R. Kumar et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4613–4618
4617
allowed to compete with a fixed concentration of a radi-
olabeled ligand, [3H]-rosiglitazone (40 nM), for the
PPARc binding.21 As the concentration of unlabeled li-
gand increased, the amount of [3H]-rosiglitazone bound
to the receptor decreased. A sigmoidal curve was plotted
as percent inhibition of [3H]-rosiglitazone vs log concen-
trations of compounds and IC50 (concn that inhibits
50% of [3H]-rosiglitazone binding) value for each com-
pound was found by nonlinear regression curve fitting
using Graph Pad Prism software.
All the compounds with methoxy substituted central
ring have lower score than corresponding unsubstituted
compounds except for 6p. The reason for the better
activity of 4-hydroxy substituted carbazole derivatives
over compounds with N-substitution can be attributed
to the fact that the 4-hydroxy substituent orients its oxy-
gen such that there is an additional H-bonding interac-
tion that is observed with this oxygen atom and a
water molecule lying in close vicinity. Hence the strate-
gic position of this group is important for its activity.
Thus with these studies we could get some insights into
the nature of binding of the ligands and these studies can
be applied for designing better molecules. Some of rep-
resentative compounds having better docking score 6a
to 6s were synthesized and radioligand binding assay
was performed. It has been observed that all the synthe-
sized compounds have inhibited [3H]-rosiglitazone bind-
ing to PPARc in dose dependent manner.
From the radioligand binding assay, it was found that
compounds 6a to 6s could inhibit the [3H]-rosiglitazone
binding to PPARc in a dose dependent manner similar
to the commercially available PPARc agonist, pioglitaz-
one (reference standard). The expansion of SAR was
done by investigating effects of replacing thiazolidinedi-
one group with pyrimidone, so that they may modulate
the binding with the receptor. Molecular docking studies
have been performed on all the molecules in order to
study their binding mode in the active sites of PPARc.
The docking results have been analyzed in terms of
FlexX docking scores obtained by the molecules (using
the Bohm scoring function, employed within FlexX).
The scores of compounds 6a to 6s are better in active
sites of the receptor and also showing good binding
affinity.
As a part of our ongoing efforts to find a potential
PPAR agonist, 6-methyl-2-oxo-1,2,3,4-tetrahydro-
pyrimidine-5-carboxylic acid derivatives have been stud-
ied for the first time and are found to be high affinity
ligands for PPARc. Further advanced pharmacological
studies on these compounds are underway.
Acknowledgments
Molecular docking studies were carried out on the syn-
thesized compounds to get insight about their binding
preferences at the active site of the receptor. Rosiglitaz-
one when docked in the active site of PPARc receptor
attained a score of À15.5 kcal/mol. It showed the
important H-bonding interactions with the residues at
the active site of PPARc. These results are on the
expected lines as rosiglitazone is a PPARc agonist.
The FlexX docking scores of the hit compounds are
listed in Table 1.
R.K. and A.M. thank CSIR and DST, New Delhi,
respectively, for their Senior Research Fellowships. We
acknowledge Department of Pharmacology and Toxi-
cology, NIPER, for radioligand binding study.
Supplementary data
Experimental details of synthesis and biological evalua-
tion are available. Supplementary data associated with
this article can be found, in the online version, at
From observed results, the following conclusions can be
drawn: The most active compound 6e fitted the best in
the active site of PPARc and attained the best score of
À23.7 kcal/mol amongst all the molecules. It showed
all the prime interactions to anchor well in the active sites
of the receptor (Fig. 1a). The 4-methoxy carbazole deriv-
ative 6c, the oxygen of methoxy group shows the hydro-
gen bonding with Ser342 and water molecule which is in
close proximity. The pyrimidone carboxylic acid group
of 6c shows double hydrogen bonding with Tyr327.
References and notes
1. Joe, M. C.; Arshag, D. M. Drugs 2000, 60, 95.
2. Rotella, D. P. J. Med. Chem. 2004, 47, 4111.
3. Ramarao, P.; Kaul, C. L. Drugs Today 1999, 35, 895.
4. Skyler, J. S. J. Med. Chem. 2004, 47, 4113.
5. Willson, T. M.; Brown, P. J.; Sternbach, D. D.; Henke, B.
R. J. Med. Chem. 2000, 43, 527.
6. Ramachandran, U.; Kumar, R.; Mittal, A. Mini-rev. Med.
Chem. 2006, 6, 563.
7. Miller, A. R.; Etgen, G. J. Expt. Opin Investig. Drugs 2003,
12, 1489.
8. Khanna, S.; Sobhia, M. E.; Bharatam, P. V. J. Med. Chem
2005, 48, 3015.
9. Ramachandran, U.; Mital, A.; Bharatum, P. V.; Khanna,
S.; Rao, P. R.; Srinivasan, K.; Kumar, R.; Chawla, H. P.
S.; Kaul, C. L.; Raichur, S.; Chakrabarti, R. Bioorg. Med.
Chem. 2004, 12, 655.
10. Kumar, R.; Ramachandran, U.; Khanna, S.; Bharatam, P.
V.; Raichur, S.; Chakrabarti, R. Bioorg. Med. Chem. 2007,
15, 1547.
In substituted 4-hydroxycarbazole compound 6e, there
is additional hydrogen bonding with NH of carbazole
ring with Ser342 and water molecule. In compound 6f
carbon chain linker is varied from 2 to 3 between the
hydrophobic region and the central aromatic unit. It
also shows additional hydrogen bonding with NH of
carbazole and Ser342 and water molecule. Compound
6s, where 4-chlorobenzophenone moiety is hydrophobic
head, shows almost same interaction of middle carbonyl
group oxygen with Ser342 and water (Fig. 1b). The extra
double hydrogen bonding is seen in 6s, between pyrimi-
done carboxylic acid group and Leu453. Similar interac-
tion in the other analogues has been observed.