Design and synthesis of novel N-sulfonyl-2-indole carboxamides as potent PPAR-γ binding agents with potential application to the treatment of osteoporosis

Article abstract of DOI:10.1016/j.bmcl.2006.08.003

The synthesis and structure-activity relationships of a novel series of N-sulfonyl-2-indole carboxamides that bind to peroxisome proliferator-activated receptor gamma (PPAR-γ) are reported. Chemical optimization of the series led to the identification of 4q (IC₅₀ = 50 nM) as a potent binding agent of PPAR-γ. Also reported is preliminary cell based data suggesting the use of these compounds in the treatment of osteoporosis.

Full text of DOI:10.1016/j.bmcl.2006.08.003

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5659–5663
Design and synthesis of novel N-sulfonyl-2-indole carboxamides
as potent PPAR-c binding agents with potential application
to the treatment of osteoporosis
Corey R. Hopkins,* Steven V. O’Neil, Michael C. Laufersweiler, Yili Wang,
Matthew Pokross, Marlene Mekel, Artem Evdokimov, Richard Walter,
Maria Kontoyianni, Maria E. Petrey, Georgios Sabatakos, Jeff T. Roesgen,
Eloise Richardson and Thomas P. Demuth, Jr.
Procter & Gamble Pharmaceuticals, Health Care Research Center, 8700 Mason-Montgomery Road, Mason OH 45040, USA
Received 12 July 2006; revised 26 July 2006; accepted 1 August 2006
Available online 21 August 2006
Abstract—The synthesis and structure–activity relationships of a novel series of N-sulfonyl-2-indole carboxamides that bind to per-
oxisome proliferator-activated receptor gamma (PPAR-c) are reported. Chemical optimization of the series led to the identification
of 4q (IC50 = 50 nM) as a potent binding agent of PPAR-c. Also reported is preliminary cell based data suggesting the use of these
compounds in the treatment of osteoporosis.
Ó 2006 Elsevier Ltd. All rights reserved.
Peroxisome proliferator-activated receptors (PPARs)
are members of the nuclear receptor superfamily.1 The
PPARs have generated much interest in the pharmaceu-
tical industry due to their effects in a widerange of ther-
apeutic areas, and a number of agonists of this class
have progressed through the clinic and to marketed
anti-diabetic drugs.2 Although the initial effort in this
area was to find agonists, recent data have suggested
that partial agonists and antagonists of PPARs (namely
PPAR-c) could be potentially beneficial in a number of
areas. Over the past several years, PPAR-c modulators
have attracted increasing attention as potential treat-
ments for osteoporosis. Recent evidence in both genetic
and pharmacological studies suggests that attenuation
of PPAR function can alter the balance between adipo-
genesis and osteogenesis, such that bone formation may
potentially be enhanced.3 In fact, using various animal
models, the thiazolidinedione, rosiglitazone (a full
PPAR-c agonist), was shown to reduce bone mineral
density and increase bone marrow adipocytes.4 Thus,
the regulation of PPAR-c activation may control differ-
entiation of mesenchymal stem cells allowing for osteo-
blast formation.5 Due to this, there have been a number
of reports in the literature regarding synthetic PPAR-c
modulators that affect adipocyte differentiation.3,6
There have been a number of reports in the literature
about indole-derived PPARc binding agents, and it
was with these reports that our scaffolds were derived.7
The synthesis of the N-sulfonyl-2-indole carboxamide
compounds is outlined in Scheme 1.8 Starting from the
appropriately substituted ethyl indole-2-carboxylate,
the 3-Ar-substituted compounds (2a–f) were synthesized
by bromination (NBS, THF) followed by N-alkylation
(K2CO3, DMF, R3-Br) to yield 1. Next, palladium-cata-
lyzed Suzuki–Miyaura cross-coupling9 (Pd(PPh3)4, 2 M
Na2CO3, EtOH, toluene, ArB(OH)2) gave the 3-Ar-sub-
stitution which was then carried on to the acid via
saponification of the ethyl ester (3 M KOH, THF/
EtOH, reflux). Finally, the target compounds, 2a–f,
were realized by coupling with a variety of aryl- or alkyl-
sulfonamides (EDCI, DMAP, Et3N, CH2Cl2) in good
overall yield (58–93%). The indoles without substitution
at the 3-position (3-H) were synthesized in an analogous
fashion. Thus, the ethyl indole-2-carboxylate was N-al-
kylated (K2CO3, DMF, R3-Br), the ester saponified
(3 M KOH, THF/EtOH, reflux) followed by coupling
Keywords: PPAR; N-Sulfonyl-2-indole carboxamides; Alkaline phos-
phatase assay; Osteoporosis.
*
Corresponding author. Present address: Sanofi-Aventis Pharmaceu-
ticals, 1041 Route 202-206, Bridgewater, NJ 08807, USA.
Tel./fax: +1 908 231 3444; e-mail: Corey.Hopkins@sanofi-aventis.
com
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.08.003

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C. R. Hopkins et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5659–5663
1. Pd(PPh3)4, 2 M Na2CO3,
R1
R2
HN S
EtOH, Toluene, 85 oC,
Br
R1B(OH)2; 86%
R
R
R
O
O
O
O
O
1.NBS, THF; quant.
O
2. 3 M KOH, THF, EtOH,
reflux, 97%
2. K2CO3, DMF,
N
O
N
N
R3
R3-Br; 86%
R3
2a-f
H
3. DMAP, EDCI, Et3N, CH2Cl2,
1
R2-SO2NH2, 58-93%
R2
HN S
1. K2CO3, DMF,
DMAP, EDCI, Et3N, CH2Cl2,
R3-Br, 32-98%
R
R
OH
O
R2-SO2NH2, 45-95%
O
O
2. 3 M KOH, THF, EtOH,
reflux, 63-80%
N
R3
4a-w
O
N
R3
3a-u
Scheme 1. Synthesis of N-sulfonyl-2-indole carboxamides.10
Table 2. PPAR-c binding11 for N-sulfonyl-2-indole carboxamides
4a–k
of an appropriate sulfonamide (EDCI, DMAP, Et3N,
CH2Cl2) giving the 3-H-substituted compounds 4a–w
in satisfactory overall yield (20–78%).
R2
R
HN
O
S
O
O
Our first efforts concentrated on evaluating 3-Ar N-sul-
fonyl-2-indole carboxamide compounds (2a–f). It was
soon apparent (Table 1) that these were very potent li-
gands for PPAR-c. Substitution at R2 made very little
impact on the binding data, as a variety of substituents
were well tolerated (i.e., aryl vs alkyl, 2a vs 2f). The only
drop in activity came when the phenyl group was substi-
tuted at the 3-position (2c, 60 nM).
N
R3
4a-w
Compound
R
R2
Ph
R3
a
IC50 (nM)
4a
4b
4c
4d
4e
4f
H
H
H
H
H
H
H
H
H
H
H
3-CF3Bn 290
3-CF3Bn 720
3-CF3Bn 280
4-F–Ph
4-Cl–Ph
3-CF3–Ph
4-CH3Ph
2-CH3Ph
2-Naphthyl
2-CF3–Ph
3-CF3Bn
80
3-CF3Bn 290
3-CF3Bn 180
However, since very similar compounds had been pub-
lished,7 our next attempt was to see what influence the
3-Ar-substituent had on the activity. With this, a num-
ber of compounds were made with R1 = H investigating
the SAR around several positions (R, R2, and R3).
While keeping R3 constant (3-CF3Bn), we first looked
at changing the sulfonamide portion (R2) to evaluate
whether the activity could be maintained as compared
to the 3-Ar-substituted compounds (Table 2). The first
compounds synthesized gave a noticeable erosion of
binding activity (2a vs 4a, 2 nM vs 290 nM). This loss
4g
4h
4i
3-CF3Bn
3-CF3Bn
90
80
80
2-(5-Chlorothiophene) 3-CF3Bn
Me
4-CF3–Ph
4j
4k
3-CF3Bn 680
4-CF3Bn 700
a IC50 determinations are the average of three determinations on three
separate days.
of activity was seen throughout most of the series with
the exception, however, of a couple of compounds.
First, the N-(3-(trifluoromethyl)phenyl)sulfonyl com-
pound (4d) showed comparable activity to its 3-Ar coun-
terpart (2c, 60 nM vs 80 nM). Other compounds that
showed similar activity include 4g–i, which all have sub-
stituents in either the ortho- or meta-position. One inter-
esting observation was the loss of activity when going
from the ortho- or meta-position to the para-position
(4d and 4h, 80 nM, vs 4k, 700 nM). Also, unlike in the
3-Ar-substituted series, an aryl substituent on the N-sul-
fonyl group was essential to maintain good binding data
(4a vs 4j, 290 nM vs 680 nM).
Table 1. PPAR-c binding11 for 3-Ar-substituted N-sulfonyl-2-indole
carboxamides 2a–f
R1
R2
R
HN
O
S
O
O
N
R3
2a-f
a
Compound
R
R1
R2
R3
IC50
(nM)
2a
2b
2c
2d
2e
2f
H
H
H
H
H
H
4-CH3OPh Ph
4-CH3OPh 4-F–Ph
4-CH3OPh 3-CF3–Ph
4-CH3OPh 2-CO2MePh 3-CF3Bn
3-CF3Bn
3
7
Having established our best N-sulfonyl substituent (3-
CF3Ph, 4d), we next evaluated the indole N-substituent
(R3) to see if there was any advantage for other groups
(Table 3). The initial compounds made were closely
related to the previously tested 3-trifluoromethylbenzyl
compound (4d). The 3-methoxy- and 3-trifluorometh-
oxybenzyl compounds (4l,m) showed similar activity
3-CF3Bn
3-CF3Bn 60
1
7
2
4-CH3OPh 2-CH3Ph
4-CH3OPh Me
3-CF3Bn
3-CF3Bn
a IC50 determinations are the average of three determinations on three
separate days.

C. R. Hopkins et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5659–5663
5661
Table 3. PPAR-c binding11 for N-sulfonyl-2-indole carboxamides 4l–t
activity than the parent R = H compound. Even small
substituents (4u, R = Cl and 4u, R = OH) showed a
marked loss of binding.
R2
R
HN
O
S
O
O
N
R3
4a-w
To better understand the binding interactions of these
sulfonyl indole carboxamides with PPAR-c, compound
2a was co-crystallized with the ligand-binding domain
of the protein (PPAR-c LBD) and the co-activator pep-
tide fragment (SRC-1), and subjected to X-ray structure
determination (PDB-id, 2HFP).13 Figure 1 illustrates
the observed binding interactions between 2a and
PPAR-c LBD. Interestingly, two molecules of com-
pound 2a were seen to span the binding pocket. Such
a 2:1 stoichiometry of binding is not typical for reported
PPAR-c modulators.14,15
a
IC50 (nM)
Compound
R
R2
R3
4l
4m
H
H
H
H
H
H
H
H
H
3-CF3–Ph
3-CF3–Ph
3-CF3–Ph
3-CF3–Ph
3-CF3–Ph
3-CF3–Ph
3-CF3–Ph
3-CF3–Ph
3-CF3–Ph
3-CH3OBn
3-CF3OBn
3-CF3PhCO
Et
140
90
4n
4o
9400
2440
400
50
4p
4q
4-CF3Bn
3-BnOBn
Bn
4r
280
380
330
90
4s
2,5-DiClBn
4-t-BuBn
4t
Rosiglitazone
512
One molecule of 2a (molecule B of Fig. 1) bound
near the Helix-12 region and was shown to make
hydrogen bonds with His449 through the carbonyl
and sulfonyl oxygens. Also noteworthy with regard
to molecule B is the close proximity to Tyr473, which
has been implicated in the mechanism of action of
PPAR-c,14a although no distinct interaction is ob-
served. The second molecule of compound 2a bound
at the opposite end of the PPAR-c LBD (molecule
A). This molecule occupied the area in PPAR-c bind-
ing pocket similar to other known compounds, such
as compound 5,12 and the N-methylaminopyridine
portion of rosiglitazone.13b Hydrogen bond interac-
tions are observed between the carbonyl and sulfonyl
oxygens of this molecule and Ser342 and Lys265. The
rest of the molecule makes productive hydrophobic
interactions in the binding domain with residues
Arg288, Leu330, and Ile341. The two bound ligand
molecules interact with one another via their benzin-
dole portions.
80
a IC50 determinations are the average of three determinations on three
separate days.
(140 nM and 90 nM, respectively). However, when the
benzyl group was changed to a benzoyl group all activity
was lost (4d vs 4n, 80 nM vs 9400 nM). Alkyl substitu-
ents were also not tolerated in this position (4o,
2400 nM). The meta-substitution again proved to be
the optimal compound; changing the 3-CF3Bn to
4-CF3Bn resulted in a significant loss of activity (4d vs
4p, 80 nM vs 400 nM). The unsubstituted benzyl group
proved to be less potent (4r, 280 nM) as well. However,
the one change that proved beneficial was increasing the
steric bulk at the meta-position of the benzyl group.
Replacing the trifluoromethyl group with a benzyloxyb-
enzyl group provided an increase in potency, albeit mod-
est (4d vs 4q, 80 nM vs 50 nM). We also evaluated the
binding data of two known PPAR-c modulators in-
house, rosiglitazone (90 nM) and compound 5 (80 nM).
Having established a series of potent ligands for PPAR-c,
we next evaluated a number of these compounds in a
cell-based assay to assess their potential to stimulate
osteogenesis. For this measurement, we used the alkaline
phosphatase (ALP) assay, where pre-osteoblastic cells
were induced to produce alkaline phosphatase, a marker
for osteoblast differentiation, on exposure to test
compounds. Potency (EC50) in this cell-based assay
would indicate that a compound could potentially exert
an osteogenic response in vivo. Such compounds may
therefore be anabolic to bone, and thus offer a novel
therapeutic utility to osteoporosis.
Lastly, we investigated substitutions on the phenyl ring
of the indole (i.e., R), due to reports in the literature
regarding this substituent.7b Keeping R2 and R3 con-
stant, we were interested to see if these compounds
would maintain the activity shown for the other com-
pounds (Table 4). Starting from commercially available
precursors we were able to obtain compounds 4u–w.
Unfortunately, these compounds showed much less
Table 4. PPAR-c binding11 for N-sulfonyl-2-indole carboxamides
4u–w
Compounds 2a, 2b, 4c, and 5 (a reported PPAR-c
antagonist) as well as rosiglitazone were evaluated in
the ALP assay (Table 5). Compared to rosiglitazone (a
PPAR-c full agonist) which was inactive in this assay,
all compounds stimulated an alkaline phosphatase re-
sponse, indicating the compounds induced osteoblast
differentiation. Parallel cell count and viability measure-
ments indicated the ALP response was not due to cell
proliferation (data not shown). The 3-Ar analog, 2a,
proved to be the most potent with an ALP
EC50 ꢀ 100 nM. An additional 3-Ar analog, 2c, along
with the 3-H analog, 4c, were active in the 1–10 lM
range.
R2
R
HN
O
S
O
O
N
R3
4a-w
a
IC50 (nM)
Compound
R
R2
R3
4u
4v
Cl
3-CF3–Ph
3-CF3–Ph
3-CF3–Ph
3-CF3Bn
3-CF3Bn
3-CF3Bn
330
770
540
OBn
OH
4w
a IC50 determinations are the average of three determinations on three
separate days.

5662
C. R. Hopkins et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5659–5663
Figure 1. X-ray crystal structure of compound 2a co-crystallized with PPAR-c LBD and SRC-1. Ligands and relevant protein residues are shown as
thick and thin sticks, respectively. The important Tyrosine 473 is highlighted in red; hydrogen bonds are represented by green dashes.
2. Henke, B. R. J. Med. Chem. 2004, 47, 4118.
Table 5. Alkaline phosphatase (ALP) EC50 data for compounds 2a,
2d, 4c, and 516
3. (a) Barak, Y.; Nelson, M. C.; Ong, E. S.; Jones, Y. Z.;
Ruiz-Lozano, P.; Chien, K. R.; Koder, A.; Evans, R. M.
Mol. Cell 1999, 4, 585; (b) Cock, T. A.; Back, J.;
Elefteriou, F.; Karsenty, G.; Kastner, P.; Chan, S.;
Auwerx, J. EMBO Rep. 2004, 5, 1; (c) Akune, T.; Ohba,
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Kadowaki, T.; Kawaguchi, H. J. Clin. Invest. 2004, 113,
846.
a
Compound
ALP EC50 (lM)
Solubility (lg/mL)
2a
2d
4c
512
0.1
5
1–10
1–10
0.1
ND
ND
ND
a EC50 determinations are the average of two determinations on two
separate days.
4. (a) Rzonca, S. O.; Suva, L. J.; Gaddy, D.; Montague, D.
C.; Lecka-Czernik, B. Endocrinology 2004, 145, 401; (b)
´
Soroceanu, M. A.; Miao1, D.; Bai, X-Y.; Su, H.;
In conclusion, we have described the synthesis and
in vitro evaluation of novel N-sulfonyl-2-indole carbox-
amides as PPAR-c binding agents. Several of these com-
pounds were found to stimulate osteoblast
differentiation in a cell-based assay, thus suggesting a
potential application for the treatment of osteoporosis.
Goltzman, D.; Karaplis, A. C. J. Endocrinol. 2004, 183,
203; (c) Sottile, V.; Seuwen, K.; Kneissel, M. Calcif. Tissue
Int. 2004, 75, 329; (d) Schwartz, A. V.; Sellmeyer, D. E.;
Feingold, K. R. Diabetes 2002, 51, abstract 237; (e) Ali, A.
A.; Weinstein, R. S.; Stewart, S. A.; Parfitt, A. M.;
Manolagas, S. C.; Jilka, R. L. Endocrinology 2005, 146,
1226; (f) Lazarenko, O. P.; Rzonca, S. O.; Suva, L. J.;
Lecka-Czernik, B. Bone 2006, 38, 74.
Acknowledgments
5. (a) Lecka-Czernik, B.; Moerman, E. J.; Grant, D. F.;
Lehmann, J. M.; Manolagas, S. C.; Jilka, R. L. Endocri-
nology 2002, 143, 2376; (b) Duque, G. Drug News
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Curr. Opin. Pharmacol. 2004, 4, 290.
6. (a) Mukherjee, R.; Hoener, P. A.; Jow, L.; Bilakovics, J.;
Klausing, K.; Mais, D. E.; Faulkner, A.; Croston, G. E.;
Paterniti, J. R., Jr. Mol. Endocrinol. 2000, 14, 1425; (b)
Camp, H. S.; Chaudhry, A.; Leff, T. Endocrinology 2001,
142, 3207.
X-ray data were collected at SER-CAT 22-ID beam line
at the Advanced Photon Source, Argonne National
Laboratory. Use of the Advanced Photon Source was
supported by the US Department of Energy, Office of
Science, Office of Basic Energy Sciences, under Contract
No. W-31-109-Eng-38. All relevant structures were sub-
mitted to the PDB, code 2HFP.
7. (a) Henke, B. R.; Adkinson, K. K.; Blanchard, S. G.;
Leesnitzer, L. M.; Mook, R. A., Jr.; Plunket, K. D.; Ray,
J. A.; Roberson, C.; Unwalla, R.; Willson, T. M. Bioorg.
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H.; Campbell, A.-M.; Qi, N.; Baryza, J. L.; Cook, J. H.
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10. Representative example. To a rt solution of acid 3q
(359.8 mg; 1.007 mmol) in CH2Cl2 (9 mL) were added
DMAP (33.0 mg; 0.270 mmol), Et3N (200 lL; 1.43 mmol),
EDCI (232.3 mg; 1.212 mmol), and 3-(trifluorometh-
yl)benzenesulfonamide (285.6 mg; 1.269 mmol). The mix-
ture was stirred at rt for 16 h. The reaction was quenched
with 1 N HCl and then extracted with EtOAc. The organic
extracts were washed with brine, dried (Na2SO4), and
concentrated in vacuo. The residue was purified by column
chromatography (34 g SiO2 pre-packed column from
AnaLogix, 50% EtOAc in hexanes eluent) to yield
489.6 mg (0.8672 mmol; 86%) of the desired N-sulfonyl-
2-indole carboxamide 4q as a colorless, amorphous solid.
1H NMR (DMSO-d6) d 8.31–8.27 (m, 2H), 8.10 (d, 1H,
J = 7.8 Hz), 7.88 (t, 1H, J = 7.8 Hz), 7.75 (d, 1H,
J = 7.8 Hz), 7.61–7.58 (m, 2 H), 7.38 (m, 6 H), 7.17 (t,
1H, J = 7.2 Hz), 7.03 (t, 1H, J = 8.1 Hz), 6.81 (dd, 1H,
J = 8.1, 2.1 Hz), 6.58 (s, 1H), 6.40 (d, 1H, J = 7.5 Hz), 5.65
(s, 2 H), 4.95 (s, 2 H). HRMS (EI) m/z calcd for
C30H23F3N2O4S (564.57), found 565.14 (M+H)+.
11. PPAR-c competitor assay. Twenty-seven nanomolar
ligand binding domain of PPAR-c (aa193-475) was added
to 2.5 nM Fluormone, a high affinity, fluorescent-tagged
PPAR-c ligand (Kd = 9 nM). The PPAR-c LBD/Fluor-
mone complex yielded a high fluorescence polarization
value (mP). Competitor compound was added to the
complex, displacing Fluormone, yielding a lower fluores-
cence polarization value. Compounds that do not compete
for PPAR-c LBD binding will not reduce the fluorescence
polarization value.
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16. Cell culture. MC3T3-E1 cells were obtained from ATCC
(CRL-2594) and were grown in standard growth medium
containing a-MEM and 10% fetal bovine serum in a
humidified atmosphere of 5% CO2 and 90% air at 37 °C.
Cells were plated at 2500 cells/well in 96 half-well plates
and were grown to confluence in standard growth media.
Once confluent, cells were cultured in differentiation
medium containing a-MEM, 10% fetal bovine serum,
25 lg/mL ascorbic acid 2-phosphate, 10 mM b-glycerol
phosphate, and compounds were resuspended in DMSO
for 6 days. Fifty nanogram per milliliter of rhBMP-2 from
R&D Systems was utilized as the positive control. Alka-
line phosphatase activity. To assess alkaline phosphatase
(ALP) activity, 10 lL Cell Titer 96Ò Aqueous One
Solution Reagent (Promega) was added to each well of
the 96 half-well plate containing 50 lL of culture medium.
The cells were incubated for 1 h in a humidified atmo-
sphere of 5% CO2 and 90% air at 37 °C. Absorbance was
read at 490 nm on a Molecular Device’s SPECTRAmax
PLUS 384 plate reader. After removing the culture
medium, cell layers were washed once with D-PBS at
room temperature. Fifty microliters ALP substrate solu-
tion (one tablet of Sigma 104Ò Phosphatase Substrate in
3.75 mL of substrate buffer containing distilled water,
50 mM glycine, and 1 mM MgCl2 (adjust to pH 10.5) was
added to each well and the cells were incubated at room
temperature for 20 min. Absorbance was read at 405 nm
on a Molecular Devices SPECTRAmax PLUS 384 plate
reader. The enzyme activity was expressed as pNP nmols/
min/million cells.
12. Thor, M.; Beierlein, K.; Dykes, G.; Gustavsson, A.-L.;
Heidrich, J.; Jendeberg, L.; Lindqvist, B.; Pegurier, C.;
Roussel, P.; Slater, M.; Svensson, S.; Sydow-Ba¨ckman,
M.; Thornstro¨m, U.; Uppenberg, J. Bioorg. Med. Chem.
Lett. 2002, 12, 3565, .
13. (a) Two microliters of protein-peptide complex solution
(10 mg/mL 206-477 His6-PPAR-c, 1 mM SRC-1 peptide
fragment) was added to two microliters of well solution
(2% Peg400, 1.6 M ammonium sulfate, and 100 mM Mes
6.5) and suspended over 1 mL of well solution in a
hanging drop crystallization setup. The X-ray coordinates