Synthesis and evaluation of aminomethyl dihydrocinnamates as a new class of PPAR ligands

Article abstract of DOI:10.1016/j.bmcl.2006.09.011

PPAR ligands with varied subtype selectivity have been synthesized using an achiral aminomethyl dihydrocinnamate template. Several compounds in this series have demonstrated potent plasma glucose and triglyceride lowering capability in rodent models of type 2 diabetes.

Full text of DOI:10.1016/j.bmcl.2006.09.011

Bioorganic & Medicinal Chemistry Letters 16 (2006) 6328–6333
Synthesis and evaluation of aminomethyl dihydrocinnamates
as a new class of PPAR ligands
Alan M. Warshawsky,* Charles A. Alt, Joseph T. Brozinick, Allen R. Harkness,
Eric D. Hawkins, James R. Henry, Donald P. Matthews, Anne R. Miller,
Elizabeth A. Misener, Chahrzad Montrose-Rafizadeh, Gary A. Rhodes,
Quanrong Shen, Jennifer A. Vance, Uko E. Udodong, Minmin Wang,
Tony Y. Zhang and Richard W. Zink
Eli Lilly and Co, Indianapolis, IN 46285, USA
Received 16 June 2006; revised 1 September 2006; accepted 6 September 2006
Available online 26 September 2006
Abstract—PPAR ligands with varied subtype selectivity have been synthesized using an achiral aminomethyl dihydrocinnamate
template. Several compounds in this series have demonstrated potent plasma glucose and triglyceride lowering capability in rodent
models of type 2 diabetes.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Modulation of the peroxisome proliferator-activated
receptors (PPARs) has been advanced as a means to
treat a number of diseases, ranging from metabolic
and cardiovascular disorders to inflammation and can-
cer.1 The PPARs belong to the superfamily of nuclear
hormone receptors, and function as ligand-activated
transcription factors through heterodimers with the ret-
inoic acid X receptor (RXR), another nuclear hormone
receptor. There are 3 known PPAR subtypes: a, d, and
c. PPARa is expressed primarily in metabolic tissues.
PPARa agonists, such as fenofibrate (1) and clofibrate
(2), are used to lower serum triglycerides and raise
HDL cholesterol in humans (Fig. 1). PPARd is ex-
pressed broadly. While preclinical studies have deter-
long to the thiazolidinedione (TZD) class of compounds
and are currently used to treat type 2 diabetes (Fig. 1).
These chiral compounds have been developed as race-
mates because of the propensity of their chiral centers
to epimerize in vivo. As illustrated by rosiglitazone, this
property leads to reduced compound efficiency since
only the (S)-enantiomer was determined to be a high
affinity ligand for PPARc.2 The TZD functionality
participates in an extensive hydrogen bonding network
within the receptor to promote functional activity.3
Carboxylic acids can also participate in this hydrogen
bonding interaction.3a The in vivo racemization
potential of chiral carboxylic acid containing com-
pounds varies by their overall structure.4 A number of
carboxylic acid-based PPARc dominant agonists, GI
262570 (5),5 BMS 298585 (6),6 and LY519818 (7)7
among others, have been advanced to clinical trials
(Fig. 2). These PPAR agonists share common structural
features: a 1,4-disubstituted benzene core and heteroat-
om substituents nestled around a terminal carboxylic
acid group.
mined
a role for PPARd in the regulation of
cholesterol, PPARd agonists have yet to reach the
market.
PPARc has been the most thoroughly studied PPAR
subtype. It is found primarily in adipose tissue, and is
expressed substantially in the liver, kidney, heart, and
skeletal muscle as well as in the colon, intestines, pancre-
as, and spleen. The selective PPARc agonists on the
market today, rosiglitazone (3) and pioglitazone (4), be-
In this communication, we describe the synthesis and
biological evaluation of a new class of PPAR agonists,
the aminomethyl dihydrocinnamates (AMCs). As part
of our overall effort to identify compounds with varied
affinities for PPARs, we recognized certain favorable
structural features of the AMC template (8) as a starting
Keyword: PPAR.
*
Corresponding author. Tel.: +1 317 277 7226; e-mail: amw@lilly.com
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.09.011

A. M. Warshawsky et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6328–6333
6329
O
O
Cl
O
O
O
O
Cl
O
Fenofibrate (1)
Clofibrate (2)
O
O
NH
NH
S
S
N
N
O
N
O
O
O
Rosiglitazone (3)
Pioglitazone (4)
Figure 1. Marketed PPAR ligands.
CO2H
O
O
N
CO2H
HN
N
N
O
O
O
O
O
O
GI 262570
BMS 298585
Farglitazar (5)
Muraglitazar (6)
CO2H
CO2H
O
O
O
O
Tailpiece O
NRR'
LY 519818
Naveglitazar (7)
AMC Template (8)
Figure 2. Non-TZD PPAR agonists in the clinic and a new PPAR ligand template.
point to develop novel PPAR ligands. This achiral tem-
plate lacks the potential for racemization and maintains
functionality for the key hydrogen bonding interactions
with the receptors. The aminomethyl substitution on the
phenyl ring permits interactions with the PPARs that
are possible with the substituents alpha or beta to the
carboxylic acid group in the compounds presented in
Figure 2.
An alternate approach for the headpiece preparation is
described in Scheme 2. Phenol 20 was brominated and
subjected to Heck coupling conditions to provide cinna-
mate 21. The aldehyde group was converted to the cor-
responding oxime; hydrogenation yielded aminomethyl
hydroxy-dihydrocinnamate 22. The amine function
was modified selectively with isopropyl chloroformate
to give carbamate 23 or with 2-pyrazine carboxylic acid
and EDC to give amide 24. Synthetic routes toward tail-
piece variations are given in Scheme 3. Aryl bromide 259
was transformed to boronate 26, which underwent Su-
zuki coupling with 2-chloropyrimidine to give 27. Alter-
nately, the benzyl ether of 25 was reacted with phenol
via palladium catalysis to provide phenyl ether 29.
Hydrogenolysis gave the primary alcohol, which was
treated with tosyl chloride to give tosylate 30. Tosylate
31 was prepared from the corresponding alcohol ob-
tained via Suzuki coupling of 25 with phenyl boronic
acid.9 Tosylation of the known thiazole alcohol5b pro-
vided 32. Reacting headpiece phenols 23 and 24 with
tailpiece tosylates 28 and 30–32 using cesium carbonate
in DMF followed by treatment with trifluoroacetic acid
gave final compounds 33–38 in good overall yields
(Scheme 4).
Compounds 15–19 and 33–38 were synthesized by the
routes detailed in Schemes 1–4. The general synthetic
strategy involved the reaction of a phenolic headpiece
and a tailpiece tosylate. The specific method used to
elaborate the aminomethyl substitution is presented in
Scheme 1. Silyl ether protection of phenol 9 and subse-
quent radical bromination using NBS gave benzyl bro-
mide 10. Treatment with potassium phthalimide
introduced the latent amino group. Heck coupling and
fluoride ion mediated silyl ether cleavage provided
hydroxycinnamate 11. Hydrogenation yielded the dihy-
drocinnamate (12), which was coupled with tailpiece
tosylate 138 using cesium carbonate in DMF. The
phthalimide protecting group was removed under mild
reductive-hydrolysis conditions to give the amine salt
14. The free amine was released with saturated aqueous
bicarbonate and treated with the appropriate electro-
phile to afford final compounds 15–19 after ester cleav-
age using trifluoroacetic acid.
AMC derivatives 15–19 and 33–38 were evaluated in
binding and co-transfection (CTF) assays to determine
intrinsic receptor affinities (IC50) and functional activities

6330
A. M. Warshawsky et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6328–6333
Br
Br
CO2t-Bu
O
a, b
c, d
e
HO
HO
TBSO
HO
N
Br
O
11
9
10
O
13
CO2t-Bu
CO2t-Bu
O
N
OTs
N
O
f, g
NPhth
NH2HOAc
12
14
15, R1 = CO2CH2Ph (85%)
16, R1 = CONCH2Ph (64%)
17, R1 = COCH2CH2Ph (91%)
18, R1 = SO2CH2Ph (75%)
19, R1 = CO2i-Pr (93%)
CO2H
O
h, i
N
O
NHR1
Scheme 1. Aminomethyl dihydrocinnamate synthesis: amino substitution variation. Reagents: (a) TBSCl, TEA, CH2Cl2 (98%); (b) NBS, AIBN,
DCE (100%); (c) PhthNK, DMF (68%); (d) 1. t-Bu acrylate, Pd(OAc)2, (o-Tol)3P, i-Pr2NEt, EtCN; 2. TBAF, THF (87%); (e) 5% Pd–C, 60–75 psi
H2, EtOAc (87%); (f) 13, Cs2CO3, DMF (71%); (g) 1—NaBH4, IPA; 2—HOAc (79%); (h) 1—aqueous NaHCO3, CH2Cl2; 2—RNCO, RSO2Cl, or
RCOCl, TEA, CH2Cl2; (i) TFA, p-anisole, CH2Cl2.
Scheme 2. Alternate headpiece synthesis. Reagents: (a) Br2, CH2Cl2 (63%); (b) tert-butyl acrylate, Pd(OAc)2, (o-Tol)3P, i-Pr2NEt, EtCN (81%); (c)
H2NOH–HCl, pyr, EtOH (55%); (d) 10% Pd–C, 50 psi H2, EtOH (87%); (e) i-PrOCOCl, TEA, CH2Cl2; (f) pyrazine-2-carboxylic acid, EDC, HOBt,
CH2Cl2.
O
O
a
b
O
B
Br
N
N
OH
OH
O
25
26
O
O
c
N
N
N
N
OH
OTs
N
N
27
28 (79%)
O
O
d,e
f, c
O
O
25
N
N
OBn
OTs
29
30 (67% for step c)
O
N
S
N
N
O
OTs
OTs
31
32 (68% form alcohol)
Scheme 3. Synthesis of tailpieces. Reagents and conditions: (a) pinacol diborane, PdCl2(dppf), KOAc, DMSO (67%); (b) 2-chloroprimidine,
PdCl2(dppf), CsF, dioxane (41%); (c) TsCl, DMAP, TEA, CH2Cl2; (d) PhCH2Br, NaH, THF (89%); (e) PhOH, Pd(OAc)2, 2-BiPhP(t-Bu)2, K3PO4,
EtOH, Tol (72%); (f) H2, Pd(OH)–C, EtOH (70%).

A. M. Warshawsky et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6328–6333
6331
CO2t-Bu
CO2H
R2
X
R4
a, b
R3OTs
+
N
HO
O
28, 30-32
HN
R2
HN
O
O
23, 24
33, R4 = p-PhPh; X = O; R2 = Oi-Pr (100 x 94%)
34, R4 = p-PhOPh; X = O; R2 = Oi-Pr (90 x 76%)
35, R4 = 2-pyrimidine-p-Ph; X = O; R2 = Oi-Pr (92 x 100%)
36, R4 = p-Ph; X = O; R2 = 2-pyrazine (100% overall)
37, R4 = p-PhPh; X = O; R2 = 2-pyrazine (79% overall)
38, R4 = N-morpholine; X = S; R2 = Oi-Pr (98 x 60%)
Scheme 4. Coupling of headpieces and tailpieces. Reagents: (a) Cs2CO3, DMF; (b) TFA, p-anisole, CH2Cl2.
(% efficacy; EC50), respectively, for each PPAR subtype
(Table 1). Compounds with efficacies greater than 50%
were considered agonists. Most of the AMC compounds
with IC50 values less than 1 lM were found to be agonists.
Data for PPARd are not included in the table, since none
of the compounds demonstrated significant binding or
efficacy.
binding affinity and functional potency were equivalent
for carbamate 15, larger differences (4- to 7-fold) were
noted with 16–18. These data suggest the potential value
in using the AMC template in PPAR ligand design.
A dramatic improvement in both binding and CTF
activities was realized by going from the benzyl to the
isopropyl carbamate substituent (15 vs 19); the 5-fold
PPARc/a selectivity in binding was maintained. Chang-
ing the amide substituent also led to a compound (36)
with a greatly improved in vitro profile. For pyrazine
carboxamide 36, the PPARc/a binding selectivity was
reduced to 3-fold.
Variations at the aminomethyl substituent were evaluat-
ed. Within an initial series of related phenyl-substituted
carbamate, urea, amide, and sulfonamide compounds
15–18, SAR trends became evident. With respect to
binding, carbamate 15 was the most potent for both
PPARc and a. The corresponding amide (17) and relat-
ed sulfonamide (18) exhibited PPARc binding similar to
carbamate 15, but showed ꢀ10-fold weaker affinity for
PPARa. The urea analog (16) showed the weakest bind-
ing for PPARc but offered the greatest selectivity versus
PPARa. PPARa functional activities among these com-
pounds derived from the cell-based CTF assay roughly
followed the trends in binding results. While the PPARc
Tailpiece modifications were evaluated also. Phenyl,
phenoxy, and 2-pyrimidinyl substitution on the tailpiece
phenyl group provided compounds (33–35 and 37) with
PPARc binding similar to that of the parent compound
but with greater PPARc/a selectivity. The 2-pyrimidine-
containing AMC (35) was found to be a potent and
selective PPARc agonist with 200-fold binding and
a,c b,c
Table 1. Binding IC50, co-transfection efficacy and EC50, and in vivo data
Compound
hPPARc
hPPARa
In vivo
d
EC50 (nM)
IC50 (nM)
EC50 (nM)
CTF %Eff
IC50 (nM)
CTF %Eff
db/db mouse
versus (3)e
ZDF rat
ED50 mpkf
15
16
17
18
19
33
34
35
36
37
38
3
91 27
706 188
161 32
72 22
115 20
2684 34
1019 186
527 64
65
24
52
56
72
92
80
85
70
84
57
100
9
2
2
2
2
4
4
2
4
6
4
3
441 124
>10,000
515 86
NC
54
5
2
0.5
1
4962 842
3390 1064
146 33
2801 127
2940 46
70 13
32
33
51
52
46
37
60
68
37
9
3
26
6
8
19 10
2
2
58 (33)
56 (49)
Not tested
0.02 0.06
0.05 0.04
0.04 0.01
47 16
50 27
50 21
3489 917
516 161
9940 60
53 17
845 66
78 11
900 143
3
2
4
1
2
Not tested
52 (36)
1
1
17 5
26 15
7
1
35 4
198 53
4
2
28
0.3
6
449 195
1495 342
>10,000
2
87 (33)
58 (49)
0.03 0.01
0.12 0.03
0.41 0.12
22
67
1
8
986 118
NC
2646 78
1
308 21
NC
1bg
>10,000
4
>10,000
35
1
a Concentration of test compound to required to displace 50% of tritiated ligand: PPARa/PPARd agonist, 2-(4-{2-[3-(2,4-difluoro-phenyl)-1-heptyl-
ureido]-ethyl}-phenoxy)-2- methyl-butyric acid and PPARc agonist, 2-methyl-2-(4-{3-[propyl-(5-pyridin-2-yl-thiophene-2-sulfonyl)-amino]-pro-
pyl}-phenoxy)-propionic acid.
b Concentration of test compound to that produces 50% of the maximal reporter activity as determined in CV-1 cells; maximum efficacy as % of
maximum efficacy of a standard: unlabeled ligands in note a.
c Mean of at least 3 determinations standard error; NC, not calculated for efficacy <20% standard at 10lM.
d Gal4-hPPARa was used to eliminate interference by endogenous PPARc.
e db/db mice were dosed orally 30 mg/kg for 7 days using 30 mg/kg rosiglitazone (3) as a standard; % normalization = [(vehicle À compound)/
(vehicle À 200)] · 100.
f ED50 values in mg/kg were calculated from the day 7 change-from-baseline data versus dose by regression-based MED analysis.
g Fenofibric acid (1b) is the active metabolite of fenofibrate (1).

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A. M. Warshawsky et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6328–6333
CTF selectivities versus PPARa. The improved gamma
selectivity of these substituted tailpieces can result from
differences in the receptor binding as seen through
molecular docking studies with 15 and 35 into PPARc
(PDB: 1fm9) and PPARa (PDB: 1k7l).10 Substitution
on the terminal phenyl ring introduced unfavorable ste-
ric interactions with the PPARa LBD Tyr334, Cys275,
and Leu254 side chains; this steric effect is absent with
the corresponding Glu343, Gly 284, and Ile262 side
chains in PPARc. Finally, incorporation of the morpho-
linothiazole-based tailpiece led to a potent PPARc
agonist (38). Compared to the phenyloxazole-based
tailpiece analog (19), AMC 38 had equivalent PPARc
binding but 11-fold greater PPARc/a selectivity.
References and notes
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2005, 54, 2460; (b) Etgen, G. J.; Mantlo, N. Curr. Top.
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Expert Opin. Investig. Drugs 2003, 12, 1489; (d) Willson,
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Nolte, R. T.; Wisely, G. B.; Westin, S.; Cobb, J. T.;
Lambert, M. H.; Kurokawa, R.; Rosenfeld, M. G.;
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J.; Lenhard, J. M.; Orband-Miller, L. A.; Miller, J. F.;
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Boswell, G. E.; Charifson, P. S.; Cobb, J. E.; Henke, B. R.;
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Leesnitzer, L. M.; Lehmann, J.; Lenhard, J. M.; Orband-
Miller, L. A.; Gray-Nunez, Y.; Parks, D. J.; Plunket, K.
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Hull-Ryde, E. A.; Lake, D. H.; Leesnitzer, L. M.;
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The functional potencies (EC50s) of these more tightly
bound ligands were consistently less than or equal to
the IC50 values, and the ratios between these values var-
ied considerably. The many parameters involved in the
CTF assay; cell type, response element, co-activators,
nuclear and cell membrane penetrability, among others;
may account for these differences. While in vitro charac-
terization helped to prioritize compounds for in vivo
study, the in vitro data did not correlate rigorously with
in vivo responses (vide infra).
Two rodent models of type 2 diabetes responsive to
PPARc modulation, the db/db mouse11a and the male
Zucker diabetic fatty (ZDF) rat,11b were used to evalu-
ate several of these AMC compounds (Table 1). In
db/db mice, compounds were dosed orally at 30 mg/kg
for 7 days, and % glucose normalizations relative to
plasma glucose levels of lean animals were determined
using rosiglitazone (3) as an internal standard in each
study. Dose–response studies in ZDF rats (7 days at
0.3, 1, 3, and 10 mg/kg) generated ED50 values. The
AMC PPARc in vitro profiles translated nicely into in vi-
vo responses. The AMC compounds tested in db/db mice
produced plasma glucose reductions similar to or sub-
stantially better than rosiglitazone. All of the AMC
compounds tested in the ZDF rat normalized plasma
glucose and were significantly more potent than rosiglit-
azone with ED50 values ranging from 0.02 to 0.12 mg/kg
versus 0.41 mg/kg, respectively. A concomitant lowering
of plasma triglycerides with reductions in plasma glu-
cose was observed in both rodent models.
6. (a) 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, R.; Chen, S.;
Devasthale, P.; Jeon, Y.; Seethala, R.; Yang, W.-P.; Ren,
J.; Zhou, M.; Biller, S.; Mookhtiar, K. A.; Wetterau, J.;
Gregg, R.; Cheng, P. T.; Hariharan, N. Diabetes 2006, 55,
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29, 1084.
7. Martin, J. A.; Brooks, D. A.; Prieto, L.; Gonzalez,
R.; Torrado, A.; Rojo, I.; Lopez de Uralde, B.;
Lamas, C.; Ferritto, R.; Dolores Martin-Ortega, M.;
Agejas, J.; Parra, F.; Rizzo, J. R.; Rhodes, G. A.;
Robey, R. L.; Alt, C. A.; Wendel, S. R.; Zhang, T.
Y.; Reifel-Miller, A.; Montrose-Rafizadeh, C.; Brozi-
nick, J. T.; Hawkins, E.; Misener, E. A.; Briere, D.
A.; Ardecky, R.; Fraser, J. D.; Warshawsky, A. M.
Bioorg. Med. Chem. Lett. 2005, 15, 51.
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Wakitani, K.; Uchida, I. J. Med. Chem. 1998, 41, 1927,
preparation of mesylate.
9. Brooks, D. A.; Etgen, G. J.; Rito, C. J.; Shuker, A. J.;
Dominianni, S. J.; Warshawsky, A. M.; Ardecky, R.;
Paterniti, J. R.; Tyhonas, J.; Karanewsky, D. S.; Kauff-
man, R. F.; Broderick, C. L.; Oldham, B. A.; Montrose-
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Differences between human and mouse PPARa have been
documented in the literature.12 The AMC compounds in
Table 1 uniformly lacked functional mouse PPARa activ-
ity as determined through CTF assays, precluding their
evaluation in our PPARa responsive animal model.
In summary, the value of the AMC template in PPAR
ligand design has been demonstrated. By varying the
substitution on the amino group and the tailpiece, com-
pounds were identified having potent PPARc binding
affinity and functional activity with varying degrees of
PPARa activity. Reductions in plasma triglycerides
and glucose by these compounds in rodents models of
type 2 diabetes sensitive to PPARc were observed.
Further SAR studies with the AMC template will be
reported in due course.

A. M. Warshawsky et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6328–6333
6333
10. Compounds were docked into the PPAR LBDs using
CDocker: Vieth, M.; Hirst, J. D.; Kolinski, A.; Brooks, C.
L., III. J. Comput. Chem. 1998, 19, 1612; Vieth, M.; Hirst,
J. D.; Dominy, B. N.; Daigler, H.; Brooks, C. L., III.
J. Comput. Chem. 1998, 19, 1623.
daily by oral gavage between 8:30 and 9:30 AM for 7 days.
The dosing vehicle was 1% w/v CMC, 0.25% Tween 80.
Blood samples were obtained 1 hour post-dose on day 7;
following centrifugation of blood samples, plasma was
used for measurements of glucose and triglyceride levels.
Statistical significance was determined by one-way
ANOVA. When statistical significance was detected with
this method, group differences were determined by
Neuman–Keuls post hoc analyses.
11. (a) db/db mouse studies. Five-week-old db/db mice were
purchased from Jackson Laboratories (Bar Harbor, ME,
USA). After a 2-week acclimation period, the mice were
pre-bled and assigned to three groups (vehicle, rosiglitaz-
one, and test compound; 5 animals per group) based on
starting plasma glucose and body weight (day À1); (b)
Zucker diabetic fatty (ZDF) rat studies. Male ZDF rats
were obtained from Charles River (Genetic Models, Inc,
Indianapolis, IN, USA) at 6 weeks of age. After a 2-week
acclimation period, rats were pre-bled and assigned to four
groups (5 animals per group; vehicle, test compound at the
stated dose, or rosiglitazone at 10.0 mg/kg/day) based on
starting plasma glucose levels and body weight (day À1).
In both animal models, compounds were administered
12. (a) Wang, M.; Winneroski, L. L.; Ardecky, R. J.; Babine,
R. E.; Brooks, D. A.; Etgen, G. J.; Hutchison, D. R.;
Kauffman, R. F.; Kunkel, A.; Mais, D. E.; Montrose-
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