Design, synthesis and molecular docking of substituted 3-hydrazinyl-3-oxo-propanamides as anti-tubercular agents

Article abstract of DOI:10.1016/j.bmcl.2014.09.080

Based on the anti-mycobacterial activity of various acid hydrazides, a series of substituted 3-hydrazinyl-3-oxo-propanamides has been designed. The target compounds have been synthesized from diethylmalonate using substituted amines and hydrazine hydrate

Full text of DOI:10.1016/j.bmcl.2014.09.080

Bioorganic & Medicinal Chemistry Letters 24 (2014) 5181–5184
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and molecular docking of substituted
3-hydrazinyl-3-oxo-propanamides as anti-tubercular agents q
⇑
Arshi Naqvi, Richa Malasoni, Akansha Srivastava, Rishi Ranjan Pandey, Anil Kumar Dwivedi
Division of Pharmaceutics, CSIR-Central Drug Research Institute, B 10/1, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226031, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on the anti-mycobacterial activity of various acid hydrazides, a series of substituted 3-hydrazinyl-
3-oxo-propanamides has been designed. The target compounds have been synthesized from
diethylmalonate using substituted amines and hydrazine hydrate in ethanol. Computational studies
and anti-tubercular activity screenings were undertaken to test their inhibitory effect on protein kinase
PknB from Mycobacterium tuberculosis. Binding poses of the compounds were energetically favorable and
showed good interactions with active site residues. Designed molecules obey the Lipinski’s rule of 5
and gave moderate to good drug likeness score. Among the sixteen compounds (1–16) taken for in silico
and in vitro studies, 3 compounds (11, 12 and 15) have shown good binding energies along with
exhibiting good anti-tubercular activity and thus may be considered as a good inhibitors of PknB.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 24 June 2014
Revised 29 August 2014
Accepted 25 September 2014
Available online 2 October 2014
Keywords:
Hydrazides
Molecular docking
Binding energy
Drug likeness score
Anti-tubercular
Mycobacterium tuberculosis (MTB) is considered to be the lead-
ing bacterial infection around the globe.1 With an estimated 8.7
million new tuberculosis (TB) cases (13% co-infected with HIV)
and 1.4 million fatalities each year,2 MTB causes more human
deaths than any other single infectious organism. The disease is
one of India’s most challenging public health problems and it
accounts for nearly one-third of the global burden. Approximately
2 million people acquire TB every year in India.3 Furthermore, MTB
strains resistance is emerging at an alarming rate to all of the first
line drugs (isoniazid, rifampicin, fluoroquinolone) and at least one
of three injectable second-line drugs (i.e., amikacin, kanamycin, or
capreomycin).4
Isonicotinic acid hydrazide (isoniazid, INH, Fig. 1) belongs to the
group of the first line antitubercular drugs which are in clinical
practice for more than 50 years. After the discovery of INH, the full
therapeutic possibilities of acid hydrazides were realized. Investi-
gations of other heterocyclic hydrazides having mono-cyclic nuclei
such as furan, thiophene, pyrrole and dicyclic nuclei such as
quinoline and isoquinoline was stimulated due to the remarkable
clinical value of INH.5 A large number of such substances have been
synthesized in pure form having differing ranges of curative
effects. With a view to establish the structural requirements for
antitubercular activity, Yale et al.6 reported the synthesis of a
number of hydrazides of the nicotinic acid hydrazide type.
A large number of hydrazides and their derivatives are reported
to possess a wide array of biological activities like antifungal,7
psychotropic,7 antituberculous,8–10 antiparasite,7,11 bacterio-
static,7,12–14 antiviral,14 insecticidal15 and anti-cancer16 activities.
Thus, these were found to be useful especially in the treatment
of inflammatory and autoimmune diseases, osteoarthritis,
respiratory diseases, tumors, cachexia, cardiovascular diseases,
fever, hemorrhage and sepsis.17
Protein kinase B (PknB) plays an important role in mammalian
cellular signaling. Mycobacterium tuberculosis PknB is an essential
receptor-like protein kinase involved in cell growth control. The
protein kinase B peptide contains two types of structural elements
(VAL 95, ARG 97) and basic residue ring constituted of glycine rich
residue. M. tuberculosis PknB is a trans-membrane Ser/Thr protein
kinase (STPK) highly conserved in Gram-positive bacteria and
apparently essential for mycobacterial viability.18 It was previously
shown that PknB is regulated by auto-phosphorylation and
de-phosphorylation by the Ser/Thr protein phosphatase PstP19,20
H
O
N
NH2
q
CDRI Communication No. 8813.
Corresponding author. Tel.: +91 522 2623405; fax: +91 522 2623938.
E-mail address: anilcdri@gmail.com (A.K. Dwivedi).
N
⇑
Figure 1. Structure of isoniazid.
http://dx.doi.org/10.1016/j.bmcl.2014.09.080
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

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A. Naqvi et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5181–5184
and work showed that PknB is predominantly expressed during
exponential growth, where it’s over expression causes morpholog-
ical changes linked to defects in cell wall synthesis and cell
division.21
All the compounds were prepared by Scheme 1. For synthesis,
substituted amines (I–XVI) were first refluxed with ethanolic
solution of diethyl malonate (XVII) for 4 h. Further refluxing
with hydrazine hydrate for another 5 h gave compounds 1–16
(58–88% yield, Table 1, Scheme 1). All the compounds (1–16) were
characterized spectroscopically. The base peak in ESI-MS was
found at M++1. In IR spectra, a strong band from 1538 to
1681 cmÀ1 indicated the presence of carbonyl of amide group.
Bands from 2806 to 2969 indicated the presence of methylene
group. In 1H NMR spectra of compounds 1–16, singlet peak
between 3.52 and 3.32 ppm, indicated two protons of the methy-
lene group (>CH2). Broad singlet between 4.32 and 4.20 ppm
was found indicating two protons attached to nitrogen atom
(–NH–NH2). Peaks between 8.91 and 6.60 ppm indicated the
presence of protons of aromatic ring of respective compounds.
Two broad singlets between 9.36 and 9.21 ppm were found
indicating –CONH– proton. 13C spectra clearly supported the
results obtained from 1H NMR spectra and also justified the
number of carbon atoms in corresponding compounds.
Therefore, there is an urgent need to develop new drugs against
MTB. To achieve this endeavor, we have synthesized some substi-
tuted malonamic acid hydrazides (1–16). The molecular docking
studies of newly synthesized compounds were carried out. Oral
bioavailability is a desirable property of compounds under investi-
gation in the drug discovery process. Lipinski’s rule-of-five is a
simple model to forecast the absorption and intestinal permeabil-
ity of a compound.22,23 The designed molecules obey the Lipinski
rule of five along with obeying some additional parameters.24
Compounds which were found promising in the docking study
were evaluated for their anti-mycobacterial activity by resazurin
micro-titre assay (REMA) method25 against M. tuberculosis H37Rv.
O
O
R
+
Table 2
H2N
O
O
Molecular docking results of the target compounds (1–16)
XVII
Mol. No.
Binding energy (kcal/mol)
Inhibition constant (
lM)
ClogP
I-XVI
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
À7.44
À6.33
À6.69
À6.78
À7.09
À6.98
À6.87
À7.03
À7.20
À6.66
À6.24
À6.88
À6.98
À7.01
À7.48
À6.46
3.53
22.87
12.48
10.79
6.38
7.68
9.21
7.01
5.25
13.13
26.51
9.04
7.59
7.32
0.80
À0.56
À0.10
À0.10
À0.63
1.37
i
1.25
O
O
À0.89
À0.89
0.82
1.55
1.02
H2NHN
N
H
R
À3.18
1-16
0.26
3.86
18.46
À0.36
À1.20
Scheme 1. Reagents and conditions: (i) [a] C2H2OH, reflux, 4 h. [b] NH2NH2ÁHCl,
reflux, 5 h.
Table 1
Characterization & anti-tubercular activity of substituted 3-hydrazinyl-3-oxo-propanamides (1–16)
O
O
S. No.
Molecular formula
Mp (°C)
% Yield
MICa
(lg/mL) H37Rv
N
H
H2NHN
R
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
3-Cl-4-F
2-NO2
3-NO2
C9H9N3O2FCl
C9H10N4O4
C9H10N4O4
C9H10N4O4
C9H9N5O6
C9H9N3O2Cl2
C9H9N3O2Cl2
C11H15N3O4
C11H15N3O4
C13H20N4O2
C13H19N3O2
C12H17N3O2
C9H11N3O5S
162–164
156–159
152–154
185–186
154–156
169–171
183–185
111–114
105–106
150–152
160–163
156–157
152–153
143–146
158–159
79.20
58.51
85.96
71.49
75.17
78.61
74.29
69.38
73.42
79.70
81.33
78.37
85.51
85.53
88.51
50
>100
>100
>100
>100
>100
>100
>100
>100
100
12.5
12.5
50
50
12.5
4-NO2
2,4-(NO2)2
3,5-(Cl)2
3,4-(Cl)2
2,5-(OCH3)2
2,4-(OCH3)2
4-N(C2H5)2
4-C4H9
4-C3H7
4-SO3H
2-COOH
4-COOH
O
C
C
10H11N3O4
10H11N3O4
O
O
H2
C
16
C8H11N3O3
151–153
60.58
>100
H2NHN
N
H
a
MIC INH: 0.02 lg/mL, RIF: 0.01 lg/mL.

A. Naqvi et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5181–5184
5183
Table 3
Lipinski’s rule of 5 with additional parameters
Mol. No.
Mol. wt
logPa
logSb [in log (moles/L)]
PSAc
72.08
HBAd
HBDe
Drug likeness score
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
245
238
238
238
283
262
262
253
253
264
249
235
273
237
237
197
0.10
À1.21
À1.09
À1.09
À1.54
0.66
À2.61
À1.45
À2.10
À2.08
À2.25
À3.07
À2.91
À1.39
À1.34
À3.21
À2.68
À2.47
À0.59
À1.10
À1.52
À0.48
3
5
5
5
7
3
3
5
5
3
3
3
6
5
5
4
4
4
4
4
4
4
4
4
4
4
4
4
5
5
5
4
À0.17
À1.04
À1.47
À1.02
À1.56
À0.91
À0.01
À0.56
À0.84
À0.34
0.59
104.46
105.46
105.46
137.84
72.08
72.08
86.56
86.56
74.82
72.08
72.08
114.49
99.79
100.49
81.15
0.54
À0.70
À0.70
0.29
0.94
0.35
À2.61
À1.18
À1.06
À1.64
0.60
0.37
0.34
0.72
À1.13
a
b
c
Solubility parameter.
Calculated lipophilicity.
Polar surface area (Å2).
d
e
Number of hydrogen bond acceptor.
Number of hydrogen bond donor.
Figure 2. Docking of active compounds (11, 12 and 15) into active site of PknB of Mycobacterium tuberculosis.

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A. Naqvi et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5181–5184
After the achievement of synthesis and characterization of the
revealed that compounds 11, 12 and 15 have minimum binding
energies with good drug likeness scores and may be considered
as a good inhibitors of PknB. Thus compounds 11, 12 and 15 have
emerged as most active amongst all the tested compounds against
Mycobacterium tuberculosis H37Rv.
compounds, we performed in silico studies. In order to expose the
specificity of the Protein Kinase PknB from Mycobacterium
tuberculosis towards the target compounds, docking approach was
carried out. Docking was used to predict the binding orientation
of drug to their protein target in order to in-turn predict the affinity
& activity of drug which includes docking of ligand to a set of grids
describing the target protein. The target compounds were docked
into the nucleotide-binding pocket of the M. tuberculosis PknB
structure (PDB: 2FUM). A Lamarckian genetic algorithm method,
implemented in the program AutoDock 4.0, was employed. The
docking of the ligand molecules (1–16) reveals that all the inhibitor
compounds are exhibiting the bonding with one or the other amino
acids in the active pockets. Theoretically all the sixteen molecules
showed very good binding energy ranging from À6.24 kcal/mol to
À7.48 kcal/mol (Table 2). Compound nos. 11 and 12 had shown
minimum binding energies of À6.24 and À6.88 kcal/mol with inhi-
bition constant 26.51 and 9.04, respectively. The ClogP value of
compound 11 is 1.55 and that of compound 12 is 1.02. The com-
pound 15 had shown minimum binding energy of À7.48 kcal/mol
with inhibition constant 3.86 and ClogP value of À0.36. The drug
candidates had shown favorable drug-like properties and follow
the Lipinski’s rule of 5 with additional parameters predicted by
Molsoft (Table 3). Thus, they are potentially interesting for further
optimization. Their drug likeness score ranges from À1.56 to 0.72.
Out of them the drug likeness score of 11 and 12 is 0.59 and 0.60,
respectively, with almost same PSA value of 72.08, 3 hydrogen bond
acceptors and 4 hydrogen bond donors. The drug likeness score of
compound no. 15 is 0.72 with approximate molecular weight of
237, logP value of À1.06, logS value of À1.52, PSA value of
100.49, 5 hydrogen bond acceptors and 5 hydrogen bond donors.
With in silico results in hand, it was thought worth-while to do
in vitro studies to support the in silico studies. The anti-mycobacte-
rial activity of the compounds (1–16) was tested and the minimum
inhibitory concentration (MIC) was determined using REMA
(resazurin microtitre assay) method.25 Among the tested com-
pounds, N-(4-butylphenyl)-3-hydrazinyl-3-oxopropanamide(11),
3-Hydrazinyl-3-oxo-N-(4-propylphenyl)propanamide(12) and
4-(3-hydrazinyl-3-oxo-propanamido)benzoic acid (15) have shown
good activity, N-(3-chloro-4-fluorophenyl)-3-hydrazinyl-3-oxo-
propanamide (1), 4-(3-Hydrazinyl-3-oxopropanamido) benzene
sulfonic acid (13) and 2-(3-Hydrazinyl-3-oxopropanamido)benzoic
acid (14) have shown moderate activity while rest of the com-
Acknowledgments
Authors are thankful to staff members of SAIF, CDRI for
the spectral data, Council of Scientific and Industrial Research,
University Grant Commission, Ministry of Health and Family
Welfare, Government of India, New Delhi, for providing financial
assistance. We are thankful to Jalma Institute of leprosy and other
mycobacterial diseases, Agra, India for anti-tubercular activity and
BioDiscovery-Solutions for future for docking studies.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.09.080.
References and notes
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pounds have MIC P100 lg/ml (Table 1).
Compound no. 11 and 12 are structurally very similar and so their
drug likeness scores, that is, 0.59 and 0.60, respectively with almost
same PSA value, hydrogen bond acceptors and donors. The com-
pound bearing butyl moiety has shown minimum binding energy
of À6.28 kcal/mol whereas the minimum binding energy is
À6.88 kcal/mol in case of isopropyl chain. Thus substitutions with
alkyl chain (butyl and iso propyl) resulted in the increased anti-
mycobacterial activity. Presence of a –COOH at para position of the
aromatic ring also increased its activity both in silico and in vitro.
Binding poses of compound nos. 11, 12 and 15 are shown in Figure 2.
In summary, a series of substituted 3-hydrazinyl-3-oxo-propan-
amides were designed and synthesized. Their inhibitory effects on
PknB of Mycobacterium tuberculosis were evaluated using in silico
followed by in vitro studies. Molecular docking studies has
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