Discovery of a novel class of 2-mercaptohexanoic acid derivatives as highly active PPARα agonists

Article abstract of DOI:10.1016/j.bmcl.2009.05.057

A novel and robust scaffold for highly active PPARα agonists based on the 2-mercaptohexanoic acid substructure is presented. Systematic structural variation of the substitution pattern of the phenolic backbone yielded detailed SAR especially of ortho and

Full text of DOI:10.1016/j.bmcl.2009.05.057

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4421–4426
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of a novel class of 2-mercaptohexanoic acid derivatives
as highly active PPARa agonists
Heiko Zettl a, Ramona Steri a, Michael Lämmerhofer b, Manfred Schubert-Zsilavecz a,
*
a Goethe-University Frankfurt, Institute of Pharmaceutical Chemistry/ZAFES/LiFF, Max-von-Laue-Str. 9, D-60348 Frankfurt/M., Germany
b University of Vienna, Department for Analytical Chemistry and Food Chemistry, Währinger Str. 38, A-1090 Vienna, Austria
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel and robust scaffold for highly active PPAR
structure is presented. Systematic structural variation of the substitution pattern of the phenolic back-
bone yielded detailed SAR especially of ortho and meta substituents. We corroborated the importance
a
agonists based on the 2-mercaptohexanoic acid sub-
Received 17 April 2009
Revised 13 May 2009
Accepted 14 May 2009
Available online 18 May 2009
of the sulfur atom as well as of the n-butyl chain for PPAR
group by preparation of carbon analogs and -unsubstituted derivatives. Compound 10 represents a low
nano molar active PPAR activator with excellent selectivity towards PPAR
a activity in the 2-mercaptohexanoic acid head
a
a
c.
Keywords:
PPAR
Ó 2009 Elsevier Ltd. All rights reserved.
2-Mercaptohexanoic acid
SAR
PPARa agonists
The peroxisome proliferator-activated receptors (PPARs) are
among today’s most prominent targets for antidiabetic and antilip-
idemic drugs. Activation of different PPAR subtypes (a, b, c) leads
to elevated expression of apolipoproteins and cellular glucose
transporters, resulting in enhanced clearance of lipids and glucose
Synthetic PPAR agonists commonly share a general structure
mimicking those of fatty acids, which serve as endogenous li-
gands.6 In general, the structural features include an acidic head
group combined with a large lipophilic backbone. Subtype selectiv-
ity may be influenced by two means: either by the introduction of
from the blood. PPAR
for the treatment of dyslipidemias, whereas PPAR
a
-selective fibrates are well established drugs
a lipophilic a-substituent or by varying size and branching of the
c-selective glit-
backbone of the molecule (Fig. 1).7
azones have been proven as valuable drugs for the treatment of
In previous studies, we used pirinixic acid (WY14,643; 1) as a
scaffold for structural optimization. By introducing alkyl chains
type 2 diabetes.1
PPARa
is mainly expressed in tissues with a high rate of fatty
in
a-position of the carboxylic acid we achieved an enhanced activ-
acid catabolism such as liver and skeletal muscle. Its activation
by fibrates has been proven to be efficient in lowering triglyceride
levels accompanied by a moderate elevation of HDL–cholesterol.
The clinical benefits are nevertheless limited, as several large clin-
ical trials failed to show a reduction in cardiovascular mortality.2,3
In respect to their pharmacological profiles, these drugs have some
significant detrimental properties: Exhibiting EC50 values in the
ity for both PPAR
a
and PPAR and identified an optimal chain
c
length of 4–6 carbons, represented by n-butyl-substituted deriva-
tive 2.8
For this study, we replaced the central pyrimidine ring of 2 by
benzene and the amine linker by a larger propane-1,3-diol moiety.
We maintained the 2-mercaptohexanoic acid substructure as the
acidic head group for the first series (Fig. 2), and systematically
varied the cyclic tail of the molecule in order to evaluate the SAR
of various alkyl and alkoxy substituents in ortho and meta posi-
tions. The 2,3-dimethylphenyl-moiety has been retained in the
micro molar range, they are neither very potent PPAR
a agonists
nor highly selective versus other PPAR subtypes.1 Thus, the ques-
tion remains if highly active and selective PPAR
a agonists would
provide better clinical prospects.4 State-of-the-art compounds,
such as the recently reported 1,3-dioxane-2-carboxylic acid deriv-
second series (Fig. 3) to evaluate the influence of the
chain (by preparing the -unsubstituted analogs) and of the sulfur
a-n-butyl-
a
ative NS220, display nano molar PPAR
a
activity in vitro and very
(by replacement with a methylene group).
promising hypolipidemic properties in mice models.5
The 2-mercaptohexanoic acid derivatives 6–21 were synthe-
sized starting with 4-mercaptophenol and ethyl 2-bromohexano-
ate as shown in Scheme 1. Ethyl 2-bromoacetate was used as the
ester component for the preparation of 21. Nucleophilic substitu-
* Corresponding author. Tel.: +49 69 79829339; fax: +49 69 79829332.
E-mail address: Schubert-zsilavecz@pharmchem.uni-frankfurt.de (M. Schubert-
Zsilavecz).
tion with the
a-bromoester first yielded the thioether derivatives
3a and 3b.9 Two equal synthetic pathways could then be used to
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.05.057

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H. Zettl et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4421–4426
Figure 1. General structure of synthetic PPAR agonists.
Figure 2. Structural development of presented compounds according to previous studies.8,15
introduce the propylene spacer via a Williamson-like ether synthe-
sis with 3-bromopropan-1-ol (step b): either by direct coupling to
3a to yield 4 or by reaction with the commercially available phenol
derivatives to yield the precursors 5a–5e. Subsequent arrangement
of the second ether function was achieved by a Mitsunobu reaction
(6a–21a).10,11 Finally, hydrolysis with lithium hydroxide gave the
desired carboxylic acids 6–21.
The carbon analogs of 11 were prepared starting with the con-
version of ethyl 2-bromohexanoate into its phosphonate derivative
22 by an Arbuzov reaction (Scheme 2).12 This was coupled via a
Wittig–Horner reaction to the aldehyde group of the precursor 24,
which was synthesized based on 4-hydroxybenzaldehyde.13 The
resulting ester 26a could be directly hydrolyzed to yield 26 or
Figure 3. SAR-investigation of the 2-mercaptohexanoic acid head group based on
compound 11.
Scheme 1. Reagents and conditions: (a) ethyl-2-bromoacetate (Z = H) or ethyl-2-bromohexanoate (Z = nBu), Et3N, CHCl3, reflux, 1 h; (b) 3-bromopropanol, K2CO3, ACN, reflux,
24-48 h; (c) DEAD or ADDP, TPP, THF, rt, 4–48 h; (d) LiOH, MeOH, H2O, 40 °C, 2 h.

H. Zettl et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4421–4426
4423
Scheme 2. Reagents and conditions: (a) 3-bromopropanol, K2CO3, ACN, reflux, 24 h; (b) triethylphosphite, reflux, overnight; (c) 4-hydroxybenzaldehyde or ethyl 3-(4-
hydroxyphenyl)propanoate, DEAD, TPP, THF, rt, 4 h; (d) NaH, THF, rt, 24 h; (e) Pd/C, H2, EtOH, rt, 24 h; (f) LiOH, MeOH, H2O, reflux, 8–20 h.
hydrogenated and afterwards hydrolyzed to yield 27. Only two
steps (Mitsunobu condensation and hydrolysis) were needed for
ents based on the various methyl analogs. We thus prepared meth-
oxy and isopropyl ortho and meta analogs. Both ortho-substituted
the preparation of the
a-unsubstituted carbon analog 25 as the
compounds (13 and 15) showed decreased PPARa activity. The
precursor molecule ethyl 3-(4-hydroxyphenyl)propanoate was
most significant loss was observed for the largest substituent
commercially available.
(15; isopropyl). meta-Methoxy substituted 14 was able to further
The final compounds were tested in vitro for PPAR transactiva-
tion (EC50 values are given in Tables 1 and 2).14 The structural ref-
erence used as the starting point was the phenyl-unsubstituted 2-
(4-(3-phenoxypropoxy)-phenylthio)hexanoic acid 6 (Table 1). This
enhance PPARa activity (EC50 0.049 lM) compared to meta-methyl
substituted 8 whereas the meta-isopropyl substitution (16) did not
cause any further improvement.
As the meta-substitution turned out to be the most eligible tool
compound itself is already a nano molar active PPAR
a
-agonist with
. This
to selectively improve PPARa activity, we replaced the methyl
group of 8 by a trifluoromethyl to yield compound 17. This modi-
an EC50 of 0.29 M and a ninefold selectivity versus PPAR
l
c
compound and all other 2-mercaptohexanoic acid derivatives
presented are PPARb-inactive. The ortho(7)-, meta(8)- and
para(9)-methyl analogs were prepared based on 6. SAR revealed
fication was the most successful structural modification of the
meta-position with a PPAR
tivity towards PPAR (EC50 4.6
the additional para-chloro substituent of 18 decreased both PPAR
and PPAR activity.
Compounds 19 and 20 represent a larger structural modifica-
tion and could be described as PPAR -preferential dual PPAR
agonists. Biphenyl-substituted 19 is the most potent synthesized
compound in regard to PPAR (EC50 0.54 M) with an approx. five-
fold remaining selectivity for PPAR . Compound 20, which bears a
quinoline-6-yl moiety, shows a very promising PPAR modulating
profile (EC50 1.3 M, maximal relative activation of 49%) combined
with a sixfold selectivity for PPAR
We recently showed the impact of the stereocentre in
a
EC50 of 0.025
lM and excellent selec-
c
lM). It was noted with interest that
a
significant improvement for the meta and para-methyl
substituted compounds shifting EC50 values to the double-digit
nanomolar range (PPAR 8: EC50 0.09 M, 9: EC50 0.085 M),
a
c
a
l
l
whereas no improvement could be observed for the ortho-methyl
derivative. Comparison of the selectivity profile of meta and para-
a
a/c
methyl derivatives (PPAR
revealed that meta-methyl substitution was able to selectively
enhance PPAR activity whereas para-methyl substitution
enhanced PPAR and PPAR activity equally.
c
8: EC50 3.1
l
M, 9: EC50 0.93
l
M)
c
l
a
a
c
a
c
l
Two vicinal methyl groups were introduced next, yielding the
3,4-dimethyl (10) and 2,3-dimethyl (11) derivatives. Both com-
a.
a-posi-
pounds were able to further enhance PPAR
a
activity compared
tion of a-n-hexyl substituted pirinixic acid derivatives on PPAR
with the mono methyl-substituted analogs. As indicated by the
activity of the meta and para-methyl analogs, the combination
3,4-dimethyl represents the most active racemic compound within
activity emphasized by in silico studies.15 Encouraged by these re-
sults and by examples found in literature,16 we separated 11 by
enantio-selective preparative HPLC into its distinct enantio-
mers.15,17,18 In vitro data of (R)-11 and (S)-11 are in line with pre-
this series (PPAR
that the 2,3-dimethyl moiety also showed significant improvement
of PPAR activity (EC50 0.056 M). In regard to PPAR , selectivity
a 10: EC50 0.021 lM). It was noted with interest
vious observations: (R)-11 (PPAR
a EC50 0.011 lM) displays
a
l
c
superior biological activity and was shown to be approx. five times
increased by the introduction of two methyl groups from factor
nine for unsubstituted phenyl (6) to factor 54 for compound 11.
According to previous studies,8 this substructure is of particular
interest (see Fig. 2). We thus maintained 11 as a scaffold for the
investigation of the stereochemistry ((R)-11 and (S)-11) as well
as for the SAR of the acidic head group of our series (21 and 25–
27).
more potent than the racemic mixture and approx. 140 times more
potent than the (S)-enantiomer. (R)-11 is the most potent PPAR
activator in this series with the highest selectivity versus PPAR
a
.
c
We again chose the 2,3-dimethyl substitution pattern of com-
pound 11 for the investigation of the SAR (Table 2) of the linker be-
tween the acidic head group and the central aromatic ring (Fig. 1).
We first prepared the
led to an attenuation of PPAR
theless, it showed an interesting PPAR-pan-agonistic profile with a
slight preference for PPAR . Obviously, PPARb activity was gained
by removal of the -substitution. Finally, the sulfur atom was re-
a-unsubstituted analog 21. This modification
Bridging of the 2,3-dimethyl substructure of 11 yielded the tet-
rahydronaphthyl derivative 12, which showed a slight decrease in
a
selectivity compared to 11; never-
PPARa
activity compared to 11. We decided to further elucidate
a
SAR of the ortho and meta position by introducing larger substitu-
a

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H. Zettl et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4421–4426
Table 1
In vitro transactivation activities for compounds 6–20 on human PPARs
O
S
∗
OH
RO
O
Compound
R
Transactivation EC50 (lM) SD (% activation compared to control)
PPAR
a
PPARb
PPAR
c
0.04
0.35
6
0.29
(76%)
(64%)
(80%)
ia
2.6
(83%)
Me
0.05
0.02
0.5
7
0.25
0.09
ia
2.3
(114%)
Me
0.41
8
ia
3.0
(96%)
Me
Me
0.03
0.12
9
0.085
0.021
(91%)
(72%)
(31% at 10
l
M)
0.93
(87%)
Me
Me
0.01
0.1
10
ia
3.5
(61%)
Me
0.019
0.004
0.22
0.83
1.44
11
(R)-11
(S)-11
0.056
0.011
1.7
(113%)
(115%)
ia
ia
ia
3.0
3.9
9.9
(82%)
(125%)
(77%)
0.18
(94%)
0.04
0.03
0.5
12
13
14
0.19
(101%)
(92%)
ia
ia
ia
8.1
1.6
1.2
(107%)
OMe
0.41
0.55
(85%)
(85%)
OMe
0.02
0.16
0.32
0.3
0.049
(85%)
Me
Me
0.43
15
16
17
2.0
(89%)
ia
ia
ia
4.1
5.3
4.6
(92%)
Me
Me
0.02
0.15
(98%)
(91%)
CF3
0.02
0.19
0.025
(75%)
(68%)

H. Zettl et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4421–4426
4425
Table 1 (continued)
Compound
R
Transactivation EC50 (lM) SD (% activation compared to control)
PPAR
a
PPARb
PPAR
c
CF3
0.02
0.4
Cl
18
19
20
0.39
(65%)
(125%)
(95%)
ia
7.8
(96%)
0.01
0.04
0.06
0.11
0.22
ia
ia
0.54
(108%)
N
0.9
1.3
(49%)
EC50/SD values were calculated using SigmaPlot2001 based on the mean values of at least three determinations. Values in brackets give the relative activation compared to
the positive control. Positive controls were GW7647 for PPAR
a
, pioglitazone for PPAR
c
and L165,041 for PPARb, each at 1 lM; ia: inactive at 10 lM. Rand X are typed in bold
as these entities display the varied substructure of the respective scaffold.
Table 2
In vitro transactivation activities for compounds 21 and 25–27 on human PPARs
O
X
OH
R
O
O
Compound
X
R
Transactivation EC50
(
lM) SD (% activation compared to control)
PPAR
a
PPARb PPARc
0.12
0.38
0.34
21
25
26
27
–S–
H
H
n-Bu
n-Bu
0.60
13.8
ia
(81%)
1.4
(52%)
(72%)
3.8
(77%)
(54%)
3.4
0.45
0.77
–CH2–
–CH@
–CH2–
(63%)
11.2
ia
15.7
ia
4.2
0.25
0.53
1.5
(73%)
ia
(120%)
EC50/SD values were calculated using SigmaPlot2001 based on the mean values of at least three determinations. Values in brackets give the relative activation compared to
the positive control. Positive controls were GW7647 for PPAR
a
, pioglitazone for PPAR
c
and L165,041 for PPARb, each at 1 lM; ia: inactive at 10 lM. Rand X are typed in bold
as these entities display the varied substructure of the respective scaffold.
placed by a methylene group and the
a-unsubstituted (25) as well
Supplementary data
as the -n-butyl-substituted (27) analog was synthesized. Methin-
a
analog 26 was obtained during synthesis. Interestingly, rigidization
by the additional exocyclic double bond caused a complete loss of
PPAR activity. Methylene analog 25 revealed an attenuated activity
for all PPAR subtypes compared to 21, whereas for n-butyl-substi-
Supplementary data (detailed synthetic procedure and analyti-
cal characterization of the compounds) associated with this article
can be found, in the online version, at doi:10.1016/j.bmcl.2009.
05.057.
tuted 27 an impressive loss could be observed only for PPAR
a
activity. Regarding the 2-mercaptohexanoic acid substructure,
References and notes
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In summary, we successfully established a novel and robust
scaffold for highly active PPAR agonists based on the 2-mercapto-
hexanoic acid substructure. By systematic structural variation, we
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a
were able to cover a broad selectivity profile from PPAR
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PPAR agonists. By preparation of carbon analogs and
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a-unsubsti-
tuted derivatives, we corroborated the importance of the sulfur
atom as well as of the n-butyl chain of our 2-mercaptohexanoic
acid head group for PPAR activity. Regarding the phenolic back-
bone of our scaffold, SAR revealed that especially substituents in
meta-position (e.g., –Me, –CF3, –OMe) were able to selectively im-
prove PPARa activity. Encouraged by these promising in vitro re-
sults, we are planning further profiling of an eligible candidate
in vivo in the near future.
Acknowledgement
We gratefully acknowledge financial support from the Else-Kro-
ener-Fresenius-Stiftung (FIRST).

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H. Zettl et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4421–4426
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seeded in 96-well plates in a density of 30,000 cells/well. Transfection was
carried out by LipofectamineTM 2000 reagent (Invitrogen) according to the
manufacturer’s protocol with pFR-Luc (Stratagene), pRL-SV40 (Promega) and
the Gal4-fusion receptor plasmids (pFA-CMV-hPPAR-LBD) of the respective
subtype. 5 h after transfection, medium was changed to DMEM without FCS,
containing 0.1% DMSO and the respective concentrations of the test
compounds. Following overnight incubation with the test compounds, cells
were assayed for reportergene activity using Dual-GloTM Luciferase Assay
System (Promega) according to the manufacturer’s protocol. Luminescence
was measured with a GENios Pro luminometer (Tecan).