A Greener Approach Synthesis and Docking Studies of Perimidine Derivatives as Potential Anticancer Agents

Article abstract of DOI:10.1002/jhet.3059

Microwave solvent-free technique was employed to the synthesis of series of 2-(1H-perimidin-2(3H)-ylidene) derivatives, 4-(1H-perimidin-2-yl)-1H-pyrazole-3-carboxamides, pyrrolo[1,2-a]perimidin-10-ones, and 8H-[1,2,4]triazolo[4,3-a]perimidine. Compared wi

Full text of DOI:10.1002/jhet.3059

Month 2017
A Greener Approach Synthesis and Docking Studies of Perimidine
Derivatives as Potential Anticancer Agents
a
b
*
Hany A. Eldeab and Ahmad F. Eweas
aDepartment of Pharmaceutical Chemistry, College of Pharmacy, Taif University, Taif, Saudi Arabia
bDepartment of Medicinal Chemistry, Pharmaceutical and Drug Industries Division, National Research Centre,
Giza, Egypt
*
E-mail: eweas1@gmail.com
Received July 28, 2017
DOI 10.1002/jhet.3059
Published online 00 Month 2017 in Wiley Online Library (wileyonlinelibrary.com).
Microwave solvent-free technique was employed to the synthesis of series of 2-(1H-perimidin-2(3H)-yl-
idene) derivatives, 4-(1H-perimidin-2-yl)-1H-pyrazole-3-carboxamides, pyrrolo[1,2-a]perimidin-10-ones,
and 8H-[1,2,4]triazolo[4,3-a]perimidine. Compared with conventional method, the obvious feature of
microwave method is higher in chemical yield, faster reaction rate, and cleaner reaction condition. The
structures of the synthesized compounds were confirmed based on their elemental analyses and spectro-
scopic data (FT-IR, 1H-NMR, 19F-NMR, 13C-NMR, and LC-MS/MS). Some of the synthesized compounds
exhibit anticancer potential against the growth of both the human breast (MCF-7) and the liver carcinoma
(HepG2) tumor cells. The most active cytotoxic perimidine derivatives were docked against topoisomerase
II to investigate their binding and DNA intercalating activity against the protein crystal structure.
J. Heterocyclic Chem., 00, 00 (2017).
INTRODUCTION
assisted synthesis of perimidine derivatives [25].
Microwave irradiation technologies originate new areas to
the synthetic chemistry in the form of new reactions that
cannot be accomplished using conventional heating and
serve a pliable tool for chemical reactions. Recently, there
has been a dramatic interest in using of microwave
irradiation as an alternative energy source to perform
organic reactions. Microwave-assisted organic synthesis
is a tool by which we can achieve goals in a few minutes
as compared with conventional heating. Current
microwave technologies are able to translate small-scale
microwave synthesis from milligram or gram scale to
multi-kilogram scale by aid of batch or continuous-flow
processing. However, many of the benefits of small-scale
microwave synthesis are lost when the processes
performed in larger batch reactors, which is one of the
disadvantages facing this methodology. Based on these
findings, we herein report a microwave-assisted synthesis
of perimidine derivatives under solvent-free conditions.
Furthermore, the new perimidine derivatives were
evaluated for their cytotoxicity against both the human
breast (MCF-7) and the liver carcinoma (HepG2) tumor
cell lines. In addition, a molecular docking study was
carried out for the highest cytotoxic compounds against
Perimidine derivatives have always attracted a great
interest because of their wide range of applications
varying from dyestuffs [1–7], fluorescent molecular
sensors [8], manufacture of polyester fibers, and as a
source of a novel carbene ligand [9]. In addition to their
biological activity including antiulcer, antimicrobial, and
antifungal properties [10–14], more recently, the DNA
binding affinity and antitumor potential of many
perimidine derivatives have been investigated [15,16].
Perimidine derivatives proved to be valuable starting
substances for the preparation of more complex
polyheterocycles [17–22]. Traditionally, synthesis of
perimidine derivatives was achieved using conventional
solvent-based synthetic routes with low to moderate
yields [23,24]. Perimidines is a condensed tricyclic ring
system with unusual electronic properties among azines
where the lone pair of a pyrrole-like nitrogen participates
in the π-system of the molecule. It shows a transfer of
electron density from the heterocyclic to the naphthalene
moiety. Therefore, perimidines have the characteristics of
both π-deficient and π-excessive systems. Recently, a few
number of reports in the literature uses microwave-
© 2017 Wiley Periodicals, Inc.

H. A. Eldeab and A. F. Eweas
Vol 000
Table 1
topoisomerase II crystal structure to investigate their
binding and DNA intercalating activity against the
protein crystal structure.
Microwave methods to synthesize of 2-(1H-perimidin-2(3H)-ylidene)
derivatives (4a–c).
Microwave synthesis
Compound
no.
Reaction time
(min)
Yield
(%)
RESULTS AND DISCUSSION
R
4a
4b
4c
CH3
C6H5
4-MeOC6H5
3
2
2
96
97
96
The synthesis of the starting materials, 1H-perimidin-
2(3H)-ylidenes (4a–c), has been previously reported from
the reactions of naphthalene-1,8-diamine (1) with the
corresponding β-keto esters
2 using the classical
approaches in solution, which is expensive and time-
consuming [25]. Our work is directed towards
establishing a facile direct method to synthesis the target
compounds 4a–c under solvent-free conditions as a new
method (Scheme 1). Short reaction times and excellent
yields (96–97%) resulted in the isolation of the products
4a–c as shown in Table 1.
C-2) at δ = 155.3 ppm. This procedure afforded product
yields of up to 90% in short time (Table 2).
Furthermore, microwave-assisted reaction under
solvent-free condition was employed to synthesis
8H-[1,2,4]triazolo[4,3-a]perimidines (14a–h), whereas
mixture of the perimidine thione 11 and the appropriate
hydrazonoyl chlorides (12) in the presence of piperidine
under controlled microwave irradiation was carried out to
afford the target compounds (14a–h) (Table 3). The
chemical reaction proceeds via the formation of the
Reaction of 1H-perimidin-2(3H)-ylidenes (4a–c) with
the appropriated hydrazonoyl chlorides 5 or 8 in the
presence of a catalytic amount of piperidine was
performed under microwave controlled conditions to
yield the corresponding 4-(1H-perimidin-2-yl)-1H-
pyrazole-3-carboxamides (7a–f) and pyrrolo[1,2-a]
perimidin-10-ones (10a–f) as single isomers, respectively.
The mechanistic pathways are described by the formation
of the intermediates (6) and (9), which undergoes in situ
elimination of water or alcohol molecule to yield the
products (7a–f) and (10a–f), respectively, as single
product (Scheme 2). The structure of the obtained
products (7a–f) and (10a–f) was confirmed on the bases
intermediates
(13),
which
undergoes
Smiles
rearrangement [26] followed by hydrogen sulfide
elimination (Scheme 3). The structure of the obtained
compounds was unambiguously established by spectral
1
data (FT-IR, H-NMR, 19F-NMR,13C-NMR, and LC-MS/
MS). In particular, the analytical data for (14d) have a
molecular formula C25H16FN5O. LC-MS (ionization
method): m/z 422 [M+1]. IR data gave signal at υ
1671 cmÀ1 corresponding to the presence of carbonyl
group. 1H-NMR (400 MHz, DMSO-d6) spectrum of
compound 14d showed one singlet at δ = 11.53 ppm
assigned for the NH groups. 19F-NMR (400 MHz,
DMSO-d6) gave multiplet signal at δ = À108.31 because
of the presence of fluorine atom, whereas 13C-NMR
(400 MHz, DMSO-d6) showed the appearance of signal
at δ = 155.7 ppm assigned for the (perimidin C-2).
1
of their elemental analyses and spectral data (FT-IR, H-
NMR, 19F-NMR, 13C-NMR, and LC-MS/MS). Thus, the
analytical data for (7b) revealed a molecular formula
C28H20FN5O. LC-MS (ionization method): m/z 462 [M
+1]. IR showed signal at υ 1643 cmÀ1 assigned for the
presence of carbonyl group. 1H-NMR (400 MHz,
DMSO-d6) spectrum of compound (7b) showed a two
singlet at δ = 12.44 and 10.83 ppm assigned for the 2NH
groups. 19F-NMR (400 MHz, DMSO-d6) gave multiplet
signal at δ = À108.49 because of the presence of fluorine
atom, whereas 13C-NMR (400 MHz, DMSO-d6) showed
the appearance of signal corresponding to the (perimidin
Cytotoxicity.
Preliminary screening showed that all
selected compounds exhibited a moderate to strong
growth inhibition activity on the tested cell lines between
0.59 and >50 μg/mL concentrations in comparison with
the known anticancer drug Doxorubicin 0.46 μg/mL
Scheme 1. Synthesis of 2-(1H-perimidin-2(3H)-ylidene) derivatives (4a–c).
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2017
Microwave Synthesis of Novel Periminde Derivatives and Screening of Their
Antitumor Activity
Scheme 2. Synthesis of 4-(1H-perimidin-2-yl)-1H-pyrazole-3-carboxamides (7a–f) and pyrrolo[1,2-a]perimidin-10-ones (10a–f).
Table 2
Scheme 3. Synthesis of 8H-[1,2,4]triazolo[4,3-a]perimidines (14a–h).
The reaction time and yield of the microwave methods to synthesize of 4-
(1H-perimidin-2-yl)-1H-pyrazole-3-carboxamides (7a–f) and pyrrolo[1,2-
a]perimidin-10-ones (10a–f).
Compound
no.
Reaction time
(min)
Yield
(%)
R1
Ar
C6H5
4-FC6H4
4-MeC6H4
4-NO2C6H4
7a
7b
7c
7d
7e
7f
10a
10b
10c
10d
10e
10f
CH3
CH3
CH3
CH3
4
4
4
5
4
4
4
3
4
4
4
4
97
95
98
93
91
94
99
93
98
94
94
92
C6H5 C6H5
C6H5 4-MeC6H4
CH3
CH3
CH3
CH3
the highest cytotoxic activity against both tested cell lines
in comparison with the reference drug. On the other
hand, compound 10c showed the lowest activity against
both cell lines with IC50 of >50 μg/mL.
C6H5
4-FC6H4
4-MeC6H4
4-NO2C6H4
Molecular docking.
In order to interpret the
C6H5 C6H5
C6H5 4-MeC6H4
topoisomerase II inhibitory activity of the most active
newly synthesized perimidine derivatives on a molecular
level, a docking study was carried out using FlexX module
in LEADIT 2.1.8 software package [BioSolveIT GmbH,
Sankt Augustin, Germany (2014)]. The complex of DNA
with human topoisomerase IIβ along with etoposide refer-
ence ligand (PDB: 3QX3) was downloaded from Protein
(Table 4) against HepG2 cell line and 0.49 to >50 μg/mL
compared with 0.426 μg/mL for the reference drug
against MCF cell line. Among all tested compounds, it
was found that compounds 7c, 14a, and 14g exhibited
Table 3
The reaction time and yield of the microwave methods to synthesize of 8H-[1,2,4]triazolo[4,3-a]perimidines (14a–h).
Compound no.
R
Ar
4-FC6H4
4-NO2C6H4
C6H5
Reaction time (min)
Yield (%)
14a
14b
14c
14d
14e
14f
COCH3
COCH3
3
4
4
4
4
4
3
3
92
95
91
92
94
91
98
95
CONHC6H5
CONHC6H5
CONHC6H5
CONHC6H5
COOC2H5
4-FC6H4
4-MeC6H4
4-NO2C6H4
4-MeC6H4
4-NO2C6H4
14g
14h
COOC2H5
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

H. A. Eldeab and A. F. Eweas
Vol 000
Table 4
perimidine derivatives have DNA binding affinity
The anticancer activity of the synthesized compounds.
according to the published data [15].
IC50 (μg/mL)
Compound no.
HepG2 cell
MCF-7 cell line
0.49
EXPERIMENTAL
7c
10a
10b
10c
14a
14d
14g
Doxorubicin
0.59
21.7
9.1
>50
0.62
18.0
1.75
0.469
42.2
Chemistry. General: Microwave synthetic protocol was
done using CEM microwave system. Melting points were
determined on (Pyrex capillary) Gallenkamp apparatus. IR
spectra were recorded with a Thermo Nicolet Nexus 470
FT-IR spectrometer in the range 4000–400 cmÀ1 on
18.2
>50
14.4
6.2
0.52
0.426
1
samples in potassium bromide disks. H-NMR spectra and
13C-NMR spectra were obtained on Varian Gemini
400 MHz FT-NMR spectrometer in DMSO-d6; chemical
shifts were recorded in δ (ppm) units, relative to Me4Si as
an internal standard. The mass spectra were recorded on
Shimadzu LCMS-QP 800 LC-MS and AB-4000 Q-trap LC-
MS/MS. Analytical data were obtained using PerkinElmer
2400 II series Carbon hydrogen and Nitrogen (CHN)
analyzer. Thin-layer chromatography (TLC) was carried out
on precoated Merck silica gel F254 plates (Merck Millipore,
Burlington, MA), and UV light was used for visualization.
Column chromatography was performed on a Merck silica
gel. The reagents were purchased from Sigma-Aldrich (St.
Louis, MO) and used without further purification.
Data Bank (PDB) server (www.rcsb.org) and was employed;
the chain A along with DNA complex was selected for the
present molecular docking studies through the extra-
precision docking mode of LEADIT 2.1.8. The docking result
of the reference drug etoposide binding energy was
À37.50 kcal/mol. Etoposide also shows hydrogen bonding
interactions with Asp479 of topo II and DAF12 of the
DNA and hydrophobic interactions with Gly478, Gly504,
and Arg503 of topo II in addition to DGF10 and DGF13 of
the DNA. On the other hand, the tested compounds 7c,
14a, and 14g scores high binding affinity towards the binding
site in topoisomerase II (3QX3) ranging from À30.05 kcal/
mol for 14a to À32.52 kcal/mol for 7c (Table 5).
General procedure for the synthesis of 2-(1H-perimidin-
2(3H)-ylidene) derivatives (4a–c). Microwave method.
A
Compound 7c the most cytotoxic active compound
against both examined cell lines showed one hydrogen
bonding with DTF9 of the DNA in addition to
hydrophobic interactions with DGF13 and DCF9 of the
DNA as well as Arg503, Gly504, and Gly778 of the
protein. Furthermore, both compounds 14a and 14g
showed very similar interaction with the target molecule,
with one hydrogen bond with DCF8 of the DNA and
hydrophobic interactions with DTF9, DAF12, and
DGF13 of the DNA site, in addition to hydrophobic
interaction with Arg503 of the protein. The 2D and 3D
poses interactions of compounds in the active site of
topoisomerase II are depicted in (Fig. 1). From these
results, we can conclude the newly synthesized
perimidine derivatives have higher affinity towards the
DNA binding site of the topoisomerase IIβ rather than the
protein itself, which is in accordance with the fact that
mixture of naphthalene-1,8-diamine (1) (0.01 mol) and
β-keto esters (2) (0.01 mol) in the presence of potassium
carbonate was irradiated at power = 200 W for an
appropriate time in a 10-mL closed vial using CEM
microwave system. After completion of the reaction, as
indicated by TLC, the obtained products were purified by
crystallization from suitable solvent to afford the products
(4a–c).
1-(1H-Perimidin-2(3H)-ylidene)propan-2-one (4a).
mp
1
261°C; IR (KBr, cmÀ1)υ 3128 (NH), 1658 (C═O); H-
NMR (400 MHz, DMSO-d6) δ = 13.21, 10.19 (s, 2H, NH),
7.19–6.40 (m, 6H, Ar-H), 4.68 (s, 1H, ═CH), 2.10 ppm
(CH3); 13C-NMR (100 MHz, DMSO-d6) δ = 191.5 (C═O),
152.4 (perimidin C-2), 134.3–104.5 (Ar-C), 80.0 (═CH),
28.6 ppm (CH3). LC-MS (ionization method): m/z 225 [M
+1]. Anal. Calcd for C14H12N2O: C, 74.98; H, 5.39; N,
12.49. Found: C, 75.11; H, 5.27; N, 12.55%.
Table 5
Docking results of the active compounds using LEADIT 2.1.8 software.
Compound no.
7c
14a
14g
Etoposide
Doxorubicin
Affinity score (kcal/mol)
Lipophilic contribution score
Clash score
Ligand entropy conformation score
À32.52
À30.05
À30.51
À37.50
À38.46
À9.38
À12.39
À14.49
À18.73
À10.68
3.36
2.58
4.68
7.69
2.70
0.00
0.00
2.80
15.40
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2017
Microwave Synthesis of Novel Periminde Derivatives and Screening of Their
Antitumor Activity
Figure 1. Possible binding modes of compounds (A) etoposide, (B) 7c, (C) 14a, and (D) 14g. [Color figure can be viewed at wileyonlinelibrary.com]
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

H. A. Eldeab and A. F. Eweas
Vol 000
2-(1H-Perimidin-2(3H)-ylidene)-1-phenylethan-1-one
DMSO-d6) δ = 12.33 (s, 1H, NH), 10.39 (s, 1H, NH),
7.80–6.28 (m, 15H, Ar-H), 2.38 ppm (s, 3H, CH3), 1.92
(s, 3H, CH3); 13C-NMR (100 MHz, DMSO-d6) δ = 170.9
(C═O), 155.4 (perimidin C-2), 145.1–107.4 (Ar-C), 25.1
(CH3), 23.8 ppm (CH3); LC-MS (ionization method): m/z
457 [M]. Anal. Calcd for C29H23N5O: C, 76.13; H, 5.07;
N, 15.31. Found: C, 76.21; H, 5.14; N, 15.17%.
(4b).
mp 248°C; IR (KBr, cmÀ1)υ 3261 (NH), 1644
1
(C═O); H-NMR (400 MHz, DMSO-d6) δ = 13.78, 10.63
(s, 2H, NH), 7.77–6.54 (m, 11H, Ar-H), 5.60 ppm (s, 1H,
═CH); 13C-NMR (100 MHz, DMSO-d6) δ = 185.3
(C═O), 154.7 (perimidin C-2), 141.3–106.6 (Ar-C),
80.5 ppm (═CH). LC-MS (ionization method): m/z 287
[M+1]. Anal. Calcd for C19H14N2O: C, 79.70; H, 4.93;
5-Methyl-1-(4-nitrophenyl)-4-(1H-perimidin-2-yl)-N-phenyl-
1H-pyrazole-3-carboxamide (7d).
mp 210°C; IR (KBr,
N, 9.78. Found: C, 79.93; H, 4.81; N, 9.89%.
cmÀ1)υ 3392, 3267 (2NH), 1678 (C═O); 1H-NMR
(400 MHz, DMSO-d6) δ = 12.27 (s, 1H, NH), 10.34 (s,
1H, NH), 8.27–6.82 (m, 15H, Ar-H), 2.04 ppm (s, 3H,
CH3); 13C-NMR (100 MHz, DMSO-d6) δ = 172.1
(C═O), 156.2 (perimidin C-2), 145.4–107.9 (Ar-C), 22.7
(CH3); LC-MS (ionization method): m/z 488 [M]. Anal.
Calcd for C28H20N6O3: C, 68.84; H, 4.13; N, 17.20.
1-(4-Methoxyphenyl)-2-(1H-perimidin-2(3H)-ylidene)ethan-
1-one (4c). mp >300°C; IR (KBr, cmÀ1)υ 3284 (NH),
1
1649 (C═O); H-NMR (400 MHz, DMSO-d6) δ = 13.91,
10.67 (s, 2H, NH), 7.90–6.62 (m, 10H, Ar-H), 5.57 (s,
1H, ═CH), 3.83 (s, 3H, OCH3); 13C-NMR (100 MHz,
DMSO-d6) δ = 184.4 (C═O), 153.5 (perimidin C-2),
141.6–106.7 (Ar-C), 77.1 ppm (═CH), 56.4 (OCH3); LC-
MS (ionization method): m/z 316 [M]. Anal. Calcd for
C20H16N2O2: C, 75.93; H, 5.10; N, 8.86. Found: C,
75.79; H, 5.23; N, 8.94%.
Found: C, 68.91; H, 4.22; N, 17.34%.
4-(1H-Perimidin-2-yl)-N,1,5-triphenyl-1H-pyrazole-3-
carboxamide (7e).
mp 178°C; IR (KBr, cmÀ1)υ 3372,
3259 (2NH), 1678 (C═O); 1H-NMR (400 MHz,
DMSO-d6) δ = 12.83 (s, 1H, NH), 10.56 (s, 1H, NH),
8.41–6.73 ppm (m, 21 H, Ar-H); 13C-NMR (100 MHz,
DMSO-d6) δ = 171.4 (C═O), 154.9 (perimidin C-2),
143.8–109.1 ppm (Ar-C); LC-MS (ionization method): m/
z 505 [M]. Anal. Calcd for C33H23N5O: C, 78.40; H,
General procedures for the synthesis of 4-(1H-perimidin-2-
yl)-1H-pyrazole-3-carboxamides (7a–f) and pyrrolo[1,2-a]
perimidin-10-ones (10a–f). Microwave method. Catalytic
amount of piperidine was added to a mixture of 2-(1H-
perimidin-2(3H)-ylidenes) (4) (0.01 mol) and the
appropriate hydrazonoyl chloride (5) or (8) (0.01 mol).
The reaction mixture was subjected to microwave
irradiation at power = 200 W for an appropriate time in a
10-mL closed vial. TLC was used to indicate the reaction
time. The obtained substances were purified by
crystallization from an appropriate solvent to yield the
4.59; N, 13.85. Found: C, 78.58; H, 4.71; N, 14.01%.
4-(1H-Perimidin-2-yl)-N,5-diphenyl-1-(p-tolyl)-1H-pyrazole-
3-carboxamide (7f). mp 191°C; IR (KBr, cmÀ1)υ 3368,
3252 (2NH), 1678 (CO); 1H-NMR (400 MHz,
DMSO-d6) δ = 12.78 (s, 1H, NH), 10.53 (s, 1H, NH),
8.44–6.70 (m, 20 H, Ar-H), 2.41 ppm (s, 3H, CH3); 13C-
NMR (100 MHz, DMSO-d6) δ = 170.8 (C═O), 155.6
(perimidin C-2), 144.2–108.8 (Ar-C), 24.8 ppm (CH3);
LC-MS (ionization method): m/z 520 [M+1]. Anal. Calcd
for C34H25N5O: C, 78.59; H, 4.85; N, 13.48. Found: C,
products (7a–f) and (10a–f).
5-Methyl-4-(1H-perimidin-2-yl)-N,1-diphenyl-1H-pyrazole-3-
carboxamide (7a). mp 182°С; IR (KBr, cmÀ1)υ 3371, 3421
(2NH), 1641 (C═O) cmÀ1; 1H-NMR (400 MHz, DMSO-d6)
δ = 12.32 (s, 1H, NH), 10.54 (s, 1H, NH), 8.24–6.34 (m,
16H, Ar H), 2.08 (s, 3H, CH3); 13C-NMR (100 MHz,
DMSO-d6) δ = 171.8 (C═O), 155.8 (perimidin C-2),
145.2–107.2 (Ar-C), 24.4 ppm (CH3); LC-MS (ionization
method): m/z 443 [M]. Anal. Calcd for C28H21N5O: C,
75.83; H, 4.77; N, 15.79. Found: C, 75.93; H, 4.83; N,
15.91%.
78.73; H, 4.73; N, 13.61%.
8-Acetyl-9-(2-phenylhydrazono)-7,9-dihydro-10H-
pyrrolo[1,2-a]perimidin-10-one (10a).
mp >300°C; IR
(KBr, cmÀ1)υ 3224, 3171 (2NH), 1707, 1654 (2C═O); 1H-
NMR (400 MHz, DMSO-d6) δ = 12.23 (s, 1H, NH), 11.81
(s, 1H, NH), 8.52–6.73 (m, 11H, Ar-H), 3.14 ppm (s, 3H,
CH3), 13C-NMR (100 MHz, DMSO-d6) δ = 185.8
(COCH3), δ = 163.7 (C═O), 159.3 (perimidin C-2), 146.5–
104.9 (Ar-C), 29.8 ppm (CH3); LC-MS (ionization
method): m/z 368 [M]. Anal. Calcd for C22H16N4O2: C,
71.73; H, 4.38; N, 15.21. Found: C, 71.87; H, 4.51; N,
1-(4-Fluorophenyl)-5-methyl-4-(1H-perimidin-2-yl)-N-
phenyl-1H-pyrazole-3-carboxamide (7b).
mp 220°C; IR
(KBr, cmÀ1)υ 3359, 3052 (2NH), 1643 (C═O); H-NMR
(400 MHz, DMSO-d6) δ = 12.44 (s, 1H, NH), 10.83 (s,
1H, NH), 8.31–6.29 (m, 15H, Ar H), 2.14 ppm (s, 3H,
CH3); 19F-NMR (400 MHz, DMSO-d6) δ = À108.46–
[À108.51] (m, 1F); 13C-NMR (400 MHz, DMSO-d6)
δ = 172.2 (C═O), 155.3 (perimidin C-2), 145.9–107.7 (Ar-
C), 22.8 ppm (CH3); LC-MS (ionization method): m/z 462
[M+1]. Anal. Calcd for C28H20FN5O: C, 72.87; H, 4.37; N,
1
15.13%.
8-Acetyl-9-(2-(4-fluorophenyl)hydrazono)-7,9-dihydro-10H-
pyrrolo[1,2-a]perimidin-10-one (10b). mp 278°C; IR (KBr,
1
cmÀ1)υ 3349, 3178 (2NH), 1691, 1644 (2CO) cmÀ1; H-
NMR (400 MHz, DMSO-d6) δ = 12.41 (s, 1H, NH),
12.20 (s, 1H, NH), 8.56–6.34 (m, 10H, Ar-H), 3.45 ppm
(s, 3H, CH3); 19F-NMR (400 MHz, DMSO-d6)
δ = À109.02–[À109.08] (m, 1F); 13C-NMR (100 MHz,
DMSO-d6) δ = 187.6 (COCH3), δ = 165.2 (C═O), 158.4
15.18. Found: C, 72.95; H, 4.45; N, 15.24%.
5-Methyl-4-(1H-perimidin-2-yl)-N-phenyl-1-(p-tolyl)-1H-
pyrazole-3-carboxamide (7c). mp 201°C; IR (KBr, cmÀ1)υ
1
3367 (NH), 3051 (NH), 1641 (CO); H-NMR (400 MHz,
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2017
Microwave Synthesis of Novel Periminde Derivatives and Screening of Their
Antitumor Activity
(perimidin C-2), 148.3–106.3 (Ar-C), 30.2 ppm (CH3);
LC-MS (ionization method): m/z 387 [M+1]. Anal. Calcd
for C22H15FN4O2: C, 68.39; H, 3.91; N, 14.50. Found: C,
68.51; H, 4.05; N, 14.37%.
8-Acetyl-9-(2-(p-tolyl)hydrazono)-7,9-dihydro-10H-
1-(8-(4-Fluorophenyl)-8H-[1,2,4]triazolo[4,3-a]perimidin-
10-yl)ethan-1-one (14a). mp 168°C; IR (KBr, cmÀ1)υ 1718
(C═O); 1H-NMR (400 MHz, DMSO-d6) δ = 8.31–6.69 (m,
10H, Ar-H), 3.04 ppm (s, 3H, CH3); 19F-NMR (400 MHz,
DMSO-d6): δ = À108.60–[À108.55] (m, 1F); 13C-NMR
(100 MHz, DMSO-d6) δ = 179.2 (C═O), 158.7 (perimidin
C-2), 147.2–123.5 (Ar-C), 25.3 ppm (CH3); LC-MS
(ionization method): m/z 345 [M+1]. Anal. Calcd for
C20H13FN4O: C, 69.76; H, 3.81; N, 16.27. Found: C,
pyrrolo[1,2-a]perimidin-10-one (10c).
mp >300°C; IR
(KBr, cmÀ1)υ 3358, 3147 (2NH), 1696, 1647 (2C═O);
1H-NMR (400 MHz, DMSO-d6) δ = 12.33 (s, 1H, NH),
11.91 (s, 1H, NH), 8.31–7.20 (m, 10H, Ar-H), 3.45 (s,
3H, CH3), 2.61 ppm (s, 3H, CH3); 13C-NMR (100 MHz,
DMSO-d6) δ = 185.8 (COCH3), δ = 162.7 (C═O), 158.4
(perimidin C-2), 144.8–107.3 (Ar-C), 30.2 (CH3),
24.7 ppm (CH3); LC-MS (ionization method): m/z 382
[M]. Anal. Calcd for C23H18N4O2: C, 72.24; H, 4.74; N,
69.82; H, 3.62; N, 16.41%.
1-(8-(4-Nitrophenyl)-8H-[1,2,4]triazolo[4,3-a]perimidin-10-
yl)ethan-1-one (14b). mp 258°C; IR (KBr, cmÀ1)υ 1721
1
(C═O); H-NMR (400 MHz, DMSO-d6) δ = 8.62–6.65
(m, 10H, Ar-H), 3.12 ppm (s, 3H, CH3); 13C-NMR
(100 MHz, DMSO-d6)
(perimidin C-2), 148.6–122.6 (Ar-C), 24.7 ppm (CH3);
LC-MS (ionization method): m/z 371 [M]. Anal. Calcd
for C20H13N5O3: C, 64.69; H, 3.53; N, 18.86. Found: C,
64.91; H, 3.70; N, 18.63%.
δ = 181.7 (C═O), 159.6
14.65. Found: C, 72.38; H, 4.85; N, 14.48%.
8-Acetyl-9-(2-(4-nitrophenyl)hydrazono)-7,9-dihydro-10H-
pyrrolo[1,2-a]perimidin-10-one (10d).
mp >300°C; IR
(KBr, cmÀ1)υ 3431, 3259 (2NH), 1712 (C═O), 1648
1
(C═O); H-NMR (400 MHz, DMSO-d6) δ = 12.24 (s,
1H, NH), 11.91 (s, 1H, NH), 8.34–7.30 (m, 10H, Ar-H),
3.18 ppm (s, 3H, CH3); 13C-NMR (100 MHz, DMSO-d6)
δ = 184.6 (COCH3), δ = 161.9 (C═O), 159.1 (perimidin
C-2), 146.2–105.9 (Ar-C), 30.7 ppm (CH3); LC-MS
(ionization method): m/z 413 [M]. Anal. Calcd for
C22H15N5O4: C, 63.92; H, 3.66; N, 16.94. Found: C,
64.05; H, 4.74; N, 17.02%.
8-Benzoyl-9-(2-phenylhydrazono)-7,9-dihydro-10H-
pyrrolo[1,2-a]perimidin-10-one (10e). mp 261°C; IR (KBr,
N,8-Diphenyl-8H-[1,2,4]triazolo[4,3-a]perimidine-10-
carboxamide (14c).
mp 272°C; IR (KBr, cmÀ1)υ 3285
(NH), 1674 (C═O); 1H-NMR (400 MHz, DMSO-d6)
δ = 11.61 (s, 1H, NH); 8.47–6.84 ppm (m, 16H, Ar-H);
13C-NMR (100 MHz, DMSO-d6) δ = 169.1 (C═O),
159.2 (perimidin C-2), 147.8–123.8 ppm (Ar-C); LC-MS
(ionization method): m/z 404 [M+1]. Anal. Calcd for
C25H17N5O: C, 74.43; H, 4.25; N, 17.36. Found: C,
74.71; H, 3.98; N, 17.41%.
cmÀ1)υ 3314, 3182 (2NH), 1725, 1658 (2C═O); 1H-
NMR (400 MHz, DMSO-d6) δ = 12.41 (s, 1H, NH),
11.70 (s, 1H, NH), 8.61–6.83 ppm (m, 16H, Ar-H); 13C-
NMR (100 MHz, DMSO-d6) δ = 187.1 (COPh),
δ = 164.2 (C═O), 158.8 (perimidin C-2), 148.5–
107.9 ppm (Ar-C); LC-MS (ionization method): m/z 431
[M+1]. Anal. Calcd for C27H18N4O2: C, 75.34; H, 4.21;
8-(4-Fluorophenyl)-N-phenyl-8H-[1,2,4]triazolo[4,3-a]
perimidine-10-carboxamide (14d).
mp 306°C; IR (KBr,
cmÀ1)υ 3247 (NH), 1671 (C═O); H-NMR (400 MHz,
DMSO-d6) δ = 11.53 (s, 1H, NH); 8.51–6.57 ppm (m,
16H, Ar-H); 19F-NMR (400 MHz, DMSO-d6):
δ = À108.27–[À108.34] (m, 1F); 13C-NMR (100 MHz,
DMSO-d6) δ = 168.8 (C═O), 158.7 (perimidin C-2),
148.1–124.1 ppm (Ar-C); LC-MS (ionization method):
m/z 422 [M+1]. Anal. Calcd for C25H16FN5O: C,
71.25; H, 3.83; N, 16.62. Found: C, 71.38; H, 4.03; N,
16.50%.
1
N, 13.02. Found: C, 75.51; H, 4.34; N, 13.24%.
8-Benzoyl-9-(2-(p-tolyl)hydrazono)-7,9-dihydro-10H-
pyrrolo[1,2-a]perimidin-10-one (10f). mp 289°C; IR (KBr,
cmÀ1)υ 3327, 3173 (2NH), 1728, 1648 (2C═O); 1H-NMR
(400 MHz, DMSO-d6) δ = 12.34 (s, 1H, NH), 11.54 (s,
1H, NH), 8.54–6.71 (m, 15H, Ar-H), 2.41 ppm (s, 3H,
CH3); 13C-NMR (100 MHz, DMSO-d6) δ = 184.8
(COPh), δ = 163.1 (C═O), 159.1 (perimidin C-2),
148.0–107.2 ppm (Ar-C); LC-MS (ionization method):
m/z 445 [M+1]. Anal. Calcd for C28H20N4O2: C, 75.66;
H, 4.54; N, 12.60. Found: C, 75.78; H, 4.52; N, 12.81%.
N-Phenyl-8-(p-tolyl)-8H-[1,2,4]triazolo[4,3-a]perimidine-10-
carboxamide (14e).
mp 250°C; IR (KBr, cmÀ1)υ 3222
(NH), 1667 (C═O); 1H-NMR (400 MHz, DMSO-d6)
δ = 11.64 (s, 1H, NH), 8.31–6.70 ppm (m, 16H, Ar-H);
13C-NMR (100 MHz, DMSO-d6) δ = 169.6 (C═O),
159.6 (perimidin C-2), 146.7–124.3 ppm (Ar-C); LC-MS
(ionization method): m/z 417 [M]. Anal. Calcd for
C26H19N5O: C, 74.80; H, 4.59; N, 16.78. Found: C,
74.93; H, 4.72; N, 16.89%.
General procedures for synthesis of 8H-[1,2,4]triazolo[4,3-
a]perimidines (14a–h). Microwave method.
Perimidine
thione (11) (0.01 mol) and the appropriate hydrazonoyl
chloride (12) (0.01 mol) were mixed in the presence of
catalytic amount of piperidine. The reaction mixture was
heated under microwave irradiation (power = 200 W)
for a suitable time in a 10-mL closed vial. The yields
were collected to give the corresponding products
(14a–h).
8-(4-Nitrophenyl)-N-phenyl-8H-[1,2,4]triazolo[4,3-a]
perimidine-10-carboxamide (14f).
mp 272°C; IR (KBr,
1
cmÀ1)υ 3209 (NH), 1661 (C═O); H-NMR (400 MHz,
DMSO-d6) δ = 11.71 (s, 1H, NH), 8.61–6.71 ppm (m,
16H, Ar-H); 13C-NMR (100 MHz, DMSO-d6) δ = 170.3
(C═O), 158.3 (perimidin C-2), 147.4–126.2 ppm (Ar-C);
LC-MS (ionization method): m/z 449 [M+1]. Anal. Calcd
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

H. A. Eldeab and A. F. Eweas
Vol 000
for C25H16N6O3: C, 66.96; H, 3.60; N, 18.74. Found: C,
added to each well for at least 30 min. The stain was
removed, and the plates were rinsed using tap water until
all excess stain is removed. Acetic acid (30%) was then
added to all wells and mixed thoroughly, and then the
absorbance of the plates was measured after gently
shaken on microplate reader, using a test wavelength of
490 nm. All results were corrected for background
absorbance detected in wells without added stain. Treated
samples were compared with the cell control in the
absence of the tested compounds. All experiments were
carried out in triplicate. The cell cytotoxic effect of each
tested compound was calculated (Table 4).
67.21; H, 3.84; N, 18.92%.
1-(8-(p-Tolyl)-8H-[1,2,4]triazolo[4,3-a]perimidin-10-yl)
propan-1-one (14g). mp 298°C; IR (KBr, cmÀ1)υ 1747
1
(C═O); H-NMR (400 MHz, DMSO-d6) δ = 8.01–6.60
(m, 10H, Ar-H), 1.81 (t, 3H, CH3), 2.70 (s, 3H, CH3),
4.72 ppm (q, 2H, CH2); 13C-NMR (100 MHz, DMSO-d6)
δ = 169.3 (C═O), 159.5 (perimidin C-2), 146.1–125.0
(Ar-C), 67.7 (CH2), 24.8 (CH3), 18.1 ppm (CH3); LC-MS
(ionization method): m/z 354 [M]. Anal. Calcd for
C22H18N4O2: C, 74.56; H, 5.12; N, 15.81. Found: C,
74.67; H, 5.27; N, 15.67%.
1-(8-(4-Nitrophenyl)-8H-[1,2,4]triazolo[4,3-a]perimidin-10-
yl)propan-1-one (14h).
mp 206°C; IR (KBr, cmÀ1)υ
1762 (C═O); 1H-NMR (400 MHz, DMSO-d6)
δ = 8.70–6.88 (m, 10H, Ar-H), 1.76 (t, 3H, CH3),
4.77 ppm (q, 2H, CH2); 13C-NMR (100 MHz,
DMSO-d6) δ = 172.2 (C═O), 158.7 (perimidin C-2),
148.7–126.4 (Ar-C), 68.3 (CH2), 17.7 ppm (CH3); LC-
MS (ionization method): m/z 402 [M+1]. Anal. Calcd
for C21H15N5O4: C, 62.84; H, 3.77; N, 17.45. Found:
C, 62.93; H, 3.58; N, 17.58%.
MOLECULAR DOCKING
All docking studies were performed using FlexX module
in LEADIT 2.1.8 software package (BioSolveIT GmbH,
2014). A set of the most active cytotoxic new perimidine
derivatives including compounds 7c, 14a, and 14g were
compiled using CHEMDRAW, and 3D structures were
constructed using CHEMBIO3D ULTRA 12.0 software
[Molecular Modeling and Analysis; Cambridge Soft
Corporation, Waltham, MA (2010)], and then they were
energetically minimized using Molecular Orbital PACkage
(MOPAC) (semi-empirical quantum mechanics), Job Type
with 100 iterations, and minimum root mean square (RMS)
gradient of 0.01 and saved as SDF MolFile (*.sdf). The
crystal structure of target enzyme topoisomerase II
complexed with DNA (PDB code = 3QX3) was retrieved
from the Protein Data Bank (http://www.rcsb.org). The
protein was loaded into LEADIT 2.1.8, and the receptor
components were chosen by selection of all chains in the
protein. Binding site was defined by choosing the native
ligand etoposide as a reference ligand to which all
coordinates were computed. Amino acids within radius 6.5
A were selected in the binding site. All chemical
ambiguities of residues were left as default. Ligand binding
was driven by enthalpy (classic triangle matching). For
scoring, all default settings were restored. Intra-ligand
clashes were computed using clash factor = 0.6. Maximum
number of solutions per iteration is 200. Maximum number
of solutions per fragmentation is 200. The base placement
method was used as a docking strategy.
CYTOTOXICITY ACTIVITY
The anticancer activity of the synthesized compounds 7,
10, and 14 was determined against the human breast cell
line (MCF-7) and the liver carcinoma cell line (HepG2),
using
the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide assay, and Doxorubicin was used as
a reference drug. Data generated were used to plot a
dose–response curve of which the concentration of test
compounds required to kill 50% of cell population (IC50)
was determined. Cytotoxic activity was expressed as the
mean IC50 of three independent experiments (Table 4)
The method applied is similar to that reported by Vijayan
et al. [27] using crystal violet stain (1%). Cells were
well plate at a cell concentration 1 × 104
seeded in 96-
cells per well in 100 μL of growth medium. Fresh
medium containing different concentrations of the test
sample was added after 24 h of seeding. Serial twofold
dilutions of the tested chemical compound were added to
confluent cell monolayers, flat-bottomed microtiter plates
using a multichannel pipette. The microtiter plates were
incubated at 37°C in a humidified incubator with 5%
CO2
for 48 h. Three wells were used for each
concentration of the test sample. Control cells were
incubated without test sample with DMSO. After
incubation of the cells for 24 h at 37°C, various
concentrations of sample (50, 25, 12.5, 6.25, 3.125, and
1.56 μg) were added, and the incubation was continued
for 48 h, and the viable cells yield was determined by a
colorimetric method. After the end of incubation period,
media were aspirated, and the crystal violet solution was
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Antitumor Activity
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Journal of Heterocyclic Chemistry
DOI 10.1002/jhet