Design and synthesis of 2-methyl-2-{4-[2-(5-methyl-2-aryloxazol-4-yl)ethoxy]phenoxy}propionic acids: A new class of dual PPARα/γ agonists

Article abstract of DOI:10.1021/jm0155188

Propionic acid derivative 8, which was designed and synthesized based on putative pharmacophores of known PPARγ- and PPARα-selective compounds, exhibits potent dual PPARα/γ agonist activity as demonstrated by in vitro binding and dose overlap in the newly

Full text of DOI:10.1021/jm0155188

J . Med. Chem. 2001, 44, 2061-2064
2061
Letters
Ch a r t 1. PPAR Agonists
Design a n d Syn th esis of
2-Meth yl-2-{4-[2-(5-m eth yl-2-a r yloxa zol-
4-yl)eth oxy]p h en oxy}p r op ion ic Acid s: A
New Cla ss of Du a l P P ARr/γ Agon ists
Dawn A. Brooks,*,† Garret J . Etgen,*,†
Christopher J . Rito,† Anthony J . Shuker,†,§
Samuel J . Dominianni,† Alan M. Warshawsky,†
Robert Ardecky,‡ J ames R. Paterniti,‡ J ohn Tyhonas,‡
Donald S. Karanewsky,‡ Raymond F. Kauffman,†
Carol L. Broderick,† Brian A. Oldham,†
Chahzrad Montrose-Rafizadeh,†
Leonard L. Winneroski,† Margaret M. Faul,† and
J ames R. McCarthy†
Lilly Research Laboratories, A Division of
Eli Lilly & Company, Lilly Corporate Center,
Indianapolis, Indiana 46285, Ligand Pharmaceuticals,
10275 Science Center Drive, San Diego, California 92121
Received March 28, 2001
Sch em e 1a
Abstr a ct: Propionic acid derivative 8, which was designed
and synthesized based on putative pharmacophores of known
PPARγ- and PPARR-selective compounds, exhibits potent dual
PPARR/γ agonist activity as demonstrated by in vitro binding
and dose overlap in the newly introduced EOB mouse model
for glucose lowering and lipid/cholesterol homeostasis.
In tr od u ction . Type 2 diabetes is a metabolic disorder
that afflicts 120 million worldwide at present and is
estimated to rise to over 200 million by the year 2010.1
In addition to the characteristic combination of insulin
resistance and insulin deficiency, the type 2 diabetic
often displays cardiovascular risk factors including
dyslipidemia (hypertriglyceridemia, low HDL, and small
dense LDL), coagulopathy (elevated fibrinogen and
plasminogen activator inhibitor-1 (PAI-1)), hyperten-
sion, and obesity. The recent publication of the United
Kingdom Prospective Diabetes Study (UKPDS)2 has
revealed that in Type 2 diabetes, intensive glucose-
lowering therapy is ineffective at reducing cardiovas-
cular complications, despite decreasing microvascular
complications such as retinopathy.
The PPARs (peroxisome proliferator activated recep-
tors) were cloned less than a decade ago and are
members of the superfamily of nuclear transcription
factors that includes the receptors for steroid, retinoid,
and thyroid hormones.3,4 The PPARs form heterodimers
with another nuclear receptor, the 9-cis-retinoic acid
receptor (RXR). This heterodimer complex interacts with
critical DNA response elements within promoter re-
a
(a) p-Br-benzaldehyde, HOAc, HCl (74%); (b) POCl3, CHCl3
(72%); (c) KCN, KI, DMF (100%); (d) KOH, 2-MeO-ethanol (60%);
(e) BH3-THF, MeOH (72%); (f) PhB(OH)2, Pd(OAc)2, PPh3 (95%);
(g) (Ts)2O, Pyr, DMAP (95%); (h) 2-(4-hydroxyphenoxy)-2-meth-
ylpropanoic acid ethyl ester, Cs2CO3, DMF (50%), NaOH, EtOH
(68%).
gions, and when activated by agonist ligand binding, it
leads to gene transcription of proteins involved in
control of lipid and carbohydrate metabolism. PPARγ
agonists (e.g., rosiglitazone (1, Avandia) and troglitazone
(2, Rezulin)) have displayed clinical utility for increasing
insulin sensitivity and improving glycemic control in
Type 2 diabetes (Chart 1). In addition, these compounds
have been shown to inhibit inflammatory activation of
macrophages,5a to promote cholesterol efflux in mac-
rophages via the LXR-ABCA1 pathway,5b and to inhibit
atherosclerosis in the ApoE deficient and LDL receptor
deficient mice.6 Seminal contributions by the Takeda
and Pfizer groups have shown that the 5-methyl-2-
phenyl-4-oxazolyl thiazolidine-2,4-diones such as 10a
give highly potent in vivo antidiabetic activities in the
ob/ ob mouse.7 Thiazolidine-2,4-diones (TZDs) are gen-
erally selective for PPARγ, although a TZD, KRP-297
(9), with activity at PPARR and PPARγ was recently
disclosed.8 The structurally related isoxazolidinedione
J TT-501 (10b) has also been recently reported with
* Corresponding authors. Chemistry: tel 1-317-433-4950; fax 1-317-
277-2035; e-mail Brooks_Dawn_A@Lilly.com. Pharmacology: tel 1-317-
276-9800; fax 1-317-276-9574; e-mail Etgen_Garret_J @Lilly.com.
† Lilly Research Laboratories.
‡ Ligand Pharmaceuticals.
§ Current address: Nutec Sciences, 1201 W. Peachtree St., Suite
800, Atlanta, GA 30309.
10.1021/jm0155188 CCC: $20.00 © 2001 American Chemical Society
Published on Web 05/23/2001

2062 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13
Letters
Ta ble 1. Binding IC50 and Cotransfection Efficacy Data: Comparison of Compounds 4-8 with Reference Compoundsa
hPPARγ
hPPARRb
no.
IC50 (nM)
CTF eff (%)
EC50 (nM)
IC50 (nM)
CTF eff (%)
EC50 (nM)
4
5
6
7
8
1
2
3
10591 ( 710
3120 ( 247
3017 ( 117
4556 ( 308
548 ( 72
6 ( 1
eff < 20%
2751 ( 91
2059 ( 243
1716 ( 398
882 ( 89
657 ( 345
2235 ( 344
eff < 20%
1090 ( 305
8 ( 1
10596 ( 2044
2033 ( 440
1445 ( 346
242 ( 50
28 ( 1
65 ( 1
69 ( 2
58 ( 6
92 ( 21
9
0
16
54 ( 1
30 ( 0
2751 ( 91
625 ( 125
717 ( 107
61.9 ( 0.98
149.5 ( 48.5
eff < 20%
eff < 20%
eff < 20%
922 ( 185
771 ( 44
42 ( 4
55 ( 5
24 ( 5
123 ( 24
100 ( 21
79 ( 1
0
174 ( 17
48 ( 2
>10000
no binding
68000
914 ( 88
4086 ( 363
1285 ( 61
no binding
463 ( 88
9
10a
109 ( 5
61 ( 2
14 ( 3
a
b
Values are the mean of three to five experiments. Gal4-hPPARR was used to eliminate interference by endogenous PPARγ receptors
in CV-1 cells.
PPARγ activity similar to troglitazone accompanied by
weak activity at PPARR.9
This also afforded the opportunity to conduct initial in
vitro screens using a displacement study (ABCD binding
assays) to determine binding IC50s and cotransfection
studies14 in CV-1 cells to demonstrate functional ef-
ficacy. The results using these primary tools to drive
the SAR efforts are shown in Table 1. Assessment of
compound 4 demonstrates the necessity of methyl
substitution R to the carboxylic acid to achieve efficacy.
Para substitution of the 2-phenyloxazole in analogue 7
provides a significant boost in binding at the hPPARR
receptor (242 nM). This breakthrough led to the syn-
thesis of the p-biphenyl-oxazole analogue 8, which binds
to both hPPARR and hPPARγ with IC50 values (∼Ki) of
174 and 548 nM, respectively. In addition, compound 8
is shown by cotransfection studies to be an effective
agonist at both of these receptors. Compound 8 is also
PPARR agonists (e.g., gemfibrozil, fenofibric acid (3)),
on the other hand, produce reductions in serum tri-
glycerides and increases in HDL cholesterol, in some
cases accompanied by reductions in serum fibrinogen.10
Moreover, PPARR receptor activation has also been
shown to produce antiinflammatory effects in vascular
cells.11 The combined profile of a dual PPARR/γ agonist
thus appears well-suited for treatment of hyperglycemia
together with prevention of cardiovascular disease in
Type 2 diabetes.12 Herein we describe our efforts to
identify a dual PPARR/γ agonist based on in vivo studies
and in vitro binding results. This research has resulted
in the identification of 8, which combines the putative
pharmacophores of known PPARγ- and PPARR-selective
agents in a single molecule with excellent dual PPARR/γ
agonist activity.
Ch em istr y. The synthesis of 8, as outlined in Scheme
1, was designed to allow flexibility in the preparation
of analogues for SAR studies. Using a modification of
the Goto et al. procedure,13 butanedione monooxime was
condensed with 4-bromobenzaldehyde, and the resulting
N-oxide (11) was converted to chloride 12 by treatment
with phosphorus oxychloride. The corresponding nitrile
was prepared by reaction of chloride 12 with potassium
cyanide in the presence of potassium iodide. Following
conversion of the nitrile to the corresponding carboxylic
acid with potassium hydroxide, the acid was reduced
to the desired alcohol (13) with borane-THF. Suzuki
coupling of alcohol 13 with phenyl boronic acid pro-
vides alcohol 14. Tosylate formation of alcohol 14 was
followed by coupling to 2-(4-hydroxyphenoxy)-2-meth-
ylpropanoic acid ethyl ester with cesium carbonate in
DMF and hydrolysis to afford 8. Analogues 4-6 were
prepared via the straightforward coupling of 5-methyl-
2-phenyl-4-oxazolyl ethanol with the appropriate (4-
hydroxyphenoxy)propanoic (or acetic) acid ethyl ester,
followed by hydrolysis to the corresponding acid. Direct
coupling of 13 to 2-(4-hydroxyphenoxy)-2-methylpro-
panoic acid ethyl ester, followed by hydrolysis, affords
compound 7.
an effective agonist at mouse PPARR (104%, EC50
)
2560 nM). This latter finding is important because of
substantial interspecies variability in ligand activation
of PPARR, in contrast to PPARγ, and the requirement
for activation of mouse PPARR in the preclinical animal
model.
Binding and cotransfection data for 8 are compared
with corresponding data for reference compounds in
Table 1. Although not as potent as rosiglitazone (1) or
Takeda’s 5-methyl-2-phenyl-4-oxazolyl thiazolidine-2,4-
dione 10a at binding to hPPARγ, compound 8 shows
efficacy comparable to rosiglitazone (1) at this receptor.
Moreover, 8 is considerably more potent at activating
and binding to hPPARR receptors than the reference
compounds, including the dual agonist KRP-297 (9).
Thus, the in vitro profile of 8 as a dual PPARR/γ agonist
distinguishes this compound from marketed compounds
now known to act on these receptors.
Specificity of 8 was examined in binding and cotrans-
fection studies using a panel of nuclear transcription
factors. For the following receptors, 8 either failed to
bind or displayed poor affinity (Ki > 10 µM) as a lig-
and: retinoic acid receptor (RARR, RARâ, RARγ), ret-
inoic acid X receptor (RXRR, RXRâ, RXRγ), glucocorti-
coid receptor, and thyroid receptor (TRR and TRâ).
Furthermore, cotransfection assays for RARR and RXRR
demonstrated a lack of agonist activity for 8 at these
receptors. Thus, binding and/or activation of nuclear
transcription factors by 8 appear to be confined to the
PPAR family of receptors.
Resu lts a n d Discu ssion . Compounds 4-8 were
prepared based on the rational design of a dual PPAR
agonist agent using the putative pharmacophores of
known PPARγ- and PPARR-selective compounds that
were developed and optimized solely by in vivo animal
studies. Cloning and expressing both the human PPARR
(hPPARR) and PPARγ (hPPARγ) receptors led to an
increased mechanistic understanding of their function.
To determine the ability of 8 to both lower hypergly-
cemia and positively impact lipid/cholesterol homeosta-
sis in a clinically relevant manner, a new diabetic
animal model, the EOB mouse,15 was created. Briefly,

Letters
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13 2063
F igu r e 1. Plasma glucose levels in EOB mice treated via oral
gavage for 7 days with fenofibrate, rosiglitazone, or compound
8. Bars represent means ( SE (n ) 5 mice per group; *p <
0.05 relative to vehicle control). Horizontal line indicates aver-
age plasma glucose levels in untreated chow fed (Purina 5001
diet) human apoA-I transgenic mice (159.4 ( 7.5 mg/dL).
F igu r e 3. Plasma triglyceride levels in EOB mice treated via
oral gavage for 7 days with fenofibrate, rosiglitazone, or
compound 8. Bars represent means ( SE (n ) 5 mice per
group; *p < 0.05 relative to vehicle control). Horizontal line
indicates average plasma triglyceride levels in untreated chow
fed (Purina 5001 diet) human apoA-I transgenic mice (167.2
( 10.6 mg/dL).
agents. In addition to normalizing plasma glucose, 8
dose-dependently elevated serum HDL cholesterol levels
(Figure 2) and reduced plasma triglycerides (Figure 3).
At the glucose normalizing dose of 30 mg/kg/d, HDL
cholesterol levels were elevated by 48.4% and plasma
triglycerides were reduced by ∼5.5-fold, relative to
control levels.
In summary, compound 8 was designed and synthe-
sized as a dual PPARR/γ agonist. This profile was
demonstrated by in vitro binding to the human and
mouse PPAR receptors and in vivo efficacy in a diabetic
animal model. Further evaluation of compound 8 is
underway.
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
tails for the binding assays, cotransfection assay, the EOB
mouse model, and the intermediate and final compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
F igu r e 2. Increase in serum HDL cholesterol levels in EOB
mice treated via oral gavage for 7 days with fenofibrate,
rosiglitazone, or compound 8. Bars represent percent HDL
cholesterol change from vehicle control for FPLC analyzed
serum samples pooled for each group (n ) 5 mice per group).
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