Thiosemicarbazones, semicarbazones, dithiocarbazates and hydrazide/hydrazones: Anti - Mycobacterium tuberculosis activity and cytotoxicity

Article abstract of DOI:10.1016/j.ejmech.2010.01.028

The aim of this study was to identify a candidate drug for the development of anti-tuberculosis therapy from previously synthesized compounds based on the thiosemicarbazones, semicarbazones, dithiocarbazates and hydrazide/hydrazones compounds. The minimal inhibitory concentration (MIC) of these compounds against Mycobacterium tuberculosis was determined. Their in vitro cytotoxicity to J774 cells (IC₅₀) was determined to establish a selectivity index (SI) (SI = IC₅₀/MIC). The best compounds were the thiosemicarbazones (2, 3 and 4) and the hydrazide/hydrazones (14, 15, 16 and 18). The results are comparable to or better than those of first line or second line drugs commonly used to treat TB, suggesting these compounds as anti-TB drug candidates.

Full text of DOI:10.1016/j.ejmech.2010.01.028

European Journal of Medicinal Chemistry 45 (2010) 1898–1905
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Thiosemicarbazones, semicarbazones, dithiocarbazates and hydrazide/
hydrazones: Anti – Mycobacterium tuberculosis activity and cytotoxicity
,
b
c
b
d
Fernando R. Pavan a , Pedro I. da S. Maia , Sergio R.A. Leite , Victor M. Deflon , Alzir A. Batista ,
*
Daisy N. Sato e, Scott G. Franzblau f, Clarice Q.F. Leite a
,
*
a Faculdade de Cieˆncias Farmaceˆuticas, Universidade Estadual Paulista, 14801-902 Araraquara, SP, Brazil
b Instituto de Qu´ımica de S˜ao Carlos, Universidade de S˜ao Paulo, 13566-590 S˜ao Carlos, SP, Brazil
c Instituto de Qu´ımica, Universidade Estadual Paulista, 14800-900 Araraquara, SP, Brazil
d Departamento de Qu´ımica, Universidade Federal de S˜ao Carlos, 13565-905 S˜ao Carlos, SP, Brazil
e
˜
˜
Instituto Adolfo Lutz, Unidade de Ribeirao Preto, 14085-410 Ribeirao Preto, SP, Brazil
f Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago, Chicago, IL, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The aim of this study was to identify a candidate drug for the development of anti-tuberculosis therapy
from previously synthesized compounds based on the thiosemicarbazones, semicarbazones, dithio-
carbazates and hydrazide/hydrazones compounds. The minimal inhibitory concentration (MIC) of these
compounds against Mycobacterium tuberculosis was determined. Their in vitro cytotoxicity to J774 cells
(IC50) was determined to establish a selectivity index (SI) (SI ¼ IC50/MIC). The best compounds were the
thiosemicarbazones (2, 3 and 4) and the hydrazide/hydrazones (14, 15, 16 and 18). The results are
comparable to or better than those of ‘‘first line’’ or ‘‘second line’’ drugs commonly used to treat TB,
suggesting these compounds as anti-TB drug candidates.
Received 3 November 2009
Received in revised form
11 January 2010
Accepted 14 January 2010
Available online 28 January 2010
Keywords:
Antimycobacterial agents
REMA
Ó 2010 Elsevier Masson SAS. All rights reserved.
Cytotoxicity
Thiosemicarbazones
Semicarbazones
Dithiocarbazates
Hydrazide/hydrazones
1. Introduction
attention, owing to their remarkable biological and pharmacolog-
ical properties, such as antibacterial, antiviral, antineoplastic, and
Mycobacterium tuberculosis (MTB), the pathogenic agent of
tuberculosis (TB), is responsible for the death of 2–3 million people
annually and for a global economic toll of w$12 billion each year
[1]. The rise of multidrug resistance (MDR) in MTB has complicated
and prolonged the treatment [2]. No new drugs have been devel-
oped specifically against mycobacteria since the 1960s and only
within the last few years have some promising drug candidates
emerged [3,4]. Thus, there is an urgent need to develop new ther-
apeutics for tuberculosis, to reduce the duration of treatment and
to provide a more effective therapy for MDR TB and for latent
tuberculosis infection [5].
antimalarial activities [6]. The antibacterial properties of TSCs have
been studied since 1946, when Domagk et al. reported their activity
against M. tuberculosis [7]. The one compound in this class that has
been widely used for treatment of tuberculosis in Africa and
South America is p-acetamidobenzaldehyde thiosemicarbazone,
commercially available as thiacetazone [8]. Semicarbazones differ
from TSCs only by replacing the sulfur atom with an oxygen atom
(X in Chart 1) and SCs also exhibit antitubercular activity against
M. tuberculosis H37Rv (ATCC 27294) [9]. A compound related to the
HZs class that has excellent anti-TB activity is isonicotinoyl hydra-
zide (INH), commercially known as isoniazid [10–12]. Finally,
although the anti-TB activity of dithiocarbazates is not yet reported,
this class of compounds is also worth investigating, in view of their
great structural similarity with TSCs and because of their good
activity against some bacteria [13].
Thiosemicarbazones (TSCs), semicarbazones (SCs), hydrazide/
hydrazones (HZs) and dithiocarbazates (DTCs) (see Chart 1) are
important classes of compounds which have long attracted
Very recently, we reported the anti-TB activity of some TSCs
derived from 2-acetylpyridine and their vanadium complexes and
it was observed that variation of the groups on the N(4) atom of the
* Corresponding authors. Tel.: þ55 (16) 33016953.
E-mail addresses: pavanfer@yahoo.com.br (F.R. Pavan), leitecqf@fcfar.unesp.br
(C.Q.F. Leite).
0223-5234/$ – see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.01.028

F.R. Pavan et al. / European Journal of Medicinal Chemistry 45 (2010) 1898–1905
1899
R2
R1
R2
R1
R2
compounds 8 and 9 are inactive. It is known that the sulfur atom
binds more strongly to soft acids as defined by Pearson [17] than
the oxygen. This suggests a soft acid character for the hypothetical
mycobacterial receptor group. All these conjectures require further
investigation. On the other hand, contradicting these consider-
ations, dithiocarbazates 11–13 showed poor activity. This promp-
ted the discontinuation of tests with this last class. Considering the
dithiocarbazate compounds derived from benzoylacetone, 12 and
13, the low activity may be explained by the differences in the
molecular structures of these compounds from the 2-acetylpyr-
idine- and dipyridylketone-TSC type compounds. Compound 12,
which is very similar to compound 13, was analyzed by X-ray
diffraction, which showed that this compound suffered cyclization
during the synthesis reaction (Scheme 1, supplementary material),
being found in its isomeric 5-hydroxypyrazolinic form instead of
the open chain form (see Chart 2). The cyclization occurs via
deprotonation of the nitrogen atom N1 and bond formation with
C12, resulting in a stereogenic center at the carbon atom (Fig. 1).
Related findings have been observed before by Casas et al. [18],
R1
N
N
N
HN
HN
HN
R3
R3
N
R3
S
X
O
S
R4
A
B
C
Chart 1. (A) Thiosemicarbazones (X ¼ S; R1,R2,R3,R4 ¼ H, alkyl or aryl), semi-
carbazones (X ¼ O; R1,R2,R3,R4 ¼ H, alkyl or aryl); (B) hydrazide hydrazones
(R1,R2,R3 ¼ alkyl or aryl); (C) Dithiocarbazates (R1,R2,R3 ¼ alkyl or aryl).
thiosemicarbazone moiety affected the biological action [14]. This
work motivated us to conduct further investigations on the
relationship between the TSC structure and anti-TB activity, by
changing the substituents at the N(4) position and the number of
donor atoms, with the purpose of obtaining compounds with
a dimeric character. Since the biological properties of TSCs are
often related to metal ion coordination, similar compounds
(SCs, HZs and DTCs) were also prepared and tested. Thus, four
different pharmacological moieties have been studied, with
different hydrocarbon branching, configurations of heteroatoms
(NNS, NNO, ONO, ONS, NNSS and NNOO) and possible charges
(mono or bi-deprotonated), in order to discover novel lead
compounds. Hence, we present here the results and discussion of
in vitro cytotoxicity and anti-M. tuberculosis activity of twenty
compounds based on the thiosemicarbazone, semicarbazone,
hydrazide/hydrazone and dithiocarbazate classes.
where some thiosemicarbazones derived from
b-dicarbonyl
compounds were reported to suffer cyclization in the presence of
a metal substrate. However, the cyclic form observed was the
pyrazolate form (see Chart 2) instead of the 5-hydroxypyrazolinic
one. Selected bond distances and angles for compound 12 and 14
are listed in Tables 2 and 3 respectively (supplementary material).
Similar results to those of the TSCs were observed for the hydra-
zide/hydrazones. The best results were obtained with the deriva-
tives of di-2-pyridyl ketone, compound 16 and 18. As with the
thiosemicarbazone compound 5, the analogous dimeric
compound 17 had presented low activity. Regarding the deriva-
2. Results and discussion
tives of isoniazid with b-diketones, the substitution of the methyl
of the acetyl group (compound 15) by a phenyl group (compound
14) lead to better activity. Semi-empirical calculation shows that
this replacement does not affect the electron density on the
oxygen atoms [16]. Therefore, a charge effect seems irrelevant in
this case. A rise in the lipophilicity of the molecule due to the
replacement of methyl by phenyl (Log P increases) [16] can justify
this increase in activity. Greater lipophilicity facilitates diffusion
through the lipid-rich bacterial wall, thus enhancing anti-
mycobacterial effectiveness. Although a method of synthesizing
compound 14 has been reported previously, its crystal structure
has not been determined up to now (Table 4, supplementary
material). The similarities with compound 12 led us to perform
crystallographic studies on 14 as well. Interestingly, differently
from 12, this compound was not found in the cyclic form. The
protonation of the nitrogen atoms N1 and N2 is experimentally
confirmed by the detection of the corresponding hydrogen atoms
in difference Fourier maps and by their involvement in intra and
inter-molecular hydrogen bonds (see Fig. 2). Thus the bonding
situation in the solid state of compound 14 recommends that it is
treated as a ‘‘hydrazide’’ rather than a ‘‘hydrazone’’, as reported
before for thiosemicarbazone/thiosemicarbazide compounds [19].
Furthermore, there is no sign of CH2 protons in the final Fourier
map, which is consistent with previous results reporting that there
is no evidence of them in the NMR spectrum of the yellow form of
compound 14 [20].
The in vitro anti-M. tuberculosis properties of the synthesized
thiosemicarbazones, semicarbazones, dithiocarbazates and
hydrazide/hydrazones (1–20) were evaluated against M. tubercu-
losis H37Rv ATCC 27294 and the results for the Minimum Inhibitory
Concentrations (MICs) are reported in Table 1. By investigating
a series of thiosemicarbazones derived from 2-acetylpyridine it
was observed that the substitution of one hydrogen atom linked to
N(4) in the thiosemicarbazone moiety by various groups caused an
improvement in their activities. Combining the results presented
here with those previously reported by our group [14], we find that
the activity of the 2-acetylpyridinethiosemicarbazones substituted
at N(4) increases in the following order of the substituent:
H < phenyl < methyl < ethyl < cyclohexyl ¼ morpholinyl (Table 1).
The cyclic disubstituted thiosemicarbazone 1 was not active up to
50 mg/mL. The acetylation of the pyridine at position R2 (see Chart
1) in the case of 2,6-diacetylpyridine-N(4)-phenyl-thio-
semicarbazone 7 did not change the activity of the 2-acetylpyr-
idine phenyl derivative Happtsc [14] (Table 2, supplementary
material). The compound bis[benzaldehyde N4(phenyl)-thio-
semicarbazone], 5, showed a lower activity than compound 7 and
Happtsc. Perhaps its dimeric structure hinders the interaction with
a supposed bacterial receptor. Additionally, the activity of Happtsc
can be improved by replacing the R1 group methyl with pyridyl,
resulting in compound 4. Assuming that the sulfur atom has a role
in the antimicrobial activity (as occurs in some parasiticidal thio-
semicarbazones) [15] and that the electronic density on this atom
increases as a consequence of this replacement, as predicted by
semi-empirical quantum-mechanical calculation [16], this
increase in activity may be explained by the consequent
strengthening of a supposed interaction between the sulfur and
a biological receptor. Indeed, when the sulfur atom of this thio-
semicarbazone is replaced with an oxygen atom (semicarbazone
10), the activity decreases. Similarity, the semicarbazone
Finally, the presence of a hydrophilic hydroxyl group in the
structure of compound 19 reduces considerably the activity,
relative to the similar compound Hapbh (2-acetylpyridine-ben-
zoylhydrazone), previously published by our group [21]. Here the
explanation is the same, since the introduction of the phenolic
group reduces drastically the lipophilicity of the compound. The
replacement of a hydroxyl group by the amino group also decreases
the activity, as shown previously (compound Hapah, 2-acetylpyr-
idine-2-amino-benzoylhydrazone) [21].

1900
F.R. Pavan et al. / European Journal of Medicinal Chemistry 45 (2010) 1898–1905
Table 1
Thiosemicarbazones, semicarbazones, dithiocarbazates and hydrazide hydrazones: anti – M. tuberculosis activity (MIC), cytotoxicity (IC50) and selectivity index (SI).
Nꢀ of
Compound
Compound
Molecular
Mass
Structure
MIC
g/mL
IC50
g/mL
SI
m
mM
m
m
M
IC50/MIC
Thiosemicarbazones
N
N
S
N
N
H
N
Haphmtsc; 2-acetylpyridine
hexamethyleneimine-thiosemicarbazone
1
276.40
ꢁ50
ꢁ180.9
1250
4522.43 ꢂ25
S
N
N
H
HapN(et)tsc; 2-acetylpyridine N4(ethyl)
thiosemicarbazone
N
H
2
3
222.31
276.40
3.13
14.08
2.82
625
625
2811.3
200
N
S
N
N
H
HapN(ch)tsc; 2-acetylpyridine
N4(cyclohexyl)thiosemicarbazone
N
0.78
0.78
2261.22 801
H
N
N
S
N
N
H
N
H
Hdpkptsc; di-2-pyridyl ketone
N4(phenyl)thiosemicarbazone
4
333.41
2.34
78.1
234.25 100
H
H
H2bbptsc; bis[benzaldehyde
N4(phenyl)-thiosemicarbazone]
5
508.66
25
49.15
1250
2457.44
50
N
S
N
N
S
N
N
N
H
H
S
N
N
Hapmotsc; 2-acetylpyridine-morpholyl-
thiosemicarbazone
N
H
N
O
6
7
264.35
312.39
0.78
2.95
ꢂ3.9 ꢂ14.75
ꢂ5
O
N
S
m-Dapptsc; mono-diacetylpyridine-
N(4)-phenyl-thiosemicarbazone
15.6
49.94
625
2000.7
40
N
N
H
N
H
N
N
S
N
Haptsc; 2-acetylpyridine-
N
H
NH2
194.26
208.28
31.3
7.8
161.12 Not evaluated
37.45 Not evaluated
thiosemicarbazonea
S
H
N
H
Hapmtsc; 2-acetylpyridine-
N
H
N
H
N(4)-methyl-thiosemicarbazonea
N
S
Happtsc; 2-acetylpyridine-
N
270.35
15.6
57.7
Not evaluated
N(4)-phenyl-thiosemicarbazonea
N
H
N
H

F.R. Pavan et al. / European Journal of Medicinal Chemistry 45 (2010) 1898–1905
1901
Table 1 (continued)
Nꢀ of
Compound
Compound
Molecular
Mass
Structure
MIC
IC50
g/mL
SI
mg/mL
mM
m
m
M
IC50/MIC
Semicarbazones
N
O
O
N
N
H
NH2
NH2
8
9
Hapsc; 2-acetylpyridine-semicarbazone
178.19
193.21
ꢁ250
ꢁ1403
ꢁ1294
312.5 1753.75
ꢂ1.25
OH
N
N
H
H2hasc; 2-hydroxy-acetophenone-
semicarbazone
ꢁ250
156.3
809
ꢂ0.6
N
N
O
N
N
H
N
H
Hdpkpsc; di-2-pyridyl ketone
N4(phenyl)semicarbazone
10
317.35
12.5
39.39
625
1969.43
50
Dithiocarbazates
OH
S
N
N
H
S
H2habdtc; S-benzyl-b-N-
(2-hydroxyphenylethylidene) dithiocarbazate
11
316.44
50
158
ꢁ1250
3950.2
25
HO
S
N
N
H2babdtc; 5-hydroxy-3-methyl-5-
phenyl-pyrazoline-1-(S-benzyldithiocarbazate)
12
342.47
ꢁ250
ꢁ730
625
1825
ꢂ2.5
S
HO
N
N
S
S
H2banbdtc; 5-hydroxy-3-methyl-5-phenyl-
pyrazoline-1-(S-p-nitrobenzyldithiocarbazate)
13
387.47
ꢁ250
ꢁ645.21
1250
3226.06
ꢂ5
NO2
Hydrazones
O
O
H
N
H2faihz; monobenzoylacetone
isonicotinoylhydrazone
14
281.31
219.24
3.13
11.13
28.51
1250
1250
4443.5
399
N
H
N
O
O
H
N
H2acihz; monoacetylacetone
isonicotinoylhydrazone
N
H
15
6.25
5701.51 200
N
(continued on next page)

1902
Table 1 (continued)
F.R. Pavan et al. / European Journal of Medicinal Chemistry 45 (2010) 1898–1905
Nꢀ of
Compound
Compound
Molecular
Mass
Structure
MIC
g/mL
IC50
g/mL
SI
m
mM
m
m
M
IC50/MIC
N
N
O
N
N
H
Hdpkihz; di-2-pyridyl ketone
isonicotinoylhydrazone
N
16
303.32
448.48
308.36
3.13
10.32
27.87
5.06
625
2060.53 200
H
H
H2bbihz; bis(benzaldehyde
isonicotinoylhydrazone
17
12.5
156.3
348.51
12.5
N
N
N
N
N
N
O
N
O
O
N
N
S
N
H
Hdpkthz; di-2-pyridyl ketone
thiophenehydrazone
18
19
1.56
625
2026.85 401
N
N
O
O
OH
OH
Hfphhz; 2-formylpyridine
2-hydroxybenzoylhydrazone
N
H
241.25
256.26
ꢁ250
ꢁ1036.3
312.5 1295.34
312.5 1219.46
ꢂ1.25
H
OH
N
H2sashz; salicylaldehyde
2-hydroxybenzoylhydrazone
N
H
20
31.25
121.95
10
H
a
Data reported previously.14
The best MIC values were given by compounds 3, 4, 6 (0.78
mL) and 18 (1.56 g/mL), which were comparable to or better than
the MIC of some ‘‘second-line’’ drugs such as streptomycin
(MIC ¼ 1.00 g/mL), ciprofloxacin (MIC ¼ 2.00 g/mL), p-amino-
salicylic acid (MIC ¼ 0.5–2.0 g/mL), ethionamide (MIC ¼ 0.63–
1.25 g/mL), gentamicin (MIC ¼
g/mL), cycloserine (MIC ¼ 12.5–50
2.0–4.0 g/mL), kanamycin
g/mL), ethambutol (MIC ¼ 0.94–1.88
(MIC ¼ 1.25–5.0 g/mL), tobramycin (MIC ¼ 4.0–8.0 g/mL), clari-
thromycin (MIC ¼ 8.0–16 g/mL) and thiacetazone (MIC ¼ 0.125–
2.0 g/mL), 14
g/mL) [22]. For the compounds 2 (MIC ¼ 3.13
(MIC ¼ 3.13 g/mL), 15 (MIC ¼ 6.25 g/mL) and 16 (MIC ¼ 3.13 g/
mL), the MIC values were comparable to cycloserine (MIC ¼ 12.5–
50 g/mL), kanamycin (MIC ¼
g/mL), gentamicin (MIC ¼ 2.0–4.0
1.25–5.0 g/mL) and clari-
g/mL), tobramycin (MIC ¼ 4.0–8.0
thromycin (MIC ¼ 8.0–16 g/mL) [22].
m
g/
1250
rifampicin, first-line drugs in current TB treatment, were also
analyzed and had IC50 values of 1250 and 156.3 g/mL, respectively.
The selectivity index (SI) of each compound was determined as
the ratio of IC50 to MIC (Table 1). According to Orme et al. [31],
candidates for new drugs must have an index equal or higher than
mg/mL), except for compound 6 (3.9 mg/mL). Isoniazid and
m
m
m
m
m
m
m
m
m
10, with MIC lower than 6.25 mg/mL and a low cytotoxicity. SI is
m
m
used to estimate the therapeutic window of a drug and to identify
drug candidates for further studies. Thus, our study identified thi-
osemicarbazones (2, 3 and 4, with SI values of 200, 801 and 100,
respectively) and hydrazide/hydrazones (14, 15, 16 and 18, with SI
of 399, 200, 200 and 401, respectively) as very promising new
tuberculosis drug candidates. The SI of compound 3, for example,
m
m
m
m
m
m
m
m
m
m
m
R2
From our results, it appears that, in general, the best activity is
found in compounds of higher lipophilicity, as observed by Maccari
et al. [11], and with NNS or NNO tridentate binding sites. Since the
classes of compounds that exhibited good activity here are known
to be good iron chelators [23–28], it is likely that the activity of
these compounds is related to their ability to bind intracellular iron,
which is considered essential to the mycobacteria [28]. It is known
that this is the mechanism of action of this class of compounds on
the protozoan causative agent of malaria [29,30].
O
R1
HO
S
O
N
S
H
S
N
N
S
N
NH
N
S
NH2
II
I
III
X
X
The cytotoxicity results (Table 1), expressed as the IC50 for the
J774 cell line, show that all of the TSCs, SCs, HZs and DTCs tested
showed very low cytotoxicity (IC50 value ranging from 78.5 to
Chart 2. (I) Open chain form of compounds 12 (X ¼ H) and 13 (X ¼ NO2); (II) 5-
hydroxypyrazolinic form of compounds 12 (X ¼ H) and 13 (X ¼ NO2); (III) Pyrazolate
form of thiosemicarbazones derived from b-dicarbonyl compounds.

F.R. Pavan et al. / European Journal of Medicinal Chemistry 45 (2010) 1898–1905
1903
activity of TSCs, SCs, HZs and DTCs against drug-resistant strains
and M. tuberculosis bacilli in a state of latency, or internalized in
macrophages, will be the next steps in this line of research.
3. Conclusions
The results evidenced that some hydrazide/hydrazones and
thiosemicarbazones, among the compounds presented here, can
be considered as promising anti-M. tuberculosis agents, since one
of them has MIC of 0.78
mg/mL and other four have MICs ranging
from 1.56 to 3.13 g/mL, with SI values equal or higher than 200,
m
being more active and less toxic than some drugs used in the
treatment of TB.
4. Experimental section
4.1. Materials for synthesis
2-acetylpyridine, formylpyridine, 4-phenyl-2,4-butanedione,
acetylacetone, di-2-pyridyl ketone, 2,6-diacetylpyridine, salicy-
laldehyde, 2-hydroxyacetophenone, benzyl isoniazid, 2-thio-
phenehydrazide, 2-hydroxybenzoylhydrazide, semicarbazide,
N(4)-phenyl-semicarbazide, N(4)-ethyl-thiosemicarbazide, N(4)-
phenyl-thiosemicarbazide and analytical reagent grade chemicals
and solvents were obtained commercially and used without further
purification. The compounds 1–3, 6, 19, 20 [14], 4, 10, 16, 18 [32], 5
[33], 8 [34], 9 [35], 11 [36], 12 [37], 14 [38] and 15 [39] were
synthesized as described previously.
4.2. Instrumentation
Fig. 1. Molecular structure of the compound 12.
IR spectra were measured as KBr pellets on a Shimadzu FTIR-
spectrometer between 400 and 4000 cmꢃ1. 1H-NMR spectra were
was 801 and this index is comparable to the SI of rifampicin
taken with
a JEOL 400 MHz multinuclear spectrometer. The
(MIC ¼ 0.2
m
g/mL, IC50 ¼ 156.3 g/mL: SI ¼ 781 [22]), which is used
m
elemental analysis was determined using a Heraeus vario EL
elemental analyzer.
together with isoniazid to treat tuberculosis. The determination of
4.3. X-ray structure determination
Single crystals of 12 and 14 suitable for X-ray analysis were
grown by slow concentration of a solution in a 1:1 mixture of
MeOH/CH2Cl2. The data were collected with Mo-K
a radiation
(l
¼ 71.073 pm) in a STOE IPDS 2T diffractometer. Standard proce-
dures were applied for data reduction and absorption correction.
The structures were solved by direct methods with the program
SHELXS-97 and refined with SHELXL-97 [40]. Idealized hydrogen
atom positions were calculated with the riding model option of
SHELXL-97 [40]. Additional crystal data and information about the
X-ray structural analyses are shown in Table 4 (supplementary
material).
4.4. Preparations
Synthesis of mono-diacetylpyridine-N(4)-phenyl-thiosemicarbazone 7.
2,6-Diacetylpyridine (1.63 g, 10.0 mmol) was dissolved in about
50 mL of a hot EtOH/H2O mixture (1:1 v/v), and 4-phenyl-
thiosemicarbazide 1.67 g, 10.0 mmol) in about 50 mL of the EtOH/
H2O mixture was slowly added. After being heated under reflux for
1 h, the creamy yellow precipitate was filtered off, washed with
EtOH and dried in a vacuum. Yield: 83%. Anal. For C16H16N4OS:
(312.39 g molꢃ1): C, 61.52; H, 5.16; N, 17.93; S, 10.26. Found: C,
61.09; H, 5.05; N, 17.78; S, 10.52 %. IR (KBr) (nmax/cmꢃ1): 3356, 3321
(s, NH), 1690 (vs, C]O), 1578 (m, C]N). 1H NMR (CDCl3, ppm): 2.48
(s, 3H, CH3), 2.71 (s, 3H, CH3C]O), 7.18–7.22 (m, 1H, Ph þ CHCl3),
7.36 (t, J ¼ 7.9, 2H, Ph), 7.63 (d, J ¼ 7.9, 2H, Ph), 7.83 (t, J ¼ 7.6,1H, Ph),
Fig. 2. Crystal and molecular structure of 14 with atom labels. Methyl and aromatic
hydrogen atoms are omitted for clarity. [N(2)/O(1) ¼ 2.598(3) Å, N(2)–H2/
O(1) ¼ 133.9ꢀ]; [N(1)/O(1b) ¼ 2.753(3) Å, N(1)–H1/O(1b) ¼ 129.4ꢀ]. Symmetry
b
operations used: ꢃx þ 1, y ꢃ 1/2, ꢃz þ 1/2.

1904
F.R. Pavan et al. / European Journal of Medicinal Chemistry 45 (2010) 1898–1905
7.99 (d, J ¼ 7.6, 2H, Ph), 8.15 (d, J ¼ 7.6, 2H, Ph), 8.84 (s, 1H, NH), 9.28
(s, 1H, NH).
maintained in Complete Medium (RPMI-1640 supplemented with
10% heat-inactivated fetal bovine serum (FBS); 100 U/mL penicillin
Synthesis of 5-hydroxy-3-methyl-5-phenyl-pyrazoline-1-(S-p-
nitrobenzyldithiocarbazate) 13. Compound 13 was synthesized by
modifying the procedure described by Crouse et al. [41], using
p-nitrobenzylchloride in place of 2-picolylchloride hydrochloride
to prepare S-4-nitrobenzyldithiocarbazate, which was than
dissolved in MeOH and added to a solution of 4-phenyl-2,4-buta-
nedione in MeOH. The mixture was refluxed for 2 h. The solution
was cooled to room temperature and a yellow precipitate was
obtained. The solid was filtered off, washed with n-hexane and
dried in air. Yield: 64 %. Anal. For C18H17N3O3S2: (387.47 g molꢃ1): C,
55.80; H, 4.42; N, 10.80; S, 16.55. Found: C, 55.13; H, 4.40; N, 10.73;
S, 16.17 %. IR (KBr) (nmax/cmꢃ1): 3363 (m, OH), 1628 (m, C]N). 1H
NMR (DMSO-d6, ppm): 2.07 (s, 3H, CH3), 3.27 (dd, 2J ¼ 19.3 Hz, 2H,
CH2), 4.44 (dd, 2J ¼ 14.3 Hz, 2H, CH2), 7.16 (s, 1H, OH), 7.23–7.26 (m,
3H, Ph), 7.32 (t, 3J ¼ 7.6 Hz, 2H, Ph), 7.59 (d, 3J ¼ 8.7 Hz, 2H, Ph-NO2),
8.14 (d, 3J ¼ 8.7 Hz, 2H, Ph-NO2).
and 100 m
g/mL streptomycin), at 37 ꢀC, in a humidified 5% CO2
atmosphere. After reaching confluence, the cells were detached and
counted. For the cytotoxicity assay, 1 ꢄ105 cells/mL were seeded in
200 mL of Complete Medium in 96-well plates (NUNC). The plates
were incubated at 37 ꢀC under a 5% CO2 atmosphere for 24 h, to
allow cell adhesion prior to drug testing. The compounds were
dissolved in DMSO and subjected to two-fold serial dilution from
1250 to 3.9 mg/mL. Cells were exposed to the compounds at various
concentrations for a 24 h-period. Resazurin solution was than
added to the cell cultures and incubated for 6 hours. Cell respira-
tion, as an indicator of cell viability, was detected by reduction of
resazurin to resorufin, whose pink colour and fluorescence
indicates cell viability. A persistent blue colour of resazurin is a sign
of cell death [43]. The fluorescence measurements (530 nm exci-
tation filter and 590 nm emission filter) were performed in
a SPECTRAfluor Plus (Tecan) microfluorimeter. The IC50 value was
defined as the highest drug concentration at which 50% of the cells
are viable relative to the control [44].
Synthesis of bis(benzaldehyde isonicotinoylhydrazone 17. Three
drops of sulfuric acid were added to a mixture of isoniazid (1.37 g,
10 mmol) and benzyl (1.05 g, 5.00 mmol) in MeOH (25 mL) and the
mixture was stirred under reflux for 4 h. The solution was left at
ꢃ15 ꢀC overnight while bright yellow needles precipitated. The
precipitate was collected by filtration, washed with cold EtOH and
n-hexane, dried. Yield: 71%. Anal. For C26H20N6O2: (448.48 g
molꢃ1): C, 69.63; H, 4.49; N, 18.74. Found: C, 70.70; H, 4.50; N, 17.98
%. IR (KBr) (nmax/cmꢃ1): 3175 (m, NH), 1659 (vs, C]O), 1593 (m,
C]N). 1H NMR (CDCl3, ppm): 7.21–7.72 (m,15H), 7.82 (d, J ¼ 5.8, 2H,
Ph), 7.91 (d, J ¼ 7.2, 1H, Ph), 8.70 (s, 2H, NH).
4.7. Selectivity index
The selectivity index (SI) was calculated by dividing IC50 by
the MIC; if the SI is ꢁ10, the compound is then investigated
further [31].
Acknowledgements
This study was supported by CNPq, Capes and Fundaça˜o de
4.5. Anti-M. tuberculosis activity assay
`
˜
Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), ref. Processes:
2008/10390-2 and 2009/06499-1. The authors are thankful to the
Prof. Dr. Ulrich Abram, Freie Universitat Berlin, Germany, for
providing necessary instrumental facilities.
The anti-M. tuberculosis activity of the compounds was deter-
mined by the Resazurin Microtiter Assay (REMA) [42]. Stock solu-
tions of the test compounds were prepared in dimethyl sulfoxide
(DMSO) and diluted in Middlebrook 7H9 broth (Difco), supple-
mented with oleic acid, albumin, dextrose and catalase (OADC
enrichment – BBL/Becton Dickinson, Sparks, MD, USA), to obtain
Appendix A. Supplementary information
Supplementary data associated with this article can be found in
the online version at doi:10.1016/j.ejmech.2010.01.028.
final drug concentration ranged from 0.15 to 250 mg/mL. The
isoniazid was dissolved in distilled water, according to the manu-
facturers’ recommendations (Difco laboratories, Detroit, MI, USA),
and used as a standard drug. M. tuberculosis H37Rv ATCC 27294 was
grown for 7 to 10 days in Middlebrook 7H9 broth supplemented
with OADC, plus 0.05% Tween 80 to avoid clumps. Suspensions
were prepared and their turbidities matched to the optical density
of the McFarland no. 1 standard. After a further dilution of 1:25 in
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