Novel 1,3,4-oxadiazole thioether derivatives targeting thymidylate synthase as dual anticancer/antimicrobial agents

Article abstract of DOI:10.1016/j.bmc.2013.02.008

A series of novel 1,3,4-oxadiazole thioether derivatives (compounds 9-44) were designed and synthesized as potential inhibitors of thymidylate synthase (TS) and as anticancer agents. The in vitro anticancer activities of these compounds were evaluated against three cancer cell lines by the MTT method. Among all the designed compounds, compound 18 bearing a nitro substituent exhibited more potent in vitro anticancer activities with IC₅₀ values of 0.7 ± 0.2, 30.0 ± 1.2, 18.3 ± 1.4 μM, respectively, which was superior to the positive control. In the further study, it was identified as the most potent inhibitor against two kinds of TS protein (for human TS and Escherichia coli TS, IC₅₀ values: 0.62 and 0.47 μM, respectively) in the TS inhibition assay in vitro and the most potent antibacterial agents with MIC (minimum inhibitory concentrations) of 1.56-3.13 μg/mL against the tested four bacterial strains. Molecular docking and 3D-QSAR study supported that compound 18 can be selected as dual antitumor/antibacterial candidate in the future study.

Full text of DOI:10.1016/j.bmc.2013.02.008

Accepted Manuscript
Novel 1,3,4-Oxadiazole Thioether Derivatives Targeting Thymidylate Synthase
as Dual Anticancer/Antimicrobial Agents
Qian-Ru Du, Dong-Dong Li, Ya-Zhou Pi, Jing-Ran Li, Jian Sun, Fei Fang, Hai-
Bin Gong, Hai-Liang Zhu
PII:
S0968-0896(13)00132-6
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.02.008
BMC 10603
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Please cite this article as: Du, Q-R., Li, D-D., Pi, Y-Z., Li, J-R., Sun, J., Fang, F., Gong, H-B., Zhu, H-L., Novel
1,3,4-Oxadiazole Thioether Derivatives Targeting Thymidylate Synthase as Dual Anticancer/Antimicrobial Agents,
Bioorganic & Medicinal Chemistry (2013), doi: http://dx.doi.org/10.1016/j.bmc.2013.02.008
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Novel 1,3,4-Oxadiazole Thioether Derivatives Targeting Thymidylate Synthase
as Dual Anticancer/Antimicrobial Agents
Qian-Ru Du a+, Dong-Dong Li a+, Ya-Zhou Pi a, Jing-Ran Li a, Jian Sun a, Fei Fang a,
Hai-Bin Gong b*, Hai-Liang Zhu a*
a State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University,
Nanjing 210093, P. R. China.
b Xuzhou Central Hospital, Xuzhou 221009, P. R. China.
[+] These authors contributed equally to this work.
* Corresponding authors. Tel./fax: +86-25-83592672;
E-mail address: zhuhl@nju.edu.cn
Abstract: A series of novel 1,3,4-oxadiazole thioether derivatives (compounds 9-44)
were designed and synthesized as potential inhibitors of thymidylate synthase (TS)
and as anticancer agents. The in vitro anticancer activities of these compounds were
evaluated against three cancer cell lines by the MTT method. Among all the designed
compounds, compound 18 bearing a nitro substituent exhibited more potent in vitro
anticancer activities with IC50 values of 0.7 0.2 μM, 30.0 1.2 μM, 18.3 1.4 μM,
respectively, which was superior to the positive control. In the further study, it was
identified as the most potent inhibitor against two kinds of TS protein (for human TS
and E. coli TS, IC50 values: 0.62 μM and 0.47 μM, respectively) in the TS inhibition
assay in vitro and the most potent antibacterial agents with MIC (minimum inhibitory
concentrations) of 1.56-3.13 μg/mL against the tested four bacterial strains. Molecular
docking and 3D-QSAR study supported that compound 18 can be selected as dual
antitumor/antibacterial candidate in the future study.
Keywords: Thymidylate Synthase; 1,3,4-Oxadiazoles; Metronidazole; X-ray
crystallography; Anticancer activities
1

1. Introduction
Thymidylate synthase (TS) has long been recognized as an important and
attractive target for chemotherapy because of its vital role in DNA biosynthesis.1 It
catalyzes the reductive methylation of 2’-deoxyuridine 5’-monophosphate (dUMP) to
2’-deoxythymidine 5’-monophosphate (dTMP), which could be converted to a
triphosphate (dTTP) by deoxyribonucleoside diphosphate kinase and subsequently be
incorporated into DNA. This reaction provides a sole de novo pathway for the
production of dTMP and the oxidization of 5,10-methylenetetrahydrofolate during
one-carbon transfer.2 TS inhibition, which leads to deoxynucleotide (dNTP) pool
imbalances, is an attractive strategy for the development of antitumor agents.3 In the
early 1950s, 5-fluorouracil (5-FU) as a pyrimidine analog which works through
irreversible inhibition of TS, was a prominent antitumor agent. The emergence of
resistance as well as the insensitivity of certain tumor types to 5-FU has triggered the
design of novel folate analogs as potential TS inhibitors and anticancer agents.4
Several compounds with dual inhibitory activity of TS and dihydrofolate reductase
(DHFR) in Figure 1, such as Raltitrexed (ZD1694) and Pemetrexed (LY231514)，
have been approved as antitumor agents5-6. These drugs that possess a benzoyl
L-glutamic acid side chain similar to natural folates could be polyglutamated by folyl
polyglutamate synthetase (FPGS) 7 to the pentaglutamate forms which could be easily
transferred into cells, leading to high intracellular concentrations of these antitumor
agents. Although polyglutamylation can increase potent inhibitory activity, it is also
involved with the toxicity in host cells and can not circumvent tumors with low levels
of FPGS.8 Facing the problems associated with classical antifolates, several strategies
have been put forward, such as designing nonclassical lipophilic antifolate analogs
that would not dependent on FPGS for their potency, or designing novel 5-FU analogs
only targeting TS. As displayed in Figure 2, the first nonclassical TS inhibitor,
AG337 (nolatrexed) has been studied under a series of clinical trials;9 Gangjee et al.
also designed several potent TS inhibitors (1-3) based on the X-ray crystal structure of
2

TS and AG337.8,10-11 Although other important successful works have been completed
in the field of nonclassical antifolate, TS will still present ample opportunities for
scientists in drug discovery field .
(Figure 1)
(Figure 2)
1,3,4-Oxadiazoles possessing a variety of biological activities12-15which belongs to
an important class of heterocyclic compounds. The widespread use of them as a
scaffold in medicinal chemistry establishes this moiety as a member of the privileged
structures class. In particular, a few of differently substituted 1,3,4-oxadiazoles have
been found to exhibit anticancer activities.16-17 Moreover, 1,3,4-oxadiazole
heterocycles are very good bioisosteres of amides and esters, which contribute
substantially to increasing pharmacological activity by participating in hydrogen
bonding interactions with the receptors.18 Nitroimidazoles are extensively used as
antimicrobial chemotherapeutics and as antiangiogenic hypoxic cell radiosensitizers.19
Their derivatives have attracted considerable attentions due to showing a tendency to
penetrate and accumulate in regions of tumors20-21 and can undergo bioreduction to
yield electrophilic substances that can attack proteins and nucleic acids.22 Importantly,
the toxicology and metabolism of nitroimidazoles, particularly metronidazole, have
been characterized.23-24 Thus, nitroimidzaoles may provide an attractive possibility for
employing these molecules as carriers to targeted delivery in cancer therapy.25-26
Recently, Swenson et al. synthesized a 10B-enriched nitroimidazole by coupling the
Cs salt of BSH (Cs2-10B12H11SH) with 1-(2-bromoethyl)-2-methyl-5-nitroimidazole
and it was used for boron neutron capture therapy of cancer.27
Based on the statements above, a bold attempt to design a series of novel
scaffold TS inhibitors by coupling 1,3,4-oxadiazole with metronidazole moiety has
been put forward for the first time. Figure 1 shows in detail the composition of these
novel TS inhibitors, in which the metronidazole part seems like a warhead that
3

directs the whole molecule to the hypoxic tissue, such as the regions of solid tumors;
while 1,3,4-oxadiazole part would presumably mimics 5-FU as a pharmacophore. In
aim to investigate whether the designed compounds could work on TS protein,
docking simulations were performed on the two known TS protein crystal structure
(PDB code: 1HVY derived from human TS, and 2KCE derived from E. coli TS).
The obtained results have been presented in Figure 3, describing the binding energy
and CDOCKER_INTERACTION_ENERGY of all the designed compounds.
Compared with the positive drug, AG337, most of the designed molecules below the
blue dash lines possess lower binding energies and interaction energies,
demonstrating these compounds are likely to exhibit more potent against both of the
two protein targets. Figure 3A showed the binding energies of designed molecules
interacting with two TS protein targets, among which two compounds 18, 37 worked
very well both in 2KCE and 1HVY proteins. Figure 3B also displayed the
alternative measure of the protein-ligand interaction by CDOCKER-
_INTERACTION_ENERGY which was used to accurately estimate the results of
the docking study.28 Taken together from the two pictures, compound 18 performed
best in the two kinds of evaluation programs, in which its binding energy and
docking energy could reached up to -92.5608, and -46.2693 kcal/mol, respectively.
Therefore, these preliminary works would serve as a modest spur to induce us to
probe these 1,3,4-oxadiazole scaffold compounds. In this manuscript, we described
the synthesis and the structure activity relationships (SAR) of a series of novel
1,3,4-oxadiazole analogs as TS inhibitors and as dual antitumor/antimicrobial agents.
Moreover, according to the binding model, we established a 3D-QSAR model to
provide a marker post for the development of novel TS inhibitors based on the
1,3,4-oxadiazole scaffold.
(Figure 3)
2. Results and discussion
2.1. Chemistry
4

The key intermediates 6 were prepared in three steps by the procedure shown in
Scheme 1 17. Esterification of the carboxylic acids with the mixture of ethanol and
concentrated sulfuric acid afforded the corresponding esters 4. The aroyl hydrazides 5
were obtained by the reaction of esters 4 with 85% hydrazine hydrate in ethanol.
Treatment of the hydrazides 5 with carbon disulfide in the presence of KOH in 95%
ethanol
under
reflux
gave
the
key
intermediates
6.
1-(2-Bromoethyl)-2-methyl-5-nitro-1H-imidazole
8
was synthesized from
metronidazole by bromizing as previously described (Scheme 2)29. The synthesis of
the target compounds 9-44 was accomplished by refluxing compounds 6 with
1-(2-bromoethyl)-2-methyl-5-nitro-1H-imidazole 8 in the presence of NaOEt in EtOH
(Scheme 3). The resulting 9-44 are all new compounds.
(Scheme 1)
(Scheme 2)
(Scheme 3)
All of the synthetic compounds have presented both satisfactory analytical and
spectroscopic data, which were in full accordance with their depicted structures.
Additionally, the structure of compound 9 was further confirmed by X-ray diffraction
and its crystal data was presented in Table 1. Besides, Figure 4 gave a perspective
view of this compound together with the atomic labeling system.
(Table 1)
(Figure 4)
2.2. Bioassays and SAR
5

All newly synthesized compounds 9-44 were evaluated for their in vitro
anticancer activities against HepG2 (human hepatoma cells), SGC-7901 (human
gastric cancer cells), and MCF-7 (human breast cancer cells) cell lines. The results
were summarized in Table 2. A number of 1,3,4-oxadiazole thioether derivatives
showed remarkable effects on anticancer activities. To our delight, all compounds
9-44 displayed potent inhibition activities against HepG2 cell lines. Among the
compounds tested, compounds 11, 17, 18, 21, 40 exhibited potent inhibitory activities
against HepG2 cell lines (IC50 < 3 μM; as for 11, IC50 = 2.2 0.2 μM; as for 17, IC50
= 1.9 0.4 μM; as for 18, IC50 = 0.7 0.2 μM; as for 21, IC50 = 0.6 0.2 μM; as for
40, IC50 = 0.9
0.3 μM), which were more potent than the positive control
5-fluorouracil (5-FU). Besides, it was worth mentioning that eight target compounds
(11, 13, 17, 19, 21, 31, 39 and 44) showed potent anticancer activities against the
other SGC-7901, and MCF-7 cell lines, all of which were more potent than the
positive control 5-FU.
(Table 2)
Generally, structure-activity relationships (SAR) at the cellular level for these
1,3,4-oxadiazole thioether derivatives demonstrated that compounds with substituents
at para position of benzene ring had a moderate increase of activity than those at
ortho position, comparing compounds (11, 13, 15, 17, 21, 23) to compounds (10, 12,
14, 16, 20, 22), respectively. Unlike these compounds, compound 19 bearing a nitro
substituent at para position of the phenyl ring was actually inferior to the
corresponding compound 18 with nitro substituent located at ortho position of the
phenyl ring, which indicated that nitro group at the proper position of the benzene
ring may play an important role on the anticancer activities. Moreover, Table 2 also
listed other designed molecules in which the phenyl ring was replaced by five kinds of
the heterocycles (furan: 33; thiophene: 34; pyridine: 35-41; naphthalene: 42, 43;
deoxygenated ring: 44). In general, most compounds exhibited modest
anti-proliferative activities against the three cancer cells except for 40, and the
6

replacement of the pyridine ring was more likely to increase compounds’ biological
activity than those of other sorts of rings. On the other hand, electron withdrawing
groups of the phenyl ring performed better than electron donating groups on
anti-proliferative activity, no matter the replacement at ortho position or para position,
and the similar influence was also observed on heterocycle substitution. Disubstituted
or three substituted donating groups could provide a little effect on improving
anti-proliferative activity when referring to 28-32. However, compounds 32, 37 were
considered as the two best compounds in the analysis of the molecular docking study
whereas they performed not well in the “wet experiment” above. In the next step,
eleven compounds including 32 and 37, would be selected and be further evaluated
for the TS inhibition assay.
(Table 3)
(Table 4)
Compounds 11, 17, 18, 21, 25, 26, 32, 37, 40, 41 and 44, were evaluated as
inhibitors of human TS and Escherichia coli TS (Table 3). All of the compounds were
moderate to potent inhibitors of human TS, in which their IC50 values were in the
range of 0.62-3.9 μM against hTS. Compounds 18 and 40 were two most potent
inhibitors of hTS and E. coli TS that comparable in potency to clinically used analog
Raltitrexed (ZD1694). These two compounds (18, 40) were approximately 15- and
10-fold more potent than Pemetrexed (LY231514), respectively. In addition, it was
noticeable that although the two predicted potent inhibitors (32, 37) showed better
inhibitory activities in the TS assay in vitro, the possible explanation for the loss in
potency of anticancer cells could be probably attributed to higher molecular weight
(as to 32, MW: 421.428) and lower ALOGP values (as to 37, ALOGP: 0.886). As
summarized in Table 3, compounds 18, 40 possessed potent inhibitory activities
against both hTS and E. coli TS, and subsequently would be picked up to test their
relative antibacterial activity. Taken together, compounds (18, 37, 40) were evaluated
7

for antibacterial activity against two Gram-negative bacterial strains: E. coli and P.
fluorescence and two Gram-positive bacterial strains: B. subtilis and S. aureus by the
MTT method. Table 4 presented in detail the antibacterial data of these dual
functional agents. Compound 18 showed the most potent antibacterial activity with
MIC (minimum inhibitory concentrations) of 1.56–3.13 μg/mL against the tested
bacterial strains, comparable in potency to clinically known antibacterial agent
kanamycin, and could be selected to improve as dual antitumor/antibacterial agent in
the future study.
(Figure 5)
A docking study of the most active compound 18 and the two TS protein was
performed to validate the structure-activity relationship (SAR) of similar compounds.
As seen in Figure 5, the structures of 18 had been docked in the crystal structure of
human TS and E. coli TS (PDB ID: 1HVY and 2KCE), respectively. Docking results
(Figure 5A and 5C) revealed that amino acid Asn 226 in the binding pocket of 1HVY
protein and three amino acids Trp80, Trp83, His207 in the binding pocket of 2KCE
protein played vital roles in the conformation of 18, which were stabilized by
Pi-Cation bond, Pi-Pi bond, and hydrogen bond that shown in 2D diagram. Besides,
the lead compound was described to stretch up in the binding site to reduce the
interaction energy.
In order to obtain a systematic SAR profile on 1,3,4-oxadiazole thioether analogs
as antitumor agents and to explore the more potent and selective TS inhibitors,
3D-QSAR model was built using the corresponding pIC50 values which were
converted from the obtained IC50 values (μM) of human TS inhibition (the way of this
transformation was derived from an online calculator developed by an indian
medicinal chemistry lab (http://www.sanjeevslab.org/tools-IC50.html) and performed
by built-in QSAR software of DS 3.1 (Discovery Studio 3.1, Accelrys, Co. Ltd). The
training and test sets were divided by the random diverse molecules method of DS 3.1,
in which the training set accounted for 80% of all the molecules while the test set was
8

set to 20%. The training set was composed of 10 agents and the relative test set
comprised 3 agents, which had been presented in Table 5. One key issue in the
development of 3D-QSAR model was to determine the active conformation and how
to align these molecules reasonably. An efficient solution in this study mainly depends
on the docking study and the reliability of this method has been documented in
previous studies.30
(Table 5)
In default situation, the alignment conformation of each molecule was the one
that possessed the lowest CDOCKER_INTERACTION_ENENGY among the twenty
docked poses. The 3D-QSAR model which generated from DS 3.1, defined the
critical regions (steric or electrostatic) affecting the binding affinity. It was a PLS
model built on 400 independent variables (conventional R2= 0.87). The observed and
predicted values and the corresponding residual values for the training set and test set
molecules in 3D-QSAR model were listed in Table 5. Moreover, their graphical
relationship was illustrated in Figure 6A, in which the plot of observed IC50 versus
the predicted results showed that this model could be used in prediction of activity for
novel 1,3,4-oxadiazole thioether derivatives as TS inhibitors.
(Figure 6)
A contour plot of the electrostatic field region favorable (in blue) or unfavorable
(red) for anticancer activity based on TS protein target were displayed in Figure 6B
while the energy grids corresponding to the favorable (in green) or unfavorable
(yellow) steric effects for the TS affinity were displayed in Figure 6C. It was widely
acceptable that a better inhibitor based on the 3D QSAR model should have a strong
Van der Waals attraction in the green areas and a polar group in the blue electrostatic
potential areas (which were dominant close to the skeleton). As expected, those potent
compounds (11, 17, 18 and so on) not only could circumvent the red subregion or the
9

unfavorable yellow steric subregion but also can get more close to the favorable blue
and green spaces. Thus, this promising model would provide a guideline to design and
optimize more effective TS inhibitors and pave the way for us in the further study.
3. Conclusion
In summary, a series of novel 1,3,4-oxadiazole thioether derivatives based on
1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole (metronidazole) scaffold have been
synthesized and the cytotoxic activities of all the compounds were assessed against
three cancer cell lines. The SAR for these designed compounds against human TS
reveals that the sort and position of the substituent(s) on the phenyl ring or
heterocycle are important. It can be simply concluded that the nature of the electron
withdrawing group at 4’-position of the phenyl ring is detrimental to potent TS
inhibition. In particular, compound 18 bearing nitro substituent exhibits more potent
in vitro anticancer activities with IC50 value of 0.7 0.2 μM, 30.0 1.2 μM, 18.3
1.4 μM, respectively, which was superior to the positive control. In the further study,
it can be identified as the most potent inhibitor against two kinds of TS proteins (as
for hTS and E. coli TS, IC50 values: 0.62 μM and 0.47μM, respectively) in the TS
assay in vitro and the most potent antibacterial agent with MIC (minimum inhibitory
concentrations) of 1.56-3.13 μg/mL against the tested four bacterial strains. Molecular
docking and 3D-QSAR study support that compound 18 can be selected as dual
antitumor/antibacterial candidate in the future study.
4. Experimental
4.1. Chemistry
All chemicals and reagents used in current study were analytical grade. The
reactions were monitored by the thin layer chromatography (TLC) on Merck
pre-coated silica GF254 plates. Melting points (uncorrected) were determined on a
XT4MP apparatus (Taike Corp., Beijing, China). ESI mass spectra were obtained on a
1
Mariner System 5304 mass spectrometer, and H NMR spectra were collected on a
10

Bruker DPX500 or DPX300 spectrometer at room temperature with TMS and solvent
signals allotted as internal standards. Chemical shifts were reported in ppm (δ).
Elemental analyses were performed on a CHN-O-Rapid instrument, and were within
0.4% of the theoretical values.
4.1.1. General procedure for the preparation of target compounds 9-44
To a stirred solution of compound 6 (2 mmol) and sodium ethoxide (0.16 g, 2.4
mmol)
in
anhydrous
ethanol
(30
mL),
1-(2-bromoethyl)-2-methyl-5-nitro-1H-imidazole 8 (0.47 g, 2 mmol) was added. The
resulting mixture was heated under reflux for 8-36 h, and the reaction was monitored
by the thin layer chromatography (TLC). Afterwards the solution was cooled to room
temperature and the organic solvent was removed in vacuo. The residue was dissolved
in dichloromethane and the organic layer was washed with water and saturated brine,
respectively. Then the organic phase was dried over anhydrous Na2SO4, filtered, and
removed in vacuo. The purification of the residue by recrystallization from anhydrous
ethanol afforded the desired compounds 9-44.
4.1.1.1.
2-((2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)thio)-5-phenyl-1,3,4-oxadiazole (9)
Light brown powder, yield: 60.9%, mp: 119-120 . 1H NMR (300 MHz, CDCl3)
δ: 2.61 (s, 3H), 3.66 (t, J = 6.9 Hz, 2H), 4.83 (t, J = 6.9 Hz, 2H), 7.49-7.59 (m, 3H),
7.99-8.03 (m, 3H). 13C NMR (100 MHz, CDCl3) δ: 162.34, 159.39, 149.72, 139.65,
133.46, 131.57, 129.06, 127.08, 126.66, 42.18, 26.59, 16.82. MS (ESI): 332.1
(C14H14N5O3S, [M+H]+). Anal. Calcd for C14H13N5O3S: C, 50.75; H, 3.95; N, 21.14;
Found: C, 50.63; H, 3.96; N, 21.17.
4.1.1.2.
2-((2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)thio)-5-(o-tolyl)-1,3,4-oxadiazole (10)
Light yellow powder, yield: 65.6%, mp: 84-85 . 1H NMR (500 MHz, CDCl3) δ:
2.63 (s, 3H), 2.73 (s, 3H), 3.69 (t, J = 6.8 Hz, 2H), 4.87 (t, J = 6.8 Hz, 2H), 7.34-7.39
11

(m, 2H), 7.46 (t, J = 7.5 Hz, 1H), 7.91 (d, J = 7.5 Hz, 1H), 8.01 (s, 1H). 13C NMR
(100 MHz, CDCl3) δ: 162.34, 149.72, 148.76, 139.65, 136.59, 133.46, 130.81, 129.37,
128.75, 128.51, 126.53, 42.18, 26.59, 20.24, 16.82. MS (ESI): 346.1 (C15H16N5O3S,
[M+H]+). Anal. Calcd for C15H15N5O3S: C, 52.16; H, 4.38; N, 20.28; Found: C, 52.24;
H, 4.35; N, 20.31.
4.1.1.3.
2-((2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)thio)-5-(p-tolyl)-1,3,4-oxadiazole (11)
Light yellow crystals, yield: 61.1%, mp: 182-183 . 1H NMR (300 MHz, CDCl3)
δ: 2.44 (s, 3H), 2.60 (s, 3H), 3.65 (t, J = 7.0 Hz, 2H), 4.83 (t, J = 7.0 Hz, 2H), 7.32 (d,
J = 8.0 Hz, 2H), 7.90 (d, J = 8.3 Hz, 2H), 7.98 (s, 1H). 13C NMR (100 MHz, CDCl3)
δ: 162.34, 159.39, 149.72, 140.60, 139.65, 133.46, 129.10, 126.99, 125.47, 42.18,
26.59, 21.13, 16.82. MS (ESI): 346.1 (C15H16N5O3S, [M+H]+). Anal. Calcd for
C15H15N5O3S: C, 52.16; H, 4.38; N, 20.28; Found: C, 52.20; H, 4.34; N, 20.22.
4.1.1.4.
2-(2-methoxyphenyl)-5-((2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)thio)-1,3,4-oxadi
azole (12)
1
Brown crystals, yield: 57.0%, mp: 155-156 . H NMR (300 MHz, CDCl3) δ:
2.60 (s, 3H), 3.64 (t, J = 7.1 Hz, 2H), 3.98 (s, 3H), 4.84 (t, J = 7.1 Hz, 2H), 7.08 (t, J
= 7.4 Hz, 2H), 7.52 (dt, J1 = 7.9 Hz, J2 =1.6 Hz, 1H), 7.88 (dd, J1 = 8.0 Hz, J2 = 1.6
Hz, 1H), 7.97 (s, 1H). 13C NMR (100 MHz, CDCl3) δ: 162.34, 158.21, 151.42, 149.72,
139.65, 133.46, 131.99, 131.60, 120.56, 118.38, 115.31, 56.79, 42.18, 26.59, 16.82.
MS (ESI): 362.1 (C15H16N5O4S, [M+H]+). Anal. Calcd for C15H15N5O4S: C, 49.85; H,
4.18; N, 19.38; Found: C, 49.76; H, 4.17; N, 19.43.
4.1.1.5.
2-(4-methoxyphenyl)-5-((2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)thio)-1,3,4-oxadi
azole (13)
1
Brown crystals, yield: 59.2%, mp: 171-172 . H NMR (300 MHz, CDCl3) δ:
12

2.61 (s, 3H), 3.64 (t, J = 7.0 Hz, 2H), 3.89 (s, 3H), 4.82 (t, J = 7.0 Hz, 2H), 7.00-7.02
(d, J = 9.0 Hz, 2H), 7.93-7.98 (m, 3H). 13C NMR (100 MHz, CDCl3) δ: 162.34,
162.19, 159.39, 149.72, 139.65, 133.46, 127.07, 121.55, 113.88, 56.04, 42.18, 26.59,
16.82. MS (ESI): 362.1 (C15H16N5O4S, [M+H]+). Anal. Calcd for C15H15N5O4S: C,
49.85; H, 4.18; N, 19.38; Found: C, 49.83; H, 4.13; N, 19.42.
4.1.1.6.
2-(5-((2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)thio)-1,3,4-oxadiazol-2-yl)aniline
(14)
Dark green crystals, yield: 72.9%, mp: 127-128 . 1H NMR (500 MHz, CDCl3)
δ: 2.62 (s, 3H), 3.68 (t, J = 7.0 Hz, 2H), 4.85 (t, J = 7.0 Hz, 2H), 5.85 (brs, 2H),
6.78-6.84 (m, 2H), 7.31 (t, J = 7.8 Hz, 1H), 7.71 (d, J = 8.0 Hz, 1H), 8.01 (s, 1H). 13C
NMR (100 MHz, CDCl3) δ: 162.34, 150.82, 149.72, 144.39, 139.65, 133.46, 132.13,
125.59, 117.62, 117.43, 108.55, 42.18, 26.59, 16.82. MS (ESI): 347.1 (C14H15N6O3S,
[M+H]+). Anal. Calcd for C14H14N6O3S: C, 48.55; H, 4.07; N, 24.26; Found: C, 48.60;
H, 4.01; N, 24.25.
4.1.1.7.
4-(5-((2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)thio)-1,3,4-oxadiazol-2-yl)aniline
(15)
1
Brilliant yellow crystals, yield: 66.7%, mp: 212-213 . H NMR (300 MHz,
CDCl3) δ: 2.59 (s, 3H), 3.62 (t, J = 7.0 Hz, 2H), 4.06 (brs, 2H), 4.81 (t, J = 7.0 Hz,
2H), 6.73 (d, J = 8.6 Hz, 2H), 7.79 (d, J = 8.4 Hz, 2H), 7.97 (s, 1H). 13C NMR (100
MHz, CDCl3) δ: 162.34, 159.39, 156.25, 149.72, 139.65, 133.46, 127.68, 118.01,
114.55, 42.18, 26.59, 16.82. MS (ESI): 347.1 (C14H15N6O3S, [M+H]+). Anal. Calcd
for C14H14N6O3S: C, 48.55; H, 4.07; N, 24.26; Found: C, 48.61; H, 4.02; N, 24.29.
4.1.1.8.
2-(2-ethoxyphenyl)-5-((2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)thio)-1,3,4-oxadia
zole (16)
13

Tan powder, yield: 66.2%, mp: 100-101 . 1H NMR (500 MHz, CDCl3) δ: 1.50
(t, J = 7.0 Hz, 3H), 2.61 (s, 3H), 3.67 (t, J = 7.0 Hz, 2H), 4.22 (q , J = 7.0 Hz, 2H),
4.86 (t, J = 7.0 Hz, 2H), 7.06-7.10 (m, 2H), 7.52 (t, J = 7.5 Hz, 1H), 7.91 (d, J = 8.0
Hz, 1H), 8.00 (s, 1H). 13C NMR (100 MHz, CDCl3) δ: 162.34, 150.13, 149.72, 139.65,
134.72, 133.46, 129.88, 128.67, 128.66, 127.56, 73.21, 58.33, 42.18, 26.59, 16.82.
MS (ESI): 376.1 (C16H18N5O4S, [M+H]+). Anal. Calcd for C16H17N5O4S: C, 51.19; H,
4.56; N, 18.66; Found: C, 51.10; H, 4.53; N, 18.69.
4.1.1.9.
2-(4-ethoxyphenyl)-5-((2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)thio)-1,3,4-oxadi
azole (17)
Light yellow crystals, yield: 67.2%, mp: 151-152 . 1H NMR (500 MHz, CDCl3)
δ: 1.48 (t, J = 7.0 Hz, 3H), 2.63 (s, 3H), 3.66 (t, J = 7.0 Hz, 2H), 4.14 (q, J = 7.0 Hz,
2H), 4.85 (t, J = 7.0 Hz, 2H), 7.02 (d, J = 9.0 Hz, 2H), 7.95 (d, J = 8.5 Hz, 2H), 8.01
(s, 1H). 13C NMR (100 MHz, CDCl3) δ: 162.34, 159.39, 149.72, 141.37, 139.65,
133.46, 130.94, 127.28, 126.95, 76.15, 58.33, 42.18, 26.59, 16.82. MS (ESI): 376.1
(C16H18N5O4S, [M+H]+). Anal. Calcd for C16H17N5O4S: C, 51.19; H, 4.56; N, 18.66;
Found: C, 51.26; H, 4.51; N, 18.71.
4.1.1.10.
2-((2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)thio)-5-(2-nitrophenyl)-1,3,4-oxadiaz
ole (18)
Light yellow powder, yield: 48.5%, mp: 144-145 . 1H NMR (500 MHz, CDCl3)
δ: 2.62 (s, 3H), 3.69 (t, J = 6.8 Hz, 2H), 4.85 (t, J = 6.8 Hz, 2H), 7.80-7.84 (m, 2H),
7.95-7.96 (m, 1H), 8.02 (s, 1H), 8.11-8.13 (m, 1H). 13C NMR (100 MHz, CDCl3) δ:
162.34, 148.27, 146.01, 139.65, 133.93, 133.46, 132.32, 126.60, 126.12, 124.02,
42.18, 26.59, 16.82. MS (ESI): 377.1 (C14H13N6O5S, [M+H]+). Anal. Calcd for
C14H12N6O5S: C, 44.68; H, 3.21; N, 22.33; Found: C, 44.83; H, 3.15; N, 22.24.
4.1.1.11.
14

2-((2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)thio)-5-(4-nitrophenyl)-1,3,4-oxadiaz
ole (19)
1
Yellow powder, yield: 63.5%, mp: 199-200 . H NMR (500 MHz, CDCl3) δ:
2.62 (s, 3H), 3.72 (t, J = 7.0 Hz, 2H), 4.86 (t, J = 7.0 Hz, 2H), 8.01 (s, 1H), 8.24 (d, J
= 8.5 Hz, 2H), 8.42 (d, J = 8.5 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ: 162.34,
159.39, 147.39, 139.65, 133.46, 132.08, 127.45, 124.68, 42.18, 26.59, 16.82. MS
(ESI): 377.1 (C14H13N6O5S, [M+H]+). Anal. Calcd for C14H12N6O5S: C, 44.68; H,
3.21; N, 22.33; Found: C, 44.81; H, 3.20; N, 22.26.
4.1.1.12.
2-(2-fluorophenyl)-5-((2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)thio)-1,3,4-oxadiaz
ole (20)
1
Grayish white powder, yield: 56.9%, mp: 110-111 . H NMR (300 MHz,
CDCl3) δ: 2.59 (s, 3H), 3.68 (t, J = 7.1 Hz, 2H), 4.85 (t, J = 7.1 Hz, 2H), 7.30-7.35
13
(m, 2H), 7.55-7.61 (m, 1H), 7.99-8.05 (m, 2H). C NMR (100 MHz, CDCl3) δ:
162.34, 161.60, 149.72, 149.13, 133.46, 127.48, 124.49, 117.68, 117.49, 42.18, 26.59,
16.82. MS (ESI): 350.1 (C14H13FN5O3S, [M+H]+). Anal. Calcd for C14H12FN5O3S: C,
48.13; H, 3.46; N, 20.05; Found: C, 48.30; H, 3.51; N, 19.97.
4.1.1.13.
2-(4-fluorophenyl)-5-((2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)thio)-1,3,4-oxadiaz
ole (21)
Light yellow crystals, yield: 43.5%, mp: 186-187 . 1H NMR (500 MHz, CDCl3)
δ: 2.63 (s, 3H), 3.68 (t, J = 6.8 Hz, 2H), 4.84 (t, J = 6.8 Hz, 2H), 7.24 (t, J = 8.5 Hz,
2H), 8.00 (s, 1H), 8.03-8.06 (m, 2H). 13C NMR (100 MHz, CDCl3) δ: 165.21, 162.34,
159.39, 149.72, 133.46, 129.70, 123.55, 116.18, 42.18, 26.59, 16.82. MS (ESI): 350.1
(C14H13FN5O3S, [M+H]+). Anal. Calcd for C14H12FN5O3S: C, 48.13; H, 3.46; N, 20.05;
Found: C, 48.02; H, 3.46; N, 20.12.
4.1.1.14.
15

2-(2-chlorophenyl)-5-((2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)thio)-1,3,4-oxadia
zole (22)
1
Reddish-brown powder, yield: 56.1%, mp: 107-108 . H NMR (300 MHz,
CDCl3) δ: 2.61 (s, 3H), 3.67 (t, J = 7.0 Hz, 2H), 4.84 (t, J = 7.0 Hz, 2H), 7.40-7.45
(m, 1H), 7.46-7.52 (m, 1H), 7.56-7.58 (m, 1H), 7.93-7.96 (m, 1H), 7.98 (s, 1H). 13C
NMR (100 MHz, CDCl3) δ: 162.34, 149.72, 149.11, 139.65, 134.38, 133.46, 131.36,
131.12, 127.60, 127.11, 126.34, 42.18, 26.59, 16.82. MS (ESI): 366.0
(C14H13ClN5O3S, [M+H]+). Anal. Calcd for C14H12ClN5O3S: C, 45.97; H, 3.31; N,
19.15; Found: C, 45.88; H, 3.32; N, 19.19.
4.1.1.15.
2-(4-chlorophenyl)-5-((2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)thio)-1,3,4-oxadi
azole (23)
Light yellow powder, yield: 62.1%, mp: 205-207 . 1H NMR (300 MHz, CDCl3)
δ: 2.61 (s, 3H), 3.66 (t, J = 7.1 Hz, 2H), 4.83 (t, J = 7.1 Hz, 2H), 7.51 (d, J = 6.9 Hz,
2H), 7.94-7.98 (m, 3H). 13C NMR (100 MHz, CDCl3) δ: 162.34, 159.39, 149.72,
139.65, 137.31, 133.46, 129.46, 128.39, 128.09, 42.18, 26.59, 16.82. MS (ESI): 366.0
(C14H13ClN5O3S, [M+H]+). Anal. Calcd for C14H12ClN5O3S: C, 45.97; H, 3.31; N,
19.15; Found: C, 46.02; H, 3.28; N, 19.11.
4.1.1.16.
2-(5-((2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)thio)-1,3,4-oxadiazol-2-yl)phenol
(24)
Light brown crystals, yield: 62.9%, mp: 159-160 . 1H NMR (300 MHz, CDCl3)
δ: 2.60 (s, 3H), 3.69 (t, J = 6.9 Hz, 2H), 4.82 (t, J = 6.9 Hz, 2H), 7.02 (t, J = 7.9 Hz,
1H), 7.14 (d, J = 7.9 Hz, 1H), 7.46 (t, J = 7.9 Hz, 1H), 7.70 (dd, J1 = 7.8 Hz, J2 = 1.7
Hz, 1H), 7.98 (s, 1H), 9.74 (s, 1H). 13C NMR (100 MHz, CDCl3) δ: 162.34, 158.44,
149.72, 133.46, 130.12, 118.80, 111.70, 42.18, 26.59, 16.82. MS (ESI): 348.1
(C14H14N5O4S, [M+H]+). Anal. Calcd for C14H13N5O4S: C, 48.41; H, 3.77; N, 20.16;
Found: C, 48.32; H, 3.75; N, 20.12.
16

4.1.1.17.
2-(4-isopropylphenyl)-5-((2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)thio)-1,3,4-ox
adiazole (25)
Light yellow crystals, yield: 67.8%, mp: 130-131 . 1H NMR (500 MHz, CDCl3)
δ: 1.31(d, J = 7.0 Hz, 6H), 2.62 (s, 3H), 2.99-3.02 (m, 1H), 3.67 (t, J = 6.8 Hz, 2H),
4.85 (t, J = 6.8 Hz, 2H), 7.39 (d, J = 8.0 Hz, 2H), 7.94-7.96 (m, 2H), 8.00 (s, 1H). 13C
NMR (100 MHz, CDCl3) δ: 162.34, 159.39, 147.31, 139.65, 133.46, 129.60, 127.49,
125.42, 42.18, 34.20, 23.37, 16.82. MS (ESI): 374.1 (C17H20N5O3S, [M+H]+). Anal.
Calcd for C17H19N5O3S: C, 54.68; H, 5.13; N, 18.75; Found: C, 54.70; H, 5.12; N,
18.78.
4.1.1.18.
2-(4-(tert-butyl)phenyl)-5-((2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)thio)-1,3,4-ox
adiazole (26)
Light yellow crystals, yield: 58.7%, mp: 167-168 . 1H NMR (300 MHz, CDCl3)
δ: 1.36 (s, 9H), 2.60 (s, 3H), 3.65 (t, J = 7.1 Hz, 2H), 4.83 (t, J = 7.1 Hz, 2H), 7.53 (d,
J = 8.4 Hz, 2H), 7.92-7.98 (m, 3H). 13C NMR (100 MHz, CDCl3) δ: 162.34, 159.39,
153.17, 149.72, 139.65, 133.46, 128.94, 126.90, 126.42, 42.18, 31.44, 26.59, 16.82.
MS (ESI): 388.1 (C18H22N5O3S, [M+H]+). Anal. Calcd for C18H21N5O3S: C, 55.80; H,
5.46; N, 18.08; Found: C, 55.71; H, 5.48; N, 18.11.
4.1.1.19.
N,N-dimethyl-4-(5-((2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)thio)-1,3,4-oxadiazol
-2-yl)aniline (27)
1
Brilliant yellow crystals, yield: 53.6%, mp: 184-185 . H NMR (300 MHz,
CDCl3) δ: 2.60 (s, 3H), 3.06 (s, 6H), 3.62 (t, J = 6.9 Hz, 2H), 4.82 (t, J = 6.9 Hz, 2H),
6.73 (d, J = 8.6 Hz, 2H), 7.84 (d, J = 8.8 Hz, 2H), 7.98 (s, 1H). 13C NMR (100 MHz,
CDCl3) δ: 162.34, 159.39, 157.16, 149.72, 133.46, 127.55, 118.60, 111.14, 42.18,
41.91, 26.59, 16.82. MS (ESI): 375.1 (C16H19N6O3S, [M+H]+). Anal. Calcd for
17

C16H18N6O3S: C, 51.33; H, 4.85; N, 22.45; Found: C, 51.25; H, 4.86; N, 22.41.
4.1.1.20.
2-methyl-6-(5-((2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)thio)-1,3,4-oxadiazol-2-
yl)phenol (28)
1
Brown crystals, yield: 60.0%, mp: 163-164 . H NMR (500 MHz, CDCl3) δ:
2.37 (s, 3H), 2.62 (s, 3H), 3.71 (t, J = 7.0 Hz, 2H), 4.85 (t, J = 7.0 Hz, 2H), 6.94 (t, J
= 7.5 Hz, 1H), 7.35 (d, J = 7.0 Hz, 1H), 7.58 (d, J = 7.5 Hz, 1H), 8.01 (s, 1H), 9.92 (s,
1H). 13C NMR (100 MHz, CDCl3) δ: 162.34, 155.83, 149.72, 139.65, 126.96, 118.86,
111.54, 42.18, 26.59, 15.70, 16.82. MS (ESI): 362.1 (C15H16N5O4S, [M+H]+). Anal.
Calcd for C15H15N5O4S: C, 49.85; H, 4.18; N, 19.38; Found: C, 50.02; H, 4.25; N,
19.31.
4.1.1.21.
5-methyl-2-(5-((2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)thio)-1,3,4-oxadiazol-2-
yl)phenol (29)
1
Brown crystals, yield: 66.1%, mp: 176-178 . H NMR (500 MHz, CDCl3) δ:
2.41 (s, 3H), 2.62 (s, 3H), 3.70 (t, J = 7.0 Hz, 2H), 4.85 (t, J = 7.0 Hz, 2H), 6.86 (d, J
= 8.0 Hz, 1H), 6.97 (s, 1H), 7.60 (d, J = 8.5 Hz, 1H), 8.01 (s, 1H), 9.69 (s, 1H). 13C
NMR (100 MHz, CDCl3) δ: 162.34, 157.81, 149.72, 139.65, 129.27, 120.30, 112.36,
42.18, 26.59, 21.21, 16.82. MS (ESI): 362.1 (C15H15N5O4S, [M+H]+). Anal. Calcd for
C15H15N5O4S: C, 49.85; H, 4.18; N, 19.38; Found: C, 49.96; H, 4.21; N, 19.29.
4.1.1.22.
5-methoxy-2-(5-((2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)thio)-1,3,4-oxadiazol-2
-yl)phenol (30)
1
Brown powder, yield: 40.8%, mp: 180-181 . H NMR (500 MHz, CDCl3) δ:
2.63 (s, 3H), 3.69 (t, J = 6.5 Hz, 2H), 3.88 (s, 3H), 4.84 (t, J = 6.5 Hz, 2H), 6.61 (d, J
= 8.5 Hz, 1H), 6.65 (s, 1H), 7.62 (d, J = 9.0 Hz, 1H), 8.02 (s, 1H), 9.88 (s, 1H). 13C
NMR (100 MHz, CDCl3) δ: 162.34, 160.53, 159.84, 149.72, 139.65, 133.46, 130.30,
18

109.80, 106.85, 103.04, 56.04, 42.18, 26.59, 16.82. MS (ESI): 378.1 (C15H16N5O5S,
[M+H]+). Anal. Calcd for C15H15N5O5S: C, 47.74; H, 4.01; N, 18.56; Found: C, 47.89;
H, 4.08; N, 18.48.
4.1.1.23.
2-(3,5-dimethoxyphenyl)-5-((2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)thio)-1,3,4-
oxadiazole (31)
Milky-white crystals, yield: 47.0%, mp: 164-166 . 1H NMR (500 MHz, CDCl3)
δ: 2.63 (s, 3H), 3.68 (t, J = 6.5 Hz, 2H), 3.87 (s, 6H), 4.85 (t, J = 6.3 Hz, 2H), 6.66 (s,
1H), 7.17 (s, 2H), 8.01 (s, 1H). 13C NMR (100 MHz, CDCl3) δ: 163.18, 162.34,
159.35, 149.72, 133.46, 125.65, 107.95, 106.36, 56.04, 42.18, 26.59, 16.82. MS (ESI):
392.1 (C16H18N5O5S, [M+H]+). Anal. Calcd for C16H17N5O5S: C, 49.10; H, 4.38; N,
17.89; Found: C, 48.91; H, 4.30; N, 17.83.
4.1.1.24.
2-((2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)thio)-5-(3,4,5-trimethoxyphenyl)-1,3,4
-oxadiazole (32)
Light yellow powder, yield: 51.2%, mp: 154-155 . 1H NMR (500 MHz, CDCl3)
δ: 2.64 (s, 3H), 3.68 (t, J = 7.3 Hz, 2H), 3.95 (s, 9H), 4.86 (t, J = 7.3 Hz, 2H), 7.26 (s,
2H), 8.01 (s, 1H). 13C NMR (100 MHz, CDCl3) δ: 162.34, 159.35, 151.61, 139.65,
133.46, 122.02, 107.03, 60.65, 56.79, 42.18, 26.59, 16.82. MS (ESI): 422.1
(C17H20N5O6S, [M+H]+). Anal. Calcd for C17H19N5O6S: C, 48.45; H, 4.54; N, 16.62;
Found: C, 48.36; H, 4.50; N, 16.71.
4.1.1.25.
2-(furan-2-yl)-5-((2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)thio)-1,3,4-oxadiazole
(33)
1
Brown crystals, yield: 68.7%, mp: 119-120 . H NMR (300 MHz, CDCl3) δ:
2.59 (s, 3H), 3.65 (t, J = 7.0 Hz, 2H), 4.82 (t, J = 7.0 Hz, 2H), 6.60-6.62 (m, 1H),
7.16 (d, J = 3.5 Hz, 1H), 7.65-7.66 (m, 1H), 7.98 (s, 1H). 13C NMR (100 MHz, CDCl3)
19

δ: 163.79, 150.65, 149.93, 149.72, 144.04, 139.65, 133.46, 112.60, 111.21, 42.18,
26.59, 16.82. MS (ESI): 322.1 (C12H12N5O4S, [M+H]+). Anal. Calcd for C12H11N5O4S:
C, 44.86; H, 3.45; N, 21.80; Found: C, 44.79; H, 3.45; N, 21.77.
4.1.1.26.
2-((2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)thio)-5-(thiophen-2-yl)-1,3,4-oxadiazo
le (34)
1
Brown crystals, yield: 63.4%, mp: 123-124 . H NMR (300 MHz, CDCl3) δ:
2.60 (s, 3H), 3.64 (t, J = 7.1 Hz, 2H), 4.81 (t, J = 7.1 Hz, 2H), 7.17-7.20 (m, 1H),
7.57-7.59 (m, 1H), 7.72-7.74 (m, 1H), 7.98 (s, 1H). 13C NMR (100 MHz, CDCl3) δ:
163.79, 163.78, 149.72, 139.65, 133.46, 131.30, 130.54, 130.05, 127.41, 42.18, 26.59,
16.82. MS (ESI): 338.0 (C12H12N5O3S2, [M+H]+). Anal. Calcd for C12H11N5O3S2: C,
42.72; H, 3.29; N, 20.76; Found: C, 42.75; H, 3.30; N, 20.80.
4.1.1.27.
2-((2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)thio)-5-(pyridin-2-yl)-1,3,4-oxadiazole
(35)
1
Brown crystals, yield: 46.2%, mp: 154-155 . H NMR (300 MHz, CDCl3) δ:
2.61 (s, 3H), 3.69 (t, J = 7.0 Hz, 2H), 4.83 (t, J = 7.0 Hz, 2H), 7.46-7.50 (m, 1H),
7.90 (dt, J1 = 7.9 Hz, J2 =1.8 Hz, 1H), 7.97 (s, 1H), 8.20 (d, J = 8.0 Hz, 1H), 8.78 (d,
J = 4.2 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ: 162.34, 161.29, 149.72, 148.47,
142.58, 139.65, 133.46, 128.49, 119.84, 42.18, 26.59, 16.82. MS (ESI): 333.1
(C13H13N6O3S, [M+H]+). Anal. Calcd for C13H12N6O3S: C, 46.98; H, 3.64; N, 25.29;
Found: C, 47.12; H, 3.56; N, 25.22.
4.1.1.28.
2-((2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)thio)-5-(pyridin-3-yl)-1,3,4-oxadiazole
(36)
1
Reddish-brown crystals, yield: 57.1%, mp: 156-158 . H NMR (300 MHz,
CDCl3) δ: 2.61 (s, 3H), 3.68 (t, J = 7.0 Hz, 2H), 4.84 (t, J = 7.0 Hz, 2H), 7.46-7.50
20

(m, 1H), 7.98 (s, 1H), 8.28-8.32 (m, 1H), 8.81 (s, 1H), 9.25 (s, 1H). 13C NMR (100
MHz, CDCl3) δ: 162.34, 149.72, 148.34, 139.65, 135.74, 133.46, 128.39, 123.63,
42.18, 26.59, 16.82. MS (ESI): 333.1 (C13H13N6O3S, [M+H]+). Anal. Calcd for
C13H12N6O3S: C, 46.98; H, 3.64; N, 25.29; Found: C, 46.92; H, 3.60; N, 25.32.
4.1.1.29.
3-(5-((2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)thio)-1,3,4-oxadiazol-2-yl)pyridin
-4-amine (37)
Light yellow crystals, yield: 45.9%, mp: 216-218 . 1H NMR (500 MHz, CDCl3)
δ: 2.63 (s, 3H), 3.71 (t, J = 7.0 Hz, 2H), 4.85 (t, J = 6.8 Hz, 2H), 6.28 (brs, 2H), 6.70
(d, J = 6.5 Hz, 1H), 8.01 (s, 1H), 8.28 (d, J = 6.0 Hz, 1H), 8.82 (s, 1H). 13C NMR
(100 MHz, CDCl3) δ: 163.37, 162.34, 151.35, 148.77, 142.51, 139.65, 133.46, 110.32,
108.89, 42.18, 26.59, 16.82. MS (ESI): 348.1 (C13H14N7O3S, [M+H]+). Anal. Calcd
for C13H13N7O3S: C, 44.95; H, 3.77; N, 28.23; Found: C, 45.09; H, 3.69; N, 28.22.
4.1.1.30.
5-(5-((2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)thio)-1,3,4-oxadiazol-2-yl)pyridin
-2-amine (38)
1
Brilliant yellow powder, yield: 48.5%, mp: 153-154 . H NMR (500 MHz,
CDCl3) δ: 2.63 (s, 3H), 3.67 (t, J = 7.0 Hz, 2H), 4.85 (t, J = 7.3 Hz, 2H), 4.94 (s, 2H),
6.61 (d, J = 8.5 Hz, 1H), 8.01 (s, 1H), 8.05 (d, J = 8.5 Hz, 1H), 8.72 (s, 1H). 13C
NMR (100 MHz, CDCl3) δ: 162.34, 161.95, 154.91, 149.72, 147.64, 139.65, 136.12,
133.46, 117.19, 106.54, 42.18, 26.59, 16.82. MS (ESI): 348.1 (C13H14N7O3S,
[M+H]+). Anal. Calcd for C13H13N7O3S: C, 44.95; H, 3.77; N, 28.23; Found: C, 44.81;
H, 3.81; N, 28.33.
4.1.1.31.
2-((2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)thio)-5-(pyridin-4-yl)-1,3,4-oxadiazole
(39)
Light yellow crystals, yield: 56.9%, mp: 172-173 . 1H NMR (300 MHz, CDCl3)
21

δ: 2.61 (s, 3H), 3.70 (t, J = 7.0 Hz, 2H), 4.83 (t, J = 7.0 Hz, 2H), 7.86-7.88 (d, J = 5.7
Hz, 2H), 7.98 (s, 1H), 8.84 (s, 2H). 13C NMR (100 MHz, CDCl3) δ: 162.34, 159.39,
150.31, 149.72, 139.65, 133.37, 133.46, 123.42, 42.18, 26.59, 16.82. MS (ESI): 333.1
(C13H13N6O3S, [M+H]+). Anal. Calcd for C13H12N6O3S: C, 46.98; H, 3.64; N, 25.29;
Found: C, 47.01; H, 3.62; N, 25.27.
4.1.1.32.
4-(5-((2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)thio)-1,3,4-oxadiazol-2-yl)pyridin
-2-amine (40)
Light yellow powder, yield: 41.9%, mp: 217-218 . 1H NMR (500 MHz, CDCl3)
δ: 2.63 (s, 3H), 3.70 (t, J = 7.0 Hz, 2H), 4.84 (t, J = 6.8 Hz, 2H), 4.88 (s, 2H), 7.11 (s,
1H), 7.23 (d, J = 5.0 Hz, 1H), 8.00 (s, 1H), 8.25 (d, J = 5.5 Hz, 1H). 13C NMR (100
MHz, CDCl3) δ: 162.34, 160.38, 159.57, 150.20, 149.72, 139.65, 134.07, 133.46,
112.98, 109.02, 42.18, 26.59, 16.82. MS (ESI): 348.1 (C13H14N7O3S, [M+H]+). Anal.
Calcd for C13H13N7O3S: C, 44.95; H, 3.77; N, 28.23; Found: C, 44.82; H, 3.80; N,
28.31.
4.1.1.33.
2-(2-chloropyridin-4-yl)-5-((2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)thio)-1,3,4-
oxadiazole (41)
Light yellow powder, yield: 57.5%, mp: 161-163 . 1H NMR (500 MHz, CDCl3)
δ: 2.63 (s, 3H), 3.73 (t, J = 7.0 Hz, 2H), 4.85 (t, J = 7.0 Hz, 2H), 7.83 (d, J = 5.5 Hz,
1H), 7.94 (s, 1H), 8.01 (s, 1H), 8.63 (d, J = 5.5 Hz, 1H). 13C NMR (100 MHz, CDCl3)
δ: 162.34, 159.57, 152.36, 151.13, 149.72, 139.65, 135.93, 133.46, 124.81, 123.09,
42.18, 26.59, 16.82. MS (ESI): 367.0 (C13H12ClN6O3S, [M+H]+). Anal. Calcd for
C13H11ClN6O3S: C, 42.57; H, 3.02; N, 22.91; Found: C, 42.70; H, 2.95; N, 22.83.
4.1.1.34.
2-((2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)thio)-5-(naphthalen-1-yl)-1,3,4-oxadi
azole (42)
22

1
Brown crystals, yield: 72.9%, mp: 155-157 . H NMR (500 MHz, CDCl3) δ:
2.67 (s, 3H), 3.74 (t, J = 7.3 Hz, 2H), 4.91 (t, J = 7.0 Hz, 2H), 7.60-7.66 (m, 2H),
7.72-7.75 (m, 1H), 7.98 (d, J = 8.5 Hz, 1H), 8.02 (s, 1H), 8.08 (d, J = 8.0 Hz, 1H),
8.17 (d, J = 8.0 Hz, 1H), 9.20 (d, J = 8.5 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ:
162.34, 152.33, 149.72, 139.65, 135.93, 133.28, 133.46, 131.10, 128.28, 128.24,
127.95, 127.34, 125.88, 125.81, 125.01, 124.39, 42.18, 26.59, 16.82. MS (ESI): 382.1
(C18H16N5O3S, [M+H]+). Anal. Calcd for C18H15N5O3S: C, 56.68; H, 3.96; N, 18.36;
Found: C, 56.71; H, 3.98; N, 18.39.
4.1.1.35.
2-((2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)thio)-5-(naphthalen-2-yl)-1,3,4-oxadi
azole (43)
Light brown powder, yield: 41.8%, mp: 167-168 . 1H NMR (500 MHz, CDCl3)
δ: 2.66 (s, 3H), 3.71 (t, J = 7.0 Hz, 2H), 4.88 (t, J = 7.0 Hz, 2H), 7.61-7.64 (m, 2H),
7.93 (d, J = 7.5 Hz, 1H), 7.98-8.02 (m, 3H), 8.12 (d, J = 8.5 Hz, 1H). 13C NMR (100
MHz, CDCl3) δ: 162.34, 160.11, 149.72, 139.65, 134.51, 134.02, 133.46, 130.64,
129.70, 129.33, 128.22, 127.00, 126.52, 125.93, 122.41, 42.18, 26.59, 16.82. MS
(ESI): 382.1 (C18H16N5O3S, [M+H]+). Anal. Calcd for C18H15N5O3S: C, 56.68; H,
3.96; N, 18.36; Found: C, 56.81; H, 4.00; N, 18.31.
4.1.1.36.
2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-5-((2-(2-methyl-5-nitro-1H-imidazol-1-yl)et
hyl)thio)-1,3,4-oxadiazole (44)
1
Grayish white crystals, yield: 60.5%, mp: 173-174 . H NMR (500 MHz,
CDCl3) δ: 2.62 (s, 3H), 3.66 (t, J = 7.3 Hz, 2H), 4.33-4.36 (m, 4H), 4.84 (t, J = 7.0
Hz, 2H), 7.00 (d, J = 8.5 Hz, 1H), 7.52-7.54 (m, 2H), 8.01 (s, 1H). 13C NMR (100
MHz, CDCl3) δ: 162.34, 159.57, 149.94, 149.72, 145.95, 139.65, 133.46, 120.41,
119.59, 117.45, 113.31, 61.31, 42.18, 26.59, 16.82. MS (ESI): 390.1 (C16H16N5O5S,
[M+H]+). Anal. Calcd for C16H15N5O5S: C, 49.35; H, 3.88; N, 17.99; Found: C, 49.51;
H, 3.91; N, 18.02.
23

4.1.2. Crystallographic Studies
X-ray single-crystal diffraction data of compound 9 were collected on a Bruker
SMARTAPEX CCD diffractometer at 293(2) K using Mo Kα radiation (λ = 0.71073
Ǻ) by the ω scan mode. The program SAINT was used for the integration of the
diffraction profiles. All the structures were solved by direct methods using the
SHELXS program of the SHELXTL package and refined by full-matrix least-squares
methods with SHELXL-97.31 All non-hydrogen atoms of compound 9 were refined
with anisotropic thermal parameters. All hydrogen atoms were placed in
geometrically idealized positions and constrained to ride on their parent atoms.
4.2. Anti-proliferation assay
The in vitro anticancer activities of the prepared compounds 9-44 against HepG2,
SGC-7901, and MCF-7 cell lines were evaluated as described in the literature32 with
some modifications. Target tumor cells grew to log phase in RPMI 1640 medium
supplemented with 10% fetal bovine serum. After reaching a dilution of 3×104 cells
mL-1 with the medium, 100 μL of the obtained cell suspension was added to each well
of 96-well culture plates. Subsequently, incubation was performed at 37 ˚C in 5%
CO2 atmosphere for 24 h before the cytotoxicity assessment. Testing samples at
pre-set concentrations were added to 6 wells with 5-FU being employed as a positive
reference. After 48 h exposure period, 25 μL of PBS containing 2.5 mg mL-1 of MTT
was added to each well. After 4 h, the medium was replaced by 150 μL DMSO to
dissolve the purple formazan crystals produced. The absorbance at 570 nm of each
well was measured on an ELISA plate reader. The data represented the mean of three
independent experiments in triplicate and were expressed as means SD. The IC50
value was defined as the concentration at which 50% of the cells could survive.
4.3. Thymidylate Synthase Assay
TS was assayed spectrophotometrically at 30° and pH 7.4 in a mixture
containing 0.1 M 2-mercaptoethanol, 0.0003 M (6R,S)-tetrahydrofolate, 0.012 M
24

formaldehyde, 0.02 M MgCl2, 0.001 M dUMP, 0.04 M TrisHCl, and 0.00075 M
NaEDTA.
This was the assay described by A. J. Wahba and M.Friedkin,33a except that the
dUMP concentration was increased 25-fold as per V. J. Davisson, W. Sirawaraporn,
and D. V. Santi.33b The reaction was initiated by the addition of an amount of enzyme
yielding a change in absorbance at 340 nm of 0.016/min in the absence of inhibitor.
The percent inhibition was determined at a minimum of four inhibitor concentrations
within 20% of the 50% point. The standard deviations for determination of the 50%
points were within 10% of the values given.
4.4. Bacterial suppressive assay
The antibacterial activity of compounds 18, 37, and 40 was tested against E. coli
ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC
6538 and Bacillus subtilis ATCC 530 using MH medium (Mueller-Hinton medium:
casein hydrolysate 17.5 g, soluble starch 1.5 g, beef extract 1000 mL). The MIC
values of the tested compounds were determined by a colorimetric method using the
dye MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide). A stock
solution of the synthesized compound (100 μg/mL) in DMSO was prepared, and
graded quantities of the tested compounds were incorporated in specified quantity of
sterilized liquid MH medium. A specified quantity of the medium containing the
compound was poured into microtitration plates. Suspension of the microorganism
was prepared to approximately 105 cfu/mL and applied to microtitration plates with
serially diluted compounds in DMSO, and then they were incubated at 37 ℃ for 24
h. After the MICs were visually determined on each of the microtitration plates, 50 μL
of PBS (phosphate buffered saline 0.01 mol/L, pH 7.4: Na2HPO4•12H2O 2.9 g,
KH2PO4 0.2 g, NaCl 8.0 g, KCl 0.2 g, distilled water 1000 mL) containing 2 mg of
MTT/mL was added to each well. Incubation was continued at room temperature for
4–5 h. The content of each well was removed, and 100 μL of isopropanol containing
5% 1 mol/L HCl was added to extract the dye. After 12 h of incubation at room
25

temperature, the optical density (OD) was measured with a microplate reader at 550
nm.
4.5. Experimental protocol of docking study
Molecular docking of compound 18 into the three dimensional X-ray structure of
human TS and E. coli TS (PDB code: 1HVY and 2KCE, respectively) was carried out
using the Discovery Studio (version 3.1) as implement through the graphical user
interface DS- CDOCKER protocol.
The three-dimensional structures of the aforementioned compounds were constructed
using Chem. 3D ultra 12.0 software [Chemical Structure Drawing Standard;
Cambridge Soft corporation, USA (2010)], then they were energetically minimized by
using MMFF94 with 5000 iterations and minimum RMS gradient of 0.10. The crystal
structures of two TS proteins complexes were retrieved from the RCSB Protein Data
Bank (http://www.rcsb.org/pdb/home/home.do). All bound waters and ligands were
eliminated from the protein and the polar hydrogen was added to the proteins.
4.6. 3D-QSAR
Ligand-based 3D-QSAR approach was performed by QSAR software of DS 3.1
(Discovery Studio 3.1, Accelrys, Co. Ltd). The training sets were composed of 29
inhibitors with the corresponding pIC50 values which were converted from the
obtained IC50 (μM), and test sets comprised 7 compounds of data sets as list in Table
5. All the definition of the descriptors can be seen in the Help of DS 3.1 software and
they were calculated by QSAR protocol of DS 3.1.34 The alignment conformation of
each molecule was the one with lowest interaction energy in the docked results of
CDOCKER (generated in part 4.5). The predictive ability of 3D-QSAR modeling
could be evaluated based on the cross-validated correlation coefficient, which
qualified the predictive ability of the models. Scrambled test (Y scrambling) was
performed to investigate the risk of chance correlations. The inhibitory potencies of
compounds were randomly reordered for 30 times and subject to leave-one-out
validation test, respectively. The models were also validated by test sets, in which the
26

compounds are not included in the training sets. Usually, one can believe that the
modeling is reliable, when the r2 for test sets is larger than 0.6, respectively.
Acknowledgements
This work was supported by Jiangsu National Science Foundation (No.
BK2009239) and the Fundamental Research Fund for the Central Universities (No.
1092020804).
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29