Design, synthesis, biological activities and DFT calculation of novel 1,2,4-triazole Schiff base derivatives

Article abstract of DOI:10.1016/j.bioorg.2018.06.030

Series of 1,2,4-triazole Schiff bases (2a-2d, 2f-2h and 3a-3h) have been designed and synthesized. The structure of title compounds was confirmed on the basis of their spectral data and elemental analysis. All the target compounds were screened for their in vitro antifungal activity and antibacterial activity. Two of the tested compounds (2a and 2b) exhibited significant antifungal activity against most fungi, especially compound 2a showed better antifungal activity than triadimefon. Meanwhile, the antibacterial activity assay also indicated compound 2a exhibited excellent antibacterial activities comparable to chloramphenicol. The SAR manifested no substitution at position 5 of the triazole ring caused an increase in activity, and 3-phenoxy phenyl group introduced in 1,2,4-triazole scaffold can enhance the antibacterial activity. The DFT calculation indicated triazole ring, S atom and benzene ring in both of the 2a and 3a make a major contribution to the activity.

Full text of DOI:10.1016/j.bioorg.2018.06.030

Accepted Manuscript
Design, synthesis, biological activities and DFT calculation of novel 1,2,4-tri-
azole Schiff base derivatives
Ru-Yi Jin, Chu-Yue Zeng, Xu-Hua Liang, Xiao-Hong Sun, Yuan-Fa Liu, Yan-
Yan Wang, Sha Zhou
PII:
S0045-2068(18)30272-4
DOI:
https://doi.org/10.1016/j.bioorg.2018.06.030
YBIOO 2410
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
19 March 2018
21 June 2018
23 June 2018
Please cite this article as: R-Y. Jin, C-Y. Zeng, X-H. Liang, X-H. Sun, Y-F. Liu, Y-Y. Wang, S. Zhou, Design,
synthesis, biological activities and DFT calculation of novel 1,2,4-triazole Schiff base derivatives, Bioorganic
Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.06.030
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Design, synthesis, biological activities and
DFT calculation of novel 1,2,4-triazole
Schiff base derivatives
＊
d
a
c
b
Ru-Yi Jin , Chu-Yue Zeng , Xu-Hua Liang , Xiao-Hong Sun ,
＊
Yuan-Fa Liue, Yan-Yan Wangf, Sha Zhoug
aCollege of Pharmacy, Shaanxi University of Chinese Medicine, Xi’an/Xianyang, China
bDepartment of Chemistry, Hong Kong University of Science and Technology, Hong Kong, China
cCollege of Biology Pharmacy and Food Engineering, Shangluo University, Shangluo, China
dSchool of Chemical Engineering, Northwest University, Xi’an, China
eSchool of Chemistry and Materials, Northwest University, Xi’an, China
fDepartment of Medcine, Ankang Vocational and Technical College, Ankang, China
gState Key Laboratory of Elemento-Organic Chemistry, Institute of Elemento-Organic Chemistry, Collaborative Innovation Center of
Chemical Science and Engineering (Tianjin), College of Chemistry, Nankai University, Tianjin, China
Abstract: Series of 1,2,4-triazole Schiff bases (2a-h and 3a-h) have been designed and
synthesized. The structure of title compounds was confirmed on the basis of their spectral data and
elemental analysis. All the target compounds were screened for their in vitro antifungal activity
and antibacterial activity. Two of the tested compounds (2a and 2b) exhibited significant
antifungal activity against most fungi, especially compound 2a showed better antifungal activity
than triadimefon. Meanwhile, the antibacterial activity assay also indicated compound 2a
exhibited excellent antibacterial activities comparable to chloramphenicol. The SAR manifested
no substitution at position 5 of the triazole ring caused an increase in activity, and 3-phenoxy
phenyl group introduced in 1,2,4-triazole scaffold can enhance the antibacterial activity. The DFT
calculation indicated triazole ring, S atom and benzene ring in both of the 2a and 3a make a major
contribution to the activity.
Keywords: Synthesis; 1,2,4-Triazole; Biological activity; DFT calculation
1. Introduction
1,2,4-Triazole derivatives due to their broad-spectrum activities have potential applications in
the fields of pesticides and medicines possessing antifungal[1], antibacterial[2], antitumor[3],
antitrypanosomal[4], antiproliferative[5], and antibiotics[6] properties. Several clinical 1,2,4-triazole
drugs were used widely in clinical therapy, for instance, voriconazole and fluconazole (Fig.1).
This study was supported by the National Natural Science Foundation of China (No. 21673179).
Corresponding authors. E-mail: xhsun888@sohu.com; E-mail: zhousha@nankai.edu.cn.
1

In addition, there are known triazole antifungal agents presently play a leading role in the
treatment of agricultural fungal infections, such as, tebuconazole, epoxiconazol, cyproconazole,
and triadimefon (Fig. 1). However, their use is problematic as both can cause side effects and have
limited efficacy, and some strains are refractory to treatment. In addition, antifungal agents usually
can’t treat bacteria at the same time. Therefore, the need for high activity, new, safer, and dual
antifungal and antibacteria agent is urgent.
Fig. 1 Triazole antifungal agents used in clinical therapy and pesticides
1,2,4-Triazole Schiff bases represented an important N-containing heterocyclic compounds
with potential bioactivity[7-10]. In our previous research, serious of 4-amino-5-substituent-1,2,4
-triazole Schiff bases were designed and synthesized. The bioassay showed parts of the
compounds exhibit excellent antifungal activity and could be the potential antifungal agent
candidates[11] (Fig.2), and the results indicated that no substituent at position Q can increase the
antifungal activity, compound (a) reached same antifungal level compared with triadimefon. The
results prompted us to systemically explore the effect on biological of different substituents at
position Q and position P, which might bring us some unknown biological properties. Researcher
found that compounds with 3-phenoxyphenyl structural feature had good antibacteria activity[12]
,
then we designed introducing 3-phenoxyphenyl into position P, which could be screening a
potential dual antifungal and antibacteria agent, meanwhile, 4-mehoxy-2,3,6-trimethyl phenyl also
has been introduced into position P, and their influence of these factors on bioactivity were been
studied.
2

Cl
S
S
HN
N
HN
N
N
N
N
N
P
F
H
Q
(b)
(a)
Fig. 2 Chemical structures of 1,2,4-triazole Schiff bases
Based on the above viewpoints, series of 4-amino-5-substituent-1,2,4-triazole-3-thione Schiff
bases were synthesized via 4-amino-5-substituent-1,2,4-triazole-3-thione (1a-h) with 3-phenoxy-
benzaldehyde and 4-methoxy 2,3,6-trimethyl benzaldehyde. Their structures were confirmed by
1
melting point, IR, H NMR,13C NMR, HRMS and elemental analysis. The crystal structure of
compound 2f was obtained by X-ray diffraction and the crystallographic data has been deposited
at the Cambridge Crystallographic Data Center, CCDC-1016443. Their antifungal activities to
pythium solani, gibberlla nicotiancola, fusarium oxysporium f.sp. niveum, and gibberlla saubinetii
were tested. Their antibacterial activities against sacillus subtilis, escherichia coli, and
staphylococcus aureus bacterial strains were presented as well. The HOMO and LUMO of
compounds 2a and 3a were calculated by DFT of Gaussian 09.
2. Results and discussion
2.1 Synthesis
All the target compounds were first reported except compound 2a, which covered in patent
used as altering lifespan of a eukaryotic organism[13]. The synthetic route of 4-amino-5-
substituent-1,2,4-triazole-3-thione Schiff bases in this experiment was outlined in Scheme 1.
Compounds 1a-1h were prepared according to literature[14-16]. The title compounds 2a-2d, 2f-2h
and 3a-h were synthesized by condensation of 4-amino-5-substituent-1,2,4-triazole-3-thione with
3-phenoxy-benzaldehyde and 4-methoxy-2,3,6-trimethyl benzaldehyde, respectively. The structure
of title compounds was confirmed on the basis of their spectral data and elemental analysis. All
spectral and analytical data were consistent with the assigned structures. The bands observed
around 2900 and 3100 cm-1 in the IR spectrum are assigned to the C–H and N–H stretching modes,
respectively. The strong peaks around 1600 cm−1 are ascribed to the C=N group of Schiff base.
The characteristic stretching vibrations v(C=S) appeared at around 1330 cm−1, meanwhile, there is
no absorption at 2565-2550 cm-1 (S–H), manifesting title compounds are mainly exist in keto
configuration, and this result is in agreement with the crystal structure (2f).
3

Scheme 1 Synthesis of 1,2,4-triazole Schiff bases. Reagents and conditions: (A) 98% H2SO4, EtOH, reflux; (B/D) NH2NH2·H2O, EtOH,
reflux; (C) CS2, KOH, rt; (E/F/I/J) EtOH, CH3COOH, reflux; (G) CH3I, K2CO3, THF, reflux; (H) POCl3, DMF, NaOH, 90℃.
2.2 Crystal structure analysis
The yellow crystal (0.39 mm × 0.30 mm × 0.14 mm) structure of compound 2f was obtained
in DMF, which was suitable for X-ray single crystal diffraction as shown in Fig. 3 with the
following crystallographic parameters: a = 9.808 (7) Å, b = 12.837 (9) Å, c = 13.937 (10) Å, α =
90°, β = 93.077 (11)°, γ = 90°, V = 1752 (2) Å3, Z = 4, Dc = 1.416 mg·m−3, μ = 0.206 mm−1, F
(000) = 776, R = 0.0502, wR = 0.1114, the final R = 0.0408 and wR = 0.1050 for 2568 observed
reflections with I > 2σ (I).
4

Fig. 3 The ORTEP diagram of the compound 2f
2.3 Antifungal activities
The antifungal activities of compounds 2a-h, 3a-h, and triadimefon against gibberlla
nicotiancola (A), pythium solani (B), gibberlla saubinetii (C), and fusarium oxysporium f. sp.
niveum (D) were listed in Table 1, the regression equation and R value were listed in supporting
information. The Antifungal activities against gibberlla nicotiancola (A): compounds 2a and 3a
reached an excellent activity with EC50 values of 0.0309 g· L-1 and 0.0012 g· L-1, respectively,
lower than that of triadimefon (0.0620 g· L-1). The EC95 values of compounds 2a and 3a are
4.6390 g·L-1 and 0.0339 g·L-1, respectively, which are also lower than that of triadimefon (7.3943
g· L-1). Antifungal activities against pythium solani (B): compound 2a showed excellent activity
with EC50 and EC95 values of 0.0038 g· L-1 and 0.4010 g· L-1 equivalent to triadimefon (EC50
=
0.0013 g· L-1, EC95 = 0.2810 g·L-1). Antifungal activities against gibberlla saubinetii (C):
compound 2a possessed excellent activity with EC50 values of 0.0087 g· L-1 and EC95 values of
2.7930 g· L-1, respectively, lower than triadimefon (EC50 = 0.0195 g· L-1, EC95 = 7.5650 g· L-1).
Compound 3a and 3d reached the similar activity level as triadimefon. Compound 2e and 3h
exhibited a good activity. Antifungal activities against fusarium oxysporium f. sp. niveum (D):
compounds 2a, 2b, 3a and 3b showed good activity, and their EC50 values are in the range of
0.0232-0.3847 g·L-1.
The bar chart of antifungal activity (EC50 < 1.4500 g·L-1) of target compounds against four
vegetable pathogens was showed in Fig. 4. Compounds 2a and 2b showed remarkable antifungal
activity against four pathogens, especially compounds 2a, which showed better antifungal activity
compared with triadimefon. Compounds 2c, 2d, 3a and 3h showed good antifungal activity against
5

four pathogens. The results indicated no substituent at position Q can increase the antifungal
activity, and this result consistents with our previous research[11]
.
Table 1 The antifungal activity results of title compounds against four vegetable pathogens
Compounds
Pathogen
EC50/(g·L-1)
EC95/(g· L-1)
A
B
C
D
0.0309
4.6390
0.0038
0.4010
2a
0.0087
2.7930
0.0232
4.5180
A
B
C
D
0.1350
0.0271
0.0479
0.2840
10.0860
17.9590
/
2b
2c
2d
2f
10.1220
A
B
C
D
A
B
C
D
A
B
C
D
A
B
C
D
A
B
C
D
0.0979
0.0913
0.9670
1.2890
0.2130
0.1720
1.3430
0.5560
1.0503
1.1260
5.8670
1.2750
20.2812
1.7565
4.0154
1.3680
0.5212
0.6345
2.0654
1.4830
359.5010
11.6890
/
/
520.6760
215.0700
227.1290
242.1160
19.6473
368.1590
779.7300
28.7520
743.7434
16.3978
44.4352
180.9040
23.0132
17.2567
12.8500
24.8100
2g
2h
3a
3b
3c
3d
3e
3f
A
B
C
D
0.0012
1.0328
0.1035
0.2287
0.0339
9.1474
3.0974
38.9045
A
B
C
D
A
B
C
D
A
B
C
D
A
B
C
D
A
B
C
D
1.5840
1.8197
1.9588
0.3847
2.5119
7.8886
1.7701
1.9072
1.4454
1.0328
0.6266
0.7610
0.2691
1.9120
1.1324
0.6374
3.0199
10.2494
35.1318
1.0294
39.7924
14.3186
19.9572
8.3387
/
/
186.1230
/
31.9007
9.1474
5.3088
30.1926
924.6982
167.3016
/
16.3305
337.7536
571.6102
/
925.1241
A
B
1.3183
7.7446
84.8203
/
6

3g
C
D
0.9057
0.5494
/
73.7055
A
B
C
D
A
B
C
D
0.2238
0.7871
0.8954
0.2679
0.0620
0.0013
0.0195
0.0079
/
622.4436
13.0610
4.6313
7.3943
0.2810
7.5650
1.0510
3h
Triadimefon
Symbols: EC50 = 50% effective concentration; EC95 = 95% effective concentration; A = gibberlla nicotiancola;
B = pythium solani; C = gibberlla saubinetii; D = fusarium oxysporium f. sp. niveum; (/) = EC50, EC95>1000 g/L.
Fig. 4 The bar chart of antifungal activity results against four vegetable pathogens (EC50 / g•L-1)
2.3 Antibacterial activities
The antibacterial activities of compounds 2a-2d, 2f-2h, 3a-3h and chloramphenicol against
sacillus subtilis, staphylococcus aureus and escherichia coli were listed in Table 2. Compounds 2a
and 2b exhibited excellent antibacterial activities comparable to chloramphenicol. In particular,
compound 2a showed remarkable antibacterial activities.
Table 2 Antibacterial activities of title compounds
Compounds
Zone of inhibition (100 ug·mL-1)
Sacillus subtilis
Staphylococcus aureus
Escherichia coli
2a
2b
2c
2d
2f
+++
++
+++
+
+++
+++
+++
+
+++
++
++
+
+
++
+
++
+
2g
2h
3a
-
++
+
+
++
+
+
7

3b
-
-
-
3c
-
-
-
3d
-
-
-
3e
-
-
-
3f
-
-
-
-
3g
3h
-
-
-
-
+
Chloramphenicol
+++
+++
+++
Symbols: zone diameter of growth inhibition: (-) = Inactive (< 6 mm); (+) = week active (6-10 mm); (++) = moderate active (10-15 mm);
(+++) = high active (15-20 mm); (+++) = Chloramphenicol.
2.4 DFT calculation
According to the frontier molecular orbital theory, HOMO and LUMO are the most important
factors effected bioactivity[17]. HOMO has the priority to provide electrons, while LUMO accepts
electrons in the first place[18]. A study of the frontier orbital energy can provide some useful
information for the active mechanism. Density functional theory (DFT) has been extensively used
to calculate a wide variety of molecular properties because of high efficiency and accuracy [19,20]
.
DFT also adequately takes into account electron correlation contributions especially in systems
containing extensive electron conjugation and/or electron lone pairs[21]
.
The energy and pictorial illustrations of the HOMO and the LUMO of representative
compounds 2a and 3a with good and moderate activity, respectively, which have been given in Fig.
5. It is showed that HOMO orbital of 2a is mainly be delocalized on triazole ring and S atom,
especially the latter, LUMO of 2a mainly contains Schiff base double bond (C=N) and
benzene ring. Both HOMO and LUMO of 3a mainly located on 1,2,4-triazole ring, Schiff base
double bond (C=N) and benzene ring. It is observed that the triazole ring, S atom and benzene ring
in both of the molecules make a major contribution to the activity. These are largely through
hydrophobic interaction obviously in the HOMO maps (especially the S atom and N atom of
position 2 of triazole ring). It is found that there are bit differences at the frontier molecular
orbitals of 2a and 3a. The HOMO of compound 2a is mainly located on the triazole ring and S
atom, 2a has a remarkable activity maybe because electron of 2a can easily transferred from
HOMO to the receptor. Compared with 2a, 3a is more stable than 2a and has a moderate activity
due to the HOMO and LUMO of 3a are wide delocalized.
8

(2a) HOMO E77 = - 0.216 a.u.
(2a) LUMO E78 = - 0.084 a.u.
(3a) HOMO E73 = - 0.215 a.u.
(3a) LUMO E74 = - 0.067 a.u.
Fig. 5 LUMO and HOMO maps for compounds 2a and 3a from DFT/B3LYP/6-311G+(d,p) calculation. The
green parts represent positive molecular orbital, and the red parts represent negative molecular orbital.
Conclusions
In summary, series of 4-amino-5-substituent-1,2,4-triazole-3-thione Schiff bases have been
1
prepared, and their structures were confirmed by melting point, IR, H NMR, 13C NMR and
elemental analyses and HRMS. The antifungal activities against gibberlla nicotiancola, pythium
solani, gibberlla saubinetii, and fusarium oxysporium f. sp. niveum were evaluated. The antifungal
activities results showed compound 2a and 2b showed excellent activity to most of the fungi.
Compound 2a had a better effective against gibberlla nicotiancola and gibberlla saubinetii than
triadimefon. Meanwhile, their antibacterial activity against sacillus subtilis, staphylococcus aureus,
and escherichia coli were also tested. Form antibacterial activity results, compounds 2a reached a
good activity as well as chloramphenicol. All bioassays showed that the no substituent at position
5 in the triazole ring was more effective than others, and 3-phenoxyphenyl group introduced in the
scaffold can enhanced the antibacterial activity. The DFT calculation results indicated triazole ring,
S atom and benzene ring in both of the 2a and 3a make a major contribution to the activity. The
present work demonstrated that compound 2a can be used as lead compound for the development
of new dual antifungal and antibacterial agent.
3. Experimental
3.1 Chemistry
9

Elemental analysis was performed with a Vario ELШ CHNOS analyzer; IR (KBr) spectra
was recorded on an IR-400 spectrophotomete; 1H NMR and 13C NMR spectra were measured with
a Varian unity INOVA-400 nuclear magnetic resonance using CDCl3 or DMSO-d6 as the solvent
with tetramethylsilane as an internal standard; mass spectra was obtained from micrOTOF-Q II
mass spectrometer; the melting point was determined on MP3 melting point apparatus; the X-ray
diffraction data were collected on a Bruker SMART-APEX II CCD diffractometer 2004.
3.2 Synthesis
3.2.1 Synthesis of 4-amino-5-substituent-1,2,4-triazolethione 1a-h
Compounds 1a-e were obtained in our previous research[11]. Compounds 1f-h were
1
synthesized by a known procedure[14,15], 1f: H NMR (400MHz, DMSO-d6) δ 8.84(2H, m),
7.20(2H, m), 3.72(2H, s); 1g: 1H NMR (400MHz, CDCl3) δ 7.75(2H, d, J = 7.6Hz), 6.97(2H, d, J
= 7.6Hz), 2.37(3H, s), 2.10(2H, s); 1h: 1H NMR (400MHz, CDCl3) 7.54(1H, t, J=7.6 Hz), 7.49(1H,
d, J=7.2 Hz), 7.12(1H, d, J=8.0 Hz), 7.04(1H, t, J=7.2 Hz), 5.49(2H, s), 3.70(3H, s).
3.2.2 Synthesis of 4-methoxy-2,3,6-trimethyl-benzaldehyde
4-Methoxy-2,3,6-trimethyl-benzaldehyde was synthesized according to the method presented
in literature[22]. 1H NMR(400MHz) δ 10.53(1H, s), 6.56(1H, s), 3.87(3H, s), 2.60(3H, s), 2.53(3H,
s), 2.15(3H, s).
3.2.3 General synthetic procedure for compounds 2a-d and 2f-h
4-Amino-5-substituent-1,2,4-triazole-3-thione (1a-d and 1f-h) (5 mmol) was added to a
solution of 3-phenoxy-benzaldehyde (5 mmol) in ethanol (20 mL) and glacial acetic acid (2 mL),
the mixture was refluxed for about 4 h. The reaction mixture was left standing overnight and then
filtration. The crude products were crystallized from absolute ethanol except compounds 2f, 2g,
and 2h, which were crystallized from DMF.
5H-4-[(3-phenoxyphenyl)methyleneamino]-3H-1,2,4-triazole-3-thione (2a): white solid, yield
= 88.3 %, mp 143.2-144.1 ℃. 1H NMR (400MHz, CDCl3) δ 10.99 (1H, s), 10.42 (1H, s), 8.04 (1H,
s), 7.55 (1H, d, J=7.6 Hz), 7.51 (1H, s), 7.44 (1H, t, J=7.6 Hz), 7.38 (2H, t, J=7.6 Hz), 7.18 (2H,
q), 7.04 (2H, d, J=7.6 Hz). 13C NMR (400MHz, DMSO) δ 117.33, 119.71, 122.94, 124.32, 124.63,
130.76, 131.47, 134.54, 138.53, 156.43, 157.97, 160.62, 163.44. IR (KBr) v/cm-1: 3113, 2933,
1554, 1491, 1293, 927, 847, 686, 561; MS: m/z 297.3473 (M+1). Elemental Anal. Calcd for
C15H12N4OS: C, 60.79; H, 4.08; N, 18.91. Found: C, 60.81; H, 4.06; N, 18.89.
10

5-Methyl-4-[(3-phenoxyphenyl)methyleneamino]-3H-1,2,4-triazole-3-thione (2b): white
solid, yield = 89.7 %, mp 154.0-155.5 ℃. 1H NMR (400MHz, CDCl3) δ 11.34 (1H, s), 10.41 (1H,
s), 7.56 ( 2H, t, J=7.6 Hz), 7.43 (1H, t, J=8.0), 7.37 (2H, t, J=7.6 Hz), 7.15 (2H, t, J=8.0 Hz), 7.04
(2H, d, J=8.0 Hz), 2.44 (3H, s). 13C NMR (400MHz, DMSO) δ 11.17, 117.61, 119.57, 122.98,
124.46, 124.53, 130.71, 131.42, 134.63, 148.83, 156.51, 157.89, 161.71, 162.95. IR (KBr) v/cm-1:
3066, 2896, 1593, 1491, 1252, 959, 850, 686, 561. MS: m/z 311.3738 (M+1). Elemental Anal.
Calcd for C16H14N4OS: C, 61.92; H, 4.55; N, 18.05. Found: C, 61.90; H, 4.54; N, 18.06.
5-Ethyl-4-[(3-phenoxyphenyl)methyleneamino]-3H-1,2,4-triazole-3-thione (2c): white solid,
1
yield = 88.0 %, mp 131.5-132.6 ℃. H NMR (400MHz, CDCl3) δ 11.31 (1H, s), 10.39 (1H, s),
7.54 (3H, t, J=7.2 Hz), 7.38 (3H, m), 7.15 (2H, d, J=7.2 Hz), 7.04 (1H, d, J=7.6 Hz), 2.82 (2H, q),
1.30 (3H, t, J=7.2 Hz). 13C NMR (400MHz, DMSO) δ 10.49, 18.73, 117.46, 119.62, 122.90,
124.39, 124.55, 130.69, 131.41, 134.65, 152.75, 156.45, 157.94, 161.83, 162.76. IR (KBr) v/cm-1:
3065, 2943, 1591, 1492, 1263, 907, 788, 655, 564. MS: 325.4002 m/z (M+1). Elemental Anal.
Calcd for C17H16N4OS: C, 62.94; H, 4.97; N, 17.27. Found: C, 62.95; H, 4.94; N, 17.26.
4-[(3-Phenoxyphenyl)methyleneamino]-5-propyl-3H-1,2,4-triazole-3-thione (2d): white solid,
1
yield = 87.8 %, mp 115.5-121.1 ℃. H NMR (400MHz, CDCl3) δ 11.31 (1H, s), 10.39 (1H, s),
7.54 (2H, t, J=11.6 Hz), 7.43 (1H, t, J=8.0 Hz), 7.37 (2H, t, J=7.6 Hz), 7.16 (2H, d, J=7.2 Hz),
7.05 (2H, d, J=7.6 Hz), 2.76 (2H, t, J=7.2 Hz), 1.75 (2H, m), 0.97 (3H, t, J=7.2 Hz). 13C NMR
(400MHz, DMSO) δ 13.89, 19.35, 26.81, 39.99, 117.18, 119.73, 122.89, 124.43, 124.62, 130.71,
131.45, 134.63, 151.60, 156.38, 158.03, 161.72, 162.73. IR (KBr) v/cm-1: 3132, 2964, 1571, 1491,
1241, 959, 843, 689. MS: m/z 339.4268 (M+1). Elemental Anal. Calcd for C18H18N4OS: C, 63.88;
H, 5.36; N, 16.56. Found: C, 63.93; H, 5.31; N, 16.51.
4-[(3-Phenoxyphenyl)methyleneamino]-5-(4-pyridyl)-3H-1,2,4-triazole-3-thione (2f): yellow
solid, yield = 83.8 %, mp > 250 ℃. 1H NMR (400 MHz, DMSO-d6) δ 9.77 (1H, s), 8.71 (2H, d, J
= 4.8 Hz), 7.80 (2H, d, J = 4.8 Hz ), 7.65 (1H, d, J = 7.6 Hz), 7.58 (1H, t, J=8.0 Hz), 7.43 (3H, t,
J=8.2 Hz ), 7.29 (1H, d, J = 8.4 Hz), 7.19 (1H, t, J = 7.6 Hz), 7.11 (2H, d, J = 8.0 Hz). IR (KBr)
v/cm-1: 3109, 2948, 1569, 1476, 1435, 1260, 948, 673, 543. MS: m/z 374.4313 (M+1). Elemental
Anal. Calcd for C20H15N5OS: C, 64.33; H, 4.05; N, 18.75. Found: C, 64.34; H, 4.03; N, 18.76.
4-[(3-Methylphenyl)methyleneamino]-5-(4-methylphenyl)-3H-1,2,4-triazole-3-thione (2g):
1
white solid, yield = 84.6 %, mp 156.0-158.0 ℃. H NMR (400 MHz, DMSO-d6) δ 9.79 (1H, s),
11

7.69 (2H, d, J=8.0 Hz), 7.62 (2H, m), 7.45 (3H, m), 7.30 (3H, d, J=7.2 Hz), 7.22 (1H, m), 7.12
(2H, d, J=7.6 Hz), 2.37 (3H,s). IR (KBr) v/cm-1: 3076, 2850, 1573, 1476, 1435, 1260, 958, 679.
MS: m/z 387.4697 (M+1), Elemental Anal. Calcd for C22H18N4OS: C, 68.37; H, 4.69; N, 14.50.
Found: C, 68.35; H, 4.68; N, 14.50.
4-[(3-Phenoxyphenyl)methyleneamino]-5-(2-methoxyphenyl)-3H-1,2,4-triazole-3-thione (2h):
1
white solid, yield = 88.3 %, mp 164.2-166.1℃. H NMR (400 MHz, DMSO-d6) δ 9.70(1H, s),
7.50(3H, m), 7.41(3H, q), 7.28(1H, s), 7.22(2H, m), 7.06(4H, t, J=7.6 Hz), 3.60(3H, s). IR (KBr)
v/cm-1: 3139, 2978, 1589, 1498, 1467, 1272, 989, 697. MS: m/z 403.4686 (M+1). Elemental Anal.
Calcd for C22H18N4O2S: C, 65.65; H, 4.51; N, 13.92. Found: C, 65.64; H, 4.51; N, 13.91.
3.2.4 General synthetic procedure for compounds 3a-3h
4-Amino-5-substituent-1,2,4-triazole-3-thione (1a-1h) (5 mmol) was added to a solution of
4-methoxy-2,3,6-trimethyl benzaldehyde (5 mmol) in ethanol (20 mL) and glacial acetic acid (2
mL), the mixture was refluxed about 4 h. The reaction mixture was filtrated when the solvent is
hot. The crude product was crystallized from DMF.
4-[(4-Mehoxy-2,3,6-trimethylphenyl)methyleneamino]-5H-3H-1,2,4-triazole-3-thione (3a):
white solid, yield = 78.3 %, mp 181.1-182.3 ℃. 1H NMR (400MHz, CDCl3) δ 11.25 (1H, s), 10.46
(1H, s), 8.03 (1H, s), 6.63 (1H, s), 3.86 (3H, s), 2.56 (3H, s), 2.48 (3H, s), 2.16 (3H, s). 13C NMR
(400MHz, DMSO) δ 11.93, 17.43, 22.41, 55.99, 111.47, 122.39, 123.07, 138.65, 139.42, 159.15,
162.44, 163.94. IR (KBr) v/cm-1: 3112, 2913, 1564, 1488, 1291, 920, 845, 680. MS: m/z
277.3574(M+1). Elemental Anal. Calcd for C13H16N4OS: C, 56.50; H, 5.84; N, 20.27. Found: C,
56.53; H, 5.80; N, 20.30.
4-[(4-Mehoxy-2,3,6-trimethylphenyl)methyleneamino]-5-methyl-3H-1,2,4-triazole-3-thione
(3b): white solid, yield = 77.5 %, mp 217.7-218.2 ℃. 1H NMR (400MHz, CDCl3) δ 11.12 (1H, s),
10.48 (1H, s), 6.64 (1H, s), 3.87 (3H, s), 2.59 (3H, s), 2.51 (3H, s), 2.45 (3H,s), 2.18 (3H, s). 13C
NMR (400MHz, DMSO) δ 11.41, 11.95, 17.49, 22.60, 56.04, 111.59, 122.55, 123.11, 138.57,
139.51, 148.75, 159.19, 161.23, 165.64. IR(KBr) v/cm-1: 3064, 2953, 1587, 1499, 1221, 967, 859,
693. MS: m/z 291.3837(M+1). Elemental Anal. Calcd for C14H18N4OS: C, 57.91; H, 6.25; N,
19.29. Found: C, 57.88; H, 6.24; N, 19.30.
5-Ethyl-4-[(4-mehoxy-2,3,6-trimethylphenyl)methyleneamino]-3H-1,2,4-triazole-3-thione
1
(3c): white solid, yield = 78.3 %, mp 188.2-189.1℃. H NMR (400MHz, CDCl3) 10.43 (1H, s),
12

6.63 (1H, s), 3.86 (3H, s), 2.80 (2H, q), 2.59 (3H, s), 2.51 (3H, s), 2.17 (3H,s), 1.33 (3H, t, J=7.2
Hz). 13C NMR (400MHz, DMSO) δ 10.44, 11.88, 17.44, 18.91, 22.61, 55.96, 111.50, 122.45,
123.07, 138.57, 139.53,152.62, 159.15, 161.32, 165.37, 165.81. IR(KBr) v/cm-1: 3056, 2932, 1583,
1454, 1280, 965, 841, 689. MS: m/z 305.4106(M+1). Elemental Anal. Calcd for C15H20N4OS: C,
59.18; H, 6.62; N, 18.41. Found: C, 59.20; H, 6.58; N, 18.39.
4-[(4-Mehoxy-2,3,6-trimethylphenyl)methyleneamino]-5-propyl-3H-1,2,4-triazole-3-thione
(3d): white solid, yield = 73.3 %, mp 170.4-171.2 ℃. 1H NMR (400MHz, CDCl3) δ 10.76 (1H, s),
10.56 (1H, s), 7.37 (1H, s), 6.76 (1H, s), 3.97 (3H, s), 2.87 (2H, t, J=7.2 Hz), 2.71 (3H, s), 2.63
(3H,s), 2.30 (3H, s), 1.89 (2H, q), 1.13 (3H, t, J=7.2 Hz). 13C NMR (400MHz, DMSO) δ 11.90,
13.91, 17.42, 19.47, 22.54, 27.06, 55.99, 111.54, 122.45, 123.12, 138.60, 139.54, 151.43, 159.20,
161.26, 165.69. IR (KBr) v/cm-1: 3102, 2927, 1581, 1460, 1271, 1007, 839, 647. MS: m/z
319.4370(M+1). Elemental Anal. Calcd for C16H22N4OS: C, 60.35; H, 6.96; N, 17.59. Found: C,
60.38; H, 6.95; N, 17.57.
5-Benzyl-4-[(4-mehoxy-2,3,6-trimethylphenyl)methyleneamino]-3H-1,2,4-triazole-3-thione
(3e): white solid, yield = 78.8 %, mp 186.6-187.4 ℃. 1H NMR (400MHz, CDCl3) δ 10.03 (1H, s),
7.30 (2H, t, J=4.8 Hz), 7.23 (3H, t, J=5.2 Hz), 6.81 (1H, s), 4.13 (2H, s), 3.83 (3H, s), 2.43 (3H, s),
2.36 (3H, s), 2.09 (3H, s). 13C NMR (400MHz, DMSO) δ 11.93, 17.43, 22.47, 31.11, 55.03,
111.56, 122.36, 123.11, 127.29, 128.98, 129.03, 135.61, 138.74, 139.60, 150.48, 159.29, 161.60,
166.13. IR(KBr) v/cm-1: 3103, 2920, 1576, 1456, 1281, 1017, 840, 695, 581. MS: m/z 367.4800
(M+1). Elemental Anal. Calcd for C20H22N4OS: C, 65.55; H, 6.05; N, 15.29. Found: C, 65.56; H,
6.01; N, 15.30.
4-[(4-Mehoxy-2,3,6-trimethylphenyl)methyleneamino]-5-(4-pyridyl)-3H-1,2,4-triazole-3-thio
ne (3f): yellow solid, yield = 75.8 %, mp > 250 ℃. 1H NMR (400MHz, DMSO-d6) δ 9.91 (1H, s),
8.83 (2H, m), 7.90 (2H, m), 6.93 (1H, s), 3.92 (3H, s), 2.56 (3H, s), 2.49 (3H,s), 2.19 (3H, s). 13C
NMR (400MHz, DMSO) δ 11.96, 17.50, 22.54, 56.12, 111.70, 121.95, 122.05, 122.66, 123.31,
133.41, 139.43, 140.11, 147.10, 147.86, 150.65, 159.68, 163.25, 168.20, 169.65. IR (KBr) v/cm-1:
3208, 2997, 1589, 1480, 1245, 998, 839, 568. MS: m/z 354.4413 (M+1). Elemental Anal. Calcd
for C18H19N5OS: C, 61.17; H, 5.42; N, 19.81. Found: C, 61.18; H, 5.40; N, 19.80.
4-[(4-Mehoxy-2,3,6-trimethylphenyl)methyleneamino]-5-(4-methylphenyl)-3H-1,2,4-triazole
1
-3-thione (3g): white solid, yield = 72.3 %, mp 210.1-211.2 ℃. H NMR (400MHz, DMSO-d6) δ
13

9.74 (1H, s), 7.68 (2H, d, J=8.0 Hz ), 7.30 (2H, d, J=8.4 Hz), 6.81 (1H, s), 3.82 (3H, s), 2.45 (3H,
s), 2.39 (3H,s), 2.34 (3H, s). IR (KBr) v/cm-1: 3090, 2908, 1579, 1432, 1223, 989, 808, 636. MS:
m/z 367.4797(M+1). Elemental Anal. Calcd for C20H22N4OS: C, 65.55; H, 6.05; N, 15.29. Found:
C, 65.52; H, 6.01; N, 15.30.
4-[(4-Mehoxy-2,3,6-trimethylphenyl)methyleneamino]-5-(2-methoxyphenyl)-3H-1,2,4-triazo
le-3-thione (3h): white solid, yield = 73.3 %, mp 168.4–170.2 ℃. 1H NMR (400MHz, DMSO-d6) δ
9.72(1H, s), 7.56(1H, t, J=7.6 Hz), 7.48(1H, d, J=7.2 Hz), 7.14(1H, d, J=8.0 Hz), 7.08(1H, t,
J=7.2 Hz), 6.75(1H, s), 3.80(3H, s), 3.70(3H, s), 2.26(3H,s), 2.24(3H, s), 2.05(3H, s). IR(KBr)
v/cm-1: 3231, 2965, 1598, 1476, 1282, 1017, 848, 688. MS: m/z 384.4794 (M+1). Elemental Anal.
Calcd for C20H22N4O2S: C, 62.80; H, 5.80; N, 14.65. Found: C, 62.83; H, 5.78; N, 14.66.
3.3 Crystal structure determination
A yellow single crystal of the title compound with dimensions of 0.39 mm × 0.30 mm × 0.14
mm was selected for X-ray diffraction analysis. The X-ray diffraction data was collected on a
Bruker SMART-APEX II CCD diffractometer[296(2)K] equipped with a graphite monochromatic
Mo Kα radiation (λ = 0.71073 Ǻ) by using -2 scan technique at room temperature. A total of
3106 reflections were collected in the range of 2.48 < θ < 25.10°, of which 2564 were independent
with Rint = 0.0383. The structure was solved by direct methods with SHELXS-97, and refined
using the full-matrix least squares method on F2 with anisotropic thermal parameters for all
non-hydrogen atoms using SHELXL-97[23,24]. All hydrogen atoms were located theoretically and
refined with riding model position parameters and fixed isotropic thermal parameters.
3.4 Antifungal activities
The antifungal activities of compounds 2a-d, 2f-h, and 3a-h were evaluated against four
plants pathogenic fungi, containing pythium solani, gibberlla nicotiancola, fusarium oxysporium
f.s.p. niveum, and gibberlla saubinetii, which are often encountered in plants. The four plants
pathogenic fungi were provided by Microbiology Institute of Shaanxi. Triadimefon was purchased
from Jiangsu Sevencontinent Green Chemical Co., Ltd. The technique of antifungal activities and
data-processing were according to the method described in literature[25]
.
3.5 Antibacterial activities
The title compounds were screened for their antibacterial activity against sacillus subtilis,
staphylococcus aureus and escherichia coli bacterial strains by disc diffusion method described in
14

literature[26]. Chloramphenicol was used as control and N,N-dimethyl formamide was used as
solvent. The discs measuring 6.00 mm in diameter were prepared from filter paper by hole
puncher and sterilized at 121˚C for 1 h. A standard inculum (1-2ⅹ107c.f.u·mL-1 0.5 McFarland
standards) was introduced onto the surface of sterile agar, and a sterile glass spreader was used for
even distribution of the inoculum. The dried sterile discs previously soaked in a known
concentration (100 ug·mL-1) of the test compounds were placed in nutrient agar medium. The
plates were labeled and incubated for 18-24 h at 37 ˚C. All tests were repeated for three times.
3.6 Density Functional Theoretical (DFT) calculation
The density functional theoretical (DFT) calculations of compounds 2a and 3a were
performed at the Becke-Lee-Parr hybrid exchange correlation three-parameter functional (B3LYP)
level with standard 6-311G+(d,p) basis set. The complete geometry optimization and vibrational
frequencies were calculated at B3LYP/6-311G+(d,p) level on the basis of the optimized structure.
Vibration analysis showed that the optimized structure was in accordance with the minimum
points on the potential energy surface. All of the convergent precisions were the system default
values, and all calculations reported in this work were carried out with the Gaussian 09
program[27]
.
Acknowledgements
The authors are grateful to the National Natural Science Foundation of China (No.21373161)
for its financial support for this study.
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17

Highlights
Series of 1,2,4-triazole Schiff bases (2a-h and 3a-h) were screened for antifungal and antibacterial
activity, biological results showed compound 2a exhibited excellent antifungal and antibacterial
activities.
The SAR manifested no substitution at position 5 of the triazole ring caused an increase in activity,
and 3-phenoxy phenyl group introduced in 1,2,4-triazole scaffold can enhance the antibacterial
activity.
The DFT calculation results indicated triazole ring, S atom and benzene ring in both of the 2a and
3a make a major contribution to the activity.
Graphical abstract:
Series of novel 1,2,4-triazole Schiff bases were synthesized and evaluated for antifungal and
antibacterial activities. The bioassay showed compound 2a has potential to developing candidate
compound. The DFT calculation indicated triazole ring, S atom and benzene ring in both of the 2a
and 3a make a major contribution to the activity.
18