Synthesis and anticancer evaluation of some novel N,O,S heterocyclic compounds pendant to 3-methyl-5-cyano-6-(3,4-dimethoxyphenyl)pyrimidine and other related fused pyrimidines

Article abstract of DOI:10.1002/jhet.3748

The key starting compound 5-cyano-6-(3,4-dimethoxyphenyl)-2-thiouracil (1) was synthesized and allowed to undergo electrophilic substitution with methyl iodide to give the corresponding 6-(3,4-dimethoxyphenyl)-4-oxo-2-thioxo-1,2,3,4-tetrahydro-pyrimidine-

Full text of DOI:10.1002/jhet.3748

Received: 20 November 2018
DOI: 10.1002/jhet.3748
Revised: 19 August 2019
Accepted: 5 September 2019
A R T I C L E
Synthesis and anticancer evaluation of some novel N,O,S
heterocyclic compounds pendant to 3-methyl-5-cyano-
6-(3,4-dimethoxyphenyl)pyrimidine and other related
fused pyrimidines
Rasha S. Gouhar1
| Mohsen M. Kamel1 | Ahmed A.F. Soliman2
1Therapeutical Chemistry Department,
National Research Centre, Giza, Egypt
Abstract
2Drug Bioassay-Cell Culture Laboratory,
Department of Pharmacognosy, National
Research Centre, Giza, Egypt
The key starting compound 5-cyano-6-(3,4-dimethoxyphenyl)-2-thiouracil (1) was
synthesized and allowed to undergo electrophilic substitution with methyl iodide
to give the corresponding 6-(3,4-dimethoxyphenyl)-4-oxo-2-thioxo-1,2,3,4-tetra-
hydro-pyrimidine-5-carbonitrile (2). Nucleophilic substitution on compound
2 with hydrazine hydrate led to the corresponding 2-hyrazinopyrimidone inter-
mediate 3. Compound 3 underwent several substitution and cyclization reac-
tions with β-ketoester, β-ketone, alkyl halides, arylisothiocyanate, or aromatic
aldehydes followed by cyclization reactions to give the corresponding N,O,S
heterocyclic compounds incorporated into pyrimidine moiety and/or the
related S-triazino[3,4-b]pyrimidine derivatives 4-18. Anticancer evaluation of
some representative examples of newly synthesized compounds was carried
out against MCF-7, HCT116 cell lines. Some of the newly synthesized com-
pounds showed significant activity.
Correspondence
Rasha S. Gouhar, Therapeutical
Chemistry Department, National
Research Centre, Giza 12613, Egypt.
Email: dr.rasha_gouhar@yahoo.com
Funding information
National Research Centre, Grant/Award
Number: 11010317
1 | INTRODUCTION
Pyrimidines are among those molecules that make life pos-
sible as being building blocks of nucleic acids DNA and
RNA. DNA damaged by exogenous physical and chemical
agents is the most common cause of cancer. Conversely,
some of the commonly used anticancer drugs kill
malignant cell by damaging their DNA. Several
substituted pyrimidine derivatives are reported to possess
anticancer activity. Broprimine I,[1] 2-amino-5-bromo-
6-phenylpyrimidin-4(3H)-one, is a well-known antineoplas-
tic agent. Also, several 2-(((1-(4-chlorobenzyl)-1H-
1,2,3-triazol-4-yl)methyl)thio)-4-((4-chlorophenyl)amino)-
6-phenylpyrimidine-5-carbonitrile (II) are recently reported[2]
as potential anticancer agents against MGC-803, EC-109,
MCF-7, and B16-F10 cell lines.
Several other pyrimidine derivatives have been reported
to possess diverse chemotherapeutic activities such as ant-
iviral, antimalarial, antiprotozoal, antifungal, and anti-
mycotic agents.[3–6] The therapeutic importance of this class
of compounds prompted as to synthesize a new series of
3-methyl-4-oxo-5-cyano-6-(3,4-dimethoxyphenyl) pyrimi-
dines and other related S-triazino[3,4-b]pyrimidines as a
continuation of previous work[7–16] in our drug research
program for anticancer evaluation against cancer cell
lines.
J Heterocyclic Chem. 2019;1–12.
wileyonlinelibrary.com/journal/jhet
© 2019 Wiley Periodicals, Inc.
1

2
GOUHAR ET AL.
aminophenol afforded the corresponding benzo(1,4)
oxazine derivatives 8a,b, respectively.
2 | RESULTS AND DISCUSSION
2.1 | Chemistry
On the other hand, reaction of
3
with
substituted arylisothiocyanate, namely, phenyl and/or ben-
zyl isothiocyanate, gave the corresponding 2-substituted
thiosemicarbazide derivatives 9a,b, respectively (Scheme 2).
Further, reactions of compounds 9a,b with chloroacetic acid
afforded the corresponding thiazolidinone derivatives 10a,b
respectively, while upon reaction with malonic acid, it gave
the thiobarbituric acid derivatives 11a,b respectively. The
structure of compound 11 was strongly confirmed by its IR
spectrum, which showed the vibrational coupling of the car-
bonyl groups (cf. Section 3). Also, reaction of 9 with phen-
acyl bromide gave the corresponding phenyl thiazolidone
derivatives 12a,b, respectively (Scheme 3).
Furthermore, reactions of compounds 3 with appropri-
ate aldehydes, namely, indole-3-carboxaldehyde and/or
naphthalene-2-carboxaldehyde, gave the corresponding
Schiff bases 13a,b, which upon cyclization with
thiosalicylic acid, it gave the corresponding benzo-
thiazinone derivatives 14a,b, respectively. On the other
hand, cyclization of the Schiff bases 13a,b using lead ace-
tate afforded the corresponding S-triazolo[4,3-a]pyrimi-
dine-6-carbonitrile 15a,b respectively (Scheme 4).
For the purpose of this study, the synthetic approach was
confined to three general schemes (1–3) to obtain the tar-
get polyfunctionally substituted pyrimidinones and the
related fused pyrimidines. The first starting compound,
6-(3,4-dimethoxyphenyl)-4-oxo-2-thioxo-1,2,3,4-tetrahydr-
opyrimidine-5-carbonitrile (1) in almost good yield was
previously prepared by our research group,[10] via the
reaction of thiourea with 3,4-dimethoxybenazldehyde
and ethylcyanoacetate in alkaline medium. Compound
1 was allowed to undergo electrophilic substitution
with methyl iodide in sodium carbonate to give the
corresponding 4-(3,4-dimethoxyphenyl)-1-methyl-2-(met-
hylthio)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile (2).
Nucleophilic substitution on compound 2 with hydrazine
hydrate led to the corresponding strategic intermediate
4-(3,4-dimethoxyphenyl)-2-hydrazinyl-1-methyl-6-oxo-1,6-
dihydro- pyrimidine-5-carbonitrile (3) (Scheme 1). Com-
pound 3 was investigated by spectral and elemental
analyses. Thus, the IR spectrum of the hydrazino deriva-
tive 3 showed the appearance of NH2 and NH bands at
3417, 3324, 3198 cm−1. Further support for the structure
Finally, upon cyclization of compound 3 with either
trimethyl orthoformate or formic acid, it gave the
corresponding S-triazolo[3,4-b]pyrimidine derivative 16
in almost good yield, while upon cyclization using
ethyl choroformate, it gave the corresponding 5-(3,4-
dimethoxyphenyl)-8-methyl-3,7-dioxo-2,3,7,8-tetrahydro-
1
of compound 3 was gained from its H-NMR spectrum,
which showed a broad singlet signals corresponding to
NH2 and NH groups at 9.90 and 10.76 ppm, respectively,
(cf. Section 3).
Compound 3 underwent several cyclization reactions
to give the corresponding N,O, and/or S heterocyclic
compounds pendant to pyrimidine moiety. Thus, reaction
of 3 with acetoacetic ester afforded the corresponding
[1,2,4]triazolo[4,3-a]pyrimidine-6-carbonitrile
Also, upon cyclization of compound 3 with acetic
anhydride, it gave the 5-(3,4-dimethoxyphenyl)-
(17).
2-(3-methylpyrazolone-1-yl-pyrimidine
derivative
4,
3,8-dimethyl-7-oxo-7,8-dihydro-[1,2,4]triazolo[4,3-a]pyrimi-
dine-6-carbonitrile (18), while upon cyclization with
while cyclization on of 3 using acetyl acetone afforded
the corresponding 2-(3,5-dimethylpyrazole-1-yl)pyrimi-
dine derivative 5. Reaction of 3 with acid anhydrides,
newly maleic anhydride, or phthalic anhydride in glacial
acetic acid for 6 hours, afforded the corresponding
pyrrolidindione and/or the phthalimido derivatives 6a,b,
respectively. Also, reaction of 3 with chloroacetylchloride
and/or chloroacetone gave the corresponding alkyl
hydrazine derivatives 7a,b respectively. Compounds 7a,b
upon cyclization with o-aminophenol and/or 4-chloro-o-
diethyl
oxalate,
it
gave
the
corresponding
triazinopyrimidinetrione derivative 19 (Scheme 5). All
newly prepared compounds were investigated by elemental
and spectroscopic analyses.
2.2 | Evaluation of cytotoxic activity
The new pyrimidinone and the related fused pyrimidine
derivatives were evaluated as cytotoxic agents against
CHO
O
O
O
CN
S
Me
CN
OEt
NH2NH2
Me
CN
HN
MeI/Na2CO3
OMe
N
NC
N
+
OMe
+
NH2
H2N
Me
O
H2N
S
N
H
S
N
N
N
OMe
H
OMe
OMe
OMe
OMe
1
OMe
2
3
SCHEME 1 Synthesis of compounds 1 to 3

GOUHAR ET AL.
3
SCHEME 2 Conversion of compound 3 into compounds 4-9
SCHEME 3 Formation of compounds 10-12

4
GOUHAR ET AL.
SCHEME 4 Formation of compounds 13-15
O
N
O
Me
HN
CN
Me
CN
N
N
Lead acetate
N
ArCHO
N
N
3
H
N
Ar
OMe
Ar
OMe
13a,b
OMe
OMe
15a,b
O
OH
SH
O
Me
O
CN
N
N
N
N
S
H
Ar
OMe
14a,b
OMe
a, Ar=
b, Ar =
N
H
O
N
Me
N
CN
CH(OMe)3
N
or
HCOOH
N
OMe
16
OMe
O
O
Me
CN
N
Cl
OEt
N
N
HN
OMe
O
OMe
O
N
17
Me
CN
N
O
H2N
N
Me
CN
H
N
OMe
3
Ac2O
N
N
OMe
N
OMe
Me
OMe
18
O
Me
CN
O
O
OEt
N
O
N
N
OEt
HN
O
OMe
SCHE ME 5 Conversion of compound 3 into
compounds 16-19
OMe
19
two types of cancer cell lines; human breast (MCF-7) and
human colon cancer cell (HCT116) using MTT assay. The
resultant data were expressed as IC50 (μg/mL) in Tables 1
and 2. To determine the safety of the new compounds,
the cytotoxic activity of the active derivatives was exam-
ined against the normal skin fibroblast BJ1. It has been
noticed that the parent hydrazino-pyrimidinone deriva-
tive 3 produced moderate cytotoxic activity against
MCF-7 cancer cells of IC50; 66.6 μg/mL. Equivalent
potency was obtained upon conjugation of the parent
pyrimidinone nucleus with thiazolidinone ring as com-
pound 10a of IC50; 66.2 μg/mL. On the other hand, the
inhibitory potency against MCF-7 cancer cells was
increased by the fused S-triazolo[4,3-a]pyrimidine deriva-
tive (compound 15) producing IC50; 50 μg/mL. The com-
bination of the basic pyrimidinone scaffold with
benzothiazinone ring (compound 14a) was favorable for
the activity. It boosted the anticancer potency approxi-
mately by 1.7 folds (IC50; 36 μg/mL) comparing with the
starting pyrimidinone 3. Furthermore, the parent

GOUHAR ET AL.
5
TABLE 1 Sample concentration range between 100 and
0.78 μg/mL using MTT assay
TABLE 2 The sample was tested against the human tumor
cell line(s)
LC50,
LC90,
LC50,
LC90,
Sample Code
μg/mL
μg/mL
Remarks
Sample Code
μg/mL
μg/mL
Remarks
12
13a
14aa
15a
--------
54.2
--------
81.3
60.1% at 100 ppm
96.5% at 100 ppm
93.2% at 100 ppm
100% at 100 ppm
39.5% at 100 ppm
97.2% at 100 ppm
34.2% at 100 ppm
61.3% at 100 ppm
84.2% at 100 ppm
49.5% at 100 ppm
63.1% at 100 ppm
29.1% at 100 ppm
67.2% at 100 ppm
100% at 100 ppm
31.2% at 100 ppm
1% at 100 ppm
0%
13
14aa
15a
--------
36.6
--------
64.8
34.1% at 100 ppm
94.2% at 100 ppm
98.2% at 100 ppm
40.1% at 100 ppm
51.2% at 100 ppm
11.4% at 100 ppm
29.5% at 100 ppm
20.1% at 100 ppm
24.3% at 100 ppm
54.2% at 100 ppm
17.1% at 100 ppm
72.3% at 100 ppm
54.2% at 100 ppm
51.3% at 100 ppm
72.3% at 100 ppm
3% at 100 ppm
56.4
89.2
50.6
79
25.3
35.9
16
--------
--------
--------
--------
--------
--------
--------
--------
66.2
--------
--------
--------
--------
--------
--------
--------
--------
109.8
--------
--------
111
16
17a
--------
49.0
--------
78.8
17
18
18
--------
--------
49.5
--------
--------
89.8
19
19
3a
6
8
6
--------
--------
--------
--------
28.7
--------
--------
--------
--------
48.1
7
7
9
10aa
8
9
10aa
11
--------
--------
66.6
12
3a
11
--------
--------
--------
--------
--------
--------
DMSO
DMSO
--------
--------
--------
--------
Negative control
Negative control
0%
Note. LC50: lethal concentration of the sample, which causes the death of
50% of cells in 48 hours. LC90: lethal concentration of the sample, which
causes the death of 90% of cells in 48 hours.
aCompounds that show positive response.
Note. LC50: lethal concentration of the sample, which causes the death of
50% of cells in 48 hours. LC90: lethal concentration of the sample, which
causes the death of 90% of cells in 48 hours.
aCompounds that show positive response.
hydrazino-pyrimidinone derivative 3 produced moderate
growth inhibitory activity against colon cancer cell of
IC50; 49.5 μg/mL, while its thiazolidinone 10a and
S-triazolo[4,3-a]pyrimidine analogues 15 remarkably
increased the activity by 1.7 and 2 folds, of IC50; 28.7 and
25.3 μg/mL, respectively. Alternatively, an observable
reduction in growth inhibition activity was detected by
the pyrimidine-Schiff base 13 and pyrimidine-benzo-
thiazinone conjugate 14 producing IC50; 54.2 and 56.4 μg/
mL, respectively comparing with the parent compound 3.
Fortunately, no active derivatives produced any cytotoxic
activity against the normal fibroblast cells confirming its
complete safety profile against the human normal cells. It
could be concluded that the tested derivatives are more
effective against human colon cancer cell line than the
human breast cancer cell lines. The fused S-
triazolo[4,3-a]pyrimidine derivative 15 produced dual
cytotoxic activity against both types of human breast and
colon cancer cell lines. Despite the benzothiazinone ring
is favorable for the anticancer potency against MCF-7
cells, the thiazolidinone one is more active against
HCT116 cancer cells. Further structural modification is
required to improve the anticancer activity of the previ-
ous newly synthesized pyrimidine compounds.
2.2.1 | Cytotoxic activity test
The sample was tested against the human tumor cell
line(s):
1. HCT116 (colon cell line)
2. MCF-7 (human Caucasian breast adenocarcinoma)
3 | EXPERIMENTAL
3.1 | Chemistry
All melting points are uncorrected and were taken on
open capillary tubes using electrothermal apparatus
9100. Elemental microanalyses were carried out at micro-
analytical unit, Central Services Laboratory, National
Research Centre, Dokki, Giza, Egypt, using Vario
Elementar and were found within + or −0.5% of the the-
oretical values. Infrared spectra were recorded on a Jasco
FT/IR-6100, Fourier transform infrared spectrometer at
per centimeter scale using KBr disc technique at the Cen-
tral Services Lab. NRC, Dokki, Giza, Egypt. 1HNMR
and 13CNMR spectra were determined by using a JEOL

6
GOUHAR ET AL.
EX-NMR Spectrometer—300 MHz and 75 MHz—at Cen-
tral Services Lab, (Faculty of Science, Cairo University,
Cairo, Egypt). Mass spectra were measured with Fin-
nigan M A T SSQ-7000 mass spectrometer at the Central
Services, NRC Dokki, Giza, Egypt. Nomenclature given
for the new compounds are according to the IUPAC
System.
3.1.3 | Synthesis of
4-(3,4-dimethoxyphenyl)-1-methyl-
2-(3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-
1-yl)-6-oxo-1,6-dihydropyrimidine-
5-carbonitrile (4)
A mixture of compound 3 (1.35 g, 0.05 mol) and ethyl
acetoacetate (0.65 mL, 0.005 mol) in ethanol (40 mL)
was refluxed for 6 hours. The solvent was concentrated
under reduced pressure and the formed precipitate,
which obtained on cooling was filtered off and dried to
give product 4 that separated as yellow crystals (chloro-
3.1.1 | Synthesis of
4-(3,4-dimethoxyphenyl)-1-methyl-
2-(methylthio)-6-oxo-
form), yield 68%. mp 170ꢀC to 172ꢀC. IR (KBr), ṽ/cm−1
:
1,6-dihydropyrimidine-5-carbonitrile (2)
2219 (CN), 1720 (C═O), 1650 (C═N).1H NMR
(DMSO):δ (ppm) 2.3 (s,3H,CH3), 2.80 (s, 3H, N-CH3),
3.30 (s, 3H, OCH3), 3.50 (s, 3H, OCH3), 4.40 (s, 2H,
CH2), 6.51 to 6.90 (m, 3H, Harom) ppm. MS (EI, 70 eV):
m/z (%) = 368 (30) (M+) Anal. for C18H17N5O4 (367.36):
calcd. C, 58.85; H, 4.66; N, 19.06; found: C, 58.96; H,
4.41; N, 19.52.
To a solution of compound 1 (2.9 g, 0.01 mol) in DMF
(30 mL), potassium carbonate (4 g) and 0.03 mol of
methyl iodide were added and stirred for 3 hours at
room temperature. The reaction mixture was diluted
with cold water and the precipitate was filtered off. The
product 2 was separated as brownish yellow crystals
(ethanol), yield 85%. mp 146ꢀC to 137ꢀC. IR (KBr),
ṽ/cm−1: 2222 (CN), 1719 (C═O).1H NMR (CDCl3): δ
(ppm) 2.90 (s, 3H, S-CH3), 3.10 (s, 3H, N-CH3), 3.20 (s,
3H, OCH3), 3.60 (s, 3H, OCH3), 6.60 to 7.10 (m, 3H,
Harom) ppm. MS (EI, 70 eV): m/z (%) = 317 (20) (M+)
Anal. for C15H15N3O3S (317.36): calcd. C, 56.77; H,
4.76; N, 13.24; S, 10.10; found: C, 57.00; H, 5.09; N,
12.74; S, 10.16.
3.1.4 | Synthesis of
4-(3,4-dimethoxyphenyl)-2-
(3,5-dimethyl-1H-pyrazol-1-yl)-1-
methyl-6-oxo-1,6-dihydropyrimidine-
5-carbonitrile (5)
The foregoing procedures for preparation of compound
4 were applied except that acetyl acetone was used
instead of ethyl acetoacetate to give product 5 as yel-
lowish brown crystals (chloroform), yield 55%. mp
196ꢀC to 197ꢀC. IR (KBr), ṽ/cm−1: 2228 (CN), 1648
3.1.2 | Synthesis of
4-(3,4-dimethoxyphenyl)-2-hydrazinyl-
1-methyl-6-oxo-1,6-dihydropyrimidine-
5-carbonitrile (3)
1
(C═N), 1667 (C═O). H NMR (DMSO): δ (ppm) 2.54 (s,
3H, CH3), 2.63 (s, 3H, CH3), 2.80 (s, 3H, N-CH3), 3.30
(s, 3H, OCH3), 3.70 (s, 3H, OCH3), 6.30 to 6.80 (m, 4H,
Harom) ppm. MS (EI, 70 eV): m/z (%) = 365 (15) (M+)
Anal. for C19H19N5O3 (365.39): calcd. C, 62.46; H,
5.24; N, 19.17; found: C, 62.68; H, 5.41; N, 19.36.
A suspension of compound 2 (3.2 g, 0.01 mol) in ethanol
(25 mL) was added with 15 mL of hydrazine hydrate, and
the reaction mixture was refluxed for 5 hours then cooled
with cold water, and the precipitate was filtered off and
washed several times with cold water to give product 3,
which was recrystallized from ethanol as brown crystals,
yield 90%. mp 152ꢀC to 154ꢀC. IR (KBr), ṽ/cm−1: 3417,
3324, 3198 (NH2, NH), 2279 (CN), 1699 (C═O).1H NMR
(CDCl3): δ (ppm) 2.70 (s, 3H, N-CH3), 3.10 (s, 3H, OCH3),
3.70 (s, 3H, OCH3), 6.50 to 7.20 (m, 3H, Harom), 9.90 (s,
2H, NH2, exchangeable), 10.76 (s, 1H, NH, exchangeable)
ppm. 13C-NMR (75 MHz, DMSO-d6) δ/ppm: 28.7 (CH3),
57.4, 58.2 (2 O-CH3), 98.2, 112.3, 112.8, 119.5, 126.9,
131.8, 146.4, 149.2, 154.1, 162.5 (Ar-C), 171.3 (C═O). MS
(EI, 70 eV): m/z (%) = 301 (80) (M+) Anal. for
C14H15N5O3 (301.30): calcd. C, 55.81; H, 5.02; N, 23.24;
found: C, 56.02; H, 4.88; N, 22.83.
3.1.5 | General procedures for synthesis
of 6a,b
A mixture of compound 3 (0.27 g, 0.001 mol) and the
appropriate acid anhydride, namely, maleic anhydride
and phthalic anhydride (0.001 mol) in glacial acetic
acid (10 mL), was heated under refluxed for 6 hours;
the reaction mixture was cooled and poured onto
ice/cold water; the formed precipitate was filtered off,
dried, and recrystallized from the proper solvent to give
compounds 6a,b.

GOUHAR ET AL.
7
4-(3,4-Dimethoxyphenyl)-2-((2,5-dioxo-
2,5-dihydro-1H-pyrrol-1-yl)amino)-1-methyl-6-oxo-
1,6-dihydropyrimidine-5-carbonitrile (6a)
N0-(5-Cyano-4-(3,4-dimethoxyphenyl)-1-methyl-
6-oxo-1,6-dihydropyrimidin-2-yl)-
3-oxobutanehydrazide (7b)
Product 6a was separated as yellow crystals (chloroform),
yield 53%. mp 176ꢀC to 178ꢀC. IR (KBr), ṽ/cm−1: 3340
(NH), 2234 (CN), 1735, 1709, 1698 (C═O), 1647 (C═N).
1H NMR (DMSO): δ (ppm) 2.80 (s, 3H, N-CH3), 3.30 (s,
3H, OCH3), 3.70 (s, 3H, OCH3), 6.20 (s, 1H, NH,
exchangeable), 6.50 to 7.37 (m, 5H, Harom) ppm. MS (EI,
70 eV): m/z (%) = 381 (15) (M+) Anal. for C18H15N5O5
(381.34): calcd. C, 56.69; H, 3.96; N, 18.37; found: C,
56.33; H, 4.40; N, 18.74.
Product 7b was separated as brown crystals (ethyl ace-
tate), yield 79%. mp 182ꢀC to 184ꢀC. IR (KBr), ṽ/cm−1
:
3343 (NH), 2227 (CN), 1720, 1680 (C═O), 1647 (C═N).
1H NMR (DMSO): δ (ppm) 2.67 (s, 3H, CH3CO), 2.80 (s,
3H, N-CH3), 3.30 (s, 3H, OCH3), 3.50 (s, 3H, OCH3), 4.50
(s, 2H, CH2), 5.80 (s, 1H, NH, exchangeable), 6.30 (s, 1H,
NH, exchangeable), 6.20 to 6.90 (m, 3H, Harom) ppm. MS
(EI, 70 eV): m/z (%) = 357 (35) (M+) Anal. for
C17H19N5O4 (357.14): calcd. C, 57.14; H, 5.36; N, 19.60;
found: C, 57.26; H, 5.01; N, 20.10.
4-(3,4-Dimethoxyphenyl)-2-((1,3-dioxoisoindolin-
2-yl)amino)-1-methyl-6-oxo-
3.1.7 | General procedures for synthesis
1,6-dihydropyrimidine-5-carbonitrile (6b)
Product 6b was separated as yellow crystals (ethanol),
yield 63%. mp 103–105ꢀC. IR (KBr), ṽ/cm−1: 3344 (NH),
2213 (CN), 1742, 1719, 1708 (C═O), 1647 (C═N). 1H
NMR (DMSO): δ (ppm) 2.65 (s, 3H, N-CH3), 3.30 (s, 3H,
OCH3), 3.80 (s, 3H, OCH3), 6.50 (s, 1H, NH, exchange-
able), 6.60 to 7.50 (m, 7H, Harom) ppm. MS (EI, 70 eV):
m/z (%) = 431 (25) (M+) Anal. for C22H17N5O5 (431.12):
calcd. C, 61.25; H, 3.97; N, 16.23; found: C, 61.09; H,
4.16; N, 15.81.
of 8a,b
A mixture of compound 7a (0.30, 0.001 mol) and the
appropriate phenol, namely, 2-amino phenol and/or
2-amino-4-chlorophenol (0.001 mol) in absolute ethanol
(10 mL), with few drops of dil.HCl. The reaction mixture
was refluxed for 10 hours; the reaction mixture was
cooled and poured onto ice/cold water; the formed pre-
cipitate was filtered off, dried, and recrystallized from
ethanol to give compounds 8a,b.
2-(2-(2H-Benzo[b][1,4]oxazin-3-yl)hydrazinyl)-
4-(3,4-dimethoxyphenyl)-1-methyl-6-oxo-
1,6-dihydropyrimidine-5-carbonitrile (8a)
3.1.6 | General procedures for synthesis
of 7a,b
Product 8a was separated as yellow crystals (ethanol),
yield 53%. mp 153ꢀC to 154ꢀC. IR (KBr), ṽ/cm−1: 3390
(NH), 2218 (CN), 1695 (C═O), 1644 (C═N). 1H NMR
(DMSO): δ (ppm): 2.80 (s, 3H, N-CH3), 3.20 (s, 3H,
OCH3), 3.70 (s, 3H, OCH3), 4.10 (s, 2H, CH2-O), 6.50 (s,
1H, NH, exchangeable), 6.92 (s, 1H, NH, exchangeable),
7.20 to 8.11 (m, 7H, Harom) ppm. MS (EI, 70 eV): m/z
(%) = 432 (13) (M+) Anal. for C22H20N6O4 (432.44): cal-
cd. C, 61.10; H, 4.66; N, 19.43; found: C, 61.31; H,
4.83; N, 19.61.
A mixture of compound 3 (0.27 g, 0.001 mol) and the
appropriate alkyl halides, namely, 2-chloroacetyl chlo-
ride and/or 2-chloroacetone (0.001 mol) in absolute
ethanol (30 mL), was refluxed for 6 hours; the reaction
mixture was cooled and poured onto ice/cold water;
the formed precipitate was filtered off, dried, and rec-
rystallized from the proper solvent to give compounds
7a,b, respectively.
2-Chloro-N0-(5-cyano-4-(3,4-dimethoxyphenyl)-
1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)
acetohydrazide (7a)
2-(2-(7-Chloro-2H-benzo[b][1,4]oxazin-3-yl)
hydrazinyl)-4-(3,4-dimethoxyphenyl)-1-methyl-
6-oxo-1,6-dihydropyrimidine-5-carbonitrile (8b)
Product 8b was separated as yellow crystals (ethanol),
yield 50%. mp 169ꢀC to 170ꢀC. IR (KBr), ṽ/cm−1: 3383
(NH), 2218 (CN), 1678 (C═O), 1638 (C═N), 1173 (C-Cl).
1H NMR (DMSO): δ (ppm) 2.70 (s, 3H, N-CH3), 3.20 (s,
3H, OCH3), 3.60 (s, 3H, OCH3), 4.60 (s, 2H, CH2), 5.60 (s,
1H, NH, exchangeable), 6.72 (s, 1H, NH, exchangeable),
6.20 to 7.50 (m, 6H, Harom) ppm. 13C-NMR (75 MHz,
DMSO-d6) δ/ppm: 28.2 (CH3), 57.6, 57.9 (2 O-CH3),
71.8(O-CH2), 95.7, 111.9, 112.3, 115.8, 116.5, 124.9, 125.1,
Product 7a was separated as brown crystals (ethanol),
yield 56%. mp 270ꢀC to 271ꢀC. IR (KBr), ṽ/cm−1: 3365
(NH), 2221 (CN), 1672, 1656 (C═O), 1648 (C═N), 773 (C-
1
Cl). H NMR (DMSO):δ (ppm) 2.70 (s, 3H, N-CH3), 3.50
(s, 3H, OCH3), 3.80 (s, 3H, OCH3), 4.50 (s, 2H, CH2), 6.10
(s, 1H, NH), 6.60 to 7.50 (m, 3H, Harom), 10.30 (s, 1H,
NH, exchangeable), ppm. MS (EI, 70 eV): m/z
(%) = 379(M + 2, 6), 377 (20) (M+) Anal. for
C16H16ClN5O4 (377.09): calcd. C, 50.87; H, 4.27; N, 18.54;
found: C, 51.09; H, 4.60; N, 18.99.

8
GOUHAR ET AL.
126.3, 128.9, 130.0, 131.8, 132.0, 136.8, 149.2, 149.7, 154.6
155.5 (Ar-C), 170.2 (C═O). MS (EI, 70 eV): m/z
(%) = 468(M + 2, 5), 466 (13) (M+) Anal. for
C22H19ClN6O4 (466.88): calcd. C, 56.60; H, 4.10; N, 18.00;
found: C, 56.84; H, 3.91; N, 18.12.
in absolute ethanol was heated under refluxed for
10 hours; the formed precipitate was filtered off, dried,
and recrystallized from the proper solvent to give com-
pounds 10a,b.
4-(3,4-Dimethoxyphenyl)-1-methyl-6-oxo-
2-(2-(4-oxo-3-phenylthiazolidin-2-ylidene)
hydrazinyl)-1,6-dihydropyrimidine-
3.1.8 | General procedures for synthesis
of 9a,b
5-carbonitrile (10a)
Product 10a was separated as yellowish brown crystals
(ethanol), yield 70%. mp 182ꢀC to 183ꢀC. IR (KBr),
ṽ/cm−1: 3449 (NH), 2318 (CN), 1725, 1677 (C═O), 1647
A mixture of compound 3 (0.27 g, 0.001 mol) and the
appropriate isothiocyanate, namely, phenyl and benzyl
isothiocyanate (0.001 mol) in dry benzene (30 mL), was
heated under refluxed for 6 hours; the formed precipitate
on cooling was filtered off, dried, and recrystallized from
the proper solvent to give compounds 9a,b.
1
(C═N). H NMR (DMSO): δ (ppm) 2.64 (s, 3H, N-CH3),
3.20 (s, 3H, OCH3), 3.70 (s, 3H, OCH3), 3.90 (s, 2H,
CH2CO), 6.50 (s, 1H, NH, exchangeable), 6.50 to 7.80 (m,
8H, Harom) ppm. MS (EI, 70 eV): m/z (%) = 476 (25) (M+)
Anal. for C23H20N6O4S (476.13): calcd. C, 57.97; H,
4.23; N, 17.64; found: C, 58.16; H, 4.07; N, 17.97.
2-(5-Cyano-4-(3,4-dimethoxyphenyl)-1-methyl-
6-oxo-1,6-dihydropyrimidin-2-yl)-N-
phenylhydrazinecarbothioamide (9a)
Product 9a was separated as yellow crystals (ethanol),
yield 58%. mp 146ꢀC to 147ꢀC. IR (KBr), ṽ/cm−1: 3426
(NH), 2230 (CN), 1689 (C═O), 1648 (C═N), 1136 (C═S).
1H NMR (DMSO): δ (ppm) 2.50 (s, 3H, N-CH3), 3.20 (s,
3H, OCH3), 3.70 (s, 3H, OCH3), 6.20 (s, 1H, NH,
exchangeable), 6.90 to 7.50 (m, 8H, Harom), 10.80 (s, 1H,
NH, exchangeable), 11.60 (s, 1H, NH, exchangeable)
ppm. MS (EI, 70 eV): m/z (%) = 437 (15) (M+1) Anal. for
C21H20N6O3S (436.13): calcd. C, 57.79; H, 4.26; N, 19.25;
found: C, 58.15; H, 4.36; N, 19.36.
2-(2-(3-Benzyl-4-oxothiazolidin-2-ylidene)
hydrazinyl)-4-(3,4-dimethoxyphenyl)-1-methyl-
6-oxo-1,6-dihydropyrimidine-5-carbonitrile (10b)
Product 10b was separated as yellowish brown crystals
(ethanol), yield 63%. mp 211ꢀC to 212ꢀC. IR (KBr),
ṽ/cm−1: 3438 (NH), 2303 (CN), 1689 (C═O), 1644 (C═N).
1H NMR (DMSO): δ (ppm) 2.78 (s, 3H, N-CH3), 3.10 (s,
3H, OCH3), 3.50 (s, 3H, OCH3), 3.80 (s, 2H, N-CH2), 4.40
(s, 2H, CH2CO), 6.30 (s, 1H, NH, exchangeable), 6.20 to
7.90 (m, 8H, Harom) ppm. MS (EI, 70 eV): m/z (%) = 490
(10) (M+) Anal. for C24H22N6O4S (490.14): calcd. C,
58.76; H, 4.52; N, 17.13; found: C, 58.88; H, 4.35; N, 17.81.
N-Benzyl-2-(5-cyano-4-(3,4-dimethoxyphenyl)-
1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)
hydrazinecarbothioamide (9b)
3.1.10 | General procedures for synthesis
Product 9b was separated as yellow crystals (ethanol), yield
65%. mp 110ꢀC to 111ꢀC. IR (KBr), ṽ/cm−1: 3419 (NH),
2288 (CN), 1686 (C═O), 1644 (C═N), 1137 (C═S). 1H NMR
(DMSO): δ (ppm) 2.90 (s, 3H, N-CH3), 3.50 (s, 3H, OCH3),
3.80 (s, 3H, OCH3), 4.70 (s, 2H, CH2), 6.30 (s, 1H, NH),
10.50 (s, 1H, NH, exchangeable), 11.80 (s, 1H, NH,
exchangeable), 6.60 to 7.80 (m, 8H, Harom) ppm. 13C-NMR
(75 MHz, DMSO-d6) δ/ppm: 26.7 (CH3), 50.3 (CH2-Ph),
57.2, 57.5 (2 O-CH3), 96.1, 112.0, 112.3, 115.5, 119.8, 126.5,
126.9, 128.1, 130.1, 138.2, 149.5, 149.8, 153.6 155.8 (Ar-C),
170.5 (C═O), 185.0 (C═S). MS (EI, 70 eV): m/z (%) = 450
(18) (M+) Anal. for C22H22N6O3S (450.52): calcd. C,
58.65; H, 4.92; N, 18.65; found: C, 58.92; H, 5.36; N, 18.36.
of 11a,b
A mixture of compounds 9a,b (0.001 mol) and malonic
acid (1.0 g, 0.01 mol) in absolute ethanol (20 mL) was
heated under refluxed for 15 hours; the reaction mixture
was cooled and poured onto ice/cold water contains few
drops of HCl; the formed precipitate was filtered off,
dried, and recrystallized from the proper solvent to give
compounds 11a,b.
4-(3,4-Dimethoxyphenyl)-2-((4,6-dioxo-3-phenyl-
2-thioxotetrahydropyrimidin-1(2H)-yl)amino)-
1-methyl-6-oxo-1,6-dihydropyrimidine-
5-carbonitrile (11a)
Product 11a was separated as yellow crystals (ethanol),
yield 58%. mp 191-192ꢀC. IR (KBr), ṽ/cm−1: 3432 (NH),
2257 (CN), 1725, 1701, 1667 (C═O), 1642 (C═N), 1161
3.1.9 | General procedures for synthesis
of 10a,b
1
A mixture of compounds 9a,b (0.001 mol) and chlo-
(C═S). H NMR (DMSO): δ (ppm) 2.59 (s, 3H, N-CH3),
roacetic acid (0.4 g, 0.01 mol) and sodium acetate (0.1 g)
3.30 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 4.20 (s, 2H, CH2),

GOUHAR ET AL.
9
5.60 (s, 1H, NH, exchangeable), 6.50 to 7.80 (m, 8H,
Harom) ppm. MS (EI, 70 eV): m/z (%) 505 (22) (M+1) Anal.
for C24H20N6O4S (504.12): calcd. C, 57.14; H, 4.00; N,
16.66; found: C, 57.48; H, 4.18; N, 16.42.
OCH3), 3.60 (s, 3H, OCH3), 4.20 (s, 2H, N-CH2), 5.67 (s,
1H, NH, exchangeable), 6.50 to 8.10 (m, 14H, Harom
)
ppm. MS (EI, 70 eV): m/z (%) = 549 (28) (M-1) Anal. for
C30H26N6O3S (550.18): calcd. C, 65.44; H, 4.76; N, 15.26;
found: C, 65.78; H, 5.04; N, 15.08.
2-((3-Benzyl-4,6-dioxo-
2-thioxotetrahydropyrimidin-1(2H)-yl)amino)-
4-(3,4-dimethoxyphenyl)-1-methyl-6-oxo-
1,6-dihydropyrimidine-5-carbonitrile (11b)
Product 11b was separated as yellow crystals (ethanol),
yield 62%. mp 177ꢀC to 178ꢀC. IR (KBr), ṽ/cm−1: 3330
(NH), 2249 (CN), 1730, 1705, 1685 (C═O), 1645 (C═N),
3.1.12 | General procedures for synthesis
of 13a,b
A mixture of compound 3 (2.7 g, 0.01 mol) and indole-
3-carboxaldehyde and/or naphthalene-2-carboxaldehyde
(0.01 mol) in absolute ethanol (30 mL) was heated under
refluxed for 6 hours; the reaction mixture was cooled, and
the formed precipitate was filtered off, dried, and rec-
rystallized from the proper solvent to give Schiff bases 13a,b.
1
1204 (C═S). H NMR (DMSO): δ (ppm) 2.80 (s, 3H, N-
CH3), 3.20 (s, 3H, OCH3), 3.60 (s, 3H, OCH3), 3.90 (s, 2H,
CH2CO), 4.50 (s, 2H, N-CH2), 5.80 (s, 1H, NH, exchange-
able), 6.63 to 7.80 (m, 8H, Harom) ppm. MS (EI, 70 eV):
m/z (%) = 517 (19) (M−1) Anal. for C25H22N6O4S
(518.14): calcd. C, 57.91; H, 4.28; N, 16.21; found: C,
58.02; H, 3.92; N, 16.43.
2-(2-((1H-Indol-3-yl)methylene)hydrazinyl)-
4-(3,4-dimethoxyphenyl)-1-methyl-6-oxo-
1,6-dihydropyrimidine-5-carbonitrile (13a)
Product 13a was separated as brownish yellow crystals
(ethanol), yield 80%. mp 232ꢀC to 233ꢀC. IR (KBr),
ṽ/cm−1: 3423 (NH), 2230 (CN), 1692 (C═O), 1648 (C═N).
1H NMR (DMSO): δ (ppm) 2.80 (s, 3H, N-CH3), 3.30 (s,
3H, OCH3), 3.80 (s, 3H, OCH3), 6.09 (s, 1H, NH,
exchangeable), 6.80 to 7.80 (m, 8H, Harom), 8.50 (s, 1H,
NH, exchangeable), 9.81(s, 1H, N═CH) ppm. 13C-NMR
(75 MHz, DMSO-d6) δ/ppm: 27.9 (CH3), 56.2, 56.7 (2 O-
CH3), 83.4, 94.6, 111.0, 111.5, 111.8, 112.2, 115.8, 119.4,
121.9, 122.5, 123.1, 126.3, 127.4, 138.1, 138.6, 148.5, 149.8,
151.2 159.8 (Ar-C), 170.5 (C═O). MS (EI, 70 eV): m/z
(%) = 428 (35) (M+) Anal. for C23H20N6O4 (428.16): cal-
cd. C, 64.48; H, 4.71; N, 19.62; found: C, 64.09; H,
4.61; N, 19.25.
3.1.11 | General procedures for synthesis
of 12a,b
A mixture of compounds 9a,b (0.001 mol) and phenacyl
bromide (1.9 g, 0.01 mol) in absolute ethanol (20 mL)
was heated under refluxed for 8 hours; the solvent evapo-
rated under reduced pressure; the formed precipitate was
collected, washed with K2CO3 solution, dried, and rec-
rystallized from the proper solvent to give com-
pounds 12a,b.
4-(3,4-Dimethoxyphenyl)-
2-(2-(3,4-diphenylthiazol-2(3H)-ylidene)
hydrazinyl)-1-methyl-6-oxo-
1,6-dihydropyrimidine-5-carbonitrile (12a)
Product 12a was separated as yellow crystals (ethanol),
yield 56%. mp 113ꢀC to 114ꢀC. IR (KBr), ṽ/cm−1: 3327
(NH), 2236 (CN), 1690 (C═O), 1619 (C═N). 1H NMR
(DMSO): δ (ppm) 2.75 (s, 3H, N-CH3), 3.30 (s, 3H,
OCH3), 3.50 (s, 3H, OCH3), 5.80 (s, 1H, NH, exchange-
able), 6.30 to 7.90 (m, 14H, Harom) ppm. MS (EI, 70 eV):
m/z (%) = 536 (13) (M+) Anal. for C29H24N6O3S (536.16):
calcd. C, 64.91; H, 4.51; N, 15.66; found: C, 65.10; H,
4.27; N, 15.39.
4-(3,4-Dimethoxyphenyl)-1-methyl-
2-(2-(naphthalen-2-ylmethylene)hydrazinyl)-
6-oxo-1,6-dihydropyrimidine-5-carbonitrile (13b)
Product 13b was separated as brownish yellow crystals
(ethanol), yield 75%. mp 101ꢀC to 102ꢀC. IR (KBr),
ṽ/cm−1: 3438 (NH), 2283 (CN), 1689 (C═O), 1644 (C═N).
1H NMR (DMSO): δ (ppm) 2.74 (s, 3H, N-CH3), 3.20 (s,
3H, OCH3), 3.70 (s, 3H, OCH3), 6.14 (s, 1H, NH,
exchangeable), 6.50 to 7.90 (m, 10H, Harom), 9.40 (s, 1H,
N═CH) ppm. MS (EI, 70 eV): m/z (%) = 439 (25) (M+)
Anal. for C25H21N5O3 (439.16): calcd. C, 68.33; H,
4.82; N, 15.94; found: C, 68.08; H, 4.49; N, 16.16.
2-(2-(3-Benzyl-4-phenylthiazol-2(3H)-ylidene)
hydrazinyl)-4-(3,4-dimethoxyphenyl)-1-methyl-
6-oxo-1,6-dihydropyrimidine-5-carbonitrile (12b)
Product 12b was separated as yellow crystals (ethanol),
yield 71%. mp 135ꢀC to 136ꢀC. IR (KBr), ṽ/cm−1: 3421
(NH), 2305 (CN), 1702 (C═O), 1648 (C═N). 1H NMR
(DMSO): δ (ppm) 2.70 (s, 3H, N-CH3), 3.30 (s, 3H,
3.1.13 | General procedures for synthesis
of 14a,b
A mixture of Schiff bases 13a,b (0.001 mol) and
thiosalicylic acid (1.54 g, 0.01 mol) in dry benzene

10
GOUHAR ET AL.
(30 mL) was heated under refluxed for 15 hours; the sol-
vent evaporated under reduced pressure; the obtained
residue was treated with petroleum ether. The solid pre-
cipitate was filtered off, washed with petroleum ether,
dried, and recrystallized from the proper solvent to give
compounds 14a,b.
(NH), 2267 (CN), 1688 (C═O), 1646 (C═N). 1H NMR
(CDCl3): δ (ppm) 2.85 (s, 3H, N-CH3), 3.50 (s, 3H, OCH3),
3.80 (s, 3H, OCH3), 4.60 (s, 1H, CH), 6.10 (s, 1H, NH,
exchangeable), 6.30 to 7.90 (m, 8H, Harom), 10.80 (s, 1H,
NH, exchangeable) ppm. MS (EI, 70 eV): m/z (%) = 429
(30) (M + 1) Anal. for C23H20N6O3 (428.16): calcd. C,
64.48; H, 4.71; N, 19.62; found: C, 64.67; H, 4.48; N, 19.94.
2-((2-(1H-Indol-3-yl)-4-oxo-2H-benzo[e][1,3]
thiazin-3(4H)-yl)amino)-4-(3,4-dimethoxyphenyl)-
1-methyl-6-oxo-1,6-dihydropyrimidine-
5-(3,4-Dimethoxyphenyl)-8-methyl-3-(naphthalen-
2-yl)-7-oxo-1,7,8,8a-tetrahydro-[1,2,4]triazolo[4,3-a]
pyrimidine-6-carbonitrile (15b)
5-carbonitrile (14a)
Product 14a was separated as yellow crystals (ethanol),
yield 78%. mp 174ꢀC to 175ꢀC. IR (KBr), ṽ/cm−1: 3429
(NH), 2210 (CN), 1698, 1686 (C═O), 1648 (C═N). 1H
NMR (DMSO): δ (ppm) 2.64 (s, 3H, N-CH3), 3.50 (s, 3H,
OCH3), 3.80 (s, 3H, OCH3), 4.10 (s, 1H, CH), 5.40 (s, 1H,
NH, exchangeable), 6.60 to 8.10 (m, 12H, Harom), 8.30 (s,
1H, NH, exchangeable) ppm. MS (EI, 70 eV): m/z
(%) = 565 (22) (M+1) Anal. for C30H24N6O4S (564.16): cal-
cd. C, 63.82; H, 4.28; N, 14.88; found: C, 64.32; H,
4.11; N, 14.07.
Product 15b was separated as yellow crystals (ethanol),
yield 64%. mp 220-221ꢀC. IR (KBr), ṽ/cm−1: 3424 (NH),
1
2272 (CN), 1683 (C═O), 1637 (C═N). H NMR (DMSO):
δ (ppm) 2.90 (s, 3H, N-CH3), 3.50 (s, 3H, OCH3), 3.80 (s,
3H, OCH3), 4.30 (s, 1H, CH), 6.70 to 8.20 (m, 10H,
Harom), 10.80 (s, 1H, NH, exchangeable) ppm. MS (EI,
70 eV): m/z (%) = 439 (25) (M+) Anal. for C25H21N5O3
(439.16): calcd. C, 68.33; H, 4.82; N, 15.94; found: C,
67.98; H, 4.93; N, 16.43.
3.1.15 | General procedures for synthesis
of compounds 16-19
4-(3,4-Dimethoxyphenyl)-1-methyl-
2-((2-(naphthalen-2-yl)-4-oxo-2H-benzo[e][1,3]
thiazin-3(4H)-yl)amino)-6-oxo-
A solution of compound 3 (1.35 g, 0.05 mol) and formic
acid, ethylchloroacetate, acetic anhydride, and/or diethyl
malonate (25 mL) was heated under refluxed for 4 hours;
the reaction mixture was cooled and poured onto ice/cold
water; the formed precipitate was filtered off, dried, and
recrystallized from the proper solvent to give compounds
16-19.
1,6-dihydropyrimidine-5-carbonitrile (14b)
Product 14b was separated as yellow crystals (methanol),
yield 69%. mp 197ꢀC to 198ꢀC. IR (KBr), ṽ/cm−1: 3430
(NH), 2220 (CN), 1690, 1675 (C═O), 1648 (C═N). 1H
NMR (DMSO): δ (ppm) 2.65 (s, 3H, N-CH3), 3.50 (s, 3H,
OCH3), 3.80 (s, 3H, OCH3), 4.20 (s, 1H, CH), 5.50 (s, 1H,
NH, exchangeable), 6.50 to 7.60 (m, 14H, Harom) ppm.
MS (EI, 70 eV): m/z (%) = 575 (20) (M+) Anal. for
C32H25N5O4S (575.16): calcd. C, 66.77; H, 4.38; N, 12.17;
found: C, 66.06; H, 4.65; N, 12.31.
5-(3,4-Dimethoxyphenyl)-8-methyl-7-oxo-
7,8-dihydro-[1,2,4]triazolo[4,3-a]pyrimidine-
6-carbonitrile (16)
Product 16 was separated as yellow crystals (methanol),
yield 75%. mp 188ꢀC to 189ꢀC. IR (KBr), ṽ/cm−1: 2250
(CN), 1673 (C═O), 1645 (C═N). 1H NMR (DMSO): δ
(ppm) 2.70 (s, 3H, N-CH3), 3.40 (s, 3H, OCH3), 3.80 (s,
3H, OCH3), 6.50 to 7.34 (m, 4H, Harom) ppm. MS (EI,
70 eV): m/z (%) = 311 (30) (M+) Anal. for C15H13N5O3
(311.10): calcd. C, 57.87; H, 4.21; N, 22.50; found: C,
57.71; H, 4.43; N, 22.00.
3.1.14 | General procedures for synthesis
of compounds 15a,b
Compounds 13a and/or 13b (0.001 mol) was dissolved in
15 mL of acetic acid with stirring at 45ꢀC; lead tetraacetate
(1.7 g) was added portion wise at interval of 3 hours. The
excess solvent evaporated; the reaction mixture was
poured onto ice/cold water; the formed precipitate was fil-
tered off, washed with water, dried, and recrystallized
from the proper solvent to give compounds 15a,b.
5-(3,4-Dimethoxyphenyl)-8-methyl-3,7-dioxo-
2,3,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrimidine-
6-carbonitrile (17)
Product 17 was separated as yellow crystals (methanol),
yield 95%. mp 232ꢀC to 233ꢀC. IR (KBr), ṽ/cm−1: 3470
(NH), 2276 (CN), 1687, 1675 (C═O), 1635 (C═N). 1H
NMR (DMSO): δ (ppm) 2.70 (s, 3H, N-CH3), 3.50 (s, 3H,
OCH3), 3.80 (s, 3H, OCH3), 6.70 to 6.90 (m, 3H, Harom),
5-(3,4-Dimethoxyphenyl)-3-(1H-indol-3-yl)-
8-methyl-7-oxo-1,7,8,8a-tetrahydro-[1,2,4]
triazolo[4,3-a]pyrimidine-6-carbonitrile (15a)
Product 15a was separated as yellow crystals (ethanol),
yield 77%. mp 280ꢀC to 281ꢀC. IR (KBr), ṽ/cm−1: 3441

GOUHAR ET AL.
11
10.66 (s, 1H, NH, exchangeable), ppm. MS (EI, 70 eV):
m/z (%) = 327 (98) (M+) Anal. for C15H13N5O4 (327.10):
calcd. C, 55.05; H, 4.00; N, 21.40; found: C, 55.49; H,
4.23; N, 21.16.
OR, USA). Media were aspirated; fresh medium (without
serum) was added, and cells were incubated either alone
(negative control) or with different concentrations of
samples to give a final concentration of (100-50-25-12.5-
6.25-3.125 -0.78 and 1.56 ug/mL). After 48 hours of incu-
bation, medium was aspirated; 40 uL of MTT salts
(2.5 μg/mL) were added to each well and incubated for
further 4 hours at 37ꢀC under 5% CO2. To stop the reac-
tion and dissolving the formed crystals, 200 μL of 10%
sodium dodecyl sulfate (SDS) in deionized water was
added to each well and incubated overnight at 37ꢀC. A
positive control, which composed of 100 μg/mL, was used
as a known cytotoxic natural agent who gives 100%
lethality under the same conditions.[18,19]
The absorbance was then measured using a micro
plate multi-well reader (Bio-Rad Laboratories Inc., model
3350, Hercules, California, USA) at 595 nm and a refer-
ence wavelength of 620 nm. A statistical significance was
tested between samples and negative control (cells with
vehicle) using independent t-test by SPSS 11 program.
DMSO is the vehicle used for dissolution of plant extracts
and its final concentration on the cells was less than
0.2%. The percentage of change in viability was calcu-
lated according to the formula: ([reading of
extract/reading of negative control] − 1) × 100. A probity
analysis was carried for IC50 and IC90 determination
using SPSS 11 program.
5-(3,4-Dimethoxyphenyl)-3,8-dimethyl-7-oxo-
7,8-dihydro-[1,2,4]triazolo[4,3-a]pyrimidine-
6-carbonitrile (18)
Product 18 was separated as brownish yellow crystals
(ethanol), yield 73%. mp 165ꢀC to 166ꢀC. IR (KBr),
1
ṽ/cm−1: 2304 (CN), 1694 (C═O), 1653 (C═N). H NMR
(DMSO): δ (ppm) 2.50 (s, 3H, CH3), 2.90 (s, 3H, N-CH3),
3.40 (s, 3H, OCH3), 3.70 (s, 3H, OCH3), 6.50–6.80 (m, 3H,
Harom) ppm. MS (EI, 70 eV): m/z (%) = 325 (25) (M+)
Anal. for C16H15N5O3 (325.12): calcd. C, 59.07; H,
4.65; N, 21.53; found: C, 58.60; H, 4.77; N, 21.03.
6-(3,4-Dimethoxyphenyl)-9-methyl-3,4,8-trioxo-
3,4,8,9-tetrahydro-2H-pyrimido[2,1-c][1,2,4]
triazine-7-carbonitrile (19)
Product 19 was separated as brownish yellow crystals
(ethanol), yield 66%. mp 219ꢀC to 220ꢀC. IR (KBr),
ṽ/cm−1: 3375 (NH), 2305 (CN), 1704, 1687 (C═O), 1644
1
(C═N). H NMR (DMSO): δ (ppm) 2.85 (s, 3H, N-CH3),
3.60 (s, 3H, OCH3), 3.83 (s, 3H, OCH3), 6.60 to 6.90 (m,
3H, Harom), 10.30 (s, 1H, NH, exchangeable), ppm. 13C-
NMR (75 MHz, DMSO-d6) δ/ppm: 26.9 (CH3), 57.1, 57.4
(2 O-CH3), 81.7, 111.1, 111.6, 115.6, 122.5, 127.5, 145.6,
149.4, 149.9, 159.6 (Ar-C), 160.1, 162.8, 164.2 (3C═O). MS
(EI, 70 eV): m/z (%) = 355 (18) (M+) Anal. for
C16H13N5O5 (355.09): calcd. C, 54.09; H, 3.69; N, 19.71;
found: C, 54.42; H, 3.86; N, 19.55.
ACKNOWLEDGMENT
The authors thank the National Research Centre for the
financial support (project number 11010317).
ORCID
4 | CYTOTOXIC ACTIVITY
Rasha S. Gouhar https://orcid.org/0000-0003-0955-
589X
Cell viability was assessed by the mitochondrial depen-
dent reduction of yellow MTT (3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium
bromide)
to
purple
formazan.[17] All the following procedures were done in a
sterile area using a Laminar flow cabinet biosafety class
II level (Baker, SG403INT, Sanford, ME, USA). Cells
were suspended in RPMI 1640 medium for MCF-7,
HCT116, and BJ1. The media were supplemented with
1% antibiotic-antimycotic mixture (a potassium penicillin
of 10 000 U/mL, a streptomycin sulfate of 10 000 μg/mL,
and an amphotericin B of 25 μg/mL), 1% L-glutamine and
10% fetal bovine serum and kept at 37ꢀC under 5% CO2.
Cells were batch cultured for 10 days then seeded at
concentration of 10 × 103 cells/well in fresh complete
growth medium in 96-well micro titer plastic plates at
37ꢀC for 24 hours under 5% CO2 using a water-jacketed
carbon dioxide incubator (Sheldon, TC2323, Cornelius,
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