–H), 1738 (C=O). H-NMR
The cell cytotoxicity of synthesized compounds was
assessed by using a 3-[4,5-dimethylthiazol-2-y]-2,5-di-
phenyltetrazoliumbromide (MTT) assay. A 100 μL of the
cell suspensions (5 × 104 cells/mL) was seeded on to the
96-well plates and cultured 24 h prior to drug treatment.
40 mM stock solution of target compounds was prepared
in DMSO. Cells were exposed to 25, 50, and 100 μM of
compounds. The plates were incubated for 72 h. After that,
the supernatant was removed carefully. MTT solution was
prepared from 5 mg/mL diluted to 1:7 in RPMI without phe-
nol red. Cells were treated with 80 μL of MTT solution for
4 h at 37 °C. The media were removed, and 200 μL DMSO
was added to each well to dissolve the formazan crystals.
After 1-h incubation at 37 °C, plates were gently shaken
for 30 min. Absorbance at 570 nm with background cor-
rection at 655 nm was measured using Bio-Rad Microplate
reader (Model 680, Bio-Rad, Hercules, CA). The percentage
of cell viability was measured, and IC50 values were calcu-
lated using CurveExpert software version 1.34 for Windows.
(400 MHz, DMSO-d6): δ (ppm) 1.19 (t, 3H, J = 7.2 Hz,
CH3), 1.42 (s, 3H, CH3), 2.18 (s, 6H, 2 × CH3), 3.86 (s,
3H, OCH3), 3.93 (s, 3H, OCH3), 4.07–4.19 (m, 2H, CH2),
5.97 (s, 1H, = CH), 6.96–7.01 (m, 8H, Ar), 7.12 (d, 1H,
J=9.2 Hz, Ar), 7.27 (d, 1H, J=9.2 Hz, Ar), 7.44 (dd, 1H,
J1 =9.0, J2 =2.8 Hz, Ar), 7.54 (dd, 1H, J1 =8.8, J2 =2.8 Hz,
Ar), 7.65–7.66 (m, 2H, Ar), 8.42 (s, 1H, NH), 9.87 (s,
1H, SO2NH), 9.98 (s, 1H, SO2NH). 13C NMR (100 MHz,
DMSO-d6): δ (ppm) 14.3, 20.7, 21.1, 56.8, 56.9, 62.4, 68.5,
107.9, 114.1, 114.2, 119.7, 120.3, 123.9, 127.0, 127.2,
128.1, 128.4, 129.8, 129.9, 133.3, 133.4, 134.6, 134.9,
135.5, 135.7, 151.1, 155.4, 167.1, 171.2. MS (EI, 70 eV),
m/z (%): 734 (M+, 0.02), 523 (9.8), 353 (94.4), 299 (20.5),
236 (53.1), 97 (64.3), 43 (base peak). Anal. Calcd for
C36H38N4O9S2: C, 58.84; H, 5.21; N, 7.62; S, 8.73; found:
C, 58.51; H, 5.17, N, 7.28.
Ethyl 1‑(3‑(N‑(3,4‑dimethylphenyl)
sulfamoyl)‑4‑methoxyphenyl)
‑4‑((3‑(N‑(3,4‑dimethyl‑phenyl)
Molecular docking study
sulfamoyl)‑4‑methoxyphenyl)amino)‑2‑methyl‑5‑oxo‑2,
5‑dihdro‑1H‑pyrrole‑2‑carboxylate (4d)
All of the compounds were docked in the active site of hCA
II in complex with saccharin (PDB entry: 2Q38). The X-ray
crystal structure of protein was retrieved from Protein Data
Bank (http://www.rcsb.org). Molecular docking studies
were performed using AutoDock 4.2 and Auto Dock Tools
1.5.4 (ADT). Water molecules and co-crystallized ligands
were removed. The polar hydrogens and Gasteiger charges
were assigned. The 3D structures of synthesized ligands
were sketched using Avogadro software and submitted to
ORCA software at DFT, B3LYP/3–21 level for optimiza-
tion. The Gasteiger charges were assigned, and the number
White solid, recrystallized from ethanol, mp > 250 °C.
1
IR (KBr) (cm−1): 3300 (N-H), 1737 (C=O). H-NMR
(400 MHz, DMSO-d6): δ (ppm) 1.19 (t, 3H, J = 7.2 Hz,
CH3), 1.42 (s, 3H, CH3), 2.08 (s, 12H, 4 × CH3), 3.87 (s,
3H, OCH3), 3.94 (s, 3H, OCH3), 4.07–4.19 (m, 2H, CH2),
5.97 (s, 1H, = CH), 6.79–6.87 (m, 3H, Ar), 6.91–6.95 (m,
3H, Ar), 7.12 (d, 1H, J=9.2 Hz, Ar), 7.27 (d, 1H, J=9.2 Hz,
Ar), 7.45 (dd, 1H, J1 =8.8, J2 =2.8 Hz, Ar), 7.55 (dd, 1H,
J1 =8.8, J2 =2.8 Hz, Ar), 7.65–7.67 (m, 2H, Ar), 8.43 (s, 1H,
1 3

Journal of the Iranian Chemical Society
of rotatable bonds was defned to create PDBQT format of
the synthesized ligands. The grid box was set at 60×60×60
with a spacing value of 0.375 Å. Lamarckian genetic algo-
rithm (LGA), with 100 GA runs, and 25,000,000 energy
evaluations for rigid docking were chosen. The other param-
eters were left as default if not stated. Docking procedure
was performed, and cluster analysis was performed by root-
mean-square deviation (RMSD ≤2 Å).
time and higher yields. The best conditions were obtained
when the reaction was performed at 80 °C (Table 1, entry 8).
The ethyl pyruvate is liquid; when it was mixed with aniline
derivatives and heated at 80 °C, the reaction mixture was
changed in liquid solution in most cases and the reaction
proceeds well under solvent-free conditions.
To explore the generality and efciency of the current
protocol, ethyl pyruvate was reacted with diferent aniline
derivatives and the corresponding compounds were synthe-
sized in good-to-high yields (Table 2).
Results and discussion
Results indicate that both electron-donating and elec-
tron-withdrawing substituents on the aniline ring were well
tolerated under optimized conditions. As it is clear from
Table 2, the reactions of anilines having substituents such
as 4-OMe, 4-Me, 3-Me, and 3,4-(Me)2 with ethyl pyruvate
were proceeded efficiently to generate the 2,5-dihydro-
1H-pyrrole-2-carboxylate derivates in good-to-high yields
(Table 2, entries 1–4). Aniline-substituted halogens (such
as 4-Cl-, 3-Cl, 4-Br-) and electron-withdrawing group (such
as 3-NO2-) converted into corresponding products in lower
yields (Table 2, entries 5–8). The low obtained yields for
such aniline derivatives may be related to lower intensity or
nucleophilicity toward condensing with ethyl pyruvate in
comparison with aniline-bearing electron-donating groups
(see mechanism Scheme 3). In addition, aniline-bearing
sulfonamides treated with ethyl pyruvate under optimized
conditions gave the corresponding products (4a-d) in good-
to-high yields (Table 2, entries 9–12). This result ofers the
potential of this method for the synthesis of diverse 2,5-dihy-
dro-1H-pyrrole-2-carboxylate products in good yield after
a short reaction time and with a facile workup procedure.
A proposed mechanistic pathway for the formation of
2,5-dihydro-1H-pyrrole-2-carboxylate derivatives is sug-
gested in Scheme 3.
Chemistry
Initially, the synthesis of the sulfonamide derivatives has
been carried out according to the reported procedure [34].
As briefy shown in Scheme 2, treatment of p-anisidine 5
with acetic anhydride aforded N-(4-methoxyphenyl)aceta-
mide 6, which was further exposed to chlorosulfonylation
with chlorosulfonic acid. The arylsulfonyl chloride interme-
diate 7 was reacted with aniline derivatives including aniline
(a), 4-chloroaniline (b), 4-methylaniline (c), and 3,4-dimeth-
ylaniline (d) in the presence of anhydrous NaHCO3 under
solvent-free conditions at room temperature. Finally, the
compounds (8a–d) were hydrolyzed with water and acid to
give aniline-bearing sulfonamide derivatives (9a–d) in high
yield and purity. (For details, please see the reference 34.)
These synthesized aniline-bearing sulfonamides as well
as aniline derivatives were employed to synthesize the target
pyrrole-2-carboxylate compounds. Then, in order to opti-
mize the reaction conditions, the two-component reaction
of 4-methoxy aniline and ethyl pyruvate was screened as a
model reaction under various solvents and catalyst-free con-
ditions. As listed in Table 1, the efect of diferent solvents
including n-hexane, dichloromethane, chloroform, ethanol,
water, dimethylformamide and solvent-free conditions was
studied under catalyst-free conditions. It was found that in
diferent solvents the reaction gave the corresponding prod-
uct in lower yield after long reaction time (Table 1, entries
1–7), whereas the model reaction was carried out at 110 °C
and lower temperatures gave the product in lower reaction
In the initial step, aniline or aniline-bearing sulfonamide
1 condensed with ethyl pyruvate 2 and gave corresponding
imine or enamine A. Next, enamine A converted into inter-
mediate B by second condensing with another ethyl pyruvate
2, which pursued by dehydration of the aldol product B,
leads to the formation of intermediate C. Michael addition
of aniline or aniline-bearing sulfonamide 1 to intermediate
Scheme 2 Synthesis of sulfonamide derivatives 9a-d
1 3

Journal of the Iranian Chemical Society
Table 1 Optimization of the reaction conditions for the synthesis of 2,5-dihydro-1H-pyrrole-2-carboxylate 3a(a)
(b)
Entry
Solvent
Conditions
Time (min)
Yield(b)
%
1
2
3
4
5
6
7
8
n-Hexane
Refux
Refux
Refux
Refux
Refux
Refux
Refux
RT
120
120
120
120
120
120
120
120
50
24
20
28
10
20
15
20
–
Dichloromethane
Chloroform
Ethyl acetate
Ethanol
Water
Dimethylformamide
Solvent-free
Solvent-free
Solvent-free
Solvent-free
9
10
11
70 °C
80 °C
110 °C
78
90
90
30
25
(a)Reaction conditions: ethyl pyruvate (2 mmol), 4-methoxy aniline (2 mmol), and in the case of solvent (3 mL). (b)Isolated yield
C afords D which then undergoes an intermolecular cycliza-
tion to form 2,5-dihydro-1H-pyrrole-2-carboxylate product
3 or 4.
point of view. However, 4-chloro- and 4-bromo-derivatives
(compounds 3e and 3g, respectively) showed the inhibitory
activity toward all tested cell lines. Therefore, the introduc-
tion of halogen atoms on phenyl ring could improve the
cytotoxic efect against all cancer cells. In the case of methyl
substituent on phenyl ring, the 3-methyl derivative 3c exhib-
ited cytotoxic activity toward all cell lines compared to the
4-methyl isomer 3b. However, the introduction of methyl
group into the 3, 4-positions of phenyl ring in compound 3d
aforded better activity against all cancer cells in comparison
with its counterparts 3b and 3c.
In vitro anticancer screening
The in vitro anticancer activity of synthesized compounds
was evaluated against three human cell lines including
MCF7, MOLT-4, and HL-60 using MTT assay. The cultured
cells were treated with diferent concentrations of the target
compounds (25, 50, and 100 µM), and cisplatin was used as
a reference drug. The cytotoxicity activity of these analogs
is presented in Table 3, as IC50 values in μM. The results
displayed that the tested compounds showed a broad range
of cell growth inhibitory activity toward tested cell lines.
As listed in Table 3, compounds 4a–d possessing sul-
fonamide moiety exhibited better cytotoxic activity against
all cell lines compared to compounds 3a–h. Among them,
compound 4d having 3,4-dimethyl substituents at sulfona-
mide moiety displayed good inhibitory activity against tested
cell lines with 19.8, 15.3 and 17.6 µM IC50 values for MCF7,
MOLT-4, and HL-60, respectively. Also, the results revealed
that the deletion of substitutions from the phenyl ring at sul-
fonamide moiety led to a decrease in the inhibitory activity.
The observed IC50 values of compounds 3a–h indicated
that the 4-methoxy and 3-nitro substituents (3a and 3h,
respectively) were not desirable from cytotoxic activity
Molecular docking study
In order to rationalize the growth inhibitory activity of syn-
thesized compounds by means of molecular docking studies,
hCA II with saccharin as cognate ligand (PDB entry: 2Q38)
was used. The docking results showed estimated free energy
of binding of compounds between −7.49 and −10.14 kcal/
mol (Table 4). The best-ranked docking conformation of
compound 4d within the active site of hCA II is shown in
Fig. 2. Further analysis of results indicates that sulfonamide
moiety in compounds 4a, 4b, 4c, and 4d is involved in addi-
tional hydrogen bonds and plays a crucial role in the growth
inhibitory activity of target compounds. The binding orienta-
tion of the sulfonamide moiety is similar to that of observed
for isothiazol-3(2H)-one 1,1-dioxide ring in saccharin as
1 3

Journal of the Iranian Chemical Society
Table 2 Synthesis of 2,5-dihydro-1H-pyrrole-2-carboxylate derivatives under solvent- and catalyst-free conditions
Entry
R or Ar
Product
Time
(min)
Yield
(%)a
O
H
N
N
OMe
MeO
1
4–MeO–
30
90
H
CO2Et
Me
3a
O
H
N
N
Me
Me
2
3
4–Me–
3–Me–
50
50
75
70
H
CO2Et
Me
3b
4
3,4–(Me)2–
60
75
5
6
7
4–Cl–
3–Cl–
4–Br–
40
40
45
40
44
68
8
9
3–O2N–
50
50
35
85
HN
SO2
NH
O2S
4–MeO–, 3–
(C6H5–
NHSO2)–
O
H
N
N
OCH3
H3CO
CO2Et
Me
4a
Cl
Cl
HN
SO2
NH
O2S
4–MeO–, 3–(4–
Cl-C6H4–
O
N
H
N
10
11
12
70
60
65
78
80
79
OCH3
NHSO2)–
H3CO
CO2Et
Me
4b
Me
Me
HN
SO2
NH
O2S
4–MeO–, 3–(4–
Me–C6H4–
O
H
N
N
OCH3
NHSO2)–
H3CO
CO2Et
Me
4c
Me
Me
Me
Me
HN
SO2
NH
O2S
4–MeO–, 3–
(3,4–(Me)2–
C6H3–NHSO2)–
O
H
N
N
OCH3
H3CO
CO2Et
Me
4d
aIsolated yield
1 3

Journal of the Iranian Chemical Society
Scheme 3 Plausible mechanism
for the synthesis of 2,5-dihydro-
1H-pyrrole-2-carboxylates
Table 3 Cell growth inhibitory activity of synthetic derivatives
co-crystal ligand. A good correlation between the docking
results and growth inhibitory activities can be observed.
assessed by the MTT reduction assay
Compound
IC50 (µM)a
MCF7
MOLT-4
HL-60
Conclusions
3a
>100
>100
>100
3b
83.7 7.2
71.5 5.7
65.9 6.6
58.5 9.8
95.2 9.1
51.4 6.5
>100
53.6 8.0
21.7 2.5
23.6 8.2
19.8 4.9
13.8 0.5
75.4 12.4
55.4 9.2
44.3 3.6
45.2 7.5
80.2 8.3
35.9 8.6
86.2 6.3
36.2 7.1
24.8 3.8
29.3 1.7
15.3 7.7
8.2 2.3
90.1 8.8
73.3 10.7
49.1 10.0
42.1 5.5
97.4 8.7
36.4 3.1
>100
42.4 6.7
32.1 5.2
40.4 9.1
17.6 4.3
9.5 1.8
In conclusion, we introduced a new route for the synthesis of
2,5-dihydro-1H-pyrrole-2-carboxylates via a one-pot, two-
component reaction of ethyl pyruvate and aniline derivatives
under solvent- and catalyst-free conditions. This method has
several advantages including eco-friendly, operational sim-
plicity, mild conditions, easy workup, and high yields of the
products. These products were investigated for their cytotox-
icity activity against MCF7, MOLT-4, and HL-60 cell lines.
Among them, compound 4d possessing sulfonamide moi-
ety displayed good inhibitory activity. Molecular docking
3c
3d
3e
3f
3g
3h
4a
4b
4c
4d
Cisplatin
aValues represent mean S.E.M.
1 3

Journal of the Iranian Chemical Society
Table 4 Molecular docking
results of the synthesized
compounds with hCA II
Compound
∆G (kcal/mol)
H-bonds
Amino acids
Distance (Å)
3a
3b
3c
3d
3e
3f
3g
3h
4a
4b
4c
4d
−8.04
−7.83
−7.81
−8.87
−7.49
−8.25
−8.06
−7.57
−8.86
−9.24
−9.12
−10.14
Tyr7, His94, Gln92
Thr200
Thr200, Pro201
Thr200
Thr200, Gln92
Thr200
Gln92, Thr200
Thr200, Gln92
3.0, 3.1, 3.2
3.1 (3.3)
2.8 (3.6), 3.3
3.1 (3.3)
3.0 (3.1), 3.3
3.2
3.3, 3.0 (3.1)
3.0 (3.1), 3.2
His94, His94, Thr199, Thr200
His94, Gln92, Asn67, Pro201
Trp5, Glu69, Glu92
3.2, 3.4, 3.0 (3.2), 2.3 (3.2)
3.3, 2.7, 2.9 (3.5), 2.9
2.8, 3.2, 3.1
His94, His119, Thr199
2.7, 3.0, 2.4 (3.1)
Fig. 2 Representation of the binding mode of the most active compound 4d in the active site of hCA II (PDB entry: 2Q38)
4. S. Yan, Y. Chen, L. Liu, N. He, J. Lin, Green Chem. 12, 2043
studies were performed to insert the target compounds into
hCA II active site to predict a probable binding mode.
(2010)
5. J. Feng, K. Ablajan, A. Sali, Tetrahedron 70, 484–489 (2014)
6. K. Ablajan, L.J. Wang, Z. Maimaiti, Y.T. Liu, Monatsh. Chem.
145, 491 (2014)
Acknowledgements We are thankful to Persian Gulf University
Research Council for partial support of this work. Also, we are thankful
to the School of Chemistry, Manchester University, for running NMRs.
7. B. Li, M.P.A. Lyle, G. Chen, J. Li, K. Hu, L. Tang, M.A. Alaoui-
Jamali, J. Webster, Bioorg. Med. Chem. 15, 4601 (2007)
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