In vivo and in vitro anti-inflammatory, antipyretic and ulcerogenic activities of pyridone and chromenopyridone derivatives, physicochemical and pharmacokinetic studies

Article abstract of DOI:10.1016/j.bioorg.2021.104742

Throughout this study, we present the victorious synthesis of a novel class of 2(1H)-pyridone molecules, bearing a 4-hydroxyphenyl moiety through a one-pot reaction of 2-cyano-N-(4-hydroxyphenyl)acetamide with cyanoacetamide, acetylacetone or ethyl acetoacetate, and their corresponding aldehydes. In addition, the chromene moiety was introduced into the pyridine skeleton through the cyclization of the cyanoacetamide 2 with salicylaldehyde, followed by treatment with malononitrile, ethyl cyanoacetate, and cyanoacetamide, in order to improve their biological behaviour. Due to their anti-inflammatory, ulcerogenic, and antipyretic characters, the target molecules have undergone in-vitro and in-vivo examination, that display promising results. Moreover, in order to predict the physicochemical and ADME traits of all synthesized compounds and standard reference drugs, paracetamol and phenylbutazone, the in-silico prediction methodology was provided.

Full text of DOI:10.1016/j.bioorg.2021.104742

Bioorganic Chemistry 109 (2021) 104742
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
In vivo and in vitro anti-inflammatory, antipyretic and ulcerogenic activities
of pyridone and chromenopyridone derivatives, physicochemical and
pharmacokinetic studies
,
Eman A. Fayeda *, Ashraf H. Bayoumib, Aya S. Salehc, Elham M. Ezz Al-Arabc,
,
**
Yousry A. Ammard
a Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy (Girls), Al-Azhar University, Cairo 11754, Egypt
b Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy (Boys), Al-Azhar University, Cairo 11754, Egypt
c National Organization for Drug Control and Research, Cairo, Egypt
d Chemistry Department, Faculty of Science (Boys), Al-Azhar University, Cairo 11754, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords:
Throughout this study, we present the victorious synthesis of a novel class of 2(1H)-pyridone molecules, bearing
a 4-hydroxyphenyl moiety through a one-pot reaction of 2-cyano-N-(4-hydroxyphenyl)acetamide with cyanoa-
cetamide, acetylacetone or ethyl acetoacetate, and their corresponding aldehydes. In addition, the chromene
moiety was introduced into the pyridine skeleton through the cyclization of the cyanoacetamide 2 with sali-
cylaldehyde, followed by treatment with malononitrile, ethyl cyanoacetate, and cyanoacetamide, in order to
improve their biological behaviour. Due to their anti-inflammatory, ulcerogenic, and antipyretic characters, the
target molecules have undergone in-vitro and in-vivo examination, that display promising results. Moreover, in
order to predict the physicochemical and ADME traits of all synthesized compounds and standard reference
drugs, paracetamol and phenylbutazone, the in-silico prediction methodology was provided.
Pyridone
Chromenopyridone
Anti-inflammatory
Antipyretic
Ulcerogenic
And physicochemical studies
1. Introduction
as an anticancer [9a–9i], antibiotic (Ciclopirox (C)) [10a-10c], and
therapeutic agents [11,12] while maintaining authorization by the
Inflammation is a self-protective procedure, effectively eliminating
the injurious stimuli as well as initiating the recovery mechanism.
However, without systematic observation, it can entail an array of dis-
eases, namely: atherosclerosis, rheumatoid arthritis, and vasomotor
rhinnorrhoea [1a–1c]. The drugs, presently employed for the manipu-
lation of pain and inflammatory conditions, are either narcotics or non-
narcotics (NSAIDs) [2], which exhibit renowned toxic and lethal effects,
such as gastric lesions, ulcer and cardiovascular. Moreover, these drugs
are alleged to have unfavorable and variable efficiency [3,4]. These
obstacles have encouraged researchers to construct novel classes of anti-
inflammatory drug candidates with higher efficiency and undisruptive
side effects. Simultaneously, heterocyclic molecules are opulent in
operating as anti-inflammatory drug-like contenders [5]. Among these
heterocyclic derivatives, pyridine scaffolds possess an exclusive station
with their exceptionally sundry biological assets [6a–6f]. Additionally,
within the pyridine derivatives’ abilities [7,8], it demonstrates behavior
United States Food and Drug Administration (FDA) for heart failure
therapy, including: Milrinone (A) and Amrinone (B), (Fig. 1)
[10a,13a–13c].
Similarly, chromene molecules garnered fascination for their
miscellaneous biological properties and medicinal applications, specif-
ically anti-inflammatory, anticancer, and antifungal [14a–14e]. As an
extension of our aforementioned work, the chromene moiety pertains
the perception of an extraordinary scaffold for the evolution of novel
drug-like nominees [15]. Presently, the prospective combination of the
pyridine and chromene moieties could unveil innovative features, which
revolutionize their medicinal behavior.
Herein, we present the synthesis of a novel series of pyridone and
chromenopyridone derivatives incorporating a 4-hydroxyphenyl moi-
ety, utilizing 2-cyano-N-(4-hydroxyphenyl)acetamide (2), which is an
superior precursor for achieving several classes of heterocyclic mole-
cules due to its highly reactive bifunctionality [16a–16i]. In an attempt
* Corresponding author.
** Corresponding author.
E-mail addresses: alfayed_e@azhar.edu.eg (E.A. Fayed), yossry@azhar.edu.eg (Y.A. Ammar).
https://doi.org/10.1016/j.bioorg.2021.104742
Received 4 July 2020; Received in revised form 5 February 2021; Accepted 9 February 2021
Available online 17 February 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

E.A. Fayed et al.
Bioorganic Chemistry 109 (2021) 104742
to discover new and valuable agents for treating inflammatory diseases,
we have replaced the acetyl group of paracetamol with additional het-
erocycles, which have been found to possess an interesting profile of
anti-inflammatory activity with significant reduction in their ulcero-
genic effect. The structure modifications proposed in the present
investigation (Fig. 2) centered mainly on analyzing the impact of linking
various polysubstituted pyridines or fused pyridines. The substitution
pattern of the target compounds includes various functionalities that
would act as hydrogen-bond forming centers, such as the carbonyl,
amide, amino, cyano, hydroxy, and carbethoxy groups. Upon the suc-
cessful preparation, the majority of the proposed compounds were
screened for their in-vitro and in-vivo anti-inflammatory, ulcerogenic,
and antipyretic activities, which illustrated optimistic results. Further-
more, the synthesized molecules have been tested with the in-silico
prediction approach in order to forecast their physicochemical drug
likeliness. Computational high-throughput “absorption, distribution,
metabolism, and excretion” (ADME) investigations are considered to be
an effectual prototype for sieving synthetic molecules for drug discovery
affairs [17a–17c]. Considering these junctures worthy, the synthesized
molecules were examined for their pharmacodynamic and pharmaco-
kinetic properties via ADME predictions.
corresponding pyridone derivatives 4a-d, which was further verified by
the spectroscopic techniques. The infrared spectrum of 4b as an instance
exhibited bands at 3447, 3366, 3307, and 3177, corresponding to the
OH, NH2, and 2NH groups, while the nitrile and carbonyl groups
appeared at 2209 and 1699 cmꢀ 1, respectively. The 1H NMR spectrum
portrayed a singlet signal at 3.83 ppm, due to the methoxy group,
whereas the three singlet signals at 7.64, 7.77, and 10.01 ppm were
assignable to NH2, 2NH, and OH, which were omitted with D2O. The
mass spectroscopic measurement showed m/z 376 (M+, 2.16) with a
base peak at m/z 109. Compound 4 was further prepared by an alter-
native synthetic procedure through the reaction of the arylidene de-
rivatives 3 with the cyanoacetamide in a refluxing piperidine/ethanolic
solution.
In continuation of this medicinal project with the purpose of
obtaining pyridine derivatives containing the hydroxyphenyl moiety,
the one-pot reaction of cyanoacetamide 2 with aldehydes and acetyla-
cetone afforded the acetyl pyridine derivative 5. Molecule 5 was
established to be acquired through the reaction of the arylidene de-
rivatives 3 with acetylacetone under similar reaction conditions,
(Scheme 2). The IR spectrum of 5 contains bands at 3396, 2214, and
1660 cmꢀ 1, ascribed to the OH, CN, and two CO functionalities,
1
This work was a continuation of our efforts towards the discovery of
bioactive molecules from different heterocycles and with different bio-
logical activities [18a–18f].
respectively. The H NMR spectrum showed two singlet resonances at
2.34 and 2.48 ppm, corresponding to the two methyl protons and other
variable singlet signal at 9.35, due to the hydroxyl proton. Subsequently,
the aromatic protons were observed as signals at 6.71–7.44 ppm.
Furthermore, the interaction of 2 with aldehydes and ethyl acetoa-
cetate in a one-pot reaction produced the consequent cyanopyrid-2-one
derivatives 6a, b. The formation of 6 may be preceded via the Michael
type addition, followed by the auto-oxidation of the intermediates.
Another synthetic methodology was employed to afford 6 as showcased
in (Scheme 2) via the mixing of the arylidene derivatives 3 with the ethyl
acetoacetate under analogous reaction environments. The elemental
analysis and spectral data were in accord with the formation of pyridone
products 6, Scheme 2. As an example, the IR analysis of 6a presented
bands at 3315, 3211, 2220, and 1646 cmꢀ 1, corresponding to the OH,
CN, and two carbonyl moieties. Additionally, its 1H NMR exhibited two
singlets at 2.48 and 3.85 ppm, due to the methyl and methoxy protons
while, the two variable resonances at 9.30 and 10.00 ppm represented
the two hydroxyl protons.
2. Results and discussion
2.1. Chemistry
This report explores the utilization of 2-cyano-N-(4-hydroxyphenyl)
acetamide (2) in the construction of novel classes of molecules with
biomedical features. The examined precursor was synthesized according
to a previous methodology [6d], via a solvent free reaction of ethyl
cyanoacetate with 4-aminophenol (1). The reactivity of compound 2
towards some carbonyl molecules was investigated, as described in
(Scheme 1). A condensation reaction of the cyanoacetamide 2 with ar-
omatic aldehydes furnished the corresponding arylidene derivatives
3a–d, where their structure identities were confirmed through their
elemental analysis and spectral data. For instance, the IR spectrum of 3b
revealed strong absorption bands at 2215 and 1674 cmꢀ 1 that are due to
the olifinic CN and the carbonyl groups, respectively. Meanwhile, the 1H
NMR spectrum of 3b displayed two new singlets at δ 3.84 and 8.13 ppm,
The incorporation of the chromene moiety into the pyridine scaffold
is illustrated in Scheme 3. N-(4-hydroxyphenyl)-2-imino-2H-chromene-
3-carboxamide (7) was prepared by condensing cyanoacetamide 2 with
salicylaldehyde, implementing piperidine as a catalyst in ethanol,
(Scheme 3). The presence of 7 was achieved through its IR spectrum,
which demonstrated the lack of a nitrile band and the presence of the OH
and NH bands at 3393, 3286, and 3211 cmꢀ 1 alongside a carbonyl band
at 1663 cmꢀ 1. The 1H NMR spectrum of 7 in DMSO‑d6 displayed a lack of
CH2, which was discovered in the starting material and revealed three
variable signals at 8.57, 9.12, and 12.50 ppm attributable to the OH and
2NH functionalities while the aromatic protons appeared as a multiplet
in the region 6.73–7.82 ppm.
assigned to the methoxy and CH protons, respectively. The 13C NMR
–
–
spectrum exhibited signals at δ 56.06, 104.20, 150.34, 154.60, 160.78,
–
–
–
and 163.04 ppm, which are characteristics of the OCH , (C ), (CH ),
–
3
–
–
–
–
(C O), (C OCH ), and C O carbon, respectively. The mass spectrum
disclosed a molec3ular ion peak at m/z = 294, which is corresponding to
the molecular formula C17H14N2O3, with a base peak at m/z = 186.
This study was extended to utilize the cyanoacetamide in the syn-
thesis of pyridine derivatives via a one-pot reaction, (Scheme 2). A
mixture of cyanoacetamide 2 and the requisite aromatic aldehydes in
ethanol and in the presence of catalytic amount of piperidine yielded the
Moreover, the resulting chromene derivative 7 contained latent
Fig. 1. Notable active derivatives of pyridone.
2

E.A. Fayed et al.
Bioorganic Chemistry 109 (2021) 104742
Fig. 2. Rational design for anti-inflammatory and antipyretic compound A, in relation to the new synthesized derivatives of pyridone.
Scheme 1. Synthesis of compounds 3a-d.
functional substituents, which carry the aptitude for further chemical
transformations as new routes for the condensation of chromene with
possible biological activity. The reaction of 2-iminochromene derivative
7 with malononitrile in a refluxing piperidine/ethanolic solution affor-
ded in each case a product with their analytical and spectral data in
sound agreement with chromene[3,4–c]-pyridine derivative 8. The IR
analysis of 8 portrayed absorption bands at 3442, 3350, 3240, 3190,
exchangeable with D2O, which were assigned to the OH, NH2, and NH
protons. The mass spectrum of compound 8 disclosed a molecular ion
peak at m/z 344 (M+; 1.22%), conforming the molecular formula
C
19H12N4O3 along with the base peak at m/z 300. The formation of 8
proceeds via the Michael addition of the active methylene group of
malononitrile to activate the double bond center in 7, yielding the
acyclic Michael adduct that spontaneously cyclized to the respective
dihydrochromeno-pyridine, which aromatized to bear the final product.
Additionally, the treatment of the chromene molecule 7 with cya-
noacetamide 2 in ethanol with the catalytic amount of piperidine
2204 and 1664 cmꢀ 1, corresponding to the OH, NH2, NH, CN, and
1
–
–
amidic C O moieties, respectively. Meanwhile, the H NMR spectrum
of 8 presented 3 singlet resonances at 6.28, 6.49, and 8.91 ppm,
3

E.A. Fayed et al.
Bioorganic Chemistry 109 (2021) 104742
Scheme 2. Utilization of cyanoacetamide 2 in one-pot reactions.
furnished the amide chromeno[3,4-c]pyridine derivative 9, (Scheme 3),
as evidenced by the analytical and spectral data. The IR assessment
exhibited the lack of the cyano group and is comprised of bands at 3365,
3164, and 1675 cmꢀ 1, corresponding to the OH, NH2, NH, and carbonyl
groups. The 1H NMR evaluation possessed singlet signals at δ 7.74,
10.32, and 11.54 ppm for NH2, NH, and OH, respectively. The remedy of
the chromene compound 7 with ethyl cyanoacetate in piperidine
/ethanol fostered the chromeno[3,4-c]pyridine derivative 10, as veri-
fied by the analytical and spectral data. The infrared spectrum of the
reaction product demonstrates the characteristic absorption bands at
3365 and 3164 cmꢀ 1 for the NH/OH groups and at 2202 cmꢀ 1 for the
2.2. Biological activity
2.2.1. Anti-inflammatory activity
Most of the newly synthesized molecules were screened for their in-
vivo anti-inflammatory activity, employing the carrageenan-stimulated
rat hind paw edema method, according to Winter et al, [6e,20]. Data
of the anti-inflammatory activity is expressed as mean ± SEM. The anti-
inflammatory behavior of the compounds 4b, c, 5, 6a, b, 7, 8, and 10 in
a 100 mg/kg ratio with a rat’s body weight were compared with the
standard Paracetamol and Phenylbutazone (100 mg/kg). The first
standard (Paracetamol) was uncovered to possess mild anti-
inflammatory activity, while the second’s (Phenylbutazone) anti-
inflammatory activity is the main.
1
cyano group. The H NMR analysis indicates the absence of the ethyl
group and appearance of two doublets at 6.79, 6.91 ppm for hydrox-
–
yphenyl Ar H, in addition to three singlet signals at 9.57, 9.93 and
The results, depicted in (Table 1) and (Figs. 3 and 4), indicated that
all investigated derivatives (4b, c, 5, 6a, b, 7, 8, and 10) presented
higher anti-inflammatory activity in assessment against Paracetamol
while the majority of derivatives exhibit moderate to high activity in
comparison to Phenylbutazone. Amongst these compounds, compound
7 displayed a better performance (16.44%, 38.6%, 41.6%, and 43.48%)
10.26 ppm representative to NH and 2OH which disappeared by D2O.
The formation of 10 is assumed to ensue via the intramolecular cycli-
zation of the Michael adduct via the loss of ethanol, followed by the
tautomerization and oxidation under reaction conditions.
4

E.A. Fayed et al.
Bioorganic Chemistry 109 (2021) 104742
Scheme 3. Synthetic pathway of chromenopyridone derivatives 8, 9, and 10.
Table 1
Anti-inflammatory activity of the newly synthesized compounds, using the carrageenan-caused rat hind paw edema method in a 100 mg/kg ratio [21,22].
Compd.
After 1st h
After 2nd h
After 3rd h
After 4th h
Mean ± SE
% of Inh. 1
Mean ± SE
% of Inh. 2
Mean ± SE
% of Inh.3
Mean ± SE
% of Inh.4
Control
1.9 ± 0.087
0.0
2.02 ± 0.081
0.0
2.252 ± 0.070
0.0
2.468 ± 0.033
1.448 ± 0.087
1.524 ± 0.070
1.336 ± 0.023 a
1.448 ± 0.096 a
1.284 ± 0.027 a,c
1.596 ± 0.065 a,b
1.34 ± 0.074 a,b
1.34 ± 0.066 a
1.576 ± 0.087 a,b
1.416 ± 0.089 a
0.0
Phenylbutazone
1.9 ± 0.079
0.72
1.676 ± 0.079
29.92
14.26
24.6
1.508 ± 0.095
41.2
25.4
42.2
35.4
28
43.32
31.49
40.64
40.32
42.52
27.24
37.32
43.48
29.44
37.8
Paracetamol
1.92 ± 0.079
ꢀ 14.64
9.28
1.676 ± 0.079
1.6 ± 0.078
4b
4c
5
2.032 ± 0.029 a,b,c
1.696 ± 0.083 a,c
1.724 ± 0.081c
1.844 ± 0.076 a,b
1.844 ± 0.072 a,b,c
1.668 ± 0.020 a,b,c
1.692 ± 0.121 a,b
1.9 ± 0.081c
1.572 ± 0.070 a c
1.676 ± 0.027 a,c
1.66 ± 0.018 a,b
1.696 ± 0.023 a,b
1.604 ± 0.074 a,b
1.448 ± 0.104 a,b,c
1.708 ± 0.111 a,b
1.696 ± 0.028 a,b
1.308 ± 0.022 a,c
1.548 ± 0.084 a,b,c
1.588 ± 0.068 a,b
1.704 ± 0.099 a,b
1.54 ± 0.087 a,b
1.396 ± 0.064 a,c
1.624 ± 0.092 a,b
1.508 ± 0.068 a
7.84
24.6
ꢀ 4.64
ꢀ 11.08
6.44
18.2
6a
6b
7
10.36
16.8
18.4
26.6
41.6
24.2
32.6
16.44
ꢀ 12.84
ꢀ 3.2
38.6
8
12.04
16.52
10
Results are expressed as mean ± SEM of 6 rats.
Statistical analysis was carried out using One Way Analysis of Variance (ANOVA) followed by Turkey-Kramer multiple comparison test.
Percentage expressed data was carried out using previous relation.
a,b,c
= Significantly different from negative control, Phenylbutazone & Paracetamol at P < 0.05 using one way ANOVA.
in the opening 4 h in evaluation against Phenylbutazone, which has a %
of inhibition (0.72, 29.92, 41.2, and 43.32). The witnessed effect was
elevated when appraised with the anti-inflammatory effect of Phenyl-
butazone at all hours, thus, verifying its long-lasting duration of the
proposed compounds. Furthermore, molecule 4b revealed significant
anti-inflammatory behavior with a % of inhibition (9.28%, 24.6%,
42.2%, and 40.64%) in the first 4 h, which was superior to the anti-
inflammatory effect of Phenylbutazone in the 3rd hour only. The up-
surge in activity may be resulting from the pyridine ring, substituted
with p-methoxyphenyl, which is an electron-donating group. Another
5

E.A. Fayed et al.
Bioorganic Chemistry 109 (2021) 104742
Fig. 3. Mean ± S.E. in the 1st, 2nd, 3rd, & 4th h for the newly synthesized derivatives. Results are expressed as mean ± SEM of 6 rats. Statistical analysis was carried
out using One Way Analysis of Variance (ANOVA) followed by Turkey-Kramer multiple comparison test. Percentage expressed data was carried out using the
previous relation. a,b,c = Significantly different from negative control, Paracetamol at P < 0.05 using one way ANOVA.
Fig. 4. Percent of inhibition in the 1st, 2nd, 3rd, & 4th h for the newly synthesized molecules.
derivative, 4c, similarly containing noteworthy anti-inflammatory ac-
Table 2
tivity, possesses an effect that is less than that of 4b in the 1st and 3rd
Anti-inflammatory activity of the newly synthesized compounds using IL-6
hours (7.84 &35.4%), equivalent in the 2nd hour (24.6%), and higher in
content inhibition.
the 4th hour (40.32%). This result may be attributed to the presence of
Compd.
X ± SE
% IL-6 inhibition
the 4-bromopheny substitution, encompassing a –I effect. Additionally,
compound 5 has anti-inflammatory activity, which progressively
amplified and achieved maximum at the 4th hour to become (42.52%).
Upon replacing the methyl group in molecule 5 with the hydroxyl group
in 6b, the activity dwindled to (37.32%) at the 4th hour; this may be due
to the + M effect of the hydroxyl group.
Control a
6.5426 ± 0.0276
0.9426 ± 0.367
0.0
Phenylbutazone b
85.592%
24.403%
65.136%
49.576%
35.175%
31.430%
41.283%
40.094%
30.523%
39.523%
Paracetamol c
4.946 ± 0.222
4b
4c
5
2.281 ± 0.333 a,b,c
3.299 ± 0.774 a,b
3.7086 ± 0.375 a,b
4. 444 ± 0.432 a,b
3.8416 ± 0.265 a,b
3.9194 ± 0.396 a,b
4.545 ± 0.585 a,b
3.9292 ± 0.405b
6a
6b
7
2.2.2. Anti-inflammatory activity evaluation (in-vitro)
Six of the most active cyanoacetamides were assessed for their in-
vitro anti-inflammatory activity, exploiting the interleukin-6 Kits, ac-
cording to Croce, et al., [23] (Table 2).
8
10
Each value (X) represents the mean of IL-6 content (pg/mL) ± standard error of
the number of animals in each group (n = 6).
From the preceding Table, it was realized that all the examined
molecules demonstrated higher anti-inflammatory behavior in the
evaluation against Paracetamol with two folds or more. On the other
hand, upon comparing with Phenylbutazone, only molecule 4b was
a,b,c
= significancy different from negative control, Phenylbutazon & Para-
cetamol at P < 0.05 using one way ANOVA.
6

E.A. Fayed et al.
Bioorganic Chemistry 109 (2021) 104742
discovered to have anti-inflammatory activity nearer to Phenylbutazone
with a % of inhibition of 65.136% while the others exhibited half-fold of
this activity. The higher activity of 4b may be due to the p-methoxy
substitution of the phenyl ring, which has a + M effect. When the p-
methoxy group, replaced by the bromo moiety in 4c which has a –I ef-
fect, the anti-inflammatory effect was decreased in appraisal with
Phenylbutazone but was still superior against Paracetamol.
submucosa (Fig. 8a). In several rats, the moderate erosion of the su-
perficial area of the mucosa was shown (Fig. 8b).
The histopathological investigation of the stomach sections given 7
indicated mild erosion in the superficial area of the mucosa and in-
flammatory infiltration in the basal area of the mucosa (Fig. 9a). Addi-
tionally, the micrographs of rats given 8 presented mild erosion in the
superficial area of the mucosa (Fig. 9b). The examination revealed that
the superficial area of the mucosa appeared like normal in some rats
(Fig. 9c). The gastric mucosa of rat stomachs receiving 10 demonstrated
the superficial area of the mucosa appeared like normal (Fig. 9d).
2.2.3. Anti-pyretic activity evaluation (in vivo)
Three of the newly synthesized cyanoacitamides were assessed for
their anti-pyretic activity, utilizing the Brewer’s yeast induced rat py-
rexia method, according to Teotino et al., [24] and Panthong et al., [25]
in 10 mL/kg of 20% yeast suspension. The results in (Table 3) and
(Fig. 5) revealed that the three analyzed derivatives 3a, b, c, and d were
active as anti-pyritic agents in evaluation against Paracetamol. Com-
pounds 3b and 3d displayed significant antipyretic activity with a
2.2.5. In-silico study
In silico, physicochemical, Pharmacokinetic / ADME and drug
liqueur characteristics were used to supply standard reference drugs
such as Paracetamol and Phenylbutazone along with all synthesized
compounds. Several different physiochemical parameters have been
reported such as molar refractivity, unique nuclear class counts, number
of rotten bonds and lipophilicity. The Topological Polar Surface Area
(TPSA) is a worthy physiochemical vector evaluated for the regulation of
drug transport characteristics.
◦
decrease in the temperature of (0.98, ꢀ 0.04, and 0.38 C) for 3b and
(0.48, 0.44, and 0.4 ◦C) for 3d at the 1st, 2nd, and 3rd hours. This ac-
tivity was higher than the anti-pyritic one of Paracetamol at the 1st hour
while bearing the same anti-pyretic activity of Paracetamol at the 2nd
and 3rd hours: so, they can be used as drugs with rapid onsets. More-
over, compound 3c demonstrated similar anti-pyretic activity of Para-
cetamol at the 1st and 2nd hours, simultaneously presenting a higher
anti-pyretic behavior in comparison to Paracetamol at the 3rd hour
with a reduction in the temperature of (0.6, 0.82, and 1.18 ◦C) at the 1st,
2nd, and 3rd hours, allowing them to be employed as drugs with delayed
onsets. Compounds 3a displayed low antipyretic activity, in comparison
to ther derivatives, with a decrease in the temperature of (0.67, 0.37,
and 0.30 ◦C) at the 1st, 2nd, and 3rd hours but still higher than Para-
cetamol. The activity of these molecules may be related to the presence
of different substitutions on the phenyl ring, which can be p-methyl, p-
methoxy, p-bromo, or p-chloro for 3a, 3b, 3c, and 3d, respectively. The
behavior is attributable to the +M effect in 3b and the –I effect in case of
3c and d.
The calculations sought to predict the absorption of all the molecules
tested by the equation reported (percentage ABS = 109-(0.345 X TPSA)
[26a-26c]. In Table 4 the physicochemical characteristics are shown. In
the chosen substances, the percentage absorption ranges from 53.22% to
81.75%. Most compounds are moderately soluble in well-mounted
water and have demonstrated moderate to excellent in-silico absorp-
tion, with 94,99 and 88,54 percent respectively of the reference drugs
and the synthesized compounds.
The predicted pharmacokinetic / ADME attributes of the derivatives
being investigated are given in (Table 5). The data reveals that all of the
tested compounds demonstrated higher gastrointestinal (GI) absorption
with the exception of molecule 9 and are P-gp (p-glycoprotein) non-
inhibitors except molecule 4b. The blood brain barrier (BBB) could
not pass most of the compounds examined.
2.2.4. Ulcerogenicity evaluation
3. Conclusion
In order to create novel, effective anti-inflammatory agents, two
groups of heterocyclic derivatives have been developed. The new de-
rivatives of pyridine and chromene-pyridine were obtained via a one-pot
cyanoacetamide precursor reaction. Our objective was to substitute the
acetyl group of Paracetamol with additional heterocycles to advance the
anti-inflammatory activity and minimize their ulcerogenic effect. Due to
various functionalities on the phenyl ring, the performed in-vivo and in-
vitro assessment demonstrated the powerful behavior of these molecules.
In addition, in-silico and ADME predictions have inspected the synthe-
sized medication compounds for their physiochemical likelihood,
pharmacodynamic and pharmacokinetic properties. According to the
described findings, the evaluated compounds are prospective drug-like
candidates.
2.2.4.1. Histopathological results. In the case of rats that received Para-
cetamol, sections of the rats’ stomach exhibited numerous gastric pits
and the normal arrangement of gastric glands (Fig. 6a). Meanwhile, rats
injected with Phenylbutazone displayed shallow ulcers with the for-
mation of new capillary blood vessels surrounding the lesions, unap-
parent gastric pits, and inflammatory inflammation in their stomachs
(Fig. 6b). The developed erosion in the superficial area of the mucosa
and a patchy of edema into the submucosa were found (Fig. 6c).
Furthermore, a shallow ulcer with the developed erosion in the super-
ficial area of the mucosa and hemorrhage was seen (Fig. 6d).
The microscopic examination of stomach portions from the rat,
supplied with molecule 4b, revealed mild erosion in the superficial area
of the mucosa (Fig. 7a) while the examination of similar areas in rats
given 4c disclosed that the superficial area of the mucosa appeared more
or less like normal (Fig. 7b).
The micrographs of rat stomach segments provided with derivative
6b illustrated mild erosion of the superficial area of the mucosa, asso-
ciated with spots of inflammatory infiltration and edema in the
Table 3
The anti-pyretic activity of the newly synthesized compounds in Scheme 1 using Brewer’s yeast-induced rat pyrexia method in 10 mL/kg.
Compd.
Normal Temp
Pyrexia
After 1 h
Decrease in Temp
After 2 h
Decrease in Temp
After 3 h
Decrease in Temp
Control a
35.96 ± 0.024
36.38 ± 0.159
30.70 ± 0.020
36.08 ± 0.156
35.92 ± 0.020
35.92 ± 0.020
37.26 ± 0.342
37.28 ± 0.318
34.42 ± 0.172
36.94 ± 0.310
36.96 ± 0.222
36.84 ± 0.183
37.18 ± 0.466
36.7 ± 0.223
34.22 ± 0.183
36.28 ± 0.253
36.66 ± 0.292
36.42 ± 0.171
0.08 ◦
0.56 ◦
0.67 ◦
0.98 ◦
C
C
C
C
37.08 ± 0.203
36.52 ± 0.096
34.30 ± 0.222
36.98 ± 0.224
36.14 ± 0.213
36.4 ± 0.202
0.18 ◦
0.76 ◦
0.37 ◦
C
C
C
37.5 ± 0.244
36.9 ± 0.0836
34.53 ± 0.340
36.56 ± 0.324
35.78 ± 0.185
36.44 ± 0.335
ꢀ 0.24 ◦
C
Paracetamol b
0.38 ◦
0.30 ◦
0.38 ◦
1.18 ◦
0.40 ◦
C
C
C
C
C
3a
3b
3c
3d
ꢀ 0.04 ◦
C
0.6 ◦
C
0.82 ◦
0.44 ◦
C
C
0.84 ◦
C
7

E.A. Fayed et al.
Bioorganic Chemistry 109 (2021) 104742
Fig. 5. The decrease in Temp. at the 1st, 2nd, 3rd & 4th h., for the newly synthesized compounds.
Fig. 6c. A micrograph of section from stomach of a rat given 2 (Phenylbuta-
zone) shows the developed erosion in the superficial area of the mucosa
(arrow), and a patchy of edema (star) into the submucosa (H & E, Scale Bar ¼
20 μm).
Fig. 6a. A micrograph of section from stomach of rat treated with 1 (para-
cetamol) shows normal arrangement of gastric glands (H & E, Scale Bar ¼
20 μm).
Fig. 6d. A micrograph of section from stomach of a rat given 2 (Phenylbuta-
zone) shows shallow ulcers with the developed erosion in the superficial area of
the mucosa and hemorrhage (arrow) (H & E, Scale Bar ¼ 20 μm).
4. Material and methods
4.1. Chemistry
All the melting points were taken on the Electrothermal LA9000
series, Digital Melting point Apparatus were uncorrected. The IR Spectra
were determined, employing the KBr disc technique on the Nikolet IR
200 FT IR Spectrophotometer at Pharmaceutical Analytical Unit, Faculty
of Pharmacy, Al-Azhar University, and the values are represented in
(cmꢀ 1). The 1H NMR and 13C NMR Spectra were recorded on a Gemini
Fig. 6b. A micrograph of section from stomach of a rat received 2 (Phenyl-
butazone) shows shallow ulcers (arrow) with formation of new capillary blood
vessels (arrowhead) surrounding the lesions, unapparent gastric pits and in-
flammatory inflammation (star) (H & E, Scale Bar ¼ 20 μm).
1
300 MHz, Mercury 400 MHz for H NMR, and 100 MHz for 13C NMR
Spectrometer at the Main Chemical Warfare Laboratories, Chemical
Warfare Department, Ministry of Defense. DMSO‑d6 was used as a
8

E.A. Fayed et al.
Bioorganic Chemistry 109 (2021) 104742
Fig. 8b. A micrograph of section from stomach of rat given 6b shows moderate
erosion (arrows) of the superficial area of the mucosa (H & E, Scale Bar ¼
Fig. 7a. A micrograph of section from stomach of rat given 4b shows mild
erosion in the superficial area of the mucosa (arrow) (H & E, Scale Bar ¼
20 μm).
20 μm).
Fig. 9a. A micrograph of section from stomach of rat given 7 shows mild
erosion (arrow) in the superficial area of the mucosa and inflammatory infil-
Fig. 7b. A micrograph of section from stomach of rat given 4c shows the su-
perficial area of the mucosa appeared more or less like normal (H & E, Scale
tration (star) in the basal area of themucosa (H & E, Scale Bar = 20 μm).
Bar ¼ 20 μm).
Fig. 9b. A micrograph of section from stomach of rat given 8 shows mild
erosion (arrow) in the superficial area of the mucosa (H & E, Scale Bar ¼
Fig. 8a. A micrograph of section from stomach of rat given 6b shows mild
erosion (short arrows) of the superficial area of the mucosa associated with
spots of inflammatory infiltration (arrows) and edema in the submucosa
20 μm).
(arrowheds) (H & E, Scale Bar ¼ 20 μm).
solvent; chemical shifts were measured in δ ppm, relative to TMS as an
internal standard. The mass spectrum was recorded at 70ev on the DI-50
unit of the Schimadzu GC/ MS- QP5050A Spectrometer at the Regional
Center for Mycology and Biotechnology (RCMB) at Al-Azhar University,
represented as m/z (relative abundance %). Furthermore, the elemental
analysis (C, H, N) was carried out at the Regional Center for Mycology
and Biotechnology, Al-Azhar University, and the values were found to be
within ±0.4% of the theoretical ones, unless otherwise indicated. The
anti-inflamatory, antipyritic and histopathological studies were done at
the National Research Center, Cairo, Egypt. The progress of the reaction
was monitored by the TLC, using the TLC sheets precoated with the UV
fluorescent silica gel Merck 60 F254 plates and was visualized, utilizing
the UV lamp.
Fig. 9c. A micrograph of section from stomach of rat given 8 shows the su-
perficial area of the mucosa appeared like normal (H & E, Scale Bar ¼ 20 m).
μ
9

E.A. Fayed et al.
Bioorganic Chemistry 109 (2021) 104742
◦
226–228 C; IR (KBr):
υ
(cmꢀ 1) = 3398 (OH), 3298 (NH), 2930 (CH-
aliph), 2215 (C N), 1674 (C O); 1H NMR: δ/ppm = 3.84 (s, 3H,
–
–
–
–
–
–
–
OCH3), 6.71 (d, 2H, J = 12 Hz Ar H), 7.12 (d, 2H, J = 12 Hz Ar H),
–
–
7.39 (d, 2H, J = 12 Hz, Ar H), 7.96 (d, 2H, J = 12 Hz Ar H), 8.13 (s,
–
–
1H, C CH), 9.29 (s, 1H, OH exchangeable by D O), 9.99 (s, 1H, NH
2
exchangeable by D2O); 13C NMR: 56.06 (OCH ), 104.20, 150.34 (C C),
–
–
3
–
–
115.52 (C N), 115.30 117.36, 122.93, 124.92, 130.24, 132.90, 154.60,
–
160.78 (Ar-C), 163.04 (C O); MS: m/z (%) = 294 (52.67%) (M +•), 186
–
–
(100) C11H8 NO2˥+; Anal. Calcd. for C17H14N2O3 (294.30): C, 69.38; H,
4.79; N, 9.52; Found: C, 69.70; H, 4.87; N, 9.68%.
4.1.1.3. 3-(4-Bromophenyl)-2-cyano-N-(4-hydroxyphenyl)acrylamide
(3c). Canary yellow powder, crystallized from ethanol; yield: 82%; m.
p.: 208–210 ◦C; IR (KBr):
υ
(cmꢀ 1) = 3327 (OH), 3301 (NH), 3054
Fig. 9d. A micrograph of section from stomach of rat given 10 shows the su-
1
–
–
–
–
–
(Ar H), 2216 (C N), 1650 (C O); H NMR: δ/ppm = 6.72 (d, 2H, J =
–
perficial area of the mucosa appeared like normal (H & E, Scale Bar ¼ 20 μm).
–
–
–
8 Hz Ar H), 7.41 (d, 2H, J = 8 Hz Ar H), 7.82 (d, 2H, J = 8 Hz Ar H),
–
–
–
7.96 (d, 2H, J = 8 Hz Ar H), 8.32 (s, 1H, C CH), 9.36 (s, 1H, OH
4.1.1. Synthesis of 3-(aryl)-2-cyano-N-(4-hydroxyphenyl) acrylamides
exchangeable by D2O), 10.21 (s, 1H, NH exchangeable by D2O); MS: m/z
(%) = 344 (15.38) (M+2), 342 (28.81) (M +•), 43 (100) CHNO˥+; Anal.
Calcd. for C16H11BrN2O2 (343.17): C, 56.00; H, 3.23; N 8.16; Found: C,
56.23; H, 3.27; N, 8.44%.
(3a–d)
A mixture of 2 (1.76 g, 0.01 mol), appropriate aldehyde derivatives
(0.01 mol) and 3 drops of piperidine was initiated by fusion then with
the addition of ethanol (30 mL). The mixture was heated under reflux for
5 h. The solution was left overnight and the obtained product was
collected by filtration and recrystallized from ethanol.
4.1.1.4. 3-(4-Chlorophenyl)-2-cyano-N-(4-hydroxyphenyl)acrylamide
(3d). Brown powder, crystallized from ethanol; yield: 78%; m.p.:
250–252 ◦C; IR (KBr):
υ
(cmꢀ 1) = 3325 (broad OH & NH), 3050 (Ar H),
–
4.1.1.1. 2-Cyano-N-(4-hydroxyphenyl)-3-(4-tollyl)acrylamide (3a). Yel-
2215 (C N), 1641 (C O); 1H NMR: δ/ppm = 6.88–7.65 (m, 11H,
–
–
–
–
–
low crystals, crystallized from dioxane; yield: 85%; m.p.: 248–250 ◦C;
–
–
Ar H, NH & C CH), 9.88 (s, 1H, OH exchangeable by D O); MS: m/z
–
2
(%) = 298 (1.08) (M +•), 109 (100) C6H6 NO˥+; Anal2. Calcd.: for
IR:
υ
/cmꢀ 1 = 3425 (OH), 3325 (NH), 3050 (Ar H), 2860 (aliphatic
–
1
–
–
–
–
CH), 2220 (C N), 1647 (C O); H NMR: δ/ppm = 2.38 (s, 3H, CH ),
–
3
C
16H11ClN2O2 (298.72): C, 64.33; H, 3.71; N 9.38; Found: C, 64.09; H,
–
–
6.71 (d, 2H, J = 8 Hz Ar H), 7.37 (d, 2H, J = 8 Hz Ar H), 7.40 (d, 2H,
3.84; N, 9.51%.
–
–
–
–
J = 8 Hz Ar H), 7.86 (d, 2H, J = 8 Hz Ar H), 8.16 (s, 1H, C CH), 9.32
(s, 1H, OH exchangeable by D2O), 10.09 (s, 1H, NH exchangeable by
4.1.2. 2-Amino-5-cyano-N-(4-hydroxy-phenyl)-6-oxo-4-
(substitutedphenyl)-1,6-dihydropyridine-3-carboxamides (4a–d)
A mixture of cyano acetanilide 2 (1.76 g, 0.01 mol), cyano acetamide
(0.84 g, 0.01 mol), the requisite aldehyde (0.01 mol) and piperidine (0.5
mL) in ethanol (20 mL) was heated under reflux for 6 h, the solid product
that obtained on cooling was collected and crystallized from the proper
solvent.
D2O); 13C NMR: 21.10 (CH ), 106.07, 150.04 (C CH), 114.99 (C N),
–
–
–
–
–
3
116.33, 120.57, 122.41, 129.59, 129.96, 132.51, 142.81, 154.13 (Ar-C),
159.94 (C O); MS: m/z (%) = 278 (93.34) (M +•), 170 (100)
–
–
C
11H8NO˥+; Anal. calcd. for C17H14N2O2 (278.31): C, 73.37; H, 5.07; N,
10.07; Found: C, 73.64; H, 5.14; N, 10.38%.
4.1.1.2. 2-Cyano-N-(4-hydroxyphenyl)-3-(4-methoxyphenyl)acrylamide
(3b). Yellow granules, crystallized from dioxane; yield: 70%; m.p.:
Table 4
Physicochemical properties of the selected compounds.
Compd. No.
Fraction Csp3a
No. of rotatable bonds
HBAb
HBDc
iLogPd
Molar Refractivity
Log Se
TPSAf
In-silico % absorption
P*
PB**
3a
3b
3c
3d
4a
4b
4c
4d
5
0.12
0.26
0.06
0.06
0.00
0.00
0.05
0.05
0.00
0.00
0.09
0.09
0.05
0.00
0.00
0.00
0.00
2
5
4
5
4
4
4
5
4
4
3
4
3
3
1
2
1
2
2
3
4
3
3
3
4
3
3
3
5
4
4
5
5
6
2
0
2
2
2
2
4
4
4
4
1
2
2
3
3
4
3
1.21
3.32
2.26
2.27
2.23
2.18
1.91
1.82
2.00
1.87
3.00
2.77
2.65
1.79
1.76
1.10
1.97
42.78
S
49.33
40.62
73.12
82.35
73.12
73.12
108.21
117.44
108.21
108.21
59.30
88.76
79.53
86.32
129.03
148.33
123.24
91.98
94.99
83.77
80.59
83.77
83.77
71.67
68.48
71.67
71.67
88.54
78.38
81.56
79.22
64.49
57.83
66.48
97.76
MS
MS
MS
MS
MS
PS
81.91
83.44
84.65
81.96
106.04
107.57
108.78
106.09
107.60
106.14
104.66
78.98
PS
PS
PS
PS
6a
6b
7
MS
MS
MS
MS
MS
MS
8
96.99
9
100.37
94.60
10
P*= Paracetamol, PB**= Phenylbutazone.
a
The ratio of sp3 hybridized carbons over the total carbon count of the molecule.
number of hydrogen bond acceptors.
b
c
d
e
f
number of hydrogen bond donors.
lipophilicity.
Water solubility (SILICOS-IT [PS = Poorly soluble, MS = Moderately Soluble, S = Soluble]).
topological polar surface area (Å2).
10

E.A. Fayed et al.
Bioorganic Chemistry 109 (2021) 104742
Table 5
Pharmacokinetic/ADME properties of the selected compounds.
Compd.
No.
Pharmacokinetic/ADME properties
i
GI abs
BBB
P-gp
CYP1A2 inhibitor
CYP2C19 inhibitor
CYP2C9 inhibitor
CYP2D6 inhibitor
CYP3A4 inhibitor
Log Kp
permeantb
substratec
a
d
e
f
g
h
P*
PB**
3a
3b
3c
3d
4a
4b
4c
4d
5
High
High
High
High
High
High
High
High
High
High
High
High
High
High
High
Low
Yes
Yes
Yes
No
Yes
Yes
No
No
No
No
Yes
No
No
No
No
No
No
No
No
No
No
No
No
No
Yes
No
No
No
No
No
No
No
No
No
No
No
Yes
No
No
Yes
Yes
No
No
No
No
Yes
No
No
No
No
No
No
No
Yes
Yes
Yes
Yes
Yes
No
No
No
No
Yes
Yes
Yes
No
No
No
No
No
Yes
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Yes
Yes
Yes
Yes
Yes
No
No
No
No
No
No
No
No
No
No
No
ꢀ 6.90
ꢀ 5.94
ꢀ 5.57
ꢀ 5.94
ꢀ 5.73
ꢀ 5.51
ꢀ 6.87
ꢀ 7.25
ꢀ 7.03
ꢀ 6.81
ꢀ 6.17
ꢀ 6.60
ꢀ 6.16
ꢀ 6.10
ꢀ 6.93
ꢀ 7.64
ꢀ 6.70
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
6a
6b
7
No
No
Yes
No
8
9
No
10
High
No
a
Gastro Intestinal absorption.
Blood Brain Barrier permeant.
P-glycoprotein substrate.
b
c
d
e
f
CYP1A2: Cytochrome P450 family 1 subfamily A member 2 (PDB:2HI4).
CYP2C19: Cytochrome P450 family 2 subfamily C member 19 (PDB:4GQS).
CYP2C9: Cytochrome P450 family 2 subfamily C member 9 (PDB:1OG2).
CYP2D6: Cytochrome P450 family 2 subfamily D member 6 (PDB:5TFT).
CYP3A4: Cytochrome P450 family 3 subfamily A member 4 (PDB:4K9T).
Skin permeation in cm/s.
g
h
i
4.1.2.1. 2-Amino-5-cyano-N-(4-hydroxyphenyl)-6-oxo-4-(4-methyl-
4.1.2.4. 2-Amino-4-(4-chlorophenyl)-5-cyano-N-(4-hydroxyphenyl)-6-
phenyl)-1,6-dihydropyridine-3-carboxamide (4a). Greenish powder,
oxo-1,6-dihydropyridine-3-carboxamide (4d). Buff powder, crystallized
crystallized from dioxane; yield: 58%; m.p.: 228-230◦C (%), IR (KBr):
υ
from ethanol; yield: 69%; m.p.: 269–270 ◦C; IR (KBr):
υ
(cmꢀ 1) = 3398,
(cmꢀ 1) = 3406 (broad OH &NH2), 3109 (NH), 3060 (Ar H), 2951
3313, 3220 (OH, NH & NH2), 2209 (C N) 1669 (C O); 1H NMR:
–
–
–
–
–
–
1
–
–
–
–
(aliphatic H), 2209 (C N), 1660 (C O); H NMR: δ/ppm = 2.31 (s, 3H,
δ/ppm = 6.61 (s, 2H, 2NH exchangeable by D2O), 6.71 (d, 2H, J = 8 Hz
–
–
–
–
Ar H), 7.31 (s, 2H, NH2 exchangeable by D2O), 7.38 (d, 2H, J = 8 Hz
CH3), 6.56 (d, 2H, J = 8 Hz Ar H), 6.69 (d, 2H, J = 8 Hz Ar H), 7.21
–
–
–
–
(m, 8H, Ar H, 2NH & NH2), 10.43 (s, 1H, OH exchangeable by D2O);
Ar H), 7.40 (d, 2H, J = 8 Hz Ar H), 7.50 (d, 2H, J = 8 Hz Ar H),
10.52 (s, 1H, OH exchangeable by D2O); MS: m/z (%) = 379 (3.68) (M
+•), 40 (100) CNO˥+; Anal. Calcd.: for C19H13ClN4O3 (380.78): C, 59.93;
H, 3.44; N, 14.71; Found: C, 60.17; H, 3.38; N, 15.03%.
MS: m/z (%) = 360 (2.68) (M +•), 40 (100) CNO˥+; Anal. Calcd.: for
C
20H16N4O3 (360.37): C, 66.66; H, 4.48; N, 15.55; Found: C, 66.40; H,
4.38; N, 15.68%.
4.1.3. 5-Acetyl-4-(4-chlorophenyl)-1-(4-hydroxyphenyl)-6-methyl-2-oxo-
1,2-dihydro-pyridine-3-carbonitrile 5
4.1.2.2. 2-Amino-5-cyano-N-(4-hydroxyphenyl)-4-(4-methoxyphenyl)-6-
oxo-1,6-dihydropyridine-3-carboxamide (4b). Brown powder, crystal-
lized from dioxane; yield: 62%; m.p.: 198–200 ◦C; IR (KBr):
υ
(cmꢀ 1) =
To a solution of cyanoacetanilide 2 (1.76 g, 0.01 mol), the requisite
aldehyde in ethanol (30 mL) and piperidine (0.5 mL) was added. The
reaction mixture was heated for 8 h. The solid product that obtained
–
3447 (OH), 3366,3307 (NH2), 3177 (NH), 3065 (Ar H), 2942–2847
1
–
–
–
(aliphatic -CH), 2209 (C N), 1699 (C O); H NMR: δ/ppm = 3.83 (s,
–
–
–
–
after cooling was collected and crystallized from dioxan.
3H, OCH3), 6.71 (d, 2H, J = 8 Hz Ar H), 7.10 (d, 2H, J = 8 Hz Ar H),
Brown powder; yield: 62%; m.p.: 98–100 C; IR (KBr):
υ
(cmꢀ 1) =
◦
7.64 (s, 2H, NH2 exchangeable by D2O), 7.77 (s, 2H, 2NH exchangeable
–
–
–
–
3396 (OH), 3060 (Ar H), 2850 (aliphatic-H), 2214 (C N), 1660
–
–
by D2O), 7.93 (d, 2H, J = 8 Hz Ar H), 8.03 (d, 2H, J = 8 Hz Ar H),
10.01 (s, 1H, OH exchangeable by D2O); MS: m/z (%) = 376 (2.16) (M
+•), 109 (100) C6H7NO˥+•; Anal. Calcd.: for C20H16N4O4 (376.37): C,
63.82; H, 4.28; N, 14.89; Found: C, 64.13; H, 4.40; N, 15.17%.
1
–
(2C O); H NMR: δ/ppm = 2.34 (s, 3H, CH ), 2.48 (s, 3H, COCH ), 6.71
–
3
3
–
–
(d, 2H, J = 8 Hz Ar H), 7.22 (d, 2H, J = 8 Hz Ar H), 7.32 (d, 2H, J = 8
–
–
Hz Ar H), 7.44 (d, 2H, J = 8 Hz Ar H), 9.35 (s, 1H, OH exchangeable
by D2O); MS: m/z (%) = 380 (0.21) (M+2), 378 (0.70) (M +•), 101 (100)
C7H3N˥+; Anal. Calcd.: for C21H15ClN2O3 (378.81): C, 66.58; H, 3.99; N,
7.40; Found: C, 66.36; H, 4.12; N, 7.68%.
4.1.2.3. 2-Amino-4-(4-bromophenyl)-5-cyano-N-(4-hydroxyphenyl)-6-
oxo-1,6-dihydropyridine-3-carboxamide (4c). Yellow crystals, crystal-
lized from ethanol; yield: 66%; m.p.: 208–210 ◦C; IR (KBr):
υ
(cmꢀ 1) =
–
–
–
4.1.4. 5-Acetyl-4-aryl-6-hydroxy-1-(4-hydroxyphenyl)-2-oxo-1,2-
dihydropyridine-3-carbonitriles (6a,b)
–
3462 (OH), 3298,3205 (NH2), 3149 (NH), 3040 (Ar H), 2214 (C N),
1
–
–
1698 (C O); H NMR: δ/ppm = 7.45 (d, 2H, J = 8 Hz Ar H), 7.56 (d,
–
The reaction was obtained through two methods,
First method:
–
–
2H, J = 8 Hz Ar H), 7.79 (d, 2H, J = 8 Hz Ar H), 7.84 (s, 2H, 2NH
exchangeable by D2O), 7.88 (s, 2H, NH2 exchangeable by D2O), 7.91 (d,
A mixture of 2 (1.76 g, 0.01 mol), aldehyde derivatives (0.01 mol),
ethyl acetoacetate (1.30 mL, 0.01 mol), and piperidine (3 drops) was
initiated by fusion, followed by the addition of ethanol (20 mL). The
mixture was heated under reflux for 2 h. Furthermore, the mixture was
cooled, and the obtained product was collected by filtration and crys-
tallized from ethanol.
–
2H, J = 8 Hz Ar H), 8.02 (s, 1H, OH exchangeable by D2O); MS: m/z
(%) = 425 (1.13) (M +•), 44 (100) CH2NO˥+; Anal. Calcd.: for
C
19H13BrN4O3 (425.24): C, 53.67; H, 3.08; N, 13.18; Found: C, 53.89; H,
3.19; N, 13.45%.
11

E.A. Fayed et al.
Bioorganic Chemistry 109 (2021) 104742
– –
δ/ppm = 7.45 (d, 2H, J = 8 Hz Ar H), 7.50 (d, 2H, Ar H), 7.54 (t, 1H,
Second method:
–
–
A mixture of 3 (b, d) (0.01 mol), ethyl acetoacetate (1.30 mL, 0.01
mol), and piperidine (3 drops) initiated by fusion with ethanol (20 mL)
added and was heated under reflux for 2 h. The mixture was cooled, and
the obtained product was collected by filtration and crystallized from
ethanol.
Ar H), 7.74 (s, 2H, NH2 exchangeable by D2O), 7.78 (t, 1H, Ar H),
–
9.00 (d, 2H, J = 8 Hz Ar H), 10.32 (s, 2H, NH2 exchangeable by D2O),
11.54 (s, 2H, NH&OH exchangeable by D2O); MS: m/z (%) = 362 (2.89)
(M +•), 41 (100) CNO˥+; Anal. Calcd.: for C19H14N4O4 (362.34): C,
62.98; H, 3.89; N, 15.46; Found: C, 62.86; H, 4.02; N, 15.68%.
4.1.4.1. 5-Acetyl-6-hydroxy-1-(4-hydroxyphenyl)-4-(4-meth-oxyphenyl)-
4.1.6.3. 2-Hydroxy-3-(4-hydroxyphenyl)-5-imino-4-oxo-4,5-dihydro-3H-
2-oxo-1,2-dihydropyridine-3-carbonitrile (6a). Yellow powder, crystal-
chromenonpyridine-1-carbonitrile (10). Brown powder, crystallized from
lized from ethanol; yield: 81%; m.p.: 270–272 ◦C; IR (KBr):
υ
(cmꢀ 1) =
ethanol; yield: 38%; m.p.: ˃ 300 ◦C; IR:
υ
/cmꢀ 1 = 3365, 3167 (2OH
1
–
–
–
–
–
3315, 3211 (2OH), 3063 (Ar H), 2975–2841 (aliphatic-H), 2220
&NH), 2202 (C N), 1662 (C O); H NMR: δ/ppm = 6.79,6.91 (2d, 4H,
–
1
–
–
–
– – –
J = 12 Hz Ar H), 7.46 (d, 1H, J = 12 Hz Ar H), 7.55 (t, 1H, Ar H),
(C N), 1664 (2C O); H NMR: δ/ppm = 2.48 (s, 3H, COCH ), 3.85 (s,
–
–
3
–
–
–
–
3H, OCH3), 6.71 (d, 2H, J = 8 Hz Ar H), 7.12 (d, 2H, J = 8 Hz Ar H),
7.77 (t, 1H, Ar H), 9.04 (d, 1H, J = 8 Hz Ar H), 9.57 (s, 1H, NH
exchangeable by D2O), 9.93, 10.26 (2 s, 2H, 2OH exchangeable by D2O);
MS: m/z (%) = 345 (7.82) (M +•), 108 (100) C6H6N˥+; Anal. Calcd.: for
C19H11N3O4 (345.31): C, 66.09; H, 3.21; N, 12.17; Found: C, 66.32; H,
3.27; N, 12.41%.
–
–
7.39 (d, 2H, J = 8 Hz Ar H), 7.97 (d, 2H, J = 8 Hz Ar H), 9.30, 10.00
(2 s, 2H, 2OH exchangeable by D2O); MS: m/z (%) = 376 (5.44) (M +•),
108 (100) C6H6NO˥+; Anal. Calcd.: for C21H16N2O5 (376.36): C, 67.02;
H, 4.28; N, 7.44; Found: C, 67.30; H, 4.34; N, 7.73%.
4.1.4.2. 5-Acetyl-4-(4-chlorophenyl)-6-hydroxy-1-(4-hydroxyphenyl)-2-
4.2. Biological activities
oxo-1,2-dihydropyridine-3-carbonitrile (6b). Light brown powder, crys-
tallized from dioxane; yield: 76%; m.p.: 226–228 ◦C; IR (KBr):
υ
(cmꢀ 1
)
4.2.1. Anti-inflammatory activity
–
–
–
–
= 3399, 3313 (2OH), 3070 (Ar H), 2947 (aliphatic-H), 2267 (C N),
Eight of the newly synthesized cyanoacitanilides were assessed for
their anti-inflammatory activity, utilizing the carrageenan-induced rat
hind paw edema method, according to Winter and coworkers [20] by
employing the 1% carrageenan solution as a phlogistic agent. This
technique is based upon the ability of the anti-inflammatory agents to
reduce the edema produced in the hind paw of rat. Edema was induced
in the left hind paw of adult male albino rats, weighing (120–150 g),
obtained from the animal house of the National Research Center, Dokki,
Egypt by the sub-planter injection of 0.1 mL of 1% carrageenan in
normal saline. Each group was composed of six animals. The equipment
used was the Dial micrometer model (120–1206 Baty, Sussex, England).
The healthy animals were kept in approved plastic cages with metal
mesh lids and bottoms at a temperature of 20 ± 2 ◦C and were exposed to
a 12 h light dark cycle. Food and water was supplied ad libitum
throughout the duration of the study. Carrageenan was prepared as a 1%
W/V solution in 0.9% saline. Animals are weighed, randomized into
groups (n = 6), and kept for 1 week to acclimatize to the laboratory
conditions. This helps to keep stress levels low, which is important for
the development of a good inflammatory response. All the animals were
marked on the tail with an indelible pen for identification. Both Para-
cetamol and the appraised molecules were dissolved in DMSO and
administered to animals at an appropriate time point before the carra-
geenan injection.
1
–
–
1679 (2C O); H NMR: δ/ppm = 2.48 (s, 3H, COCH ), 6.68 (d, 4H, J =
–
3
–
8 Hz Ar H), 7.28 (d, 4H, J = 8 Hz Ar H), 9.26, 10.01 (2 s, 2H, 2OH
exchangeable by D2O); MS: m/z (%) = 382 (5.60) (M+2), 380 (15.18)
(M +•), 135 (100) C8H9NO˥+; Anal. Calcd.: for C20H13ClN2O4 (380.78):
C, 63.08; H, 3.44; N, 7.36; Found: C, 63.24; H, 3.65; N, 7.61%.
4.1.5. N-(4-Hydroxyphenyl)-2-imino-2H-chromene-3-carboxamide (7)
A mixture of cyanoacetanilide 2 (1.76 g, 0.01 mol), salicyaldehyde
(1.22 mL, 0.01 mol) and (0.5 mL) of piperidine was initiated by fusion,
ensued by the addition of ethanol (20 mL) and heated under reflux for 2
h. The solution was left overnight; the obtained product was collected by
filtration and crystallized from ethanol.
Yellow powder; yield: 83%; m.p.: 197–199 C; IR:
υ
/cmꢀ 1 = 3393
◦
1
–
–
–
(OH), 3286, 3211 (2NH), 3026 (Ar H), 1663 (C O); H NMR: δ/ppm
–
–
–
–
–
= 6.73–7.82 (m, 8H, Ar H), 8.50 (s, 1H, CH C), 8.57 (s, 1H, C NH
exchangeable by D2O), 9.17 (s, 1H, OH exchangeable by D2O), 12.50 (s,
1H, NH exchangeable by D2O); MS: m/z (%) = 280 (100) (M +•); Anal.
Calcd.: for C16H12N2O3 (280.28): C, 68.56; H, 4.32; N, 9.99; Found: C,
68.31; H, 4.46; N, 10.23%.
4.1.6. 3-(4-Hydroxyphenyl)-5-imino-4-oxo-4,5-dihydro-3H-chromeno
[3,4-c]pyridines (8–10)
A mixture of compound 7 (2.8 g, 0.01 mol) and malononitrile, cya-
noacetamide or ethyl cyanoacetate (0.01 mol), and piperidine (0.5 mL)
in ethanol (30 mL) was heated under reflux for 3 h. The solid product
was collected by filtration and crystallized from suitable solvent.
Animals were divided randomly into groups of six. One group was
kept as a -ve control receiving the vehicle (DMSO) only. Another two
groups were kept as a +ve control receiving standard drugs dissolved in
DMSO orally. Effects of the unknown compounds were evaluated against
the reference compound, whose pharmacology and action in this model
are known. The NSAID such as Paracetamol and Phenylbutazone (100
mg/kg per-orally) was used [21,22]. The investigated groups received
the analyzed drugs at 100 mg/kg dissolved in DMSO orally. The volumes
of the pre-injection paws were measured immediately prior to the
carrageenan injection. A 1% solution of carrageenan in 0.9% saline was
injected subcutaneously into the plantar region of the right hind paw
carrageenan, and the control paw volumes were measured 1hr after the
carrageenan injection and then hourly from 1 to 4 h to measure the
decrease of the paw volumes by using a micrometric caliper. The size of
edema was expressed as the increase in the thickness in mm after the
carrageenan injection.
4.1.6.1. 2-Amino-3-(4-hydroxyphenyl)-5-imino-4-oxo-4,5-dihydro-3H-
chromeno[3,4-c]pyridine-1-carbonitrile (8). Brown powder, crystallized
from ethanol; yield: 52%; m.p. ˃ 300 ◦C; IR:
υ
/cmꢀ 1 = 3442 (OH), 3350
1
–
–
–
–
–
(NH), 3240 & 3190 (NH2), 3040 (Ar H), 2204 (C N), 1664 (C O); H
–
NMR: δ/ppm = 6.28 (s, 2H, NH2 exchangeable by D2O), 6.49 (s, 1H, NH
–
exchangeable by D2O), 6.68 (d, 2H, J = 8 Hz Ar H), 6.95 (d, 2H, J = 8
–
–
–
Hz Ar H), 7.43 (d, 1H, J = 8 Hz Ar H), 7.51,7.75 (2 t, 2H, Ar H), 7.83
–
(s, 1H, OH exchangeable by D2O), 8.91 (d, 1H, J = 8 Hz Ar H); MS: m/z
(%) = 344 (1.22) (M +•), 300 (100) C18H11N3O2˥+; Anal. Calcd.: for
C
19H12N4O3 (344.32): C, 66.28; H, 3.51; N, 16.27; Found: C, 66.45; H,
3.60; N, 16.48%.
4.1.6.2. 2-Amino-3-(4-hydroxyphenyl)-5-imino-4-oxo-4,5-dihydro-3H-
4.2.1.1. Calculations. The inhibition (%) of edema was calculated for
chromeno[3,4-c]pyridine-1-carboxamide (9). Orange yellow powder,
each drug, employing the following relation:
crystallized from ethanol; yield: 38%; m.p.:˃ 300 ◦C; IR:
υ
/cmꢀ 1 = 3365
1
–
–
Percentage of inhibition = PC ꢀ PT × 100/PC
(OH), 3164 (broad NH &2NH2), 3040 (Ar H), 1675 (2C O); H NMR:
–
12

E.A. Fayed et al.
Bioorganic Chemistry 109 (2021) 104742
rectal temperatures were determined before and hourly intervals up to 3
h after drugs administration.
where PC = increase in paw thickness of the control group and PT =
increase in paw thickness of the treatment groups [27].
4.2.3.1. Statistical analysis. The experimental data were expressed as
Mean ± SEM. The statistical analysis was carried out by One Way
Analysis of Variance (ANOVA), followed by the Turkey-Kramer multiple
comparison tests to calculate the significance in the difference between
treatments. The statistical analyses were performed, applying the prism
graph pad computer program (version 5.00). P < 0.05 was taken as an
acute off value for significance.
4.2.1.2. Statistical analysis. The experimental data were expressed as
Mean ± SEM. The statistical analysis was carried out by a One Way
Analysis of Variance (ANOVA), followed by the Turkey-Kramer multiple
comparison tests to calculate the significance of the difference between
treatments. The statistical analysis was performed, using the prism
graph pad computer program (version 5.00). P < 0.05 was taken as an
acute off value for significance.
4.2.4. Ulcerogenic activity screening
4.2.2. Anti-Inflammatory activity screening (In-Vitro)
Three of the newly-synthesized active cyanoacitanilides were
assessed for their in-vitro anti-inflammatory activity, exploiting the
interleukin-6 Kits according to Croce and coworkers [23]. The assay
employs an antibody specific for IL-6. Standards, samples, and bio-
tinylated anti IL-6 were pipetted into the wells, and the IL-6 present in a
sample was captured by the biotinylated IL-6 specific detection anti-
body. The HRP-conjugated streptavidin (avidin-peroxidase) and TMB
(3, 3′, 5, and 5′-tetramethyl benzidine) substrate solution were pipetted
to the wells, resulting in a color development proportional to the amount
of the IL-6 bound. The stop solution (2 N H2SO4) changes the color from
blue to yellow, and the intensity of the color is measured at 450 nm.
4.2.4.1. Histopathological studies. A longitudinal section of the gastric
tissue was taken from the anterior part of the stomach and fixed in a 10%
formalin solution. After 24 h of the fixation followed by the embedding
in a paraffin wax, it was cut into sections of 5 µm onto a glass slide and
stained with hematoxylin-eosin for the histological assessment of the
gastric mucosa [30].
4.2.5. Method of Computation: In silico study
In the present study, all the synthesized derivatives as well as the
standard reference drugs i.e. Paracetamol and Phenylbutazone were
subjected to screening by the Swiss ADMET website (http://www.sib.
swiss) interface, provoking the in-silico ADME characteristics and
examining their aptitude to exhibit drug-likeliness [26a–26c].
4.2.2.1. Preparation of blood samples. After 4 h of the carrageenan in-
jection, five samples of blood were collected from each group under light
anesthesia by diethyl ether, according to the method of Cocchetto and
Bjornsson, utilizing the orbital sinus technique [28,29]. The serum was
separated by centrifugation at 4000 rpm (round per minute), using the
cooling centrifuge for 15 min, stored in an eppendorf tube in a
refrigerator.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
4.2.2.2. Calculation of the results. The standard curve, Fig. 4, was con-
structed to determine the amount of IL-6 in an unknown sample. The
standard curve was generated by plotting the average absorbance at 450
nm for each of the standard concentrations on the (Y) axis versus the
corresponding IL-6 concentration (pg/mL) on the (X) axis. The following
curve is obtained for the various IL-6 standards over the range of 0–500
pg/mL.
References
[1] (a) H.I. Mohammad, Z.A. Iffat, T.S. Mohammad, J. Pharm. Pharm. Sci. 5 (2013)
541–554;
(b) P.M. Henson, R.C. Murphy, Mediators of the Inflammatory Process, Vol. 6,
Elsevier, Amsterdam, Amsterdam, 1989;
(c) M.N. Gambhire, S.S. Wankhede, A.R. Juvekar, J. Young Pharm 1 (2009)
220–224.
[2] K. Afshar, S. Jafari, A.J. Marks, A. Eftekhari, A.E. MacNeily, Cochrane Database
Syst Rev (2015) 1–124, https://doi.org/10.1002/14651858.CD006027.pub2.
4.2.3. Anti-pyretic activity screening (in-vivo)
́
́
[3] R. Rodriguez-Monguio, M.J. Otero, J. Rovira, Pharmacoeconomics 21 (2003)
623–650, https://doi.org/10.2165/00019053-200321090-00002.
[4] A. Ahmadiani, M. Fereidoni, S. Semnanian, M. Kamalinejad, S. Saremi,
J. Ethanopharmacol. 61 (1998) 229–235, https://doi.org/10.1016/s0378-8741
(98)00043-9.
Three of the newly synthesized cyanoacitanilides were assessed for
their anti-pyretic activity, using the Brewer’s yeast-induced rat pyrexia
method, according to Teotino, Panthong, and coworkers [24,25] in 10
mL/kg of 20% yeast suspension. The healthy animals were kept in
approved plastic cages with metal mesh lids and bottoms at a temper-
[5] A.F. Pozharskii, A. Soldatenkov, A.R. Katritzky, Heterocycles in Life and Society:
An Introduction to Heterocyclic Chemistry, Biochemistry, and Applications, second
ed., Wiley, 2011.
◦
ature of 20 ± 2 C and were exposed to a 12 h light dark cycle. Hy-
[6] (a) L. Yet, Privileged Structures in Drug Discovery: Medicinal Chemistry and
Synthesis, Wiley, 2018, pp. 481–523;
perthermia was induced in rats, employing the Brewer’s yeast induced
rat pyrexia method, according to Panthong, Teotino, and coworkers.
Male rats, weighing (120–150 g), were used for the experiment. Animals
were fasted overnight and during the entire duration of the experiment
but given water ad libitum. The initial rectal temperatures were recor-
ded before the induction of pyrexia with a lubricated digital thermom-
eter inserted about 3 cm into the rectum of each rat. To induce pyrexia,
the rats were given 10 mL/kg of a 20% aqueous suspension of brewer’s
yeast subcutaneously. After 19 h, animals that presented an increase of
not less than 0.5 ◦C in the rectal temperature were selected for the
experiment. Animals were divided randomly into eleven groups of five
animals each. One group was kept as negative control, receiving the
vehicle (DMSO) orally. Another group was kept as positive control,
receiving the standard drug dissolved in DMSO orally. The effects of the
unknown compounds are assessed to the reference molecule whose
pharmacological action in this model is known. The selected NSAID
Paracetamol (100 mg/kg per-orally) was utilized. Eight groups received
the tested drugs orally at 100 mg/kg dissolved in DMSO orally. The
(b) A.A. Altaf, A. Shahzad, Z. Gul, N. Rasool, A. Badshah, B. Lal, E.A. Khan, J. Drug
Design Med. Chem. 1 (2015) 1–11, https://doi.org/10.11648/j.
jddmc.20150101.11;
(c) R.D. Taylor, M. MacCoss, A.D.G. Lawson, Rings in drugs, J. Med. Chem. 57
(2014) 5845–5859, https://doi.org/10.1021/jm4017625;
(d) Y.A. Ammar, E.A. Fayed, A.H. Bayoumi, M.A. Saleh, M.E. El-Araby, Am. J.
PharmTech Res. 5 (2015) 2249–3387;
(e) E.A. Fayed, A.H. Bayoumi, M.A. Saleh, Y.A. Ammar Der, Pharma Chem. 8
(2016) 495–508.(f)Y.A. Ammar, M.A. Salem, E.A. Fayed, M.H. Helal, M.S.A. El-
Gaby, H. Kh. Thabet, Synthetic Commun. doi: 10.1080/00397911.2017.1328066.
[7] E. Lukevits, Chem. Hetero. Comp. 31 (1995) 639–650, https://doi.org/10.1007/
bf01169065.
[8] A. Chicca, R. Berg, H.J. Jessen, N. Marck, F. Schmid, P. Burch, J. Gertsch,
K. Gademann, Bioorg. Med. Chem. 25 (2017) 6102–6114, https://doi.org/
10.1016/j.bmc.2017.02.031.
[9] (a) M.V. Goryaeva, Y.V. Burgart, Y.S. Kudyakova, M.A. Ezhikova, M.I. Kodess, V.
I. Saloutin, Eur. J. Org. Chem. (2017) 3986–3991, https://doi.org/10.1002/
ejoc.201700683;
(b) W.M. Eldehna, G.S. Hassan, S.T. Al-Rashood, T. Al-Warhi, A.E. Altyar, H.
M. Alkahtani, A.A. Almehizia, H.A. Abdel-Aziz, J. Enzyme Inhib. Med. Chem. 34
(1) (2019) 322–332, https://doi.org/10.1080/14756366.2018.1547286;
(c) A. Mrozek-Wilczkiewicz, K. Malarz, M. Rejmund, J. Polanski, R. Musiol, Eur. J.
13

E.A. Fayed et al.
Bioorganic Chemistry 109 (2021) 104742
Med. Chem. 171 (2019) 180–194, https://doi.org/10.1016/j.ejmech.2019.03.027;
(f) Y.A. Ammar, M.A. Mohsan, Y.A. Mohamed, H.K.H. Thabet, M.S.A. El- Gaby,
Heterocycl. Commun. 19 (2013) 195–200, https://doi.org/10.1515/hc-2013-
0032;
(d) M.F. Ansari, D. Idrees, M.I. Hassan, K. Ahmad, F. Avecilla, A. Azam, Eur. J.
Med. Chem. 144 (2018) 544–556, https://doi.org/10.1016/j.ejmech.2017.12.049;
(e) N.M. Ghanema, F. Farouka, Riham F. Georgeb, S.E.S. Abbasb, O.M. Ossama,
M. El-Badry, Biooirg. Chem. 80 (2018) 565–576, https://doi.org/10.1016/j.
bioorg.2018.07.00;
(g) Y.A. Ammar, S.Y. Abbas, M.M. Ghorab, M.S. Al Said, Tetrahedron Lett. 57
(2016) 275–277, https://doi.org/10.1016/j.tetlet.2015.11.098;
(h) Y.A. Ammar, A.G. Al-Sehemi, M.M. Ali, Y.A. Mohamed, H.Kh. Thabet, M.S.
A. Al-gaby, Hetrocycl. lett. 5 (2015) 157–167;
(f) Y.-K. Shi, B. Wang, X.-L. Shi, Y.-D. Zhao, B. Yu, H.-M. Liu, Eur. J. Med. Chem.
145 (2018) 11–22, https://doi.org/10.1016/j.ejmech.2017.12.094;
(g) S. Prachayasittikul, R. Pingaew, A. Worachartcheewan, N. Sinthupoom,
V. Prachayasittikul, S. Ruchirawat, Mini-Rev. Med. Chem. 17 (2017) 869–901,
https://doi.org/10.2174/1389557516666160923125801;
(i) Y.A. Ammar, M.S.A. El-Gaby, M.A. Salem, Arabian J. Chem. 7 (2014) 615–622,
https://doi.org/10.1016/j.arabjc.2013.11.026.
[17] (a) C.A. Lipinski, J. Pharmacol. Tox. Methods. 44 (2000) 235–249, https://doi.
org/10.1016/s1056-8719(00)00107-6;
(h) C.W. Fu, Y.J. Hsieh, T.T. Chang, C.L. Chen, C.Y. Yang, A. Liao, P.W. Hsiao, W.
S. Li, Eur. J. Med. Chem. 104 (2015) 165–181;
(b) D. Butina, M.D. Segall, K. Frankcombe, Drug Discovery Today. 7 (2002)
S83–S88, https://doi.org/10.1016/S1359-6446(02)02288-2;
(c) D. Zhang, G. Luo, X. Ding, C. Lu, Acta Pharm. Sin. B 2 (6) (2012) 549–561,
https://doi.org/10.1016/j.apsb.2012.10.004.
(i) S. Zheng, Q. Zhong, M. Mottamal, Q. Zhang, C. Zhang, E. LeMelle, H. McFerrin,
G. Wang, J. Med. Chem. 57 (2014) 3369–3381, https://doi.org/10.1021/
jm500002k.
[18] (a) S.A. El-Kalyoubi, E.A. Fayed, A.S. Abdel-Razek, Chem. Central J. 11 (1) (2017)
1–13, https://doi.org/10.1186/s13065-017-0294-0;
´
[10] (a) J.A.D. Good, J. Silver, C. Nuñez-Otero, W. Bahnan, K.S. Krishnan, O. Salin,
¨
¨
P. Engstrom, R. Svensson, P. Artursson, A. Gylfe, S. Bergstrom, F. Almqvist, J. Med.
Chem. 59 (2016) 2094–2108, https://doi.org/10.1021/acs.jmedchem.5b01759;
(b) K.M. Carlson-Banning, A. Chou, Z. Liu, R.J. Hamill, Y. Song, L. Zechiedrich,
PLoS One 8 (2013), e69646, https://doi.org/10.1002/ejoc.201700683;
(c) A. Subissi, D. Monti, G. Togni, F. Mailland, Drugs 70 (2010) 2133–2152,
https://doi.org/10.2165/11538110-000000000-00000.
(b)S. Elkalyoubi, E.J. Fayed, Chem. Res. 40(12) 2016, doi: 10.3184/
174751916X14798125870610. (c) E.A. Fayed, S.I. Eissa, A.H. Bayoumi, N.
A. Gohar, A.B.M. Mehany, Y.A. Ammar, Mol. Diver. 23 (1) (2019) 165–181,
https://doi.org/10.1007/s11030-018-9865-9;
(d) Y.A. Ammar, E.A. Fayed, A.H. Bayoumi, Res. Chem. Intermed. (2017), https://
doi.org/10.1007/s11164-017-3019-z;
´
´
´
ˇ
[11] D. Mijin, B.B. Nedeljkovic, B. Bozic, I. Kivrlija, J. Ladarevic, C. Uscumlíc, Turk. J.
Chem. 42 (2018) 896–907, https://doi.org/10.3906/kim-1711-97.
[12] H. Jia, Y. Song, J. Yu, P. Zhan, D. Rai, X. Liang, C. Ma, X. Liu, Eur. J. Med. Chem.
136 (2017) 144–153, https://doi.org/10.1016/j.ejmech.2017.04.048.
[13] (a) M. Ravinder, B. Mahendar, S. Mattapally, K.V. Hamsini, T.N. Reddy, C. Rohit,
K. Srinivas, S.K. Banerjee, V.J. Rao, Bioorg. Med. Chem. Lett. 22 (2012)
6010–6015, https://doi.org/10.1016/j.bmcl.2012.05.019;
(e) E.A. Fayed, H.Y. Der Ahmed, Pharma Chemica 8 (13) (2016) 77–90;
(f) B.A. AbdelFattah, M. Khalifa, A.H. El-Sehrawi, E. Fayed, A. Bayoumi, Lett.
Drug Des. Discov. 8 (4) (2011) 330–338, https://doi.org/10.2174/
157018011794839448.
[20] C.A. Wintere, D.A. Risley, G.W. Nuss, Exp. Biol. Med. 111 (1962) 544–547, https://
doi.org/10.1016/j.molliq.2018.05.071.
[21] K.P.P. Peiris, B.K. Ashok, J. Ind, Nat. Prod. Res. 2 (2011), https://doi.org/10.3390/
molecules23071727.
(b) F.F. Fleming, L. Yao, P.C. Ravikumar, L. Funk, B.C. Shook, J. Med. Chem. 53
(2010) 7902–7917, https://doi.org/10.1021/jm100762r;
[22] G. Mariappan, B.P. Saha, L. Sutharson, A. Singh, S. Garg, L. Pandey, D. Kumar,
Saudi Pharm. J. 19 (2011) 115–122, https://doi.org/10.1016/j.jsps.2011.01.00.
[23] M.A. Croce, T.C. Fabian, J.H. Patton, S.M. Melton, M. Moore, L.L. Trenthem,
J. Trauma 45 (1998) 273–282, https://doi.org/10.1097/00005373-199808000-
00012.
(c) P. Fossa, G. Menozzi, P. Dorigo, M. Floreani, L. Mosti, Bioorg. Med. Chem. 11
(2003) 4749–4759, https://doi.org/10.1016/S0968-0896(03)00528-5.
[14] (a) S.L. Wang, Y.C. Tsai, S.L. Fu, M.J. Cheng, M.I. Chung, J.J. Chen, Molecules 23
(2018) 289–300, https://doi.org/10.3390/molecules23020289;
(b) O. Pontes, M. Costa, F. Santos, B. Sampaio-Marques, T. Dias, P. Ludovico,
F. Baltazar, F. Proença, Eur. J. Med. Chem. 157 (2018) 101–114;
(c) N.D. Than, D.S. Hai, V.T. Ngoc Bich, P.T. Thu Hien, N.T. Ky Duven, N.T. Mai,
T.T. Dung, V.N. Toan, H.T. Kim Van, L.H. Dang, D.N. Toan, T.T. Thanh van, Eur. J.
Med. Chem 167 (2019) 454–474, https://doi.org/10.1016/j.ejmech.2019.01.060.
(d)E.A. Fayed, R. Sabour, M.F. Harras, A.B.M. Mehany, Med. Chem. Res. doi
10.1007/s00044-019-02373-x.
[24] U.M. Teotino, L.P. Friz, A. Gandini, D.D. Bella, J. Med. Chem. 6 (1963) 248–250,
https://doi.org/10.1021/jm00339a007.
[25] A. Panthong, P. Norkaew, D. Kanjanapothi, T. Taesotikul, N. Anantachoke,
V. Reutrakul, J Ethnopharmacol. 4 (111) (2007) 335–340, https://doi.org/
10.1016/j.jep.2006.11.038.
[26] (a) F.F. Al-blewi, N. Rezki, A. Naqvi, H. Husna Qutb Uddin, S. Al-Sodies,
M. Messali, M.R. Aouad, S.A. Bardaweel, Int. J. Mol. Sci. 20 (2019) 2865–2884,
https://doi.org/10.3390/ijms20122865;
[15] S. Khode, V. Maddi, P. Aragade, M. Palkar, P.K. Ronad, Sh. Mamledesai, A.H.
M. Thippeswamy, D. Satyanarayana, Eur. J. Med. Chem. 44 (2009) 1682–1688.
[16] (a) V.D. Dyachenko, R.P. Tkachiov, O.S. Bityukova, Russ. J. Org. Chem. 44 (2008)
1565–1579, https://doi.org/10.1134/S1070428008110018;
(b) E.A. Fayed, Y.A. Ammar, A. Ragab, N.A. Gohar, A.B.M. Mehany, A.M. Farrag,
Bioorg. Chem. 100 (2020), https://doi.org/10.1016/j.bioorg.2020.103951;
(c) Y.A. Ammar, A.A. Farag, A.M. Ali, S.A. Hessein, A.A. Askar, E.A. Fayed, D.
M. Elsisi, A. Ragab, Bioorg. Chem. 99 (2020), https://doi.org/10.1016/j.
bioorg.2020.103841.
(b) A.A. Fadda, M.M. Mukhtar, H.M. Refat, Am. J. Org. Chem. 2 (2012) 32–40,
https://doi.org/10.5923/j.ajoc.20120202.06;
(c) A.A. Farag, S.N. Abd-Alrahman, G.F. Ahmed, R.M. Ammar, Y.A. Ammar, S.
Y. Abbas, Arch. Pharm. Chem. Life Sci. 345 (2012) 703–712, https://doi.org/
10.1002/ardp.201200014;
[27] P. Das, S. Mekap, S. Pani, R. Sethi, P. Nayak, Der Pharmacia Lettre 2 (2010)
151–154.
[28] D.M. Cocchetto, T.D. Bjornsson, J. Phar. Sci. 72 (1983) 465–492, https://doi.org/
10.1002/jps.2600720503.
(d) Y.A. Ammar, M.M. Aly, A.G. Al-Sehemi, M.A. Salem, M.S.A. El-Gaby, J. Chin.
Chem. Soc. 56 (2009) 1064–1071, https://doi.org/10.1002/jccs.200900154;
(e) Y.A. Ammar, S. Bondock, A.G. Al-Sehemi, M.S.A. El-Gaby, A.M. Fouda, H.
Kh. Thabet, Turk. J. Chem. 35 (2011) 893–903, https://doi.org/10.3906/kim-
1104.54;
[29] H.S. Sanford, Science 119 (1954) 100, https://doi.org/10.1126/
science.119.3081.100.
[30] R.A.B. Drury, E.A. Wallington, Carleton’s Histological Technique, fourth ed.,
Oxford University Press, 1980.
14