792 Letters in Drug Design & Discovery, 2011, 8, 792-810

Isonicotinic Acid Hydrazide Derivatives: Synthesis, Antimycobacterial,

Antiviral, Antimicrobial Activity and QSAR Studies
Vikramjeet Judge1, Balasubramanian Narasimhan*,2, Munish Ahuja1, Dharmarajan Sriram3 and
Perumal Yogeeswari3

1
Department of Pharmaceutical Sciences, Guru Jambheshwar University of Science and Technology, Hisar 125001,
India
2
Faculty of Pharmaceutical Sciences, Maharshi Dayanand University, Rohtak 124001, India
3
Medicinal Chemistry Research Laboratory, Pharmacy Group, Birla Institute of Technology and Science, Hyderabad
500078, India
Received June 20, 2011: Revised July 19, 2011: Accepted July 19, 2011

Abstract: A series of isonicotinic acid hydrazide derivatives (1-14) was synthesized and tested for their in vitro antimy-
cobacterial activity against Mycobacterium tuberculosis and the compounds with bromo, N2-2,4-hexadienoyl, N2-lauryl
and N2-octadecanoyl groups were found to be the most effective, especially isonicotinic acid-N'-octadecanoyl hydrazide
(12) was more active than the standard drug isoniazid. The results of antiviral activity testing showed that none of the
tested compounds were active at subtoxic concentrations. The synthesized compounds were also screened for their antim-
icrobial potential against S. aureus, B. subtilis, E. coli, C. albicans and A. niger and the results indicated that compounds
4, 11, 12 and 14 were found to be active with Benzoic acid N'-(4-hydroxy-3,5-dimethoxy-benzylidene)-N-(pyridine-4-
carbonyl)-hydrazide (14) having highest antimicrobial potential. The QSAR studies indicated the importance of topologi-
cal parameters 3 and
1 in governing the antimicrobial activity of synthesized derivatives.
Keywords: Acylated isoniazid derivatives, Antimycobacterial, Antiviral, Antimicrobial, QSAR.

INTRODUCTION basic objectives involved in the development of new antitu-

Tuberculosis (TB), a contagious disease caused by My-
cobacterium tuberculosis, is one of the leading causes of
death in the world. The world health organization (WHO)
has reported that about 33% of the world’s population is in-
fected with TB, and has predicted that by the year 2020 there
will be one billion new active cases if new anti-TB drugs or
treatments are not developed [1]. The worldwide appearance
of drug-resistant strains of M. tuberculosis, along with social
problems such as homelessness, increased poverty and im-
migration, significantly contributed to this resurgence in both
developing and industrialized countries [2]. In most parts of
the world, we are limited to the combination of five drugs to
treat TB effectively, namely rifampicin, isoniazid (INH),
ethambutol, streptomycin and pyrazinamide. Problems in the
chemotherapy of tuberculosis arise when patients develop
bacterial resistance to any of these first line drugs and second
line drugs, such as ethionamide, aminosalicylic acid, cy-
closerine, amikacin, kanamycin and capreomycin are too
toxic and cannot be employed simultaneously [3]. The global
resurgence of TB has been favored by: (a) the diffusion of
the human immunodeficiency virus (HIV) and the deadly
synergy of TB and nontubercular mycobacterial infections
with HIV; (b) the widespread emergence of resistant strains
of Mycobacterium tuberculosis, which are insensitive to one
or more of the first line anti-TB drugs [4]. There are three
*Address correspondence to this author at the Faculty of Pharmaceutical
Sciences, Maharshi Dayanand University, Rohtak 124001, India; Tel:
No+91-1262-272535; Fax: +91-1262-274133;
E-mail: naru2000us@yahoo.com
bercular drugs: to reduce the total duration of treatment, to
improve the efficacy against MDR-TB and to provide more
effective treatment of latent tuberculosis infection [5].
There is a rapidly growing crisis in the clinical manage-
ment of life-threatening infectious disease caused by multi-
antibiotic-resistant (MAR) strains of pathogenic Gram-
positive (Gr+) bacteria. Solving this problem will depend, in
part, on the development of chemotherapeutic agents which
selectively attack new bacterial targets [6]. The rapid devel-
opment of drug resistance, the unsatisfactory status of pre-
sent treatments of bacterial and fungal infections and the
drug side effects limit the usage of most antimicrobial agents
[7]. Growing drug resistance among bacterial pathogens
drives the need for new agents effective against them. Ide-
ally, such antibacterial agents hit essential proteins that (i)
appear in as many bacterial pathogens as possible but (ii)
differ significantly from related mammalian proteins, to
yield a broad spectrum of activity with minimal mechanism-
based toxicity [8].
Acquired immunodeficiency syndrome (AIDS) is a fatal
disorder for which no complete and successful chemotherapy
has been developed so far. Human immunodeficiency virus
subtype 1 (HIV-1), a retrovirus of the lentivirus family, has
been found to be prevalent in causing this disease. HIV-1
produces a progressive immunosuppression by destruction of
CD4+ T lymphocytes (‘helper’ cells, which lead attack
against infections), and results in opportunistic infections
and death [9]. The replicative cycle of HIV is comprised of
ten steps that may be adequate targets for chemotherapeuti-
cal intervention. Most of the substances identified as anti-

1570-1808/11 $58.00+.00 © 2011 Bentham Science Publishers

Synthesis and Antimicrobial Evaluation of Isonicotinic Acid Hydrazides
HIV agents interfere with one of these steps of HIV replica-
tive cycle. These steps are: (1) viral adsorption to the cell
membrane, (2) fusion between the viral envelope and the cell
membrane, (3) uncoating of the viral nucleocapsid, (4) re-
verse transcription of the viral RNA to proviral DNA, (5)
integration of the proviral DNA into the cellular genome, (6)
DNA replication, (7) transcription of the proviral DNA to
RNA, (8) translation of the viral precursor mRNA to mature
mRNA, (9) maturation of the viral precursor proteins by pro-
teolysis, myristoylation, and glycosylation, and (10) bud-
ding, virion assembly, and release. Step 4, a key step in the
replicative cycle of retroviruses, which makes it distinct
from the replicative cycle of other viruses, is the reverse
transcription catalyzed by reverse transcriptase. Another
target for therapeutic intervention is step 9, particularly the
proteolysis of precursor proteins by HIV protease. The ma-
jority of chemotherapeutic strategies have, therefore, focused
on the development of retroviral enzyme inhibitors [10].
The main paradigm of medicinal chemistry is that bio-
logical activity, as well as physical, physicochemical and
chemical properties, of organic compounds depends on their
molecular structure [11]. Quantitative structure-activity rela-
tionships (QSAR) correlate, within congeneric series of
compounds, affinities of ligands to their binding sites, inhibi-
tion constants, rate constants, and other biological activities,
either with certain structural features (Free Wilson analysis)
or with atomic, group or molecular properties, such as lipo-
philicity, polarizability, electronic and steric properties
(Hansch analysis) [12]. The use of computational approaches
for the estimation of the activity of various molecules as
drug candidates prior to their synthesis accelerates drug dis-
covery procedure. The purpose in quantitative structure ac-
tivity relationship (QSAR) methodology is to construct a
relationship between physicochemical properties as inde-
pendent and bioactivity of ligands as a dependent variable.
Physicochemical properties could be obtained as descriptors
and then in silico methods are applied to manipulate the in-
formation, remove noise and derive useful information [13].
Isoniazid (INH) is the most frequently prescribed primary
prophylactic and chemotherapeutic drug against M. tubercu-
losis. Enzymatic acylation of the antitubercular isoniazid
(INH) by N-acetyl transferases (NATs) reduces the therapeu-
tic effectiveness of the drug. Because it represents a major
metabolic pathway for INH in human beings, such acetyla-
tion has serious consequences for tuberculosis treatment
regimens. Among patients in whom this process is efficient,
the “rapid acetylators,” the resultant chronic underdosing of
INH may give rise to the development of resistance, as well
as inadequate therapy. NATs occur in mycobacteria and in
their mammalian hosts. It is thought that the resultant chemi-
cal change prevents the activation of INH that is required for
proper drug action. A recombinant NAT from M. tuberculo-
sis acetylates INH in vitro. When the corresponding NAT
gene is over expressed in a suitable INH-susceptible host,
Mycobacterium smegmatis, the resultant organism becomes
more resistant to INH. Resistance to INH in mycobacteria
can thus be related to increased expression of NAT. Not
much work has been done previously to characterize the anti-
tubercular properties of other N2- acylisoniazids. The appre-
ciable in vitro and in vivo antimycobacterial activities of
acylated derivatives of isoniazid have been reported by
Letters in Drug Design & Discovery, 2011, Vol. 8, No. 9 793
Hearn and Cynamon [14]. The isoniazid derivatives have
also been reported to possess antimicrobial activity [15-17].
In order to bring sharper focus on the activties of acylated
derivatives of INH and in pursuit of achieving this goal, our
research efforts focused on the synthesis, antimicrobial, an-
timycobacterial, antiviral and QSAR studies [18-20], here-
with we report the synthesis, antimycobacterial, anti-HIV,
antimicrobial and QSAR studies of isonicotinic acid hydraz-
ide derivatives.
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
chromatography 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 (
, ppm) downfield from
tetramethylsilane (internal standard). Infrared (IR) spectra
were recorded on a Shimadzu FTIR spectrometer.
General Procedure for Synthesis of Ester
The mixture of isonicotinic acid (0.1 mol) and ethanol (in
excess) was refluxed with sulphuric 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 ice
cold water and excess of 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 appropriate time. The
reaction mixture was then cooled and the precipitated solid
was washed with water, dried and recrystallized from etha-
nol.
General Procedure for the Synthesis of N2-acyl Isonico-
tinic Acid Hydrazides (1-12)
The different carboxylic acids (0.01 mol) were refluxed
with thionyl chloride (0.015 mol) for three hours. After re-
fluxing was complete, the excess thionyl chloride was dis-
tilled off to get the respective acyl chlorides. The isonicotinic
acid hydrazide (0.01 mol) was suspended in dichloromethane
and acyl chloride (0.01 mol) was added dropwise to this so-
lution with constant stirring on a magnetic stirrer for 3-4 h.
The product obtained after evaporation of dichloromethane
was recrystallized from ethanol.
4-Methyl-benzoic acid N'-(pyridine-4-carbonyl)-hydrazide
(1)
Mp (°C) 166-169; Yield– 78.90%; 1H NMR (400 MHz,
DMSO)
ppm: 2.40-2.41 (s, 3H, CH3), 7.07-7.31 (m, 4H,
CH of phenyl ring), 8.34-8.38 (d, 2H, CH of C3 and C5 of
794 Letters in Drug Design & Discovery, 2011, Vol. 8, No. 9
pyridine ring), 9.00-9.02 (d, 2H, CH of C2 and C6 of pyridine
ring), 10.65 (s, 1H, NH proton of N2), 11.26 (s, 1H, NH pro-
ton of N1). IR (KBr pellets)  cm-1 : 3147.10 (NH str., am-
ide), 3028.65 (CH str., aromatic), 1662.11 (C=O str., Nicoti-
noyl), 1611.27 (C=O str., Acyl), 1490.17 (C=C str., skeletal
phenyl nucleus), 1280.84 (C-N str., coupled vibrations am-
ide), 834.68 (CH out of plane bending, 4-substituted pyri-
dine), 750.61 (OCN deformations, amide IV band), 631.54
(OCN deformations, amide VI band).
4-Methoxy-benzoic acid N'-(pyridine-4-carbonyl)-hydra-
zide (3)
Mp (°C) 220-223; Yield– 78.45%; 1H NMR (400 MHz,
DMSO)
ppm: 3.84-3.86 (s, 3H, OCH3), 6.92-6.95 (m, 2H,
CH of C3 and C5 of phenyl ring), 7.90-7.93 (d, 2H, CH of C2
and C6 of phenyl ring), 8.30-8.32 (d, 2H, CH of C3 and C5 of
pyridine ring), 9.08-9.09 (d, 2H, CH of C2 and C6 of pyridine
ring), 10.65 (s, 1H, NH proton of N2), 11.30 (s, 1H, NH pro-
ton of N1). IR (KBr pellets)  cm-1 : 3151.43 (NH str., am-
ide), 3054.68 (CH str., aromatic), 1685.94 (C=O str., Nicoti-
noyl), 1662.69 (C=O str., Acyl), 1499.45 (C=C str., skeletal
phenyl nucleus), 1286.45 (C-N str., coupled vibrations am-
ide), 1262.49 (C-O-C str., aralkyl ether) 838.52 (CH out of
plane bending, 4-substituted pyridine), 754.83 (OCN defor-
mations, amide IV band), 631.58 (OCN deformations, amide
VI band).
3-Nitro-benzoic acid N'-(pyridine-4-carbonyl)-hydrazide (4)
Mp (°C) 230-233; Yield– 72.59%; 1H NMR (400 MHz,
DMSO)
ppm: 6.98-7.46 (m, 4H, CH of phenyl ring), 7.90-
7.93 (d, 2H, CH of C3 and C5 of pyridine ring), 8.78-8.79 (d,
2H, CH of C2 and C6 of pyridine ring), 9.64 (s, 1H, NH pro-
ton of N2), 10.86 (s, 1H, NH proton of N1). IR (KBr pellets)
 cm-1: 3160.20 (NH str., amide), 3084.68 (CH str., aro-
matic), 1715.92 (C=O str., Nicotinoyl), 1636.19 (C=O str.,
Acyl), 1514.80 (NO2 asym. str., aromatic nitro group),
1363.25 (NO2 sym. str., aromatic nitro group), 1273.09 (C-N
str., coupled vibrations amide), 826.45 (CH out of plane
bending, 4-substituted pyridine), 747.91 (OCN deformations,
amide IV band), 624.43 (OCN deformations, amide VI
band).
5-Chloro-2-hydroxy-benzoic acid N'-(pyridine-4-carbonyl)-
hydrazide (7)
Mp (°C) 232-235; Yield– 63.81%; 1H NMR (400 MHz,
DMSO)
ppm: 6.96 (s,1 H, OH), 7.46-8.01 (m, 3H, CH of
phenyl ring), 8.31-8.33 (d, 2H, CH of C3 and C5 of pyridine
ring), 9.04-9.05 (d, 2H, CH of C2 and C6 of pyridine ring),
10.90 (s, 1H, NH proton of N2), 11.60 (s, 1H, NH proton of
N1). IR (KBr pellets)  cm-1: 3153.75 (NH str., amide),
3039.94 (CH str., aromatic), 1675.25 (C=O str., Nicotinoyl),
1603.88 (C=O str., Acyl), 1485.25 (C=C str., skeletal phenyl
nucleus), 1220.99 (C-N str., coupled vibrations amide),
835.21 (CH out of plane bending, 4-substituted pyridine),
750.34 (C-Cl str., aromatic), 648.11 (OCN deformations,
amide IV band), 535.27 (OCN deformations, amide VI
band).
Isonicotinic acid N'-hexa-2,4-dienoyl-hydrazide (10)
Mp (°C) 194-197; Yield– 65.48%; 1H NMR (400 MHz,
DMSO)
ppm: 1.85-1.89 (t, 3H, CH3), 6.05-6.26 (m, 3H,
CH of C3, C4 and C5 of 2,4-hexadienoyl chain), 7.24-7.26 (m,
Judge et al.
1H, CH of C2 of 2,4-hexadienoyl chain), 8.20-8.21 (d, 2H,
CH of C3 and C5 of pyridine ring), 8.85-8.88 (d, 2H, CH of
C2 and C6 of pyridine ring), 10.31 (s, 1H, NH proton of N2),
11.13 (s, 1H, NH proton of N1). IR (KBr pellets)  cm-1:
3239.22 (NH str., amide), 3000.28 (CH str., aliphatic),
1690.08 (C=O str., Nicotinoyl), 1659.87 (C=O str., Acyl),
1500.36 (C=C str., skeletal phenyl nucleus), 1457.73 (CH
bending, CH3), 1247.73 (C-N str., coupled vibrations amide),
818.95 (CH out of plane bending, 4-substituted pyridine),
739.94 (OCN deformations, amide IV band), 673.57 (OCN
deformations, amide VI band).
General Procedure for the Synthesis of Hydrazone of
Isonicotinic Acid Hydrazide (13)
Isonicotinic acid hydrazide (0.05 mol) and syringldehyde
(0.06 mol) in ethanol was refluxed for 5-6 h. At the comple-
tion of reaction the precipitates start to appear which were
filtered off and recrystallized from ethanol.
Isonicotinic acid (4-hydroxy-3,5-dimethoxy-benzylidene)-
hydrazide (13)
Mp (°C) 212-215; Yield– 78.90%; 1H NMR (400 MHz,
DMSO)
ppm: 3.91 (s, 6H, OCH3), 6.78 (s, 1H, OH), 7.02
(s, 2H, CH of C2 and C6 of dimethoxy phenyl ring), 7.84-
7.85 (d, 2H, CH of C3 and C5 of pyridine ring), 8.34 (s, 1H,
CH of N=CH), 8.75-8.77 (d, 2H, CH of C2 and C6 of pyri-
dine ring), 11.72 (s, 1H, NH). IR (KBr pellets)  cm-1:
3190.40 (NH str., amide), 3025.48 (CH str., aromatic),
1641.49 (C=O str., Nicotinoyl), 1587.48 (C=C str., skeletal
phenyl nucleus), 1220.99 (C-O-C str., aralkyl ether), 1116.83
(C-N str., coupled vibrations amide), 833.28 (CH out of
plane bending, 4-substituted pyridine), 547.81 (OCN defor-
mations, amide VI band).
General Procedure for the Synthesis of Benzoic Acid N'-
(4-hydroxy-3,5-dimethoxy-benzylidene)-N-(pyridine-4-
carbonyl)-hydrazide (14)
Isonicotinic acid (4-hydroxy-3,5-dimethoxy-benzyli-
dene)-hydrazide (13) was dissolved in dichloromethane
(0.001 mol) and a solution of benzoyl chloride (0.001 mol),
obtained by the reaction of benzoic acid with thionyl chlo-
ride, in dichloromethane was added drop wise and kept for
stirring on a magnetic stirrer till the evaporation of di-
chloromethane. The product obtained was checked for purity
by TLC and recrystallized from ethanol.
Benzoic acid N'-(4-hydroxy-3,5-dimethoxy-benzylidene)-N-
(pyridine-4-carbonyl)-hydrazide (14)
Mp (°C) 218-221; Yield– 58.90%; 1H NMR (400 MHz,
DMSO)
ppm: 3.92 (s, 6H, OCH3), 6.75 (s, 1H, OH), 7.04-
7.64 (m, 5H, CH of C6H5), 7.99-8.02 (s, 2H, CH of C2 and C6
of dimethoxy phenyl ring), 8.19-8.21 (d, 2H, CH of C3 and
C5 of pyridine ring), 8.48 (s, 1H, CH of N=CH), 8.84-8.85
(d, 2H, CH of C2 and C6 of pyridine ring). IR (KBr pellets) 
cm-1: 3211.662 (amide I band), 3067.91 (CH str., aromatic),
1655.96 (C=O str., Nicotinoyl), 1597.13 (C=O str., benzoyl),
1468.86 (C=C str., skeletal phenyl nucleus), 1236.42 (C-N
str., coupled vibrations amide), 832.32 (CH out of plane
bending, 4-substituted pyridine), 665.47 (OCN deformations,
amide VI band).
Synthesis and Antimicrobial Evaluation of Isonicotinic Acid Hydrazides
Evaluation of Antimycobacterial Activity
All compounds were screened for their in vitro antimy-
cobacterial 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 [21]. The minimum inhibitory concentration
(MIC) is defined as the minimum concentration of com-
pound required to give complete inhibition of bacterial
growth.
Evaluation of Antimicrobial Activity (Determination of
Minimum Inhibitory Concentration)
The antimicrobial activity was performed against Gram-
positive bacteria: S. aureus, B. sublitis, Gram-negative bacte-
rium: E. coli and fungal strains: C. albicans and A. niger by
tube dilution method [22]. Dilutions of test and standard
compounds [norfloxacin (antibacterial) and fluconazole
(antifungal)] were prepared in double strength nutrient broth
– I.P. (bacteria) and Sabouraud dextrose broth I.P. [23]
(fungi). The samples were incubated at 37 °C for 24 h (bac-
teria), at 25 °C for 7 d (A. niger) and at 37 °C 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).
QSAR Studies
Preparation of Data Set
An Intel (R) Core (TM) 2 Duo personal computer (CPU
T 6600 at 2.20 GHz) with windows XP operating system
was used. To obtain a QSAR model, the compounds must be
represented by molecular descriptors that retain as much
structure information as possible. The descriptors were gen-
erated as follows: The structures of acylated isonicotinic acid
hydrazide derivatives were first pre-optimized with the Mo-
lecular Mechanics Force Field (MM+) procedure included in
Hyperchem 6.03 [24] and the resulting geometries are further
refined by means of the semiempirical method PM3 (Para-
metric Method-3). We chose a gradient norm limit of 0.01
kcal/A˚ for the geometry optimization. The output files were
used for producing constitutional, topological, electrostatic,
and semiempirical descriptors using TSAR 3.3 software for
Windows [25]. These quantitative descriptors contain infor-
mation on the inter atom connections and the shape, branch-
ing, symmetry, distribution of charge and quantum-chemical
properties of the molecule. Further, the regression analysis
was performed using the SPSS software package [26]. The
predictive powers of the equation were validated by determi-
nation of cross-validated r2 (q2) using leave one out (LOO)
cross-validation method [27]. According to LOO method,
the predictive powers of the equations were validated by
leave one out (LOO) cross validation method, where a model
is built with N-1 compounds and Nth compound is predicted.
Each compound is left out of the model derivation and pre-
dicted in turn. An indication of the performance is obtained
from cross-validated q2) method which is defined as
q2 = 1 – (Ypredicted – Yactual)2 / (Yactual – Ymean)2
Letters in Drug Design & Discovery, 2011, Vol. 8, No. 9 795
where,Ypredicted, Yactual and Ymean are predicted, actual
and mean values of target property (pMIC) respectively.
(Ypredicted – Yactual)2 is predictive residual error sum of
squares.
Calculation of Statistical Parameters [28, 29]
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)
These parameters were calculated from the following
equations.
PE = 2(1- r2)/3
n
Where, r, correlation coefficient and n, number of com-
pounds used.
LSE = (Yobs-Ycalc)2
Where, Yobs and Ycalc are the observed and calculated
values.
LOF = LSE/{1-(C+d · p/n)}2
Where, LSE, least square error; C, number of descriptors
+1; p, number of independent parameters; n, number of
compounds used; d, smoothing parameter which controls the
bias in the scoring factor between equations with different
number of terms and was kept 1.0.
SEP =
LSE/n
The Quality value, Q is given by Q = r/Se
Where, Q, Quality value; r, correlation coefficient and
Se, standard error.
The predictive ability of QSAR models was also quanti-
fied in terms of q2, which is defined as
q2 = 1- {(Yobs-Ycalc)2/ (Yobs-Ymean)2}
The resubstitution test statistical parameters [30] viz. re-
gression coefficient (r), root mean square error (RMSE) and
absolute average error (e) were calculated as: Regression
coefficient (r) =
{ 1 – ((Ycalc - Yobs)2/ (Ycalc - Ymean)2)}
The Absolute average error (e) is used to illustrate the
predictive accuracy more explicitly and given by
Absolute average error (e) = {|Ycalc - Yobs|}/n
The root mean square error (RMSE) is calculated
mathematically as:
Root mean square error (RMSE) =
{(Ycalc - Yobs)2}/n
The low value of PE, LSE, LOF and SEP and high value
of Q and q2 are the essential criteria for qualifying the model
as the best one. Variation Inflation Factor [31] is employed
to determine the multicolinearity between the physicochemi-
cal parameters. The VIF value is calculated as VIF = 1/1-r2,
Where, r2 is the squared multiple correlation coefficient of
one parameter effect on the remaining parameter. VIF values
greater than 5 indicate the presence of unacceptably large
multicolinearity between parameters in the correlation.
796 Letters in Drug Design & Discovery, 2011, Vol. 8, No. 9 Judge et al.

Theory of Stepwise Multiple Linear Regression (MLR)
[32]
Multiple linear regression (MLR) is the most widely used
multiple linear modeling techniques. Following is the regres-
sion equation:
Y = b 0 + b1X1 + b 2X2 + . . . . + bnXn
In this equation, Y is the property, that is, the dependent
variable; X1–Xn represents the specific descriptors, while b1–
bn represents the coefficient of those descriptors, and b0 is
the intercept of this equation. As is well known, MLR cannot
be used to model complex data, since in most cases the num-
ber of explanatory variables exceeds the number of objects.
Therefore, it is often used in combination with the stepwise
procedure for variable selection. The forward stepwise re-
gression procedure consists simply in a step-by-step addition
of the best descriptors to the model that leads to the smallest
standard deviation(s), until there is no other variable outside
the equation that satisfies the selection criteria.
Evaluation of Anti-HIV Activity
The anti-HIV activity and cytotoxicity were evaluated
against HIV-1 strain IIIB and HIV-2 ROD in MT-4 cell cul-
tures using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide (MTT) method [33]. Briefly, virus
stocks were titrated in MT-4 cells and expressed as the 50%
cell culture infective dose (CCID50). MT-4 cells were sus-
pended in culture medium at 1 x 105 cells/ml and infected
with HIV at a multiplicity of infection of 0.02. Immediately
after viral infection, 100
l of the cell suspension was placed
in each well of a flat-bottomed microtiter tray containing

O O O NH NH
O O
O
OH SOCl2 Cl
O
N 9

CHO
Cl
O OH OC2H5 R5 R1 O
O NHNH2 O
O
O NHN
R4 R2 CH R1
C2H5OH NH2NH2 N N
R3 O CH R1
R5 R2
N N R5 R2
N
N R4 R3
R4 R3
N
13
14

COOH
COCl O
Y Y O
R1 SOCl2 R1
R5 R5 O NH NH Y O NH NH
RCOOH RCOCl
R5 R1 R
R4 X R2 R4 X R2
R3
R3 N R4 X R2
N
R3
10-12
1-8

Comp. R1 R2 R3 R4 R5 X Y R

1 H H CH3 H H - - -
2 H H C6H5 H H - - -
3 H H OCH3 H H - - -
4 H NO2 H H H - - -
5 Br H H H H - - -
6 H H Cl H H - CH2 -
7 OH H H Cl H - - -
8 H H - H H N - -
10 - - - - - - - -CH=CH-CH=CH-CH 3
11 - - - - - - - -C10H 20CH 3
12 - - - - - - - -C16H 32CH 3
13 H OCH3 OH OCH3 H - - -
14 H OCH3 OH OCH3 H - - -

Scheme 1. Synthetic route followed for the synthesis of isonicotinic acid hydrazide derivatives.
Synthesis and Antimicrobial Evaluation of Isonicotinic Acid Hydrazides Letters in Drug Design & Discovery, 2011, Vol. 8, No. 9 797

various concentrations of the test compounds. After a 4-d
incubation period at 37 °C, the number of viable cells was
determined using the MTT method. Compounds were tested
in parallel for cytotoxic effects in uninfected MT-4 cells.
RESULTS AND DISCUSSION
Chemistry
The acylated isonicotinic acid hydrazide derivatives were
synthesized as represented by 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 acylated isonicotinic acid
hydrazide derivatives (1-12) were synthesized by the reac-
tion of isonicotinic acid hydrazide, suspended in dichloro-
methane, with respective acyl chlorides which in turn were
obtained by refluxing the respective acids with thionyl chlo-
ride. The hydrazone of isonicotinic acid hydrazide (13) was
synthesized by refluxing Isonicotinic acid hydrazide with
syringldehyde in ethanol and compounds 14 was prepared by
the reaction of benzoyl chloride with solution of compound
13 (in dichloromethane). The target compounds were ob-
tained in appreciable yield and their physicochemical charac-
teristics are presented in Table 1.
The purity of acylated isonicotinic acid hydrazide deriva-
tives was assessed by single spot TLC on silica gel-G plates
and their IR and NMR spectra were found in agreement with
their assigned molecular structures. The two doublet signals,
in the region
7.90 – 9.09 ppm, each having an integral
equivalent to two protons corresponds the presence of four
CH protons of the pyridine ring in compounds 1, 3, 4, 7, 8,
10, 13 and 14. The singlet signals observed around
10 ppm
and
11 ppm represents the NH protons at N2 and N1 posi-
tion of the hydrazide portion in compounds 1, 3, 4, 7, 8, and
10. This clearly signifies that the acyl group was attached to
the isonicotinic acid hydrazide by replacing one of the two
protons of NH2 group. The singlet signal having equivalent
to three protons at
2.40 ppm depicted the presence of CH 3
group compound 1. In compound 2, a singlet signal at
3.84
ppm revealed the presence of a OCH3 group on the phenyl
ring in its structure. The appearance of signals for three aro-
matic protons and a singlet at
6.96 ppm confirmed that the
disubstituted phenyl ring attached as N2-substituent has got
OH group as one of the two substituents, thus confirming the
structure of compound 7. The presence of signals for unsatu-
rated aliphatic chain confirmed that 2,4-hexadienoyl sub-
stituent was present in compound 10. The formation of ben-
zylidene hydrazide (compound 13) was confirmed by the
singlet signal for N=CH linkage at
8.34 ppm and the pres-
ence of dimethoxy group in its structure was revealed by the
singlet signal at
3.91 ppm. The structure of benzoic acid
N'-(4-hydroxy-3,5-dimethoxy-benzylidene)-N-(pyridine-4-
carbonyl) hydrazide (14) was confirmed by the disappear-
ance of signals for the NH protons thus demonstrating that
both the NH protons have been replaced.
The vibrational frequencies corresponding to two C=O
functional groups appeared in the IR spectra of compounds
1, 3, 4, 7, 8, and 10 revealed the fact that the acyl group has
been added to the isonicotinic acid hydrazide molecule. The

Table 1. Physicochemical Properties and Antimycobacterial Activity of Synthesized Isonicotinic Acid Hydrazide Derivatives

Comp. Mol. Formula Mol. Wt. M.p. (°C) Rf* % yield MIC MTB-H37Rv (
M x 10-3)

1 C14H 13N3 O2 255 166-169 0.91 78.90 49.0

2 C19H 15N3 O2 317 210-213 0.65 81.35 19.7
3 C14H 13N3 O3 271 220-223 0.79 78.45 23.1
4 C13H 10N4 O4 286 230-233 0.71 72.59 43.7
5 C13H 10N3 O2Br 320 166-169 0.76 53.68 9.8
6 C14H 12N3 O2Cl 289 226-229 0.66 45.79 43.3
7 C13H 10N3 O3Cl 291 232-235 0.72 63.81 43.0
8 C12H 10N4 O2 242 Above 242 0.69 73.56 51.7
9 C18H 15N3 O3 321 212-215 0.51 45.92 38.9
10 C12H 13N3 O2 231 218-221 0.67 81.49 13.5
11 C18H 29N3 O2 319 140-143 0.58 68.32 4.9
12 C24H 41N3 O2 403 186-189 0.52 41.29 1.9
13 C15H 15N3 O4 301 212-215 0.72 78.90 21.0
14 C22H 19N3 O5 405 218-221 0.42 58.9 >62.0
INH 2.04
a
ETB 15.31
CFL 9.4
a
RIF 0.24
*Mobile phase: Ethanol.
a
D. Sriram, P. Yogeeswari, P. Dhakla, P. Senthilkumar, D., Banerjee, Bioorg. Med. Chem. Lett. 17 (2007) 1888-1891.
798 Letters in Drug Design & Discovery, 2011, Vol. 8, No. 9
amide I band was observed in the spectra of compounds 1, 3,
4, 7, 8, 10, and 13 in the region 3200-3100 cm-1. The pres-
ence of an aromatic and aliphatic portion in the synthesized
compounds was depicted by the presence of peaks slightly
above and below 3000 cm-1. The OCN deformations corre-
sponding to amide IV and amide VI bands were observed
around 750-720 cm-1 and 650-630 cm-1 in the IR spectra of
synthesized compounds. The skeletal C=C- stretching bands
(aromatic) were observed around 1513-1466 cm-1 in the
spectra of the synthesized compounds which represents the
presence of aromatic nucleus in their structure. The C-Cl
stretching band observed at 750.34 cm-1 that depicted the
presence of chloro group on aromatic nucleus in the molecu-
lar structure of compound 7. The C-O-C stretching band, in
compound 13, for aralkyl ether observed at 1220.99 cm-1
confirmed the presence of OCH3 group attached to an aro-
matic nucleus.
Antimycobacterial Activity
The in vitro antitubercular activity of synthesized acy-
lated isonicotinic acid hydrazides against Mycobacterium
tuberculosis (MTB) was carried out in Middlebrook
7H11agar medium supplemented with OADC (Oleic Acid
and Dextrose as Carbon source) by agar dilution method and
the results are presented in Table 1. At the commencement of
this study in the preliminary screening, compound (1), isoni-
cotinic acid hydrazide with a p-methyl substituted phenyl
ring attached at N2-position through carbonyl function, dis-
played poor antimycobacterial activity with a MIC of 49 x
10-3 μM. Therefore, compound 1 was taken as a lead mole-
cule and we planned to improve its antimycobacterial activ-
ity by altering the substituent on phenyl ring. The first step
towards lead optimization was incorporation of a benzene
ring as a substituent (C6H5) on phenyl ring. The addition of
benzene ring as a substituent greatly improved the antituber-
cular activity evidenced by the increase in the activity of
compound 2 (MIC = 19.7 x 10-3 μM) in comparison to 1.
Then we replaced the phenyl group with 4-methoxy substitu-
ent (Compound 3) on phenyl nucleus gave a molecule en-
dowed with appreciable antimycobacterial activity (MIC =
23.1 x 10-3 μM). Then electron withdrawing group were
taken as substituent on the phenyl nucleus and we synthe-
sized compound 4 having 3-nitro substitution but the com-
pound 4 was found to be less active with MIC of 43.7 x 10-3
μM. The next modification in the structure was addition of
electronegative bromo substituent on the phenyl nucleus that
resulted in the synthesis of compound 5, which has 2-bromo
substitution on phenyl nucleus. This molecule (compound 5)
was also found to be highly active antimycobacterial agent
with MIC of 9.8 x 10-3 μM. In compound 6, the 4-
chlorophenyl acetyl group was added as substituent on N2-
position of isonicotinic acid hydrazide, but this modification
leads to decrease in activity with MIC falling to 43.3 x 10-3
μM. Therefore, the idea of addition of acetyl group as a
linker was dropped and next change was addition of 2-OH-5-
Cl as substituent on the phenyl nucleus that resulted in the
synthesis of compound 7 but the antimycobacterial activity
of this molecule was also low with MIC of 43.0 x 10-3 μM.
Then we planned to add some hetero atom in the phenyl nu-
cleus and synthesized compound 8 having hetero atom nitro-
gen at 4th position of the phenyl nucleus. This idea also has
Judge et al.
gone in vain and compound 8 was found to be least active,
with MIC of 51.7 x 10-3 μM among the synthesized com-
pounds. In compound 9, the napthoxy acetyl group was
taken as N2-substituent and this compound has got some
improvement in activity with MIC of 38.9 x 10-3 μM. The
next modification was the replacement of phenyl nucleus
with aliphatic chain of carbon atoms that resulted in com-
pound 10, which has 2,4-hexadienoyl group as substituent
attached to the N2 position of isonicotinic acid hydrazide and
the synthesized molecule was found to be highly active with
MIC 13.5 x 10-3 μM. Then we added lauryl aliphatic chain as
N2 substituent, in compound 11, and got a molecule with still
improved antimycobacterial activity having MIC of 4.9 x10-3
μM. In compound 12, stearoyl group (acyl group of stearic
acid) was taken as substituent on N2 of hydrazide and the
resultant molecule was the most active antimycobacterial
agent with MIC of 1.9 x 10-3 μM, which was lowest among
the derivatives synthesized in this series.
From the results of antimycobacterial activity the follow-
ing conclusions regarding structure activity relationship
(SAR) can be drawn:
1. The replacement of the phenyl nucleus with long ali-
phatic chain (Compound 10, 11 and 12) gave the
molecules with highly improved antimycobacterial
activity. This is similar to one of our previous report
in which an increase in antimicrobial activity was ob-
served with long aliphatic chain compounds [34].
2. In contrast with Tripathi and his coworkers [35] who
stated that the OH group at ortho position leads to a
measurable change in activity of the compounds, the
presence of the OH group at ortho position (Com-
pound 7) decreased the activity of the compounds.
3. The presence of electron withdrawing bromo group
on the phenyl ring increases the antitubercular activ-
ity as evidenced by the high antimycobacterial activ-
ity of compounds 5. The role of electron withdrawing
group in improving activity is supported by the earlier
studies [36].
4. In contrast with above fact, the electron withdrawing
group nitro present at 3rd position (compound 4) de-
creased the antimycobacterial activity.
5. The results of antimycobacterial activity depicted that
the presence of electron donating group, OCH3, en-
hanced the antitubercular activity of the synthesized
derivatives (Compound 3). This is in concordance
with the findings of Emami and coworkers who ob-
served increase in activity by adding OCH3 group
[37].
6. The presence of electron withdrawing bromo group in
compound 5 enhances the growth inhibition potential
of the molecule, which is similar to the earlier results
[38] where it was observed that the presence of
bromo and chloro substituents on the benzene ring in-
creases the activity.
7. The compound 11 and 12 having high logP values
were found to be more active than other synthesized
derivatives which is similar to the findings of Sriram
and coworkers [39] who observed that increased lipo-
Synthesis and Antimicrobial Evaluation of Isonicotinic Acid Hydrazides Letters in Drug Design & Discovery, 2011, Vol. 8, No. 9 799

philicity of 1-[(4-sub)phenyl]-3-(4-{1-[(pyridine-4-
carbonyl)hydrazono]ethyl}phenyl thiourea rendered
them more capable of penetrating various biomem-
branes.
Based on the SAR results, we have synthesized a test set
of isonicotinic acid hydrazide derivatives (13, 14) so as to
validate the SAR findings by the results of antimycobacterial
activity of test set compounds. The hydrazones of isonico-
tinic acid hydrazide have been reported to be active antimy-
cobacterial agents. So, hydrazone of isonicotinic acid hy-
drazide, compound 13 having 4-OH-3,5-OCH3 substituent
on phenyl nucleus, was synthesized and this compound was
found to be moderately active with MIC of 21.0 x 10-3 μM.
The fact that presence of phenyl substituent increased the
antimycobacterial activity of isoniazid derivatives, lead us to
the synthesis of compound 14 in which the N1-proton of 13
was replaced with benzoyl nucleus and this substitution de-
creased the antimycobacterial activity (MIC) to > 62.0 x 10-3
μM. It can be seen from these results that presence of at least
one NH group is necessary for a molecule to be an active
antimycobacterial agent by forming a hydrogen bond at the
mycobacterial target site.
In conclusion, it can be stated that long alkyl chain sub-
stituted derivatives were highly active antimycobacterial
agents with compound 12 having stearoyl substituent on N2-
position (MIC = 1.9 x 10-3 μM) was more active than isoni-
azid (MIC = 2.04 x 10-3 μM). This may be due to the fact
that mycolic acid that constitutes the mycobacterial cell wall
contains long chain stearyl group in its molecular structure
and compounds derived from long alky chains may act as
false substrate for the Mycobacterium and get incorporated
in the bacterial cell wall and give rise to a pseudo cell wall
and also carries the isonicotinic acid hydrazide attached to its
structure in to the bacterial cell thus leading to an increased
uptake of isonicotinic acid hydrazide by the Mycobacterium
which may be cause of lysis of Mycobacterium and in-
creased antimycobacterial potential of saturated long chain
acylated isonicotinic acid hydrazide derivatives.
Cytostatic, Cytotoxic and Antiviral Activity
The anti-HIV activity and cytotoxicity were evaluated
against HIV-1 strain IIIB and HIV-2 ROD in MT-4 cell cul-
tures and generally, none of the compounds were inhibitory
to HIV viruses tested at subtoxic concentrations (Table 2).
Antibacterial and Antifungal Activity
The synthesized acylated isonicotinic acid hydrazide de-
rivatives [1-14] were evaluated, in vitro, for their antibacte-
rial activity against Gram positive Staphylococcus aureus,
Bacillus subtilis, Gram negative Escherichia coli and anti-
fungal activity against Candida albicans and Aspergillus
niger by the serial dilution method [22] using norfloxacin
and fluconazole as reference standards for antibacterial and
antifungal activity, respectively, and the results are presented
in Table 3.
The results of antibacterial activity against B. subtilis in-
dicated that all the synthesized derivatives have higher anti-
bacterial potential than isoniazid. In particular the com-
pounds 4 and 12 were found to be the most effective antibac-

Table 2. Anti-HIV Potential of Synthesized Isonicotinic Acid Hydrazide Derivatives in MT-4 Cellsa

Compound EC50 (
g/ml) CC50 b (
g/ml)

HIV-1 (IIIb) HIV-2 (ROD)

1 >112.00 >112.00
112.00

2 >102.65 >102.65 102.65
3 >110.77 >110.77 110.77 ± 9.19
4 >122.00 >122.00
122.00
5 >125.00 >125.00 >125.00
6 >125.00 >125.00 >125.00
7 >4.72 >4.72 4.72 ± 1.68
8 >103.00 <103.00 103.00 ± 9.76
9 >94.95 >94.95 >94.95± 3.28
10 >90.10 >90.10
90.10
11 >39.35 >39.35 39.35± 3.17
12 >13.81 >13.81 >13.81 ± 9.67
13 >105.00 >105.00
105.00
14 >125.00 >125.00 >125.00
Nevirapine 0.050 ± 0.011 >4.0 >4.0
Zidovudine 0.002 ± 0.001 0.002 ± 0.001 >25
a
50% Effective concentration or compound concentration required to inhibit virus-induced cytopathicity by 50%. b50% Cytotoxic concentration or compound concentration required
to reduce MT-4 cell viability by 50%.
NT- not tested.
800 Letters in Drug Design & Discovery, 2011, Vol. 8, No. 9 Judge et al.

terial agents having pMICbs values 2.96 and 2.21
M, re-
spectively. In case of S. aureus compounds 2 and 3 have
shown marked antibacterial potential at pMICsa values 2.40
and 2.64
M, respectively. For antibacterial activity against
E. coli compounds 4, 5, 9 and 11 had shown better antibacte-
rial activity with pMICec values of 2.66, 2.41, 2.41 and 2.41

M, respectively. The antibacterial potential of compound 4
was found to be better than the standard drug norfloxacin.
The antifungal activity against C. albicans revealed that
compounds 6, 7, 9 and 11 were the potential candidates hav-
ing pMICca values 2.67, 2.67, 2.71 and 2.71
M, respectively
and their antifungal activity was higher than the standard
drug fluconazole. In case of A. niger compounds 5, 9, 11 and
12 were found to be active with pMICan values 2.41, 2.41,
2.41 and 2.51
M, respectively.
It can be observed from the results presented in Table 3
that the synthesized acylated isonicotinic acid hydrazide de-
rivatives have higher antifungal potential particularly against
C. albicans. The compounds were also found to be more
active against Gram negative E. coli as compared to Gram-
positive B. subtilis and S. aureus.
Structure Activity Relationship
From the results of antimicrobial activity, the following
structure activity the relationship can be drawn:
1. The antifungal potential of acylated isonicotinic acid
hydrazide derivatives was higher than the antibacte-
rial activity, which shows that different structural re-
quirements are essential for antibacterial and antifun-
gal activity. The results are similar to earlier ones
[40].
2. Similar to the observations of Guven and coworkers
[41], the presence of substitutent at ortho position in-
creased the antibacterial and antifungal potential of
the compounds which can be seen from the antimi-
crobial activity of compound 5, and 7.
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 3). This fact is supported by the
previous results [37].
4. The synthesized derivatives were more active towards
the Gram-negative bacterium E. coli than Gram-
positive B. subtilis and S. aureus. This finding is in
agreement with the studies of Sbardella and cowork-
ers [42].
5. It was observed from the results of antimicrobial ac-
tivity that the introduction of phenyl group as sub-
stituent in compound 2 in comparison to 4-methyl
substituent (compound 1) enhanced the growth inhibi-
tion potential of synthesized derivative.
6. Compounds 11 and 12 having long aliphatic chains as
substituent were found to be most active antimicro-
bial agent among the synthesized derivatives. This is
similar to one of our previous reports [34].

Table 3. Antibacterial and Antifungal Potential (
M/mL) of Synthesized Isonicotinic Acid Hydrazide Derivatives

Comp. pMIC bs pMICsa pMICec pMICca pMICan pMIC b pMICf pMICam

Training set
1 2.01 2.01 2.01 2.31 2.31 2.01 2.31 2.13
2 2.10 2.40 2.40 2.40 2.40 2.30 2.40 2.34
3 2.04 2.64 2.34 2.34 2.34 2.34 2.34 2.34
4 2.96 2.36 2.66 2.36 2.36 2.66 2.36 2.54
5 2.11 2.11 2.41 2.41 2.41 2.21 2.41 2.29
6 2.06 2.06 2.36 2.67 2.36 2.16 2.52 2.30
7 2.07 2.07 2.37 2.67 2.37 2.17 2.52 2.31
8 1.99 1.99 1.99 2.59 2.29 1.99 2.44 2.17
9 2.11 2.11 2.41 2.71 2.41 2.21 2.56 2.35
10 1.97 1.97 2.27 2.27 2.27 2.07 2.27 2.15
11 1.80 2.11 2.41 2.71 2.41 2.11 2.56 2.29
12 2.21 2.21 2.21 2.21 2.51 2.21 2.36 2.27
INH 1.74 2.04 1.74 2.34 2.04 1.84 2.19 1.98
S.D.a 0.29 0.20 0.24 0.18 0.11 0.20 0.11 0.14
Test set
13 2.38 2.98 2.38 2.68 2.38 2.58 2.53 2.56
14 2.51 2.51 2.51 3.11 2.51 2.51 2.81 2.63
Std. 2.61* 2.61* 2.61* 2.64** 2.64** 2.61 2.64 2.62
*Norfloxacin; **Fluconazole; a Standard deviation.
Synthesis and Antimicrobial Evaluation of Isonicotinic Acid Hydrazides
7. The presence of electron withdrawing groups on the
phenyl ring increases the antitubercular activity as
evidenced by the high antimycobacterial activity of
compounds 4 and 5. The role of electron withdrawing
group in improving activity is supported earlier study
[36].
The SAR findings of the antimycobacterial and antimi-
crobial activity are summarized in Fig. (1).
The synthesized derivatives of test set were also evalu-
ated for their antimicrobial activity against all the strains
tested above. The results of antimicrobial activity studies
revealed that compound 14 was highly active against all the
five microorganisms tested and its antifungal potential
against Candida albicans was better than the standard drug
fluconazole. Compound 13 was also active antimicrobial
agent especially against S. aureus for which its antibacterial
activity was higher than the standard drug norfloxacin.
QSAR Studies
Development of One-Target QSAR Model
QSAR analysis is an area of computational research
which builds models of biological activity using physico-
chemical properties of a series of compounds. QSAR ap-
proach is based on the assumption that the behavior of a
compound, expressed by any measured activity which is
correlated with the molecular features of the compound,
termed descriptors. After calculation of the molecular de-
scriptors, the commonly used linear methods, such as linear
regression (LR) and multiple linear regression (MLR), can
be used in the development of a relationship between the
structure descriptors and the activity [32]. By definition, a
model is not reality and does not replace reality (e.g., ex-
periments and observations). A model considers some se-
lected and relevant elements of a phenomenon and attempts
to mimic their behavior in such a way that predictions can be
made. A model is useful in those situations in which either
O Increases antimicrobial activity
H H
O N N C
N
Essential for
antimycobacterial activity
Replacement with small
alipahtic alkyl chain
Fig. (1). Structural requirements for the antimycobacterial and antimicrobial activity of synthesized isonicotinic acid hydrazide derivatives.
Letters in Drug Design & Discovery, 2011, Vol. 8, No. 9 801
we do not want or we cannot carry out direct experiments.
Thus, the key features of a model are (a) the phenomenologi-
cal elements that it represents and (b) the accuracy of the
representation [43]. To obtain suitable equations two factors
were taken into account [44].
1. A ratio of compounds to variables greater than 5.
2. An intercorrelation among the independent variables
smaller than 0.6.
In order to identify the substituent effect on the antimi-
crobial activity, quantitative structure activity relationship
(QSAR) studies between the in vitro antimicrobial activity
and descriptors coding for lipophilic, electronic, steric and
topological properties of the 12 synthesized N2-acyl isonico-
tinic acid hydrazide derivatives were undertaken, using the
linear free energy relationship model (LFER) described by
Hansch and Fujita [45]. Biological activity data determined
as MIC values was first transformed into pMIC values (i.e. –
log MIC) to get all the values positive, normal distribution of
errors and to get linear free energy relationship of these data
with physicochemical properties and used as dependent vari-
able in QSAR study.
The thermodynamic, spatial, electronic and topological
parameters calculated for QSAR analysis are presented in
Table 4. Thermodynamic parameters describe free energy
change during drug receptor complex formation. Spatial pa-
rameters are the quantified steric features of drug molecules
required for its complimentary fit with receptor. Electronic
parameters describe weak non-covalent bonding between
drug molecules and receptor [46]. The different molecular
descriptors (independent variables) like log of octanol–water
partition coefficient (log P), molar refractivity (MR), Kier’s
molecular connectivity (0
, 0
v, 1
, 1
v, 2
, 2
v) and shape
(1, 2, 3, 
1, 
2, 
3) topological indices, Randic topo-
logical index (R), Balaban topological index (J), Wiener
topological index (W), Total energy (Te), energies of highest
occupied molecular orbital (HOMO) and lowest unoccupied
molecular orbital (LUMO), dipole moment (μ), nuclear re-
Replacement with long Increases antimycobacterial activity
alipahtic alkyl chain
Increases antimicrobial activity
Increases antimycobacterial activity
Br
Y
Increases antimycobacterial activity
NO2
Increases antimicrobial activity
R'
Decreases antimycobacterial activity
OH
X Increases antimicrobial activity
Increases antimycobacterial activity
Cl
Increases antibacterial activity
Decreases antimycobacterial activity
CH3
Decreases antibacterial activity
Decreases antimycobacterial activity
Decreases antimicrobial activity
802 Letters in Drug Design & Discovery, 2011, Vol. 8, No. 9 Judge et al.

pulsion energy (Nu.E) and electronic energy (Ele.E), calcu-
lated for isoniazid derivatives are presented in Table 4 [47-
52]. Units of the energies and dipole were electron volts
(eV), and atomic units (a.u.), respectively [53].
For Hansch analysis, regression was performed using
pMIC values as dependent variables and calculated parame-
ters as independent variables. From a large number of equa-
tions obtained, some of the best QSAR equations are given
below. These equations were generated in stepwise manner
by forward selection method starting with best single vari-
able and adding further significant variable according to their
contribution to the model.
In the present study, a data set of 13 compounds (12 acy-
lated isonicotinic acid hydrazide derivatives along with
isoniazid itself) was subjected to linear free energy regres-
sion analysis for model generation. During the regression
analysis studies it was observed that the response values of
compound 3 were outside the limits of response values of
other synthesized N2-acyl isonicotinic acid hydrazide deriva-
tives. Thus compound 3 was designated as an outlier and it
was not involved in the data set for QSAR model generation.
In multivariate statistics, it is common to define three types
of outliers [54].
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.
2. X outliers. Briefly, 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 sam-
ples. They are substances for which the reference
value of response is invalid.
A compound was considered as an outlier for deriving a
particular model when the residual value exceeded twice the
standard error of estimate of the model [55]. In light of the
above guidelines, Compound 3 was considered as outlier
because its response values [antimicrobial activity] were
outside the range in comparison to the other compounds in-
cluded in the present study.
Based on the colinearity problem among the descriptors
and their contribution towards the biological activity, differ-
ent descriptors and/or a combination of descriptors were sub-
jected to linear regressions using SPSS. Preliminary analysis
was carried out in terms of correlation analysis. A correlation
matrix constructed for antibacterial activity against S. aureus
is presented in Table 5. The correlations of different molecu-
lar descriptors with antibacterial and antifungal activity are
presented in Table 6. The high interrelationship was ob-
served between 0
and 1
(r = 0.996), 0
and Ele.E (r = -
0.996), and low interrelationship was observed between log
P and 3
(r = -0.017). The correlation matrix indicated that
topological parameters (1
, 3
and
1), total energy (Te) and
energy of lowest unoccupied molecular orbital (LUMO)
governs the antimicrobial activity of the synthesized com-
pounds against various tested strains. The statistical quality
of the models was gauged by the parameters like correlation
coefficient (r) or squared correlation coefficient (r2), standard
error of estimate (s), variance ratio (F). In order to corrobo-
rate the validity of the derived QSAR models, leave-one-out
(LOO) method was used. Sum of squared prediction errors
called predictive residual sum of squares (PRESS) statistic is
calculated as the sum of squares of the differences between
predicted and observed values of the activity. Standard de-
viation of prediction (Spress), the cross-validated correlation
coefficient (q2) and standard deviation error of predictions
(SDEP) were calculated for each model and taken as an es-
timate of the predictability of the models.

Table 4. Value of Selected Descriptors Used in the Regression Analysis

Comp. log P MR 0

1

2

3


1 W Te LUMO HOMO

1 2.033 70.530 13.665 9.165 7.950 1.093 15.390 808.000 -3231.200 -0.693 -9.773
2 3.250 90.625 16.778 11.737 10.003 1.138 18.781 1592.000 -3898.170 -0.780 -9.267
3 1.313 71.952 14.372 9.703 8.119 1.009 16.372 946.000 -3551.200 -0.696 -9.443
4 1.519 72.814 15.242 10.075 8.861 1.305 17.355 1050.000 -3906.130 -1.442 -10.375
5 2.357 73.112 13.665 9.182 7.856 1.010 15.390 784.000 -3414.810 -0.729 -9.952
6 2.016 74.843 14.372 9.648 8.420 1.184 16.372 968.000 -3591.250 -0.872 -9.465
7 1.799 71.988 14.535 9.575 8.489 1.299 16.372 896.000 -3756.010 -0.770 -9.613
8 0.718 63.300 12.795 8.771 7.328 0.805 14.410 689.000 -3140.250 -0.861 -10.276
9 2.158 87.728 16.778 11.720 10.119 1.229 18.781 1632.000 -4090.400 -0.921 -8.615
10 1.289 65.956 12.510 8.236 6.554 0.691 15.059 638.000 -2974.690 -0.725 -9.459
11 3.847 91.352 16.753 11.236 8.675 0.691 21.044 1714.000 -3966.730 -0.801 -10.203
12 6.225 118.958 20.996 14.236 10.796 0.691 27.035 3618.000 -4901.740 -0.821 -10.254
INH 0.020 36.934 7.397 4.843 3.784 0.402 8.100 121.000 -1804.620 -0.594 -10.310
13 1.789 80.177 15.949 10.651 8.839 1.132 18.340 1205.000 -4026.800 -0.691 -8.473
14 3.700 109.915 21.510 14.545 12.289 1.584 24.639 2493.000 -5297.280 -0.643 -8.594
Synthesis and Antimicrobial Evaluation of Isonicotinic Acid Hydrazides Letters in Drug Design & Discovery, 2011, Vol. 8, No. 9 803

Model 1: The QSAR model represented by Eq. 1 corre-
lates the antifungal activity of synthesized acylated isonico-
tinic acid hydrazide derivatives against A. niger with mo-
lecular descriptors and demonstrated that the antifungal po-
tential is governed by the total energy (Te) of the molecules
(Eq.1).
ot-QSAR model for antifungal activity against A. niger
pMICan = -0.00015 Te + 1.824
2
n = 12 r = 0.956 q = 0.818 s = 0.036 F = 105.92
Here and thereafter, n - number of data points, r - correla-
tion coefficient, q2 - cross validated r2 obtained by leave one
out method, s - standard error of the estimate and F - Fischer
statistics.
For antifungal activity against A. niger, the developed
QSAR model (Eq. 1) describes the importance of total en-
ergy (Te) of the molecule. The total energy (Te) calculated
by semiemperical methods can be used as a measure of non-
specific interactions of the drug with its target site i.e. the
total energies of protonated and neutral molecule, can be
considered as a good measure of hydrogen bonds (the higher
the energy, the stronger the bond) and can be used to deter-
mine the correct localization of the most favorable hydrogen
bond acceptor site [56]. The results presented in the Table 3
are in concordance with the model expressed by Eq. 1 and
values of Te shown in Table 4 which depicts that compounds
9 and 12 having lowest Te values (-4090.400 and -4901.740)
have got the highest antifungal potential (2.41 and 2.51). The
value of r2 for Eq. 1 is 0.914 which means the QSAR model
is able to predict the 91.40% of variance.
To systematically assess a prediction algorithm, a reliable
validation is required. Usually, a predictive method is evalu-
ated by the predictive results for a training data set and test-
ing data set, respectively. According to the statistical termi-
nology, the former is called a test of resubstitution, and the
latter is a test of cross-validation. By the test of resubstitu-
tion, the value of each compound in a training data set is
predicted using the rules derived from the same set. Al-
though this test gives somewhat optimistic error estimate
because the same compounds are used to derive the predic-
tion rules and to predict themselves, the resubstitution test is
absolutely necessary due to its ability of reflecting the self-
consistency of a given method. On the other hand, a cross-
validation test for an independent testing data set is also
needed because it can reflect the generalizing effectiveness
Eq. 1 of a predictive method. Of various cross-validation tests, the
LOO test is thought to be a reliable one. Both tests of resub-
stitution and LOO [30] were used to evaluate the prediction
of models developed in the current study. In the resubstitu-
tion test, three statistical parameters viz. regression coeffi-
cient (r), root mean square error (RMSE) and absolute aver-
age error (e
) were used to evaluate the performance. The
QSAR model expressed by Eq. 1 was cross validated by its
high q2 values (q2 = 0.818) obtained with leave one out
(LOO) method. The value of q2 greater than 0.5 is the basic
requirement for qualifying a QSAR model to be valid one
[57]. The comparison of observed and predicted antibacterial
activities is presented in Table 7. It can be seen from the
results that the observed and predicted antibacterial activities
lie close to each other as evidenced by their low residual
values (Table 7). The plots of observed, predicted and resid-
ual pMIC activity values were also developed to check the
statistical validity of QSAR models. The plot of predicted
pMICan against observed pMICan (Fig. 2) also favors the
model expressed by Eq. 1. Further, the plot of observed
pMICan Vs residual pMICan (Fig. 3) indicated that there was
no systemic error in model development as the propagation
of error was observed on both sides of zero [31].
Equations 2 - 4 were developed to predict the antibacte-
rial activity of acylated isonicotinic acid hydrazide deriva-
tives against B. subtilis and E. coli.
ot-QSAR model for antibacterial activity against B. subtilis
pMICbs = - 1.315 LUMO + 0.997 Eq. 2
2
n = 12 r = 0.914 q = 0.635 s = 0.129 F = 50.81

Table 5. Correlation Matrix for Antifungal Activity of Synthesized Isonicotinic Acid Hydrazide Derivatives Against A. niger

log P MR 0
 1
 2
 3

1

1 Te Ele.E LUMO HOMO pMICan

log P 1.000
MR 0.946 1.000
0
 0.892 0.985 1.000
1
 0.881 0.984 0.996 1.000
2
 0.759 0.913 0.958 0.966 1.000
3
 -0.017 0.202 0.332 0.334 0.562 1.000

1 0.937 0.986 0.981 0.969 0.884 0.163 1.000

1 0.953 0.971 0.953 0.936 0.828 0.070 0.992 1.000
Te -0.832 -0.948 -0.986 -0.976 -0.966 -0.439 -0.952 -0.920 1.000
Ele.E -0.902 -0.982 -0.996 -0.992 -0.946 -0.303 -0.982 -0.959 0.983 1.000
LUMO -0.008 -0.189 -0.324 -0.304 -0.397 -0.522 -0.261 -0.202 0.419 0.330 1.000
HOMO -0.027 0.168 0.179 0.223 0.345 0.486 0.063 -0.012 -0.178 -0.131 0.138 1.000
pMICan 0.799 0.911 0.938 0.931 0.940 0.479 0.893 0.861 -0.956 -0.922 -0.316 0.222 1.000
804 Letters in Drug Design & Discovery, 2011, Vol. 8, No. 9 Judge et al.

Table 6. Correlation of Molecular Descriptors with Antimicrobial Activity of Synthesized Isonicotinic Acid Hydrazide Derivatives

Mol. Descriptor pMIC bs pMICsa pMICec pMICca pMICan pMIC b pMICf pMICam

log P 0.088 0.422 0.327 -0.144 0.799 0.277 0.278 0.333

MR 0.227 0.472 0.495 0.006 0.911 0.428 0.450 0.521
0
 0.348 0.517 0.581 0.066 0.938 0.535 0.511 0.633

0 v
0.203 0.421 0.478 0.024 0.915 0.397 0.467 0.500
1
 0.324 0.527 0.561 0.081 0.931 0.517 0.520 0.621

1 v
0.152 0.405 0.403 0.000 0.866 0.337 0.423 0.434
2
 0.427 0.568 0.659 0.175 0.940 0.618 0.598 0.734

2 v
0.172 0.419 0.434 0.039 0.895 0.363 0.469 0.471
3
 0.581 0.376 0.669 0.356 0.479 0.654 0.518 0.736

3 v
0.293 0.178 0.495 0.359 0.409 0.391 0.487 0.499
1 0.277 0.448 0.501 -0.004 0.893 0.451 0.433 0.535
2 0.105 0.302 0.287 -0.120 0.733 0.242 0.264 0.298
3 0.045 0.213 0.201 -0.156 0.647 0.157 0.193 0.201

1 0.214 0.387 0.438 -0.017 0.861 0.379 0.408 0.464

2 0.057 0.252 0.233 -0.124 0.695 0.185 0.242 0.241

3 0.013 0.184 0.162 -0.156 0.619 0.117 0.180 0.162
R 0.324 0.527 0.561 0.081 0.931 0.517 0.520 0.621
J -0.339 -0.435 -0.421 -0.376 -0.483 -0.443 -0.534 -0.561
W 0.187 0.442 0.283 -0.133 0.757 0.314 0.265 0.360
Te -0.439 -0.524 -0.664 -0.140 -0.956 -0.616 -0.579 -0.726
Ele.E -0.355 -0.539 -0.557 -0.055 -0.922 -0.534 -0.494 -0.627
Nu.E 0.344 0.540 0.543 0.045 0.915 0.522 0.483 0.613
LUMO -0.914 -0.560 -0.656 -0.076 -0.316 -0.860 -0.215 -0.813
HOMO -0.136 -0.021 0.277 0.348 0.222 0.040 0.386 0.163

0.543 0.360 0.716 0.128 0.674 0.652 0.433 0.706

ot-QSAR model for antibacterial activity against E. coli
pMICec = 0.197
+ 1.492 Eq. 3
2
n = 12 r = 0.716 q = 0.292 s = 0.182 F = 10.51
ot-QSAR model for antibacterial activity against E. coli de-
veloped by MLR
pMICecMLR = 0.506 3 + 0.022 1 + 1.402 Eq. 4
2
n = 12 r = 0.778 q = 0.102 s = 0.172 F = 6.90
Model 2: The model expressed by Eq. 2 demonstrated
that antibacterial activity against B. subtilis is governed by
the energy of lowest unoccupied molecular orbital (LUMO)
of the molecule. The electronic parameter LUMO which
denote the energy of the lowest unoccupied molecular or-
bital, directly relates to the electron affinity and characterizes
the susceptibility of the molecule towards an attack by nu-
cleophiles [58]. The model expressed by Eq. 2, having high r
and q2 values, has good predictability as evidenced by the
low residual values in Table 7.
Model 3: In case of E. coli, the model expressed by Eq. 3
depicted the importance of dipole moment (
) in describing
the antibacterial activity of synthesized derivatives. In this
case the bi-parametric model expressed by Eq. 4, obtained by
multiple linear regression demonstrated that a combination
of third order molecular connectivity index (3) and Kier’s
first order shape topological index (1) best describes the
antibacterial activity of the synthesized derivatives which
can be seen from the low residual values obtained by Eq. 4
(Table 7). Statistically valid models were not obtained for
antibacterial activity against S. aureus and antifungal activity
against C. albicans.
As in case of Eq. 1, the high q2 values (q2>0.5) supported
the validity of developed QSAR models described by Eq. 2.
The cross validated correlation coefficient (q2 > 0.5) values
obtained for best QSAR model indicated their reliability in
predicting the antimicrobial activity of synthesized com-
pounds. In case of Eq. 3 and Eq. 4, the value of q2 is less
than 0.5 for the developed QSAR models, which shows that
the developed model is an invalid one. But according to the
recommendations of Kim and his coworkers. [59], the re-
gression models are acceptable if the value of standard de-
viation (SD, Table 3) is not much larger than 0.3. As the
Synthesis and Antimicrobial Evaluation of Isonicotinic Acid Hydrazides Letters in Drug Design & Discovery, 2011, Vol. 8, No. 9 805

Table 7. Comparison of Observed and Predicted Antibacterial and Antifungal Activity Obtained by ot-QSAR Model

Comp. pMICan (Eq. 1) pMIC bs (Eq. 2) pMICecMLR (Eq. 4)

Obs. Pre. Res. Obs. Pre. Res. Obs. Pre. Res.

Training set
1 2.31 2.30 0.01 2.01 1.91 0.10 2.01 2.30 -0.29
2 2.40 2.40 0.00 2.10 2.02 0.08 2.40 2.40 0.00
4 2.36 2.40 -0.04 2.96 2.89 0.07 2.66 2.45 0.21
5 2.41 2.32 0.09 2.11 1.96 0.15 2.41 2.26 0.15
6 2.36 2.35 0.01 2.06 2.14 -0.08 2.36 2.37 -0.01
7 2.37 2.37 0.00 2.07 2.01 0.06 2.37 2.43 -0.06
8 2.29 2.28 0.01 1.99 2.13 -0.14 1.99 2.13 -0.14
9 2.41 2.42 -0.01 2.11 2.21 -0.10 2.41 2.44 -0.03
10 2.27 2.26 0.01 1.97 1.95 0.02 2.27 2.09 0.18
11 2.41 2.41 0.00 1.80 2.05 -0.25 2.41 2.22 0.19
12 2.51 2.54 -0.03 2.21 2.08 0.13 2.21 2.36 -0.15
INH 2.04 2.09 -0.05 1.74 1.78 -0.04 1.74 1.79 -0.05
Test set
13 2.38 2.37 0.01 2.38 2.09 0.29 2.38 2.31 0.07
14 2.51 2.55 -0.04 2.51 2.07 0.44 2.51 2.56 -0.05

value of standard deviation (0.24) in this case is less than model lies in its predictive ability even though the activity
0.3, so the developed QSAR models are valid. data are in the narrow range. Further, recent literature reveals
that the QSAR have been applied to describe the relationship
2.6 between narrow range of biological activity and physico-
chemical properties of the molecules [61-64]. When biologi-
2.5
cal activity data lies in the narrow range, the presence of
minimum standard deviation of the biological activity justi-
fies its use in QSAR studies [31, 61]. The minimum standard
Predicted pMICan

2.4 deviation (Table 3) observed in the antimicrobial activity

data justifies its use in QSAR studies. The value of PRESS
2.3 (predictive residual sum of squares which is also termed as
least square error (LSE)) < sum of squares of response val-
2.2 .10

.08
2.1

.06
2.0
Residual pMICan

2.0 2.1 2.2 2.3 2.4 2.5 2.6

.04

Observed pMICan
.02
Fig. (2). Plot of observed pMICan against the predicted pMICan for
the linear regression model developed by Eq. 1. 0.00

-.02
Generally for QSAR studies, the biological activities of
compounds should span 2-3 orders of magnitude. But in the
-.04
present study the range of antibacterial and antifungal activi-
ties of the synthesized compounds is within one order of -.06
magnitude. It is important to note that the predictability of 2.0 2.1 2.2 2.3 2.4 2.5 2.6

the QSAR models developed in the present study is high

Observed pMICan
which is evidenced by their low residual values. This is in
agreement with results suggested by the literature report Fig. (3). Plot of residual pMICan against the experimental pMICan
[60], where is was stated that the reliability of the QSAR for the linear regression model developed by Eq. 1.
806 Letters in Drug Design & Discovery, 2011, Vol. 8, No. 9 Judge et al.

ues (SSY) is an indicative of statistical significance of pre-
diction of QSAR models [65] which can be easily depicted
by their values listed in Table 8. In 2D-QSAR, the internal
consistency of the models using q2 is assessed with the ratio
RQR = q2/r2 [66]. Considering RQR for the QSAR models
represented by Eq. 1, 2 and 4 (Table 9), it is pleasing that the
lowest value is RQR = 0.169 for QSAR Model 3 and there is
a gradual, roughly monotonic increase across the series to
RQR = 0.895. Thus the QSAR models presented here are
statistically robust. The low value of probable error of the
coefficient of correlation (PE), least square error (LSE),
Friedman’s lack of fit measure (LOF), standard error of pre-
diction (SEP), and SSY (sum of squares of response values),
Standard deviation of prediction (Spress), and standard de-
viation error of predictions (SDEP), root mean square error
(RMSE) and absolute average error (e
) and high values of
quality value (Q), r2 and the cross-validated correlation coef-
ficient (q2) (Table 8 and Table 9) revealed the statistical sig-
nificance of the model described by Eq. 1, 2 and 4. Also the
value VIF < 5 indicates that MLR models expressed by Eq.
4, 7 and 9 are statistically valid [31].
Development of Multi-Target QSAR Model
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 spe-
cies. The ot-QSAR models, which are almost in the whole
literature, become unpractical or at less complicated to use
when we have to predict to each compound results for more
than one target. In these cases we have to develop one ot-
QSAR for each target. However, very recently the interest
has been increased in development of multi-target QSAR
(mt- QSAR) models. In opposition 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 antibacterial and antifungal activity [67-71].
In the present study we have attempted to develop three
different types of mt-QSAR models viz. mt-QSAR model for
describing antibacterial activity of synthesized compounds
against S. aureus, B. subtilis and E. coli, mt-QSAR model
for describing antifungal activity of synthesized compounds
against C. albicans and A. niger as well a common mt-
QSAR model for describing the antimicrobial (overall anti-
bacterial and antifungal) activity of synthesized isoniazid
derivatives against all the above mentioned microorganisms.
In order to develop mt-QSAR models, initially we have
calculated the average antibacterial activity, antifungal activ-
ity and antimicrobial activity values of N2-acyl isonicotinic
acid hydrazide derivatives which are presented in Table 3.
These average activity values were also correlated with the
molecular descriptors of synthesized compounds (Table 6).
Model 4: The mt-QSAR model for antibacterial activity
depicted the importance of energy of lowest unoccupied mo-
lecular orbital (LUMO) in describing the antibacterial activ-
ity of synthesized acylated isonicotinic acid hydrazide de-
rivatives, and presented in the model expressed by Eq. 5.
mt-QSAR model for antibacterial activity
pMICb = 0.818 LUMO + 1.480 Eq. 5
2
n = 12 r = 0.860 q = 0.671 s = 0.107 F = 28.33
Model 5: The mt-QSAR model for antifungal activity
revealed the importance of first order molecular connectivity
index (1) in describing antifungal activity of synthesized
derivatives (Eq. 6) and and the multiple linear regression

Table 8. PE, LSE, LOF, SEP, Q and SSY Values Calculated for the Derived Models for Modeling Antimicrobial Activity of Syn-
thesized Isonicotinic Acid Hydrazide Derivatives

S. No. Descriptor PE LSE (PRESS) LOF SEP Q SSY

Eq. 1 Te 0.017 0.014 0.0011 0.010 26.56 0.027

Eq. 2 LUMO, 0.032 0.165 0.0135 0.034 7.09 0.369
Eq. 4 3
, 1 0.076 0.268 0.0132 0.043 4.52 0.607
Eq. 5 LUMO 0.050 0.113 0.0090 0.028 8.03 0.145
Eq. 7 1
, W 0.095 0.075 0.0037 0.023 7.74 0.216
Eq. 9 3
, 1 0.054 0.062 0.0031 0.021 10.34 0.105

Table 9. RMSE, e
, SPress, SDEP, VIF, r2 and RQR Values Calculated for the Derived Models for Modeling Antimicrobial Activity
of Synthesized Isonicotinic Acid Hydrazide Derivatives

S.No. Descriptor e
RMSE SPress SDEP VIF r2 RQR

Eq. 1 Te 0.022 0.034 0.110 0.035 - 0.914 0.895

Eq. 2 LUMO, 0.102 0.117 0.276 0.122 - 0.835 0.760
Eq. 4 3
, 1 0.122 0.149 0.193 0.156 2.53 0.605 0.169
Eq. 5 LUMO 0.072 0.097 0.172 0.101 - 0.740 0.907
Eq. 7 1
, W 0.071 0.079 0.084 0.082 2.03 0.507 0.809
Eq. 9 3
, 1 0.046 0.072 0.117 0.075 3.56 0.719 0.711
Synthesis and Antimicrobial Evaluation of Isonicotinic Acid Hydrazides Letters in Drug Design & Discovery, 2011, Vol. 8, No. 9 807

model obtained by the combination of first order molecular
connectivity index (1) and wiener topological index (W)
best describes the antifungal activity and is expressed by Eq.
7.
mt-QSAR model for antifungal activity
pMICf = 0.027 1 + 2.145
2
n = 12 r = 0.520 q = -0.502 s = 0.106 F = 3.70
mt-QSAR model for antifungal activity developed by MLR
pMICf = 0.080 1 – 0.00015 W + 1.785
2
n = 12 r = 0.712 q = -0.410 s = 0.092 F = 4.65
Model 6: The mt-QSAR model for antimicrobial activity
depicted that third order molecular connectivity index (3)
effectively describes the overall antimicrobial activity of the
synthesized derivatives and MLR model was developed by
the combination of third order molecular connectivity index
(3) with Kier’s first order shape topological index (1) and
the models are represented by Eq. 8 and Eq. 9 respectively.
mt-QSAR model for antimicrobial activity
pMICam = 0.348 3 + 1.925
2 dictions (SDEP), root mean square error (RMSE) and abso-
n = 12 r = 0.736 q = 0.306 s = 0.099 F = 11.83
mt-QSAR model for antimicrobial activity developed by MLR
pMICamMLR = 0.315 3 + 0.0133 1 + 1.731
2 described by Eq. 5, 7 and 9. Similarly Considering RQR for
n = 12 r = 0.848 q = 0.511 s = 0.082 F = 11.49
It was observed from mt-QSAR models [Eq. 5-9] that the
antibacterial, antifungal and overall antimicrobial activity of
synthesized acylated isonicotinic acid hydrazide derivatives
is governed by topological and energy parameters, viz. en-
ergy of lowest unoccupied molecular orbital (LUMO) de-
scribes the antibacterial activity and combination of first
order molecular connectivity index (1) and wiener topologi-
cal index (W) best describes the antifungal potential of the
acylated isonicotinic acid hydrazide derivatives and the
combination of third order molecular connectivity index (3)
Eq. 6 with Kier’s first order shape topological index (1) effec-
tively describes the overall antimicrobial activity. The devel-
oped mt-QSAR models were statistically valid as they had
the high r and q2 values and low residual values. The residual
Eq. 7 values obtained by mt-QSAR models are presented in Table
10. As in the case of ot-QSAR models, the plot of predicted
pMICamMLR against observed pMICamMLR (Fig. 4) also favors
the developed model expressed by Eq. 8. Further, the plot of
observed pMICamMLR Vs residual pMICamMLR (Fig. 5) indi-
cated that there was no systemic error in model development
as the propagation of error was observed on both sides of
zero i.e. both positive and negative residual values were ob-
served. The low value of probable error of the coefficient of
correlation (PE), least square error (LSE), Friedman’s lack of
fit measure (LOF), standard error of prediction (SEP), and
SSY (sum of squares of response values), Standard deviation
Eq. 8 of prediction (Spress), and standard deviation error of pre-
lute average error (e
) and high value of quality value (Q)
and the cross-validated correlation coefficient (q2) (Table 8
Eq. 9 and Table 9) revealed the statistical significance of the model
the QSAR models represented by Eq. 5, 7 and 9 (Table 9), it
was observed that QSAR models has got the RQR value of
0.711 - 0.907. Also the value VIF < 5 indicates that MLR
models expressed by Eq. 7 and 9 are statistically valid. Thus,
here also the QSAR models are statistically robust.

Table 10. Comparison of Observed and Predicted Antibacterial, Antifungal and Antimicrobial Activity Obtained by mt-QSAR
Model

Comp. pMIC b (Eq. 5) pMICf (Eq. 7) pMICamMLR (Eq. 9)

Obs. Pre. Res. Obs. Pre. Res. Obs. Pre. Res.

Training set
1 2.01 2.05 -0.04 2.31 2.41 -0.10 2.13 2.28 -0.15
2 2.30 2.12 0.18 2.40 2.50 -0.10 2.34 2.34 0.00
4 2.66 2.66 0.00 2.36 2.45 -0.09 2.54 2.37 0.17
5 2.21 2.08 0.13 2.41 2.42 -0.01 2.29 2.25 0.04
6 2.16 2.19 -0.03 2.52 2.43 0.09 2.30 2.32 -0.02
7 2.17 2.11 0.06 2.52 2.43 0.09 2.31 2.36 -0.05
8 1.99 2.18 -0.19 2.44 2.40 0.04 2.17 2.18 -0.01
9 2.21 2.23 -0.02 2.56 2.49 0.07 2.35 2.37 -0.02
10 2.07 2.07 0.00 2.27 2.36 -0.09 2.15 2.15 0.00
11 2.11 2.13 -0.02 2.56 2.44 0.12 2.29 2.23 0.06
12 2.21 2.15 0.06 2.36 2.39 -0.03 2.27 2.31 -0.04
INH 1.84 1.97 -0.13 2.19 2.17 0.02 1.98 1.97 0.01
Test set
13 2.58 2.05 0.53 2.53 2.48 0.05 2.56 2.33 0.23
14 2.51 2.01 0.50 2.81 2.68 0.13 2.63 2.57 0.06
808 Letters in Drug Design & Discovery, 2011, Vol. 8, No. 9 Judge et al.

2.4 benzoic acid N'-(4-hydroxy-3,5-dimethoxy-benzylidene)-N-

(pyridine-4-carbonyl)-hydrazide (14) were the most active
antimicrobial agents. To find out the correlation between
2.3 physicochemical parameters and antibacterial and antifungal
Predicted pMICamMLR

activity of acylated isonicotinic acid hydrazide derivatives,

QSAR investigation was performed by development of one
2.2 target and multi target models. The QSAR studies indicated
the importance of topological parameters third order molecu-
lar connectivity index (3) and Kier’s first order shape topo-
2.1 logical index (1) in governing the antimicrobial activity of
acylated isonicotinic acid hydrazide derivatives.
2.0
ACKNOWLEDGEMENTS
1.9 The author (VJ) is thankful to Department of Technical
1.9 2.0 2.1 2.2 2.3 2.4 2.5 2.6 Education, Government of Haryana (India) for providing
fellowship to carry out the research project. Antiviral activity
Observed pMICamMLR tests were carried out at the Rega Institute for Medical Re-
Fig. (4). Plot of observed pMICamMLR against the predicted pMI-
search (Leuven, Belgium).
CamMLR for the multiple linear regression model developed by
Eq. 8. DECLARATION OF INTEREST
The authors report no conflicts of interest. The authors
.2
alone are responsible for the content and writing of the pa-
per.

.1
Residual pMICamMLR

DISCLOSURE
The part of information included in this chapter has been
previously published in MEDICINAL CHEMISTRY
0.0 RESEARCH DOI: 10.1007/s00044-011- 9662-9.

-.1
-.2
1.9 2.0 2.1 2.2 2.3
Observed pMICamMLR
Fig. (5). Plot of residual pMICamMLR against the experimental
pMICamMLR for the multiple linear regression model developed
by Eq. 8.
CONCLUSION
A series of isonicotinic acid hydrazide derivatives (1-14)
was synthesized and tested for their in vitro antimycobacte-
rial activity against Mycobacterium tuberculosis and the
compounds with bromo, N2-2,4-hexadienoyl, N2-lauryl and
N2-octadecanoyl groups were found to be the most effective.
The results of antiviral activity testing showed that none of
the tested compounds was active against HIV viruses. The
synthesized 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 com-
pounds 3-nitro-benzoic acid N'-(pyridine-4-carbonyl)-
hydrazide (4), isonicotinic acid N'-dodecanoyl-hydrazide
(11), Isonicotinic acid N'-octadecanoyl-hydrazide (12), and
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