Synthesis of gatifloxacin derivatives and their biological activities against Mycobacterium leprae and Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.bmc.2012.12.011

Novel 3′-piperazinyl derivatives of the 8-hydrogeno and 8-methoxy-6-fluoro-1-cyclopropyl-4-quinolone-3-carboxylic acid scaffolds were designed, synthesized and characterized by ¹H, ¹³C and ¹⁹F NMR, and HRMS. The activity of these derivatives against pathogenic mycobacteria (M. leprae and M. tuberculosis), wild-type (WT) strains or strains harboring mutations implicated in quinolone resistance, were determined by measuring drug concentrations inhibiting cell growth (MIC) and/or DNA supercoiling by DNA gyrase (IC₅₀), or inducing 25% DNA cleavage by DNA gyrase (CC₂₅). Compound 4 (with a methoxy in R₈ and a secondary carbamate in R₃′) and compound 5 (with a hydrogen in R₈ and an ethyl ester in R₃′) displayed biological activities close to those of ofloxacin but inferior to those of gatifloxacin and moxifloxacin against M. tuberculosis and M. leprae WT DNA gyrases, whereas all of the compounds were less active in inhibiting M. tuberculosis growth and M. leprae mutant DNA gyrases. Since R₃′ substitutions have been poorly investigated previously, our results may help to design new quinolone derivatives in the future.

Full text of DOI:10.1016/j.bmc.2012.12.011

Bioorganic & Medicinal Chemistry 21 (2013) 948–956
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of gatifloxacin derivatives and their biological activities
against Mycobacterium leprae and Mycobacterium tuberculosis
Catherine Gomez a, Prishila Ponien a, Nawal Serradji a, Aazdine Lamouri a, Alix Pantel b,c, Estelle Capton b,
Vincent Jarlier b,c,d, Guillaume Anquetin a, , Alexandra Aubry b,c,d,
⇑
⇑
a Université Paris Diderot, Sorbonne Paris Cité, ITODYS, UMR CNRS 7086, 15 rue J-A de Baïf, 75205 Paris Cedex 13, France
b UPMC Université Paris 06, ER5 (EA 1541), laboratoire de Bactériologie-Hygiène, F-75005 Paris, France
c AP-HP, Hôpital Pitié-Salpêtrière, laboratoire de Bactériologie-Hygiène, F-75013 Paris, France
d Centre National de Référence des Mycobactéries et de la Résistance des Mycobactéries aux Antituberculeux, F-75013 Paris, France
a r t i c l e i n f o
a b s t r a c t
Novel 30-piperazinyl derivatives of the 8-hydrogeno and 8-methoxy-6-fluoro-1-cyclopropyl-4-
quinolone-3-carboxylic acid scaffolds were designed, synthesized and characterized by 1H, 13C and 19F
NMR, and HRMS. The activity of these derivatives against pathogenic mycobacteria (M. leprae and M.
tuberculosis), wild-type (WT) strains or strains harboring mutations implicated in quinolone resistance,
were determined by measuring drug concentrations inhibiting cell growth (MIC) and/or DNA supercoiling
Article history:
Received 12 October 2012
Revised 29 November 2012
Accepted 3 December 2012
Available online 20 December 2012
by DNA gyrase (IC50), or inducing 25% DNA cleavage by DNA gyrase (CC25). Compound 4 (with a methoxy
Keywords:
Quinolones
Mycobacterium leprae
Mycobacterium tuberculosis
DNA gyrase
0
in R8 and a secondary carbamate in R30) and compound 5 (with a hydrogen in R8 and an ethyl ester in R3
)
displayed biological activities close to those of ofloxacin but inferior to those of gatifloxacin and moxiflox-
acin against M. tuberculosis and M. leprae WT DNA gyrases, whereas all of the compounds were less active
0
in inhibiting M. tuberculosis growth and M. leprae mutant DNA gyrases. Since R3 substitutions have been
poorly investigated previously, our results may help to design new quinolone derivatives in the future.
SAR
Gatifloxacin
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
and the Final Push strategy to eliminate leprosy3 emphasize the
need to increase surveillance, control, and treatment efforts. The
Controlling leprosy and tuberculosis (TB), two communicable
diseases caused by Mycobacterium leprae and Mycobacterium tuber-
culosis, respectively, is often challenging due to the emergence of
multidrug-resistant strains.1 The World Health Assembly decided,
in 1991, to ‘eliminate leprosy as a public health problem’ by the
year 2000. After an important decrease in the number of new cases
of leprosy detected each year worldwide, from 763,000 in 2000 to
299,000 in 2005, the decrease was gradual since 2005, with
228,000 new cases at the end of 2010.1 In conjunction with this
still high number of new cases, leprosy resistant to dapsone and
rifampin therapy emerged. Similarly, TB affecting millions of peo-
ple worldwide remains a public health challenge, partly due to
the emergence of multidrug-resistant tuberculosis (MDR-TB), that
is, TB resistant to both isoniazid and rifampin, which currently rep-
resents a serious obstacle to TB control. As a consequence of the
threat of drug-resistant TB and leprosy, the WHO Stop TB Strategy2
development of new antimycobacterial drugs should have two
objectives: shorten treatment to improve compliance and maintain
activity against drug-resistant strains. Quinolones are good candi-
dates for the development of more powerful agents against leprosy
and tuberculosis.4 They play a critical role in the treatment of
MDR-TB and drug-resistant leprosy. Moxifloxacin and gatifloxacin
(Fig. 1) are the most active quinolones against M. tuberculosis and
M. leprae, and are under evaluation to shorten the treatment of
TB and leprosy caused by susceptible mycobacteria.4 Unfortu-
nately, MDR-TB strains resistant also to quinolones have emerged,
0
leading to virtually untreatable eXtensively Drug Resistant0 (XDR)
tuberculosis. Cases of leprosy resistant to dapsone, rifampin and
quinolones have been reported also.5 In the future, quinolone resis-
tance will likely increase in TB and leprosy due to (i) poor MDR-TB
management, (ii) increasing use of quinolones in susceptible TB,
and (iii) wide use of quinolones for empirical treatment of a larger
range of non-mycobacterial infections such as urinary and respira-
tory tract infections, diarrhea and typhoid fever, which are com-
mon infections in the areas of high prevalence of tuberculosis
and leprosy.6,7
⇑
Corresponding authors. Tel.: +33 1 57 27 72 57; fax: +33 1 57 27 72 63 (G.A.).
Present address: Laboratoire de Bactériologie, Faculté de Médecine Pierre et Marie
Curie, 91 Boulevard de l’hôpital, 75634 Paris Cedex 13, France. Tel.: +33 1 40 77 97
46; fax: +33 1 45 82 75 77 (A.A.).
Quinolones act by forming a reversible ternary complex with
their bacterial targets (DNA gyrase and topoisomerase IV) and
DNA, blocking bacterial growth and chromosome fragmentation,
E-mail addresses: guillaume.anquetin@univ-paris-diderot.fr (G. Anquetin),
alexandra.aubry@upmc.fr (A. Aubry).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.12.011

C. Gomez et al. / Bioorg. Med. Chem. 21 (2013) 948–956
949
O
N
O
N
O
5
8
F
COOH
4
1
F
COOH
F
COOH
10
9
6
3
2
H
N 7
N
N
N
1'
OMe
HN
O
N
2'
Me
Me
4'
H
NH
3'
Ciprofloxacin
Ofloxacin
Moxifloxacin
Compound
R3'
CH3
R4'
H
R8
OMe
O
Gatifloxacin
1
F
COOH
CH2OC(O)NEt2
H
H
2
N
N
H
H
OMe
H
N
R8
CH2OC(O)NHBu
3
R4'
4
H
H
H
H
H
Et
Et
OMe
H
R3'
5
COOEt
COOH
General structure of
gatifloxacin
and
6
OMe
H
compounds
1-10
7
8
OMe
H
9
10
OMe
Figure 1. General structure of ciprofloxacin, ofloxacin, moxifloxacin, gatifloxacin and compounds 1–10.
which leads to irreversible lethal damage.8–10 Mutations in the sole
target of M. tuberculosis and M. leprae, that is, DNA gyrase (GyrA2-
GyrB2), are the prominent established mechanisms of acquired
resistance to quinolones. Recently we established structure activity
relationships (SAR) for quinolones with respect to M. leprae and M.
tuberculosis.11–13 These data underline the importance of (i) a
cyclopropyl substituent at the N1 position, (ii) a fluorine atom at
the R6 position (except for garenoxacin), (iii) a substituted carbon
atom at the C8 position (except for ciprofloxacin), and (iv) a cycle
or a bicycle containing a nitrogen atom at the R7 position (Fig. 1).
Despite the fact that modifications at the R7 position have been
extensively studied, the few modifications of the piperazine core
began with 3-hydrogeno (11) and 3-methoxy (12) 2,4,5-trifluoro-
benzoic acids. The first four steps of the synthesis were adapted
from earlier work.21,22 After conversion of the carboxylic acid to
the corresponding acyl chloride, the key step was the reaction be-
tween ethyl 3-(diethylamino) acrylate and the corresponding trif-
luorobenzoyl chloride in the presence of triethyl amine (Scheme 1).
After transaminolysis with cyclopropylamine, the quinolone ring
formation proceeded smoothly with potassium carbonate in hot
DMF to afford 13 and 14 with good yields (62% and 65%, respec-
tively, after 4 steps).
Due to the poor reactivity of the C7–F bond towards nucleophilic
aromatic substitution, the C7 atom of 13 and 14 was rendered more
electrophile by the coordination of a Lewis acid, –BF2, onto both car-
bonyl functions (Scheme 1). In a recently described procedure,
1.2 equiv of K2CO3 and 1.4 equiv of BF3ꢀEt2O23 instead of 10 equiv
of BF3ꢀEt2O were used.21 Using this improved non-chromatographic
process, the difluoro–boron complexes 15 and 16, required for the
nucleophilic aromatic substitutions, were obtained with good yield
(89% and 69%, respectively). Finally, compounds 1–6 were obtained
by coupling 15 and 16 with various piperazines24 in refluxing aceto-
nitrile for 1–7 days with poor to moderate yield (Table 1) in agree-
ment with published data.17,22 In our case, the poor yield observed
for compounds 2–4 (approximately 20%) can be correlated to the
steric hindrance between the large substituent on the piperazine
0
that have been investigated were restricted to R3 = Me, diMe, Et,
CH2OH, CH2F, CH2CN, CH2N(CH3)2 and C6H5.14–17 The R7 position
is fundamental for biological activity as illustrated by two recent
crystal structures of topoisomerase IV with DNA and moxifloxa-
cin.18,19 The M. tuberculosis DNA gyrase reaction core resembles
closely the core of these structures.20 The bulky substituent in R7,
which occupies a large and solvent-accessible pocket of DNA gyr-
ase, interacts with the beginning of the
a2 helix (residues 498–
502 of GyrB) of one of the three regions of the TOPRIM domain
which are part of the quinolone-binding pocket. These observa-
tions prompted us to select gatifloxacin, that contains a 30-methyl
piperazine in R7 (Fig. 1), as the starting compound for the search of
more potent quinolones against M. leprae and M. tuberculosis in or-
der to take advantage of the interaction between R7 and the large
quinolone-binding pocket of DNA gyrase. Hence, the methyl group
of gatifloxacin was replaced by different functional groups (with
F2
B
O
N
O
N
O
0
R3 being an ethyl ester, carboxylic acid, secondary or tertiary car-
F
F
COOH
a-d
F
F
COOEt
e
F
F
bamates) in order to assess the impact of the functionalization of
O
0
this R3 -position on the antimycobacterial activity of those novel
F
gatifloxacin derivatives (Fig. 1).
R8
R8
R8
2. Results and discussion
2.1. Chemistry
11: R8 = H
13: R8 = H
15: R8 = H
12: R8 = OMe
14: R8 = OMe
16: R8 = OMe
Scheme 1. Synthetic pathway to difluoro–boron complexes 15 and 16. Reagents
and conditions: (a) oxalyl chloride, DMF, DCM, 24 h; (b) Et2N–CH@CH–COOEt, Et3N,
toluene, 90 °C, 5h (75–79%); (c) Et2O/EtOH:2:1, cyclopropylamine, 25 °C, 3h; (d)
DMF, K2CO3, 90 °C, 5h (82–83%); (e) BF3ꢀEt2O, THF, K2CO3 (69–89%).
Compounds 1–6 and 7–10 were prepared as shown in Scheme 1
and Table 1, and Table 2, respectively. The synthetic investigations

950
C. Gomez et al. / Bioorg. Med. Chem. 21 (2013) 948–956
Table 1
Synthesis of compounds 1–6
Moreover, the use of microwaves to shorten reaction time and im-
prove yield,17,25 in order to obtain compounds 1–6, did not prove
to be efficient. Concerning the synthesis of compounds 7–10 (Ta-
ble 2), 7–8 were directly obtained by basic hydrolysis of 5 and 6,
respectively. Recently, quinolones bearing an ethyl group at the
O
O
N
F
F
COOBF2
F
COOH
0
N4 position were shown to reduce the emergence of resistant mu-
a-b
NH
N
N
tants.26 Hence, we synthesized N4 –Et quinolones (9–10) in order
0
R8
HN
.2HCl
R8
0
to assess the impact of N4 -ethylation on their biological activity
HN
R3'
against quinolone-resistant strains. Thus, to begin with, compounds
5Et and 6Et were obtained after alkylation of both secondary amine
and carboxylic acid (Table 2). The last step was the basic hydrolysis
of both ethyl ester groups to obtain the corresponding dicarboxylic
acids 9–10 with moderate to good yields.
15: R8 = H
16
: R8 = OMe
R3'
1-6
0
Compounds
R8
R3
Yield (%)
1
2
3
4
5
6
H
OMe
H
OMe
H
OMe
CH2OC(O)NEt2
CH2OC(O)NEt2
CH2OC(O)NHBu
CH2OC(O)NHBu
COOEt
55
23
23
18
47
51
2.2. Antimicrobial activity
In order to explore the importance of substituents in the R30 position
on the piperazine rings, compounds 1–10 were compared to well
known quinolones (ofloxacin, ciprofloxacin, moxifloxacin and gatiflox-
acin). The ten compounds were screened for their capacity to inhibit (i)
the growth of M. tuberculosis (MIC) and (ii) the supercoiling activity of
wild-type (WT) M. tuberculosis and M. leprae DNA gyrases (IC50); then
(iii) they were screened for their capacity to induce 25% cleavage
(CC25) of DNA from M. leprae WT and mutant (G89C and A91 V) strains.
Residues 89 and 91 correspond to amino acids 88 and 90, and 81 and
83, in the M. tuberculosis and Escherichia coli numbering systems,
respectively. The results are shown in Table 3.
COOEt
Reagents and conditions: (a) CH3CN, NEt3, reflux, 1–7 days, Only for 5: (b) MeOH,
NEt3, reflux, 24 h.
ring (carbamate function) and the quinolone core (especially for
R8 = OMe). But, due to the steric hindrance, this nucleophilic aro-
matic substitution appeared to be regiospecific and prevented us
from time-consuming and unnecessary selective protection and
deprotection of the N4 secondary amine of the piperazine ring. To
confirm this assessment, we used a selectively N1 -protected
0
0
0
piperazine, 17, bearing the smallest functional group in R3 (i.e.,
2.2.1. MIC determination
COOEt), and the less hindered difluoro–boron complex, that is, 15
(Scheme 2). After 70 h at reflux in acetonitrile, no reaction occurred
between 15 and 17. This absence of reactivity confirmed the ob-
served regiospecificity of the nucleophilic substitution of N1 , due
to the steric hindrance caused by bulky substituent’s in R3 .
The compounds were screened for their in vitro activity
against M. tuberculosis H37Rv comparatively to the in vitro activ-
ity of well known quinolones. Compound 5, with a MIC of 4
0
0
lg/mL, was the most potent inhibitor in the series. Indeed,
Table 2
Synthesis of compounds 7–10
O
O
F
COOH
F
COOH
.HCl
a
N
N
N
N
HN
R8
HN
R8
COOEt
: R8 = H
: R8 = OMe
COOH
5
6
7: R8 = H
b
8: R8 = OMe
O
O
N
F
COOEt
F
COOH
.HCl
a
N
N
N
N
R8
N
R8
Et
Et
COOEt
COOH
9: R8 = H
5Et: R8 = H
10: R8 = OMe
6Et: R8 = OMe
0
0
Compounds
R8
R3
R4
Yield (%)
5Et
6Et
7
8
9
H
OMe
H
OMe
H
OMe
COOEt
COOEt
COOH
COOH
COOH
COOH
Et
Et
H
H
Et
Et
39
44
56
78
75
41
10
Reagents and conditions: (a) CH3OH/H2O:4:1, LiOH, rt, 1–5 days; (b) DMF, Cs2CO3 or NaHCO3, EtI, 60 °C, 1–3 days.

C. Gomez et al. / Bioorg. Med. Chem. 21 (2013) 948–956
951
O
N
O
N
F
F
COOBF2
F
COOH
a
EtOOC
N
NH
COOEt
+
N
x
N
Bn
Bn
15
17
Scheme 2. Synthetic pathway explored to selectively obtain novel 20-substituted piperazinyl quinolones. Reagents and conditions: (a) CH3CN, NEt3, reflux, 70 h.
Table 3
Activities of compounds 1–10 inhibiting M. leprae DNA cleavable complex formation (CC25) with wild-type and modified DNA gyrase, M. tuberculosis H37Rv growth (MIC), and
DNA supercoiling of DNA gyrase (IC50
)
O
O
O
N
F
COOH
F
COOH
F
COOH
H
N
N
N
N
N
OMe
N
R8
N
O
R4'
Me
Me
H
NH
R3'
Moxifloxacin
Ofloxacin
General structure for
ciprofloxacin, gatifloxacin
and
compounds 1-10
cLogPa
M. tuberculosis
M. leprae
0
0
Compound
R3
R4
R8
MIC (l
g/mL)
IC50
(l
g/mL)
IC50
WT
(lg/mL)
CC25
WT
(l
g/mL)
A91 V
G89C
Gatifloxacin
Moxifloxacin
Ciprofloxacin
Ofloxacin
1
2
3
4
5
6
7
8
9
CH3
—
H
H
H
—
H
Me
H
H
H
H
H
H
OMe
OMe
H
Bridge C1–C8
H
OMe
H
OMe
H
OMe
H
OMe
H
ꢁ0.27
ꢁ1.50
ꢁ0.73
ꢁ0.51
ꢁ0.22
0.86
0.125
0.5
0.5
1
128
32
16
8
3
2
6
6
3
2
2
8
1
2
2
10
>240
60
30
10
20
80
30
27
>240
>240
15
12
30
>240
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
CH2OC(O)NEt2
CH2OC(O)NEt2
CH2OC(O)NHBu
CH2OC(O)NHBu
COOEt
COOEt
COOH
COOH
COOH
265
50
30
17
27
21
20
16
48
87
118
20
22
10
20
17
30
20
35
55
>240
no
ꢁ0.33
0.76
>240
>240
>240
>240
>240
No
ꢁ1.35
ꢁ0.27
ꢁ1.42
ꢁ1.48
ꢁ0.28
ꢁ0.34
4
>32
32
16
32
32
H
H
Et
Et
10
COOH
OMe
>240
No: not observable.
>240: observable but 25% of cut was not reached.
a
cLogP values were calculated using ChemDrawUltra10.0.
compared to that of ofloxacin, the MICs of compounds 5 and 4
were four to eightfold higher, whereas the MICs of compounds
1–3 and 6–10 were at least 16-fold higher. With R8 = H, and con-
served for MICs, considering the nature of the substitution in R8,
compounds harboring a OMe were more potent than their hydro-
geno counterparts, except for compound 10 where it resulted in
a small decrease of the inhibitory activity.
0
sidering the functional group in R3 , the rank of order for antimy-
cobacterial activity decreased from COOEt (as in 5),
CH2OC(O)NHBu (as in 3), COOH (as in 7 and 9), to CH2OC(O)NEt2
(as in 1). For R8 = OMe, the rank of order was similar except that
2.2.3. Drug-inducible DNA cleavage by M. leprae gyrase
With M. leprae WT DNA gyrase, compounds 3–5 and 7–8
showed the best induction of DNA cleavage at CC25s below
0
the compound with COOEt (as in 6) in R3 was the least active
(Table 3). Considering the nature of the substitution in R8, com-
pounds harboring OMe were more potent than their hydrogeno
counterparts, except for compound 6.
30
10
l
l
g/mL. Compound 4 was as active as ofloxacin with a CC25 of
g/mL (Table 3), whereas compounds 1, 6, 9 and 10 had CC25
s
ranging from 8-fold higher (6) to undeterminable (1 and 9–10).
CC25 values varied depending on the nature of the 30 substituent
on the piperazine ring. For R8 = H, CC25 values decreased in the or-
der: CH2OC(O)NHBu (as in 3), COOEt (as in 5), and COOH (as in 7),
to CH2OC(O)NEt2 (as in 1) and COOH (with R4 = Et as in 9) while
for R8 = OMe they decreased in the order: CH2OC(O)NHBu (as in
2.2.2. Inhibition of M. tuberculosis and M. leprae wild-type DNA
gyrases
Among the compounds tested against M. tuberculosis and M. lep-
rae WT DNA gyrases, compounds 3–8 proved to be the most effi-
0
cient inhibitors, with an IC50 630
l
g/mL. IC50s varied depending
4) and COOH (as in 8), to CH2OC(O)NEt2 (as in 2) and COOEt (as
in 6), and to COOH (with R4 = Et as in 10). In contrast to what
0
on the nature of the 30 substituent on the piperazine ring. For
R8 = H, IC50s decreased in the order CH2OC(O)NHBu (as in 3), COOEt
(as in 5), and COOH (as in 7), to CH2OC(O)NEt2 (as in 1) and COOH
was observed for MICs and IC50s, no clear conclusion concerning
cleavage activity can be drawn from the nature of the substitution
0
0
(with R4 = Et as in 9) while for R8 = OMe, IC50s decreased in the or-
in R8. Indeed, with carbamates in R3 (i.e., 1–4), compounds bearing
der CH2OC(O)NHBu (as in 4), COOEt (as in 6), and COOH (as in 8), to
CH2OC(O)NEt2 (as in 2) and COOH (with R4 = Et as in 10). As ob-
a OMe in R8 were equally or more active than their hydrogeno
counterparts, whereas with a carboxylic acid function in R3 (i.e.,
0
0

952
C. Gomez et al. / Bioorg. Med. Chem. 21 (2013) 948–956
7–10), the presence or absence of a OMe did not make any differ-
ence, and compound 5 with an ethyl ester function in R3 was four-
could be compensated for by a better cell-wall penetration, as sug-
gested by their higher clogP values.
0
times more active than its OMe derivative 6.
Regarding mutated M. leprae DNA gyrases, all compounds were
equally poorly active against the two mutated strains (Table 3).
It was noticed that the concentration of quinolones required to
inhibit DNA supercoiling by gyrase is substantially higher than
that required to inhibit growth, as observed previously,13,30 due
to the poisoning effect of quinolones interacting with the
topoisomerases.
2.3. General discussion of structure–activity relationships
Finally, the DNA cleavage assay was thought to be more rele-
vant than the supercoiling inhibition assay for measurement of
the activity of a gyrase inhibitor.27,31 However, it seems that this
conclusion does not apply to M. leprae, as the effective quinolone
concentrations measured by the DNA cleavage assay were slightly
different from those measured by the supercoiling assay and were
less well correlated with the concentrations inhibiting M. tubercu-
losis growth. The DNA gyrase supercoiling inhibition assay and
DNA gyrase cleavable-complex assay are distinct in that the former
measures catalytic inhibition, whereas the latter probes an estab-
lished equilibrium between the ternary DNA–enzyme–drug com-
plexes in which the DNA is either broken or intact.31
Considering all biological results, the most successful substitu-
tions at R30 were secondary carbamates (4) and ethyl ester (5) func-
0
tions, whereas a tertiary carbamate function in R3 (1–2) and
0
ethylation in N4 (9–10) abolished activity. Concerning the impact
of the number of hydrogen bond donors/acceptors or of lipophilic-
ity (Table 3) on the biological results, a few trends can be observed.
Concerning carbamates, compounds with secondary carbamates
were more potent than tertiary carbamates in all assays. Indeed,
as compounds 1 and 3, and compounds 2 and 4, had the same lipo-
philicity, the enhanced activities of compounds 3 and 4 were
linked to secondary carbamates, which was due to their hydrogen
atom that might play the role of a hydrogen bond donor. But the
0
lack of a hydrogen bond donor in R3 did not abolish all activity,
2.5. Conclusions
as compound 5, substituted with an ethyl ester function at the
0
R3 position, proved to be very active. Concerning carboxylic acid
In conclusion, this work describes the synthesis, characteriza-
tion, and evaluation, using M. tuberculosis and M. leprae, of ten
gatifloxacin derivatives obtained by reaction of 8-hydrogeno (15)
and 8-methoxy (16) 1-cyclopropyl-6,7-difluoro-1,4-dihydro-4-
oxoquinoline-3-carboxylato-difluoro–boron with several substi-
tuted piperazines. Taking into account all biological results, none
of our gatifloxacin derivatives were more active than existing quin-
olones (Table 3). With biological activities close to those of ofloxa-
cin, the two most active compounds were 4 (R8 = OMe and
0
function, compounds not ethylated at N4 (7–8) were more active
than compounds that were (9–10), and they were as active as sec-
ondary carbamates (3–4), except with respect to the MIC for M.
0
tuberculosis. Finally, ethylation at N4 did not enhance biological
activities against M. leprae with gyrase mutations implicated in
quinolone resistance.
2.4. Correlation between MICs, IC50s and CC25
s
0
0
R3 = CH2OC(O)NEt2) and 5 (R8 = H and R3 = COOEt). It is evident
then that a OMe substituent in R8 is not mandatory in the search
for potent antimycobacterial quinolones. Finally, since R30 substitu-
tions have been poorly investigated to date and never with the
substituents explored here, our results will help the future design
of new quinolones. Indeed, our results prove that piperazines can
The concentrations of compounds that inhibited 50% of the DNA
supercoiling activity of the M. tuberculosis DNA gyrase were well
correlated with the concentrations required to inhibit 50% of the
corresponding activity of M. leprae (Fig. 2, R2 = 0.95).
On the other hand, MICs and IC50s for M. tuberculosis were not
proportional (data not shown). This absence of nonproportionality
has been noted previsouly,13,17,27 presumably reflecting differences
in the cell-permeating properties and accumulation of the different
quinolones.28 Penetration of the M. tuberculosis cell wall by quino-
lones has not been evaluated yet, because the study of the myco-
0
be functionalized in R3 in various ways (e.g., by secondary carba-
mate or ester functions) without a deleterious effect on the biolog-
0
ical activity. Moreover, as the N4 –H bond has a crucial importance
0
in the quinolone backbone, this R3 substitution offers a supple-
bacterial cell wall is still
a
difficult and uncertain task.28
mentary possibility for the synthesis of quinolone–drug conju-
gates, quinolone hybrids or quinolone prodrugs.
Nevertheless, penetration of the M. tuberculosis cell wall seems to
be at least 100-fold less efficient than that of E. coli.29 Thus, the
0
3. Materials and methods
3.1. Reagents
MICs of compounds 7–10, which differed only by N4 -ethylation
and the substitution in R8, were similar but the IC50 values varied:
0
the IC50s of compounds 9 and 10, ethylated in N4 , were at least
twice as high as those of 7 and 8, which were not ethylated in
N4 . Ethyl substitution decreased bacterial target affinity, which
0
The following four quinolones were provided by their corre-
sponding manufacturers: gatifloxacin (Grünenthal, Levallois-Perret,
France); ciprofloxacin and moxifloxacin (Bayer Pharma, Puteaux,
France); and ofloxacin (Sigma–Aldrich Chimie, Saint Quentin
Fallavier, France).
140
R² = 0.953
120
100
80
60
40
20
0
3.2. In vitro antimicrobial activity
M. tuberculosis H37Rv was grown on Löwenstein-Jensen med-
ium. MICs were determined by the proportion method as described
previously.32 Briefly, 103 and 105 CFU were spread onto 7H11 agar
supplemented with 10% oleic acid–albumin–dextrose-catalase and
containing serial twofold dilutions of the compound2. Colonies
were enumerated after 21–30 days of incubation at 37 °C. The
MIC was defined as the drug concentration at which the bacterial
growth was reduced to 1% or less of that of the drug-free control
culture.33
0
50
100
150
200
250
300
IC50 M.tuberculosis (µg/mL)
Figure 2. Correlation between IC50 against M. leprae and IC50 against M. tuberculosis.

C. Gomez et al. / Bioorg. Med. Chem. 21 (2013) 948–956
953
3.3. DNA supercoiling assay
D2O or DMSO-d6 as solvent, on a BRUKER AC 300 or 400 spectrom-
eter at 300 or 400 MHz for 1H, 75 or 100 MHz for 13C and 282 or
377 MHz for 19F spectra. Chemical shifts (d) were expressed in
parts per million relative to the signal indirectly (i) to CHCl3 (d
7.27 for 1H and (ii) to CDCl3 (d 77.2) for 13C and directly (iii) to
CFCl3 (internal standard) (d 0.0) for 19F. Chemical shifts are given
in ppm and peak multiplicities are designated as follows: s, singlet;
br s, broad singlet; d, doublet; dd, doublet of doublet; t, triplet; q,
quadruplet; quint, quintuplet; m, multiplet. High resolution mass
spectra (HRMS) of the ten final compounds (1–10) were obtained
Recently, we developed an in vitro assay that replaces the time-
consuming mouse footpad system for antimicrobial evaluation in
M. leprae.11 M. tuberculosis and M. leprae DNA gyrase was purified
as described previously.11,13 The reaction mixture (total volume,
30
7.5], 25 mM KCl, 6 mM magnesium acetate, 2 mM spermidine,
4 mM dithiothreitol, bovine serum albumin [0.36 g/mL], 10 mM
potassium glutamate, 1 mM ATP [pH 8.0]) and relaxed pBR322
DNA (0.4 g) as the substrate. Gyrase proteins (300 ng of GyrA
ll) contained DNA gyrase assay buffer (40 mM Tris–HCl [pH
l
0
l
from the Service Central d0analyse de Solaize0 (Centre Nationale
and 250 ng of GyrB) were mixed in the presence of increasing con-
centrations of quinolones for 1 h at 37 °C for M. tuberculosis and 2 h
at 30 °C for M. leprae. Reactions were terminated by the addition of
50% glycerol containing 0.25% bromophenol blue, and the total
reaction mixture was subjected to electrophoresis in a 1% agarose
gel in 0.5ꢂ TBE (Tris–borate–EDTA, pH 8.3) buffer. After electro-
phoresis for 5.5 h at 50 V, the gel was stained with ethidium bro-
de la Recherche Scientifique) and were recorded on a Waters spec-
trometer using electrospray ionization-TOF (ESI-TOF).
The synthesis of compounds 13–16 has been previously de-
scribed.21,23,34 Compounds 1–10 were synthesized according to
procedures described below and were dissolved in DMSO or NaOH
for biological tests.
mide (0.7
lg/mL). The inhibitory effect of quinolones on DNA
3.6.2. Ethyl 1-cyclopropyl-7-(30-(ethoxycarbonyl)-40-
gyrase was assessed by determining the concentration of drug re-
quired to inhibit the supercoiling activity of the enzyme by 50%
(IC50). Supercoiling activity was assessed by tracing the brightness
of the bands corresponding to the supercoiled pBR322 DNA with
Molecular Analyst software (Bio-Rad).
ethylpiperazin-1-yl)-6-fluoro-4-oxo-1,4-dihydroquinoline-3-
carboxylate 5Et
A solution of 5 (0.107 g, 0.26 mmol) in 10 mL of DMF, NaHCO3
(61.2 mg, 0.73 mmol) and iodoethane (0.074 mL, 0.925 mmol)
was vigorously stirred at 90 °C during 60 h. DCM and water were
added, after extraction with DCM (3 ꢂ 30 mL), the organic layers
were collected, washed with water (3 ꢂ 30 mL), dried over MgSO4,
filtered and evaporated. The oily residue was purified by flash
chromatography on silica gel (DCM/MeOH: 96:4) affording the
product 5Et as a colorless oil (44 mg, 0.102 mmol, 39%).
Note: 5Et was obtained as a mixture with the corresponding car-
boxylic acid in C3, which was not a problem as the next step con-
sisted in the hydrolysis of both ester functions.
3.4. DNA cleavage assay
M. leprae DNA gyrases were purified as described previ-
ously.11,12 The reaction mixture (total volume, 20
lL) contained
the DNA gyrase assay buffer (40 mM Tris–HCl [pH 7.5], 25 mM
KCl, 20 mM magnesium acetate, 2 mM spermidine, 4 mM dithio-
threitol, 0.1
[0.36 g/mL], 3 mM ATP [pH 8.0]) and supercoiled pBR322 DNA
(0.4 g) as the substrate. Three hundred nanograms of M. leprae
lg/mL of yeast tRNA, bovine serum albumin
l
l
1H NMR (CDCl3): d 1.07–1.11 (m, 5H, CH2(cPr) and NCH2CH3),
1.24–1.28 (m, 5H, CH2(cPr) and OCH2CH3), 1.36 (t, 3H, OCH2CH3,
0
GyrA and 250 ng of M. leprae GyrB were mixed in the presence of
increasing concentrations of quinolones for 1 h at 30 °C. Three
microliters of 2% SDS were added to separate the free DNA from
JH–H = 7.14Hz), 2.57 (m, 2H, H2 and NCH), 2.79 (m, 1H, NCH),
0
0
0
0
3.29–3.47 (m, 7H, H2 , H3 , H5 and H6 ), 4.17 (q, 2H, COO(CH2)CH3,
JH–H = 7.1Hz), 4.20 (q, 2H, COOCH2, JH–H = 7.1Hz), 4.36 (q, 2H,
COOCH2, JH–H = 7.1Hz), 7.23 (d, 1H, H8, JH–F = 5.9Hz), 8.00 (d, 1H,
H5, JH–F = 13.1Hz), 8.45 (s, 1H, H2).
the cleaved DNA covalently linked to DNA gyrase, and 3
lL of a
1
l
g/mL solution of proteinase K was added to remove the cova-
lently bound GyrA protein. Incubation was continued for 30 min
at 37 °C. The reactions were stopped as described above for super-
coiling. The DNA products were examined by agarose gel electro-
phoresis, and the drug concentration that resulted in 25% DNA
cleavage (CC25) was determined.
19F NMR (CDCl3) d ꢁ123.9 s, 1F, F6).
3.6.3. Ethyl 1-cyclopropyl-7-(30-(ethoxycarbonyl)-40-
ethylpiperazin-1-yl)-6-fluoro-8-methoxy-4-oxo-1,4-
dihydroquinoline-3-carboxylate 6Et
A solution of 6 (152 mg, 0.35 mmol) in 10 mL of DMF, Cs2CO3
(392 mg, 1.20 mmol) and iodoethane (0.20 mL, 0.315 mmol) was
vigorously stirred at 60 °C during 19 h. DCM and water were
added, after extraction with DCM (3 ꢂ 30 mL), the organic layers
were collected, washed with water (3 ꢂ 30 mL), dried over MgSO4,
filtered and evaporated. The oily residue was purified by flash
chromatography on silica gel (DCM/MeOH: 96:4) affording the
product 6Et as a brown oil (75 mg, 0.153 mmol, 44%). 1H NMR
(CDCl3): d 0.89 (m, 2H, CH2(cPr)), 1.07–1.12 (m, 5H, NCH2CH3
and CH2(cPr)), 1.21 (t, 2H, OCH2, JH–H = 7.0Hz), 1.34 (t, 2H, OCH2,
3.5. Correlation between MICs, IC50s and CC25
s
The relationships between the MICs and IC50s or CC50s were as-
sessed by estimating a linear regression between two components.
The strength of this relationship was quantified by the R2 coeffi-
cient and displayed graphically by the regression line.
3.6. Synthesis
0
3.6.1. General chemistry methods
JH–H = 7.0Hz), 2.54 (m, 2H, H2 and NCH2), 2.75–2.80 (m, 1H,
0
0
0
All materials were obtained from commercial suppliers and
used without further purification. Thin-layer chromatography
was performed on TLC plastic sheets of silica gel 60F254 (layer
thickness 0.2 mm) from Merck. Column chromatography purifica-
tion was carried out on silica gel 60 (70–230 mesh ASTM, Merck).
Melting points were determined either on a digital melting point
apparatus (Electrothermal IA 8103) and are uncorrected or on a
Kofler bench type WME (Wagner & Munz). IR, 1H, 19F and 13C
NMR spectra were used to confirm the structures of all compounds.
IR spectra were recorded on a Perkin Elmer Spectrum 100 FT-IR
spectrometer and NMR spectra were recorded, using CDCl3, CD3CN,
NCH2), 3.25–3.30 (m, 1H, H2 ) 3.30–3.35 (m, 3H, H3 and 2H6 ),
0
3.53–3.56 (m, 1H, H5 ), 3.69 (s, 3H, OCH3), 3.83 (quint., 1H,
CH(cPr)), 4.15 (q, 2H, OCH2, JH–H = 7.1Hz), 4.32 (q, 2H, OCH2,
JH–H = 7.1Hz), 7.81 (d, 1H, H5, JH–F = 12.4Hz), 8.51 (s, 1H, H2). 19F
NMR (CDCl3) d ꢁ121.6 s, 1F, F6). 13C NMR (CDCl3): 9.3 and 9.4
0
(2CH2(cPr)), 14.2 and 14.4 (2 OCH2CH3), 39.4 (CH(cPr)), 49.0 (C2 ),
0
0
49.6 (NCH2), 50.7 (C5 ), 53.3 (C6 ), 60.7 and 60.8 (2 OCH2), 62.6
3
0
(OCH3), 63.5 (C3 ), 108.9 (d, C5, JC–F = 23.3 Hz), 109.9 (C3), 125.6
3
(d, C10
,
3JC–F = 8.0 Hz), 132.8 (s, C9), 137.5 (d, C8, JC–F = 12.0 Hz),
1
145.7 (s, C7), 150.6 (s, C2), 155.8 (d, C6, JC–F = 257.1 Hz), 165.6
(COOH), 166.2 and 171.4 (2 COOEt), 172.7 (d, C4, JC–F = 2.2 Hz).
4

954
C. Gomez et al. / Bioorg. Med. Chem. 21 (2013) 948–956
3.6.4. General procedure for the synthesis of compounds (1–6)
A mixture of the suitable difluoro–boron quinolone 15–16
(1.0 equiv) and the suitable piperazine (2.3 equiv) and dry NEt3
(5.0 equiv) was stirred at refluxed of dry CH3CN (3 mL) between
one day and one week. After evaporation, the crude residue was ta-
ken up in a 1:1 CHCl3/H2O mixture. After extraction, the organic
layer was washed with water (2 ꢂ 5 mL) and dried over MgSO4.
The oily residue was purified by flash chromatography (CHCl3/
MeOH: 10:0 to 9:1) and then by recrystallization (H2O/CH3CN:
9:1) and lyophilization to afford the desired target compounds
(1–6).
above, starting from 15 (149 mg, 0.48mmol) and piperazin-2-
ylmethyl butyl carbamate (356 mg, 1.24 mmol). 1H NMR (CD3CN)
3
d 0.84–0.88 (m, 2H, CH2(cPr)), 0.86 (appt, 3H, CH3, JH–H = 7.2 Hz),
0
1.22–1.47 (m, 6H, CH2(cPr) and CH2), 2.86–2.95 (m, 1H, H2 ),
0
0
0
0
2.99–3.14 (m, 4H, H6 , H5 , NCH2), 3.16–3.24 (m, 2H, H2 , H5 ),
0
0
0
3.26–3.33 (m, 1H, H3 ), 3.56–3.71 (m, 3H, H2 , H6 , CH(cPr)), 4.05–
4
4.15 (m, 2H, OCH2), 7.54 (d, 1H, H8, JH–F = 6.9 Hz), 7.90 (d, 1H,
H5, JH–F = 13.4 Hz), 8.73 (s, 1H, H2), 14.85 (br s, 1H, OH). 19F
3
NMR (CD3CN) d ꢁ121.2 s, 1F, F6). 13C NMR (CD3CN) d 8.6 (s,
CH2(cPr)), 14.1 (s, CH3), 20.6 and 32.5 (s, CH2), 36.9 (s, CH(cPr)),
0
0
0
0
41.3 (s, NCH2), 44.7 (s, C5 ), 49.9 (s, C6 ), 52.1 (s, C2 ), 54.6 (s, C3 ),
3
65.1 (s, OCH2), 107.7 (d, C8, JC–F = 4.0 Hz), 108.1 (s, C3), 112.4 (d,
C5, JC–F = 23.2 Hz), 120.9 (d, C10
2
3.6.4.1. 1-Cyclopropyl-7-(30-((diethylcarbamoyloxy)methyl)pip-
erazin-1-yl)-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic
,
3JC–F = 8.0 Hz), 135.5 (s, C9),
1
140.5 (s, C7), 149.5 (s, C2), 156.2 (d, C6, JC–F = 247.7 Hz), 157.8 (s,
NC(O)O), 174.9 (s, COOH), 178.1 (s, C4). HR-ESMS: m/z: calculated
for C23H30FN4O5: 461.2200; found 461.2178 [M+H]+ IR (ATR):
2960 (OH acide), 1718 (ester), 1624 (amide), 1488 (C@C), 1303
(C–N, C–C, C–O), 1266 (C–N, C–C, C–O), 1102 (C–F). Mp = 225 °C.
acid (1).
Compound 1 was synthesized (yellow powder,
114 mg, 0.25 mmol, 55%) according to the general procedure de-
scribed above, starting from 15 (140 mg, 0.45 mmol) and pipera-
zin-2-ylmethyl diethyl carbamate (297 mg, 1.03 mmol). 1H NMR
(CD3CN) d 1.10–1.13 (m, 8H, CH2(cPr) and 2CH3), 1.27–1.34 (m,
3
2H, CH2(cPr)), 2.76 (app t, 1H, H2 , JH–H = 10.7 Hz), 2.93–3.02 (m,
3.6.4.4.
cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-
3-carboxylic acid (4). Compound 4 was synthesized (yellow
7-(30-((Butylcarbamoyloxy)methyl)piperazin-1-yl)-1-
0
0
0
0
0
2H, H6 and H5 ), 3.09–3.20 (m, 2H, H3 and H5 ), 3.27 (q, 4H,
3
0
0
NCH2, JH–H = 7.0 Hz), 3.58–3.71 (m, 3H, H2 ,H6 and CH(cPr)), 4.04
3
4
(d, 2H, OCH2, JH–H = 5.8 Hz), 7.54 (d, 1H, H8, JH–F = 7.4 Hz), 7.94
powder, 35 mg, 0.072 mmol, 18%) according to the general proce-
dure described above, starting from 16 (138 mg, 0.40 mmol) and
piperazin-2-ylmethyl butyl carbamate (300 mg, 1.05 mmol). 1H
3
(d, 1H, H5, JH–F = 13.4 Hz), 8.70 (s, 1H, H2), 15.18 (br s, 1H, OH).
19F NMR (CD3CN) d ꢁ123.0 s, 1F, F6). 13C NMR (CD3CN) d 9.2 (s,
3
CH2(cPr)), 14.3 and 15.1 (s, 2CH3), 37.3 (s, CH(cPr)), 42.8 and 43.1
NMR (CD3CN) d 0.90 (app. t, 3H, CH3, JH–H = 7.3 Hz), 0.94–0.99
4
4
0
0
0
(s, 2CH2), 46.3 (s, C5 ), 52.2 (d, C6 , JC–F = 4.6 Hz), 54.6 (d, C2 , JC–F
= 4.4 Hz), 55.6 (s, C3 ), 67.7 (s, OCH2), 107.9 (d, C8, JC–F = 3.4 Hz),
109.0 (s, C3), 112.8 (d, C5, JC–F = 23.3 Hz), 120.9 (d, C10
= 7.8 Hz), 141.2 (s, C9), 147.6 (d, C7, JC–F = 10.0 Hz), 149.6 (s, C2),
(m, 2H, CH2(cPr)), 1.11–1.48 (m, 6H, CH2(cPr) and 2 CH2), 2.94–
3
0
0
0
0
3.15 (m, 6H, H5 , H6 and NCH2), 3.19–3.29 (m, 1H, H3 ), 3.36–3.49
2
3JC–F
(m, 2H, H2 ), 3.95–4.01 (m, 1H, CH(cPr)), 4.08–4.16 (m, 2H,
0
,
2
3
OCH2), 7.80 (d, 1H, H5, JH–F = 12.5 Hz), 8.75 (s, 1H, H2), 14.90 (br
1
155.2 (d, C6, JC–F = 247.7 Hz), 157.0 (s, NC(O)O), 168.2 (s, COOH),
s, 1H, OH). 19F NMR (CD2Cl2) d ꢁ120.1 s, 1F, F6). 13C NMR (CD2Cl2)
4
178.8 (d, C4, JC–F = 2.4 Hz). HR-ESMS: m/z: calculated for
d 9.8 (s, 2CH2(cPr)), 13.9 (s, CH3), 20.3 and 32.4 (s, 2CH2), 40.9 (s,
23H30FN4O5: 461.2200; found 461.2174 [M+H]+ IR (ATR): 2988
CH(cPr)), 41.2 (s, NCH2), 46.1 (s, C5 ), 51.6 (s, C6 ), 51.7 (s, C2 ),
0
0
0
C
2
0
(OH acide), 1696 (ester), 1625 (amide), 1483 (C@C), 1456 (C@C),
1385 (C–N, C–C, C–O), 1268 (C–N, C–C, C–O), 1171 (C–N, C–C,
C–O), 1072 (C–F). Mp = 124 °C.
55.1 (s, C3 ), 62.9 (s, OCH3) 66.1 (s, OCH2), 107.6 (d, C5, JC–F
= 24.5 Hz), 108.1 (s, C3), 122.3 (d, C10
,
3JC–F = 8.8 Hz), 134.5 (s, C9),
2
3
140.0 (d, C7, JC–F = 11.4 Hz), 146.1 (d, C8, JC–F = 5.1 Hz) 150.4 (s,
C2), 156.6 (d, C6, JC–F = 251.0 Hz), 156.5 (s, NC(O)O), 166.9 (s,
1
3
3.6.4.2. 1-Cyclopropyl-7-(30-((diethylcarbamoyloxy)methyl)pip-
erazin-1-yl)-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-
COOH), 177.5 (d, C4, JC–F = 3.0 Hz). HR-ESMS: m/z: calculated for
C
24H32FN4O6: 491.2306; found 491.2298 [M+H]+ IR (ATR): 3320
3-carboxylic acid (2).
Compound 2 was synthesized (yellow
(OH acide), 1714 (ester), 1616 (cetone), 1442 (C@C), 1316 (C–N,
C–C, C–O), 1270 (C–N, C–C, C–O), 1246 (C–N, C–C, C–O), 1057
(C–F). Mp = 104 °C.
powder, 50 mg, 0.10 mmol, 23%) according to the general proce-
dure described above, starting from 16 (150 mg, 0.44 mmol) and
piperazin-2-ylmethyl diethyl carbamate (292 mg, 1.02 mmol). 1H
NMR (CD3CN) d 0.85–0.89 (m, 2H, CH2(cPr)), 0.95–1.06 (m, 8H,
3.6.4.5.
fluoro-4-oxo-1,4-dihydroquinoline-carboxylic
(5). Compound 5 was synthesized (yellow powder, 103 mg,
1-Cyclopropyl-7-(30-ethoxycarbonylpiperazin-1-yl)-6-
acid
0
0
2CH3 and CH2(cPr)), 2.87–2.98 (m, 3H, H2 and 2H5 ), 3.05–3.20
3
0
0
(m, 2H, H3 and H6 ), 3.18 (q, 4H, 2CH2, JH–H = 7.0 Hz), 3.32 (d,
3
3
0
0
1H, H6 , JH–H = 10.8 Hz), 3.43 (d, 1H, H2 , JH–H = 11.5 Hz), 3.68 (s,
3H, OCH3), 3.91–3.96 (m, 2H, OCH2), 4.00–4.07 (m, 1H, CH(cPr)),
0.24 mmol, 47%) according to the general procedure described
above, starting from 15 (158 mg, 0.51 mmol) and ethyl-piperazi-
nyl-2-carboxylate (304 mg, 1.32 mmol). 1H NMR (CD3CN) d 1.02–
3
7.71 (d, 1H, H5, JH–F = 12.4 Hz), 8.66 (s, 1H, H2), 14.85 (br s, 1H,
OH). 19F NMR (CD3CN) d ꢁ121.3 s, 1F, F6). 13C NMR (CD3CN) d 9.9
1.10 (m, 2H, CH2(cPr)), 1.18 (t, 3H, CH3, JH–H = 7.0 Hz), 1.26–1.28
3
0
0
(s, 2CH2(cPr)), 13.7 and 14.5 (s, 2CH3), 41.7 (s, CH(cPr)), 42.1 and
(m, 2H, CH2(cPr)), 3.02–3.15 (m, 1H, H5 ), 3.19–3.33 (m, 2H, H6
4
0
0
0
0
0
0
42.6 (s, CH2), 46.6 (s, C5 ), 52.1 (d, C6 , JC–F = 17.2 Hz), 54.7 (d, C2 ,
and H5 ), 3.39–3.50 (m, 2H, H2 and H6 ), 3.53–3.63 (m, 1H, CH(cPr)),
4
3
3
0
0
0
JC–F = 14.6 Hz), 55.6 (s, C3 ), 63.5 (s, OCH3), 66.9 (s, OCH2), 107.9
3.72 (d, 1H, H2 , JH–H = 12.4 Hz), 3.94 (d, 1H, H3 , JH–H = 4.8 Hz),
2
3
4
(d, C5, JC–F = 23.3 Hz), 108.2 (s, C3), 122.6 (d, C10
,
3JC–F = 8.9 Hz),
4.15 (q, 2H, CH2, JH–H = 7.0 Hz), 7.49 (d, 1H, H8, JH–F = 7.3 Hz),
2
3
135.6 (s, C9), 140.7 (d, C7, JC–F = 11.6 Hz), 147.2 (s, C8), 151.6 (s,
C2), 156.5 (s, NC(O)O), 157.2 (d, C6, JC–F = 249.0 Hz), 167.3 (s,
7.84 (d, 1H, H5, JH–F = 13.0 Hz), 8.60 (s, 1H, H2). 19F NMR (CD3CN)
1
d ꢁ123.3 s, 1F, F6), ꢁ151,6 s, 0.44F, BF2). 13C NMR (CD3CN) d 8.7 (s,
0
0
C(O)O), 178.1 (s, C4). HR-ESMS: m/z: calculated for C24H32FN4O6:
491.2306; found 491.2279 [M+H]+ IR (ATR): 2975 (OH acide),
1728 (ester), 1689 (cetone), 1617 (amide), 1438 (C@C), 1315 (C–
N, C–C, C–O), 1272 (C–N, C–C, C–O), 1172 (C–N, C–C, C–O), 1058
(C–F). Mp = 100 °C.
CH2(cPr)), 14.5 (s, CH3), 36.8 (s, CH(cPr)), 44.2 (s, C5 ), 49,7 (d, C6 ,
4
4
0
0
JC–F = 3.1 Hz), 51.9 (d, C2 , JC–F = 5.2 Hz), 57.3 (s, C3 ), 62.8 (s,
CH2), 107.9 (d, C8, JC–F = 3.0 Hz), 108.4 (s, C3), 112.4 (d, C5, JC–F
= 23.2 Hz), 120.9 (s, C10
3
2
,
3JC–F = 7.9 Hz), 140.4 (s, C9), 146.0 (d, C7,
2JC–F = 10.5 Hz), 149.2 (s, C2), 154.6 (d, C6, JC–F = 247.9 Hz), 167.6
(s, COOH), 170.6 (s, COOEt), 178.0 (s, C4). HR-ESMS: m/z: calculated
for C20H23FN3O5: 404.1621; found 404.1610 [M+H]+ IR (ATR): 3002
(OH acide), 1730 (ester), 1628 (cetone), 1492 (C@C), 1458 (C@C),
1381 (C–N, C–C, C–O), 1337 (C–N, C–C, C–O), 1268 (C–N, C–C,
C–O), 1055 (C–F). Mp >300 °C.
1
3.6.4.3.
cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic
acid (3). Compound 3 was synthesized (beige powder, 51 mg,
0.11 mmol, 23%) according to the general procedure described
7-(30-((Butylcarbamoyloxy)methyl)piperazin-1-yl)-1-

C. Gomez et al. / Bioorg. Med. Chem. 21 (2013) 948–956
955
3.6.4.6. 1-Cyclopropyl-7-(30-ethylcarbonylpiperazin-1-yl)-6-flu-
oro-8-methoxy-4-oxo-1,4-dihydroquinoline-carboxylic acid
(6). Compound 6 was synthesized (yellow powder, 97 mg,
3.6.5.3. 1-Cyclopropyl-7-(30-carboxy-40-ethylpiperazin-1-yl)-6-
fluoro-4-oxo-1,4-dihydroquinoline carboxylic acid
(9). Compound 9 was synthesized (white powder, 20.6 mg,
0.22 mmol, 51%) according to the general procedure described
above, starting from 16 (152 mg, 0.44 mmol) and ethyl-piperazi-
nyl-2-carboxylate (266 mg, 1.15 mmol). 1H NMR (CD3CN) d 0.91–
0.047 mmol, 75%) according to the general procedure described
above, starting from 5Et (29 mg, 0.063 mmol). 1H RMN (D2O/
CD3CN: 9:1): d 1.46 (m, 2H, CH2(cPr)), 1.66 (m, 5H, NCH2(CH3)),
and CH2(cPr)), 3.60–3.70 (m, 2H, NCH and NCH(CH3)), 3.83 (m,
3H, NCH2 and NCH(CH3)), 4.02 (m, 1H, CH(cPr)), 4.18 (m, 2H),
4.33 (m, 1H), 4.48 (m, 1H), 7.87 (d, 1H, H8, JH–H = 5.6Hz), 8.05 (d,
1H, H5, JH–H = 12.8Hz), 9.02 (s, 1H, H2). 19F NMR (D2O/CD3CN:
9:1) d ꢁ121.2 s, 1F, F6). 13C RMN (D2O/CD3CN: 9:1): 7.2 (CH2(cPr)),
8.5 (NCH2(CH3)), 35.9 (CH(cPr)), 45.4, 48.7, 48.9, 50.7 (NCH2(CH3)),
1.01 (m, 2H, CH2(cPr)), 1.08–1.12 (m, 2H, CH2(cPr)), 1.16 (t, 3H,
3
0
CH3, JH–H = 7.1 Hz), 2.99–3.05 (m, 1H, H4 ), 3.18–3.22 (m, 1H,
0
0
0
H4 ), 3.31–3.36 (m, 2H, H3 ), 3.45–3.53 (m, 1H, H2 ), 3.66–3.74 (m,
0
0
5H, OCH3, H1 and H2 ), 4.00 (quint., 1H, CH(cPr)), 4.15 (q, 2H,
3
3
CH2, JH–H = 7.1 Hz), 7.85 (d, 1H, H5, JH–F = 11.2 Hz), 8.79 (s, 1H,
H2). 19F NMR (CD3CN) d ꢁ120.8 s, 1F, F6). 13C NMR (CD3CN) d 9.6
2
0
and 9.9 (s, 2CH2(cPr)), 14.4 (s, CH3), 40.7 (s, CH(cPr)), 44.9 (s, C5 ),
67.1 (NCH(COOH) 105.9 (C3), 106.8 (C8), 111.1 (d, C5, JC–F
4
4
0
0
0
51.3 (d, C6 , JC–F = 3.6 Hz), 52.9 (d, C2 , JC–F = 4.6 Hz), 57.6 (s, C3 ),
61.7 (s, CH2), 62.2 (s, OCH3), 107.7 (s, C3), 108.4 (d, C5, JC–F
= 23.4 Hz), 122.7 (d, C10
C7, JC–F = 10.4 Hz), 146.0 (d, C8, JC–F = 5.6 Hz), 150.2 (s, C2), 156.2
= 23.4 Hz), 119.3 (C10), 139.1 (C9), 143.4 (C7), 148.3 (C2), 153.1 (d,
2
1
C6, JC–F = 253.89 Hz), 168.4 (COOH), 176.4 (C4). HR-ESMS: m/z:
,
3JC–F = 11.4 Hz), 134.0 (s, C9), 139.5 (d,
calculated for C20H23FN3O5: 404.1621; found 404.1611 [M+H]+
2
3
Mp = 208–209 °C.
1
(d, C6, JC–F = 248.1 Hz), 166.9 (s, COOH), 171.2 (s, COOEt), 177.2
(s, C4). HR-ESMS: m/z: calculated for C21H25FN3O6: 434.1727;
found 434.1717 [M+H]+ IR (ATR): 3348 (COO–H), 1742 (ester),
1617 (ketone), 1579 (N–H), 1462 (C@C), 1446 (C@C), 1290 (C–N,
C–C, C–O), 1090 (C–N, C–C, C–O), 1058 (C–F). Mp >300 °C.
3.6.5.4. 1-Cyclopropyl-7-(30-carboxy-40-ethylpiperazin-1-yl)-6-
fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline carboxylic acid
(10).
Compound 10 was synthesized (white powder, 55 mg,
0.117 mmol, 41%) according to the general procedure described
above, starting from 6Et (75 mg, 0.286 mmol). 1H RMN (D2O/
CD3CN: 9:1): 1.26 (m, 2H, CH2(cPr)), 1.47 (m, 2H, CH2(cPr)), 1.62
(t, 3H, NCH2(CH3)), JH–H = 7.1Hz), 3.60–3.70 (m, 3H, NCH and
NCH2(CH3)), 3.80–3.90 (m, 3H), 4.02 (s, 3H, OCH3), 4.10–4.20 (m,
2H), 4.36 (m, 1H CH(cPr)), 7.81 (d, 1H, H5, JH–H = 12.8Hz), 9.06 (s,
1H, H2). 19F NMR (D2O/CD3CN: 9:1) d ꢁ119.6 s, 1F, F6). 13C RMN
(D2O/CD3CN: 9:1): 8.4 (CH2(cPr)), 8.7 (NCH2(CH3)), 40.8 (CH(cPr)),
46.4, 49.8, 50.9 (NCH2), 62.5 (s, OCH3), 105.7 (C3), 106.6 (d, C5,
2JC–F = 23.8 Hz), 122.0 (C10), 133.8 (C9), 137.5 (C8), 143.4 (C7),
3.6.5. General procedure for the synthesis of compounds (7–10)
LiOH (10 equiv) was added to a solution of the suitable quino-
lone (1.0 equiv) in MeOH/H2O: 8:2, and the resulting mixture
was stirred during 3 days at room temperature. After addition of
HCl (1M) until pH = 1, the precipitate was filtered and dried afford-
ing the desired targeted compounds (7–10).
3.6.5.1. 7-(30-Carboxypiperazin-1-yl)-1-cyclopropyl-6-fluoro-4-
1
oxo-1,4-dihydroquinoline-3-carboxylic acid (7).
Compound
150.9 (C2), 155.7 (d, C6, JC–F = 250.7 Hz), 168.2 (COOH), 176.5 (C4,
7
was synthesized (white powder, 65 mg, 0.149 mmol, 56%)
4JC–F = 3.1Hz). HR-ESMS: m/z: calculated for C21H25FN3O6:
434.1727; found 434.1714 [M+H]+ Mp = 201–202 °C.
according to the general procedure described above, starting from
5 (107 mg, 0.265 mmol). 1H NMR (D2O/CD3CN: 9:1): d 1.11 (m, 2H,
0
0
CH2(cPr)), 1.36 (m, 2H, CH2(cPr)), 3.35 (m, 2H, H5 and H2 ), 3.53 (m,
Acknowledgements
0
0
0
0
2H, CH(cPr) and H6 ), 3.72 (m, 2H, H6 and H5 ), 3.96 (m, 1H, H2 ),
0
4.06 (m, 1H, H3 ), 7.63 (d, 1H, H8, JH–F = 7.3Hz), 7.92 (d, 1H, H5,
The authors wish to thank The Order of MALTA grants for lep-
rosy research for research funding (postdoctoral fellowship for
CG). We are grateful to Aurélie Chauffour for her precious help
with MIC determinations for M. tuberculosis and Rachid Boudjelloul
for technical assistance. We thank Ekkehard Collatz and Judith
Crane, native English-speaking colleagues, for careful reading of
the manuscript.
JH–F = 13.1Hz), 8.80 (s, 1H, H2). 19F NMR (D2O/CD3CN: 9:1) d
ꢁ121.0 s, 1F, F6). 13C NMR (D2O/CD3CN: 9:1): 6.7 (s, 2CH2(cPr)),
0
0
0
33.5 (s, CH(cPr)), 37.1 (s, C5 ), 43.1 (s, C6 ), 47.0 (s, C2 ), 57.0 (s,
3
2
0
C3 ), 107.6 (s, C3), 108.5 (d, C8, JC–F = 2.23 Hz), 112.5 (d, C5, JC–F
= 23.3 Hz), 121.1 (d, C10
,
3JC–F = 8.0 Hz), 140.3 (s, C9), 145.2 (d, C7,
2JC–F = 10.0 Hz), 149.7 (s, C2), 154.5 (d, C6, JC–F = 250.0 Hz), 169.1
1
4
(COOH), 170.0 (COOH), 178.0 (d, C4, JC–F = 2.0 Hz). HR-ESMS: m/
z: calculated for C18H19FN3O5: 376.1308; found 376.1303 [M+H]+.
Mp = 219–222 °C.
References and notes
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above, starting from 6 (151 mg, 0.348 mmol). 1H RMN (D2O/
CD3CN: 9:1): d 0.95 (m, 2H, CH2(cPr)), 1.14 (m, 2H, CH2(cPr)),
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