Synthesis and biological evaluation of novel pyrrolidine acid analogs as potent dual PPARα/γ agonists

Article abstract of DOI:10.1016/j.bmcl.2015.01.066

The design, synthesis and structure-activity relationships of a novel series of 3,4-disubstituted pyrrolidine acid analogs as PPAR ligands is outlined. In both the 1,3- and 1,4-oxybenzyl pyrrolidine acid series, the preferred stereochemistry was shown to be the cis-3R,4S isomer, as exemplified by the potent dual PPARα/γ agonists 3k and 4i. The N-4-trifluoromethyl-pyrimidinyl pyrrolidine acid analog 4i was efficacious in lowering fasting glucose and triglyceride levels in diabetic db/db mice.

Full text of DOI:10.1016/j.bmcl.2015.01.066

Accepted Manuscript
Synthesis and biological evaluation of novel pyrrolidine acid analogs as potent
dual PPARα/γ agonists
Hao Zhang, Charles Z. Ding, Zhi Lai, Sean S. Chen, Pratik Devasthale, Tim
Herpin, George Morton, Fucheng Qu, Denis Ryono, Rebecca Smirk, Wei Wang,
Shung Wu, Xiang-Xang Ye, Yi-Xin Li, Atsu Apedo, Dennis Farrelly, Tao
Wang, Liqun Gu, Nathan Morgan, Neil Flynn, Cuixia Chu, Lori Kunselman,
Jonathan Lippy, Kenneth Locke, Kevin Omalley, Thomas Harrity, Michael Cap,
Lisa Zhang, Vinayak Hosagrahara, Pathanjali Kadiyala, Carrie Xu, Arthur M.
Doweyko, Robert Zahler, Narayanan Hariharan, Peter T.W. Cheng
PII:
S0960-894X(15)00087-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.01.066
BMCL 22406
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
28 November 2014
27 January 2015
29 January 2015
Please cite this article as: Zhang, H., Ding, C.Z., Lai, Z., Chen, S.S., Devasthale, P., Herpin, T., Morton, G., Qu, F.,
Ryono, D., Smirk, R., Wang, W., Wu, S., Ye, X-X., Li, Y-X., Apedo, A., Farrelly, D., Wang, T., Gu, L., Morgan,
N., Flynn, N., Chu, C., Kunselman, L., Lippy, J., Locke, K., Omalley, K., Harrity, T., Cap, M., Zhang, L.,
Hosagrahara, V., Kadiyala, P., Xu, C., Doweyko, A.M., Zahler, R., Hariharan, N., Cheng, P.T.W., Synthesis and
biological evaluation of novel pyrrolidine acid analogs as potent dual PPARα/γ agonists, Bioorganic & Medicinal
Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.01.066
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Synthesis and biological evaluation of novel pyrrolidine acid analogs as potent dual
PPARα/γ agonists
Hao Zhanga*, Charles Z. Dinga, Zhi Laia, Sean S. Chena, Pratik Devasthalea, Tim Herpina, George Mortona,
Fucheng Qua, Denis Ryonoa, Rebecca Smirka, Wei Wanga, Shung Wua, Xiang-Xang Yea, Yi-Xin Lif, Atsu
Apedof, Dennis Farrellyb, Tao Wangc, Liqun Gub, Nathan Morganb, Neil Flynnb, Cuixia Chub, Lori
Kunselmanb, Jonathan Lippyc, Kenneth Lockec, Kevin Omalleyc, Thomas Harrityb, Michael Capb, Lisa
Zhangd, Vinayak Hosagraharad, Pathanjali Kadiyalad, Carrie Xud, Arthur M. Doweykoe, Robert Zahlera,
Narayanan Hariharanb, Peter T. W. Chenga,*
a
b
Metabolic Diseases Chemistry, Bristol-Myers Squibb Research and Development (R & D), Princeton, NJ 08543-5400, USA; Metabolic Diseases Biology,
Bristol-Myers Squibb R & D, Princeton, NJ 08543-5400, USA; c Lead Evaluation, Bristol-Myers Squibb R & D, Princeton, NJ 08543-5400, USA; d Metabolism
e
and Pharmacokinetics, Bristol-Myers Squibb R & D, Princeton, NJ 08543-5400, USA; Computer-Assisted Drug Design, Bristol-Myers Squibb R & D,
Princeton, NJ 08543-5400, USA; f Discovery Analytical Sciences, Bristol-Myers Squibb R & D, Princeton, NJ 08543-5400, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
The design, synthesis and structure-activity relationships of a novel series of 3,4-
disubstituted pyrrolidine acid analogs as PPAR ligands is outlined. In both the 1,3-
and 1,4-oxybenzyl pyrrolidine acid series, the preferred stereochemistry was shown
to be the cis-3R, 4S isomer, as exemplified by the potent dual PPARα/γ agonists 3k
and 4i. The N-4-trifluoromethyl-pyrimidinyl pyrrolidine acid analog 4i was
efficacious in lowering fasting glucose and triglyceride levels in diabetic db/db mice
Revised
Accepted
Available online
Keywords:
Peroxisome Proliferator-Activated Receptor
dual agonists
pyrrolidine acid
2014 Elsevier Ltd. All rights reserved
.
type 2 diabetes
The PPARs (Peroxisome Proliferator-Activated Receptors) are
a group of nuclear hormone receptors which function as
transcription factors in the regulation of genes involved in
glucose and lipid fatty acid metabolism as well as vessel wall
function.1 Three PPAR subtypes have been identified: PPARα, γ
and δ. PPARα is predominantly expressed in catabolically active
tissues such as liver, heart, kidney, and muscle. PPARα has been
shown to be intimately involved in the uptake and β-oxidation of
free fatty acids as well as lipoprotein metabolism.2 The clinically
used PPARα agonists comprise the fibrate class of drugs
(including fenofibrate3 and gemfibrozil4), which elevate HDL
cholesterol and lower triglyceride and LDL cholesterol levels.
PPARγ, which is mainly expressed in adipose tissue, regulates
insulin sensitivity, glucose and free fatty acid utilization as well
as adipocyte differentiation.5 The clinically used PPARγ agonists
encompass the thiazolidinedione (TZD) class of antidiabetic
drugs such as rosiglitazone6 and pioglitazone.7 An attractive
hypothesis has been that combining PPARγ and PPARα agonist
activities in a single compound would result in an excellent anti-
diabetic agent based on the projected synergistic improvements
in insulin sensitivity and normalization of glucose metabolism as
well as the amelioration of the characteristic dyslipidemia
associated with type 2 diabetes.
As previously described, the initial structure-activity
relationship (SAR) exploration of the oxybenzylglycine series of
PPARα/γ dual agonists resulted in the discovery of the Phase III
clinical compound Muraglitazar I.8 Compound I has good in
vitro functional activity at both human PPARα and PPARγ
(αEC50 = 0.32 µM and γ EC50 = 0.11 µM in CV-1 cells; αEC50
=
1.41 µM & γ EC50 = 0.035 µM in HEK293 cells) and
demonstrated excellent efficacy in animal models of type 2
diabetes and the associated dyslipidemia. Our subsequent goal in
the program was to discover backup PPARα/γ dual agonists with
significantly differentiated in vitro/in vivo profiles from I (while
retaining in vivo efficacy and safety). We initially investigated
structurally differentiated analogs of muraglitazar by
conformationally constraining the “head-piece” N-carbamoyl
glycine moiety of I. This included the structural motif where the
benzylic methylene is cyclized onto the glycine α-carbon to form
a 2,5-disubstituted pyrrolidine acid, as exemplified by II, which
is a relatively potent and highly selective PPARγ agonist; analogs
from this series have demonstrated good activity both in vitro and
in vivo. To further explore the scope of the SAR of this

interesting pyrrolidine acid series, we decided to investigate the
effect of shifting both the 5-aryl and 2-carboxylic acid
substituents of the pyrrolidine ring of II into the corresponding 4-
and 3-positions (i.e. structure III). We report here the exploration
of the SAR of this 3,4-disubstituted pyrrolidine acid series
(analogs 1a-6c, Fig. 1). This effort resulted in the discovery of a
number of analogs which have potent dual PPARα and PPARγ
agonist in vitro activity (both in binding and functional assays).
Hydrogenolysis of the N-benzyl group gave the free amine
pyrrolidine ester 11. Reaction of pyrrolidine ester 11 with
appropriate chloroformates provided the corresponding
pyrrolidine carbamates. Acid-mediated deprotection of the ethyl
ester then provided the desired cis-substituted pyrrolidine-
carbamate acid analogs (1a-d; 3a, 3d, 3i, 3j; 5a, 5b, 5d, 5e). The
cis pyrrolidine acetic acid analogs 2a, 2b, 2e and 2f were
synthesized by carrying corresponding pyrroridine acids through
a standard Arndt-Eistert homologation sequence.
Alternatively, standard Horner-Emmons reaction of aldehyde 8
gave the trans-alkenyl ester 12 after chromatography.13 The
trans-alkenyl ester 12 was subjected to the same acid-mediated
1,3 dipolar cycloaddition with N-TMS-methyl N-methoxymethyl
benzylamine as in Scheme 1 to give the corresponding trans-
substituted N-benzyl pyrrolidine-ester 13. Analogous to the
sequence described in Scheme 1, hydrogenolysis of the N-benzyl
pyrrolidine ester 13 gave the pyrrolidine-ester 14, which was
reacted with the appropriate chloroformates, then deprotected
under acidic conditions to provide the trans-substituted
pyrrolidine-carbamate acids (1e, 1f; 3g, 3h; 5c, 5f, 5g). The trans
pyrrolidine acetic acids (2c, 2d; 6a, 6b) were synthesized from
corresponding pyrroridine acids by Arndt-Eister homologation
(Scheme 2).
Figure 1. Design of 3,4-disubstituted pyrrolidine acids as PPARα/γ agonists.
The synthesis of the racemic cis-3,4-disubstituted pyrrolidine
acid analogs is shown in Scheme 1.9 Oxidation of alcohol 710
with pyridinium chlorochromate (PCC) gave the aldehyde 8,
which was subjected to reaction with a bis-trifluoroethyl
phosphonate Horner-Emmons reagent to give preferentially the
cis-alkenyl ester 9.11 The cis-alkenyl ester 9 was then reacted
with N-trimethylsilylmethyl N-methoxymethyl benzylamine
under acidic conditions in a 1,3-dipolar cycloaddition to give the
corresponding cis-N-benzyl pyrrolidine ester 10.12
Scheme 2. Reagents and conditions: (a) toluene, reflux; (b) TFA; (c) H2, 10%
Pd/C, acetic acid; (d) i. NaHCO3 ii. HCl; (e) i. (COCl)2; ii. CH2N2, iii.
PhCO2Ag, (C2H5)3N; iv. LiOH/THF
The synthesis of the corresponding heteroaryl-substituted
pyrrolidine acids is shown in Scheme 3. Copper-mediated N-
arylation of the pyrrolidine ester 11 with an appropriate
heteroaryl halide furnished the corresponding N-heteroaryl
pyrrolidine ester.14
Scheme 1. Reagents and conditions: Reagents and conditions: (a) PCC; (b)
(CF3CH2O)2P(O)CH2CO2Et, KHMDS, 18-crown-6, THF, -78oC; (c) TFA; (d)
H2, 10%Pd/C, acetic acid; (e) i. R'OCOCl/NaHCO3 ii. HCl; (f) i. (COCl)2, ii.
CH2N2, iii. PhCO2Ag, (C2H5)3N; iv. LiOH/THF
Scheme 3. Reagents and conditions: (a) i. CuI, K3PO4, 2,6-lutidine; ii. HCl;
(b) i. DIPEA ii. HCl
Deprotection of this ester under acidic conditions (necessitated

Deprotection was deliberately carried out under basic conditions
in order to induce partial epimerization of C-3 on the pyrrolidine
ring. This protocol provided a mixture of the chiral cis
pyrrolidine-acid 4i (3R, 4S) and the corresponding trans
pyrrolidine-acid 4l (3S, 4S) which were separated by preparative
HPLC; their structures were fully characterized by extensive 1D
and 2D-NMR studies.
due to partial epimerization of the stereochemistry of the α-
carbon of the ester under basic hydrolytic conditions) then
provided the racemic cis-aryl-substituted pyrrolidine acids (4a,
4m, 4n). Alternatively, reaction of pyrrolidine ester 11 with
activated halo-heteroaryls such as 2-chloro-4-(trifluoromethyl)
pyrimidines in the presence of Hunig’s base followed by acid-
mediated ester hydrolysis provided the pyrimidine substituted
pyrrolidine acid analogs (4f, 4g, and 4h).
The corresponding cis (3R, 4S) pyrrolidine-acid analog 4d (in
the 1,3-oxybenzyl series) was prepared by the same synthetic
sequence as for 4i except that acidic hydrolysis was used as the
last step to avoid epimerization. Using this general methodology,
we have also synthesized the corresponding reference
enantiomers 4e (cis 3S,4R), 4j (cis 3S,4R), and 4k (trans 3R,4R)
by simply using the opposite L-camphorsultam chiral auxiliary.
Alternatively, the racemic pyrrolidine-ester 11 was also
separated by chiral HPLC (on a Chiralpak AD column [5 cm x 50
cm, 20 µm] using an isocratic eluting system of 20% MeOH/
EtOH(1:1) + 80% heptanes + 0.1% Et2NH) to provide the two
individual enantiomers 21 and 22. Reaction of optically pure
pyrrolidines 21 and 22 with chloroformates followed by acid-
mediated ester hydrolysis provided the optically pure pyrrolidine-
carbamate acid analogs 3b, 3c, 3e, 3f, and 3k respectively
(Scheme 5). The absolute configurations of the individual
enantiomers from the chiral separation were determined by
correlation with the enantiomers of known absolute
stereochemistry obtained via asymmetric synthesis as described
above. The homologated cis chiral pyrrolidine acetic acids 6c
was also synthesized by Arndt-Eistert reaction with 3b. The
absolute stereochemistry of analog 3e (cis 3R,4S) was also
separately confirmed by Vibrational Circular Dichroism(VCD).16
The absolute stereochemistry of the enantiomers of the 1,4-
phenyl cis-pyrrolidine acid analogs 4j (cis 3S,4R) or 4i (cis
3R,4S) and 4l (trans 3S,4S) or 4k (trans 3R,4R) was determined
by asymmetric synthesis as shown in Scheme 4. The key step in
the synthesis is a chiral 1,3-dipolar cycloaddition using D-
Camphorsultam as the chiral auxiliary (Xc).15 Me3Al-mediated
reaction of methyl 2-(bis(2,2,2-trifluoroethoxy) phosphoryl)
acetate 15 with D-camphorsultam gave the chiral Horner-
Emmons reagent 16, which was reacted with aldehyde 8 to give
the predominantly (Z)-alkene ester 17. TFA-mediated 1,3-
dipolar cycloaddition of chiral alkene-ester 17 with N-
trimethylsilylmethyl N-methoxymethyl benzylamine provided a
~4:1 mixture of diastereoisomers, as determined by HPLC
analysis. The major product, the chiral cis-pyrrolidine ester 18,
was readily isolated by column chromatography in 73% yield.
The predicted absolute stereochemistry of the 2 chiral centers of
18 is (3R, 4S), based on the 1,3-dipolar cycloaddition proceeding
via the accepted transition state for the cycloaddition reaction of
D-camphorsultam derivatives.15 Hydrogenolysis of 18 gave the
pyrrolidine ester 19, which was then reacted with 2-chloro-4-
(trifluoromethyl)pyrimidine in the presence of Hunig’s base to
provide the N-pyrimidinyl pyrrolidine ester 20.
Scheme 5. Reagents and conditions: (a) Chiral separation using AD column,
20% (MeOH/EtOH) in heptane + 0.1% DEA; (b) i. R'OC(O)Cl/NaHCO3 ii.
20%HCl/HOAc; (c) i. (COCl)2; ii. CH2N2, iii. PhCO2Ag, (C2H5)3N; iv.
LiOH/THF; (d) i. R'OC(O)Cl/NaHCO3 ii. 20% HCl/HOAc
An alternative route for the asymmetric synthesis of pyrrolidine
acids is shown in Scheme 6. Reaction of maleic anhydride 23
with (R)-methylbenzylamine and (COCl)2/Et3N in CH2Cl2
provided maleimide 24 in 55%. This chiral maleimide underwent
1,3-dipolar cycloaddition with N-trimethylsilylmethyl-N-
methoxymethyl benzylamine to give the corresponding N-benzyl
pyrrolidine-ester 25. Stereoselective reaction of meso-imide 25
(from the less-hindered face of the imide) with a protected 4-
oxyaryl Grignard reagent gives an intermediate hydroxy lactam
26,17 which is immediately reduced with NaBH4 followed by
thermally-induced intramolecular cyclization to give the lactone
27 in 61% yield (98% ee). Deprotection of the THP group of 27
Scheme 4. Reagents and conditions: (a) AlMe3; (b) KHMDS, 18-crown-6,
THF, -78oC; (c) TFA; (d) H2, 10%Pd/C, acetic acid; (e) DIPEA; (f)
LiOH/THF

Table 1. In vitro PPAR α and γ activities of phenyl pyrrolidine acid analogs 1
provided the phenol-lactone 27, followed by base-mediated
alkylation of the phenol 27 with phenyloxazole-ethanol mesylate
28 to provide the alkylated phenol lactone 28. Hydrogenolysis of
the N-benzyl group with concomitant cleavage of the benzylic
lactone C-O bond provided the chiral pyrrolidine acid 29.
Subsequent
reaction
of
29
with
2-chloro-4-
(trifluoromethyl)pyrimidine provided the chiral N-pyrimidinyl
pyrrolidine acid 4i;18 the absolute stereochemistry of 4i was
correlated with material from the alternative asymmetric
synthesis described above.
Oxy-
γEC50
(µM)
αEC50
(µM)
3,4-
Stereo-
γIC50
(µM)
αIC50
(µM)
benzyl
Substi-
tution
R
Cpd
chemistry
IA (%)
IA (%)
24
(58%)
20
(52%)
1a
1,3-
1,3-
-Et
>10
>10
cis
5.65
(35%)
1.65
(54%)
1b
1c
1d
1e
1f
-Ph
-Et
-Ph
-Et
-Ph
>10
>10
4.47
>10
5.86
>10
>10
>10
>10
2.86
cis
cis
>32
> 15
>15
1,4-
1,4-
1,4-
1,4-
4.18
(33%)
cis
>32
> 15
trans
trans
0.01
2.58
(99%)
(58%)
IA = intrinsic activity
Addition of a methylene linker between the pyrrolidine ring
and the carboxylic acid, as illustrated by analogs 2a-2f, resulted
in dramatic changes in their PPAR in vitro activities (Table 2). In
the 1,3-oxyphenyl series, the racemic homologated cis
pyrrolidine carbamate-acids (2a
&
increases in potency at both PPARα
2b) showed ≥10-fold
versus the
&
γ
corresponding pyrrolidine acid analogs (e.g. 1a and 1b).
However, the trans pyrrolidine analogs 2c & 2d only showed
modest PPARα and γ agonist activities. In the 1,3-oxyphenyl
series, the homologated racemic cis pyrrolidine carbamate
analogs (2e & 2f) also did not show any improvement in
PPARα and γ agonist activities versus 1c and 1d.
Scheme 6. Reagents and conditions: (a) i. (R)-α-methyl benzyl amine, ii.
(COCl)2/CH2Cl2’iii. TEA; (b) TFA; (c) THF; (d) i. NaBH4, ii. toluene, reflux;
(e) i. p-TsOH in DCM, ii. K2CO3 in CH3CN; (f) H2, 10%Pd/C, acetic acid; (g)
DIPEA
Table 2. In vitro PPAR α and γ activities of phenyl pyrrolidine-acetic acid
analogs 2
The PPARα and PPARγ in vitro activities (binding affinity19 as
well as transactivation activity20) of representative analogs from
these two novel pyrrolidine-acid series are shown in Tables 1-5.
In an earlier SAR study21 on a structurally analogous azole acid
series (derived from constraining the carbamate moiety of 1), we
showed that PPARα/γ activities were modulated by: 1) the linker
length between the central phenyl ring and the azole core and 2)
1,3 vs. 1,4-substitution on the central phenyl ring. We sought to
determine the importance of these structural features in the
pyrrolidine acid series.
Oxy-
γEC50
αEC50
3,4-
Stereo-
chemistry
γIC50
(µM)
αIC50
(µM)
benzyl
Substi-
tution
R
Cpd
(µM)
(µM)
IA (%)
IA (%)
0.02
1.13
2a
2b
2c
1,3-
1,3-
1,3-
-Et
-Ph
-Et
0.91
0.24
5.46
0.99
4.02
>10
cis
cis
(85%)
(70%)
4.80
(7%)
3.55
(18%)
Our initial SAR studies with pyrrolidine acid analogs 1a-1f are
shown in Table 1. Comparison of the relative PPAR activities of
both the 1,3- & 1,4-oxyphenyl series of racemic cis- or trans-
pyrrolidine carbamate-acids (1a~1e) showed that they displayed
very weak in vitro activities at both PPARα and PPARγ. The
only compound to show significant PPARγ agonist activity was
the racemic trans-pyrrolidine phenyl carbamate acid analog 1f in
the 1,4-oxyphenyl series. The analogs in this series have their
two key pharmacophores (oxyphenyl & carboxylic acid) in fixed
conformations at the 3- and 4-positions of the pyrrolidine ring,
and are most likely not in optimal position in the PPARα or γ
binding sites in the ligand-binding domain. We postulated that
the introduction of additonal flexibility into these two key
substituents on the pyrrolidine ring would be necessary to
improve their PPARα/γ in vitro activity.
9.68
8.10
trans
(104%)
(88%)
14.6
(51%)
11.7
(17%)
2d
2e
2f
1,3-
1,4-
1,4-
-Ph
-Et
-Ph
1.82
>10
8.31
>10
>10
>15
trans
cis
4.18
(33%)
>15
cis
We next explored the effects of inserting a methylene spacer
between the oxyphenyl substituent and the pyrrolidine ring
(analogs 3a-3k in Table 3). In the 1,3-oxyphenyl series, the

corresponding oxybenzyl analogs (i.e. 3a vs 1a, 3d vs 1b)
showed that increasing the distance not only dramatically
improved PPARα/γ binding affinities, but also resulted in more
potent functional activity at both receptors (from 5- to 30-fold at
PPARγ and >1500-fold at PPARα for these 2 pairs of analogs).
We then examined the effects of the relative & absolute
stereochemistry of the 3- and 4- substituents on the pyrrolidine
ring on PPAR potency. The (3R, 4S) enantiomers (3b & 3e) in
the 1,3-oxybenzyl cis-pyrrolidine carbamate-acid series are very
potent PPARα agonists (EC50 = 3-4 nM), but the ethyl carbamate
3b is a much more potent PPARγ agonist (γEC50 = 9 nM), than
the phenyl carbamate 3e (γEC50 = 2.0 µM). Both of the cis (3R,
4S) enantiomers (3b & 3e) are much more potent at PPARα/γ
than the corresponding cis (3S, 4R) enantiomers 3c & 3f (3f
shows activity only at PPARα).
preferred absolute stereochemistry for both binding affinity and
optimal functional activity at PPARα and PPARγ.
Table 4. In vitro PPAR α and γ activities of benzyl pyrrolidine-acid analogs 4
3,4-
Stereo-
Chemis-
try
Oxy-
benzyl
Substi-
tution
γEC50
αEC50
γIC50
(µM)
αIC50
(µM)
Ar
Cpd
(µM)
(µM)
IA (%)
IA (%)
3.41
0.02
4a
4b
1,3-
1,3-
Ph
Ph
0.22
0.08
0.04
0.07
cis
(24%)
(107%)
cis
3.44
(29%)
0.05
(152%)
Table 3. In vitro PPAR α and γ activities of benzyl pyrrolidine-acid analogs 3
(3R, 4S)
cis
0.68
(75%)
4c
1,3-
1,3-
Ph
0.14
0.04
>10
0.32
0.02
(3S, 4R)
cis
F3C
2.50
(75%)
0.014
(107%)
N
N
Oxy-
4d
γEC50
αEC50
(3R, 4S)
3,4-
Stereo-
chemistry
γIC50
(µM)
αIC50
(µM)
benzyl
Substi
-tution
R
Cpd
(µM)
(µM)
IA (%)
IA (%)
F3
C
cis
N
N
4e
4f
1,3-
1,4-
1,4-
1,4-
1,4-
1,4-
1,4-
1,4-
1,4-
1,4-
0.11
2.04
0.45
0.03
0.02
1.53
3.57
1.16
0.59
0.05
>10
1.12
>10
3.98
0.74
0.96
>10
1.12
>50
2.19
5.01
>10
0.75
0.01
3a
3b
1,3-
1,3-
Et
Et
1.90
1.51
0.17
0.26
(3S, 4R)
cis
(58%)
(170%)
cis
0.009
0.003
N
N
1.06
(67%)
2.91
(58%)
(75%)
(127%)
cis
cis
cis
(3R, 4S)
cis
10.9
3c
3d
3e
1,3-
1,3-
1,3-
Et
Ph
Ph
4.28
1.33
0.49
>10
>10
0.22
0.07
(27%)
(3S, 4R)
N
N
0.63
(49%)
0.17
(93%)
4g
4h
4i
1.25
(27%)
0.01
(165%)
cis
F3
C
N
N
0.05
0.03
cis
2.0
0.004
(107%)
(110%)
(44%)
(130%)
(3R, 4S)
cis
13.3
(9%)
0.95
(93%)
3f
1,3-
Ph
0.59
0.66
F3
C
cis
0.03
(85%)
0.09
(108%)
N
N
(3S, 4R)
(3R, 4S)
2.85
0.15
3g
3h
3i
1,3-
1,3-
1,4-
1,4-
Et
Ph
Et
8.11
3.63
2.68
0.08
2.12
3.09
2.02
0.61
trans
trans
cis
(105%)
(96%)
F3C
cis
N
N
4j
>10
>30
>30
>10
6.17
(59%)
2.59
(74%)
(3S, 4R)
0.30
0.006
F3
C
trans
1.0(21
%)
N
N
(76%)
(96%)
4k
4l
(3R, 4R)
0.64
(105%)
0.001
(125%)
3j
Ph
cis
F3C
trans
N
N
4.41(21
%)
cis
0.03
(93%)
0.005
(98%)
(3S, 4S)
3k
1,4-
Ph
0.08
0.18
(3R, 4S)
N
2.08
(52%)
2.48
(44%)
The SAR in the 1,4-oxybenzyl series correlates with the 1,3-
oxybenzyl series; the racemic cis pyrrolidine carbamates 3i and
3j have moderate PPARγ activity (EC50 = 0.30-0.64 µM), but are
very potent PPARα agonists (EC50 = 1-6 nM; >50-fold α vs. γ
selectivity). Of the two enantiomers of the cis pyrrolidine
carbamate-acid 3i, the preferred (3R,4S) analog 3k, as expected,
4m
4n
cis
cis
O
F3
C
N
O
1.47
(20%)
>30
is a very potent PPARα/γ dual agonist (γ EC50 = 30 nM; αEC50
5 nM). Based on these results, for this pyrrolidine acid series, the
cis-(3R,4S) absolute stereochemistry is established to be the
=
In this oxybenzyl pyrrolidine acid series, we also replaced the
N-carbamoyl moiety with various N-aryl substituents (which had
provided very potent analogs in the related triazole acid series21)

(4a-4n; Table 4). In the 1,3-oxybenzyl series, the racemic cis N-
phenyl pyrrolidine acid 4a is a potent PPARα agonist (EC50 = 20
nM), but is >170-fold less active at PPARγ (EC50 = 3.44 µM). As
observed previously with the N-carbamoyl-substuted pyrrolidine
analogs, the (3R,4S) enantiomer 4b is ~13-fold more potent at
PPARα than the corresponding (3S,4R) enantiomer 4c. The
enantiomeric pair of 4-trifluoromethyl pyrimidine analogs 4d and
4e also showed very similar PPAR activities to 4b and 4c. The
more active (3R,4S) enantiomer 4d is a potent PPARα selective
dual agonist (αEC50 = 14 nM; α/γ selectivity = >170-fold). In the
racemic cis 1,4-oxybenzyl pyrrolidine acid series series, the
effect of the 4-pyrimidinyl substituent on PPAR activity was
examined. The parent unsubstituted pyrimidine analog 4f showed
corresponding analogs in the oxybenzyl pyrrolidine series (e.g.
compare 5a, 5b, 5d, and 5e vs 3a, 3d, 3i, 3j respectively). These
analogs are generally potent PPARα agonists, but are
significantly weaker (3- to 20-fold) vs. PPARγ. In contrast,
analogs in the corresponding trans-oxyphenethyl series (e.g. 5c)
show significantly reduced (300-fold) PPARα agonist activity.
Significantly, in the trans-(1,4-oxyphenethyl) pyrrolidine acid
series, we were able to identify the N-carbamate analogs 5f and
5g as potent, highly selective PPARα agonists (without
significant PPARγ functional activity).
Finally, we explored the effects of inserting a methylene linker
between the pyrrolidine ring and the carboxylic acid in the 1,3-
oxybenzyl-pyrrolidine acid series (Table 6). In the trans-
oxybenzyl pyrrolidine acetic acid series, the ethyl carbamate
analog 6a is a very potent, highly selective PPARα full agonist
(EC50 = 6 nM). However, the corresponding phenyl carbamate
analog 6b is >10-fold less potent (EC50 = 0.85 µM) and is,
moreover, a partial PPARα agonist. Both 6a and 6b show
minimal PPARγ agonist functional activity. In the cis-oxybenzyl
series, even the preferred (3R,4S) enantiomer 6c only showed
weak, partial PPARα agonist activity (EC50 = 2.87 µM; intrinsic
activity = 32%). Overall, we were unable to identify any
balanced PPARα/γ dual agonists in this oxybenzyl pyrrolidine
acetic acid series. Interestingly, in our structurally related triazole
acid series of PPARα/γ agonists20, the optimal compounds were
oxybenzyl triazole acetic acids (analogous in structure to 6),
whereas in the pyrroldine acid series, the optimal structures are
oxybenzyl pyrrolidine acids 3 & 4; this is a rather striking
divergence in the SAR between these two series.
modest activity at both PPARα (EC50 = 2.91 µM) and γ (EC50
=
1.06 µM). Addition of a 4-methyl group on the pyrimidine (4g)
improved PPARα activity (EC50 = 170 nM; 17-fold vs. 4f) but
PPARγ activity was relatively unchanged. The most potent
analog in this series was the 4-trifluoromethyl pyrimidine 4h,
which is a potent, balanced PPAR α (EC50 = 30 nM) and γ (EC50
= 50 nM) dual agonist. As expected, the (3R,4S) enantiomer 4i
retains all the PPARα/γ agonist activity of 4h, whereas the
(3S,4R) enantiomer 4j was inactive at both PPARα and γ.
Interestingly, in the corresponding trans 1,4-oxybenzyl
pyrrolidine acid series, both the (3R, 4R) and (3S, 4S)
enantiomers 4k and 4l showed only weak PPAR functional
activities. Other N-heteroaryl substituents, such as N-2-
benzoxazole and 4-trifluoromethyl oxazole, also provided very
weakly active analogs (4m and 4n respectively).
Based on the improved PPARα/γ activities of the oxybenzyl
pyrrolidine acid series vs the oxyphenyl pyrrolidine acids, we
then examined the effect of further extending the linker between
the oxyphenyl group and the pyrrolidine ring. Insertion of
another methylene group resulted in a series of oxyphenethyl
pyrrolidine-acid analogs as shown in Table 5.
Table 6. In vitro PPAR α and γ activities of oxybenzyl-pyrrolidine-acetic
acid analogs 6
Table 5. In vitro PPAR α and γ activities of phenethyl-pyrrolidine-acid
analogs 5
γEC50
αEC50
3,4-
Stereo-
γIC50
(µM)
αIC50
(µM)
Oxybenzyl
substitution
(µM)
IA
R
(µM)
Cpd
chemistry
IA (%)
(%)
0.006
(143%)
6a
6b
1,3-
1,3-
-Et
-Ph
8.11
0.21
>10
>10
2.12
3.09
trans
trans
γEC50
αEC50
3,4-
γIC50
(µM)
αIC50
(µM)
Oxybenzyl
substitution
Stereo-
chemistry
R
Cpd
(µM)
(µM)
IA (%)
IA (%)
0.85
(44%)
0.71
0.20
5a
5b
5c
5d
5e
5f
1,3-
1,3-
1,3-
1,4-
1,4-
1,4-
1,4-
Et
Ph
Ph
Et
0.49
0.21
0.52
0.69
0.09
1.59
0.34
2.69
1.06
>10
3.27
0.48
1.81
3.25
cis
cis
(26%)
(117%)
cis
2.87
6c
1,3-
-Et
2.83
>10
5.55
(32%)
(3R, 4S)
0.63
(126%)
0.008
(113%)
Among these compounds, we further evaluated the N-phenyl
carbamate analog 3k and the 4-trifluoromethyl N-pyrimidine
analog 4i (from the 1,4-oxybenzyl cis-3R,4S-pyrrolidine acid
series), both of which have significantly improved in vitro
potency at PPARγ (> 10-fold for both) and at PPARα (>10-fold
for 3k and >100-fold for 4i) versus muraglitazar I (PPARα EC50
= 1.41 µM, and PPARγ EC50 = 35 nM in HEK293 cells).
1.17
2.60
trans
cis
(45%)
(30%)
0.54
(64%)
0.02
(133%)
0.16
0.01
Ph
Et
cis
(62%)
(77%)
0.01
(81%)
The in vivo antidiabetic and lipid-lowering activities of 3k and
4i were characterized in a 14-day study in female db/db mice.22
The data are shown in Table 7. The N-4-trifluoromethyl
pyrimidinyl pyrrolidine acid 4i (administered orally at a 10
mg/kg dose once daily) showed excellent efficacy in this diabetic
animal model, significantly reducing levels of both fasting
plasma glucose (-37%) as well as fasting triglycerides (-48%).
>10
>10
trans
trans
0.98
5g
Ph
(89%)
Overall both the cis 1,3 and 1,4-oxyphenylethyl pyrrolidine
analogs have very comparable PPARα/γ activities to the

Interestingly, the structurally closely related N-phenyl carbamoyl
pyrrolidine acid 3k (also dosed orally at 10 mg/kg/day), which
also lowering fasting glucose comparably (-30%), did not
significantly lower fasting plasma triglyceride levels (-12%), in
spite of being actually more potent at PPARα (EC50 = 5 nM) than
4i (EC50 = 90 nM) in the functional assay.
oxybenzylglycine 3,4-disubstituted pyrrolidine acid series will be
reported separately.
Acknowledgments
We thank the BMS Discovery Synthesis Group and the BMS
Discovery Analytical Sciences Department for support. We thank
Dr. Jeff Robl for careful proofreading of this manuscript.
Table 7. 14-day In vivo efficacy study of 3k & 4i (10 mg/kg/day
administered p.o.) in 8-10 week old female db/db mice
Treatment
10 mg/kg/day
Fasting
Glucose
(mg/dL)
Fasting
Triglycerides
(mg/dL)
References and notes
after 14 days
after 14 days
1. For general reviews on PPARs: (a) Sternbach, D. D. Annual Rep.
Med. Chem. 2003, 38, 71; Cheng, P. T. W.; Mukherjee, R. Minirev.
Med. Chem. 2005, 5, 741; (e) Cheng, P. T. W., Kick, E., Mukherjee,
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4i
240 18 (-37%)*
91 6 (-48%)*
143 16
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2. Duval, C.; Muller, M.; Kersten, S.; Biochim. Biophys. Acta, 2007,
1771, 961; Zandberg, F., Plutzky, J.; Biochim. Biophys. Acta, 2007,
1771, 972.
3k
355 26 (-30%)*
127
10 (-12%)
*p value <0.05
3. Saurav, A.; Kaushik, M.; Mohiuddin, S. M.; Exp. Opin.
Pharmacother., 2012, 13, 717.
4. Todd, P. A.; Ward, A.; Drugs 1988, 36, 314.
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Analog 4i also showed an acceptable profile in a set of standard
in vitro liabilities (metabolic stability in human/mouse/rat
microsomes, hERG and other ion channels). In a panel of 6
standard Cytochrome P450 (CYP450) inhibition assays, 4i only
showed modest activity at CYP2C9 (IC50 = 3.2 µM) and
CYP2C19 (IC50 = 8.8 µM). In a rat pharmacokinetics study,
(dosed orally at 10 mg/kg in a solution of 40% polyethylene
glycol, 10% Cremaphor and 50% aqueous phosphate buffer), 4i
had good oral bioavailability (65%) and reasonable plasma/tissue
distribution, as reflected by the Vss (volume of distribution) of
0.47 L/kg. However, the compound had a relatively short half-
life of 1.54 h.
6. Malinowski, J. M.; Bolesta, S.; Clin. Ther., 2000, 22, 1151.
7. Derosa, G.; Drugs, 2010, 70, 1945.
8.
(a) Devasthale, P. V.; Chen, S.; Jeon, Y.; Qu, F.; Shao, C.; Wang,
W.; Zhang, H.; Farrelly, D.; Golla, R.; Grover, G.; Harrity, T.; Ma,
Z.; Moore, L.; Ren, J.; Seethala, R.; Cheng, L.; Sleph, P.; Sun, W.;
Tieman, A.; Wetterau, J. R.; Doweyko, A.; Chandrasena, G.;
Chang, S. Y.; Humphreys, W. G.; Sasseville, V. G.; Biller, S. A.;
Ryono, D. E.; Selan, F.; Hariharan, N.; Cheng, P. T. W.; J. Med.
Chem. 2005, 48, 2248; (b) Devasthale, P. V.; Chen, S.; Jeon, Y.;
Qu, F.; Ryono, D. E.; Wang, W.; Zhang, H.; Cheng, L.; Farrelly,
D.; Golla, R.\; Grover, G.; Ma, Z.; Moore, L.; Seethala, R.; Sun,
W.; Doweyko, A. M.; Chandrasena, G.; Sleph, P.; Hariharan, N.;
Cheng, P. T. W.; Bioorg. Med. Chem. Lett., 2007, 17, 2312; (c)
Harrity, T.; Farrelly, D.; Tieman, A.; Chu, C.; Kunselman, L.; Gu,
L.; Ponticiello, R.; Cap, M.; Qu, F.; Shao, C.; Wang, W.; Zhang,
H.; Fenderson, W.; Chen, S.; Devasthale, P.; Jeon, Y.; Seethala, R.;
Yang, W.-P.; Ren, J.; Zhou, M.; Ryono, D.; Biller, S.; Mookhtiar,
K. A.; Wetterau, J.; Gregg, R.; Cheng, P. T.; Hariharan, N.;
Diabetes, 2006, 55, 240; (d) Kendall, D. M.; Rubin, C. J.;
Mohideen, P.; Ledeine, J.-M.; Belder, R.; Gross, J.; Norwood, P.;
O’Mahony, M.; Sall, K.; Sloan, G.; Roberts, A.; Fiedorek, F. T.;
DeFronzo, R. A. Diabetes Care 2006, 29, 1016.
In conclusion, the structure-activity relationships of several
series of novel 1,3- and 1,4-oxyphenyl 3,4-disubstituted
pyrrolidine acids (analogs 1a-6c) have been explored, resulting in
the discovery of analogs with a wide range of PPARγ and α
agonist activities, in particular the potent PPARα/γ dual agonists
3a-3k and 4a-4n. From these SAR studies, we have found that:
among the different permutations of spacing the oxyphenyl and
carboxylic acid pharmacophores on the pyrrolidine ring: 1) the
oxybenzyl pyrrolidine acid series overall provided the best
balance of PPARα/γ functional activities and 2) the cis-
substituted pyrrolidine acids are preferred versus the trans series,
and 3) N-carbamoyl and N-aryl-substituted oxybenzyl
pyrrolidine acid analogs provide potent, balanced PPARα/γ dual
agonists. The analogs in the 1,3-oxybenzylglycine pyrrolidine
acid series are relatively α-selective PPARα/γ agonists, whereas
analogs in the 1,4-oxybenzylglycine pyrrolidine acid series have
relatively more balanced PPARγ and PPARα agonist activities.
In the 1,4-oxybenzylglycine 3,4-disubstituted pyrrolidine acid
series, a set of N-pyrimidinyl & N-carbamoyl pyrrolidine analogs
was identified as particularly promising PPARα/γ dual agonists.
In both of these sub-series, the cis enantiomer (3R,4S absolute
stereochemistry) was found to be preferred for optimal PPARα/γ
functional activity and binding affinities. The cis enantiomer N-
pyrimidine analog 4i and the N-phenyl carbamate 3k both
display very potent, balanced PPARα/γ agonist activities in vitro
and were characterized in chronic efficacy studies. The N-
pyrimidinyl pyrrolidine acid analog 4i showed efficacious
glycemic & triglyceride lowering in a 14-day chronic study in
db/db mice. Additional SAR studies will be needed to further
optimize the in vitro liability/ADME profile of 4i. The SAR of
9. Detailed experimental procedures for the synthesis of the
compounds in this article can be found in WO 2004004665.
10. (a) Cheng, P. T.; Jeon, Y.; Wang, W. US Patent 7105556 (2006);
(b) Cheng, P. T.; Jeon, Y.; Wang, W. poster presented at the
National Medicinal Chemistry Symposium 2006; (c) Cheng, P. T.;
Chen, S.; Devasthale, P.; Ding, C. Z.; Herpin, T. F.; Wu, S.; Zhang,
H.; Wang, W.; Ye, X.-Y.; US Patent 7951793 (2011).
11. Still, W.C., Gennari, C. Tetrahedron Lett., 1985, 24, 4405.
12. Padwa, A.; Dent, W., Org. Synth. 1989, 67, 133.
13. “Preparation of Alkenes, A Practical Approach”, J.M.J. Williams,
Ed., Chapter 2, “The Wittig reaction and related methods”, N.J.
Lawrence, Oxford University Press, 1996.
14. Artis Klapars, Jon C. Antilla, Xiaohua Huang, and Stephen L.
Buchwald J. Am. Chem. Soc., 2001, 123, 7727.
15. (a) Karlsson, S. and Hogberg, H.-E., Tetrahedron: Asymmetry 2001,
12, 1975; (b) Carey, J. S., J. Org. Chem. 2001, 66, 2526.
16. Freedman, T. B.; Cao, X.; Phillips, L. M.; Cheng, P. T. W.;
Dalterio, R., Shu, Y.-Z.; Zhang, H.; Zhao, N.; Shukla, R. B.;
Tymiak, A.; Gozo, S. K.; Nafie, L. A.; Gougoutas, J. Z.; Chirality,
2006, 18, 746.
17. Mueller, R. H.; Wang, S.; Pansegrau,P. D.; Jannotti, J. Q.; Poss, M.
A.; Thottathil, J. K.; Singh, J.; Humora, M. J.; Kissick, T. P.;
Boyhan B.; Org. Process. Res. Dev. 1997, 1, 14.
18. [M + H]+ = 553.2; [α](0.96w/v%)MeOH589 = -70.8°; Analytical chiral
HPLC: Chiralpak AD chiral column 4.6 X 250 mm, 10 µm;
Isocratic solvent system: 3:7 A:B, where Solvent A = 100% IPA +
0.1% TFA; Solvent B = 100% heptane Detection = 254 nm; Flow
rate = 2 mL/min; Retention time = 6.7 min. 1H NMR (CD3OD): δ
PPARα-selective
agonists
derived
from
the
1,3-

8.41 (m, 1H), 7.85 (m, 2 H), 7.36 (m, 3 H), 7.00 (d, J = 9 Hz, 2 H),
6.76 (m, 3 H), 4.13 (m, 2 H), 3.81 (m, 1H), 3.62 (m, 1 H), 3.51 (m,
2 H), 3.18 (m, 1 H), 2.87 (m, 2H), 2.73 (m, 2 H), 2.41 (m, 1 H),
2.28 (s, 3H)
19. Seethala, R., Golla, R., Ma, Z., Cheng, L., Zhang, H., O’Malley,
K., Lippy, J., Zhang, L., Hariharan N., and Cheng, P. T. W., Anal.
Biochem., 2007, 363, 263.
20. In vitro PPAR agonist functional assays were performed by
transiently transfecting GAL4-hPPARα-LBD or GAL4-hPPARγ-
LBD constructs respectively into HEK293 cells stably expressing 5
copies of GAL4RE-Luciferase. Data were normalized for efficacy
at 1 µM to known agonists (rosiglitazone for hPPARγ and GW-
2331 for hPPARα). Agonist binding results in an increase in
luciferase enzyme activity which can be monitored by measuring
luminescence upon cell lysing and the addition of luciferin
substrate. EC50 values (µM) for PPARα or γ agonist activity were
calculated as the concentration of the test ligand (µM) required for
the half-maximal fold induction of HEK293 cells. The “intrinsic
activity” of a test ligand is defined as its activity at 1 µM (expressed
as a percentage) relative to the activity of the primary standards
(GW2331 for PPARα and rosiglitazone for PPARγ respectively,
both tested at 1 µM).
21. Zhang, H.; Ryono, D. E.; Devasthale, P.; Wang, W.; O'Malley, K.;
Farrelly, D.; Gu, L.; Harrity, T.; Cap, M.; Chu, C.; Kunselman, L.;
Morgan, N.; Flynn, N.; Moore, L.; Hosagrahara, V.; Zhang, L.;
Kadiyala, P.; Xu, C.; Doweyko, A. M.; Bell, A.; Chang, C.;
Muckelbauer, J.; Zahler, R.; Ryono, D. E.; Hariharan, N.; Cheng, P.
T. W.; Bioorg. Med. Chem. Lett. 2009, 19, 1451.
22. db/db mice, 8-10 weeks old, fed normal chow ad lib, were orally
dosed with compound and/or vehicle (5% N-methyl pyrrolidinone,
20% PEG-400, 20 mM aqueous sodium phosphate buffer, pH=8)
once daily for 14 days. Blood samples were drawn from the tail
vein on the 7th day after overnight fasting, and plasma analysis was
performed on a COBAS automated analyzer. After 14 days, fasted
animals were sacrificed. Blood and tissue samples were collected
for clinical biochemistry analyses.

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Synthesis and biological evaluation of novel
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Hao Zhang, Peter T. W. Cheng
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