Design, synthesis and biological evaluation of new 2-aminothiazole scaffolds as phosphodiesterase type 5 regulators and COX-1/COX-2 inhibitors

Article abstract of DOI:10.1039/d0ra05561a

A new series of 2-aminothiazole derivatives was designed and prepared as phosphodiesterase type 5 (PDE5) regulators and COX-1/COX-2 inhibitors. The screening of the synthesized compounds for PDE5 activity was carried out using sildenafil as a reference drug. Strikingly, compounds 23a and 23c were found to have a complete inhibitory effect on PDE5 (100%) at 10 μM without causing hypotension and the limited side effect of PDE5 inhibitors, suggest a distinctive therapeutic role of these derivatives in erectile dysfunction. On the other hand, compounds 5a, 17, 21 and 23b increased the PDE5 activity (PDE5 enhancers) at 10 μM. In addition, the study includes the screening of the COX-1/COX-2 inhibition induced by the synthesized compounds. All tested compounds have an inhibitory effect against COX-1 activity (IC50 = 1.00-6.34 μM range) and COX-2 activity (IC50 = 0.09-0.71 μM range). Moreover, a molecular docking study was implemented to reveal the binding interactions of potent compounds in the binding sites of PDE5 (PDB ID 2H42), COX-1 and COX-2 (PDB ID 3LN1) enzymes. For the interaction with the PDE5 enzyme, activator compounds had a strong binding mode (HB with Gln817:A) than inhibitory derivatives. Both types of compounds are considered as PDE5 regulators. This novel finding will encourage us to discover a new pharmacological application of small chemical entities as the PDE5 enhancer, or will lower side effects as PDE5 inhibitors. All active compounds adopted the Y-shape along the COX-2 active site. This journal is

Full text of DOI:10.1039/d0ra05561a

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Design, synthesis and biological evaluation of new
2-aminothiazole scaffolds as phosphodiesterase
type 5 regulators and COX-1/COX-2 inhibitors†
Cite this: RSC Adv., 2020, 10, 29723
a
Abdel Haleem M. Hussein,a Ahmed A. Khames,a Abu-Bakr A. El-Adasy,
Ahmed A. Atalla,a Mohamed Abdel-Rady,b Mohamed I. A. Hassan,c
Mohamed T. M. Nemrd and Yaseen A. A. M. Elshaier
*
e
A new series of 2-aminothiazole derivatives was designed and prepared as phosphodiesterase type 5 (PDE5)
regulators and COX-1/COX-2 inhibitors. The screening of the synthesized compounds for PDE5 activity was
carried out using sildenafil as a reference drug. Strikingly, compounds 23a and 23c were found to have
a complete inhibitory effect on PDE5 (100%) at 10 mM without causing hypotension and the limited side
effect of PDE5 inhibitors, suggest
a distinctive therapeutic role of these derivatives in erectile
dysfunction. On the other hand, compounds 5a, 17, 21 and 23b increased the PDE5 activity (PDE5
enhancers) at 10 mM. In addition, the study includes the screening of the COX-1/COX-2 inhibition
induced by the synthesized compounds. All tested compounds have an inhibitory effect against COX-1
activity (IC50 ¼ 1.00–6.34 mM range) and COX-2 activity (IC50 ¼ 0.09–0.71 mM range). Moreover,
a molecular docking study was implemented to reveal the binding interactions of potent compounds in
the binding sites of PDE5 (PDB ID 2H42), COX-1 and COX-2 (PDB ID 3LN1) enzymes. For the interaction
with the PDE5 enzyme, activator compounds had a strong binding mode (HB with Gln817:A) than
inhibitory derivatives. Both types of compounds are considered as PDE5 regulators. This novel finding
will encourage us to discover a new pharmacological application of small chemical entities as the PDE5
enhancer, or will lower side effects as PDE5 inhibitors. All active compounds adopted the Y-shape along
the COX-2 active site.
Received 25th June 2020
Accepted 23rd July 2020
DOI: 10.1039/d0ra05561a
rsc.li/rsc-advances
that chronic inammation is the main factor in benign prostatic
hyperplasia (BPH) progression. The use of anti-inammatory
1. Introduction
agents such as steroids and cyclooxygenase-2 (COX-2) inhibitors
have been reported as treatment for prostate diseases.3 It has been
reported that PDE5 inhibitors (PDE5-Is) relax the smooth muscle
bers of the bladder and prostate by NO/cGMPc signaling, and
then reduce the hyperactivity of the autonomic nervous system.4 As
a result, we can say that phosphodiesterase-5 inhibitors can be
used to treat prostatic inammation. The literature survey revealed
that sildenal (Fig. 1) (as a PDE5 inhibitor) has been shown to be
benecial in the early phases of inammation and vascular
remodeling in a pulmonary arterial hypertension (PAH). The
administration of sildenal signicantly reduced the inamma-
tion in the acute stage of the disease and prevented pulmonary
arterial remodeling. These results revealed that sildenal has
a direct anti-inammatory effect, in addition to its vasodilatation
and anti-proliferative effects.5 The anti-inammatory effect of
PDE5 inhibitors was conrmed by another study in the rat model
of colitis.6 On the other hand, PDE activators (biochemicals that
activate PDE) have many applications in biochemical and physio-
logical research. For example, calmodulin (a multifunctional
intermediate calcium-binding messenger protein expressed in all
eukaryotic cells) is an activator of NOS, PDE and CaMK.7 Another
Phosphodiesterase 5 inhibitors and the COX activity have recently
emerged and the phosphodiesterase type 5 inhibitors (PDE5-Is)
have been reported as potential therapy for neuroinammatory
and neurodegenerative diseases.1 PDE5-Is produce an anti-
inammatory and neuroprotective action by enhancing the
expression of nitric oxide synthases and in the accumulation of
cGMP, in addition to activating protein kinase G (PKG), which play
an important role in the development of several neurodiseases,
such as Alzheimer's disease (AD), Parkinson's disease (PD), and
multiple sclerosis (MS).2 Moreover, clinical experiments indicate
aDepartment of Chemistry, Faculty of Science, Al-Azhar University, Assiut 71524, Egypt.
E-mail: yaseen.elshaier@fop.usc.edu.eg
bDepartment of Chemistry, Faculty of Science, Assiut University, Assiut 71516, Egypt
cDepartment of Pharmacology & Toxicology, Faculty of Pharmacy, Al-Azhar University,
Assiut Branch, Assiut, 71524 Egypt
dPharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Cairo
University, Kasr El-Eini Street 11562, Cairo, Egypt
eOrganic & Medicinal Chemistry Department, Faculty of Pharmacy, University of Sadat
City, Menoua, 32958, Egypt
† Electronic supplementary information (ESI) available. See DOI:
10.1039/d0ra05561a
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example of PDE activators is UCR1C, a novel activator of PDE4 long suggested. In the comparison of 23b with celecoxib (also illus-
isoforms, where PDE4 activation by UCR1C attenuates car- trated), the replacement of methyl group with aryl moiety is
diomyocyte hypertrophy.8 Regulatory molecules are chemical suggested.
entities that either increase or inhibit enzyme activity. Compounds
Finally, we hypothesize the synthesis of new derivatives of
that enhance the activity of an enzyme are called activators, while aminothiazole, especially with the general formula in Fig. 2.
those that decrease the activity of an enzyme are called inhibitors.
Different modications in sildenal pharmacophoric
features were performed in our previous studies for the design
of new pyrazolo[3,4-d]pyrimidin-4-(5H)-one derivatives as
inhibitors of phosphodiesterase enzyme type 5 (PDE5) as sil-
denal analogues.9,10 Most of the explored tested compounds
2. Results and discussion
2.1. Chemistry
As we previously reported,11–14 3-oxobutanamide scan be utilized
as
a precursor in the synthesis of several heterocyclic
showed the same binding mechanism as vardenal, and some
of these compounds showed promising activity in comparison
to sildenal in the pharmacological studies (Fig. 1). The results
indicated that compounds with a styryl moiety or those with
a longer chain alkyl group are the most active derivatives.9,10
Based on these biological interests, in addition to our
successful synthesis of fused moieties using 3-oxobutanamide
derivatives as starting materials,11–14 it was of interest to prepare
a series of 2-aminothiazole derivatives (2, 5a,b, 7, 11, 13, 16–19,
22, 23a–c) using 3-oxobutanamides as a precursor. We investi-
gated their PDE5 regulatory activity, in addition to the COX-1
and COX-2 inhibitory activity (Fig. 2). Furthermore, a docking
study was carried out for all synthesized compounds to predict
their binding mode with the PDE5 compared to sildenal and
avanal (Fig. 1) as standard PDE5 inhibitors. In the case of COX-
1 and COX-2, the binding mode of the compounds was
compared to celecoxib as the COX inhibitor (Fig. 1).
substances. In the current work, we explored the reaction of 3-
oxobutanamides 1 with some electrophilic and nucleophilic
reagents. Thus, the treatment of 1 with sodium nitrite in acetic
ꢀ
acid at 0 C afforded the 2-hydroxyimino-3-oxo-N-thiazole-2-yl-
butyramide 2.15 The structure of compound 2 was conrmed
by spectroscopic data. Moreover, the reduction of compound 2
with zinc dust in acetic acid furnished the intermediate 2-
amino-3-oxo-N-thiazole-2yl- butyramide 3 (not isolated), which
converted directly to the pyrrole derivatives 5a,b on treatment
with an equimolar amount of ethyl acetoacetate and acetyl
acetone, respectively, through the intermediate 4.15 Compound
5 may proceed by condensation of the carbonyl group in active
methylene to amino function in compound 3 to form the
Schiff's base intermediate 4, which under intramolecular
cyclization by loss of water, forms the nal product 5a,b
(Scheme 1).
Similarly, when compound 1 is allowed to react with
hydroxylamine hydrochloride in reuxing ethanol in the pres-
ence of sodium acetate, the pyrazole derivative 7 was obtained
through the unisolated oxime 6. The synthetic potential of 1
toward a variety of nucleophilic regents was also investigated.
Thus, the boiling of compound 1 in sodium ethoxide solution
following the addition of an equimolar amount of ethyl
In order to nd a new drug candidate with selective COX-2
inhibition and PDE5 inhibitors, a comparative shape align-
ment was carried out to synthesize a new lead candidate.
Regarding PDE5 inhibition, the comparative shape alignment
between sildenal and avanal with enamide 23c showed that
the replacement of the methyl group with an aryl moiety is
Fig. 1 Structures of PDE5 inhibitors and COX-2 inhibitory reference drugs.
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Fig. 2 Rational for the design of more potent PDE5 inhibitors and selective COX-2.
chloroacetate did not yield the expected 9, and only ethyl 4-oxo- 7.2 Hz assigned to CH3, triplet signals at d ¼ 2.78 ppm, J ¼
4-(thiazol-2-ylamino) butanoate 11 was obtained.16 Compound 6.4 Hz, d ¼ 2.87 ppm, J ¼ 6.4 Hz assigned for 2CH2, quartet
11 is assumed to proceed by condensation of ethyl chloroacetate signal at d ¼ 4.13 ppm, J ¼ 7.2 Hz assigned to CH2 ester, doublet
to the sodium salt of compound 1, forming the (not isolated) signals at d ¼ 6.96 ppm, d ¼ 7.47 ppm assigned to 2 thiazole H-4,
intermediate 10 by loss of a HCl molecule and subsequent loss H-5, and a singlet signal at d ¼ 11.12 ppm for the NH group. In
of an acetaldehyde molecule to form 11. Compound 11 was the 1H,1H COSY spectrum for compound 11, triplet protons for
1
conrmed based on its compatible spectroscopic data (IR, H the methyl group at d ¼ 1.23 ppm showed J3 correlation with
NMR, 1H,1H COSY, 13C NMR, DEPT-135 of 13C NMR, NOE of 13
NMR). H NMR revealed a triplet signal at d ¼ 1.23 ppm, J ¼ moiety. At the same time, the methylene protons at d ¼
C
quartet methylene protons at d ¼ 4.13 ppm to indicate an ethyl
1
Scheme 1
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Scheme 2
2.78 ppm exhibited J3 coupling with methylene protons at d ¼ disappearance of one quaternary carbon atom signal at d ¼
1
2.87 ppm, In the H,1H COSY spectrum for compound 11, the 159.95 ppm and the presence of the sp2 and sp3 carbons at d ¼
methylene at d ¼ 4.3 ppm showed a correlation to the aromatic (+)14.11 ppm (q), d ¼ (ꢁ)28.77 ppm (t), d ¼ (ꢁ)30.66 ppm (t), d ¼
proton at d ¼ 7.47 ppm to indicate these two protons in the (ꢁ)60.91 ppm (t), d ¼ (+)113.45 ppm (d), and d ¼ (+)136.30 ppm
same direction. The 13C NMR of compound 11 showed a signal (d). In the NOE (Nuclear Overhauser Effect) of 13C NMR,
at d ¼ 14.17 ppm (q) for CH3, signals at d ¼ 28.84 ppm (t), d ¼ a quartet methyl carbon at d ¼ 12.17–16.04 ppm and a triplet
30.74 ppm (t), d ¼ 60.95 ppm (t), for the 3CH2 carbons, signals at methylene carbon at d ¼ 59.49–62.55 ppm were observed. At the
d ¼ 113.50 ppm (d), d ¼ 136.36 ppm (d), for the 2CH carbons, same time, 2 methylene groups at d ¼ 27.49–31.95 ppm were
signals at d ¼ 159.95 ppm (s), d ¼ 169.64 ppm (s, C]O), and d ¼ overlapped together. In the aromatic region, a doublet carbon at
172.39 ppm (s, C]O). The (DEPT-135) of 13C NMR showed the d ¼ 112.39–114.84 ppm was overlapped with a doublet carbon in
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Scheme 3
the range of d ¼ 135.63–137.47 ppm for the thiazole ring, and
Similarly, the addition of an equimolar amount of ethyl
the quaternary carbon at d ¼ 169.57 ppm (s), and d ¼ chloroformate to the boiling solution of compound 1 in sodium
172.32 ppm (s) for the (2C]O) group.
ethoxide gave the unexpected thiazol-2-yl-carbamic acid ethyl
Scheme 4
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Table 1 Effect of tested compounds on PDE5 (10 mM) and MABP, and consensus scores against PDE5a,b,c
PDE5 activity (consensus
scores)
Comp. no.
Comp. structure
MABP
115##
2
53Y (122)
222[ (28)
5a
5b
108##
15[ (49)
98
7
75Y (126)
81#
11
17
3.5[ (100)
371[ (110)
85&
87&
21
4290[
118##
23a
100Y (57)
108
23b
23c
15[ (50)
110##
100Y (48)
100
Control (DMSO)
Nitroprusside
Sildenal
—
—
Fig. 1
—
93.10 ꢂ 1.55
52.10 ꢂ 1.11###
72.50 ꢂ 2.9##
100Y
a
Data were expressed as means ꢂ SE (n ¼ 4). b Multiple comparisons were done using the one way ANOVA, followed by Tukey–Kramer as the post-
ANOVA test. c #: Signicantly different from the control group at P < 0.05. &: Signicantly different from the sildenal group at P < 0.05.
ester 13 rather than the expected 3-oxo-2-(thiazole-2-yl- CH3, quartet signal at d ¼ 4.30 ppm, J ¼ 6.8 Hz, doublet signal at
carbamoyl)-butyric acid ethyl ester 12. Compound 13 was d ¼ 6.91 ppm, d ¼ 7.39 ppm assigned to 2 thiazole-H, and
established on the basis of the spectroscopic data. 1H NMR a singlet signal at d ¼ 12.81 ppm assigned to the NH group.
revealed a triplet signal at d ¼ 1.37 ppm, J ¼ 6.8 Hz assigned to 1H,1H COSY showed a strong J3 coupling between the triplet
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2-amino-thiazole, which react directly with ethylchloroformate
present in the reaction medium to give compound 13 (Scheme
2).
On the other hand, a reaction of compound 1 with a-chlor-
oacetylchloride afforded the pyrrole derivative 16 through the
intermediates 14 and 15. In the same manner, the reaction of
compound 1 with acetyl chloride in benzene under reuxing
gave the adduct 17. Compound 17 was conrmed by its reaction
with nucleophilic reagents (such as hydrazine hydrate) and urea
to obtain some azole and azine derivatives. So, the treatment of
compound 17 with hydrazine hydrate in ethanol under reux
afforded the corresponding pyrazole derivative 18. In the same
way, the pyrimidine derivative 19 was synthesized by reaction of
compound 17 and urea in ethanol under reux (Scheme 3).
In contrast, the behavior of compound 1 with 2-hydroxy
benzaldehyde in pyridine solution under reux gave the ben-
zopyrane derivative 21 through the intermediate 20. However,
under the reaction conditions, the quinoline derivative 22 was
obtained through a Dimroth rearrangement of 21. On the other
hand, the reaction of compound 1 with aromatic amines was
also investigated. So, it reacts with aromatic amines (like
aniline, p-anisidine and p-chloroaniline) to give the adducts
23a–c with the loss of water. Structures 23a–c were conrmed on
Fig. 3 Line chart representing the PDE5 regulatory activity (either
increase or decrease in PDE5%) of the tested compounds.
methyl protons at d ¼ 1.36 ppm and quartet methylene protons
at d ¼ 4.30 ppm to conrm the ethyl group. 13C NMR showed
signals at d ¼ 14.47 ppm (q), d ¼ 62.13 ppm (t), d ¼ 112.33 ppm
(d), d ¼ 136.71 ppm (d), d ¼ 154.02 ppm (s), and d ¼ 161.94 ppm
(C]O) group. (DEPT-135) of 13C NMR recorded the appearance
of only the sp3 and sp2 carbons at d ¼ (+)14.46 ppm (q), d ¼ (ꢁ)
62.13 ppm (t), d ¼ (+)112.31 ppm (d), and d ¼ (+)136.70 ppm (d).
The NOE (Nuclear Overhauser Effect) of 13C NMR spectra
showed two doublet aromatic methene carbons at d ¼ 111.31–
113.18 ppm and d ¼ 135.74–137.60 ppm. The coupling constant
difference indicates that they are geminal protons. At the same
time, the triplet methylene carbon at d ¼ 60.66–63.61 ppm and
quartet methyl carbon at d ¼ 12.57–16.37 ppm were observed
and the quaternary carbon at d ¼ 154.02 ppm (s), and at d ¼
162.05 (s) for the (C]O) group. Structural elucidation of
compound 13 came from its synthesis by another method,
conducting the believes 2-aminothiazole and ethyl chlor-
oformate in boiling solution of ethanolic sodium ethoxide (mp;
mx.mp and TLC). We suggest that compound 13 proceeds by
the addition of ethyl chloroformate to 8 (the unisolated sodium
salt of compound 1). The structure was broken and freed from
1
the basis of spectroscopic data. In the H,1H COSY spectrum,
the protons A–B system showed a strong J3 coupling. At the
same time, a doublet proton H4 at d ¼ 6.86 ppm showed
a strong J3 correlation with a doublet proton at d ¼ 7.37 ppm for
the thiazole ring (Scheme 4).
2. 2. Pharmacological evaluation
2. 2. 1 Phosphodiesterase 5 interaction and antihyperten-
sive activity. The vasorelaxant effects of the tested compounds
are presented in Table 1. The data showed that the MABP values
of compounds 2, 5a, 21 and 23b are signicantly higher than
those of the control, while the values of compounds 5b, 23a and
23c are higher than the control values in a nonsignicant
manner. In addition, the values of compounds 11 and 17 are
signicantly higher than the sildenal values, but insigni-
cantly lower than the control values. In contrast, the value of
compound 7 is signicantly lower than that of the control, and
insignicantly higher than the sildenal one. In addition, the
Table 2 In vitro COX-1 and COX-2 inhibitory activity of the target
compounds and reference drugsa,b
Comp. no.
COX-1 IC50a (mM)
COX-2 IC50a (mM)
COX-2 S.I.b
2
5a
5b
7
11
17
21
23a
1.5
1.6
1.3
6.34
1.7
1.4
1.0
6.30
5.74
6.33
7.21
0.11
0.10
0.24
0.63
0.21
0.09
0.33
0.71
0.56
0.61
0.83
13.64
16
5.4
10.06
8.1
15.56
3.03
8.87
10.25
10.38
8.68
23b
23c
Celecoxib
a
The concentration of the test compound that produces 50% inhibition
of COX-1, COX-2 enzyme activity. The result is a mean of two values
b
obtained by the assay of enzyme kits. The in vitro COX-2 selectivity
Fig. 4 Chart representing the COX-2 inhibitory activity of the tested
index (COX-1/COX-2).
compounds against celecoxib as a reference.
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results of the PDE5 regulatory activity of the tested compounds imine tautomerism. This, of course, will increase the compound
against sildenal are summarized in Table 1, and graphically binding with the specic amino acid cles inside the active site
presented in Fig. 3. The compounds are categorized as three domains. We speculate that, according to the strength of the
groups based on their action against PDE5: (a) activators, which compound interaction with PDE5, a conformational change of the
are compound 21 (PDE5 4290% enhancement), compound 17 enzyme will occur. Therefore, the activity uctuated from
(PDE5 371% enhancement), and compound 5a (PDE5 222% enhancement to inhibition.
enhancement); (b) weak regulators (activators), which are
2. 2. 2 In vitro anti-inammatory activity. The prepared
compounds 5b and 23b (PDE5 15% enhancement), and compounds were assayed against COX-1 and COX-2 using a color-
compound 11 (PDE5 3.5% enhancement); and nally (c) imetric enzyme immunoassay (EIA) kit (Cayman Chemicals Inc.,
inhibitors, which are compounds 23a and 23b (PDE5 100% Ann Arbor, MI, USA) to screen the isozyme-specic inhibition. The
inhibition), compound
compound 2 (PDE5 53% inhibition).
7
(PDE5 75% inhibition), and potency of the testing compounds was estimated as the concen-
tration causing 50% enzyme inhibition (IC50). The in vitro selective
The mean arterial blood pressure (MABP) results and that of index (SI) COX-1/COX-2 isozyme inhibition studies determined the
PDE5 revealed that compounds 23a and 23c have a good chance to ability of all synthesized compounds in inhibiting ovine COX-1 and
be used in erectile dysfunction without the development of the human recombinant COX-2.17
common side effect, hypotension, that is associated with the use of
The data (Table 2) showed that the target compounds have
such drugs to manage erectile dysfunction. In contrast, higher inhibitory activities against both COX-1 isozyme (IC50
¼
compounds 5a, 7, and 21 illustrated the enhancement in PDE5 1.00–6.34 mM range) and COX-2 isozyme (IC50 ¼ 0.09–0.71 mM
activity (Table 1). Other compounds, such as 5b, 11, 23b, showed range), with COX-2 selectivity indexes in the range of 3.03 to 16
very low activity (either in inhibition or enhancements). In in comparison with the COX-2 selective reference drug celecoxib
a comparison study between compounds 5a and 5b or 23a and 23c (COX-1 IC50 ¼ 7.21 mM, COX-2 IC50 ¼ 0.83 mM and S.I. ¼ 8.68).
to 23b, it is clear that any change in pharmacophoric feature in In addition, the COX-2 inhibitory activity of the tested
such types of compounds has a great effect on the PDE5 activity. compounds against celecoxib is graphically represented in
Notably, compound 21 has a remarkable effect on PDE5 as Fig. 4.
a strong activator. This behavior could be attributed to the pres-
In the comparison between the compound activities on
ence of a fused heterocyclic system (chromene ring) linked with PDE5 and anti-inammatory effect, all compounds have COX-2
the 2-aminothiazole moiety, and the capability to form an amine– selectivity whether they are an activator or inhibitor to PDE5.
Fig. 5 Visual representation by Vida. (A) Sildenafil in the catalytic domain of PDE5 (PDB code: 2H24); (B) 23a in the catalytic domain of PDE5 (PDB
code: 2H24); (C) 23a, 23c overlay; (D) shape alignment of 23c with sildenafil.
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However, an urge need for further study in the future to describe shown in Fig. 5B. Compound 23c forms a hydrophobic–hydro-
the exact molecular mechanism that underlies the anti- phobic interaction with the receptor. Both compounds adopted
inammatory effect and selectivity index to the COX-2 a complete overlay with the same orientation of the thiazole
isoenzyme.
moiety. The sulfur atom was oriented towards the Gln: 817, as
shown in Fig. 5C. This mode is different from that of the analogue
23b, which will be presented later. Both compounds 2 and 7
showed a low consensus score among the tested compounds (48,
51, respectively), and formed HBs and hydrophobic interactions,
respectively. The shape alignment between 23c and sildenal, as
represented in Fig. 5D, or vardenal emphasize the required
modication of the synthesized compounds to get more potent
derivatives at a lower dose.
The novelty in this work arose from some compounds with
an enhancement effect on PDE5. The docking protocol was
applied to understand this action and subsequently, the
development of new drugs as PDE enhancers, which will open
the gate for new pharmacological applications.
Concerning other compounds with an enhancement activity
on PDE5, these compounds showed strong HBs formation with
the same catalytic site of PDE5. Compound 21 has the highest
enhancement value for PDE5 activity with a consensus score of
23. It forms a strong HB with Gln: 81A, as shown in Fig. 6A. In
addition, it has high similarity with compound 5a in its binding
mode and pose, as shown in Fig. 6B. For the small molecule,
compound 17, it forms two strong HBs with Gln: 817A. The
enamide 23b has a hydrophobic–hydrophobic interaction with
2.3. Docking study
2. 3. 1 PDE5 interaction. All molecular docking studies
were implemented using OpenEye scientic soware academic
license.18 All compounds were generated in the most stable
conformer as performed by the Omega application. The study
implemented different human phosphodiesterase-5 (PDE5) X-
ray structures. Each was complexed with its co-crystallized
ligand sildenal, vardenal or tadalal (ID: 1TBF, 1XP0,
1XOZ, and 2H42), and were retrieved from the Protein Data
Bank.19 However, the study focused on PDB ID 2H42.20
In order to validate our docking study, the docking protocol was
continued with the redocking of the reported PDE5 inhibitor drugs
sildenal, vardenal, tadalal and avanal with the catalytic
domain receptor of PDE5 (PDB: ID: 2h42).20 All drugs showed
a similar binding mode and pose, in particular, the co-crystallized
ligand drug, sildenal. Sildenal forms two HBs with Gln: 817:A
through its lactam functionality (Fig. 5A). This docking pattern is
similar to the reported co-crystallized pose. Both enamides (23a
and 23c), with 100% PDE5 inhibition, interact with the receptor
with a similar pose. However, compound 23a forms a HB with the
key amino acid Gln: 817:A through its carbonyl group (acceptor), as
Fig. 6 Visual representation as visualized by Vida. (A) Compound 21 in the catalytic domain of PDE5 (PDB code: 2H24); (B) compound 5a in the
catalytic domain of PDE5 (PDB code: 2H24); (C) compound 23b with a hydrophobic–hydrophobic interaction without the formation of a HB; (D)
overlay between compounds 5a and 21.
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Fig. 7 (A) Celecoxib with the COX-2 active site (PD: ID 3IN1); (B) enamide 23c; (C) 23a and 23b overlay; (D) Y shape of enamide (blue) inside the
receptor overlay with celecoxib (pink colour).
the receptor in which the nitrogen atom of the thiazole moiety is was examined. The synthesized compounds adopted two
oriented toward Gln: 817, as shown in Fig. 6C. This behavior different patterns against PDE5. Compounds 23a and 23b
differs from the other enamide 23a. Both compounds 5a and 21 exhibited 100% inhibition of PDE5 at 10 mM without a notable
occupied the receptor domain with high similarity in mode and decrease in MABP. This kind of compound has the benet over
pose, as shown in Fig. 6D.
current drugs and PDE5 inhibitors, which have a hypotensive
2. 3. 2 COX-2 inhibition. The docking started point was effect as a drawback. In contrast, compounds 5a, 21, 17 and 23b
determined by examining the interaction of the standard drug increased the PDE5 activity (PDE5 enhancers) at 10 mM. Both
celecoxib with the COX-2 enzyme. Celecoxib was found to interact types of compounds are considered PDE5 regulators. To the
with the receptor of PDB ID: 3LN1,21 taking its known pose with the best of our knowledge, this is the rst report of small molecules
Y shape (pyrazole core with vicinal diaryl pharmacophores), as as PDE5 enhancers with the hope of nding a pharmacological
shown in Fig. 7A, with a best consensus score of 0.00.
application. All compounds showed anti-inammatory activity
All examined compounds interacted with the COX-2 enzyme and based on the COX-1/COX-2 Ic50% values.
docked inside the receptor. Some of these compounds exhibited
All tested compounds have an inhibitory effect against the
binding poses that were similar to that of celecoxib. Enamide 23c COX-1 activity (IC50 ¼ 1.00–6.34 mM range) and COX-2 activity
has the highest consensus score value (12) with the formation of the (IC50 ¼ 0.09–0.71 mM range). The selectivity indexes of these
hydrophobic–hydrophobic interaction, as shown in Fig. 7B. Enam- compounds to the COX-2 isoenzyme fall in the range of 3.03 to
ides 23a and 23b with consensus scores of 19 and 14, respectively, 16 in comparison with celecoxib, a selective COX-2 inhibitor
adopted binding poses with the formation of HB with Tyr 341:A, as (COX-1 IC50 ¼ 7.21 mM, COX-2 IC50 ¼ 0.83 mM and S.I. ¼ 8.68).
shown in Fig. 7C. From a medicinal chemistry perspective, the
We envisioned that there is a relationship between the
enamides adopted the Y shape inside the receptor, as illustrated in strength of the compound interaction and their effect on the
Fig. 7D. Enamide 23b and celecoxib interacted through HBs with enzyme from inhibition, ranging from nothing to activation.
the amino acids cle of Tyr 341:A, Leu 338:A, and Hist 75:A.
4. Experimental
4.1. Chemistry
3. Conclusion
New aminothiazole derivatives having different functionalities Melting points were uncorrected and determined on a Stuart
were synthesized and their activity with PDE5 and COX-1/COX-2 melting point apparatus (Stuart Scientic, Redhill, UK).
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Elemental analyses of C, H and N were performed on a Perkin- 117.12, 125.34, 137.30, 142.41, 156.39, 162.86, 164.83; Dept 135
Elmer 2400 analyzer (Perkin-Elmer, Norwalk, CT, USA). The IR NMR d 12.17, 14.36, 14.75, 59.81, 113.15, 125.34; anal. calcd for
spectra (KBr) were measured on a Shimadzu IR 435 spectro-
C13H15N3O3S (293.34): C, 53.23; H, 5.15; N, 14.32; S, 10.93%,
1
photometer and the values are represented in cmꢁ1. H NMR found: C, 53.25; H, 5.12; N, 14.30; S, 10.91%.
(400 MHz) and 13C NMR (75 MHz) spectra were carried out
4.1.4 4-Acetyl-3,5-dimethyl-N-(thiazol-2-yl)-1H-pyrrole-2-
using a Varian Gemini 300-BB spectrophotometer (Bruker, carboxamide 5b. Brown cꢁry1stals from aqua's ethanol; yield 56%;
Munich, Germany). Splitting patterns were designated as mp 220 ꢀC; IR (KBr) n cm ¼ 3404 (NH), 3086 (CH arom.), 2918
follows: s: singlet; d: doublet; t: triplet; m: multiplet, and (CH aliph.); 1687 (C]O); 1H NMR (DMSO-d6): d 2.39 (s, 3H,
chemical shi values were recorded in ppm on a scale. Mass CH3), 2.51 (s, 3H, CH3), 2.57 (s, 3H, CH3), 7.22 (d, 1H, CH-
spectra were run on a Shimadzu Qp-2010 plus. The progress of thiazole, J ¼ 3.6 Hz), 7.51 (d, 1H, CH-thiazole, J ¼ 3.6 Hz),
the reactions was monitored by TLC using TLC sheets pre- 11.79 (s, 2H, 2NH); 13C NMR (DMSO-d6): d 12.43, 14.79, 31.21,
coated with UV orescent silica gel (Merck 60 F 254), and was 113.30, 119.01, 122.68, 129.03, 137.14, 137.88, 158.56, 194.40;
visualized using a UV lamp. The chemicals used were supplied MS (relative intensity) m/z: 265.9 (M2+, 93.3%); anal. calcd for
from Acros (New Jersey, USA).
C12H13N3O2S (263.32): C, 54.74; H, 4.98; N, 15.96; S, 12.18%,
4.1.1 Preparation of 2-(hydroxyimino)-3-oxo-N-(thiazol-2-yl) found: C, 54.76; H, 4.95; N, 15.94; S, 12.16%.
butanamide (2). A mixture of 3-oxobutanamides 1 (0.03 mol) in
4.1.5 Preparation of 3-methyl-1-(thiazol-2-yl)-1H-pyrazol-5-
100 ml of glacial acetic acid was cooled in an efficient freezing ol 7. A mixture of 1 (0.01 mol) and hydroxyl amine hydrochlo-
ꢀ
mixture to 5 C, and a cold solution of (0.0147 mole) sodium ride (0.01 mol) was reuxed in ethanol containing fused sodium
nitrite in 5 ml water was added dropwise with vigorous stirring acetate for 8 h, and then 30 ml of glacial acetic acid was stirred
ꢀ
at such a rate that the temperature remained between 5–7 C. and portions of (0.5 gram atoms) of zinc dust were added. Aer
With efficient cooling, about one-half hour was required to add the addition was completed, the mixture was heated by a burner
the nitrite. The mixture was stirred for one-half hour longer, and reuxed for one hour. While still hot, the contents of the
and then allowed to stand for 4 h. During this time, it warmed ask were decanted from the remaining zinc into a beaker
up to room temperature. Finally, the solid product formed was containing 500 ml water, which was vigorously stirred. The zinc
collected by ltration and recrystallized from aqua's ethanol to residue was washed with two 50 ml portions of hot glacial acetic
give 2.
acid, which were also decanted into the water. Aer standing
1
ꢀ
Brown crystals; yield, 65%; mp 182 C; H NMR (DMSO-d6): overnight, the crude product was ltered by suction, washed on
d2.40 (s, 3H, CH3), 7.28 (d, 1H, CH-thiazole, J ¼ 0.8 Hz), 7.50 (d, the lter with two 500 ml portions of water, and dried in air to
1H, CH-thiazole, J ¼ 1.6 Hz), 12.54 (s, 1H, NH), 13.06 (s, 1H, N– a constant weight, and then le to cool. The solid product
OH); 13C NMR (DMSO-d6): d 25.28, 114.17, 137.85, 152.16, formed was ltered off and recrystallized from aqua's ethanol to
156.77, 160.18, 194.35; Dept 135 NMR: d 25.22, 114.10, 137.85; give 7.
MS (relative intensity) m/z: 214.11 (M+, 7.8%); anal. calcd for
C7H7N3O3S (213.21): C, 39.43; H, 3.31; N, 19.71; S, 15.04%, 3426 (OH), 3027 (CH arom.), 2949 (CH aliph.); 1H NMR (DMSO-
found: C, 39.41; H, 3.33; N, 19.72; S, 15.05%. d6): d 2.15 (s, 3H, CH3), 5.65 (s, 1H, CH pyrazole), 7.05 (d, 1H,
Dark red crystals; yield: 48%, mp 290 C; IR (KBr) n cm
¼
ꢁ1
ꢀ
4.1.2 General procedure for the synthesis of compounds CH-thiazole, J ¼ 1.6 Hz), 7.38 (d, 1H, CH-thiazole, J ¼ 1.6 Hz),
5a,b. To a solution of 2 (0.01 mol) in 50 ml of glacial acetic acid, 11.77 (br. S, 1H, OH); anal. calcd for C7H7N3OS (181.21): C,
portions of (0.5 gram atoms) of zinc dust were added quickly. It 46.40; H, 3.89; N, 23.19; S, 17.69%, found: C, 46.41; H, 3.88; N,
was stirred until the liquid boiled, and then frequently enough 23.20; S, 17.66%.
to keep it boiling. 0.01 mol of ethyl acetoacetate and acetyl
4.1.6 Preparation of ethyl 4-oxo-4-(thiazol-2-ylamino)
acetone were added. Aer the addition was completed, the butanoate 11. To a solution of compound 1 (0.01 mol) in
mixture was heated by a burner and reuxed for one hour. 30 ml of absolute ethanol containing 0.01 mol of clean sodium
While still hot, the contents of the ask were decanted from the metal, 0.01 mol of ethyl chloroacetate was added slowly. The
remaining zinc into a beaker containing 500 ml of water, which reaction mixture was reuxed for 6 h and le to cool, then
was vigorously stirred. The zinc residue was washed with two poured onto ice/water containing a few drops of HCl. The
50 ml portions of hot glacial acetic acid, which were also dec- formed solid product was collected by ltration and recrystal-
anted into the water. Aer standing overnight, the crude lized from aqua's ethanol to give 11.
ꢁ1
ꢀ
product was ltered by suction, washed on the lter with two
White crystals; yield: 56%, mp ¼ 154 C; IR (KBr) n cm
¼
500 ml portions of water, and dried in air to a constant weight to 3435 (NH), 3082 (CH arom.), 2922 (CH aliph.), 1726, 1688 (2C]
1
give 5a,b.
4.1.3 Ethyl
O); H NMR (CDCl3-d1): d 1.23 (t, 3H, CH3, J ¼ 7.2 Hz), 2.78 (t,
2,4-dimethyl-5-(thiazol-2-ylcarbamoyl)-1H- 2H, CH2, J ¼ 6.4 Hz), 2.87 (t, 2H, CH2, J ¼ 6.4 Hz), 4.13 (q, 2H,
pyrrole-3-carboxylate 5a. Dark red crꢁys1tals from aqua's ethanol; CH2, J ¼ 7.2 Hz), 6.96 (d, 1H, CH-thiazole, J ¼ 3.2 Hz), 7.47 (d,
yield 45%; mp 147 ꢀC; IR (KBr) n cm ¼ 3450 (2NH), 3081 (CH 1H, CH-thiazole, J ¼ 4 Hz), 11.12 (s, 1H, NH); 13C NMR (CDCl3-
arom.), 2921 (CH aliph.), 1657 (C]O); 1H NMR (CDCl3-d1): d1): d 14.17, 28.84, 30.74, 60.95, 113.50, 136.36, 159.95, 169.64,
d 1.33 (t, 3H, CH3, J ¼ 7.2 Hz), 2.58 (s, 3H, CH3), 2.69 (s, 3H, 172.39; Dept 135 NMR d 14.11, 28.77, 30.66, 60.91, 113.45,
CH3), 4.24 (q, 2H, CH2, J ¼ 7.2 Hz), 7.07 (d, 1H, CH-thiazole, J ¼ 136.30; NOE NMR d 12.17-16.04 (q, CH3), 27.49–31.95 (m,
4.4 Hz), 7.51 (d, 1H, CH-thiazole, J ¼ 4.4 Hz), 11.12 (s, 2H, NH); 2CH2), 59.49 (t, CH2), 112.39 (d, CH), 135.63 (d, CH); 159.7 8 (s),
13C NMR (CDCl3-d1): d 12.17, 14.36, 14.75, 59.77, 113.17, 114.81, 169.57 (s), 172.32 (s); anal. calcd for C9H12N2O3S (228.27): C,
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47.35; H, 5.30; N, 12.27; S, 14.05%, found: C, 47.33; H, 5.31; N, NMR d 18.99, 20.07, 113.75, 114.16, 137.54; NOE NMR: d 18.15-
12.29; S, 14.04%. 20.75, 112.92, 113.19, 136.65, 161.60, 166.41, 171.75, 177.62;
4.1.7 Preparation of ethyl thiazol-2-ylcarbamate 13. A anal. calcd for C9H10N2O3S (226.25): C, 47.78; H, 4.45; N, 12.38;
mixture of 3-oxobutanamide 1 (0.01 mol), ethyl chloroformate S, 14.17%, found: C, 47.76; H, 4.47; N, 12.39; S, 14.15%.
(0.01 mol) in sodium ethoxide (20 ml) was reuxed for 12 h, and
4.1.10 Preparation of 3,5-dimethyl-N-(thiazol-2-yl)-1H-
then le to cool. It was poured onto ice/water, which was pyrazole-4-carboxamide 18. A mixture of 17 (0.01 mol) and an
vigorously stirred. The solid product formed was collected by excess of hydrazine hydrate was reuxed for 6 h, and then le to
ltration and recrystallized from aqua's ethanol to give 13ꢁ. 1
cool. The solid product formed was collected by ltration and
recrystallized from ethanol to give 18.
Brown crystals; yield; 58%, mp 142 ꢀC; IR (KBr) n cm
¼
3423 (NH), 3080 (CH arom.), 2977 (CH aliph.), 1722, 1644 (2C]
Brown crystals; yield: 57%, mp 210 ꢀC; 1H NMR (DMSO-d6):
O); 1H NMR (CDCl3-d1): d 1.37 (t, 3H, CH3, J ¼ 6.8 Hz), 4.30 (q, d 2.31 (s, 3H, CH3), 2.59 (s, 3H, CH3), 7.29 (d, 1H, CH-thiazole, J
2H, CH2, J ¼ 6.8 Hz), 6.91 (d, 1H, CH-thiazole, J ¼ 3.6 Hz), 7.39 ¼ 3.2 Hz), 7.52 (d, 1H, CH-thiazole, J ¼ 3.6 Hz), 9.69 (s, 1H, NH),
(d, 1H, CH-thiazole, J ¼ 4 Hz), 12.81 (s, 1H, NH); 13C NMR 12.67 (s, 1H, NH); 13C NMR (DMSO-d6): d 19.01, 20.09, 114.18,
(CDCl3-d1): d 14.47, 62.13, 112.33, 136.71, 154.02, 161.94; Dept 137.52, 157.14, 161.56, 166.07, 171.82, 177.62; Dept 135 NMR
135 NMR d 14.46 (q), 62.13, 112.31, 136.70; NOE NMR d 12.57 (q, d 18.99, 20.07, 114.16, 137.59; MS (relative intensity) m/z: 223.23
CH3); 60.66 (t, CH2); 111.31 (d, CH); 135.74 (d, CH); 154.02 (s); (M1+, 2.8%); anal. calcd for C9H10N4OS (222.27): C, 48.63; H,
162.05 (s); anal. calcd for C6H8N2O2S (172.2): C, 41.85; H, 4.53; N, 25.21; S, 14.43%, found: C, 48.61; H, 4.50; N, 25.24; S,
4.68; N, 16.27; S, 18.62%, found: C, 41.88; H, 4.67; N, 16.29; S, 14.46%.
18.60%.
4.1.11 Preparation of 4,6-dimethyl-2-oxo-N-(thiazol-2-yl)-
4.1.8 Preparation of 1-(2,4-dihydroxy-1-(thiazol-2-yl)-1H- 1,2-dihydropyrimid-ine-5-carboxamide 19. A mixture of 17
pyrrol-3-yl)ethanone 16. A mixture of 1 (0.01 mol), 2-chlor- (0.01 mol) and urea (0.01 mol) in ethanol (30 ml) was reuxed
oacetyl chloride (0.01 mol) in ethanol (30 ml) was treated with for 6 h, and then le to cool. The solid product formed was
a few drops of piperidine and reuxed for 6 h, and then le to collected by ltration and recrystallized from ethanol to give 19.
cool. The solid product formed was collected by ltration and
recrystallized from ethanol to give 16.
Brown crystals; yield: 53%, mp 235 ꢀC; 1H NMR (DMSO-d6) (d
ppm); d 2.07 (s, 3H, CH3), 2.36 (s, 3H, CH3), 7.28 (d, 1H, CH-
1
ꢀ
Brown crystals; yield: 48%, mp 270 C; H NMR (DMSO-d6): thiazole, J ¼ 0.8 Hz), 7.50 (d, 1H, CH-thiazole, J ¼ 1.6 Hz),
d 2.41 (s, 3H, CH3), 5.001 (s, 2H, 2OH), 6.08 (s, 1H, CH-pyrrole), 8.76 (s, 1H, NH), 12.81 (s, 1H, NH); 13C NMR (DMSO-d6): d 17.07,
7.30 (d, 1H, CH-thiazole, J ¼ 4.8 Hz), 7.76 (d, 1H, CH-thiazole, J 20.07, 114.25, 137.53, 157.35, 161.52, 166.39, 171.96 (s), 177.48,
¼ 5.2 Hz); 13C NMR (DMSO-d6): d 18.18, 109.66, 109.86, 123.37, 196.29; anal. calcd for C10H10N4O2S (250.28): C, 47.99; H,
146.57, 153.17, 165.13, 167.27, 196.28 (s, C]O); Dept 135 NMR 4.03; N, 22.39; S, 12.81%, found: C, 47.96; H, 4.07; N, 22.40; S,
d 18.04, 109.55, 109.73, 123.24; anal. calcd for C9H8N2O3S 12.78%.
(224.24): C, 48.21; H, 3.60; N, 12.49; S, 14.30%, found: C, 48.22;
H, 3.63; N, 12.50; S, 14.31%.
4.1.12 Preparation of 3-acetyl-1-(thiazol-2-yl)quinolin-
2(1H)-one 22. A mixture of 1 (0.01 mol) and 2-hydroxy benzal-
4.1.9 Preparation
of
2-acetyl-3-oxo-N-(thiazol-2-yl) dehyde (0.01 mol) was reuxed for 5 h in pyridine. The solid
butanamide 17. A mixture of (0.01 mol) of magnesium turn- product formed was collected by ltration and recrystallized
ings, (0.01 mol) of 3-oxobutanamide 1, 20 ml of benzene (dried from benzene to give 22.
1
ꢀ
over sodium), and (0.015 mol) of acetyl chloride was heated
Yellow crystals; yield: 45%, mp 110 C; H NMR (CDCl3-d1):
under reux for 12 h in a 100 ml round-bottomed ask. This d 2.70 (s, 3H, CH3), 7.30 (d, 1H, CH-thiazole), 7.33 (d, 1H, CH-
ask was provided with a condenser closed by a calcium chlo- thiazole), 7.35 (d, 1H, Ar–H), 7.61–7.62 (m, 1H, Ar-H), 7.63–
ride tube and supported in an oil bath (85–90 ꢀC). The reaction 7.65 (m, 1H, Ar-H), 7.65 (s, 1H, Ar-H), 8.49 (s, 1H, CH-quinoline);
mixture was cooled in an ice bath, and the liquid portion was 13C NMR (CDCl3-d1): d 30.74, 114.24, 117.26, 124.83, 129.80,
decanted into a separating funnel. The residue le in the ask 131.17, 138.57, 139.90, 142.34, 143.97, 156.05, 161.36, 177.05,
aer decantation was washed twice with 10 ml portions of ether, 198.71; anal. calcd for C14H10N2O2S (270.31): C, 62.21; H,
and the ethereal solution was poured over ice. The ether–water 3.73; N, 10.36; S, 11.86%, found: C, 62.24; H, 3.76; N, 10.32; S,
mixture was then added to the benzene solution in the sepa- 11.83%.
rating funnel, and the mixture was shaken thoroughly. The
4.1.13 General procedure for the synthesis of compounds
aqueous layer was drawn off and discarded. The benzene–ether 23a–c. In a round-bottomed ask attached to a Dean and Stark
solution was washed once with 10 ml of 5% sodium bicarbonate constant water separator, which was connected to a reux
solution, and once with 50 ml of water. Finally, it was dried over condenser, was placed a mixture of 3-oxobutanamide 1 (0.05
calcium chloride. The ether and benzene were removed by mol), 0.05 mol of aromatic amines, 100 ml of benzene, and 1 ml
distillation from a water bath. The solid product formed was of glacial acetic acid. The ask was heated in an oil bath at
ꢀ
recrystallized from ethanol to give 17.
about 125 C, and the water which distilled out of the mixture
1
ꢀ
Brown crystals; yield: 46%, mp 187 C; H NMR (DMSO-d6): with the reuxing benzene was removed at intervals. Reuxing
d 2.31 (s, 3H, CH3), 2.59 (s, 3H, CH3), 6.43 (s, 1H, CH), 7.29 (d, was continued until no more water separated, and then for an
1H, CH-thiazole, J ¼ 3.2 Hz), 7.52 (d, 1H, CH-thiazole, J ¼ 3.6 additional 30 minutes. The benzene was then distilled under
Hz), 13.20 (s, 1H, NH); 13C NMR (DMSO-d6): d 19.01, 20.09, reduced pressure. The solid product formed was ltered off and
113.76, 114.18, 137.54, 161.56, 166.37, 171.82, 177.62; Dept 135 recrystallized from aqua's ethanol to give 23a–c.
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4.1.14 Preparation of (Z)-3-(phenylamino)-N-(thiazol-2-yl)
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and ltered through a nylon-66 lter, 0.2 mm (Rainin Corpo-
ration). The clear ltrate obtained may be used directly for
HPLC assay or stored at ꢁ20 C. A blank with protein, denatu-
but-2-enamide 23a. Yellow crystals; yield 74%; mp 220 ꢀC;
IR cmꢁ1 n ¼ 3192, 3136 (2NH), 3008 (CH arom.), 2914 (CH
aliph.), 1644 (C]O); 1H NMR (DMSO-d6) (d ppm); d 2.03 (s, 3H,
CH3), 5.05 (s, 1H, Ali CH), 7.11 (d, 1H, CH-thiazole, J ¼ 4 Hz),
7.17–7.14 (m, 5H, Ar-H), 7.45 (d, 1H, CH-thiazole, J ¼ 3.6 Hz),
11.03 (s, 1H, NH), 11.65 (s, 1H, NH); 13C NMR (DMSO-d6)
d 20.20, 87.88, 112.41, 123.60, 124.58, 129.21, 137.50, 138.93,
157.94, 158.55, 166.74; Dept 135 NMR d 20.15, 87.83, 112.38,
123.55, 124.55, 129.16, 137.47; MS (relative intensity) m/z:
261.46 (M2+, 3.6%); anal. calcd for C13H13N3OS (259.33): C,
60.21; H, 5.05; N, 16.20; S, 12.36%, found: C, 60.22; H, 5.07; N,
16.19; S, 12.35%.
4.1.15 Preparation of (Z)-3-((4-methoxyphenyl)amino)-N-
(thiazol-2-yl)but-2-en-amide 23b. Green crystals; yield 60%; mp
¼ 235 ꢀC; IR (KBr) cmꢁ1 n ¼ 3208, 3124 (2NH), 3011 (CH arom.),
2955 (CH aliph.), 1652 (C]O); 1H NMR (DMSO-d6) (d ppm):
d 1.90 (s, 3H, CH3), 3.73 (s, 3H, OCH3), 4.93 (s, 1H, Ali CH), 6.89
(d, 2H, Ar–H, J ¼ 8.8 Hz), 7.04 (d, 1H, CH-thiazole, J ¼ 3.6 Hz),
7.11 (d, 2H, Ar–H, J ¼ 8.8 Hz), 7.38 (d, 1H, CH thiazole, J ¼ 3.6
Hz), 10.38 (s, 1H, NH), 11.51 (s, 1H, NH); 13C NMR (DMSO-d6):
d 19.95, 55.26, 86.51, 112.26, 114.25, 126.10, 131.63, 137.45,
156.84, 158.91, 166.77; Dept 135 NMR d 19.90, 55.21, 86.46,
112.21, 114.25, 126.05, 137.40; anal. calcd for C14H15N3O2S
(289.35): C, 58.11; H, 5.53; N, 14.52; S, 11.08%, found: C, 58.13;
H, 5.55; N, 14.50; S, 11.09%.
4.1.16 Preparation of (Z)-3-((4-chlorophenyl)amino)-N-
(thiazol-2-yl)but-2-enamide 23c. Yellow crystals; yield 68%; mp
¼ 190 ꢀC; IR (KBr) n cmꢁ1 ¼ 3242, 3208 (2N–H), 3056 (CH
arom.), 2923 (CH aliph.), 1649 (C]O); 1H NMR (CDCl3-d1) (d
ppm): d 1.97 (s, 3H, CH3), 4.88 (s, 1H, Ali CH), 6.86 (d, 1H,
thiazole, J ¼ 2.8 Hz), 6.99 (d, 2H, Ar-H, J ¼ 8 Hz), 7.24 (d, 2H, Ar-
H, J ¼ 8 Hz), 7.37 (d, 1H, thiazole, J ¼ 2.8 Hz), 10.86 (s, 1H, NH);
11.96 (s, 1H, NH); 13C NMR (CDCl3-d1) (d ppm): d 20.45, 87.83,
112.34, 125.90, 129.24, 131.04, 133.73, 137.34, 159.78, 161.03,
166.36; Dept 135 NMR d C 20.45, 87.81, 112.34, 126.00, 129.23,
133.62; NOE NMR d 19.81–22.37 (q), 86.97–88.58 (d), 111.34–
113.36 (d), 159.86–161.05 (d), 125.19–137.24 (m, s) 166.31 (s,
C]O); anal. calcd for C13H12ClN3OS (293.77): C, 53.15; H,
4.12; N, 14.30; Cl, 12.07; S, 10.91%, found: C, 53.11; H, 4.12; N,
14.31; Cl, 12.09; S, 10.90%.
ꢀ
rated by boiling water bath for 3 min, with and without
substrate was performed. Both incubation time and enzyme
concentration were adjusted so that no more than 25% of the
substrate was hydrolyzed under the assay conditions. The
chromatographic system was an Agilent Technologies 1200
Series, G1315D DAD. The column used was a Zorbax Eclipse. A
rapid resolution 4.69 ꢃ 150 mm 3.5 mM particle size. The
mobile phase employed for the separation (isocratic elution)
consisted of 200 mM ammonium acetate at pH 6.0 with 2%
acetonitrile (v/v). The ow rate was 1.5 cm3 minꢁ1 and the
detector DAD was at 254 nm. The injection volume was 30 mm3.
Peak identities were conrmed by co-elution with standards,
and all assays were done in duplicate.
4.2.2 Arterial blood pressure measurement
4.2.2.1 Experimental animals. Regardless of sex factor,
White New Zealand rabbits weighing 2–2.5 kg were obtained
from the animal house of the Faculty of Medicine, Assiut
University. The animals were housed for one week to adapt to
the environmental conditions. Rabbits were fed a standard diet
and allowed free access to water. “All animal procedures were
performed in accordance with the Guidelines for Care and Use
of Laboratory Animals of Faculty of Pharmacy, Minia University
and approved by the Animal Ethics Committee” of the Faculty of
Pharmacy, Minia University.
4.2.2.2 Experimental design. Fiy-two of normotensive white
New Zealand rabbits (2–2.5 kg) were selected randomly, and
used in this study (four rabbits for each group) to investigate the
possible vasorelaxant effect of the selected compounds. Using
pentobarbital sodium (30 mg kgꢁ1, i.v.), normotensive rabbits
were anesthetized. Then, they were held on their backs with legs
xed and heads pinned down, and the trachea was exposed to
cut before proper cannulation was done.
4.2.2.3 Data recoding. Aer separation, the two muscle
bundles of the sternomastoid and the sternothyroid, the
common carotid artery lying in the neck between the lateral
bundles of muscle (longus capitis), and the trachea was exposed
and carefully freed from the nerves, veins and connective tissue.
The heparinized cannula was carefully inserted and rmly tied
via ne thread. Then, the cannula was connected to the pres-
sure transducer using a universal oscillograph (Harvard appa-
ratus, ser. no. K10542) for blood pressure recording. The
4.2. Pharmacological assessment
4.2.1 Phosphodiesterase 5 enzyme activity assay. The external jugular vein was also tightly cannulated for adminis-
standard enzymatic reaction mixture (total volume of 200 ml) tration of tested compounds (dissolved in DMSO), followed by
contained 100 ml M Tris–HCl buffer (pH 8.3), 10 mM MgCl2, heparinized saline (0.90% w/v NaCl).
10 mM KCl at 37 ꢀC. Alfa casein (2 mg) was used as a carrier for
The mean arterial blood pressure was calculated using the
protein precipitation when the protein concentration was low. formula:
The PDE A1 enzyme sample had a nal protein concentration of
0.5 mg mlꢁ1
.
A concentration of 10 mM of the agents under
22
MABP ¼ DBP + 1/3(SBP ꢁ DBP).
study (sildenal analogues) was prepared in dimethyl sulfoxide
(DMSO) and pre-incubated in the enzymatic mixture for 5 min
at room temperature. The reaction was initiated by the addition
of the substrate cGMP (5 mM) at 35 ꢀC for 30 min. The reaction
was nished by transferring the reaction mixture tubes in
a boiling water bath for 3 min. The sample was then centrifuged
where MDP ¼ mean arterial blood pressure, DBP ¼ diastolic
blood pressure, SBP ¼ systolic blood pressure.
4.2.2.4 Statistical analysis and data interpretation. Statistical
analysis was done using GraphPad Prism 5.0 soware (Graph-
Pad, San Diego, CA, USA). Data were represented as the mean ꢂ
This journal is © The Royal Society of Chemistry 2020
RSC Adv., 2020, 10, 29723–29736 | 29735

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Paper
SE. Multiple comparisons were carried out using the one-way
ANOVA with the Tukey–Kramer test as a multiple comparison
post-ANOVA test. P < 0.05 was selected to refer to statistical
signicance.
7 F. C. Stevens, Can. J. Biochem. Cell Biol., 1983, 61(8), 906–910.
8 L. Wang, B. T. Burmeister, K. R. Johnsona, G. S. Baillie,
A. V. Karginov, R. A. Skidgel, J. P. O'Bryan and
G. K. Carnegie, Cell. Signalling, 2015, 27, 908–922.
4.2.3 In vitro anti-inammatory activity screening. The in
vitro COX-1/COX-2 inhibition was detected using an enzyme
9 M. A. Shaaban, A. A. Radwan, Y. A. M. M. Elshaier and
B. A. Abdelwahab, Saudi Pharm. J., 2009, 17, 109–129.
immunoassay (EIA) kit (Cayman Chemical, Ann Arbor, MI) as 10 Y. A. M. M. Elshaier, ACS Midwest Regional, St. Joseph, MO,
the reported procedure for screening the isozyme-specic
October 2015; 21–24, Missouri, USA, ABSTRACT ID:
inhibition. The potency of the tested compounds was deter-
2361553, oral presentation, 10.
mined as the concentration causing 50% inhibition of enzyme 11 A. M. Hussein, M. M. Othman, M. A. M. Gad-Elkareem,
activity (IC50). Also, the COX-2 selectivity indexes (S.I. values)
were calculated as IC50 (COX-1)/IC50 (COX-2), and compared
M. El-Adasy and A. A. Khames, Elixir Org. Chem., 2017, 106,
46634–46643.
with that of the standard drug, celecoxib, which is a selective 12 A. M. Hussein, M. El-Adasy, A. A. Khames, A. A. Atalla and
COX-2 inhibitor.17
M. Abdel-Rady, ChemistrySelect, 2017, 2, 1625–1629.
13 A. M. Hussein, A. A. Khames, A. A. Abu-Bakr, M. El-Adasy,
M. A. M. Gad-Elkareem and M. M. Othman, Int. J. Pharm.
Sci., 2015, 5(1), 864–874.
14 A. M. Hussein, M. A. M. Gad-ElkareemA, A. Abu-Bakr, M. El-
Adasy, A. A. Khames and M. M. Othman, Int. J. Org. Chem.,
2012, 2, 341–351.
4.3. Docking study
The study was performed according to our previous method
using OpenEye soware.23
Conflicts of interest
15 H. Fischer, Org. Synth., 1941, 21, 67.
16 H. Adkins, N. Isbell and B. Wojcik, Org. Synth., 1934, 14, 38.
17 B. Roschek Jr, R. C. Fink, D. Li, M. McMichael, C. M. Tower,
R. D. Smith and R. S. Alberte, J. Med. Food, 2009, 12, 615–623.
18 https://www.eyesopen.com/, 15-6-2020.
There are no conicts to declare.
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29736 | RSC Adv., 2020, 10, 29723–29736
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