Synthesis and antiproliferative activity of multisubstituted N-fused heterocycles against the Hep-G2 cancer cell line

Article abstract of DOI:10.1007/s00706-011-0469-7

Pyrrolo[1,2-a]imidazole and pyrrolo[2,1-b]thiazole derivatives were synthesized in a one-pot procedure by [3 + 2] cycloaddition reactions of the corresponding imidazolium ylides and thiazolium ylides with an alkene followed by oxidative aromatization of t

Full text of DOI:10.1007/s00706-011-0469-7

Monatsh Chem (2011) 142:521–528
DOI 10.1007/s00706-011-0469-7
ORIGINAL PAPER
Synthesis and antiproliferative activity of multisubstituted
N-fused heterocycles against the Hep-G2 cancer cell line
•
Yong-Miao Shen Peng-Cheng Lv Ming-Zhu Zhang
•
•
•
•
•
Hui-Quan Xiao Li-Ping Deng Hai-Liang Zhu
Chen-Ze Qi
Received: 27 March 2010 / Accepted: 19 February 2011 / Published online: 28 April 2011
Ó Springer-Verlag 2011
Abstract Pyrrolo[1,2-a]imidazole and pyrrolo[2,1-b]thia-
zole derivatives were synthesized in a one-pot procedure by
[3 ? 2] cycloaddition reactions of the corresponding
imidazolium ylides and thiazolium ylides with an alkene
followed by oxidative aromatization of the primary
cycloadducts under the action of the mild oxidant tetra-
kispyridinecobalt(II) dichromate. Antiproliferative activity
of 14 new bicyclic N-fused heterocycles against the human
hepatocellular liver carcinoma (Hep-G2) cell line were
examined by the MTT method. Some of the compounds
showed favorable antiproliferative activity, especially
compound 3i displayed potent antiproliferative activities
with an IC50 value of 0.36 lg/cm3.
Introduction
Heteroaromatic molecules with an N-fused pyrrole unit
or their reduced analogues are an important structural
motif, many of which are biologically active substances
[1–3]. In particular, the fused 5–5 bicyclic structural
frameworks of pyrrolo[2,1-b]thiazole [3] and pyrrolo[1,2-
a]imidazole with a bridgehead nitrogen atom are widely
found in naturally occurring and synthetic biologically
active molecules. The pyrrolo[2,1-b]thiazole derivatives
display a wide range of biological activities such as
antileukemic [4], anticonvulsant [5], anti-inflammatory
[6, 7], antagonistic [8], and hypoglycemic activities [9].
Some of the pyrrolo[1,2-a]imidazole derivatives are
potent kinase inhibitors with improved affinity and
selectivity, and are useful in designing new kinase
inhibitors [10–12]. Pyrrolo[1,2-a]imidazoles also serve as
potent partial agonists of the a1A adrenergic receptor
with good selectivity over the a1B, a1D, and a2A
receptor subtypes [13]. As a result, the synthesis of
pyrrolo[2,1-b]thiazole [14–19] and pyrrolo[1,2-a]imidaz-
ole [20–23] derivatives has attracted much recent
research interest.
Keywords N-fused heterocycles ꢀ
Antiproliferative activity ꢀ Structure–activity relationship ꢀ
Hep-G2 cell line
Electronic supplementary material The online version of this
article (doi:10.1007/s00706-011-0469-7) contains supplementary
material, which is available to authorized users.
Hepatocellular carcinoma (HCC) is the most commonly
seen histological type of primary liver carcinoma. The
worldwide incidence of HCC is one of the highest of all
cancer types [24–27]. However, there are few reports on
the search for potential drugs with antitumor activity
against HCC, and the bioactivity of pyrrolo[2,1-b]thiazoles
and pyrrolo[1,2-a]imidazoles against HCC [28] has not
been investigated. With the aim of searching for new
pharmaceuticals against HCC, a series of multifunctional-
ized pyrrolo[2,1-b]thiazoles and pyrrolo[1,2-a]imidazoles
were synthesized and their activity in inhibiting the Hep-
G2 cell line was examined.
Y.-M. Shen ꢀ M.-Z. Zhang ꢀ H.-Q. Xiao ꢀ L.-P. Deng ꢀ
C.-Z. Qi (&)
Institute of Chemistry and Chemical Engineering,
Shaoxing University, Shaoxing 312000,
People’s Republic of China
e-mail: shenyongmiao@usx.edu.cn
P.-C. Lv ꢀ H.-L. Zhu
State Key Laboratory of Pharmaceutical Biotechnology,
Nanjing University, Nanjing 210093,
People’s Republic of China
123

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Y.-M. Shen et al.
Results and discussion
triethylamine and TPCD afforded the pyrrolo[1,2-a]imida-
zoles 5a–5e (Scheme 4, Table 2). The only difference in
these cases is that the ylides were prepared in situ from
N-methylimidazole and bromopropanone prior to the 1,3-
cycloadditions without separation because these imidazoli-
um salts are highly hygroscopic. Beside the normal product
5, we obtained the corresponding indolizine derivatives 50 as
by-products (Fig. 2).
Synthesis and chemical characterization
1,3-Dipolar cycloadditions of thiazolium ylides derived
from the thiazolium salts 1a–1c [29] (Table 1, Scheme 1)
with the electron deficient alkenes 2a–2e were carried out
in N,N-dimethylformamide (DMF) solution in the presence
of triethylamine as a base and tetrakispyridinecobalt(II)
dichromate [Py4Co(HCrO4)2] (TPCD) [30] as an oxidant.
During the reactions, the primary cycloadducts, tetrahy-
dropyrrolo[2,1-b]thiazoles, were oxidatively dehydrogenated
by TPCD to give the products. Pyrrolo[2,1-b]thiazoles 3a–3i
were prepared from simple starting materials in satisfactory
yields by similar one-pot reactions (Scheme 2, Table 2).
The structures of the products were assigned on the basis
of their spectral (IR, 1H and 13C NMR, MS) and elemental
analysis data and were further confirmed by X-ray crys-
tallographic analyses of product 3c (Fig. 1).
Table 2 Synthesis of pyrrolo[2,1-b]thiazoles
Product
R
R1
R2
Yield (%)
3a
3b
3c
3d
3e
3f
Cyclopropyl
Cyclopropyl
Cyclopropyl
CH3
H
CN
52
49
28
49
23
16
79
38
51
H
CO2CH3
CO2CH3
CN
CO2CH3
H
CH3
H
CO2CH3
CO2Et
CN
CH3
CO2Et
It can be seen from Table 2 that the yield of compound
3g (79%) in which R is a phenyl group is significantly
higher than those when R is a cyclopropyl group or methyl
group. The yield of compound 3h is lower than 3g because
the ester group is hydrolyzed under the reaction conditions
to give the by-product 3h0 (Scheme 3).
3g
3h
3i
Ph
H
H
Ph
CO2CH3
Cyclopropyl
–CO–NPh–CO–
Similarly, the 1,3-dipolar cycloadditions of imidazolium
ylides derived from 1-methylimidazole and b-bromoketones
4a and 4b with the alkenes 2a, 2b, and 2d in the presence of
Table 1 Yields and melting points of the thiazolium salts
Compd.
R
m.p. (°C) (lit.)
Yield (%)
1a
1b
1c
Cyclopropyl
180–181
62
94
81
CH3
Ph
172–174
215 (210 [29])
R
O
H3C
O
EtOAc
N
H3C
Br
+
1a R=cyclopropyl
1b R=CH3
Br
N
R
S
1c R=Ph
S
Fig. 1 Oak Ridge thermal ellipsoid plot (ORTEP) drawing of
compound 3c
Scheme 1
R
O
R
O
R1
H3C
Br
TPCD,NEt3,DMF
90 °C, 5-10h
H3C
N
N
+
R1HC CHR2
R2
S
1
2
S
2a
2b
R =H, R =CN
1
2
R =H, R =CO2CH3
2c R1=R2=CO2CH3
2d R1=R2=CO2Et
3
1
2
2e
R ,R =-CO-NPh-CO-
Scheme 2
Scheme 3
123

Synthesis and antiproliferative activity of multisubstituted N-fused heterocycles
523
Scheme 4
R
N
O
R
O
O
N
R1
TPCD,NEt3,DMF
90 °C, 5-10h
Br
+
+
R1HC CHR2
R
R1
N
+
N
R2
CH3
N
1
2
2a
R =H, R =CN
4a R=cyclopropyl
4b R=CH3
R2
2b R1=H, R2=CO2CH3
2d R1=R2=CO2Et
CH3
5
5'
Table 3 Synthesis of pyrrolo[1,2-a]imidazoles using TPCD and
MnO2
Compd.
R
R1
R2
Yield (%)
TPCD
MnO2
5a
5a0
5b
5b0
5c
5c0
5d
5d0
5e
Cyclopropyl
Cyclopropyl
CH3
H
CN
21
6
16
–
H
CO2CH3
CN
20
8
15
–
H
17
18
12
15
9
11
–
CH3
H
CO2CH3
CO2Et
10
–
CH3
CO2Et
7
5e0
10
–
Table 4 Antiproliferative effect of pyrrolo[2,1-b]thiazoles and pyr-
rolo[1,2-a]imidazoles against Hep-G2
Fig. 2 ORTEP drawing of compound 5b0
Compd.
IC50 (lg/cm3)
Compd.
IC50 (lg/cm3)
3a
1.1 0.6
37 0.6
1.4 0.6
19 0.9
25 0.7
37 0.6
3.8 0.3
0.25 0.07
3h
3i
5.2 0.6
0.36 0.09
15 0.9
31 0.8
26 0.9
23 0.9
35 0.9
–
N-Methylimidazole (pKa = 7.33) is a stronger base than
pyridine (pKa = 5.2) [31]. The exchange between the
imidazole in the imidazolium ylides and the pyridine in
TPCD results in the formation of pyridinium ylides which
took part in cycloadditions with the alkenes to give the
indolizine derivatives 50. Therefore, when we used freshly
prepared manganese dioxide as an alternative oxidant to
replace TPCD, the products 50 were no longer obtained
(Table 3). However, in this case, the yields of the com-
pounds 5 were not improved. Therefore, TPCD seems to be
a more suitable oxidant in these reactions because it is
more easily prepared and can be stored for a longer time
without losing activity.
3b
3c
5a
5b
5c
5d
5e
–
3d
3e
3f
3g
5-Fluorouracil
cell death with respect to the control culture) are reported in
Table 4.
As shown in Table 4, compound 3i, which contains a
cyclopropylcarbonyl group and N-phenylmaleimide group,
exhibits the most potent antiproliferative activity with an
IC50 value of 0.36 lg/cm3, which is comparable to the
reference compound 5-fluorouracil (IC50 0.25 lg/cm3).
The pyrrolo[2,1-b]thiazole compounds usually have better
antiproliferative activity than the pyrrolo[1,2-a]imidazole
derivatives. Furthermore, pyrrolo[2,1-b]thiazole deriva-
tives with a 7-CN group showed better activity than the
corresponding 7-ester compounds (e.g., 3a vs. 3b, 3d vs.
3e, 3g vs. 3h). The same trend was also found for the
pyrrolo[1,2-a]imidazole series (e.g., 5a vs. 5b, 5c vs. 5d).
Antiproliferative activity against the Hep-G2 cell line
The in vitro antiproliferative activities of the synthe-
sized pyrrolo[2,1-b]thiazoles and pyrrolo[1,2-a]imidazoles
against the human liver cancer cell line Hep-G2 were studied
by applying the MTT colorimetric assay. Compounds were
tested over a range of concentrations from 0.1 to 40 lg/cm3,
and the calculated IC50 values (the IC50 value refers to the
concentration (lg/cm3) of a compound able to cause 50%
123

524
Y.-M. Shen et al.
General procedure for the synthesis of 1a–1c
Conclusion
A mixture of 9.9 g 4-methylthiazole (100 mmol) and
16.3 g 2-bromo-1-cyclopropylethanone (100 mmol) in
50 cm3 EtOAc was stirred at room temperature for 0.5 h.
After it stood for another 48 h, the precipitated solid was
collected and rinsed with 50 cm3 EtOAc to give 1a. Salts
1b and 1c were prepared by the same procedure and
directly used in the next step without any further
purification.
A series of pyrrolo[1,2-a]imidazole and pyrrolo[2,1-b]thi-
azole derivatives were synthesized in a one-pot procedure.
Antiproliferative activity of the 14 new bicyclic N-fused
heterocycles (compounds 3a–3i and 5a–5e) against the
human hepatocellular liver carcinoma (Hep-G2) cell line
was examined. Some of the compounds (e.g., 3a, 3c, and
3i) show favorable antiproliferative activity, with com-
pound 3i, which contains a cyclopropylcarbonyl group and
an N-phenylmaleimide group, having the strongest activity
with an IC50 value of 0.36 lg/cm3.
General procedure for the synthesis of 3a–3i
A mixture of the thiazolium salt (10 mmol), olefin
(40 mmol), 2 cm3 triethylamine, and 4 g TPCD in 40 cm3
DMF was heated at 90 °C for 5–10 h. The reaction course
was monitored by TLC. After the reaction was completed,
the solution was cooled and the reaction mixture was
poured into an aqueous hydrochloric acid solution (5%,
100 cm3). The precipitated crude product was collected by
filtration and further purified by silica gel column chro-
matography with petroleum ether (b.p. 60–90 °C)/ethyl
acetate as eluents (gradient elution).
Experimental
General methods
Melting points were measured on a X-4 (Taike Corp.,
1
Beijing, China) microscopic melting point apparatus. H
NMR spectra were recorded on a Bruker ACF-400 spec-
trometer with CDCl3 as solvent unless otherwise specified.
13C NMR spectra were measured on a Bruker ACF-400
spectrometer at 100 MHz with CDCl3 as solvent. The
chemical shifts (d) are reported in ppm relative to the
residual undeuterated solvent signal, and coupling con-
stants (J) are given in Hz. IR spectra were measured with a
Nicolet FT-IR 5DX spectrometer by attenuated total
reflection Fourier transform infrared (ATR-FTIR) spec-
troscopy. Mass spectra (EI) were recorded with a VG ZAB-
HS spectrometer. Elemental analyses were obtained using a
Heraeus CHN-O-Rapid analyzer, and results agreed with
calculated values.
General procedure for the synthesis of 5a–5e
A mixture of 1-methyl-1H-imidazole (10 mmol), bromo-
propanone (10 mmol), olefin (40 mmol), 2 cm3 triethy-
lamine, and 4 g TPCD in 40 cm3 DMF was heated at 90 °C
for 5–10 h. The reaction course was monitored by TLC.
After the reaction was completed, the solution was cooled
and the reaction mixture was poured into an aqueous
hydrochloric acid solution (5%, 100 cm3). The precipitated
crude product was collected by filtration and further purified
by silica gel column chromatography with petroleum ether
(b.p. 60–90 °C)/ethyl acetate as eluents (gradient elution).
For X-ray crystallographic analysis, the X-ray diffrac-
tion intensities and the unit cell parameters were
determined on a Bruker SMART APEXII CCD diffrac-
tometer employing graphite-monochromated (Mo-Ka)
3-(2-Cyclopropyl-2-oxoethyl)-4-methylthiazol-3-ium
bromide (1a, C9H12NOS)
.
White powder; IR (ATR): vꢀ = 3,131, 1,660 cm-1 1H
˚
radiation (k = 0.71073 A) and operating in the x/2h scan
mode. Data collection and cell refinement were performed
with APEX2 software. Structures were solved by direct
methods and refined by full-matrix least-squares on F2 with
SHELXTL. Non-hydrogen atoms were refined by aniso-
tropic displacement parameters, and the positions of all
hydrogen atoms were fixed geometrically and included in
estimated positions using a riding model. Crystallographic
data (excluding structure factors) for the structures in this
paper have been deposited with the Cambridge Crystallo-
graphic Data Centre as supplementary publication numbers
CCDC 745819 and 745820. Copies of the data can be
obtained, free of charge, on application to CCDC, 12 Union
Road, Cambridge, CB2 1EZ, UK (fax: ?44 1223 336033
or e-mail: deposit@ccdc. cam.ac.uk).
NMR (D2O, 400 MHz): d = 1.32 (t, 2H, J = 2.0 Hz), 1.32
(t, 2H, J = 3.2 Hz), 2.44 (t, 1H, J = 2.9 Hz), 2.54 (s, 3H),
4.80 (s, 2H), 5.94 (s, 1H), 7.99 (s, 1H) ppm; 13C NMR
(D2O, 100 MHz): d = 12.2, 13.1, 36.2, 60.5, 120.6, 146.9,
159.4, 202.2 ppm.
4-Methyl-3-(2-oxopropyl)thiazol-3-ium bromide
(1b, C7H10NOS)
.
White powder; IR (ATR): vꢀ = 3,016, 1,723 cm-1 1H
NMR (D2O, 400 MHz): d = 2.55 (s, 6H), 5.79 (s, 2H),
8.00 (s, 1H), 9.89 (s, 1H) ppm; 13C NMR (D2O, 100 MHz):
d = 12.1, 19.1, 60.4, 120.5, 146.9, 159.4, 204.6 ppm.
123

Synthesis and antiproliferative activity of multisubstituted N-fused heterocycles
525
4-Methyl-3-(2-oxo-2-phenylethyl)thiazol-3-ium bromide
(1c) [29]
5-Acetyl-3-methylpyrrolo[2,1-b]thiazole-7-carbonitrile
(3d, C10H8N2OS)
1
White needles. H NMR (D2O, 400 MHz): d = 2.59 (s,
Yellow powder; m.p.: 198–199 °C; IR (ATR): vꢀ = 3,133,
1
3H), 4.80 (s, 2H), 6.41 (d, 1H, J = 7.6 Hz), 7.77 (t, 2H,
J = 7.6 Hz), 7.94 (t, 1H, J = 7.2 Hz), 8.06 (s, 1H), 8.22
(d, 2H, J = 7.6 Hz) ppm; 13C NMR (D2O, 100 MHz):
d = 12.2, 58.4, 120.7, 128.5, 129.2, 132.8, 135.6, 147.2,
192.2 ppm.
3,098, 2,221, 1,652, 952, 734 cm-1; H NMR (400 MHz,
CDCl3): d = 2.50 (s, 3H), 2.72 (s, 3H), 6.60 (s, 1H), 7.51
(s, 1H) ppm; 13C NMR (100 MHz, CDCl3): d = 17.4,
27.3, 83.9, 110.9, 114.6, 127.9, 136.3, 147.1 ppm; MS: m/
z (%) = 204 (M?, 25), 189 (100), 161 (8).
5-Cyclopropylcarbonyl-3-methylpyrrolo[2,1-b]thiazole-
7-carbonitrile (3a, C12H10N2OS)
Methyl 5-acetyl-3-methylpyrrolo[2,1-b]thiazole-
7-carboxylate (3e, C11H11NO3S)
Yellow powder; m.p.: 158–159 °C; IR (ATR): vꢀ = 3,127,
Yellow powder; m.p.: 142–144 °C; IR (ATR): vꢀ = 3,100,
1
3,010, 2,219, 1,657, 1,452, 965, 735 cm-1
;
1H NMR
2,949, 2,362, 1,702, 1,665, 1,221, 943, 758, 705 cm-1; H
(400 MHz, CDCl3): d = 0.99–1.04 (m, 2H), 1.16–1.20 (m,
2H), 2.40–2.46 (m, 1H), 2.65 (s, 3H), 6.59 (s, 1H), 7.67 (s,
1H) ppm; 13C NMR (100 MHz, CDCl3): d = 10.8, 17.1,
18.3, 83.7, 110.8, 114.8, 127.0, 128.3, 136.0, 146.6,
187.7 ppm; MS: m/z (%) = 230 (M?, 100), 189 (44),
175 (7), 161 (8).
NMR (400 MHz, CDCl3): d = 2.50 (s, 3H), 2.73 (d, 3H,
J = 0.8 Hz), 3.90 (s, 3H), 6.56 (s, 1H), 7.69 (d, 1H,
J = 1.2 Hz) ppm; 13C NMR (100 MHz, CDCl3):
d = 17.4, 27.1, 51.6, 105.6, 111.3, 125.9, 127.9, 135.9,
146.7, 163.6, 185.5 ppm; MS: m/z (%) = 237 (M?, 43),
222 (100), 206 (12).
Methyl 5-cyclopropylcarbonyl-3-methylpyrrolo[2,1-b]-
thiazole-7-carboxylate (3b, C13H13NO3S)
Diethyl 5-acetyl-3-methylpyrrolo[2,1-b]thiazole-6,7-
dicarboxylate (3f, C15H17NO5S)
Yellow powder; m.p.: 176 °C; IR (ATR): vꢀ = 3,086,
Yellow powder; m.p.: 114–115 °C; IR (ATR): vꢀ = 3,104,
1
2,932, 1,708, 1,643, 1,223, 966, 765, 734 cm-1; H NMR
2,923, 1,727, 1,696, 1,662, 1,421, 1,208, 966, 778 cm-1
;
(400 MHz, CDCl3): d = 0.97 (s, 2H), 1.15 (s, 2H), 2.50 (s,
1H), 2.67 (s, 3H), 3.91 (s, 3H), 6.56 (s, 1H), 7.86 (s,
1H) ppm; 13C NMR (100 MHz, CDCl3): d = 10.4, 17.1,
18.0, 51.6, 105.6, 111.2, 125.1, 128.4, 135.7, 146.2, 163.8,
188.0 ppm; MS: m/z (%) = 263 (M?, 100), 222 (39), 204
(14), 164 (7).
1H NMR (400 MHz, CDCl3): d = 1.37 (t, 3H,
J = 7.0 Hz), 1.43 (t, 3H, J = 7.2 Hz), 2.47 (s, 3H), 2.58
(s, 3H), 4.35 (q, 2H, J = 7.1 Hz), 4.47 (q, 2H,
J = 7.2 Hz), 6.62 (s, 1H) ppm; 13C NMR (100 MHz,
CDCl3): d = 14.0, 14.4, 17.1, 28.2, 60.6, 62.4, 103.9,
112.3, 125.1, 129.6, 135.6, 145.1, 162.1, 165.6, 186.1 ppm;
MS: m/z (%) = 323 (M?, 31), 277 (47), 234 (22), 177
(100).
Diethyl 5-cyclopropylcarbonyl-3-methylpyrrolo[2,1-b]-
thiazole-6,7-dicarboxylate (3c, C15H15NO5S)
Yellow powder; m.p.: 118–119 °C; IR (ATR): vꢀ = 2,957,
2,360, 1,720, 1,662, 1,219, 739 cm-1; 1H NMR (400 MHz,
CDCl3): d = 0.98–1.03 (m, 2H), 1.22–1.26 (m, 2H), 2.37
(d, 1H, J = 3.9 Hz), 2.45 (d, 3H, J = 0.8 Hz), 3.90 (s,
3H), 3.97 (s, 3H), 6.62 (d, 1H, J = 0.8 Hz) ppm; 13C NMR
(100 MHz, CDCl3): d = 11.9, 16.4, 20.1, 51.8, 53.0,
103.5, 112.1, 125.7, 128.1, 134.7, 143.9, 162.7, 166.0,
190.0 ppm; MS: m/z (%) = 321 (M?, 100), 289 (92), 258
(23), 203 (32), 175 (23).
5-Benzoyl-3-methylpyrrolo[2,1-b]thiazole-7-carbonitrile
(3g, C15H10N2OS)
Yellow powder; m.p.: 177–179 °C; IR (ATR): vꢀ = 3,087,
1
2,925, 2,225, 1,628, 910, 709 cm-1; H NMR (400 MHz,
CDCl3): d = 2.66 (s, 3H), 6.69 (s, 1H), 7.24 (s, 1H), 7.52
(t, 2H, J = 7.6 Hz), 7.63 (t, 1H, J = 7.4 Hz), 7.91 (d, 2H,
J = 7.6 Hz) ppm; 13C NMR (100 MHz, CDCl3):
d = 16.5, 83.8, 111.0, 114.7, 126.6, 128.5, 129.78,
129.82, 132.9, 135.6, 137.9, 146.6, 182.5 ppm; MS: m/
z (%) = 266 (M?, 100), 265 (75), 189 (19), 105 (18), 77
(38).
X-ray structure analysis: C15H15NO5S, M = 243.25.
˚
Orthorhombic, space group Iba2, a = 10.9785(16) A,
˚
˚
b = 34.405(6) A, c = 8.0074(13) A, a = 90°, b = 90°,
Methyl 5-benzoyl-3-methylpyrrolo[2,1-b]thiazole-7-
carboxylate (3h, C16H13NO3S)
3
-3
˚
c = 90°, V = 3024.6(8) A , Z = 8, Dc = 1.411 g cm
,
F(000) = 1,344.0, absorption coefficient 0.237 mm-1
,
Yellow powder; m.p.: 139–141 °C; IR (ATR): vꢀ = 3,098,
scan range for data collection 1.93 B h B 27.52°, 9,467
measured reflections, 3,028 independent reflections, 2,248
reflections with I [ 2r(I), Rint = 0.0262, 200 refinable
parameters, R[F2 [ 2r(F2)] = 0.0361, wR2(F2) = 0.0990.
2,929, 1,696, 1,633, 1,246, 901, 700 cm-1 1H NMR
;
(400 MHz, CDCl3): d = 2.68 (d, 3H, J = 0.8 Hz), 3.88 (s,
3H), 6.67 (d, 1H, J = 0.8 Hz), 7.41 (d, 1H, J = 0.8 Hz),
7.48–7.52 (m, 2H), 7.58-7.62 (m, 1H), 7.91 (d, 2H,
123

526
Y.-M. Shen et al.
J = 1.6 Hz) ppm; 13C NMR (100 MHz, CDCl3): d = 16.6,
29.7, 51.6, 105.8, 111.4, 126.9, 128.1, 128.4, 129.8, 132.4,
135.4, 138.6, 146.4, 163.7, 183.1 ppm; MS: m/z (%) = 299
(M?, 100), 298 (24), 268 (19), 222 (12), 105 (11), 77 (20).
Methyl 5-cyclopropylcarbonyl-1-methyl-1H-pyrrolo-
[1,2-a]imidazole-7-carboxylate (5b, C13H14N2O3)
Yellow powder; m.p.: 115–117 °C; IR (ATR): vꢀ = 2,949,
1,689, 1,611, 1,496, 1,203, 1,089, 686 cm-1 1H NMR
;
(400 MHz, CDCl3): d = 0.89–0.93 (m, 2H), 1.14–1.17 (m,
2H), 2.39 (d, 1H, J = 3.9 Hz), 3.84 (s, 3H), 4.12 (s, 3H),
6.77 (d, 1H, J = 0.8 Hz), 7.69 (s, 1H), 7.94 (d, 1H,
J = 2.4 Hz) ppm; 13C NMR (100 MHz, CDCl3): d = 9.5,
16.3, 35.6, 51.0, 92.5, 110.9, 118.8, 122.6, 124.0, 140.1,
164.1, 187.8 ppm; MS: m/z (%) = 246 (M?, 100), 215 (7),
205 (25).
5-Benzoyl-3-methylpyrrolo[2,1-b]thiazole-7-carboxylic
acid (3h0, C15H11NO3S)
Yellow powder; m.p.: 115–117 °C; IR (ATR): vꢀ = 3,262,
2,955, 1,726, 1,629, 1,556, 1,215, 759, 698 cm-1; 1H NMR
(400 MHz, CDCl3): d = 3.85 (s, 3H), 7.31 (q, 1H,
J = 1.3 Hz), 7.49–7.53 (m, 2H), 7.59–7.63 (m, 1H), 7.72
(q, 1H, J = 1.6 Hz), 7.91–7.94 (m, 2H), 10.11 (s, 1H)
ppm; 13C NMR (100 MHz, CDCl3): d = 51.46, 51.51,
118.5, 119.3, 128.57, 128.63, 128.7, 129.0, 131.5, 132.5,
Methyl 1-cyclopropylcarbonylindolizine-3-carboxylate
(5b0, C14H13NO3)
137.4, 164.3, 185.0 ppm; MS: m/z (%) = 241 (M?
-
White powder; m.p.: 135–136 °C; IR (ATR): vꢀ = 3,113,
COOH, 0.14), 229 (100), 198 (86), 170 (19), 152 (20), 120
(41), 105 (13), 77 (21).
2,951, 1,697, 1,618, 756 cm-1 1H NMR (400 MHz,
;
CDCl3): d = 0.97–1.02 (m, 2H), 1.21–1.25 (m, 2H),
2.56–2.62 (m, 1H), 3.94 (s, 3H), 6.99 (t, 1H, J = 7.0 Hz),
7.38 (t, 1H, J = 7.8 Hz), 8.17 (s, 1H), 8.34 (d, 1H,
J = 9.2 Hz), 9.90 (d, 1H, J = 6.8 Hz) ppm; 13C NMR
(100 MHz, CDCl3): d = 10.3, 18.0, 51.3, 105.4, 115.0,
119.2, 123.3, 125.4, 127.0, 129.1, 139.2, 164.5, 189.8 ppm;
MS: m/z (%) = 243 (M?, 100), 212 (10), 202 (19).
5-Cyclopropylcarbonyl-3-methyl-7-phenyl-6H-pyrrolo-
[30,40:3,4]pyrrolo-[2,1-b]thiazole-6,8(7H)-dione
(3i, C19H14N2O3S)
Yellow powder; m.p.: 177–179 °C; IR (ATR): vꢀ = 2,925,
2,853, 1,757, 1,699, 1,656, 895, 761, 730, 699 cm-1; H
1
NMR (400 MHz, CDCl3): d = 1.12–1.17 (m, 2H),
1.22–1.26 (m, 2H), 2.59 (d, 3H, 1.2), 3.37–3.43 (m, 1H),
6.71 (d, 1H, J = 1.2 Hz), 7.37–7.42 (m, 3H), 7.47–7.51
(m, 2H) ppm; 13C NMR (100 MHz, CDCl3): d = 12.7,
17.0, 20.7, 108.9, 112.9, 124.7, 127.1, 128.0, 129.0, 130.2,
132.3, 135.6, 136.4, 161.5, 163.2, 189.8 ppm; MS: m/
z (%) = 350 (M?, 100), 322 (18), 258 (14), 203 (14).
X-ray structure analysis: C14H13NO3, M = 243.25.
ꢀ
˚
Triclinic, space group P1, a = 7.2548(6) A, b = 8.2019(8)
˚
˚
A, c = 11.0324(10) A, a = 89.636(7), b = 71.956(6),
3
˚
c = 70.945(6), V = 586.69(9) A , Z = 2, Dc = 1.377
g cm-3, F(000) = 256.0, absorption coefficient 0.098
mm-1
, scan range for data collection 1.95 B h B
27.62°, 9,170 measured reflections, 2,715 independent
reflections, 1,821 reflections with I [ 2r(I), Rint = 0.0262,
163 refinable parameters, R[F2 [ 2r(F2)] = 0.0452,
wR2(F2) = 0.1367.
5-Cyclopropylcarbonyl-1-methyl-1H-pyrrolo[1,2-a]-
imidazole-7-carbonitrile (5a, C12H11N3O)
Yellow powder; m.p.: 168–170 °C; IR (ATR): vꢀ = 3,163,
2,919, 2,207, 1,620, 1,604, 1,498, 702 cm-1 1H NMR
;
(400 MHz, CDCl3): d = 0.95 (q, 2H, J = 3.6 Hz), 1.17 (t,
2H, J = 3.6 Hz), 2.29–2.35 (m, 1H), 3.88 (s, 3H), 6.84 (s,
1H), 7.47 (s, 1H), 7.94 (s, 1H) ppm; 13C NMR (100 MHz,
CDCl3): d = 9.9, 16.5, 33.5, 67.0, 111.5, 116.2, 119.6,
122.3, 124.2, 140.6, 187.7 ppm; MS: m/z (%) = 213 (M?,
100), 172 (38), 144 (31).
5-Acetyl-1-methyl-1H-pyrrolo[1,2-a]imidazole-7-
carbonitrile (5c, C10H9N3O)
Yellow powder; m.p.: 188–190 °C; IR (ATR): vꢀ = 3,107,
2,203, 1,626, 1,501, 703 cm-1 1H NMR (400 MHz,
;
CDCl3): d = 2.40 (s, 3H), 3.89 (s, 3H), 6.85 (q, 1H,
J = 0.9 Hz), 7.33 (d, 1H, J = 1.2 Hz), 7.96 (d, 1H, J =
2.4 Hz) ppm; 13C NMR (100 MHz, CDCl3): d = 25.0,
33.5, 67.1, 111.6, 116.0, 119.3, 122.3, 125.0, 185.4 ppm;
MS: m/z (%) = 187 (M?, 60), 172 (100), 144 (74).
1-Cyclopropylcarbonylindolizine-3-carbonitrile
(5a0, C13H10N2O)
White crystals; m.p.: 163–164 °C; IR (ATR): vꢀ = 3,114,
2,924, 2,213, 1,637, 1,628, 942, 758 cm-1
;
1H NMR
1-Acetylindolizine-3-carbonitrile (5c0, C11H8N2O)
(400 MHz, CDCl3): d = 1.04 (s, 2H), 1.25 (s, 2H), 2.51 (s,
1H), 7.06 (s, 1H), 7.42 (s, 1H), 7.79 (s, 1H), 7.94 (s, 1H),
9.90 (br, 1H) ppm; 13C NMR (100 MHz, CDCl3):
d = 10.7, 18.2, 84.4, 115.3, 115.6, 117.4, 123.8, 125.7,
127.2, 129.5, 140.7, 189.5 ppm; MS: m/z (%) = 210 (M?,
100), 169 (46), 141 (15), 114 (8).
White powder; m.p.: 199–201 °C; IR (ATR): vꢀ = 3,116,
2,925, 2,218, 1,647, 1,206, 753 cm-1; 1H NMR (400 MHz,
CDCl3): d = 2.59 (s, 3H), 7.08–7.11 (m, 1H), 7.45 (dd, 1H,
J = 8.4, 7.2 Hz), 7.78 (d, 1H, J = 1.6 Hz), 7.81 (s, 1H),
9.90 (d, 1H, J = 7.2 Hz) ppm; 13C NMR (100 MHz,
CDCl3): d = 27.3, 84.3, 115.2, 115.8, 117.4, 123.2,
123

Synthesis and antiproliferative activity of multisubstituted N-fused heterocycles
527
126.4, 127.5, 129.5, 140.8, 187.4 ppm; MS: m/z (%) = 184
(M?, 31), 169 (100), 141 (23), 114 (6).
Antiproliferative activities assay
The antiproliferative activities of the pyrrolo[2,1-b]thiazole
and pyrrolo[1,2-a]imidazole derivatives were determined
using a standard MTT-based colorimetric assay (Sigma).
Briefly, cell lines were seeded at a density of 7 9 103 cells/
well in 96-well microtiter plates (Costar). After 12 h,
exponentially growing cells were exposed to the indicated
compounds at final concentrations ranging from 0.1 to
40 lg/cm3. After 48 h, cell survival was determined by the
addition of an MTT solution (25 mm3 of 4 mg/cm3 MTT in
PBS). After 6 h, 100 mm3 of 10% SDS in 0.01 N HCl was
added, and the plates were incubated at 37 °C for a further
12 h; optical absorbance was measured at 570 nm on an
LX300 Epson Diagnostic microplate reader. Survival ratios
are expressed in percentages with respect to untreated cells.
IC50 values were determined from replicates of six wells
from at least two independent experiments.
Methyl 5-acetyl-1-methyl-1H-pyrrolo[1,2-a]imidazole-
7-carboxylate (5d, C11H12N2O3)
Yellow powder; m.p.: 158–160 °C; IR (ATR): vꢀ = 3,093,
2,955, 1,686, 1,619, 1,198, 691 cm-1; 1H NMR (400 MHz,
CDCl3): d = 2.42 (s, 3H), 3.84 (s, 3H), 4.12 (s, 3H), 6.79
(s, 1H), 7.55 (s, 1H), 7.96 (d, 1H, J = 2.0 Hz) ppm; 13C
NMR (100 MHz, CDCl3): d = 24.9, 35.6, 51.1, 92.7,
111.0, 118.4, 122.7, 125.0, 140.2, 164.0, 185.5 ppm; MS:
m/z (%) = 220 (M?, 100), 205 (81), 189 (21), 177 (8).
Methyl 1-acetylindolizine-3-carboxylate
(5d0, C12H11NO3)
White crystals; m.p.: 151–152 °C; IR (ATR): vꢀ = 3,116,
2,952, 1,694, 1,623, 1206, 745 cm-1; 1H NMR (400 MHz,
CDCl3): d = 2.60 (s, 3H), 3.94 (s, 3H), 7.02–7.06 (m, 1H),
7.39–7.43 (m, 1H), 8.01 (s, 1H), 8.35 (d, 1H, J = 9.2 Hz),
9.90–9.92 (m, 1H) ppm; 13C NMR (100 MHz, CDCl3):
d = 27.2, 51.2, 105.4, 115.2, 119.3, 122.8, 126.1, 127.2,
129.1, 139.3, 164.4, 187.8 ppm; MS: m/z (%) = 217 (M?,
100), 202 (86), 186 (67), 174 (8).
Acknowledgments The work was supported by the National Nat-
ural Science Foundation of China (NSFC, 30772627) and Zhejiang
Provincial Natural Science Foundation (Y4080395, Y4080481).
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Yellow powder; m.p.: 78–80 °C; IR (ATR): vꢀ = 2,984,
1,724, 1,689, 1,628, 1,200, 730, 694 cm-1 1H NMR
;
(400 MHz, CDCl3): d = 1.34 (t, 3H, J = 7.2 Hz), 1.43 (t,
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