Design, synthesis, chemical characterization, biological evaluation, and docking study of new 1,3,4-oxadiazole homonucleoside analogs

Article abstract of DOI:10.1080/15257770.2020.1761982

Herein, we report the synthetic strategies and characterization of some novel 1,3,4-oxadiazole homonucleoside analogs that are relevant to potential antitumor and cytotoxic activities. The structure of all compounds is confirmed using various spectroscopic methods such as ¹H-NMR, ¹³C-NMR, HRMS, and FTIR. These compounds were evaluated against three human cancer cell lines (MCF-7, SKBR3, and HL60 Cell Line). Preliminary investigations showed that the cytotoxic activity was markedly dependent on the nucleobase. Introduction of 5-Iodouracil 4g and theobromine 6b proved to be extremely beneficial even they were more potent than the reference drug (DOX). Also, the synthesized compounds were tested for their antiviral activities against the human varicella-zoster virus (VZV). The product 4h was (6-azauracil derivative) more potent to the reference (acyclovir) against the deficient TK ? VZV strain by about 2-fold. Finally, molecular docking suggested that the anticancer activities of compounds 6b and 4g mediated by inhibiting dual proteins EGFR/HER2 with low micromolar inhibition constant Ki range. The 1,3,4-oxadiazole homonucleosides showed a strong affinity to binding sites of target proteins by forming H-bond, carbon–hydrogen bond, Pi-anion, Pi-sulfur, Pi-sigma, alkyl, and Pi-alkyl interactions.

Full text of DOI:10.1080/15257770.2020.1761982

Nucleosides, Nucleotides & Nucleic Acids
ISSN: 1525-7770 (Print) 1532-2335 (Online) Journal homepage: https://www.tandfonline.com/loi/lncn20
Design, synthesis, chemical characterization,
biological evaluation, and docking study of new
1,3,4-oxadiazole homonucleoside analogs
Az-Eddine El Mansouri, Mohamed Maatallah, Hassan Ait Benhassou,
Abdeladim Moumen, Ahmad Mehdi, Robert Snoeck, Graciela Andrei,
Mohamed Zahouily & Hassan B. Lazrek
To cite this article: Az-Eddine El Mansouri, Mohamed Maatallah, Hassan Ait Benhassou,
Abdeladim Moumen, Ahmad Mehdi, Robert Snoeck, Graciela Andrei, Mohamed Zahouily & Hassan
B. Lazrek (2020): Design, synthesis, chemical characterization, biological evaluation, and docking
study of new 1,3,4-oxadiazole homonucleoside analogs, Nucleosides, Nucleotides & Nucleic Acids,
DOI: 10.1080/15257770.2020.1761982
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NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
https://doi.org/10.1080/15257770.2020.1761982
Design, synthesis, chemical characterization, biological
evaluation, and docking study of new 1,3,4-oxadiazole
homonucleoside analogs
Az-Eddine El Mansouria,b, Mohamed Maatallahc, Hassan Ait Benhassoud,
Abdeladim Moumend, Ahmad Mehdie, Robert Snoeckf, Graciela Andreif,
Mohamed Zahouilyb,d, and Hassan B. Lazreka
aLaboratory of Biomolecular and Medicinal Chemistry, Faculty of Science Semlalia, Cadi Ayyad
b
ꢀ
University, Marrakech, Morocco; Laboratoire de Materiaux, Catalyse & Valorisation des
ꢀ
ꢀ
Ressources Naturelles, URAC 24, Faculte des Sciences et Techniques, Universite Hassan II,
c
ꢀ
Casablanca, Morocco; Laboratoire de Chimie theorique, Faculty of Science Semlalia, Cadi Ayyad
d
e
University, Marrakech, Morocco; MAScIR Medical Biotechnology, Rabat, Morocco; Institut
ꢀ
Charles Gerhardt Montpellier UMR 5253 CNRS-UM-ENSCM, Universite de Montpellier, Montpellier
cedex 5, France; Rega Institute for Medical Research, KU Leuven, Leuven, Belgium
f
ABSTRACT
ARTICLE HISTORY
Received 6 April 2020
Accepted 24 April 2020
Herein, we report the synthetic strategies and characterization
of some novel 1,3,4-oxadiazole homonucleoside analogs that
are relevant to potential antitumor and cytotoxic activities.
The structure of all compounds is confirmed using various
spectroscopic methods such as 1H-NMR, 13C-NMR, HRMS, and
FTIR. These compounds were evaluated against three human
cancer cell lines (MCF-7, SKBR3, and HL60 Cell Line).
Preliminary investigations showed that the cytotoxic activity
was markedly dependent on the nucleobase. Introduction of
5-Iodouracil 4g and theobromine 6b proved to be extremely
beneficial even they were more potent than the reference
drug (DOX). Also, the synthesized compounds were tested for
their antiviral activities against the human varicella-zoster virus
(VZV). The product 4h was (6-azauracil derivative) more potent
to the reference (acyclovir) against the deficient TK ꢀ VZV
strain by about 2-fold. Finally, molecular docking suggested
that the anticancer activities of compounds 6b and 4g
mediated by inhibiting dual proteins EGFR/HER2 with low
micromolar inhibition constant Ki range. The 1,3,4-oxadiazole
homonucleosides showed a strong affinity to binding sites of
target proteins by forming H-bond, carbon–hydrogen bond,
Pi-anion, Pi-sulfur, Pi-sigma, alkyl, and Pi-alkyl interactions.
KEYWORDS
1,3,4-Oxadiazole;
homonucleosides; anti-
cancer activity; antiviral
activity; molecular docking
Introduction
The treatment of cancer and viral diseases persists as one of the major
challenges to modern medicine. Moreover, cancer is the most common dis-
ease in the modern era and causes the most deaths (one in eight deaths)
CONTACT Hassan B. Lazrek
hblazrek50@gmail.com
Laboratory of Biomolecular and Medicinal Chemistry,
Faculty of Science Semlalia, Cadi Ayyad University, Marrakech 40000, Morocco.
Supplemental data for this article can be accessed at https://doi.org/10.1080/15257770.2020.1761982.
ß 2020 Taylor & Francis Group, LLC

2
A.-E. EL MANSOURI ET AL.
Figure 1. Design of 1,3,4-oxadiazole homonucleoside analogs.
worldwide.[1] Indeed, the receptor tyrosine kinases (RTKs) are considered
attractive targets for driven therapy in cancer.[2,3] Among these receptors,
epidermal growth factor receptor (EGFR) and tyrosine kinase domain of
human epidermal growth factor receptors (HER-2) deserve special atten-
tion. The activation of EGFR and HER2 is an essential step for the initi-
ation and progression of breast cancer.[4] Moreover, EGFR/HER2
heterodimers are usually formed, and they are considered to be substantial
targets for cancer therapy.[5–7] As a result, the dual inhibition of EGFR and
HER2 proteins prevents heterodimerization and generally blocks drug
resistance.[5] Indeed, several dual EGFR/HER2 inhibitors containing pyr-
imidine moiety were reported in the literature. The lapatinib P1 (Fig. 1)
was the first dual EGFR/HER2 inhibitor to be approved by the FDA in
2007 for breast cancer.[8] The pyrimidine derivative P2 was developed as
an efficient dual EGFR/HER2 inhibitors.[9] Also, several fused pyrimidines

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
3
with other heterocycles such as pyrrole, thiene, and azepine (Fig. 1) were
found to be high inhibitors of EGFR/HER2.[10–12]
Pyrimidine and purine nucleoside analogs are widely used in the chemo-
therapy of cancer and viral diseases for almost 50 years.[13–16] Among the
possible modifications of nucleosides, the introduction of a methylene
bridge between a nucleobase and the sugar moiety led to the formation of
homonucleosides.[17] For instance, the homonucleoside analogs P3–5 were
reported in the literature with antiviral activities against herpes simplex
virus,[18] vaccinia virus,[19] and influenza type virus AH1N1.[20] On the
other hand, since the discovery of Lamivudine P6 and Emtricitabine P7 as
anti-HIV drugs (Fig. 1), significant interest has been committed toward the
chemistry of heterocyclic nucleosides.[21] The later were obtained through
the substitution of the furanose moiety by another heterocycle.[22] Also,
various heterocyclic nucleoside analogs with dioxolane,[23] dithiolane,[24]
and pyrazoline[25] were reported.
Recently, the combination of a nucleobase and heterocycle in one single
molecule via the introduction of the methylene bridge (heterocycle homo-
nucleosides) was described. Piotrowska et al. were synthesized and eval-
uated a novel c-Lactam homonucleoside analogs for their antiviral and
anticancer activities, compound P8 (Fig. 1) showed mild IC50 range toward
pancreatic adenocarcinoma cells (Capan-1).[26] Also, a new series of isoxa-
zolidine homonucleosides P9 (Fig. 1) was prepared with no significant anti-
viral activities.[27] Moreover, various 1,2,3-triazole homonucleoside analogs
P10 (Fig. 1) were synthesized and evaluated for their biological
activities.[28–31]
The oxadiazole chemistry has been developed extensively and is still
developing.[32] The –N ¼ C–O– linkage can react with the nucleophilic cen-
ters of malignant cells and is responsible for its biological activity.[33] Their
electronic and charge-transporting properties ensure the connection with
targets through non-covalent interactions. In other cases, oxadiazole moi-
eties have been shown to act as a flat, aromatic linker to place substituents
in the appropriate orientation,[34] as well as modulating molecular proper-
ties by positioning them in the periphery of the molecule.[35] Furthermore,
oxadiazoles have been described as bioisosteres for carboxylic acids, esters,
and carboxamides.[36]These characteristics of the 1,3,4-oxadiazole ring
resulted in the diverse pharmaceutical applications as antiviral (Raltegravir
P11)[37,38] and anticancer (Zibotentan P12)[39,40] drugs (Fig. 1).
Taking into consideration the biological importance of homonucleosides,
heterocyclic nucleosides, and 1,3,4-oxadiazole derivatives, we report the
design, synthesis, and biological activity of the novel 1,3,4-oxadiazole
homonucleoside analogs as anticancer and antiviral agents. Moreover,
based on the similarity between EGFR/HER2 inhibitors and our products

4
A.-E. EL MANSOURI ET AL.
(pyrimidine or fused pyrimidine), the molecular docking study on these
two proteins was performed to explore the anticancer activity at the
molecular level and to investigate the mode of action of the most
potent compounds.
Results and discussion
Chemistry
Several methods for the synthesis of both symmetrical and asymmetrical
1,3,4-oxadiazoles have been reported. One of the most popular ways
involves cyclodehydration of 1,2-diacylhydrazines using phosphorous oxy-
chloride (POCl3).[38] Next, the second method is the cyclization of 1,2-diac-
ylhydrazines with thionyl chloride (SOCl2).[41] Moreover, different
activating agents for cyclization of diacylhydrazines such as 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDC), 1,10-carbonyldiimidazole (CDI),
and silica-supported dichlorophosphate were also described for the cyclode-
hydration of this intermediate.[42–44] Yet, there are still disadvantages asso-
ciated with these methodologies, such as harsh reaction conditions, the use
of expensive reagents, and limited scalability. To synthesize the desired
products, we adopted the following strategy. First, we prepared 1,3,4-oxa-
diazoles by cyclodehydration of diacylhydrazines. Then, these chemical
units were used as an alkylating agent for pyrimidines and purines. The
hydrazides 2a,b were synthesized via the condensation of ester 1a,b and
hydrazine in ethanol at reflux temperature. Then, dehydrating reagent
POCl3 was used in the reaction of acylhydrazides 2a,b with chloroacetic
acids to give 1,3,4-oxadiazole derivatives 3a,b.[45] The conditions described
in this literature were modified: we showed no need to use POCl3 as a solv-
ent. Therefore, the amount of POCl3 was reduced to 3 equivalents and
dichloroethane (DCE) was used as a solvent (Scheme 1).
Scheme 1. Preparation of alkylating agents 3a–b.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
5
Table 1. Preparation of pyrimidine homonucleoside analogs 4a–j using Hilbert–Johnson
modified reaction.
4c, R=CH3, 80%
4d, R=F, 40%
4e, R=Cl, 38%
4f, R=Br, 39%
4g, R=I, 35%
4a, R=H, 84%
4b R=OCH3, 85%
4h, R=H, 52%
4i, R=CH3, 56%
4j, 91%
The preparation of homonucleoside analogs 4a–j was carried out through
Hilbert–Johnson modified reaction.[46] First, the silylated pyrimidines
were synthesized using the ammonium sulfate and hexamethyldisilazane
(HMDS). Then, the alkylating agents 3a,b, potassium iodide and acetonitrile
were added to the mixture (Table 1). The novel compounds 1,3,4-oxadiazole-
pyrimidines were prepared with good yields except the 5-halouracils 4d–g.
This result can be explained by the electron-withdrawing effect of halogen in
position C5 (halogen decreases the nucleophilicity of N1). In the aim to
increase the yield of the N1-alkylation of 5-halouracils, we changed the
synthesis strategy by using K2CO3 in DMF (Table 2). Thus, the desired
products 4d–g was prepared with improved yields (48–51%).
The conditions applied to synthesize pyrimidine hybrids did not work to
prepare purines 6a–c. Thereafter, the synthetic strategy of N-alkylated
purines was changed to basic medium (K2CO3/DMF). For the N-alkylation
of theophylline 5a and theobromine 5b, the desired products (6a, 6b) were
obtained with excellent yields. However, when adenine 5c was used
the yield decreased (Table 3).

6
A.-E. EL MANSOURI ET AL.
Table 2. Preparation of C5-halopyrimidine homonucleosides 4d–g using basic medium.
4d, X=F, 51%
4e, X=Cl, 49%
4f, X=Br, 49%
4g, X=I, 48%
Table 3. Preparation of purine homonucleoside analogs 6a–c.
6a, 83%
6b, 86%
6c, 34%
Biological assays
Cytotoxic activity
As described in Fig. 2, compounds 4e, 4g, 6b have the most prominent
effect by compromising the cell growth of MCF7 and SKBR3 cell lines.
However, 6b was the most potent molecule in terms of growth inhibition
(Fig. 2). On the other hand, no growth inhibition has been observed after
the treatment of leukemia cancer cells HL60 by these compounds except 4j
in comparison to the reference drug. Generally, breast cancer cell lines
were the most sensitive to the synthesized compounds. About broad-spec-
trum of anticancer activity, a close examination of the data presented in
Fig. 2 reveals interesting structure-activity relationship and substituent
effect on the cytotoxic potential. The compound 6b with theobromine as
nucleobase showed better cytotoxic activity than all other hybrids against
breast cancer cell lines, followed by hybrid 5-Iodouracil as nucleobase 4g.
Careful analysis of the in vitro cytotoxic evaluation also indicates that the
linkage of 5-substituted uracil is more active than the 6-azauracil deriva-
tives. Furthermore, cytotoxicity was enhanced by changing the halogen

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
7
Figure 2. Cytotoxicity effect of synthesized compounds in 10 mM concentration on human
MCF-7, SKBR3, and HL60 cells.
Table 4. Activity of compounds 4a–j and 6a–c against varicella-zoster virus (VZV) in human
embryonic lung (HEL).
Antiviral activity EC50 (mM)a
Cytotoxicity (mM)
Compounds
TK þ VZV strain
TK ꢀ VZV strain
Cell morphologyb
4a
4b
4c
4d
4e
4g
4h
4i
4j
6a
6b
6c
Acyclovir
Brivudin
>100
>100
>100
>100
>100
>100
39.39
>100
>100
>100
>100
>100
2.75
>100
>100
>100
>100
>100
>100
49.67
>100
>100
>100
>100
>100
103.23
35.14
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>350
>300
0.011
aEffective concentration required to reduce virus plaque formation by 50%. Virus input was 20 plaque-forming
units (PFU).
bMinimum cytotoxic concentration that causes a microscopically detectable alteration of cell morphology.
group in 5 positions of the uracil by decreasing the electronegativity as evi-
dent in changing fluorine to iodine. Moreover, changing xanthine (6a–b)
to adenine (6c) remarkably decreases cytotoxicity.
Antiviral activity
All the synthesized compounds were tested for their antiviral activities
against the human varicella-zoster virus (VZV), both wild-type and thymi-
dine kinase-deficient TKꢀ (Table 4). The antiviral activities of synthesized
pyrimidine-1,3,4-oxadiazole hybrid molecules were lower than the antiviral
activities of reference compound acyclovir and brivudin against the
TK þ VZV strain, except 4h (6-azauracil derivative), showed better activity

8
A.-E. EL MANSOURI ET AL.
Table 5. Results of molecular docking.
Protein
EGFR
Ligand
Estimated binding energy (kcal/mol)
Estimated inhibition constant Ki (mM)
4g
6b
4g
6b
ꢀ8.05
ꢀ7.5
1.25
3.18
0.45
0.35
HER2
ꢀ8.66
ꢀ8.81
in comparison to acyclovir against the TK ꢀ VZV strain by 2-fold. As the
TK ꢀ VZV strain is known for resistance to conventional antiviral drugs,
pyrimidine-1,3,4-oxadiazole 4h can serve as a promising lead candidate for
further study. All synthesized compounds showed quantifiable cytotoxicity.
Molecular docking
The molecular docking study was performed to understand the mode of
action and to explore the anticancer activity at the molecular level. Indeed,
taking into account the importance of these dual inhibitors and their simi-
larity to our products (pyrimidine or fused pyrimidine), we have chosen to
dock the most active compounds 4g and 6b into the active site of EGFR
(PDB: 1m17) and HER2 (PDB: 3PP0).
Before determining the validity of the docking protocol, first, the self-
docking experiments were carried out. The root-mean-square deviations
(RMSD) between the predicted and the native poses found to be 1.74 Å
(1M17; ERLOTINIB) and 0.54 Å (3PP0; 2-{2-[4-({5-chloro-6-[3-(trifluoro-
methyl)phenoxy]pyridin-3-yl}c1amino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]e-
thoxy}ethanol). These results indicated that the adopted docking protocol is
good for the reproduction of the native poses (<2Å).
The docking results are summarized in Table 5 and Figs. 3–6 (see Tables
S1 and S2 in supplementary data). Concerning EGFR, ligands 4g and 6b
are bound deeply inside the active site with the estimated free energy of
binding ꢀ8.05 and ꢀ7.5 kcal/mol, respectively. Also, the compounds 4g
and 6b inhibit EGFR protein with estimated inhibition constants (Ki): 1.25
and 3.18 mM, respectively. The product 4g interacted with amino acid resi-
dues of EGFR by one hydrogen bond LYS721—O ¼ C4 (2.071 Å). Also,
compound 6b showed one H-bound between MET769 and N9 (1.995 Å).
On the other hand, HER2 interacts with ligands 4g and 6b with the esti-
mated free energy of binding ꢀ8.66 and ꢀ8.81 kcal/mol, respectively. Then,
these compounds inhibit HER2 protein with estimated inhibition constants
of 0.45 and 0.35 mM, respectively. Besides, product 4g exhibited two hydro-
gen bonds THR862—O ¼ C4 (2,424 Å) and ASP863—Noxadiazole(2,634 Å).
While, ligand 6b interacted with HER2 by showing three H-binding inter-
actions
MET801—N ¼ C9(2,104 Å),
THR862—O ¼ C2(1,962 Å),
and
ASP863—Ooxadiazole(2,181 Å). Furthermore, the products 4g and 6b interact

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
9
Figure 3. 2D interactions of compound 4g in the binding sites of EGFR protein (PDB ID: 1m17).
Figure 4. 2D interactions of compound 6b in the binding sites of EGFR protein (PDB ID: 1m17).

10
A.-E. EL MANSOURI ET AL.
Figure 5. 2D interactions of compound 4g in the binding sites of HER2 protein (PDB ID: 3pp0).
Figure 6. 2D interactions of compound 6b in the binding sites of HER2 protein (PDB ID: 3pp0).
with EGFR and HER by other interactions such as carbon–hydrogen bond,
Pi-anion, Pi-sulfur, Pi-sigma, alkyl, and Pi-alkyl.
Conclusion
A series of 1,3,4-oxadiazole-pyrimidines and purines hybrids (4a–j and
6a–c) have been designed and synthesized by employing silyl

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
11
Hilbert–Johnson modified reaction pyrimidines and basic medium for
purines. The in vitro anti-cancer activity of all the synthesized compounds
against breast (SKBR3 and MCF7) and leukemia cancer (HL60) cells was
evaluated. Compounds 4g and 6b have shown promising results in com-
parison with the marketed drug doxorubicin. Moreover, the hybrid 4h was
more potent than the reference (acyclovir) against the deficient TK ꢀ VZV
strain by about 2-fold. Also, molecular docking suggested that the anti-
cancer activities of compounds 6b and 4g mediated by inhibiting dual pro-
teins EGFR/HER2 at low micromolar inhibition constant Ki range.
Therefore, these compounds can serve as a promising lead candidate for
further study.
Experimental
Chemistry
General
€
Melting points were measured using a Buchi B-545 digital capillary melting
point apparatus and used without correction. Reactions were checked with
TLC using aluminum sheets with silica gel 60 F254 from Merck. Infrared
spectra (IR) were recorded on a PerkinElmer VERTEX 70 FTIR spectrom-
1
eter covering field 400–4000 cmꢀ1. The spectra of H NMR and 13C NMR
were recorded in solution in DMSO-d6 on a Bruker Advance 300 spec-
trometer at 300 and 75 MHz, respectively. The chemical shifts are expressed
in parts per million (ppm) by using DMSO-d6 as internal reference. The
multiplicities of the signals are indicated by the following abbreviations: s,
singlet; d, doublet; t, triplet; q, quadruplet; and m, multiplet, and coupling
constants 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-Aldrich.
General procedure for the preparation of alkylating agent 3a–b[47]
Brief, a round bottom flask (25 mL) equipped with a condenser has added
a mixture of ester 1a–b (10 mmol), hydrazine hydrate (11 mmol, 0.5 ml)
and ethanol (5 ml). The mixture was refluxed for 3 hours. The solvent was
evaporated under vacuum and the hydrazide was recrystallized using etha-
nol to afford the desired product with 70% for 2a and 87% for 2b. Then,
chloroacetic acid (11 mmol, 1,04 g), phosphorus oxychloride (30 mmol,
2.8 ml) and DCE (20 ml) were mixed in 100 ml round bottom flask and
heated at 80 ꢁC for 2 hours. After, carboxylic acid hydrazide (2a or 2b) was
added to the mixture and heated for 12 hours. After completion of the
reaction, water (40 ml) was added and the pH was adjusted to 7–8 with

12
A.-E. EL MANSOURI ET AL.
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 3a in 68% and 3b
in 74%.
General procedure for the preparation of compounds 4a–j
Method A. A mixture of the appropriate pyrimidine (1 mmol) and ammo-
nium sulfate (0.10 mmol, 13 mg) in HMDS (1.5 ml) was refluxed until clear
solution was obtained. Then, the appropriate alkylating agent 3a–b
(1.5 mmol), KI (0.75 mmol, 124 mg) and acetonitrile (2.5 ml) were added.
Reaction mixture was heated at 85 ꢁC for 12 hours. After completion of the
reaction (monitored by TLC), the reaction mixture was diluted with
dichloromethane and evaporated to dryness. The residue was purified by
flash chromatography eluting with (CH2Cl2/MeOH ¼ 99:1).
Method B. A mixture of 5-halopyrimidine (1 mmol), alkylating agent 3a
(1.1 mmol, 215 mg) and potassium carbonate (0.5 mmol, 69 mg) in DMF
(10 ml) was heated at 90 ꢁC for 2 hours. Then the DMF was evaporated under
reduced pressure to dryness. The residue was purified by flash chromatography
eluting with (CH2Cl2/MeOH ¼ 99:1) to obtain pure product 4d–g.
1-((5-phenyl-1,3,4-oxadiazol-2-yl)methyl)pyrimidine-2,4(1H,3H)-dione (4a).
white solid, Yield: 84%. Rf ¼ 0.5 (CH2Cl2/CH3OH: 9.6/0.4). Mp (ꢁC) 247–249.
NMR 1H: 5.25 ppm (s, 2H, CH2-N); 5.7 ppm (1H, d, J ¼ 7.9 Hz, C5H);
7.63 ppm (3H, m, HPh); 7.81 ppm (d, J ¼ 7.9 Hz, 1H, C6H); 7.99 ppm (m, 2H,
HPh); 11,5ppm (s,1H, N-H). NMR 13C: 42 ppm (CH2); 106 ppm (C5); 123 ppm
(CPh); 126 (CPh); 129 (CPh); 132 ppm (CPh); 146 ppm (C6); 151 (C2); 163 (C4);
164 (O-C¼N); 165 (O-C¼N). IR (KBr, cmꢀ1) 1708 (C¼O), 1606 (C¼N),
1503 (C ¼ C), 1122(C-O-C). HRMS Calculated [M þ H]þ 271.0831, Found
[M þ H]þ 271.0845.
1-((5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-yl)methyl)pyrimidine-2,4(1H,3H)-
dione (4b) white solid, Yield: 85%. Rf ¼ 0.51 (CH2Cl2/CH3OH: 9.6/0.4). Mp
1
(ꢁC) 233–235. NMR H: 3.8 ppm (3H, s, CH3-O); 5.2 ppm (2H, s, CH2-N);
5.7 ppm (1H, d, J ¼ 7.9 Hz, C5-H); 7.17 ppm (2H, dd, HPh); 7.8 ppm (1H, d,
J¼ 7.9 Hz, C6-H); 7.92 ppm (2H, dd, HPh); 11.5ppm (1H, s, N-H). NMR 13C:
42 ppm (CH2); 55 ppm (CH3-O); 102 ppm (C5); 115 (CPh); 126 (CPh); 129
(CPh); 132 ppm (CPh); 146 ppm (C6); 151 ppm (C2); 162 (C4); 164 (O-C¼N);
165 (O-C¼N). IR (KBr, cmꢀ1): 3106 (Csp3), 1714 (C¼O), 1608 (C¼N), 1502
(C ¼ C), 1171(C-O-C). HRMS Calculated [M þ H]þ: 301.0982 Found
[M þ H]þ 301.0992.
5-methyl-1-((5-phenyl-1,3,4-oxadiazol-2-yl)methyl)pyrimidine-2,4(1H,3H)-dione
(4c) white solid, Yield: 80%. Rf ¼ 0.56 (CH2Cl2/CH3OH: 9.6/0.4). Mp (ꢁC)
1
241–243. NMR H: 1.8pppm (s, 3H, CH3); 5.2 ppm (2H, s, CH2); 7.65 ppm (3H,

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
13
m, HPh); 7.69 ppm (1H, d, J ¼ 8.2 Hz, C6-H); 7.99 ppm (2H, m, HPh); 11.5 ppm
(1H, s, N-H). NMR 13C: 12 ppm (CH3); 42 ppm (CH2); 110 ppm (C5); 123 ppm
(CPh); 126 (CPh); 129 (CPh); 132 ppm (CPh); 141 ppm (C6); 151 ppm (C2); 163
(C4); 164 (O-C¼N); 165 (O-C¼N). IR (KBr, cmꢀ1) 1699 (C¼O), 1652 (C¼O),
1609 (C¼N), 1555 (C ¼ C), 1154 (C-O-C). HRMS Calculated [M þ H]þ:
301.0931 Found [M þ H]þ 301.0951
5-fluoro-1-((5-phenyl-1,3,4-oxadiazol-2-yl)methyl)pyrimidine-2,4(1H,3H)-
dione (4d) white solid, Yield: 40/51% (method A/B). Rf ¼ 0.36 (CH2Cl2/
1
CH3OH: 9.6/0.4). Mp (ꢁC) 216–218. NMR H: 5.22 ppm (2H, s, CH2);
7.62 ppm (3H, m, HPh); 7.98 ppm (2H, m, HPh); 8.24 ppm (1H, d,
J ¼ 8.24 Hz, C6-H). 12.14 (1H, s, NH). NMR 13C: 42.24 ppm (CH2); 123 .06
(CPh); 126.56 (CPh); 129.41 (CPh); 129.66 (d, 2.4 Hz, C6); 132 ppm (CPh);
139.82 ppm (d, J ¼ 229Hz, C6); 149.44 ppm (C2); 157.32 (d, J ¼ 26Hz, C4);
162.17 (O-C¼N); 164.51 (O-C¼N). IR (KBr, cmꢀ1) 1693 (C¼O), 1606
(C¼N), 1554 (C ¼ C), 1173 C-O-C. HRMS Calculated [M þ H]þ: 289.0731
Found [M þ H]þ: 289.0751
5-chloro-1-((5-phenyl-1,3,4-oxadiazol-2-yl)methyl)pyrimidine-2,4(1H,3H)-dione
(4e) white solid, Yield: 38/49% (method A/B). Rf ¼ 0.39 (CH2Cl2/CH3OH: 9.6/
1
0.4). Mp (ꢁC) 233–235. NMR H: 5.33 ppm (2H, s, CH2); 7.6 ppm (3H, m, HPh);
8.28 ppm (2H, m, HPh); 8,8ppm (1H, s, C6-H). NMR 13C: 42 (CH2); 106 ppm
(C5); 123 (CPh); 126 (CPh); 129 (CPh); 132 ppm (CPh); 142 ppm (C6); 149 ppm
(C2); 159 (C4); 162 (O-C¼N); 164 (O-C¼N). IR (KBr, cmꢀ1) 1724 (C¼O), 1666
(C¼O), 1631 (C¼N), 1551 (C ¼ C), 1244 (C-O-C). HRMS Calculated [M þ H]þ:
305.0436 Found [M þ H]þ: 305.0451.
5-bromo-1-((5-phenyl-1,3,4-oxadiazol-2-yl)methyl)
pyrimidine-2,4(1H,3H)-
dione (4f) white solid, Yield: 39/49% (method A/B). Rf ¼ 0.4 (CH2Cl2/CH3OH:
1
9.6/0.4). Mp (ꢁC) 258–260. NMR H: 5.35 ppm (2H, s, CH2); 7.6 ppm (3H, m,
HPh); 8.1 ppm (2H, m, HPh); 8.5 ppm (1H, s, C6-H); 11.8 (1, s, NH). NMR 13C:
42.36 ppm (CH2); 95.5 ppm (C5); 123.01 (CPh); 126.54 (CPh); 129.46 (CPh);
132.14 ppm (CPh); 144.95 ppm (C6); 150.13 ppm (C2); 159.57 (C4); 162.25 (O-
C¼N); 164.48 (O-C¼N). IR (KBr, cmꢀ1) 1693 (C¼O), 1606 (C¼N), 1554
(C ¼ C), 1173 (C-O-C). HRMS Calculated [M þ H]þ: 348.9931 Found
[M þ H]þ 348.9940.
5-iodo-1-((5-phenyl-1,3,4-oxadiazol-2-yl)methyl)pyrimidine-2,4(1H,3H)-dione
(4g) white solid, Yield: 35/48% (method A/B). R ¼ 0.42 (CH2Cl2/
1
CH3OH: 9.6/0.4). Mp (ꢁC) 282–284. NMR H: 5.3 ppm (2H, s, CH2);
7.64 ppm (3H, m, HPh); 8 ppm (2H, m, HPh); 8.38 ppm (1H, s, C6-H);
11.85 ppm (1H, s, N-H). NMR 13C: 42 ppm (CH2); 70 ppm (C5);
123 ppm (CPh); 126 (CPh); 129 (CPh); 133 ppm (CPh); 150 ppm (C6);
151 ppm (C2); 161 (C4); 162 (O-C¼N); 164 (O-C¼N). IR (KBr, cm-1)
1707 (C¼O), 1614 (C¼N), 1554 (C ¼ C), 1246 (C-O-C). HRMS
Calculated [M þ H] : 396.9792 Found [M þ H] 396.9798.
þ
þ

14
A.-E. EL MANSOURI ET AL.
2-((5-phenyl-1,3,4-oxadiazol-2-yl)methyl)-1,2,4-triazine-3,5(2H,4H)-dione
(4h) white solid, Yield: 52%. R ¼ 0.38 (CH2Cl2/CH3OH: 9.6/0.4). Mp (ꢁC)
244–246. NMR 1H: 5.4 ppm (2H, s, CH2-N); 7.6 ppm (3H, m, HPh);
7.96 ppm (2H, m, HPh); 8.1 ppm (1H, s, C5-H). NMR 13C: 43 ppm (CH2);
107 ppm (C5); 123 ppm (CPh); 126 (CPh); 129 (CPh); 133 ppm (CPh);
143 ppm (C2); 150 (C4); 163 (O-C¼N); 165 (O-C¼N). IR (KBr, cmꢀ1)
3406 (N-H), 1693 (C¼O), 1555 (C ¼ C), 1112(C-O-C). HRMS Calculated
[M þ H]þ: 272.0778 Found [M þ H]þ 272.0790.
6-methyl-2-((5-phenyl-1,3,4-oxadiazol-2-yl)methyl)-1,2,4-triazine-3,5(2H,4H)-
dione (4i) white solid, Yield: 56%. R ¼ 0.36 (CH2Cl2/CH3OH: 9.6/0.4). Mp
1
(ꢁC) 241–243. NMR H: 2.15pppm (s, 3H, CH3); 5.28ppm (2H, s, CH2);
7.36 ppm (3H, m, HPh); 7.99 ppm (2H, m, HPh); 12.3 (NH). NMR 13C:
16.49 ppm (CH3); 34.08 ppm (CH2); 123 .06 (CPh); 126.53 (CPh); 129.37 (CPh);
132.02 ppm (CPh); 141.91 ppm (C5); 148.9 ppm (C2); 155.9 ppm (C4);
161.74 ppm (O-C¼N); 164.31 ppm (O-C¼N). IR (KBr, cmꢀ1) 1649 (C¼O),
1607 (C¼N), 1554 (C ¼ C), 1173 (C-O-C). HRMS Calculated [M þ H]þ:
286.0935 Found [M þ H]þ 286.0943.
4-amino-1-((5-phenyl-1,3,4-oxadiazol-2-yl)methyl)pyrimidin-2(1H)-one (4j)
white solid, Yield: 91%. R ¼ 0.3 (CH2Cl2/CH3OH: 9/1). Mp (ꢁC) 281–283.
1
NMR H: 5.24 (2H, s, CH2); 5.86 ppm (1H, d, C5-H); 7.47 (2H, s, NH2);
7.62 ppm (3H, m, HPh); 7.83 (1H, d, C6-H); 8.6 ppm (2H, m, HPh). NMR 13C:
43.24 (CH2); 94.37 (C5); 123.11 (CPh); 126.47 (CPh); 129.42 (CPh); 132.03 ppm
(CPh); 146.62 (C6); 153.73 (C2); 162.96 ppm (O-C¼N); 164.21 ppm (O-C¼N);
164.92 (C4). IR (KBr, cmꢀ1): 3414 (NH2), 1640 (C¼O), 1628 (C¼N and
C¼ C), 1195 (C-N). HRMS Calculated: [M þ H]þ: 270.0986 Found
[M þ H]þ: 270.0995.
General procedure for the preparation of compounds 6a–c
A mixture of purine (1 mmol), alkylating agent 3a (1.5 mmol, 294 mg) and
potassium carbonate (1 mmol, 138 mg) in DMF (10 ml) was heated at 90 ꢁC
for 4 h. Then the DMF was evaporated under reduced pressure to dryness.
The residue was purified by flash chromatography eluting with CH2Cl2/
MeOH to obtain pure desired product 6a–c.
1,3-dimethyl-7-((5-phenyl-1,3,4-oxadiazol-2-yl)methyl)-1H-purine-2,6(3H,7H)-
dione (6a) white solid, Yield: 83%. R ¼ 0.61 (CH2Cl2/CH3OH: 9.6/0.4). Mp (ꢁC)
1
200–202. NMR H: 3.2 ppm (3H, s, CH3-N); 3.5 ppm (3H, s, CH3-N); 5.9 ppm
(2H, s, CH2-N); 7.61 ppm (3H, m, HPh); 8 ppm (2H, m, HPh); 8.3 ppm (1H, s,
13
C8-H). NMR C: 29 ppm (CH3-N); 33 ppm (CH3-N); 38 (CH2); 107 ppm (C5);
124 (CPh); 126 (CPh); 129 (CPh); 132 ppm (CPh); 142 (C8); 148 (C6); 151.5 (C4);
163 (O-C¼N); 165 ppm (O-C¼N). IR (KBr, cmꢀ1) 1706 (C¼O), 1664 (C¼O),
1605 (C¼N), 1549 (C ¼ C), 1188(C-O-C). HRMS Calculated [M þ H]þ:
339.1200 Found [M þ H]þ 339.1205.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
15
3,7-dimethyl-1-((5-phenyl-1,3,4-oxadiazol-2-yl)methyl)-1H-purine-2,6(3H,7H)-
dione (6b) white solid, Yield: 86%. R ¼ 0.59 (CH2Cl2/CH3OH: 9.6/0.4). Mp (ꢁC)
1
204–206. NMR H: 3.45 ppm (3H, s, CH3-N); 3.9 ppm (3H, s, CH3-N); 5.4 ppm
(2H, s, CH2-N); 7.6 ppm (3H, m, HPh); 7.97 ppm (2H, m, HPh); 8.1 ppm (1H, s,
13
C8-H). NMR C: 27 ppm (CH3-N); 29 ppm (CH3-N); 41 (CH2); 107 ppm (C5);
123 (CPh); 127 (CPh); 130 (CPh); 133 ppm (CPh); 144 (C8); 148 (C6); 151.5 (C4);
163 (O-C¼N); 165 ppm (O-C¼N). IR (KBr, cm-1) 1706 (C¼O), 1664 (C¼O),
1605 (C¼N), 1549 (C ¼ C), 1188(C-O-C). HRMS Calculated [M þ H]þ:
339.1200 Found [M þ H]þ 339.1207
9-((5-phenyl-1,3,4-oxadiazol-2-yl)methyl)-9H-purin-6-amine (6c) white
solid, Yield: 34%. R ¼ 0.5 (CH2Cl2/CH3OH: 9/1). Mp (ꢁC) 241–243. NMR
1H: 5.8 ppm (2H, s, CH2-N); 7.45 ppm (2H, s, NH2); 7.6 ppm (3H, m, HPh);
7.9 ppm (2H, m, HPh); 8.17 ppm (1H, s, C8H); 8.26 ppm (1H, s, C3H).
NMR 13C: 39 (CH2); 119 (C5); 123 .06 (CPh); 126.53 (CPh); 129.37 (CPh);
132.02 ppm (CPh); 142 (C8); 151 (C6); 153 (C2); 157 (C4); 161.74 ppm (O-
C¼N); 164.31 ppm (O-C¼N). IR (KBr, cmꢀ1) 3258 (N-H), 1605 (C¼N),
1547 (C ¼ C), 1162(C-O-C). HRMS Calculated [M þ H]þ: 294.1098 Found
[M þ H]þ 294.1102.
Biological data
Cytotoxicity activity. Cell culture. Two human breast cancer cells lines
SKBR3, MCF7 and a human leukemia cell line, HL60 used in this work
were purchased from the American Type Culture Collection (ATCC,
Manassas, VA, USA). Cells were cultured in Iscove’s modified Dulbecco’s
media supplemented with 10% (v/v) heat-inactivated fetal bovine serum
(FBS) and 1% penicillin–streptomycin. Cells were maintained at 37 ꢁC and
5% CO2 in a humid environment. All experiments were performed with
cells in the exponential growth phase.
Cell proliferation and cytotoxicity assay. SKBR3, MCF7 and HL60 cells were
seeded in a 96-multiwell plate at a density of 5000 cells/well for 24 hours.
The cells were then treated with the chemically synthesized molecules 4a–j
and 6a–c at a concentration of 10 mM for 18 hours. before the WST-8 test.
Cells treated with Doxorubicin were used as positive control and non-
treated cells as a negative control. The CCK-8 assay was used to measure
the cytotoxicity of the compounds. After incubation, CCK-8 solution
(10 mL) was added to each well, followed by incubation for 2 hours at
37 ꢁC. A multiplate reader (Multiskan FC photometer Thermo Scientific,
Langenselbold, Germany) determined the absorbance at 450 nm. Cell viabil-
ity was expressed as a percentage of the control (untreated) cells by using
the following equation:

16
A.-E. EL MANSOURI ET AL.
ODꢀuntreated cell – ODꢀtreated cells=ODꢀuntreated cell ꢂ 100
.
For each compound concentration, the mean values of the absorbance rates
from three wells were calculated. In fact, WST-8 is reduced by dehydrogen-
ase activities in cells to give a yellow-color formazan dye, which is soluble
in the tissue culture media. The amount of the formazan dye, generated by
the activities of dehydrogenases in cells, is directly proportional to the
number of living cells.
Antiviral activity assays. The antiviral assays were based on inhibition of
virus-induced cytopathicity or plaque formation in human embryonic lung
(HEL) cells against VZV (thymidine kinase wild-type (TKþ) (Oka strain)
and TK-deficient (TKꢀ) (07-1 strain). Confluent cell cultures in microtiter
96-well plates were infected with 20 plates forming units (PFU) for VZV.
After 2-hour adsorption, the viral inoculum was removed and the cell cul-
tures were incubated with fresh medium in the presence of varying concen-
trations of the test compounds. Viral plaque formation for VZV or viral
cytopathic effect (for HCMV) was recorded after, respectively, 5- and 7-day
post-infection. Antiviral activity was expressed as the EC50 or compound
concentration required to reduce viral plaque formation (VZV) by 50%.
Alternatively, the cytostatic activity of the test compounds was measured
based on the inhibition of cell growth.
HEL cells were seeded at a rate of 5 ꢂ 103 cells/well into 96-well micro-
titer plates and allowed to proliferate for 24 hours. Then, medium contain-
ing different concentrations of the test compounds was added. After 3 days
of incubation at 37 ꢁC, the cell number was determined with a Coulter
counter. The cytostatic concentration was calculated as the CC50, or the
compound concentration required to reduce cell proliferation by 50% rela-
tive to the number of cells in the untreated controls.
Molecular docking
In silico computational docking studies were performed using AutoDock
4.2.[48] The X-ray crystallographic structures of HER2 and EGFR were
downloaded from the RCSB Protein Data Bank (PDB) ID: 1PP0[49] and
1M17.[50] The proteins were prepared separately by removing water and
co-crystallized ligands bound with the proteins to make receptor free of
any ligand before docking. Then, Polar hydrogens and Gastieger charges
were added using the MGL Tools and proteins saved in PDBQT format.
Ligand structures were created separately using ChemDraw Ultra 12.0,
energy minimized in Chem3D, torsional bonds of ligands were set flexible
3 and saved in PDBQT format. Next, the receptor was kept rigid, the grid

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
17
covering all the amino acid residues present inside the active site of pro-
teins was built for HER2 (grid box size of 50 Å ꢂ 50 Å ꢂ 50 Å with a spac-
ing of 0.375 Å between the grid points and centered at 17.882 (x), 15.918
(y), and 28.054 (z)) and for EGFR (grid box size of 72 Å ꢂ 44 Å ꢂ 60 Å
with a spacing of 0.375 Å between the grid points and centered at 24.21
(x), 0.49 (y), and 59.2 (z)). The best conformers were searched by the
Lamarckian genetic algorithm (LGA), the population size was set to 150
and maximum number of energy evaluation was set to 250,000,00. Finally,
the results were analyzed and visualized by the discovery studio.
ORCID
Hassan B. Lazrek
http://orcid.org/0000-0002-3849-9092
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