Cyclization of N-benzyl cyanoacetamide: Novel synthesis and biological activity of pyrrole, pyrimidine, and pyran derivatives

Article abstract of DOI:10.1002/jhet.3892

Heteroannulation of N-Benzyl cyanoacetamide 1 to a new series of heterocycles has been developed. Thus, reaction of 1 with different polarized π systems afforded pyrrolo 4, pyridone 6, pyridine 8, and diazapene 10 derivatives, respectively. N-Benzyl cyano

Full text of DOI:10.1002/jhet.3892

Received: 26 September 2019
DOI: 10.1002/jhet.3892
Revised: 11 December 2019
Accepted: 23 December 2019
A R T I C L E
Cyclization of N-benzyl cyanoacetamide: Novel synthesis
and biological activity of pyrrole, pyrimidine, and pyran
derivatives
Eman O. Hamed1
| Mohamed G. Assy1 | Adel M. Shalaby2 | Rawda E. Sayed1
1Department of Chemistry, Faculty of
Science, Zagazig University, Zagazig,
Egypt
Abstract
Heteroannulation of N-Benzyl cyanoacetamide 1 to a new series of hetero-
cycles has been developed. Thus, reaction of 1 with different polarized π sys-
tems afforded pyrrolo 4, pyridone 6, pyridine 8, and diazapene 10
derivatives, respectively. N-Benzyl cyanoacetamide that undergo condensa-
tion reaction with salicylaldehyde yielded pyran derivative 11. Nitrosation
of 11 furnished condensed pyran 13. Compound 11 reacted with benzalde-
hyde, carbon disulfide (cyclizing agent), and ammonium thiocyanate to pro-
vide pyrane 17, thiazine 18, and thiourea 20 derivatives, respectively.
Cinnamoyl isothiocyanate was reacted with compound 11 to produce non-
isolable thiourea derivative 21. The newly synthesized compounds have
been characterized by infrared (IR), proton nuclear magnetic resonance (1H
NMR), and carbon nuclear magnetic resonance (13C NMR) spectral data.
The compounds were then evaluated for antibacterial and anticancer
activities.
2Central Laboratory for Revolution
Research the Abbasid Fish, Zagazig, Egypt
Correspondence
Eman O. Hamed, Department of
Chemistry, Faculty of Science, Zagazig
University, Zagazig 44519, Egypt.
Email: dremanomar54@gmail.com
1 | INTRODUCTION
heterocyclic compounds. N-Benzyl cyanoacetamide
(Figure 1) contains multilateral reactive centers as
Heterocycles constitute the largest part of organic chem-
istry. They have immense applications in biological and
industrial fields. For example, numerous natural (eg, qui-
nine, atropine, codeine, morphine, and theophylline) and
synthetic (eg, diazepam, chlorpromazine, antipyrine, and
isoniazid) drugs are heterocycles. Moreover, heterocycles
are the main constitute of dyes (eg, mauveine), herbicides
(eg, paraquat), luminophores (eg, acridine orange), and
pesticides (eg, diazinon). Synthesis of a wide range of het-
erocycle including compounds has been a research inter-
est for long time, and numerous approaches are
described in the literature. Introduction of new, atom
economic, and efficient routes for this chemistry branch
is currently a popular research interest. Among the new
synthetic routes are the uses of cyano acetamide as a
reactive precursor for synthesis of multifunctional
β-functional nitrile (C N), amide moiety (NH C O),
and α-CH acid, which are favorable reactive centers to
react with numerous nucleophiles and electrophiles pro-
viding heterocyclic systems with different ring sizes[1–8]
that exhibit pharmaceuticals,[9,10] antibacterial,[11]
anticoagulant,[12]
antifungacidal,[13]
antihistaminic
agent,[14] antileishhumanial agent,[15] antimicrobial
agent,[16] herbicides,[17] and dyes[18,19] properties.
Recently, our research interest directed to design and
synthesis of biologically active heterocyclic systems from
readily available reagents.[20,21] Herein, we hope to use a
facile and direct approach to synthesize a novel series of
heterocyclic systems (namely, pyridine, pyrrole, furan,
thiazine, benzopyrane, and pyrazole) utilizing N-benzyl
cyanoacetamide as simple synthetic synthon via addition
reactions involving polarized π systems.
J Heterocyclic Chem. 2020;1–10.
wileyonlinelibrary.com/journal/jhet
© 2020 Wiley Periodicals, Inc.
1

2
HAMED ET AL.
the sp3 aliphatic protons system in addition to
OH. Parent peak for heterocyclic 6 was observed at
140.13 ppm, in addition to carbon signals that was
detected at 165 ppm for sp2 of C O and sp of CN at
140 ppm. Benzylidene ethyl cyanoacetate underwent
heterocyclization, affording N-benzylpyridone 7 followed
H
N
NC
O
FIGURE 1 Favorable reactive centers of N-benzyl
1
by enolization providing 8 (Scheme 1). Thus, H NMR
cyanoacetamide
leads to 4 H pyridine at 6.62 and also to no C O absorp-
tion, where CN peak was located at 2210 cm−1. Spectral
analysis was in agreement with the proved structure.
Cyclization of 1 to diazepne derivative 10 was achieved
via base-mediated addition of carbanion of 1 to acyl
heteroallene electrophilic carbon followed by addition of
nucleophilic benzylic carbanion to oxo-electrophilic car-
bon and subsequent dehydration (Scheme 1). The struc-
ture was achieved by the presence of C N, C O, and SH
functions in IR spectrum, in addition to the absence of
benzyl CH2 protons and in addition to carbon signals that
were detected at 165 ppm for sp2 C O and sp of C N at
2 | RESULTS AND DISCUSSION
Compounds of types 4, 6, 8, and 10 were prepared as a
result of Michael-type addition of activated methylene of
N-phenyl cyanoacetamide to different polarized π sys-
tems. Reaction of 1 with maleic anhydride afforded 4 via
initial non-isolable Michael adduct 2 that undergo intra-
molecular cyclodehydration, cyanohydrolysis, and then
evolution of CO2 (Scheme 1).
The structure assigned for product 4 is confirmed by
analytical and spectral data. The infrared (IR) spectrum
of 4 provided C O broad absorption peaks at 1643 cm−1
and also displayed downfield signal at 8.71 for OH group,
aromatic multiplet in region δ = 7.26 to 7.35 ppm, and
doublet of CH2 in addition to methylene proton that was
observed at 2.23 ppm. Carbon nuclear magnetic reso-
nance (13C NMR) indicated the presence of sp2 C O and
SP C N at δ = 162.64 and 139.02 ppm, respectively. Con-
jugated addition of 1 to benzal acetophenone: the reac-
182 ppm in IR spectra at 1645 and 2257 cm−1
respectively.
,
The target pyran derivative 11 was obtained from the
condensation reaction of N-benzylcyanoacetamide 1 with
salicylaldehyde followed by intramolecular addition of
nucleophilic OH to CN function (Scheme 2). Nitrosation
of 11 provided condensed pyran 13, the reaction started
by formation of non-isolable N-nitroso derivative
followed by losing of H2O providing 13; non-oxazine
derivative 15 was observed (Scheme 2).
Compound 11 displayed deshielded signal at 10.69
and 8.90 for NH protons. Also, C O frequency appeared
at 1673 cm−1 in addition to carbon signal of C O that
was observed at 163.73 ppm. Pyrazolo pyran 13 contained
signal for NH2 at 9.11 ppm, and carbonyl frequency was
tion
started
with
formation
of
non-isolable
acyclic Michael adduct 5 followed by intramolecular
nucleophilic addition of imino gp to C O, affording
N-benzylpyridine derivatives 6 (Scheme 1); absorption
frequencies of C N and C O were observed at 2410 and
1621 cm−1, respectively. Proton nuclear magnetic reso-
nance (1H NMR) spectrum of the compound 6 showed
SCHEME 1

HAMED ET AL.
3
Ph
O
SCHEME 2
N
N
O
Ph
N=O
N
NH2
O
O
NH
O
O
O
12
Ph
13
NaNO3
H
NC
Ph
N
H
Ph
N
H
Ph
+
OH
O
NH
NH
N
HON
1
NH
11
O
O
NH
O
O
NH
14
15
O
O
N
N
Ph
N
H
Ph
H
-H2
O
O
N
N
O
N
O
O
S
S
O
AC2O/PhCH
Ph
=CHCONCS
Ph
Ph
N
H
Ph
N
H
22
23
O
O
NH
NH
N
reflux 4h
Ph
S
O
O
21
11
N
Ph
O
Ph
O
N
N
24
SCHEME 3
located at 1655 cm−1; carbonyl carbon signal was
detected at 161.74 ppm.
3 | EXPERIMENTAL
3.1 | Chemistry
The target 17 showed signals at 9.13 and 9.11 for NH2
protons; carbonyl absorption was located at 1699 cm−1
,
while 13C leads to carbonyl carbon at 161. ppm. Using
carbon disulfide as cyclizing agent provided thiazine
derivative 19 presumably via the non-isolable thiazine 18
that suffers air oxidation (Scheme 3). IR spectrum of
compound 19 provided NH, C O and C S absorption
peaks at 3300, 1695 cm−1, and 1284 cm−1. Also, com-
pound 19 shows δ = 9.13 ppm for NH proton, and double
doublet at 4.54 and 4.50 ppm for benzylic CH2. 13C NMR
showed sp2 of C O at 161 ppm. Compound 11 was added
to ammonium thiocyanate to provide thiourea derivative
20 (Scheme 3). Compound 20 revealed downfield signal
for NH and NH2 in addition to C S and C O sp2 carbon
signals at 161.75 and 154.36 ppm, respectively.
Cinnamoyl isothiocyanate was reacted with exocyclic
nucleophilic nitrogen pyrimidine derivative of 11 to produce
non-isolable thiourea derivative 21 that cyclized to produce
thiazine derivative 23, and non-24 was isolated (Scheme 4).
IR of the target 23 led to the presence of NH and CO in
addition to C N. 1H NMR showed signals at δ = 10.43 ppm
for NH and δ = 7.36 to 7.20 ppm for ArH'S, and double
doublet at δ = 4.53 to 4.38 ppm for benzylic protons. 13C
NMR indicate C O sp2 carbon at 163 and 153 ppm.
Melting points were measured using an Electro thermal
IA 9100 apparatus with open capillary tubes and are
uncorrected. All experiments were carried out using dry-
ing solvents. Products were purified by crystallization.
The IR spectra (KBr disk) were recorded on a Pye
Unicom Sp-3-300 or a Shimadzu FTIR 8101 PC infrared
spectrophotometer. The 1H/(13C) NMR spectra were
recorded at Varian Mercury VX-300 NMR 300 (75.4)
MHz spectrometer using dimethyl sulfoxide (DMSO)-d6
as a solvent. All chemical shifts were expressed on the δ
(ppm) scale using tetramethylsilane (TMS) as an internal
standard reference. The coupling constant (Ј) values are
given in Hz. Mass spectrometer and analytical data were
obtained from the Microanalysis Center at Cairo Univer-
sity, Giza, Egypt.
3.1.1 | 6-Benzyl-2-hydroxy-4, 6-dihydro-
5H-furo[2,3-b] pyrrole-5-one (4)
A solution of maleic anhydride (0.01 mol), compound
1 (0.01 mol), and tetraethylammonium (TEA) (three

4
HAMED ET AL.
Ph
Ph
H
N
Ph
Ph
O
N
O
1,5 H shift
ACOH /PhCOH
NH2
O
O
NH
reflux 4h
17
16
S
Ph
O
S
N
S
O
Ph
N
S
EtOH/NaOH/
2CS2
O
NH
N
H
Ph
NH
O
reflux 6h
O
O
NH
18
19
11
O
ACOH/
Ph
NH2
N
H
NH4SCN
N
reflux 4h
O
S
20
SCHEME 4
drops) in dimethylformamide (DMF) 20 mL was
refluxed for 6 hours and then poured onto diluted gla-
cial acetic acid. The formed solid was filtered off,
washed with water, dried, and crystallized from glacial
acetic acid to give brown powder of 4 in 79% yield. mp
128-132ꢀC; IR (KBr) νmax 3299 (OH) broad, 1643
Calcd. for C25H22N2O2: C, 78.50; H, 5.80; N, 7.33; O,
8.37. Found: C, 78.00; H, 5.70; N, 7.20.
3.1.3 | 1-Benzyl-2,6-dihydroxy-4-phenyl-
1,4-dihydropyridine-3,5 dicarbonitrile (8)
(C O), and 1542 (C C) cm−1 1H NMR (300 MHz,
;
DMSO-d6) δ (ppm) 8.0-7.3 (S, 1H, OH), 7.26-7.35 (m,
6H, ArH's), 3.33 (S, 1H, CH), 2.5 (S, 2H, CH2Ph), 2.2 (d,
4H, 2CH2); 13C NMR (300 MHz, DMSO-d6) δ (ppm)
162.64, 139.02, 128.84, 127.85, 116.67, 43.14, 39.98,
25.77; Anal. Calcd. for C13H11NO3: C, 68.12; H, 4.84; N,
6.11; O, 20.92. Found: C, 68.00; H, 4.85; N, 6.00.
A
solution of (Z)-2-benzylidene-3-oxopentanenitrile
(0.01 mol), compound (0.01 mol), and ethoxide
1
(0.01 mol) in absolute ethanol (20 mL) was refluxed for
6 hours, then poured into diluted glacial acetic acid
hydrochloric acid, stirring till the formation of precipitate
was filtered off, and then crystallized from ethanol to give
pale yellow crystal of 8 in 97% yield; mp 158-162ꢀC, IR
(KBr) vmax 3429 (OH), 2210 (CN), and1621 (C O) cm−1
,
3.1.2 | 1-Benzyl-6-hydroxy-2-oxo-
4,6-diphenylpiperidine-3-carbonitrile (6)
1H NMR (300 MHz, DMSO-d6) δ (ppm) 11.87 (S, 1H,
OH), 7.46-6.63 (m, 10H, ArH's), 4.7 (d, 1H, CH,
J = 0.6 HZ), 4.60 (d, 1H, CH, J = 29.6 HZ), 4.10 (dd, 2H,
CH2, J = 5.6 HZ); 13C NMR (300 MHz, DMSO-d6) δ
(ppm) 182.36, 168.03, 166.15, 163.18, 149.10, 144.45,
134.3, 131.13, 129.47, 128.64, 120.58, 117.35; Anal. Calcd.
for C20H15N3O2: C, 72.93; H, 4.59; N, 12.76; O, 9.72.
Found: C, 73.00; H, 4.50; N, 12.00.
A mixture of benzal acetophenone (0.01 mol), com-
pound 1 (0.01 mol), and three drops (TEA) was
refluxed in butanol (20 mL) for 6 hours and then con-
centrated and acidified by hydrochloric acid (HCl). The
formed precipitate was filtered off, washed in water,
dried, and crystallized from butanol to give off white
crystals of 6 in 80% yield. mp 134-138ꢀC, IR (KBr) vmax
3320 (OH), 2241(CN), and 1621(CO) cm−1
;
1H NMR
3.1.4 | 5-oxo-3-phenyl-2-[(E)-
(300 MHz, DMSO-d6) δ (ppm) 8.94 (S, 1H, OH),
8.91-6.85 (m, 15H, ArH's), 4.3-3.84 (m, 6H, sp3H); 13C
NMR (300 MHz, DMSO-d6) δ (ppm) 197.41, 164.27,
140.13, 138.84, 136.73, 133.88, 129.23, 128.86, 128.62,
128.40, 128.30, 127.98, 127.82, 127.55, 127.41; Anal.
2-phenylethenyl]-7-sulfonyl-4,5-dihydro-1H-
1,4-diazepine-6-carbonitrile (10)
A mixture of cinnamoyl isothiocyanate (0.01 mol) and
compound 1 (0.01 mole) was refluxed for 4 hours in dry

HAMED ET AL.
5
acetone in presence of three drops (TEA) and then
poured into diluted glacial acetic acid, and the formed
precipitate was crystallized from glacial acetic acid to
give yellow crystal of 10 in 95% yield. mp 208-212ꢀC, IR
(KBr) vmax 3303 (NH), 2257 (C N), 1645 (C O), 1548
(C C), and 1333 cm−1(SH), 1H NMR (300 MHz,
DMSO-d6) δ (ppm) 11.19 (S, 1H, SH), 9.81 (S, 1H, NH),
9.47(S, 1H, NH), 7.94-6.82 (m, 12H, ArH's, +2H styrly),
13C NMR (300 MHz, DMSO-d6) δ (ppm) 182.36, 166.15,
149.1, 144.39, 134.05, 131.13, 129.36, 128, 120.58,
117.35, 40.40, 39.57); Anal. Calcd. for C20H15N3OS: C,
69.54; H, 4.38; N, 12.17; O, 4.63; S, 9.28. Found: C,
69.50; H, 4.27; N, 12.17; O, 4.63; S, 9.20.
116.61, 43.22, 40.41, 39.58. Anal. Calcd. for
C17H13N3O2: C, 70.09; H, 3.80; N, 14.43; O, 11.00.
Found: C, 70.00; H, 4.50, N, 14.50.
3.1.7 | 4-Amino-2-benzyl-
1-phenylchromeno[3,4-c] pyrrol-3(2H)-
one (17)
A mixture of compound 11 (0.01 mol) and benzalde-
hyde (0.01 mol) was refluxed in glacial acetic acid
(20 mL) for 6 hours and then poured stepwise into ice
water, and the precipitate obtained was filtered off,
washed in water, dried, and crystallized from glacial
acetic acid to give pale yellow powder of 17 in 75%
yield, mp 158-162ꢀC; IR (KBr) vmax 3308 (NH2), 1699
3.1.5 | N-Benzyl-2-imino-2H-chromene-
3-carboxamide (11)
(C O), and 1654 (C C) cm−1 1H NMR (300 MHz,
;
DMSO-d6) δ (ppm) 9.13-9.11 (S, 1H, NH2), 7.99-7.25 (m,
14H, ArH's), 4.55-4.54 (d, 2H, J = 6.0 HZ); 13C NMR
(300 MHz, DMSO-d6) δ (ppm) 161.73, 154.36, 154.36,
148, 139.37, 134.56, 130.72, 128.86, 127.87, 125.60,
119.57, 116.61, 43.22, 40.21, 39.58; Anal. Calcd. for
C24H18N2O2: C, 78.66; H, 4.95; N, 7.65; O, 8.73.
Found: C, 78.70; H, 5.00, N, 7.6
A solution of compound 1 (0.01 mol), salicylaldehyde
(0.01 mol), and sodium ethoxide (0.01 mole) in abso-
lute ethanol (20 mL) was refluxed for 8 hours, and the
reddish solution was poured into diluted HCl. The
formed reddish-brown precipitate was filtered off,
washed with water, dried, and crystallized from abso-
lute ethanol to give reddish-brown powder of 11 in 96%
yield. mp 148-152ꢀC, IR (KBr) vmax 3307 (NH), 1673
1
(C O), 1641 (C N), and 1608 (C C) cm−1; H NMR
3.1.8 | 3-Benzyl-5-imino-2-thioxo-2,
3-dihydro-4H,5H-chromeno[3,4-e][1,3]
thiazin-4-one (19)
(300 MHz, DMSO-d6) δ (ppm) 10.699 (S, 1H, C NH),
8.992 (S, 1H, CONH), 8.50-6.90 (m, 10H, ArH's),
4.54-4.53 (dd, 1H, CH2, J = 5.2 HZ), 4.39-4.38 (dd,
1H,CH2, J = 5.2 HZ); 13C NMR (300 MHz, DMSO-d6) δ
(ppm) 140.18, 135, 133.44, 128.86, 128.35, 127.81,
127.14, 124.54, 120.70, 119.83, 116.61, 115.36, 112.64,
43.44, 40.19, 39.36; Anal. Calcd. for C17H14N2O2: C,
73.36; H, 5.07; N, 10.068; O, 11.5. Found: C, 73.00; H,
5.00; N, 10.1.
A solution of compound 11 (0.01 mol) and carbon disul-
fide (0.02 mol) was refluxed in presence TEA (0.01 mol)
in ethanol (20 mL) for 6 hours and then poured into
diluted glacial acetic acid; the formed precipitate was fil-
tered off, washed in water, dried, and crystallized from
ethanol to give pale brown powder of 19 in 96% yield; mp
298-302ꢀC; IR (KBr) vmax 3300 (NH), 1695(C O), and
1607 (C N) cm−1 1H NMR (300 MHz, DMSO-d6) δ
;
3.1.6 | 4-Amino-2-benzylchromeno[4,3-c]
pyrazol-3(2H)-one (13)
(ppm) 9.13 (S, 1H, NH), 7.99-7.24 (m, 9H, ArH's),
4.55-4.54 (d, 2H, CH2, J = 6.0 HZ); 13C NMR (300 MHz,
DMSO-d6) δ (ppm) 161.74, 160.81, 154.36, 148, 139.37,
134.56, 130.72, 128.86, 127.86, 125.60, 119.58, 116.61,
43.22, 40.21, 39.58; Anal. Calcd. for C18H12N2O2S2: C,
61.34; H, 3.43; N, 7.95; O, 9.08; S, 18.20. Found: C,
61.30; H, 3.50; N, 8.00; S, 18.00.
A solution of compound 11 (0.01 mol) and sodium nitrate
(0.01 mole) was stirred in the presence of glacial acetic
acid (20 mL) for 6 hours and then poured stepwise into
ice water; the formed precipitate was filtered off, washed
in water, dried, and crystallized from glacial acetic acid
to give pale yellow powder of 13 in 85% yield. mp
163-167ꢀC, IR (KBr) νmax 3250 (NH2), 1655 (C O), and
3.1.9 | N-Benzyl-
2-(carbamothioylimino)-2H-chromene-
3-carboxamide (20)
1610 (C C) cm−1
;
1H NMR (300 MHz, DMSO-d6) δ
(ppm) 9.12 (S, 2H, NH2), 7.99-7.26 (m, 9H, ArH's),
4.55-4.54 (d, 2H, CH2, J = 5.6 HZ), 13C NMR (300 MHz,
DMSO-d6) δ (ppm) 161.74, 160.81, 154.36, 148, 139.38,
134.56, 13.72, 128.86, 127.86, 125.60, 119.58, 118.95,
A solution of compound 11 (0.01 mol) and ammonium
thiocyanate (0.01 mol) in glacial acetic acid (20 mL) was

6
HAMED ET AL.
refluxed for 8 hours, then poured stepwise into ice water,
and the formed was stirred. It was filtered off, washed in
water, dried solid, and was crystallized from glacial acetic
acid to give dark brown of 20 in 78% yield; mp
168-172ꢀC; IR (KBr) vmax 3410 (NH), 3264 (NH2), 1678
concentration (MIC) was realized by the preparation of a
series of dilutions of 1/2 of the synthetic product to test on
liquid medium (microdilution). The minimum bactericidal
concentration (MBC) was regarded as being the lowest
concentration, in product tested, having shown an absence
of growth. According to our study, compounds 17 and 19
have an inhibitory activity on the S. aureus, E. coli,
S. faecalis, B. subtilis, N. gonorrhoeae, P. aeruginosa,
A. flavus, and C. albicans strains.
(C O), 1607 (C C), and 1354 (C S) cm−1 1H NMR
;
(300 MHz, DMSO-d6) δ (ppm) 9.99 (S, 1H, NH), 8.88-8.86
(S, 2H, NH2, J = 5.2 HZ), 7.35-7.20 (m, 10H, ArH's),
5.11-5.06 (d, 2H, CH2Ph, J = 20HZ); 13C NMR (300 MHz,
DMSO-d6) δ (ppm) 161.75, 154.36, 147.99, 139.37, 138.30,
130.72, 128.86, 128.04, 127.28, 12.60, 119.59, 117.56,
116.61, 43.22, 39.97, 23.79; Anal. Calcd. for
C18H15N3O2S: C, 64.07; H, 4.48; N, 12.46; O, 9.48; S, 9.51.
Found: C, 64.00; H, 4.40; N, 12.40; S, 9.40.
3.3 | Biological activity (sensitivity tests)
by Kirby-Bauer method
Antimicrobial activity of the tested samples was deter-
mined using a modified Kirby-Bauer disk diffusion
method.[22] Briefly, 100 μL of the test bacteria/fungi were
grown in 10 mL of fresh media until they reached a count
of approximately107 cells per milliliter for bacteria or
105 cells per milliliter for fungi.[23] A 100 μL of microbial
suspension was spread onto agar plates corresponding to
the broth in which they were maintained. Isolated colo-
nies of each organism that might be playing a pathogenic
role should be selected from primary agar plates and
tested for susceptibility by disk diffusion method.[24,25] Of
the many media available, National Committee for Clini-
cal Laboratory Standards (NCCLS) recommends Mueller-
Hinton agar because it results in good batch-to-batch
reproducibility. Disc diffusion method for filamentous
fungi was tested by using approved standard method
(M38-A) developed by the study of David, Vishnu,
Espinel, and Daniel[26] for evaluating the susceptibilities
of filamentous fungi to antifungal agents. Disc diffusion
method for yeasts developed by using approved standard
method (M44-P) by the study of Julia, George, Max, and
Wendy.[27] Plates were inoculated with filamentous fungi
such as A. flavus at 25ꢀC for 48 hours. Gram-positive
bacteria such as S. aureus and B. subtilis and Gram-
negative bacteria such as E. coli and P. aeuroginosa
were incubated at 35 to 37ꢀC for 24 to 48 hours, and
yeast such as C. albicans was incubated at 30ꢀC for
24 to 48 hours, and then the diameters of the inhibition
zones were measured in millimeters.[22] Standard disks of
ampicillin (antibacterial agent) and amphotericin B (anti-
fungal agent) served as positive controls for antimicrobial
activity, but filter disks impregnated with 10 μL of solvent
(distilled water, chloroform, and DMSO) were used as a
negative control. The agar used is Meuller-Hinton agar
that is rigorously tested for composition and pH. Further,
the depth of the agar in the plate is a factor to be consid-
ered in the disk diffusion method. This method is well
documented, and standard zones of inhibition have been
determined for susceptible and resistant values. Blank
3.1.10 | N-Benzyl-2-imino-N-(4-oxo-
6-phenyl-5,6-dihydro-4H-1,3-thiazin-2-yl)-
2H-chromene-3-carboxamide (23)
The solution of compound 11 (0.01 mol) and
cinnamoyl isothiocyanate (0.01 mol) in dioxane
(20 mL) was refluxed for 8 hours and then poured into
ice water; the obtainable solid was filtered off, washed
with water, dried, and crystallized to form beige pow-
der of 23 in 95% yield; mp 178-182ꢀC; IR (KBr) vmax
3367 (NH), 1726 (C O) and 1337 (C S) cm−1; 1H NMR
(300 MHz, DMSO-d6) δ (ppm) 10.43 (S, 1H, NH),
7.36-7.20 (m, 16H, ArH's), 5.75 (S, 1H, CH), 4.84 (S,
1H, NH), 4.53-4.38 (d, 2H, CH2, J = 5.2 HZ); 13C NMR
(300 MHz, DMSO-d6) δ (ppm) 163.25, 153.06, 151.30,
138.68, 138.37, 129.01, 128.62, 127.71, 127.25, 73.77,
45.36, 40.20, 39.37; Anal. Calcd. for C27H19N3O3S: C,
70.42; H, 4.01; N, 8.801; O, 10.051; S, 6.72. Found: C,
70.40; H, 4.00; N, 8.90; S, 6.72.
3.2 | Biological activity
3.2.1 | Antibacterial activity
The synthesized compound was tested for its in vitro
antibacterial activity against the Gram-positive and the
Gram-negative bacteria, Staphylococcus aureus, Streptococ-
cus faecalis, Bacillus subtilis, Escherichia coli, Neisseria
gonorrhoeae, Pseudomonas aeruginosa, Aspergillus flavus
(fungus), and Candida albicans (fungus), using the liquid
serial dilutions method for determination of minimal
inhibitory concentration (MIC). The latter is defined
according to the Antibiogram Committee of a modified
Kirby-Bauer disk diffusion method.[22] As being the lowest
concentration that results in the inhibition of visible bacte-
rial growth, the determination of the minimum inhibitory

HAMED ET AL.
7
paper disks (Schleicher & Schuell, Spain) with a diameter
of 8.0 mm were impregnated with 10 μ of tested concen-
tration of the stock solutions. When a filter paper disk
impregnated with a tested chemical is placed on agar, the
chemical will diffuse from the disk into the agar. This dif-
fusion will place the chemical in the agar only around the
disk. The solubility of the chemical and its molecular size
will determine the size of the area of chemical infiltration
around the disk. If an organism is placed on the agar, it
will not grow in the area around the disk if it is susceptible
to the chemical. This area of no growth around the disk is
known as a “zone of inhibition” or “clear zone.” For the
disk diffusion, the zone diameters were measured with
slipping calipers of the National Committee for Clinical
Laboratory Standards.[27] Agar-based methods such as
Etest and disk diffusion can be good alternatives because
they are simpler and faster than broth-based
methods.[28,29]
Crystal violet stain (1%). It composed of 0.5% (w/v) crystal
violet and 50% methanol then made up to volume with
ddH2O and filtered through a Whatman No.1 filter paper.
Cell line propagation. The cells were propagated in
Dulbecco's modified Eagle's medium (DMEM) sup-
plemented with 10% heat-inactivated fetal bovine serum,
1% L-glutamine, HEPES buffer, and 50-μg/ml gen-
tamycin. All cells were maintained at 37ꢀC in a humidi-
fied atmosphere with 5% CO2 and were subcultured two
times a week.
Cytotoxicity evaluation using viability assay. For cytotoxic-
ity assay, the cells were seeded in 96-well plate at a cell
concentration of 1 × 104 cells per well in 100 μL of growth
medium. Fresh medium containing different concentra-
tions of the test sample was added after 24 hours of
seeding. Serial two-fold dilutions of the tested chemical
Sample
Inhibition Zone Diameter (mm/mg Sample)
Bacterial Species
G+
Fungi
G-
Bacillus
subtilis
Staphylococcus
aureus
Streptococcus
faecalis
Escherichia
coli
Neisseria
gonorrhoeae
Pseudomonas
aeruginosa
Aspergillus flavus
(Fungus)
Candida
albicans
(Fungus)
Standard
Ampicillin
(Antibacterial
26
21
27
25
28
26
—
—
Agent)
Amphotericin B
—
—
—
—
—
—
17
21
(Antifungal Agent)
Control: DMSO
0.0
0.0
0.0
13
0.0
0.0
0.0
14
9
0.0
0.0
0.0
14
0.0
0.0
0.0
13
9
0.0
0.0
0.0
12
0.0
0.0
0.0
13
9
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
15
11
13
17
19
13
0.0
0.0
0.0
3.3.1 | Anticancer activity
compound were added to confluent cell monolayers dis-
pensed into 96-well, flat-bottomed microtiter plates[30]
using a multichannel pipette. The microtiter plates were
incubated at 37ꢀC in a humidified incubator with 5% CO2
for a period of 48 hours. Three wells were used for each
concentration of the test sample. Control cells were incu-
bated without test sample and with or without DMSO.
The little percentage of DMSO present in the wells (maxi-
mal 0.1%) was found not to affect the experiment. After
incubation of the cells for at 37ꢀC, various concentrations
of sample were added, and the incubation was continued
for 24 hours, and viable cells yield was determined by a
colorimetric method.
Evaluation of cytotoxic effects of certain chemical
compound
Mammalian cell lines. HepG-2 cells (human hepatocellu-
lar cancer cell line) and HCT-116 cells (human colon can-
cer cell line) were obtained from VACSERA Tissue
Culture Unit.
Chemicals used. DMSO, crystal violet, and trypan blue
dye were purchased from Sigma.[30] Fetal Bovine
serum, DMEM, RPMI-1640, HEPES buffer solution, L-
glutamine, gentamycin, and 0.25% Trypsin-ethylene
diamine tetraacetic acid (EDTA) were purchased from
Lonza.
In brief, after the end of the incubation period, media
were aspirated, and the crystal violet solution (1%) was

8
HAMED ET AL.
added to each well for at least 30 minutes. The stain was
removed, and the plates were rinsed using tap water until
all excess stain is removed. Glacial acetic acid (30%) was
then added to all wells and mixed thoroughly, and then
the absorbance of the plates were measured after it was
gently shaken on Microplate reader (TECAN, Inc.), 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 experi-
ments were carried out in triplicate. The cell cytotoxic
effect of each tested compound was calculated. The optical
density was measured with the microplate reader[31] to
determine the number of viable cells, and the percentage
of viability was calculated as [(ODt/ODc)]×100%, where
ODt is the mean optical density of wells treated with the
tested sample, and ODc is the mean optical density of
untreated cells. The relation between surviving cells and
drug concentration is plotted to get the survival curve of
each tumor cell line after treatment with the specified
compound. The 50% inhibitory concentration (IC50), the
concentration required to cause toxic effects in 50% of
intact cells, was estimated from graphic plots of the dose
response curve for each concentration using Graphpad
Prism software (San Diego, CA. USA).
HepG-2 cells (human hepatocellular cancer cell line)
after a continuous exposure for 48 hours. The results
were introduced in Table 1 and Figure 2.
HepG-2
71.4
100
50
0
60.6
27.2
17
18.8
19
0.36
doxorubicin
11
13
Compound
FIGURE 2 The inhibitory activities against HepG-2 cell lin
TABLE 3 Effect of the obtained compounds on the growth of
HCT-116 cell line
GI50, μmol L−1
Compound
HCT-116
0.49
Doxorubicin
11
13
17
19
55.5
49.5
29.1
Antitumor activity tests against HepG-2 cells
28.4
The effect of the newly synthesized compounds 11,
13, 17, and 19 was evaluated on the in vitro growth of
TABLE 1 Effect of the obtained compounds on the growth of
HepG-2 cell line
GI50, μmol L−1
Compound
HepG-2
0.36
Doxorubicin
11
13
17
19
71.4
60.6
27.2
18.8
FIGURE 3 The cytotoxic activities against HepG-2 cell line
TABLE 2 Evaluation of cytotoxicity of the obtained compounds against HepG-2 cell line
Viability Rate (%)
0
3.9
7.8
15.6
31.25
62.5
125
250
500
IC50
Compound
(μg/mL)
(μg/mL)
(μg/mL)
(μg/mL)
(μg/mL)
(μg/mL)
(μg/mL)
(μg/mL)
(μg/mL)
(mg/mL)
Doxorubicin
100
100
100
100
100
25.59
99.64
99.72
87.26
75.23
20.81
94.27
94.86
76.49
66.91
18.13
85.41
86.73
63.91
52.67
13.05
69.23
72.94
45.51
39.72
6.13
4.22
2.70
1.72
8.92
11.79
5.92
3.97
0.36
71.4
60.6
27.2
18.8
11
13
17
19
51.88
48.51
32.73
30.46
38.75
31.27
20.56
17.89
23.67
23.82
11.78
7.43

HAMED ET AL.
9
TABLE 4 Evaluation of cytotoxicity of the obtained compounds against HCT-116 cell line
Viability Rate (%)
0
3.9
7.8
15.6
31.25
62.5
125
250
500
IC50
Compound
(μg/mL)
(μg/mL)
(μg/mL)
(μg/mL)
(μg/mL)
(μg/mL)
(μg/mL)
(μg/mL)
(μg/mL)
(mg/mL)
Doxorubicin
100
100
100
100
100
28.86
98.06
98.06
85.19
92.37
24.82
92.42
91.42
74.93
81.46
19.38
78.15
79.23
62.37
68.92
11.04
62.59
60.18
48.06
45.87
6.51
46.37
42.75
34.59
36.45
4.86
34.62
29.83
23.65
20.38
3.36
19.21
17.56
12.14
8.67
2.08
7.34
6.94
5.38
4.59
0.49
55.5
49.5
29.1
28.4
11
13
17
19
Results are given in concentrations that were able
to cause 50% of cell growth inhibition (GI50) after a
continuous exposure of 48 hours and to show means
SEM of three-independent experiments performed in
duplicate. All the examined compounds were able to
inhibit the growth of the tested human tumor cell line
in a dose-dependent manner. The results indicated
through Table 1 and Figure 2 revealed that compound
19 showed high inhibitory effects against HepG-2 cell
line, which is less than the corresponding reference
doxorubicin. Compound 11 showed the lowest inhibi-
tory effect, while the compounds 13 and 17 exhibited
a moderate growth inhibitory effect. The cytotoxic
and antitumor activities of prepared compounds 11,
13, 17, and 19 were tested against HepG-2 cells line.
The inhibitory activities were detected by using differ-
ent concentrations of the tested compounds (0, 3.9,
7.8, 15.6, 31.25, 62.5, 125, 250, and 500 μg), and the
viability cells (%) were determined by colorimetric
method. Also, the (IC50) was calculated as shown in
Table 2 and Figure 3.
HCT-116
55.5
49.5
60
29.1
28.4
40
20
0
0.49
doxorubicin
11
13
Compound
17
19
FIGURE 4 The inhibitory activities against HCT-116 cell line
Results presented in Table 2 and Figure 3 revealed
that compounds 19 and 17 have strong cytotoxic anti-
tumor activity, and compound 13 and 11 have moder-
ate cytotoxic antitumor activity against HepG-2
cell line.
FIGURE 5 The cytotoxic activities against HCT-116 cell line
Antitumor activity tests against HCT-116 cells
The effect of the newly synthesized compounds 11, 13,
17, and 19 was evaluated on the in vitro growth of HCT-
116 cells (colon carcinoma cancer cell line) after a contin-
uous exposure for 48 hours. The results were introduced
in Table 3 and Figure 4.
Results are given in concentrations that were able to
cause 50% of cell growth inhibition (GI50) after a contin-
uous exposure of 48 hours and to show means SEM of
three-independent experiments performed in duplicate.
All the examined compounds were able to inhibit the
growth of the tested human tumor cell line in a dose-
dependent manner. The results indicated through
Table 3 and Figure 4 revealed that compound 19 showed
high inhibitory effects against HCT-116 cell line, which is
less than the corresponding reference doxorubicin. Com-
pound 11 showed the lowest inhibitory effect, while the
compounds 13 and 17 exhibited a moderate growth
inhibitory effect. The cytotoxic and antitumor activities of
prepared compounds 11, 13, 17, and 19 were tested
against HepG-2 cells line. The inhibitory activities were
detected by using different concentrations of the tested
compounds (0, 3.9, 7.8, 15.6, 31.25, 62.5, 125, 250, and
500 μg), and the viability cells (%) were determined by
colorimetric method. Also, the (IC50) was calculated as
shown in Table 4 and Figure 5.

10
HAMED ET AL.
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How to cite this article: Hamed EO, Assy MG,
Shalaby AM, Sayed RE. Cyclization of N-benzyl
cyanoacetamide: Novel synthesis and biological
activity of pyrrole, pyrimidine, and pyran
derivatives. J Heterocyclic Chem. 2020;1–10. https://
doi.org/10.1002/jhet.3892