Discovery of Cysteine and Its Derivatives as Novel Antiviral and Antifungal Agents

Abstract

Based on the structure of the natural product cysteine, a series of thiazolidine-4-carboxylic acids were designed and synthesized. All target compounds bearing thiazolidine-4-carboxylic acid were characterized by 1H-NMR, 13C-NMR, and HRMS techniques. The antiviral and antifungal activities of cysteine and its derivatives were evaluated in vitro and in vivo. The results of anti-TMV activity revealed that all compounds exhibited moderate to excellent activities against tobacco mosaic virus (TMV) at the concentration of 500 μg/mL. The compounds cysteine (1), 3–4, 7, 10, 13, 20,23, and 24 displayed higher anti-TMV activities than the commercial plant virucide ribavirin (inhibitory rate: 40, 40, and 38% at 500 μg/mL for inactivation, curative, and protection activity in vivo, respectively), especially compound 3 (inhibitory rate: 51%, 47%, and 49% at 500 μg/mL for inactivation, curative, and protection activity in vivo, respectively) with excellent antiviral activity emerged as a new antiviral candidate. Antiviral mechanism research by TEM exhibited that compound 3 could inhibit virus assembly by aggregated the 20S protein disk. Molecular docking results revealed that compound 3 with higher antiviral activities than that of compound 24 did show stronger interaction with TMV CP. Further fungicidal activity tests against 14 kinds of phytopathogenic fungi revealed that these cysteine derivatives displayed broad-spectrum fungicidal activities. Compound 16 exhibited higher antifungal activities against Cercospora arachidicola Hori and Alternaria solani than commercial fungicides carbendazim and chlorothalonil, which emerged as a new candidate for fungicidal research.

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molecules
Article
Discovery of Cysteine and Its Derivatives as Novel Antiviral
and Antifungal Agents
Shan Yang 1,† , Tienan Wang 1, †, Yanan Zhou 1, Li Shi 1, Aidang Lu 1, * and Ziwen Wang 2,*


Citation: Yang, S.; Wang, T.; Zhou, Y.;
Shi, L.; Lu, A.; Wang, Z. Discovery of
Cysteine and Its Derivatives as Novel
Antiviral and Antifungal Agents.
Molecules 2021,26, 383.
https://doi.org/10.3390/molecules2
6020383)
Academic Editor: Jih-Jung Chen
Received: 23 December 2020
Accepted: 11 January 2021
Published: 13 January 2021
Publisher’s Note: MDPI stays neu-
tral with regard to jurisdictional clai-
ms in published maps and institutio-
nal affiliations.
Copyright: © 2021 by the authors. Li-
censee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and con-
ditions of the Creative Commons At-
tribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1School of Chemical Engineering and Technology, Hebei University of Technology, Tianjin 300130, China;
17865513875@163.com (S.Y.); cdwangtienan@163.com (T.W.); zyn990611@163.com (Y.Z.);
s18310724767@163.com (L.S.)
2
Tianjin Key Laboratory of Structure and Performance for Functional Molecules, College of Chemistry, Tianjin
Normal University, Tianjin 300387, China
*Correspondence: luaidang@hebut.edu.cn (A.L.); hxxywzw@tjnu.edu.cn (Z.W.); Tel.: +86-22-60202812 (A.L.);
+86-22-23766531 (Z.W.)
† These authors contributed equally to this work.
Abstract:
Based on the structure of the natural product cysteine, a series of thiazolidine-4-carboxylic
acids were designed and synthesized. All target compounds bearing thiazolidine-4-carboxylic acid
were characterized by
1
H-NMR,
13
C-NMR, and HRMS techniques. The antiviral and antifungal
activities of cysteine and its derivatives were evaluated
in vitro
and
in vivo
. The results of anti-TMV
activity revealed that all compounds exhibited moderate to excellent activities against tobacco mosaic
virus (TMV) at the concentration of 500
µ
g/mL. The compounds cysteine (
1
),
3
–
4
,
7
,
10
,
13
,
20
,
23
, and
24
displayed higher anti-TMV activities than the commercial plant virucide ribavirin (inhibitory rate:
40, 40, and 38% at 500
µ
g/mL for inactivation, curative, and protection activity
in vivo
, respectively),
especially compound 3(inhibitory rate: 51%, 47%, and 49% at 500 µg/mL for inactivation, curative,
and protection activity
in vivo
, respectively) with excellent antiviral activity emerged as a new
antiviral candidate. Antiviral mechanism research by TEM exhibited that compound
3
could inhibit
virus assembly by aggregated the 20S protein disk. Molecular docking results revealed that compound
3
with higher antiviral activities than that of compound
24
did show stronger interaction with
TMV CP. Further fungicidal activity tests against 14 kinds of phytopathogenic fungi revealed that
these cysteine derivatives displayed broad-spectrum fungicidal activities. Compound
16
exhibited
higher antifungal activities against Cercospora arachidicola Hori and Alternaria solani than commercial
fungicides carbendazim and chlorothalonil, which emerged as a new candidate for fungicidal
research.
Keywords:
natural product; cysteine and its derivatives; anti-TMV activity; antifungal activity; mode
of action; molecular docking
1. Introduction
Fungal and viral pathogens can induce various of plant diseases such as those caus-
ing brown spots on peanut leaves, ring rots on stem and fruit of apples, gray mold on
cucumbers and grapes, leading to huge losses to agriculture and horticulture production
and threatening food security [
1
]. Tobacco mosaic virus (TMV), known as “plant cancer”,
not only can seriously harm tobacco, but also can infect potato, pepper, tomato, eggplant,
nightshade, and more than 400 other plant species [
2
,
3
]. The long-term, large-scale use of
traditional high-toxic pesticides not only forces bacteria and viruses to develop resistance,
but also poses a threat to human health. The development of new antifungal and antiviral
agents with unique mode of action is becoming more and more urgent for plant protection
and agricultural production [4–6].
Amino acids are substances that exist widely in Nature. Cysteine is the only sulfur-
containing amino acid with the ability to form disulfide bonds. Due to the bigger atomic
Molecules 2021,26, 383. https://doi.org/10.3390/molecules26020383 https://www.mdpi.com/journal/molecules

Molecules 2021,26, 383 2 of 16
radius of sulfur and the lower dissociation energy of the S
−
H bond, the thiol group of
cysteine possesses the ability to perform both nucleophilic and redox-active functions which
are unfeasible for the other natural amino acids [
7
–
9
]. Cysteine, with its easily modified
molecular structure, has attracted attention from biological and chemical scientists. A
series of biological activities of cysteine and its derivatives have been reported, such as
cytotoxicity [
10
], neurotoxicity [
11
–
13
], oxidant activity [
14
], accelerated DNA oxidative
damage [15], and so on.
Heterocycles are an important framework for the development of new drugs, es-
pecially S-containing heterocycles which have been found to have the ability to induce
apoptosis of various cells [
16
]. Thiazolidine drugs containing N and S atoms can exert
drug effects through various mechanisms of action, such as inhibiting neuraminidase of
influenza A virus [
17
], inhibiting protein synthesis [
18
], accelerating cell apoptosis [
16
,
19
],
enhancing antioxidant capacity [
7
], immune stimulation [
20
], etc. Some thiazolidine-
4-carboxylic acid derivatives can also oxidatively cleave DNA and interact with metal
ions [
21
]. Some thiazolidine derivatives have been used in a variety of synthetic mod-
ifications due to their simple structure, diverse biological characteristics, and excellent
environmental compatibility [22,23].
It is an important research direction for us to find effective antiviral candidates based
on natural products [
24
]. In our previous work, a series of amino acid gossypol Schiff
bases were designed and synthesized. The structure-activity relationship revealed that
both the carboxy group and substituents in amino acids had significant effect on anti-
TMV [
25
]. Combined with our existing work experience and the above findings, we made
systematic research on cysteine and its derivatives (Figure 1) in the present work. All
synthetic compounds were characterized by
1
H-NMR,
13
C-NMR, and HRMS. The antiviral
and antifungal activities of these compounds and the structure-activity relationship were
evaluated systematically.
Molecules 2021, 26, x FOR PEER REVIEW 2 of 16
Amino acids are substances that exist widely in Nature. Cysteine is the only sulfur-
containing amino acid with the ability to form disulfide bonds. Due to the bigger atomic
radius of sulfur and the lower dissociation energy of the S−H bond, the thiol group of
cysteine possesses the ability to perform both nucleophilic and redox-active functions
which are unfeasible for the other natural amino acids [7–9]. Cysteine, with its easily mod-
ified molecular structure, has attracted attention from biological and chemical scientists.
A series of biological activities of cysteine and its derivatives have been reported, such as
cytotoxicity [10], neurotoxicity [11–13], oxidant activity [14], accelerated DNA oxidative
damage [15], and so on.
Heterocycles are an important framework for the development of new drugs, espe-
cially S-containing heterocycles which have been found to have the ability to induce apop-
tosis of various cells [16]. Thiazolidine drugs containing N and S atoms can exert drug
effects through various mechanisms of action, such as inhibiting neuraminidase of influ-
enza A virus [17], inhibiting protein synthesis [18], accelerating cell apoptosis [16,19], en-
hancing antioxidant capacity [7], immune stimulation [20], etc. Some thiazolidine-4-car-
boxylic acid derivatives can also oxidatively cleave DNA and interact with metal ions [21].
Some thiazolidine derivatives have been used in a variety of synthetic modifications due
to their simple structure, diverse biological characteristics, and excellent environmental
compatibility [22,23].
It is an important research direction for us to find effective antiviral candidates based
on natural products [24]. In our previous work, a series of amino acid gossypol Schiff
bases were designed and synthesized. The structure-activity relationship revealed that
both the carboxy group and substituents in amino acids had significant effect on anti-TMV
[25]. Combined with our existing work experience and the above findings, we made sys-
tematic research on cysteine and its derivatives (Figure 1) in the present work. All syn-
thetic compounds were characterized by 1H-NMR, 13C-NMR, and HRMS. The antiviral
and antifungal activities of these compounds and the structure-activity relationship were
evaluated systematically.
Figure 1. Design of cysteine derivatives.
2. Results
2.1. Chemistry
Cysteine and its derivatives 1–7 (Figure 2) were purchased directly. A series of thia-
zole ring containing L-cysteine derivatives 8–24 were designed and synthesized from L-
cysteine and different substituted aldehydes (Scheme 1) by a one-pot method [7,26].
Figure 1. Design of cysteine derivatives.
2. Results
2.1. Chemistry
Cysteine and its derivatives
1
–
7
(Figure 2) were purchased directly. A series of thiazole
ring containing L-cysteine derivatives
8
–
24
were designed and synthesized from L-cysteine
and different substituted aldehydes (Scheme 1) by a one-pot method [7,26].

Molecules 2021,26, 383 3 of 16
Molecules 2021, 26, x FOR PEER REVIEW 3 of 16
Figure 2. Structures of compounds 1–7.
Scheme 1. Synthesis of 8–24.
2.2. Phytotoxic Activity
The phytotoxicity-activity tests revealed that cysteine and its derivatives were safe
for testing on plants at 500 μg/mL. The detailed test procedures can be seen in our previ-
ous reports [5,27]. The detailed test procedures can also be found in the Supplementary
Materials.
2.3. Antiviral Activity
2.3.1. In Vitro Anti-TMV Activity
The anti-TMV activities of cysteine and its derivatives 1–24 are listed in Table 1 with
the commercial drugs ribavirin and ningnanmycin as controls. Compounds 1–7 had better
anti-TMV activities than ribavirin. Among compounds 1–7, compounds 3 (inhibitory rate:
48% at 500 μg/mL) and 4 (inhibitory rate: 45% at 500 μg/mL) showed better anti-TMV
activities than the others. At the concentration of 100 μg/mL, compounds 3 (inhibitory
rate: 13%) and 4 (inhibitory rate: 12%) also displayed better anti-TMV activities than the
commercial plant virucide ribavirin (inhibitory rate: 7%). Most of the cysteine derivatives
Figure 2. Structures of compounds 1–7.
Molecules 2021, 26, x FOR PEER REVIEW 3 of 16
Figure 2. Structures of compounds 1–7.
Scheme 1. Synthesis of 8–24.
2.2. Phytotoxic Activity
The phytotoxicity-activity tests revealed that cysteine and its derivatives were safe
for testing on plants at 500 μg/mL. The detailed test procedures can be seen in our previ-
ous reports [5,27]. The detailed test procedures can also be found in the Supplementary
Materials.
2.3. Antiviral Activity
2.3.1. In Vitro Anti-TMV Activity
The anti-TMV activities of cysteine and its derivatives 1–24 are listed in Table 1 with
the commercial drugs ribavirin and ningnanmycin as controls. Compounds 1–7 had better
anti-TMV activities than ribavirin. Among compounds 1–7, compounds 3 (inhibitory rate:
48% at 500 μg/mL) and 4 (inhibitory rate: 45% at 500 μg/mL) showed better anti-TMV
activities than the others. At the concentration of 100 μg/mL, compounds 3 (inhibitory
rate: 13%) and 4 (inhibitory rate: 12%) also displayed better anti-TMV activities than the
commercial plant virucide ribavirin (inhibitory rate: 7%). Most of the cysteine derivatives
Scheme 1. Synthesis of 8–24.
2.2. Phytotoxic Activity
The phytotoxicity-activity tests revealed that cysteine and its derivatives were safe for
testing on plants at 500
µ
g/mL. The detailed test procedures can be seen in our previous
reports [
5
,
27
]. The detailed test procedures can also be found in the Supplementary Materials.
2.3. Antiviral Activity
2.3.1. In Vitro Anti-TMV Activity
The anti-TMV activities of cysteine and its derivatives
1
–
24
are listed in Table 1with the
commercial drugs ribavirin and ningnanmycin as controls. Compounds
1
–
7
had better anti-
TMV activities than ribavirin. Among compounds
1
–
7
, compounds
3
(inhibitory rate: 48%
at 500
µ
g/mL) and
4
(inhibitory rate: 45% at 500
µ
g/mL) showed better anti-TMV activities
than the others. At the concentration of 100
µ
g/mL, compounds
3
(inhibitory rate: 13%)
and
4
(inhibitory rate: 12%) also displayed better anti-TMV activities than the commercial
plant virucide ribavirin (inhibitory rate: 7%). Most of the cysteine derivatives
8
–
24
with
thiazolidine structure exhibited better anti-TMV activities than that of the commercial plant

Molecules 2021,26, 383 4 of 16
virucide ribavirin. Especially, compounds
23
(inhibitory rate: 46% at 500
µ
g/mL) and
24
(inhibitory rate: 45% at 500
µ
g/mL) showed 10% higher anti-TMV activity than ribavirin
(inhibitory rate: 35% at 500 µg/mL).
Table 1. In vitro antiviral activity of compounds 1–24 against TMV.
Compd. Concn (µg/mL) Inhibition Rate (%) aCompd. Concn (µg/mL) Inhibition Rate (%) a
1-D 500 39 ±313 500 43 ±1
100 13 ±2 100 17 ±1
1-L 500 41 ±214 500 18 ±2
100 11 ±1 100 0
2-D 500 38 ±215 500 34 ±1
100 15 ±2 100 12 ±1
2-L 500 40 ±116 500 40 ±2
100 16 ±2 100 16 ±2
3500 48 ±117 500 39 ±1
100 13 ±2 100 14 ±2
4500 45 ±218 500 24 ±2
100 12 ±1 100 0
5500 42 ±219 500 32 ±2
100 10 ±1 100 0
6500 40 ±220 500 43 ±2
100 20 ±1 100 18 ±2
7500 44 ±221 500 35 ±2
100 22 ±1 100 11 ±1
8500 33 ±222 500 33 ±1
100 9 ±2 100 10 ±1
9500 35 ±123 500 46 ±1
100 12 ±2 100 17 ±2
10 500 39 ±224 500 45 ±1
100 13 ±1 100 19 ±1
11 500 43 ±1Ribavirin 500 35 ±1
100 19 ±1 100 7 ±1
12 500 38 ±2Ningnanmycin 500 61 ±2
100 16 ±1 100 23 ±2
aAverage of three replicates. All results are expressed as mean ±SD.
2.3.2. In Vivo Anti-TMV Activity
In vivo
anti-TMV activity includes three test modes: inactivation, curative, and protec-
tion. As shown in Table 2, most of the compounds also displayed higher
in vivo
activities
than ribavirin. Compound
3
displayed the best anti-TMV activity at 500
µ
g/mL (inactiva-
tion activity, 51%; curative activity, 47%; protection activity, 49%), which is significantly
higher than that of ribavirin (inactivation activity, 40%; curative activity, 40%; protection
activity, 38%).
2.4. Mode of Action Studies
2.4.1. Preliminary Mode of Action
Preliminary mode of action revealed that these compounds can inhibit the assembly
of TMV. The detailed method was described in the literature [
24
] and also can be found in
the Supporting Materials.

Molecules 2021,26, 383 5 of 16
Table 2. In Vivo antiviral activity of compounds 1–24 against TMV.
Compd. Concn (µg/mL) Inactivation Effect (%) aCurative Effect (%) aProtection Effect (%) a
1-D 500 44 ±2 38 ±2 37 ±1
100 18 ±3 15 ±2 14 ±1
1-L 500 43 ±1 42 ±2 40 ±1
100 20 ±2 16 ±1 13 ±2
2-D 500 37 ±2 41 ±3 36 ±1
100 12 ±1 13 ±1 15 ±2
2-L 500 39 ±1 40 ±2 38 ±2
100 11 ±2 14 ±1 12 ±1
3500 51 ±2 47 ±2 49 ±2
100 26 ±1 23±1 25 ±1
4500 45 ±1 43 ±1 46 ±1
100 18 ±2 20 ±2 22 ±2
5500 40 ±1 39 ±2 37 ±2
100 13 ±1 16 ±1 16 ±1
6500 38 ±2 40 ±2 35 ±2
100 16 ±2 16 ±1 16 ±1
7500 44 ±1 42 ±2 41 ±2
100 20 ±2 16 ±1 18 ±1
8500 37 ±2 33 ±2 35 ±2
100 11 ±1 10 ±1 11 ±1
9500 36 ±2 35 ±1 34 ±1
100 10 ±1 9 ±1 9 ±1
10 500 41 ±2 40 ±2 41 ±2
100 10 ±1 10 ±1 13 ±1
11 500 40 ±2 39 ±2 40 ±2
100 15 ±1 17 ±1 12 ±1
12 500 43 ±2 36 ±2 41 ±2
100 14 ±1 13 ±1 15 ±1
13 500 49 ±2 46 ±2 44 ±2
100 18 ±2 15 ±2 13 ±2
14 500 21 ±2 17 ±2 15 ±2
100 0 0 0
15 500 28 ±2 33 ±2 25 ±2
100 9 ±1 11 ±1 7 ±1
16 500 42 ±1 37 ±1 35 ±1
100 0 0 0
17 500 40 ±1 36 ±1 35 ±1
100 0 0 0
18 500 24 ±2 23 ±2 20 ±2
100 0 0 0
19 500 34 ±2 30 ±2 36 ±2
100 0 0 0
20 500 47 ±2 45 ±2 43 ±2
100 21 ±2 19 ±2 14 ±2
21 500 37 ±2 40 ±2 34 ±2
100 11 ±2 15 ±2 11 ±2
22 500 33 ±2 37 ±2 32 ±2
100 10 ±2 13 ±2 9 ±2

Molecules 2021,26, 383 6 of 16
Table 2. Cont.
Compd. Concn (µg/mL) Inactivation Effect (%) aCurative Effect (%) aProtection Effect (%) a
23 500 48 ±1 49 ±1 47 ±1
100 19 ±2 15 ±2 17 ±2
24 500 47 ±1 42 ±1 43 ±1
100 15 ±1 17 ±1 15 ±1
Ribavirin 500 40 ±1 40 ±1 38 ±1
100 13 ±1 15 ±1 10 ±1
Ningnanmycin
500 56 ±2 53 ±2 59 ±1
100 28 ±1 24 ±1 30 ±1
aAverage of three replicates. All results are expressed as mean ±SD.
2.4.2. Docking Studies
To further study the mechanism of the interaction between cysteines and TMV CP,
we chose AutoDock Vina 1.1.2 for molecular docking [
28
]. The docking poses are ranked
according to their docking sites and the lowest binding energy of macromolecule-ligand
complex is considered being the best. It can be proved H-bond interaction and strong
binding affinity between cysteines and TMV CP.
2.5. Fungicidal Activity
2.5.1. In Vitro Fungicidal Activity
We further tested the inhibitory effects of compounds
1
–
24
on 14 common agricultural
pathogens at a concentration of 50
µ
g/mL using fungicidal growth rate assay [
27
,
29
] with
commercial fungicidal agents chlorothalonil and carbendazim as controls. All compounds
showed broad-spectrum fungicidal activities (Table 3). Compound
16
exhibited higher
antifungal activities against Cercospora arachidicola Hori (inhibitory rate: 71%) and Alternaria
solani (inhibitory rate: 58%) than commercial fungicides carbendazim and chlorothalonil.
Compound 16 had an inhibitory rate of 83% against Physalospora piricola.
2.5.2. In Vivo Fungicidal Activity
Compounds
1
–
24
were further tested
in vivo
fungicidal activity at a concentration of
200
µ
g/mL using standard methods [
29
] with azoxystrobin as a control against Blumeria
graminis f.sp. tritici,Sclerotinia sclerotiorum,Botrytis cinereal,Rhizoctonia solani,Corynespora
cassiicola, and Phytophthora capsica 6 kinds of pathogenic fungi. As shown in Table 4, most of
these compounds also displayed broad-spectrum
in vivo
fungicidal activities. Compounds
12
and
18
showed 20% inhibition rate against Rhizoctonia solani. Compound
12
exhibited
higher activity than other compounds against Botrytis cinereal. The inhibition rate of
compound 18 is more than 20% against Corynespora cassiicola.

Molecules 2021,26, 383 7 of 16
Table 3. In vitro fungicidal activities of compounds 1–24 against 14 kinds of fungi.
Compd. Fungicidal Activity (%) aat 50 µg/mL
F.C bC.H bP.P bR.C bB.M bW.A bF.M bA.S bF.G bP.I bP.C bS.S bB.C bR.S b
1-D 18 ±2 7 ±1 30 ±2 9 ±1 10 ±2 11 ±3 9 ±1 37 ±3 16 ±2 8 ±2 27 ±3 20 ±1 16 ±2 11 ±1
1-L 16 ±1 5 ±1 35 ±1 11 ±1 7 ±2 16 ±1 6 ±1 35 ±1 10 ±2 10 ±1 22 ±1 25 ±1 18 ±1 12 ±1
2-D 13 ±1 19 ±2 25 ±2 12 ±1 26 ±2 30 ±2 16 ±1 42 ±1 24 ±1 13 ±1 16 ±1 31 ±3 24 ±1 17 ±2
2-L 18 ±1 13 ±2 29 ±2 15 ±1 21 ±2 33 ±2 19 ±1 49 ±2 26 ±1 13 ±1 17 ±1 36 ±2 21 ±1 14 ±1
311 ±1 13 ±1 22 ±1 26 ±2 10 ±1 11 ±1 13 ±1 39 ±1 13 ±1 15 ±1 21 ±1 23 ±1 17 ±1 16 ±1
49±1 7 ±1 31 ±1 16 ±1 13 ±2 22 ±1 16 ±1 36 ±1 13 ±2 15 ±2 12 ±1 19 ±1 10 ±1 17 ±2
523 ±1 16 ±2 35 ±1 10 ±1 23 ±2 16 ±1 24 ±1 42 ±1 10 ±1 13 ±1 17 ±1 29 ±1 14 ±1 13 ±2
612 ±1 18 ±1 41 ±1 36 ±1 26 ±2 19 ±2 15 ±1 43 ±1 13 ±1 11 ±1 17 ±1 21 ±1 8 ±1 13 ±1
737 ±1 15 ±1 32 ±1 42 ±1 25 ±1 19 ±1 23 ±1 46 ±1 17 ±1 13 ±1 34 ±1 19 ±1 22 ±1 17 ±1
810 ±1 14 ±1 14 ±1 9 ±1 7 ±1 16 ±1 6 ±2 42 ±1 42 ±1 16 ±1 12 ±1 19 ±2 14 ±1 6 ±1
916 ±1 14 ±1 17 ±1 5 ±1 17 ±1 12 ±1 18 ±1 39 ±1 32 ±2 10 ±2 6 ±1 25 ±1 13 ±1 8 ±1
10 29 ±1 52 ±2 62 ±1 28 ±1 14 ±2 32 ±1 18 ±2 39 ±1 32 ±1 10 ±1 12 ±1 13 ±1 9 ±1 16 ±1
11 19 ±2 14 ±1 24 ±1 12 ±1 7 ±1 16 ±1 18 ±2 39 ±2 13±10 ±1 10 ±1 31 ±1 4 ±1 23 ±1
12 29 ±1 19 ±1 10 ±1 11 ±2 14 ±2 24 ±2 24 ±1 23 ±1 10 ±1 13 ±2 14 ±1 19 ±2 13 ±1 12 ±1
13 26 ±1 10 ±1 38 ±2 9 ±1 17 ±1 16 ±1 12 ±1 39 ±1 10 ±1 3 ±1 12 ±1 25 ±1 11 ±1 6 ±1
14 13 ±1 0 17 ±1 0 10 ±1 12 ±1 6 ±1 42 ±1 16 ±1 7 ±1 8 ±1 25 ±1 13 ±1 8 ±1
15 19 ±2 10 ±2 20 ±2 18 ±1 7 ±1 16 ±2 12 ±2 27 ±1 10 ±1 13 ±1 4 ±1 19 ±1 9 ±1 12 ±1
16 32 ±1 71 ±1 83 ±1 54 ±1 45 ±1 16 ±2 18 ±1 58 ±1 32 ±1 10 ±1 31 ±2 50 ±1 48 ±1 29 ±1
17 16 ±1 10 ±1 38 ±1 18 ±1 10 ±1 20 ±1 12 ±1 31 ±1 10 ±1 13 ±2 16 ±1 44 ±2 18 ±1 8 ±1
18 16 ±1 48 ±1 45 ±2 23 ±2 10 ±1 20 ±1 18 ±1 27 ±1 19 ±1 10 ±1 20 ±1 31 ±1 7 ±1 12 ±1
19 48 ±2 33 ±1 45 ±1 19 ±1 10 ±1 36 ±1 35 ±1 35 ±2 13 ±2 7 ±1 29 ±1 13 ±1 11 ±1 6 ±1
20 26 ±1 10 ±1 21 ±1 12 ±1 3 ±2 16 ±1 12 ±1 39 ±2 16 ±1 10 ±1 6 ±1 19 ±1 7 ±1 6 ±1
21 19 ±2 0 17 ±1 28 ±1 14 ±2 20 ±1 24 ±1 46 ±1 32 ±1 10 ±2 22 ±1 25 ±1 4 ±1 12 ±1
22 16 ±1 10 ±1 31 ±1 14 ±1 7 ±1 12 ±1 12 ±2 39 ±1 32 ±1 16 ±1 16 ±1 19 ±1 21 ±1 12 ±1
23 23 ±1 19 ±1 21 ±1 9 ±1 10 ±1 16 ±1 29 ±1 35 ±1 16 ±1 13 ±1 6 ±1 31 ±1 21 ±1 12 ±1
24 19 ±1 19 ±1 35 ±1 12 ±1 7 ±1 16 ±1 18 ±1 50 ±1 42 ±1 3 ±1 6 ±1 31 ±1 27 ±2 14 ±1
Chlorothalonil c100 69 ±1 89 ±1 100 91 ±1 95 ±1 100 56 ±1 100 100 55 ±2 100 100 100
Carbendazim c100 53 ±1 100 100 100 100 71 ±1 56 ±2 88 ±2 83 ±1 90 ±2 100 96 ±1 100
a
Average of three replicates. All results are expressed as mean
±
SD.
b
F.C, Fusarium oxysporium f. sp. Cucumeris, C.H, Cercospora arachidicola Hori, P.P, Physalospora piricola, R.C, Rhizoctonia cerealis, B.M, Bipolaris
maydis, W.A, Watermelon anthracnose, F.M, Fusarium moniliforme, A.S, Alternaria solani, F.G, Fusarium graminearum, P.I, Phytophthora infestans, P.C, Phytophthora capsica, S.S, Sclerotinia sclerotiorum, B.C, Botrytis cinereal,
R.S, Rhizoctonia solani.cThe commercial agricultural fungicides were used for comparison of antifungal activity.

Molecules 2021,26, 383 8 of 16
Table 4. In vivo fungicidal activities of compounds 1–24 against six kinds of fungi.
Compd. Inhibition Rate a(%) at 200 µg/mL
B.G bS.S bB.C bR.S bC.C bP.C b
1-D 0 12 ±2 5 ±1 9 ±2 10 ±1 5 ±1
1-L 0 11 ±1 7 ±2 7 ±1 11 ±2 5 ±1
2-D 0 8 ±2 12 ±2 8 ±2 13 ±2 12 ±1
2-L 0 10 ±2 11 ±1 7 ±2 17 ±2 13 ±1
30 7 ±2 13 ±1 8 ±2 13 ±1 13 ±1
40 10 ±1 11 ±2 0 13 ±2 0
50 19 ±3 4 ±1 11 ±1 5 ±1 12 ±2
60 16 ±1 15 ±2 8 ±2 11 ±1 7 ±1
70 10 ±2 12 ±1 16 ±1 13 ±2 5 ±1
80 4 ±1 9 ±1 0 6 ±2 5 ±1
90 11 ±1 5 ±1 0 0 0
10 0 11 ±3 12 ±2 14 ±1 10 ±1 0
11 0 16 ±1 1 ±1 11 ±3 17 ±3 0
12 0 11 ±3 7 ±3 20 ±1 13 ±2 5 ±1
13 0 17 ±1 6 ±1 0 13 ±2 5 ±1
14 0 11 ±2 7 ±1 0 11 ±1 0
15 0 11 ±2 5 ±1 11 ±2 4 ±1 0
16 0 19 ±1 26 ±1 16 ±2 13 ±1 10 ±1
17 0 17 ±2 15 ±2 0 0 5 ±2
18 0 4 ±1 3 ±1 20 ±1 26 ±1 5 ±1
19 20 ±2 4 ±1 11 ±2 0 20 ±2 10 ±2
20 0 17 ±2 11 ±2 0 20 ±1 0
21 0 7 ±1 12 ±2 0 11 ±2 5 ±1
22 0 16 ±2 15 ±2 5 ±1 8 ±1 0
23 0 10 ±2 9 ±1 0 12 ±2 5 ±1
24 0 11 ±1 15 ±2 0 22 ±1 0
azoxystrobin c81 ±2 100 100 75 ±2 80 ±1 85 ±2
a
Average of three replicates. All results are expressed as mean
±
SD.
b
B.G, Blumeria graminis f.sp. tritici, S.S,
Sclerotinia sclerotiorum, B.C, Botrytis cinereal, R.S, Rhizoctonia solani, C.C, Corynespora cassiicola, P.C, Phytophthora
capsica.cThe commercial agricultural fungicides were used for comparison of antifungal activity.
3. Discussion
3.1. Synthesis
Compounds
8
–
24
bearing a thiazolidine ring based on L-cysteine were synthesized
under basic conditions by a one-pot method in high to nearly quantitative yields. The
cyclization of aldehydes with cysteine has been reported in the literature under conditions
using a water/ethanol mixture (50:50, v:v) [
7
,
26
]. To promote the precipitation of the
product from the reaction system and improve the yield of the reaction, the amount of
ethanol was reduced based on references. The yields of compounds
8
–
24
ranged from 82%
to 99%. Two nucleophilic attacks of the aldehyde produced the closed ring structure and led
to the generation of a new uncontrolled chiral center. Thus, compounds
8
–
24
are obtained
as diastereomeric mixtures [
7
]. Although the 2R, 4Rand 2S,4Risomers were mixed, the
distinctive singlet around 5.5 ppm of the hydrogen on C-2 gave a clearly distinguishable
ratio of the isomers [7].
3.2. Phytotoxic Activity
Healthy growing 5–6 leaf stage tobaccos (Nicotiana tabacum var Xanthi nc) were slected
for phytotoxic activity tests. The fresh solutions (500
µ
g/mL) of compounds
1
–
24
were
gently smeared on the leaves. One hour after treatment, all the treated tobacco leaves were
observed intact. The growth of tobacco leaves was continuously observed and calculated
the weight after 3, 7, and 10 days, respectively. Encouragingly, none of the compounds
were toxic to tobacco leaves.

Molecules 2021,26, 383 9 of 16
3.3. Structure-Activity Relationship of the Antiviral Activity
As the results of Table 1, compounds
1
–
7
had better anti-TMV activities than ribavirin.
Chirality has no obvious effect on the antiviral activities of these compounds (inhibitory
effect:
1
-
D≈1
-
L
,
2-D ≈2
-
L
). Therefore, in the follow-up study, we only used L-cysteine
derivatives to explore the effect of different substituents. The antiviral activity was in-
creased slightly when the S atom and N atom of cysteine had substituents (antiviral activity:
3
>
4
>
7
>
1
,
2
,
5
,
6
). Cyclization is an important method to improve molecular stability and
biological activity. A series of thiazolidine-4-carboxylic acid-containing compounds
8
–
24
were designed and synthesized. As shown in Table 1, most of the designed compounds
displayed better antiviral activities than ribavirin. For the substituted benzene compounds
8
–
17
, the electron-withdrawing group substitution at the para position of the benzene ring
is beneficial to the improvement of biological activity (inhibitory effect:
11
>
16
>
10
,
17
>
9
>
8
). When introduction of electron-donating groups such as CH
3
O (
12
), CH
3
(
13
) into
the para position of the benzene ring, the anti-TMV activity is also improved (inhibitory
effect:
13
>
12
>
15
>
8
,
17
>
9
>
8
). However, the activity is sharply declined after the
introduction of -OH at the para-position of the benzene ring (
14
). When the 2-position of
thiazolidine structure is heterocyclic groups, such as thiophene (
18
), furan (
19
), pyridine
(
20
), only compound
20
displayed better anti-TMV activity. Compounds
21
–
24
with the
aliphatic groups at 2-position of thiazolidine exhibited moderate to excellent anti-TMV
activities, the introduction of long-chain fat groups (
23
), and cyclohexyl groups (
24
) at
2-position of thiazolidine can lead to an increase in activity (23 >24 >35 >22).
Just like the antiviral activity
in vitro
, compounds
2−24
displayed moderate to good
in vivo
activities (Table 2), and the activities of compounds
3
,
4
,
7
,
13
,
23
,
24
are significantly
higher than that of cysteine. In particular, compound
3
showed excellent activity against
TMV (inhibitory rate: 51%, 47%, and 49% at 500
µ
g/mL for inactivation, curative, and
protection activity
in vivo
, respectively). Compounds
4
,
7
,
10
,
13
,
20
,
23
, and
24
displayed
higher anti-TMV activities than the commercial plant virucide ribavirin (inhibitory rate:
40, 40, and 38% at 500
µ
g/mL for inactivation, curative, and protection activity
in vivo
,
respectively). Compounds
2
,
10
,
12
,
16
exhibited approximate anti-TMV activities as
cysteine. The structure-activity relationship revealed that the substitutions of S atom and
N atom had a great influence on the anti-TMV activity.
3.4. Study on the Mechanism of Anti-TMV Activity
3.4.1. Preliminary Mode of Action
Considering structural features and biological activity, compound
3
was chosen to
study the mechanism of anti-TMV activity. The results showed that 20S CP and TMV RNA
could assembled into TMV particles of about 300 nm in length, and dimethyl sulfoxide
(DMSO) had no effect on the assembly (panels A and B of Figure 3). Compound
3
can
cause a reduction in the length and number of TMV particles, indicating that it can inhibit
the assembly of the viruses (panel C of Figure 3). The interaction experiment between
compound
3
and 20S CP was also designed. The TMV protein can form the homogeneous-
dispersed disc structure (Figure 4A), and a small amount of DMSO has no effect on the
formation (Figure 4B). As seen in Figure 4C, compound
3
can lead to polymerization of
TMV CP.
3.4.2. Molecular Docking Study
Molecular docking studies were performed to explore the binding sites of cysteine
derivatives on TMV CP. Compounds
3
,
23
, and
24
were selected for molecular docking
with TMV CP (PDB code 1EI7). The results showed that compound
3
has lain into the TMV
CP activity pocket of SER 255, ASN 73, and GLN 257 (Figure 5A). Compound
3
forms five
conventional hydrogen bonds with the active site of SER 255 (1.9 Å and 2.3 Å), GLN 257
(2.2 Å and 2.8 Å), and ASN 73 (2.4 Å) (Figure 5A). Compound
23
forms three conventional
hydrogen bonds with the active site of SER 255 (2.4 Å), GLN 257 (1.2 Å), and ASN 73 (2.4 Å)
(Figure 5B). As seen in Figure 5C, compound
24
forms two conventional hydrogen bonds

Molecules 2021,26, 383 10 of 16
with ASN 73 (2.1 Å) and GLY 137 (2.5 Å). The molecular docking results indicate that these
compounds interact with CP through hydrogen bonding. The results of molecular docking
also showed that compound
3
had more binding sites with TMV CP and shorter hydrogen
bond distance. The stronger the interaction with TMV CP, the greater influence on the
assembly of TMV, and the higher the inhibition rate. This result is consistent with the
activity test.
Molecules 2021, 26, x FOR PEER REVIEW 9 of 16
Considering structural features and biological activity, compound 3 was chosen to
study the mechanism of anti-TMV activity. The results showed that 20S CP and TMV RNA
could assembled into TMV particles of about 300 nm in length, and dimethyl sulfoxide
(DMSO) had no effect on the assembly (panels A and B of Figure 3). Compound 3 can
cause a reduction in the length and number of TMV particles, indicating that it can inhibit
the assembly of the viruses (panel C of Figure 3). The interaction experiment between
compound 3 and 20S CP was also designed. The TMV protein can form the homogeneous-
dispersed disc structure (Figure 4A), and a small amount of DMSO has no effect on the
formation (Figure 4B). As seen in Figure 4C, compound 3 can lead to polymerization of
TMV CP.
(A) (B) (C)
Figure 3. TMV rod assembly inhibition of compound 3 and NK0209: (A) 20S CP disk + RNA (500 nm scale bar), (B) 20S
CP disk + RNA + 1/100 DMSO (500 nm scale bar), (C) 20S CP disk + RNA + 10 μM 3 (500 nm scale bar).
(A) (B) (C)
Figure 4. 20S CP disk assembly inhibition of compound 3 (100 nm scale bar): (A) CP, (B) CP + 1/100 DMSO (100 nm scale
bar), (C) CP + 10 μM compound 3 (200 nm scale bar).
3.4.2. Molecular Docking Study
Molecular docking studies were performed to explore the binding sites of cysteine
derivatives on TMV CP. Compounds 3, 23, and 24 were selected for molecular docking
with TMV CP (PDB code 1EI7). The results showed that compound 3 has lain into the
TMV CP activity pocket of SER 255, ASN 73, and GLN 257 (Figure 5A). Compound 3
forms five conventional hydrogen bonds with the active site of SER 255 (1.9 Å and 2.3 Å),
GLN 257 (2.2 Å and 2.8 Å), and ASN 73 (2.4 Å) (Figure 5A). Compound 23 forms three
conventional hydrogen bonds with the active site of SER 255 (2.4 Å), GLN 257 (1.2 Å), and
ASN 73 (2.4 Å) (Figure 5B). As seen in Figure 5C, compound 24 forms two conventional
hydrogen bonds with ASN 73 (2.1 Å) and GLY 137 (2.5 Å). The molecular docking results
indicate that these compounds interact with CP through hydrogen bonding. The results
of molecular docking also showed that compound 3 had more binding sites with TMV CP
Figure 3.
TMV rod assembly inhibition of compound
3
and NK0209: (
A
) 20S CP disk + RNA (500 nm scale bar), (
B
) 20S CP
disk + RNA + 1/100 DMSO (500 nm scale bar), (C) 20S CP disk + RNA + 10 µM3(500 nm scale bar).
Molecules 2021, 26, x FOR PEER REVIEW 9 of 16
Considering structural features and biological activity, compound 3 was chosen to
study the mechanism of anti-TMV activity. The results showed that 20S CP and TMV RNA
could assembled into TMV particles of about 300 nm in length, and dimethyl sulfoxide
(DMSO) had no effect on the assembly (panels A and B of Figure 3). Compound 3 can
cause a reduction in the length and number of TMV particles, indicating that it can inhibit
the assembly of the viruses (panel C of Figure 3). The interaction experiment between
compound 3 and 20S CP was also designed. The TMV protein can form the homogeneous-
dispersed disc structure (Figure 4A), and a small amount of DMSO has no effect on the
formation (Figure 4B). As seen in Figure 4C, compound 3 can lead to polymerization of
TMV CP.
(A) (B) (C)
Figure 3. TMV rod assembly inhibition of compound 3 and NK0209: (A) 20S CP disk + RNA (500 nm scale bar), (B) 20S
CP disk + RNA + 1/100 DMSO (500 nm scale bar), (C) 20S CP disk + RNA + 10 μM 3 (500 nm scale bar).
(A) (B) (C)
Figure 4. 20S CP disk assembly inhibition of compound 3 (100 nm scale bar): (A) CP, (B) CP + 1/100 DMSO (100 nm scale
bar), (C) CP + 10 μM compound 3 (200 nm scale bar).
3.4.2. Molecular Docking Study
Molecular docking studies were performed to explore the binding sites of cysteine
derivatives on TMV CP. Compounds 3, 23, and 24 were selected for molecular docking
with TMV CP (PDB code 1EI7). The results showed that compound 3 has lain into the
TMV CP activity pocket of SER 255, ASN 73, and GLN 257 (Figure 5A). Compound 3
forms five conventional hydrogen bonds with the active site of SER 255 (1.9 Å and 2.3 Å),
GLN 257 (2.2 Å and 2.8 Å), and ASN 73 (2.4 Å) (Figure 5A). Compound 23 forms three
conventional hydrogen bonds with the active site of SER 255 (2.4 Å), GLN 257 (1.2 Å), and
ASN 73 (2.4 Å) (Figure 5B). As seen in Figure 5C, compound 24 forms two conventional
hydrogen bonds with ASN 73 (2.1 Å) and GLY 137 (2.5 Å). The molecular docking results
indicate that these compounds interact with CP through hydrogen bonding. The results
of molecular docking also showed that compound 3 had more binding sites with TMV CP
Figure 4.
20S CP disk assembly inhibition of compound
3
(100 nm scale bar): (
A
) CP, (
B
) CP + 1/100 DMSO (100 nm scale
bar), (C) CP + 10 µM compound 3(200 nm scale bar).
3.5. Structure-Activity Relationship of the Fungicidal Activity
As the results showed in Table 3, all compounds exhibited broad-spectrum fungicidal
activities
in vitro
. Kapachery et al. [
30
] reported that N-acetylcysteine (NAC, compound
7
)
affected four kinds of bacteria (Aeromonas hydrophila,Pseudomonas putida,Stenotrophomonas
sp., and Serratia marcescens) at the concentration of 1.5 mg/mL which were isolated from
polluted reverse osmosis membrane. Perez-Giraldo et al. [
31
] confirmed that NAC could
control bacterial biofilm formation on medical catheters. NAC has been widely used as a
mucolytic agent in the treatment of chronic bronchitis [
32
]. However, the activity of cysteine
and its derivatives on plant phytopathogenic fungi was not significant. The inhibitory
effect of NAC on 14 plant pathogens was less than 50%. Changing the substituents of N, S,
and O atoms in cysteine can slightly raise its anti-plant pathogen activity. Encouraged by
the antifungal activities of compound
16
, compounds
1
–
24
were further evaluated
in vivo
fungicidal activity. The modification of cysteine had some effect on its fungicidal activity
in vivo
. It can be seen from the results of fungicidal activity that cysteine and its derivatives
have broad-spectrum activity, but with a low to moderate degree of activity.

Molecules 2021,26, 383 11 of 16
Molecules 2021, 26, x FOR PEER REVIEW 10 of 16
and shorter hydrogen bond distance. The stronger the interaction with TMV CP, the
greater influence on the assembly of TMV, and the higher the inhibition rate. This result
is consistent with the activity test.
Figure 5. Molecule docking results of (A) compound 3, (B) compound 23, (C) compound 24 with TMV CP.
3.5. Structure-Activity Relationship of the Fungicidal Activity
As the results showed in Table 3, all compounds exhibited broad-spectrum fungi-
cidal activities in vitro. Kapachery et al. [30] reported that N-acetylcysteine (NAC, com-
pound 7) affected four kinds of bacteria (Aeromonas hydrophila, Pseudomonas putida, Steno-
trophomonas sp., and Serratia marcescens) at the concentration of 1.5 mg/mL which were
isolated from polluted reverse osmosis membrane. Perez-Giraldo et al. [31] confirmed that
NAC could control bacterial biofilm formation on medical catheters. NAC has been
widely used as a mucolytic agent in the treatment of chronic bronchitis [32]. However, the
activity of cysteine and its derivatives on plant phytopathogenic fungi was not significant.
The inhibitory effect of NAC on 14 plant pathogens was less than 50%. Changing the sub-
stituents of N, S, and O atoms in cysteine can slightly raise its anti-plant pathogen activity.
Encouraged by the antifungal activities of compound 16, compounds 1–24 were further
evaluated in vivo fungicidal activity. The modification of cysteine had some effect on its
fungicidal activity in vivo. It can be seen from the results of fungicidal activity that cyste-
ine and its derivatives have broad-spectrum activity, but with a low to moderate degree
of activity.
4. Materials and Methods
4.1. General Procedures
4.1.1. Instruments
The melting points of the products were determined on an X-4 binocular microscope
(Gongyi Yuhua Instrument Co., Gongyi, China) and are not corrected. NMR spectra were
acquired with a 400 MHz (100 MHz for 13C) instrument (Bruker, Billerica, MA, USA) at
room temperature. Chemical shifts were measured relative to residual solvent peaks of
DMSO-d6 as internal standards (1H: δ = 2.5 and 3.3 ppm; 13C: δ = 39.9 ppm). The following
abbreviations are used to designate chemical shift multiplicities: s = singlet, d = doublet,
dd = doublet of doublets, t = triplet, m = multiplet, and brs = broad singlet. HRMS data
were recorded with a QFT-ESI instrument (Varian, Palo Alto, CA, USA). All reagents were
of analytical reagent (AR) grade or chemically pure (CR). Compounds 1–7 (AR) were pur-
chased from Shanghai Bidepharm Co., Ltd. (Shanghai, China).
4.1.2. Synthesis of Compounds 8–24
Figure 5. Molecule docking results of (A) compound 3, (B) compound 23, (C) compound 24 with TMV CP.
4. Materials and Methods
4.1. General Procedures
4.1.1. Instruments
The melting points of the products were determined on an X-4 binocular microscope
(Gongyi Yuhua Instrument Co., Gongyi, China) and are not corrected. NMR spectra were
acquired with a 400 MHz (100 MHz for
13
C) instrument (Bruker, Billerica, MA, USA) at
room temperature. Chemical shifts were measured relative to residual solvent peaks of
DMSO-d
6
as internal standards (
1
H:
δ
= 2.5 and 3.3 ppm;
13
C:
δ
= 39.9 ppm). The following
abbreviations are used to designate chemical shift multiplicities: s = singlet, d = doublet,
dd = doublet of doublets, t = triplet, m = multiplet, and brs = broad singlet. HRMS data
were recorded with a QFT-ESI instrument (Varian, Palo Alto, CA, USA). All reagents were
of analytical reagent (AR) grade or chemically pure (CR). Compounds
1
–
7
(AR) were
purchased from Shanghai Bidepharm Co., Ltd. (Shanghai, China).
4.1.2. Synthesis of Compounds 8–24
L-Cysteine (3.63 g, 30 mmol) was dissolved in a mixed solvent of water (50 mL) and
EtOH (6 mL). Then the solution of corresponding aldehydes (1.0 equiv.) in EtOH (15 mL)
was added. The mixture was stirred at 25
◦
C for 6 h, filtered, washed with water, and dried
to afford compounds 8–24 [7,26].
(2RS,4R)-2-Phenyl-1,3-thiazolidine-4-carboxylic acid (
8
). White solid, 93% yield, m.p.
155
−
157
◦
C (lit. [
33
] 158
−
159
◦
C);
1
H-NMR (DMSO-d
6
): 7.25–7.52 (m, 5H, Ar-
H
), 5.67 (s,
0.5H, Ar-C
H
), 5.50 (s, 0.5H, Ar-C
H
), 4.24 (dd, J= 4.4 and 6.8 Hz, 0.5H, CH
2
C
H
), 3.90 (dd,
J= 7.6 and 8.4 Hz, 0.5H, CH
2
C
H
), 3.38 (dd, J= 7.2 and 10.0 Hz, 0.5H, C
H2
), 3.30 (dd, J=
7.2 and 10.4 Hz, 0.5H, C
H2
), 3.14 (dd, J= 4.8 and 10.4 Hz, 0.5H, C
H2
), 3.08 (t, J= 8.8 Hz,
0.5H, C
H2
);
13
C-NMR (DMSO-d
6
): 172.9, 172.2, 141.2, 138.9, 128.5, 128.3, 127.6, 127.2, 126.9,
71.7, 71.1, 65.4, 64.8, 38.4, 37.9; HRMS (ESI) m/z calc’d for C
10
H
11
NO
2
S [M + H]
+
: 209.0510,
found 209.0503.
(2RS,4R)-2-(4-Fluorophenyl)-1,3-thiazolidine-4-carboxylic acid (
9
). White solid, 82% yield,
m.p. 153–155
◦
C (lit. [
34
] 166
◦
C);
1
H-NMR (DMSO-d
6
): 7.48–7.60 (m, 2H, Ar-
H
), 7.13–7.22
(m, 2H, Ar-
H
), 5.67 (s, 0.6H, Ar-C
H
), 5.51 (s, 0.4H, Ar-C
H
), 4.21 (dd, J= 4.8 and 6.8 Hz,
0.6H, CH
2
C
H
), 3.89 (t, J= 7.6 Hz, 0.4H, CH
2
C
H
), 3.36 (dd, J= 7.2 and 10.0 Hz, 0.5H, C
H2
),
3.30 (dd, J= 7.2 and 10.0 Hz, 0.7H, C
H2
), 3.13 (dd, J= 4.4 and 10.0 Hz, 0.6H, C
H2
), 3.09 (t, J
= 9.2 Hz, 0.5H, C
H2
);
13
C-NMR (DMSO-d
6
): 173.4, 172.6, 163.5, 163.1, 161.1, 160.7, 138.0,
135.8, 130.0, 129.9, 129.4, 115.8, 115.6, 115.5, 115.3, 71.4, 70.7, 66.0, 65.3, 38.8, 38.4; HRMS
(ESI) m/z calc’d for C10H10FNO2S [M + H]+: 227.0416, found 227.0421.

Molecules 2021,26, 383 12 of 16
(2RS,4R)-2-(4-Bromophenyl)-1,3-thiazolidine-4-carboxylic acid (
10
). White solid, 87% yield,
m.p. 158–161
◦
C (lit. [
34
] 165–166
◦
C);
1
H-NMR (DMSO-d
6
): 7.38–7.58 (m, 4H, Ar-
H
), 5.67
(s, 0.6H, Ar-C
H
), 5.49 (s, 0.4H, Ar-C
H
), 4.17 (dd, J= 4.8 and 6.8 Hz, 0.6H, CH
2
C
H
), 3.90
(dd, J= 7.2 and 8.8 Hz, 0.4H, CH
2
C
H
), 3.35 (dd, J= 6.8 and 10.0 Hz, 0.6H, C
H2
), 3.29 (dd, J
= 7.2 and 10.4 Hz, 0.7H, C
H2
), 3.12 (t, J= 5.4 Hz, 0.6H, C
H2
), 3.08 (t, J= 7.2 Hz, 0.4H, C
H2
);
13
C-NMR (DMSO-d
6
): 173.3, 172.5, 141.6, 139.1, 131.8, 131.6, 130.1, 129.6, 130.0, 71.3, 70.6,
66.0, 65.3, 38.8, 38.5; HRMS (ESI) m/z calc’d for C
10
H
10
BrNO
2
S [M + H]
+
: 288.9616, found
288.9614.
(2RS,4R)-2-(4-Trifluoromethylphenyl)-1,3-thiazolidine-4-carboxylic acid (
11
). White solid,
99% yield, m.p. 145–147
◦
C;
1
H-NMR (DMSO-d
6
): 7.63–7.77 (m, 4H, Ar-
H
), 5.81 (s, 0.6H,
Ar-C
H
), 5.61 (s, 0.4H, Ar-C
H
), 4.15 (t, J= 6.0 Hz, 0.6H, CH
2
C
H
), 3.95 (dd, J= 7.2 and 8.4
Hz, 0.4H, CH2CH), 3.37 (dd, J= 6.8 and 10.0 Hz, 0.4H, CH2), 3.31 (dd, J= 7.2 and 10.4 Hz,
0.6H, C
H2
), 3.08–3.12 (m, 1H, C
H2
);
13
C-NMR (DMSO-d
6
): 173.2, 172.5, 147.3, 128.7, 128.0,
125.8, 125.7, 125.6, 71.1, 70.4, 66.1, 65.3, 38.6, 38.5; HRMS (ESI) m/z calc’d for C
11
H
10
F
3
NO
2
S
[M +H]+: 277.0384, found 277.0383.
(2RS,4R)-2-(4-Methoxyphenyl)-1,3-thiazolidine-4-carboxylic acid (
12
). White solid, 93%
yield, m.p. 153–156
◦
C (lit. [
33
] 163–164
◦
C);
1
H-NMR (DMSO-d
6
): 7.44 (d, J= 8.8 Hz, 1H,
Ar-
H
), 7.37 (d, J= 8.8 Hz, 1H, Ar-
H
), 6.92 (d, J= 8.8 Hz, 1H, Ar-
H
), 6.88 (d, J= 8.4 Hz,
1H, Ar-
H
), 5.60 (s, 0.5H, Ar-C
H
), 5.45 (s, 0.5H, Ar-C
H
), 4.25 (dd, J= 4.0 and 6.8 Hz, 0.5H,
CH
2
C
H
), 3.87 (dd, J= 7.2 and 8.8 Hz, 0.5H, CH
2
C
H
), 3.75 (s, 1.5H, C
H3
), 3.74 (s, 1.5H, C
H3
),
3.36 (dd, J= 7.2 and 10.0 Hz, 0.5H, C
H2
), 3.28 (dd, J= 7.2 and 10.0 Hz, 0.5H, C
H2
), 3.15 (dd,
J= 4.0 and 10.4 Hz, 0.5H, C
H2
), 3.07 (t, J= 8.8 Hz, 0.5H, C
H2
);
13
C-NMR (DMSO-d
6
): 173.1,
172.3, 159.2, 158.7, 132.8, 130.7, 128.5, 128.3, 114.5, 113.8, 113.6, 71.5, 70.9, 65.4, 64.8, 55.1,
55.0, 38.5, 37.9; HRMS (ESI) m/z calc’d for C
11
H
13
NO
3
S [M + H]
+
: 239.0616, found 239.0612.
(2RS,4R)-2-(4-Methylphenyl)-1,3-thiazolidine-4-carboxylic acid (
13
). White solid, 99%
yield, m.p. 146–148
◦
C (lit. [
34
] 163.2–163.7
◦
C);
1
H-NMR (DMSO-d
6
): 7.39 (d, J= 8.0 Hz,
1H, Ar-
H
), 7.32 (d, J= 8.0 Hz, 1H, Ar-
H
), 7.17 (d, J= 7.6 Hz, 1H, Ar-
H
), 7.13 (d, J= 7.6
Hz, 1H, Ar-
H
), 5.61 (s, 0.5H, Ar-C
H
), 5.46 (s, 0.5H, Ar-C
H
), 4.24 (dd, J= 4.4 and 6.8 Hz,
0.5H, CH
2
C
H
), 3.87 (dd, J= 7.6 and 8.8 Hz, 0.5H, CH
2
C
H
), 3.36 (dd, J= 6.8 and 10.0 Hz,
0.5H, C
H2
), 3.28 (dd, J= 6.8 and 10.0 Hz, 0.5H, C
H2
), 3.14 (dd, J= 4.0 and 10.0 Hz, 0.5H,
C
H2
), 3.07 (t, J= 8.8 Hz, 0.5H, C
H2
), 2.30 (s, 1.5H, C
H3
), 2.28 (s, 1.5H, C
H3
);
13
C-NMR
(DMSO-d
6
): 173.5, 172.8, 138.5, 138.1, 137.3, 136.3, 130.2, 130.1, 129.4, 129.2, 127.6, 127.4,
72.2, 71.6, 65.9, 65.3, 38.9, 38.4, 21.2, 21.1; HRMS (ESI) m/z calc’d for C
11
H
13
NO
2
S [M + H]
+
:
223.0667, found 223.0673.
(2RS,4R)-2-(4-Hydroxyphenyl)-1,3-thiazolidine-4-carboxylic acid (
14
). White solid, 81%
yield, m.p. 161–164
◦
C (lit. [
35
] 167–1169
◦
C);
1
H-NMR (DMSO-d
6
): 9.51 (br s, 1H, O
H
),
7.33 (d, J= 8.8 Hz, 1H, Ar-
H
), 7.26 (d, J= 8.0 Hz, 1H, Ar-
H
), 6.75 (d, J= 8.0 Hz, 1H, Ar-
H
),
6.72 (d, J= 8.8 Hz, 1H, Ar-
H
), 5.55 (s, 0.5H, Ar-C
H
), 5.41 (s, 0.5H, Ar-C
H
), 4.26 (dd, J= 3.6
and 6.8 Hz, 0.5H, CH
2
C
H
), 3.86 (t, J= 7.6 Hz, 0.5H, CH
2
C
H
), 3.36 (dd, J= 6.8 and 10.0 Hz,
0.5H, C
H2
), 3.28 (dd, J= 6.8 and 10.0 Hz, 0.5H, C
H2
), 3.16 (dd, J= 3.6 and 10.4 Hz, 0.5H,
C
H2
), 3.06 (t, J= 9.2 Hz, 0.5H, C
H2
);
13
C-NMR (DMSO-d
6
): 173.1, 172.3, 157.4, 156.9, 130.7,
128.8, 128.5, 128.3, 115.1, 114.9, 71.8, 71.2, 65.2, 64.7, 38.5, 37.8; HRMS (ESI) m/z calc’d for
C10H11NO3S [M + H]+: 225.0460, found 225.0463.
(2RS,4R)-2-(3-Hydroxy-4-methoxyphenyl)-1,3-thiazolidine-4-carboxylic acid (
15
). White
solid, 93% yield, m.p. 170–173
◦
C;
1
H-NMR (DMSO-d
6
): 9.03 (br s, 1H, O
H
), 7.12 (s, 0.5H,
Ar-
H
), 7.11 (s, 0.5H, Ar-
H
), 6.83–6.90 (m, 1H, Ar-
H
), 6.69–6.74 (m, 1H, Ar-
H
), 5.53 (s, 0.5H,
Ar-C
H
), 5.40 (s, 0.5H, Ar-C
H
), 4.29 (dd, J= 3.6 and 6.8 Hz, 0.5H, CH
2
C
H
), 3.83 (dd, J=
7.2 and 8.8 Hz, 0.5H, CH
2
C
H
), 3.77 (s, 1.5H), 3.76 (s, 0.5H, CH
2
C
H
), 3.33 (dd, J= 7.2 and
10.0 Hz, 0.5H, C
H2
), 3.28 (dd, J= 7.2 and 10.0 Hz, 0.5H, C
H2
), 3.16 (dd, J= 3.6 and 10.4 Hz,
0.5H, C
H2
), 3.06 (t, J= 9.2 Hz, 0.5H, C
H2
);
13
C-NMR (DMSO-d
6
): 173.7, 172.8, 148.0, 147.8,
147.1, 146.6, 131.7, 129.9, 120.4, 120.1, 115.6, 115.4, 111.9, 111.7, 72.6, 72.0, 66.0, 65.3, 56.1,
56.0, 38.9, 38.3; HRMS (ESI) m/z calc’d for C
11
H
13
NO
4
S [M + H]
+
: 255.0565, found 255.0573.

Molecules 2021,26, 383 13 of 16
(2RS,4R)-2-(4-Nitrophenyl)-1,3-thiazolidine-4-carboxylic acid (
16
). White solid, 99% yield,
m.p. 94–97
◦
C (lit. [
34
] 95–97
◦
C);
1
H-NMR (DMSO-d
6
): 8.17–8.23 (m, 2H, Ar-
H
), 7.80 (d, J
= 8.8 Hz, 0.8H, Ar-
H
), 7.67 (d, J= 8.8 Hz, 1H, Ar-
H
), 5.87 (s, 0.6H, Ar-C
H
), 5.66 (s, 0.4H,
Ar-C
H
), 4.12 (t, J= 6.4 Hz, 0.7H, CH
2
C
H
), 3.97 (dd, J= 6.8 and 9.2 Hz, 0.5H, CH
2
C
H
), 3.37
(dd, J= 6.8 and 10.0 Hz, 0.7H, C
H2
), 3.32 (dd, J= 6.8 and 10.0 Hz, 0.8H, C
H2
), 3.06–3.13 (m,
1H, C
H2
);
13
C-NMR (DMSO-d
6
): 173.1, 172.5, 150.6, 147.7, 147.1, 129.1, 128.3, 124.0 123.9,
70.6, 69.9, 66.2, 65.4, 38.6, 38.5; HRMS (ESI) m/z calc’d for C
10
H
10
N
2
O
4
S [M + H]
+
: 254.0361,
found 254.0367.
(2RS,4R)-2-(3-Nitrophenyl)-1,3-thiazolidine-4-carboxylic acid (
17
). White solid, 99% yield,
m.p. 90–93
◦
C;
1
H-NMR (DMSO-d
6
): 8.46 (s, 0.4H, Ar-
H
), 8.29 (s, 0.6H, Ar-
H
), 8.17 (dd, J=
1.6 and 8.0 Hz, 0.4H, Ar-
H
), 8.12 (dd, J= 1.6 and 8.4 Hz, 0.6H, Ar-
H
), 7.96 (d, J= 8.0 Hz,
0.4H, Ar-
H
), 7.87 (d, J= 7.6 Hz, 0.6H, Ar-
H
), 7.60–7.67 (m, 1H, Ar-
H
), 5.86 (s, 0.6H, Ar-C
H
),
5.67 (s, 0.4H, Ar-C
H
), 4.14 (t, J= 6.0 Hz, 0.6H), 3.95 (dd, J= 7.2 and 8.4 Hz, 0.5H, CH
2
C
H
),
3.31–3.38 (m, 1H, C
H2
), 3.09–3.15 (m, 1H, C
H2
);
13
C-NMR (DMSO-d
6
): 173.1, 172.4, 148.2,
148.1, 145.1, 142.5, 134.8, 134.3, 130.4, 130.3, 70.6, 69.9, 66.3, 65.2, 38.6, 38.5; HRMS (ESI) m/z
calc’d for C10H10N2O4S [M + H]+: 254.0361, found 254.0365.
(2RS,4R)-2-(Thiophen-2-yl)thiazolidine-4-carboxylic acid (
18
). White solid, 99% yield, m.p.
144–147
◦
C (lit. [
36
] 146–147
◦
C);
1
H-NMR (DMSO-d
6
): 7.51 (dd, J= 0.8 and 4.8 Hz, 0.3H,
Ar-
H
), 7.42 (dd, J= 0.8 and 5.2 Hz, 0.6H, Ar-
H
), 7.20 (d, J= 3.2 Hz, 0.4H, Ar-
H
), 7.06 (d, J=
3.6 Hz, 0.6H, Ar-
H
), 6.99 (dd, J= 3.2 and 4.8 Hz, 0.3H, Ar-
H
), 6.95 (dd, J= 3.6 and 4.2 Hz,
0.6H, Ar-
H
), 5.94 (s, 0.7H, Ar-C
H
), 5.75 (s, 0.4H, Ar-C
H
), 4.07 (t, J= 6.4 Hz, 0.7H, CH
2
C
H
),
3.91 (dd, J= 7.2 and 8.8 Hz, 0.4H, CH
2
C
H
), 3.31–3.39 (m, 1H, C
H2
), 3.03–3.11 (m, 1H, C
H2
);
13
C-NMR (DMSO-d
6
): 172.6, 172.2, 147.1, 142.8, 126.8, 126.7, 126.2, 125.4, 125.2, 66.6, 66.1,
65.4, 64.5, 38.5, 38.0; HRMS (ESI) m/z calc’d for C
8
H
9
NO
2
S
2
[M + H]
+
: 215.0075, found
215.0079.
(2RS,4R)-2-(Furan-2-yl)thiazolidine-4-carboxylic acid (
19
). White solid, 99% yield, m.p.
137–140
◦
C (lit. [
33
] 137–138
◦
C);
1
H-NMR (DMSO-d
6
): 7.65–7.66 (m, 0.3H, Ar-
H
), 7.58–7.59
(m, 0.6H, Ar-
H
), 6.50 (d, J= 3.2 Hz, 0.3H, Ar-
H
), 6.43–6.44 (m, 0.3H, Ar-
H
), 6.37–6.38 (m,
0.6H, Ar-
H
), 6.34–6.35 (m, 0.6H, Ar-
H
), 5.74 (s, 0.7H, Ar-C
H
), 5.61 (s, 0.4H, Ar-C
H
), 4.11
(t, J= 6.4 Hz, 0.7H, CH
2
C
H
), 3.87 (dd, J= 6.8 and 8.8 Hz, 0.4H, CH
2
C
H
), 3.27–3.37 (m,
1H, C
H2
), 2.97–3.01 (m, 1H, C
H2
);
13
C-NMR (DMSO-d
6
): 173.0, 172.7, 154.9, 151.8, 143.4,
142.9, 111.1, 110.8, 108.0, 106.8, 65.9, 65.3, 64.7, 64.4, 38.6, 38.3; HRMS (ESI) m/z calc’d for
C8H9NO3S [M + H]+: 119.0303, found 119.0305.
(2RS,4R)-2-(Pyridin-4-yl)thiazolidine-4-carboxylic acid (
20
). White solid, 93% yield, m.p.
163–165
◦
C (lit. [
34
] 175–176
◦
C);
1
H-NMR (DMSO-d
6
): 8.55 (d, J= 6.0 Hz, 0.4H, Ar-
H
), 8.50
(d, J= 6.1 Hz, 1.6H, Ar-
H
), 7.52 (d, J= 6.0 Hz, 0.4H, Ar-
H
), 7.40 (d, J= 6.0 Hz, 1.6H, Ar-
H
),
5.75 (s, 0.8H, Ar-C
H
), 5.54 (s, 0.2H, Ar-C
H
), 4.10 (t, J= 6.3 Hz, 0.8H, CH
2
C
H
), 3.97 (dd, J=
6.9 and 8.8 Hz, 0.2H, CH
2
C
H
), 3.36 (dd, J= 6.9 and 10.0 Hz, 0.2H, C
H2
), 3.30 (dd, J= 6.8
and 10.2 Hz, 0.8H, C
H2
), 3.04–3.10 (m, 1H, C
H2
);
13
C-NMR (DMSO-d
6
): 173.0, 172.5, 151.9,
151.1, 150.1, 149.8, 122.3, 122.1, 70.2, 69.5, 66.2, 65.3, 38.6, 38.4; HRMS (ESI) m/z calc’d for
C9H10N2O2S [M + H]+: 210.0463, found 210.0460.
(2RS,4R)-2-Benzylthiazolidine-4-carboxylic acid (
21
). White solid, 93% yield, m.p. 152–
155
◦
C (lit. [
35
] 165–166
◦
C);
1
H-NMR (DMSO-d
6
): 7.18–7.30 (m, 5H, Ar-
H
), 4.81 (t, J= 7.0
Hz, 0.7H, SC
H
), 4.65 (t, J= 6.8 Hz, 0.3H, SC
H
), 4.15 (dd, J= 5.6 and 6.7 Hz, 0.7H, CH
2
C
H
),
3.72 (dd, J= 6.9 and 9.2 Hz, 0.3H, CH
2
C
H
), 3.07–3.27 (m, 2H, CH
2
C
H
and Ar-
CH2
), 3.00
(dd, J= 7.3 and 13.7 Hz, 0.3H, CH
2
C
H
), 2.92 (dd, J= 5.4 and 10.2 Hz, 0.7H, CH
2
C
H
),
2.75–2.85 (m, 1H, Ar-
CH2
);
13
C-NMR (DMSO-d
6
): 173.3, 172.7, 139.5, 139.3, 129.6, 129.4,
128.7, 128.6, 126.9, 126.7, 72.4, 71.8, 65.7, 64.6, 43.3, 41.1, 37.8, 37.6; HRMS (ESI) m/z calc’d
for C11H13NO2S [M + H]+: 223.0667, found 223.0662.
(2RS,4R)-2-Propylthiazolidine-4-carboxylic acid (
22
). White solid, 93% yield, m.p. 190–
192
◦
C;
1
H-NMR (DMSO-d
6
): 4.56 (t, J= 6.6 Hz, 0.5H, SC
H
), 4.42 (t, J= 7.2 Hz, 0.5H, SC
H
),
4.07 (dd, J= 6.7 Hz and 5.4 Hz, 0.5H, SCH
2
C
H
), 3.71 (dd, J= 7.0 and 9.0 Hz, 0.5H, SCH
2
C
H
),
3.20 (dd, J= 7.0 and 9.9 Hz, 0.5H, SC
H2
), 3.09 (dd, J= 7.0 and 10.2 Hz, 0.5H, SC
H2
), 2.94

Molecules 2021,26, 383 14 of 16
(dd, J= 5.1 and 10.2 Hz, 0.5H, SC
H2
), 2.76 (t, J= 9.4 Hz, 0.5H, SC
H2
), 1.85–1.93 (m, 0.5H,
CH
2
C
H2
CH), 1.65–1.77 (m, 1H, CH
2
C
H2
CH), 1.49–1.56 (m, 0.5H, CH
2
C
H2
CH), 1.35–1.43
(m, 2H, CH
2
C
H2
), 0.86–0.91 (m, 3H, C
H3
);
13
C-NMR (DMSO-d
6
): 173.3, 172.8, 71.2, 70.4,
65.6, 64.6, 39.3, 39.2, 37.4, 37.0, 21.4, 21.1, 14.3, 14.2; HRMS (ESI) m/z calc’d for C
7
H
13
NO
2
S
[M + H]+: 175.0667, found 175.0670.
(2RS,4R)-2-Heptylthiazolidine-4-carboxylic acid (
23
). White solid, 93% yield, m.p. 136–
139
◦
C (lit. [
37
] 159–160
◦
C);
1
H-NMR (DMSO-d
6
): 4.54 (t, J= 6.6 Hz, 0.5H, SC
H
), 4.40 (t, J=
6.7 Hz, 0.5H, SC
H
), 4.06 (t, J= 6.1 Hz, 0.5H, SCH
2
C
H
), 3.70 (t, J= 8.5 Hz, 0.5H, SCH
2
C
H
),
3.19 (t, J= 9.6 Hz, 0.5H, SC
H2
), 3.08 (t, J= 9.8 Hz, 0.5H, SC
H2
), 2.93 (dd, J= 4.9 and 9.8 Hz,
0.5H, SC
H2
), 2.75 (t, J= 9.6 Hz, 0.5H, SC
H2
), 1.85–1.92 (m, 0.5H, CH
2
C
H2
CH), 1.69–1.76 (m,
1H, CH
2
C
H2
CH), 1.50–1.67 (m, 0.5H, CH
2
C
H2
CH), 1.24–1.48 (m, 10H, CH
2
C
H2
), 0.84–0.86
(m, 3H, C
H3
);
13
C-NMR (DMSO-d
6
): 173.3, 172.8, 71.5, 70.8, 65.7, 64.6, 37.4, 37.1, 37.0, 35.3,
31.7, 29.3, 29.2, 29.1, 29.0, 28.1, 27.9, 22.5, 14.4; HRMS (ESI) m/z calc’d for C
11
H
21
NO
2
S [M +
H]+: 231.1293, found 231.1294.
(2RS,4R)-2-Cyclohexylthiazolidine-4-carboxylic acid (
24
). White solid, 82% yield, m.p.184–
187
◦
C;
1
H-NMR (DMSO-d
6
): 4.01–4.02 (m, 0.4H, SC
H
), 3.65–3.69 (m, 0.6H, SC
H
), 3.13–3.16
(m, 0.6H, SC
H2
CH), 2.98–3.01 (m, 0.4H, SC
H2
CH), 2.87–2.89 (m, 0.4H, SC
H2
CH), 2.65–2.68
(m, 0.6H, SC
H2
CH), 1.95–1.97 (m, 1H, CH
2
C
H
CH
2
), 1.40–1.66 (m, 4H, CH
2
C
H2
CH), 0.98–
1.15 (m, 6H, CH
2
C
H2
CH
2
);
13
C-NMR (DMSO-d
6
): 173.4, 172.9, 77.4, 76.6, 65.7, 64.8, 44.1,
43.1, 36.7, 31.7, 31.6, 30.1, 26.4, 26.0, 25.9, 25.8; HRMS (ESI) m/z calc’d for C
10
H
17
NO
2
S [M +
H]+: 215.0980, found 215.0978.
4.2. Biological Assays
Each test was repeated three times at 25
±
1
◦
C. Active effect expressed in percentage
scale of 0–100 (0: no activity; 100: total inhibited). Specific test methods for the anti-TMV
and fungicidal activities were carried out by the literature method [
5
,
27
], Detailed bioassay
procedures for the anti-TMV and fungicidal activities were described in the literature and
can be seen in the Supplementary Materials.
5. Conclusions
Based on the structure of natural product cysteine, a series of cysteine and its deriva-
tives were designed, synthesized, and evaluated for their antiviral and antifungal activities
in vitro
and
in vivo
. By studying the influence of O, N, and S atom substituents of cysteine,
it was found that some compounds had excellent anti-TMV activity. The preliminary mode
of action studies exhibited that compound
3
can hold back virus assembly by aggregating
the 20S protein disk. We further study the binding sites of the interaction between cysteines
and TMV CP by molecular docking. Further fungicidal activity tests against 14 kinds of
phytopathogenic fungi revealed that these cysteine derivatives displayed broad-spectrum
fungicidal activities. In this work, cysteine and its derivatives are found to be potential
inhibitors against plant viruses and plant pathogens.
Supplementary Materials:
The following are available online, Supplementary data (Detailed bio-
assay procedures for the anti-TMV and fungicidal activities; mode of action studies; copies of
1
H &
13C-NMR spectra) can be found in the online version.
Author Contributions:
Project administration, supervision, Z.W. and A.L.; writing—original draft,
A.L., chemical methodology, S.Y. and T.W., biological methodology, L.S. and Y.Z.; docking studies,
Z.W. All authors have read and agreed to the published version of the manuscript.
Funding:
This research was funded by the Natural Science Fundation of Hebei Province (B2020202028).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.

Molecules 2021,26, 383 15 of 16
Acknowledgments:
The authors also acknowledge the State Key Laboratory of Elemento-Organic
Chemistry (Nankai University) for biological activity test.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds 8–24 are available from the authors.
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