Synthesis and?characterization of?novel hydrazide-hydrazones and?the?study of?their?structure-antituberculosis activity

Article abstract of DOI:10.1016/j.ejmech.2006.06.009

A series of hydrazide-hydrazones, based on a series of 4-substituted benzoic acid, were synthesized, and their structures were elucidated and screened for the antituberculosis activity against Mycobacterium tuberculosis H37Rv with the help of the BACTEC 460 radiometric system. Compound 3, 4-fluorobenzoic acid [((5-nitro)thiophen-2yl) methylene]hydrazide showed the highest inhibitory activity in this series. The search of pharmacophores was done by means of the Electronic-Topological Method (ETM). The model developed in this study is supposed to be applied to the design, preparation and screening of new compounds of similar structure in order to further test and optimize the model with the eventual goal of preparing new anti-tubercular agents.

Full text of DOI:10.1016/j.ejmech.2006.06.009

European Journal of Medicinal Chemistry 41 (2006) 1253–1261
http://france.elsevier.com/direct/ejmech
Original article
Synthesis and characterization of novel hydrazide–hydrazones
and the study of their structure–antituberculosis activity
Koçyiğit-Kaymakçıoğlu Bediaa, Oruç Elçina, Unsalan Sedaa, Kandemirli Fatmab, Shvets Nathalyc,
a,
*
c,d,
*
Rollas Sevim , Anatholy Dimoglo
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Marmara University, Haydarpaşa 34668, Istanbul, Turkey
b Department of Chemistry, Kocaeli University, Umuttepe, 41380, Kocaeli, Turkey
c Gebze Institute of Technology, Cayirova, Gebze 41400, Kocaeli, Turkey
d Institute of Applied Physics, Academy of Sciences, MD2028 Kishinev, Moldova
Received 1 February 2006; received in revised form 31 March 2006; accepted 15 June 2006
Available online 17 August 2006
Abstract
A series of hydrazide–hydrazones, based on a series of 4-substituted benzoic acid, were synthesized, and their structures were elucidated and
screened for the antituberculosis activity against Mycobacterium tuberculosis H37Rv with the help of the BACTEC 460 radiometric system.
Compound 3, 4-fluorobenzoic acid [((5-nitro)thiophen-2yl) methylene]hydrazide showed the highest inhibitory activity in this series. The search
of pharmacophores was done by means of the Electronic-Topological Method (ETM). The model developed in this study is supposed to be
applied to the design, preparation and screening of new compounds of similar structure in order to further test and optimize the model with
the eventual goal of preparing new anti-tubercular agents.
© 2006 Elsevier Masson SAS. All rights reserved.
Keywords: Antituberculosis activity; Hydrazide–hydrazones; Structure–activity relationships
1. Introduction
[5–7], antiinflammatory [8,9], antimalarial [10] and antituber-
culosis activities [11–17]. We described previously the antimi-
crobial profile of hydrazide–hydrazone derivatives of 4-
fluorobenzoic acide, some of which have exhibited a remark-
able antimicrobial activity [18]. With the aim of obtaining new
antimycobacterial compounds we synthesized a series of 4-
substituted benzoic acid (substituted methylene) (3–31) hydra-
zide derivatives in which the nitrophenyl/furanyl moiety of the
previously described compounds was replaced by the substi-
tuted phenyl, furanyl, pyrolyl or thiophenyl moiety.
Tuberculosis is presently regarded as the most dangerous
infective disease world-wide and one of the major AIDS-
associated infections. The simultaneous presence of HIV infec-
tion, the spread of drug resistant strains of Mycobacterium
tuberculosis, and the scarce compliance with the lengthy com-
plex therapies often complicate the treatment of tuberculosis
[1]. Therefore, the search for new antituberculosis agents is
required. This requirement had an impact on our further work
on the synthesis and search for some new hydrazide–hydra-
zones with the antituberculosis activity.
The named compounds were evaluated for the antimycobac-
terial activity; on account of biological profile of some pre-
viously synthesized hydrazide–hydrazones [18], the new
hydrazide–hydrazone derivatives were tested in vitro as to
their activities against M. tuberculosis H37Rv.
It is well known that the hydrazone group plays an impor-
tant role for the antimicrobial activity. Furthermore, a number
of hydrazide–hydrazone derivatives have been claimed to pos-
sess interesting antibacterial–antifungal [2–4], anticonvulsant
The present study used the Electronic-Topological Method
(ETM) to build the system capable of predicting antituberculo-
sis activities. The ETM is a structure-based approach for study-
ing structure–activity relationships (SAR) of molecules [19].
As known, more informative molecular descriptions in terms
* Corresponding authors.
E-mail addresses: sevim@sevimrollas.com (K.-K. Bedia),
dimoglo@gyte.edu.tr (A. Dimoglo).
0223-5234/$ - see front matter © 2006 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.06.009

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K.-K. Bedia et al. / European Journal of Medicinal Chemistry 41 (2006) 1253–1261
of their structural and electron parameters cause better results
in pattern recognition and separation of molecules by the activ-
ity levels. ETM is capable of taking into account individual
properties of separate atoms and bonds that may be crucial
for revealing details of interactions between a biologic receptor
and an active molecule. A number of studies have been
reported that use the ETM to find SAR models involving a
representative list of activities and thousands of compounds
belonging to different chemical classes [20–23]. The ETM
approach and software have both undergone considerable
development [24–26]. The new ETM-based system capable
of unifying Web-based SAR resources and using Internet com-
munications in the framework of a project, ETOSAR, makes
the ETM a valuable tool for SAR studies and provides rules
for the synthesis of new potentially active compounds [27–29].
4, which was selected for the level 2 screening, did not possess
a MIC less than 6.25 μg/ml. In addition, IC50 and selectivity
index value (SI) were determined 1.28, 0.41 for compound 3;
0.48, 0.62 μg/ml for compound 5; 0.41, 0.13 for compound 6,
respectively.
3. Results and discussion
3.1. The search for pharmacophores (Ph) and anti-
pharmacophores (APh) by using ETM
The compounds under study (29 molecules) are shown in
Table 1. Molecules were classified as either active or inactive
ones. Conformational analysis and quantum chemistry calcula-
tions were carried out by means of the molecular mechanics
method (MMP2) and the semi-empirical quantum chemistry
method (AM1), respectively [35]. All obtained data were
arranged as matrices called electronic-topological matrices of
contiguity (ETMC). Diagonal elements of the matrices reflect
one or more possible atomic properties (represented by a sepa-
rate value or by a vector of characteristics). Off-diagonal ele-
ments characterize bonds between pairs of atoms, if they exist,
or distances otherwise. The matrices are symmetric relative to
their diagonals that is why only their upper triangles are used
in calculations. For the sake of simplification, the ETM calcu-
lations generally use only by one property for atoms and
bonds. In cases when more than by one property for atoms
and bonds is needed, the ETM calculations can be repeated
separately for each pair of atom–bond properties. In our case,
effective charges on atoms are taken as diagonal elements, and
the values of Wiberg’s index represent off-diagonal elements
corresponding to bonds; if no bond, then off-diagonal elements
are distances for corresponding pairs of atoms in 3D-space.
The computational part of the ETM is a sequence of the
following steps [36–38]:
2. Chemistry and biological activity testing
The synthesis of starting compounds, 4-substituted benzoic
acid hydrazide derivatives 2a–d was carried out by treating 4-
substituted phenyl benzoate 1a–d with hydrazine hydrate (see
[30] for the procedure), while 4-nitrobenzoic acid hydrazide 2e
was prepared by the reaction of 4-nitrobenzoyl chloride with
hydrazine hydrate. The hydrazones 3–31 (Table 1) were pre-
pared through the synthetic pathway shown in Scheme 1, from
the reaction of equimolar amounts of properly 4-substituted
benzoic acid hydrazide with substituted aldehydes in the pre-
sence of ethanol.
Physicochemical and spectroscopic characterization of the
hydrazones 6, 7 [17], 8, 10, 15 [18], 13, 30 [31] and 29 [32]
as well as of compounds 21, 31 [33] have been previously
described. The other newly described here hydrazone com-
pounds were isolated in satisfactory yields (40–97%) and pur-
ified (recrystallized with the use of ethanol). Their chemical
1
structures were confirmed by H-NMR, mass (MS-ES), and
elemental analysis. The hydrazone –CH= and –NH– signal of
novel synthesized compounds appeared as a singlet at
8.16–8.67 and 10.45–12.25 ppm, respectively. The signal of
aromatic protons was also found in the expected field.
● conformational analysis;
● quantum-chemistry calculations;
● ETMC formation;
The in vitro antimycobacterial evaluation was performed
according to the TAACF antituberculosis drug discovery pro-
gram [34]. The primary screening data for all compounds were
shown in Table 1. The results of the study on the antitubercu-
losis activity showed that compound 3 exhibited highest inhi-
bition (99%) at a constant concentration level (6.25 μg/ml)
against M. tuberculosis H37Rv. Compounds 3–7
effecting ≥ 90% inhibition in the primary screen at 6.25 μg/ml
were re-tested at lower concentration against M. tuberculosis
H37Rv using microplate alamar blue assay (MABA) to deter-
mine the actual minimum inhibitory concentration (MIC) in a
broth microdilution assay. The MIC is defined as the lowest
concentration effecting the 90% reduction in fluorescence, rela-
tive to controls. Compound 5 demonstrated potent inhibitory
activity against M. tuberculosis H37Rv by a MIC value of
0.78 μg/ml. Compound 3, 6 and 7 demonstrated same inhibi-
tory activity by a MIC value of 3.13 μg/ml, whereas compound
● the search for structural features, responsible for a com-
pound’s activity/inactivity (the features being fragments of
molecular structures are referenced as pharmacophores/anti-
pharmacophores, correspondingly).
To find pharmacophores, a template active compound and
the rest of the compound set are compared as weighted graphs.
To find anti-pharmacophores, an inactive compound is used as
a template for the comparison. By this, molecular flexibility is
taken into account when comparing atomic and bond weights
for any two matrices. The essential part of the ETM is repre-
sented just by the last two steps.
The main advantages of the ETM are conditioned by the
fact that its molecular descriptions reflect a molecule’s electro-
nic and 3D conformational properties and, at the same time,
they do not depend on the numbers and sorts of atoms.

K.-K. Bedia et al. / European Journal of Medicinal Chemistry 41 (2006) 1253–1261
1255
Table 1
Antituberculosis activities of compounds (3–31)
Compounds
R
Ar
Inhibition
(%)
99
Compounds
R
Ar
Inhibition (%)
17
3
–F
18
–F
4
5
6
7
–F
98
98
96
96
19
20
21
22
–F
–F
–H
–F
13
9
–NO2
–Cl
–H
9
7
8
9
–F
–F
72
57
23
24
–F
–F
5
5
10
–F
39
25
–F
4
11
12
–F
–F
36
35
26
27
–F
–F
3
0
13
14
–Cl
–F
34
28
28
29
–F
–F
0
0
15
16
17
–F
–F
–F
25
23
18
30
31
–Br
0
0
–NO2
MIC (μg/ml) < 6.25 for compounds 3–7, and MIC > 6.25 for the rest of compounds.
Thus, for each compound (active or inactive) taken as a tem-
plate, its ETMC was compared with the ETMCs of the rest of
compounds in both classes of the series under study. The com-
parison resulted in a few common structural fragments for the
two cases. The fragments were found as submatrices of template
ETMCs (they will be referenced as Electron-Topological Subma-
trices of Contiguity, or ETSCs). Consequently, all pharmaco-
phores and anti-pharmacophores found from the ETM calcula-
tions form a system for the activity identification [39–42].
For the series studied, the system includes 10 pharmaco-
phores and 10 anti-pharmacophores. From compounds 3 and
5 chosen as active template compounds, activity features 1
and 2 (or the Ph1 and Ph2 pharmacophores) were found.
They are shown in Fig. 1a,b by their corresponding ETSCs,
which contain electronic-topological characteristics of these
pharmacophores.
As seen from the Ph1 and Ph2 pharmacophore’s structures,
they consists of seven atoms for Ph1 (S13, C14, C15, N16, F19,

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K.-K. Bedia et al. / European Journal of Medicinal Chemistry 41 (2006) 1253–1261
Scheme 1. Synthetic pathway for compounds 1–31. R: H (a), Br (b), Cl (c), F
(d), NO2 (e).
Ar: Substituted phenyl/thiophenyl, furanyl, pyrolyl. Reagents and conditions:
(a) NaOH; (b) NH2NH2 , CH3OH; (c) RCHO, C2H5OH.
O20, O21) and seven atoms for Ph2 (C7, C10, C12, C13, C15,
C16, O19). Its submatrix is found after setting allowable limits
for the search for equivalent matrix elements. The limits are δ1
=
0.05 (diagonal elements) and δ2
=
0.15 (off-diagonal
ones). To evaluate the probability (PA) of occurrence of a phar-
macophore (Phi) in the series under study, one common criter-
ion for structural methods is used as given by the following
equation
PA ðPhiÞ ¼ ðn1 þ 1Þ=ðn1 þ n2 þ 2Þ
(1)
where n1, n2 are numbers of active/inactive compounds,
respectively, that contain the Ph. For an anti-pharmacophore
(APhi), (n2 + 1) is to be used at place of (n1 + 1) in Eq. (1).
The pharmacophore found from the ETM-calculations is rea-
lized in five high-active compounds (3–5), and the probability
PA of its realization in this class is about 0.86. The remaining
pharmacophores, Ph3–Ph10, have been found analogously, and
the probabilities of their realization in the class of active com-
pounds vary between 0.83 and 0.86.
To determine anti-pharmacophores (‘break of activity’),
ETMCs of inactive compounds were taken as templates. Ten
anti-pharmacophores, APh1–APh10, were found in total. The
ETSCs that correspond to APh1and APh2 are given in Fig. 2a,
b along with structure of the corresponding template after
which the anti-pharmacophores have been found.
As seen from Fig. 2a, APh1 (based on the inactive template
27) consists of six atoms. APh1 is present in 22 inactive mole-
cules, thus the probability of its realization is 0.96. Analysis of
submatrices that correspond to APh1 and APh2 but are calcu-
lated relative to different template compounds, has shown their
close similarity.
The highest occupied molecular orbitals (HOMO) and the
lowest unoccupied molecular orbitals (LUMO), called also
frontier orbitals, may well play an important role in the
Fig. 1. Submatrices of the Ph1 and Ph2 pharmacophores (template compounds
3(a), 5(b)) and their corresponding structures.
donor-acceptor interaction of a substance with the correspond-
ing receptors. Analysis of LUMOs for the compounds contain-
ing Ph1, and Ph2 has shown that atoms with the highest values
of the atomic orbital coefficients are mainly those atoms that
enter into the fragments. Graphical representation of LUMO
orbitals is given in Fig. 3.
LUMO orbital for the template compound 3 (Fig. 3) con-
sists of orbitals belonging to those atoms that form furanyl
group and, partially, phenyl ring. Atoms representing Ph1 are
mostly those that deposit considerably to the LUMO orbitals.
Similar situation can be observed in the case of template com-
pound 5. In contrast to Ph1, LUMO orbital of APh1 consists of
atoms of phenyl rings and hydrazide group. All the said sug-
gests again an important role of these atoms in the substrate-
receptor interaction.
Even inconsiderable changes in the molecular structure can
affect the values of the corresponding ETMC and, in this way,

K.-K. Bedia et al. / European Journal of Medicinal Chemistry 41 (2006) 1253–1261
1257
Fig. 3. A 3D-view of LUMO orbitals for template compounds 3, 5, 27 and 29.
Fig. 2. Submatrices of the APh1 and APh2 anti-pharmacophores (template
compounds 27(a), 29(b)), and their corresponding structures.
Fig. 4. Comparison of active and inactive molecules.
influence the compound’s activity. For the template compound
3 some examples are given, which demonstrate the effects of
its modifications on its activity (Fig. 4). As it was said above,
the presence of thiophen ring and a voluminous electronegative
group attached to it (NO2, Br) is of great importance for the
activity manifestation. Thus, when the atom of sulfur (com-
pound 3) is replaced by the oxygen atom, the thiophen ring
transforms to furane ring, and activity of the resulting com-
pound 8 is 27% lower, in comparison to the compound 3.
The absence of the NO2 group in the thiophen ring deactivates
compounds considerably (see compound 17). Activity
decreases by 81%. Analogous situation is observed when com-
pound 4 transforms to compound 13.
The sort of halogen in para-position to aromatic ring does
not influence substantially the compound’s activity (see com-
pounds 3, 5–7 in Table 2, Ph1).
Along with this, if NO2-group in the thiophen ring is absent,
activity decreases abruptly with the substituent replacement
(compounds 13, 17, 21, 30). The thiophen ring replacement
by phenyl, pyridine, and pyrole ring causes the decrease of
activity as well (see compounds 4, 11, 15, 19, 28).
When comparing the structures of the pharmacophores and
anti-pharmacophores, one should pay close attention to the dif-
ferences in their spatial and electronic characteristics.

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K.-K. Bedia et al. / European Journal of Medicinal Chemistry 41 (2006) 1253–1261
Table 2
Substituents in para-position to aromatic ring and their influence on the compound’s activity
1. R in phenyl, –NO2 thiophene
MIC, %
–F (compound 3)
99
–NO2 (compound 5)
98
–Cl (compound 6)
96
–H (compound 7)
96
2. R in phenyl,–NO2 is absent
MIC, %
–F (compound 17)
18
–Br (compound 30), –NO2 (compound 31)
0
–Cl (compound 13)
34
–H (compound 21)
9
To test the system’s capability of the antituberculosis activ-
ity prognostication, we modeled a few compounds with analo-
gous structures. After their geometries had been optimized and
their electronic structures had been calculated, ETMC matrices
were formed. In concordance with the set of pharmacophores
found, these compounds were classified by their activities as
active (“+”) and inactive (“−”). Structures of these compounds
and their activities are presented in Table 3. For the compounds
T1–T6, their predicted activities MIC > 70% (i.e. they are
active compounds), while for the compounds T7–T12 their
expected MIC values are < 20% (inactive compounds).
Thus, the complex of pharmacophores and anti-
pharmacophores taken as a whole plays an important role in
the activities prediction as well as in the search for new
drugs. The set of activity/inactivity fragments found as a result
of this study forms a basis for the development of a system for
the antituberculosis activity prediction.
can include both pharmacophores and anti-pharmacophores.
The model developed in this study is supposed to be applied
to the design, preparation and screening of new compounds of
similar structure in order to further test and optimize the model
with the eventual goal of preparing new anti-tubercular agents.
5. Experimental
All chemicals and solvents were purchased from Merck,
Aldrich, and Fluka. Melting points (m.p.) were determined
with a Buchi 530 capillary apparatus and were uncorrected.
1H-NMR spectra were recorded in DMSO on a Bruker
AVANC-DPX-400 spectrometer in DMSO-d6 and chemical
shifts were given in δ ppm with tetramethylsilane (TMS). The
splitting patterns of 1H-NMR were designed as follows: s:
singlet, d: doublet, t: triplet, q: quarlet, m. multiplet. The
Mass spectrometry was performed using Agilent 1100 MSD
spectrometer in the electrospray mode. All new compounds
were analyzed for C, H, N and the results were in an acceptable
range (1H-NMR, mass and elemental analysis were provided
by the Scientific and Technical Research Council of Turkey,
TÜBITAK).
4. Conclusions
A number of hydrazide–hydrazones were designed, synthe-
sized, and screened for antituberculosis activity against
M. tuberculosis H37Rv. A systematic SAR study of the com-
pounds was performed through the application of the ETM
relative to their experimentally measured antituberculosis activ-
ity. Data obtained from conformational analysis and quantum-
chemistry calculations were used to form electronic-topological
matrices. These matrices were effectively used in the search for
a system of pharmacophores and anti-pharmacophores capable
of effective separation of compounds from the examination set
into groups of active and inactive compounds. Low-active
molecules are badly responsive to the activity prognostication,
because they form a buffer zone consisting of compounds that
5.1. Antituberculosis activity
The primary screen is conducted at 6.25 μg/ml (or molar
equivalent of highest molecular weight compound in a series
of congeners) against M. tuberculosis H37Rv (ATCC 27294) in
BACTEC 12B medium using the MABA. Compounds exhibit-
ing fluorescence are tested in the BACTEC 460-radiometric
system. Compounds effecting < 90% inhibition in the primary
screen (MIC > 6.25 μg/ml) are not generally evaluated further.
Table 3
Compounds of the examining set and their activities (predicted)
Compounds
T1
R
Ar
Activity
+
Compounds
T7
R
Ar
Activity
–
–OCH3
–OCH3
T2
T3
T4
T5
T6
–CH3
–NH2
–N(CH3)2
–OH
+
+
+
+
+
T8
–CH3
–NH2
–N(CH3)2
–OH
–
–
–
–
–
T9
T10
T11
T12
–CN
–CN

K.-K. Bedia et al. / European Journal of Medicinal Chemistry 41 (2006) 1253–1261
1259
Determination of MIC: Compounds demonstrating at least
90% inhibition in the primary screen are re-tested at lower con-
centrations against M. tuberculosis H37Rv to determine the
actual MIC in the MABA. The MIC is defined as the lowest
concentration effecting a reduction in fluorescence of 90%
relative to controls.
C12H8N4O5S: C, 45.00; H, 2.52; N, 17.49; S, 10.01. Found: C,
44.99; H, 2.40; N, 17.37; S, 9.58. MS(ES): m/z 321 (MH+).
5.4.4. 4-Fluorobenzoic acid [(3-
chlorophenyl)methylene]hydrazide (9)
Yield 80%, m.p. 165 °C. 1H-NMR (DMSO-d6, 400 MHz) δ
(ppm) 7.38 (t, 2H, ortho-protons to F), 7.49 (d, 2H, J: 4.31 Hz,
H4 and H6 protons), 7.70 (t, 1H, H5 proton), 7.79 (s, 1H, H2
proton), 7.99–8.02 (m, 2H, meta-protons to F), 8.43 (s, 1H,
=C–H), 12.02 (s, 1H, –NH). Anal. Calcd. for C14H10ClFN2O:
C, 60.77; H, 3.64; N, 10.12. Found: C, 59.22; H, 4.18; N, 9.58.
MS(ES): m/z 277 (MH+), 279 (MH+ + 2).
5.2. Synthesis of 4-substituted phenyl benzoate (1)
To the solution of 0.1 mol of phenol in 100 ml of sodium
hydroxide (10%), 0.1 mol of 4-fluorobenzoyl chloride was
added and stirred for 30 min. The solid product was washed
with distilled water and crystallized from ethanol [30].
5.4.5. 4-Fluorobenzoic acid [(4-
bromophenyl)methylene]hydrazide (11)
5.3. Synthesis of 4-substituted benzoic acid hydrazide (2)
Yield 53%, m.p. 196 °C. 1H-NMR (DMSO-d6, 400 MHz) δ
(ppm) 7.35–8.01 (m, 8H, ArH), 8.42 (s, 1H, =CH–), 11.93 (s,
1H, –NH). Anal. Calcd. for C14H10BrFN2O.H2O: C, 49.70; H,
3.55; N, 8.28. Found: C, 49.14; H, 3.40; N, 8.25. MS(ES): m/z
321 (MH+), 323 (MH+ + 2).
To the solution of 0.05 mol of 1 in 3 ml of methanol was
added 0.1 mol of 99% hydrazine hydrate. The mixture was
refluxed on a water bath for 30 min. After cooling, the preci-
pitate was collected, washed with distilled water, and recrystal-
lized from ethanol [18].
5.4.6. 4-Fluorobenzoic acid [(2-
fluorophenyl)methylene]hydrazide (12)
5.4. Synthesis of 4-substitutedbenzoic acid
(substitutedmethylene) hydrazides (3–31)
Yield 80%, m.p. 215 °C. 1H-NMR (DMSO-d6, 400 MHz) δ
(ppm) 7.95–8.04 (m, 8H, ArH), 8.60 (s, 1H, –CH=), 11.89(s,
1H, –NH). Anal. Calcd. for C14H10F2N2O: C, 64.61; H, 3.87;
N, 10.76. Found: C, 63.92; H, 3.74; N, 10.65. MS(ES): m/z
261 (MH+).
A solution of 0.005 mol of substituted aldehydes in ethanol
was added a solution of 0.005 mol of 2a–e and isoniazide in
50 ml of ethanol. The mixture was refluxed on a water bath for
2–2.5 h. After cooling the mixture, the precipitate was filtered,
dried and recrystallized from ethanol [11,18].
5.4.7. 4-Fluorobenzoic acid [(4-
chlorophenyl)methylene]hydrazide (14)
5.4.1. 4-Fluorobenzoic acid [((5-nitro)thiophen-2-yl)
methylene]hydrazide (3)
Yield 75%, m.p. 182 °C. 1H-NMR (DMSO-d6, 400 MHz) δ
(ppm) 7.38 (t, 2H, ortho-protons to F), 7.54 (d, 2H, J: 8.28 Hz,
ortho-protons to Cl), 7.77 (d, 2H, J: 8.28 Hz, meta-protons to
Cl), 7.98–8.02 (m, 2H, meta-protons to F), 8.45 (s, 1H, =C–H),
11.95 (s, 1H, –NH). Anal. Calcd. for C14H10ClFN2O: C, 60.77;
H, 3.64; N, 10.12. Found: C, 59.80; H, 3.60; N, 9.55. MS(ES):
m/z 277 (MH+), 279 (MH+ + 2).
Yield 87%, m.p. 275 °C. 1H-NMR (DMSO-d6, 400 MHz) δ
(ppm) 7.39 (t, 2H, ortho-protons to F), 7.59 (d, 1H, J: 4.22 Hz,
H3 proton of thiophen), 7.95–8.05 (m, 2H, meta-protons to F),
8.15 (d, 1H, J: 4.33 Hz, H4 proton of thiophen), 8.67 (s, 1H,
=C–H), 12.25 (s, 1H, –NH). Anal. Calcd. for C12H8FN3O3S:
C, 49.14; H, 2.73; N, 14.33; S, 10.92. Found: C, 48.97; H,
2.63; N, 14.15; S, 10.46. MS(ES): 294 (MH+).
5.4.8. 4-Fluorobenzoic acid [(4-
dimethylaminocinnamyl)methylene]hydrazide (16)
5.4.2. 4-Fluorobenzoic acid [(5-bromothiophen-2-yl)
methylene]hydrazide (4)
Yield 92%, m.p. 235–237 °C. 1H-NMR (DMSO-d6,
400 MHz) δ (ppm) 2.96 (s, 6H, –CH3 protons of tert.amine),
6.72 (d, 2H, J: 8.75 Hz, ortho-protons to tert.amine), 6.76–6.82
(2d, 1H, =CH–CH=CH–), 6.92 (d, 1H, =CH–CH=CH–), 7.36
(t, 2H, ortho-protons to F), 7.45 (d, 1H, J: 8.75 Hz, meta-
protons to tert.amine), 7.95–7.98 (m, 2H, meta-protons to F),
8.16 (d, 1H, J: 9.17 Hz, –CH=), 11.59 (s, 1H, –NH). Anal.
Calcd. for C18H18FN3O: C, 70.13; H, 6.20; N, 12.91. Found:
C, 69.52; H, 5.76; N, 13.40. MS(ES): m/z 312 (MH+).
Yield 93%, m.p. 190 °C. 1H-NMR (DMSO-d6, 400 MHz) δ
(ppm) 7.19–7.89 (m, 6H, ArH), 8.49 (s, 1H, =C–H), 11.81 (s,
1H, –NH). Anal. Calcd. for C12H8BrFN2OS.H2O: C, 41.73; H,
2.89; N, 8.11; S, 9.27. Found: C, 41.65; H, 2.57; N, 8.06; S,
9.41. MS(ES): m/z 327 (MH+), 329 (MH+ + 2).
5.4.3. 4-Nitrobenzoic acid [((5-nitro)thiophen-2-yl)methylene]
hydrazide (5)
Yield 84%, m.p. 240 °C. 1H-NMR (DMSO-d6, 400 MHz) δ
(ppm) 7.64 (d, 1H, J: 4.27 Hz, H3 proton of thiophen),
8.14–8.16 (m, 3H, H4 proton of thiophen and meta-protons to
NO2), 8.40 (d, 2H, J: 8.66 Hz, ortho-protons to NO2), 8.69 (s,
1H, =C–H), 12.51 (s, 1H, –NH). Anal. Calcd. for
5.4.9. 4-Fluorobenzoic acid [(thiophen-2-yl)methylene]
hydrazide (17)
Yield 90%, m.p. 238 °C. 1H-NMR (DMSO-d6, 400 MHz) δ
(ppm) 7.16 (t, 1H, H4 proton of thiophen), 7.37 (t, 2H, ortho-
protons to F), 7.49 (d, 1H, J: 3.13 Hz, H3 proton of thiophen),

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K.-K. Bedia et al. / European Journal of Medicinal Chemistry 41 (2006) 1253–1261
7.69 (d, 1H, J: 4.81 Hz, H5 proton of thiophen), 7.96–8.00 (m,
2H, meta-protons to F) 8.66 (s, 1H, =C–H), 11.83 (s, 1H, –
NH). Anal. Calcd. for C12H9FN2OS: C, 58.05; H, 3.65; N,
11.28; S, 12.92. Found: C, 57.74; H, 3.46; N, 11.30; S,
12.78. MS(ES): m/z 249 (MH+).
5.4.15. 4-Fluorobenzoic acid [(2-furanyl)methylene]hydrazide
(24)
Yield 82%, m.p. 193–194 °C. 1H-NMR (DMSO-d6,
400 MHz) δ (ppm) 6.56 (s, 1H, H4 proton of furan), 6.86 (d,
1H, J: 3.10 Hz, H3 proton of furan), 7.28 (t, 2H, ortho-protons
to F), 7.77 (s, 1H, H5 proton of furan), 7.87–7.90 (m, 2H,
meta-protons to F), 8.24 (s, 1H, –CH=), 11.72 (s, 1H, –NH).
Anal. Calcd. for C12H9FN2O2: C, 62.07; H, 3.91; N, 12.06.
Found: C, 61.61; H, 3.58; N, 12.09. MS(ES): m/z 233
(MH+).
5.4.10. 4-Fluorobenzoic acid [(2-
hydroxyphenyl)methylene]hydrazide (18)
Yield 82%, m.p. 190 °C. 1H-NMR (DMSO-d6, 400 MHz) δ
(ppm) 6.96 (t, 2H, ortho-protons to F), 7.29–7.33 (m, 1H, H5
proton), 7.39 (t, 2H, H4 and H6 protons), 7.56 (d, 1H, J:
8.77 Hz, H3 proton), 8.01–8.04 (m, 2H, meta-protons to F),
8.65 (s, 1H, =C–H), 11.25 (s, 1H, –OH), 12.13 (s, 1H, –NH).
Anal. Calcd. for C14H11FN2O2: C, 65.11; H, 4.29; N, 10.85.
Found: C, 64.36; H, 4.15; N, 10.28. MS(ES): m/z 259 (MH+).
5.4.16. 4-Fluorobenzoic acid [(benzyl)methylene]hydrazide
(25)
Yield 40%, m.p. 153 °C. 1H-NMR (DMSO-d6, 400 MHz) δ
(ppm) 4.49 (s, 2H, –CH2–), 7.28–7.88 (m, 9H, aromatic pro-
tons), 8.30 (s, 1H, =CH–), 11.90 (s, 1H, –NH). Anal. Calcd.
for C15H13FN2O: C, 70.30; H, 5.11; N, 10.93. Found: C,
69.29; H, 5.14; N, 10.78. MS(ES): m/z 257 (MH+).
5.4.11. 4-Fluorobenzoic acid [(pyridyl-4-yl)methylene]
hydrazide (19)
Yield 80%, m.p. 224 °C. 1H-NMR (DMSO-d6, 400 MHz) δ
(ppm) 7.28–8.58 (m, 8H, ArH), 8.35 (s, 1H, =C–H), 12.05 (s,
1H, –NH). Anal. Calcd. for C13H10FN3O: C, 64.19; H, 4.14;
N, 17.28. Found: C, 63.34; H, 4.86; N, 17.21. MS(ES): m/z
244 (MH+).
5.4.17. 4-Fluorobenzoic acid [(4-hydroxy-3-methoxyphenyl)
methylene]hydrazide (26)
Yield 82%, m.p. 183 °C. 1H-NMR (DMSO-d6, 400 MHz) δ
(ppm) 3.84 (s, 3H, CH3 protons), 6.85 (d, 1H, J: 8.11 Hz, H5
proton), 7.09 (d, 1H, J: 8.09 Hz, H6 proton), 7.32–7.39 (m, 3H,
H2 proton and ortho-protons to F), 7.96–8.00 (m, 2H, meta-
protons to F), 8.34 (s, 1H, –CH=), 9.57 (s, 1H, –OH), 11.48
(s, 1H, –NH). Anal. Calcd. for C15H13FN2O3: C, 62.50; H,
4.55; N, 9.72. Found: C, 62.25 H; 4.03; N, 9.76. MS(ES): m/
z 289 (MH+).
5.4.12. 4-Fluorobenzoic acid [(4-
hydroxyphenyl)methylene]hydrazide (20)
Yield 65%, m.p. 260 °C. 1H-NMR (DMSO-d6, 400 MHz) δ
(ppm) 6.85 (d, 2H, J: 8.44 Hz, ortho-protons to OH), 7.36 (t,
2H, ortho-protons to F), 7.57 (d, 2H, J: 8.44 Hz, meta-protons
to OH), 7.96–8.00 (m, 2H, meta-protons to F), 8.34 (s, 1H, –
CH=), 9.94 (s, 1H, –OH), 11.67 (s, 1H, –NH). Anal. Calcd. for
C14H11FN2O2: C, 65.11; H, 4.29; N, 10.85. Found: C, 65.29;
H, 4.52; N, 10.95. MS(ES): m/z 259 (MH+).
5.4.18. 4-Fluorobenzoic acid [(4-
fluorophenyl)methylene]hydrazide (27)
Yield 90%, m.p. 184–185 °C. 1H-NMR (DMSO-d6,
400 MHz) δ (ppm) 7.31 (t, 2H, meta-protons to –CONH),
7.37 (t, 2H, meta-protons to –N=CH), 7.78–7.82 (m, 2H,
ortho-protons to –CONH), 7.98–8.02 (m, 2H, ortho-protons
to –N=CH), 8.45 (s, 1H, –CH=), 11.89 (s, 1H, –NH). Anal.
Calcd. for C14H10F2N2O: C, 64.61; H, 3.87; N, 10.76. Found:
C, 64.75; H, 4.04; N, 10.15. MS(ES): m/z 261 (MH+).
5.4.13. 4-Fluorobenzoic acid [(4-hydroxy-3-ethoxyphenyl)
methylene]hydrazide (22)
Yield 66%, m.p. 215 °C. 1H-NMR (DMSO-d6, 400 MHz) δ
(ppm) 1.37 (t, 3H, –CH2CH3), 4.08 (q, 2H, –CH2CH3), 6.87 (t,
1H, H5 proton), 7.09 (d, 1H, J: 8.12 Hz, H6 proton), 7.30 (s,
1H, H2 proton), 7.36 (t, 2H, ortho-protons to F), 7.96–8.00 (m,
2H, meta-protons to F), 8.32 (s, 1H, =CH–), 9.50 (s, 1H, –
OH), 11.68 (s, 1H, –NH). Anal. Calcd. for C16H15FN2O3: C,
63.57; H, 5.00; N, 9.27. Found: C, 63.93; H, 4.85; N, 9.29. MS
(ES): m/z 303 (MH+).
5.4.19. 4-Fluorobenzoic acid [(pyrole-2-yl)methylene]
hydrazide (28)
Yield 92%, m.p. 217 °C. 1H-NMR (DMSO-d6, 400 MHz) δ
(ppm) 6.06 (s, 1H, H4 proton of pyrole), 6.40 (s, 1H, H3 proton
of pyrole), 6.83 (s, 1H, H5 proton of pyrole), 7.26 (t, 2H,
ortho-protons to F), 7.86–7.90 (m, 2H, meta-protons to F),
8.18 (s, 1H, =C–H), 11.45 (s, 2H, –NH of hydrazone and –
NH of pyrole). Anal. Calcd. for C12H10FN3O.H2O: C, 57.83;
H, 4.81; N, 16.87. Found: C, 57.78; H, 4.82; N, 16.96. MS
(ES): m/z 232 (MH+).
5.4.14. 4-Fluorobenzoic acid [(4-
dimethylaminophenyl)methylene]hydrazide (23)
Yield 80%, m.p. 183–184 °C. 1H-NMR (DMSO-d6,
400 MHz) δ (ppm) 2.98 (s, 6H, CH3 protons of tert.amine),
6.76 (d, 2H, J: 8.85 Hz, ortho-protons to tert.amine), 7.35 (t,
2H, ortho-protons to F), 7.55 (d, 2H, J: 8.85 Hz, meta-protons
to tert.amine), 7.96–7.99 (m, 2H, meta-protons to F), 8.31 (s,
1H, –CH=), 11.57 (s, 1H, –NH). Anal. Calcd. for
C16H16FN3O: C, 67.35; H, 5.65; N, 14.73. Found: C, 66.86; H,
5.08; N, 14.03. MS(ES): m/z 286 (MH+).
5.4.20. 2-[(Fluorobenzoyl)hydrazono]-1,3-dihydro-indol-3-
one (29)
Yield 95%, m.p. 283 °C. 1H-NMR (DMSO-d6, 400 MHz) δ
(ppm) 6.87 (d, 1H, J: 7.83 Hz, H7 proton of indole), 7.03 (t,
1H, H6 proton of indole), 7.30–7.39 (m, 3H, ortho-protons to F

K.-K. Bedia et al. / European Journal of Medicinal Chemistry 41 (2006) 1253–1261
1261
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indole), 7.86–7.90 (m, 2H, meta-protons to F), 11.15 (s, 1H, –
NH), 13.89 (s, 1H, –NH of indole). Anal. Calcd. for
C15H10FN3O2: C, 63.60; H, 3.56; N, 14.83. Found: C, 63.58;
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The primary screen is conducted at 6.25 μg/ml (or molar
equivalent of highest molecular weight compound in a series
of congeners) against M. tuberculosis H37Rv (ATCC 27294) in
BACTEC 12B medium using the MABA. Compounds exhibit-
ing fluorescence are tested in the BACTEC 460-radiometric
system. Compounds effecting < 90% inhibition in the primary
screen (MIC > 6.25 μg/ml) are not generally evaluated further.
Determination of MIC: Compounds demonstrating at least
90% inhibition in the primary screen are re-tested at lower con-
centrations against M. tuberculosis H37Rv to determine the
actual MIC in the MABA. The MIC is defined as the lowest
concentration effecting a reduction in fluorescence of 90%
relative to controls.
Determination of 50% inhibitory concentrations (IC50):
Concurrent with the determination of MICs, compounds are
tested for cytotoxicity (IC50) in vero cells at concentrations
less than or equal to 62.5 μg/ml or 10 times the mıc for
m. tuberculosis H37Rv. after 72 hours exposure, viability is
assessed on the basis of cellular conversion of mtt into a for-
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Acknowledgements
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This research was supported by The Research Fund of Mar-
mara University, project number SAG-BGS-270605-0138. We
thank Dr. Joseph A. Maddry from Tuberculosis Antimicrobial
Acquisition and Coordinating Facility (TAACF) Southern
Research Institute for his assistance.
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