New fluorine-containing hydrazones active against MDR-tuberculosis

Article abstract of DOI:10.1016/j.ejmech.2011.07.052

Several new fluorine-containing hydrazones were synthesized and screened for their in vitro antimycobacterial activity. Nine of these derivatives have shown a remarkable activity against MDR-TB strain with MIC 0.5 μg/mL and high value of selectivity index (SI). Compound 3h with the highest SI (1268.58) was used for stability evaluation with putative metabolites (ciprofloxacin and formylciprofloxacin) detection. Compound 3h was stable at pH 7.4 of aqueous buffer and rat plasma, in acidic buffers (at pH 3 and 5) slow decomposition was observed. Interestingly, no formylciprofloxacin was detected in the solution, and only slightly increased concentration of ciprofloxacin was observed instead. Trifluoromethyl hydrazones 3f and 3g exhibited the best activity also against two strains of Mycobacterium kansasii (MIC 1-4 μmol/L). All evaluated compounds were found to be non-cytotoxic.

Full text of DOI:10.1016/j.ejmech.2011.07.052

European Journal of Medicinal Chemistry 46 (2011) 4937e4945
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
New fluorine-containing hydrazones active against MDR-tuberculosis
a
b
b
c
c
d
ꢀ
ꢀ
}
ꢀ
ꢀ
Eva Vavríková , Slovenko Polanc , Marijan Kocevar , Kata Horváti , Szilvia Bosze , Jirina Stolaríková ,
a
a,
*
ꢀ
ꢀ
Katerina Vávrová , Jarmila Vinsová
a Charles University, Faculty of Pharmacy, Department of Inorganic and Organic Chemistry, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic
b
ꢀ
ꢀ
University of Ljubljana, Faculty of Chemistry and Chemical Technology, Askerceva 5, SI-1000 Ljubljana, Slovenia
c Eötvös Loránd University, Research Group of Peptide Chemistry, Hungarian Academy of Science, Pázmány Péter Sétány 1/A, Budapest, H-1117, Hungary
d Institute of Public Health, Centre of Hygienic Laboratories, Partyzánské nám. 7, 702 00 Ostrava, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
Several new fluorine-containing hydrazones were synthesized and screened for their in vitro anti-
mycobacterial activity. Nine of these derivatives have shown a remarkable activity against MDR-TB strain
with MIC 0.5 mg/mL and high value of selectivity index (SI). Compound 3h with the highest SI (1268.58)
was used for stability evaluation with putative metabolites (ciprofloxacin and formylciprofloxacin)
detection. Compound 3h was stable at pH 7.4 of aqueous buffer and rat plasma, in acidic buffers (at pH 3
and 5) slow decomposition was observed. Interestingly, no formylciprofloxacin was detected in the
solution, and only slightly increased concentration of ciprofloxacin was observed instead. Tri-
fluoromethyl hydrazones 3f and 3g exhibited the best activity also against two strains of Mycobacterium
Received 28 April 2011
Received in revised form
27 July 2011
Accepted 29 July 2011
Available online 5 August 2011
Keywords:
Tuberculosis
Multidrug-resistant tuberculosis
Hydrazone
kansasii (MIC 1e4
m
mol/L). All evaluated compounds were found to be non-cytotoxic.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Antitubercular drug
In vitro activity
1. Introduction
for antimicrobial activity [6]. Substituted carbohydrazone moiety
has been found as a good pharmacophore group for many antitu-
berculosis active molecules [7e10]. In our previous study we
described the possibilities of synergic effect of two components
[11]. Namely, C1 fragment that originated from hydrazones was
found useful for the formation of CeN bond with the appropriate
amines as nucleophiles [12]. It is assumed that the methine bridge
as a linker of both parts is gradually hydrolyzed to release active
molecule or molecules from their “depot” form. The combined
molecule thus serves as a prodrug which can increase bioavail-
ability, passing through the membrane, targeting to the active site
and protecting against multidrug-resistance. Involvement of fluo-
roquinolones as second line antituberculotics to the potential active
molecule seems to be promising. Antimycobacterial activity of
ciprofloxacin (CPX) or norfloxacin (NFX) connected with another
active partner was proven as advantageous [4]. Implementation of
the fluorine atom to the molecule was also beneficial as it improves
the antimycobacterial activity as well as the lipophilicity [6]. The
benzoic acid analogues of isoniazid was successfully applied to
obtain the properly substituted 4,5-dihydro-1H-pyrazol-3-yl]-2-
methylphenol derivatives showing activity against both suscep-
Increasing number of multidrug-resistant tuberculosis (MDR-
TB), extensively drug-resistant (XDR-TB) and most recently emer-
gence of totally resistant (TDR) tuberculosis (TB) is alarming.
Tuberculosis is still one of the major causes of bacterial infections
and death in the world as 9.4 million incident cases of TB was
estimated and 1.7 million of deaths from TB were determined in
2009 [1]. In 2006, WHO developed the Global plan named “The
Stop TB Strategy”. Its goal is to reduce dramatically the global
burden of TB till 2015 by ensuring all patients. The strategy also
supports the development of new and effective tools to prevent,
detect and treat TB. Other object is to achieve universal access to
high-quality diagnosis and patient-centred treatment or to protect
poor and vulnerable populations from TB, TB/HIV and MDR-TB. The
main target is an elimination of TB as a public health problem till
2050 [2].
Modifications of first or second line antituberculotic drugs are
widely applied [3]. A connection of two active parts is one of the
possible approaches in the drug design giving a prodrug form [4,5].
It is well known that the hydrazone group plays an important role
tible and INH-resistant strains with a MIC value of 0.62 mg/mL [13].
Thus, we have designed and synthesised other new kind of
“double” active molecule having fluorinated hydrazide of benzoic
* Corresponding author. Tel.: þ420 495067343; fax: þ420 495067166.
ꢀ
E-mail address: vinsova@faf.cuni.cz (J. Vinsová).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.07.052

ꢀ
E. Vavríková et al. / European Journal of Medicinal Chemistry 46 (2011) 4937e4945
4938
acid as an isoniazid isostere, connected by methine linker with the
first or second line antituberculotic drugs. We expected possible
enhanced activity and slow release in the cell. All compounds were
tested on mycobacterial inhibition against one tuberculosis strain
Mycobacterium tuberculosis, three non-tuberculous mycobacterial
strains and one MDR-TB strain.
2.3. In vitro cytotoxicity
Cytotoxicity of the most active compounds was determined on
human hepatocellular carcinoma cells HepG2, PBMC (Peripheral
Blood Mononuclear Cells) and human SH-SY5Y neuroblastoma cells
by MTT assay for cellular toxicity. IC50 values in mmol/L are pre-
sented in Table 3. Values of selectivity index (SI) indicate rate
between IC50 of HepG2 cytotoxicity and MIC M. tuberculosis are
presented in Table 1. If the values of SI ꢀ 10, compounds are
considered for further screening [14]. IC50 of tested compounds are
within the range of 0.0373e1.28 mmol/L. Selectivity index calcu-
lated for MDR-TB for compounds 3de3i exhibit high values.
2. Results and discussion
2.1. Chemistry
The synthesis of new potential antitubercular drugs involves
two steps. The appropriate hydrazide of substituted benzoic acid
(1) reacted with diethoxymethyl acetate in acetonitrile to give
ethyl benzoylhydrazonoformate (2). The later was treated with
N-nucleophile to afford (3) (Scheme 1). p-Aminosalicylic acid (PAS,
A-H), ciprofloxain (CPX, B-H) and norfloxacin (NFX, C-H) were used
as N-nucleophiles.
Isoniazid (INH) is not a suitable N-nucleophile for similar reac-
tions as mentioned above. An attempt to substitute the ethoxy
group in 2 with INH resulted in a partial transfer of the entire
ethoxymethylene group from 2 to INH thus giving a mixture of two
hydrazonoformates and two hydrazides.
Slightly different approach was applied for the synthesis of
pyrazinecarboxamide (PZA, D-H) derivatives (Scheme 2). The
starting carboxamide was first transformed with N,N-dime-
thylformamide dimethyl acetal to N-(dimethylaminomethylene)
pyrazine-2-carboxamide [3], which reacted with a molecule of the
appropriate hydrazide (1) to give N-(benzoylhydrazonomethyl)
pyrazine-2-carboxamide (4).
2.4. Stability of 3h in aqueous buffers and rat plasma
To get more information about the stability of compounds 3 we
studied the ciprofloxacin-containing derivative 3h as follows. First,
an HPLC method for a simultaneous determination of 3h and its
putative metabolites, ciprofloxacin and formylciprofloxacin was
developed. Due to a high lipophilicity of 3h and its low solubility
in aqueous environments, fluorescence detection was selected.
The excitation/emission wavelengths were set according to those
published for ciprofloxacin [15]. Although it was possible to detect
the second decomposition product, i.e. 4-fluorobenzoic hydrazide
by simultaneous UV detection as well, the detector response was
too low to detect it at the concentrations needed for dissolving the
parent 3h in the aqueous buffers. Nevertheless, a simple isocratic
method using C18 reversed phase silica was able to separate 3h
and its putative metabolites, ciprofloxacin and formylcipro-
floxacin. To decrease the analysis time, we have used a monolithic
column and a flow rate of 2.5 mL/min yielding retention times of
1.1, 3.8 and 4.8 min for ciprofloxacin, 3h and formylciprofloxacin,
respectively.
2.2. Antimycobacterial evaluation
The stability of 3h at different pH values is given in Fig. 1. At pH
7.4, the compound was stable; no significant decomposition was
observed during the 48 h experiment. In both acidic buffers (pH 3
and 5), the concentration of 3h decreased, accompanied by
a proportional increase in the concentration of ciprofloxacin. The
half life values (with their 95% confidence intervals) were 2.9
(2.8e3.1) h and 11.8 (9.6e15.2) h at pH 3 and 5, respectively, and the
plateau was reached at 0.5 (ꢁ1.1e2.0) % and 20.5 (14.0e27.1) %
concentration. Interestingly, no formylciprofloxacin was detected
in the samples.
In vitro antimycobacterial activity was evaluated against M.
tuberculosis H37Rv (ATCC 27294), Mycobacterium kansasii CNTC My
235/80, Mycobacterium kansasii 6509/96 and Mycobacterium avium
CNTC 330/88. Minimal inhibitory concentration (MIC) is the lowest
concentration of a substance at which the inhibition of the growth
of Mycobacterium occurs. Compounds 3ae3l were tested also
against MDR Mycobacterium tuberculosis A8 241 culture which is
resistant at least to isoniazid and rifampicin (Table 1). MIC of
compounds 3f and 3g (containing trifluoromethyl group and CPX)
was found as the lowest against both strains of Mycobacterium
kansasii in comparison to the other compounds and the standard
INH (Table 2).
To be able to detect 3h, ciprofloxacin and formylciprofloxacin
in plasma, gradient analysis had to be employed to separate the
most polar analyte ciprofloxacin from the endogenous plasma
R
O
O
R
R
H-NR1R2
+
+
O
O
N
NR1R2
N
O
NH2
O
N
H
O
N
O
N
H
H
3
2
1
R = F, CF3
R1R2N- =
O
N
O
N
COOH
OH
F
COOH
F
COOH
N
N
-N
-N
NH-
A-
B-
C-
Scheme 1. Synthesis of PAS-, CPX- and NFX-derivatives.

ꢀ
E. Vavríková et al. / European Journal of Medicinal Chemistry 46 (2011) 4937e4945
4939
O
R
N
N
N
R
N
N
H
N
+
N
N
O
N
H
NH2
O
N
H
O
4
1
N
N
-HN
O
D-
Scheme 2. PZA-derivatives preparation.
components. For this initial experiment, we used acetonitrile
precipitation for the preparation of the sample, because we found
satisfactory recovery values (more than 95%) for both 3h and
ciprofloxacin. For ciprofloxacin, this value is in accordance with
a previous study [16]. All three analytes were well separated from
the endogenous plasma components with the retention times of
ciprofloxacin, 3h and formylciprofloxacin being 4.2, 6.2 and
6.7 min, respectively. Blank plasma samples were analysed and no
interference peaks were found at the retention times of the ana-
lytes of interest. We did not find any interest of developing solid
phase extraction to get cleaner plasma samples or to find a suit-
able internal standard, because after the initial experiments, we
did not see any statistically significant decomposition of 3h.
After 48 h incubation, 87 ꢂ 3% of the parent compound was
still present. One experiment (in triplicate) was prolonged to 96 h.
At this time, 82 ꢂ 3% of 3h was found, together with a propor-
tional increase in ciprofloxacin concentration; again formylcipro-
floxacin was not detected. This slight decomposition in
concentration of 3h was not statistically different from that
observed in the buffer at pH 7.4. Thus, no enzymatic decomposi-
tion occurs in rat plasma.
4. Experimental
4.1. Synthesis
Chemicals were obtained from SigmaeAldrich Co. Melting
points were determined on a Kofler micro-hot-stage and are
uncorrected. Elemental analyses (C, H, N) were performed with
a PerkineElmer 2400 CHNS/O analyzer. Infrared spectra were
recorded on a Bio-Rad FTS 3000 MX spectrometer in KBr pellets.
NMR spectra were measured in CF3CO2D or DMSO-d6 solutions on
a Bruker Avance 300 (300 MHz for 1H and 75.5 MHz for 13C). The
chemical shifts (d), are given in ppm, related to tetramethylsilane
(TMS) as an internal standard. The coupling constants (J) are
reported in Hz. The reactions were monitored and the purity of the
products was checked by TLC (Fluka silica gel/TLC cards 60 PF254).
Mass spectra were recorded with a VG-Analytical AutospecQ
instrument.
4.1.1. General procedure for the synthesis of ethyl
benzoylhydrazonoformate 2ae2e (for the atom numbering scheme
for 1H NMR spectra assignment, see Fig. 2)
Diethoxymethyl acetate (243 mg, 1.5 mmol) was added at room
temperature to the mixture of a substituted benzoic acid hydrazide
1 (1 mmol) in acetonitrile (10 mL). Reaction mixture was then
3. Conclusion
In this study, new fluorine-containing hydrazones with anti-
mycobacterial properties were prepared and investigated.
Compounds 3ae3i have shown higher activity against MDR-TB than
INH, CPX and NFX and their MIC values are lower on MDR-TB than on
M. tuberculosis. These results might suggest that compounds 3ae3i
can be effective against MDR-TB infection. Prepared compounds are
non-toxic in MIC concentrations for human hepatocytes, PBMC cells
and human SH-SY5Y neuroblastoma cells. The highest selectivity
index for MDR-TB was found to be 1268.58 for 1-cyclopropyl-6-
fluoro-4-oxo-7-{4-[{[4-(trifluormethyl)benzoyl]hydrazono}methyl]
piperazin-1-yl}-1,4-dihydroquinoline-3-carboxylic acid (3h). The
toxicity values show that the compound 3h does not exhibit
a significant cytotoxic effect on tested cells within the examined
range of concentrations. An evaluation of its stability towards
hydrolysis has shown that the compound is stable at neutral pH, thus
improving the bioavailability to the target site. Compounds 3f and 3g
(having trifluoromethylbenzoyl group and CPX in the molecule) were
found as the most active against M. kansasii 235/80 and M. kansasii
isolated from patient in comparison to the other compounds as well
as the standard INH. It is evident that the compounds, containing CPX
and either 4-fluorobenzoyl or 3-fluorobenzoyl moiety (3h, 3i),
showed thebest selectivityindexes which were tentimes higher than
SI of CPX in the case of MDR-TB A8 241.
Table 1
Antimycobacterial evaluation on M. tuberculosis cultures.
R
R1R2N MIC [
m
g/mL]
SI for MTB SI for MDR-TB
H37Rv
A8 241
MTB
H37Rv
ATCC 27294
MDR-TB
A8 241
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
4-CF3
3-CF3
4-F
3-F
2-F
4-CF3
3-CF3
4-F
3-F
4-CF3
4-F
A
A
A
A
A
B
4
4
4
2
2
1
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
5
NT
5
NT
NT
1
NT
NT
NT
NT
NT
NT
148.25
192.06
189.21
87.79
634.29
402.38
3.98
NT
592.64
767.77
378.41
351.15
1268.58
804.76
3.98
NT
B
2
B
1
B
1
C
5
C
C
NT
6
3-F
4-CF3
3-F
31.67
NT
38
NT
4a
D
D
e
e
e
e
NT
NT
0.01
0.2
0.5
5
4b
INH
PAS
CPX
NFX
NT
NT
e
NT
NT
e
0.25
1.5
10
NT
NT
e
333.33
31.85
110.37
15.87
e
NT e not tested.

ꢀ
E. Vavríková et al. / European Journal of Medicinal Chemistry 46 (2011) 4937e4945
4940
Table 2
Antimycobacterial evaluation on non-tuberculous mycobacterial cultures.
R
R1R2N
MIC [mmol/L]
M. avium
330/88
14 d
M. kansasii
235/80
7 d
M. kansasii
6509/96
7 d
21 d
14 d
125
125
62.5
62.5
32
2
2
4
4
4
32
32
250
250
>250
1000
2
16
>1000
21 d
250
125
62.5
14 d
21 d
125
62.5
32
32
62.5
2
2
16
4
8
62.5
32
500
>500
4
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
4-CF3
3-CF3
4-F
3-F
2-F
4-CF3
3-CF3
4-F
3-F
4-CF3
4-F
3-F
4-CF3
3-F
e
A
A
A
A
A
B
B
B
B
C
32
250
32
32
32
125
500
125
62.5
62.5
62.5
250
125
250
500
250
250
>1000
>500
>250
125
62,5
250
62.5
32
32
32
32
2
2
4
4
2
16
16
250
250
>250
125
1
62.5
62.5
32
32
32
1
1
8
4
2
32
16
250
250
2
32
1
2
62,5
62.5
32
32
62.5
1
1
8
4
4
62.5
16
500
>500
4
125
1
8
62.5
62.5
4
4
8
32
250
62.5
125
500
125
125
>1000
>500
>250
32
8
8
C
C
62.5
62.5
250
>500
>250
>1000
2
4a
D
D
e
e
e
e
e
4b
INH
PAS
CPX
NFX
PZA
e
500
2
8
e
62.5
125
500
e
8
500
16
>1000
e
>1000
125
1000
1000
NT e not tested.
stirred for 10 min at room temperature and evaporated to dryness.
A crude product was recrystallized from the appropriate solvent.
H2, H6), 7.92 (m,1H, H4), 7.74 (m,1H, H5), 4.17 (q, J ¼ 7.1, 2H, CH2), 1.29 (t,
J ¼ 7.1 Hz, 3H, CH3). MS (EI): 261 (m/z (M þ H)þ). HRMS Calcd for
(C11H11F3N2O2
þ
H): 261.0851. Found: 261.0838. Anal. Calcd for
4.1.1.1. Ethyl [4-(trifluoromethyl)benzoyl]hydrazonoformate (2a). Yield
93%; mp 148e150 ꢃC (EtOH). IR (KBr): 3231, 1628, 1332, 1268, 1234,
C11H11F3N2O2 (260.21): C, 50.77; H, 4.26; N, 10.77. Found: C, 49.90; H,
3.90; N, 12.20.
1171, 1130, 1067, 976, 772 cmꢁ1 1H (DMSO-d6, 300 MHz):
d 11.24 (s,
1H, NH), 8.37 (s, 1H, CH]N), 8.02 (d, J ¼ 8.1 Hz, 2H, H2, H6), 7.85 (m,
2H, H3, H5), 4.18 (q, J ¼ 7.1 Hz, 2H, CH2), 1.29 (t, J ¼ 7.1 Hz, 3H, CH3).
13C NMR (DMSO-d6, 75 MHz): 162.3, 157.2, 138.6, 129.5, 129.0, 126.2,
124.8, 63.5, 15.0. MS (EI): 261 (m/z (M þ H)þ). Anal. Calcd for
C11H11F3N2O2 (260.21): C, 50.77; H, 4.26; N, 10.77. Found: C, 50.90; H,
4.01; N, 10.81.
4.1.1.3. Ethyl 4-(fluorobenzoyl)hydrazonoformate (2c). Yield 88%;
mp 138e139 ꢃC (ethyl acetate/diethyl ether). IR (KBr): 3215, 3061,
2986, 1658, 1617, 1563, 1506, 1363, 1319, 1250, 1229, 1159, 1115,
1065, 1013, 848 cm-1. 1H NMR (DMSO-d6, 300 MHz):
d 11.05 (s, H,
NH), 8.37 (s, 1H, CH]N), 7.90 (m, 2H, H2, H6), 7.31 (m, 2H, H3, H5),
4.15 (q, J ¼ 7.0 Hz, 2H, CH2), 1.29 (t, J ¼ 7.0 Hz, 3H, CH3). 13C NMR
(DMSO-d6, 75 MHz): 163.8, 161.6, 155.9, 130.3, 129.8, 115.3, 67.2,
15.4. MS (EI): 211 (m/z (M þ H)þ). Anal. Calcd for C10H11FN2O2
(210.20): C, 57.14; H, 5.27; N, 13.33. Found: C, 56.88; H, 5.11; N,
13.53.
4.1.1.2. Ethyl [3-(trifluoromethyl)benzoyl]hydrazonoformate (2b). Yield
91%; mp 127e130 ꢃC (ethyl acetate/hexane). IR (KBr): 3204, 3075, 1661,
1614, 1566, 1372, 1338, 1314,1267, 1165, 1109, 1073, 814 cmꢁ1 1H NMR
(DMSO-d6, 300 MHz): d 11.25 (s,1H, NH), 8.37 (s,1H, CH]N), 8.13 (m, 2H,
4.1.1.4. Ethyl 3-(fluorobenzoyl)hydrazonoformate (2d). Yield 95%;
mp 114e116 ꢃC (acetonitrile). IR (KBr): 3203, 3074, 1655, 1616, 1559,
1479, 1442, 1364, 1312, 1248, 1221, 1127, 1069, 1015, 973, 849,
Table 3
820 cmꢁ1 1H NMR (DMSO-d6, 300 MHz):
d 11.10 (s, 1H, NH), 8.36 (s,
In vitro cytotoxicity on human cell cultures.
R
R1R2N
HepG2 IC50
[mmol/L]
PBMC IC50
SY5Y IC50
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
4a
4b
INH
CPX
NFX
4-CF3
3-CF3
4-F
3-F
2-F
4-CF3
3-CF3
4-F
3-F
4-CF3
4-F
A
A
A
A
A
B
B
B
B
C
C
C
D
D
e
e
e
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
0.107
NT
> 0.430
NT
NT
NT
>0,5
>0,5
0.934
1.210
0.347
>0.322
>1.280
>0.812
>0.0373
>0.797
>0.393
>0.624
>0.130
>1
0.934
1.210
0.347
>0.322
>0.763
>0.305
0.262
>0.331
>0.393
NT
3-F
4-CF3
3-F
>0.305
w4.67
>0,5
e
e
>0,5
>0,5
e
>0,5
Fig. 1. Stability of 3h in aqueous buffers at pH 7.4, 5 and 3. Data are presented as
means ꢂ S.D, n ¼ 3. Where no error bars are visible, they are smaller than the symbol.
NT e not tested.

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E. Vavríková et al. / European Journal of Medicinal Chemistry 46 (2011) 4937e4945
4941
1H, CH]N), 7.60 (m, 3H, H2, H4, H6), 7.39 (m, 1H, H5), 4.15 (q,
J ¼ 7.0 Hz, 2H, CH2), 1.29 (t, J ¼ 7.0 Hz, 3H, CH3). MS (EI): (m/z
(M þ H)þ). HRMS Calcd for (C10H11FN2O2 þ H): 211.0883. Found:
211.0885. Anal. Calcd for C10H11FN2O2 (210.20): C, 57.14; H, 5.27; N,
13.33. Found: C, 56.71; H, 5.20; N, 13.85.
(367.28): C, 52.32; H, 3.29; N, 11.44. Found: C, 52.04; H, 3.25; N,
11.27.
4.1.2.2. 2-Hydroxy-4-[(3-trifluoromethylbenzoyl)hydrazonomethyla-
mino]benzoic acid (3b). Yield 63%; mp 201e203 ꢃC (acetonitrile). IR
(KBr): 3415, 3216, 3069, 1638, 1583, 1555, 1336, 1279, 1237, 1161,
4.1.1.5. Ethyl 2-(fluorobenzoyl)hydrazonoformate (2e). Yield 93%;
mp 101e105 ꢃC (ethyl acetate/hexane). IR (KBr): 3220, 3079, 1655,
1612, 1562, 1476, 1365, 1318, 1251, 1217, 1100, 1065, 909, 753 cmꢁ1
1128, 1072, 972, 773 cmꢁ1 1H NMR (DMSO-d6, 300 MHz):
d 11.90
(broad,1H, COOH),11.24 (s,1H, NHN ¼ ), 9.91 (s,1H, OH), 8.62 (s,1H,
CH]N), 8.17 (m, 2H, H20, H60), 7.92 (s, 1H, NH), 7.76 (m, 2H, H40,
H50), 7.68 (d, J ¼ 7.8 Hz,1H, H6), 6.84 (d, J ¼ 2.0 Hz,1H, H3), 6.66 (dd,
J ¼ 7.8, 2.0 Hz, 1H, H5). 13C NMR (DMSO-d6, 75 MHz): 172.7, 163.7,
161.3, 148.2, 146.6, 135.8, 132.3, 130.7, 128.5, 126.7, 124.5, 123.1,
119.5,108.2, 106.3,102.8. MS (EI) 368 (m/z (M þ H)þ). Anal. Calcd for
C16H12F3N3O4 (367.28): C, 52.32; H, 3.29; N, 11.44. Found: C, 52.40;
H, 3.18; N, 11.27.
1H NMR (DMSO-d6, 300 MHz):
d 10.98 (s, 1H, NH), 8.28 (s, 1H, CH]
N), 7.55 (m, 2H, H4, H6), 7.29 (m, 2H, H3, H5), 4.14 (q, J ¼ 7.1 Hz, 2H,
CH2), 1.28 (t, J ¼ 7.1 Hz, 3H, CH3). MS (EI) (m/z (M þ H)þ). HRMS
Calcd for (C10H11FN2O2 þ H): 211.0883. Found: 211.0873. Anal. Calcd
for C10H11FN2O2 (210.20): C, 57.14; H, 5.27; N, 13.33. Found: C,
56.93; H, 4.91; N, 13.98.
4.1.2. General procedure for the synthesis of derivatives of 4-amino-
2-hydroxybenzoic acid 3a-3e (for the atom numbering scheme for
1H NMR spectra assignment, see Fig. 2)
4-Amino-2-hydroxybenzoic acid (1 mmol) was dissolved in
acetonitrile (15 mL) at room temperature. Then, a solution of the
corresponding ethyl benzoylhydrazonoformate (1 mmol) in
acetonitrile (4 mL) was added. The reaction mixture was stirred at
room temperature for 24 h. The solid material was filtered off, dried
in the air, suspended in acetonitrile (10 mL), refluxed for 5 min, and
the product was filtered off from a hot suspension.
4.1.2.3. 4-[(4-Fluorobenzoyl)hydrazonomethylamino]-2-
hydroxybenzoic acid (3c). Yield 60%; mp 194.9e195.5 ꢃC (acetoni-
trile). IR (KBr): 3231, 3072, 1638, 1551, 1507, 1308, 1268, 1234, 1161,
971, 776 cmꢁ1 1H NMR (DMSO-d6, 300 MHz):
d 11.70 (broad, 1H,
COOH), 11.04 (s, 1H, NHN ¼ ), 9.86 (s, 1H, OH), 8.59 (s, 1H, CH]N),
7.93 (dd, J ¼ 8.6, 5.6 Hz, 2H, H20, H60), 7.71 (s, 1H, NH), 7.67 (d,
J ¼ 8.6 Hz, 1H, H6), 7.34 (dd, J ¼ 17.4, 8.6 Hz, 2H, H30, H50), 6.83 (d,
J ¼ 1.7 Hz,1H, H3), 6.65 (dd, J ¼ 8.6,1.7 Hz,1H, H5). 13C NMR (DMSO-
d6, 75 MHz): 167.3, 162.2, 160.9, 147.4, 131.5, 130.5, 129.7, 115.5,
115.2, 107.3, 105.2, 101.7, 100.6. MS (EI) 318 (m/z (M þ H)þ). Anal.
Calcd for C15H12FN3O4 (317.27): C, 56.78; H, 3.81; N, 13.24. Found: C,
56.59; H, 3.89; N, 13.04.
4.1.2.1. 2-Hydroxy-4-[(4-trifluoromethylbenzoyl)hydrazonomethyla-
mino]benzoic acid (3a). Yield 45%; mp 250e252 ꢃC (acetonitrile). IR
(KBr): 3214, 1657, 1616, 1562, 1328, 1250, 1171, 1121, 1072, 1021, 907,
4.1.2.4. 4-[(3-Fluorobenzoyl)hydrazonomethylamino]-2-
854 cmꢁ1 1H NMR (DMSO-d6, 300 MHz):
d
11.80 (broad, COOH),
hydroxybenzoic acid (3d). Yield 71%; mp 178.3e179.2 ꢃC (acetoni-
trile). IR (KBr): 3228, 1642, 1583, 1549, 1439, 1270, 1161, 972, 838,
11.23 (s, 1H, NHN ¼ ), 9.94 (s, 1H, OH), 8.60 (s, 1H, CH]N), 8.06 (d,
J ¼ 8.1 Hz, 2H, H20, H60), 7.88 (m, 2H, H30, H50), 7.8 (broad, NH), 7.69
(d, J ¼ 8.0 Hz, 1H, H6), 6.86 (d, J ¼ 1.9 Hz, 1H, H3), 6.66 (dd, J ¼ 8.0,
1.9 Hz, 1H, H5). 13C NMR (DMSO-d6, 75 MHz): 172.7, 163.7, 161.6,
148.2, 146.6, 138.7, 132.4, 128.9, 126.3, 124.8, 123.0, 108.3, 106.2,
102.8. MS (EI) 368 (m/z (M þ H)þ). Anal. Calcd for C16H12F3N3O4
774 cmꢁ1 1H NMR (DMSO-d6, 300 MHz):
d 11.80 (broad, 1H, COOH),
11.09 (s, 1H, NHN]), 9.89 (s, 1H, OH), 8.59 (s, 1H, CH]N), 7.63 (m,
3H, H6, H20, H60), 7.56 (m, 1H, H50), 7.40 (m, 1H, H40), 6.83 (d,
J ¼ 2.0 Hz, 1H, H3), 6.76 (s, 1H, NH), 6.65 (dd, J ¼ 8.7, 2.0 Hz, 1H, H5).
13C NMR (DMSO-d6, 75 MHz): 171.8, 163.6, 162.9, 160.6, 147.4, 145.5,
4'
4
5'
6'
3'
5
3R1,R2
R1,R2
6
2'
2
H
3
N
N
OH
N
O
O
N
H
O
N
H
5
COOH
6
O
N
O
N
5
4'
5
4'
F
COOH
5'
F
COOH
5'
6'
3'
3'
R1,R2
R1,R2
2'
2
6'
2'
2
N
N
8
8
N
N
N
N
O
N
H
O
N
H
4'
O
N
5
5'
6'
3'
F
COOH
R1,R2
2'
N
N
3
5
6
2
H
N
N
8
N
O
N
H
N
OHC
O
Fig. 2. Atom numbering scheme for 1H NMR spectra assignment.

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E. Vavríková et al. / European Journal of Medicinal Chemistry 46 (2011) 4937e4945
4942
136.5, 131.5, 130.5, 123.3, 118.1, 114.1, 107.3, 105.4, 101.9. MS (EI) 318
(m/z (M þ H)þ). Anal. Calcd for C15H12FN3O4 (317.27): C, 56.78; H,
3.81; N, 13.24. Found: C, 56.48; H, 3.54; N, 12.96.
1725, 1675, 1627, 1505, 1468, 1335, 1260, 1237, 1020, 943, 848 cmꢁ1
1H NMR (DMSO-d6, 300 MHz):
10.76 (s, 1H, NH), 8.69 (s, 1H, CH]
d
N), 8.05 (s, 1H, H2), 7.96 (d, J ¼ 13.2, 1H, H5), 7.88 (m, 2H, H20, H60),
7.63 (d, J ¼ 7.6, 1H, H8), 7.30 (dd, J ¼ 7.8, 7.8 Hz, 2H, H30, H50), 3.85
(m, 1H, CH - cyclopropyl), 3.53 (m, 4H, 2 ꢄ CH2 - piperazinyl), 3.40
(m, 4H, 2 ꢄ CH2 - piperazinyl), 1.33 (m, 2H, CH2 - cyclopropyl), 1.21
(m, 2H, CH2 - cyclopropyl), COOH not observed (exchanged). 13C
NMR (CF3COOD, 75 MHz): 172.5, 171.8, 167.7, 160.4, 159.0, 155.6,
151.3, 150.3, 143.5, 132.6, 126.5, 119.2, 117.2, 114.1, 107.2, 105.3, 54.2,
50.7, 40.6, 9.7. MS (EI) 496 (m/z (M þ H)þ). Anal. Calcd for
C25H23F2N5O4 (495.48): C, 60.60; H, 4.68; N, 14.13. Found: C, 60.30;
H, 4.49; N, 13.97.
4.1.2.5. 4-[(2-Fluorobenzoyl)hydrazonomethylamino]-2-
hydroxybenzoic acid (3e). Yield 54%; mp 195e196 ꢃC (acetonitrile).
IR (KBr): 3408, 3223, 3067, 2479, 1641, 1610, 1555 cmꢁ1 1H NMR
(DMSO-d6, 300 MHz):
d 11.70 (broad, 1H, COOH), 10.94 (s, 1H,
NHN ¼ ), 9.88 (s, 1H, OH), 8.51 (s, 1H, CH]N), 7,67 (m, 3H, H6, H40,
H60), 7.55 (m, 1H, H30), 7.32 (m, 2H, H50, NH), 6.87 (d, J ¼ 2.0 Hz, 1H,
H3), 6.66 (dd, 1H, J ¼ 8.7, 2.0 Hz, H5). 13C NMR (DMSO-d6, 75 MHz):
172.7, 163.7, 159.93, 159.91, 148.2, 145.8, 133.2, 132.3, 131.0, 125.4,
124.5, 117.0, 108.3, 106.2, 102.8. MS (EI) 318 (m/z (M þ H)þ). Anal.
Calcd for C15H12FN3O4 (317.27): C, 56.78; H, 3.81; N,13.24. Found: C,
56.47; H, 3.52; N, 12.82.
4.1.3.4. 1-Cyclopropyl-6-fluoro-4-oxo-7-{4-[3-(fluorobenzoylhy-
drazono)methyl]piperazin-1-yl}-1,4-dihydroquinoline-3-carboxylic
acid (3i). Yield 44%; mp 235e237 ꢃC (acetonitrile). IR (KBr): 3297,
3050, 2823, 1722, 1677, 1629 cmꢁ1 1H NMR (DMSO-d6, 300 MHz):
4.1.3. General procedure for the synthesis of ciprofloxacin
derivatives 3fe3i (for the atom numbering scheme for 1H NMR
spectra assignment, see Fig. 2)
d
15.1 (broad, 1H, COOH), 10.81 (s, 1H, NH), 8.66 (s, 1H, CH]N), 8.06
(s, 1H, H2), 7.95 (d, 1H, J ¼ 13.2 Hz, H5), 7.55 (m, 4H, H8, H20, H40,
H60), 7.36 (ddd, J ¼ 8.5, 8.2, 1.9 Hz, 1H, H50), 3.85 (m, 1H, CH -
cyclopropyl), 3.54 (m, 4H, 2 ꢄ CH2 - piperazinyl), 3.39 (m, 4H,
2 ꢄ CH2 - piperazinyl), 1.34 (m, 2H, CH2 - cyclopropyl), 1.21 (m, 2H,
CH2 - cyclopropyl). 13C NMR (DMSO-d6, 75 MHz): 177.2,166.8,164.5,
161.5, 161.2, 154.7, 148.9, 145.9, 140.0, 137.7, 137.7, 133.3, 131.3, 124.0,
114.8, 114.5, 112.1, 107.7, 50.0, 45.8, 36.8, 8.5. MS (EI) 496 (m/z
(M þ H)þ). Anal. Calcd for C25H23F2N5O4 (495.48): C, 60.60; H, 4.68;
N, 14.13. Found: C, 60.39; H, 4.67; N, 14.05.
Ciprofloxacin (331 mg, 1 mmol) was dissolved in acetonitrile
(15 mL) and glacial acetic acid (2.4 mL) at 50 ꢃC. Then, ethyl ben-
zoylhydrazonoformate 2 (1 mmol) was added and the reaction
mixture was stirred at 50 ꢃC for 4 h. After this time the temperature
was decreased to the room temperature and the reaction mixture
was stirred for 30 min. The crystals were filtered off and washed
with diethyl ether (20 mL). A solid material was refluxed in 5 mL of
acetonitrile for 5 min and filtered off.
4.1.3.1. 1-Cyclopropyl-6-fluoro-4-oxo-7-{4-[4-(trifluormethylbenzoyl)]
hydrazonomethyl]piperazin-1-yl}-1,4-dihydroquinoline-3-carboxylic
acid (3f). Yield 19%; mp 236e238 ꢃC (acetonitrile). IR (KBr): 3513,
3469, 1709, 1663, 1622, 1495, 1463, 1320, 1261, 1238, 1133, 1062,
4.1.4. General procedure for the synthesis of norfloxacin derivatives
3je3l (for the atom numbering scheme for 1H NMR spectra
assignment, see Fig. 2)
Norfloxacin (319 mg, 1 mmol) was dissolved in acetonitrile
(12 mL) and glacial acetic acid (2.5 mL) at 50 ꢃC, followed by the
addition of ethyl benzoylhydrazonoformate (1 mmol). The reaction
mixture was stirred for 5 h at 50 ꢃC and after this time, the
temperature was slowly decreased to the room temperature. After
stirring at room temperature for 40 min, the solid was filtered off
and washed with diethyl ether (25 mL). Crystals were dried in the
air, suspended in acetonitrile (10 mL) and refluxed for 10 min. The
product was filtered off from a hot suspension.
1014 cmꢁ1 1H NMR (DMSO-d6, 300 MHz):
d 14.80 (broad,1H, COOH),
10.95 (s, 1H, NH), 8.68 (s, 1H, CH]N), 8.07 (s, 1H, H2), 8.01 (d,
J ¼ 8.1 Hz, 2H, H20, H60), 7.95 (d, J ¼ 13.2 Hz, 1H, H5), 7.85 (d,
J ¼ 8.1 Hz, 2H, H30, H50), 7.63 (d, J ¼ 7.5 Hz, 1H, H8), 3.85 (m, 1H, CH -
cyclopropyl), 3.55 (m, 4H, 2 ꢄ CH2 - piperazinyl), 3.40 (m, 4H,
2 ꢄ CH2 - piperazinyl), 1.34 (m, 2H, CH2 - cyclopropyl), 1.20 (m, 2H,
CH2 - cyclopropyl). 13C NMR (DMSO-d6, 75 MHz): 176.3, 165.9, 160.7,
154.6, 153.9, 151.3, 147.9, 145.0, 139.1, 138.3, 130.9, 127.9, 126.1, 122.2,
118.9, 111.1, 106.8, 49.1, 44.9, 35.9, 7.6. MS (EI) 545 (m/z (M)þ). Anal.
Calcd for C26H23F4N5O4 (545.49): C, 57.25; H, 4.25; N,12.84. Found: C,
55.29; H, 4.44; N, 12.30. C26H23F4N5O4þH2O calculated: C, 55.42; H,
4.47; N, 12.43.
4.1.4.1. 1-Ethyl-6-fluoro-4-oxo-7-{4-[4-(trifluoromethylbenzoyl)
hydrazonomethyl]piperazin-1-yl}-1,4-dihydroquinoline-3-carboxylic
acid (3j). Yield 56%; mp 259e260 ꢃC (acetonitrile). IR (KBr): 3194,
1726, 1621, 1482, 1328, 1265, 1239, 1110, 1068, 1019, 860 cmꢁ1 1H
4.1.3.2. 1-Cyclopropyl-6-fluoro-4-oxo-7-{4-[3-(tri-
NMR (DMSO-d6, 300 MHz):): d 15.27 (s, 1H, COOH), 10.96 (s, 1H,
fluoromethylbenzoyl)hydrazonomethyl]piperazin-1-yl}-1,4-
dihydroquinoline-3-carboxylic acid (3g). Yield 51%; mp 238e239 ꢃC
(acetonitrile). IR (KBr): 3268, 3063, 1728, 1676, 1628, 1467, 1333,
1260, 1237, 1120, 1020, 937, 812, 747, 701 cmꢁ1 1H NMR (DMSO-d6,
NH), 8.96 (s, 1H, CH]N), 8.08 (s, 1H, H2), 7.96 (m, 3H, H5, H30, H50),
7.85 (d, J ¼ 7.8 Hz, 2H, H20, H60), 7.25 (d, J ¼ 7.2 Hz, 1H, H8), 4.61 (q,
J ¼ 7.0 Hz, 2H, CH2CH3), 3.53 (m, 4H, 2 ꢄ CH2 - piperazinyl), 3.40 (m,
4H, 2 ꢄ CH2 - piperazinyl), 1.44 (t, J ¼ 7.0 Hz, 3H, CH2CH3). 13C NMR
(DMSO-d6, 75 MHz): 176.1, 166.1, 160.7, 154.5, 154.0, 151.3, 148.5,
145.4, 138.3, 137.1, 130.5, 127.9, 126.1, 119.5, 111.4, 107.1, 106.4, 49.2,
49.1, 45.0, 14.4. MS (EI) 533 (m/z (M)þ). Anal. Calcd for
C25H23F4N5O4þH2O: C, 54.45; H, 4.57; N, 12.70. Found: C, 54.46; H,
4.64; N, 12.63.
300 MHz): d 15.00 (broad, 1H, COOH), 10.98 (s, 1H, NH), 8.68 (s, 1H,
CH]N), 8.1 (m, 3H, H2, H20, H60), 7.94 (d, J ¼ 13.2 Hz, 1H, H5), 7.89
(m, 1H, H40), 7.72 (dd, J ¼ 7.8, 7.8 Hz, 1H, H50), 7.63 (d, J ¼ 7.5 Hz, 1H,
H8), 3.85 (m, 1H, CH - cyclopropyl), 3.54 (m, 4H, 2 ꢄ CH2 - piper-
azinyl), 3.41 (m, 4H, 2 ꢄ CH2 - piperazinyl), 1.33 (m, 2H, CH2
cyclopropyl), 1.21 (m, 2H, CH2 cyclopropyl). 13C NMR (CF3COOD,
75 MHz): 168.3, 167.7, 160.4, 155.7, 154.4, 151.0, 146.6, 138.9, 130.1,
128.2, 127.5, 126.9, 122.4, 119.0, 114.6, 110.9, 109.3, 102.6, 100.8, 49.7,
43.7, 36.0, 5.1. MS (EI) 546 (m/z (M þ H)þ). Anal. Calcd for
C26H23F4N5O4 (545.49): C, 57.25; H, 4.25; N, 12.84. Found: C, 57.12;
H, 4.33; N, 13.13.
4.1.4.2. 1-Ethyl-6-fluoro-4-oxo-7-{4-[4-(fluorobenzoyl)hydrazono-
methyl]piperazin-1-yl}-1,4-dihydroquinoline-3-carboxylic acid (3k).
Yield 44%; mp 270e272 ꢃC (ethyl acetate/acetonitrile). IR (KBr):
3260, 3048, 1727, 1669, 1625, 1474, 1260, 1237, 1157, 1112, 1020,
852 cmꢁ1 1H NMR (DMSO-d6, 300 MHz):
d 15.35 (s, 1H, COOH),
10.75 (s, 1H, NH), 8.97 (s, 1H, CH]N), 8.04 (s, 1H, H2), 7.97 (d,
J ¼ 13.1, 1H, H5), 7.88 (m, 2H, H20, H60), 7.28 (m, 3H, H8, H30, H50),
4.62 (q, J ¼ 7.0 Hz, 2H, CH2CH3), 3.51 (m, 4H, 2 ꢄ CH2 - piperazinyl),
3.39 (m, 4H, 2 ꢄ CH2 - piperazinyl), 1.44 (t, J ¼ 7.0 Hz, 3H, CH2CH3).
4.1.3.3. 1-Cyclopropyl-6-fluoro-4-oxo-7-{4-[4-(fluorobenzoyl)hydra-
zonomethyl]piperazin-1-yl}-1,4-dihydroquinoline-3-carboxylic acid
(3h). Yield 52%; mp 256e260 ꢃC (acetonitrile). IR (KBr): 3279, 3047,

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4943
13C NMR (CF3COOD, 75 MHz): 167.6, 164.8, 163.1, 160.4, 155.8, 154.3,
150.8, 145.7, 136.8, 128.1, 121.9, 114.2, 113.3, 109.4, 101.9, 101.1, 50.4,
49.7, 43.6, 10.5. MS (EI) 484 (m/z (M þ H)þ). Anal. Calcd for
C24H23F2N5O4 (483.47): C, 59.62; H, 4.80; N, 14.49. Found: C, 59.37;
H, 4.67; N, 14.33.
C13H10FN5O2 (287.25): C, 54.36; H, 3.51; N, 24.38. Found: C, 53.89;
H, 3.79; N, 23.98.
4.1.7. 1-Cyclopropyl-6-fluoro-7-(4-formylpiperazin-1-yl)-4-oxo-
1,4-dihydroquinoline-3-carboxylic acid
The compound was prepared following the procedure described
for the synthesis of similar ciprofloxacin derivative [17]. A mixture
of acetic anhydride (224 mg, 2.2 mmol) and formic acid (78 mg,
1.7 mmol) was heated at 50 ꢃC for 20 min, then ciprofloxacin
(351 mg, 1.1 mmol) was added and the mixture was heated at 80 ꢃC
for 2.5 h. Upon cooling, distilled water (2 mL) was slowly dropped
to the reaction mixture, and crystals were filtered off. For the atom
numbering scheme for 1H NMR spectrum assignment, see Fig. 2.
Yield 77%; mp 281e283 ꢃC (methanol). IR (ATR): 2866, 1726,
1666, 1624, 1547, 1439, 1264, 1235, 1009, 937, 803 cmꢁ1 1H NMR
4.1.4.3. 1-Ethyl-6-fluoro-4-oxo-7-{4-[3-(fluorobenzoyl)hydrazono-
methyl]piperazin-1-yl}-1,4-dihydroquinoline-3-carboxylic
acid
(3l). Yield 49%; mp 251e253 ꢃC (acetonitrile). IR (KBr): 3267, 3055,
2875, 2817, 1724, 1670, 1628 cmꢁ1 1H NMR (DMSO-d6, 300 MHz):
d
15.29 (s, 1H, COOH), 10.81 (s, 1H, NH), 8.97 (s, 1H, CH]N), 8.05 (s,
1H, H2), 7.96 (d, J ¼ 13.2 Hz, 1H, H5), 7.57 (m, 3H, H20, H30, H60), 7.36
(ddd, J ¼ 8.3, 8.1, 2.09 Hz, 1H, H50), 7.25 (d, J ¼ 7.3 Hz, 1H, H8), 4.61
(q, J ¼ 7.1 Hz, 2H, CH2CH3), 3.53 (m, 4H, 2 ꢄ CH2 - piperazinyl), 3.38
(m, 4H, 2 ꢄ CH2 - piperazinyl), 1.44 (t, J ¼ 7.1 Hz, 3H, CH2CH3). 13
C
NMR (CF3COOD, 75 MHz): 168.4, 168.3, 163.2, 159.9, 156.5, 153.3,
146.8, 146.5, 137.5, 129.5, 128.7, 121.5, 120.2, 114.0, 113.0, 110.6, 102.7,
101.9, 51.2, 50.5, 44.4, 11.3. MS (EI) 484 (m/z (M þ H)þ). Anal. Calcd
for C24H23F2N5O4 (483.47): C, 59.62; H, 4.80; N, 14.49. Found: C,
59.33; H, 4.78; N, 14.44.
(DMSO-d6, 300 MHz): d 8.92 (s, 1H, CHO), 8.23 (s, 1H, H2), 7.96 (d,
J ¼ 12.3 Hz, 1H, H5), 7.56 (d, J ¼ 7.6 Hz, 1H, H8), 3.81 (m, 1H, CH -
cyclopropyl), 3.62 (m, 4H, 2 ꢄ CH2 - piperazinyl), 3.46 (m, 4H,
2 ꢄ CH2 - piperazinyl), 1.38 (m, 2H, CH2 - cyclopropyl), 1.16 (m, 2H,
CH2 - cyclopropyl), COOH not observed (exchanged). 13C NMR
(DMSO-d6, 75 MHz): 176.6, 166.1, 161.2, 148.3, 145.2, 139.3, 119.3,
112.0, 107.3, 107.0, 50.4, 49.3, 44.6, 36.1, 7.8. Anal. Calcd for
C18H18FN3O4 (359.35): C, 60.16; H, 5.05; N, 11.69. Found: C, 60.03; H,
5.17; N, 11.74.
4.1.5. N-{2-[4-(Trifluoromethyl)benzoyl]hydrazonomethyl}
pyrazine-2-carboxamide (4a)
N-[(Dimethylamino)methylene]pyrazine-2-carboxamide
(178 mg, 1 mmol) was dissolved in acetonitrile (5 mL) at room
temperature followed by the addition of a solution of 4-(tri-
fluoromethyl)benzohydrazide 1 (204 mg, 1 mmol) in acetonitrile
(5 mL). The reaction mixture was stirred for 4.5 h at room
temperature. The crystals were filtered off and dried in the air,
suspended in methanol (10 mL) at room temperature, filtered off
and dried in the air. For the atom numbering scheme for 1H NMR
spectrum assignment, see Fig. 2.
4.2. In vitro functional assays
4.2.1. In vitro antimycobacterial evaluation on M. avium 330/88,
M. kansasii 235/80 and M. kansasii 6509/96 cultures
In vitro antimycobacterial activity was evaluated against Myco-
bacterium kansasii CNTC My 235/80, M. kansasii 6509/96 and
Mycobacterium avium CNTC 330/88. All strains were obtained from
the Czech National Collection of Type Cultures (CNCTC) with
exception of M. kansasii 6509/96, which is a clinical isolate. Anti-
mycobacterial activity was measured in Sula semisynthetic
medium (SEVAC, Prague) at 37 ꢃC. Compounds were dissolved in
dimethyl sulfoxide solution (max 5% DMSO in water) and added to
the medium within a concentration range 250, 125, 62, 31, 16, 8, 4,2
Yield 17%; mp 242e244 ꢃC (methanol). IR (KBr): 3267, 1679,
1627, 1556, 1501, 1329, 1163, 1109, 1021, 921 cmꢁ1 1H NMR (DMSO-
d6, 300 MHz): 11.71 (s, 1H, NHN ¼ ), 11.35 (d, J ¼ 6.0 Hz, 1H,
CHꢁNHCO), 9.26 (d, J ¼ 1.3 Hz, 1H, H3), 9.08 (d, J ¼ 6.0 Hz, 1H,
CHꢁNHCO), 8.94 (d, J ¼ 2.4 Hz, 1H, H5), 8.82 (dd, J ¼ 2.4, 1.3 Hz, 1H,
H6), 8.11 (d, J ¼ 7.5 Hz, 2H, H20, H60), 7.91 (d, J ¼ 7.5 Hz, 2H, H30, H50).
13C NMR (CF3COOD, 75 MHz): 165.9, 162.9, 159.5, 145.8, 144.4,138.7,
135.4, 128.3, 125.8, 123.8, 122.5, 116.6. MS (EI) 337 (m/z (M)þ). Anal.
Calcd for C14H10F3N5O2 (337.26): C, 49.86; H, 2.99; N, 20.77. Found:
C, 49.72; H, 3.09; N, 20.61.
and 1 mmol/L. Minimal inhibitory concentration (MIC) was deter-
mined after incubation at 37 ꢃC for 7, 14 and 21 days. The MIC is the
lowest concentration of a substance at which the inhibition of the
growth of mycobacterium occurs.
4.1.6. N-{[2-(3-Fluorobenzoyl)hydrazinyl]methylene}pyrazine-2-
carboxamide (4b)
4.2.2. In vitro antimycobacterial evaluation on M. tuberculosis
H37Rv and MDR-TB A8 241
N-[(Dimethylamino)methylene]pyrazine-2-carboxamide
(267 mg, 1.5 mmol) was dissolved in acetonitrile (10 mL) at room
temperature. The solution was warmed up to 50 ꢃC. The catalyst
(pyridinium p-toluenesulphonate polymer-bound, 427 mg,
1.5 mmol) was added to the solution, followed by the addition of 3-
fluorobenzohydrazide (231 mg, 1.5 mmol). The reaction mixture
was stirred for 4 h at 50 ꢃC, then the temperature was decreased to
room temperature and stirring was continued for 16 h at the same
temperature. Then, the solid was filtered off and washed with
methanol (4 ꢄ 10 mL). The filtrate was evaporated to dryness to
afford 4b. For the atom numbering scheme for 1H NMR spectrum
assignment, see Fig. 2.
In vitro antimycobacterial activity of the compounds was
determined on M. tuberculosis H37Rv and MDR-TB A8 (resistant to at
least INH and RIF) in Sula semisynthetic medium, which was
prepared in-house [18e20] at pH 6.5 by serial dilution. The test
compounds were added to the medium as DMSO solutions (max 2%
DMSO). Minimal inhibitory concentration (MIC) was determined
after incubation at 37 ꢃC for 28 days. MIC was the lowest concen-
tration of a compound at which the visible inhibition of the growth
of the bacteria occurred. The activities of the tested compounds
were confirmed using a colony forming unit (CFU) determination
by subculturing from the Sula medium onto drug-free Löwenstein-
Jensen solid medium. The samples were incubated for further 28
days [19,21,22]. The experiments were repeated at least two times
with similar results.
Yield 16%; mp 200e202 ꢃC (methanol). IR (KBr): 3293, 3245,
3082, 2360, 1683, 1625, 1552 cmꢁ1 1H NMR (DMSO-d6, 300 MHz):
d
11.57 (s, 1H, NHN]), 11.32 (d, J ¼ 9.7 Hz, 1H, CHꢁNHCO), 9.26 (d,
J ¼ 1.4 Hz, 1H, H3), 9.06 (d, J ¼ 9.7 Hz, 1H, CHꢁNHCO), 8.93 (d,
J ¼ 2.5 Hz, 1H, H6), 8.81 (dd, J ¼ 2.5, 1.4 Hz, 1H, H5), 7.72 (m, 2H, H20,
H60), 7.58 (m, 1H, H50), 7.43 (m, 1H, H40). 13C NMR (DMSO-d6,
75 MHz): 163.4, 162.3, 161.6, 149.1, 148.6, 144.5, 144.1, 141.4, 136.1,
131.0, 124.0, 118.8, 114.6. MS (EI) 288 (m/z (M þ H)þ). Anal. Calcd for
4.2.3. In vitro cytotoxicity of compounds by MTT assay
HepG2 human hepatoma cells (ATCC HB-8065) and freshly
prepared human PBMC (peripheral blood mononuclear cells) [23]
were cultured in RPMI-1640 medium without phenol red supple-
mented with 10% fetal calf serum (FCS), 2 mM L-glutamine and

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E. Vavríková et al. / European Journal of Medicinal Chemistry 46 (2011) 4937e4945
4944
160
m
g/mL gentamycin [24,25]. SH-SY5Y human neuroblastoma cell
For the determination of the stability of 3h in aqueous buffers,
a simple isocratic method using 20% acetonitrile in 50 mM phos-
phate buffer pH 2.0 as a mobile phase at a flow rate 2.5 mL/min and
a monolitic Chromolith Performance column (RP-18e 100e4.6 mm,
Merck, Darmstadt, Germany) with the same precolumn (RP-18e
10e4.6 mm) maintained at 30 ꢃC was employed to separate 3h
from its putative metabolites ciprofloxacin and formylciprofloxacin.
The compounds were detected by both UV at 280 nm (scanned
from 230 to 350 nm) and fluorescence (excitation/emission set at
276/442 nm) [15]. Fluorescence was used for quantification. The
retention times of ciprofloxacin, 3h and formylciprofloxacin were
1.1, 3.8 and 4.8 min, respectively. The length of the analysis was
5.5 min.
line was grown in DMEM medium without phenol red containing
10% FCS, 2 mM -glutamine, 160 g/mL gentamycin and 1%
L
m
nonessential amino acids (NEAA) [26,27]. Cell cultures were
maintained at 37 ꢃC, 5% CO2 in water-saturated atmosphere.
Cells were plated into 96-well plate with initial cell number of
5 ꢄ 103 per well (in the case of SH-SY5Y 7.5 ꢄ 103, PBMC 2.0 ꢄ 105
cells/well). After 24 h incubation at 37 ꢃC prior to the experiment,
cells were treated with compounds in 100 mL serum free medium
overnight. Control cells were treated with serum free medium. Four
parallel measurements were performed in all cases.
After overnight incubation at 37 ꢃC, the cell viability was deter-
mined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT)-assay [28,29]. Then 45
m
L MTT-solution (2 mg/mL)
For the determination of the stability of 3h in rat plasma,
a gradient method was employed. Solvent A was 50 mM phosphate
buffer pH 2.0 and solvent B was acetonitrile. The mobile phase
contained 11% B for 3 min, 25% B for 4 min and 11% B for 3 min with
total analysis time of 10 min. The column and precolumn and the
detection conditions were the same as above.
The calibration curves were constructed by spiking the buffers
or plasma with known amounts of the analytes at the concentration
levels from 0.1 to 5 nmol/mL. Both methods were validated and
were within the acceptance criteria for accuracy, precision and
linearity recommended by the FDA Guidance for Industry for Bio-
analytical Method Validation (2001).
was added to each well. The respiratory chain and other electron
transport systems [30] reduce MTT and thereby form non-water-
soluble violet formazan crystals within the cell [31]. The amount
of these crystals can be determined spectrophotometrically and
serves to estimate the number of mitochondria and hence the
number of living cells in the well [32]. After 4 h of incubation, cells
were centrifuged for 5 min (2000 rpm) and supernatant was
removed. The obtained formazan crystals were dissolved in 50 or
100
at
mL DMSO and optical density (OD) of the samples was measured
¼ 540 and 620 nm using ELISA Reader (iEMS Reader, Labsystems,
l
Finland). OD620 values were subtracted from OD540 values. The
percent of cytotoxicity was calculated using the following equation:
Acknowledgements
Cytotoxicityð%Þ ¼ ½1 ꢁ ðODtreated=ODcontrolÞꢅx100;
This work was financially supported by the Research project
MSM 0021620822, IGA NS 10367-3, by grants from the Hungarian
National Science Fund (OTKA 68358) and National Office for
Research and Technology (NKFP_07_1-TB_INTER-HU). The financial
support from the Slovenian Reasearch Agency (projects P1-0230-
103 and BI-CZ/10-11-005) is also acknowledged. We would like to
where ODtreated and ODcontrol correspond to the optical densities
of the treated and the control cells, respectively. In each case two
independent experiments were carried out with 4e8 parallel
measurements. The 50% inhibitory concentration (IC50) values were
determined from the doseeresponse curves. The curves were
defined using MicrocalTM Origin1 (version 7.5) software.
ꢀ
ꢀ
thank Dr. Bogdan Kralj and Dr. Dusan Zigon (Mass Spectrometry
ꢀ
Center, Jozef Stefan Institute, Ljubljana, Slovenia) for recording the
mass spectra. This work was also partially supported with infra-
structure of the EN-FIST Centre of Excellence, Ljubljana, Slovenia.
Dr. Zsolt Datki (Department of Medical Chemistry, University of
Szeged, Szeged, Hungary) is acknowledged for a kind donation of
SH-SY5Y human neuroblastoma cell line.
4.3. Stability of 3h in aqueous buffers and rat plasma
The hydrolytic stability of 3h was first evaluated in aqueous
buffers at pH 7.4, 5.0, and 3.0 as follows: 20
mL of stock solution of
3h in dimethyl sulfoxide (0.15 mol/mL) was added to 980
m
mL of
100 mM buffers (phosphate for pH 3 and 7 and acetate buffer for pH
5) yielding 3 nmol/mL solutions. Samples of the individual solu-
tions were analyzed for the content of 3h, ciprofloxacin and for-
mylciprofloxacin at predetermined time intervals up to 48 h. The
experiment was performed in triplicate.
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