Design, synthesis, and pharmacological characterization of some 2-substituted-3-phenyl-quinazolin-4(3H)-one derivatives as phosphodiesterase inhibitors

Article abstract of DOI:10.1002/ardp.202100051

Some 3-phenyl-quinazolin-4(3H)-one-2-thioethers (3a–e, 5a,b, 7a–e, 9a–d, 10a–d, and 12) along with 2-aminoquinazoline derivatives 13a–c were prepared and screened for their in vitro phosphodiesterase (PDE) inhibitory activity. Some compounds such as 7d,e,

Full text of DOI:10.1002/ardp.202100051

Received: 3 February 2021
Revised: 14 April 2021
Accepted: 16 April 2021
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DOI: 10.1002/ardp.202100051
F U L L P A P E R
Design, synthesis, and pharmacological characterization of
some 2‐substituted‐3‐phenyl‐quinazolin‐4(3H)‐one
derivatives as phosphodiesterase inhibitors
Kamilia M. Amin1 | Gehan H. Hegazy1 | Riham F. George1
| Nahla R. Ibrahim1
|
Nada M. Mohamed2
1Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, Cairo University, Cairo,
Egypt
Abstract
Some 3‐phenyl‐quinazolin‐4(3H)‐one‐2‐thioethers (3a–e, 5a,b, 7a–e, 9a–d, 10a–d,
and 12) along with 2‐aminoquinazoline derivatives 13a–c were prepared and
screened for their in vitro phosphodiesterase (PDE) inhibitory activity. Some com-
pounds such as 7d,e, 9a,b,d, 10a,d, and 13b exhibited promising activity as com-
pared with the non‐selective PDE inhibitor IBMX. This inhibitory activity was
validated by molecular docking in the active site of PDE7A and PDE4 to investigate
their selectivity. Furthermore, the most active compound 10d (IC50 = 1.15 μM)
was tested in vivo using behavioral tests. Compound 10d was able to pass the
blood–brain barrier and improve scopolamine‐induced cognitive deficits. Therefore,
this core can be considered as a promising scaffold for further optimization to
obtain new compounds with better PDE7A selective inhibition.
2Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, Modern University for
Technology and Information MTI, Cairo, Egypt
Correspondence
Riham F. George, Department of
Pharmaceutical Chemistry, Faculty of
Pharmacy, Cairo University, Cairo 11562,
Egypt.
Email: rihamfgeorge@gmail.com
K E Y W O R D S
behavioral tests, molecular docking, phosphodiesterase, quinazoline
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1
INTRODUCTION
directed attention to customized selective PDE4D inhibitors that
have promising activity to restore memory impairment without in-
Phosphodiesterases (PDEs) are enzymes that catalyze the break-
down of cyclic purine nucleotides (cAMP/cGMP) to 5ʹ‐AMP and
5ʹ‐GMP through hydrolyzing their 3ʹ phosphate bond.[1] The PDEs
are classified into 11 different subfamilies where PDE1, PDE4, PDE7,
and PDE10 are highly expressed in brain. Therefore, increasing
cAMP level could be involved in the management of neurodegen-
erative diseases and interfering with both the inflammatory and
neurotransmitters' cascades. Thus, selective inhibition of these PDEs
could represent a novel approach to treat several central nervous
system (CNS) disorders[2,3] such as depression, ischemia–reperfusion
injury, and Alzheimer's disease.[4–6] To date, most of research on PDE
inhibitors was centered on the PDE4 family owing to their promising
results in neuroprotection.[7–9] However, their limited clinical use
due to significant side effects such as emesis and sedation[10]
ducing emesis.[11] Furthermore, searching for another cAMP‐specific
PDE family such as PDE7 has been started to show up where several
PDE7A inhibitors have been reported in the literature.[12–14] These
inhibitors have been of great interest not only due to their promising
results in neuroprotection and low potential for clinically significant
side effects but also due to their catalytic domain similarity with
PDE4.[15] This similarity makes it desirable to develop either dual
PDE4/7 inhibitors or selective PDE7 inhibitors.[16]
When designing potential candidates for PDE7A inhibition, the two
essential elements for binding should be taken into consideration. First,
stacking against the conserved phenylalanine residue necessitates the
presence of an aromatic system. Consequently, most of the subse-
quently designed PDE7 inhibitors had a bicyclic heteroaromatic scaffold
containing a pyrimidine ring that resembled the natural substrate cAMP.
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Second, forming a hydrogen bond with the invariant glutamine residue,
where the presence of a group that can form H‐bond at position 4 of the
pyrimidine ring is essential.[17] In this emerging and challenging scenario,
many scaffolds were used in the design of PDE7 inhibitors such as
thieno[3,2‐d]pyrimidines,[12–14] benzothiadiazine, benzothienothiadiazine
derivatives,[18,19] spiroquinazolinones, sulfonamide, and thiadiazole
analogs.[20,21] Furthermore, a ligand‐based virtual screening, using the
descriptors generated by CODES program,[22] pointed out the quina-
zoline derivatives as new PDE7 inhibitors.[23] After a preliminary syn-
thetic optimization, new and potent quinazoline derivatives as S14 (1)
and a thienopyrimidine derivative (II) were obtained.[17] Moreover,
alkylation of sulfur at position 2 of the pyrimidine resulted in higher
potencies, as mentioned by Castaño et al.[17] Upon comparing the
PDE7A1 inhibition of the synthesized 2‐thioxoquinazolinones (IIIa–c)
with the 2‐methylthioquinazolinones (IVa–c) that showed IC50 = 1–10
and 0.24–1 µM, respectively, almost 10‐fold increment in the activity
was observed. In addition, the in silico data predicted that these com-
pounds could cross the blood–brain barrier (BBB) and had anti‐
inflammatory properties (Figure 1).
variable substituents at position 2. Consequently, it seemed of in-
terest to prepare compounds 3a–e and 5a,b aiming to deduce the
effect of the alkyl spacer length of the 2‐mercapto side chain on the
activity. Similarly, 7a–e was a comparable series that could reveal
whether the aromatic amides differ from the alicyclic amides of 3a–e
regarding the activity and, on the contrary, whether the amide group
was important for potency as compared with 9a–d. Moreover, mo-
tivated by the positive impact of the oxime as a functional group and
based on the suggestion that oximes were capable of interacting with
the substrate‐binding pocket of PDE4[27] where they could gain ac-
cess to the CNS despite being hydrophilic probably by carrier‐
mediated transport[28], some new oxime derivatives 10a–d and their
hydrazone bioisosteres 12a–d were prepared to investigate the ef-
fect of this modification on the obtained inhibitory activity. Finally,
compounds 13a–c were designed to evaluate the effect of additional
heterocycles as pyrrolidindione, pyrroledione, and isoindolinedione
at position 2 on the obtained activity (Figure 2).
All the target compounds were screened for their in vitro
PDE inhibitory activity and their activity was validated by
molecular docking in the active site of PDE7A, PDE4B, and
PDE4D to predict their selectivity and to highlight the key
enzyme–inhibitor interactions. Furthermore, the most active
compound was subjected to in vivo behavioral tests to confirm
its ability to pass the BBB and improve cognitive activity in
mice model.
Furthermore, many reported potent PDE7 inhibitors were sub-
stituted at position 2 of the pyrimidine ring with –NH‐containing
moieties as in compound V.[12] This pharmacophoric feature was
found to enhance the activity probably by assisting the binding to
PDE7A through the formation of an H‐bond with one water molecule
in the active site, as configured from the 2D ligand interaction of V
with the catalytic domain of PDE7A.[12]
On the basis of the aforementioned data that confirmed the
importance of quinazoline as a promising core in the design of PDE7
inhibitors due to its high potency and BBB permeability,[24] in addi-
tion to its ease of chemical accessibility,[25,26] quinazolin‐4(3H)‐one
scaffold was adopted for the synthesis of various 2‐substituted
quinazolinone derivatives bearing different side chains with a variety
of functional groups as potential candidates for PDE7 inhibition with
expected neuroprotective activity.
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2
RESULTS AND DISCUSSION
Chemistry
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2.1
The target compounds were synthesized as depicted in Schemes 1
and 2. All reactions were performed via S‐alkylation of 2‐thioxo‐3‐
phenylquinazolin‐4(3H)‐one (1)[29,30] through nucleophilic substitu-
tion SN2 of different reagents that afforded the required S‐alkylated
products rather than N‐alkylated analogs due to higher nucleophilicity
Thus, 2‐thioxo‐3‐phenyl‐quinazolin‐4(3H)‐one S14 (1) was taken
as a key starting material to obtain different derivatives by using
FIGURE 1 Structures of some PDE7 inhibitors (1, II–V)

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FIGURE 2 The structures of the lead
compound and the target compounds
of –SH group than –NH moiety. Thus, the reaction of 1 with the
prepared acyl halide derivatives 2a–e[31–36] resulted in the formation
of thioethers 3a–e. This was achieved using different solvents and
catalysts to obtain the required derivative in a higher yield. Thus,
sodium ethoxide in absolute ethanol was used for 3a, whereas anhy-
drous potassium carbonate in dry dimethylformamide resulted in
compounds 3b,d,e. However, anhydrous potassium carbonate in dry
acetone was used with the addition of catalytic amount of potassium
iodide to afford 3c. The formation of 3a–e was confirmed by spectral
data where infrared (IR) spectra revealed the disappearance of NH
stretching band at 3244 cm−1 and the presence of an additional car-
bonyl band at 1685–1663 cm−1. Moreover, 1H nuclear magnetic re-
sonance (NMR) spectra showed a singlet signal at δ 4.13–4.23 ppm
attributed to CH2 protons along with the aliphatic protons of the
introduced alicyclic moieties. The 13C NMR spectra of 3a–e confirmed
their structures through the presence of signals at δ 29.3–36.2,
160.4–161.6, and 165.4–166.2 ppm assigned to –CH2S and 2 –C═O,
respectively, in addition to signals of the alicyclic amines' carbons and
aromatic carbons. Also, the mass spectrum of compound 3a showed a
molecular ion peak at m/z 381.49, which was consistent with its mo-
lecular weight.
Similarly, compounds 5a,b were prepared under the same con-
ditions adopted for the preparation of 3a and 3c, using the prepared
propionyl chloride derivatives 4a and 4b,[37] respectively. The
structures of 5a,b were confirmed by spectroscopic data where their
IR spectra revealed the appearance of aliphatic –CH stretching
bands at 2981–2927 cm−1 and additional C═O stretching bands of
the side chain at 1693 and 1685 cm−1 for compounds 5a and 5b,
respectively. Furthermore, their 1H NMR spectra displayed the ap-
pearance of the two characteristic triplet signals of the propyl side
chain at δ 2.85–2.91 and 3.48–3.49 ppm in addition to the other
expected signals of the morpholine and phenyl piperazine protons.
13C NMR spectra of 5a,b exhibited two signals for the ethylene
carbons at δ 27.8–27.9 and 33.2–33.3 ppm, and signals of the mor-
pholine and piperazine carbons at δ 45.7, 66.9, and 41.6–50.0 ppm,
respectively. Besides, their 13C NMR spectra showed two signals
corresponding to the carbonyl carbons at
δ 160.4–161.8 and
166.5–168.6 ppm along with those of the aromatic carbons.
Likewise, the targeted compounds 7a–e were obtained upon
heating reactant 1 with the prepared aromatic chloroacetamides
6a–e[38–40] in dimethylformamide in the presence of potassium car-
bonate as a base catalyst. IR spectra of 7a–e expressed NH

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SCHEME 1 The synthetic pathways for compounds 3a–e, 5a,b, and 7a–e. Reaction conditions and reagents: (a) 2a, NaOEt, EtOH for 3a;
2b,d,e, K2CO3, DMF, heat, 24h for 3b,d,e; 2c, K2CO3, KI, dry acetone, heat, 48h for 3c; (b) 4a, NaOEt, EtOH, heat, 2h for 5a; 4b, K2CO3, KI, dry
acetone, heat, 48h for 5b; (c) K2CO3, DMF, heat, 8‐10h
(a)
(b)
(c)
(d)
SCHEME 2 The synthetic pathways for compounds 9a–d, 10a–d, 11, 12, and 13a–c. Reaction conditions and reagents: (a) K2CO3, dry
acetone, reflux, 24h; (b) NH2OH.HCl, ethanol, reflux, 12h; (c) NH2NH2.H2O, ethanol, reflux, 12h; (d) appropriate acid anhydride, glacial acetic
acid, reflux, 16h

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stretching bands in the range of 3341–3287 cm−1 and C═O
stretching band for the aromatic acetamide derivatives in the range
of 1663–1655 cm−1. Moreover, their 1H NMR spectra revealed the
presence of the methylene singlet signal at δ 3.90–4.01 ppm, along
with an exchangeable NH at δ 9.68–10.01 ppm in addition to in-
crease in aromatic signals assigned for aromatic protons. Further-
more, 13C NMR spectrum of 7e, as a representative example,
exhibited methylene carbon signal at δ 36.7 ppm while that of the
methoxy at δ 55.5 ppm. The two signals corresponding to the car-
bonyl carbons appeared at δ 160.8 and 166.2 ppm, besides those of
the aromatic carbons. Also, the mass spectrum of compound 7b
showed a molecular ion peak at m/z 421.08, which was consistent
with its molecular weight (Scheme 1).
by spectral data; IR spectrum displayed the presence of a forked
stretching band at 3580 and 3472 cm−1 corresponding to NH2 group
and another band at 3306 cm−1 for the NH group. Its 1H NMR
spectrum displayed two singlet signals at δ 4.39 and 6.90 ppm, which
both disappeared upon deuteration corresponding to NH2 and NH,
respectively, along with the normal pattern of other protons. How-
ever, confirmation of the resultant product 12 relied on its spectral
data; IR spectrum revealed the presence of a forked stretching band at
3256 and 3125 cm−1 corresponding to NH2 group and the dis-
appearance of one of the two carbonyl stretching bands of 9d at
1670 cm−1 1H NMR displayed a singlet signal at δ 6.97 ppm of the
.
amino group, which disappeared upon deuteration. 13C NMR spec-
trum showed the appearance of two signals of the imine and carbonyl
carbons at δ 157.8 and 161.2 ppm, respectively. Aliphatic carbons
appeared at δ 25.2 and 55.6 ppm for the methylene and methoxy
groups, respectively. Also, the mass spectrum of compound 12 showed
a molecular ion peak at m/z 416.46, which was consistent with its
molecular weight. Furthermore, synthesis of the desired 13a–c was
achieved by aminolysis of 2‐hydrazinyl‐3‐phenylquinazolin‐4(3H)‐one
(11) through refluxing with the appropriate acid anhydride in glacial
acetic acid. Structures of 13a–c were confirmed by spectral data;
IR spectra showed the disappearance of the forked band corre-
sponding to NH2 group while retaining NH band in the range of
3395–3217 cm−1, in addition to the appearance of extra two C═O
bands that overlapped the C═O of the quinazolinone ring in the range
of 1744–1694 cm−1. The mass spectrum of compound 13a showed a
molecular ion peak at m/z 334.14, which was consistent with its mo-
lecular weight.
Alkylation of the aromatic thiol group of compound 1 with the
prepared phenacyl bromide derivatives 8a–d[41] could be achieved
using anhydrous potassium carbonate in dry acetone to give the
reported compounds 9a–d[42] (Scheme 2). Structures of compounds
9a–d were confirmed by spectral data as follows: IR spectra showed
the presence of aliphatic CH stretching at 2955–2909 cm−1, in ad-
dition to an extra (C═O) band at 1686–1670 cm−1 1H NMR spectra
.
revealed singlet signal at δ 4.53–4.62 ppm corresponding to the
methylene protons and another singlet signal corresponding to the
methoxy protons at δ 3.93 ppm in case of compound 9d in addition to
the increase in aromatic protons. 13C NMR spectrum of 9d displayed
signals at δ 39.5 and 55.6 ppm due to methylene and methoxy car-
bons, respectively, along with two signals at δ 164.0 and 192.1 ppm
for the two carbonyl groups of the quinazolinone ring and ethanone
side chain, respectively. The oxime derivatives 10a–d were obtained
from reactions of 9a–d with hydroxylamine hydrochloride in abso-
lute ethanol without using a catalyst. Formation of those novel
compounds was confirmed by spectral data where IR spectra
showed the presence of the characteristic OH stretching bands
in the range of 3217–3198 cm−1along with the disappearance of one
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2.2
Biological evaluation
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2.2.1
In vitro PDE inhibitory activity
of the two carbonyl bands of compounds 9a–d at 1686–1678 cm−1
.
1H NMR spectra revealed the presence of exchangeable OH
signals at δ 11.57–11.95 ppm, which disappeared upon deuteration.
13C NMR spectrum of compounds 10a–d showed a signal at
25.3–31.1 ppm for the methylene carbon, two signals at 157.1–157.4
and 161.1–161.2 ppm for the imine carbon and amidic carbonyl
carbon, respectively, in addition to the normal pattern of the aro-
matic carbons of the compounds. Also, the mass spectrum of com-
pound 10b showed a molecular ion peak at m/z 466.22, which was
consistent with its molecular weight.
All the newly synthesized compounds were evaluated for their pre-
liminary in vitro PDE inhibitory activity using Abcam PDE Activity
Assay Kit (ab139460), a colorimetric non‐radioactive assay designed
in a microplate format. IBMX, a non‐selective PDE inhibitor, was
used as a reference standard to validate the PDE inhibitory activity
rather than to compare the selectivity. The in vitro testing assay was
carried out at the confirmatory diagnostic unit, Vacsera, Egypt, and
the results as IC50 (µM) values are represented in Table 1.
From the obtained results, it can be observed that most of the
synthesized compounds showed inhibitory activity in the range of
IC50 values (1.15–4102.00 µM), compared with IBMX as a reference
standard with IC50 4.68 µM.
It is noteworthy that compound 11 was obtained accidentally
while intending the synthesis of the hydrazone derivatives from
series 9a–d upon their reaction with hydrazine hydrate in ethanol.
They all were cleaved at the S‐alkyl side chain to give the hydrazinyl
derivative 11, except the methoxy‐substituted derivative 9d that
gave the desired hydrazone 12. This could be explained in a way that
the electron‐donating effect of the methoxy in 9d stabilized the
molecule and hindered the nucleophilic substitution, whereas the
other halogenated and unsubstituted derivatives lack this property,
which facilitates the substitution. The structure of 11 was confirmed
From the presented results in Table 1, the following
structure–activity relationships could be concluded. The presence
of two carbon spacers between the alicyclic amines and
2‐thioxoquinazolin‐4‐one in compounds 3a–e resulted in mild‐to‐
potent inhibition with IC50 8.76–2262 µM, where the substituted
piperazine derivatives 3c–e exhibited promising activity (IC50
=
67.55, 40.08, and 8.76 µM, respectively), compared with their

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TABLE 1 PDE inhibition activity (IC50, µM) for the synthesized
compounds
10c. This may be due to the steric hindrance resulted from the larger
bromide atomic size than the chloride. Furthermore, the hydrazone
derivative 12 exhibited lower activity (IC50 = 8.06 µM) than its oxime
analog 10d (IC50 = 1.15 µM). The 2‐hydrazino derivative 11 exhibited
moderate activity (IC50 = 187.89 µM) that was enhanced by the in-
corporation of cyclic diketonic heterocycles as compounds 13a–c.
The highest activity was obtained from compound 13b (IC50 = 3.64
µM), followed by 13c (IC50 = 9.11 µM) and 13a (IC50 = 11.52 µM).
Compound ID
IC50 (µM)
4.70 0.30
Compound ID
IC50 (µM)
1.58 0.06
1
9b
3a
3b
3c
3d
3e
5a
5b
7a
7b
7c
7d
7e
9a
2262 20.00
316.80 5.10
67.55 3.15
40.08 2.56
8.76 0.50
9c
10.07 0.90
4.22 0.09
2.61 0.07
77.50 4.25
5.60 0.14
1.15 0.07
187.89 1.90
8.06 0.60
11.52 1.10
3.64 0.14
9.11 0.75
4.68 0.19
9d
10a
10b
10c
10d
11
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2.2.2
In vivo behavioral tests
381.50 4.58
354.70 3.95
4102.00 32.10
12.98 0.86
17.02 1.20
3.85 0.09
It was reported that inhibition of PDE7 reduced the cognitive im-
pairment in a mouse model of Alzheimer's disease.[43] Therefore,
compound 10d, which showed the best results in both the in vitro
PDE study in addition to in silico molecular docking, was evaluated
for its ability to ameliorate impaired learning and memory functions
in vivo. Memory impairment was induced by administering scopola-
mine, and then Y‐maze test[44] and step‐through passive avoidance
test[45] were performed. Donepezil was used as a positive control.
12
13a
13b
13c
IBMX
1.30 0.03
1.70 0.06
Abbreviation: PDE, phosphodiesterase.
Y‐maze test
In this test, it was observed from Figure 3a that the scopolamine
model group exhibited lower percentage of alternations than the
control group (% alternations = 52.71 and 87.36, respectively). Mice
groups treated with donepezil, IBMX, and compound 10d showed a
comparable increase in the percentage of alternations (83.79, 89.1,
and 79.9, respectively).
morpholine 3a or piperidine 3b congeners (IC50 = 2262 and
316.80 µM, respectively).
In contrast, elongation of the spacer in 3a–e by an extra carbon
atom as in compounds 5a,b resulted in higher activity only in the case
of morpholine derivative 5a, whereas the 4‐phenyl piperazine analog
5b revealed lower activity than its 2‐oxoethyl derivative 3d. This may
be due to the steric hindrance of the phenyl piperazinyl moiety.
The incorporation of aromatic amides in 7a–e resulted in high
activity in some compounds, especially in case of substituted deri-
vatives having electron‐donating moieties as in 7d,e (IC50 = 3.85 and
1.30 µM), followed by those substituted with electron‐withdrawing
groups, 7b,c (IC50 = 12.98 and 17.02 µM). It was found that the
position of the electron‐withdrawing substituent was more favorable
to be in para position than in meta position. The unsubstituted de-
rivative 7a exhibited no activity (IC50 = 4102 µM). Moreover, the
(a)
obtained activity was greatly enhanced in the case of 9a–d (IC50
=
1.58–10.07 µM). It was found that the electronegativity of the sub-
stituents had no absolute impact on the activity. The most active
compound in this series was compound 9b with the bromo sub-
stituent (IC50 = 1.58 µM), followed by the unsubstituted derivative 9a
(IC50 = 1.70 µM) and the 4‐methoxy analog 9d (IC50 = 4.22 µM). In
contrast, the least active one was the chloro‐substituted derivative
9c (IC50 = 10.07 µM).
(b)
However, the oxime derivatives 10a–d revealed the highest
activity in case of electron‐donating substituted derivative 10d
(IC50 = 1.15 µM), followed by the unsubstituted analog 10a (IC50
=
2.61 µM) and finally the electron‐withdrawing substituted deriva-
tives 10b,c (IC50 = 77.5 and 5.6 µM, respectively). It was obvious that
the bromo derivative 10b had lower activity than its chloro analog
FIGURE 3 (a) Percentage of spontaneous alterations in the
Y‐maze test. (b) The transfer latency time (s) for the step‐through
passive avoidance test

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Step‐through passive avoidance test
Moreover, the defending effect of compound 10d against memory
deficit was detected using the step‐through passive avoidance test.
The transfer latency time (TLT) was measured and illustrated in
Figure 3b. In comparison with the control, the TLT of scopolamine‐
treated group was significantly (TLT = 164.6 s) reduced than that of
the control (TLT = 235.8 s). Meanwhile, treatment with donepezil,
IBMX, and compound 10d (TLT = 218.6, 221.6, and 197 s) reversed
the scopolamine‐lowered TLT compared with the model group. These
results demonstrated that compound 10d was able to pass the BBB
and improve scopolamine‐induced cognitive deficit.
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2.3
Molecular docking study
Molecular docking simulations were performed for the most potent
compounds 7d,e, 9a,b,d, 10a,d, and 13b to investigate their binding
mode to PDE7A, PDE4B, and PDE4D active site key amino acid re-
sidues. It also predicted the compounds' possible conformation and
orientation within each binding site. Furthermore, molecular docking
was used to evaluate the selectivity of the active compounds regarding
PDE4 that exhibits similar binding site amino acid sequence.[46] As
reported, PDE4B has a key role in inflammation cascade; thus, its in-
hibition caused appreciable anti‐inflammatory response in neurode-
generative disease with enhancement of cognitive performance.[47]
Moreover, inhibition of PDE4D was correlated with cognitive perfor-
mance enhancement in Alzheimer's disease.[48] However, non‐selective
inhibition of PDE4s or either PDE4B or PDE4D had been linked to
many side effects such as nausea, emesis, and other gastrointestinal
tract disturbances.[49] Consequently, inhibition of PDE4B and/or
PDE4D isoenzymes could be advantageous in treating neurodegen-
erative disorders.
FIGURE 4 Molecular docking of 10d showing the two main
interactions with the catalytic domain of PDE7A using PDB 1ZKL, where
(a) and (b) are the two‐ and three‐dimensional presentations,
respectively. IBMX and 10d are shown in magenta and green stick
model, respectively, where the hydrogen bond formed with Gln 413 is
represented with a green dotted line with its accompanied distance in Å
active site of the PDE7A catalytic domain. The most potent deriva-
tive 10d with IC50 1.15 µM bound to the binding site with the lowest
energy score, followed by 7e, 9b, 9a, 10a, giving −9.23, −8.99, −8.65,
−8.76, and −8.22 kcal/mol, respectively, which complied with their in
vitro biological results. However, despite having relatively higher
energy score than 7b and 9d, compound 13b caused higher PDE7A
inhibition according to the in vitro data presented in Table 1. This
could be explained by 13b capability of interacting with three of the
crucial amino acid residues versus interacting with only two by 7b
and 9d, as presented in Table 2 and Figure S2.
|
2.3.1
Molecular docking study of PDE7A
The PDB file 1ZKL was used to evaluate the possible binding con-
formations of the active compounds versus ligand IBMX.[50] Docking
protocol validation was done to obtain the lowest possible RMSD
(0.58 Å) with the highest energy score (−6.77 kcal/mol), as shown in
Figure S1.
According to the performed docking study, the most potent com-
pounds 7d,e, 9a,b,d, 10a,d, and 13b showed a common predicted binding
pattern to the PDE7A binding site, as shown in Figures 4 and S2; energy
scores and binding interactions are presented in Table 2.
In addition to having lower energy score than IBMX, 10d man-
aged to show the two main required interactions by arene stacking
against Phe 416 and forming H‐bond with Gln 413 to inhibit PDE7A,
which was translated into its potent in vitro inhibitory result,
achieving the lowest IC50 value 1.15 μM among the active deriva-
tives. Another explanation for the achieved potency of 10d was the
hydrogen bond strength formed with Gln 413, which was strong and
comparable to IBMX in terms of distance 2.38 and 2.23 Å, respec-
tively, as shown in Figure 4.[52] Another explanation for accom-
plishing such good in vitro inhibition was that 10d had a bulkier
structure than IBMX, which accommodated most of the binding site
volume of PDE7A catalytic domain, as presented in Figure 5, which
All the active compounds exhibited arene–arene stacking against
the crucial Phe 416 and displayed other hydrophobic/hydrogen
bonds with other residues of the catalytic domain binding site that
explained their detected in vitro PDE7A inhibitory activity.[51]
Moreover, the active derivatives bound to the catalytic domain
with lower energy scores than IBMX, giving a range of −9.23 to
−7.40 kcal/mol compared with −6.77 kcal/mol, which uncovered a
significant correlation between their potency and their binding to the

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TABLE 2 The molecular docking data of compounds 7d,e, 9a,b,d,
10a,d, and 13b on PDE7A using PDB 1ZKL in comparison to the
co‐crystallized ligand IBMX
Energy
score in
Interacting amino Type of
Compound ID kcal/mol
acid residues
interaction
IBMX
−6.77
Phe 416
Arene–arene
H‐bond donor
Gln 413
7d
7e
−7.92
−8.99
Phe 416, Phe 384 Arene–arene
Phe 416 Arene–arene
Asn 260, Gln 261 Arene–H
His 256
Phe 416
H‐bond donor
Arene–arene
9a
9b
−8.76
−8.65
FIGURE 5 Three‐dimensional presentation of both IBMX in
magenta color and 10d in green inside the active site of PDE7A using
PDB 1ZKL, where hydrophobic and polar regions are shown in red
and blue, respectively, with exposed white areas
Val 380, Phe 384, Arene–H
His 256
Mg+2
Ion contact
Phe 416
Arene–arene
high interest to find if these new derivatives have another inhibitory
activity toward other PDEs. Therefore, in silico evaluation of possible
binding to both PDE4B and 4D was performed using PDB crystals
2QYL and 1OYN for PDE4B and PDE4D, respectively.[53,54] Both
molecular docking protocols were validated at the beginning of the
study, as shown in Figure S3.
Asn 260, Phe 384 Arene–H
Gly 258
H‐bond donor
9d
−7.78
−8.22
Phe 416
Arene–arene
H‐bond donor
Arene–arene
Arene–H
His 256
10a
Phe 416
Reported data of PDE4B exposed the essential interactions for
its inhibition, which were stacking against Phe 446 together with
hydrogen bond formation with Ile 410 and/or Gln 443 of the hy-
drophobic adenine pocket.[55] Moreover, published data demon-
strated the binding of cAMP to Ile 410 among other residues in the
hydrophobic pocket.[56] Therefore, if the new derivatives managed to
interact with either of the mentioned amino acid residues, then, hy-
pothetically, a PDE4B inhibition could be achieved.
Phe 384, His 256
Thr 321
H‐bond donor
Arene–arene
H‐bond donor
Arene–arene
Arene–H
10d
13b
−9.23
−7.40
Phe 416
Gln 413
Phe 416
Val 380, Ile 323
Derivatives 7d,e, 9a,b,d, 10a,d, and 13b were docked to PDB
2QYL, giving the binding results shown in Table 3 and Figures 6 and S4.
As demonstrated in Table 3, derivatives 7d,e, 9a,b,d, 10a,d, and
13b showed score range −7.74 to −6.34 kcal/mol, which was not as
good as NPV (−11.11 kcal/mol). However, all those derivatives could
interact with one or more of the three crucial amino acids of PDB4B
mentioned earlier. For instance, all PDE7A active derivatives bound
to Ile 410 through arene–H bond except 13b. Furthermore, three out
of the eight derivatives stacked against Phe 446 using arene–arene
interaction.
could hinder the access of PDE7 natural substrate cAMP. As shown
in Figure 5, most of the binding site is hydrophobic in nature with
few polar regions, and 10d could align in accordance to hydro-
phobicity, filling most of the binding site volume as compared with
the smaller structure of IBMX.
Although other active derivatives 7d,e, 9a,b,d, 10a, and 13b
showed no direct interaction with the conserved Gln 413 residue, they
interacted with other residues of the catalytic binding site such as Phe
384, Val 380, and Ile 323 in addition to the crucial Phe 416. Reported
data of thioxoquinazoline derivatives revealed the possibility of in-
hibiting PDE7 by binding to Phe 416 and other amino acids of the
catalytic domain without the necessity of interacting with Gln 413.[17]
The best inhibition outcome was with 13b that showed the best
score value −7.74 kcal/mol and engaged in two out of the three
crucial interactions with residues Phe 446 and Gln 443 in addition to
a third arene–hydrogen bond with Phe 414. Interestingly, the formed
H‐bond between 13b and Gln 443 was approximately as strong as
the one formed by ligand NPV regarding distance and energy, giving
2.93 Å, −2.0 kcal/mol and 3.04 Å, −1.9 kcal/mol, respectively, as
shown in Figure 6.
|
2.3.2
Molecular docking study of PDE4B and
PDE4D
Due to the nonspecific hydrophobic interaction of the active deri-
vatives with PDE7A and the resemblance with PDE4,[46] it was of
Interestingly, the highest PDE7A inhibitor 10d was not as much
inhibitor to PDE4B considering the docking score comparing to NPV

AMIN ET AL.
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|
TABLE 3 Molecular docking results of 7d,e, 9a,b,d, 10a,d, and
13b on PDE4B using PDB 2QYL in comparison to the co‐crystallized
ligand NPV
Moreover, molecular docking of the most active compounds into
the active site of PDE7A, PDE4B, and PDE4D highlighted that the
obtained activity may be due to a preferential selective inhibition
of PDE7A. Therefore, those compounds can be subjected for
further future in vitro PDE7A inhibition assays to confirm this
assumption.
Energy score
Interacting amino
acid residues
Type of
Compound in kcal/mol
interaction
NPV
−11.11
Ile 410
Gln 443
Ile 410
Ile 410
Arene–H
H‐donor
Arene–H
Arene–H
Arene–H
7d
7e
9a
−7.10
−7.13
−6.45
|
4
EXPERIMENTAL
Chemistry
|
Ile 410, Phe 414,
Asn 283
4.1
|
4.1.1
General
9b
9d
−6.91
−7.45
Ile 410, Asn 283
Ile 410
Arene–H
Arene–H
Melting points were determined by the open capillary tube method
using Stuart SMP3 digital melting point apparatus and they were
uncorrected. Elemental microanalyses were carried out at the Re-
gional Center for Mycology and Biotechnology, Al‐Azhar University.
IR spectra were done on Shimadzu IR spectrometer 8400S and ex-
pressed in wavenumber (υmax) cm−1. The 1H NMR spectra (see the
Supporting Information) were measured in chloroform (CDCl3) or
dimethyl sulfoxide (DMSO‐d6) on Bruker AVANCE ӀӀӀ 400 MHz
FT‐NMR spectrophotometer. Chemical shifts were recorded in δ as
parts per million (ppm) downfield from tetramethylsilane as the in-
ternal standard. 13C NMR spectra (see the Supporting Information)
were recorded at 100 MHz. Mass spectra were performed on Hew-
lett Packard (HP)‐5988 mass spectrometer at 70 eV at the Regional
Center for Mycology and Biotechnology, Al‐Azhar University. Reac-
tions were monitored by thin‐layer chromatography (TLC) using
Macherey‐Nagel Alugram Sil G/UV 254 silica gel plates and
chloroform–ethanol (9.5:0.5) as an eluting system. The spots were
visualized using a Vilber Lourmet lamp at λ = 254 and 365 nm.
Compounds 1,[29,30] 2a–e,[31–36] 4a,b,[37] 6a–e,[38–40] and 8a–d[41]
were prepared according to their reported procedures.
Phe 446
Arene–arene
Arene–H
10a
10d
−6.34
−7.11
Ile 410, Phe 414,
Asn 283
Ile 410, Phe 414
Phe 446
Arene–H
Arene–arene
Arene–arene
H‐donor
13b
−7.74
Phe 446
Gln 443
Phe 414
Arene–H
(−7.11 and −11.11 kcal/mol, respectively); yet, it showed typical
arene–arene stacking against Phe 446 and arene–hydrogen binding
with both Ile 410 and Phe 414.
Considering PDE4D, its inhibition required the interaction with
the main conserved residues Gln 369 and Phe 372, as demonstrated
by the PDEs non‐selective inhibitor IBMX and the selective PDE4
inhibitor rolipram.[46,54] None of the PDE7A active compounds could
interact with the conserved or other binding site residues of PDE4D,
as shown in Figure S5.
The InChI codes of the investigated compounds, together with
some biological activity data, are provided as Supporting Information.
Giving the previously discussed molecular docking data, the
promising compounds leaned toward PDE7A inhibition rather than
PDE4B and PDE4D, which requires further future in vitro biological
evaluation to support this assumption.
2‐{[2‐Oxo‐2‐(morpholin‐1‐yl)ethyl]thio}‐3‐phenylquinazolin‐4(3H)‐
one (3a)[57]
To a solution of sodium ethoxide (0.94 g, 13.83 mmol) in absolute
ethanol (15.00 ml), compound 1 (1.75 g, 6.88 mmol) was added, fol-
lowed by 2‐chloro‐1‐morpholinylethan‐1‐one 2a (0.23 g, 13.83 mmol).
The reaction mixture was stirred at 60°C for 2 h. The reaction mixture
was evaporated under vacuum, diluted with ethyl acetate, and washed
with water, and the resultant white solid was filtered and recrystallized
|
3
CONCLUSION
Twenty‐five 4‐quinazolinone derivatives were synthesized and
screened for their inhibitory activity against PDE using the non‐
selective inhibitor, IBMX, as a reference standard. The most active
compounds were 7d,e, 9a,b,d, 10a,d, and 13b comprising a wide
range of substitution at position 2, suggesting that this core can be
considered as a promising scaffold for further structural optimi-
zation to obtain new compounds with higher PDE inhibitory ac-
tivity. Furthermore, the most active compound 10d was confirmed
for its neuroprotective activity through the behavioral in vivo
tests, the Y‐maze test, and step‐through passive avoidance test.
from benzene. Yield: 55%; m.p.: 197–200°C; IR (cm−1
, KBr):
3059–3044 (CH aromatic), 2990–2920 (CH aliphatic), 1678 (2C═O
bands), 1651 (C═N), 1604–1543 (C═C); 1H NMR (CDCl3, 400 Hz, δ,
ppm): 3.64–3.76 (m, 8H, morpholine), 4.13 (s, 2H, CH2), 7.35–7.37 (m,
2H, Ar–H), 7.42 (t, J = 7.40 Hz, 1H, Ar–H), 7.55–7.57 (m, 4H, Ar–H),
7.73–7.77 (m, 1H, Ar–H), 8.25 (dd, Ja = 1.36, Jb = 7.96 Hz, 1H, Ar–H);
13C NMR (CDCl3, 100 MHz, δ, ppm): 35.1 (CH2), 42.6 (CH2–N), 46.6
(CH2–N), 66.6 (CH2–O), 66.8 (CH2–O), 119.9, 126.0, 126.1, 126.7,

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FIGURE 6 (a, b) Two‐ and three‐dimensional presentation of molecular docking results of 10d and 13b, respectively, with PDE4B using PDB
2QYL, where 10d and 13b are shown in green, whereas the crystallized ligand is shown in magenta stick model
127.5, 129.0, 129.7, 129.9, 130.0, 134.7, 135.5, 147.5, 156.6 (Ar car-
piperidine), 4.23 (s, 2H, CH2), 7.35–7.37 (m, 2H, Ar–H), 7.41–7.45
(m, 1H, Ar–H), 7.54–7.58 (m, 3H, Ar–H), 7.66 (d, J = 7.84 Hz, 1H,
Ar–H), 7.73–7.78 (m, 1H, Ar–H), 8.26 (dd, Ja = 1.16, Jb = 7.84 Hz,
1H, Ar–H); 13C NMR (CDCl3, 100 MHz, δ, ppm): 24.4, 25.5, 26.6
(3C of piperidine), 36.2 (CH2), 43.4 (C–N piperidine), 47.4 (C–N
piperidine), 119.9, 126.0, 127.3, 128.3, 129.2, 129.8, 130.1, 134.7,
135.6, 147.5, 157.2 (Ar carbons), 161.6 and 165.4 (2C═O); anal.
calcd. for C21H21N3O2S: C, 66.47; H, 5.58; N, 11.07%; found: C,
66.30; H, 5.72; N, 11.25%.
bons), 161.6 and 166.2 (2C═O).
|
4.1.2
General procedure for the synthesis of
compounds 3b,d,e
A
mixture of compound 1 (1.00 g, 3.93 mmol), a suitable
2‐chloroethan‐1‐one derivative 2b, 2d, or 2e (3.93 mmol), and po-
tassium carbonate (0.54 g, 3.93 mmol) in dry dimethylformamide
(15.00 ml) was heated under reflux. The reaction was monitored
periodically by TLC. After complete reaction, it was left to cool,
poured on crushed ice/water, and stirred for 1 h. The produced solid
was separated by filtration and recrystallized from either benzene
for 3b and 3e or methanol for 3d.
2‐{[2‐Oxo‐2‐(4‐phenylpiperazin‐1‐yl)ethyl]thio}‐3‐phenylquinazolin‐
4(3H)‐one (3d)[58]
Yield: 60%; m.p.: 187–188°C; IR (cm−1, KBr): 3089 (CH aromatic),
2930 (CH aliphatic), 1678 (2C═O), 1651 (C═N), 1600–1543 (C═C);
1H NMR (CDCl3, 400 Hz, δ, ppm): 3.27–3.32 (m, 4H, N(CH2)2 piper-
azine), 3.85–3.89 (m, 4H, N(CH2)2 piperazine), 4.20 (s, 2H, CH2),
6.98–7.02 (m, 3H, Ar–H), 7.32–7.38 (m, 4H, Ar–H), 7.41–7.45 (m, 1H,
Ar–H), 7.56–7.59 (m, 4H, Ar–H), 7.72–7.76 (m, 1H, Ar–H), 8.26 (dd,
Ja = 1.20, Jb = 7.96 Hz, 1H, Ar–H); 13C NMR (CDCl3, 100 MHz, δ,
ppm): 35.4 (CH2), 42.1, 46.0, 49.6, 50.1 (piperazine carbons), 116.9,
119.9, 126.0, 126.1, 127.4, 128.4, 129.2, 129.4, 129.9, 130.2, 134.7,
135.5, 147.6, 156.7 (Ar carbons), 161.6 and 166.0 (2C═O).
2‐{[2‐Oxo‐2‐(piperidin‐1‐yl)ethyl]thio}‐3‐phenylquinazolin‐4(3H)‐
one (3b)
Yield: 93%; m.p.: 83–84°C; IR (cm−1, KBr): 3039–3005 (CH aro-
matic), 2936–2905 (CH aliphatic), 1686 (2C═O bands), 1635
(C═N), 1608–1543 (C═C); 1H NMR (CDCl3, 400 Hz, δ, ppm):
1.57–1.69 (m, 6H, 3CH2 piperidine), 3.59 (s, 4H, N(CH2)2

AMIN ET AL.
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Ethyl 4‐{2‐[(4‐oxo‐3‐phenyl‐3,4‐dihydroquinazolin‐2yl)thio]acetyl}‐
piperazine‐1‐carboxylate (3e)[59]
2‐{[3‐Oxo‐3‐(4‐phenylpiperazin‐1‐yl)propyl]thio}‐3phenylquinazolin‐
4(3H)‐one (5b)
Yield: 70%; m.p.: 137–140°C; IR (cm−1, KBr): 3070–3028 (CH aro-
matic), 2924 (CH aliphatic), 1663 (3C═O), 1624 (C═N), 1593–1531
(C═C); 1H NMR (CDCl3, 400 Hz, δ, ppm): 1.30 (t, J = 7.08 Hz, 3H,
CH3), 3.50–3.69 (m, 8H, piperazine H), 4.16 (s, 2H, CH2), 4.20 (q,
J = 7.16 Hz, 2H, CH2), 7.34–7.37 (m, 2H, Ar–H), 7.43 (t, J = 7.16 Hz,
1H, Ar–H), 7.54–7.59 (m, 4H, Ar–H), 7.73–7.77 (m, 1H, Ar–H), 8.26
(dd, Ja = 1.08, Jb = 7.88 Hz, 1H, Ar–H).
To solution of 1 (1.00 g, 3.93 mmol) and anhydrous potassium car-
bonate (1.08 g, 7.86 mmol) in dry acetone (20 ml), 3‐chloro‐1‐
(phenylpiperazine)propan‐1‐one (4b) (1.19 g, 4.72 mmol) was added,
followed by a catalytic amount of potassium iodide. The mixture was
heated under reflux with continuous stirring for 48 h, and then it was
poured on ice/water mixture and scratched. The obtained solid was
filtered off and dried. Yield: 54%; m.p.: 147–148°C; IR (cm−1, KBr):
3062–3035 (CH aromatic), 2981–2927 (CH aliphatic), 1685 (2C═O),
1643 (C═N), 1573–1543 (C═C); 1H NMR (CDCl3, 400 Hz, δ, ppm):
2.91 (t, J = 7.12 Hz, 2H, CH2), 3.23 (br s, 4H, N(CH2)2 piperazine),
3.49 (t, J = 7.32 Hz, 2H, CH2), 3.78–3.89 (m, 4H, N(CH2)2 piperazine),
7.02–7.06 (m, 3H, Ar–H), 7.31–7.36 (m, 4H, Ar–H), 7.42–7.46 (m, 1H,
Ar–H), 7.53–7.56 (m, 3H, Ar–H), 7.60 (d, J = 8.0 Hz, 1H, Ar–H),
7.74–7.78 (m, 1H, Ar–H), 8.27 (dd, Ja = 1.16, Jb = 7.92 Hz, 1H, Ar–H);
13C NMR (CDCl3, 100 MHz, δ, ppm): 27.9 (SCH2), 33.2 (CH2–CO),
41.6, 45.3, 49.6, 50.0 (piperazine carbons), 116.9, 119.9, 121.0,
125.9, 126.1, 129.2, 129.3, 129.7, 130.0, 134.7, 135.8, 147.8, 150.6,
157.4 (Ar carbons), 161.8 and 168.6 (2C═O); MS (m/z): 470.27 [M]+,
471.27 [M+1]+, 472.27 [M+2]+; anal. calcd. for C27H26N4O2S: C,
68.91; H, 5.57; N, 11.91%; found C, 68.84; H, 5.71; N, 12.18%.
2‐{[2‐Oxo‐2‐(4‐methylpiperazin‐1‐yl)ethyl]thio}‐3‐phenylquinazolin‐
4(3H)‐one (3c)
To a mixture of 1 (1.00 g, 3.93 mmol), anhydrous potassium carbo-
nate (1.08 g, 7.86 mmol), and 2c (0.69 g, 3.93 mmol) in dry acetone
(20.00 ml), a catalytic amount of potassium iodide was added. The
mixture was refluxed with continuous stirring for 48 h. It was poured
into ice/water mixure. The obtained solid was filtered off and washed
with diluted potassium hydroxide to dissolve any traces from com-
pound 1. Yield: 57%; m.p.: 184–186°C; IR (cm−1, KBr): 3090–3021
(CH aromatic), 2986–2916 (CH aliphatic), 1678 (2C═O), 1643
(C═N), 1604–1551 (C═C); 1H NMR (CDCl3, 400 Hz, δ, ppm): 2.35 (s,
3H, CH3), 2.41–2.52 (m, 4H, N(CH2)2 piperazine), 3.67–3.72 (m, 4H,
N(CH2)2 piperazine), 4.14 (s, 2H, CH2), 7.35–7.37 (m, 2H, Ar–H), 7.42
(t, J = 7.68 Hz, 1H, Ar–H), 7.55–7.59 (m, 4H, Ar–H), 7.74 (t, J = 7.16,
1H, Ar–H), 8.25 (d, J = 7.88 Hz, 1H, Ar–H); 13C NMR (CDCl3,
100 MHz, δ, ppm): 35.4 (CH2), 42.0 (CH3–N), 45.8, 45.9, 54.4, 55.0
(piperazine carbons), 119.9, 126.0, 127.4, 129.2, 129.8, 130.2, 134.7,
135.5, 147.6, 156.7 (Ar carbons), 161.6 and 165.9 (2C═O); anal.
calcd. for C21H22N4O2S: C, 63.94; H, 5.62; N, 14.20%; found C,
63.76; H, 5.88; N, 14.37%.
|
4.1.3
General procedure for the preparation of
compounds 7a–e
A mixture of compound 1 (1.00 g, 3.93 mmol), a suitable 2‐chloro‐N‐
(3/4‐substituted phenyl)acetamide derivative (6a–e) (3.93 mmol),
and potassium carbonate (1.08 g, 7.86 mmol) in dry dimethylforma-
mide (10 ml) was heated under reflux for 8–10 h. After completion of
reaction, it was allowed to cool and poured on crushed ice/water
mixture. The produced solid was separated by filtration, dried, and
recrystallized from either absolute ethanol or benzene.
2‐[(3‐Morpholin‐1‐yl‐3‐oxopropyl)thio]‐3‐phenylquinazolin‐4(3H)‐
one (5a)
To a solution of sodium ethoxide (0.27 g, 3.93 mmol) in absolute
ethanol (15 ml), compound 1 (1.00 g, 3.93 mmol) was added, fol-
lowed by 3‐chloro‐1‐(morpholino)propan‐1‐one (4a) (0.83 g,
4.72 mmol). The reaction mixture was stirred at 60°C for 2 h. The
solvent was evaporated, the residue was diluted with ethyl acet-
ate, and washed with water. The resultant off‐white solid was
filtered and recrystallized from benzene; yield: 75%; m.p.:
175–176°C; IR (cm−1, KBr): 3070–3017 (CH aromatic), 2974 (CH
aliphatic), 1693 (C═O), 1639 (C═N), 1608–1550 (C═C); 1H NMR
(CDCl3, 400 Hz, δ, ppm): 2.85 (t, J = 7.12 Hz, 2H, CH2), 3.48 (t,
J = 7.20 Hz, 2H, CH2), 3.53 (t, J = 3.96 Hz, 2H, CH2), 3.64–3.69 (m,
6H, 3CH2), 7.31–7.33 (m, 2H, Ar–H), 7.44 (t, J = 7.20 Hz, 1H,
Ar–H), 7.55–7.56 (m, 3H, Ar–H), 7.62 (d, J = 8.08 Hz, 1H, Ar–H),
7.74–7.78 (m, 1H, Ar–H), 8.26 (dd, Ja = 1.08, Jb = 7.92 Hz, 1H,
Ar–H); 13C NMR (DMSO‐d6, 100 MHz, δ, ppm): 27.8 (SCH2), 33.3
(CH2CO), 45.7 (N(CH2)2 morpholine), 66.9 (O(CH2)2 morpholine),
116.1, 116.4, 124.4, 128.3, 128.5, 128.7, 129.3, 129.6, 135.5,
139.2, 147.5 (aromatic carbons), 160.4 and 166.5 (2C═O); anal.
calcd. for C21H17N3O3S: C, 63.78; H, 5.35; N, 10.63%; found C,
64.02; H, 5.48; N, 10.87%.
2‐[(4‐Oxo‐3‐phenyl‐3,4‐dihydroquinazolin‐2‐yl)thio]‐N‐
phenylacetamide (7a)[60]
Yield: 89%; m.p.: 196–197°C; IR (cm−1
, KBr): 3294 (NH str.),
3070–3017 (CH aromatic), 2978–2912 (CH aliphatic), 1685 and
1660 (2C═O), 1600 (C═N), 1550–1531 (C═C); 1H NMR (CDCl3,
400 Hz, δ, ppm): 4.01 (s, 2H, CH2), 7.10 (t, J = 7.36 Hz, 1H, Ar–H),
7.31–7.37 (m, 4H, Ar–H), 7.51–7.60 (m, 6H, Ar–H), 7.76–7.89 (m, 2H,
Ar–H), 8.34 (d, J = 7.80 Hz, 1H, Ar–H), 9.97 (s, 1H, NH exchanged
with D2O).
N‐(4‐Chlorophenyl)‐2‐[(4‐oxo‐3‐phenyl‐3,4‐dihydroquinazolin‐2‐yl)
thio]acetamide (7b)
Yield: 92%; m.p.: 230–231°C; IR (cm−1, KBr): 3341 (NH str.), 3080
(CH aromatic), 2940 (CH aliphatic), 1693 and 1663 (2C═O), 1597
(C═N), 1551 (C═C); 1H NMR (CDCl3, 400 Hz, δ, ppm): 3.93 (s, 2H,
CH2), 7.25–7.29 (m, 2H, Ar–H), 7.36–7.37 (m, 2H, Ar–H), 7.44–7.46
(m, 2H, Ar–H), 7.53–7.60 (m, 4H, Ar–H), 7.75 (d, J = 8.08 Hz, 1H,
Ar–H), 7.86–7.88 (m, 1H, Ar–H), 8.33 (dd, Ja = 0.96, Jb = 7.88 Hz, 1H,

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Ar–H), 10.0 (s, 1H, NH exchanged with D2O); 13C NMR (CDCl3,
100 MHz, δ, ppm): 36.6 (CH2), 120.0, 120.6, 120.7, 125.0, 126.9,
128.0, 129.0, 129.06, 129.1, 130.0, 130.6, 135.2, 135.5, 136.6, 146.8,
158.8 (Ar carbons), 161.1 and 166.7 (2C═O); MS (m/z): 421.08 [M]+,
423.53 [M+2]+; anal. calcd. for C22H16ClN3O2S: C, 62.63; H, 3.82; Cl,
8.40; N, 9.96%; found: C, 62.74; H, 3.96; N, 10.23%.
equivalent amount of the appropriate reagent 8a–d (3.93 mmol). The
mixture was heated with continuous stirring under reflux for 24 h
and filtered while hot. The precipitate was washed with water and
recrystallized from ethanol to yield compounds 9a–d.[42]
2‐[(2‐Oxo‐2‐phenylethyl)thio]‐3‐phenylquinazolin‐4(3H)‐one (9a)
Yield: 86%; m.p.: 211–212°C, IR (cm−1, KBr): 3059–3001 (CH aro-
matic), 2909 (CH aliphatic), 1686 (2C═O), 1609 (C═N), 1574–1547
(C═C); 1H NMR (CDCl3, 400 Hz, δ, ppm): 4.62 (s, 2H, CH2), 7.26 (d,
J = 8.16 Hz, 1H, Ar–H), 7.29–7.43 (m, 3H, Ar–H), 7.54–7.69 (m, 7H,
Ar–H), 8.11 (d, J = 7.32 Hz, 2H, Ar–H), 8.23 (dd, Ja = 1.12, Jb = 7.92 Hz,
1H, Ar–H).
N‐(3‐Fluorophenyl)‐2‐[(4‐oxo‐3‐phenyl‐3,4‐dihydroquinazolin‐2‐yl)‐
thio]acetamide (7c)
Yield: 87%; m.p.: 119–121°C; IR (cm−1, KBr): 3260 (NH str.),
3066–3055 (CH aromatic), 2986–2931 (CH aliphatic), 1685 and
1662 (2C═O), 1612 (C═N), 1554 (C═C); 1H NMR (CDCl3, 400 Hz, δ,
ppm): 3.90 (s, 2H, CH2), 6.79 (t, J = 8.24 Hz, 1H, Ar–H), 7.08 (t,
J = 8.32 Hz, 1H, Ar–H), 7.21–7.29 (m, 1H, Ar–H), 7.31–7.37 (m, 2H,
Ar–H), 7.43–7.59 (m, 5H, Ar–H), 7.72 (d, J = 8.08 Hz, 1H, Ar–H), 7.87
(t, J = 8.32 Hz, 1H, Ar–H), 8.30–8.40 (m, 1H, Ar–H), 10.01 (s, 1H, NH,
exchanged with D2O); 13C NMR (CDCl3, 100 MHz, δ, ppm): 36.7
(CH2), 106.9, 107.2, 110.8, 111.0, 114.7, 120.0, 125.1, 126.9, 127.0,
127.9, 128.6, 129.0, 130.0, 130.6, 135.2, 135.5, 139.5, 139.6, 146.8,
158.7, 161.1, 162.6, 164.2 (Ar carbons), 161.1 and 166.9 (2C═O);
anal. calcd. for C22H16FN3O2S: C, 65.17; H, 3.98; N, 10.36%; found:
C, 65.41; H, 4.15; N, 10.49%.
2‐{[2‐(4‐Bromophenyl)‐2‐oxoethyl]thio}‐3‐phenylquinazolin‐4(3H)‐
one (9b)
Yield: 72%; m.p.: 206–207°C; IR (cm−1, KBr): 3067–3013 (CH aro-
matic), 2955 (CH aliphatic), 1686 (2C═O), 1605 (C═N), 1585–1543
(C═C); 1H NMR (CDCl3, 400 Hz, δ, ppm): 4.53 (s, 2H, CH2), 7.22 (d,
J = 8.16 Hz, 1H, Ar–H), 7.38–7.42 (m, 3H, Ar–H), 7.58–7.71 (m, 6H,
Ar–H), 7.98 (d, J = 8.48 Hz, 2H, Ar–H), 8.23 (dd, Ja = 0.84, Jb = 7.84 Hz,
1H, Ar–H).
2‐{[2‐(4‐Chlorophenyl)‐2‐oxoethyl]thio}‐3‐phenylquinazolin‐4(3H)‐
one (9c)
2‐[(4‐Oxo‐3‐phenyl‐3,4‐dihydroquinazolin‐2‐yl)thio]‐N‐(p‐tolyl)‐
acetamide (7d)[60]
Yield: 88%; m.p.: 208–210°C; IR (cm−1, KBr): 3067 (CH aromatic),
2955 (CH aliphatic), 1678 (2C═O), 1605 (C═N), 1551 (C═C); 1H
NMR (CDCl3, 400 Hz, δ, ppm): 4.54 (s, 2H, CH2), 7.22 (d, J = 8.16 Hz,
1H, Ar–H), 7.39 (t, J = 7.64 Hz, 3H, Ar–H), 7.41–7.64 (m, 6H, Ar–3H),
8.05 (d, J = 8.56 Hz, 2H, Ar–H), 8.23 (dd, Ja = 1.2, Jb = 7.92 Hz,
1H, Ar–H).
Yield: 90%; m.p.: 205–208°C; IR (cm−1, KBr): 3294 (NH str.), 3066
(CH aromatic), 2966–2905 (CH aliphatic), 1686 and 1663 (2C═O),
1604 (C═N), 1554–1535 (C═C); 1H NMR (CDCl3, 400 Hz, δ, ppm):
2.31 (s, 3H, CH3), 3.91 (s, 2H, CH2), 7.10 (d, J = 8.16 Hz, 2H, Ar–H),
7.29–7.60 (m, 8H, Ar–H), 7.74 (d, J = 8.12 Hz, 1H, Ar–H), 7.86 (t,
J = 6.44 Hz, 1H, Ar–H), 8.32 (d, J = 7.88 Hz, 1H, Ar–H), 9.72 (s, 1H,
NH, exchanged with D2O).
2‐{[2‐(4‐Methoxyphenyl)‐2‐oxoethyl]thio}‐3‐phenylquinazolin‐4(3H)‐
one (9d)
N‐(4‐Methoxyphenyl)‐2‐[(4‐oxo‐3‐phenyl‐3,4‐dihydroquinazolin‐2‐
yl)thio]acetamide (7e)[60]
Yield: 84%; m.p.: 195–196°C; IR (cm−1, KBr): 3055 (CH aromatic),
2924 (CH aliphatic), 1670 (2C═O), 1597 (C═N), 1574–1543 (C═C);
1H NMR (CDCl3,400 Hz, δ, ppm): 3.93 (s, 3H, OCH3), 4.60 (s, 2H,
CH2), 7.02 (d, J = 8.84 Hz, 2H, Ar–H), 7.29–7.42 (m, 4H, Ar–H),
7.51–7.69 (m, 4H, Ar–H), 8.09 (d, J = 8.84 Hz, 2H, Ar–H), 8.23 (dd,
Ja = 1.00, Jb = 7.80 Hz, 1H, Ar–H); 13C NMR (CDCl3, 100 MHz, δ,
ppm): 39.5 (CH2), 55.6 (OCH3), 113.9, 119.9, 125.9, 126.0, 127.3,
129.2, 129.3, 129.8, 130.2, 130.9, 134.6, 135.8, 147.5, 156.5, 161.7
(Ar carbons), 164.0 and 192.1 (2C═O).
Yield: 84%; m.p.: 189–191°C; IR (cm−1, KBr): 3287 (NH str.),
3059–3009 (CH aromatic), 2962–2905 (CH aliphatic), 1685 and
1655 (2C═O), 1601 (C═N), 1578–1551 (C═C); 1H NMR (CDCl3,
400 Hz, δ, ppm): 3.79 (s, 3H, OCH3), 3.91 (s, 2H, CH2), 6.84 (d,
J = 8.76 Hz, 2H, Ar–H), 7.36–7.41 (m, 4H, Ar–H), 7.53–7.59 (m, 4H,
Ar–H), 7.74 (d, J = 8.04 Hz, 1H, Ar–H), 7.87 (t, J = 7.32 Hz, 1H, Ar–H),
8.32 (d, J = 7.68 Hz, 1H, Ar–H), 9.68 (s, 1H, NH, exchanged with
D2O); 13C NMR (CDCl3, 100 MHz, δ, ppm): 36.7 (CH2), 55.5 (OCH3),
114.2, 119.8, 121.2, 124.6, 127.0, 127.1, 127.9, 128.9, 130.0, 130.7,
131.2, 135.1, 135.5, 146.0, 156.4 (Ar carbons), 160.8 and
166.2 (2C═O).
|
4.1.5
General procedure for the preparation of
compounds 10a–d
A mixture of the appropriate derivative 9a–d (2.50 mmol) and
hydroxylamine hydrochloride (0.17 g, 2.50 mmol) in absolute
ethanol (15.00 ml) was heated under reflux for up to 12 h. The re-
action mixture was then cooled and filtered. The resulting solid was
washed with water, dried, and recrystallized from benzene to yield
compounds 10a–d.
|
4.1.4
General procedure for the preparation of
compounds 9a–d
To a mixture of 1 (1.00 g, 3.93 mmol) in dry acetone (15 ml), dry
potassium carbonate was added (0.54 g, 3.93 mmol), followed by an

AMIN ET AL.
13 of 16
|
2‐{[2‐(Hydroxyimino)‐2‐phenylethyl]thio}‐3‐phenylquinazolin‐4(3H)‐
one (10a)[61]
11.59 (s, 1H, OH exchanged with D2O); 13C NMR (DMSO‐d6,
100 MHz, δ, ppm): 25.5 (CH2), 55.7 (OCH3), 114.4, 120.0, 126.4,
126.5, 127.1, 127.3, 127.8, 128.8, 129.9, 130.0, 130.4, 135.5, 136.1,
147.5, 152.1, 160.5 (Ar carbons), 157.4 (C═N), 161.2 (C═O); anal.
calcd. for C23H19N3O3S: C, 66.17; H, 4.59; N, 10.07%; found C,
66.15; H, 5.08; N, 9.81%.
Yield: 41%; m.p.: 206–207°C; IR (cm−1, KBr): 3198 (OH), 3059–3005
(CH aromatic), 2940 (CH aliphatic), 1728 (C═O), 1667 (C═N),
1607–1547 (C═C). 1H NMR (DMSO‐d6, 400 Hz, δ, ppm): 4.75 (s, 2H,
CH2), 7.10, 7.21 (2d, J = 8.08 Hz, 1H, Ar–H), 7.24, 7.33 (2d,
J = 8.80 Hz, 2H, Ar–H), 7.43, 7.49 (2t, J = 7.28 Hz, 2H, Ar–H),
7.59–7.65 (m, 3H, Ar–H), 7.69–7.74 (m, 2H, Ar–H), 7.93 (d,
J = 7.60 Hz, 1H, Ar–H), 8.05 (dd, Ja = 0.96, Jb = 7.88 Hz, 1H, Ar–H),
8.09 (d, J = 7.28 Hz, 2H, Ar–H), 11.57 (s, 1H, OH exchanged with
D2O); 13C NMR (DMSO‐d6, 100 MHz, δ, ppm): 31.10 (CH2), 115.7,
119.9, 123.0, 126.0, 126.5, 127.1, 129.3, 129.6, 130.1, 130.5, 133.9,
135.3, 135.6, 136.2, 136.9, 140.3, 147.4, 150.7 (Ar carbons), 157.2
(C═N), 161.1 (C═O).
|
4.1.6
General procedure for the preparation of
compounds 11, 12
A mixture of the appropriate derivative 9a–d (2.50 mmol) and hy-
drazine hydrate (0.25 ml, 5.00 mmol) in absolute ethanol (15.00 ml)
was heated under reflux for up to 12 h. Then, the reaction mixture
was cooled and filtered, and the resulting solid was recrystallized
from ethanol to yield compound 11 (in case of compounds 9a–c) or
12 (in case of compound 9d).
2‐{[2‐(4‐Bromophenyl)‐2‐(hydroxyimino)ethyl]thio}‐3‐
phenylquinazolin‐4(3H)‐one (10b)
Yield: 65%; m.p.: 220–221°C; IR (cm−1, KBr): 3198 (OH), 3063–3001
(CH aromatic), 2924 (CH aliphatic), 1728 (C═O), 1647 (C═N), 1543
(C═C); 1H NMR (DMSO‐d6, 400 Hz, δ, ppm): 4.51 (s, 2H, CH2),
7.42–7.44 (m, 2H, Ar–H), 7.48–7.55 (m, 4H, Ar–H), 7.60 (d,
J = 8.56 Hz, 2H, Ar–H), 7.67 (d, J = 7.88 Hz, 1H, Ar–H), 7.74 (d,
J = 8.56 Hz, 2H, Ar–H), 7.85–7.89 (m, 1H, Ar–H), 8.08 (dd, Ja = 1.20,
Jb = 7.92 Hz, 1H, Ar–H), 11.95 (s, 1H, OH exchanged with D2O); 13C
NMR (DMSO‐d6, 100 MHz, δ, ppm): 25.3 (CH2), 120.0, 123.0, 126.4,
126.6, 127.1, 128.5, 129.9, 130.0, 130.4, 131.9, 134.3, 135.5, 136.1,
147.5, 152.0 (Ar carbons), 157.1 (C═N), 161.2 (C═O); MS (m/
z) = 466.22: [M]+, 467.42 [M+1]+; anal. calcd. for C22H16BrN3O2S: C,
56.66; H, 3.46; N, 9.01%; found: C, 56.92; H, 3.62; N, 9.26%.
2‐Hydrazinyl‐3‐phenylquinazolin‐4(3H)‐one (11)[30,62]
Yield: 33%; m.p.: 158–160°C as reported; IR (cm−1, KBr): 3580 and
3472 (NH2), 3306 (NH), 3017 (CH aromatic), 2947 (CH aliphatic),
1651 (C═O), 1604 (C═N), 1582–1562 (C═C); 1H NMR (DMSO‐d6,
400 MHz, δ, ppm): 4.39 (s, 2H, NH2, exchanged with D2O), 6.86 (s,
1H, NH, exchanged with D2O), 7.16–7.19 (m, 1H, Ar–H), 7.31–7.41
(m, 3H, Ar–H), 7.51–7.68 (m, 4H, Ar–H), 7.93 (t, J = 7.62 Hz,
1H, Ar–H).
2‐{[2‐Hydrazono‐2‐(4‐methoxyphenyl)ethyl]thio}‐3‐
phenylquinazolin‐4(3H)‐one (12)
Yield: 36%; m.p.: 171–172°C; IR (cm−1, KBr): 3256 and 3125 (NH2),
3044–3009 (CH aromatic), 2963–2920 (CH aliphatic), 1701 (C═O),
1651 (NH bending), 1605 (C═N), 1578–1543 (C═C); 1H NMR
(DMSO‐d6, 400 MHz, δ, ppm): 3.73 (s, 3H, OCH3), 4.44 (s, 2H, CH2),
6.86 (d, J = 8.92 Hz, 2H, Ar–H), 6.97 (s, 2H, NH2, exchanged with
D2O), 7.32 (d, J = 7.20 Hz, 2H, Ar–H), 7.39 (d, J = 7.76 Hz, 1H, Ar–H),
7.42–7.44 (m, 2H, Ar–H), 7.53–7.56 (m, 3H, Ar–H), 7.69 (d,
J = 8.00 Hz, 1H, Ar–H), 7.87–7.93 (m, 1H, Ar–H), 8.11 (dd, Ja = 1.20,
Jb = 7.96 Hz, 1H, Ar–H); 13C NMR (DMSO‐d6, 100 MHz, δ, ppm): 25.2
(CH2), 55.6 (OCH3), 114.1, 120.0, 126.2, 126.5, 126.6, 127.2, 129.5,
129.8, 130.0, 130.3, 130.4, 130.7, 135.0, 135.5, 136.2, 139.0, 147.4,
159.2 (Ar carbons), 157.8 (C═N), 161.2 (C═O); MS (m/z): 416.46
[M]+; anal. calcd. for C23H20N4O2S: C, 66.33; H, 4.84; N, 13.45%;
found: C, 66.54; H, 4.97; N, 13.28%.
2‐{[2‐(4‐Chlorophenyl)‐2‐(hydroxyimino)ethyl]thio}‐3‐
phenylquinazolin‐4(3H)‐one (10c)
Yield: 52%; m.p.: 224–225°C; IR (cm−1, KBr): 3217 (OH), 3098–3005
(CH aromatic), 2912 (CH aliphatic), 1686 (C═O), 1605 (C═N), 1543
(C═C); 1H NMR (DMSO‐d6, 400 Hz, δ, ppm): 4.52 (s, 2H, CH2),
7.42–7.44 (m, 2H, Ar–H), 7.46–7.50 (m, 3H, Ar–H), 7.52–7.55 (m, 3H,
Ar–H), 7.67 (d, J = 8.04 Hz, 1H, Ar–H), 7.81 (d, J = 8.60 Hz, 2H, Ar–H),
7.85–7.89 (m, 1H, Ar–H), 8.08 (dd, Ja = 0.92, Jb = 7.88 Hz, 1H, Ar–H),
11.95 (s, 1H, OH exchanged with D2O); 13C NMR (DMSO‐d6,
100 MHz, δ, ppm): 25.4 (CH2), 120.0, 126.4, 126.6, 127.1, 128.3,
129.0, 129.9, 130.0, 130.4, 133.9, 134.3, 135.5, 136.1, 147.5, 151.9
(Ar carbons), 157.1 (C═N), 161.2 (C═O); anal. calcd. for
C
22H16ClN3O2S: C, 62.63; H, 3.82; N, 9.96%; found C, 62.86; H, 4.09;
N, 9.85%.
|
General procedure for the preparation of
2‐{[2‐(Hydroxyimino)‐2‐(4‐methoxyphenyl)ethyl]thio}‐3‐
4.1.7
phenylquinazolin‐4(3H)‐one (10d)
compounds 13a–c
Yield: 36%; m.p.: 216–217°C; IR (cm−1, KBr): 3210 (OH), 3059–3001
(CH aromatic), 2920 (CH aliphatic), 1694 (C═O), 1647 (C═N),
1605–1543 (C═C); 1H NMR (DMSO‐d6, 400 Hz, δ, ppm): 3.77 (s, 3H,
OCH3), 4.52 (s, 2H, CH2), 6.95 (d, J = 8.88 Hz, 2H, Ar–H), 7.42–7.44
(m, 2H, Ar–H), 7.49–7.55 (m, 4H, Ar–H), 7.70–7.74 (m, 3H, Ar–H),
7.86–7.90 (m, 1H, Ar–H), 8.09 (dd, Ja = 1.00, Jb = 7.92 Hz, 1H, Ar–H),
To a solution of compound 11 (1.00 g, 3.96 mmol) in glacial acetic
acid, 3.96 mmol of the appropriate acid anhydride (succinic, maleic,
and phthalic) was added. The reaction mixture was heated under
reflux for up to 16 h, and then it was poured on ice/water mixture,
filtered off, and dried.

14 of 16
AMIN ET AL.
|
1‐[(4‐Oxo‐3‐phenyl‐3,4‐dihydroquinazolin‐2‐yl)amino]pyrrolidine‐
2,5‐dione (13a)
3ʹ,5ʹ‐cAMP substrate, IBMX inhibitor, 5ʹ‐AMP standards were
stored on ice. Furthermore, 20 U/ml solution of PDE was pre-
pared by adding 200 µl of cold assay buffer to one of the vials of
lyophilized enzyme. Green Assay Reagent was warmed to room
temperature. The tested compounds were dissolved in DMSO to
prepare a stock solution, which was then diluted to four con-
centrations ranging from 1 to 1000 ng/ml, immediately before
use. Also, 20 µl of cAMP substrate (0.5 mM) was added in each
well, followed by 5 µl of PDE assay buffer in each well, except
control wells in which 15 µl was added. Then, 10 µl of 5ʹ‐
nucleotidase (undiluted, 5 kU/µl) was added per well, and 10 µl of
each of the tested compounds in DMSO (1–1000 ng/ml) was
added to each well except the control wells. The reactions star-
ted by adding 5 µl of PDE enzyme (4 mU/µl) in each well and
incubation of the plate for 20 min at 37°C.
Yield: 87%; m.p.: 277–278°C; IR (cm−1, KBr): 3372 (NH str.), 3059
(CH aromatic), 2974–2943 (CH aliphatic), 1732–1694 (3C═O), 1609
(NH bending), 1593–1566 (C═C); 1H NMR (DMSO‐d6, 400 MHz, δ,
ppm): 2.91 (t, J = 6.68 Hz, 2H, CH2), 3.48 (t, J = 6.76 Hz, 2H, CH2),
7.28–7.34 (m, 2H, Ar–H), 7.50–7.67 (m, 2H, Ar–H), 7.70–7.74 (m, 1H,
Ar–H), 7.93–8.02 (m, 2H, Ar–H), 8.10 (d, J = 8.52 Hz, 1H, Ar–H), 8.30
(dd, Ja = 1.16, Jb = 7.80 Hz, 1H, Ar–H), 10.5 (s, 1H, NH, exchanged
with D2O); 13C NMR (DMSO‐d6, 100 MHz, δ, ppm): 24.5 (CH2), 30.4
(CH2), 116.6, 118.0, 127.0, 129.2, 129.5, 129.7, 129.9, 134.4, 135.7,
146.0, 148.5, 149.6, and 150.0 (Ar carbons), 158.8, 173.7, and 174.6
(three carbonyl carbons); MS (m/z): 334.14 [M]+; anal. calcd. for
C
18H14N4O3: C, 64.66; H, 4.22; N, 16.76%; found: C, 64.52; H, 4.36;
N, 16.94%.
Then, termination of the reactions was achieved by adding
100 µl Green Assay Reagent, and the color was allowed to de-
velop for 20 min before reading on the microplate reader at
620 nm. A standard curve was prepared from seven dilutions of
PDE standard (1000 ng/ml).
1‐[(4‐Oxo‐3‐phenyl‐3,4‐dihydroquinazolin‐2‐yl)amino]‐1H‐pyrrole‐
2,5‐dione (13b)
Yield: 91%; m.p.: 304–308°C; IR (cm−1, KBr): 3395 (NH), 3060 (CH
aromatic), 2931 (CH aliphatic), 1700 (3C═O), 1601 (NH bending),
1566–1543 (C═C); 1H NMR (DMSO‐d6, 400 MHz, δ, ppm): 6.58 (s,
2H, olefinic H), 7.50–7.66 (m, 7H, 6 Ar–H + NH exchanged with D2O),
7.98 (t, J = 7.60 Hz, 1H, Ar–H), 8.12 (d, J = 8.40 Hz, 1H, Ar–H), 8.29 (d,
J = 7.60 Hz, 1H, Ar–H); 13C NMR (DMSO‐d6, 100 MHz, δ, ppm):
116.5, 118.0, 127.0, 129.2, 129.3, 129.7, 129.8, 134.5, 135.6, 135.7,
146.0, and 149.6 (Ar carbons + two olefinic carbons), 158.9 and
172.5 (three carbonyl carbons); anal. calcd. for C18H12N4O3: C,
65.06; H, 3.64; N, 16.86%; found: C, 65.33; H, 3.51; N, 17.12%.
PDE activity is calculated according to the following equation:
%Inhibition = [1 − ΔODTestCpd/ΔODControl] × 100
where ΔODTest Cpd is the optical density value of a test compound
well at 0 min subtracted from the optical density value of a test
compound well at 20 min. ΔODControl is the optical density value of
the control well at 0 min subtracted from the optical density value of
the control well at 20 min.
2‐[(4‐Oxo‐3‐phenyl‐3,4‐dihydroquinazolin‐2‐yl)amino]isoindoline‐
1,3‐dione (13c)
|
4.2.2
In vivo behavioral tests
Yield: 83%; m.p.: 270–271°C; IR (cm−1
, KBr): 3217 (NH),
3059–3013 (CH aromatic), 2981–2932 (CH aliphatic),
1744–1713 (3C═O), 1663 (NH bending), 1597–1566 (C═C); 1H
NMR (DMSO‐d6, 400 MHz, δ, ppm): 7.24–7.40 (m, 2H, Ar–H),
7.50–7.69 (m, 5H, Ar–H), 7.84 (s, 2H, Ar–H), 7.92 (t, J = 8.00 Hz,
2H, Ar–H), 8.03 (d, J = 8.32 Hz, 1H, Ar–H), 8.22 (d, J = 7.64 Hz, 1H,
Ar–H), 12.10 (s, 1H, NH, exchanged with D2O); 13C NMR (DMSO‐
d6, 100 MHz, δ, ppm): 118.0, 123.2, 125.3, 128.0, 128.9, 129.2,
129.3, 129.5, 129.7, 129.8, 131.2, 131.9, 133.3, 134.5, 135.6,
136.7, 146.0, 149.6, and 158.8 (Ar carbons), 165.8, 169.1, and
172.5 (three carbonyl carbons); anal. calcd. for C22H14N4O3: C,
69.10; H, 3.69; N, 14.65%; found: C, 68.98; H, 3.62; N, 14.54%.
The procedures of the in vivo experiments applied in the beha-
vioral tests were approved by The Commission on the Ethics of
Scientific Research, Faculty of Pharmacy, Minia University with
number 52/2019. Adult Swiss Albino mice (8−10 weeks old,
weight 25−30 g) were kept in the animal house in well‐ventilated
cages with free access to standard forage. Animals adapted for 1
week in a room with controlled temperature (25 2°C), humidity
(60 10%), and lighting (12 h light–dark cycle). Food and water
were provided ad libitum throughout the experiment. The mice
were divided into five groups:
• Control (saline only).
• Model (scopolamine).
|
4.2
Biological evaluation
• Donepezil (scopolamine plus donepezil).
• IBMX (scopolamine plus compound IBMX).
• 10d (scopolamine plus compound 10d).
|
4.2.1
In vitro PDE inhibitory activity
All the newly synthesized compounds were evaluated for their in
vitro PDE inhibitory activity using Abcam PDE Activity Assay Kit
(ab139460), a colorimetric, nonradioactive assay designed in a
microplate format. Thaw assay buffer, 5ʹ‐nucleotidase, the
All drugs were dissolved in saline, and treatments were given in-
traperitoneally in equimolar doses to donepezil (5 mg/kg) 30 min before
memory impairment induction by scopolamine (0.5 mg/kg, ip).

AMIN ET AL.
15 of 16
|
Y‐maze test[44]
4.3.2
Molecular docking of PDE4B and PDE4D
|
Y‐maze composed of three equally spaced horizontal arms (120°,
45 cm long, and 16 cm high). Each mouse was placed into one of
the three arms and allowed to move freely between the arms of
the maze. The sequence of arm entries and the number of arm
entries were recorded. The spontaneous alternation is a measure
of the memory performance and calculated from the following
equation: % Alternation = [Number of alternations/(total arm
entries) − 2] × 100.
This study considered two PDB files 2QYL and 1OYN for PDE4B and
PDE4D, respectively.[53,54] Water molecules were removed from
both PDB files while keeping metal ions during the molecular
docking evaluation. For both PDB files, MMFF94x was used as the
forcefield, Triangle Matcher as the placement methodology, and
London dG and GBVI/WSA dG as the refinement rescoring 1 and 2,
respectively.
Step‐through passive avoidance test[45]
CONFLICTS OF INTERESTS
It was carried out in an apparatus that was divided into two
distinct chambers. The illuminated chamber, free from electric
stimuli, was connected to a dark compartment with electrifiable
grid floor. Both chambers were separated by a sliding door. Then,
the mice underwent two separate trials: a training trial and a test
trial. The training trial was performed by placing each mouse in
the illuminated chamber, and the mouse was allowed to get fa-
miliar with it. Next, the sliding door was opened to allow entry
into the dark compartment. When the animal stepped completely
into the dark chamber, the door was closed and the animal re-
ceived an electric foot shock (24 V, 0.5 mA) for 2 s. Latency time
to enter the dark compartment was recorded, which measures
the working memory. Twenty‐four hours after the training trial, a
test trial was performed, but no electric foot shock was applied.
The authors declare that there are no conflicts of interests.
ORCID
Riham F. George
http://orcid.org/0000-0002-2493-1315
http://orcid.org/0000-0001-5954-668X
Nada M. Mohamed
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How to cite this article: K. M. Amin, G. H. Hegazy, R. F.
George, N. R. Ibrahim, N. M. Mohamed. Design, synthesis,
and pharmacological characterization of some 2‐substituted‐
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