N′-[4-[(Substituted imino)methyl]benzylidene]-substituted benzohydrazides: Synthesis, antimicrobial, antiviral, and anticancer evaluation, and QSAR studies

Monatsh Chem (2013) 144:825–849

DOI 10.1007/s00706-012-0877-3

ORIGINAL PAPER

N⁰-[4-[(Substituted imino)methyl]benzylidene]-substituted

benzohydrazides: synthesis, antimicrobial, antiviral,

and anticancer evaluation, and QSAR studies

•

Pradeep Kumar Balasubramanian Narasimhan Kalavathy Ramasamy

•

Vasudevan Mani Rakesh Kumar Mishra Abu Bakar Abdul Majeed

Erik De Clercq

Received: 24 April 2012 / Accepted: 12 October 2012 / Published online: 1 December 2012

ꢀ Springer-Verlag Wien 2012

Abstract A variety of N⁰-[4-[(substituted imino)methyl]-

benzylidene]-substituted benzohydrazides have been syn-

thesized and evaluated for antimicrobial and anticancer

potential. Results from testing of antimicrobial activity

indicated the most potent antimicrobial agents had

pMIC_am= 1.51. The synthesized compounds were bacte-

riostatic and fungistatic in action. Results from evaluation

of antiviral activity indicated that none of the synthesized

hydrazide derivatives inhibited viral replication at sub-

toxic concentrations. Results from anti-HIV screening

against HIV-2 strain ROD indicated that one compound

was more potent (IC₅₀C 1 lg/cm³) than the standard drug

nevirapine (IC₅₀C 4 lg/cm³) and another was equipotent

(IC₅₀C 4 lg/cm³). The most effective anticancer agent

against both HCT116 and MCF7 cancer cell lines had

IC₅₀= 19 and 18 lg/cm³, respectively. QSAR analysis

indicated the importance of Wiener index (W) and energy

of the lowest unoccupied molecular orbital (LUMO) in

describing the antimicrobial activity of the synthesized

compounds.

Keywords Hydrazides ꢀ Antimicrobial ꢀ Antiviral ꢀ

Anticancer ꢀ QSAR

Introduction

The dramatically rising prevalence of multi-drug-resistant

microbial infections in the past few decades has become a

serious health care problem. In particular, the emergence of

multi-drug resistant strains of Gram-positive bacterial

pathogens, for example methicillin-resistant Staphylococ-

cus aureus and Staphylococcus epidermis and vancomycin-

resistant Enterococcus is a problem of ever-increasing

signiﬁcance. One way of overcoming this challenge is

control of the use of currently marketed antibiotics; another

is the development of novel antimicrobial agents. Conse-

quently, the search for new antimicrobial agents will

remain an important and challenging task for medicinal

chemists [1].

P. Kumar ꢀ B. Narasimhan (&)

Faculty of Pharmaceutical Sciences,

Maharshi Dayanand University, Rohtak 124001, India

e-mail: naru2000us@yahoo.com

K. Ramasamy

Acquired immune deﬁciency syndrome (AIDS) has

become a global pandemic and has claimed more lives than

any other disease. According to the 2008 UNAIDS report

released by the WHO, 33 million people were diagnosed as

human immunodeﬁciency virus (HIV)-positive in 2007 [2].

Human T lymphocytes are target cells for HIV replication,

and the MT-4 cell line is used for the screening of anti-HIV

agents. In recent years, much attention has been devoted to

the search for effective chemotherapeutic agents for inhi-

bition of HIV with minimum side effects [3].

Collaborative Drug Discovery Research Group,

Faculty of Pharmacy, Campus Puncak Alam,

Universiti Teknologi MARA (UiTM), Bandar Puncak Alam,

42300 Shan Alam, Selangor, Malaysia

V. Mani ꢀ R. K. Mishra ꢀ A. B. A. Majeed

Brain Research Laboratory, Faculty of Pharmacy,

Campus Puncak Alam, Universiti Teknologi MARA (UiTM),

Bandar Puncak Alam, 42300 Shan Alam, Selangor, Malaysia

E. De Clercq

Laboratory of Virology and Chemotherapy, Rega Institute

for Medical Research, Minderbroedersstraat 10, 3000 Louvain,

Belgium

Cancer is a disease of worldwide importance. Its inci-

dence in the developed countries is increasing, and its

123

826

P. Kumar et al.

R²

R⁴

R¹

R⁵

Scheme 1

R²

R⁴

R¹

R⁵

R²

R⁴

R¹

R⁵

O

NH₂NH₂.H₂O

EtOH

R³

OH

OEt

NHNH₂

NH₂

X¹

OHC

CHO

X⁵

X⁴

R²

R¹

R⁵

X²

N

X¹

R²

R⁴

R¹

R⁵

X³

O

X⁵

X²

O

R³

NHN

X⁴

X³

NHN

R⁴

1-42

R-NH₂

NR

R²

R⁴

R¹

R⁵

O

R³

NHN

43-48

mortality is ranked second in the order of causes of

death. A similar tendency is observed in the developing

world: gradual improvement in life expectancy is associ-

ated with elevated cancer incidence and mortality.

Malignancy and its consecutive burden is therefore a

global problem resulting in widespread interest in cancer

therapy [4].

Results and discussion

Chemistry

Synthesis of the target compounds (1–48) was performed

as outlined in Scheme 1. All the compounds were high-

melting-point solids (m.p. [ 300 ꢁC). The structures of the

compounds 1–48 were ascertained on the basis of their

consistent IR, NMR, and mass spectral characteristics and

results from elemental (C, H, N) analysis, all of which were

in full agreement with their assigned molecular structures.

The synthesis of novel pharmacologically active mole-

cules with reduced toxicity is of prime interest. Recently,

quantitative structure–activity relationships (QSAR) have

gained importance in medicinal chemistry. QSAR are

predictive tools used for preliminary evaluation of the

activity of chemical compounds by use of computer-aided

models. Use of QSAR has increased the probability of

success in the drug-discovery process and reduced the time

and cost involved [5].

Antimicrobial activity

The synthesized compounds were screened for their

in-vitro antibacterial activity against S. aureus, Bacillus

subtilis, and Escherichia coli and antifungal activity

against Candida albicans and Aspergillus niger by the tube

dilution method [19] using norﬂoxacin and ﬂuconazole as

reference standards for antibacterial and antifungal activ-

ity, respectively; the results are presented in Table 1.

Among the synthesized compounds, N⁰-[4-[(2-chloro-4-

nitrophenylimino)methyl]benzylidene]-4-chlorobenzohyd-

razide (6), N⁰-[4-[(2-chloro-4-nitrophenylimino)methyl]

benzylidene]-4-aminobenzohydrazide (33), and N’-[4-[(2-

chloro-4-nitrophenylimino)methyl]benzylidene]-4-hydroxy-

benzohydrazide (37) were found to be effective against

S. aureus, having pMIC_savalues of 1.25, 1.23, and 1.23,

respectively. Against B. subtilis, E. coli, and C. albicans,

compounds 6, 33, and 37 emerged as the most effective

antimicrobial agents with pMIC values of 1.55, 1.53, and

Hydrazones have attracted the attention of many

chemists owing to their wide range of biological activity.

Literature reports reveal that chemistry and biology of

hydrazones have been intensively investigated during the

past decade. Hydrazone derivatives have been found to

have antimicrobial [6], anticancer [7], antimycobacterial

[8], antimalarial [9], antioxidant [10], anticonvulsant [11],

analgesic [12], anti-inﬂammatory [13], and antidiabetic

[14] activity.

Motivated by these facts, and in continuation of our

research efforts in synthesis, evaluation of antimicrobial

activity, and QSAR studies [15–18], we report herein

the synthesis, antimicrobial, antiviral, and anticancer

evaluation, and QSAR studies of N⁰-[4-[(substituted

imino)methyl]benzylidene]-substituted benzohydrazides.

123

N⁰-[4-[(Substituted imino)methyl]benzylidene]-substituted benzohydrazides

827

Table 1 Antimicrobial activity (pMIC/lM cm^-3) of N⁰-[4-[(substituted imino)methyl]benzylidene]-substituted benzohydrazides

Comp.

pMICsa

pMICbs

pMICec

pMICca

pMICan

pMICab

pMICaf

pMICam

1

1.16

1.20

1.19

1.25

1.21

1.14

1.18

1.19

1.17

1.14

1.18

1.17

1.13

1.18

1.17

1.18

1.14

1.18

1.17

1.21

1.17

1.21

1.19

1.18

1.19

1.21

1.23

1.18

1.19

1.23

1.19

1.18

1.16

1.20

1.11

1.15

1.09

1.05

1.20

1.09

0.04

2.61*

1.16

1.20

1.49

1.19

1.49

1.55

1.51

1.21

1.14

1.18

1.19

1.17

1.14

1.18

1.17

1.13

1.48

1.47

1.48

1.44

1.48

1.47

1.51

1.47

1.51

1.49

1.18

1.19

1.49

1.21

1.53

1.48

1.49

1.53

1.49

1.48

1.46

1.50

1.41

1.45

1.09

1.35

1.50

1.39

0.15

2.61*

1.46

1.50

1.49

1.55

1.51

1.44

1.48

1.49

1.47

1.44

1.48

1.47

1.44

1.48

1.47

1.48

1.44

1.48

1.47

1.51

1.47

1.51

1.49

1.48

1.49

1.51

1.53

1.48

1.49

1.53

1.49

1.18

1.48

1.46

1.50

1.41

1.45

1.09

1.35

1.50

1.39

0.08

2.61*

1.46

1.50

1.49

1.55

1.51

1.44

1.48

1.49

1.47

1.44

1.48

1.47

1.44

1.48

1.47

1.48

1.44

1.48

1.47

1.51

1.47

1.51

1.49

1.48

1.49

1.51

1.53

1.48

1.49

1.53

1.49

1.48

1.46

1.50

1.41

1.45

1.39

1.35

1.50

1.39

0.04

2.64**

1.46

1.50

1.49

1.55

1.51

1.44

1.48

1.49

1.47

1.44

1.48

1.47

1.44

1.48

1.47

1.48

1.44

1.48

1.47

1.51

1.81

1.77

1.81

1.79

1.78

1.79

1.51

1.53

1.48

1.19

1.49

1.53

1.49

1.48

1.16

1.50

1.41

1.45

1.39

1.05

1.50

1.39

0.15

2.64**

1.26

1.30

1.39

1.29

1.39

1.45

1.41

1.31

1.24

1.28

1.29

1.27

1.24

1.28

1.27

1.24

1.38

1.37

1.38

1.34

1.38

1.37

1.41

1.37

1.41

1.39

1.28

1.29

1.39

1.31

1.43

1.38

1.39

1.43

1.39

1.28

1.38

1.36

1.40

1.31

1.35

1.09

1.25

1.40

1.29

0.07

–

1.46

1.50

1.49

1.55

1.51

1.44

1.48

1.49

1.47

1.44

1.48

1.47

1.44

1.48

1.47

1.48

1.44

1.48

1.47

1.51

1.66

1.62

1.66

1.64

1.63

1.64

1.51

1.53

1.48

1.34

1.49

1.53

1.49

1.48

1.31

1.50

1.41

1.45

1.39

1.20

1.50

1.39

0.09

–

1.34

1.38

1.43

1.37

1.43

1.49

1.45

1.39

1.32

1.36

1.37

1.35

1.32

1.36

1.35

1.32

1.42

1.41

1.42

1.38

1.42

1.41

1.45

1.51

1.47

1.51

1.49

1.42

1.43

1.49

1.39

1.47

1.42

1.37

1.43

1.47

1.43

1.36

1.42

1.34

1.44

1.35

1.39

1.21

1.23

1.44

1.33

0.06

–

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

SD

Std.

* Norﬂoxacin

** Fluconazole

123

828

P. Kumar et al.

Table 2 Minimum bactericidal/fungicidal concentrations of N⁰-[4-[(substituted imino)methyl]benzylidene]-substituted benzohydrazides

Comp.

Minimum bactericidal/fungicidal concentration/lM cm^-3

S. aureus

B. subtilis

E. coli

C. albicans

A. niger

1

[0.14

[0.13

[0.11

[0.12

[0.15

0.13

[0.14

[0.13

0.13

[0.14

[0.13

[0.11

[0.12

[0.15

[0.13

[0.15

[0.13

[0.14

[0.15

[0.13

[0.14

[0.13

[0.15

[0.13

[0.12

[0.13

[0.12

[0.13

[0.12

[0.13

[0.12

[0.13

[0.14

[0.13

[0.16

[0.14

[0.16

[0.18

[0.13

[0.16

0.019^a

[0.14

[0.13

[0.11

[0.12

0.15

0.14

[0.13

[0.11

0.12

2

3

4

5

[0.13

[0.11

[0.12

0.12

6

7

8

[0.12

[0.15

0.13

9

[0.15

0.13

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

Standard

[0.13

0.13

[0.13

[0.15

0.07

[0.13

[0.15

[0.13

0.14

[0.13

[0.15

[0.13

0.14

[0.15

[0.13

[0.14

0.15

[0.14

[0.15

[0.13

[0.14

[0.13

[0.14

[0.13

[0.12

[0.13

0.06

[0.14

[0.15

[0.13

[0.14

0.13

[0.14

[0.15

[0.13

[0.14

[0.13

[0.15

[0.13

[0.12

[0.13

[0.12

[0.13

0.13

[0.13

[0.14

[0.13

[0.15

[0.13

[0.12

[0.13

[0.12

[0.13

[0.12

[0.13

[0.12

[0.13

[0.14

[0.13

[0.16

[0.14

[0.16

[0.18

[0.13

[0.16

0.040^b

0.15

0.13

[0.12

[0.13

[0.12

[0.13

[0.12

[0.13

[0.12

[0.13

[0.14

[0.13

[0.16

[0.14

[0.16

[0.18

[0.13

0.16

[0.13

0.13

[0.13

[0.12

[0.13

[0.12

[0.13

[0.14

[0.13

[0.16

[0.14

[0.16

[0.18

[0.13

0.16

[0.13

[0.12

[0.13

0.12

0.13

[0.13

[0.14

[0.13

[0.16

[0.14

[0.16

[0.18

[0.13

[0.16

0.040^b

0.019^a

a

Norﬂoxacin

b

Fluconazole

123

N⁰-[4-[(Substituted imino)methyl]benzylidene]-substituted benzohydrazides

829

Table 3 Anti-feline corona virus (FIPV) and anti-feline herpes virus activity and cytotoxicity of the synthesized hydrazide derivatives in CRFK

cell cultures

EC₅₀^b/lg cm^-3

Comp.

CC₅₀^a/lg cm^-3EC₅₀^b/lg cm^-3

Feline corona virus Feline herpes virus

a

Comp. CC₅₀

/

lg cm^-3

Feline corona virus Feline herpes virus

1

[100

34.1

[100

[20

[100

[20

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

HHA

UDA

[100

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

[100

7.6

1.8

[100

1.1

43.7

Ganciclovir (lM) [100

0.8

0.7

CRFK cells Crandell–Rees feline kidney cells

a

50 % Cytotoxic concentration, as determined by measuring the cell viability with the colorimetric formazan-based MTS assay

b

50 % Effective concentration, or concentration resulting in 50 % inhibition of virus-induced cytopathic effect, as determined by measuring the

cell viability with the colorimetric formazan-based MTS assay

1.53, respectively (Table 1). N⁰-[4-[(4-Chlorophenylimi-

no)methyl]benzylidene]-3-nitrobenzohydrazide (25) and

N⁰-[4-[(2-chlorophenylimino)methyl]benzylidene]-4-nitro-

benzohydrazide (27) were found to be effective against

A. niger with a pMIC value of 1.81 (for both 25 and 27) and

proved the most effective antimicrobial agents with a

pMIC_amvalue of 1.51.

Antiviral activity

The antiviral assays were based on inhibition of virus-

induced cytopathicity in CRFK, HEL, Vero, HeLa, and

MT-4 cell cultures. The results of the antiviral evaluation

are given in Tables 3, 4, 5 and 6. None of the synthesized

hydrazide derivatives inhibited viral replication at sub-

toxic concentrations.

In general, results from minimum bactericidal concen-

tration (MBC) and minimum fungicidal concentration

(MFC) studies (Table 2) revealed that the synthesized

compounds were bacteriostatic and fungistatic in action,

because their MFC and MBC values were threefold higher

than their MIC values (a drug is considered to be bacter-

iostatic/fungistatic when its MFC and MBC values are

threefold higher than its MIC value) [20].

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 [21]; the

123

830

P. Kumar et al.

Table 4 Cytotoxicity and antiviral activity of the synthesized hydrazide derivatives in HEL cell cultures

Comp.

Minimum cytotoxic

concentration^a/lg cm^-3

EC₅₀^b/lg cm^-3

Herpes simplex

virus-1 (KOS)

Herpes simplex

virus-2 (G)

Vaccinia

virus

Vesicular

stomatitis

virus

Herpes simplex virus-1

TK-KOS ACV^r

1

[100

100

[100

[20

[100

[20

[100

[20

[100

[20

[100

[20

2

3

[100

100

[100

[20

[100

[20

[100

[20

[100

[20

[100

[20

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

100

[20

100

[20

[100

100

[100

[20

[100

[20

[100

[20

[100

[20

[100

[20

[100

123

N⁰-[4-[(Substituted imino)methyl]benzylidene]-substituted benzohydrazides

Table 4 continued

831

Comp.

Minimum cytotoxic

concentration^a/lg cm^-3

EC₅₀^b/lg cm^-3

Herpes simplex

virus-1 (KOS)

Herpes simplex

virus-2 (G)

Vaccinia

virus

Vesicular

stomatitis

virus

Herpes simplex virus-1

TK-KOS ACV^r

47

100

[100

[250

[100

[20

[100

0.03

1.2

[20

[100

96

[20

[100

29

[20

[100

[250

[100

[20

[100

250

2

48

Brivudin (lM)

Cidofovir (lM)

Acyclovir (lM)

Ganciclovir (lM)

a

1.2

10

0.4

[250

[100

183

20

0.05

0.08

Amount required to cause a microscopically detectable alteration of normal cell morphology

b

Amount required to reduce virus-induced cytopathogenicity by 50 %

results are presented in Table 7. The results indicated that

increases antifungal activity against A. niger. It is impor-

tant to note that the incubation temperature was the same

for all three bacterial species and C. albicans (a fungus).

These similar incubation conditions may be the reason for

the higher activity of compounds 6, 33, and 37 against

C. albicans and the bacterial species. The effect of the

electron-withdrawing group in improving antimicrobial

activity is supported by the studies of Sharma et al. [23].

compound 29 (IC₅₀C 1 lg/cm³) was more potent than the

standard drug nevirapine (IC₅₀C 4 lg/cm³) against the

HIV-2 strain ROD; compound 39 (IC₅₀C 4 lg/cm³) was

found to be equipotent.

Anticancer activity

The anticancer activity of the synthesized hydrazide

derivatives against human colon cancer (HCT116) and

breast cancer (MCF7) cell lines was determined by use of

the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium

bromide (MTT) assay [22]; the results are presented in

Table 8.

1. The presence of an electron-releasing group on the

benzoic acid structure (13, 29, and 39) increases the

anti-HIV activity of the synthesized compounds.

2. The presence of an electron-releasing group on the

benzoic acid structure (13, 15, 16, 20, and 22)

increases the anticancer potential of the synthesized

compounds. The effect of an electron-releasing group

in improving the anticancer potential of benzodiaze-

pine derivatives is similar to the observation of Kamal

et al. [24].

In general, the synthesized compounds had poor anti-

cancer potential. Compounds 13, 20, 22, and 48 were

effective against HCT116 cancer cell lines with IC₅₀values

of 40, 19, 30, and 30 lg/cm³, respectively, and compounds

15, 16, and 20 were effective against MCF7 cancer cell

lines with IC₅₀values of 35, 25, and 18 lg/cm³,

respectively.

3. The presence of an imino moiety does not improve the

antimicrobial and anticancer potential of the synthe-

sized compounds except for the propylimino moiety

(48) which improved anticancer activity against human

colon cancer cell lines (HCT 116).

The results from antimicrobial, antiviral, and anticancer

evaluation revealed that the nature of the substituents has a

substantial effect on the biological activity of the target

hydrazones; the structure–activity relationships (SAR)

discussed below were deduced.

4. The presence of the naphthalene-1-ylimino moiety

(47) in the synthesized compounds does not improve

antimicrobial and anticancer potential.

5. The aforementioned results indicate that different

structural requirements are essential for a compound

to be selected as antibacterial or antifungal agent. This

is similar to results obtained by Sortino et al. [25].

Structure–activity relationships

Results of antimicrobial screening indicated that presence

of electron-withdrawing 2-chloro-4-nitro substituents on

the phenylimino structure of compounds 6, 33, and 37

increases antimicrobial activity against S. aureus, B. sub-

tilis, E. coli, and C. albicans, whereas the presence of an

electron-withdrawing nitro group on the benzoic acid

structure and a chloro group on the phenylimino structure

QSAR studies

To identify the effects of substituents on antimicrobial

activity, quantitative structure–activity relationship (QSAR)

123

832

P. Kumar et al.

Table 5 Cytotoxicity and antiviral activity of the synthesized

hydrazide derivatives in HeLa cell cultures

Table 5 continued

Comp.

Minimum

cytotoxic

EC₅₀^b/lg cm^-3

Vesicular Coxsackie Respiratory

Comp.

Minimum

cytotoxic

concentration^a/

lg cm^-3

EC₅₀^b/lg cm^-3

concentration^a/

lg cm^-3

Vesicular Coxsackie Respiratory

stomatitis virus B4

virus

stomatitis virus B4

virus

syncytial

virus

syncytial

virus

47

[100

20

[100

34

[100

4

1

100

[20

48

2

DS-5000

3

C20

[20

(S)-DHPA [250

(lM)

[250

4

C20

[20

5

C20

[20

Ribavirin

(lM)

[250

4

22

10

6

C20

[20

a

7

C20

[20

Amount required to cause a microscopically detectable alteration of

normal cell morphology

8

C20

[20

b

Amount required to reduce virus-induced cytopathogenicity by

9

C20

[20

50 %

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

33

34

35

36

37

38

39

40

41

42

43

44

45

46

C20

[20

C20

[20

studies were undertaken, using the linear free energy rela-

tionship (LFER) model described by Hansch and Fujita [26].

In this study, all synthesized N⁰-[4-[(substituted

imino)methyl]benzylidene]-substituted benzohydrazides

(1–48) were used for linear regression model generation.

The standard drugs norﬂoxacin and ﬂuconazole were not

used for model development because they differ in structure

from the synthesized compounds. Biological activity data

determined as MIC values were ﬁrst transformed into pMIC

values (i.e. -log MIC, Table 1); these were used as depen-

dent variables in the QSAR study. The different molecular

descriptors (independent variables), for example the loga-

rithm of the octanol–water partition coefﬁcient (log P),

molar refractivity (MR), Kier’s molecular connectivity (⁰v,

⁰v^v, ¹v, ¹v^v, ²v, ²v^v) and shape (j₁, ja₁, ja₂, ja₃) topological

indices, the Randic topological index (R), the Balaban

topological index (J), the Wiener topological index (W), total

energy (Te), energies of highest occupied molecular orbital

(HOMO) and lowest unoccupied molecular orbital (LUMO),

dipole moment (l) and electronic energy (Ele. E) [26–31]

were calculated for the hydrazide derivatives. Some of the

values obtained are presented in Table 9.

C20

[20

100

[20

100

[20

[100

C20

[100

[20

[100

[20

[100

[20

[100

[50

[100

[50

[100

[50

[100

[50

[100

Our earlier studies [15–17] indicated that multi-target

QSAR (mt-QSAR) models are more suitable than

one-target QSAR (ot-QSAR) models for describing anti-

microbial activity. In this study, therefore, we developed

multi-target QSAR models to describe the antimicrobial

activity of compounds 1–48.

For ot-QSAR models one should use ﬁve different

equations with different errors to predict the activity of a

new compound against ﬁve microbial species. Use of

ot-QSAR models, which are used almost throughout the

literature, were not practical, however, when we had to

predict each compound’s action against more than one

target. In those cases we had to develop one ot-QSAR

model for each target.

123

N⁰-[4-[(Substituted imino)methyl]benzylidene]-substituted benzohydrazides

833

Table 6 Cytotoxicity and antiviral activity of the synthesized hydrazide derivatives in Vero cell cultures

Comp.

Minimum cytotoxic

concentration^a/lg cm^-3

EC₅₀^b/lg cm^-3

Para-inﬂuenza-3

virus

Reovirus-1

Sindbis virus

Coxsackie virus B4

Punta Toro virus

1

[100

100

[100

[20

[100

[20

[100

[20

[100

[20

[100

[20

2

3

4

5

6

100

[20

7

[100

100

[100

[20

[100

[20

[100

[20

[100

[20

[100

[20

8

9

C20

[20

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

100

[20

100

[20

100

[20

100

[20

100

[20

[100

100

[100

[20

[100

[20

[100

[20

[100

[20

[100

[20

100

[20

100

[20

[100

123

834

P. Kumar et al.

Table 6 continued

Comp.

Minimum cytotoxic

concentration^a/lg cm^-3

EC₅₀^b/lg cm^-3

Para-inﬂuenza-3

virus

Reovirus-1

Sindbis virus

Coxsackie virus B4

Punta Toro virus

47

100

[20

[100

[250

50

[20

20

[20

96

[20

100

48

DS-5000

[100

[250

[100

[250

(S)-DHPA (lM)

Ribavirin (lM)

a

[250

50

Amount required to cause a microscopically detectable alteration of normal cell morphology

b

Amount required to reduce virus-induced cytopathogenicity by 50 %

where n is the number of data points, r is the correlation

coefﬁcient, q²is the cross validated r²obtained by the

leave-one-out (LOO) method, s is the standard error of the

estimate, and F is the Fischer statistic.

However, very recently interest has increased in the

development of multi-target QSAR (mt-QSAR) models. As

opposed 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 [32–35].

Because the coefﬁcient of Te in Eq. (1) is negative,

antibacterial activity of synthesized compounds will

decrease with increasing Te value. This is evidenced by the

In this study, we attempted to develop three different

types of mt-QSAR model, viz. an mt-QSAR model to

describe the antibacterial activity of the synthesized com-

pounds against S. aureus, B. subtilis, and E. coli, an

mt-QSAR model to describe the antifungal activity of the

synthesized compounds against C. albicans and A. niger,

and a common mt-QSAR model to describe the antimi-

crobial (overall antibacterial and antifungal) activity of the

synthesized hydrazide derivatives against all the above

mentioned microorganisms.

high antibacterial activity of compound

6

(pMI-

C_ab= 1.45 lM/cm³) which has a low Te value (Te =

-5,411.83).

To improve the value of the correlation coefﬁcient (r),

the electronic property total energy (Te) was coupled with

the lipophilicity log P; as a result the r value increased

from 0.664 to 0.706 (Eq. 2).

MLR-mt-QSAR model for antibacterial activity:

pMIC_ab¼ ꢁ0:020 log P ꢁ 0:000096Te þ 0:791

To develop mt-QSAR models, initially we calculated the

average antibacterial, antifungal, and antimicrobial activity

of the hydrazide derivatives (Table 1). These average anti-

bacterial activity values were correlated with the molecular

descriptors of the synthesized compounds (Table 10).

A high interrelationship was observed among topologi-

cal indices, the Wiener index (W), and the Randic index

(R, r = 0.989), and a low interrelationship was observed

between the lipophilic parameter log P and the electronic

property energy of the highest occupied molecular orbital

(HOMO) (r = 0.046). Correlation of average antibacterial,

antifungal, and antimicrobial activity values with different

molecular descriptors is shown in Table 11.

n ¼ 48 r ¼ 0:706 q²¼ 0:203 s ¼ 0:0511 F ¼ 22:35:

ð2Þ

The developed model was cross validated by the LOO

method [36]. The q²value is\0.5 (Eq. 2), which showed that

the developed model is invalid. According to the

recommendations of Kim et al. [37], however, regression

models are acceptable if the value of the standard deviation

(SD, Table 1) is \0.3. Because the value of standard

deviation is \0.3, the developed model (Eq. 2) is a valid

one. Furthermore, the observed and predicted antibacterial

activity values are close to each other (Table 12), so the

mt-QSAR model for antibacterial activity (Eq. 2) is a valid

one. The plot of predicted pMIC_abagainst observed pMIC_ab

(Fig. 1) also favours the developed model expressed by

Eq. (2). Further, the plot of observed pMIC_abversus residual

pMIC_ab(Fig. 2) indicated that there was no systemic error in

model development because the propagation of error was

observed on both sides of zero [38].

From the correlation matrix (Table 10), it was observed

that the electronic property total energy (Te) dominated

description of the antibacterial activity of the synthesized

compounds (Eq. 1).

LR-mt-QSAR model for antibacterial activity:

pMIC_ab¼ ꢁ0:00011Te þ 0:830

n ¼ 48 r ¼ 0:664 q²¼ 0:385 s ¼ 0:0533 F ¼ 36:21

ð1Þ

Kier’s second-order shape index (j₂) was found to be

the most dominating descriptor explaining the antifungal

123

N⁰-[4-[(Substituted imino)methyl]benzylidene]-substituted benzohydrazides

835

Table 7 Anti-HIV potential of the synthesized compounds in MT-4

cells

Table 7 continued

Comp.

Avg. IC₅₀/lg cm^-3

Avg. CC₅₀

lg cm^-3

/

Comp.

Avg. IC₅₀/lg cm^-3

Avg. CC₅₀

lg cm^-3

/

HIV-1

(IIIB)

HIV-2

(ROD)

HIV-1

(IIIB)

HIV-2

(ROD)

47

[59

59

[113

[4.00

[25

1

[53

[56

[54

[49

[37

[26

[47

[53

[22

[57

[5

[53

[56

[54

[49

[37

[2

53

48

[113

2

Nevirapine

0.047

[4.00

0.0016

3

56

Azidothymidine,

zidovudine, retrovir

0.001

4

54

5

49

Dideoxycytidine

0.29

2.9

0.30

4.6

[20

[50

6

49

Dideoxyinosine, didanosine

7

37

8

26

9

[4

47

Table 8 Cytotoxicity (IC₅₀) of the synthesized compounds against

human colon cell line HCT116 and breast cancer cell line MCF7

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

[53

[22

[57

[5

53

22

Comp. IC₅₀/lg cm^-3

HCT 116 MCF 7

Comp. IC₅₀/lg cm^-3

HCT 116 MCF 7

57

5

[10

[82

[12

[67

[102

[73

[45

[50

[65

[109

[41

[90

[77

[78

[42

[1

[10

[8

10

1

300

200

350

200

300

200

60

90

100

175

120

175

200

NA

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

150

450

300

150

82

2

[12

[67

[102

[45

[50

[65

[104

[41

[90

[77

[78

[42

[1

12

3

[1,000

60

210

67

4

NA

C102

C73

C45

50

5

50

NA

6

NA

400

7

100

NA

8

550

150

[1,000

175

300

NA

[1,000

60

NA

65

9

200

C109

41

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

5-FU

60

[1,000

NA

70

[1,000

NA

C90

77

70

40

NA

78

[1,000

110

150

110

200

190

19

[1,000

35

60

[1,000

122

C42

1

NA

25

NA

[1,000

125

[68

[58

[62

[15

[125

[10

[12

[86

[125

[4

[68

[58

[62

[15

[125

[102

[125

[86

[125

[4

68

250

200

18

80

58

70

NA

62

700

112

15

300

110

[125

102

[125

86

270

30

200

120

200

100

80

122

[1,000

350

NA

60

NA

110

6

30

400

[125

4

0.67 5-FU

6

0.67

Data are mean values from three replicates

NA, not able to obtain IC₅₀after three independent tests

[17

[59

[64

[73

[74

[70

[69

[17

[59

[64

[73

[74

[70

[69

17

59

64

73

74

activity of the synthesized substituted hydrazide deriva-

tives (Table 11).

70

69

LR-mt-QSAR model for antifungal activity:

123

836

P. Kumar et al.

Table 9 Values of selected descriptors used in QSAR studies of N⁰-[4-[(substituted imino)methyl]benzylidene]-substituted benzohydrazides

Comp.

log P

j₁

j₂

R

W

Te

LUMO

HOMO

l

1

5.72

6.24

6.65

6.19

5.67

4.92

5.43

4.87

5.85

4.42

4.94

5.35

5.67

6.19

6.60

6.19

4.92

5.43

5.85

5.67

5.15

5.67

4.37

4.94

4.37

4.84

4.89

5.43

4.87

5.39

4.87

5.43

4.56

5.08

3.94

4.51

2.29

2.98

5.42

3.55

20.73

21.70

22.68

24.64

23.66

20.73

21.70

23.66

22.68

20.73

21.70

22.68

20.73

21.70

22.68

21.70

20.73

21.70

22.68

23.66

22.68

23.66

21.70

23.66

24.64

21.70

23.66

24.64

23.66

21.70

22.68

20.31

21.70

19.33

17.36

23.17

19.33

11.11

11.25

11.41

12.30

12.14

11.11

11.25

12.14

11.41

11.11

11.25

11.41

11.11

11.25

11.41

11.25

11.11

11.25

11.41

12.14

12.00

12.14

11.25

12.14

12.30

11.25

12.14

12.30

12.14

11.25

11.41

11.58

11.87

10.78

9.21

12.70

13.11

13.52

13.51

14.42

14.02

14.01

12.72

13.13

14.02

13.52

12.70

13.10

13.51

12.70

13.11

13.51

13.10

12.70

13.11

13.51

14.02

14.01

13.61

14.02

14.01

13.10

14.01

14.02

14.42

13.10

14.02

14.01

14.42

14.01

13.10

13.11

13.10

13.51

11.69

13.20

11.19

10.19

14.69

11.19

2,192.00

2,412.00

2,655.00

2,676.00

2,657.00

3,262.00

2,908.00

2,968.00

2,154.00

2,372.00

2,984.00

2,615.00

2,192.00

2,432.00

2,676.00

2,657.00

2,192.00

2,412.00

2,676.00

2,412.00

2,192.00

2,412.00

2,657.00

2,914.00

2,958.00

2,718.00

2,974.00

3,028.00

2,452.00

2,968.00

2,908.00

3,205.00

3,262.00

2,432.00

2,908.00

3,028.00

3,262.00

2,968.00

2,452.00

2,412.00

2,432.00

2,695.00

1,785.00

2,492.00

1,554.00

1,160.00

3,174.00

1,554.00

-4,220.71

-4,580.78

-4,532.36

-4,532.43

-5,411.83

-5,051.73

-5,051.81

-4,181.28

-4,541.35

-5,012.41

-4,493.00

-4,081.72

-4,441.82

-4,393.43

-4,016.52

-4,376.57

-4,328.23

-4,376.60

-4,181.26

-4,541.31

-4,492.96

-5,051.70

-5,051.75

-4,691.64

-5,051.70

-4,912.84

-4,441.83

-4,912.57

-4,912.49

-5,068.67

-5,272.84

-4,541.35

-5,012.26

-5,012.37

-5,372.37

-5,012.35

-4,541.37

-4,541.41

-4,553.12

-4,913.13

-3,882.10

-4,237.44

-3,891.18

-3,578.57

-4,621.09

-3,726.27

-0.85

-0.87

-0.79

-0.80

-1.54

-1.12

-1.23

-0.86

-0.88

-1.46

-0.79

-0.67

-0.79

-0.64

-0.72

-0.79

-0.70

-0.84

-0.74

-0.80

-0.71

-1.46

-1.49

-1.64

-1.65

-1.37

-0.81

-1.21

-1.01

-1.12

-1.45

-0.85

-1.08

-1.41

-1.50

-1.19

-0.87

-0.94

-0.80

-0.92

-0.43

-0.46

-0.52

-0.51

-0.80

-0.43

-8.66

-8.78

-8.65

-8.56

-8.62

-9.15

-9.01

-9.03

-8.69

-8.80

-9.15

-8.64

-8.59

-8.69

-8.47

-8.52

-8.65

-8.70

-8.51

-8.76

-8.66

-8.72

-8.58

-8.91

-8.89

-8.87

-8.93

-8.91

-8.65

-8.82

-8.83

-8.82

-8.92

-8.77

-8.93

-9.05

-9.07

-8.96

-8.72

-8.80

-8.71

-8.72

-8.66

-8.69

-8.73

-8.66

-8.31

-8.66

4.92

2

5.88

4.62

4.81

5.18

8.02

4.25

6.79

5.62

6.48

8.50

5.88

6.19

7.65

5.74

6.13

6.30

6.88

5.78

7.18

5.05

5.74

5.03

4.87

4.32

4.26

4.61

11.23

7.45

8.33

8.99

9.59

11.12

6.51

4.77

9.29

9.38

7.97

6.04

6.61

8.13

8.84

5.44

5.92

6.48

5.77

6.22

5.47

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

45

42

43

44

46

47

48

11.74

10.78

123

N⁰-[4-[(Substituted imino)methyl]benzylidene]-substituted benzohydrazides

837

Table 10 Correlation matrix for the antibacterial activity of N⁰-[4-[(substituted imino)methyl]benzylidene]-substituted benzohydrazides

pMIC_ab

log P

j₂

R

W

Te

LUMO

HOMO

l

pMIC_ab

log P

j₂

1.000

0.453

1.000

0.572

0.258

1.000

0.636

0.530

0.874

1.000

0.651

0.468

0.891

0.989

1.000

-0.664

-0.342

-0.862

-0.892

-0.924

1.000

-0.546

-0.207

-0.746

-0.697

-0.743

0.862

-0.379

0.046

0.159

-0.264

0.332

-0.596

-0.390

-0.455

0.703

R

0.319

W

0.384

Te

-0.421

-0.320

-0.373

1.000

LUMO

HOMO

l

1.000

0.791

1.000

correlated to Kier’s second-order shape index (j₂), i.e.

the antifungal activity of the compounds will increase with

increasing j₂value. High antifungal activity of compounds

25 and 27 (pMIC_af= 1.66) having a high j₂value

(j₂= 12.14) illustrates the positive correlation. Very

useful topological indices of the second generation are

the j indices of molecular shape and ﬂexibility [39].

According to Kier, the shape of a molecule may be

partitioned into attributes, each described by the count of

bonds of various path lengths. The basis for devising a

relative index of shape is given by the relationship between

Table 11 Correlation of different molecular descriptors with the

antimicrobial activity of substituted hydrazide derivatives

pMIC_ab

pMIC_af

pMIC_am

log P

MR

⁰v

⁰v^v

¹v

¹v^v

²v

²v^v

³v

0.453

0.553

0.644

0.582

0.636

0.573

0.637

0.531

0.590

0.401

0.641

0.572

0.407

0.650

0.566

0.340

0.636

-0.409

0.651

-0.664

-0.643

0.633

0.379

-0.546

-0.379

0.159

0.322

0.617

0.639

0.583

0.648

0.585

0.627

0.516

0.529

0.354

0.645

0.667

0.562

0.643

0.652

0.478

0.648

-0.473

0.647

-0.603

-0.612

0.607

0.317

-0.629

-0.317

0.219

0.473

0.698

0.770

0.699

0.770

0.693

0.759

0.628

0.675

0.456

0.771

0.737

0.572

0.776

0.725

0.482

0.770

-0.525

0.778

-0.763

-0.755

0.745

0.421

-0.700

-0.421

0.223

l

the number of paths of length l in the molecule i, P_i, and

reference values based on molecules with a given number

³v^v

l

of atoms, n, in which the values of P are maximum and

l

j₁

minimum, P_maxand P_min. The ﬁrst-order shape attribute,

j₁, is given by the expression:

j₂

j₃

2

1

2

j₁¼ nðn ꢁ 1Þ =ð P_iÞ :

ja₁

ja₂

ja₃

R

The second and third-order kappa indices are deﬁned as

follows:

2

j₂¼ ðn ꢁ 1Þðn ꢁ 2Þ =ð P_iÞ :

J

Coupling of the topological index Kier’s second order

shape index (j₂) with the electronic property energy of the

lowest unoccupied molecular orbital (LUMO) resulted in

an increase in the r value from 0.667 to 0.695 (Eq. 4).

MLR-mt-QSAR model for antifungal activity:

W

Te

Ele. E

Nu. E

I.P.

LUMO

HOMO

l

pMIC_af¼ ꢁ0:067j₂ꢁ 0:0076 Lumo þ 0:641

n ¼ 48 r ¼ 0:695 q²¼ 0:191 s ¼ 0:063 F ¼ 21:07:

ð4Þ

Although the q²value derived for Eq. (4) by the LOO

method was \0.5, which made the model invalid, the low

residual values observed for antifungal activity predictions

using Eq. (4) (Table 12) and low standard deviation

(Table 1) resulted in the QSAR model for antifungal

activity being a valid one.

pMIC_af¼ 0:1007j₂þ 0:332

n ¼ 48 r ¼ 0:667 q²¼ 0:389 s ¼ 0:065 F ¼ 36:84:

ð3Þ

It is clearly evident from Eq. (3) that the antifungal

activity of the synthesized compounds is positively

123

838

P. Kumar et al.

Table 12 Comparison of observed and predicted antimicrobial activity obtained by mt-QSAR models

Comp.

pMIC_ab/lM cm^-3

pMIC_af/lM cm^-3

pMIC_am/lM cm^-3

Obs.

Pre.

Res.

Obs.

Pre.

Res.

0.01

Obs.

Pre.

Res.

1

1.26

1.30

1.39

1.29

1.39

1.45

1.41

1.31

1.24

1.28

1.29

1.27

1.24

1.28

1.27

1.24

1.38

1.37

1.38

1.34

1.38

1.37

1.41

1.37

1.41

1.39

1.28

1.29

1.39

1.31

1.43

1.38

1.39

1.43

1.39

1.28

1.38

1.36

1.40

1.31

1.35

1.09

1.25

1.40

1.29

1.31

1.36

1.44

1.39

1.29

1.34

1.37

1.34

1.27

1.32

1.29

1.34

1.29

1.34

1.39

1.35

1.39

1.35

1.32

1.35

1.37

1.40

1.34

1.37

1.42

1.37

1.34

1.32

1.36

1.24

1.29

1.21

1.19

1.34

1.22

-0.05

-0.06

0.03

1.46

1.50

1.49

1.55

1.51

1.44

1.48

1.49

1.47

1.44

1.48

1.47

1.44

1.48

1.47

1.48

1.44

1.48

1.47

1.51

1.66

1.62

1.66

1.64

1.63

1.64

1.51

1.53

1.48

1.34

1.49

1.53

1.49

1.48

1.31

1.50

1.41

1.45

1.39

1.20

1.50

1.39

1.45

1.46

1.47

1.58

1.54

1.55

1.45

1.46

1.57

1.47

1.44

1.46

1.45

1.44

1.46

1.44

1.46

1.57

1.58

1.56

1.46

1.55

1.53

1.55

1.58

1.46

1.54

1.56

1.58

1.55

1.46

1.47

1.46

1.48

1.45

1.47

1.40

1.30

1.49

1.40

1.34

1.38

1.43

1.37

1.43

1.49

1.45

1.39

1.32

1.36

1.37

1.35

1.32

1.36

1.35

1.32

1.42

1.41

1.42

1.38

1.42

1.41

1.45

1.51

1.47

1.51

1.49

1.42

1.43

1.49

1.39

1.47

1.42

1.37

1.43

1.47

1.43

1.36

1.42

1.34

1.44

1.35

1.39

1.21

1.23

1.44

1.33

1.36

1.38

1.39

1.40

1.39

1.48

1.43

1.44

1.36

1.38

1.46

1.39

1.35

1.38

1.39

1.35

1.37

1.39

1.38

1.35

1.37

1.39

1.45

1.44

1.46

1.45

1.38

1.44

1.43

1.46

1.48

1.38

1.43

1.46

1.48

1.44

1.38

1.40

1.31

1.36

1.29

1.26

1.44

1.29

-0.02

0.00

2

0.04

0.02

3

0.04

4

-0.07

0.03

0.02

-0.03

0.04

5

0.02

6

0.01

-0.03

-0.04

-0.01

0.02

0.01

7

0.02

8

-0.08

-0.05

-0.06

-0.08

-0.07

-0.03

-0.04

-0.05

0.04

-0.05

-0.04

-0.02

-0.09

-0.04

-0.03

-0.02

-0.04

-0.03

0.05

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

45

42

43

44

46

47

48

-0.08

0.00

0.02

0.01

0.02

0.00

0.02

0.03

0.01

0.02

0.04

0.02

0.04

0.05

0.00

0.03

0.04

0.02

0.05

0.03

0.01

0.02

-0.06

0.09

0.00

0.02

0.06

0.02

0.05

0.03

0.02

0.08

0.05

0.04

0.08

0.04

-0.04

-0.06

0.04

0.17

0.04

0.09

-0.01

0.06

0.11

-0.06

0.03

-0.04

-0.05

0.02

-0.07

-0.01

0.04

0.02

-0.20

-0.07

-0.05

-0.06

0.02

-0.06

-0.03

-0.01

-0.02

0.04

0.02

0.01

0.02

-0.06

0.04

0.01

0.04

-0.15

0.02

-0.04

0.04

0.07

-0.04

-0.02

-0.01

-0.10

0.01

0.04

0.06

0.03

-0.12

0.06

-0.08

-0.03

0.00

0.06

0.07

-0.01

0.04

123

N⁰-[4-[(Substituted imino)methyl]benzylidene]-substituted benzohydrazides

839

1.5

.2

.1

1.4

1.3

-.0

1.2

1.1

-.1

-.2

1.0

1.1

1.2

1.3

1.4

1.5

1.0

1.1

1.2

1.3

1.4

1.5

Observed pMIC_ab/µM cm^-3

Fig. 1 Plot of predicted pMIC_abvalues against observed pMIC_ab

values for linear regression model expressed by Eq. (2)

Fig. 2 Plot of residual pMIC_abvalues against observed pMIC_ab

values for linear regression model expressed by Eq. (2)

The antimicrobial activity of the synthesized hydrazide

derivatives was best explained by a topological index, the

Wiener index (W) (Table 11).

derivatives with a nucleophilic amino acid residue, for

example cysteine, of microorganisms [40].

The validity and predictability of the QSAR model for

antimicrobial activity, i.e. Eq. (6) is evidenced by the high

q²value (q²[ 0.5) obtained by the LOO method and the

low residual activity values (Table 12). In summary,

QSAR analysis indicated the importance of the topological

index, the Wiener index (W), and the electronic property,

the energy of the lowest unoccupied molecular orbital

(LUMO), in describing the antimicrobial activity of N⁰-[4-

[(substituted imino)methyl]benzylidene]-substituted benzo-

hydrazides.

LR-mt-QSAR model for antimicrobial activity:

pMIC_am¼ ꢁ0:0001W þ 1:114

n ¼ 48 r ¼ 0:778 q²¼ 0:567 s ¼ 0:040 F ¼ 70:62:

ð5Þ

The Wiener index W = W(G) of G is deﬁned as the half

sum of the elements of the distance matrix:

X

W ¼ WðGÞ ¼ 1=2

ðDÞij i ¼ 1; j ¼ 1

Generally, for QSAR studies, the biological activity of

the compounds should span two to three orders of magni-

tude but in this study the range of antimicrobial activity of

the synthesized compounds is within one order of magni-

tude. This is similar to results obtained by Bajaj et al. [41]

who stated that the reliability of the QSAR model lies in its

predictive ability even though the activity data are in a

narrow range. When biological activity data is in a narrow

range, a low standard deviation of the biological activity

justiﬁes its use in QSAR studies [42]. The low standard

deviation observed in the antimicrobial activity data in

Table 1 justiﬁes its use in QSAR studies.

where (D)ij is the ijth element of the distance matrix which

denotes the shortest graph-theoretical distance between

sites i and j of G [31].

Similar to the antifungal activity, the antimicrobial

activity of the synthesized compounds also positively

correlated with the Wiener index (W) (Table 11). Addition

of the electronic property energy of the lowest unoccupied

molecular orbital (LUMO) to the topological index

W resulted in the best model for describing antimicrobial

activity of the synthesized compounds (Eq. 6).

MLR-mt-QSAR model for antimicrobial activity:

pMIC_am¼ ꢁ0:0008W þ 0:052LUMO þ 1:138

n ¼ 48 r ¼ 0:799 q²¼ 0:582 s ¼ 0:039 F ¼ 39:70:

ð6Þ

The electronic property LUMO, which denotes the

energy of the lowest unoccupied molecular orbital, is

directly related to electron afﬁnity and characterizes the

sensitivity of the molecule to attack by a nucleophile. The

contribution of LUMO to describing antimicrobial activity

may be attributed to the interaction of hydrazide

Conclusion

N⁰-[4-[(Substituted

imino)methyl]benzylidene]-substituted

benzohydrazides were synthesized and evaluated for their

antimicrobial, antiviral, and anticancer potential. Antimicro-

bial activity results revealed that N⁰-[4-[(4-chloropheny-

limino)methyl]benzylidene]-3-nitrobenzohydrazide (25) and

N⁰-[4-[(2-chlorophenylimino)methyl]benzylidene]-4-nitro

123

840

P. Kumar et al.

benzohydrazide (27) (pMIC_am= 1.51) were the most

potent antimicrobial agents. In general, the synthesized

compounds were bacteriostatic and fungistatic in action,

because their MFC and MBC values were threefold higher

than their MIC values. None of the synthesized compounds

inhibited viral replication at sub-toxic concentrations. The

results of anti-HIV screening against HIV-2 strain ROD

indicated that compound 29 (IC₅₀C 1 lg/cm³) was more

potent than the standard drug nevirapine (IC₅₀C 4 lg/cm³)

and that 39 (IC₅₀C 4 lg/cm³) was equipotent. Compound

20 was found to be the most effective anticancer agent

against both HCT116 and MCF7 cancer cell lines, with

IC₅₀values of 19 and 18 lg/cm³, respectively. QSAR

analysis indicated the importance of a topological index,

the Wiener index (W), and an electronic property, the

energy of the lowest unoccupied molecular orbital

(LUMO), in describing the antimicrobial activity of the

synthesized compounds.

hydrate (99 %, 0.015 mol) was added to an ethanolic

solution of ester (0.01 mol) and the mixture was heated

under reﬂux for 5 h. The reaction mixture was then cooled

and the precipitate was isolated by ﬁltration, washed with

water, dried, and recrystallized from ethanol.

A solution of 6.70 g terephthaldehyde (50 mmol) in

50 cm³ethanol was added to a solution of the substituted

benzoic acid hydrazide (synthesized above; 0.05 mol) in

50 cm³ethanol. The mixture was heated under reﬂux for

5 h. The reaction mixture was then left to cool at room

temperature and the precipitated hydrazone was isolated by

ﬁltration, dried, and recrystallized from ethanol.

A solution of 50 mmol of the above synthesized

hydrazone in 50 cm³DMF was added to a solution of

50 mmol substituted anilines/amines/naphthalen-1-amine

in 50 cm³DMF. The mixture was heated under reﬂux for

5 h followed by cooling in an ice bath and the resulting

product was isolated by ﬁltration and puriﬁed.

N⁰-[4-[(Phenylimino)methyl]benzylidene]-4-chlorobenzoic

acid hydrazide (1, C₂₁H₁₆ClN₃O)

Experimental

ꢀ

Yield: 64.1 %; TLC: R_f= 0.57; IR (KBr): m = 1,649

(C=O str., secondary amide), 1,506 (C=C skeletal str.,

phenyl nucleus), 820 (C–H out of plane bending, 1,4-

disubstituted benzene ring), 730 (C–Cl str., ArCl), 706

(C–H out of plane bending, monosubstituted benzene ring)

Starting materials were obtained from commercial sources

and were used without further puriﬁcation. Reaction pro-

gress was observed by thin-layer chromatography on

commercial silica gel plates (Merck silica gel F₂₅₄on

aluminum sheets) with chloroform–acetone 9:1 as mobile

phase. Melting points were determined in open capillary

tubes on a Sonar melting point apparatus. Nuclear magnetic

resonance (¹H and ¹³C NMR) spectra were determined

using a Bruker Avance II 400 NMR spectrometer and are

expressed in parts per million (d, ppm) downﬁeld from the

internal standard, TMS. Compounds were dissolved in

appropriate deuterated solvents. NMR data are provided

with multiplicity (s, singlet; d, doublet; t, triplet; m, mul-

tiplet) and number of protons. Infrared (IR) spectra were

recorded on a Varian Resolutions Pro FTIR spectrometer.

Elemental analysis was performed on a Perkin–Elmer 2400

C, H, N analyzer. Mass spectra were taken on a Waters

Micromass Q-ToF Micro instrument.

1

cm^-1; H NMR (MeOH-d₄): d = 8.32 (s, 1H, –CH=N),

7.92 (d, 2H, 4-chlorophenyl), 7.58 (d, 2H, benzylidene),

7.46 (m, 4H, 4-chlorophenyl, benzylidene), 7.29 (m, 5H,

phenylimino) ppm; ¹³C NMR (DMSO-d₆): d = 163.3,

160.2, 153.5, 153.1, 143.1, 137.5, 136.0, 132.1, 130.4,

130.2, 129.5, 129.3, 128.5, 127.6, 127.6, 123.4, 122.5 ppm;

MS (ES?, ToF): m/z = 363 ([M ? 1]^?).

N⁰-[4-[(2-Chlorophenylimino)methyl]benzylidene]-4-chlo-

robenzoic acid hydrazide (2, C₂₁H₁₅Cl₂N₃O)

ꢀ

Yield: 61.2 %; TLC: R_f= 0.75; IR (KBr): m = 1,649

(C=O str., secondary amide), 1,506 (C=C skeletal str.,

phenyl nucleus), 815 (C–H out of plane bending, 1,4-

disubstituted benzene ring), 754 (C–H out of plane

bending, 1,2-disubstituted benzene ring), 730 (C–Cl str.,

ArCl) cm^-^{1 1}H NMR (MeOH-d₄): d = 8.42 (s, 1H,

;

General procedure for synthesis of N⁰-[4-[(substituted

imino)methyl]benzylidene]-substituted benzohydrazides

1–48

–CH=N), 7.93 (m, 4H, 4-chlorophenyl, benzylidene), 7.93

(d, 2H, benzylidene), 7.59 (d, 2H, 4-chlorophenyl), 7.35

(m, 4H, 2-chlorophenyl) ppm; ¹³C NMR (DMSO-d₆):

d = 163.0, 160.5, 143.4, 143.3, 137.4, 136.2, 132.4, 129.6,

129.1 ppm; MS (ES?, ToF): m/z = 397 ([M ? 1]^?).

A mixture of 80 mmol substituted benzoic acid and

34.04 g (43.14 cm³, 0.74 mol) ethanol was heated under

reﬂux in the presence of sulfuric acid until completion of

the reaction. When the reaction was complete, the reaction

mixture was added to 200 cm³ice-cold water and the ester

formed was extracted with 50 cm³ether. The ether layer

was separated and, on evaporation, yielded the crude ester

which was then recrystallized from alcohol. Hydrazine

N⁰-[4-[(2,3-Dimethylphenylimino)methyl]benzylidene]-4-

chlorobenzoic acid hydrazide (3, C₂₃H₂₀ClN₃O)

ꢀ

Yield: 65.5 %; TLC: R_f= 0.79; IR (KBr): m = 1,650

(C=O str., secondary amide), 1,506 (C=C skeletal str.,

phenyl nucleus), 819 (C–H out of plane bending, 1,4-

disubstituted benzene ring), 730 (C–C out of plane

123

N⁰-[4-[(Substituted imino)methyl]benzylidene]-substituted benzohydrazides

841

bending, 1,2,3-trisubstituted benzene ring), 707 (C–Cl str.,

ArCl) cm^-1

N⁰-[4-[(2-Nitrophenylimino)methyl]benzylidene]-4-chloro-

benzoic acid hydrazide (7, C₂₁H₁₅ClN₄O₃)

;

¹H NMR (MeOH-d₄): d = 8.12 (s, 1H,

ꢀ

–CH=N), 7.96 (m, 4H, 4-chlorophenyl, benzylidene), 7.83

(d, 2H, benzylidene), 7.44 (d, 2H, 4-chlorophenyl), 7.02

(m, 3H, 2,3-dimethylphenyl), 2.40 (s, 6H, Ar(CH₃)₂) ppm;

¹³C NMR (DMSO-d₆): d = 163.6, 160.5, 151.3, 143.0,

137.3, 136.4, 132.4, 129.2, 129.0, 128.8, 127.7, 119.0,

17.6, 9.2 ppm; MS (ES?, ToF): m/z = 391 ([M ? 1]^?).

Yield: 63.5 %; TLC: R_f= 0.69; IR (KBr): m = 1,649

(C=O str., secondary amide), 1,548 (asym. str., NO₂), 1,514

(C=C skeletal str., phenyl nucleus), 814 (C–H out of plane

bending, 1,4-disubstituted benzene ring), 752 (C–H out of

plane bending, 1,2-disubstituted benzene ring) cm^-1; H

1

NMR (MeOH-d₄): d = 8.72 (s, 1H, –CH=N), 8.44 (d, 1H,

ArNO₂), 8.04 (d, 2H, benzylidene), 7.99 (m, 4H, 4-chlo-

rophenyl, benzylidene), 7.70 (m, 1H, ArNO₂), 7.59 (d, 2H,

ArNO₂), 7.28 (d, 2H, 4-chlorophenyl) ppm; ¹³C NMR

(DMSO-d₆): d = 129.6, 129.3, 128.3, 127.5, 119.8, 109.9,

107.3 ppm; MS (ES?, ToF): m/z = 408 ([M ? 1]^?).

N⁰-[4-[(2,4-Dimethylphenylimino)methyl]benzylidene]-4-

chlorobenzoic acid hydrazide (4, C₂₃H₂₀ClN₃O)

ꢀ

Yield: 68.3 %; TLC: R_f= 0.52; IR (KBr): m = 1,650

(C=O str., secondary amide), 1,506 (C=C skeletal str.,

phenyl nucleus), 839 (C–H out of plane bending, 1,2,4-

trisubstituted benzene ring), 818 (C–H out of plane

bending, 1,4-disubstituted benzene ring), 730 (C–Cl str.,

N⁰-[4-[(3-Nitrophenylimino)methyl]benzylidene]-4-chloro-

benzoic acid hydrazide (8, C₂₁H₁₅ClN₄O₃)

ArCl) cm^-1

;

¹H NMR (MeOH-d₄): d = 8.57 (s, 1H,

Yield: 66.9 %; TLC: R_f= 0.54; IR (KBr): m = 1,649

ꢀ

–CH=N), 7.94 (m, 4H, 4-chlorophenyl, benzylidene), 7.81

(d, 2H, benzylidene), 7.47 (d, 2H, 4-chlorophenyl), 7.01 (s,

1H, phenylimino), 7.00 (d, 2H, phenylimino), 2.36 (s, 6H,

Ar(CH₃)₂) ppm; ¹³C NMR (DMSO-d₆): d = 163.5, 160.1,

148.5, 143.1, 137.2, 136.9, 136.3, 132.7, 132.4, 130.7,

129.4, 129.3, 127.1, 122.3 ppm; MS (ES?, ToF): m/

z = 391 ([M ? 1]^?).

(C=N str., CH=N), 1,632 (C=O str., secondary amide),

1,520 (NO₂asym. str., Ar-NO₂), 812 (C–H out of plane

bending, 1,4-disubstituted benzene ring), 738 (C–Cl str.,

ArCl), 693 (C–C out of plane bending, 1,3-disubstituted

1

benzene ring) cm^-1; H NMR (MeOH-d₄): d = 8.44 (s,

1H, –CH=N), 8.17 (d, 2H, ArNO₂), 7.98 (d, 2H, benzyl-

idene), 7.70 (m, 4H, 4-chlorophenyl, benzylidene), 7.60 (d,

2H, ArNO₂), 7.58 (d, 2H, 4-chlorophenyl), 7.53 (m, 1H,

ArNO₂) ppm; ¹³C NMR (DMSO-d₆): d = 191.6, 149.5,

139.8, 129.7, 129.4, 128.4, 127.7, 120.1, 110.5, 107.8 ppm;

MS (ES?, ToF): m/z = 408 ([M ? 1]^?).

N⁰-[4-[(2,5-Dimethylphenylimino)methyl]benzylidene]-4-

chlorobenzoic acid hydrazide (5, C₂₃H₂₀ClN₃O)

ꢀ

Yield: 75.3 %; TLC: R_f= 0.55; IR (KBr): m = 1,649 (C=O

str., secondary amide), 1,506 (C=C skeletal str., phenyl

nucleus), 815 (C–H out of plane bending, 1,4-disubstituted

benzene ring), 730 (C–Cl str., ArCl) cm^-1

N⁰-[4-[(Phenylimino)methyl]benzylidene]-2-hydroxybenzo-

ic acid hydrazide (9, C₂₁H₁₇N₃O₂)

;

¹H NMR

ꢀ

(MeOH-d₄): d = 8.22 (s, 1H, –CH=N), 7.93 (m, 4H,

4-chlorophenyl, benzylidene), 7.83 (d, 2H, benzylidene),

7.46 (d, 2H, 4-chlorophenyl), 7.01 (s, 1H, phenylimino),

Yield: 62.6 %; TLC: R_f= 0.51; IR (KBr): m = 1,650

(C=N str., CH=N), 1,625 (C=O str., secondary amide),

1,585 (C=C skeletal str., phenyl nucleus), 1,359 (C–O str.

and O–H in plane bending, phenol), 740 (C–H out of plane

bending, 1,2-disubstituted benzene ring), 692 (C–C out of

7.00 (d, 2H, phenylimino), 2.69 (s, 6H, Ar(CH₃)₂) ppm; ¹³

C

NMR (DMSO-d₆): d = 163.6, 160.3, 151.0, 143.4, 137.3,

136.8, 136.0, 132.0, 130.2, 129.5, 129.1, 127.7, 123.9, 24.3,

15.6 ppm; MS (ES?, ToF): m/z = 391 ([M ? 1]^?).

plane bending, monosubstituted benzene ring) cm^-1; H

1

NMR (MeOH-d₄): d = 15.42, 8.44 (s, 1H, –CH=N), 7.96

(d, 2H, benzylidene), 7.83 (d, 1H, 2-hydroxyphenyl), 7.82

(d, 2H, benzylidene), 7.40 (m, 1H, 2-hydroxyphenyl), 7.27

(m, 5H, phenylimino), 7.11 (m, 1H, 2-hydroxyphenyl),

6.94 (d, 1H, 2-hydroxyphenyl) ppm; ¹³C NMR (DMSO-

d₆): d = 163.2, 160.4, 160.3, 159.1, 153.3, 143.2, 136.3,

133.5, 130.2, 129.8, 129.0, 127.3, 122.0, 121.7, 119.8 ppm;

MS (ES?, ToF): m/z = 344 ([M ? 1]^?).

N⁰-[4-[(2-Chloro-4-nitrophenylimino)methyl]benzylidene]-

4-chlorobenzoic acid hydrazide (6, C₂₁H₁₄Cl₂N₄O₃)

ꢀ

Yield: 72.0 %; TLC: R_f= 0.61; IR (KBr): m = 1,650

(C=O str., secondary amide), 1,545 (NO₂asym. str.,

Ar–NO₂), 1,506 (C=C skeletal str., phenyl nucleus), 839

(C–H out of plane bending, 1,2,4-trisubstituted benzene

ring), 818 (C–H out of plane bending, 1,4-disubstituted

benzene ring), 730 (C–Cl str., ArCl) cm^-1

;

¹H NMR

N⁰-[4-[(2-Chlorophenylimino)methyl]benzylidene]-2-hy-

droxybenzoic acid hydrazide (10, C₂₁H₁₆ClN₃O₂)

(MeOH-d₄): d = 8.65 (s, 1H, –CH=N), 8.29 (s, 1H,

ArClNO₂), 8.11 (d, 1H, ArClNO₂), 7.94 (m, 2H, benzyli-

dene), 7.89 (m, 4H, 4-chlorophenyl, benzylidene), 7.59 (d,

1H, ArClNO₂), 7.45 (d, 2H, 4-chlorophenyl) ppm; ¹³C NMR

(DMSO-d₆): d = 163.4, 160.1, 149.3, 148.4, 143.5, 137.4,

136.3, 132.3, 129.2, 129.0, 128.7, 125.0, 124.1, 120.6 ppm;

MS (ES?, ToF): m/z = 442 ([M ? 1]^?).

ꢀ

Yield: 68.1 %; TLC: R_f= 0.67; IR (KBr): m = 1,650

(C=N str., CH=N), 1,626 (C=O str., secondary amide),

1,585 (C=C skeletal str., phenyl nucleus), 1,359 (C–O str.

and O–H in plane bending, phenol), 740 (C–H out of plane

bending, 1,2-disubstituted benzene ring), 695 (C–Cl str.,

ArCl) cm^-1

;

¹H NMR (MeOH-d₄): d = 8.41 (s, 1H,

123

842

P. Kumar et al.

–CH=N), 7.81 (d, 2H, benzylidene), 7.81 (d, 1H,

2-hydroxyphenyl), 7.63 (d, 2H, benzylidene), 7.32 (m,

4H, phenylimino), 7.31 (m, 1H, 2-hydroxyphenyl), 7.10

(m, 1H, 2-hydroxyphenyl), 6.72 (d, 1H, 2-hydroxyphenyl)

ppm; ¹³C NMR (DMSO-d₆): d = 165.3, 159.7, 147.9,

133.8, 129.6, 128.1, 127.6, 127.5, 118.6, 117.3, 114.8 ppm;

MS (ES?, ToF): m/z = 379 ([M ? 1]^?).

5H, phenylimino), 6.99 (d, 2H, 4-aminophenyl) ppm; ¹³C

NMR (DMSO-d₆): d = 163.3, 160.0, 153.3, 151.6, 143.1,

136.1, 130.1, 129.2, 128.3, 127.0, 124.1, 122.4, 116.0 ppm;

MS (ES?, ToF): m/z = 343 ([M ? 1]^?).

N⁰-[4-[(3-Chlorophenylimino)methyl]benzylidene]-4-ami-

nobenzoic acid hydrazide (14, C₂₁H₁₇ClN₄O)

ꢀ

Yield: 73.8 %; TLC: R_f= 0.55; IR (KBr): m = 1,675

N⁰-[4-[(4-Nitrophenylimino)methyl]benzylidene]-2-hy-

droxybenzoic acid hydrazide (11, C₂₁H₁₆N₄O₄)

(C=N str., CH=N), 1,624 (C=O str., secondary amide),

1,584 (C=C skeletal str., phenyl nucleus), 1,303 (C–N str.,

Ar-NH₂), 837 (C–H out of plane bending, 1,4-disubstituted

benzene ring), 730 (C–Cl str., ArCl), 682 (C–C out of plane

ꢀ

Yield: 69.3 %; TLC: R_f= 0.56; IR (KBr): m = 1,626

(C=O str., secondary amide), 1,546 (asym. str., NO₂), 1,492

(C=C skeletal str., phenyl nucleus), 1,360 (C–O str. and O–

H in plane bending, phenol), 829 (C–H out of plane

bending, 1,4-disubstituted benzene ring), 740 (C–H out of

1

bending, 1,3-disubstituted benzene ring) cm^-1; H NMR

(MeOH-d₄): d = 8.68 (s, 1H, –CH=N), 7.93 (d, 2H,

benzylidene), 7.80 (d, 2H, benzylidene), 7.74 (d, 2H,

4-aminophenyl), 7.34 (s, 1H, phenylimino), 7.26 (d,

2H, phenylimino), 7.02 (m, 1H, phenylimino), 6.64 (d,

2H, 4-aminophenyl) ppm; ¹³C NMR (DMSO-d₆): d =

163.1, 160.3, 154.6, 151.8, 143.1, 136.0, 135.9, 131.7,

129.1, 128.4, 127.3, 124.2, 122.7, 120.2, 116.2 ppm; MS

(ES?, ToF): m/z = 378 ([M ? 1]^?).

1

plane bending, 1,2-disubstituted benzene ring) cm^-1; H

NMR (MeOH-d₄): d = 8.43 (s, 1H, –CH=N), 8.22 (d,

2H, phenylimino), 8.00 (d, 2H, benzylidene), 7.93 (d, 2H,

benzylidene), 7.80 (d, 1H, 2-hydroxyphenyl), 7.51 (d, 2H,

phenylimino), 7.44 (m, 1H, 2-hydroxyphenyl), 7.00 (m,

1H, 2-hydroxyphenyl), 6.97 (d, 1H, 2-hydroxyphenyl)

ppm; ¹³C NMR (DMSO-d₆): d = 163.0, 160.7, 160.2,

159.3, 159.1, 146.9, 143.3, 136.5, 133.8, 129.9, 129.2,

123.5, 122.2, 121.5, 119.5 ppm; MS (ES?, ToF): m/

z = 389 ([M ? 1]^?).

N⁰-[4-[(2,4-Dimethylphenylimino)methyl]benzylidene]-4-

aminobenzoic acid hydrazide (15, C₂₃H₂₂N₄O)

ꢀ

Yield: 49.7 %; TLC: R_f= 0.67; IR (KBr): m = 1,706 (C=N

str., CH=N), 1,623 (C=O str., secondary amide), 1,592 (C=C

skeletal str., phenyl nucleus), 1,308 (C–N str., Ar-NH₂), 884

(C–H out of plane bending, 1,2,4-trisubstituted benzene

ring), 802 (C–H out of plane bending, 1,4-disubstituted

benzene ring) cm^-1; ¹H NMR (MeOH-d₄): d = 8.57 (s, 1H,

–CH=N), 7.93 (d, 2H, benzylidene), 7.72 (d, 2H, 4-amino-

phenyl), 7.72 (d, 2H, benzylidene), 7.10 (d, 2H,

phenylimino), 6.94 (s, 1H, phenylimino), 6.68 (d, 2H,

4-aminophenyl), 2.37 (s, 6H, Ar(CH₃)₂) ppm; ¹³C

NMR (DMSO-d₆): d = 163.3, 160.5, 160.2, 151.6, 148.2,

143.2, 136.6, 136.2, 132.4, 130.7, 129.4, 128.2, 127.0, 124.0,

116.3, 112.2, 24.8 ppm; MS (ES?, ToF): m/z = 371

([M ? 1]^?).

N⁰-[4-[(2,5-Dimethylphenylimino)methyl]benzylidene]-2-

hydroxybenzoic acid hydrazide (12, C₂₃H₂₁N₃O₂)

ꢀ

Yield: 62.2 %; TLC: R_f= 0.63; IR (KBr): m = 1,650

(C=N str., CH=N), 1,626 (C=O str., secondary amide),

1,584 (C=C skeletal str., phenyl nucleus), 1,370 (C–O str.

and O–H in plane bending, phenol), 740 (C–H out of plane

1

bending, 1,2-disubstituted benzene ring) cm^-1; H NMR

(MeOH-d₄): d = 9.84 (s, 1H, phenylimino), 8.28 (s, 1H,

–CH=N), 7.93 (d, 2H, benzylidene), 7.85 (d, 2H, benzyl-

idene), 7.81 (d, 1H, 2-hydroxyphenyl), 7.46 (m, 1H,

2-hydroxyphenyl), 7.01 (m, 1H, 2-hydroxyphenyl), 7.00

(d, 2H, phenylimino), 6.94 (d, 1H, 2-hydroxyphenyl), 2.68

(s, 6H, Ar(CH₃)₂) ppm; ¹³C NMR (DMSO-d₆): d = 163.1,

160.4, 159.2, 151.2, 136.7, 133.6, 130.1, 129.9, 127.8,

123.5, 121.7, 119.6, 24.2, 15.8 ppm; MS (ES?, ToF): m/

z = 372 ([M ? 1]^?).

N⁰-[4-[(2,5-Dimethylphenylimino)methyl]benzylidene]-4-

aminobenzoic acid hydrazide (16, C₂₃H₂₂N₄O)

ꢀ

Yield: 66.2 %; TLC: R_f= 0.69; IR (KBr): m = 1,677

(C=N str., CH=N), 1,618 (C=O str., secondary amide),

1,571 (C=C skeletal str., phenyl nucleus), 1,292 (C–N str.,

Ar-NH₂), 804 (C–H out of plane bending, 1,4-disubstituted

N⁰-[4-[(Phenylimino)methyl]benzylidene]-4-aminobenzoic

acid hydrazide (13, C₂₁H₁₈N₄O)

1

benzene ring) cm^-1; H NMR (MeOH-d₄): d = 8.52 (s,

ꢀ

Yield: 61.0 %; TLC: R_f= 0.72; IR (KBr): m = 1,707 (C=N

str., CH=N), 1,616 (C=O str., secondary amide), 1,584

(C=C skeletal str., phenyl nucleus), 1,310 (C–N str.,

Ar–NH₂), 829 (C–H out of plane bending, 1,4-disubstituted

benzene ring), 693 (C–H out of plane bending, monosub-

1H, –CH=N), 7.93 (d, 2H, benzylidene), 7.82 (d, 2H,

benzylidene), 7.72 (d, 2H, 4-aminophenyl), 6.99 (d,

2H, phenylimino), 6.94 (s, 1H, phenylimino), 6.86 (d,

2H, 4-aminophenyl), 2.37 (s, 6H, Ar(CH₃)₂) ppm; ¹³C

NMR (DMSO-d₆): d = 158.8, 149.9, 138.5, 135.8, 129.3,

128.8, 128.4, 126.5, 118.2 ppm; MS (ES?, ToF): m/z =

371 ([M ? 1]^?).

stituted benzene ring) cm^-1

;

¹H NMR (MeOH-d₄):

d = 8.67 (s, 1H, –CH=N), 8.10 (d, 2H, benzylidene), 7.92

(d, 2H, benzylidene), 7.59 (d, 2H, 4-aminophenyl), 7.33 (m,

123

N⁰-[4-[(Substituted imino)methyl]benzylidene]-substituted benzohydrazides

843

N⁰-[4-[(Phenylimino)methyl]benzylidene]-4-methylbenzoic

acid hydrazide (17, C₂₂H₁₉N₃O)

3-methylbenzohydrazide), 7.46 (d, 1H, 3-methylbenzohyd-

razide), 7.33(s, 1H, phenylimino), 7.29(m, 1H, phenylimino),

7.21 (d, 2H, phenylimino), 7.03 (m, 1H, 3-methylbenzohyd-

razide), 2.47 (s, 3H, ArCH₃) ppm; ¹³C NMR (DMSO-d₆):

d = 163.4, 160.7, 154.6, 143.1, 138.3, 136.6, 135.5, 134.2,

132.7, 131.2, 129.8, 128.5, 127.5, 127.4, 124.6, 122.8, 120.3,

24.7 ppm; MS (ES?, ToF): m/z = 377 ([M ? 1]^?).

ꢀ

Yield: 64.0 %; TLC: R_f= 0.38; IR (KBr): m = 1,690 (C=N

str., CH=N), 1,617 (C=O str., secondary amide), 1,507 (C=C

skeletal str., phenyl nucleus), 827 (C–H out of plane bending,

1,4-disubstituted benzene ring) cm^-1; ¹H NMR (MeOH-d₄):

d = 8.42 (s, 1H, –N=CH), 7.98 (d, 2H, benzylidene), 7.86 (d,

2H, 4-methylbenzohydrazide), 7.83 (d, 2H, benzylidene),

7.65 (m, 5H, phenylimino), 7.22 (d, 2H, 4-methylbenzohyd-

razide) ppm; ¹³C NMR (DMSO-d₆): d = 163.2, 160.2,

153.5, 143.1, 141.4, 136.3, 130.3, 129.5, 129.4, 127.6, 127.5,

122.4, 24.6 ppm; MS (ES?, ToF): m/z = 342 ([M ? 1]^?).

N⁰-[4-[(Phenylimino)methyl]benzylidene]-4-hydroxybenzo-

ic acid hydrazide (21, C₂₁H₁₇N₃O₂)

ꢀ

Yield: 65.7 %; TLC: R_f= 0.64; IR (KBr): m = 1,697

(C=N str., CH=N), 1,618 (C=O str., secondary amide),

1,584 (C=C skeletal str., phenyl nucleus), 1,388 (C–O str.

and O–H in plane bending, phenol), 827 (C–H out of plane

bending, 1,4-disubstituted benzene ring), 691 (C–C out of

N⁰-[4-[(2-Chlorophenylimino)methyl]benzylidene]-4-meth-

ylbenzoic acid hydrazide (18, C₂₂H₁₈ClN₃O)

1

Yield: 68.4 %; TLC: R_f= 0.73; IR (KBr): m = 1,646

plane bending, monosubstituted benzene ring) cm^-1; H

ꢀ

(C=N str., CH=N), 1,611 (C=O str., secondary amide),

1,502 (C=C skeletal str., phenyl nucleus), 830 (C–H out of

plane bending, 1,4-disubstituted benzene ring), 745 (C–H

out of plane bending, 1,2-disubstituted benzene ring), 704

(C–Cl str., ArCl) cm^-1; ¹H NMR (MeOH-d₄): d = 7.96 (s,

1H, –CH=N), 7.94 (d, 2H, benzylidene), 7.90 (d, 2H,

benzylidene), 7.88 (d, 2H, 4-methylbenzohydrazide), 7.40

(d, 2H, 4-methylbenzohydrazide), 7.40 (d, 3H, phenylimi-

no), 7.22 (m, 1H, phenylimino), 2.46 (s, 3H, ArCH₃) ppm;

¹³C NMR (DMSO-d₆): d = 163.0, 160.2, 143.4, 143.4,

141.1, 136.5, 130.5, 129.7, 129.3, 128.7, 127.5, 127.4,

124.7, 12.5 ppm; MS (ES?, ToF): m/z = 377 ([M ? 1]^?).

NMR (MeOH-d₄): d = 8.09 (s, 1H, –CH=N), 7.95 (d, 2H,

benzylidene), 7.85 (d, 2H, benzylidene), 7.79 (d, 2H,

4-hydroxybenzohydrazide), 7.32 (m, 5H, phenylimino),

6.85 (d, 2H, 4-hydroxybenzohydrazide) ppm; ¹³C NMR

(DMSO-d₆): d = 163.2, 161.6, 160.3, 153.2, 143.3, 136.2,

130.3, 129.4, 128.7, 128.5, 127.5, 122.4, 116.2 ppm; MS

(ES?, ToF): m/z = 344 ([M ? 1]^?).

N⁰-[4-[(2-Chlorophenylimino)methyl]benzylidene]-4-hy-

droxybenzoic acid hydrazide (22, C₂₁H₁₆ClN₃O₂)

ꢀ

Yield: 78.2 %; TLC: R_f= 0.62; IR (KBr): m = 1,687 (C=N

str., CH=N), 1,620 (C=O str., secondary amide), 1,576 (C=C

skeletal str., phenyl nucleus), 1,392 (C–O str. and O–H in

plane bending, phenol), 817 (C–H out of plane bending, 1,4-

disubstituted benzene ring), 772 (C–H out of plane bending,

N⁰-[4-[(2,4-Dimethylphenylimino)methyl]benzylidene]-4-

methylbenzoic acid hydrazide (19, C₂₄H₂₃N₃O)

Yield: 61.7 %; TLC: R_f= 0.50; IR (KBr): m = 1,652

1,2-disubstituted benzene ring), 718 (C–Cl str., ArCl) cm^-1

;

ꢀ

(C=N str., CH=N), 1,630 (C=O str., secondary amide),

1,505 (C=C skeletal str., phenyl nucleus), 917 (C–H out of

plane bending, 1,2,4-trisubstituted benzene ring), 832 (C–H

¹H NMR (MeOH-d₄): d = 8.37 (s, 1H, –CH=N), 7.93 (d, 2H,

benzylidene), 7.81 (d, 2H, benzylidene), 7.76 (d, 2H,

4-hydroxybenzohydrazide), 7.33 (m, 3H, phenylimino),

7.22 (m, 1H, phenylimino), 6.84 (d, 2H, 4-hydrox-

ybenzohydrazide) ppm; ¹³C NMR (DMSO-d₆): d = 163.0,

161.3, 160.4, 151.4, 143.4, 143.1, 136.9, 136.3, 130.3, 130.2,

129.5, 128.6, 128.6, 128.6, 127.8, 127.5, 123.9, 123.3, 116.0,

24.2, 15.8 ppm; MS (ES?, ToF): m/z = 379 ([M ? 1]^?).

out of plane bending, 1,4-disubstituted benzene ring) cm^-1

;

¹H NMR (MeOH-d₄): d = 8.00 (s, 1H, –N=CH), 7.99 (d,

2H, benzylidene), 7.87 (d, 2H, 4-methylbenzohydrazide),

7.86 (d, 2H, benzylidene), 7.39 (d, 2H, 4-methylbenzohyd-

razide), 6.84 (d, 2H, phenylimino), 6.08 (s, 1H,

phenylimino), 2.36 (s, 6H, –Ar(CH₃)₂) ppm; ¹³C NMR

(DMSO-d₆): d = 163.3, 160.5, 160.4, 148.3, 143.6, 141.0,

136.8, 136.7, 132.3, 130.7, 129.5, 129.4, 127.4, 127.2,

122.2, 24.9 ppm; MS (ES?, ToF): m/z = 370 ([M ? 1]^?).

N⁰-[4-[(2,5-Dimethylphenylimino)methyl]benzylidene]-4-

hydroxybenzoic acid hydrazide (23, C₂₃H₂₁N₃O₂)

ꢀ

Yield: 81.5 %; TLC: R_f= 0.57; IR (KBr): m = 1,688

(C=N str., CH=N), 1,620 (C=O str., secondary amide),

1,576 (C=C skeletal str., phenyl nucleus), 1,391 (C–O str.

and O–H in plane bending, phenol), 824 (C–H out of plane

N⁰-[4-[(3-Chlorophenylimino)methyl]benzylidene]-3-meth-

ylbenzoic acid hydrazide (20, C₂₂H₁₈ClN₃O)

1

Yield: 62.3 %; TLC: R_f= 0.82; IR (KBr): m = 1,656 (C=N

bending, 1,4-disubstituted benzene ring) cm^-1; H NMR

ꢀ

str., CH=N), 1,619 (C=O str., secondary amide), 1,506 (C=C

skeletal str., phenyl nucleus), 732 (C–Cl str., ArCl), 682

(C–C out of plane bending, 1,3-disubstituted benzene

(MeOH-d₄): d = 8.01 (s, 1H, –CH=N), 7.91 (d, 2H,

benzylidene), 7.85 (d, 2H, benzylidene), 7.76 (d, 2H,

4-hydroxybenzohydrazide), 7.48 (d, 2H, 4-hydrox-

ybenzohydrazide), 7.32 (d, 2H, phenylimino), 7.02 (s,

1H, phenylimino), 2.36 (s, 6H, –Ar(CH₃)₂) ppm; ¹³C NMR

(DMSO-d₆): d = 163.2, 161.5, 160.1, 143.5, 136.1, 129.7,

ring) cm^-^{1 1}H NMR (MeOH-d₄): d = 8.00 (s, 1H,

;

–CH=N), 7.96 (d, 1H, 3-methylbenzohydrazide), 7.96 (d,

2H, benzylidene), 7.81 (d, 2H, benzylidene), 7.78 (s, 1H,

123

844

P. Kumar et al.

128.7, 128.4, 116.2 ppm; MS (ES?, ToF): m/z = 372

([M ? 1]^?).

skeletal str., phenyl nucleus), 821 (C–H out of plane

bending, 1,4-disubstituted benzene ring), 702 (C–Cl str.,

ArCl) cm^-^{1 1}H NMR (MeOH-d₄): d = 8.57 (s, 1H,

;

N⁰-[4-[(2-Chlorophenylimino)methyl]benzylidene]-3-nitro-

benzoic acid hydrazide (24, C₂₁H₁₅ClN₄O₃)

–N=CH), 8.32 (d, 2H, 4-nitrobenzohydrazide), 8.01 (d, 2H,

4-nitrobenzohydrazide), 8.00 (d, 2H, benzylidene), 7.76 (d,

2H, benzylidene), 7.38 (m, 3H, phenylimino), 7.22 (m, 1H,

phenylimino) ppm; ¹³C NMR (DMSO-d₆): d = 163.3, 162.1,

148.9, 143.1, 135.3, 133.8, 129.8, 129.7, 129.2, 124.2, 123.4,

122.6 ppm; MS (ES?, ToF): m/z = 408 ([M ? 1]^?).

ꢀ

Yield: 84.0 %; TLC: R_f= 0.81; IR (KBr): m = 1,650 (C=N

str., CH=N), 1,614 (C=O str., secondary amide), 1,550

(asym. str., NO₂), 1,504 (C=C skeletal str., phenyl nucleus),

768 (C–H out of plane bending, 1,2-disubstituted benzene

ring), 753 (C–Cl str., ArCl), 721 (C–C out of plane bending,

1,3-disubstituted benzene ring) cm^-1; ¹H NMR (MeOH-d₄):

d = 8.92 (s, 1H, 3-nitrobenzohydrazide), 8.61 (d, 1H,

3-nitrobenzohydrazide), 8.54 (d, 1H, 3-nitrobenzohydraz-

ide), 8.44 (s, 1H, –CH=N), 7.95 (d, 2H, benzylidene), 7.89

(m, 1H, 3-nitrobenzohydrazide), 7.63 (d, 2H, benzylidene),

7.36 (m, 3H, phenylimino), 7.21 (m, 1H, phenylimino) ppm;

¹³C NMR (DMSO-d₆): d = 163.2, 160.2, 148.6, 143.3,

143.1, 136.3, 135.2, 133.9, 130.4, 129.9, 129.4, 128.7, 127.5,

124.1, 123.3, 122.5 ppm; MS (ES?, ToF): m/z = 408

([M ? 1]^?).

N⁰-[4-[(4-Nitrophenylimino)methyl]benzylidene]-4-amino-

benzoic acid hydrazide (28, C₂₁H₁₇N₅O₃)

ꢀ

Yield: 83.2 %; TLC: R_f= 0.42; IR (KBr): m = 1,689 (C=N

str., CH=N), 1,619 (C=O str., secondary amide), 1,505

(asym. str., NO₂), 1,275 (C–N str., Ar–NH₂), 823 (C–H out of

1

plane bending, 1,4-disubstituted benzene ring) cm^-1; H

NMR (MeOH-d₄): d = 8.33 (s, 1H, –N=CH), 8.12 (d, 2H,

phenylimino), 7.89 (d, 2H, benzylidene), 7.84 (d, 2H, ben-

zylidene), 7.70 (d, 2H, 4-aminophenyl), 7.60 (d, 2H,

phenylimino), 6.66 (d, 2H, 4-aminophenyl) ppm; ¹³C

NMR (DMSO-d₆): d = 163.4, 160.1, 159.5, 151.8, 146.8,

143.2, 136.0, 129.3, 128.4, 124.0, 123.4, 123.3, 122.7, 122.5,

116.1 ppm; MS (ES?, ToF): m/z = 388 ([M ? 1]^?).

N⁰-[4-[(4-Chlorophenylimino)methyl]benzylidene]-3-nitro-

benzoic acid hydrazide (25, C₂₁H₁₅ClN₄O₃)

ꢀ

Yield: 76.7 %; TLC: R_f= 0.54; IR (KBr): m = 1,651 (C=N

str., CH=N), 1,614 (C=O str., secondary amide), 1,552

(asym. str., NO₂), 1,506 (C=C skeletal str., phenyl nucleus),

815 (C–H out of plane bending, 1,4-disubstituted benzene

N⁰-[4-[(4-Chlorophenylimino)methyl]benzylidene]-4-ami-

nobenzoic acid hydrazide (29, C₂₁H₁₇ClN₄O)

ꢀ

Yield: 66.7 %; TLC: R_f= 0.44; IR (KBr): m = 1,700

1

ring), 721 (C–Cl str., ArCl) cm^-1; H NMR (MeOH-d₄):

(C=N str., CH=N), 1,618 (C=O str., secondary amide),

1,274 (C–N str., Ar–NH₂), 828 (C–H out of plane bending,

1,4-disubstituted benzene ring), 729 (C–Cl str., ArCl)

d = 8.90 (s, 1H, 3-nitrobenzohydrazide), 8.43 (d, 1H,

3-nitrobenzohydrazide), 8.42 (s, 1H, –CH=N), 8.07 (d, 1H,

3-nitrobenzohydrazide), 7.97 (d, 2H, benzylidene), 7.82 (m,

1H, 3-nitrobenzohydrazide), 7.61 (d, 2H, benzylidene), 7.31

(d, 2H, phenylimino), 7.19 (d, 2H, phenylimino) ppm; ¹³C

NMR (DMSO-d₆): d = 163.3, 160.0, 151.0, 148.9, 143.1,

136.0, 135.3, 133.8, 132.6, 130.1, 129.7, 129.5, 124.2, 123.8,

122.6 ppm; MS (ES?, ToF): m/z = 408 ([M ? 1]^?).

1

cm^-1; H NMR (MeOH-d₄): d = 8.33 (s, 1H, –N=CH),

8.12 (d, 2H, phenylimino), 7.98 (d, 2H, benzylidene), 7.83

(d, 2H, benzylidene), 7.74 (d, 2H, 4-aminophenyl), 7.60 (d,

2H, phenylimino), 6.64 (d, 2H, 4-aminophenyl), 4.31 (s,

2H, –ArNH₂) ppm; ¹³C NMR (DMSO-d₆): d = 163.7,

160.4, 151.5, 151.5, 143.5, 136.0, 132.6, 130.4, 130.3,

129.1, 128.6, 124.3, 123.9, 123.5, 116.4 ppm; MS (ES?,

ToF): m/z = 378 ([M ? 1]^?).

N⁰-[4-[(Phenylimino)methyl]benzylidene]-4-nitrobenzoic

acid hydrazide (26, C₂₁H₁₆N₄O₃)

ꢀ

Yield: 69.3 %; TLC: R_f= 0.70; IR (KBr): m = 1,657 (C=N

N⁰-[4-[(3-Nitrophenylimino)methyl]benzylidene]-4-amino-

benzoic acid hydrazide (30, C₂₁H₁₇N₅O₃)

str., CH=N), 1,561 (asym. str., NO₂), 1,516 (C=C skeletal

str., phenyl nucleus), 820 (C–H out of plane bending, 1,4-

disubstituted benzene ring), 702 (C–C out of plane bending,

monosubstituted benzene ring) cm^-1; ¹H NMR (MeOH-d₄):

d = 8.42 (d, 2H, 4-nitrobenzohydrazide), 8.42 (s, 1H,

–CH=N), 8.19 (d, 2H, 4-nitrobenzohydrazide), 8.01 (d, 2H,

benzylidene), 7.89 (d, 2H, benzylidene), 7.19 (m, 5H,

phenylimino) ppm; ¹³C NMR (DMSO-d₆): d = 161.9,

149.8, 141.8, 129.6, 129.0, 128.8, 127.5, 123.3, 122.6,

121.2 ppm; MS (ES?, ToF): m/z = 373 ([M ? 1]^?).

ꢀ

Yield: 67.4 %; TLC: R_f= 0.53; IR (KBr): m = 1,698 (C=N

str., CH=N), 1,619 (C=O str., secondary amide), 1,524 (asym.

str., NO₂), 1,274 (C–N str., Ar–NH₂), 826 (C–H out of plane

bending, 1,4-disubstituted benzene ring), 697 (C–C out of

plane bending, 1,3-disubstitutedbenzenering) cm^-1;¹HNMR

(MeOH-d₄): d = 8.42 (s, 1H, –N=CH), 8.31 (s, 1H, phenyli-

mino), 8.11 (d, 1H, phenylimino), 7.99 (d, 2H, benzylidene),

7.85 (d, 2H, benzylidene), 7.79 (d, 1H, phenylimino), 7.78 (d,

2H, 4-aminophenyl), 7.50 (m, 1H, phenylimino), 6.67 (d, 2H,

4-aminophenyl), 4.29(s, 2H, –NH₂ofArNH₂) ppm; ¹³C NMR

(DMSO-d₆): d = 163.1, 160.3, 154.3, 151.6, 149.6, 143.3,

136.1, 131.2, 129.4, 128.7, 128.0, 124.2, 119.9, 117.3,

116.3 ppm; MS (ES?, ToF): m/z = 388 ([M ? 1]^?).

N⁰-[4-[(2-Chlorophenylimino)methyl]benzylidene]-4-nitro-

benzoic acid hydrazide (27, C₂₁H₁₅ClN₄O₃)

ꢀ

Yield: 67.4 %; TLC: R_f= 0.78; IR (KBr): m = 1,657

(C=N str., CH=N), 1,562 (asym. str., NO₂), 1,515 (C=C

123

N⁰-[4-[(Substituted imino)methyl]benzylidene]-substituted benzohydrazides

845

N⁰-[4-[(2-Nitrophenylimino)methyl]benzylidene]-4-amino-

benzoic acid hydrazide (31, C₂₁H₁₇N₅O₃)

1,581 (C=C skeletal str., phenyl nucleus), 1,390 (C–O str.

and O–H in plane bending, phenol), 826 (C–H out of plane

bending, 1,4-disubstituted benzene ring), 710 (C–Cl str.,

Ar–Cl), 683 (C–C out of plane bending, 1,3-disubstituted

ꢀ

Yield: 60.5 %; TLC: R_f= 0.56; IR (KBr): m = 1,697

(C=N str., CH=N), 1,619 (C=O str., secondary amide),

1,504 (asym. str., NO₂), 1,275 (C–N str., Ar–NH₂), 826

(C–H out of plane bending, 1,4-disubstituted benzene ring),

744 (C–H out of plane bending, 1,2-disubstituted benzene

1

benzene ring) cm^-1; H NMR (MeOH-d₄): d = 8.65 (s,

1H, –CH=N), 7.98 (d, 2H, benzylidene), 7.89 (m, 1H,

benzylidene), 7.76 (d, 2H, 4-hydroxyphenyl), 7.55 (s,

1H, phenylimino), 7.21 (d, 2H, phenylimino), 7.12 (d, 1H,

phenylimino), 6.84 (d, 2H, 4-hydroxyphenyl) ppm; ¹³C

NMR (DMSO-d₆): d = 163.6, 161.7, 160.5, 154.9, 143.4,

136.6, 135.8, 131.6, 129.0, 128.9, 128.5, 127.6, 122.9,

120.7, 116.3 ppm; MS (ES?, ToF): m/z = 379

([M ? 1]^?).

ring) cm^-^{1 1}H NMR (MeOH-d₄): d = 8.52 (d, 2H,

;

phenylimino), 8.26 (s, 1H, –N=CH), 8.11 (d, 1H, phenyli-

mino), 7.98 (d, 2H, benzylidene), 7.85 (d, 2H, benzylidene),

7.79 (m, 1H, phenylimino), 7.77 (d, 2H, 4-aminophenyl),

7.38 (m, 1H, phenylimino), 6.66 (d, 2H, 4-aminophenyl),

4.32 (s, 2H, –NH₂of ArNH₂) ppm; ¹³C NMR (DMSO-d₆):

d = 163.0, 160.0, 151.8, 148.5, 143.5, 141.8, 136.4, 136.3,

129.6, 128.3, 128.2, 124.0, 123.3, 122.6, 116.5 ppm; MS

(ES?, ToF): m/z = 388 ([M ? 1]^?).

N⁰-[4-[(2-Nitrophenylimino)methyl]benzylidene]-4-hy-

droxybenzoic acid hydrazide (35, C₂₁H₁₆N₄O₄)

ꢀ

Yield: 65.6 %; TLC: R_f= 0.38; IR (KBr): m = 1,699

(C=N str., CH=N), 1,620 (C=O str., secondary amide),

1,511 (asym. str., NO₂), 1,391 (C–O str. and O–H in plane

bending, phenol), 828 (C–H out of plane bending, 1,4-

disubstituted benzene ring), 754 (C–H out of plane

N⁰-[4-[(2-Methyl-5-nitrophenylimino)methyl]benzylidene]-

4-aminobenzoic acid hydrazide (32, C₂₂H₁₉N₅O₃)

ꢀ

Yield: 40.6 %; TLC: R_f= 0.54; IR (KBr): m = 1,701

(C=N str., CH=N), 1,623 (C=O str., secondary amide),

1,509 (asym. str., NO₂), 1,270 (C–N str., Ar–NH₂), 829

(C–H out of plane bending, 1,4-disubstituted benzene ring)

1

bending, 1,2-disubstituted benzene ring) cm^-1; H NMR

(MeOH-d₄): d = 8.22 (s, 1H, –N=CH), 8.00 (d, 1H,

phenylimino), 7.88 (d, 2H, benzylidene), 7.77 (m, 1H,

benzylidene), 7.77 (m, 1H, phenylimino), 7.62 (d, 2H,

4-hydroxyphenyl), 7.53 (m, 2H, phenylimino), 6.64 (d, 2H,

4-hydroxyphenyl), 5.22 (s, 1H, –ArOH) ppm; ¹³C NMR

(DMSO-d₆): d = 163.9, 161.8, 160.2, 148.8, 148.6, 143.7,

136.9, 136.3, 129.4, 128.8, 128.4, 128.4, 123.5, 122.4,

116.4 ppm; MS (ES?, ToF): m/z = 389 ([M ? 1]^?).

1

cm^-1; H NMR (MeOH-d₄): d = 8.46 (s, 1H, –N=CHAr),

8.32 (d, 1H, phenylimino), 8.21 (d, 2H, phenylimino), 7.93

(d, 2H, benzylidene), 7.83 (d, 2H, benzylidene), 7.72 (d,

2H, 4-aminophenyl), 6.64 (d, 2H, 4-aminophenyl), 2.07 (s,

3H, –ArCH₃) ppm; ¹³C NMR (DMSO-d₆): d = 163.1,

160.4, 152.4, 151.3, 146.9, 143.6, 136.7, 136.0, 131.6,

129.5, 128.0, 124.1, 119.4, 117.2, 116.3, 15.5 ppm; MS

(ES?, ToF): m/z = 402 ([M ? 1]^?).

N⁰-[4-[(4-Nitrophenylimino)methyl]benzylidene]-4-hy-

droxybenzoic acid hydrazide (36, C₂₁H₁₆N₄O₄)

N⁰-[4-[(2-Chloro-4-nitrophenylimino)methyl]benzylidene]-

4-aminobenzoic acid hydrazide (33, C₂₁H₁₆ClN₅O₃)

ꢀ

Yield: 79.0 %; TLC: R_f= 0.59; IR (KBr): m = 1,695

(C=N str., CH=N), 1,618 (C=O str., secondary amide),

1,506 (asym. str., NO₂), 1,345 (C–O str. and O–H in plane

bending, phenol), 823 (C–H out of plane bending, 1,4-

ꢀ

Yield: 63.7 %; TLC: R_f= 0.36; IR (KBr): m = 1,703

(C=N str., CH=N), 1,620 (C=O str., secondary amide),

1,505 (asym. str., NO₂), 1,272 (C–N str., Ar–NH₂), 885

(C–H out of plane bending, 1,2,4-trisubstituted benzene

ring), 828 (C–H out of plane bending, 1,4-disubstituted

1

disubstituted benzene ring) cm^-1; H NMR (MeOH-d₄):

d = 8.35 (s, 1H, –N=CH), 8.06 (d, 2H, phenylimino), 7.99

(d, 2H, benzylidene), 7.89 (m, 1H, benzylidene), 7.70 (d,

2H, 4-hydroxyphenyl), 7.50 (d, 2H, phenylimino), 6.63 (d,

2H, 4-hydroxyphenyl) ppm; ¹³C NMR (DMSO-d₆):

d = 163.3, 161.8, 160.4, 159.4, 146.8, 143.2, 136.4,

136.3, 129.7, 129.5, 129.5, 129.2, 128.8, 128.7, 126.6,

123.5, 123.4, 122.7, 122.6, 116.4, 116.3 ppm; MS (ES?,

ToF): m/z = 389 ([M ? 1]^?).

benzene ring), 723 (C–Cl str., Ar–Cl) cm^-^{1 1}H NMR

;

(MeOH-d₄): d = 8.65 (s, 1H, –CH=N), 8.22 (s, 1H,

phenylimino), 8.11 (d, 1H, phenylimino), 8.05 (d, 2H, ben-

zylidene), 7.84 (d, 2H, benzylidene), 7.76 (d, 2H,

4-aminophenyl), 7.55 (d, 1H, phenylimino), 6.65 (d, 2H,

4-aminophenyl), 4.31 (s, 2H, –ArNH₂) ppm; ¹³C NMR

(DMSO-d₆): d = 163.5, 160.5, 151.1, 149.5, 148.6, 143.8,

136.1, 129.3, 128.9, 128.2, 125.4, 124.7, 124.5, 120.4,

116.4 ppm; MS (ES?, ToF): m/z = 423 ([M ? 1]^?).

N⁰-[4-[(2-Chloro-4-nitrophenylimino)methyl]benzylidene]-

4-hydroxybenzoic acid hydrazide (37, C₂₁H₁₅ClN₄O₄)

ꢀ

Yield: 61.4 %; TLC: R_f= 0.51; IR (KBr): m = 1,696

N⁰-[4-[(3-Chlorophenylimino)methyl]benzylidene]-4-hy-

droxybenzoic acid hydrazide (34, C₂₁H₁₆ClN₃O₂)

(C=N str., CH=N), 1,619 (C=O str., secondary amide),

1,506 (asym. str., NO₂), 1,391 (C–O str. and O–H in plane

bending, phenol), 874 (C–H out of plane bending, 1,2,4-

trisubstituted benzene ring), 825 (C–H out of plane

ꢀ

Yield: 60.9 %; TLC: R_f= 0.57; IR (KBr): m = 1,699

(C=N str., CH=N), 1,618 (C=O str., secondary amide),

123

846

P. Kumar et al.

bending, 1,4-disubstituted benzene ring), 745 (C–Cl str.,

Ar–Cl) cm^-^{1 1}H NMR (MeOH-d₄): d = 8.48 (s, 1H,

;

(d, 2H, phenylimino), 7.00 (m, 1H, 2-hydroxyphenyl), 6.89

(d, 1H, 2-hydroxyphenyl) ppm; ¹³C NMR (DMSO-d₆):

d = 163.2, 160.6, 160.5, 159.0, 151.4, 143.4, 136.5, 133.7,

132.6, 130.5, 130.0, 129.6, 129.2, 123.9, 123.5, 121.8,

119.5 ppm; MS (ES?, ToF): m/z = 379 ([M ? 1]^?).

–CH=N), 8.13 (s, 1H, phenylimino), 8.01 (d, 1H, phenyli-

mino), 7.89 (d, 2H, benzylidene), 7.80 (m, 1H, benzylidene),

7.72 (d, 2H, 4-hydroxyphenyl), 7.62 (d, 1H, phenylimino),

6.65 (d, 2H, 4-hydroxyphenyl) ppm; ¹³C NMR (DMSO-d₆):

d = 163.6, 161.6, 160.0, 149.7, 148.5, 143.5, 136.8, 129.3,

128.9, 128.9, 128.6, 125.3, 124.8, 120.9, 116.8 ppm; MS

(ES?, ToF): m/z = 424 ([M ? 1]^?).

N⁰-[4-[(3-Fluorophenylimino)methyl]benzylidene]-4-ami-

nobenzoic acid hydrazide (41, C₂₁H₁₇FN₄O)

ꢀ

Yield: 62.3 %; TLC: R_f= 0.48; IR (KBr): m = 1,698

(C=N str., CH=N), 1,618 (C=O str., secondary amide),

1,508 (C=C skeletal str., phenyl), 1,297 (C–N str.,

Ar–NH₂), 1,013 (C–F str., Ar–F), 828 (C–H out of plane

bending, 1,4-disubstituted benzene ring), 698 (C–C out of

N⁰-[4-[(3-Nitrophenylimino)methyl]benzylidene]-4-hy-

droxybenzoic acid hydrazide (38, C₂₁H₁₆N₄O₄)

ꢀ

Yield: 80.0 %; TLC: R_f= 0.40; IR (KBr): m = 1,698 (C=N

1

str., CH=N), 1,618 (C=O str., secondary amide), 1,524

(asym. str., NO₂), 1,389 (C–O str. and O–H in plane bending,

phenol), 825 (C–H out of plane bending, 1,4-disubstituted

benzene ring), 697 (C–C out of plane bending, 1,3-disubsti-

plane bending, 1,3-disubstituted benzene ring) cm^-1; H

NMR (MeOH-d₄): d = 8.44 (s, 1H, –CH=N), 7.93 (d, 2H,

benzylidene), 7.82 (d, 2H, benzylidene), 7.61 (d, 2H, 4-ami-

nophenyl), 7.29 (s, 1H, phenylimino), 7.01 (d, 2H,

phenylimino), 7.01 (m, 1H, phenylimino), 6.67 (d, 2H,

4-aminophenyl) ppm; ¹³C NMR (DMSO-d₆): d = 164.4,

163.4, 160.5, 154.9, 151.8, 143.9, 136.2, 131.6, 129.7,

128.3, 124.0, 117.7, 116.5, 114.3, 109.5 ppm; MS (ES?,

ToF): m/z = 361 ([M ? 1]^?).

1

tuted benzene ring) cm^-1; H NMR (MeOH-d₄): d = 8.41

(s, 1H, –N=CH), 8.23 (s, 1H, phenylimino), 8.22 (d, 1H,

phenylimino), 7.89 (d, 2H, benzylidene), 7.84 (d, 1H,

phenylimino), 7.80 (m, 1H, benzylidene), 7.72 (d, 2H,

4-hydroxyphenyl), 7.51 (m, 1H, phenylimino), 6.65 (d, 2H,

4-hydroxyphenyl), 5.23 (s, 1H, –ArOH) ppm; ¹³C NMR

(DMSO-d₆): d = 163.3, 161.5, 160.2, 154.3, 149.8, 143.2,

136.6, 129.5, 129.4, 128.7, 128.6, 128.4, 119.9, 117.3,

116.9 ppm; MS (ES?, ToF): m/z = 389 ([M ? 1]^?).

N⁰-[4-[(3-Chloro-4-ﬂuorophenylimino)methyl]benzyli-

dene]-4-aminobenzoic acid hydrazide

(42, C₂₁H₁₆ClFN₄O)

ꢀ

Yield: 69.1 %; TLC: R_f= 0.58; IR (KBr): m = 1,696

N⁰-[4-[(4-Chlorophenylimino)methyl]benzylidene]-4-hy-

droxybenzoic acid hydrazide (39, C₂₁H₁₆ClN₃O₂)

(C=N str., CH=N), 1,618 (C=O str., secondary amide),

1,503 (skeletal str., phenyl), 1,297 (C–N str., Ar–NH₂),

1,014 (C–F str., Ar–F), 827 (C–H out of plane bending,

1,4-disubstituted benzene ring), 728 (C–Cl str., Ar–Cl)

ꢀ

Yield: 82.7 %; TLC: R_f= 0.42; IR (KBr): m = 1,698

(C=N str., CH=N), 1,618 (C=O str., secondary amide),

1,508 (C=C skeletal str., phenyl), 1,390 (C–O str. and O–H

in plane bending, phenol), 826 (C–H out of plane bending,

1,4-disubstituted benzene ring), 730 (C–Cl str., Ar–Cl)

cm^-1; ¹H NMR (MeOH-d₄): d = 8.10 (s, 1H, CH=N), 7.91

(d, 2H, benzylidene), 7.89 (m, 1H, benzylidene), 7.76 (d,

2H, 4-hydroxyphenyl), 7.34 (d, 2H, phenylimino), 7.26 (d,

2H, phenylimino), 6.86 (d, 2H, 4-hydroxyphenyl), 5.50 (s,

1H, –ArOH) ppm; ¹³C NMR (DMSO-d₆): d = 163.9,

161.4, 160.4, 151.5, 143.8, 136.4, 132.6, 130.5, 130.4,

129.7, 128.9, 128.8, 123.9, 123.4, 116.5 ppm; MS (ES?,

ToF): m/z = 379 ([M ? 1]^?).

1

cm^-1; H NMR (MeOH-d₄): d = 8.12 (s, 1H, –CH=N),

7.94 (d, 2H, benzylidene), 7.82 (d, 2H, benzylidene), 7.80

(d, 2H, 4-aminophenyl), 7.29 (s, 1H, phenylimino), 7.01 (d,

1H, phenylimino), 6.73 (d, 1H, phenylimino), 6.69 (d, 2H,

4-aminophenyl) ppm; ¹³C NMR (DMSO-d₆): d = 163.7,

161.5, 160.6, 151.6, 150.4, 143.7, 136.4, 129.9, 128.1,

124.4, 124.3, 122.3, 122.2, 118.3, 116.2 ppm; MS (ES?,

ToF): m/z = 396 ([M ? 1]^?).

N⁰-[4-[(Butylimino)methyl]benzylidene]-4-aminobenzoic

acid hydrazide (43, C₁₉H₂₂N₄O)

ꢀ

Yield: 71.5 %; TLC: R_f= 0.58; IR (KBr): m = 1,697 (C=N

N⁰-[4-[(4-Chlorophenylimino)methyl]benzylidene]-2-hy-

droxybenzoic acid hydrazide (40, C₂₁H₁₆ClN₃O₂)

str., CH=N), 1,618 (C=O str., secondary amide), 1,509

(skeletal str., phenyl), 1,463 (CH₃asym. bending, R–CH₃),

1,297 (C–N str., Ar–NH₂), 827 (C–H out of plane bending,

ꢀ

Yield: 70.5 %; TLC: R_f= 0.60; IR (KBr): m = 1,652

1

(C=N str., CH=N), 1,619 (C=O str., secondary amide),

1,377 (C–O str. and O–H in plane bending, phenol), 827

(C–H out of plane bending, 1,4-disubstituted benzene ring),

738 (C–H out of plane bending, 1,2-disubstituted benzene

1,4-disubstituted benzene ring) cm^-1; H NMR (MeOH-

d₄): d = 8.09 (s, 1H, CH=N), 7.98 (d, 2H, benzylidene),

7.81 (d, 2H, benzylidene), 7.76 (d, 2H, 4-aminophenyl),

6.77 (d, 2H, 4-aminophenyl), 3.52 (t, 2H, –C₄H₉), 1.58 (m,

2H, –C₄H₉), 1.32 (m, 2H, –C₄H₉), 0.93 (t, 3H, –C₄H₉) ppm;

¹³C NMR (DMSO-d₆): d = 163.9, 160.3, 151.7, 143.5,

136.5, 129.8, 128.3, 124.1, 116.4, 55.9, 33.8, 20.1,

13.6 ppm; MS (ES?, ToF): m/z = 323 ([M ? 1]^?).

1

ring), 691 (C–Cl str., Ar–Cl) cm^-1; H NMR (MeOH-d₄):

d = 8.44 (s, 1H, –CH=N), 7.91 (d, 2H, benzylidene), 7.89

(m, 1H, benzylidene), 7.65 (d, 1H, 2-hydroxyphenyl), 7.35

(m, 1H, 2-hydroxyphenyl), 7.30 (d, 2H, phenylimino), 7.22

123

N⁰-[4-[(Substituted imino)methyl]benzylidene]-substituted benzohydrazides

847

N⁰-[4-[(Benzylimino)methyl]benzylidene]-4-aminobenzoic

acid hydrazide (44, C₂₂H₂₀N₄O)

benzylidene), 7.77 (d, 2H, naphthalene), 7.75 (d, 2H,

benzylidene), 7.43 (d, 2H, 4-aminophenyl), 7.26 (m, 5H,

naphthalene), 6.65 (d, 2H, 4-aminophenyl) ppm; ¹³C NMR

(DMSO-d₆): d = 163.5, 160.3, 152.3, 151.2, 143.5, 136.4,

135.4, 129.9, 128.6, 128.5, 128.4, 127.7, 127.7, 126.5, 126.3,

126.2, 124.4, 116.4, 115.4 ppm; MS (ES?, ToF): m/z = 393

([M ? 1]^?).

ꢀ

Yield: 63.3 %; TLC: R_f= 0.62; IR (KBr): m = 1,696

(C=N str., CH=N), 1,618 (C=O str., secondary amide),

1,509 (skeletal str., phenyl), 1,297 (C–N str., Ar–NH₂), 828

(C–H out of plane bending, 1,4-disubstituted benzene ring),

731 (C–H out of plane bending, monosubstituted benzene

ring) cm^-^{1 1}H NMR (MeOH-d₄): d = 8.18 (s, 1H,

;

N⁰-[4-[(Propylimino)methyl]benzylidene]-4-aminobenzoic

acid hydrazide (48, C₁₈H₂₀N₄O)

–CH=N), 7.94 (d, 2H, benzylidene), 7.68 (d, 2H, benzyl-

idene), 7.68 (d, 2H, phenylimino), 7.35 (d, 2H,

4-aminophenyl), 7.24 (d, 2H, phenylimino), 7.11 (m, 1H,

phenylimino), 6.82 (d, 2H, 4-aminophenyl) ppm; ¹³C NMR

(DMSO-d₆): d = 163.6, 160.4, 151.8, 143.6, 138.7, 136.6,

129.9, 129.3, 129.2, 128.8, 128.6, 128.5, 125.5, 124.0,

116.6, 58.5 ppm; MS (ES?, ToF): m/z = 357 ([M ? 1]^?).

ꢀ

Yield: 63.5 %; TLC: R_f= 0.30; IR (KBr): m = 1,696

(C=N str., CH=N), 1,618 (C=O str., secondary amide),

1,511 (C=C skeletal str., phenyl nucleus), 1,464 (CH₃

asym. bending, R–CH₃), 1,276 (C–N str., Ar–NH₂), 827

(C–H out of plane bending, 1,4-disubstituted benzene ring)

cm^-^{1 1}H NMR (MeOH-d₄): d = 7.95 (d, 2H, benzyli-

;

N⁰-[4-[(2-Hydroxyethylimino)methyl]benzylidene]-4-ami-

nobenzoic acid hydrazide (45, C₁₇H₁₈N₄O₂)

dene), 7.93 (s, 1H, –CH=N), 7.45 (d, 2H, benzylidene),

7.32 (d, 2H, 4-aminophenyl), 6.85 (d, 2H, 4-aminophenyl),

3.66 (t, 2H, –C₃H₇), 0.94 (t, 3H, –C₃H₇) ppm; ¹³C NMR

(DMSO-d₆): d = 163.7, 160.4, 151.6, 143.5, 136.5, 129.7,

128.5, 124.2, 116.3, 58.5, 24.9, 11.4 ppm; MS (ES?, ToF):

m/z = 309 ([M ? 1]^?).

ꢀ

Yield: 66.4 %; TLC: R_f= 0.54; IR (KBr): m = 1,667

(C=N str., CH=N), 1,618 (C=O str., secondary amide),

1,510 (skeletal str., phenyl), 1,297 (C–N str., Ar–NH₂),

1,279 (C–O str. and O–H in plane bending, primary

alcohol), 828 (C–H out of plane bending, 1,4-disubstituted

1

benzene ring) cm^-1; H NMR (MeOH-d₄): d = 8.19 (s,

Evaluation of antimicrobial activity: determination

of MIC

1H, –CH=N), 7.96 (d, 2H, benzylidene), 7.81 (d, 2H,

benzylidene), 7.78 (d, 2H, 4-aminophenyl), 6.67 (d,

2H, 4-aminophenyl), 3.86 (m, 2H, N-C₂H₄OH), 2.08 (t,

1H, –N–C₂H₄OH) ppm; ¹³C NMR (DMSO-d₆): d = 163.7,

160.4, 151.5, 143.6, 136.6, 129.7, 128.5, 124.3, 116.6,

64.3, 59.0 ppm; MS (ES?, ToF): m/z = 311 ([M ? 1]^?).

The antimicrobial activity of the synthesized compounds

was evaluated against Gram-positive bacteria Staphylo-

coccus aureus MTCC 2901 and Bacillus subtilis MTCC

2063, the Gram-negative bacterium Escherichia coli

MTCC 1652, and fungal strains Candida albicans MTCC

227 and Aspergillus niger MTCC 8189, using the tube-

dilution method [19]. Dilutions of test and standard com-

pounds were prepared in double-strength nutrient broth I.P.

(bacteria) or Sabouraud dextrose broth I.P. (fungi) [43].

The samples were incubated at 37 ꢁC for 24 h (bacteria),

at 25 ꢁC for 7 days (A. niger), or at 37 ꢁC for 48 h

(C. albicans) and the results were recorded in terms of MIC.

N⁰-[4-[(Hydroxyimino)methyl]benzylidene]-4-aminoben-

zoic acid hydrazide (46, C₁₅H₁₄N₄O₂)

ꢀ

Yield: 64.2 %; TLC: R_f= 0.54; IR (KBr): m = 1,692

(C=N str., CH=N), 1,618 (C=O str., secondary amide),

1,508 (C=C skeletal str., phenyl nucleus), 1,321 (C–N str.,

Ar–NH₂), 1,297 (C=O str. and O–H in-plane bending,

primary alcohol), 829 (C–H out of plane bending, 1,4-

1

disubstituted benzene ring) cm^-1; H NMR (MeOH-d₄):

d = 8.01 (s, 1H, –CH=N), 7.93 (d, 2H, benzylidene), 7.77

(d, 2H, benzylidene), 7.75 (d, 2H, 4-aminophenyl), 6.65 (d,

2H, 4-aminophenyl), 2.07 (s, 1H, –N–OH) ppm; ¹³C NMR

(DMSO-d₆): d = 163.6, 151.1, 148.0, 143.7, 136.6, 136.4,

129.5, 129.4, 129.3, 129.3, 128.5, 124.1, 116.3 ppm; MS

(ES?, ToF): m/z = 283 ([M ? 1]^?).

Determination of MBC/MFC

The minimum bactericidal concentration (MBC) and

minimum fungicidal concentration (MFC) were determined

by subculturing 100 mm³of culture from each tube (which

remained clear in the MIC determination) on fresh med-

ium. MBC and MFC values are the lowest concentrations

of compound that produce a 99.9 % end point reduction

[44].

N⁰-[4-[(Naphthalen-1-ylimino)methyl]benzylidene]-4-ami-

nobenzoic acid hydrazide (47, C₂₅H₂₀N₄O)

ꢀ

Yield: 67.3 %; TLC: R_f= 0.64; IR (KBr): m = 1,696 (C=N

str., CH=N), 1,617 (C=O str., secondary amide), 1,514 (C=C

skeletal str., phenyl nucleus), 1,321 (C–N str., Ar–NH₂), 826

(C–H out of plane bending, 1,4-disubstituted benzene ring),

Antiviral assays

1

771 (C–H out of plane bending, 1-naphthalenyl) cm^-1; H

The antiviral assays (except anti-human immunodeﬁ-

ciency virus (HIV) assay) were based on inhibition of

NMR (MeOH-d₄): d = 7.99 (s, 1H, –CH=N), 7.96 (d, 2H,

123

848

P. Kumar et al.

virus-induced cytopathicity in CRFK (feline corona virus

and feline herpes virus), HEL (herpes simplex virus type 1

(HSV-1), HSV-2 (G), vaccinia virus, and vesicular sto-

matitis virus), Vero (parainﬂuenza-3, reovirus-1, Sindbis,

Coxsackie B4, and Punta Toro virus), and HeLa (vesicular

stomatitis virus, Coxsackie virus B4, and respiratory syn-

cytial virus) cell cultures. Conﬂuent cell cultures in

microtiter 96-well plates were inoculated with 100 cell

culture inhibitory dose-50 (CCID₅₀) of the virus (1

CCID₅₀, being the virus dose infecting 50 % of the cell

cultures) in the presence of different concentrations (100,

20, 4, … lg/cm³) of the test compounds. Viral cytopath-

icity was recorded as soon as it reached completion in the

control virus-infected cell cultures that were not treated

with the test compounds.

expressed as the mean IC₅₀from three independent

experiments.

QSAR studies

The structures of 1–48 were ﬁrst pre-optimized by use of

MM^?procedure included in Hyperchem 6.03 [45] and the

resulting geometries were further reﬁned by means of the

semiempirical method PM3 (Parametric Method-3). We

˚

chose a gradient norm limit of 0.04 kJ/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 [46]. Further, the

regression analysis was performed by use of the SPSS

software package [47].

Acknowledgments The antiviral assays were performed by the

Laboratory of Virology and Chemotherapy at the Rega Institute for

Medical Research, KU Leuven, Leuven, Belgium.

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 by use of the 3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide (MTT) method. Brieﬂy, 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 10⁵cells/cm³and

infected with HIV at a multiplicity of infection of 0.02.

Immediately after viral infection, 100 mm³of the cell

suspension was placed in wells of a ﬂat-bottomed micro-

titer tray containing different concentrations of the test

compounds. After incubation for four days at 37 ꢁC, the

number of viable cells was determined by use of the MTT

method. Compounds were tested in parallel for cytotoxic

effects in uninfected MT-4 cells.

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Article Doi

N′-[4-[(Substituted imino)methyl]benzylidene]-substituted benzohydrazides: Synthesis, antimicrobial, antiviral, and anticancer evaluation, and QSAR studies

DOI: 10.1007/s00706-012-0877-3

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