New 7-[4-(4-(un)substituted)piperazine-1-carbonyl]-piperazin-1-yl] derivatives of fluoroquinolone: Synthesis and antimicrobial evaluation

Article abstract of DOI:10.3390/molecules18077557

Fluoroquinolones have been a class of important synthetic antimicrobial agents broadly and effectively used in clinic for infectious diseases. In this study, the synthesis of a range of fluoroquinolone derivatives with 4-(carbopiperazin-1-yl)piperazinyl moieties at the C7 position and their inhibition of bacterial pathogens commonly disseminated in hospital environment were described. The results indicated that a 7-[4-(4-(benzoyl)carbopiperazin-1- yl)]piperazinyl derivative 5h and two 7-[4-(4- (benzenesulfonyl)carbopiperazin- 1-yl)]piperazinyl derivatives 5k and 5l showed more promising growth inhibition of ciprofloxacin-resistant P. aeruginosa (CRPA) with MIC values as low as 16 μg/mL which is 16-fold more potent than ciprofloxacin, while most of other derivatives maintained potency against methicillin-resistant Staphylococcus aureus (MRSA).

Full text of DOI:10.3390/molecules18077557

Molecules 2013, 18, 7557-7569; doi:10.3390/molecules18077557
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
New 7-[4-(4-(un)Substituted)piperazine-1-carbonyl]-
piperazin-1-yl] Derivatives of Fluoroquinolone: Synthesis and
Antimicrobial Evaluation
Po-Ting Chen 1, Wen-Po Lin 2, An-Rong Lee 1 and Ming-Kuan Hu 1,*
1
School of Pharmacy, National Defense Medical Center, 161 Min-Chuan East Road, Section 6,
Taipei 114, Taiwan; E-Mails: wawaspider@yahoo.com.tw (P.-T.C.);
lar@ndmctsgh.edu.tw (A.-R.L.)
2
Department of Microbiology and Immunology, National Defense Medical Center,
161 Min-Chuan East Road, Section 6, Taipei 114, Taiwan; E-Mail: ae3843@ndmctsgh.edu.tw
* Author to whom correspondence should be addressed: E-Mail: hmk@ndmctsgh.edu.tw;
Tel.: +886-2-8792-6030; Fax: +886-2-8792-3169.
Received: 5 June 2013; in revised form: 26 June 2013 / Accepted: 26 June 2013 /
Published: 27 June 2013
Abstract: Fluoroquinolones have been a class of important synthetic antimicrobial
agents broadly and effectively used in clinic for infectious diseases. In this study, the
synthesis of a range of fluoroquinolone derivatives with 4-(carbopiperazin-1-yl)piperazinyl
moieties at the C7 position and their inhibition of bacterial pathogens commonly
disseminated in hospital environment were described. The results indicated that a
7-[4-(4-(benzoyl)carbopiperazin-1-yl)]piperazinyl derivative 5h and two 7-[4-(4-
(benzenesulfonyl)carbopiperazin-1-yl)]piperazinyl derivatives 5k and 5l showed more
promising growth inhibition of ciprofloxacin-resistant P. aeruginosa (CRPA) with MIC
values as low as 16 μg/mL which is 16-fold more potent than ciprofloxacin, while most of
other derivatives maintained potency against methicillin-resistant Staphylococcus aureus
(MRSA).
Keywords: antimicrobial agents; ciprofloxacin; ciprofloxacin-resistant Pseudomonas
aeruginosa; methicillin-resistant Staphylococcus aureus

Molecules 2013, 18
7558
1. Introduction
Despite advances in drug development among pharmaceutical companies, it has proved relatively
difficult to achieve breakthroughs in the discovery of new antimicrobial agents with new targets.
Currently, one of the practical approaches to these challenges is direct manipulation of the structure of
exsiting antibacterial agents to improve antimicrobial potency and efficacy. Fluoroquinolones are a
type of important synthetic antibacterial agents used broadly and effectively in clinic for infectious
diseases [1,2]. They possess excellent activities against Gram-negative and relatively moderate against
Gram-positive bacteria. Although certain adverse events still remain during the use of fluoroquinolones
for therapies, fluoroquinolones are still one of the major antimicrobial agents with many advances for
clinical use, and much continuous effort has gone into the structural modification of the fluoroquinolone
framework to provide newer congeners with improved potency and efficacy to conquer the
fluoroquinolone-resistant pathogens commonly encountered in the hospital environment [3–5].
Based on the struture-activity relationships of fluoroquinolones that have been well addressed,
substituents at the 7-position of the fluoroquinolone skeleton greatly influence their antimicrobial
spectrum, potency, and even safety [6]. With the piperazinyl moieties in ciprofloxacin, levofloxacin, and
sparfloxacin, the basicity and lipophilicity of each moiety dominate their ability to penetrate into cell
walls and widen their activity spectrum. From the structure-activity point of view, the variety of
piperazinyl substituents at the 7-position of fluoroquinolone agents has disclosed the looseness of
the binding pocket of the targeted type II topoisomerases and established the groundwork for
further modification toward new fluoroquinolone agents useful against certain clinically resistant
organisms [7–10].
In our research program on antimicrobial agents, we recently developed a series of ciprofloxacin
derivatives bearing a 3-carboxamate moiety, which were found to exhibit comparable activities against
certain Gram-negative organisms [11]. In this study, we manipulated the piperazinyl moiety at the
7-position of ciprofloxacin with an additional carbopiperazinyl group and introduced various N-benzoyl
and N-benzenesulfonyl substituents to the carbopiperazinyl moiety to improve the relative antimicrobial
activities against certain resistant species commonly disseminated in the hospital environment.
2. Results and Discussion
2.1. Synthesis
The synthesis of the novel 7-(4-carbopiperazin-1-yl)piperazinyl derivatives of fluoroquinolone was
described in Scheme 1. Ciprofloxacin (2) was economically prepared from condensation of
1-cyclopropyl-7-chloro-6-fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylic acid (1) and piperazine in
dimethylsulfoxide with the assistance of microwave irradiation (100 W, 2 h) [12]. The manipulation of
the piperazinyl moiety at the 7-position of ciprofloxacin with an additional carbopiperazinyl group was
conducted in situ with triphosgene and N-protected piperazine to give 7-(4-carbopiperazin-1-yl)
intermediate 3 in moderate yields. Heterogenous hydrogenation (5% Pd on activated carbon) of 3
followed by aroylation or benzenesulfonation of the resulting free amine led to a range of
7-[4-(4-(aroyl)carbopiperazin-1-yl)]piperazinyl and 7-[4-(4-(benzenesulfonyl)carbopiperazin-1-yl)]-

Molecules 2013, 18
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piperazinyl derivatives 5a–m in good yields (55~85%) after purification by silica gel chromatography.
The chemical structures and log P values of these new ciprofloxacin derivatives are listed in Table 1.
Scheme 1. Synthesis of 7-(4-substituted 4-(carbopiperazin-1-yl))piperazinyl derivatives
of fluoroquinolone.
Reagents and conditions: (i) microwaves (100 W, 2h), dimethylsulfoxide (ii) triphosgene,
triethylamine, N-benzyloxycarbonyl-piperazine (iii) H2(g), 5% Pd/C; (iv) aroyl- or benzenesulfonyl
chlorides, triethylamine, room temperature.
2.2. Inhibition of Bacterial Growth
For the evaluation of the antibacterial activities of these 7-(4-carbopiperazin-1-yl) derivatives of
ciprofloxacin, the minimal inhibitory concentrations (MICs) that prevent visible growth of bacteria
were determined by a standard broth microdilution method [13]. The MIC values of the synthetic
ciprofloxacin derivatives along with the standard drugs tested according to this approach are reported
in Table 2. In the first phase of screening against conventional Gram-negative bacteria, most of the
7-(4-carbopiperazin-1-yl) derivatives exhibited substantial activities, similar to the prevailing
fluoroquinolones, against E. coli, beta-lactamase-producing E. coli (pUC18), and P. aeruginosa, with
MICs of less than 0.016 μg/mL. We distinctively observed the ability of these new derivatives against
a clinically isolated ciprofloxacin-resistant (cipror) strain of P. aeruginosa (CRPA) and found that five
ciprofloxacin derivatives (compounds 5h–l) exhibited improved growth inhibition against CRPA
compared to ciprofloxacin, with MICs in the 16 μg/mL to 64 μg/mL range. When tested on the
Gram-positive B. subtilis, two benzoyl (compounds 5f, 5h) and three benzenesulfonyl derivatives
(compounds 5i–k) displayed moderate in vitro activities, with MICs ranging from 1 μg/mL to 4 μg/mL,
while pyridocarbonyl analogues 5a and 5b and methoxybenzoyl analogues 5d and 5e were apparently

Molecules 2013, 18
7560
inactive (MIC ≥ 256 μg/mL). However, most of the synthesized derivatives showed excellent activities
similar to ciprofloxacin against MRSA (MIC < 0.016 μg/mL). Only four of them (compounds 3, 4, and
5l–m) displayed moderate potency (MIC values, 4 μg/mL~16 μg/mL). These results revealed that the
increased lipophilicity with the additional 4-((4-substituted)carbopiperazin-1-yl) moieties at the
7-position of ciprofloxacin improves the abilities of the fluoroquinolone derivatives to penetrate the
microbial cell wall, thus improving their antimicrobial activities, especially against CRPA and also
maintaining potency on the tested Gram-negative species. Recently, Huang and co-workers reported
that 7-triazolylpiperidinyl fluoroquinolone derivatives showed comparable antibacterial activity
compared to ciprofloxacin [14]. Our series of ciprofloxacin derivatives featuring an additional
carbopiperazinyl moiety at the 7-position exhibited potent antimicrobial activities against certain
clinical isolates of Gram-negative strains.
Table 1. Structure, yield, and lipophilicity of the synthesized ciprofloxacin derivatives.
O
F
CO2H
N
N
N
R
N
N
O
Compound
3
R
yield
log P a
Compound
5g
R
yield
log P a
O
MeO
O
O
55%
2.42
78%
1.99
MeO
OMe
O
H
4
80%
62%
55%
67%
0.08
1.19
0.99
1.97
5h
5i
65%
76%
78%
80%
2.10
1.83
2.33
2.84
MeO
F
O
S
O
O
5a
5b
5c
N
O
O
S
Cl
N
5j
O
O
O
S
F3CO
5k
OMe
O
O
S
MeO
O
F3C
F3C
5d
5e
82%
85%
1.98
2.17
5l
78%
76%
2.61
2.81
O
O
S
O
MeO
MeO
5m
O
O
5f
73%
1.90
Ciprofloxacin
75%
0.50
MeO
a Calculated log P values were obtained from the ALogPS 2.1 program [15].

Molecules 2013, 18
Table 2. In vitro MIC values of ciprofloxacin derivatives against certain G(+)-
7561
and G(−)-strains.
MIC (μg/mL)
Compound
E. coli
P. aeru.
(cipror)
>256
>256
>256
>256
128
E. coli
P. aeru.
MRSA
B. subtilis
(pUC18)
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
0.063
3
4
0.063
0.063
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
64
256
>256
>256
>256
16
64
5a
5b
5c
5d
5e
5f
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
0.063
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
4
>256
>256
128
>256
>256
4
5g
5h
5i
>256
16
>256
4
64
4
5j
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
64
1
5k
5l
16
1
16
>256
16
5m
>256
>256
>256
64
Ciprofloxacin <0.016
Norfloxacin <0.016
<0.016
<0.016
<0.016
0.5
In order to examine whether these derivatives possessed bactericidal activities against these selected
bacterial species, we further measured the minimum bactericidal concentrations (MBCs) based on each
MIC by a dilution method as previously described [16]. The MBC values of these derivatives
according to the method are given in Table 3. Most of the synthesized compounds generally showed
less bactericidal activities against Gram-negative bacteria than the prevailing ciprofloxacin and
norfloxacin, yet two benzenesulfonyl derivatives (compound 5j and 5k) exhibited substantial potency,
with MBCs less than 0.016 μg/mL against E. coli. From the screening on the clinically emerging
β-lactamase-producing E. coli, the MBCs of the derivatives 5a and 5c–f were in the range from 0.016
μg/mL to 0.063 μg/mL. These results indicated that the synthesized derivatives with 4-benzoyl
substituents at the 7-(4-carbopiperazin-1-yl) position showed stronger bactericidal effects against
Gram-negative species than those analogs with benzenesulfonyl moieties. Concerning the screening on
Gram-positive bacterial strains, most derivatives showed only moderate bactericidal activities on B.
subtilis, probably because the unique capsular membrane component of B. subtilis was an obstacle to
the membrane penetration of the tested compounds and resulted in the observed decreased bactericidal
activities. They were also less active than ciprofloxacin against MRSA. The results suggested that in
these derivatives the presence of additional moieties at the 7-position of ciprofloxacin seems not to be
beneficial to their bactericidal abilities against the tested Gram-positive species.

Molecules 2013, 18
Table 3. In vitro MBC values of ciprofloxacin derivatives against certain G(+)-
7562
and G(-)-strains.
MBC (μg/mL)
E. coli
(pUC18)
0.25
0.25
<0.016
1
P. aeru.
E. coli
P. aeru.
MRSA
B. subtilis
(cipror)
>256
>256
>256
>256
256
3
128
128
4
256
>256
128
64
>256
16
4
256
5a
5b
5c
5d
5e
5f
5g
5h
5i
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
128
0.25
1
16
<0.016
0.063
0.063
0.063
0.25
0.25
0.25
1
16
16
>256
>256
256
128
64
4
16
>256
>256
>256
128
16
16
>256
>256
>256
>256
>256
>256
>256
16
16
16
4
5j
5k
5l
<0.016
<0.016
64
>256
>256
>256
4
1
4
1
>256
5m
128
0.063
<0.016
<0.016
>256
0.125
4
>256
>256
>256
>256
0.031
4
16
0.125
0.5
Ciprofloxacin
Norfloxacin
<0.016
<0.016
3. Experimental
3.1. General
All reagents and solvents were commercial materials and were used directly unless otherwise noted.
DMF was dehydrated over 4 Å molecular sieves. Reactions were monitored by thin layer
chromatography using Echo silica gel F254 plates visualized under UV irradiation along with staining
with phosphomolybdic acid/heat, or iodine. Melting points were recorded on a Thomas Hoover
capillary melting point apparatus in open capillary tubes and are uncorrected. NMR spectra were
1
recorded on a Varian Gemini instrument at 300 MHz for H and at 75 MHz for 13C. Fast atom
bombardment mass spectra (FABMS) were acquired on a Finnigan Mat 95S mass spectrometer.
Chromatography refers to flash chromatography on silica gel (silica gel 60, 230–400 mesh ASTM, E.
Merck, Darmstadt, Germany).
3.2. Synthesis
7-(4-[4-(Benzyloxycarbonyl)piperazino]carbopiperazino)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-3-
quinolinecarboxylic acid (3). A solution of N-(benzyloxycarbonyl)piperazine (220 mg, 1 mmol) in
dichloromethane (10 mL) was slowly added to the stirred solution of triphosgene (110 g, 0.37 mmol)
in dichloromethane (2 mL) over a period of 30 min using a syringe pump. After 30 further min of
stirring, a solution of 2 (398 mg, 1.2 mmol) and diisopropylethylamine (DIEA, 0.38 mL, 2.2 mmol) in
DCM/EtOH (120 mL, 4:1) was added in one portion. The reaction mixture was stirred for 2 hours at

Molecules 2013, 18
7563
room temperature. After evaporation of solvent under vacuum, the residue was purified by silica gel
chromatography to give 3 (318 mg, 55%); tlc Rf = 0.30 (DCM/EtOH = 20:1); mp 215–216 °C; UV
1
λmax (DCM/EtOH = 3:2) nm (log ε) 240.4 (9.22); H-NMR (DMSO-d6) δ 8.67 (s, 1H, C2-H), 7.90 (d,
J = 13.2 Hz, 1H, C5-H), 7.56 (d, J = 7.5 Hz, 1H, C8-H), 7.37–7.31 (m, 5H, Ar-H), 5.09 (s, 2H,
-O-CH2-Ar), 3.78–3.82 (m, 1H, cyclopropyl), 3.15–3.40 (m, 16H, piperazinyl), 1.17–1.32 (m, 4H,
cyclopropyl); 13C-NMR (CDCl3) δ 8.1, 35.2, 41.4, 41.5, 44.3, 44.6, 48.1, 49.3, 49.6, 50.3, 69.5, 105.4,
108.4, 111.1, 112.8, 118.8, 127.3, 128.2, 128.8, 129.6, 136.9, 137.8, 146.5, 149.8, 153.4, 161.2, 165.3,
168.6, 176,2; FABMS: m/z 578 [M+H]+.
1-Cyclopropyl-6-fluoro-4-oxo-7-[4-(piperazin-1-yl)carbopiperazino]-1,4-dihydro-3-quinolinecarboxyl
ic acid (4). To a solution of 3 (289 mg, 0.5 mmol) and 10% Pd/C in DCM/EtOH (50 mL, 1:1) was
charged with H2 at 1 atm and stirred at room temperature for 1 h. The catalyst was filtered off through
celite. After evaporation of solvent under vacuum, the residue was recrystallized from DCM/acetone to
give 4 (177 mg, 80%); tlc Rf = 0.10 (DCM/EtOH = 9:1); mp 196–197 °C; UV λmax (DCM/EtOH = 3:2)
1
nm (log ε) 238.8 (9.11); H-NMR (DMSO-d6) δ 9.10 (s, 1H, br, CH2-NH-CH2), 8.77 (s, 1H, C2-H),
7.91 (d, J = 13.2 Hz, 1H, C5-H), 7.55 (d, J = 7.5 Hz, 1H, C8-H), 3.78–3.82 (m, 1H, cyclopropyl),
3.08–3.40 (m, 16H, piperazinyl), 1.16–1.31 (m, 4H, cyclopropyl); 13C-NMR (CDCl3) δ 8.1, 35.2, 40.2,
41.6, 43.6, 44.6, 48.2, 49.2, 49.6, 50.3, 106.2, 108.4, 112.8, 118.8, 137.8, 146.5, 149.8, 153.4, 161.2,
166.6, 176,2; FABMS: m/z 444 [M+H]+.
General procedure for the synthesis of 1-cyclopropyl-6-fluoro-4-oxo-7-(4-[4-(substituted benzoyl or
benzenesulfonyl) piperazino]carbopiperazino)-1,4-dihydro-3-quinolinecarboxylic acids 5a–m.
To a solution of amine 4 (0.67 g, 1.5 mmol) in DCM/EtOH (150 mL, 4:1) was added triethylamine
(0.4 mL, 3.0 mmol) and the appropriate aroyl or benzenesulfonyl halide (1.2 mmol). The mixture was
stirred at room temperature under argon for several hours depending on the completion of the reaction,
which was checked by tlc. After evaporation of solvent under vacuum, the residue was purified by
silica gel chromatography and recrystallized from an appropriate solvent to give the title products.
1-Cyclopropyl-6-fluoro-4-oxo-7-(4-[4-(3-pyridylcarbonyl)piperazino]carbopiperazino)-1,4-dihydro-3-
quinolinecarboxylic acid (5a). Amine 4 (0.67 g, 1.5 mmol) was treated with nicotinyl chloride (0.17 g,
1.2 mmol) to give 5a (0.41 g, 62%) as a white solid; tlc Rf = 0.18 (DCM/EtOH = 20 : 1); mp
219–220 °C; UV λmax (DCM/EtOH = 3:2) nm (log ε): 242.6 (9.20); 1H-NMR (CDCl3) δ 8.63–8.65 (m,
2H, Ar-H), 8.61 (s, 1H, C2-H), 7.84 (d, J = 12.9 Hz, 1H, C5-H), 7.73–7.76 (m, 1H, Ar-H), 7.35–7.39
(m, 1H, Ar-H), 7.32 (d, J = 7.2 Hz, 1H, C8-H), 3.76–3.80 (m, 1H, cyclopropyl), 3.32–3.53 (m, 16H,
13
piperazinyl), 1.17–1.40 (m, 4H, cyclopropyl); C-NMR (CDCl3) δ 8.1, 35.3, 41.3, 41.9, 44.4, 44.8,
48.8, 49.2, 49.6, 50.1, 105.1, 107.2, 109.4, 112.0, 112.3, 119.8, 127.1, 138.0, 144.6, 145.0, 147.2,
149.8, 153.6, 160.6, 166.0, 168.8, 176,6; FABMS: m/z 549 [M+H]+; HRFABMS: calcd for
C28H30FN6O5 [M+H]+ 549.2264, found 549.2268.
1-Cyclopropyl-6-fluoro-4-oxo-7-(4-[4-(4-pyridylcarbonyl)piperazino]carbopiperazino)-1,4-dihydro-3-
quinolinecarboxylic acid (5b). Amine 4 (0.67 g, 1.5 mmol) was treated with isonicotinyl chloride
(0.17 g, 1.2 mmol) to give 5b (0.36 g, 55%) as a white solid; tlc Rf = 0.18 (DCM/EtOH = 20:1); mp

Molecules 2013, 18
7564
217–218 °C; UV λmax (DCM/EtOH = 3:2) nm (log ε): 239.4 (9.20); 1H-NMR (CDCl3) δ 8.74–8.65 (m,
2H, Ar-H), 8.67 (s, 1H, C2-H), 7.91 (d, J = 12.9 Hz, 1H, C5-H), 7.34 (d, J = 7.2 Hz, 1H, C8-H),
7.30–7.35 (m, 2H, Ar-H), 3.78–3.82 (m, 1H, cyclopropyl), 3.32–3.54 (m, 16H, piperazinyl), 1.18–1.40
13
(m, 4H, cyclopropyl); C-NMR (CDCl3) δ 8.2, 35.3, 41.6, 41.9, 44.3, 44.5, 47.9, 49.3, 49.6, 50.4,
105.8, 106.9, 108.7, 111.3, 112.2, 118.8, 127.3, 137.8, 144.2, 146.5, 147.4, 149.8, 153.4, 161.2, 165.3,
168.2, 176,3; FABMS: m/z 549 [M+H]+; HRFABMS: calcd for C28H30FN6O5 [M+H]+ 549.2264, found
549.2262.
1-Cyclopropyl-6-fluoro-7-(4-[4-(2-methoxybenzoyl)piperazino]carbopiperazino)-4-oxo-1,4-dihydro-3-
quinolinecarboxylic acid (5c). Amine 4 (0.67 g, 1.5 mmol) was treated with 2-methoxybenzoyl
chloride (0.2 g, 1.2 mmol) to give 5c (0.46 g, 67%) as a white solid; tlc Rf = 0.33 (DCM/EtOH = 20:1);
1
mp 240–241 °C; UV λmax (DCM/EtOH = 3:2) nm (log ε): 241.8 (9.27); H-NMR (CDCl3) δ 8.66 (s,
1H, C2-H), 7.90 (d, J = 12.9 Hz, 1H, C5-H), 7.34 (d, J = 7.2 Hz, 1H, C8-H), 6.89–7.38 (m, 4H, Ar-H),
3.82 (s, 3H, -OCH3), 3.78–3.85 (m, 1H, cyclopropyl), 3.26–3.53 (m, 16H, piperazinyl), 1.17–1.39 (m,
13
4H, cyclopropyl); C NMR (CDCl3) δ 8.2, 35.3, 41.6, 41.9, 44.3, 44.5, 47.9, 49.3, 49.6, 50.4, 54.2,
107.1, 108.7, 111.3, 112.2, 114.6, 118.8, 120.8, 127.3, 128.2, 129.2, 137.8, 146.5, 149.8, 153.4, 160.2,
161.2, 165.3, 168.6, 176,2; FABMS: m/z 578 [M+H]+; HRFABMS: calcd for C30H33FN5O6 [M+H]+
578.2417, found 578.2420.
1-Cyclopropyl-6-fluoro-7-(4-[4-(3-methoxybenzoyl)piperazino]carbopiperazino)-4-oxo-1,4-dihydro-
3-quinolinecarboxylic acid (5d). Amine 4 (0.67 g, 1.5 mmol) was treated with 3-methoxybenzoyl
chloride (0.2 g, 1.2 mmol) to give 5d (0.57 g, 82%) as a white solid; tlc Rf = 0.34 (DCM/EtOH = 20:1);
1
mp 236–237 °C; UV λmax (DCM/EtOH = 3:2) nm (log ε): 240.7 (9.27); H-NMR (CDCl3) δ 8.70 (s,
1H, C2-H), 7.95 (d, J = 12.9 Hz, 1H, C5-H), 7.35 (d, J = 7.2 Hz, 1H, C8-H), 7.29–7.35 (m, 1H, Ar-H),
6.92–6.98 (m, 3H, Ar-H), 3.82 (s, 3H, -OCH3), 3.78–3.82 (m, 1H, cyclopropyl), 3.26–3.54 (m, 16H,
piperazinyl), 1.1–1.40 (m, 4H, cyclopropyl); 13C-NMR (CDCl3) δ 8.2, 35.1, 41.4, 41.9, 44.3, 44.5, 47.9,
49.2, 49.6, 50.3, 53.7, 107.2, 107.9, 111.5, 112.2, 114.6, 118.8, 127.3, 128.2, 129.2, 137.8, 146.4, 149.8,
153.4, 160.2, 161.2, 165.6, 168.4, 176,2; FABMS: m/z 578 [M+H]+, HRFABMS: calcd for
C30H33FN5O6 [M+H]+ 578.2417, found 578.2413.
1-Cyclopropyl-6-fluoro-7-(4-[4-(4-methoxybenzoyl)piperazino]carbopiperazino)-4-oxo-1,4-dihydro-3-
quinolinecarboxylic acid (5e). Amine 4 (0.67 g, 1.5 mmol) was treated with 4-methoxybenzoyl
chloride (0.2 g, 1.2 mmol) to give 5e (0.59 g, 85%) as a white solid; tlc Rf = 0.34 (DCM/EtOH = 20:1);
1
mp 224–225 °C; UV λmax (DCM/EtOH = 3:2) nm (log ε): 239.0 (9.23); H-NMR (CDCl3) δ 8.60 (s,
1H, C2-H), 7.83 (d, J = 12.9 Hz, 1H, C5-H), 7.35 (dd, J = 6.9, 2.1 Hz, 2H, Ar-H), 7.32 (d, J = 7.2 Hz,
1H, C8-H), 6.88 (dd, J = 6.9, 2.1 Hz, 2H, Ar-H), 3.80 (s, 3H, -OCH3), 3.78–3.82 (m, 1H, cyclopropyl),
3.30–3.62 (m, 16H, piperazinyl), 1.16–1.40 (m, 4H, cyclopropyl); 13C-NMR (CDCl3) δ 8.2, 35.2, 41.7,
41.2, 44.6, 44.9, 47.3, 49.2, 49.6, 50.3, 53.2, 107.3, 107.9, 110.8, 111.6, 114.7, 118.8, 127.5, 128.2,
129.2, 137.8, 146.4, 148.2, 153.4, 160.2, 161.6, 165.8, 168.3, 176,2; FABMS: m/z 578 [M+H]+,
HRFABMS: calcd for C30H33FN5O6 [M+H]+ 578.2417, found 578.2422.
1-Cyclopropyl-7-(4-[4-(3,5-dimethoxybenzoyl)piperazino]carbopiperazino)-6-fluoro-4-oxo-1,4-dihydr
o-3-quinolinecarboxylic acid (5f). Amine 4 (0.67 g, 1.5 mmol) was treated with 3,5-dimethoxybenzoyl

Molecules 2013, 18
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chloride (0.24 g, 1.2 mmol) to give 5f (0.53 g, 73%) as a white solid; tlc Rf = 0.38 (DCM/EtOH =
20:1); mp 233–234 °C; UV λmax (DCM/EtOH = 3:2) nm (log ε): 240.4 (9.25); 1H-NMR (CDCl3) δ 8.61
(s, 1H, C2-H), 7.84 (d, J = 12.9 Hz, 1H, C5-H), 7.32 (d, J = 6.9 Hz, 1H, C8-H), 6.46–6.48 (m, 3H,
Ar-H), 3.77 (s, 6H, -OCH3), 3.78–3.82 (m, 1H, cyclopropyl), 3.30–3.65 (m, 16H, piperazinyl),
1.17–1.39 (m, 4H, cyclopropyl); 13C-NMR (CDCl3) δ 8.1, 35.2, 41.6, 41.2, 44.6, 44.8, 47.3, 49.5, 49.2,
50.3, 53.4, 53.8, 106.8, 107.9, 110.8, 112.4, 114.7, 118.9, 127.5, 128.1, 137.8, 146.4, 148.2, 153.4,
159.8, 160.4, 161.4, 165.8, 168.2, 176,2; FABMS: m/z 608 [M+H]+, HRFABMS: calcd for
C31H35FN5O7 [M+H]+ 608.2523, found 608.2527.
1-Cyclopropyl-7-(4-[4-(3,4-dimethoxybenzoyl)piperazino]carbopiperazino)-6-fluoro-4-oxo-1,4-dihydr
o-3-quinolinecarboxylic acid (5g). Amine 4 (0.67 g, 1.5 mmol) was treated with 3,4-dimethoxybenzoyl
chloride (0.24 g, 1.2 mmol) to give 5g (0.57 g, 78%) as a white solid; tlc Rf = 0.38 (DCM/EtOH =
20:1); mp 257–258 °C; UV λmax (DCM/EtOH = 3:2) nm (log ε): 243.8 (9.28); 1H-NMR (CDCl3) δ 8.72
(s, 1H, C2-H), δ 7.98 (d, J = 12.9 Hz, 1H, C5-H), δ 7.36 (d, J = 7.2 Hz, 1H, C8-H), δ 6.99 (d, J = 1.8 Hz,
1H, Ar-H), δ 6.98 (dd, J = 9.9 Hz, 1.8 Hz, 1H, Ar-H), 6.87(d, J = 9.9 Hz, 1H, Ar-H), 3.91 (s, 3H,
-OCH3), 3.90 (s, 3H, -OCH3), 3.66–3.68 (m, 1H, cyclopropyl), 3.33–3.55 (m, 16H, piperazinyl),
1.19–1.40 (m, 4H, cyclopropyl); 13C-NMR (CDCl3) δ 8.2, 35.3, 41.2, 41.2, 44.8, 44.8, 48.1, 49.5, 49.2,
50.3, 53.4, 54.2, 106.8, 107.9, 110.8, 112.4, 114.7, 119.1, 128.5, 128.1, 137.2, 146.3, 149.5, 153.4,
159.1, 160.6, 161.2, 165.8, 168.2, 176,4; FAB MS: m/z 608 [M+H]+; HRFABMS: calcd for
C31H35FN5O7 [M+H]+ 608.2523, found 608.2525.
1-Cyclopropyl-7-(4-[4-(2,4-dimethoxybenzoyl)piperazino]carbopiperazino)-6-fluoro-4-oxo-1,4-dihydro-
3-quinolinecarboxylic acid (5h). Amine 4 (0.67 g, 1.5 mmol) was treated with 2,4-dimethoxybenzoyl
chloride (0.24 g, 1.2 mmol) to give 5h (0.47 g, 65%) as a white solid; tlc Rf = 0.47 (DCM/EtOH =
1
20:1); mp 244–245 °C; UV λmax (DCM/EtOH = 3 : 2) nm (log ε): 240.4 (9.29); H-NMR (CDCl3) δ
8.64 (s, 1H, C2-H), 7.88 (d, J = 12.9 Hz, 1H, C5-H), 7.33 (d, J = 7.2 Hz, 1H, C8-H), 7.17 (d, J = 8.4 Hz,
1H, Ar-H), 6.50 (dd, J = 8.4 Hz, 2.1 Hz, 1H, Ar-H), 6.43 (d, J = 2.1 Hz, 1H, Ar-H), 3.80 (s, 3H,
-OCH3), 3.79 (s, 3H, -OCH3), 3.78–3.82 (m, 1H, cyclopropyl), 3.25–3.52 (m, 16H, piperaznyl),
1.18–1.39 (m, 4H, cyclopropyl); 13C-NMR (CDCl3) δ 8.2, 35.3, 41.2, 41.1, 44.6, 44.8, 47.3, 49.4, 49.2,
50.3, 53.4, 53.7, 106.8, 107.9, 110.8, 112.4, 114.7, 118.9, 127.2, 128.5, 137.8, 146.4, 148.2, 153.4,
159.3, 160.3, 161.2, 165.7, 168.1, 176,3; FABMS: m/z 608 [M+H]+; HRFABMS: calcd for
C31H35FN5O7 [M+H]+ 608.2523, found 608.2519.
1-Cyclopropyl-6-fluoro-7-(4-[4-(4-fluorobenzenesulfonyl)piperazino]carbopiperazino)-4-oxo-1,4-dihy
dro-3-quinolinecarboxylic acid (5i). Amine 4 (0.67g, 1.5 mmol) was treated with 4-fluoro-
benzenesulfonyl chloride (0.23 g, 1.2 mmol) to give 5i (0.55 g, 76%) as a white solid; tlc Rf = 0.42
(DCM/EtOH = 20:1); mp 268–269 °C; UV λmax (DCM/EtOH = 3:2) nm (log ε): 241.1 (9.24); 1H-NMR
(CDCl3) δ 8.76 (s, 1H, C2-H), 8.02 (d, J = 12.9 Hz, 1H, C5-H), 7.77 (dt, J = 4.8 Hz, 2.1 Hz, 2H, Ar-H),
7.34 (d, J = 7.2 Hz, 1H, C8-H), 7.25 (dd, J = 4.8 Hz, 2.1 Hz, 2H, Ar-H), 3.02–3.51 (m, 17H,
13
piperazinyl, cyclopropyl), 1.20–1.40 (m, 4H, cyclopropyl); C-NMR (CDCl3) δ 8.1, 35.3, 41.4, 41.9,
44.3, 44.5, 47.9, 49.3, 49.6, 50.4, 107.1, 108.7, 111.3, 112.2, 112.6, 114.4, 127.3, 128.2, 137.8, 138.6,

Molecules 2013, 18
7566
146.5, 149.8, 153.4, 158.2, 161.9, 165.8, 176,5; FABMS: m/z 602 [M+H]+; HRFABMS: calcd for
C28H30F2N5O6S [M+H]+ 602.1887, found 602.1885.
7-(4-[4-(4-Chlorobenzenesulfonyl)piperazino]carbopiperazino)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihy
dro-3-quinolinecarboxylic acid (5j). Amine 4 (0.67 g, 1.5 mmol) was treated with
4-chlorobenzenesulfonyl chloride (0.25 g, 1.2 mmol) to give 5j (0.58 g, 78%) as a white solid; tlc Rf =
0.42 (DCM/EtOH = 20:1); mp 274–275 °C; UV λmax (DCM/EtOH = 3:2) nm (log ε): 242.5 (9.29);
1H-NMR (CDCl3) δ 8.74 (s, 1H, C2-H), 8.00 (d, J = 12.9 Hz, 1H, C5-H), 7.69 (d, J = 8.4 Hz, 2H, Ar-H),
7.53 (d, J = 8.4 Hz, 2H, Ar-H), 7.33 (d, J = 7.2 Hz, 1H, C8-H), 3.04–3.47 (m, 17H, piperazinyl,
13
cyclopropyl), 1.20–1.40 (m, 4H, cyclopropyl); C-NMR (CDCl3) δ 8.1, 35.3, 41.4, 41.9, 44.3, 44.5,
47.9, 49.3, 49.6, 50.4, 107.1, 108.7, 111.3, 112.6, 127.3, 128.2, 128.9, 129.3, 135.6, 137.8, 138.6,
146.5, 149.8, 153.4, 161.9, 165.8, 176,4; FABMS: m/z 618 [M+H]+; HRFABMS: calcd for
C28H30FClN5O6S [M+H]+ 618.1592, found 618.1589.
1-Cyclopropyl-6-fluoro-4-oxo-7-(4-[4-(4-trifluoromethoxybenzenesulfonyl)piperazino]carbopiperazin
o)-1,4-dihydro-3-quinolinecarboxylic acid (5k). Amine 4 (0.67 g, 1.5 mmol) was treated with
4-trifluoromethoxybenzenesulfonyl chloride (0.31 g, 1.2 mmol) to give 5k (0.64 g, 80%) as a white
solid; tlc Rf = 0.40 (DCM/EtOH = 20:1); mp 248–249 °C; UV λmax (DCM/EtOH = 3 : 2) nm (log ε):
239.2 (9.33); 1H-NMR (CDCl3) δ 8.77 (s, 1H, C2-H), 8.03 (d, J = 12.9 Hz, 1H, C5-H), 7.81 (d, J = 8.7
Hz, 2H, Ar-H), 7.38 (d, J = 8.7 Hz, 2H, Ar-H), 7.35 (d, J = 7.2 Hz, 1H, C8-H), 3.06–3.46 (m, 17H,
13
piperazinyl, cyclopropyl), 1.19–1.40 (m, 4H, cyclopropyl); C-NMR (CDCl3) δ 8.2, 35.2, 41.6, 41.9,
44.2, 44.5, 47.9, 49.8, 49.5, 50.4, 107.2, 108.7, 111.3, 112.6, 114.7, 119.3, 127.3, 128.2, 137.8, 138.6,
146.5, 149.2, 151.8, 158.2, 160.4, 161.5, 165.4, 176,2; FABMS: m/z 668 [M+H]+; HRFABMS: calcd
for C29H30F4N5O7S [M+H]+ 668.1804, found 668.1802.
1-Cyclopropyl-6-fluoro-4-oxo-7-(4-[4-(4-trifluoromethylbenzenesulfonyl)piperazino]carbopiperazino)
-1,4-dihydro-3-quinolinecarboxylic acid (5l). Amine 4 (0.67 g, 1.5 mmol) was treated with
4-trifluoromethylbenzenesulfonyl chloride (0.29 g, 1.2 mmol) to give 5l (0.61 g, 78%); tlc Rf = 0.40
(DCM/EtOH = 20:1); mp 167–168 °C; UV λmax (DCM/EtOH = 3:2) nm (log ε): 241.1 (9.28); 1H-NMR
(DMSO-d6 with a drop of D2O) δ 8.64 (s, 1H, C2-H), 8.04 (d, J = 8.4 Hz, 2H, Ar-H), 7.96 (d, J = 8.4
Hz, 2H, Ar-H), 7.87 (d, J = 13.2 Hz, 1H, C5-H), 7.51 (d, J = 7.5 Hz, 1H, C8-H), 3.74–3.80 (m, 1H,
13
cyclopropyl), 2.96–3.30 (m, 16H, piperazinyl), 1.12–1.30 (m, 4H, cyclopropyl); C-NMR (CDCl3) δ
8.2, 35.1, 41.6, 41.9, 44.3, 44.5, 47.9, 49.3, 49.6, 50.4, 107.3, 108.7, 111.3, 112.2, 124.5, 125.6, 126.2,
127.3, 128.2, 130.8, 137.8, 138.6, 145.7, 149.2, 158.7, 161.3, 165.4, 176,2; FABMS: m/z 652 [M+H]+;
HRFABMS: calcd for C29H30F4N5O6S [M+H]+ 652.1855, found 652.1853.
1-Cyclopropyl-6-fluoro-4-oxo-7-(4-[4-(3-trifluoromethylbenzenesulfonyl)piperazino]carbopiperazino)
-1,4-dihydro-3-quinolinecarboxylic acid (5m). Amine 4 (0.67 g, 1.5 mmol) was treated with
3-trifluoromethylbenzenesulfonyl chloride (0.29 g, 1.2 mmol) to give 5m (0.59 g, 76%) as a white
solid; tlc Rf = 0.37 (DCM/EtOH = 20:1); mp 267–268 °C; UV λmax (DCM/EtOH = 3:2) nm (log ε):
1
239.2 (9.32); H-NMR (DMSO-d6 with a drop of D2O) δ 8.64 (s, 1H, C2-H), 8.15 (d, J = 7.5 Hz, 1H,
Ar-H), δ 8.07 (d, J = 7.8 Hz, 1H, Ar-H), δ 7.97 (s, 1H, Ar-H), δ 7.92 (dd, J = 7.8 Hz, 7.5 Hz, 1H, Ar-H),
7.89 (d, J = 13.2 Hz, 1H, C5-H), 7.52 (d, J = 7.5 Hz, 1H, C8-H), 3.76 ~ δ 3.81 (m, 1H, cyclopropyl),

Molecules 2013, 18
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2.97–3.30 (m, 16H, piperazinyl), 1.13–1.30 (m, 4H, cyclopropyl); 13C-NMR (CDCl3) δ 8.1, 35.4, 41.2,
41.9, 44.4, 44.9, 47.5, 49.3, 49.6, 50.4, 107.3, 108.1, 111.5, 112.2, 124.1, 125.2, 126.6, 127.4, 128.1,
130.2, 137.8, 138.6, 145.1, 149.6, 158.7, 161.6, 165.2, 176,2; FABMS: m/z 652 [M+H]+; HRFABMS:
calcd for C29H30F4N5O6S [M+H]+ 652.1855, found 652.1852.
3.3. Antimicrobial Susceptibility Testing
Escherichia coli BCRC 13084 and Pseudomonas aeruginosa ATCC 27853 were obtained from the
Culture Collection and Research Center (CCRC), Hsin-Chu, Taiwan, R.O.C. Clinical isolates of
Bacillus subtilis, methicillin-resistant Staphylococcus aureus (MRSA) and ciprofloxacin-resistant
(cipror) Pseudomonas aeruginosa (CRPA) were obtained from the Culture Collection of Tri-Service
General Hospital, Taipei, Taiwan. Escherichia coli JM109 harboring plasmid pUC 18 (E. coli/pUC18)
was kindly provided by Department of Microbiology and Immunology, National Defense Medical
Center, Taiwan and used as a beta-lactamase-producing clone. All bacteria were stored in Luria-Bertani
broth (Difco Laboratories, Detroit, MI, USA) with 15% glycerol (vol/vol) at −80 °C.
To determine the efficacies of minimum inhibitory and bactericidal concentrations (MICs and
MBCs) of the synthetic derivatives of ciprofloxacin, a wide range of Gram-negative (E. coli,
E. coli/pUC18, and Pseudomonas aeruginosa) and Gram-positive (MRSA and Bacillus subtilis)
bacterial species were used by the broth microdilution technique as described by the Clinical and
Laboratory Standards Institute (CLSI) methodology [14]. In addition to the synthesized compounds, the
prevailing antibiotics, including norfloxacin and ciprofloxacin (Sigma Chemical Co., St. Louis, MO,
USA) were included for comparison. Serial two-fold dilutions, ranging from 0.016 to 256 μg/mL, for
each antibiotic in Mueller-Hinton broth (Difco) were prepared in 96-well flat-bottom polystyrene
microtiter plates. Each compound for each organism with an inoculum size of 2 × 105 CFUs was carried
out and cultures were incubated at 37 °C for 24 h. The MICs were recorded as the lowest concentration
that produced inhibition of visible growth after overnight incubation. Each experiment was performed
independently three times. MBCs were obtained by sampling ten-microliter cultures from each well and
streaking onto the surface of Mueller-Hinton agar plates. After overnight incubation at 37 °C, colonies
were counted and the MBCs, defined as the concentration at which did not show any bacterial growth
after incubation during MIC assay, were determined [15]. Each experiment was also performed
independently three times.
4. Conclusions
Modifications to the structure of ciprofloxacin at the 7-position has provided 7-(4-carbo-
piperazin-4-yl) derivatives with improved antimicrobial activities compared to the prevailing
ciprofloxacin. 7-[4-Carbopiperazin-4-(3,5-dimethoxybenzoyl)-yl] derivative 5h and 7-[4-carbo-
piperazin-4-(4-trifluoromethoxybenzenesulfonyl)-yl] derivative 5k showed an impressive selective
potency against a clinic isolate of CRPA and also maintained activities against MRSA, demonstrating
that the delicate manipulation at the 7-position of the fluoroquinolone framework can still be a suitable
ways to obtain new, broad spectrum fluoroquinolones, against especially the Gram-negative pathogens
and certain drug-resistant strains.

Molecules 2013, 18
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Acknowledgements
This work was supported by the National Defense Medical Center (DOD 98–11-03) and Minister of
Economic Affair (99-EC-17-A-20-S1-028) of the Republic of China. High-resolution mass spectra
analyses performed by the National Science Council Regional Instruments Center at National Taiwan
University and National Chiao Tung University are gratefully acknowledged.
Conflicts of Interest
The authors declare no conflict of interest.
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© 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).