# Structural Development Studies of Subtype-Selective Ligands for Peroxisome Proliferator-Activated Receptors (PPARs) Based on the 3,4-Disubstituted Phenylpropanoic Acid Scaffold as a Versatile Template

Authors: Hiroyuki Miyachi; Yuichi Hashimoto
Journal: PPAR Research
 (2008)

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## Abstract

Improvements in our understanding of the functions of the nuclear receptor peroxisome proliferator-activated receptor (PPAR) subtypes as pleiotropic regulators of biological responses, including lipid, lipoprotein, glucose homeostasis, inflammation, differentiation and proliferation of various cancer cells, and memory, have provided an opportunity to develop novel PPAR ligands with characteristic subtype selectivity. Such ligands are not only chemical tools to investigate the functions of PPARs, but also candidates for the treatment of PPAR-mediated diseases, including metabolic syndrome, inflammation, dementia, and cancer. This minireview summarizes our work on the design, synthesis, and pharmacological evaluation of subtype-selective PPAR agonists based on the use of 3,4-disubstituted phenylpropanoic acid as a versatile template.


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## Body

## 1. Nuclear Receptors

The nuclear receptors (NRs)
form a superfamily of ligand-dependent transcription factors that control
diverse aspects of reproduction, development, homeostasis, immune function, and
so on. This superfamily includes the
known receptors for steroid hormones, thyroid hormones, retinoid receptors and
vitamin D receptor, as well as a large number of orphan receptors. The
structures of NRs are composed of several functionally important regions
(designated A to F). The N-terminal A/B region contains a transcriptional
activation function-1 (AF-1) motif that works independently of ligand binding.
The central DNA-binding region (C region) is highly conserved among the NRs and
contains two zinc-finger motifs that make contact with specific nucleotide
sequences, termed hormone response elements. The C-terminal part, which
consists of the D, E, and F regions, is required for ligand binding and
receptor dimerization. In most receptors, this region also contains a second
highly conserved transcriptional activation function-2 (AF-2) motif, which is
important for ligand-dependent transcription.Based on the elucidated human genome
sequence, 48 NRs are speculated to exist in humans [1]. However, the ligands
have been identified for only 20 to 25 of them. The others are so-called orphan
receptors, whose endogenous ligands are not known [2, 3]. Among the NRs, much
attention has been focused on the peroxisome proliferator-activated receptors
(PPARs) over the past two decades.


## 2. Peroxisome Proliferator-Activated Receptors

PPARs
are activated by endogenous saturated and unsaturated fatty acids and their
metabolites and synthetic ligands [4]. Three subtypes have been isolated to
date: PPARα (NR1C1), PPARδ (NR1C2), and PPARγ (NR1C3),
and each of them appears to be differentially expressed in a tissue-specific
manner. PPARα is mostly expressed in tissues involved in lipid oxidation, such
as liver, kidney, skeletal, cardiac muscle, and adrenal glands. PPARγ is
expressed in adipose tissue, macrophages, and vascular smooth muscles. In
contrast to the specific distribution of PPARα and PPARγ, PPARδ is ubiquitously expressed [5].Upon
ligand binding, PPARs heterodimerize with another nuclear receptor partner,
retinoid X receptor (RXR), and the heterodimers regulate gene(s) expression by
binding to specific consensus DNA sequences, called peroxisome proliferator
responsive elements. These elements are a direct repeat of the hexameric AGGTCA
recognition motif, separated by one nucleotide (DR1), present in the promoter
region of the target genes [6].


## 3. PPARs as Regulators of Metabolic Homeostasis

Each
of the PPAR subtypes plays a pivotal role in lipid, lipoprotein, and glucose
homeostasis. PPARα regulates genes
involved in fatty acid uptake (such as fatty acid binding protein, FABP), β-oxidation (acyl-CoA oxidase), and ω-oxidation (cytochrome P450). It downregulates
apolipoprotein C-III, a protein that inhibits triglyceride hydrolysis by
lipoprotein lipase, and it also regulates genes involved in reverse
cholesterol transport, such as apolipoprotein A-I and
apolipoprotein A-II [7]. PPARγ is a master regulator of adipocyte
differentiation, but more recent molecular-biological studies have indicated
that its activation is also linked to the expression of many important genes
that affect energy metabolism, such as TNF-α, leptin, and adiponectin genes [8]. PPARδ is the least well-defined subtype among the PPARs, but recent
biological study has disclosed that its activation significantly
increases HDL cholesterol levels, and it influences glycemic control in a
primate model of metabolic syndrome [9–11]. Furthermore, its activation markedly improved
glucose tolerance and insulin resistance in ob/ob mice, although the underlying
mechanism remains unclear [12].


## 4. PPARs as Templates for Development of Versatile Regulators

Research in the field of PPAR
biology and/or pharmacology is attracting enormous interest, and the range of
therapeutic potential for PPAR agonists is rapidly expanding well beyond lipid,
lipoprotein, and glucose homeostasis. For example,
ligand-mediated PPARα activation induces expression and
activation of antioxidant enzymes, such as superoxide dismutase (SOD) and
glutathione peroxidase (GTP) [13]. Therefore, PPARα activation blocks the synthesis and
release of inflammatory cytokines, such as IL-6 and TNF-α. PPARγ activation attenuates the expression
of inducible nitric oxide (iNOS) and cyclooxygenase-2 (COX-2), as well as the
production of proinflammatory cytokines [14]. Considering that PPARs are also expressed in neurons and in astrocytes,
both PPARα and PPARγ are expected to be useful as pharmacological targets for neuroprotection
in stroke and neurodegenerative diseases.PPARγ was initially noted to be highly expressed in adipose
tissue, but later studies demonstrated that PPARγ was also expressed widely in tumors originated from
various organs. Ligand-mediated
activation of PPARγ inhibits cell
proliferation and/or induces apoptosis or terminal differentiation, by
upregulating the expression of cyclin-dependent kinase (CDK) inhibitors,
including P18, P21, and P27 [15]. PPARγ also promotes cell cycle arrest by inhibiting CDK
activity in several tumor cell lines [16].Angiogenesis, the formation of new blood
vessels, is a critical step in solid tumor growth. PPARγ activation inhibits the expression of at least three
important genes involved in the angiogenic processes, that is, VEGF, VEGF receptor
1, and urokinase plasminogen activator (uPA) [17]. Therefore, PPARγ is considered as a therapeutic target for certain human malignancies.Based on the findings that the glitazone-class antidiabetic
agents and fibrate-class antidyslipidemic agents are ligands of PPARγ and PPARα, respectively, much research
interest has been focused on these two metabolic NR subtypes as therapeutic
targets for the treatment of diabetes and dyslipidemia. In contrast, although
PPARδ is ubiquitously
distributed in a wide range of tissues and cells, research interest in PPARδ has been limited. However,
after 2001, the availability of PPARδ˜ knockout animals and selective ligands prompted us to
examine the involvement of PPARδ in fatty acid metabolism, insulin resistance, reverse cholesterol transport,
inflammation, and so on. Furthermore, molecular-pharmacological
studies have indicated that PPARδ is also involved in other
biological functions, extending beyond metabolic homeostasis. PPARδ is reported to play critical role(s) in wound healing.
After tissue damage resulting from chemical, mechanical, and biological injury,
the injured cells release proinflammatory cytokines [18, 19]. These stimulate
PPARδ expression,
coordinating transcriptional upregulation of integrin-linked kinase and
3-phosphoinositide-dependent kinase (PDK), and repress the expression of
phosphatase and tensin homolog 10 (PTEN) [20]. As a consequence, the PKBα activity is increased and apoptotic cascades are repressed. The
resulting increased resistance to cell death helps to maintain a sufficient
number of viable wound keratinocytes for re-epithelization. Therefore, PPARδ is expected to be a therapeutic target for tissue injury.The above examples remind us that PPARs are pleiotropic
NRs, and the subtypes are unique, though somewhat overlapping, therapeutic
targets for the treatment of not only metabolic homeostasis, but also
inflammation, cancer, neurodegeneration, wounds, and so forth. PPAR ligands
clearly have enormous potential as therapeutic agents, and the range of
possible applications has certainly not yet been fully explored.From a basic research point of view, it is of
primary importance to develop potent and PPAR subtype-selective ligands as
chemical tools to investigate individual PPAR functions in detail. Furthermore, from a medicinal chemical point
of view, it is also important to consider PPAR dual agonists (which can
activate two out of three PPAR subtypes effectively), such as PPARα/δ-, PPARα/γ-, and PPARδ/γ dual agonists, and PPAR-pan agonists (which can activate all three PPAR subtypes), since
these may exhibit additive and/or synergistic pharmacological effects.In
this minireview, we focus on our structural development studies to create PPARα-selective PPARα/δ-dual and PPARδ-selective agonists,
using the 3,4-disubstituted phenylpropanoic acid template as a common
structure.


## 5. Our Working Hypothesis Concerning the Ligand Superfamily Concept

We have been engaged in
structural development studies of NR ligands (agonists and antagonists) for
over ten years, based on our working hypothesis concerning the NR ligand
superfamily concept [21]. Broadly speaking, the structural/functional features
of NRs are similar, even though there are many kinds of NRs (48 in humans), that is, NRs generally consist of an aminoterminal region which has a
ligand-independent transcriptional activation function (AF-1), a DNA-binding
domain (DBD) with a motif structure of two zinc fingers which has a
sequence-specific DNA (response element: RE)-binding function, and a large
carboxyl-terminal region (ligand-binding domain: LBD), which has a specific
ligand-binding function/dimerization function/ligand-dependent activation
function (AF-2) [22]. Thus, NRs are probably derived from a single ancestral
protein, and may have structurally evolved in order to fit various kinds of
endogenous NR ligands. We speculated that similar evolution of NR ligands would
have occurred from an ancestral ligand to form a superfamily, even though NR
ligands now have diverse functions. Based on this working hypothesis, we divided the structure of NR ligands
into two portions. One is a common hydrophobic framework that fits into the
ligand binding pocket of the basic ancestral protein, and the other is a
characteristic structural motif which provides NR selectivity. If this
hypothesis is correct, it is easily deduced that the hydrophobic backbones of
various NR ligands should be exchangeable with each other to construct
structurally new NR ligands.Numerous previous reports
have indicated that PPAR subtype-selective agonists have certain unique
structures associated with the subtype selectivity. For example,
thiazolidine-2,4-dione, and related structures, as exemplified by pioglitazone
(1), for PPARγ selective agonists, 2,2-dialkyl
(usually dimethyl) phenoxyacetic acid structure, exemplified by fenofibrate (2), for PPARα-selective
agonists, and 2,2-unsubstituted phenoxyacetic acid structure, exemplified by
GW-501516 (3), for PPARδ-selective
agonist (Figure 1). But, based on our working hypothesis, we anticipated that
various kinds of subtype-selective, dual-, and pan-agonists could be created by
using a common chemical framework as a template.Figure 1
Structures of: (1) PPARγ agonist pioglitazone, (2)
PPARα agonist fenofibrate, (3) PPARδ agonist GW-501516, and (4) dual
PPARγ/α agonist
KRP-297.


## 6. PPARα-Selective Agonist: KCL

We
designed and synthesized a series of substituted phenylpropanoic acid
derivatives in order to
develop structurally new human PPARα-selective agonists, using KRP-297 (4),
a unique thiazolidine-2,4-dione derivative with PPARγ/α dual agonist activity,
as a lead compound for
the preparation of antidyslipidemic agents [23–25]. Although KRP-297 belongs structurally
to the glitazones (thiazolidine-2,4-dione class insulin sensitizers), it binds
directly to and activates not only PPARγ, but also PPARα with
almost equal affinity (its affinity for PPARγ and PPARα is approximately 0.23 and 0.33 μM, resp., and it transactivates
PPARγ and PPARα with effective concentrations of 0.8
and 1.0 μM, resp.) [24].
This is a characteristic feature of classical glitazones, including
troglitazone (5), pioglitazone (1), and rosiglitazone (6), which were reported to bind to and
activate selectively the PPARγ subtype.
The reason why they exhibit dual-ligand nature was unclear, but we anticipated
that the replacement of the thiazolidine-2,4-dione ring structure of KRP-297
with another acidic functionality, such as a carboxyl group, which is usually used
in fibrates, might favor PPARα selectivity and that further chemical
modification might improve the potency and selectivity for the PPARα subtype. Structurally, KRP-297 can be regarded as having
three key regions: (i) the acidic head group, (ii) the linking group, and (iii)
the hydrophobic tail group. We performed chemical modification of the (i) to
(ii) parts of the molecule to understand the structure-activity relationship in
detail. After synthesizing numerous compounds, we obtained KCL, (S)-2-[4-methoxy-3-(4-trifluoromethylbenzylcarbamoyl)phenylmethyl]butyric
acid (S)-11 [26–29] as a potent and PPARα-selective
agonist. The
synthetic route to KCL is shown in Scheme 1. Our SAR results can be summarized
as follows. (i) The distance between the carboxyl group and the right-side
benzene ring in the compound is important. (ii) The introduction of alkyl substituents
at the α-position of the carboxyl group strikingly affected PPARα transactivation activity and subtype selectivity, and the
ethyl group is the most favorable (Table 1). (iii) Stereochemistry at the α-position of the ethyl group is crucial and the (S)-configuration
is preferable (Figure 2). (iv) The length of the linking group is important for
potency, and a three-atom unit with an amide group such as –CH2–NH–CO–  is preferable (Table 2).Table 1
SAR 1: effect of the acidic partial structure
in the present series of compounds.
Transactivation activityEC50 (nM)(a)No.RPPARαPPARδPPARγ7COOHia(b)iaia8CH2COOHiaiaia9(CH2)2COOH1300iaia10CH2CH(Me)COOH(c)2402800ia11CH2CH(Et)COOH(c)403600100012CH2CH(n-Pr)COOH(c)3602400ia13CH2CH(i-Pr)COOH(c)290iaia14CH2CH(n-Bu)COOH(c)1000ia250015CH2CH(Ph)COOH(c)iaiaia16CH2CH(OMe)COOH(c)230iaia17CH2CH(OEt)COOH(c)16003000280018CH2CH(OPh)COOH(c)iaiaia19CH2C(Me)2COOH2900iaia20CH2C(Et)2COOH2800iaia21CH2CH(SEt)COOH(c)16003000280022CH2CH(SPh)COOH(c)iaiaia23CH2CH(SBn)COOH(c)iaiaiaKRP-2971000ia800(a) Compounds were screened for agonist
activity on PPAR-GAL4 chimeric receptors in transiently transfected CHO-K1
cells as described. EC50 value is
the molar concentration of the test compound that causes 50% of the maximal
reporter activity,
(b) “ia” means inactive at the concentration of 10 μM,
(c) assayed as a racemate.Table 2
SAR 2: effect of
the linker partial structure in the present series of compounds.
Transactivation activityEC50 (nM)(a)No.APPARαPPARδPPARγ11CH2NHCO(c)403600100024NHCO(c)6000ia(b)ia25(CH2)2NHCO(c)7401500ia26CH2N(Me)CO(c)iaiaia27CH2CONH(c)130iaia28NHCONH(c)iaiaia29{NHCOCH2}(c)20iaia30CH2CH2CO(c)320ia740031{CH2CH2CH2}(c)860640ia32CH2CH2O(c)680400ia33{CONHCH2}(c)401201000034{CH2NHCH2}(c)49008000ia(a), (b), (c) See
footnotes of Table 1.Figure 2
Dose-response curves of each enantiomer for PPARα transactivation.Scheme 1
Chemical structure of our PPARα-selective agonist KCL, and the synthetic
route. Reagents and conditions: (a) BnBr, KHCO3,
DMF, rt., (b) MeI, K2CO3, DMF, rt. (c) NaBH4,
EtOH, rt. (d) PBr3, ether, 0°C, (e) (1) (R)-4-benzyl-3-butyryloxazolidin-2-one, LiHMDS,
THF, −40°C–−10°C, (2) benzyl
5-bromomethyl-2-methoxybenzoate, THF, −40°C–−10°C, (f) H2,
10% Pd–C, AcOEt, rt., (g) (1) ethyl chloroformate, TEA, THF, −10°C, (2)
4-trifluoromethylbenzylamine, THF, −10°C -rt., (h) LiOH H2O,
H2O2, THF–H2O, 0°C.(Although compound 29 exhibited potent and PPARα-selective
activity, we did not go forward with this compound, because our experience
indicated that it would be metabolically
unstable; consequently it was not expected to exhibit potent or prolonged in
vivo activity, despite its potency in vitro.)It is important to
note that KCL exhibits distinct species-dependence in transactivation for PPARα.
Although species dependency of some PPARα agonists was reported previously, the
degree of selectivity was low [30, 31]. However, the species-selectivity of
KCL is extremely high: KCL activated
human, dog, and rat PPARα with EC50 values of 0.06,
0.16, and 5.2 μM,
respectively (Table 3). KCL exhibited
species preference for humans and its transactivation activity for PPARα was approximately 100-fold and 30-fold less potent in
rats than in humans and in dogs, respectively.Table 3
Species
differences in the transactivation of PPARα isoforms.
Transactivation activityEC50 (nM)(a)Compd.HumanDogRatKCL601605200fenofibrate410005000049000(a) See footnote of Table 1.This apparent species difference was reported to result from specific interaction between the 272-aminoacid,
isoleucine (Ile272), which is located on the helix 3 region of the human PPARα LBD and the hydrophobic tail part of KCL [32–34]. The
corresponding aminoacid residue in the rat PPARα LBD is sterically more bulky phenylalanine.
KCL was reported to reduce plasma triglyceride levels
>100-fold more potently in dogs than in rats, which is consistent with the
in vitro assay data. Clinical studies of a KCL-related compound are under way.


## 7. PPARα/δ-Dual Agonist: TIPP-401

We next planned to
develop a PPARα/δ-dual agonist, which would effectively activate both PPARα and PPARδ, because pharmacological
evidence indicated that PPARα regulates the
expression of genes encoding proteins involved in lipid and lipoprotein
homeostasis, and subsequent pharmacological findings for PPARδ demonstrated that it also plays a key role in
lipid metabolism and insulin resistance. Furthermore, PPARδ plays a key role in foam cell and macrophage
activation in atherosclerosis.The metabolic function(s) of PPARδ seem to be mainly targeted to adipose tissue and smooth muscle, via fatty
acid oxidation and energy uncoupling. If this were the case, we expected that a compound
which can effectively activate both PPARα and PPARδ might have additive and/or
synergistic positive effect(s) in the treatment of metabolic syndrome, by
modulating both hepatic fatty acid oxidation through PPARα, and fatty acid oxidation
and energy uncoupling in muscle and adipose tissue through PPARδ. In addition,
in 2004, there were only a few examples of PPARα/δ dual agonists in the
literature, including compounds 35 [35], 36 [36], and 37 [37] (Figure 3), and their
activities seemed rather low and their structural variety poor. Therefore,
there was considerable interest in creating novel PPARα/δ dual agonists from
both basic scientific and clinical points of view. We expected that small
manipulations of the structure of KCL would affect the activities towards both PPARα and PPARδ.
Therefore, we reconsidered the SAR of PPARα-selective KCL derivatives.Figure 3
Structures of the dual PPARα/δ agonists.As the linking group of the KCL series, we found that a
three-atom unit with an amide group such as –CH2–NH–CO– was preferable for potency and
selectivity against PPARα, and these compounds
did not exhibit remarkable PPARδ activity. We noted that a flexible linker,
such as –CH2–CH2–CH2– (31) or –CH2–CH2–O– (32), decreased PPARα transactivation
activity, but also resulted in the appearance of PPARδ transactivation
activity. Therefore, we focused our attention on a hybrid-type linker, that is, –CO–NH–CH2– (33), and found that this linker
increased both PPARα and PPARδ transactivation activity to some extent, as
compared with the amide-type (–CH2–NH–CO–) linker in KCL (Table 2).We selected 33 as the next lead compound, and performed further chemical modifications,
focusing especially on the hydrophobic tail part of the molecule, and obtained
a PPARα/δ-dual agonist termed
TIPP-401 ((S)-2-{3-[(2-fluoro-4-trifluoromethylbenzoylamino)methyl]-4-methoxybenzyl}butyric
acid). The synthetic route is summarized in Scheme 2. We found that the
introduction of a fluorine atom affected the PPAR transactivation activity or
selectivity (Table 4). Compounds 38 and 39, which have a fluorine atom
at the ortho- or meta-position of benzene in the hydrophobic tail part,
respectively, exhibited more potent PPARα and PPARδ transactivation activities
than those of the nonfluorinated compound. The position of the distal benzene
substituents is crucial, since compound 40,
which has a fluorine atom at the para-position and a trifluoromethyl group
at the meta-position, showed
considerably decreased PPAR transactivation activity. This is consistent with
the previously obtained SAR result that steric bulkiness at the para-position is an important factor for
potent PPARα transactivation activity.Table 4
In vitro functional PPAR transactivation activity of
substituted phenylpropanoic acids.
Transactivation activityEC50 (nM)(a)No.XYstereoPPARαPPARδPPARγ334-CF3Hrac192002600384-CF32-Frac10242200394-CF33-Frac11516000403-F4-CF3rac2502000ia(b)414-CF32-FS10121900424-CF33-FS12234900434-CF33-FR150840ia(b)(a)
Compounds were screened for agonist activity on PPAR-GAL4 chimeric receptors in
transiently transfected HEK-293 cells. The EC50 value is
the molar concentration of the test
compound that causes 50% of the maximal reporter activity,
(b) “ia” means
inactive at the concentration of 10 μM.Scheme 2
Chemical structure of our PPARα/δ-dual agonist TIPP-401, and the synthetic route. Reagents and
conditions: (i) BH3-tetrahydrofuran,
THF, 0°C, (j) activated MnO2, CH2Cl2, r.t., (k)
2-fluoro-4-trifluoromethylbenzamide, triethylsilane, trifluoroacetic acid,
toluene, reflux, (l) LiOH H2O, H2O2,
THF-H2O, 0°C.Considering these results, we prepared
optically active derivatives, 41 (TIPP-401), 42, and 43. As can be seen from
Table4, a clear enantio-dependency of the transactivation activity towards the
PPARα and PPARδ isoforms was found. Compound 42, which has (S)
configuration, exhibited potent transactivation activity towards both PPARα and PPARδ, while the antipodal (R)
isomer 43 exhibited far less
potency. Therefore, we concluded that both PPARα and PPARδ transactivation
activities reside almost exclusively in the (S)-enantiomer, and both TIPP-401 and 42 show dual-agonist activity toward PPARα and PPARδ. The synthetic route to TIPP-401 is
shown in Scheme 2.In order to investigate the nuclear receptor
selectivity (cross-reactivity) of the representative compound TIPP-401, we
determined the transactivation activity of TIPP-401 on representative nuclear receptors (PPARs, VDR, FXR, LXRα, RARα,
and RXRα). As can be seen from Figure 4, TIPP-401 seems to be specific for PPARα and PPARδ because it did not
significantly activate VDR, PPARγ, LXRα, RARα, or RXRα at concentrations up to
1 μM under the experimental conditions used. These results indicate that
although the ligand binding domains of nuclear receptors are similar, there are
distinct structural requirements for preferential
binding to both PPARα and PPARδ.Figure 4
Cross-nuclear receptor reactivity of 0.1μM, and 1 μM TIPP-401.


## 8. PPARδ-Selective Agonist: TIPP-204

Our next target was a PPARδ-selective agonist. As
described above, the availability of PPARδ˜ knockout animals and selective
ligands, especially GW-50151 (3),
developed by GlaxoSmithKline, prompted to examine the involvement of PPARδ in
fatty acid metabolism, insulin resistance, reverse cholesterol transport,
inflammation, and so on. For example, ligand-mediated PPARδ activation significantly increased HDL cholesterol
levels, possibly in association with decreased lipoprotein lipase
activity, in insulin-resistant middle-aged obese rhesus monkeys [38]. In a primate model of the metabolic syndrome,
PPARδ activation
lowered
plasma insulin levels, without any adverse effect on glycemic
control [38]. Similarly, in the case of ob/ob mice, PPARδ activation markedly improved
glucose tolerance and insulin resistance [38]. All these observations suggest that PPARδ may be
an effective target for the treatment of metabolic syndrome.Several
PPARδ˜ selective agonists (44–48) have been reported in the
literature (Figure 5) after the disclosure of GW-501516 (3), though most are derivatives of GW-501516 (3) and L-165041 (44)
(Merck’s compound), that is, (2-methyl)phenoxyacetic acid derivatives [39–44]. As a part of our continuing research directed toward the
structural development of characteristic subtype-selective PPAR agonists, we
planned to construct phenylpropanoic acid-type PPARδ˜ selective agonists,
based on the PPARα/δ dual agonist, TIPP-401 as a lead compound.Figure 5
Structures of representative, recently reported PPARδ-selectiv agonists.To create PPARδ-selective agonists, we took into
account the results of X-ray crystallographic analyses of PPARδ complexed with
a natural unsaturated fatty acid, eicosapentaenoic acid (EPA) [45].
The PPAR
ligand-binding pocket forms a large Y-shaped cavity which extends from the
C-terminal helix to the β-sheet lying between helix 3 and helix 6.
EPA binds to the cavity in two distinct
conformations, that is, tail-up and tail-down conformations. The
carboxyl group and the first eight carbon units take almost the same
configuration in both conformations. However, the distal hydrophobic tail part
of the tail-up conformer of EPA was bent upwards into the upper cavity of the
Y-shaped pocket, while in the tail-down conformer, the hydrophobic tail part
was bent downwards into the bottom cavity of the Y-shaped pocket. Contrary to
the case of PPARδ, none of the PPARα and PPARγ agonists whose binding
structures have been solved by X-ray crystallography takes the tail-up
conformation, although the reason for this is not known. However, we speculated that the aminoacid(s)
forming the entrance to the upper cavity might be bulkier in PPARα and PPARγ than in PPARδ. We previously suggested that our PPARα-selective agonist KCL
might take a tail-down conformation, based on molecular modeling studies of the
KCL-PPARα complex and the results of site-directed mutagenesis studies of PPARα with our PPAR agonists. The PPARα/δ dual agonist TIPP-401 was also considered
to dock into the downward cavity of PPARα, because Ile272, which is located on
the lower half of helix 3, is also critical for potent PPARα transactivation by
TIPP-401.Based
on these insights, we hypothesized that if we could connect one more sterically
bulky hydrophobic side chain to the backbone of TIPP-401, directed towards the
upper cavity of PPARδ, it should have the effect of strengthening the PPARδ activity, while weakening the PPARα activity.
Based on our previously reported binding model of KCL, a methoxy group
at the 4-position was expected to be directed towards the upper cavity, so we
prepared various 3-(4-alkoxyphenyl)propanoic acids, and found the compound
termed TIPP-204 ((S)-2-{3-[(2-fluoro-4-trifluoromethylbenzoylamino)methyl]-4-buthoxy- benzyl}butyric
acid). The structure and the method of preparation are summarized in Scheme 3.
We obtained clear SAR confirming that the subtype-selectivity largely depends
on the nature of the substituents, as expected.Scheme 3
The synthetic
route to TIPP-204. Reagents and
conditions: (m) nBuI, K2CO3, DMF, rt., (n) NaBH4,
EtOH, rt., (o) PBr3, ether, 0°C, (p) (1) (R)-4-benzyl-3-butyryloxazolidin-2-one, LiHMDS,
THF, −40°C–−10°C, (2) benzyl
5-bromomethyl-2-butoxybenzoate, THF, −40°C–−10°C, (q) H2,
10% Pd–c, AcOEt, rt., (r) BH3-tetrahydrofuran, THF, 0°C, (s) activated MnO2,
CH2Cl2, rt., (t) 2-fluoro-4-trifluoromethylbenzamide,
triethylsilane, trifluoroacetic acid, toluene, reflux, (u) LiOH H2O,
H2O2, THF-H2O, 0°C.That is, as regards PPARα, introduction of a short-chain
alkoxy group was found to be favorable for the transactivation activity, that
is, the ethoxy (50) and methoxy (49) derivatives exhibited the most
potent activity. In contrast, a longer alkoxy group was preferable in the case
of PPARδ transactivation activity, and the n-butoxy
(52) and n-propoxy (51)
derivatives were the most potent (Table 5). These results are consistent with
the working hypothesis that the shape and the environment of the hydrophobic
cavity hosting the alkoxy group at the para-position
differ somewhat among these PPAR subtypes. These compounds were basically weak
agonists for the PPARγ subtype, because each compound exhibited an EC50 value of micromolar order or less.Table 5
PPARs
transactivation activity of the present series of compounds.
Transactivation activityEC50 (nM)(a)No.R1R2R3stereoPPARαPPARδPPARγ49HMeEtrac18170260050HEtEtrac1215180051Hn-PrEtrac776.8130052Hn-BuEtrac5204.8130053Hn-HexylEtrac120045230054HBnEtrac>10000110620055FMeEtrac8.221220056Fn-PrEtrac411.765057Fn-BUEtrac2801.9140058HMeHrac2103000>1000059HMeMerac19210>1000060HMen Prrac70120360061HMen BUrac230700240062FMen Prrac2940150063FMen BUrac140220100064Fn-BUn Prrac8205.1100065Fn-BUEtS2500.91110066Fn-BUEtR6208.27600GW-50151610001.88600(a)
Compounds were screened for agonist activity on PPAR-GAL4 chimeric receptors in
transiently transfected HEK-293 cells. The EC50 value is
the molar concentration of the test
compound that causes 50% of the maximal reporter activity.As described above, the introduction of a fluorine atom on
the distal benzene ring, especially at the 2-position, was found to enhance the
PPAR transactivation activity, as in TIPP-401. Therefore, we prepared
fluorinated compounds 55–57. As expected, these compounds exhibited enhanced PPARα and PPARδ transactivation activities as compared with the nonfluorinated compounds. We
found that the PPARδ transactivation activity of 57 is comparable with that of GW-501516 in our assay system, and
the selectivity indexes for PPARδ over both PPARα and PPARγ are more than
100-fold (Table 5).As mentioned above, the
substituent at theα-position of the carboxyl group is also important for the
potency in the case of PPARα, and therefore we investigated the effect of
substitution at this position in the present series. As regards PPARα,
introduction of an ethyl group (49)
or a methyl group (59) was favorable
for the transactivation activity, and further elongation of the substituent
decreased the activity. Similarly, an ethyl group (49) or an n-propyl group
(60) was favorable for PPARδ transactivation activity, and further elongation of the substituent decreased
the activity (Table 5). These results may mean that the shape and the
environment of the cavity hosting the alkyl group located at the α-position of
the carboxyl group are similar in PPARα and PPARδ.Considering
these results, we then prepared the optically active derivatives 65 (S-isomer)(TIPP-204) and 66 (R-isomer). As expected, clear
enantio-dependency of the transactivation activity towards the PPAR subtypes
was found, and TIPP-204, which has (S)-configuration,
exhibited more potent transactivation activity than the antipodal (R)-isomer, 66 (Table 5). Therefore, we concluded that the
activity also resides primarily in the (S)-enantiomer,
but the degree of the enantio-selectivity is less apparent than in the case of
the PPARα/δ dual agonist, TIPP-401. TIPP-204 exhibited extremely potent PPARδ transactivation activity, comparable with or even superior to that of the known
PPARδ-selective agonist GW-501516, and its PPAR subtype selectivity was also
high (Figure 6).Figure 6
Dose-dependency
of TIPP-204 for transactivation of PPARs.To investigate the nuclear receptor
selectivity (cross-reactivity) of TIPP-204, we analyzed the transactivation
activity of TIPP-204 on
representative nuclear receptors (PPARs, VDR, FXR, LXRα, RARα, and RXRα) in the
same way as with TIPP-401 (Figure 7). TIPP-204 seems to be specific for PPARδ (and to a lesser extent  PPARα) because it
did not significantly activate VDR, PPARγ, LXRα, RARα, or RXRα at
concentrations up to 300 nM (more than 300-fold higher concentration as
compared to the EC50 of TIPP-204) under the experimental conditions
used. These results indicate that,
although the ligand-binding domains of nuclear receptors are similar, there are
distinct structural requirements for preferential
binding of TIPP-204 to PPARδ.Figure 7
Nuclear receptor selectivity (cross reactivity) of TIPP-204.We
have successfully obtained a potent and selective, structurally novel PPARδ agonist, TIPP-204. In order to investigate the
structure-activity relationship, and the reason for the PPARδ selectivity, we analyzed the three-dimensional
structure-activity relationship by means of the comparative molecular field
analysis (CoMFA) method, and a molecular modeling study (Figures 8–10). (Comparative molecular field analysis (CoMFA) was used to construct a three-dimensional quantitative structure-activity relationships model. The atomic charges of each conformer were calculated using the semiempirical method MNDO with electrostatic potential-derived point charges (MNDO/ESP) in MOPAC2002. Conventional CoMFA was performed using the QSAR option implemented in the SYBYL package. CoMFA fields were derived in a 3D cubic lattice with a grid spacing of 2 Å and extending 4 Å beyond the aligned molecules in all directions. CoMFA steric (Lennard-Jones 6-12 potential) field energies and CoMFA electrostatic (Coulombic potential) fields were calculated using a probe atom with the van der Waals properties of sp3 carbon and a charge of +1.0. CoMFA electrostatic fields were calculated with a distance-dependent dielectric at each lattice point. The SYBYL energy cutoff of 30 kcal/mol was used. In CoMFA calculation, potential functions (a Lennard-Jones potential and a Coulombic potential) are very steep near the van der Waals surface, causing rapid change, so that the use of cut-off values is required. The poses of ligands generated from the Glide program were used to carry out partial-least-square (PLS) regression analyses. The CoMFA fields were used as independent variables and the logarithm of the reciprocal of EC50 was used as the dependent variable in PLS regression analyses. The optimal number of components in the PLS model was determined using the cross-validated coefficient r2 (called q2) values obtained from the leave-one-out cross-validation technique. The PLS model with the highest q2 values was then selected to derive 3D QSAR models and the poses of ligands with respect to the PPARδ LBD.)
Comparison of the CoMFA counterplots with the crystal structure of the PPARδ ligand-binding domain provided information
about how structural changes of the agonists affect their activities. As can be
seen in Figure 8, hydrogen-bonding interaction was observed between carbonyl
oxygen of the reversed-amide type linker and threonine 288 (T288) of PPARδ (Figure 8 left panel), while such a
hydrogen-bonding interaction was not found between carbonyl oxygen of the
amide-type linker and T288 (Figure 8 right panel). This might be one of the
reasons why the change of the linker from amide type to reversed-amide type
enhanced the PPARδ transactivation
activity by 10-fold.Predicted mode of binding the amide derivative and the reversed-amide derivative to PPARδ. Orange: hydrophobic aminoacids. Green: hydrophilic aminoacids. Magenta: amide derivative and reversed-amide derivative. Hydrogen bonds are shown as yellow dotted lines.
(a)(b)Comparison of the CoMFA counterplot of the steric field based on TIPP-401 (right) and the superimposition of TIPP-401 on the ligand-binding domain of PPARδ (left).
The CoMFA steric counter map is shown in green and yellow. Green: areas in which bulky atomic groups are sterically favorable for the activity.  Yellow: areas in which bulky atomic groups are unfavorable for the activity.
(a)(b)Figure 10
Predicted
mode of binding of TIPP-401 to PPARδ and PPARα.
Upper
figures represent van der Waals surface contact of ligand-binding cavity, and
the lower figure represent detailed component aminoacids.Figure 11
SAR
summary of the present series of compounds.In this CoMFA model (Figure9), the
introduction of a sterically bulky group near the methoxy group at the
4-position in the present series favors the activity, and this was deduced to
be related to the presence of the upper cavity in the Y-shaped ligand-binding
domain of PPARδ. Based on our molecular
modeling (Figure 10), we speculated that the side-chain butoxy group of
TIPP-204 fits into the upper cavity of hPPARδ formed
by the hydrophobic aminoacids V334, L339, and I364. In the case of hPPARα, the corresponding cavity is composed of
sterically bulkier aminoacids, M325, M330, and M355. Indeed, the volume of the
hPPARα upper cavity was calculated to
be only one third of that of the hPPARδ upper
cavity, and we considered that it might not readily accommodate the bulky
butoxy group of TIPP-204. In order to confirm this idea, an X-ray
crystallographic analysis in combination with molecular modeling is in progress.


## 9. Concluding Remarks and Future Directions

In this minireview, we have
described our PPAR ligands based on 3,4-disubstituted phenylpropanoic acid
structure as a versatile template for subtype-selective PPARs ligands, based on
our ligand superfamily working hypothesis. We also describe their
pharmacological evaluation. We succeeded
in obtaining three kinds of subtype-selective PPARs ligands, that is, the PPARα selective agonist KCL, the PPARα/δ-dual agonist TIPP-401, and the PPARδ selective agonist TIPP-204. The
structure-activity relationships among ligands for PPARα and PPARδ were well
charcterized, and are summarized in Figure 10.Basically, our series of
compounds showed weak activity against the PPARγ subtype. However, considering
the moderately high-sequence similarity among PPARs, it should be possible to
obtain greater activity. So, we are conducting further chemical modification
studies directed towards PPARγ activity. Some novel structural requirements for PPARγ activity have
already been identified. We expect to report 3,4-disubstituted phenylpropanoic
acid-type PPAR-pan agonists in the near future.



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*Source: 689859-2008-06-15.xml*