Synthesis and antiproliferative activity of indolizine derivatives incorporating a cyclopropylcarbonyl group against Hep-G2 cancer cell line

Article abstract of DOI:10.1016/j.ejmech.2010.02.056

Indolizine and annulated indolizine derivatives incorporating a cyclopropylcarbonyl group were synthesized in a one pot procedure by the tanden reactions of [3+2] cycloaddition of the corresponding N-ylide with electron deficient alkene. Seventeen indolizine derivatives were reported for the first time. All the compounds were examined for their antiproliferative activity against the human hepatocellular liver carcinoma (Hep-G2) cell line by MTT method. Among the compounds tested, 5a, 5d, 5g and 5j showed the most favorable activities with IC₅₀ values of 0.39, 0.48, 0.29 and 0.20 μg/mL. Especially, compound 5j displayed potent antiproliferative activities with IC₅₀ value of 0.20 μg/mL, and showed significant EGFR kinase inhibitory activity with IC₅₀ value of 0.085 mM. Docking simulations of 5j were carried out to illustrate the binding mode of the molecular into the EGFR active site.

Full text of DOI:10.1016/j.ejmech.2010.02.056

European Journal of Medicinal Chemistry 45 (2010) 3184e3190
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and antiproliferative activity of indolizine derivatives incorporating
a cyclopropylcarbonyl group against Hep-G2 cancer cell line
,
,
*
Yong-Miao Shen a b, Peng-Cheng Lv a, Wu Chen a, Peng-Gang Liu a, Ming-Zhu Zhang b, Hai-Liang Zhu a
a State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China
b Institute of Chemistry and Chemical Engineering, Shaoxing University, Shaoxing 312000, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Indolizine and annulated indolizine derivatives incorporating
a cyclopropylcarbonyl group were
Received 21 August 2009
Received in revised form
21 December 2009
Accepted 23 February 2010
Available online 1 March 2010
synthesized in a one pot procedure by the tanden reactions of [3 þ 2] cycloaddition of the corresponding
N-ylide with electron deficient alkene. Seventeen indolizine derivatives were reported for the first time.
All the compounds were examined for their antiproliferative activity against the human hepatocellular
liver carcinoma (Hep-G2) cell line by MTT method. Among the compounds tested, 5a, 5d, 5g and 5j
showed the most favorable activities with IC50 values of 0.39, 0.48, 0.29 and 0.20
compound 5j displayed potent antiproliferative activities with IC50 value of 0.20 g/mL, and showed
significant EGFR kinase inhibitory activity with IC50 value of 0.085 M. Docking simulations of 5j were
carried out to illustrate the binding mode of the molecular into the EGFR active site.
Ó 2010 Elsevier Masson SAS. All rights reserved.
mg/mL. Especially,
m
Keywords:
m
Indolizine derivatives
Antiproliferative activities
Hep-G2 cell line
EGFR
Structure-activity relationship
Docking simulations
1. Introduction
replacement of an alicyclic chain or a larger alicyclic ring often
showed beneficial effect to the bioactivity of the original targets.
However, there were only a few scatter report on indolizine
derivatives containing a cyclopropyl group and their biological
activity are rarely investigated [22,23]. The hepatocellular carci-
noma (HCC) is the most often seen histological type of primary liver
carcinoma and is one of the cancer types of highest incidence in the
world [24]. Although there were several reports on the antitumor
activity of indolizines, their bioactivity against HCC have not been
investigated. With the aim of searching for new pharmaceuticals
against HCC, we designed and synthesized a series of indolizines
and annulated indolizines incorporating a cyclopropylcarbonyl
group and examined their activity against the Hep-G2 cell line.
Receptor protein tyrosine kinases play a key role in signal trans-
duction pathways that regulate cell division and differentiation.
Among the growth factor receptor kinases, EGFR kinase (also known
as erb-B1 or HER-1) is important in cancer deregulation of growth-
factor signaling due to hyperactivation of EGFR is seen in several
cancer types. Compounds that inhibit the kinase activity of EGFR
after binding of its cognate ligand are of potential interest as new
therapeutic antitumor agents [25,26]. Docking simulations of
compound 5j were carried out to give structural insights into the
binding mode with the epidermal growth factor receptor tyrosine
kinase (EGFR TK), to illustrate the antiproliferative activities against
the Hep-G2 cancer cell line. In the binding model, compound 5j is
The indolizine and hydrogenated indolizine structures are found
in many alkaloids such as amorine, crythraline, swainsonine,
cryptaustoline, cryptowoline [1,2], camptothesin [3], nuevamine
[4,5], etc. These natural and many synthetic indolizine derivatives
have been found to have a variety of biological activity, such as
anti-inflammatory [6,7], antiviral [8], aromatase inhibitory [9],
analgestic [10], antitumor [11,12] activities. Besides, some of them
are potent antioxidants inhibiting lipid peroxidation in vitro
[13e15]. As a result, indolizine derivatives have now become
important target compounds in developing new pharmaceuticals
for the treatment of cancer, cardiovascular diseases [16], and HIV
infections [17,18].
Meanwhile, the cyclopropane is a noteworthy structural motif
because of its own biological activity and its effect to modify the
bioactivity of the parent compounds [19,20]. As a rigid yet chemi-
cally relatively stable structural unit, cyclopropane is sterically
smaller than isopropyl and less sterically demanding than germinal
dimethyl. Because of these characteristics, cyclopropyl was more
and more to be taken into consideration in designing new
pharmaceuticals [21]. The introduction of a cyclopropyl or its
* Corresponding author. Tel./fax: þ86 25 8359 2672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.02.056

Y.-M. Shen et al. / European Journal of Medicinal Chemistry 45 (2010) 3184e3190
3185
Table 1
nicely bound to the amino hydrogen of Met 769, with the eCN
Yield and melting point of the pyridinium salts.
forming a more optimal H-bond interaction, and carbonyl group of
compound 5j also forms hydrogen bond with the side chain amino
hydrogen of Lys 721. This molecular docking result, along with the
data of EGFR inhibition assay, suggesting that compound 5j is
a potential inhibitor of EGFR with potent antiproliferative activity.
Iminium salts
R
m.p. (ꢀC)
Yield (%)
2a
2b
2c
2d
2e
2f
3
H
4-CH3
2-Br
2-CH3,5-Br
2,3-benzo
3,4-benzo
e
160e162
172e173
174e175
166e168
180e181
187e188
208e209
97
98
49
62
61
99
98
2. Result and discussion
2.1. Synthesis of 3-Cyclopropylcarbonylindolizines
Reaction of cyclopropylbromomethylmethanone with the pyri-
dine derivatives in ethyl acetate or under solvent free conditions
afforded the pyridinium salts in moderate to high yield. (Scheme 1
and Table 1).
2.2. Biological assay
The in vitro antiproliferative activities of the above synthesized
indolizines were studied on the human liver cancer cell line Hep-G2
by applying the MTT colorimetric assay. Compounds were tested
over a range of concentrations from 0.1 to 40
calculated IC50 values, that is, the concentration (
a compound able to cause 50% of cell death with respect to the
control culture, are reported in Table 3.
The results in Table 3 show that several of the products (5a,
5d, 5g and 5j) strongly exhibit the most potent activity with IC50
The [3 þ 2] cycloadditions of the in situ prepared pyridinium
ylide with the electron deficient alkene were carried out in DMF
solution in the presence of triethylamine as a base and the oxidant
tetrakispyridinecobalt(II) dichromate [Py4Co(HCrO4)2] (TPCD) [27]
as a dehedrogenative reagent. The primary cycloadduct tetrahy-
droindolizine was oxidatively dehydrogenated in situ by TPCD to
give the final product. By these one pot tandem reactions,
3-cyclopropylcarbonylindolizines (5ae5p) and the yet unknown
imidazo[2,1-f]pyrrolo[1,2-b]pyridazine derivatives(6a, 6b) were
prepared (Schemes 2 and 3, Table 2). The structures of the products
are based on their spectral (IR, 1H and 13C NMR, MS) and elemental
analysis data and are further confirmed by an X-ray crystallo-
graphic analysis of product 5e (Fig. 1).
It is seen in Table 2 that when R2 is a cyano group, the yields of
the products (5a, 5e, 5h, 5j, 5l, 5n and 6a) are significantly higher
than those when R2 is an ester group (5b, 5c, 5f, 5g, 5i, 5k, 5m, 5o,
5p and 6b). This is because the dipolarophiles methyl acrylate and
dimethyl maleate are prone to polymerize under heating. As
a matter of fact, a trimer of the dimethyl maleate (trimethyl
1,3,5-benzene tricarboxylate) was isolated from the reaction
mixture after column chromatography.
The cycloadditions are regioselective, with the electron with-
drawing group (the cyano or the ester group) bonded to the Cortho
atom in the pyridinium ylide to give the 1-cyano (1-alkox-
ycarbonyl) 3-cyclopropylcarbonyl indolizines and their annulated
products. This regioselectivity can be rationalized by considering
the frontier molecular orbital (FMO) interactions [28] in the two
reactants as shown in Fig. 2. It can be seen that in these 1,3-dipolar
cycloadditions, in the predominant HOMO(ylide)-LUMO(alkene)
interactions, maximum positive FMO overlap indeed result in the
actually observed regioselectivity.
It is also noted that by using maleimide as the dipolarophile,
beside the normal product 5d, the indolizinecarboxamide product
5d0 (Scheme 4) was also obtained. This may derive from decar-
boxylative hydrolysis of 5d in the basic reaction media under
heating. It is known that the indolizine carboxylic acid derivatives
are difficult to undergo aminolysis reaction [29], therefore, our
finding of formation of 5d0 from 5d might provide a route for the
access of the indolizinecarboxamides after the reaction conditions
are further improved and optimized.
m
g/mL, and the
mg/mL) of
values of 0.39, 0.48, 0.29 and 0.2 mg/mL. It is also seen that as
a general trend, the indolizines with a 1eCN group usually have
better antiproliferative activity than the corresponding analog
with a 1-ester functionality (eg. 5a vs. 5b, 5e vs. 5f, 5h vs 5i, 5l vs.
5m, 6a vs. 6b). The presence of methyl group and halo atom has
significant influence on the anticancer activity. Furthermore, the
carboxamide group (5d IC50 ¼ 0.48
m
g/mL and 5d0 IC50 ¼ 0.56
mg/
mL) can significantly increase the activity. The benzo fusion of
the indolizines at either 5,6- (5l and 5m) or 7,8- (5n an 5o)
positions showed no better antiproliferative activities against
Hep-G2 and these annulated indolizines turned out to be rather
weaker antiproliferative agents. Compounds 6a and 6b show the
same trend. Furthermore, compound 5j was evaluated for their
ability to inhibit EGFR tyrosine kinases. The results (Table 4)
indicated that compound 5j exhibited potent activity with IC50
values of 0.085
Iressa.
mM, which was comparable to the positive control
2.3. Binding model of compound 5j into EGFR
Molecular docking of the most potent antiproliferative
compound 5j into ATP binding site of EGFR kinase was performed
on the binding model based on the EGFR complex structure
(1M17.pdb). The binding model of compound 5j and EGFR is
depicted in Fig. 3. In the binding model, compound 5j is nicely
bound to the region with the eCN group project toward the
amino hydrogen of Met 769, with the eCN forming a more
optimal H-bond interaction, and carbonyl group of compound 5j
also forms hydrogen bond with the side chain amino hydrogen of
Lys 721. This molecular docking result suggesting that compound
5j is a potential inhibitor of EGFR with potent antiproliferative
activity.
N
O
R
O
N
R
N
N
Br
O
Br
N
+
Br
+
N
N
N
Br
O
1a
1a
2
3
Scheme 1. Synthesis of pyridinium salts.

3186
Y.-M. Shen et al. / European Journal of Medicinal Chemistry 45 (2010) 3184e3190
R
R2
R
R1
TPCD,NEt3,DMF
90 ,5-10h
N
+
R1HC CHR2
N
Br
O
O
4
2
5
5a
5b
R=H R1=H R2=CN
R=H R1=H R2=CO2CH3
5c R=H R1=CO2Et R2=CO2Et
5e R=7-CH3 R1=H R2=CN
5d R=H R1=1,2-N-phenylmaleimide
5f R=7-CH3 R1=H R2=CO2CH3
5h R=5-Br R1=H R2=CN
5g R=7-CH3 R1=CO2Et R2=CO2Et
5i R=5-Br R1=H R2=CO2CH3
5j R=5-CH3,8-Br R1=H R2=CN
5k
5l
R=5-CH3,8-Br R1=H R2=CO2CH3
R=5,6-benzo R1=H R2=CN
5m
5n
R=5,6-benzo R1=H R2=CO2CH3
R=7,8-benzo R1=H R2=CN
5o
5p
R=7,8-benzo R1=H R2=CO2CH3
R=7,8-benzo R1=CO2CH3 R2=CO2CH3
Scheme 2. Synthesis of compounds 5ae5p.
3. Conclusions
spectrometer. Elemental analyses were obtained using a Heraeus
CHN-O-Rapid analyzer. For X-ray Crystallographic analysis, the X-
ray diffraction intensities and the unit cell parameters were
determined on a Brucker SMART APEXII CCD diffractometer
In summary, indolizine and annulated indolizine derivatives
incorporating a cyclopropylcarbonyl group are synthesized in a one
pot procedure by the tanden reactions of [3 þ 2] cycloaddition of
the corresponding N-ylide with electron deficient alkene and the
dehydroaromatization of the primary cycloadduct under the action
of the mild oxidant TPCD. Antiproliferative experiment against the
human hepatocellular liver carcinoma (Hep-G2) cell line were
examined with 5-fluorouracil as a reference compound. Several of
the synthesized indolizines (5a, 5d, 5g and 5j) show strong anti-
cancer activity comparable with 5-fluorouracil. Therefore, the
cyclopropyl incorporated indolizines turned out to be promising
candidates in developing new agents against the hepatocellular
carcinoma (HCC). Molecular docking result suggesting that
compound 5j is a potential inhibitor of EGFR with potent anti-
proliferative activity. Further investigation on the structure-activity
relationship on this new type of HCC inhibiting compounds is being
undertaken.
employing
graphite-monochromated (Mo-K
a)
radiation
(
l
¼ 0.71073 Å) and operating in the
u/2q scan mode. Data collec-
tion 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 anisotropic displacement parameters, and the
positions of all H-atoms were fixed geometrically and included in
estimated positions using a riding model.
4.2. General procedure for the synthesis of the pyridinium salts
A
mixture of pyridine (7.9 g, 100 mmol) and cyclo-
propylbromomethylmethanone (12 g, 100 mmol) in EtOAc (50 mL)
was stirred at room temperature for 0.5 h. After it stood for another
48 h, the precipitated solid was collected and rinsed with EtOAc
(50 mL) to give 2a. Salts 2be2f, 3 were prepared by the same
procedure and they were directly used in the next step without any
further purification.
4. Experimental section
4.1. Chemistry
Melting points were measured on an X-4 (Taike Corp., Beijing,
China) microscopic melting point apparatus and are uncorrected.
1H NMR spectra were recorded on a Bruker ACF-400 spectrometer
with CDCl3 as solvent unless otherwise specified. 13C NMR spectra
were measured on a Bruker ACF-400 spectrometer at 100 MHz
Table 2
Synthesis of the 3-Cyclopropylcarbonylindolizines and 6-Cyclopropylcarbonyl-
imidazo[2,1-f]pyrrolo[1,2-b]pyridazines.
Compd.
R
R1
R2
Yield (%)
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
6a
6b
H
H
H
H
7-CH3
7-CH3
7-CH3
5-Br
H
H
CO2Et
CN
CO2CH3
CO2Et
73
42
58
51a
70
35
50
48
46
75
50
57
55
93
66
76
50
40
with CDCl3 as solvent. The chemical shifts (d) are reported in ppm
relative to the residual undeuterated solvent signal, and coupling
constants (J) are given in Hz. IR spectra were taken with a Nicolet
FT-IR 5DX spectrometer for samples were characterized by atten-
uated total reflection Fourier transform infrared (ATR-FTIR)
spectroscopy. Mass spectra (EI) were recorded with a VG ZAB-HS
1,2-N-phenylmaleimide
H
H
CO2Et
H
H
H
H
H
H
H
H
CN
CO2CH3
CO2Et
CN
CO2CH3
CN
CO2CH3
CN
CO2CH3
CN
5-Br
5-CH3,8-Br
5-CH3,8-Br
5,6-benzo
5,6-benzo
7,8-benzo
7,8-benzo
7,8-benzo
e
N
N
N
N
TPCD,NEt3,DMF
90 ,5-10h
R2
N
N
+
R1HC CHR2
Br
O
CO2CH3
CO2CH3
CN
R1
CO2CH3
H
H
O
4a R1=H, R2=CN
3
6
4b R1=H, R2=CO2CH3
e
CO2CH3
a
Scheme 3. Synthesis of compounds 6a and 6b.
Another product 5d0 was also obtained in 26% yield (vide infra).

Y.-M. Shen et al. / European Journal of Medicinal Chemistry 45 (2010) 3184e3190
3187
O
O
Ph
N
NHPh
O
N
N
O
O
5d(51%)
5d'(26%)
Scheme 4. Chemical structures of compounds 5d and 5d0.
4.3.3. Diethyl 3-cyclopropylcarbonylindolizine-1,2-dicarboxylate
(5c)
White crystal. m.p. 69e70 ꢀC.IR
n
: 2988, 1728, 1690, 1622, 1207,
963, 784, 756 cmꢁ1. 1H NMR (CDCl3, 400 MHz):
d
0.95e0.99(m, 2H,
CH2), 1.25e1.29(m, 2H, CH2), 1.39e1.44(m, 6H, CH3), 2.34e2.41
(m, 1H, CH), 4.40(q, 2H, J ¼ 7.1 Hz, OCH2), 4.48(q, 2H, J ¼ 7.2 Hz,
OCH2), 7.03(t, 1H, J ¼ 7.0 Hz, py-H), 7.41(t, 1H, J ¼ 7.8 Hz, py-H), 8.36
(d, 1H, J ¼ 8.8 Hz, py-H), 9.82(d, 1H, J ¼ 7.2 Hz, py-H). 13C NMR
Fig. 1. ORTEP drawing of compound 5e.
4.3. General procedure for the synthesis and purification of the
(CDCl3, 100 MHz):
d 11.0, 14.0, 14.4, 19.1, 60.5, 62.3, 103.8, 115.8,
indolizines
119.7, 127.7, 129.036, 130.8, 133.6, 137.6, 163.1, 167.0, 190.1. MS m/z
(%) ¼ 329 (Mþ, 100), 227 (12). C18H19NO5 (329.35): calcd. C 65.60,
H 5.79, N 4.27; Found C 65.64, H 5.81, N, 4.25.
A mixture of the pyridinium salt (10 mmol), acrylonitrile
(40 mmol), triethylamine (2 ml) and TPCD (4 g) in DMF (40 ml) was
heated at 90 ꢀC for 5 hours. After cooling, the reaction mixture was
poured into an aqueous hydrochloric acid solution (5%, 100 ml), the
precipitated crude product was collected by filtration and further
purified by silica gel column chromatography with petroleum ether
(bp 60e90 ꢀC)-ethyl acetate as eluents.
4.3.4. 4-Cyclopropylcarbonyl-2-methyl-1,3-dioxo-1H-pyrrolo[3,4-
a]indolizine (5d)
Yellow powder. m.p.211e212 ꢀC. IR (ATR)
n
: 3124, 2961, 1760,
1699, 1628, 1358, 1062, 912, 808, 748, 696 cmꢁ1 1H NMR (CDCl3,
400 MHz): 1.10e1.15(m, 2H, CH2), 1.26e1.30(m, 2H, CH2), 3.81-
.
d
3.87(m, 1H, CH), 7.10e7.13(m, 1H, py-H), 7.39e7.46(m, 3H, Ar-H),
7.47e7.53(m, 3H, Ar-H, py-H), 8.00(d, 1H, J ¼ 8.8 Hz, py-H), 10.05
4.3.1. 3-Cyclopropylcarbonyl-1-indolizinecarbonitrile (5a)
White powder. m.p. 163e164 ꢀC. IR (ATR)
n
: 3114, 2924, 2213,
(d, 1H, J ¼ 7.2 Hz, py-H). 13C NMR (CDCl3, 100 MHz):
d 12.2, 19.9,
1637, 1628, 942, 758 cmꢁ1. 1H NMR (CDCl3, 400 MHz):
d
1.04(s, 2H,
110.6, 116.7, 118.6, 118.8, 127.1, 128.0, 128.6, 129.0, 130.1, 130.6, 131.9,
132.3, 162.5, 163.9, 191.6. MS m/z (%) ¼ 330 (Mþ, 100), 245 (5), 238
(21). C20H14N2O3 (330.34): calcd. C 72.05, H 4.24, N 8.44; Found
C 72.72, H 4.27, N 8.48.
CH2), 1.25(s, 2H, CH2), 2.51(s, 1H, CH), 7.06(s, 1H, py-H), 7.42(s, 1H,
py-H), 7.79(s, 1H, pyrrole-H), 7.94(s, 1H, py-H), 9.90(s, 1H, py-H). 13C
NMR (CDCl3, 100 MHz):
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. MS m/z (%) ¼ 210 (Mþ, 100), 169
(46), 141 (15), 114 (8). C13H10N2O (210.23): calcd. C 74.25, H 4.80, N
13.30; Found C 74.27, H 4.79, N 13.33.
4.3.5. 3-Cyclopropylcarbonyl-N-phenyl-1-indolizinecarboxamide
(5d0)
Yellow powder. m.p.213e215 ꢀC. IR (ATR)
n
: 3354, 3107, 2926,
2359, 1625, 1594, 1536, 1526, 1487, 1442, 1215, 1109, 947, 777, 756,
695 cmꢁ1 1H NMR (CDCl3, 400 MHz):
0.99e1.04(m, 2H, CH2),
4.3.2. Methyl 3-cyclopropylcarbonylindolizine-1-carboxylate (5b)
Yellow powder. m.p.135e136 ꢀC. IR (ATR)
n
: 3113, 2951, 1697,
1618, 756 cmꢁ1 1H NMR (CDCl3, 400 MHz):
. d 0.97e1.02(m, 2H,
.
d
1.26e1.29(m, 2H, CH2), 2.58(q, 1H, J ¼ 4.1 Hz, CH), 7.01e7.04(m, 1H,
py-H), 7.13e7.17(m, 1H, py-H), 7.36e7.41(m, 3H, Ar-H), 7.66(dd, 2H,
J ¼ 8.2, 1 Hz, Ar-H), 7.69(s, 1H, py-H), 7.97(s, 1H, pyrrole-H),
8.54e8.57(m, 1H, N-H), 9.91(p, 1H, J ¼ 2 Hz, py-H). 13C NMR
CH2), 1.21e1.25(m, 2H, CH2), 2.56e2.62(m, 1H, CH), 3.94(s, 3H,
OCH3), 6.99(t, 1H, J ¼ 7.0 Hz, py-H), 7.38(t, 1H, J ¼ 7.8 Hz, py-H), 8.17
(s, 1H, pyrrole-H), 8.34(d, 1H, J ¼ 9.2 Hz, py-H), 9.90(d, 1H,
J ¼ 6.8 Hz, py-H). 13C NMR (CDCl3, 100 MHz):
d 10.3, 18.0, 51.3, 105.4,
(CDCl3, 100 MHz):
d 10.3, 10.4, 18.1, 108.9, 115.4, 119.8, 120.2, 120.6,
115.0, 119.2, 123.3, 125.4, 127.0, 129.087, 139.2, 164.5, 189.8. MS m/z
(%) ¼ 243 (Mþ, 100), 212 (10), 202 (19). C14H13NO3 (243.26): calcd.
C 69.07, H 5.43, N 19.77; Found C 69.12, H 5.39, N 5.76.
122.9, 124.2, 126.8, 128.9, 129.1, 138.2, 139.2, 162.3, 189.4. MS m/z
(%) ¼ 304 (Mþ, 6), 212 (100). C19H16N2O2 (304.34): calcd. C 74.93, H
5.34, N 9.23; Found C 74.98, H 5.30, N 9.20.
Table 3
The antiproliferative effect of the indolizines against Hep-G2.
Compd.
IC50
(
m
g/mL)
Compd.
IC50(mg/mL)
-0.20279
-0.01952
-0.20325
5a
5b
5c
5d
5d0
5e
5f
5g
5h
5i
0.32 ꢂ 0.9
6.6 ꢂ 1.6
32 ꢂ 0.9
5j
5k
5l
5m
5n
5o
5p
6a
6b
0.20 ꢂ 0.09
1.1 ꢂ 0.6
2.1 ꢂ 0.9
4.6 ꢂ 1.6
4.3 ꢂ 1.9
9.3 ꢂ 1.3
27 ꢂ 0.9
15 ꢂ 0.9
25 ꢂ 0.7
-0.42025
LUMO
N
0.53712
0.48 ꢂ 0.05
0.56 ꢂ 0.07
21 ꢂ 0.6
0.41222
N
25 ꢂ 0.7
O
0.29 ꢂ 0.09
1.8 ꢂ 0.6
2.5 ꢂ 0.5
HOMO
Fig. 2. FMOs in the pyridinium ylide and in acrylonitrile.

3188
Y.-M. Shen et al. / European Journal of Medicinal Chemistry 45 (2010) 3184e3190
Table 4
4.3.8. Diethyl 3-Cyclopropylcarbonyl-7-methyl-indolizine-1,2-
Inhibition of EGFR kinase compound 5j.
dicarboxylate (5g)
White powder. m.p.65e66 ꢀC. IR (ATR)
n
: 2985, 1733, 1688, 1613,
0.93e0.97
Compound
EGFR Inhibition IC50 (mM)
1202, 920, 806, 783 cmꢁ1. 1H NMR (CDCl3, 400 MHz):
d
5j
Iressa
0.085 ꢂ 0.007
0.033 ꢂ 0.001
(m, 2H, CH2), 1.23e1.27(m, 2H, CH2), 1.41(q, 6H, J ¼ 6.9 Hz, CH3),
2.31e2.38(m, 1H, CH), 2.46(s, 3H, CH3), 4.38(q, 2H, J ¼ 7.2 Hz, CH2),
4.46(q, 2H, J ¼ 7.2, CH2), 6.86(dd,1H, J ¼ 7.4, 1.4 Hz, py-H), 8.15(s,1H,
py-H), 9.71(d, 1H, J ¼ 7.2 Hz, py-H). 13C NMR (CDCl3, 100 MHz):
4.3.6. 3-Cyclopropylcarbonyl-7-methyl-1-indolizinecarbonitrile
d
10.8, 14.0, 14.4, 18.9, 21.6, 60.3, 62.3, 102.7, 118.3, 120.3, 128.5,
(5e)
White powder. m.p.149e150 ꢀC. IR (ATR)
n
: 3114, 2924, 2213,
130.9, 133.6, 138.2, 139.4, 163.2, 167.1, 189.8. MS m/z (%) ¼ 343
(Mþ, 100), 241 (5), 184 (3). C19H21NO5 (343.37): calcd. C 66.40,
H 6.15, N 4.10; Found C 66.46, H 6.16; N 4.08.
1637, 1628, 942, 758 cmꢁ1. 1H NMR (CDCl3, 400 MHz):
d
1.02(d, 2H,
J ¼ 3.6 Hz, CH2), 1.23(s, 2H, CH2), 2.48(s, 4H, CH, CH3), 6.88(d, 1H,
J ¼ 6.4 Hz, py-H), 7.54(s, 1H, py-H), 7.87(s, 1H, pyrrole-H), 9.75
(d, 1H, J ¼ 6.8 Hz, py-H). 13C NMR (CDCl3, 100 MHz):
d 10.6, 18.0,
4.3.9. 5-Bromo-3-cyclopropylcarbonyl-1-indolizinecarbonitrile
(5h)
21.4, 83.0, 115.7, 116.1, 118.1, 123.3, 125.832, 128.8, 139.0, 141.2, 189.2.
MS m/z (%) ¼ 224 (Mþ, 100), 183 (32), 155 (19). C14H12N2O (224.26):
calcd. C 74.91, H 5.38, N 12.52; Found C 74.98, H 5.39, N 12.49. X-ray
structure analysis: C14H12N2O, M ¼ 224.26. Triclinic, space group
Yellow powder. m.p.105e106 ꢀC. IR (ATR)
n
: 3076, 2223, 1660,
949, 788 cmꢁ1. 1H NMR (CDCl3, 400 MHz):
d 1.08e1.14(m, 2H, CH2),
1.30e1.34(m, 2H, CH2), 2.47e2.53(m, 1H, CH), 7.24(dd, 1H, J ¼ 8.6,
7.4 Hz, py-H), 7.31(dd, 1H, J ¼ 7.2, 0.8 Hz, py-H), 7.80(dd, 1H, J ¼ 8.4,
1.2 Hz, py-H), 7.83(s, 1H, pyrrole-H). 13C NMR (CDCl3, 100 MHz):
P-1, a ¼ 7.1397(8), b ¼ 11.6103(13), c ¼ 14.6499(15) Å,
a
¼ 76.201(7),
b
¼ 86.261(7),
g
¼ 87.924(7), V ¼ 1176.6(2) Å, Z ¼ 4, Dc ¼ 1.266 -
g cmꢁ3, F(000) ¼ 472.0, absorption coefficient 0.082 mmꢁ1, scan
d
11.6, 20.4, 85.0, 114.9, 116.7, 118.5, 122.1, 125.4, 126.4, 129.1, 143.8,
188.8. MS m/z (%) ¼ 290 (Mþ, 2), 209 (100), 179 (13), 169 (15), 140
(18). C13H9BrN2O (289.13): calcd. C 53.03, H 3.16, N 9.71; Found
C 54.00, H 3.14, N 9.69.
range for data collection 1.43 ꢃ
q
ꢃ 27.63ꢀ, 17,716 measured reflec-
tions, 5390 independent reflections, 3088 reflections with I > 2s(I),
Rint ¼ 0.0308, 307 refinable parameters, R[F2 > 2
s
(F2)] ¼ 0.0450,
wR2 (F2) ¼ 0.1347.
4.3.10. Methyl 5-bromo-3-cyclopropylcarbonyl-indolizine-1-
carboxylate (5i)
4.3.7. Methyl 3-cyclopropylcarbony-7-methyllindolizine-1-
White powder. m.p.85e86 ꢀC. IR (ATR)
n
: 3111, 2947, 1704, 1655,
carboxylate (5f)
946, 786, 729 cmꢁ1. 1H NMR (CDCl3, 400 MHz):
d
1.05e1.09(m, 2H,
White powder. m.p.154e155 ꢀC. IR (ATR)
n
: 2988, 1728, 1622,
962, 756 cmꢁ1. 1H NMR (CDCl3, 400 MHz):
d 0.97(s, 2H, CH2), 1.21
CH2), 1.27e1.31(m, 2H, CH2), 2.56(m, 1H, CH), 3.94(s, 3H, CH3), 7.20
(t, 1H, J ¼ 8.0 Hz, py-H), 7.25(s, 1H, py-H), 8.08(s, 1H, pyrrole-H),
(s, 2H, CH2), 2.46(s, 3H, CH3), 2.56(s, 1H, CH), 3.93(s, 3H, OCH3), 6.84
8.41(d, 1H, J ¼ 8.4 Hz, py-H). 13C NMR (CDCl3, 100 MHz):
d 11.1,
(s, 1H, py-H), 8.12(s, 2H, pyrrole-H, py-H), 9.78(s, 1H, py-H). 13C
20.0, 51.4, 105.7, 118.0, 118.5, 121.7, 125.7, 126.4, 128.5, 142.6, 164.1,
188.8. MS m/z (%) ¼ 321 (Mþ, 0.5), 243 (63), 242 (100), 202 (14).
C14H12BrNO3 (322.15): calcd. C 52.17, H 3.71, N 4.37; Found C 52.20,
H 3.75, N 4.35.
NMR (CDCl3, 100 MHz):
d 10.1, 17.8, 21.5, 51.2, 104.2, 117.5, 118.0,
123,0, 125.7, 128.5, 138.7, 139.7, 164.7, 189.5. MS m/z (%) ¼ 257
(Mþ, 100), 226 (8), 216 (15). C15H15NO3 (257.28):calcd. C 69.98,
H 5.83, N 5.46; Found C 70.02, H 5.88, N 5.44.
Fig. 3. Molecular docking modeling of compound 5j with EGFR kinase.

Y.-M. Shen et al. / European Journal of Medicinal Chemistry 45 (2010) 3184e3190
3189
4.3.11. 8-Bromo-3-cyclopropylcarbonyl-5-methyl-1-
(Mþ, 100), 262 (8), 252 (11). C18H15NO3 (293.32): calcd. C 73.64,
H 5.10, N 4.81; Found C 73.71, H 5.15, N 4.78.
indolizinecarbonitrile (5j)
Yellow powder. m.p.133e135 ꢀC.IR (ATR)
n
: 3100, 2214, 1647,
957, 746 cmꢁ1. 1H NMR (CDCl3, 400 MHz):
d
1.09e1.14(m, 2H, CH2),
4.3.17. Dimethyl 3-cyclopropylcarbonylpyrrolo[1,2-a]isoquinoline-
1.25e1.29(m, 2H, CH2), 2.48(s, 3H, CH3), 2.54e2.59(m, 1H, CH), 6.72
(d, 1H, J ¼ 7.6 Hz, py-H), 7.52(d, 1H, J ¼ 7.6 Hz, py-H), 7.98(s, 1H,
1,2- dicarboxylate (5p)
Yellow crystal. m.p.115e116 ꢀC. IR (ATR)
n
: 2954, 1739, 1707,
pyrrole-H). 13C NMR (CDCl3, 100 MHz):
d
11.7, 20.0, 22.9, 86.3, 109.9,
1644, 989, 799, 756 cmꢁ1. 1H NMR (CDCl3, 400 MHz):
d
1.00e1.05
115.9, 117.0, 28.1, 128.8, 130.5, 138.8, 139.5, 189.2. MS m/z (%) ¼ 304
(Mþ, 80), 302 (100), 287 (75), 285 (67), 273 (66), 261 (21), 127 (20).
C14H11BrN2O (303.15): calcd. C 55.35, H 3.68, N 9.42; Found C 55.47,
H 3.66, N 9.24.
(m, 2H, CH2), 1.30e1.34(m, 2H, CH2), 2.39(m, 1H, CH), 3.97(d, 6H,
J ¼ 3.2, OCH3), 7.16(d, 1H, J ¼ 7.6 Hz, Ar-H), 7.60(q, 2H, J ¼ 3.1 Hz,
Ar-H), 7.70(q, 1H, J ¼ 3.1 Hz, pyrrole-H), 9.17(q, 1H, J ¼ 3.2 Hz, py-H),
9.23(d, 1H, J ¼ 7.6 Hz, py-H). 13C NMR (CDCl3, 100 MHz):
d 11.9, 20.6,
52.3, 52.9, 108.8, 115.9, 123.0, 124.2, 124.5, 126.8, 126.9, 128.0, 128.2,
129.2, 130.0, 133.3, 165.0, 166.9, 191.8. MS m/z (%) ¼ 351 (Mþ, 100),
310 (11), 296 (8). C20H17NO5 (351.35): calcd. C 68.33, H 4.90, N 3.96;
Found C 68.37, H 4.88, N 3.99.
4.3.12. Methyl 8-bromo-3-cyclopropylcarbonyl-5-methyl-
indolizine-1-carboxylate (5k)
Yellow crystal. m.p.107e108 ꢀC. IR (ATR)
n
: 2951, 1714, 1652, 960,
777 cmꢁ1 1H NMR (CDCl3, 400 MHz):
. d 1.03e1.08(m, 2H, CH2),
1.21e1.25(m, 2H, CH2), 2.48(s, 3H, CH3), 2.61(m, 1H, CH), 3.94(s, 3H,
OCH3), 6.66(d, 1H, J ¼ 8.0 Hz, py-H), 7.53(d, 1H, J ¼ 7.6 Hz, py-H),
4.3.18. 6-Cyclopropylcarbonyl-imidazo[2,1-f]pyrrolo[1,2-b]
pyridazine-8-carboxylate-3-carbonitrile (6a)
8.08(s, 1H, pyrrole-H). 13C NMR (CDCl3, 100 MHz):
d
11.1, 19.7,
Red powder. m.p.230e232 ꢀC IR (ATR)
n
: 3143, 2924, 2194, 1601,
23.0, 51.9, 108.8, 109.2, 116.2, 126.8, 127.7, 131.1, 136.7, 138.9, 164.5,
188.9. MS m/z (%) ¼ 337(Mþ, 74), 335(100), 318(53), 172(22).
C15H14BrNO3 (336.18): calcd. C 53.48, H 4.25, N 4.21; Found
C, 53.59; H, 4.20; N, 4.17.
1217, 791, 731, 681 cmꢁ1
.
1H NMR (CDCl3, 400 MHz):
d
0.84e0.94
(m, 2H, CH2),1.06e1.12(m, 2H, CH2), 2.05e2.12(m,1H, CH), 5.53(d,1H,
J ¼ 5.6 Hz, imidazole-H), 7.24(s, 1H, pyrrole-H), 7.37(t, 1H, J ¼ 4.0 Hz,
imidazole-H), 7.46(d, 1H, J ¼ 5.6 Hz, pyridazine-H), 8.79(d, 1H,
J ¼ 2.0 Hz, pyridazine-H). 13C NMR (CDCl3, 100 MHz):
d 9.9, 15.1, 76.1,
4.3.13. 1-CyclopropylcarbonylPyrrolo[1,2-a]quinoline-3-
carbonitrile (5l)
96.5,115.2,117.6,117.6,121.3,134.903,136.3,139.1,152.3,186.7. MS m/z
(%) ¼ 250 (Mþ, 100), 221 (5), 181 (16). C14H10N4O (250.26): calcd.
C 67.11, H 4.00, N 22.41; Found C, 67.19; H, 4.03; N, 22.39.
Yellow powder. m.p.161e162 ꢀC. 1H NMR (CDCl3, 400 MHz):
d
1.14(d, 2H, J ¼ 4.0 Hz, CH2), 1.36(s, 2H, CH2), 2.61(s, 1H, CH), 7.52
(t, 1H, J ¼ 6.8 Hz, Ar-H), 7.61e7.69(m, 3H, Ar-H), 7.80(d, 1H,
J ¼ 6.4 Hz, py-H), 7.89(s, 1H, py-H), 8.16(d, 1H, J ¼ 8.0 Hz, pyrrole-H).
4.3.19. Methyl 6-Cyclopropylcarbonyl- imidazo[2,1-f]pyrrolo[1,2-b]
pyridazine-8-carboxylate (6b)
Red powder. m.p.231e233 ꢀC. IR (ATR)
n
: 3197, 2923, 1684, 1599,
4.3.14. Methyl 1-cyclopropylcarbonylPyrrolo[1,2-a]quinoline-3-
1212, 728 cmꢁ1. 1H NMR (CDCl3, 400 MHz):
d
0.91(t, 2H, J ¼ 3.6 Hz,
carboxylate (5m)
CH2), 1.08(s, 2H, CH2), 2.28(m, 1H, CH), 3.80(s, 3H, OCH3), 6.49
(d, 1H, J ¼ 5.2 Hz, imidazole-H), 7.39(s, 1H, pyrrole-H), 7.53(s, 1H,
imidazole-H), 7.89(s, 1H, pyridazine-H), 8.86(s, 1H, pyridazine-H).
Yellow powder. m.p.174e175 ꢀC. IR (ATR)
n
: 2943, 1698, 1644,
940, 815, 742 cmꢁ1. 1H NMR (CDCl3, 400 MHz):
d
0.91(s, 2H, CH2),
1.32(s, 2H, CH2), 2.68(s, 1H, CH), 3.95(s, 3H, OCH3), 7.47(s, 1H, Ar-H),
7.58(s, 1H, Ar-H), 7.64(s, 1H, Ar-H), 7.77(s, 1H, Ar-H), 8.13(s, 2H,
13C NMR (CDCl3, 100 MHz):
d 9.8, 15.1, 51.1, 95.5, 99.5, 114.8, 117.1,
120.1, 134.4, 134.9, 150.9, 165.6, 187.5. MS m/z (%) ¼ 283 (Mþ, 100),
214 (4), 156 (3). C15H13N3O3 (283.28): calcd. C 63.56, H 4.66,
N 14.80; Found C 63.60, H 4.63, N 14.83.
pyrrole-H), 8.29(s, 1H, py-H). 13C NMR (CDCl3, 100 MHz):
d 11.2,
19.6, 51.4, 107.3, 117.6, 120.5, 125.4, 127.0, 128.4, 128.6, 128.8, 129.8,
133.5, 140.0, 164.5, 190.5. MS m/z (%) ¼ 293 (Mþ, 100), 252 (25), 194
(5). C18H15NO3 (293.32): calcd. C 73.66, H 5.24, N 4.76; Found
C 73.71, H 5.15, N 4.78.
4.4. Antiproliferative activities assay
The antiproliferative activities of indolizine derivatives were
determined using a standard (MTT)-based colorimetric assay
(Sigma). Briefly, cell lines were seeded at a density of 7 ꢄ 103 cells/
well in 96-well microtiter plates (Costar). After 12 h, exponentially
growing cells were exposed to the indicated compounds at final
4.3.15. 3-Cyclopropylcarbonylpyrrolo[1,2-a]isoquinoline-1-
carbonitrile (5n)
Pink powder. m.p.190e191 ꢀC.IR (ATR)
n
: 3110, 2925, 2218, 1625,
796, 685 cmꢁ1. 1H NMR (CDCl3, 400 MHz):
d 1.05e1.09(m, 2H, CH2),
1.26e1.30(m, 2H, CH2), 2.53e2.59(m, 1H, CH), 7.22(d, 1H, J ¼ 7.6 Hz,
Ar-H), 7.66e7.73(m, 2H, Ar-H), 7.76e7.79(m, 1H, Ar-H), 7.91(s, 1H,
pyrrole-H), 8.97(dd, 1H, J ¼ 6.8, 2.0 Hz, py-H), 9.59(d, 1H, J ¼ 7.2 Hz,
concentrations ranging from 0.1 to 40
was determined by the addition of an MTT solution (25
mL MTT in PBS). After 6 h,100 L of 10% SDS in 0.01 N HCl was added,
m
g/mL. After 48 h, cell survival
m
L of 4 mg/
m
py-H). 13C NMR (CDCl3, 100 MHz):
d
11.0, 18.5, 85.5, 115.6, 117.3,
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 repli-
cates of 6 wells from at least two independent experiments.
123.6, 124.0, 125.3, 125.4, 127.1, 128.7, 129.5, 129.8, 137.2, 190.1.
MS m/z (%) ¼ 260 (Mþ, 100), 219 (36), 191 (21), 164 (17). C17H12N2O
(260.29): calcd. C 78.40, H 4.63, N 10.78; Found C 78.44, H 4.65,
N 10.76.
4.3.16. Methyl 3-cyclopropylcarbonylpyrrolo[1,2-a]isoquinoline-1-
4.5. EGFR inhibitory assay
carboxylate (5o)
Yellow powder. m.p.157e158 ꢀC. IR (ATR)
n
: 3133, 1708, 1634,
The EGFR kinase assay methods used are the same as our
previous papers [25,26].
991, 756 cmꢁ1. 1H NMR (CDCl3, 400 MHz):
d 1.03(s, 2H, CH2), 1.26
(s, 2H, CH2), 2.66(s, 1H, CH), 3.98(s, 3H, OCH3), 7.21(d, 1H, J ¼ 5.6 Hz,
Ar-H), 7.69(d, 3H, J ¼ 32.4 Hz, Ar-H), 8.25(s, 1H, pyrrole-H), 9.71
(d, 1H, J ¼ 5.6 Hz, py-H), 9.86(d, 1H, J ¼ 5.6 Hz, py-H). 13C NMR
4.6. Molecular Docking modeling
(CDCl3, 100 MHz):
d
10.7, 18.4, 51.8, 109.5, 115.4, 124.1, 124.6, 125.3,
Molecular docking of compound 5j into the three-dimensional
126.6, 127.7, 128.1, 129.2, 130.3, 136.3, 165.1, 190.4. MS m/z (%) ¼ 293
EGFR complex structure (1M17.pdb, downloaded from the PDB)

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Y.-M. Shen et al. / European Journal of Medicinal Chemistry 45 (2010) 3184e3190
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