Design, synthesis, and antihypertensive activity of new pyrimidine derivatives endowing new pharmacophores

Article abstract of DOI:10.1007/s00044-019-02289-6

A new series of achiral pyrimidine derivatives based on nifedipine-like structure was designed and synthesized. These pyrimidyl derivatives contained hydrazine, hydrazones, acetohydrazide, differently substituted benzylidene functionalities, benzosulfohyd

Full text of DOI:10.1007/s00044-019-02289-6

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-019-02289-6
ORIGINAL RESEARCH
Design, synthesis, and antihypertensive activity of new pyrimidine
derivatives endowing new pharmacophores
Ahmed M. Farghaly1 Omaima M. AboulWafa1 Yaseen A. M. Elshaier2 Waleed A. Badawi3 Haridy H. Haridy4
Heba A. E. Mubarak5
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Received: 22 October 2018 / Accepted: 6 January 2019
© Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
A new series of achiral pyrimidine derivatives based on nifedipine-like structure was designed and synthesized. These
pyrimidyl derivatives contained hydrazine, hydrazones, acetohydrazide, differently substituted benzylidene functionalities,
benzosulfohydrazine, various heterocycles such as pyrazole, pyrazolidinedione, thiazoline, and thiazolidinone rings, and
fused ring systems such as triazolopyrimidine and pyrimidotriazine rings. Compounds 5a, 5b, 11b, 8b, 9b–d, and 15b
showed a decrease in mean arterial rabbit blood pressure (MABP) ranging from 51.4 to 78.2 mmHg in rabbits in comparison
with nifedipine-treated rabbits. Among these derivatives, compounds 5a, 5b, 9b, and 9c were found to exhibit calcium
channel blockade activity on preparations of rabbit aortae. They exhibited relaxation in the range of 89.2% to 74.4% in
comparison to nifedipine (57.6%) as well as a decrease in heart rate. Histopathological effect of compounds 5a,b on the
expression of endothelial nitric oxide synthase (eNOS) was also examined on rat aorta. An intense expression of eNOS
immune staining in aortic endothelium was seen for compound 5b indicating that it lowered blood pressure via activation of
eNOS expression in aorta.
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Keywords Nifedipine Pyrimidines ENOS Antihypertensive Histopathology SAR
Introduction
Organization 2011). Hypertension affects billions of people
all around the world despite the great effort and progress in
developing new drugs targeting hypertension (European
Society of Hypertension 2013). Of the several classes of
antihypertensive agents that have found their way in clinical
use, calcium channel blockers (CCBs) have received
increasing attention in recent years. They are highly
recommended and are widely used as the first line anti-
hypertensive therapy with few contraindications in pre-
vention and treatment of CVDs (Lopez et al. 2006; Ozawa
et al. 2006; De la Sierra and Ruilope 2007). To this day,
1,4-dihydropyridines (DHPs) are still the most potent group
of CCBs with nifedipine (Adalat®) (Vater et al. 1972) (Fig. 1)
being the initial drug treatment alone or in combination with
other classes in arterial hypertension. Nifedipine, the pro-
totype DHP, since its introduction into clinical medicine in
1975, has been modified to numerous second, third, and
fourth generation commercial products for improving
duration of activity, potency, and safety profiles (Ozawa
et al. 2006; Yousef et al. 2005; Catterall et al. 2003; Bossert
and Vater 1989). Further structure modification of the DHP
core focused on the aza-analogs of DHPs resulting in the
introduction of the potent dihydropyrimidine (DHPM)
Cardiovascular diseases (CVDs), including coronary heart
diseases, hypertension, and peripheral artery diseases are
the main cause of worldwide mortality (World Stroke
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-019-02289-6) contains supplementary
material, which is available to authorized users.
* Omaima M. AboulWafa
omaimawafa@yahoo.com
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Alexandria University, Alexandria 21215, Egypt
2
Department of Organic and Medicinal Chemistry, Faculty of
Pharmacy, University of Sadat City, Monofia 32897, Egypt
3
Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Damanhour University, Damanhour, Egypt
4
Department of Pharmacology, Faculty of Medicine, Al-Azhar
University (Assiut Branch), Assiut 71524, Egypt
5
Department of Histology and Cell Biology, Faculty of Medicine,
Assiut University, Assiut, Egypt

Medicinal Chemistry Research
of interest were derivatized at N3 and were chosen to
comprise various functionalities as potent and selective
candidates to investigate the effect of structural variations
on activity modulation (Teleb et al. 2017a, b) (Fig. 1).
Based on their biological results, these moieties revealed
potent (Teleb et al. 2017a, b) and selective activity against
hypertension.
Nifedipine
SQ32547
As an extension of our previous investigations and with
the aim to generate new antihypertensive agents with
desirable potency and drug-like properties, our interest was
focused on novel fully aromatized pyrimidine derivatives.
They were designed via replacing the pyridine core present
in nifedipine by the bioisosteric pyrimidine which possesses
significant and privileged bioactivity and pharmacokinetic
properties. As shown in Fig. 2, pyrimidine moiety is
favorably used as anchor points and served as a critical
moiety responsible for H-bond interactions. According to
bioisosterism and molecular hybridization (Fig. 2), sub-
stituted pyrimidine, taken as lead compound, was modified
at position 2 assuming that derivatization at that position of
the pyrimidine backbone does not affect the left-hand side
of the molecule essential for activity. This may give rise to
fruitful potentially active compounds that would be of great
interest. Furthermore, substitution at C2 of the pyrimidine
ring system would prevent its inactivation by hydroxylation
and conjugation reactions (138–141). Therefore, it was
believed that pyrimidine derivatives bearing hydrazine
group at C2 would provide a valuable matrix for pyrimidine
functionalized calcium channel antagonism scaffold with
potential application in treatment of hypertension.
SQ32926
our previous work
Fig. 1 Nifedipine and lead aza analogs of DHPs
derivatives, which show a very similar pharmacological
profile to classical DHPs antihypertensive modulators
(Kappe 1998; Grover et al. 1995; Rovnyak et al. 1995).
These inherently asymmetric DHPM derivatives are not
only very potent antihypertensive modulators, but have also
been studied extensively to expand the existing structure–
activity relationship (SAR) and to get further insight into
molecular interactions at the receptor level (Kappe 1998;
Grover et al. 1995; Rovnyak et al. 1995; De Fatima et al.
2015). In general, conformational features previously
reported for DHP calcium channel modulators (Bikker and
Weaver 1993; Gaudio et al. 1994; Palmer and Andersen
1996) were also preserved for DHPMs. The most notable
difference between DHPs and DHPMs, however, is the
extreme flattening of the boat-type DHPM ring around N1 as
a result of amide type bonds present in the pyrimidine core
nucleus. Over the past years, several lead DHPMs have
been developed among which SQ 32926 and SQ 32547
(Horovitz 1990; Negwer 1994) (Fig. 1) have been currently
identified to possess antihypertensive properties. They were
effective orally and found to be superior in potency
and duration of antihypertensive activity to classical DHP
drugs (Bikker and Weaver 1993; Kappe et al. 1997; Jain
et al. 2008).
Our attention was focused on numerous pyrimidine and
dihydropyrimidine derivatives bearing electron donating
groups on the C4 phenyl ring that were developed and
tested for biological activity (Alam et al. 2010; Kaur and
Kaur 2013; Enseleit et al. 2010; Jeanneau et al. 1992) and
some of them have revealed calcium channel blockade
activity.
In our lab, we have explored N-containing pyrimidine
scaffold and their activity profiles and we have reported,
recently, a series of DHPM hybrids (Teleb et al. 2017a, b)
(Fig. 1) as CCB for treatment of hypertension. Compounds
Molecular design of the compounds based on pyrimidine
scaffold consisted principally of
4 pharmacophores
unchanged in all synthesized compounds: 1—Pyrimidine
ring essential for coordination at the receptor level.
2—Phenyl ring usually substituted with an electron donat-
ing group as OCH3 connected to non-chiral C4 pyrimidine
by one atom length NH linker. 3—Additional lipophilic
group at C6 position either methyl or phenyl ring and a CN
moiety introduced at C5 instead of the traditional ester
functional group while retaining its electron withdrawing
properties trying to provide a strong H-bonding site. Mod-
ification at C4 by substituting the aromatic p-substituted
aniline group for o-nitrophenyl moiety and hydrophobic
methyl/phenyl groups at C6 could be of further effect to
optimize the inhibitory potency. The phenyl ring could
enable the molecule to make additional π–π interaction with
aromatic or heteroaromatic amino acids residues in receptor
binding site. 4—Additional hydrophilic group at C2, par-
ticularly –NHNH2 group, substituted or unsubstituted, is a
biologically active pharmacophore that has been a part of
clinically used drugs as hydralazine (Ellershaw and Gurney
2001). In order to evaluate the necessity of heterocyclic

Medicinal Chemistry Research
Fig. 2 Rationale for the designed
compounds: bioisosterism and
molecular pharmacophore
hybridization
Tail = pharmacophores including hydrophobic, H-bond acceptor/donor points, heterocycles
attached or fused to pyrimidine nucleus
substitution for activity, we included several fused or
attached moieties as a part of the tail to establish new SAR
regarding the type and size of those moieties. In the target
structures, the active −NHNH2 group was retained along
with various functionalities at NH at the right hand side of
the pyrimidine core and with either H-bond donor or
acceptor properties to examine the influence of different
size, polarity, electronic properties, flexibility, and con-
formational freedom of molecules on the inhibitory activity
of the target molecules. Moreover, for further assessment of
activity, we adopted a conformation restricted approach by
adopting the strategy to link or fuse other heterocyclic
moieties with the pyrimidine template in search of new lead
molecules as potential drug candidates. Heterocyclic por-
tions of these compounds could act as versatile building
block to introduce different new functional groups, to
anchor these rings into optimal space for binding, and may
imply an important role in antihypertensive activity.
Therefore, it was our intention to synthesize ring systems as
pyrazole, pyrazolidinedione, thiazoline, and thiazolidinone
rings in addition to fused ring systems as triazolopyrimidine
and pyrimidotriazine rings to study the effect of increasing
bulkiness, planarity, and polarity of such fused ring systems
on their calcium antagonistic effects and specificity.
The principal features defining SARs in the present study
are represented in Fig. 3.
Materials and methods
Chemistry
Experimental
All melting points were uncorrected and were determined in
open-glass capillaries using an electrothermal melting point
apparatus (Stuart Scientific, Model SMP1, UK). IR spectra
were recorded (KBr) on a Shimadzu Infrared Spectro-
photometer IR 470 at the Faculty of Pharmacy, Assiut
1
University. Nuclear magnetic resonance spectra, H NMR
and 13C NMR, were taken using Bruker Avance III appa-
ratus 400 MHz; Central Laboratory Unit, Faculty of Phar-
macy, Cairo and Bruker DRX 400 MHz, Central Laboratory
Unit, Beni-suef Universities for H and 101 MHz for 13C
1
NMR, Varian EM-360L NMR spectrophotometer 60 MHz,
Varian, USA, Faculty of Pharmacy, Assiut University,
Varian Mercury VX 300 BB Spectrophotometer; Faculty of
1
Science, Cairo University 300 MHz for H and 75.45 MHz
for 13C NMR, Bruker apparatus DRX 400 MHz; Dakota
State University, Brookings, SD, USA for 1H and 101 MHz
for 13C NMR. TMS was used as an internal reference and
chemical shifts are expressed in δ units (ppm). Mass spectra
(MS) were run on a gas chromatograph/mass spectrometer
Shimadzu GCMS-Qp2010 plus, single quad (70 eV),
Regional Centre for Mycology and Biotechnology, Al-
Azhar University, Nasr City, Cairo, and ThermoFinnigan
MAT 95 XL, LR and HR work, EI, electrospray type: high
resolution double-focusing magnetic sector; ESI: ESI II
spray head LC/MS ThermoFinnigan LCQ Advantage Ion
Trap, LR electrospray type: Ion Trap, University of South
Herein, we report the synthesis of rationally designed
fully aromatic pyrimidine multifunctional derivatives.
Antihypertensive evaluation and preliminary SAR study of
these compounds are also described. Newly designed
compounds were synthesized straightforward as depicted in
Schemes 1–3.

Medicinal Chemistry Research
Fig. 3 Chemical target of the
present research work
Maintains Hydrophobic
interaction
4-Chloro-6-methyl-2-(methylthio)-pyrimidine-5-carbonitrile
(3a) Yield 93%; mp 88–90 °C; IR v/cm−1: 3300 (NH),
2215 (C≡N), 1614 (C=N), 1604 (C=C–Ar), 1263, 1134
(vas and vs C–O–C).
Dakota, USA. All the analytical data were obtained from
Microanalytical Data Unit at Cairo University, Cairo, Egypt
and all results were in an acceptable range.
General method for the synthesis of 2-(methylthio)-4-
methyl/phenyl-6-oxo-1,6-dihydropyrimidine-5-carbonitriles
(2a,b)
4-Chloro-6-phenyl-2-(methylthio)-pyrimidine-5-carbonitrile
(3b) Yield 94%; mp 141–140 °C; IR v/cm−1: 3312 (NH),
2214 (C≡N), 1614 (C=N), 1572 (C=C–Ar), 1293, 1089
(vas and vs C–O–C).
A mixture of ethyl 2-cyano-3-phenylacrylate (1a) (Hussein
et al. 1985) or ethyl 2-cyano-3-ethoxybutenoate (1b) (Yang
et al. 2012) (10 mmol), S-methylisothiourea sulfate (11.0
mmol), and anhydrous sodium acetate (2.0 g, 24.4 mmol) in
DMF (1 ml) was heated at 70 °C for 1 h. The reaction
mixture was poured onto ice-cold H2O and the formed
precipitate was filtered and crystallized from EtOH (Hussein
et al. 1985; Abdel-Aziz et al. 2011).
General method for the synthesis of 4-(4-methoxyanilino)-
6-methyl/phenyl-2-(methylthio)-pyrimidine-5-carbonitriles
(4a,b)
A mixture of 3a,b (2.86 mmol), 4-methoxyaniline (0.36 g,
2.88 mmol) and anhydrous K2CO3 (0.4 g, 5.76 mmol) in
EtOH (15 ml) was heated under reflux for 5 h. It was then
cooled, filtered and the filtrate was concentrated to half its
volume. The obtained precipitate was filtered and crystal-
lized from EtOH-H2O (3:1 v/v).
2-(Methylthio)-4-methyl-6-oxo-1,6-dihydropyrimidine-5-
carbonitrile (2a) Yield 40%; mp 140–142 °C; IR v/cm−1:
3427 (NH), 2227 (C≡N), 1557 (C=N), 1495 (C=C–Ar); 1H-
NMR (DMSO-d6, 400 MHz) δ/ppm: 8.31 (s, br, 1H, NH),
2.25 (s, 3H, S-CH3), 2.11 (s, 3H, CH3) (Hussein et al. 1985).
4-(4-Methoxyanilino)-6-methyl-2-(methylthio)-pyrimidine-
5-carbonitrile (4a) Yield 71%; mp 158–160 °C; IR v/cm–1:
3300 (NH), 2214 (C≡N), 1614 (C=N), 1604 (C=C–Ar),
1293, 1134 (vas and vs C–O–C); 1H NMR (CDCl3, 60 MHz)
δ/ppm: 7.49 (d, 2H, Ar-Hs, o- to NH-Ar, J = 12.6 Hz), 7.21
(s, br, 1H, NH), 6.90 (d, 2H, Ar-Hs, o- to OCH3, J = 12.0
Hz), 3.83 (s, 3H, OCH3), 2.58 (s, 3H, S-CH3), 2.50 (s, 3H,
CH3); EIMS m/z 286 (M+., 100), 271 (33), 270 (25), 238
(13); Anal. Calcd. for C14H14N4OS: (286.35): C, 58.72; H,
4.93; N, 19.72. Found: C, 58.97; H, 4.98; N, 19.57.
2-(Methylthio)-4-phenyl-6-oxo-1,6-dihydropyrimidine-5-car-
bonitrile (2b) Yield 60%; mp 276–277 °C; IR v/cm−1
:
3412 (NH), 2228 (C≡N), 1593 (C=N), 1573 (C=C–Ar)
(Hussein et al. 1985; Abdel-Aziz et al. 2011).
General method for the synthesis of 4-chloro-6-methyl/
phenyl-2-(methylthio)-pyrimidine-5-carbonitriles (3a,b)
A mixture of 2a,b (1.49 mmol), POCl3 (0.4 ml, 4 mmol) and
N,N-dimethylaniline (0.2 ml, 1.58 mmol) was heated at 60 °
C for 2 h. After completion of the reaction as indicated by
TLC, the mixture was poured onto ice-cold H2O and the
formed precipitate was filtered and crystallized from EtOH
(Vasudevan et al. 2012, 2014).
4-(4-Methoxyanilino)-6-phenyl-2-(methylthio)-pyrimidine-
5-carbonitrile (4b) Yield 75%; mp 190–192 °C; IR v/cm–1:
3312 (NH), 2215 (C≡N), 1622 (C=N), 1572 (C=C–Ar),
1263, 1089 (vas and vs C–O–C); H NMR (CDCl3, 400
MHz) δ/ppm: 7.97 (d, 2H, C2,6-Hs of phenyl ring, J = 8.0
1

Medicinal Chemistry Research
Scheme 1 Synthetic pathway to
hydrazine derivatives 5a,b
Hz), 7.46–7.55 (m, 4H, C3,4,5-Hs of phenyl ring, + NH-
Ar), 7.25 (d, 2H, Ar-Hs, o- to NH-Ar, J = 6.0 Hz), 6.91 (d,
2H, Ar-Hs, o- to OCH3, J = 9.2 Hz), 3.82 (s, 3H, OCH3),
2.50 (s, 3H,S-CH3); Anal. Calcd. for C19H16N4OS (348.42):
C, 65.50; H, 4.93; N, 16.08. Found: C, 65.78; H, 4.98; N,
16.34.
65.05; H, 4.85; N, 25.29. Found: C, 65.17; H, 4.91; N,
25.43.
General method for the synthesis of 8-(4-Methoxyanilino)-
6-methyl-3-aryl-4H-pyrimido[2,1-c][1,2,4]-triazine-7-
carbonitriles (6a–c)
General method for the synthesis of 2-hydrazinyl-6-methyl/
phenyl-4-(4-methoxyanilino)-pyrimidine-5-carbonitriles (5a,b)
The appropriately substituted phenacyl bromide (1 mmol)
was added to a solution of the hydrazine derivative 5a (0.27
g, 1 mmol) in absolute EtOH (5 ml). The reaction mixture
was heated under reflux for 2 h, concentrated to half its
volume then left to cool to RT. The obtained precipitate was
filtered, washed with petroleum ether (40°–60°), dried and
crystallized from EtOH.
A mixture of 4a,b (0.24 mmol), and hydrazine hydrate
(0.012 g, 0.24 mmol) in dioxane (1 ml) was heated under
reflux for 4 h. The reaction mixture was concentrated to half
its volume and the obtained precipitate was filtered and
crystallized from EtOH.
8-(4-Methoxyanilino)-6-methyl-3-(4-bromophenyl)-4H-pyri-
mido[2,1-c][1,2,4]-triazine-7-carbonitrile (6a) Yield 60%;
mp 200–203 °C; IR v/cm−1: 3565 (NH), 2223 (C≡N), 1617
(C=N), 1576 (C=C–Ar), 1254, 1026 (vas and vs C–O–C)
1H NMR (DMSO-d6, 400 MHz) δ/ppm: 12.63 (s, 1H, NH-
triazine), 10.77 (s, 1H, NH-Ar), 7.83 (d, 2H, Ar-Hs, o- to
Br, J = 8.68 Hz), 7.75 (d, 2H, Ar-Hs, m- to Br, J = 8.68
Hz), 7.44 (d, 2H, Ar-Hs, o- to NH-Ar, J = 8.72 Hz), 7.02 (d,
2H, Ar-Hs, o- to OCH3, J = 9.0 Hz), 5.32 (s, 2H, C4-H2),
3.80 (s, 3H, OCH3), 2.82 (s, 3H, CH3); 13C NMR (DMSO-
d6, 101 MHz) δ/ppm: 168.6 (CN), 160.0 (C-4 of 4-bromo-
phenyl), 159.2 (C-7), 159.0 (C-9a), 148.7 (C-6, C-1 of 4-
methoxyaniline), 145.1 (C-8), 134.2 (C-3, C-5 of 4-bro-
mophenyl), 132.8 (C-3), 130.0 (C-4 of 4-methoxyaniline),
129.3 (C-2, C-6 of 4-methoxyaniline), 126.4 (C-2, C-6 of 4-
bromophenyl), 124.8 (C-1 of 4-bromophenyl), 114.0 (C-3,
C-5 of 4-methoxyaniline), 55.39 (OCH3), 44.9 (C-4), 18.9
(C6-CH3); HRESIMS M+ +1 at m/z 449.0729 (100) and M
+ + 3 at m/z 451.0712 (94) corresponding to
C21H1779BrN6O and C21H1781BrN6O radical cation frag-
ments; calculated = 448 (450); Anal. Calcd for
2-Hydrazinyl-6-methyl-4-(4-methoxyanilino)-pyrimidine-5-
carbonitrile (5a) Yield 50%; mp 190–192 °C; IR v/cm−1
:
3326 (NH), 3215 (NH + NH2), 2188 (C≡N), 1610 (C=N),
1490 (C=C–Ar), 1180, 1094 (vas and vs C–O–C); 1H NMR
(DMSO-d6, 60 MHz) δ/ppm: 8.60 (s, br, 1H, C2-NH), 8.35
(s, br, 1H, NH-Ar), 7.63 (d, 2H, Ar-Hs, o- to NH-Ar, J =
11.4 Hz), 6.91 (d, 2H, Ar-Hs, o- to OCH3, J = 11.4 Hz),
3.88 (s, 3H, OCH3), 2.44 (s, 3H, CH3); Anal. Calcd. for
C13H14N6O (270.29): C, 57.77; H, 5.22; N, 31.09. Found:
C, 57.96; H, 5.34; N, 31.36.
2-Hydrazinyl-6-phenyl-4-(4-methoxyanilino)-pyrimidine-5-
carbonitrile (5b) Yield 55%; mp 215–217 °C; IR v/cm−1
:
3287 (NH), 3270 (NH + NH2), 2188 (C≡N), 1612 (C=N),
1582 (C=C–Ar), 1251, 1032 (vas and vs C–O–C); 1H NMR
(DMSO-d6, 400 MHz) δ/ppm: 9.12 (s, br, 1H, C2-NH, D2O
exchangeable), 8.96 (s, br, 1H, NH-Ar, D2O exchangeable),
7.53–7.90 (m, 7H, Ar-Hs), 6.90 (d, 2H, Ar-Hs, o- to OCH3,
J = 8.92 Hz), 4.38 (s, 2H, NH2, D2O exchangeable), 3.76 (s,
3H, OCH3); Anal. Calcd. for C18H16N6O (332.36): C,

Medicinal Chemistry Research
C21H17BrN6O (449.30): C, 56.14; H, 3.81; N, 18.70. Found:
C, 56.30; H, 3.78; N, 18.86.
300 MHz) δ/ppm: 9.87 (s, br, 1H, NH-Ar, D2O-exchange-
able), 9.50 (d, 1H, triazole NH, J = 10.5 Hz, D2O-
exchangeable), 9.21 (s, br, 1H, pyrimidine N-H, D2O-
exchangeable), 8.02 (s, 1H, C3-H), 7.50 (d, 2H, Ar-Hs, o- to
NH-Ar, J = 6.9 Hz), 6.87 (d, 2H, Ar-Hs, o- to OCH3, J =
6.9 Hz), 3.74 (s, 3H, OCH3), 2.37 (s, 3H, CH3); 13C NMR
(DMSO-d6, 75 MHz) δ/ppm: 171.7 (CN), 167.2 (C-6), 161.5
(C-5), 160.3 (C-9), 159.8 (C-7), 131.0 (C-1 of 4-methox-
yaniline), 125.2 (C-3), 123.9 (C-4 of 4-methoxyaniline),
113.5, 113.1 (C-5,C-3 of 4-methoxyaniline), 116.2, 116.1
(C-6, C-2 of 4-methoxyaniline), 55.1 (OCH3), 23.0 (C-5-
CH3); Anal. Calcd. for C14H12N6O (280.28): C, 59.99; H,
4.32; N, 29.98. Found: C, 60.14; H, 4.39; N, 30.16.
8-(4-Methoxyanilino)-6-methyl-3-(4-chlorophenyl)-4H-pyri-
mido[2,1-c][1,2,4]-triazine-7-carbonitrile (6b) Yield 52%;
mp 193–195 °C; IR v/cm−1: 3399 (NH), 2223 (C≡N), 1624
(C=N), 1509 (C=C–Ar), 1232, 1090 (vas and vs C–O–C);
1H NMR (DMSO-d6, 400 MHz) δ ppm: 12.64 (s, 1H, NH-
triazine), 10.78 (s, 1H, NH-Ar,), 7.91 (d, 2H, Ar-Hs, o- to
Cl, J = 7.6 Hz), 7.60 (d, 2H, Ar-Hs, m- to Cl, J = 8.0 Hz),
7.45 (d, 2H, Ar-Hs, o- to NH-Ar, J = 8.0 Hz), 7.02 (d, 2H,
Ar-Hs, o- to OCH3, J = 8.4 Hz), 5.34 (s, 2H, C4-H2), 3.79
(s, 3H, OCH3), 2.83 (s, 3H, CH3); Anal. Calcd. for
C21H17ClN6O (404.85): C, 62.30; H, 4.23; N, 20.76. Found:
C, 62.57; H, 4.30; N, 20.92.
7-(4-Methoxyanilino)-3,5-dimethyl-[1,2,4]triazolo[4,3-a]pyr-
imidine-6-carbonitrile (7b) Yield 32%; mp 185–187 °C;
IR v/cm−1: 3315 (NH), 2216 (C≡N), 1626 (C=N), 1577
(C=C–Ar), 1254, 1022 (vas and vs C–O–C); 1H NMR
(DMSO-d6, 400 MHz) δ/ppm: 9.78 (s, br, 1H, NH-Ar, D2O-
exchangeable), 9.28 (s, 1H, triazole N-H), 9.15 (s, 1H,
pyrimidine N-H, D2O-exchangeable), 7.50 (d, 2H, Ar-Hs,
o- to NH-Ar, J = 8.8 Hz), 6.85 (d, 2H, Ar-Hs, o- to OCH3,
J = 8.4 Hz), 3.74 (s, 3H, OCH3), 2.36 (s, 3H, C5-CH3), 1.85
(s, 3H, C3-CH3); Anal. Calcd. for C15H14N6O (294.12): C,
61.21; H, 4.79; N, 28.55. Found: C, 61.42; H, 4.87; N,
28.17.
8-(4-Methoxyanilino)-6-methyl-3-(4-nitrophenyl)-4H-pyri-
mido[2,1-c][1,2,4]-triazine-7-carbonitrile (6c) Yield 65%;
mp 205–207 °C; IR v/cm−1: 3565 (NH), 2230 (C≡N), 1635
(C=N), 1507 (C=C–Ar), 1457, 1346 (vas and vs NO2),
1
1254, 1032 (vas and vs C–O–C); H NMR (DMSO-d6, 400
MHz) δ/ppm: 12.80 (s, br, 1H, NH- triazine, D2O
exchangeable), 10.85 (s, br, 1H, NH-Ar, D2O exchange-
able), 8.36 (d, 2H, Ar-Hs, o- to NO2, J = 8.84 Hz), 8.14 (d,
2H, Ar-Hs, m- to NO2, J = 8.8 Hz), 7.44 (d, 2H, Ar-Hs, o-
to NH-Ar, J = 8.68 Hz), 7.03 (d, 2H, Ar-Hs, o- to OCH3, J
= 8.88 Hz), 5.38 (s, 2H, C4-H2), 3.80 (s, 3H, OCH3), 2.83
(s, 3H, CH3); Anal. Calcd. for C21H17N7O3 (415.14): C,
60.72; H, 4.12; N, 23.60. Found: C, 60.89; H, 4.14; N,
23.84.
7-(4-Methoxyanilino)-3-phenyl-5-methyl-[1,2,4]triazolo[4,3-a]
pyrimidine-6-carbonitrile (7c) Yield 40%; mp 203–202 °C;
IR v/cm−1: 3340 (NH), 2212 (C≡N), 16211 (C=N), 1585
(C=C–Ar), 1249, 1042 (vas and vs C–O–C); 1H NMR
(DMSO-d6, 400 MHz) δ/ppm: 10.84 (s, 1H, NH-Ar, D2O-
General method for the synthesis of 7-(4-methoxyanilino)-
3,5-disubstituted-[1,2,4]triazolo[4,3-a]pyrimidine-6-
carbonitriles (7a–f)
exchangeable), 8.25 (dd, 2H, C2,6-Hs of C3-phenyl, JAM
=
7.9 Hz, JAX = 2.3 Hz), 7.56–7.61 (m, 3H, C3,4,5-Hs of C3-
phenyl), 7.41 (d, 2H, Ar-Hs, o- to NH-Ar, J = 8.84 Hz),
7.02 (d, 2H, Ar-Hs, o- to OCH3, J = 8.88 Hz), 3.82 (s, 3H,
OCH3), 2.51 (s, 3H, C5-CH3); EIMS m/z: 357 (M+., 25),
356 (99), 341 (70), 185 (67), 92 (32), 77 (100), 76 (31);
Anal. Calcd. for C20H16N6O (356.14): C, 67.40; H, 4.53; N,
23.58. Found: C, 67.64; H, 4.59; N, 23.71.
For 7a,b,d,e: A solution of the hydrazine derivative 5a,b (1
mmol) and the appropriate aliphatic acid (5 ml) was heated
under reflux for 7 h. The reaction mixture was concentrated
to a small volume and diluted with ice-cold H2O. The
obtained precipitate was filtered, washed with H2O, dried
and crystallized from EtOH/H2O.
For 7c,f: A solution of the hydrazine derivative 5a,b (1
mmol), benzoic acid (0.122 g, 1 mmol) and POCl3 (0.4 ml)
was heated under reflux for 4 h. After cooling, the reaction
mixture was poured onto ice-cold H2O and neutralized with
a saturated solution of NaHCO3. The precipitate was col-
lected by filtration, washed with cold H2O and recrystallized
from EtOH.
7-(4-Methoxyanilino)-5-phenyl-[1,2,4]triazolo[4,3-a]pyrimi-
dine-6-carbonitrile (7d) Yield 20%; mp 222–220 °C; IR v/
cm−1: 3199 (NH), 2212 (C≡N), 1624 (C=N), 1583 (C=C–
1
Ar), 1248, 1042 (vas and vs C–O–C); H NMR (DMSO-d6,
400 MHz) δ/ppm: 9.79 (s, br, 1H, NH-Ar, D2O-exchange-
able), 9.58 (d, br, 1H, triazole NH, J = 10 Hz, D2O-
exchangeable), 9.30 (s, br, 1H, pyrimidine NH, D2O-
exchangeable), 8.01 (s, 1H, C3-H), 7.81 (d, 2H, Ar-Hs, o-
to NH-Ar, J = 7.68 Hz), 7.53–7.57 (m, 5H, Ar-Hs), 6.90 (d,
dist, 2H, Ar-Hs, o- to OCH3, J = 7.92 Hz), 3.76 (s, 3H,
OCH3); Anal. Calcd. for C19H14N6O (342.12): C, 66.66; H,
4.12; N, 24.55. Found: C, 66.81; H, 4.09; N, 24.69.
7-(4-Methoxyanilino)-5-methyl-[1,2,4]triazolo[4,3-a]pyrimi-
dine-6-carbonitrile (7a) Yield 23%; mp 178–180 °C; IR v/
cm−1: 3213 (NH), 2202 (C≡N), 1626 (C=N), 1581 (C=C–
1
Ar), 1231, 1033 (vas and vs C–O–C); H NMR (DMSO-d6,

Medicinal Chemistry Research
7-(4-Methoxyanilino)-3-methyl-5-phenyl-[1,2,4]triazolo[4,3-
a]pyrimidine-6-carbonitrile (7e) Yield 40%; mp 213–215 °
C; IR v/cm−1: 3270 (NH), 2208 (C≡N), 1618 (C=N), 1580
(C=C–Ar), 1245, 1038 (vas and vs C–O–C); 1H NMR
(DMSO-d6, 400 MHz) δ/ppm: 9.77 (s, br, 1H, NH-Ar), 9.50
(s, 1H, triazole H), 9.30 (s, 1H, triazole N-H), 7.81 (d, 2H,
C2,6-Hs of C-3-phenyl, J = 5.2 Hz), 7.51–7.57 (m, 3H,
C3,4,5-Hs of C-5-phenyl), 7.53 (d, 2H, Ar-Hs, o- to NH-Ar,
J = 8.8 Hz), 6.89 (d, 2H, Ar-Hs, o- to OCH3, J = 8.8 Hz),
3.75 (s, 3H, OCH3), 1.87 (s, 3H, CH3); Anal. Calcd. for
C20H16N6O (356.14): C, 67.40; H, 4.53; N, 23.58. Found:
C, 67.63; H, 4.59; N, 23.74.
1H, NH-Ar), 7.88 (d, 2H, Ar-Hs, J = 6.80 Hz), 7.79 (s, dist,
2H, pyrazole-NH2), 7.55–7.79 (m, 3H, Ar-Hs), 7.44 (d, 2H,
Ar-Hs, o- to NH-Ar, J = 8.68 Hz), 7.00 (d, 2H, Ar-Hs, o- to
OCH3, J = 8.72 Hz), 3.81 (s, 3H, OCH3), 2.16 (s, 3H, C3-
CH3-pyrazole); EIMS m/z: 422 (M+., 49), 421 (36), 357
(54), 333 (22), 332 (100), 317 (33), 302 (30); Anal. Calcd.
for C23H18N8O (422.16): C, 65.39; H, 4.29; N, 26.53.
Found: C, 65.61; H, 4.32; N, 26.79.
General method for the synthesis of 2-(4-Cyano-5-oxo-3-
aryl-4,5-dihydro-1H-pyrazol-1-yl)-6-methyl/phenyl-4-(4-
methoxyanilino)-pyrimidine-5-carbonitriles (9a–d)
7-(4-Methoxyanilino)-3,5-diphenyl-[1,2,4]triazolo[4,3-a]pyri-
midine-6-carbonitrile (7f) Yield 43%; mp 287–289 °C; IR
v/cm−1: 3346 (NH), 2212 (C≡N), 1603 (C=N), 1573
(C=C–Ar), 1252, 1029 (vas and vs C–O–C); 1H NMR
(DMSO-d6, 400 MHz) δ/ppm: 11.00 (s, br, 1H, NH-Ar),
7.83, 8.76 (2d, 4H, Ar-Hs of C-5-phenyl, J = 6.0 Hz), 7.54–
7.75 (m, 6H, 5 Ar-Hs of C-3-phenyl + 1 Ar-H of C-5-
phenyl), 7.49 (d, 2H, Ar-Hs, o- to NH-Ar, J = 8.0 Hz), 7.03
(d, 2H, Ar-Hs, o- to OCH3, J = 7.6 Hz), 3.80 (s, 3H,
OCH3); Anal. Calcd. for C25H18N6O (418.15): C, 71.76; H,
4.34; N, 20.08. Found: C, 71.89; H, 4.41; N, 20.29.
A mixture of the hydrazine derivative 5a,b (0.5 mmol) and
ethyl 2-cyano-3-phenyl acrylate 1a or ethyl 2-cyano-3-
methoxyphenylacrylate(143) 1b (0.1 g, 0.5 mmol) in absolute
EtOH (5 ml) was heated under reflux for 5 h and left to cool
to RT. The separated products were filtered, washed with
petroleum ether dried and crystallized from EtOH.
2-(4-Cyano-5-oxo-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-6-
methyl-4-(4-methoxyanilino)-pyrimidine-5-carbonitrile
(9a) Yield 40%; mp 170–172 °C; IR v/cm−1: 3275 (OH),
3187 (NH), 2360, 2213 (2 × C≡N), 1606 (C=N), 1561
(C=C–Ar), 1233, 1028 (vas and vs C–O–C); 1H NMR
(DMSO-d6, 400 MHz) δ/ppm: 11.60 (s, br, 1H, OH, C5-
OH-pyrazole), 9.23 (s, br, 1H, NH-Ar), 8.16 (s, 1H, C4-H-
pyrazole), 7.69 (d, 2H, Ar-Hs, o- to NH-Ar, J = 6.52 Hz),
7.37–7.46 (m, 5H, Ar-Hs of C-3-pyrazole), 6.93 (d, 2H, Ar-
Hs, o- to OCH3, J = 8.72 Hz), 3.79 (s, 3H, OCH3), 2.43 (s,
3H, CH3); 13C NMR (DMSO-d6, 101 MHz) δ/ppm: 172.7
(2 × CN), 160.9 (C5), 159.3 (C2), 156.3 (C6), 143.7 (C4),
135.4 (C-1, C-4 of 4-methoxyaniline), 132.3 (C-3, C-5-
pyrazole), 129.9 (C4-pyrazole), 129.2 (C-2, C-6 of 4-
methoxyaniline), 127.0 (C-3, C-5 of 4-methoxyaniline),
124.4 (C-1, C-4 of phenyl at C3-pyrazole), 117.0 (C-2, C-6
of phenyl at C3-pyrazole), 113.8 (C-3, C-5 of phenyl at C3-
pyrazole), 55.7 (OCH3), 23.6 (C6-CH3); EIMS m/z 423 (M
+., 10), 382 (16), 357 (22), 325 (24), 312 (35), 309 (20), 296
(23), 286 (100), 281 (28), 280 (63), 244 (85), 210 (35), 207
(38), 201 (40), 191 (37), 190 (38), 184 (30), 181 (47), 178
(24), 176 (25), 163 (63), 145 (35), 144 (95), 141 (38), 121
(60); Anal. Calcd. for C23H17N7O (423.43): C, 65.24; H,
4.05; N, 23.16. Found: C, 65.39; H, 4.11; N, 23.42.
General method for the synthesis of 2-(5-Amino-4-cyano-3-
methyl-1H-pyrazol-1-yl)-6-methyl/phenyl-4-(4-
methoxyanilino)-pyrimidine-5-carbonitriles (8a,b)
A mixture of the hydrazine derivative 5a,b (0.5 mmol) and
2-(1-ethoxy-ethylidene)malononitrile (1b) (Yang et al.
2012) (0.13 g, 0.5 mmol) in absolute EtOH (5 ml) was
heated under reflux for 5 h and left to cool to RT. The
separated products were filtered, washed with petroleum
ether, dried, and crystallized from EtOH.
2-(5-Amino-4-cyano-3-methyl-1H-pyrazol-1-yl)-6-methyl-4-
(4-methoxyanilino)-pyrimidine-5-carbonitrile (8a) Yield
23%; mp 140–143 °C; IR v/cm−1: 3389 (NH), 2221 (C≡N),
1635 (C=N), 1598 (C=C–Ar), 1258, 1030 (vas and vs C–O–
1
C); H NMR (DMSO-d6, 400 MHz) δ/ppm: 10.04 (s, br,
1H, NH-Ar), 9.24 (s, br, 2H, pyrazole-NH2), 7.83 (m, 2H,
Ar-Hs, o- to NH-Ar), 6.94 (m, 2H, Ar-Hs, o- to OCH3),
3.76 (s, 3H, OCH3), 2.41 (s, 3H, C6-CH3-pyrimidine), 2.15
(s, 3H, C3-CH3-pyrazole); Anal. Calcd. for C18H16N8O
(360.37): C, 59.99; H, 4.48; N, 31.09. Found: C, 60.18; H,
4.57; N, 31.34.
2-(4-Cyano-5-oxo-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyr-
azol-1-yl)-6-methyl-4-(4-methoxyanilino)-pyrimidine-5-car-
bonitrile (9b) Yield 45%; mp 188–190 °C; IR v/cm−1
:
2-(5-Amino-4-cyano-3-methyl-1H-pyrazol-1-yl)-6-phenyl-4-
(4-methoxyanilino)-pyrimidine-5-carbonitrile (8b) Yield
21%; mp 192–190 °C; IR v/cm−1: 3315 (NH), 2216 (C≡N),
1635 (C=N), 1578 (C=C–Ar), 1248, 1035 (vas and vs C–O–
3282 (OH), 3280 (NH), 2320, 2215 (2 × C≡N), 1619
(C=N), 1577 (C=C–Ar), 1244, 1035 (vas and vs C–O–C);
1H NMR (DMSO-d6, 400 MHz) δ/ppm: 11.60 (s, br, 1H,
OH, C5-OH-pyrazole, D2O exchangeable), 9.18 (s, br, 1H,
NH-Ar, D2O exchangeable), 8.11 (s, 1H, C4-H-pyrazole),
1
C); H NMR (DMSO-d6, 400 MHz) δ/ppm: 10.15 (s, br,

Medicinal Chemistry Research
7.61–7.91 (m, 4H, Ar-Hs of 4-methoxyphenyl at C3-pyr-
azole), 7.00 (d, 2H, Ar-Hs, o- to NH-Ar, J = 8.36 Hz), 6.93
(d, 2H, Ar-Hs, o- to OCH3, J = 8.8 Hz), 3.78 (s, 6H, 2 ×
OCH3), 2.41 (s, 3H, CH3); 13C NMR (DMSO-d6, 101 MHz)
δ/ppm: 172.0 (2 × CN), 160.9 (pyrazole-C4 + C5), 160.8
(C5), 159.2 (C2), 156.1 (C6), 143.8 (C4), 132.5 (C1 of 4-
methoxyaniline), 128.5 (pyrazole-C3), 128.0 (C-1 of 4-
methoxyphenyl at C3-pyrazole), 124.2 (C-4 of 4-methox-
yaniline + C4 of 4-methoxyphenyl at C3-pyrazole), 117.1
(C-2, C-6 of 4-methoxyphenyl at C3-pyrazole), 114.7 (C-2,
C-6 of 4-methoxyaniline), 113.8 (C-3, C-5 of 4-
methoxyphenyl at C3-pyrazole + C-3, C-5 of 4-methox-
yaniline), 55.8, 55.7 (2 × OCH3), 23.6 (C-6-CH3); EIMS m/
z: 453 (M+., 2), 390 (24), 389 (100), 388 (39), 387 (35), 386
(23); Anal. Calcd. for C24H19N7O3 (453.45): C, 63.57; H,
4.22; N, 21.62. Found: C, 63.80; H, 4.27; N, 21.74.
J = 7.72 Hz), 6.96 (d, 2H, Ar-Hs, o- to OCH3, J = 8.88 Hz),
3.82, 3.81 (2s, 6H, 2 × OCH3); 13C NMR (DMSO-d6, 101
MHz) δ/ppm: 172.8, 169.5 (2 × CN), 161.6 (C4-pyrazole),
160.6 (C5), 156.5 (C2), 153.1 (C6), 148.1 (C4), 131.4 (C5-
pyrazole), 129.8 (C3-pyrazole), 128.5 (C1 of 4-methox-
yaniline), 126.1 (C1 of phenyl at C6-pyrimidine), 124.8 (C-
2, C-6 of phenyl at C6-pyrimidine), 121.0 (C4 of 4-meth-
oxyaniline + C4 of 4-methoxyphenyl at C3-pyrazole),
120.9 (C-3, C-5 of phenyl at C6-pyrimidine), 116.5 (C1 of
4-methoxyphenyl at C3-pyrazole), 114.1 (C-2, C-6 of 4-
methoxyaniline + C-2, C-6 of 4-methoxyphenyl at C3-
pyrazole), 113.8 (C-3, C-5 of 4-methoxyaniline, C-3, C-5 of
4-methoxyphenyl at C3-pyrazole), 82.2 (C4 of phenyl at
C6-pyrimidine), 55.7 (OCH3), 30.4 (OCH3); Anal. Calcd.
for C29H21N7O3 (515.52): C, 67.56; H, 4.11; N, 19.02.
Found: C, 67.74; H, 4.18; N, 19.26.
2-(4-Cyano-5-oxo-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-6-
phenyl-4-(4-methoxyanilino)-pyrimidine-5-carbonitrile
(9c) Yield 41%; mp 191–193 °C; IR v/cm−1: 3400 (OH),
3305 (NH), 2222, 2200 (2 × C≡N), 1621 (C=N), 1584
(C=C–Ar), 1231, 1028 (vas and vs C–O–C); 1H NMR
(DMSO-d6, 400 MHz) δ/ppm: 11.79 (s, br, 1H, OH, C5-
OH-pyrazole), 9.33 (s, br, 1H, NH-Ar), 8.21 (s, 1H, C4-H-
pyrazole), 7.45–7.96 (m, 12H, Ar-Hs, 2 × 5-phenyl-Hs + 2
Ar-Hs, o- to NH-Ar), 6.97 (d, 2H, Ar-Hs, o- to OCH3, J =
6.64 Hz), 3.81 (s, 3H, OCH3); 13C NMR (DMSO-d6, 101
MHz) δ/ppm: 180.1 (2 × CN), 160.7 (C5), 159.6 (C2),
157.5 (C6), 142.0 (C4), 138.5 (C-1 of 4-methoxyaniline),
135.5 (C-4, C-5 pyrazole), 132.0 (C4 of 4-methoxyaniline),
129.8 (C-1 of phenyl at C3-pyrazole + C3-pyrazole), 129.1
(C-2, C-6 of 4-methoxyaniline), 128.5 (C-1 of phenyl at C6-
pyrimidine), 128.3 (C-2, C-6 of phenyl at C6-pyrimidine),
126.5 (C-2, C-6 of phenyl at C3-pyrazole), 124.4 (C-3, C-5
of phenyl at C6-pyrimidine), 117.1 (C-3, C-5 of 4-meth-
oxyaniline), 113.2 (C-3, C-5 of phenyl at C3-pyrazole),
83.75 (C4 of phenyl at C3-pyrazole + C4 of phenyl at C-6-
pyrimidine), 55.19 (OCH3); EIMS m/z: 486 (M+., 2), 317
(23), 316 (22), 199 (24), 175 (34), 171 (41), 158 (28), 144
(25), 141 (49), 140 (50), 131 (23), 114 (22), 111 (70), 110
(27), 109 (23), 66 (100); Anal. Calcd. for C28H19N7O2
(485.50): C, 69.27; H, 3.94; N, 20.20. Found: C, 69.67; H,
3.76; N, 20.52.
General method for the synthesis of 2-(5-Amino-4-cyano-
1H-pyrazol-1-yl)-6-methyl/phenyl-4-(4-methoxyanilino)-
pyrimidine-5-carbonitriles (10a,b)
A mixture of the hydrazine derivative 5a,b (0.5 mmol) and
2-(ethoxymethylene) malonitrile (Ding et al. 1987) (0.061 g,
0.5 mmol) in absolute EtOH (5 ml) was heated under reflux
for 5 h and left to cool to RT. The separated product was
filtered, washed with petroleum ether, dried, and crystal-
lized from EtOH.
2-(5-Amino-4-cyano-1H-pyrazol-1-yl)-6-methyl-4-(4-meth-
oxy-anilino)-pyrimidine-5-carbonitrile (10a) Yield 23%;
mp 122–123 °C; IR v/cm−1: 3384 (NH), 3309 (NH2), 2210
(C≡N), 1635 (C=N), 1564 (C=C–Ar), 1256, 1025 (vas and
1
vs C–O–C); H NMR (DMSO-d6, 300 MHz) δ/ppm: 10.06
(s, 1H, NH-Ar), 7.86 (s, 2H, pyrazole-NH2), 7.84 (s, 1H,
C3-H pyrazole), 7.43 (d, 2H, Ar-Hs, o- to NH-Ar, J = 8.76
Hz), 6.97 (d, 2H, Ar-Hs, o- to OCH3, J = 8.88 Hz), 3.80 (s,
3H, OCH3), 2.56 (s, 3H, CH3); 13C NMR (DMSO-d6, 75
MHz) δ/ppm: 172.7 (2 × CN), 160.6 (C5), 157.0 (C4-pyr-
azole), 155.8 (C2), 154.1 (C6), 143.0 (C-4, C-1 of 4-
methoxyaniline), 129.7 (C5-pyrazole), 125.9 (C4 of 4-
methoxyaniline), 114.5 (C-2, C-6 of 4-methoxyaniline),
114.0 (C-3, C-5 of 4-methoxyaniline), 72.6 (C3-pyrazole),
55.2 (OCH3), 23.3 (C6-CH3); EIMS m/z: 347 (M+., 23), 346
(100), 331 (66), 197 (22), 196 (22), 92 (22); Anal. Calcd.
for C17H14N8O (346.13): C, 58.95; H, 4.07; N, 32.35.
Found: C, 59.18; H, 4.12; N, 32.49.
2-(4-Cyano-5-oxo-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyr-
azol-1-yl)-6-phenyl-4-(4-methoxyanilino)-pyrimidine-5-car-
bonitrile (9d) Yield 43%; mp 201–203 °C; IR v/cm−1
:
3331 (OH), 3187 (NH), 2305, 2206 (2 × C≡N), 1608
(C=N), 1586 (C=C–Ar), 1247, 1031 (vas and vs C–O–C);
1H NMR (DMSO-d6, 400 MHz) δ/ppm: 11.65 (s, br, 1H,
OH, C5-OH-pyrazole, D2O exchangeable), 9.28 (s, br, 1H,
NH-Ar, D2O exchangeable), 8.15 (s, 1H, C4-H-pyrazole),
7.56–7.96 (m, 9H, Ar-Hs), 7.02 (d, 2H, Ar-Hs, o- to NH-Ar,
2-(5-Amino-4-cyano-1H-pyrazol-1-yl)-6-phenyl-4-(4-meth-
oxy-anilino)-pyrimidine-5-carbonitrile (10b) Yield 21%;
mp 200–201 °C; IR v/cm−1: 3384 (NH), 3300 (NH2), 2223
(C≡N), 1644 (C=N), 1564 (C=C–Ar), 1247, 1040 (vas and
vs C–O–C); H NMR (DMSO-d6, 400 MHz) δ/ppm: 10.06
(s, 1H, NH-Ar, D2O-exchangeable), 7.91(d, 2H, Ar-Hs of
1

Medicinal Chemistry Research
C6-phenyl, J = 6.88 Hz), 7.87 (s, 1H, C3-H pyrazole), 7.84,
(s, 2H, pyrazole-NH2, D2O-exchangeable), 7.59–7.64 (m,
3H, Ar-Hs of C6-phenyl), 7.46 (d, 2H, Ar-Hs, o- to NH-Ar,
J = 8.60 Hz), 7.00 (d, 2H, Ar-Hs, o- to OCH3, J = 8.64 Hz),
3.81 (s, 3H, OCH3); Anal. Calcd. for C22H16N8O (408.42):
C, 64.70; H, 3.95; N, 27.44. Found: C, 64.94; H, 3.93; N,
27.61.
167 (39), 165 (28), 158 (82), 157 (37), 156 (30), 155 (31),
153 (89), 151 (42), 148 (41), 147 (35), 142 (28), 141 (45),
137 (40), 136 (100), 134 (43), 133(38), 129 (31), 127 (36),
120 (33), 119 (56), 115 (32), 114 (34), 108 (40), 107 (409),
105 (44), 104 (93), 103 (31), 102 (48); Anal. Calcd. for
C23H17N9O (435.44): C, 63.44; H, 3.94; N, 28.95. Found:
C, 63.71; H, 3.92; N, 27.17.
General method for the synthesis of 2-(4-amino-1H-
pyrazolo[3,4-d] pyrimidin-1-yl)-6-methyl/phenyl-4-(4-
methoxyanilino)-pyrimidine-5-carbonitriles (11a,b)
General method for the synthesis of 4-(4-methoxyanilino)-
6-methyl-2-[2-(substituted thiocarbamoyl)hydrazinyl]-
pyrimidine-5-carbonitriles (12a,b)
A mixture of 2-(5-amino-4-cyano-1H-pyrazol-1-yl)-4-(4-
methoxyanilino)-6-substituted-pyrimidine-5-carbonitrile
10a,b (1 mmol) and formamide (2 ml) was heated under
reflux for 2–3 h. The solution was cooled to RT and poured
onto H2O. The obtained precipitate was filtered, dried and
crystallized from EtOH.
The appropriate isothiocyanate derivative (1 mmol) was
added to a well stirred suspension of the hydrazine deri-
vative 5a (0.27 g, 1 mmol) in absolute EtOH (20 ml). The
reaction mixture was stirred at RT for 2 h during which time
dissolution and reprecipitation occurred. The obtained pre-
cipitate was filtered, dried and crystallized from EtOH.
2-(4-Amino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-6-methyl-4-
(4-methoxyanilino)-pyrimidine-5-carbonitrile (11a) Yield
70%; mp 199–197 °C; IR v/cm−1: 3499 (NH2), 3294 (NH),
2213 (C≡N), 1636 (C=N), 1549 (C=C–Ar), 1248, 1033
4-(4-Methoxyanilino)-6-methyl-2-[2-phenylthiocarbamoyl-
hydrazinyl]-pyrimidine-5-carbonitrile (12a) Yield 86%;
mp 130–132 °C; IR v/cm−1: 3400 (NH), 3268 (NH), 2226
(C≡N), 1700 (C=N), 1597 (C=C–Ar), 1551, 1334, 1199,
1039 (NH-C=S I, II, III, IV mixed vibrational bands), 1266,
1
(vas and vs C–O–C); H NMR (DMSO-d6, 400 MHz) δ/
1
ppm: 8.83 (s, 1H, NH-Ar, D2O exchangeable), 7.45 (d, 2H,
Ar-Hs, o- to NH-Ar, J = 9.0 Hz), 7.00 (s, br, 2H, NH2,
D2O-exchangeable), 6.87 (d, 2H, Ar-Hs, o- to OCH3, J =
9.0 Hz), 3.74 (s, 3H, OCH3), 2.30 (s, 3H, CH3); Anal.
Calcd. for C18H15N9O (373.37): C, 57.90; H, 4.05; N,
33.76. Found: C, 58.08; H, 4.12; N, 33.94.
1094 (vas and vs C–O–C); H NMR (DMSO-d6, 400 MHz)
δ/ppm: 11.85 (s, 1H, NHNHC=S, D2O-exchangeable),
10.28 (s, 1H, C2-NH, D2O-exchangeable), 9.90 (s, 1H,
NHC6H5, D2O-exchangeable), 9.70 (s, br, 1H, C4-NH-Ar,
D2O-exchangeable), 7.14–7.55 (m, 5H, Ar-Hs of phe-
nylthiocarbamoyl), 7.54 (d, 2H, Ar-Hs, o- to NH-Ar, J =
7.76 Hz), 7.15 (d, 2H, Ar-Hs, o- to OCH3, J = 7.36 Hz),
3.33 (s, 3H, O-CH3), 2.32 (s, 3H, CH3); Anal. Calcd. for
C20H19N7OS (405.14): C, 59.24; H, 4.72; N, 24.18. Found:
C, 59.51; H, 4.79; N, 24.42.
2-(4-Amino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-6-phenyl-4-
(4-methoxyanilino)-pyrimidine-5-carbonitrile (11b) Yield
75%; mp 205–207 °C; IR v/cm−1: 3491(NH2), 3296 (NH),
2211 (C≡N), 1649 (C=N), 1560 (C=C–Ar), 1242, 1034
1
(vas and vs C–O–C); H NMR (DMSO-d6, 400 MHz) δ/
4-(4-Methoxyanilino)-6-methyl-2-[2-(4-nitrophenyl)thiocar-
bamoylhydrazinyl]-pyrimidine-5-carbonitrile (12b) Yield
88%; mp 140–142 °C; IR v/cm−1: 3495 (NH), 3292 (NH),
2230 (C≡N), 1706 (C=N), 1640 (C=C–Ar), 1535, 1340
(vas and vs NO2), 1560, 1356, 1204, 1094 (NH-C=S I, II,
III, IV mixed vibrational bands), 1270, 1111 (vas and vs C–
ppm: 8.94 (s, 1H, NH-Ar, D2O exchangeable), 7.78 (d, dist,
2H, Ar-Hs, o- to NH-Ar, J = 9.16 Hz), 7.48–7.56 (m, 5H,
Ar-Hs of C6-phenyl), 7.21 (s, br, 2H, NH2, D2O-
exchangeable), 6.91 (d, 2H, Ar-Hs, o- to OCH3, J = 8.92
Hz), 3.78 (s, 3H, OCH3); 13C NMR (DMSO-d6, 400 MHz)
δ/ppm: 170.5 (CN), 163.1 (C5), 162.6 (C4 of pyr-
azolopyrimidine), 156.5 (C2), 137.6 (C4), 132.0 (C1 of 4-
methoxyaniline), 130.8 (C6, C-1 of C6-phenyl), 129.1 (C-
3a, C-7a of pyrazolopyrimidine), 128.9 (C6 of pyr-
azolopyrimidine), 128.8 (C-2, C-6 of C6-phenyl), 128.7 (C-
3, C-5 of C6-phenyl), 125.6 (C-4 of 4-methoxyaniline + C-
4 of C6-phenyl), 118.1 (C-2, C-6 of 4-methoxyaniline),
114.0 (C-3, C-5 of 4-methoxyaniline), 78.0 (C3), 55.7
(OCH3); EIMS m/z: M+. absent, 434 (M+.-1, 2), 330 (26),
300 (29), 287 (26), 243 (23), 234 (28), 230 (25), 199 (28),
198 (23), 195 (33), 181 (26), 171 (40), 169 (26), 168 (29),
1
O–C); H NMR (DMSO-d6, 400 MHz) δ/ppm: 13.20 (s,
1H, NHC6H4-NO2, D2O-exchangeable), 11.62 (s, 1H,
NHNHC=S NHC6H4, D2O-exchangeable), 10.87 (s, 1H,
C2-NH, D2O-exchangeable), 9.60 (s, br, 1H, C4-NH-Ar,
D2O-exchangeable), 8.25 (d, 2H, Ar-Hs o- to NO2, J =
7.08 Hz), 8.23 (d, 2H, Ar-Hs m- to NO2, J = 7.08 Hz), 7.80
(d, 2H, Ar-Hs, o- to NH-Ar, J = 9.20 Hz), 6.60 (d, 2H, Ar-
Hs, o- to OCH3, J = 9.20 Hz), 3.43 (s, 3H, O-CH3), 2.32 (s,
3H, CH3); Anal. Calcd. for C20H18N8O3S (450.12): C,
53.32; H, 4.03; N, 24.87. Found: C, 53.59; H, 4.07; N,
25.03.

Medicinal Chemistry Research
General method for the synthesis of 6-methyl-4-(4-
methoxyanilino)-2-(2-(4-oxo-3-arylthiazolidin-2-ylidene)
hydrazinyl)pyrimidine-5-carbonitriles (13a,b)
nitrophenyl), 155.2 (thiazolidinone-C2), 142.5 (C6), 130.0
(C4), 125.2 (C-3, C-5 of thiazolidinone-N-nitrophenyl),
124.7 (C-2, C-6 of thiazolidinone-N-nitrophenyl), 124.8
(C1 of 4-methoxyaniline), 121.5 (C-2, C-6 of 4-methox-
yaniline), 116.0 (C4 of 4-methoxyaniline), 113.5 (C-3, C-5
of 4-methoxyaniline), 82.2 (thiazolidinone-C5), 55.3
(OCH3), 23.0 (CH3); HRESIMS: M+.+1 491.12363 (100)
(calculated mass = 490.49); Anal. Calcd. for C22H18N8O4S
(490.49): C, 53.87; H, 3.70; N, 22.85. Found: C, 54.03; H,
3.74; N, 23.03.
Ethyl bromoacetate (0.167 g, 1 mmol) and anhydrous
NaOAc (0.12 g, 1.5 mmol) in EtOH (5 ml) were added to
the solution of the appropriate thiosemicarbazide derivative
12a,b and the mixture was heated under reflux for 3 h,
concentrated to half its volume, allowed to attain RT and
poured onto ice-cold H2O (10 ml). The obtained precipitate
was filtered, dried and crystallized from EtOH.
General method for the synthesis of 6-methyl-4-(4-
methoxyanilino)-2-(3,4-diarylthiazol-2(3H)-
ylidenehydrazinyl)pyrimidine-5-carbonitriles (14a–c, 15a–c)
6-Methyl-4-(4-methoxyanilino)-2-(2-(4-oxo-3-phenylthiazo-
lidin-2-ylidene) hydrazinyl)pyrimidine-5-carbonitrile (13a)
Yield 22%; mp 199–201 °C; IR v/cm−1: 3359 (NH), 2211
(C≡N), 1742 (C=O), 1651 (C=N), 1585 (C=C–Ar), 1234,
The properly 4-substituted phenacyl bromide (1 mmol) was
added to a solution of the corresponding thiosemicarbazide
derivative 12a,b and sodium acetate (0.164 g, 2 mmol) in
EtOH (5 ml) and the reaction mixture was heated under
reflux for 2 h, concentrated to half its volume then left to
cool to RT. The obtained precipitate was filtered, washed
with petroleum ether (40o–60o), dried and crystallized from
EtOH.
1
1166 (vas and vs C–O–C); H NMR (DMSO-d6, 400 MHz)
δ/ppm: 10.42 (s, br, 1H, C2-NH), 9.37 (s, 1H, NH-Ar),
7.08–7.50 (m, 5H, Ar-H of thiazolidinone-N-phenyl), 6.89
(d, 2H, Ar-Hs, o- to NH-Ar, J = 8.68 Hz), 6.68 (d, 2H, Ar-
Hs, o- to OCH3, J = 7.64 Hz), 4.18 (d, 1H, thiazolidinone-
C5-HB, J = 7.68 Hz), 3.77 (d, 1H, thiazolidinone-C5-HA, J
= 7.6 Hz), 3.72 (s, 3H, OCH3), 2.43 (s, 3H, CH3); 13C NMR
(DMSO-d6, 101 MHz) δ/ppm: 172.8 (CN), 169.5 (C=O),
161.1 (C5), 160.6 (C2), 156.6 (C1 of thiazolidinone-N-
phenyl), 153.0 (thiazolidinone-C2), 148.1 (C6), 131.4 (C4),
129.8 (C1 of 4-methoxyaniline), 124.8 (C-2, C-6 of thia-
zolidinone-N-phenyl), 120.9 (C-2, C-6 of 4-methoxyani-
line), 116.5 (C-4 of phenyl), 114.1 (C-3, C-5 of
thiazolidinone-N-phenyl), 113.8 (C-3, C-5 of 4-methox-
yaniline), 82.2 (C4 of thiazolidinone-N-phenyl), 55.7
(OCH3), 30.4 (thiazolidinone-C5), 23.5 (CH3); EIMS: 445
(M+., 31), 240 (100), 239 (53), 225 (23), 197 (16), 196 (22),
135 (13), 108 (22), 92 (17), 77 (49); Anal. Calcd. for
C22H19N7O2S (445.50): C, 59.31; H, 4.30; N, 22.01. Found:
C, 59.48; H, 4.38; N, 22.19.
6-Methyl-4-(4-methoxyanilino)-2-(3-phenyl-4-(4-bromophe-
nyl)thiazol-2(3H)-ylidene hydrazinyl)pyrimidine-5-carboni-
trile (14a) Yield 22%; mp 178–180 °C; IR v/cm−1: 3320
(NH), 2215 (C≡N), 1636 (C=N), 1570 (C=C–Ar), 1230,
1
1114 (vas and vs C–O–C); H NMR (DMSO-d6, 400 MHz)
δ/ppm: 9.77, 9.60 (2s, 2 × 1H, C2-NH), 9.31, 8.84 (2s, 2 ×
1H, NH-Ar), 6.85–8.18 (hump, 26 H, Ar-Hs), 6.79, 6.77
(2s, 2 × 1H, thiazoline-H), 3.76, 3.73 (2s, 2 × 3H, 2 ×
OCH3), 2.34, 2.30 (2s, 2 × 3H, 2 × CH3); HRESIMS: M+ +
1 = 584.08429 (9), M+ + 3 = 586.07745 (8); calculated
mass = 583 (585); Anal. Calcd. for C28H22BrN7OS
(584.49): C, 57.54; H, 3.79; N, 16.77. Found: C, 57.70; H,
3.82; N, 16.89.
6-Methyl-4-(4-methoxyanilino)-2-(2-(4-oxo-3-(4-nitrophe-
nyl)thiazolidin-2-ylidene) hydrazinyl)pyrimidine-5-carboni-
trile (13b) Yield 20%; mp 203–205 °C; IR v/cm−1: 3399
(NH), 2213 (C≡N), 1740 (C=O), 1651 (C=N), 1585
(C=C–Ar), 1489, 1339 (vas and vs NO2), 124, 1176 (vas and
6-Methyl-4-(4-methoxyanilino)-2-(3-phenyl-4-(4-chlorophe-
nyl)thiazol-2(3H)-ylidene hydrazinyl)pyrimidine-5-carboni-
trile (14b) Yield 23%; mp 150–152 °C; IR v/cm−1: 3293
(NH), 2209 (C≡N), 1636 (C=N), 1573 (C=C–Ar), 1510,
1
1
vs C–O-C); H NMR (DMSO-d6, 400 MHz) δ/ppm: 10.54
1040 (vas and vs C–O–C); H NMR (DMSO-d6, 400 MHz)
(s, 1H, C2-NH), 9.44 (s, 1H, NH-Ar), 8.18 (d, 2H, Ar-Hs,
o- to NO2, J = 8.0 Hz), 7.41 (d, 2H, Ar-Hs, o- to NH-Ar, J
= 8.0 Hz), 6.94 (d, 2H, Ar-Hs, m- to NO2, J = 8 Hz), 6.85
(d, 2H, Ar-Hs, o- to OCH3, J = 8.0 Hz), 4.29 (d, 1H, thia-
zolidinone-C5-HB, J = 15.0 Hz), 3.85 (d, 1H, thiazolidi-
none-C5-HA, J = 16.0 Hz), 3.73 (s, 3H, OCH3), 2.43 (s, 3H,
CH3); 13C NMR (DMSO-d6, 101 MHz) δ/ppm: 171.3 (CN),
168.8 (C=O), 160.0 (C5), 159.8 (C4 of thiazolidinone-N-
nitrophenyl), 159.1 (C2), 156.6 (C1 of thiazolidinone-N-
δ/ppm: 9.77, 9.52 (2s, 2 × 1H, C2-NH), 9.38, 8.84 (2s, 2 ×
1H, NH-Ar), 6.74–7.67 (hump, 26 H, Ar-Hs), 6.50, 6.41
(2s, 2 × 1H, thiazoline-H), 3.76, 3.73 (2s, 2 × 3H, 2 ×
OCH3), 2.34, 2.29 (2s, 2 × 3H, 2 × CH3); 13C NMR
(DMSO-d6, 101 MHz) δ/ppm: 172.7, 172.0 (CN), 163.0,
162.2 (C5), 161.4, 160.9 (C2), 157.0, 156.7 (C6), 155.9,
155.7 (thiazoline-C2), 151.0, 150.9 (C4 of 4-chlorophenyl),
140.8, 140.6 (C1 of thiazoline-2-(or 3-)-phenyl), 133.4,
133.3 (C4), 133.2, 131.9 (C1 of 4-methoxyaniline), 130.0,

Medicinal Chemistry Research
129.5 (C-3, C-5 of 4-chlorophenyl), 129.4, 129.3 (C-2, C-6
of thiazoline-2-(or 3-)-phenyl), 128.5, 127.9 (C1 of 4-
chlorophenyl), 125.7, 125.6 (C-2, C-6 of 4-chlorophenyl),
123.8, 123.7 (C-2, C-6 of 4-methoxyaniline), 121.60, 121.5
(C-3, C-5 of thiazoline-2-(or 3-)-phenyl), 116.7, 116.4 (C4
of 4-methoxyaniline), 114.5, 114.1 (C4 of thiazoline-2-(or
3-)-phenyl), 113.9, 113.7 (C-3, C-5 of 4-methoxyaniline),
92.7, 91.5 (thiazoline-C4), 82.1, 81.3 (thiazoline-C5), 55.8,
55.7 (OCH3), 23.4 (CH3); HRESIMS: M+ + 1 = 540.1364
(100), M+ + 3 = 542.1340 (39) (calculated mass = 540.04);
Anal. Calcd. for C28H22ClN7OS (540.04): C, 62.27; H,
4.11; N, 18.16. Found: C, 62.41; H, 4.17; N, 18.34.
6-Methyl-4-(4-methoxyanilino)-2-(3-(4-nitrophenyl)-4-(4-
chlorophenyl)thiazol-2(3H)-ylidenehydrazinyl)pyrimidine-5-
carbonitrile (15b) Yield 25%; mp 185–186 °C; IR v/cm−1
3293 (NH), 2209 (C≡N), 1610 (C=N), 1573 (C=C–Ar),
1415, 1338 (vas and vs NO2), 1230, 1034 (vas and vs C–O–
:
1
C); H NMR (DMSO-d6, 400 MHz) δ/ppm: 9.86, 9.45 (2s,
2 × 1H, C2-NH), 9.41, 9.13 (2s, 2 × 1H, NH-Ar), 6.96–8.21
(hump, 24H, Ar-Hs), 6.50 (s, 1H, thiazoline-H), 3.73, 3.70
(2s, 2 × 3H, 2 × OCH3), 2.60, 2.30 (2s, 2 × 3H, 2 × CH3);
EIMS m/z: 586 (M+., 17), 584 (43), 449 (26), 448 (21), 447
(71), 417 (19), 416 (73), 401 (41), 391 (13), 389 (19), 331
(30), 286 (14), 285 (13), 284 (33), 255 (57), 254 (64), 240
(100), 239 (93), 225 (25), 224 (20), 218 (14), 214 (20), 211
(24), 197 (32), 170 (40), 168 (90), 139 (33), 134 (27), 133
(41), 108 (27), 90 (32); Anal. Calcd. for C28H21ClN8O3S
(584.11): C, 57.48; H 3.62; N, 19.15. Found: C, 57.62; H,
3.60; N, 19.38.
6-Methyl-4-(4-methoxyanilino)-2-(3-phenyl-4-(4-nitrophe-
nyl)-2(3H)-ylidene-hydrazinyl)pyrimidine-5-carbonitrile
(14c) Yield 25%; mp 166–167 °C; IR v/cm−1: 3411 (NH),
2213 (C≡N), 1636 (C=N), 1593 (C=C–Ar), 1415, 1347
1
(vas and vs NO2), 1237, 1114 (vas and vs C–O–C); H NMR
(DMSO-d6, 400 MHz) δ/ppm: 9.83, 9.43, (2s, 2 × 1H, C2-
NH), 9.29, 8.95 (2s, 2 × 1H, NH-Ar), 6.53–8.38 (hump, 28
H, thiazoline-H + Ar-Hs), 3.76, 3.74 (2s, 2 × 3H, 2 ×
OCH3), 2.35, 2.29 (2s, 2 × 3H, 2 × CH3); HRESIMS: M+ +
1 = 551.15948 (12) (calculated mass = 550.59); Anal.
Calcd. for C28H22N8O3S (550.59): C, 61.08; H, 4.03; N,
20.35. Found: C, 61.35; H, 4.11; N, 20.57.
6-Methyl-4-(4-methoxyanilino)-2-(3,4-(4-nitrophenyl)thia-
zol-2(3H)-ylidenehydrazinyl)pyrimidine-5-carbonitrile
(15c) Yield 26%; mp 190–191 °C; IR v/cm−1: 3411 (NH),
2213 (C≡N), 1636 (C=N), 1593 (C=C–Ar), 1509, 1347
(vas and vs NO2), 1237–1230, 1114–1034 (vas and vs C–O–
1
C); H NMR (DMSO-d6, 400 MHz) δ/ppm: 9.88, 9.73 (2s,
2 × 1H, C2-NH), 9.44, 9.21 (2s, 2 × 1H, NH-Ar), 6.97–8.24
(hump, 24H, Ar-Hs), 6.66 (s, 1H, thiazoline-H), 3.74, 3.72
(2s, 2 × 3H, 2 × OCH3), 2.33, 2.28 (2s, 2 × 3H, 2 × CH3);
Anal. Calcd. for C28H21N9O5S (595.59): C, 56.47; H, 3.55;
N, 21.17. Found: C, 56.59; H, 3.59; N, 21.39.
6-Methyl-4-(4-methoxyanilino)-2-(3-(4-nitrophenyl)-4-(4-
bromophenyl)thiazol-2(3H)-ylidenehydrazinyl)pyrimidine-
5-carbonitrile (15a) Yield 23%; mp 183–182 °C; IR v/cm−1:
3305 (NH), 2210 (C≡N), 1620 (C=N), 1580 (C=C–Ar),
1515, 1342 (vas and vs NO2), 1235, 1040 (vas and vs C–
O–C); 1H NMR (DMSO-d6, 400 MHz) δ/ppm: 10.67, 10.16
(2s, 2 × 1H, C2-NH), 9.41, 9.13 (2s, 2 × 1H, NH-Ar), 6.88–
8.19 (hump, 24H, Ar-Hs), 6.70, 6.45 (2s, 2 × 1H, thiazoline-
H), 3.76, 3.74 (2s, 2 × 3H, 2 × OCH3), 2.34, 2.29 (2s, 2 ×
3H, 2 × CH3); 13C NMR (DMSO-d6, 101 MHz) δ/ppm:
172.5, 172.1 (CN), 162.6, 161.9 (C5), 161.5, 161.1 (C2),
157.5, 157.3 (C6), 157.0, 156.9 (thiazoline-C2), 143.5,
143.3 (C4 of 4-bromophenyl), 140.9, 140.5 (C4 of thiazo-
line-2-(or 3-)-4-nitrophenyl), 131.7, 131.5 (C4), 130.9,
130.7 (C1 of 4-methoxyaniline), 129.8, 129.6 (C-3, C-5 of
thiazoline-2-(or 3-)-4-nitrophenyl), 126.1, 125.8 (C-3, C-5
of 4-bromophenyl), 125.6, 125.5 (C1 of 4-bromophenyl),
122.5, 122.4 (C-2, C-6 of thiazoline-2-(or 3-)-4-nitrophe-
nyl), 122.3, 122.2 (C-2, C-6 of 4-bromophenyl), 122.1,
121.8 (C-2, C-6 of 4-methoxyaniline), 116.7, 114.0 (C4 of
4-methoxyaniline), 114.5, 114.1 (C1 of thiazoline-2-(or
3-)-4-nitrophenyl), 114.0, 113.8 (C-3, C-5 of 4-methox-
yaniline), 93.3, 92.0 (thiazoline-C4), 82.0, 81.4 (thiazoline-
C5), 55.8, 55.7 (OCH3), 23.4 (CH3); Anal. Calcd. for
C28H21BrN8O3S (629.49): C, 53.42; H, 3.36; N, 17.80.
Found: C, 53.69; H, 3.34; N, 18.02.
Pharmacological studies
Drugs, chemicals, and materials
Nifedipine was obtained from Amriya Pharmaceutical
Industries, Alexandria-Cairo Desert Road Km 25, Amriya,
Alexandria, Egypt. Other chemicals such as pentobarbital
sodium, heparin, dimethylsulfoxide (DMSO), freshly mixed
heparinized saline, physiological solutions were of analy-
tical grade. i.v. Catheters were of 20GA, 22 GA, 24 GA
calibers.
Animals
White New Zealand rabbits (2–2.5 kg) of either sex were
obtained from Animal House, Faculty of Medicine, Assiut
University. Rabbits were kept in cages in Faculty of Med-
icine, Al-Azhar University, Assiut Branch, Assiut, housed
at ordinary RT, exposed to natural daily light-dark cycles,
fed with standard laboratory diet and allowed for tap H2O
ad libitum.

Medicinal Chemistry Research
In vivo experiments
concentration and 10 min exposure time. Between admin-
istrations of the individual substances, the preparation was
washed until the initial situation had been re-established and
the potassium chloride contractions were induced. The
contractions were enrolled by FTO3 force–displacement
transducer, and recorded on a polygraph (Kaur and Kaur
2013; Girgis et al. 2006, 2008). When response reached its
plateau, response of aorta rings to solutions of 5a, 5b, 9b,
and 9c was done and each ring was serially washed after
obtaining the maximum response to reach the baseline and
equilibrated. At this point, another compound solution was
added.
Forty-two normotensive white New Zealand rabbits (2–2.5
kg) of either sex were used for this assay and six rabbits
were used for each group.
In vivo assessment: hypotensive activity on
normotensive anesthetized rabbits
Normotensive rabbits were anesthetized with pentobarbital
sodium (30 mg/kg, i.v.). They were placed on their backs on
the operating table while legs were fixed and heads pinned
down. Trachea were exposed and cut and plastic cannula
were inserted and firmly tied in place. The common carotid
artery lying in the neck between the lateral bundle of muscle
(longus capitis) and the trachea was exposed by separating
the two muscle bundles of the sternomastoid and the ster-
nothyroid. The carotid artery was separated from the nerves
and veins and carefully freed of connective tissue. A venous
cannula filled with heparin to prevent thrombus formations
was carefully inserted and a thread was tied on it. The
cannula was connected to a pressure transducer via uni-
versal oscillograph (Harvard apparatus, ser. No. K10542)
for blood pressure records. On the opposite side, a venous
cannula was inserted into the external jugular vein and tied
with thread for injection of 29 tested compounds followed
by heparinized saline (0.90% w/v NaCl). The mean arterial
blood pressure was calculated using the formula: MAP =
DBP + 1/3 (SBP − DBP), where MDP = mean arterial
blood pressure, DBP = diastolic blood pressure, and SBP =
systolic blood pressure.
Effect of test compounds on the rabbits heart rates
Hearts of rabbits were prepared according to the previously
reported method (Spáčilová 2007; Burn 1952). Aorta was
dissected off, freed from all other connecting vessels,
carefully tied and cut. Threads were attached to the ventricle
by means of a small clip and connected to a special trans-
ducer via universal oscillograph (Harvard apparatus, ser.
No. K10542) for heart activity records. Test compounds 5a,
5b, 9b, and 9c were added to the cannulae containing the
perfusion fluid and entered aorta to reach the coronaries.
Aorta was tied into the glass cannula of the perfusion
apparatus. Oxygenated Locke solution at 37 °C was allowed
in from a reservoir maintained at a constant pressure. That
keeps the aortic valve closed while the fluid can pass
through the coronary vessels and exit from the inferior vena
cava to be collected in a container (Spáčilová 2007; Burn
1952).
In vitro experiments
In vitro preliminary screening of eNOS
activation in aorta and histopathological
changes
Seven white New Zealand rabbits (2–2.5 kg) of either sex
were starved and allowed free access to H2O for 24 h prior
to experiments then slaughtered at the day of experiment to
isolate the heart and aorta.
Materials and method
Calcium channel blockade activity
Chemicals
Calcium channel blockade activity procedure was carried
out in accordance to the previously reported technique
(Kaur and Kaur 2013; Girgis et al. 2006, 2008). Com-
pounds 5a, 5b, 9b, and 9c which elicited high hypotensive
activity were used for assessment of the calcium channel
blockade activity. In order to check for antagonistic effects,
contractions were induced with potassium chloride (67
mmol/L). After thorough washing out, this process was
repeated until the amplitude of the contraction became
constant. The substances to be tested were investigated
using the single-dose technique. KCl contractions were
Endothelial nitric oxide synthase antibody (eNOS) was
purchased from Thermo Scientific Co.
Animals
Eight adult male rats weighing about 220–250 g were used
in the present work. They were purchased from Assiut
University Animal Breeding Unit. Animals were kept in a
controlled light room with photoperiod of 12 h dark and 12
h light (dark–light cycle 12:12) with lights ON from 6:00 to
18:00 h at a temperature of 28 + 2 °C. All animals were
given free access to standard laboratory chow and tap water.
induced after addition of the substances at 10−4
M

Medicinal Chemistry Research
Rats were randomly divided into 4 groups, 2 rats each.
Group (I) served as control group and used for hematoxylin
and eosin staining and eNOS immunostaining while Group
(II) and (III) received compounds 5a and 5b respectively by
intra-peritoneal injection in a dose of 30 mg/kg body weight
for eNOS immunostaining. Finally, Group (IV) served as
positive control and received L-NAME an inhibitor for
eNOS synthesis in a dose of 30 mg/kg body weight by intra-
peritoneal injection for eNOS immunostaining.
described (Scheme 1). 2-Methylthio derivatives 2a,b were
prepared by heating a mixture of compounds 1a or 1b
(Jones 1952), S-methylthiourea sulfate and anhydrous
AcONa in DMF. Heating compounds 2a,b with 2.5 molar
equivalents of POCl3 at RT in presence of N,N-dimethyla-
niline furnished compounds 3a,b. This modified method
was better than the reported one (Ding et al. 1987) avoiding
the use of excess of POCl3. Compounds 4a,b were prepared
by heating under reflux the corresponding 4-chloro deriva-
tives 3a,b with 4-methoxyaniline in EtOH in presence of
K2CO3 (Atwal et al. 1987). Hydrazine hydrate was allowed
to react with the S-methyl derivatives 4a,b, where upon the
good leaving sulfanyl group was successfully displaced
(Spáčilová et al. 2007) furnishing the 2-hydrazinyl deriva-
tives 5a,b (Scheme 1).
Tissue preparation
Animals were anesthetized with ether, fixed by intracardiac
perfusion with 10% formaldehyde. Aorta specimens were
rapidly dissected immediately, and immersed into 10%
formaldehyde solution to continue fixation 2 more days (pH
7.2). Specimens were processed to prepare paraffin blocks
through dehydration in ascending series of EtOH, cleared in
methyl benzoate and embedded in paraffin wax. Paraffin
sections (5 μm) were cut using a microtome mounted on
glass slides, for histological examination with hematoxylin–
eosin stain and immunohistochemical staining.
It was of interest to extend the scope of such valuable
transformation allowing the introduction of considerable
functionalities at C2 of Biginelli derived pyrimidine core. In
this context, hydrazine derivatives 5a,b reacted with dif-
ferent reagents to install a variety of heterocycles linked
with pyrimidine system as illustrated in Schemes 2 and 3.
Addition of phenacyl bromides to the key intermediate 5a in
EtOH furnished the desired compounds 6a–c, Scheme 2.
Triazolopyrimidine derivatives 7a–f were successfully pre-
pared by heating the hydrazines 5a,b with aliphatic car-
boxylic acids where in situ cyclization occurred. In case of
the less reactive benzoic acid, POCl3 was necessary to
catalyze the reaction, Scheme 2. Compounds 8a,b and 10a,
b were obtained in a fairly good yield upon treatment of the
hydrazines 5a,b with appropriate acceptors as 2-(1-ethox-
yethylene)malononitrile (Jones 1952) or 2-(ethox-
ymethylene)malononitrile (Ding et al. 1987), in boiling
EtOH, Scheme 2. Heating under reflux compounds 10a,b
with excess formamide gave the corresponding 2-4-amino-
1H-pyrazolo[3,4-d]pyrimidine derivatives 11a,b, Scheme 2.
A mixture of the hydrazine derivatives 5a,b and ethyl 2-
cyano-3-arylacrylates (Hussain et al. 1985) were reacted in
boiling EtOH, however, unlike previously evidenced, the
second attack of N1 of hydrazine took place on the carbonyl
ester rather on the nitrile C giving rise to the postulated
pyrazolonenitrile product 9a–d instead of the pyrazole ester
initially reported in literature (Aggarwal et al. 2011; Bakr
and Kamal 2012). Confirmatory evidence for structures 9a–
Histological and histopathological examinations
Immunohistochemistry
For eNOS immunohistochemistry: After fixation in 10%
neutral formalin for 2 days, dehydration, clearing, and
embedding in paraffin soon followed. Paraffin sections were
cut at 5 μm and stained with avidin–biotin peroxidase
technique. Sections underwent deparaffinization and rehy-
dration by descending grades of EtOH (100, 90, and 70%).
They were treated with 0.01 M citrate buffer (pH 6.0) for
10 min to unmask antigen and incubated in 0.3% H2O2 for
30 min to abolish endogenous peroxidase activity before
blocking with 5% horse serum for 1–2 h. Slides were
incubated with the primary antibody (1:100 monoclonal
mouse anti-eNOS) at 4 °C for 18–20 h, then washed and
incubated with biotinylated secondary antibodies and then
with avidin–biotin complex. Finally, sections were devel-
oped with diaminobenzidine (DAB) chromogen. Slides
were counter stained with Mayer's hematoxylin, dehy-
drated, cleared, and mounted. eNOS positive cells appeared
with brown cytoplasm while nuclei appeared blue.
1
d was proved by spectral data (IR, H NMR and 13C NMR
and MS).
Under neutral conditions, the hydrazine derivatives 5a,b
were allowed to react with phenyl or p-nitrophenyl iso-
thiocyanate affording products 12a,b in good yields
(Scheme 3). Condensation of 12a,b with ethyl bromoacetate
in boiling EtOH containing anhydrous NaOAc afforded the
corresponding thiazolidinone derivatives 13a,b, Scheme 3.
It was postulated that reaction proceeded via S-alkylation
followed by elimination of H2O and cyclization. Reacting
Results and discussion
Chemistry
The designed pyrimidine derivatives were obtained through
the synthesis of hydrazinyl pyrimidine derivatives 5a,b as

Medicinal Chemistry Research
Scheme 2 Synthetic pathways
to pyrimidotriazines 6a–c,
triazolopyrimidines 7a–f,
pyrazolylpyrimidines 8a,b, 9a–
d, and 10a,b and
pyrazolopyrimidinylpyrimidines
11a,b
Scheme 3 Synthetic pathways
to thiosemicarbazide derivatives
12a,b, thiazolidinone derivatives
13a,b, thiazoline derivatives
14a–c and 15a–c
12a,b with various phenacyl bromides provided the thia-
zoline derivatives 14a–c and 15a–c. However, 2 positional
isomers might be possible (Bonde and Gaikwad 2004). H
Pharmacological screening: calcium channel
blockade activity
1
NMR analysis confirmed the presence of a mixture of the 2
1
isomers as indicated by duplication of many of H and 13C
NMR signals.
Several in vitro and in vivo techniques are available for
evaluating calcium channel blocking activity (Vogel 2008).

Medicinal Chemistry Research
Table 1 Effect of nifedipine and selected test compounds (mg/kg, i.v.)
in normotensive anesthetized rabbits represented by change in MAP
(mmHg)
100
80
60
40
20
0
Cpd No.
ABP (mmHg) as
mean SE
Cpd No. ABP (mmHg) as
mean SE
Normal
92.2 0.86
9c
51.8 0.37*#
56 0.70*#
91.6 0.81#
71.8 0.37*#
91.8 0.66#
78.2 0.66*#
92.0 0.54#
Nifedipine 84.2 0.58*
9d
5a
5b
7b
7f
51.4 0.74*#
51.4 0.6*#
91.6 0.81#
91.4 0.8#
66.6 0.55*#
55.2 0.86*#
10c
11b
14c
15b
15c
Fig. 4 Effect of nifedipine and compounds 5a, 5b, 8b and 9b–d, 11b
on MABP
8b
9b
51.4–78.2 mmHg in rabbits in comparison with nifedipine-
treated rabbits. Among the compounds with hypotensive
effects, we selected compounds 5a, 5b, 9b, and 9c for
further assessment as CCBs.
Each value represents the mean SE (standard error) of 6 experiments
Nifedipine (0.01 M) caused 8 mmHg drop in arterial blood pressure
Results were significant at P < 0.01 according to Tukey–Kramer test
*Significant difference from the normal rabbits (P < 0.01)
Calcium channel blockade activity
#Significant difference from rabbits treated with nifedipine (P < 0.01)
As previously reported by other investigators (Zorkun et al.
2006), smooth muscles depend on calcium influx for con-
traction and although their underlying mechanism is
somewhat different, inhibition of calcium channel influx
into smooth muscles by CCBs leads to relaxation. There-
fore, compounds 5a, 5b, 9b, and 9c which elicited high
hypotensive activity were further evaluated for their calcium
channel blockade activity as indicated by their effects on
KCl-stimulated rabbit aortae. Nifedipine was included in all
tests as the reference drug (Table 2 and Fig. 5). Results
obtained from nifedipine, 5a, 5b, 9b, and 9c, indicated that
the relaxant response of the aorta of the rabbits treated with
test compounds increased significantly (P < 0.01) in com-
parison to nifedipine-treated rabbits aortae, as shown in
Table 2 and Fig. 5. As shown, the maximum relaxation
values of all tested compounds were much more superior
when compared with that of nifedipine. In particular, the
hydrazine derivatives 5a and 5b exhibited the highest
activities showing 89.2% relaxation while all other tested
compounds 9b and 9c showed 77–74.4% relation in com-
parison with nifedipine (57.6% relaxation).
In vitro studies are considered rapid useful tools for primary
screening and various smooth muscle preparations such as
rabbit jejunum (Taqvi et al. 2006), rat ileum (Taqvi et al.
2006), rat colon (Hadizadeh et al. 2008), and guinea pig
trachea (Yu et al. 1994) were utilized as testing models.
Moreover, whole animal experiments provide reliable
assessment of their various in vivo effects. For evaluation of
their cardiovascular activity, the main therapeutic value of
CCBs, different models were employed, of which mon-
itoring their effect on arterial blood pressure in hypertensive
or normotensive dogs (Guarneri et al. 1997) or rabbits
(Jeanneau et al. 1992).
In the current pharmacological assessment, three phases
were established for in vitro and in vivo evaluation of the
newly synthesized compounds: (1) Thirteen selected com-
pounds were investigated for preliminary evaluation of
hypotensive activity in normotensive anesthetized rabbits.
(2) Active compounds from the previous screening were
tested for calcium channel blockade on preparations of
rabbit aortae. (3) Further, the same active compounds were
evaluated for their effect on the rabbits heart rate.
Effect of nifedipine and compounds 5a, 5b, 9b, and
9c on heart rates
Effect of tested compounds on mean arterial blood
pressure (MABP)
Active candidates that elicited significant lowering of blood
pressure in normotensive rabbits in particular compounds
5a, 5b, 9b, and 9c were further evaluated for their effects on
heart rates. Results of this study showed that, there is no
significant difference (P > 0.05) between heart rates of
nifedipine-treated rabbits and normal rabbits, but there were
a significant difference (P < 0.01) on the heart rates between
normal and nifedipine treated rabbits from one side and
rabbits treated with test compounds from the other side. The
Preliminary results of biological evaluation for synthesized
compounds on mean arterial blood pressure (MABP) were
examined and presented in Table 1 and Fig. 4. Among the
tested compounds, results showed that MABP in rabbits
treated with nifedipine decreased (84.2 0.58 mmHg) sig-
nificantly (P < 0.01) in comparison with normal rabbits
(92.2 0.86 mmHg). Compounds 5a, 5b, 8b, 9b–d, 11b,
and 15b showed a decrease in MABP in the range

Medicinal Chemistry Research
Table 2 Effect of nifedipine and
compounds 5a, 5b, 9b, and 9c
on the relaxant response of
rabbit aortae
Groups
Nifedipine
57.6 3.20
5a
5b
9b
9c
Relaxation%
89.2 2.4#
89.2 2.41#
77.4 3.12#
77.2 3#
Each value represents the mean SE (standard error) of 6 experiments
#Significant difference from rabbits treated with nifedipine (P < 0.01)
endothelium-derived NO may be involved in preventing the
progression of age-related vascular diseases (Moncada and
Higgs 2006, Pacher et al. 2007, Vanhoutte 2009; Vanhoutte
et al. 2009).
Immuno-staining of the aortic sections of the four groups
showed the followings changes.
100
80
60
40
20
0
Light microscopic examination of aortic section of the
control rat group stained with hematoxylin and eosin
showed the three layers of the aortic wall (Fig. 7). Control
group showed mild immune-positive staining in the aortic
endothelial cells of the intima and very few in adventitia
(Fig. 8). In this study, compounds 5a and 5b were evaluated
upon modulation blood pressure in an animal model of
hypertension through its effect upon eNOS expression in
the endothelium of aorta. Compounds 5a and 5b showed
dense staining of the aortic endothelium in comparison to
control group (Figs 9 and 10) respectively. The adventitia of
5a and 5b treated groups had immune-positive staining in
the endothelial lining of blood vessels but seems to be more
intense in the phenyl 5b treated group (Figs 9 and 10).
Compound 5b induced increase in eNOS expression in the
aorta as confirmed histologically in this study (Fig. 10).
Intense eNOS expression localized to the aortic endothe-
lium was observed with compound 5b more than compound
5a (Fig. 9). L-NAME (Nω-Nitro-L-arginine methyl ester
hydrochloride) treated group as inhibitor of eNOS (positive
control) showed no immunostaining of eNOS in the endo-
thelial cells or other parts of the aorta (Fig. 11).
Fig. 5 Effect of nifedipine and compounds 5a, 5b, 9b, and 9c on the
relaxant response of rabbit aortae
effect of the test compounds on rabbit heart rate indicated
that all compounds possessed a significant mean percentage
decrease on rabbit's heart rate as shown in Table 3 and
Fig. 6.
Correlation between in vivo hypotensive activity in
normotensive anesthetized rabbits (Table 1) and in vitro
calcium channel blockade activity as indicated by relaxant
response of rabbit aortae (Table 2) indicated that com-
pounds 5a, 5b, 9b, 9c exhibited promising lowering on
MABP, calcium channel blocking activity accompanied by
decrease in heart rate while compounds 8b, 8d elicited good
lowering on MABP. In addition, compounds 7b, 8a, 11b,
15b showed moderate lowering on MABP. Such com-
pounds could be further investigated to constitute a special
class of compounds for treatment of hypotension. Mean-
while, 6c, 7b, 7f, 14c, 15b, 15c could be considered as
inactive compounds.
Structure–activity relationship (SAR) studies
As can be seen in the most active compounds, 5a, 5b, the
most favorable structural elements contributing favorably to
binding –NHNH2 is capable of acting as H-bond acceptor/
donor bound in a complementary region of the receptor
containing amino acid residues with opposite features.
Compound bearing a C6-methyl group 5a exhibited a slight
increase in inhibition activity. These results implied that
electron donating groups at this position seemed to be
beneficial to inhibitory activity (more than phenyl group).
Thus, it was reasonable to presume that size of substituent at
C6 is important to the activity of the target compounds.
Besides, the promising activity of some compounds con-
taining attached heterocyclic pyrazoline ring as 9b, 9c
indicates that, while steric effects are important character-
istics for activity, electronic and lipophilic effects are
Histopathological studies
The endothelium of blood vessels including aorta plays a
modulator role in the basal and dynamic regulation of blood
vessel diameter by releasing endothelium-derived nitric
oxide (NO) (Zhao et al. 2015). In the blood vessel wall,
eNOS has a protective function in the cardiovascular system
attributable to NO production. Once NO is produced in
endothelial cells, it diffuses across the vascular smooth
muscle cell membranes and, through a special mechanism,
promotes vascular relaxation (Zhao et al. 2013). In addition,

Medicinal Chemistry Research
Table 3 Effect of nifedipine and
compounds 5a, 5b, 9b, and 9c
on heart rates
Groups
Normal
Nifedipine
74.6 1.07
5a
5b
9b
9c
Heart rate/min
76 1.30
54 0.70*#
52 0.70*#
61.2 0.58*#
60 0.70*#
Each value represents the mean SE (standard error) of 6 experiments
*Significant difference from the normal rabbits (P < 0.01)
#Significant difference from rabbits treated with nifedipine (P < 0.01)
80
70
60
50
40
30
20
10
0
Fig. 6 Effect of nifedipine and compounds 5a, 5b, 9b, and 9c on the
heart rates
Fig. 8 Photomicrograph of a control adult rat aorta immunostained
with anti-eNOS, a marker for e-NOS enzyme, showing immuno-
positive staining of the endothelium (eNOS immunostaining ×400).
Inset: few immune-positive stained endothelial cells of adventitial
blood vessels (↑) (eNOS immunostaining ×1000)
Fig. 7 Photomicrograph of a control adult rat aorta showing intimal
endothelium (E) layer, the media (M), and the adventitia (A). H&E
(400)
consistent also to contribute well for activity as the het-
erocyclic moiety might play a favorable role in overall
binding and therefore in recognition area on the receptor.
Such compounds, bearing a non-chiral planar pyrimidine
ring would adopt the same total volume and polar surface
area present in similar DHPs and DHPMs known to exhibit
antihypertensive activity and indicated that introducing
polar substituents at this position (NH) near the vicinity of
C4 may lead to even better activity. Since all compounds
have a common scaffold in their structures, observed SAR
is mainly due to the variation of other functionalities on this
Fig. 9 Photomicrograph of adult rat aorta treated with 5a and immu-
nostained with anti-eNOS showing immune-positive staining of
endothelium (E) more or less similar to control rat section (eNOS
immune-staining ×400). Inset: more immune-positive stained endo-
thelial cells of adventitial blood vessels (brown color) (eNOS immu-
nostaining ×1000)
scaffold. Variation of the tail on the main scaffold at C2 is
responsible for variation in activity potential although all
structural features are taking part in the activity. Derivatives
including versatile functionalities at position 2 varied

Medicinal Chemistry Research
activity. Correlating the significant hypotensive and vaso-
dilating activities of compounds 5a,b bearing a hydrazine
moiety at C2, led to the investigation of the mechanism of
vasodilatation of other antihypertensive hydrazines. In
particular, Hydralazine (Apresoline®), has been in clinical
use as an anti-hypertensive agent for nearly 5 decades, but
its mechanism of action remained poorly understood. It
causes vasodilation as a result of a direct action on vascular
smooth muscle cells at an intracellular site to cause vasor-
elaxation (Ellershaw and Gurney 2001). It was previously
shown to be a directly acting vasodilator, evoking a slowly
developing, long-lived relaxation in rabbit aorta rings. It
causes vasodilation as a result of a direct action on vascular
smooth muscle cells at an intracellular site to cause vasor-
elaxation (Ellershaw and Gurney 2001). It does not act as a
calcium antagonist, because it is a poor inhibitor of vaso-
constriction caused by elevated extracellular K+ (Khayyal
et al. 1981; Higashio and Kuroda 1988; Gurney and Allam
1995) and its effectiveness at inhibiting phenylephrine-
induced contraction is little affected by removing extra-
cellular Ca2+ (Higashio and Kuroda 1988; Gurney and
Allam 1995; Orallo et al. 1991).
Fig. 10 Photomicrograph of adult rat aorta-treated with 5b and
immunostained with anti-eNOS showing dense immune-positive
staining of endothelium (E) than control rat section (eNOS immu-
nostaining ×400). Inset: dense immune-positive stained endothelial
cells of adventitial blood vessels (brown color) (eNOS immunostain-
ing ×1000)
Conclusion
In summary, the present work is an extension of an ongoing
effort toward the development and identification of new
molecules with antihypertensive activity. In the present
investigation, bioisosterism, and hybrid pharmacophore-
based drug design led to the identification of multi-
functional non-chiral C4-pyrimidine derivatives with anti-
hypertensive potency. Compounds 5a, 5b, 9b, 9c exhibited
promising lowering on MABP, calcium channel blocking
activity, and decrease in heart rate. It could be concluded
clearly that, synthesized pyrimidine derivatives containing
C5-CN, C6-methyl (or phenyl) and C4-p-methoxyanilino
moieties, showed variable activities. The impact of struc-
tural variation on activity was examined leading to that
changes in C5-, C6-, and C4-positions could be important in
receptor binding.
Fig. 11 Photomicrograph of adult rat aorta; a positive control (treated
with L-NAME drug) immunostained with anti-eNOS, a marker for e-
NOS, showing no immuno-staining (eNOS immunostaining ×400)
between simple flexible hydrazine derivatives, planar or
non-planar aromatic heterocycles, and fused ring systems.
Variation of substituents on C2-pyrimidine ring can achieve
a host of biological actions, through subtle electron density
changes in the substituent framework. Other factors
including absorption, transport, and site of intracellular
action might also be of importance.
This study also demonstrates that compounds 5a and 5b
are NO-mediated vascular relaxation via aortic eNOS acti-
vation. This may lead to decreased BP in a hypertensive
animal model. The 2 compounds exhibited excellent
bioactivity profiles indicating that they might be multi-
functional lead compounds for the purpose of improving
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Ethics approval All procedures performed involving animals were in
accordance with the ethical standards. The experimental protocol was
approved by Animal Care and Use Committee, Faculty of Pharmacy,
Alexandria University (ACUC Project Number 16/1).
Publisher’s note: Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.

Medicinal Chemistry Research
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