Investigation on 4-amino-5-substituent-1,2,4-triazole-3-thione Schiff bases an antifungal drug by characterization (spectroscopic, XRD), biological activities, molecular docking studies and electrostatic potential (ESP)

Article abstract of DOI:10.1016/j.molstruc.2019.07.013

Four novel Schiff bases 4-(2,4-dinitrobenzylideneamino)-5-m-tolyl-2H-1,2,4-triazole-3(4H)-thione) (F₁), 4-(2,4-dinitrobenzylideneamino)-5-(2-methoxyphenyl)-2H-1,2,4-triazole-3(4H)-thione) (F₂), 4-(2,4-dinitrobenzylideneamino)-5-(3-methoxyphenyl)-2H-1,2,4-triazole-3(4H)-thione) (F₃) and 4-(2,4-dinitrobenzylideneamino)-5-(4-methoxyphenyl)-2H-1,2,4-triazole-3(4H)-thione) (F₄) were prepared as new antifungal compounds contributing 4-Amino-5-Substituent-1,2,4-Triazole-3-Thione and 2,4-dinitrobenzaldehyde via a condensation reaction under the mild conditions with excellent yields. The structures of compounds were characterized by elemental analysis (EA), FT-IR, ¹H NMR, ¹³C NMR spectra and X-ray analysis. In addition, the compounds were screened for in vitro biological activity, and the bioassay results indicated that the newly synthesized compounds showed different in vitro antifungal activities against five plant fungi. Particularly, compound F₃ (EC₅₀ = 11.16 mg/L) was found to be the most active respectively against Wheat gibberellic, even more effective than Fluconazole (EC₅₀ = 16.03 mg/L). The active compounds were further evaluated for enzyme inhibition efficacy against the receptor CYP51 by docking. Meanwhile, an explicit surface analysis on compounds were carried out theoretically using the wave function analyzer (Multiwfn 3.4.1 software) in order to study the reactivity of the compounds.

Full text of DOI:10.1016/j.molstruc.2019.07.013

Accepted Manuscript
Investigation on 4-amino-5-substituent-1,2,4-triazole-3-thione Schiff bases an
antifungal drug by characterization (spectroscopic, XRD), biological activities,
molecular docking studies and electrostatic potential (ESP)
Shaojie Wu, Wenhui Zhang, Le Qi, Yinghui Ren, Haixia Ma
PII:
S0022-2860(19)30847-6
DOI:
https://doi.org/10.1016/j.molstruc.2019.07.013
MOLSTR 26766
Reference:
To appear in: Journal of Molecular Structure
Received Date: 30 May 2019
Revised Date: 30 June 2019
Accepted Date: 3 July 2019
Please cite this article as: S. Wu, W. Zhang, L. Qi, Y. Ren, H. Ma, Investigation on 4-amino-5-
substituent-1,2,4-triazole-3-thione Schiff bases an antifungal drug by characterization (spectroscopic,
XRD), biological activities, molecular docking studies and electrostatic potential (ESP), Journal of
Molecular Structure (2019), doi: https://doi.org/10.1016/j.molstruc.2019.07.013.
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ACCEPTED MANUSCRIPT
Investigation on 4-amino-5-substituent-1,2,4-triazole-3-thione Schiff
bases an antifungal drug by characterization (spectroscopic, XRD)
,
biological activities, molecular docking studies and Electrostatic
potential (ESP)
Shaojie Wua, Wenhui Zhanga, Le Qia, Yinghui Rena,*, Haixia Maa
aSchool of Chemical Engineering, Northwest University, Xi'an, Shaanxi, 710069, People's Republic of China
Abstractꢀ
Four
novel
Schiff
bases
(F1),
(F2),
4-(2,4-dinitrobenzylideneamino)-5-m-tolyl-2H-1,2,4-triazole-3(4H)-thione)
4-(2,4-dinitrobenzylideneamino)-5-(2-methoxyphenyl)-2H-1,2,4-triazole-3(4H)-thione)
4-(2,4-dinitrobenzylideneamino)-5-(3-methoxyphenyl)-2H-1,2,4-triazole-3(4H)-thione) (F3) and
4-(2,4-dinitrobenzylideneamino)-5-(4-methoxyphenyl)-2H-1,2,4-triazole-3(4H)-thione) (F4)
were prepared as new antifungal compounds contributing
4-Amino-5-Substituent-1,2,4-Triazole-3-Thione and 2,4-dinitrobenzaldehyde via a condensation
reaction under the mild conditions with excellent yields. The structures of compounds were
1
characterized by elemental analysis (EA), FT-IR, H NMR, 13C NMR spectra and X-ray analysis.
In addition, the compounds were screened for in vitro biological activity, and the bioassay results
indicated that the newly synthesized compounds showed different in vitro antifungal activities
against five plant fungi. Particularly, compound F3 (EC50=11.16 mg/L) was found to be the most
active respectively against Wheat gibberellic, even more effective than Fluconazole (EC50=16.03
mg/L). The active compounds were further evaluated for enzyme inhibition efficacy against the
receptor CYP51 by docking. Meanwhile, an explicit surface analysis on compounds were carried
out theoretically using the wave function analyser (Multiwfn 3.4.1 software) in order to study the
reactivity of the compounds.
Keywords:Antifungal activities; 1,2,4-Triazoles; Molecular docking; ESP.
*
Corresponding author: Yinghui Ren.
E-mail address: nwuryh@163.com.

ACCEPTED MANUSCRIPT
1. Introduction
The chemistry of 1,2,4-Triazole compounds has attracted widespread attention due to their
diverse biological activities [1], and the 1,2,4-Triazole containing amino group is also important
for obtaining various Schiff bases with well-established antimicrobial properties as well.
1,2,4-Triazole moieties are indeed known as important polar pharmacophore, both as a donor to
hydrogen bond and as a acceptor, and thus readily to interact with a variety of partners in
biological systems [2]. Particularly, they are indispensable part in almost all kinds of
agrochemicals [3-4]. Several well-known pesticides containing 1,2,4-Triazole are Triadimefon [5],
Itraconazole, Voriconazole, Fluconazole, Diniconazole and Tebuconazol [6] (Fig. 1).
1,2,4-Triazole has an ambident nucleophilic centers and is a good starting material for the
synthesis of several interesting N and S-bridged heterocycles [7]. Therefore, 1,2,4-Triazole is of
significance for the design of new molecular entities with unique structural features.
N
N
O
N
F
OH
Cl
O
N
N
N
N
N
O
Cl
O
F
N
N
N
N
F
N
Itraconazole
Voriconazole
N
N
N
N
N
N
OH
Cl
F
O
N
N
N
Cl
N
OH
N
N
O
Cl
N
N
N
HO
Cl
F
Triadimefon
Diniconazole
Tebuconazol
Fluconazole
Fig. 1
.
The structures of some commercial fungicides containing 1,2,4-Triazole
Schiff bases are the compounds containing imine groups (–C=N–), which are formed by the
condensation products of ketones or aldehydes with primary amines [8-9]. This family of
compounds is used as starting materials in the synthesis of important drugs, including antifungal
[8-11], antileishmanial [12-13], antimicrobial [14-15], anticancer [16], anti-inflammatory [17-18],
antibacterial [17] and so on. Schiff bases of aliphatic aldehydes are relatively unstable and are
readily polymerized while those of aromatic aldehydes, having an effective conjugation system,
are more stable [20]. The chemistry of 1,2,4-Triazole compounds has attracted widespread
attention due to their diverse biological activities, especially as antifungal agents[10-11,20].
Introduction of 1,2,4-Triazole in the Schiff bases could effectively improves its biological activity,
so the demand and share of 1,2,4-Triazole Schiff bases compounds in agriculture is increasing
[21].
Prompted by these observations, as part of our search in view to develop an environmentally
benign synthetic route for the rapid generation of Schiff bases linked 1,2,4-Triazole library
compounds to obtain basic information about the structural requirements for biological activity,
we synthesized 4-Amino-5-Substituted-1,2,4-Triazole-3-Thione Schiff bases (F1, F2, F3 and F4)
using
4-Amino-5-Substituted-1,2,4-Triazole-3-Thione
and
aromatic
aldehyde
(2,4-dinitrobenzaldehyde) under refluxing. The obtained compounds were characterized by
1
elemental analysis, and the spectral methods (FT-IR, H NMR, 13C NMR and XRD). The
crystallographic data has been deposited at the Cambridge Crystallographic Data Center,

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CCDC-1903135 (F2), 1903136 (F1), 1903137 (F3), 1915700 (F4). The in vitro biological activity of
the compounds against five phytopathogenic fungi (Wheat gibberellic (I), Physalospora piricola
(II), Potato dry rot fungus (III), Glomerella cingulata (IV) and Maize rough dwarf bacteria (V))
have been investigated. The standard drug (SD) used in this paper was Fluconazole, an effective
antibacterial agent that contains 1,2,4-Triazole, which is commonly used at present [22]. Moreover,
the compounds and Fluconazole were docked with cytochrome P450 14a-desmethylase (CYP51)
(PDB ID: 6CR2) were also studied. In addition, Becke3-Lee-Yang-Parr (B3LYP) [23] level with
6-31G basis set using Gaussian 09ꢀW [24] program package was employed for the theoretical
calculation, and the distribution of electrons and the reactive sites on the surface of the title
compounds were analysed using Electrostatic potential (ESP) of Multiwfn 3.4.1 wave function
analyzer [25].
2. Experimental
2.1 Chemistry
All used chemicals are of analytical grade. Wheat gibberellic (I), Physalospora piricola (II),
Potato dry rot fungus (III), Glomerella cingulata (IV), and Maize rough dwarf bacteria (V) were
provided by Collage of life Sciences, Northwest A&F University. Fluconazole was purchased
from Shanghai Siyu Chemical Technology Co. Ltd.
Melting point was determined with SGWX-4B melting point apparatus. Elemental analysis of C,
H and N was performed on a Vario EL III elemental analyzer from ELEMENTAR Company.
Fourier infrared spectrum (FT-IR) were recorded in the range of 4000-400 cm‒1 on an EQUINOX
55 FT-IR spectrometer from Bruker Company. 1H NMR and 13C NMR spectroscopy was recorded
with AV400 NMR spectrometer (Bruker) and proton chemical shifts are recorded in ppm relative
to tetramethylsilane (TMS) as an internal standard using DMSO-d6 as solvent. The crystal
structure was measured on a Bruker SMART APEX CCD X-ray diffractometer.
2.2. Synthesis of 4-Amino-5- Substituent-1,2,4-Triazole-3-Thione Schiff bases (F)
2.2.1. Synthesis of 4-Amino-5-Substituent-1,2,4-Triazole-3-Thione (E)
The intermediate for 4-Amino-5-Substituent-1,2,4-Triazole-3-Thione (E) in this manuscript
was prepared by the four-step reaction of benzoic acid with different substitutions, which includes
esterification, hydrazidation, salt formation, and cyclization. The products were purified by
absolute ethanol, and the synthetic route was outlined according to the literature (shown in
Scheme 1) [26-28].
EtOH
98% H2SO4
EtOH
NH2NH2 H2O
O
.
O
C
OEt
O
C
NHNH2
C
reflux,6h
reflux,6h
R
R
OH
R
(A)
(C)
(B)
EtOH
CS2,KOH
reflux,4h
O
EtOH
N
.
NH
NH2NH2 H2O
S
C
R
N
reflux,4h
R
NHNHCK
S
H2N
(E)
Scheme 1. The synthesis of the intermediates E.
(D)
2.2.2. General synthetic procedure for compounds F1, F2, F3 and F4
All of the title compounds are first reported. 4-Amino-5-Substituent-1,2,4-Triazole-3-Thione

ACCEPTED MANUSCRIPT
(E) (5 mmol) was added to the solution of 2,4-dinitrobenzaldehyde (5 mmol) in glacial acetic acid
(10 mL), the mixture was refluxed for about 4 h. The reaction mixture was left standing overnight
and then filtrated (Scheme 2). The title compounds F1, F2, F3 and F4 were obtained after
recrystallization with absolute ethanol [29-31].
H
N
F R: =3-CH
1 3
O
N
N
2
S
N
N
F R: =2 O- CH
NH
NO
CH3COOH
2
3
N
N
2
O HC
NO
CH
2
+
reflux
R
F R: =3O- CH
R
S
3
3
H2N
NO
F R: =4O- CH
(F)
2
4
3
(E)
Scheme 2. The synthesis of the Schiff bases F.
4-(2,4-dinitrobenzylideneamino)-5-m-tolyl-2H-1,2,4-triazole-3(4H)-thione (F1): Orange
solid, yield = 78.1 %, m.p. 236.8-237.3 °C. 1H NMR (400 MHz, DMSO-d6) δ : 14.38 (s, 1H, N-H),
10.81 (s, 1H, azomethine), 8.87 (d, J = 2.3 Hz, 1H, Ar-H), 8.67 (dd, J = 8.6, 2.3 Hz, 1H, Ar-H),
8.29 (d, J = 8.6 Hz, 1H, Ar-H), 7.74-7.54 (m, 2H, Ar-H), 7.51-7.28 (m, 2H, Ar-H), 2.38 (s, 3H,
CH3). 13C NMR (101 MHz, DMSO) δ: 162.27, 157.13, 149.39, 148.93, 148.60, 138.08, 132.15,
131.45, 130.48, 129.05, 128.55, 128.18, 125.87, 124.97, 120.37, 20.89. IR (KBr) v/cm-1: 3086,
2938, 1612, 1529, 1343, 1273. Elemental Anal. Calcd for C16H12N6O4S: C, 50.00; H, 3.15; N,
21.86. Found: C, 50.03; H, 3.16; N, 21.89.
4-(2,4-dinitrobenzylideneamino)-5-(2-methoxyphenyl)-2H-1,2,4-triazole-3(4H)-thione (F2) :
Orange solid, yield = 75.3 %, m.p. 229.4-230.5 °C. 1H NMR (400 MHz, DMSO-d6) δ : 14.34 (s,
1H, N-H), 10.82 (s, 1H, azomethine), 8.82 (s, 1H,Ar-H), 8.62 (s, 1H, Ar-H), 8.00 (s, 1H, Ar-H),
7.59 (s, 1H,Ar-H), 7.52 (s, 1H, Ar-H), 7.19 (s, 1H, Ar-H), 7.10 (s, 1H, Ar-H), 3.73 (s, 3H, OCH3).
13C NMR (101 MHz, DMSO) δ: 161.90, 157.64, 155.15, 148.75, 148.71, 148.41, 132.95, 132.61,
131.34, 130.30, 128.31, 120.45, 120.33, 114.08, 111.71, 55.66. IR (KBr) v/cm-1: 3092, 2906, 1593,
1516, 1343, 1266. Elemental Anal. Calcd for C16H12N6O5S: C, 48.00; H, 3.02; N, 20.99. Found: C,
48.02; H, 3.03; N, 20.11.
4-(2,4-dinitrobenzylideneamino)-5-(3-methoxyphenyl)-2H-1,2,4-triazole-3(4H)-thione (F3) :
light yellow solid, yield = 81.1 %, m.p. 193.5-194.3 °C. 1H NMR (400 MHz, DMSO-d6) δ : 14.43
(s, 1H, N-H), 10.78 (s, 1H, azomethine), 8.87 (d, J = 2.3 Hz, 1H, Ar-H), 8.67 (dd, J = 8.4, 2.2 Hz,
1H, Ar-H), 8.32 (d, J = 8.6 Hz, 1H, Ar-H), 7.54-7.28 (m, 1H, Ar-H), 7.14 (m, J = 8.0, 2.4, 1.2 Hz,
3H, Ar-H), 3.77 (s, 3H, OCH3). 13C NMR (101 MHz, DMSO) δ: 162.34, 159.12, 157.47, 149.08,
149.00, 148.66, 132.12, 130.40, 129.91, 128.23, 126.22, 120.95, 120.41, 116.75, 113.86, 55.31. IR
(KBr) v/cm-1: 3111, 2977, 1610, 1516, 1336, 1278. Elemental Anal. Calcd for C16H12N6O5S: C,
48.00; H, 3.02; N, 20.99. Found: C, 48.03; H, 3.02; N, 20.12.
4-(2,4-dinitrobenzylideneamino)-5-(4-methoxyphenyl)-2H-1,2,4-triazole-3(4H)-thione (F4) :
orange solid, yield= 78.5 %; m.p. 227.3-228.1 °C. 1H NMR (400 MHz, DMSO-d6) δ: 14.29 (d, J =
10.2 Hz, 1H, N-H), 10.95–10.55 (m, 1H, azomethine), 9.00–8.73 (m, 1H, Ar-H), 8.65 (d, J = 8.8 Hz,
1H, Ar-H), 8.40–8.11 (m, 1H, Ar-H), 7.79 (d, J = 10.3 Hz, 2H, Ar-H), 7.07 (d, J = 9.0 Hz, 2H, Ar-H),
4.00–3.57 (m, 3H, OCH3). 13C NMR (101 MHz, DMSO-d6) δ: 162.03, 161.09, 156.74, 149.24, 148.88,
148.60, 132.26, 130.48, 130.28, 128.20, 120.35, 117.25, 114.14, 55.35. IR (KBr) v/cm-1: 3105, 2929,
1606, 1508, 1343, 1253. Elemental Anal. Calcd for C16H12N6O5S: C, 48.00; H, 3.02; N, 20.99.
Found: C, 48.12; H, 3.21; N, 21.02.
2.3. Crystal structure determination
The suitable crystal of each compound was measured on a Bruker SMART APEX CCD X-ray

ACCEPTED MANUSCRIPT
diffractometer using graphite-monochromated Mo-Kα radiation (λ = 0.71073 Å) at 296.15 K. The
crystal was kept at 296.15 K during data collection. The crystal structures were solved by using
the direct methods with Olex2 program [32, 33]. The non-hydrogen atoms were refined by the
full-matrix-block leasts quares method on F2 with anisotropic thermal parameters by SHELXL
program [34]. All hydrogen atoms were located theoretically and refined with riding model
position parameters and fixed isotropic thermal parameters. Relevant crystal data and the
refinement detail are listed in Table 1.
Table 1. Crystal data, data collection and structure refinement for F1, F2, F3 and F4.
Identification code
F1
C16H12N6O4S
384.38
F2
C16H12N6O5S
400.38
F3
C33H26Cl2N12O10S2
885.68
F4
C16H12N6O5S
400.38
Empirical formula
Formula weight
Temperature/K
296.15
296.15
296.15
296.15
Crystal system
monoclinic
P21/n
monoclinic
P21/n
monoclinic
P21/c
triclinic
Space group
P-1
a/Å
12.524(4)
6.648(2)
20.270(7)
90
10.9666(18)
7.8499(13)
20.610(3)
90
10.984(3)
10.708(3)
18.998(4)
90
7.1221(17)
10.702(3)
12.558(3)
66.662(4)
79.922(4)
74.539(4)
844.4(3)
b/Å
c/Å
α/°
β/°
91.869(6)
90
95.986(3)
90
120.229(11)
90
γ/°
Volume/Å3
1686.9(10)
4
1764.6(5)
4
1930.6(9)
2
Z
2
ρcalcg/cm3
ꢁ/mm-1
1.513
1.507
1.524
1.575
0.230
0.227
0.350
0.238
F(000)
792.0
824.0
908.0
412.0
Crystal size/mm3
Radiation
0.34 × 0.29 × 0.15
MoKα (λ = 0.71073)
3.768 to 51.494
0.3 × 0.3 × 0.25
MoKα (λ = 0.71073)
3.974 to 52.546
0.30 × 0.25 × 0.20
MoKα (λ = 0.71073)
4.542 to 50.052
0.21 × 0.2 × 0.19
MoKα (λ = 0.71073)
4.418 to 52.358
2θ range for data collection/°
-12 ꢁ h ꢁ 13, -9
ꢁ k ꢁ 9, -18 ꢁ l ꢁ
25
-15 ꢁ h ꢁ 14, -4 ꢁ
k ꢁ 8, -23 ꢁ l ꢁ 24
-13 ≤ h ≤ 11, -12 ≤ k ≤
12, -10 ≤ l ≤ 22
-6 ≤ h ≤ 8, -11 ≤ k ≤
13, -14 ≤ l ≤ 15
Index ranges
Reflections collected
8549
9270
9495
4572
3213 [Rint = 0.0739,
Rsigma = 0.0925]
3540 [Rint = 0.0364,
Rsigma = 0.0416]
3415 [Rint = 0.0896,
Rsigma = 0.1195]
3267 [Rint = 0.0225,
Rsigma = 0.0597]
Independent reflections
Data/restraints/parameters
Goodness-of-fit on F2
3213/49/264
0.967
3540/36/277
1.027
3415/1/281
0.925
3267/1/257
0.983
R1 = 0.0556, wR2 =
0.1086
R1 = 0.0470, wR2 =
0.1102
R1 = 0.0517, wR2 =
0.0942
R1 = 0.0549, wR2 =
0.1368
Final R indexes [I>=2σ (I)]
R1 = 0.1301, wR2 =
0.1355
R1 = 0.0720, wR2 =
0.1226
R1 = 0.1588, wR2 =
0.1294
R1 = 0.0927, wR2 =
0.1595
Final R indexes [all data]
Largest diff. peak/hole / e Å-3
0.20/-0.19
0.23/-0.24
0.20/-0.22
0.28/-0.19
2.4. Biological activities

ACCEPTED MANUSCRIPT
The in vitro antifungal activity of compounds (F1, F2, F3 and F4) against Wheat gibberellic (I),
Physalospora piricola (II), Potato dry rot fungus (III), Glomerella cingulata (IV), and Maize
rough dwarf bacteria (V) were tested according to the references [35-36], and the bioactivity were
evaluated using the mycelium growth rate test. The test compounds and SD were dissolved in
DMF to form four concentrations of 2, 4, 8 and 16 mg/L, respectively. All the tests were carried
out in triplicates. The antifungal activity of the compounds was measured by the percentage of
inhibition calculated according to the diameter of inhibition zone. The formula for calculating
the inhibition rate is as follows [37]:
diameter of control - diameter of test compounds
ʚ ʛ
Inhibition rate % =
×100%
diameter of control
The technique of antifungal activities and data processing (Toxicity regression equation, EC50,
and Correlation coefficent) were carried out according to the method described in literature [38].
2.5. Electrostatic Potential
Electrostatic potential (ESP) is widely used to predict the nucleophilic and electrophilic active
sites of compounds, based on the fact that the positive region of the electrostatic potential on one
molecular surface tends to interact with the negative region of another molecule, and the more
positive the positive, the more negative the negative value, the stronger the tendency. [39].
The complete geometry optimization and vibrational frequencies were calculated at
B3LYP/6-31G on the basis of the optimized structure. The MEP analyses was finished by
Multiwfn 3.4.1 [25], which is a multifunctional wave function analysis program. And the MEP
map was rendered by VMD 1.9.1 program [40] based on the outputs of Multiwfn 3.4.1.
3. Results and discussion
3.1. Spectroscopic Analysis
The title compounds are first reported, and the structure were confirmed on the basis of
elemental analysis and spectral data. All spectral and analytical data were consistent with the
assigned structures. The FT-IR spectra of four compounds all exhibit one N-H stretching band at
around 3000-3100 cm-1 (3086 cm-1 for F1, 3092 cm-1 for F2, 3111 cm-1 for F3, 3105 cm-1 for F4),
two anti-symmetric and symmetric NO2 stretching bands at around 1500 and 1250 cm-1 (1529 and
1273 cm-1 for F1, 1516 and 1266 cm-1 for F2, 1516 and 1278 cm-1 for F3, 1508 and 1253 cm-1 for
F4 ), and one C=N stretching band at around 1600 cm-1 (1612 cm-1 for F1, 1593 cm-1 for F2, 1610
cm-1 for F3, 1606 cm-1 for F4). Moreover, there are an obvious N-N stretching vibration (around
1200-1300 cm-1) and a broad CH3 or OCH3 stretching vibration (around 2900 cm-1) in the IR
spectrum of four compounds. The FT-IR and NMR spectra are attached in Fig. S1-12.
3.2. Crystal structure analysis
The structures of compound F1, F2, F3 and F4 were further confirmed by single crystal X-ray
diffraction analysis (Fig.2-5). The most significant of bond lengths, bond angles and torsion angles
are presented in Table 2 and others are attached in Table S1-12.

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Fig. 2. X-ray structure of compound F1.
Fig. 3. X-ray structure of compound F2.
Fig. 4. X-ray structure of compound F3.
Fig. 5. X-ray structure of compound F4.
For F1, F2, F3 and F4, the torsional angle (C(11)-C(10)-C(9)-N(6)) between the substituted
benzene ring and the triazole ring are 41.7(5)°, 45.2(3)°, 136.5(5)° and -152.7(3)°, respectively,
which indicates the two rings are not coplanar in the molecular structure. The values of torsional
angle are 177.7(3)°, 176.8(2)°, -176.6(3)° and 176.6(3)° for N(4)-N(3)-C(7)-C(3) fragment of F1,
F2, F3 and F4 which indicates the molecules have good planarity and there is a common
phenomenon. that the C=N double bond of each compound is in the (E)-configuration structure
[41].The bond lengths of N(3)-C(7) are 1.253(4) Å, 1.258(2) Å, 1.271(5) Å and 1.250(4) Å,
indicating that there is a decrease in pi electron delocalization, which is also consistent with the
reported amino Schiff bases [42-43]. Meanwhile, the azomethine bond length of F3 (1.271(5) Å) is
longer than others, which may be mainly due to the decease of density of electron cloud by the
strong electron-withdrawing (methoxy group) at m-positon.
Table 2. Selected Bond Lengths (Å) and Bond Angles (
°
)
of the compounds
Compounds
F1
F2
F3
F4
Bond Length
S(1)-C(8)
N(3)-C(7)
N(4)-N(3)
O(5)-C(16)
1.668(3)
1.253(4)
1.387(3)
—
1.674(2)
1.258(2)
1.399(2)
1.427(3)
1.671(4)
1.271(5)
1.381(4)
1.435(4)
1.673(3)
1.250(4)
1.391(3)
1.421(4)
Bond Angle
N(4)-C(9)-C(10)
126.0(3)
127.9(2)
124.4(4)
127.8(2)

ACCEPTED MANUSCRIPT
C(7)-N(3)-N(4)
Torsional Angle
120.1(3)
116.2(2)
117.4(3)
120.8(3)
C(11)-C(10)-C(9)-N(6)
N(4)-N(3)-C(7)-C(2)
C(9)-N(4)-N(3)-C(7)
41.7(5)
177.7(3)
-173.0(3)
45.2(3)
176.8(2)
-155.9(2)
136.5(5)
-176.6(3)
154.4(4)
-152.7(3)
176.6(3)
-175.6(3)
Moreover, the existence of two intra-molecular hydrogen bonds of C(7)-H(7)…S(1) and
C(7)-H(7)…O(4) in three compounds could stabilize the molecular structures by forming
six-membered ring. Besides, unlike the compound F2 and F3, there is an extra C(3)-H(3)…N(3)
hydrogen bond in compound F1 (F4) which also form a five-membered ring, meanwhile the
compound F4 have another C(11)-H(11)…N(3) hydrogen bond form a six-membered ring.
Therefore, the compound F1 and F4 more stable than the others (seen in Fig. 2-5 and Table 3).
Table 3. Hydrogen Bond interactions in F1, F2, F3 and F4.
d(D–H)/ Å
D–H
F1
A
d(H A)/ Å
d(D A)/ Å
DHA/°
N(5)-H(5) S(1)#1
C(16)-H(16C) O(2)#2
C(3)-H(3) O(1)#3
C(6)-H(6) O(3A)#4
C(3)-H(3)…N(3)
C(7)-H(7)…S(1)
C(7)-H(7)…O(4A)
F2
0.86
0.96
0.93
0.93
0.93
0.93
0.93
2.47
2.48
2.46
2.51
2.41
2.47
2.23
3.318(3)
3.411(7)
3.106(4)
3.430(7)
2.731(4)
3.200(4)
2.798(6)
171.0
163.0
127.0
170.0
100.0
135.0
119.0
N(5)-H(5) S(1)#5
C(3)-H(3) O(1)#6
C(7)-H(7) S(1)
C(7)-H(7)…O(4)
C(13)-H(13) …O4(A) #7
C(14)-H(14) …O4(A) #7
F3
0.83
0.93
0.93
0.93
0.93
0.93
2.49
2.48
2.54
2.18
2.34
2.54
3.317(2)
3.197(4)
3.193(3)
2.719(4)
2.986(7)
3.077(7)
175.3
134.0
127.0
116.0
126.0
117.0
N(5)-H(5) S(1)#8
C(3)-H(3) O(1)#9
C(6)-H(6) Cl(2)#10
C(17)-H(17B) O(2)#11
C(7)-H(7)…S(1)
C(7)-H(7)…O(4)
F4
0.86
0.93
0.93
0.97
0.93
0.93
2.42
2.57
2.81
2.56
2.61
2.32
3.275(5)
3.350(7)
3.721(17)
3.287(11)
3.198(4)
2.789(5)
171.0
141.0
167.0
132.0
122.0
111.0
N(5)-H(5) S(1)#12
C(4)-H(4) O(1)#13
C(16)-H(16A)…O(2)#13
C(14)-H(14)…O(4)#14
C(16)-H(16B)…O(4)#15
C(6)-H(6) O(3)#5
C(3)-H(3) N(3)
C(7)-H(7)…O(4)
0.87
0.93
0.96
0.93
0.96
0.93
0.93
0.93
2.49
2.44
2.57
2.58
2.56
2.52
2.43
2.20
3.351(3)
3.208(4)
3.505(5)
3.469(4)
3.249(5)
3.445(4)
2.745(5)
2.779(4)
173.0
141.0
164.0
159.0
129.0
176.0
100.0
119.0

ACCEPTED MANUSCRIPT
C(7)-H(7)…S(1)
0.93
0.93
2.43
2.49
3.179(4)
3.016(4)
137.0
116.0
C(11)-H(11)…N(3)
Symmetry transformations: #1:1-x, 2-y, -z; #2: 1.5-x, 1.5+y, 0.5-z; #3:1.5-x,0.5+y,0.5-z; #4:0.5-x,-0.5+y,0.5-z; #5:1-x, 1-y, -z; #6: 0.5-x,
-0/5+y, 0.5-z; #7:-1+x, y, z; #8:1-x, -y, -z; #9:-x, -0.5+y, 0.5-z; #10: -x, 2-y, -z; #11: -x, 2-y, -z; #12: 3-x, -y, 1-z; #13: -x, 1-y, 1-z; #14: x, y,
1+z; #15: -1+x, y, 1+z.
In addition, the 2D network of all compounds showing the interactions of intermolecular
hydrogen-bonds is exhibited in Fig. 6. The unit cell diagram of F1, F2 and F4 showed that the
hydrogen bond N(5)-H(5) S(1) links two dimers and the distance between the donor and acceptor
atoms were 3.318(3) Å, 3.318(3) Å and 3.351(3) Å (Table 3), meanwhile the compound F3 also
showed that the hydrogen bond N(5)-H(5) S(1) with the distance (3.275(5) Å). However, because
of the special position of dichloromethane, it have a certain interaction force with the molecules of
the compounds, as indicated by Fig. 6(c).
(a)
(b)
(c)

ACCEPTED MANUSCRIPT
(d)
Fig. 6. The 2D network of F1 (a), F2 (b), F3 (c) and F4 (d) showing the intermolecular hydrogen-bond interactions (the dashed lines)
3.3. Biological activities
The in vitro antifungal screening of compounds in inhibiting the mycelial growth of five test
fungi at different concentrations (2, 4, 8, 16 mg/L) were tested. The data are the mean of three
replicate tests performed with each antifungal compound. The inhibitory rates of the title
compounds at 16 mg/L are listed in Table 4, and the other concentrations results are presented in
Table. S13.
Table 4. Biological activities of the title compounds.
Antifungal activity, inhibition rate ( %, 16 mg/L)
Compd.
I
II
III
IV
V
F1
48.09
47.19
57.37
42.64
53.21
40.46
33.13
38.61
34.20
61.05
40.84
36.75
33.71
54.93
60.95
51.34
53.09
53.85
49.79
57.71
52.87
56.13
54.01
44.66
55.04
F2
F3
F4
Fluconazole
As indicated in Table 4, the compounds exhibited different in vitro antifungal activities against
five test fungi at 16 mg/L. Among the fungi tested, the compounds against Glomerella cingulata
(IV), and Maize rough dwarf bacteria (V) showed the more significant antifungal activity than
that of others. Especially, the inhibition rate of F2 (56.13 %) against Maize rough dwarf bacteria
(V) is more favourable than that of commercial controls Fluconazole (55.04 %) (Fig. 7 1a). For
Wheat gibberellic (I), the inhibition activity of compounds F3 (57.37%) is better than that of
Fluconazole (53.21 %) (Fig. 7 1b). Compounds F1, F2, F3 and F4 possessed the higher inhibition
activity with 51.34 %, 53.09 %, 53.85 % and 49.79 % against Glomerella cingulata (IV).
Unfortunately, the compounds have the weaker effect against Physalospora piricola (II) and
Potato dry rot fungus (III).
Fig. 7. The effect of compounds F2 to Maize rough dwarf bacteria (left: a) and F3 to Wheat gibberellic (right: b) at 2, 4, 8, 16 mg/L.
(Fluconazole as a reference (1a, 1b))
Given all that, the further structural optimizations could be made for the discovery and

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development of novel fungicides in the future. Therefore, the toxicity regression equation, the
C
orrelation coefficient (R2) and the value of EC50 for all the compounds against Wheat gibberellic
(I), Physalospora piricola (II), Pythium solani (III), Glomerella cingulata (IV), and Maize rough
dwarf bacteria (V) were listed in Table 5.
Table 5. The toxicity regression equation, the R2 and EC50 value of F1, F2, F3 and F4.
Fungi
Compd.
Toxicity regression equation
y = 1.6886x + 2.8781
y = 1.0073x + 3.6766
y = 0.9381x + 4.0171
y = 0.8926x + 3.5655
y = 0.9438x + 3.8628
y = 1.1566x + 3.3315
y = 1.0754x + 3.2311
y = 1.0987x + 3.3658
y = 1.0225x + 3.5386
y = 1.0792x + 4.0532
y = 1.1617x + 3.3470
y = 1.3746x + 3.0731
y = 1.0987x + 3.0928
y = 0.8020x + 4.1517
y = 1.4155x + 3.6690
y = 1.4316x + 3.2346
y = 1.5719x + 3.0891
y = 1.7216x + 2.9614
y = 1.2688x + 3.4383
y = 1.2172x + 3.8120
y = 1.2685x + 3.4382
y = 1.0539x + 3.7763
y = 1.5142x + 3.1396
y = 1.1404x + 3.5028
y = 1.3861x + 3.4970
Correlation coefficent, R2
0.9938
EC50/(mg·L‒1)
18.05
20.60
11.16
40.46
16.03
27.71
44.21
30.72
26.87
7.54
F1
F2
0.9720
I
F3
0.9737
F4
0.9726
Fluconazole
0.9980
F1
0.9924
F2
0.9815
II
III
IV
V
F3
0.9954
F4
0.9806
Fluconazole
0.9614
F1
0.9917
26.48
25.14
54.43
11.42
8.71
F2
0.9827
F3
0.9779
F4
0.9995
Fluconazole
0.9904
F1
0.9801
17.11
16.43
15.28
17.02
9.46
F2
0.9729
F3
0.9784
F4
0.9917
Fluconazole
0.9824
F1
0.9780
17.03
14.49
16.93
20.55
12.14
F2
0.9866
F3
F4
0.9824
0.9938
Fluconazole
0.9929
*
Symbols: EC50 values (mg/L): 50 % effective concentration.
We can see that the compounds F3 exhibits the excellent antifungal activity against Wheat
gibberellic (I) with EC50 value of 11.16 mg/L (Fluconazole, EC50 16.03 mg/L). Instead, the
compounds against Physalospora piricola (II) and Pythium solani (III) possessed higher EC50
values than that of other fungi (Table 5), which has the consistency with the results in Table 1.
Particularly, the bar chart of EC50 value against Wheat gibberellic (I), Glomerella cingulata (IV)
and Maize rough dwarf bacteria (V) was shown in Fig. 8.
From the Fig. 8, we could clearly see that only compound F3 for Wheat gibberellic (I) shows
an EC50 value lower than Fluconazole, whereas compound F1, F2 and F4 possessed higher value
than Fluconazole, especially the compound F4. For Glomerella cingulata (IV): The order of EC50
values of compounds was F1 > F4 > F2 > F3 > Fluconazole, and weaker than other fungi. And for
Maize rough dwarf bacteria (V): the compounds F2 was better than that of others, but still lower
than that of Fluconazole.

ACCEPTED MANUSCRIPT
In general, only the 3-OCH3 (F3) substituent compound showed broad spectrum antifungal
activity, which has the potential to be further researched as antifungal agent.
F1
F2
F3
F4
40
35
30
25
20
15
10
5
Fluconazole
0
I
VI
V
Fig. 8. The bar chart of EC50 value against three fungi.
3.4. Molecular docking
To rationalize the potency of the active compounds as antifungal agents, the title compounds
and Fluconazole were docked against cytochrome P450 14a-desmethylase (CYP51) from
Fusarium graminearum (PDB ID: 6CR2) [44], which was get from protein data bank (RCSB)
using AutoDock 4.2 software [45-46]. The results obtained (the .dlg file results) were viewed
through the Discovery Studio 4.5 Visualizer [47-48] and summarized in Table 6 and Fig. 9-13.
Table 6. Docking results of F1, F2, F3 and F4 against 6CR2.
Compound
Binding Energy
(kcal/mol)
-8.33
Internal Energy
(kcal/mol)
-2.91
Torsional Energy
(kcal/mol)
1.79
Unbound Energy
(kcal/mol)
-2.91
Intermolecular Energy
Ki(ꢁM)a
ID(s)
(kcal/mol)
-10.12
-9.97
F1
3.08
3.01
F2
-8.18
-3.02
1.79
-3.02
F3
F4
-8.97
3.26
-10.46
-9.47
-2.98
1.49
-2.98
-7.98
3.05
-3.04
1.49
-3.04
Fluconazole
-5.06
195.31
-6.85
-4.47
1.79
-4.47
a): Estimated inhibition constant, Ki (ꢁM).
The docking results were listed in Table 6, including binding energy, inhibition constant (Ki),
and other energy. The Fig. 9-13 showed amino acid residues that have H-bond interactions with
the ligands and hydrophobic interactions for each compounds and Fluconazole. As can be seen
from the results, the hydrogen bonds interactions were formed among all compounds and amino
acid residues, and showed the hydrogen bonding and hydrophobic interactions of the ligand with
the active site of residues. The 3D diagrams (Figs. 9a-13a) showed the binding sites of the ligands
within the receptor 6CR2. The molecules interacted with the active sites (amino acids) of receptor
6CR2 through van der Waals, conventional hydrogen bonds, carbon-hydrogen bond, Pi-Alkyl,
Pi-Donor hydrogen bond, Pi-Sulphur, Pi-Sigma and much more (Figs. 9b-13b). In addition, there
were residues PRO A: 455, TYR A: 136, PHE A: 456, CYS A: 463, ILE A: 376, ILE A: 373, HIS
A: 461, ALA A: 307 and ALA A: 458 around the active pocket. Among the four molecules, the

ACCEPTED MANUSCRIPT
docking binding energy displayed best values in compound F3 (-8.97 kcal/mol) undering the
Fluconazole (-5.06 kcal/mol), which may be attributed to the existence of 3-phenoxyphenyl group,
and the results correspond to the biological activities.
In Fig. 9b, the NO2 and NH groups of compound F1 formed hydrogen bond with residues PRO
A: 455 and TYR A: 136. Also, the Pi-alkyl and Pi-Sulfur type of interaction observed between
aromatic rings of F1 and ILE A: 376, CYS A: 463. There is a hydrogen bonding interaction
between NO2 group of compound F2 and TYR A: 136. Van der Waals, carbon-hydrogen bond and
Pi-Donor hydrogen bond type of interactions were indicated with different amino acids of chain A
with F2 (Fig. 10b). Moreover, the hydrogen bonds were observed between the NO2 and NH groups
of F3 and CYS A: 463, PRO A: 455. The aromatic rings interacted with receptor by forming
Pi-Sigma and Amide-Pi Stacked with ILE A: 376, ILE A: 373, HIS A: 461, PHE A: 456 (Fig. 11b).
And then, the N=CH group of compound F4 formed hydrogen bond with residues CYS A: 436
(Fig. 12b). In addition, the Cl-atom in Fluconazole interacted through conventional H-bond with
ARG A: 378 and CYS A: 463 (Fig. 13b).
The four compounds all bind well to the receptor protein (6CR2) in different type of bond, And
above results indicated the selectivity of the potent ligand molecules as CYP51 inhibitors.
(a)
(b)
Fig. 9. (a) 3D model of ligand F1 bonded with active site of 6CR2 receptor. (b) 2D model of interaction between amino acid residues and
F1 with types of bond.
(a)
(b)
Fig. 10. (a) 3D model of ligand F2 bonded with active site of 6CR2 receptor. (b) 2D model of interaction between amino acid residues and
F2 with types of bond.

ACCEPTED MANUSCRIPT
(a)
(b)
Fig. 11. (a) 3D model of ligand F3 bonded with active site of 6CR2 receptor. (b) 2D model of interaction between amino acid residues and
F3 with types of bond.
Fig. 12. (a) 3D model of ligand F4 bonded with active site of 6CR2 receptor. (b) 2D model of interaction between amino acid residues and
Fluconazole with types of bond.
(a)
(b)
Fig. 13. (a) 3D model of ligand Fluconazole bonded with active site of 6CR2 receptor. (b) 2D model of interaction between amino acid
residues and Fluconazole with types of bond.
3.5. Electrostatic potential analyses
Electrostatic potential (ESP) mapped along with surface extrema and the graph of surface area
plotted against different ESP ranges of the title compounds are shown in Fig. 14 (left). Red (blue)
area represent surface local minima (maxima) of ESP, and the orange (cyan) beads on the

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molecular surface are also represent maximum (minimum) values, respectively.
In the Fig. 14 (left), the largest negative value are found in the oxygen atom of the nitro-group
(-91.38, -87.34, -81.99 and -30.01 kcal/mol), which corresponds to the global minimum on the
surface of the compounds, which is attributed to the lone pair of oxygen atoms. The global
maximum appears on the surface of the phenyl ring, the ESP at those point is much larger (80.89,
87.31, 95.93 and 32.36 kcal/mol) than that others. Particularly, there are two maximum points
(134.48, 130.57 kcal/mol) at the triazole ring on the ESP surface of F1. From the Fig. 14 (right), it
can be seen that there is a large portion of the molecular surface with small ESP value, namely
from −20 to 20ꢀkcal/mol. There are abundant clouds below the phenyl ring, corresponding to the
positive part of each compound; the negative part is mainly from S and H atom of the triazole ring;
the near-neutral part represents the boundary region between the negative and positive parts.
In conclusion, the ESP-mapped of F1, F2, F3, and F4 indicated the active sites of the reaction.
Because of the different substituents, their effects on the reaction activity are different. Especially,
For compound F4 (Fig. 14), we can see that the area of the electrostatic potential is concentrated in
the range of -20 to 20 kcal/mol, indicating that compound F4 has higher stability than others, so its
corresponding biological activity is relatively weaker, which is consistent with the results of
biological experiments and molecular docking.
F1
F2
F3

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F4
Fig. 14. ESP maps and the surface areas for each ESP range for compounds
4. Conclusions
In summary, four compounds of 4-Amino-5-Substituent-1,2,4-Triazole-3-Thione Schiff bases
have been prepared, and their structures were confirmed by melting point, elemental analyses, IR,
1H NMR, 13C NMR and XRD. The biological activity of compounds were evaluated including
antifungal experiments and molecular docking simulations. The antifungal activities against
Wheat gibberellic (I), Physalospora piricola (II), Pythium solani (III), Glomerella cingulata (IV),
and Maize rough dwarf bacteria (V) were evaluated. The results of antifungal activities showed
that the compounds have a different spectrum of biological activity to fungi. And compounds F3
held excellent antifungal activity against Wheat gibberellic (I) with EC50 value of 11.16 mg/L. All
bioassays showed that the 3-OCH3 substituent have a broad spectrum of antifungal properties than
others. And the docking showed that the synthesized compounds has the lower binding energy
against the enzyme cytochrome P450 14a-desmethylase (CYP51) than Fluconazole. Meanwhile,
in order to further understand the nucleophilic and electrophilic active sites of compounds, the
electron distribution and reactive sites on the surface of the title compounds were analyzed by ESP,
which also made the four compounds more persuasive as antifungal agents.
Acknowledgements
This work is funded by the Provincial Key-point Natural Science Foundation of Shaanxi, China
(No.2016JZ003) and the Innovation and Creativity Funds for Graduates in Northwest University,
China (No.YZZ17137).
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ACCEPTED MANUSCRIPT
Highlights
1. High purity of the product and get theirs crystal structure.
2. Laboratory biological activity experiment of mycelial growth rate method
3. Molecular docking used to analyze the mechanism of biological activity
4. Electrostatic potential (ESP) mapped analysis by Wave function