Synthesis, antimycobacterial, antiviral, antimicrobial activities, and QSAR studies of isonicotinic acid-1-(substituted phenyl)-ethylidene/cycloheptylidene hydrazides

Article abstract of DOI:10.1007/s00044-011-9705-2

A series of isonicotinic acid-1-(substituted phenyl)-ethylidene/ cycloheptylidene hydrazide derivatives (1-12) was tested for their, in vitro antimycobacterial activity against Mycobacterium tuberculosis, and compound 2 was found to be more active than isoniazid. The antiviral screening results indicated that none of the tested compounds was active against a broad variety of DNA and RNA viruses at subtoxic concentrations, except compounds 8 and 10 that proved to be active against DNA viruses at concentrations close to their cytostatic potential. The synthesized compounds were also screened for their antimicrobial potential against S. aureus, B. subtilis, E. coli, C. albicans and A. niger, and the results indicated that compounds having Br, OCH3 and Cl groups were highly active. The multi-target QSAR models indicated the importance of lipophilic (log P) and topological parameters (3vv) in describing the antimicrobial activity.

Full text of DOI:10.1007/s00044-011-9705-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:1935–1952
DOI 10.1007/s00044-011-9705-2
ORIGINAL RESEARCH
Synthesis, antimycobacterial, antiviral, antimicrobial activities,
and QSAR studies of isonicotinic acid-1-(substituted phenyl)-
ethylidene/cycloheptylidene hydrazides
•
Vikramjeet Judge Balasubramanian Narasimhan Munish Ahuja
•
•
•
Dharmarajan Sriram Perumal Yogeeswari Erik De Clercq
•
•
•
Christophe Pannecouque Jan Balzarini
Received: 7 April 2011 / Accepted: 11 June 2011 / Published online: 9 July 2011
ꢀ Springer Science+Business Media, LLC 2011
Abstract A series of isonicotinic acid-1-(substituted
phenyl)-ethylidene/cycloheptylidene hydrazide derivatives
(1–12) was tested for their, in vitro antimycobacterial
activity against Mycobacterium tuberculosis, and com-
pound 2 was found to be more active than isoniazid. The
antiviral screening results indicated that none of the tested
compounds was active against a broad variety of DNA and
RNA viruses at subtoxic concentrations, except compounds
8 and 10 that proved to be active against DNA viruses at
concentrations close to their cytostatic potential. The syn-
thesized compounds were also screened for their antimi-
crobial potential against S. aureus, B. subtilis, E. coli,
C. albicans and A. niger, and the results indicated that
compounds having Br, OCH3 and Cl groups were highly
active. The multi-target QSAR models indicated the
importance of lipophilic (log P) and topological parameters
(3vv) in describing the antimicrobial activity.
Keywords Hydrazide derivatives ꢀ Antimycobacterial ꢀ
Antiviral ꢀ Antimicrobial ꢀ QSAR
Introduction
During the past few decades, the human population has
been affected with life-threatening infectious diseases
caused by multidrug-resistant Gram-positive and Gram-
negative pathogenic bacteria which had increased at an
alarming rate around the world. For this reason, new
classes of antibacterial agents with novel mechanisms are
crucially needed to combat with the multidrug-resistant
infections (Bayrak et al., 2009a).
Tuberculosis (TB) is one of the leading infectious causes
of death in the world and has re-emanated as a growing
global health problem. This is not only because of the lack
of proper therapeutic agents for its treatment but also
because of development of drug-resistant strains (Kamal
et al., 2006). The resurgence of TB over the last 15 years,
even in industrialized countries where it had almost been
eradicated, has been favored by the pathogenic synergy
with human immunodeficiency virus (HIV) infection. In
fact, TB and other atypical mycobacterias are now diseases
frequently associated with AIDS; HIV infection signifi-
cantly increases the risk that new or latent TB infections
will progress to active diseases (Shahar Yar et al., 2007).
However, powerful new antitubercular drugs with new
mechanisms of action have not been developed over the
last 40 years. In the developing countries, the annual
infection rate is 20–50 times greater than in the developed
countries, and this prevailing high level shows little or no
downward trend. It is expected that development of new
effective anti-TB drugs will bring various outcomes, viz.,
shortening the total duration of therapy, reducing the total
V. Judge ꢀ M. Ahuja
Department of Pharmaceutical Sciences, Guru Jambheswar
University of Science and Technology, Hisar 125001, India
B. Narasimhan (&)
Faculty of Pharmaceutical Sciences, Maharshi Dayanand
University, Rohtak 124001, India
e-mail: naru2000us@yahoo.com
D. Sriram ꢀ P. Yogeeswari
Medicinal Chemistry Research Laboratory, Pharmacy Group,
Birla Institute of Technology and Science, Hyderabad 500078,
India
E. De Clercq ꢀ C. Pannecouque ꢀ J. Balzarini
Laboratory of Virology & Chemotherapy, Rega Institute
for Medical Research, Minderbroedersstraat 10, 3000 Leuven,
Belgium
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Med Chem Res (2012) 21:1935–1952
expenditure and treatment of multidrug-resistant tubercu-
losis (MDR-TB) by single dosage regimen (Maccari et al.,
2002).
QSAR studies of Isonicotinic acid-1-(substituted phenyl)-
ethylidene/cycloheptylidene hydrazides.
Over the last decade, great strides have been made in the
treatment of HIV infection through use of drug combina-
tions known as highly active anti-retroviral therapy
(HAART). HAART regimens usually include three or
more drugs with inhibitory activity against either HIV
reverse transcriptase (RT) or HIV protease. Currently, most
HAART regimens include a backbone of two nucleos(t)ide
reverse-transcriptase inhibitors (N(t)RTIs) with a third
agent added from the non-nucleoside reverse-transcrip-
tase inhibitor (NNRTI) or protease inhibitor classes (PI)
(Boojamra et al., 2008). Although combination therapies
have proven to decrease HIV-related mortality, there is still
a greater need for development of novel antiretroviral
agents due to the emergence of multi-drug resistance,
which is a major challenge to successful therapy for indi-
viduals infected with HIV (Kucukguzel et al., 2008).
A large number of constitutional, topological, geomet-
ric, electrostatic, and quantum indices were introduced
with the aim to express the chemical structure in a
numerical form. Such structural descriptors can be uti-
lized to model physical, chemical, or biological properties
with quantitative structure–property relationships (QSPRs)
and quantitative structure–activity relationships (QSARs)
(Ivanciuc et al., 2002). Spatial descriptors describe the
molecule’s ‘‘solvent-accessible’’ surface areas and their
charges. Electronic descriptors describe the electron ori-
entation and charge. Topological descriptors are based on
graphic/structure concepts and geometric features such as
shape, size, and branching. Thermodynamic descriptors
describe energy of molecules and their conversions.
Quantum mechanical descriptors are calculated using semi-
empirical methods that are likely to be more accurate (Sahu
et al., 2007).
Experimental
Melting points were determined in open capillary tubes on
a Sonar melting point apparatus and are uncorrected.
Reaction progress was monitored by thin layer chroma-
tography on silica gel sheets (Merck silica gel-G), and the
purity of the compounds was ascertained by single spot on
TLC sheet. 1H nuclear magnetic resonance (1H NMR)
spectra were recorded in Bruker Avance II 400 NMR
spectrometer using appropriate deuterated solvents and are
expressed in parts per million (d, ppm) downfield from
tetramethylsilane (internal standard). Infrared (IR) spectra
were recorded on a Shimadzu FTIR spectrometer.
General procedure for synthesis of ester of isonicotinic
acid
The mixture of isonicotinic acid (0.1 mol) and ethanol (in
excess) was refluxed with sulfuric acid (1–2 ml) till the
completion of reaction monitored by TLC on silica gel G
plates. Then, the reaction mixture was added to 200 ml of
ice cold water, and excess of the acid was neutralized by a
solution of sodium bicarbonate. The crude ester was
extracted with ether. The ether layer was separated, and
ester was obtained on evaporation of ether layer.
General procedure for the synthesis of isonicotinic acid
hydrazide
The ethanolic solution of ester (0.01 mol) and hydrazine-
hydrate (0.015 mol) was refluxed for 6–7 h. The reaction
mixture was then cooled, and the precipitates were washed
with water, dried, and recrystallized from ethanol.
Isoniazid is a prodrug that targets mycolic acid bio-
synthesis and is activated inside the mycobacterial cell.
Isoniazid is activated either due to the isonicotinic acyl
anion (Shoeb et al., 1985) or radical (Johnson and Schultz,
1994) by KatG, a catalase–peroxidase enzyme (Zhang
et al., 1992). When activated by the catalase–peroxidase
katG, INH attaches itself to NADH to form a covalent
adduct INH-NAD. This is the actual drug that binds the
inhA enzyme and inhibits its function (Khasnobis et al.,
2002). The antimicrobial activity of isoniazid derivatives
have been investigated very recently (Bayrak et al., 2009a;
Bayrak et al., 2009b; Rodriguez-Arguelles et al., 2007).
Overwhelmed by all these facts and in continuation of our
research endowed toward the synthesis, antimicrobial, an-
timycobacterial, antiviral and QSAR studies (Kumar et al.,
2010a, b, 2007; Judge et al., 2010), we hereby report
the synthesis, antitubercular, anti-HIV, antimicrobial, and
General procedure for the synthesis of isonicotinic acid-
1-(substituted phenyl)-ethylidene/cycloheptylidene
hydrazides (1–12)
The solution of isonicotinic acid hydrazide (0.01 mol) and
appropriate substituted acetophenone/cycloheptanone (0.01
mol), in ethanol, was refluxed for 4–5 h. The precipitates
obtained were filtered off, washed, and recrystallized from
ethanol.
Isonicotinic acid [1-(4-bromo-phenyl)-ethylidene]-
hydrazide (2)
Mp (ꢁC) 237–240; Yield 72.6%; 1H NMR (400 MHz,
CDCl3) d ppm: 2.19 (s, 3H, CH3), 7.52–7.65 (m, 4H, CH of
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Med Chem Res (2012) 21:1935–1952
1937
Isonicotinic acid [1-(2,4-dimethoxy-phenyl)-ethylidene]-
hydrazide (6)
phenyl ring), 7.78–7.91 (d, 2H, CH of C3 and C5 of pyri-
dine ring; J = 12), 8.78–8.81 (d, 2H, CH of C2 and C6 of
pyridine ring; J = 12), and 11.71 (s, 1H, NH); 13CNMR
(d ppm): 11.4, 121.6, 121.9, 125.4, 129.3, 130.5, 130.8,
131.4,141.6, 149.0, 154.2, and 168.4; IR (KBr pellets) m
cm-1: 3186.54 (NH str., amide), 3074.66 (CH str., aro-
matic), 1670.43 (C=O str., amide), 1600.99 (C=C str.,
skeletal phenyl nucleus), 825.57 (CH out-of-plane bending,
4-substituted pyridine), and 597.96 (C–Br str., aromatic);
CHN analysis calc.(found): C, 52.85 (52.19); H, 3.80
(3.65); N, 13.21 (13.32); O, 5.03 (4.91); and Br, 25.11
(24.95).
Mp (ꢁC) 120–123; Yield 59.1%; 1H NMR (400 MHz,
DMSO) d ppm: 2.07 (s, 3H, CH3), 3.79 (s, 6H, OCH3),
6.58 (s, 1H, CH of C3 of phenyl ring), 7.31–7.34 (d, 1H,
CH of C5 of phenyl ring; J = 12), 7.40–7.42 (d, 1H, CH of
C6 of phenyl ring; J = 8), 7.69–7.71 (d, 2H, CH of C3 and
C5 of pyridine ring; J = 8), 8.60–8.63 (d, 2H, CH of C2
and C6 of pyridine ring; J = 12), and 10.72 (s, 1H, NH);
13CNMR (d ppm): 11.8, 55.6, 55.9, 100.3, 106.5, 109.8,
122.2, 122.7, 130.8, 142.0, 149.2, 149.6, 153.5, 163.1,
164.8, and 169.2; IR (KBr pellets) m cm-1: 3105.53 (NH
str., amide), 1683.63 (C=O str., amide), 1307.79 (C–O–C
str., aralkyl ether), and 792.78 (CH out of plane bending,
4-substituted pyridine); CHN analysis calc.(found): C,
64.20 (64.32); H, 5.72 (5.51); N, 14.04 (13.89); and O,
16.04 (16.23).
Isonicotinic acid [1-(2,4-dichloro-phenyl)-ethylidene]-
hydrazide (3)
Mp (ꢁC) 175–178; Yield 55.4%; 1H NMR (400 MHz,
DMSO) d ppm: 2.35 (s, 3H, CH3), 7.25–7.46 (m, 3H, CH
of C3, C5 and C6 of dichloro phenyl ring), 7.74–7.75 (d,
2H, CH of C3 and C5 of pyridine ring; J = 4), 8.70–8.71
(d, 2H, CH of C2 and C6 of pyridine ring; J = 4), and 11.00
(s, 1H, NH); 13CNMR (d ppm): 11.2, 121.3, 121.8,
126.5,128.9, 129.3, 134.2, 136.7, 141.2, 149.5, 154.7, and
169.1; IR (KBr pellets) m cm-1: 3187.51 (NH str., amide),
3031.26 (CH str., aromatic), 1661.75 (C=O str., amide),
1559.51 (C=C str., skeletal phenyl nucleus), 816.89 (CH
out of plane bending, 4-substituted pyridine), and 666.43
(C–Cl str., aromatic); CHN analysis calc.(found): C, 54.57
(54.38); H, 3.60 (3.81); N, 13.64 (13.49); O, 5.19 (5.03);
Cl, and 23.01 (22.87).
Isonicotinic acid [1-(4-hydroxy-phenyl)-ethylidene]-
hydrazide (9)
1
Mp (ꢁC) Above 242; Yield 76.0%; H NMR (400 MHz,
DMSO) d ppm: 2.15 (s, 3H, CH3), 6.75–6.77 (s, 1H, OH),
6.83–6.87 (d, 2H, CH of C3 and C5 of phenyl ring; J = 16),
7.74–7.76 (d, 2H, CH of C2 and C6 of phenyl ring; J = 8),
7.80–7.84 (d, 2H, CH of C3 and C5 of pyridine ring;
J = 16), 8.75–8.76 (d, 2H, CH of C2 and C6 of pyridine
ring; J = 4), and 10.69 (s, 1H, NH); 13CNMR (d ppm):
11.5, 114.2, 114.5,121.4, 121.9, 123.1,129.4, 129.7,
141.5,149.3, 154.1, 159.2, and 168.3; IR (KBr pellets) m
cm-1: 3666.84 (OH str., Phenol), 3279.13 (NH str., amide),
3068.88 (CH str., aromatic), 2795.94 (CH3 str. asymmetric,
ArCH3), 2685.99 (CH3 str. symmetric, ArCH3), 1647.28
(C=O str., amide), and 1532.51 (C=C str., skeletal phenyl
nucleus), 826.53 (CH out of plane bending, 4-substituted
pyridine); CHN analysis calc.(found): C, 65.87 (65.63); H,
5.13 (5.01); N, 16.46 (16.23); and O, 12.54 (12.38).
Isonicotinic acid [1-(4-nitro-phenyl)-ethylidene]-hydrazide
(4)
1
Mp (ꢁC) Above 242; Yield 87.3%; H NMR (400 MHz,
DMSO) d ppm: 2.14 (s, 3H, CH3), 7.62–7.81 (m, 4H,
CH of phenyl ring), 8.24–8.26 (d, 2H, CH of C3 and C5
of pyridine ring; J = 8), 8.68–8.77 (d, 2H, CH of C2 and
C6 of pyridine ring), and 11.15 (s, 1H, NH); 13CNMR (d
ppm): 11.4, 121.4, 121.8, 122.7, 123.2, 128.7, 129.3,
136.9, 141.8, 149.1, 150.2, 154.2, and 168.5; IR (KBr
pellets) m cm-1: 3191.36 (NH str., amide), 3091.06 (CH
str., aromatic), 2937.71 (CH str., aliphatic), 1671.39
(C=O str., amide), 1577.84 (C=C str., skeletal phenyl
nucleus), 1508.40 (NO2 str. asymmetric, aromatic nitro
group), 1350.23 (NO2 str. symmetric, aromatic nitro
group), and 843.89 (CH out of plane bending, 4-substi-
tuted pyridine); CHN analysis calc.(found): C, 59.15
(58.91); H, 4.25 (4.11); N, 19.71 (19.87); O, and 16.88
(16.63).
Evaluation of antimycobacterial activity
All compounds were screened for their in vitro antimyco-
bacterial activity against MTB, in Middlebrook 7H11agar
medium supplemented with OADC by agar dilution
method similar to that recommended by the National
Committee for Clinical Laboratory Standards for the
determination of MIC in triplicate (National Committee for
Clinical Laboratory Standards, 1995). The minimum
inhibitory concentration (MIC) is defined as the minimum
concentration of compound required to give complete
inhibition of bacterial growth.
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Med Chem Res (2012) 21:1935–1952
2
LOF ¼ LSE=f1 ꢁ ðC þ dp=nÞg
Evaluation of antimicrobial activity (determination
of minimum inhibitory concentration)
where, LSE is the least square error; C is the number of
descriptors ?1; p is the number of independent parameters;
n is the number of compounds used; and d is the smoothing
parameter which controls the bias in the scoring factor
between equations with different number of terms, and was
kept 1.0.
The antimicrobial activity was performed against Gram-
positive bacteria: S. aureus and B. subtilis; Gram-negative
bacterium: E. coli; and fungal strains: C. albicans and
A. niger by tube dilution method (Cappucino and Sherman,
1999). Dilutions of test and standard compounds [nor-
floxacin (antibacterial) and fluconazole (antifungal)] were
prepared in double strength nutrient broth—I.P. (bacteria),
and Sabouraud dextrose broth I.P. (fungi) (Pharmacopoeia
of India, 2007). The samples were incubated at 37ꢁC for 24
h (bacteria), at 25ꢁC for 7 days (A. niger) and at 37ꢁC for
48 h (C. albicans), respectively, and the results were
recorded in terms of MIC (the lowest concentration of test
substance which inhibited the growth of microorganisms).
p
SEP ¼ LSE=n
The Quality value, Q is given by
Q ¼ r=Se
where, Q is the Quality value, r is the correlation coeffi-
cient, and Se is standard error.
The predictive ability of QSAR models was also quan-
tified in terms of q2, which is defined as
2
2
q2 ¼ 1 ꢁ fRðYobs ꢁ YcalcÞ =RðYobs ꢁ YmeanÞ g
QSAR studies
The low values of PE, LSE, LOF, and SEP and the high
values of Q and q2 are the essential criteria for qualifying
the model as the best one.
The structures of isonicotinic acid-1-(substituted phenyl)-
ethylidene/cycloheptylidene hydrazide derivatives were
first pre-optimized with the Molecular Mechanics Force
Field (MM?) procedure included in Hyperchem 6.03
(Hyperchem 6.0, 1993), and the resulting geometries are
further refined by means of the semiempirical method PM3
(Parametric Method-3). We chose a gradient norm limit of
Evaluation of anti-HIV activity
The anti-HIV activity and cytotoxicity were evaluated
against HIV-1 strain IIIB and HIV-2 strain ROD in MT-4
cell cultures using the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) method (Pauwels
et al., 1988). In brief, virus stocks were titrated in MT-4
cells and expressed as the 50% cell culture infective dose
(CCID50). MT-4 cells were suspended in culture medium at
1 9 105 cells/ml and infected with HIV at a multiplicity of
infection of 0.02. Immediately after viral infection, 100 ll
of the cell suspension was placed in each well of a flat-
bottomed microtiter tray containing various concentrations
of the test compounds. After a 4-day incubation period at
37ꢁC, the number of viable cells was determined using the
MTT method. Compounds were tested in parallel for
cytotoxic effects in uninfected MT-4 cells.
˚
0.01 kcal/A for the geometry optimization. The lowest
energy structure was used for each molecule to calculate
physicochemical properties using TSAR 3.3 software for
Windows (TSAR 3D Version 3.3, 2000). Further, the
regression analysis was performed using the SPSS software
package (SPSS for Windows, 1999).
Calculation of statistical parameters
The developed QSAR models were validated by the cal-
culation of following statistical parameters : probable error
of the coefficient of correlation (PE), least square error
(LSE), Friedman’s lack of fit measure (LOF), standard
error of prediction (SEP), quality value (Q), and SSY (sum
of squares of response values) (Mandloi et al., 2005;
Pinheiro et al., 2004).
Antiviral assays
The antiviral assays [except anti-HIV assays] were based
on inhibition of virus-induced cytopathicity in HEL [herpes
simplex virus type 1 (HSV-1), HSV-2 (G), vaccinia virus,
and vesicular stomatitis virus], Vero (parainfluenza-3,
reovirus-1, Sindbis, Coxsackie B4, and Punta Toro virus),
HeLa (vesicular stomatitis virus, Coxsackie virus B4, and
respiratory syncytial virus) cell cultures. Confluent cell
cultures in microtiter 96-well plates were inoculated with
100-cell culture inhibitory dose-50 (CCID50) of virus (1
CCID50 being the virus dose to infect 50% of the cell
These parameters were calculated from the following
equations:
p
PE ¼ 2ð1 ꢁ r2Þ=3 n
where, r is the correlation coefficient, and n is the number
of compounds used.
2
LSE ¼ RðYobs ꢁ Ycalc
where, Yobs and Ycalc are the observed and calculated
Þ
values.
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Med Chem Res (2012) 21:1935–1952
1939
cultures) in the presence of varying concentrations (100,
20, 4,… lg/ml) of the test compounds. Viral cytopathicity
was recorded as soon as it reached completion in the
control virus-infected cell cultures that were not treated
with the test compounds.
yield of some synthetic compounds may be attributed to any
one or more of the following reasons (Furniss et al., 1998):
(a) the reaction may be reversible and position of equilibrium
is unfavorable to the product; (b) the incursion of side
reactions leading to the formation of by-products; (c) the
premature work-up of the reaction before its completion;
(d) the volatilization of products during reaction or work-up;
(e) the loss of product due to incomplete extraction, ineffi-
cient crystallization, or other work-up procedures; (f) the
presence of contaminants in the reactants or reagents leading
to a less efficient reaction. In addition to the above facts, the
compound 11 has been derived from cycloheptanone which
is a cyclic ketone, due to which it may be less reactive toward
the reaction with hydrazide which may have led to the lower
yield. In compound 12, where Br group is present at ortho
position, the electronegative Br group creates difficulty in
the release of ketonic oxygen as an anion and, hence, lowers
the reaction rate that may have led to the lesser yield of this
compound. The physicochemical characteristics of synthe-
sized compounds are presented in Table 1.
Results and discussion
Chemistry
The synthesis of target compounds was carried out as
depicted in Scheme 1. The isonicotinic acid was refluxed
with ethanol in the presence of sulphuric acid to get the
ethyl ester of isonicotinic acid. The ester thus obtained was
refluxed with hydrazine hydrate to obtain the isonicotinic
acid hydrazide. The isonicotinoyl hydrazide was refluxed
with various substituted acetophenones/cycloheptanone to
yield the target isonicotinic acid-1-(substituted phenyl)-
ethylidene/cycloheptylidene hydrazides (1–12). The low
CH3
R1
NH
NH2
OH
Scheme 1 Synthetic route
OC2H5
CH3
O
O
O
O
followed for the synthesis of
isonicotinic acid-1-(substituted
phenyl)-ethylidene/
cycloheptylidene hydrazide
derivatives
N
R5
H
R1
N
O
R5
R4
R2
NH2NH2
R3
C2H5OH
H+
R2
R4
R3
N
N
N
N
1-10, 12
O
NH
N
O
N
11
Comp.
R1
R2
H
H
H
H
R3
Cl
R4
H
R5
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
1
2
Br
H
Cl
H
3
NO2
H
H
4
NO2
H
H
5
OCH3
OCH3
OH
H
OCH3
H
H
6
H
OCH3
H
7
H
H
8
H
OH
OH
H
H
9
OH
Br
H
H
10
12
H
H
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Med Chem Res (2012) 21:1935–1952
Table 1 Physicochemical properties and antimycobacterial activity of synthesized isonicotinic acid-1-(substituted phenyl)-ethylidene/cyclo-
heptylidene hydrazide derivatives
Comp.
Mol. formula
Mol. wt.
Melting pt. (ꢁC)
Rfa
% yield
MIC MTB-H37Rv
(lM 9 10-3
)
1
C14H12N3OCl
273
318
308
284
284
299
299
255
255
271
231
318
226–229
237–240
175–178
379–382
208–211
120–123
125–128
223–226
336–339
235–238
115–118
Semisolid
0.48
0.46
0.43
0.51
0.49
0.64
0.67
0.72
0.75
0.71
0.42
0.53
69.3
72.6
55.4
87.3
82.5
59.1
77.3
63.8
76.0
51.4
40.2
49.6
3
2
2
C
C
C
C
C
C
C
C
C
C
C
14H12N3OBr
14H11N3OCl2
14H12N4O3
14H12N4O3
16H17N3O3
16H17N3O3
14H13N3O2
14H13N3O2
14H13N3O3
13H17N3O
3
3
4
\3
5
5
6
10
21
25
49
46
3
7
8
9
10
11
12
INH
ETBb
CFL
RIFb
a
14H12N3OBr
5
2.04
15.31
9.4
0.24
Mobile phase: Ethanol
b
Sriram et al. (2007)
The structures of synthesized compounds were con-
characteristic of the amide linkage. In compounds 2, 3, 4,
6, and 9, the characteristic NH stretching band of a sec-
ondary amide was observed around 3200–3100 cm-1. The
presence of peaks slightly above and below 3000 cm-1
indicates the presence of an aromatic and aliphatic portions
in synthesized compounds, respectively. The skeletal
C=C– stretching bands were observed around 1500 cm-1
in the spectra of the synthesized compounds which repre-
sents the presence of aromatic groups. In compound 6, the
firmed on the basis of their consistent IR and NMR spectral
data. The presence of singlet signal above d 10.69 ppm in
compounds 2, 3, 4, 6, and 9 depicted the presence of NH
proton of hydrazide linkage in the synthesized derivatives.
The presence of singlet signal around d 2.07–2.35 ppm
revealed the presence of CH3 group of acetophenones in
the synthesized compounds. In all the synthesized com-
pounds, two doublet signals were observed at two different
d values, with the one at higher d value corresponding to C2
and C6 CH protons as they are in close proximity of the
electronegative nitrogen atom in the ring and the doublet
signal at lower d represents the remaining two protons of
the isonicotinyl nucleus. The singlet signal having six
protons due to OCH3 at d 3.79 ppm in compound 6 con-
firmed the presence of a dimethoxy group in the synthe-
sized compound. The appearance of signal for four
aromatic protons in compounds 2, 4, and 9 and signals for
three aromatic protons in compounds 3 and 6 confirmed the
presence of a monosubstituted and disubstituted phenyl
nucleus in the synthesized compound, respectively. The
singlet signal at d 4.46 ppm due to OH protons in com-
pound 9 confirmed the presence of hydroxyl group in its
structure.
C–O–C stretching band was observed at 1307.79 cm-1
,
which revealed the presence of methoxy group in its
structure.
The CH out-of-plane bending observed around vibra-
tional frequency of 860–814 cm-1 indicated the presence
of a 4-substituted pyridine ring in the structure of synthe-
sized compounds. The characteristic C–Br str. and C–Cl
str. bands were observed at 597.96 and 666.43 cm-1 in
compounds 2 and 3, respectively. The presence of OH
group in compound 9 was confirmed by a stretching band
at 3666.84 cm-1. The asymmetric NO2 str. at 1508.40
cm-1 and symmetric NO2 str. at 1350.23 cm-1 confirmed
the presence of an aromatic nitro group in compound 4.
Antimycobacterial activity
The presence of C=O functional group was demon-
strated by the appearance of stretching band around 1670
cm-1, in compounds 2, 3, 4, 6, and 9, which is the
The in vitro antitubercular activity against Mycobacterium
tuberculosis (MTB) was carried out in Middlebrook
123

Med Chem Res (2012) 21:1935–1952
1941
evidenced by the high antimycobacterial activity of
compounds 1–5. The role of electron-withdrawing
group in improving antimicrobial activities is sup-
ported by the studies of Sharma et al. (2004)
7H11agar medium supplemented with OADC by agar
dilution method, and the results are presented in Table 1.
Compound 1, having chloro substitution at para position,
had shown excellent antimycobacterial activity having
MIC of 3 9 10-3 lM. Based on these results, we planned
to substitute some other electronegative atom in the mol-
ecules, and synthesized compound 2, having p-bromo
substitution on the phenyl nucleus; it showed improved
antimycobacterial activity with MIC value 2 9 10-3 lM,
but when bromo substituent was added in ortho position
(compound 12), a fall in activity was observed with MIC of
5 9 10-3 lM, showing that para substitution favors the
antimycobacterial activity. As there was an improvement
of antimycobacterial activity with electronegative substit-
uents, this stimulated us to substitute the phenyl nucleus
with two electronegative atoms in compound 3, having
chloro substituents at ortho and para positions, but
improvement in activity was not observed, and it showed
antimycobacterial activity at 2 9 10-3 lM. Then, we
planned to introduce more electron-withdrawing nitro
group as substituent in the synthesized molecules and came
out with compounds 4 and 5 having nitro substitution at
para and meta positions, respectively. Compound 4 was
highly active with an MIC value \3 9 10-3 lM, and
compound 5 had an MIC value of 5 9 10-3 lM, which
indicates that an electron-withdrawing group at meta
position decreased the activity. The next change in the
substituent was the addition of an electron-donating
methoxy group in compounds 6 and 7, but, unfortunately,
this change led to a fall in antimycobacterial activity.
Compound 6 having 2,4-dimethoxy substituent had MIC
of 10 9 10-3 lM and compound 7 had an MIC of
21 9 10-3 lM. Then, we added hydroxyl substituent in
the phenyl nucleus and synthesized compounds 8, 9, and
10. Again, a steep fall in activity was observed upon
addition of a hydroxy substituent in the phenyl ring, in
compounds 8, 9, and 10, at ortho, para, and ortho-para
positions, respectively. In compound 11, the phenyl
nucleus was replaced with cycloheptyl ring, and a highly
active molecule with an MIC of 3 9 10-3 lM was
obtained. It is seen from the results of antimycobacterial
activity that compounds 1–5 and 11 were found to be
highly active with MIC values \5 9 10-3 lM, which is
well below the MIC values of standard drugs ciprofloxacin
and ethambutol. Compound 2 was found to be a more
active antimycobacterial agent than isoniazid, and com-
pound 3 was also found to be equally active.
2. The presence of OH group at ortho position (Com-
pound 8) increases the antimycobacterial activity in
comparison to the presence of OH group at para
position (Compound 9). This is in concordance with
Tripathi et al. (2006) who stated that OH group at
ortho position leads to a measurable change in activity
of the compounds.
3. The substitution of phenyl ring with electron-donating
OCH3 (Compound 5) decreases the antimycobacterial
activity. These results are similar to the reports of
Sriram et al. (2007) who observed that the addition of
methoxy group decreases the antimycobacterial activ-
ity of N-hydroxythiosemicarbazones.
4. The replacement of benzylidene ring with cyclohepty-
lidene ring (compound 11) improves the antimycobac-
terial activity of the molecule.
5. The presence of halo groups at para position improved
the antitubercular activity of the synthesized com-
pounds. This is similar to the results observed by
Mamolo et al. (2004) who stated that the presence of
bromo, chloro, and phenyl substituents, at the para
position of the benzene ring improved the antituber-
cular activity.
6. The presence of electron-withdrawing bromo group in
compound 2 enhances the growth-inhibiting potency of
the molecule, which is similar to the results observed
by Masunari and Tavares (2007) who stated that the
presence of bromo and chloro substituents, at the para
position of the benzene ring increases the activity.
7. Compound 5 with nitro substitution at para position
was more active than nitro substitution at ortho
position (compound 6).
Cytostatic, cytotoxic and antiviral activity
In general, none of the compounds was inhibitory to a
variety of DNA and RNA viruses tested at subtoxic con-
centrations (Tables 2, 3, 4, 5), except for compounds 8 and
10 that showed activities against HSV-1, HSV-2, and VV.
Although the compounds were not measurably toxic
against the HEL cell cultures (in which the anti-DNA virus
activity was performed), they showed pronounced toxicity
in several other cell types. Also, they were very cytostatic
against HEL cell proliferation. Therefore, it is currently
unclear whether the anti-DNA virus activity noticed is due
to a specific antiviral activity or to a (more likely) indirect
antiviral effect due to a cellular cytostatic potential of the
compounds.
From the results of antimycobacterial activity, the fol-
lowing conclusions regarding structure–activity relation-
ship (SAR) can be drawn:
1. The presence of electron-withdrawing groups on the
phenyl ring increases the antitubercular activity as
123

1942
Med Chem Res (2012) 21:1935–1952
Antibacterial and antifungal activities
Table 2 Anti-HIV potential of synthesized isonicotinic acid-
1-(substituted phenyl)-ethylidene/cycloheptylidene hydrazide deriva-
tives in MT-4 cells
The in vitro antimicrobial activity of synthesized com-
pounds were determined by tube dilution method using
norfloxacin and fluconazole as reference standards for
antibacterial and antifungal activities, respectively, and the
results are presented in Table 6.
Compound
EC50 (lg/ml)
CCa50 (lg/ml)
HIV-1 (IIIb)
HIV-2 (ROD)
1
[88
[88
88 15
43 4.6
38 14
77 7.3
63 5.9
12 0.96
88 15
0.50 0.07
95 15
9.7 4.4
86 10
–
2
[43
[43
The results for antibacterial activity against B. subtilis
indicated that compound 12 was the most active among all
the synthesized derivatives as well as the standard drug
norfloxacin. In particular, the compounds 6 and 12 were
found to be the most effective antibacterial agents having
pMICbs values 2.38 and 2.71 lM, respectively. In case of
S. aureus, compounds 1, 6, and 12 have shown marked
antibacterial potential at pMICsa values 2.34, 2.38, and
2.41 lM, respectively. For antibacterial activity against
E. coli compounds 2, 7, and 12 were found to be effective
with pMICec values of 2.41, 2.68, and 2.41 lM, respec-
tively. The antibacterial potential of compound 7 against
E. coli was higher than the activity of norfloxacin standard.
The antifungal activity against C. albicans demonstrated
that compounds 3, 6, and 12 were the potential candidates
having pMICca values 2.69, 2.68, and 2.71 lM, respec-
tively. All these three compounds (3, 6, and 12) have better
antifungal activities than the standard drug fluconazole, and
3
[38
[38
4
[77
[77
5
[63
[63
6
[12
[12
7
[88
[88
8
[0.50
[95
[0.50
[95
9
10
[9.7
[9.7
[86
11
[86
NT
12
NT
Nevirapine
Zidovudine
0.050 0.011
0.002 0.001
[4.0
0.002 0.001
[4.0
[25
50% Effective concentration or compound concentration required to
inhibit virus-induced cytopathicity by 50%
NT not tested
a
50% Cytotoxic concentration or compound concentration required
to reduce MT-4 cell viability by 50%
Table 3 Cytotoxicity and anti-DNA virus activity of synthesized isonicotinic acid-1-(substituted phenyl)-ethylidene/cycloheptylidene hydrazide
derivatives in HEL cell cultures
Compound
CCa50
Minimum cytotoxic
concentrationb
(lg/ml)
ECc50 (lg/ml)
Herpes simplex
virus-1 (KOS)
Herpes simplex
virus-2 (G)
Vaccinia
virus
Herpes simplex
virus-1 TK-
KOS ACVr
VZV
(OKA/07-1)
HCMV
(AD-169/
Davis)
1
20
100
[20
[20
[100
[100
[20
[100
[100
1.4
[20
[20
[100
[100
[20
[100
[100
1.3
[20
[20
[100
[100
[20
[100
[100
3.5
[20
[20
[100
[100
[20
[100
[100
4
[20
[20
[20
[20
[4
[20
[20
[20
[20
[20
[20
[100
[0.8
[20
[4
2
51
C20
3
11
[100
[100
100
4
54
5
13
6
10
[100
[100
100
[20
[100
1.8–3.1
[20
8–10
[100
NT
7
100
0.40
91
8
9
100
[20
2.5
[20
2.0
[20
2.5
[20
2
10
11
12
2.1
[100
[100 [100
NT NT
[100
NT
[100
NT
[100
NT
[100
NT
[100
NT
NT Not tested
a
Required to inhibit HEL cell proliferation by 50%
b
c
Required to cause a microscopically detectable alteration of normal cell morphology
Required to reduce virus-induced cytopathicity by 50%
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Med Chem Res (2012) 21:1935–1952
1943
Table 4 Cytotoxicity and anti-
RNA virus activity of
synthesized isonicotinic acid-1-
(substituted phenyl)-ethylidene/
cycloheptylidene hydrazide
derivatives in HeLa cell cultures
Compound
Minimum cytotoxic
concentrationa (lg/ml)
ECb50 (lg/ml)
Vesicular
stomatitis virus
Coxsackie
virus B4
Respiratory
syncytial virus
1
100
20
[20
[4
[20
[4
[20
[4
2
3
100
100
20
[20
[20
[4
[20
[20
[4
[20
[20
[4
4
5
6
20
[4
[4
[4
7
[100
1
[100
[0.2
[20
[0.8
[100
NT
[100
[0.2
[20
[0.8
[100
NT
[100
[0.2
[20
[0.8
[100
NT
NT Not tested
a
Required to cause a
8
9
100
4
microscopically detectable
alteration of normal cell
morphology
10
11
12
[100
NT
b
Required to reduce virus-
induced cytopathicity by 50%
Table 5 Cytotoxicity and anti-
RNA virus activity of
synthesized isonicotinic acid-1-
(substituted phenyl)-ethylidene/
cycloheptylidene hydrazide
derivatives in Vero cell cultures
Compound Minimum cytotoxic ECb50 (lg/ml)
concentrationa
Para influenza-3 Reovirus-1 Sindbis Coxsackie Punta
virus
(lg/ml)
virus
virus B4
Toro virus
1
100
C20
100
100
20
[20
[20
[20
[20
[4
[20
[20
[20
[20
[4
[20
[20
[20
[20
[4
[20
[20
[20
[20
[4
[20
[20
[20
[20
[4
2
3
4
5
6
100
[100
C4
[20
[100
[4
[20
[100
[4
[20
[100
[4
[20
[100
[4
[20
[100
[4
7
8
a
Required to cause a
9
C20
20
[20
[4
[20
[4
[20
[4
[20
[4
[20
[4
microscopically detectable
alteration of normal cell
morphology
10
11
12
[100
NT
[100
NT
[100
NT
[100
NT
[100
NT
[100
NT
b
Required to reduce virus-
induced cytopathicity by 50%
compound 10 had shown the antifungal potential equiva-
lent to the standard. In case of A. niger, compounds 2 and
12 were found to be active with pMICan value of 2.41 lM
each, which is better than the parent drug isonicotinic acid
hydrazide.
These results of antimicrobial activity can be represented
as follows:
C. albicans [ A. niger [ E. coli [ B. subtilis [
S. aureus
It is evident from the results presented in Table 6 that
the synthesized isonicotinic acid-1-(substituted phenyl)-
ethylidene/cycloheptylidene hydrazide derivatives have
shown marked antimicrobial potential against the tested
strains, with some of the synthesized compounds exhibiting
their antimicrobial activity higher than the standard drugs
norfloxacin and fluconazole. The isonicotinic acid-1-
(substituted phenyl)-ethylidene/cycloheptylidene hydrazide
derivatives were found to be more active against fungal
strains C. albicans and A. niger, and Gram negative E. coli
as compared with Gram-positive B. subtilis and S. aureus.
Structure activity relationship
From the results of antimicrobial activity, the following
SARs can be drawn:
1. The presence of substituent at ortho position increases
the antibacterial and antifungal potentials of the
compounds which are seen from the antimicrobial
activity of compounds 6, 8, 10, and 12. This fact is
supported by the observations of Guven et al. (2007).
123

1944
Med Chem Res (2012) 21:1935–1952
Table 6 Antibacterial and antifungal potentials (lM/ml) of synthesized isonicotinic acid-1-(substituted phenyl)-ethylidene/cycloheptylidene
hydrazide derivatives
Comp.
pMICbs
pMICsa
pMICec
pMICca
pMICan
pMICb
pMICf
pMICam
1
2.04
2.10
2.09
2.06
2.06
2.38
2.08
2.01
2.01
2.04
1.66
2.71
1.74
0.25
2.61b
2.34
2.10
2.09
2.06
2.06
2.38
2.08
2.01
2.01
2.04
1.66
2.41
2.04
0.20
2.61b
2.34
2.41
2.39
2.36
2.36
2.08
2.68
2.31
2.01
2.04
1.36
2.41
1.74
0.33
2.61b
2.34
2.41
2.69
2.36
2.36
2.68
2.38
2.61
2.31
2.64
1.66
2.71
2.34
0.29
2.64c
2.34
2.41
2.39
2.36
2.36
2.38
2.38
2.31
2.31
2.34
1.66
2.41
2.04
0.21
2.64c
2.24
2.20
2.19
2.16
2.16
2.28
2.28
2.11
2.01
2.04
1.56
2.51
1.84
0.23
2.61
2.34
2.41
2.54
2.36
2.36
2.53
2.38
2.46
2.31
2.49
1.66
2.56
2.19
0.24
2.64
2.28
2.29
2.33
2.24
2.24
2.38
2.32
2.25
2.13
2.22
1.60
2.53
1.98
0.22
2.62
2
3
4
5
6
7
8
9
10
11
12
INH
S.D.a
Std.
a
Standard deviation
b
c
Norfloxacin
Fluconazole
2. The compounds have shown marked antifungal
potential as compared to antibacterial activity, which
shows that different structural requirements are essen-
tial for antibacterial and antifungal activity. These
results are similar to those of Sortino et al. (2007).
3. The results of antimicrobial activity depicted that the
presence of electron-donating group, OCH3, enhanced
the antimicrobial activity of the synthesized deriva-
tives (Compound 6 and 7). This is supported by the
findings of Emami et al. (2008).
results observed by Masunari and Tavares (2007) who
stated that the presence of bromo and chloro substit-
uents, at the para position of the benzene ring showed
marked antibacterial activity against S. aureus.
The SAR findings of the antimycobacterial and antimi-
crobial activity are summarized in Fig. 1.
QSAR studies
4. The replacement of benzylidene nucleus with cycloh-
eptylidene nucleus (Compound 11) leads to a decline
in antimicrobial activity. This fact indicates that
phenyl nucleus is necessary for antimicrobial activity.
5. The synthesized derivatives were more active toward
the Gram-negative bacterium E. coli than Gram-
positive B. subtilis and S. aureus. This finding is in
concordance with Sbardella et al. (2004).
Development of one-target QSAR model
In order to identify the substituent effect on the antimi-
crobial activity, QSAR studies between the in vitro activity
and descriptors coding for lipophilic, electronic, steric, and
topological properties of the 12 synthesized isonicotinic
acid-1-(substituted
phenyl)-ethylidene/cycloheptylidene
hydrazide derivatives were undertaken, using the linear
free-energy relationship model (LFER) described by Han-
sch and Fujita (1964). Biological activity data determined
as MIC values was first converted into pMIC values (i.e.,
-log MIC) and used as dependent variable in QSAR study.
The different molecular descriptors (independent vari-
ables) like log of octanol–water partition coefficient (log
P), molar refractivity (MR), Kier’s molecular connectivity
6. The results of antimicrobial activity showed that ortho-
and para-disubstituted chloro derivative (compound 3)
has higher antimicrobial potency in comparison to
monochloro-substituted (compound 1) derivative except
against S. aureus. The higher antimicrobial potential of
compound 3 may be due to the presence of an extra
electron-withdrawing chloro group in its structure.
0
1
1
2
2
7. The presence of electron-withdrawing bromo group in
compounds 2 and 12 enhances the growth inhibition
potency of isonicotinic acid-1-(substituted phenyl)-
ethylidene hydrazide derivatives. This is similar to the
(0v, vv, v, vv, v, vv) and shape (j1, j2, j3, ja1, ja2,
ja3) topological indices, Randic topological index (R),
Balaban topological index (J), Wiener topological index
(W), Total energy (Te), energies of the highest occupied
123

Med Chem Res (2012) 21:1935–1952
1945
Fig. 1 Structural requirements
for the antimycobacterial and
antimicrobial activity of
synthesized isonicotinic acid-1-
(substituted phenyl)-ethylidene/
cycloheptylidene hydrazide
derivatives
Increases the antimycobacterial activity
Decreases the antimicrobial activity
Replacement with
cycloheptylidene ring
CH3
H
N
N
Increases the antimicrobial
and antimycobacterial activity
Cl/Br
NO2
O
R
Increases the antimycobacterial
activity
Decrease the antimicrobial
activity
N
Decreases the antimycobacterial
activity
OCH3
Increases the antimicrobial
activity
Essential for antimycobacterial
activity
Decreases the antimycobacterial
activity
OH
Increases the antifungal
activity
Decreases the antibacterial
activity
Table 7 Values of selected descriptors used in the regression analysis
Comp. log P MR j1
0v 0vv 1v 2v 3v 3vv
ja1
W
Te
Ele.E
LUMO HOMO
1
2.730 74.173 13.665 11.117 9.165 7.950 1.093 0.529 15.390 14.175 808.000 -3270.560 -18896.800 -0.651 -9.146
3.004 76.991 13.665 11.922 9.165 7.950 1.093 0.663 15.390 14.361 808.000 -3250.060 -18820.300 -0.689 -9.191
3.248 78.978 14.535 12.235 9.575 8.489 1.299 0.690 16.372 15.439 907.000 -3630.570 -20981.400 -0.644 -9.425
2.165 76.693 15.242 11.185 10.075 8.849 1.305 0.441 17.355 15.419 1086.000 -3741.310 -22189.900 -1.243 -9.690
2.165 76.693 15.242 11.185 10.075 8.861 1.305 0.441 17.355 15.419 1050.000 -3741.320 -22346.000 -1.088 -9.608
1.706 82.295 15.949 12.661 10.651 8.850 1.145 0.445 18.340 16.755 1175.000 -3862.080 -25136.500 -0.471 -8.787
1.706 82.295 15.949 12.661 10.651 8.850 1.145 0.445 18.340 16.755 1155.000 -3862.030 -25191.400 -0.491 -8.629
1.927 71.062 13.665 10.369 9.182 7.856 1.010 0.383 15.390 13.852 784.000 -3231.030 -19401.700 -0.660 -9.003
1.927 71.062 13.665 10.369 9.165 7.950 1.093 0.404 15.390 13.852 808.000 -3231.060 -19021.300 -0.578 -8.849
1.643 72.756 14.535 10.739 9.575 8.489 1.299 0.457 16.372 14.792 907.000 -3551.560 -21288.300 -0.500 -8.857
2.781 65.756 11.924 9.855 8.360 6.810 0.606 0.246 13.432 12.700 584.000 -2839.070 -17511.600 -0.506 -9.361
3.004 76.991 13.665 11.922 9.182 7.856 1.010 0.607 15.390 14.361 784.000 -3250.110 -19638.300 -0.337 -9.660
0.020 36.934 7.397 5.242 4.843 3.784 0.402 0.134 8.100 7.249 121.000 -1804.500 -8006.920 -0.511 -10.427
2
3
4
5
6
7
8
9
10
11
12a
INH
a
Outlier
molecular orbital (HOMO) and the lowest unoccupied
molecular orbital (LUMO), dipole moment (l), nuclear
repulsion energy (Nu.E), and electronic energy (Ele.E),
calculated for isoniazid derivatives are presented in
Table 7 (Hansch et al., 1973; Kier and Hall, 1976; Randic
1975; Balaban 1982; Wiener 1947; Randic 1993). Units of
the energies and dipole were electron volts (eV), and
atomic units (a.u.), respectively (Dai et al., 1999).
In the present study, a dataset of 13 compounds (12
isonicotinic acid-1-(substituted phenyl)-ethylidene/cyclo-
heptylidene hydrazide derivatives along with isoniazid
itself) was subjected to linear/multiple linear free-energy
regression analysis for model generation. During the
regression analysis studies, it was observed that the
response values of compound 12 were outside the limits
of response values of other synthesized isonicotinic acid-
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1946
Med Chem Res (2012) 21:1935–1952
1-(substituted phenyl)-ethylidene/cycloheptylidene hydra-
zide derivatives. Thus, compound 12 was designated as an
outlier, and it was not involved in the dataset for QSAR
model generation. In multivariate statistics, it is common to
define three types of outliers (Furusjo et al., 2006):
The antifungal activity of synthesized isonicotinic
acid-1-(substituted
phenyl)-ethylidene/cycloheptylidene
hydrazide derivatives against A. niger is governed by the
third order molecular connectivity topological index (3v)
(Eq. 1).
1. X/Y relation outliers are substances for which the
relationship between the descriptors (X variables) and
the dependent variables (Y variables) is not the same as
in the (rest of the) training data.
QSAR model for antifungal activity against A. niger
pMICan ¼ 0:6083v þ 1:625
2. X outliers. In brief, a substance is an X outlier if the
molecular descriptors for this substance do not lie in
the same range as the (rest of the) training data.
3. Y outliers are only defined for training or test samples.
They are substances for which the reference value of
response is invalid.
n ¼ 12; r ¼ 0:802; q2 ¼ 0:108; s ¼ 0:135; F ¼ 18:04
ð1Þ
where and henceforth, n is the number of data points, r is
the correlation coefficient, q2 is the cross-validated r2
obtained by leave-one-out (LOO) method, s is the standard
error of the estimate, and F is the Fischer statistics.
In light of the above guidelines, compound 12 was
considered as outlier because its response values [antimi-
crobial activity] were outside the range in comparison to
the other compounds included in the present study.
For antifungal activity against A. niger, the developed
QSAR model (Eq. 1) describes the importance of third-
order molecular connectivity topological index (3v). Topo-
logical indices are numerical quantifiers of molecular
topology and are sensitive to bonding pattern, symmetry,
content of heteroatom as well as degree of complexity of
atomic neighborhoods (Lather and Madan, 2005). The
third-order molecular connectivity topological index (3v)
represents the molecules with highly branched structure. In
this case, the positive correlation was observed between 3v
and antifungal activity against A. niger, and the results
presented in the Table 9 are in concordance with the model
expressed by Eq. 1. It can be seen from Table 7 that
Preliminary analysis was carried out in terms of corre-
lation analysis. A correlation matrix constructed for anti-
bacterial activity against A. niger is presented in Table 8.
The correlations of different molecular descriptors with
antibacterial and antifungal activities are presented in
Table 9. In general, high colinearity (r [ 0.8) was observed
between different parameters. The high interrelationship
was observed between 0v and j1 (r = 0.999), and the
low interrelationship was observed between 3vv and l
(r = 0.220). The correlation matrix indicated the predom-
inance of topological parameters in describing the antimi-
crobial activity of the synthesized compounds.
3
compounds 2, 3, 6, and 7 having high v values (1.093,
1.299, 1.145, and 1.145) have got the highest antibacterial
Table 8 Correlation matrix for antifungal activity of synthesized isonicotinic acid-1-(substituted phenyl)-ethylidene/cycloheptylidene hydrazide
derivatives against A. niger
log P
0v
0vv
1v
1vv
2v
2vv
3v
3vv
j1
W
Te
l
Nu.E pMICan
log P
0v
0vv
1v
1vv
2v
2vv
3v
1.000
0.504
0.682
0.538
0.788
0.576
0.875
0.469
0.740
0.486
0.413
1.000
0.943
0.996
0.893
0.991
0.827
0.893
0.647
0.999
0.989
1.000
0.952
0.971
0.937
0.943
0.805
0.779
0.941
0.913
1.000
0.920
0.987
0.852
0.856
0.625
0.995
0.979
1.000
0.904
0.982
0.722
0.722
0.889
0.844
1.000
0.856
0.920
0.691
0.985
0.965
1.000
0.712
0.808
0.818
0.764
1.000
3vv
0.767
0.881
0.872
1.000
j1
0.629
0.591
1.000
0.992
W
1.000
Te
-0.493 -0.995 -0.932 -0.985 -0.875 -0.985 -0.814 -0.910 -0.656 -0.994 -0.989
1.000
l
0.395
0.435
0.125
0.496
0.981
0.627
0.376
0.927
0.565
0.502
0.985
0.566
0.423
0.884
0.379
0.541
0.952
0.624
0.395
0.795
0.380
0.497
0.803
0.802
0.220
0.541
0.704
0.490
0.986
0.620
0.501 -0.486 1.000
Nu.E
pMICan
0.980 -0.975 0.419 1.000
0.638 -0.637 0.213 0.523 1.000
123

Med Chem Res (2012) 21:1935–1952
1947
Table 9 Correlation of molecular descriptors with antimicrobial (antibacterial and antifungal) activity
Mol. descriptor
pMICbs
pMICsa
pMICec
pMICca
pMICan
pMICb
pMICf
pMICam
log P
MR
0v
0vv
1v
1vv
2v
2vv
3v
0.201
0.741
0.772
0.746
0.737
0.591
0.734
0.546
0.758
0.649
0.774
0.701
0.762
0.659
0.737
-0.178
0.797
-0.772
-0.744
-0.119
0.543
0.230
0.738
-0.054
0.329
0.351
0.348
0.305
0.166
0.309
0.150
0.425
0.439
0.355
0.268
0.340
0.229
0.305
0.191
0.397
-0.357
-0.311
-0.010
0.219
-0.041
0.304
0.255
0.622
0.636
0.617
0.591
0.463
0.624
0.460
0.723
0.691
0.633
0.532
0.614
0.481
0.591
-0.134
0.645
-0.644
-0.567
-0.363
0.325
0.227
0.554
-0.122
0.302
0.371
0.307
0.301
0.115
0.352
0.119
0.568
0.494
0.363
0.206
0.338
0.148
0.301
0.257
0.379
-0.398
-0.320
0.002
0.236
-0.090
0.307
0.125
0.580
0.627
0.565
0.566
0.379
0.624
0.380
0.802
0.704
0.620
0.478
0.586
0.402
0.566
-0.052
0.638
-0.637
-0.539
-0.285
0.366
0.213
0.523
0.185
0.658
0.681
0.661
0.632
0.478
0.652
0.459
0.747
0.700
0.681
0.579
0.663
0.526
0.632
-0.072
0.707
-0.688
-0.623
-0.242
0.402
0.183
0.612
-0.014
0.444
0.505
0.439
0.436
0.242
0.493
0.244
0.701
0.613
0.497
0.341
0.468
0.272
0.436
0.127
0.515
-0.526
-0.435
-0.130
0.306
0.045
0.420
0.106
0.585
0.625
0.586
0.566
0.390
0.602
0.381
0.751
0.687
0.621
0.492
0.598
0.431
0.566
0.012
0.644
-0.638
-0.559
-0.200
0.373
0.128
0.546
3vv
j1
j2
ja1
ja2
R
J
W
Te
Ele.E
LUMO
HOMO
l
Nu.E
Table 10 Comparison of observed and predicted antibacterial and antifungal activities obtained by ot-QSAR model
Comp.
pMICan (Eq. 1)
pMICbs (Eq. 2)
pMICec (Eq. 3)
pMICca (Eq. 5)
Obs.
Pre.
Res.
0.05
Obs.
Pre.
Res.
0.04
Obs.
Pre.
Res.
0.14
Obs.
Pre.
Res.
1
2.34
2.41
2.39
2.36
2.36
2.38
2.38
2.31
2.31
2.34
1.66
2.04
2.29
2.29
2.41
2.42
2.42
2.32
2.32
2.24
2.29
2.41
1.99
1.87
2.04
2.10
2.09
2.06
2.06
2.38
2.08
2.01
2.01
2.04
1.66
1.74
2.00
2.00
2.05
2.14
2.12
2.18
2.17
1.99
2.00
2.05
1.89
1.66
2.34
2.41
2.39
2.36
2.36
2.08
2.68
2.31
2.01
2.04
1.36
1.74
2.20
2.20
2.38
2.39
2.39
2.24
2.24
2.12
2.20
2.38
1.76
1.58
2.34
2.41
2.69
2.36
2.36
2.68
2.38
2.61
2.31
2.64
1.66
2.34
2.43
2.42
2.64
2.24
2.53
2.48
2.56
2.32
2.36
2.66
2.04
2.11
-0.09
-0.01
0.05
2
0.12
-0.02
-0.06
-0.06
0.06
0.10
0.04
0.21
0.01
3
4
-0.08
-0.06
0.20
-0.03
-0.03
-0.16
0.44
0.12
5
-0.17
0.20
6
7
0.06
-0.09
0.02
-0.18
0.29
8
0.07
0.19
9
0.02
0.01
-0.19
-0.34
-0.40
0.16
-0.05
-0.02
-0.38
0.23
10
11
INH
-0.07
-0.33
0.17
-0.01
-0.23
0.08
potential (2.41, 2.39, 2.38, and 2.38). The compound 11
3
Tropsha, 2002). In this case, the q2 (Eq. 1) value is less
than 0.5, which shows that the developed model is an
invalid one. However, according to the recommendations
of Kim et al. (2007), the regression models are acceptable
if the value of standard deviation (SD, Table 6) is not much
larger than 0.3. As the value of standard deviation in case
of (Eq. 1) is less than 0.3 i.e., 0.21, so the developed model
having the least v value (0.606) has got the minimum
antifungal activity (1.66).
The QSAR model expressed by Eq. 1 was cross vali-
dated by q2 values obtained with LOO method. The value
of q2 greater than 0.5 is the basic requirement for quali-
fying a QSAR model as the valid one (Golbraikh and
123

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Med Chem Res (2012) 21:1935–1952
The model expressed by Eq. 2 demonstrated that antibac-
terial activity against B. subtilis is governed by the Wiener
topological index (W). The Wiener topological index
(W) was introduced by Wiener (Wiener, 1947) to demon-
strate the correlations between the physicochemical prop-
erties of organic compounds and the topological structure
of their molecular graphs in terms of sum of distances
between any two carbon atoms in the molecule.
Table 11 PE, LSE, LOF, SEP, VIF, Q, and SSY values calculated
for the derived models for modeling antimicrobial activity of syn-
thesized isonicotinic acid-1-(substituted phenyl)-ethylidene/cyclo-
heptylidene hydrazide derivatives
S. no.
Descriptor PE
LSE
LOF
SEP
Q
Eq. 1
Eq. 2
Eq. 3
Eq. 5
Eq. 6
Eq. 8
Eq. 9
3v
W
3v
3v
3v
0.0687 0.1797 0.0154 0.0353 5.941
0.0702 0.1312 0.0112 0.0302 6.991
0.0919 0.6582 0.0564 0.0676 2.813
0.0949 0.4107 0.0072 0.0534 3.342
0.0851 0.2162 0.0185 0.0387 5.116
As the coefficient of Wiener topological index (W) in
Eq. 2 is positive, the antibacterial activity against B. sub-
tilis will increase with increase in the Wiener topological
index (W) values. This is clearly evident from Table 6 that
compounds 4, 5, 6, and 7 having high W values of 1086.00,
1050.00, 1175.00, and 1155.00, respectively (Table 7),
have got the highest antibacterial activity values of 2.06,
2.06, 2.38, and 2.08 respectively. Similarly, compound 11
having minimum W value 584.00 (Table 7) has got
the minimum antibacterial activity against B. subtilis
(Table 6).
log P, vv 0.0274 0.0874 0.0045 0.0246 9.449
3v
3
0.0839 0.2155 0.0185 0.0387 5.109
Eq. 10 log P, vv 0.0327 0.0860 0.0044 0.0244 9.392
3
is a valid one. The comparisons of the observed and pre-
dicted antifungal activities are presented in Table 10. It is
evident from the results that the observed and the predicted
antibacterial activities lie close to each other as indicated
by their low residual values (Table 10). The low residual
activity values also support the validity of the QSAR model
described by Eq. 1. The low values of PE, LSE, LOF, and
SEP, and the high values of Q, (Table 11) revealed the
statistical significance of the model described by Eq. 1.
Equations 2–5 were developed to predict the antibacterial
and antifungal activities of isonicotinic acid-1-(substituted
phenyl)-ethylidene/cycloheptylidene hydrazide derivatives
against B. subtilis, E. coli and C. albicans.
The model described by Eq. 3 demonstrated the
importance of third order molecular connectivity topolog-
ical index (3v) in describing the antibacterial activity
against E. coli. The positive correlation of the molecular
descriptor with antibacterial activity reveals that increase in
3
the value of v (Table 7) will lead to an increase in anti-
bacterial activity against E. coli. The QSAR model for
antifungal activity (Eq. 4) against C. albicans depicted that
third-order molecular connectivity topological index (3v)
best describes the antifungal activity of synthesized
derivatives. The model expressed by Eq. 4 has got low r
value which stimulated us to go for the development of
multiparametric models using multiple linear regression
(MLR) analysis. Using the MLR analysis, we came out
with Eq. 5, where combination of third-order molecular
connectivity topological index (3v) with dipole moment (l)
increased the r value from 0.568 to 0.712.
QSAR model for antibacterial activity against B. Subtilis
pMICbs ¼ 0:000498W þ 1:599
n ¼ 12; r ¼ 0:797; q2 ¼ 0:338; s ¼ 0:114; F ¼ 17:40
ð2Þ
QSAR model for antibacterial activity against E. coli
Similar to Eq. 1, Eqs. 2–5 have also got low q2 values,
hence in these cases also, the low standard deviation sup-
ports the validity of developed QSAR models. The low
residual values presented in Table 10 also support the fact
that models expressed by Eqs. 2–5 are valid ones. The low
values of PE, LSE, LOF, and SEP and high value of Q,
(Table 11) revealed the statistical significance of the model
described by Eqs. 2, 3, and 5. Statistically significant
models were not obtained for antibacterial activity against
S. aureus.
pMICec ¼ 0:8993v þ 1:215
n ¼ 12; r ¼ 0:723; q2 ¼ 0:215; s ¼ 0:257; F ¼ 12:95
ð3Þ
QSAR model for antifungal activity against C. albicans
pMICca ¼ 0:5483v þ 1:814
n ¼ 12; r ¼ 0:568; q2 ¼ ꢁ0:754; s ¼ 0:237; F ¼ 4:76
ð4Þ
In general, for QSAR studies, the biological activities of
compounds should span 2–3 orders of magnitude. How-
ever, in the present study, the range of antibacterial and
antifungal activities of the synthesized compounds is
within one order of magnitude. It is important to note that
the predictability of the QSAR models developed in the
present study is high as evidenced by their low residual
QSAR model for antifungal activity against C. albicans
obtained by MLR
pMICcaMLR ¼ 0:7863v ꢁ ꢁ0:107l þ 2:054
n ¼ 12; r ¼ 0:712; q2 ¼ ꢁ0:742; s ¼ 0:213; F ¼ 4:62
ð5Þ
123

Med Chem Res (2012) 21:1935–1952
1949
values. This is in accordance with results suggested by the
Bajaj et al. (2005), who stated that the reliability of the
QSAR model lies in its predictive ability, even though
the activity data are in the narrow range. Further, the
recent literature reveals that the QSAR have been applied
to describe the relationship between narrow range of
biological activity and physicochemical properties of the
molecules (Narasimhan et al., 2007; Sharma et al., 2006;
Hatya et al., 2006). When biological activity data lie in
the narrow range, the presence of minimum standard
deviation of the biological activity justifies its use in
QSAR studies (Kumar et al., 2007; Narasimhan et al.,
2007). The minimum standard deviation (Table 6)
observed in the antimicrobial activity data justifies its use
in QSAR studies.
topological index (3v) in describing the antibacterial
activity of synthesized isonicotinic acid-1-(substituted
phenyl)-ethylidene/cycloheptylidene hydrazide derivatives,
represented as the model expressed by Eq. 6.
mt-QSAR model for antibacterial activity
pMICb ¼ 0:5483v þ 1:505
n ¼ 12; r ¼ 0:747; q2 ¼ 0:181; s ¼ 0:146; F ¼ 12:63
ð6Þ
The mt-QSAR model for antifungal activity demonstrated
the importance of third-order molecular connectivity
topological index (3v) in describing the antifungal activity
of the synthesized isonicotinic acid-1-(substituted phenyl)-
ethylidene/cycloheptylidene hydrazide derivatives repre-
sented by Eq. 7.
Development of multi-target QSAR model
mt-QSAR model for antifungal activity
According to the above ot-QSAR models, one should use
five different equations with different errors to predict the
activity of a new compound against the five microbial
species. The ot-QSAR models, which are almost in the
whole literature, become impracticable, or at worse, too
complicated to use when we have to predict to each com-
pound the results for more than one target. However, very
recently, the interest has become increased in the devel-
opment of multi-target QSAR (mt-QSAR) models. In
contrast to ot-QSAR, the mt-QSAR model is a single
equation that considers the nature of molecular descriptors
which are common and essential for describing the anti-
bacterial and antifungal activity (Prado-Prado et al., 2008;
Gonzalez-Diaz et al., 2008; Cruz-Monteagudo et al., 2007;
Gonzalez-Diaz et al., 2007; Gonzalez-Diaz and Prado-
Prado, 2008).
pMICf ¼ 0:5783v þ 1:719
n ¼ 12; r ¼ 0:701; q2 ¼ ꢁ0:298; s ¼ 0:175; F ¼ 9:68
ð7Þ
The model for antifungal activity developed by linear
regression has got r value of 0.701, but, if we go for
multiple linear regression analysis, the r value increased to
0.926, when we combined log P and valence third-order
molecular connectivity topological index (3vv), and the
model is represented by Eq. 8
mt-QSAR model for antifungal activity by MLR
pMICam ¼ ꢁ0:286 log P þ 2:1173vv þ 2:000
n ¼ 12; r ¼ 0:926; q2 ¼ ꢁ0:374; s ¼ 0:098; F ¼ 27:23
ð8Þ
In the present study, we have attempted to develop three
different types of mt-QSAR models: mt-QSAR model for
describing the antibacterial activity of the synthesized
compounds against S. aureus, B. subtilis, and E. coli,;
mt-QSAR model for describing the antifungal activity
of the synthesized derivatives against C. albicans and
A. niger; and common mt-QSAR model for describing the
antimicrobial (overall antibacterial and antifungal) activity
of the synthesized isonicotinic acid-1-(substituted phenyl)-
ethylidene/cycloheptylidene hydrazide derivatives.
Similarly, the overall antimicrobial activity of synthesized
isonicotinic acid-1-(substituted phenyl)-ethylidene/cyclo-
heptylidene hydrazide derivatives is also governed by
third-order molecular connectivity topological index (3v),
and represented in the model expressed by Eq. 9
mt-QSAR model for antimicrobial activity
pMICam ¼ 0:5603v þ 1:591
In order to develop mt-QSAR models, initially, we have
calculated the average antibacterial, antifungal, and anti-
microbial activity values of isonicotinic acid-1-(substituted
phenyl)-ethylidene/cycloheptylidene hydrazide derivatives
which are presented in Table 6. These average activity
values were also correlated with the molecular descriptors
of synthesized compounds (Table 9).
n ¼ 12; r ¼ 0:751; q2 ¼ 0:018; s ¼ 0:147; F ¼ 12:93
ð9Þ
Similarly, if we go for model generation by multiple linear
regression analysis, the r value increased to 0.911 when we
combined log P and valence third-order molecular con-
nectivity topological index (3vv), and the model is repre-
sented by Eq. 10
The mt-QSAR model for antibacterial activity displayed
the importance of the third-order molecular connectivity
123

1950
Med Chem Res (2012) 21:1935–1952
Table 12 Comparison of observed and predicted antibacterial and antifungal activity obtained by mt-QSAR model
Comp.
pMICb (Eq. 6)
pMICf (Eq. 8)
pMICam (Eq. 9)
pMICamMLR (Eq. 10)
Obs.
Pre.
Res.
0.14
Obs.
Pre.
Res.
0.00
Obs.
Pre.
Res.
0.08
Obs.
Pre.
Res.
0.07
1
2.24
2.20
2.19
2.16
2.16
2.28
2.28
2.11
2.01
2.04
1.56
1.84
2.10
2.10
2.22
2.22
2.22
2.13
2.13
2.06
2.10
2.22
1.84
1.73
2.34
2.41
2.54
2.36
2.36
2.53
2.38
2.46
2.31
2.49
1.66
2.19
2.34
2.55
2.53
2.32
2.32
2.46
2.46
2.26
2.30
2.50
1.73
2.28
2.28
2.29
2.33
2.24
2.24
2.38
2.32
2.25
2.13
2.22
1.60
1.98
2.20
2.20
2.32
2.32
2.32
2.23
2.23
2.16
2.20
2.32
1.93
1.82
2.28
2.29
2.33
2.24
2.24
2.38
2.32
2.25
2.13
2.22
1.60
1.98
2.21
2.40
2.40
2.17
2.17
2.28
2.28
2.12
2.16
2.32
1.67
2.08
2
0.10
-0.03
-0.06
-0.06
0.15
-0.14
0.01
0.09
0.01
-0.11
-0.07
0.07
3
4
0.04
-0.08
-0.08
0.15
5
0.04
0.07
6
0.07
0.10
7
0.15
-0.08
0.20
0.09
0.04
8
0.05
0.09
0.13
9
-0.09
-0.18
-0.28
0.11
0.01
-0.07
-0.10
-0.33
0.16
-0.03
-0.10
-0.07
-0.10
10
11
INH
-0.01
-0.07
-0.09
mt-QSAR model for antimicrobial activity by MLR
results of antiviral activity testing showed that none of the
tested compounds was active at subtoxic concentrations.
The anti-DNA virus activity of compounds 8 and 10 may
likely be due to indirect cytostatic activity. The synthesized
compounds were also screened for their antimicrobial
potentials against S. aureus, B. subtilis, E. coli, C. albicans,
and A. niger, and the results of antimicrobial activity
indicated that compounds having Br, OCH3, and Cl groups
were highly active antimicrobial agents with some of them
exhibiting activities better than the standard compounds,
norfloxacin and fluconazole. To understand the relationship
between physicochemical parameters and antibacterial and
antifungal activity of isonicotinic acid-1-(substituted phe-
nyl)-ethylidene/cycloheptylidene hydrazide derivatives,
QSAR investigation was performed by the development of
one-target and multi-target models. The multi-target model
was found to be effective in describing the antimicrobial
activity of isonicotinic acid-1-(substituted phenyl)-ethyli-
dene/cycloheptylidene hydrazide derivatives in comparison
to the one-target models and indicated the importance of
MLR models involving log P and valence third-order
molecular connectivity topological index (3vv) in describ-
ing antifungal and overall antimicrobial activities, and the
third-order molecular connectivity topological index (3v)
best describes the antibacterial potentials of isonicotinic
pMICam ¼ ꢁ0:223 log P þ 1:8723vv þ 1:829
n ¼ 12; r ¼ 0:911; q2 ¼ ꢁ0:204; s ¼ 0:097; F ¼ 22:04
ð10Þ
The mt-QSAR models (Eqs. 6–10) for the antibacterial,
antifungal, and overall antimicrobial activities of synthe-
sized isonicotinic acid-1-(substituted phenyl)-ethylidene/
cycloheptylidene hydrazide derivatives depicted the fact
that topological parameters, viz. third-order molecular
connectivity topological index (3v), govern all these activ-
ities. It was also observed that antifungal antimicrobial
activity of the synthesized isonicotinic acid-1-(substituted
phenyl)-ethylidene/cycloheptylidene hydrazide derivatives
are best explained by MLR models involving molecular
descriptors log P and valence third-order molecular con-
nectivity topological index (3vv). The developed mt-QSAR
models were statistically valid as they had the high r and
low residual values (Table 12). The low values of PE, LSE,
LOFs and SEP and the high value of Q, (Table 11) revealed
the statistical significance of the model described by
Eqs. 6–10.
Conclusion
acid-1-(substituted
phenyl)-ethylidene/cycloheptylidene
hydrazide derivatives.
A series of isonicotinic acid-1-(substituted phenyl)-ethyli-
dene/cycloheptylidene hydrazide derivatives (1–12) was
synthesized and tested for their in vitro antimycobacterial
activity against Mycobacterium tuberculosis, and the
compounds with Cl, Br, NO2 groups and cycloheptylidene
ring were found to be active ones. Compound 2 was found
to be more active antimycobacterial agent than isoniazid,
and compound 3 was also found to be equally active. The
Acknowledgments One of the authors (VJ) is thankful to the
Department of Technical Education, Government of Haryana (India)
for providing fellowship to carry out the research project. The authors
would like to thank Mrs. Leentje Persoons, Mrs. Frieda De Meyer,
Mrs. Kristien Erven, Mr. Kris Uyttersprot, Mrs. Anita Camps, Mrs.
Lies Van den Heurck, Mr. Steven Carmans, and Mrs. Leen Ingels,
Rega Institute for Medical Research, Belgium for their excellent
technical assistance in the evaluation of antiviral activity. Financial
123

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