Oxadiazolo pyrrolidine carboxamides as enoyl-ACP reductase inhibitors: Design, synthesis and antitubercular activity screening

Article abstract of DOI:10.1007/s00044-012-0340-3

Pyrrolidine carboxamides have been recognized as potent direct enoyl-acyl carrier protein reductase inhibitors. In our study, the search for new pyrrolidine carboxamides incorporated with various heterocyclic moieties, possessing antitubercular activities is been done. A series of oxadiazolyl pyrrolidine carboxamides (2a-e) were synthesized by reacting pyrrolidine carboxylic acid and oxadiazole amines using HBTU as amide forming agent. The purity of the synthesized compounds was confirmed by physical characterization like melting point and thin layer chromatography. The functional group identification was done based on spectral characterization utilizing, FT-IR, ¹H NMR, ¹³C NMR, mass spectral data and elemental analysis. Invitro antitubercular screening was performed by alamar blue assay method on mycobacterium tuberculosis H37Rv.

Full text of DOI:10.1007/s00044-012-0340-3

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:3428–3433
DOI 10.1007/s00044-012-0340-3
ORIGINAL RESEARCH
Oxadiazolo pyrrolidine carboxamides as enoyl-ACP reductase
inhibitors: design, synthesis and antitubercular activity screening
George Sonia Thengungal Kochupappy Ravi
•
Received: 29 May 2012 / Accepted: 7 November 2012 / Published online: 23 November 2012
Ó Springer Science+Business Media New York 2012
Abstract Pyrrolidine carboxamides have been recognized
as potent direct enoyl-acyl carrier protein reductase inhib-
itors. In our study, the search for new pyrrolidine carbox-
amides incorporated with various heterocyclic moieties,
possessing antitubercular activities is been done. A series of
oxadiazolyl pyrrolidine carboxamides (2a–e) were synthe-
sized by reacting pyrrolidine carboxylic acid and oxadiaz-
ole amines using HBTU as amide forming agent. The purity
of the synthesized compounds was confirmed by physical
characterization like melting point and thin layer chroma-
and the prevalence of tuberculosis as opportunistic infec-
tion associated with AIDS have made the situation even
worse (Espinal and Dye, 2005; Mondal and Jain, 2007).
Therefore, it is essential to identify a potential antituber-
cular lead to combat the present scenario. On drug dis-
covery perspective, one of the key targets for antitubercular
drug research is the enzymes involved in cell wall bio-
synthesis. Mycolic acid biosynthesis is essential for the
building up of cell wall in mycobacteria and related species
(Kolattukudy et al., 1997). The first line antitubercular
drug, isoniazid (INH) owes its mechanism of action
attributed to the inhibition of the biosynthesis of mycolic
acid (Winder and Collins, 1970). The enzyme, enoyl-acyl
carrier protein (ACP) reductase of the Mycobacterium
tuberculosis, encoded by the gene InhA, of the type II fatty
acid biosynthesis pathway (FASII) is identified as one of
the key enzyme for the biosynthesis of mycolic acids
(Banerjee et al., 1994). Though INH has its mechanism of
action attributed to the inhibition of InhA, it requires initial
activation by the catalase peroxidase enzyme, KatG (Richa
et al., 2003). The activated INH binds covalently to the
nicotinamide ring of the NADH bond within the active
site of the enzyme forming a tight INH–NADH adduct
(Rozwarski et al., 1998). But the effectiveness of INH as an
enoyl-ACP reductase inhibitor has not been well appreci-
ated due to the resistance developed by the organism owing
to the mutations in KatG (Chih-Jen et al., 2003).Conse-
quently the developments of new leads that can directly
inhibit InhA by bye-passing this activation step, has
promising potential as novel drugs for combating tuber-
culosis. Thus, the research is now focused on the devel-
opment of new Mtb InhA enoyl-ACP reductase inhibitors.
Triclosan prodrugs, pyrrolidine carboxamides, pyrazole
derivatives, etc., are now identified as having Mtb
InhA enoyl-ACP reductase inhibition (Heath et al., 1998;
tography. The functional group identification was done
1
based on spectral characterization utilizing, FT-IR,
H
1
3
NMR, C NMR, mass spectral data and elemental analysis.
Invitro antitubercular screening was performed by alamar
blue assay method on mycobacterium tuberculosis H37Rv.
Keywords Pyrrolidine carboxamides Á Oxadiazole Á
Docking Á Enoyl-acyl carrier protein (ACP) Á
Reductase inhibitors Á Antitubercular activities
Introduction
Tuberculosis is estimated as one among the most dreaded
diseases affecting mankind. Despite the ravaging increase
of tuberculosis worldwide, no new effective drug has
struck the market for the past 40 years. The re-emergence
of tuberculosis as multidrug resistant tuberculosis (MDR-
TB) and extensively drug resistant tuberculosis (XDR-TB)
G. Sonia (&) Á T. K. Ravi
Department of Pharmaceutical Chemistry, College of Pharmacy,
Sri Ramakrishna Institute of Paramedical Sciences, Coimbatore
641 044, India
e-mail: soniashenjeev@gmail.com
1
23

Med Chem Res (2013) 22:3428–3433
3429
Kuo et al., 2003). 1, 3, 4-Oxadiazoles are well known
compounds exhibiting antitubercular activity (Maria et al.,
(http://www.rcsb.pdb.org). The enzyme was refined to
remove all hetero atoms and water and Kollmann charges
were added. The optimized ligand structures were energy
minimized, Gasteiger charges and polar hydrogens were
added and torsion was set.
2
005; Foks et al., 2004).
Though afore said compounds are identified as possessing
antitubercular activity by inhibiting enoyl-ACP reductase
enzyme, none of them are into the market and are still in the
research phase. Therefore, we had undertaken the design and
synthesis of direct enoyl-ACP reductase inhibitors. Thus this
study involves the design of novel oxadiazole incorporated
pyrrolidine carboxamides, as possible enoyl-ACP reductase
inhibitors, and their invitro antitubercular activity screening
through alamar blue assay technique.
Docking simulations
The energy minimized ligands were subjected to docking
studies in order to predict its interaction with the key
binding sites on the enzyme. Initially Grid map was set with
˚
60 points and a spacing of 0.375 A was set. Lamarckian
genetic algorithm was used, with a maximum number of
2
5
5,000,000 energy evaluations and a maximum number of
,000 generations, for each run and 150 docking runs were
Materials and methods
performed. The log p values and the binding energies
obtained through in silico studies are depicted in Table 1.
The snap shots of the docked structures are shown in Fig. 1.
Insilico studies
Virtual screening was performed utilizing iGEMDOCK v.2
(
http://gemdock.life.nctu.edu.tw/dock/). The hits were
Chemistry
selected from the ZINC database (http://zinc.docking.org/)
and the optimization of the lead was done utilizing
Molinspiration server and Accelerys Accord for Excel
The melting points reported in the study were determined
on a MR-VIS (MR08190508) instrument. IR spectra were
done on KBr pellets on Shimadzu FTIR 8400S spectro-
(http://www.molinspiration.com/cgi-bin/properties; Accelerys
Software Inc., San Diego.) Auto Dock 4.2 was the software
used for docking (http://autodock.scripps.edu/).
1
13
photometer. H NMR and C NMR spectra were recorded
on a Bruker 300 MHz NMR spectrometer. Mass spectra
were obtained on a Shimadzu LCMS-2010 EV. The ele-
mental analysis was done on VarioEL III CHNS and the
analysis data (C, H, N) were within the range of ± 0.4 % of
the calculated values. Completion of the reaction was
monitored by thin layer chromatography on perforated
sheets of silica gel-G using iodine vapor for detection.
The synthetic pathway is enumerated in the Scheme 1.
The pyrrolidine-3-carboxylic acid 1 was formed by the
reaction of itaconic acid and aniline, when the ring closure
taken place has resulted in the pyrrolidine ring formation
Selection of the target and lead
The target enzyme selected was enoyl-acyl protein reduc-
tase (InhA) and the hits were obtained from the zinc
database. The selection of lead from the hits was based on
the virtual screening technique using iGEMDOCK v.2. A
small molecular library of 10,000 hits was constructed
utilizing ZINC database and was docked into the enzyme
and the potential leads were discovered. Pyrrolidine car-
boxamide was selected as the potential lead.
(
Paytash et al., 1950). Prior to the synthesis of oxadiazole
amine, aryl semicarbazones were synthesized by the reac-
tion of aromatic aldehyde and semicarbazide hydrochloride
Lead optimization
(
Hania, 2009). This semicarbazones on reaction with bro-
The selected lead was substituted with various substituents
and the insilico lead optimization was done to evaluate its
pharmacokinetic profile. Drug likeness evaluated through
Molinspiration server showed the selected leads to be
orally bioavailable and the ADMEtox data evaluated
through Accelerys Accord for Excel predicted the designed
leads to have good pharmacokinetic profile.
mine in presence of sodium acetate underwent oxidative
cyclization through a nitrilimine intermediate, resulting
in the formation of oxadiazole amine 2a–e (Mulwad
and Chaskar, 2006). The peptide forming reagents like
Table 1 Log p value and binding energy of designed compounds
S. no
Comp code
Log p
Binding energy (kcal/mol)
Docking studies
1.
2.
3.
4.
5.
3a
3b
3c
3d
3e
4.632
4.853
2.386
4.325
3.692
-9.96
-8.28
-7.86
-9.89
-8.08
Enzyme and ligand preparation
The X-ray crystal structure of the enzyme Mtb InhA
was obtained from Protein Data Bank (PDB ID: 2H7I)
123

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Med Chem Res (2013) 22:3428–3433
benzaldehyde (2.12 g, 0.02 mol) in 15 ml ethanol was
added by stirring. After shaking for few minutes the mix-
ture was left undisturbed and the precipitate obtained was
collected and purified by recrystallization from ethanol.
The prepared semicarbazone (0.82 g, 0.005 mol) and
anhydrous sodium acetate (6.0 g, 0.073 mol) were made
into a slurry by adding 17.5 ml of acetic acid. To the slurry,
bromine (0.8 ml, 2.51 g, 0.016 mol) in acetic acid (5 ml)
was added drop wise by stirring. After addition, the stirring
was continued for another 2 h after which the slurry was
poured to cold water by occasional stirring. The solid
obtained was filtered, washed with water, dried, and puri-
fied by recrystallisation using absolute ethanol. m.p.:
2
39.5 °C. The rest of the derivatives were prepared in the
similar fashion.
Synthesis of 5-oxo-1-phenyl-N-(5-substitutedphenyl-1,3,4-
oxadiazol-2-yl)pyrrolidine-3-carboxamides (3a–e)
2-Amino-5-(4-substituted
phenyl)-1,3,4-oxadiazole
(0.0018 mol) and 5-oxo-1-phenyl pyrrolidine-3-carboxylic
acid (0.39 g, 0.0019 mol) were dissolved in 58 ml of dry
DMF. HBTU (0.87 g, 0.0023 mol) and DIEA (0.93 ml,
Fig. 1 Snap shots of the docked ligands 3a (a) and 3d (b) bound with
the InhA enzyme 2H7I; the ligands are represented in green and
hydrogen bond by dotted green lines; ligand is found bound with
Tyr158 and NAD (Color figure online)
0.0053 mol) were added and stirred for 5 h at 23 °C. The
reaction was quenched by adding brine. The resulting
mixture was extracted with ethyl acetate (3 9 50 ml). The
combined ethyl acetate layer was washed with 1N HCl then
with saturated sodium bicarbonate, followed by brine. The
organic layer was dried over anhydrous sodium sulphate.
The residue left out after concentrating the ethylacetate in
vacuo was recrystallised from dichloromethane methanol
mixture. The purity was established by single spot on the
TLC plates. The solvent system used was acetone:benzene
(4:6).
2
-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexa-
fluoro phosphate (HBTU) and diisopropyl ethylamine
DIEA) used in the reaction of pyrrolidine carboxylic acid
(
and oxadiazole amine, resulted in the formation of title
compounds, 5-oxo-1-phenyl-N-(5-substitutedphenyl-1,3,
4
-oxa-diazol-2-yl)pyrrolidine-3-carboxamides 3a–e.
Synthesis of 5-oxo-1-phenylpyrrolidine-3-carboxylic
acid (1)
A mixture of itaconic acid (13.10 g, 0.1 mol), aniline
5-Oxo-1-phenyl-N-(5-phenyl-1,3,4-oxadiazol-2-yl)
pyrrolidine-3-carboxamide (3a)
(
9.20 ml, 0.1 mol) and water (200 ml) were refluxed for
h or until the odor of amine became faint after which the
1
reaction mixture was chilled for 1 h. The product obtained
was filtered, washed with cold water and dissolved in dilute
sodium hydroxide. The clear solution was treated with
charcoal and filtered hot and acidified with dilute hydro-
chloric acid. The precipitate obtained was filtered, dried,
and recrystallised from water to obtain white needle like
crystals. m.p.: 189.4 °C.
Yield: 60 %; m.p. 236.4 °C. Spectroscopic analysis:
-
1
IR(KBr) mmax/cm
1,660.77, 1,597.11, 1,498.22, 1,074.39, 1,041.75, 744.55;
:
3,488.63, 3,113.21, 1,734.06,
1
H NMR (DMSO-d , 300 MHz,d ppm): 2.820–2.962 (dd,
6
2H, CH2 pyrrolidine), 3.614 (s, 1H, CH pyrrolidine),
4.136–4.273 (dd, 2H, CH pyrrolidine), 7.282–7.668 (m,
2
1
3
10H, Ar–H), 9.426 (s, 1H, NH amide); C NMR (DMSO-
d6, 300 MHz, d ppm): 38.61 (C3 of pyrrolidine), 45.63 (C4
of pyrrolidine), 53.89 (C5 of pyrrolidine), 123.18–134.85
(aromatic carbons), 138.65 (C1 of phenyl attached to N of
pyrrolidine), 156.51 (C5 of oxadiazole), 163.96 (C2 of
oxadiazole), 170.56 (C=O of amide),171.23 (C=O of pyr-
Synthesis of 2-amino-5-(4-substituted phenyl)-1,3,4-
oxadiazoles (2a–e)
To an aqueous solution of semicarbazide hydrochloride
?
(
1.11 g, 0.01 mol) and sodium acetate (8.203 g, 0.1 mol),
rolidine); (M?1) ion peak at 349.
1
23

Med Chem Res (2013) 22:3428–3433
3431
Scheme 1 Formation of
oxadiazolo pyrrolidine
O
OH
carboxamides by the reaction of
pyrrolidine carboxylic acid 1
and oxadiazole amines 2a–e
O
OH
NH2
N
H C
2
45-60min, reflux
O
+
HO
O
1
itaconic acid
aniline
5
-oxo-1-phenylpyrrolidine-3-carboxylic acid
O
O
NH
NH2
N
sodium acetate
H
+
H N
2
NH2
O
R
NH
R
aromatic aldehyde
semicarbazide
4- substituted phenyl semicarbazone
Br /AcOH,2h stirring
2
N
N
NH2
O
2a-e
R
5-(4-substituted-phenyl)-1,3,4-oxadiazol-2-amine
O
N
N
N
OH
N
NH2
NH
O
R
O
HBTU/DIEA/DMF
o
O
N
+
O
5
h,stirring ,23
N
R
5
-(4-substituted-phenyl)-1,3,4-oxadiazol-2-amine
O
3a-e
-oxo-1-phenyl-N-[5-(4-substituted phenyl)-1,3,4-oxadiazol-2-yl]pyrrolidine-3-carboxamide
R = H,Cl,CH ,OCH ,NO
5
3
3
2
N-[5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-yl]-5-oxo-1-
phenylpyrrolidine-3-carboxamide (3b)
oxadiazole), 169.96 (C=O of amide), 171.20 (C=O of
?
pyrrolidine); M ion peak at 382.
Yield: 56 %; m.p. 268.3 °C. Spectroscopic analysis:
IR(KBr) mmax/cm : 3,488.63, 113.28,1,734.06, 1,660.77,
N-[5-(4-Methylphenyl)-1,3,4-oxadiazol-2-yl]-5-oxo-1-
phenylpyrrolidine-3-carboxamide (3c)
-
1
1
,595.18, 1,487.17, 1,091.75, 1,074.39, 1,041.75,744.55;
1
H NMR (DMSO-d , 300 MHz, d ppm) : 2.355–2.972 (dd,
Yield: 57 %; m.p. 277.8. Spectroscopic analysis: IR(KBr)
1
6
-
2
H, CH2 pyrrolidine), 3.663 (s, 1H, CH pyrrolidine),
mmax/cm
1,595.18, 1,504.12, 1,091.75, 1,074.39, 1,031.95, 742.62;
:
3,380.97, 3,099.71, 1,725.91,1,654.98,
4
.236–4.573 (dd, 2H, CH pyrrolidine), 7.282–7.868 (m,
2
1
0H, Ar–H), 9.166 (s, 1H, NH amide); C NMR (DMSO-
3
1
1
H NMR (DMSO-d , 300 MHz, d ppm): 2.53 (s, 3H, CH
6
3
d6, 300 MHz,d ppm): 38.62 (C3 of pyrrolidine), 45.87 (C4
of pyrrolidine), 53.26 (C5 of pyrrolidine), 123.19–134.85
attached to phenyl ring),2.867–2.972 (dd, 2H, CH pyr-
2
rolidine), 3.663 (s, 1H, CH pyrrolidine), 4.035–4.573 (dd,
(aromatic carbons), 139.65 (C1 of phenyl attached to N of
pyrrolidine), 156.50 (C5 of oxadiazole), 163.96 (C2 of
2H, CH pyrrolidine), 7.282–7.845 (m, 9H, Ar–H), 8.943
2
?
(s, 1H, NH, amide); (M-1) ion peak at 361.
123

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Med Chem Res (2013) 22:3428–3433
N-[5-(4-Methoxyphenyl)-1,3,4-oxadiazol-2-yl]-5-oxo-1-
phenylpyrrolidine-3-carboxamide (3d)
Antitubercular alamar blue assay
The drug concentrations of 0.2–100 lg/ml in dimethyl
formamide (DMF) were made in serial twofold dilutions in
a 96-well microtitre plate using 100 ll 7H9-S. A growth
control with no drug and a sterility control without
inoculums were included in each plate. The wells con-
taining 100 ll of the diluted drug were inoculated with
0.005 ml of the 1:5-diluted culture and sealed in plastic
bags and kept for incubation at 37 °C. The control wells
were also inoculated in the same manner. Control tubes
were tested after 7 days by adding 0.02 ml of alamar blue
solution and 0.05 ml of 5 % Tween 20 solution and incu-
bating it for 2 h at 50 °C to determine whether the drug-
containing tubes were ready to be read. After the color
change was observed from blue to pink after 2 h of incu-
bation, then tween20 and alamar blue were added to the
drug-containing wells and these were incubated for 2 h at
50 °C before the MIC was read. The MIC was defined as
the lowest concentration of drug that prevented a color
change. Isoniazid was used as the positive control. The
MIC data is depicted in Table 2.
Yield: 64 %; m.p. 240.9. Spectroscopic analysis: IR(KBr)
-
1
mmax/cm
,606.76, 1,504.53, 1,076.32), 1,022.31, 742.62; H NMR
DMSO-d , 300 MHz, d ppm): 2.533–2.972 (dd, 2H, CH2
:
3,323.46, 3,134.43, 1,725.99, 1,654.98,
1
1
(
6
pyrrolidine), 3.666 (s, 1H, CH pyrrolidine),4.235 (s, 3H,
OCH attached to phenyl ring) 4.266–4.831 (dd, 2H, CH2
3
pyrrolidine), 7.272–7.955 (m, 9H, Ar–H), 9.328 (s, 1H, NH
?
amide); M ion peak at 378.
N-[5-(4-Nitrophenyl)-1,3,4-oxadiazol-2-yl]-5-oxo-1-
phenylpyrrolidine-3-carboxamide (3e)
Yield: 70 %; m.p. 212.2. Spectroscopic analysis: IR(KBr)
-
1
mmax/cm
:
3,311.26, 3,107.43, 1,730.21, 1,658.84,
1
,597.11, 1,489.10, 1,444.73, 1,074.39, 1,049.31, 740.69;
1
H NMR (DMSO-d , 300 MHz, d ppm): 2.406–2.685 (dd,
6
2
H, CH2 pyrrolidine), 2.936 (s, 1H, CH pyrrolidine),
4
9
3
.234–4.489 (dd, 2H, CH pyrrolidine), 7.379–7.826 (m,
2
?
H, Ar–H), 9.138 (s, 1H, NH amide); (M-1) ion peak at
92.
Biological activity
Results and discussion
Antitubercular activity
This study involved the design of ligands as M. tubercu-
losis enoyl-acyl carrier protein reductase (InhA) inhibitors.
Virtual screening technique utilized for the study helped in
selecting pyrrolidine carboxamide as the lead for inhibiting
M. tuberculosis enoyl-acyl carrier protein reductase (InhA).
Aiming to obtain ligands of good pharmacokinetic profile,
the pyrrolidine carboxamides were substituted with various
substitutions and their insilico lead optimization was done.
The synthesized compounds were screened for their
in vitro antitubercular activity against M. tuberculosis
H37Rv MTCC 300 by alamar blue assay method (Yajko
et al., 1995).
Cultivation of the organism
4-substituted aryl oxadiazole amines incorporated to the
Mycobacterium tuberculosis H37Rv was maintained on
Lowenstein–Jensen medium. 37.3 g of LJ medium was
suspended in 600 ml water containing 12.5 ml glycerol.
After autoclaving at 15 lbs for 15 min, it was mixed uni-
formly with whole egg emulsion. It was distributed in
screw capped tubes and arranged in slant position. The
medium was coagulated in an insipissator or water bath at
pyrrolidine carboxylic acid was found to show good
pharmacokinetic profile. The optimized ligands were
docked to the binding sites of the enzyme InhA to evaluate
their binding affinity. The docking results revealed the
successful binding of the ligands with the key binding sites
Tyr158 and NAD (Xin et al., 2006). The best binding
8
5–90 °C for 45 min. Then the organism was inoculated
Table 2 Antitubercular activity of compounds synthesized
and kept for growth for 21 days at 37 °C.
S. no
Comp code
MIC (lg/ml)
Preparation of inocula for antimycobacterial screening
1.
2.
3.
4.
5.
6.
a
3a
3b
3c
3d
3e
12.5
25
Inocula were prepared by growing strains of M. tubercu-
losis (maintained on LJ medium) in 7H9 broth (Middle-
brook 7H9 broth base supplemented with 0.5 ml of Tween
50
3.125
25
8
0 and 10 % ADC enrichment per liter) to the turbidity
a
Std
0.25
equal to that of a no. 1 McFarland standard and diluting the
culture to 1:5 in broth.
INH
1
23

Med Chem Res (2013) 22:3428–3433
3433
affinity was shown by 3a and 3d with a binding energy of
-
of some (4-phenylpiperazin-1-ylmethyl)-1,3,4-oxadiazole and
(4-phenylpiperazin-1-ylmethyl)-1,2,4-triazole derivatives. Acta
Pol Pharm 61(6):473–476
9.96 and -9.89 kcal/mol. The binding energy of standard
ligand, cyclohexyl pyrrolidine carboxamide was found to
be -8.89 kcal/mol. When oxadiazole ring was incorpo-
rated in the place of earlier reported phenyl ring, in the
formation of pyrrolidine carboxamides, the binding affinity
of the ligand to the enzyme was found to be improved. The
designed series of oxadiazolyl pyrrolidine carboxamides
were synthesized by the condensation of aryl substituted
oxadiazole amines with pyrrolidine carboxylic acid. All the
derivatives were obtained in good yields and were char-
acterized by the spectral data. The antitubercular activity
screening revealed that compounds 3a and 3d showed
significant activity with MIC of 12.5 and 3.125 lg/ml,
respectively. The derivatives 3b and 3e showed moderate
activity with MIC of 25 lg/ml.
In conclusion, the new series of oxadiazolo pyrrolidine
carboxamides, designed as enoyl-ACP reductase inhibitors,
were synthesized and were screened for their antitubercular
potential. The derivatives showed significant activity. The
activity profile obtained, revealed that the incorporation of
oxadiazole moiety in the pyrrolidine carboxylic amides has
shown significant effect. The optimum substitution at dif-
ferent positions of the oxadiazole can result in the devel-
opment of more potent compounds.
Hania MM (2009) Synthesis and antibacterial activity of some
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