Discovery of benzotriazole-azo-phenol/aniline derivatives as antifungal agents

Article abstract of DOI:10.1016/j.bmcl.2017.11.032

A series of benzotriazole-azo-phenol/aniline derivatives were prepared and evaluated for their antifungal activities against six phytopathogenic fungi such as Fusarium graminearum, Fusarium solani, Alternaria alternate, Valsa mali, Botrytis cinerea, and Curvularia lunata. Among them, compounds IIf, IIn, and IIr showed a broad-spectrum of potent antifungal activities. Especially some compounds displayed 3.5–10.8 folds more potent activities than carbendazim against A. alternata and C. lunata. Notably, compounds IIc, IIm, and IIr exhibited good protective and therapeutic effects against B. cinerea at 200 μg/mL. Their structure-activity relationships were also discussed.

Full text of DOI:10.1016/j.bmcl.2017.11.032

Accepted Manuscript
Discovery of benzotriazole-azo-phenol/aniline derivatives as antifungal agents
Min Lv, Jingchun Ma, Qin Li, Hui Xu
PII:
S0960-894X(17)31119-8
https://doi.org/10.1016/j.bmcl.2017.11.032
BMCL 25438
DOI:
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Bioorganic & Medicinal Chemistry Letters
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Accepted Date:
23 October 2017
15 November 2017
16 November 2017
Please cite this article as: Lv, M., Ma, J., Li, Q., Xu, H., Discovery of benzotriazole-azo-phenol/aniline derivatives
as antifungal agents, Bioorganic & Medicinal Chemistry Letters (2017), doi: https://doi.org/10.1016/j.bmcl.
2
017.11.032
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Discovery of benzotriazole-azo-phenol/aniline derivatives as
antifungal agents
a
Min Lv , Jingchun Ma , Qin Li , Hui Xu
a
a
a,b,
*
a
Research Institute of Pesticidal Design & Synthesis, College of Plant Protection,
Northwest A&F University, Yangling 712100, Shaanxi Province, China.
b
Shaanxi Key Laboratory of Natural Products & Chemical Biology, College of
Chemistry and Pharmacy, Northwest A&F University, Yangling 712100, Shaanxi
Province, China
*Corresponding author. Tel./fax: +86 29 87091952. E-mail address:
orgxuhui@nwsuaf.edu.cn (H. Xu)
1

Abstract: A series of benzotriazole-azo-phenol/aniline derivatives were prepared and
evaluated for their antifungal activities against six phytopathogenic fungi such as
Fusarium graminearum, Fusarium solani, Alternaria alternate, Valsa mali, Botrytis
cinerea, and Curvularia lunata. Among them, compounds IIf, IIn, and IIr showed a
broad-spectrum of potent antifungal activities. Especially some compounds displayed
3.5-10.8 folds more potent activities than carbendazim against A. alternata and C.
lunata. Notably, compounds IIc, IIm, and IIr exhibited good protective and
therapeutic effects against B. cinerea at 200 µg/mL. Their structure-activity
relationships were also discussed.
Keywords: Benzotriazole; Benzotriazole-azo-phenol; Benzotriazole-azo-aniline;
Antifungal activity; Therapeutic effect; Protective effects
2

Fungicides play a key role in the integrated management of diseases in
crops, however, the appearance of resistance in the target pathogens has resulted in
1
limiting the efficacy and useful lifetime of fungicides. Therefore, development of
new alternatives for effective control of those agricultural diseases is highly
2
desirable.
part B
HO
part A
R2
N
N
N
R1
N
N
N
H
N
H
benzotriazole
I
part B
H N
2
HO
R
N
N
R
N
N
N
N
N
N
H
N
N
H
II-B
II-A
Figure 1. The chemical structures of benzotriazole, benzimidazole-azo-phenols (I),
benzotriazole-azo-phenols (II-A), and benzotriazole-azo-anilines (II-B).
Many compounds containing the benzotriazole nucleus (Figure 1) display a
3
variety of biological properties including CK2 inhibitory activity, antifungal
4
activity, antimicrobial activity,
-6
7-10
7 11
antiparasitic activity, antiviral activity, and
7
antitumor activity. Previously, we synthesized a series of benzimidazole-zao-phenol
derivaives (I, Figure 1) as antifungal agents, and found that some compounds
exhibited the potent antifungal activities against five phytopathogenic fungi such as
Fusarium graminearum, Alternaria solani, Valsa mali, Botrytis cinerea, and
12
Curvularia lunata. In continuation of our program aimed at the discovery and
3

1
3-16
development of pesticidal agents,
here we prepared
a
series of
benzotriazole-azo-phenol hybrids (II-A, Figure 1) as antifungal agents by
incorporating the benzotriazole nucleus with the fragment B of I; meanwhile, a series
of benzotriazole-azo-aniline derivatives (II-B, Figure 1), where the amino group was
substituted for the hydroxy of II-A, were also synthesized as antifungal agents. Their
antifungal activities were tested against six phytopathogenic fungi, such as Fusarium
graminearum, Fusarium solani, Alternaria alternate, Valsa mali, Botrytis cinerea, and
Curvularia lunata.
Scheme 1 Synthesis of 5-aminobenzotriazole (3).
Scheme 2 Synthesis of intermediates 6a-f.
4

R2
=
NH2
N
N
NH2
9a: H
9d: 4-Cl
N
H
9
b: 2-CH3 9e: 4-Br
3
9c: 2-Cl
R2
R2
IIr-x
NH2
NaNO2, HCl
OH
6a-f, 8k
NaOH/EtOH
o
N
IIk: R2 = H
0
~5
C
N
N
2
N
N
IIl:
R
= CH
3
o
IIk-m
0
~5 C, 3~6 h
+
-
N
H
IIm: R2 = Cl
HO
N2 Cl
R2
N
9a-e
8k
N
NaOH/EtOH
R3
N
H
0~5 oC, 3~6 h
7
N
IIn
IIo
: R2 = Cl
2
N
N
4
a,b
:
R = Br
N
N
NH2
N
NaOH/EtOH
~5 C, 4 h or 3 h
NaOH/EtOH
N
H
N
0~5 oC,3~6 h
IIn-o
o
0
8a-j
N
H
IIp,q
IIp: R3 = 1-OH
IIq: R3 = 2-OH
R1
R1
OH
R1
N
N
=
N
N
N
8
8
8
8
a: H
8b: 2-CH3
N
OH
II : R1 = 4-C2H5
N
N
H
OH
N
c:3-CH3
e: 4-Cl
8d: 4-CH3
IId-j
IIa-c
8f: 4-C(CH3)3
N
H
1
g: 4-OCH3 8h: 4-C H5
IId
: R = 4-CH3
h
2
IIa: R1 = H
R1
a-j
1
1
8i: 3-OH
8j: 4-OH
IIe: R = 4-Cl
IIi: R = 5-OH
1
8
IIb: R = 2-CH3
IIc: R1 = 3-CH3
IIf: R = 4-C(CH3)3 IIj: R1 = 4-OH
1
IIg: R1 = 4-OCH3
N
N
NH
OH
O
O
OH
N
N
N
N
N
N
N
N
N
N
H
IIs
N
HO
N
IIr
H
H
N
N
N
O
OH
O
O
O
N
N
N
N
N
IIu
N
IIt
OH
N
H
H
N
O
O
OH
N
H
O
N
N
N
N
N
O
N
IIw
N
N
IIv
OH
O
N
N
O
N
N
H
N
OH
IIx
Scheme 3 Synthesis of benzotriazole-azo-phenol/aniline derivatives (IIa-x).
As shown in Scheme 1, firstly, 4-nitro-o-phenylenediamine (1) reacted with
sodium nitrite in the presence of acetic acid to afford 5-nitrobenzotriazole (2), which
was further reduced by SnCl ·2H O to give 5-aminobenzotriazole (3). On the other
hand, 1-naphthol (4a) or 2-naphthol (4b) reacted with 1,3-dibromopropane or
2
2
1,4-dibromobutane to give 5a–c. Then compounds 6a–f were obtained by reaction of
5

5a-c with resorcinol or hydroquinone (Scheme 2). Finally, as shown in Scheme 3,
o
compound 3 reacted with concentrated hydrochloric acid and sodium nitrite at 0-5 C
to give the corresponding benzenediazonium chloride (7), which further reacted with
o
the different phenols/anilines (4a,b; 6a–f; 8a–k or 9a–e) at 0-5 C for 3-6 h to give
benzotriazole-azo-phenol/aniline derivatives (IIa–x). Their structures were well
1
characterized by H NMR, HRMS, IR and mp (see Supplementary data).
Table 1
Antifungal activities of IIa-x against six phytopathogenic fungi at 100 µg /mL
a
Antifungal activities (inhibition, %)
Compounds
V. mali
C. lunata
F. solani
A.alternata F. graminearum B. cinerea
b
2
3
40.3gh
10.2n
52.0def
22.4k
48.5fg
43.9fg
7.7l
36.8ij
26.9kl
39.0hij
40.4ghij
41.3ghij
49.3de
17.5m
77.6a
21.2jk
9.3lm
50.8de
24.4ij
45.1efg
36.3gh
15.0kl
83.4b
39.1jk
33.8klm
45.7hi
43.1ij
66.3d
47.8o
63.7def
30.5m
57.9fg
2.0o
IIa
62.1ef
48.5no
76.4b
IIb
IIc
56.3de
51.7efg
32.5lm
82.1a
68.3cd
36.3kl
37.8jkl
82.8c
IId
IIe
63.7e
64.0e
IIf
72.8c
72.1c
IIg
32.7j
24.7kl
18.4m
10.8n
31.1hi
53.9de
23.3ijk
9.5lm
36.3gh
31.6hi
34.7h
33.1klm
59.9cd
28.5m
13.8o
48.7no
57.5ghi
52.4klm
21.2q
9.9n
IIh
IIi
58.7d
2.6l
32.5lm
36.3klm
9.9n
IIj
8.9l
10.6n
IIk
IIl
39.8ghi
35.7hij
40.3ghi
55.6de
30.6jk
54.6de
35.2hij
83.2b
43.9gh
44.3gh
39.3hi
36.1hij
52.1ef
16.1l
45.7efg
28.3kl
43.0fgh
59.5c
55.6def
37.8jkl
49.0gh
76.2b
60.5fg
49.7mno
58.4gh
77.1b
53.0gh
29.9m
69.3cd
69.0cd
85.3b
IIm
IIn
IIo
60.5d
62.7b
36.3gh
52.3de
37.3gh
68.6c
73.5b
78.8b
IIp
IIq
IIr
23.2l
41.5ij
62.1ef
58.9gh
51.1lmn
49.8mno
56.5hij
55.2ijk
44.1p
49.7hi
42.9ijk
90.8b
35.4j
38.4jkl
64.1c
63.9b
IIs
40.7ghij
23.6l
35.9h
56.2de
64.9c
42.9ijk
52.8gh
58.5efg
43.9ij
IIt
36.6h
IIu
IIv
42.6fghi
29.6k
38.9gh
17.3kl
48.0ef
3.3m
59.4cd
38.8jk
72.1b
IIw
IIx
48.1def
14.4mn
51.4cde
53.6jkl
18.7q
65.1de
30.4lm
100.0a
19.6n
carbendazim
90.8a
91.7a
54.8efg
100.0a
6

a
b
Note: Values are the mean of three replicates. Multiple range test using Duncan’s test ( p
0.05). The same letters denote treatments not significantly different from each other.
As shown in Table 1, their antifungal activities of
<
benzotriazole-azo-phenol/aniline derivatives (IIa–x) were tested in vitro at 100
µg/mL against six phytopathogenic fungi such as Fusarium graminearum,
Alternaria alternate, Fusarium solani, Valsa mali, Botrytis cinerea, and
Curvularia lunata. Carbendazim, a commercial agricultural fungicide, was used as
a positive control. Analysis of variance (ANOVA) was followed by Duncan’s post
test, which was conducted using SPSS 20 for Windows 7. Among all derivatives,
compounds IIa, IIf, IIh, IIn, IIp, IIr, and IIw showed the good antifungal activity
against V. mali; compounds IIf, IIn, IIo, and IIr showed the good antifungal
activity against C. lunata; compounds IIa, IIf, IIh, IIn, IIp, and IIr showed the
good antifungal activity against F. solani; compounds IIc, IId, IIf, IIh, IIk, IIn,
IIo, IIr–u, and IIw showed the good antifungal activity against A. alternata;
compounds 2, IIa, IIc–f, IIh, IIi, IIk, IIm–r, IIt, IIu, and IIw showed the good
antifungal activity against F. graminearum; compounds 2, IIa, IIc, IIf, IIk,
IIm–o, IIr, IIt, IIu, and IIw showed the good antifungal activity against B.
cinerea. Especially compounds IIf, IIn, and IIr showed a broad-spectrum of
antifungal activities against six tested phytopathogenic fungi.
To benzotriazole-azo-phenol derivatives (IIa–j), compound IIf containing the
4
-tert-butyl and 1-hydroxy groups on the phenyl ring, showed an excellent and a
broad-spectrum of antifungal activities against six tested phytopathogenic fungi.
7

The inhibition rates of IIf at 100 µg/mL against V. mali, C. lunata, F. solani, A.
alternata, F. graminearum, and B. cinerea were 72.8%, 77.6%, 83.4%, 82.1%,
7
2.1%, and 82.8%, respectively. However, when other groups such as methyl,
chlorine atom, methoxy, ethyl, and hydroxy were at the 4-position of the phenyl
ring, the antifungal activities of the corresponding compounds were generally not
good (Table 1). It demonstrated that the tert-butyl group at the 4-position of the
phenyl ring was very important for the activities. This result was the same with
1
2
that of benzimidazole-zao-phenol derivaives.
To benzotriazole-azo-aniline
derivatives (IIk–o), compound IIn containing the 4-Cl and 1-amino groups on the
phenyl ring, showed a broad-spectrum of antifungal activities against six tested
phytopathogenic fungi. The inhibition rates of IIn at 100 µg/mL against V. mali, C.
lunata, F. solani, A. alternata, F. graminearum, and B. cinerea were 55.6%,
5
9.5%, 60.5%, 76.2%, 77.1%, and 69.0%, respectively. Whereas the inhibition
rates of IIm (containing the 3-Cl and 1-amino groups on the phenyl ring) at 100
µg/mL against V. mali, C. lunata, F. solani, A. alternata, F. graminearum, and B.
cinerea were 40.3%, 43.0%, 34.7%, 49.0%, 58.4%, and 69.3%, respectively. It
suggested that the position of the amino group on the phenyl ring was also
essential for the activities. In general, the activities of 1-naphthol derivative were
better than those of 2-naphthol one. It was noteworthy that
benzotriazole-azo-2,2'-biphenol derivative (IIr) exhibited the good and
broad-spectrum of antifungal activities against six phytopathogenic fungi. The
inhibition rates of IIr at 100 µg/mL against V. mali, C. lunata, F. solani, A.
8

alternata, F. graminearum, and B. cinerea were 83.2%, 63.9%, 68.6%, 64.1%,
5
1
1.1%, and 90.8%, respectively. To IIu–x, compounds IIu and IIw (containing
-OH and 5-alkylxoy) generally showed more potent activities than compounds
IIv and IIx (containing 1-OH and 4-alkylxoy).
Table 2
EC50 values of some selected compounds against six phytopathogenic fungi.
a
EC50 (µmol/mL)
Compounds
carbendazim
F. solani
B. cinerea
A. alternate
V. mali
F. graminearum
C. lunata
0.002
0.004
0.54
0.005
0.002
0.56
IIc
IIf
/
0.07
0.07
/
0.14
0.15
0.14
/
/
0.10
/
0.14
0.13
0.16
0.12
IIk
IIm
IIn
IIo
IIr
IIs
IIt
/
/
/
/
/
0.16
0.09
0.12
0.05
/
/
/
/
0.17
0.10
0.13
0.09
0.08
0.10
0.08
0.05
/
/
0.14
/
/
/
0.20
0.16
0.11
0.15
/
/
/
/
/
/
0.14
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
IIu
IIw
2
/
0.07
0.21
a
50% effective concentration: concentration of compound that inhibits the fungi growth by 50%.
As described in Table 2, subsequently, the EC50 values of potent compounds
IIc, IIf, IIk, IIm–o, IIr–u, IIw, and 2 against some phytopathogenic fungi were
further tested. Among them, the EC50 values of IIf, IIn, and IIr against F. Solani
were 0.14, 0.17, and 0.11 µmol/mL, respectively; the EC50 values of IIc, IIf,
IIm–o, IIr, IIw, and 2 against B. cinerea were 0.07, 0.07, 0.16, 0.09, 0.12, 0.05,
0
.07, and 0.21 µmol/mL, respectively; the EC50 values of IIc, IIf, IIk, IIn, IIo,
IIr–u, and IIw against A. alternata were 0.14, 0.15, 0.14, 0.10, 0.13, 0.09, 0.08,
0
.10, 0.08, and 0.05 µmol/mL, respectively. Obviously, compounds IIc, IIf, IIk,
9

IIn, IIo, IIr–u, and IIw displayed 3.5-10.8 folds more potent activities than
carbendazim (EC50 value: 0.54 µmol/mL) against A. alternata. The EC50 values of
IIf, and IIr against V. mali were 0.13, and 0.15 µmol/mL, respectively; the EC50
values of IIf, IIo, and IIr against C. lunata were 0.12, 0.16, and 0.14 µmol/mL,
respectively. Whereas the EC50 value of carbendazim against C. lunata was 0.56
µmol/mL. That is, against C. lunata, compounds IIf, IIo, and IIr exhibited 3.5-4.6
folds more potent activities than carbendazim. Meanwhile, effects of compounds
IIf and IIr on the growth of V.mali and B. cinerea at different concentrations were
shown in Figures 2 and 3, respectively.
1
0

Figure 2. Effects of compound IIf (MJC-42) on the growth of Valsa mali at different
concentrations (CK: blank control group).
11

Figure 3. Effects of compound IIr (MJC-82) on the growth of Botrytis cinerea at
different concentrations (CK: blank control group).
Finally, as shown in Table 3, the protective and therapeutic effects of potent
1
7
compounds IIc, IIf, IIm–o, IIr, and 2 against B. cinerea were conducted.
Compounds IIc, IIm, and IIr showed good protective and therapeutic effects
against B. cinerea at 200 µg/mL. Especially the protective and therapeutic effects
of compound IIc were 60.0% and 70.7%, respectively. Therapeutic effects of
compounds IIc, IIf, IIm–o, IIr, and 2 against B. cinerea at 200 µg/mL were
1
2

depicted in Figure 4.
Table 3
Protective and therapeutic effects of compounds 2, IIc, IIf, IIm-o and IIr against B.
cinerea at 200 µg /mL
a
Compounds
Protective effect (%)
27.3 ± 1.1
60.0 ± 1.9
69.4 ± 1.1
72.8 ± 1.6
45.4 ± 1.1
36.4 ± 1.3
72.8 ± 0.4
82.5 ± 1.1
Therapeutic effect (%)
56.1 ± 1.0
70.7 ± 2.5
29.7 ± 1.8
56.1 ± 3.2
40.7 ± 2.4
61.2 ± 3.4
48.8 ± 2.4
78.3 ± 0.4
2
IIc
IIf
IIm
IIn
IIo
IIr
carbendazim
a
Note: Values are the mean ± SE of three replicates.
Figure 4. Therapeutic effects of compounds IIc, IIf, IIm-o, IIr, and 2 against
Botrytis cinerea at 200 µg/mL (CK: blank control group; CB: carbendazim).
In conclusion, we have synthesized a series of benzotriazole-azo-phenol/aniline
1
3

derivatives and evaluated for their antifungal activities against six phytopathogenic
fungi. Among them, compounds IIf, IIn, and IIr exhibited the excellent and a
broad-spectrum of antifungal activities; especially against A. alternata and C. lunata,
some derivatives showed 3.5-10.8 folds more promising activities than carbendazim.
Moreover, three compounds such as IIc, IIm, and IIr displayed good protective and
therapeutic effects against B. cinerea. Their structure-activity relationships were also
observed. This will pave the way for further preparation and application of
benzotriazole-azo-phenol/aniline derivatives as antifungal agents.
Acknowledgments
The present research was partly supported by National Key R&D Program of
China (No. 2017YFD0200506), and Special Funds of Central Colleges Basic
Scientific Research Operating Expenses (No.2452015096).
Supplementary data
Supplementary data (spectral data, and the protocol used for antifungal studies)
associated with this article can be found, in the online version.
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Graphic abstract
Discovery of benzotriazole-azo-phenol/aniline derivatives as antifungal agents
Min Lv, Jingchun Ma, Qin Li, Hui Xu*
1
6