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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.
Mohan, Sahoo Biswa
