New series of isoniazid hydrazones linked with electron-withdrawing substituents

Article abstract of DOI:10.1016/j.ejmech.2011.09.054

A series of new isoniazid hydrazones was synthesized by two procedures. In the first isoniazid was activated with diethoxymethyl acetate and condensed with the appropriate anilines. Alternatively, substituted anilines were activated by diethoxymethyl acet

Full text of DOI:10.1016/j.ejmech.2011.09.054

European Journal of Medicinal Chemistry 46 (2011) 5902e5909
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journal homepage: http://www.elsevier.com/locate/ejmech
Original article
New series of isoniazid hydrazones linked with electron-withdrawing
substituents
a
b
b
b
c
c
ꢀ
ꢀ
ꢀ
}
Eva Vavríková , Slovenko Polanc , Marijan Kocevar , Janez Kosmrlj , Kata Horváti , Szilvia Bosze ,
d
e
a,
*
ꢀ
ꢀ
ꢀ
ꢀ
Jirina Stolaríková , Ales Imramovský , Jarmila Vinsová
a Charles University, Faculty of Pharmacy, Department of Inorganic and Organic Chemistry, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic
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
e University of Pardubice, Faculty of Chemical Technology, Studentská 573, 532 10 Pardubice, Czech Republic
b
ꢀ
ꢀ
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new isoniazid hydrazones was synthesized by two procedures. In the first isoniazid was
activated with diethoxymethyl acetate and condensed with the appropriate anilines. Alternatively,
substituted anilines were activated by diethoxymethyl acetate and subsequently condensed with
isoniazid. NMR study confirmed that both synthetic approaches gave the same tautomer. All compounds
were screened for in vitro antimycobacterial activity. Most of them exhibited the same activity against
Received 12 July 2011
Received in revised form
26 September 2011
Accepted 29 September 2011
Available online 13 October 2011
Mycobacterium tuberculosis (MIC 1
kansasii 325/80 (MIC 0.125e0.250
m
mol Lꢀ1) as isoniazid (INH), better activity against Mycobacterium
m
mol Lꢀ1), high value of selectivity index (SI) and IC50 between 0.0218
Keywords:
Tuberculosis
Isoniazid
and 0.326 mmol Lꢀ1. Compound 2o with the best SI was used as a model compound for the stability test
and was found to be stable at neutral pH, but under acidic conditions it slowly hydrolysed.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Hydrazone
Antitubercular drug
In vitro activity
1. Introduction
these derivatives against M. tuberculosis has not yet exceeded that
of INH [4]. To overcome the resistance, combination of INH mole-
Tuberculosis (TB) remains a major cause of mortality throughout
the world. Resistance of Mycobacterium tuberculosis to antituber-
culosis drugs becomes very serious problem [1]. Besides MDR-TB, in
2006 more dangerous form of M. tuberculosis resistant against first
line antituberculosis drugs, quinolones, and one of the second line
anti-TB injectable drugs, called XDR-TB, was observed. The most
dangerous bacteria, resistant against both the first and the second
line TB drugs were presented in 2009 and are called totally resistant
TDR-TB [2]. These facts clearly indicate the need for the develop-
ment of new antituberculosis agents [3].
cule with other active molecules is frequently applied [5]. We are
interested in isonicotinoyl hydrazones as an effective pharmaco-
phore. Hydrazones have been demonstrated to posses many
interesting biological activities including antimicrobial, anticon-
vulsant, antitubercular, antitumor and others [6]. Several hydra-
zones of 2-pyrazinecarbohydrazide were recently designed as
analogues of pyrazinamide, another first line drug for TB treatment,
to reduce the toxicity [7e10]. In our previous study we described
the possible synergistic effect of two components [11], thus we
expected the same for the newly synthesized series. The linkage
between isonicotinoyl hydrazone and an appropriate partner by
methine bridge can be gradually hydrolyzed to release active
molecules [12]. The combined molecules could serve as prodrugs
with an increase of bioavalibility and membrane permeability,
targeting to the active site and protecting against multidrug-
resistance. An introduction of electron-withdrawing substituents
into the molecule of antitubercular active compounds generally
increases activity as well as lipophilicity, which is important for the
transport through the mycobacterial cell [4].
Isonicotinic acid hydrazide (isoniazid, INH) belongs to the group
of the first line antituberculosis drugs being in clinical practice over
50 years. Chemical modifications of isonicotinic acid hydrazide
were performed on all parts of the molecule, but the activity of
* Corresponding author. Tel.: þ420 495067343; fax: þ420 495067166.
ꢀ
E-mail addresses: evavavrikova@seznam.cz (E. Vavríková), slovenko.polanc@
ꢀ
fkkt.uni-lj.si (S. Polanc), marijan.kocevar@fkkt.uni-lj.si (M. Kocevar), janez.
ꢀ
kosmrlj@fkkt.uni-lj.si (J. Kosmrlj), khorvati@gmail.com (K. Horváti), szilvia.bosze@
This work was aimed at enhancing the antimycobacterial
activity of INH by conjugation with aniline group having electron-
}
ꢀ
gmail.com (S. Bosze), jirina.stolarikova@zu.cz (J. Stolaríková), ales.imramovsky@
ꢀ
upce.cz (A. Imramovský), 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.2011.09.054

ꢀ
E. Vavríková et al. / European Journal of Medicinal Chemistry 46 (2011) 5902e5909
5903
withdrawing substituents. Potential increases in biological effect
of substituents were calculated from SAR studies [4]. INH was
linked with monosubstituted or disubstituted anilines through
a CH fragment into N-phenyl-N0-(pyridin-4-ylcarbonyl)hydrazo-
noformamide derivatives. All compounds were tested on myco-
bacterial inhibition against one tuberculous strain M. tuberculosis
and three non-tuberculous mycobacterial strainseMycobacterium
avium and two strains of Mycobacterium kansasii; one of them was
isolated from the patient.
2. Results and discussion
2.1. Chemistry
Two different approaches were used for the preparation of N-
phenyl-N0-(pyridin-4-ylcarbonyl)hydrazonoformamide (2). Method
A involved the reaction of isoniazid (INH) with diethoxymethyl
acetate into N0-isonicotinoylethoxymethylene hydrazone (1), which
was subsequently reacted with the appropriate aniline derivative
(Scheme 1).
Fig. 1. Aromatic part of 1H NMR and 13C NMR spectra of crude 2a recorded in DMSO-d6
with the peak assignment of the resonances belonging to E-2a (plain figures) and Z-2a
(italic figures). For atom numbering scheme, see Fig. 2. Asterisk denotes unidentified
minor product (see text).
Some chloro-, bromo- and nitroanilines did not react with the
compound 1 by the Method A. In these cases an alternative
approach was applied that involved the reaction of the aniline
derivatives with diethoxymethyl acetate into 3, which upon
substitution with INH provided the desired compounds 2 (Method
B, Scheme 1).
For the above-mentioned major two species present in the
crude sample of 2a all the expected structural elements could
clearly be identified, i.e., the hydrazonoformamide chain, func-
tionalized with the isonicotinoyl and the 3-hydroxyphenyl group at
N-200 and N-500, respectively. The major differences in proton,
carbon and nitrogen resonances belonging to these two molecules
were observed for the hydrazonoformamide atoms, whereas the
resonances of the remaining atoms appeared at similar values. For
both species, however, the 1He13C HMBC spectrum exhibited cross
peak of NH proton, assigned as H-500, to both C-20 and C-60 carbon
atoms of the 3-hydroxyphenyl group (Fig. 2, Tables 1 and 2). Along
with the other spectral features, collected in Tables 1 and 2, the
above observations confirmed the hydrazonoformamide structures
of E-2a and Z-2a, and ruled out the possibility of the tautomeric
form 2a0 in any of these two molecules. The assignment of E-2a
stereochemistry of the compound was corroborated by NOE cross
The hydrazonoformamide structure of compounds
2 was
confirmed by NMR spectroscopy. In proton NMR spectrum of crude
2a in DMSO-d6 solution two sets of resonances were observed with
the relative integrals of 1:0.3 (Fig. 1) and were assigned to E-2a and
Z-2a (Fig. 2). The assignment was based on the distinct correlations
in 1He1H COSY, 1He13C HSQC, 1He13C HMBC and 1He15N HMBC
spectra. The data are summarized in Tables 1 and 2. The structure of
the minor compound that can be seen from Fig. 1 could not be
elucidated because in 2D NMR spectra the corresponding low-
intensity cross peaks were scrambled in the noise.
Scheme 1. The synthesis of hydrazonoformamides (2).

ꢀ
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E. Vavríková et al. / European Journal of Medicinal Chemistry 46 (2011) 5902e5909
Fig. 2. The structures of isomeric E-2a and Z-2a with selected 1He13C HMBC correlations (plain arrow) and NOEs (dotted arrow), and the structure of tautomeric 2a’.
peak between H-200 and H-400 in the 1He1H NOESY spectrum
(Fig. 2).
Based on the integration, the 1:0.3 ratio of E-2a and Z-2a
remained unchanged if the spectra were recorded in DMSO-d6 at
different temperatures in the range of 290e330 K thus indicating
that these isomers are most probably not in a dynamic equilibrium
but should have been formed during the preparation. Interestingly,
the spectra of the samples prepared either by the Approach A or the
Approach B shown in Scheme 1 are completely identical.
In contrast to 2a, the NMR spectra of other hydrazonoforma-
mides 2 generally showed one major set of resonances, presumably
belonging to E-stereoisomers. This was supported by the distinct
15N NMR chemical shifts of 324.1, 161.1, 252.2 and 106.2 ppm for N-
1, N200, N300 and N500 nitrogen atoms, respectively, in the compound
E-2f, which is in good agreement with that of E-2a (Table 1).
compounds 2 showed the same activity as INH, whereas several
derivatives had even better activity against M. kansasii 235/80. In
general, halogenated derivatives exhibited lower MIC values.
Compound 2o was the most active for all the tested strains and was
used as a model compound for further evaluation. The best MIC
values are emphasized in Table 3.
One may expect an enhancing antibacterial activity of the
conjugated aniline molecule having halogens like Cl, Br, F; nitro
group, trifluoromethyl group, hydroxy and methoxy group. The
contribution of these substituents is evident for many hydrazones
[4]. Here, in general, none of them increased the activity in
comparison with INH against M. tuberculosis. The activity seems to
be based predominantly on the isoniazid core. All derivatives
exhibited the same or slightly lower effectiveness in comparison
with INH. Better results gave hydrazones against M. avium and
M. kansasii. Namely, most of them are more active than INH; the
best representatives derived from disubstituted anilines (2k, 2l,
2o). The most active molecule 2o having two halogens (4-Br, 3-F)
showed MIC 62.5 against M. kansasii after 7 days.
2.2. Antimycobacterial evaluation
In vitro antimycobacterial activity was evaluated against
M. tuberculosis CNTC My 331/88 (ATCC 27294), M. avium CNTC 330/
88, M. kansasii CNTC My 235/80 and M. kansasii 6509/96 (isolated
from patient). Minimal inhibitory concentration (MIC) is the lowest
concentration of the substance at which the inhibition of the
growth of Mycobacterium occurs (Table 3). Most of the synthetized
An incorporation of halogen atoms increases the lipophilicity of
the molecule. Whereas mycobacterial cell wall is very lipophilic, the
contribution of these lipophilic substituents plays an important
role. On the other hand, the hydroxy (2a, 2b) or methoxy (2c, 2d)
Table 2
Table 1
1H, 13C and 15N NMR chemical shifts, 1He1H COSY, 1He13C HSQC, 1He13C HMBC and
1He15N HMBC for Z-2a. For atom numbering scheme, see Fig. 2.
1H, 13C and 15N NMR chemical shifts, 1He1H COSY, 1He13C HSQC, 1He13C HMBC and
1He15N HMBC for E-2a. For atom numbering scheme, see Fig. 2.
C
dC
H
dH (multiplicity,
J/Hza)
COSYb
HSQCb 1He13C HMBCb
C
dC
H
dH (multiplicity,
J/Hza)
COSYb
HSQCb 1He13C HMBCb
2(6) 149.95 2 (6) 8.74 (d, nd c
3(5) 121.58 3 (5) 7.82 (d, 5.1)
4
)
H-3(5)
H-2(6)
H-3(5) H-3(5)
H-2,5 (3,6)
H-2,6
2(6) 150.08 2 (6) 8.74 (d, 5.8)
3(5) 121.07 3 (5) 7.76 (d, 5.5)
4
H-3(5)
H-2(6)
H-2 H-3,6 (2,5)
H-3(5) H-2,5 (3,6)
H-20, 50, 400
141.20
141.20
10
20
30
40
50
60
100
400
141.26
102.81 20
158.27
H-50,400
10
20
30
40
50
60
100
400
141.81
103.00 20
158.04
6.54 (br s)
H-60,40
H-20,50
H-20
H-40,60,500
H-20,40,50
H-20,60
6.68 (br s)
H-40
H-20
H-60,40
H-20,50,40
H-20,60
109.20 40
130.16 50
106.23 60
161.19
6.41 (d, 8.1)
H-40
H-50
H-60
108.33 40
129.81 50
106.62 60
159.84
6.34 (d, 8.0)
H-20,60,50 H-40
7.08 (dd, 8.0, 8.0) H-60,40
7.04 (dd, 8.0, 8.0) H-20,40
H-50
H-60
6.61 (d, 8.0)
H-20,50
H-20,40,500
H-3,5,200
H-200
6.65 (d, 8.1)
8.46 (d, 8.7)
H-40,50
H-500
H-20,40,500
H-3,5,200
H-200
140.68 400
7.53 (d, 11.6)
H-200,500 H-400
147.32 400
H-400
N
1
200
300
500
dN
1He15N HMBCb
H-2,3,5,6
H-200,d 400
H-400
N
1
200
300
500
dN
1He15N HMBCb
H-2,3,5,6
H-200, c400
H-400
323.7
323.7
157.2
236.2
110.8
200
500
10.52 (br s)
H-400
H-400
161.1
249.2
105.7
200
500
11.14 (br s)
9.41 (d, 8.9)
8.97 (d, 11.6)
H-400
c
H-400
H-400,500
a
J values are quoted as directly measured from the spectra.
Unambiguous correlations are listed.
Not determined because of overlap with other resonances.
a
b
c
b
c
J values are quoted as directly measured from the spectra.
Unambiguous correlations are listed.
Observed as a doublet due to incompletely suppressed direct NH correlations.
d
Observed as a doublet due to incompletely suppressed direct NH correlations.

ꢀ
E. Vavríková et al. / European Journal of Medicinal Chemistry 46 (2011) 5902e5909
5905
Table 3
Antimycobacterial evaluation on M. tuberculosis, M. avium and M. kansasii cultures.
R1
R2
MIC [m ]
mol Lꢀ1
M. tuberculosis
M. avium 330/88
M. kansasii 235/80
M. kansasii 6509/96
331/88
14 d
21 d
14 d
21 d
7 d
14 d
21 d
7 d
14 d
21 d
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2r
3-OH
4-OH
3-OCH3
4-OCH3
4-CF3
3-F
4-F
3-Cl
3-Br
4-Cl
5-Cl
3-Cl
3-F
3-Cl
4-Br
3-CF3
4-Cl
4-Br
3-NO2
e
e
2
4
2
2
2
1
1
1
1
2
2
1
2
1
1
1
1
2
2
1
2
8
2
4
2
1
2
1
1
4
2
2
2
2
1
1
1
4
2
1
500
500
500
500
500
500
250
500
250
250
500
250
250
250
250
500
500
500
250
>250
500
500
500
500
1000
1000
500
1000
500
500
500
250
250
500
250
1000
1000
500
250
250
250
250
250
125
250
125
250
250
125
250
250
250
62.5
125
125
125
125
>250
1000
500
500
500
500
1000
1000
500
1000
500
250
500
500
1000
250
250
250
250
250
1000
500
16
32
16
16
32
16
8
16
8
16
8
32
62.5
16
16
32
16
8
32
62.5
16
32
32
16
8
16
16
32
32
16
32
32
16
32
32
32
16
4
e
e
1000
1000
1000
>1000
>1000
500
1000
500
250
e
e
e
e
e
16
8
e
2-OH
2-OH
4-Cl
4-F
4-F
3-F
e
16
16
16
16
16
8
16
32
32
16
4
500
8
8
1000
>1000
250
1000
1000
500
16
8
16
16
32
8
e
2s
2t
INH
e
e
500
>250
500
>250
e
>250
2
substituents seem to be less effective as their contribution to the
lipophilicity is lower.
Two compounds (2k, 2o), the most active against M. kansasii,
were used for the hepatotoxicity evaluation. It is evident that the
contribution of halogenated aromatic rings has a positive influence
on the reduction of INH hepatotoxicity.
over the time (
l
¼ 267 nm) was selected to construct a curve of
decomposition as the time dependence on absorbance and the
observed pseudo-first-order rate constants kobs were calculated
(Table 5). Graphical representation of the results, the observed rate
constants (kobs) as a function of pH, is shown in Fig. 3.
Fig. 4 shows an illustrative example of kobs determination for
compound 2o in 0.01 M hydrochloric acid solution (pH 2) at 37 ꢂC.
This figure shows the time evolution of UVevis spectra recorded in
the range of 200e500 nm. The inset represents time dependence of
2.3. In vitro cytotoxicity
absorbance extracted from the spectra at
l
¼ 267 nm. The observed
Cytotoxicity of the most active compounds 2k and 2o was
determined on human hepatocellular carcinoma cells HepG2 and
PBMC (Peripheral Blood Mononuclear Cells) by MTT assay
for cellular toxicity. IC50 values in mmol Lꢀ1 are presented in
Table 4. Values of selectivity index (SI) indicate rate between IC50 of
HepG2 cytotoxicity and MIC M. tuberculosis. The compounds with
SI ꢁ 10 are considered as promising candidates for further
screening [13]. IC50 of the tested compounds were in the range
pseudo-first-order rate constant kobs of 2.85 ꢃ 10ꢀ3 sꢀ1 was
calculated from this absorbance-time dependence.
3. Experimental
3.1. Synthesis
of 0.0218e0.326 mmol Lꢀ1
. Selectivity index calculated for
All chemicals were obtained from SigmaeAldrich Co. Melting
points were determined on the Büchi Melting Point apparatus B-
540. Elemental analyses (C, H, N) were performed with a Per-
kineElmer 2400 CHNS/O analyzer. Infrared spectra were recorded
M. tuberculosis for compound 2o exhibits high value of 162.
2.4. Stability measurement
The stability of the most active compound, 2o, was quantita-
tively studied by UVevis spectroscopy at 37 ꢂC on ca.
5 ꢃ 10ꢀ5 mol Lꢀ1 solutions. Measurements were carried out in
water solutions of HCl, KOH and in appropriate buffer solutions to
cover the pH range of 2e13 (Table 5). For each pH the kinetic
measurements were carried out where changes in UVevis spectra
were recorded. Wavelength with the highest change of absorbance
Table 5
Stability of compound 2o in presented solutions and its rate constant kobs
.
Buffer or solution
pH
2.00
kobs (sꢀ1
)
HCl 0.01 M
HCl 0.007 M
HCl 0.005 M
HCl 0.001 M
0.0028500
0.0019200
0.0017000
0.0005140
0.0003220
0.0004250
0.0001140
0.0000710
0.0000130
0.0000120
0.0000550
0.0000445
0.0001100
0.0000691
2.16
2.30
3.00
3.30
3.35
4.55
5.81
7.23
7.86
8.92
9.71
11.70
13.00
HCl 0.0005 M
Methoxyacetate buffer
Acetate buffer
Phosphate buffer
Imidazole buffer
N-Methylmorpholine buffer
TRIS buffer
Carbonate buffer
KOH 0.005 M
KOH 0.1 M
Table 4
Cytotoxicity evaluation of compounds 2k and 2o.
R1
R2
HepG2 IC50
PBMC IC50
SI for M. tuberculosis.
331/88
[mmol Lꢀ1
]
2k
2o
INH
5-Cl
3-F
e
2-OH
4-Br
e
0.0218
0.162
>1
0.075
0.326
w4.67
10.9
162
e

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E. Vavríková et al. / European Journal of Medicinal Chemistry 46 (2011) 5902e5909
5906
generated with ACD/ChemSketch 12.01 and structures were drawn
with ChemBioDraw Ultra 11.0 and are formatted as ACS Document
1996.
3.1.1. General procedures for the synthesis of hydrazonoformamides
2
3.1.1.1. Method A. Compound 1: Diethoxymethyl acetate (15 mmol)
was added to a stirred solution of isoniazid (10 mmol) in acetoni-
trile (100 mL) at 55 ꢂC. The reaction mixture was stirred for 30 min
at the same temperature. The solvent was evaporated in vacuo and
the crude product was washed with diethyl ether (2 ꢃ 20 mL). The
product, ethyl isonicotinoylhydrazonoformate 1, was dried in the
air and recrystallized from acetonitrile [11].
Compound 2: A solution of substituted aniline (1 mmol) in
ethanol (1 mL) was added to a stirred solution of ethyl iso-
nicotinoylhydrazonoformate 1 (1 mmol) in ethanol (10 mL) at
55 ꢂC. The mixture was stirred for 6 h at the same temperature and
for 20 h at room temperature. The product 2 was collected by
filtration and recrystallized from the appropriate solvent.
Fig. 3. Dependence of the observed rate constants (kobs, sꢀ1) on pH measured at 37 ꢂ
C
in hydrochloric acid (C), buffers (B) and potassium hydroxide (:) (Table 5). The ionic
strength I ¼ 1 mol Lꢀ1
.
3.1.1.2. Method B. Compound 3: Diethoxymethyl acetate (411 mg,
3 mmol) was added to a stirred solution of substituted aniline
(2 mmol) in acetonitrile (20 mL) at 40 ꢂC. The reaction mixture was
stirred for 30 min at the same temperature. The crude product 3
was filtered off and used without further purification for the
following reactions.
Compound 2: A solution of the intermediate 3 (1 mmol) in
acetonitrile (1 mL) was added to a stirred solution of isoniazid
(137 mg, 1 mmol) in acetonitrile (10 mL) at 55 ꢂC. The mixture was
stirred for 6 h at the same temperature and for 20 h at room
temperature. The precipitate was collected by filtration and
recrystallized from the appropriate solvent.
on a Bio-Rad FTS 3000 MX spectrometer in KBr pellets. NMR
spectra were measured on a Bruker Avance 300 operating at
300 MHz (1H) and 75 MHz (13C), and Bruker Avance III 500 MHz
NMR instrument operating at 500 MHz (1H), 125 MHz (13C) and
50 MHz (15N). The proton and carbon spectra were referenced to
TMS as internal standard. The nitrogen chemical shifts were
extracted from 1He15N HMBC spectra. The reported 15N chemical
shifts were measured with respect to external 90% CH3NO2 in CDCl3
and converted to the
d
15N (liq. NH3) ¼ 0 ppm scale using the
relation:
d
15N (CH3NO2) ¼
d
15N (liq. NH3) þ 380.50 ppm. Chemical
shifts are given on the
d scale (ppm). Coupling constants (J) are
given in Hz. Multiplicities are indicated as follows: s (singlet),
d (doublet), t (triplet), q (quartet), m (multiplet), or br (broadened).
Atom numbering scheme for 1H NMR spectra assignment is shown
in Fig. 2. Phase sensitive NOESY with gradient pulses in mixing
time, of 2a, was recorded in DMSO-d6 at 296 K with mixing time of
300 ms and relaxation delay of 2 s. Names of compounds were
3.1.2. Data of compounds 2
3.1.2.1. N-(3-Hydroxyphenyl)-N0-(pyridin-4-ylcarbonyl)hydrazono-
formamide (2a). Yield 54% (Method A), 57% (Method B); mp
190e192 ꢂC (ethanol). IR (KBr): 3226, 3085, 3030, 2871, 2601, 2488,
1669, 1631, 1599, 1544, 1497, 1445, 1414, 1366, 1306, 1247, 1164,
1062, 1003, 964, 909, 849, 764, 697, 688 cmꢀ1. Anal. Calcd for
C13H12N4O2 (256.26): C, 60.93; H, 4.72; N, 22.86. Found: C, 61.08; H,
5.02; N, 21.98.
3.1.2.2. N-(4-Hydroxyphenyl)-N0-(pyridin-4-ylcarbonyl)hydrazono-
formamide (2b). Yield 54% (Method A), 59% (Method B); mp
190e192 ꢂC (ethanol). IR (KBr): 3212, 3150, 3069, 2610, 1668, 1634,
1601, 1676, 1549, 1519, 1458, 1412, 1366, 1314, 1247, 1170, 1105, 1059,
1005, 1067, 909, 826, 755, 690 cmꢀ1. 1H NMR (DMSO-d6, 300 MHz):
d
11.06 (s, 1H, NH), 9.12 (s, 1H, NH), 8.71 (d, J ¼ 4.9 Hz, 2H, H2, H6),
8.26 (s, 1H, CH), 7.79 (d, J ¼ 5.1 Hz, 2H, H3, H5), 7.45 (s, 1H, OH), 7.19
(d, J ¼ 7.7 Hz, 2H, H20,H60), 6.70 (d, J ¼ 8.1 Hz, 2H, H30, H50). 13C NMR
(DMSO-d6, 75 MHz):
d 160.2, 152.3, 150.3 (2C), 148.4, 141.7, 132.9,
121.4 (2C), 118.4 (2C), 115.8 (2C). Anal. Calcd for C13H12N4O2
(256.26): C, 60.93; H, 4.72; N, 21.86. Found: C, 60.81; H, 5.15; N,
21.41.
3.1.2.3. N-(3-Methoxyphenyl)-N0-(pyridin-4-ylcarbonyl)hydrazono-
formamide (2c). Yield 55% (Method A); mp 164e166 ꢂC (ethanol).
IR (KBr): 3041, 2360, 1671, 1645, 1634, 1621, 1600, 1549, 1500, 1474,
1464, 1410, 1360, 1317, 1288, 1230, 1159, 1047, 968, 907, 840, 776,
755, 689 cmꢀ1. 1H NMR (DMSO-d6, 300 MHz):
d 11.16 (s, 1H, NH),
9.47 (s, 1H, NH), 8.73 (d, J ¼ 5.8 Hz, 2H, H2, H6), 8.47 (d, J ¼ 4.9 Hz,
1H, CH), 7.8 (d, J ¼ 5.6 Hz, 2H, H3, H5), 7.16 (t, J ¼ 8.1 Hz, 1H), 6.93 (s,
1H), 6.78 (d, J ¼ 7.9 Hz, 1H), 6.49 (d, J ¼ 8.1 Hz, 1H), 3.36 (s, CH3). 13
C
Fig. 4. The changes in UVevis spectra for the decomposition of compound 2o in
0.01 M hydrochloric acid solution at 37 ꢂC. The inset represents time dependence of
absorbance at 267 nm [s1/2 ¼ (243 ꢄ 4) s].
NMR (DMSO-d6, 75 MHz):
d 160.3, 150.4 (2C), 147.5, 142.2, 141.5,
130.2, 121.9, 121.4 (2C), 108.7, 106.7, 102.3, 55.2. Anal. Calcd for

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E. Vavríková et al. / European Journal of Medicinal Chemistry 46 (2011) 5902e5909
5907
C14H14N4O2 (270.36): C, 62.21; H, 5.22; N, 20.73. Found: C, 62.14; H,
5.46; N, 20.92.
1548, 1481, 1408, 1378, 1314, 1283, 1219, 1070, 992, 908, 839, 763,
701, 681 cmꢀ1. 1H NMR (DMSO-d6, 300 MHz):
11.21 (s, 1H, NH),
d
9.60 (s, 1H, NH), 8.73 (d, J ¼ 5.6 Hz, 2H, H2, H6), 8.43 (d, J ¼ 7.0 Hz,
3.1.2.4. N-(4-Methoxyphenyl)-N0-(pyridin-4-ylcarbonyl)hydrazono-
formamide (2d). Yield 29% (Method A); mp 170e172 ꢂC (ethanol). IR
(KBr): 3421, 3208, 2836, 1667, 1620, 1549, 1535, 1515, 1466, 1365, 1319,
1308, 1290, 1247, 1179, 1035, 828, 685, 670 cmꢀ1. 1H NMR (DMSO-d6,
1H, CH), 7.79 (d, J ¼ 5.8 Hz, 2H, H3, H5), 7.60 (s, 1H), 7.42 (m, 1H),
7.21 (m, 1H), 7.07 (m, 1H). 13C NMR (DMSO-d6, 75 MHz):
d 160.3,
150.4 (2C), 147.1, 142.6, 141.4, 131.2, 123.8, 122.4, 121.4 (2C), 118.7,
115.3. Anal. Calcd for C13H11BrN4O (319.16): C, 48.92; H, 3.47; N,
17.55. Found: C, 49.24; H, 3.86; N, 17.91.
300 MHz):
d
11.13 (s, 1H, NH), 9.27 (s, 1H, NH), 8.71 (d, J ¼ 5.7 Hz, 2H,
H2, H6), 8.35 (s, 1H, CH), 7.78 (d, J ¼ 5.5 Hz, 2H, H3, H5), 7.29 (d,
J ¼ 7.7 Hz, 2H, H20,H60), 6.85 (d, J ¼ 8.0 Hz, 2H, H30, H50), 3.70 (s, CH3).
3.1.2.10. N-(4-Cchloro-2-hydroxyphenyl)-N0-(pyridin-4-ylcarbonyl)
hydrazonoformamide (2j). Yield 30% (Method A); mp 191 ꢂC
(ethanol). IR (KBr): 3193, 1693, 1633, 1549, 1488, 1411, 1360, 1317,
13C NMR (DMSO-d6, 75 MHz):
d 160.1, 154.2, 150.4 (2C), 148.1, 141.6,
134.3, 121.4 (2C), 118.1 (2C), 114.6 (2C). Anal. Calcd for C14H14N4O2
(270.36): C, 62.21; H, 5.22; N, 20.73. Found: C, 62.40; H, 5.51; N, 20.82.
1261, 1222, 1061, 1023, 1002, 848, 682 cmꢀ1 1H NMR (DMSO-d6,
.
300 MHz):
d
11.02 (s, 1H, NH), 9.04 (s, 1H, NH), 8.73 (d, J ¼ 2.8 Hz,
3.1.2.5. N0-(Pyridin-4-ylcarbonyl)-N-[4-(trifluoromethyl)phenyl]
hydrazonoformamide (2e). Yield 42% (Method A); mp 191e193 ꢂC
(acetonitrile). IR (KBr): 3424, 3234, 3051, 2360, 1651, 1635, 1615,
1549, 1530,1491,1458,1412,1328,1270,1194,1164,1115,1068,1014,
2H, H2, H6), 8.36 (s, 1H, CH), 8.14 (s, 1H), 7.94 (s, 1H, OH), 7.76 (d,
J ¼ 3.9 Hz, 2H, H3, H5), 7.54 (m, 1H), 6.84 (m, 1H). 13C NMR (DMSO-
d6, 75 MHz): d 160.9, 151.6 (2C),147.8, 143.9, 141.2, 130.2, 122.7, 121.0
(2C), 119.2, 115.4, 107.3. Anal. Calcd for C13H11N4O2Cl (291.72): C,
53.71; H, 3.81; N, 19.27. Found: C, 54.02; H, 4.06; N, 18.83.
834, 753, 669 cmꢀ1. 1H NMR (DMSO-d6, 300 MHz):
d 11.09 (s, 1H,
NH), 8.71 (d, J ¼ 4.5 Hz, 3H, NH, H2, H6), 8.32 (s, 1H, CH), 7.78 (m,
2H, H3, H5), 7.30 (d, J ¼ 8.0 Hz, 2H, H20, H60), 6.87 (d, J ¼ 8.2 Hz, 2H,
3.1.2.11. N-(5-Chloro-2-hydroxyphenyl)-N0-(pyridin-4-ylcarbonyl)
hydrazonoformamide (2k). Yield 43% (Method A); mp 207e208 ꢂC
(ethanol). IR (KBr): 3219, 1695, 1635, 1587, 1542, 1515, 1413, 1389,
H30, H50). 13C NMR (DMSO-d6, 75 MHz):
d 160.1, 154.2 (2C), 150.3,
148.0, 141.6, 134.3, 121.9, 121.4 (2C), 118.1 (2C), 114.6 (2C). Anal.
Calcd for C14H11F3N4O (308.27): C, 54.55; H, 3.60; N,18.18. Found: C,
54.81; H, 3.93; N, 18.44.
1324, 1285, 1060, 1010, 848, 684 cmꢀ1 1H NMR (DMSO-d6,
.
300 MHz):
d
11.27 (s, 1H, NH), 9.03 (s, 1H, NH), 8.72 (d, J ¼ 4.7 Hz,
2H, H2, H6), 8.31 (d, J ¼ 17.9 Hz, 1H, CH), 8.15 (s, 1H), 7.77 (d,
J ¼ 4.9 Hz, 2H, H3, H5), 7.59 (d, J ¼ 5.5 Hz, 1H), 7.54 (s, 1H, OH), 6.81
3.1.2.6. N-(3-Fluorophenyl)-N0-(pyridin-4-ylcarbonyl)hydrazono-
formamide (2f). Yield 62% (Method A), 80% (Method B); mp
180e182 ꢂC (ethanol). IR (KBr): 3208, 1693, 1651, 1614, 1596, 1548,
1549, 1496, 1463, 1408, 1366, 1320, 1267, 1218, 1152, 1060, 1001, 970,
(m, 1H). 13C NMR (DMSO-d6, 75 MHz):
d 160.6, 150.6 (2C), 147.8,
144.9, 141.5, 130.2, 122.9, 121.4 (2C), 118.2, 115.4, 108.3. Anal. Calcd
for C13H11N4O2Cl (291.72): C, 53.71; H, 3.81; N, 19.27. Found: C,
53.39; H, 3.61; N, 19.55.
908, 845, 776, 682 cmꢀ1. 1H NMR (DMSO-d6, 300 MHz):
d 11.22 (s,
1H, NH), 9.62 (s, 1H, NH), 8.79 (d, J ¼ 4.2 Hz, 2H, H2, H6), 8.42 (s, 1H,
CH), 7.73 (d, J ¼ 5.1 Hz, 2H, H3, H5), 7.30 (m, 2H), 7.03 (d, J ¼ 6.7 Hz,
3.1.2.12. N-(3,4-Dichlorophenyl)-N0-(pyridin-4-ylcarbonyl)hydrazo-
noformamide (2l). Yield 33% (Method A), 32% (Method B); mp
192 ꢂC (acetonitrile). IR (KBr): 3443, 2988, 1693, 1633, 1549, 1487,
1411, 1360, 1316, 1262, 1061, 1023, 849, 684 cmꢀ1. 1H NMR (DMSO-
1H), 6.71 (t, J ¼ 5.9 Hz, 1H). 13C NMR (DMSO-d6, 75 MHz):
d 164.5,
160.4, 150.4 (2C), 147.1,142.9, 141.4,130.9, 121.4 (2C), 112.5,107.5 (q),
103.4 (q). Anal. Calcd for C13H11FN4O (258.25): C, 60.46; H, 4.29; N,
21.69. Found: C, 60.29; H, 4.06; N, 21.31.
d6, 300 MHz):
d
11.02 (s, 1H, NH), 9.78 (s, 1H, NH), 8.73 (d, J ¼ 4.9 Hz,
2H, H2, H6), 8.54 (s, 1H, CH), 8.25 (s, 1H), 7.76 (s, 1H), 7.54 (d,
3.1.2.7. N-(4-Fluorophenyl)-N0-(pyridin-4-ylcarbonyl)hydrazono-
formamide (2g). Yield 39% (Method A), 61% (Method B); mp
191e193 ꢂC (ethanol). IR (KBr): 3213, 3128, 3068, 2830, 1667, 1621,
1549, 1539, 1515, 1410, 1358, 1296, 1222, 1155, 1005, 965, 830, 754,
J ¼ 4.9 Hz, 2H, H3, H5), 7.02 (m, 1H). 13C NMR (DMSO-d6, 75 MHz):
d
160.5, 150.7 (2C), 147.2, 142.3, 140.1, 131.8, 130.2, 122.9, 121.2 (2C),
116.9,110.0. Anal. Calcd for C13H10N4OCl2 (309.16): C, 50.51; H, 3.26;
N, 18.12. Found: C, 50.12; H, 3.66; N, 18.47.
671 cmꢀ1. 1H NMR (DMSO-d6, 300 MHz):
d 11.15 (s, 1H, NH), 9.03 (s,
1H, NH), 8.69 (d, J ¼ 4.6 Hz, 2H, H2, H6), 8.38 (s, 1H, CH), 7.76 (d,
3.1.2.13. N-(3,4-Difluorophenyl)-N0-(pyridin-4-ylcarbonyl)hydrazo-
noformamide (2m). Yield 24% (Method A), 30% (Method B); mp
193 ꢂC (ethanol). IR (KBr): 3189, 2915, 1694, 1633, 1557, 1487, 1411,
1361, 1261, 1183, 1151, 1061, 1023, 1003, 920, 848, 752, 682 cmꢀ1. 1H
J ¼ 5.0 Hz, 2H, H3, H5), 7.38 (d, J ¼ 6.4 Hz, 2H, H20, H60), 7.12 (d,
J ¼ 6.9 Hz, 2H, H30, H50). 13C NMR (DMSO-d6, 75 MHz):
d 160.3,
158.77, 150.3 (2C), 147.7, 141.5, 137.4, 121.4 (2C), 118.2 (2C), 115.7 (q,
2C). Anal. Calcd for C13H11FN4O (258.25): C, 60.46; H, 4.29; N, 21.69.
Found: C, 60.76; H, 4.05; N, 21.39.
NMR (DMSO-d6, 300 MHz): d 11.09 (s, 1H, NH), 9.80 (s, 1H, NH), 8.73
(d, J ¼ 5.4 Hz, 2H, H2, H6), 8.54 (s, 1H, CH), 8.36 (m, 1H), 7.76 (s, 1H),
7.53 (d, J ¼ 5.8 Hz, 2H, H3, H5), 7.10 (m, 1H). 13C NMR (DMSO-d6,
3.1.2.8. N-(3-Chlorophenyl)-N0-(pyridin-4-ylcarbonyl)hydrazono-
formamide (2h). Yield 54% (Method A), 77% (Method B); mp
176e178 ꢂC (ethanol). IR (KBr): 3208, 1692, 1650, 1616, 1593, 1548,
1549,1502,1463,1403,1366,1320,1265,1218,1154,1060,1006, 970,
75 MHz): d 161.7, 151.2 (2C), 148.1, 141.9, 140.5, 132.2, 131.4, 122.6,
121.2 (2C), 117.9, 112.1. Anal. Calcd for C13H10N4OF2 (277.26): C,
56.52; H, 3.65; N, 20.28. Found: C, 56.12; H, 3.91; N, 19.89.
908, 845, 759, 682 cmꢀ1. 1H NMR (DMSO-d6, 300 MHz):
d
11.21 (s,
3.1.2.14. N-(3-Chloro-4-fluorophenyl)-N0-(pyridin-4-ylcarbonyl)
hydrazonoformamide (2n). Yield 22% (Method A), 45% (Method B);
mp 193e195 ꢂC (ethanol). IR (KBr): 3200, 2915, 1686, 1632, 1602,
1H, NH), 9.56 (s, 1H, NH), 8.77 (d, J ¼ 5.1 Hz, 2H, H2, H6), 8.46 (s, 1H,
CH), 7.71 (d, J ¼ 5.6 Hz, 2H, H3, H5), 7.36 (m, 2H), 7.03 (d, J ¼ 7.1 Hz,
1H), 6.92 (m, 1H). 13C NMR (DMSO-d6, 75 MHz):
d
160.1, 150.2 (2C),
1549, 1489, 1411, 1360, 1261, 1222, 1062, 905, 848, 682 cmꢀ1
.
1H
147.1, 142.8, 141.4, 130.9, 123.7, 122.3, 121.4 (2C), 118.2, 115.4. Anal.
Calcd for C13H11ClN4O (274.71): C, 56.84; H, 4.04; N, 20.40. Found:
C, 56.62; H, 4.36; N, 20.26.
NMR (DMSO-d6, 300 MHz): d 11.03 (s, 1H, NH), 9.78 (s, 1H, NH),
8.74 (d, J ¼ 4.9 Hz, 2H, H2, H6), 8.38 (s, 1H, CH), 8.14 (m, 1H), 7.76
(s, 1H), 7.53 (d, J ¼ 4.9 Hz, 2H, H3, H5), 6.99 (m, 1H). 13C NMR
(DMSO-d6, 75 MHz):
d 160.8, 158.1, 150.2 (2C), 147.0, 145.7, 140.4,
3.1.2.9. N-(3-Bromophenyl)-N0-(pyridin-4-ylcarbonyl)hydrazono-
formamide (2i). Yield 60% (Method A), 80% (Method B); mp
178e180 ꢂC (ethanol). IR (KBr): 3201, 3050, 2892, 1673, 1632, 1593,
133.4, 126.8, 121.5 (2C), 115.8, 110.9. Anal. Calcd for C13H10N4OClF
(293.71): C, 53.35; H, 3.44; N, 19.14. Found: C, 53.74; H, 3.09; N,
19.46.

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5908
3.1.2.15. N-(4-Bromo-3-fluorophenyl)-N0-(pyridin-4-ylcarbonyl)
hydrazonoformamide (2o). Yield 12% (Method A), 17% (Method B);
mp 193e195 ꢂC (ethanol). IR (KBr): 3201, 2989, 1686, 1631, 1602,
1548,1489,1411,1359,1261,1222,1062, 905, 848, 682 cmꢀ1.1H NMR
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 semi-synthetic
medium (SEVAC, Prague) at 37 ꢂC. Compounds were dissolved in
dimethyl sulfoxide solution (max. 5% DMSO in water) and added to
the medium within a concentration range 1000, 500, 250, 125, 62,
(DMSO-d6, 300 MHz):
d 11.03 (s, 1H, NH), 9.80 (s, 1H, NH), 8.74 (d,
J ¼ 4.5 Hz, 2H, H2, H6), 8.40 (d, J ¼ 13.2 Hz, 1H, CH), 8.15 (s, 1H), 7.77
(d, J ¼ 5.5 Hz, 2H, H3, H5), 7.54 (m, 1H), 7.00 (m, 1H). 13C NMR
31, 16, 8, 4, 2 and 1 m
mol Lꢀ1. Minimal inhibitory concentration
(DMSO-d6, 75 MHz):
d
160.4, 157.4, 150.4 (2C), 146.7, 142.3, 141.1,
(MIC) was determined after incubation at 37 ꢂC for 7, 14 and 21
days.
133.8, 121.4 (2C), 114.1, 105.0, 98.3. Anal. Calcd for C13H10N4OBrF
(338.17): C, 46.31; H, 2.99; N,16.62. Found: C, 46.01; H, 3.39; N,16.41.
3.2.2. In vitro cytotoxicity of compounds by MTT assay
3.1.2.16. N0-(Pyridin-4-ylcarbonyl)-N-[3-(trifluoromethyl)phenyl]
hydrazonoformamide (2p). Yield 71% (Method B); mp 189e191 ꢂC
(acetonitrile). IR (KBr): 3240, 3100, 3041, 1657, 1633, 1600, 1559,
1545,1497,1478,1340,1324,1263,1206,1188,1172,1132,1105,1072,
999, 901, 881, 833, 791, 710, 697, 670, 639 cmꢀ1. 1H NMR (DMSO-d6,
HepG2 human hepatoma cells (ATCC HB-8065) and freshly
prepared human PBMC (peripheral blood mononuclear cells) [14]
were cultured in RPMI-1640 medium without phenol red supple-
mented with 10% fetal calf serum (FCS), 2 mM
160
g mLꢀ1 gentamycin [15,16]. Cell cultures were maintained at
37 ꢂC, 5% CO2 in water-saturated atmosphere.
L-glutamine and
m
300 MHz):
d
11.25 (s, 1H, NH), 9.78 (d, J ¼ 6.0 Hz, 1H, NH), 8.74 (d,
J ¼ 5.8 Hz, 2H, H2, H6), 8.52 (d, J ¼ 5.6 Hz,1H, CH), 7.84 (d, J ¼ 4.2 Hz,
Cells were plated into 96-well plate with initial cell number of
5 ꢃ103 per well (in the case of PBMC 2.0 ꢃ 105 cells/well). After 24 h
incubation at 37 ꢂC prior to the experiment, cells were treated with
1H), 7.76 (d, J ¼ 5.7 Hz, 2H, H3, H5), 7.52 (m, 2H), 7.24 (d, J ¼ 6.9 Hz,
1H). 13C NMR (DMSO-d6, 75 MHz):
d 160.3, 150.4 (2C), 147.1, 141.7,
141.3, 130.4, 130.0, 124.2 (q, J ¼ 259.6 Hz, CF3), 121.4 (2C), 120.1,
117.5, 112.5. Anal. Calcd for C14H11F3N4O (308.27): C, 54.55; H, 3.60;
N, 18.18. Found: C, 54.54; H, 3.25; N, 18.51.
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 ꢂC, the cell viability was deter-
mined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
3.1.2.17. N-(4-Chlorophenyl)-N0-(pyridin-4-ylcarbonyl)hydrazono-
formamide (2r). Yield 34% (Method B); mp 178e180 ꢂC (ethanol). IR
(KBr): 3234, 1634, 1586, 1494, 1407, 1314, 1289, 1262, 1093, 1011,
bromide (MTT)-assay [17,18]. Then 45
mL
MTT-solution
(2 mg mLꢀ1) was added to each well. The respiratory chain [14]
and other electron transport systems [19] reduce MTT and
thereby form non-water-soluble violet formazan crystals within the
cell [20]. The amount of these crystals can be determined spectro-
photometrically and serves to estimate the number of mitochondria
and hence the number of living cells in the well [21]. After 4 h of
incubation, cells were centrifuged for 5 min (2000 rpm) and
supernatant was removed. The obtained formazan crystals were
820, 675 cmꢀ1. 1H NMR (DMSO-d6, 300 MHz):
d 11.20 (s, 1H, NH),
9.57 (d, J ¼ 6.4 Hz, 1H, NH), 8.73 (d, J ¼ 4.4 Hz, 2H, H2, H6), 8.42 (d,
J ¼ 6.0 Hz,1H, CH), 7.75 (d, J ¼ 4.5 Hz, 2H, H3, H5), 7.34 (d, J ¼ 4.4 Hz,
2H, H20, H60), 7.20 (d, J ¼ 8.7 Hz, 2H, H30, H50). 13C NMR (DMSO-d6,
75 MHz):
d 160.3, 150.4 (2C), 147.2, 141.4, 139.9, 129.1 (2C), 124.8,
121.4 (2C),118.1 (2C). Anal. Calcd for C13H11N4OCl (274.71): C, 56.84;
H, 4.04; N, 20.40. Found: C, 56.90; H, 4.31; N, 20.49.
dissolved in 50 or 100
samples was measured at
Reader (iEMS Reader, Labsystems, Finland). OD620 values were
subtracted from OD540 values. The percent of cytotoxicity was
calculated using the following equation:
mL DMSO and optical density (OD) of the
3.1.2.18. N-(4-Bromophenyl)-N0-(pyridin-4-ylcarbonyl)hydrazono-
formamide (2s). Yield 49% (Method B); mp 186e188 ꢂC (ethanol). IR
(KBr): 3220,1699,1631,1586,1548,1491,1313,1258,1075,1004, 846,
l
¼ 540 nm and
l
¼ 620 nm using ELISA
815, 693, 676 cmꢀ1.1H NMR (DMSO-d6, 300 MHz):
d 11.21 (s,1H, NH),
9.58 (d, J ¼ 7.1 Hz, 1H, NH), 8.72 (d, J ¼ 6.1 Hz, 2H, H2, H6), 8.42 (d,
J ¼ 6.7 Hz,1H, CH), 7.74 (d, J ¼ 6.0 Hz, 2H, H3, H5), 7.44 (d, J ¼ 8.9 Hz,
2H, H20, H60), 7.29 (d, J ¼ 8.9 Hz, 2H, H30, H50). 13C NMR (DMSO-d6,
Cytotoxicity (%) ¼ [1 e (ODtreated/ODcontrol)] ꢃ 100;
75 MHz):
d
160.4,150.5 (2C),147.2,141.5,140.3,132.1 (2C),121.5 (2C),
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 dose-response curves. The curves were
defined using MicrocalTM Origin1 (version 7.5) software.
118.6 (2C), 112.7. Anal. Calcd for C13H11N4OBr (319.17): C, 48.92; H,
3.47; N, 17.55. Found: C, 49.20; H, 3.81; N, 17.89.
3.1.2.19. N-(3-Nitrophenyl)-N0-(pyridin-4-ylcarbonyl)hydrazono-
formamide (2t). Yield 39% (Method B); mp 190e191 ꢂC (ethanol). IR
(KBr): 3232, 3080,1641,1636,1530,1350,1265,1209,1149,1068, 998,
840, 736, 678 cmꢀ1.1H NMR (DMSO-d6, 300 MHz):
d
11.29 (s,1H, NH),
3.3. Stability testing
9.93 (d, J ¼ 7.3 Hz, 1H, NH), 8.74 (d, J ¼ 6.0 Hz, 2H, H2, H6), 8.56 (d,
J ¼ 7.2 Hz, 1H, CH), 8.27 (s, 1H), 7.84 (d, J ¼ 5.3 Hz, 1H), 7.77 (d,
J ¼ 1.9 Hz, 2H, H3, H5), 7.69 (d, J ¼ 7.6 Hz, 1H), 7.58 (m, 1H). 13C NMR
All of the kinetic measurements were carried out in a 1 cm
closable cell using a HewlettePackard 8453 diode array spectro-
photometer at 37 ꢄ 0.1 ꢂC. The observed pseudo-first-order rate
constants kobs were calculated from absorbance-time dependences
(DMSO-d6, 75 MHz):
d 160.4, 150.4 (2C), 148.7, 146.8, 142.1, 141.3,
130.6, 122.7, 121.4 (2C), 115.8, 110.4. Anal. Calcd for C13H11N5O3
(285.26): C, 54.74; H, 3.89; N, 24.55. Found: C, 54.49; H, 4.12; N, 24.43.
at the wavelength of
l
¼ 267 nm and at the substrate concentra-
tions of ca. 5 ꢃ 10ꢀ5 mol Lꢀ1. Ionic strength I ¼ 1 mol Lꢀ1 was
adjusted by KCl. In all kinetic runs, the standard deviation in the fit
was always less than 1% of the quoted value and was more usually
between 0.2 and 0.4% of the quoted value. The pH of individual
buffers was measured using PHM 93 Radiometer Copenhagen
apparatus equipped with glass electrode. Redistilled water,
commercially available substituted acetic acids, amines, and
potassium chloride (p.a.) for adjustment of ionic strength of buffer
solutions were used.
3.2. In vitro biological assays
3.2.1. In vitro antimycobacterial evaluation on M. tuberculosis 331/
88, M. avium 330/88, M. kansasii 235/80 and M. kansasii 6509/96
cultures
In vitro antimycobacterial activity was evaluated against
M. tuberculosis CNTC My 331/88, M. avium CNTC 330/88, M. kansasii
CNTC My 235/80 and M. kansasii 6509/96. All strains were obtained

ꢀ
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5909
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4. Conclusion
A new series of isoniazid hydrazones linked by the CH fragment
with substituted anilines possessing electron-withdrawing
substituents were prepared by two synthetic approaches. Nearly
all compounds exhibited the same inhibitory effect as a standard
(INH, MIC 1
(MIC 62.5e125
bromo-3-fluorophenyl)-N0-(pyridin-4-ylcarbonyl)hydrazonoforma-
mide (2o) (M. tuberculosis 1
mol Lꢀ1; M. avium 250 mol Lꢀ1
M. kansasii 62.5
mol Lꢀ1; M. kansasii clinical isolate 8 mol Lꢀ1).
m
mol Lꢀ1), they were more active against M. kansasii
m
mol Lꢀ1). The best activity was observed for N-(4-
m
m
;
m
m
This compound is stable under neutral pH, slowly hydrolyses under
acidic conditions and exhibits promising selectivity index (SI ¼ 162)
that predetermines it for further screening.
[10] M.L. Ferreira, A.L.P. Candea, M.G.M.O. Henriques, C.R. Kaiser, M.V.N. de Souza,
Evaluation of substituted benzaldehydes against Mycobacterium tuberculosis,
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Acknowledgements
ꢀ
ꢀ
This work was financially supported by the Research project
MSM 0021620822, MSM 0021627501, 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 Research
Agency (projects P1-0230-103 and BI-CZ/10-11-005) is also
acknowledged. This work was also partially supported with infra-
structure of the EN-FIST Centre of Excellence, Ljubljana, Slovenia.
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