Synthesis, characterization and anti mycobacterial activity of novel hydrazones

Article abstract of DOI:10.13005/ojc/330140

The hydrazone Schiff base analogues namely benzoic acid (4-allyloxy-benzylidene)-hydrazide and its series were designed and synthesized. The structures of newly synthesized compounds were characterized by analytical methods and spectral analysis and subjected to antibacterial and docking studies. The synthesized molecules were subjected to molecular docking studies using enoyl-acyl-carrier protein reductase (NADH) from mycobacterium tuberculosis as the receptor. The docking results confirm the binding affinity of the synthesized compounds with the selected receptor. Preliminary in-vitro anti bacterial studies were carried out with Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria. Benzoic acid (4-allyloxy-benzylidene)-hydrazide was found to be most potent anti tuberculosis agent at 200 and 100μg per ml, the percentage of inhibition was 98.7% and 97.9%. The designed synthesized molecules were considered for evaluation for the molecular docking studies of their binding pattern with enoyl-acyl-carrier protein reductase from Mycobacterium tuberculosis. The antimycobacterial screening was performed against MTB H37Rv an isoniazid-resistant clinical isolate of MTB was used for the study.

Full text of DOI:10.13005/ojc/330140

ISSN: 0970-020 X
CODEN: OJCHEG
017, Vol. 33, No. (1):
Pg. 335-345
ORIENTAL JOURNAL OF CHEMISTRY
An International Open Free Access, Peer Reviewed Research Journal
2
www.orientjchem.org
Synthesis, Characterization and
Anti Mycobacterial Activity of Novel Hydrazones
S KULANꢀAI THERESE* and G GEETHAMALIKA
Department of Chemistry, Nirmala College for Women, Coimbatore, Tamil Nadu, India.
*
Corresponding author E-mail: kulandaifspm@gmail.com
http://dx.doi.org/10.13005/ojc/330140
(Received: December 23, 2016; Accepted: January 01, 2017)
AꢁSTRACT
The hydrazone Schiff base analogues namely benzoic acid (4-allyloxy-benzylidene)-hydrazide
and its series were designed and synthesized. The structures of newly synthesized compounds
were characterized by analytical methods and spectral analysis and subjected to antibacterial and
docking studies. The synthesized molecules were subjected to molecular docking studies using
enoyl-acyl–carrier protein reductase (NADH) from mycobacterium tuberculosis as the receptor.The
docking results confirm the binding affinity of the synthesized compounds with the selected receptor.
Preliminary in-vitro anti bacterial studies were carried out with Gram-positive (Staphylococcus aureus)
and Gram-negative (Escherichia coli) bacteria.Benzoic acid (4-allyloxy-benzylidene)-hydrazide was
found to be most potent anti tuberculosis agent at 200 and 100µg per ml, the percentage of inhibition
was 98.7% and 97.9%.The designed synthesized molecules were considered for evaluation for the
molecular docking studies of their binding pattern with enoyl-acyl-carrier protein reductase from
Mycobacterium tuberculosis. The antimycobacterial screening was performed against MTB H37Rv
an isoniazid-resistant clinical isolate of MTB was used for the study.
Keywords: hydrazones, spectral studies, docking and anti mycobacterial activity.
INTROꢀUCTION
properties. They have been used as a synthetic
intermediate to produce different type of drugs in the
treatment of different chronic illnesses. Hydrazides
used in medicine include the anti-tuberculosis drug
isoniazid and the antihypertensive and peripheral
vasodilator drug hydralsane, whose hydrochloride is
an effective drug for emergency reduction of blood
pressure in hypertensive crisis. In the same way the
hydrazine derivatives have been widely used in the
Tuberculosis (TB) is a worldwide pandemic
caused by different species of mycobacteria.
Mycobacterium tuberculosis is a successful
pathogen, which causes tuberculosis and is the
greatest single infectious cause of death worldwide,
1
killing approximately two million people annually .
Hydrazides are highly reactive bases with reducing

336
THERESE & GEETHAMAꢀIꢁA, Orient. J. Chem., Vol. 33(1), 335-345 (2017)
treatment of tuberculosis and mental disorder.They compounds were checked by TꢀC. Further the
can also be used as antimicrobial, antihypertensive, product formed was washed with petroleum ether,
2-11
antimalarial and antitumoral agents
.
dried, recrystallized and characterized by spectral
analysis.
Schiff bases are considered to be among
the most important group of compounds in medicinal
Ethanolicsolutionof4-allyloxybenzaldehyde
chemistry due to their preparative accessibility, (10 m mol) was added to a 10 m mol of ethanolic
1
2
structural variety and wide biological profile . solution of benzohydrazides and the reaction mixture
The synthesized benzohydrazides were potent was stirred for 30 minutes at room temperature in
1
3
antimicrobial and anticancer agents . Numbers the presence of glacial acetic acid as catalyst. After
of hydrazide-hydrazone derivatives possess refluxing for 3-5 h the completion of the reaction was
1
4-21
antituberculosis activity
. The present study is monitored by TꢀC.The reaction mixture was cooled
aimed to carry out design, synthesis, in silico and and placed in refrigerator overnight. The resulting
in-vitro antimicrobial studies for the series of benzoic solid was filtered and washed with petroleum ether
acid (4-allyloxy-benzylidene)-hydrazides.
and recrystallised with ethanol.
ExpERIMENTAL
In silico Charaterisation
Thesynthesizedcompoundswereassessed
All the chemicals and solvents used in silico for their biological activity using PASS
2
3
are of analytical grade and were purchased from server . The three dimensional structure of the
Royal scientific company and used without further protein / enzymes was retrieved from Protein Data
24
purification. TꢀC was run on the silica coated Bank .The compound under study was docked with
25
aluminium sheets (TꢀC silica gel 60 F254, Analytical the identified receptor using Autodock software and
chromatography) and visualized in low UV light. IR further analyzed for interactions using Pymol.
spectra in ꢁBr pellets were recorded on the FT-IR
1
Perkin Elmer Spectrum spectrophotometer. H NMR Antimicrobial activity
1
3
and C NMR spectra were recorded on a 300 M Hz
Antibacterial screening of the synthesized
spectrometer with DMSO-d6 as solvent and TMS compounds was evaluated against Staphylococcus
as internal reference. Chemical shifts are quoted as aureus and Escherichia coli, and the zone of
dppm and the coupling constants J in Hz in signals inhibition was measured in mm. The broth media
are described as s (singlet), d (doublet), t (triplet), was inoculated and grown at 37ºC for 18 h to revive
m (multiplet) and b (broad). Melting points were the bacteria from the stock cultures.The agar plates
determined on a Sigma melting point apparatus with wells were inoculated by spreading evenly with
without corrections. GC MS mass spectrum was 18h old bacterial cultures (100 µl, 10-4 cfu). After 20
taken in JEOꢀ GCMATE II GC-MS with Data system min, various concentrations of the compound and the
in a high resolution, double focusing instrument.
antibiotic were filled in the wells. Finally, the plates
were incubated at 37ºC for 24 h and the inhibition
26
General ꢂrocedure for the synthesis of zone was observed .
benzohydrazides (3a-e)
The substituted benzoic acid (2.46 m mol) Luciferase reꢂorter ꢂhage (LRp) assay (27)
was refluxed with methanol in sulphuric acid for 4h.
The MTB H37Rv and clinical isolate of
The ester formed reacts with hydrazine and gets MTB were grown in Middlebrook 7H9 complete
22
converted to benzohydrazides by hydrozinolysis .
medium 12 with and without test sample for 3 days
at 37°C. ꢀRP assay was done using concentrations
Synthesis of ꢁenzoic acid (4-allyloꢃy- of 200, 100 and 50 µg/mꢀ of test samples. 50µꢀ
benzylidene)-hydrazide (5a-e) bacterial suspension equivalent to MacFarlands.
Benzaldehyde undergoes nucleophilic No.2 standard was added to 400 µꢀ of 7H9 with
addition reaction with aromatic hydrazides resulting and without the test compound. For each sample,
in the formation of hydrazones. The completion of two drug-free controls and two drug concentrations
reaction and the homogeneity of the synthesized were prepared and this set up was incubated for 72

THERESE & GEETHAMAꢀIꢁA, Orient. J. Chem., Vol. 33(1), 335-345 (2017)
337
h at 37°C. After incubation, 50µꢀ of the high titer
luciferase reporter phage (phAETRC202) and 40
a broth microdilution method (28) in Middlebrook
7H9 medium supplemented with OADC, with a
final inoculum of 1x10 cfu/mꢀ. The compounds
7
µꢀ of 0.1 M CaCl were added to all the vials and
2
this set up was incubated at 37°C for 4 h. After
incubation, 100 µꢀ of the mixture was taken from
each tube into a ꢀuminometer cuvette and an equal
amount of working D-luciferin (0.3 mM in 0.05 M
sodium citrate buffer, pH 4.5) solution was added.
The Relative ꢀight Units (RꢀU) was measured after
were dissolved in DMF (1.25 mg/ mꢀ) and used as
stock solution. Concentrations ranging from 1250
to 1 µg/mꢀ were used to assess the effectiveness
of the compounds. After inoculation the microtiter
tubes were incubated at 37°C for 72 h, and the
growth inhibition was recorded for 14 and 21 days,
respectively. The MIC value represents the lowest
dilution of the compound at which no bacterial growth
was detected.
1
0s of integration in the ꢀuminometer. Readings
were recorded in duplicate for each sample and the
mean was calculated. The percentage reduction in
the RꢀU was calculated for each test sample and
compared with that of control.
RESULTS ANꢀ ꢀISCUSSION
ꢁ
roth microdilution method
TheMICforMTBH37Rvandisoniazid(INH)
ꢁenzoic acid (4-allyloꢃy-benzylidene)-
hydrazide (5a) was prepared by the above
mentioned method. benzohydrazide (0.136g
resistant clinical isolate of MTB was determined using
0
(
.001mol 1 equivalent) 4-allyloxybenzaldehyde
0.15ml 0.001mol 1 equivalent), pale yellow
amorphous solid, yield:90%. m.p. 118-120°C. FT-IR
Table 1: Activity ꢂrediction for the
synthesized comꢂounds
-
1
(mmax ꢁBr pellets, cm ) 935(N-N) str, 1256 (C-O-C),
Comꢂound
code
Activity
pa
pi
1
1542 (C=C), 1650 (C=N) str, 3032 (C-H) str. H NMR
(
CDCl , 300 MHz) dH: 5.29-5.45 (dd, 1.2Hz, 2 H),
3
5
2
.9-6.1(m, 1H), 4.57(d, 2H, 5.1Hz), 6.9(d, 8.1Hz,
5
5
5
5
5
a
b
c
d
e
Antituberculosic
Antituberculosic
Antituberculosic
Antituberculosic
Antituberculosic
0.831
0.827
0.794
0.781
0.805
0.003
0.003
0.003
0.003
0.003
H), 7.7 (d, 6.6Hz, 2H), 8.23 (1H, s), 9.2 (1H, s), 7.8
13
(
d, 6.6 Hz, 2H), 7.4-7.5 (m, 3H). C NMR (DMSO
3
1
00 MHz): 160, 132, 131, 129, 128, 127, 126, 117,
14 and 68. m/z = 315 (M+1). Molecular Formula
C H N O .
17
16
2
2
O
COOCH
3
NH2
COOH
NH NH .H O
N
CH OH
2
2
2
3
H
+
R
1
CH
3
OH
R
1
+
R
1
H /heat
1a-e
2a-e
3a-e
1
2
3
a: R1 = H, 1b: R1 = Cl, 1c: R1 = CH , 1d: R1 = OCH , 1e: R1 = OH
3
3
3
a: R1 = H, 2b: R1 = Cl, 2c: R1 = CH , 2d: R1 = OCH , 2e: R1 = OH
3
a: R1 = H, 3b: R1 = Cl, 3c: R1 = CH , 3d: R1 = OCH , 3e: R1 = OH
3
3
Scheme 1: Synthesis of ꢁenzohydrazide from substituted benzoic acid
O
O
N
O
O
N
NH
2
H
N
R
1
H
OHC
R
1
+
3a-e
4
5a-e
5
a: R1 = H, 5b: R1 = Cl, 5c: R1 = CH , 5d: R1 = OCH , 5e: R1 = OH
3
3
Scheme 2: Synthesis of ꢁenzoic acid (4-allyloꢃy-benzylidene)-hydrazides

338
THERESE & GEETHAMAꢀIꢁA, Orient. J. Chem., Vol. 33(1), 335-345 (2017)
Fig 1: ꢁinding interactions
Fig 2: ꢁinding interactions
between 4-chloro-benzoic
acid (4-allyloꢃy-benzylidene)-
hydrazide with the receꢂtor
Fig 3: ꢁinding interactions
between ꢁenzoic acid
4-allyloꢃy-benzylidene)-
between 4-methyl-benzoic
acid (4-allyloꢃy-benylidene)-
hydrazide with the receꢂtor
(
hydrazide with the receꢂtor
Fig 4: ꢁinding interactions
between 4-methoꢃy-benzoic
acid (4-allyloꢃy-benylidene)-
hydrazide with the receꢂtor
Fig 5: ꢁinding interactions
between 4- hydroꢃy -benzoic
acid (4-allyloꢃy-benylidene)-
hydrazide with the receꢂtor
Fig 6: ꢁinding interactions
between isoniazid with the
receꢂtor
Table 2a: ꢀocking results of ꢁenzoic acid (4-allyloꢃy-benzylidene)-hydrazide with the receꢂtor
pose ꢁ.E
L.E IC (nM) Int.E
Vdw Elec.E Total.IE Tor.E Unb.E No. of
Hydrogen
H bonds bond
1
-11.81 -0.56
2.22
-13.6 -13.59 -0.01 -0.92 1.79 -0.92
1
Met98:HN:
ꢀig1:O
:
2
3
4
5
-10.79 -0.51 12.37 -12.58 -12.58
0.0
-0.81 1.79 -0.81
0
0
0
1
-
-
-
-10.73 -0.51 13.61 -12.52 -12.49 -0.03 -0.97 1.79 -0.97
-10.72 -0.51 13.94 -12.51 -12.52 0.01 -1.03 1.79 -1.03
-10.68 -0.51 14.91 -12.47 -12.42 -0.05 -1.14 1.79 -1.14
Ile194:HN:
:
ꢀig1:N
6
7
-10.44 -0.5
22.27 -12.23 -12.2 -0.03 -1.07 1.79 -1.07
0
1
-
-10.33 -0.49 26.91 -12.12 -12.04 -0.07 -0.95 1.79 -0.95
Tyr158:HH:
:
ꢀig1:O
ꢀig1:N:
Ala201:
Oꢀig1:N:
Ala201:O
Ile194:HN:
ꢀig1:N
Tyr158:HH:
ꢀig1:N
8
-9.84 -0.47 61.29 -11.63 -11.62 -0.01 -0.85 1.79 -0.85
2
:
:
9
1
-9.82 -0.47 63.87 -11.61 -11.58 -0.03 -1.23 1.79 -1.23
1
1
:
0
-9.56 -0.46
98.8
-11.35 -11.26 -0.09 -1.18 1.79 -1.18
:

THERESE & GEETHAMAꢀIꢁA, Orient. J. Chem., Vol. 33(1), 335-345 (2017)
339
4
-chloro-benzoic acid (4-allyloꢃy-
4-methyl-benzoic acid (4-allyloꢃy-
benylidene)-hydrazide (5c) was prepared by the
above mentioned method.4-methyl benzohydrazide
(0.15g0.001mol1equivalent)4-allyloxybenzaldehyde
(0.15ml 0.001mol 1 equivalent), yellow amorphous
b e n z y l i d e n e ) - h y d r a z i d e ( 5 b ) w a s
prepared by the above mentioned method.
-chlorobenzohydrazide (0.171g 0.001mol 1
equivalent)4-allyloxybenzaldehyde(0.15ml0.001mol
4
1
9
3
equivalent), pale yellow amorphous solid, yield:
2%. m.p. 158-160°C. FT-IR (umax ꢁBr pellets, cm )
434 or 3283 (N-H), 2930Ar (C-H) str, 2849 Ali (CH)
solid, yield:88%.m.p.168ÚC.FT-IR (u ꢁBr pellets,
max
-
1
-1
1
cm ) 937 (N=N), 1654 (C=O), 1526 (C=N). H NMR
(DMSO, 300 MHz) dH: 5.23- 5.24 (d, 2H), 5.89(s,
1H), 4.61 (2H), 6.8(d, 2H), 7.5(d, 2H), 8.1(s, 1H),
str, 1652 (C=O) str, 1506 (C=N) str, DMSO, 300
MHz) dH: 5.2-5.3 (d, d, 2H1.5Hz), 5.9-6.1 (CH 1H
m), 4.63 (d 2H, 5.1 Hz), 7.0(d, 2H, 8.7 Hz), 7.61 (d,
13
8(s, 1H), 7.83(d, 2H), 7.24(d, 2H), 2.35(S, 3H).
C
NMR (DMSO 300 MHz) 115, 137, 75, 164, 114, 130,
123, 154, 127, 130.5, 129, 141 and 20. m/z = 292
(M+1). Molecular Formula C H N O .
2
H, 8.7Hz), 5.3-5.4 (d, d 1.5-1.8Hz, 1H), 8.38 (1H,
13
s), 7.9 (d, 2H, 8.7Hz), 7.6 (d, 2H, 9 Hz). C NMR
DMSO 300 MHz) 161, 159, 147 136, 133, 132,
29, 128, 128, 117, 115 and 68. m/z = 315 (M+1).
18
18
2
2
(
1
4-methoꢃy-benzoic acid (4-allyloꢃy-
benylidenehydrazide (5d) was prepared
by the above mentioned method. 4-methoxy
Molecular Formula C H ClN O .
17
15
2
2
Table 2b: ꢀocking results of 4-chloro-benzoic acid
4-allyloꢃy-benzylidene)-hydrazide with the receꢂtor
(
pose ꢁ.E
L.E
IC
nM)
Int.E
Vdw
Elec. Total. Tor.E Unb. No. of Hydrogen bond
(
E
IE
E
H bonds
1
2
-11.87 -0.54 1.99 -13.66 -13.66 -0.01 -0.85 1.79
-11.31 -0.51 5.09 -13.1 -13.11 0.01 -0.81 1.79
-0.85
3
ꢀig1:O::4oim:
Ala201:Oꢀig1:N:
:
4oim:Ala201:
Oꢀig1:N::4oim:
Ala201:O
ꢀig1:O::Gly205:
Oꢀig1:N::Ala201
-0.81
3
:
:
Oꢀig1:N:
Ala201:O
3
4
-11.07 -0.5 7.67 -12.86 -12.89 0.03 -0.83 1.79
-11.04 -0.5 8.12 -12.83 -12.86 0.04 -0.79 1.79
-0.83
-0.79
1
3
ꢀig1:O::Gly96:O
ꢀig1:O::Ala201:
Oꢀig1:O::Gly96:
Oꢀig1:N:
:
Met98:O
5
6
-11.01 -0.5 8.44 -12.8 -12.8
-10.54 -0.48 18.78 -12.33 -12.34 0.01 -0.63 1.79
0.0
-0.81 1.79
-0.81
-0.63
0
1
-
Met98:HN:
:
ꢀig1:O
7
8
9
1
-10.51 -0.48 19.66 -12.3 -12.36 0.05 -0.54 1.79
-10.42 -0.47 22.98 -12.21 -12.18 -0.03 -0.73 1.79
-0.54
-0.73
-0.8
0
1
1
3
-
ꢀig1:O::Ile194:O
ꢀig1:N::Ala201:O
Ala157:HN::ꢀig1
-9.86 -0.45 58.91 -11.65 -11.63 -0.02 -0.8
1.79
0
-8.71 -0.4 414.7 -10.5 -10.47 -0.02 -0.78 1.79
-0.78
:
N,NAsn 159:
HD21::ꢀig1:
Oꢀig1:N:
:
Asn159:OD1

340
THERESE & GEETHAMAꢀIꢁA, Orient. J. Chem., Vol. 33(1), 335-345 (2017)
benzohydrazide (0.166g 0.001mol 1 equivalent)
-allyloxybenzaldehyde (0.15ml 0.001mol 1 (4-allyloxy-benzylidene)-hydrazide), (4-chloro-
equivalent), light yellow amorphous solid, yield: benzoic acid (4-allyloxy-benzylidene)-hydrazide),
Synthesized compounds (Benzoic acid
4
-
1
8
8%. m.p.162ÚC. FT-IR (umax ꢁBr pellets, cm ) were subjected to a preliminary anti bacterial
1
640 (C=N), 937 (N-N), 1410 (C-O), 2918 Ar (C-H). screening against the strain gram positive
1
H NMR (DMSO, 300 MHz) dH: 5.23- 5.24 (d, 2H), (Staphylococcus aureus) and Gram-negative
5
.89(s, 1H), 4.61 (2H), 6.8(d, 2H), 7.5(d, 2H), 8.1(s, (Escherichia coli) bacteria to assess the antibacterial
H), 8(s, 1H), 7.84(d, 2H), 6.95(d, 2H), 3.73(s, 3H). susceptibility of the test compounds.The compounds
C NMR (DMSO 300 MHz) 115, 137, 75, 164, 114, showed good inhibition at lower concentration.
30, 123, 154, 125, 128, 114, 165 and 56.m/z = 292
1
1
3
1
(
M+1). Molecular Formula C H N O .
It is evident from table-1 that the compound
18
18
2
2
5
a exhibits maximum anti-tuberculosis activity
4
- hydroꢃy -benzoic acid (4-allyloꢃy- among all the study compounds as its probability of
benzylidene)- hydrazide (5e) was prepared being active (Pa) value is 0.831 and Probability of
by the above mentioned method. 4-hydroxy being inactive (Pi) is only 0.003. Also all the other
benzohydrazide (0.152g 0.001mol 1 equivalent) compounds demonstrate anti-tuberculosic activity
4
-allyloxybenzaldehyde (0.15ml 0.001mol 1 with Pa between 0.827 and 0.781. Hence, all the
equivalent), yellow amorphous solid, yield: 84%. study compounds were docked with the target
-
1
m.p. 178ÚC. FT-IR (umax ꢁBr pellets, cm ) 1535 Enoyl-[acyl-carrier-protein] reductase [NADH] from
1
(
C=C), 1625 (C=N), 3250 (N-H), 1658 (C=O).
H
Mycobacterium tuberculosis. The interaction of the
NMR (DMSO, 300 MHz) dH: 5.23- 5.24 (d, 2H), synthesized compound with the target was evaluated
5
.89(s, 1H), 4.61 (2H), 6.8(d, 2H), 7.5(d, 2H), 8.1(s, using Docking studies. The docking scores along
H), 8(s, 1H), 7.78 (d, 2H), 6.91 (d, 2H), 5 (S, 1H). with the other parameters for the different poses of
C NMR (DMSO 300 MHz) 115, 137, 75, 164, 114, the docked molecules are presented in Tables 2(a)
30, 123, 154, 126, 128, 115 and 160. m/z = 310 – 2(e).
1
1
3
1
(
M+1). Molecular Formula C H N O .
18 18 2 3
Table 2c: ꢀocking results of 4-methyl-benzoic acid
4-allyloꢃy-benylidene)- hydrazide with the receꢂtor
(
pose ꢁ.E
L.E
IC
Int.E
Vdw Elec. Total. Tor.E Unb.E No. of
Hydrogen
(nM)
E
IE
H bonds bond
1
-11.37 -0.52 4.64 -13.46 -13.48 0.03
-0.8
2.09
-0.8
2
ꢀIG1:N:
:
:
MET98:OꢀIG1
N::MET98:O
2
3
4
-11.17 -0.51 6.48 -13.26 -13.25 -0.01 -0.85 2.09 -0.85
-10.69 -0.49 14.59 -12.78 -12.78 0.0 -0.85 2.09 -0.85
-10.61 -0.48 16.73 -12.7 -12.68 -0.02 -0.91 2.09 -0.91
0
0
2
-
-
ꢀIG1:O:
:
MET98:
OMET98:HN:
ꢀIG1:O
:
5
6
-10.4 -0.47 23.61 -12.49 -12.47 -0.03 -0.92 2.09 -0.92
-10.3 -0.47 28.01 -12.39 -12.34 -0.05 -0.83 2.09 -0.83
0
1
-
IꢀE194:HN:
:
ꢀIG1:O
7
8
-10.02 -0.46 45.14 -12.11 -12.08 -0.02 -0.94 2.09 -0.94
-9.98 -0.45 48.37 -12.07 -12.01 -0.05 -1.09 2.09 -1.09
0
1
-
TYR158:HH:
:
ꢀIG1:O
9
1
-9.74 -0.44 72.73 -11.83 -11.72 -0.11 -1.05 2.09 -1.05
-9.7 -0.44 77.03 -11.79 -11.76 -0.03 -0.94 2.09 -0.94
0
0
-
-
0

THERESE & GEETHAMAꢀIꢁA, Orient. J. Chem., Vol. 33(1), 335-345 (2017)
341
The docking results of Enoyl-[acyl-carrier-
protein] reductase [NADH] from Mycobacterium
tuberculosis with the ligand Benzoic acid (4-allyloxy-
benzylidene)-hydrazide is presented in table 2a.
The binding energies of the different binding
conformations range between -11.81 and -9.56 kcal/
mol. Among the ten different docked conformations
conformation 8 had a binding energy of -9.84 kcal/
mol with 2 hydrogen bonds between the ligand
Benzoic acid (4-allyloxy-benzylidene)-hydrazide
and Ala 201. The conformation 1, Conformation 5,
Conformation 7, Conformation 9 and Conformation
The table 2b presents the ten different
binding conformations of Enoyl-[acyl-carrier-protein]
reductase [NADH] from Mycobacterium tuberculosis
with the ligand 4-chloro-benzoic acid (4-allyloxy-
benzylidene)-hydrazide. The binding energy
ranges from -11.87 kcal/mol to -8.71 kcal/mol. The
conformations 1,2,4 and 10 had three hydrogen
bonds between the receptor, Enoyl-[acyl-carrier-
protein] reductase [NADH] and ligand 4-chloro-
benzoic acid (4-allyloxy-benzylidene)-hydrazide. In
the first binding conformation, all the three hydrogen
bonds are formed between the ligand and Alanine
201 in Enoyl-[acyl-carrier-protein] reductase [NADH]
protein. But in the second binding conformation two
hydrogen bonds are formed between the ligand and
Alanine 201 and the third hydrogen bond is formed
between the ligand and Glycine 205 in the receptor.
Alternatively in the binding conformation 4, Alanine
10 had only one hydrogen bond formed with binding
energies -11.81, -10.68, -10.33, -9.82 and -9.56
kcal/mol respectively. Hence, conformation 8 with 2
hydrogen bonds and binding energy of -9.84 kcal/
mol is considered the best docked conformation and
represented in figure-1
201, Glycine 96 and Methionine 98 in the receptor
Table 2d: ꢀocking results of 4-methoꢃy-benzoic acid
4-allyloꢃy-benylidene)- hydrazide with the receꢂtor
(
pose ꢁ.E
L.E
IC
nM)
Int.E
Vdw Elec. Total. Tor.E Unb.E No. of Hydrogen
IE H bonds bond
(
E
1
2
3
-11.89 -0.52
-10.96 -0.48
1.93 -13.98 -13.97 -0.01 -0.85 2.09
-0.85
-0.82
-0.85
1
1
2
ꢀIG1:N:
AꢀA201:O
ꢀIG1:O:
PRO156:O
ꢀIG:N::AꢀA201:
OꢀIG1:N:
:
9.29 -13.05 -13.09 0.04
-0.82 2.09
-0.85 2.09
:
-10.89 -0.47 10.41 -12.98 -12.98 0.0
-10.8 -0.47 12.02 -12.89 -12.89 0.0
:
AꢀA201:O
4
-0.91 2.09
-0.91
4
ꢀIG1:N::AꢀA201
:
:
OꢀIG1:O:
ASN106:
OD1ꢀIG1:O:
GꢀY96:OꢀIG1:
:
O::AꢀA201:O
5
6
-10.62 -0.46
16.4 -12.71 -12.67 -0.03 -0.87 2.09
-0.87
-0.93
0
1
-
-10.41 -0.45 23.36 -12.5 -12.46 -0.04 -0.93 2.09
ꢀIG1:O:
:
MET98:O
ꢀIG1:O:
PRO156:O
MET98:HN:
ꢀIG1:O
ꢀIG1:O:
AꢀA201:O
7
8
9
1
-10.16 -0.44 35.45 -12.25 -12.27 0.02
-9.7 -0.42 77.07 -11.79 -11.67 -0.12 -0.84 2.09
-9.41 -0.41 125.97 -11.5 -11.46 -0.04 -0.9 2.09
-8.98 -0.39 259.88 -11.07 -10.94 -0.13 -0.85 2.09
-0.73 2.09
-0.73
-0.84
-0.9
1
1
1
0
:
:
:
0
-0.85
-

342
THERESE & GEETHAMAꢀIꢁA, Orient. J. Chem., Vol. 33(1), 335-345 (2017)
form one hydrogen bond each with the ligand. binding energy and three hydrogen bonds is chosen
Finally, the conformation 10 had 3 hydrogen bonds as the best possible orientation which is displayed in
formed each between Alanine 157, Asparagine 159, Figure-2
Asparagine 159 and the ligand with a binding energy
of -8.71 kcal/mol. Among the ten different binding
The table 2c depicts the different binding
conformations, the conformation 1 with the least orientations of the ligand ꢁ1b with the receptor
Table 2e: ꢀocking results of 4- hydroꢃy -benzoic acid
(4-allyloꢃy-benylidene)- hydrazide with the receꢂtor
pose ꢁ.E
L.E
IC
nM)
Int.E
Vdw Elec. Total. Tor.E Unb.E No. of Hydrogen
IE H bonds bond
(
E
1
-11.35 -0.52
4.83 -13.14 -13.12 -0.02 -0.61 1.79
-0.61
1
ꢀIG:N:
AꢀA201:O
:
2
3
4
5
-10.26 -0.47 30.34- -12.05 -12.03 -0.02 -0.81 1.79
-10.25 -0.47 30.41 -12.04 -12.08 0.03 -0.81 1.79
9.97 -0.45 49.19 -11.76 -11.74 -0.02 -0.58 1.79
-9.95 -0.45 50.58 -11.74 -11.74 -0.01 -0.64 1.79
-0.81
-0.81
-0.58
-0.64
0
0
0
2
-
-
-
ꢀIG1:N::AꢀA201
:
:
OꢀIG1:O:
ASN106:OD1
6
7
8
-9.78 -0.44 67.28 -11.57 -11.59 0.02
-9.76 -0.44 70.34 -11.55 -11.62 0.07
-9.74 -0.44 72.95 -11.53 -11.49 -0.04 -0.77 1.79
-0.73 1.79
-0.79 1.79
-0.73
-0.79
-0.77
0
1
2
-
ꢀIG:O::MET98:O
ꢀIG1:O::MET98:
OMET98:HN:
:
ꢀIG1:O
ꢀIG1:N:
AꢀA201:O
9
1
-9.73 -0.44 73.19 -11.52 -11.53 0.01
-0.84 1.79
-0.84
-0.77
1
0
:
0
-7.1 -0.32 6.29 -8.89 -8.84 -0.05 -0.77 1.79
-
Table 2f: ꢀocking results of isoniazid with the receꢂtor
pose ꢁ.E
L.E
IC
nM)
Int.E
Vdw Elec. Total. Tor.E Unb.E No. of Hydrogen
IE H bonds bond
(
E
1
2
3
4
5
6
7
-6.73 -0.67 11.61 -7.03
-6.73 -0.67 11.69 -7.03 -6.99 -0.04 -0.09 0.3
-6.71 -0.67 12.11 -7.01 -6.97 -0.03 -0.09 0.3
-6.67 -0.67 12.94 -6.97 -6.98 0.01
-6.67 -0.67 13.01 -6.96 -6.98 0.02
-6.54 -0.65 16.15 -6.84 -6.73 -0.11 -0.09 0.3
-6.04 -0.60 37.35 -6.34 -6.26 -0.08 -0.09 0.3
-7.0 -0.04 -0.09 0.3
-0.09
-0.09
-0.09
-0.09
-0.09
-0.09
-0.09
1
1
1
1
1
0
2
Iso:O::Ile 202:O
Iso:O::Ile 202:O
Iso:O::Ile 202:O
Iso:O::Ala 201:O
Iso:O::Ala 201:O
-
Tyr158:HH::Iso:O
Ile194:HN::Iso:N
Tyr158:HH::Iso:
OIle194:HN:
-0.09 0.3
-0.09 0.3
8
-6.04 -0.60 37.62 -6.33 -6.25 -0.08 -0.09 0.3
-0.09
2
:
Iso:N
9
1
-6.00 -0.60 39.78 -6.30 -6.21 -0.09 -0.09 0.3
-5.83 -0.58 53.14 -6.13 -6.06 -0.07 -0.09 0.3
-0.09
-0.09
1
1
Ile194:HN::Iso:N
Ile194:HN::Iso:O
0

THERESE & GEETHAMAꢀIꢁA, Orient. J. Chem., Vol. 33(1), 335-345 (2017)
343
9
had one hydrogen bond between the receptor,
Table 3: Antimicrobial activity
data of 5a and5b by LpR assay
Enoyl-[acyl-carrier-protein]reductase [NADH] and
ligand 5e, whereas conformations 5 and 8 had two
hydrogen bonds. Among the ten different binding
conformations, the conformation 5 with binding
energy of -9.95 kcal/mol and two hydrogen bonds
is chosen as the best possible orientation which is
displayed in Figure 5
Comꢂ.
code
Conc.
(mL)
CFU
(col/mL)
%
inhibition
6
5a
10
3.2x10
19.1
0
0
2
5
0
0
The docking results of Enoyl-[acyl-carrier-
protein] reductase [NADH] from Mycobacterium
tuberculosis with the standard drug Isoniazid is
presented in table 2f. The binding energies of the
different binding conformations range between
-6.73 and -5.83 kcal/mol. Among the ten different
docked conformations conformation 7 and 8 show
2
100
00
6
5
b
4.4x10
97.9
98.7
6
2
4
00
00
-
2.4x10
6
Control-1
Control-2
5.8x10
5.0x10
-
-
6
-
2
hydrogen bonds between the drug and Tyr158
Control-1 -H37Rv
control-2 -H37Rv
and Ile194. The conformation 1, Conformation 2,
Conformation 3, Conformation 4 and Conformation
5
had only one hydrogen bond formed with binding
Enoyl-[acyl-carrier-protein] reductase [NADH] from
Mycobacterium tuberculosis. Poses 1 and 4 have
two hydrogen bonds formed by ‘O’ of Met98 and ‘N’
and ‘O’ atoms in the ligand. Poses 6 and 8 had one
hydrogen bond each between‘H’atom of Ile 194 and
Tyr 158 respectively with the ‘O’ atom of the ꢀigand.
Among the ten different binding conformations, the
conformation 1 with binding energy of -11.37 kcal/
mol and two hydrogen bonds is chosen as the best
possible orientation which is displayed in Figure 3.
energies -11.81, -10.68, -10.33, -9.82 and -9.56
kcal/mol respectively. Hence, conformation 8 with 2
hydrogen bonds and binding energy of -9.84 kcal/
mol is considered the best docked conformation and
represented in Figure-6
Synthesized compounds were evaluated
for antimycobacterial activity using ꢀuciferase
Reporter Phase (ꢀRP) assay on MTB H37Rv and
one isoniazid-resistant (INH-resistant) clinical isolate
at National Institute for Research in Tuberculosis
The table 2d presents the ten different
binding conformations of Enoyl-[acyl-carrier-protein]
reductase [NADH] from Mycobacterium tuberculosis
with the ligand 5d. The binding energy ranges from
(
NIRT), Chennai (28).
The percentage growth inhibition was
recorded at the concentrations of 200, 100 and 50
µg/mꢀ (Table 3). Seeing the good inhibition of the
synthesized compound further biological assays
were conducted to determine their MIC values on
different strains of Mycobacterium.
-
2
11.89 kcal/mol to -8.98 kcal/mol. The poses 1,
, 6, 7, 8 and 9 had one hydrogen bond between
the receptor, Enoyl-[acyl-carrier-protein]reductase
NADH] and ligand 5d. But pose 3 and 4 had 2 and
[
four hydrogen bonds between the receptor and
the ligand. Hence, among the ten different binding
conformations, the conformation 4 with the binding
energy of -10.8 and 4 hydrogen bonds is chosen as
the best possible orientation which is displayed in
Figure 4.
Results of these investigations indicate that
some of the test compounds exhibited significant
antimycobacterial activity along with antibacterial
activity against all the tested strains.
CONCLUSION
The table 2e presents the ten different
binding conformations of Enoyl-[acyl-carrier-protein]
reductase [NADH] from Mycobacterium tuberculosis
with the ligand 5e. The binding energy ranges from
A series of hydrazone Schiff base
analogues were synthesized in good yield adopting
standard procedures. The compounds were
-
11.35 kcal/mol to -7.1 kcal/mol.The poses 1, 7 and

THERESE & GEETHAMAꢀIꢁA, Orient. J. Chem., Vol. 33(1), 335-345 (2017)
344
characterized by spectral studies. Docking studies
of the above mentioned compounds revealed scope
for antibacterial activity especially anti-tuberculosis
activity. Further, all the study compounds exhibit a
binding better than the existing drug isoniazid for
Tuberculosis. This was further confirmed using in
vivo studies where compound 4-chloro-benzoic acid
(4-allyloxy-benzylidene)-hydrazide had maximum
inhibition (97.9%) 100µmꢀ. Hence, it can be
concluded from the study that compound 4-chloro-
benzoic acid (4-allyloxy-benzylidene)-hydrazide
can be further evaluated for its efficiency in treating
Tuberculosis.
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