Novel 4-(morpholin-4-yl)-N′-(arylidene)benzohydrazides: Synthesis, antimycobacterial activity and QSAR investigations

Article abstract of DOI:10.1016/j.ejmech.2009.04.023

A series of 4-(morpholin-4-yl)-N′-(arylidene)benzohydrazides were synthesized using appropriate synthetic route. Antimycobacterial activity of the synthesized compounds (5a-5j) was carried out and percentage reduction in relative light units (RLU) was calculated using luciferase reporter phages (LRP) assay. Percentage reduction in relative light units (RLU) for isoniazid was also calculated. The test compounds showed significant antitubercular activity against Mycobacterium tuberculosis H37Rv and clinical isolates: S, H, R, and E resistant M. tuberculosis, when tested in vitro. Quantitative structure-activity relationship (QSAR) investigation with 2D-QSAR analysis was applied to find a correlation between different experimental or calculated physicochemical parameters of the compounds studied and 3D-QSAR analysis and to indicate the exact steric and electronic requirements in the ranges at various positions around pharmacophore. In general Schiff bases exhibit antimycobacterial activity and morpholine ring is important for antimicrobial activity. So we have synthesized 10 different 4-(morpholin-4-yl)-N′-(arylidene)benzohydrazides. The structures of new compounds were characterized by TLC, FTIR, ¹H NMR, mass spectral data and elemental analysis. Amongst the compounds tested 5d and 5c were found to be the most potent, while 5i, 5e, and 5j were found to have an average activity against M. tuberculosis H37Rv and 5a, 5f, 5h, 5g, and 5b were found to have a greater activity against clinical isolates: S, H, R, and E resistant M. tuberculosis as compared to M. tuberculosis H37Rv.

Full text of DOI:10.1016/j.ejmech.2009.04.023

European Journal of Medicinal Chemistry 44 (2009) 3954–3960
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel 4-(morpholin-4-yl)-N0-(arylidene)benzohydrazides: Synthesis,
antimycobacterial activity and QSAR investigations
,
a
b
a
Vyankatesh Raparti a, Trupti Chitre a , Kailas Bothara , Vanaja Kumar , Sudarshan Dangre ,
*
Chetan Khachane a, Suraj Gore a, Bhavana Deshmane a
a Department of Pharmaceutical Chemistry, A.I.S.S.M.S. College of Pharmacy, Near RTO, Kennedy Road, Pune 411001, Maharashtra, India
b Tuberculosis Research Centre (ICMR), Chetpet, Chennai 600031, Tamilnadu, India
a r t i c l e i n f o
a b s t r a c t
series of 4-(morpholin-4-yl)-N0-(arylidene)benzohydrazides were synthesized using appropriate
Article history:
A
Received 12 August 2008
Received in revised form
8 April 2009
Accepted 14 April 2009
Available online 22 April 2009
synthetic route. Antimycobacterial activity of the synthesized compounds (5a–5j) was carried out and
percentage reduction in relative light units (RLU) was calculated using luciferase reporter phages (LRP)
assay. Percentage reduction in relative light units (RLU) for isoniazid was also calculated. The test
compounds showed significant antitubercular activity against Mycobacterium tuberculosis H37Rv and
clinical isolates: S, H, R, and E resistant M. tuberculosis, when tested in vitro.
Quantitative structure–activity relationship (QSAR) investigation with 2D-QSAR analysis was applied to
find a correlation between different experimental or calculated physicochemical parameters of the
compounds studied and 3D-QSAR analysis and to indicate the exact steric and electronic requirements in
the ranges at various positions around pharmacophore.
In general Schiff bases exhibit antimycobacterial activity and morpholine ring is important for antimi-
crobial activity. So we have synthesized 10 different 4-(morpholin-4-yl)-N0-(arylidene)benzohydrazides.
The structures of new compounds were characterized by TLC, FTIR, 1H NMR, mass spectral data and
elemental analysis.
Keywords:
Morpholine
Antimycobacterial
Schiff base
2D-QSAR
3D-QSAR
Amongst the compounds tested 5d and 5c were found to be the most potent, while 5i, 5e, and 5j were
found to have an average activity against M. tuberculosis H37Rv and 5a, 5f, 5h, 5g, and 5b were found to
have a greater activity against clinical isolates: S, H, R, and E resistant M. tuberculosis as compared to
M. tuberculosis H37Rv.
Ó 2009 Published by Elsevier Masson SAS.
1. Introduction
Furthermore, treatment of infectious diseases is more difficult in
immunodeficient patients such as those infected with human
The deterioration of human population due to the enhanced
prevalence of infectious diseases is becoming worldwide
immunodeficiency virus (HIV) [9]. Recent studies showed that the
application of appropriate dosage regimen with highly potent
antimicrobial agents not only eradicates bacterial growth but also
minimizes the probability of resistance formation [10–12]. The
biochemical basis of both intrinsic and acquired resistance are now
known [13–15] and has contributed significantly towards the
design of new entities by rational strategies that can be used to
counteract the resistance. The development of new potential drugs
will be one of the possible solutions to treat various infectious
diseases with multi-drug treatment over a long period of time.
Schiff bases exhibit a wide variety of biological activities such as
antimycobacterial [16–18], antimicrobial [19], and anticonvulsant
[20], etc. Literature survey reveals that morpholine ring is important
for antimicrobial activity [21]. In view of the fact that morpholine
ring is important for antimicrobial activity and Schiff bases exhibit
antimycobacterial activity, we have synthesized some novel
a
problem. Over the last few years, tuberculosis is retrieving its place
among these infectious diseases and today, nearly one-third of the
world’s population is infected with Mycobacterium tuberculosis
with approximately three million patients deceasing every year [1].
The contemporary treatment of these infectious diseases involves
administration of multi-drug regimen over a long period of time
[2,3], which lead to patient noncompliance and rapid emergence of
multi-drug resistance strain [4–7]. The resistance problem
demands to seek antimycobacterial agents effective against path-
ogenic microorganisms resistant to current treatment [8].
* Corresponding author. Tel.: þ91 20 26058204; fax: þ91 20 26058208; mobile:
þ919860993381.
E-mail address: chitre_13@yahoo.co.in (T. Chitre).
0223-5234/$ – see front matter Ó 2009 Published by Elsevier Masson SAS.
doi:10.1016/j.ejmech.2009.04.023

V. Raparti et al. / European Journal of Medicinal Chemistry 44 (2009) 3954–3960
3955
4-(morpholin-4-yl)-N0-(arylidene)benzohydrazides with the aim of
obtaining the new antimycobacterial agents which will be active
against some of the resistant strains. Thus in the present
investigation, 10 different 4-(morpholin-4-yl)-N0-(arylidene)-
benzohydrazides were synthesized and evaluated for their anti-
mycobacterial activity.
stretching bands at 1705–1677 cmꢀ1 and N–H stretching bands at
3362–3236 cmꢀ1. In the proton NMR spectral data, all protons were
seen according to the expected chemical shift and integral values.
The aromatic protons appeared as multiplet peaks within the range
6.8–7.8
d ppm, singlet signals derived from hydrazide (–NH–NH2)
structure appeared at 4.23 ppm. The 1H NMR spectra of compounds
(4) and (5a–5j) displayed broad singlet due to NH proton of amide
(probably due to their ability to get exchanged with D2O) and each
signal showing integration for one proton. For the compounds (5a–
5j), the signals belonging to benzylidene group were observed at
aromatic region, while the signals belonging to –NHNH2 dis-
appeared indicating functionalization of hydrazide to hydrazones
with substituted aromatic aldehydes.
2. Results and discussion
2.1. Chemistry
The synthetic route used to synthesize title compounds is out-
lined in Scheme 1. 4-(Morpholin-4-yl)benzonitrile (1) and was
prepared according to the method reported in the literature, using
4-chlorobenzonitrile as a starting material. The 4-(morpholin-4-
yl)benzoic acid (2) was prepared by basic hydrolysis of 4-(mor-
pholin-4-yl)benzonitrile (1). 4-(Morpholin-4-yl)benzoic acid
hydrazide (4) was prepared by hydrazination of 4-(morpholin-4-
yl)benzoic acid chloride (3) which was prepared by reacting 4-
(morpholin-4-yl)benzoic acid (2) with thionyl chloride. It was
condensed with different substituted aromatic aldehydes in
methanol to give corresponding 4-(morpholin-4-yl)-N0-(arylide-
ne)benzohydrazides (5a–5j) in very good yields. The structures of
various synthesized compounds were assigned on the basis of
different chromatographic, spectral studies and qualitative and
quantitative organic analytical studies. The physical data, FTIR, 1H
NMR and mass spectral data for all the synthesized compounds are
reported in experimental protocols.
2.2. Biological investigation
All the newly synthesized Schiff bases were assayed in vitro for
antitubercular activity against M. tuberculosis H37Rv and clinical
isolates: S, H, R, and E resistant M. tuberculosis. In case of anti-
mycobacterial activity % reduction in relative light units (RLU) was
calculated using luciferase reporter phages (LRP) assay at four
different concentrations (0.05, 0.5, 5, and 10 mg/ml) using isoniazid
as a reference standard and the observed percentage inhibition is
tabulated in Table 1. A compound is considered to be an anti-
mycobacterial agent if fifty percent reduction in the relative light
units (RLU) is observed when compared to the control using
a luminometer.
The obtained results revealed that the nature of the substituent
and substitution pattern on the benzene ring may have a considerable
impact on the antitubercular activity of the synthesized compounds.
Literature survey reveals that electron-withdrawing or donating
groups amend the lipophilicity of the test compounds, which in turn
The FTIR spectra of the Schiff bases exhibited very similar
features and showed the expected bands for the characteristic
groups which are present in the compounds such as C–H and the
C]N stretching vibrations. The Schiff bases (5a–5j) showed C]O
120°C
CN
Cl
O
NH +
O
N
CN
Reflux for 12 h
4-chlorobenzonitrile
1
Morpholine
NaOH
Reflux for 5 h
O
C
O
SOCl2
O
N
OH
O
N
C
Cl
Reflux for 6 h
3
2
MeOH
Reflux for 4 h
NH2NH2
O
O
O
C
H
N
Ar
C
H
H
N
O
N
NH2
C
N
O
N
Reflux for 7-8 h
MeOH
CH
Ar
5a-5j
4
Scheme 1. Synthetic route for title compounds 5a–5j.

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V. Raparti et al. / European Journal of Medicinal Chemistry 44 (2009) 3954–3960
Table 1
% Reduction in relative light units (RLU) against M. tuberculosis H37Rv and clinical isolates: S, H, R, and E resistant M. tuberculosis.
Sr. No.
Comp
M. tuberculosis H37Rv
Clinical isolates: S, H, R, and E resistant M. tuberculosis
% Reduction in relative light units (RLU)
0.05
m
g/ml
0.5
mg/ml
5
mg/ml
10
m
g/ml
0.05
m
g/ml
0.5
m
g/ml
5
m
g/ml
10 mg/ml
1
2
3
4
5
6
7
8
5a
5b
5c
5d
5e
5f
5g
5h
5i
15.04
9.118
78.09
96.78
51.79
30.04
20.85
24.19
63.16
49.68
96.00
96.90
98.04
98.02
98.22
77.81
96.24
98.09
97.29
98.96
96.51
98.42
98.41
98.40
98.38
98.98
97.53
98.10
98.49
97.75
97.82
97.59
99.01
97.55
98.40
99.26
99.09
97.24
97.18
98.82
98.18
97.32
97.77
98.99
58.54
29.92
67.15
83.22
43.92
56.53
48.14
48.49
55.70
35.83
84.37
86.92
87.17
89.91
86.33
73.58
82.97
90.72
87.24
85.67
96.88
89.6
92.52
90.07
91.19
93.28
88.08
89.33
93.18
90.83
91.61
90.29
95.85
93.72
88.86
94.29
95.09
88.45
90.36
95.02
91.37
92.10
90.24
94.70
9
10
11
5j
INH
alters permeability across the bacterial cell membrane. Further, the
presence of electron-withdrawing groups (both halogen) in 5d and 5c
showed maximum antimycobacterial activity.
From the above equation it is clear that the alignment inde-
pendent descriptor T_2_N_5 contributes negatively for the anti-
mycobacterial activity against M. tuberculosis H37Rv which
corresponds to number of double bonded atoms (i.e. any double
bonded atom, T_2) separated from nitrogen atom by 5 bonds.
The regression equation so obtained will be useful for the
prediction of biological activities of the designed series of
compounds, in future. The 2D-QSAR study for the synthesized
compounds was carried out using multiple linear regression (MLR)
method to study the structural activity relationship. In all, 10
compounds along with the standard were used for the 2D-QSAR
studies. The compounds were divided into training set (8) and test
set (2) respectively using manual selection method by considering
the biological and chemical variation.
2.2.1. Antimycobacterial activity
Amongst the compounds tested 5d and 5c were found to be the
most potent, while 5i, 5e, and 5j were found to have an average
activity against M. tuberculosis H37Rv.
Amongst the compounds tested 5a, 5f, 5h, and 5g were found to
have a greater activity against clinical isolates: S, H, R, and E
resistant M. tuberculosis as compared to M. tuberculosis H37Rv.
2.3. 2D-QSAR study
The antimycobacterial activities of the compounds tested, pre-
sented in pIC50 (Table 2), were used in the 2D-QSAR studies. pIC50
values were calculated using the following equation.
2.4. 3D-QSAR study
The 3-D QSAR studies were carried out using the Step Wise K
Nearest Neighbour Molecular Field Analysis [(SW) kNN MFA]
method which can be discussed as follows.
pIC50 ¼ ꢀlog C þ log it
For the kNN MFA method, the alignment of the molecules was
carried out using Field Fit alignment. KNN-MFA models were
generated using 10 molecules.
where C ¼ molar concentration ¼ [concentration ( g/ml) ꢁ 0.001/
m
molecular weight]
log it ¼ log½%inhibition=100 ꢀ %inhibitionꢂ
The most common structure was used as a template for the
alignment of all the analogues.
A 2D-QSAR study was performed using multiple linear regres-
sion (MLR) method. The results obtained are presented in Table 2.
The residuals (actual–predicted activity) were found to be
minimal and are presented in Table 3.
Also the graph for actual vs predicted activity for the series is
plotted in Graph 1 which shows good correlation.
The regression equation obtained for the different series of
compounds is given below.
The careful analysis of comparison of biological activities (pIC50),
predicted activities for training and test set molecules indicate very
less significant differences (lower values of residuals). Therefore it
can be said that the predictive abilities of SW kNN MFA model is
good. This comment is supported by the statistical values presented
in Table 6.
Table 3
pIC50ðM: tuberculosis H37RvÞ ¼ ꢀ0:4739T 2 N 5 þ 3:1198
Predicted activity obtained from multiple linear regressions of 2D-QSAR study.
(1)
Comp
M. tuberculosis H37Rv
n ¼ 8, r2 ¼ 0.7346, q2 ¼ 0.6523, F-test ¼ 16.6048.
Biological activity pIC50
Predicted activity
Residual
5a
5b
5c
5d
5e
5f
5g
5h
5i
ꢀ0.89956
ꢀ1.1445
0.4057
ꢀ0.143408
ꢀ1.082856
ꢀ0.756152
ꢀ0.061644
0.549108
Table 2
Results of 2D-QSAR equation obtained by multiple linear regression method.
ꢀ0.143408
a
1.3312
Sr. No.
Statistical parameter and contributing descriptors
Results
ꢀ0.1157
ꢀ0.5135
ꢀ0.7253
ꢀ0.643
0.0877
ꢀ0.143408
ꢀ0.143408
ꢀ0.613132
ꢀ0.143408
ꢀ0.143408
ꢀ0.143408
1.265764
0.027708
ꢀ0.370092
ꢀ0.112168
ꢀ0.499592
0.231108
1
2
3
4
5
6
7
8
r2
0.7346
0.6523
0.6637
q2
Pred_r2
Pred_r2SE
F-test
0.4173
16.6048
0.01000
0.43524
1.81367
5j
INH
ꢀ0.1503
ꢀ0.006892
Alpha
1.2249
ꢀ0.040864
Best-Ran_q2
Z-score
INH – isoniazid.
a
Outlier.

V. Raparti et al. / European Journal of Medicinal Chemistry 44 (2009) 3954–3960
3957
In all, it can be said that the results obtained by 3D kNN MFA
studies by SW method are in agreement with the general
requirements of selective and ideal pharmacophore required for
potential antimycobacterial activity. The graph for actual vs pre-
dicted activity for the series is plotted in Graph 2, which shows
good correlation.
the determination of percentage of C, H, and N. Purity of the
compounds was checked on TLC plates using silica gel G as
stationary phase and iodine vapours as visualizing agent. The
4-chlorobenzonitrile was purchased from S.D. Fine-Chem Ltd,
Mumbai.
Result of 3D data points generated, gives information of steric
and electronic interaction energy of a probe atom with each
molecule at the grid points. This leads to identification of various
molecular features responsible for activity variation and hence aid
in design of novel potential molecules. In addition, it provides
a mathematical model, which can be used to quantitatively predict
the activity of newly designed molecules.
4.1. Synthesis of 4-(morpholin-4-yl)benzonitrile (1)
The compound, 4-(morpholin-4-yl)benzonitrile was prepared
as per procedure reported in literature [22] and was purified by
recrystallization from 50% aqueous ethanol. Yield: 53.28% (solid);
mp 82–83 ꢃC. FTIR (KBr) cmꢀ1: 3050 (Ar C–H); 2980 (C–H); 2215
(C^N); 1495 (C]C); 1115 (C–O–C). 1H NMR chemical shifts at
Fig. 2 shows the relative position and the range of the corre-
sponding important electrostatic field in the model generated.
Negative range indicates that negative electrostatic potential is
favorable for increase in the activity and hence a more electro-
negative substituent group is preferred in that region. So from the
3D counter plot i.e. Fig. 2 obtained using the SW kNN MFA method
it was found that electronegative atoms like –Cl, –F, were essential
for the potential antimycobacterial activity.
(300 MHz, CDCl3, d ppm): 3.28 (t, 4H, CH2); 3.75 (t, 4H, CH2); 6.78
(dd, 2H, phenyl); 7.52 (dd, 2H, phenyl). Mass spectra of compound
exhibited molecular ion peak at m/z 188 (Mþ).
4.2. Synthesis of 4-(morpholin-4-yl)benzoic acid (2)
The compound, 4-(morpholin-4-yl)benzoic acid was prepared
as per procedure reported in literature [22]. Yield: 98.34% (solid);
mp 275-277 ꢃC. FTIR (KBr) cmꢀ1: 3241 (O-H); 3065 (Ar C–H); 2992
(C–H); 1694 (C]O); 1467 (C]C); 1113 (C–O–C). 1H NMR chemical
3. Conclusion
shifts at (300 MHz, CDCl3,
d ppm): 3.25 (t, 4H, CH2); 3.62 (t, 4H,
In the present investigation, 10 different 4-(morpholin-4-yl)-N0-
(arylidene)benzohydrazides were synthesized and evaluated for
their antimycobacterial activity. Most compounds exhibited
significant antimycobacterial activity. A remarkable activity was
found in compound 5d carrying 4-fluro moiety. Compound 5c with
4-chloro substitution also exhibited good antimycobacterial
activity. Amongst the compounds tested 5d and 5c were found to
be most potent, while 5i, 5e, and 5j were found to have an average
activity against M. tuberculosis H37Rv and 5a, 5f and 5h were found
to have good activity against clinical isolates: S, H, R, and E resistant
M. tuberculosis as compared to M. tuberculosis H37Rv.
From the detailed analysis of the results of above studies, we
conclude that antimycobacterial activity of the synthesized
compounds significantly depends on the presence of electronega-
tive group. The result obtained show that out of the synthesized
and tested compounds, especially 5d, 5c, 5i, 5a, 5f and 5h may be
considered promising for the development of new anti-
mycobacterial agents. Various physicochemical indices are helpful
for the understanding of microbiological results, as shown by our
2D- and 3D-QSAR study. As the process of antimycobacterial
activity is not clearly defined in terms of the target bio-
macromolecules, the 2D-QSAR results could not be addressed to
a concrete drug–receptor interaction, but they can reveal trends in
the relationship between ligand structures and their activities for
our set of antimycobacterial agents. Analysis of 3D counter plot
generated in 3D-QSAR study provides details on the fine relation-
ship linking structure and activity and offers clues for structural
modifications that can improve the activity. These trends should
prove to be an essential guide for the future work.
CH2); 6.82 (dd, 2H, phenyl); 7.68 (dd, 2H, phenyl); 12.23 (s, 1H, OH).
Mass spectra of compound exhibited molecular ion peak at
m/z 207 (Mþ).
4.3. Synthesis of 4-(morpholin-4-yl)benzoyl chloride [23,24] (3)
To 4-(morpholin-4-yl)benzoic acid (2) (0.01 mol), thionyl chlo-
ride (0.015 mol) was added and refluxed on water bath for 6 h.
Excess of thionyl chloride was removed under reduced pressure
and the residue so collected was used for the next step.
Yield: 79.6% (Solid); mp 130–132 ꢃC. FTIR (KBr) cmꢀ1: 3077
(Ar C–H); 2972 (C–H); 1774 (C]O); 1475 (C]C); 1120 (C–O–C); 870
(C–Cl). 1H NMR chemical shifts at (300 MHz, CDCl3,
d ppm): 3.31
(t, 4H, CH2); 3.6 (t, 4H, CH2); 6.79 (dd, 2H, phenyl); 7.71 (dd, 2H,
phenyl). Mass spectra of compound exhibited molecular ion peak at
m/z 225.5 (Mþ).
4.4. Synthesis of 4-(morpholin-4-yl)benzoic acid hydrazide (4)
To the solution of 4-(morpholin-4-yl)benzoic acid chloride (3)
(0.05 mol) in 75 ml of methanol, 99% hydrazine hydrate (0.1 mol)
was added and the mixture was refluxed on water bath for 4 h.
After cooling, the precipitate was collected, washed with distilled
water and recrystallized from ethanol [25].
Yield: 95.23% (solid); mp 282–284 ꢃC. FTIR (KBr) cmꢀ1: 3325
(N–H); 3102 (Ar C–H); 2988 (C–H); 1638 (C]O); 1455 (C]C); 1115
(C–O–C). 1H NMR chemical shifts at (300 MHz, CDCl3,
d ppm): 3.28
(t, 4H, CH2); 3.69 (t, 4H, CH2); 4.23 (s, 2H, NH2); 6.98–7.12 (m, 4H,
phenyl); 8.27 (s, 1H, NH). Mass spectra of compound exhibited
molecular ion peak at m/z 221 (Mþ), 222 (M þ 1).
4. Experimental
4.5. General procedure for the preparation of 4-(morpholin-4-yl)-
Melting points were determined by open capillary tubes and are
uncorrected. FTIR spectra of the powdered compounds were
recorded using KBr on a Jasco FTIR V 430þ spectrometer using
Diffuse Reflectance Attachment and are reported in cmꢀ1 and 1H
NMR spectra were recorded on a Varian Mercury YH300 (300 MHz
FT NMR) spectrophotometer using TMS as an internal reference
N0-(arylidene)benzohydrazides (5a–5J)
The 4-(morpholin-4-yl)-N0-(arylidene)benzohydrazides were
prepared as per the reported procedure [15,20], and purified by
recrystallization from ethanol.
(chemical shift represented in
d
ppm). Mass spectra were recorded
4.5.1. 4-(Morpholin-4-yl)-N0-(furan-2-ylidene)benzohydrazide
Yield: 94.1% (solid); mp 224–226 ꢃC. FTIR (KBr) cmꢀ1: 3285
(N–H); 3085 (Ar C–H); 2972 (C–H); 2860 (N]CH); 1695 (C]O);
on GC–MS QP5050A System (benchtop quadrupole mass spectro-
photometer). Microanalyses of compounds were also performed for

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V. Raparti et al. / European Journal of Medicinal Chemistry 44 (2009) 3954–3960
1585 (C]N); 1459 (C]C); 1108 (C–O–C). 1H NMR chemical shifts at
(300 MHz, CDCl3, ppm): 3.3 (t, 4H, CH2); 3.7 (t, 4H, CH2);); 6.28–
d
6.33 (dd, 1H, furanyl, J3,4 ¼ 3.5 Hz, J3,5 ¼ 0.8 Hz); 6.38–6.44 (dd, 1H,
furanyl J3,5 ¼ 3.5 Hz, J4,5 ¼ 3.5 Hz); 6.52–6.59 (m, 4H, phenyl); 7.36–
7.42 (dd, 1H, furanyl, J3,5 ¼ 0.8 Hz, J4,5 ¼ 3.7 Hz); 8.66 (s, 1H, CH);
11.12 (bs, 1H, NH). Mass spectra of compound exhibited molecular
ion peak at m/z 354 (Mþ), 355 (M þ 1) Anal. Calcd for C16H17O3N3: C,
64.21; H, 5.84; N, 16.05. Found: C, 64.83; H, 5.04; N, 16.83.
4.5.2. 4-(Morpholin-4-yl)-N0-(3-nitrobenzylidene)benzohydrazide
Yield: 87.5% (solid); mp 157–159 ꢃC. FTIR (KBr) cmꢀ1: 3288 (N–
H); 3065 (Ar C–H); 2976 (C–H); 2862 (N]CH); 1698 (C]O); 1605
(C]N); 1532 (asymm NO2); 1460 (C]C); 1347 (symm NO2); 1111
(C–O–C). 1H NMR chemical shifts at (300 MHz, CDCl3,
d ppm): 3.22
(t, 4H, CH2); 3.62 (t, 4H, CH2); 6.94–7.1 (m, 4H, phenyl); 7.22–7.29
(m, 4H, phenyl); 8.86 (s, 1H, CH); 11.28 (bs, 1H, NH). Mass spectra of
compound exhibited molecular ion peak at m/z 354 (Mþ), 355
(M þ 1) Anal. Calcd for C18H18O4N4: C, 61.01; H, 5.08; N, 15.81.
Found: C, 61.55; H, 5.86; N, 15.12.
Fig. 1. Overlain of all 4-(morpholin-4-yl)-N0-(arylidene)benzohydrazides aligned using
alignment tool of V-Life MDS.
4.5.7. 4-(Morpholin-4-yl)-N0-(4-(N,N-dimethylamino)benzylidene)
benzohydrazide
4.5.3. 4-(Morpholin-4-yl)-N0-(4-chlorobenzylidene)benzohydrazide
Yield: 85.5% (solid); mp 216–218 ꢃC. FTIR (KBr) cmꢀ1: 3380 (N–
H); 3092 (Ar C–H); 2982 (C–H); 2864 (N]CH); 1701 (C]O); 1615
(C]N); 1462 (C]C); 1116 (C–O–C); 766 (C–Cl). 1H NMR chemical
Yield: 92.1% (solid); mp 165–167 ꢃC. FTIR (KBr) cmꢀ1: 3245 (N–
H); 3061(Ar C–H); 2975 (C–H); 2830 (N]CH); 1680 (C]O); 1572
(C]N); 1454 (N (CH3)2); 1446 (C]C); 1105 (C–O–C). 1H NMR
chemical shifts at (300 MHz, CDCl3,
d ppm): 3.27 (t, 4H, CH2); 3.54
shifts at (300 MHz, CDCl3,
d ppm): 3.26 (t, 4H, CH2); 3.79 (t, 4H,
(t, 4H, CH2); 7.1–7.18 (m, 4H, phenyl); 7.2–7.34 (m, 4H, phenyl); 8.45
(s, 1H, CH); 10.6 (bs, 1H, NH). Mass spectra of compound exhibited
molecular ion peak at m/z 352 (Mþ), 353 (M þ 1). Anal. Calcd for
C20H24O2N4: C, 68.18; H, 6,82; N, 15.09. Found: C, 68.70; H, 6.42; N,
15.52.
CH2); 7.20-7.23 (m, 4H, phenyl); 7.50-7.68 (m, 4H, phenyl); 8.91
(s, 1H, CH); 11.36 (bs, 1H, NH). Mass spectra of compound exhibited
molecular ion peak at m/z 343.5 (Mþ), 344.5 (M þ 1). Anal. Calcd for
C18H18O2N3Cl: C, 62.88; H, 5.24; N, 12.33. Found: C, 62.24; H, 5.06;
N, 12.19.
4.5.8. 4-(Morpholin-4-yl)-N0-(4-
methylbenzylidene)benzohydrazide
4.5.4. 4-(Morpholin-4-yl)-N0-(4-fluorobenzylidene)benzohydrazide
Yield: 85.6% (solid); mp 216–218 ꢃC. FTIR (KBr) cmꢀ1: 3289 (N–
H); 3085 (Ar C–H); 2986 (C–H); 2868 (N]CH); 1705 (C]O); 1621
(C]N); 1465 (C]C); 1231 (C–F); 1120 (C–O–C). 1H NMR chemical
Yield: 63.9% (solid); mp 138–140 ꢃC. FTIR (KBr) cmꢀ1: 3244 (N–
H); 3059 (Ar C–H); 2981 (C–H); 2896 (N]CH); 1678 (C]O); 1569
(C]N); 1450 (C]C); 1108 (C–O–C), 741 (C–H). 1H NMR chemical
shifts at (300 MHz, CDCl3,
d ppm): 3.32 (t, 4H, CH2); 3.81 (t, 4H,
shifts at (300 MHz, CDCl3,
d ppm): 3.12 (t, 4H, CH2); 3.48 (t, 4H,
CH2); 7.49–7.58 (m, 4H, phenyl); 7.68–7.79 (m, 4H, phenyl); 8.95
(s, 1H, CH); 12.1 (bs, 1H, NH). Mass spectra of compound exhibited
molecular ion peak at m/z 327 (Mþ), 328 (M þ 1). Anal. Calcd for
C18H18O2N3F: C, 66.06; H, 5.51; N, 12.84. Found: C, 66.28; H, 5.66;
N, 12.13.
CH2); 6.94–7.16 (m, 4H, phenyl); 7.32–7.45 (m, 4H, phenyl); 8.48 (s,
1H, CH); 10.71 (bs, 1H, NH). Mass spectra of compound exhibited
molecular ion peak at m/z 323 (Mþ), 324 (M þ 1). Anal. Calcd for
C19H21O2N3: C, 70.58; H, 6.50; N, 13.00. Found: C, 70.02; H, 6.72; N,
13.66.
4.5.5. 4-(Morpholin-4-yl)-N0-(4-
4.5.9. 4-(Morpholin-4-yl)-N0-(3-phenylallylidene)benzohydrazide
Yield: 88.1% (solid); mp 186–188 ꢃC. FTIR (KBr) cmꢀ1: 3269 (N–
H); 3076 (Ar C–H); 2988 (C–H); 2856 (N ¼ CH); 1688 (C]O); 1582
(C]N); 1462 (C]C); 1113 (C–O–C). 1H NMR chemical shifts at
hydroxybenzylidene)benzohydrazide
Yield: 81.8% (solid); mp 148–150 ꢃC. FTIR (KBr) cmꢀ1: 3289 (O–
H); 3255 (N–H); 3056 (Ar C–H); 2974 (C–H); 2830 (N]CH); 1670
(C]O); 1569 (C]N); 1449 (C]C); 1106 (C–O–C). 1H NMR chemical
(300 MHz, CDCl3,
d ppm): 3.34 (t, 4H, CH2); 3.58 (t, 4H, CH2); 6.95-
shifts at (300 MHz, CDCl3,
d ppm): 3.26 (t, 4H, CH2); 3.51 (t, 4H,
6.98 (d, 1H, J ¼ 6.5 Hz, CH]CH-Ph); 7.43-7.48 (m, 4H, phenyl);
7.55–7.61 (d, 1H, J ¼ 2.2 Hz, N]CH–CH); 8.71 (s, 1H, CH); 10.09 (bs,
1H, NH). Mass spectra of compound exhibited molecular ion peak at
m/z 335 (Mþ), 336 (M þ 1). Anal. Calcd for C20H21O2N3: C, 71.64; H,
6.27; N, 12.54. Found: C, 71.05; H, 6.42; N, 12.88.
CH2); 6.94–7.16 (m, 4H, phenyl); 7.39–7.46 (m, 4H, phenyl); 8.42
(s, 1H, CH); 10.12 (bs, 1H, NH). Mass spectra of compound exhibited
molecular ion peak at m/z 325 (Mþ), 326 (M þ 1). Anal. Calcd for
C18H19O3N3: C, 66.46; H, 5.85; N, 12.92. Found: C, 66.12; H, 5.05;
N, 12.21.
4.5.10. 4-(Morpholin-4-yl)-N0-(3,4,5-trimethoxybenzylidene)
benzohydrazide
4.5.6. 4-(Morpholin-4-yl)-N0-(4-
methoxybenzylidene)benzohydrazide
Yield: 74.1% (solid); mp 209–211 ꢃC. FTIR (KBr) cmꢀ1: 3272 (N–
H); 3079 (Ar C–H); 2992 (C–H); 2864 (N]CH); 1690 (C]O); 1594
(C]N); 1464 (C]C); 1176 (Ar C–O); 1116 (C–O–C). 1H NMR chem-
Yield: 73.9% (solid); mp 233–235 ꢃC. FTIR (KBr) cmꢀ1: 3265 (N–
H); 3072 (Ar C–H); 2982 (C–H); 2844 (N]CH); 1678 (C]O); 1672
(C]N); 1451 (C]C); 1174 (Ar C–O); 1109 (C–O–C). 1H NMR chem-
ical shifts at (300 MHz, CDCl3, d ppm): 3.38 (t, 4H, CH2); 3.59 (t, 4H,
ical shifts at (300 MHz, CDCl3, d ppm): 3.36 (t, 4H, CH2); 3.66 (t, 4H,
CH2); 7.4–7.46 (m, 4H, phenyl); 7.65–7.68 (m, 4H, phenyl); 8.76 (s,
1H, CH); 11.22 (bs, 1H, NH). Mass spectra of compound exhibited
molecular ion peak at m/z 339 (Mþ), 340 (M þ 1). Anal. Calcd for
C21H25O5N3: C, 63.16; H, 6.27; N, 10.53. Found: C, 63.78; H, 6.89; N,
10.84.
CH2); 6.91–7.12 (m, 4H, phenyl); 7.31–7.36 (m, 4H, phenyl); 8.65
(s, 1H, CH); 11.2 (bs, 1H, NH). Mass spectra of compound exhibited
molecular ion peak at m/z 339 (Mþ), 340 (M þ 1). Anal. Calcd for
C19H21O3N3: C, 67.26; H, 6.19; N, 12.38. Found: C, 67.02; H, 6.82;
N, 12.76.

V. Raparti et al. / European Journal of Medicinal Chemistry 44 (2009) 3954–3960
3959
Table 5
Predicted activity of test set of molecules for 3D-QSAR study.
Sr. No.
Molecule no.
Biological activity pIC50
SW-kNN MFA
Predicted activity
Residual
1.
2.
5g
5j
ꢀ0.7253
ꢀ0.1503
ꢀ0.714470
ꢀ0.311808
ꢀ1.4397
ꢀ0.4621
Molecular Force Field (MMFF) method [27] with distance depen-
dent-dielectric function and energy gradient of 0.001 kcal/mol Å.
Alignment of the compounds is a very important feature for any
3D-QSAR method and so was for of Step Wise (SW) kNN MFA
method [28]. The overlain of the aligned molecules using MMFF
method is shown in Fig. 1. The initial conformations were selected
and minimized using the Powell method till root-mean-square
deviation of 0.001 kcal/mol Å was achieved.
The compounds were divided into training set (8) and test set
(2) respectively using manual selection method by considering the
biological and chemical variation. Training set and test set used for
developing kNN MFA models using SW kNN MFA model are shown
in Tables 4 and 5 respectively. All 8 molecules in the training set
were considered as observations to generate QSAR equation using
the Step Wise (SW) kNN MFA method.
Fig. 2. Stereo view of the molecular rectangular field grid around the superposed
4-(morpholin-4-yl)-N0-(arylidene)benzohydrazides series of Molecular units using SW
kNN MFA Method.
5. Biological evaluation
All the newly synthesized 4-(morpholin-4-yl)-N0-(arylidene)-
benzohydrazides were assayed in vitro for antitubercular activity
against M. tuberculosis H37Rv and clinical isolates: S, H, R, and
E resistant M. tuberculosis. In case of antimycobacterial activity,
percentage reduction in relative light units (RLU) were calculated
using luciferase reporter phages (LRP) assay [29–31] using isoniazid
as a reference standard.
4.6. 2D-QSAR studies
All computational studies were performed using V-Life Mole-
cule Design Suite version 3.0 [26]. The compounds were con-
structed using the fragments in the V-Life molecular database with
standard bond lengths and bond angles. Geometry optimization
was carried out using standard Merck Molecular Force Filed
(MMFF) method with distance dependant dielectric function set to
1.00 and energy gradient of 0.001 kcal/mol. The synthesized
compounds (10) along with the standard were used during the 2D-
QSAR study.
5.1. Luciferase reporter phages (LRP) assay [29,30]
Fifty-microliter bacterial suspension equivalent to MacFarlands
No.2 standard was added to 400 ml of G7H9 with and without the
In case of QSAR study performed for synthesized compounds
and reference standard against M. tuberculosis H37Rv, expressed as
pIC50, two compounds were used in test set, viz., 5g and 5h and
remaining eight compounds were used in training set and
compound 5d was considered as an outlier.
test compound. For each sample, two drug-free controls and four
drug concentrations were prepared and this setup was incubated
for 72 h at 37 ꢃC. After incubation 50
m
l of the high titer luciferase
l of 0.1 M CaCl2 were added to
all the vials and this setup was incubated at 37 ꢃC for 4 h. After
incubation, 100 l of the mixture was taken from each tube into
a luminometer cuvette and equal amount of working -luciferin
reporter phage (phAE129) and 400
m
m
D
4.7. 3D-QSAR studies
(0.3 mM in 0.05 M sodium citrate buffer, pH 4.5) solution was
added. The RLU was measured after 10 s of integration in the
Luminometer (Monolight 2010). Duplicate readings were recorded
for each sample and the mean was calculated. The percentage
reduction in the RLU was calculated for each test sample and
compared with the control. The experiment was repeated when the
mean RLU of the control was less than 1000.
All computational studies were performed using V-Life Molec-
ular Design Software Version 3.0 [26]. The compounds were con-
structed using the fragments in the V-life molecular milder
database with standard bond lengths and bond angles and geom-
etry optimization was carried out using the standard Merck
Table 4
Predicted activity of training set of molecules for 3D-QSAR study.
Table 6
Statistical parameters giving details of SW kNN MFA method.
Sr. No.
Molecule no.
Biological activity pIC50
SW-kNN MFA
Predicted activity
Residual
Sr. No.
Parameter
SW kNN MFA method
1.
2.
3.
4.
5.
6.
7.
8.
5a
5b
5c
5d
5e
5f
ꢀ0.89956
ꢀ1.1445
0.4057
ꢀ0.705814
ꢀ0.528838
0.485445
0.164762
ꢀ0.1937
ꢀ0.6157
ꢀ0.0797
1.1665
ꢀ0.3324
ꢀ0.0916
ꢀ1.5429
ꢀ0.4873
1
2
3
4
5
6
q2
0.5071
0.9102
0.5614
0.1619
5
Pred r2
q2_SE
1.3312
Pred_r2 SE
k nearest neighbour
Contributing descriptor
Electrostatic
Steric
ꢀ0.1157
ꢀ0.5135
ꢀ0.643
0.0877
0.216743
ꢀ0.421828
ꢀ0.899957
0.575019
5h
5i
E_189 (ꢀ3.5059 0.1088)
–

3960
V. Raparti et al. / European Journal of Medicinal Chemistry 44 (2009) 3954–3960
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The authors are thankful to The Principal, AISSMS College of
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also thankful to Mrs. S.H. Rao. The authors also wish to thank
Shimadzu Analytical Centre and Dept. of Chemistry, University of
Pune, for providing facilities for spectral studies of compounds and
Sun Pharma Advance Research Centre (SPARC), Baroda, for per-
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