New 4-phenylcoumarin derivatives as potent 3C protease inhibitors: Design, synthesis, anti-HAV effect and molecular modeling

Article abstract of DOI:10.1016/j.ejmech.2019.02.048

A new series of 4-phenylcoumarin derivatives was synthesized starting from (2-oxo-4-phenyl-2H-chromen-7-yloxy) acetic acid hydrazide 3. Evaluation of the target compounds for their antiviral activity against hepatitis A virus revealed that the ethylthiose

Full text of DOI:10.1016/j.ejmech.2019.02.048

Accepted Manuscript
New 4-phenylcoumarin derivatives as potent 3C protease inhibitors: Design,
synthesis, anti-HAV effect and molecular modeling
Asmaa F. Kassem, Rasha Z. Batran, Eman M.H. Abbas, Samia A. Elseginy,
Mohamed N. Shaheen, El-Mahdy M. El-Mahdy
PII:
S0223-5234(19)30162-X
DOI:
https://doi.org/10.1016/j.ejmech.2019.02.048
EJMECH 11140
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 November 2018
Revised Date: 31 January 2019
Accepted Date: 14 February 2019
Please cite this article as: A.F. Kassem, R.Z. Batran, E.M.H. Abbas, S.A. Elseginy, M.N. Shaheen, E.-
M.M. El-Mahdy, New 4-phenylcoumarin derivatives as potent 3C protease inhibitors: Design, synthesis,
anti-HAV effect and molecular modeling, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.02.048.
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Graphical Abstract
New 4-phenylcoumarin derivatives as potent 3C protease inhibitors: Design,
synthesis, anti-HAV effect and molecular modeling
S
H
N
N
N
O
O
O
H
H
O
7b
H
N
N
O
O
O
O
Cl
14b
H
N
N
O
O
O
O
Br
14c
7b
14b
10.7µg/mL, 91
(virustatic)
(viral replication)
0.104 µM
14c
HAV(IC50, TI)
3.1µg/mL, 83
(virucidal)
8.5 µg/mL, 88
(virustatic)
(viral adsorption)
0.217 µM
HAV 3C pro ( Ki)
HRV 3C pro( IC50)
1.903 µM
16.1µM
4.13 µM
6.3 µM

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New 4-phenylcoumarin derivatives as potent 3C protease inhibitors:
Design, synthesis, anti-HAV effect and molecular modeling
Asmaa F. Kassem,a Rasha Z. Batran,b* Eman M.H. Abbas,a Samia A.
Elseginy,c Mohamed N. Shaheen,d El-Mahdy M. El-Mahdyd
a
Chemistry of Natural and Microbial Products Department, Pharmaceutical and
Drug Industries Research Division, National Research Centre, 33 El Bohouth St.,
Dokki, Giza, p.o.box 12622, Egypt
b Chemistry of Natural Compounds Department, Pharmaceutical and Drug Industries
Research Division, National Research Centre, 33 El Bohouth St., Dokki, Giza, p.o.
box 12622, Egypt
c
Green Chemistry Department, Chemical Industries Research Division, National
Research Centre, 33 El Bohouth St., Dokki, Giza, p.o. box 12622, Egypt
d
Environmental Virology laboratory, Water pollution Research Department,
Environmental Research Division, National Research Centre, 33 El Bohouth St.,
Dokki, Giza, p.o. box 12622, Egypt
*Corresponding author: rasha_batran@yahoo.com
Abstract
A new series of 4-phenylcoumarin derivatives was synthesized starting from
(2-oxo-4-phenyl-2H-chromen-7-yloxy) acetic acid hydrazide 3. Evaluation of the
target compounds for their antiviral activity against hepatitis A virus revealed that the
ethylthiosemicarbazide derivative 7b was the most potent virucidal agent
(IC50=3.1µg/mL, TI = 83). The Schiff′s bases 14c and 14b demonstrated the highest
virustatic effects against viral adsorption and replication, respectively (14c; IC50=8.5
µg/mL, TI = 88 and 14b; IC50=10.7µg/mL, TI = 91). Furthermore, compounds 7b,
14b and 14c were tested against HAV 3C protease and showed significant inhibition
effects (Ki= 1.903, 0.104 and 0.217 µM, respectively). The remarkable inhibitory
effect expressed by the three target compounds against HAV 3C protease prompted us
to expand our research on HRV 3C protease, a structurally related enzyme of the
same family, and interestingly, the three target compounds displayed significant
inhibitory effect against HRV 3C protease (IC50= 16.10, 4.13 and 6.30µM,
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respectively). Moreover, the active compounds 7b, 14b and 14c were docked within
the pocket site of HAV 3C protease (PDB code: 2HAL) illustrating a strong H-profile
with the key amino acids Gly170 and Cys172 similar to the co-crystallized ligand.
Furthermore, 3D-pharmacophore and quantitative structure activity relationship
(QSAR) models were generated to explore the structural requirements for the
observed antiviral activity.
Keywords: 4-phenylcoumarins; heterocyclic; anti-HAV; 3C protease; docking;
QSAR; 3D pharmacophore.
1.
Introduction
Picornaviruses, family Picornaviridae, are positive-strand non enveloped
RNA viruses comprising important human pathogens such as human rhinovirus
(HRV), poliovirus (PV), encephalomyocarditis virus and hepatitis A virus (HAV)[1].
Regulated proteolysis is an essential feature in the processing of the picornaviral
polyprotein (250 kDa) that is crucial for replication process [2]. As such,
picornaviruses evolve highly specific proteases, 3C proteases (3C pro) sharing similar
spatial structures. These proteases are responsible for processing the polyprotein
precursor and also cleaving certain cellular factors needed for transcription and
translation processes as well as nucleocytoplasmic trafficking in order to modulate
pro
cell physiology for viral replication thus 3C
play a principle role in the viral life
cycle which might expose viral proteases as selective targets for antiviral therapy [3].
Hepatitis A is a highly contagious liver disease caused by hepatitis A virus
(HAV). It is a self-limited acute disease usually transmitted via fecal-oral route.
Infection with HAV causes fever, weakness, malaise, myalgias, arthralgias, anorexia,
vomiting, and nausea [4-6]. Fulminant hepatitis A is a rare but devastating
complication of HAV infection that leads to acute liver failure (ALF) which is a
potential lethal disease [7]. Elderly patients and those with chronic liver disease are at
a higher risk for fulminant hepatitis A [8]. Although millions of infection cases are
yearly reported, with much higher incidence in developing countries, there are no
commercially available anti-HAV drugs till date. Similar to other picornaviruses,
pro
HAV genome encodes a main processing protease, assigned HAV 3C (HAV 3C
)
which is a nonstructural cysteine protein responsible for the cleavage process within
the viral polyprotein which is critical for the replication process.
The chromene scaffold represents a functional core structure in various
biologically active anti-picornaviral agents and 3C protease inhibitors. Flavonoids, 2-
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phenyl-4H-chromen-4-ones, as luteoloside [9], quercetin [10], rutin and fisetin [11]
have been reported for their antiviral effectiveness against EV71 which was mediated
by the inhibition of 3C protease enzymatic activity and hence blocking viral
replication. Recently, synthetic chromene analogues have been considered as
promising biodynamic pharmacophores for designing and development of anti-
picornaviral therapeutic agents [12-16].
Coumarins (2H-chromen-2-ones) are recognized as a privileged bioactive
scaffold for designing new agents with high affinity and specificity to various
molecular targets, [17-20] especially as antiviral agents [21-25]. 4-Phenylcoumarins
(neoflavones), bioisosteres of flavonoids, gained great attention in the past few
decades as lead target structures for the discovery of new antiviral agents [26,27].
Although the antiviral effectiveness of various coumarin compounds has already been
published, the anti-HAV potential of coumarin derivatives remains largely
unexplored.
In view of these observations, momentous attention has been paid to make use
of the structural isosterism between the 4-phenylcoumarin scaffold and the flavonid
structural core to develop new modified 4-phenylcoumarin-based drug candidates as
new 3C protease inhibitors.
We report herein the synthesis of new 4-phenylcoumarin derivatives
hybridized with acyclic thiosemicarbazide and imine functionalities as well as various
five membered azahetrocyclic rings, reported for their anti-HAV effect, such as
pyrrol, pyrazole, oxadiazole, thiadiazole and triazole moieties and the study of their
antiviral activities against hepatitis A [28-34]. The most active anti-HAV derivatives
were further assessed for their inhibitory potential towards HAV 3C pro
.
Molecular docking was performed for the most active anti-HAV candidates
within the pocket site of HAV 3C protease (PDB code: 2HAL).Moreover, QSAR and
pharmacophore methodology were used to identify the structural features required for
the antiviral properties of these new series. In addition, drug likeness properties of the
new series were investigated in order to identify the oral bioavailability of the new 4-
phenylcoumarin derivatives.
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2.
Results and discussion
2.1.
Chemistry
The 4-phenyl-7-oxycoumarin derivatives 1-4, 5a-c, 6, 7a,b, 8a,b , 9a,b, 10-12,
13a-c and 14a-c were efficiently synthesized according to the procedures outlined in
schemes 1−4.
A facile synthesis of the key intermediate hydrazide 3 [35] involves the
reaction of the ester compound 2 [35] and hydrazine hydrate in ethyl alcohol at room
temperature (Scheme 1).
The 5-thioxo-1,3,4-oxadiazole derivative 4 [35] was prepared by refluxing the
hydrazide 3 with carbon disulfide in pyridine. Compound 4 was further S-alkyled
with dimethylaminoethyl chloride hydrochloride, diethylaminoethyl chloride
hydrochloride and/or 2-morpholinoethyl chloride hydrochloride, in refluxing dry
acetone in the presence of anhydrous potassium carbonate, to afford the target
dimethylaminoethylthio-1,3,4-oxadiazole 5a, diethylaminoethylthio-1,3,4-oxadiazole
5b and morpholinoethylthio-1,3,4-oxadiazoles 5c, respectively. On the other hand,
hydrazinolysis of the 5-thioxo-1,3,4-oxadiazole derivative 4 with hydrazine hydrate in
refluxing ethyl alcohol afforded the 4-amino-5-mercapto-1,2,4-triazole derivative 6
(Scheme 2).
The thiosemicarbazide derivatives 7a,b [17] were obtained through the
condensation of the hydrazide 3 with methylisothiocyanate and
/
or
ethylisothiocyanate, respectively. Heterocyclization of the thiosemicarbazides 7a,b
with H2SO4 gave the corresponding aminothiadiazoles 8a,b, respectively. While,
condensing thiosemicarbazide derivatives 7a,b with 2N potassium hydroxide solution
afforded the corresponding N-(methyl/ ethyl)-5-thioxo-1,2,4-triazole derivatives 9a,b.
The 7-((5-(2-morpholinoethylthio)-4-ethyl-4H-1,2,4-triazol-3-yl) methoxy)-4-phenyl-
2H-chromen-2-one 10 was prepared via refluxing 4-ethyl-5-thioxo-1,2,4-triazole 9b
with 2-morpholinoethyl chloride hydrochloride, in dry acetone in the presence of
anhydrous potassium carbonate (Scheme3).
Cyclocondensation of the hydrazide 3 with acetylacetone afforded the
corresponding 3,5-dimethylpyrazole 11. The target 2-(4-phenylcoumarin-7-yloxy)-N-
(1,3-dioxoisoindolin-2-yl) acetamide 12 [35] was obtained by the condensation of the
hydrazide 3 with phthalic anhydride in acetic acid, while refluxing the hydrazide 3
with isatin derivatives, namely; isatin, 6-chloroisatin and / or 6-bromoisatin, yielded
the corresponding oxoindolin-3ylidene acetohydrazides 13a-c. Moreover, the Schiff’s
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bases 14 a-c were obtained by the condensation of the hydrazide compound 3 with
acetophenone derivatives, namely; acetophenone, 4-chloroacetophenone and/ or 4-
bromoacetophenone (Scheme 4). The structures of the new compounds were
characterized by means of IR, 1HNMR, 13CNMR and EI-MS.
2.2. Biology
2.2.1. Antiviral Screening
In this work we reported the anti-HAV activity of the synthesized target
compounds. The cytotoxicity test was conducted to determine the safe doses which
could be used in the antiviral assays. Safe concentrations were used to determine the
antiviral effect of the compounds through three different assays to understand the
mechanism by which these compounds can inhibit the virus infection.
In virucidal experiment, the tested compounds showed therapeutic index
ranging from 0.2 to 83, where five compounds 1, 4, 7b, 9a and 13c displayed
therapeutic index higher than 50 (TI = 70.8, 78, 83, 57, 73, respectively) (Table 1).
The target compounds may exert virucidal activity through the interaction with the
viral capsid changing its configuration and causing inability of the virus to bind to the
cell receptor. The bioactive compounds may also interact with the capsid proteins
resulting in the prevention of the virus entry into host cells. In the pre-treatment assay,
the compounds showed therapeutic index ranging from 0.2 to 88 where 5b, 7a, 12 and
14c compounds exhibited therapeutic index higher than 50 (TI = 77, 80, 72, and 88,
respectively) (Table 1). These findings indicate that the target compounds inhibited
virus infection through the effect on virus adsorption to the cell surface. These
compounds may interfere with virus binding by blocking the cellular receptors and
hence lead to the anti-HAV effect. During the post-treatment assay, the compounds
showed therapeutic index ranging from 0.1 to 91 where 4, 10, 13c and 14b showed
strong antiviral activity with TI ≥ 50 (TI = 50, 70, 52 and 91, respectively) (Table 1).
In this experiment, the virus inhibition may be due to the interaction of the target
compounds with one or more viral enzyme needed to complete the replication cycle
or due to the interference with one step or more in the viral life cycle.
2.2.2. Inhibition of HAV 3C protease
In this study, the most active compounds against hepatitis A 7b, 14b and 14 c
were further screened to identify potent inhibitors of the picornaviral HAV 3C pro. The
three compounds showed promising inhibitory effect against HAV 3Cpro with Ki
values of 1.903, 0.104 and 0.217 µM, respectively (Table 2).
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Both the 4-chlorophenyl Schiff's base 14b and the 4-bromophenyl congener
14c showed significant inhibitory constants at low micromolar concentrations (Ki=
0.104 and 0.217 µM, respectively) indicating strong binding to HAV 3Cpro.
2.2.3. Inhibition of HRV 3C protease
The significant inhibitory effect of compounds 7b, 14b and 14c against HAV
3Cpro encouraged us to expand our research area to discover novel 3C protease
inhibitors, we have tested the inhibitory effect of the three target compounds 7b, 14b
and 14 c against another structurally related picornaviral 3C protease, HRV 3C pro as a
screening procedure and guidance for future investigation on a panel of the same
family proteases as an important step in anti-picornaviral drug discovery. The three
compounds showed promising inhibition effects with IC50 values of 16.10, 4.13 and
6.30 µM, respectively, almost with similar IC50 values to that displayed against HAV
pro
3C
(Table 2). The aforementioned results indicate that the target compounds are
non selective 3C protease inhibitors as such could be important lead compounds for
future discovery of broad spectrum 3C protease inhibitors.
2.3. Structure activity relationship
The 7-hydroxy4-phenylcoumarin 1 showed high virucidal effect with IC50=
4.8µg/ml and TI= 70.8. The activity was diminished for the ester 2 and the hydrazide
derivative 3 (IC50= 1285.7, 3925µg/ml, TI= 0.7, 0.2). The post treatment assay
revealed that compound 2 can moderately affect viral replication (IC50=25.7µg/ml,
TI= 35).
The thioxo-1,3,4-oxadiazole 4 elicited significant virucidal potency (IC50= 4.7
µg/ml, TI= 78) and remarkable effect on viral replication (IC50=7.34µg/ml, TI= 50).
Furthermore, S-alkylation of the thioxo-1,3,4-oxadiazolyl derivative 4 with
dimethylaminoethyl moiety 5a decreases both the virucidal effect (IC50=44.8µg/ml,
TI= 20) and the viral replication potency (IC50 = 35.8µg/ml, TI= 25). Although the
diethylaminoethyl derivative 5b was devoid of any virucidal effect (IC50=81.6 µg/ml,
TI= 3), it exhibited potent effect on viral adsorption (IC50= 3.18 µg/ml, TI= 77) and
moderate effect on viral replication (IC50= 0.75 µg/ml, TI= 32).The morpholinoethyl
derivative 5c showed decreased anti-HAV effect for both virucidal activity and effect
on viral adsorption (IC50= 9.9µg/ml, TI= 25), and diminished effect on viral
replication (IC50= 622.5µg/ml, TI= 0.4).
The thiosemicarbazides 7a,b demonstrated potent anti-HAV activities. The
ethylthiosemicarbazide derivative 7b was the most potent virucidal agent
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(IC50=3.1µg/ml, TI= 83). It demonstrated higher antiviral effect than the standard
drug acyclovir (IC50= 35.6 µg/ml, TI= 80).While, the methyl congener 7a showed
significant effect on viral adsorption (IC50= 11.88 µg/ml, TI= 80). On the other hand,
cyclization of the thiosemicarbazides into aminothiadiazoles 8a,b completely
diminished the antiviral activity.
Within the cyclized triazole series both N-methyl/ and N-ethyl thioxotriazoles
9a,b showed the highest virucidal effects (IC50= 11.3, 12.9 µg/ml, TI= 57,46) while,
the 7-((5-(2-morpholinoethylthio)-4-ethyl-4H-1,2,4-triazol-3-yl) methoxy)- 4-
phenylcoumarin 10 was significantly the most potent antiviral agent affecting viral
replication(IC50= 13.4µg/ml, TI= 70). The N-amino-mercaptotriazole 6 was devoid of
any anti-HAV effect.
The 3,5-dimethylpyrazole derivative 11 displayed decreased virucidal effect
with IC50= 37.6µg/ml and TI= 20. It also displayed weak effect on viral replication
(IC50= 44.3µg/ml, TI= 17).
The target bromoindolyl-4-phenylcoumarin 13c exhibited remarkable
improved anti-HAV properties. It demonstrated significant virucidal effect (IC50=
13µg/ml, TI= 73) and remarkable effects on both viral replication (IC50= 18.3 µg/ml,
TI= 52) and adsorption (IC50= 21.6 µg/ml, TI= 44). The isoindole derivative 12
elicited high antiviral potency through its effect on viral adsorption (IC50=13 µg/ml,
TI= 72). On the other hand the unsubstituted indolyl derivative 13a and the
chloroindolyl-4-phenylcoumarin 13b completely lacked any anti-viral effects against
HAV.
Additionally, the 4-chlorophenyl Schiff's base 14b was the most potent anti-
HAV agent affecting viral replication among all the synthesized compounds (IC50=
10.7µg/ml, TI= 91), with comparable activity to the standard drug Acyclovir (IC50=
30.6 µg/ml, TI= 93).It had moderate virucidal effect (IC50= 30.5µg/ml, TI= 32) along
with decreased effect on viral adsorption (IC50= 44.4µg/ml, TI= 22). Furthermore, the
4-bromophenyl congener 14c proved to be the most potent compound affecting viral
adsorption (IC50= 8.5 µg/ml, TI= 88) with higher activity than the standard drug
(IC50= 35.5 µg/ml, TI= 85), while it had weak effect on viral replication
(IC50=62.5µg/ml, TI= 12).The antiviral activity was completely diminished for the
unsubstituted phenyl Schiff's base derivative 14a.
2.4. Molecular modeling study
2.4.1. Molecular docking
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The crystal structure of HAV 3C protease (PDB code: 2CXV) [36] revealed
that the enzyme binds to the substrate at four or five residues (P4 to P1; Leu, Arg, Thr
and Gln) [37]. The mode of action proceeds through a nucleophilic attack of Cys172
thiolate on the substrate carbonyl oxygen with base assistance from His44. Also,
hydrogen bond between His191 and carbonyl oxygen in the side chain of glutamine
(P1) was shown in this crystal structure [37]. In 2006 Jiang et al [38], solved the
crystal structure of HAV 3C with tetrapeptidyl-based methyl ketone inhibitor at high
resolution (PDB:2HAL) [36], the tetrapeptide portion of these inhibitors are
analogous to the residues at P4 to P1 in the natural substrate position. Tetrapeptidyl
inhibitor (N-acety-Leucyl-phenylalanyl-phenylalanyl-glutamate) formed two covalent
bonds between P1-Glutamine and Cys172, and H-bond with Gly170 [38] (Fig.1). P3-
phenylalanine formed two H-bonds with Val144 and Gly194 (Fig.1), P4-Leucin
contributed by two H-bonds with Val144 and Gly194 (Fig. 1, Table 3).
In our modeling study we docked the three active compounds 7b, 14b and 14c
within the pocket site of HAV 3C protease (PDB code: 2HAL), MOE [39] software
was used to build the compounds and minimize the energy while docking studies
were carried out with Autodock4 [40].
The docking results showed that compound 7b illustrated interactions with the
key residues Gly170 and Cys 172 and formed four H-bonds in a similar manner to the
tetrapeptidyl inhibitor where, O atom of C=O of compound 7b formed two hydrogen
bonds with SH-Cys172 and additional two H-bonds one with NH of Cys172 and one
with NH –Gly170 (Fig.2A), the four H-bonds formed net-like structure that stabilized
the compound within the binding pocket. The C=O of coumarin moiety formed H-
bond with NH of Gly194 which is another key amino acid at a distance of 2.9Å
(Fig.2B). Unexpectedly the sulfur atom of the linker side chain received H from NH-
Hist191, this amino acid showed an important interaction with C=O of glutamine
(P1), in the previous crystal structure (PDB:code 2CXV). The inhibitory activity of
compound 7b was attributed to these H-bonds and the hydrophobic interaction with
Pro169 and Ala193 (Fig2, Table 3).
Compounds 14b and 14c are analogues, the difference was only in the p-
phenyl substituent and this explains the similarity between the two compounds in the
interactions and fitting within the binding pocket of HAV 3C protease. Oxygen atom
of C=O moiety of compounds 14b and 14c showed the same H-profiles; O atom
formed two H- bonds with the SH-Cys172, the most important amino acid for the
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activity. Also, O atom formed two H bonds with NH-Cys172 and NH-Gly172. The 4-
phenyl coumarin moiety showed hydrophobic interactions with Leu168, pro169,
Ala193 and the hydrophobic part of Gly194 and Gly195 (Figs. 3 and 4, Table 3).
2.4.2. Binding Modes of compounds 7b, 14b and 14c
To further predict the possible binding mode for each compound within the
pocket site of HAV 3C protease, the binding free energy (∆GBind) and binding affinity
pKd were determined (Table 4).
The binding mode of the three compounds was represented in Figure 5.
Compound 7b (Ki = 1.903 µM) showed good binding mode as 4-phenylcoumarin
moiety occupied the same position as P2-phenylalanine and P3-phenylalanine, and the
side chain filled the same space similar to P1-glutamine. It had the highest ∆G Bind and
binding affinity pKd (∆G Bind= -9.15 kcal/mol, pKd= 6.78). Compounds 14b and 14c
(Ki=0.104 and 0.217µM, respectively) exhibited the same binding mode as P1-
glutamine of the tetrapeptidyl inhibitor displaying comparable ∆GBind and binding
affinity pKd (∆G= -7.44 and -7.47 kcal/mol, pKd= 5.51 and 5.53).
It could be seen that the three inhibitors 7b, 14b and 14c illustrated a strong H-
profile with the key amino acids Gly170 and Cys172 in a similar manner to the
tetrapeptidyl inhibitor; the co-crystallized ligand which explains the inhibitory activity
of these compounds and displayed relative favorable binding free energy and binding
affinity. These results were in agreement with the biological findings and it proved the
importance of Cys172 residue for enzyme activity. The potential interactions of
compounds 7b, 14b and 14c within the active site of HAV 3C protease provide
significant rationale as to the mechanism by which these compounds suppress HAV
3C protease activity. Nevertheless, experimental analysis is required to validate the
predicted binding mode of the three compounds.
2.4.3. QSAR studies
QSAR studies were carried out for the coumarin derivatives that showed a
virucidal inhibitory activity. The data set was divided randomly as training set (18
compounds) and a test set (5 compounds) with their measured pIC50 (-log IC50).The
molecular operating environment (MOE) software [41] was employed for the
calculation of the physicochemical descriptors of the molecules. 2D and 3D QSAR
descriptors were calculated; 2D descriptors include physical properties, subdivided
subsurface areas, adjacency and distance and partial charge descriptors. 3D
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descriptors include potential Energy, MOPAC, surface Area, volume and shape
descriptors and conformation dependent charge descriptors.
Partial least square (PLS) method was used to perform the study. The models
underwent validation study by internal methods via leave one-out cross-validation
(LOO), r2 (squared correlation coefficient), r2 prediction (predictive squared
correlation coefficient), Z-outlier score and residuals between the experimental and
predicted activity [42].
In this study volume surface vsurf_Wp (3D descriptor), density (3D
descriptor) and opr_brigid (2D descriptor), contribute for the best predicted activity
and the highest r2 (0.8). vsurf_Wp descriptor describes the polar volume of the
molecules [43], in general, Wp1-4 account for dispersion forces and polarizability
while Wp5-8 account for polar and hydrogen bond donor/ acceptor regions [43].
Additionally, opr_brigid descriptor describes the number of rigid bonds.
QSAR models were represented graphically by scattering plots of the
experimental versus the predicted bioactivity (Fig.6). Partial least square (PLS)
method was used to build the QSAR models, r2=0.891, root mean square error
(RMSE) = 0.200, cross validation r2=0.671 and cross validation RMSE= 0.351.
In conclusion, the virucidal activity of the synthesized compounds is
correlated negatively with vsurf_Wp4, vsurf_Wp6, and opr_rigid., but is correlated
positively with density. The virucidal activity decreased with the increase in the H-
bond donor/or acceptor groups on molecules′ surface and with the increase in the
polarizability of the molecules. Also, the virucidal activity decreased with the increase
in the number of rigid bonds of the molecules. On the other hand, the antiviral activity
increased with the rise in density. It is interesting to note that although compound 7b
had higher vsurf_Wp4 and vsurf-Wp6, it possessed the best virucidal effect which
may be due to its lower number of rigid bonds. These findings are relevant for the
design of novel antiviral agents and these structure-based approaches may lead to
improved virucidal activity for the next generation.
QSAR models were validated internally applying leave-1-out cross-validation
where r2 (squared correlation coefficient value) is 0.891, cross validation r2=0.671 and
RMSE = 0.200 which is a small error value. Validation was also employed by
measuring the residuals between the experimental activity and the predicted activity
(Table 5). It is noticeable that the residuals between the predicted antiviral activity
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and the experimental one are very close, indicating the reliability and effectiveness of
the established QSAR mode. Also, Z-outlier values of the training set were less than 2
2.4.4. Pharmacophore modeling
In this study, pharmacophore generation was performed using MOE software,
the pharmacophore query editor protocol was used to generate the pharmacophore
from a set of ligands with known antiviral activity values. The pharmacophore
features which were used are hydrogen bond acceptor (HBA), hydrogen bond donor
(HBD), hydrophobic interactions (HYD) and ring aromatic (RA). Different
pharmacophore models (hypothesis) were generated via flexible aligning of different
conformations of the best five compounds with the best antiviral activity. The top
pharmacophore was generated including seven features; two HBA, one HBD, three
RA, and one HYD (Fig.7). The constraint distances and angles between
pharmacophore features are represented in table 6.
Compound 7b and 4 are represented with the best generated pharmacophore
model (Fig.8). Seven features of the pharmacophore were considered, where
compound 7b (Fig.8A) fitted with six features out of seven, where carbonyl group
and oxygen atom fitted with two features (HBA), coumarin moiety and phenyl group
fitted with three (RA) and NH fitted with (HBD). While compound 4 fitted with five
features out of seven, where carbonyl group and oxygen atom fitted with two features
(HBA), coumarin moiety and phenyl group fitted with three (RA).
The results of pharmacophore supported the results of QSAR analysis as the
pharmacophore features include only one HBD and 2HDA. QSAR results suggested
that the antiviral activity decreased with the increase in the number of H-bond donors
or acceptors.
2.4.5. Drug likeness properties
The twenty three synthesized compounds were assessed against Lipinski’s rule
[44]. Briefly, the rule is based on the observation that most orally administered drugs
have a molecular weight (MW) of 500 or less, five or fewer hydrogen bond donor
sites, ten or fewer hydrogen bond acceptor sites and Log P should not be greater than
5. In addition, the polar surface area (PSA), was calculated, since it is another key
property that has been linked to the drug bioavailability. Thus, passively absorbed
molecules with a PSA >140 Å are thought to have low oral bioavailability [45]. The
results showed that our compounds fulfilled Lipinski’s rule expect compound 13c
which violated the rule in the MW and compounds 5c, 9a,b and 14a-c that broke the
11

ACCEPTED MANUSCRIPT
rule in the logP by a little difference, whilst the active compounds 1,4,5c,7b and 9a
fulfilled Lipinski’s rule (Table 7). Although the active compound 13c broke the rule,
it is still expected to be orally bioactive, because the rule states that an orally active
drug has no more than one violation of rule of five or Lipinski’s rule. In addition, the
compounds also fulfilled the topological surface area. The most active compound 7b
fulfilled the Lipinski’s rule and PSA which indicates that it has a good oral
bioavailability.
3. Conclusion
A new series of 4-phenylcoumarin derivatives was synthesized and the target
compounds were evaluated for their antiviral effect against hepatitis A virus through
studying their virucidal activity, effect on viral adsorption and effect on viral
replication in the host cell. The ethylthiosemicarbazide derivative 7b displayed the
highest virucidal effect among the tested target compounds (IC50=3.1µg/ml, TI = 83).
The 4-bromophenyl Schiff′s base 14c was the most potent compound affecting viral
adsorption (IC50=8.5 µg/ml, TI = 88) while the 4-chlorophenyl congener 14b showed
the most significant potential on viral replication (IC50=10.7µg/ml, TI = 91).
Furthermore, compounds 7b, 14c and 14b were tested against HAV 3C protease
revealing significant inhibition effects (IC50= 17.78, 7.19 and 5.94 µM, respectively).
The three compounds showed promising inhibition effect against the
pro
structurally related protease HRV 3C
with IC50 values of 16.1, 4.13and 6.3µM,
respectively. On the other hand, molecular docking study was performed between the
three inhibitors 7b, 14b and 14c and HAV 3C protease, which illustrated a strong H-
profile with the key amino acids Gly170 and Cys172 in a similar manner to the co-
crystalized ligand tetrapeptidyl inhibitor which explains the inhibitory activity of
these compounds. The combined analysis of QSAR and 3D pharmacophore revealed
significant information on the structural requirement for promising antiviral activity.
Several QSAR models were generated and the best one showed r2=0.891, root mean
square error (RMSE) = 0.200. The best four descriptors of the selected model were
vsurf_Wp4, vsurf-Wp6, density and opr_brigid. The generated 3D pharmacophore
showed 7 features; two HBA, one HBD, three RA, and one HYD. The molecular
modeling results may have a significant role in proposing further optimization
directions for designing more potent second generation antiviral agents. Docking and
pharmacophore modeling study proved the importance of coumarin moiety in the
binding mode of the promising compounds 7b, 14a and 14c within the active site of
12

ACCEPTED MANUSCRIPT
HAV3C protease and hence their promising enzymatic inhibitory activity. Based on
QSAR results, optimization of 4-phenylcoumarin derivatives could be achieved by
designing new derivatives with more hydrophobic characters by introducing less polar
substituents and also, linking 4-phenylcouamrin scaffold to flexible substituent
groups. The bioactive candidates 7b, 14a and 14c are predicted to have good oral
bioavailability according to Lipinski’s rule and PSA.
4. Experimental
4.1. Chemistry
All melting points were uncorrected and measured using Electrothermal IA
9000 apparatus. Infrared spectra were measured by Nexus 670 FT-IR FT-Raman
1
spectrometer using KBr discs at National Research Center, Cairo, Egypt. HNMR
spectra were determined using Varian mercury 300 MHz spectrometer at Micro-
Analytical Laboratory, Central Services Laboratory, Faculty of Science, Cairo
University, Egypt and Bruker 400 MHz spectrometer at Faculty of Pharmacy, Ain
Shams University, Egypt, using TMS as the internal standard. 13CNMR spectra were
determined using Varian mercury 300 MHz spectrometer and 400 MHz spectrometer.
The mass spectra were recorded on GCMS-QP 1000EX Shimadzu Gas
Chromatography MS Spectrometer. 13CNMR, Mass Spectra and the elemental
analyses were performed at Micro-Analytical Laboratory, Central Services
Laboratory, Faculty of Science, Cairo University, Egypt. The reactions were followed
by TLC (silica gel, aluminum sheets 60 F254, Merck) using chloroform-methanol
(9.5:0.5 v/v) as eluent and sprayed with iodine-potassium iodide reagent. The purity
of the newly synthesized compounds was assessed by TLC and elemental analysis and
was found to be higher than 95%. Compounds 1[46], 2 [47], 3 [35], 4 [35], 6 [35] and
12 [35] were previously prepared.
General procedure for the preparation of 7-((5-[(2-(dimethylamino)ethylthio), 2-
(diethylamino)ethylthio) and / or (2-morpholinoethylthio)]-1,3,4-oxadiazol-2-yl)
methoxy)-4-phenyl-2H-chromen-2-one ( 5a-c)
A mixture of 4 (3.52g, 0.01 mol), dimethylaminoethyl chloride hydrochloride,
diethylaminoethyl chloride hydrochloride, and/or 4-(2-chloroethyl) morpholine
hydrochloride (0.03mol) and anhydrous potassium carbonate (4.2 g, 0.03 mol) in dry
acetone (100 mL) was refluxed while stirring for 10−15 hours. The mixture was
filtered while hot, the filtrate was concentrated, and water was added to precipitate the
desired target compounds which were recrystallized from acetone/water.
13

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7-((5-(2-(Dimethylamino)ethylthio)-1,3,4-oxadiazol-2-yl)methoxy)-4-phenyl-2H-
chromen-2-one ( 5a)
Off white powder, yield 76 %, mp 122-4°C.Anal.Calcd.: C, 62.40; H, 5.00; N, 9.92;
S, 7.57. Found: C, 62.55; H, 5.18; N, 10.12; S, 7.67. IR (cm-1, KBr): 3067 (CH
aromatic stretching), 2944, 2932 (CH aliphatic stretching), 1726 (C=O, α-pyrone).
1HNMR (300MHz, DMSO-d6, δ, ppm): 2.17 (6H, s, 2CH3N), 2.58-2.62 (2H, t,
CH2N), 3.38-3.41 (2H, t, CH2S), 5.55 (2H, s, OCH2), 6.29 (1H, s, H-3 coumarin),
7.27-7.59 (8H, m, Ar−H). 13CNMR (75MHz, DMSO-d6, δ, ppm): 30.8, 44.7, 57.2,
59.9, 102.4, 112.1, 112.8, 128.0, 128.4, 128.9, 129.7, 134.9, 154.9, 155.2, 159.7,
160.2, 162.9, 165.6. EI-MS (m/z) for C22H21N3O4S (423.13): 423 [M]+ (0.53%), 72
(100.00%).
7-((5-(2-(Diethylamino)ethylthio)-1,3,4-oxadiazol-2-yl)methoxy)-4-phenyl-2H-
chromen-2-one ( 5b)
White powder, yield70%, mp 78−80°C. Anal. Calcd.: C, 63.84; H, 5.58; N, 9.31; S,
7.10. Found: C, 63.64; H, 5.45; N, 9.19; S, 6.98. IR (cm-1, KBr): 3061 (CH aromatic
1
stretching), 2956, 2936 (CH aliphatic stretching), 1727 (C=O, α-pyrone). HNMR
(300MHz, DMSO-d6, δ, ppm): 0.90-0.95 (6H, t, 2CH3CH2N), 2.45-2.49 (4H, q,
2CH3CH2N), 2.73-2.77 (2H, t, CH2N), 3.34-3.38 (2H, t, CH2S), 5.55 (2H, s, OCH2),
6.29 (1H, s, H-3 coumarin), 7.02-7.59 (8H, m, Ar−H). 13CNMR (75MHz, DMSO-d6,
δ, ppm): 14.2, 30.9, 44.7, 57.2, 59.9, 102.4, 112.1, 112.8, 127.9, 128.4, 128.8, 129.6,
134.9, 154.9, 155.5, 159.7, 160.2, 162.9, 165.6. EI-MS (m/z) for C24H25N3O4S
(451.16): 451 [M]+ (0.14%), 86 (100.00%).
7-((5-(2-Morpholinoethylthio)-1,3,4-oxadiazol-2-yl)methoxy)-4-phenyl-2H-
chromen-2-one ( 5c)
White powder, yield 70%, mp 120-2 °C. Anal. Calcd.: C, 61.92; H, 4.98; N, 9.03; S,
6.89. Found: C, 62.24; H, 5.10; N, 9.19; S, 6.95. IR (cm-1, KBr): 3060 (CH aromatic
1
stretching), 2943, 2932 (CH aliphatic stretching), 1729 (C=O, α-pyrone). HNMR
(300MHz, CDCl3, δ, ppm): 2.49-2.53 (4H, t, 2 CH2N morpholine), 2.76- 2.81 (2H, t,
CH2N), 3.43-3.48 (2H, t, CH2S), 3.67-3.71 (4H, t, 2 CH2O morpholine), 5.30 (2H, s,
OCH2), 6.27 (1H, s, H-3 coumarin), 6.89−7.54 (8H, m, Ar−H). 13CNMR (75MHz,
DMSO-d6, δ, ppm): 30.5, 48.7, 54.2, 60.6, 61.1, 102.4, 111.7, 112.5, 112.9, 127.8,
128.3, 128.7, 129.5, 134.8, 151.7, 154.9, 155.1, 159.7, 160.5, 166.1. EI-MS (m/z) for
C24H23N3O5S (465.14): 465 [M]+ (0.21%), 100 (100%).
14

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General Procedure for the preparation of 1-(2-(2-oxo-4-phenyl-2H-chromen-7-
yloxy)acetyl)-4-methyl and / or ethylthiosemicarbazide 7a,b.
To a solution of the hydrazide 3 (3.1g, 0.01 mol) in hot absolute ethanol,
methylisothiocyanate and/or ethylisothiocyanate (0.01mol) was added. The mixture
was refluxed for 5-7 hours and the separated solid was filtered, washed with ethanol
and recrystallized from acetic acid.
1-(2-(2-Oxo-4-phenyl-2H-chromen-7-yloxy)acetyl)-4-methylthiosemicarbazide
7a [17].
White powder, yield 85%, mp 217−9°C. Anal. Calcd.: C, 59.52; H, 4.47; N, 10.96; S,
8.36. Found: C, 59.64; H, 4.63; N, 11.19; S, 8.48. IR (cm-1, KBr): 3399, 3376, 3310
(3NH), 1702 (C=O, α-pyrone), 1670 (C=O), 1242 (C=S). 1HNMR (300MHz, DMSO-
d6, δ, ppm): 2.88-2.89 (3H, d, CH3), 4.74 (2H, s, OCH2), 6.26 (1H, s, H-3 coumarin),
6.81−7.57 (8H, m, Ar−H), 7.83-7.85 (1H, q, NHCH3, D2O exchangeable), 10.18 (1H,
s, NH, D2O exchangeable), 13.91(1H, s, NH, D2O exchangeable). EI-MS (m/z) for
C19H17N3O4S (383.09): 383 [M]+ (13%), 163 (100%).
1-(2-(2-Oxo-4-phenyl-2H-chromen-7-yloxy)acetyl)-4-ethylthiosemicarbazide
7b [17] .
White powder, yield 80%, mp 219−21°C. Anal. Calcd.: C, 60.44; H, 4.82; N, 10.57;
S, 8.07. Found: C, 60.34; H, 4.71; N, 10.49; S, 7.91. IR (cm-1, KBr): 3385, 3376,
3325, (3NH), 1708 (C=O, α-pyrone), 1675 (C=O), 1242 (C=S).1HNMR (300MHz,
DMSO-d6, δ, ppm): 1.05-1.09 (3H, t, CH3), 3.47-3.49 (2H, m, CH2), 4.73 (2H, s,
OCH2), 6.26 (1H, s, H-3 coumarin), 6.99−7.57 (8H, m, Ar−H), 7.99-8.02 (1H, t,
NHCH2, D2O exchangeable), 9.19 (1H, s, NH, D2O exchangeable), 10.08 (1H, s, NH,
D2O exchangeable). EI-MS (m/z) for C20H19N3O4S (397.11): 397 [M]+ (3%), 161
(100%).
Preparation of 7-((5-((methyl and /or ethyl) amino)-1,3,4-thiadiazol-2-
yl)methoxy)-4-phenyl-2H-chromen-2-one 8a,b.
An appropriate thiosemicarbazide 7a and/or 7b (0.001 mol) was dissolved in
concentrated sulphuric acid (1.6 mL), cooled and allowed to stand for 30 minutes at
0˚C while stirring. The reaction mixture was added gradually to crushed ice. The
separated solid was filtered off, washed with water till acid free, dried and
recrystallized from acetic acid.
7-((5-(Methylamino)-1,3,4-thiadiazol-2-yl)methoxy)-4-phenyl-2H-chromen-2-one
8a.
15

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White powder, yield 75%, mp 180-2°C. Anal. Calcd.: C, 62.45; H, 4.14; N,11.50; S,
8.78. Found: C, 62.35; H, 4.01; N, 11.39; S, 8.67. IR (cm-1, KBr): 3303 (NH), 1676
1
(C=O, α-pyrone). HNMR (400MHz, DMSO-d6, δ, ppm): 2.88-2.89 (3H, d, CH3),
5.45 (2H, s, OCH2), 6.27 (1H, s, H-3 coumarin), 6.99−7.58 (8H, m, Ar−H), 7.82-7.86
13
(1H, q, NH, D2O exchangeable). CNMR (75MHz, DMSO-d6, δ, ppm): 30.9, 64.8,
102.4, 111.8, 112.5, 112.9, 128.0, 128.4, 128.9, 129.7, 134.9, 152.2, 154.9, 155.3,
159.8, 160.6, 169.9. EI-MS (m/z) for C19H15N3O3S (365.08): 365 [M]+ (2.83%), 128
(100.00%).
7-((5-(Ethylamino)-1,3,4-thiadiazol-2-yl)methoxy)-4-phenyl-2H-chromen-2-one
8b.
White powder, yield 85%, mp 188-90°C. Anal. Calcd.: C, 63.31; H, 4.52; N, 11.07; S,
8.45. Found: C, 63.20; H, 4.42; N, 10.97; S, 8.38. IR (cm-1, KBr): 3305 (NH), 1670
1
(C=O, α-pyrone). HNMR (300MHz, DMSO-d6, δ, ppm): 1.14-1.19 (3H, t, CH3),
3.26-3.29 (2H, m, CH2), 5.44 (2H, s, OCH2), 6.26 (1H, s, H-3 coumarin), 6.99−7.56
(8H, m, Ar−H), 7.82-7.85 (1H, t, NH, D2O exchangeable). 13CNMR (75MHz,
DMSO-d6, δ, ppm): 14.2, 37.9, 64.8, 102.5, 111.8, 112.5, 112.9, 127.9, 128.4, 128.8,
129.6, 134.9, 152.2, 154.9, 155.3, 159.8, 160.6, 169.9. EI-MS (m/z) for C20H17N3O3S
(379.1): 380 [M+1]+ (22.23%), 379 [M]+ (7.3%), 189 (100%).
Preparation 7-((4-methyl and / or ethyl)-5-thioxo-1,2,4-triazol-3-yl)methoxy)-4-
phenyl-2H-chromen-2-one 9a,b.
A solution of 7a and / or 7b (0.01 mol) in 2N KOH (30 ml) was refluxed for
4-6 hours. The mixture was cooled, acidified with dil. HCl, filtered, washed with
water and recrystallized from ethanol.
7-((4-Methyl-5-thioxo-1,2,4-triazol-3-yl)methoxy)-4-phenyl-2H-chromen-2-one
9a.
White powder, yield 75%, mp 240−2°C. Anal. Calcd.: C, 62.45; H, 4.14; N,
11.50; S, 8.78. Found: C, 62.57; H, 4.25; N, 11.67; S, 8.89. IR (cm-1, KBr): 3350
1
(NH), 1685 (C=O, α-pyrone). HNMR (300MHz, DMSO-d6, δ, ppm): 3.51 (3H, s,
CH3), 5.39 (2H, s, OCH2), 6.28 (1H, s, H-3 coumarin), 6.81−7.56 (8H, m, Ar−H),
13
13.80 (1H, s, NH, D2O exchangeable). CNMR (75MHz, DMSO-d6, δ, ppm): 30.5,
61.1, 102.4, 111.7, 112.5, 112.8, 127.8, 128.3, 128.7, 129.5, 134.8, 151.7, 154.9,
155.1, 159.7, 160.5, 168.2. EI-MS (m/z) for C19H15N3O3S (365.08): 365 [M]+
(43.96%), 238 (100.00%).
7-((4-Ethyl-5-thioxo-1,2,4-triazol-3-yl)methoxy)-4-phenyl-2H-chromen-2-one 9b.
16

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White powder, yield 85%, mp 194-6 °C. Anal. Calcd.: C, 63.31; H, 4.52; N,
11.07; S, 8.45. Found: C, 63.21; H, 4.41; N, 10.98; S, 8.39. IR (cm-1, KBr): 3351
(NH), 1687 (C=O, α-pyrone). 1HNMR (400MHz, DMSO-d6, δ, ppm): 1.25-1.29 (3H,
t, CH3), 4.02-4.07 (2H, q, CH2), 5.41 (2H, s, OCH2), 6.29 (1H, s, H-3 coumarin),
7.03−7.57 (8H, m, Ar−H), 13.92 (1H, s, NH, D2O exchangeable). 13CNMR (100MHz,
DMSO-d6, δ, ppm): 13.8, 29.8, 60.8, 102.9, 112.4, 113.2, 113.4, 128.5, 128.9, 129.3,
130.2, 135.3, 147.8, 155.5, 155.7, 160.3, 160.6, 167.7. EI-MS (m/z) for C20H17N3O3S
(379.1): 379 [M]+ (23.14%), 211(100.00%).
Preparation
of
7-((5-(2-morpholinoethylthio)-4-ethyl-4H-1,2,4-triazol-3-yl)
methoxy)-4-phenyl-2H-chromen-2-one 10.
A mixture of 9b
(0.38g, 0.001 mol), 4-(2-chloroethyl) morpholine
hydrochloride (0.56g, 0.003 mol ) and anhydrous potassium carbonate in dry acetone
(100 ml) was refluxed while stirring for 10-15 hours. The mixture was filtered while
hot, the filtrate was concentrated and water was added to precipitate the desired target
compounds which were recrystallized from acetone/water.
White powder, yield 78%, mp 150-2°C. Anal. Calcd.: C, 63.40; H, 5.73; N, 11.37; S,
6.51. Found: C, 63.28; H, 5.61; N, 11.26; S, 6.42. IR (cm-1, KBr): 2949- 2931 (CH
1
aliphatic stretching), 1698 (C=O, α-pyrone). HNMR (300MHz, DMSO-d6, δ, ppm):
1.25-1.30 (3H, t, CH3CH2), 2.33-2.36 (4H, t, 2CH2N morpholine), 2.59-2.64 (2H, t,
CH2N), 3.31-3.36 (2H, t, CH2S), 3.47-3.49 (4H, t, 2CH2O morpholine), 4.02-4.05
(2H, q, CH2CH3), 5.46 (2H, s, OCH2), 6.27 (1H, s, H-3 coumarin), 7.02−7.58 (8H, m,
Ar−H). 13CNMR (100MHz, DMSO-d6, δ, ppm): 15.6, 30.5, 53.3, 53.4, 57.9, 60.8,
66.5, 102.8, 112.3, 112.9, 113.4, 126.7, 128.4, 128.5, 128.9, 129.3, 130.2, 135.3,
151.0, 151.6, 155.5, 155.7, 160.3, 160.9. EI-MS (m/z) for C26H28N4O4S (492.18): 492
[M]+ (0.39%), 113 (100%).
Preparation of 7-(2-(3,5-dimethyl-1H-pyrazol-1-yl)-2-oxoethoxy)-4-phenyl-2H-
chromen-2-one 11.
A mixture of the hydrazide 3 (0.31g, 0.001 mol) and acetylacetone (0.001 mol,
0.1 mL) in 20 mL absolute ethanol was refluxed for about 12 hours while stirring. The
formed precipitate after cooling was filtered, dried and recrystallized from ethyl
alcohol.
White powder, yield 79%, mp 164-166 °C. Anal. Calcd.: C, 70.58; H, 4.85; N,
7.48. Found: C, 70.69; H, 4.91; N, 7.53. IR (cm-1, KBr): 1707 (C=O, α-pyrone), 1678
1
(C=O amide). HNMR (300MHz, DMSO-d6, δ, ppm): 2.22 (3H, s, CH3-pyrazole),
17

ACCEPTED MANUSCRIPT
2.47 (3H, s, CH3-pyrazole), 5.63 (2H, s, OCH2), 6.23 (1H, s, CH-pyrazole), 6.25 (1H,
s, H-3 coumarin), 6.98- 7.57 (8H, m, Ar−H). 13CNMR (75MHz, DMSO-d6, δ, ppm):
13.5, 13.5, 66.5, 102.3, 111.3, 111.6, 112.4, 112.6, 127.8, 128.4, 128.8, 129.6, 134.98,
143.7, 152.5, 155.0, 155.3, 159.8, 161.7, 167.4. EI-MS (m/z) for C22H18N2O4
(374.13): 374 [M]+ (28.2 %), 109 (100%).
Preparation of 2-(2-oxo-4-phenyl-2H-chromen-7-yloxy)-N'-(5-(un)substituted-2-
oxoindolin-3-ylidene) acetohydrazide 13a-c
A mixture of hydrazide 3 (3.1g, 0.01 mol) and the appropriate isatin
compounds, namely, isatin, 5-chloroisatin and/or 5-bromoisatin (0.01 mol) in absolute
ethanol (30 mL) containing glacial acetic acid (1mL) was refluxed while stirring for
8-10 hours. The reaction mixture was cooled and the formed solid was collected by
filtration and recrystallized from acetic acid to give the target compounds.
2-(2-Oxo-4-phenyl-2H-chromen-7-yloxy)-N'-(2-oxoindolin-3-ylidene)
acetohydrazide 13a
Yellow powder, yield 80%, mp 273-5°C. Anal. Calcd.: C, 68.33; H, 3.90; N, 9.56.
Found: C, 68.28; H, 3.81; N, 9.48. IR (cm-1, KBr): 3367, 3311 (2NH), 1707 (C=O, α-
1
pyrone), 1689 (C=O, isatin), 1616 (C=O, hydrazide). HNMR (300MHz, DMSO-d6,
δ, ppm): 5.01 (2H, s, OCH2), 6.26 (1H, s, H-3 coumarin), 6.93–7.59 (12H, m, Ar-H),
11.25 (1H, s, NH, D2O exchangeable), 13.81 (1H, s, NH, D2O exchangeable).
13CNMR (100MHz, DMSO-d6, δ, ppm): 67.7, 102.7, 111.7, 112.4, 113.2, 120.1,
121.5, 123.1, 128.5, 128.9, 129.2, 129.3, 130.2, 132.4, 135.4, 143.1, 155.4, 155.7,
160.3, 162.9. EI-MS (m/z) for C25H17N3O5 (439.12): 439 [M]+ (6.2 %), 175 (100%).
2-(2-Oxo-4-phenyl-2H-chromen-7-yloxy)-N'-(5-chloro-2-oxoindolin-3-
ylidene)acetohydrazide 13b
Orange crystals, yield 85%, mp 118-20°C. Anal. Calcd.: C, 63.37; H, 3.40; N, 8.87.
Found: C, 63.29; H, 3.33; N, 8.78. IR (cm-1, KBr): 3371, 3325(2NH), 1711 (C=O, α-
1
pyrone), 1689 (C=O, isatin), 1615 (C=O, hydrazide). HNMR (300MHz, DMSO-d6,
δ, ppm): 5.03 (2H, s, OCH2), 6.24 (1H, s, H-3 coumarin), 6.88–7.56 (11H, m, Ar-H),
11.23 (1H, s, NH, D2O exchangeable), 13.66 (1H, s, NH, D2O exchangeable).
13CNMR (75MHz, DMSO-d6, δ, ppm): 66.5, 102.5, 111.9, 112.6, 113.0, 120.9, 120.9,
127.6, 128.2, 128.3, 128.7, 129.5, 129.8, 131.3, 134.9, 137.5, 143.7, 155.1, 155.3,
159.9, 160.6, 168.5. EI-MS (m/z) for C25H16ClN3O5 (473.08): 475/473 [M+2]+/ [M]+
(8.97/23.72%), 209 (100.00%).
18

ACCEPTED MANUSCRIPT
2-(2-Oxo-4-phenyl-2H-chromen-7-yloxy)-N'-(5-bromo-2-oxoindolin-3-
ylidene)acetohydrazide 13c
Orange crystals, yield 82%, mp 120-2°C. Anal. Calcd.: C, 57.93; H, 3.11; N, 8.11.
Found: C, 58.05; H, 3.22; N, 8.20. IR (cm-1, KBr): 3373, 3329 (2NH), 1710 (C=O, α-
1
pyrone), 1687 (C=O, isatin), 1618 (C=O, hydrazide). HNMR (300MHz, DMSO-d6,
δ, ppm): 5.02 (2H, s, OCH2), 6.26 (1H, s, H-3 coumarin), 6.90–7.57 (11H, m, Ar-H),
11.25 (1H, s, NH, D2O exchangeable), 13.78 (1H, s, NH, D2O exchangeable).
13CNMR (75MHz, DMSO-d6, δ, ppm): 66.40, 101.9, 111.7, 112.5, 112.9, 120.4,
120.5, 123.0, 128.2, 128.3, 128.7, 129.5, 129.8, 131.3, 134.8, 137.5, 144.8, 154.9,
155.2, 159.7, 160.4, 166.1. EI-MS (m/z) for C25H16BrN3O5 (517.03): 519/517
[M+2]+/ [M]+ (1.29/ 1.24%), 57 (100.00%).
Preparation
of
2-(2-Oxo-4-phenyl-2H-chromen-7-yloxy)-N'-(1-(4-
(un)substituted phenyl) ethylidene) acetohydrazide 14a-c
A mixture of hydrazide compound 3 (3.1g, 0.01 mol) and acetophenone
compounds,
namely;
acetophenone,
4-chloroacetophenone
and/or
4-
bromoacetophenone (0.01 mol ) in absolute ethanol in the presence of glacial acetic
acid (2mL), was refluxed for 6-8 hours, the formed precipitate was filtered off, dried
and recrystallized form acetic acid.
2-(2-Oxo-4-phenyl-2H-chromen-7-yloxy)-N'-(1-phenylethylidene)acetohydrazide
14a
White powder, yield 90%, mp 218-20 °C. Anal. Calcd.: C, 72.80; H, 4.89; N,6.79.
Found: C, 72.75; H, 4.82; N, 6.68. IR (cm-1, KBr): 3180 (NH), 1717 (C=O, α-pyrone),
1
1682 (C=O). HNMR (300MHz, DMSO-d6, δ, ppm): 2.33 (3H, s, CH3), 5.33 (2H, s,
OCH2), 6.22 (1H, s, H-3 coumarin), 6.99-7.87 (13H, m, Ar−H), 10.82 (1H, s, NH,
D2O exchangeable). 13CNMR (75MHz, DMSO-d6, δ, ppm): 13.3, 65.8, 101.9, 111.7,
112.5, 112.9, 127.6, 128.2, 128.3, 128.8, 129.5, 129.8, 131.3, 134.8, 137.8, 148.7,
154.8, 155.0, 159.7, 160.4, 168.6. EI-MS (m/z) for C25H20N2O4 (412.14): 414 [M+2]+
(21.54%), 412 [M]+ (12.4%), 219 (100%).
2-(2-Oxo-4-phenyl-2H-chromen-7-yloxy)-N'-(1-(4-chlorophenyl)ethylidene)
acetohydrazide 14b
White powder, yield 85 %, mp 258-9°C. Anal. Calcd.: C, 67.19; H, 4.29; N, 6.27.
Found: C, 67.25; H, 4.33; N, 6.36. IR (cm-1, KBr): 3188 (NH), 1710 (C=O, α-pyrone),
1
1686 (C=O). HNMR (300MHz, DMSO-d6, δ, ppm): 2.27 (3H, s, CH3), 5.35 (2H, s,
OCH2), 6.24 (1H, s, H-3 coumarin), 6.99−7.87 (12H, m, Ar−H), 10.95 (1H, s, NH,
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D2O exchangeable).13CNMR (100MHz, DMSO-d6, δ, ppm): 13.9, 66.2, 102.5, 111.9,
112.4, 113.2, 128.2, 128.5, 128.8, 128.9, 129.3, 130.1, 134.4, 135.5, 137.2, 147.9,
155.6, 155.7, 160.4, 162.0, 169.7. EI-MS (m/z) for C25H19ClN2O4 (446.1): 448/446
[M+2]+/ [M]+ (26.03/66.69%), 209 (100.00%).
2-(2-Oxo-4-phenyl-2H-chromen-7-yloxy)-N'-(1-(4-bromophenyl)ethylidene)
acetohydrazide 14c
White powder, yield 95%, mp 265-7°C. Anal. Calcd.: C, 61.11; H, 3.90; N, 5.70.
Found: C, 61.21; H, 3.98; N, 5.81. IR (cm-1, KBr): 3200 (NH), 1719 (C=O, α-pyrone),
1
1688 (C=O). HNMR (300MHz, DMSO-d6, δ, ppm): 2.34 (3H, s, CH3), 5.38 (2H, s,
OCH2), 6.23 (1H, s, H-3 coumarin), 6.97−7.88 (12H, m, Ar−H), 10.57 (1H, s, NH,
D2O exchangeable). 13CNMR (75MHz, DMSO-d6, δ, ppm): 13.9, 66.4, 101.9, 111.9,
112.6, 113.0, 127.6, 128.2, 128.3, 128.8, 129.5, 129.8, 131.3, 134.9, 137.9, 148.7,
154.9, 155.2, 159.7, 163.1, 171.1. EI-MS (m/z) for C25H19BrN2O4 (490.05): 492/490
[M+2]+/ [M]+ (30.99/ 30.71%), 253 (100.00%).
4.2. Biology
4.2.1. Anti-HAV assay
4.2.1.1. Cell culture and virus
A fetal rhesus monkey kidney cell line (Frhk-4), Purchased from VACSERA-
Egypt, cultured in DMEM (Dulbecco's Modified Eagle Medium) enriched with 10%
of FBS (fetal bovine serum), 100 units/ml, 100µg/ml streptomycin, penicillin under
5% CO2 incubator. Hepatitis A virus (HAV) prepared in ten-fold dilution was
inoculated onto cell lines in 96 well plate. After 72 h of inoculation, the cytopathic
effect was investigated under inverted microscope and TCID50/0.1ml (50% tissue
culture infectious doses/0.1 ml) was estimated according to Karber method [48], then
the tittered stock distributed into small aliquots and stored at – 80oC until used.
4.2.1.2. Cytotoxicity assay
The cytotoxicity of the compounds on Frhk-4 cell lines was performed by
MTT [3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay, as
described by Nabil et al [49]. In brief, the cells at concentration 5 X 103 cells / well
were seeded in 96- well plates. After incubation period for 24 hours, the cell medium
was discarded and the cell monolayers were incubated with various concentrations
(ranging from 7.8 - 1000 µg/mL) of each compound followed by incubation period for
o
additional 48 hours at 37 C under 5% CO2 atmosphere. The compounds were
removed and 100 µL of MTT solution (5 mg/mL) was added for 4 hours at 37°C.
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After removing MTT, 50 µL dimethyl sulfoxide (DMSO) was added to cells. After 30
minutes at 37°C, the optical density was checked at 540 nm using a multiwell ELISA
reader. The CC50 (50% cytotoxic concentration) was estimated as (K - B / K) x 100,
where K and B are the mean of three OD540 of untreated and treated cells with
compounds, respectively.
4.2.1.3. Antiviral effect of the tested compounds by MTT method
Frhk-4cell lines were seeded in 96- well plates and after 24 hours incubation,
the cell medium was removed from the wells and the antiviral experiment was
conducted in three different approaches.
4.2.1.3.1. Virucidal assay
The virus (106 log10 TCID50/0.1 ml) was mixed and incubated with equal
volume of various non-toxic concentrations of each compound for 1 hour at 37°C.
Then 100 µl of the previous mixture was added and incubated with cell monolayers
for 1hour. After that, the mixture was removed from wells and replaced with culture
medium.
4.2.1.3.2. Pre-treatment assay
The cell monolayers grown in 24-well plates were treated and incubated with
various non-toxic concentrations of each compound for 24 hours at 37°C under 5%
CO2 atmosphere. The compound concentrations were removed and replaced with
virus suspension. After 1 hour at 37°C, then viral suspension was removed and
replaced with culture medium.
4.2.1.3.3. Post-treatment assay
The confluent cell monolayers were infected with virus suspension. After 1 hour, the
unbound virus was removed and replaced with test medium containing various non-
toxic concentrations of each compounds.
In the three experiments, positive control (virus-infected cells) and negative
control wells (only cells with culture medium) were included. After incubating all
plates for 72 hours under 5% CO2 atmosphere at 37oC. The mean of three OD540 of
treated cells, positive control, and negative control were read on multiwall ELISA
reader then IC50 (the 50% inhibitory concentration) was estimated as [(K-V)/(C-V)
x100], where K, V and C refer to the mean three absorbance values of treated cells,
positive control, and negative control, respectively. The therapeutic index (TI) for
each compound was calculated by dividing of CC50 / IC50.
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4.2.2. HAV 3Cpro determination assay
4.2.2.1. Protein expression and purification
The HAV 3Cpro was produced heterologously using Escherichia coli
BL21(DE3) pLysS containing pHAV-3CEX according to the reported procedures
[50].
4.2.2.2. Fluorogenic peptide assay
The steady-state proteolytic activities of HAV 3C was measured using the
fluorogenic peptide substrate Dabcyl-GLRTQSND (edans) G. The standard assay was
performed using 0.1 M potassium phosphate, pH 7.5, 2 mM EDTA containing 0.1 µM
protease and 10–75 µM peptide at 37 °C. Substrate (10 µM) and inhibitor (0.25–10
µM) were pre-warmed for three minutes in black 96-well microplates in a total
volume of 200 µl before the reaction was started with the addition of enzyme (λex=
355 nm, λem= 460 nm). Initial inhibitor screening and IC50 determination for HAV 3C
pro were done using a 96-well plate and a Victor 2 fluorescence plate reader [50]. The
Ki value for each inhibitor candidate was calculated using the Cheng and Prusoff
equation [51].
4.2.3. HRV 3Cpro determination assay
Inhibition activities of compounds 7b, 14b and 14c against HRV 3Cpro were
measured colorimetrically using ab211089 HRV 3C Protease Inhibitor Screening Kit
according to the manufacturer instructions.
All materials and prepared reagents were equilibrated to room temperature
prior to use. The reaction wells were set up where, the screening sample wells contain
10 µL of the tested compounds, the enzyme control wells contain 10 µL assay buffer
and the solvent control wells contain 10 µL solvent. The HRV 3C protease enzyme
solution (50 µL/well) was prepared by adding 2 µL of HRV 3C− protease to 48 µL of
assay buffer, mixed well and added into each well then the plate was incubated at
room temperature for 15 minutes. Additionally, 40 µL of the substrate mixture was
prepared by adding 5 µL of HRV 3C− substrate to 35 µL of assay buffer, added to
each well and mixed well. The absorbance was measured at OD 405 nm in kinetic
mode for 1 – 2 hours at 37°C.
4.3.
Molecular modeling
4.3.1. Molecular docking
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The crystal structure of HAV 3C protease in complex with tetrapeptidyl
methyl ketone was retrieved from The RCSB DAT Bank (PDB:2HAL). Ligand
structures were built with MOE2008 software, and the energy was minimized using
the MMFF94x force field. Docking studies were carried out using autdock4 software
where, the native ligand was re-docked to investigate the validity of the docking
protocol, the docking site was considered using the position of the tetrapeptidyl
inhibitor and grid box was set to -6.457, -6.566, 36.559 dimensions. The photos were
created using Pymol software. Binding free energy (∆GBind) and binding affinity pKd
were calculated via PlayMolecule.org [52].
4.3.2. QSAR analysis
QSAR models were generated by QSAR software of MOE 2008, the
structures were built using builder tool the energy minimization was performed using
MMFF94x force field. The pIC50 values were calculated using (–log IC50 . A large
)
number of 2D and 3D descriptors were calculated, predicted PIC50 for the data set
were calculated and QSAR models were validated by using the r2, RMSE and residual
values between expected and predicted PIC50.
4.3.3. Pharmacophore generation
The pharmacophore was built using (MOE;2008.10 software), flexible
alignment protocol was applied for the best five active antiviral compounds, the
molecules were adjusted and resulted in the best alignment mode. Pharmacophore
query editor was used to create the pharmacophore features. The features were
neither permissive nor excessively restrictive, the scheme of annotation was selected
to be ”Unified”, the model of building was set ”Consensus method”, the tolerance was
set 0.32 Å and the threshold was set to 99%.
Acknowledgments
The authors extend their deepest appreciation to Dr. Essam Rashwan, head of
confirmatory diagnostic unit, Vacsera-Egypt, for helping in performing the HAV
3Cpro and HRV 3Cpro determination assays.
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