Synthesis of novel 1,3,4-oxadiazole derivatives containing diamides as promising antibacterial and antiviral agents

Article abstract of DOI:10.1007/s11164-017-2980-x

In this paper, a variety of novel 1,3,4-oxadiazole derivatives possessing diamides were synthesized and tested for their antibacterial and antiviral activity. Preliminary antibacterial assays indicated that some intermediates and title compounds displayed

Full text of DOI:10.1007/s11164-017-2980-x

Res Chem Intermed
DOI 10.1007/s11164-017-2980-x
Synthesis of novel 1,3,4-oxadiazole derivatives
containing diamides as promising antibacterial
and antiviral agents
Pei-Yi Wang1 Wu-Bin Shao1 Hai-Tao Xue1,2 He-Shu Fang1
•
•
•
•
Jian Zhou1 Zhi-Bing Wu1 Bao-An Song1 Song Yang1
•
•
•
Received: 17 February 2017 / Accepted: 6 May 2017
Ó Springer Science+Business Media Dordrecht 2017
Abstract In this paper, a variety of novel 1,3,4-oxadiazole derivatives possessing
diamides were synthesized and tested for their antibacterial and antiviral activity.
Preliminary antibacterial assays indicated that some intermediates and title com-
pounds displayed excellent inhibition effects against plant pathogens Xanthomonas
oryzae pv. oryzae (Xoo) and Xanthomonas axonopodis pv. citri (Xac). Further
studies revealed that compound H15 exhibited the strongest activities against Xoo
and Xac with minimal EC50 values of 0.7 and 5.9 lg/mL, respectively. Antiviral
bioassays suggested that some of these structures displayed appreciable curative
activities and moderate protective effects against tobacco mosaic virus (TMV)
in vivo. Among them, compound H8 exerted the best chemotherapeutic effect
against TMV with the curative rate of 60.0% at 500 lg/mL, which was comparable
with those of commercial agricultural antiviral agent ningnanmycin (54.2%). Given
their significant biological activities, this kind of compound could serve as new
leading compounds in the study of antibacterial and antiviral chemotherapy.
Keywords 1,3,4-Oxadiazole Á Diamides Á Synthesis Á Antibacterial Á Antiviral
Electronic supplementary material The online version of this article (doi:10.1007/s11164-017-2980-x)
contains supplementary material, which is available to authorized users.
Pei-Yi Wang and Wu-Bin Shao authors have contributed equally to this work.
&
&
1
Bao-An Song
songbaoan22@yahoo.com
Song Yang
jhzx.msm@gmail.com
State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key
Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Center
for R&D of Fine Chemicals of Guizhou University, Guiyang 550025, China
2
School of Pharmaceutical Sciences, Guizhou University, Guiyang 550025, China
123

P.-Y. Wang et al.
Introduction
Agricultural disease-causing bacteria as ‘‘plant killers’’ can cause necrotic lesions
on leaves, stems, and/or fruits, which consequently result in a significant loss in
agricultural output [1–3]. Xanthomonas oryzae pv. oryzae (Xoo) is a gram-negative
bacteria causing bacterial leaf blight of rice which is one of the serious worldwide
diseases of rice in most of the rice growing countries [4–6]. As another important
gram-negative plant-pathogenic bacteria, Xanthomonas axonopodis pv. citri (Xac),
can cause an extremely persistent disease-citrus bacterial canker through entering
the natural openings (stomata) or wounds of plants in all producing countries [7–9].
Therefore, management of crops against spoilage by invading pathogenetic bacteria
is a necessity. Using chemicals to address these bacterial diseases has become one
of the effective measures [10, 11]. To date, great progress has been made in the
exploration and development of antibacterial agents, and has led to a few of them
being commercialized, such as bismerthiazol (BT) and thiodiazole copper (TC).
However, these agents cannot meet the desired requirements due to their poor
efficiency and/or bacterial resistance [12, 13]. Thus, a search for new types of
inhibitors with excellent bioactivities is urgently needed in pesticide science.
Tobacco mosaic virus (TMV), as a classical pathogenic virus, can infect nine plant
families, and at least 125 individual species including tobacco, tomato, pepper, potato,
and cucumber [14]. Infected plants by TMV can give characteristic patterns with
‘‘mosaic’’-like mottling and discoloration on the leaves, which consequently affect the
quality and output of agricultural products every year [15, 16]. In the past decades, a
few of the TMV-inhibitors have been commercialized, such as ningnanmycin (NNM)
and ribavirin. They gave curative rates of 56.0 and 38.5% at a high dosage of 500 lg/
mL, respectively. Moreover, their photosensitivity and/or water stickiness have
become a major limitation for their field application [17, 18]. Obviously, exploring
new high-efficient agents is required in antiviral drug discovery.
Natural substructures possess advantages over other general building blocks in
the construction of bioactive molecules for several reasons, such as low mammalian
toxicity, impressive biodegradability, the specific ability to the target species,
desirable biological activities, as well as good biocompatibility [19, 20]. Oxadi-
azole, a natural skeleton, is usually utilized as the bioisosteric replacement of the
amide bonds [21–24]. This scaffold was commonly observed with increased
hydrolytic and metabolic stability, and improved pharmacokinetic performance
in vivo, which endows it an important structural motif for the pharmaceutical
industry [25, 26]. From the literature review, compounds bearing oxadiazole have
displayed an impressive array of biological activities including antitumor,
herbicidal, insecticidal, and antifungal activities [24, 27–30]. Particularly, their
antibacterial and antiviral activities have attracted the interests of chemists [31–34].
For example, Khalilullah and co-workers had evaluated the antimicrobial activity of
a series of 1,3,4-oxadizole derivatives containing 1,4-benzodioxane ring, and found
that some of the tested compounds showed better antibacterial and antifungal
activities than those of chloramphenicol and fluconazole [35]. Barral and co-
workers reported a series of 5-phenyl-2-[2-(2-thienyl)ethenyl]-1,3,4-oxadiazole
123

Synthesis of novel 1,3,4-oxadiazole derivatives containing…
Fig. 1 Design strategy of the target compound
derivatives exhibiting submicromolar activity on the four dengue virus serotypes
[36]. Neda and co-workers had synthesized a series of novel 1,2,4-oxadiazoles, and
found that this kind of structure exhibited antitumor activities towards 12 cancer cell
lines [37]. Meanwhile, it is noted that the synthesis of oxadiazole motif is
economical and facile, which has made it become the promising pharmacophore for
the construction of bioactive molecules [38]. As another crucial building block,
diamide bonds, existing in many natural products, have been proved to possess
diverse chemotherapeutic potentials of their derivatives [39–46]. Moreover, many
compounds containing diamide bonds have been commercialized and used as
pesticides, such as chlorantraniliprole, flubendiamide (insecticides), and fenpyraza-
mine (fungicide). In view of the aforementioned facts, the design of compounds
possessing both oxadiazole pharmacophore and diamide bonds might result in
bioactive molecules with better efficacy owing to the synergistic effect of these
valuable moieties. Herein, a series of 1,3,4-oxadiazole derivatives bearing diamides
were designed and synthesized, in which the diamides were placed at the 5-postion
of 1,3,4-oxadiazole. All the title compounds were examined for their antibacterial
and antiviral activities. To the best of our knowledge, no studies have so far reported
the usage of this kind of compound against pathogenic Xoo, Xac, and TMV (Fig. 1).
Methods and materials
Chemistry
All the chemicals were purchased from Aladdin, and used as received. The organic
solvents were distilled before used. Melting points of the compounds were
determined on a XT-4 binocular microscope (Beijing Tech Instrument Co., China).
NMR spectra were obtained by using a JEOL-ECX-500 apparatus. Chemical shifts
were reported in parts per million (ppm) down field from TMS with the solvent
resonance as the internal standard. Coupling constants (J) were reported in Hz and
referred to apparent peak multiplications.
General synthetic procedures for the intermediates and target compounds
N-(2-hydrazinyl-2-oxoethyl)-2-nitrobenzamide (C) 2-Nitrobenzoic acid (3.59 g,
21.5 mmol) and SOCl2 (15 mL) were refluxed for 5 h. The solvent was removed
123

P.-Y. Wang et al.
under reduced pressure, and re-dissolved by dry tetrahydrofuran (THF, 8 mL). It
was added dropwise into a solution of glycine ethyl ester hydrochloride (3.00 g,
21.5 mmol) and triethylamine (TEA, 7.45 mL, 53.7 mmol) in dry THF (30 mL) on
ice bath, and then the mixture was stirred at room temperature for 8 h. After that, the
solvent was removed under reduced pressure, and re-dissolved by ethyl acetate. The
organic layer was washed by water, brine, dried with sodium sulfate, filtered, and
followed by the removal of the solvent under vacuum, and re-dissolved in 20 mL
ethanol containing 3 mL 80% hydrazine hydrate. The mixture was refluxed for
12 h. The solvent was removed under reduced pressure, and the precipitate was
washed with water to give a light yellow solid (C), yield 56.8%, m. p. 206–207 °C.
1H NMR (500 MHz, DMSO-d6) d 9.04 (s, 1H, NHNH2), 8.91 (t, J = 5.8 Hz, 1H,
NHCH2), 7.99 (dd, J = 7.9, 1.4 Hz, 1H, phenyl-H), 7.76 (td, J = 7.5, 1.2 Hz, 1H,
phenyl-H), 7.68–7.64 (m, 2H, phenyl-H), 4.23 (s, 2H, NH2), 3.79 (d, J = 5.8 Hz,
2H, NHCH2); 13C NMR (126 MHz, DMSO-d6) d 167.7, 165.7, 147.1, 133.4, 131.9,
130.9, 129.3, 124.0, 41.2; MS (ESI): m/z = 239 [M ? H?].
N-(2-hydrazinyl-2-oxoethyl)-3-nitrobenzamide (C0) The synthesis of C0 was
carried out by the same method of C, and the difference is that 3-nitrobenzoic
acid was used to replace 2-nitrobenzoic acid, a light yellow solid, yield 90.7%, m.
1
p. 160–161 °C. H NMR (500 MHz, DMSO-D6) d 9.23 (s, 1H, NHNH2), 9.16
(t, J = 5.8 Hz, 1H, NHCH2), 8.71 (s, 1H, phenyl-H), 8.37 (dd, J = 8.2, 2.2 Hz, 1H,
phenyl-H), 8.29 (d, J = 7.7 Hz, 1H, phenyl-H), 7.77 (t, J = 8.0 Hz, 1H, phenyl-H),
4.26 (s, 2H, NH2), 3.88 (d, J = 5.8 Hz, 2H, NHCH2); 13C NMR (126 MHz, DMSO-
d6) d 168.1, 164.6, 147.7, 135.6, 133.9, 130.1, 126.0, 122.3; MS (ESI): m/z = 239
[M ? H?].
The synthesis of D, D0, E, and E0 were carried out by our previous methods [47].
N-((5-mercapto-1,3,4-oxadiazol-2-yl)methyl)-2-nitrobenzamide (D)
A
yellow
1
solid, yield 54.2%, m. p. [230 °C; H NMR (500 MHz, DMSO-d6) d 14.52 (s,
1H, SH), 9.44 (t, J = 5.6 Hz, 1H, NH), 8.07 (dd, J = 8.1, 0.8 Hz, 1H, phenyl-H),
7.83 (td, J = 7.5, 1.0 Hz, 1H, phenyl-H), 7.73 (td, J = 8.0, 1.3 Hz, 1H, phenyl-H),
7.63 (dd, J = 7.6, 1.2 Hz, 1H, phenyl-H), 4.54 (d, J = 5.7 Hz, 2H, N–CH2); 13C
NMR (126 MHz, DMSO-d6) 178.0, 165.9, 160.8, 146.9, 133.8, 131.4, 131.3, 129.1,
124.3, 34.4; MS (ESI): m/z = 281 [M ? H?].
N-((5-mercapto-1,3,4-oxadiazol-2-yl)methyl)-3-nitrobenzamide (D0)
A
yellow
1
solid, yield 75.2%, m. p. 144–145 °C; H NMR (500 MHz, DMSO-d6) d 14.50
(s, 1H, SH), 9.60 (t, J = 5.4 Hz, 1H, NHCH2), 8.77–8.64 (m, 1H, phenyl-H), 8.42
(ddd, J = 8.2, 2.3, 1.0 Hz, 1H, phenyl-H), 8.35–8.26 (m, 1H, phenyl-H), 7.81
(t, J = 8.0 Hz, 1H, phenyl-H), 4.60 (d, J = 5.5 Hz, 2H, NHCH2); 13C NMR
(126 MHz, DMSO-d6) d 177.9, 164.5, 161.1, 147.9, 134.5, 133.8, 130.4, 126.5,
122.1, 34.8; MS (ESI): m/z = 281 [M ? H?].
N-((5-(methylthio)-1,3,4-oxadiazol-2-yl)methyl)-2-nitrobenzamide (E) A yellow
1
solid, yield 31.4%, m. p. 108–109 °C; H NMR (500 MHz, CDCl3) d 7.92 (dd,
J = 8.1, 0.8 Hz, 1H, phenyl-H), 7.76 (t, J = 5.7 Hz, 1H, NH), 7.59 (td, J = 7.5,
0.9 Hz, 1H, phenyl-H), 7.52–7.47 (m, 2H, phenyl-H), 4.68 (d, J = 5.9 Hz, 2H),
123

Synthesis of novel 1,3,4-oxadiazole derivatives containing…
2.58 (s, 3H, CH3); 13C NMR (126 MHz, CDCl3) d 167.0, 166.2, 164.2, 146.3, 133.9,
131.8, 130.9, 129.0, 124.5, 34.8, 14.6; MS (ESI): m/z = 295 [M ? H?].
N-((5-(methylthio)-1,3,4-oxadiazol-2-yl)methyl)-3-nitrobenzamide (E0) A yellow
1
solid, yield 58.1%, m. p. 129–130 °C; H NMR (500 MHz, CDCl3) d 8.66 (t, J =
1.9 Hz, 1H, phenyl-H), 8.34 (ddd, J = 8.2, 2.2, 1.0 Hz, 1H, phenyl-H), 8.20–8.16
(m, 1H, phenyl-H), 7.91 (t, J = 5.5 Hz, 1H, NH), 7.61 (t, J = 8.0 Hz, 1H, phenyl-
H), 4.88 (d, J = 5.8 Hz, 2H, N–CH2), 2.69 (s, 3H, CH3); 13C NMR (126 MHz,
CDCl3) d 166.7, 165.2, 164.7, 148.3, 134.8, 133.5, 130.0, 126.6, 122.4, 35.2, 14.7;
MS (ESI): m/z = 295 [M ? H?].
2-Amino-N-((5-(methylthio)-1,3,4-oxadiazol-2-yl)methyl)benzamide (F) Interme-
diate E (0.50 g, 1.7 mmol) was added into an ethanol solution (20 mL) containing
zinc powder (1.11 g, 17.0 mmol) and NH4Cl (0.91 g, 17.0 mmol), then the mixture
was refluxed for 10 h. After that, the mixture was filtrated, and solvent was removed
under reduced pressure, the pure compound (F) could be obtained by column
chromatography using (CH2Cl2:EtOAc = 4:1) as the eluent, a yellow solid, yield
47.3%, m. p. 120–121 °C. 1H NMR (500 MHz, CDCl3) d 7.39 (dd, J = 7.9, 1.3 Hz,
1H, phenyl-H), 7.26–7.22 (m, 1H, phenyl-H), 6.70–6.65 (m, 3H, phenyl-H&NH),
5.55 (s, 2H, NH2), 4.81 (d, J = 5.7 Hz, 2H, N–CH2), 2.72 (s, 3H, CH3); 13C NMR
(126 MHz, CDCl3) d 169.2, 166.3, 164.7, 149.2, 133.2, 127.5, 117.6, 116.9, 114.6,
34.7, 14.8; MS (ESI): m/z = 265 [M ? H?].
3-Amino-N-((5-(methylthio)-1,3,4-oxadiazol-2-yl)methyl)benzamide (F0) The syn-
thesis of F0 was carried out as synthesis of F, a white solid, yield 39.0%, m.
p. 128–129 °C; 1H NMR (500 MHz, CDCl3) d 7.19 (t, J = 7.8 Hz, 1H, phenyl-H),
7.15 (t, J = 2.0 Hz, 1H, phenyl-H), 7.13–7.09 (m, 1H, phenyl-H), 7.00 (t, J =
5.1 Hz, 1H, NH), 6.80 (ddd, J = 7.9, 2.4, 0.9 Hz, 1H, phenyl-H), 4.82 (d,
J = 5.7 Hz, 2H, N–CH2), 2.69 (s, 3H, CH3); 13C NMR (126 MHz, CDCl3) d 167.9,
166.3, 164.8, 147.0, 134.5, 129.7, 118.6, 116.8, 114.0, 35.0, 14.7; MS (ESI):
m/z = 265 [M ? H?].
2-Acetamido-N-((5-(methylthio)-1,3,4-oxadiazol-2-yl)methyl)benzamide (G1) Acetylchlo-
ride (0.09 g, 1.14 mmol) was added into a solution of F (0.20 g, 0.76 mmol) and
TEA (0.2 mL) in dry THF (8 mL), then the mixture was stirred at room temperature
for 12 h. After that, the solvent was removed under reduced pressure, and
re-dissolved by ethyl acetate. The organic layer was washed by water, brine, dried
with sodium sulfate, filtered, and followed by the removal of the solvent under
vacuum. The pure compound (G1) could be obtained by column chromatography
using (CH2Cl2:CH3OH = 20:1) as the eluent, a white solid, yield 11.4%, m.
1
p. 143–144 °C. H NMR (500 MHz, CDCl3) d 10.84 (s, 1H, NHCOCH3), 8.52
(d, J = 7.8 Hz, 1H, phenyl-H), 7.55 (d, J = 7.4 Hz, 1H, phenyl-H), 7.46 (t, J =
7.5 Hz, 1H, phenyl-H), 7.35 (s, 1H, NH–CH2), 7.07–7.04 (m, 1H, phenyl-H), 4.79
(d, J = 5.5 Hz, 2H, NCH2), 2.71 (s, 3H, SCH3), 2.18 (s, 3H, COCH3); 13C NMR
(126 MHz, CDCl3) d 169.3, 169.2, 166.5, 164.3, 139.7, 133.3, 127.1, 123.0, 121.7,
119.3, 34.9, 25.5, 14.7; MS (ESI): m/z = 307 [M ? H?].
The synthesis of G2, G20 and G3 were carried out by synthetic protocols of G1.
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P.-Y. Wang et al.
N-((5-(methylthio)-1,3,4-oxadiazol-2-yl)methyl)-2-propionamidobenzamide
(G2) A white solid, yield 60.9%, m. p. 121–122 °C; 1H NMR (500 MHz, CDCl3)
d 10.87 (s, 1H, NHCOCH2), 8.59 (d, J = 8.3 Hz, 1H, phenyl-H), 7.55 (d,
J = 7.9 Hz, 1H, phenyl-H), 7.48 (t, J = 7.9 Hz, 1H, phenyl-H), 7.19 (t, J =
5.0 Hz, 1H, NHCH2), 7.06 (t, J = 7.7 Hz, 1H, phenyl-H), 4.81 (d, J = 5.7 Hz, 2H,
N–CH2), 2.71 (s, 3H, SCH3), 2.43 (q, J = 7.6 Hz, 2H, COCH2), 1.24 (t, J =
7.6 Hz, 3H, COCH2CH3); 13C NMR (126 MHz, CDCl3) d 173.0, 169.2, 166.5,
164.2, 139.9, 133.3, 127.0, 122.8, 121.7, 119.2, 34.9, 31.6, 14.7, 9.7; MS (ESI):
m/z = 321 [M ? H?].
N-((5-(methylthio)-1,3,4-oxadiazol-2-yl)methyl)-3-propionamidobenzamide (G20)
A
1
light yellow solid, yield 72.3%, m. p. 128–129 °C; H NMR (500 MHz, CDCl3) d
8.67 (s, 1H, NHCOCH2), 8.16 (t, J = 5.7 Hz, 1H, NHCH2), 7.79 (s, 1H, phenyl-H),
7.72 (d, J = 8.1 Hz, 1H, phenyl-H), 7.44 (d, J = 7.7 Hz, 1H, phenyl-H), 7.19 (t,
J = 7.9 Hz, 1H, phenyl-H), 4.73 (d, J = 5.8 Hz, 2H, N–CH2), 2.62 (s, 3H, SCH3),
2.33 (q, J = 7.5 Hz, 2H, COCH2), 1.13 (t, J = 7.5 Hz, 3H, COCH2CH3); 13C NMR
(126 MHz, CDCl3) d 173.3, 167.9, 166.2, 165.1, 138.7, 133.7, 129.2, 123.6, 122.8,
118.6, 35.0, 30.5, 14.6, 9.7; MS (ESI): m/z = 321 [M ? H?].
2-benzamido-N-((5-(methylthio)-1,3,4-oxadiazol-2-yl)methyl)benzamide (G3)
A
1
white solid, yield 27.4%, m. p. 216–217 °C; H NMR (500 MHz, DMSO-d6) d
12.19 (s, 1H, NHCO), 9.57 (t, J = 5.3 Hz, 1H, NHCH2), 8.63 (d, J = 8.5 Hz, 1H,
phenyl-H), 7.92–7.56 (m, 7H, phenyl-H), 7.24 (t, J = 7.8 Hz, 1H, phenyl-H), 4.73
(d, J = 5.4 Hz, 2H, CH2), 2.66 (s, 1H, SCH3); 13C NMR (126 MHz, DMSO-d6) d
169.0, 164.8, 164.7, 164.5, 139.4, 134.5, 132.8, 132.1, 129.0, 128.3, 126.9, 123.1,
120.6, 119.8, 34.4, 14.3; MS (ESI): m/z = 369 [M ? H?].
1-(5-((2-(((5-(methylthio)-1,3,4-oxadiazol-2-yl)methyl)carbamoyl)phenyl)amino)-5-
oxopentyl)pyridin-1-ium bromide (H4) 5-bromopentanoyl chloride (0.45 mmol)
was added into a mixture of intermediate F (0.12 g, 0.45 mmol) and TEA
(0.91 mmol) in 10 mL CH2Cl2. Then it was stirred at room temperature for 5 h.
After that, the solvent was removed under reduced pressure, and re-dissolved by
ethyl acetate. The organic layer was washed by water, brine, dried with sodium
sulfate, filtered, and followed by the removal of the solvent under vacuum. The
crude product was incubated in 5 mL pyridine at 50 °C for 12 h. After that, the
extra pyridine was removed under reduced pressure. The crude residue was further
purified by flash column chromatography on a silica gel using CH2Cl2 and CH3OH
(6:1) as the eluent to afford the desired product (H4), a yellow liquid, yield 41.2%.
1H NMR (500 MHz, CD3OD) d 9.05 (d, J = 5.6 Hz, 2H, pyridine-H), 8.59
(t, J = 7.9 Hz, 1H, pyridine-H), 8.18 (d, J = 8.2 Hz, 1H, phenyl-H), 8.12 (t, J =
7.0 Hz, 2H, pyridine-H), 7.68 (d, J = 9.3 Hz, 1H, phenyl-H), 7.48 (t, J = 7.9 Hz,
1H, phenyl-H), 7.19 (t, J = 8.1 Hz, 1H, phenyl-H), 4.72 (s, 2H, NH–CH2), 4.69
(t, J = 7.5 Hz, 2H, N–CH2), 2.70 (s, 3H, SCH3), 2.48 (t, J = 7.1 Hz, 2H, COCH2),
2.13–2.05 (m, 2H, NCH2CH2), 1.79–1.70 (m, 2H, CH2); 13C NMR (126 MHz,
CD3OD) d 173.2, 171.2, 167.7, 166.5, 146.9, 146.0, 139.2, 133.3, 129.6, 129.1,
125.0, 123.8, 123.2, 62.7, 37.4, 35.6, 31.7, 22.9, 14.7; MS (ESI): m/z = 426
[M - Br].
123

Synthesis of novel 1,3,4-oxadiazole derivatives containing…
The synthesis of Hn (n = 5, 6, 7, 8, 9, 10, 15) and Hn0 (n = 10, 15) were carried
out by synthetic protocols of H4.
1-(6-((2-(((5-(methylthio)-1,3,4-oxadiazol-2-yl)methyl)carbamoyl)phenyl)amino)-6-
1
oxohexyl)pyridin-1-ium bromide (H5) A yellow liquid, yield 42.0%; H NMR
(500 MHz, CD3OD) d 9.04 (d, J = 6.2 Hz, 2H, pyridine-H), 8.58 (t, J = 7.8 Hz,
1H, pyridine-H), 8.26 (d, J = 8.3 Hz, 1H, phenyl-H), 8.10 (t, J = 7.0 Hz, 2H,
pyridine-H), 7.73 (d, J = 9.3 Hz, 1H, phenyl-H), 7.51 (t, J = 8.6 Hz, 1H, phenyl-
H), 7.21 (t, J = 7.9 Hz, 1H, phenyl-H), 4.76 (s, 2H, NHCH2), 4.68 (t, J = 7.5 Hz,
2H, N–CH2), 2.71 (s, 3H, SCH3), 2.43 (t, J = 7.2 Hz, 2H, COCH2), 2.12–2.04 (m,
2H, NCH2CH2), 1.81–1.73 (m, 2H, CH2), 1.49–1.42 (m, 2H, CH2); 13C NMR
(126 MHz, CD3OD) d 173.8, 171.2, 167.6, 166.6, 146.8, 146.0, 139.5, 133.4, 129.5,
129.1, 124.8, 123.3, 122.9, 62.8, 38.1, 35.6, 31.9, 26.3, 25.7, 14.7; MS (ESI):
m/z = 440 [M - Br].
1-(7-((2-(((5-(methylthio)-1,3,4-oxadiazol-2-yl)methyl)carbamoyl)phenyl)amino)-7-
1
oxoheptyl)pyridin-1-ium bromide (H6) A yellow liquid, yield 62.2%; H NMR
(500 MHz, CD3OD) d 9.04 (d, J = 5.6 Hz, 2H, pyridine-H), 8.58 (t, J = 8.4 Hz,
1H, pyridine-H), 8.29 (d, J = 8.3 Hz, 1H, phenyl-H), 8.11 (t, J = 7.0 Hz, 2H,
pyridine-H), 7.73 (d, J = 9.2 Hz, 1H, phenyl-H), 7.50 (t, J = 8.5 Hz, 1H, phenyl-
H), 7.20 (t, J = 7.6 Hz, 1H, phenyl-H), 4.76 (s, 2H, NHCH2), 4.67 (t, J = 7.5 Hz,
2H, N–CH2), 2.71 (s, 3H, SCH3), 2.40 (t, J = 7.3 Hz, 2H, COCH2), 2.08–2.01 (m,
2H, NCH2CH2), 1.75–1.67 (m, 2H, CH2), 1.49–1.40 (m, 4H, 2CH2); 13C NMR
(126 MHz, CD3OD) d 174.1, 171.1, 167.6, 166.6, 146.8, 145.9, 139.6, 133.4, 129.5,
129.1, 124.7, 123.1, 122.9, 62.9, 38.4, 35.6, 32.1, 29.3, 26.7, 26.1, 14.7; MS (ESI):
m/z = 454 [M - Br].
1-(8-((2-(((5-(methylthio)-1,3,4-oxadiazol-2-yl)methyl)carbamoyl)phenyl)amino)-8-
1
oxooctyl)pyridin-1-ium bromide (H7) A yellow liquid, yield 19.1%; H NMR
(500 MHz, CD3OD) d 9.02 (d, J = 6.3 Hz, 2H, pyridine-H), 8.60 (t, J = 8.4 Hz,
1H, pyridine-H), 8.32 (d, J = 8.5 Hz, 1H, phenyl-H), 8.12 (t, J = 6.9 Hz, 2H,
pyridine-H), 7.72 (d, J = 7.8 Hz, 1H, phenyl-H), 7.51 (t, J = 9.2 Hz, 1H, phenyl-
H), 7.20 (t, J = 7.6 Hz, 1H, phenyl-H), 4.75 (s, 2H, NHCH2), 4.65 (t, J = 7.6 Hz,
2H, N–CH2), 2.72 (d, J = 1.2 Hz, 3H, SCH3), 2.40 (t, J = 7.2 Hz, 2H, COCH2),
2.13–1.97 (m, 2H, NCH2CH2), 1.78–1.60 (m, 2H, CH2), 1.45–1.40 (m, 6H, 3CH2);
13C NMR (126 MHz, CDCl3) d 172.7, 169.5, 165.7, 165.0, 145.0, 144.9, 144.8.
139.8, 132.8, 128.5, 123.1, 121.5, 119.7, 62.3, 38.4, 35.1, 31.8, 29.8, 28.1, 25.6,
25.1, 14.8; MS (ESI): m/z = 468 [M - Br].
1-(9-((2-(((5-(methylthio)-1,3,4-oxadiazol-2-yl)methyl)carbamoyl)phenyl)amino)-9-
1
oxononyl)pyridin-1-ium bromide (H8) A yellow liquid, yield 28.6%; H NMR
(500 MHz, CD3OD) d 9.03 (d, J = 5.6 Hz, 2H, pyridine-H), 8.59 (t, J = 7.9 Hz,
1H, pyridine-H), 8.31 (d, J = 8.3 Hz, 1H, phenyl-H), 8.12 (t, J = 6.9 Hz, 2H,
pyridine-H), 7.73 (d, J = 7.9 Hz, 1H, phenyl-H), 7.50 (t, J = 7.8 Hz, 1H, phenyl-
H), 7.19 (t, J = 8.0 Hz, 1H, phenyl-H), 4.76 (s, 2H, NHCH2), 4.65 (t, J = 7.6 Hz,
2H, N–CH2), 2.71 (s, 3H, SCH3), 2.38 (t, J = 7.4 Hz, 2H, COCH2), 2.04 (m, 2H,
NCH2CH2), 1.76–1.60 (m, 2H, CH2), 1.38 (m, 8H, 4CH2); 13C NMR (126 MHz,
123

P.-Y. Wang et al.
CD3OD) d 174.3, 171.1, 167.6, 166.5, 146.8, 145.9, 139.7, 133.4, 129.5, 129.1,
124.7, 122.9, 122.8, 63.1, 38.7, 35.6, 32.4, 29.9, 29.8, 29.7, 26.9, 26.5, 14.7; MS
(ESI): m/z = 482 [M - Br].
1-(10-((2-(((5-(methylthio)-1,3,4-oxadiazol-2-yl)methyl)carbamoyl)phenyl)amino)-
10-oxodecyl)pyridin-1-ium bromide (H9) A yellow liquid, yield 36.4%; 1H NMR
(500 MHz, CDCl3) d 11.17 (s, 1H, NHCO), 9.17 (d, J = 5.9 Hz, 2H, pyridine-H),
8.78 (t, J = 5.6 Hz, 1H, NHCH2), 8.50 (d, J = 8.3 Hz, 1H, phenyl-H), 8.39 (t,
J = 7.4 Hz, 1H, pyridine-H), 8.03 (t, J = 7.0 Hz, 2H, pyridine-H), 7.97 (d,
J = 7.4 Hz, 1H, phenyl-H), 7.41 (t, J = 7.9 Hz, 1H, phenyl-H), 7.06 (t,
J = 7.7 Hz, 1H, phenyl-H), 4.83 (d, J = 5.7 Hz, 2H, NHCH2), 4.71 (t,
J = 7.6 Hz, 2H, NCH2), 2.69 (s, 3H, SCH3), 2.39 (t, J = 6.9 Hz, 2H, COCH2),
1.89 (dt, J = 14.6, 7.2 Hz, 2H, NCH2CH2), 1.70 (dt, J = 14.0, 7.0 Hz, 2H,
COCH2CH2), 1.27–1.23 (m, 10H, 5CH2); 13C NMR (126 MHz, CDCl3) d 172.7,
169.5, 165.7, 164.9, 145.0, 144.9, 139.8, 132.8, 128.5, 123.1, 121.5, 119.7, 62.3,
38.4, 35.1, 31.8, 29.8, 28.3, 28.1, 28.0, 25.6, 25.1, 14.8; MS (ESI): m/z = 496
[M - Br].
1-(11-((2-(((5-(methylthio)-1,3,4-oxadiazol-2-yl)methyl)carbamoyl)phenyl)amino)-
11-oxoundecyl)pyridin-1-ium bromide (H10) A light yellow solid, yield 38.7%,
m. p. 105–106 °C; 1H NMR (500 MHz, CD3OD) d 9.04 (d, J = 5.3 Hz, 2H,
pyridine-H), 8.60 (t, J = 7.8 Hz, 1H, pyridine-H), 8.31 (d, J = 8.3 Hz, 2H,
pyridine-H), 8.12 (t, J = 7.1 Hz, 1H, phenyl-H), 7.73 (d, J = 7.9 Hz, 1H, phenyl-
H), 7.50 (t, J = 8.6 Hz, 1H, phenyl-H), 7.19 (t, J = 7.6 Hz, 1H, phenyl-H), 4.76 (s,
2H, NHCH2), 4.65 (t, J = 7.5 Hz, 2H, N–CH2), 2.71 (s, 3H, SCH3), 2.38 (t,
J = 7.5 Hz, 2H, COCH2), 2.10–1.91 (m, 2H, NCH2CH2), 1.75–1.59 (m, 2H,
COCH2CH2), 1.37–1.31 (m, 12H, 6CH2); 13C NMR (126 MHz, CD3OD) d 174.4,
171.1, 167.5, 166.5, 146.8, 145.9, 139.7, 133.4, 129.5, 129.1, 124.7, 122.9, 122.8,
63.1, 38.8, 35.6, 32.4, 30.3, 30.2, 30.1, 29.9, 27.1, 26.6, 14.7; MS (ESI): m/z = 510
[M - Br].
1-(11-((3-(((5-(methylthio)-1,3,4-oxadiazol-2-yl)methyl)carbamoyl)phenyl)amino)-
11-oxoundecyl)pyridin-1-ium bromide (H100) A white solid, yield 57.6%, m.
p. 163–164 °C; 1H NMR (500 MHz, CD3OD) d 9.04 (d, J = 5.8 Hz, 2H, pyridine-
H), 8.59 (t, J = 7.8 Hz, 1H, pyridine-H), 8.12 (t, J = 7.0 Hz, 3H, pyridine-H &
phenyl-H), 7.73 (d, J = 8.0 Hz, 1H, phenyl-H), 7.57 (d, J = 7.8 Hz, 1H, phenyl-
H), 7.41 (t, J = 7.9 Hz, 1H, phenyl-H), 4.77 (s, 2H, NHCH2), 4.65 (t, J = 7.5 Hz,
2H, NCH2), 2.70 (s, 3H, SCH3), 2.40 (t, J = 7.4 Hz, 2H, COCH2), 2.07–1.91 (m,
2H, NCH2CH2), 1.75–1.60 (m, 2H, COCH2CH2), 1.36–1.31 (m, 12H, 6CH2); 13C
NMR (126 MHz, CD3OD) d 174.8, 169.9, 167.5, 166.6, 146.8, 145.9, 140.4, 135.4,
130.1, 129.5, 124.6, 123.7, 120.3, 63.1, 37.9, 35.7, 32.4, 30.2, 30.1, 29.9, 27.1, 26.7,
14.7; MS (ESI): m/z = 510 [M - Br].
1-(16-((2-(((5-(methylthio)-1,3,4-oxadiazol-2-yl)methyl)carbamoyl)phenyl)amino)-
16-oxohexadecyl)pyridin-1-ium bromide (H15) A colourless liquid, yield 44.0%;
1H NMR (500 MHz, CD3OD) d 9.01 (d, J = 5.7 Hz, 2H, pyridine-H), 8.60
(t, J = 7.8 Hz, 1H, pyridine-H), 8.32 (d, J = 8.3 Hz, 1H, phenyl-H), 8.12
123

Synthesis of novel 1,3,4-oxadiazole derivatives containing…
(t, J = 7.0 Hz, 2H, pyridine-H), 7.71 (d, J = 7.9 Hz, 1H, phenyl-H), 7.50 (t,
J = 8.0 Hz, 1H, phenyl-H), 7.19 (t, J = 7.5 Hz, 1H, phenyl-H), 4.76 (s, 2H,
NHCH2), 4.64 (t, J = 7.5 Hz, 2H, NCH2), 2.72 (s, 3H, SCH3), 2.39 (t, J = 7.6 Hz,
2H, COCH2), 2.06–1.98 (m, 2H, NCH2CH2), 1.73–1.65 (m, 2H, COCH2CH2),
1.43–1.24 (m, 22H, 11CH2); 13C NMR (126 MHz, CD3OD) d 166.6, 146.9, 145.9,
133.4, 129.5, 129.0, 124.7, 123.0, 122.9, 63.1, 35.5, 32.5, 30.7, 30.6, 30.5, 30.4,
30.3, 30.2, 30.1, 27.2, 26.7 (s), 14.7; MS (ESI): m/z = 580 [M - Br].
1-(16-((3-(((5-(methylthio)-1,3,4-oxadiazol-2-yl)methyl)carbamoyl)phenyl)amino)-
16-oxohexadecyl)pyridin-1-ium bromide (H150) A white solid, yield 45.8%, m.
p. 123–124 °C; 1H NMR (500 MHz, CD3OD) d 9.03 (d, J = 6.0 Hz, 2H, pyridine-
H), 8.60 (t, J = 7.8 Hz, 1H, pyridine-H), 8.14–8.10 (m, 3H, pyridine-H & phenyl-
H), 7.73 (d, J = 9.0 Hz, 1H, phenyl-H), 7.57 (d, J = 7.7 Hz, 1H, phenyl-H), 7.42
(t, J = 7.9 Hz, 1H, phenyl-H), 4.77 (s, 2H, NHCH2), 4.65 (t, J = 7.4 Hz, 2H, N–
CH2), 2.71 (s, 3H, SCH3), 2.39 (t, J = 7.4 Hz, 2H, COCH2), 2.10–1.93 (m, 2H,
NCH2CH2), 1.77–1.58 (m, 2H, COCH2CH2), 1.37–1.27 (m, 22H, 11CH2); 13C
NMR (126 MHz, CD3OD) d 174.9, 170.0, 167.6, 166.6, 146.8, 145.9, 140.4, 135.4,
130.1, 129.5, 124.6, 123.7, 120.3, 63.1, 38.0, 35.7, 32.5, 30.6, 30.5, 30.4, 30.3, 30.2,
30.1, 27.1, 26.8, 14.7; MS (ESI): m/z = 580 [M - Br].
In vitro antibacterial bioassay
In our study, all the synthesized target compounds were evaluated for their
antibacterial activities against Xoo and Xac by the turbidimeter test in vitro.
Dimethylsulfoxide in sterile distilled water served as a blank control. Bismerthiazol
and Thiodiazole Copper served as positive controls. Approximately 40 lL of
solvent NB (1.5 g beef extract, 2.5 g peptone, 0.5 g yeast powder, 5.0 g glucose,
and 500 mL distilled water; pH 7.0–7.2) containing Xoo (or Xac), incubated in the
phase of logarithmic growth, was added to 5 mL of solvent NB containing different
concentrations of the test compounds and positive control, such as 100, 50 lg/mL
(for preliminary bioassay), 80, 40, 20, 10, 5 lg/mL, 20, 10, 5, 2.5, 1.25 lg/mL, or
1.25, 0.625, 0.3125, 0.156, 0.078 lg/mL (for EC50 detection, depend on the
bioactivity of different compounds, the concentration was chosen as two times the
decline trend). The inoculated test tubes were incubated at 28 1 °C and
continuously shaken at 180 rpm for 24–48 h until the bacteria were incubated in the
logarithmic growth phase. The growth of the cultures was monitored on a
microplate reader by measuring the optical density at 595 nm (OD595) given by
turbidity corrected values = ODbacterial wilt - ODno bacterial wilt, and the inhibition
rate I was calculated by I = (C - T)/C 9 100%. C is the corrected turbidity values
of bacterial growth on untreated NB (blank control), and T is the corrected turbidity
values of bacterial growth on treated NB. By using the SPSS 17.0 software and the
obtained inhibition rates at different concentrations, a regression equation was
provided. The results of antibacterial activities (expressed by EC50) against Xoo and
Xac were calculated from the equation. The experiment was repeated three times.
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P.-Y. Wang et al.
In vivo antiviral bioassay
Curative activity of the target compounds against TMV in vivo
Growing Nicotiana tabacum L. leaves of the same age were selected. The leaves
were inoculated with TMV (concentration of 6 9 10-3 mg/mL) by dipping and
brushing the whole leaves, which had previously been scattered with silicon carbide.
The leaves were then washed with water after inoculation for 0.5 h. The compound
solution was smeared on the left side of the leaves, and the solvent was smeared on
the right side as the control. The number of local lesions was counted and recorded
3–4 days after inoculation. Three replicates were set up for each.
Protection activity of the target compounds against TMV in vivo
The compound solutions were smeared on the left side of the N. tabacum L. leaves,
and the solvents were smeared on the right side as the control sample for growing N.
tabacum L. leaves. After 12 h, crude TMV (concentration of 6 9 10-3 mg/mL) was
inoculated on whole leaves at the same concentration on each side of the leaves,
which were previously scattered with silicon carbide. After 0.5 h, the leaves were
washed with water and then dried. The number of local lesions was recorded
3–4 days after inoculation. Three replicates were used for each compound. The
inhibitory rate of the compound was calculated according to the following formula:
Inhibition rate¼½ðaverage local lesion number of controlðnot treated with compoundÞ
Àaverage local lesion number smeared with drugsÞ=
average local lesion number of control
ðnot treated with compoundފÂ100%:
Results and discussion
The structure and synthesis of title compounds (G1–G3 and Hn) are illustrated in
Scheme 1. Ethyl 2-(2-nitrobenzamido)acetate (B) was synthesized from 2-nitroben-
zoic acid (A) by chlorination, followed by treatment with glycine ethyl ester
hydrochloride. The reaction of B and hydrazine hydrate in ethanol gave the desired
intermediate N-(2-hydrazinyl-2-oxoethyl)-2-nitrobenzamide (C) that was then
treated with carbon disulfide in the presence of potassium hydroxide to form N-
((5-mercapto-1,3,4-oxadiazol-2-yl)methyl)-2-nitrobenzamide (D). Subsequently, a
substitution reaction with dimethyl sulfate to give N-((5-(methylthio)-1,3,4-oxadi-
azol-2-yl)methyl)-2-nitrobenzamide (E) that was dealt with by zinc and ammonium
chloride to give an important immediate (F) bearing an amino group. The target
molecules (G1–G3) were obtained via treating F with substituted acyl chloride.
Intermediate (F) was reacted by two-step consecutive reactions with bromide-
tailored acyl chloride and pyridine to provide title compounds (Hn, n = 4, 5, 6, 7, 8,
123

Synthesis of novel 1,3,4-oxadiazole derivatives containing…
Scheme 1 Synthetic route of the target compounds (G1–G3 and Hn)
1
9, 10, 15). All the structures were confirmed by H NMR, 13C NMR, and MS
(detailed NMR spectra see supplementary data).
Turbidimeter tests [48, 49] was carried out to evaluate their antibacterial
activities toward pathogenic bacteria Xoo and Xac in vitro, and the commercial
antibacterial agents (BT and TC) were used as positive controls under the same
conditions. As shown in Table 1, some intermediates and most of the target
compounds displayed certain growth suppression effects against Xoo and Xac at 100
or 50 lg/mL. Among them, compounds D, F, H10, and H15 exerted the strongest
antibacterial effect toward Xoo with inhibition rates of 99.3, 86.5, 90.5, and 99.7%
at 100 lg/mL, respectively, which were better than those of BT (56.1%) or TC
(43.1%). Moreover, the intermediate D and compound H15 remained the best
antibacterial functions against Xoo with the rate above 98% as the concentrate
decreased two fold. Further studies indicated that compounds D and H15 showed
admirable inhibition effects against Xoo with EC50 values of 10.1 and 0.7 lg/mL
(Table 2), respectively, which were quite superior to those of BT (EC50 = 92.6 lg/
mL) or TC (EC50 = 121.8 lg/mL). Compounds G2, H4, H6, and H15 gave the
inhibition rates of 98.0, 67.6, 70.0, and 99.8% against Xac at 100 lg/mL,
respectively, which were better than that of TC (56.0%) at 100 lg/mL. It was noted
that only compound H15 still maintained the potent inhibition effect against Xac
with the rate of 99.8% at 50 lg/mL. EC50 value for H15 against Xac was
determined as 5.9 lg/mL. Based on the above bioassays, this kind of compound
could be further studied as alternative antibacterial agents.
Preliminary structure-antibacterial activity relationship analysis toward Xoo and
Xac was elucidated according to the bioassay results. It is worth mentioning that the
123

P.-Y. Wang et al.
Table 1 Inhibition rates (%) of
compounds (D, D0, E, E0, F, F0,
G1–G3, G20, Hn, H100, and
H150) against Xoo and Xac
in vitro
Compounds Xoo
100 lg/mL 50 lg/mL
99.3 0.7 98.5 1.1 3.9 5.5
Xac
100 lg/mL 50 lg/mL
D
0
E
34.7 3.9 26.2 4.1 13.8 2.9 12.6 5.6
86.5 4.4 33.7 7.2 34.1 3.4 14.0 4.7
F
G1
G2
G3
H4
H5
H6
H7
H8
H9
H10
H15
D0
25.8 1.8 20.2 9.0 14.5 7.2
0
37.8 2.9 26.0 2.9 98.0 2.5 20.1 1.7
68.3 5.3 19.2 6.7 13.8 2.9 12.6 5.6
39.6 4.6 34.8 4.3 67.6 2.3 32.5 4.8
19.2 6.7 18.5 6.5 32.0 3.3 20.5 0.7
30.7 7.2 21.4 4.8 70.0 1.5 29.3 1.9
26.9 3.1 25.1 4.3 33.8 1.7 22.6 6.9
38.4 3.0 34.2 1.8 53.3 4.5 5.7 3.2
39.3 6.4 32.8 2.0
0
0
90.5 4.6 45.5 4.9 29.9 5.1 4.3 2.1
99.7 1.7 98.3 2.7 99.8 0.3 99.8 0.3
99.2 0.2 99.0 0.2 28.5 5.0 21.1 0.8
88.8 2.9 55.5 5.4 28.8 6.9 18.4 5.3
63.0 4.0 33.1 1.9 17.6 1.5 10.9 3.8
E0
F0
G20
H100
H150
BT
TC
61.2 1.9 39.3 4.8 5.0 1.7
4.1 0.5
74.0 0.8 42.1 3.8 71.5 1.0 44.5 3.2
86.8 3.9 56.8 5.2 11.6 1.4 9.4 0.7
53.7 1.2 35.3 3.4
–
–
43.1 3.2 28.5 1.7 56.0 1.2 37.6 2.3
Table 2 EC50 values of target compounds against pathogenic bacteria Xoo and Xac
Compd. Xoo
Regression equation
y = 1.578x ? 4.175 0.96
Xac
r
EC50 (lg/mL) Regression equation
r
EC50 (lg/mL)
D
10.1 4.6
0.7 0.5
–
–
–
H15
D0
H150
BT
TC
y = 1.282x ? 5.197 0.97
y = 0.861x ? 4.063 0.99
y = 1.166x ? 3.032 0.99
y = 1.499x ? 2.052 0.98
y = 1.617x ? 3.752 0.95 5.9 0.1
12.2 1.1
48.7 1.7
92.6 2.1
–
–
–
–
–
–
–
–
–
y = 1.540x ? 1.788 0.98 121.8 3.6
y = 2.153x ? 0.938 0.98 77.0 2.0
substituents had a great impact on bioactivity. The bioactivity against Xoo was
dramatically decreased by introducing an aliphatic group (–CH3, E, 34.7%) on the
mercapto group (–SH, D, 99.3%), indicating that the sulfhydryl group might own
the specific ability to bind the target species of bacteria. An increased bioactivity
was observed when the nitro-group (–NO2, E, 34.7%) was reduced into an amino
group (–NH2, F, 86.5%), suggesting a hydrogen-bond donor is favourable for the
123

Synthesis of novel 1,3,4-oxadiazole derivatives containing…
bioactivity. The anti-Xoo activity was improved about two fold when the aromatic
ring (phenyl group, G3, 68.3%) replaced the aliphatic substituents (–CH3, G1,
25.8%; –CH2CH3, G2, 37.8%) in the molecules, probably attributed to the
molecular lipid solubility. It was noted that the inhibition effect was enhanced with
increasing the alkyl length (n = 15, H15, 99.7%; n = 10, H10, 90.5%; n = 9, H9,
39.3%; n = 8, H8, 38.4%; n = 7, H7, 26.9%), might be ascribed to the enhanced
molecular hydrophobicity. In contrast, a suitable aliphatic substituent (–CH2CH3,
G2, 98.0%) showed better anti-Xac activity than those of other aliphatic (–CH3, G1,
14.5%) or aromatic (phenyl group, G3, 13.8%) substituents. The anti-Xac effect
reached the maximum when n was 15, indicating that a suitable hydrophobicity/
hydrophilicity balance was gained for H15 against Xac (Fig. 2).
In order to optimize the molecular structure and investigate the position of the
amide bond toward bioactivity, intermediates (D0, E0, F0) and target compounds
(G20, H100, H150) were selectively synthesized by using 3-nitrobenzoic acid as the
starting material in instead of 2-nitrobenzoic acid. All the compounds were tested
for their antibacterial activities under the same conditions. As indicated in Tables 1
and 2, intermediate D0 almost maintained the same anti-Xoo effect with EC50 value
of 12.2 lg/mL (D, 10.1 lg/mL), while intermediate E0 gave improved anti-Xoo
activity with an inhibition rate of 88.8% at 100 lg/mL (E, 34.7%). A decreased
bioactivity against Xoo was observed for compounds H10 (from 90.5 to 74.0%,
100 lg/mL) and H15 (EC50 value from 0.7 to 28.9 lg/mL). Meanwhile, it was
disadvantageous to anti-Xac activity when the substituent at the meta-position of the
benzene ring, as indicated by the comparison of compounds H150 (11.6%, 100 lg/
mL) and H15 (99.8%, 100 lg/mL). The above fact revealed that the position of the
amide bond on the benzene ring did have a significant impact on the bioactivity.
The anti-TMV activity of the intermediates and all title compounds were tested
in vivo as previously described [50, 51], and the commercial agent NNM was chosen
as the positive control. As shown in Table 3, most of the tested compounds displayed
certain curative and protective activities against TMV at 500 lg/mL. Among them,
compounds H6, H8, and H15 displayed the strongest curative activity with the
Fig. 2 Structures of compounds D0, E0, F0, G20, H100, H150 with crystal structure of F0
123

P.-Y. Wang et al.
Table 3 Anti-TMV activities of compounds (D, D0, E, E0, F, F0, G1–G3, G20, Hn, H100, and H150) at
500 lg/mL in vivo
Compounds
Rate (%)
Compounds
Rate (%)
Curative
activity
Protective
activity
Curative
activity
Protective
activity
D
38.5 1.2
36.9 5.1
26.8 5.2
22.3 6.4
47.2 2.8
44.8 9.5
7.1 1.7
35.2 3.1
14.4 2.9
54.5 2.9
54.6 5.2
38.8 4.5
36.8 0.8
51.2 7.6
27.8 5.5
42.9 2.5
23.5 1.1
65.7 2.2
H8
H9
H10
H15
D0
E0
F0
G20
H100
H150
60.0 5.6
26.9 2.9
48.7 5.1
51.9 3.0
41.8 1.0
17.5 1.2
17.7 1.2
49.3 2.0
33.9 1.3
35.3 2.3
–
36.4 1.0
43.3 3.0
25.2 2.9
45.6 4.2
41.7 1.7
32.2 1.6
42.6 2.2
19.6 2.4
20.2 1.0
19.3 0.8
E
F
G1
G2
G3
H4
H5
H6
H7
NNM
37.4 3.5
50.6 4.7
37.1 3.3
54.2 2.9
therapeutic rates of 50.6, 60.0, and 51.9%, respectively, which were comparable with
that of NNM (54.2%). Meanwhile, compounds F, G1 and H4 displayed moderate
protective effects with values of 54.5, 54.6, and 51.2%, respectively, which were
lower than that of NNM (65.7%). The structure–activity relationship analysis was
elucidated based on the preliminary bioassays. For compounds H4, H6, H8, and
H10, the curative activity reached the maximum with n = 6, suggesting that the
appropriate hydrophobicity/hydrophility ratio of a molecule was advantageous to the
bioactivity. The position of substituents also affected the anti-TMV activity, as
indicated by the comparison of curative activity of compounds H10 (48.7%) and
H100 (33.9%), H15 (51.9%) and H150 (35.3%), revealing that the amide bond at the
ortho-position of the benzene ring might be beneficial for anti-TMV activity.
Conclusion
In summary, two series of novel 1,3,4-oxadiazole derivatives bearing diamides were
fabricated and evaluated their antibacterial and antiviral activity. Antibacterial
assays indicated that intermediates D and D0 showed excellent inhibition effects
against Xoo with EC50 values of 10.1 and 12.2 lg/mL, respectively, which were
quite superior to those of commercial antibacterial agents BT (EC50 = 92.6 lg/mL)
or TC (EC50 = 121.8 lg/mL). Title compound H15 exhibited the best activities
against Xoo and Xac with minimal EC50 values of 0.7 and 5.9 lg/mL, respectively.
Meanwhile, antiviral bioassays suggested compound H8 exerted the best curative
activity against TMV with a rate of 60.0% at 500 lg/mL, which was comparable
with that of commercial agent NNM (54.2%). Given their potent bioactivities, these
kinds of compounds could be further studied as promising antibacterial and antiviral
candidates.
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Synthesis of novel 1,3,4-oxadiazole derivatives containing…
Acknowledgements We acknowledge the financial support of the Key Technologies R&D Program
(2014BAD23B01), National Natural Science Foundation of China (21372052, 21662009), the Research
Project of Ministry of Education of China (20135201110005), and Scientific and Technological Projects
of Guizhou Province [2016]1029.
References
1. C.M. Hopkins, F.F. White, S.H. Choi, A. Guo, J.E. Leach, Mol. Plant Microbe Interact. 5, 451 (1992)
2. B. Li, B.P. Liu, C.L. Shan, M. Ibrahim, Y.H. Lou, Y.L. Wang, G.L. Xie, H.Y. Li, G.S. Sun, Pest
Manag. Sci. 69, 312 (2013)
3. J.H. Graham, T.R. Gottwald, J. Cubero, A.D.S. Achor, Mol. Plant Pathol. 5, 1 (2004)
4. X.F. Zhu, Y. Xu, D. Peng, Y. Zhang, T.T. Huang, J.X. Wang, M.G. Zhou, Crop Prot. 47, 24 (2013)
5. P. Li, L. Shi, L. Yang, X.W. Chen, F. Wu, Q.C. Shi, W.M. Xu, M. He, D.Y. Hu, B.A. Song, Bioorg.
Med. Chem. Lett. 24, 1677 (2014)
6. Y. Chen, X. Yang, C.Y. Gu, A.F. Zhang, Y. Zhang, W.X. Wang, T.C. Gao, J. Yao, S.K. Yuan, Ann.
Appl. Biol. 166, 129 (2015)
7. L.S. Murate, A.G. de Oliveira, A.Y. Higashi, A.R. Barazetti, A.S. Simionato, C.S. da Silva, G.C.
Simo˜es, I.M.O. dos Santos, M.R. Ferreira, M.V.T. Cely, M.O.P. Navarro, V.F. de Freitas, M.A.
Nogueira, J.C.P. de Mello, R.P.L.G. Andrade Jr., Agric. Sci. 6, 295 (2015)
8. A. Piazza, T. Zimaro, B.S. Garavaglia, F.A. Ficarra, L. Thomas, C. Marondedze, R. Feil, J.E. Lunn,
C. Gehring, J. Ottado, N. Gottig, J. Exp. Bot. 66, 2795 (2015)
9. A.G. de Oliveira, L.S. Murate, F.R. Spago, L. de Paula Lopes, J.P. Oliveira Beranger, J.A.B.S. de
Martin, M.A. Nogueira, J.C.P. de Mello, C.G.T. de Jesus Andrade, G. Andrade, Biol. Control 56, 125
(2011)
10. A. Hazra, S. Mondal, A. Maity, S. Naskar, P. Saha, R. Paira, K.B. Sahu, P. Paira, S. Ghosh, C. Sinha,
A. Samanta, S. Banerjee, N.B. Mondal, Eur. J. Med. Chem. 46, 2132 (2011)
11. H. Khalilullah, M.J. Ahsan, M. Hedaitullah, S. Khan, B. Ahmed, Mini Rev. Med. Chem. 12, 789
(2012)
12. P. Li, L. Shi, M. Gao, X. Yang, W. Xue, L.H. Jin, D.Y. Hu, B.A. Song, Bioorg. Med. Chem. Lett. 25,
481 (2015)
13. S. Perumalsamy, M. Bharani, M. Sudha, P. Nagarajan, L. Arul, R. Saraswathi, P. Balasubramanian, J.
Ramalingam, Plant Breed. 129, 400 (2010)
14. M. Wu, G.F. Han, Z.W. Wang, Y.X. Liu, Q.M. Wang, J. Agric. Food Chem. 61, 1030 (2013)
15. W.G. Zhao, J.G. Wang, Z.M. Li, Z. Yang, Bioorg. Med. Chem. Lett. 16, 6107 (2006)
16. B. Su, C.L. Cai, M. Deng, Q.M. Wang, J. Agric. Food Chem. 64, 2039 (2016)
17. Z.W. Wang, P. Wei, L.Z. Wang, Q.M. Wang, J. Agric. Food Chem. 60, 10212 (2012)
18. G.Y. Huang, C. Cui, Z.P. Wang, Y.Q. Li, L.X. Xiong, L.Z. Wang, S.J. Yu, Z.M. Li, W.G. Zhao,
Chem. Cent. J. 7, 33 (2013)
19. Z. Li, P. Zhan, X. Liu, Mini Rev. Med. Chem. 11, 1130 (2011)
20. X.H. Gan, D.Y. Hu, P. Li, J. Wu, X.W. Chen, W. Xue, B.A. Song, Pest Manag. Sci. 72, 534 (2016)
21. Z. Jakopin, M.S. Dolenc, Curr. Org. Chem. 12, 850 (2008)
22. C.V. Maftei, E. Fodor, P.G. Jones, M. Freytag, M.H. Franz, G. Kelter, H.-H. Fiebig, M. Tamm, I.
Neda, Eur. J. Med. Chem. 101, 431 (2015)
23. E. Spink, D.R. Ding, Z.H. Peng, M.A. Boudreau, E. Leemans, E. Lastochkin, W. Song, K. Licht-
enwalter, P.I. O’Daniel, S.A. Testero, H.L. Pi, V.A. Schroeder, W.R. Wolter, N.T. Antunes, M.A.
Suckow, S. Vakulenko, M. Chang, S. Mobashery, J. Med. Chem. 58, 1380 (2015)
24. E. Leemans, K.V. Mahasenan, M. Kumarasiri, E. Spink, D.R. Ding, P.I. O’Daniel, M.A. Boudreau,
E. Lastochkin, S.A. Testero, T. Yamaguchi, M. Lee, D. Hesek, J.F. Fisher, M. Chang, S. Mobashery,
Bioorg. Med. Chem. Lett. 26, 1011 (2016)
25. A. Vaidya, S. Jain, P. Jain, P. Jain, N. Tiwari, R. Jain, R. Jain, A.K. Jain, R.K. Agrawal, Mini Rev.
Med. Chem. 16, 825 (2016)
26. S.C. Karad, V.B. Purohit, J.R. Avalani, N.H. Sapariya, D.K. Raval, RSC Adv. 6, 41532 (2016)
27. Q.Z. Zheng, X.M. Zhang, Y. Xu, K. Cheng, Q.C. Jiao, H.L. Zhu, Bioorg. Med. Chem. 18, 7836
(2010)
28. R.A. Rane, S.D. Gutte, N.U. Sahu, Bioorg. Med. Chem. Lett. 22, 6429 (2012)
123

P.-Y. Wang et al.
29. A.H. Shridhar, J. Keshavayya, S.K. Peethambar, H.J. Hoskeri, Arab. J. Chem. 9, S1643 (2016)
30. S.T. Dhumal, A.R. Deshmukh, M.R. Bhosle, V.M. Khedkar, L.U. Nawale, D. Sarkar, R.A. Mane,
Bioorg. Med. Chem. Lett. 26, 3646 (2016)
31. B.A. Johns, J.G. Weatherhead, S.H. Allen, J.B. Thompson, E.P. Garvey, S.A. Foster, J.L. Jeffrey,
W.H. Miller, Bioorg. Med. Chem. Lett. 19, 1807 (2009)
32. P. Neeraja, S. Srinivas, K. Mukkanti, P.K. Dubey, S. Pal, Bioorg. Med. Chem. Lett. 26, 5212 (2016)
33. V. Ravichandran, S. Shalini, K. Sundram, A.D. Sokkalingam, Eur. J. Med. Chem. 45, 2791 (2010)
34. T. Pal, R.R. Somani, P.Y. Shirodkar, U.V. Bhanushali, Med. Chem. Res. 20, 1550 (2011)
35. H. Khalilullah, S. Khan, M.S. Nomani, B. Ahmed, Arab. J. Chem. 9, S1029 (2016)
36. F. Benmansour, C. Eydoux, G. Querat, X. de Lamballerie, B. Canard, K. Alvarez, J.C. Guillemot, K.
Barral, Eur. J. Med. Chem. 109, 146 (2016)
37. C.V. Maftei, E. Fodor, P.G. Jones, C.G. Daniliuc, M.H. Franz, G. Kelter, H.-H. Fiebig, M. Tamm, I.
Neda, Tetrahedron 72, 1185 (2016)
38. M.S. Behalo, RSC Adv. 6, 103132 (2016)
39. Z.B. Yang, D.Y. Hu, S. Zeng, B.A. Song, Bioorg. Med. Chem. Lett. 26, 1161 (2016)
´
¨
40. J.A. Good, J. Silver, C. Nun˜ez-Otero, W. Bahnan, K.S. Krishnan, O. Salin, P. Engstrom, R. Svensson,
˚
¨
P. Artursson, A. Gylfe, S. Bergstrom, F. Almqvist, J. Med. Chem. 59, 2094 (2016)
41. P. Teng, D. Huo, A. Nimmagadda, J. Wu, F. She, M. Su, X. Lin, J. Yan, A. Cao, C. Xi, Y. Hu, J. Cai,
J. Med. Chem. 59, 7877 (2016)
42. I. Neda, H.-J. Plinta, A. Fischer, P.G. Jones, R. Schmutzler, J. Fluor. Chem. 71, 65 (1995)
43. H.-J. Plinta, I. Neda, R. Schmutzler, Z. Naturforsch. B 49, 100 (1994)
44. R. Sonnenburg, I. Neda, A. Fischer, P.G. Jones, R. Schmutzler, Z. Naturforsch. B 49, 788 (1994)
45. I. Neda, H.-J. Plinta, R. Schmutzler, Z. Naturforsch. B 48, 333 (1993)
46. I. Neda, T. Kaukorat, R. Schmutzler, Phosphorus Sulfur 84, 205 (1993)
47. P.Y. Wang, L. Zhou, J. Zhou, Z.B. Wu, W. Xue, B.A. Song, S. Yang, Bioorg. Med. Chem. Lett. 26,
1214 (2016)
48. P.Y. Wang, M.N. Gao, L. Zhou, Z.B. Wu, D.Y. Hu, J. Hu, S. Yang, Bioorg. Med. Chem. Lett. 26,
1236 (2016)
49. X. Wang, J. Yin, L. Shi, G. Zhang, B.A. Song, Eur. J. Med. Chem. 77, 65 (2014)
50. W.M. Xu, S.Z. Li, M. He, S. Yang, X.Y. Li, P. Li, Bioorg. Med. Chem. Lett. 23, 5821 (2013)
51. Y. Han, Y. Ding, D.D. Xie, D.Y. Hu, P. Li, X.Y. Li, W. Xue, L.H. Jin, B.A. Song, Eur. J. Med.
Chem. 92, 732 (2015)
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