New PPARγ ligands based on barbituric acid: Virtual screening, synthesis and receptor binding studies

Article abstract of DOI:10.1016/j.bmcl.2008.08.028

A new series of PPARγ ligands based on barbituric acid (BA) has been designed employing virtual screening and molecular docking approach. To validate the computational approach, designed molecules were synthesized and evaluated in in vitro radioligand bin

Full text of DOI:10.1016/j.bmcl.2008.08.028

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4959–4962
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
New PPAR
c
ligands based on barbituric acid: Virtual screening, synthesis
and receptor binding studies
Sandeep Sundriyal a, Bhoomi Viswanad b, Poduri Ramarao b, Asit K. Chakraborti a, Prasad V. Bharatam a,
*
a Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research, Sector 67, S.A.S. Nagar, Punjab 160 062, India
b Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, Sector 67, S.A.S. Nagar, Punjab 160 062, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of PPAR
c
ligands based on barbituric acid (BA) has been designed employing virtual screen-
Received 15 May 2008
Revised 7 August 2008
Accepted 11 August 2008
Available online 14 August 2008
ing and molecular docking approach. To validate the computational approach, designed molecules were
synthesized and evaluated in in vitro radioligand binding studies. Out of the total 14 molecules, 6 were
found to bind to the murine PPAR
c
with IC50 ranging from 0.1 to 2.5
lM as compared to reference stan-
dard, pioglitazone (IC50 = 0.7 M).
l
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
PPAR
c
Virtual screening
Docking
Diabetes
Barbituric acid
Rosiglitazone
Peroxisome proliferator-activated receptor (PPAR) belongs to
the nuclear hormone receptor (NHR) superfamily.1 Three subtypes,
PPARa, PPARc and PPARd, for this receptor have been identified
regard.9 In continuation of our programme on design and synthesis
of new PPAR agents,10 we hereby report a virtual screening ap-
proach for this purpose, together with the experimental results.
and found to be important targets for the treatment of type 2 dia-
betes, dyslipidemia, atherosclerosis, etc.2 The molecules belonging
to fibrate class of drugs, such as clofibrate (1) and fenofibrate (2),
A survey of various classes of PPARc agonists, 3D-QSAR studies
and crystal structure information reveals that the pharmacophoric
features of these agents essentially consists of three parts: (i) an
acidic head group, (ii) central aromatic region and (iii) a lipophilic
side chain (Fig. 1).11 The TZD ring of rosiglitazone is reported to
make three important H-bonds with His323, Tyr473 and His449
that are important for the activation of the receptor.11b This obser-
vation was instrumental in the design of a variety of non-TZD PPAR
agonists based on free carboxyl group, oxazolidinedione, tetra-
zoles, etc.12 However, such reports are limited and most of the re-
are known to act as PPAR
a
agonists (Scheme 1).3 Thiazolidinedi-
ones (TZDs) class of insulin sensitizers, synthesized in early
1980s,4 were later found to mediate hypoglycaemic effect through
PPARc 5
. Currently, rosiglitazone (3) and pioglitazone (4) are clini-
cally available TZDs for the treatment of type 2 diabetes. Recently,
PPARd is also being studied as a target for the treatment of obes-
ity.6 Out of the three isoforms, PPAR
c
and its agonists have been
studied extensively. In addition, the beneficial effects of PPAR li-
c
ported PPARc agonists still belong to either ‘glitazone’ class
gands have also been established for the treatment of inflamma-
(possessing TZD ring) or ‘glitazar’ class (possessing free carboxylic
acid). Thus, we set our objective to explore newer acidic head
tory conditions and cancer.7 However, many of the reported
PPAR
c
ligands are associated with serious adverse effects such as
groups for designing novel series of PPARc ligands. It was realized
fluid retention, weight gain, pro-carcinogenicity and hepatotoxic-
ity.8 Recently, use of rosiglitazone has been shown to be correlated
with the increased incidences of heart attacks in patients.8c Thus,
continued efforts are required to design and discover novel and
safe ligands for these therapeutically valuable targets. The multiple
that virtual screening, an important computational technique that
can help in discovering newer scaffolds with desired features, can
be employed for this purpose.13 In fact, one of the recent reports
suggests that this approach can be successfully applied for the dis-
covery of novel PPARc
agonists.14
activating ligands such as PPAR
tive PPAR modulators (SPPARMs) are also being developed in this
a
/
c
, PPAR
c
/d, PPAR
a
/
c
/d and selec-
To start with, the bioactive (co-crystallized) conformation of
rosiglitazone was extracted from the protein crystal structure
(PDB code 2PRG) and a query was generated using the ‘View
Hypothesis’ workbench of Catalyst program.15 Based on the
ligand-protein interactions, different features were assigned to
* Corresponding author. Tel.: +91 172 2214684; fax: +91 172 2214692.
E-mail address: pvbharatam@niper.ac.in (P.V. Bharatam).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.08.028

4960
S. Sundriyal et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4959–4962
O
O
O
O
Cl
O
O
Cl
Clofibrate, 1
Fenofibrate, 2
O
O
O
NH
NH
N
S
S
O
N
O
O
O
N
Rosiglitazone, 3
Pioglitazone, 4
Scheme 1.
Figure 1. Design of barbituric acid derivatives as PPARc ligands employing virtual screening approach.
rosiglitazone molecule that includes (i) two hydrogen bond accep-
tor (HBA) features corresponding to the two carbonyls of TZD ring
and one hydrogen bond donor (HBD), related to the –NH of the TZD
ring. (ii) hydrophobic aromatic (HYDaromatic) feature was as-
signed to the central aromatic region and (iii) HYDaromatic feature
was also assigned to the pyridine ring in the lipophilic side chain
(Fig. 1). These features were merged with the shape and hypothesis
query, generated using the co-crystallized conformation of the ros-
iglitazone molecule leading to hypo-1. The later was used as a filter
to screen NCI (total compounds = 238,819) and Maybridge (total
compounds = 59,652) databases of small molecules for hits.
A total of 46 hits were obtained from NCI and 13 from the May-
bridge. All hits were inspected visually individually to observe
mappings of the molecules to various chemical features of the
hypo-1. The analysis revealed that derivatives of barbituric acid
(BA) such as NCI 0685357 can perfectly map to the hypo-1
(Fig. 1). The BA ring mapped to the important HBA and HBD fea-
tures, while other aromatic rings mapped to the two HYDaromatic
features (Fig. 1). To the best of our knowledge, this ring has not
ring overlaid the TZD ring and maintained the essential H-bonding
interactions with His323, His449, Tyr473 and Gln286 as shown in
Figure 2.
been reported as a substitute for the TZD ring in PPARc agonists.
To further support the hypothesis, FlexX-based docking (imple-
mented in SYBYL6.9)16 was performed on a few designed deriva-
Figure 2. One of the designed barbituric acid derivative (8n) docked in the active
tives of BA into the active site of the PPAR
c (PDB code 2PRG).
site of PPARc (2PRG). The co-crystallized ligand is shown in green colour for
The docking results showed that the HBD and HBA features of BA
comparison while interacting amino acids (labeled in red) are shown in magenta.

S. Sundriyal et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4959–4962
4961
Table 1
Hence, taking clues from this virtual screening exercise, we de-
Structure and IC50 values of the final molecules
signed a variety of molecules with BA as acidic head group keeping
a central aromatic ring and varying lipophilic side chain (Fig. 1).
While developing synthetic strategy for this class of molecules, an-
other advantage of using BA ring in place of TZD was observed.
Both TZD and BA derivatives can be obtained by the Knovenagel
condensation of an aromatic aldehyde with the corresponding
acidic ring (TZD or BA) in the final step (Scheme 2). In case of
TZD, the condensation results in the formation of mixture of ‘E’
and ‘Z’ isomers which may be difficult to separate.10b On the other
hand, condensation of BA ring with aromatic aldehydes leads to a
single product owing to the symmetry in the BA moiety. Thus,
for the synthesis of different aromatic aldehydes, the first step
adopted was the reaction between the p- or m-hydroxy benzalde-
hyde and 1,2-dibromoethane or 1,3-dibromopropane to give differ-
ent monobromo products (3). The later were further reacted with
different substituted phenols or ‘NH’ containing heterocyles under
the basic conditions to yield the O- or N-alkylated aldehydes (4a–4j
and 5a–5c). The para analogue of the hit molecule NCI0685357
(Fig. 1) was also synthesized from the corresponding aldehyde
(6) that was obtained by reacting p-hydroxy benzaldehyde and
benzyl bromide. Finally, all aromatic aldehydes were condensed
with BA (7) by a reported procedure17 to yield final products
(8a–8n). All the final products were characterized using Mass, 1H
NMR and 13C NMR spectroscopy.18
O
R
O
O
meta or para
substitution
NH
N
H
O
Compound
R
IC50
i.a.
(
lM)
FlexX score
8a
À28.0
O
O
H3C
8b
i.a.
0.2
0.8
À25.5
À27.6
À27.5
8c
O
NC
NC
8d*,#
O
8e
i.a.
2.5
À27.1
À28.1
O
NC
Final molecules (8a–8n) were evaluated in vitro for their ability
to bind to murine PPARc in a standard radioligand binding assay
(Table 1).19 Out of the total 14 molecules, 6 were found to be active
in this assay. However, these molecules were found to act as partial
inhibitors in this assay except for 8d and 8f which inhibited nearly
100% binding of [3H]-rosiglitazone (see Supplementary informa-
tion). The molecule 8n was found to be most potent with IC50 of
8f*
O
O2N
8g
8h
0.2
i.a.
À25.5
À26.8
O
MeO
Br
O
CHO
CHO
n
a
Br
Br
n
Br
+
8i
8j
i.a.
i.a.
À27.6
À27.3
HO
O
O
3 eq.
3
meta or para
n = 2 or 3
1
2
O
N
b
N
N
c
HN
CHO
CHO
8k
8l
i.a.
0.5
À30.8
À34.0
n
n
5a-5c
O
Ar
O
O
O
4a-4j
N
N
CHO
CHO
Br
+
b
O
OH
8m
8n
i.a.
0.1
À32.6
À20.7
N
CHO
6
O
O
NH
d
NH
O
+
N
O
R
O
H
O
N
H
O
O
4a-4j,
5a-5c,6
O
8a-n
7
R
Pioglitazone (reference standard) IC50 = 0.7
lM.
i.a., inactive (less than 20% inhibition at 10
l
M).
Scheme 2. Reagents and conditions: (a) K2CO3, DMF, 50 °C, 3–5 h, 40–72%;
(b) ArOH, K2CO3, DMF, reflux, 2–4 h, 52–93%; (c) NaH, DMF, 0 °C-RT, 4–5 h,
63–85%; (d) MeOH, RT, 2–6 h, 76–92%.
*
Inhibited nearly 100% binding of [3H]-rosiglitazone.
#
Meta substitution (all other compounds have para substitution).

4962
S. Sundriyal et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4959–4962
Sternbach, D. D.; Kliewer, S. A.; Hansen, B. C.; Willson, T. M. Proc. Natl. Acad. Sci.
0.1 lM. However, experimentally observed IC50 values of these
U.S.A. 2001, 98, 5306.
molecules did not correlate with the docking scores. This may be
due to the inability of some of the compounds to penetrate the adi-
pocyte cells under the given bioassay conditions for their inappro-
priate physicochemical properties. In addition, the problem may
arise due to the limitation of docking programme that considers
only the flexibility of the ligand and not the protein. Nonetheless,
experimental findings demonstrate the proof of concept for the
adopted virtual screening approach and show that BA ring can be
employed as the acidic group for the design of novel class of PPAR
ligands.
In conclusion, computer-aided design of a novel series of PPAR
ligands based on barbituric acid has been reported. Preliminary
synthetic and receptor binding studies were taken up to validate
the adopted computer-aided design strategy. The synthesized mol-
ecules were indeed found to bind to the receptor with IC50 values
comparable to the reference standard, pioglitazone.
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Acknowledgment
Authors thank Department of Science and Technology (DST),
New Delhi, for providing financial assistance.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.08.028.
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