Synthesis, in vitro antitrypanosomal and antibacterial activity of phenoxy, phenylthio or benzyloxy substituted quinolones

Article abstract of DOI:10.1016/j.bmcl.2008.11.078

Chagas' disease, caused by Trypanosoma cruzi (T. cruzi), is one of the most serious parasitic diseases in Latin America. The currently available chemotherapy, based on nifurtimox or benznidazole, is unsatisfactory due to the limited efficacy in the preval

Full text of DOI:10.1016/j.bmcl.2008.11.078

Bioorganic & Medicinal Chemistry Letters 19 (2009) 986–989
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, in vitro antitrypanosomal and antibacterial activity of phenoxy,
phenylthio or benzyloxy substituted quinolones
a,
c
Xiang Ma a,b, , Weicheng Zhou , Reto Brun
*
*
a State Key Lab of New Drug & Pharmaceutical Process, Shanghai Institute of Pharmaceutical Industry, 1111 North Zhongshan Road, Shanghai 200437, PR China
b Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, B1k E5 #02-09, Singapore 117576, Singapore
c Medical Parasitology and Infection Biology, Parasite Chemotherapy, Swiss Tropical Institute, Socinstrasse 57, CH-4002 Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 June 2008
Revised 28 October 2008
Accepted 19 November 2008
Available online 24 November 2008
Chagas’ disease, caused by Trypanosoma cruzi (T. cruzi), is one of the most serious parasitic diseases in
Latin America. The currently available chemotherapy, based on nifurtimox or benznidazole, is unsatisfac-
tory due to the limited efficacy in the prevalent chronic stage of the disease and toxic side effects. In order
to address these deficiencies, a series of quinolones based novel molecules have been synthesized and
evaluated as potential antitrypanosomal agents. The most active analogue 10 inhibited T. cruzi with an
IC50 of 1.3
antitrypanosomal agents.
lg/mL. The results of this study have implications in the development of novel quinolone’s
Keywords:
Chagas’ disease
Trypanosoma cruzi
Quinolones
Ó 2008 Elsevier Ltd. All rights reserved.
Antitrypanosomal
Antibacterial activity
Chagas’ disease, caused by Trypanosoma cruzi (T. cruzi), is one of
the most serious parasitic diseases in Latin America. It is a poten-
tially fatal, chronic illness that currently affects about 18–20 mil-
lion people and another 100 million people are at risk of
acquiring the disease.1,2 Although sequencing of T. cruzi genome
was completed,3 neither prospect of antitrypanosomal vaccine
nor a new drug has been developed yet to prevent or treat Chagas’
disease. The medication for Chagas’ disease has been made entirely
by chemotherapy.4,5 The currently available chemotherapy is
based on two agents introduced in the market in the 1970s: nifur-
the synthesis of an antimalarial agent chloroquine.7 In the 1980s,
medicinal chemists discovered that specific chemical modifica-
tions of nalidixic acid dramatically improved the antibacterial
spectrum of activity. One of these modifications was the addition
of fluorine (the source of the name ‘fluoroquinolone’). In 1986, nor-
floxacin became the first fluoroquinolone marketed for human use,
closely followed by ciprofloxacin in 1987. Since 1986, more than 20
fluoroquinolones have been approved by FDA and most of those re-
main on the market.8
The antibacterial activity generated by fluoroquinolones is
caused by the inhibition of two bacterial enzymes: DNA gyrase (a
topoisomerase enzyme in bacteria) and topoisomerase IV enzyme.8
The general function of topoisomerases is to facilitate the uncoiling
of DNA during DNA replication, as well as to facilitate the recoiling
and packaging of DNA once DNA replication has occurred.9 Like
bacteria, kinetoplastid protozoa have unique and complex types
of mitochondrial DNA, composed of thousands of topologically
interlocked circular DNA molecules, creating a high demand for
topoisomerase enzymes activity.10 Targeting topoisomerases may
be a way to follow in searching for new chemotherapeutic agents
against the diseases caused by trypanosoma, such as Chagas’ dis-
ease, where a high rate of T. cruzi proliferation and unique aspects
of the kDNA replication were involved.11
timox
(4-(5-nitrofurfurylindenamino)-3-methylthiomorpholine
1,1-dioxide, Lampit, discontinued by Bayer), a nitrofuran deriva-
tive; and benznidazole (N-benzyl-2-(2-nitro-1H-imidazol-1-
yl)acetamide, Rochagan, Roche), a nitroimidazole derivative.5 Due
to limited efficacy in the prevalent chronic stage of the disease
and toxic side effects, the need for the discovery of new therapeutic
agents to treat Chagas’ disease is clear and urgent.
The quinolones and fluoroquinolones constitute a major class of
antibacterial chemotherapeutic agents, which have a broad spec-
trum of activity against bacteria, mycobacteria, parasites, and other
diseases.6 The history of quinolones began in the late 1950s, when
Lesher accidentally discovered nalidixic acid, 1-ethyl-1,4-dihydro-
7-methyl-1,8-naphthylidine-3-carboxylic acid, as a by-product in
A number of studies reported that fluoroquinolones have activ-
ity in vitro or in vivo against trypanosomes or Leishmania (a genus
of trypanosome protozoa).12–21 However, only limited in vitro
activity against T. cruzi was observed for the fluoroquinolones
and quinolones in clinical use.12–15 The investigators found that
* Corresponding authors. Tel.: +65 6516 2657; fax: +65 6779 1554 (X.M.), tel.:
+86 21 55514600; fax: +86 21 35052484 (W.Z.).
E-mail addresses: chemx@nus.edu.sg (X. Ma), profzhouwc@yahoo.com.cn (W.
Zhou).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.11.078

X. Ma et al. / Bioorg. Med. Chem. Lett. 19 (2009) 986–989
987
the presence of nalidixic acid at the concentration of 200
l
g/mL re-
the same condition, reaction with 1-fluoroethyl quinolone Ic gave
N1 dehydrofluoride 7,8-dibenzyloxy derivative 11.24 However,
when this condition was applied to Ib, a mixture was formed
and the attempts of isolation were failed. Two sets of 1H NMR spec-
tral data from the mixture with different H–F coupling constants
on 5-H of the quinolone suggest the formation of a nearly equal
amount of 7,8-disubstituted and 6,7-disubstituted products. This
could be due to the steric effect of N-cyclopropyl group which hin-
dered the leaving of fluoride at position 8. Some of these deriva-
tives were further elaborated: esterification of carboxylic acids 4,
5 and 10 was accomplished by the addition of SOCl2 in methanol
or ethanol to afford methyl or ethyl ester 12–17; and in the other
way, treatment of compound 4 or 5 with 1,1-carbonyldiimidazole
(CDI), followed by reacting with NH3, gave 18 or 20. Further dehy-
dration of the amide 18 by POCl3 gave 3-cyanoquinolone 19.
During the preparation of 7,8-disubstituted derivatives, some
mono or tri-substituted by-products were also formed and perked
our interests, since some antibacterial quinolones are substituted
only at position 7 and with fluorines at position 6 and 8. This phe-
nomenon prompted us to synthesize 7-monosubstituted quino-
lones to explore how such modifications would affect the
antitrypanosomal activity. The 7-substitution of Ia or Ib by phenol
in the presence of NaH/DMSO at room temperature provided 21
and 22. In addition, the monosubstitution at 7-position of Ia or
Ib by thiophenol or benzyl alcohol was achieved with 3 N KOH at
50 °C to give 23–25. Furthermore, displacement of all the fluorine
atoms at a higher temperature by above three nucleophiles gave
tri-substituted derivatives 26–28.
sulted in 60% inhibition of T. cruzi epimastigote proliferation,15 and
another study showed that ciprofloxacin exhibited limited activity
against T. cruzi, clearing amastigotes from 26% of macrophages at
the concentration of 250 l
M.14 A combination of a fluorine atom
at position 6 (see Table 1 for fluoroquinolone numbering) and a
5- or 6-membered heterocycle that contains peripheral nitrogens
at position 7 in quinolones often resulted in a broad and potent
antimicrobial activity. However, this combination may not be nec-
essary for their antitrypanosomal activity against T. cruzi. There
were no reports for this class of compounds using non-nitrogen
containing heterocyclic substitutions for their activity against T.
cruzi. Herein, as a part of our ongoing research program to discover
new classes of antitrypanosomal agents, we developed a small li-
brary of quinolone derivatives which did not contain nitrogen het-
erocyclic substitution at position 7 of quinolone moiety for their
in vitro antitrypanosomal activity against T. cruzi. In this letter,
the potential of quinolones substituted with a non-nitrogen group
such as phenoxy, phenylthio or benzyloxy at position 6, 7 or 8 as
antitrypanosomal agents against T. cruzi was investigated. And
antibacterial activity was also tested to determine the selectivity
between the trypanosoma and bacterial species.
A series of phenoxy, benzyloxy and phenylthio quinolones ana-
logues 1–28 listed in Table 1 were synthesized as outlined in
Scheme 1. 6,7,8-Trifluoro-1,4-dihydroquinoline-3-carboxylic acid
(Ia, Ib or Ic),22 a crucial intermediate for the synthesis of antibac-
terial quinolones, was treated with excess phenol or thiophenol
with NaH in DMSO to give 7,8-disubstituted derivatives 1–9. Using
benzyl alcohol as a nucleophile, the substitution on Ia was taken
place under aqueous alkali condition (8 N KOH) to give 10.23 Under
Compounds 1–28 were characterized for the antitrypanosomal
activity against T. cruzi,25 and the results are summarized in Table
Table 1
In vitro antitrypanosomal activity against T. cruzi of quinolone derivatives
O
4
5
R6
R7
R3
3
6
2
7
N
1
8
R8 R1
Compound
R1
R3
R6
R7
R8
Yield (%)
IC50 (lg/mL)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
c-Pr
C2H5
CH@CH2
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
c-Pr
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOMe
COOEt
COOMe
COOEt
COOMe
COOEt
CONH2
CN
CONH2
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
4-CH3-PhO
4-OH-PhO
2,4-Dichloro-PhO
PhO
4-CH3-PhO
4-OH-PhO
2,4-Dichloro-PhO
PhO
50.8
23.9
43.3
56.7
62.6
59.0
52.1
43.7
36.6
31.6
33.7
82.3
43.7
66.1
75.0
38.3
31.6
77.9
60.0
86.4
63.2
63.8
76.4
74.7
84.2
28.3
35.5
28.3
19.4
57.1
12.7
>90
19.3
6.2
16.3
52.7
17.8
1.3
6.2
>90
1.7
2.4
6.0
PhS
PhS
4-F-PhS
4-Cl-PhS
4-CH3-PhS
PhS
PhCH2O
PhCH2O
PhO
PhO
PhS
PhS
PhCH2O
PhCH2O
PhO
PhO
PhS
4-CH3-PhO
PhO
PhS
PhCH2O
PhCH2O
4-CH3-PhO
PhS
4-F-PhS
4-Cl-PhS
4-CH3-PhS
PhS
PhCH2O
PhCH2O
PhO
PhO
PhS
PhS
PhCH2O
PhCH2O
PhO
PhO
PhS
F
F
F
F
3.8
1.7
4.0
>90
>90
18.3
>90
34.6
18.3
>90
>90
39.9
2.5
C2H5
C2H5
c-Pr
C2H5
C2H5
C2H5
F
F
4-CH3-PhO
PhS
PhCH2O
4-CH3-PhO
PhS
PhCH2O
PhCH2O
Benznidazole
0.83

988
X. Ma et al. / Bioorg. Med. Chem. Lett. 19 (2009) 986–989
O
4
O
O
5
F
F
COOH
3
F
COOH
F
CO2Me (Et)
6
b
d
2
R7
R7
N
N
7
N
8
1
R1
R8 R1
R8 R1
F
1-8, 10, R1=ethyl
9, R1=cyclopropyl
11, R1=ethenyl
Ia, R1=ethyl
Ib, R1=cyclopropyl
Ic, R1=FCH2CH2
a
12-17
O
F
COOH
e
R7
N
c
O
O
F
R1
O
F
CN
F
CONH2
f
R6
R7
COOH
21-25
R7
R7
N
N
R8 R1
N
R8 R1
R8 R1
18, 20
19
26-28
Scheme 1. Reagents and conditions: (a) BnOH, 3 N KOH, 50 °C, or ArOH, NaH/DMSO, rt, or ArSH, 2 N KOH, 50 °C; (b) BnOH, 8 N KOH, 80 °C, or ArOH, NaH/DMSO, 60 °C, or
ArSH, NaH/DMSO, rt; (c) BnOH, 12 N KOH, reflux, or ArOH, NaH/DMSO, 140 °C, or ArSH, NaH/DMSO, 50 °C; (d) SOCl2, MeOH or EtOH, reflux; (e) i—1,10-carbonyldimidazole
(CDI), DMF, 100 °C; ii—NH3, methyl-2-pyrrolidinone, rt; (f) i—POCl3, methyl-2-pyrrolidinone, rt; ii—NaHCO3, pH = 8.
Table 2
1. Most 7,8-disubstituted quinolones exhibited significant inhibi-
tory activity against T. cruzi as exemplified by 10, 13 and 17.
Among them, the 7,8-dibenzyloxy fluoroquinolone 10 exhibited
In vitro antibacterial activity of some 7-(substituted) quinolone derivatives
Strain
MIC (lg/mL)
the most potent activity with an IC50 value of 1.3
ment of the N-ethyl group of 10 with ethenyl resulted in 11 with a
decreased activity (IC50 = 6.2 g/mL). Esterification of 3-carboxylic
group (compound 16 or 17) also resulted in a decreased potency.
The 7,8-diphenoxy compound 4 was less potent than the analogues
1–3 in which the phenyl group was substituted with methyl, hy-
droxyl or chloro group. When 4 was esterified with ethanol, the
lg/mL. Replace-
21
22
23
24
25
Lomefloxacin
S. aureus
S. pneumoniae
S. albus
S. epidermidis
E. faecalis
0.78
0.39
12.5
6.25
>25
>25
25
12.5
6.25
>25
25
>25
>25
25
6.25
25
0.39
0.098
1.56
1.56
12.5
>25
3.13
1.56
1.56
>25
6.25
6.25
12.5
3.13
1.56
3.13
3.13
1.56
6.25
12.5
3.13
0.78
>25
3.13
>25
>25
>25
12.5
12.5
>25
>25
>25
>25
>25
12.5
>25
>25
12.5
>25
>25
6.25
1.56
>25
25
>25
>25
>25
12.5
25
>25
>25
>25
>25
>25
6.25
>25
25
1.56
1.56
6.25
1.56
12.5
6.25
1.56
1.56
1.56
>25
6.25
6.25
6.25
6.25
0.78
3.13
1.56
1.56
3.13
6.25
0.78
0.195
3.13
0.78
25
l
c
-Streptococcus
6.25
E. coli
S. sonnei
S. boydii
S.flexneri
P. mirabilis
P. vulgaris
P. aeruginosa
M. morganii
K. pneumoniae
S. enteritidis
S. typhimurium
C. freundii
0.098
0.098
0.098
0.78
0.39
0.78
activity of 13 increased greatly with an IC50 value of 1.7
and a surprising difference was found for the methyl ester 12
(IC50 > 90 g/mL). An increase in inhibitory activity was also found
lg/mL,
l
on 3-carboxylic amide derivative 18. However, when the amide
was transformed into nitrile, compound 19 showed no improve-
ment in potency over the acid 4. For the phenylthio analogues,
incorporation of a fluoro atom at 4-position of phenyl ring as in
compound 6 increased activity by nearly 3-fold when compared
to unsubstituted derivative 5. It suggests that the electro-with-
drawing property of the 4-substituent in the phenyl ring is impor-
tant, which is corroborated by increased activity of 7 with a chloro
group and decreased activity of 8 with a methyl group in the 4-po-
sition of phenyl ring. Esterification of 5 enhanced the inhibition,
3.13
0.195
0.098
0.098
0.195
0.098
6.25
25
12.5
25
25
>25
>25
A. hydrophila
S. marcescens
>25
0.195
and the activity of methyl ester 14 with IC50 value of 2.4
lg/mL
with a cyclopropyl group at 1-position was more active than the
reference agent lomefloxacin against Staphylococcus aureus, or
Streptococcus pneumoniae. Compound 21 exhibited potent activity
against Gram-positive organisms S. aureus and S. pneumoniae com-
parable to lomefloxacin. The antibacterial property of these com-
pounds seems to show no correlation to the antitrypanosomal
activity, and a Spearman’s correlation showed that the correlation
between the IC50 against T. cruzi and MICs against S. aureus
was more potent than ethyl ester 15 (6.0 g/mL). However, modi-
fication of 3-carboxylic acid into amide 20 resulted in the depletion
of activity. Good inhibitory activity was also seen in 6,7,8-triben-
zyloxy substituted derivatives (28, IC50 = 2.5 lg/mL), although the
6,7,8-trisubstitutions were less favorable in other substituted
patterns.
The minimum inhibitory concentrations (MICs) of the quino-
lones 1–28 against several representative Gram-negative and
Gram-positive organisms are summarized in Table 2, along with
data of lomefloxacin for comparison. MICs were determined by
an agar dilution method as outlined by the Clinical and Laboratory
Standards Institute (CLSI).26 The MIC was defined as the lowest
concentration resulting in inhibition of visible bacterial growth
after incubation at 37 °C for 24 h. Among all the 28 tested com-
pounds, only those 7-monosubstituted compounds 21–25 showed
l
(q
= ꢀ0.263, p = 0.195) and S. pneumoniae ( = ꢀ0.253, p = 0.176)
q
was statistically not significant.27 Since none of these compounds
showed good antitrypanosomal activity against T. cruzi, this small
library of quinolones exhibited great selectivity on trypanosoma.
In summary, a series of phenoxy, phenylthio and benzyloxy
substituted quinolones were synthesized and evaluated for the
inhibitory activity against T. cruzi and antibacterial activity. Most
compounds exhibited moderate to high inhibitory activity
in vitro against T. cruzi. Among them, the 7,8-dibenzyloxy quino-
lone 10 demonstrated the most significant antitrypanosomal activ-
significant antibacterial activities (MIC < 25 lg/mL), and the activ-
ities against Gram-positive organisms of these compounds were
better than that against Gram-negative organisms. Compound 22

X. Ma et al. / Bioorg. Med. Chem. Lett. 19 (2009) 986–989
989
10. Douc-Rasy, S.; Kayser, A.; Riou, J.-F.; Riou, G. Proc. Natl. Acad. Sci. U.S.A. 1986, 83,
7152.
11. Das, A.; Dasgupta, A.; Sengupta, T.; Majumder, H. K. Trends Parasitol. 2004, 20,
381.
12. Gonzales-Perdomo, M.; De Castro, S. L.; Meirelles, M. N. L.; Spohr, U.;
Goldenberg, S. Mem. Inst. Oswaldo Cruz 1986, 81, 93.
13. Betbeder, D.; Hutchinson, D. W.; Baltz, T.; Cros, S. Med. Sci. Res. 1988, 16, 141.
14. Croft, S. L.; Hogg, J. Trans. R. Soc. Trop. Med. Hyg. 1988, 82, 856.
15. Gonzales-Perdomo, M.; De Castro, S. L.; Meirelles, M. N. S. L.; Goldenberg, S.
Antimicrob. Agents Chemother. 1990, 34, 1707.
16. Raether, W.; Seidenath, H.; Hofmann, J. Parasitol. Res. 1989, 75, 412.
17. Sanguigni, S.; Marangi, M.; Gramiccia, M.; Orsini, S.; Paparo, B. S.; Nicodemo,
G.; Gradoni, L. G. Mal. Infett. Parassit. 1993, 45, 447.
18. Savoia, D.; Biglino, S.; Cestaro, A.; Zucca, M. Eur. Bull. Drug Res. 1993, 2, 135.
19. Nenortas, E.; Kulikowicz, T.; Burri, C.; Shapiro, T. A. Antimicrob. Agents
Chemother. 2003, 47, 3015.
ity against T. cruzi, which was comparable to the positive control,
benznidazole. On the other hand, compounds 21–25 also showed
some antibacterial activity, and inhibitory activity of 22 against
two Gram-positive bacteria was even higher than the reference
lomefloxacin. However, the inhibitory activities of these com-
pounds against T. cruzi and bacteria were unrelated. Except com-
pounds 21–25, most compounds in this library showed great
selectivity on T. cruzi to test bacteria. Hence, these compounds
are promising candidates for further efficacy evaluation against T.
cruzi. Besides that, this work should also provide significant oppor-
tunity for the discovery of novel quinolones as antitrypanosomal
agents.
20. Keiser, J.; Burri, C. Trop. Med. Int. Health 2001, 6, 369.
21. Nenortas, E.; Burri, C.; Shapiro, T. A. Antimicrob. Agents Chemother. 1999, 43,
2066.
Acknowledgments
22. Zhang, Z.; Yu, A.; Zhou, W. Bioorg. Med. Chem. 2007, 15, 7274.
23. 7,8-Bis-benzyloxy-1-ethyl-6-fluoro-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid
We acknowledge the financial support from the United Nations
Development Program/World Health Organization/Special Pro-
gram for Research and Training in Tropical Diseases. We thank
the Research and Development Center of Xinchang Pharmaceutical
Ltd for the generosity in providing the intermediates for this study.
(10): Yield 270 mg (31.6%) of
a
white solid. Mp: 161–163 °C. 1H NMR
(400 MHz, CDCl3): d 1.29 (3H, t, J = 6.8 Hz, CH3), 4.50 (2H, q, J = 6.8 Hz, CH2),
5.22 (2H, s, CH2), 5.37 (2H, s, CH2), 7.38 (10H, m, Ph), 8.08 (1H, d, JHF = 10.0 Hz,
5-H), 8.56 (1H, s, 2-H), 14.70 (1H, br s, COOH). IR cmꢀ1: 3040 (
(tC@N), 1050 (t
t
ArCAH), 2223
C–F); EI-MS m/z: 447 (M+), 403 (M+–CO2); Anal. Calcd for
C26H22FNO5: C, 69.79; H, 4.96; N, 3.13. Found: C, 69.55; H, 4.97; N, 3.14.
24. 7,8-Bis-benzyloxy-6-fluoro-4-oxo-1-vinyl-1,4-dihydro-quinoline-3-carboxylic acid
a
white solid. Mp: 144–146 °C. 1H NMR
Supplementary data
(11): Yield 287 mg (33.7%) of
(400 MHz, CDCl3): d 4.98 (2H, s, CH2), 5.19 (1H, dd, J = 1.6 Hz, J = 7.6 Hz,
@CH2), 5.33 (2H, s, CH2), 5.47 (1H, dd, J = 1.6 Hz, J = 15.2 Hz, @CH2), 7.36 (10H,
m, Ph), 7.53 (1H, dd, J = 7.6 Hz, J = 15.2 Hz, ACH@), 8.04 (1H, d, JHF = 11.2 Hz, 5-
H), 8.68 (1H, s, 2-H), 14.49 (1H, br s, COOH). Ana; Calcd for C26H20FNO5: C,
70.11; H, 4.53; N, 3.14. Found: C, 69.86; H, 4.43; N, 3.03.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.11.078.
25. Rat skeletal myoblasts (L-6 cells) were seeded in 96-well microtiter plates at
References and notes
2000 cells per well per 100
-glutamine. After 24 h, 5000 trypomastigotes of T. cruzi (Tulahuen strain
C2C4) containing the b-galactosidase (Lac Z) gene were added in 100 L per
lL in RMPI 1640 medium with 10% FBS and 2 mM
L
1. World Health Organization. WHO Technical Report Series 905, Geneva, 2002.
2. Zerba, E. N. WHO Position Paper, 1999.
3. El-Sayed, N. M.; Myler, P. J.; Bartholomeu, D. C.; Nilsson, D.; Aggarwal, G., et al
Science 2005, 309, 409.
4. Schofield, C. J.; Jannin, J.; Salvatella, R. Trends Parasitol. 2006, 22, 583.
5. Urbina, J. A. Curr. Pharm. Des. 2002, 8, 287.
6. Gootz, T. D.; Brighty, K. E. In The Quinolones; Andriole, V. T., Ed., Second Ed.;
Academic Press: San Diego, 1998; p 29.
7. Lesher, G. Y.; Froelich, E. J.; Gruet, M. D.; Bailey, J. H.; Brundage, R. P. J. Med.
Chem. 1962, 5, 1063.
8. De Souza, M. V. N. Mini-Rev. Med. Chem. 2005, 5, 1009.
9. Champoux, J. J. Ann. Rev. Biochem. 2001, 70, 369.
l
well with 2ꢁ of a serial drug dilution. The plates incubated for 4 days. For IC50
value determination, the substrate CPRG/Nonidet was added to the wells and
the color reaction which developed during the following 2–4 h was read
photometrically at 540 nm. From the sigmoidal inhibition curves IC50 values
were calculated. The experiment was carried out in triplicate.
26. MICs were determined as described by the NCCLS (see National Committee for
Clinical Laboratory Standards. Performance Standards for Antimicrobial
Susceptibility Testing: 11th Informational Supplement, National Committee for
Clinical Laboratory Standards: Wayne, PA, 2001; Vol. 21, M100-S11.
27. Statistical analysis was performed with the Spearman test (two tails) using
SPSS version 16.0.