Synthesis, antifungal activity, and 3d-qsar study of novel nopol-derived 1,3,4-thiadiazole-thiourea compounds

Article abstract of DOI:10.3390/molecules26061708

A series of novel nopol derivatives bearing the 1,3,4-thiadiazole-thiourea moiety were designed and synthesized by multi-step reactions in search of potent natural product-based antifungal agents. Their structures were confirmed by FT-IR, NMR, ESI-MS, and elemental analysis. Antifungal activity of the target compounds was preliminarily evaluated by in vitro methods against Fusarium oxysporum f. sp. cucumerinum, Cercospora arachidicola, Physalospora piricola, Alternaria solani, Gibberella zeae, Rhizoeotnia solani, Bipolaris maydis, and Colleterichum orbicalare at 50 μg/mL. All the target compounds exhibited better antifungal activity against P. piricola, C. arachidicola, and A. solani. Compound 6j (R = m, p-Cl Ph) showed the best broad-spectrum antifungal activity against all the tested fungi. Compounds 6c (R = m-Me Ph), 6q (R = i-Pr), and 6i (R = p-Cl Ph) had inhibition rates of 86.1%, 86.1%, and 80.2%, respectively, against P. piricola, much better than that of the positive control chlorothalonil. Moreover, compounds 6h (R = m-Cl Ph) and 6n (R = o-CF₃ Ph) held inhibition rates of 80.6% and 79.0% against C. arachidicola and G. zeae, respectively, much better than that of the commercial fungicide chlorothalonil. In order to design more effective antifungal compounds against A. solani, analysis of the three-dimensional quantitative structure–activity relationship (3D-QSAR) was carried out using the CoMFA method, and a reasonable and effective 3D-QSAR model (r² = 0.992, q² = 0.753) has been established. Furthermore, some intriguing structure–activity relationships were found and are discussed by theoretical calculation.

Full text of DOI:10.3390/molecules26061708

molecules
Article
Synthesis, Antifungal Activity, and 3D-QSAR Study of Novel
Nopol-Derived 1,3,4-Thiadiazole-Thiourea Compounds
Ming Chen, Wen-Gui Duan *, Gui-Shan Lin *, Zhong-Tian Fan and Xiu Wang
School of Chemistry and Chemical Engineering, Guangxi University, Nanning 530004, China;
chenminggxu@163.com (M.C.); fztt187@163.com (Z.-T.F.); wangxiumt2015@163.com (X.W.)
* Correspondence: wgduan@gxu.edu.cn (W.-G.D.); gslin@gxu.edu.cn (G.-S.L.);
Tel./Fax: +86-771-323-3718 (W.-G.D.)
Abstract: A series of novel nopol derivatives bearing the 1,3,4-thiadiazole-thiourea moiety were
designed and synthesized by multi-step reactions in search of potent natural product-based antifungal
agents. Their structures were confirmed by FT-IR, NMR, ESI-MS, and elemental analysis. Antifungal
activity of the target compounds was preliminarily evaluated by in vitro methods against Fusarium
oxysporum f. sp. cucumerinum, Cercospora arachidicola, Physalospora piricola, Alternaria solani, Gibberella
zeae, Rhizoeotnia solani, Bipolaris maydis, and Colleterichum orbicalare at 50 µg/mL. All the target
compounds exhibited better antifungal activity against P. piricola, C. arachidicola, and A. solani.
Compound 6j (R = m, p-Cl Ph) showed the best broad-spectrum antifungal activity against all
the tested fungi. Compounds 6c (R = m-Me Ph), 6q (R = i-Pr), and 6i (R = p-Cl Ph) had inhibition
rates of 86.1%, 86.1%, and 80.2%, respectively, against P. piricola, much better than that of the positive
control chlorothalonil. Moreover, compounds 6h (R = m-Cl Ph) and 6n (R = o-CF3 Ph) held inhibition
rates of 80.6% and 79.0% against C. arachidicola and G. zeae, respectively, much better than that of the
commercial fungicide chlorothalonil. In order to design more effective antifungal compounds against
A. solani, analysis of the three-dimensional quantitative structure–activity relationship (3D-QSAR)
was carried out using the CoMFA method, and a reasonable and effective 3D-QSAR model (r2 = 0.992,
q2 = 0.753) has been established. Furthermore, some intriguing structure–activity relationships were
found and are discussed by theoretical calculation.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Chen, M.; Duan, W.-G.; Lin,
G.-S.; Fan, Z.-T.; Wang, X. Synthesis,
Antifungal Activity, and 3D-QSAR
Study of Novel Nopol-Derived
1,3,4-Thiadiazole-Thiourea
Compounds. Molecules 2021, 26, 1708.
https://doi.org/10.3390/
molecules26061708
Keywords: β-pinene; nopol; 1,3,4-thiadiazole; thiourea; antifungal activity; 3D-QSAR
Academic Editors: David Díez and
María Ángeles Castro
1. Introduction
Received: 25 February 2021
Accepted: 17 March 2021
Published: 18 March 2021
Turpentine oil, which was obtained by the steam distillation of the oleoresin exudate
from living pine trees, is an extremely abundant natural biomass resource in nature, and is
widely used in the synthesis of fine chemicals such as food additives, pharmaceuticals,
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
agrochemicals, and flavors [
as high as about 30% in raw turpentine oils [
to convert β-pinene into high value-added fine chemicals.
Nopol, with the chemical name of 2-(6,6-dimethyl-2-bicyclo [3.1.1] hept-2-enyl) ethanol,
is an optically active bicyclic primary alcohol, and contains three reactive functional groups,
including a hydroxyl group, a carbon–carbon double bond, and a four-membered ring. It is
1
]. As one of the major components,
β-pinene content can be
2]. Therefore, it is an important research topic
usually prepared by a Prins condensation reaction of β-pinene with paraformaldehyde and
is generally used in the flavor and fragrance industries, in the agrochemical industry to
produce pesticides, as well as in the manufacture of soaps, detergents, polishes, and other
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
household products [
3
]. Nopol and its derivatives were found to exhibit a broad spec-
], antitumor [ ], and repellent [
] (see examples in
trum of bioactivities, such as insecticidal, antifungal [
4
5
6]
characteristics, as well as for treatment of irritable bowel syndrome [
Figure 1).
7
Molecules 2021, 26, 1708. https://doi.org/10.3390/molecules26061708
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 1708
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Figure 1. Bioactive molecules containing nopol, 1,3,4-thiadiazole and thiourea moieties.
On the other hand, 1,3,4-thiadiazole derivatives exhibit various biological properties,
such as insecticidal [ ], herbicidal [ ], antibacterial [10 12], antiviral [13], antifungal [14], an-
titumor [15 16] and plant growth regulation [17] activities (see examples in Figure 1). In ad-
dition, thiourea derivatives have considerable applications in agriculture, gold leaching
processes, analytical chemistry, and medicine [18 20]. Compounds with thiourea scaffolds
hold diverse pharmacological properties such as insecticidal [21], antifungal [22], herbi-
8
9
–
,
–
cidal [23], antibacterial [24], antitubercular [25], antithyroid [26], and antimicrobial [27
activities (see examples in Figure 1).
]
In continuation of our long-term focus on natural product-based bioactive com-
pounds [28 32], a series of novel nopol-derived 1,3,4-thiadiazole-thiourea compounds were
–
synthesized by integrating bioactive 1,3,4-thiadiazole and thiourea moieties into the molec-
ular skeleton of nopol. Structural characterization, antifungal evaluation, and structure–
activity relationships of the title compounds were determined as well.
2. Results and Discussion
2.1. Synthesis and Characterization
As illustrated in Scheme 1, nopol (
) with paraformaldehyde, and further oxidized to obtain nopol aldehyde (
nopol-based thiosemicarbazone ( ) was prepared by a condensation reaction of compound
with thiosemicarbazide, followed by a ring-closing reaction to produce 5-nopyl-2-amino-
1,3,4-thiadiazole ( ). Lastly, a series of target compounds, 6a 6r, were synthesized by the
2) was prepared by the Prins reaction of
β
-pinene
(
1
3). Then,
4
3
5
–
nucleophilic addition reaction of compound 5 with different isothiocyanates.
The structures of the target compounds were characterized by means of FT-IR, 1H-
NMR, 13C-NMR, ESI-MS, and elemental analysis. In the FT-IR spectra, the target com-
pounds exhibited moderate absorption bands at 3294–3330 cm−1, which were assigned
to the N–H stretching vibration. The weak absorption bands at 1632–1671 cm−1 were at-
tributed to the stretching vibrations of the unsaturated C=C in the nopol moiety. The strong
absorption bands at 1524–1586 cm−1 and the moderate absorption bands at 646–660 cm−1
were assigned to the vibrations of C=N and C–S–C in 1,3,4-thiadiazole moiety, respec-
tively. The three strong absorption bands around 1361–1440 cm−1, 1326–1390 cm−1
,
1
and 1077–1091 cm−1 confirmed the presence of thiourea groups. In the H-NMR spec-

Molecules 2021, 26, 1708
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tra, the olefinic protons of nopol scaffolds showed signals at about 5.48–5.54 ppm, and the
other protons bonded to the saturated carbons of the nopol moiety displayed signals in the
range of 0.75–3.74 ppm. Signals of the N–H protons were located at δ13.27–14.49 ppm and
7.54–10.93 ppm, respectively. The 13C-NMR spectra of the target compounds showed peaks
for the olefinic carbons of the nopol moiety at 120.4–121.2 ppm, and the other saturated
carbons displayed signals in the region of 21.1–45.3 ppm. For the 1,3,4-thiadiazole moiety,
the signals at 157.0–172.0 ppm and 143.7–161.9 ppm were assigned to the carbons of C=N.
The carbons of C=S in the thiourea moiety displayed the signals at 177.2–188.0 ppm. Their
molecular weights and the C, H, and N element ratios were confirmed by ESI-MS and
elemental analysis, respectively (see Supplementary Materials).
Scheme 1. Synthesis of nopol-derived 1,3,4-thiadiazole-thiourea compounds 6a
–
6r. Reagents and
) Dess–Martin periodinane, NaHCO3,
) I2, K2CO3, 1,4-dioxane, 80 ◦C, 6 h;
conditions: (a b
) (CH2O)n, ZnCl2, 75 ◦C, 1 h; 110 ◦C, 12 h; (
◦
0 C, 4 h; (
c
) thiosemicarbazide, H2O, EtOH, 65 ◦C, 15 min; (
d
◦
(e) RNCS, NaOH, 1,4-dioxane, 90 C, 8–12 h.
2.2. Antifungal Activity
The antifungal activity of the target compounds 6a–6r was evaluated by in vitro
method against fusarium wilt on cucumber (Fusarium oxysporum f. sp. cucumerinum),
speckle on peanut (Cercospora arachidicola), apple ring rot (Physalospora piricola), tomato
early blight (Alternaria solani), wheat scab (Gibberella zeae), rice sheath blight (Rhizoeotnia
solani), corn southern leaf blight (Bipolaris maydis), and watermelon anthracnose (Col-
leterichum orbicalare) at concentrations of 50
chlorothalonil as positive control. The results are listed in Table 1.
It was found that, at 50 g/mL, the target compounds showed certain antifungal
µg/mL, using the commercial antifungal agent
µ
activity against the eight tested fungi. On the whole, all the target compounds exhibited
better antifungal activity against P. piricola, C. arachidicola, and A. solani, with average
inhibition rates of 65.7%, 64.3%, and 64.2%, respectively. Compound 6j (R = m, p-Cl Ph)
showed the best and broad-spectrum antifungal activity against all the tested fungi, with
an average inhibition activity of 51.9%. Compounds 6c (R = m-Me Ph), 6q (R = i-Pr), and
6i (R = p-Cl Ph) had inhibition rates of 86.1%, 86.1%, and 80.2%, respectively, against
P. piricola, much better than the of the positive control chlorothalonil. Besides, compounds
6h (R = m-Cl Ph) and 6n (R = o-CF3 Ph) held inhibition rates of 80.6% and 79.0% against
C. arachidicola and G. zeae, respectively, much better than that of the positive control
chlorothalonil. In order to investigate the structure–activity relationship, 3D-QSAR studies
and theoretical calculations were subsequently performed.

Molecules 2021, 26, 1708
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Table 1. Antifungal activity of the target compounds 6a–6r at 50 µg/mL.
Inhibition Rate (%) against the Tested Fungi
Compounds
F. oxysporum f.
sp. cucumerinum
C. arachidicola
P. piricola
A. solani
G. zeae
R. solani
B. myadis
C. orbicalare
Average (I)
6a (R = Ph)
6b (R = o-Me Ph)
6c (R = m-Me Ph)
6d (R = p-Me Ph)
6e (R = m-OMe Ph)
6f (R = p-OMe Ph)
6g (R = p-F Ph)
6h (R = m-Cl Ph)
6i (R = p-Cl Ph)
6j (R = m, p-Cl Ph)
6k (R = o-Br Ph)
6l (R = m-Br Ph)
6m (R = p-Br Ph)
6n (R = o-CF3 Ph)
6o (R = m-NO2 Ph)
6p (R = p-NO2 Ph)
6q (R = i-Pr)
34.1
37.8
28.5
30.4
28.5
21.1
21.1
30.5
29.8
36.1
32.2
26.7
34.1
34.1
26.7
35.9
34.1
28.5
30.6
100
64.3
67.1
52.9
50.0
64.3
50.0
70.0
80.6
69.3
66.3
64.3
52.9
67.1
61.4
72.9
72.9
70.0
61.4
64.3
73.3
64.3
75.2
86.1
66.5
55.7
68.7
64.3
55.5
80.2
66.3
75.2
51.3
64.3
57.8
53.5
36.1
86.1
75.2
65.7
75.0
60.6
72.3
61.3
33.1
65.0
50.9
77.3
69.7
61.3
70.3
69.0
71.0
61.3
72.8
70.9
63.8
60.6
63.8
64.2
73.9
54.8
47.9
56.6
47.9
58.3
39.3
56.6
56.8
58.1
65.4
47.9
30.7
27.2
79.0
56.6
51.4
58.3
51.4
52.5
73.1
19.8
19.8
17.3
53.9
14.9
17.3
17.3
19.3
22.3
21.8
14.9
19.8
17.3
16.1
17.3
17.3
17.3
14.9
19.9
96.1
37.7
29.1
33.4
24.9
24.9
22.8
27.0
36.5
38.4
39.7
35.5
35.5
29.1
31.3
29.1
35.5
22.8
24.9
31.0
90.4
35.5
35.5
25.7
29.6
27.6
25.7
27.6
33.6
28.7
49.3
33.5
29.6
31.6
37.5
25.7
37.5
29.6
27.6
31.7
91.3
46.4
48.1
45.2
42.0
42.4
37.0
45.2
47.8
48.5
51.9
46.6
39.7
41.5
48.8
44.1
43.8
47.4
43.5
-
6r (R = n-Bu)
Average (II)
Chlorothalonil
-
2.3. CoMFA Analysis
The 3D-QSAR analysis of the antifungal activity against A. solani of the target com-
pounds was carried out by the CoMFA method. In this work, fifteen target compounds in
the training set and one compound in the test set are presented in Table 2. A predictive 3D-
QSAR model with the value of non-validated correlational coefficient (r2 = 0.992), the Fis-
cher ratio (F = 294.200), standard error of estimate (S = 0.021) and the cross-validated corre-
lational coefficient (q2 = 0.753) was established (Table 3). As shown in Figure 2, the Scatter
plot of predicted ED values vs experimental ED values is presented, and all data were
concentrated near the X = Y line, indicating that the 3D-QSAR model was reasonable
and effective.
Table 2. The ED values of experimental and predicted activities.
ED[a]
Compounds
MW
ED
Residue
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p *
370.13
384.14
384.14
384.14
400.14
400.14
388.12
404.09
404.09
438.05
448.04
448.04
448.04
438.12
415.11
415.11
−2.381
−2.168
−2.385
−2.890
−2.333
−2.587
−2.057
−2.245
−2.407
−2.267
−2.304
−2.262
−2.452
−2.214
−2.231
−2.372
−2.368
−2.197
−2.401
−2.907
−2.317
−2.578
−2.079
−2.246
−2.400
−2.282
−2.264
−2.260
−2.443
−2.225
−2.218
−2.360
−0.013
0.029
0.016
0.017
−0.016
−0.009
0.022
0.001
−0.007
0.015
−0.040
−0.002
−0.009
0.011
−0.013
−0.012
ED: experimental value; ED[a]: predictive value; *: test-set compound.
Table 3. Summary of CoMFA analysis.
Contribution (%)
q2
r2
S
F
Steric
Electrostatic
CoMFA
0.753
0.992
0.021
294.200
52.9
47.1
q2: cross-validated correlational coefficient; r2: non-validated correlational coefficient; S: standard error of estimate;
F: the Fischer ratio.

Molecules 2021, 26, 1708
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Figure 2. Predicted ED values of CoMFA model vs experimental ED value.
The electrostatic and steric contribution maps of CoMFA are shown in Figure 3.
The contribution rate of the electrostatic field was 47.1% while the steric field was 52.9%,
which revealed that the steric field was the major contributor to the increase in the an-
tifungal activity against A. Solani. In Figure 3a, there were some green regions located
around the 2-position and 3-position of the benzene ring; the green area indicated that the
introduction of large groups was beneficial to increasing the antifungal activity. For exam-
ple, compounds 6c (R = m-Me Ph), 6h (R = m-Cl Ph), 6l (R = m-Br Ph), 6n (R = o-CF3 Ph)
and 6o (R = m-NO2 Ph) displayed higher antifungal activity than compound 6a (R = Ph)
.
In Figure 3b, the electrostatic field contours were displayed in two distinguishing colors.
The blue area indicated that the introduction of electron-donating groups was benefi-
cial to improving activity, and the red area indicated that the introduction of electron-
withdrawing groups was beneficial to improving activity. Therefore, the introduction
of electron-donating groups at the 2-position and electron-withdrawing groups at the
4-position of benzene rings were both favorable for antifungal activity. For instance, com-
pound 6b (R = o-Me Ph) exhibited better antifungal activity than that of 6k (R = o-Br Ph)
,
compounds 6g (R = p-F Ph), and 6i (R = p-Cl Ph), and 6m (R = p-Br Ph) possessed a higher
inhibitory rate than 6d (R = p-Me Ph) and 6f (R = p-OMe Ph).
The established CoMFA model could be used to predict the activity of new candidate
compounds prior to their synthesis. Herein, based on 3D-QSAR analysis above, two novel
unsynthesized compounds (Figure 4) were designed and the predicted ED values were
calculated by the established CoMFA model. As a result, the designed compounds
A
(ED[a]
=
−
1.790) and
B
(
ED[a] = −1.806) showed potential excellent antifungal activities
with inhibition rates of 87.9% and 88.2%, respectively, which deserved further study.

Molecules 2021, 26, 1708
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(a)
(b)
Figure 3. Contours of CoMFA analysis: (a) contours of steric contribution are represented in yellow and green; (b) contours
of electrostatic contribution are represented in red and blue.
(A)
(B)
Figure 4. The proposed new molecules based on the established CoMFA model: 1-(5-((6,6-dimethylbicyclo[3.1.1]hept-2-en-
2-yl)methyl)-1,3,4-thiadiazol-2-yl)-3-(4-fluoro-2-methyl-3-nitrophenyl) thiourea ( ); 1-(3-bromo-4-fluoro-2-methylphenyl)-3-
A
(5-((6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)-1,3,4-thiadiazol-2-yl) thiourea (B).
2.4. Theoretical Calculation and Analysis
Among the title compounds, disubstituted compound 6j (R = m, p-Cl Ph) showed the
best broad-spectrum antifungal activity with an average inhibition activity of 51.9% against
all the tested fungi. Its corresponding unsubstituted compound 6a (R = Ph), and mono-
substituted compounds 6h (R = m-Cl Ph) and 6i (R = p-Cl Ph) showed lower activity,
with average inhibition rates of 46.4%, 47.8%, 48.5%, respectively. According to the fron-
tier molecular orbital theory, HOMO can provide electrons, while LUMO readily accepts
electrons; thus, these two frontier orbitals can affect the bioactivity of compounds [33].
Meanwhile, the properties of molecule such as electrostatic potentials (ESPs) and dipole
moments can also affect the bioactivity of compounds. Therefore, the frontier molecular
orbitals, ESPs, and dipole moments for compounds 6a (R = Ph), 6h (R = m-Cl Ph), 6i
(R = p-Cl Ph), and 6j (R = m, p-Cl Ph) were calculated by means of DFT/B3LYP/6-31G (d,
p) in the Gaussian 09 package on the Supercomputing Platform at Guangxi University,
and the result was viewed using the GaussView 5 software [34]. The result is showed
in Figure 5.

Molecules 2021, 26, 1708
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Figure 5. Frontier molecular orbitals and electrostatic potentials (ESPs) of compounds 6a (R = Ph): HOMO (a), LUMO (b),
and ESP (c); 6h (R = m-Cl Ph): HOMO (d), LUMO (e), and ESP (f); 6i (R = p-Cl Ph): HOMO (g), LUMO (h), and ESP (i);
mboxboldmath6j (R = m, p-Cl Ph): HOMO (j), LUMO (k), and ESP (l).
As shown in Figure 5a,d,g,j, a large portion of the HOMO was located on the thiourea
group and the substituted benzene ring, with similar distribution and phase for the four
compounds. The LUMO for these compounds was located on the 1,3,4-thiadiazole ring,
the thiourea group and the substituted benzene ring, and shared the same case (Figure 5b,e,h,k)
The dipole moments for the compounds 6a (R = Ph), 6h (R = m-Cl Ph), 6i (R = p-Cl Ph) and
6j (R = m, p-Cl Ph) were 5.7380 D, 6.5806 D, 7.6854 D and 8.2789 D, respectively, which
showed corresponding correlation with the rank of their antifungal activity. The dipole
moment had a correlation with the ESP of molecules. The ESPs for these compounds
are shown in Figure 5c,f,i,l. The chlorine atoms of these molecules displayed a negative
electrostatic potential. The chlorine atoms at meta and para positions of the benzene ring
.

Molecules 2021, 26, 1708
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could increase the dipole moment value, resulting in the improvement of antifungal activity.
These results would be useful for further investigating in these compounds.
3. Experimental Section
3.1. General Information
HPLC analysis was conducted on Waters 1525 HPLC instrument (Waters Co., Ltd.,
Milford, MA, USA) equipped with Waters 2998 PDA detector and column C18 5 µm
(
4.6 mm × 150 mm). GC analysis was conducted on Agilent 6890 GC (Agilent Technologies
Inc., Santa Clara, CA, USA) equipped with column HP-1 (30 m, 0.530 mm, 0.88 m) and FID.
µ
Melting points were recorded using an MP420 automatic melting point apparatus (Hanon
Instruments Co., Ltd., Jinan, China) and were not corrected. IR spectra were recorded on a
Nicolet iS50 FT-IR spectrometer (Thermo Scientific Co., Ltd., Madison, WI, USA) by the KBr
pellet method. NMR spectra were determined in a CDCl3 or DMSO-d6 solvent on a Bruker
Avance III HD 500 MHz/600 MHz spectrometer (Switzerland Bruker Co., Ltd., Zurich,
Switzerland), and chemical shifts are expressed in (δ) ppm downfield relative to TMS as an
internal standard. MS spectra were obtained by means of the electrospray ionization (ESI)
method on the TSQ Quantum Access MAX HPLC-MS instrument (Thermo Scientific Co.,
Ltd., Waltham, MA, USA). Elemental analyses were measured using a PE 2400 II elemental
analyzer (Perkin-Elmer Instruments Co., Ltd., Waltham, MA, USA). β-Pinene (GC purity
98%) was provided by Jiangxi Cedar Natural Medicinal Oil Co., Ltd., Jian, China. Other
reagents were purchased from commercial suppliers and used as received.
3.2. Synthesis of Nopol (2)
β
-Pinene 1 (133.50 g, 980 mmol) and paraformaldehyde (42.84 g, 476 mmol) were
mixed and stirred at 75 ◦C. Then, ZnCl2 (1.50 g, 11.0 mmol) was slowly added and stirred
◦
for 1 h. Afterwards, the reaction mixture was stirred at 110 C for 12 h. The reaction
process was monitored by TLC. When the reaction was completed, the mixture was cooled
to room temperature. The organic layer was separated, washed three times with water,
dried over anhydrous Na2SO4, and distilled in a vacuum to give compound
transparent liquid. Yield 60%; FT-IR (KBr, cm−1): 3369 (–OH), 3030 (=CH), 1471 (CH2), 1386,
1368 (C(CH3)2), 1048 (C–O); 1H-NMR (600 MHz, CDCl3):
5.33 (dt, J = 4.2, 1.3 Hz, 1H),
2 as a colorless
δ
3.60 (d, J = 5.4 Hz, 2H), 2.38 (dt, J = 8.6, 5.6 Hz, 1H), 2.28 (d, J = 17.5 Hz, 1H), 2.24–2.19 (m,
3H), 2.12–2.08 (m, 1H), 2.03 (td, J = 5.6, 1.4 Hz), 1.49 (s, 1H), 1.27 (s, 3H), 1.14 (d, J = 8.6 Hz),
0.84 (s, 3H); 13C-NMR (151 MHz, CDCl3):
26.3, 21.2.
δ 144.7, 119.4, 60.0, 45.6, 40.7, 40.2, 37.9, 31.8, 31.4,
3.3. Synthesis of Nopol Aldehyde (3)
Compound
Nopol (1.66 g, 10 mmol), Dess–Martin periodinane (5.51g, 13 mmol) and NaHCO3 (2.18 g,
26 mmol) were dissolved in CH2Cl2 (40 mL) and stirred at 0 ◦C for 4 h. Upon completion,
the filtrate was collected by filtration and washed three times with 5% Na2S2O3 (3
3 was prepared according to a method described in the literature [35].
2
×
20 mL), then dried over anhydrous Na2SO4, and evaporated in vacuo to give compound
3
as a colorless transparent liquid. Yield 75%; FT-IR (KBr, cm−1): 3030 (C=C–H), 2768, 2733
(O=C–H), 1671 (C=O), 1623 (C=C); ESI-MS m/z: 165.08 ([M + H+]).
3.4. Synthesis of Nopol-Based Thiosemicarbazone (4)
Compound 4 was prepared by the method presented in our previous work [36].
To a stirred solution of thiosemicarbazide (0.91 g, 10 mmol) in distilled water (20.0 mL),
a solution of nopol aldehyde 3 (1.64 g, 10 mmol) in anhydrous ethanol (20.0 mL) was added
dropwise at 65 ◦C in 15 min. The reaction mixture was cooled to room temperature and
filtrated. The resulting filter cake was washed several times with a mixture of distilled
water and ethanol (distilled water–ethanol = 1:1, v/v) to give the crude product, which
was purified by recrystallization in ethanol to obtain compound
4 as a white solid. Yield
85%; m.p.154.2–156.8 ◦C; FT-IR (KBr, cm−1): 3412, 3257, 3158 (N–H), 3030 (C=C–H), 1603

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(C=C), 1535 (C=N), 1462, 1366, 1070 (N–C=S); 1H-NMR (600 MHz, CDCl3)
δ
10.02 (s, 1H),
7.23 (t, J = 5.6 Hz, 1H), 7.08 (s, 1H), 6.49 (s, 1H), 5.32 (s, 1H), 2.89 (d, J = 5.4 Hz, 2H), 2.36
(dt, J = 8.5, 5.7 Hz, 1H), 2.23 (d, J = 25.7 Hz, 2H), 2.08 (s, 1H), 1.99 (t, J = 5.5 Hz, 1H), 1.25 (s,
3H), 1.15 (d, J = 8.6 Hz, 1H), 0.81 (s, 3H); 13C-NMR (151 MHz, CDCl3)
δ 178.1, 146.3, 142.5,
120.2, 45.5, 40.6, 39.8, 38.1, 31.6, 31.4, 26.2, 21.2; ESI-MS m/z: 238.13 ([M + H+]).
3.5. Synthesis of 5-Nopyl-2-amino-1,3,4-thiadiazole (5)
As described in the literature [37], compound
4 (0.71 g, 3 mmol), iodine (0.91 g,
3.6 mmol), and potassium carbonate (1.24 g, 9 mmol) were added into dioxane (25 mL) at
80 ◦C and stirred for 6 h. The reaction process was monitored by TLC. Upon completion,
the reaction mixture was cooled to room temperature, reacted with 5% Na2S2O3 (20 mL),
and extracted with EtOAc. The upper layer with the desired organic compound was
collected, dried over anhydrous Na2SO4, and evaporated in vacuo to obtain compound
5
as a brown solid. Yield 65%; m.p.167.9–169.1 ◦C; FT-IR (KBr, cm−1): 3297, 3101 (N–H),
3006 (=C–H), 1642 (C=C), 1520 (C=N); 1H-NMR (600 MHz, DMSO)
δ 7.00 (s, 2H), 5.42 (s,
1H), 3.46 (q, J = 14.9 Hz, 2H), 2.35 (dt, J = 8.4, 5.6 Hz, 1H), 2.21 (dd, J = 52.9, 17.7 Hz, 2H),
2.05 (s, 1H), 2.02 (t, J = 5.2 Hz, 1H), 1.21 (s, 3H), 1.07 (d, J = 8.5 Hz, 1H), 0.75 (s, 3H); 13C-
NMR (151 MHz, DMSO)
δ 169.1, 156.7, 144.6, 119.6, 45.0, 40.4, 37.9, 37.5, 32.0, 31.3, 26.4,
21.4; ESI-MS m/z: 236.10 ([M + H+]).
3.6. General Procedure for Synthesis of Nopol-Based 1,3,4-Thiadiazole-thiourea Compiunds (6a–6r)
To a mixture of compound
5 (0.24 g, 1 mmol) and sodium hydroxide (0.04 g, 1 mmol)
in dry dioxane (20 mL), substituted isothiocyanate (1.2 mmol) was added slowl◦y with
vigorous stirring at room temperature. Then, the reaction mixture was heated to 90 C and
stirred for 8–12 h. After the reaction was completed, the residual sodium hydroxide was
removed through filtration. The filtrate was concentrated in vacuo and the residue was
purified by silica gel chromatography to afford the target compounds 6a–6r in yields of
45%–70%.
Compound(6a): 1-(5-((6,6-Dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)-1,3,4-thiadiazol-
2-yl)-3-phenylthiourea. White solid. Yield: 45.0%, m.p. 180.0–181.2 ◦C; FT-IR (KBr, cm−1):
3315 (N-H), 3024 (=C–H), 1667 (C=C), 1599, 1502 (Ar–C=C), 1553 (C=N), 1440, 1326, 1081
(N–C=S), 658 (C–S–C); 1H-NMR (500 MHz, DMSO)
δ 13.99 (s, 1H, N–H), 10.40 (s, 1H, N–H),
7.68 (d, J = 7.9 Hz, 2H, C15-H, C19-H), 7.31 (t, J = 7.7 Hz, 2H, C16-H, C18-H), 7.08 (s, 1H,
C17-H), 5.50 (s, 1H, C3-H), 3.58–3.50 (m, 2H, C10-H), 2.36 (dt, J = 8.4, 5.6 Hz, 1H, C1-H), 2.23
(dd, J = 49.3, 17.7 Hz, 2H, C4-Ha, C7-Ha), 2.05 (d, J = 4.9 Hz, 2H, C7-Hb, C5-H), 1.22 (s, 3H,
C9-CH3), 1.11 (d, J = 8.5 Hz, 1H, C4-Hb), 0.76 (s, 3H, C8-CH3); 13C-NMR (126 MHz, DMSO)
δ
184.6, 170.1, 156.7, 143.6, 140.2, 128.8, 124.3, 122.9, 120.6, 45.1, 40.4, 38.0, 37.6, 31.9, 31.4,
26.4, 21.4; ESI-MS m/z: 371.28 ([M + H+]). Anal. calcd. for C19H22N4S2: C, 61.59; H, 5.98;
N, 15.12; Found: C, 61.56; H, 5.95; N:15.10.
Compound(6b): 1-(5-((6,6-Dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)-1,3,4-thiadiazol-
2-yl)-3-(o-tolyl) thiourea. White solid. Yield: 69.0%, m.p. 178.6–179.4 ◦C; FT-IR (KBr, cm−1):
3325, 3307 (N–H), 3022 (=C–H), 1632 (C=C), 1586, 1500 (Ar–C=C), 1524 (C=N), 1434, 1366,
1081 (N–C=S), 654 (C–S–C); 1H-NMR (500 MHz, DMSO) δ 13.27 (s, 1H, N–H), 9.98 (s, 1H,
N–H), 7.32–7.22 (m, 2H, C16-H, C18-H), 7.21–7.14 (m, 2H, C19-H, C17-H), 5.48 (s, 1H, C3-H),
3.53 (q, J = 15.4 Hz, 2H, C10-H), 2.35 (dt, J = 8.4, 5.6 Hz, 1H, C1-H), 2.30–2.15 (m, 5H, C4-Ha,
C7-Ha, C20-CH3), 2.04 (d, J = 4.9 Hz, 2H, C7-Hb, C5-H), 1.22 (s, 3H, C9-CH3), 1.10 (d, J =
8.4 Hz, 1H, C4-Hb), 0.76 (s, 3H, C8-CH3); 13C-NMR (126 MHz, DMSO)
δ 185.1, 170.6, 157.2,
143.8, 138.3, 134.8, 130.7, 128.1, 127.0, 126.5, 120.4, 45.1, 40.4, 38.0, 37.4, 31.9, 31.4, 26.4, 21.4,
18.3; ESI-MS m/z: 385.28 ([M + H+]). Anal. calcd. for C20H24N4S2: C, 62.47; H, 6.29; N,
14.57; Found: C, 62.46; H, 6.29; N:14.50.
Compound(6c): 1-(5-((6,6-Dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)-1,3,4-thiadiazol-
2-yl)-3-(m-tolyl) thiourea. White solid. Yield: 50.3%, m.p. 182.6–184.6 ◦C; FT-IR (KBr, cm−1):
3330 (N-H), 3035 (=C-H), 1663 (C=C), 1611, 1495 (Ar-C=C), 1555 (C=N), 1438, 1359, 1089
(N-C=S), 660 (C-S-C); 1H-NMR (500 MHz, DMSO)
δ 13.91 (s, 1H, N-H), 10.33 (s, 1H, N-H),

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7.47 (d, J = 9.4 Hz, 2H, C15-H, C19-H), 7.18 (t, J = 7.7 Hz, 1H, C18-H), 6.90 (d, J = 6.6 Hz, 1H,
C17-H), 5.50 (s, 1H, C3-H), 3.58–3.50 (m, 2H, C10-H), 2.36 (dt, J = 8.4, 5.6 Hz, 1H, C1-H), 2.28
(d, J = 15.2 Hz, 4H, C4-Ha, C20-CH3), 2.18 (d, J = 17.6 Hz, 1H, C7-Ha), 2.05 (d, J = 5.4 Hz, 2H,
C7-Hb, C5-H), 1.22 (s, 3H, C9-CH3), 1.11 (d, J = 8.5 Hz, 1H, C4-Hb), 0.76 (s, 3H, C8-CH3);
13C-NMR (126 MHz, DMSO)
δ 184.6, 170.1, 156.7, 143.6, 140.1, 138.0, 128.6, 125.1, 123.6,
112.8, 120.5, 45.1, 40.4, 38.0, 37.6, 31.9, 31.4, 26.4, 21.6, 21.4; ESI-MS m/z: 385.29 ([M + H+]).
Anal. calcd. for C20H24N4S2:C, 62.47; H, 6.29; N, 14.57; Found: C, 62.45; H, 6.27; N:14.52.
Compound(6d): 1-(5-((6,6-Dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)-1,3,4-thiadiazol-
2-yl)-3-(p-tolyl) thiourea
3321 (N–H), 3035 (=C-H), 1663 (C=C), 1601, 1512 (Ar–C=C), 1545 (C=N), 1436, 1368, 1081
(N–C=S), 652 (C-S-C); 1H-NMR (500 MHz, DMSO)
14.01 (s, 1H, N–H), 10.33 (s, 1H, N–H),
.
White solid. Yield: 61.2%, m.p. 185.5–187.0 ◦C; FT-IR (KBr, cm−1):
δ
7.52 (d, J = 8.3 Hz, 2H, C16-H, C18-H), 7.11 (d, J = 8.0 Hz, 2H, C15-H, C19-H), 5.50 (s, 1H,
C3-H), 3.57–3.49 (m, 2H, C10-H), 2.36 (dt, J = 8.5, 5.6 Hz, 1H, C1-H), 2.28 (d, J = 15.8 Hz, 4H,
C4-Ha, C20-CH3), 2.18 (d, J = 17.7 Hz, 1H, C7-Ha), 2.05 (d, J = 4.5 Hz, 2H, C7-Hb, C5-H),
1.22 (s, 3H, C9-CH3), 1.10 (d, J = 8.5 Hz, 1H, C4-Hb), 0.76 (s, 3H, C8-CH3); 13C-NMR (126
MHz, DMSO)
δ 184.6, 170.6, 156.4, 143.6, 137.6, 133.5, 129.2, 123.1, 120.5, 45.1, 40.4, 38.0,
37.6, 31.9, 31.4, 26.4, 21.4, 21.0; ESI-MS m/z: 385.29 ([M + H+]). Anal. calcd. for C20H24N4S2:
C, 62.47; H, 6.29; N, 14.57; Found: C, 62.42; H, 6.28; N:14.51.
Compound(6e): 1-(5-((6,6-Dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)-1,3,4-thiadiazol-
2-yl)-3-(3-methoxyphenyl) thiourea. White solid. Yield: 64.6%, m.p. 152.0–155.0 ◦C; FT-IR
(KBr, cm−1): 3323 (N–H), 3030 (=C–H), 1652 (C=C), 1599, 1495 (Ar–C=C), 1535 (C=N), 1434,
1359, 1079 (N–C=S), 652 (C–S–C); 1H-NMR (500 MHz, DMSO)
δ 14.18 (s, 1H, N-H), 10.37 (s,
1H, N–H), 7.37 (s, 1H, C15-H), 7.26 (d, J = 8.0 Hz, 1H, C18-H), 7.20 (t, J = 8.1 Hz, 1H, C19-H),
6.66 (d, J = 6.5 Hz, 1H, C17-H), 5.51 (s, 1H, C3-H), 3.74 (s, 3H, C20-OCH3), 3.58–3.50 (m, 2H,
C10-H), 2.36 (dt, J = 8.5, 5.6 Hz, 1H, C1-H), 2.24 (dd, J = 49.5, 17.7 Hz, 2H, C4-Ha, C7-Ha),
2.08–2.03 (m, 2H, C7-Hb, C5-H), 1.22 (s, 3H, C9-CH3), 1.11 (d, J = 8.5 Hz, 1H, C4-Hb), 0.76 (s,
3H, C8-CH3); 13C-NMR (126 MHz, DMSO)
δ 184.7, 170.3, 159.7, 156.7, 143.6, 141.3, 129.5,
120.6, 114.9, 109.6, 108.5, 55.5, 45.1, 40.4, 38.0, 37.6, 31.9, 31.4, 26.4, 21.4; ESI-MS m/z: 401.29
([M + H+]). Anal. calcd. for C20H24N4OS2:C, 59.97; H, 6.04; N, 13.99; Found: C, 59.92; H,
6.00; N:13.92.
Compound(6f): 1-(5-((6,6-Dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)-1,3,4-thiadiazol-
2-yl)-3-(4-methoxyphenyl) thiourea. White solid. Yield: 70.0%, m.p. 185.1–185.8 ◦C; FT-IR
(KBr, cm−1): 3319 (N-H), 3024 (=C-H), 1665 (C=C), 1601, 1510 (Ar-C=C), 1553 (C=N), 1438,
1372, 1077 (N-C=S), 652 (C-S-C); 1H-NMR (500 MHz, DMSO)
δ 13.74 (s, 1H, N-H), 10.27 (s,
1H, N-H), 7.51 (d, J = 8.9 Hz, 2H, C15-H, C19-H), 6.89 (dd, J = 8.7, 1.9 Hz, 2H, C16-H, C18-H),
5.49 (s, 1H, C3-H), 3.74 (s, 3H, C20-OCH3), 3.53 (q, J = 15.4 Hz, 2H, C10-H), 2.36 (dt, J = 8.4,
5.6 Hz, 1H, C1-H), 2.23 (dd, J = 49.5, 17.7 Hz, 2H, C4-Ha, C7-Ha), 2.05 (d, J = 5.1 Hz, 2H,
C7-Hb, C5-H), 1.22 (s, 3H, C9-CH3), 1.10 (d, J = 8.5 Hz, 1H, C4-Hb), 0.76 (s, 3H, C8-CH3);
13C-NMR (126 MHz, DMSO)
δ 180.7, 157.0, 156.6, 143.7, 133.0, 132.7, 126.5, 125.8, 120.5,
114.1, 114.0, 55.7, 45.1, 40.4, 38.0, 37.5, 31.9, 31.4, 26.4, 21.4; ESI-MS m/z: 401.31 ([M + H+]).
Anal. calcd. for C20H24N4OS2: C, 59.97; H, 6.04; N, 13.99; Found: C, 59.93; H, 6.02; N:13.95.
Compound(6g): 1-(5-((6,6-Dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)-1,3,4-thiadiazol-
2-yl)-3-(4-fluorophenyl) thiourea. White solid. Yield: 55.5%, m.p. 192.6–193.6 ◦C; FT-IR (KBr,
cm−1): 3323 (N–H), 3012 (=C–H), 1671 (C=C), 1613, 1510 (Ar–C=C), 1561 (C=N), 1436, 1363,
1079 (N–C=S), 652 (C–S–C); 1H-NMR (500 MHz, DMSO)
δ 14.03 (s, 1H, N–H), 10.42 (s, 1H,
N–H), 7.68–7.64 (m, 2H, C15-H, C19-H), 7.13 (t, J = 8.2 Hz, 2H, C16-H, C18-H), 5.50 (s, 1H,
C3-H), 3.57–3.50 (m, 2H, C10-H), 2.35 (dt, J = 10.4, 5.3 Hz, 1H, C1-H), 2.23 (dd, J = 49.0,
17.7 Hz, 2H, C4-Ha, C7-Ha), 2.04 (d, J = 4.4 Hz, 2H, C7-Hb, C5-H), 1.21 (s, 3H, C9-CH3),
1.10 (d, J = 8.5 Hz, 1H, C4-Hb), 0.75 (s, 3H, C8-CH3); 13C-NMR (126 MHz, DMSO)
δ 184.8,
170.6, 159.0 (d, J = 259.56 Hz), 156.7, 143.6, 136.6, 124.9, 120.6, 115.4 (d, J = 21.42 Hz), 45.1,
40.4, 38.0, 37.6, 31.9, 31.4, 26.4, 21.4; ESI-MS m/z: 389.26 ([M + H+]). Anal. calcd. for
C19H21FN4S2: C, 58.74; H, 5.45; N 14.42; Found: C, 58.70; H, 5.43; N:13.41.
Compound(6h): 1-(3-Chlorophenyl)-3-(5-((6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)meth
◦
yl)-1,3,4-thiadiazol-2-yl) thiourea. White solid. Yield: 64.8%, m.p. 181.3–183.2 C; FT-IR (KBr,

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cm−1): 3323 (N–H), 3022 (=C–H), 1663 (C=C), 1592, 1485 (Ar–C=C), 1547 (C=N), 1434,
1357, 1077 (N–C=S), 656 (C-S-C); 1H-NMR (500 MHz, DMSO)
δ 14.35 (s, 1H, N–H), 10.52
(s, 1H, N–H), 7.86 (s, 1H, C15-H), 7.69 (d, J = 8.0 Hz, 1H, C18-H), 7.31 (t, J = 8.1 Hz, 1H,
C19-H), 7.10 (d, J = 6.8 Hz, 1H, C17-H), 5.52 (s, 1H, C3-H), 3.59–3.52 (m, 2H, C10-H), 2.37 (dt,
J = 8.4, 5.6 Hz, 1H, C1-H), 2.24 (dd, J = 49.7, 17.7 Hz, 2H, C4-Ha, C7-Ha), 2.06 (d, J = 4.9 Hz,
2H, C7-Hb, C5-H), 1.22 (s, 3H, C9-CH3), 1.11 (d, J = 8.5 Hz, 1H, Hb-4), 0.76 (s, 3H, H-8);
13C-NMR (126 MHz, DMSO)
δ 185.4, 170.6, 156.7, 143.5, 141.7, 138.4, 133.2, 130.4, 123.6,
121.8, 120.7, 45.1, 40.4, 38.0, 37.6, 31.9, 31.4, 26.4, 21.4; ESI-MS m/z: 405.22 ([M + H+]). Anal.
calcd. for C19H21ClN4S2: C, 56.35; H, 5.23; N, 13.83; Found: C, 56.31; H, 5.20; N:13.82.
Compound(6i): 1-(4-Chlorophenyl)-3-(5-((6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)meth
◦
yl)-1,3,4-thiadiazol-2-yl) thiourea. White solid. Yield: 68.4%, m.p. 193.9–196.3 C; FT-IR (KBr,
cm−1): 3319 (N–H), 3037 (=C–H), 1663 (C=C), 1597, 1495 (Ar–C=C), 1549 (C=N), 1436, 1361,
1091 (N–C=S), 652 (C–S–C); 1H-NMR (500 MHz, DMSO)
δ 14.23 (s, 1H, N–H), 10.50 (s, 1H,
N–H), 7.74 (d, J = 8.6 Hz, 2H, C15-H, C19-H), 7.34 (d, J = 8.1 Hz, 2H, C16-H, C18-H), 5.51 (s,
1H, C3-H), 3.58–3.51 (m, 2H, C10-H), 2.37 (dt, J = 8.3, 5.6 Hz, 1H, C1-H), 2.24 (dd, J = 49.9,
17.7 Hz, 2H, C4-Ha, C7-Ha), 2.05 (d, J = 4.7 Hz, 2H, C7-Hb, C5-H), 1.22 (s, 3H, C9-CH3),
1.11 (d, J = 8.5 Hz, 1H, C4-Hb), 0.76 (s, 3H, C8-H); 13C-NMR (126 MHz, DMSO)
δ 185.0,
170.6, 156.6, 143.5, 139.2, 128.6, 127.7, 124.1, 120.6, 45.1, 40.4, 38.0, 37.6, 31.9, 31.4, 26.4, 21.4;
ESI-MS m/z: 405.26 ([M + H+]). Anal. calcd. for C19H21ClN4S2: C, 56.35; H, 5.23; N, 13.83;
Found: C, 56.33; H, 5.21; N:13.80.
Compound(6j): 1-(3,4-Dichlorophenyl)-3-(5-((6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)
methyl)-1,3,4-thiadiazol-2-yl) thiourea. White solid. Yield: 70.0%, m.p. 203.6–205.1 ◦C; FT-IR
(KBr, cm−1): 3311 (N–H), 3024 (=C–H), 1663 (C=C), 1588, 1475 (Ar–C=C), 1539 (C=N), 1434,
1355, 1083 (N–C=S), 646 (C–S–C); 1H-NMR (500 MHz, DMSO)
δ 14.39 (s, 1H, N–H), 10.59
(s, 1H, N–H), 8.09 (s, 1H, C15-H), 7.73 (dd, J = 8.7, 1.6 Hz, 1H, C18-H), 7.52 (d, J = 8.8 Hz, 1H,
C19-H), 5.52 (s, 1H, C3-H), 3.60–3.52 (m, 2H, C10-H), 2.36 (dt, J = 8.4, 5.6 Hz, 1H, C1-H), 2.24
(dd, J = 49.7, 17.7 Hz, 2H, C4-Ha, C7-Ha), 2.05 (d, J = 5.1 Hz, 2H, C7-Hb, C5-H), 1.22 (s, 3H,
C9-CH3), 1.11 (d, J = 8.5 Hz, 1H, C4-Hb), 0.76 (s, 3H, C8-CH3); 13C-NMR (126 MHz, DMSO)
δ
185.4, 170.9, 156.8, 143.5, 140.4, 131.1, 130.6, 125.3, 123.3, 122.2, 120.7, 45.1, 40.3, 38.0, 37.6,
31.9, 31.4, 26.4, 21.4; ESI-MS m/z: 439.19 ([M + H+]). Anal. calcd. for C19H20Cl2N4S2: C,
51.93; H, 4.59; N, 12.75; Found: C, 51.92; H, 4.56; N:12.75.
Compound(6k): 1-(2-Bromophenyl)-3-(5-((6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)meth
yl)-1,3,4-thiadiazol-2-yl) thiourea. White solid. Yield: 61.8%, m.p.162.9–164.4 ◦C; FT-IR (KBr,
cm−1): 3294 (N–H), 3030 (=C–H), 1640 (C=C), 1578, 1438 (Ar–C=C), 1528 (C=N), 1361, 1330,
1081 (N–C=S), 652 (C–S–C); 1H-NMR (500 MHz, CDCl3)
δ 14.21 (s, 1H, N–H), 9.01 (s, 1H,
N–H), 7.88 (d, J = 8.0 Hz, 1H, C16-H), 7.66 (d, J = 8.0 Hz, 1H, C19-H), 7.38 (t, J = 7.7 Hz, 1H,
C18-H), 7.20–7.15 (m, 1H, C17-H), 5.48 (s, 1H, C3-H), 3.65–3.58 (m, 2H, C10-H), 2.36–2.32
(m, 1H, C1-H), 2.31–2.20 (m, 2H, C4-Ha, C7-Ha), 2.07 (d, J = 5.1 Hz, 2H, C7-Hb, C5-H),
1.22 (s, 3H, C9-CH3), 1.16 (d, J = 8.7 Hz, 1H, C4-Hb), 0.80 (s, 3H, C8-CH3); 13C- NMR (126
MHz, CDCl3) δ 179.6, 165.5, 161.5, 143.1, 136.7, 133.0, 129.0, 128.2, 127.6, 121.1, 120.0, 45.3,
40.4, 38.1, 37.4, 31.9, 31.3, 26.1, 21.1; ESI-MS m/z: 449.18 ([M + H+]). Anal. calcd. for
C19H21BrN4S2: C, 50.78; H, 4.71; N, 12.47; Found: C, 50.75; H, 4.66; N:12.43.
Compound(6l): 1-(3-Bromophenyl)-3-(5-((6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)meth
◦
yl)-1,3,4-thiadiazol-2-yl) thiourea. White solid. Yield: 54.2%, m.p.178.1–179.3 C; FT-IR
(
KBr, cm−1): 3330 (N–H), 3026 (=C–H), 1663 (C=C), 1588, 1481 (Ar–C=C), 1545 (C=N), 1434,
1357, 1081 (N–C=S), 652 (C-S–C); 1H-NMR (500 MHz, CDCl3)
δ 14.49 (s, 1H, N–H), 9.31 (s,
1H, N–H), 7.99 (s, 1H, C15-H), 7.79 (d, J = 7.4 Hz, 1H, C17-H), 7.36 (d, J = 7.8 Hz, 1H, C19-H),
7.26 (dd, J = 10.4, 5.5 Hz, C18-H), 5.54 (s, 1H, C3-H), 3.71–3.61 (m, 2H, C10-H), 2.38 (dt,
J = 8.7, 5.7 Hz, 1H, C1-H), 2.35–2.22 (m, 2H, C4-Ha, C7-Ha), 2.10 (d, J = 4.8 Hz, 2H, C7-Hb,
C5-H), 1.24 (s, 3H, C9-CH3), 1.19 (d, J = 8.7 Hz, 1H, C4-Hb), 0.81 (s, 3H, C8-CH3); 13C-NMR
(126 MHz, CDCl3) δ 177.2, 164.7, 161.9, 143.1, 139.8, 130.0, 128.7, 126.1, 122.2, 121.6, 121.2,
45.3, 40.4, 38.1, 37.3, 31.9, 31.3, 26.1, 21.1; ESI-MS m/z: 449.22 ([M + H+]). Anal. calcd. for
C19H21BrN4S2: C, 50.78; H, 4.71; N, 12.47; Found: C, 50.77; H, 4.69; N:12.46.

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Compound(6m): 1-(4-Bromophenyl)-3-(5-((6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)meth
◦
yl)-1,3,4-thiadiazol-2-yl) thiourea. White solid. Yield: 60.4%, m.p. 205.3–206.7 C; FT-IR
(
KBr, cm−1): 3319 (N–H), 3037 (=C–H), 1663 (C=C), 1603, 1590, 1489 (Ar–C=C), 1547 (C=N),
1436, 1361, 1077 (N–C=S), 656 (C–S–C); 1H-NMR (500 MHz, DMSO)
δ 14.21 (s, 1H, N–H),
10.49 (s, 1H, N–H), 7.69 (d, J = 8.8 Hz, 2H, C15-H, C19-H), 7.46 (d, J = 8.5 Hz, 2H, C16-H,
C18-H), 5.51 (s, 1H, C3-H), 3.58–3.50 (m, 2H, C10-H), 2.36 (dt, J = 8.4, 5.6 Hz, 1H, C1-H),
2.23 (dd, J = 49.7, 17.7 Hz, 2H, C4-Ha, C7-Ha), 2.06–2.03 (m, 2H, C7-Hb, C5-H), 1.22 (s, 3H,
C9-CH3), 1.11 (d, J = 8.5 Hz, 1H, C4-Hb), 0.75 (s, 3H, C8-CH3); 13C-NMR (126 MHz, DMSO)
δ
184.8, 170.9, 156.6, 143.5, 139.6, 131.5, 124.4, 120.6, 115.8, 45.1, 40.4, 38.0, 37.6, 31.9, 31.4,
26.4, 21.4; ESI-MS m/z: 449.20 ([M + H+]). Anal. calcd. for C19H21BrN4S2: C, 50.78; H, 4.71;
N, 12.47; Found: C, 50.74; H, 4.68; N:12.42.
Compound(6n): 1-(5-((6,6-Dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)-1,3,4-thiadiazol-
2-yl)-3-(2-(trifluoromethyl) phenyl) thiourea. Yellow solid. Yield: 45.5%, m.p. 159.7–161.3 ◦C
;
FT-IR (KBr, cm−1): 3305 (N-H), 3039 (=C–H), 1634 (C=C), 1609, 1526 (Ar–C=C), 1586 (C=N),
1436, 1357, 1081 (N–C=S), 656 (C-S-C); 1H-NMR (500 MHz, DMSO)
δ
13.82 (s, 1H, N–H),
10.04 (s, 1H, N–H), 7.74 (d, J = 7.8 Hz, 1H, C16-H), 7.69 (t, J = 7.7 Hz, 1H, C17-H), 7.55
(d, J = 5.7 Hz, 1H, C18-H), 7.49 (t, J = 7.6 Hz, 1H, C19-H), 5.50–5.48 (m, 1H, C3-H), 3.58–
3.50 (m, 2H, C10-H), 2.36 (dt, J = 8.5, 5.6 Hz, 1H, C1-H), 2.23 (d, J = 32.0 Hz, 2H, C4-Ha,
C7-Ha), 2.05–2.03 (m, 2H, C7-Hb, C5-H), 1.22 (s, 3H, C9-CH3), 1.10 (d, J = 8.5 Hz, 1H,
C4-Hb), 0.76 (s, 3H, C8-CH3); 13C-NMR (126 MHz, DMSO)
δ 188.0, 172.0, 143.7, 133.3, 132.2,
127.5 (d, J = 63.9 Hz), 126.7 (q, J = 31.2, 30.7 Hz),126.4, 126.1 (q, J = 4.9 Hz) 125.7, 124.0 (d,
J = 273.5 Hz), 120.5, 45.1, 40.4, 38.0, 37.4, 31.9, 31.4, 26.4, 21.4; ESI-MS m/z: 439.29 ([M +
H+]). Anal. calcd. for C20H21F3N4S2: C, 54.78; H, 4.83; N, 12.78; Found: C, 54.77; H, 4.80;
N:12.76.
Compound(6o): 1-(5-((6,6-Dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)-1,3,4-thiadiazol-
2-yl)-3-(3-nitrophenyl) thiourea. Yellow solid. Yield: 68.7%, m.p. 184.8–188.3 ◦C; FT-IR (KBr,
cm−1): 3319 (N–H), 3024 (=C–H), 1665 (C=C), 1599, 1533 (Ar–C=C), 1557 (C=N), 1432, 1357,
1079 (N–C=S), 652 (C–S–C); 1H-NMR (500 MHz, DMSO)
δ 14.25 (s, 1H, N–H), 10.79 (s, 1H,
N–H), 8.61 (s, 1H, C15-H), 8.21 (d, J = 7.3 Hz, 1H, C17-H), 7.89 (d, J = 7.1 Hz, 1H, C19-H),
7.58 (t, J = 8.0 Hz, 1H, C18-H), 5.51 (s, 1H, C3-H), 3.60–3.52 (m, 2H, C10-H), 2.38–2.34 (m,
1H, C1-H), 2.23 (dd, J = 49.4, 17.6 Hz, 2H, C4-Ha, C7-Ha), 2.05 (d, J = 4.1 Hz, 2H, C7-Hb,
C5-H), 1.21 (s, 3H, C9-CH3), 1.11 (d, J = 8.3 Hz, 1H, C4-Hb), 0.75 (s, 3H, C8-CH3); 13C-NMR
(126 MHz, DMSO) δ 185.5, 170.1, 156.8, 148.2, 143.4, 141.4, 130.1, 128.4, 120.7, 118.2, 116.5,
45.1, 40.4, 38.0, 37.6, 31.9, 31.4, 26.4, 21.4; ESI-MS m/z: 416.21 ([M + H+]). Anal. calcd. for
C19H21N5O2S2:C, 54.92; H, 5.09; N, 16.85; Found: C, 54.90; H, 5.05; N:16.84.
Compound(6p): 1-(5-((6,6-Dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)-1,3,4-thiadiazol-
2-yl)-3-(4-nitrophenyl) thiourea. Yellow solid. Yield: 54.9%, m.p. 212.6–214.3 ◦C; FT-IR (KBr,
cm−1): 3315 (N–H), 3030 (=C–H), 1665 (C=C), 1599, 1508 (Ar–C=C), 1564 (C=N), 1434, 1355,
1081 (N–C=S), 646 (C–S–C); 1H-NMR (500 MHz, DMSO)
δ 14.49 (s, 1H, N–H), 10.93 (s,
1H, N–H), 8.16 (d, J = 9.2 Hz, 2H, C16-H, C18-H), 8.07 (d, J = 9.2 Hz, 2H, C15-H, C19-H),
5.52 (s, 1H, C3-H), 3.58 (q, J = 15.5 Hz, 2H, C10-H), 2.37 (dt, J = 8.2, 5.6 Hz, 1H, C1-H), 2.24
(dd, J = 49.6
, 17.7 Hz, 2H, C4-Ha, C7-Ha), 2.06 (d, J = 4.9 Hz, 2H, C7-Hb, C5-H), 1.22 (s, 3H,
C9-CH3), 1.11 (d, J = 8.5 Hz, 1H, C4-Hb), 0.76 (s, 3H, C8-CH3); 13C-NMR (126 MHz, DMSO)
δ
185.5, 170.7, 157.1, 146.4, 143.4, 142.1, 124.9, 121.1, 120.8, 45.2, 40.4, 38.0, 37.6, 31.9, 31.4,
26.4, 21.4; ESI-MS m/z: 416.28 ([M + H+]). Anal. calcd. for C19H21N5O2S2: C, 54.92; H, 5.09;
N, 16.85; Found: C, 54.89; H, 5.05; N:16.81.
Compound(6q): 1-(5-((6,6-Dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)-1,3,4-thiadiazol-
2-yl)-3-isopropylthiourea. White solid. Yield: 48.9%, m.p. 188.5–190.5 ◦C; FT-IR (KBr, cm−1):
3315 (N–H), 3037 (=C–H), 1636 (C=C), 1535 (C=N), 1434, 1380, 1081 (N–C=S), 652 (C–S–C);
1H-NMR (500 MHz, CDCl3)
δ 14.14 (s, 1H, N-H), 7.54 (s, 1H, N-H), 5.48 (s, 1H, C3-H),
4.53 (dq, J = 13.1, 6.4 Hz, 1H, C14-H), 3.65–3.53 (m, 2H, C10-H), 2.38 (dt, J = 8.4, 5.6 Hz,
1H, C1-H), 2.34–2.20 (m, 2H, C4-Ha, C7-Ha), 2.08 (d, J = 4.5 Hz, 2H, C7-Hb, C5-H), 1.33 (d,
J = 6.3 Hz, 6H, C15-H, C16-H), 1.24 (s, 3H, C9-CH3), 1.19 (d, J = 8.7 Hz, 1H, C4-Hb), 0.80 (s,
3H, C8-CH3); 13C NMR (126 MHz, CDCl3)
δ 177.4, 164.6, 161.2, 143.4, 120.8, 100,0 47.2, 45.3,

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40.5, 38.1, 37.3, 31.9, 31.3, 26.1, 22.1, 21.1; ESI-MS m/z:337.27([M + H+]). Anal. calcd. for
C16H24N4S2: C, 57.11; H, 7.19; N, 16.65; Found: C, 57.10; H, 7.16; N:16.60.
Compound(6r): 1-Butyl-3-(5-((6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)-1,3,4-thi
◦
adiazol-2-yl) thiourea. White solid. Yield: 64.0%, m.p. 152.8–153.8 C; FT-IR (KBr, cm−1):3325
(N-H), 3041 (=C-H), 1636 (C=C), 1537 (C=N), 1436, 1390, 1079 (N-C=S), 652 (C-S-C); 1H-
NMR (500 MHz, CDCl3)
δ 14.28 (s, 1H, N–H), 7.61 (s, 1H, N–H), 5.49 (s, 1H, C3-H), 3.69
(dd, J = 12.1, 6.4 Hz, 2H, C14-H), 3.60 (q, J = 15.5 Hz, 2H, C10-H), 2.38 (dt, J = 8.6, 5.6 Hz,
1H, C1-H), 2.34–2.22 (m, 2H, C15-H), 2.08 (dd, J = 11.4, 5.7 Hz, 2H, C4-Ha, C7-Ha), 1.72–1.68
(m, 2H, C7-Hb, C5-H), 1.48 (dd, J = 14.9, 7.4 Hz, 2H, C16-H), 1.24 (s, 3H, C9-CH3), 1.19
(d, J = 8.7 Hz, 1H, C4-Hb), 0.97 (t, J = 7.3 Hz, 3H, C17-H), 0.81 (s, 3H, C8-CH3); 13C-NMR
(
126 MHz, CDCl3) δ 178.6, 164.7, 161.3, 143.4, 120.8, 45.2, 40.5, 38.1, 37.3, 31.9, 31.3, 30.6,
26.1, 21.1, 20.1, 13.8; ESI-MS m/z: 351.31([M + H+]). Anal. calcd. for C17H26N4S2:C, 58.25;
H, 7.48; N, 15.98; Found: C, 58.20; H, 7.46; N:15.95.
3.7. Antifungal Activity Test
Testing of the primary biological activity was performed in an isolated culture. Under
sterile conditions, the tested compound was dissolved in acetone. Sorporl-144 (200
emulsifier was added to dilute the solution to 500 g/mL. Then, 1 mL solution of the tested
compound was poured into a culture plate, followed by the addition of 9 mL PSA culture
medium. The final mass concentration of the title compound was 50 g/mL. A bacterium
µg/mL)
µ
µ
tray of 5 mm in diameter cut along the external edge of the mycelium was transferred to the
flat surface containing the tested compound and arranged in an equilateral triangular style
◦
in duplicate. The culture plates were cultivated at 24
±
1 C and the extended diameters of
the circles of mycelium were measured after 48 h. The relative inhibition of the circle of
mycelium compared with the blank assay was calculated by use of the equation:
Relative inhibitory rate (%) = (CK − PT)/CK × 100%
where CK is the extended diameter of the circle of mycelium during the blank assay and
PT is the extended diameter of the circle of mycelium during testing.
3.8. 3D-QSAR Analysis
Molecular modeling was performed using SYBYL-X 2.1.1 software (Tripos, Inc.,
St. Louis, MO, USA). According to a report in the literature [38], the antifungal activ-
ity against A. solani was expressed in terms of activity factor (ED) by the formula:
ED = log {I/[(100 − I) × MW]}
where I is the percent inhibition at 50 µg/mL and MW was the molecular weight of the
tested compounds.
Complete conformational optimization of each structure was performed using a
conjugate gradient procedure based on the Tripos force field and Gasteiger–Hückel charges.
Compound 6g (R = p-F Ph) was used as a template to build the other molecular structures.
According to the common skeleton marked with an asterisk shown in Figure 6, fifteen
optimized molecules containing benzene ring were superimposed (Figure 7). The values of
the CoMFA field were automatically calculated by the SYBYL/CoMFA routine. A predictive
3D-QSAR model was established using CoMFA descriptors as independent variables and
ED values as dependent variables. The cross-validation with the leave-one-out mothed was
carried out to obtain the cross-validated q2 and the optimal number of components. Then,
a non-cross-validation analysis under the optimal number of components was performed.
The modeling capability was indicated by the correlation coefficient squared r2, and the
prediction capability was judged by the r2 and q2.

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Figure 6. The asterisk skeleton of title compounds.
Figure 7. Superposition modes of compounds.
4. Conclusions
Eighteen novel nopol-derived 1,3,4-thiadiazole-thiourea compounds were synthesized,
characterized, and evaluated for their antifungal activity. As a result, at 50 g/mL, all the
µ
target compounds exhibited better antifungal activity against P. piricola, C. arachidicola, and
A. solani. Compound 6j (R = m, p-Cl Ph) showed the best broad-spectrum antifungal activity
against all the tested fungi. Compounds 6c (R = m-Me Ph), 6q (R = i-Pr), and 6i (R = p-Cl Ph)
had inhibition rates of 86.1%, 86.1%, and 80.2%, respectively, against P. piricola, much better
than that of the positive control chlorothalonil. Besides, compounds 6h (R = m-Cl Ph) and
6n (R = o-CF3 Ph) held inhibition rates of 80.6% and 79.0% against C. arachidicola and G. zeae,
respectively, much better than that of the commercial fungicide chlorothalonil. Therefore,
these new analogs can serve as starting points for additional antifungal studies. In order to

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design more effective antifungal compounds, a reasonable and effective 3D-QSAR model
had been established, and two new molecules with modification on phenyl groups were
proposed. Furthermore, some intriguing structure–activity relationships were found and
discussed by theoretical calculations.
Supplementary Materials: Supplementary materials are available online. Figures S1–S85: FT-IR,
1H-NMR, 13C-NMR, and ESI-MS spectra of compounds 2, 3, 4, 5, and 6a–6r.
Author Contributions: M.C. carried out the experimental work, participated in the discussion of
biological activities, and wrote the paper; W.-G.D. and G.-S.L. constructed the target compound
structure, designed the experimental scheme, directed and supervised the whole experimentation,
3D-QSAR study and theoretical calculation, discussed the biological activities, and revised the paper;
Z.-T.F. and X.W. participated in the synthesis of target compounds and the discussion of biological
activities. All authors have read and agreed to the published version of the manuscript.
Funding: This work was financially supported by the National Natural Science Foundation of China
(No. 31870556).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained within the article or the supplementary data file.
Acknowledgments: The authors are grateful to the State Key Laboratory of Element-Organic Chem-
istry, Nankai University, China, for the bioassay test.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are available from the authors.
References
1.
Unnikrishnan, R.P.; Endalkachew, S.D. Mesoporous iron phosphate as an active, selective and recyclable catalyst for the synthesis
of nopol by Prins condensation. Chem. Commun. 2004, 7, 826–827.
2.
3.
Neuenschwander, U.; Meier, E.; Hermans, I. Peculiarities of β-pinene autoxidation. ChemSusChem 2011, 4, 1613–1621. [CrossRef]
Wang, J.; Jaenicke, S.; Chuah, G.K.; Hua, W.; Yue, Y.; Gao, Z. Acidity and porosity modulation of MWW type zeolites for nopol
production by Prins condensation. Catal. Commun. 2011, 12, 1131–1135. [CrossRef]
4.
5.
6.
Xu, L.F.; Xiao, Z.Q.; Wang, P.; Fan, G.R.; Chen, J.Z.; Chen, S.X.; Wang, Z.D. The synthesis and antifungal activity of cyclic acetal
hydronopic aldehyde derivatives. J. Jiangxi Norm. Univ. 2014, 38, 472–475.
Jin, L.L.; Xiao, Z.Q.; Chen, J.Z.; Fan, G.R.; Wang, P.; Wang, Z.D.; Chen, S.X. The synthesis and structural analysis for hydronopyl
tri-alkyl ammonium halides. J. Jiangxi Norm. Univ. 2015, 39, 484–487.
Wang, Z.D.; Song, J.; Chen, J.Z.; Song, Z.Q.; Shang, S.B.; Jiang, Z.K.; Han, Z.J. QSAR study of mosquito repellents from terpenoid
with a six-member-ring. Bioorg. Med. Chem. Lett. 2008, 18, 2854–2859. [CrossRef]
7.
8.
Baronnet, R. Terpene Derivatives. U.S. Patent 3,845,048, 14 November 1972.
Behrouzi, F.M.; Poorrajab, F.; Ardestani, S.K.; Emami, S.; Shafiree, A.; Foroumadi, A. Synthesis and in vitro anti-leishmanial
activity of 1-[5-(5-nitrofuran-2-yl)-1,3,4-thiadiazol-2-yl]- and 1-[5-(5-nitrothiophen-2-yl)-1,3,4-thiadiazol-2-yl]-4-aroylpiperazines.
Bioorg. Med. Chem. 2008, 16, 4509–4515. [CrossRef] [PubMed]
9.
Li, Z.S.; Wang, W.M.; Lu, W.; Niu, C.W.; Li, Y.H.; Li, Z.M.; Wang, J.G. Synthesis and biological evaluation of nonsymmetrical
aromatic disulfides as novel inhibitors of acetohydroxyacid synthase. Bioorg. Med. Chem. Lett. 2013, 23, 3723–3727. [CrossRef]
[PubMed]
10. Banday, M.R.; Rauf, A. Synthesis and evaluation of in vitro antibacterial activity of novel 2,5-disubstituted-1,3,4-thiadiazoles
from fatty acids. Chin. Chem. Lett. 2008, 19, 1427–1430. [CrossRef]
11. Padmavathi, V.; Reddy, G.S.; Padmaja, A.; Kondaiah, P.; Ali-Shazia. Synthesis, antimicrobial and cytotoxic activities of 1,3,4-
oxadiazoles, 1,3,4-thiadiazoles and 1,2,4-triazoles. Eur. J. Med. Chem. 2009, 44, 2106–2112. [CrossRef]
12. Bansode, S.; Kamble, R. Synthesis of novel 2-(3’-aryl-sydnon-4’-ylidene)-5’-substituted-[1,3,4]-thiadiazolylamines and [1,3,4]
-thiadiazol-2’-yl-3-oxo- [1,2,4]-triazoles as antimicrobial agents. Med. Chem. Res. 2012, 21, 867–873. [CrossRef]
13. Hamad, N.S.; Al-Haidery, N.H.; Al-Masoudi, I.A.; Sabri, M.; Sabri, L.; Al-Masoudi, N.A. Amino Acid Derivatives, Part 4:
Synthesis and anti-HIV activity of new naphthalene derivatives. Arch. Pharm. Chem. Life Sci. 2010, 343, 397–403. [CrossRef]
[PubMed]
14. Sipkina, N.Y.; Polyakova, M.V.; Yakovlev, I.P. Development and Validation of a new HPLC Method for quantifcation of a novel
antifungal drug based on 1,3,4-thiadiazole and its impurities. Chromatographia 2019, 82, 1633–1639. [CrossRef]

Molecules 2021, 26, 1708
16 of 16
15. Hamama, W.S.; Gouda, M.A.; Badr, M.H.; Zoorob, H.H. Synthesis, antioxidant, and antitumor evaluation of certain new
N-substituted-2-amino-1,3,4-thiadiazoles. Med. Chem. Res. 2013, 22, 3556–3565. [CrossRef]
16. Zhu, H.M.; Qin, J.H.; Ouyang, G.P. Synthesis and antitumor activities of novel 4-(5-N-substituted-1,3,4-thiadiazole- 2-
thiol)benzo[4,5] furo [3,2-d] pyrimidine derivatives. Chin. J. Syn. Chem. 2012, 20, 156–160.
17. Tan, X.H.; Song, X.J.; Wang, Y.G. Synthesis and biological activity of N-[5-(3-pyridyl)-1,3,4-thiadiazol-2-y1]-N’-(substituted
phenyl)ureas. J. Cent. Chin. Norm. Univ. 2008, 42, 62–64.
18. Wahid, A.; Basra, S.M.A.; Farooq, M. Thiourea: A molecule with immense biological significance for plants. Int. J. Agric. Biol.
2017, 19, 911–920. [CrossRef]
19. Rizki, I.N.; Tanaka, Y.; Okibe, N. Thiourea bioleaching for gold recycling from e-waste. Waste Manag. 2019, 84, 158–165. [CrossRef]
20. Shakeel, A.; Altaf, A.A.; Qureshi, A.M.; Badshah, A. Thiourea derivatives in drug design and medicinal chemistry: A short review.
J. Drug Des. Med. Chem. 2016, 2, 10–20. [CrossRef]
21. Xu, X.Y.; Qian, X.H.; Li, Z.; Huang, Q.C.; Chen, G. Synthesis and insecticidal activity of new substituted N-aryl-N’-benzoylthiourea
compounds. J. Fluorine. Chem. 2003, 121, 51–54. [CrossRef]
22. Keche, A.P.; Hatnapure, G.D.; Tale, R.H.; Rodge, A.H.; Birajdar, S.S.; Kamble, V.M. A novel pyrimidine derivatives with aryl urea,
thiourea and sulfonamide moieties: Synthesis, anti-inflam matory and antimicrobial evaluation. Bioorg. Med. Chem. Lett. 2012, 22,
3445–3448. [CrossRef] [PubMed]
23. Ma, S.J.; Wu, L.L.; Liu, M.; Huang, Y.D.; Wang, Y.M. Asymmetric aza-Michael additions of 4-nitrophthalimide to nitroalkenes and
preliminary study of the products for herbicidal activities. Tetrahedron 2013, 69, 2613–2618. [CrossRef]
24. Abbas, S.Y.; El-Sharief, M.A.M.S.; Basyouni, W.M.; Fakhr, I.M.I.; El-Gammal, E.W. Thiourea derivatives incorporating a hippuric
acid moiety: Synthesis and evaluation of antibacterial and antifungal activities. Eur. J. Med. Chem. 2013, 64, 111–120. [CrossRef]
[PubMed]
25. Rajesh, S.M.; Kumar, R.S.; Libertsen, L.A.; Perumal, S.; Yogeeswari, P.; Sriram, D. A green expedient synthesis of pyridopyrimidine-
2-thiones and their antitubercular activity. Bioorg. Med. Chem. Lett. 2011, 21, 3012–3016. [CrossRef] [PubMed]
26. Bhabak, K.P.; Bhowmick, D. Synthesis and structural characterization of some trisulfide analoges of thiouracil-based antithyroid
drugs. J. Mol. Struct. 2012, 1022, 16–24. [CrossRef]
27. Suhas, R.; Chandrashekar, S.; Gowda, D.C. Synthesis of uriedo and thiouriedo derivatives of peptide conjugated heterocycles—
A new class of promising antimicrobials. Eur. J. Med. Chem. 2012, 48, 179–191. [CrossRef]
28. Hu, Q.; Lin, G.S.; Duan, W.G.; Huang, M.; Lei, F.H. Synthesis and biological activity of novel (Z)- and (E)-verbenone oxime esters.
Molecules 2017, 22, 1678. [CrossRef]
29. Kang, G.Q.; Duan, W.G.; Lin, G.S.; Yu, Y.P.; Wang, X.Y.; Lu, S.Z. Synthesis of bioactive compounds from 3-carene (II): Synthesis,
antifungal activity and 3D-QSAR study of (Z)- and (E)-3-caren-5-one oxime sulfonates. Molecules 2019, 24, 477. [CrossRef]
30. Yu, Y.P.; Duan, W.G.; Lin, G.S.; Kang, G.Q.; Wang, X.Y.; Lei, F.H. Synthesis, biological activity and three-dimensional quantitative
structure-activity relationship (3D-QSAR) study of novel 4-methyl-1,2,4-triazole-thioethers containing gem- dimethylcyclo-
propane ring. Chin. J. Org. Chem. 2020, 40, 1647–1657. [CrossRef]
31. Chen, N.Y.; Duan, W.G.; Lin, G.S.; Liu, L.Z.; Zhang, R.; Li, D.P. Synthesis and antifungal activity of dehydroabietic acid-based
1,3,4-thiadiazole-thiazolidinone compounds. Mol. Divers. 2016, 20, 897–905. [CrossRef]
32. Lin, G.S.; Bai, X.; Duan, W.G.; Cen, B.; Huang, M.; Lu, Z.S. High value-added application of sustainable natural forest product
α
-pinene: Synthesis of myrtenal oxime esters as potential KARI inhibitors. ACS Sustain. Chem. Eng. 2019, 7, 7862–7868. [CrossRef]
33. Wang, B.L.; Shi, Y.X.; Zhang, S.J.; Ma, Y.; Wang, H.X.; Zhang, L.Y.; Wei, W.; Liu, X.H.; Li, Y.H.; Li, Z.M.; et al. Syntheses, biological
activities and SAR studies of novel carboxamide compounds containing piperazine and arylsulfonyl moieties. Eur. J. Med. Chem.
2016, 117, 167–178. [CrossRef]
34. Zhu, X.P.; Lin, G.S.; Duan, W.G.; Li, Q.M.; Li, F.Y.; Lu, S.Z. Synthesis and antiproliferative evaluation of novel longifolene-derived
tetralone derivatives bearing 1,2,4-triazole moiety. Molecules 2020, 25, 986. [CrossRef] [PubMed]
35. Ghorai, P.; Dussault, P.H.; Hu, C.H. Synthesis of spiro-bisperoxyketals. Org. Lett. 2008, 10, 2401–2404. [CrossRef]
36. Lin, G.S.; Duan, W.G.; Li, Z.S.; Bai, X.; Hu, Q.; Lei, F.H. Synthesis and properties of myrtenal-based thiadiazole-amide compounds.
Fine. Chem. 2017, 34, 588–595.
37. Javid, M.T.; Rahim, F.; Taha, M.; Rehman, H.U.; Nawaz, M.; Wadood, A.; Imran, S.; Uddin, I.; Mosaddik, A.; Khan, K.M. Synthesis,
in vitro alpha-glucosidase inhibitory potential and molecular docking study of thiadiazole analogs. Bioorg. Chem. 2018, 78,
201–209. [CrossRef] [PubMed]
38. Liu, X.H.; Shi, Y.X.; Ma, Y.; Zhang, C.Y.; Dong, W.L.; Pan, L.; Wang, B.L.; Li, B.J.; Li, Z.M. Synthesis, antifungal activities and
3D-QSAR study of N-(5-substituted-1,3,4-thiadiazol-2-yl)cyclopropane- carboxamides. Eur. J. Med. Chem. 2009, 44, 2782–2786.
[CrossRef]