Synthesis, antimicrobial evaluation and QSAR studies of 3-ethoxy-4-hydroxybenzylidene/4-nitrobenzylidene hydrazides

Article abstract of DOI:10.1016/j.cclet.2011.06.014

A series of 3-ethoxy-4-hydroxybenzylidene/4-nitrobenzylidene hydrazides (1-20) was synthesized and tested for in vitro antimicrobial activity. The results of antimicrobial studies indicated that the compounds having dinitro, methoxy, hydroxy and nitro substituents on phenyl ring of the aromatic acids were most active ones. The QSAR investigation indicated the importance of the topological parameter, third order molecular connectivity index ( ³χ) in describing the antimicrobial activity of synthesized hydrazides.

Full text of DOI:10.1016/j.cclet.2011.06.014

Available online at www.sciencedirect.com
Chinese Chemical Letters 22 (2011) 1293–1296
www.elsevier.com/locate/cclet
Synthesis, antimicrobial evaluation and QSAR studies of
3-ethoxy-4-hydroxybenzylidene/4-nitrobenzylidene hydrazides
Davinder Kumar a, Archana Kapoor a, Ananda Thangadurai b,
Pradeep Kumar c, Balasubramanian Narasimhan c,
*
a Department of Pharmaceutical Sciences, Guru Jambheshwar University of Science and Technology, Hisar 125001, India
b Department of Pharmaceutical Chemistry, Swamy Vivekanandha College of Pharmacy, Tiruchengode 637 205, India
c Faculty of Pharmaceutical Sciences, Maharshi Dayanand University, Rohtak 124001, India
Received 31 March 2011
Available online 28 July 2011
Abstract
A series of 3-ethoxy-4-hydroxybenzylidene/4-nitrobenzylidene hydrazides (1–20) was synthesized and tested for in vitro
antimicrobial activity. The results of antimicrobial studies indicated that the compounds having dinitro, methoxy, hydroxy and nitro
substituents on phenyl ring of the aromatic acids were most active ones. The QSAR investigation indicated the importance of the
topological parameter, third order molecular connectivity index (3x) in describing the antimicrobial activity of synthesized hydrazides.
# 2011 Balasubramanian Narasimhan. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Keywords: Benzylidene hydrazides; Synthesis; Antimicrobial; mt-QSAR; LOO method
As pathogenic microorganisms continuously evolve mechanisms of resistance to current antimicrobial agents, the
discovery of novel and potent bactericides is the best way to overcome bacterial resistance [1]. It is well known in the
literature that the hydrazone group possesses antimicrobial [2], antitubercular [3], anticonvulsant [4], anti-
inflammatory [5], and antileishmanial activities [6]. Quantitative structure–activity relationship (QSAR) studies
represent a non-experimental part of drug design encompassing the study of both structure–activity and structure–
property relations in broad sense [7]. Overwhelmed by the above facts and in continuation to our work in describing
biological activity and QSAR study [8,9], we hereby report the synthesis, antimicrobial evaluation and QSAR studies
of substituted benzylidene hydrazides.
1. Experimental
1.1. General procedure for synthesis of hydrazones (1–20)
The mixture of corresponding acid (0.08 mol) and ethanol (0.74 mol) refluxed in presence of sulphuric acid till the
completion of reaction. Then the reaction mixture was added to 200 mL ice cold water and the ester formed was
separated by extracting it with ether (50 mL) and followed by the evaporation of ether. The hydrazine-hydrate (99%)
* Corresponding author.
E-mail address: naru2000us@yahoo.com (B. Narasimhan).
1001-8417/$ – see front matter # 2011 Balasubramanian Narasimhan. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
doi:10.1016/j.cclet.2011.06.014

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D. Kumar et al. / Chinese Chemical Letters 22 (2011) 1293–1296
(0.015 mol) was added to ethanolic solution of ester (0.01 mol) synthesized above and refluxed for 5 h. The reaction
mixture was then cooled, and the precipitated hydrazide was separated and recrystallized from ethanol. A solution of
0.01 mol of above hydrazide and 0.01 mol of appropriate aldehyde, in ethanol, was heated under reflux for 4–5 h. The
precipitate obtained was filtered off, washed with water and recrystallized from ethanol.
Compound 9: 1H NMR (400 MHz, CDCl3): d 6.53–7.07 (m, 6H, Ar–OH and Ar–(OCH3)2), 1.33–1.36 (t, 3H, terminal
CH3 ofOCH2CH3), 4.04–4.07(q, 2H, CH2 ofOCH2CH3), 8.2(s, 1H, NHofCO–NH), 3.75(s, 6H, OCH3 ofAr–(OCH3)2),
5.2 (1H, S, OH of Ar–OH). Compound 10: 1H NMR (400 MHz, CDCl3): d 6.71–7.08 (m, 3H, Ar–OH), 9.28–9.36 (m, 3H,
Ar–(NO2)2), 1.38–1.41 (t, 3H, terminal CH3 of OCH2CH3), 3.96–3.98 (q, 2H, CH2 of OCH2CH3), 8.0 (s, 1H, NH of CO–
NH), 4.92 (s, 1H, OH of Ar–OH); Compound 19: 1H NMR (400 MHz, CDCl3): d 6.68–7.10 (m, 3H, Ar–(OCH3)2), 7.94–
1
8.27 (m, 3H, Ar–(NO2)), 3.92 (s, 6H, OCH3 of Ar–(OCH3)2), 8.4 (s, 1H, NH of CO–NH); Compound 20: H NMR
(400 MHz, CDCl3): d 8.07–8.41 (m, 4H, ArH of ArNO2), 9.17–9.23 (s, 3H, ArH of Ar(NO2)2), 10.17 (s, 1H, NH).
1.2. Evaluation of antimicrobial activity-determination of MIC
The antimicrobial activity was performed against Gram-positive bacteria: Staphylcococcus aureus, Bacillus
sublitis, Gram-negative bacterium: Escherichia coli and fungal strains: Candida albicans and Aspergillus niger by
tube dilution method [10] using double strength nutrient broth – I.P. (bacteria) or Sabouraud dextrose broth I.P. (fungi)
as media [11]. The samples were incubated at 37 8C for 24 h (bacteria), at 25 8C for 7 d (A. niger) and at 37 8C for 48 h
(C. albicans), and the results were recorded in terms of MIC.
1.3. QSAR studies
The structure of synthesized hydrazides was optimized by energy minimization and the physicochemical properties
were calculated using TSAR 3.3 software for Windows [12]. Further, the regression analysis was performed using the
SPSS software package [13]. The predictive powers of the equations were validated by leave one out (LOO) cross
validation method [14].
2. Results and discussion
The synthesis of target compounds was carried out as outlined in Scheme 1. All the compounds were obtained in
appreciable yield and their spectral and elemental analysis data are found in agreement with the assigned molecular
structures. The synthesized compounds (1–20) were screened for their in vitro antimicrobial activity and the results are
presented inTable 1. Itcanbeseenfrom theresults ofantimicrobialactivitythat theactivityincreaseswithincreaseinchain
length of acid portion of synthesized compounds (1–4 and 11–14). This is similar to one of our previous reports [9]. The
presence of electron withdrawing group (NO2) in compounds 10 and 20 makes them highly active antimicrobial agents.
The role ofelectronwithdrawing group inincreasing the antimicrobialactivityis similarto the results ofSharma etal. [15].
2.1. Development of multi-target QSAR model
QSAR studies were undertaken using linear free energy relationship (LFER) model of Hansch and Fujita [16].
Biological activity data determined as MIC values was first transformed to pMIC on molar basis, which was used as
dependent variable in QSAR study. The mt-QSAR model is a single equation that considers the nature of molecular
descriptors which are common and essential for describing the antimicrobial activity [17]. In the present study we have
developed mt-QSAR models for antibacterial, antifungal and antimicrobial activities of substituted benzylidene
hydrazides by taking average antibacterial, antifungal and antimicrobial activities respectively.
pMICb ¼ 0:4193x þ 0:873
(1)
(n = 20, r = 0.812, q2 = 0.571, s = 0.110, F = 34.80).
The mt-QSAR model described by Eq. (1) depicts the importance of third order molecular connectivity index (3x)
for antibacterial activity of substituted benzylidene hydrazides. We have attempted to combine different parameters so
as to increase the regression coefficient (r value), by multiple linear regressions.

D. Kumar et al. / Chinese Chemical Letters 22 (2011) 1293–1296
1295
R5
R4
R3
H
O
O
C
C NHN CH
R
R1
O
C
O
C
R5
R4
R1
NH2NH2.H2O
R2
EtOH
O
C
R
OEt
R
OH
R
NHNH2
H2SO4
R2
R3
1-5, 11-15
X
X
X
X
2
1
O
H
O
X
X
X
X
X
2
X
X
Y
NHN CH
1
R
R
R
2
1
1
X
X
X
3
1
5
4
2
1
O
O
EtOH
O
R
R
R
NH NH .OH
5
4
2
2
R
2
X
Y
OH
X
Y
OEt
X
Y
NHNH
2
3
3
4
5
3
R
3
H SO
2
4
R
2
X
X
X
4
5
4
5
4
5
R
3
6-10, 16-20
Compd.
R
R1
H
H
H
H
H
H
H
H
H
H
R2
R3
R4
H
H
H
H
H
H
H
H
H
H
R5
H
H
H
H
H
H
H
H
H
H
Compd.
Y
X1
H
H
H
H
H
H
H
H
H
H
X2
H
X3
H
X4
H
X5
R1
R2
R3
R4
R5
H
H
H
H
H
H
H
H
H
H
CH3(CH2)9CH2-
CH3(CH2)11CH2-
CH3(CH2)13CH2-
CH3(CH2)15CH2-
CH3(CH=CH)2-
CH3(CH2)9CH2-
CH3(CH2)11CH2-
CH3(CH2)13CH2-
CH3(CH2)15CH2-
CH3(CH=CH)2-
OEt
OEt
OEt
OEt
OEt
H
OH
OH
OH
OH
OH
NO2
NO2
NO2
NO2
NO2
CH=CH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OEt
OEt
OEt
OEt
OEt
H
OH
OH
OH
OH
OH
H
H
H
H
H
H
H
H
H
H
1
6
−
CH3
H
H
H
2
7
−
CH3
H
H
3
8
4
9
−
OCH3
NO2
H
CH3
H
OCH3
NO2
OCH3
NO2
H
5
10
16
17
18
19
20
−
H
11
12
13
14
15
CH=CH
H
NO2
NO2
NO2
NO2
NO2
H
−
−
−
−
H
H
H
OCH3
NO2
H
H
CH3
H
H
H
H
H
H
H
Scheme 1. Synthetic route followed to obtain target compounds from aliphatic or aromatic acids.
Table 1
Antimicrobial activity (mmol/mL) of synthesized substituted benzylidene hydrazides.
Compd.
pMICsa
pMICbs
pMICec
pMICca
pMICan
pMICam
pMICb
pMICf
1
1.16
1.19
1.32
1.55
1.04
1.09
1.08
1.08
1.14
1.18
1.14
1.18
1.21
1.24
1.02
1.07
1.05
1.05
1.12
1.46
0.14
2.61b
1.16
1.19
1.63
1.25
1.34
1.09
1.38
1.38
1.44
2.08
1.44
1.18
1.21
1.30
1.02
1.67
1.36
1.66
1.70
2.06
0.29
2.61b
1.16
1.49
1.52
1.42
1.65
1.70
1.38
1.61
1.75
2.08
1.14
1.18
1.51
1.54
1.32
1.68
1.67
1.36
1.70
2.06
0.26
2.61b
1.46
1.19
1.47
1.57
1.04
1.09
1.08
1.38
1.14
1.78
1.14
1.48
1.41
1.54
1.02
1.07
1.05
1.05
1.42
1.46
0.23
2.64c
1.76
1.49
1.22
1.55
1.60
1.51
1.47
1.48
1.44
2.08
1.44
1.48
1.51
1.84
1.92
1.07
1.66
1.96
2.02
2.06
0.28
2.64c
1.34
1.31
1.43
1.47
1.33
1.30
1.28
1.39
1.38
1.84
1.26
1.30
1.37
1.49
1.26
1.31
1.36
1.42
1.59
1.82
0.17
2.61
1.16
1.29
1.49
1.41
1.34
1.29
1.28
1.36
1.44
1.78
1.24
1.18
1.31
1.36
1.12
1.47
1.36
1.36
1.51
1.86
0.18
2.64
1.61
1.34
1.35
1.56
1.32
1.30
1.28
1.43
1.29
1.93
1.29
1.48
1.46
1.69
1.47
1.07
1.36
1.51
1.72
1.76
0.20
2.62
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
S.D.a
Std.
a
Standard deviation.
b
c
Ciprofloxacin.
Fluconazole.
pMICb ¼ 0:4953x þ 0:000061W þ 0:647
(2)
(n = 20, r = 0.889, q2 = 0.611, s = 0.089, F = 31.95).
In this model the Wiener index (W) [18] in addition to third order molecular connectivity index 3x increased the
regression coefficient (r) value from 0.812 to 0.889 and q2 from 0.571 to 0.611. It is important to note that there is no

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D. Kumar et al. / Chinese Chemical Letters 22 (2011) 1293–1296
colinearity between W and 3x (r = 0.376). As regression coefficient, r > 0.8 and q2 > 0.5, so the model expressed by
Eq. (2) is valid one.
pMICf ¼ ꢀ0:00016Te þ 0:738
(3)
(n = 20, r = 0.544, q2 = 0.015, s = 0.175, F = 7.56).
In case of mt-QSAR model for antifungal activity, the molecular descriptor total energy (Te) may moderately
influence the antifungal activity of synthesized hydrazides in comparison to the other parameters. The coefficient of
total energy (Eq. (3)) is negative which implies the antifungal activity will increase with decrease in total energy.
An attempt was made to develop a common QSAR model describing the antibacterial and antifungal activities of
synthesized substituted benzylidene hydrazides.
pMICam ¼ 0:3693x þ 0:967
(4)
(n = 20, r = 0.794, q2 = 0.579, s = 0.103, F = 30.8).
The model developed by linear regression analysis for antimicrobial activity (Eq. (4)) depicts the importance of
third order molecular connectivity index (3x) in describing the antimicrobial activity.
pMICam ¼ 0:005MR þ 0:4743x þ 0:375
(5)
(n = 20, r = 0.917, q2 = 0.729, s = 0.069, F = 44.64).
When third order molecular connectivity index (3x) was combined to molecular refractivity (MR) we observed a
huge increase in regression coefficient from 0.794 to 0.917 and q2 from 0.579 to 0.729. This signifies that there is no
colinearity between MR and 3x (r = 0.444). It can be observed from Eqs. (1)–(5) that third order molecular
connectivity index (3x,), is highly important in describing the antimicrobial activities of synthesized benzylidene
hydrazides.
3. Conclusion
A series of 3-ethoxy-4-hydroxy benzylidene/4-nitrobenzylidene hydrazides (1–20) was synthesized and tested for
antimicrobial activity. The results of antimicrobial studies indicated that the compounds having dinitro, methoxy,
hydroxy and nitro substituents on phenyl ring of the aromatic acids were most active ones. The QSAR investigation
indicated the importance of third order molecular connectivity index (3x) in describing the antimicrobial activity of
synthesized hydrazides.
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