Novel 3-[4-alkoxy-3-(1H-tetrazol-1-yl) phenyl]-1,2,4-oxadiazol-5(4H)-ones as promising xanthine oxidase inhibitors: Design, synthesis and biological evaluation

Article abstract of DOI:10.1016/j.bioorg.2019.103564

Xanthine oxidase (XO) is a critical target for the therapy of hyperuricemia and gout. In this study, a number of 3-[4-alkoxy-3-(1H-tetrazol-1-yl) phenyl]-1,2,4-oxadiazol-5(4H)-ones (3a–3w) were newly designed by a bioisosteric replacement and hybrid strategy with the hope of obtaining novel and effective nonpurine XO inhibitors. Subsequently, these compounds were synthesized through a three-step procedure, with good yields. In addition, the in vitro bovine XO inhibitions were measured by spectrophotometric determination of uric acid formation at 295 nm using allopurinol as a positive control. As a result, compound 3j was found to be the most potent XO inhibitor, with an IC₅₀ value of 0.121 μM, which was approximately 63-fold more potent than allopurinol, and the analysis of the structure-activity relationships indicated that the hydrophobic group at 4′-position was essential for inhibitory potency. Additionally, the molecular modeling results showed that the 1,2,4-oxadiazol-5(4H)-one moiety binds to XO active site via various hydrogen bonds with Arg880 and Thr1010. Moreover, the compound 3j was demonstrated to be a mixed-type nonpurine XO inhibitor. Furthermore, the hypouricemic studies on a rat model, induced by potassium oxonate, demonstrated that serum uric acid levels could be effectually reduced by compound 3j at an oral dose of 15 mg/kg. Therefore, compound 3j could be a promising lead compound for the treatment of hyperuricemia and gout.

Full text of DOI:10.1016/j.bioorg.2019.103564

Journal Pre-proofs
Novel 3-[4-alkoxy-3-(1H-tetrazol-1-yl) phenyl]-1,2,4-oxadiazol-5(4H)-ones
as promising xanthine oxidase inhibitors: design, synthesis and biological
evaluation
Jun Gao, Zhaofeng Zhang, Bing Zhang, Qing Mao, Xiwen Dai, Qian Zou, Yu
Lei, Yao Feng, Shaojie Wang
PII:
S0045-2068(19)31902-9
DOI:
https://doi.org/10.1016/j.bioorg.2019.103564
YBIOO 103564
Reference:
To appear in:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
8 November 2019
27 December 2019
30 December 2019
Please cite this article as: J. Gao, Z. Zhang, B. Zhang, Q. Mao, X. Dai, Q. Zou, Y. Lei, Y. Feng, S. Wang, Novel
3-[4-alkoxy-3-(1H-tetrazol-1-yl) phenyl]-1,2,4-oxadiazol-5(4H)-ones as promising xanthine oxidase inhibitors:
design, synthesis and biological evaluation, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.
2019.103564
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Novel 3-[4-alkoxy-3-(1H-tetrazol-1-yl) phenyl]-1,2,4-oxadiazol-5(4H)-ones as promising
xanthine oxidase inhibitors: design, synthesis and biological evaluation
Jun Gao a, 1, Zhaofeng Zhang a, 1, Bing Zhang a, Qing Mao a, Xiwen Dai a, Qian Zou b, Yu Lei a, Yao
Feng a, Shaojie Wang a, *
a Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, School of
Pharmaceutical Engineering, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe
District, Shenyang, 110016, PR China
b
Wuya College of Innovation, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe
District, Shenyang, 110016, PR China
*Corresponding author
Tel/Fax: +86-24-43520230, E-mail: Wangshaojie@syphu.edu.cn (S. J. Wang).
1 These two authors contributed equally to this work.
1

ABSTRACT
Xanthine oxidase (XO) is a critical target for the therapy of hyperuricemia and gout. In this
study, a number of 3-[4-alkoxy-3-(1H-tetrazol-1-yl) phenyl]-1,2,4-oxadiazol-5(4H)-ones (3a-3w)
were newly designed by a bioisosteric replacement and hybrid strategy with the hope of obtaining
novel and effective nonpurine XO inhibitors. Subsequently, these compounds were synthesized
through a three-step procedure, with good yields. In addition, the in vitro bovine XO inhibitions
were measured by spectrophotometric determination of uric acid formation at 295 nm using
allopurinol as a positive control. As a result, compound 3j was found to be the most potent XO
inhibitor, with an IC50 value of 0.121 µM, which was approximately 63-fold more potent than
allopurinol, and the analysis of the structure-activity relationships indicated that the hydrophobic
group at 4′-position was essential for inhibitory potency. Additionally, the molecular modeling
results showed that the 1,2,4-oxadiazol-5(4H)-one moiety binds to XO active site via various
hydrogen bonds with Arg880 and Thr1010. Moreover, the compound 3j was demonstrated to be a
mixed-type nonpurine XO inhibitor. Furthermore, the hypouricemic studies on a rat model, induced
by potassium oxonate, demonstrated that serum uric acid levels could be effectually reduced by
compound 3j at an oral dose of 15 mg/kg. Therefore, compound 3j could be a promising lead
compound for the treatment of hyperuricemia and gout.
Keywords: Xanthine oxidase; Inhibitor; 1,2,4-Oxadiazol-5(4H)-one; Synthesis; Biological
evaluation
2

1. Introduction
Xanthine oxidase (XO), a versatile molybdoflavoprotein, is a rate-limiting and critical enzyme
involved in biosynthesis of uric acid in humans [1-4], and the overproduction of uric acid is
associated with a variety of pathological progressions such as gout, inflammation, ischemia-
reperfusion damage, cardiovascular diseases, renal diseases and even cancer [5-8]. Therefore, XO
has been considered as a promising target for treating hyperuricemia, gout and other associated
pathological conditions [3, 9].
As an analogue of hypoxanthine, allopurinol is a prototypical XO inhibitor that has been used
clinically as an anti-gout drug for decades due to its obvious curative effects [3]. However, the
purine scaffold of allopurinol and its analogues can be metabolized by hypoxanthine-guanine
phosphoribosyltransferase (HGPRT) at the N-1 position, leading to the production of nucleotide
analogues, such as allopurinol-1-ribonucleotide, which further lead to interference with normal
purine and pyrimidine metabolism [2]. As a result, some severe side effects, such as skin rash,
eosinophilia, hypersensitivity with fever, gastrointestinal distress and renal toxicity, are produced
by allopurinol and other purine-type XO inhibitors [3, 10]. Accordingly, extensive research has been
carried out to obtain nonpurine compounds with more potent XO inhibitory potency and fewer side
effects. In recent years, many nonpurine-based XO inhibitors with different structural classes have
been published, including pyrazoles [11], imidazoles [12, 13], isoxazoles [14], selenazoles [15],
pyrimidines [16-18], isocytosines [19-21], thiazoles [22-25], furans [26], 5-aryl-1H-tetrazoles [27],
carboxylated aurone derivatives [28], dihydropyrazolo quinazoline derivatives [29] and 1,2,3-
triazole derivatives [30, 31]. In addition, other XO inhibitors with various chemotypes have also
been recently developed, such as flavonoid derivatives [32, 33], coumarin derivatives [34-36],
salvianolic acid C [37], caffeic acid phenethyl ester derivatives [38], barbiturate derivatives [39]
and chalcones [8, 40]. As a result, two nonpurine XO inhibitors, febuxostat and topiroxostat, were
approved for the treatment of hyperuricemia and gout by the US Food and Drug Administration
(FDA) and the Japan Pharmaceutical and Food Safety Bureau in 2009 and 2013, respectively [41,
42]. Unfortunately, due to higher overall rates of major cardiovascular events compared with
allopurinol, a black box warning was mandated for febuxostat by the FDA in February 2019 [43].
3

Therefore, there is a growing demand for novel nonpurine XO inhibitors developed to the therapy
of hyperuricemia, gout and related complications [18, 44].
As an important pharmacophore, the pyrimidinone moiety of allopurinol plays an important role
in binding to the XO active site [45-47]. Accordingly, retaining the pyrimidinone moiety and
discarding the pyrazole ring of allopurinol not only maintains the interaction between pyrimidinone
and XO but also excludes the possibility of allopurinol being converted to unnatural nucleotides by
HGPRT at the N-1 position; this strategy was adopted for the successful design of Y-700 [11].
Meanwhile, 1,2,4-oxadiazol-5(4H)-one is a planar acidic heterocycle (pKa = ~6-7) [48, 49], and it
can be considered as a bioisostere of pyrimidinone. Therefore, it is reasonable to replace the
pyrimidinone moiety with 1,2,4-oxadiazol-5(4H)-one to design novel nonpurine XO inhibitors [50-
52].
In our previous report, 2-[3-(1H-tetrazol-1-yl) phenyl] pyrimidones (Fig. 1) with a tetrazole
moiety targeting the subpocket of the XO active cavity were found to show remarkable XO
inhibitory potency through hydrogen bonding with Asn768 and Lys771 in the subpocket [17], which
inspired us to further develop novel nonpurine XO inhibitors based on the tetrazole group that
targets the subpocket. Therefore, the left part of 2-[3-(1H-tetrazol-1-yl) phenyl] pyrimidones was
kept and hybridized with the 1,2,4-oxadiazol-5(4H)-one moiety to design a range of 3-[4-alkoxy-3-
(1H-tetrazol-1-yl) phenyl]-1,2,4-oxadiazol-5(4H)-ones (3b-3v) with the hope of obtaining novel
and effective nonpurine XO inhibitors. Moreover, in order to further investigate the influence of the
4′-alkoxy and 3′-tetrazole group on the inhibitory potency, the compound 3-[3-(1H-tetrazol-1-yl)
phenyl]-1,2,4-oxadiazol-5(4H)-one (3a) and 3-(4-cyclohexylmethoxy phenyl)-1,2,4-oxadiazol-
5(4H)-one (3w) were also prepared and evaluated. Furthermore, enzyme kinetic studies, molecular
modeling studies and in vivo hypouricemic studies were also performed to evaluate the
representative compound 3j.
4

Fig. 1. Design strategy.
2. Results and discussion
2.1. Chemistry
The reaction sequence for the synthesis of target compounds is shown in Scheme 1.
Commercially available 3-amino-benzonitrile was cyclized with sodium azide and triethyl
orthoformate in glacial acetic acid to provide 3-(1H-tetrazol-1-yl) benzonitrile (1a) in moderate
yield. The starting material 4-hydroxy-3-(1H-tetrazol-1-yl) benzonitrile was synthesized from 4-
hydroxybenzonitrile based on the methods studied in our previous research [17]. The hydroxy group
was alkylated with alkyl halides to give good yields of 4-alkoxy-3-(1H-tetrazol-1-yl) benzonitriles
(1b-1v) and 4-cyclohexylmethoxy-benzonitrile (1w), respectively. Furthermore, the intermediates
1a-1w were reacted with hydroxylamine hydrochloride and sodium carbonate in dimethyl sulfoxide
(DMSO) to furnish N'-hydroxy-benzimidamide derivatives (2a-2w), which were further cyclized in
the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and N,N'-carbonyldiimidazole (CDI) in
dichloromethane, providing target compounds 3-[4-alkoxy-3-(1H-tetrazol-1-yl) phenyl]-1,2,4-
oxadiazol-5(4H)-ones (3a-3w) with good yields.
5

Scheme 1. Reagents and conditions: i. Triethyl orthoformate, NaN3, glacial acetic acid, 60°C; ii.
H2NOH·HCl, Na2CO3, DMSO, 80°C; iii. CDI, DBU, CH2Cl2, 30°C; iv. RCl or RBr, K2CO3, KI,
N,N-dimethylformamide, 60-80°C.
2.2. Biological evaluation
2.2.1. Xanthine oxidase inhibitory activity
All the target compounds 3a-3w have been evaluated for in vitro bovine XO inhibitory potency
with allopurinol as the positive control. As a result, almost all compounds (Table 1) exhibited potent
XO inhibitory potency, with IC50 values between 0.121 µM and 3.282 µM, except 3a (not active,
<50% inhibition at 10 μM). Especially, compound 3j, with a cyclohexylmethoxy moiety substituted
6

at the 4-position of the phenyl group, emerged as the strongest XO inhibitor (IC50 = 0.121 µM),
being approximately 63-fold more potent than allopurinol (IC50 = 7.590 µM).
Table 1. In vitro XO inhibitory potency of designed compounds
O
HN
O
N
R
R1
3a-3w
Compd.
3a
R
H
R1
IC50 (µM)a
Compd.
3m
3n
R
R1
IC50 (µM)a
n.a.b
O
O
N
N
0.315 ± 0.0166
0.160 ± 0.0145
0.151 ± 0.0066
0.149 ± 0.0131
0.206 ± 0.0154
0.157 ± 0.0153
0.334 ± 0.0197
0.233 ± 0.0066
0.185 ± 0.0035
0.215 ± 0.0186
3.282  0.1201
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
3.212 ± 0.0961
0.345 ± 0.0047
0.514 ± 0.0265
0.173 ± 0.0056
0.232 ± 0.0145
0.245 ± 0.0360
0.303 ± 0.0194
0.315 ± 0.0156
0.121 ± 0.0177
0.146 ± 0.0645
3b
3c
O
N
N
N
N
N
N
N
N
N
N
N
N
N
O
N
N
N
N
N
N
N
N
N
N
O
3o
N
N
O
N
F
N
O
O
3d
3e
3p
N
N
N
N
Cl
Br
O
3q
N
N
O
N
N
O
3f
3r
N
N
O
N
N
O
O
3g
3s
O
N
N
N
N
F
O
3h
3i
3t
O
N
N
N
N
Cl
Br
O
O
3u
N
N
O
N
N
O
3j
3v
N
N
N
N
O
O
3k
3w
H
N
N
7

O
N
0.208 ± 0.0106
3l
Allopurinol
7.590  0.215
N
N
N
a. All values are expressed as the mean ± standard error of the mean of triplicate determinations.
b. n.a.: Not active (<50% inhibition at 10 μM).
As shown in Table 1, compounds 3b-3w (IC50 values between 0.121 µM and 3.282 µM),
substituted with different alkoxy groups at the 4-position, had different degrees of XO inhibitory
potency. However, XO inhibition was not observed for the unsubstituted compound 3a, which
suggested that alkoxy substitution at the 4-position of the benzene ring was favorable for the target
compounds to produce XO inhibitory potency. Specifically, the inhibitory potencies of compounds
3c (IC50 = 0.345 µM), 3d (IC50 = 0.514 µM) and 3e (IC50 = 0.173 µM), bearing iso-propoxy, iso-
butoxy and iso-pentyloxy substituents at the 4-position, were approximately 9.3-fold, 6.2-fold and
18.6-fold stronger than that of the methoxy substituted compound 3b (IC50 = 3.212 µM), and the
inhibitory potency of compound 3j (IC50 = 0.121 µM), with a cyclohexylmethoxy substituent, was
approximately 2.5-fold and 2.6-fold better than those of the cyclopropylmethoxy- and
cyclopentyloxy-substituted compounds 3h (IC50 = 0.303 µM) and 3i (IC50 = 0.315 µM), respectively,
which indicated that increasing the size of the saturated alkoxy or the cycloalkoxy groups at the 4-
position was good for the XO inhibitory potency. This tendency was similar to those of imidazoles
[12] and selenazoles [15].
When the cyclohexylmethoxy group of compound 3j (IC50 = 0.121 µM) was changed to a
benzyloxy group to give compound 3k (IC50 = 0.146 μM), the inhibitory potency was maintained,
indicating that this change had no significant effect on potency. In addition, methyl, tert-butyl,
methoxy, and halogen (F, Cl and Br) groups were introduced at the meta or para position of the
benzyloxy group to obtain compounds 3l-3v to explore the possibility of improving the inhibitory
potency. As a result, the inhibitory potency of compound 3m (IC50 = 0.315 µM), bearing a tert-
butyl substituent at the para position of the benzyloxy group, was approximately 2.2-fold weaker
than that of compound 3k (IC50 = 0.146 µM), implying that the steric hindrance created by the tert-
butyl group could damage the inhibitory potency, which was in accordance with the conclusion of
8

pyrimidone derivatives [16, 17]. Meanwhile, it was also found that the electron-withdrawing or
electron-donating substituents, including fluoro, chloro, bromo, methyl and methoxy groups, at the
para position of the benzyloxy group did not significantly change the XO inhibitory potency
compared to the unsubstituted compound 3k. However, the presence of methoxy (3n) and fluoro
(3o) substituents at the para position of the benzyloxy group led to an approximately 2.1-fold and
1.5-fold increased potency compared to compounds substituted at the meta position (3n vs 3s, 3o
vs 3t, IC50 = 0.160 µM, 0.334 µM, 0.151 µM and 0.233 µM, respectively). These results implied
that a methoxy group or a fluorine atom substituted at the para position of the benzyloxy group was
beneficial for the XO inhibitory potency.
Notably, compound 3w, without a tetrazole group, possessed an IC50 value of 3.282 µM, which
was approximately 27-fold weaker than that of compound 3j (IC50 = 0.121 µM), which had a
tetrazole group substituent at the 3-position, suggesting that the tetrazole moiety substituent at the
3-position was crucial for the XO inhibitory potency.
2.2.2. Molecular docking
To further explore the binding mode of newly designed compounds with XO and to rationalize
the structure-activity relationships observed in this study, molecular modeling of the representative
compound 3j was performed. As shown in Fig. 2, allopurinol was accommodated in the active site
through hydrogen bonding with Glu802, - stacking interactions with Phe914 and Phe1009, and
hydrophobic interactions with other hydrophobic amino acids such as Leu873, Val1011, Leu1014
and Ala1078; and compound 3j was inserted into the active cavity, as expected. In the deepest part
of the binding site, the 1,2,4-oxadiazol-5(4H)-one moiety binds to XO active site via various
hydrogen bonds where the carbonyl group interacts with the guanidine group of Arg880 through
two hydrogen bonds, and the N-4 atom interacts with the hydroxyl group of Thr1010 through one
hydrogen bond, which was similar to the interaction mode of 5-aryl-1H-tetrazoles [27]. In addition,
the tetrazole group at the 3-position of the phenyl moiety formed two hydrogen bonds with Asn768
and Lys771 in the subpocket of XO, which was similar to our previous report [17]. At the same
time, the 1,2,4-oxadiazol-5(4H)-one ring was sandwiched between the amino acid residues of
9

Phe914 and Phe1009 through “face-to-face” and “face-to-edge” - stacking interactions, which
was consistent with the interactions of allopurinol and isocytosine derivatives with XO [19].
Furthermore, several hydrophobic interactions were also observed at the entrance of the XO active
pocket, such as Leu648, Phe649, Leu873, Val1011, Leu1014, Pro1076 and Ala1079. Therefore,
compound 3j showed significantly more binding interactions compared with the standard drug
allopurinol. These observations could provide a rational explanation for the increased potency of
compound 3j compared to that of allopurinol.
Fig. 2. The binding modes of allopurinol and compound 3j within the active cavity of XO. (a) View
of superimposed docking conformations for allopurinol (orange) and compound 3j (green) binding
to the active site of XO. The ligands are shown as a line and the protein is shown as a surface (PDB
code 1N5X, gray). (b) The binding interactions of allopurinol with the amino acid residues. (c) The
binding modes of compound 3j and the active site of XO. The π-π stacking interactions and
hydrogen bonds are depicted by violet and green dashed lines, respectively. The amino acid residues
of XO interacting with ligands are shown as cyan lines.
10

2.2.3. Enzyme inhibitory kinetics
Enzyme kinetics were also studied to explore the action mode of the representative compound
3j. The Lineweaver-Burk plot analysis (Fig. 3) revealed that compound 3j acted as a mixed-type
XO inhibitor, where Km, Vmax and the slope were all affected by the inhibitor, which was different
from the competitive inhibition mode of allopurinol [53]. As shown in Fig. 3, the inhibitor increased
the Km and slope (Km/Vmax) but decreased the Vmax. The Km and Vmax values in the presence of 0 to
0.848 μM 3j were 87.32, 206.27, 444.17, 558.57, 621.70 μM, and 0.186, 0.152, 0.089, 0.067, 0.045
μM/min, respectively. In addition, the equilibrium constants for binding with the free enzyme (Ki)
and enzyme-substrate complex (Kis) were calculated to be 0.081 μM and 0.333 μM, respectively,
implying that the inhibitor preferentially bound to the free enzyme rather than to the enzyme-
substrate complex [54-56]. Moreover, a dose-dependent inhibition curve of XO by compound 3j
was also exhibited in Fig. 4.
Fig. 3. Lineweaver-Burk plots of XO in the presence of compound 3j.
11

Fig. 4. Dose-dependent inhibition curve of XO by compound 3j (means ± SD, n = 3).
2.2.4. Hypouricemic effect in vivo
The in vivo hypouricemic effects of compound 3j (15 mg/kg) were further investigated and
compared with allopurinol (5 mg/kg, Fig. 5). As expected, the serum uric acid levels of the model
group were markedly increased compared with the blank group (P < 0.0001) after 1 h of drug
administration, implying that the model was successfully established by intraperitoneal injection of
potassium oxonate (300 mg/kg). Moreover, compound 3j showed an apparent reduction of 14.2%
in uric acid levels (P < 0.05) at 3 h and a reduction of 14.1% in the AUC (uric acid, 1-8 h) (P < 0.0001)
compared with the model group at an oral dose of 15 mg/kg. Thus, the results of the hypouricemic
effect in vivo suggested that compound 3j could be a promising lead compound for the treatment of
hyperuricemia.
Fig. 5. Anti-hyperuricemic effect of compound 3j and allopurinol. (a) Time-course changes of
12

serum uric acid levels after 1-8 h of treatment. (b) Serum uric acid levels after 3h of treatment. (c)
The AUC of serum uric acid after 1-8 h of treatment. The data is shown as the mean  SD. (n=6),
*P < 0.05 and ****P < 0.0001 vs model.
3. Conclusion
In summary, a number of 3-[4-alkoxy-3-(1H-tetrazol-1-yl) phenyl]-1,2,4-oxadiazol-5(4H)-ones
(3a-3w) were designed by a bioisosteric replacement and hybrid strategy and these compounds were
subsequently synthesized via a three-step procedure with good yields. In particular, compound 3j
was identified as the strongest XO inhibitor, with an IC50 value of 0.121 µM, which was
approximately 63-fold more potent than allopurinol, and the structure-activity relationships
indicated that the hydrophobic group at 4-position of phenyl group was critical for inhibitory
potency. In addition, the result of molecular modeling study on compound 3j showed that the 1,2,4-
oxadiazol-5(4H)-one moiety binds to XO active site via various hydrogen bonds where the carbonyl
group interacts with the guanidine group of Arg880 through two hydrogen bonds, and the N-4 atom
interacts with the hydroxyl group of Thr1010 through one hydrogen bond, which provide a
reasonable explanation for the structure-activity relationships observed in this study. Moreover, the
representative compound 3j was demonstrated to be a mixed-type nonpurine XO inhibitor.
Furthermore, compound 3j was effective in reducing the serum uric acid levels at an oral dose of
15 mg/kg. Therefore, compound 3j could be a promising lead compound for the treatment of
hyperuricemia and gout, and further detailed studies on compound 3j are under progress.
4. Experimental section
4.1. Chemistry
Unless otherwise noted, the solvents and reagents, obtained from commercial companies, were
used directly without further purification. All reactions were run using a Teflon-coated magnetic
stir bar on the benchtop. Reactions that were run at elevated temperatures were run in an oil bath at
13

the indicated temperature. The reaction progresses were monitored by thin-layer chromatography
(TLC) with a fluorescence indicator at 254 nm. Column chromatography was carried out on silica
gel (200–300 mesh) from Qingdao Ocean Chemicals (Qingdao, Shandong, China). The purity of
1
the target compounds was determined by HPLC to be over 95%. H NMR and 13C NMR spectra
were measured on a Bruker 400 MHz spectrometer or a Bruker 600 MHz spectrometer (Bruker Inc.,
Billerica, USA). Chemical shifts were recorded in δ units using tetramethylsilane (TMS) as a
reference and DMSO-d6 as a solvent. Low resolution mass spectrometry (MS) were recorded with
an Agilent 1100 ion trap liquid chromatography-mass spectrometer. High-resolution mass spectra
(HRMS) data were taken with Agilent Q-TOF system. Melting points were determined on a Büchi
melting point apparatus (B-540) uncorrected. Infra-red (IR) spectra were recorded on Bruker IR-
27G spectrometer with KBr pellets.
4.1.1. Synthesis of 3-(1H-tetrazol-1-yl) benzonitrile (1a)
A mixture of 3-amino-benzonitrile (10 g, 0.08 mol), triethyl orthoformate (15.1 g, 0.10 mol),
sodium azide (5.5 g, 0.08 mol) and AcOH (50 mL) was stirred at 60°C under N2 atmosphere for 24
h. At the end of the reaction, monitored by TLC, the precipitate was filtered, washed with water,
and then recrystallized from 40% methanol to afford the brown powder. yield: 67.2%; M.p.: 141.6-
142.5oC；1H NMR (400 MHz, DMSO-d6) δ 10.17 (s, 1H), 8.50 (t, J = 1.9 Hz, 1H), 8.30 (dd, J =
8.3, 2.2 Hz, 1H), 8.06 (dt, J = 7.8, 1.3 Hz, 1H), 7.89 (t, J = 8.0 Hz, 1H); ESI-MS m/z 172.06 [M+H]+.
4.1.2. General protocol for the synthesis of 4-alkoxy-3-(1H-tetrazol-1-yl) benzonitriles (1b-1v)
and 4-cyclohexylmethoxy-benzonitrile (1w)
A 500 mL three-necked flask was charged with 4-hydroxy-3-(1H-tetrazol-1-yl) benzonitrile (or
4-hydroxybenzonitrile, 80 mmol), alkyl halides (100 mmol), potassium iodide (8 mmol), and
potassium carbonate (160 mmol) in 80 mL N,N-dimethylformamide. The reaction was stirred at 60-
80°C overnight. After complete, the reaction was quenched by cautions, dropwise addition of water,
then poured into cold saturated sodium chloride and stirred 30 min. The solution was extracted with
14

ethyl acetate, washed with brine, dried over anhydrous Na2SO4 and concentrated in reduced pressure.
The crude product was purified by flash column chromatography to afford the title compounds.
4-Methoxy-3-(1H-tetrazol-1-yl) benzonitrile (1b)
A gray powder; yield 60.9.%; M.p 172.3°C-174.1°C; 1H NMR (600 MHz, DMSO-d6) δ 9.85 (s,
1H), 8.28 (d, J = 2.1 Hz, 1H), 8.12 (dd, J = 8.8, 2.1 Hz, 1H), 7.56 (d, J = 8.8 Hz, 1H), 3.96 (s, 3H);
ESI-MS m/z 202.4 [M+H]+, 224.4 [M+Na]+.
4-Isopropoxy-3-(1H-tetrazol-1-yl) benzonitrile (1c)
A red powder; yield 70.7%; M.p 135.6°C-136.6°C; 1H NMR (600 MHz, DMSO-d6) δ 9.79 (s,
1H), 8.26 (d, J = 2.1 Hz, 1H), 8.07 (dd, J = 8.8, 2.1 Hz, 1H), 7.58 (d, J = 8.9 Hz, 1H), 4.92 (hept, J
= 6.0 Hz, 1H), 1.27 (d, J = 6.0 Hz, 6H); ESI-MS m/z 480.8 [2M+Na]+.
4-Isobutoxy-3-(1H-tetrazol-1-yl) benzonitrile (1d)
1
A white powder; yield 72.2%; M.p 139.2°C-140.6°C; H NMR (600 MHz, DMSO-d6) δ 9.79
(s, 1H), 8.28 (d, J = 2.1 Hz, 1H), 8.10 (dd, J = 8.8, 2.1 Hz, 1H), 7.54 (d, J = 8.8 Hz, 1H), 3.98 (d, J
= 6.3 Hz, 2H), 1.95 (hept, J = 6.6 Hz, 1H), 0.84 (d, J = 6.7 Hz, 6H); ESI-MS m/z 244.4 [M+H]+.
4-Isopentyloxy-3-(1H-tetrazol-1-yl) benzonitrile (1e)
A white powder; yield 73.5%; M.p 108.7°C-109°C; 1H NMR (600 MHz, DMSO-d6) δ 9.79 (s,
1H), 8.27 (d, J = 2.1 Hz, 1H), 8.10 (dd, J = 8.8, 2.1 Hz, 1H), 7.57 (d, J = 8.8 Hz, 1H), 4.22 (t, J =
6.3 Hz, 2H), 1.62 – 1.54 (m, 3H), 0.83 (d, J = 6.3 Hz, 6H); ESI-MS m/z 258.3 [M+H]+.
4-(3-Methylbut-2-en-1-yl) oxy-3-(1H-tetrazol-1-yl) benzonitrile (1f)
1
A light pink powder; yield 62.4%; M.p 126.3°C-127.2°C; H NMR (600 MHz, DMSO-d6) δ
9.80 (s, 1H), 8.27 (d, J = 2.2 Hz, 1H), 8.10 (dd, J = 8.7, 2.2 Hz, 1H), 7.56 (d, J = 8.8 Hz, 1H), 5.41
– 5.34 (m, 1H), 4.79 (d, J = 6.7 Hz, 2H), 1.72 (s, 3H), 1.68 (s, 3H); ESI-MS m/z 256.2 [M+H]+,
278.2 [M+Na]+.
15

4-(Prop-2-yn-1-yloxy)-3-(1H-tetrazol-1-yl) benzonitrile (1g)
1
A white powder; yield 60.5%; M.p 138.4°C-138.9°C; H NMR (600 MHz, DMSO-d6) δ 9.83
(s, 1H), 8.37 – 8.27 (m, 1H), 8.21 – 8.12 (m, 1H), 7.61 (d, J = 8.8 Hz, 1H), 5.08 (s, 2H), 3.74 (s,
1H); ESI-MS m/z 226.05 [M+H]+.
4-Cyclopropylmethoxy-3-(1H-tetrazol-1-yl) benzonitrile (1h)
1
A white powder; yield 71.2%; M.p 139.1°C-140.2°C; H NMR (600 MHz, DMSO-d6) δ 9.81
(s, 1H), 8.28 (d, J = 2.1 Hz, 1H), 8.07 (dd, J = 8.7, 2.1 Hz, 1H), 7.52 (d, J = 8.8 Hz, 1H), 4.09 (d, J
= 7.1 Hz, 2H), 1.24 – 1.15 (m, 1H), 0.56 – 0.50 (m, 2H), 0.33 – 0.28 (m, 2H); ESI-MS m/z 242.07
[M+H]+.
4-Cyclopentyloxy-3-(1H-tetrazol-1-yl) benzonitrile (1i)
A white powder; yield 67.9%; M.p 148°C-149.1°C; 1H NMR (600 MHz, DMSO-d6) δ 9.76 (s,
1H), 8.27 (d, J = 2.1 Hz, 1H), 8.08 (dd, J = 8.8, 2.2 Hz, 1H), 7.55 (d, J = 8.8 Hz, 1H), 5.12 (tt, J =
5.6, 2.5 Hz, 1H), 1.95 – 1.87 (m, J = 5.2 Hz, 2H), 1.71 – 1.66 (m, 2H), 1.59 – 1.52 (m, 4H); ESI-
MS m/z 256.3 [M+H]+.
4-Cyclohexylmethoxy-3-(1H-tetrazol-1-yl) benzonitrile (1j)
1
A light pink powder; yield 63.5%; M.p 120.6°C-122.2°C; H NMR (600 MHz, DMSO-d6) δ
9.78 (s, 1H), 8.28 (d, J = 2.1 Hz, 1H), 8.10 (dd, J = 8.8, 2.1 Hz, 1H), 7.55 (d, J = 8.8 Hz, 1H), 4.01
(d, J = 6.2 Hz, 2H), 1.72 – 1.55 (m, 6H), 1.23 – 1.14 (m, 2H), 1.11 – 1.04 (m, 1H), 0.95 – 0.88 (m,
2H); ESI-MS m/z 284.11 [M+H]+.
4-Benzyloxy-3-(1H-tetrazol-1-yl) benzonitrile (1k)
A light red powder; yield 68.8%; M.p 148.3°C-149°C; 1H NMR (600 MHz, DMSO-d6) δ 9.83
(s, 1H), 8.31 (d, J = 2.1 Hz, 1H), 8.12 (dd, J = 8.8, 2.1 Hz, 1H), 7.63 (d, J = 8.8 Hz, 1H), 7.41 – 7.31
(m, 5H), 5.36 (s, 2H); ESI-MS m/z 576.8 [2M+Na]+.
4-(4-Methylbenzyl) oxy-3-(1H-tetrazol-1-yl) benzonitrile (1l)
16

A red powder; yield 58.2%; M.p 161.2°C-162.5 °C; 1H NMR (600 MHz, DMSO-d6) δ 9.80 (s,
1H), 8.29 (d, J = 2.1 Hz, 1H), 8.11 (dd, J = 8.8, 2.1 Hz, 1H), 7.62 (d, J = 8.8 Hz, 1H), 7.26 (d, J =
8.0 Hz, 2H), 7.18 (d, J = 7.8 Hz, 2H), 5.29 (s, 2H), 2.28 (s, 3H); ESI-MS m/z 604.6 [2M+Na]+.
4-[4-(Tert-butyl) benzyl] oxy-3-(1H-tetrazol-1-yl) benzonitrile (1m)
A white powder; yield 65%; M.p 135.6°C-136.6°C; 1H NMR (600 MHz, DMSO-d6) δ 9.84 (s,
1H), 8.30 (d, J = 2.1 Hz, 1H), 8.12 (dd, J = 8.8, 2.2 Hz, 1H), 7.65 (d, J = 8.9 Hz, 1H), 7.42 – 7.38
(m, 2H), 7.32 – 7.29 (m, 2H), 5.32 (s, 2H), 1.26 (s, 9H); ESI-MS m/z 356.4 [M+Na]+.
4-(4-methoxybenzyl) oxy-3-(1H-tetrazol-1-yl) benzonitrile (1n)
1
A white powder; yield 67.7%; M.p 148.9°C-150.2°C; H NMR (600 MHz, DMSO-d6) δ 9.79
(s, 1H), 8.29 (d, J = 2.0 Hz, 1H), 8.12 (dd, J = 8.8, 2.1 Hz, 1H), 7.64 (d, J = 8.8 Hz, 1H), 7.33 (d, J
= 8.6 Hz, 2H), 6.93 (d, J = 8.6 Hz, 2H), 5.26 (s, 2H), 3.74 (s, 3H); ESI-MS m/z 330.2 [M+Na]+.
4-(4-Fluorobenzyl) oxy-3-(1H-tetrazol-1-yl) benzonitrile (1o)
A light red powder; yield 73.6%; M.p 160.7°C-161.6°C; 1H NMR (600 MHz, DMSO-d6) δ 9.84
(s, 1H), 8.31 (d, J = 2.0 Hz, 1H), 8.12 (dd, J = 8.8, 2.0 Hz, 1H), 7.61 (d, J = 8.8 Hz, 1H), 7.45 (d, J
= 8.4 Hz, 2H), 7.41 (d, J = 8.4 Hz, 2H), 5.35 (s, 2H); ESI-MS m/z 296.4 [M+H]+, 318.2 [M+Na]+.
4-(4-Chlorobenzyl) oxy-3-(1H-tetrazol-1-yl) benzonitrile (1p)
A white powder; yield 58%; M.p 195.9°C-197°C; 1H NMR (600 MHz, DMSO-d6) δ 9.83 (s, 1H),
8.30 (d, J = 2.0 Hz, 1H), 8.12 (dd, J = 8.8, 2.0 Hz, 1H), 7.63 (d, J = 8.8 Hz, 1H), 7.45 (dd, J = 8.4,
5.6 Hz, 2H), 7.21 (t, J = 8.8 Hz, 2H), 5.34 (s, 2H); ESI-MS m/z 312.06 [M+H]+.
4-(4-Bromobenzyl) oxy-3-(1H-tetrazol-1-yl) benzonitrile (1q)
A light red powder; yield 64.1%; M.p 181.3°C-183.6°C; 1H NMR (600 MHz, DMSO-d6) δ 9.84
(s, 1H), 8.31 (d, J = 1.8 Hz, 1H), 8.12 (dd, J = 8.8, 1.8 Hz, 1H), 7.61 (d, J = 8.8 Hz, 1H), 7.58 (d, J
= 8.3 Hz, 2H), 7.34 (d, J = 8.2 Hz, 2H), 5.34 (s, 2H); ESI-MS m/z 356.04, 358.03 [M+H]+.
17

4-(3-Methylbenzyl) oxy-3-(1H-tetrazol-1-yl) benzonitrile (1r)
A red powder; yield 74.4%; M.p 154.3°C-155.1 °C; 1H NMR (400 MHz, DMSO-d6) δ 9.82 (s,
1H), 8.30 (d, J = 2.0 Hz, 1H), 8.12 (dd, J = 8.8, 2.1 Hz, 1H), 7.62 (d, J = 8.8 Hz, 1H), 7.26 (t, J =
7.5 Hz, 1H), 7.19 – 7.12 (m, 3H), 5.31 (s, 2H); ESI-MS m/z 292.07 [M+H]+.
4-(3-Methoxybenzyl) oxy-3-(1H-tetrazol-1-yl) benzonitrile (1s)
A white powder; yield 53.7%; M.p 134.4°C-135.6 °C; 1H NMR (400 MHz, DMSO-d6) δ 9.85
(s, 1H), 8.31 (d, J = 2.1 Hz, 1H), 8.12 (dd, J = 8.8, 2.1 Hz, 1H), 7.61 (d, J = 8.8 Hz, 1H), 7.28 (t,
1H), 6.97 – 6.85 (m, 3H), 5.33 (s, 2H), 3.73 (s, 3H); ESI-MS m/z 308.3 [M+H]+, 330.2 [M+Na]+.
4-(3-Fluorobenzyl) oxy-3-(1H-tetrazol-1-yl) benzonitrile (1t)
A white powder; yield 74.3%; M.p 142.6°C-144.7 °C; 1H NMR (400 MHz, DMSO-d6) δ 9.87
(s, 1H), 8.32 (d, J = 2.1 Hz, 1H), 8.13 (dd, J = 8.8, 2.2 Hz, 1H), 7.61 (d, J = 8.8 Hz, 1H), 7.44 (td, J
= 7.8, 5.9 Hz, 1H), 7.28 – 7.11 (m, 3H), 5.39 (s, 2H); ESI-MS m/z 296.3 [M+H]+.
4-(3-Chlorobenzyl) oxy-3-(1H-tetrazol-1-yl) benzonitrile (1u)
A light red powder; yield 57.4%; M.p 157.2°C-158.5 °C; 1H NMR (600 MHz, DMSO-d6) δ 9.88
(s, 1H), 8.32 (d, J = 2.2 Hz, 1H), 8.14 (dd, J = 8.8, 2.1 Hz, 1H), 7.61 (d, J = 8.8 Hz, 1H), 7.46 (d, J
= 2.0 Hz, 1H), 7.44 – 7.39 (m, 2H), 7.35 – 7.33 (m, 1H), 5.37 (s, 2H); ESI-MS m/z 312.04 [M+H]+.
4-(3-Bromobenzyl) oxy-3-(1H-tetrazol-1-yl) benzonitrile (1v).
A light red powder; yield 61.8%; M.p 161.9°C-162.2°C; 1H NMR (600 MHz, DMSO-d6) δ 9.87
(s, 1H), 8.32 (d, J = 2.1 Hz, 1H), 8.14 (dd, J = 8.8, 2.1 Hz, 1H), 7.64 – 7.57 (m, 2H), 7.54 (dt, J =
7.5, 1.8 Hz, 1H), 7.40 – 7.31 (m, 2H), 5.36 (s, 2H); ESI-MS m/z 378.2 [M+Na]+.
4-Cyclohexylmethoxy benzonitrile (1w)
A powder white; yield 71.8%; 1H NMR (600 MHz, DMSO-d6) δ 7.74 (dd, J = 9.0, 2.8 Hz, 2H),
7.09 (dd, J = 9.0, 2.8 Hz, 2H), 3.86 (dd, J = 6.4, 2.8 Hz, 2H), 1.89 – 1.59 (m, 6H), 1.28 – 1.15 (m,
3H), 1.08 – 1.01 (m, 2H); ESI-MS m/z 216.14 [M+H]+.
18

4.1.3. General protocol for the synthesis of N'-hydroxy-3-(1H-tetrazol-1-yl) benzimidamide
(2a), N'-hydroxy-4-alyoxy-3-(1H-tetrazol-1-yl) benzimidamides (2b-2v), and N'-hydroxy-4-
alyoxy benzimidamide (2w)
Benzonitrile (1a, 1b-1v and 1w, 30 mmol), hydroxylamine (36 mmol) and Na2CO3 (60 mmol)
were suspended in 30 mL of DMSO. The reaction mixture was stirred at 80°C for 2-3 hour and
monitored by TLC. The solvent was cooled to room temperature and poured into cold water with
stirring after the completion of the reaction. The resulting precipitate was filtered, washed with
water and dried to obtain the corresponding title compounds.
N'-Hydroxy-3-(1H-tetrazol-1-yl) benzimidamide (2a)
A white powder; yield 81.6%; M.p 188.9°C-189.2°C; 1H NMR (400 MHz, DMSO-d6) δ 10.16
(s, 1H), 9.92 (s, 1H), 8.23 (t, J = 1.9 Hz, 1H), 7.93 (dd, J = 7.9, 2.2 Hz, 1H), 7.91 – 7.86 (m, 1H),
7.67 (t, J = 8.0 Hz, 1H), 6.03 (s, 2H); ESI-MS m/z 227.9 [M+Na]+.
N'-Hydroxy-4-methoxy-3-(1H-tetrazol-1-yl) benzimidamide (2b)
1
A white powder; yield 86.4%; M.p 183.8°C-183.9°C; H NMR (400 MHz, DMSO-d6) δ 9.82
(s, 1H), 9.68 (s, 1H), 7.98 (d, J = 2.2 Hz, 1H), 7.91 (dd, J = 8.8, 2.2 Hz, 1H), 7.38 (d, J = 8.8 Hz,
1H), 5.91 (s, 2H), 3.90 (s, 3H); ESI-MS m/z 235.4 [M+H]+, 257.3 [M+Na]+.
N'-Hydroxy-4-isopropoxy-3-(1H-tetrazol-1-yl) benzimidamide (2c)
A white powder; yield 75.5%; M.p 182.6°C-183°C; 1H NMR (400 MHz, DMSO-d6) δ 9.75 (s,
1H), 9.67 (s, 1H), 7.96 (d, J = 2.3 Hz, 1H), 7.87 (dd, J = 8.8, 2.3 Hz, 1H), 7.39 (d, J = 8.9 Hz, 1H),
5.90 (s, 2H), 4.80 (hept, J = 6.1 Hz, 1H), 1.25 (d, J = 6.0 Hz, 6H); ESI-MS m/z 263.3[M+H]+,
285.2[M+Na]+.
N'-Hydroxy-4-isobutoxy-3-(1H-tetrazol-1-yl) benzimidamide (2d)
A white powder; yield 80%; M.p 172.6°C-174.1°C; 1H NMR (400 MHz, DMSO-d6) δ 9.76 (s,
19

1H), 9.67 (s, 1H), 7.95 (d, J = 2.2 Hz, 1H), 7.89 (dd, J = 8.8, 2.2 Hz, 1H), 7.37 (d, J = 8.8 Hz, 1H),
5.90 (s, 2H), 3.91 (d, J = 6.4 Hz, 2H), 1.95 (hept, J = 6.6 Hz, 1H), 0.85 (d, J = 6.7 Hz, 6H); ESI-MS
m/z 275.3 [M-H]-.
N'-Hydroxy-4-isopentyloxy-3-(1H-tetrazol-1-yl) benzimidamide (2e)
1
A white powder; yield 80.7%; M.p 170.1°C-171.2°C; H NMR (400 MHz, DMSO-d6) δ 9.75
(s, 1H), 9.67 (s, 1H), 7.95 (d, J = 2.2 Hz, 1H), 7.89 (dd, J = 8.8, 2.2 Hz, 1H), 7.39 (d, J = 8.8 Hz,
1H), 5.90 (s, 2H), 4.15 (t, J = 6.3 Hz, 2H), 1.65 – 1.53 (m, 3H), 0.85 (d, J = 6.2 Hz, 6H); ESI-MS
m/z 291.6 [M+H]+.
N'-Hydroxy-4-(3-methylbut-2-en-1-yl) oxy-3-(1H-tetrazol-1yl) benzimidamide (2f)
1
A white powder; yield 78.8%; M.p 175.4°C-176.8°C; H NMR (400 MHz, DMSO-d6) δ 9.76
(s, 1H), 9.68 (s, 1H), 7.96 (d, J = 2.2 Hz, 1H), 7.88 (dd, J = 8.8, 2.3 Hz, 1H), 7.39 (d, J = 8.8 Hz,
1H), 5.90 (s, 2H), 5.41 – 5.33 (m, 1H), 4.70 (d, J = 6.7 Hz, 2H), 1.72 (s, 3H), 1.67 (s, 3H); ESI-MS
m/z 289.5 [M+H]+, 311.4 [M+Na]+.
N'-Hydroxy-4-(prop-2-yn-1-yl) oxy-3-(1H-tetrazol-1-yl) benzimidamide (2g)
1
A white powder; yield 77.5%; M.p 156.4°C-157.2°C; H NMR (400 MHz, DMSO-d6) δ 9.79
(s, 1H), 9.71 (s, 1H), 7.98 (d, J = 2.2 Hz, 1H), 7.91 (dd, J = 8.8, 2.2 Hz, 1H), 7.45 (d, J = 8.8 Hz,
1H), 5.92 (s, 2H), 4.98 (d, J = 2.4 Hz, 2H), 3.66 (t, J = 2.4 Hz, 1H); ESI-MS m/z 517.0 [2M+H]+.
N'-Hydroxy-4-cyclopropylmethoxy-3-(1H-tetrazol-1-yl) benzimidamide (2h)
1
A white powder; yield 73.4%; M.p 175.2°C-176.1°C; H NMR (400 MHz, DMSO-d6) δ 9.79
(s, 1H), 9.67 (s, 1H), 7.98 (d, J = 2.2 Hz, 1H), 7.87 (dd, J = 8.8, 2.2 Hz, 1H), 7.35 (d, J = 8.8 Hz,
1H), 5.90 (s, 2H), 4.01 (d, J = 7.0 Hz, 2H), 1.24 – 1.14 (m, 1H), 0.56 – 0.47 (m, 2H), 0.31 – 0.28
(m, 2H); ESI-MS m/z 275.07 [M+H]+.
N'-Hydroxy-4-cyclopentyloxy-3-(1H-tetrazol-1-yl) benzimidamide (2i)
1
A white powder; yield 69.2%; M.p 178.1°C-178.9°C; H NMR (400 MHz, DMSO-d6) δ 9.72
20

(s, 1H), 9.68 (s, 1H), 7.96 (d, J = 2.2 Hz, 1H), 7.88 (dd, J = 8.8, 2.3 Hz, 1H), 7.37 (d, J = 8.8 Hz,
1H), 5.90 (s, 2H), 5.02 (tt, J = 5.6, 2.5 Hz, 1H), 1.94 – 1.82 (m, 2H), 1.72 – 1.65 (m, 2H), 1.58 –
1.51 (m, 4H); ESI-MS m/z 289.6 [M+H]+.
N'-Hydroxy-4-cyclohexylmethoxy-3-(1H-tetrazol-1-yl) benzimidamide (2j)
1
A white powder; yield 77.1%; M.p 186.7°C-186.9°C; H NMR (400 MHz, DMSO-d6) δ 9.75
(s, 1H), 9.68 (s, 1H), 7.96 (d, J = 2.2 Hz, 1H), 7.89 (dd, J = 8.8, 2.3 Hz, 1H), 7.37 (d, J = 8.9 Hz,
1H), 5.91 (s, 2H), 3.94 (d, J = 6.0 Hz, 2H), 1.72 – 1.57 (m, 6H), 1.30 – 1.04 (m, 3H), 0.98 – 0.88
(m, 2H); ESI-MS m/z 317.7 [M+H]+, 633.1 [2M+H]+.
N'-Hydroxy-4-benzyloxy-3-(1H-tetrazol-1-yl) benzimidamide (2k)
A white powder; yield 83.8%; M.p 195°C-195.8°C; ESI-MS m/z 311.6 [M+H]+. 1H NMR (400
MHz, DMSO-d6) δ 9.82 (s, 1H), 9.71 (s, 1H), 7.99 (d, J = 2.2 Hz, 1H), 7.90 (dd, J = 8.8, 2.3 Hz,
1H), 7.47 (d, J = 8.9 Hz, 1H), 7.38 (d, J = 4.4 Hz, 4H), 7.36 – 7.30 (m, 1H), 5.93 (s, 2H), 5.29 (s,
2H); ESI-MS m/z 311.6 [M+H]+.
N'-Hydroxy-4-(4-methylbenzyl) oxy-3-(1H-tetrazol-1-yl) benzimidamide (2l)
1
A white powder; yield 83.7%; M.p 199.8°C-200.1°C; H NMR (400 MHz, DMSO-d6) δ 9.80
(s, 1H), 9.73 (s, 1H), 8.00 (d, J = 2.2 Hz, 1H), 7.91 (dd, J = 8.8, 2.3 Hz, 1H), 7.46 (d, J = 8.8 Hz,
1H), 7.28 (d, J = 7.7 Hz, 2H), 7.18 (d, J = 7.7 Hz, 2H), 5.94 (s, 2H), 5.23 (s, 2H), 2.29 (s, 3H); ESI-
MS m/z 649.0 [2M+H]+.
N'-Hydroxy-4-[4-(tert-butyl) benzyl] oxy-3-(1H-tetrazol-1-yl) benzimidamide (2m)
1
A white powder; yield 74.5%; M.p 194.8°C-195.5°C; H NMR (400 MHz, DMSO-d6) δ 9.82
(s, 1H), 9.74 (s, 1H), 7.99 (d, J = 2.2 Hz, 1H), 7.90 (dd, J = 8.8, 2.3 Hz, 1H), 7.48 (d, J = 8.9 Hz,
1H), 7.42 – 7.36 (m, 2H), 7.30 (d, J = 8.2 Hz, 2H), 5.92 (s, 2H), 5.24 (s, 2H), 1.27 (s, 9H); ESI-MS
m/z 367.4 [M+H]+.
N'-Hydroxy-4-(4-methoxybenzyl) oxy-3-(1H-tetrazol-1-yl) benzimidamide (2n)
21

1
A white powder; yield 75.1%; M.p 189.4°C-190.5°C; H NMR (400 MHz, DMSO-d6) δ 9.77
(s, 1H), 9.71 (s, 1H), 7.99 (d, J = 2.2 Hz, 1H), 7.90 (dd, J = 8.8, 2.3 Hz, 1H), 7.48 (d, J = 8.9 Hz,
1H), 7.37 – 7.29 (m, 2H), 6.97 – 6.89 (m, 2H), 5.93 (s, 2H), 5.20 (s, 2H), 3.75 (s, 3H); ESI-MS m/z
363.3 [M+Na]+.
N'-Hydroxy-4-(4-fluorobenzyl) oxy-3-(1H-tetrazol-1-yl) benzimidamide (2o)
1
A white powder; yield 79.2%; M.p 198.8°C-199.4°C; H NMR (400 MHz, DMSO-d6) δ 9.80
(s, 1H), 9.70 (s, 1H), 7.98 (d, J = 2.2 Hz, 1H), 7.90 (dd, J = 8.8, 2.2 Hz, 1H), 7.49 – 7.41 (m, 3H),
7.25 – 7.18 (m, 2H), 5.92 (s, 2H), 5.26 (s, 2H); ESI-MS m/z 329.3 [M+H]+.
N'-Hydroxy-4-(4-chlorobenzyl) oxy-3-(1H-tetrazol-1-yl) benzimidamide (2p)
1
A white powder; yield 76.7%; M.p 206.7°C-207.9°C; H NMR (400 MHz, DMSO-d6) δ 9.82
(s, 1H), 9.70 (s, 1H), 7.98 (d, J = 2.3 Hz, 1H), 7.89 (dd, J = 8.8, 2.3 Hz, 1H), 7.47 – 7.39 (m, 5H),
5.92 (s, 2H), 5.28 (s, 2H); ESI-MS m/z 345.5 [M+H]+.
N'-Hydroxy-4-(4-bromobenzyl) oxy-3-(1H-tetrazol-1-yl) benzimidamide (2q)
1
A white powder; yield 85.3%; M.p 183.1°C-183.5°C; H NMR (400 MHz, DMSO-d6) δ 9.82
(s, 1H), 9.70 (s, 1H), 7.98 (d, J = 2.2 Hz, 1H), 7.89 (dd, J = 8.7, 2.3 Hz, 1H), 7.58 (d, J = 8.0 Hz,
2H), 7.44 (d, J = 8.9 Hz, 1H), 7.34 (d, J = 8.1 Hz, 2H), 5.92 (s, 2H), 5.26 (s, 2H); ESI-MS m/z 390.9
[M+H]+.
N'-Hydroxy-4-(3-methylbenzyl) oxy-3-(1H-tetrazol-1-yl) benzimidamide (2r)
1
A white powder; yield 79.8%; M.p 192.3°C-192.5°C; H NMR (400 MHz, DMSO-d6) δ 9.81
(s, 1H), 9.70 (s, 1H), 7.98 (d, J = 2.2 Hz, 1H), 7.89 (dd, J = 8.8, 2.2 Hz, 1H), 7.45 (d, J = 8.8 Hz,
1H), 7.26 (t, J = 7.5 Hz, 1H), 7.18 – 7.11 (m, 3H), 5.92 (s, 2H), 5.24 (s, 2H); ESI-MS m/z 325.8
[M+H]+.
N'-Hydroxy-4-(3-methoxybenzyl) oxy-3-(1H-tetrazol-1-yl) benzimidamide (2s)
1
A white powder; yield 74.5%; M.p 181.6°C-182.1°C; H NMR (400 MHz, DMSO-d6) δ 9.84
22

(s, 1H), 9.69 (s, 1H), 7.98 (d, J = 2.2 Hz, 1H), 7.89 (dd, J = 8.8, 2.2 Hz, 1H), 7.45 (d, J = 8.9 Hz,
1H), 7.28 (t, J = 8.0 Hz, 1H), 6.97 – 6.90 (m, 2H), 6.88 (dd, J = 8.0, 2.5 Hz, 1H), 5.91 (s, 2H), 5.25
(s, 2H), 3.74 (s, 3H); ESI-MS m/z 681.1 [2M+H]+.
N'-Hydroxy-4-(3-fluorobenzyl) oxy-3-(1H-tetrazol-1-yl) benzimidamide (2t)
A white powder; yield 80%; M.p 189.5°C-189.8°C; 1H NMR (400 MHz, DMSO-d6) δ 9.85 (s,
1H), 9.70 (s, 1H), 7.99 (d, J = 2.2 Hz, 1H), 7.90 (dd, J = 8.8, 2.2 Hz, 1H), 7.47 – 7.38 (m, 2H), 7.25
– 7.12 (m, 3H), 5.92 (s, 2H), 5.30 (s, 2H); ESI-MS m/z 329.6 [M+H]+.
N'-Hydroxy-4-(3-chlorobenzyl) oxy-3-(1H-tetrazol-1-yl) benzimidamide (2u)
1
A white powder; yield 73.5%; M.p 186.5°C-187.4°C; H NMR (400 MHz, DMSO-d6) δ 9.86
(s, 1H), 9.72 (s, 1H), 8.00 (d, J = 2.2 Hz, 1H), 7.91 (dd, J = 8.8, 2.3 Hz, 1H), 7.48 – 7.35 (m, 4H),
7.34 (dd, J = 6.5, 2.1 Hz, 1H), 5.93 (s, 2H), 5.30 (s, 2H); ESI-MS m/z 345.4 [M+H]+, 367.1 [M+Na]+.
N'-Hydroxy-4-(3-bromobenzyl) oxy-3-(1H-tetrazol-1-yl) benzimidamide (2v)
1
A white powder; yield 82.5%; M.p 181.4°C-181.6°C; H NMR (400 MHz, DMSO-d6) δ 9.85
(s, 1H), 9.71 (s, 1H), 7.99 (d, J = 2.2 Hz, 1H), 7.90 (dd, J = 8.8, 2.2 Hz, 1H), 7.60 (t, J = 1.8 Hz,
1H), 7.53 (dt, J = 7.4, 1.9 Hz, 1H), 7.44 (d, J = 8.8 Hz, 1H), 7.40 – 7.31 (m, 2H), 5.92 (s, 2H), 5.29
(s, 2H); ESI-MS m/z 391.4 [M+H]+.
N'-Hydroxy-4-cyclohexylmethoxy benzimidamide (2w)
A white powder; yield 78.2%; 1H NMR (600 MHz, DMSO-d6) δ 9.44 (s, 1H), 7.61 – 7.55 (m, 2H),
6.93 – 6.87 (m, 2H), 5.68 (s, 2H), 3.79 (d, J = 6.4 Hz, 2H), 1.82 – 1.62 (m, 6H), 1.29 – 1.12 (m,
3H), 1.07 – 1.00 (m, 2H); ESI-MS m/z 249.15 [M+H]+.
4.1.4. General protocol for synthesis of 3-[4-alkoxy-3-(1H-tetrazol-1-yl) phenyl]-1,2,4-
oxadiazol-5(4H)-ones (3a-3w)
N'-Hydroxy benzimidamide (2a, 2b-2v and 2w, 20 mmol) and DBU (40 mmol) were dissolved
23

in dichloromethane (20 mL) at 30°C. Thereafter, CDI (22 mmol) was added to the reaction mixture,
which was stirred at 30°C for 2 h. The solvent was evaporated in vacuo after the completion of the
reaction, monitored by TLC. Ice water was added to the residue, and the pH was brought to 2 with
2 M hydrochloric acid. The resulting precipitate was filtered, dried, washed with water and
recrystallized with anhydrous methanol to obtain the corresponding target compounds.
3-[3-(1H-Tetrazol-1-yl) phenyl]-1,2,4-oxadiazol-5(4H)-one (3a)
A white crystalline powder; yield 82.8%; M.p 219.3°C-220.1°C; IR (KBr, cm-1): 3195.5, 3123.0,
1776.1, 1631.1, 1570.7, 1522.2, 1467.5; 1H NMR (400 MHz, DMSO-d6) δ 13.17 (s, 1H), 10.18 (s,
1H), 8.38 (t, J = 1.9 Hz, 1H), 8.18 (dd, J = 8.0, 2.2 Hz, 1H), 8.01 – 7.95 (m, 1H), 7.88 (t, J = 8.0
Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 160.36, 157.05, 142.90, 134.89, 131.69, 127.48, 125.68,
124.87, 118.97; ESI-HRMS calcd. for C9H5N6O2 [M-H]- 229.0479 found 229.0501.
3-[4-Methoxy-3-(1H-tetrazol-1-yl) phenyl]-1,2,4-oxadiazol-5(4H)-one (3b)
A white crystalline powder; yield 75.2%; M.p 228.9°C-229.5°C; IR (KBr, cm-1): 3454.4, 3162.4,
1778.8, 1619.9, 1493.2, 1439.2; 1H NMR (400 MHz, DMSO-d6) δ 12.80 (s, 1H), 9.89 (s, 1H), 8.17
(d, J = 2.2 Hz, 1H), 8.02 (dd, J = 8.8, 2.2 Hz, 1H), 7.59 (d, J = 8.8 Hz, 1H), 3.99 (s, 3H); 13C NMR
(100 MHz, DMSO-d6) δ 160.52, 156.81, 154.65, 145.16, 129.80, 123.79, 123.46, 116.71, 114.57,
57.42; ESI-HRMS calcd. for C10H7N6O3 [M-H]- 259.0585, found 259.0600.
3-[4-Isopropoxy-3-(1H-tetrazol-1-yl) phenyl]-1,2,4-oxadiazol-5(4H)-one (3c)
A white crystalline powder; yield 71%; M.p 209.5°C-211°C; IR (KBr, cm-1): 3080.7, 2968.9,
1783.9, 1620.0, 1497.0, 1462.4; 1H NMR (400 MHz, DMSO-d6) δ 12.99 (s, 1H), 9.82 (s, 1H), 8.16
(d, J = 2.2 Hz, 1H), 7.99 (dd, J = 8.9, 2.2 Hz, 1H), 7.62 (d, J = 8.9 Hz, 1H), 4.92 (hept, J = 6.1 Hz,
1H), 1.31 (d, J = 6.1 Hz, 6H); 13C NMR (100 MHz, DMSO-d6) δ 160.30, 156.67, 153.08, 145.15,
129.71, 124.16, 124.07, 116.21, 116.09, 72.85, 21.79 (2C); ESI-HRMS calcd. for C12H11N6O3 [M-
H]- 287.0898, found 287.0910.
3-[4-Isobutoxy-3-(1H-tetrazol-1-yl) phenyl]-1,2,4-oxadiazol-5(4H)-one (3d)
24

A white crystalline powder; yield 65.5%; M.p 179.6°C-180.3°C; IR (KBr, cm-1): 3457.3, 3170.2,
2963.1, 2879.0, 1778.5, 1621.8, 1533.1, 1462.2; 1H NMR (400 MHz, DMSO-d6) δ 12.98 (s, 1H),
9.81 (s, 1H), 8.14 (d, J = 2.2 Hz, 1H), 8.01 (dd, J = 8.8, 2.3 Hz, 1H), 7.59 (d, J = 8.9 Hz, 1H), 3.99
(d, J = 6.4 Hz, 2H), 1.99 (hept, J = 6.6 Hz, 1H), 0.87 (d, J = 6.7 Hz, 6H); 13C NMR (100 MHz,
DMSO-d6) δ 160.31, 156.70, 154.64, 145.31, 130.08, 124.39, 123.59, 116.42, 115.26, 75.79, 27.86,
19.15 (2C); ESI-HRMS calcd. for C13H13N6O3 [M-H]- 301.1054, found 301.1070.
3-[4-Isopentyloxy-3-(1H-tetrazol-1-yl) phenyl]-1,2,4-oxadiazol-5(4H)-one (3e)
A white crystalline powder; yield 67.3%; M.p 187.6°C-188.6°C; IR (KBr, cm-1): 3459.7, 3160.5,
2958.0, 2884.7, 1788.1, 1620.6, 1500.8, 1459.5; 1H NMR (400 MHz, DMSO-d6) δ 12.98 (s, 1H),
9.81 (s, 1H), 8.14 (d, J = 2.3 Hz, 1H), 8.01 (dd, J = 8.8, 2.3 Hz, 1H), 7.61 (d, J = 8.8 Hz, 1H), 4.23
(t, J = 6.1 Hz, 2H), 1.67 – 1.57 (m, 3H), 0.86 (d, J = 6.1 Hz, 6H); 13C NMR (100 MHz, DMSO-d6)
δ 160.28, 156.69, 154.37, 145.22, 129.96, 124.16, 123.57, 116.40, 115.29, 68.62, 37.22, 25.04,
22.75 (2C); ESI-HRMS calcd. for C14H15N6O3 [M-H]- 315.1211, found 315.1227.
3-[4-(3-Methylbut-2-en-1-yl) oxy-3-(1H-tetrazol-1-yl) phenyl]-1,2,4-oxadiazol-5(4H)-one (3f)
A white crystalline powder; yield 64.5%; M.p 184.2°C-184.4°C; IR (KBr, cm-1): 3460.4, 3096.0,
1789.6, 1617.7, 1527.8, 1462.0; 1H NMR (400 MHz, DMSO-d6) δ 12.98 (s, 1H), 9.81 (s, 1H), 8.16
(d, J = 2.3 Hz, 1H), 8.00 (dd, J = 8.8, 2.3 Hz, 1H), 7.60 (d, J = 8.9 Hz, 1H), 5.47 – 5.39 (m, 1H),
4.79 (d, J = 6.7 Hz, 2H), 1.74 (s, 3H), 1.70 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 160.25,
156.64, 153.96, 145.11, 139.44, 129.69, 123.96, 123.70, 118.81, 116.44, 115.65, 66.89, 25.84, 18.55;
ESI-HRMS calcd. for C14H15N6O3 [M+H]+ 315.1201, found 315.1170.
3-[4-(Prop-2-yn-1-yl) oxy-3-(1H-tetrazol-1-yl) phenyl]-1,2,4-oxadiazol-5(4H)-one (3g)
A white, amorphous powder; yield 61.8%; M.p 200.6°C-201.1°C; IR (KBr, cm-1): 3459.6,
1
3267.5, 3139.7, 2122.5, 1793.6, 1622.0, 1495.4, 1455.2; H NMR (400 MHz, DMSO-d6) δ 13.02
(s, 1H), 9.85 (s, 1H), 8.18 (d, J = 2.2 Hz, 1H), 8.05 (dd, J = 8.8, 2.3 Hz, 1H), 7.65 (d, J = 8.8 Hz,
1H), 5.08 (d, J = 2.5 Hz, 2H), 3.72 (t, J = 2.4 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 160.30,
156.65, 152.85, 145.26, 129.75, 124.41, 123.91, 117.44, 115.93, 80.29, 78.22, 57.77; ESI-HRMS
25

calcd. for C12H7N6O3 [M-H]- 283.0585, found 283.0595.
3-[4-Cyclopropylmethoxy-3-(1H-tetrazol-1-yl) phenyl]-1,2,4-oxadiazol-5(4H)-one (3h)
A white crystalline powder; yield 67.7%; M.p 186.1°C-186.5°C; IR (KBr, cm-1): 3456.5, 3150.0,
3080.1, 2942.8, 1793.9, 1619.2, 1501.9, 1459.2; 1H NMR (400 MHz, DMSO-d6) δ 12.98 (s, 1H),
9.84 (s, 1H), 8.17 (d, J = 2.2 Hz, 1H), 7.99 (dd, J = 8.8, 2.2 Hz, 1H), 7.57 (d, J = 8.9 Hz, 1H), 4.10
(d, J = 7.1 Hz, 2H), 1.29 – 1.18 (m, 1H), 0.58 – 0.51 (m, 2H), 0.36 – 0.30 (m, 2H); 13C NMR (100
MHz, DMSO-d6) δ 160.28, 156.70, 154.12, 145.14, 129.77, 123.89, 123.66, 116.45, 115.58, 74.49,
10.02, 3.43 (2C); ESI-HRMS calcd. for C13H13N6O3 [M+H]+ 301.1044, found 301.1067.
3-[4-Cyclopentyloxy-3-(1H-tetrazol-1-yl) phenyl]-1,2,4-oxadiazol-5(4H)-one (3i)
A white crystalline powder; yield 80.6%; M.p 214.5°C-215°C; IR (KBr, cm-1): 3460.1, 3155.1,
3076.4, 2970.5, 1774.6, 1618.6, 1495.1, 1462.5; 1H NMR (400 MHz, DMSO-d6) δ 12.96 (s, 1H),
9.74 (s, 1H), 8.13 (d, J = 2.2 Hz, 1H), 7.96 (dd, J = 8.8, 2.3 Hz, 1H), 7.55 (d, J = 8.8 Hz, 1H), 5.09
(tt, J = 5.5, 2.5 Hz, 1H), 1.97 – 1.88 (m, 2H), 1.78 – 1.65 (m, 2H), 1.61 – 1.53 (m, 4H); 13C NMR
(100 MHz, DMSO-d6) δ 160.28, 156.66, 153.25, 145.07, 129.69, 124.14, 124.10, 116.20, 116.17,
81.98, 32.49 (2C), 23.81 (2C); ESI-HRMS calcd. for C14H15N6O3 [M+H]+ 315.1201, found
315.1228.
3-[4-Cyclohexylmethoxy-3-(1H-tetrazol-1-yl) phenyl]-1,2,4-oxadiazol-5(4H)-one (3j)
A white crystalline powder; yield 73.5%; M.p 191.1°C-192.5°C; IR (KBr, cm-1): 3458.7, 3086.8,
2929.2, 2849.5, 1785.6, 1619.8, 1496.0, 1457.5; 1H NMR (400 MHz, DMSO-d6) δ 12.95 (s, 1H),
9.79 (s, 1H), 8.14 (s, 1H), 8.00 (d, J = 8.7 Hz, 1H), 7.58 (d, J = 8.8 Hz, 1H), 4.02 (d, J = 6.2 Hz,
2H), 1.81 – 1.56 (m, 6H), 1.25 – 1.05 (m, 3H), 1.00 – 0.91 (m, 2H); 13C NMR (100 MHz, DMSO-
d6) δ 160.31, 156.68, 154.54, 145.22, 129.97, 124.22, 123.56, 116.39, 115.21, 74.86, 37.06, 29.31
(2C), 26.35, 25.57 (2C); ESI-HRMS calcd. for C16H17N6O3 [M-H]- 341.1367, found 341.1382.
3-[4-Benzyloxy-3-(1H-tetrazol-1-yl) phenyl]-1,2,4-oxadiazol-5(4H)-one (3k)
A white crystalline powder; yield 52.5%; M.p 211.7°C-212.4°C; IR (KBr, cm-1): 3167.6, 3092.8,
26

1789.1, 1616.0, 1529.1, 1483.1, 1459.4; 1H NMR (400 MHz, DMSO-d6) δ 12.87 (s, 1H), 9.86 (s,
1H), 8.17 (d, J = 2.2 Hz, 1H), 8.02 (dd, J = 8.8, 2.2 Hz, 1H), 7.67 (d, J = 8.8 Hz, 1H), 7.44 – 7.32
(m, 5H), 5.36 (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 160.76, 157.00, 153.90, 145.24, 135.95,
129.88, 129.05 (2C), 128.76, 128.07 (2C), 124.31, 123.78, 117.14, 115.74, 71.32; ESI-HRMS calcd.
for C16H11N6O3 [M-H]- 335.0898, found 335.0909.
3-[4-(4-Methylbenzyloxy)-3-(1H-tetrazol-1-yl) phenyl]-1,2,4-oxadiazol-5(4H)-one (3l)
A white, amorphous powder; yield 53.6%; M.p 217.8°C-218.4°C; IR (KBr, cm-1): 3458.5,
1
3152.3, 3083.2, 2926.0, 1774.8, 1618.3, 1498.4, 1460.4; H NMR (400 MHz, DMSO-d6) δ 12.93
(s, 1H), 9.82 (s, 1H), 8.17 (d, J = 2.3 Hz, 1H), 8.00 (dd, J = 8.8, 2.3 Hz, 1H), 7.65 (d, J = 8.8 Hz,
1H), 7.30 (d, J = 7.7 Hz, 2H), 7.18 (d, J = 7.7 Hz, 2H), 5.30 (s, 2H), 2.29 (s, 3H); 13C NMR (100
MHz, DMSO-d6) δ 160.52, 156.84, 153.96, 145.21, 138.17, 132.87, 129.86, 129.60 (2C), 128.28
(2C), 124.27, 123.78, 116.91, 115.79, 71.30, 21.22; ESI-HRMS calcd. for C17H13N6O3 [M-H]-
349.1054, found 349.1063.
3-[4-(4-Tert-butylbenzyloxy)-3-(1H-tetrazol-1-yl) phenyl]-1,2,4-oxadiazol-5(4H)-one (3m)
A white, amorphous powder; yield 79.8%; M.p 182.4°C-183.2°C; IR (KBr, cm-1): 3509.2,
3133.6, 2965.1, 1782.2, 1619.8, 1495.5, 1463.0; 1H NMR (400 MHz, DMSO-d6) δ 12.69 (s, 1H),
9.86 (s, 1H), 8.17 (d, J = 2.2 Hz, 1H), 8.03 (dd, J = 8.8, 2.2 Hz, 1H), 7.69 (d, J = 8.8 Hz, 1H), 7.41
(d, J = 8.2 Hz, 2H), 7.33 (d, J = 8.1 Hz, 2H), 5.32 (s, 2H), 1.27 (s, 9H); 13C NMR (100 MHz, DMSO-
d6) δ 160.53, 156.83, 154.00, 151.27, 145.24, 132.92, 129.89, 127.93 (2C), 125.79 (2C), 124.30,
123.75, 116.92, 115.71, 71.14, 34.78, 31.51 (3C); ESI-HRMS calcd. for C20H19N6O3 [M-H]-
391.1524, found 391.1537.
3-[4-(4-Methoxy-benzyloxy)-3-(1H-tetrazol-1-yl) phenyl]-1,2,4-oxadiazol-5(4H)-one (3n)
A white, amorphous powder; yield 75%; M.p 196.2°C-197.1°C; IR (KBr, cm-1): 3453.8,
3079.1, 1775.2, 1615.0, 1517.6, 1496.3, 1454.2; 1H NMR (400 MHz, DMSO-d6) δ 12.85 (s, 1H),
9.81 (s, 1H), 8.17 (d, J = 2.2 Hz, 1H), 8.02 (dd, J = 8.8, 2.3 Hz, 1H), 7.68 (d, J = 8.9 Hz, 1H), 7.41
– 7.31 (m, 2H), 7.00 – 6.90 (m, 2H), 5.28 (s, 2H), 3.76 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ
27

160.63, 159.80, 156.90, 153.88, 145.13, 130.08 (2C), 129.78, 127.70, 124.12, 123.77, 116.92,
115.78, 114.43 (2C), 71.23, 55.55; ESI-HRMS calcd. for C17H13N6O4 [M-H]- 365.1003, found
365.1007.
3-[4-(4-Fluorobenzyloxy)-3-(1H-tetrazol-1-yl) phenyl]-1,2,4-oxadiazol-5(4H)-one (3o)
A white, amorphous powder; yield 69%; M.p 214.2°C-214.9°C; IR (KBr, cm-1): 3450.2, 3094.7,
1784.3, 1617.7, 1514.8, 1461.0; 1H NMR (400 MHz, DMSO-d6) δ 13.01 (s, 1H), 9.85 (s, 1H), 8.18
(d, J = 2.2 Hz, 1H), 8.03 (dd, J = 8.8, 2.2 Hz, 1H), 7.69 (d, J = 8.9 Hz, 1H), 7.53 – 7.44 (m, 2H),
7.29 – 7.19 (m, 2H), 5.35 (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 159.88, 156.18, 153.21, 144.71,
134.45, 133.03, 129.54 (2C), 129.36, 128.60 (2C), 123.75, 123.34, 116.54, 115.23, 70.09; ESI-
HRMS calcd. for C16H10FN6O3 [M-H]- 353.0803, found 353.0821.
3-[4-(4-Chlorobenzyloxy)-3-(1H-tetrazol-1-yl) phenyl]-1,2,4-oxadiazol-5(4H)-one (3p)
A white, amorphous powder; yield 74.4%; M.p 211.8°C-212.5°C; IR (KBr, cm-1): 3459.0,
1
3157.1, 3081.7, 2938.8, 1766.9, 1618.2, 1496.0, 1440.2; H NMR (400 MHz, DMSO-d6) δ 12.81
(s, 1H), 9.84 (s, 1H), 8.17 (s, 1H), 8.00 (d, J = 8.8 Hz, 1H), 7.63 (d, J = 8.7 Hz, 1H), 7.43 (s, 4H),
5.35 (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 160.32, 156.62, 153.66, 145.15, 134.89, 133.47,
129.98 (2C), 129.80, 129.04 (2C), 124.19, 123.78, 116.99, 115.67, 70.53; ESI-HRMS calcd. for
C16H10ClN6O3 [M-H]- 369.0508, found 369.0512.
3-[4-(4-Bromobenzyloxy)-3-(1H-tetrazol-1-yl) phenyl]-1,2,4-oxadiazol-5(4H)-one (3q)
A white, amorphous powder; yield 85.8%; M.p 209.3°C-210.3°C; IR (KBr, cm-1): 3457.8,
3124.9, 3083.3, 2939.5, 1773.2, 1631.5, 1492.9, 1461.7, 1443.0; 1H NMR (400 MHz, DMSO-d6) δ
12.99 (s, 1H), 9.86 (s, 1H), 8.17 (d, J = 2.2 Hz, 1H), 8.02 (dd, J = 8.8, 2.3 Hz, 1H), 7.65 (d, J = 8.9
Hz, 1H), 7.63 – 7.55 (m, 2H), 7.42 – 7.34 (m, 2H), 5.34 (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ
160.26, 156.64, 153.80, 145.26, 135.39, 132.00(2C), 130.33(2C), 129.93, 124.39, 123.81, 122.01,
116.95, 115.78, 70.54; ESI-HRMS calcd. for C16H12BrN6O3 [M-H]+ 415.0149, found 415.0149.
3-[4-(3-Methybenzyloxy)-3-(1H-tetrazol-1-yl) phenyl]-1,2,4-oxadiazol-5(4H)-one (3r)
28

A white, amorphous powder; yield 74.3%; M.p 210.4°C-210.6°C; IR (KBr, cm-1): 3163.6,
3094.6, 1787.2, 1616.7, 1528.4, 1481.0, 1463.2; 1H NMR (400 MHz, DMSO-d6) δ 12.88 (s, 1H),
9.86 (s, 1H), 8.19 (d, J = 2.2 Hz, 1H), 8.06 – 7.93 (m, 1H), 7.66 (d, J = 8.8 Hz, 1H), 7.28 (t, J = 7.5
Hz, 1H), 7.24 – 7.12 (m, 3H), 5.32 (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 160.51, 156.79,
153.90, 145.20, 138.24, 135.83, 129.84, 129.37, 128.94, 128.61, 125.11, 124.20, 123.80, 116.96,
115.73, 71.40, 21.39; ESI-HRMS calcd. for C17H13N6O3 [M-H]- 349.1054, found 349.1073.
3-[4-(3-Methoxy-benzyloxy)-3-(1H-tetrazol-1-yl) phenyl]-1,2,4-oxadiazol-5(4H)-one (3s)
A white, amorphous powder; yield 75.4%; M.p 204.8°C-205.9°C; IR (KBr, cm-1): 3460.1,
3155.1, 3076.4, 2970.5, 1774.6, 1618.6, 1495.1, 1462.5, 1436.9; 1H NMR (400 MHz, DMSO-d6) δ
12.96 (s, 1H), 9.88 (s, 1H), 8.17 (d, J = 2.3 Hz, 1H), 8.02 (dd, J = 8.8, 2.3 Hz, 1H), 7.65 (d, J = 8.8
Hz, 1H), 7.29 (t, J = 8.0 Hz, 1H), 6.96 (d, J = 6.5 Hz, 2H), 6.89 (dd, J = 8.3, 2.4 Hz, 1H), 5.33 (s,
2H), 3.74 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 160.33, 159.85, 156.68, 153.95, 145.28, 137.49,
130.15, 129.92, 124.32, 123.81, 119.96, 116.89, 115.72, 114.30, 113.30, 71.13, 55.48; ESI-HRMS
calcd. for C17H13N6O4 [M-H]- 365.1003, found 365.1011.
3-[4-(3-Fluorobenzyloxy)-3-(1H-tetrazol-1-yl) phenyl]-1,2,4-oxadiazol-5(4H)-one (3t)
A white, amorphous powder; yield 77.4%; M.p 216.7°C-218°C; IR (KBr, cm-1): 3456.5, 3170.6,
3094.5, 1793.3, 1617.5, 1530.0, 1485.6, 1456.2; 1H NMR (400 MHz, DMSO-d6) δ 12.66 (s, 1H),
9.91 (s, 1H), 8.19 (s, 1H), 8.03 (d, J = 8.8 Hz, 1H), 7.65 (d, J = 8.8 Hz, 1H), 7.45 (q, J = 7.5 Hz,
1H), 7.26 (d, J = 8.3 Hz, 2H), 7.18 (t, J = 8.8 Hz, 1H), 5.39 (s, 2H); 13C NMR (100 MHz, DMSO-
d6) δ 160.05, 156.32, 153.20, 144.78, 138.22, 131.11, 130.73, 130.25, 129.43, 126.48, 123.85,
123.33, 121.79, 116.71, 115.20, 69.87; ESI-HRMS calcd. for C16H10FN6O3 [M-H]- 353.0803, found
353.0810.
3-[4-(3-Chlorobenzyloxy)-3-(1H-tetrazol-1-yl) phenyl]-1,2,4-oxadiazol-5(4H)-one (3u)
A white, amorphous powder; yield 80.5%; M.p 210.7°C-211.3°C; IR (KBr, cm-1): 3454.1,
3096.2, 1789.2, 1617.2, 1529.5, 1480.4, 1438.5; 1H NMR (400 MHz, DMSO-d6) δ 12.86 (s, 1H),
9.91 (s, 1H), 8.19 (d, J = 2.3 Hz, 1H), 8.04 (dd, J = 8.8, 2.3 Hz, 1H), 7.66 (d, J = 8.9 Hz, 1H), 7.50
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