Discovery of novel furo[2,3-d]pyrimidin-2-one–1,3,4-oxadiazole hybrid derivatives as dual antiviral and anticancer agents that induce apoptosis

Article abstract of DOI:10.1002/ardp.202100146

A new series of furo[2,3-d]pyrimidine–1,3,4-oxadiazole hybrid derivatives were synthesized via an environmentally friendly, multistep synthetic tool and a one-pot Songoashira-heterocyclization protocol using, for the first time, nanostructured palladium pyrophosphate (Na₂PdP₂O₇) as a heterogeneous catalyst. Compounds 9a–c exhibited broad-spectrum activity with low micromolar EC₅₀ values toward wild and mutant varicella-zoster virus (VZV) strains. Compound 9b was up to threefold more potent than the reference drug acyclovir against thymidine kinase-deficient VZV strains. Importantly, derivative 9b was not cytostatic at the maximum tested concentration (CC₅₀ > 100 μM) and had an acceptable selectivity index value of up to 7.8. Moreover, all synthesized 1,3,4-oxadiazole hybrids were evaluated for their cytotoxic activity in four human cancer cell lines: fibrosarcoma (HT-1080), breast (MCF-7 and MDA-MB-231), and lung carcinoma (A549). Data showed that compound 8f exhibits moderate cytotoxicity, with IC₅₀ values ranging from 13.89 to 19.43 μM. Besides, compound 8f induced apoptosis through caspase 3/7 activation, cell death independently of the mitochondrial pathway, and cell cycle arrest in the S phase for HT1080 cells and the G1/M phase for A549 cells. Finally, the molecular docking study confirmed that the anticancer activity of the synthesized compounds is mediated by the activation of caspase 3.

Full text of DOI:10.1002/ardp.202100146

Received: 17 April 2021
Revised: 19 May 2021
Accepted: 22 May 2021
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DOI: 10.1002/ardp.202100146
F U L L P A P E R
Discovery of novel furo[2,3‐d]pyrimidin‐2‐one–1,3,
4‐oxadiazole hybrid derivatives as dual antiviral and
anticancer agents that induce apoptosis
Az‐eddine El Mansouri1,2 | Ali Oubella3 | Karim Dânoun4 | Mehdi Ahmad5
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Johan Neyts6 | Dirk Jochmans6 | Robert Snoeck6 | Graciela Andrei7
Hamid Morjani7 | Mohamed Zahouily2 | Hassan B. Lazrek1
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1Laboratory of Biomolecular and Medicinal
Chemistry, Chemistry Department, Faculty of
Science Semlalia, University Cadi Ayyad,
Marrakesh, Morocco
Abstract
A new series of furo[2,3‐d]pyrimidine–1,3,4‐oxadiazole hybrid derivatives were
synthesized via an environmentally friendly, multistep synthetic tool and a one‐pot
Songoashira‐heterocyclization protocol using, for the first time, nanostructured
palladium pyrophosphate (Na2PdP2O7) as a heterogeneous catalyst. Compounds
9a–c exhibited broad‐spectrum activity with low micromolar EC50 values toward
wild and mutant varicella‐zoster virus (VZV) strains. Compound 9b was up to
threefold more potent than the reference drug acyclovir against thymidine kinase‐
deficient VZV strains. Importantly, derivative 9b was not cytostatic at the maximum
tested concentration (CC50 > 100 µM) and had an acceptable selectivity index value
of up to 7.8. Moreover, all synthesized 1,3,4‐oxadiazole hybrids were evaluated for
their cytotoxic activity in four human cancer cell lines: fibrosarcoma (HT‐1080),
breast (MCF‐7 and MDA‐MB‐231), and lung carcinoma (A549). Data showed that
compound 8f exhibits moderate cytotoxicity, with IC50 values ranging from 13.89 to
19.43 µM. Besides, compound 8f induced apoptosis through caspase 3/7 activation,
cell death independently of the mitochondrial pathway, and cell cycle arrest in the S
phase for HT1080 cells and the G1/M phase for A549 cells. Finally, the molecular
docking study confirmed that the anticancer activity of the synthesized compounds
is mediated by the activation of caspase 3.
2Laboratoire de Matériaux, Catalyse &
Valorisation des Ressources Naturelles, URAC
24, Department de chimie, Faculté des
Sciences et Techniques, Université Hassan II,
Casablanca, Morocco
3Laboratoire de Synthese Organique et de
Physico‐Chimie Moleculaire, Departement de
Chimie, Faculté des Sciences Semlalia,
Marrakech, Morocco
4MASCIR Foundation,, Rabat Design, Rue
Mohamed El Jazouli, Madinat El Irfane, 10100
Rabat, Morocco, Rabat, Morocco
5ICGM, Université Montpellier, CNRS,
ENSCM, Montpellier, France
6Rega Institute for Medical Research, KU
Leuven, Leuven, Belgium
7BioSpecT—EA7506, UFR de Pharmacie,
Reims, France
Correspondence
Hassan B. Lazrek, Laboratory of Biomolecular
and Medicinal Chemistry, Chemistry
Department, Faculty of Science Semlalia,
University Cadi Ayyad, 40000 Marrakesh,
Morocco.
K E Y W O R D S
Email: hblazrek50@gmail.com
1,3,4‐oxadiazole, anticancer, antiviral, apoptosis, furopyrimidine, heterogeneous catalyst,
hybrid molecules, molecular docking
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1
INTRODUCTION
virus (VZV) infections can be life‐threatening and can lead to sig-
nificant morbidity and mortality for immunocompromised in-
The treatment of cancer and viral diseases remains one of the major
challenges to modern medicine. Cancer is the second most common
cause of death after heart disease, and one in eight deaths in the
world is due to cancer.[1] On the contrary, virus infections consist of
one of the primary health problems. For instance, varicella‐zoster
dividuals, such as cancer patients.[2,3] Indeed, the discovery of new
anticancer and antiviral compounds remains a vital element in che-
motherapy because of undesirable effects, drug resistance, and re-
duced bioavailability of the currently available therapeutics.[4]
Apoptosis (programmed cell death) is a fundamental biological
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Arch Pharm. 2021;e2100146.
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© 2021 Deutsche Pharmazeutische Gesellschaft
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process that allows killing and removal of tumor cells and infected
cells with pathogens.[5] This process is mediated by the sequential
activation of a family of cysteinyl–aspartate‐specific proteases (cas-
pases).[6] Among these, caspases 3 and 7 are the crucial effector
enzymes in the execution of apoptosis.[7] Therefore, apoptosis be-
comes one of the attractive targets for developing new anticancer
agents.[8] Similarly, apoptotic induction of virally infected cells is an
antiviral mechanism to eliminate the viruses.[9] Brazeau and cow-
orkers demonstrated that the death of VZV‐infected human mela-
noma (MeWo) cells appears to be caused by apoptosis, accompanied
by the activation of the caspase cascade.[10] Moreover, immune
system stimulation due to HIV infection can be the cause of apop-
tosis in CD4 and CD8 T‐immune cells.[11]
The compounds with the sugar 2'‐deoxyribose are nontoxic and highly
effective inhibitors of the VZV,[16] and analogs bearing di‐deoxyribose or
acyclic fragments inhibited human cytomegalovirus.[17] Moreover, a bi-
cyclic nucleoside analog (CF‐1743 I, Figure 1) has high antiviral activity
against the VZV with EC50 below 1 nM.[18] Valnivudine (FV‐100 free
base), a prodrug of CF‐1743 I, is an orally active antiherpes zoster (HZ)
nucleoside analog and is rapidly and extensively converted into CF‐1743
in vivo.[19] Meanwhile, the substitution of sugar 2'‐deoxyribose by
methyl‐1,2,3‐triazole leads to improved anticancer activities. Indeed,
Gregorić et al.[20] reported that furo[2,3‐d]pyrimidine‐2‐one–1,2,3‐
triazole hybrid II exhibited promising cytostatic properties against he-
patocellular carcinoma (HepG2) and cervical carcinoma cells, with higher
potency than the reference drug 5‐fluorouracil. Also, Hossam et al.[21]
have shown that compound III exhibited antitumor activity comparable
to that of gefitinib. This effect could be mediated at least partly via
activation of apoptosis and product III increases the level of caspase 3 by
more than twofold.[21]
Pyrimidine derivatives play an essential role in antiviral and antic-
ancer therapy, being among the first chemotherapeutic agents used in
the medical treatment of cancer diseases and viral infections.[12–14]
Furo[2,3‐d]pyrimidines were obtained by coupling a pyrimidine ring with
a furan heterocycle. Bicyclic furo[2,3‐d]pyrimidine nucleosides were de-
veloped for the first time by McGuigan et al.[15] as herpesvirus inhibitors.
1,3,4‐Oxadiazole is another widely used pharmacophore in
medicinal chemistry due to its broad spectrum of biological activities.
FIGURE 1 Design of the new furopyrimidine hybrid derivatives

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This heterocycle is used in the treatment of viral infections (ralte-
gravir) [22] and cancer diseases (zibotentan).[23] Furthermore, several
1,3,4‐oxadiazole derivatives were reported to show apoptotic ef-
fects, including the activation of caspase enzymes. Sankhe et al. have
synthesized compound IV (Figure 1), which induces apoptosis
through the activation of caspase 3.[24] A series of 1,3,4‐oxadiazoles
were investigated for their anticancer potential against HCC cells,
and product V was identified as the lead compound, which induced
apoptosis by the activation of caspase 3/7.[25]
(PPh3)4[20,33–37]; other catalysts were described in the literature
such as PdCl2(PPh3)2,[38] Na2PdCl4/TXPTS,[39] and Pd(dppf)Cl2.[40]
However, the described methods suffer from several limitations,
including the use of air‐ and moisture‐sensitive catalytic systems
(phosphines), difficulty in the separation of palladium from the
reaction, and the use of toxic reagents and ligands.[41] On the
contrary, metal pyrophosphates (MP2O7) have attracted consider-
able attention due to their attractive properties in a wide range of
applications such as catalytic systems.[42] However, only a few
studies have been reported in the literature describing the use of
palladium phosphate as a catalyst.[43] For instance, our group re-
ported that nanostructured palladium pyrophosphate (Na2PdP2O7)
exhibited high catalytic activity in Suzuki–Miyaura cross‐
coupling.[44] To the best of our knowledge, the use of nanos-
tructured pyrophosphate palladium as a heterogeneous catalyst for
the preparation of furo[2,3‐d]pyrimidines through the Sonogashira
coupling reaction has not been reported in the literature.
In our previous work,[26,27]
a series of 1,3,4‐oxadiazole‐
pyrimidine hybrids (VI, VII) were synthesized and some compounds
exhibited anticancer and anti‐VZV activities. In continuation of our
research work and based on the importance of furopyrimidines and
1,3,4‐oxadiazole, we used the pharmacophore hybridization method
(with the capacity to overcome resistance, enhance selectivity, and
improve biological activities).[28] Besides, we also considered the
following points: (a) By introducing a furo‐fused heterocycle; (b) in-
troducing a chlorine atom into the phenyl group; (c) by improving the
lipophilicity of alcohol by esterification; and (d) adding a pharmaco-
phore group such as ibuprofen or phenol, a new series of furo[2,3‐d]
pyrimidine‐2‐one–1,3,4‐oxadiazole hybrid analogs are synthesized.
Also, we describe a synthetic methodology for the construction of
furo[2,3‐d]pyrimidine‐2‐one through the one‐pot Sonogashira‐
heterocyclization method using, for the first time, nanostructured
pyrophosphate palladium Na2PdP2O7 as a highly efficient hetero-
geneous catalyst. Moreover, the prepared compounds were
evaluated for their in vitro antiviral and selective activities against
VZV, cytomegalovirus (CMV), yellow fever (YFV), chikungunya
(CHIKV), human Immunodeficiency (HIV‐1 and HIV‐2), and human
enterovirus (EV), as well as anticancer activities in HT‐1080, A‐549,
MCF‐7, and MDA‐MB‐231 cell lines. The most effective compound
was evaluated for its effects on annexin V, mitochondrial membrane
potential, caspase 3/7, and cell cycle. Finally, molecular docking was
used to study the interactions of these compounds with caspase‐3
protein.
In the present work, we described a new free ligand‐catalyzed
method for the preparation of furopyrimidine using a heterogeneous
palladium catalyst, which is easy to separate from the reaction
medium (Schemes 1–4). Indeed, we developed an environmentally
friendly and multistep synthetic tool to directly obtain a new series
of furo[2,3‐d]pyrimidine‐2‐one–1,3,4‐oxadiazole hybrid derivatives.
The preparation of furo[2,3‐d]pyrimidines is described through the
one‐pot Sonogashira‐cyclization synthesis using Na2PdP2O7 as a
heterogeneous catalyst. To synthesize the desired products, we
adopted the following strategy: the esterification of benzoic acid
derivatives 1a,b using methanol and SOCl2 afforded the esters 2a,b.
The latter reacted with hydrazine in ethanol to give hydrazides 3a–d.
Then, the mixture of chloroacetic acid, products 3a–d and phos-
phorus oxychloride (POCl3) afforded oxadiazoles 4a–d.[26] After that,
these chemicals were used as alkylating agents to react with io-
douracil (5) to prepare 1,3,4‐oxadiazole–pyrimidin‐2,4‐dione hybrids
6a–d via the modified Hilbert–Johnson reaction.[45] The 1,3,4‐
oxadiazole homonucleoside analogs were obtained in 29–35% of the
yield. On the contrary, compounds 7a–d were obtained over the
alkylation reaction of amine or alcohol derivatives by propargyl
bromide in a basic medium (K2CO3). Later, furopyrimidines were
prepared from 5‐iodouracils 6a–d and propargylated products 7a–f
via a Na2PdP2O7‐catalyzed cross‐coupling reaction. The optimal
conditions were investigated by changing the solvent, base, and their
amount. It has been found that the use of acetonitrile/t‐BuOH and
three equivalents of triethanolamine gave the desired product with
an improved yield of 56% (Entry 8, Table 1). These conditions were
generalized on the reaction of 5‐iodouracils 6a–d with propargyl
derivatives 7a–d to obtain furo[2,3‐d]pyrimidine‐2‐one–1,3,4‐
oxadiazole analogs 8a–h in the yield ranging from 47% to 81%.
Finally, to increase the lipophilicity of furopyrimidine 8a, the
esterification was performed using the activating agent dicyclohex-
ylcarbodiimide (DCC) and different carboxylic acids including ben-
zoic acid, amino acid boc‐L‐valine, and ibuprofen to afford esters
9a–c in moderate yields. The choice of these pharmacophore groups
was based on the following reasons: (i) Derivatives of benzoic acid
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2
RESULTS AND DISCUSSION
Chemistry
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2.1
Considering the biological importance of furopyrimidine deriva-
tives, the need for the development of an efficient synthetic
methodology persists. Therefore, Sonogashira–palladium‐catalyzed
coupling of 5‐halide uracil derivatives and alkynes, followed by in-
tramolecular cyclization, is the most common method to access a
wide variety of furo[2,3‐d]pyrimidine derivatives.[29,30] This process
is one of the most important, powerful, and versatile methods
in modern synthetic organic chemistry for constructing
carbon–carbon bonds.[29,31] The development of a one‐pot reaction
makes the process economic by avoiding an additional purification
step (without the isolation of the intermediate alkyne).[32] Indeed,
the most heterogeneous catalyst used in this reaction is Pd

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SCHEME 1 Synthesis of compounds 4a–d,
6a–d, and 7a–d. Reagents and conditions: (i)
SOCl2, MeOH, reflux, 6 h, (ii) NH2NH2, EtOH,
reflux, 3 h, (iii) ClCH2CO2H, POCl3, DCE, 12 h,
(iv) HMDS, (NH4)2SO4, 120°C, 3 h, (v) 4a‐d, KI,
80°C, 12 h, and (vi) K2CO3, acetone,
reflux, 12 h
SCHEME 2 Preparation of
furopyrimidine 8a
are considered as a class of simple aromatic acids known for their
pharmacological properties,[46] (ii) amino acids are widely used for
the development of prodrugs to improve several properties, namely,
increased bioavailability, decreased toxicity of the parent drug, ac-
curate delivery to target tissues or organs, and prevention of fast
metabolism,[47] and (iii) ibuprofen is a widely used drug and has many
applications such as for the treatment of viral respiratory
infections.[48]
[2,3‐d]pyrimidin‐2‐one–1,3,4‐oxadiazole analogs (9a–c) were lower
than that of reference drug acyclovir (ACV) against the TK+ VZV
strain and they were better than that of ACV against the TK− VZV
strain. Remarkably, compound 9b was up to threefold more potent
than the reference drug against TK− VZV strains. Importantly, 9b has
lower cytotoxicity (CC50 > 100 µM) and an acceptable selectivity in-
dex value of up to 7.8 in comparison to acyclovir. Indeed, the product
9a containing the benzoic acid moiety was about twofold more po-
tent than compound 9c (acyclic acid) against the TK+ VZV strain.
Also, compound 9b containing the ibuprofen pharmacophore group
was more active than product 9c against the TK− VZV strain. We
observed that the acid‐containing phenyl group increased the anti‐
VZV activity. Thus, the introduction of a substituted phenyl group
may improve the antiviral properties. By comparing compound 8a
with molecules 9a–c, the protection of the OH with the acyl group
increased the antiviral activity. This fact is explained by the high
lipophilicity of the protected compounds 9a–c. However, lipophilicity
is an essential feature of molecules in the pharmaceutical, bio-
chemical, and medical chemistry fields.[49] This feature is a physico-
chemical parameter that controls the affinity of a molecule for
binding sites and the transport through biological membranes.[50,51]
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2.2
Pharmacology/biology
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2.2.1
In vitro antiviral activity
All the new molecules 8a–h and 9a–c were tested for their antiviral
activities against the human VZV, both wild‐type (TK+) and thymi-
dine kinase‐deficient (TK−), in human embryonic lung (HEL) cells.
Compounds 9a–c were the most potent and the other molecules did
not show measurable activity against VZV. The growth inhibition of
HEL cells was only studied for the compounds that exhibited an
antiviral activity lower than 20 µM. The antiviral activities of furo

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SCHEME 3 Generalization of the reaction
SCHEME 4 Esterification reaction of
furopyrimidine 8a
In general, biological potency is often accompanied by increased
molecular lipophilicity.[52] Lipophilicity represented by the octanol/
water partition coefficients (ClogP) and the products with high
CLogP are more lipophilic. In this study, partition coefficients were
predicted using ChemDraw. As reported in Table 2, the introduction
of the acyl group increases the lipophilicity of furopyrimidines. One
possible explanation for the low activity of 8a is the cell permeability
due to the low lipophilicity.
All compounds were also tested against several other viruses
such as human cytomegalovirus (HCMV) (AD‐169 and Davis strain)
in human embryonic lung (HEL cells), yellow fever (YFV‐17D strain)
(HEL cells), chikungunya (CHIKV‐899 strain) (VeroE6 cells), human
immunodeficiency (HIV‐1 IIIb strain HIV‐2 ROD) (MT‐4 cells), and
human enterovirus 71 (EV‐71 BrCr strain) (Vero cells). All tested
compounds had EC50 higher than 100 µM, except compound 9a,
which was active against the Davis strain with EC50 equal 54.69 µM.

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TABLE 1 Optimization of the
preparation of furopyrimidine 8a
Entry
Solvent
Base
Equiv of base
Yield (%)
1
2
3
4
5
6
7
8
9
toluene
DIPEA
DIPEA
DIPEA
DIPEA
DIPEA
DIPEA
K2CO3
TEA
3
20
acetonitrile
3
38
acetonitrile/water (3/1)
acetonitrile/MeOH (3/1)
acetonitrile/PrOH (3/1)
acetonitrile/t‐BuOH (3/1)
acetonitrile/t‐BuOH (3/1)
acetonitrile/t‐BuOH (3/1)
acetonitrile/t‐BuOH (3/1)
3
12
3
46
3
48
3
53
3
no reaction
3
56
32
TEA
1.5
Note: Optimal conditions: Acetonitrile/t‐BuOH 3:1 (10 ml), TEA (3 equiv), Na2PdP2O7 (0.1 equiv), CuI
(0.3 equiv), 80°C, and 12 h.
Abbreviations: DIPEA, N,N‐diisopropylethylamine; TEA, triethanolamine.
TABLE 2 Antiviral and selectivity activity against the varicella‐zoster virus (VZV) in human embryonic lung cells
Antiviral activity EC50 (µM)a
Cytotoxicity (µM)
Cell morphology
Selectivity Indexd
TK+ VZV strain
TK+ VZV strain
TK− VZV strain
Cell growth
TK− VZV strain
c
Compound
OKA
07‐1
(MCC)b
>100
>100
>100
>100
>440
(CC50
)
CC50/EC50
ND
ClogP
−1.333
1.502
3.416
0.96
8a
>100
>100
ND
ND
9a
14.67 2.21
18.18 1.82
25.4 13.7
2.48 1.06
18.34 10.255
12.82 0.19
18.08 6.38
41.69 3.24
44.42 1.07
>100
3.03
2.44
>7.8
2.58
>10.55
9b
>5.52
1.83
9c
46.66 1.84
>440
Acyclovir
>177.41
–
Abbreviations: ND, not determined; OKA, Oka strain; TK+, thymidine kinase wild‐type; TK−, thymidine kinase‐deficient.
aEffective concentration required to reduce virus plaque formation by 50%. Virus input was 20 plaque‐forming units.
bMinimum cytotoxic concentration that causes a microscopically detectable alteration of cell morphology.
cCytostatic concentration required to reduce cell growth by 50%.
dRatio: CC50/EC50
.
TABLE 3 In vitro anticancer activity
(IC50) of 1,3,4‐oxadiazole hybrids (8a–i
and 9a–c) against HT1080, A549, MCF7,
and MDA‐MB231 cells
Product
HT‐1080
A‐549
MCF‐7
MDA‐MB‐231
26.30 1.05
34.11 0.92
29.34 0.13
>100
8a
19.17 0.21
46.31 1.86
34.94 1.43
35.54 2.54
19.03 0.68
13.89 0.22
18.98 0.63
17.21 1.48
18.33 2.51
19.39 1.21
29.21 2.82
6.36 0.38
23.60 0.56
28.93 0.83
36.76 1.89
75.41 5.81
21.28 2.52
15.98 0.18
21.03 3.11
27.19 3.90
23.34 1.27
28.44 0.88
36.01 3.09
4.96 0.07
22.22 0.32
37.19 1.30
>100
8b
8c
8d
55.03 3.25
20.31 3.09
19.43 0.48
28.09 2.85
37.39 4.52
50.34 6.46
35.98 1.16
35.60 3.88
5.46 0.44
8e
25.45 0.90
17.32 1.07
18.54 1.45
33.21 1.03
28.02 0.34
39.02 1.72
32.77 2.60
5.13 0.31
8f
8g
8h
9a
9b
9c
Doxorubicin

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2.2.2
In vitro anticancer screening
(8f) with the phenyl moiety at the 5‐position of the 1,3,
4‐oxadiazole heterocycle and dimethylphenoxymethyl at the 6‐
position of the furopyrimidine base was the most potent, with
IC50 values of 13.89 0.22, 15.98 0.18, 19.43 0.48, and
17.32 1.07 μM against HT‐1080, A‐549, MCF‐7, and MDA‐MB‐
23, respectively.
All the newly synthesized products were evaluated for their cy-
totoxicity in human HT‐1080, A‐549, MCF‐7, and MDA‐MB‐23
cell lines with doxorubicin as the positive control using
the MTT assay. The antiproliferative activities of the
furopyrimidine–1,3,4‐oxadiazole hybrid derivatives are shown in
Table 3. First, the structure–activity relationship study focused
on the variation of substituted phenyl at the 5‐attachment of
1,3,4‐oxadiazole. The substitution of hydrogen in phenyl by a
methyl group (8b) at the para or chlorine atom at ortho and para
positions (8c–d) decreased the anticancer potency. Then, we
retained the nonsubstituted phenyl moiety (8a) and we have
replaced OH at position 6 of the furopyrimidine base with an
aniline group to give compound 8g, which showed moderately
decreased activity in all human cancer cell lines, whereas product
8h, possessing a thymine nucleobase at the same position, ex-
hibited less anticancer activity in comparison to compound 8a.
Next, the OH had different carboxyl functions to afford com-
pounds 9a–c; product 9a has the same antiproliferative activity
as furopyrimidine 8a against HT‐1080 and A‐549. Finally, the
replacement of the hydrogen atom of OH with phenyl (8e, ether
bound) and 3,5‐dimethylphenyl (8f) increased the anticancer
activity in all cell lines. In summary, the hybrid furopyrimidine
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2.2.3
Annexin V/7‐AAD staining to detect
apoptosis
The most potent compound 8f was tested for its apoptotic induction
activity using flow cytometric annexin V/7‐AAD dual staining ana-
lysis. The HT‐1080 and A‐549 cancer cell lines were treated with
15 µM of the most potent compound 8f for 24 h to study the
apoptosis‐dependent cell death. The results (Figure 2) showed an
increase in total apoptosis in HT‐1080 by about 21‐fold (from 1.28%
to 26.47%) compared to the control untreated cancer cells. In-
dividually, early and late apoptosis increased by approximately 6‐fold
and 43‐fold, respectively. Also, compound 8f induced apoptosis in
A‐549 by ninefold from 1.76% to 15.71% (early apoptosis
1.47–4.31% and late apoptosis 0.29–11.4%). In conclusion, hybrid
derivative 8f showed the ability to induce apoptosis‐dependent cell
death in HT‐1080 and A‐549 cancer cell lines.
FIGURE 2 Annexin V‐FITC/7‐AAD double staining for detection of apoptosis in HT‐1080 and A‐549 cells after treatment with 15 µM of
compound 8f as well as control (dimethyl sulfoxide) for 24 h. Compound 8f induces apoptosis

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2.2.4
Effect of compound 8f on the mitochondrial
7.81% 0.38% to 13.685% 2.51%. In summary, these data are in
agreement with those obtained on annexin V staining and suggest
that the product 8f induced apoptosis through caspase 3/7 in both
cells, especially in the HT‐1080 cancer line.
membrane potential (MMP)
The dysfunction of MMP was also reported to involve induction of
apoptosis. In this study, mitochondrial dysfunction was determined
based on the MMP index that was measured by flow cytometry using
the Muse MitoPotential Kit (Millipore‐Merck). HT‐1080 and A‐549
cells were treated with compound 8f at 15 µM concentration for
24 h. As shown in Figure 3, compound 8f induced an increase in the
MMP index. In fact, in the case of HT‐1080 cells, the rate of dead
cells with high MMP increased from 5.605% 1.963% to
20.03% 0.064%. Similarly, in A‐549 cells, this rate increased from
5.840% 1.501% to 19.51% 1.5%. This suggests that compound 8f
can induce cell death independently of the mitochondrial pathway.
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2.2.6
Effect of compound 8f on cell cycle
distribution
Flow cytometry was used to explore the effects of compound 8f on
cell cycle progression. HT‐1080 and A‐549 cells were treated with
15 µM of the compound for 24 h. As shown in Figure 5, the treatment
of HT‐1080 cells with compound 8f increased cell cycle arrest in the
S phase, resulting in an increase in the cell population in the S phase
(10.25% 0.05%) compared to the control cells (13.3% 0.1%).
Moreover, treatment of A‐549 cells with compound 8f increased cell
cycle arrest in the G1M phase from 11.57% 0.15% to 15.8% 0.2%
compared to the untreated cells. These results demonstrate that in
HT‐1080 and A‐549 cells, cell cycle arrest occurs in the S and G1M
phases, which contributes to the cytotoxicity of the compound 8f.
|
2.2.5
Effect of compound 8f on caspase‐3/‐7
activity
The mechanism of apoptosis induction by the most active product 8f
in HT‐1080 and A‐549 was investigated by the determination of
caspase‐3/‐7 percentages. The cells were treated with 15 µM of
compound 8f for 24 h; all the results are summarized in Figure 4. In
the HT‐1080 cancer cell, furopyrimidine 8f increased the percentage
of caspase 3/7 by about fourfold from 7.185% 0.72% to
29.44% 0.42% in comparison to the control. Similarly, the product
8f also induced caspase 3/7 by increasing its percentage from
|
2.3
Molecular docking
With the aim of continuing the investigation of apoptosis induction
mechanism via the caspase activation, in silico molecular docking was
studied. Also, taking into account the docking study of oxadiazole
FIGURE 3 Flow cytometric analysis (FACS) of the mitochondrial membrane potential in HT‐1080 and A‐549 cells treated with compound 8f
at 15 µM concentration for 24 h (data represent mean SD of three individual experiments)

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FIGURE 4 Induction of caspase 3/7
activity in response to compound 8f. HT‐1080
and A‐549 cells were treated with 15 µM of
the desired compound as well as dimethyl
sulfoxide (control) for 24 h (data represent
mean SD of three individual experiments)
FIGURE 5 Flow cytometry analysis of cell cycle phase distribution in HT‐1080 and A‐549 cells after treatment of 15 µM of compound 8f in
comparison with the control for 24 h. Compound 8f induced S‐phase arrest for HT1080 and G1M arrest for A549 cells (data represent
mean SD of three individual experiments)

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derivatives as potent caspase‐3 activators,[27,53,54] compound 8f was
selected and docked into the binding site of caspase 3 (PDB ID:
1RE1). Before this, to determine the validity of our docking protocol,
self‐docking experiments were carried out. The root‐mean‐square
deviations between the predicted and the native poses were found
to be 0.95 Å. These results indicated that the adopted docking pro-
tocol is good for the reproduction of the native poses (<2 Å).
The docking results of the oxadiazole 8f with caspase 3 are sum-
marized in Figures 6–8 and in the Supporting Information Table. Com-
pound 8f could be docked into the active site of caspase 3 with the
estimated free energy of binding −10.4 kcal/mol. The docked pose of 8f
was surrounded by a partially hydrophobic cavity, especially the phenyl
and dimethylphenyl moieties (Figure 6). This ligand formed six H bonds
with the amino acid residues ARG179 (2.534 Å), ARG179 (2.306 Å),
HIS237 (1.827 Å), GLN283 (2.034 Å), ARG341 (2.368 Å), and ARG341
cancer cell lines HT‐1080, A‐549, MCF‐7, and MDA‐MB‐23. The
mechanism of the antiproliferative effect on HT‐1080 and A‐549 was
proapoptotic through activation of caspase 3/7, cell death in-
dependently of the mitochondrial pathway, and cell arrest in the S
phase for HT1080 and the G1M phase for A‐549. Finally, molecular
docking showed that compound 8f entered deep into the active site
of caspase 3 by forming stable complexes (H‐bond, carbon–hydrogen
bond, etc.). These results suggested that the anticancer activity of
the product 8f could be at least partially mediated by binding to
caspase 3.
|
4
EXPERIMENTAL
Chemistry
|
4.1
(2.477 Å). Also, other interactions were observed, such as
a
|
General
carbon–hydrogen bond, π–cation, π–donor hydrogen bond, π–sulfur, π–π
T‐shaped, and π–alkyl. This result is in agreement with the literature, in
which it has been reported that these binding interactions are funda-
mental for caspase 3 activation.[27,54–57] In summary, the binding affinities
and the interactions were in good agreement with apoptosis induction
via caspase activation. The ligands showed heterogeneity in binding
modes, including H‐bond interactions. Besides, the promising activity of
compound 8f could be mediated by binding to active sites of caspase 3.
4.1.1
Melting points were measured using a Büchi B‐545 digital capillary
melting point apparatus and used without correction. Reactions were
checked with thin‐layer chromatography (TLC) using aluminum
sheets with silica gel 60 F254 from Merck. Infrared (IR) spectra were
recorded on a Perkin‐Elmer VERTEX 70 FTIR spectrometer covering
field 400–4000 cm−1. The spectra of 1H NMR (nuclear magnetic re-
sonance) and 13C NMR (see the Supporting Information) were re-
corded in solution in DMSO‐d6 or CDCl3 on a Bruker Advance 300
spectrometer at 300 and 75 MHz, respectively. The chemical shifts
are expressed in parts per million (ppm) using DMSO‐d6 as the in-
ternal reference. The multiplicities of the signals are indicated by the
following abbreviations: s, singlet; d, doublet; t, triplet; q, quadruplet;
and m, multiplet. Coupling constants J are expressed in Hertz. Mass
spectra were collected using an API 3200 LC/MS/MS system,
equipped with an ESI source. The chemical reagents used in synthesis
were purchased from Fluka, Sigma, and Aldrich.
|
3
CONCLUSIONS
In conclusion, a new free ligand‐catalyzed method for the prepara-
tion of furopyrimidine using heterogeneous pyrophosphate palla-
dium Na2PdP2O7 has been reported. A novel series of furo[2,3‐d]
pyrimidine‐2‐one‐1,3,4‐oxadiazole hybrid derivatives were synthe-
sized for the first time as antiviral and anticancer agents. Furopyr-
imidine 9b was up to threefold more potent than the reference drug
against TK− VZV and was not cytostatic at the maximum tested
concentration (CC50 > 100 µM). Besides, among the synthesized de-
rivatives, 8f showed the best cytotoxic activity against four human
The InChI codes of the investigated compounds, together with
some biological activity data, are provided in the Supporting
Information.
FIGURE 6 Position of ligand 8f in the
hydrophobic cavity of caspase 3

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FIGURE 7 Three‐dimensional interactions
of compound 8f with the amino acid residues
of caspase 3
FIGURE 8 Two‐dimensional interactions
of compound 8f with the amino acid residues
of caspase 3
|
4.1.2
General procedure for the synthesis of
purified by column chromatography to afford esters 2a–d. In the
second step, to a round‐bottom flask (25 ml) equipped with a
condenser, a mixture of esters 2a–d (10 mmol), hydrazine hydrate
(11 mmol, 0.5 ml), and ethanol (5 ml) was added. The mixture was
refluxed for 3 h. The solvent was evaporated under vacuum and
the hydrazide was recrystallized using ethanol to afford the
desired products 3a–d. Then, chloroacetic acid (11 mmol, 1.04 g),
alkylating agents 4a–d
First, 10 mmol carboxylic acid 1a–d was taken in 15 ml of methanol,
and thionyl chloride was added dropwise for 10 min at 0°C. Then,
the mixture was stirred at room temperature for 3 h and methanol
was evaporated under vacuum, extracted with ethyl acetate, and

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phosphorus oxychloride (30 mmol, 2.8 ml), and DCE (20 ml) were
mixed in a 100 ml round‐bottom flask and heated at 80°C for 2 h.
Afterward, carboxylic acid hydrazide (3a–d) was added to the
mixture and heated for 12 h. After completion of the reaction,
water (40 ml) was added and the pH was adjusted to 7–8 with
sodium hydrogen carbonate and extracted with dichloromethane.
The organic layer was dried (Na2SO4), evaporated, and purified by
column chromatography (silica gel, 5% EtOAc in hexane) to give the
alkylating agents 4a–d.
(1.5 mmol), KI (0.75 mmol, 124 mg), and acetonitrile (2.5 ml) were
added. The reaction mixture was heated at 85°C for 12 h. After
completion of the reaction (monitored by TLC), the mixture was di-
luted with dichloromethane and evaporated to dryness. The residue
was purified by flash chromatography by eluting with (CH2Cl2/
MeOH: 99:1).
5‐Iodo‐1‐[(5‐phenyl‐1,3,4‐oxadiazol‐2‐yl)methyl]pyrimidine‐
2,4(1H,3H)‐dione (6a)
Yield = 35%. Rf = 0.42 (CH2Cl2/CH3OH [9.6:0.4]). Mp (°C) 282–284.
IR (KBr) ʋ (cm−1): 1707 (C═O), 1614 (C═N), 1554 (C═C), and 1246
(C–O–C). 1H NMR (300 MHz, DMSO‐d6) δ (ppm): 5.3 (s, 2H, H‐7);
7.64 (m, 3H, HPh); 8 (m, 2H, HPh); 8.38 (s, 1H, H‐6); 11.85 (s, 1H, H‐3).
13C NMR (75 MHz, DMSO‐d6) δ (ppm): 42 (C‐7); 70 (C‐5); 123
(CPh, IV); 126 (CPh, IV); 129 (CPh, IV); 133 (CPh, IV); 150 (C‐6); 151 (C‐2);
161 (C‐4); 162, 164 (C‐8, C‐9).
2‐(Chloromethyl)‐5‐phenyl‐1,3,4‐oxadiazole (4a)
Yield = 68%. Rf = 0.4 (9:1 [hexane/AcOEt]). Mp (°C) 105–107. IR
(KBr) ʋ (cm−1): 3029 (Csp2H), 2972 (Csp3H), 1552 (C═N), 1017
(C–O–C), and 746 (C–Cl). 1H NMR (300 MHz, CDCl3) δ (ppm): 5.16
(s, 2H, H‐1); 7.6 (m, 3H, HPh); 8.03 (m, 2H, HPh). 13C NMR (75 MHz,
CDCl3) δ (ppm): 33.23 (C‐1); 122.85 (CPh, IV); 126.65 (CPh, III);
129.53 (CPh, III); 132.38 (CPh, III); 162.88, 164.95 (C‐2, C‐3).
5‐Iodo‐1‐{[5‐(p‐tolyl)‐1,3,4‐oxadiazol‐2‐yl]methyl}pyrimidine‐
2,4(1H,3H)‐dione (6b)
2‐(Chloromethyl)‐5‐(4‐methylphenyl)‐1,3,4‐oxadiazole (4b)
Yield = 75%. Rf = 0.46 (9:1 [hexane/AcOEt]). Mp (°C) 112–114. IR
(KBr) ʋ (cm−1): 3020 (Csp2H), 2965 (Csp3H), 1562 (C═N), 1018
(C–O–‐C), and 760 (C–Cl). 1H NMR (300 MHz, CDCl3) δ (ppm): 2.49
(s, 3H, H‐4); 5.58 (s, 2H, H‐1); 7.39 (d, 3JH‐H = 8.1 Hz, 2H, HPh); 7.82
(d, 3JH‐H = 8.1 Hz, 2H, HPh). 13C NMR (75 MHz, CDCl3) δ (ppm): 21.64
(C‐4): 39.12 (C‐1); 115.04 (CPh, IV); 126.87 (CPh, III); 130.50 (CPh, III);
132.85 (CPh, IV); 162.81, 164.77 (C‐2, C‐3).
Yield = 35%. Rf = 0.45 (CH2Cl2/CH3OH [9.6:0.4]). Mp (°C)
289–291. IR (KBr) ʋ (cm−1): 1706 (C═O), 1619 (C═N), 1548
(C═C), and 1242 (C–O–C). 1H NMR (300 MHz, DMSO‐d6) δ
(ppm): 2.42 (s, 1H, H‐10); 5.38 (s, 2H, H‐7); 7.33 (d, 3JH‐H = 7.8 Hz,
2H, HPh); 7.97 (d, 3JH‐H = 7.8 Hz, 2H, HPh); 8.24 (1H, s, H‐6); 11.72
(1H, s, H‐3). 13C NMR (75 MHz, DMSO‐d6) δ (ppm): 21.18 (C‐10);
45.36 (C‐7); 71.39 (C‐5); 115.54 (CPh, IV); 126.35 (CPh, III); 130.62
(CPh, III); 132.83 (CPh, IV); 150.14 (C‐6); 150.96 (C‐2); 161.93 (C‐4);
162.30, 164.91 (C‐8, C‐9).
2‐(Chloromethyl)‐5‐(4‐chlorophenyl)‐1,3,4‐oxadiazole (4c)
Yield = 68%. Rf = 0.34 (9:1 [hexane/AcOEt]). Mp (°C) 83–85. IR (KBr)
ʋ (cm−1): 3021 (Csp2H), 2964 (Csp3H), 1568 (C═N), 1011 (C–O–C),
and 756 (C–Cl). 1H NMR (300 MHz, CDCl3) δ (ppm): 5.15 (s, 2H, H‐1);
7.66 (t, 3JH‐H = 7.5 Hz, 2H, HPh); 8.01 (d, 3JH‐H = 7.5 Hz, 2H, HPh). 13C
NMR (75 MHz, CDCl3) δ (ppm): 36.23 (C‐1); 121.09 (CPh, IV); 124.62
(CPh, III); 125.37 (CPh, III); 136.45 (CPh, IV); 162.28, 164.12 (C‐2, C‐3).
1‐{[5‐(4‐Chlorophenyl)‐1,3,4‐oxadiazol‐2‐yl]methyl}‐5‐
iodopyrimidine‐2,4(1H,3H)‐dione (6c)
Yield = 32%. Rf = 0.41 (CH2Cl2/CH3OH [9.6:0.4]). Mp (°C)
290–292. IR (KBr) ʋ (cm−1): 1704 (C═O), 1618 (C═N), 1541
(C═C), and 1239 (C–O–C). 1H NMR (300 MHz, DMSO‐d6) δ
(ppm): 5.12 (s, 2H, H‐7); 7.25 (d, 3JH‐H = 8 Hz, 2H, HPh); 8.04 (d,
3JH‐H = 8 Hz, 2H, HPh); 8.31 (1H, s, H‐6); 11.69 (1H, s, H‐3). 13C
NMR (75 MHz, DMSO‐d6) δ (ppm): 44.86 (C‐7); 71.43 (C‐5);
116.11 (CPh, IV); 126.80 (CPh, III); 130.14 (CPh, III); 133.25 (CPh, IV);
150.07 (C‐6); 151.48 (C‐2); 162.36 (C‐4); 162.87, 165.35 (C‐8,
C‐9).
2‐(Chloromethyl)‐5‐(2‐chlorophenyl)‐1,3,4‐oxadiazole (4d)
Yield = 70%. Rf = 0.35 (9:1 [hexane/AcOEt]). Mp (°C) 91‐93. IR (KBr)
ʋ (cm−1): 3018 (Csp2H), 2964 (Csp3H), 1554 (C═N), 1013 (C–O–C),
and 753 (C–Cl). 1H NMR (300 MHz, CDCl3) δ (ppm): 5.22 (s, 2H, H‐1);
7.5 (t, 3JH‐H = 7.6 Hz, 1H, HPh); 7.62 (d, 3JH‐H = 7.2 Hz, 1H, HPh); 7.69
(t, 3JH‐H = 7.2 Hz, 1H, HPh); 7.93 (t, 3JH‐H = 7.6 Hz, 1H, HPh). NMR 13
C
(75 MHz, CDCl3) δ (ppm): 35.41 (C‐1); 122.65 (CPh, IV); 127.82
(CPh, III); 131.52 (CPh, III); 131.83 (CPh, III); 132.37 (CPh, III); 133.98
(CPh, IV); 162.35, 164.22 (C‐2, C‐3).
1‐{[5‐(2‐Chlorophenyl)‐1,3,4‐oxadiazol‐2‐yl]methyl}‐5‐
iodopyrimidine‐2,4(1H,3H)‐dione (6d)
Yield = 29%. Rf = 0.42 (CH2Cl2/CH3OH [9.6:0.4]). Mp (°C) 289–291.
IR (KBr) ʋ (cm−1): 1704 (C═O), 1614 (C═N), 1542 (C═C), and 1231
(C–O–C). 1H NMR (300 MHz, DMSO d6) δ (ppm): 5.20 (s, 2H, H‐7);
7.38 (t, 3JH‐H = 7.6 Hz, 1H, HPh); 7.52 (d, 3JH‐H = 7.5 Hz, 1H, HPh); 7.78
(t, 3JH‐H = 7.6 Hz, 1H, HPh); 8.04 (t, 3JH‐H = 8 Hz, 1H, HPh); 8.29 (1H, s,
H‐6); 11.71 (1H, s, H‐3). 13C NMR (75 MHz, DMSO d6) δ (ppm): 45.25
(C‐7); 72.89 (C‐5); 121.74 (CPh, IV); 127.33 (CPh, III); 131.81 (CPh, III);
132.05 (CPh, III); 132.20 (CPh, III); 133.63 (CPh, IV); 150.41 (C‐6); 151.96
(C‐2); 162.29 (C‐4); 163.27, 165.56 (C‐8, C‐9).
|
4.1.3
General procedure for the synthesis of the
alkylated 5‐iodouracil derivatives 6a–d
A mixture of 5‐iodouracil 5 (1 mmol, 238 mg) and ammonium sulfate
(0.10 mmol, 13 mg) in HMDS (1.5 ml) was refluxed until a clear so-
lution was obtained. Then, the appropriate alkylating agent 4a–d

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4.1.4
General procedure for the synthesis of the
(C═N), and 1228 (C–O–C). 1H NMR (300 MHz, DMSO‐d6) δ (ppm):
4.45 (d, 3JH‐H = 5.7 Hz, 2H, CH2–O); 5.53 (s, 2H, CH2–N); 5.61 (t, 3JH‐
H = 5.7 Hz, 1H, OH); 6.68 (s, 1H, H‐5); 7.66 (d, 3JH‐H = 8.4 Hz, 2H, Hph);
7.97 (d, 3JH‐H = 8.4 Hz, 2H, Hph); 8.72 (s, 1H, H‐4). 13C NMR (75 MHz,
DMSO‐d6) δ (ppm): 46.00 (CH2–N); 56.36 (CH2–O); 101.38 (C‐5);
107.38 (C‐4a); 122.44 (CPh, IV); 128.93 (CPh, III); 130.24 (CPh, III);
137.48 (Cph, IV); 143.89 (C‐4); 154.93 (C‐2); 158.46 (C‐7a); 163.34
(O–C═N); 164.17 (O–C═N); 172.70 (C‐6). HRMS calculated for
propargylated derivatives 7a–d
A mixture of phenol or amine derivative (1 mmol), propargyl
bromide (1.1 mmol), and potassium carbonate (0.5 mmol, 69 mg)
in DMF (10 ml) was heated at 90°C for 2 h. Then, the DMF was
evaporated under reduced pressure to dryness. The residue was
purified by flash chromatography to obtain pure product 7a–d.
C
16H12ClN4O4+: 359.0547, found: 359.0557.
|
4.1.5
General procedure for the synthesis of furo
3‐{[5‐(2‐Chlorophenyl)‐1,3,4‐oxadiazol‐2‐yl]methyl}‐6‐
[2,3‐d]pyrimidin‐2(3H)‐ones 8a–h
(hydroxymethyl)furo[2,3‐d]pyrimidin‐2(3H)‐one (8d)
Yield = 75%. Rf = 0.35 (CH2Cl2/MeOH [9:1]). Mp (°C): 176–178. IR
(KBr) ʋ (cm−1): 3438 (OH), 3051 (Csp2H), 2847 (Csp3H), 1687 (C═O),
1611 (C═N), and 1165 (C–O–C). 1H NMR (300 MHz, DMSO‐d6) δ
(ppm): 4.45 (d, 3JH‐H = 6 Hz, 2H, CH2–O); 5.55 (s, 2H, CH2–N); 5.60 (t,
3JH‐H = 6 Hz, 1H, OH); 6.68 (s, 1H, H‐5); 7.55 (t.d, 3JH‐H = 7.5 Hz, JH‐
H = 7.8 Hz, 1H, HPh); 7.64 (t.d, 3JH‐H = 8.1 Hz 3JH‐H = 7.8 Hz, 1H, HPh);
The appropriate 5‐iodouracil analog 6a–d (0.4 mmol), propargylated de-
rivative 7a–d (1.2 mmol), CuI (0.12 mmol, 23 mg), and Na2PdP2O7
(0.04 mmol, 13 mg) were dissolved in CH3CN/t‐BuOH: 3/1 (4 ml). The
N,N‐diisopropylethylamine (3.6 mmol, 0.6 ml) was added slowly at 0°C,
and then the mixture was stirred at 70°C for 12 h. The solvent was
evaporated under reduced pressure and the residue was purified using
column chromatography (CH2Cl2–MeOH) to obtain pure products 8a–h.
3
7.64 (d.d, 3JH‐H = 8.1 Hz 3JH‐H = 7.5 Hz, 1H, HPh); 7.70 (d.d, JH‐
H = 8.1 Hz, 3JH‐H = 7.8 Hz, 1H, HPh); 7.93 (d.d, 3JH‐H = 7.8 Hz JH‐
3
3
H = 8.1 Hz, 1H, HPh); 8.73 (s, 1H, H‐4). 13C NMR (75 MHz, DMSO‐d6)
δ (ppm): 46.02 (CH2–N); 56.36 (CH2–O); 101.34 (C‐5); 107.36 (C‐4a);
122.75 (CPh, IV); 128.52 (CPh, III); 131.74 (CPh, III); 131.84 (CPh, III); 132.35
3‐[(5‐Phenyl‐1,3,4‐oxadiazol‐2‐yl)methyl]‐6‐(hydroxymethyl)furo‐
[2,3‐d]pyrimidin‐2(3H)‐one (8a)
Yield = 75%. Rf = 0.32 (CH2Cl2/MeOH [9:1]). Mp (°C): 186–187. IR (KBr)
ʋ (cm−1): 3339 (OH), 3051 (Csp2H), 2847 (Csp3H), 1687 (C═O), 1611
(C=N), 1240 (C–O–C). Rf = 0.32 (CH2Cl2/MeOH [9:1]). 1H NMR
(300 MHz, DMSO‐d6) δ (ppm): 4.52 (d, 3JH‐H = 6 Hz, 2H, CH2–O); 5.49 (s,
2H, CH2–N); 5.63 (t, 3JH‐H = 6 Hz, 1H, OH); 6.67 (s, 1H, H‐5); 7.48 (m, 3H,
(Cph, IV); 133.98 (CPh, III); 143.90 (C‐4); 154.92 (C‐2); 158.48 (C‐7a);
+
163.01 (O–C═N); 163.55 (O–C═N); 172.71 (C‐6). C16H12ClN4O4
:
359.0547, found: 359.0555.
6‐(Phenoxymethyl)‐3‐[(5‐phenyl‐1,3,4‐oxadiazol‐2‐yl)methyl]furo‐
[2,3‐d]pyrimidin‐2(3H)‐one (8e)
H
ph); 8.04 (d, 3JH‐H = 8 Hz, 2H, HPh); 8.79 (s, 1H, H‐4). 13C NMR (75 MHz,
DMSO‐d6) δ (ppm): 46.03 (CH2–N); 56.31 (CH2–O); 104.30 (C‐5); 107.38
(C‐4a); 123.19 (CPh, IV); 127.36 (CPh, III); 129.02 (CPh, III); 132.27 (CPh, III);
143.83 (C‐4); 154.92 (C‐2); 158.46 (C‐7a); 162.92 (O–C═N); 165.21
(O–C═N); 172.77 (C–6). HRMS calculated for C16H13N4O4+: 325.0937,
found 325.0947.
Yield = 59%. Rf = 0.56 (CH2Cl2/MeOH [9.5:0.5]). Mp (°C): 230–232.
IR (KBr) ʋ (cm−1): 3079 (Csp2H), 2941 (Csp3H), 1686 (C═O), 1575
(C═N), and 1228 (C–O–C). 1H NMR (300 MHz, DMSO‐d6) δ (ppm):
5.16 (s, 2H, CH2–O); 5.54 (s, 2H, CH2–N); 6.99 (m, 4H, H‐5 + 3Hph);
7.31 (m, 2H, HPh); 7.59 (m, 3H, Hph); 7.96 (m, 2H, HPh); 8.82 (s, 1H,
H‐4). 13C NMR (75 MHz, DMSO‐d6) δ (ppm): 45.39 (CH2‐N); 61.72
(CH2–O); 104.51 (C‐5); 106.23 (C‐4a); 114.92 (CPh, III); 121.31 (CPh,
III); 123.02 (CPh, IV); 126.52 (CPh, III); 129.41 (CPh, III); 129.51 (CPh, III);
132.08 (CPh, III); 144.416 (C‐4); 152.426 (Cph, IV–O); 154.629 (C‐2);
157.629 (C‐7a); 162.414 (O–C═N); 164.341 (O–C═N); 171.98 (C‐6).
HRMS calculated for C22H17N4O4+: 401.1258, found: 401.1250.
6‐(Hydroxymethyl)‐3‐{[5‐(p‐tolyl)‐1,3,4‐oxadiazol‐2‐yl]methyl}furo‐
[2,3‐d]pyrimidin‐2(3H)‐one (8b)
Yield = 74%. Rf = 0.36 (CH2Cl2/MeOH [9:1]). Mp (°C): 218‐220. IR (KBr) ʋ
(cm−1): 3387 (OH), 3033 (Csp2H), 2964 (Csp3H), 1675 (C═O), 1621
(C═N), and 1162 (C–O–C). 1H NMR (300 MHz, DMSO‐d6) δ (ppm): 2.38
(s, 3H, CH3); 4.46 (d, 3JH‐H = 5.7 Hz, 2H, CH2–O); 5.52 (s, 2H, CH2–N);
5.81 (t, 3JH‐H = 5.7 Hz, 1H, OH); 6.68 (s, 1H, H‐5); 7.39 (d, 3JH‐H = 8.1 Hz,
2H, Hph); 7.85 (d, 3JH‐H = 8.1 Hz, 2H, Hph); 8.72 (s, 1H, H‐4). 13C NMR
(75 MHz, DMSO‐d6) δ (ppm): 21.69 (CH3); 46.01 (CH2–N); 56.38
(CH2–O); 101.37 (C‐5); 107.36 (C‐4a); 120.85 (CPh, IV); 127.08 (CPh, III);
130.60 (CPh, III); 142.93 (Cph, IV); 143.88 (C‐4); 154.92 (C‐2); 158.45 (C‐7a);
162.89 (O–C═N); 165.02 (O–C═N); 172.70 (C‐6). HRMS calculated for
6‐[(3,5‐Dimethylphenoxy)methyl]‐3‐[(5‐phenyl‐1,3,4‐oxadiazol‐2‐yl)
memethyl]furo[2,3‐d]pyrimidin‐2(3H)‐one (8f)
Yield = 61%. Rf = 0.59 (CH2Cl2/MeOH [9.5:0.5]). Mp (°C): 219–212.
IR (KBr) ʋ (cm−1): 3022 (Csp2H), 2954 (Csp3H), 1675 (C═O), 1578
(C═N), and 1157 (C–O–C). 1H NMR (300 MHz, DMSO‐d6) δ (ppm):
2.83 (s, 6H, CH3); 4.51 (s, 2H, CH2–O); 4.96 (s, 2H, CH2–N); 6.05 (s,
1H, Hph); 6.12 (s, 2H, Hph); 6.41 (s, 1H, H‐5); 7.04 (m, 3H, Hph); 7.4 (d,
3JH‐H = 7.8 Hz, 2H, Hph); 8.26 (s, 1H, H‐4). 13C NMR (75 MHz, DMSO‐
d6) δ (ppm): 20.99 (CH3); 45.50 (CH2–N); 61.47 (CH2–O); 104.44
(C‐5); 106.36 (C‐4a); 112.51 (CPh, III); 122.91 (CPh, III); 123.15 (CPh, IV);
126.54 (CPh, III); 129.48 (CPh, III); 132.18 (CPh, III); 138.76 (CPh, IV);
144.51 (C‐4); 152.58 (C‐2); 157.58 (C‐7a); 162.48 (O–C═N); 164.37
C
17H15N4O4+: 339.1088, found: 339.1105.
3‐{[5‐(4‐Chlorophenyl)‐1,3,4‐oxadiazol‐2‐yl]methyl}‐6‐
(hydroxymethyl)furo[2,3‐d]pyrimidin‐2(3H)‐one (8c)
Yield = 72%. Rf = 0.34 (CH2Cl2/MeOH [9:1]). Mp (°C): 230–232. IR
(KBr) ʋ (cm−1): 3079 (Csp2H), 2941 (Csp3H), 1686 (C═O), 1575

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EL MANSOURI ET AL.
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+
(O–C═N); 172.09 (C‐6). HRMS calculated for C24H21N4O4
:
HPh); 8.02 (m, 4H, Hph); 8.27 (s, 1H, H‐4). 13C NMR (75 MHz, CDCl3) δ
(ppm): 44.59 (CH2–N); 58.15 (CH2–O); 103.98 (C‐5); 107.36 (C‐4a);
123.06 (CPh, IV); 127.14 (CPh, III); 128.53 (CPh, III); 129.13 (CPh, III); 129.84
(CPh, III); 132.25 (CPh, III); 133.56 (CPh, III); 140.86 (C‐4); 150.01 (Cph, IV);
153.16 (C‐2); 154.82 (C‐7a); 162.96 (O–C═N); 164.13 (O–C═N); 165.86
(O–C═O); 172.40 (C‐6). HRMS calculated for C23H17N4O5+: 429.1199,
found: 429.1205.
429.1563, found: 429.1571.
3‐[(5‐Phenyl‐1,3,4‐oxadiazol‐2‐yl)methyl]‐6‐[(phenylamino)methyl]‐
furo[2,3‐d]pyrimidin‐2(3H)‐one (8g)
Yield = 49%. Rf = 0.48 (CH2Cl2/MeOH [9.5:0.5]). Mp (°C): 217–219. IR
(KBr) ʋ (cm−1): 3432 (NH), 3040 (Csp2H), 2844 (Csp3H), 1685 (C═O),
1571 (C═N), and 1163 (C–O–C). 1H NMR (300 MHz, DMSO‐d6) δ (ppm):
3.90 (d, 3JH‐H = 6 Hz, 2H, CH2–NH); 5.06 (s, 2H, CH2–N); 5.79 (t, JH‐
{2‐Oxo‐3‐[(5‐phenyl‐1,3,4‐oxadiazol‐2‐yl)methyl]‐2,3‐dihydrofuro‐
[2,3‐d]pyrimidin‐6‐yl}methyl 2‐(4‐isobutylphenyl)propanoate (9b)
Yield = 77%. Rf = 0.63 (CH2Cl2/MeOH [9.5:0.5]). Mp (°C): 162–164. IR
(KBr) ʋ (cm−1): 3034 (Csp2H), 2970 (Csp3H), 1735 (C═O), 1672 (C═O),
1566 (C═N), and 1172 (C–O–C). 1H NMR (300 MHz, CDCl3) δ (ppm):
0.88 (d, 3JH‐H = 6.6 Hz, 6H, CH3–CH–CH2); 1.51 (d, 3JH‐H = 7.2 Hz,
3H, CH3–CH); 1.84 (s, 3JH‐H = 6.6 Hz, 1H, CH3–CH–CH3); 1.84 (d,
3JH‐H = 6.6 Hz, 2H, CH2–CH); 3.76 (q, 3JH‐H = 7.2 Hz, 1H, CH–CH3); 5.01
3
H = 6 Hz, 1H, NH); 6.12 (t, 3JH‐H = 7.2 Hz, 1H, HPh); 6.20 (s, 1H, H‐5); 6.23
(d, 3JH‐H = 7.8 Hz, 2H, HPh); 6.63 (t, 3JH‐H = 7.5 Hz, 2H, HPh); 7.158 (m, 3H,
HPh); 7.510 (dd, 3JH‐H = 7.2 Hz 3JH‐H = 8.1 Hz, 2H, HPh); 8.217 (s, 1H, H4).
13C NMR (75 MHz, DMSO‐d6) δ (ppm): 40.51 (CH2); 45.50 (CH2); 101.24
(C‐5); 107.00 (C‐4a); 112.58 (CPh, III); 116.62 (CPh, III); 123.15 (CPh, IV);
126.68 (CPh, III); 129.03 (CPh, III); 129.60 (CPh, III); 132.27 (CPh, III); 143.04
(C‐4); 147.98 (Cph, IV–N); 154.41 (C‐2); 156.29 (C‐7a); 162.70 (O–C═N);
+
3
164.48 (O–C═N); 172.17 (C‐6). HRMS calculated for C22H18N5O3
:
(AB‐system, 3JH‐H = 14 Hz, 1H, (CH2)a–O); 5.08 (AB‐system, JH‐
400.1410, found: 400.1419.
H = 14 Hz, 1H, (CH2)b–O); 5.55 (s, 2H, CH2‐N); 6.34 (s, 1H, H‐5); 7.09
(d, 3JH‐H = 8.1 Hz, 2H, HPh); 7.20 (d, 3JH‐H = 8.1 Hz, 2H, HPh); 7.52 (m, 3H,
5‐Methyl‐1‐{[2‐oxo‐3‐((5‐phenyl‐1,3,4‐oxadiazol‐2‐yl)methyl)‐2,3‐
dihydrofuro[2,3‐d]pyrimidin‐6‐yl]methyl}pyrimidine‐2,4(1H,3H)‐
dione (8h)
H
ph); 8.09 (d.d, 3JH‐H = 8.4 Hz 3JH‐H = 7.8 Hz, 2H, HPh); 8.14 (s, 1H, H‐4).
13C NMR (75 MHz, CDCl3) δ (ppm): 18.33 (CH3); 22.35 (CH3); 30.14
(CH); 44.35 (CH2); 44.90 (CH); 44.98 (CH2); 57.94 (CH2–O); 103.16 (C‐5);
108.08 (C‐4a); 123.06 (CPh, IV); 127.16 (CPh, III); 129.13 (CPh, III); 129.44
(CPh, III); 132.27 (CPh, III); 136.96 (CPh, IV); 140.26 (C‐4); 140.86 (CPh, IV);
153.25 (C‐2); 154.75 (C‐7a); 161.29 (O–C═N); 166.07 (O–C═N); 172.33
(C‐6); 173.98 (O–C═O). HRMS calculated for C26H30N5O6+: 508.2191,
found: 508.2198.
Yield = 81%. Rf = 0.24 (CH2Cl2/MeOH [9:1]). Mp (°C): 237–239. IR (KBr)
ʋ (cm−1): 3433 (NH), 3036 (Csp2H), 2826 (Csp3H), 1672 (C═O), 1570
(C═N), and 1176 (C–O–C). 1H NMR (DMSO‐d6, 300 MHz) δ (ppm): 1.77
(s, 3H, CH3); 4.93 (s, 2H, CH2–N); 5.54 (s, 2H, CH2–N); 6.81 (s, 1H, H‐5);
7.61 (m, 4H, HPh+ H6'); 7.98 (d, 3JH‐H = 6.9 Hz, 2H, HPh); 8.77 (s, 1H, H4);
11.41 (s, 1H, NH). 13C NMR (DMSO‐d6, 75 MHz) δ (ppm): 11.93 (CH3);
43.35 (CH2–N); 45.42 (CH2–N); 103.18 (C‐5); 106.43 (C‐5'); 109.35
(C‐4a); 122.97 (CPh, IV); 126.53 (CPh, III); 129.45 (CPh, III); 132.13 (CPh, III);
140.56 (C‐6'); 144.03 (C‐4); 150.64 (C‐4'); 152.01 (C‐2); 154.16 (C‐7a);
162.47 (O–C═N); 164.15 (O–C═N); 164.32 (C‐2'); 171.88 (C‐6). HRMS
calculated for C21H17N6O5+: 433.1260, found: 433.1267.
{2‐Oxo‐3‐[(5‐phenyl‐1,3,4‐oxadiazol‐2‐yl)methyl]‐2,3‐dihydrofuro‐
[2,3‐d]pyrimidin‐6‐yl}methyl pivaloylvalinate (9c)
Yield = 69%. Rf = 0.55 (CH2Cl2/MeOH [9.5:0.5]). Mp (°C): 168–170. IR
(KBr): 3031 (Csp2H), 2969 (Csp3H), 1733 (C═O), 1687 (C═O), 1565
(C═N), and 1170 (C–O–C). 1H NMR (300 MHz, CDCl3) δ (ppm): 0.88
(d, 3JH‐H = 6.9 Hz, 3H, CH3‐CH); 0.95 (d, 3JH‐H = 6.9 Hz, 3H, CH3‐CH);
1.45 (s, 9H, CH3–C); 2.14 (m, 1H, CH–CH3); 4.24 (m, 1H, CH–N); 5.07
(AB‐system, 3JH‐H = 13.8 Hz, 1H, (CH2)a–O); 5.17 (AB‐system,
3JH‐H = 13.8 Hz, 1H, (CH2)b‐O); 5.57 (s, 2H, CH2–N); 6.57 (s, 1H,
H‐5); 7.09 (d, 3JH‐H = 8.1 Hz, 2H, HPh); 7.53 (m, 3H, Hph); 8.01 (d,
3JH‐H = 8.1 Hz, 2H, HPh); 8.25 (s, 1H, H‐4). 13C NMR (75 MHz, CDCl3) δ
(ppm): 17.61 (CH3–CH); 19.01 (CH3–CH); 28.29 (CH3–C); 31.10
(CH–CH3); 44.56 (CH2–N); 58.11 (CH2‐O); 58.67 (N–CH–CO); 104.04
(C‐5); 107.92 (C‐4a); 123.06 (CPh, IV); 127.12 (CPh, III); 129.13 (CPh, III);
132.26 (CPh, III); 141.02 (C‐4); 152.54 (C‐2); 154.79 (C‐7a); 161.356
(O–C═N); 165.99 (O–C═N); 171.84 (C‐6); 172.34 (O–C═O). HRMS
calculated for C29H29N4O5+: 513.2138, found: 513.2146.
|
4.1.6
General procedure for the synthesis of
furopyrimidine esters 9a–c
Carboxylic acid (0.2 mmol), DCC (0.22 mmol, 45 mg), and furopyrimidine
8a (0.2 mmol, 65 mg) in dichloromethane/DMF (2:1 ml) with catalytic
amounts of 4‐dimethylaminopyridine (DMAP) were stirred mechanically
at room temperature for 12 h. N,N‐Dicyclohexylurea formed was filtered
off. The filtrate was washed with 5% acetic acid (12 ml) and water (12 ml)
and dried over anhydrous sodium sulfate. The solvent was removed
under reduced pressure and the residue was chromatographed over a
column of silica gel using CH2Cl2/MeOH (99:1, v/v) as an eluent.
|
{2‐Oxo‐3‐[(5‐phenyl‐1,3,4‐oxadiazol‐2‐yl)methyl]‐2,3‐dihydrofuro‐
[2,3‐d]pyrimidin‐6‐yl}methyl benzoate (9a)
4.2
Pharmacological/biological assays
|
Yield = 64%. Rf = 0.59 (CH2Cl2/MeOH [9.5:0.5]). Mp (°C): 145–147. IR
(KBr) ʋ (cm−1): 3034 (Csp2H), 2970 (Csp3H), 1738 (C═O), 1681 (C═O),
1568 (C═N), and 1156 (C–O–C). 1H NMR (300 MHz, CDCl3) δ (ppm):
5.30 (s, 2H, CH2–O); 5.57 (s, 2H, CH2–N); 6.62 (s, 1H, H‐5); 7.45 (m, 6H,
4.2.1
Antiviral activity evaluation
The antiviral assays were based on inhibition of virus‐induced cyto-
pathic or plaque formation in HEL cells against the VZV, TK+ (Oka

EL MANSOURI ET AL.
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|
strain), and TK─ (07‐1 strain) and against HCMV AD‐169 and Davis
strains. Confluent cell cultures in microtiter 96‐well plates were in-
fected with 100 (for HCMV) with 20 plate‐forming units for VZV.
After 2 h adsorption, the viral inoculum was removed and the cell
cultures were incubated with fresh medium in the presence of
varying concentrations of the test compounds. Viral plaque forma-
tion for VZV or the viral cytopathic effect (for HCMV) was recorded
after, respectively, 5 and 7 days postinfection. Antiviral activity was
expressed as the EC50 or compound concentration required to re-
duce the viral cytopathic effect (HCMV) or viral plaque formation
(VZV) by 50%. Alternatively, the cytostatic activity of the test
compounds was measured based on inhibition of cell growth. HEL
cells were seeded at a rate of 5 × 103 cells/well into 96‐well micro-
titer plates and allowed to proliferate for 24 h. Then, medium con-
taining different concentrations of the test compounds was added.
After 3 days of incubation at 37°C, the cell number was determined
using a Coulter counter. The cytostatic concentration was calculated
as CC50, or the compound concentration required to reduce cell
proliferation by 50% relative to the number of cells in the untreated
controls.
at 4°C, before adding 100 µl of annexin V‐binding buffer staining
(Annexin V Apoptosis Detection KIT with 7‐AAD; Millipore‐Merck).
The cells were then incubated for 20 min at room temperature in the
dark. Apoptotic cells were evaluated using a Muse Cell Analyzer
(Millipore‐Merck).
Mitochondrial membrane potential measurement
The Muse MitoPotential Kit was used to determine the mitochon-
drial membrane potential (Millipore‐Merck). The cells were treated
with compound 8f at a concentration of 5 µM. After harvesting and
washing, the cells were resuspended in 5 × 105 cells/ml in the assay
buffer. Then, to each 100 µl cell suspension, 90 µl of the MitoPo-
tential working solution was added. After 20 min incubation at 37°C,
5 μl of the Muse MitoPotential 7‐AAD reagent was added to each
sample. After 5 min incubation, cells were analyzed using the Muse
flow cytometer (Millipore‐Merck).
Caspase‐3/‐7 activity
Cells were treated with 15 µM concentration of the compound 8f.
After 24 h incubation, cells were then harvested and washed twice
with PBS at 4°C. After staining with the Caspase Assay Kit (Promega)
and incubation for 30 min, caspase‐3 activity was analyzed according
to the manufacturer's instructions and using the Muse Cell Analyzer
(Millipore‐Merck).
|
4.2.2
Anticancer activity evaluation
Cell culture
Four cancer cell lines HT‐1080 fibrosarcoma, A‐549 lung carcinoma,
MCF‐7, and MDA‐MB‐231 breast adenocarcinoma were kindly
provided by Dr. P. Coursaget from INSERM (Tours, France). A‐549,
MCF‐7, and MDA‐MB‐231 cells were cultured in Dulbecco's modified
Eagle's medium, whereas HT‐1080 cells were cultured in MEM
(minimum essential medium) culture media, both supplemented with
10% (v/v) fetal bovine serum and 1% penicillin–streptomycin. The cells
were incubated at 37°C in a humidified atmosphere with 5% CO2.
Cell cycle assay
Cells were treated with 15 µM of the compound 8f for 24 h. The cells
were then harvested and washed with PBS. The collected cells
(106 cells) were suspended in 70% ice‐cold ethanol at −20°C for
fixation. After washing, the cells were pelleted by gentle cen-
trifugation and suspended in 1 ml of staining buffer (100 mM Tris, pH
7.4, 150 mM NaCl, 1 mM CaCl2, 0.5 mM MgCl2, 0.1% Nonidet P‐40)
containing 3 µM propidium iodide (Millipore‐Merck). The cells were
then incubated for 15 min at room temperature and analyzed for cell
cycle phase distribution using the Muse Cell Analyzer (Millipore‐
Merck).
Antiproliferative activity
Cells were seeded in 96‐well plates at a density of 5 × 103 cells/well
for 24 h. Subsequently, cells were treated with various concentra-
tions of compounds 8a–h and 9a–c for 24 h. Doxorubicin (TEVA
Pharma S.A.) was used as a positive control. Then, cell growth was
analyzed using the MTT (3[4,5‐dimethylthiazol‐2‐yl‐]2,5‐diphenyl
tetrazolium bromide) assay. Cells were incubated with 5 mg/ml of
MTT for 4 h at +37°C. The supernatant was then discarded before
dissolving the product of the reaction of formazan with 150 µl of
DMSO. The absorbance was then measured at 570 nm wavelength
using a microplate reader (Thermo Fisher Scientific). IC50 was esti-
mated using GraphPad Prism7.
|
4.3
Molecular docking
In silico computational docking studies were performed using
AutoDock 4.2.[58] The X‐ray crystallographic structure of caspase
3 was downloaded from the RCSB Protein Data Bank (PDB) ID:
1RE1.[59] The protein was prepared separately by removing wa-
ter and cocrystalized ligands bound with the protein to make
remove any ligand from the receptor free before docking. Then,
polar hydrogen and Gastieger charges were added using the MGL
Tools and proteins saved in PDBQT format. The ligand structure
was created separately using ChemDraw Ultra 12.0, energy was
minimized in Chem3D, and torsional bonds of the ligand were set
to be flexible and saved in PDBQT format.[60] Next, the receptor
was kept rigid and a grid covering all the amino acid residues
present inside the active site of proteins was built (grid box size
Annexin V‐binding assay
Cells were seeded at a density of 2 × 105/well and incubated over-
night at 37°C. The cells were then treated with compound 8f at a
concentration of 15 µM for 24 h. Cells were then harvested and
washed twice with phosphate‐buffered saline (PBS) at 4°C. Then, the
cells were centrifuged at 300g for 5 min and washed twice with PBS

16 of 17
EL MANSOURI ET AL.
|
of 50 × 50 × 50 Å with a spacing of 0.375 Å between the grid
points and centered at 35.58 [x], 94.397 [y], and 17.54 [z]). The
best conformers were searched using the Lamarckian genetic
algorithm, the population size was set to 150, and the maximum
number of energy evaluations was set to 25,000,000. Finally, the
results were analyzed and visualized using Discovery Studio.
[20] T. Gregorić, M. Sedić, P. Grbčić, A. Tomljenović Paravić,
S. Kraljević Pavelić, M. Cetina, R. Vianello, S. Raić‐Malić, Eur. J. Med.
Chem. 2017, 125, 1247.
[21] M. Hossam, D. S. Lasheen, N. S. M. Ismail, A. Esmat, A. M. Mansour,
A. N. B. Singab, K. A. M. Abouzid, Eur. J. Med. Chem. 2018, 144, 330.
[22] C. S. de Oliveira, B. F. Lira, J. M. Barbosa‐Filho, J. G. Lorenzo,
P. F. de Athayde‐Filho, Molecules 2012, 17, 10192.
[23] S. S. De, M. P. Khambete, M. S. Degani, Bioorg. Med. Chem. Lett.
2019, 29, 1999.
ACKNOWLEDGMENTS
[24] N. M. Sankhe, E. Durgashivaprasad, N. Gopalan Kutty,
J. Venkata Rao, K. Narayanan, N. Kumar, P. Jain, N. Udupa,
P. Vasanth Raj, Arab. J. Chem. 2019, 12, 2548.
The authors would like to thank the technical staff of the University of
Montpellier, Montpellier, France, the University Cadi Ayyad Marrakech,
and Faculty of Sciences and Technologies Mohammedia, University
Hassan II Casablanca, Morocco, for running the spectroscopic analysis.
The authors wish to express their sincere thanks to M. Brecht Dirix and
Mrs. Leentje Persoons for evaluation of the antiviral activities (Rega In-
stitute, Leuven, Belgium).
[25] C. D., Mohan, N. C., Anilkumar, S., Rangappa, M. K., Shanmugam,
S., Mishra, A., Chinnathambi, S. A., Alharbi, A., Bhattacharjee,
G., Sethi, A. P., Kumar, Basappa, K. S., Rangappa, Front. Oncol. 2018,
8, 42.
[26] A.‐E. El Mansouri, M. Maatallah, H. Ait Benhassou, A. Moumen,
A. Mehdi, R. Snoeck, G. Andrei, M. Zahouily, H. B. Lazrek,
Nucleosides Nucleotides Nucleic Acids 2020, 39, 1088.
[27] A.‐e El Mansouri, A. Oubella, A. Mehdi, M. Y. AitItto, M. Zahouily,
H. Morjani, H. B. Lazrek, Bioorg. Chem. 2020, 108, 104558.
[28] F. Gao, T. Wang, J. Xiao, G. Huang, Eur. J. Med. Chem. 2019, 173,
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CONFLICT OF INTERESTS
The authors declare that there are no conflicts of interest.
[29] R. Chinchilla, C. Najera, Chem. Rev. 2007, 107, 874.
[30] Z. Janeba, A. Holý, R. Pohl, R. Snoeck, G. Andrei, E. de Clercq,
J. Balzarini, Can. J. Chem. 2010, 88, 628.
ORCID
Hassan B. Lazrek
http://orcid.org/0000-0002-3849-9092
[31] R. Chinchilla, C. Najera, Chem. Soc. Rev. 2011, 40, 5084.
[32] A. Behr, A. J. Vorholt, K. A. Ostrowski, T. Seidensticker, Green
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