Discovery of cysteine and its derivatives as novel antiviral and antifungal agents

Article abstract of DOI:10.3390/molecules26020383

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 by¹ H-NMR,¹³ C-NMR, and

Full text of DOI:10.3390/molecules26020383

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,
*
1
School 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.)
Tianjin Key Laboratory of Structure and Performance for Functional Molecules, College of Chemistry, Tianjin
Normal University, Tianjin 300387, China
2
*
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
1
were characterized by H-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
24 displayed higher anti-TMV activities than the commercial plant virucide ribavirin (inhibitory rate:
40, 40, and 38% at 500 g/mL for inactivation, curative, and protection activity in vivo, respectively),
µg/mL. The compounds cysteine (1), 3–4, 7, 10, 13, 20, 23, and
µ
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
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
antiviral candidate. Antiviral mechanism research by TEM exhibited that compound
virus assembly by aggregated the 20S protein disk. Molecular docking results revealed that compound
with higher antiviral activities than that of compound 24 did show stronger interaction with
3 could inhibit
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.
3
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.
https://doi.org/10.3390/molecules2
6020383)
Academic Editor: Jih-Jung Chen
Received: 23 December 2020
Accepted: 11 January 2021
Published: 13 January 2021
Keywords: natural product; cysteine and its derivatives; anti-TMV activity; antifungal activity; mode
of action; molecular docking
Publisher’s Note: MDPI stays neu-
tral with regard to jurisdictional clai-
ms in published maps and institutio-
nal affiliations.
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 [
not only can seriously harm tobacco, but also can infect potato, pepper, tomato, eggplant,
nightshade, and more than 400 other plant species [ ]. 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].
1]. Tobacco mosaic virus (TMV), known as “plant cancer”,
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/).
2,3
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
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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 [ ]. Cysteine, with its easily modified
7–9
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 [ ], immune stimulation [20], etc. Some thiazolidine-
7
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 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
ring containing L-cysteine derivatives
and different substituted aldehydes (Scheme 1) by a one-pot method [7,26].
1
–7
(Figure 2) were purchased directly. A series of thiazole
8
–24 were designed and synthesized from L-cysteine

Molecules 2021, 26, 383
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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 g/mL. The detailed test procedures can be seen in our previous
reports [ 27]. The detailed test procedures can also be found in the Supplementary Materials.
µ
5,
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
had better anti-
(inhibitory rate: 48%
g/mL) showed better anti-TMV activities
g/mL, compounds (inhibitory rate: 13%)
(inhibitory rate: 12%) also displayed better anti-TMV activities than the commercial
plant virucide ribavirin (inhibitory rate: 7%). Most of the cysteine derivatives 24 with
thiazolidine structure exhibited better anti-TMV activities than that of the commercial plant
commercial drugs ribavirin and ningnanmycin as controls. Compounds
TMV activities than ribavirin. Among compounds , compounds
at 500 g/mL) and (inhibitory rate: 45% at 500
than the others. At the concentration of 100
and
1–7
1–7
3
µ
4
µ
µ
3
4
8–

Molecules 2021, 26, 383
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virucide ribavirin. Especially, compounds 23 (inhibitory rate: 46% at 500
(inhibitory rate: 45% at 500
µ
g/mL) and 24
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.
1-D
Concn (µg/mL)
Inhibition Rate (%) a
Compd.
13
Concn (µg/mL)
Inhibition Rate (%) a
500
100
39 ± 3
500
100
43 ± 1
13 ± 2
17 ± 1
500
100
41 ± 2
11 ± 1
500
100
18 ± 2
1-L
2-D
2-L
3
14
15
16
17
18
19
20
21
22
23
24
0
500
100
38 ± 2
500
100
34 ± 1
15 ± 2
12 ± 1
500
100
40 ± 1
16 ± 2
500
100
40 ± 2
16 ± 2
500
100
48 ± 1
500
100
39 ± 1
13 ± 2
14 ± 2
500
100
45 ± 2
12 ± 1
500
100
24 ± 2
4
0
500
100
42 ± 2
500
100
32 ± 2
5
10 ± 1
0
500
100
40 ± 2
20 ± 1
500
100
43 ± 2
6
18 ± 2
500
100
44 ± 2
500
100
35 ± 2
11 ± 1
7
22 ± 1
500
100
33 ± 2
9 ± 2
500
100
33 ± 1
8
10 ± 1
500
100
35 ± 1
500
100
46 ± 1
17 ± 2
9
12 ± 2
500
100
39 ± 2
13 ± 1
500
100
45 ± 1
10
11
12
19 ± 1
500
100
43 ± 1
500
100
35 ± 1
7 ± 1
Ribavirin
19 ± 1
500
100
a
38 ± 2
16 ± 1
500
100
61 ± 2
Ningnanmycin
23 ± 2
Average 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 µg/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.

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Table 2. In Vivo antiviral activity of compounds 1–24 against TMV.
Compd.
1-D
Concn (µg/mL)
Inactivation Effect (%) a
Curative Effect (%) a
Protection Effect (%) a
500
100
44 ± 2
38 ± 2
37 ± 1
18 ± 3
15 ± 2
14 ± 1
500
100
43 ± 1
20 ± 2
42 ± 2
16 ± 1
40 ± 1
13 ± 2
1-L
2-D
2-L
3
500
100
37 ± 2
41 ± 3
36 ± 1
12 ± 1
13 ± 1
15 ± 2
500
100
39 ± 1
11 ± 2
40 ± 2
14 ± 1
38 ± 2
12 ± 1
500
100
51 ± 2
47 ± 2
49 ± 2
26 ± 1
23± 1
25 ± 1
500
100
45 ± 1
18 ± 2
43 ± 1
20 ± 2
46 ± 1
22 ± 2
4
500
100
40 ± 1
39 ± 2
37 ± 2
5
13 ± 1
16 ± 1
16 ± 1
500
100
38 ± 2
16 ± 2
40 ± 2
16 ± 1
35 ± 2
16 ± 1
6
500
100
44 ± 1
42 ± 2
41 ± 2
7
20 ± 2
16 ± 1
18 ± 1
500
100
37 ± 2
11 ± 1
33 ± 2
10 ± 1
35 ± 2
11 ± 1
8
500
100
36 ± 2
35 ± 1
34 ± 1
9
10 ± 1
9 ± 1
9 ± 1
500
100
41 ± 2
10 ± 1
40 ± 2
10 ± 1
41 ± 2
13 ± 1
10
11
12
13
14
15
16
17
18
19
20
21
22
500
100
40 ± 2
39 ± 2
40 ± 2
15 ± 1
17 ± 1
12 ± 1
500
100
43 ± 2
14 ± 1
36 ± 2
13 ± 1
41 ± 2
15 ± 1
500
100
49 ± 2
46 ± 2
44 ± 2
18 ± 2
15 ± 2
13 ± 2
500
100
21 ± 2
17 ± 2
15 ± 2
0
0
0
500
100
28 ± 2
33 ± 2
25 ± 2
9 ± 1
11 ± 1
7 ± 1
500
100
42 ± 1
37 ± 1
35 ± 1
0
0
0
500
100
40 ± 1
36 ± 1
35 ± 1
0
0
0
500
100
24 ± 2
23 ± 2
20 ± 2
0
0
0
500
100
34 ± 2
30 ± 2
36 ± 2
0
0
0
500
100
47 ± 2
45 ± 2
43 ± 2
21 ± 2
19 ± 2
14 ± 2
500
100
37 ± 2
11 ± 2
40 ± 2
15 ± 2
34 ± 2
11 ± 2
500
100
33 ± 2
37 ± 2
32 ± 2
10 ± 2
13 ± 2
9 ± 2

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Table 2. Cont.
Inactivation Effect (%) a
Compd.
23
Concn (µg/mL)
Curative Effect (%) a
Protection Effect (%) a
500
100
48 ± 1
49 ± 1
47 ± 1
19 ± 2
15 ± 2
17 ± 2
500
100
47 ± 1
15 ± 1
42 ± 1
17 ± 1
43 ± 1
15 ± 1
24
500
100
40 ± 1
40 ± 1
38 ± 1
Ribavirin
13 ± 1
15 ± 1
10 ± 1
500
100
a
56 ± 2
28 ± 1
53 ± 2
24 ± 1
59 ± 1
30 ± 1
Ningnanmycin
Average 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
29] with
pathogens at a concentration of 50 g/mL using fungicidal growth rate assay [27
µ
,
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
µg/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.

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Table 3. In vitro fungicidal activities of compounds 1–24 against 14 kinds of fungi.
Fungicidal Activity (%) a at 50 µg/mL
Compd.
F.C b
C.H b
P.P b
R.C b
B.M b
W.A b
F.M b
A.S b
F.G b
P.I b
P.C b
S.S b
B.C b
R.S b
1-D
1-L
2-D
2-L
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
18 ± 2
16 ± 1
13 ± 1
18 ± 1
11 ± 1
9 ± 1
7 ± 1
5 ± 1
30 ± 2
35 ± 1
25 ± 2
29 ± 2
22 ± 1
31 ± 1
35 ± 1
41 ± 1
32 ± 1
14 ± 1
17 ± 1
62 ± 1
24 ± 1
10 ± 1
38 ± 2
17 ± 1
20 ± 2
83 ± 1
38 ± 1
45 ± 2
45 ± 1
21 ± 1
17 ± 1
31 ± 1
21 ± 1
35 ± 1
89 ± 1
100
9 ± 1
11 ± 1
12 ± 1
15 ± 1
26 ± 2
16 ± 1
10 ± 1
36 ± 1
42 ± 1
9 ± 1
10 ± 2
7 ± 2
11 ± 3
16 ± 1
30 ± 2
33 ± 2
11 ± 1
22 ± 1
16 ± 1
19 ± 2
19 ± 1
16 ± 1
12 ± 1
32 ± 1
16 ± 1
24 ± 2
16 ± 1
12 ± 1
16 ± 2
16 ± 2
20 ± 1
20 ± 1
36 ± 1
16 ± 1
20 ± 1
12 ± 1
16 ± 1
16 ± 1
95 ± 1
100
9 ± 1
6 ± 1
37 ± 3
35 ± 1
42 ± 1
49 ± 2
39 ± 1
36 ± 1
42 ± 1
43 ± 1
46 ± 1
42 ± 1
39 ± 1
39 ± 1
39 ± 2
23 ± 1
39 ± 1
42 ± 1
27 ± 1
58 ± 1
31 ± 1
27 ± 1
35 ± 2
39 ± 2
46 ± 1
39 ± 1
35 ± 1
50 ± 1
56 ± 1
56 ± 2
16 ± 2
10 ± 2
24 ± 1
26 ± 1
13 ± 1
13 ± 2
10 ± 1
13 ± 1
17 ± 1
42 ± 1
32 ± 2
32 ± 1
13±
8 ± 2
10 ± 1
13 ± 1
13 ± 1
15 ± 1
15 ± 2
13 ± 1
11 ± 1
13 ± 1
16 ± 1
10 ± 2
10 ± 1
10 ± 1
13 ± 2
3 ± 1
27 ± 3
22 ± 1
16 ± 1
17 ± 1
21 ± 1
12 ± 1
17 ± 1
17 ± 1
34 ± 1
12 ± 1
6 ± 1
20 ± 1
25 ± 1
31 ± 3
36 ± 2
23 ± 1
19 ± 1
29 ± 1
21 ± 1
19 ± 1
19 ± 2
25 ± 1
13 ± 1
31 ± 1
19 ± 2
25 ± 1
25 ± 1
19 ± 1
50 ± 1
44 ± 2
31 ± 1
13 ± 1
19 ± 1
25 ± 1
19 ± 1
31 ± 1
31 ± 1
100
16 ± 2
18 ± 1
24 ± 1
21 ± 1
17 ± 1
10 ± 1
14 ± 1
8 ± 1
11 ± 1
12 ± 1
17 ± 2
14 ± 1
16 ± 1
17 ± 2
13 ± 2
13 ± 1
17 ± 1
6 ± 1
19 ± 2
13 ± 2
13 ± 1
7 ± 1
26 ± 2
21 ± 2
10 ± 1
13 ± 2
23 ± 2
26 ± 2
25 ± 1
7 ± 1
16 ± 1
19 ± 1
13 ± 1
16 ± 1
24 ± 1
15 ± 1
23 ± 1
6 ± 2
23 ± 1
12 ± 1
37 ± 1
10 ± 1
16 ± 1
29 ± 1
19 ± 2
29 ± 1
26 ± 1
13 ± 1
19 ± 2
32 ± 1
16 ± 1
16 ± 1
48 ± 2
26 ± 1
19 ± 2
16 ± 1
23 ± 1
19 ± 1
100
16 ± 2
18 ± 1
15 ± 1
14 ± 1
14 ± 1
52 ± 2
14 ± 1
19 ± 1
10 ± 1
0
10 ± 2
71 ± 1
10 ± 1
48 ± 1
33 ± 1
10 ± 1
0
22 ± 1
14 ± 1
13 ± 1
9 ± 1
5 ± 1
17 ± 1
14 ± 2
7 ± 1
18 ± 1
18 ± 2
18 ± 2
24 ± 1
12 ± 1
6 ± 1
8 ± 1
28 ± 1
12 ± 1
11 ± 2
9 ± 1
12 ± 1
10 ± 1
14 ± 1
12 ± 1
8 ± 1
16 ± 1
23 ± 1
12 ± 1
6 ± 1
4 ± 1
14 ± 2
17 ± 1
10 ± 1
7 ± 1
10 ± 1
10 ± 1
16 ± 1
10 ± 1
32 ± 1
10 ± 1
19 ± 1
13 ± 2
16 ± 1
32 ± 1
32 ± 1
16 ± 1
42 ± 1
100
13 ± 1
11 ± 1
13 ± 1
9 ± 1
0
7 ± 1
8 ± 1
18 ± 1
54 ± 1
18 ± 1
23 ± 2
19 ± 1
12 ± 1
28 ± 1
14 ± 1
9 ± 1
12 ± 2
18 ± 1
12 ± 1
18 ± 1
35 ± 1
12 ± 1
24 ± 1
12 ± 2
29 ± 1
18 ± 1
100
13 ± 1
10 ± 1
13 ± 2
10 ± 1
7 ± 1
4 ± 1
12 ± 1
29 ± 1
8 ± 1
45 ± 1
10 ± 1
10 ± 1
10 ± 1
3 ± 2
31 ± 2
16 ± 1
20 ± 1
29 ± 1
6 ± 1
48 ± 1
18 ± 1
7 ± 1
12 ± 1
6 ± 1
11 ± 1
7 ± 1
10 ± 1
10 ± 2
16 ± 1
13 ± 1
3 ± 1
6 ± 1
14 ± 2
7 ± 1
22 ± 1
16 ± 1
6 ± 1
4 ± 1
12 ± 1
12 ± 1
12 ± 1
14 ± 1
100
10 ± 1
19 ± 1
19 ± 1
69 ± 1
53 ± 1
21 ± 1
21 ± 1
27 ± 2
100
23
24
10 ± 1
7 ± 1
12 ± 1
6 ± 1
Chlorothalonil c
Carbendazim c
a
100
91 ± 1
100
55 ± 2
90 ± 2
100
100
100
71 ± 1
88 ± 2
83 ± 1
100
96 ± 1
100
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. The commercial agricultural fungicides were used for comparison of antifungal activity.
c

Molecules 2021, 26, 383
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Table 4. In vivo fungicidal activities of compounds 1–24 against six kinds of fungi.
Inhibition Rate a (%) at 200 µg/mL
Compd.
B.G b
S.S b
B.C b
R.S b
C.C b
P.C b
1-D
1-L
2-D
2-L
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
12 ± 2
11 ± 1
8 ± 2
5 ± 1
7 ± 2
9 ± 2
7 ± 1
8 ± 2
7 ± 2
8 ± 2
0
11 ± 1
8 ± 2
16 ± 1
0
10 ± 1
11 ± 2
13 ± 2
17 ± 2
13 ± 1
13 ± 2
5 ± 1
5 ± 1
5 ± 1
12 ± 1
13 ± 1
13 ± 1
0
12 ± 2
7 ± 1
5 ± 1
5 ± 1
0
12 ± 2
11 ± 1
13 ± 1
11 ± 2
4 ± 1
10 ± 2
7 ± 2
10 ± 1
19 ± 3
16 ± 1
10 ± 2
4 ± 1
15 ± 2
12 ± 1
9 ± 1
11 ± 1
13 ± 2
6 ± 2
11 ± 1
11 ± 3
16 ± 1
11 ± 3
17 ± 1
11 ± 2
11 ± 2
19 ± 1
17 ± 2
4 ± 1
5 ± 1
0
0
12 ± 2
1 ± 1
14 ± 1
11 ± 3
20 ± 1
0
10 ± 1
17 ± 3
13 ± 2
13 ± 2
11 ± 1
4 ± 1
0
0
7 ± 3
5 ± 1
5 ± 1
0
6 ± 1
7 ± 1
0
0
0
0
0
5 ± 1
11 ± 2
16 ± 2
0
0
26 ± 1
15 ± 2
3 ± 1
13 ± 1
10 ± 1
5 ± 2
5 ± 1
10 ± 2
0
0
20 ± 1
26 ± 1
20 ± 2
20 ± 1
11 ± 2
8 ± 1
20 ± 2
4 ± 1
11 ± 2
11 ± 2
12 ± 2
15 ± 2
9 ± 1
0
0
17 ± 2
7 ± 1
0
0
0
0
0
0
5 ± 1
16 ± 2
10 ± 2
11 ± 1
100
5 ± 1
0
23
24
0
12 ± 2
22 ± 1
80 ± 1
5 ± 1
0
85 ± 2
15 ± 2
100
0
azoxystrobin c
81 ± 2
75 ± 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
c
capsica. The 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) [
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 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 24 are obtained
as diastereomeric mixtures [ ]. Although the 2R, 4R and 2S,4R isomers were mixed, the
7,26]. To promote the precipitation of the
8–
8–
7
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 µg/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 had better anti-TMV activities than ribavirin.
Chirality has no obvious effect on the antiviral activities of these compounds (inhibitory
effect: ). Therefore, in the follow-up study, we only used L-cysteine
1–7
1
-
D ≈ 1
-
L
,
2-D ≈ 2
-L
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 24
8
–
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
). When introduction of electron-donating groups such as CH3O (12), CH3 (13) into
the para position of the benzene ring, the anti-TMV activity is also improved (inhibitory
effect: 13 12 15 17 ). However, the activity is sharply declined after the
>
>
,
>
9
> 8
>
>
>
8,
> 9 > 8
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
showed excellent activity against
g/mL for inactivation, curative, and
protection activity in vivo, respectively). Compounds 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 g/mL for inactivation, curative, and protection activity in vivo
respectively). Compounds 10 12 16 exhibited approximate anti-TMV activities as
, 23, 24 are significantly
higher than that of cysteine. In particular, compound
TMV (inhibitory rate: 51%, 47%, and 49% at 500
3
µ
4
,
7
,
,
,
,
µ
,
2,
,
,
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
TMV CP.
3 can lead to polymerization of
3.4.2. Molecular Docking Study
Molecular docking studies were performed to explore the binding sites of cysteine
derivatives on TMV CP. Compounds 23, and 24 were selected for molecular docking
with TMV CP (PDB code 1EI7). The results showed that compound has lain into the TMV
CP activity pocket of SER 255, ASN 73, and GLN 257 (Figure 5A). Compound forms five
3,
3
3
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
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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.
(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
bar), (C) CP + 10 µM compound 3 (200 nm scale bar).
3
(100 nm scale bar): (A) CP, (B) CP + 1/100 DMSO (100 nm scale
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.

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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 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 δ = 39.9 ppm). The following
= 2.5 and 3.3 ppm; 13C:
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
purchased from Shanghai Bidepharm Co., Ltd. (Shanghai, China).
1–7 (AR) were
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 (
157 ◦C (lit. [33] 158 159 ◦C); 1H-NMR (DMSO-d6): 7.25–7.52 (m, 5H, Ar-
0.5H, Ar-C ), 5.50 (s, 0.5H, Ar-C ), 4.24 (dd, J = 4.4 and 6.8 Hz, 0.5H, CH2C
J = 7.6 and 8.4 Hz, 0.5H, CH2C ), 3.38 (dd, J = 7.2 and 10.0 Hz, 0.5H, CH2), 3.30 (dd, J =
7.2 and 10.4 Hz, 0.5H, CH2), 3.14 (dd, J = 4.8 and 10.4 Hz, 0.5H, CH2), 3.08 (t, J = 8.8 Hz,
8
). White solid, 93% yield, m.p.
), 5.67 (s,
), 3.90 (dd,
155
−
−
H
H
H
H
H
0.5H, CH
2); 13C-NMR (DMSO-d6): 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 C10H11NO2S [M + H]+: 209.0510,
found 209.0503.
(2RS,4R)-2-(4-Fluorophenyl)-1,3-thiazolidine-4-carboxylic acid (
m.p. 153–155 ◦C (lit. [34] 166 ◦C); 1H-NMR (DMSO-d6): 7.48–7.60 (m, 2H, Ar-
(m, 2H, Ar- ), 5.67 (s, 0.6H, Ar-C ), 5.51 (s, 0.4H, Ar-C ), 4.21 (dd, J = 4.8 and 6.8 Hz,
0.6H, CH2C ), 3.89 (t, J = 7.6 Hz, 0.4H, CH2C ), 3.36 (dd, J = 7.2 and 10.0 Hz, 0.5H, C 2),
9
). White solid, 82% yield,
H), 7.13–7.22
H
H
H
H
H
H
3.30 (dd, J = 7.2 and 10.0 Hz, 0.7H, CH2), 3.13 (dd, J = 4.4 and 10.0 Hz, 0.6H, CH2), 3.09 (t, J
= 9.2 Hz, 0.5H, CH2); 13C-NMR (DMSO-d6): 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.

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(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); 1H-NMR (DMSO-d6): 7.38–7.58 (m, 4H, Ar-
H
), 5.67
), 3.90
), 3.35 (dd, J = 6.8 and 10.0 Hz, 0.6H, CH2), 3.29 (dd, J
(s, 0.6H, Ar-CH), 5.49 (s, 0.4H, Ar-CH), 4.17 (dd, J = 4.8 and 6.8 Hz, 0.6H, CH2CH
(dd, J = 7.2 and 8.8 Hz, 0.4H, CH2C
H
= 7.2 and 10.4 Hz, 0.7H, CH2), 3.12 (t, J = 5.4 Hz, 0.6H, CH2), 3.08 (t, J = 7.2 Hz, 0.4H, CH2);
13C-NMR (DMSO-d6): 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 C10H10BrNO2S [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; 1H-NMR (DMSO-d6): 7.63–7.77 (m, 4H, Ar-
Ar-CH), 5.61 (s, 0.4H, Ar-CH), 4.15 (t, J = 6.0 Hz, 0.6H, CH2CH
H
), 5.81 (s, 0.6H,
), 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 2), 3.08–3.12 (m, 1H, C
2); 13C-NMR (DMSO-d6): 173.2, 172.5, 147.3, 128.7, 128.0,
H
H
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 C11H10F3NO2S
[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); 1H-NMR (DMSO-d6): 7.44 (d, J = 8.8 Hz, 1H,
Ar-
1H, Ar-
CH2C
3.36 (dd, J = 7.2 and 10.0 Hz, 0.5H, C
J = 4.0 and 10.4 Hz, 0.5H, C 2), 3.07 (t, J = 8.8 Hz, 0.5H, C
H
), 7.37 (d, J = 8.8 Hz, 1H, Ar-
), 5.60 (s, 0.5H, Ar-C ), 5.45 (s, 0.5H, Ar-C
), 3.87 (dd, J = 7.2 and 8.8 Hz, 0.5H, CH2C ), 3.75 (s, 1.5H, C
2), 3.28 (dd, J = 7.2 and 10.0 Hz, 0.5H, C
2); 13C-NMR (DMSO-d6): 173.1,
H
), 6.92 (d, J = 8.8 Hz, 1H, Ar-
), 4.25 (dd, J = 4.0 and 6.8 Hz, 0.5H,
3), 3.74 (s, 1.5H, C 3),
2), 3.15 (dd,
H), 6.88 (d, J = 8.4 Hz,
H
H
H
H
H
H
H
H
H
H
H
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 C11H13NO3S [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); 1H-NMR (DMSO-d6): 7.39 (d, J = 8.0 Hz,
1H, Ar-
Hz, 1H, Ar-
0.5H, CH2C
0.5H, CH2), 3.28 (dd, J = 6.8 and 10.0 Hz, 0.5H, CH2), 3.14 (dd, J = 4.0 and 10.0 Hz, 0.5H,
H2), 3.07 (t, J = 8.8 Hz, 0.5H, CH2), 2.30 (s, 1.5H, CH3), 2.28 (s, 1.5H, CH3); 13C-NMR
H
), 7.32 (d, J = 8.0 Hz, 1H, Ar-
), 5.61 (s, 0.5H, Ar-C ), 5.46 (s, 0.5H, Ar-C
), 3.87 (dd, J = 7.6 and 8.8 Hz, 0.5H, CH2C
H
), 7.17 (d, J = 7.6 Hz, 1H, Ar-
), 4.24 (dd, J = 4.4 and 6.8 Hz,
), 3.36 (dd, J = 6.8 and 10.0 Hz,
H), 7.13 (d, J = 7.6
H
H
H
H
H
C
(DMSO-d6): 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 C11H13NO2S [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); 1H-NMR (DMSO-d6): 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-
), 5.55 (s, 0.5H, Ar-C ), 5.41 (s, 0.5H, Ar-C
), 3.36 (dd, J = 6.8 and 10.0 Hz,
0.5H, CH2), 3.28 (dd, J = 6.8 and 10.0 Hz, 0.5H, CH2), 3.16 (dd, J = 3.6 and 10.4 Hz, 0.5H,
H2), 3.06 (t, J = 9.2 Hz, 0.5H, CH2); 13C-NMR (DMSO-d6): 173.1, 172.3, 157.4, 156.9, 130.7,
H), 6.75 (d, J = 8.0 Hz, 1H, Ar-
H
),
6.72 (d, J = 8.8 Hz, 1H, Ar-
H
H
H), 4.26 (dd, J = 3.6
and 6.8 Hz, 0.5H, CH2CH), 3.86 (t, J = 7.6 Hz, 0.5H, CH2CH
C
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; 1H-NMR (DMSO-d6): 9.03 (br s, 1H, O
H), 7.12 (s, 0.5H,
Ar-
Ar-C
7.2 and 8.8 Hz, 0.5H, CH2C
H
H
), 7.11 (s, 0.5H, Ar-
), 5.40 (s, 0.5H, Ar-C
H
), 6.83–6.90 (m, 1H, Ar-
H
H
H
), 6.69–6.74 (m, 1H, Ar-
), 4.29 (dd, J = 3.6 and 6.8 Hz, 0.5H, CH2C
), 3.77 (s, 1.5H), 3.76 (s, 0.5H, CH2C
H
), 5.53 (s, 0.5H,
H), 3.83 (dd, J =
H
), 3.33 (dd, J = 7.2 and
10.0 Hz, 0.5H, CH2), 3.28 (dd, J = 7.2 and 10.0 Hz, 0.5H, CH2), 3.16 (dd, J = 3.6 and 10.4 Hz,
0.5H, CH2), 3.06 (t, J = 9.2 Hz, 0.5H, CH2); 13C-NMR (DMSO-d6): 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 C11H13NO4S [M + H]+: 255.0565, found 255.0573.

Molecules 2021, 26, 383
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(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); 1H-NMR (DMSO-d6): 8.17–8.23 (m, 2H, Ar-
H
), 7.80 (d, J
), 5.66 (s, 0.4H,
), 3.97 (dd, J = 6.8 and 9.2 Hz, 0.5H, CH2C ), 3.37
2), 3.32 (dd, J = 6.8 and 10.0 Hz, 0.8H, C 2), 3.06–3.13 (m,
= 8.8 Hz, 0.8H, Ar-
Ar-C ), 4.12 (t, J = 6.4 Hz, 0.7H, CH2C
(dd, J = 6.8 and 10.0 Hz, 0.7H, C
H), 7.67 (d, J = 8.8 Hz, 1H, Ar-H), 5.87 (s, 0.6H, Ar-CH
H
H
H
H
H
1H, CH2); 13C-NMR (DMSO-d6): 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 C10H10N2O4S [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; 1H-NMR (DMSO-d6): 8.46 (s, 0.4H, Ar-
H
), 8.29 (s, 0.6H, Ar-
), 8.12 (dd, J = 1.6 and 8.4 Hz, 0.6H, Ar- ), 7.96 (d, J = 8.0 Hz,
), 7.87 (d, J = 7.6 Hz, 0.6H, Ar- ), 7.60–7.67 (m, 1H, Ar- ), 5.86 (s, 0.6H, Ar-C
5.67 (s, 0.4H, Ar-C
H), 8.17 (dd, J =
1.6 and 8.0 Hz, 0.4H, Ar-
H
H
0.4H, Ar-
H
H
H
H),
),
H
), 4.14 (t, J = 6.0 Hz, 0.6H), 3.95 (dd, J = 7.2 and 8.4 Hz, 0.5H, CH2C
H
3.31–3.38 (m, 1H, CH2), 3.09–3.15 (m, 1H, CH2); 13C-NMR (DMSO-d6): 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); 1H-NMR (DMSO-d6): 7.51 (dd, J = 0.8 and 4.8 Hz, 0.3H,
Ar-
3.6 Hz, 0.6H, Ar-
0.6H, Ar- ), 5.94 (s, 0.7H, Ar-C
3.91 (dd, J = 7.2 and 8.8 Hz, 0.4H, CH2C
H
), 7.42 (dd, J = 0.8 and 5.2 Hz, 0.6H, Ar-
), 6.99 (dd, J = 3.2 and 4.8 Hz, 0.3H, Ar-
), 5.75 (s, 0.4H, Ar-C ), 4.07 (t, J = 6.4 Hz, 0.7H, CH2C
), 3.31–3.39 (m, 1H, C 2), 3.03–3.11 (m, 1H, CH
H
), 7.20 (d, J = 3.2 Hz, 0.4H, Ar-
), 6.95 (dd, J = 3.6 and 4.2 Hz,
),
2);
H), 7.06 (d, J =
H
H
H
H
H
H
H
H
13C-NMR (DMSO-d6): 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 C8H9NO2S2 [M + H]+: 215.0075, found
215.0079.
(2R◦S,4R)-2-(Furan-2-yl)◦thiazolidine-4-carboxylic acid (19). White solid, 99% yield, m.p.
137–140 C (lit. [33] 137–138 C); 1H-NMR (DMSO-d6): 7.65–7.66 (m, 0.3H, Ar-
H), 7.58–7.59
(m, 0.6H, Ar-
0.6H, Ar- ), 6.34–6.35 (m, 0.6H, Ar-
(t, J = 6.4 Hz, 0.7H, CH2C
H
), 6.50 (d, J = 3.2 Hz, 0.3H, Ar-
), 5.74 (s, 0.7H, Ar-C
), 3.87 (dd, J = 6.8 and 8.8 Hz, 0.4H, CH2C
H
), 6.43–6.44 (m, 0.3H, Ar-
), 5.61 (s, 0.4H, Ar-C
), 3.27–3.37 (m,
H
), 6.37–6.38 (m,
H
H
H
H
), 4.11
H
H
1H, CH2), 2.97–3.01 (m, 1H, CH2); 13C-NMR (DMSO-d6): 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); 1H-NMR (DMSO-d6): 8.55 (d, J = 6.0 Hz, 0.4H, Ar-
H
), 8.50
), 7.40 (d, J = 6.0 Hz, 1.6H, Ar- ),
), 3.97 (dd, J =
(d, J = 6.1 Hz, 1.6H, Ar-
5.75 (s, 0.8H, Ar-C ), 5.54 (s, 0.2H, Ar-C
6.9 and 8.8 Hz, 0.2H, CH2C
H
), 7.52 (d, J = 6.0 Hz, 0.4H, Ar-
H
H
H
H), 4.10 (t, J = 6.3 Hz, 0.8H, CH2CH
H
), 3.36 (dd, J = 6.9 and 10.0 Hz, 0.2H, CH2), 3.30 (dd, J = 6.8
and 10.2 Hz, 0.8H, CH2), 3.04–3.10 (m, 1H, CH2); 13C-NMR (DMSO-d6): 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); 1H-NMR (DMSO-d6): 7.18–7.30 (m, 5H, Ar-
H), 4.81 (t, J = 7.0
Hz, 0.7H, SC
3.72 (dd, J = 6.9 and 9.2 Hz, 0.3H, CH2C
(dd, J = 7.3 and 13.7 Hz, 0.3H, CH2C
H
), 4.65 (t, J = 6.8 Hz, 0.3H, SC
), 3.07–3.27 (m, 2H, CH2C
), 2.92 (dd, J = 5.4 and 10.2 Hz, 0.7H, CH2C
H
), 4.15 (dd, J = 5.6 and 6.7 Hz, 0.7H, CH2C
H
),
H
H
H
and Ar-CH2), 3.00
H
),
2.75–2.85 (m, 1H, Ar-CH2); 13C-NMR (DMSO-d6): 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; 1H-NMR (DMSO-d6): 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, SCH2C
H), 3.71 (dd, J = 7.0 and 9.0 Hz, 0.5H, SCH2C
H
),
3.20 (dd, J = 7.0 and 9.9 Hz, 0.5H, SCH2), 3.09 (dd, J = 7.0 and 10.2 Hz, 0.5H, SCH2), 2.94

Molecules 2021, 26, 383
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(dd, J = 5.1 and 10.2 Hz, 0.5H, SCH2), 2.76 (t, J = 9.4 Hz, 0.5H, SCH2), 1.85–1.93 (m, 0.5H,
CH2CH2CH), 1.65–1.77 (m, 1H, CH2CH2CH), 1.49–1.56 (m, 0.5H, CH2CH2CH), 1.35–1.43
(m, 2H, CH2CH2), 0.86–0.91 (m, 3H, CH3); 13C-NMR (DMSO-d6): 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 C7H13NO2S
[M + H]+: 175.0667, found 175.0670.
◦(2RS,4R)-2-Heptylt◦hiazolidine-4-carboxylic acid (23). White solid, 93% yield, m.p. 136–
139 C (lit. [37] 159–160 C); 1H-NMR (DMSO-d6): 4.54 (t, J = 6.6 Hz, 0.5H, SC
H), 4.40 (t, J =
6.7 Hz, 0.5H, SC
3.19 (t, J = 9.6 Hz, 0.5H, SC
0.5H, SC 2), 2.75 (t, J = 9.6 Hz, 0.5H, SC
1H, CH2C 2CH), 1.50–1.67 (m, 0.5H, CH2C
H
), 4.06 (t, J = 6.1 Hz, 0.5H, SCH2C
2), 3.08 (t, J = 9.8 Hz, 0.5H, SC
2), 1.85–1.92 (m, 0.5H, CH2C
2CH), 1.24–1.48 (m, 10H, CH2CH
H
), 3.70 (t, J = 8.5 Hz, 0.5H, SCH2C
2), 2.93 (dd, J = 4.9 and 9.8 Hz,
2CH), 1.69–1.76 (m,
2), 0.84–0.86
H),
H
H
H
H
H
H
H
(m, 3H, CH3); 13C-NMR (DMSO-d6): 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 C11H21NO2S [M +
H]+: 231.1293, found 231.1294.
(2RS,4R)-2-Cyclohexylthiazolidine-4-carboxylic acid (24). White solid, 82% yield, m.p.184–
◦
187 C; 1H-NMR (DMSO-d6): 4.01–4.02 (m, 0.4H, SC
H), 3.65–3.69 (m, 0.6H, SCH), 3.13–3.16
(m, 0.6H, SCH2CH), 2.98–3.01 (m, 0.4H, SCH2CH), 2.87–2.89 (m, 0.4H, SCH2CH), 2.65–2.68
(m, 0.6H, SCH2CH), 1.95–1.97 (m, 1H, CH2CHCH2), 1.40–1.66 (m, 4H, CH2CH2CH), 0.98–
1.15 (m, 6H, CH2CH2CH2); 13C-NMR (DMSO-d6): 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 C10H17NO2S [M +
H]+: 215.0980, found 215.0978.
4.2. Biological Assays
◦
Each test was repeated three times at 25
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 [ 27], Detailed bioassay
±
1 C. Active effect expressed in percentage
5,
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 1H &
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
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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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