7-((4-Substituted)piperazin-1-yl) derivatives of ciprofloxacin: Synthesis and in vitro biological evaluation as potential antitumor agents

Article abstract of DOI:10.1016/j.bmc.2009.06.053

Ciprofloxacin (CP), an antibiotic has been shown to have antiproliferative and apoptotic activities in several cancer cell lines. Moreover, several reports have highlighted the interest of increasing the lipophilicity to improve the antitumor efficacy. Th

Full text of DOI:10.1016/j.bmc.2009.06.053

Bioorganic & Medicinal Chemistry 17 (2009) 5396–5407
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
7-((4-Substituted)piperazin-1-yl) derivatives of ciprofloxacin: Synthesis
and in vitro biological evaluation as potential antitumor agents
a
b
c
b
Joëlle Azéma a, , Brigitte Guidetti , Janique Dewelle , Benjamin Le Calve , Tatjana Mijatovic ,
*
Alexander Korolyov a, Julie Vaysse a, Myriam Malet-Martino a, Robert Martino a, Robert Kiss c
a Université de Toulouse, UPS, Laboratoire de Synthèse et Physico-Chimie de Molécules d’Intérêt Biologique (SPCMIB), Groupe de RMN et Synthèse Biomédicales,
118 route de Narbonne, 31062 Toulouse cedex 9, France
b Unibioscreen SA, 40 Avenue Joseph Wybran, 1070 Bruxelles, Belgium
c Laboratoire de Toxicologie, Institut de Pharmacie, Université Libre de Bruxelles, Bruxelles, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Ciprofloxacin (CP), an antibiotic has been shown to have antiproliferative and apoptotic activities in sev-
eral cancer cell lines. Moreover, several reports have highlighted the interest of increasing the lipophil-
icity to improve the antitumor efficacy. These studies have led us to synthesize new CP derivatives of
various lipophilicities and to evaluate their activity in five human cancer cell lines. With an easy and
cost-efficient procedure, 31 7-((4-substituted)piperazin-1-yl) derivatives of CP were prepared that dis-
Received 26 March 2009
Revised 19 June 2009
Accepted 22 June 2009
Available online 27 June 2009
played IC50 values ranging from
lM to mM concentrations and are non-toxic in vivo in healthy mice
Keywords:
Fluoroquinolones
Lipophilic ciprofloxacin derivatives
In vitro biological evaluation
Cancer cell lines
as shown by their maximal tolerated dose (MTD) indices >80 mg/kg. Several derivatives displayed higher
in vitro antitumor activity than parent CP however this was not dependent on the lipophilicity of the sub-
stituent. Among all synthesized derivatives, the most potent were 2 and 6h whose IC50 values were
610
l
M in three (derivative 2) or four (derivative 6h) cancer cell lines.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
involvement of different cell cycle molecules.10 The fact that deriv-
atives of the FQ class of drugs showed topoisomerase II inhibitory
activity has provided a rationale for quinolone-based drug design
in the search for novel anticancer agents.
Ciprofloxacin (CP, 1; Fig. 1), a commonly used broad-spectrum
fluoroquinolone (FQ) antibiotic with low side effects, has been
shown to have antiproliferative and apoptotic activities in several
cancer cell lines.1–6 Several other FQ derivatives including ofloxa-
cin, levofloxacin, and fleroxacin have also been shown to inhibit
the growth of transitional cell bladder carcinoma cell lines.4,5,7
All FQs induced nearly similar types of morphological alterations,
that is, some cells became rounded, detached and showed cell
membrane blebbing, a typical morphological change indicating ini-
tiation of apoptotic processes.6 Induction of apoptosis also resulted
from CP treatment of human transitional cells of bladder, colorec-
tal, or prostate carcinoma.1–3 In a cell free system, Hussy et al.8
demonstrated the inhibitory activity of several FQs against mam-
malian DNA topoisomerases I and II and DNA polymerase and
reported that CP was the most potent FQ inhibitor of these
enzymes. The mechanism by which FQs exert their growth inhibi-
tory effect and lead to cell death is not fully understood but it has
been suggested that these events may be mediated through intrin-
sic apoptotic pathway9 or through induction of cell cycle arrest by
Moreover, several reports have provided evidence in support of
increasing the lipophilicity of compounds to improve their antitu-
mor efficacy. Increasing lipophilic substituents at C-7 of campto-
thecin led to the discovery of gimatecan that is currently in a
Phase II clinical trial.11 Sodium butyrate and the more lipophilic
valproic acid are being investigated in Phase I and II clinical trials
as histone deacetylase inhibitors.12 A positive correlation between
lipophilicity, in vitro antitumor activity and rate of drug uptake
was observed with platinum(II) complexes tested on lung carci-
noma and leukemia cell lines.13 The antiproliferative activity of
bis-quinolinium choline kinase inhibitors against the HT-29 colon
cancer cell line has also been improved with a higher lipophilicity
of the substituents.14
We thus initiated a screening program to search for new FQ
derivatives as potential antitumor agents. In the current study,
we report the synthesis of new derivatives of CP and their antipro-
liferative properties on five human cancer cell lines. First, 7-
(substituted-piperazin-1-yl) derivatives of CP with non bulky
substituents were synthesized, that is, the 7-(4-(2-chloroace-
tyl)piperazin-1-yl) 2 and the 7-(4-(tert-butoxycarbonyl)piperazin-
1-yl) 3 derivatives of CP (Scheme 1). As these modifications led
to enhanced in vitro antitumor activity compared to CP, we chose
Abbreviations: CP, ciprofloxacine; FQ, fluoroquinolone; MTD, maximum toler-
ated dose.
* Corresponding author. Tel.: +33 5 61556107; fax: +33 5 61557625.
E-mail address: azema@chimie.ups-tlse.fr (J. Azéma).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.06.053

J. Azéma et al. / Bioorg. Med. Chem. 17 (2009) 5396–5407
5397
Figure 1. Chemical structures of ciprofloxacine, 6,8-difluoroquinolones (A-65282,5; CP-115,953,6 and WIN572947), quinobenzoxazines (A-621768 and A-852269), and
cytotoxic quinolone CX-3543.
to extend the synthesis to create more lipophilic derivatives. So,
the 7-(4-(2-chloroacetyl)piperazin-1-yl) derivative 2 of CP was
used as a precursor of a 7-(4-(2-oxoethylalcanoate)piperazin-1-
yl) homologous series 4 (Scheme 2). We also synthesized two other
homologous series of CP, that is, a 7-(4-(alkoxycarbonyl)piperazin-
zin-1-yl) 3 derivatives were easily prepared in one step. Acylation
of 1 with chloroacetyl chloride or reaction of 1 with Boc2O in diox-
ane/NaOH aqueous solution (2 M) led to compounds 2 or 3 in 73%
and 74% yields after purification, respectively (Scheme 1, Table 1).
The homologous series of 7-(4-(2-oxoethylalcanoate)piperazin-1-
yl) derivatives of CP was prepared by a subsequent condensation
of commercially available carboxylic acids with 2 in dimethylform-
amide to produce the corresponding compounds 4a–j in yields
ranging from 27% to 75% (Scheme 2, Table 1). The 7-(4-(alkoxycar-
bonyl)piperazin-1-yl) derivatives were prepared in methylene
chloride by condensation of 1 with commercially available alkyl-
chloroformates to produce compounds 5a–f in yields ranging from
1-yl) series
5 and a 7-(4-(alkanoyl)piperazin-1-yl) series 6
(Schemes 3 and 4).
2. Chemistry
Starting from commercially available CP 1, the 7-(4-(2-chloro-
acetyl)piperazin-1-yl) 2 and the 7-(4-(tert-butoxycarbonyl)pipera-
Scheme 1. Preparation of the 7-(4-(2-chloroacetyl)piperazin-1-yl) 2 and 7-(4-(tert-butoxy-carbonyl)piperazin-1-yl) 3 derivatives of ciprofloxacin. Reagents and conditions:
(a) Et3N, ClCH2COCl, CH2Cl2, 0 °C–rt; (b) Boc2O, dioxane/NaOH aq solution 2 M, 0 °C–rt.
Scheme 2. Preparation of 7-(4-(2-oxoethylalcanoate)piperazin-1-yl) derivatives 4a–j from 7-(4-(2-chloroacetyl)piperazin-1-yl) derivative 2 of ciprofloxacin. Reagents and
conditions: CH3(CH2)nCOOH, K2CO3, DMF, 100 °C.
Scheme 3. Preparation of 7-(4-(alkoxycarbonyl)piperazin-1-yl) derivatives 5a–f of ciprofloxacin. Reagents and conditions: ROCOCl, CH2Cl2, 0 °C–rt.

5398
J. Azéma et al. / Bioorg. Med. Chem. 17 (2009) 5396–5407
the aim to study stabilities of synthesized derivatives, compound 8
was prepared in two steps. First, acylation of 1 with benzyloxyace-
tyl chloride results in a 46% yield the 7-(4-(2-benzyloxyacetyl)pip-
erazin-1-yl) derivative
7 that was submitted to a catalytic
hydrogenation to give 8 in a 60% yield after purification (Scheme
5). The stability of compounds 2, 3, 4b–d, 4h, 5a–c, 6f, 6h, and
6k was determined using HPLC after the incubation of each
compound in phosphate buffer (pH 7.4) at 37 °C for six days.
3. Pharmacological evaluation
3.1. In vitro determination of the drug-induced inhibition of
human cancer cell line growth
Scheme 4. Preparation of 7-(4-(alkanoyl)piperazin-1-yl) derivatives 6a–l of cipro-
floxacin. Reagents and conditions: RCOCl, Et3N, CH2Cl2, 0 °C–rt.
The in vitro antitumor activity of compounds 2, 3, 4a–j, 5a–f,
6a–l, 8, and CP 1 have been determined for prostate (PC-3),
glioblastoma (U373-MG), colorectal (LoVo), NSCLC (A549), and
breast (MCF-7) human cancer cell lines. The results are summa-
rized in Table 1. For each compound, nine concentrations were
tested for five days on each cancer cell line. We made use of the
MTT colorimetric assay, which indirectly assesses the effect of
the potentially anticancer compounds on the overall growth of
33% to 66% (Scheme 3, Table 1). The 7-(4-(alkanoyl)piperazin-1-yl)
derivatives 6a–l were obtained by acylation of 1 with commer-
cially available acylchlorides in good yields (45–90%) (Scheme 4,
Table 1). Some derivatives of the same series were difficult to pur-
ify, which explains the differences among yields.
Hydrolysis of the synthesized CP derivatives could lead to CP or
to the 7-(4-(2-hydroxyacetyl)piperazin-1-yl) derivative 8. So, with
Table 1
Synthesized compounds: structures, yields of reaction, lipophilicity, stability, and IC50
O
N
O
F
OH
N
N
R
a
R
Compound number
Yieldsb (%)
Clog Pc
Stabilityd (%)
IC50
LoVo
89
(lM)
PC-3
143
U373-MG
A549
MCF-7
H
1e
2
3
À1.14
0.78
3.06
0.74
1.80
2.86
3.92
4.44
4.97
5.50
6.03
7.09
8.15
2.34
3.40
5.52
6.05
6.58
7.63
0.17
0.69
1.22
1.75
1.40
2.81
3.87
4.40
5.45
6.51
7.57
3.90
0.38
1
96
4
4
0.3
2
280 11
10 0.2
18 0.2
476 13
23 0.2
13 0.3
COCH2Cl
C(O)OC(CH3)3
73
74
60
63
75
45
27
47
43
46
41
46
66
42
37
33
41
38
88
75
76
64
90
50
61
45
66
65
58
50
26
>99
>99
8
0.1
26 0.6
176
17 0.3
40 0.4
268
613
27 0.8
29 0.4
267
317
35 0.9
COCH2OCOCH3
COCH2OCO(CH2)2CH3
COCH2OCO(CH2)4CH3
COCH2OCO(CH2)6CH3
COCH2OCO(CH2)7CH3
COCH2OCO(CH2)8CH3
COCH2OCO(CH2)9CH3
COCH2OCO(CH2)10CH3
COCH2OCO(CH2)12CH3
COCH2OCO(CH2)14CH3
C(O)OCH2CH3
C(O)O(CH2)3CH3
C(O)O(CH2)7CH3
C(O)O(CH2)8CH3
C(O)O(CH2)9CH3
C(O)O(CH2)11CH3
COCH3
COCH2CH3
CO(CH2)2CH3
CO(CH2)3CH3
COC(CH3)3
CO(CH2)5CH3
CO(CH2)7CH3
CO(CH2)8CH3
4a
4b
4c
4d
4e
4f
4g
4h
4i
2
99
2
4
6
373
456
20 0.3
16 0.2
8
5
279
1000 11
72
818 17
6
100
>99
>97
715 10
14 0.1
23 0.3
257
416
nd
586
6
28 0.7
53 0.7
2
4
8
272
291
7
5
2
2
216
273
1
4
255
353
3
4
>1000
49
828 13
39 0.7
766 12
>1000
15 0.2
742 18
816 17
621 12
828 14
56 0.8
455 12
14 0.2
>98
3
0.1
2
745 14
30 0.4
385 14
748 23
119 0.6
4j
75
2
112
1
5a
5b
5c
5d
5e
5f
6a
6b
6c
6d
6e
6f
6g
6h
6i
100
100
100
228
3
272
52 1
43
30 0.1
32 0.1
347
323
306
148
87
8
>1000
42 1.3
21 0.5
9
0.3
31 0.5
79 0.7
24 0.6
30 0.5
24 0.7
217
680 33
352
85
73 0.7
246
779 21
1
23
1
283
401 44
94
535 13
9
38
44
2
5
35+2
20
1
2
7
6
8
6
2
273
7
200
3
584 14
402 21
697 18
64 0.8
509 11
808 12
803 15
727 17
7
279
104
86
6
2
2
2
172
316 11
85
9
2
5
4
67 3
729
93
1
100
100
9
793 14
30 0.4
0.2
45 0.9
101 0.9
290
41 0.6
408 12
754 12
26 0.3
20 0.4
7
4
4
94
0.1
0.1
0.1
1
24 0.3
0.2
18 0.2
205
6
3
7
0.1
0.1
0.1
5
7
CO(CH2)10CH3
CO(CH2)12CH3
CO(CH2)14CH3
–COCH2C6H5
108
96
2
1
6j
6k
6l
3
56 0.4
65 0.7
29 0.3
100
114 0.6
243
433 19
16 0.1
34 0.7
4
145
1
2
32 0.5
COCH2OH
8
294
4
456
8
985 26
a
IC50 values were determined using the MTT colorimetric assay on five human cancer cell lines (PC-3: prostate cancer, U373-MG: glioblastoma, LoVo: colon cancer, A549:
lung cancer and MCF-7: breast cancer) treated for 5 days.
b
Yields after preparative chromatography on silica (see Section 6).
Lipophilicity was evaluated by in silico calculation. Software-predicted lipophilicity was estimated by means of the software Clog P (ChemDraw Ultra 8.0). The Clog P
c
predictor is based on topological structure descriptors and was developed with artificial neural networks.
d
Stability studies were carried out using HPLC analysis after incubation of each compound in phosphate buffer (pH 7.4) at 37 °C for 6 days.
Commercially available from Fluka.
e

J. Azéma et al. / Bioorg. Med. Chem. 17 (2009) 5396–5407
5399
Scheme 5. Preparation of 7-(4-(2-hydroxyacetyl)piperazin-1-yl) derivative 8 of ciprofloxacin. Reagents and conditions: (a) Et3N, C6H5CH2OCH2COCl, CH2Cl2, 0 °C–rt; (b) H2,
Pd/C, DMF, rt.
adherent cell lines.15,16 The IC50 values, which are the concentra-
tions that reduce the mean growth value of the cells by 50%, were
determined for each drug and compared to the mean control
growth value, and are listed in Table 1.
was observed for compounds 5d and 5e when compared to 3
(Table 1).
Twelve 7-(4-(alkanoyl)piperazin-1-yl) derivatives (6a–l) with
different chain lengths from one to 15 carbon atoms (Clog P rang-
ing from 0.17 to 7.57) have been synthesized and evaluated for
their in vitro antitumor activities. Only three compounds (6g, 6h,
3.2. In vivo maximum tolerated dose (MTD) in healthy mice
and 6i) showed ICs50 6 15 lM (Table 1). The most potent
The in vivo tolerance of compounds 1, 2, and 3 as well as that of
those derivatives from series 5 and 6 with the lowest or highest
in vitro activity (5a–c, 6f, 6h, 6k) was determined as the MTD
index, which represents the highest single dose of the compound
that can be administered ip to experimental groups of three
healthy mice per group over a maximum period of 28 days without
causing their death.17
compound against all cancer cell lines was 6h, which exhibited
IC50 values from 13- to 93-fold lower than that of CP (Table 1).
In vivo tolerance was determined for compounds 1, 2, 3, 5a, 5b,
5c, 6f, 6h, and 6k, all of which displayed MTD indices >80 mg/kg.
5. Discussion
Among all the compounds tested in the current study, 2 and 6h
were the most potent antitumor agents. However, their antitumor
activity is weak relative to natural compounds such as taxol,16,18
vincristine,19 or sodium pump inhibitors,18,20 whose IC50 values
in vitro are in the nM range. In addition, all the compounds that
have been tested in vivo for tolerance displayed MTD indices
>80 mg/kg.
4. Results
The 31 synthesized 7-(4-(substituted)piperazin-1-yl) deriva-
tives and the native pharmacophore CP 1, evaluated in vitro for
their antitumor activity, displayed IC50 values ranging from
to mM concentrations.
lM
Compounds 2 and 3 showed higher antitumor activity in vitro
than the native pharmacophore CP with mean IC50 values of 9
and 48 lM, respectively, compared to 250 lM for 1. Compounds
2 and 3 exhibited IC50 values 5–28-fold and 2–37-fold lower than
that of CP, respectively, depending on human cancer cell lines
(Table 1).
As indicated in the Introduction, the mechanisms of action by
which some FQ antibiotics, including CP, exert antitumor effects
on various human cancer cell lines were determined to be through
pro-apoptotic effects.1–3,6,8 However, it must be emphasized that
these time- and dose-dependent pro-apoptotic effects were
observed when CP was evaluated within a 600–1500 lM concentra-
Starting from compound 2, 10 7-(4-(2-oxoethylalcanoate)pip-
erazin-1-yl) derivatives (4a–j) with increasing lipophilicity of the
alkyl chain bearing one to 15 carbon atoms (Clog P ranging from
0.74 to 8.15) have been synthesized. No correlation can be
observed between in vitro antitumor activity and chain length
(Table 1). Among these 10 compounds, only three derivatives (4c
(mean IC50: 26 lM); 4d (32 lM); 4h (37 lM)) showed higher activ-
ities than 1 and 3 but none of them showed higher activity when
compared to 2. The most interesting compound of this series was
4h whose IC50 value on the prostate cancer cell line (3 lM) was
47-fold lower than that of 1. Stability tests of compounds 4c, 4d,
and 4h in phosphate buffer at pH 7.4 have shown no degradation
into 8. As 8 has a very weak in vitro antitumor activity (mean
IC50: 484 lM), the observed antitumor activities recorded for 4c,
4d, and 4h are thus not related to a degradation of these
tionrange, whileinvitrogrowthinhibitionvaluesoftenoccuratlow-
er, or even much lower, doses as we detail in Table 2. The possibility
thus remains that some FQ derivatives exert their antitumor activity
throughnon-apoptotic-dependent mechanisms. Wewill investigate
in the near future whether or not the most active compounds we
synthesized in the current study induce sustained pro-autophagic-
and/or lysosomal membrane permeabilization-related cell death
instead of a pro-apoptotic-related cell death, such as we recently
demonstrated for various compounds including for example a CXC
chemokine pan-antagonist,21,22 sodium pump antagonists,19,23 and
temozolomide in the context of galectin-1 deficiency.24
In addition, flow cytometric analysis of bladder cancer cells
treated with CP showed that these cells were arrested in the S/
G2-M phases of the cell cycle, suggesting CP-mediated effects on
cancer cell cycle kinetics.1 Evaluation of the antiproliferative activ-
ity of some FQ antibiotics, including CP, on a human non-small-cell
lung carcinoma (NSCLC) cell line in culture has demonstrated that
the treatment induced morphological alterations, inhibited cellular
proliferation, increased population doubling time, and reduced sat-
uration density.6
compounds.
Due to the higher antitumor activity observed in vitro for com-
pound 3 as compared to CP, six 7-(4-(alkoxycarbonyl)piperazin-1-
yl) derivatives (5a–f) with different length chains from two to 12
carbon atoms (Clog P ranging from 2.34 to 7.63) have been
prepared. No correlation between the activity and the chain length
could again be established (Table 1). Indeed, compounds 5a and 5f
were not active against human cancer cells (Table 1). In addition,
compared with 3, no pharmacological benefit (in terms of
in vitro antitumor activity) was observed for compounds 5b–e
(Table 1). Compounds 5b and 5c present in vitro antitumor activity
against the five cancer cell lines that is similar to 3. Moreover, a
significant loss of activity against the glioblastoma cancer cell line
Usually, we employ computer-assisted phase contrast micros-
copy to grossly decipher the mechanisms of action of novel
antitumor drugs.21–23,25 However, CP was reported early on to be
photounstable.26 Mass-spectrometry evidence has suggested that
a photodimer of unknown structure is formed,27 while further stud-
ies have led to the isolation of products resulting from the degrada-
tion of the piperazinyl side chain.28 Later on, defluorination was
observed together with the photolytic replacement of the C-6 fluo-

5400
J. Azéma et al. / Bioorg. Med. Chem. 17 (2009) 5396–5407
Table 2
In vitro cell proliferation and postulated mechanisms of action of ciprofloxacin and cytotoxic quinolones (data from literature)
Compound
Cell lines and in vitro IC50
growth inhibitory values
(
lM)
Postulated mechanism of action
Ciprofloxacin
F
O
N
O
HeLa epithelial (300,42 80043
MG63 osteosarcoma (48043
Chinese Hamster V79 (>100044
K-562 leukemia (>15045
NCI-H460 NSC lung (606)
)
Inhibitor of the catalytic activity of the topoisomerase II
OH
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Activity against topoisomerase II (inhibition of DNA relaxation) = 104 l
M42
)
N
120 l
M of ciprofloxacin increases the percentage of apoptotic cells from 17% to 35%6
)
HN
)
O
N
O
F
OH
OH
ꢀ
ꢀ
CHO ovary (938
VpmR-5 mutant CHO ovary (1238
)
Enhance topoisomerase II-mediated DNA cleavage
Activity against topoisomerase II (inhibition of DNA relaxation): 60 l
M38
ꢀ
)
F
HO
O
N
O
F
ꢀ
ꢀ
CHO ovary (2038
VpmR-5 mutant CHO ovary (4038
)
Enhance topoisomerase II-mediated DNA cleavage
Activity against topoisomerase II (inhibition of DNA relaxation): 130 l
M38
ꢀ
)
HO
ꢀ
ꢀ
A-549 lung (1.346
HT-29 colon (0.5,46 0.2,34 0.3,47
0.535
HCT-8 colon (0.346
MCF-7 breast (0.8,46 0.735
A546 breast (0.747
DU-145 prostate (0.2,34 0.535
P-388 leukemia (0.0547
8226/S myeloma (0.335
)
Both a topoisomerase II poison and a catalytic inhibitor
ꢀ
Conversion of catenated to decatenated kDNA by human topoisomerase II: 0.5
M48
Concentration of DNA unwinding: 50
l l
M,34 0.5 M,47
3
l
)
ꢀ
l
M34
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
)
)
)
)
)
)
O
O
F
OH
N
N
O
ꢀ
ꢀ
B16 Melanoma (0.2 (S), 0.02 (R))34 Unwind DNA and catalytic inhibitor of topoisomerase II
ꢀ
Conversion of catenated to decatenated kDNA by human topoisomerase II: 0.5 lM (S), 0.2 lM
H2N
MDA-231 Breast (0.08 (S), 0.005
(R)34
(R))34
ꢀ
Concentration of DNA unwinding: 10 lM (S), 3 l
M (R)34
ꢀ
H226 non-small cell lung (0.03 (S),
0.01 (R))34
Stereoisomers (S) and (R)
ꢀ
ꢀ
HT-29 colon (0.05 (S), 0.03 (R))34
DU-145 prostate (0.06 (S), 0.03
(R))34
O
N
O
F
OH
N
O
ꢀ
ꢀ
MCF-7 breast (1 (S), 0.5 (R))48
Major mechanism of action for stereoisomer (S): Topoisomerase II poisonMajor mechanism of
H2N
8226/S myeloma (0.7 (S), 0.3 (R))45 action for stereoisomer (R): G-quadruplex interaction.
ꢀ
G-quadruplex interaction polymerase stop assay: IC50 = 0.7 lM (S); 0.06 l
M (R)48
Stereoisomers (S) and (R)
rine atom by a hydroxyl group.29 This photounstability thus pre-
cluded the use of quantitative videomicroscopy to investigate the
mechanisms of action of the compounds associated with the highest
anti-tumor activity in the current study. We will proceed with a
whole genomic approach as we did previously with respect to other
types of anti-cancer agents.22,30 However, before proceeding with
such experiments, we must still select a lead compound through
the characterization of in vivo activity on a broad panel of biologi-
cally aggressive human xenograft models.19,21–23
Frominitialapproachesto define structure–activityrelationships
for cytotoxic quinolones derived from structural modifications of
native CP, it became evident that no single position has a controlling
effect on the observed in vitro antitumor activity.31,32 A combination
of various structural modifications on several positions of the native
FQ, as shown by the structures of cytotoxic quinolones depicted in
Figure 1, contributes to enhance cytotoxicity (IC50 values on several
human and murine cell lines ranged from nM to
lM concentra-
tions).33–37 For example, it has been shown that the introduction of

J. Azéma et al. / Bioorg. Med. Chem. 17 (2009) 5396–5407
5401
Table 3
In vitro cell proliferation inhibition of ciprofloxacin and some other fluoroquinolones (data from literature)
FQ
In vitro IC50 (l
M) growth inhibitory valuesa
HeLa
MG63
NCI-H460
MCF-7
A431
EJ
SW480
KB
SKMEL
MC3T3-E1
NIH 3T3
study)
Ciprofloxacin
Norfloxacin
Enoxacin
Levofloxacin
Lomefloxacin
Moxifloxacin
Trovafloxacin
Sparfloxacin
Gatifloxacin
Tosufloxacin
800,43 27051
39051
48043
—
606
199,40 76(this
23840
202,40 13552
191,40 7052
175,40 15052
20240
21040
17840
12840
16840
15940
160,40 17752
163,40 15052
137,40 15452
19640
19440
19640
12049
—
>100050
>100050
866
366
1526
17051
19340
22049
41051
32043
48350
12250
23043
<1.249
26551
40051
3251
a
HeLa: epithelial, MG63: osteosarcoma, NCI-H460: NSC lung, MCF-7: breast carcinoma, A431: epidermo carcinoma, EJ: bladder carcinoma, W480: colon carcinoma, KB:
cervical carcinoma, SKMEL: melanoma, MC3T3-E1: osteoblastic, NIH 3T3: fibroblast.
a C-8 fluorine in CP-115,953 enhanced the cytotoxicity by twofold
against wild type CHO cells.38 Nevertheless, we chose to synthesize
compounds with single modification on C-7 of CP as it has been
observed a more efficient in vitro growth inhibition in a panel of can-
cer cell lines treated with norfloxacin or CP thus structurally modi-
(<70 lm) by means of the solvent systems indicated. Melting
points were determined using a Kofler type system and are uncor-
rected. 1H NMR spectra were recorded on a Bruker AC-300 spec-
trometer. Data are reported in the following order: chemical shift
d in ppm, signal multiplicity, number of protons and value(s) of
coupling constant(s). 13C NMR spectra were recorded on a Bruker
Avance 300 spectrometer. The methylene groups of alkyl chains
are reported in Greek alphabetical order starting from CH3. The
other attributions are reported according to the numbering in
Scheme 1. Elemental analyses were carried out by the ‘Service
Commun de Microanalyse élémentaire UPS-INP’ in Toulouse. Mass
spectra were recorded on a Perkin Elmer SCIEX API 100 operating
in chemical ionization (NH3) mode or on a Nermag R10-10 operat-
ing in Fast Atom Bombardment (MNBA) mode.
fied (IC50 ranging from 3 to >100 l
M).39,40 It can be seen from the
data presented in Table 1 that the IC50 values measured for our com-
pounds are in the same order of magnitude as those previously
reported. Moreover, the breakage of the zwitterionic properties of
quinolones by modifying the C-7 basic substituent could change
the electron distribution and thus the protonation equilibria,31 and
eventually the transmembrane passage. Finally, QSAR analysis of
7-(pyrrolidin-1-yl)-4-oxo-1-(thiazol-2-yl)-1,4-dihydro-1,8-naph-
thyridine-3-carboxylic acid derivatives, structurally similar to FQ,
has demonstrated that bulky substituents with low electron den-
sity (e.g., alkyl groups) on the C-7 position enhanced the antitumor
activity.41
6.1.1. 7-(4-(2-Chloroacetyl)piperazin-1-yl)-1-cyclopropyl-6-
fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylic acid (2)
Several of the novel FQ derivatives of this current study displayed
higher in vitro antitumor activity than parent CP or other more
recent FQ (Table 3). By increasing lipophilicity of CP, we could expect
an increase in the affinity of the compounds for the cell membrane
and thusaneasier penetrationinto thecell. However, if substitutions
on the piperazinyl group of CP can lead to an increase in antitumor
activity, this is not dependent on the lipophilicity of the substituent
sincethemost potentcompounds 2 and 6h haveClog P valuesof 0.78
and 4.40, respectively. In comparing each homologous series or each
cancer cell line independently, no correlation between Clog P and
IC50 values can be observed. However, in comparing the same chain
length butwith a differentfunctional linkerto CP, thealkanoylderiv-
atives 6 seem to be more potent than the alkoxycarbonyl 5 or
oxoethylalcanoate 4 derivatives. For example, the most potent anti-
proliferative derivative 6h, with a chain length of nine carbon atoms,
is more active than the corresponding alkoxycarbonyl 5d or oxy-
ethylalcanoate 4f derivatives.
In conclusion, we have synthesized, with an easy and cost-effi-
cient procedure, 31 7-((4-substitued)piperazin-1-yl) derivatives of
CP that are non-toxic as shown by their MTD values. Among all these
derivatives, two of them, that is, compounds 2 and 6h, showed
potent in vitro antitumor activity. This study thus demonstrates that
the cytotoxicityof CP can be positively modulated through the intro-
duction of simple substituents on the piperazinyl group.
CP 1 (3.0 g, 9 mmol) and triethylamine (1.3 mL, 9 mmol) were
stirred in 40 mL of dry methylene chloride at 0 °C for 15 min. Chloro-
acetylchloride(1.1 mL, 14 mmol)was addeddropwise. Afterstirring
at 0 °C for 15 min and at room temperature for 1 h, the mixture was
filtered and the resulting solids were washed with water and meth-
ylene chloride. The aqueous layer was extracted with 3 Â 30 mL of
CH2Cl2. The organic layers were collected, dried over MgSO4, and
concentrated. The remaining residue was purified by silica gel chro-
matography (CH2Cl2–MeOH 2%) to yield the desired product (2.68 g,
73%) as a pale yellow solid. Mp >260 °C. 1H NMR (DMSO-d6) d 1.19 (t,
2H, 3J = 7.2 Hz, CH2 (12)), 1.32 (t, 2H, 3J = 6.9 Hz, CH2 (13)), 3.37 (m,
4H, CH2 (16)), 3.70 (m, 4H, CH2 (15)), 3.82 (tt, 1H, 3J = 7.2 Hz,
3J = 6.9 Hz, CH (11)), 4.45 (s, 2H, CH2–Cl), 7.58 (d, 1H, 4JH–F = 7.6 Hz,
3
CH (8)), 7.92 (d, 1H, JH–F = 13.2 Hz, CH (5)), 8.66 (s, 1H, CH (2)),
14.7 (s, 1H, –COOH). 13C NMR (DMSO-d6) d 176.2 (d, 4JC–F = 2.6 Hz,
1
C4), 165.7 (C14), 166.7 (C17), 152.8 (d, JC–F = 249.3 Hz, C6), 147.9
2
(C2), 144.6 (d, JC–F = 10.2 Hz, C7), 138.9 (C9), 118.7 (d,
2
3JC–F = 7.7 Hz, C10), 110.8 (d, JC–F = 23.1 Hz, C5), 106.6 (C3), 106.4
(d, 3JC–F = 3.1 Hz, C8), 49.3 (C15), 48.9 (C15), 44.8 (C16), 41.6 (CH2-
Cl), 41.2 (C16), 35.8 (C11), 7.4 (C12,13). FAB-MS m/z 408 [MH+]. Anal.
Calcd for C19H19ClFN3O4Á0.4H2O: C, 54.99; H, 4.81; N, 10.12. Found:
C, 54.90; H, 4.65; N, 10.15.
6.1.2. 7-(4-(tert-Butoxycarbonyl)piperazin-1-yl)-1-cyclopropyl-
6-fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylic acid (3)
To a solution of CP 1 (0.5 g, 1.5 mmol) in 10 mL of dioxane,
sodium hydroxide (0.9 g, 22 mmol) in 10 mL of water was added
at 0 °C. A solution of di-tert-butyl dicarbonate (0.36 g, 1.65 mmol)
in 10 mL of dioxane was then added dropwise and the reaction
mixture was stirred at 0 °C for 1 h and at room temperature for
24 h. Dioxane was concentrated and 50 mL of water were added.
The pH of the aqueous solution was adjusted to 3–4 with a 20%
6. Experimental
6.1. Chemistry
All reagents were purchased from commercial sources and used
as supplied without further purification. CP was purchased from
Fluka (>98%). Chromatography was performed on silica gel

5402
J. Azéma et al. / Bioorg. Med. Chem. 17 (2009) 5396–5407
aqueous solution of monohydrate citric acid. The obtained precip-
itate was filtered and washed twice with ethanol and diethyl ether.
The remaining residue was purified by silica gel chromatography
(CH2Cl2–MeOH 2%) to yield the desired product (0.49 g, 74%) as a
pale yellow solid. Mp >260 °C. 1H NMR (CDCl3) d 1.14 (m, 2H,
CH2 (12)), 1.33 (m, 2H, CH2 (13)), 1.49 (s, 9H, CH3), 3.22 (m, 4H,
CH2 (16)), 3.50 (m, 1H, CH (11)), 3.60 (m, 4H, CH2 (15)), 7.29 (d,
(C11), 35.4 (CH2 b), 18.4 (CH2 a), 13.6 (CH3), 8.2 (C12,13). CI/NH3-MS
(positive mode) m/z 459.2 [M], 460.2 [MH+]. Anal. Calcd for
C23H26FN3O6Á0.5H2O: C, 58.97; H, 5.81; N, 8.97. Found: C, 59.02; H,
5.83; N, 9.01.
6.1.3.3. 7-(4-(2-Oxoethylhexanoate)piperazin-1-yl)-1-cyclopro-
pyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylic acid (4c). A
purification by silica gel chromatography (AcOEt–MeOH 1%) provided
the desired compound (268 mg, 75%) as a pale yellow solid. Mp 167 °C.
1H NMR (CDCl3) d 0.88 (t, 3H, 3J = 6.9 Hz, CH3), 1.19 (m, 2H, CH2 (12)),
4
3
1H, JH–F = 7.2 Hz, CH (8)), 7.93 (d, 1H, JH–F = 12.9 Hz, CH (5)),
8.67 (s, 1H, CH (2)), 14.88 (s, 1H, –COOH). 13C NMR (CDCl3) d
4
176.8 (d, JC–F = 2.5 Hz, C4), 166.6 (C14), 154.9 (C17), 153.4 (d, 1J
2
C–F = 249.1 Hz, C6), 148.5 (C2), 145.3 (d, JC–F = 10.2 Hz, C7), 139.7
1.35 (m, 6H, CH2 (13,a c),
,b), 1.67 (qt, 2H, 3J = 6.9 Hz, 3J = 7.5 Hz, CH2
3
2
(C9), 119.4 (d, JC–F = 7.2 Hz, C10), 111.6 (d, JC–F = 23.5 Hz, C5),
2.43 (t, 2H, 3J = 7.5 Hz, CH2 d), 3.35 (m, 4H, CH2 (16)), 3.55 (m, 1H, CH
(11)), 3.66 (m, 2H, CH2 (15)), 3.86 (m, 2H, CH2 (15)), 4.77 (s, 2H, –O–
3
107.6 (C3), 107.1 (d, JC–F = 3.0 Hz, C8), 80.3 (C(CH3)3), 49.9 (C15),
4
49.5 (C15), 45.5 (C16), 41.9 (C16), 35.4 (C11), 28.5 (CH3), 8.1
(C12,13). FAB-MS m/z 432 [MH+]. Anal. Calcd for
C22H26FN3O5Á0.5H2O: C, 59.99; H, 6.17; N, 9.54. Found: C, 59.93;
H, 6.01; N, 9.52.
CH2–CO–), 7.34 (d, 1H, JH–F = 7.2 Hz, CH (8)), 7.90 (d, 1H,
3JH–F = 12.9 Hz, CH (5)), 8.65 (s, 1H, CH (2)), 14.82 (s, 1H, –COOH). 13
C
NMR (CDCl3) d 176.9 (d,4JC–F = 2.3 Hz, C4), 173.5 (CH2dCO–), 166.7
1
(C14), 165.3 (C17), 153.5 (d, JC–F = 249.7 Hz, C6), 147.5 (C2), 145.3
(d, 2JC–F = 10.5 Hz, C7), 138.9 (C9), 120.2 (d, 3JC–F = 7.5 Hz, C10), 112.4
2
3
6.1.3. General procedure for the preparation of 7-(4-(2-
oxoethylalcanoate)piperazin-1-yl) derivatives of CP (4a–j)
To a solution of carboxylic acid (0.74 mmol) in dry dimethyl-
formamide (10 mL), potassium carbonate (117 mg, 0.85 mmol)
was added at room temperature and the reaction mixture was stir-
red for 1 h at 110 °C, after which compound 2 (0.3 g, 0.74 mmol)
was added. The mixture was stirred for 4 h at 110 °C. DMF was
evaporated in vacuo. For compounds 4a–e, the residue was diluted
with 20 mL of water. The aqueous layer was extracted with
4 Â 20 mL of ethyl acetate. The organic layers were collected, dried
over MgSO4, and evaporated. The remaining residue was purified
by silica gel chromatography. For compounds 4f–j, purification
was done directly after evaporation in vacuo of DMF.
(d, JC–F = 23.2 Hz, C5), 108.0 (C3), 105.3 (d, JC–F = 3.0 Hz, C8), 60.9
(–O–CH2–CO–), 49.9 (C15), 49.3 (C15), 44.4 (C16), 41.5 (C16), 35.4
(C11), 33.9 (CH2 d), 31.2 (CH2
c), 24.5 (CH2 b), 22.3 (CH2 a), 13.9
(CH3), 8.2 (C12,13). CI/NH3-MS (positive mode) m/z 487.2 [M], 488.2
[MH+]. Anal. Calcd for C25H30FN3O6Á0.4H2O: C, 60.69; H, 6.27; N,
8.49. Found: C, 60.8; H, 6.30; N, 8.52.
6.1.3.4. 7-(4-(2-Oxoethyloctanoate)piperazin-1-yl)-1-cyclopro-
pyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylic acid (4d). A
purification by silica gel chromatography (AcOEt–MeOH 1%) provided
the desired compound (171 mg, 45%) as a white solid. Mp 176 °C. 1H
NMR (CDCl3) d 0.86 (t, 3H, 3J = 6.9 Hz, CH3), 1.30 (m, 12H, CH2
(12,13,
a
Àd), 1.67 (tt, 2H, 3J = 7.2 Hz, 3J = 7.5 Hz, CH2
e), 2.45 (t, 2H,
3J = 7.5 Hz, CH2 f), 3.35 (m, 4H, CH2 (16)), 3.55 (tt, 1H, 3J = 7.2 Hz,
3J = 6.9 Hz, CH (11)), 3.66 (m, 2H, CH2 (15)), 3.86 (m, 2H, CH2 (15)),
4.78 (s, 2H, –O–CH2–CO–), 7.35 (d, 1H,4JH–F = 7.2 Hz, CH (8)), 7.94 (d,
1H, 3JH–F = 12.6 Hz, CH (5)), 8.68 (s, 1H, CH (2)), 14.84 (s, 1H, –COOH).
6.1.3.1. 7-(4-(2-Oxoethylethanoate)piperazin-1-yl)-1-cyclopro-
pyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylic acid (4a). A
purification by silica gel chromatography (CH2Cl2–MeOH 1–2%) pro-
vided the desired compound (194 mg, 60%) as a white solid. Mp
>260 °C. 1H NMR (DMSO-d6) d 1.18 (m, 2H, CH2 (12)), 1.30 (m, 2H,
CH2 (13)), 2.09 (s, 3H, CH3), 3.33 (m, 4H, CH2 (16)), 3.66 (m, 4H, CH2
(15)), 3.81 (m, 1H, CH (11)), 4.84 (s, 2H, –O–CH2–CO–), 7.58 (d, 1H,
13C NMR (CDCl3) d 176.7 (d, JC–F = 2.2 Hz, C4), 173.4 (CH2fCO–),
4
1
166.6 (C14), 165.3 (C17), 153.8 (d, JC–F = 251.3 Hz, C6), 147.4 (C2),
145.2 (d, 2JC–F = 10.6 Hz, C7), 138.9 (C9), 119.8 (d, 3JC–F = 6.8 Hz, C10),
2
3
112.0 (d, JC–F = 23.4 Hz, C5), 107.7 (C3), 105.3 (d, JC–F = 3.0 Hz, C8),
60.9 (–O–CH2–CO–), 49.7 (C15), 49.2 (C15), 44.4 (C16), 41.5 (C16),
3
4JH–F = 7.5 Hz, CH (8)), 7.91 (d, 1H, JH–F = 13.2 Hz, CH (5)), 8.66 (s,
1H, CH (2)), 15.10 (s, 1H, –COOH). 13C NMR (DMSO-d6) d 176.9 (d,
35.4 (C11), 33.9 (CH2 f), 31.6 (CH2
(CH2 b), 22.6 (CH2
e), 29.0 (CH2 d), 28.9 (CH2 c), 24.8
4JC–F = 2.6 Hz, C4), 173.2 (CH3CO–), 166.6 (C14), 165.2 (C17), 153.6
a
), 14.0 (CH3), 8.2 (C12,13). CI/NH3-MS (positive
1
2
(d, JC–F = 250.2 Hz, C6), 147.5 (C2), 145.3 (d, JC–F = 10.5 Hz, C7),
138.8 (C9), 120.1 (d, 3JC–F = 7.5 Hz, C10), 112.5 (d, 2JC–F = 23.5 Hz, C5),
mode) m/z 515.3 [M], 516.3 [MH+]. Anal. Calcd for
C27H34FN3O6Á0.5H2O: C, 61.82; H, 6.72; N, 8.01. Found: C, 61.81; H,
6.70; N, 8.12.
3
108.1 (C3), 105.3 (d, JC–F = 2.9 Hz, C8), 60.9 (–O–CH2–CO–), 49.9
(C15), 49.2 (C15), 44.4 (C16), 41.5 (C16), 35.5 (C11), 19.1 (CH3), 8.1
(C12,13). CI/NH3-MS(positivemode)m/z431.1 [M],432.1[MH+].Anal.
Calcd for C21H22FN3O6Á0.2H2O: C, 57.98; H, 5.19; N, 9.65. Found: C,
58.01; H, 5.18; N, 9.67.
6.1.3.5. 7-(4-(2-Oxoethylnonanoate)piperazin-1-yl)-1-cyclopro-
pyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylic acid (4e). A
purification by silica gel chromatography (AcOEt–MeOH 0.05%) pro-
vided the desired compound (100 mg, 27%) as a white solid. Mp
136 °C. 1H NMR (CDCl3) d 0.86 (t, 3H, 3J = 6.5 Hz, CH3), 1.25 (m, 14H,
6.1.3.2. 7-(4-(2-Oxoethylbutanoate)piperazin-1-yl)-1-cyclopro-
pyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylic acid (4b). A
purification by silica gel chromatography (AcOEt–MeOH 1%) provided
thedesiredcompound(213 mg, 63%)asapaleyellowsolid.Mp211 °C.
1H NMR (CDCl3) d 0.97 (t, 3H, 3J = 7.5 Hz, CH3), 1.20 (m, 2H, CH2 (12)),
CH2 (12,13,
2H, 3J = 7.5 Hz, CH2
3.66 (m, 2H, CH2 (15)), 3.86 (m, 2H, CH2 (15)), 4.78 (s, 2H, –O–CH2–
aÀ
e
), 1.65 (tt, 2H, 3J = 7.2 Hz, 3J = 7.5 Hz, CH2 f), 2.44 (t,
g), 3.35 (m, 4H, CH2 (16)), 3.55 (m, 1H, CH (11)),
4
3
CO–), 7.34 (d, 1H, JH–F = 6.9 Hz, CH (8)), 7.91 (d, 1H, JH–F = 12.9 Hz,
1.39 (m, 2H, CH2 (13)), 1.69 (sext, 2H, 3J = 7.5 Hz, CH2
a), 2.42 (t, 2H,
CH (5)), 8.67 (s, 1H, CH (2)), 14.88 (s, 1H, –COOH). 13C NMR (CDCl3) d
3J = 7.5 Hz, CH2 b), 3.35 (m, 4H, CH2 (16)), 3.55 (m, 1H, CH (11)), 3.66
(m, 2H, CH2 (16)), 3.85 (m, 2H, CH2 (15)), 4.78 (s, 2H, –O–CH2–CO–),
176.8 (d, JC–F = 2.5 Hz, C4), 173.4 (CH2 CO–), 166.7 (C14), 165.3
4
g
1
2
(C17), 153.5 (d, JC–F = 251.3 Hz, C6), 147.5 (C2), 145.2 (d, JC–
4
3
7.34 (d, 1H, JH–F = 7.2 Hz, CH (8)), 7.90 (d, 1H, JH–F = 12.9 Hz, CH
F = 10.5 Hz, C7), 138.9 (C9), 120.1 (d, 3JC–F = 7.8 Hz, C10), 112.3 (d, 2JC–
(5)), 8.65 (s, 1H, CH (2)), 14.82 (s, 1H, –COOH). 13C NMR (CDCl3) d
176.9 (d, JC–F = 3.0 Hz, C4), 173.3 (CH2bCO–), 166.7 (C14), 165.3
F = 23.2 Hz, C5), 107.9 (C3), 105.2 (d, JC–F = 2.9 Hz, C8), 60.9 (–O–
3
4
CH2–CO–), 49.8 (C15), 49.2 (C15), 44.4 (C16), 41.5 (C16), 35.3 (C11),
1
(C17), 153.5 (d, JC–F = 249.7 Hz, C6), 147.5 (C2), 145.3 (d,
33.8 (CH2
g), 31.7 (CH2 f), 29.1 (CH2 e), 29.0 (CH2 c,d), 24.8 (CH2 b),
2JC–F = 10.5 Hz, C7), 138.9 (C9), 120.2 (d, 3JC–F = 7.5 Hz, C10), 112.4 (d,
22.6 (CH2 a), 14.0 (CH3), 8.2 (C12,13). CI/NH3-MS (positive mode) m/
3
2JC–F = 23.2 Hz, C5), 108.0 (C3), 105.3 (d, JC–F = 3.0 Hz, C8), 60.9
z 529.3 [M], 530.3 [MH+]. Anal. Calcd for C28H36FN3O6Á0.5H2O: C,
(–O–CH2–CO–), 49.9 (C15), 49.3 (C15), 44.4 (C16), 41.5 (C16), 35.8
62.44; H, 6.92; N, 7.80. Found: C, 62.49; H, 6.95; N, 7.83.

J. Azéma et al. / Bioorg. Med. Chem. 17 (2009) 5396–5407
5403
6.1.3.6. 7-(4-(2-Oxoethyldecanoate)piperazin-1-yl)-1-cyclopro-
pyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylic acid (4f). A
purification by silica gel chromatography (AcOEt–MeOH 0.05%) pro-
vided the desired compound (190 mg, 47%) as a white solid. Mp
162 °C. 1H NMR (CDCl3) d 0.86 (t, 3H, 3J = 6.6 Hz, CH3), 1.30 (m, 16H,
purification by silica gel chromatography (AcOEt) provided the desired
compound (180 mg, 41%) as a pale yellow solid. Mp 170 °C. 1H NMR
(CDCl3) d 0.84 (t, 3H, 3J = 6.9 Hz, CH3), 1.22 (m, 24H, CH2 (12,13,
a
À
j
),
1.65 (tt, 2H, J = 6.9 Hz, J = 7.5 Hz, CH2 k), 2.42 (t, 2H, J = 7.5 Hz, CH2
), 3.35 (m, 4H, CH2 (16)), 3.55 (tt, 1H, 3J = 7.2 Hz, 3J = 6.6 Hz, CH (11)),
3
3
3
l
CH2 (12,13,
a
Àf), 1.66 (tt, 2H, 3J = 7.2 Hz, 3J = 7.5 Hz, CH2
g
), 2.44 (t,
3.66 (m, 2H, CH2 (15)), 3.85 (m, 2H, CH2 (15)), 4.77 (s, 2H, –O–CH2–
CO–), 7.33 (d, 1H, 4JH–F = 7.2 Hz, CH (8)), 7.84 (d, 1H, 3JH–F = 12.9 Hz, CH
(5)), 8.61 (s, 1H, CH (2)), 14.82 (s, 1H, –COOH). 13C NMR (CDCl3) d 176.7
2H, 3J = 7.5 Hz, CH2 h), 3.34 (m, 4H, CH2 16), 3.39 (m, 1H, CH 11), 3.66
(m, 2H, CH2 15), 3.86 (m, 2H, CH2 15), 4.78 (s, 2H,
4
–O–CH2–CO–), 7.35 (d, 1H, JH–F = 6.6 Hz, CH 8), 7.94 (d, 1H,
(d, 4JC–F = 2.6 Hz, C4), 173.4 (CH2 CO–), 166.6 (C14), 165.3 (C17), 153.5
l
3JH–F = 12.9 Hz, CH 5), 8.69 (s, 1H, CH 2), 14.86 (s, 1H, –COOH). 13C
(d, JC–F = 251.3 Hz, C6), 147.4 (C2), 145.2 (d, JC–F = 10.4 Hz, C7), 138.9
(C9), 120.0 (d, JC–F = 7.8 Hz, C10), 112.2 (d, JC–F = 23.3 Hz, C5), 107.9
(C3), 105.3 (d, JC–F = 3.0 Hz, C8), 60.9 (–O–CH2–CO–), 49.8 (C15), 49.2
1
2
4
3
2
NMR (CDCl3) d 176.9 (d, JC–F = 2.6 Hz, C4), 173.4 (CH2hCO–), 166.7
1
3
(C14), 165.3 (C17), 153.5 (d, JC–F = 251.2 Hz, C6), 147.5 (C2), 145.2
(d, 2JC–F = 10.5 Hz, C7), 138.9 (C9), 120.2 (d, 3JC–F = 7.8 Hz, C10), 112.4
(C15), 44.4 (C16), 41.5 (C16), 35.4 (C11), 33.9 (CH2
(CH2 ), 29.65 (CH2 ,h), 29.6 (CH2 ), 29.4 (CH2 f), 29.3 (CH2
(CH2 d), 29.1 (CH2 ), 24.8 (CH2 b), 22.7 (CH2 ), 14.1 (CH3), 8.2 (C12,13).
l), 31.9 (CH2 k), 29.7
2
3
(d, JC–F = 23.2 Hz, C5), 108.0 (C3), 105.2 (d, JC–F = 2.9 Hz, C8), 60.9
(–O–CH2–CO–), 49.9 (C15), 49.2 (C15), 44.4 (C16), 41.5 (C16), 35.3
j
i
g
e), 29.2
c
a
(C11), 33.9 (CH2 h), 31.8 (CH2
g), 29.3 (CH2 f), 29.2 (CH2 d,e), 29.1
CI/NH3-MS (positive mode) m/z 599.3 [M], 600.3 [MH+]. Anal. Calcd for
C33H46FN3O6Á0.5H2O: C, 65.15; H, 7.79; N, 6.91. Found: C, 65.06; H,
7.78; N, 7.04.
(CH2 ), 24.8 (CH2 b), 22.6 (CH2
c
a), 14.0 (CH3), 8.2 (C12,13). CI/NH3-
MS (positive mode) m/z 543.3 [M], 544.3 [MH+]. Anal. Calcd for
C29H38FN3O6Á0.25H2O: C, 63.54; H, 7.08; N, 7.67. Found: C, 63.58; H,
7.10; N, 7.69.
6.1.3.10. 7-(4-(2-Oxoethylhexadecanoate)piperazin-1-yl)-1-cyclo
propyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylic
acid
6.1.3.7. 7-(4-(2-Oxoethylundecanoate)piperazin-1-yl)-1-cyclopro-
pyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylic acid (4g). A
purification by silica gel chromatography (AcOEt–MeOH 0.05%) pro-
vided the desired compound (178 mg, 43%) as a white solid. Mp
(4j). A purification by silica gel chromatography (AcOEt) provided
the desired compound (213 mg, 46%) as a pale yellow solid. Mp
164 °C. 1H NMR (CDCl3) d 0.84 (t, 3H, 3J = 6.6 Hz, CH3), 1.22 (m, 28H,
CH2 (12,13,
a l m), 2.42 (t,
), 1.63 (tt, 2H, 3J = 6.9 Hz, 3J = 7.5 Hz, CH2
À
166 °C. 1H NMR (CDCl3) d 0.86 (t, 3H, 3J = 6.3 Hz, CH3), 1.25 (m, 18H,
2H, 3J = 7.5 Hz, CH2 n), 3.35 (m, 4H, CH2 (16)), 3.55 (m, 1H, CH (11)),
3.66 (m, 2H, CH2 (15)), 3.85 (m, 2H, CH2 (15)), 4.77 (s, 2H, –O–CH2–
CO–), 7.33 (d, 1H, 4JH–F = 7.0 Hz, CH (8)), 7.85 (d, 1H, 3JH–F = 12.9 Hz,
CH (5)), 8.61 (s, 1H, CH (2)), 14.86 (s, 1H, –COOH). 13C NMR (CDCl3)
3
CH2 (12,13,
a
À
g
), 1.69 (m, 2H, CH2 h), 2.45 (t, 2H, J = 7.5 Hz, CH2
i),
3.38 (m, 4H, CH2 (16)), 3.55 (m, 1H, CH (11)), 3.66 (m, 2H, CH2 (15)),
3.87 (m, 2H, CH2 (15)), 4.78 (s, 2H, –O–CH2–CO–), 7.35 (d, 1H,
4JH–F = 6.6 Hz, CH (8)), 7.97 (d, 1H, 3JH–F = 12.9 Hz, CH (5)), 8.71 (s, 1H,
d 176.7 (d, JC–F = 2.6 Hz, C4), 173.3 (CH2nCO–), 166.5 (C14), 165.2
4
4
1
2
CH (2)), 14.85 (s, 1H, –COOH). 13C NMR (CDCl3) d 176.9 (d, JC–F
=
(C17), 153.4 (d, JC–F = 251.3 Hz, C6), 147.3 (C2), 145.1 (d, JC–F =
3
2.6 Hz, C4), 173.5 (CH2 CO–), 166.7 (C14), 165.3 (C17), 153.5 (d,
10.6 Hz, C7), 138.7 (C9), 119.9 (d, JC–F = 7.7 Hz, C10), 112.2 (d,
2JC–F = 23.3 Hz, C5), 107.8 (C3), 105.2 (d, 3JC–F = 2.9 Hz, C8), 60.9 (–O–
CH2–CO–), 49.8 (C15), 49.2 (C15), 44.3 (C16), 41.4 (C16), 35.3 (C11),
i
2
1JC–F = 251.2 Hz, C6), 147.5 (C2), 145.2 (d, JC–F = 10.6 Hz, C7), 138.9
(C9), 120.3 (d, 3JC–F = 7.8 Hz, C10), 112.5 (d, 2JC–F = 23.3 Hz, C5), 108.1
(C3), 105.2 (d, 3JC–F = 3.0 Hz, C8), 60.9 (–O–CH2–CO–), 49.9 (C15), 49.2
33.8 (CH2 n), 31.8 (CH2
m), 29.6 (CH2
j,k,l), 29.55 (CH2 h,i), 29.5 (CH2
(C15), 44.4 (C16), 41.5 (C16), 35.3 (C11), 33.9 (CH2
29.5 (CH2 ), 29.4 (CH2 f), 29.3 (CH2 ), 29.2 (CH2 d), 29.1 (CH2
24.8 (CH2 b), 22.6 (CH2 ), 14.1 (CH3), 8.2 (C12,13). CI/NH3-MS
i
), 31.8 (CH2 h),
g
), 29.3 (CH2 f), 29.2 (CH2 ), 29.1 (CH2 d), 29.0 (CH2 c
e
), 24.7 (CH2 b),
g
e
c),
22.6 (CH2 a), 14.0 (CH3), 8.2 (C12,13). CI/NH3-MS (positive mode) m/
a
z 627.4 [M], 628.4 [MH+]. Anal. Calcd for C35H50FN3O6Á0.4H2O: C,
(positive mode) m/z 557.3 [M], 558.3 [MH+]. Anal. Calcd for
C30H40FN3O6Á0.5H2O: C, 63.59; H, 7.29; N, 7.41. Found: C, 63.61; H,
7.30; N, 7.43.
66.24; H, 8.07; N, 6.62. Found: C, 66.23; H, 8.12; N, 6.83.
6.1.4. General procedure for the preparation of 7-(4-
(alkoxycarbonyl)piperazin-1-yl) derivatives of CP (5a–f)
To a solution of CP 1 (0.5 g, 1.5 mmol) in methylene chloride
(20 mL) at 0 °C was added dropwise alkyl chloroformate
(3.0 mmol) over a 15 min period. The reaction was stirred at 0 °C
for 15 min and then 1 h at room temperature. The mixture was
quenched by an aqueous solution of 1 M HCl (50 mL). The aqueous
layer was extracted once with 30 mL of CH2Cl2. The organic layers
were collected, dried over Na2SO4, and concentrated. The remain-
ing residue was purified by silica gel chromatography.
6.1.3.8. 7-(4-(2-Oxoethyldodecanoate)piperazin-1-yl)-1-cyclo-
propyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylic acid
(4h). A purification by silica gel chromatography (AcOEt–MeOH
0.5%) provided the desired compound (193 mg, 46%) as a pale yel-
low solid. Mp 169 °C. 1H NMR (CDCl3) d 0.84 (t, 3H, 3J = 6.9 Hz,
CH3), 1.22 (m, 20H, CH2 (12,13,
a
Àh), 1.65 (tt, 2H, 3J = 7.2 Hz,
3J = 7.5 Hz, CH2 ), 2.42 (t, 2H, 3J = 7.5 Hz, CH2
i j), 3.35 (m, 4H,
CH2 (16)), 3.55 (tt, 1H, 3J = 7.2 Hz, 3J = 6.9 Hz, CH (11)), 3.66 (m,
2H, CH2 (15)), 3.85 (m, 2H, CH2 (15)), 4.77 (s, 2H, –O–CH2–CO–),
4
3
7.33 (d, 1H, JH–F = 6.9 Hz, CH (8)), 7.84 (d, 1H, JH–F = 12.6 Hz, CH
6.1.4.1. 7-[4-(Ethoxycarbonyl)piperazin-1-yl]-1-cyclopropyl-6-
fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylic acid (5a). A
purification by silica gel chromatography (CH2Cl2–MeOH 1%) pro-
vided the desired compound (401 mg, 66%) as a pale yellow solid.
Mp >260 °C. 1H NMR (CDCl3) d 1.20 (m, 2H, CH2 (12)), 1.29 (t, 3H,
3J = 7.0 Hz, CH3), 1.39 (m, 2H, CH2 (13)), 3.28 (m, 4H, CH2 (16)), 3.54
(5)), 8.61 (s, 1H, CH (2)), 14.82 (s, 1H, –COOH). 13C NMR (CDCl3)
4
d 176.7 (d, JC–F = 2.6 Hz, C4), 173.3 (CH2 CO–), 166.5 (C14), 165.2
j
1
(C17), 153.4 (d, JC–F = 251.3 Hz, C6), 147.3 (C2), 145.1 (d,
2JC–F = 10.4 Hz, C7), 138.8 (C9), 119.8 (d, JC–F = 7.7 Hz, C10), 112.0
3
2
3
(d, JC–F = 23.3 Hz, C5), 107.8 (C3), 105.2 (d, JC–F = 2.9 Hz, C8),
60.9 (–O–CH2–CO–), 49.8 (C15), 49.2 (C15), 44.3 (C16), 41.4
(m, 1H, CH2 (11)), 3.72 (m, 4H, CH2 (15)), 4.18 (q, 2H, 3J = 7.0 Hz,
4
(C16), 35.3 (C11), 33.8 (CH2
(CH2 f), 29.2 (CH2 ), 29.1 (CH2 d), 29.0 (CH2
(CH2
j
), 31.8 (CH2
i
), 29.5 (CH2 h,
g
), 29.3
CH2
a
), 7.37 (d, 1H, JH–F = 7.2 Hz, CH (8)), 8.04 (d, 1H,
e
c), 24.7 (CH2 b), 22.6
3JH–F = 12.9 Hz, CH (5)), 8.78 (s, 1H, CH (2)), 15.02 (s, 1H, –COOH).
4
a
), 14.0 (CH3), 8.2 (C12,13). CI/NH3-MS (positive mode) m/z
13C NMR (CDCl3) d 176.9 (d, JC–F = 3.0 Hz, C4), 166.7 (C14), 155.4
1
571.3 [M], 572.3 [MH+]. Anal. Calcd for C31H42FN3O6Á0.25H2O: C,
(C17), 153.5 (d, JC–F = 249.5 Hz, C6), 147.5 (C2), 145.4 (d,
3
64.66; H, 7.44; N, 7.30. Found: C, 64.67; H, 7.58; N, 7.32.
2JC–F = 10.5 Hz, C7), 138.9 (C9), 120.1 (d, JC–F = 7.5 Hz, C10), 112.4
2
3
(d, JC–F = 23.0 Hz, C5), 108.1 (C3), 105.1 (d, JC–F = 3.2 Hz, C8),
65.6 (CH2 ), 49.6 (C15), 43.4 (C16), 29.9 (C11), 14.1 (CH3), 8.2
(C12,13). CI/NH3-MS (positive mode) m/z 403.2 [M], 404.2 [MH+].
6.1.3.9. 7-(4-(2-Oxoethyltetradecanoate)piperazin-1-yl)-1-cyclopro-
pyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylic acid (4i). A
a

5404
J. Azéma et al. / Bioorg. Med. Chem. 17 (2009) 5396–5407
Anal. Calcd for C20H22FN3O5: C, 59.55; H, 5.50; N, 10.42. Found: C,
59.60; H, 5.41; N, 10.45.
3JH–F = 12.9 Hz, CH (5)), 8.54 (s, 1H, CH (2)), 14.80 (s, 1H, –COOH).
4
13C NMR (CDCl3) d 176.8 (d, JC–F = 2.6 Hz, C4), 166.7 (C14), 155.4
1
2
(C17), 153.5 (d, JC–F = 251.2 Hz, C6), 147.3 (C2), 145.7 (d, JC–F
=
3
6.1.4.2. 7-[4-(Butoxycarbonyl)piperazin-1-yl]-1-cyclopropyl-6-
fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylic acid (5b). A puri-
fication by silica gel chromatography (CH2Cl2–MeOH 2%) provided
the desired compound (290 mg, 42%) as a pale yellow solid. Mp
240 °C. 1H NMR (CDCl3) d 0.95 (t, 3H, 3J = 7.5 Hz, CH3), 1.20 (m, 2H,
10.2 Hz, C7), 138.9 (C9), 119.8 (d, JC–F = 8.0 Hz, C10), 112.2 (d,
2JC–F = 23.2 Hz, C5), 107.9 (C3), 105.1 (d, 3JC–F = 3.0 Hz, C8), 65.9 (CH2
i
), 49.6 (C15), 49.5 (C15), 43.4 (C16), 43.3 (C16), 35.3 (C11), 31.8
(CH2 h), 29.5 (CH2 ), 29.4 (CH2 f), 29.3 (CH2 ), 29.2 (CH2 d), 28.9
(CH2 ), 25.9 (CH2 b), 22.6 (CH2 ), 14.1 (CH3), 8.2 (C12,13). CI/NH3-
g
e
c
a
CH2 (12)), 1.40 (m, 4H, CH2 (13,
a
)), 1.65 (m, 2H, CH2 b), 3.30 (m, 4H,
MS (positive mode) m/z 515.3 [M], 516.3 [MH+]. Anal. Calcd for
C28H38FN3O5: C, 65.22; H, 7.43; N, 8.15. Found: C, 65.29; H, 7.40; N,
8.21.
CH2 16), 3.55 (tt, 1H, 3J = 6.9 Hz, 3J = 7.2 Hz, CH (11)), 3.72 (m, 4H,
CH2 15), 4.13 (t, 2H, 3J = 6.6 Hz, CH2
c
), 7.37 (d, 1H, 4JH–F = 7.2 Hz, CH
3
(8)), 8.00 (d, 1H, JH–F = 12.9 Hz, CH (5)), 8.73 (s, 1H, CH (2)), 14.91
(s, 1H, –COOH). 13C NMR (CDCl3) d 176.8 (d, 4JC–F = 2.6 Hz, C4), 166.7
6.1.4.6. 7-[4-((Dodecyloxy)carbonyl)piperazin-1-yl]-1-cyclopro-
pyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylic acid (5f). A
purification by silica gel chromatography (CH2Cl2–MeOH 1%) pro-
vided the desired compound (310 mg, 38%) as a pale yellow solid.
Mp 140 °C. 1H NMR (CDCl3) d 0.81 (t, 3H, 3J = 6.6 Hz, CH3), 1.19 (m,
1
(C14), 155.4 (C17), 153.5 (d, JC–F = 251.3 Hz, C6), 147.4 (C2), 145.6
2
3
(d, JC–F = 10.4 Hz, C7), 138.9 (C9), 120.0 (d, JC–F = 7.9 Hz, C10),
112.2 (d, 2JC–F = 23.4 Hz, C5), 107.9 (C3), 105.1 (d, 3JC–F = 3.2 Hz, C8),
65.6 (CH2
(CH2
c), 49.6 (C15), 43.4 (C16), 35.3 (C11), 31.0 (CH2 b), 19.1
a
), 13.7 (CH3), 8.2 (C12,13). CI/NH3-MS (positive mode) m/z
22H, CH2 (12,13,
a i j), 3.24 (m, 4H,
), 1.59 (m, 2H, 3J = 6.9 Hz, CH2
À
431.4 [M], 432.4 [MH+]. Anal. Calcd for C22H26FN3O5: C, 61.24; H,
6.07; N, 9.74. Found: C, 61.50; H, 6.42; N, 9.51.
CH2 (16)), 3.48 (m, 1H, CH (11)), 3.66 (m, 4H, CH2 (15)), 4.06 (t, 2H,
3J = 6.9 Hz, CH2 k), 7.29 (d, 1H, JH–F = 7.2 Hz, CH (8)), 7.94 (d, 1H,
4
3JH–F = 12.9 Hz, CH (5)), 8.67 (s, 1H, CH (2)), 14.83 (s, 1H, –COOH).
4
6.1.4.3. 7-[4-((Octyloxy)carbonyl)piperazin-1-yl]-1-cyclopropyl-6-
fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylic acid (5c). A purifi-
cation by silica gel chromatography (CH2Cl2–MeOH 1%) provided the
desired compound (270 mg, 37%) as a pale yellow solid. Mp 154 °C.
1H NMR (CDCl3) d 0.88 (t, 3H, 3J = 6.9 Hz, CH3), 1.27 (m, 14H, CH2
13C NMR (CDCl3) d 176.8 (d, JC–F = 2.2 Hz, C4), 166.7 (C14), 155.4
1
2
(C17), 153.5 (d, JC–F = 250.2 Hz, C6), 147.3 (C2), 145.7 (d, JC–F
=
=
9.7 Hz, C7), 138.9 (C9), 119.8 (d, 3JC–F = 8.1 Hz, C10), 112.2 (d, 2JC–F
3
23.2 Hz, C5), 107.9 (C3), 105.1 (d, JC–F = 3.0 Hz, C8), 65.9 (CH2 k),
49.6 (C15), 43.4 (C16), 35.3 (C11), 31.8 (CH2
j
), 29.7 (CH2 ), 29.6
i
(12,13,
a
À
e
), 1.64 (m, 2H, CH2 f), 3.30 (m, 4H, CH2 (16)), 3.54 (m, 1H,
(CH2 h), 29.5 (CH2
g
), 29.3 (CH2 f), 29.2 (CH2
e
), 28.9 (CH2 c,d), 25.9
3
CH (11)), 3.71 (m, 4H, CH2 (15)), 4.12 (t, 2H, J = 6.6 Hz, CH2
g), 7.34
(CH2 b), 22.6 (CH2 a), 14.1 (CH3), 8.2 (C12,13). CI/NH3-MS (positive
(d, 1H, 4JH–F = 7.2 Hz, CH (8)), 7.99 (d, 1H, 3JH–F = 12.9 Hz, CH (5)), 8.73
(s, 1H, CH (2)), 14.90 (s, 1H, –COOH). 13C NMR (CDCl3) d 177.0 (d,
4JC–F = 2.6 Hz, C4), 166.8 (C14), 155.4 (C17), 153.6 (d, 1JC–F = 251.3 Hz,
mode) m/z 543.4 [M], 544.3 [MH+]. Anal. Calcd for C30H42FN3O5: C,
66.28; H, 7.79; N, 7.73. Found: C, 66.25; H, 7.82; N, 7.77.
2
C6), 147.5 (C2), 145.7 (d, JC–F = 10.3 Hz, C7), 138.9 (C9), 120.2 (d,
6.1.5. General procedure for the preparation of 7-(4-
(alkanoyl)piperazin-1-yl) derivatives of ciprofloxacin (6a–j)
CP 1 (0.5 g, 1.5 mmol) and triethylamine (0.23 mL, 1.65 mmol)
were stirred in 20 mL of methylene chloride at 0 °C for 15 min.
Acylchloride (3.75 mmol) was added dropwise. After stirring at
0 °C for 15 min and then at room temperature for 1 h, 20 mL of
water were added to the mixture. The aqueous layer was extracted
once with CH2Cl2, and the organic phase was washed with water
(20 mL). The organic layer was dried over Na2SO4 and concen-
trated. The remaining residue was purified by silica gel
chromatography.
3JC–F = 7.9 Hz, C10), 112.5 (d, 2JC–F = 23.4 Hz, C5), 108.2 (C3), 105.0 (d,
3JC–F = 3.2 Hz, C8), 66.0 (CH2
g), 49.7 (C15), 49.6 (C15), 43.4 (C16),
43.3 (C16), 35.3 (C11), 31.8 (CH2 f), 29.2 (CH2 ), 29.1 (CH2 d), 28.9
e
(CH2 c), 25.9 (CH2 b), 22.6 (CH2 a), 14.1 (CH3), 8.2 (C12,13). CI/NH3-
MS (positive mode) m/z 487.3 [M], 488.3 [MH+]. Anal. Calcd for
C26H34FN3O5: C, 64.05;H, 7.03; N, 8.62. Found:C, 63.99; H, 7.09; N, 8.66.
6.1.4.4. 7-[4-((Nonyloxy)carbonyl)piperazin-1-yl]-1-cyclopropyl-
6-fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylic acid (5d). A
purification by silica gel chromatography (CH2Cl2–MeOH 1%) pro-
vided the desired compound (253 mg, 33%) as a pale yellow solid.
Mp 157 °C. 1H NMR (CDCl3) d 0.84 (m, 3H, CH3), 1.25 (m, 16H, CH2
6.1.5.1. 7-(4-Acetylpiperazin-1-yl)-1-cyclopropyl-6-fluoro-1,4-
dihydro-4-oxoquinoline-3-carboxylic acid (6a). A purification by
silica gel chromatography (CH2Cl2–MeOH 2%) provided the desired
compound (500 mg, 88%) as a pale yellow solid. Mp >260 °C. 1H
NMR (DMSO-d6) d 1.19 (m, 2H, CH2 (12)), 1.32 (m, 2H, CH2 (13)),
2.07 (s, 3H, CH3), 3.30 (m, 2H, CH2 (16)), 3.36 (m, 2H, CH2 (16)),
3.67 (m, 4H, CH2 (15)), 3.82 (tt, 1H, 3J = 7.5 Hz, 3J = 6.9 Hz, CH
(12,13,
a
Àf), 1.63 (m, 2H, CH2
g), 3.29 (m, 4H, CH2 (16)), 3.54 (m,
1H, CH (11)), 3.69 (m, 4H, CH2 (15)), 4.09 (t, 2H, 3J = 6.6 Hz, CH2 h),
4
3
7.32 (d, 1H, JH–F = 6.7 Hz, CH (8)), 7.85 (d, 1H, JH–F = 12.9 Hz, CH
(5)), 8.62 (s, 1H, CH (2)), 14.85 (s, 1H, –COOH). 13C NMR (CDCl3) d
4
176.8 (d, JC–F = 2.6 Hz, C4), 166.6 (C14), 155.4 (C17), 153.5 (d,
1JC–F = 251.4 Hz, C6), 147.3 (C2), 145.6 (d, JC–F = 10.3 Hz, C7), 138.9
2
3
2
4
3
(C9), 119.8 (d, JC–F = 7.8 Hz, C10), 112.1 (d, JC–F = 23.4 Hz, C5),
(11)), 7.57 (t, 1H, JH–F = 7.5 Hz, CH (8)), 7.93 (d, 1H, JH–F =
107.8 (C3), 105.0 (d, 3JC–F = 3.1 Hz, C8), 65.9 (CH2 h), 49.6 (C15), 49.5
13.5 Hz, CH (5)), 8.66 (s, 1H, CH (2)), 15.09 (s, 1H, –COOH). 13C
4
(C15), 43.3 (C16), 43.2 (C16), 35.3 (C11), 31.8 (CH2
29.2 (CH2 ), 29.1 (CH2 d), 28.9 (CH2 ), 25.9 (CH2 b), 22.6 (CH2
14.0 (CH3), 8.1 (C12,13). CI/NH3-MS (positive mode) m/z 501.3 [M],
502.3 [MH+]. Anal. Calcd for C27H36FN3O5: C, 64.65; H, 7.23; N, 8.38.
Found: C, 64.69; H, 7.20; N, 8.41.
g), 29.4 (CH2 f),
NMR (DMSO-d6) d 177.6 (d, JC–F = 2.5 Hz, C4), 174.8 (C17), 169.1
1
2
e
c
a),
(C14), 152.3 (d, JC–F = 249.5 Hz, C6), 147.5 (C2), 143.3 (d, JC–F =
3
10.0 Hz, C7), 138.2 (C9), 122.2 (d, JC–F = 7.2 Hz, C10), 111.5 (d,
2JC–F = 23.2 Hz, C5), 109.1 (C3), 105.8 (d, JC–F = 3.0 Hz, C8), 50.1
3
(C15), 49.7 (C15), 45.7 (C16), 40.9 (C16), 34.3 (C11), 13.8 (CH3),
8.2 (C12,13). CI/NH3-MS (positive mode) m/z 373.3 [M], 374.3
[MH+]. Anal. Calcd for C19H20FN3O4: C, 61.12; H, 5.40; N, 11.25.
Found: C, 61.15; H, 5.42; N, 11.27.
6.1.4.5. 7-[4-((Decyloxy)carbonyl)piperazin-1-yl]-1-cyclopropyl-
6-fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylic acid (5e). A
purification by silica gel chromatography (CH2Cl2–MeOH 1%) pro-
vided the desired compound (319 mg, 41%) as a pale yellow solid.
Mp 152 °C. 1H NMR (CDCl3) d 0.82 (t, 3H, 3J = 6.6 Hz, CH3), 1.24 (m,
6.1.5.2. 7-(4-Propionylpiperazin-1-yl)-1-cyclopropyl-6-fluoro-
1,4-dihydro-4-oxoquinoline-3-carboxylic acid (6b). A purifica-
tion by silica gel chromatography (CH2Cl2–MeOH 2%) provided
the desired compound (435 mg, 75%) as a pale yellow solid. Mp
258 °C. 1H NMR (CDCl3) d 1.19 (t, 5H, 3J = 7.5 Hz, CH3, CH2 (12)),
18H, CH2 (12,13,
aÀ
g
), 1.57 (m, 2H, 3J = 6.6 Hz, CH2 h), 3.23 (m, 4H,
CH2 (16)), 3.48 (m, 1H, CH (11)), 3.63 (m, 4H, CH2 (15)), 4.02 (t, 2H,
3J = 6.6 Hz, CH2
i), 7.25 (d, 1H, JH–F = 6.9 Hz, CH (8)), 7.78 (d, 1H,
4

J. Azéma et al. / Bioorg. Med. Chem. 17 (2009) 5396–5407
5405
3
1.40 (m, 2H, CH2 (13)), 2.41 (q, 2H, 3J = 7.5 Hz, CH2
a
), 3.33 (m, 4H,
C5), 108.2 (C3), 105.0 (d, JC–F = 3.2 Hz, C8), 49.9 (C15), 49.8 (C15),
44.8 (C16), 38.7 (C(CH3)3), 35.3 (C11), 28.4 (CH3), 8.2 (C12,13). CI/
NH3-MS (positive mode) m/z 415.4 [M], 416.4 [MH+]. Anal. Calcd
for C22H26FN3O4Á0.2H2O: C, 63.05; H, 6.35; N, 10.03. Found: C,
62.95; H, 6.40; N, 10.03.
CH2 (16)), 3.54 (tt, 1H, 3J = 7.2 Hz, 3J = 6.9 Hz, CH (11)), 3.80 (m, 4H,
4
CH2 (15)), 7.37 (d, 1H, JH–F = 6.9 Hz, CH (8)), 8.00 (d, 1H,
3JH–F = 12.9 Hz, CH (5)), 8.75 (s, 1H, CH (2)), 14.95 (s, 1H, –COOH).
4
13C NMR (CDCl3) d 176.9 (d, JC–F = 2.6 Hz, C4), 172.4 (C17), 166.8
1
2
(C14), 153.6 (d, JC–F = 251.4 Hz, C6), 147.5 (C2), 145.3 (d, JC–F
=
3
10.4 Hz, C7), 138.9 (C9), 120.2 (d, JC–F = 7.8 Hz, C10), 112.5 (d,
6.1.5.6. 7-(4-Heptanoylpiperazin-1-yl)-1-cyclopropyl-6-fluoro-
1,4-dihydro-4-oxoquinoline-3-carboxylic acid (6f). A purifica-
tion by silica gel chromatography (CH2Cl2–MeOH 2%) provided
the desired compound (320 mg, 47%) as a pale yellow solid. Mp
175 °C. 1H NMR (CDCl3) d 0.88 (t, 3H, 3J = 6.9 Hz, CH3), 1.32 (m,
3
2JC–F = 23.3 Hz, C5), 108.2 (C3), 105.2 (d, JC–F = 3.1 Hz, C8), 49.6
(C15), 41.1 (C16), 35.3 (C11), 26.4 (CH2 a), 9.4 (CH3), 8.3 (C12,13).
CI/NH3-MS (positive mode) m/z 387.3 [M], 388.3 [MH+]. Anal. Calcd
for C20H22FN3O4Á0.2H2O: C, 61.43; H, 5.77; N, 10.74. Found: C,
61.33; H, 5.80; N, 10.76.
10H, CH2 (12,13,
2.38 (t, 2H, 3J = 7.5 Hz, CH2
1H, 3J = 6.9 Hz, 3J = 7.2 Hz, CH (11)), 3.80 (m, 4H, CH2 (15)), 7.37
a
Àc
)), 1.65 (tt, 2H, 3J = 6.9 Hz, 3J = 7.5 Hz, CH2 d),
), 3.33 (m, 4H, CH2 (16)), 3.55 (tt,
e
6.1.5.3. 7-(4-Butyrylpiperazin-1-yl)-1-cyclopropyl-6-fluoro-1,4-
dihydro-4-oxoquinoline-3-carboxylic acid (6c). A purification by
silica gel chromatography (CH2Cl2–MeOH 1–2%) provided the
desired compound (460 mg, 76%) as a pale yellow solid. Mp
>260 °C. 1H NMR (CDCl3) d 0.99 (t, 3H, 3J = 7.2 Hz, CH3), 1.20
(m, 2H, CH2 (12)), 1.39 (m, 2H, CH2 (13)), 1.71 (tq, 2H,
4
3
(d, 1H, JH–F = 7.2 Hz, CH (8)), 7.96 (d, 1H, JH–F = 12.9 Hz, CH (5)),
8.70 (s, 1H, CH (2)), 14.97 (s, 1H, –COOH). 13C NMR (CDCl3) d
4
176.8 (d, JC–F = 2.3 Hz, C4), 172.0 (C17), 166.8 (C14), 153.4 (d,
1JC–F = 251.3 Hz, C6), 147.4 (C2), 145.3 (d, JC–F = 10.4 Hz, C7),
2
3
2
138.9 (C9), 119.8 (d, JC–F = 8.0 Hz, C10), 112.2 (d, JC–F = 23.2 Hz,
3
3J = 7.2 Hz, 3J = 7.8 Hz, CH2
a
), 2.37 (t, 2H, 3J = 7.8 Hz, CH2 b),
C5), 107.8 (C3), 105.0 (d, JC–F = 3.2 Hz, C8), 50.1 (C15), 49.3 (C15),
3.33 (m, 4H, CH2 (16)), 3.55 (m, 1H, CH (11)), 3.80 (m, 4H,
45.3 (C16), 41.1 (C16), 35.3 (C11), 33.2 (CH2
e
), 31.5 (CH2 d), 29.0
4
CH2 (15)), 7.37 (d, 1H, JH–F = 7.2 Hz, CH (8)), 7.99 (d, 1H,
(CH2 c), 25.2 (CH2 b), 22.4 (CH2 a), 14.0 (CH3), 8.2 (C12,13). CI/
3JH–F = 12.9 Hz, CH (5)), 8.73 (s, 1H, CH (2)), 14.88 (s, 1H, –
NH3-MS (positive mode) m/z 443.4 [M], 444.4 [MH+]. Anal. Calcd
for C24H30FN3O4: C, 64.99; H, 6.82; N, 9.47. Found: C, 65.17; H,
7.34; N, 8.98.
4
COOH). 13C NMR (CDCl3) d 177.0 (d, JC–F = 2.6 Hz, C4), 171.7
1
(C17), 166.8 (C14), 153.6 (d, JC–F = 251.2 Hz, C6), 147.5 (C2),
2
3
145.4 (d, JC–F = 10.5 Hz, C7), 139.0 (C9), 120.2 (d, JC–F = 7.7 Hz,
2
3
C10), 112.6 (d, JC–F = 23.4 Hz, C5), 108.2 (C3), 105.0 (d, JC–
F = 3.2 Hz, C8), 50.3 (C15), 49.5 (C15), 45.4 (C16), 41.1 (C16),
6.1.5.7. 7-(4-Octanoylpiperazin-1-yl)-1-cyclopropyl-6-fluoro-
1,4-dihydro-4-oxoquinoline-3-carboxylic acid (6g). A purifica-
tion by silica gel chromatography (CH2Cl2–MeOH 2%) provided
the desired compound (420 mg, 61%) as a pale yellow solid. Mp
148 °C. 1H NMR (CDCl3) d 0.81 (t, 3H, 3J = 6.6 Hz, CH3), 1.26 (m,
35.3 (C11), 35.1 (CH2 b), 18.7 (CH2
a), 14.0 (CH3), 8.2 (C12,13).
CI/NH3-MS (positive mode) m/z 401.3 [M], 402.3 [MH+]. Anal.
Calcd for C21H24FN3O4Á0.5H2O: C, 61.45; H, 6.13; N, 10.23.
Found: C, 61.38; H, 6.04; N, 10.28.
14H, CH2 (12,13,
a
À
e
g
)), 1.59 (m, 2H, 3J = 7.5 Hz, CH2 f), 2.32 (t,
), 3.27 (m, 4H, CH2 (16)), 3.49 (m, 1H, CH
2H, 3J = 7.5 Hz, CH2
6.1.5.4. 7-(4-Pentanoylpiperazin-1-yl)-1-cyclopropyl-6-fluoro-
1,4-dihydro-4-oxoquinoline-3-carboxylic acid (6d). A purifica-
tion by silica gel chromatography (CH2Cl2–MeOH 2%) provided
the desired compound (500 mg, 64%) as a pale yellow solid. Mp
258 °C. 1H NMR (CDCl3) d 0.94 (t, 3H, 3J = 7.2 Hz, CH3), 1.21 (m,
(11)), 3.67 (m, 2H, CH2 (15)), 3.80 (m, 2H, CH2 (15)), 7.30 (d, 1H,
3
4JH–F = 6.0 Hz, CH (8)), 7.90 (d, 1H, JH–F = 15.0 Hz, CH (5)), 8.64 (s,
1H, CH (2)), 14.90 (s, 1H, –COOH). 13C NMR (CDCl3) d 176.7 (d,
1
4JC–F = 2.2 Hz, C4), 171.9 (C17), 166.7 (C14), 153.5 (d, JC–F
=
2
249.7 Hz, C6), 147.3 (C2), 145.4 (d, JC–F = 10.5 Hz, C7), 138.9 (C9),
119.7 (d, JC–F = 8.2 Hz, C10), 112.1 (d, JC–F = 23.2 Hz, C5), 107.8
(C3), 105.1 (d, JC–F = 3.0 Hz, C8), 50.1 (C15), 49.4 (C15), 45.3
3
2
2H, CH2 (12)), 1.38 (m, 4H, CH2 (13,
a
)), 1.65 (tt, 2H, 3J = 7.2 Hz,
3J = 7.5 Hz, CH2 b), 2.39 (t, 2H, 3J = 7.5 Hz, CH2
c
), 3.33 (m, 4H,
3
CH2 (16)), 3.55 (tt, 1H, 3J = 6.9 Hz, 3J = 7.2 Hz, CH (11)), 3.80 (m,
(C16), 41.1 (C16), 35.4 (C11), 33.2 (CH2
g
), 31.6 (CH2 f), 29.4
4
4H, CH2 (15)), 7.36 (d, 1H, JH–F = 7.2 Hz, CH (8)), 7.98 (d, 1H,
(CH2 e), 29.1 (CH2 c,d), 25.2 (CH2 b), 22.6 (CH2 a), 14.1 (CH3), 8.2
3JH–F = 12.9 Hz, CH (5)), 8.72 (s, 1H, CH (2)), 14.95 (s, 1H, –COOH).
(C12,13). CI/NH3-MS (positive mode) m/z 471.3 [M], 472.3 [MH+].
Anal. Calcd for C26H34FN3O4: C, 66.22; H, 7.27; N, 8.91. Found: C,
66.19; H, 7.44; N, 8.98.
4
13C NMR (CDCl3) d 176.8 (d, JC–F = 2.6 Hz, C4), 171.8 (C17), 166.6
1
2
(C14), 153.5 (d, JC–F = 251.3 Hz, C6), 147.4 (C2), 145.3 (d, JC–F
=
3
10.4 Hz, C7), 138.9 (C9), 119.9 (d, JC–F = 7.8 Hz, C10), 112.2 (d,
2JC–F = 23.4 Hz, C5), 107.9 (C3), 105.1 (d, JC–F = 3.2 Hz, C8), 50.1
6.1.5.8. 7-(4-Decanoylpiperazin-1-yl)-1-cyclopropyl-6-fluoro-
1,4-dihydro-4-oxoquinoline-3-carboxylic acid (6h). A purifica-
tion by silica gel chromatography (CH2Cl2–MeOH 1–2%) provided
the desired compound (330 mg, 45%) as a pale yellow solid. Mp
146 °C. 1H NMR (CDCl3) d 0.84 (t, 3H, 3J = 6.9 Hz, CH3), 1.30 (m,
3
(C15), 49.4 (C15), 45.4 (C16), 41.1 (C16), 35.3 (C11), 32.9 (CH2 c),
27.3 (CH2 b), 22.5 (CH2
a), 13.8 (CH3), 8.2 (C12,13). CI/NH3-MS
(positive mode) m/z 415.4 [M], 416.4 [MH+]. Anal. Calcd for
C22H26FN3O4: C, 63.60; H, 6.31; N, 10.11. Found: C, 63.60; H,
6.82; N, 9.53.
16H, CH2 (12,13,
a
Àf)), 1.62 (tq, 2H, 3J = 7.8 Hz, 3J = 6.9 Hz, CH2
g),
2.37 (t, 2H, 3J = 7.8 Hz, CH2 h), 3.28 (m, 2H, CH2 (16)), 3.35 (m, 2H,
CH2 (16)), 3.55 (tt, 1H, 3J = 7.2 Hz, 3J = 6.9 Hz, CH (11)), 3.71 (m, 2H,
6.1.5.5. 7-[4-(Pivaloyl)piperazin-1-yl]-1-cyclopropyl-6-fluoro-
1,4-dihydro-4-oxoquinoline-3-carboxylic acid (6e). A purifica-
tion by silica gel chromatography (CH2Cl2–MeOH 2%) provided
the desired compound (560 mg, 90%) as a pale yellow solid. Mp
>260 °C. 1H NMR (CDCl3) d 1.21 (m, 2H, CH2 (12)), 1.33 (s, 9H,
CH3), 1.40 (m, 2H, CH2 (13)), 3.32 (m, 4H, CH2 (16)), 3.54 (tt, 1H,
3J = 7.2 Hz, 3J = 7.5 Hz, CH (11)), 3.90 (m, 4H, CH2 (15)), 7.37 (d,
4
CH2 (15)), 3.85 (m, 2H, CH2 (15)), 7.33 (d, 1H, JH–F = 7.2 Hz, CH
(8)), 7.86 (d, 1H, 3JH–F = 12.9 Hz, CH (5)), 8.64 (s, 1H, CH (2)), 14.95
(s, 1H, –COOH). 13C NMR (CDCl3) d 176.7 (d, JC–F = 2.6 Hz, C4),
4
171.9 (C17), 166.7 (C14), 153.5 (d, 1JC–F = 251.3 Hz, C6), 147.4 (C2),
2
3
145.3 (d, JC–F = 10.4 Hz, C7), 138.9 (C9), 119.9 (d, JC–F = 7.9 Hz,
2
3
C10), 112.2 (d, JC–F = 23.4 Hz, C5), 107.9 (C3), 105.1 (d, JC–F
3.2 Hz, C8), 50.2 (C15), 49.4 (C15), 45.3 (C16), 41.1 (C16), 35.3
(C11), 33.2 (CH2 h), 31.8 (CH2 ), 29.4 (CH2 d, ,f), 29.2 (CH2 ), 25.3
=
4
3
1H, JH–F = 7.2 Hz, CH (8)), 8.01 (d, 1H, JH–F = 12.9 Hz, CH (5)),
8.74 (s, 1H, CH (2)), 14.90 (s, 1H, COOH). 13C NMR (CDCl3) d
g
e
c
4
177.0 (d, JC–F = 2.5 Hz, C4), 176.7 (C17), 166.8 (C14), 153.6 (d,
(CH2 b), 22.6 (CH2 a), 14.1 (CH3), 8.2 (C12,13). CI/NH3-MS (positive
1JC–F = 251.3 Hz, C6), 147.5 (C2), 145.4 (d, JC–F = 10.4 Hz, C7),
mode) m/z 485.6 [M], 486.6 [MH+]. Anal. Calcd for C27H36FN3O4: C,
2
3
2
138.9 (C9), 120.2 (d, JC–F = 7.7 Hz, C10), 112.5 (d, JC–F = 23.4 Hz,
66.78; H, 7.47; N, 8.65. Found: C, 67.39; H, 8.01; N, 8.31.

5406
J. Azéma et al. / Bioorg. Med. Chem. 17 (2009) 5396–5407
6.1.5.9. 7-(4-Dodecanoylpiperazin-1-yl)-1-cyclopropyl-6-flu-
oro-1,4-dihydro-4-oxoquinoline-3-carboxylic acid (6i). A purifi-
cation by silica gel chromatography (CH2Cl2–MeOH 1–1.5%)
provided the desired compound (508 mg, 66%) as a pale yellow so-
lid. Mp 149 °C. 1H NMR (CDCl3) d 0.81 (t, 3H, 3J = 6.3 Hz, CH3), 1.20
(m, 2H, CH2 (16)), 3.27 (m, 2H, CH2 (16)), 3.69 (s, 1H, CH2 (11)), 3.69
(m, 2H, CH2 (15)), 3.81 (s, 2H, CH2Ph), 3.90 (s, 2H, CH2 (15)), 7.29 (m,
3
6H, CH (8, ar)), 7.99 (d, 1H, JH–F = 12.9 Hz, CH (5)), 8.75 (s, 1H, CH
(2)), 14.88 (s, 1H, –COOH). No 13C NMR was available for this product
due to its lack of solubility. CI/NH3-MS (positive mode) m/z 449.4
[M], 450.4 [MH+]. Anal. Calcd for C25H24FN3O4Á0.2H2O: C, 66.27; H,
5.42; N, 9.27. Found: C, 66.39; H, 5.77; N, 8.61.
(m, 20H, CH2 (12,13,
a
Àh)), 1.60 (m, 2H, 3J = 7.5 Hz, CH2
i), 2.32 (t,
2H, 3J = 7.5 Hz, CH2
j), 3.28 (m, 4H, CH2 (16)), 3.49 (m, 1H, CH
(11)), 3.67 (m, 2H, CH2 (15)), 3.80 (m, 2H, CH2 (15)), 7.30 (d, 1H,
3
4JH–F = 7.2 Hz, CH (8)), 7.90 (d, 1H, JH–F = 12.9 Hz, CH (5)), 8.65 (s,
6.1.6. 7-(4-(2-Benzyloxyacetyl)piperazin-1-yl)-1-cyclopropyl-6-
fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylic acid (7)
1H, CH (2)), 14.80 (s, 1H, –COOH). 13C NMR (CDCl3) d 176.7 (d, 4J
C–F = 2.2 Hz, C4), 171.9 (C17), 166.7 (C14), 153.5 (d,
CP 1 (1.2 g, 3.6 mmol) and triethylamine (0.51 mL, 5.42 mmol)
were stirred in 20 mL of dry methylene chloride at 0 °C for
15 min. Benzyloxyacetyl chloride (1 g, 5.4 mmol) was added drop-
wise. After stirring at 0 °C for 15 min and then at room tempera-
ture for 2 h, the mixture was filtered and the resulting solids
were washed with water and methylene chloride. The aqueous
layer was extracted with 3 Â 20 mL of CH2Cl2. After extraction,
the organic layers were dried over MgSO4 and concentrated. The
remaining residue was purified by silica gel chromatography
(CH2Cl2–MeOH 2%) to yield the desired product (0.73 g, 43%) as a
pale yellow solid. Mp 210 °C. 1H NMR (CDCl3) d 1.19 (m, 2H, CH2
(12)), 1.37 (m, 2H, CH2 (13)), 3.30 (m, 4H, CH2 (16)), 3.52 (tt, 1H,
3J = 6.9 Hz, 3J = 7.2 Hz, CH (11)), 3.76 (m, 2H, CH2 (15)), 3.86 (m,
2H, CH2 (15)), 4.25 (s, 2H, Ph-CH2–O–), 4.62 (s, 2H, –O–CH2–CO–),
1JC–F = 249.7 Hz, C6), 147.4 (C2), 145.3 (d, JC–F = 10.5 Hz, C7),
2
3
2
138.9 (C9), 119.8 (d, JC–F = 7.5 Hz, C10), 112.2 (d, JC–F = 23.2 Hz,
C5), 107.8 (C3), 105.1 (d, JC–F = 3.7 Hz, C(8)), 50.2 (C15), 49.4
3
(C15), 45.3 (C16), 41.1 (C16), 35.4 (C11), 33.2 (CH2
j
), 31.8 (CH2
), 29.5 (CH2 f), 29.4 (CH2 ), 29.3
), 14.1 (CH3) 8.2
i), 29.7 (CH2 h), 29.6 (CH2
g
e
(CH2 d), 29.2 (CH2
c
), 25.2 (CH2 b), 22.6 (CH2 a
(C12,13). CI/NH3-MS (positive mode) m/z 513.4 [M], 514.4 [MH+].
Anal. Calcd for C29H40FN3O4Á0.5H2O: C, 66.64; H, 7.90; N, 8.04.
Found: C, 66.67; H, 7.84; N, 8.08.
6.1.5.10. 7-(4-Tetradecanoylpiperazin-1-yl)-1-cyclopropyl-6-flu-
oro-1,4-dihydro-4-oxoquinoline-3-carboxylic acid (6j). A purifica-
tion by silica gel chromatography (CH2Cl2–MeOH 1–2%) provided the
desired compound (490 mg, 65%) as a paleyellow solid. Mp148 °C. 1H
NMR (CDCl3) d 0.86 (t, 3H, 3J = 6.6 Hz, CH3), 1.25 (m, 24H, CH2
3
7.35 (m, 6H, CH (8,ar)), 8.00 (d, 1H, JH–F = 12.9 Hz, CH (5)), 8.73
(s, 1H, CH (2)), 14.90 (s, 1H, –COOH). 13C NMR (CDCl3) d 176.7 (d,
1
(12,13,
3J = 7.5 Hz, CH2
3J = 7.2 Hz, CH (11)), 3.72 (m, 2H, CH2 (15)), 3.86 (m, 2H, CH2 (15)),
aÀj
)), 1.67 (tt, 2H, 3J = 7.5 Hz, 3J = 6.9 Hz, CH2 k), 2.37 (t, 2H,
), 3.32 (m, 4H, CH2 (16)), 3.55 (tt, 1H, 3J = 6.9 Hz,
4JC–F = 2.6 Hz, C4), 167.7 (C17), 166.6 (C14), 153.4 (d, JC–
2
l
F = 251.3 Hz, C6), 147.3 (C2), 145.2 (d, JC–F = 10.4 Hz, C7), 138.8
(C9), 136.9 (Cq ar), 128.4 (C ar), 128.0 (C ar), 127.9 (C ar), 119.8
4
3
3
2
7.34 (d, 1H, JH–F = 6.9 Hz, CH (8)), 7.95 (d, 1H, JH–F = 12.9 Hz, CH
(d, JC–F = 7.8 Hz, C10), 112.1 (d, JC–F = 23.4 Hz, C5), 107.7 (C3),
(5)), 8.70 (s, 1H, CH (2)), 14.88 (s, 1H, –COOH). 13C NMR (CDCl3) d
105.1 (d, JC–F = 3.2 Hz, C8), 73.2 (–O–CH2–CO), 69.5 (–CH2-Ph),
3
4
176.9 (d, JC–F = 2.6 Hz, C4), 171.9 (C17), 166.7 (C14), 153.5 (d,
50.0 (C15), 49.3 (C15), 44.8 (C16), 41.3 (C16), 35.3 (C11), 8.1
(C12,13). CI/NH3-MS (positive mode) m/z 479.3 [M], 480.3 [MH+].
Anal. Calcd for C26H26FN3O5Á0.2H2O: C, 64.65; H, 5.51; N, 8.70.
Found: C, 64.67; H, 5.50; N, 8.64.
1JC–F = 251.2 Hz, C6), 147.4 (C2), 145.4 (d, JC–F = 10.4 Hz, C7), 138.9
2
3
2
(C9), 120.1 (d, JC–F = 7.9 Hz, C10), 112.4 (d, JC–F = 23.4 Hz, C5),
108.1 (C3), 105.0 (d, JC–F = 3.1 Hz, C8), 50.2 (C15), 49.4 (C15), 45.3
3
(C16), 41.0 (C16), 35.3 (C11), 33.2 (CH2
,h, ), 29.5 (CH2 f, ), 29.4 (CH2 d), 29.3 (CH2
(CH2
l
), 31.9 (CH2 k), 29.6 (CH2
j,i
g
a
e
c), 25.3 (CH2 b), 22.6
6.1.7. 7-(4-(2-Hydroxyacetyl)piperazin-1-yl)-1-cyclopropyl-6-
fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylic acid (8)
), 14.1 (CH3), 8.2 (C12,13). CI/NH3-MS (positive mode) m/z
541.4 [M], 542.4 [MH+]. Anal. Calcd for C31H44FN3O4: C, 68.73; H,
8.19; N, 7.76. Found: C, 68.69; H, 8.21; N, 7.73.
Compound 7 (0.52 g, 1.09 mmol) was dissolved in hot dimethyl-
formamide. After cooling at room temperature, palladium on char-
coal (10%, 116 mg, 0.1 mmol) was added. After two evacuations
and fillings with hydrogen, the mixture was hydrogenated for 96 h
at room temperature under atmospheric pressure. The catalyst
was filtered through a pad of Celite. Celite was washed with methy-
lene chloride and solvents were evaporated in vacuo. The remaining
residue was purified by silica gel chromatography (CH2Cl2–MeOH
1%) to yield the desired product (175 mg, 60%) as a white solid. Mp
>260 °C. 1H NMR (DMSO-d6) d 1.19 (m, 2H, CH2 (12)), 1.32 (m, 2H,
CH2 (13)), 3.30 (m, 4H, CH2 (16)), 3.70 (m, 4H, CH2 (15)), 3.82 (tt,
1H, 3J = 6.9 Hz, 3J = 7.5 Hz, CH (11)), 4.16 (d, 2H, 3J = 6.0 Hz, –CH2OH),
4.70 (t, 1H, 3J = 6.0 Hz, –OH), 7.57 (d, 1H, 4JH–F = 7.5 Hz, CH (8)), 7.92
6.1.5.11. 7-(4-Hexadecanoylpiperazin-1-yl)-1-cyclopropyl-6-
fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylic acid (6k). A
purification by silica gel chromatography (CH2Cl2–MeOH 1–2%)
provided the desired compound (298 mg, 58%) as a pale yellow so-
lid. Mp 147 °C. 1H NMR (CDCl3) d 0.86 (t, 3H, 3J = 6.6 Hz, CH3), 1.32
(m, 28H, CH2 (12,13,
), 2.37 (t, 2H, 3J = 7.5 Hz, CH2 n), 3.32 (m, 4H, CH2 (16)), 3.55 (m,
1H, CH (11)), 3.71 (m, 2H, CH2 (15)), 3.86 (m, 2H, CH2 (15)), 7.34
aÀ
l
)), 1.65 (tt, 2H, 3J = 7.5 Hz, 3J = 7.2 Hz, CH2
m
4
3
(d, 1H, JH–F = 7.2 Hz, CH (8)), 7.94 (d, 1H, JH–F = 12.9 Hz, CH (5)),
8.68 (s, 1H, CH (2)), 14.86 (s, 1H, –COOH). 13C NMR (CDCl3) d
4
3
176.9 (d, JC–F = 2.6 Hz, C4), 171.9 (C17), 166.7 (C14), 153.5 (d,
(d, 1H, JH–F = 13.2 Hz, CH (5)), 8.60 (s, 1H, CH (2)), 15.20 (s, 1H,
1JC–F = 251.3 Hz, C6), 147.4 (C2), 145.4 (d, JC–F = 10.4 Hz, C7),
–COOH). 13C NMR (DMSO-d6) d 176.2 (d, JC–F = 2.6 Hz, C4), 165.7
2
4
3
2
138.9 (C9), 120.0 (d, JC–F = 7.8 Hz, C10), 112.3 (d, JC–F = 23.2 Hz,
C5), 108.0 (C3), 105.0 (d, JC–F = 3.1 Hz, C8), 50.2 (C15), 49.3 (C15),
(C17), 164.7 (C14), 152.8 (d, 1JC–F = 249.5 Hz, C6), 147.9 (C2), 144.6
3
2
3
(d, JC–F = 10.2 Hz, C7), 138.9 (C9), 118.7 (d, JC–F = 7.7 Hz, C10),
2
3
45.3 (C16), 41.0 (C16), 35.3 (C11), 33.2 (CH2 n), 31.9 (CH2 ), 29.6
m
110.8 (d, JC–F = 23.1 Hz, C5), 106.7 (C3), 106.5 (d, JC–F = 3.0 Hz,
C8), 60.0 (–CH2OH), 49.3 (C15), 48.9 (C15), 44.8 (C16), 41.6 (C16),
35.7 (C11), 7.4 (C12,13). CI/NH3-MS (negative mode) m/z 389.2
[M]. Anal. Calcd for C19H20FN3O5Á1H2O: C, 56.02; H, 5.44; N, 10.31.
Found: C, 56.12; H, 5.62; N, 10.34.
(CH2 ,k, ,h, ), 29.5 (CH2 f, ), 29.4 (CH2 d), 29.3 (CH2 c), 25.3
l
j,i
g
e
(CH2 b), 22.6 (CH2 a), 14.0 (CH3), 8.2 (C12,13). CI/NH3-MS (positive
mode) m/z 569.4 [M], 570.4 [MH+]. Anal. Calcd for C33H48FN3O4: C,
69.57; H, 8.49; N, 7.38. Found: C, 69.61; H, 8.41; N, 7.43.
6.1.5.12. 1-Cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-[4-(2-phen-
ylacetyl)piperazin-1-yl] quinoline-3-carboxylic acid (6l). A purifica-
tion by silica gel chromatography (CH2Cl2–MeOH 2%) provided the
desired compound (340 mg, 50%) as a pale yellow solid. Mp 252 °C.
1H NMR (CDCl3) d 1.19 (m, 2H, CH2 (12)), 1.38 (m, 2H, CH2 (13)), 3.12
6.2. Stability tests
HPLC analysis was performed on a Waters system (Waters
Associates Inc., Milford, MA, USA) consisting of a 600 controller
pump, a PDA996 diode array detector, a 717 plus autosampler,

J. Azéma et al. / Bioorg. Med. Chem. 17 (2009) 5396–5407
5407
12. Carew, J. S.; Giles, F. J.; Nawrocki, S. T. Cancer Lett. 2008, 269, 7.
and a Lisa 30 Ecrosas (ICS) oven at 30 °C. The instrument was
controlled by the Empower software. The experiments were car-
ried out on a C18 Luna (PhenomenexÒ) reverse-phase column
13. Silva, H.; Valério Barra, C.; França da Costa, C.; de Almeida, M. V.; César, E. T.;
Silveira, J. N.; Garnier-Suillerot, A.; Silva de Paula, F. C.; Pereira-Maia, E. C.;
Fontes, A. P. J. Inorg. Biochem. 2008, 102, 767.
14. Sánchez-Martín, R.; Campos, J. M.; Conejo-García, A.; Cruz-López, O.; Báñez-
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Med. Chem. 2005, 48, 3354.
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2002, 45, 3765.
16. Van Quaquebeke, E.; Simon, G.; André, A.; Dewelle, J.; Yazidi, M. E.; Bruyneel,
F.; Tuti, J.; Nacoulma, O.; Guissou, P.; Decaestecker, C.; Braekman, J. C.; Kiss, R.;
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Gailly, P.; Kondo, S.; Blanco, G.; Kiss, R. Neurosurgery 2008, 62, 211.
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(length 100 mm  3 mm i.d., 3
lm particle size) eluted with a mo-
bile phase consisting of various linear gradients of acetonitrile (Sol-
vent A)/water (+0.1% TFA) (Solvent B) at a flow rate of 0.6 mL/min.
The eluting solvents were prepared daily and degassed with he-
lium during analyses. The detection was set at 280 nm. All injec-
tions (10
lL) were made in duplicate. In a typical experiment,
100 L of a 1 mM solution of tested compound in DMSO was di-
l
luted with phosphate buffer (pH 7.4) to a final volume of 1 mL.
An aliquot was discarded and kept at 4 °C (t0). The remaining solu-
tion placed in a sealed tube was heated in a water bath at 37 °C for
6 days (t6d). To evaluate the rate of degradation of the tested com-
pounds, CP 1 (degradation product expected for 3, 5a–c, 6f, 6h, and
6k) and compound 8 (degradation product expected for 2, 4b–d,
and 4h) were injected. Their respective retention times were 2.6
and 1.6 min. For compounds whose stability was less than 100%,
the ratio [compound peak area/(compound peak + degradation
product peak) area] was calculated.
6.3. Biology
6.3.1. Evaluation of in vitro cell proliferation by means of the
MTT colorimetric assay
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The assessment of cell population growth is based on the capa-
bility of living cells to reduce the yellow product MTT (3-[4,5-
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occurring in the mitochondria.15,16 The five cell lines were incu-
bated for 24 h in 96-microwell plates (at a concentration of 104
to 3 Â 104 cells/mL culture medium depending on the cell type)
to ensure adequate plating prior to cell growth determination.
The number of living cells after 120 h of culture in the presence
or absence (control) of the various drugs is directly proportional
to the intensity of the blue color, measured by spectrophotometry
using a 680XR microplate reader (Bio-Rad Laboratories Inc, Hercu-
les, CA) at a wavelength of 570 nm (with a reference at 630 nm).
Each experiment was carried out in hexaplicate. Nine concentra-
tions ranging from 10À3 to 10À7 M (with semi-log decrease in con-
centration) were tested for each of the compounds under study.
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