Design, synthesis and activity against Toxoplasma gondii, Plasmodium spp., and Mycobacterium tuberculosis of new 6-fluoroquinolones

Article abstract of DOI:10.1016/j.ejmech.2006.07.003

This paper reports on the rational design of a series of new 6-fluoroquinolones by QSAR analysis against Toxoplasma (T.) gondii, their synthesis, their biological evaluation against T. gondii and Plasmodium (P.) spp., and their effect on Mycobacterium (M.) tuberculosis DNA gyrase and growth inhibition. Of the 12 computer-designed 8-ethyl(or methoxy)- and 5-ethyl-8-methoxy-6-fluoroquinolones predicted to be active against T. gondii, we succeeded in the synthesis of four 6-fluoro-8-methoxy-quinolones. The four 6-fluoro-8-methoxy-quinolones are active on T. gondii but only one is as active as predicted. One of these four compounds appears to be an antiparasitical drug of great potential with inhibitory activities comparable to or higher than that of trovafloxacin, gatifloxacin, and moxifloxacin. They also inhibit DNA supercoiling by M. tuberculosis gyrase with an efficiency comparable to that of the most active quinolones but are poor inhibitors of M. tuberculosis growth.

Full text of DOI:10.1016/j.ejmech.2006.07.003

European Journal of Medicinal Chemistry 41 (2006) 1478e1493
http://www.elsevier.com/locate/ejmech
Short communication
Design, synthesis and activity against Toxoplasma gondii, Plasmodium
spp., and Mycobacterium tuberculosis of new 6-fluoroquinolones
Guillaume Anquetin a, Jacques Greiner a, Nassira Mahmoudi b,c, Maud Santillana-Hayat b,
Rafael Gozalbes b,1, Khemais Farhati c, Francis Derouin b, Alexandra Aubry d,
Emmanuelle Cambau e, Pierre Vierling a,
*
a Laboratoire de Chimie Bioorganique UMR-CNRS 6001, Universite´ de Nice-Sophia Antipolis, Parc Valrose, 06108 Nice Cedex 2, France
b Laboratoire de Parasitologie et Mycologie, Universite´ Paris 7, Assistance Publique-Hoˆpitaux de Paris,
15 rue de l’Ecole de Me´decine, 75006 Paris, France
c INSERM 511, Immunobiologie Cellulaire et Mole´culaire des Infections Parasitaires, CHU Pitie´-Salpe´trie`re,
Assistance Publique-Hoˆpitaux de Paris, 75013 Paris, France
d Laboratoire de Recherche sur les Antibiotiques, INSERM 0004, Universite´ Pierre et Marie Curie, Paris, France
e Laboratoire de Bacte´riologie-Virologie-Hygie`ne, CHU Henri Mondor, Faculte´ de Me´decine de Cre´teil,
Universite´ Paris 12, 51 Avenue Mare´chal de Lattre de Tassigny, 94010 Cre´teil Cedex, France
Received 28 February 2006; received in revised form 28 June 2006; accepted 3 July 2006
Available online 25 September 2006
Abstract
This paper reports on the rational design of a series of new 6-fluoroquinolones by QSAR analysis against Toxoplasma (T.) gondii, their syn-
thesis, their biological evaluation against T. gondii and Plasmodium (P.) spp., and their effect on Mycobacterium (M.) tuberculosis DNA gyrase
and growth inhibition. Of the 12 computer-designed 8-ethyl(or methoxy)- and 5-ethyl-8-methoxy-6-fluoroquinolones predicted to be active
against T. gondii, we succeeded in the synthesis of four 6-fluoro-8-methoxy-quinolones. The four 6-fluoro-8-methoxy-quinolones are active
on T. gondii but only one is as active as predicted. One of these four compounds appears to be an antiparasitical drug of great potential
with inhibitory activities comparable to or higher than that of trovafloxacin, gatifloxacin, and moxifloxacin. They also inhibit DNA supercoiling
by M. tuberculosis gyrase with an efficiency comparable to that of the most active quinolones but are poor inhibitors of M. tuberculosis growth.
Ó 2006 Elsevier Masson SAS. All rights reserved.
Keywords: Fluoroquinolone; 6-Fluoro-8-methoxy-quinolone; Toxoplasma gondii; Plasmodium spp.; Mycobacterium tuberculosis; Antiparasitical; Antibacterial;
Malaria; Toxoplasmosis; QSAR
1. Introduction
World Health Organization, one third of human population
is infected by Mycobacterium (M.) tuberculosis and around
two million people die from tuberculosis every year. The treat-
ment of tuberculosis (isoniazid and rifampicin) [1] is long and
observance is therefore a problem. Tuberculosis associated
with AIDS (due to HIV infection) forms a fatal combination.
Tuberculosis is currently responsible for 13% of the number
of deaths due to HIV infection [2]. Toxoplasmosis, as well,
is an opportunistic disease frequently associated with AIDS
[3]. Toxoplasma (T.) gondii is the parasite responsible for
toxoplasmosis [4]. Almost half of the human population is in-
fected by this parasite, and even if most seropositive people do
Owing to the emergence and alarming spread of bacterial,
parasitical and viral strains that are resistant against the drugs
used at present in clinics, the discovery of new therapeutical
targets and the development of new antibacterial, antiparasiti-
cal and antiviral drugs are urgently needed. According to the
* Corresponding author. Tel.: þ33 4 92 07 61 43; fax: þ33 4 92 07 61 51.
E-mail address: vierling@unice.fr (P. Vierling).
1
Present address: CEREP, Molecular Modeling Department, 128 rue
Danton, 92500 Rueil Malmaison, France.
0223-5234/$ - see front matter Ó 2006 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.07.003

G. Anquetin et al. / European Journal of Medicinal Chemistry 41 (2006) 1478e1493
1479
not develop any symptom of this disease, its transmission to
immunocompromised patients such as AIDS patients is life
threatening. The other problem concerns its transmission to
the fetus, which can result in malformations or stillborns. Che-
motherapeutic treatment against toxoplasmosis is limited by
side effects or poor absorption of efficient drugs. Plasmodium
(P.) spp., which are parasitic protozoans belonging to the same
apicomplexan phylum as T. gondii, are also of great concern as
they are responsible for another fatal disease, i.e. malaria. In-
deed, it represents two million deaths per year and 90% are
due to Plasmodium falciparum [5]. Numerous strains are resis-
tant to the most frequently used antimalarial drugs, i.e. chloro-
quine and pyrimethamine/sulfadoxine [6,7].
In the search of new therapeutical targets and new anti-
infective agents, fluoroquinolones (see generic structure in
Table 1 with R6 ¼ F and examples in Table 2) are particu-
larly interesting because of their broad spectrum of activity
against various bacteria, mycobacteria, and parasites [4,8e
15]. Quinolones are bactericidal by interfering with two es-
sential bacterial enzymes, DNA topoisomerases II (DNA
gyrase) and IV, which are enzymes involved in DNA repli-
cation, decatenation, recombination and repair [11,16e19].
There are clues that quinolones act on similar targets of
T. gondii and P. falciparum, i.e. the plastids which are cir-
cular DNA episomes located within the apicoplast organelle
of both parasites, and through similar pathways, although no
definitive evidence has been provided up to now [15]. Since
the first antibacterial quinolone, i.e. nalidixic acid which
was isolated in 1962 [20], more than ten thousands of qui-
nolones have been patented, and the successive chemical
modifications improved considerably their potency and spec-
trum of activity [for reviews, see Refs. [8e14]] and parasit-
ical responses [15,21e24].
The most often used, relatively safe and well-tolerated 6-
fluoroquinolones as antibacterials include norfloxacin (NFX),
ofloxacin (OFX), ciprofloxacin (CPFX), levofloxacin
(LVFX), moxifloxacin (MXFX), and gatifloxacin (GTFX).
OFX was used as second-line agent against M. tuberculosis
[25]. Furthermore, MXFX, which turns out to be as efficient
as rifampicin and isoniazid [26], was recently suggested by
the American Thoracic Society to be used against tuberculosis,
as well as GTFX and LVFX. MXFX and GTFX with grepa-
floxacin (GPFX) and trovafloxacin (TVFX) account for among
the most powerful antiparasitical fluoroquinolones known so
far (for the chemical structure of MXFX, GTFX, GPFX and
TVFX, see Table 2) [22,23]. However, GPFX and TVFX
were taken off from the market more or less shortly after
launch. If a huge number of (fluoro)quinolones differing by
the nature of the R7 substituent were elaborated enabling the
establishment of structure/antibacterial [27], antituberculosis
[28], or antiparasitical relationships [15,22,24], only a few
R5 or/and R8 substituted 6-fluoroquinolones are known, prob-
ably due to the difficult access to precursors of these
derivatives.
Table 1
Chemical structure of the targeted ‘‘virtual’’ 6-fluoroquinolones and their predicted anti-Toxoplasma gondii activity (the atom numbering is used for the description
of the NMR data)
Compound number
R7
R1
R5
R8
Predicted IC50
(mg/mL)
a Series
1
2
3
4
6A-C
cP
cP
H
H
H
H
MeO
MeO
MeO
MeO
0.4
0.5
0.3
0.3
6AM-C
6A-C
6AM-C
dFP
dFP
b Series
c Series
5
6
7
8
6A-C
cP
cP
Et
Et
Et
Et
MeO
MeO
MeO
MeO
0.5
0.6
0.4
0.3
6AM-C
6A-C
6AM-C
dFP
dFP
9
6A-C
cP
cP
H
H
H
H
Et
Et
Et
Et
0.4
0.5
0.3
0.3
10
11
12
6AM-C
6A-C
6AM-C
dFP
dFP

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G. Anquetin et al. / European Journal of Medicinal Chemistry 41 (2006) 1478e1493
Table 2
Structural features and in vitro anti-T. gondii activities (IC50), antimalarial activities (IC50) against blood stages of P. falciparum and hepatic stages of P. yoelii
yoelii, and M. tuberculosis DNA gyrase (IC50) and growth inhibition (MIC) of quinolones 1-4 in comparison with those of already known quinolones
R5
R5
R5
O
O
O
R6
R7
R6
R7
CO2H
H
CO2H
CO2H
H
N
N
N
R7
N
N
R1
H
N
R1
R8 R1
Structure A
(quinolone)
Structure B
(naphthyridone)
Structure C
Quinolones
Base
Substituent
Inhibitory concentrations (mg/mL)
R1a
R5
R6
R7b
R8
T. gondii
P. falciparum
P. yoelii yoelii
M. tuberculosis
RH (IC50
)
NF54-Rc (IC50
)
265Byd* (IC50
)
DNA gyrase (IC50
)
MICe
1
A
A
A
A
A
A
B
B
A
A
A
B
B
C
cP
cP
H
F
F
F
F
F
F
F
F
F
F
F
F
F
6A-C
OMe
OMe
OMe
OMe
H
1.3
30
4.1f
5.1f
2.4f
40f
0.6i
4.3i
19
56
45
45*
>100
>100g
>100g
4.4g*
53g
3.5
6
64
64
0.12h
0.5h
1h
0.25h
2
Gatifloxacin (GTFX)
H
6AM-C
3Me-Pi
PpP
cP
cP
H
11g
18g
3.1g
74g
e
3h
Moxifloxacin (MXFX)
Grepafloxacin (GPFX)
H
4.5h
16h
2h
cP
cP
Me
NH2
H
3Me-Pi
dMe-Pi
6A-C
Sparfloxacin (SPFX)
FQ4
F
cP
cP
e
e
e
e
FQ9
3
H
6AM-C
6A-C
e
e
e
dFP
dFP
dFP
dFP
dFP
Et
H
OMe
OMe
H
62
24
>100
>100
e
20
15
e
15h
e
256
128
e
16h
e
4
FQ6
H
6AM-C
6A-C
6A-C
22
H
1.1i
0.4f
4.3i
26f
e
Trovafloxacin (TVFX)
FQ11
Piromidic acid
H
9.2g
e
31g*
e
H
6AM-C
P
H
14g
22g*
e
e
R'
H
H
H
R = NH2
6A-C
R' = H; R" = Me 3Me-Pi
R' = R" = Me dMe-Pi
P
PpP
N
H
N
N
N
N
N
R = CH2NH2 6AM-C
R
H
R''
a
dFP ¼ 2,4-Difluorophenyl; cP ¼ cyclopropyl.
b
6A-C ¼ 6-Amino-3-azabicyclo[3.1.0]hexan-3-yl,6AM-C ¼ 6-(aminomethyl)-C, 3Me-Pi ¼ 3-methyl-piperazin-1-yl, dMe-Pi ¼ 3,4-dimethyl-Pi, P ¼ pyrrolidin-1-yl;
PpP ¼ piperidinopyrrolidinyl.
c
Against blood stages of chloroquine-resistant (NF54-R) strain.
Against hepatic stages, symbol *indicates that in these cases only inhibition was also associated with an effect on schizont size.
d
e
MIC: minimum inhibitory concentration.
Data from Ref. [22].
f
g
Data from Ref. [23].
Data from Ref. [28].
Data from Ref. [21].
h
i
In the search for new potent antiparasitical fluoroquino-
lones, a QSAR analysis by molecular connectivity of a series
of quinolones active against T. gondii was performed
[22,24]. This analysis led to the design of R5- and/or R8-
substituted 6-fluoroquinolones which were predicted to dis-
play higher or at least comparable biological activities to
those of already known fluoroquinolones. Among the virtu-
ally computer designed, potentially active derivatives, we se-
lected those presented in Table 1 (compounds 1e12). Their
structures are all original combinations of the R1, R5, R7
and/or R8 substituents found in the most anti-T. gondii active
quinolones MXFX, GTFX, GPFX, TVFX, FQ4, FQ6, FQ9
and FQ11 (see structures in Table 2). Moreover, the ethyl
group in R5 or in R8 position has never been explored to
our knowledge.
This paper is dedicated to (i) the rational design, by
QSAR analysis against T. gondii, of the new series of fluoro-
quinolones shown in Table 1, (ii) the detailed synthesis of de-
rivatives 1e4, (iii) our unsuccessful attempts to prepare
compounds 5e12, and (iv) the results of in vitro biological
tests, including antiparasitical activity against T. gondii, blood
stages of P. falciparum and hepatic stages of Plasmodium
yoelii yoelii, and antibacterial activity against M. tuberculosis
(DNA gyrase and growth inhibition). Part of this work was
briefly reported as a preliminary communication [24].
2. Chemistry
The retrosynthetic scheme to obtain the targeted R5- and/or
R8-substituted 6-fluoroquinolones starting from commercially

G. Anquetin et al. / European Journal of Medicinal Chemistry 41 (2006) 1478e1493
1481
available derivatives 13 and 14 is shown in Scheme 1. The en-
try to quinolin-4-one nucleus relies on an intramolecular cycli-
zation step in enaminone intermediates 19. Such intermediates
were used for the building-up of thousands of quinolone ana-
logs known to date, though other peculiar alternatives have
also been explored [19,29,30]. The approach to 19 from acid
17 is more classically performed through compounds of type
22 (route II) [19,29]. However, we used the alternative route
I where the enaminone part was introduced in two steps
from 17 using an (N-alkyl) acrylate derivative [30e33]. More-
over, this method is easier to implement (milder conditions)
and more efficient than the one depicted in route II. The appro-
priate starting acids 13 and 14 were thus needed to get the key
acids 17, precursors of the target 6-fluoro-8-methoxy-quino-
lones (a and b series) and 6-fluoro-8-ethyl-quinolones (c se-
ries) listed in Table 1 (see Scheme 1). These acids 13 and
14 can be converted into 15 which contains (i) the ethyl or me-
thoxy group in the 3 position (R8 for the position numbering
see Scheme 1), (ii) the fluor atom in the 5 position essential
for the bioactivity of the target 6-fluoro-8-ethyl(or methoxy)-
quinolones, (iii) two fluor atoms in the 2 and 4 positions, the
former being necessary for the building of the heterocyclic nu-
cleus, the latter for the regioselective introduction of the amino
derivative moiety (e.g. R7) through its nucleophilic displace-
ment by suitable amines [19,29] and (iv) an aromatic CeH
(position 6), which can be further used to introduce the R5 sub-
stituent (i.e. the ethyl radical), thus generating the R5 and R8
substituted synthons 17, precursor of the target 6-fluoro-5-
ethyl-8-methoxy-quinolones (b series).
preparation of R5- and/or R8-substituted 17-type derivatives
[34e36]. The 6-amino-3-aza-bicyclo[3.1.0]hexane 23 and its
methyleneamino analog 24 (Fig. 1), which constitute the
amino R7 moiety of the target molecules, were synthesized
from commercial N-benzylmaleimide in eight steps (about
10% overall yield) according to the published procedures
[24,37,38].
Of the targeted R5/R8-6-fluoroquinolones 1e12 listed in
Table 1, we succeeded only in the synthesis of the 6-fluoro-
8-methoxy-quinolones 1e4 (a series). These compounds
were prepared in seven steps from 17a in 25e30% overall
yield (Scheme 2). For the building of the key quinolone cycle
20a, acid 17a was first converted into its acid chloride
derivative which was then reacted with ethyl 3-(diethyla-
mino)-2(E )-propenoate [39], thus affording the N,N-diethyl
enaminone intermediate 18a. Transaminolysis of 18a with cyclo-
propylamine or 2,4-difluorophenylamine followed by cyclization
of the resulting cyclopropyl- or 2,4-difluorophenyl-enaminone
intermediates 19a1 or 19a2 afforded 20a1 or 20a2, respec-
tively, in 65% yield. These two steps were performed in
1
a one-pot process and H NMR monitoring showed that com-
pounds 19 consisted of E/Z mixture (data not shown). For cy-
clization, the mild K2CO3 base was preferred to the most
frequently used NaH [36,40], which in our hands led to numer-
ous by-products. Noticeably, this three-step two-pot process
was more efficient than the two-step one-pot procedure
[31,41,42] consisting of the condensation of the acid chloride
derivative of 17 with ethyl 3-(cyclopropylamino)-2(E )-prope-
noate or ethyl 3-(2,4-difluorophenylamino)-2(E )-propenoate,
followed by cyclization. Actually in these particular cases,
the condensation step gave essentially the amide compounds
of type 25 (example given in Scheme 5, data not shown),
rather than the corresponding enaminone compound of type
19, thus preventing all further cyclization steps. Next the intro-
duction of the R7 substituent onto 20a1 and 20a2 could only
be achieved provided the C-7 position was activated, as
The detailed synthetic pathway to the 6-fluoroquinolone
targets of series a, b, and c starting from synthons of type
17 is depicted in Schemes 2e4, respectively. The syntheses
of the synthons 17 wherein R5/R8 is H/methoxy (i.e. 17a),
ethyl/methoxy (i.e. 17b), and H/ethyl (i.e. 17c) from 13 and
14 have been described elsewhere [34]. They were adapted
from procedures that were elaborated in the literature for the
Scheme 1. Retrosynthetic pathway to the targeted quinolin-4-one derivatives 1e12.

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G. Anquetin et al. / European Journal of Medicinal Chemistry 41 (2006) 1478e1493
Scheme 2. Synthetic pathways to the quinolin-4-one derivatives 1e4 (a series), compounds 1e4: (i) oxalyl chloride, then Et2NCH]CHCO2Et, NEt3, toluene,
90 ꢀC, 5 h; (ii) R1NH2, 1:2 EtOH/Et2O rt, 3 h; (iii) K2CO3, DMF, 100 ꢀC, 5 h; (iv) BF3$Et2O, THF, reflux; (v) amine 23 or 24, CH3CN, seven days, reflux;
(vi) 1:1 CH2Cl2/TFA; cP ¼ cyclopropyl; dFP ¼ 2,4-difluorophenyl.
reported in the literature for similar analogs [36,43e45].
Indeed, no reaction occurred between 20a1 and 20a2 and
the heterobicyclic amines 23 or 24 (Fig. 1). Compounds
20a1 and 20a2 were thus activated as their boron difluoride
derivatives 21a1 and 21a2, respectively, by reaction with
boron trifluoride etherate in refluxing THF. The successive
but nevertheless time-consuming (seven days) nucleophilic
displacement of the C-7 fluorine atom in 21a1 (resp. 21a2)
with 2.5e3 equiv of amines 23 or 24 followed by N-Boc
deprotection using an excess of 1:1 TFA/CH2Cl2 gave the
desired fluoroquinolones 1 or 2 (resp. 3 or 4), as their TFA
salts in 40e44% overall yield from 20a1 and 20a2.
Concerning the 5-ethyl-8-methoxy quinolones 5e8 (b se-
ries, Scheme 3), all our attempts to obtain the ethyl R5-
substituted enaminone derivative 18b or b-ketoester 22b
from 17b, which constitutes the entry to these quinolones,
b: 5-ethyl-8-methoxyquinolone series
Et
O
O
Route I
F
F
i)
OEt
F
Et2N
O
N
Et
O
O
N
18b
Et
Et
O
OMe
F
COOH
F
F
F
F
OH
OEt
F
H
N
OMe R1
OMe R1
OMe
Et
O
F
O
17b
20b
5-8
R2
F
F
ii)
H
OEt
Route II
22b
OMe
Scheme 3. Synthetic pathways explored to the quinolin-4-one derivatives 5e8 (b series): (i) oxalyl chloride, then Et2NCH]CHCO2Et, NEt3, toluene, 90 ꢀC, 5 h;
(ii) HO2CCH2CO2Et, n-BuLi (2 equiv).

G. Anquetin et al. / European Journal of Medicinal Chemistry 41 (2006) 1478e1493
1483
c: 8-ethyl-quinolone series
O
F
O
O
O
O
O
COOH
F
F
F
F
F
F
F
F
F
OEt
OEt
NHR1
E/Z
i)
ii)
iii)
OEt
F
Et2N
N
Et R1
Et
Et
Et
17c
18c
19c1
20c1: R1 = cP
20c2: R1 = dFP
F2
19c2
B
O
O
O
O
F
iv)
v)
F
F
OH
O
9-12
N
R1
H
N
N
R1
Et
Et
R2
H
9-Boc: R1 = cP; R2 = NHBoc
10-Boc: R1 = cP; R2 = CH2NHBoc
11-Boc: R1 = dFP; R2 = NHBoc
21c1: R1 = cP
21c2: R1 = dFP
12-Boc: R1 = dFP; R2 = CH2NHBoc
Scheme 4. Synthetic pathways explored to the quinolin-4-one derivatives 9e12 (c series). See Scheme 2 for (i)e(v). cP ¼ cyclopropyl; dFP ¼ 2,4-difluorophenyl.
failed. Indeed, the acid chloride of the ethyl-methoxy disubsti-
tuted acid 17b led, under the same conditions as used for the
synthesis of quinolones 20a (and also 20c, see below), to
a complex mixture from which we could not isolate the ex-
pected 18b (Scheme 3, route I), thus prohibiting the building
of the key quinolone cycle 20b, and, consequently, the access
to the targeted quinolones 5e8 of series b. The more classical
route II shown in Scheme 3, which involves the intermediary
preparation of 22b, by reacting the previous acid chloride with
monoethylmalonate was also unsuccessful.
23 or 24. Indeed, reacting 20c2 or 21c2 with 23 or 24 led to the
decarboxylation of 20c2 or 21c2 giving 26c1 or 26c2 (Fig. 2),
while derivative 21c1, under the same conditions, was inert. It
should be further noticed that the aromatic substitution of the
C-7 fluorine in 21c1 was observed when 21c1 was reacted
with piperazine. Thus, 25% yield of the 7-piperazine com-
pound 27 (Fig. 2), i.e. the analog of 9-Boc and 10-Boc which
were expected from the reaction between 21c1 and amines 23
or 24, were obtained (data not shown), indicating that the nu-
cleophilicity of the entering amine is of importance.
Concerning the synthesis of the 8-ethyl-quinolones 9e12 (c
series, Scheme 4), we were fully successful in building the key
quinolones 20c1 and 20c2 from 17c using the same strategy as
that described above for the synthesis of their 21a1 and 21a2
analogs. However, and in sharp contrast with the a series, the
aromatic substitution of the C-7 fluorine in compounds 20c1
and 20c2 (Scheme 2) whether in the presence of a Lewis
acid such as LiCl or Al(OTf)3, or activated as their BF2-
derivatives 21c1 and 21c2 could not be performed with amines
The chemical structures of fluoroquinolones 1e4 (as their
TFA salts) and of all intermediates shown in Schemes 2 and 4
1
1
were unambiguously attested by H, 13C, 19F NMR, He13C
DEPT, HSQC, ESI-high resolution mass spectrometry, and
1
by comparison with the H, 13C and 19F NMR data reported
for similar analogs [46]. The cyclization, and consequently
the quinolone structures of 20a1, 20a2, 20c1, and 20c2,
were established by the presence of two fluor resonances in
their 19F NMR spectra (as compared to three for 18) and the
deshielding of the H-2 proton (w0.5e1 ppm) with respect to
1
the chemical shift of the vinylic one in the H NMR spectra
of 18. The introduction of the azabicyclic moieties into 1e4
H
N
H
N
O
O
F
F
F
F
F
i)
ii)
OH
N
H
H
H
H
CHCO2Et
F
NH
OMe
OMe
CH2NHBoc
17a
25a1
NHBoc
23
CO2Et
24
Fig. 1. Structure of Boc-protected 6-amino-3-aza-bicyclo[3.1.0]hexane 23 and
its 6-aminomethyl analog 24.
Scheme 5. Formation of amide 25a1: (i) oxalyl chloride, CH2Cl2 rt, 24 h; (ii)
toluene, 90 ꢀC, 5 h.

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G. Anquetin et al. / European Journal of Medicinal Chemistry 41 (2006) 1478e1493
and for their predicted high anti-T. gondii activities within the
O
N
O
O
0.3e0.6 mg/mL range. For their design, we used, as topologi-
cal indices (TIs), the E-state indices, which are specific for
each atom and reflect the electronic and topological atomic
features taking into account the interaction with the rest of
the molecule [53]. These TIs were related to the anti-T. gondii
activity of known quinolones, thus providing some insights
into the substitutions leading to higher anti-T. gondii activity.
Computational screening was then used to select new quino-
lones with improved efficacy. Virtual structures were designed
around the quinolone chemotype by using a home-made soft-
ware that allows the introduction of virtual radicals on the
most active quinolones (i.e. TVFX, GPFX, GTFX and
MXFX with IC50 values below 5 mg/mL) and their systematic
combination, thus leading to a ‘‘virtual combinatorial library’’
of compounds. Their TIs were calculated, and linear discrim-
inant analysis (LDA) and multilinear regression (MLR) equa-
tions were used to determine their activity/inactivity and IC50
values, respectively.
The originality of the selected 12 6-fluoroquinolones listed in
Table 1 lies in the combination of R1, R5, R7 and R8 substituents
which are found in the most potent anti-T. gondii TVFX, FQ4
and FQ9 naphthyridones [21], and MXFX, GTFX, GPFX quino-
lones (see structures in Table 2). Indeed, these fluoroquinolones
contain on a quinolone skeleton (i) as R1, the cyclopropyl moiety
of GTFX, MXFX, and GPFX, or the 2,4-difluorophenyl one of
TVFX, FQ6 and FQ11, (ii) as R7, the azabicyclohexyl (6A-C or
6AM-C) substituent of TVFX, FQ6 and FQ11, (iii) as R8, the
methoxy group of MXFX and GTFX, and (iv) as R5 and/or R8,
the ethyl radical which has up to now not been investigated. Al-
though R5 or/and R8-substituted 6-fluoroquinolones have been
the focus of intensive search, R5 or R8 substitutions are restricted
to F, Cl, Br, NH2, Me, MeO, EtO, MeS, OH, F2CHO, or CF3O
[29,35,36,40,43,54,55]. R5-andR8-substituted 6-fluoroquinolones
are also scarce and R5/R8 substitutions are limited to Me/F, Me/Cl,
Me/Me, Me/OMe, Cl/Me, NH2/Me, NHMe/Me, NMe2/Me, NH2/
OMe, NH2/OEt, NH2/F, NH2/Cl [35,40,43,56e58].
F
F
F
OH
N
N
R1
Et
Et
HN
26c1 R1 = cP
26c2 R1 = dFP
27
Fig. 2. Structure of decarboxylated by-product derivatives 26c1 and 26c2, and
7-piperazinyl-1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-4-oxo-3-quinoli-
necarboxylic acid, 27; cP ¼ cyclopropyl; dFP ¼ 2,4-difluorophenyl.
was attested by the presence of their characteristic 1H and 13
C
1
patterns. Its location in R7 was confirmed by H NMR which
showed the presence of a doublet for H-5 at 7.4e7.7 ppm with
3
J w 13.0e13.5 Hz, characteristic of a JHeF coupling. Con-
cerning the degree of purity of the target fluoroquinolones
1e4, the samples used for the determination of their antipara-
sitical and antibacterial activities (see Section 3) were ob-
tained by recrystallization from appropriate solvents, and
analyzed by two distinct HPLC systems which showed the ab-
sence of any trace of impurities.
3. Pharmacology
The antiparasitical activities of quinolones 1e4 were eval-
uated in vitro against T. gondii (RH strain), blood stages of
P. falciparum (chloroquine-resistant strain NF54-R), and he-
patic stages of P. yoelii yoelii (265BY strain) according to
the published procedures [22,23]. The quinolones 1e4 were
also assessed in vitro for their ability to inhibit M. tuberculosis
DNA gyrase activity (IC50) and the growth of M. tuberculosis
(MIC). Table 2 lists all the data from these biological evalua-
tions together with those of selected (fluoro)quinolones and
naphthyridones taken from literature for comparison and de-
termined strictly under the same conditions.
It is further expected that the original R1, R5, R7 and/or R8
combinations provide not only active antiparasitical/antibacte-
rial drugs but also new drugs displaying reduced side effects as
compared with TVFX and GPFX which were withdrawn from
the market owing to hepatotoxicity, phototoxicity and/or CNS
reactions [59]. Lower toxic side effects are more particularly
expected for the derivatives containing a methoxy as R8. In-
deed, this substituent in MXFX and GTFX contributed favor-
ably to the reduction of phototoxicity [19], the selection of less
resistant strains [60], and to their activity on resistant strains
[61,62].
4. Results and discussion
4.1. Design rationale
Numerous structureeactivity relationships concerning qui-
nolones have been examined to rationalize the various biolog-
ical activities of quinolones as well as to design new powerful
compounds [19,47e50]. In our search for new potent antipar-
asitical fluoroquinolones, we used the previously described
QSAR analysis by molecular connectivity that was performed
on quinolones active against T. gondii [22] and validated on
TVFX analogs [21] for which a good agreement was observed
between the predicted and experimental IC50 values
[22,51,52]. This analysis by molecular topology and virtual
computational techniques led to the design of virtual structures
which were expected to display higher or at least comparable
anti-T. gondii activities to those of already known fluoroquino-
lones. Among these virtually active structures, we selected the
structures shown in Table 1 for the feasibility of their synthesis
4.2. Anti-T. gondii activity
The four fluoroquinolones 1e4, which were predicted to
display an anti-T. gondii activity, were indeed found to be ac-
tive. However, only compound 1 had an activity (1.3 mg/mL)
close to that predicted (0.4 mg/mL), while the other three de-
rivatives were less active than predicted. It should be noted
that the anti-T. gondii activity of 1 is in the range found for

G. Anquetin et al. / European Journal of Medicinal Chemistry 41 (2006) 1478e1493
1485
the most potent quinolones, e.g. TVFX, GPFX, FQ4, and FQ6
(see Table 2). The data from these evaluations show the useful-
ness and limits of the predictive QSAR models based on mo-
lecular topology and multilinear regression analysis which led
to the identification of the basic chemical structures of quino-
lones responsible for anti-toxoplasmosis activity and to the de-
sign of quinolones 1e4.
however, along 3-MePi (as in GTFX) w PpP (as in
MXFX) > 6AM-C (as in 2) w 6A-C (as in 1) for anti-P. falcipa-
rum, and (iii), 6A-C (as in 1) > 6AM-C (as in 2), 3-MePi (as in
GTFX), or PpP (as in MXFX) for anti-P. yoelii yoleii. In the 2,4-
difluorophenyl-quinolone series, the contribution of 6A-C to
anti-T. gondii activity is almost comparable to that of 6AM-C
(IC50 3 w 4) while in the 2,4-difluorophenyl-naphtyridone se-
ries, replacement of 6A-C by 6AM-C induces a substantial de-
crease of activity (IC50 TVFX < FQ11).
4.3. Anti-P. falciparum and P. yoelii yoelii activities
Concerning R1 SAR, replacement of cyclopropyl for 2,4-di-
fluorophenyl is also contrasting, in line with literature, and de-
pends on R7 and on the nature of the antiparasitical activity:
when R7 is 6A-C, this replacement decreases anti-T. gondii and
anti-P. yoleii yoleii activities (IC50 1 < 3) but is rather of low inci-
dence on anti-P. falciparum activity (IC50 1 w 3); when R7 is
6AM-C, it increases anti-P. falciparum activity (IC50 2 > 4) and
is rather of low incidence on anti-T. gondii activity (IC50 2 w 4).
The four quinolones 1e4 display a moderate activity against
blood stages of the chloroquine-resistant strain (NF54-R) of P.
falciparum (IC50 range 24e62 mg/mL). Their activity is much
lower than that of the most active quinolones, i.e. GPFX and
TVFX. Moreover, of these four quinolones, only 1 is active
(45 mg/mL) against the hepatic stages of P. yoelii yoelii, and in-
terestingly shows an alteration of P. yoelii yoelii schizonts.
Among the 30 quinolones tested in the literature against chloro-
quine-sensitive and resistant blood stages of P. falciparum, and
hepatic stages of P. yoelii yoelii, only fluoroquinolone 1 and
three other quinolones, i.e. GPFX, TVFX, and piromidic acid,
were associated with a marked morphology alteration, number
and size reduction of the P. yoelii yoelii schizonts [23].
The analysis of these data indicates that the predictive QSAR
models established for T. gondii cannot be reliably extended for
the design of highly active antimalarial drugs. Although all the
four quinolones that were active against T. gondii were also
found to be active against the chloroquine-resistant blood stages
of P. falciparum, only one of them displayed inhibitory effects
against the hepatic stages of P. yoelii yoelii.
4.5. Antibacterial activity against M. tuberculosis and
SAR study
The quinolones 1e4 were found to inhibit DNA gyrase and
growth of M. tuberculosis (see data Table 2). If they display
a DNA gyrase activity comparable to that of commercially
available fluoroquinolones, two of them demonstrated an ac-
tivity close to the most active compounds, e.g. MXFX,
SPFX and GTFX. Our data confirm that anti-M. tuberculosis
activity is higher for the cyclopropyl fluoroquinolone series
(i.e. 1, 2) than for the 2,4-difluorophenyl series (i.e. 3, 4), in
line with literature (GTFX, MXFX vs TVFX) [28]. Interest-
ingly, it appears also that the nature of substituent R7 (6A-C,
6AM-C, 3Me-Pi, PpP) has almost no impact on DNA gyrase
inhibition (IC50 1 w 2 w GTFX, MXFX).
4.4. SAR studies
MICs of all the four compounds werevery high (>64 mg/mL)
and they account for among the lowest active quinolones for in-
hibiting the growth of M. tuberculosis. Moreover, no correlation
was found for quinolones 1e4 between DNA gyrase and growth
inhibition of M. tuberculosis, in contrast to the other quinolones
listed in Table 2. This could be due to either a poor bacterial up-
take of quinolones 1e4, as a result of low diffusion across the
cell wall or efficient efflux. Instability or degradation of these
compounds during MIC determination experiment, which re-
quires incubation at 37 ꢀC for 21e30 days, seems unlikely as
they were found to be very stable under even more drastic con-
ditions. However, we may observe that MIC is indeed increasing
along 3Me-Pi (as in GTFX) < PpP (as in MXFX) < 6A-C (as in
1/resp. 3) ¼ 6AM-C (as in 2/resp. 4), indicating that the high
MIC of 1e4 and consequently their poor bacterial uptake could
mainly be related to the R7 6A-C and 6MA-C azabicyclic rings.
These results further indicate that MIC measurements are essen-
tial to determine whether a quinolone is a promising antituber-
culosis agent or not.
Concerning SAR at position 8 (CeOMe, CeH or N),
the analysis of the experimental IC50 values of 1e4 and of
structurally-related quinolones published in literature indicates
that the anti-T. gondii and anti-P. falciparum activities are
increasing, in a homogenous series, when replacing at position 8
CeOMe by CeH or by N. Indeed, in the 1-difluorophenyl-
substituted quinolone series (3, 4, FQ6, TVFX and FQ11),
the replacement of C-OMe in 3 (resp. 4) by CeH as in FQ6
or by N as in TVFX (resp. FQ11) induces a substantial de-
crease of the IC50 values [3 (resp. 4) > FQ6 > TVFX (resp.
FQ11) for anti-T. gondii; 3 (resp. 4) > TVFX (resp. FQ11)
for anti-P. falciparum]. A similar trend but to a much lesser
extent is also found in the cyclopropyl series (IC50 of
1 > FQ4; 2 > FQ9). That at position 8 a CeOMe is likely
less suited for antiparasitical activity than CeH or N seems
to be supported by the fact that among the six fluoroquino-
lones possessing a methoxy group as R8 (1e4, GTFX, and
MXFX), only one (i.e. 1) was found to be active, though mod-
erately, against P. yoelii yoelii.
Concerning R7 SAR, our data with those of literature indicate
that the contribution of R7 to antiparasitical activity is also con-
trasting. In the 1-cyclopropyl-quinolone series, it decreases
along (i) 6A-C (as in 1) w 3-MePi (as in GTFX) w PpP (as in
MXFX) > 6AM-C (as in 2), for anti-T. gondii, (ii), it varies,
5. Conclusion
The synthesis of the four new 8-methoxy-substituted fluo-
roquinolones 1e4 was performed with 15e18% overall yield

1486
G. Anquetin et al. / European Journal of Medicinal Chemistry 41 (2006) 1478e1493
in seven steps from commercially available acid materials.
Unfortunately, the synthetic strategy applied for 1e4 was
unsuccessful for the preparation of the corresponding 8-ethyl-
substituted fluoroquinolones 9e12, the introduction of the
substituent R7 through an aromatic nucleophilic substitution
of the C-7 fluorine atom in synthons 20c1,c2 or 21c1,c2 being
the most difficult step to achieve. It was also unsuccessful for
the 5-ethyl-8-methoxy analogs 5e8, the main difficulty being
here the condensation step leading to the key enaminone 18b
or malonate 22, from the 6-ethyl-3-methoxy synthon 17b.
The biological results show the usefulness and limits of our
predictive QSAR models for T. gondii. Indeed, the four com-
puter-designed fluoroquinolones were active on T. gondii but
only one of these derivatives, i.e. 1, was as active as predicted,
showing that the models remain of interest to direct the synthesis
of active anti-T. gondii quinolones. Concerning anti-Plasmo-
dium spp. activity, the four compounds are active against blood
stages of P. falciparum though at high concentration and one of
them, i.e. 1, is also inhibitory for hepatic stages associated with
an effect on schizont size reduction. However, further studies
are necessary to define more adapted and specific QSAR models
for the design of new antimalarial drugs. Moreover, fluoroqui-
nolones 1e4 inhibit DNA supercoiling by M. tuberculosis gyr-
ase, the IC50 of 1 and 2 being below 6 mg/mL. This promising
antituberculosis activity was unfortunately invalidated by their
poor inhibition of M. tuberculosis growth as shown by their
high MIC values. These results confirm the SAR deduced
from previous study [28]. Nevertheless, it would be worth to ex-
plore the pharmacological properties and safety profile of 1
which is active against T. gondii, as well as against both eryth-
rocytic and hepatic stages of P. falciparum, and against hepatic
P. yoelii yoelii schizonts, thus appearing to be an antiparasitical
drug with great potential.
grepafloxacin, gatifloxacin and moxifloxacin. Furthermore,
11 trovafloxacin analogs experimentally tested [21] were sub-
mitted to the QSAR model, and good agreement was observed
between the predicted and experimental IC50 values.
We then used as TIs, the E-state indices, which are specific
for each atom and reflect the electronic and topological atomic
features taking into account the interaction with the rest of the
molecule [53]. These TIs were related to the anti-T. gondii ac-
tivity of 24 known quinolones, thus providing some insights
about the substituents leading to higher anti-Toxoplasma activ-
ity. Computational screening was finally used to select new
quinolones with improved efficacy. Virtual structures were de-
signed by the omission or substitution of some radicals on the
most active quinolones tested (trovafloxacin, grepafloxacin,
gatifloxacin and moxifloxacin, with IC50 values below 5 mg/L).
Their TIs were calculated, and LDA and MLR equations were
used to determine their activity/inactivity and IC50 values,
respectively. The LDA and MLR models were then used to
identify the pharmacophoric structures responsible for anti-
Toxoplasma activity of quinolones [22].
6.2. Chemistry
6.2.1. General methods, reagents and starting materials
Reactions were conducted under an anhydrous nitrogen at-
mosphere using freshly distilled and dry solvents. Anhydrous
solvents were prepared by standard methods. Column chroma-
tography purifications were carried out on Silica Gel 60 (E.
Merck, 70e230 mesh). The purity of all new compounds was
checked by thin-layer chromatography (TLC) and NMR. All re-
actions were monitored by TLC analyses on precoated Silica
Gel F254 plates (E. Merck) with detection by UV and by
KMnO4 (0.5% in 1 N aq NaOH solution, w/v) or charring
with ninhydrin (0.3% in MeOH containing 3 vol% of acetic
acid, w/v) or with 50% methanolesulfuric acid solution. The
purity of the final products (>99.5%) was checked by HPLC
analyses (flow of 1 mL minꢁ1) using a Waters 996 photodiode
array detector apparatus (PDA, UV detector from 195 to
290 nm) using a Lichrospher 100 RP-18 (5 mm)-packed column
(250 ꢂ 4 mm) and two solvent systems, i.e. solvent A [isocratic
H2O/CH3CN (65:35) 0.1% TFA] or solvent B [gradient H2O/
CH3CN (from 80:20 to 0:100) 0.1% TFA over 30 min]. Melting
points, determined with an Electrothermal model 3100 appara-
tus, are uncorrected. The 1H, 13C, and (1H decoupled) 19F NMR
spectra were recorded with a Bruker AC 200 spectrometer at
200, 50.3, and 188.3 MHz, respectively. Chemical shifts (d)
were expressed in parts per million relative to the signal indi-
rectly (i) to CHCl3 (d 7.27) for 1H and (ii) to CDCl3 (d 77.16)
for 13C, and directly (iii) to CFCl3 (internal standard) (d 0.0)
for 19F. Coupling constants are expressed in hertz, and multiplic-
ities are referred to as s (singlet), br s (broad singlet), d (doublet),
t (triplet), and m (multiplet). Concerning the description of the
NMR spectra, the atom numbering is indicated in Table 1. Elec-
tron-spray ionization mass spectra in positive mode [ESI(þ)
MS] were recorded on a Finnigan MAT TSQ 7000 apparatus
equipped with an atmospheric pressure ionization source. The
high resolution mass spectrometry (HRMS) analyses were
6. Materials and methods
6.1. Design of quinolones
The mathematical QSAR models used for the design of the
fluoroquinolones listed in Table 1 and for the prediction of
their anti-T. gondii activity are detailed elsewhere [22,51,52].
Briefly, these models were based on the numerical description
of a set of ‘‘training’’ compounds by topological indices (TIs)
and the application of the statistical LDA technique. The ob-
tained QSAR-LDA equations were applied to 24 known quino-
lones and the results indicate theoretical activity against T.
gondii to be in good agreement with the experimentally in vitro
IC50 data. These latter data were used for developing a new
QSAR model by multilinear regression (MLR). The equation
thus obtained accurately matched experimental and calculated
IC50 values (r2 ¼ 0.87), and a very good predictive capacity of
the model was confirmed by the cross-validation test (rc2v
¼
0.74). From both the experiments and the mathematical model,
four fluoroquinolones emerged as being more active than the
other compounds, as their IC50 values were below 10 mg/L.
Trovafloxacin was the most active drug, with experimental
and calculated IC50 values below 0.5 mg/L, followed by

G. Anquetin et al. / European Journal of Medicinal Chemistry 41 (2006) 1478e1493
1487
´
performed by the ‘‘Service Commun de Spectrometrie de
Masse’’ at the Institut de Chimie des Substances Naturelles,
Gif sur Yvette, France.
hexane/AcOEt, UV); 1H NMR (CDCl3) d 0.77 (t, 3H,
3
OCH2CH3, JHeH ¼ 7.1 Hz), 0.84 (br s, 3H, NCH2CH3), 1.08
(br s, 3H, NCH2CH3), 3.25 (br s, 4H, NCH2CH3), 3.76 (q, 2H,
3
N-Benzylmaleimide, boron trifluoride diethyl etherate, trie-
thylamine, trifluoroacetic acid (TFA) and oxalyl chloride were
purchased from Aldrich. Ethyl propiolate, cyclopropylamine
and 2,4-difluoraniline were obtained from Fluka.
2,4,5-Trifluoro-3-methoxybenzoic acid 17a was prepared
from 3,4,5,6-tetrafluorophthalic acid (Lancaster) as described
in literature [36,63]. The 3-ethyl-2,4,5-trifluorobenzoic acid
17b was synthesized from 2,4,5-trifluorobenzoic acid (Lancas-
ter) according to literature [34].
The synthesis of (1a,5a,6a)-6-tert-butoxycarbonylamino-3-
azabicyclo[3.1.0]hexane23and(1a,5a,6a)-6-(tert-butoxycarbo-
nyl)aminomethyl-3-azabicyclo[3.1.0]hexane 24 was performed
from N-benzylmaleimide in eight steps (about 10% overall yield)
according to published procedures [37,38].
OCH2CH3, JHeH ¼ 7.1 Hz), 3.78 (s, 3H, OCH3), 6.87 (ddd,
3
4
4
1H, HAr, JHeF ¼ 10.1 Hz, JHeF ¼ 8.5 Hz, JHeF ¼ 6.0 Hz),
7.53 (s, 1H, C]CHN); 19F NMR (CDCl3) d ꢁ135.1 (dd, F2,
5JFeF ¼ 13.8 Hz, JFeF ¼ 7.6 Hz), ꢁ141.5 (dd, F5, JFeF
4
3
5
3
¼ 20.6 Hz, JFeF ¼ 13.7 Hz), ꢁ149.2 (br d, F4, JFeF
¼ 16.5 Hz); 13C NMR (CDCl3) d 10.8, 14.2 (br s, NCH2CH3),
13.4 (OCH2CH3), 45.0, 53.8 (br s, NCH2CH3), 59.4 (OCH2CH3),
4
61.5 (t, OCH3, JCeF ¼ 3.5 Hz), 101.6 (C]CHN), 109.5
2
3
(dd, C6, JCeF ¼ 20.1 Hz, JCeF ¼ 3.3 Hz), 126.2 (ddd, C1,
2JCeF ¼ 14.8 Hz, JCeF ¼ 5.6 Hz, JCeF ¼ 3.9 Hz), 137.1
3
4
2
2
3
(ddd, C3, JCeF ¼ 16.6 Hz, JCeF ¼ 11.1 Hz, JCeF ¼ 2.1 Hz),
1
2
3
145.1 (ddd, C4, JCeF ¼ 253.2 Hz, JCeF ¼ 15.2 Hz, JCeF
¼
1
2
5.3 Hz), 146.6 (ddd, C5, JCeF ¼ 246.1 Hz, JCeF ¼ 11.5 Hz,
4JCeF ¼ 3.3 Hz), 149.1 (dt, C2, JCeF ¼ 248.8 Hz, JCeF
¼
1
3
Compound 23: Mp ¼ 110e111 ꢀC; Rf ¼ 0.35 (90:10:1
CHCl3/MeOH/NH4OH, UV, ninhydrin); 1H NMR (CDCl3)
d 1.42 [s, 10H, NH and C(CH3)3], 1.54 (s, 2H, H1 and H5),
2.26 (br s, 1H, H6), 2.88 and 3.10 (AB system, 4H, H2 and
4JCeF ¼ 3.3 Hz), 154.4 (C]CHN), 167.3 [C(O)O], 184.5
[C(O)C].
6.2.2.2. Ethyl a(E )-[(diethylamino)methylene]-3-ethyl-2,4,5-
trifluoro-b-oxo-benzenepropanoate, 18c. The procedure as de-
scribed for 6, when applied to 17c (524 mg, 2.58 mmol),
0.35 mL (4.0 mmol) of oxalyl chloride, 1.4 mL (10 mmol) of
Et3N and 790 mg (4.61 mmol) of ethyl 3-(diethylamino)-2E-
propenoate gave, after silica gel chromatography (90:10 to
75:25 hexane/AcOEt), 750 mg (2.10 mmol, 80%) of 18c as
H4, JHeH ¼ 11.6 Hz), 4.69 (s, 1H, NHBoc); 13C NMR
2
(CDCl3) d 26.4 (C1 and C5), 28.5 [C(CH3)3], 30.4 (C6), 48.8
(C2 and C4), 79.7 [C(CH3)3], 156.4 [NC(O)].
Compound 24: Rf ¼ 0.30 (1:1 AcOEt/MeOH, UV, ninhy-
drin); ESI-MS (positive mode): (M þ H)þ ¼ 213.3 (calcd for
1
C11H20N2O2 212.15); H NMR (CDCl3) d 0.70 (tt, 1H, H6,
a colourless oil. Rf ¼ 0.25 (7:3 hexane/AcOEt, UV); 1H
3JHeH ¼ 6.9 Hz, JHeH ¼ 3.4 Hz), 1.21 (m, 2H, H1 and H5),
3
3
NMR (CDCl3)
d
0.84 (t, 3H, OCH2CH3, JHeH
¼
1.34 [s, 9H, C(CH3)3], 1.88 (br s, 1H, NH), 2.78e2.96
(m, 6H, H2, H4 and CH2NHBoc), 4.92 (br s, 1H, NHBoc);
13C NMR (CDCl3) d 20.4 (C6), 21.1 (C1 and C5), 28.2
[C(CH3)3], 41.4 (CH2NHBoc), 46.7 (C2 and C4), 79.0
[C(CH3)3], 155.9 [NHC(O)].
7.1 Hz), 0.90 (br s, 3H, NCH2CH3), 1.08 (t, 3H, CH3(Ar),
3JHeH ¼ 7.6 Hz), 1.10 (br s, 3H, NCH2CH3), 2.59 (q, 2H,
3
CH2(Ar), JHeH ¼ 7.5 Hz), 3.36 (br s, 4H, 2NCH2CH3), 3.86
3
(q, 2H, OCH2CH3, JHeH ¼ 7.1 Hz), 7.16 (td, 1H, H(Ar),
3JHeF ¼ 4JHeF ¼ 9.2 Hz, JHeF ¼ 6.6 Hz), 7.64 (s, 1H,
4
The synthesis of ethyl 3-(diethylamino)-2(E )-propenoate
was carried out by heating an equimolar amount of diethyl-
amine and ethyl propiolate in acetonitrile as described in
Ref. [39]. After solvent evaporation, the Michael adduct was
used without further purification [1H NMR (CDCl3) d 4.55
5
C]CHN); 19F NMR (CDCl3) d ꢁ122.8 (dd, 1F, F2, JFeF
¼
4
15.8 Hz, JFeF ¼ 6.9 Hz), ꢁ137.1 (br s, 1F, F4), ꢁ143.4 (dd,
F5, JFeF ¼ 21.3 Hz, JFeF ¼ 15.8 Hz); 13C NMR (CDCl3)
3
5
d 11.2, 14.5 (2br s, 2NCH2CH3), 13.7 (2s, CH3(Ar) and
OCH2CH3), 16.1 [CH2(Ar)], 45.3, 54.1 (2br s, 2NCH2CH3),
3
(d, 1H, HC]CHN, JHeH ¼ 13.1 Hz), and 7.42 (d, 1H,
2
59.7 (OCH2CH3), 102.1 (C]CHN), 114.3 (dd, C6, JCeF
¼
3
HC]CHN, JHeH ¼ 13.1 Hz)].
3
2
19.9 Hz, JCeF ¼ 3.1 Hz), 121.1 (dd, C3, JCeF ¼ 23.8 Hz,
2
3
2JCeF ¼ 17.2 Hz), 126.3 (dt, C1, JCeF ¼ 17.2 Hz, JCeF
¼
¼
6.2.2. Synthesis of a-enamino-b-ketoesters
1
2
4JCeF ¼ 4.4 Hz), 146.5 (ddd, C5, JCeF ¼ 245.1 Hz, JCeF
4
1
13.5 Hz, JCeF ¼ 3.3 Hz), 149.8 (ddd, C4, JCeF ¼ 251.0 Hz,
6.2.2.1. Ethyl a(E )-[(diethylamino)methylene]-2,4,5-trifluoro-
3-methoxy-b-oxo-benzenepropanoate, 18a. A CH2Cl2 solution
(30 mL) of 17a (1.36 g, 6.60 mmol), oxalyl chloride (0.80 mL,
9.17 mmol) and five drops of DMF was stirred for 24 h at room
temperature. The reaction mixture was then subjected to con-
centrated evaporation under reduced pressure, solubilized in tol-
uene (15 mL) and added dropwise to a toluene solution (15 mL)
of triethylamine (3 mL, 16.5 mmol) and ethyl 3-(diethyla-
mino)-2E-propenoate (1.29 g, 7.54 mmol). After 5 h of stirring
at 90 ꢀC, the cooled reaction mixture was washed with water.
The organic layer was dried over Na2SO4, filtered and evapo-
rated. The crude residue was purified by flash chromatography
on silica gel (95:5 to 60:40 hexane/AcOEt) to give 18a
(1.88 g, 5.22 mmol, 79%) as a colourless oil. Rf ¼ 0.25 (7:3
2JCeF ¼ 14.3 Hz, JCeF ¼ 9.2 Hz), 153.8 (ddd, C2, JCeF
3
1
¼ 247.7 Hz,
3JCeF ¼ 7.0 Hz,
4JCeF ¼ 2.4 Hz),
154.5
(C]CHN), 167.9 [C(O)O], 185.7 [C(O)C].
6.2.3. Synthesis of ethyl 6,7-difluoro-8-substituted
quinoline-carboxylates (cyclization step)
6.2.3.1. Ethyl 1-cyclopropyl-6,7-difluoro-1,4-dihydro-8-me-
thoxy-4-oxo-3-quinoline-carboxylate, 20a1. Compound 18a
(850 mg, 2.37 mmol) in 1:2 EtOH/Et2O (20 mL) was added
to cyclopropylamine (0.38 mL, 5.48 mmol). After 3 h of
stirring at room temperature, the reaction mixture was evapo-
rated under reduced pressure. The oily residue containing

1488
G. Anquetin et al. / European Journal of Medicinal Chemistry 41 (2006) 1478e1493
3
3
19a1 (1H NMR monitoring) was dissolved in DMF (20 mL)
and K2CO3 (1.32 g, 9.57 mmol) was then added. After 5 h
of stirring at 100 ꢀC, cold water (3 mL) was added. The yellow
precipitate was filtered and dried affording 20a1 (627 mg,
1.94 mmol, 82%). Rf ¼ 0.60 (97:3 CHCl3/MeOH, UV);
2.8 Hz), 3.88 (tt, 1H, CH(cPr), JHeH ¼ 7.0 Hz, JHeH ¼
3.5 Hz), 4.35 (q, 2H, OCH2CH3, 3JHeH ¼ 7.1 Hz), 8.09 (t, 1H,
H5, JHeF ¼ 4JHeF ¼ 9.6 Hz), 8.64 (s, 1H, H2); 19F NMR
3
3
(CDCl3) d ꢁ131.0 and ꢁ138.5 (2d, 1F, F6 and F7, JFeF
¼
22.7 Hz); 13C NMR (CDCl3) d 11.1 [2 CH2(cPr)], 14.4
1
3
mp ¼ 183e184 ꢀC; H NMR (CDCl3) d 1.04 and 1.19 [2m,
(OCH2CH3), 15.3 [CH3(Ar)], 19.3 (dd, CH2(Ar), JCeF
¼
3
4
4H, CH2(cPr)], 1.37 (t, 3H, OCH2CH3, JHeH ¼ 7.1 Hz),
7.2 Hz, JCeF ¼ 1.7 Hz), 38.9 [CH(cPr)], 60.9 (OCH2CH3),
3
3
2
3
3.97 [tt, 1H, CH(cPr), JHeH ¼ 7.5 Hz, JHeH ¼ 3.7 Hz],
110.6 (C3), 112.4 (dd, C5, JCeF ¼ 18.9 Hz, JCeF ¼ 2.8 Hz),
124.1 (d, C8, 2JCeF ¼ 15.4 Hz), 127.2 (dd, C10, 3JCeF ¼ 5.1 Hz,
5
4.07 (d, 3H, OCH3, JHeF ¼ 1.9 Hz), 4.35 (q, 2H, OCH2CH3,
3
4
3JHeH ¼ 7.1 Hz), 7.97 (dd, 1H, H5, 3JHeF ¼ 10.0 Hz,
4JHeF ¼ 8.8 Hz), 8.56 (s, 1H, H2); 19F NMR (CDCl3) d ꢁ136.9
4JCeF ¼ 2.2 Hz), 136.9 (dd, C9, JCeF ¼ 5.3 Hz, JCeF
¼
1
2
2.0 Hz), 148.6 (dd, C6, JCeF ¼ 250.7 Hz, JCeF ¼ 15.9 Hz),
3
1
2
and ꢁ145.1 (2d, 2F, F6 and F7, JFeF ¼ 21.3 Hz); 13C NMR
152.2 (C2), 152.3 (dd, C7, JCeF ¼ 250.3 Hz, JCeF ¼
(CDCl3) d 9.2 [CH2(cPr)], 14.5 (OCH2CH3), 39.8 [CH(cPr)],
14.5 Hz), 165.0 [C(O)O], 173.0 (C4).
4
61.1 (OCH2CH3), 62.9 (d, OCH3, JCeF ¼ 7.7 Hz), 108.7 (dd,
2
3
C5, JCeF ¼ 18.7 Hz, JCeF ¼ 1.1 Hz), 110.1 (C3), 126.1 (dd,
6.2.3.4. Ethyl 8-ethyl-1-(2,4-difluorophenyl)-6,7-difluoro-1,4-
dihydro-4-oxo-3-quinoline-carboxylate, 20c2. The procedure
described for 20a1 when applied to 11 (438 mg, 1.23 mmol),
2,4-difluoraniline (0.40 mL, 3.97 mmol), and K2CO3 (606 mg,
4.38 mmol) gave after work-up 20c2 (388 mg, 0.99 mmol,
80%) as an orange solid. Rf ¼ 0.65 (97:3 CHCl3/MeOH,
UV); mp ¼ 172e174 ꢀC; 1H NMR (CDCl3) d 0.79 (t, 3H,
3
4
3
C10, JCeF ¼ 5.9 Hz, JCeF ¼ 1.8 Hz), 131.6 (dd, C9, JCeF
¼
4
2
3.7 Hz, JCeF ¼ 2.2 Hz), 140.4 (d, C8, JCeF ¼ 12.1 Hz), 148.2
1
2
(dd, C7, JCeF ¼ 253.7 Hz, JCeF ¼ 15.6 Hz), 149.2 (dd, C6,
1JCeF ¼ 251.4 Hz, 2JCeF ¼ 12.4 Hz), 150.7 (C2), 165.2 [C(O)O],
172.3 (C4).
3
3
6.2.3.2. Ethyl 1-(2,4-difluorophenyl)-6,7-difluoro-1,4-dihydro-8-me-
thoxy-4-oxo-3-quinoline-carboxylate, 20a2. The procedure described
for 20a1 when applied first to 18a (672 mg, 1.87 mmol) and 2,4-di-
fluoroaniline (0.40 mL, 3.97 mmol) for 24 h at room temperature,
then to DMF (15 mL) and K2CO3 (978 mg, 7.08 mmol), led to
20a2 (611 mg, 1.55 mmol, 83%) as a yellow solid. Rf ¼ 0.65
CH3(Ar), JHeH ¼ 7.4 Hz), 1.28 (t, 3H, OCH2CH3, JHeH
¼
7.1 Hz), 2.17 [m, 2H, CH2(Ar)], 4.26 (q, 2H, OCH2CH3,
3JHeH ¼ 7.1 Hz), 7.04e7.10 [m, 2H, H(Ar)], 7.70 [m, 1H,
H(Ar)], 8.03 (t, 1H, H5, JHeF ¼ 4JHeF ¼ 9.6 Hz), 8.18 (s,
3
1H, H2); 19F NMR (CDCl3) d ꢁ104.5 and ꢁ116.0 (2d, 2F,
4
0
0
F2 and F4 , JFeF ¼ 8.5 Hz), ꢁ130.0 and ꢁ137.8 (2d, 2F, F6
1
and F7, JFeF ¼ 22.7 Hz); 13C NMR (CDCl3)
d 13.8
¼
3
(97:3 CHCl3/MeOH, UV); mp ¼ 186e187 ꢀC; H NMR (CDCl3)
3
3
d 1.30 (t, 3H, OCH2CH3, JHeH ¼ 7.1 Hz), 3.46 (d, 3H, OCH3,
[CH3(Ar)], 14.2 (OCH2CH3), 18.2 (dd, CH2(Ar), JCeF
3
9
5JHeF ¼ 1.5 Hz), 4.26 (qd, 2H, OCH2CH3, JHeH ¼ 7.1 Hz, JHeF
4
0
6.6 Hz, JCeF ¼ 1.1 Hz), 61.0 (OCH2CH3), 105.9 (dd, C3 ,
2JCeF ¼ 26.5 Hz, JCeF ¼ 22.9 Hz), 111.0 (C3), 112.4 (dd,
2
¼ 1.3 Hz), 7.00e7.07 [m, 2H, H(Ar)], 7.73 [td, 1H, H(Ar),
J ¼ 8.6 Hz, J ¼ 5.7 Hz], 7.87 (dd, 1H, H5, 3JHeF ¼ 10.2 Hz, JHeF
4
C5, 2JCeF ¼ 18.7 Hz, 3JCeF ¼ 2.6 Hz), 113.2 (dd, C5 , JCeF
¼
2
0
¼ 8.5 Hz), 8.16 (s, 1H, H2); 19F NMR (CDCl3) d ꢁ106.5 and
4
2
22.7 Hz, JCeF ¼ 4.0 Hz), 123.5 (d, C8, JCeF ¼ 16.1 Hz),
4
3
4
0
0
ꢁ117.2 (2d, 2F, F2 and F4 , JFeF ¼ 8.2 Hz), ꢁ136.4 and ꢁ144.8
126.6 (dd, C10, JCeF ¼ 5.1 Hz, JCeF ¼ 2.2 Hz), 128.1 (dd,
(2d, 2F, F6 and F7, JFeF ¼ 21.3 Hz); 13C NMR (CDCl3) d 14.3
3
2
4
3
0
0
C1 , JCeF ¼ 12.8 Hz, JCeF ¼ 4.4 Hz), 129.9 (d, C6 , JCeF
¼
4
3
4
(OCH2CH3), 61.1 (OCH2CH3), 62.0 (d, OCH3, JCeF ¼ 7.0 Hz),
10.2 Hz), 135.8 (dd, C9, JCeF ¼ 5.7 Hz, JCeF ¼ 2.0 Hz),
2
2
1
2
0
104.7 (dd, C3 , JCeF ¼ 26.7 Hz, JCeF ¼ 23.1 Hz), 108.4 (d, C5,
148.7 (dd, C6, JCeF ¼ 251.4 Hz, JCeF ¼ 15.7 Hz), 152.2
2
2
1
2
0
JCeF ¼ 19.0 Hz), 110.9 (C3), 112.0 (dd, C5 , JCeF
¼
(C2), 152.8 (dd, C7, JCeF ¼ 251.4 Hz, JCeF ¼ 14.3 Hz),
4
3
4
1
3
0
0
22.7 Hz, JCeF ¼ 4.0 Hz), 125.0 (dd, C10, JCeF ¼ 6.2 Hz, JCeF
157.5 (dd, C2 or C4 , JCeF ¼ 255.4 Hz, JCeF ¼ 12.6 Hz),
2
4
1
3
0
0
0
¼ 2.2 Hz), 128.7 (dd, C1 , JCeF ¼ 13.5 Hz, JCeF ¼ 4.4 Hz),
163.3 (dd, C2 or C4 , JCeF ¼ 255.4 Hz, JCeF ¼ 11.0 Hz),
3
3
0
128.9 (d, C6 , JCeF ¼ 10.6 Hz), 130.9 (dd, C9, JCeF ¼ 4.0 Hz,
164.2 [C(O)O], 172.8 (C4).
4JCeF ¼ 2.2 Hz), 139.2 (dd, C8, JCeF ¼ 12.4 Hz, JCeF ¼ 1.1 Hz),
2
3
1
2
148.0 (dd, C7, JCeF ¼ 254.9 Hz, JCeF ¼ 15.5 Hz), 149.2 (dd, C6,
6.2.4. Synthesis of boron complexes
1
2
0
JCeF ¼ 252.3 Hz, JCeF ¼ 12.3 Hz), 151.2 (C2), 157.6 (dd, C2 or
1
3
0
0
0
C4 , JCeF ¼ 254.0 Hz, JCeF ¼ 12.8 Hz), 162.8 (dd, C2 or C4 ,
6.2.4.1. (1-Cyclopropyl-6,7-difluoro-1,4-dihydro-8-methoxy-4-
oxo-3-quinoline-carboxylato-O3,O4)difluoro-boron,
1JCeF ¼ 253.2 Hz, 3JCeF ¼ 11.0 Hz), 164.2 [C(O)O], 172.1 (br s, C4).
21a1.
BF3$Et2O (1.5 mL, 11.8 mmol) was added to 20a1 (321 mg,
0.99 mmol) in suspension in THF (20 mL). After refluxing
for 48 h, the clear reaction mixture was evaporated under re-
duced pressure. The crude oily residue was successively
washed with Et2O, CHCl3, and water affording 21a1 as a white
solid (265 mg, 0.77 mmol, 78%). Rf ¼ 0.40 (97:3 CHCl3/MeOH,
UV); mp ¼ 221e223 ꢀC; ESI-MS (positive mode):
(M þ H)þ ¼ 344.2 (calcd for C14H10BF4NO4 343.06); 1H
NMR (CD3CN) d 1.25e1.37 [m, 4H, 2CH2(cPr)], 4.19 (d, 3H,
OCH3, 5JHeF ¼ 2.4 Hz), 4.48 (tt, 1H, CH(cPr), 3JHeH ¼ 7.3 Hz,
6.2.3.3. Ethyl 1-cyclopropyl-8-ethyl-6,7-difluoro-1,4-dihydro-
4-oxo-3-quinoline-carboxylate, 20c1. The procedure described
for 20a1 when applied to 18c (195 mg, 0.55 mmol), cyclopropyl-
amine (0.10 mL, 1.42 mmol), and K2CO3 (300 mg, 2.17 mmol)
gave after work-up 20c1 (148 mg, 0.46 mmol, 83%) as a yellow
solid. Rf ¼ 0.65 (96:4 CHCl3/MeOH, UV); mp ¼ 202e204 ꢀC;
1H NMR (CDCl3) d 1.01 and 1.22 [2m, 4H, CH2(cPr)], 1.20 (br t,
3H, CH3(Ar), 3JHeH ¼ 7.5 Hz), 1.37 (t, 3H, OCH2CH3, 3JHeH
¼
3
4
7.1 Hz), 3.39 (qd, 2H, CH2(Ar), JHeH ¼ 7.5 Hz, JHeF
¼

G. Anquetin et al. / European Journal of Medicinal Chemistry 41 (2006) 1478e1493
1489
3
4
3JHeH ¼ 3.8 Hz), 8.17 (dd, 1H, H5, JHeF ¼ 9.8 Hz, JHeF
¼
1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylic acid, 1-
Boc. A solution of 23 (78 mg, 0.39 mmol) and 21a1
(52 mg, 0.15 mmol) in CH3CN (2 mL) was refluxed for
one week. After evaporation under reduced pressure, the
crude residue was partitioned in 1:1 CHCl3/H2O. The or-
8.1 Hz), 9.17 (s, 1H, H2); 19F NMR (CD3CN) d ꢁ131.7 and
3
ꢁ139.0 (2d, 2F, F6 and F7, JFeF ¼ 19.9 Hz), ꢁ144.0 (s, 0.5F,
10BF2), ꢁ144.1 (s, 2.4F, 11BF2).
6.2.4.2.
(1-(2,4-Difluorophenyl)-6,7-difluoro-1,4-dihydro-8-
ganic layer was evaporated leading to a yellow oil
methoxy-4-oxo-3-quinolinecarboxylato-O3,O4)difluoro-boron,
21a2. Similarly, 20a2 (499 mg, 1.26 mmol) when reacted
with BF3$Et2O (2.0 mL, 15.80 mmol) in THF (25 mL)
afforded after work-up 21a2 (407 mg, 0.98 mmol, 78%)
as a white solid. Rf ¼ 0.50 (97:3 CHCl3/MeOH, UV);
(97 mg) consisting of a mixture of 10-Boc (difluoro-boron
complex of 1-Boc) and 21a1 in 75:25 ratio (TLC and 19F
NMR monitoring). This oily residue dissolved in EtOH
(2 mL) and triethylamine (0.9 mL, 6.4 mmol) was stirred
at 80 ꢀC for 48 h. After evaporation, the crude residue ob-
tained was poured into 2 N NaOH and filtered. The precip-
itate was dissolved in CHCl3. The organic layer was washed
with water, dried over Na2SO4, filtered and evaporated to
afford 1-Boc (52 mg, 0.10 mmol, 72%) as a yellow oil.
Rf ¼ 0.50 (94:6 CHCl3/MeOH, UV); 1H NMR (CDCl3)
1
mp ¼ 227e228 ꢀC; H NMR (CD3CN) d 3.54 (d, 3H, OCH3,
5JHeF ¼ 2.0 Hz), 7.26 [m, 2H, H(Ar)], 7.70 [m, 1H, H(Ar)],
3
4
8.30 (dd, 1H, H5, JHeF ¼ 9.7 Hz, JHeF ¼ 8.0 Hz), 9.06 (s,
1H, H2); 19F NMR (DMSO-d6) d ꢁ105.6 and ꢁ116.9 (2d,
4
0
0
2F, F2 and F4 , JFeF ¼ 8.9 Hz), ꢁ128.5 and ꢁ135.9 (2d, 2F,
3
F6 and F7, JFeF ¼ 22.7 Hz), 139.0 and 140.2 (AB system,
d 0.95 and 1.18 [m, 4H, CH2(cPr)], 1.42 [s, 9H,
0.6F, 10BF2, JFeF ¼ 71.5 Hz), 139.1 and 140.3 (AB system,
2.0F, 11BF2, JFeF ¼ 71.5 Hz).
C(CH3)3], 1.78 (s, 2H, H1 and H5 ), 2.52 (s, 1H, H6 ),
00
00
00
00
3.55 (s, 3H, OCH3), 3.62 and 3.89 (AB system, 4H, H2
2
00
and H4 , JHeH ¼ 11.1 Hz), 3.99 [m, 1H, CH(cPr)], 4.87
3
6.2.4.3. (1-Cyclopropyl-8-ethyl-6,7-difluoro-1,4-dihydro-4-oxo-3-
quinoline-carboxylato-O3,O4)difluoro-boron, 21c1. Similarly,
20c1 (117 mg, 0.364 mmol) and BF3$Et2O (0.60 mL,
4.74 mmol) in THF (5 mL) afforded after work-up 21c1
(97 mg, 0.28 mmol, 78%) as a white solid. Rf ¼ 0.50 (96:4
CHCl3/MeOH, UV); mp ¼ 274e276 ꢀC; 1H NMR (CD3CN)
(br s, 1H, NHBoc), 7.72 (d, 1H, H5, JHeF ¼ 13.3 Hz),
8.74 (s, 1H, H2), 14.89 (br s, 1H, COOH); 19F NMR
(CDCl3) d ꢁ118.4 (s, 1F, F6); 13C NMR (CDCl3) d 9.6
00
[CH2(cPr)], 24.5 (C1 and C5 ), 28.4 [C(CH3)3], 31.8
00
4
00
00
00
(C6 ), 40.6 [CH(cPr)], 51.8 (d, C2 and C4 , JCeF
7.3 Hz), 61.7 (OCH3), 79.8 [C(CH3)3], 107.7 (C3), 107.9
¼
3
2
3
d 1.16 (br t, 5H, CH2(cPr) and CH3(Ar), JHeH ¼ 7.5 Hz), 1.38
(d, C5, JCeF ¼ 24.2 Hz), 120.3 (d, C10, JCeF ¼ 9.2 Hz),
3
2
[m, 2H, CH2(cPr)], 3.60 (qd, 2H, CH2(Ar), JHeH ¼ 7.5 Hz,
134.2 (C9), 136.5 (d, C7, JCeF ¼ 11.3 Hz), 143.8 (d, C8,
4JHeF ¼ 3.3 Hz), 4.49 (tt, 1H, CH(cPr), JHeH ¼ 7.0 Hz,
3JCeF ¼ 6.6 Hz), 149.9 (C2), 155.1 (d, C6, 1JCeF
3
3JHeH ¼ 3.5 Hz), 8.23 (t, 1H, H5, JHeF ¼ 4JHeF ¼ 9.1 Hz),
¼ 252.1 Hz), 156.4 [NHC(O)O], 166.9 [C(O)O], 176.9 (d,
3
9.24 (s, 1H, H2); 19F NMR (CD3CN) d ꢁ121.8 and ꢁ131.8
C4, JCeF ¼ 2.9 Hz).
4
(2d, 2F, F6 and F7, JFeF ¼ 21.3 Hz), ꢁ142.2 (s, 0.4F, 10BF2),
6.2.5.1.2. Synthesis of 1 (as its 2.6 TFA salt). The Boc de-
protection of 1-Boc (22 mg, 0.0465 mmol) was achieved in
1:1 CH2Cl2/TFA mixture (4 mL) at 0 ꢀC for 2 h. After evap-
oration, several additions of hexane and evaporations were
performed to eliminate excess TFA. The crude residue
was successively washed with Et2O and CH2Cl2. Recrystal-
lization (9:1 H2O/CH3CN) followed by lyophilization af-
forded 1 (21 mg, 0.037 mmol, 79%) as a white solid.
Rf ¼ 0.20 (98:2 CHCl3/MeOH, UV); HPLC: Rt ¼ 5.2 min
(solvent A) and 11.8 min (solvent B); 1H NMR (1:4
CD3CN/D2O) d 0.93 and 1.13 [m, 4H, CH2(cPr)], 2.04 (s,
3
ꢁ142.3 (s, 1.9F, 11BF2).
6.2.4.4. (8-Ethyl-1-(2,4-difluorophenyl)-6,7-difluoro-1,4-dihy-
dro-4-oxo-3-quinolinecarboxylato-O3,O4) difluoro-boron, 21c2.
Similarly, 20c2 (285 mg, 0.725 mmol) and BF3$Et2O
(1.20 mL, 9.44 mmol) in THF (25 mL) afforded 21c2
(152 mg, 0.37 mmol, 51%) as a white solid. Rf ¼ 0.50 (92:8
1
CHCl3/MeOH, UV); mp ¼ 279e281 ꢀC; H NMR (CD3CN)
3
d 0.87 (t, 3H, CH3(Ar), JHeH ¼ 7.5 Hz), 2.35 [m, 2H,
CH2(Ar)], 7.30 [m, 2H, H(Ar)], 7.80 [m, 1H, H(Ar)], 8.48 (t,
1H, H5, JHeF ¼ 4JHeF ¼ 9.1 Hz), 9.09 (s, 1H, H2); 19F NMR
3
00
2H, H1 and H5 ), 2.54 (s, 1H, H6 ), 3.52 (s, 3H, OCH3),
00
00
4
0
0
00
00
and H4 ,
(DMSO-d6) d ꢁ103.2 and ꢁ116.3 (2d, 2F, F2 and F4 , JFeF
¼
¼
3.59 and 3.82 (AB system, 4H, H2
3
9.3 Hz), ꢁ119.4 and ꢁ130.4 (d, 1F, F6 and F7, JFeF
2JHeH ¼ 9.7 Hz), 4.10 [m, 1H, CH(cPr)], 7.63 (d, 1H, H5,
3JHeF ¼ 13.1 Hz), 8.75 (s, 1H, H2); 19F NMR (1:4
CD3CN/D2O) d ꢁ74.4 (s, 4.8F, TFA), ꢁ117.4 (s, 1F, F6);
21.3 Hz), 141.6 and 142.4 (AB system, 0.5F, 10BF2, JFeF
¼ 72.9 Hz), 141.7 and 142.5 (AB system, 2.0F, 11BF2, JFeF
¼ 72.9 Hz).
13C NMR (1:4 CD3CN/D2O) d 9.3 [CH2(cPr)], 20.9 (C1
00
00
and C5 ), 29.9 (C6 ), 41.1 [CH(cPr)], 51.2 (d, C2 and
00
00
4
00
6.2.5. Synthesis of 7-amino-derived-6-fluoro-8-methoxy-qui-
nolinecarboxylic acids, 1e4
C4 , JCeF ¼ 5.6 Hz), 62.4 (OCH3), 106.6 (C3), 106.7 (d,
2
3
C5, JCeF ¼ 24.1 Hz), 119.9 (d, C10, JCeF ¼ 8.0 Hz), 134.5
2
(C9), 136.1 (d, C7, JCeF ¼ 11.4 Hz), 144.8 (d, C8,
1
6.2.5.1. 7-[(1a,5a,6a)-6-Amino-3-azabicyclo[3.1.0]hex-3-yl]-
1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-4-oxo-3-quino-
linecarboxylic acid, 1
6.2.5.1.1. 1-Cyclopropyl-7-[(1a,5a,6a)-6-[[(1,1-dimethyle-
thoxy)carbonyl]amino]-3-azabicyclo[3.1.0]hex-3-yl]-6-fluoro-
3JCeF ¼ 6.9 Hz), 150.8 (C2), 154.9 (d, C6, JCeF
¼
2
250.1 Hz), 158.8 (q, CF3CO2H, JCeF ¼ 31.7 Hz), 166.3
[C(O)O], 176.5 (C4). ESI-MS (positive mode): (M þ H)þ ¼
374.2, (M þ Na)þ ¼ 396.2, in agreement with the mass cal-
culated for M ¼ C19H20FN3O4 (373.14). ESI-HRMS

1490
G. Anquetin et al. / European Journal of Medicinal Chemistry 41 (2006) 1478e1493
(positive mode): 374.1516 is in agreement with the mass
calculated for (M þ H) ¼ C19H21FN3O4 (374.1516).
388.1656 is in agreement with the mass calculated for
(M þ H) ¼ C20H23FN3O4 (386.1673).
6.2.5.2. 7-[(1a,5a,6a)-6-(Aminomethyl)-3-azabicyclo[3.1.0]-
hex-3-yl]-1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-4-
oxo-3-quinolinecarboxylic acid, 2
6.2.5.3. 7-[(1a,5a,6a)-6-Amino-3-azabicyclo[3.1.0]hex-3-yl]-
1-(2,4-difluoro-phenyl)-6-fluoro-1,4-dihydro-8-methoxy-4-oxo-
3-quinolinecarboxylic acid, 3
6.2.5.2.1. 1-Cyclopropyl-7-[(1a,5a,6a)-6-[[[(1,1-dimethy-
lethoxy)carbonyl]amino]-methyl]-3-azabicyclo[3.1.0]hex-3-
yl]-6-fluoro-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarbox-
ylic acid, 2-Boc. A solution of 24 (445 mg, 2.10 mmol) and
21a1 (157 mg, 0.46 mmol) in CH3CN (14 mL) was refluxed
for one week. After evaporation, the crude residue was dis-
solved in a 1:1 CHCl3/H2O mixture. The organic layer was ex-
tracted, dried over Na2SO4, filtered and evaporated. The
yellow oily residue was purified by flash chromatography on
silica gel (100:0 to 96:4 CHCl3/MeOH) affording 2-Boc
6.2.5.3.1. 1-(2,4-Difluorophenyl)-7-[(1a,5a,6a)-6-[[(1,1-di-
methylethoxy)carbonyl]-amino]-3-azabicyclo[3.1.0]hex-3-yl]-
6-fluoro-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylic
acid, 3-Boc. The procedure, as described for 2-Boc, when applied
to 23 (189 mg, 0.95 mmol) and 21a1 (157 mg, 0.38 mmol) in
CH3CN (8 mL) gave, after work-up and flash chromatography,
3-Boc (151 mg, 0.277 mmol, 73%) as a colourless oil. Rf ¼ 0.70
(9:1 CHCl3/MeOH, UV); 1H NMR (CDCl3) d 1.42 [s, 9H,
00
00
C(CH3)3], 1.72 (s, 2H, H1 and H5 ), 2.45 (s, 1H, H6 ), 3.07 (s,
00
2
00
00
3H, OCH3), 3.38 [d, 1H, H2 or H4 endo or exo, JHeH
00
00
(180 mg, 0.37 mmol, 81%) as a colourless oil. Rf ¼ 0.85 (9:1
¼ 10.1 Hz], 3.64 (s, 2H, H2 or H4 endo or exo), 3.94 [d, 1H,
1
CHCl3/MeOH, UV); H NMR (CDCl3) d 0.70 (tt, 1H, H6 ,
2
00
00 00
H2 or H4 endo or exo, JHeH ¼ 10.4 Hz], 4.76 (br s, 1H, NHBoc),
7.03 [m, 2H, H(Ar)], 7.45 (td, 1H, H(Ar), J ¼ 8.7 Hz, J ¼ 5.6 Hz),
7.85 (d, 1H, H5,3JHeF ¼ 13.3 Hz), 8.45 (s, 1H, H2), 14.73 (br s, 1H,
3
3JHeH ¼ 3.4 Hz, JHeH ¼ 6.9 Hz), 0.90 and 1.13 [2m, 4H,
00
CH2(cPr)], 1.33 [s, 9H, C(CH3)3], 1.47 (s, 2H, H1 and H5 ),
00
COOH); 19F NMR (CDCl3) d ꢁ106.7 and ꢁ117.3 (2d, 2F, F2 and
0
3.02 (m, 2H, CH2NHBoc), 3.49 (s, 3H, OCH3), 3.53 (m, 2H,
F4 , JFeF ¼ 8.2 Hz), ꢁ117.8 (br s, 1F, F6); 13C NMR (CDCl3)
4
00
H2 and H4 exo or endo), 3.71 (d, 2H, H2 and H4 exo or
00
00
00
0
2
3
00 00 00 00
d 24.4 (C1 and C5 ), 28.4 [C(CH3)3], 31.8 (C6 ), 51.4 (d, C2 or
endo, JHeH ¼ 10.2 Hz), 3.95 (tt, 1H, CH(cPr), JHeH
3
4
4
00
00
00
¼ 3.6 Hz, JHeH ¼ 7.0 Hz), 4.93 (br s, 1H, NHBoc), 7.59 (d,
C4 , JCeF ¼ 7.0 Hz), 52.1 (d, C4 or C2 , JCeF ¼ 7.7 Hz), 60.8
3
2
0
1H, H5, JHeF ¼ 13.4 Hz), 8.65 (s, 1H, H2), 14.86 (br s, 1H,
(OCH3), 79.9 [C(CH3)3], 104.9 (dd, C3 , JCeF ¼ 26.7 Hz,
COOH); 19F NMR (CDCl3) d ꢁ118.7 (s, 1F, F6); 13C NMR
2JCeF ¼ 23.1 Hz), 108.1 (d, C5, JCeF ¼ 24.2 Hz), 108.5 (C3),
2
111.9 (dd, C5 , JCeF ¼ 22.9 Hz, 4JCeF ¼ 3.8 Hz), 119.8 (d, C10,
2
00
(CDCl3) d 9.2 [CH2(cPr)], 20.7 (C6 ), 21.0 (C1 and C5 ),
00
00
0
3
3
0
0
28.1 [C(CH3)3], 40.3 (CH2NHBoc), 41.9 [CH(cPr)], 51.9 (d,
4
JCeF ¼ 9.2 Hz), 127.3 (d, C6 , JCeF ¼ 10.3 Hz), 129.2 (dd, C1 ,
4
00
00
C2 and C4 , JCeF ¼ 7.3 Hz), 61.3 (OCH3), 78.9 [C(CH3)3],
2JCeF ¼ 13.5 Hz, JCeF ¼ 4.4 Hz), 133.0 (C9), 136.5 (d, C7,
2
3
107.1 (C3), 107.3 (d, C5, JCeF ¼ 24.2 Hz), 119.4 (d, C10,
2JCeF ¼ 11.3 Hz), 142.5 (d, C8, JCeF ¼ 7.0 Hz), 150.6 (C2),
4JCeF ¼ 9.2 Hz), 133.9 (C9), 136.7 (d, C7, JCeF ¼ 11.0 Hz),
155.4 (d, C6, JCeF ¼ 252.5 Hz), 156.4 [NHC(O)O], 157.3 (dd,
2
1
3
1
C2 or C4 , JCeF ¼ 254.3 Hz, 3JCeF ¼ 12.4 Hz), 162.8 (dd, C2 or
0
0
0
143.3 (d, C8, JCeF ¼ 6.6 Hz), 149.4 (C2), 154.6 (d, C6,
1JCeF ¼ 252.1 Hz), 155.7 [NHC(O)O], 166.6 [C(O)O], 176.5
C4 , JCeF ¼ 253.6 Hz, JCeF ¼ 11.2 Hz), 166.6 [C(O)O], 177.4
1
3
0
(d, C4, JCeF ¼ 3.3 Hz).
(d, C4, JCeF ¼ 2.9 Hz).
6.2.5.2.2. Synthesis of 2 (as its 0.6 TFA salt). Likewise, the
Boc deprotection procedure when applied to 2-Boc (114 mg,
0.234 mmol) afforded, after recrystallization (9:1 H2O/
CH3CN) and lyophilization, 2 as a white solid (103 mg,
0.148 mmol, 63%). Rf ¼ 0.35 (80:20 CHCl3/MeOH, UV);
HPLC: Rt ¼ 7.4 min (solvent A) and 13.2 min (solvent B);
1H NMR (1:9 CD3CN/D2O) d 0.93 [m, 2H, CH2(cPr)],
6.2.5.3.2. Synthesis of 3 (as its 1.6 TFA salt). Likewise, 3-
Boc (41 mg, 0.0753 mmol) gave, after Boc deprotection, recrys-
tallization (9:1 H2O/CH3CN) and lyophilization, 3 (33 mg,
0.053 mmol, 70%) as a pale yellow solid. Rf ¼ 0.30 (84:14:2
CHCl3/MeOH/H2O, UV); HPLC: Rt ¼ 9.0 min (solvent A) and
1
14.6 min (solvent B); H NMR (1:4 CD3CN/D2O) d 1.98 (m,
00
2H, H1 and H5 ), 2.46 (br s, 1H, H6 ), 2.97 (s, 3H, OCH3),
00
00
00
1.13 [m, 3H, CH2(cPr) and H6 ], 1.66 (s, 2H, H1 and
00
00
00
3.28 and 3.57 (2d, 1H each, H2 or H4 endo or exo, JHeH
¼
3
00
00
7.8 Hz), 3.45 and 3.82 (2d, 1H each, H2 or H4 endo or exo,
00
H5 ), 2.88 (d, 2H, CH2NHBoc, JHeH ¼ 7.5 Hz), 3.49 (s,
3
00
00
3H, OCH3), 3.53 and 3.75 (AB system, 4H, H2 and H4 ,
JHeH ¼ 9.1 Hz), 7.11 [m, 2H, H(Ar)], 7.50 (d, 1H, H5, JHeF
2JHeH ¼ 10.4 Hz), 4.13 (tt, 1H, CH(cPr), JHeH ¼ 7.2 Hz,
¼ 13.0 Hz), 7.63 [m, 1H, H(Ar)], 8.84 (s, 1H, H2); 19F NMR
3
3JHeH ¼ 3.6 Hz), 7.45 (d, 1H, H5, JHeF ¼ 13.4 Hz), 8.74
(1:4 CD3CN/D2O) d ꢁ74.4 (s, 4.8F, TFA), ꢁ106.7 and ꢁ117.9
3
(s, 1H, H2); 19F NMR (1:9 CD3CN/D2O) d ꢁ74.4 (s, 1.8F,
TFA), ꢁ117.2 (s, 1F, F6); 13C NMR (1:9 CD3CN/D2O)
(2d, 2F, F2 and F4 , JFeF ¼ 7.9 Hz), ꢁ117.5 (s, 1F, F6); 13C
4
0
0
00
NMR (1:4 CD3CN/D2O) d 21.5 and 21.6 (C1 and C5 ), 30.7
00
4
00
00
00
d 9.9 [CH2(cPr)], 18.6 (s, C6 ), 22.6 (C1 and C5 ), 42.2
00
00
00
00
00
(C6 ), 51.5 (d, C2 or C4 , JCeF ¼ 6.9 Hz), 52.2 (d, C4 or C2 ,
4
2
00
00
0
and 42.5 [CH(cPr) and CH2NH2], 52.7 (d, C2 and C4 ,
JCeF ¼ 6.9 Hz), 61.8 (s, OCH3), 105.1 (dd, C3 , JCeF ¼
4JCeF ¼ 7.0 Hz), 62.7 (OCH3), 107.0 (C3), 107.5 (d, C5,
27.0 Hz, JCeF ¼ 23.5 Hz), 107.6 (d, C5, JCeF ¼ 24.1 Hz),
2
2
2JCeF ¼ 24.2 Hz), 119.9 (d, C10, JCeF ¼ 8.0 Hz), 135.7
107.8 (C3), 112.7 (d, C5 , JCeF ¼ 21.8 Hz), 120.0 (d, C10,
3
2
0
2
3
3
3
0
(C9), 138.4 (d, C7, JCeF ¼ 11.4 Hz), 145.1 (d, C8, JCeF
¼
JCeF ¼ 7.4 Hz), 128.7 (d, C6 , JCeF ¼ 10.7 Hz), 129.5 (dd,
1
2
4
0
7.0 Hz), 151.8 (C2), 156.1 (d, C6, JCeF ¼ 250.7 Hz), 169.5
[C(O)O], 177.6 (C4). ESI-MS (positive mode): (M þ H)þ
¼ 388.2 is in agreement with the mass calculated for
M ¼ C20H22FN3O4 (387.16). ESI-HRMS (positive mode):
C1 , JCeF ¼ 13.2 Hz, JCeF ¼ 4.0 Hz), 133.8 (C9), 137.3 (d,
2
3
C7, JCeF ¼ 11.5 Hz), 143.7 (d, C8, JCeF ¼ 6.9 Hz), 152.1
1
0
(C2), 156.1 (d, C6, JCeF ¼ 251.0 Hz), 157.7 (dd, C2 or
1
3
0
0
C4 , JCeF ¼ 251.3 Hz, JCeF ¼ 12.6 Hz), 162.5 (dd, C2 or

G. Anquetin et al. / European Journal of Medicinal Chemistry 41 (2006) 1478e1493
1491
1
3
2
0
C4 , JCeF ¼ 251.3 Hz, JCeF ¼ 10.9 Hz), 168.2 [C(O)O], 178.0
(C4). ESI-MS (positive mode): (M þ H)þ ¼ 446.1 is in agree-
ment with the mass calculated for M ¼ C22H18F3N3O4
(445.12). ESI-HRMS (positive mode): 446.1313 is in agreement
with the mass calculated for (M þ H) ¼ C22H19F3N3O4
(446.1328).
25.2 Hz), 107.9 (d, C5, JCeF ¼ 21.8 Hz), 108.1 (C3), 112.8
2
4
0
(dd, C5 , JCeF ¼ 20.7 Hz, JCeF ¼ 4.4 Hz), 119.9 (d, C10,
3
3
0
JCeF ¼ 3.3 Hz), 128.8 (d, C6 , JCeF ¼ 8.0 Hz), 129.8 (dd,
2
4
0
C1 , JCeF ¼ 13.5 Hz, JCeF ¼ 4.8 Hz), 134.2 (C9), 138.2 (d,
2
3
C7, JCeF ¼ 12.6 Hz), 143.4 (d, C8, JCeF ¼ 6.6 Hz), 152.3
1
0
0
(C2), 156.2 (d, C6, JCeF ¼ 253.5 Hz), 157.9 (d, C2 or C4 ,
1
1
0
0
JCeF ¼ 251.0 Hz), 165.6 (d, C2 or C4 , JCeF ¼ 250.0 Hz),
6.2.5.4. 7-[(1a,5a,6a)-6-(Aminomethyl)-3-azabicyclo[3.1.0]-
hex-3-yl]-1-(2,4-difluorophenyl)-6-fluoro-1,4-dihydro-8-me-
thoxy-4-oxo-3-quinolinecarboxylic acid, 4
6.2.5.4.1. 1-(2,4-Difluorophenyl)-7-[(1a,5a,6a)-6-[[[(1,1-
dimethylethoxy)carbonyl]-amino]methyl]-3-azabicyclo[3.1.0]-
hex-3-yl]-6-fluoro-1,4-dihydro-8-methoxy-4-oxo-3-quinoline-
carboxylic acid, 4-Boc. The procedure, as described for 2-
Boc, when applied to 24 (211 mg, 0.99 mmol) and 21a2
(145 mg, 0.35 mmol) gave after work-up and flash chromatog-
raphy 4-Boc (173 mg, 0.31 mmol, 89%) as a colourless oil.
168.7 [C(O)O], 178.1 (C4). ESI-MS (positive mode):
(M þ H)þ ¼ 460.2, (M þ Na)þ ¼ 482.2 is in agreement with
the mass calculated for M ¼ C23H20F3N3O4 (459.14). ESI-
HRMS (positive mode): 460.1488 is in agreement with the
mass calculated for (M þ H) ¼ C23H21F3N3O4 (460.1484).
6.3. Biological assays
6.3.1. In vitro antiparasitical activities
1
Rf ¼ 0.75 (9:1 CHCl3/MeOH, UV); H NMR (CDCl3) d 0.94
6.3.1.1. Anti-T. gondii activity. Briefly, following a previously
described method [22], the virulent RH strain of T. gondii was
maintained in mice by intraperitoneal passage every two days.
For each experiment, tachyzoites were collected from the peri-
toneal cavity of infected mice then resuspended in physiolog-
ical saline. Tissue cultures and drug tests were carried out
using MRC5 fibroblast tissue cultures. Confluent monolayers
prepared in 96-well tissue culture plates were inoculated
with 2000 fresh tachyzoites. After 4 h, drugs at various con-
centrations were added into the culture medium and culture
plates were incubated for an additional 72 h. Each culture
plate comprised eight negative control (without T. gondii)
and eight positive control wells (without drug). After incuba-
tion, the plates were examined microscopically for cytopathic
effects and thereafter fixed with cold methanol for 5 min.
Toxoplasma growth was assessed by enzyme linked immuno-
assay (ELISA) performed directly on the fixed cultures using
a peroxidase labeled monoclonal antibody directed against
the SAG-1 surface protein of T. gondii. After addition of the
substrate, spectrophotometric readings were recorded at
a wavelength of 405 nm with blank on the negative control
well. For each well, the results were expressed as optical den-
sity (OD) values. The effect of each drug at various concentra-
tions was described by plotting the OD values as a function of
the logarithm of the concentration and a linear regression
model was used to summarize the concentrationeeffect rela-
tionship and to determine the IC50.
3
3
00
(tt, 1H, H6 , JHeH ¼ 3.4 Hz, JHeH ¼ 6.8 Hz), 1.37 [s, 9H,
00
00
C(CH3)3], 1.56 (s, 2H, H1 and H5 ), 3.03 (s, 3H, OCH3),
00
00
3.40e3.80 (m, 6H, H2 , H4 and CH2NHBoc), 4.79 (br s,
1H, NHBoc), 6.95 [m, 2H, H(Ar)], 7.49 [m, 1H, H(Ar)],
3
7.73 (d, 1H, H5, JHeF ¼ 13.4 Hz), 8.38 (s, 1H, H2), 14.69
(br s, 1H, COOH); 19F NMR (CDCl3) d ꢁ106.8 and ꢁ117.4
4
0
0
(2d, 2F, F2 and F4 , JFeF ¼ 8.3 Hz), ꢁ117.6 (br s, 1F, F6);
13C NMR (CDCl3) d 20.9 and 21.1 (C1 and C5 ), 21.2
00
00
00
00
(C6 ), 28.4 [C(CH3)3], 42.2 (CH2NHBoc), 51.7 (d, C2 or
4
4
00
00
00
C4 , JCeF ¼ 7.3 Hz), 52.5 (d, C4 or C2 , JCeF ¼ 7.2 Hz),
2
0
60.7 (OCH3), 79.3 [C(CH3)3], 104.7 (dd, C3 , JCeF
¼
2
2
26.7 Hz, JCeF ¼ 22.7 Hz), 107.8 (d, C5, JCeF ¼ 24.5 Hz),
2
4
0
108.2 (C3), 111.7 (dd, C5 , JCeF ¼ 22.9 Hz, JCeF ¼ 3.8 Hz),
3
3
0
119.8 (d, C10, JCeF ¼ 9.2 Hz), 127.3 (d, C6 , JCeF
¼
2
4
0
9.5 Hz), 129.1 (dd, C1 , JCeF ¼ 13.4 Hz, JCeF ¼ 4.2 Hz),
2
132.9 (C9), 137.0 (d, C7, JCeF ¼ 11.0 Hz), 142.2 (d, C8,
3JCeF ¼ 7.0 Hz), 150.4 (C2), 155.2 (d, C6, JCeF ¼ 252.5 Hz),
1
1
0
0
155.9 [NHC(O)O], 157.1 (dd, C2 or C4 , JCeF
¼
¼
3
1
0
0
254.3 Hz, JCeF ¼ 12.4 Hz), 162.7 (dd, C2 or C4 , JCeF
3
252.9 Hz, JCeF ¼ 11.0 Hz), 166.4 [C(O)O], 177.1 (d, C4,
JCeF ¼ 3.3 Hz).
6.2.5.4.2. Synthesis of 4 (as its 1.2 TFA salt). Likewise,
compound 4-Boc (63 mg, 0.113 mmol) gave, after Boc
deprotection, recrystallization (9:1 H2O/CH3CN) and lyophili-
zation, 4 (43 mg, 0.072 mmol, 64%) as a pale yellow solid.
Rf ¼ 0.20 (84:14:2 CHCl3/MeOH/H2O, UV); HPLC: Rt ¼
14.0 min (solvent A) and 16.1 min (solvent B); mp ¼ 126e
1
130 ꢀC; H NMR (1:4 CD3CN/D2O) d 1.00 (m, 1H, H6 ),
6.3.1.2. Anti-P. falciparum activity. Cultures of the NF54-R
chloroquine-resistant derived from the NF54 strain were main-
tained in continuous culture according to Ref. [64]. The in vi-
tro activities of the drugs were evaluated by using the methods
described in Ref. [65]. Two hundred microliters of ring stage
parasitized erythrocytes (parasitemia, 0.5%; hematocrit,
1.8%) were distributed in 96-well plates preloaded with nine
concentrations (0.025e166 mg/mL) of each drug in triplicate
and with serial dilutions of chloroquine in positive control
wells. After 72 h, [3H] hypoxanthine was added to each well
and then plates were incubated for an additional 24 h. Para-
sites were harvested, and incorporation of radioactivity was
00
3
00
00
1.59 (s, 2H, H1 and H5 ), 2.83 (d, 2H, CH2NH2, JHeH
5.8 Hz), 2.98 (s, 3H, OCH3), 3.27 and 3.59 (2d, 1H each,
¼
00
00
H2 or H4 endo or exo, JHeH ¼ 9.5 Hz), 3.38 and 3.82 (2d,
00
00
1H each, H2 or H4 endo or exo, JHeH ¼ 10.0 Hz), 7.13 [m,
3
2H, H (Ar)], 7.66 [m, 1H, H (Ar)], 7.73 (d, 1H, H5, JHe
¼ 13.4 Hz), 8.48 (s, 1H, H2); 19F NMR (1:4 CD3CN/D2O)
F
0
d ꢁ74.4 (s, 3.6F, TFA), ꢁ107.3 and ꢁ118.1 (2d, 2F, F2 and
F4 , JFeF ¼ 7.6 Hz), ꢁ116.2 (s, 1F, F6); 13C NMR (1:4
4
0
00
CD3CN/D2O) d 18.4 (C6 ), 22.3 and 22.4 (C1 and C5 ),
00
00
4
00
00
42.2 (CH2NH2), 52.0 (d, C2 or C4 , JCeF ¼ 5.7 Hz), 52.9
2
00
00
0
(br s, C4 or C2 ), 61.7 (OCH3), 105.2 (t, C3 , JCeF
¼

1492
G. Anquetin et al. / European Journal of Medicinal Chemistry 41 (2006) 1478e1493
determined by liquid scintillation counting. Experiments were
repeated twice.
MIC determination. MIC values were defined as the lowest
concentration of quinolone that inhibited more than 99% of
the bacterial growth.
6.3.1.3. Anti-P. yoelii yoelii activity. Mouse hepatocyte cul-
tures were prepared in Lab-Tek slides as described in
Ref. [66] and then incubated for 24 h before sporozoite inoc-
ulation. Sporozoites were obtained by dissection of the sali-
vary glands of Anopheles stephensi mosquitoes infected with
P. yoelii yoelii (265 By). Dilutions of drugs were made in cul-
ture medium (1e100 mg/mL), and 8 ꢂ 104 sporozoites of P.
yoelii yoelii were added to hepatocyte cultures. Each drug
was tested in four replicates. The culture medium containing
the drug was renewed every 24 h, thus maintaining a correct
concentration. Cultures were incubated for 48 h for P. yoelii
yoelii and then fixed with cold methanol. Schizonts were eval-
uated using an immunofluorescence test with an anti-HSP-i72
polyclonal antibody. Both the numbers and size of schizonts in
the culture were taken into account to assess drug activity. Ex-
periments were repeated twice.
Acknowledgements
We thank the Centre National de la Recherche Scientifique
(CNRS) for financial support, and MNERT (G.A.) for grant.
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pH 7.5), 25 mM KCl, 6 mM magnesium acetate, 2 mM sper-
midine, 4 mM dithiothreitol, 0.1 mg of Escherichia coli
tRNA per mL, bovine serum albumin (0.36 mg/mL),
100 mM potassium glutamate, 1 mM ATP (pH 8) and relaxed
pBR322 DNA (0.4 mg) as the substrate were incubated with or
without increasing concentrations of quinolones at 37 ꢀC for
1 h. Reactions were terminated by the addition of 50% glyc-
erol containing 0.25% bromophenol blue, and the total reac-
tion mixture was subjected to electrophoresis in a 1%
agarose gel in 0.5ꢂ TBE buffer. After running for 5.5 h at
50 V, the gel was stained with ethidium bromide (0.7 mg/mL).
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DNA gyrase that converted 400 ng of relaxed pBR322 to
the supercoiled form in 1 h at 37 ꢀC. DNA gyrase of E. coli
was used as a positive control for the assay procedures and
buffer. Inhibition of the supercoiling activity of the recombi-
nant DNA gyrase was performed by the following method.
A reaction mixture containing 1 U of purified DNA gyrase
and increasing concentrations of quinolones was incubated
as describe above. The inhibitory effect of quinolones on
DNA gyrase was assayed by determining the concentration
of drug required to inhibit the supercoiling activity of the en-
zyme by 50% (IC50).
[13] P.M. Hawkey, J. Antimicrob. Chemother. 51 (2003) 29e35.
[14] J. Ruiz, J. Antimicrob. Chemother. 51 (2003) 1109e1117.
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