1,3,4-oxadiazole/chalcone hybrids: Design, synthesis, and inhibition of leukemia cell growth and EGFR, Src, IL-6 and STAT3 activities

Article abstract of DOI:10.1016/j.bioorg.2018.11.032

A new series of 1,3,4-oxadiazole/chalcone hybrids was designed, synthesized, identified with different spectroscopic techniques and biologically evaluated as inhibitors of EGFR, Src, and IL-6. The synthesized compounds showed promising anticancer activity, particularly against leukemia, with 8v being the most potent. The synthesized compounds exhibited strong to moderate cytotoxic activities against K-562, KG-1a, and Jurkat leukemia cell lines in MTT assays. Compound 8v showed the strongest cytotoxic activity with IC₅₀ of 1.95 μM, 2.36 μM and 3.45 μM against K-562, Jurkat and KG-1a leukemia cell lines, respectively. Moreover; the synthesized compounds inhibited EGFR, Src, and IL-6. Compound 8v was most effective at inhibiting EGFR (IC₅₀ = 0.24 μM), Src (IC₅₀ = 0.96 μM), and IL-6 (% of control = 20%). Additionally, most of the compounds decreased STAT3 activation.

Full text of DOI:10.1016/j.bioorg.2018.11.032

Accepted Manuscript
1,3,4-Oxadiazole/chalcone hybrids: Design, synthesis, and inhibition of leuke-
mia cell growth and EGFR, Src, IL-6 and STAT3 activities
Marwa Ali A. Fathi, Amer Ali Abd El-Hafeez, Dalia Abdelhamid, Samar H.
Abbas, Monica M. Montano, Mohamed Abdel-Aziz
PII:
S0045-2068(18)30757-0
DOI:
https://doi.org/10.1016/j.bioorg.2018.11.032
YBIOO 2640
Reference:
Bioorganic Chemistry
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24 July 2018
19 November 2018
20 November 2018
Please cite this article as: M. Ali A. Fathi, A. Ali Abd El-Hafeez, D. Abdelhamid, S.H. Abbas, M.M. Montano, M.
Abdel-Aziz, 1,3,4-Oxadiazole/chalcone hybrids: Design, synthesis, and inhibition of leukemia cell growth and
EGFR, Src, IL-6 and STAT3 activities, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.
2018.11.032
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1,3,4-Oxadiazole/chalcone hybrids: Design, synthesis, and inhibition
of leukemia cell growth and EGFR, Src, IL-6 and STAT3 activities
Marwa Ali A. Fathia,¶, Amer Ali Abd El-Hafeezb,c,d,¶,*, Dalia Abdelhamida, Samar H.
Abbasa, *, Monica M. Montano d, Mohamed Abdel-Aziza
aMedicinal Chemistry Department, Faculty of Pharmacy, Minia University, Minia
61519, Egypt.
bPharmacology and Experimental Oncology Unit, Cancer Biology Department,
National Cancer Institute, Cairo University, Cairo, 11796, Egypt.
cPharmacotherapy Department, Graduate School of Biomedical and Health Sciences,
Hiroshima University, Hiroshima 734-8553, Japan,
dPharmacology Department, Case Western Reserve University School of Medicine,
10900 Euclid Avenue, Cleveland, Ohio 44106, USA.
* Corresponding Author
Samar H. Abbas; Phone: (+20) 100-542-4005. Fax: (+20) 086-236-9075. E-mail:
samar_hafez@mu.edu.eg, drsamar77@hotmail.com.
* Corresponding author
Amer Ali Abd El-Hafeez; Phone: (+81)-70-7566-3891, Fax: (+81)-82-424-7000, E-
mail: amer.ali@nci.cu.edu.eg
¶ Both authors contributed equally to this work.
1

Abstract
A new series of 1,3,4-oxadiazole/chalcone hybrids was designed, synthesized,
identified with different spectroscopic techniques and biologically evaluated as
inhibitors of EGFR, Src, and IL-6. The synthesized compounds showed promising
anticancer activity, particularly against leukemia, with 8v being the most potent. The
synthesized compounds exhibited strong to moderate cytotoxic activities against K-
562, KG-1a, and Jurkat leukemia cell lines in MTT assays. Compound 8v showed the
strongest cytotoxic activity with IC50 of 1.95 µM, 2.36 µM and 3.45 µM against K-
562, Jurkat and KG-1a leukemia cell lines, respectively. Moreover; the synthesized
compounds inhibited EGFR, Src, and IL-6. Compound 8v was most effective at
inhibiting EGFR (IC50= 0.24 μM), Src (IC50= 0.96 μM), and IL-6 (% of control=
20%). Additionally, most of the compounds decreased STAT3 activation.
Key words: Leukemia; oxadiazole; chalcone, tyrosine kinases; cytokines; STAT3
1. Introduction
Leukemia is a type of cancer that starts in the blood-forming cells of the bone marrow
[1]. Chemotherapeutic agents are found to be effective in killing leukemic cells,
however, the 5-year overall survival is very poor [2]. Most patients relapse by tumor
regrowth are initiated by chemoresistant leukemia cells. Potential bases for
therapeutic resistance in leukemia treatment have been reported [3-5]. The signal
transducers and activators of transcription 3 (STAT3) is a critical signaling
intermediate in leukemia cells. STAT3 can be activated via phosphorylation by
tyrosine kinases such as EGFR and Src and cytokines such as IL-6 [6]. The activation
of STAT3 in leukemia is associated with poor prognosis [7]. Furthermore,
development of chemo-resistant leukemic cells has been attributed to activation of the
STAT3 pathway [8-11]. Consequently, significant effort has been committed towards
the development of drugs that target STAT3 activators, and includes gefitinib (EGFR
inhibitor) [12], dasatinib (Src inhibitor) [13] and tocilizumab (IL-6 inhibitor) [14, 15].
Unfortunately, the targeting of STAT3 activators such as EGFR, Src or IL-6
individually resulted only in moderate clinical efficacy [16-18]. Moreover, resistance
develops due to the cross-talk between the pathways that activates STAT3, thus
bypassing the inhibition of any one of the activators [8, 19-21]. Recently, efforts have
shifted toward the generation of drug candidates that can target two STAT3
activators. For example, Lin et al [22] synthesized oxazolo[4,5-g]quinazolin-2(1H)-
2

ones derivatives as dual EGFR and Src inhibitors. Additionally, a series of azaacridine
derivatives were synthesized as dual EGFR and Src inhibitors [23]. These dual EGFR
and Src inhibitors have the potential to overcome the resistance of leukemia cells to
STAT3 inhibition. However, STAT3 activation could occur via pathways other than
EGFR and Src, such as IL-6 [24-26]. Dual EGFR and Src inhibition may not be
sufficient for sustained inhibition of STAT3 activation. Therefore, the development of
drugs that target EGFR, Src, and IL-6 may offer a therapeutic advantage.
Oxadiazole derivatives are typical heterocyclic compounds that have exhibited
potential as anticancer therapeutics [27-32]. The anti-cancer activities of 1,3,4-
oxadiazole involve different mechanisms, and included inhibition of matrix
metalloproteinase-9 [33], tubulin polymerization [34], growth factors [30], NF-κB
signaling [35], Bcl-2 [36], cell cycle progression at G2/M phase [37], thymidine
phosphorylase [38, 39], and STAT3 [40-44]. The potential antitumor effects of
oxadiazole derivatives occur mainly via the inhibition of STAT3 pathways directly
[40] or indirectly [41-44]. A library of 1,3,4-oxadiazole-2-carboxamide compounds
was registered in a U.S. patent with the general formula I (Fig. 1). These compounds
inhibited STAT3 at a concentration of 100 μM, as well as inhibited growth of several
cancer cell lines, including MDA-MB-468 and human lymphoma cells (SCC-3) [45].
The 2, 4'-bis mercapto-1,3,4-oxadiazole diphenylamine derivative IIa-c (Fig. 1)
showed more potent anticancer activity with IC50 less than or equal to 2µM and
inhibited EGFR tyrosine kinase activity [41]. The 1,3,4-oxadiazole linked
benzimidazole derivative III a-b inhibited the EGFR receptor at 0.081 and 0.098 µM,
and were more cytotoxic than 5-fluorouracil [42]. The 1,3,4-oxadiazole-2-thione
derivative IV was the most potent Src kinase inhibitor among 384 tested compounds,
with IC50 equal to 1.9 µM [44]. The 1,3,4-oxadiazole derivative V was also effective
in inhibiting the release of IL-1, IL-6 and IL-10 in ConA-stimulated mouse lymph
node cells [43].
3

Fig. 1. Structures of Compounds I-V.
On the other hand, chalcones are precursors of flavonoids and isoflavonoids that
exhibited anti-cancer activities, which may be attributed to the inhibition of different
molecular targets [46] including EGFR [47, 48], Src family protein [49], IL-6 [50],
and others [51]. For example, caspase-3 levels were significantly increased while
STAT3 levels were decreased in the leukemia HL60 cell line upon treatment with
chalcones VIa-b (Fig. 2) [52]. Additionally, Butein VII (Fig. 2) exerted anticancer
effects in human hepatocellular carcinoma through suppression of constitutive and IL-
6-induced STAT3 activation, as well as the inhibition of c-Src, JAK1, and JAK2
activation [53]. Moreover, Cardomin VIII (Fig. 2) exhibited significant anticancer
effects in prostate cancer by suppression of STAT3 phosphorylation, nuclear
translocation, DNA binding ability, and inhibition of STAT3 dimerization.
Computational modeling indicated that Cardomin VIII can bind to the Src Homology
2 domain [54]. Several chalcones were derivatized with different bioactive
heterocyclic moieties with the goal of generating anticancer agents with increased
selectivity, ability to overcome drug resistance, and decreased side effects. 1,2,4-
4

Triazole/chalcone hybrid IX (Fig. 2) induced caspase-3 dependent apoptosis in A549
cells through both extrinsic and intrinsic pathways [51]. Additionally, pyrazolo[1,5-
a]pyrimidine/chalcone hybrid X showed (Fig. 2) promising anti-proliferative activity
and downregulated EGFR, p-EGFR, STAT3, and p-STAT3 in MDA-MB-231 cells
[55]. Moreover; benzo[c]furan-chalcone derivative XI (Fig. 2) exhibited significant
cytotoxicity in MCF-7 cell lines via inhibition of tubulin polymerization (IC50 = 5.51
× 10-5 μM) and EGFR-TK phosphorylation (IC50 = 0.09 μM) [56]. The
triazoloquinoxaline-chalcone hybrids XIIa-b also inhibited EGFR-TK in the
submicromolar range (0.039 to 0.083 µM) [57]. Benzimidazole/ chalcone hybrids
XIIIa-b (Fig. 2) are EGFR antagonist that exhibited promising cytotoxicity against
the HCT116 and H460 cell lines with IC50 ranging from 6.83 -12.52 µM [58].
Fig. 2. Structures of compounds VI-XIII.
Based on the studies discussed above, the present study involved the design and
synthesis of a series of 1,3,4-oxadiazole/chalcone hybrids (Fig. 3) with the combined
5

abilities to inhibit EGFR, Src, and IL-6. The hybrids were designed such that the
oxadiazole or chalcone moieties possess a methoxy group (s) or chlorine as electron
donating or as electron withdrawing substituents; respectively. The synthesized
compounds were evaluated for their cytotoxic activities against different cancer cell
lines, including three different leukemia cell lines (K-562, KG-1a, and Jurkat).
Moreover, their inhibitory activities against EGFR, Src, IL-6, and STAT3 were
evaluated.
Fig. 3. Pharmacophore of the target compounds.
2. Results and discussion
2.1. Chemistry
The target compounds and intermediates were prepared as outlined in Scheme 1.
Alkylation of oxadiazole derivatives 4a-e with acetylated chalcone derivatives 7a–e
was achieved in acetonitrile in the presence of TEA to generate the target compounds
1
8a-x in a good yield ranging from 51 % to 89% [59]. H NMR and 13C NMR
1
confirmed the formation of target compounds. H NMR spectra of compounds 8a-x
showed a singlet signal at δ: 4.09-4.80 ppm related to (S-CH2-CO). Furthermore, the
two chalcone protons appear at the aromatic region as doublet signals at δ: 7.81-7.98
ppm and 7.39-7.78 ppm with coupling constant J = 15.00-16.00 Hz. The amide proton
NH appears as singlet signals at δ 9.74-11.25 ppm. The 13C NMR spectra of
compounds 8a-x revealed the presence of two carbonyl groups appearing at δ: 187.89-
189.05 ppm and δ: 166.02-169.22 ppm related to C=O of chalcone and N-C=O, and a
characteristic signal of SCH2 appears at 36.31-37.43 ppm.
6

Compd.
8a
R
H
H
H
H
R1
H
4-Cl
Compd.
8m
8n
R
R1
3,4-di-OCH3
3,4,5-tri-OCH3
H
4-OCH3
4-OCH3
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
3,4-di-OCH3
3,4,5-tri-OCH3
H
3,4-di-OCH3
3,4-di-OCH3
3,4-di-OCH3
3,4-di-OCH3
3,4-di-OCH3
3,4,5-tri-OCH3
3,4,5-tri-OCH3
3,4,5-tri-OCH3
3,4,5-tri-OCH3
8o
8p
8q
8r
8s
8t
8u
8v
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-OCH3
4-OCH3
4-OCH3
4-OCH3
3,4-di-OCH3
3,4,5-tri-OCH3
H
4-Cl
4-OCH3
3,4-di-OCH3
4-Cl
4-OCH3
3,4-di-OCH3
3,4,5-tri-OCH3
H
4-Cl
4-OCH3
8w
8x
3,4,5-tri-OCH3 3,4,5-tri-OCH3
i) EtOH, Conc H2SO4, reflux for 12-18 h. (75.7-87.2 %). ii) NH2NH2H2O (97%),
EtOH, reflux 5-8 h, (72.7-80.2 %). iii) 1- CS2, KOH, EtOH, reflux 12 h.; 2- Conc
HCl, (60.0-87.0%). iv) p-aminoacetophenone, KOH (60%), EtOH, Stirring 4 h,
(67.3-82.9%). v) BrCH2COBr, CH2Cl2, K2CO3, H2O, Stirring overnight, (69.7-
78%).
Scheme1: Synthesis of the target compounds 8a-x.
7

2.2. Biological evaluation
2.2.1. Cytotoxic assays
2.2.1.1. In vitro one dose anticancer assay
All the twenty-four synthesized compounds (8a-x) were selected by the National
Cancer Institute (NCI), USA for in vitro anticancer screening. Compounds were
screened at 10 M dose using the Sulforhodamine B colorimetric assay against the
full NCI panel of 60 cell lines consisting of nine tumor subpanels, which include
leukemia, lung, colon, melanoma, renal, prostate, CNS, ovarian, and breast cancer cell
lines.
The compounds tested exhibited promising anticancer activities (Table 1 and 2
supporting information) against human cancer cells, particularly leukemia cell lines.
Among the synthesized compounds, 8a, 8n, 8p, and 8v were the most potent (Mean
growth inhibition % = 37.77, 27.29, 28.20, and 132.29; respectively).
Compound 8v exhibited potent cytotoxicities against most of the tested cell lines, with
complete cell death observed for 45 used cell lines and % growth inhibition ranging
from 101.51- 191.80% (Table 2 supporting information).
Six compounds, 8a, 8d, 8m, 8n, 8s, and 8v exhibited significant anticancer activity
against K-562 cell line with % growth inhibition ranging from 35.80 to 152.18%.
Additionally; compounds 8a, 8d, 8e, 8j, 8k-n, 8p, 8s, 8v, and 8x showed anticancer
activity against RPMI-8226 cell line with % growth inhibition ranging from 31.29-
143.74%. Compounds 8a, 8d, 8e, 8i-k, 8m, 8n, 8p, 8s, 8v and 8x exhibited good
anticancer activity against SR cell line with % growth inhibition ranging from 32.85 –
160.49%.
On the other hand, compounds 8a, 8d, 8l-n, and 8p exhibited only moderate
anticancer activity against MOLT-4 cell line with % growth inhibition ranging from
31.88– 108.34%.
2.2.1.2 In vitro five-dose anticancer assay
Compound 8v was selected for advanced five-dose testing against the full panel of 58
human tumor cell lines. All of the 58 cell lines, representing nine tumor subpanels,
were incubated at five different concentrations of (0.01, 0.1, 1, 10 and 100 µM). The
data indicate that compound 8v exhibited broad-spectrum antitumor activity with GI50
ranging from 0.32 to 11 µM, with selectivity ratio ranging between 0.65-1.20 at GI50
level (Table 1). Moreover, compound 8v showed the ability to inhibit growth of all
8

the cell lines tested, with TGI concentration ranging from 1.28 to 23.60 μM.
Compound 8v exhibited LC50 with concentrations ranging from 5.28 to > 100 μM.
Table 1. GI50 and TGI of compound 8v against the panel of 58 cell lines representing 9
different cancers tested using NCI's in vitro five dose anticancer assay.
Panel/Cell Line
GI50 TGI Panel/Cell Line
(µM) (µM)
GI50 TGI
(µM) (µM)
Leukemia
Melanoma
1.84
2.00
2.05
1.73
1.86
4.56
4.10
5.27
4.31
4.01
0.32
1.68
1.60
1.70
1.79
1.74
2.14
2.16
1.73
1.28
3.45
3.30
3.40
4.82
3.40
5.24
4.69
3.43
CCRF-CEM
HL-60(TB)
MOLT-4
RPMI-8226
SR
Non-Small Cell Lung Cancer
A549/ATCC
EKVX
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
1.84
3.53
3.98
1.79
2.13
1.93
2.67
1.88
1.59
2.00
1.55
4.53
4.23
5.69
3.97
2.98
3.85
3.21
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
CNS cancer
SF-268
UACC-62
Ovarian Cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal Cancer
786-0
A498
ACHN
RXF 393
SN12C
TK-10
1.54
1.78
2.00
1.72
2.32
2.42
2.42
3.75
3.38
4.62
3.30
5.73
7.03
5.68
3.84
3.65
1.94
3.22
3.62
3.40
3.60
1.94
1.88
0.50
1.63
1.83
1.77
1.86
1.70
11.00
1.72
1.74
1.83
1.52
1.46
3.36
23.60
3.22
3.54
3.57
3.03
2.97
1.79
3.96
UO-31
1.77
1.64
1.78
1.47
1.68
3.35
3.03
3.56
3.15
3.18
SF-295
SF-539
SNB-19
SNB-75
U251
Prostate Cancer
PC-3
DU-145
Breast cancer
MCF7
MDA-MB231/ATCC
HS 578T
BT-549
T-47D
1.27
1.82
2.65
1.40
2.05
2.10
3.68
3.50
7.76
2.81
5.08
5.02
1.75
1.75
3.30
3.21
MDA-MB-468
9

2.2.1.3 In vitro anti-proliferative assay using K-562, KG-1a, and Jurkat cells
The cytotoxic activities of the synthesized compounds were determined using three
different leukemia cell lines (K-562, KG-1a, and Jurkat) and evaluated using MTT
assays. Results presented in Table 2 indicate that most of the synthesized 1,3,4-
oxadiazole/chalcone derivatives exhibited strong to moderate cytotoxic activities
against the three leukemia cell lines. The relative sensitivities of leukemia cells
toward the synthesized compounds were K-562 > Jurkat > KG-1a. Consistent with the
NCI results, 8v showed the strongest cytotoxic activity among the derivatives
synthesized, with the relative sensitivities of K-562 (IC50 = 1.95 µM) > Jurkat (IC50 =
2.36 µM) > KG-1a (IC50 = 3.45 µM).
Our structure-activity relationship (SAR) analyses indicate that the type of
substitution in both phenyl rings is essential for anticancer activity. For anticancer
activity, the substitution of phenyl ring carrying the oxadiazole moiety should be
either H, p-methoxy or 3,4,5-trimethoxy group (s), with an effective substitution order
of 3,4,5-trimethoxy groups > H > p-methoxy group. Also, the more effective
substitutions of the chalcone phenyl ring are either H, p-methoxy or 3,4,5-trimethoxy
group (s). For optimal activity, R must be 3,4,5-trimethoxy groups and R1 must be
methoxy group in p-position. Thus for good anti-cancer activity, the two phenyl rings
should be either unsubstituted or one of the rings should carry p-methoxy group,
while the second ring should contain 3,4,5-trimethoxy groups.
10

Table 2. Cytotoxic activity of oxadiazole/chalcone hybrids, 8a-x, against three
leukemia cell lines.
(IC50 µM) ± SD
Compound
K-562
KG-1a
Jurkat
7.21±0.35
8.85±0.22
8.47±1.03
8a
8b
75.35±4.61
69.54±5.15
22.84±1.32
93.31±5.21
82.27±3.08
13.05±1.65
72.14±3.32
55.34±4.89
22.61±2.34
58.42±3.75
35.22±2.05
23.11±0.28
10.25±0.94
58.37±5.45
16.09±1.08
40.22±2.82
>100
82.24±4.56
95.22±3.88
31.34±1.05
>100
78.71±2.13
75.22±1.25
28.28±0.62
>100
8c
8d
8e
91.54±4.15
55.64±1.34
84.64±5.21
78.95±3.02
25.22±0.48
71.07±1.22
38.32±2.45
31.54±0.42
15.97±0.96
65.64±2.31
22.52±1.07
45.29±2.31
93.08±1.14
32.12±0.64
84.57±1.28
95.69±2.48
3.45±0.25
>100
88.61±1.35
41.21±0.91
78.06±2.10
62.56±1.37
31.13±0.46
62.67±2.71
33.28±1.22
28.36±0.85
13.15±0.08
94.09±5.31
17.24±0.79
31.94±1.30
>100
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
8q
8r
15.14±0.25
73.25±3.72
84.22±4.36
1.95±0.18
90.34±2.91
62.15±3.07
3.21±0.45
25.14±0.45
76.36±1.31
>100
8s
8t
8u
2.36±0.47
>100
8v
8w
8x
71.48±1.18
12.15±3.41
69.17±0.15
15.12±1.35
Cisplatin
11

2.2.2. 1,3,4-oxadiazole/chalcone hybrids inhibited EGFR and Src kinase activities
and protein expression.
The oxadiazole/chalcone hybrids 8a-x inhibited EGFR kinase activity with relatively
lower concentrations than that required for the inhibition of Src Kinase activity. The
effects of synthesized compounds on EGFR and Src tyrosine kinases are presented in
Table 3. Compounds 8a, 8n and 8v displayed the highest inhibitory activities against
EGFR (IC50 = 0.24-2.35 μM) as well as Src (IC50 = 0.96-6.24 μM). While compounds
8b, 8g-8m, 8p, 8q, 8s, 8t, and 8x exhibited moderate inhibitory activities against
EGFR (IC50 = 10.24-35.15 μM) and Src (IC50 = 12.5-44.1 μM). On the other hand,
compounds 8c, 8e, 8f, 8o, 8r, 8u, and 8w showed the lowest activity against EGFR
(IC50 = 48.34-71.15 μM) and Src (IC50 = 50.32-76.2 μM).
Table 3. EGFR and Src kinases inhibitory activity of 1,3,4-oxadiazole/chalcone hybrids.
EGFR
Src
EGFR
Src
Compound
Compound
(IC50 µM)
1.23±0.25
35.15±1.36
64.35±3.56
11.22±1.25
59.35±5.63
64.68±3.65
12.67±1.25
30.25±2.35
29.34±3.65
11.47±1.24
18.34±2.35
12.34±1.35
10.24±0.36
(IC50 µM)
4.5±1.2
(IC50 µM)
2.35±0.95
71.15±3.25
13.26±0.65
11.38±1.54
63.25±2.36
12.35±0.8
22.34±2.35
55.45±4.35
0.24±0.035
48.34±2.36
20.5±1.35
0.8±0.23
(IC50 µM)
6.24±0.86
51.24±5.6
13.67±0.89
29.48±1.23
76.2±3.65
15.34±1.25
40.21±1.34
53.09±5.6
0.96±0.2
8a
8b
8c
8d
8e
8f
8n
44.1±3.5
8o
57.3±2.4
8p
13.41±1.6
74.4±3.6
8q
8r
50.32±3.5
22.14±2.15
41.36±4.5
43.67±3.5
25.34±1.56
33.64±2.35
24.98±3.46
12.5±0.3
8s
8g
8h
8i
8t
8u
8v
60.34±2.5
35.12±3.26
< 0.001
8j
8w
8k
8l
8x
Dasatinib
Gefitinib
0.023±0.004
1.25±0.15
8m
To confirm the ability of the synthesized compounds to inhibit EGFR and Src, we
evaluated the effects of the most potent compound 8v, on the expression levels of
EGFR, p-EGFR, Src and p-Src in K562 cells using Western blot analysis. As shown
in Fig.4, compound 8v downregulated the protein expression levels of p-EGFR,
12

EGFR, p-Src and Src in a dose-dependent manner. Therefore, it was reasonable to
conclude that the anti-proliferative effect of compound 8v may be attributed to the
inhibition of EGFR and Src.
A
B
8v (µM)
0.2
0.8
1.6
1
p-EGFR
EGFR
*
*
Control
0.2 µM
0.8 µM
1.6 µM
0.5
**
p-Src
Src
*
**
**
**
*
**
**
0
p-EGFR
EGFR
p-Src
Src
GAPDH
Fig.4. (A) Western blotting analysis of the expression of EGFR, p-EGFR, Src and p-
Src in K562 cells treated with 0, 0.2, 0.8, or 1.6 μM oxadiazole chalcone hybrid 8v.
GAPDH served as a loading control. (B) The protein expression of EGFR, p-EGFR,
Src and p-Src after treatment with different concentrations of compound 8v. The
values are expressed as the mean ± SD based on three different experiments. *, P <
0.05 and **, P < 0.005 indicate a significant difference compared with vehicle treated
cells.
2.2.3. 1,3,4-Oxadiazole/chalcone hybrids inhibited IL-6 production
K562 cells were treated with 10 µM of 1,3,4-oxadiazole/chalcone hybrids 8a-x for 24
h, after which media were collected to determine IL-6 protein levels. As illustrated in
Fig. 5A, 1,3,4-oxadiazole/chalcone hybrids decreased IL-6 protein levels. Consistent
with relative effectiveness of the compounds in downregulating EGFR and Src,
compounds 8a, 8n, and 8v were found to be most effective at downregulating IL-6
levels (% of control = 20-23%). Compounds, 8b, 8g-8m, 8p, 8q, 8s, 8t, and 8x
induced moderate inhibition of IL-6 levels (% of control = 30-75%). On the other
hand, compounds 8c, 8e, 8f, 8o, 8r, 8u, and 8w did not significantly downregulate IL-
6 levels (% of control = 82-98%). The initial downregulation of the levels of IL-6
13

protein were observed 8 hours after treatment with compound 8v, and IL-6 levels
were still downregulated after 48 hours Fig. 5B.
Fig. 5. (A) IL-6 protein expression in K-562 cells 24 h after treatment with 10 µM
oxadiazole/chalcone derivatives (8a-x) or dexamethasone relative to vehicle treated
cells (B) Time course effect of 8v on IL-6 protein expression in K-562 cells treated
with 10 µM oxadiazole/chalcone derivative 8v. The values are expressed as the mean
± SD based on three different experiments. *, P < 0.05 and **, P < 0.005 indicate a
significant difference compared to vehicle treated cells.
14

2.2.4. 1,3,4-Oxadiazole/chalcone hybrids inhibited STAT3 activity
Because 1,3,4-oxadiazole/chalcone derivatives inhibited EGFR, Src, and IL-6, which
a upstream activators of STAT3, the effect of the synthesized compounds on STAT3
activation was evaluated. The results presented in Fig. 6 indicate that 1,3,4-
oxadiazole/chalcone derivatives 8a-x inhibited STAT3 activation. Compounds 8a, 8n,
and 8v were the most effective at inhibiting STAT3 activity. Compounds 8b, 8g-8m,
8p, 8q, 8s, 8t, and 8x exhibited moderate activities, and the remaining compounds
showed low or no activities. Our results also confirmed the type of substitution on
phenyl rings influenced activity. For effective STAT3 inhibition, the two phenyl ring
should be either unsubstituted or one ring should carry p-methoxy group while the
second ring should contain 3,4,5-trimethoxy groups.
Fig. 6. STAT3 activation assay in K-562 cells treated with 10 µM oxadiazole/chalcone
derivatives (8a-x), WP1066, or vehicle for 24 h. The values are expressed as the mean ± SD
based on three different experiments. *, P < 0.05 and **, P < 0.005 indicate a significant
difference relative to vehicle treated cells.
15

3. Conclusion
A series of novel 1,3,4-oxadiazole/chalcone hybrids was prepared and identified using
different spectroscopic techniques. The compounds we generated showed significant
anticancer activity, particularly against leukemia. Compounds 8a, 8n, and 8v showed
the highest cytotoxicity activity against leukemia cell lines K-562 among the
compounds tested. The IC50 for the compounds were in the range of 1.95 - 10.25 µM,
compared to cisplatin that has an IC50 of 3.21 μM. In vitro (one dose) anticancer and
MTT assays indicated that compound 8v exhibited the highest activities against
human cancer cells. Compounds, 8a, 8n and 8v displayed the most potent or effective
inhibitory activity against EGFR (IC50 = 0.24-2.35μM), Src (IC50 = 0.96-6.24μM), and
IL-6 (% of control = 20-23%). Compounds 8a, 8n, and 8v are also the most effective
at inhibiting STAT3. It is possible that inhibition of EGFR, Src, and IL-6 by 1,3,4-
oxadiazole/chalcone hybrids play roles in the inhibition of STAT3. The inhibition of
STAT3 support the potential of 1,3,4-oxadiazole/chalcone hybrids in the treatment of
leukemia and prevention of the development of resistance in leukemia cells. However,
further studies using in vivo models are needed to verify the potential of 1,3,4-
oxadiazole/chalcone hybrids as anticancer agents.
4. EXPERIMENTAL SECTION
4.1.Chemistry section:
 Analytical grade chemicals and solvents were used. The progress of the
reactions was monitored by thin layer chromatography pre-coated Merck silica
gel 60 F254 aluminum sheets.
 Melting points were determined on Stuart electro-thermal melting point
apparatus in degree Celsius and were uncorrected.

1H spectra were recorded on Burker AG, Switzerland, 500 MHz, Faculty of
Pharmaceutical Sciences, Umm Al-Qura University, Mecca, Saudi Arabia;
chemical shift (δ) were expressed in ppm relative to TMS (δ=0 PPM) as
internal standard and CDCl3 or DMSO-d6 as a solvent. Chemical shifts (δ)
were expressed in parts per million (ppm) and coupling constants (J) were
expressed in Hertz. The signals were designated as follows: s, singlet; d,
doublet; t, triplet; m, multiplet.
16


13C spectra were recorded on Burker AG, Switzerland and 125 MHz, faculty
of Pharmaceutical Sciences, Umm Al-Qura University, Mecca, Saudi Arabia
using TMS as the reference standard and CDCl3 or DMSO-d6 as a solvent.
Chemical shifts (δ) were expressed in parts per million (ppm).
 LC/MS/MS were carried out using Agilent UPLC/MS/MS 1260 infinity II
with 6420 Triple quad LC/MS detector at Faculty of Pharmacy, Minia
University, Minia, Egypt
 The purity of the compounds was checked by HPLC.
 Elemental analyses were recorded on Shimadzu GC/ MS-QP5050A, The
Regional center for Mycology and Biotechnology, Al-Azhar University, Egypt.
4.1.1. General procedure for the synthesis of substituted ethylbenzoate 2a-e [60].
A mixture of the appropriate substituted benzoic acid 1a-e (10 mmol), absolute
ethanol (20 mL), and concentrated sulfuric acid (2 mL) was heated under reflux for
12-18 h. Excess solvent was removed under reduced pressure. The residue was
extracted with ether (2 X 50 mL) and washed with saturated NaHCO3 (2 X 20 mL).
The ether layer was dried over anhydrous magnesium sulfate, and ether was
evaporated under vacuum to produce the ethyl ester derivatives 2a-e.
4.1.2. General procedure for the synthesis of substituted benzohydrazides 3a-e
[61, 62].
Hydrazine hydrate (97 %, 30 mmol, 1.5 mL) was added to a solution of the isolated
esters 2a–e (10 mmol) in ethanol (20 mL), and the mixture was heated at reflux for 5-
8 h. After cooling, the resulting precipitate was filtered off, washed with water, dried,
and crystallized from ethanol.
Benzohydrazide (3a) [62].
White solid (77.2 % yield); mp 110-112oC (Reported mp = 112-114 oC).
4-Chloro-benzohydrazide (3b) [63].
White solid (79.3% yield); mp 195-197 oC (Reported mp = 189-191oC).
4-Methoxybenohydrazide (3c) [62].
White solid (80.2 % yield); mp 134-137 ºC (Reported mp =136-140ºC)
17

3,4-Dimethoxybenzohydrazide (3d) [62].
White solid (72.7 % yield); mp 145-146 ºC as reported.
3,4,5-Trimethoxybenzohydrazide (3e) [62].
White solid (72.9% yield); mp 158-160oC as reported.
4.1.3. General procedure for the synthesis of 5-aryl--1,3,4-oxadiazole-2(3H)-
thione derivatives (4a-e) [43, 64].
A mixture of the benzohydrazides 3a-e (0.05 mol), potassium hydroxide (0.05 mol,
2.81g) and carbon disulfide (0.17 mol, 12.94 g) in 50 mL of absolute ethanol was
heated at reflux with stirring for 12 h. The solvent was evaporated under vacuum. The
resulting solid was dissolved in water and acidified with 10% HCl. The resulting
precipitate was filtered off, washed with water, dried, and recrystallized from ethanol
to generate the corresponding oxadiazole derivatives 4a-e [64].
5-Phenyl-1,3,4-oxadiazole-2(3H)-thione (4a) [65].
White solid (76 % yield); mp 215-216oC (Reported mp = 218-220oC).
5-(4-Chlorophenyl) -1,3,4-oxadiazole-2(3H)-thione (4b) [65].
Yellowish white solid (87 % yield); mp 172-173oC (Reported mp = 176-178oC).
5-(4-Methoxyphenyl)-1,3,4-oxadiazole-2(3H)-thione (4c) [65].
White solid (78 % yield); mp 192-194oC (Reported mp = 198-200oC).
5-(3,4-Dimethoxyphenyl)-1,3,4-oxadiazole-2(3H)-thione (4d) [66].
Yellowish white solid (60 % yield); mp 238-239oC (Reported mp = 237-238oC).
5-(3,4,5-Trimethoxyphenyl)-1,3,4-oxadiazole-2(3H)-thione (4e) [67].
Yellowish white solid (79 % yield); mp 192-193oC (Reported mp = 186-188oC).
4.1.4. General procedure for the synthesis of 1-(4-aminophenyl)-3-arylprop-2-en-
1-one (6a-d) [68].
An equimolar amount of p-aminoacetophenone (0.1 mol, 13.51 g) and the appropriate
aldehyde 5a-d (0.1 mol), were dissolved in a minimum amount of ethanol, and
aqueous NaOH (0.25 mol, 60%) was then added dropwise. The reaction mixture was
stirred in ice bath for 30 min then at rt, until the precipitate was formed within 3 h.
The precipitate was filtered off and washed thoroughly with cold distilled water and
18

cold methanol (2 x 20 mL). The product was recrystallized from absolute ethanol. The
structure of the product was confirmed by mp.
1-(4-Aminophenyl)-3-phenyl prop-2-en-1-one (6a) [59]
Buff solid (77.8 % yield); mp 156-157oC (Reported mp = 157-158oC).
1-(4-Aminophenyl)-3-(4-chlorophenyl)prop-2-en-1-one (6b) [68].
Yellow solid (80.3 % yield); mp 159-160oC (Reported mp = 158-159oC).
1-(4-Aminophenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (6c) [68].
Yellow solid (67.3 % yield); mp 115-116oC (Reported mp = 114-115oC).
1-(4-Aminophenyl)-3-(3,4-dimethoxyphenyl)prop-2-en-1-one (6d) [68].
Yellow solid (82.9 % yield); mp 159-160oC (Reported mp = 158-160oC).
1-(4-Aminophenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (6e) [68].
Yellow solid (70.4 % yield); mp 166-168oC as reported.
4.1.5. General procedure for the synthesis of 2-bromo-N-(4-(3-
arylacryloyl)phenyl)acetamides (7a-e) [59].
To a mixture of the appropriate chalcone 6a-e (6.30 mmol) in dichloromethane (20
mL) and potassium carbonate (1.302 mmol, 0.18g) in 100 mL water in an ice bath,
bromoacetyl bromide (9.20 mmol, 1.856 g) in 30 mL dichloromethane was added in a
dropwise manner with stirring over 30 min. Stirring was continued for 2 h at 0ᴼC and
at rt overnight. The reaction mixture was extracted with dichloromethane (2 x 60 mL)
and the organic layer was washed with distilled water (2 x 40 mL), dried over
anhydrous sodium sulfate, filtered, evaporated under vacuum and the residue was
recrystallized from ethanol.
2-Bromo-N-(4-((E)-3-phenylacryloyl)phenyl)acetamide (7a) [59].
Pale yellow powder (70.0 % yield); mp 157-159oC (Reported mp = 157-158oC).
2-Bromo-N-(4-(E)-3-(4-chlorophenyl)acryloyl)phenyl)acetamide (7b) [68].
Pale yellow powder (71.5 % yield); mp 191-192oC (Reported mp = 190-192oC).
2-Bromo-N-(4-((E)-3-(4-methoxyphenyl)acryloyl)phenyl)acetamide (7c) [59].
Pale orange crystal (69.70 % yield); mp 155-156oC (Reported mp = 160-162oC).
2-Bromo-N-(4-((E)-3-(3,4-dimethoxyphenyl)acryloyl)phenyl)acetamide (7d) [59].
19

Yellow powder (71.0 % yield); mp 150-151oC (Reported mp= 149-150oC).
2-Bromo-N-(4-((E)-3-(3,4,5-trimethoxyphenyl)acryloyl)phenyl)acetamide (7e)
[59].
Yellow powder (78.0 % yield); mp 166-167oC (Reported mp= 160-162oC).
4.1.6. General Procedure for the synthesis of 2-(5-phenyl-1,3,4-oxadiazole-2-
ylthio)-N-(4-((E)-3-phenylacryloyl)phenyl)acetamide derivatives (8a-x).
TEA (0.18 mmol, 0.018 g) was added as a base to an equimolar mixture of 4a-e (0.09
mmol) and compound 7a-e (0.09 mmol) in acetonitrile. The reaction mixture was
stirred at room temperature until the precipitate formed. The resulting precipitate was
filtrated off, and the precipitate was then crystallized with acetonitrile to afford the
target compounds [59].
4.1.6.1. N-(4-(3-Phenyl)acryloyl)phenyl-2-((5-phenyl-1,3,4-oxadiazol-2-yl)thio)-
acetamide 8a
1
White solid (0.39g, 89 % yield); mp 199-201°C; H NMR (500 MHz, DMSO-d6) -
ppm)s, 1H, NH), 8.18 (d, 2H, J = 8.0 Hz, Ar-H), -(m, 3H, 2
Ar-H and CH=CH), 7.88-7.87 (m, 2H, Ar-H), 7.80 (d, 2H, J = 8.0 Hz, Ar-H), 7.73
(d, 1H, J = 16.0 Hz, CH=CH), 7.61 (d, 2H, J = 6.0 Hz Ar-H), 7.58 (d, 2H, J = 6.0
Hz, Ar-H), 7.49-7.46 (m, 2H, Ar-H), 4.42 (s, 2H, CH2);13C NMR (125 MHz, DMSO-
d6)ppm)
LC−
MS m/z 440.00 [M − H]−; Anal. Calcd for C25H19N3O3S: C, 68.01; H, 4.34; N, 9.52;
S, 7.26. Found: C, 68.17; H, 4.52; N, 9.80; S, 7.35.
4.1.6.2. N-(4-(3-(4-Chlorophenyl)acryloyl)phenyl)-2-((5-phenyl-1,3,4-oxadiazol-
2-yl)thio)acetamide 8b
1
Yellow solid (0.29g, 63 % yield) ; mp 250-252°C; H NMR (500 MHz, DMSO-d6)
(ppm)s, 1H, NH), 8.20 (d, 2H, J = 6.6 Hz, Ar-H), 8.00-7.97 (m, 2H, Ar-
H), 7.96-7.93 (m, 3H, 2 Ar-H and CH=CH), 7.79 (d, 2H, J = 6.8 Hz, Ar-H), 7.72 (d,
1H, J = 15.5 Hz, CH=CH ), 7.62-7.58 (m, 3H, Ar-H), 7.54 (d, 2H, J = 6.6 Hz, Ar-H),
4.42 (s, 2H, CH2); 13C NMR (125 MHz, DMSO-d6)ppm): 187.92, 166.13, 165.67,
20

163.81, 143.56, 142.51, 135.47, 134.23, 133.07, 132.55, 131.02, 130.56, 129.91,
129.43, 126.85, 123.42, 123.16, 119.07, 37.39 LC−MS m/z 475.10 [M]+; Anal.
Calcd for C25H18ClN3O3S: C, 63.09; H, 3.81; N, 8.83; S, 6.74. Found: C, 63.23; H,
3.97; N, 9.06; S, 6.88.
4.1.6.3. N-(4-(3-(3,4-Dimethoxyphenyl)acryloyl)phenyl)-2-((5-phenyl-1,3,4-
oxadiazol-2-yl)thio)acetamide 8c
1
Yellow solid (0.25g, 51% yield) mp 230-231°C; H NMR (500 MHz, DMSO-d6)
ppm )s, 1H, NH), 8.19 (d, 2H, J = 6.4 Hz, Ar-H), 7.97-7.96 (m, 2H, Ar-
H), 7.84 (d, 1H, J = 15.2 Hz, CH=CH), 7.80 (d, 2H, J = 6.4 Hz, Ar-H), 7.70 (d, 1H, J
= 15.2 Hz, CH=CH), 7.63-7.57 (m, 3H, Ar-H), 7.55 (s, 1H, Ar-H), 7.39 (d, 1H, J =
6.0 Hz, Ar-H), 7.03 (d, 1H J = 6.0 Hz Ar-H), 4.42 (s, 2H, CH2), 3.87 (s, 3H, OCH3),
13
3.83 (s, 3H, OCH3); C NMR (125 MHz, DMSO-d6) ppm): 187.89, 166.08,
165.67, 163.82, 151.69, 149.49, 144.53, 143.29, 133.47, 132.55, 130.38, 129.91,
128.05, 126.85, 124.41, 123.42, 119.90, 119.02, 112.01, 111.12, 56.22, 56.07, 37.40;
LC−MS m/z 501.90 [M + H]+; Anal. Calcd for C27H23N3O5S: C, 64.66; H, 4.62; N,
8.38; S, 6.39. Found: C, 64.39; H, 4.80; N, 8.54; S, 6.52.
4.1.6.4.
N-(4-(3-(3.4.5-Trimethoxyphenyl)acryloyl)phenyl)-2-((5-phenyl-1,3,4-
oxadiazol-2-yl)thio)acetamide 8d
Yellow crystals (0.30g, 58 % yield ) mp 261-262°C; 1H NMR (500 MHz, DMSO-d6)
ppms, 1H, NH), 8.20 (d, 2H, J = 7.5 Hz, Ar-H), 7.97 (d, 2H, J = 5.2 Hz,
Ar-H), (d, 1H, J = 15.5 Hz, CH=CH), 7.81 (d, 2H, J = 7.5 Hz, Ar-H), 7.69 (d,
1H, J = 15.5 Hz, CH=CH), 7.65-7.58 (m, 3H Ar-H ), 7.23 (s, 2H, Ar-H ), 4.43 (s, 2H,
CH2), 3.87 (s, 6H, 2OCH3), 3.72 (s, 3H, OCH3); 13C NMR (125 MHz, DMSO-
d6)ppm)

 LC−MS m/z 529.90 [M – H]−; Anal. Calcd for C28H25N3O6S: C, 63.26; H,
4.74; N, 7.90; S, 6.03. Found: C, 63.50; H, 4.87; N, 8.21; S, 6.11.
21

4.1.6.5.
2-((5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-yl)thio)-N-(4-cinnamoyl
phenyl)acetamide 8e
1
Yellow solid (0.30g, 63.3% yield) mp 230-231°C; H NMR (500 MHz, DMSO-d6)
ppm): 10.90 (s, 1H, NH), 8.19 (d, 2H, J = 7.0 Hz, Ar-H), 7.99-7.97 (m, 2H, Ar-H),
7.93-7.90 (m, 3H, 2 Ar-H and CH=CH), 7.80 (d, 2H, J = 7.0 Hz, Ar-H), 7.74 (d, 1H,
J = 15.5 Hz, CH=CH), 7.66 (d, 2H, J = 6.5 Hz, Ar-H), 7.49-7.44 (m, 3H, Ar-H), 4.43
(s, 2H, CH2); 13CNMR (125 MHz, DMSO-d6)(ppm): 188.05, 166.08, 164.92,
164.11, 143.99, 143.49, 137.24, 135.23, 133.17, 131.02, 130.50, 130.09, 129.39,
129.31, 128.66, 122.41, 122.32, 119.08, 37.38; LC−MS m/z 474.10 [M – H]−; Anal.
Calcd for C25H18ClN3O3S: C, 63.09; H, 3.81; N, 8.83; S, 6.74. Found: C, 63.28; H,
3.75; N, 8.97; S, 6.82.
4.1.6.6.
2-((5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-yl)thio)-N-(4-(3-(4-chloro-
phenyl)acryloyl)phenyl)acetamide 8f
1
Yellow solid (0.39g, 77.0% yield) mp 181-182°C; H NMR (500 MHz, DMSO-d6)
ppm)s, 1H, NH), 8.18 (d, 2H, J = 8.0 Hz, Ar-H ), 7.98-7.97 (m, 3H, 2 Ar-
H and CH=CH ), 7.93 (d, 2H, J = 8.0 Hz, Ar-H), 7.78 (d, 2H, J = 8.0 Hz, Ar-H),
7.71 (d, 1H, J = 15.0 Hz, CH=CH), 7.66 (d, 2H, J =7.5Hz, Ar-H), 7.53 (d, 2H J =
8.0Hz, Ar-H), 4.42 (s, 2H, CH2); 13C NMR (125 MHz, DMSO-d6) ppm): 187.93,
166.09, 164.92, 164.11, 137.25, 135.47, 134.21, 133.06, 131.02, 130.56, 130.09,
129.47, 129.43, 129.01, 128.66, 123.14, 122.31, 119.06, 37.38; LC−MS m/z 509.90
[M + H]+; Anal. Calcd for C25H17Cl2N3O3S: C, 58.83; H, 3.36; N, 8.23; S, 6.28.
Found: C, 59.17; H, 3.42; N, 8.04; S, 6.12.
4.1.6.7.
2-(5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-ylthio)-N-(4-(3-(4-methoxy-
phenyl)acryloyl)phenyl)acetamide 8g
1
Yellow crystals (0.31g, 62 % yield) mp 227-228°C; H NMR (500 MHz DMSO-d6)
ppm)s, 1H, NH), 8.17 (d, 2H, J = 6.0 Hz, Ar-H), 7.98 (d, 2H, J = 5.7 Hz,
Ar-H), 7.86 (d, 2H, J = 5.7 Hz, Ar-H),, 7.79-7.77 (m, 3H, 2 Ar-H + CH=CH), 7.71
(d, 1H, J = 15.00 Hz, CH=CH), 7.67 (d, 2H, J = 6.0 Hz, Ar-H), 7.03 (d, 2H, J = 5.9
13
Hz, Ar-H), 4.43 (s, 2H, CH2), 3.83 (s, 3H, OCH3); C NMR (125 MHz, DMSO-d6)
ppm): 188.83, 166.02, 164.92, 164.12, 161.98, 161.77, 144.95, 143.98, 143.26,
139.02, 131.41, 131.19, 130.33, 130.08, 128.64, 122.31, 119.03, 114.87, 55.84,
22

37.37; LC−MS m/z 504.00 [M – H]−; Anal. Calcd for C26H20ClN3O4S: C, 61.72; H,
3.98; N, 8.30; S, 6.34. Found: C, 61.95; H, 4.12; N, 8.59; S, 6.51.
4.1.6.8. 2-(5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-ylthio)-N-(4-(3-(3,4-dimethoxy-
phenyl)acryloyl)phenyl)acetamide 8h
1
Yellow solid (0.38g, 71.4% yield) mp 215-216°C; H NMR (500 MHz, CDCl3)
ppm)s, 1H, NH), 8.19 (d, 2H, J = 6.6 Hz, Ar-H), 7.98 (d, 2H, J = 6.1 Hz,
Ar-H), 7.84 (d, 1H, J = 15.3 Hz, CH=CH), 7.79 (d, 2H, J = 6.6 Hz, Ar-H), 7.71-7.66
(m, 3H, 2 Ar-H + CH=CH), 7.55 (s, 1H, Ar-H), 7.39 ( d, 1H, J = 6.1 Hz, Ar-H),
7.03 (d, 1H, J = 6.1 Hz, Ar-H), 4.43 (s, 2H, CH2), 3.87 (s, 3H, OCH3), 3.83 (s, 3H,
OCH3); 13CNMR (125 MHz, CDCl3) ppm): 187.90, 166.02, 164.93, 164.13,
151.69, 149.49, 144.53, 143.27, 137.25, 133.47, 130.38, 130.09, 128.66, 128.05,
124.41, 122.32, 119.90, 119.02, 112.02, 111.12, 56.22, 56.07, 37.40; LC−MS m/z
534.00 [M – H]−; Anal. Calcd for C27H22ClN3O5S: C, 60.50; H, 4.14; N, 7.84; S, 5.98.
Found: C, 60.38; H, 4.27; N, 8.11; S, 6.15.
4.1.6.9.
2-(5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-ylthio)-N-(4-(3-(3,4,5-
trimethoxyphenyl)acryloyl)phenyl)acetamide 8i
Yellow crystals ( 0.40g, 71% yield ) mp 261-262°C; 1H NMR (500 MHz,,DMSO-d6
ppm)10.85 (s, 1H, NH), (d, 2H, J = 7.1 Hz, Ar-H), 7.98 (d, 2H, J = 6.9
Hz, Ar-H), 7.91 (d, 1H, J = 15.4, CH=CH), 7.80 (d, 2H, J = 7.1 Hz, Ar-H), 7.71-
7.67 (m, 3H, 2 Ar-H + CH=CH), 7.24 (s, 2H, Ar-H), 4.43 (s, 2H, CH2), 3.87 (s, 6H,
2OCH3), 3.72 (s, 3H ,OCH3); 13C NMR (125 MHz, DMSO-d6) ppm): 187.96,
166.05, 164.93, 164.13, 153.58, 144.52, 143.41, 140.05, 137.25, 133.29, 130.78,
130.49, 130.10, 128.66, 122.32, 121.53, 119.03, 106.96, 60.62, 57.30, 56.61, 37.41;
Anal. Calcd for C28H24ClN3O6S: C, 59.41; H, 4.27; N, 7.42; S, 5.67. Found: C, 59.58;
H, 4.35; N, 7.60; S, 5.74.
4.1.6.10.
2-(5-(4-Methoxyphenyl)-1,3,4-oxadiazol-2-ylthio)-N-(4-(3-phenyl
acryloyl)phenyl)acetamide 8j
1
Yellow solid (0.39g, 84% yield) mp 195-197°C; H NMR (500 MHz, CDCl3)
ppm)9.90 (s, 1H, NH), (d, 2H, J = 7.0 Hz, Ar-H), 7.96 (d, 2H, J = 7.0
23

Hz, Ar-H), 7.81 (d, 1H, J = 15.0 Hz, CH=CH), 7.77-7.75 (m, 1H, Ar-H), 7.66 (d,
2H, J = 6.0 Hz, Ar-H), 7.53 (d, 1H, J = 15.0, CH=CH), 7.45- 7.42 (m, 4H, Ar-H),
7.02 (d, 2H, J = 6.0 Hz, Ar-H), 4.11 (s, 2H, CH2) 3.90 (s, 3H, OCH3); 13C NMR
,
(125 MHz, CDCl3) ppm)189.01, 166.56, 165.84, 164.44, 162.84, 144.59, 141.96,
130.49, 129.92, 128.97, 128.75, 128.47, 121.83, 119.36, 115.17, 114.83, 114.73,
114.12, 55.56, 36.50; Anal. Calcd for C26H21N3O4S: C, 66.23; H, 4.49; N, 8.91; S,
6.80. Found: C, 66.06; H, 4.62; N, 9.18; S, 6.59.
4.1.6.11.
2-(5-(4-Methoxyphenyl)-1,3,4-oxadiazol-2-ylthio)-N-(4-(3-(4-chloro-
phenyl)acryloyl)phenyl)acetamide 8k
1
Yellow solid (0.37g, 75% yield) mp 250-251°C; H NMR (500 MHz, DMSO-d6)
ppm)10.83 (s, 1H, NH), (d, 2H, J = 6.3 Hz, Ar-H), 7.98 (d, 1H, J = 16.0
Hz, CH=CH), 7.94 (d, 2H, J = 5.7 Hz, Ar-H), 7.89 (d, 2H, J = 6.2 Hz, Ar-H), 7.79
(d, 2H, J = 6.2 Hz, Ar-H), 7.72 (d, 1H, J = 16.0 Hz, CH=CH), 7.54 (d, 2H, J = 5.7
Hz, Ar-H), 7.12 (d, 2H, J = 6.3 Hz, Ar-H), 4.39 (s, 2H, CH2), 3.84 (s, 3H, OCH3);
13C NMR (125 MHz, DMSO-d6) ppm)188.04, 166.11, 165.61, 163.62,
153.92, 144.01, 143.48, 141.02, 135.21, 133.17, 131.03, 130.50, 129.40, 129.31,
122.38, 119.06, 118.59, 104.23, 56.59, 37.41; LC−MS m/z 504.00 [M − H]−; Anal.
Calcd for C26H20 ClN3O4S: C, 61.72; H, 3.98; N, 8.30; S, 6.34. Found: C, 61.97; H,
4.11; N, 8.54; S, 6.47.
4.1.6.12.
2-(5-(4-Methoxyphenyl)-1,3,4-oxadiazol-2-ylthio)-N-(4-((E)-3-(4-
methoxyphenyl)acryloyl)phenyl)acetamide 8l
1
Yellow solid (0.37g, 75% yield) mp 183-185°C; H NMR (500 MHz, DMSO-d6)
ppm )10.86 (s, 1H, NH), (d, 2H, J = 8.0 Hz, Ar-H), 7.86 (d, 2H, J = 7.5 Hz,
Ar-H), 7.81 (d, 2H, J = 8.0 Hz, Ar-H), 7.75-7.71 (m, 3H, Ar-H and CH=CH), 7.68 (d,
1H, J = 15.0 Hz , CH=CH) 7.08 (d, 2H, J = 8.0 Hz, Ar-H), 7.00 (d, 2H, J = 7.5 Hz,
13
Ar-H), 4.32 (s, 2H, CH2), 3.82 (s, 3H, OCH3), 3.80 (s, 3H, OCH3); C NMR (125
MHz, DMSO-d6) ppm): 187.91, 166.15, 165.63, 162.94, 162.53, 161.77, 143.90,
143.32, 133.43, 131.20, 130.33, 128.72, 127.81, 119.03,115.74, 115.33,
114.87LC−MS m/z 500.10 [M − H]−; Anal. Calcd for
24

C27H23N3O5S: C, 64.66; H, 4.62; N, 8.38; S, 6.39. Found: C, 64.49; H, 4.80; N, 8.57;
S, 6.21.
4.1.6.13.
2-(5-(4-Methoxyphenyl)-1,3,4-oxadiazol-2-ylthio)-N-(4-3-(3,4-
dimethoxyphenyl)acryloyl)phenyl)acetamide 8m
Yellow crystals (0.40g, 76.9% yield) mp 193-195°C; 1H NMR (500 MHz, DMSO-d6)
ppm )10.83 (s, 1H, NH), (d, 2H, J = 7.3 Hz, Ar-H), 7.90 (d, 2H, J = 7.6
Hz, Ar-H), 7.84 (d, 1H, J = 15.2 Hz, CH=CH), 7.79 (d, 2H, J = 7.3 Hz, Ar-H), 7.70
(d, 1H, J = 15.2 Hz, CH=CH), 7.55 (s, 1H, Ar-H), 7.39 (d, 1H, J = 7.1 Hz, Ar-
H), 7.12 (d, 2H, J = 7.6 Hz, Ar-H), 7.03 (d, 1H, J = 7.1 Hz, Ar-H), 4.39 (s, 2H,
CH2) 3.87 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 3.83 (s, 3H, OCH3); 13C NMR (125
,
MHz, DMSO-d6) ppm) 187.90, 166.13, 165.64, 162.95, 162.55, 151.70, 149.49,
144.53, 143.30, 133.46, 130.38, 128.73, 128.05, 124.41, 119.90, 119.02, 115.75,
115.36, 112.02, 111.13, 56.22, 56.08, 56.01, 37.38; Anal. Calcd for C28H25N3O6S:
C, 63.26; H, 4.74; N, 7.90; S, 6.03. Found: C, 63.58; H, 4.89; N, 7.81; S, 6.22.
4.1.6.14.
2-(5-(4-Methoxyphenyl)-1,3,4-oxadiazol-2-ylthio)-N-(4-(3-(3,4,5-tri-
methoxyphenyl)acryloyl)phenyl)acetamide 8n
Yellow solid (0.43g, 83.3% yield ) mp 220-221°C; 1H NMR (500 MHz, DMSO-d6)
ppm)10.84 (s, 1H, NH), (d, 2H, J = 5.7 Hz, Ar-H), 7.91-7.89 (m, 3H, 2 Ar-
H and CH=CH), 7.80 (d, 2H, J = 5.5 Hz, Ar-H), 7.69 (d, 1H, J = 15.3 Hz, CH=CH),
7.24 (s, 2H, Ar-H), 7.12 (d, 2H, J = 5.7 Hz, Ar-H), 4.40 (s, 2H, CH2), 3.87 (s, 6H, 2
OCH3), 3.85 (s, 3H, OCH3), 3.72 (s, 3H, OCH3); 13C NMR (125 MHz, DMSO-d6)
ppm)187.95, 166.17, 165.63, 162.96, 162.54, 153.58, 144.53, 143.46, 140.10,
133.26, 130.78, 130.50, 128.72, 121.51, 119.01, 115.74, 115.35, 106.93, 56.59,
55.99, 53.91, 37.37; LC−MS m/z 560.10 [M − H]−; Anal. Calcd for C29H27N3O7S:
C, 62.02; H, 4.85; N, 7.48; S, 5.71. Found: C, 61.89; H, 4.97; N, 7.67; S, 5.84.
4.1.6.15. N-(4-Cinnamoylphenyl)-2-((5-(3,4-dimethoxyphenyl)-1,3,4-oxadiazol-2-
yl)thio)acetamide 8o
1
Yellow powder (0.41 g, 83.3% yield) mp 200-202°C; H NMR (500 MHz, DMSO-
d6) ppm)11.25 (s, 1H, NH), (d, 2H, J = 7.3 Hz, Ar-H), 7.96 (d, 1H, J = 15.3
Hz, CH=CH), 7.91-7.88 (m, 3H, Ar-H), 7.84 (d, 2H, J = 7.3 Hz, Ar-H), 7.73 (d, 1H,
25

J = 15.3 Hz, CH=CH), 7.55 (d, 1H, J = 7.7 Hz, Ar-H), 7.48-7.45 (m, 3H, Ar-H),
7.13 (d, 1H, J = 7.7 Hz, Ar-H), 4.45 (s, 2H, CH2), 3.84 (s, 3H, OCH3), 3.82 (s, 3H,
13
OCH3); C NMR (125 MHz, DMSO-d6)ppm)188.05, 166.19, 165.72, 163.00,
152.34, 149.54, 143.99, 143.54, 135.21, 131.02, 130.48, 129.39, 129.31, 122.39,
120.45, 119.60, 119.06, 115.65, 112.46, 109.45, 56.49, 56.18, 37.36; LC−MS m/z
500.10 [M − H]−; Anal. Calcd for C27H23N3O5S: C, 64.66; H, 4.62; N, 8.38; S, 6.39.
Found: C, 64.89; H, 4.76; N, 8.62; S, 6.50.
4.1.6.16. N-(4-(3-(4-chlorophenyl)acryloyl)phenyl)-2-((5-(3,4-dimethoxyphenyl)-
1,3,4-oxadiazol-2-yl)thio)acetamide 8p
1
Pale yellow powder (0.32g, 60.7% yield) mp 239-241°C; H NMR (500 MHz,
DMSO-d6) ppm)10.88 ( s, 1H, NH), (d, 2H, J = 8.1 Hz, Ar-H), 7.97 (d, 1H,
J = 15.6 Hz, CH=CH), 7.93 (d, 2H, J = 7.8 Hz, Ar-H), 7.78 (d, 2H, J = 8.1 Hz, Ar-
H), 7.71 (d, 1H, J =15.6 Hz, CH=CH), 7.53 (d, 3H, J = 8.4 Hz, Ar-H), 7.42 (s, 1H,
Ar-H), 7.12 (d, 1H, J = 8.4 Hz, Ar-H), 4.39 (s, 2H, CH2), 3.83 (s ,3H ,OCH3), 3.80 (s,
3H, OCH3); 13C NMR (125 MHz, DMSO-d6)ppm)187.94, 166.19, 165.73,
163.01, 152.33, 149.52, 143.57, 142.53, 135.48, 134.19, 131.01, 130.55, 129.60,
129.43, 123.10, 120.45, 119.06, 115.62, 112.44, 109.40, 56.17, 56.08, 37.36; Anal.
Calcd for C27H22ClN3O5S: C, 60.50; H, 4.14; N, 7.84; S, 5.98. Found: C, 60.76; H,
4.23; N, 8.11; S, 6.09.
4.1.6.17.
2-((5-(3,4-Dimethoxyphenyl)-1,3,4-oxadiazol-2-yl)thio)-N-(4-(3-(4-
methoxyphenyl)acryloyl)phenyl)acetamide 8q
1
Yellow solid (0.34g, 65.3% yield) mp 189-190°C; H NMR (500 MHz, DMSO-d6 )
ppm)10.82 (s, 1H, NH), (d, 2H, J = 6.4 Hz, Ar-H), 7.86 (d, 2H, J = 6.0 Hz,
Ar-H), 7.80- 7.77 (m, 3H, 2 Ar-H + CH=CH), 7.71 (d, 1H, J = 15.3 Hz, CH=CH),
7.54 (d, 1H, J = 6.3 Hz, Ar-H), 7.43 (s, 1H, Ar-H), 7.13 (d, 1H, J = 6.3, Ar-H), 7.03
(d, 2H, J = 6.4 Hz, Ar-H), 4.40 (s, 2H, CH2 ), 3.84 (s, 6H, 2 OCH3), 3.82 (s, 3H,
OCH3); 13C NMR (125 MHz, DMSO-d6)ppm)187.69, 166.23, 165.72, 162.99,
161.76, 152.32, 149.52, 143.97, 143.42, 133.37, 131.21, 130.28, 127.84, 120.47,
119.86, 119.04, 115.64, 114.88, 112.46, 109.46, 56.19, 56.13, 55.86, 37.26; LC−MS
m/z 530.20 [M − H]−; Anal. Calcd for C28H25N3O6S: C, 63.26; H, 4.74; N, 7.90; S,
6.03. Found: C, 63.14; H, 4.89; N, 8.16; S, 6.15.
26

4.1.6.18. 2-((5-(3,4-Dimethoxyphenyl)-1,3,4-oxadiazol-2-yl)thio)-N-(4-(3-(3,4-di-
methoxyphenyl)acryloyl)phenyl)acetamide 8r
Yellow solid (0.37 g, 66.0% yield) mp 213-214°C; 1H NMR (500 MHz, DMSO-d6)
ppm)10.83 (s, 1H, NH), (d, 2H, J = 6.1 Hz, Ar-H), 7.84 (d, 1H, J = 15.2
Hz, CH=CH), (d, 2H, J = 6.1 Hz, Ar-H), 7.70 (d, 1H, J = 15.2 Hz, CH=CH),
7.55-7.51 (m, 2H, Ar-H), 7.43 (s, 1H, Ar-H), 7.39 (d, 1H, J = 5.8 Hz, Ar-H), 7.13 (d,
1H, J = 6.6 Hz, Ar-H), 7.03 (d, 1H, J = 5.8, Ar-H), 4.40 (s, 2H, CH2), 3.87 (s, 3H,
OCH3), 3.84 (s, 3H, OCH3), 3.82 (s, 6H, 2 OCH3); 13C NMR (DMSO-d6)ppm)
187.90, 166.11, 165.73, 163.02, 152.36, 151.69, 149.56, 149.49, 144.53, 143.30,
133.47, 130.37, 128.05, 124.40, 120.45, 119.89, 119.01, 115.66, 112.47, 112.01,
111.12, 109.47, 56.22, 56.19, 56.11, 56.07, 37.42; LC−MS m/z 562.00 [M + H]+;
Anal. Calcd for C29H27N3O7S: C, 62.02; H, 4.85; N, 7.48; S, 5.71. Found: C, 62.27;
H, 4.97; N, 7.62; S, 5.89.
4.1.6.19. 2-((5-(3,4-Dimethoxyphenyl)-1,3,4-oxadiazol-2-yl)thio)-N-(4-(3-(3,4,5-
trimethoxyphenyl)acryloyl)phenyl)acetamide 8s
1
Yellow solid (0.42g, 72.2% yield) mp 210-212°C; H NMR (500 MHz, CDCl3)
ppm)9.86 (s, 1H, NH), (d, 2H, J = 8.0 Hz, Ar-H), 7.75-7.70 (m, 3H, 2 Ar-H,
CH=CH), 7.60 (d, 1H, J = 7.0 Hz, Ar-H), 7.53 (s, 1H, Ar-H), 7.40 (d, 1H, J = 15.0
Hz, CH=CH), 6.99 (d, 1H, J = 8.0, Ar-H), 6.87 (s, 2H, Ar-H), 4.09 (s, 2H ,CH2),
3.98 (s, 6H, 2OCH3), 3.94 (s, 6H, 2 OCH3), 3.92 (s, 3H, OCH3); 13C NMR (125
MHz, CDCl3)ppm)189.05, 166.62, 165.87, 164.53, 153.48, 152.51, 149.47,
144.83, 141.86, 140.35, 134.15, 130.42, 129.93, 121.19, 120.61, 119.33, 115.26,
111.21, 109.14, 105.60, 61.04, 56.25, 56.21, 56.13, 36.35; LC−MS m/z 590.20 [M −
H]−; Anal. Calcd for C30H29N3O8S: C, 60.90; H, 4.94; N, 7.10; S, 5.42. Found: C,
61.23; H, 4.89; N, 7.24; S, 5.58.
4.1.6.20. N-(4-Cinnamoylphenyl)-2-((5-(3,4,5-trimethoxyphenyl)-1,3,4-oxadiazol-
2-yl)thio)acetamide 8t
1
Pale yellow crystals (0.38g,71.8% yield) mp 219-221°C; H NMR (500 MHz,
DMSO-d6) ppm)10.84 (s, 1H, NH), (d, 2H, J = 6.7 Hz, Ar-H), 7.95 (d, 1H,
J = 15.5 Hz, CH=CH), 7.90-7.88 (m, 2H, Ar), 7.79 (d, 2H, J = 6.7 Hz, Ar-H), 7.74
27

(d, 1H, J = 15.5 Hz, CH=CH), 7.49-7.45 (m, 3H, Ar-H), 7.22 (s, 2H, Ar-H), 4.42 (s,
2H, CH2), 3.84 (s, 6H, 2OCH3), 3.74 (s, 3H, OCH3), 13C NMR (125 MHz,
CDCl3)ppm)188.98, 166.54, 165.74, 164.83, 153.80, 144.64, 141.89, 141.61,
130.52, 129.92, 129.50, 128.97, 128.46, 121.79, 120.06, 119.31, 117.85, 104.11,
61.07, 56.43, 36.39; LC−MS m/z 531.10 [M]−; Anal. Calcd for C28H25N3O6S:
C, 63.26; H, 4.74; N, 7.90; S, 6.03. Found: C, 63.54; H, 4.65; N, 8.12; S, 6.15.
4.1.6.21.
N-(4-(3-(4-Chlorophenyl)acryloyl)phenyl)-2-((5-(3,4,5-trimethoxy-
phenyl)-1,3,4-oxadiazol-2-yl)thio)acetamide 8u
Yellow solid (0.49g, 87.5% yield) mp 259-261°C; 1H NMR (500 MHz, DMSO-d6)
ppm)10.85 (s, 1H, NH), (d, 2H, J = 6.6 Hz, Ar-H), 7.98 (d, 1H, J = 16.0
Hz, CH=CH), 7.94 (d, 2H, J = 6.4 Hz, Ar-H), 7.79 (d, 2H, J = 6.4 Hz, Ar-H), 7.72
(d, 1H, J = 16.0 Hz, CH=CH), 7.54 (d, 2H, J = 6.6 Hz, Ar-H), 7.22 (s, 2H, Ar-H),
4.80 (s, 2H, CH2), 3.97 (s, 6H, 2 OCH3), 3.95 (s, 3H, OCH3), 13C NMR (125 MHz,
DMSO-d6)ppm) 187.91, 166.12, 165.62, 163.62, 153.93, 143.56, 142.52, 141.03,
135.47, 134.22, 133.08, 131.02, 130.55, 129.43, 123.14, 119.05, 118.59, 104.25,
60.71, 56.60, 37.43; LC−MS m/z 564.10 [M − H]−; Anal. Calcd for C28H24ClN3O6S:
C, 59.41; H, 4.27; N, 7.42; S, 5.67. Found: C, 59.32; H, 4.35; N, 7.68; S, 5.73.
4.1.6.22.
N-(4-(3-(4-Methoxyphenyl)acryloyl)phenyl)-2-((5-(3,4,5-trimethoxy-
phenyl)-1,3,4-oxadiazol-2-yl)thio)acetamide 8v
Yellow crystals (0.47g,84% yield) mp 211-212°C; 1H NMR (500 MHz, DMSO-d6)
ppm)10.83 (s, 1H, NH), (d, 2H, J = 6.9 Hz, Ar-H), 7.86-7.82 (m, 3H, 2
Ar-H + CH=CH), 7.78 (d, 2H, J = 7.6 Hz, Ar-H), 7.71 (d, 1H, J = 15.2 Hz,
CH=CH), 7.22 (s, 2H, Ar-H), 7.03 (d, 2H, J = 6.9, Ar-H), 4.42 (s, 2H, CH2), 3.84 (s,
9H, 3OCH3), 3.74 (s, 3H, OCH3);13C NMR (125 MHz, DMSO-d6)ppm)187.96,
169.22, 165.75, 165.02, 161.78, 153.90, 144.00, 143.22, 140.82, 133.50, 131.21,
130.26, 127.84, 119.84, 119.21, 118.83, 114.88, 104.13, 60.69, 56.62, 55.86, 37.37;
LC−MS m/z 561.90 [M + H]+; Anal. Calcd for C29H27N3O7S: C, 62.02; H, 4.85; N,
7.48; S, 5.71. Found: C, 62.19; H, 4.98; N, 7.67; S, 5.92.
4.1.6.23.
N-(4-(3-(3,4-Dimethoxyphenyl)acryloyl)phenyl)-2-((5-(3,4,5-
trimethoxyphenyl)-1,3,4-oxadiazol-2-yl)thio)acetamide 8w
28

Yellow solid (0.32g, 55.0% yield) mp 168-170°C; 1H NMR (500 MHz, DMSO-d6)
ppm)10.98 (s, 1H, NH), (d, 2H, J = 8.0 Hz, Ar-H), 7.83-7.79 (m, 3H, 2 Ar-
H + CH=CH), 7.80 (d, 1H, J =15.0 Hz, CH=CH), 7.53 (s, 1H, Ar-H), 7.38 (d, 1H,
J = 8.0 Hz Ar-H), 7.21 (s, 2H, Ar-H), 7.01 (d, 1H J = 8.0 Hz, Ar- H), 4.43 (s, 2H,
13
CH2), 3.86 (s, 6H, 2 OCH3), 3.83 (s, 6H, 2 OCH3), 3.73 (s, 3H, OCH3); C NMR
(125 MHz, DMSO-d6)ppm)188.00, 166.12, 165.61, 163.62, 153.92, 151.68,
149.47, 144.53, 143.35, 141.00, 133.43, 130.35, 128.03, 124.41, 119.88, 119.01,
118.59, 112.00, 111.10, 104.23,  Anal. Calcd for
C30H29N3O8S: C, 60.90; H, 4.94; N, 7.10; S, 5.42. Found: C, 61.23; H, 5.12; N, 7.37;
S, 5.39.
4.1.6.24. 2-((5-(3,4,5-Trimethoxyphenyl)-1,3,4-oxadiazol-2-yl)thio)-N-(4-(3-(3,4,5-
trimethoxyphenyl)acryloyl)phenyl)acetamide 8x
1
Yellow solid (0.49g, 80% yield) mp 196-198°C; H NMR (500 MHz, CDCl3)
ppm)9.74 (s, 1H, NH), (d, 2H, J = 8.1 Hz, Ar-H), 7.74-7.70 (m, 3H, 2 Ar-
H + CH=CH), 7.39 (d, 1H, J = 15.6 Hz, CH=CH), 7.24 (s, 2H, Ar-H), 6.87 (s, 2H,
Ar-H), 4.09 (s, 2H, CH2), 3.95 (s, 6H, 2 OCH3), 3.94 (s, 9H, 3 OCH3), 3.92 (s, 3H,
OCH3), 13C NMR (125 MHz, CDCl3ppm)189.03, 166.57, 165.77, 164.79,
153.79, 153.49, 144.84, 141.80, 141.58, 140.44, 134.22, 130.41, 129.91, 121.18,
119.31, 117.87, 105.69, 104.08, 61.07, 61.02, 56.41, 56.25, 36.31; LC−MS m/z
620.10 [M − H]−; Anal. Calcd for C31H31N3O9S: C, 59.89; H, 5.03; N, 6.76; S, 5.16.
Found: C, 59.71; H, 5.21; N, 6.94; S, 5.24.
4.2.Biology section 
Cell culture and reagents
Human leukemia cell lines (K-562, KG1a, and Jurkat) were obtained from the
American Type Culture Collection (ATCC; Manassas, VA, USA) and were cultured
in Iscove's Modified Dulbecco's Medium (IMDM, Sigma-Aldrich) or Roswell Park
Memorial Institute Medium (RPMI 1640, Sigma-Aldrich), containing 10% fetal
bovine serum (FBS; Sigma-Aldrich) in a humidified atmosphere with 5% CO2 at
37°C. Cisplatin, dasatinib, dexamethasone, gefitinib, and WP1066 were purchased
from Sigma-Aldrich. All chemicals used in this study were analytical or cell-culture
grade.
29