Design, synthesis and anticancer activity evaluation of some novel pyrrolo[1,2-a]azepine derivatives

Article abstract of DOI:10.1002/ardp.201400004

A novel series of pyrrolo[1,2-a]azepine derivatives 3-7 were synthesized and their structures were confirmed by spectral and elemental analyses. Antitumor activity evaluation of these compounds was carried out against liver (HepG2), breast (MCF7), and colon (HCT116) cancer cell lines using the sulforhodamine-B (SRB) assay method and doxorubicin as reference standard. Compounds 3 and 6 were found to be more potent than doxorubicin against HepG2 cells, with IC₅₀ values of 4, 1.6 and 10.8 nM, respectively. Moreover, compounds 3 and 7 showed broad-spectrum anticancer activity against all the tested cell lines, and their IC₅₀ values were in the nanomolar range (4-44.2 nM and 20.7-45.4 nM, respectively). The 2-benzoylamino derivative of pyrrolo[1,2-a]azepine 5b was the most potent one against MCF7 cells (IC₅₀ of 10.7 nM); however, the 2-(2-chloro-acetylamino)- pyrroloazepine derivative 6 was the most potent against the HCT116 cell line, with an IC₅₀ value of 21.1 nM. The novel compounds were docked into the active site of cyclin-dependent kinase 2 (CDK2) to explore the ability of these compounds to interact with these kinases. All compounds showed a lower binding score energy than the reference ligand. New pyrrolo[1,2-a]azepine derivatives 3-7 were synthesized and their antitumor activities were evaluated against liver, breast, and colon cancer cell lines. Compounds 3 and 7 showed broad-spectrum anticancer activity against all tested cell lines, with IC ₅₀ values in the nanomolar range. The ability of the new compounds to interact with cyclin-dependent kinases was also explored.

Full text of DOI:10.1002/ardp.201400004

Arch. Pharm. Chem. Life Sci. 2014, 347, 1–8
1
Full Paper
Design, Synthesis and Anticancer Activity Evaluation of
Some Novel Pyrrolo[1,2-a]azepine Derivatives
Amany Belal1,2
1
Pharmaceutical Chemistry Department, College of Pharmacy, Taif University, Taif, Kingdom of Saudi Arabia
Faculty of Pharmacy, Medicinal Chemistry Department, Beni-Suef University, Beni-Suef, Egypt
2
A novel series of pyrrolo[1,2-a]azepine derivatives 3–7 were synthesized and their structures were
confirmed by spectral and elemental analyses. Antitumor activity evaluation of these compounds was
carried out against liver (HepG2), breast (MCF7), and colon (HCT116) cancer cell lines using the
sulforhodamine-B (SRB) assay method and doxorubicin as reference standard. Compounds 3 and 6 were
found to be more potent than doxorubicin against HepG2 cells, with IC50 values of 4, 1.6 and 10.8 nM,
respectively. Moreover, compounds 3 and 7 showed broad-spectrum anticancer activity against all the
tested cell lines, and their IC50 values were in the nanomolar range (4–44.2 nM and 20.7–45.4 nM,
respectively). The 2-benzoylamino derivative of pyrrolo[1,2-a]azepine 5b was the most potent one
against MCF7 cells (IC50 of 10.7 nM); however, the 2-(2-chloro-acetylamino)-pyrroloazepine derivative 6
was the most potent against the HCT116 cell line, with an IC50 value of 21.1 nM. The novel compounds
were docked into the active site of cyclin-dependent kinase 2 (CDK2) to explore the ability of these
compounds to interact with these kinases. All compounds showed a lower binding score energy than
the reference ligand.
Keywords: Antitumor / Pyrimidopyrroloazepine / Pyrrolo[1,2-a]azepine / Pyrroloazepine derivatives
Received: January 3, 2014; Revised: February 3, 2014; Accepted: February 18, 2014
DOI 10.1002/ardp.201400004
Introduction
family plants. Herbal extracts of these plants have been used
for thousands of years in folk medicine in East Asia as cough
suppressant, antibacterial, antifungal, antitubercular and
antiparasitic [3–5]. Lindsay and Pyne [6] reported the synthesis
of 5,7-heterocyclic ring system (1H-pyrrolo[1,2-a]azepine) as an
analogue for 5,5- and 5,6-heterocyclic systems to be explored
as a potential glycosidase inhibitor. Croomine alkaloid
that has a pyrrolo[1,2-a]azepine scaffold showed antitussive
activity with an ID50 value of 0.18 mmol/kg using citric acid-
induced guinea pig cough model [7]. Muchowski et al. [8]
reported the synthesis and anti-inflammatory activity of some
3H-pyrrolo[1,2-a]azepine-1-carboxylic acid derivatives as an
analog for pyrrolo[1,2-a]pyrrole-1-carboxylic acid system. All
these findings proved the importance of the pyrrolo[1,2-a]-
azepine in medicinal chemistry. It proved to be a useful
scaffold for the design of several analogs of pharmacological
interest.
Cancer is a malignant neoplasm characterized by unregulated
cell growth, in which cell division and growth are uncontrol-
lable and cancer cells can invade nearby tissues of the body
and even can spread to more distant organs and tissues
through blood stream or the lymphatic system [1]. Cancer is a
major health problem threatening people worldwide. More
than 200 types of cancers can affect human beings, with 25% of
deaths in the United States because of cancer. Moreover, the
expected number of new cancer cases and cancer death cases
in the United States in 2013 is 1,660,290 and 580,350,
respectively [2]. The aforementioned facts call for continuous
search for new antitumor active agents.
Pyrrolo[1,2-a]azepine represents a core for over 80 alkaloids
known as stemona alkaloids separated from Stemonaceae
It is observed also from the literature that pyrrolo[1,2-a]-
azepine is the main scaffold of some antitumor active agents.
The most important examples for compounds bearing the
pyrrolo[1,2-a]azepine skeleton are esters of cephalotaxine,
Correspondence: Dr. Amany Belal, Faculty of Pharmacy, Medicinal
Chemistry Department, Beni-Suef University, Beni-Suef 62514, Egypt.
E-mail: abilalmoh1@yahoo.com
Fax: þ20 82 2317958
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A. Belal
Arch. Pharm. Chem. Life Sci. 2014, 347, 1–8
tein, decrease vinblastine efflux in multidrug-resistant
human cervical carcinoma KB-V1, and increase their sensitiv-
ity to paclitaxel, vinblastine, and doxorubicin [15, 16]. Based
on these findings, we report in this work the synthesis of
novel compounds containing the pyrrolo[1,2-a]azepine scaf-
fold 3–7 to evaluate their antitumor activity, hoping to obtain
a new series of antitumor active agents. Docking studies
against cyclin-dependent kinase 2 (CDK2) are also performed
using a PDB file (2VTO) and MOE program for a better
understanding of the antitumor results and to investigate the
ability of these compounds to act as inhibitors for this
receptor.
Results and discussion
Chemistry
Figure 1. Antitumor active agents with pyrrolo[1,2-a]azepine
The aim of this work was to develop novel derivatives having a
pyrrolo[1,2-a]azepine scaffold to evaluate their anticancer
activity. Compounds 1 and 2 were obtained from azepan-2-one
and aniline, respectively, as the previously reported methods
[17, 18]; heating both compounds under reflux for 24 h in
dry acetone and in presence of dry potassium carbonate
afforded compound 3 directly, as shown in Scheme 1. The
IR spectrum of compound 3 showed absorption bands for
cyano, NH2, NH, and carbonyl group; in addition, the 1H NMR
spectrum revealed characteristic signals for 5CH2 of
azepan, aromatic protons signal at 7.11–7.55 ppm, and two
singlets at 8.3 and 9.3 corresponding to NH2 and NH,
respectively. Reaction of the obtained pyrrolo[1,2-a]azepine
derivative 3 (Scheme 2) with equimolar amount of p-chloro
or p-nitro benzaldehyde in absolute ethanol in presence
of glacial acetic acid afforded the benzylidene derivatives
of pyrrolo[1,2-a]azepine 4a and 4b. The 1H NMR spectrum of 4a
scaffold.
which showed significant activity as anti-leukemic agents [9].
Homoharringtonine 1, the antitumor alkaloid (Fig. 1), has
reached phase I/II in the United States in clinical investigation
against myeloid leukemia, its side effects still a major
limitation [10–12]. Acylating 3-OH group of cephalotaxine
revealed compound 2 (Fig. 1) that showed significant
cytotoxicity against several human hematopoietic and solid
tumor cell lines [13]. The alkaloid 3 (Fig. 1) bearing a
pyrrolo[1,2-a]azepine scaffold was designed and synthesized
as a paclitaxel mimic and proved to be a potent compound
against several drug-resistant and drug-sensitive human
cancer cell lines [14]. Moreover, stemofoline 4 (Fig. 1) proved
to have the ability of increasing the intracellular P-glycopro-
Scheme 1. Synthesis of compound 3.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 1–8
Pyrroloazepines as Anticancer Active Agents
3
Scheme 2. Reagents and conditions: (a) p-
Substituted benzaldehyde, five drops of glacial
acetic acid, absolute ethanol, reflux 10 h. (b) Acid
chloride, dry benzene, stirring at RT for 24 h. (c)
Chloroacetylchloride, dioxan, stirred at RT for
12 h. (d) Piperidine, absolute ethanol, reflux 24 h.
and 4b showed the appearance of additional aromatic protons
revealed disappearance of the cyano group, and 1H NMR
spectrum showed the appearance of two multiplets at 2.1 and
3.1 ppm for piperidine protons in addition to a singlet signal
at 4 ppm corresponding to CH2-piperidine. The proposed
mechanism for prymidine derivative 7 formation is shown
in Scheme 3 [19].
–
and two singlets at 9.9 and 10.1 ppm corresponding to N CH
–
of 4a and 4b, respectively. Acylated derivatives of pyrrolo[1,2-
a]azepine 5a and 5b were obtained through reaction of
compound 3 with acetyl or benzoyl chloride. IR spectrum of
both compounds showed an additional absorption band for
1
carbonyl group, H NMR spectrum of 5a showed a singlet of
3H at 2.2 corresponding to CH3 and that of 5b showed
Pharmacological screening
additional aromatic protons that prove benzoylation of
In this study, cytotoxic activity of the novel compounds was
evaluated in vitro against liver (HepG2), breast (MCF7), and
colon (HCT116) cancer cell lines, using doxorubicin as a
reference drug. The survival curve was obtained by plotting
surviving fraction of cancer cells against drug concentration.
The concentration of the tested compounds that causes 50%
inhibition of viable cells (IC50) was calculated. Data repre-
sented in Table 1 show the IC50 values for the tested
compounds and doxorubicin in mg and mM. All the tested
compound
3
amino group. 2-(2-Chloro-acetylamino)-
pyrrolo[1,2-a]azepine derivative 6 was obtained by reacting
compound 3 with chloroacetylchloride in dioxan at room
1
temperature, H NMR spectrum of compound 6 showed the
appearance of a singlet signal at 4 ppm for CH2Cl in addition
to 5CH2 of azepan and aromatic proton signals. Compound 6
was refluxed in absolute ethanol with excess piperidine to
give pyrimido pyrroloazepine derivative 7. The IR spectrum
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Arch. Pharm. Chem. Life Sci. 2014, 347, 1–8
Scheme 3. Mechanism of formation of compound 7.
compounds exhibited good activity against all three tested
cell lines. The most potent activity against HepG2 cell line was
shown by compounds 3 and 6. They were more potent than
doxorubicin itself, followed by pyrimido pyrroloazepine
derivative 7 with an IC50 value of 20.7 nM. Compounds 5b,
5a, 7, 4b, and 3 were shown to be the most potent against
MCF7 cell line in a sequence mode with IC50 range (10.7–
44.2 nM). The most active compounds against HCT116 cell line
were 6 > 3 > 7 > 5b according to their IC50 values that were
expressed also in nanomoles. From these observed results, it
can be concluded that:
(1) Compounds based on pyrroloazepine as a main nucleus
showed a very good and potent activity against HepG2,
MCF7, and HCT116 cell lines and this represents a
promising scaffold in cancer chemotherapy.
(2) The unsubstituted amino group of pyrroloazepine
derivative 3 and pyrimidopyrroloazepine 7 showed a
broad-spectrum anticancer activity against all the three
tested cell lines, their IC50 expressed in a nanomolar
range. Moreover, compound 3 was more potent than
doxorubicin against HepG2 cancer cells.
and 21.1 nM, respectively, and showed a more potent
activity than doxorubicin against HepG2 cell line.
(4) Benzoylation of the 2-amino group of compound 3
revealed a compound 5b with high potency against two
cancer cell lines MCF7 and HCT116, and showed to be the
most potent one against MCF7 cell line with IC50 value of
10.7 nM. However, acylating this amino group with acetyl
moiety afforded compound 5a with high selectivity
against MCF7 cancer cells; its IC50 is 15.7 nM.
(5) p-Nitro benzylidene derivative of pyrroloazepine 4b
showed to be more potent than the p-chloro derivative
4a with high selectivity against MCF7 cell line (IC50
34 nM); the p-chloro derivative 4a was the least active one
of the tested compounds.
(6) In addition to docking results against CDK2 receptor that
revealed the ability of all the synthesized compounds to
quick fit this receptor with lower score energy than the
ligand itself (Table 2), the best fitting score energy was
shown by the broad-spectrum compound 7 followed by
the most active compound against HepG2 cell line 6, then
compounds 4b and 5b that showed great activity specially
against MCF7 cell line. Compound 4a showed a better
score energy than both 3 and 5a; however, its activity was
the least one of them and this could be explained by
(3) Acylating the free amino group of pyrroloazepine 3 with
chloro acetyl group afforded the most potent compound 6
against both HepG2 and HCT116 cell lines; its IC50 was 1.6
Table 1. In vitro anticancer activity of the synthesized
pyrroloazepines.
Table 2. Binding energy scores, amino acid interactions, and
hydrogen bond length of the docked compounds on the active
site of cyclin-dependent kinase 2 (CDK2).
HepG2
(liver cancer
cell line)
MCF7
HCT116
Compound
no.
(breast cancer (colon cancer
cell line)
cell line)
Compound
no.
S
Amino acid
residue
H bond
length A
(kcal/mol)
IC50 mg (mM)
3
4a
4b
5a
5b
6
7
1.17 (0.0040)
87.25 (0.2093) 106.33 (0.2551)
13.0 (0.0442)
8.025 (0.0273)
108.2 (0.2595)
116.2 (0.2718)
38.58 (0.1147)
22.51 (0.0565)
7.81 (0.0211)
19.06 (0.0454)
5.88 (0.0101)
3
ꢀ15.6194
ꢀ17.3536
ꢀ18.1355
ꢀ13.9417
ꢀ18.0423
ꢀ18.7142
ꢀ19.2410
ꢀ6.4785
lIe10
Lys89
Lys89
2.78
2.82
3.25
4a
4b
5a
5b
6
101.44 (0.2373)
47.07 (0.1399)
106.44 (0.2671)
0.61 (0.0016)
8.671 (0.0207)
6.26 (0.0108)
14.54 (0.0340)
5.27 (0.0157)
4.25 (0.0107)
51.06 (0.1377)
11.54 (0.0275)
4.92 (0.0085)
Leu83
2.74
Lys89, lIe10
Lys89, lIe10
Lys89, Asp86
Leu83, Glu81
2.71, 2.67
3.17, 2.81
2.37, 1.82
2.77, 3.11, 2.61
7
LZ8
Doxorubicin
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Arch. Pharm. Chem. Life Sci. 2014, 347, 1–8
Pyrroloazepines as Anticancer Active Agents
5
amino acid interactions as compounds 3 and 5a form a
hydrogen bonding with lIe10 and Leu83, respectively, but
compound 4a formed a hydrogen bonding with Lys89.
and showed a better score energy than the reference ligand, it
suggests the idea of being able to act as inhibitors for this
receptor. The best score energy was shown by compound 7
(S) ¼ ꢀ19.2410 kcal/mol that interacted with two amino acids
(Fig. 3) Lys89 and Asp86 with hydrogen bonding of 2.37 and
2.7 Å, respectively, followed by compound 6 with score energy
(S) ¼ ꢀ18.7142 kcal/mol and interactions with two amino
acids, Lys89 with hydrogen bond of 3.17 Å and Ile10 with
hydrogen bond of 2.81 Å (Fig. 4).
Molecular docking
Cyclin-dependent kinases (CDKs) are key regulatory protein
kinases in cell cycle progression. Inhibition of these cell cycle
proteins has proved to be an effective strategy in the
development of new anticancer active agents [20]. The newly
synthesized compounds 3–7 were docked into ATP binding Conclusion
site of CDK2 in a trial to suggest their mechanism of action
and explore their ability to act as inhibitors for these kinases.
The PDB file 2VTO was used to perform docking simulation.
This file contains CDK2 co-crystallized with LZ8 [21]. Molecu-
lar docking was achieved by Molecular Operating Environ-
ment software 10.2008 (MOE) provided by the Chemical
Computing Group, Canada. Verification step was performed
by redocking of the co-crystallized ligand LZ8 into the active
site of CDK2 with root mean standard deviation (RMDS)
¼ 1.2854 and the score energy was (S) ¼ ꢀ6.4785 kcal/mol. LZ8
showed three interactions with the active site of CDK2: two
hydrogen bonds with Leu83 of 2.77 and 3.11 Å and one
hydrogen bond with Glu81 of 2.61 Å (Fig. 2). The obtained data
for all the novel docked compounds, energy score (S), amino
acid interactions, and hydrogen bond length, are presented in
Table 2. All compounds 3–7 were fit to the active site of CDK2
Pyrroloazepines proved to be a promising scaffold for
designing new anticancer active agents as noticed during
this work. All the synthesized pyrroloazepines 3–7 showed a
potent inhibitory activity against liver (HepG2), breast (MCF7),
and colon (HCT116) cancer cell lines. Compound 3 and the
pyrimidopyrroloazepine 7 showed a broad-spectrum anti-
cancer activity against all the tested cell lines and their IC50
values were expressed in nanomoles. Moreover, compound 6,
which was the most potent against HepG2 and HCT116, and
compound 3 were found to be more potent than doxorubicin
against HepG2 cells; their IC50 was 1.6 and 4 nM, respectively.
The benzoylated derivative of pyrroloazepine 5b showed to be
the most active one against MCF7 cells with an IC50 value of
10.7 nM. All the synthesized compounds when docked into
the active site of CDK2 showed to quick fit this site with less
Figure 2. LZ8 ligand co-crystallized with CDK2.
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A. Belal
Arch. Pharm. Chem. Life Sci. 2014, 347, 1–8
Figure 3. 3D interactions of compound 7 with
CDK2.
Figure 4. 3D interactions of compound 6 with
CDK2.
energy score than the reference ligand LZ8 and this may help
suggesting their mechanism as CDK2 inhibitors. All the
obtained information from this work might be helpful in
design and synthesis of new chemotherapeutic active agents
based on pyrroloazepine scaffold.
were recorded in CDCl3 and on a Varian Mercury spectrometer
(300 and 400 MHz), and 13C NMR (DMSO-d6) spectra were recorded
at 100.62 MHz at the aforementioned research center in Cairo
University and the magnetic resonance unit at Beni-Suef
University. Chemical shifts are expressed in values (ppm) and
tetramethylsilane (TMS) is the internal standard. Addition of
D2O was used to confirm the exchangeable protons.
Experimental
Amino-1-cyano-6,7,8,9-tetrahydro-5H-pyrrolo[1,2-a]-
azepine-3-carboxylic acid phenylamide 3
Chemistry
To a mixture of 1 (0.16 g, 1 mmol) and 2 (0.17 g, 1 mmol) in dry
acetone (20 mL), dry potassium carbonate (0.5 g) was added and
the reaction mixture was refluxed for 24 h. The solvent was
removed under vacuum; the obtained residue was washed with
water, filtered, and crystallized from ethanol to give white solid,
0.2 g, 68% yield, m.p. 148–150°C. IR: nmax./cmꢀ1 3066 (CH
Compounds 1 and 2 were prepared according to the previously
reported procedures [17, 18]. Melting points were detected using
Electrothermal Stuart 5MP3 digital melting point apparatus and
were uncorrected. Kieselgel 0.25 mm, 60 G F 254, Merck Silica gel
plates were used for thin layer chromatography (TLC). The
running solvent system was chloroform/methanol (9:1) and
ultraviolet light was used to detect the spots. Infrared (IR) spectra
(KBr disc) were performed on FT-IR spectrophotometer. IR and
elemental microanalyses were performed at the Microanalytical
Center, Faculty of Science, Cairo University; the 1H NMR spectra
–
–
aromatic), 3266, 3163 (NH2, NH), 2202 (CN), 1591 (C O).
1H NMR (CDCl3, 400 MHz): d 1.47–1.83 (3m, 6H, azepane), 2.69
(t, 2H, azepane), 3.40 (t, 2H, azepane), 7.11–7.55 (m, 5H, aromatic
protons), 8.32 (s, 2H, NH2 exchangeable with D2O), 9.34 (s, 1H, NH
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Arch. Pharm. Chem. Life Sci. 2014, 347, 1–8
Pyrroloazepines as Anticancer Active Agents
7
exchangeable with D2O). 13C NMR (DMSO-d6): 24.5, 28.1, 29.5,
31.2, 45.1, 47.2, 116.2, 117.7, 120.2, 125.6, 128.9, 129.3, 177.4.
Anal. calcd. for C17H18N4O(294.35): C, 69.37; H, 6.16; N, 19.03.
Found: C, 69.81; H, 6.10; N, 19.43.
for C19H20N4O2 (336.39): C, 67.84; H, 5.99; N, 16.66. Found: C,
67.89; H, 6.04; N, 16.44.
Benzoylamino-1-cyano-6,7,8,9-tetrahydro-5H-pyrrolo-
[1,2-a]azepine-3-carboxylic acid phenylamide 5b
General procedure for the preparation of 4a and 4b
To a solution of 3 (0.3 g, 2 mmol) in absolute ethanol (20 mL), the
appropriate aldehyde (2 mmol) and five drops of glacial acetic
acid were added; the reaction mixture was heated under reflux
for 10 h. Solvent was removed under vacuum and the obtained
solid was crystallized from ethanol/water.
Compound 5b was prepared from compound 3 and benzoyl
chloride. White crystals, 0.36 g, 45% yield, m.p. 139–141°C. IR:
nmax./cmꢀ1 3265, 3162 (2NH), 3066 (CH aromatic), 2202 (CN),
1
–
1695, 1591 (2C O). H NMR (CDCl , 400 MHz): d 1.66–1.97 (3m, 6H,
–
3
azepane), 2.72 (t, 2H, azepane), 3.42 (t, 2H, azepane), 7.28–7.62 (m,
8H, aromatic protons), 8.09 (m, 2H, aromatic protons), 8.60, 10.01
(2s, 2H, 2NH exchangeable with D2O). 13C NMR (DMSO-d6): 24.5,
28.1, 29.5, 31.2, 45.1, 47.2, 116.2, 117.7, 125.6, 128, 128.9, 129.2,
129.3, 129.7, 131.2, 133.2, 167.7, 177.4. Anal. calcd. for
C24H22N4O2 (398.46): C, 72.34; H, 5.57; N, 14.06. Found: C,
72.47; H, 5.63; N, 14.56.
2-[(4-Chloro-benzylidene)-amino]-1-cyano-6,7,8,9-
tetrahydro-5H-pyrrolo[1,2-a]azepine-3-carboxylic acid
phenylamide 4a
Compound 4a was prepared from 3 and 4-chlorobenzaldehyde.
Yellowish green crystals, 0.35 g, 41% yield, m.p. 153–155°C. IR:
2-(2-Chloro-acetylamino)-1-cyano-6,7,8,9-tetrahydro-5H-
nmax./cmꢀ1 3163 (NH), 3062 (CH aromatic), 2202 (CN), 1649 (C O).
–
–
1H NMR (CDCl3, 400 MHz): d 1.63–1.78 (3m, 6H, azepane), 2.70
(t, 2H, azepane), 3.41 (t, 2H, azepane), 7.28–7.47 (m, 9H, aromatic
protons), 9.10 (s, 1H, NH exchangeable with D2O), 9.98 (s, 1H,
pyrrolo[1,2-a]azepine-3-carboxylic acid phenylamide 6
To a solution of compound 3 (0.3 g, 2 mmol) in dioxan (20 mL)
chloro acetyl chloride (0.22 g, 2 mmol) was added dropwise; the
reaction mixture was stirred at room temperature for 12 h, then
evaporated under vacuum. The obtained white precipitate was
recrystallized from acetone/ethanol mixture, 0.30 g, 40% yield,
m.p. 143–145°C. IR: nmax./cmꢀ1 3265, 3158 (2NH), 2202 (CN), 1592
N CH). 13C NMR (DMSO-d6): 24.5, 28.1, 29.5, 31.2, 45.1, 47.2, 53.2,
–
–
116.2, 117.7, 125.6, 127.1, 129.3, 129.8, 177.4. Anal. calcd. for
C24H21ClN4O (416.90): C, 69.14; H, 5.08; N, 13.44. Found: C, 69.63;
H, 5.45; N, 13.55.
(br. 2C O), 1629 (C N). 1H NMR (CDCl3, 400 MHz): d 1.64–1.79
–
–
–
–
(3m, 6H, azepane), 2.70 (t, 2H, azepane), 3.41 (t, 2H, azepane), 4.09
(s, 2H, CH2Cl), 7.15–7.56 (m, 5H, aromatic protons), 8.32 (s, 2H,
2NH exchangeable with D2O). 13C NMR (DMSO-d6): 24.5, 28.1, 29.5,
31.2, 44, 45.1, 47.2, 116.2, 117.7, 119.8, 124.2, 125.6, 127.1, 129.2,
129.3, 138.9, 140.6, 165, 177.4. Anal. calcd. for C19H19ClN4O2
(370.83): C, 61.54; H, 5.16; N, 15.11. Found: C, 61.18; H, 5.63;
N, 15.35.
1-Cyano-2-[(4-nitro-benzylidene)-amino]-6,7,8,9-
tetrahydro-5H-pyrrolo[1,2-a]azepine-3-carboxylic acid
phenylamide 4b
Compound 4b was obtained from compound 3 and 4-nitro-
benzaldehyde. yellow crystals, 0.40 g, 46% yield, m.p. 163–165°C.
IR: nmax./cmꢀ1 3163 (NH), 3068 (CH aromatic), 2202 (CN), 1706
1
–
(C O). H NMR (CDCl , 400 MHz): d 1.62–1.90 (3m, 6H, azepane),
–
3
4-Oxo-2-piperidin-1-ylmethyl-4,5,6,7,8,9-hexahydro-3H-
1,3,9a-triaza-benzo[a]azulene-10-carboxylic acid
phenylamide 7
2.72 (t, 2H, azepane), 3.41 (t, 2H, azepane), 7.16–7.62 (m, 5H,
aromatic protons), 8.08–8.42 (m, 4H, aromatic protons), 9.26
(s, 1H, NH exchangeable with D O), 10.17 (s, 1H, N CH). 13C NMR
–
–
2
(DMSO-d6): 24.5, 26.5, 28.1, 29.5, 45.1, 116.2, 117.7, 123.8, 124.7,
125.6, 126, 126.1, 127.1, 128.5, 129.1, 129.3, 131, 140.3, 141.2,
164.5, 177.4. Anal. calcd. for C24H21N5O3 (427.46): C, 67.44;
H, 4.95; N, 16.38. Found: C, 67.24; H, 5.09; N, 16.79.
Compound 6 (0.37 g, 1 mmol) was refluxed with piperidine
(0.426 g, 5 mmol) in absolute ethanol for 24 h; the reaction
mixture was cooled to room temperature, and the obtained white
precipitate was filtered and recrystallized from ethanol. 0.30 g,
71% yield, m.p. 155–157°C. IR: nmax./cmꢀ1 3433, 3266 (2NH), 3056
(CH aromatic), 1651, 1591 (2C O). 1H NMR (CDCl3, 300 MHz):
–
–
General procedure for the preparation of 5a and 5b
Compound 3 (0.3 g, 2 mmol) was dissolved in dry benzene (20 mL)
and the appropriate acid chloride (2 mmol) was added; the
reaction mixture was stirred at room temperature for 24 h, and
the solvent was evaporated under vacuum. The obtained product
was crystallized from methanol.
d 1.66–1.86 (3m, 6H, azepane), 2.10 (m, 4H, piperidine), 2.21 (t, 2H,
azepane), 3.10 (m, 4H, piperidine), 3.65 (t, 2H, azepane), 4.03
(s, 2H, CH2-piperidine), 7.27–7.10 (m, 5H, aromatic protons), 9.02,
10.90 (2s, 2H, 2NH exchangeable with D2O). 13C NMR (DMSO-d6):
22.1, 22.5, 24.5, 28.1, 29.5, 31.2, 44, 45.1, 47.2, 53.1, 53.2, 116.2,
117.7, 119.8, 125.6, 127.1, 129.2, 129.3, 164.5, 177.4. Anal. calcd.
for C24H29N5O2 (419.52): C, 68.71; H, 6.97; N, 16.69. Found: C,
68.48; H, 6.63; N, 16.35.
2-Acetylamino-1-cyano-6,7,8,9-tetrahydro-5H-pyrrolo-
[1,2-a]azepine-3-carboxylic acid phenylamide 5a
Compound 5a was prepared from 3 and acetyl chloride. White
solid, 0.34 g, 50% yield. IR: nmax./cmꢀ1 3265, 3163 (2NH), 2202
Pharmacological studies
(CN), 1591 (br. 2C O). 1H NMR (CDCl3, 400 MHz): d 1.60–1.90
Cytotoxicity of the newly synthesized compounds was evaluated
using sulforhodamine-B (SRB) assay method that was previously
reported by Skehan et al. [22]. HepG2, MCF7, and HCT116 cancer
cell lines were obtained from the American Type Culture
Collection (ATCC, Minisota, USA) through the Tissue Culture
Unit, The Egyptian Organization for Biological Products and
–
–
(3m, 6H, azepane), 2.23 (s, 3H, CH3), 2.86 (t, 2H, azepane), 3.60
(t, 2H, azepane), 4.55 (s, 1H, NH exchangeable with D2O), 7.10–
7.64 (m, 5H, aromatic protons), 9.81 (s, 1H, NH exchangeable with
D2O). 13C NMR (DMSO-d6): 23.1, 26.4, 28.1, 31.2, 45.1, 47.2, 116.2,
117.7, 119.9, 124.3, 125.6, 127.1, 129.2, 129.3, 177.4. Anal. calcd.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

8
A. Belal
Arch. Pharm. Chem. Life Sci. 2014, 347, 1–8
Vaccines (Vacsera, Egypt). Anticancer activity evaluation was
performed at the Center for Genetic Engineering, Al-Azhar
University, Cairo, Egypt. Reagents and chemicals were purchased
from Sigma–Aldrich Chemical Company (St. Louis, MO, USA).
Cells were seeded for 24 h in 96-well microtiter plates at a
concentration of 1000–2000cells/well, 100 mL/well; then cells were
incubated for 48 h with various concentrations (0, 6.25, 12.5, 25,
50, and 100 mg/mL) of the tested compounds and doxorubicin.
Three wells were used for each concentration. After incubation for
48 h, the cells were fixed with 10% trichloroacetic acid 150 mL/well
for 1 h at 4°C and washed with distilled water three times. Wells
were stained for 10–30 min at room temperature with 0.4% SRB
and dissolved in 1% acetic acid 70 mL/well. They were then washed
with acetic acid 1% to remove unbound dye till colorless drainage
was obtained. The plates were subjected to air drying for 24 h not
exposed to UV. The dye was solubilized with 150 mL/well of 10 mM
Tris–EDTA (pH 7.4) for 5 min on a shaker at 1600rpm. The optical
density (OD) of each well was measured spectrophotometrically at
545 nm with an ELISA microplate reader. The percent of surviving
cells was calculated and plotted against different concentrations
of the tested compounds to obtain the survival curve. The IC50
values were calculated using sigmoidal concentration–response
curve fitting models (Sigmaplot software).
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Molecular docking
All the molecular docking studies were performed using an Intel
Core i5 processor 2.5 GHz, memory (RAM) 4 GB Windows XP
operating system and Molecular Operating Environment (MOE,
10.2008) software. 3D structure of the newly synthesized
compounds was generated from their 2D structures; then energy
minimization was performed using MOE with an RMSD gradient
of 0.05 kcal/mol Å; MMFF94X force field and partial charges were
automatically calculated. Co-crystallized structure for CDK2 with
LZ8 (2VTO) [23] was obtained from protein data bank (http://www.
rcsb.org/pdb/home/home.do) and was refined for docking. Ligand
was removed from the active site, hydrogens were added, and
MOE Alpha Site Finder was used to detect the active site. Then the
obtained model was saved as MOE file and docking process was
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This work was financially supported by Taif University, Taif, Saudi
Arabia, Grant No. 1–434–2358.
[21] P. G. Wyatt, A. J. Woodhead, V. Berdini, J. A. Boulstridge,
M. G. Carr, D. M. Cross, D. J. Davis, L. A. Devine, T. R. Early,
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The author has declared no conflict of interest.
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ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com