Design, synthesis and anticancer activity of new monastrol analogues bearing 1,3,4-oxadiazole moiety

Article abstract of DOI:10.1016/j.ejmech.2017.06.026

A series of dihydropyrimidine (DHPM) derivatives bearing 1,3,4-oxadiazole moiety was designed and synthesized as monastrol analogues. The new compounds were screened for their cytotoxic activity toward 60 cancer cell lines according to NCI (USA) protocol. Seven compounds were further examined against the most sensitive cell lines, leukemia HL-60(TB) and MOLT-4. The most active compounds were 9m against HL-60(TB) (IC₅₀ = 56 nM) and 9n against MOLT-4 (IC₅₀ = 80 nM), more potent than monastrol (IC₅₀ = 147 and 215 nM, respectively). Cell cycle analysis of HL-60(TB) cells treated with 9m and MOLT-4 cells treated with 9n showed cell cycle arrest at G2/M phase and pro-apoptotic activity as indicated by annexin V-FITC staining.

Full text of DOI:10.1016/j.ejmech.2017.06.026

Accepted Manuscript
Design, synthesis and anticancer activity of new monastrol analogues bearing 1,3,4-
oxadiazole moiety
Fatma A.F. Ragab, Sahar M. Abou-Seri, Salah A. Abdel-Aziz, Abdallah M. Alfayomy,
Mohamed aboelmagd
PII:
S0223-5234(17)30472-5
10.1016/j.ejmech.2017.06.026
EJMECH 9521
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 March 2017
Revised Date: 1 June 2017
Accepted Date: 14 June 2017
Please cite this article as: F.A.F. Ragab, S.M. Abou-Seri, S.A. Abdel-Aziz, A.M. Alfayomy, M.
aboelmagd, Design, synthesis and anticancer activity of new monastrol analogues bearing 1,3,4-
oxadiazole moiety, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.06.026.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Design, synthesis and anticancer activity of new monastrol analogues
bearing 1,3,4-oxadiazole moiety
1
1
2
Fatma A.F. Ragab , Sahar M. Abou-Seri , Salah A. Abdel-Aziz , Abdallah M.
2
,*
3,4
Alfayomy , Mohamed aboelmagd
A series of dihydropyrimidines (DHPMs) bearing 1,3,4-oxadiazole moiety were
designed and synthesized as monastrol analogues. Compound 9i, 9k, 9m and 9n
possessed significant activity against HL-60(TB) and MOLT-4 compared to monastrol.
R
O
O
N
O
N
S
Cl
H
N
N
IC50(µM)
HL-60(TB)
0.103
0.143
0.056
0.153
MOLT-4
0.623
0.086
1.788
0.080
0.215
9
9
9
9
i: R = 4-Cl
k: R = 4-Br
m: R = 3-Cl
n: R = 2,4-di Cl
monastrol
0.147

1
ACCEPTED MANUSCRIPT
Design, synthesis and anticancer activity of new monastrol analogues
bearing 1,3,4-oxadiazole moiety
1
1
2
Fatma A.F. Ragab , Sahar M. Abou-Seri , Salah A. Abdel-Aziz , Abdallah M.
2
,*
3,4
Alfayomy , Mohamed Aboelmagd
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University,
Kasr El-Aini Street, Cairo, P.O. Box, 11562, Egypt
2
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Al-Azhar
University, Assiut, 71524, Egypt
3
National Center for Natural Products Research, School of Pharmacy, The University
of Mississippi, University, MS 38677, USA.
4
Pharmacognosy Department, National Research Center, Dokki, Giza, 12622, Egypt
Abstract
A series of dihydropyrimidine (DHPM) derivatives bearing 1,3,4-oxadiazole moiety
was designed and synthesized as monastrol analogues. The new compounds were
screened for their cytotoxic activity toward 60 cancer cell lines according to NCI
(USA) protocol. Seven compounds were further examined against the most sensitive
cell lines, leukemia HL-60(TB) and MOLT-4. The most active compounds were 9m
against HL-60(TB) (IC = 56 nM) and 9n against MOLT-4 (IC = 80 nM), more
5
0
50
potent than monastrol (IC = 147 and 215 nM, respectively). Cell cycle analysis of
50
HL-60(TB) cells treated with 9m and MOLT-4 cells treated with 9n showed cell
cycle arrest at G2/M phase and pro-apoptotic activity as indicated by annexin V-FITC
staining.
Keywords: Dihydropyrimidine, 1,3,4-Oxadiazole, Anticancer activity, Cell cycle
arrest, Apoptosis
*
Corresponding author: Abdallah M. Alfayomy, E-mail: ph_alfayomy@yahoo.com ;
Tel. +201099688027; Fax: +20882181191
1
. Introduction
The interest in the anticancer properties of dihydropyrimidines (DHPMs) has
been increased since 1999, when monastrol (Fig 1) was discovered. Monastrol is a
small molecule that inhibits the motility of the mitotic kinesin Eg5 (motor protein
which is required for assembling and maintaining the separation of the half spindles)
so inhibits the mitotic machinery and arrests the cells at G2/M phase [1, 2]. When

2
ACCEPTED MANUSCRIPT
mitotic arrest is imposed for prolonged periods cells often undergo programmed cell
death, known as apoptosis [2]. Drugs that inhibit kinesins are being developed as
anticancer agents with the hope that they will inhibit proliferation of tumor cells
without having adverse effect on the nervous system. Eg5 inhibitors in general have
moderate toxic side effect on the neurons overtime than taxol, a drug commonly used
for cancer therapy [3, 4].
However, since the antimitotic activity of monastrol is not very high, attempts
to develop more potent and specific inhibitors of kinesin Eg5 based on monastrol
scaffold were done [5-8]. Several compounds were synthesized and evaluated for their
antitumor activity, among them some compounds showed potent activity such as
dimethylenastron [9], vasastrol VS-83 [10], fluorastrol [11], Mon-97 [12]. In addition
the Hybrid of DHPM with coumarin which exhibited selective antibreast cancer
activity [13] (Fig 1).
(Fig. 1)
Since combination of two or more active structural moieties can possibly
augment the bioactivity, so it was of interest to hybridize DHPM nucleus with 1,3,4-
oxadiazole fragment through a methylthio bridge hoping to develop potent anticancer
agents. The 1,3,4-oxadiazole moiety was chosen as compounds bearing 1,3,4-
oxadiazole as zibotentan and compounds I-III have been reported to exhibit a
significant anticancer activity [14-16] (Fig. 2), hence the incorporation of this moiety
in the designed compounds may give new effective antitumor agents.
(Fig. 2)
2
2
. Results and discussion
.1. Chemistry
The target hybrids have been synthesized by a convergent synthesis including
condensation of 6-methyl-4-aryl-1,2,3,4-tetrahydropyrimidine-2(1H)-thione
derivatives 8a-l and 2-(chloromethyl)-5-aryl-1,3,4-oxadiazole derivatives 4a-d
Scheme 1). The reaction occurred by the attack of the more nucleophilic sulfur atom
(
rather than the less nucleophilic nitrogen atom.
scheme 1
The synthetic route for 2-(chloromethyl)-5-aryl-1,3,4-oxadiazole derivatives
4
a-d was illustrated in Scheme 2. Substituted aromatic esters 2a-d were synthesized
by esterification of the corresponding acids 1a-d with ethanol in the presence of

3
ACCEPTED MANUSCRIPT
catalytic amount of sulfuric acid. The synthesized esters 2a-d were converted to the
corresponding acid hydrazides 3a-d by refluxing with hydrazine hydrate in ethanol
[
17-20]. Acid hydrazides 3a-d were cyclized to 2-(chloromethyl)-5-aryl-1,3,4-
oxadiazole derivatives 4a-d through reaction with monochloroacetic acid in
phosphorus oxychloride [21, 22].
scheme 2
On the other hand, DHPM derivatives 8a-l were conveniently prepared via a
widely used multicomponent reaction (MCR), of Biginelli type, it involved the
cyclocondensation of araldehydes 5, thiourea 6 and acetylacetone or ethyl
acetoacetate 7 in presence of concentrated hydrochloric acid to give the corresponding
6-methyl-4-aryl-1,2,3,4-tetrahydropyrimidine-2(1H)-thione derivatives 8a-l (Scheme
3) [23, 24]. Which were S-alkylated with the appropriate 2-(chloromethyl)-5-aryl-
1,3,4-oxadiazole derivatives 4a-d in the presence of triethyl amine and potassium
iodide to yield the target compounds 9a-r.
scheme 3
H-NMR spectra of compounds 9a-f, 9h-k, 9o, 9q and 9r showed an average
1
spectrum in which appeared three singlet signals at δ 10.6-10.9 ppm (NH proton D O
2
exchangeable), δ 6.0-6.4 ppm (C4-H) and δ 2.2-2.4 ppm for (C6-CH ). In addition,
3
aromatic protons appeared at δ 6.8-7.9 ppm. Also compounds 9a-f showed a singlet
signal at δ 2.2-2.3 ppm (COCH ) while, compounds 9h-k, 9o, 9q and 9r showed a
3
characteristic triplet-quartet pattern at δ 0.9-1.2 ppm (CH -CH ) and δ 3.9-4.2 ppm
2
3
(
CH -CH ). The geminal hydrogens of SCH appeared as doublets around 4.36 and
2 3 2
4
.45 ppm (J = 17.4 Hz). This was further confirmed by HSQC spectra of compound
(9k) which showed that these two hydrogens are attached to the carbon appeared at δ
2
8.90 ppm. This geminal coupling was attributed to the fact that the two hydrogens
are diastereomeric protons as a result of the presence of asymmetric center at C4. On
1
the other hand, HNMR spectra of compounds 9m and 9n revealed the presence of
two tautomeric species, 1,4- and 1,6-dihydropyrimidines (Fig. 3), which may be
attributed to the effect of the substituent at C4-phenyl ring on tautomerization [25-29].
1
Similarly HNMR spectra of compounds 9g, 9l and 9p in a mixture of DMSO-d and
6
trifluroacetic acid (TFA) revealed separate signals for each tautomeric form. This
effect may be due to hindering of the proton transfer process by the formation of H-
bonded complexes with the solvent [25, 26].

4
ACCEPTED MANUSCRIPT
(
Fig. 3)
CNMR spectra of compounds 9a-f revealed the presence of carbonyl carbon
ketone) at the range δ 195.53-196.32 ppm while, compounds 9h-k, 9o, 9q and 9r
1
3
(
13
showed carbonyl carbon (ester) at the range δ 165.02-174.80 ppm. CNMR spectra
of compound 9n in DMSO-d and compounds 9g, 9l, 9m, 9p and 9r in a mixture of
6
DMSO-d and trifluroacetic acid showed separate signals for each tautomeric form.
6
2
.2. Biological evaluation
2
.2.1. In vitro cytotoxic screening
The newly synthesized compounds (9a-r) were submitted to Developmental
Therapeutic Program-National Cancer Institute, Bethesda, USA (www.dtp.nci.nih.gov)
to be screened for their in vitro anticancer activity. Thirteen compounds were selected
to be evaluated at one dose (10 µM) primary anticancer assay towards a panel of
approximately 60 cancer cell lines. The human tumor cell lines were derived from nine
different cancer types: leukemia, melanoma, lung, colon, CNS, ovarian, renal, prostate
and breast cancers. Screening results for each compound were reported as the
percentage growth of the treated cells compared to the untreated control cells and
presented as growth inhibition percent (GI%) caused by the test compounds (Table 1).
Table 1
The obtained data revealed that, compounds 9i, 9k, 9m and 9n showed broad
spectrum cytotoxic activity with various degrees of growth inhibition against the
tested tumor cell lines. Compounds 9i, 9k and 9n showed potent activity against
leukemia cell lines HL-60(TB) and MOLT-4 with GI% ranges of 77.47-106.39 and
8
8.75-92.98, respectively. Compound 9m showed potent activity against leukemia
HL-60(TB) cell line and non small cell lung cancer NCI-H522 with GI% values 80.42
and 74.59 % respectively. Also compound 9n showed potent activity against leukemia
CCRF-CEM; non small cell lung cancer HOP-92 and colon cancer HCT-116 cell lines
with GI% range of 71.24-73.64. In addition, compounds 9i and 9k showed moderate
activity against leukemia cell lines CCRF-CEM, RPMI-8226, SR; colon cancer HCT-
1
16; melanoma SK-MEL-5; renal cancer UO-31; prostate cancer PC-3; breast cancer
MCF-7 and T-47D with GI% range of 47.61-64.22. Moreover compound 9m showed
moderate activity against colon cancer HCT-116 and breast cancer T-47D with GI%
values 56.51 and 69.24%, respectively. Compound 9n showed moderate activity
against leukemia SR; CNS cancer SF-295; ovarian cancer IGROV1; renal cancer

5
ACCEPTED MANUSCRIPT
RXF 393, UO-31; prostate cancer PC-3; breast cancer MCF-7, MDA-MB-231/ATCC
and T-47D with GI% range 52.64-68.03.
It is worth noting that, leukemia HL-60(TB) and MOLT-4 were the most
sensitive cancer cell lines to the effect of compounds 9i, 9k, 9m and 9n with GI%
ranges of 77.47-106.39 and 44.97-92.98, respectively (Fig. 4).
(Fig. 4)
2
.2.2. In vitro IC evaluation
50
Compounds 9i, 9k, 9l, 9m, 9n, 9p and 9r were further evaluated by
determining the median growth inhibitory concentration (IC ) against the most
5
0
sensitive cell lines, HL-60(TB) and MOLT-4, by the MTT assay using monastrol as a
positive control [30]. This permitted a good structure activity relationship study.
Table 2
The results in Table 2 revealed that, the monastrol analogues 9i, 9k, 9m and
9
n which demonstrated the best cytotoxic activity against both cell lines were
substituted with ethyl carboxylate function at C5 of DHPM nucleus. In addition, the
C4 substituent markedly affected the cytotoxic activity. The most potent derivative
against HL-60(TB) cell line was 9m (IC50 = 0.056 µM) bearing a 3-chlorophenyl
substituent, showed more inhibitory activity than its congeners 9i (IC = 0.103 µM),
5
0
9
k (IC = 0.143 µM) and 9n (IC = 0.153 µM) with 4-chlorophenyl, 4-bromophenyl
50 50
or 2,4-dichlorophenyl substituent, respectively. On the other hand, the most potent
derivatives against MOLT-4 were 9n (IC = 0.080 µM) and 9k (IC = 0.086 µM)
5
0
50
having a 2,4-dichlorophenyl or 4-bromophenyl substituent, respectively. While 9i
IC50 = 0.623 µM) and 9m (IC50 = 1.778 µM) with 4-chlorophenyl and 3-
chlorophenyl substituents showed good inhibitory activity, this activity was less than
n and 9k. Interestingly, compound 9l bearing 2-chlorophenyl substituent displayed
(
9
no activity against both HL-60(TB) and MOLT-4 (IC50 = 193 and 255 µM,
respectively). Furthermore, the cytotoxic activity was influenced by the C5 substituent
at 1,3,4-oxadiazole ring. It was observed that, all the active derivatives 9i, 9k, 9m and
9
n having 4-chlorophenyl substituent. Changing this substituent with phenyl (9r) or
-methylphenyl (9p) abolished the activity (IC of 9r and 9p = 74.4 and 220 µM
4
5
0
against HL-60(TB) and 344 and 538 µM against MOLT-4).
Finally, comparing the activity of 9i, 9k, 9m and 9n with the standard drug
monastrol against HL60(TB) cell line, compounds 9m and 9i were more potent than

6
ACCEPTED MANUSCRIPT
monastrol by about 2.5 and 1.5 fold (IC = 0.056, 0.103 and 0.147 µM, respectively),
5
0
while compounds 9k and 9n were almost equipotent to monastrol (IC = 0.143 and
5
0
0
.153 µM, respectively). Regarding MOLT-4 cell line, compounds 9k and 9n
possessed significant activity which was about 3 times more active than monastrol
IC = 0.086, 0.080 and 0.215 µM, respectively), while compounds 9i and 9m were
(
5
0
less active than monastrol (IC = 0.623 and 1.788 µM, respectively).
5
0
To study the effect of lipophilicity on the activity, the log P values were
calculated using molinspiration cheminformatics web services [31] and listed in Table
2
. It was found that, the active compounds 9i, 9k, 9m and 9n showed log P values
ranging from 5.51-6.14. The other three inactive compounds 9l, 9p and 9r showed
log P values ≤ 5.48, which suggest that log P is not a determining factor for cytotoxic
activity.
2
.2.3. Cell cycle analysis
The effect of compound 9m on HL-60(TB) cells, 9n on MOLT-4 cells and
monastrol on both cell lines have been studied by flow cytometric analysis. Treatment
of HL-60(TB) cells with twice IC concentration of 9m (0.112 µM) or of monastrol
5
0
(0.294 µM) for 24 h resulted in significant alterations in cell cycle phases (Fig. 5).
There was a significant increase in the percentage of apoptotic cells at the pre-G
phase, (24.72% on exposure to 9m and 6.89% for monastrol) compared to control
(1.43%). Concurrent reduction in the percentage of cells at G0/G1 phase to 32.88%
for 9m and 61.23% for monastrol compared to control (68.43%). Also, a significant
increase in the cells at G2/M phase to 14.81% in case of 9m and 9.98% for monastrol
was detected compared to control (7.29%). Similarly, exposure of MOLT-4 cells to
0
.160 µM of 9n (twice IC ) or 0.430 µM of monastrol (twice IC ) for 24 h increased
50 50
the population of cells at the pre-G phase from 0.88% to 17.83% and 8.63%
respectively, whereas G0/G1 cells significantly reduced from 74.22% to 38.52% in
case of 9n and 55.72% in case of monastrol as compared to control. Also, a
significant increase in the cells at G2/M phase to 26.61% with 9n and 13.92% with
monastrol compared to control (4.35%). No significant difference was found in the
percent of cells in the S phase, (Fig. 6).
Hence, it can be concluded that compounds 9m and 9n inhibit the cell
proliferation through cell cycle arrest at G2/M phase, which in turn induces cell death
by apoptosis. In agreement with the cytotoxicity assay results, apoptosis was more

7
ACCEPTED MANUSCRIPT
pronounced in HL-60(TB) and MOLT-4 cells treated with 9m or 9n than those treated
with monastrol.
(Fig. 5)
(Fig. 6)
2
.2.4. Annexin V-FITC apoptosis assay
Further evaluation of the pro-apoptotic effect of compounds 9m and 9n was
carried out using Annexin V-FITC and propidium iodide double staining. Flow
cytometric analysis of the differential binding of the cells to annexin V-FITC and
propidium iodide showed a remarkable effect in the proportion of apoptotic cells. HL-
6
0(TB) cells treated with 0.112 µM of 9m (twice IC ) or 0.294 µM of monastrol
50
(
twice IC ) showed an increase in the percent of annexin V-FITC positive apoptotic
50
cells (UR+LR) to 23.76% (19.3 folds) for 9m and 6.26% (5.1 folds) for monastrol
compared to control (1.23%) (Fig. 7). On the other hand MOLT-4 cells treated with
0
.160 µM of 9n (twice IC ) or 0.430 µM of monastrol (twice IC ) showed an
50 50
increase in the percent of annexin V-FITC positive apoptotic cells (UR+LR) to
6.37% (21 folds) for 9n and 7.60% (10 folds) for monastrol compared to control
0.77%) (Fig. 8). These results proved that the anti-proliferative effect of compounds
m and 9n is attributed to their potent pro-apoptotic activity and further supported our
1
(
9
finding that HL-60(TB) and MOLT-4 cells are more sensitive to the synthesized
compounds than to monastrol.
(
Fig. 7)
Fig. 8)
(
2
.2.4. Docking study
It is well known that the monastrol exerts its antitumor activity by inhibition
of mitotic kinesin Eg5. Cell cycle analysis of HL-60(TB) and MOLT-4 cells treated
with compounds 9m and 9n respectively revealed G2/M phase arrest followed by
apoptosis similar to monastrol. This observation suggests that kinesin Eg5 could be a
potential target for the synthesized monastrol analogues. Therefore a molecular
docking study of 9m and 9n into the crystal structure of Eg5 motor domain in
complex with the allosteric inhibitor monastrol (PDB ID code 1Q0B) [32] was
performed using Molecular Operating Environment (MOE version 2008.10) [33]. The
most stable docking model was selected according to the best scored conformation
predicted by the MOE scoring function. To validate the docking protocol, the co-
crystallized ligand was redocked into Eg5 allosteric binding site and the docking pose

8
ACCEPTED MANUSCRIPT
was compared with the initial pose using root mean square deviation (RMSD).
Monastrol docked almost at the same position (RMSD = 0.837Å) with docking
energy score of -11.74 kcal/mol.
Analysis of the molecular docking results showed that, compounds 9m and 9n
could fit into the allosteric binding site with docking energy scores of -14.73 and -
1
5.76 kcal/mol, respectively (Fig. 9). The DHPM ring occupied the same position as
monastrol. The DHPM N-1 showed the potential to be involved in a key H-bond
interaction with the main chain carbonyl of Glu116. In addition, the N-3 of DHPM
ring formed a water mediated H-bond with Leu214. The substituted chlorophenyl
moiety at position 4 of DHPM ring pointed toward the solvent region but maintained
hydrophobic interaction with the side chains of Ala218 and/or Tyr211. Unlike
monastrol, the 4-chlorophenyl-1,3,4-oxadiazole moiety projected into the core of the
protein, where the oxadiazole ring was involved in π...cation interaction with side
chain of Arg221, while the 4-chlorophenyl rest occupied a hydrophobic pocket
bordered by the side chains of Met115, Ile136, Ser237, Val238, Phe239, Leu263,
Val264 and Asp265. Moreover, halogen bonding between the 4-chlorine atom and the
ͦ
main chain carbonyl oxygen of Leu263 (2.82 Å) and Val264 (≤3.5 Å) could justify
the importance of 4-chlorophenyl substituent on the oxadiazole for cytotoxic activity
(Fig. 9)
3
. Conclusion
Certain new hybrids of DHPM and 1,3,4-oxadiazole have been synthesized as
monastrol analogues and investigated for their in vitro cytotoxic activity. Compounds
i, 9k, 9m and 9n featuring a p-chlorophenyl rest on the oxadiazole ring, ethyl
9
carboxylate function at C5 and m/p-chloro(bromo)phenyl substituent at C4 of DHPM
nucleus, showed the highest cytotoxic activity against leukemia HL-60(TB) and
MOLT-4 cell lines with IC ranges = 0.056-0.153 and 0.080-1.788 µm, respectively,
5
0
which exceed or equipotent to monastrol. Cell cycle analysis of HL-60(TB) cells
treated with 9m and MOLT-4 cells treated with 9n revealed a significant G2/M phase
arrest coupled with an increase in the percentage of cells in pre-G phase, which is
indicative of apoptosis. The pro-apoptotic activity of 9m and 9n was inferred by the
significant increase in the percentage of annexin V-FITC-positive apoptotic cells.

9
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4
4
. Experimental
.1. Chemistry
Unless otherwise noted, all materials were obtained from commercial suppliers and
used without further purification. Reactions were monitored by TLC (Kieselgel 60
F254 precoated plates, E. Merck, Germany), the spots were detected by exposure to
UV lamp at λ 254nm. Melting points were determined on an electro thermal melting
1
13
point apparatus (Stuart Scientific Co.) and were uncorrected. HNMR and CNMR
spectra were determined in DMSO-d and TFA used with some compounds for
6
solubility. NMR instruments are Bruker Avance DRX 500 MHz spectrometer; 3 mm
inverse, dual and HPLC flow probes with gradient variable temperature control (vtc),
Bruker Avance DRX 400 MHz spectrometer; 3 mm inverse probe, Department of
BioMolecular Sciences, the university of Mississippi, USA and BRUKER Avance III
4
00 MHz NMR spectrometer, Faculty of Pharmacy, Beni-Suif University, Egypt.
Chemical shifts δ are reported in parts per million (ppm) downfield from
tetramethylsilane (TMS) internal standard. Elemental microanalyses were performed
at the Regional Center for Mycology and Biotechnology, Al-Azhar University, Egypt.
Infrared Spectra were recorded on Shimadzu-FTIR spectrophotometer, Faculty of
-1
Pharmacy, Cairo University, Egypt and expressed in wave number (cm ), using
potassium bromide discs.
Preparation and analytical data of compounds 4a-d [16, 22] and 8a-l [8, 23, 34,
3
5] were as reported.
4
.1 Synthesis of compounds 9a-r
A mixture of 6-methyl-5-(ethoxy or methyl)carbonyl-4-(substituted)phenyl-2-
thioxo-1,2,3,4-tetrahydropyrimidine 8a-l (2 mmol), 2-(chloromethyl)-5-(substituted)
phenyl-1,3,4-oxadiazole 4a-d (2 mmol), potassium iodide (2 mmol), and triethyl
amine (2.5 mmole), was refluxed for 4h in absolute ethanol (20 ml). The reaction
mixture was poured onto crushed ice (40 g), acidified with acetic acid (2 ml). The
deposited precipitate was filtered, washed with cold water, dried, and crystallized
from methanol/DMF mixture.
4
.1.1. 1-(2-(((5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)methyl)thio)-6-methyl-4-
phenyl-1,4-dihydropyrimidin-5-yl)ethanone (9a).
-1
Yield: 76%; Melting point: 196-198 °C; IR (KBr) νmax/cm 3452, 3163, 169o, 1651.
1
H NMR (400 MHz, DMSO-d ) δ 10.83 (s, 1H, N-H), 7.86 (d, J = 8.4 Hz, 2H, Ar-H),
6

1
0
ACCEPTED MANUSCRIPT
7
6
.56 (d, J = 8.4 Hz, 2H, Ar-H), 7.42 (d, J = 7.4 Hz, 2H, Ar-H), 7.27 (m, 3H, Ar-H),
.26 (s, 1H C4-H), 4.45 (d, J = 17.5 Hz, 1H, S-CH ), 4.36 (d, J = 17.4 Hz, 1H, S-
2
1
3
CH ), 2.35 (s, 3H, C6-CH ), 2.25 (s, 3H, CO-CH ). C NMR (100 MHz, DMSO-d )
2
3
3
6
δ 195.92, 162.16, 161.91, 154.09, 152.01, 140.28, 136.41, 132.29, 130.04, 128.42,
28.19, 127.98, 115.57, 54.86, 48.64, 30.89, 28.26, 23.77. Anal. Calcd for
C H ClN O S (438.93):C, 60.20; H, 4.36; N, 12.76. Found: C, 60.47; H, 4.49; N,
1
22
19
4
2
1
3.02.
.1.2. 1-(2-(((5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)methyl)thio)-4-(4-
4
fluorophenyl)-6-methyl-1,4-dihydropyrimidin-5-yl)ethanone (9b).
-1
Yield: 72%; Melting point: 186-188 °C; IR (KBr) νmax/cm 3421, 3170, 1680, 1639.
1
H NMR (400 MHz, DMSO-d ) δ 10.86 (s, 1H, NH), 7.87 (d, J = 8.3 Hz, 2H, Ar-H),
6
7
.57 (d, J = 8.3 Hz, 2H, Ar-H), 7.48 (dd, J = 7.7, 5.8 Hz, 2H, Ar-H), 7.14 (t, J = 8.6
Hz, 2H, Ar-H), 6.24 (s, 1H, C4-H), 4.46 (d, J = 17.5 Hz, 1H, S-CH ), 4.37 (d, J = 17.4
2
1
3
Hz, 1H, S-CH ), 2.37 (s, 3H, C6-CH ), 2.26 (s, 3H, CO-CH ). C NMR (100 MHz,
2
3
3
DMSO-d +TFA) δ 195.53, 167.00, 163.93, 163.36, 161.48, 148.89, 142.11, 137.32,
6
1
1
34.45, 132.10, 131.80, 131.75, 130.20, 128.76, 115.75, 115.54, 56.05, 31.10, 30.83,
9.10. Anal. Calcd for C H ClFN O S (456.92):C, 57.83; H, 3.97; N, 12.26. Found:
2
2
18
4
2
C, 58.17; H, 4.48; N, 12.50.
4
.1.3. 1-(4-(4-chlorophenyl)-2-(((5-(4-chlorophenyl)-1,3,4-oxadiazol-2-
yl)methyl)thio)-6-methyl-1,4-dihydropyrimidin-5-yl)ethanone (9c).
-1
Yield: 83%; Melting point: 198-200 °C; IR (KBr) νmax/cm 3371, 3170, 1697, 1660,
1
1
651. H NMR (400 MHz, DMSO-d ) δ 10.85 (s, 1H, NH), 7.86 (d, J = 8.3 Hz, 2H,
6
Ar-H), 7.55 (d, J = 8.5 Hz, 2H, Ar-H), 7.44 (d, J = 8.3 Hz, 2H, Ar-H), 7.36 (d, J = 8.3
Hz, 2H, Ar-H), 6.22 (s, 1H, C4-H), 4.45 (d, J = 17.4 Hz, 1H, S-CH ), 4.36 (d, J = 17.4
2
1
3
Hz, 1H, S-CH ), 2.36 (s, 3H, C6-CH ), 2.26 (s, 3H, CO-CH ). C NMR (100 MHz,
2
3
3
DMSO-d ) δ 196.19, 162.71, 162.44, 154.16, 152.83, 139.58, 136.89, 133.28, 132.60,
6
1
30.46, 130.05, 128.88, 128.79, 115.68, 54.79, 31.44, 28.69, 24.33. Anal. Calcd for
C H Cl N O S (473.37):C, 55.82; H, 3.83; N, 11.84. Found: C, 56.13; H, 3.9o; N,
22
18
2
4
2
1
2.16.
.1.4. 1-(2-(((5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)methyl)thio)-4-(4-
4
methoxyphenyl)-6-methyl-1,4-dihydropyrimidin-5-yl)ethanone (9d).
-1
Yield: 85%; Melting point: 218-220 °C; IR (KBr) νmax/cm 3367, 3163, 1693, 1651.
1
H NMR (400 MHz, DMSO-d ) δ 10.83 (s, 1H, NH), 7.85 (d, J = 8.1 Hz, 2H, Ar-H),
6

1
1
ACCEPTED MANUSCRIPT
7
2
1
.57 (d, J = 8.3 Hz, 2H, Ar-H), 7.35 (d, J = 8.2 Hz, 2H, Ar-H), 6.86 (d, J = 8.4 Hz,
H, Ar-H), 6.20 (s, 1H, C4-H), 4.43 (d, J = 17.5 Hz, 1H, S-CH ), 4.33 (d, J = 17.4 Hz,
2
13
H, S-CH ), 3.70 (s, 3H, O-CH ), 2.34 (s, 3H, C6-CH ), 2.22 (s, 3H, CO-CH ). C
2
3
3
3
NMR (100 MHz, DMSO-d ) δ 196.32, 162.79, 162.09, 159.52, 154.39, 152.15,
6
1
36.85, 132.70, 132.63, 130.05, 129.97, 128.93, 115.88, 114.12, 55.51, 54.78, 31.14,
8.58, 24.09. Anal. Calcd for C H ClN O S (468.96):C, 58.91; H, 4.51; N, 11.95.
2
2
3
21
4
3
Found: C, 59.12; H, 4.39; N, 12.21.
4
.1.5. 1-(4-(4-fluorophenyl)-2-(((5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-
yl)methyl)thio)-6-methyl-1,4-dihydropyrimidin-5-yl)ethanone (9e).
-1
Yield: 65%; Melting point: 210-212 °C; IR (KBr) νmax/cm 3394, 5178, 1693,1658.
1
H NMR (400 MHz, DMSO-d ) δ 10.64 (s, 1H, NH), 7.82 (d, J = 8.7 Hz, 2H, Ar-H),
6
7
.47 (t, J = 8.9 Hz, 2H, Ar-H), 7.14 (t, J = 8.9 Hz, 2H, Ar-H), 7.03 (d, J = 8.4 Hz, 2H,
Ar-H), 6.24 (s, 1H, C4-H), 4.46 (d, J = 17.3 Hz, 1H, S-CH ), 4.36 (d, J = 17.4 Hz, 1H,
2
13
S-CH ), 3.82 (s, 3H, O-CH ), 2.37 (s, 3H, C6-CH ), 2.26 (s, 3H, CO-CH ). C NMR
2
3
3
3
(
100 MHz, DMSO-d ) δ 196.25, 163.42, 163.24, 162.38, 162.33, 160.99, 152.82,
6
1
3
1
36.99, 130.70, 130.67, 130.02, 125.94, 115.73, 115.68, 115.46, 114.04, 55.86, 54.58,
1.37, 28.60, 24.28. Anal. Calcd for C H FN O S (452.50):C, 61.05; H, 4.68; N,
23
21
4
3
2.38. Found: C, 60.89; H, 4.76; N, 12.70.
.1.6. 1-(4-(4-fluorophenyl)-6-methyl-2-(((5-(p-tolyl)-1,3,4-oxadiazol-2-
4
yl)methyl)thio)-1,4-dihydropyrimidin-5-yl)ethanone (9f).
-1
Yield: 70%; Melting point: 184-186 °C; IR (KBr) νmax/cm 3417, 3170, 169o, 1670,
1
1
643. H NMR (400 MHz, DMSO-d ) δ 10.71 (s, 1H, NH), 7.76 (d, J = 7.6 Hz, 2H,
6
Ar-H), 7.49 (t, 2H, J = 7.6 Hz, Ar-H), 7.30 (d, J = 8.0 Hz, 2H, Ar-H), 7.14 (t, J = 8.6
Hz, 2H, Ar-H), 6.26 (s, 1H, C4-H), 4.47 (d, J = 17.4 Hz, 1H, S-CH ), 4.36 (d, J = 17.4
2
1
3
Hz, 1H, S-CH ), 2.37 and 2.36 (2s, 6H, C6-CH , Ar-CH ), 2.26 (s, 3H, CO-CH ). C
2
3
3
3
NMR (100 MHz, DMSO-d ) δ 196.24, 163.63, 163.43, 162.37, 161.00, 153.67,
6
1
52.83, 142.05, 136.97, 131.03, 130.79, 130.71, 129.33, 128.14, 115.68, 115.47,
4.58, 31.38, 28.62, 24.30, 21.47. Anal. Calcd for C H FN O S (436.50):C, 63.29;
5
2
3
21
4
2
H, 4.85; N, 12.84. Found: C, 63.14; H, 4.99; N, 13.17.
4
.1.7. Ethyl 2-(((5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)methyl)thio)-6-methyl-4-
phenyl-1,4-dihydropyrimidine-5-carboxylate (9g).
-1
Yield: 83%; Melting point: 190-192 °C; IR (KBr) νmax/cm 3417, 3178,1720, 1697,
1
1
651. H NMR (400 MHz, DMSO-d +TFA) δ 10.21 and 9.11 (2s, 1H, NH,), 7.89-
6

1
2
ACCEPTED MANUSCRIPT
7
3
.20 (m, 9H, Ar-H), 6.26 and 5.59 (2s, 1H, C4-H), 5.06– 4.55 (m, 2H, S-CH ), 4.17-
2
.92 (m, 2H, CH -CH ), 2.43 and 2.23 (2s, 3H, C6-CH ), 1.19-1.04 (m, 3H, CH -
2
3
3
2
13
CH3). C NMR (100 MHz, DMSO-d6+TFA) δ 165.77, 165.60, 152.70, 148.53,
1
5
4
45.25, 138.35, 129.87, 129.20, 128.80, 128.67, 127.59, 126.67, 99.89, 61.10, 59.54,
4.49, 17.99, 14.11, 13.86. Anal. Calcd for C H ClN O S (468.96):C, 58.91; H,
2
3
21
4
3
.51; N, 11.95. Found: C, 59.20; H, 4.57; N, 12.23.
.1.8. Ethyl 2-(((5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)methyl)thio)-4-(4-
4
fluorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxylate (9h).
-1
Yield: 84%; Melting point: 210-212 °C; IR (KBr) νmax/cm 3394, 3178, 1708, 1693,
1
1
651. H NMR (400 MHz, DMSO-d ) δ 10.81 (s, 1H, NH), 7.85 (d, J = 8.4 Hz, 2H,
6
Ar-H), 7.57 (d, J = 8.4 Hz, 2H, Ar-H), 7.44 (dd, J = 8.0, 5.7 Hz, 2H, Ar-H), 7.14 (t, J
=
=
8.7 Hz, 2H, Ar-H), 6.12 (s, 1H, C4-H), 4.45 (d, J = 17.5 Hz, 1H, S-CH ), 4.35 (d, J
2
17.4 Hz, 1H, S-CH ), 4.02 (q, J = 7.1 Hz, 2H, CH -CH ), 2.34 (s, 3H, C6-CH ),
2
2
3
3
1
3
1
1
1
.11 (t, J = 7.1 Hz, 3H, CH -CH ). C NMR (100 MHz, DMSO-d ) δ 165.53, 162.59,
2 3 6
62.54, 154.21, 153.80, 137.34, 136.83, 132.65, 130.59, 130.51, 130.03, 128.89,
15.65, 115.44, 106.10, 60.27, 54.98, 28.58, 23.05, 14.44. Anal. Calcd for
C H ClFN O S (486.95):C, 56.73; H, 4.14; N, 11.51. Found: C, 57.01; H, 4.23; N,
23
20
4
3
1
1.84.
.1.9. Ethyl 4-(4-chlorophenyl)-2-(((5-(4-chlorophenyl)-1,3,4-oxadiazol-2-
4
yl)methyl)thio)-6-methyl-1,4-dihydropyrimidine-5-carboxylate (9i).
-1
Yield: 80%; Melting point: 204-206 °C; IR (KBr) νmax/cm 3402, 3174, 1708, 1693,
1
1
651. H NMR (400 MHz, DMSO-d ) δ 10.82 (s, 1H, NH), 7.85 (d, J = 8.3 Hz, 2H,
6
Ar-H), 7.57 (d, J = 8.4 Hz, 2H, Ar-H), 7.41 (dd, J = 8.8, 8.4 Hz, 4H, Ar-H), 6.10 (s,
H, C -H), 4.46 (d, J = 17.4 Hz, 1H, S-CH ), 4.36 (d, J = 17.4 Hz, 1H, S-CH ), 4.03
1
4
2
2
(
q, J = 5.2 Hz, 2H, CH -CH ), 2.34 (s, 3H, , C6-CH ), 1.12 (t, J = 7.1 Hz, 3H, CH -
2 3 3 2
13
CH ). C NMR (100 MHz, DMSO-d ) δ 165.02, 162.17, 153.72, 153.44, 139.52,
3
6
1
2
36.36, 132.78, 132.16, 129.82, 129.55, 128.41, 128.30, 105.37, 59.85, 54.69, 28.11,
2.66, 13.97. Anal. Calcd for C H Cl N O S (503.40):C, 54.88; H, 4.00; N, 11.13.
2
3
20
2
4
3
Found: C, 55.13; H, 3.94; N, 11.36.
4
.1.10. Ethyl 2-(((5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)methyl)thio)-4-(4-
methoxyphenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxylate (9j).
-1
Yield: 62%; Melting point: 208-210 °C; IR (KBr) νmax/cm 3394, 3174, 1715, 1693,
1
1
647. H NMR (400 MHz, DMSO-d ) δ 10.79 (s, 1H, NH), 7.85 (d, J = 8.0 Hz, 2H,
6

1
3
ACCEPTED MANUSCRIPT
Ar-H), 7.58 (d, J = 8.1 Hz, 2H, Ar-H), 7.32 (d, J = 8.0 Hz, 2H, Ar-H), 6.87 (d, J = 8.1
Hz, 2H, Ar-H), 6.09 (s, 1H, C4-H), 4.44 (d, J = 17.3 Hz, 1H, S-CH ), 4.35 (d, J = 17.2
2
Hz, 1H, S-CH ), 4.03 (q, 2H, J = 4 Hz CH -CH ), 3.71 (s, 3H, OCH ), 2.34 (s, 3H,
2
2
3
3
C6-CH ), 1.13 (t, J = 7.1 Hz, 3H, CH -CH ). Anal. Calcd for C H ClN O S
3
2
3
24 23
4
4
(498.98):C, 57.77; H, 4.65; N, 11.23. Found: C, 58.04; H, 4.79; N, 11.58.
4
.1.11. Ethyl 4-(4-bromophenyl)-2-(((5-(4-chlorophenyl)-1,3,4-oxadiazol-2-
yl)methyl)thio)-6-methyl-1,4-dihydropyrimidine-5-carboxylate (9k).
-1
Yield: 83%; Melting point: 190-192 °C; IR (KBr) νmax/cm 3402, 3170, 1712, 1693,
1
1
651. H NMR (400 MHz, DMSO-d ) δ 10.82 (s, 1H, NH), 7.84 (d, J = 8.3 Hz, 2H,
6
Ar-H), 7.58 (d, J = 8.3 Hz, 2H, Ar-H), 7.53 (d, J = 8.1 Hz, 2H, Ar-H), 7.35 (d, J = 8.1
Hz, 2H, Ar-H), 6.09 (s, 1H, C4-H), 4.45 (d, J = 17.4 Hz, 1H, S-CH ), 4.35 (d, J = 17.4
2
Hz, 1H, S-CH ), 4.04 (q, J = 3.6 Hz, 2H, CH -CH ), 2.34 (s, 3H, C6-CH ), 1.13 (t, J
2
2
3
3
1
3
=
7.1 Hz, 3H, CH -CH ). C NMR (100 MHz, DMSO-d ) δ 165.49, 162.69, 162.59,
2 3 6
1
1
54.17, 153.91, 140.40, 136.82, 132.63, 131.72, 130.60, 130.04, 128.90, 121.90,
05.77, 60.36, 55.22, 28.60, 23.10, 14.46. Anal. Calcd for C H BrClN O S
23
20
4
3
(547.85):C, 50.42; H, 3.68; N, 10.23. Found: C, 50.35; H, 3.81; N, 10.59.
4
.1.12. Ethyl 4-(2-chlorophenyl)-2-(((5-(4-chlorophenyl)-1,3,4-oxadiazol-2-
yl)methyl)thio)-6-methyl-1,4-dihydropyrimidine-5-carboxylate (9l).
-1
Yield: 85%; Melting point: 194-196 °C; IR (KBr) νmax/cm 3417, 3167,1716,
1
1
693,1658. H NMR (500 MHz, DMSO-d ) δ 10.73 and 9.82 (2s, 1H, NH), 7.82-7.06
6
(
m, 8H, Ar-H), 6.38, 5.84 and 5.76 (3s, 1H, C4-H), 4.55-4.27 (m, 2H, S-CH ), 4.09-
2
3
.85 (m, 2H, CH -CH ), 2.29 and 2.26 (2s, 3H, C6-CH ), 1.13-0.85 (m, 3H, CH -
2 3 3 2
13
CH ). C NMR (100 MHz, DMSO-d ) δ 174.80, 165.43, 160.54, 157.54, 141.42,
3
6
1
1
1
37.66, 137.50, 130.79, 130.48, 130.02, 129.56, 128.99, 128.56, 128.35, 127.96,
27.71, 121.93, 105.18, 78.30, 61.10, 59.53, 58.54, 57.57, 25.43, 16.87, 16.58, 13.46,
3.31. Anal. Calcd for C H Cl N O S (503.40):C, 54.88; H, 4.00; N, 11.13. Found:
2
3
20
2
4
3
C, 55.12; H, 3.87; N, 11.40.
4
.1.13. Ethyl 4-(3-chlorophenyl)-2-(((5-(4-chlorophenyl)-1,3,4-oxadiazol-2-
yl)methyl)thio)-6-methyl-1,4-dihydropyrimidine-5-carboxylate (9m).
-1
Yield: 88%; Melting point: 184-186 °C; IR (KBr) νmax/cm 3417, 3167, 1716, 1693,
1
1
8
2
654. H NMR (400 MHz, DMSO-d ) δ 10.72 and 9.82 (2s, 1H, NH), 7.82-7.12 (m,
6
H, Ar-H), 6.38 and 5.85 (2s, 1H, C4-H), 4.55-4.26 (m, 2H, S-CH ), 4.02-3.85 (m,
2
1
3
H, CH -CH ), 2.30 and 2.26 (2s, 3H, C6-CH ), 1.13-0.95 (m, 3H, CH -CH ). C
2
3
3
2
3

1
4
ACCEPTED MANUSCRIPT
NMR (125 MHz, DMSO-d +TFA) δ 165.61, 165.33, 164.26, 163.65, 162.21, 160.50,
6
1
52.14, 149.19, 141.76, 132.20, 129.99, 129.65, 129.13, 128.99, 128.29, 127.69,
22.05, 105.19, 98.52, 61.02, 59.36, 52.93, 52.02, 25.52, 17.62, 16.64, 13.57. Anal.
1
Calcd for C H Cl N O S (503.40):C, 54.88; H, 4.00; N, 11.13. Found: C, 55.08; H,
2
3
20
2
4
3
4
.04; N, 11.34.
.1.14. Ethyl 2-(((5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)methyl)thio)-4-(2,4-
4
dichlorophenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxylate (9n).
-1
Yield: 80%; Melting point: 148-150 °C; IR (KBr) νmax/cm 3394, 3178, 1689, 1658.
1
H NMR (400 MHz, DMSO-d ) δ 10.74 and 9.91 (2s, 1H NH), 7.83-7.11 (m, 7H, Ar-
6
H), 6.33 and 5.78 (2s, 1H, C4-H), 4.57-4.28 (m, 2H, S-CH ), 4.23-3.85 (m, 2H, CH -
2
2
13
CH ), 2.29 and 2.27 (2s, 3H, C6-CH ), 1.13-0.95 (m, 3H, CH -CH ). C NMR (100
3
3
2
3
MHz, DMSO-d ) δ 165.97, 165.42, 164.79, 163.15, 162.61, 162.06, 155.17, 153.49,
6
1
1
5
41.49, 137.35, 136.97, 136.78, 133.99, 133.78, 132.79, 132.59, 130.40, 130.00,
29.91, 129.56, 128.83, 128.30, 128.01, 127.58, 122.53, 104.61, 60.27, 59.54, 56.52,
4.96, 28.21, 23.78, 23.07, 17.82, 14.46, 14.36. Anal. Calcd for C H Cl N O S
2
3
19
3
4
3
(537.85):C, 51.36; H, 3.56; N, 10.42. Found: C, 51.80; H, 3.67; N, 10.75.
4
.1.15. Ethyl 4-(4-fluorophenyl)-2-(((5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-
yl)methyl)thio)-6-methyl-1,4-dihydropyrimidine-5-carboxylate (9o).
-1
Yield: 95%; Melting point: 220-222°C; IR (KBr) νmax/cm 3390, 3178, 1693, 1654.
1
H NMR (400 MHz, DMSO-d ) δ 10.60 (s, 1H, NH), 7.81 (d, J = 8.7 Hz, 2H, Ar-H),
6
7
.44 (t, J = 7.7 Hz, 2H, Ar-H), 7.15 (t, J = 7.7 Hz, 2H, Ar-H), 7.03 (d, J = 8.7 Hz, 2H,
Ar-H), 6.13 (s, 1H, C4-H), 4.45 (d, J = 17.4 Hz, 1H, S-CH ), 4.35 (d, J = 17.3 Hz, 1H,
2
S-CH ), 4.03 (q, J = 7.1 Hz, 2H, CH -CH ), 3.82 (s, 3H, OCH ), 2.34 (s, 3H, C6-
2
2
3
3
1
3
CH ), 1.11 (t, J = 7.1 Hz, 3H, CH -CH ). C NMR (125 MHz, DMSO-d ) δ 165.59,
3
2
3
6
1
1
63.23, 163.20, 162.65, 153.92, 153.58, 130.57, 130.50, 130.03, 125.90, 115.64,
15.47, 114.05, 105.95, 60.28, 55.87, 54.89, 28.56, 23.06, 14.45. Anal. Calcd for
C H FN O S (482.53):C, 59.74; H, 4.80; N, 11.61. Found: C, 60.02; H, 4.89; N,
24
23
4
4
1
1.87.
.1.16. Ethyl 4-(4-chlorophenyl)-6-methyl-2-(((5-(p-tolyl)-1,3,4-oxadiazol-2-
4
yl)methyl)thio)-1,4-dihydropyrimidine-5-carboxylate (9p).
-1
Yield: 79%; Melting point: 194-196 °C; IR (KBr) νmax/cm 3367, 3178, 1715,
1
1
693,1651. H NMR (400 MHz, DMSO-d +TFA) δ 8.25 (s, 1H, NH), 7.65-7.09 (m,
6
8
H, Ar-H), 6.27 and 5.54 (2s, 1H, C4-H), 4.68-4.44 (m, 2H, S-CH ), 3.93 (m, 2H,
2

1
5
ACCEPTED MANUSCRIPT
CH -CH ), 2.37 and 2.33 (2s, 3H, C6-CH ), 2.24 and 2.26 (2s, 3H, Ar-CH ), 1.01-
2
3
3
3
1
3
0
1
1
1
1
1
.97 (m, 3H, CH -CH ). C NMR (100 MHz, DMSO-d +TFA) δ 168.44, 166.68,
2 3 6
65.50, 163.96, 162.10, 153.12, 148.02, 143.75, 143.56, 143.08, 139.07, 134.19,
32.63, 129.84, 129.55, 129.29, 129.01, 128.94, 128.68, 128.46, 128.38, 127.78,
27.61, 126.60, 105.51, 100.03, 60.84, 59.62, 54.70, 54.16, 33.54, 20.61, 17.61,
6.62, 13.48, 13.25. Anal. Calcd for C H ClN O S (482.98):C, 59.68; H, 4.80; N,
2
4
23
4
3
1.60. Found: C, 59.91; H, 4.87; N, 11.89.
.1.17. Ethyl 4-(4-fluorophenyl)-6-methyl-2-(((5-(p-tolyl)-1,3,4-oxadiazol-2-
4
yl)methyl)thio)-1,4-dihydropyrimidine-5-carboxylate (9q).
-1
Yield: 74%; Melting point: 202-204 °C; IR (KBr) νmax/cm 3387, 3186, 1715, 1693,
1
1
651. H NMR (400 MHz, DMSO-d ) δ 10.66 (s, 1H, NH), 7.74 (d, J = 7.5 Hz, 2H,
6
Ar-H), 7.45 (dd, , J = 8, 4,9 Hz, 2H, Ar-H), 7.30 (d, J = 7.9 Hz, 2H, Ar-H), 7.15 (t, J
=
=
8.4 Hz, 2H, Ar-H), 6.14 (s, 1H, C4-H), 4.46 (d, J = 17.4 Hz, 1H, S-CH ), 4.35 (d, J
2
17.4 Hz, 1H, S-CH ), 4.03 (q, J = 6 Hz, 2H, CH -CH ), 2.36, 2.35 (2s, 6H, C6-CH ,
2
2
3
3
1
3
Ar-CH ), 1.12 (t, J = 7.1 Hz, 3H, CH CH ). C NMR (100 MHz, DMSO-d ) δ
3
2
3
6
1
1
1
6
65.57, 163.54, 162.59, 161.02, 153.87, 153.74, 142.01, 137.41, 131.03, 130.59,
30.52, 129.31, 128.12, 115.64, 115.42, 106.02, 60.27, 54.96, 28.53, 23.06, 21.46,
4.44. Anal. Calcd for C H FN O S (466.53):C, 61.79; H, 4.97; N, 12.01. Found: C,
2
4
23
4
3
2.04; H, 5.08; N, 12.34.
.1.18. Ethyl 4-(4-chlorophenyl)-6-methyl-2-(((5-phenyl-1,3,4-oxadiazol-2-
4
yl)methyl)thio)-1,4-dihydropyrimidine-5-carboxylate (9r).
-1
Yield: 89%; Melting point: 170-172 °C; IR (KBr) νmax/cm 3394, 3170, 1716,1693,
1
1
654. H NMR (400 MHz, DMSO-d ) δ 10.78 (s, 1H, NH), 7.85 (d, J = 7.2 Hz, 2H,
6
Ar-H), 7.55-7.38 (m, 7H, Ar-H), 6.15 (s, 1H, C4-H), 4.49 (d, J = 17.4 Hz, 1H, S-
CH ), 4.38 (d, J = 17.3 Hz, 1H, S-CH ), 4.04 (q, J = 6.3 Hz, 2H, CH -CH ), 2.37 (s,
2
2
2
3
13
3
H, C6-CH ), 1.12 (t, J = 7.1 Hz, 3H, CH -CH ). C NMR (100 MHz, DMSO-
3 2 3
d6+TFA) δ 167.62, 165.15, 164.55, 163.89, 163.57, 162.03, 160.34, 147.48, 143.43,
142.37, 138.91, 137.02, 134.36, 134.18, 133.34, 132.20, 132.02, 131.17, 129.34,
129.03, 128.85, 128.54, 128.41, 128.24, 126.64, 123.26, 106.69, 105.64, 61.03, 60.87,
56.64, 54.77, 30.99, 25.61, 17.39, 16.82, 13.64, 13.47. Anal. Calcd for
C H ClN O S (468.96):C, 58.91; H, 4.51; N, 11.95. Found: C, 59.18; H, 4.63; N,
23
21
4
3
1
2.32.

1
6
ACCEPTED MANUSCRIPT
4
4
.2. Biological evaluation
.2.1. Preliminary in vitro anticancer screening [36]
The human tumor cell lines of the cancer screening panel are grown in RPMI
640 medium containing 5% fetal bovine serum and 2 mM L-glutamine. For a typical
1
screening experiment, cells are inoculated into 96 well microtiter plates in 100 µl at
plating densities ranging from 5,000 to 40,000 cells/well depending on the doubling
time of individual cell lines. After cell inoculation, the microtiter plates are incubated
at 37° C, 5 % CO2, 95 % air and 100 % relative humidity for 24 h prior to addition of
experimental drugs. After 24 h, two plates of each cell line are fixed in situ with TCA,
to represent a measurement of the cell population for each cell line at the time of drug
addition. Experimental drugs are solubilized in dimethyl sulfoxide at 400-fold the
desired final maximum test concentration and stored frozen prior to use. At the time
of drug addition, an aliquot of frozen concentrate is thawed and diluted to twice the
desired final maximum test concentration with complete medium containing 50 µg/ml
gentamicin. Aliquots of 100 µl of these different drug dilutions are added to the
appropriate microtiter wells already containing 100 µl of medium, resulting in the
required final drug concentrations 10 µM. Controls, containing only phosphate buffer
saline and DMSO at identical dilutions, were prepared in the same manner. Following
drug addition, the plates are incubated for an additional 48 h at 37°C, 5 % CO2, 95 %
air, and 100 % relative humidity. For adherent cells, the assay is terminated by the
addition of cold TCA. Cells are fixed in situ by the gentle addition of 50 µl of cold 50
%
(w/v) TCA (final concentration, 10 % TCA) and incubated for 60 minutes at 4°C.
The supernatant is discarded, and the plates are washed five times with tap water and
air dried. Sulforhodamine B (SRB) solution (100 µl) at 0.4 % (w/v) in 1 % acetic acid
is added to each well, and plates are incubated for 10 minutes at room temperature.
After staining, unbound dye is removed by washing five times with 1 % acetic acid
and the plates are air dried. Bound stain is subsequently solubilized with 10 mM
trizma base, and the absorbance is read on an automated plate reader at a wavelength
of 515 nm. For suspension cells, the methodology is the same except that the assay is
terminated by fixing settled cells at the bottom of the wells by gently adding 50 µl of
8
0 % TCA (final concentration, 16 % TCA). Using the seven absorbance
measurements, the percentage growth of the treated cells is calculated compared to
the untreated control cells.

1
7
ACCEPTED MANUSCRIPT
4
.2.2. MTT assay for cell viability
2
+
2+
HL-60(TB) and MOLT-4 cells were trypsinized and washed with Ca /Mg
4
free PBS (pH 7.2). Cells were adjusted to 1.2-1.8 X 10 cells/ml with Dulbecco’s
Modified Eagle’s Medium (DMEM) (Invitrogen Life Technologies) supplemented
with 10% FBS (Hyclone, Logan, UT,USA), 10 µg/ml of insulin (Sigma), 1%
penicillin/streptomycin and plated (100µl/well) in 96-well cell culture plate overnight
at 37 °C with 5% CO and 95% humidity. One hundred micro liters of serial 10-fold
2
diluted sterile tested compounds were added to final concentrations of 0.01 - 100 µM.
Culture medium was used as negative control. Cultures were incubated for 24h.
Supernatants were discarded, 50µl/well of methylthiazolyldiphenyl-tetrazolium
bromide (MTT) solution (5mg/ml in PBS, Sigma) was added and incubated for 4 h at
3
7°C with 5% CO . Acid-isopropanol (100 µl of 0.04 N HCI in isopropanol) was
2
added to all wells and mixed thoroughly to dissolve the dark blue crystals. After a few
minutes at room temperature to ensure that all crystals were dissolved. The
absorbance were read on Robonik P2000 spectrophotometer at a wavelength of 490
nm and DMEM used as a blank control. Results from eight separate experiments were
recorded and the percentage of viable cells was calculated.
4
.2.3. DNA-flow cytometry analysis
6
HL-60(TB) cells at a density of 1 × 10 cells were exposed to 0.112 µM of
9
m, 0.294 µM of monastrol or to DMSO (0.002%), as a control for 24 h. Also,
6
MOLT-4 cells at a density of 1×10 cells were exposed to 0.160 µM of 9n, 0.430 µM
of monastrol or DMSO (0.002%), as a control for 24 h. The cells were detached by
trypsinization, washed in ice-cold PBS, fixed in ice cold 70% ethanol. Keep the cells
in ethanol for at least 2h at 4°C then harvested by centrifugation. Subsequently, cells
were washed in PBS and then stained using Cycle TEST™ plus DNA Reagent Kit
(ab139418_Propidium Iodide Flow Cytometry Kit ) (Becton Dickinson Biosciences,
San Jose, CA, USA). Cell cycle distribution was determined using a FACSCalibur
flow cytometer (Becton Dickinson Biosciences, San Jose, CA, USA) and analyzed
using Cell Quest software (Becton Dickinson).
4
.2.4. Annexin V-FITC apoptosis assay
Apoptotic cells were analyzed by the Annexin V-FITC Apoptosis Detection
Kit (BD Biosciences, USA). HL-60(TB) cells were cultured and incubated for 24 h in
the presence of 0.112 µM of 9m, 0.294 µM of monastrol or DMSO (0.002%), as a

1
8
ACCEPTED MANUSCRIPT
control. Also, MOLT-4 cells were cultured in the same manner in the presence of
.160 µM of 9n, 0.430 µM of monastrol or DMSO (0.002%), as a control and
0
incubated for 24 h. Cells were collected, washed twice with PBS, and centrifuged.
6
Thereafter, 1 × 10 cells/ml were stained with a combination of fluorescein
isothiocyanate (FITC), annexin V and propidium iodide (PI) and The reaction was
allowed to proceed in the dark for 30 min at room temperature then analyzed by flow
cytometry on FACSCalibur (BD Biosciences, San Jose, CA, USA) without gating
restrictions using 10,000 cells. Quadrant analysis of co-ordinate dot plots was
performed with Cell Quest software. Unstained cells were used to adjust the
photomultiplier voltage and for compensation setting adjustment in order to eliminate
spectral overlap between the FL1 and FL2 signals.
4
.2.5. Docking study
A docking study was carried out using Molecular Operating Environment
MOE version 2008.10) [33]. For this purpose, the crystal structure of kinesin Eg5
(
with the allosteric inhibitor monastrol (PDB ID code 1Q0B) [32] was obtained from
the Protein Data Bank in order to prepare the protein for docking studies. The docking
procedure was followed using the standard protocol implemented in MOE 2008.10
and the geometry of the resulting complexes was studied using MOE's Pose Viewer
utility. The enzyme was prepared for docking as follows: 1) the co-crystallized ligand
and water molecules were removed. 2) The enzyme was 3D protonated, where
hydrogen atoms were added at their standard geometry, the partial charges were
computed and the system was optimized. Flexible ligand–rigid receptor docking of
the most stable conformers was done with MOE-DOCK using pharmacophore as the
placement method and London dG as the scoring function. The obtained poses were
subjected to forcefield refinement using the same scoring function. Thirty of the most
stable docking models for each ligand were retained with the best scored
conformation.
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ACCEPTED MANUSCRIPT
Table 1: Percentage growth inhibition (GI %) of in vitro subpanel tumor cell lines at 10 µM concentration of the
selected compounds
Subpanel
cancer cell
Lines
a
% Growth Inhibition (GI %)
9h 9i 9j
20.86 15.72 11.86 12.41 16.90 60.26 12.91 56.59 38.24 72.75 15.35 14.14
9
b
9c
9e
9f
9g
9k
9m
9n
9o
9q
Leukemia
CCRF-CEM
-
HL-60(TB) 11.46 40.27 15.19 19.19 37.98 23.54 78.56
-
-
-
77.47 80.42 106.39 11.98 25.23
K-562
MOLT-4
-
-
11.06
24.38 10.94
-
-
-
25.18 14.37 44.25
31.29 20.43 92.98
39.28 27.47
91.71 44.97 88.75
nt
nt
-
-
-
14.71
21.58
27.18
RPMI-8226 13.78 32.90 14.04 16.28 27.31 25.84 64.22 18.28 61.64 46.38
SR
Non-small cell lung cancer
19.06 37.79 13.33 19.06 24.95 16.66 57.11 18.07 61.18 16.40 62.06 15.94 26.48
A549/ATCC
EKVX
HOP-62
-
-
-
-
21.43 15.59
-
-
-
-
28.98 11.65 31.03 11.47 37.63 29.49 46.55 12.73 17.30
-
-
14.78
27.83
-
-
19.85
-
-
-
28.70
-
22.77
-
-
-
11.59 19.35
26.49 16.54
13.11 25.10 17.48 12.02
HOP-92
40.14 27.82 35.92 17.56 42.20 48.97 73.64 21.41 37.02
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
11.55 11.45
-
-
-
-
10.38 11.43 10.62 30.13 11.80 21.10
-
-
-
-
20.97 10.43 10.11
-
-
-
-
11.96
-
-
-
-
-
-
-
-
-
-
21.21
17.66
21.05
-
-
-
-
24.28
13.56
19.58
33.95
21.12
40.76
-
-
-
-
10.32
-
15.59 20.91 17.98 14.06 54.12 17.47 31.06
32.44 74.59 49.82 26.46 22.44
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
10.43
-
36.75 21.67 53.81
21.77 15.78 32.40
22.34 10.31 24.77
-
-
20.44
20.09
-
-
-
-
-
-
-
22.39 29.56
nt
-
-
-
-
-
-
-
-
-
10.31 15.54
-
13.10 25.75
51.36 56.51 71.24
34.91 29.54 44.67
21.28 46.44 46.90
23.97 15.98 36.59
18.61
11.34
12.44
-
-
-
-
-
11.02
HT29
KM12
SW-620
-
-
-
10.32
-
-
-
30.34
14.08
-
-
33.77
-
CNS cancer
SF-268
SF-295
SF-539
SNB-19
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
23.41
-
-
-
-
14.02
25.89
12.27
-
-
-
13.34
26.44 25.46 52.77
11.33 13.64
-
26.67
-
-
-
-
12.12
-
-
16.63 13.41
11.69 11.64 27.01 12.46 28.03 10.36 27.06 11.42 10.14
26.28 18.85 32.31 16.82 15.90 17.53 13.80
SNB-75
U251
16.81 28.09 17.32
-
-
-
14.75 15.34
21.19 10.10 26.13 10.11 34.26
-
34.72
-
15.05
Melanoma
LOX IMVI
MALME-
-
-
-
-
-
-
-
-
-
-
-
-
11.11
-
-
-
-
-
-
23.14
18.32
30.70
30.41
-
-
-
-
-
-
25.05 17.70 41.74
16.93 27.21
26.03 26.23 34.81
-
-
-
-
-
-
-
-
-
-
11.59 12.35
-
3
M
M14
MDA-MB-
-
-
-
13.44
19.50
-
-
-
34.94
-
-
30.97
4
35
SK-MEL-2
34.49
39.92 39.96

SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
-
-
-
-
-
-
-
-
-
L
-
-
20.17
-
18.71
-
-
-
-
-
-
ACCEPTED MANUSCRIPT
-
-
-
12.47 59.11
13.06
-
-
48.61
19.72
14.52
-
25.90
-
27.00 22.19 31.92 11.82
22.65 27.72 10.80 15.37 34.86 10.72 50.13 15.28 44.70 26.55 48.43
-
17.05
Ovarian cancer
IGROV1
15.40 23.39
-
-
10.96
-
-
-
-
17.14 37.71 17.80 34.55 14.57 56.12
-
-
-
-
-
13.94
OVCAR-3
OVCAR-4
OVCAR-5
-
-
-
-
-
-
-
-
-
29.21
29.97
10.36
-
-
-
-
22.52 17.27 37.17
-
-
-
12.15
-
10.44
-
30.44 16.90 47.27
-
-
-
-
-
10.46 10.99
OVCAR-8
NCI/ADR-
RES
14.50
20.40 11.68 25.22
27.29 33.66 45.78
17.83
-
-
-
-
-
-
-
-
-
-
30.11
17.40
-
-
32.47 21.28 45.36
-
-
-
SK-OV-3
18.32
25.65
17.97 29.89
-
14.16
Renal cancer
7
86-0
16.45 12.60
-
-
-
15.43 31.37 10.65 16.59 18.77 15.12 43.05 34.81
-
-
-
20.25
-
18.97
A498
-
-
-
-
-
-
-
12.30 19.26
35.91
-
-
26.84
30.28
-
-
22.28
42.28
nt
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
13.52
-
16.90 21.07 29.93 11.99 22.29 17.50 38.39 17.80 32.79 35.15
24.85 24.69
-
-
-
-
-
-
-
-
-
-
-
32.30
-
-
-
-
25.02
-
20.09 30.29 20.65
-
-
56.62 14.49
34.63
-
12.96
14.75
-
-
19.87
28.35
-
14.57 13.06
14.37 30.79 13.90 22.48
26.15 43.64 29.70 16.36 19.39 22.21 51.33 20.31 47.61 35.91 62.80 21.73
Prostate cancer
PC-3
18.54 31.07 18.71 14.65 42.25 23.42 51.24 17.95 51.21 39.17 54.90 12.47 26.79
DU-145
-
-
-
-
-
-
12.85
-
15.73 13.49 26.88
-
-
Breast cancer
MCF7
MDA-MB-
10.61 16.39 10.52
7.04 27.00 14.36
-
-
-
18.23 13.30 51.01
-
55.17 31.98 68.03
-
-
14.04
14.66
1
-
-
-
37.29 10.22 40.23 18.93 58.31
16.33 12.87 32.90
24.70 16.90 20.24 46.85 10.30
2
31/ATCC
HS 578T
BT-549
-
-
-
-
-
-
-
-
-
-
-
-
15.07
13.94 42.66
12.76
T-47D
24.09 39.49 28.87 25.59 53.79 27.10 50.15 15.16 52.02 69.24 52.64 33.30 32.56
MDA-MB-
1
1.27 10.76 14.67 17.07 51.05 11.59 18.07
-
-
-
41.80
-
-
4
68
a
-, GI <10%; nt, not tested; L, compound proved lethal to the cancer cell line.

ACCEPTED MANUSCRIPT
Table 2: IC againest HL-60(TB) and MOLT-4 cancer cell lines
5
0
9
i-r
a
IC µm ± SE
1
2
50
Compound
R
R
R
log P
HL-60(TB)
MOLT-4
9
9
9
9
9
9
9
i
Cl
Cl
Cl
Cl
Cl
CH
OC
OC
OC
OC
OC
OC
2
2
2
2
2
2
2
H
5
H
5
H
5
H
5
H
5
H
5
H
5
4-Cl
0.103 ± 0.017
0.143 ± 0.015
193 ± 5.39
0.623 ± 0.010
0.086 ± 0.007
255 ± 6.01
5.53
5.66
5.48
5.51
6.14
5.30
k
l
4-Br
2-Cl
m
n
p
r
3-Cl
0.056 ± 0.007
0.153 ± 0.021
220 ± 6.53
1.788 ± 0.009
0.080 ± 0.017
538 ± 7,46
2,4-diCl
4-Cl
3
H
_
OC
_
4-Cl
_
74.4 ± 4.17
344 ± 8.09
4.85
_
monastrol
0.147 ± 0.008
0.215 ± 0.013
a
IC values are the mean ± S.E. of eight separate experiments
5
0

ACCEPTED MANUSCRIPT
Figure 1: Examples of some DHPM-based anticancer agents

ACCEPTED MANUSCRIPT
Figure 2: Examples of some 1,3,4-oxadiazole-based anticancer agents

ACCEPTED MANUSCRIPT
Figure 3: Possible tautomerism of dihydropyrimidine derivatives.

ACCEPTED MANUSCRIPT
Figure 4: Inhibitory effects of the tested compounds at 10 µM concentration against Leukemia HL-
6
0(TB) and MOLT-4 cell lines.