Design, synthesis and in vitro antibacterial/antifungal evaluation of novel 1-ethyl-6-fluoro-1,4-dihydro-4-oxo-7(1-piperazinyl)quinoline-3-carboxylic acid derivatives

Article abstract of DOI:10.1016/j.ejmech.2009.05.028

A series of 1-ethyl-6-fluoro-1,4-dihydro-4-oxo-7(1-piperazinyl)quinoline-3-carboxylic acid (norfloxacin) derivatives were prepared according to the principle of combinating bioactive substructures and tested for their activities against five plant pathogenic bacteria and three fungi in vitro. The preliminary bioassays indicated that almost all synthesized target compounds retained the antibacterial activities of norfloxacin and had some antifungal activities as carboxylic acid amide compounds. The activities of compounds 1 and 22 against Xanthomonas oryzae were better than norfloxacin and all tested compounds had better antibacterial activities as compared to the agricultural streptomycin sulfate (a commercial bactericide) against X. oryzae, Xanthomonas axonopodis and Erwinia aroideae. Additionally, compounds 2 and 20 displayed good antifungal activities against Rhizoctonia solani and their inhibition of growth reached 83% and 94% respectively at the concentration of 200 mg/L.

Full text of DOI:10.1016/j.ejmech.2009.05.028

European Journal of Medicinal Chemistry 44 (2009) 4726–4733
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Design, synthesis and in vitro antibacterial/antifungal evaluation of novel
1-ethyl-6-fluoro-1,4-dihydro-4-oxo-7(1-piperazinyl)quinoline-3-carboxylic
acid derivatives
Zhiyi Yu a, Guanying Shi b, Qiu Sun a, Hong Jin c, Yun Teng b, Ke Tao b, Guoping Zhou a, Wei Liu a,
,
Fang Wen c, Taiping Hou b
*
a West China School of Pharmacy, Sichuan University, Chengdu 610064, PR China
b Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, Sichuan 610064, PR China
c Key Laboratory of Green Chemistry and Technology of Ministry of Education, College of Chemistry, Sichuan University, Chengdu, Sichuan 610064, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 1-ethyl-6-fluoro-1,4-dihydro-4-oxo-7(1-piperazinyl)quinoline-3-carboxylic acid (norfloxacin)
derivatives were prepared according to the principle of combinating bioactive substructures and tested
for their activities against five plant pathogenic bacteria and three fungi in vitro. The preliminary
bioassays indicated that almost all synthesized target compounds retained the antibacterial activities of
norfloxacin and had some antifungal activities as carboxylic acid amide compounds. The activities of
compounds 1 and 22 against Xanthomonas oryzae were better than norfloxacin and all tested compounds
had better antibacterial activities as compared to the agricultural streptomycin sulfate (a commercial
bactericide) against X. oryzae, Xanthomonas axonopodis and Erwinia aroideae. Additionally, compounds 2
and 20 displayed good antifungal activities against Rhizoctonia solani and their inhibition of growth
reached 83% and 94% respectively at the concentration of 200 mg/L.
Received 30 April 2009
Received in revised form
25 May 2009
Accepted 26 May 2009
Available online 6 June 2009
Keywords:
Norfloxacin
Carboxylic acid amide
Synthesis
Antibacterial activity
Antifungal activity
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
fluoroquinolones, and it has activities against a broad spectrum of
gram-negative and certain gram-positive bacteria [1,8]. Although
Ever since the discovery of the first marketed quinolone, nali-
dixic acid (a naphthyridine) in 1962 by Lesher et al. [1,2], numerous
structural modifications have been made in the quinoline nucleus
in order to produce agents with a broader spectrum of activity and
applicability [3]. The real breakthrough didn’t come until the 1980s
with the introduction of a fluorine molecule into this basic nucleus
at position R6 renaming the compound fluoroquinolone [3,4]. Since
then, fluoroquinolones have been widely and rapidly adopted in the
treatment of ocular infections, with topical, intravitreal and
systemic routes of administration being used, because they have
been shown in most situations to be equivalent to combination
therapies and they are also effective against polyresistant organ-
isms such as Pseudomonas aeruginosa and Enterobacteriaceae
[5–7]. Norfloxacin [1-ethyl-6-fluoro-1,4-dihydro-4-oxo-7(1-piper-
azineyl)quinoline-3-carboxylic acid], a 6-fluorinated compound
with a piperazine ring at position R7, is one of the commonly used
norfloxacin has already been widely used and studied in veterinary
and human medicine [9], its application to pesticide science has not
been reported to our knowledge.
In the agricultural chemistry field, carboxylic acid amide
fungicides have played an important role in pesticide science. Since
the first carboxylic acid amide fungicide carboxin was discovered
by von Schmeling and Kulla [10], a series of carboxylic acid amide
fungicides with novel structures have successively emerged [11].
These carboxylic acid amide fungicides can inhibit the growth of
pathogens and cause their eventual death by interfering with the
pathogens’ respiration [12,13]. Nowadays, they have been mainly
used to control diseases caused by plant pathogenic fungi and some
of them can also control diseases caused by plant pathogenic
bacteria [12].
In this context, the principle of combinating bioactive
substructures was used in this study in order to integrate nor-
floxacin with carboxylic acid amide fungicides to search for novel
lead compounds with a broader antimicrobial spectrum and better
biological activities (Fig. 1 shown as an example in which furcar-
banil is a marketed fungicide). Herein, different kinds of carboxylic
* Corresponding author. Tel.: þ86 28 85410992.
E-mail address: houtp@scu.edu.cn (T. Hou).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.05.028

Z. Yu et al. / European Journal of Medicinal Chemistry 44 (2009) 4726–4733
4727
2.2. Biological activity
O
N
Compounds 1–28 and norfloxacin were evaluated in vitro to
explore their antibacterial activities using the agar well-diffusion
method [20,21] and their antifungal activities were also studied
using the plate growth rate method [22,23].
5
8
COOH
4
1
F
6
7
O
3
2
NH
N
HN
O
2.2.1. Antibacterial activity
The antibacterial activities of all tested compounds against
X. oryzae, X. axonopodis, E. aroideae, B. subtilis and R. solanacearum
were screened at a concentration of 50 mg/L using the agar well-
diffusion method. The results were calculated and expressed as
diameters of inhibition to bacteria (Table 2).
As shown in Table 2, all the title compounds (1–28) retained the
antibacterial activities of norfloxacin (the parent compound) in
vitro. In particular, compounds 1 and 22 had better antibacterial
activities than norfloxacin against X. oryzae. In addition, the anti-
bacterial activities of all compounds, including norfloxacin, against
X. oryzae, X. axonopodis and E. aroideae were better than the agri-
cultural streptomycin sulfate, a commercial bactericide.
norfloxacin
furcarbanil
O
N
COOH
F
N
N
O
O
4
The diameters of inhibition of compounds 1–28 against
X. oryzae ranged from 4.44 to 22.94 mm at a concentration of
50 mg/L, while that of norfloxacin was 22.19 mm. The preliminary
bioassay results indicated that compounds 2–21 and 23–28 had
lower activities than norfloxacin, but compounds 1 and 22 were
more active than the parent compound. Furthermore, all
compounds tested had better activities than the agricultural
streptomycin sulfate in vitro.
When it comes to X. axonopodis and E. aroideae, the preliminary
bioassay results indicated a lower antibacterial activity by tested
compounds 1–28 than norfloxacin at a concentration of 50 mg/L.
However, all of them were more active than the agricultural
streptomycin sulfate in vitro. The diameters of inhibition of
compound 22 against X. axonopodis and E. aroideae were 15.83 and
17.85 mm respectively, and the values were close to norfloxacin
whose inhibition zones were 17.74 and 21.52 mm respectively.
Compounds 1, 22, 24 and 25, for which the diameters of inhi-
bition against B. subtilis were 22.49, 20.66, 20.08 and 22.74 mm
respectively, displayed similar activities as compared with the
agricultural streptomycin sulfate (the diameter of inhibition for it
was 24.99 mm). Nevertheless, all the other compounds tested had
much lower activities than the agricultural streptomycin sulfate. In
addition, the preliminary test results also showed that all synthe-
sized compounds, including the agricultural streptomycin sulfate,
were less active than norfloxacin.
Fig. 1. Design of compound 4.
acids regarded as pharmacophores among carboxylic acid amide
fungicides have been connected to the amino group in C7-piper-
azinyl-[norfloxacin] to give a series of novel compounds (Table 1)
and a detailed discussion of the methodology used to prepare the
aforementioned compounds is given. Their bioactivities against five
plant pathogenic bacteria (Xanthomonas oryzae, Xanthomonas
axonopodis, Erwinia aroideae, Bacillus subtilis and Ralstonia sol-
anacearum) and three plant pathogenic fungi (Rhizoctonia solani,
Gibberella zeae and Valsa mali) were also evaluated, and the results
are discussed subsequently.
2. Results and discussion
2.1. Chemistry
The intermediates 1a, 2a, 3a–3e and 4a–4i used in this study
were synthesized by Method 1 [14], 2 [15], 3 [16] or 4 [17,18]
depicted in Scheme 1 according to the literatures and they were
used in the subsequent reactions without further purification. 3-
Methylbenzofuran-2-carboxylic acid and 2,4,5-trimethylfuran-3-
carboxylic acid were synthesized in other studies by our laboratory,
and the other carboxylic acids were purchased from commercial
sources.
The activities of all synthesized compounds were poor against
R. solanacearum and much lower than the agricultural streptomycin
sulfate. At the same time, norfloxacin had a better activity than both
the title compounds and the agricultural streptomycin sulfate.
The synthesis pathways of all target compounds are shown in
Schemes 2 and 3. Their 1H NMR data and melting points are listed
in the synthesis procedures of the title compounds. Compounds 1–
8, 10–28 were prepared by the method shown in Scheme 2
successfully. Nevertheless, compound 9 couldn’t be synthesized by
the same method since the nicotinoyl chloride synthesized in
Scheme 2 existed in the form of salt and was not suitable for the
next reaction condition. However, compound 9 could be synthe-
sized using the method expressed in Scheme 3 and the yield was
relatively good [19]. Before the procedure shown in Scheme 2 was
discovered, we had tried many other ways to synthesize these
compounds such as by reacting in a sodium hydroxide solution or
tetrahydrofuran and triethylamine solution with carboxylic acid
chloride and norfloxacin and so on. Unfortunately, almost all of
them ended in failure because of too many by-products and poor
yields of the target compounds.
2.2.2. Antifungal activity
Synthesized target compounds 1–28 and norfloxacin were
screened for their anti-plant pathogenic fungi activities in vitro and
the inhibition of growth of the antifungal agents at 200 mg/L is
summarized in Table 3.
The result of the target compounds’ activities against R. solani in
vitro showed that almost all the target compounds except
compounds 13, 21 and 25 were more active than norfloxacin, and
the inhibition of growth by compounds 2 and 20 was up to 83% and
94% respectively which indicated that their activities were rela-
tively close to carbendazim (its inhibition of growth was 100%),
a commercial fungicide.
Against G. zeae and V. mali, the preliminary bioassay results
indicated that the activities of all tested compounds were not very
good as compared to carbendazim,
a commercial fungicide.

4728
Z. Yu et al. / European Journal of Medicinal Chemistry 44 (2009) 4726–4733
Table 1
Synthesized norfloxacin derivatives.
O
F
COOH
N
N
N
R
Compound
R
Compound
R
Compound
R
Cl
O
O
O
O
1
11
21
O
Cl
Cl
O
O
O
O
2
3
12
13
22
23
O
Cl
O
O
O
O
Br
O
O
O
O
Br
O
O
4
14
24
O
O
O
OH
O
O
5
6
7
15
16
17
25
26
27
O
Br
O
O
O
O
O
F
O
O
O
O
O
Cl
O
Cl
O
O
O
O
8
9
18
19
28
O
Cl
O
O
N
O
O
10
20
O
O

Z. Yu et al. / European Journal of Medicinal Chemistry 44 (2009) 4726–4733
4729
Scheme 1. Preparation of intermediates.
Scheme 2. Synthesis pathway of compounds 1–8, 10–28.

4730
Z. Yu et al. / European Journal of Medicinal Chemistry 44 (2009) 4726–4733
Scheme 3. Synthesis pathway of compound 9.
Table 2
were even better than norfloxacin against X. oryzae at 50 mg/L.
Furthermore, most of the title compounds had better antibacterial
activities as compared with the agricultural streptomycin sulfate
(a commercial bactericide) and better antifungal activities than
norfloxacin. Meanwhile, the inhibition of growth by compounds 2
and 20 against R. solani reached 83% and 94% respectively at
a concentration of 200 mg/L. In addition, although the synthesized
compounds don’t possess both good antibacterial and antifungal
activities, these preliminary results are promising and beneficial for
further studies in developing a new sort of widely used antimi-
crobial agents in the agricultural chemistry field. Moreover, further
design and biological evaluation of these compounds are ongoing
in our laboratory.
Antibacterial activities of compounds 1–28 and norfloxacin at 50 mg/L.
Compound
Inhibition zone (diameter in mm)
X. oryzae X. axonopodis E. aroideae B. subtilis R. solanacearum
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
22.94
18.10
13.02
18.10
12.69
8.35
15.49
10.24
12.08
11.66
11.08
8.66
13.85
11.19
13.69
11.74
12.44
11.02
5.94
22.49
16.99
18.91
15.74
13.91
11.16
6.99
9.81
6.97
6.72
8.39
5.31
8.56
3.56
5.72
1.06
4.89
6.22
4.67
4.97
10.89
7.47
3.31
6.31
0.50
4.64
0
5.44
5.24
15.27
7.94
12.91
6.58
14.94
9.19
19.43
8.74
15.35
14.19
17.19
11.94
5.81
14.99
10.74
10.41
10.99
6.41
13.77
13.44
11.77
12.94
8.52
17.33
15.33
14.16
17.74
10.79
12.66
13.91
17.58
9.91
4. Experimental protocols
8.02
10.74
11.08
12.49
8.16
12.08
6.83
8.94
11.02
14.52
10.02
11.69
5.35
13.27
15.19
8.52
10.85
8.02
4.1. Chemistry
4.1.1. Materials
15.16
8.91
2.08
All chemicals and solvents were purchased from commercial
sources unless otherwise specified. All bacteria and fungi were
obtained from the Institute of Pesticide and Crop Protection,
Sichuan University. Melting points were determined using an X-4
micro-melting point apparatus (Beijing second optical instrument
factory, PR China) and are uncorrected. 1H NMR spectra were
recorded in deuterochloroform solution on a Bruker 400 MHz
spectrometer, using tetramethylsilane (TMS) as an internal stan-
dard. HRMS data were obtained on an ESI-TOFMS instrument
(Agilent 6210).
4.44
5.91
2.92
0
22.69
16.85
16.52
16.69
19.69
17.69
9.35
15.83
14.24
14.58
14.41
12.99
12.91
9.99
17.85
16.00
16.19
16.52
14.60
16.60
10.44
21.52
0.50
20.66
18.99
20.08
22.74
17.08
18.49
13.16
30.49
24.99
0
5.92
8.97
8.14
2.06
3.22
1.47
0
19.31
17.27
0
Norfloxacin
Streptomycin
DMSO
22.19
0.81
17.74
0.20
0
0
0
‘‘Streptomycin’’ represents the agricultural streptomycin sulfate.
4.1.2. Synthesis of target compounds
4.1.2.1. Preparation of carboxylic acid chlorides. Thionyl chloride
(15 mL) was added into the corresponding acid (5 mmol), and then
the mixture was heated under reflux for 2 h with the help of
a drying tube filled with anhydrous calcium chloride. When the
reaction was completed, the excess thionyl chloride was removed
under reduced pressure. The crude products were used in the
subsequent reactions without further purification.
Nevertheless, the data in Table 3 showed that almost half of the
tested compounds (such as 7 and 24) had better antifungal activi-
ties than norfloxacin against G. zeae, and the inhibition of growth
by compounds 4, 5, 7, 22–24 and 26 was also higher than nor-
floxacin against V. mali.
3. Conclusion
4.1.2.2. General procedures for compounds 1–8 and 10–28. A
mixture of sodium hydroxide (0.7 g, 17.5 mmol), 1,4-dioxane
(15 mL), water (15 mL), and norfloxacin (1.6 g, 5 mmol) was dis-
solved and cooled to 0 ꢀC in an ice bath. Under stirring, carboxylic
acid chloride synthesized in ‘‘Section 4.1.2.1’’ was added dropwise
to the mixture with the temperature maintained below 3 ꢀC.
Afterwards, the resulting reaction mixture was stirred for another
5 h at room temperature. Then the mixture was poured into ice-
water and acidified with hydrochloric acid to pH 4. Finally, the
generated precipitates were collected by filtration, washed
In summary, a series of novel 1-ethyl-6-fluoro-1,4-dihydro-4-
oxo-7(1-piperazinyl) quinoline-3-carboxylic acid (norfloxacin)
derivatives have been synthesized by employing a convenient and
practical process and tested for their antibacterial activities against
X. oryzae, X. axonopodis, E. aroideae, B. subtilis and R. solanacearum
and antifungal activities against R. solani, G. zeae and V. mali. The
above biological test results showed that all the synthesized target
compounds had kept the antibacterial activities of norfloxacin (the
synthetic precursor), and the activities of compounds 1 and 22

Z. Yu et al. / European Journal of Medicinal Chemistry 44 (2009) 4726–4733
4731
Table 3
Antifungal activities of compounds 1–28 and norfloxacin at 200 mg/L.
Compound
Inhibition of growth (%)
Compound
Inhibition of growth (%)
R. solani
G. zeae
V. mali
R. solani
G. zeae
V. mali
1
2
3
4
5
6
7
8
14
83
47
16
65
17
65
20
12
30
34
3
7
25
26
25
24
7
8
25
27
29
40
9
33
7
17
8
19
12
7
14
9
16
17
18
19
20
21
22
23
24
25
26
27
52
60
72
29
94
0
25
5
15
0
36
21
5
0
100
15
17
5
5
1
12
10
17
9
5
6
42
41
44
17
37
16
0
2
45
8
23
27
34
15
28
27
12
18
100
9
11
17
20
10
14
28
6
10
11
12
13
14
15
0
53
49
28
Norfloxacin
Carbendazim
27
95
successively with water and dried. The pure products 1–8 and 10–
28 were obtained by recrystallization in DMF.
(t, J ¼ 7.3 Hz, 3H); HRMS (ESI) for C26H24FN3O5 ([M þ H]þ): found
478.1773, calcd. 478.1773.
Compound 1: white crystals, yield: 55%, m.p. 295–296 ꢀC; 1H
Compound 8: white crystals, yield: 86%, m.p.198–199 ꢀC; 1H
NMR (400 MHz, CDCl3,
d
ppm): 14.98 (s, 1H), 8.70 (s, 1H), 8.13 (d,
NMR (400 MHz, CDCl3, d ppm): 15.00 (s, 1H), 8.69 (s, 1H), 8.12 (d,
J ¼ 12.8 Hz, 1H), 7.51–7.54 (m, 1H), 7.11 (d, J ¼ 3.4 Hz, 1H), 6.87 (d,
J ¼ 6.8 Hz,1H), 6.53 (dd, J ¼ 1.7 Hz,1H), 4.33 (q, J ¼ 7.3 Hz, 2H), 3.99–
4.16 (m, 4H), 3.39 (t, J ¼ 5.0 Hz, 4H), 1.60 (t, J ¼ 7.3 Hz, 3H); HRMS
(ESI) for C21H20FN3O5 ([M þ H]þ): found 414.1464, calcd. 414.1460.
Compound 2: brown crystals, yield: 93%, m.p. 211–212 ꢀC; 1H
J ¼ 12.8 Hz, 1H), 7.52 (d, J ¼ 15.0 Hz, 1H), 7.47 (d, J ¼ 1.7 Hz, 1H), 6.86
(d, J ¼ 6.8 Hz, 1H), 6.83 (d, J ¼ 15.0 Hz, 1H), 6.60 (d, J ¼ 3.4 Hz, 1H),
6.49 (dd, J ¼ 1.7 Hz, 1H), 4.33 (q, J ¼ 7.2 Hz, 2H), 3.83–4.06 (m, 4H),
3.28–3.44 (m, 4H), 1.60 (t, J ¼ 7.2 Hz, 3H); HRMS (ESI) for
C23H22FN3O5 ([M þ H]þ): found 440.1618, calcd. 440.1616.
NMR (400 MHz, CDCl3,
d
ppm): 15.01 (s, 1H), 8.69 (s, 1H), 8.11 (d,
Compound 10: yellow crystals, yield: 55%, m.p. 236–238 ꢀC; 1H
J ¼ 12.8 Hz,1H), 7.46 (d, J ¼ 15.0 Hz,1H), 6.86 (d, J ¼ 6.8 Hz,1H), 6.73
(d, J ¼ 15.0 Hz, 1H), 6.49 (d, J ¼ 3.3 Hz, 1H), 6.09 (d, J ¼ 3.3 Hz, 1H),
4.33 (q, J ¼ 7.3 Hz, 2H), 3.78–4.03 (m, 4H), 3.28–3.43 (m, 4H), 2.36
(s, 3H), 1.60 (t, J ¼ 7.3 Hz, 3H); HRMS (ESI) for C24H24FN3O5
([M þ H]þ): found 454.1780, calcd. 454.1773.
NMR (400 MHz, CDCl3, d ppm): 14.96 (s, 1H), 8.69 (s, 1H), 8.11 (d,
J ¼ 12.8 Hz, 1H), 7.27–7.36 (m, 2H), 6.94–7.06 (m, 3H), 6.82 (d,
J ¼ 6.8 Hz, 1H), 4.76 (s, 2H), 4.31 (q, J ¼ 7.2 Hz, 2H), 3.83–3.92 (m,
4H), 3.21–3.36 (m, 4H),1.60 (t, J ¼ 7.2 Hz, 3H); HRMS (ESI) for
C24H24FN3O5 ([M þ H]þ): found 454.1774, calcd. 454.1773.
Compound 3: white crystals, yield: 56%, m.p. 229–230 ꢀC; 1H
Compound 11: green crystals, yield: 27%, m.p. 232–233 ꢀC; 1H
NMR (400 MHz, CDCl3,
d
ppm): 15.00 (s, 1H), 8.69 (s, 1H), 8.12 (d,
NMR (400 MHz, CDCl3, d ppm): 14.95 (s, 1H), 8.68 (s, 1H), 8.10 (d,
J ¼ 12.8 Hz, 1H), 7.09 (d, J ¼ 3.5 Hz, 1H), 6.87 (d, J ¼ 6.9 Hz, 1H), 6.48
(d, J ¼ 3.5 Hz, 1H), 4.33 (q, J ¼ 7.3 Hz, 2H), 3.99–4.14 (m, 4H), 3.40 (t,
J ¼ 5.1 Hz, 4H), 1.59 (t, J ¼ 7.3 Hz, 3H); HRMS (ESI) for
C21H19BrFN3O5 ([M þ H]þ): found 492.0565, calcd. 492.0565.
Compound 4: brown crystals, yield: 71%, m.p. 224–225 ꢀC; 1H
J ¼ 12.8 Hz, 1H), 7.11–7.22 (m, 2H), 6.86–6.96 (m, 2H), 6.82 (d,
J ¼ 6.8 Hz,1H), 4.77 (s, 2H), 4.32 (q, J ¼ 7.2 Hz, 2H), 3.89 (t, J ¼ 4.9 Hz,
4H), 3.20–3.37 (m, 4H), 2.26 (s, 3H), 1.57 (t, J ¼ 7.2 Hz, 3H); HRMS
(ESI) for C25H26FN3O5 ([M þ H]þ): found 468.1930, calcd. 468.1929.
Compound 12: light yellow crystals, yield: 21%, m.p. 249–
NMR (400 MHz, CDCl3,
d
ppm): 14.98 (s, 1H), 8.69 (s, 1H), 8.11 (d,
250 ꢀC; 1H NMR (400 MHz, CDCl3,
d ppm): 14.95 (s, 1H), 8.70 (s, 1H),
J ¼ 12.8 Hz, 1H), 6.85 (d, J ¼ 6.8 Hz, 1H), 5.95 (s, 1H), 4.32 (q,
J ¼ 7.2 Hz, 2H), 3.79–4.01 (m, 4H), 3.23–3.37 (m, 4H), 2.37 (s, 3H),
2.26 (s, 3H), 1.61 (t, J ¼ 7.2 Hz, 3H); HRMS (ESI) for C23H24FN3O5
([M þ H]þ): found 442.1776, calcd. 442.1773.
8.12 (d, J ¼ 12.8 Hz, 1H), 7.28–7.43 (m, 2H), 6.93–7.08 (m, 2H), 6.83
(d, J ¼ 6.9 Hz, 1H), 4.84 (s, 2H), 4.32 (q, J ¼ 7.1 Hz, 2H), 3.83–3.97 (m,
4H), 3.21–3.38 (m, 4H), 1.59 (t, J ¼ 7.1 Hz, 3H); HRMS (ESI) for
C24H23ClFN3O5 ([M þ H]þ): found 488.1384, calcd. 488.1383.
Compound 13: yellowish green crystals, yield: 58%, m.p. 241–
Compound 5: yellow crystals, yield: 68%, m.p. 266–268 ꢀC; 1H
NMR (400 MHz, CDCl3,
d
ppm): 14.99 (s, 1H), 8.68 (s, 1H), 8.09 (d,
242 ꢀC; 1H NMR (400 MHz, CDCl3,
d ppm): 14.97 (s, 1H), 8.69 (s, 1H),
J ¼ 12.8 Hz,1H), 6.86 (d, J ¼ 6.8 Hz,1H), 4.33 (q, J ¼ 7.2 Hz, 2H), 3.12–
4.12 (m, 8H), 2.27 (s, 3H), 1.92 (s, 3H), 1.65 (s, 3H), 1.60 (t, J ¼ 7.2 Hz,
3H); HRMS (ESI) for C24H26FN3O5 ([M þ H]þ): found 456.1932,
calcd. 456.1929.
8.11 (d, J ¼ 12.8 Hz, 1H), 6.88–7.04 (m, 4H), 6.82 (d, J ¼ 6.8 Hz, 1H),
4.80 (s, 2H), 4.32 (q, J ¼ 7.2 Hz, 2H), 3.88–3.97 (m, 4H), 3.87 (s, 3H),
3.25–3.36 (m, 4H), 1.59 (t, J ¼ 7.2 Hz, 3H); HRMS (ESI) for
C25H26FN3O6 ([M þ H]þ): found 484.1884, calcd. 484.1878.
Compound 6: white crystals, yield: 65%, m.p. 278–279 ꢀC; 1H
Compound 14: light yellow crystals, yield: 20%, m.p. 173–174 ꢀC;
NMR (400 MHz, CDCl3,
d
ppm): 14.98 (s, 1H), 8.70 (s, 1H), 8.14 (d,
1H NMR (400 MHz, CDCl3,
d ppm): 14.96 (s, 1H), 8.69 (s, 1H), 8.10 (d,
J ¼ 12.8 Hz, 1H), 7.67–7.71 (m, 1H), 7.52–7.57 (m, 1H), 7.43–7.47 (m,
1H), 7.42 (s, 1H), 7.30–7.35 (m, 1H), 6.89 (d, J ¼ 6.8 Hz, 1H), 4.34 (q,
J ¼ 7.2 Hz, 2H), 4.04–4.23 (m, 4H), 3.43 (t, J ¼ 5.0 Hz, 4H), 1.61 (t,
J ¼ 7.2 Hz, 3H); HRMS (ESI) for C25H22FN3O5 ([M þ H]þ): found
464.1616, calcd. 464.1616.
J ¼ 12.8 Hz, 1H), 7.32 (d, J ¼ 8.8 Hz, 2H), 6.90 (d, J ¼ 8.8 Hz, 2H), 6.83
(d, J ¼ 6.8 Hz, 1H), 4.73 (s, 2H), 4.32 (q, J ¼ 7.2 Hz, 2H), 3.83–3.93 (m,
4H), 3.26–3.37 (m, 4H), 1.59 (t, J ¼ 7.2 Hz, 3H), 1.29 (s, 9H); HRMS
(ESI) for C28H32FN3O5 ([M þ H]þ): found 510.2402, calcd. 510.2399.
Compound 15: light green crystals, yield: 24%, m.p. 248–249 ꢀC;
Compound 7: light yellow crystals, yield: 88%, m.p. 140–141 ꢀC;
1H NMR (400 MHz, CDCl3,
d ppm): 14.94 (s, 1H), 8.69 (s, 1H), 8.12 (d,
1H NMR (400 MHz, CDCl3,
d
ppm): 15.00 (s, 1H), 8.70 (s, 1H), 8.12 (d,
J ¼ 12.8 Hz, 1H), 7.40 (d, J ¼ 9.0 Hz, 2H), 6.86 (d, J ¼ 9.0 Hz, 2H), 6.82
(d, J ¼ 6.8 Hz, 1H), 4.74 (s, 2H), 4.32 (q, J ¼ 7.3 Hz, 2H), 3.82–3.90 (m,
4H), 3.22–3.35 (m, 4H), 1.59 (t, J ¼ 7.3 Hz, 3H); HRMS (ESI) for
C24H23BrFN3O5 ([M þ H]þ): found 532.0876, calcd. 532.0878.
J ¼ 12.8 Hz, 1H), 7.61–7.65 (m, 1H), 7.47–7.50 (m, 1H), 7.40–7.46
(m,1H), 7.30–7.36 (m, 1H), 6.89 (d, J ¼ 6.8 Hz,1H), 4.34 (q, J ¼ 7.3 Hz,
2H), 4.04 (t, J ¼ 5.0 Hz, 4H), 3.42 (t, J ¼ 5.0 Hz, 4H), 2.51 (s, 3H), 1.61

4732
Z. Yu et al. / European Journal of Medicinal Chemistry 44 (2009) 4726–4733
Compound 16: green crystals, yield: 19%, m.p. 232–234 ꢀC; 1H
NMR (400 MHz, CDCl3, ppm): 14.94 (s, 1H), 8.70 (s, 1H), 8.12 (d,
2H), 6.85 (d, J ¼ 6.8 Hz, 1H), 4.32 (q, J ¼ 7.0 Hz, 2H), 3.18–4.18 (m,
d
8H), 2.39 (s, 3H),1.58 (t, J ¼ 7.0 Hz, 3H); HRMS (ESI) for C24H24FN3O4
J ¼ 12.7 Hz, 1H), 6.73–7.04 (m, 5H), 4.73 (s, 2H), 4.32 (q, J ¼ 7.2 Hz,
2H), 3.81–3.92 (m, 4H), 3.19–3.42 (m, 4H), 1.61 (t, J ¼ 7.2 Hz, 3H);
HRMS (ESI) for C24H23F2N3O5 ([M þ H]þ): found 472.1679, calcd.
472.1679.
([M þ H]þ): found 438.1826, calcd. 438.1824.
Compound 27: green crystals, yield: 41%, m.p. 242–243 ꢀC; 1H
NMR (400 MHz, CDCl3,
d ppm): 14.95 (s, 1H), 8.69 (s, 1H), 8.11 (d,
J ¼ 12.8 Hz, 1H), 7.38–7.46 (m, 4H), 6.86 (d, J ¼ 6.7 Hz, 1H), 4.32 (q,
J ¼ 7.2 Hz, 2H), 3.59–4.12 (m, 4H), 3.24–3.46 (m, 4H), 1.60 (t,
J ¼ 7.2 Hz, 3H); HRMS (ESI) for C23H21ClFN3O4 ([M þ H]þ): found
458.1278, calcd. 458.1277.
Compound 17: brown crystals, yield: 35%, m.p. 217–218 ꢀC; 1H
NMR (400 MHz, CDCl3,
d ppm): 14.96 (s, 1H), 8.68 (s, 1H), 8.09 (d,
J ¼ 12.8 Hz, 1H), 6.79–6.95 (m, 5H), 4.71 (s, 2H), 4.32 (q, J ¼ 7.1 Hz,
2H), 3.87 (t, J ¼ 4.9 Hz, 4H), 3.77 (s, 3H), 3.24–3.36 (m, 4H), 1.59 (t,
J ¼ 7.1 Hz, 3H); HRMS (ESI) for C25H26FN3O6 ([M þ H]þ): found
484.1878, calcd. 484.1878.
Compound 28: light yellow crystals, yield: 63%, m.p. 288–
289 ꢀC; 1H NMR (400 MHz, CDCl3,
d ppm): 14.95 (s, 1H), 8.68 (s, 1H),
8.09 (d, J ¼ 12.8 Hz, 1H), 7.99–8.04 (m, 1H), 7.87–7.92 (m, 1H), 7.78–
7.83 (m,1H), 7.50–7.60 (m, 2H), 7.41–7.47 (m,1H), 7.35–7.40 (m,1H),
6.77 (d, J ¼ 6.8 Hz, 1H), 4.29 (q, J ¼ 7.3 Hz, 2H), 4.23 (s, 2H), 3.96 (t,
J ¼ 4.9 Hz, 2H), 3.69 (t, J ¼ 4.9 Hz, 2H), 3.29 (t, J ¼ 4.9 Hz, 2H), 3.12 (t,
J ¼ 4.9 Hz, 2H), 1.58 (t, J ¼ 7.3 Hz, 3H); HRMS (ESI) for C28H26FN3O4
([M þ H]þ): found 488.1983, calcd. 488.1980.
Compound 18: light green crystals, yield: 26%, m.p. 243–244 ꢀC;
1H NMR (400 MHz, CDCl3,
d ppm): 14.94 (s, 1H), 8.69 (s, 1H), 8.12 (d,
J ¼ 12.7 Hz, 1H), 7.39 (s, 1H), 7.21 (d, J ¼ 8.8 Hz, 1H), 6.99 (d,
J ¼ 8.8 Hz, 1H), 6.83 (d, J ¼ 6.3 Hz, 1H), 4.82 (s, 2H), 4.33 (q,
J ¼ 7.1 Hz, 2H), 3.82–3.95 (m, 4H), 3.21–3.39 (m, 4H), 1.60 (t,
J ¼ 7.1 Hz, 3H); HRMS (ESI) for C24H22Cl2FN3O5 ([M þ H]þ): found
522.0994, calcd. 522.0993.
4.1.2.3. Synthesis of compound 9. Nicotinoyl chloride prepared
under procedure ‘‘Section 4.1.2.1’’ was a light yellow solid. After
cooling to room temperature, norfloxacin (1.6 g, 5 mmol) and
pyridine (20 mL) were added. Then the reaction mixture was
vigorously stirred for 10 h at ambient temperature. When
completed, the final mixture was concentrated under reduced
pressure and then poured into ice-water and neutralized with
hydrochloric acid. The precipitates were found, collected by filtra-
tion, washed with water, dried and recrystallized from DMF to give
the title compound 9.
Compound 19: yellow crystals, yield: 80%, m.p. 252–254 ꢀC; 1H
NMR (400 MHz, CDCl3,
d ppm): 14.99 (s, 1H), 8.69 (s, 1H), 8.12 (d,
J ¼ 12.8 Hz, 1H), 7.74 (d, J ¼ 15.4 Hz, 1H), 7.50–7.59 (m, 2H), 7.35–
7.44 (m, 3H), 6.91 (d, J ¼ 15.4 Hz,1H), 6.86 (d, J ¼ 6.7 Hz,1H), 4.33 (q,
J ¼ 7.1 Hz, 2H), 3.83–4.09 (m, 4H), 3.26–3.46 (m, 4H), 1.58 (t,
J ¼ 7.1 Hz, 3H); HRMS (ESI) for C25H24FN3O4 ([M þ H]þ): found
450.1829, calcd. 450.1824.
Compound 20: brown crystals, yield: 62%, m.p. 248–249 ꢀC; 1H
NMR (400 MHz, CDCl3,
d ppm): 15.00 (s, 1H), 8.69 (s, 1H), 8.12 (d,
J ¼ 12.7 Hz, 1H), 7.71 (d, J ¼ 15.2 Hz, 1H), 7.50 (d, J ¼ 8.3 Hz, 2H),
6.82–7.02 (m, 3H), 6.78 (d, J ¼ 15.2 Hz, 1H), 4.32 (q, J ¼ 7.2 Hz, 2H),
3.87–4.06 (m, 4H), 3.85 (s, 3H), 3.23–3.46 (m, 4H), 1.60 (t, J ¼ 7.2 Hz,
3H); HRMS (ESI) for C26H26FN3O5 ([M þ H]þ): found 480.1931, calcd.
480.1929.
Compound 9: light green crystals, yield: 52%, m.p. 198–200 ꢀC;
1H NMR (400 MHz, CDCl3,
d ppm): 14.95 (s, 1H), 8.67–8.77 (m, 3H),
8.01–8.22 (m, 2H), 7.38–7.46 (m, 1H), 6.75–7.02 (m, 1H), 3.17–4.46
(m, 10H), 1.62–1.68 (m, 3H); HRMS (ESI) for C22H21FN4O4
([M þ H]þ): found 425.1619, calcd. 425.1620.
Compound 21: light green crystals, yield: 65%, m.p. 265–266 ꢀC;
1H NMR (400 MHz, CDCl3,
d ppm): 14.96 (s, 1H), 8.69 (s, 1H), 8.13 (d,
4.2. Biological assay
J ¼ 12.8 Hz, 1H), 7.99 (d, J ¼ 15.6 Hz, 1H), 7.54 (d, J ¼ 8.4 Hz, 1H), 7.46
(s, 1H), 7.27–7.30 (m, 1H), 6.84–6.92 (m, 2H), 4.33 (q, J ¼ 7.2 Hz, 2H),
3.77–4.05 (m, 4H), 3.25–3.46 (m, 4H), 1.58 (t, J ¼ 7.2 Hz, 3H); HRMS
(ESI) for C25H22Cl2FN3O4 ([M þ H]þ): found 518.1049, calcd. 518.1044.
Compound 22: light green crystals, yield: 68%, m.p. 263–264 ꢀC;
4.2.1. The assay of antibacterial activity
The antibacterial activities of the tested compounds against
X. oryzae, X. axonopodis, E. aroideae, B. subtilis and R. solanacearum
were screened using the agar well-diffusion method [20,21].
Using a sterile pipette, 0.8 mL of twenty-four-hour-old bacteria
inoculums was added to 60 mL of the sterile molten agar which had
been cooled to 45 ꢀC, mixed well and poured into three sterile Petri
dishes (each dish given 20 mL). After the agar solidified, the wells
were dug in the media with the help of a sterile metallic borer with
centers at least 24 mm apart. The recommended concentration of
the tested sample (50 mg/L using DMSO as the solvent) was added
into respective wells. Other wells were supplemented with DMSO
and the agricultural streptomycin sulfate serving as negative and
positive controls, respectively. Each sample was repeated three
times. The plates were incubated immediately at 37 ꢀC for 24 h. The
diameters (mm) of inhibition were measured and used to express
the antibacterial activities of the tested compounds.
1H NMR (400 MHz, CDCl3,
d ppm): 14.97 (s, 1H), 8.67 (s, 1H), 8.07 (d,
J ¼ 12.8 Hz, 1H), 7.42–7.53 (m, 5H), 6.86 (d, J ¼ 6.8 Hz, 1H), 4.33 (q,
J ¼ 7.2 Hz, 2H), 3.11–4.20 (m, 8H), 1.59 (t, J ¼ 7.2 Hz, 3H); HRMS (ESI)
for C23H22FN3O4 ([M þ H]þ): found 424.1680, calcd. 424.1667.
Compound 23: light yellowcrystals, yield: 70%, m.p. 262–263 ꢀC;
1H NMR (400 MHz, CDCl3,
d ppm): 14.96 (s, 1H), 8.68 (s, 1H), 8.09 (d,
J ¼ 12.8 Hz, 1H), 7.31–7.48 (m, 4H), 6.86 (d, J ¼ 6.8 Hz, 1H), 4.33 (q,
J ¼ 7.1 Hz, 2H), 3.23–4.01 (m, 8H), 1.59 (t, J ¼ 7.1 Hz, 3H); HRMS (ESI)
for C23H21ClFN3O4 ([M þ H]þ): found 458.1277, calcd. 458.1277.
Compound 24: light green crystals, yield: 59%, m.p. 269–270 ꢀC;
1H NMR (400 MHz, CDCl3,
d ppm): 14.96 (s, 1H), 8.68 (s, 1H), 8.09 (d,
J ¼ 12.8 Hz, 1H), 7.59–7.65 (m, 1H), 7.37–7.46 (m, 1H), 7.27–7.34 (m,
2H), 6.86 (d, J ¼ 6.8 Hz, 1H), 4.33 (q, J ¼ 7.2 Hz, 2H), 3.23–4.23 (m,
8H), 1.59 (t, J ¼ 7.2 Hz, 3H); HRMS (ESI) for C23H21BrFN3O4
([M þ H]þ): found 502.0772, calcd. 502.0772.
Compound 25: green crystals, yield: 32%, m.p. 286–287 ꢀC; 1H
4.2.2. The assay of antifungal activity
NMR (400 MHz, CDCl3,
d
ppm): 14.94 (s, 1H), 9.57 (s, 1H), 8.70 (s,
Compounds 1–28 and norfloxacin were screened for their anti-
plant pathogenic fungi activities against R. solani, G. zeae and V. mali
in vitro using the plate growth rate method [22,23].
Each tested compound was dissolved in DMSO, added to the
sterile culture medium (PDA) at 45 ꢀC, mixed to homogeneity and
transferred to sterile Petri dishes to solidify. For primary screenings,
compounds were used at a concentration of 200 mg/L. At the same
time, carbendazim (a commercial fungicide) and one equivalent of
DMSO were used as positive and negative controls, respectively.
1H), 8.14 (d, J ¼ 12.8 Hz, 1H), 7.36–7.42 (m, 1H), 7.28–7.33 (m, 1H),
7.03–7.08 (m,1H), 6.88–6.94 (m,1H), 6.87 (d, J ¼ 6.9 Hz,1H), 4.33 (q,
J ¼ 7.3 Hz, 2H), 4.01 (t, J ¼ 5.0 Hz, 4H), 3.37 (t, J ¼ 5.0 Hz, 4H), 1.60 (t,
J ¼ 7.3 Hz, 3H); HRMS (ESI) for C23H22FN3O5 ([M þ H]þ): found
440.1616, calcd. 440.1616.
Compound 26: brownish yellow crystals, yield: 91%, m.p. 243–
244 ꢀC; 1H NMR (400 MHz, CDCl3,
d
ppm): 14.98 (s,1H), 8.66 (s, 1H),
8.06 (d, J ¼ 12.8 Hz, 1H), 7.36 (d, J ¼ 7.9 Hz, 2H), 7.24 (d, J ¼ 7.9 Hz,

Z. Yu et al. / European Journal of Medicinal Chemistry 44 (2009) 4726–4733
4733
[3] F. Belal, A.A. Al-Majed, A.M. Al-Obaid, Talanta 50 (1999) 765–786.
[4] X. Ma, W.C. Zhou, R. Brun, Bioorg. Med. Chem. Lett. 19 (2009) 986–989.
[5] A. Smith, P.M. Pennefathe, S.B. Kaye, C.A. Hart, Drugs 61 (2001) 747–761.
[6] S.X. Stevens, B.D. Fouraker, H.G. Jensen, Arch. Ophthalmol. 109 (1991)
1737–1743.
[7] E.E.J. Ligtovoet, T. Wickerhoff-Minoggio, J. Antimicrob. Chemother. 16 (1985)
485–490.
[8] R.A. Robson, Int. J. Antimicrob. Agents 2 (1992) 3–10.
Afterwards, a mycelium agar disc (5 mm diameter) of the target
fungi was placed in the center of PDA plates, and then the plates
were incubated at 28 ꢀC in the dark until the target fungi used as
the negative control covered the plate’s surface. Then the diameters
of all fungi in the cultures were measured and the results were
reported as the inhibition of growth following Abbott’s formula: [(%
living in control ꢁ % living in treatment)/% living in control ꢂ 100]
[24]. Each compound was tested three times.
´
˜
˜
´
´
´
[9] J.A. Murillo, A. Alan˜on Molina, A. Munoz de la Pena, I. Duran Meras, A. Jimenez
Giro´n, J. Fluoresc. 17 (2007) 481–491.
[10] B.V. Schmeling, M. Kulla NO.3722 152, Science (1996) 659–660.
[11] J.C. Yang, J.B. Zhang, B.S. Chai, C.L. Liu, Agrochemicals 47 (2008) 16–19.
[12] Q.C. Huang, World Pestic. 26 (2004) 23–27.
Acknowledgments
[13] P. Leroux, Pestic. Sci. 47 (1996) 191–197.
[14] C.W. Shoppee, J. Chem. Soc., Perkin Trans. 1 (1985) 45–52.
[15] R.C. Fuson, J. Wayne Kneisley, E.W. Kaiser, Org. Synth. 24 (1944) 33–36.
[16] S. Rajagopalan, P.V.A. Raman, Org. Synth. 25 (1945) 51–52.
[17] N.V. Hayes, G.E.K. Branch, J. Am. Chem. Soc. 65 (1943) 1555–1556.
[18] C. Frederick, J. Am. Chem. Soc. 53 (1931) 304.
This study was supported by the Hi-Tech Research and Devel-
opment of China (No. 2006AA10A209) and the National Natural
Science Foundation of China (Grant Nos. 30871657, 20572076). We
also thank the Analytical and Testing Center of Sichuan University
for the NMR spectroscopic analysis and the National Institute of
Measurement and Testing Technology for HRMS analysis.
[19] X.Y. Xiong, Y. Zou, Y. Liu, M.G. Zhao, Y.G. Zhong, Chin. J. Med. Chem. 12 (2002)
86–88.
[20] A. Rehman, M.I. Choudhary, W.J. Thomsen, Bioassay Techniques for Drug
Development, Harwood Academic Publishers, Amsterdam, 2001, pp. 16–7.
[21] O. Prakash, R. Kumar, R. Sehrawat, Eur. J. Med. Chem. 44 (2009) 1763–1767.
[22] W. Huang, G.F. Yang, Bioorg. Med. Chem. 14 (2006) 8280–8285.
[23] Q. Chen, X.L. Zhu, L.L. Jiang, Z.M. Liu, G.F. Yang, Eur. J. Med. Chem. 43 (2008)
595–603.
References
[1] P.C. Appelbaum, P.A. Hunter, Int. J. Antimicrob. Agents 16 (2000) 5–15.
[2] G.Y. Lescher, E.D. Froelich, M.D. Gruet, J.H. Bailey, R.P. Brundage, J. Med. Pharm.
Chem. 5 (1962) 1063–1068.
[24] Y. Abou-Jawdah, H. Sobh, A. Salameh, J. Agric. Food Chem. 50 (2002) 3208–
3213.