Synthesis and herbicidal evaluation of novel benzothiazole derivatives as potential inhibitors of D1 protease

Article abstract of DOI:10.1016/j.bmcl.2016.01.087

D1 protease is a C-terminal processing protease that has been predicted to be an ideal herbicidal target. Three novel series of benzothiazole derivatives were synthesized and evaluated for their herbicidal activities against Brassica napus (rape) and Echinochloa crusgalli (barnyard grass). The preliminary bioassay indicated that most of the synthesized compounds possess promising D1 protease inhibitory activities and considerable herbicidal activities. Molecular docking was performed to position representative compounds into the active site of D1 protease to determine a probable binding model.

Full text of DOI:10.1016/j.bmcl.2016.01.087

Accepted Manuscript
Synthesis and herbicidal evaluation of novel benzothiazole derivatives as po-
tential inhibitors of D1 protease
Tonghui Huang, Jie Sun, Lin An, Lixian Zhang, Cuiping Han
PII:
S0960-894X(16)30097-X
http://dx.doi.org/10.1016/j.bmcl.2016.01.087
BMCL 23581
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
15 October 2015
17 January 2016
30 January 2016
Please cite this article as: Huang, T., Sun, J., An, L., Zhang, L., Han, C., Synthesis and herbicidal evaluation of
novel benzothiazole derivatives as potential inhibitors of D1 protease, Bioorganic & Medicinal Chemistry Letters
(2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.01.087
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphical Abstract
To create your abstract, type over the instructions in the template box below.
Fonts or abstract dimensions should not be changed or altered.
Synthesis and herbicidal evaluation of novel
benzothiazole derivatives as potential
inhibitors of D1 protease
Leave this area blank for abstract info.
Tonghui Huang, Jie Sun, Lin An, Lixian Zhang, Cuiping Han

Bioorganic & Medicinal Chemistry Letters
jour nal homepage: w ww.elsevier. com
Synthesis and herbicidal evaluation of novel benzothiazole derivatives as
potential inhibitors of D1 protease
Tonghui Huanga, b∗, Jie Suna, Lin Ana, Lixian Zhanga, Cuiping Hanc
aJiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, School of Pharmacy, Xuzhou Medical College, Xuzhou, Jiangsu 221004, PR China
bKey Laboratory of Pesticide and Chemical Biology of Ministry of Education, Central China Normal University, Wuhan 430079, PR China
cSchool of Medical Imaging, Xuzhou Medical College, Jiangsu, Xuzhou 221004, PR China
ARTICLE INFO
ABSTRACT
Article history:
Received
D1 protease is a C-terminal processing protease that has been predicted to be an ideal herbicidal
target. Three novel series of benzothiazole derivatives were synthesized and evaluated for their
herbicidal activities against Brassica napus (rape) and Echinochloa crusgalli (barnyard
grass). The preliminary bioassay indicated that most of the synthesized compounds possess
promising D1 protease inhibitory activities and considerable herbicidal activities. Molecular
docking was performed to position representative compounds into the active site of D1 protease
to determine a probable binding model.
Revised
Accepted
Available online
Keywords:
Benzothiazoles
D1 protease inhibitor
Molecular docking
Herbicidal activities
Synthesis
2009 Elsevier Ltd. All rights reserved.
The photosystem II D1 C-terminal processing protease (D1
protease, CtpA) is encoded by CtpA gene and responsible for
cleaving D1 precursor protein to form mature D1 protein in the
chloroplast of green plants.1,2 This process is essential for the
assembly of a manganese cluster and consequent light-mediated
water oxidation in photosystem II, which is crucial for plant
growth.3,4 Diner et al.5 reported that knockout of the CtpA gene
results in a non-photoautotrophic phenotype due to an inability to
form mature D1. Thus, the inhibitors of the D1 protease that
target the photosynthetic apparatus of plants could constitute a
broad new class of herbicides.6 Moreover, D1 protease has high
homology and lower abundance than D1 protein in the thylakoids
of higher plants.7 It is thus likely that much lower amounts of an
effective D1 protease inhibitor would be required than the widely
applied herbicides targeting D1 protein.8 The frequent use of
herbicides has resulted in many resistant weed species and no
rhodanine dimers, ketoheterocycles, benzoxazinones, CF3
peptides and imidazoles were evaluated for their inhibitory
activities against both recombinant and native spinach CtpA.
Zhang and co-workers7 designed and synthesized a series of
novel 4-(4-(5-methyl-3-arylisoxazol-4-yl)thiazol-2-yl)piperidyl
carboxamides and thiocarboxamides as potential inhibitors
targeting D1 protease in plants. In this paper, we intend to
develop novel and effective inhibitors against D1 protease.
herbicide with
a new molecular target sites has been
commercialized in the last 20 years.9,10 Identifying and
synthesizing new compounds that target D1 protease in plants
may be
a
valuable strategy for developing efficient
and environmentally friendly herbicides.11
Although the X-ray crystal structure of the D1 protease was
reported in 2000, relatively few small molecule inhibitors of D1
protease have been reported.12 Duff and co-workers6 described a
screening assay that led to the discovery of five novel chemical
Figure 1. Design strategy of the target compounds.
In the work of Duff and co-workers described above, a chiral
alpha-ketoheterocycle compound bearing a benzothiazole moiety
(benzothiazole ketone), was identified as a potential inhibitor of
D1 protease and was shown to possess significant herbicidal
classes of CtpA inhibitors. These lead compounds, including
———
∗ Corresponding author. Tel.: +86-516-83262137; fax: +86-516-83262251; e-mail: tonghhuang@xzmc.edu.cn.

activity. However, it should be noted that only the racemate was
evaluated and the single active enantiomer was not reported in
the literature.6 The active site of D1 protease consists of two
hydrophobic pockets, which are formed by the residues Phe-140,
Leu-152, Leu-212, Tyr-213, Val-324, Val-337, Ile-339, Ile-348,
Tyr-349, Val-376, Ile-400, Val-403 and Tyr-419.12 As shown in
Figure 1, a proposed pharmacophore model consisting of a
benzothiazole moiety and two suitably substituted phenyl rings
bound to a linker was hypothesised on the basis of the crystal
structure of D1 protease. To increase the structural diversity and
discover more potent inhibitors of D1 protease, three novel
benzothiazoles were designed and synthesized based on the
structure of lead compound and topological regions of D1
protease (Figure 1). Their herbicidal activities against Brassica
napus (rape) and Echinochloa crusgalli (barnyard grass) were
evaluated. Preliminary HPLC enzyme assays7 were also
performed to evaluate the native spinach D1 protease inhibitory
activities. Additionally, molecular docking was carried out to
investigate the possible binding mode of typical compounds with
D1 protease.13, 14
Schemes 1 and 2. As shown in Scheme 1, commercially available
benzothiazole 1 was converted to benzothiazol-2-yllithium in the
presence of n-BuLi at low temperature. Benzothiazol-2-yllithium
was not isolated and directly reacted with single enantiomers of
Boc-Glycine ethyl ester derivatives 2 to afford the intermediates
3. Boc group on the intermediates 3 were removed with
trifluoroacetic acid (TFA) in dichloromethane (DCM) at room
temperature, followed by amidation in the presence of 1-
[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-
b]pyridinium 3-oxid hexafluorophosphate (HATU) and
triethylamine (TEA) to afford the desired substituted
benzothiazole ketone derivatives 4.
As shown in Scheme 2, the intermediates 7 were obtained by
the reaction of commercially available benzothiazolethiol 5 with
single enantiomers of Boc-glycinol derivatives 6 in the presence
of diethyl azodicarboxylate (DEAD) and triarylphosphines (PPh3)
at room temperature. The intermediates 7 further underwent N-
Boc deprotection and amidation to give intermediate
benzothiazole sulfide derivatives 8. The resulted intermediates 8
were then reacted with m-chloroperoxybenzoic acid (m-CPBA)
in DCM at room temperature to afford the corresponding
benzothiazole sulfone derivatives 9.
The general synthesis of substituted benzothiazole ketones,
benzothiazole sulfides and benzothiazole sulfones are outlined in
Scheme 1. Reagents and conditions: a) n-BuLi, THF, -78 °C; b) THF, 5-10 °C; c) (i) TFA, DCM; (ii) carboxylic acid, HATU, TEA, DMF, rt.
Scheme 2. Reagents and conditions: a) DEAD, PPh3, THF, rt; b) (i) TFA, CH2Cl2; (ii) carboxylic acid, HATU, TEA, DMF, rt; c) m-CPBA, CH2Cl2, rt.
All the synthesized compounds 4a–4m, 8a–8h and 9a–9h
were evaluated for herbicidal activities against barnyard grass
and rape at dosages of 100 mg/L and 10 mg/L according to a
were higher than their corresponding S enantiomers. The
herbicidal activities of benzothiazole sulfone derivatives are
higher than the corresponding benzothiazole sulfide derivatives,
which have higher logP values. This implies that the lipophilicity
may contribute to the variation. For example, the benzothiazole
sulfone derivative 9g (R1= benzyl, R2= 2,4-difluorophenyl; 87;
74; 47; 38; logP = 4.82) displayed higher herbicidal activities
than the corresponding benzothiazole sulfide derivative 8g (R1 =
benzyl, R2 = 2,4-difluorophenyl; 62; 41; 29; 27; logP = 5.94). In
addition, 4i, 4j, 4k, 4l, 9e, 9f, 9g and 9h showed stronger
inhibition of the growth of the dicotyledon rape than that of the
monocotyledon barnyard grass and exhibited a relative selectivity
at the same concentration.
previously reported procedure.15,16 Atrazine,
a commercial
herbicide, was used as a control. The parent compound 4m was
used as another control. The in vitro inhibitory activities of these
compounds were further evaluated against the native spinach D1
protease. The octanol-water partition coefficient logP for these
compounds was calculated using the online Molinspiration logP
calculator.17 For the convenience of structure-activity relationship
analysis, compounds 4a–4m, 8a–8h and 9a–9h were defined as
benzothiazole ketone derivatives, benzothiazole sulfide
derivatives and benzothiazole sulfone derivatives, respectively.
The bioassay results (Table 1) indicate that most of the
synthesized compounds showed moderate to good herbicidal
activities inhibiting the growth of barnyard grass and rape at a
dosage of 100 mg/L. For instance, 4k, 4l, 9g and 9h exhibited
inhibitory rates of >80% to the growth of rape at a concentration
of 100 mg/L. However, when the concentration was decreased to
10 mg/L, the inhibitory activities of 9g and 9h decreased
obviously in comparison with the commercial herbicide
Atrazine. In general, the herbicidal activities of R enantiomers
The in vitro inhibitory activities of these compounds against
native spinach D1 protease were obtained with an HPLC assay
method7 by employing a mimic polypeptide substrate 24-mer
oligopeptide (S24). From the data in Table 1, we can conclude
that the sequence of inhibitory activities against D1 protease is
benzothiazole ketone derivatives
> benzothiazole sulfone
derivatives > benzothiazole sulfide derivatives. For example, the
benzothiazole ketone derivative 4g (R1 = Bz, R2 = 2,4-
difluorophenyl, R enantiomer) displayed better D1 protease

inhibitory activity than the corresponding benzothiazole sulfone
derivative 9g, while the benzothiazole sulfide derivative 8g
showed the least inhibitory activity with IC50 value of 32.5 µM.
In addition, the R-enantiomer was found to have more potent D1
protease inhibitory activities as compared to its corresponding S-
enantiomer and racemate. For example, compound 4g (R
enantiomer) showed a better D1 protease inhibitory activity with
an IC50 value of 3.2 µM than its corresponding S enantiomer 4h
(IC50 value 36.3 µM) and racemate 4m (IC50 value 28.5 µM).
Among these three series of compounds, replacing the R2 group
with t-BuO group reduced the inhibition of D1 protease. For
example, 4a–4d, 8a–8d, 9a–9d showed D1 protease inhibitory
activities with IC50 values more than 50 µM.
Table 1 LogP measurements, in vitro inhibitory activities of D1 protease and herbicidal activities (inhibition rate/%).
Relative inhibition
Ctpa IC50
(µM)
Comp.
Config.
R1
R2
LogP
Rape
Barnyard grass
100 mg/L 10 mg/L 100 mg/L 10 mg/L
4a
4b
4c
4d
4e
4f
R
Me
Me
Bz
Bz
Bz
Bz
Bz
Bz
Bz
Bz
Bz
Bz
Ph
Ph
Bz
Bz
Bz
Bz
Bz
Bz
Ph
Ph
Bz
Bz
Bz
Bz
Bz
Bz
Bz
t-BuO
t-BuO
>50
>50
>50
>50
29.4
44.3
3.2
4.58
4.58
4.69
4.69
4.40
4.40
4.49
4.49
4.82
4.82
3.85
3.85
5.48
5.48
5.69
5.69
5.85
5.85
5.94
5.94
4.36
4.36
4.57
4.57
4.73
4.73
4.82
4.82
4.49
2.55
39
38
50
43
61
50
79
62
75
72
97
95
48
37
61
48
66
43
62
50
53
44
61
50
78
72
87
90
74
95
21
17
24
31
34
28
59
42
66
55
95
83
24
20
29
25
22
28
41
43
33
21
38
33
53
45
74
70
42
86
41
34
48
43
53
42
68
47
69
57
46
50
49
41
49
51
47
33
29
34
48
46
63
60
39
50
47
44
53
85
17
9
S
R
t-BuO
24
12
30
29
52
37
43
26
20
32
11
0
S
t-BuO
R
4-fluorophenyl
4-fluorophenyl
2,4-difluorophenyl
2,4-difluorophenyl
3,4-dimethoxyphenyl
3,4-dimethoxyphenyl
(2,4-dichlorophenoxy)methyl
(2,4-dichlorophenoxy)methyl
t-BuO
S
4g
4h
4i
R
S
36.3
24.9
41.3
2.5
R
4j
S
4k
4l
R
S
10.4
>50
>50
>50
>50
>50
>50
32.5
48.6
>50
>50
>50
>50
34.6
46.1
14.5
31.4
28.5
>50
8a
8b
8c
8d
8e
8f
R
S
t-BuO
R
t-BuO
20
6
S
t-BuO
R
4-fluorophenyl
4-fluorophenyl
2,4-difluorophenyl
2,4-difluorophenyl
t-BuO
18
19
27
21
36
31
40
38
26
24
38
39
46
76
S
8g
8h
9a
9b
9c
9d
9e
9f
R
S
R
S
t-BuO
R
t-BuO
S
t-BuO
R
4-fluorophenyl
4-fluorophenyl
2,4-difluorophenyl
2,4-difluorophenyl
2,4-difluorophenyl
S
9g
9h
4m
R
S
Racemate
Atrazine
The growth inhibition phenotype of typical compound 4k on
rape (Brassica campestris L.) and barnyard grass (Echinochloca
crusgalli L.) at dosages of 10 mg/L and 100 mg/L is shown in
Figures 2 B and C. The controls obtained from the inhibition by
Atrazine (Figures 2 D and E) and blank (Figure 2 A) are also
shown for comparison. Obviously, both the compound 4k and
Atrazine exhibit phytotoxicity against rape and barnyard grass
either at the dosage of 100 mg/L or 10 mg/L. The stunted growth
or chlorotic phenotype (Figures 2 B and C) may be attributed to

the inhibition of D1 protease and affected the functions of
photosystem II in test plants.
Figure 2. Comparison of the effects of compound 4k and Atrazine on plants growth. (A) Wild-type rape and barnyard grass grown on regular medium. All plants
grow normally. (B) Wild-type rape and barnyard grass grown on medium containing 100 ppm of compound 4k. Most plants show stunted growth or chlorotic
phenotype. (C) Compound 4k (10 ppm). (D) Atrazine (100 ppm). (E) Atrazine (10 ppm).
D1 protease has a high homology in organisms and the crystal
structure of D1 protease of higher plants has not been reported.
To investigate the interactions of the benzothiazole derivatives
with the active site of D1 protease, the three-dimensional
structure of D1 protease of spinach was homology modeled based
on the crystal structures of D1 protease from green alga
Scenedesmus obliquus (PDB: 1FC6).12 The synthesized
compounds were energy minimized and docked into the active
site of D1 protease, which is formed by residues of Phe-140, Leu-
152, Leu-212, Tyr-213, Leu-339, Leu-399, Ile-400 and Tyr-419.
The docked representative compounds 4e, 4f, 4g, 4e, 4h, 9e, 9f,
9g, 9h clustered in the T shaped active site (Figure 3). These
compounds are shaped like a clover leaf and exhibited similar
conformations and binding modes. The benzene rings of the
compounds are stabilized by π–π interactions with Tyr-213 in the
binding pocket. A hydrogen bond is formed between the N atom
of benzothiazole moiety of the typical compounds and the side
chain amide of Ser-216, thus binding the benzothiazole moiety in
a
hydrophobic pocket. The molecular docking model for
compounds 4g and 4h may explain the molecular basis of
stereoselectivity. Compound 4g has a higher docking score and
superior inhibitory activity in comparison to 4h, which is to a
certain extent consistent with the in vivo herbicidal activities. As
shown in Figures 4 A and B, the R stereoisomer of 4g forms
interactions between the carbonyl group and the critical amino
acid residue Lys-397, while the orientation of the carboxyl of the
S stereoisomer 4h was opposite to that of Lys-397 resulting in a
decreased binding energy and a lower docking score. In addition,
a hydrogen-bond interaction between the benzothiazole sulfone
derivatives 9g and the side chain of Ser-372 is missing in the
Figure 4C, we speculate that the missing interaction may
contribute to the lower activity of 9g than compound 4g.
Figure 4. (A) Molecular docking model for compound 4g (yellow and stick)
with the active site of D1 protein, highlighting the hydrogen bonds (red
dashed lines) coordination between the compound 4g and amino acid residues
Ser-216, Lys-397 and Ser-372. (B) Molecular docking model for compound
4h (green and stick) with the active site of D1 protein, highlighting the
hydrogen bonds (red dashed lines) coordination between the compound 4h
and the amino acid residues Ser-216, Ile-167 and Ser-372. (C) Molecular
docking model for compound 9g (yellow and stick) with the bonding pocket
of D1protein, the hydrogen bond (red dashed lines) were formed between the
compound 9g and amino acid residues Ser-216, Ile-167 and Lys-397.
Figure 3. Overlay of D1 protease with compounds of 4e, 4g, 8e, 9e, 9g
(yellow), 4f, 8h, 9f, 9h (green). Molecular surface of the binding site with
rainbow colors formed by residues Phe-140, Leu-152, Leu-212, Tyr-213,
Leu-399, Ile-400, and Tyr-419.
In conclusion, three new series of benzothiazole derivatives
were synthesized and evaluated for their inhibitory activities
against native spinach D1 protease and herbicidal activities

against barnyard grass and rape. The preliminary enzyme assay
results indicated that the R enantiomers possess better D1
protease inhibitory activities than the S enantiomers. Most of the
synthesized compounds showed moderate to good herbicidal
activities at dosages of 100 mg/L and 10 mg/L.
Molecular docking revealed the probable binding model and
molecular basis of binding stereoselectivity. Further investigation
on the structural optimization of these compounds to improve the
herbicidal activities and the in vitro activities against D1 protease
is still in progress in our laboratory.
Acknowledgments
The authors are grateful to the financial support from National
Natural Science Foundation of China (No. 81302629).
Supplementary data
Supplementary data (synthetic procedure and characterization
for all the new compounds, general procedures for the biological
activity) associated with this article can be found, in the online
version, at http://dx.doi.org/xxxx.
References and notes
1. Mabbitt, P. D.; Wilbanks, S. M.; Eaton-Rye, J. J. Plant Physiol.
Biochem. 2014, 81, 96.
2. Kuviková, S.; Tichý, M.; Komenda, J. P. Photochem. Photobiol.
Sci. 2005, 4, 1044
3. Che, Y.; Fu, A.; Hou, X.; McDonald, K.; Buchanan, B. B.; Huang,
W.; Luan, S. Proc. Natl. Acad. Sci. 2013, 110, 16247.
4. Zhou, X.; Hu X.; Li, J.; Wang, N. Mol. Plant-Microbe Interact.
2015, 28, 143.
5. Trost, J. T.; Chisholm, D. A.; Jordan, D. B.; Diner, B. A. J. Biol.
Chem. 1997, 272, 20348.
6. Duff, S. M.; Chen, Y. C. S.; Fabbri, B. J.; Yalamanchili, G.;
Hamper, B. C.; Walker, D. M., et al. Pesticide biochemistry and
physiology, 2007, 88, 1.
7. Hu, D. J.; Liu, S. F.; Huang, T. H.; Tu, H. Y.; Zhang, A. D.
Molecules, 2009, 14, 1288.
8. Chen, Y. C. S.; Fabbri, B. J.; CaJacob, C. A.; Anderson, J. C.;
Duff, S. M. Weed Sci. 2009, 55, 283.
9. Liu, W.; Yuan, G.; Du, L.; Guo, W.; Li, L.; Bi, Y.; Wang,
J. Pest. Biochem. Physiol. 2015, 117, 31.
10. Zhang, H.; Wang, Q.; Ning, X.; Hang, H.; Ma, J.; Yang, X.;
Wang, X.; Wang, P. G., et al. J. Agric. Food Chem. 2015, 63,
3734.
11. Tresch, S. Plant Sci. 2013, 212, 60.
12. Liao, D. I.; Qian, J.; Chisholm, D. A.; Jordan, D. B.; Diner, B. A.
Nat. Struct. Mol. Biol. 2000, 7, 749.
13. Heo, J.; Shin, H.; Lee, J.; Kim, T.; Inn, K. S.; Kim, N. J. Bioorg.
Med. Chem. lett. 2015, 25, 3694.
14. Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew, R.
K.; Goodsell, D. S.; Olson, A. J. J. Comput. Chem. 2009, 30, 2785.
15. Li, Z. S.; Wang, W. M.; Lu, W.; Niu, C. W.; Li, Y. H.; Li, Z. M.;
Wang, J. G. Bioorg. Med. Chem. Lett. 2013, 23, 3723.
16. Huang, T. H.; Tu, H. Y.; Liu, M.; Hou, C. J.; Zhang, A. D. Chin. J.
O. C. 2011, 31, 891.
17. Molinspiration
Cheminformatics.
http://www.molinspiration.com (accessed on 30 September 2015).
Available
online: