Synthesis and evaluation of isonicotinoyl hydrazone derivatives as antimycobacterial and anticancer agents

Article abstract of DOI:10.1007/s00044-013-0632-2

A new series of isonicotinoyl hydrazone derivatives (3a-3o) have been synthesized, characterized and evaluated for in vitro antimycobacterial activity against M. tuberculosis H37Rv and two clinical isolates using tetrazolium microplate assay (TEMA). Some

Full text of DOI:10.1007/s00044-013-0632-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0632-2
ORIGINAL RESEARCH
Synthesis and evaluation of isonicotinoyl hydrazone derivatives
as antimycobacterial and anticancer agents
•
H. S. Naveen Kumar Thaigarajan Parumasivam
•
•
Fatimah Jumaat Pazilah Ibrahim
•
•
Mohd. Zaini Asmawi Amirin Sadikun
Received: 28 December 2012 / Accepted: 20 May 2013
Ó Springer Science+Business Media New York 2013
Abstract A new series of isonicotinoyl hydrazone
derivatives (3a–3o) have been synthesized, characterized
and evaluated for in vitro antimycobacterial activity against
M. tuberculosis H37Rv and two clinical isolates using
tetrazolium microplate assay (TEMA). Some of these
compounds showed moderate to good antimycobacterial
activity at micro molar concentrations. Among them, 3k
and 3m were the most potent analogues with an inhibition
concentration at 0.59 and 0.65 lM, respectively, against M.
tuberculosis H37Rv compared to parent drug, isoniazid
(0.57 lM). Additionally, all the synthesized compounds
were subjected to in vitro anticancer activity against human
colorectal cancer cell lines (HCT 116). Compounds 3b and
3l displayed antiproliferative activity at inhibitory con-
centration 3.1 and 0.29 lM, respectively, when compared
to standard, 5-fluorouracil (5 lM). These results can be
considered as an important start point for the rational
design of new leads for antitubercular and anticancer drug
discovery.
tuberculosis (M.tb) and mostly affects young adults in the
developing world (WHO, 2011). The World Health Orga-
nization (WHO) reported (in year 2010) an estimate of
8.5–9.2 million cases and 1.2–1.5 million deaths (including
deaths from TB among HIV-positive patients) (WHO). The
emergence of multi-drug resistant (MDR) and extensively
drug-resistant (XDR) strains of M.tb, particularly in settings
where TB patients are co-infected with human immunode-
ficiency virus (HIV), poses a serious threat (Mital et al.,
2006). In spite of the increasing worldwide incidence of TB,
no new drugs have been brought to the market over the past
four decades. According to WHO’s TB treatment guide-
lines, chemotherapy for TB-infected patient is based on the
combined use of multiple chemotherapeutic agents: isoni-
azid (INH), rifampin (RIF), pyrazinamide (PZA), strepto-
mycin (SM) and ethambutol (EMB), over a period of
6–10 months (Whitney and Wainberg, 2002; Ellner et al.,
1991).Therefore, to combat the M.tb and its resistant strains
there is an urgent need to develop novel anti-tubercular
drugs, which are safe and effective.
Keywords Isonicotinoyl hydrazone ꢀ
Antimycobactreial activity ꢀ Anticancer activity ꢀ
Tertrazolium microplate assay
Cancer is a disease characterized by disruption of con-
trolling mechanisms that govern cell proliferation and dif-
ferentiation. Malignancy is caused by abnormalities in cells,
which might be either inherited or due to exposure to
chemicals, radiation, or even infectious agents. The rapid
proliferation of abnormal cells that grows beyond the usual
boundaries, and then invades adjoining parts of the body
and spread to other organs is one of the characteristic fea-
tures of cancer. This process is referred to as metastasis, and
it is the major cause of death from cancer. Lungs, stomach,
liver, colon and breast are the most commonly affected
organs, which lead to death every year (7.9 million deaths).
It was projected that worldwide death toll from cancer is
continuing to rise, with an estimated 12 million deaths by
2030 (WHO, 2012). The colon cancer is the third most
Introduction
Tuberculosis (TB) is a contagious chronic granulomatous
airborne disease ubiquitously caused by Mycobacterium
H. S. Naveen Kumar (&) ꢀ T. Parumasivam ꢀ F. Jumaat ꢀ
P. Ibrahim ꢀ Mohd. Z. Asmawi ꢀ A. Sadikun
School of Pharmaceutical Sciences, University of Science
Malaysia, 11800 Minden, Pulau Pinang, Malaysia
e-mail: naviusm@gmail.com
123

Med Chem Res
common malignancy worldwide (Parkin et al., 2005). The
increase in the death toll year after year due to cancer, have
made clear that there is an urgent need for development of
newer and more powerful chemotherapeutic agents that can
eradicate cancer cells without harming normal tissues.
Isoniazid (INH) showed wide interest because of their
diverse biological and clinical applications. Recent years,
hydrazide–hydrazone derivatives have attracted interest
due to their wide range of applications in medicinal
chemistry. Generally, presence of azometine (–NHN=CH–)
moiety with these compounds contributes to its biological
activity (Rollas and Ku¨c¸u¨kgu¨zel, 2007). Schwab and Tus-
chl (2003) conducted in vitro studies on the toxicity of INH
in different cell lines showed that, INH induces cytotox-
icity via apoptosis, and can lead to a significant disturbance
of the cell cycle in mammalian cells.
reported procedure (Lourenaso et al., 2008). Reaction
mixtures were maintained at room temperature, leading to
the desired compounds (3a–3o) in 60–79 % yields
(Table 1). Progress of the reaction was monitored by thin
layer chromatography, and spots were visualized under UV
light as well as iodine chamber containing iodine vapours.
All the compounds were identified by their UV, IR, NMR,
Mass spectroscopy, X-ray crystallography as well as ele-
mental analysis. The elemental analysis results were within
0.4 % of the theoretical values.
In general, IR spectra showed the C=O peak at 1634–1687
cm-1, NH stretching vibrations at 3052–3290 cm-1 and N=C
stretching at 1581–1630 cm-1. In the nuclear magnetic res-
onancespectra (1H NMR) the signals of the respectiveprotons
of the synthesized compounds were verified on the basis of
their chemicalshifts, multiplicities and coupling constants (J).
The spectra showed the hydrazide (NH) proton as a singlet
around d 12.54–11.73 ppm and the imine proton (N=C–H) at
d 9.48–8.38 ppm. The 13C NMR spectrum showed the C=O
signals between d 163.4–158.0 ppm and C=N signals d
149.2–142.9 ppm. The crystals suitable for the X-ray crys-
tallography analysis were grown using different solvents by
slow evaporation. The X-ray crystallographic structures of
most of the synthesized compounds were reported (Nav-
eenkumar et al., 2009; 2010a,b,c,d,e,f,g,h,i,j). The lipophi-
licitiesoftheall synthesizedcompoundsandthestandarddrug
(INH) were expressed as clog P values, and were determined
using ChemBioDraw ultra 11.0 software (Table 2). All the
compounds exhibited higher lipophilicity compared to INH.
Recently, several novel hydrazine and hydrazones deriva-
tives showed strong in vitro anticancer activity on carcinoma
cells (Demirbas et al., 2004; Sztanke et al., 2008; Vicini et al.,
2006). Apart from antitumor activity, several hydrazone
derivatives also exhibited a broad spectrum of biological
activity, suchasantimicrobial, anticonvulsant, antidepressant,
˘
analgesic, anti-inflammatory (Kaymakc¸ıoglu et al., 2009)
and antimycobacterial activity (Maccari et al., 2005; Sinha
˘
et al., 2005; Kaymakc¸ıoglu et al., 2006; Gurkok et al., Gur-
kok et al. 2009; Pavan et al., 2010).
Prompted by the above discussion, our rationale was to
synthesize and characterise new isonicotinoyl hydrazone
derivatives and examine their efficacy against M.tb H37Rv
and two drug sensitive virulent clinical isolates strains
(MTB-1 and MTB-2). Simultaneously, we also present the
structure activity relationship and in vitro antiproliferative
activity against human colorectal cancer cell line (HCT 116).
Pharmacology
Antimycobacterial activity
The antimycobacterial activities of compounds 3a–o were
assessed against M.tb H37Rv (ATCC-25618) and two
clinical isolates (MTB-1 and MTB-2), using calorimetric
tetrazolium microplate assay method (TEMA) in triplicate
with slight modification. This methodology is found to be
reliable, faster and inexpensive method for the determina-
tion MICs (minimum inhibitory concentration) of M.tb
strains (Mshana et al., 1998; Caviedes et al., 2002). The
Results and discussion
Chemistry
The synthesis of isonicotinoyl hydrazone derivatives
involved the reaction (Fig. 1) between appropriately
substituted benzaldehydes and isoniazid, by previously
R
CHO
O
O
N
NHNH2
N
H
R
N
EtOH/H2O
N
Isoniazid
3a-o
Fig. 1 Synthetic route used for the preparation of isonicotinoyl hydrazone derivatives (3a–o)
123

Med Chem Res
Table 1 Chemical structures and physical constants of isonicotinoyl hydrazone derivatives (3a–o)
Sample code
Chemical structure
Melting point (°C)
Yield (%)
79
3a
238–239
O
O
O
N
N
H
N
O
3b
186–187
75
N
N
H
N
O
OH
O
3c
3d
3e
215–217
209–210
196–197
69
74
68
O
N
N
H
N
F
F
F
O
C
N
N
H
N
O
N
N
H
N
O
O
N
N
3f
194–196
206–208
177–179
69
67
73
O
H
N
O
O
O
3g
3h
O
N
N
H
N
O
O
N
N
H
N
123

Med Chem Res
Table 1 continued
Sample code
Chemical structure
Melting point (°C)
Yield (%)
78
3i
176–178
F
F
F
O
N
N
H
F
F
N
F
3j
211–213
73
O
O
N
N
H
N
O
O
N
3k
3l
201–203
258–260
76
70
N
H
N
O
O
N
N
H
N
OH
O
3m
3n
191–193
240–242
72
78
N
N
H
N
F
OH
OH
O
C
N
N
H
N
OH
HO
OH
O
C
N
3o
238–240
72
N
H
N
OH
MICs of all the individual compounds were reported in
Table 2. The MIC was defined as the lowest drug con-
centration, required for the complete inhibition of bacterial
growth and expressed in lM (micro molar).
The results showed that compounds (3a–o) exhibited
antimycobacterial activity between 18.29 and 0.28 lM
against all the tested stains. Synthesized derivatives [3f, 3g,
3h, 3j, 3k and 3m] and INH were equipotent against the
tested M.tb H37Rv and two clinical isolates. Compound 3k
was the most potent derivative with MIC 0.59 lM against
M.tb, and 0.28 lM against MTB-1&2. Unfortunately, clog
P values haven’t shown a linear correlation with the
biological activity in this series of compounds. This result
indicated that lipophilicity of the tested compounds is not
sole criteria affecting their anti-tubercular activity.
From the results of in vitro antimycobacterial activity
the following conclusions regarding structure activity
relationship (SAR) can be drawn:
1. We observed that, in most of the active compounds (3f,
3g, 3j, and 3k), the presence of electron donating
groups (–OCH3, –CH3, –OCH2CH3) on the substituted
aromatic ring increased the anti-tubercular activity.
These results were in concordance with the findings of
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Med Chem Res
Table 2 The in vitro activity of
isonicotinoyl hydrazone
derivatives (3a–o) against M.tb
H37Rv strain (ATCC-25618,
two clinical isolates) and
anticancer activity (HCT 116
Cells)
Compound
MICa
MIC Clinical isolates (lM)
IC50 (lM)
clog Pb
M.tb H37Rv (lM)
MTB-1
MTB-2
(HCT 116 cell lines)
3aa
3ba
3c
[13.83
3.77
13.83
1.9
13.83
0.93
0.53
0.57
1.15
0.24
0.24
0.35
0.21
0.26
0.28
[17.16
0.32
9.14
4.57
0.28
–
17.2
3.1
3.13
3.02
1.28
1.61
2.51
0.54
0.89
1.52
2.58
1.78
1.78
3.24
1.4
2.11
1.04
1.11
1.15
0.5
13.4
23.9
17.1
22.3
25.9
15.5
11.6
16
3da
2.25
3e
3fa
3ga
3ha
3ia
3ja
3ka
4.67
0.98
0.98
0.5
1.42
0.35
3.46
0.53
0.28
17.16
0.65
9.14
4.57
0.28
–
13.84
1.03
MIC minimum inhibitory
concentration, IC50 median
lethal dose against Human
colorectal cancer cell lines
(HCT 116), 5-FU 5-fluorouracil
0.59
13.7
0.29
26.8
10.4
28.4
–
3l
[17.16
0.65
3m
3na
3oa
INH
5-FU
a
[18.29
9.14
0.99
1.55
-0.66
-0.57
X-ray crystal structures were
published in Acta
Crystallographica (section E)
b
clog P was calculated using
Chem BioDraw Ultra 11.00
0.57
–
5
other researchers, who observed similar activity by
introduction of –OCH3 group (Emami et al., 2008;
Ferreira et al., 2008).
activity compared to ethoxy group (compound 3k) at
the same position.
6. Slight decrease in the antitubercular activity was
observed (compound 3j), when the methyl group at
3rd position of aromatic ring was replaced with a
methoxy group (compound 3f).
2. In contrast with above fact, the electron withdrawing
group (–F) present at ortho position (compound 3m) of
phenyl ring showed an increase in antimycobacterial
activity. The role of electron withdrawing group in
improving activity is supported by the earlier studies
(Lourenaso et al., 2008; Junior et al., 2005; Sharma
et al., 2004). On the other hand, additional fluoro groups
at position 4th and 5th position of substitute ring
(compound 3d) had witnessed a decrease in the activity.
3. The replacement of the phenyl ring with aliphatic
chain (compound 3h), gave the molecule with highly
improved antimycobacterial activity. This is similar to
one of a previous report by Narasimhan et al., (2007),
in which an increase in antimicrobial activity was
observed with long aliphatic chain compounds.
Anticancer activity
All the synthesized isonicotinoyl hydrazone derivatives
(3a–o) were evaluated for their in vitro anticancer effect via
the standard MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide) (Mosmann, 1983), against
human colorectal cell lines (HCT 116). MTT assay is a
standard colorimetric assay for measuring cell growth.
Compound 5-fluorouracil (5FU) was used as positive con-
trol. IC50 values (in lM) were summarized in Table 2, to
represent the concentration inducing a 50 % decrease of cell
growth after 72 h incubation. HCT116 cells were treated
with 5 lM of 5FU alone for 72 h. Similarly, HCT 116 cell
lines were continuously exposed to test compounds 3a–3o
for 72 h, and their effects on cellular viability were calcu-
lated. The profiles of percentage inhibition of cell prolifer-
ation against the concentration of the test compound were
established to calculate IC50 values of each derivative.
Percentage inhibition of cell proliferation was determined at
different concentrations of the test compounds ranging from
1.5625 to 100 lM. Figures 2a, b, c and 3a, b, c, depict the
4. The replacement of –OH group from 3rd position of the
phenyl ring (compound 3n) to 6th position (compound
3o) showed a significant increase in antimycobacterial
activity. The presence of the –OH group at ortho position
(compound 3l, 3n and 3o) showed decreased in anti-
tubercular activity of the compounds, which was in
contrast with Tripathi et al. (2006), who stated that
the –OH group at ortho position leads to a measurable
change in the activity of the compounds.
5. The presence of benzoyloxy group at ortho position of
aromatic ring (compound 3b) showed decreased
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Med Chem Res
Fig. 2 MTT assay results of isonicotinoyl hydrazone derivatives
against the HCT 116 cell line. a Compounds 3a–e. b Compounds 3f–
j. c Compounds 3k–o
Fig. 3 The effects of increasing concentration of isonicotinoyl
hydrazone derivatives on the percentage inhibition of cell prolifer-
ation. a Compounds 3a–e. b Compounds 3f–j. c Compounds 3k–o
effect of concentration of test compounds on HCT 116 cell
lines after 72 h incubation time.
Conclusion
All the tested compounds displayed moderate to excel-
lent antiproliferative effect against tested cell line, with
IC50 values in the range 0.29–28.4 lM. Among them,
compounds 3b and 3l exhibited excellent antitumor activity
(IC50 = 3.1 and 0.29 lM, respectively). Interestingly, 3l
being 17 times more potent than standard 5FU could be
easily used as a lead to be developed as a novel more
potent anticancer agent. Overall, the results obtained from
this study showed a limited correlation between the lipo-
philicity (clog P) and anticancer properties of the synthe-
sized compounds.
In the search for effective and selective antitubercular and
anticancer agents, we synthesized a series of isonicotinoyl
hydrazone derivatives. All the newly synthesized derivatives
were characterized and evaluated for in vitro antimycobac-
terial and anticancer activity. The antimycobacterial results
indicated that compounds 3k and 3m found to be most
effective against both M.tb H37Rv and two clinical isolates
compared to INH. The anticancer screening results of this
series of compounds indicated that compounds 3b and 3l
displayed superior antiproliferative activity in the low micro
molar range as compared to 5FU against HCT116 cell line.
123

Med Chem Res
Analytical data for compounds 3a–3o
These compounds looks like promising lead for further
modification for the design and development of more supe-
rior antimycobacterial and anticancer agents with high
selectivity and enhanced activity.
(E)-N0-(3-(benzyloxy-4-methoxybenzylidene) isonicotinohyd-
razide (3a) Pale yellow solid; UV (kmax):328 nm. IR
(cm-1; KBr) 3289(NH), 1671(C=O), 1597(C=N). 1H NMR
(DMSO-d6, 400 MHz) d:11.96 (s, 1H, CONH), 8.79–8.77
[dd, 2H, J = 5.88 Hz, 2 Hz, (Pr–a–CH)], 8.38 (s, 1H,
N=CH), 7.82–7.80 [dd, 2H, J = 5.92 Hz, 2.16 Hz, (Pr–b–
CH)], 7.50–7.27 (m, 7H, Ar–H), 7.10–7.07 [d, 1H,
J = 11.2 Hz, (Ar–H)], 5.15 (s, 2H, O–CH2), 3.83 (s, 3H,
CH3). 13C NMR (DMSO-d6, 400 MHz) d: 162.26, 152.22,
151.16, 150.06, 148.99, 141.50, 137.74, 129.27, 128.76,
128.72, 127.54, 123.13, 122.35, 112.35, 111.44, 70.84,
56.57. MS–ESI m/z(%)-[M?H]? 362.20 (100). Anal.
Calcd. for C21H19N3O3 (361.14) C, 69.79; H, 5.30; N,
11.63; found C, 69.71; H, 5.15; N, 11.70.
Experimental
Chemistry
All substituted benzaldehydes were obtained from Aldrich
Chemical Company (USA). HPLC grade solvents were
used for the reaction. Reagents and all solvents were ana-
lytically pure and were used without further purification.
All the melting points (m.p. °C) were uncorrected and
determined in open capillary tubes using digital melting
point apparatus 9100 Electrothermal (UK). Pre-coated
aluminium sheets silica gel 60 F254 (Merck, Darmstadt
Germany), TLC plates were used for monitoring the pro-
gress of the reaction, using dichloromethane/methanol as a
mobile phase. UV spectra were taken dissolving com-
pounds in methanol and recorded by using a Perkin-Elmer
Lambda 45 UV spectrophotometer (USA). Infrared (FT-
IR) spectra were recorded on Nicolet FT-IR Spectrometer
(USA) apparatus, using the KBr pellets method and
intensity of signals reported in wave numbers (cm-1).
Nuclear Magnetic resonance was recorded on Bruker
Fourier transform instrument in dimethyl sulfoxide-d6,
recorded at 400 MHz (1H NMR and 13C NMR). The
chemical shifts are reported in parts per million delta (d)
using tetramethylsilane (TMS) as reference standard. Mass
spectra were recorded with Agilent 1100 Series LC/MS
(liquid chromatography/mass spectrometry) using electro-
spray ionization (ESI). The elemental analyses were
recorded on Perkin-Elmer 2400 Series II CHN Elemental
Analyzer. Names for all of the new compounds were given
with the help of ChemBiodraw ultra 11.0.
(E)-N0-(2-(benzyloxy) benzylidene) isonicotinohydrazide
(3b) White solid; UV (kmax): 328 nm. IR (cm-1; KBr)
3228 (NH), 1670 (C=O), 1597(C=N). 1H NMR (DMSO-d6,
400 MHz) d:12.11 (s, 1H, –CONH), 8.83 (s, 1H, N=CH),
8.77–8.75 [dd, 2H, J = 5.88 Hz, 2.20 Hz (Pr–a–CH)],
7.93–7.90 [dd, 1H, J = 10.32 Hz, 2.24 Hz,(Ar–H)],
7.82–7.80 [dd, 2H, J = 5.88 Hz, 2.20 Hz (Pr–b–CH)],
7.53–7.32(m, 6H, Ar–H), 7.24–7.21(d, 1H, J = 10.52,(Ar–
H)), 7.07–7.02 [t, 1H, J = 9.82 Hz, (Ar–H)], 5.20 (s, 2H,
O–CH2). 13C NMR (DMSO-d6, 400 MHz) d:158.02,
151.01,145.38, 137.66,132.47, 129.34, 128.78, 128.56,
126.80, 123.69, 122.38, 121.95,114.36, 70.99. MS–ESI m/
z (%)-[M?Na]? 354.10(100).Anal. Calcd for C20H17 N3O2
(331.13) C, 72.49; H, 5.17; N, 12.68; found C, 72.83; H,
4.91; N, 12.81.
(E)-N0-((E)-3-(4-hydroxy-3-methoxyphenyl) allylidene)ison-
icotinohydrazide(3c) Yellow solid; UV (kmax): 353 nm.
IR (cm-1; KBr) 3228 (NH), 1650 (C=O), 1597 (C=N).
1HNMR (DMSO-d6, 400 MHz) d: 11.86 (s, 1H, CONH),
9.41 (s, 1H, OH), 8.78–8.76 [dd, 2H, J = 4.56 Hz, 1.4 Hz,
(Pr–a–CH)], 8.21–8.18 (dd, 1H, J = 6 Hz, 2.66 Hz,
N=CH), 7.81–7.79 [dd, 2H, J = 4.46 Hz, 1.58 Hz, (Pr–b–
CH)], 7.25 (s, 1H, Ar–H), 7.04–7.01 [dd, 1H, J = 8.18 Hz,
1.70 Hz, (Ar–CH)], 6.97–6.96 (m, 2H, =CH), 6.79–6.77 [d,
1H, J = 8.12 Hz, (Ar–H)], 3.82 (s, 3H, OCH3). 13C NMR
(DMSO-d6, 400 MHz) d; 162.11,152.36, 151.13, 148.87,
148.78, 141.44, 141.35, 128.36,123.25,122.34, 122.30,
General procedure for the synthesis of isonicotinoyl
hydrazone derivatives (3a–3o)
Isoniazid (1.0 equiv.) was made to react with appropriate
benzaldehyde 2a–2o (1.0 equiv.) in ethanol/water (20 mL).
Initially isoniazid was dissolved in water and a solution of
respective benzaldehyde in ethanol (Fig. 1) solution was
added. The reaction mixture was stirred for 1–5 h at room
temperature. The reaction mixture was concentrated under
reduced pressure, and given a cold ethanol wash followed
by ethyl ether. It was further recrystallized with ethanol to
obtain pure derivatives (3a–3o).
?
116.43, 111.18, 56.51. MS–ESI m/z (%)-[M?H] 298.2
(100). Anal. Calcd for C16H15N3O3 (297.11) C, 64.64; H,
5.09; N, 14.13; found C, 64.56; H, 4.86; N, 14.21.
123

Med Chem Res
(E)-N0-(2,4,5-trifluorobenzylidene) isonicotinohydrazide (3d)
White solid; UV (kmax): 309 nm. IR (cm-1; KBr) 3203
(NH), 1679 (C=O), 1622 (C=N). 1HNMR (DMSO-d6,
400 MHz) d: 12.25 (s, 1H, –CONH), 8.80–8.78 [dd, 2H,
J = 5.9 Hz, 2.18 Hz, (Pr–a–CH)], 8.60 (s, 1H, N=CH),
7.82–7.80 [dd, 2H, J = 5.9 Hz, 2.14 Hz, (Pr–b–CH)],
7.78–7.65 (m, 2H, Ar–H). 13C NMR (DMSO-d6,
400 MHz) d; 162.50, 158.37, 155.89, 151.25, 148.71,
146.29, 140.88, 122.34, 119.90, 114.52, 108.12.MS–ESI
m/z (%)-[M ? H]? 280.0 (100). Anal. Calcd for
C13H8F3N3O (279.06) C, 55.92; H, 2.89; N, 15.05; found
C, 55.53; H, 2.49; N, 14.97.
(E)-N0-((E)-2-methylpent-2-enylidene) isonicotinohydraz-
ide (3h) White solid; UV (kmax): 282 nm. IR (cm-1
;
KBr) 3052 (NH), 1667 (C=O), 1630 (C=N). 1H NMR
(DMSO-d6, 400 MHz) d:11.73 (s, 1H, CONH), 8.77–8.75
[dd, 2H, J = 5.8 Hz, 2.2 Hz,(Pr–a–CH)], 8.03 (s, 1H,
N=CH),7.78–7.76 [dd, 2H, J = 5.8 Hz, 2.2 Hz, (Pr–b–
CH)], 5.94–5.91 J = 4.98 Hz, [t, 1H, (=CH)], 2.29–2.19
[p, 2H, J = 19.84 Hz (–CH2)], 1.8 (s, 3H, –CH3),
1.03–0.98 [t, 3H, J = 10.02 Hz,(CH3)]. 13C NMR (DMSO-
d6, 400 MHz) d; 162.22, 154.90, 151.12, 143.41, 141.57,
133.38, 122.32, 22.11, 14.24, 11.84. MS–ESI m/z (%)-
[M?Na]? 240.10 (100). Anal. Calcd for C12H15 N3O
(217.12) C, 66.34; H, 6.96; N, 19.34; found C, 66.28; H,
6.86; N, 19.54.
(E)-N0-(2,4,6-trimethylbenzylidene) isonicotinohydrazide
(3e) White solid; UV (kmax): 302 nm.IR (cm-1; KBr) 3191
(NH), 1650 (C=O), 1601 (C=N). 1H NMR (DMSO-d6,
400 MHz) d: 11.92 (s, 1H, –CONH), 8.79–8.77 [dd, 2H,
J = 5.86 Hz, 2.22 Hz, (Pr–a–CH)], 8.76 (s, 1H,
N=CH),7.83–7.81 [dd, 2H, J = 5.86 Hz, 2.2 Hz, (Pr–b–
CH)], 6.93(s, 2H, Ar–H), 2.42(s, 3H, CH3), 2.24(s, 3H, CH3),
2.18(s, 3H, CH3). 13C NMR(DMSO-d6, 400 MHz)d; 162.14,
151.16, 150.04, 141.47, 139.44, 138.39, 130.35, 128.74,
123.36, 109.98, 21.91, 21.56. MS–ESI m/z (%)-[M?Na]?
290.10 (100). Anal. Calcd for C16H17 N3O (267.14) C, 71.89;
H, 6.41; N, 15.72; found C, 71.86; H, 6.08; N, 15.76.
(E)-N0-(2, 4-bis (trifluoromethyl) benzylidene) ison-
icotinohydrazide (3i) White solid; UV (kmax): 305 nm.IR
1
(cm-1; KBr) 3187 (NH), 1663 (C=O), 1556 (C=N). H
NMR (DMSO-d6, 400 MHz) d: 12.53 (s, 1H;–CONH),
8.86 (s, 1H, N=CH), 8.82–8.80 [d, 2H, J = 7.92 Hz, (Pr–
a–CH)], 8.46–8.43 [d, 1H, J = 11.28 Hz, (Ar–H)],
8.18–8.15 [d, 1H, J = 11.76 Hz, (Ar–H)], 8.09 (s, 1H,
Ar–H), 7.85–7.83 [dd, 2H, J = 6 Hz, 2.08 Hz, (Pr–b–
CH)]. 13C NMR (DMSO-d6, 400 MHz) d; 162.89, 151.28,
143.22, 140.73, 136.84, 131.09, 130.65, 129.12, 125.94,
123.99, 122.38, 118.83. MS–ESI m/z (%)-[M?Na]?
384.1(100). Anal. Calcd for C15H9 F6N3O (361.06) C,
49.87; H, 2.51; N, 11.63; found C, 49.48; H, 2.12; N, 11.40.
(E)-N0-(2,3,4-trimethoxybenzylidene) isonicotinohydrazide
(3f) White solid; UV (kmax): 305 nm.IR (cm-1; KBr) 3183
(NH), 1679 (C=O), 1614 (C=N). 1H NMR (DMSO-d6,
400 MHz) d:12.01(s, 1H, –CONH), 8.78–8.76 [d, 2H,
J = 7.96 Hz, (Pr–a–CH)], 8.65 (s, 1H, N=CH), 7.84–7.82
[dd, 2H, J = 6 Hz, 2.08 Hz, (Pr–b–CH)], 7.65–7.62 (d, 1H,
J = 11.8 Hz, Ar–H), 6.95–6.92 (d, 1H, J = 11.92 Hz, Ar–
H), 3.85 (s, 3H, –OCH3), 3.84 (s, 3H, OCH3), 3.76 (s, 3H,
OCH3). 13C NMR (DMSO-d6, 400 MHz) d; 162.26, 156.28,
153.65, 151.16, 145.44, 142.39, 141.50, 122.35, 121.54,
120.93, 109.60, 62.70, 61.32, 56.84. MS–ESI m/z (%)-
[M?H]? 316.20(100). Anal. Calcd for C16H17N3O4 (315.12)
C, 60.94; H, 5.43; N, 13.33; found C, 60.95; H, 5.13; N, 13.40.
(E)-N0-(2,4-dimethoxy-3-methylbenzylidene) isonicotinohyd-
razide (3j) White solid; UV (kmax): 324 nm.IR (cm-1
;
KBr) 3207 (NH), 1654 (C=O), 1597 (C=N). 1H NMR
(DMSO-d6, 400 MHz) d: 11.97 (s, 1H, –CONH), 8.78–
8.76 [dd, 2H, J = 5.88 Hz, 2.20 Hz, (Pr–a–CH)], 8.65
(s, 1H, N=CH), 7.83–7.81 [dd, 2H, J = 5.90 Hz, 2.22 Hz,
(Pr–b–CH)], 7.79–7.76 [d, 1H, J = 11.64 Hz, (Ar–H)],
6.92–6.89 [d, 1H, J = 11.84 Hz, (Ar–H)], 3.84 [s, 3H,
(–OCH3)], 3.71 [s, 3H,(–OCH3)], 2.08 [s, 3H, (–CH3)]. 13
C
NMR (DMSO-d6, 400 MHz) d; 162.13, 161.07, 159.28,
151.14, 145.96, 141.47, 124.98, 122.34, 120.26, 119.63,
108.29, 62.90, 56.67, 9.55. MS–ESI m/z (%)-[M?Na]?
322.1(100). Anal. Calcd for C16H17N3O3 (299.13) C,
64.20; H, 5.72; N, 14.04; found C, 64.14; H, 5.31; N, 14.13.
(E)-N0-(2, 4, 5-trimethoxybenzylidene) isonicotinohydraz-
ide (3g) White solid; UV (kmax): 355 nm. IR(cm-1;KBr)
3290 (NH), 1654 (C=O), 1597 (C=N). H NMR (DMSO-
1
d6, 400 MHz) d :11.92 (s, 1H, –CONH), 8.77–8.76 [dd,
2H, J = 4.5 Hz, 1.52 Hz, (Pr–a–CH)], 8.75(s, 1H, N=CH),
7.83–7.82 [dd, 2H, J = 4.5 Hz, 1.58 Hz, (Pr–b–CH)], 7.36
(s, 1H, Ar–H), 6.76 (s, 1H, Ar–H), 3.87–3.76 (m, 9H,
–OCH3).13C NMR (DMSO-d6, 400 MHz) d; 161.98,
154.49, 153.24, 151.11, 145.53, 144.15, 141.43, 122.30,
114.12, 108.64, 98.76, 57.40, 56.79, 56.67. MS–ESI
m/z (%)-[M?H]? 316.10 (100). Anal. Calcd for C16H17
N3O4 (315.12) C, 60.94; H, 5.43; N, 13.33; found C, 60.94;
H, 5.74; N, 13.39.
(E)-N0-(2-ethoxybenzylidene) isonicotinohydrazide (3k)
White solid; UV (kmax): 329 nm.IR (cm-1; KBr) 3187
(NH), 1654 (C=O), 1597 (C=N). 1H NMR(DMSO-d6,
400 MHz) d:12.09 (s, 1H, CONH), 8.83 (s, 1H, N=CH),
8.79–8.77 [dd, 2H, J = 5.88 Hz, 2.20 Hz (Pr–a–CH)],
7.90–7.87 (dd, 1H, J = 10.32 Hz, 2.24 Hz, Ar–H),
7.85–7.83 [dd, 2H, J = 5.86 Hz, 2.22 Hz, (Pr–b–CH)],
7.44–7.38 [td, 1H, J = 10.44 Hz, 2.32 Hz, (Ar–H)],
7.10–7.08 [d, 1H, J = 10.88 Hz, (Ar–H)], 7.04–6.99
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Med Chem Res
(E)-N0-(2,4,6-trihydroxybenzylidene) isonicotinohydrazide
(3o) Brown solid; UV (kmax): 338 nm. IR(cm-1; KBr)
3403 (OH), 3207 (NH), 1634 (C=O), 1581 (C=N). 1H
NMR (DMSO-d6, 400 MHz) d: 12.12 (s, 1H, –CONH),
11.08 (s, 2H, –OH), 9.91 (s, 1H, OH), 8.82 (s, 1H, N=CH),
8.79–8.77 [d, 2H, J = 7.6 Hz, (Pr–a–CH)], 7.84–7.82 [dd,
2H, J = 5.94 Hz, 2.2 Hz, (Pr–b–CH)], 5.85 (s, 2H, Ar–
H).13C NMR (DMSO-d6, 400 MHz) d; 162.81, 161.56,
160.76, 149.21, 151.10, 141.02, 122.18, 100.06, 95.52.
MS–ESI m/z (%)-[M?H]? 274.10 (100). Anal. Calcd for
C13H11N3O4 (273.07) C, 57.14; H, 4.06; N, 15.38; found C,
57.10; H, 4.10; N, 15.34.
[t, 1H, J = 10 Hz, (Ar–H)], 4.15–4.08 [q, 2H,
J = 9.28 Hz, (–CH2)], 1.41–1.36 [t, 3H, J = 9.28 Hz,
(–CH3)]. 13C NMR (DMSO-d6, 400 MHz) d; 162.13, 158.25,
150.99, 145.77, 141.62, 132.51, 126.66, 123.54, 122.37,
121.63, 114.06, 65.02, 15.45. MS–ESI m/z (%)-[M?Na]?
292.1(100). Anal. Calcd for C15H15N3O2 (269.12) C, 66.90;
H, 5.61; N, 15.60; found C, 66.81; H, 5.37; N, 15.64.
(E)-N0-((2-hydroxynaphthalen-1-yl) methylene)isonicotinohyd-
razide (3l) Yellow solid; UV (kmax): 367 nm. IR (cm-1
;
KBr) 3215 (NH), 1679 (C=O), 1626(C=N). 1H NMR
(DMSO-d6, 400 MHz) d:12.54 (s, 1H, CONH), 12.40 (s,
1H, OH), 9.48 (s, 1H, N=CH), 8.84–8.83 [d, 2H,
J = 4.68 Hz, (Pr–a–CH)], 8.32–8.30 [d, 1H, J = 8.48
Hz, (Ar–H)], 7.95–7.90 (m, 2H, Ar–H), 7.89–7.88 [d, 2H,
J = 4.6 Hz, (Pr–b–CH)], 7.64–7.60 (t, 1H, J = 7.64 Hz,
Ar–H), 7.43–7.39 (t, 1H, J = 6.98 Hz, Ar–H), 7.26–7.23
(d, 1H, J = 8.96 Hz, Ar–H).13C NMR (DMSO-d6,
400 MHz) d; 161.90, 159.04, 151.34, 148.45, 140.66,
134.00, 132.46, 129.85, 128.77, 128.72, 124.48, 122.30,
121.81, 119.68, 109.38. MS–ESI m/z (%)-[M?H]? 292.10
(100). Anal. Calcd for C17H13N3O2 (291.10) C, 70.09; H,
4.50; N, 14.42; found C, 70.05; H, 4.17; N, 14.50.
Biological assays
Antimycobacterial activity
Drug solution preparation INH was obtained commer-
cially from Sigma-Aldrich Chemical Company, United
Kingdom. INH stock solution was prepared by dissolving
in distilled water to obtain a concentration of 1 mg mL-1
.
The derivatives were dissolved in DMSO to obtain a stock
solution of 1 mg mL-1. These stock solutions were sub-
sequently diluted with distilled water on the day of
experiment to attain the desired working concentrations
(5–0.0195 lg mL-1) and then sterilised by filtration using
a cellulose membrane of 0.22 lm pore size.
(E)-N0-(2-fluorobenzyliden) isonicotinohydrazide (3m)
Pale yellow solid; UV (kmax): 305 nm. IR (cm-1; KBr)
3187 (NH), 1687(C=O), 1610 (C=N). 1H NMR (DMSO-d6,
400 MHz) d: 12.19 (s, 1H, –CONH), 8.80–8.78 [d, 2H,
J = 5.94 Hz, 2.14 Hz, (Pr–a–CH)], 8.71 (s, 1H, N=CH),
7.98–7.93 [td, 1H, J = 10.12 Hz, 2.24 Hz (Ar–H)],
7.84–7.82 [dd, 2H, J = 5.90 Hz, 2.22 Hz (Pr–b–CH)],
7.67–7.47 (m, 2H, Ar–H), 7.33–7.28 (t, 1H, J = 11.24 Hz,
Ar–H).13C NMR (DMSO-d6, 400 MHz) d; 163.44, 160.12,
151.05, 142.93, 141.43, 133.04, 132.93, 127.49, 125.76,
122.54, 116.66. MS–ESI m/z (%)-[M?H]? 244.0 (100).
Anal. Calcd for C13 H10 FN3O (243.08) C, 64.19; H, 4.14;
N, 17.28; found C, 64.07; H, 3.72; N, 17.25.
Inoculum preparation Mycobacterium tuberculosis,
strain H37Rv (ATCC 25618) and two clinical isolates MTB
1&2 (obtained from School of Medical Sciences, Univer-
sity of Science Malaysia) were used for the in vitro anti-
mycobacterial activity. The mycobacterial inoculums were
prepared from a log phase culture in Middlebrook 7H9
broth (Difco, USA) supplemented with albumin, dextrose,
and catalase (ADC) (Difco, USA), and its turbidity was
adjusted to McFarland standard no. 1. This bacterial sus-
pension was then further diluted 1:25 in Middlebrook 7H9
broth with 0.2 % glycerol and OADC (oleic acid, albumin,
dextrose and catalase) enrichment. (Difco, USA).
(E)-N0-(2, 3, 4-trihydroxybenzylidene) isonicotinohydraz-
ide (3n) Pale brown solid; UV (kmax): 331 nm.IR (cm-1
;
KBr) 3383 (OH), 3256 (NH), 1659 (C=O), 1618 (C=N). 1H
NMR (DMSO-d6, 400 MHz) d: 12.18 (s, 1H, –CONH),
11.29 (s, 1H, –OH), 9.58 (s, 1H, –OH), 8.80–8.78 [dd, 2H,
J = 5.92 Hz, 2.2 Hz, (Pr–a–CH)], 8.56 (s, 1H, OH), 8.49
(s, 1H, N=CH), 7.84–7.82 [dd, 2H, J = 5.88 Hz, 2.2 Hz,
(Pr–b–CH)], 6.85–6.82 [d, 1H, J = 11.4 Hz (Ar–H)],
6.42–6.39 [d, 1H, J = 11.28 Hz, (Ar–H)]. 13C NMR
(DMSO-d6, 400 MHz) d; 162.50, 152.36, 151.14, 149.97,
148.58, 141.05, 133.70, 122.23, 111.81, 108.76. MS–ESI
m/z (%)-[M?Na]? 296.10 (100). Anal. Calcd for C13H11
N3 O4 (273.07) C, 57.14; H, 4.06; N, 15.38; found C,
57.11; H, 4.09; N, 15.36.
In vitro anti-tuberculosis activity screening The antimy-
cobacterial activity was performed by a colorimetric tetra-
zolium microplate assay (TEMA) as described by Caviedes
et al. (2002) with some modifications. The assay was done
in 96-well sterile microplates. Each derivative was tested
thrice in triplicates. First, 200 lL of sterile distilled water
was added into the outer wells to prevent dehydration,
followed by the addition of 100 lL of the Middle brook
7H9 broth supplemented with 10 % OADC was added into
wells 3, until 11 in rows B to G. An amount of 100 lL of
each derivative working solution (10 lg mL-1) was added
in triplicate into wells in columns 2 and 3. The solutions
123

Med Chem Res
were then serially diluted with multichannel pipette from
wells in columns 3 to 4 until wells 10. The last 100 lL of
solutions from wells in column 10 were discarded. Finally,
100 lL of bacterial suspension was added into all the test
wells. The wells in column 11 served as drug-free growth
controls. Isoniazid, a standard anti-TB drug was used as a
positive control. The test concentrations of drugs were
ranged from 5 to 0.0195 lg mL-1. The plates were sealed
and incubated at 37 °C in 8 % CO2, for 5 days. On day 5,
50 lL of tetrazolium–tween 80 mixture [3-(4,5-dimethyl-
thiazol-2-yl)-2,5 diphenyl-tetrazolium bromide with a
concentration of 1 mg mL-1 in absolute ethanol and 10 %
tween 80 at 1:1] was added to well B11, and incubated for
24 h. If well B11 turned from yellow to purple, the reagent
(tetrazolium–tween 80 mixtures) was added to all wells and
incubated for another 24 h and the results were read visu-
ally on the following day. If the well B11 remained yellow,
the plates were incubated for another 24 h, tetrazolium–
tween 80 mixture was added to well C11 and incubated for
additional 24 h. If the C11 remained yellow, incubation was
continued, and tetrazolim–tween 80 mixture was added to
wells D11, E11, F11 and G11 on day 9, 11, 13 and 15,
respectively. The minimum inhibitory concentration there-
fore, was defined as the lowest drug concentration that
prevented a colour change of tetrazolium dye from yellow
to purple.
into the desired concentrations from the stock and the
plates were incubated at 37 °C with an internal atmosphere
of 5 % CO2 for 72 h. After this treatment period, 20 lL of
MTT reagent was added into each well and incubated again
for 4 h, followed by the addition of 50 lL of MTT lysis
solution (DMSO) into the wells. The plates were further
incubated for 5 min in CO2 incubator, and read at 570 and
620 nm wavelengths using a standard ELISA microplate
reader (Ascent Multiskan). Data was recorded and analysed
for the assessment of the effects of test substance on cell
viability and growth inhibition. The percentage of growth
inhibition was calculated from the optical density (OD) that
was obtained from MTT assay, i.e. hundredth multiples of
the subtracted OD value of control and survived cells over
the OD of control cells.
Acknowledgments The authors wish to thank Suriyati Mohamed
and Dr. Siti Suraiya Md. Noor, Department of Medical Microbiology
and Parasitology, School of Medical Sciences, University of Science
Malaysia (USM) for providing MTB clinical isolates. The authors also
wish to thank EMAN Testing and Research Laboratory, School of
Pharmaceutical Sciences, USM for assisting in conducting in vitro
anticancer assay. This research is supported by USM under the
Research University Grant (1001/PFARMASI/815005). Naveen
Kumar and Thaigarajan are thankful to University of Science
Malaysia, for providing USM-fellowship.
References
Anticancer study
Caviedes L, Delgado J, Gilman RH (2002) Tetrazolium microplate
assay as a rapid and inexpensive colorimetric method for
determination of antibiotic susceptibility of Mycobacterium
tuberculosis. J Clin Microbiol 40:1873–1874
Demirbas N, Karaoglu SA, Demirbas A, Sancak K (2004) Synthesis
and antimicrobial activities of some new 1-(5-phenylamino-
[1,3,4] thiadiazol-2-yl) methyl-5-oxo-[1,2,4] triazole and 1-(4-
phenyl-5-thioxo-[1,2,4] triazol-3-yl) methyl-5-oxo-[1,2,4] tria-
zole derivatives. Eur J Med Chem 39:793–804
Ellner JJ, Goldberger MJ, Parenti DM (1991) Mycobacterium avium
infection and AIDS: a therapeutic dilemma in rapid evolution.
J Infect Dis 163:1326–1335
Emami S, Foroumadi A, Lotfali ME, Rajabalian S, Ebrahimi A,
Farahyar S, Shafiee A (2008) 2-Hydroxyphenacyl azoles and
related azolium derivatives as antifungal agents. Bioorg Med
Chem Lett 18:141–146
Ferreira ML, de Cardoso F, Laura N, Goncalves RSB, da Silva ET,
Lourenco MCS, Vicente FR, de Souza MVN (2008) Synthesis
and antitubercular evaluation of N-[(E)-(hydroxy, methoxy and
ethoxy-substituted-phenyl) methylidene] isonicotinohydrazide
derivatives. Lett Drug Des Discov 5:137–140
Gurkok G, Altanlar N, Suzen S (2009) Investigation of antimicrobial
activities of indole-3-aldehyde hydrazide/hydrazone derivatives.
Chemotherapy 55:15–19
Junior IN, Lourenco M, Henriques MGMO, Ferreira B, Vasconcelos
TRA, Peralta MA, de Oliveira PSM, Wardell SMSV, de Souza
MVN (2005) Synthesis and anti-mycobacterial activity of
N-[(E)-(disubstituted-phenyl) methylidene]isonicotino-hydrazide
derivatives. Lett Drug Des Discovery 2:563–566
Preparation of cell culture Initially, cells (HCT 116) were
allowed to grow under optimal incubator conditions. Cells
that have reached a confluency of 70–80 % were chosen for
cell plating purposes. The old medium was aspirated out of
the plate, and the cells were washed 2–3 times using sterile
phosphate buffered saline (PBS) (pH 7.4). Following this,
trypsin was added and distributed evenly onto cell surfaces.
Cells were incubated at 37 °C in 5 % CO2 for 1 min. Then,
the flasks containing the cells were gently tapped to aid cell
segregation and observed under an inverted microscope (if
cells segregation is not satisfying, the cells will be incubated
for another minute). Trypsin activity was inhibited by adding
5 mL of fresh complete media (10 % FBS). Cells were
counted and diluted to get a final concentration of
2.5 9 105 cells mL-1, and inoculated into wells (100 lL
cells/well). Finally, plates containing the cells were incu-
bated at 37 °C with an internal atmosphere of 5 % CO2.
MTT Assay Cancer cells (100 lL cells/well, 1.5 9 105
cells mL-1) were inoculated in wells of microtitre plates.
Then the plate is incubated in a CO2 incubator for over-
night in order to allow the cell for attachment. One hundred
micro litre of test substance was added into each well
containing the cells. Test substance was diluted with media
˘
Kaymakc¸ıoglu BK, Oruc E, Unsalan S, Kandemirli F, Shvets N,
Rollas S, Dimoglo A (2006) Synthesis and characterization of
123

Med Chem Res
novel hydrazide–hydrazones and the study of their structure
antituberculosis activity. Eur J Med Chem 41:1253–1261
methoxyphenyl)allylidene]isonicotinohydrazide. Acta Crystal-
logr Sect E: Struct Rep Online 66:o291–o291
˘
Kaymakc¸ıoglu BK, Emre EEO, Unsalan S, Rollas
S
(2009)
Naveenkumar HS, Sadikun A, Ibrahim P, Yeap CS, Fun HK (2010h)
(E)-N’-(2,4,5-Trifluoro benzylidene)isonicotinohydrazide mono-
hydrate. Acta Crystallogr Sect E: Struct Rep Online 66:o579–
o579
Naveenkumar HS, Sadikun A, Ibrahim P, Yeap CS, Fun HK (2010i) (E)-
N’-(2,4,5-Trimethoxybenzylidene)isonicotinohydrazide dihydrate.
Acta Crystallogr Sect E: Struct Rep Online 66:o1235–o1236
Naveenkumar HS, Sadikun A, Ibrahim P, Yeap CS, Fun HK (2010j) Bis
{(E)-N’-[2,4-bis(trifluoro methyl)benzylidene]isonicotinohyd-
razide}monohydrate. Acta Crystallogr Sect E: Struct Rep Online
66:o1918–o1919
Antituberculosis activity of hydrazones derived from 4-fluoro-
benzoic acid hydrazide. Med Chem Res 18:277–286
Lourenaso MCS, Ferreira ML, de Souza MVN, Peralta MA,
Vasconcelos TRA, Henriques MGMO (2008) Synthesis and
antimycobacterial activity of (E)-N0-(mono substituted benzyli-
dene)isonicotinohydrazide derivatives. Eur J Med Chem 43:
1344–1347
Maccari R, Ottana R, Vigorita MG (2005) In vitro advanced
antimycobacterial screening of isoniazid related hydrazones,
hydrazides and cyanoboranes: part 14. Bioorg Med Chem Lett
15:2509–2513
Mital A, Negi VS, Ramachandran U (2006) Synthesis and antimy-
cobacterial activities of certain trifluoromethyl-aminoquinoline
derivatives. ARKIVOC 10:220–227
Mosmann T (1983) Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxicity assays.
J Immunol Methods 65:55–63
Parkin DM, Bray F, Ferlay J, Pisani P (2005) Global cancer statistics,
2002. CA Cancer J Clin 55:74–108
Pavan FR, da S. Maia PI, Leite SRA, Deflon VM, Batista AA, Sato
DN, Franzblau SG, Leite CQF (2010) Thiosemicarbazones,
semicarbazones, dithiocarbazates and hydrazide/hydrazones:
anti mycobacterium tuberculosis activity and cytotoxicity. Eur
J Med Chem 45:1898–1905
˚
Mshana RN, Tadesse G, Abate G, Hakan M (1998) Use of 3-(4,5-
Rollas S, Ku¨c¸u¨kgu¨zel S¸G (2007) Biological activities of hydrazone
derivatives. Molecules 12:1910–1939
dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide for
rapid detection of rifampin-resistant Mycobacterium tuberculo-
sis. J Clin Microbiol 36:121–1214
Schwab CE, Tuschl H (2003) In vitro studies on the toxicity of
isoniazid in different cell lines. Hum Exp Toxicol 22:607–615
Sharma P, Rane N, Gurram VK (2004) Synthesis and QSAR studies
of pyrimido(4,5-d)pyrimidine-2,5-dione derivatives as potential
antimicrobial agents. Bioorg Med Chem Lett 14:4185–4190
Sinha N, Jain S, Tilekar A, Upadhayaya RS, Kishore N, Jana HG
(2005) Synthesis of isonicotinic acid N0-arylidene-N-[2-oxo-2-
(4-aryl-piperazin-1-yl)-ethyl]-hydrazides as antituberculo sis
agents. Bioorg Med Chem Lett 15:1573–1576
Narasimhan B, Narang R, Judge V, Ohlan S, Ohlan R (2007) Synthesis,
antimicrobial and QSAR studies of substituted anilides. ARKI-
VOC 15:112–126
Naveenkumar HS, Sadikun A, Ibrahim P, Loh WS, Fun HK (2009)
(E)-N0-(3-Benzyloxy-4- methoxy benzylidene)isonicotinohyd-
razide. Acta Crystallogr Sect E: Struct Rep Online 65:o2540–
o2541
Naveenkumar HS, Sadikun A, Ibrahim P, Goh JH, Fun HK (2010a)
(E)-N’-(2,4,6-Trimethyl benzylidene)isonicotinohydrazide. Acta
Crystallogr Sect E: Struct Rep Online 66:o1493–o1493
Naveenkumar HS, Sadikun A, Ibrahim P, Goh JH, Fun HK (2010b)
(E)-N’-(2,3,4-Trihydroxy benzylidene) isonicotinohydrazide
dihydrate. Acta Crystallogr Sect E: Struct Rep Online 66:
o3017–o3018
Naveenkumar HS, Sadikun A, Ibrahim P, Hemamalini M, Fun HK
(2010c) (E)-N’-(2-Benzyloxy benzylidene)isonicotinohydrazide
methanol solvate monohydrate. Acta Crystallogr Sect E: Struct
Rep Online 66:o1337–o1338
Naveenkumar HS, Sadikun A, Ibrahim P, Loh WS, Fun HK (2010d)
(E)-N’-(2, 4, 6-Trihydroxy benzylidene)isonicotinohydrazide
sesquihydrate. Acta Crystallogr Sect E: Struct Rep Online
66:o1202–o1203
Naveenkumar HS, Sadikun A, Ibrahim P, Loh WS, Fun HK (2010e)
(E)-N’-[(E)-2-Methylpent-2-enylidene]isonicotinohydrazide.
Acta Crystallogr Sect E: Struct Rep Online 66:o1489–o1490
Naveenkumar HS, Sadikun A, Ibrahim P, Yeap CS, Fun HK (2010f)
(E)-N’-(2,3,4-Trimethoxy benzylidene) isonicotinohydrazide.
Acta Crystallogr Sect E: Struct Rep Online 66:o1231–o1232
Naveenkumar HS, Sadikun A, Ibrahim P, Quah CK, Fun HK (2010g)
(E)-N’-[(E)-3-(4-Hydroxy-3-
Sztanke K, Tuzimski T, Rzymowska J, Pasternak K, Kandefer-
Szersze AM (2008) Synthesis, determination of the lipophilicity,
anticancer and antimicrobial properties of some fused 1,2,4-
triazole derivatives. Eur J Med Chem 43:404–419
Tripathi RP, Saxena N, Tiwari VK, Verma SS, Chaturvedi V, Manju
YK, Srivastva AK, Gaikwad A, Sinha S (2006) Synthesis and
antitubercular activity of substituted phenylmethyl- and pyridyl-
methyl amines. Bioorg Med Chem 14:8186–8196
Vicini P, Incerti M, Doytchinova IA, La Colla P, Busonera B, Loddo
R (2006) Synthesis and anti proliferative activity of benzo
[d] isothiazole hydrazones. Eur J Med Chem 41:624–632
Whitney JB, Wainberg MA (2002) Isoniazid, the frontline of
resistance in Mycobacterium tuberculosis. McGill J Med 6:
114–123
World Health Organization Cancer Fact Sheet (2012) http://www.
who.int/mediacentre/factsheets/fs297/en/. Accessed 29 Oct 2012
World Health Organization http://www.who.int/topics/tuberculosis/
en/. Accessed 5 Aug 2012
World Health Organization Tuberculosis Fact Sheet (2011)
http://www.who.int/tb/publications/2011/factsheet_tb_2011.pdf.
Accessed 19 Sept 2012
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