Synthesis of isosteric selenium analog of the PPARβ/δ agonist GW501516 and comparison of biological activity

Article abstract of DOI:10.1016/j.bmcl.2010.05.094

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors and members of the nuclear hormone receptor superfamily. Herein, we describe an efficient synthesis of a novel isosteric selenium analog of the highly specific PPARβ/δ ligand 2-methyl-4-((4-methyl-2-(4-trifluoromethylphenyl)-1,3-thiazol-5-yl)-meth ylsulfanyl)phenoxy-acetic acid (GW501516; 1). The study examined the efficiency of the novel selenium analog 2-methyl-4-((4-methyl-2-(4-trifluoromethylphenyl)-1,3-selenazol-5-yl)-me thylsulfanyl)phenoxy-acetic acid (2) to activate PPARβ/δ and the effect of ligand activation of PPARβ/δ on cell proliferation and target gene expression in human HaCaT keratinocytes. The results showed that similar to GW501516, the Se-analog 2 increased expression of the known PPARβ/δ target gene angiopoietin-like protein 4 (ANGPTL4); the compound 2 was comparable in efficacy as compared to GW501516. Consistent with a large body of evidence, the Se-analog inhibited cell proliferation in HaCaT keratinocytes similar to that observed with GW501516. In summary, the novel Se-analog 2 has been developed as a potent PPARβ/δ ligand that may possess additional anti-cancer properties of selenium.

Full text of DOI:10.1016/j.bmcl.2010.05.094

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4050–4052
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of isosteric selenium analog of the PPARb/d agonist GW501516
and comparison of biological activity
a
b
b
a
Arun K. Sharma a, , Ugir Hossain Sk , Pengfei He , Jeffrey M. Peters , Shantu Amin
*
a Department of Pharmacology, Chemical Carcinogenesis and Chemoprevention Program of Penn State Hershey Cancer Institute,
Penn State College of Medicine, CH72, 500 University Drive, Hershey, PA 17033, USA
b Department of Veterinary and Biomedical Sciences and The Center of Molecular Toxicology and Carcinogenesis, The Pennsylvania State University, University Park, PA 16802, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors and
members of the nuclear hormone receptor superfamily. Herein, we describe an efficient synthesis of a novel
isosteric selenium analog of the highly specific PPARb/d ligand 2-methyl-4-((4-methyl-2-(4-trifluorometh-
ylphenyl)-1,3-thiazol-5-yl)-methylsulfanyl)phenoxy-acetic acid (GW501516; 1). The study examined the
efficiency of the novel selenium analog 2-methyl-4-((4-methyl-2-(4-trifluoromethylphenyl)-1,3-sele-
nazol-5-yl)-methylsulfanyl)phenoxy-acetic acid (2) to activate PPARb/d and the effect of ligand activation
of PPARb/d on cell proliferation and target gene expression in human HaCaT keratinocytes. The results
showed that similar to GW501516, the Se-analog 2 increased expression of the known PPARb/d target gene
angiopoietin-like protein 4 (ANGPTL4); the compound 2 was comparable in efficacy as compared to
GW501516. Consistent with a large body of evidence, the Se-analog inhibited cell proliferation in HaCaT
keratinocytes similar to that observed with GW501516. In summary, the novel Se-analog 2 has been devel-
oped as a potent PPARb/d ligand that may possess additional anti-cancer properties of selenium.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 26 April 2010
Accepted 21 May 2010
Available online 27 May 2010
Keywords:
PPARb/d
Agonist
Selenium analog
Angiopoietin-like protein 4
Peroxisome proliferator-activated receptors (PPARs) are ligand-
activated transcription factors and members of the nuclear hor-
We hypothesized that isosteric substitution of the sulfur atom
in GW501516 by selenium not only would retain the structural
features required for its efficient binding to the receptor and hence
its efficiency as a PPARb/d ligand, but would also possess additional
anti-cancer properties of selenium. The hypothesis was based on
our own recent studies13,14 and literature reports15 demonstrating
that compared to sulfur structural analogs, selenium compounds
are more potent anti-cancer agents. In addition, there is a large
body of evidence showing selenium to be effective for inhibiting
tumorigenesis in both animal models and epidemiological stud-
ies.16–19 Furthermore, since both sulfur and selenium, exhibit sim-
ilar oxidation states, the difference in size of the atom (from sulfur
to selenium) was not expected to alter the geometry of the mole-
cule significant enough to affect its binding to PPARb/d ligand.
Therefore, we developed the selenium analog 2 (Fig. 1) and com-
pared its efficacy as a PPARb/d ligand and as inhibitor of cell prolif-
eration as compared to GW501516.
GW501516 used in this study was synthesized as described
previously.20 The selenium analog 2-methyl-4-((4-methyl-2-(4-tri-
fluoromethylphenyl)-1,3-selenazol-5-yl)-methylsulfanyl)phenoxy-
acetic acid (2) was synthesized as outlined in Scheme 1.21 The key
precursor 2 was synthesized by treating o-cresol (3) with potassium
selenocyanate in the presence of potassium bromide and bromine,
in methanol. Selenocyanate 4 was reduced with sodium borohy-
dride to generate corresponding selenol in situ that was treated
with thiazole 5, synthesized following a reported method,10 in a
mone receptor superfamily, and exist in three isoforms, PPAR
PPAR , and PPARb (also referred to as PPARd and PPARb/d). Each
of these regulates tissue-specific target genes involved in many
biological processes.1,2 PPAR
and PPAR are the molecular targets
of a number of marketed drugs, for example, the fibrate class of
hypolipidemic drugs3 (PPAR
) and the thiazolidinedione class of
insulin-sensitizing drugs4 (PPAR
). In contrast to PPAR and PPAR
a,
c
a
c
a
c
a
c
there are no marketed drugs that target PPARb/d and considerably
less is known about the biological role of PPARb/d. Although ligand
activation of PPARb/d is known to increase serum high-density
lipoprotein cholesterol, increase skeletal muscle fatty acid catabo-
lism, and improve insulin sensitivity,5,6 its role in tumorigenesis,
apoptosis, and cell proliferation remains controversial.7–9 Exten-
sive structure-based drug design and combinatorial chemistry
studies have recently led to the identification of two highly selec-
tive PPARd agonists GW501516 (1) and its analog GW0742.10,11 Gi-
ven the pharmacological potential of these PPARb/d agonists,
which have been examined in clinical trials,12 it is critical to design
novel analogs that not only efficiently bind selectively to PPARb/d
but have enhanced anti-cancer activity.
* Corresponding author. Tel.: +1 717 531 0003x285016; fax: +1 717 531 0244.
E-mail address: aks14@psu.edu (A.K. Sharma).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.094

A. K. Sharma et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4050–4052
4051
O
Me
O
HO
S
N
S
CF3
Me
1. GW501516
O
Me
O
HO
S
Se
Figure 2. Modulation of gene expression by ligand activation of PPARb/d in HaCaT
keratinocytes. HaCaT cells were treated for 8 h with the indicated concentration of
GW501516 or selenium analog 2. Quantitative real-time PCR was performed to
examine the expression of mRNA encoding ANGPTL4 normalized to mRNA encoding
GAPDH. Values are the average-fold change compared with control treatment and
represent means SEM. Values with different letters are significantly different,
P < 0.05, as determined by ANOVA and Bonferroni’s multiple comparison test.
CF3
N
Me
2. Se-analog
Figure 1. Structures of GW501516 (1) and its novel selenium analog (2).
Several studies have shown that ligand activation of PPARb/d can
induce terminal differentiation of keratinocytes and epithelium7,8
and can inhibit cell growth in epithelium and other cell types,
including keratinocytes, colonocytes, cardiomyocytes, lung fibro-
blasts, and cancer cell lines.7–9 A few reports, however, also suggest
that ligand activation of PPARb/d can potentiate cell growth.7–9 We
have previously demonstrated that ligand activation of PPARb/d
inhibits keratinocyte proliferation through PPARb/d-dependent
mechanisms.22,23 To examine the effect of novel synthetic PPARb/d
ligand 2 on cell growth, HaCaT keratinocyte cell proliferation was
quantified in the presence of either GW501516 (1) or its selenium
analog 2.24 Inhibition of HaCaT cell proliferation was observed with
one pot reaction to give the adduct 6 in excellent yield. Compound 6
on treatment with bromoethyl acetate in the presence of cesium
carbonate resulted in the formation of ester 7 which was hydrolyzed
following a literature method20 using lithium hydroxide to yield
the desired Se derivative 2. The product was purified by silica gel
column chromatography and characterized on the basis of NMR
and high-resolution mass spectral data.
We and many other laboratories have previously demonstrated
that GW501516 and other specific PPARb/d ligands increase the
expression of ANGPTL4.7–9,22 To evaluate if the Se-analog is a spe-
cific ligand for PPARb/d, we examined the expression of the known
PPARb/d-dependent target gene ANGPTL4, and compared it with
the highly specific PPARb/d ligand GW501516. HaCaT cells were
10 lM concentrations of both GW501516 and its Se-analog 2 after
72 h of exposure. The inhibition was slightly more pronounced with
the Se-analog 2 (Fig. 3A and B). These data support the hypothesis
and suggests that the presence of selenium has the potential to make
the compound more cytotoxic.
In summary, a novel isosteric selenium analog of GW501516
has been developed. The synthetic strategy is highly efficient and
gave high overall yield. The new compound 2 is a specific PPARb/
d ligand as it exhibited a marked induction of the target gene ANG-
PTL4. Furthermore, the new analog inhibited keratinocyte prolifer-
ation slightly better than GW501516. The new Se-analog could
treated for either 8 h with 0.2 lM GW501516 or its novel selenium
analog 2. Quantitative real-time PCR was performed to examine
the expression of mRNA encoding ANGPTL4 normalized to mRNA
encoding GAPDH. The mRNA encoding the PPARb/d target gene
ANGPTL4 was induced by both GW501516 and selenium derivative
2 (Fig. 2). The selenium analog led to a similar induction of the tar-
get gene ANGPTL4 as compared to GW501516. This suggests that
the novel selenium derivative 2, designed by isosterically replacing
sulfur in GW501516 by selenium, retained the property of being a
specific PPARb/d ligand.
Me
HO
Me
Me
1. NaBH4, EtOH, 0 oC
2.
HO
KSeCN, KBr, Br2
MeOH, 0 oC
HO
S
Se
CF3
S
Cl
SeCN
N
CF3
Me
3
N
4
Me
6
5
One Pot
O
Me
O
Me
O
O
HO
MeO
CsCO3
O
LiOH
S
Se
Me
S
N
Se
Me
CF3
CF3
N
Br
MeO
2
7
Scheme 1. Synthesis of 2-methyl-4-((4-methyl-2-(4-trifluoromethylphenyl)-1,3-selenazol-5-yl)-methylsulfanyl)phenoxy-acetic acid (2).

4052
A. K. Sharma et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4050–4052
13. Sharma, A. K.; Sharma, A.; Desai, D.; Madhunapantula, S. V.; Huh, S. J.;
Robertson, G. P.; Amin, S. J. Med. Chem. 2008, 51, 7820.
14. Sharma, A.; Sharma, A. K.; Madhunapantula, S. V.; Desai, D.; Huh, S. J.; Mosca,
P.; Amin, S.; Robertson, G. P. Clin. Cancer Res. 2009, 15, 1674.
15. Ip, C.; Ganther, H. E. Carcinogenesis 1992, 13, 1167.
16. El-Bayoumy, K. The Role of Selenium in Cancer Prevention. In Cancer Principles
and Practice of Oncology, 4th ed.; J.B. Lippincott: Philadelphia, 1991; p 1.
17. Clark, L. C.; Combs, G. F., Jr.; Turnbull, B. W.; Slate, E. H.; Chalker, D. K.; Chow, J.;
Davis, L. S.; Glover, R. A.; Graham, G. F.; Gross, E. G.; Krongrad, A.; Lesher, J. L.,
Jr.; Park, H. K.; Sanders, B. B., Jr.; Smith, C. L.; Taylor, J. R. JAMA 1996, 276, 1957.
18. Combs, G. F., Jr.; Lu, J. Selenium as a Cancer Preventive Agent; Kluwer Academic
Publishers: Dordrecht, The Netherlands, 2001. p 205.
19. Combs, G. F., Jr.; Gray, W. P. Pharmacol. Ther. 1998, 79, 179.
20. Wei, Z. L.; Kozikowski, A. P. J. Org. Chem. 2003, 68, 9116.
21. Synthesis of 2-Methyl-4-[[[4-methyl-2-[4-(trifluoromethyl)phenyl]-thiazol-
5-yl]methyl]selenyl]phenoxy]acetic acid (2). To a stirred solution of o-cresol
(3) (5.4 g, 50 mmol), potassium selenocyanate (23.0 g, 160 mmol), and
methanol (40 mL) at 0 °C was added a solution of potassium bromide (5.95 g,
50 mmol) and bromine (8.0 g, 2.65 mL, 50 mmol) in methanol (60 mL). The
mixture was stirred for 3 h and then diluted with saturated NaHCO3 solution.
The mixture was extracted with CH2Cl2 (3 Â 150 mL), and the organic phases
were combined, washed with brine, dried (MgSO4), and concentrated. The
residue was purified by silica gel column chromatography (ethyl acetate/
hexanes 1:4) to give 2-methyl-4-selenocyanatophenol (4) as a light yellow
solid (8.8 g, 83%). 1H NMR (500 MHz, CDCl3) d 7.33 (d, 1H, J = 2.4 Hz), 7.26 (dd,
1H, J = 8.4, 2.4 Hz), 6.79 (d, 1H, J = 8.4 Hz), 6.00 (br s, 1H), 2.23 (s, 3H); 13C NMR
(125 MHz, CDCl3) 156.3, 135.1, 131.5, 126.9, 116.6, 112.6, 112.5, 15.8. To a
solution of 4 (2.12 g, 10 mmol) in ethanol at 0 °C under nitrogen atmosphere,
was added sodium borohydride (1.9 g, 50 mmol) portion wise. The solution
turns yellow and then colorless within 5 min. To this mixture at 0 °C was then
added 5 (2.91 g, 10 mmol), the reaction was warmed to room temperature, and
stirred for 3 h. The solvent was concentrated to about 10 mL under reduced
pressure, diluted with ethyl acetate and washed twice with water. The organic
layer was dried (MgSO4), filtered, and the solvent was evaporated to give a pale
yellow solid which was further purified by silica gel column chromatography
(ethyl acetate/hexanes 1:3) to yield 4.1 g (92%) of 2-methyl-4-[[[4-methyl-2-
[4-(trifluoromethyl) phenyl]thiazol-5-yl]methyl]selenyl]phenol (6) as a white
solid. Mp138–139 °C. 1H NMR (500 MHz, CDCl3) d 2.08 (s, 3H, CH3), 2.21 (s, 3H,
CH3), 4.11 (s, 2H, SeCH2), 6.63 (d, J = 8.0 Hz, 1H), 7.12 (dd, J = 8.0 and 1.5 Hz,
1H), 7.30 (d, J = 1.5 Hz, 1H), 7.67 (d, J = 8.5 Hz, 1H), 7.98 (d, J = 8.0 Hz, 1H);
HRMS (ESI) m/z calcd for C19H16F3NOSSeÁH+, 444.0143; found: 444.0137. To a
stirred solution of 6 (1.33 g, 3.0 mmol) in CH3CN at room temperature was
added Cs2CO3 (1.47 g, 4.5 mmol) followed by methyl bromoacetate (0.6 g,
0.36 mL, 3.9 mmol) and the reaction mixture was stirred for 2 h. Water was
added to the reaction mixture and it was extracted with EtOAc (2Â 100 mL).
The organic layers were combined and washed with water again, dried over
MgSO4 and concentrated. The crude solid thus obtained was purified by silica
gel column chromatography (ethyl acetate/hexane 1:4) to yield 1.44 g (93%) of
Figure 3. Ligand activation of PPARb/d inhibits cell proliferation of HaCaT kerat-
inocytes. HaCaT cells were treated with either GW501516 (A) or selenium analog 2
(B) with the indicated concentration of ligand (arrow) in the presence of culture
medium with serum and cell number was quantified. Values represent mean-
s
SEM. *Significantly different values (P < 0.05) from vehicle (DMSO) at the
particular time point, as determined by ANOVA and Bonferroni’s multiple compar-
ison test.
therefore be used a chemical tool to study the function of the ubiq-
uitously expressed PPARb/d.
Acknowledgments
Methyl
[2-methyl-4-[[[4-methyl-2-[4-(trifluoromethyl)-phenyl]thiazol-5-
yl]methyl]selenyl]phenoxy]acetate (7) as a white solid. Mp107–108 °C. 1H
NMR (500 MHz, CDCl3) d 2.19 (s, 3H, CH3), 2.26 (s, 3H, CH3), 3.82 (s, 3H, OCH3),
4.15 (s, 2H, SeCH2), 4.67 (s, 2H, OCH2), 6.59 (d, J = 8.5 Hz, 1H), 7.26 (dd, J = 8.5
and 1.5 Hz, 1H), 7.33 (d, J = 1.5 Hz, 1H), 7.69 (d, J = 8.0 Hz, 1H), 8.00 (d,
This study was supported by the Penn State Hershey Cancer
Institute of the Penn State College of Medicine. The authors thank
Solution Phase NMR Facility at Core Research Facilities of the Penn
State College of Medicine for recording of NMR spectra.
J = 8.0 Hz, 1H); HRMS (ESI) m/z calcd for C22H20F3NO3SSeÁH+ 516.0354; found:
,
516.0373. To a solution of 7 (1.0 g, 1.95 mmol) in 30 mL of THF and 20 mL of
H2O at 0 °C was added slowly 2.0 mL (4.0 mmol) of 2.0 M LiOH and the reaction
mixture was stirred at 0 °C for 1 h. The reaction mixture was diluted with
water (30 mL), acidified with 0.5 M NaHSO4 (7 mL), and extracted with a mixed
solvent of EtOAc and THF (3:1, 4Â 50 mL). The combined organic fractions
were briefly dried over Na2SO4 and concentrated. The residue was purified by
silica gel column chromatography with CH2Cl2/MeOH (95:5) to yield 0.89 g
(91%) of 2 as a white solid. Mp 144–145 °C. 1H NMR (500 MHz, CDCl3) d 2.08 (s,
3H, CH3), 2.24 (s, 3H, CH3), 4.13 (s, 2H, SeCH2), 4.70 (s, 2H, OCH2), 6.61 (d,
J = 8.5 Hz, 1H), 7.21 (dd, J = 8.5 and 2.0 Hz, 1H), 7.29 (d, J = 2.0 Hz, 1H), 7.69 (d,
J = 8.0 Hz, 1H), 8.01 (d, J = 8.0 Hz, 1H); HRMS (ESI) m/z calcd for
References and notes
1. Lee, C. H.; Olson, P.; Evans, R. M. Endocrinology 2003, 144, 2201.
2. Peraza, M. A.; Burdick, A. D.; Marin, H. E.; Gonzalez, F. J.; Peters, J. M. Toxicol. Sci.
2006, 90, 269.
3. Peters, J. M.; Cheung, C.; Gonzalez, F. J. J. Mol. Med. 2005, 83, 774.
4. Willson, T. M.; Brown, P. J.; Sternbach, D. D.; Henke, B. R. J. Med. Chem. 2000, 43,
527.
5. Lee, C. H.; Olson, P.; Hevener, A.; Mehl, I.; Chong, L. W.; Olefsky, J. M.; Gonzalez,
F. J.; Ham, J.; Kang, H.; Peters, J. M.; Evans, R. M. Proc. Natl. Acad. Sci. U.S.A. 2006,
103, 3444.
6. Grimaldi, P. A. Biochim. Biophys. Acta 2007, 1771, 983.
7. Burdick, A. D.; Kim, D. J.; Peraza, M. A.; Gonzalez, F. J.; Peters, J. M. Cell. Signalling
2006, 18, 9.
8. Peters, J. M.; Hollingshead, H. E.; Gonzalez, F. J. Clin. Sci. (Lond.) 2008, 115, 107.
9. Peters, J. M.; Gonzalez, F. J. Biochim. Biophys. Acta 2009, 1796, 230.
10. Sznaidman, M. L.; Haffner, C. D.; Maloney, P. R.; Fivush, A.; Chao, E.; Goreham,
D.; Sierra, M. L.; LeGrumelec, C.; Xu, H. E.; Montana, V. G.; Lambert, M. H.;
Willson, T. M.; Oliver, W. R., Jr.; Sternbach, D. D. Bioorg. Med. Chem. Lett. 2003,
13, 1517.
11. Oliver, W. R., Jr.; Shenk, J. L.; Snaith, M. R.; Russell, C. S.; Plunket, K. D.; Bodkin,
N. L.; Lewis, M. C.; Winegar, D. A.; Sznaidman, M. L.; Lambert, M. H.; Xu, H. E.;
Sternbach, D. D.; Kliewer, S. A.; Hansen, B. C.; Willson, T. M. Proc. Natl. Acad. Sci.
U.S.A. 2001, 98, 5306.
C
21H18F3NO3SSeÁH+, 502.0197; found: 502.0222.
22. Borland, M. G.; Foreman, J. E.; Girroir, E. E.; Zolfaghari, R.; Sharma, A. K.; Amin,
S.; Gonzalez, F. J.; Ross, A. C.; Peters, J. M. Mol. Pharmacol. 2008, 74, 1429.
23. Kim, D. J.; Bility, M. T.; Billin, A. N.; Willson, T. M.; Gonzalez, F. J.; Peters, J. M.
Cell Death Differ. 2006, 13, 53.
24. Cell proliferation analyses: Cell proliferation was examined as previously
described.21 Briefly, HaCaT keratinocytes were plated on a 12-well plate at a
density of 20,000 cells/well 24 h before cell counting at time 0. Cell
proliferation was determined using a Z1 Coulter particle counter (Beckman–
Coulter, Hialeah, FL). Cells were then cultured for 24 h before ligand treatment.
After this 24-h period, cells were maintained in DMEM with or without serum
and treated with control (DMSO), GW0742 or GW501516 for up to an
additional 72 h period. The concentration of GW0742 and GW501516 used for
all experiments ranged from 0.1 to 10.0 lM, because these concentrations have
been shown to specifically activate PPARb/d in case of GW501516.22 Cell
number was quantified every 24 h. Triplicate samples for each treatment were
used for each time point, and each replicate was counted three times.
12. Pelton, P. Curr. Opin. Investig. Drugs 2006, 7, 360.