Synthesis of a novel human PPARδ selective agonist and its stimulatory effect on oligodendrocyte differentiation

Article abstract of DOI:10.1016/j.bmcl.2010.11.030

We successfully synthesized a novel peroxisome proliferator-activated receptor (PPAR)δ selective agonist, namely, compound 20, with a characteristic benzisoxazole ring. Compound 20 exhibited potent human PPARδ transactivation activity and high δ selectivity. Further, it stimulated differentiation of primary oligodendrocyte precursor cells in vitro, indicating that it may be an effective drug in the treatment of demyelinating disorders such as multiple sclerosis.

Full text of DOI:10.1016/j.bmcl.2010.11.030

Bioorganic & Medicinal Chemistry Letters 21 (2011) 240–244
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of a novel human PPARd selective agonist and its stimulatory effect
on oligodendrocyte differentiation
⇑
Shogo Sakuma , Tsuyoshi Endo, Takashi Kanda, Hideki Nakamura, Satomi Yamasaki, Tomio Yamakawa
Discovery Research Laboratories, Nippon Chemiphar Co Ltd, 1-22, Hikokawado, Misato, Saitama 341-0005, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 May 2010
Revised 29 October 2010
Accepted 4 November 2010
Available online 11 November 2010
We successfully synthesized
a novel peroxisome proliferator-activated receptor (PPAR)d selective
agonist, namely, compound 20, with a characteristic benzisoxazole ring. Compound 20 exhibited potent
human PPARd transactivation activity and high d selectivity. Further, it stimulated differentiation of pri-
mary oligodendrocyte precursor cells in vitro, indicating that it may be an effective drug in the treatment
of demyelinating disorders such as multiple sclerosis.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Multiple sclerosis
PPARd agonist
Compound 20
Benzisoxazole ring
Oligodendrocyte
Multiple sclerosis (MS) is an idiopathic disease suspected to be
caused by an autoimmune disorder: the immune response is direc-
ted at the central nervous system (CNS; the brain and spinal cord),
resulting in demyelination.1 This disease usually develops in young
adults, and it is more common in females.2 Its prevalence ranges
between 2 and 150 per 100,000 individuals.3 However, there is
no known cure for MS. Many patients do well without therapy,
especially since many drugs have serious side effects and some
pose significant risks. However, 3 forms of b-interferon have now
been approved by the Food and Drug Administration (FDA) of
USA for the treatment of relapsing-remitting MS. b-Interferon has
been shown to reduce the number of exacerbations and thus slow
the progression of physical disability. If and when attacks occur,
they tend to be shorter and less severe. In clinical trials, monoclo-
nal antibody therapy with natalizumab was shown to significantly
reduce the frequency of attacks in people with relapsing forms of
MS, and this drug was approved for marketing by the FDA in
2004. However, in 2005 the drug manufacturer voluntarily sus-
pended marketing of the drug after several reports of significant
adverse events. Although steroids do not affect the course of MS
over time, they can reduce the duration and severity of attacks in
some patients. Other drugs, including amantadine, pemoline, and
the experimental drug aminopyridine, may reduce fatigue in some
patients.
Although optic symptoms usually improve even without treat-
ment, a short course of treatment with intravenous methylprednis-
olone followedby oral steroidtreatmentis sometimes administered.
As mentioned above, drugs for MS treatment that are currently
in circulation present various problems in terms of effectiveness,
administration routes, and even safety. Therefore, basic research
on the possibility of developing peroxisome proliferator-activated
receptor (PPAR)d agonists as new candidates for MS treatment
was conducted using data from recent studies.
PPARs are nuclear receptors that function after heterodimeriza-
tion with other nuclear receptors, such as retinoid X receptors
(RXRs). They regulate the transcription of the PPAR target gene
cluster by identifying specific gene sequences and binding to them.
Three PPAR subtypes, designated as PPAR
a
, PPARd (b), and PPAR
c,
have been identified. Recent advances in PPAR
a
and PPAR
c
research have revealed important roles of these PPARs in lipid
and glucose metabolism. However, the functions of PPARd are
not well understood. The availability of PPARd transgenic mice
and the discovery of PPARd agonists, especially GW-5015164 and
L-1650415 (Fig. 1), have stimulated PPARd research, and many
reports have shown that PPARd activation leads to interesting
effects such as antiobesity, improved lipid metabolism, and wound
healing.6–10
PPAR is being closely monitored as the molecular target of a
novel treatment agent for lifestyle-related diseases caused by
glycolipidoses, and many molecular ligands have been studied.
Abbreviations: MS, multiple sclerosis; CNS, central nervous system; OPCs,
oligodendrocyte precursor cells; PPAR, peroxisome proliferator-activated receptor.
However, the use of PPAR
c agonist poses many challenges such
as occurrence of side effects in ischemic cardiac disease cases
and carcinogenic effects. Therefore, many companies have stopped
⇑
Corresponding author. Tel.: +81 48 952 4311; fax: +81 48 952 0743.
E-mail address: s-sakuma@chemiphar.co.jp (S. Sakuma).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.11.030

S. Sakuma et al. / Bioorg. Med. Chem. Lett. 21 (2011) 240–244
241
demyelination of the CNS. In the CNS tissue of MS patients, oligo-
dendrocyte precursor cells (OPCs) are found to survive the demye-
linating damage in MS, but they apparently fail to proliferate and
differentiate.12 Therefore, potent and selective PPARd agonists,
which can stimulate oligodendrocyte differentiation, are expected
to be a possible remedy for dysmyelination and demyelinating
diseases.
Figure 1. Structures of representative PPARd agonists (1: GW-501516 and 2:
L-165041).
The synthesis of compound 20 and its key intermediates 8 and
15 is described below. The key intermediate 8 was synthesized as
shown in Scheme 1. Compound 4, which was obtained by reacting
ethyl isobutyrylacetate (3) with hydroxylamine, was reduced by
zinc to produce the amine form and then treated with 2,4-dic-
hlorobenzoyl chloride by the Schotten–Baumann method to obtain
compound 5. Compound 5 was treated with phosphorus oxychlo-
ride to obtain compound 6, an oxazole derivative. This three-step
procedure to synthesize compound 6 was achieved with a 43%
yield. Compound 6 was treated with LAH to obtain the alcohol
compound 7, which was then treated with thionyl chloride to pro-
duce 4-chloromethyl-2-(2,4-dichlorophenyl)-5-isopropyloxazole
(8).13 The key intermediate 15 was synthesized as shown in
Scheme 2. Aniline 10, obtained by catalytic hydrogenation of
4-methyl-3-nitrophenol (9), was acetylated to produce 11. In the
next step, compound 11 was treated with AlCl3 at a high tempera-
ture to produce compound 12 with a 79% yield. Compound 12 was
treated with hydroxylamine to obtain compound 13, which was
treated with acetic acid anhydride to obtain compound 14 and
then treated with pyridine to obtain the key intermediate 6-ace-
toamido-3,5-dimethyl benzisoxazole (15).14 Compound 20 was
synthesized as shown in Scheme 3. Compounds 8 and 15 were con-
densed with LDA to obtain compound 16, which was then treated
with hydrochloric acid to produce compound 17. This compound
was then treated with sodium nitrite and then hydrolyzed with
manufacturing PPAR agonist as a treatment agent for lifestyle-
related diseases. Further, a high level of safety needs to be assured
during long-term administration of a new PPAR agonist to patients.
Taken together, the development of a PPAR agonist as a treatment
agent for lifestyle-related diseases comes at a huge cost and risk.
On the other hand, in recent years, research on the pleiotropic ac-
tion of PPAR has shown that PPARd has a wide tissue distribution,
suggesting that PPARd agonist may also be effective against condi-
tions other than lifestyle-related diseases. A study was therefore
carried out with an aim to determine conditions other than
lifestyle-related diseases that could possibly be managed by the
effective use of the PPARd agonist. The results of this study showed
that intractable multiple sclerosis could be a candidate disease.
Currently, only few drugs have been found to be effective against
multiple sclerosis. We therefore think that a revolutionary devel-
opment of a new drug could be possible if a short-term clinical trial
of the PPARd agonist shows the effectiveness of this agent.
PPARd agonists have been reported to have an oligodendrocyte
differentiation stimulating effect.11 Oligodendrocytes are members
of the glial cell family and form the central myelin sheath. Any dis-
ease of the nervous system in which the myelin sheath of neurons
is damaged is considered as a demyelinating disorder. The most
common demyelinating disorder is MS, which is characterized by
Scheme 1. Synthesis of compound 8. Reagents and conditions: (a) NaNO2, AcOH–H2O, 0 °C, 100%; (b) (1) Zn, 30% H2SO4, 0 °C and (2) 2,4-dichlorobenzoylchloride, 0 °C to rt;
(c) POCl3, Ph–H, reflux, three-steps 43%; (d) LAH, THF, 0 °C, 96%; (e) SOCl2, Ph–H, reflux, 100%.
Scheme 2. Synthesis of compound 15. Reagents and conditions: (a) H2, Pd–C, EtOH, rt, quantitative yield; (b) Ac2O, pyridine, rt, 71%; (c) AlCl3, 130 °C, 79%; (d) NH2OHÁHCl,
EtOH, rt, 91%; (e) Ac2O, 130 °C, 94%; (f) pyridine, reflux, 86%.

242
S. Sakuma et al. / Bioorg. Med. Chem. Lett. 21 (2011) 240–244
Scheme 3. Synthesis of compound 20. Reagents and conditions: (a) LDA, THF, À78 °C, 45%; (b) 3 M HCl, AcOH, reflux, 83%; (c) (1) NaNO2, 25% H2SO4, 0 °C and (2) 75% H2SO4,
120 °C, 43%; (d) ethyl bromoacetate, K2CO3, acetone, rt, 96%; (e) 1 M NaOH, EtOH, rt, 76%.
Table 2
Table 1
PPARs transactivation activity of compound 20 as assessed by cell-based transacti-
PPARs transactivation activity of compound 20 and representative PPARs agonists as
vation assay, using the full-length receptor (full-length hPPAR + hRXRa)
assessed by a cell-based transactivation assay, using GAL4 DBD-hPPAR LBD
Compound
Transactivation activity EC50 (lM)
Compound
Transactivation activity EC50
(lM)
PPAR
a
PPAR
>10
c
PPARd
PPAR
>10
0.010
>10
a
PPARc
PPARd
20
>10
0.0045
20
>10
>10
0.10
4.1
0.025
2.6
>10
GW-590735
Rosiglitazone
GW-501516
0.99
0.0017
agonist), and 0.1
respectively.
lM GW-501516 (PPARd selective agonist),
The calculated 50% effective concentration (EC50) of compound
20 confirmed that this compound had potent hPPARd transactiva-
tion activity (EC50 = 0.025 lM) and a high d selectivity (Table 1)
(Fig. 2). In the cell-based assay using the full-length receptor and
peroxisome proliferator response element, compound 20 acted as
a complete human PPARd agonist with an EC50 of 4.5 nM. With
the full-length human receptor, compound 20 showed about
75% sulfuric acid to produce compound 18. Compound 18 was con-
densed with ethyl bromoacetate in the presence of potassium car-
bonate in acetone to obtain compound 19, which was then treated
with sodium hydroxide in ethanol to obtain compound 20.15
The human PPAR transactivation activity of compound 20 was
evaluated by the following method. The mammalian expression
vectors used were pSG5-GAL4-hPPAR
pSG5-GAL4-hPPARd, which express the ligand binding domains
of human PPAR , PPAR , and PPARd; each of these vectors were
a, pSG5-GAL4-hPPARc, and
2000-fold selectivity for PPARa and PPARc (Table 2) (Fig. 3).
This research was conducted on the basis of the forecast that if
the PPARd agonist stimulated the differentiation of Adult Oliogo-
dendrocyte Precursor Cells (Adult OPCs) inside the brain of
patients with MC, it could regenerate myelin. With these Adult
OPCs as the target cells, the OPCs of the brain of a rat fetus were
used as the model. As the OPCs differentiate into mature oliogo-
dendrocyte in the cell lineage, they specifically develop proteins.
Among the proteins, O1 and MBP were selected as the differentia-
tion markers. Within the cell lineage, these proteins exist in the
relatively late stage; in other words, they reach the mature oliogo-
dendrocyte stage just before myelin is formed in the immature
a
c
fused to the yeast transcription factor GAL4 DNA binding do-
main.16 Each receptor expression vector and the UASx4-TK-LUC17
reporter plasmid were co-transferred into CV-1 cells (kidney fibro-
blasts isolated from an African green monkey). After the addition of
compound 20 (0.001–100
and luciferase activity was measured. PPAR
transactivation activity of the test compounds was calculated rela-
tive to the luciferase activity induced by 1
M GW-59073518
(PPAR selective agonist), 10 M rosiglitazone (PPAR selective
lM), CV-1 cells were incubated for 40 h,
a
, PPAR , and PPARd
c
l
a
l
c
Figure 2. PPAR transactivation activities of compound 20 in the cell-based
Figure 3. PPAR transactivation activities of compound 20 in the cell-based
transactivation assay, using GAL4 DBD-hPPAR LBD.
transactivation assay, using full-length hPPAR + hRXRa.

S. Sakuma et al. / Bioorg. Med. Chem. Lett. 21 (2011) 240–244
243
Figure 4. Oligodendrocyte differentiation stimulating effect of compound 20. Seven-day-old primary oligodendrocyte cultures were immunostained with anti-O1 antibody.
Compound 20 was added to the cultures 3 days prior to fixation. In the cultures treated with compound 20, an increase in the number of stained cells, membrane sheet
formation, and increased cell diameter were observed.
oliogodendrocyte. For this evaluation, O1 was mainly used. The
evaluation methods and an outline of the results are described
below.
rat fetal brains by an immunostaining method and confirmed to
have a strong stimulatory effect on oligodendrocyte differentiation.
The lack of mutagenicity of compound 20 has previously been con-
firmed with Ame’s test; moreover, it passed the hERG test. We be-
lieve that compound 20 may be an effective drug for the treatment
of demyelinating diseases such as MS.
In vitro tests were conducted to evaluate the stimulatory effects
of compound 20 on the differentiation of OPCs. These cells were
prepared from the primary mixed cell cultures of the fetal brain
cortex from Wistar rats,19 and an OPC-rich fraction was prepared
using the Percoll gradient separation technique. After 4 days of cul-
ture, OPCs were treated with the test compounds. The differentia-
tion stimulatory effect was evaluated by immunostaining.
Compound 20 was dissolved in dimethylsulfoxide (DMSO), and
the final concentration of DMSO in the medium was 0.01%. Each
dissolved compound was added to the culture medium such that
Acknowledgements
The authors would like to thank Dr. Masui for extending support
during the length of our research period. We are also deeply grateful
to Mr. Kobayashi and Mr. Tashiro for their contribution to the
synthesis and analysis of compound 20 and also to Dr. Asou from
the Tokyo Metropolitan Institute of Gerontology for the guidance re-
lated to the establishment of a system to assess oligodendrocytes.
the final concentration was 0.1 lM. For the control groups, 0.01%
DMSO was added to the culture medium. The cells on a cover glass
were incubated for 3 days at 37 °C under a 5% CO2 atmosphere.
Thereafter, the cells were immunostained using an anti-oligoden-
drocyte marker O1 antibody and placed on a glass slide. Random
slide images of the specimens were selected and processed, and
the areas of stained cells were measured. The mean stained area
of the control specimen was considered as 100%, and the mean
stained area of the treated specimens was calculated relative to
this control value. The degree of differentiation was determined
on the basis of the increase in the number of stained cells, increase
in the diameter of stained cells, and membrane sheet formation.
The images of the stained cells in the control and test-com-
pound-treated specimens are shown in Figure 4. As compared to
References and notes
1. Compston, A.; Coles, A. Lancet 2008, 372, 1502.
2. Debouverie, M.; Pittion-Vouyovitch, S.; Louis, S.; Guillemin, F. Eur. J. Neurol.
2008, 9, 916.
3. Rosati, G. Neurol. Sci. 2001, 2, 117.
4. Oliver, W. R.; Sheck, J. L.; Sanith, M. R.; Russell, D. A.; Sznaidman, M. L.; Xu, H.
E.; Sternbatch, D. D.; Kliewer, S. A.; Hansen, B. C.; Wilson, T. M. Proc. Natl. Acad.
Sci. U.S.A. 2001, 98, 5306.
5. Berger, J.; Leibowitz, M. D.; Doebber, T. W.; Elbrecht, A.; Zhang, B.; Zhou, G.;
Biswas, C.; Cullinan, C. A.; Hayes, N. S.; Li, Y.; Tanen, M.; Ventre, J.; Wu, M. S.;
Berger, G. D.; Mosley, R.; Marquis, R.; Santini, C.; Sahoo, S. P.; Tolman, R. L.;
Smith, R. G.; Moller, D. E. J. Biol. Chem. 1999, 274, 6718.
6. Tanaka, T.; Yamamoto, J.; Iwasaki, S.; Asaba, H.; Hamura, H.; Ikeda, Y.;
Watanabe, M.; Magoori, K.; Ioka, R. X.; Tachibana, K.; Watanabe, Y.; Uchimura,
Y.; Sumi, K.; Iguchi, H.; Ito, S.; Doi, T.; Hamakubo, T.; Auwerx, J.; Yanagisawa,
M.; Kodama, T.; Sakai, J. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 15924.
7. Wang, Y. X.; Lee, G. H.; Tiep, S.; Yu, R. T.; Ham, J.; Kang, H.; Evans, R. M. Cell
2003, 113, 159.
the control cells, the cells treated with 0.1
showed a greater area of staining.
lM of compound 20
In conclusion, the results of this study show that the novel
PPARd agonist compound 20, which has a characteristic benzisox-
azole ring, has strong hPPARd transactivation activity and high d
selectivity. Compound 20 was examined in vitro using OPCs from
8. Dressel, U.; Allen, T. L.; Pippal, J. B.; Rohde, P. R.; Lau, P.; Muscat, G. E. Mol.
Endocrinol. 2003, 17, 2477.

244
S. Sakuma et al. / Bioorg. Med. Chem. Lett. 21 (2011) 240–244
9. Luquet, S.; Lopez-Soriano, J.; Holst, D.; Fredenrich, A.; Melki, J.; Rassoulzadegan,
9 Hz), 7.52 (d, 1H, J = 2 Hz), 7.88 (d, 1H, J = 9 Hz).
M.; Grimaldi, P. A. FASEB J. 2003, 17, 22994.
10. Chong, H. C.; Tan, M. J.; Philippe, V.; Tan, S. H.; Tan, C. K.; Ku, C. W.; Goh, Y. Y.;
Wahli, W.; Michalik, L.; Tan, N. S. J. Cell Biol. 2009, 184, 817.
Mp (dec) 182–184 °C. HRMS calcd for C24H22Cl2N2O5: 488.0906 [M]+, found
488.0885.
16. Lehmann, J. M.; Lenhard, J. M.; Oliver, B. B.; Ringold, G. M.; Kliever, S. A. J. Biol.
Chem. 1997, 272, 3406.
11. Saluja, I.; Granneman, J. G.; Skoff, R. P. Glia 2001, 33, 191.
12. Wolswijk, G. J. Neurosci. 1998, 18, 601.
17. Forman, B. M.; Umesono, K.; Chen, J.; Evans, R. M. Cell 1995, 81, 541.
18. Sierra, M. L.; Beneton, V.; Boullay, A. B.; Boyer, T.; Brewster, A. G.; Donche, F.;
Forest, M. C.; Fouchet, M. H.; Gellibert, F. J.; Grillot, D. A.; Lambert, M. H.;
Laroze, A.; Le Grumelec, C.; Linget, J. M.; Montana, V. G.; Nguyen, V. L.;
Nicodème, E.; Patel, V.; Penfornis, A.; Pineau, O.; Pohin, D.; Potvain, F.; Poulain,
G.; Ruault, C. B.; Saunders, M.; Toum, J.; Xu, H. E.; Xu, R. X.; Pianetti, P. M. J. Med.
Chem. 2007, 50, 685.
13. 4-Chloromethyl-2-(2,4-dichlorophenyl)-5-isopropyloxazole (8) 1H NMR
(CDCl3, 400 MHz) d: 1.34 (d, 6H, J = 7 Hz), 3.0–3.2 (m, 1H), 4.63 (s, 2H), 7.33
(dd, 1H, J = 2 Hz, 9 Hz), 7.51 (d, 1H, J = 2 Hz), 7.92 (d, 1H, J = 9 Hz).
14. 6-Acetoamido-3,5-dimethylbenzisoxazole (15) 1H NMR (CDCl3, 400 MHz) d:
2.26 (s, 3H), 2.38 (s, 3H), 2.53 (s, 3H), 7.14 (s, 1H), 7.39 (s, 1H), 8.34 (s, 1H).
15. [3-[2-[2-(2,4-Dichloro)
phenyl-4-isopropyl-4-oxazolyl]ethyl]-5-methyl-1,2-
benzisoxazol-6-yloxy]acetic acid (20) 1H NMR (CDCl3, 400 MHz) d: 1.12 (d,
6H, J = 7 Hz), 2.24 (s, 3H), 2.9–3.0 (m, 1H), 3.04 (dd, 2H, J = 7 Hz, 8 Hz), 3.27 (dd,
2H, J = 7 Hz, 8 Hz), 4.74 (s, 2H), 6.82 (s, 1H), 7.20 (s, 1H), 7.33 (dd, 1H, J = 2 Hz,
19. Yoshimura, K.; Sakurai, Y.; Nishimura, D.; Tsuruo, Y.; Nomura, M.; Kawato, S.;
Seiwa, C.; Iguchi, T.; Itoh, K.; Asou, H. J. Neurosci. Res. 1998, 54, 79.