Microwave-assisted synthesis, molecular docking and antitubercular activity of 1,2,3,4-tetrahydropyrimidine-5-carbonitrile derivatives

Article abstract of DOI:10.1016/j.bmcl.2012.10.032

Based on bioisosteric similarities with isoniazid, a series of 1,2,3,4-tetrahydropyrimidine-5-carbonitrile derivatives has been designed. The target compounds have been synthesized by multicomponent reaction which involves one-pot organic reactions using ethylcyanoacetate, urea/thiourea and arylaldehydes in presence of ethanolic K₂CO₃. Two methodologies, conventional and microwave-assisted, have been adopted for the synthesis. The later strategy gave high yields in less than 10 min as compared to long hours using the former approach. Molecular docking of the target compounds into the enzyme Mycobacterium tuberculosis enoyl reductase (InhA) revealed important structural information on the plausible binding interactions. Major binding interactions were found to be of dispersion type (residues Tyr158, Ile215, Met103 and Met199) and a hydrogen bond with Tyr158. Binding poses of the all the compounds were energetically favorable and showed good interactions with the active site residues. Few selected compounds were also evaluated for antitubercular activity in vitro against drug-sensitive M. tuberculosis H37Rv strain and clinically isolated S, H, R and E resistant M. tuberculosis by luciferase reporter phage (LRP) assay method. Some compounds displayed promising antimycobacterial activity comparable or less than the standard drugs isoniazid and rifampicin.

Full text of DOI:10.1016/j.bmcl.2012.10.032

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7539–7542
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Microwave-assisted synthesis, molecular docking and antitubercular activity
of 1,2,3,4-tetrahydropyrimidine-5-carbonitrile derivatives
Sahoo Biswa Mohan a, , B. V. V. Ravi Kumar a, S. C. Dinda b, D. Naik a, S. Prabu Seenivasan c, Vanaja Kumar c,
⇑
Dharmarajsinh N. Rana d, Pathik S. Brahmkshatriya e
a Department of Pharmaceutical Chemistry, Roland Institute of Pharmaceutical Sciences, Khodasinghi, Berhampur 760 010, India
b School of Pharmaceutical Education and Research, Berhampur University, Odisha, India
c Tuberculosis Research Centre, V.R. Ramanathan Road, Chennai 600 031, India
d Department of Chemistry, Gujarat University, Ahmedabad, India
e Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic and Center for Biomolecules and Complex Systems, Flemingovo nám. 2, 166 10, Prague 6,
Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on bioisosteric similarities with isoniazid, a series of 1,2,3,4-tetrahydropyrimidine-5-carbonitrile
derivatives has been designed. The target compounds have been synthesized by multicomponent reac-
tion which involves one-pot organic reactions using ethylcyanoacetate, urea/thiourea and arylaldehydes
in presence of ethanolic K2CO3. Two methodologies, conventional and microwave-assisted, have been
adopted for the synthesis. The later strategy gave high yields in less than 10 min as compared to long
hours using the former approach. Molecular docking of the target compounds into the enzyme Mycobac-
terium tuberculosis enoyl reductase (InhA) revealed important structural information on the plausible
binding interactions. Major binding interactions were found to be of dispersion type (residues Tyr158,
Ile215, Met103 and Met199) and a hydrogen bond with Tyr158. Binding poses of the all the compounds
were energetically favorable and showed good interactions with the active site residues. Few selected
compounds were also evaluated for antitubercular activity in vitro against drug-sensitive M. tuberculosis
H37Rv strain and clinically isolated S, H, R and E resistant M. tuberculosis by luciferase reporter phage
(LRP) assay method. Some compounds displayed promising antimycobacterial activity comparable or less
than the standard drugs isoniazid and rifampicin.
Received 28 April 2012
Revised 24 September 2012
Accepted 5 October 2012
Available online 13 October 2012
Keywords:
Antitubercular
Binding interactions
Luciferase reporter phage (LRP) assay
Microwave-assisted
Molecular docking
Ó 2012 Elsevier Ltd. All rights reserved.
Mycobacterium tuberculosis (MTB) is the underlying microor-
ganism causing tuberculosis (TB) in humans. It is considered as
the leading bacterial infectious agent.1 Tuberculosis usually attacks
the lungs but can also affect other parts of the body. It spreads
through the air by patient’s cough, sneeze, or spit.2 India accounts
for nearly one-third of the global burden of tuberculosis and the
disease is one of India’s most challenging public health problems.
Approximately 2 million people acquire TB every year in India.3
Furthermore, it is alarming to see the emergence of MTB strains
resistant to all of the first line drugs (leading to the multi drug
resistant TB, MDR–TB) and to isoniazid, rifampin, fluoroquinolone
and at least one of three injectable second-line drugs (i.e., amika-
cin, kanamycin, or capreomycin) (leading to the extensive drug-
resistant TB, XDR–TB).4 All the above facts reveal that there is an
urgent need for the development of new potent drug molecules
with unique divergent structure and novel mode of action which
can be effective against resistant strains of mycobacteria. Isonicot-
inic acid hydrazide (isoniazid, INH) belongs to the group of the first
line antitubercular drugs being in clinical practice over 50 years.
INH is believed to kill mycobacteria by inhibiting the biosynthesis
of mycolic acids, critical component of the cell wall.5 Thus, it was
thought of interest to replace the pyridine ring of INH with its
pyrimidine bioisostere (Scheme 1). This bioisosteric replacement
followed molecular docking of the designed compounds into crys-
tal structure of Mycobacterium tuberculosis enoyl reductase (InhA)
(PDB Code 2H7I).6 The present enzyme was chosen over a large
number of other enoyl reductases as it has better resolution
(1.62 Å) over many others (>2.0 Å). Compounds which were
found promising in the docking study were evaluated for their
antimycobacterial activity by luciferase reporter phage (LRP) assay
method against M. tuberculosis H37Rv and clinically isolated S, H, R
and E resistant M. tuberculosis at two concentrations (100 and
500 lg/ml).
Multi-component reactions (MCRs) constitute a highly valuable
synthetic tool for the construction of polyfunctionalized heterocy-
clic compounds required for drug discovery programmes.7,8 Thus, a
series of 1,2,3,4-tetrahydropyrimidine-5-carbonitrile derivatives
were synthesized based on multicomponent reaction (MCR) which
involves one-pot organic reactions (Scheme 1).9 This reaction
⇑
Corresponding author. Tel.: +91 9040442719.
E-mail address: biswamohan81@gmail.com (S.B. Mohan).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.10.032

7540
S. B. Mohan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7539–7542
O
CHO
NC
O
X
K2CO3, ethanol
NH
NC
+
+
R
OC2H5
H2N
NH2
conventional (reflux, 4-8 h)
or
MW (210W, 5-10 min)
N
H
X
R
1: X = O
2: X = S
Scheme 1. Synthetic route for the synthesis of title compounds.
satisfactory rmsd values (<2 Å), Glide pose was better
(rmsd = 0.47 Å, Fig. 1a). Hence, we used Glide XP protocol for dock-
ing the target compounds. Since, all the target compounds
contained the same core moiety (1,2,3,4-tetrahydropyrimidine-5-
carbonitrile) with very little modifications on the phenyl rings,
the binding energy (Glide-XP-Score) were found to be very similar
(À5 to À6, Table s1, Supplementary data). We know from the
crystal geometry of 2H7I ligand (1-cyclohexyl-5-oxo-N-pheny-
pyrrolidine-3-carboxamide) that the interactions are chiefly of dis-
persion type. The ligand is buried inside a hydrophobic pocket and
is less exposed to solvent. The cavity is made up of some nonpolar
amino acids like Tyr158, Ile215, Met103 and Met199. The ligand
also makes a hydrogen bond with Tyr158. All these interactions
are shown in Figure 1a. Next, we analyzed the binding interactions
of the target compounds with the neighboring residues. It was
encouraging to see that the ligand occupied essentially the same
pocket and preferentially also retained all the interactions men-
tioned above with some additional interactions. For example, com-
pound 2g showed an additional interaction, that is, halogen bond
with backbone oxygen of Pro156 (Fig. 1b). However, INH had a
very low score (À3.6). Hence, we thought of rationalizing the
low score of INH. Figure 1 shows the fit of 2g (panel c) and INH
(panel d) into the active site of the enzyme, which suggest that
the target compounds better fit the cavity as compare to a INH,
being much smaller in size.
Table 1
Chemical structures and physical properties of target compounds
O
NC
NH
N
H
X
R
Compd. No.
X
R
mp (°C)
Yield (%)
Time (min)
Ca
Mb
C
M
1a
1b
1c
1d
1e
1f
1g
1h
1i
2a
2b
2c
2d
2e
2f
O
O
O
O
O
O
O
O
O
S
S
S
S
S
S
S
H
4-F
275–280
255–260
290–295
223–228
235–241
277–281
285–288
290–293
248–252
285–287
195–200
185–190
275–280
205–208
250–252
242–245
61
61
60
59
61
57
58
57
56
66
65
63
61
61
58
59
83
81
81
79
82
78
80
79
77
85
85
82
80
83
79
81
240
360
300
420
360
300
360
480
480
240
360
300
420
360
300
360
5
7
6
8
7
7
9
10
8
5
9
7
8
6
7
8
2-Cl
3-Cl
4-Cl
3-Br
4-Br
4-OH
3-OH,4-OCH3
H
4-F
2-Cl
3-Cl
4-Cl
3-Br
4-Br
2g
a
C — Conventional.
M — Microwave.
b
After satisfactory results of the docking of target molecules into
the Mycobacterium tuberculosis enoyl reductase enzyme, we
selected a few candidates (which were found potent in silico) for
the evaluation of the antimycobacterial activity. LRP assay is a ra-
pid, inexpensive and less laborious method for high throughput
involves two mechanisms such as Knoevenagel condensation and
Michael addition. First, various arylaldehydes react with ethyl-
cyanoacetate in presence of ethanolic K2CO3 to produce an inter-
mediate by Knoevenagel condensation reaction. Then, the
intermediate reacts with urea/thiourea via Michael addition to
produce corresponding pyrimidine derivatives.10,11
screening of compounds for their antimycobacterial activity.15
A
compound is considered to be an antimycobacterial agent if 50%
reduction in the Relative Light Units (RLU) is observed when
compared to the control using a luminometer.16,17 Results of the
antimycobacterial activity are summarized in Table 2.
Table 1 represents the summary of all the compounds synthe-
sized. Clearly, the microwave approach proved to be fast and clean.
The yields were found to be quite high (75–85%) as compared to
conventional synthesis (55–65%). We believe that the most notice-
able advancement was the speed with which the reaction pro-
ceeded. Reactions under microwave were completed within just
5–10 minutes, being 50–70 times faster than the conventional
methodology.
Next, we were interested to investigate the biological profile of
the target compounds. Considering reasonable structural similar-
ity of the target compounds with the blockbuster antitubercular
agent INH, we decided to dock the target compounds into the ac-
tive site of the molecular target of INH, Mycobacterium tuberculosis
enoyl reductase (InhA). First, in order to identify proper docking
protocol, we independently used two different programs: Dock
6.512 and Glide.13 We docked the native ligand of Mycobacterium
tuberculosis enoyl reductase (PDB code 2H7I) and investigated
the root mean square deviation (rmsd) between the crystal geom-
etry and the docked pose. While both the docking programs gave
In summary, we designed a series of 1,2,3,4-tetrahydropyrimi-
dine-5-carbonitrile derivatives and synthesized them using con-
ventional and microwave-assisted one pot multicomponent
synthesis. Microwave-assisted synthesis proved to be a better syn-
thetic approach due to better yields and short reaction times. In or-
der to investigate how the target compounds bind to target, they
were docked into one of the plausible target Mycobacterium tuber-
culosis enoyl reductase. Most of the target compounds occupied
energetiacally more favorable position in the active site cavity than
isoniazid. However, none of the compounds (although few being
decently active) was found to be better in their in vitro antimyco-
bacterial activity as compared to isoniazid, when tested using lucif-
erase reporter phage assay against M. tuberculosis H37Rv and
clinical isolates S, H, R, and E resistant M. tuberculosis. The inaccu-
racy to predict isoniazid as potent inhibitor of M. tuberculosis enoyl
reductase in silico by docking than the target compounds might be
due to the well-known issues with the docking (sampling and
forcefield-based scoring function) or different molecular target of
the synthesized compounds, or both.

S. B. Mohan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7539–7542
7541
Figure 1. A close-up view of the binding interactions of the docked ligands in Mycobacterium tuberculosis enoyl reductase (PDB code 2H7I).6 (a) Overlay of the native ligand
with the crystal structure; (b) compounds 2g. The fit of the compounds 2g (c) and INH (d) (spheres) in the active site. Color coding: Ligand carbon in yellow (docked
geometry) or green (crystal geometry), protein carbon in cyan, nitrogen in blue, oxygen in red, bromine in brown, sulfur in light brown, hydrogen in white. The figure was
prepared using Pymol, ver. 0.99.14
Table 2
Antimycobacterial activity of the target compounds
Compd. No.
%Reduction in RLU
M. tuberculosis H37Rv
Clinical isolate: S, H, R and E resistant M. tuberculosis
100 g/mL 500 g/mL
100
lg/mL
500
lg/mL
l
l
1b
1d
1i
2e
46.49
54.28
33.40
46.21
75.73
82.58
98.42
44.20
61.02
48.72
65.23
82.04
38.38
50.10
25.47
61.35
38.03
29.19
89.6
47.86
52.77
33.49
66.65
74.91
2g
Rifampicin (2
Isoniazid (0.5
l
l
g/mL)
g/mL)

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S. B. Mohan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7539–7542
4. Goldman, R. C.; Plumley, K. V.; Laughon, B. E. Infect. Disord. Drug Targets 2007, 7,
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Supplementary data
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