Synthesis and anti-phytopathogenic activity of 8-hydroxyquinoline derivatives

Article abstract of DOI:10.1039/c9ra05712a

Phytopathogenic fungi have become a serious threat to the quality of agricultural products, food security and human health globally, necessitating the need to discover new antifungal agents with de novo chemical scaffolds and high efficiency. A series of 8-hydroxyquinoline derivatives were designed and synthesized, and their antifungal activity was evaluated against five phytopathogenic fungi. In vitro assays revealed that most of the tested compounds remarkably impacted the five target fungi and their inhibitory activities were better than that of the positive control azoxystrobin. Compound 2, in particular, exhibited the highest potency among all the tested compounds, with an EC₅₀ of 0.0021, 0.0016, 0.0124, 0.0059 and 0.0120 mM respectively against B. cinerea, S. sclerotiorum, F. graminearum, F. oxysporum and M. oryzae, followed by compound 5c. The morphological observations of optical microscopy and scanning electron microscopy revealed that compounds 2 and 5c caused mycelial abnormalities of S. sclerotiorum. Futhermore, the results of in vivo antifungal activity of compounds 2 and 5c against S. sclerotiorum showed that 5c possessed stronger protective and curative activity than that of 2, and the curative effects of 5c at 40 and 80 μg mL^-1 (84.18% and 95.44%) were better than those of azoxystrobin (77.32% and 83.59%). Therefore, compounds 2 and 5c are expected to be novel lead structures for the development of new fungicides.

Full text of DOI:10.1039/c9ra05712a

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Synthesis and anti-phytopathogenic activity of 8-
hydroxyquinoline derivatives†
Cite this: RSC Adv., 2019, 9, 30087
a
Xiao-Dan Yin,
Yu Sun,a Raymond Kobla Lawoe,a Guan-Zhou Yang,a
Ying-Qian Liu, Xiao-Fei Shang,ab Hua Liu,a Yu-Dong Yang,a Jia-Kai Zhua
a
*
and Xiao-Ling Huanga
Phytopathogenic fungi have become a serious threat to the quality of agricultural products, food security
and human health globally, necessitating the need to discover new antifungal agents with de novo
chemical scaffolds and high efficiency. A series of 8-hydroxyquinoline derivatives were designed and
synthesized, and their antifungal activity was evaluated against five phytopathogenic fungi. In vitro assays
revealed that most of the tested compounds remarkably impacted the five target fungi and their
inhibitory activities were better than that of the positive control azoxystrobin. Compound 2, in particular,
exhibited the highest potency among all the tested compounds, with an EC50 of 0.0021, 0.0016, 0.0124,
0.0059 and 0.0120 mM respectively against B. cinerea, S. sclerotiorum, F. graminearum, F. oxysporum
and M. oryzae, followed by compound 5c. The morphological observations of optical microscopy and
scanning electron microscopy revealed that compounds 2 and 5c caused mycelial abnormalities of S.
sclerotiorum. Futhermore, the results of in vivo antifungal activity of compounds 2 and 5c against S.
sclerotiorum showed that 5c possessed stronger protective and curative activity than that of 2, and the
curative effects of 5c at 40 and 80 mg mLꢀ1 (84.18% and 95.44%) were better than those of azoxystrobin
(77.32% and 83.59%). Therefore, compounds 2 and 5c are expected to be novel lead structures for the
development of new fungicides.
Received 24th July 2019
Accepted 16th September 2019
DOI: 10.1039/c9ra05712a
rsc.li/rsc-advances
N-Heterocycle plays a key role in drug design.9 Quinoline and
its derivatives from natural products or synthetic biologically
1. Introduction
active sources are indispensable heterocyclic compounds
endowed with a broad spectrum of pharmacological proper-
ties.10–13 Amidst quinoline core compounds, 8-hydroxyquinoline
(HQ) has become a privileged scaffold for the design and
synthesis of novel drug candidates due to its broad biological
activities,14–17 such as cytotoxic,18–20 antifungal,20,21 antibacte-
rial,22,23 antilarial,24 and anti-HIV.25 The mode of action of HQ
is related to many factors, according to reports, chelation with
metal ions appears to be crucial because metal ions are cofac-
tors for many physiologically active enzymes.17,26,27
Highly destructive phytopathogenic fungi S. sclerotiorum, B.
cinerea, F. graminearum, F. oxysporum and M. oryzae have
garnered considerable research attention owing to their typical
pathogenic characteristics. In this investigation, we chose HQ as
a primer molecule and introduced the nitro group into the HQ
scaffold (Fig. 1). Motivated by compound 2 displaying superb
antifungal activity than HQ and the positive control azoxystrobin
against the ve target phytopathogens tested, we further struc-
turally derivatized compound 2. Interestingly, preliminary work
showed that the 2-position modication of compound 2 resulted
in a dramatic decrease in antifungal activity, whereas the 7-
position modication of compound 2 with identical groups led to
improved or comparable antifungal activity with HQ (ESI†). Thus,
Phytopathogenic fungi are oen the main culprits in causing
immense decrease in yield and quality of agricultural products,
leading to serious losses in global agricultural and horticultural
production, hence posing a great threat to global food secu-
rity.1,2 More importantly, many phytopathogenic fungi can
produce mycotoxins that are pernicious to human and animal
health.1 Therefore, various antifungal agents have been
discovered, developed and used for a long time to guarantee
wholesome crops, increases in crop yields and economic
benets.3,4 However, excessive use and misuse of many tradi-
tional antifungal agents have led to heightened resistance in
target phytopathogenic fungi, residual toxicity, and even envi-
ronmental pollution in recent decades.5–7 The above concerns
have called for discovery and development of novel antifungal
compounds with lower application dose, higher efficiency and
selectivity, unique mode of action and environmental
compatibility.3,8
aSchool of Pharmacy, Lanzhou University, Lanzhou 730000, People's Republic of
China. E-mail: yqliu@lzu.edu.cn; Fax: +86-931-8915685; Tel: +86-931-8915686
bLanzhou Institute of Husbandry and Pharmaceutical Sciences, Chinese Academy of
Agricultural Sciences, 335 Jiangouyan, Lanzhou 730050, P. R. China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c9ra05712a
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a series of HQ derivatives were synthesized to investigate their conveniently assembled in a one-step synthesis according to
antifungal potential and structural activity relationships (SAR). classic Mannich reaction (Fig. 1). Compounds 4a–4o were
Furthermore, optical microscope and scanning electron micros- prepared by reuxing 2 with formaldehyde and corresponding
copy observations and effects of 8-hydroxyquinoline derivatives aliphatic amine in ethanol.29 Compounds 5a–5p were synthe-
against S. sclerotiorum in vivo were performed to evaluate the sized by reuxing compound 2 with formaldehyde and appro-
antifungal properties of these compounds.
priate aromatic piperazine in pyridine.23 All the synthesized
compounds were puried by recrystallization from absolute
ethanol. Structures of all the synthesized compounds were
1
supported by spectral data including H NMR, 13C NMR, and
2. Results and discussion
2.1. Chemistry
HRMS as reported in Experimental section.
The starting material 8-hydroxyquinoline (1) employed in the
preparation of compound 2 was obtained from Sun Chemical
2.2. Antifungal activity
Technology (Shanghai). Stirring the starting material 1 in The target compounds were evaluated for their antifungal
concentrated HCl with NaNO2 aqueous solution at 0 ꢁC, the activity against ve economically important phytopathogenic
precipitates formed were ltered, and added to a mixture of fungi B. cinerea, S. sclerotiorum, F. graminearum, F. oxysporum
concentrated HNO3 and water (v/v ¼ 3 : 2) at 17 ꢁC to be and M. oryzae. The preliminary antifungal activity screening of
transformed into 2 (Fig. 1).28 Compounds 4a–4o and 5a–5q were the target compounds was determined at 25 and 50 mg mLꢀ1
Fig. 1 Reagents and conditions: (i) NaNO2, concentrated HCl, 0 ꢁC, 1 h; HNO3 : H2O (3 : 2), 17 ꢁC, 75 min; (ii) EtOH, reflux, 24 h; (iii) pyridine,
50 ꢁC, 30 min.
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Table 1 Antifungal activity of 8-hydroxyquinoline derivatives at 50, 25 mg mLꢀ1
Average inhibition rate ꢂ SD (%) (n ¼ 3)
Compd.a
HQ
2
Concb (mg mLꢀ1
)
B. C.c
S. S.c
F. G.c
F. O.c
M. O.c
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
97.92 ꢂ 0.72
85.39 ꢂ 0.65
100.00 ꢂ 0.00
98.33 ꢂ 0.72
100.00 ꢂ 0.00
93.00 ꢂ 0.41
100.00 ꢂ 0.00
85.08 ꢂ 0.49
26.31 ꢂ 0.19
29.71 ꢂ 0.16
100.00 ꢂ 0.00
97.24 ꢂ 0.01
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
99.58 ꢂ 0.72
100.00 ꢂ 0.00
97.88 ꢂ 0.31
100.00 ꢂ 0.00
100.00 ꢂ 0.00
99.58 ꢂ 0.72
77.14 ꢂ 0.74
100.00 ꢂ 0.00
98.33 ꢂ 0.72
100.00 ꢂ 0.00
98.33 ꢂ 0.72
52.33 ꢂ 0.86
0.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
86.04 ꢂ 0.97
100.00 ꢂ 0.00
97.92 ꢂ 0.72
100.00 ꢂ 0.00
89.80 ꢂ 0.49
0.00 ꢂ 0.00
100.00 ꢂ 0.00
77.19 ꢂ 0.31
100.00 ꢂ 0.00
100.00 ꢂ 0.00
0.00 ꢂ 0.00
0.00 ꢂ 0.00
85.00 ꢂ 0.01
78.75 ꢂ 0.01
0.00 ꢂ 0.00
0.00 ꢂ 0.00
12.92 ꢂ 0.01
0.00 ꢂ 0.00
47.50 ꢂ 0.02
19.17 ꢂ 0.02
0.00 ꢂ 0.00
0.00 ꢂ 0.00
0.00 ꢂ 0.00
0.00 ꢂ 0.00
0.00 ꢂ 0.00
0.00 ꢂ 0.00
10.42 ꢂ 0.01
0.00 ꢂ 0.00
18.33 ꢂ 0.01
0.00 ꢂ 0.00
00.00 ꢂ 0.00
0.00 ꢂ 0.00
93.33 ꢂ 0.01
0.00 ꢂ 0.00
21.67 ꢂ 0.03
12.50 ꢂ 0.03
28.75 ꢂ 0.03
3.33 ꢂ 0.03
0.00 ꢂ 0.00
0.00 ꢂ 0.00
9.58 ꢂ 0.03
0.00 ꢂ 0.00
97.92 ꢂ 0.01
93.79 ꢂ 0.01
95.42 ꢂ 0.01
92.79 ꢂ 0.01
94.17 ꢂ 0.03
85.21 ꢂ 0.01
16.67 ꢂ 0.02
7.08 ꢂ 0.01
99.17 ꢂ 0.01
88.24 ꢂ 0.01
98.75 ꢂ 0.01
91.27 ꢂ 0.00
98.33 ꢂ 0.01
88.86 ꢂ 0.02
100.00 ꢂ 0.00
89.50 ꢂ 0.00
99.17 ꢂ 0.01
80.87 ꢂ 0.02
97.85 ꢂ 0.05
57.08 ꢂ 0.01
93.75 ꢂ 0.01
91.21 ꢂ 0.00
34.58 ꢂ 0.03
23.33 ꢂ 0.02
48.33 ꢂ 0.02
73.01 ꢂ 0.33
36.29 ꢂ 0.67
100.00 ꢂ 0.00
100.00 ꢂ 0.00
36.00 ꢂ 0.74
24.89 ꢂ 0.72
93.78 ꢂ 0.72
79.56 ꢂ 0.72
25.78 ꢂ 0.72
16.44 ꢂ 0.72
28.44 ꢂ 0.44
15.33 ꢂ 0.63
31.25 ꢂ 0.02
14.89 ꢂ 0.95
33.73 ꢂ 0.25
16.67 ꢂ 0.62
20.00 ꢂ 0.36
13.76 ꢂ 0.74
18.22 ꢂ 0.60
4.89 ꢂ 0.72
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
31.56 ꢂ 0.72
11.11 ꢂ 0.72
100.00 ꢂ 0.00
74.44 ꢂ 0.95
35.56 ꢂ 0.72
17.56 ꢂ 0.95
27.11 ꢂ 0.34
12.44 ꢂ 0.72
27.11 ꢂ 0.07
11.56 ꢂ 0.72
40.44 ꢂ 0.72
33.78 ꢂ 0.72
18.67 ꢂ 0.17
0.00 ꢂ 0.00
4a
4b
4c
4d
4e
0.00 ꢂ 0.00
4f
100.00 ꢂ 0.00
97.92 ꢂ 0.72
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
97.92 ꢂ 0.72
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
0.00 ꢂ 0.00
4g
4h
4i
29.78 ꢂ 0.15
11.11 ꢂ 0.72
32.44 ꢂ 0.60
20.89 ꢂ 0.72
28.89 ꢂ 0.91
16.22 ꢂ 0.36
27.56 ꢂ 0.60
17.78 ꢂ 0.72
34.67 ꢂ 0.25
20.53 ꢂ 0.66
47.56 ꢂ 0.60
41.96 ꢂ 0.63
36.89 ꢂ 0.72
25.78 ꢂ 0.72
33.78 ꢂ 0.91
27.08 ꢂ 0.75
48.89 ꢂ 0.73
38.22 ꢂ 0.72
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
98.17 ꢂ 0.48
100.00 ꢂ 0.00
100.00 ꢂ 0.00
66.22 ꢂ 0.61
59.11 ꢂ 0.72
84.00 ꢂ 0.03
80.69 ꢂ 0.73
100.00 ꢂ 0.00
98.88 ꢂ 0.60
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
44.89 ꢂ 0.44
31.16 ꢂ 0.69
99.13 ꢂ 0.41
94.92 ꢂ 0.65
83.11 ꢂ 0.72
76.67 ꢂ 0.63
63.56 ꢂ 0.91
43.11 ꢂ 0.91
19.99 ꢂ 0.01
44.89 ꢂ 0.72
26.00 ꢂ 0.62
32.89 ꢂ 0.72
23.02 ꢂ 0.80
78.67 ꢂ 0.05
43.56 ꢂ 0.72
75.56 ꢂ 0.91
52.67 ꢂ 0.62
36.00 ꢂ 0.25
19.56 ꢂ 0.72
32.89 ꢂ 0.72
18.22 ꢂ 0.72
20.89 ꢂ 0.44
0.00 ꢂ 0.00
4j
4k
4l
4m
4n
4o
5a
5b
5c
0.00 ꢂ 0.00
99.11 ꢂ 0.09
97.92 ꢂ 0.18
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
97.85 ꢂ 0.08
90.68 ꢂ 0.73
97.92 ꢂ 0.72
97.91 ꢂ 0.39
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
98.33 ꢂ 0.72
100.00 ꢂ 0.00
95.78 ꢂ 0.12
83.11 ꢂ 0.70
86.46 ꢂ 0.53
88.66 ꢂ 0.39
99.14 ꢂ 0.07
97.25 ꢂ 0.77
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
91.06 ꢂ 0.39
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
97.92 ꢂ 0.72
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
93.03 ꢂ 0.80
91.63 ꢂ 0.53
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
44.89 ꢂ 0.60
34.09 ꢂ 0.64
100.00 ꢂ 0.00
80.34 ꢂ 0.82
99.56 ꢂ 0.72
75.96 ꢂ 0.69
100.00 ꢂ 0.00
95.94 ꢂ 0.16
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
60.00 ꢂ 0.17
32.89 ꢂ 0.72
100.00 ꢂ 0.70
97.67 ꢂ 0.93
86.67 ꢂ 0.17
79.16 ꢂ 0.79
73.75 ꢂ 0.50
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
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Table 1 (Contd.)
Average inhibition rate ꢂ SD (%) (n ¼ 3)
Compd.a
Concb (mg mLꢀ1
)
B. C.c
S. S.c
F. G.c
F. O.c
0.00 ꢂ 0.00
M. O.c
25
50
25
50
25
50
25
50
25
81.50 ꢂ 0.36
87.00 ꢂ 0.00
85.82 ꢂ 0.81
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
98.33 ꢂ 0.72
47.88 ꢂ 0.43
27.00 ꢂ 0.23
97.36 ꢂ 0.92
90.55 ꢂ 0.00
77.30 ꢂ 0.23
100.00 ꢂ 0.00
100.00 ꢂ 0.00
100.00 ꢂ 0.00
98.13 ꢂ 0.63
45.57 ꢂ 0.29
30.43 ꢂ 0.98
24.17 ꢂ 0.05
39.17 ꢂ 0.01
4.17 ꢂ 0.02
73.75 ꢂ 0.01
44.17 ꢂ 0.02
11.25 ꢂ 0.03
0.00 ꢂ 0.00
61.85 ꢂ 0.06
57.32 ꢂ 0.28
55.11 ꢂ 0.72
32.89 ꢂ 0.52
23.33 ꢂ 0.62
31.56 ꢂ 0.71
23.33 ꢂ 0.62
34.22 ꢂ 0.60
20.44 ꢂ 0.72
33.31 ꢂ 0.25
29.49 ꢂ 0.70
5o
37.78 ꢂ 0.72
28.75 ꢂ 0.72
54.67 ꢂ 0.25
41.62 ꢂ 0.83
47.56 ꢂ 0.72
43.56 ꢂ 0.72
36.42 ꢂ 0.43
31.87 ꢂ 0.61
5p
5q
ASBd
a
b
c
Compd.: compound. Conc: concentration. B. C.: Botrytis cinerea. S. S.: Sclerotinia sclerotiorum. F. G.: Fusarium graminearum. F. O.: Fusarium
oxysporum. f. sp. vasinfectum. M. O.: Magnaporthe oryzae. d ASB: azoxystrobin.
respectively, and the test results were shown in Table 1. Satis-
factorily, compound 2 exhibited the most potent antifungal
activity against the ve test strains, and the inhibition rate
reached 100% for each strain at 25 mg mLꢀ1. Additionally,
excluding 4e and 5a, most of the tested compounds presented
the better antifungal activity against B. cinerea and S. scle-
rotiorum than that of the positive control azoxystrobin. Most of
the compounds 5a–5q (5b, 5c, 5d, 5f, 5g, 5h, 5i, 5j, 5l) demon-
strated moderate to remarkable antifungal activity against F.
graminearum, F. oxysporum and M. oryzae, with inhibitory rates
ranging from 76% to 100% (25 mg mLꢀ1). However, compounds
4a–4o showed weak activity against the three fungi. To further
explore the antifungal potential of the synthesized compounds,
the most active compounds (whose inhibition rates >50% at 25
mg mLꢀ1) in Table 1 were selected to determine their EC50 values
against the ve fungal strains.
Results of antifungal evaluation (Table 2) indicated that 31
out of the 33 tested compounds showed moderate to strong
inhibitory activity against B. cinerea with EC50 values of 0.0021–
0.0827 mM, which were higher than the positive control azox-
ystrobin (EC50 ¼ 0.3551 mM), and 19 of compounds with EC50
values of 0.0021–0.0330 mM demonstrated superior activity
than HQ (EC50 ¼ 0.0331 mM). The antifungal activity of 31 out
of the 33 tested compounds against S. sclerotiorum was better
than azoxystrobin (EC50 ¼ 0.1629 mM), with EC50 values
between 0.0016 and 0.0636 mM. Compound 2 showed the
greatest activity, with EC50 value of 0.0016 mM, followed by 5c
(EC50 ¼ 0.0030 mM) respectively. 10 of the tested compounds
showed higher activity against F. graminearum, with EC50 values
of 0.0124–0.0211 mM, than HQ (EC50 ¼ 0.0931 mM) and azox-
ystrobin (EC50 ¼ 0.0229 mM) respectively. Furthermore, 10 out
of 33 of the tested compounds exhibited stronger activity
against F. oxysporum than HQ (EC50 ¼ 0.1840 mM) and azox-
ystrobin (EC50 ¼ 0.1265 mM) respectively, with EC50 values of
0.0059–0.0365 mM. Similarly, antifungal activity of 10 of the
synthesized compounds against M. oryzae was better than HQ
(EC50 ¼ 0.0964 mM) and azoxystrobin (EC50 > 10.0000 mM)
respectively, with EC50 values of 0.0120–0.0159 mM. The above
results revealed that compound 2 was the most effective
compound, followed by compound 5c.
2.3. Structure–activity relationships
Different substituent groups were introduced into the
compound 2 scaffold to synthesize different analogs of 2 and
Table 2 EC50 of series 8-hydroxyquinoline derivatives against five
phytopathogenic fungi (mM)
EC50
Compd.a
B. C.b
S. S.b
F. G.b
F. O.b
M. O.b
HQ
2
0.0331
0.0021
0.0827
0.0165
0.0537
0.0536
0.0317
0.0444
0.0298
0.0496
0.0356
0.0277
0.0222
0.0790
0.0285
0.0330
0.0386
0.0124
0.0150
0.0349
0.0328
0.0172
0.0623
0.0307
0.0359
0.0348
0.0233
0.0232
0.0125
0.0325
0.0192
0.0241
0.3551
0.0181
0.0016
0.0424
0.0355
0.0490
0.0636
0.0468
0.0443
0.0434
0.0483
0.0215
0.0432
0.0234
0.0593
0.0211
0.0361
0.0362
0.0030
0.0205
0.0343
0.0320
0.0195
0.0463
0.0184
0.0458
0.0306
0.0145
0.0417
0.0190
0.0419
0.0328
0.0324
0.1629
0.0931
0.0124
—
—
—
—
—
—
—
—
—
—
—
—
—
—
0.0190
0.0140
0.0167
—
0.0192
0.0228
0.0193
0.0183
0.0211
—
0.0211
—
—
—
—
0.1840
0.0059
—
—
—
—
—
—
—
—
—
—
—
—
—
—
0.0226
0.0146
0.0208
—
0.0348
0.0365
0.0233
0.0200
0.0206
—
0.0233
—
—
—
—
0.0964
0.0120
—
—
—
—
—
—
—
—
—
—
—
—
—
—
0.0156
0.0140
0.0150
—
0.0313
0.0154
0.0142
0.0156
0.0159
—
0.0144
—
—
—
—
4a
4b
4c
4d
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
ASBc
—
0.0229
—
0.1265
—
>10.0000
a
b
Compd.: compound. B. C.: Botrytis cinerea. S. S.: Sclerotinia
sclerotiorum. F. G.: Fusarium graminearum. F. O.: Fusarium oxysporum.
f. sp. vasinfectum. M. O.: Magnaporthe oryzae. c ASB: azoxystrobin.
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Fig. 2 Optical microscope and scanning electron micrographs of the hyphae of S. sclerotiorum grown on PDA medium with DMSO or
compounds 2, 5c at 25 ꢁC. Optical microscope: (A) untreated control, 0.5% DMSO, ꢃ400; (B) compound 2 at 0.0016 mM (EC50) treatment,
ꢃ400; (C) compound 5c at 0.0030 mM (EC50) treatment, ꢃ400; scanning electron microscopy: (D) untreated control, 0.5% DMSO, ꢃ1500; (E)
after 72 h compound 2 at 0.0016 mM (EC50) treatment, ꢃ1500; (F) after 72 h compound 5c at 0.0030 mM (EC50) treatment, ꢃ1500.
their structure activity relationships (SAR) were investigated. 8-hydroxyquinoline derivative 5a showed weak activity against
The values indicated in Table 2, it was inferred that the presence B. cinerea and S. sclerotiorum. Through analysis of the EC50 data
of tertiary or secondary amine on the synthesized compound in Table 2, revealed that unlike compounds 4a–4o, most of the
was essential for the antifungal activity of the tested compounds tested compounds 5a–5q bearing aromatic piperazine substit-
4a–4o against B. cinerea and S. sclerotiorum. When NHR1R2 was uent groups showed remarkable inhibitory activity against F.
directly substituted with aliphatic ring, the corresponding graminearum, F. oxysporum and M. oryzae respectively. From
target compound displayed a very poor antifungal activity as these observations, it was inferred that when the aromatic ring
exemplied by 4e. In contrast, when the NHR1R2 was replaced of aromatic piperazine on the target compound was benzene
by substituted phenylpiperazines, a spike in antifungal activity ring, the target compound demonstrated great antifungal
was observed for the corresponding phenylpiperazine deriva- activity. However, the presence of other aromatic rings, such as
tives (5b–5q). It was therefore extrapolated that, the presence of pyridine, pyrimidine and pyrazine on the target compounds 5o,
substituted phenylpiperazine derivatives played a pivotal role in 5p and 5q respectively, led to no antifungal activity. On the
their impressive antifungal activity. However, benzylpiperazine other hand, it was telling that the number of substituent groups
Table 3 Protective and curative activity of compounds 2 and 5c in vivo
Curative effect
Protective effect
Lesion length
(mm ꢂ SD)
Compd.a
Concentration (mg mLꢀ1
)
Control efficacy (%)
Lesion length (mm ꢂ SD)
Control efficacy (%)
2
80
40
20
80
40
20
80
40
20
—
6.17 ꢂ 0.38
8.86 ꢂ 0.74
12.80 ꢂ 0.95
5.44 ꢂ 0.40
6.53 ꢂ 0.65
12.71 ꢂ 0.96
6.58 ꢂ 0.98
7.19 ꢂ 0.59
9.54 ꢂ 0.94
18.04 ꢂ 0.76
87.91
60.05
19.15
95.44
84.18
20.14
83.59
77.32
52.94
—
5.87 ꢂ 0.72
7.08 ꢂ 0.92
9.27 ꢂ 0.55
5.85 ꢂ 0.69
7.06 ꢂ 0.92
8.66 ꢂ 0.98
5.83 ꢂ 0.52
5.86 ꢂ 0.52
6.78 ꢂ 0.54
20.53 ꢂ 0.75
90.93
78.29
55.39
91.09
78.42
61.68
91.32
91.02
81.35
—
5c
ASBb
Control
a
b
Compd.: compound. ASB: azoxystrobin.
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(mono- versus di-) on the phenyl ring also impacted antifungal antifungal activity against the three fungi. In addition, the
activity of the target compounds against the various fungi introduction of halogen atoms such as F and Cl on phenyl-
tested. Premised on the above observation, direct pairwise piperazine augmented antifungal activity compounds 5i, 5j and
comparisons of antifungal activity of the tested compounds (5j 5l (EC50 values were <0.0250 mM) against the three fungi.
versus 5k, 5l versus 5m) against the three fungi were analyzed in
terms of their EC50 values. It was discovered that the mono-
2.4. Effects of compounds 2 and 5c on hyphal morphology of
substituted compounds 5j and 5l exhibited better antifungal
S. sclerotiorum
activity than their 2,4-dihalogenated counterparts 5k and 5m
respectively. This observation showed that the number of
substituents on the phenyl ring played a key role in inhibitory
activity of the tested compounds. In order to determine the
optimum position for mono-substitution on the phenyl ring,
comparisons of compounds 5c versus 5d; 5f versus 5g; 5i versus
5j were examined, and it was found that substitution at the
ortho position (compounds 5c, 5i and 5f) conferred greater
antifungal activity than substitution at the meta or para position
(5d, 5j and 5g). Interestingly, the tested compounds bearing
electron-donating groups such as methyl and methoxy on the
aromatic piperazine (5c, 5d, 5g and 5h), exhibited remarkable
The hyphae morphology of S. sclerotiorum treated with EC50 of
the two most effective compounds 2 and 5c were observed by
optical microscope (Fig. 2A, B and C) and scanning electron
microscopy (Fig. 2D, E and F), respectively. Satisfactorily, the
two experiments yielded consistent results. The mycelia of the
control group displayed a normal morphology with smooth,
linear, regular, homogeneous and a constant diameter (Fig. 2A
and D). However, mycelial morphology of the two tested
compounds treated showed signicant alteration. Hyphae of S.
sclerotiorum treated with the EC50 of compound 2 were
abnormal, with distinct bulges (Fig. 2B), correspondingly,
Fig. 3 In vivo protective efficacy of compounds 2, 5c and azoxystrobin against S. sclerotiorum on rape leaves.
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Table 5 The antibacterial spectrum of compound 2 against eight plant
pathogenic bacteria
scanning electron microscope observation results showed that
hyphae become clear swelling or shrinking (Fig. 2E); the hyphae
treated with the EC50 of compound 5c appeared obviously
contort and wrinkle in the observation of optical microscope
(Fig. 2C), further than that, scanning electron microscope
observation results showed that hyphae appeared shriveled and
collapsed (Fig. 2F). From the above observations, it was inferred
that the two compounds destroyed the cell membrane and wall
of S. sclerotiorum, culminating in the death of hyphae.
MIC (mM)
Streptomycin
Bacterium
Compound 2
sulfate
Acidovorax avenae subsp. citrulli
Agrobacterium tumefaciens
Erwinia carotovora
Pseudomonas syringae pv. actinidiae
Pseudomonas syringae pv. lachrymans
Pseudomanas solanacearum
Xanthomonas oryzae pv. oryzae
Xanthomonas oryzae pv. oryzicola
0.0263
0.1578
0.1578
0.1578
0.1578
0.3155
0.2104
0.2104
0.0412
0.1372
0.1372
0.0069
0.0206
—
2.5. Effects of compounds 2 and 5c against S. sclerotiorum in
vivo
—
—
The results of pot experiments showed that compounds 2 and
5c exhibited moderate to excellent curative and protective
effects in vivo (Table 3). Three conclusions were deduced from
the data in Table 3: rstly, the curative and protective effects of
the two compounds exhibited concentration-dependent prop-
erties; secondly, compound 5c possessed stronger protective
and curative activity than that of 2; thirdly, the curative effects of
compounds 5c and 2 at 80 mg mLꢀ1 (95.44%, 87.91%) were
better than the control azoxystrobin (83.59%) and the protective
effects of compounds 5c and 2 at 80 mg mLꢀ1 (91.09%, 90.93%)
were close to that of azoxystrobin (91.32%). Underivatized
compound 2 exhibited better activity in vitro, but the compound
5c possessed superior activity in vivo, it was therefore extrapo-
lated that the introduction of 1-(2-methylphenyl)piperazine
signicantly improved the absorbability of compound 2 scaffold
in plants. From pictures in Fig. 3, it was deduced that
compounds 2 and 5c demonstrated no obvious phytotoxicity on
oilseed rape leaves at a high concentration (80 mg mLꢀ1), which
were benign to the oilseed rape.
and 0.0043 mmol, respectively (Table 4). Additionally, the
inhibitory activity of compound 2 against eight agricultural
pathogenic bacteria (Table 5) was explored. The data in Table 5
indicated that compound 2 exhibited inhibitory effects against
the plant pathogenic bacteria tested, and the activity against
Acidovorax avenae subsp. citrulli, Pseudomanas solanacearum,
Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv.
oryzicola was higher than that of the control streptomycin
sulfate. The above results showed that compound 2 has a broad
spectrum of antifungal and antibacterial activities.
3. Conclusion
A series of 8-hydroxyquinoline derivatives were designed,
synthesized and their antifungal activity was evaluated against
ve phytopathogenic fungi. Most of the tested compounds
exhibited stronger antifungal activity against the ve fungi than
the primer molecule HQ. Especially, compound 2 demonstrated
the best antifungal activity in vitro against B. cinerea, S. scle-
rotiorum, F. graminearum, F. oxysporum and M. oryzae with EC50
values of 0.0021, 0.0016, 0.0124, 0.0059 and 0.0120 mM
respectively, followed by 5c. Moreover, compound 5c exhibited
better protective and curative activity than that of compound 2
in vivo, and the curative effects of compounds 5c and 2 at 80 mg
mLꢀ1 (95.44%, 87.91%) respectively were better than the posi-
tive control azoxystrobin (83.59%), compounds 2 and 5c effec-
tively controlled the disease development in S. sclerotiorum
infected oilseed rape in vivo, indicating great potential of these
two compounds to control fungal diseases. The obvious tera-
togenic effect of compounds 2 and 5c on hyphal morphology of
S. sclerotiorum will provide valuable insights into understanding
the antifungal mechanism of 8-hydroxyquinoline derivatives.
Additionally, compound 2 also displayed remarkable activity
against eight agricultural pathogenic bacteria.
2.6. Antifungal and antibacterial spectrum of compound 2
Compound 2 possessed the highest antifungal activity in vitro
among all of 8-hydroxyquinoline derivatives, making it a prom-
ising lead compound for the development of novel antifungal
agents. Hence, the antifungal spectrum of this compound was
investigated and the results revealed that compound 2 showed
impressive antifungal activity against many deleterious fungal
pathogens, including Rhizoctonia solani, Mycosphaerlla melonis,
Phyllosticta zeae, Colletotrichum gossypii, Phytophthora capsici
and Pythium aphanidermatum. When it came to M. melonis, C.
gossypii, P. capsici and P. aphanidermatum, compound 2 revealed
excellent antifungal activity with EC50 of 0.0081, 0.0068, 0.0019
Table 4 The antifungal spectrum of compound 2 against six plant
pathogenic fungi
Fungi
EC50 (mM)
95% Cl
R. solani
M. melonis
P. zeae
C. gossypii
P. capsici
P. aphanidermatum
0.0149
0.0081
0.0127
0.0068
0.0019
0.0043
0.0120–0.0185
0.0059–0.0111
0.0077–0.0208
0.0058–0.0081
0.0016–0.0023
0.0034–0.0055
4. Experimental section
4.1. General methods
All reactions were performed using commercially available
regents without further purication. Thin-layer chromatog-
raphy (TLC) was employed to monitor all reactions and column
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chromatography was performed with silica gel (Qingdao formed was ltered. The crude product was puried by recrys-
Haiyang Chemical Co., Ltd., Qingdao, China). Melting points tallization from 1 : 1 EtOH–H2O to yield the nal product.
(mp) were determined using glass capillary tubes on a WRS-2U
4.2.2.1. 7-((4-Methylpiperazin-1-yl)methyl)-5-nitroquinolin-8-
1
ꢁ
melting point apparatus (Shanghai Precision Instrument Co., ol (4a). Yellow solid; yield 66%; mp 206–208 C; H NMR (400
Ltd., Shanghai, China) and were uncorrected. Mass spectrom- MHz, chloroform-d, d ppm): 2.36 (s, 3H, CH3), 2.62–2.77 (m, 8H,
etry was performed using ESI mode on a Bruker Daltonics piperazine), 3.98 (s, 2H, CH2), 7.66 (dd, J ¼ 8.9, 4.1 Hz, 1H, ArH),
APEXII49e spectrometer (Bruker Daltonics Inc., Billerica, MA, 8.42 (s, 1H, ArH), 8.96 (d, J ¼ 4.0 Hz, 1H, ArH), 9.28 (d, J ¼
US). Nuclear magnetic resonance (1H NMR and 13C NMR) 8.8 Hz, 1H, ArH). 13C NMR (100 MHz, DMSO-d6, d ppm): 42.34,
spectra were recorded at 400 and 100 MHZ on a Bruker AM-400 48.45, 48.45, 50.52, 53.47, 53.47, 114.57, 117.46, 123.96, 126.39,
(Bruker Company, Billerica, MA, and US.) spectrometer with 132.88, 135.11, 136.69, 147.99, 158.85. HRMS calcd for
TMS as an internal standard.
C
15H18N4O3, [M + H]+, 303.1412; found 303.2014.
The commercial fungicide azoxystrobin (analytical grade,
4.2.2.2. 7-((4-Ethylpiperazin-1-yl)methyl)-5-nitroquinolin-8-ol
98% purity) provided by Jiangsu Balling Agrochemical Co., Ltd. (4b). Yellow solid; yield 90%; mp 187–189 ꢁC; 1H NMR (400
Jiangying, China was used as a positive control in vitro experi- MHz, DMSO-d6, d ppm): 1.06 (t, J ¼ 7.1 Hz, 3H, CH3), 2.74–2.84
ment. And the commercial bactericide streptomycin sulfate (m, 8H, 4CH2piperazine), 3.08 (s, 2H, CH2), 3.91 (s, 2H, CH2), 7.61
(analytical grade, 98% purity) (J&K) was used as a positive (dd, J ¼ 8.9, 4.1 Hz, 1H, ArH), 8.52 (s, 1H, ArH), 8.69 (d, J ¼
control in minimal inhibitory concentration (MIC) test of 4.0 Hz, 1H, ArH), 9.32 (d, J ¼ 8.8 Hz, 1H, ArH). 13C NMR (100
compound 2 against the eight phytopathogenic bacterial.
MHz, DMSO-d6, d ppm): 9.42, 49.64, 49.64, 49.64, 50.98, 53.84,
Botrytis cinerea, Sclerotinia sclerotiorum, Fusarium graminea- 53.84, 115.29, 118.22, 123.04, 126.02, 131.71, 134.20, 137.06,
rum, Fusarium oxysporum f. sp. vasinfectum, Magnaporthe oryzae, 148.71, 159.17. HRMS calcd for C16H20N4O3, [M + H]+, 317.1569;
Rhizoctonia solani, Mycosphaerlla melonis, Phyllosticta zeae, found 317.2228.
Colletotrichum gossypii, Phytophthora capsici and Pythium apha-
4.2.2.3. 5-Nitro-7-(piperidin-1-ylmethyl)quinolin-8-ol
(4c).
nidermatum were provided by the Institute of Plant Protection, Yellow solid; yield 90%; mp 206–207 ꢁC; 1H NMR (400 MHz,
Gansu Academy of Agricultural Science, and Lanzhou, China. DMSO-d6, d ppm): 1.53–1.72 (m, 2H, CH2piperidine), 1.75 (t, J ¼
Acidovorax avenae subsp. citrulli, Agrobacterium tumefaciens, 5.8 Hz, 4H, 2CH2piperidine), 3.13 (t, J ¼ 4.8 Hz, 4H, 2CH2piperidine),
Erwinia carotovora, Pseudomonas syringae pv. actinidiae, Pseu- 4.17 (s, 2H, CH2), 7.55 (dd, J ¼ 8.7, 4.1 Hz, 1H, ArH), 8.58 (s, 1H,
domonas syringae pv. lachrymans, Pseudomanas solanacearum, ArH), 8.60 (d, J ¼ 3.0 Hz, 1H, ArH), 9.31 (d, J ¼ 8.8, 1H, ArH). 13
C
Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. NMR (100 MHz, DMSO-d6, d ppm): 21.55, 22.96, 22.96, 52.48,
oryzicola, which were obtained from Shenyang Research Insti- 52.48, 53.69, 112.13, 124.11, 126.50, 132.48, 133.33, 135.06,
tute of Chemical Industry, and Shenyang, China.
136.74, 148.08, 162.83. HRMS calcd for C15H17N3O3, [M + H]+,
288.1303; found 288.2205.
4.2.2.4. 7-(Morpholinomethyl)-5-nitroquinolin-8-ol
(4d).
Yellow solid; yield 89%; mp 219–220 ꢁC; 1H NMR (400 MHz,
4.2. Synthesis and characterization of compounds
4.2.1. The preparation of compound 2. 8-Hydroxyquinoline chloroform-d, d ppm): 2.71 (t, J ¼ 3.4 Hz, 4H, 2CH2morpholine),
(1.0 mmol) was dissolved in suitable concentrated HCl in 3.83 (t, J ¼ 4.7 Hz, 4H, 2CH2morpholine), 3.97 (s, 2H, CH2), 7.69
a 50 mL ask, the mixture was cooled to 0 ꢁC, followed by (dd, J ¼ 8.9, 4.2 Hz, 1H, ArH), 8.52 (s, 1H, ArH), 8.96 (d, J ¼
dropwise addition of NaNO2 (1.5 mmol) aqueous solution into 4.0 Hz, 1H, ArH), 9.28 (d, J ¼ 8.9 Hz, 1H, ArH). 13C NMR (100
it. The yellow precipitate was ltered and washed with cold MHz, DMSO-d6, d ppm): 51.58, 51.58, 53.99, 63.74, 63.74,
water to give 8-hydroxy-5-nitrosoquinoline. Aer vacuum 117.71, 124.27, 126.56, 132.30, 133.46, 135.27, 136.67, 147.94,
drying, 8-hydroxy-5-nitrosoquinoline powder was added to 163.05. HRMS calcd for C14H15N3O4, [M + H]+, 290.1096; found
a mixture of concentrated HNO3 and water (v/v ¼ 3 : 2) at 17 ꢁC. 290.1586.
The nitrosoquinoline was rapidly converted to the insoluble
4.2.2.5. 7-((Cyclohexylamino)methyl)-5-nitroquinolin-8-ol
nitro compound. The mixture was diluted with water aer (4e). Yellow solid; yield 85%; mp 206–207 ꢁC; 1H NMR (400
75 min stirring, cooled to 0 ꢁC and made alkaline with sodium MHz, chloroform-d, d ppm): 2.53–1.31 (m, 10H, 5CH2cyclohexane),
acetate. The product was washed with water and recrystallized 3.13 (s, 1H, CH), 4.05 (s, 2H, CH2), 7.55 (dd, J ¼ 8.7, 4.1 Hz, 1H,
from ethanol. Yellow solid; yield 65%; mp 226–228 ꢁC; 1H NMR ArH), 7.92 (s, 1H, ArH), 8.62 (d, J ¼ 3.0 Hz, 1H, ArH), 9.28 (d, J ¼
(400 MHz, DMSO-d6, d ppm): 7.17 (d, J ¼ 8.8 Hz, 1H, ArH), 7.86 8.9 Hz, 1H, ArH). 13C NMR (100 MHz, DMSO-d6, d ppm): 24.39,
(dd, J ¼ 8.8, 4.0 Hz, 1H, ArH), 8.52 (d, J ¼ 8.8 Hz, 1H, ArH), 8.99 24.39, 25.17, 29.12, 29.12, 42.08, 56.95, 117.46, 126.32, 126.32,
(d, J ¼ 4.0 Hz, 1H, ArH), 9.18 (d, J ¼ 8.8 Hz, 1H, ArH). 13C NMR 131.94, 132.83, 134.76, 136.60, 148.50, 161.60. HRMS calcd for
(100 MHz, DMSO-d6, d ppm): 110.45, 122.97, 125.67, 129.51,
132.84, 135.51, 137.72, 149.62, 161.09. HRMS calcd for
C9H6N2O3, [M + H]+, 190.0378; found 190.2645.
C
16H19N3O3, [M + H]+, 302.1460; found 302.2065.
4.2.2.6. 7-((Benzylamino)methyl)-5-nitroquinolin-8-ol
(4f).
Yellow solid; yield 50%; mp 204–207 ꢁC; 1H NMR (400 MHz,
4.2.2. General procedure for preparation of target chloroform-d, d ppm): 4.07 (s, 4H, 2CH2), 4.80 (s, NH), 7.46–7.33
compounds 4a–4o. In a 50 mL ask, compound 2 (1 mmol) was (m, 5H, ArH), 8.57 (dd, J ¼ 8.9, 4.1 Hz, 1H, ArH), 8.88 (s, 1H,
mixed with formaldehyde (4.5 mmol) in 20 mL dry ethanol, the ArH), 9.24 (d, J ¼ 8.8 Hz, 1H, ArH), 9.32 (d, J ¼ 8.8 Hz, 1H, ArH).
desired amine (1.1 mmol) was dropped into this solution. The 13C NMR (100 MHz, DMSO-d6, d ppm): 53.82, 59.41, 114.24,
mixture was reuxed at 80 ꢁC for 24 h and the precipitate 117.12, 119.99, 124.29, 126.30, 129.09, 129.45, 130.49, 131.50,
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132.57, 134.50, 136.88, 145.63, 148.35, 158.69. HRMS calcd for d ppm): 42.67, 42.67, 54.60, 112.72, 124.26, 126.52, 132.24,
C
17H15N3O3, [M + H]+, 310.1147; found 310.1745.
4.2.2.7. 7-(Pyrrolidin-1-ylmethyl)-5-nitroquinolin-8-ol
133.04, 135.31, 136.62, 147.91, 162.91. HRMS calcd for
C
4.2.2.13. 7-((Dipropylamino)methyl)-5-nitroquinolin-8-ol
(4g).
12H13N3O3, [M + H]+, 248.0990; found 248.1636.
Yellow-green solid; yield 80%; mp 218–218 ꢁC; 1H NMR (400
MHz, DMSO-d6, d ppm): 1.73 (t, J ¼ 5.9 Hz, 4H, 2CH2pyrrole), 3.11 (4m). Yellow solid; yield 14%; mp 156–158 ꢁC; 1H NMR (400
(t, J ¼ 5.5 Hz, 4H, 2CH2pyrrole), 4.17 (s, 2H, CH2), 7.56 (dd, J ¼ 8.7, MHz, DMSO-d6, d ppm): 0.88 (t, J ¼ 3.2 Hz, 6H, 2CH3), 1.72 (t, J ¼
4.1 Hz, 1H, ArH), 8.59 (s, 1H, ArH), 8.61 (d, J ¼ 4.0 Hz, 1H, ArH), 12.0 Hz, 4H, 2CH2), 3.07–2.91 (m, 4H, 2CH2), 4.25 (s, 2H, CH2),
9.33 (d, J ¼ 8.7 Hz, 1H, ArH). 13C NMR (100 MHz, DMSO-d6, 7.58 (dd, J ¼ 9.0, 4.1 Hz, 1H, ArH), 8.58 (s, 1H, ArH), 8.65 (d, J ¼
d ppm): 22.96, 22.96, 51.59, 53.73, 53.73, 113.65, 124.07, 126.47, 4.0 Hz, 1H, ArH), 9.29 (d, J ¼ 8.9 Hz, 1H, ArH). 13C NMR (100
132.43, 132.43, 135.17, 136.65, 148.08, 162.40. HRMS calcd for MHz, DMSO-d6, d ppm): 11.47, 11.47, 17.35, 17.35, 54.16, 54.16,
C
14H15N3O3, [M + H]+, 274.1147; found 274.1811.
4.2.2.8. 7-(((Cyclopropylmethyl)amino)methyl)-5-
54.78, 114.45, 124.94, 126.44, 132.47, 134.05, 136.59, 141.92,
146.53, 150.06. HRMS calcd for C16H21N3O3, [M + H]+, 304.1616;
nitroquinolin-8-ol (4h). Yellow solid; yield 44%; mp 219–219 ꢁC; found 204.2135.
1H NMR (400 MHz, DMSO-d6, d ppm): 0.35 (d, J ¼ 5.6 Hz, 2H,
4.2.2.14. 7-((Isopropylamino)methyl)-5-nitroquinolin-8-ol
CH2cyclopropnane), 0.57 (d, J ¼ 7.6 Hz, 2H, CH2cyclopropnane), 1.13 (4n). Yellow solid; yield 90%; mp 207–211 ꢁC; 1H NMR (400
(s, 1H, CHcyclopropnane), 2.87 (d, J ¼ 7.4 Hz, 2H, CH2), 4.13 (s, 2H, MHz, DMSO-d6, d ppm): 1.67–0.88 (m, 6H, 2CH3), 3.60 (s, 1H,
CH2), 7.54 (dd, J ¼ 8.7, 4.1 Hz, 1H, ArH), 8.55 (s, 1H, ArH), 8.62 CH), 4.08 (s, 2H, CH2), 4.57 (s, 1H, NH), 7.56 (s, 1H, ArH), 8.62
(d, J ¼ 3.0 Hz, 1H, ArH), 9.34 (d, J ¼ 8.7 Hz, 1H, ArH). 13C NMR (d, J ¼ 17.7 Hz, 2H, ArH), 9.37 (d, J ¼ 9.3 Hz, 1H, ArH). 13C NMR
(100 MHz, DMSO-d6, d ppm): 4.42, 4.42, 7.54, 44.80, 51.83, (100 MHz, DMSO-d6, d ppm): 19.12, 19.12, 42.30, 50.52, 117.18,
114.46, 116.21, 123.99, 126.16, 132.14, 134.51, 137.28, 148.21, 123.62, 126.30, 131.85, 132.90, 134.73, 136.62, 148.54, 161.49.
158.64.
HRMS calcd for C13H15N3O3, [M + H]+, 262.1147; found
HRMS calcd for C14H15N3O3, [M + H]+, 274.1147; found 262.1806.
274.1638.
4.2.2.9. 7-((Ethylamino)methyl)-5-nitroquinolin-8-ol
4.2.2.15. 7-((tert-Butylamino)methyl)-5-nitroquinolin-8-ol
(4i). (4o). Yellow solid; yield 90%; mp 215–216 ꢁC; 1H NMR (400
Yellow solid; yield 90%; mp 210–214 ꢁC; 1H NMR (400 MHz, MHz, DMSO-d6, d ppm): 1.44 (s, 9H, 3CH3), 4.01 (s, 2H, CH2),
DMSO-d6, d ppm): 1.25 (d, J ¼ 9.1 Hz, 3H, CH3), 1.74 (s, 2H, 7.44 (dd, J ¼ 8.7, 4.1 Hz, 1H, ArH), 8.46 (s, 1H, ArH), 8.56 (d, J ¼
CH2), 4.31 (s, 2H, CH2), 7.78 (dd, J ¼ 8.7, 4.1 Hz, 1H, ArH), 8.62 4.2 Hz, 1H, ArH), 9.23 (d, J ¼ 9.2 Hz, 1H, ArH). 13C NMR (100
(s, 1H, ArH), 8.95 (d, J ¼ 4.0 Hz, 1H, ArH), 9.21 (d, J ¼ 9.6 Hz, 1H, MHz, DMSO-d6, d ppm): 25.62, 25.62, 25.6, 55.31, 57.38, 114.22,
ArH). 13C NMR (100 MHz, DMSO-d6, d ppm): 11.41, 42.52, 44.36, 117.10, 123.62, 126.33, 131.90, 134.63, 136.69, 148.69, 158.67.
114.33, 123.68, 126.31, 131.89, 132.70, 134.68, 136.72, 148.49, HRMS calcd for C14H17N3O3, [M + H]+, 276.1303; found
161.81. HRMS calcd for C12H13N3O3, [M + H]+, 248.0990; found 276.1962.
248.1824.
4.2.2.10. 7-((Propylamino)methyl)-5-nitroquinolin-8-ol
4.2.3. General procedure for preparation of target
(4j). compounds 5a–5q. The formaldehyde (3.75 mmol) and corre-
Yellow solid; yield 90%; mp 204–205 ꢁC; 1H NMR (400 MHz, sponding aromatic piperazine (1 mmol) were added to a 50 mL
DMSO-d6, d ppm): 0.91 (d, J ¼ 7.7 Hz, 3H, CH3), 1.83–1.57 (m, round-bottom ask, the mixture was stirred at 0 ꢁC to give
2H, CH2), 2.89 (d, J ¼ 8.2 Hz, 2H, CH2), 4.08 (s, 2H, CH2), 7.54 a white precipitate. The precipitate was added to the compound
ꢁ
(dd, J ¼ 8.7, 4.1 Hz, 1H, ArH), 8.55 (s, 1H, ArH), 8.61 (d, J ¼ 2 (1 mmol) dissolved in pyridine at 50 C, a yellow precipitate
4.0 Hz, 1H, ArH), 9.34 (d, J ¼ 8.8 Hz, 1H, ArH). 13C NMR (100 was formed aer a few minutes. Aer 30–40 minutes, the
MHz, DMSO-d6, d ppm): 11.38, 19.45, 44.76, 48.85, 114.26, precipitate was ltered through a Buchner funnel. The crude
123.68, 126.34, 131.96, 132.77, 134.76, 136.66, 148.47, 161.75. product was puried by recrystallization from 2 : 1 EtOH–H2O
HRMS calcd for C13H15N3O3, [M + H]+, 262.1147; found to yield the nal product.
262.1171.
4.2.2.11. 7-((Butylamino)methyl)-5-nitroquinolin-8-ol
4.2.3.1. 7-((4-Benzylpiperazin-1-yl)methyl)-5-nitroquinolin-8-
(4k). ol (5a). Yellow solid; yield 50%; mp 204–207 C; H NMR (400
1
ꢁ
Yellow solid; yield 90%; mp 194–196 ꢁC; 1H NMR (400 MHz, MHz, DMSO-d6, d ppm): 2.62 (t, J ¼ 4.8 Hz, 4H, 2CH2piperazine),
DMSO-d6, d ppm): 0.89 (t, J ¼ 7.4 Hz, 3H, CH3), 1.30–1.37 (m, 2.99 (t, J ¼ 4.4 Hz, 4H, 2CH2piperazine), 3.57 (s, 2H, CH2), 4.07 (s,
2H, CH2), 1.64 (s, 2H, CH2), 2.94 (t, J ¼ 7.8 Hz, 2H, CH2), 4.08 (s, 2H, CH2), 7.53–7.09 (m, 5H, ArH), 7.65 (dd, J ¼ 8.7, 4.2 Hz, 1H,
2H, CH2), 7.55 (dd, J ¼ 8.7, 4.1 Hz, 1H, ArH), 8.55 (s, 1H, ArH), ArH), 8.57 (s, 1H, ArH), 8.73 (d, J ¼ 4.1 Hz, 1H, ArH), 9.25 (d, J ¼
8.61 (d, J ¼ 4.0 Hz, 1H, ArH), 9.35 (d, J ¼ 8.7 Hz, 1H, ArH). 13
C
8.3 Hz, 1H, ArH). 13C NMR (100 MHz, DMSO-d6, d ppm): 49.44,
NMR (100 MHz, DMSO-d6, d ppm): 13.96, 19.72, 27.91, 44.79, 49.69, 53.68, 59.41, 115.33, 118.26, 123.26, 126.11, 129.21,
47.05, 114.29, 123.69, 126.34, 131.96, 132.74, 134.77, 136.65, 129.21, 129.60, 131.25, 131.25, 132.01, 133.29, 134.38, 136.96,
148.46, 161.75. HRMS calcd for C14H17N3O3, [M + H]+, 276.1303; 148.54, 158.80. HRMS calcd for C21H22N4O3, [M + H]+, 379.1725;
found 276.1975.
found 379.2229.
4.2.2.12. 7-((Dimethylamino)methyl)-5-nitroquinolin-8-ol (4l).
4.2.3.2. 7-((4-Phenylpiperazin-1-yl)methyl)-5-nitroquinolin-8-
Yellow solid; yield 75%; mp 210–212 ꢁC; 1H NMR (400 MHz, ol (5b). Yellow solid; yield 21%; mp 197–199 C; H NMR (400
DMSO-d6, d ppm): 2.70 (s, 6H, 2CH3), 4.15 (s, 2H, CH2), 7.57 (d, J MHz, DMSO-d6, d ppm): 2.98 (t, J ¼ 4.8 Hz, 4H, 2CH2piperazine),
¼ 4.1 Hz, 1H, ArH), 8.58 (s, 1H, ArH), 8.64 (d, J ¼ 3.4 Hz, 1H, 3.29 (t, J ¼ 4.8 Hz, 4H, 2CH2piperazine), 4.06 (s, 2H, CH2), 6.81 (t, J
ArH), 9.38 (d, J ¼ 8.7, 1H, ArH). 13C NMR (100 MHz, DMSO-d6, ¼ 7.2 Hz, 1H, ArH), 6.95 (d, J ¼ 8.2 Hz, 2H, ArH), 7.23 (t, J ¼
1
ꢁ
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7.8 Hz, 2H, ArH), 7.73 (dd, J ¼ 8.7, 4.1 Hz, 1H, ArH), 8.64 (s, 1H, 1H, ArH), 6.53 (d, J ¼ 8.4 Hz, 1H, ArH), 7.12 (t, J ¼ 8.2 Hz, 1H,
ArH), 8.84 (d, J ¼ 4.7 Hz, 1H, ArH), 9.26 (d, J ¼ 8.7 Hz, 1H, ArH). ArH), 7.68 (dd, J ¼ 8.8, 4.2 Hz, 1H, ArH), 8.60 (s, 1H, ArH), 8.78
13C NMR (100 MHz, DMSO-d6, d ppm): 7.52, 7.52, 44.47, 44.47, (d, J ¼ 3.9 Hz, 1H, ArH), 9.23 (d, J ¼ 8.8 Hz, 1H, ArH). 13C NMR
51.90, 114.35, 114.35, 114.98, 117.90, 123.62, 126.32, 131.87, (100 MHz, DMSO-d6, d ppm): 47.27, 47.27, 52.23, 52.23, 55.37,
131.87, 132.81, 134.76, 136.61, 148.46, 158.61, 158.96, 161.58. 56.20, 102.29, 105.01, 108.67, 111.95, 116.70, 124.68, 125.08,
HRMS calcd for C20H20N4O3, [M + H]+, 365.1569; found 130.17, 132.78, 135.30, 147.66, 152.12, 158.63, 159.99, 160.68.
365.2229.
4.2.3.3. 7-((4-(o-Tolyl)piperazin-1-yl)methyl)-5-nitroquinolin-
8-ol (5c). Yellow solid; yield 44%; mp 188–189 ꢁC; 1H NMR (400
HRMS calcd for C21H22N4O4, [M + H]+, 395.1675; found
395.2348.
4.2.3.8. 7-((4-(4-Methoxyphenyl)piperazin-1-yl)methyl)-5-
MHz, DMSO-d6, d ppm): 2.25 (s, 3H, CH3), 3.15–2.99 (m, 8H, nitroquinolin-8-ol (5h). Yellow solid; yield 64%; mp 172–173 ꢁC;
4CH2piperazine), 4.14 (s, 2H, CH), 7.03–6.96 (m, 2H, ArH), 7.19– 1H NMR (400 MHz, DMSO-d6, d ppm): 3.00 (t, J ¼ 4.0 Hz, 4H,
7.12 (m, 2H, ArH), 7.66 (dd, J ¼ 8.8, 4.2 Hz, 1H, ArH), 8.62 (s, 1H, 2CH2piperazine), 3.18 (t, J ¼ 4.6 Hz, 4H, 2CH2piperazine), 3.68 (s, 3H,
ArH), 8.76 (d, J ¼ 4.1 Hz, 1H, ArH), 9.24 (d, J ¼ 8.8 Hz, 1H, ArH). OCH3), 4.07 (s, 2H, CH2), 6.99–6.78 (m, 4H, ArH), 7.72 (dd, J ¼
13C NMR (100 MHz, DMSO-d6, d ppm): 17.88, 48.76, 48.76, 8.8, 4.2 Hz, 1H, ArH), 8.63 (s, 1H, ArH), 8.81 (d, J ¼ 4.2 Hz, 1H,
52.14, 52.14, 53.61, 111.99, 114.83, 117.73, 119.42, 124.26, ArH), 9.26 (d, J ¼ 8.8 Hz, 1H, ArH). 13C NMR (100 MHz, DMSO-
126.55, 127.16, 131.45, 132.54, 133.51, 135.23, 136.72, 148.00, d6, d ppm): 47.32, 47.32, 51.46, 51.46, 53.53, 55.66, 111.96,
150.16, 163.00. HRMS calcd for C21H22N4O3, [M + H]+, 379.1725; 114.85, 114.85, 118.49, 118.49, 124.29, 126.57, 132.31, 133.52,
found 379.1820.
4.2.3.4. 7-((4-(m-Tolyl)piperazin-1-yl)methyl)-5-nitroquinolin-
8-ol (5d). Yellow solid; yield 81%; mp 187–188 ꢁC; 1H NMR (400
135.32, 136.68, 144.15, 147.94, 154.16, 163.06. HRMS calcd for
C
4.2.3.9. 7-((4-(2-Fluorophenyl)piperazin-1-yl)methyl)-5-
21H22N4O4, [M + H]+, 395.1675; found 395.2326.
MHz, DMSO-d6, d ppm): 2.25 (s, 3H, CH3), 3.03 (t, J ¼ 5.0 Hz, 4H, nitroquinolin-8-ol (5i). Yellow solid; yield 68%; mp 160.0–161 ꢁC;
2CH2piperazine), 3.30 (t, J ¼ 5.4 Hz, 4H, 2CH2piperazine), 4.07 (s, 2H, 1H NMR (400 MHz, DMSO-d6,
d ppm): 2.96 (s, 4H,
CH2), 6.63 (d, J ¼ 7.4 Hz, 1H, ArH), 6.77 (s, 1H, ArH), 7.10 (t, J ¼ 2CH2piperazine), 3.16 (t, J ¼ 5.4 Hz, 4H, 2CH2piperazine), 4.02 (s, 2H,
7.8 Hz, 1H, ArH), 7.39 (d, J ¼ 13.4 Hz, 1H, ArH), 7.68 (dd, J ¼ 8.8, CH2), 7.39 (dd, J ¼ 7.5, 5.1 Hz, 4H, ArH), 8.58 (s, 1H, ArH), 8.63
4.1 Hz, 1H, ArH), 8.60 (s, 1H, ArH), 8.77 (d, J ¼ 4.1 Hz, 1H, ArH), (s, 1H, ArH), 8.87 (s, 1H, ArH), 9.26 (d, J ¼ 9.0 Hz, 1H, ArH). 13
C
9.23 (d, J ¼ 8.7 Hz, 1H). 13C NMR (100 MHz, DMSO-d6, d ppm): NMR (100 MHz, DMSO-d6, d ppm): 47.55, 47.55, 51.57, 51.57,
21.80, 46.01, 46.01, 51.26, 51.26, 53.53, 111.96, 113.60, 117.01, 53.65, 111.89, 114.65, 117.55, 120.11, 125.45, 126.56, 127.39,
121.22, 124.28, 126.55, 129.41, 132.30, 133.53, 135.28, 136.69, 132.34, 133.52, 135.29, 136.69, 143.29, 145.80, 147.94, 163.05.
138.75, 147.94, 149.95, 163.07. HRMS calcd for C21H22N4O3, [M HRMS calcd for C20H19FN4O3, [M + H]+, 383.1475; found
+ H]+, 379.1725; found 379.2224.
4.2.3.5. 7-((4-(p-Tolyl)piperazin-1-yl)methyl)-5-nitroquinolin-
383.2014.
4.2.3.10. 7-((4-(4-Fluorophenyl)piperazin-1-yl)methyl)-5-
1
8-ol (5e). Yellow solid; yield 51%; mp 206–206 ꢁC; H NMR (400 nitroquinolin-8-ol (5j). Yellow solid; yield 55%; mp 168–171 C;
MHz, DMSO-d6, d ppm): 2.20 (s, 3H, CH3), 2.94 (t, J ¼ 5.3 Hz, 4H, 1H NMR (400 MHz, DMSO-d6, d ppm): 3.02 (t, J ¼ 3.2 Hz, 4H,
2CH2piperazine), 3.21 (t, J ¼ 5.5 Hz, 4H, 2CH2piperazine), 4.03 (s, 2H, 2CH2piperazine), 3.27 (t, 4H, 2CH2piperazine), 4.06 (s, 2H, CH2), 6.97
CH2), 6.85 (d, J ¼ 8.3 Hz, 2H, ArH), 7.03 (d, J ¼ 8.3 Hz, 2H, ArH), (t, J ¼ 4.5 Hz, 2H, ArH), 7.06 (t, J ¼ 8.9 Hz, 2H, ArH), 7.70 (dd, J ¼
7.73 (dd, J ¼ 8.9, 4.2 Hz, 1H, ArH), 8.64 (s, 1H, ArH), 8.83 (s, 1H, 8.8, 4.1 Hz, 1H, ArH), 8.61 (s, 1H, ArH), 8.79 (d, J ¼ 2.5 Hz, 1H),
ArH), 9.28 (d, J ¼ 8.9 Hz, 1H, ArH). 13C NMR (100 MHz, DMSO- 9.22 (d, J ¼ 8.8 Hz, 1H). 13C NMR (100 MHz, DMSO-d6, d ppm):
d6, d ppm): 20.48, 46.42, 46.42, 51.28, 51.28, 53.52, 111.96, 48.89, 50.97, 50.97, 53.71, 53.74, 115.09, 118.0, 118.00, 118.00,
116.65, 116.65, 117.94, 124.28, 126.56, 129.40, 130.00, 130.00, 120.92, 123.38, 126.16, 133.19, 133.19, 134.54, 136.92, 148.45,
132.29, 133.53, 135.29, 136.70, 147.83, 163.08. HRMS calcd for 158.88, 159.23, 161.53. HRMS calcd for C20H19FN4O3, [M + H]+,
ꢁ
C
21H22N4O3, [M + H]+, 379.1725; found 379.1804.
383.1475; found 383.2049.
4.2.3.6. 7-((4-(2-Methoxyphenyl)piperazin-1-yl)methyl)-5-
4.2.3.11. 7-((4-(2,4-Diuorophenyl)piperazin-1-yl)methyl)-5-
nitroquinolin-8-ol (5f). Yellow solid; yield 79%; mp 196–201 C; nitroquinolin-8-ol (5k). Yellow solid; yield 62%; mp 197–203 ꢁC;
1H NMR (400 MHz, DMSO-d6, d ppm): 3.09–3.15 (m, 8H, 1H NMR (400 MHz, DMSO-d6, d ppm): 2.96–2.54 (m, 8H,
4CH2piperazine), 3.78 (s, 3H, OCH3), 4.13 (s, 2H, CH2), 7.15–6.76 4CH2piperazine), 3.92 (s, 2H, CH2), 7.39 (d, J ¼ 6.8 Hz, 1H, ArH),
(m, 4H, ArH), 7.68 (dd, J ¼ 8.8, 4.1 Hz, 1H, ArH), 8.62 (s, 1H, 7.61 (dd, J ¼ 8.7, 4.1 Hz, 1H, ArH), 7.79 (s, 1H, ArH), 8.52 (s, 1H,
ArH), 8.76 (d, J ¼ 5.8 Hz, 1H, ArH), 9.26 (d, J ¼ 10.4 Hz, 1H, ArH). ArH), 8.69 (s, 1H, ArH), 9.31 (d, J ¼ 8.8 Hz, 1H, ArH). 13C NMR
13C NMR (100 MHz, DMSO-d6, d ppm): 45.91, 45.91, 51.19, (100 MHz, DMSO-d6, d ppm): 48.84, 48.84, 50.98, 50.98, 53.67,
51.19, 53.52, 55.42, 102.73, 105.58, 108.84, 111.95, 117.76, 111.95, 114.85, 117.76, 117.76, 120.67, 123.51, 126.23, 126.23,
124.29, 126.56, 130.34, 132.31, 133.53, 135.30, 136.69, 147.94, 132.31, 133.05, 134.70, 136.86, 148.36, 159.14, 161.74. HRMS
151.26, 160.73. HRMS calcd for C21H22N4O4, [M + H]+, 395.1675; calcd for C20H18F2N4O3, [M + H]+, 401.1381; found 401.2038.
ꢁ
found 395.2277.
4.2.3.7. 7-((4-(3-Methoxyphenyl)piperazin-1-yl)methyl)-5-
4.2.3.12. 7-((4-(4-Chlorophenyl)piperazin-1-yl)methyl)-5-
nitroquinolin-8-ol (5l). Yellow solid; yield 61%; mp 192–197 C;
ꢁ
nitroquinolin-8-ol (5g). Yellow solid; yield 79%; mp 177–178 C; 1H NMR (400 MHz, DMSO-d6, d ppm): 2.99 (t, J ¼ 5.0 Hz, 4H,
1H NMR (400 MHz, DMSO-d6, d ppm): 3.01 (t, J ¼ 5.2 Hz, 4H, 2CH2piperazine), 3.32 (t, J ¼ 4.4 Hz, 4H, 2CH2piperazine), 4.04 (s, 2H,
2CH2piperazine), 3.32 (t, J ¼ 5.1 Hz, 4H, 2CH2piperazine), 3.71 (s, 3H, CH2), 6.96 (d, J ¼ 8.6 Hz, 2H, ArH), 7.24 (d, J ¼ 8.9 Hz, 2H, ArH),
OCH3), 4.05 (s, 2H, CH2), 6.40 (d, J ¼ 8.1 Hz, 1H, ArH), 6.47 (s, 7.71 (dd, J ¼ 8.8, 4.1 Hz, 1H, ArH), 8.60 (s, 1H, ArH), 8.80 (d, J ¼
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4.1 Hz, 1H, ArH), 9.21 (d, J ¼ 8.9 Hz, 1H, ArH). 13C NMR (100 ArH), 8.16–8.05 (m, 1H, ArH), 8.35 (d, J ¼ 2.3 Hz, 1H, ArH), 8.62
MHz, DMSO-d6, d ppm): 45.81, 45.81, 51.08, 51.08, 53.55, (s, 1H, ArH), 8.84 (d, J ¼ 2.5 Hz, 1H, ArH), 9.21 (d, J ¼ 8.8 Hz, 1H,
111.93, 114.86, 117.94, 117.94, 124.04, 126.57, 129.28, 129.28, ArH). 13C NMR (100 MHz, DMSO-d6, d ppm): 41.74, 41.74, 50.77,
132.27, 133.53, 135.34, 136.68, 147.91, 148.78, 163.10. HRMS 50.77, 53.69, 111.48, 114.33, 117.21, 127.65, 132.11, 133.48,
calcd for C20H19ClN4O3, [M + H]+, 399.1146; found 399.1839.
4.2.3.13. 7-((4-(2,4-Dichlorophenyl)piperazin-1-yl)methyl)-5-
nitroquinolin-8-ol (5m). Yellow solid; yield 83%, mp 165–165 ꢁC;
1H NMR (400 MHz, DMSO-d6, d ppm): 2.84 (t, J ¼ 4.0 Hz, 4H,
2CH2piperazine), 3.31 (t, J ¼ 4.2 Hz, 4H, 2CH2piperazine), 3.95 (s, 2H,
CH2), 6.94 (d, J ¼ 4.5 Hz, 1H, ArH), 7.15 (d, J ¼ 4.8 Hz, 1H, ArH),
7.39 (s, 1H, ArH), 7.77 (dd, J ¼ 8.8, 4.1 Hz, 1H, ArH), 8.60 (s, 1H,
ArH), 8.86 (d, J ¼ 2.5 Hz, 1H, ArH), 9.25 (d, J ¼ 9.0 Hz, 1H, ArH).
13C NMR (100 MHz, DMSO-d6, d ppm): 45.34, 45.34, 50.87,
50.87, 53.59, 111.90, 116.31, 117.45, 121.28, 124.33, 126.58,
127.40, 131.11, 132.12, 133.55, 135.36, 136.67, 147.90, 149.64,
158.66. HRMS calcd for C20H18Cl2N4O3, [M + H]+, 434.0726;
found 434.3588.
135.24, 141.99, 142.64, 146.65, 147.92, 154.21, 158.75. HRMS
calcd for C18H18N6O3, [M + H]+, 367.1474; found 367.2580.
4.3. Antifungal activity
The in vitro antifungal activity against B. cinerea, S. sclerotiorum,
F. graminearum, F. oxysporum, M. oryzae, R. solani, M. melonis, P.
zeae, C. gossypii, P. capsici and P. aphanidermatum were assayed by
mycelium linear growth rate method as previously reported.1 The
strains were removed from their storage tubes and grown on
ꢁ
potato dextrose agar (PDA) mediums for one week at 25 C to
allow the mycelia growth to be used for antifungal assays. The
tested compounds were dissolved in DMSO and water containing
Tween-80, and then added to the PDA to obtain mediums with
different drug concentrations. The nal concentration of DMSO
was 0.5% because it had been shown to have no signicant effect
on the growth of the tested fungi. Azoxystrobin with different
concentrations in the PDA mediums containing 0.5% DMSO (v/v)
and 0.5% DMSO in the PDA medium were used as positive
control and blank control respectively. A 5 mm diameter disc of
fungus cut from subcultured Petri dishes was placed at the center
of Petri dishes which contained PDA mediums with different
drug concentrations. The diameter of mycelia was measured
when the fungi in the blank control completely covered the Petri
dish. The inhibition percentages were calculated using the
formula:30 I (%) ¼ ([(C ꢀ d) ꢀ (T ꢀ d)])/((C ꢀ d)) ꢃ 100, where d is
diameter of the cut fungus (5 mm), I is the inhibition (%), and C
and T are the average colony diameters of the mycelium of the
blank control and treatment respectively.
Aer the preliminary antifungal activity screening,
compounds with better activity were selected to further deter-
mine their medium effective concentrations (EC50) according to
the same methods described above. A series of PDA mediums
containing 50, 25, 10, 5, 2.5 mg mLꢀ1 respectively of the tested
compounds were prepared. Azoxystrobin was used as a positive
control and 0.5% DMSO as a blank control respectively. Each
test was performed in triplicate.
4.2.3.14. 5-Nitro-7-((4-(4-nitrophenyl)piperazin-1-yl)methyl)
1
ꢁ
quinolin-8-ol (5n). Yellow solid; yield 73%; mp 176–176 C; H
NMR (400 MHz, DMSO-d6, d ppm): 2.81 (t, J ¼ 5.1 Hz, 4H,
2CH2piperazine), 3.57 (t, J ¼ 5.0 Hz, 4H, 2CH2piperazine), 3.95 (s, 2H,
CH2), 7.05 (d, J ¼ 9.5 Hz, 2H, ArH), 7.39 (dd, J ¼ 7.7, 5.7 Hz, 1H,
ArH), 7.95–7.67 (m, 2H, ArH), 8.64 (s, 1H, ArH), 8.92 (d, J ¼
10.5 Hz, 1H, ArH), 9.22 (d, J ¼ 9.2 Hz, 1H, ArH). 13C NMR (100
MHz, DMSO-d6, d ppm): 44.18, 44.18, 50.77, 50.77, 53.65,
112.96, 113.94, 113.94, 124.31, 126.10, 126.10, 128.39, 132.26,
133.52, 135.30, 136.71, 138.44, 147.91, 154.14, 163.13. HRMS
calcd for C20H19N5O5, [M + H]+, 410.1420; found 410.2100.
4.2.3.15. 5-Nitro-7-((4-(pyridin-2-yl)piperazin-1-yl)methyl)
1
ꢁ
quinolin-8-ol (5o). Yellow solid; yield 88%; mp 190–191 C; H
NMR (400 MHz, DMSO-d6, d ppm): 2.97 (t, J ¼ 5.1 Hz, 4H,
2CH2piperazine), 3.68 (t, J ¼ 4.8 Hz, 4H, 2CH2piperazine), 4.05 (s, 2H,
CH2), 6.68 (dd, J ¼ 7.2, 4.8 Hz, 1H, ArH), 6.87 (d, J ¼ 8.6 Hz, 1H,
ArH), 7.39 (t, 7.36, 1H, ArH), 7.70 (dd, J ¼ 8.8, 4.2 Hz, 1H, ArH),
7.79 (t, J ¼ 6.7 Hz, 1H, ArH), 8.61 (s, 1H, ArH), 8.79 (d, J ¼ 4.0 Hz,
1H, ArH), 9.22 (d, J ¼ 8.7 Hz, 1H, ArH). 13C NMR (100 MHz,
DMSO-d6, d ppm): 42.64, 42.64, 50.87, 50.87, 53.64, 109.30,
111.9, 114.53, 117.81, 127.14, 132.33, 133.48, 135.27, 136.70,
139.67, 143.84, 146.01, 147.94, 163.05. HRMS calcd for
C
19H19N5O3, [M + H]+, 366.1521; found 366.2084.
4.2.3.16. 5-Nitro-7-((4-(pyrimidin-2-yl)piperazin-1-yl)methyl)
1
ꢁ
quinolin-8-ol (5p). Yellow solid; yield 94%; mp 191–192 C; H
NMR (400 MHz, DMSO-d6, d ppm): 2.95 (t, J ¼ 5.2 Hz, 4H,
2CH2piperazine), 3.92 (t, J ¼ 5.2 Hz, 4H, 2CH2piperazine), 4.02 (s, 2H,
4.4. Effects of compounds 2 and 5c on hyphal morphology of
S. sclerotiorum
CH2), 6.68 (t, J ¼ 4.7 Hz, 1H, ArH), 7.39 (t, J ¼ 4.7 Hz, 1H, ArH), A mycelial disk (5 mm diameter) was taken from the periphery
7.69 (dd, J ¼ 8.6, 4.0 Hz, 1H, ArH), 8.39 (d, J ¼ 4.7 Hz, 1H, ArH), of the colony grown on PDA mediums containing EC50 (0.0016
8.58 (s, 1H, ArH), 8.79 (d, J ¼ 4.1 Hz, 1H, ArH), 9.19 (d, J ¼ mM) of compound 2 and EC50 (0.0030 mM) of compound 5c
8.8 Hz, 1H, ArH). 13C NMR (100 MHz, DMSO-d6, d ppm): 40.55, respectively. The samples were inoculated to microscope slides
40.55, 51.01, 51.01, 53.68, 111.93, 124.24, 127.28, 132.38, on the rst day, and observed the mycelial morphology by
133.44, 135.21, 136.70, 143.51, 145.45, 147.97, 158.60, 158.60, optical microscope (Motic AE31E) on the third day, respectively.
161.09. HRMS calcd for C18H18N6O3, [M + H]+, 367.1474; found Scanning electron microscopy observations on the hyphae of S.
367.2326.
4.2.3.17. 5-Nitro-7-((4-(pyrazin-2-yl)piperazin-1-yl)methyl)
quinolin-8-ol (5q). Yellow solid; yield 53%; mp 206–207 C; H from the periphery of the colony grown on PDA mediums con-
NMR (400 MHz, DMSO-d6, d ppm): 2.89 (t, J ¼ 5.2 Hz, 4H, taining EC50 (0.0016 mM) of compound 2 and EC50 (0.0030 mM)
2CH2piperazine), 3.72 (t, J ¼ 5.2 Hz, 4H, 2CH2piperazine), 3.99 (s, 2H, of compound 5c respectively. Samples were xed in 2.5%
CH2), 7.39 (dd, J ¼ 7.6, 5.7 Hz, 1H, ArH), 7.87 (d, J ¼ 2.6 Hz, 1H, glutaraldehyde for 24 h at room temperature, and were washed
sclerotiorum were conducted according to the method of
previous studies.32 A mycelial disk (5 mm diameter) was taken
1
ꢁ
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for 15 min with 0.1 mol Lꢀ1 phosphate buffer for three times,
followed another 1 h xation in 1% OsO4 solution. The speci-
mens were dehydrated in a grated ethanol series (20%, 50%,
80% and 100% respectively, 5 min for each alcohol dilution).
Aer drying at critical point and gold coating, SEM observations
were carried out with a scanning electron microscope (Hitachi,
S-3400N, Japan) at an accelerating voltage of 15.0 kV.
Acknowledgements
This work was supported nancially by the National Natural
Science Foundation of China (21672092, 21877056) and the
National Key Research and Development Program of China
(2017YFD0201404); Support was also supplied by the Key
Program for International S&T Cooperation Projects of China
Gansu Province (18YF1WA115).
4.5. Effects of compounds 2 and 5c against S. sclerotiorum in
vivo
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Conflicts of interest
The authors state no conict of interest.
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