Synthesis and structure-activity relationships of thiadiazole-derivatives as potent and orally active peroxisome proliferator-activated receptors α/δ dual agonists

Article abstract of DOI:10.1016/j.bmc.2007.12.005

Replacement of the methyl-thiazole moiety of GW501516 (a PPARδ selective agonist) with [1,2,4]thiadiazole gave compound 21 which unexpectedly displayed submicromolar potency as a partial agonist at PPARα in addition to the high potency at PPARδ. A structu

Full text of DOI:10.1016/j.bmc.2007.12.005

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 3321–3341
Synthesis and structure–activity relationships of
thiadiazole-derivatives as potent and orally active peroxisome
proliferator-activated receptors a/d dual agonists
Lan Shen, Yan Zhang, Aihua Wang, Ellen Sieber-McMaster, Xiaoli Chen,
Patricia Pelton, June Z. Xu, Maria Yang, Peifang Zhu, Lubing Zhou,
Michael Reuman, Zhiyong Hu, Ronald Russell, Alan C. Gibbs, Hamish Ross,
Keith Demarest, William V. Murray and Gee-Hong Kuo*
Drug Discovery Division, Johnson & Johnson Pharmaceutical Research and Development, L.L.C. Cedarbrook Corporate Center,
8 Clarke Dr. Cranbury, NJ 08512, USA
Received 29 October 2007; revised 30 November 2007; accepted 4 December 2007
Available online 8 December 2007
Abstract—Replacement of the methyl-thiazole moiety of GW501516 (a PPARd selective agonist) with [1,2,4]thiadiazole gave com-
pound 21 which unexpectedly displayed submicromolar potency as a partial agonist at PPARa in addition to the high potency at
PPARd. A structure–activity relationships study of 21 resulted in the identification of 40 as a potent and selective PPARa/d dual
agonist. Compound 40 and its close analogs represent a new series of PPARa/d dual agonists. The high potency, high selectivity,
significant gene induction, excellent PK profiles, low P450 inhibition or induction, and good in vivo efficacy in four animal models
support 40 being selected as a pre-clinical study candidate, and may render 40 as a valuable pharmacological tool in elucidating the
complex roles of PPARa/d dual agonists, and the potential usage for the treatment of metabolic syndrome.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
The peroxisome proliferator-activated receptors
(PPARs) belong to the nuclear hormone receptor super-
family and consist of three members, PPARa, PPARc,
and PPARd. PPARa is expressed mainly in tissues in-
volved in lipid oxidation such as liver, kidney, adrenal
glands, cardiac muscle, and skeletal muscles. PPARa
regulates the expression of genes involved in lipid
metabolism.7 The fibrate drugs, such as fenofibrate 90
and ciprofibrate 91 (Fig. 1), have been used for the clin-
ical treatment of dyslipidemia by lowering serum triglyc-
erides, free fatty acid (FFA) levels and raising HDL-C
since the 1970s.8 Several studies have provided evidence
that the hypolipidemic effect of the fibrate drugs is
attributed to the activation of PPARa.9 PPARc is ex-
pressed in adipose tissue, macrophages, and vascular
smooth muscles.7 PPARc was identified as a key regula-
tor for insulin sensitivity, and two PPARc agonists, ros-
iglitazone 92 and pioglitazone 93, have been used for the
clinical treatment of Type 2 diabetes since 1999.10 In
contrast, the biological role of PPARd and the potential
clinical utility of its ligand was unclear, in part, due to its
broad tissue expression11 and lack of good chemical
tools to study its pharmacology. Recently, a potent
Cardiovascular disease (CVD) is the most common
cause of morbidity and mortality in developed nations.1
Atherogenic dyslipidemia, characterized by an abnor-
mal circulating lipid profile including low levels of high
density lipoprotein cholesterol (HDL-C), elevated levels
of small-dense low density lipoprotein cholesterol
(LDL-C) or elevated triglycerides (TG), is often found
in patients who are obese, have type 2 diabetes or the
metabolic syndrome.2–4 These individuals are at high
risk for premature CVD. While LDL-C is prone to accu-
mulate in the arterial wall leading to the formation of
atherosclerotic cholesterol-laden foam cells,5 HDL-C
may play a protective role in removing excess cholesterol
from peripheral cells and return it to the liver.6
Keywords: Thiadiazole; Nuclear receptor; Gene induction; Metabolic
syndrome.
*
Corresponding author. Tel.: +1 609 655 6967; e-mail:
gkuo@prdus.jnj.com
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.12.005

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L. Shen et al. / Bioorg. Med. Chem. 16 (2008) 3321–3341
O
O
O
O
O
O
Cl
HO
O
HN
O
HN
O
Cl
Cl
S
N
S
O
O
N
N
O
90 (Fenofibrate)
91 (Ciprofibrate)
92 (Rosiglitazone)
93 (Pioglitazone)
O
O
O
F
O
O
F
Cl
HO
CF3
O
S
HO
N
H
HO
Cl
H
S
N
N
Cl
N
OCH3
CF3
O
96
94 (GW501516)
95 (GW2433)
Figure 1. Structures of representative PPAR agonists: PPARa (90, 91), PPARc (92, 93), PPARd (94), and PPARa/d (95, 96).
and selective PPARd agonist 94 (GW501516, phase-II
clinical trial) was developed and shown to increase plas-
ma HDL-C levels together with decreasing LDL-C and
triglycerides in obese and dyslipidemic rhesus mon-
keys.12 In addition, while one study supported an ath-
eroprotective effect of PPARd agonists in LDLRꢀ/ꢀ
mice,13 other data suggested that PPARd activation
may attenuate the metabolic syndrome including obes-
ity.14 To effectively target dyslipidemia to reduce the risk
of cardiovascular diseases, it may be beneficial to acti-
vate PPARa and PPARd simultaneously through a sin-
gle molecule. To date, there are only two series of
PPARa/d dual agonists (95 (GW2433)15 and 9616) with
in vitro data reported. A preliminary report17 from
our laboratory described the finding of [1,2,4]thiadia-
zole-derivatives as a new series of potent and orally ac-
tive PPARa/d dual agonists, however, the best
compound was found to have P450 induction issue later.
This article describes a full report of the SAR study and
identification of a potent and orally active PPARa/d
dual agonist that lacks P450 induction, therefore, being
selected as a pre-clinical study candidate.
CF3
CF3
i
ii
O
1
2
CF3
CF3
iii
Br
N
S
N
S
S
3
4
EtO2C
O
CF3
HO2C
O
SH
5
iv, v
N
S
6
Scheme 1. Reagents and conditions: (i) EtMgBr, Ac2O, THF, 20–
35 ꢁC, 94%; (ii) H2NOSO3H, NaSH, H2O, 70%; (iii) (PhCO)2O2, NBS,
CCl4, reflux, 42%; (iv) 5, CH3CN, Cs2CO3; (v) NaOH, MeOHꢀH2O,
75% (two steps).
CF3
CF3
2. Chemistry
O
i
ii
EtO2C
O
N
S
S N
The synthesis of the first target molecule 6 is shown in
Scheme 1. Trapping the anion of alkyne 1 with acetic
anhydride at 20–35 ꢁC gave 2. Cyclocondensation of 2
with hydroxylamine-O-sulfonic acid at 20 ꢁC provided
the isothiazole 3. Bromination of 3 with NBS and ben-
zoyl peroxide in refluxing CCl4 gave the bromide 4. S-
alkylation of 4 with thiophenoxide 5, followed by base
hydrolysis of the ethyl ester, provided the desired target
molecule 6. The synthesis of the isothiazole 10 is shown
in Scheme 2. Treatment of [1,3,4]-oxathiazol-2-one 717
with ethyl propiolate at 160 ꢁC with the extrusion of
CO2 gave isothiazole-5-carboxylic acid ethyl ester 8
and isothiazole-4-carboxylic acid ethyl ester 11 in almost
1:1 ratio. Reduction of the ethyl ester 8 with LAH re-
sulted in the formation of primary alcohol 9. Conver-
sion of 9 to its mesylate, followed by S-alkylation with
5 and base hydrolysis of the ethyl ester, gave the target
10. In the meanwhile, reduction of the ethyl ester 11
(Scheme 3) with NaBH4 at 20 ꢁC yielded the primary
alcohol 12. Conversion of 12 to its mesylate, followed
by S-alkylation and base hydrolysis, provided the target
7
8
CF3
iii
iv, v
CF3
HO2C
O
S
HO
S
N
S N
9
10
Scheme 2. Reagents and conditions: (i) ethyl propiolate, 1,2-dichloro-
benzene, 160 ꢁC, 31%; (ii) LAH, THF; (iii) MsCl, Et3N, CH2Cl2; (iv) 5,
CH3CN, Cs2CO3; (v) NaOH, MeOHꢀH2O, 39% (four steps).
13. The synthesis of the isoxazole 16 is shown in Scheme
4. Cyclocondensation of chloro-oxime 1418 with propar-
gyl chloride at 20 ꢁC gave isoxazole 15. S-alkylation of
15 with 5, followed by base hydrolysis, provided the tar-
get 16. The synthesis of reverse-isoxazole 20 is shown in
Scheme 5. Cyclocondensation of 2,4-dioxo-ester 1719
with hydroxylamine at 78 ꢁC yielded isoxazole-3-car-
boxylic acid ethyl ester 18. Reduction of 18 with LAH
at ꢀ78 ꢁC gave the primary alcohol 19. Conversion of

L. Shen et al. / Bioorg. Med. Chem. 16 (2008) 3321–3341
3323
R2
CF3
CF3
EtO2C
S
R3
R1
R2
O
i
ii
O
R3
R1
O
N
S
N
i
O
ii
N
S
7
1
1
H2NOC
25, R1 = CF3, R2 = F, R3 = H
31, R1 = OCF3, R2 = Cl, R3 = H
36, R1 = Cl, R2 = Cl, R3 = H
41, R1 = Cl, R2 = H, R3 = Cl
46, R1 = Me, R2 = Me, R3 = H
51, R1 = Me, R2 = Cl, R3 = H
26, R1 = CF3, R2 = F, R3 = H
32, R1 = OCF3, R2 = Cl, R3 = H
37, R1 = Cl, R2 = Cl, R3 = H
42, R1 = Cl, R2 = H, R3 = Cl
47, R1 = Me, R2 = Me, R3 = H
52, R1 = Me, R2 = Cl, R3 = H
CF3
HO
CF3
iii
iv, v
HO2C
O
S
N
N
S
S
12
13
R2
R2
Scheme 3. Reagents and conditions: (i) ethyl propiolate, 1,2-dichloro-
benzene, 160 ꢁC, 36%; (ii) NaBH4, EtOH, 93%; (iii) MsCl, Et3N,
CH2Cl2; (iv) 5, CH3CN, Cs2CO3; (v) NaOH, MeOHꢀH2O, 72% (three
steps).
R3
R1
R3
R1
N
N
EtO2C
iii
iv
HO
N
N
S
S
27, R1 = CF3, R2 = F, R3 = H
33, R1 = OCF3, R2 = Cl, R3 = H
38, R1 = Cl, R2 = Cl, R3 = H
43, R1 = Cl, R2 = H, R3 = Cl
48, R1 = Me, R2 = Me, R3 = H
53, R1 = Me, R2 = Cl, R3 = H
28, R1 = CF3, R2 = F, R3 = H
CF3
CF3
34, R1 = OCF3, R2 = Cl, R3 = H
39, R1 = Cl, R2 = Cl, R3 = H
44, R1 = Cl, R2 = H, R3 = Cl
49, R1 = Me, R2 = Me, R3 = H
54, R1 = Me, R2 = Cl, R3 = H
Cl
i
ii
Cl
N
iii
O
N
HO
14
15
CF3
HO2C
O
R2
EtO2C
29
v, vi
O
R3
N
R1
HO2C
O
S
SH
O
N
S
16
S
N
30, R1 = CF3, R2 = F, R3 = H
35, R1 = OCF3, R2 = Cl, R3 = H
40, R1 = Cl, R2 = Cl, R3 = H
45, R1 = Cl, R2 = H, R3 = Cl
50, R1 = Me, R2 = Me, R3 = H
55, R1 = Me, R2 = Cl, R3 = H
Scheme 4. Reagents and conditions: (i) propargyl chloride, CH2Cl2,
Et3N, 64%; (ii) 5, CH3CN, Cs2CO3; (iii) NaOH, MeOHꢀH2O, 91%
(two steps).
CF3
i
CF3
EtO2C
ii
EtO2C
Scheme 6. Reagents and conditions: (i) chlorocarbonylsulfenyl chlo-
ride, toluene, 60 ꢁC, 47–96%; (ii) ethyl cyanoformate, 1,2-dichloroben-
zene, 160 ꢁC, 36–91%; (iii) NaBH4, EtOH, 56–93%; (iv) PPh3, CBr4,
CH2Cl2; (v) 29, CH3CN, Cs2CO3; (vi) NaOH, MeOHꢀH2O, 20–67%
(three steps).
O
O
N
O
17
18
CF3
CF3
HO2C
O
iii
S
sation of 58 with 4-chloro-benzonitrile at 160 ꢁC with
the extrusion of CO2 gave [1,2,4]thiadiazole-3-carbox-
ylic acid ethyl ester 59. Reduction of 59 to its primary
alcohol 60, followed by the conversion of 60 to the bro-
mide 61, S-alkylation with 29, and base hydrolysis,
yielded the desired target 62. Analog 66 was prepared
in the same route. The synthesis of two-methylene-link
thiadiazole analog 70 is shown in Scheme 8. Cyclocon-
densation of 7 with ethyl cyanoacetate at 160 ꢁC pro-
vided thiadiazole 67. Reduction of 67 with NaBH4
gave the alcohol 68, bromination of 68 yielded the bro-
mide 69, S-alkylation of 69, followed by base hydrolysis,
gave target 70. Analog 75 was prepared in the same
route. The synthesis of oxygen-link thiadiazole 79 is
shown in Scheme 9. Conversion of the primary alcohol
28 to its bromide, followed by O-alkylation with 78,
and base hydrolysis, provided the target 79. The analogs
80–83 were all prepared in the same route. The synthesis
of des-methyl thiadiazole 86 is shown in Scheme 10.
Conversion of the alcohol 8417 to its mesylate, followed
by S-alkylation with 4-mercaptophenol, gave phenol 85.
O-alkylation of 85 with t-BuO2C(CH3)2CBr at 70 ꢁC
HO
iv, v
N
O
N O
19
20
Scheme 5. Reagents and conditions: (i) NH2OH, HCl, EtOH, 78 ꢁC,
91%; (ii) LAH, THF, ꢀ78 ꢁC, 45%; (iii) MsCl, Et3N, CH2Cl2; (iv) 5,
CH3CN, Cs2CO3; (v) NaOH, MeOHꢀH2O, 78% (three steps).
19 to its mesylate, followed by S-alkylation with 5, and
base hydrolysis of the ester, provided isoxazole target
20. The synthesis of [1,2,4]thiadiazole 30 is shown in
Scheme 6. Reaction of substituted benzamide 25 with
chlorocarbonylsulfenyl chloride at 60 ꢁC yielded
[1,3,4]oxathiazol-2-one 26. Cyclocondensation of 26
with ethyl cyanoformate at 160 ꢁC gave [1,2,4]thiadia-
zole-5-carboxylic acid ethyl ester 27. Reduction of 27
with NaBH4 provided primary alcohol 28. Conversion
of 28 to the bromide, S-alkylation of the bromide with
29,17 followed by base hydrolysis, gave the desired target
30. The analogs 35, 40, 45, 50, and 55 were all prepared
by the same method as that of 30. The synthesis of re-
verse-thiadiazole 62 is shown in Scheme 7. Cycloconden-

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L. Shen et al. / Bioorg. Med. Chem. 16 (2008) 3321–3341
R2
R2
R1
R1
EtO2C
78
ii, iii
O
O
EtO2C
i
ii
N
O
EtO2C
N
OH
i
HO
N
S
N
S
N
S
59, R1 = Cl, R2 = H
28, R1 = CF3, R2 = F
58
63, R1 = Cl, R2 = Cl
34, R1 = OCF3, R2 = Cl
39, R1 = Cl, R2 = Cl
49, R1 = Me, R2 = Me
54, R1 = Me, R2 = Cl
R2
R2
R1
R1
iv, v
N
N
iii
HO
Br
R2
N
60, R1 = Cl, R2 = H
64, R1 = Cl, R2 = Cl
S
N
S
R1
HO2C
O
61, R1 = Cl, R2 = H
N
65, R1 = Cl, R2 = Cl
O
S
N
R2
79, R1 = CF3, R2 = F
80, R1 = OCF3, R2 = Cl
81, R1 = Cl, R2 = Cl
82, R1 = Me, R2 = Me
83, R1 = Me, R2 = Cl
R1
HO2C
O
N
S
N
S
62, R1 = Cl, R2 = H
66, R1 = Cl, R2 = Cl
Scheme 9. Reagents: (i) PPh3, CBr4, CH2Cl2; (ii) 78, CH3CN, Cs2CO3;
(iii) NaOH, MeOHꢀH2O, 15–51% (three steps).
Scheme 7. Reagents and conditions: (i) R1R2-benzonitrile, 1,2-dichlo-
robenzene, 160 ꢁC, 13–31%; (ii) NaBH4, EtOH, 84–97%; (iii) PPh3,
CBr4, CH2Cl2, 0–20 ꢁC, 83–84%; (iv) 29, CH3CN, Cs2CO3; (v) NaOH,
MeOHꢀH2O, 46–68% (two steps).
CF3
HO
CF3
i, ii
N
N
HO
S
R2
S
N
R2
S
N
R1
R1
84
85
i
ii
O
N
O
EtO2C
HO2C
O
CF3
N
S
S
N
iii, iv
N
67, R1 = CF3, R2 = H
7, R1 = CF3, R2 = H
71, R1 = Cl, R2 = Cl
S
72, R1 = Cl, R2 = Cl
S
N
86
R2
R2
Scheme 10. Reagents and conditions: (i) MsCl, Et3N, CH2Cl2; (ii) 4-
mercapto-phenol, CH3CN, Cs2CO3, 83% (two steps); (iii) NaH,
t-BuO2C(CH3)2CBr, THF, 70 ꢁC; (iv) TFA, CH2Cl2, 15% (two steps).
R1
iii
R1
iv, v
N
N
HO
Br
S
N
S N
68, R1 = CF3, R2 = H
69, R1 = CF3, R2 = H
MeO2C
HO2C
O
73, R1 = Cl, R2 = Cl
74, R1 = Cl, R2 = Cl
i, ii
STips
R2
R1
88
HO2C
O
Cl
N
O
Cl
N
S
S
N
N
S
70, R1 = CF3, R2 = H
75, R1 = Cl, R2 = Cl
S
89
Scheme 11. Reagents and conditions: (i) 39A, TBAF, THF, 0 ꢁC, 93%;
(ii) NaOH, MeOHꢀH2O, 35%.
Scheme 8. Reagents and conditions: (i) ethyl cyanoacetate, 1,2-
dichlorobenzene, 160 ꢁC, 12–14%; (ii) NaBH4, EtOH, 64%; (iii)
PPh3, CBr4, CH2Cl2, 0–20 ꢁC, 44–86%; (iv) 29, CH3CN, Cs2CO3; (v)
NaOH, MeOHꢀH2O, 35–40% (two steps).
3. Results and discussion
3.1. Lead generation
provided 86-tert-butyl ester, that was deprotected under
acidic condition to give the target 86. The synthesis of
cyclopentyl-analog 89 is shown in Scheme 11. Treatment
of silyl-protected thiophenol 8817 with the bromide of 39
at 0 ꢁC in the presence of TBAF gave 89-methyl ester.
Base hydrolysis of the ester provided the target 89.
During this study, HEK293 cells were grown in
DMEM/F-12 medium supplemented with 10% FBS
and glutamine. The cells were co-transfected with
DNA constructs containing the ligand-binding domains

L. Shen et al. / Bioorg. Med. Chem. 16 (2008) 3321–3341
3325
Table 1. Activities in human PPAR co-transfection assaysa
of either PPARa, c or d-Gal4 chimeric receptors. The
steady-Glo luciferase assay kit was used for measuring
luciferase reporter activity. Percent efficacies for
PPARa, PPARd, and PPARc activation were reported
relative to PPARa agonist ((S)-2-{2-[1-ethyl-3-(4-tri-
fluoro-methoxyphenyl)ureido]indan-5-ylsulfanyl}-2-meth-
ylpropionic acid, compound 2.1),20 PPARd agonist
({4-[2-methoxy-3-(4-trifluoromethyl-phenoxy)-propylsul-
fanyl]-2-methyl-phenoxy}-acetic acid, compound 2),21
and PPARc agonist (rosiglitazone), respectively.
O
O
HO
CF3
S
b
Heter
a
b
b
EC50 (nM)
Compound
Heter
PPARac
(% resp.)
PPARdd
(% resp.)
a
a
b
A primary goal of our PPAR research programs has
been the identification of a functionally potent and
selective PPARd agonist with optimal in vivo efficacy,
and maximum safety margin. Since 5 is the first reported
potent and selective PPARd agonist, we were interested
in conducting structure–activity relationship (SAR)
studies with respect to 5 as the lead molecule. It seems
the methyl-substituted thiazole of 5 may contribute un-
iquely to its high functional potency and selectivity at
PPARd, therefore, we first screened various heteroaryls
or heterocycles in human PPAR co-transfection assays
to see if we could uncover any new series that may meet
our program goal. Replacement of the methylthiazole of
5 with isothiazole gave compound 6. As compared to 5
(EC50 = 1 nM at PPARd and EC50 = 1100 nM at
PPARa),22 6 displayed very similar potency at PPARd
(EC50 = 2 nM, 96%, Table 1) but much weaker potency
at PPARa (EC50 > 3000 nM). It seems 6 represents an-
other potent and selective PPARd agonist series. Mean-
while, we also synthesized the reverse-isothiazole 10.
With the interchanged positions of nitrogen and sulfur
in the ring, compound 10 exhibited slightly lower po-
tency at PPARd (EC50 = 5 nM, 94%) and lower selectiv-
ity against PPARa (EC50 = 1290 nM, 43%) compared to
6. Changing the substitution pattern on the isothiazole
ring from [3,5]- (as in 10) to [3,4]-positions gave 13.
The potency of 13 displayed at PPARd was decreased
significantly (EC50 = 150 nM). Therefore, to enhance
PPARd potency, the polar carboxyl group may prefer
to orient away from the hydrophobic 4-CF3-phenyl
group as much as possible. Replacement of the methyl-
thiazole of 5 with isoxazole gave 16. Compound 16 dis-
played ꢁ4-fold weaker potency at PPARd
(EC50 = 20 nM) and even lower activity at PPARa
(EC50 > 3000 nM) compared to isothiazole 10
(EC50 = 5 nM at PPARd, EC50 = 1290 nM at PPARa).
The reverse-isoxazole 20 exhibited high potency at
PPARd (EC50 = 2 nM, 80%) and modest potency at
PPARa (EC50 = 2208 nM, 41%). Overall, we demon-
strated that both isothiazole 6 and isoxazole 20 dis-
played comparable potency and selectivity at PPARd
compared to methylthiazole 5. Continuing replacement
of the methylthiazole of 5 with [1,2,4]thiadiazole gave
21.17 Unexpectedly, thiadiazole 21 exhibited submi-
cromolar potency at PPARa as a partial agonist
(EC50 = 468 nM, 42%) in addition to the good potency
observed at PPARd (EC50 = 10 nM, 79%).
6
>3000
2 (96)
N S
a
b
10
13
16
20
21
1290 (43)
>3000
5 (94)
S
N
a
150 (86)
20 (100)
2 (80)
b
S N
a
b
b
b
>3000
O N
a
2208 (41)
468 (42)
N O
N
a
10 (79)
S
N
a All compounds displayed EC50 > 3000 nM at PPARc.
b EC50 is the concentration of test compounds needed to induce 50% of
the maximum luciferase activity. The EC50 value is the average of
more than two separate tests.
c The internal PPARa agonist (compound 2.1)20 was used as the
standard.
d The internal PPARd agonist (compound 2)21 was used as the
standard.
pears to be beneficial for many PPARa agonists,
including 1, 2, and 6, we prepared the geminal dimethyl
analog of 21 first. Indeed, 22 displayed ꢁ6-fold higher
potency at PPARa (EC50 = 79 nM, 51%, Table 2),
slightly better potency at PPARd (EC50 = 6 nM, 76%),
and moderate potency with low activity at PPARc
(EC50 = 407 nM, 25%) compared to 21. Replacing 4-
CF3 of 22 with 4-OCF3 gave 23 with another 2-fold
higher potency observed at PPARa (EC50 = 33 nM,
44%), 2-fold higher potency at PPARd (EC50 = 3 nM,
73%)
but
abolished
activity
at
PPARc
(EC50 > 3000 nM) compared to 22. Replacing 4-CF3 of
22 with 4-Cl gave 24 with 2-fold reduced potency ob-
served at PPARa (EC50 = 186 nM, 38%), 3-fold reduced
potency at PPARd (EC50 = 18 nM, 72%), and abolished
activity at PPARc (EC50 > 3000 nM) compared to 22.
Interestingly, installation of a substituent at the 3-posi-
tion of the phenyl ring in addition to the original 4-sub-
stituent seems to further enhance the potency at PPARa
while maintaining the high potency at PPARd. For
example, adding a 3-F substituent to 22 gave 3-F,4-
CF3 analog 30. Analog 30 displayed 2-fold higher po-
tency at PPARa (EC50 = 34 nM, 53%) compared to 22
(EC50 = 79 nM, 51%) and maintained the equally high
potency at PPARd (EC50 = 5 nM, 68% for 30 versus
EC50 = 6 nM, 76% for 22), and similar low activity at
In order to identify a dual PPARa/d agonist with desired
efficacy and safety profiles, we decided to focus the SAR
study on the new [1,2,4]thiadiazole 21 series. Since gem-
inal dimethyl substitution of the acidic head moiety ap-

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L. Shen et al. / Bioorg. Med. Chem. 16 (2008) 3321–3341
Table 2. Activities in human PPAR co-transfection assays
O
R2
R3
O
HO
R1
N
S
S
N
R1
R2
R3
a
EC50 (nM)
Compound
PPARa (% resp.)
PPARd (% resp.)
PPARcb (% resp.)
22
23
24
30
35
40
45
50
55
CF3
OCF3
Cl
H
H
H
H
H
H
H
Cl
H
H
79 (51)
33 (44)
186 (38)
34 (53)
28 (73)
100 (56)
476 (33)
115 (45)
70 (74)
6 (76)
3 (73)
407 (25)
>3000
>3000
H
H
18 (72)
5 (68)
3 (68)
CF3
OCF3
Cl
F
640 (22)
>3000
>3000
Cl
Cl
H
8 (65)
Cl
102 (63)
93 (66)
25 (65)
>3000
>3000
669 (23)
CH3
CH3
CH3
Cl
a EC50 is the concentration of test compounds needed to induce 50% of the maximum luciferase activity. The EC50 value is the average of more than
two separate tests.
b Rosiglitazone was used as the standard.
Table 3. Activities in human PPAR co-transfection assays
PPARc (EC50 = 640 nM, 22%). Installation of a 3-Cl
substituent to 23 gave 3-Cl,4-OCF3 analog 35. Com-
pound 35 also exhibited higher potency and even higher
O
R2
O
HO
activity at PPARa (EC50 = 28 nM, 73%) compared to 23
(EC50 = 33 nM, 44%), and the same potency at PPARd
(EC50 = 3 nM, 68% for 35 versus EC50 = 3 nM, 73% for
23). Consistently, addition of the 3-Cl substituent to 24
gave 3-Cl,4-Cl analog 40 that also displayed higher po-
tency at PPARa (EC50 = 100 nM, 56%) compared to 24
(EC50 = 186 nM, 38%), and even higher potency at
PPARd (EC50 = 8 nM, 65% for 40 versus EC50 = 18 nM,
72% for 24). On the other hand, installation of a 2-sub-
stituent in addition to the 4-substituent of the phenyl
ring reduced the potency at both PPARa and PPARd.
For example, compound 45 with 2-Cl,4-Cl substituents
displayed lower potency at PPARa (EC50 = 476 nM,
33%) and PPARd (EC50 = 102 nM, 63%) compared to
24 (EC50 = 186 nM, 38% at PPARa and EC50 = 18 nM,
72% at PPARd). In order to expand SAR around these
PPARa/d dual agonists, we also prepared 3-CH3,4-CH3
analog 50 and 3-Cl,4-CH3 analog 55. Compound 50 dis-
played significant potency at PPARa (EC50 = 115 nM,
45%), but reduced potency at PPARd (EC50 = 93 nM,
66%) compared to 30, 35 or 40. Compound 55 showed
better potency at both PPARa (EC50 = 70 nM, 74%)
and PPARd (EC50 = 25 nM, 65%) compared to 50, but
not as selective against PPARc (EC50 = 669 nM, 23%).
Overall, 23, 24, 35, 40, and 50 represented interesting
potent PPARa/d dual agonists.
R1
N
S
N S
a
EC50 (nM)
Compound
R1
R2
PPARa
(% resp.)
PPARd
(% resp.)
PPARc
(% resp.)
56
57
62
66
CF3
OCF3
Cl
H
H
H
Cl
425 (38)
656 (47)
>3000
26 (67)
58 (55)
237 (62)
229 (58)
>3000
>3000
>3000
>3000
Cl
>3000
a EC50 is the concentration of test compounds needed to induce 50% of
the maximum luciferase activity. The EC50 value is the average of
more than two separate tests.
pared to that of 22 and 23 in the thiadiazole series,
respectively. In addition, both 4-Cl analog 62 and 3-
Cl,4-Cl analog 66 in the reverse-thiadiazole series be-
haved as PPARd selective agonists only (EC50 = 237 nM
for 62 and EC50 = 229 nM for 66), with no activity ob-
served at PPARa or PPARc. Based on these four exam-
ples, it is suggested that the orientation of
[1,2,4]thiadiazole analogs may have more favorable
interactions with the ligand-binding pockets of PPARa
and PPARd than that of the reverse-[1,2,4]thiadiazole
analogs shown in Table 3. We also briefly examined
the impact of chain length of the molecules on PPAR
activities. Insertion of a additional methylene unit into
22 gave 70. Compound 70 showed higher potency at
PPARa (EC50 = 37 nM, 65%, Table 4), higher potency
at PPARd (EC50 = 2 nM, 66%), and slightly higher
activity at PPARc (EC50 = 465 nM, 35%) compared to
22 (Table 2). The two-carbon analog of 40 (75) was also
prepared. Interestingly, 75 displayed similar potency at
PPARa (EC50 = 123 nM, 71%), similar potency at
PPARd (EC50 = 9 nM, 71%), and higher potency at
We next examined the reverse-[1,2,4]thiadiazole series to
see if the specific orientation of the five-membered thia-
diazole ring is critical to the potency or selectivity ob-
served at PPARs. Consistently, the CF3 analog 56 and
OCF3 analog 57 in the reverse-thiadiazole series dis-
played 5- to 20-fold lower potency at PPARa
(EC50 = 425 nM for 56 and EC50 = 656 nM for 57, Ta-
ble 3), and 4- to 19-fold lower potency at PPARd
(EC50 = 26 nM for 56 and EC50 = 58 nM for 57) com-

L. Shen et al. / Bioorg. Med. Chem. 16 (2008) 3321–3341
3327
Table 4. Activities in human PPAR co-transfection assays
R2
displayed modest improvement of potency at PPARa
(EC50 = 86 nM, 65%) over 4-CF3 analog 76
(EC50 = 94 nM, 71%), 3-Cl,4-OCF3 analog 80 only im-
proved the activity at PPARa (EC50 = 97 nM, 101%)
compared to the corresponding 4-OCF3 analog 77
(EC50 = 94 nM, 80%). On the other hand, the oxygen-
link 3,4-Cl2 analog 81, 3,4-(CH3)2 analog 82, and 3-
Cl,4-CH3 analog 83 all displayed 4- to 6-fold lower po-
tency but higher activity at PPARa (EC50 = 504 nM,
83% for 81, 643 nM, 58% for 82, and 297 nM, 75% for
83) and 6- to 14-fold lower potency at PPARd
(EC50 = 114 nM, 75% for 81, 534 nM, 60% for 82, and
148 nM, 85% for 83) than the corresponding sulfur-link
analogs. Overall, replacement of the sulfur atom with
the oxygen atom in the linker, in general, resulted in
the reduced potency at both PPARa and PPARd, but
increased activity at PPARa. This replacement also dis-
played either decreased potency or no impact at PPARc.
In order to confirm the critical role for potency of the
methyl group on the left-hand-side phenyl ring, we pre-
pared the des-methyl analog of 22. Indeed, 86 showed 2-
fold lower potency and weaker activity at PPARa
(EC50 = 155 nM, 31%, Table 6), ꢁ3-fold lower potency
at PPARd (EC50 = 20 nM, 70%), and abolished activity
at PPARc (EC50 > 3000 nM) compared to 22. Finally,
we wished to examine the steric tolerance of substituents
at the a-position of the critical carboxyl group beyond
the geminal-dimethyl group. Therefore, a cyclopentyl
group was installed, and compound 87 displayed 22-fold
reduced potency at PPARa (EC50 = 736 nM, 31%), over
R1
O
O
HO
N
N
S
S
a
EC50 (nM)
Compound
R1
R2
PPARa
(% resp.)
PPARd
(% resp.)
PPARc
(% resp.)
70
75
CF3
Cl
H
Cl
37 (65)
123 (71)
2 (66)
9 (71)
465 (35)
815 (21)
a EC50 is the concentration of test compounds needed to induce 50% of
the maximum luciferase activity. The EC50 value is the average of
more than two separate tests.
PPARc (EC50 = 815 nM, 21%) compared to 40 (Table
2). These two examples demonstrate that elongation of
chain length might be the direction to pursue if PPAR
pan agonists are desired.
Since oxygen may behave as a bioisostere of sulfur in
some cases, we were interested in synthesizing oxygen-
link analogs. The oxygen-link 4-CF3 analog 76 dis-
played comparable potency with higher activity at
PPARa (EC50 = 94 nM, 71%, Table 5), higher potency
at PPARd (EC50 = 3 nM, 79%), but abolished activity
at PPARc (EC50 > 3000 nM) compared to that of sul-
fur-link CF3 analog 22. The oxygen-link OCF3 analog
77 showed ꢁ3-fold lower potency but higher activity
at PPARa (EC50 = 94 nM, 80%), 4-fold lower potency
but higher activity at PPARd (EC50 = 12 nM, 83%)
compared to sulfur-link 23. Based on these two exam-
ples, it seems the oxygen-link analogs exhibited slightly
lower potency but stronger activity at PPARa. Since
we learned that the 3,4-disubstituted analogs tend to ex-
hibit higher potency at PPARa than the corresponding
4-substituted analogs in the sulfur-link series (Table 2),
we prepared several 3,4-disubstituted analogs in the oxy-
gen-link series with the hope to increase the potency at
PPARa as well. However, while 3-F,4-CF3 analog 79
333-fold
decreased
potency
at
PPARd
(EC50 > 1000 nM) compared to 23 (Table 2). This detri-
mental effect was further confirmed in 3,4-dichloro ana-
log 89 where the activities at all three PPAR subtypes
were significantly diminished (EC50 > 3000 nM for
PPARa, >1000 nM for PPARd, and >3000 nM for
PPARc). In addition, there was no cross-activity with
representative compounds, such as 23 and 40 against
several nuclear hormone receptors such as farnesoid X
receptor (FXR), liver X receptor (LXR), retinoic acid
X receptor (RXR), glucocorticoid receptor (GR), and
retinoic acid receptor (RAR). In summary, when com-
pared to PPARd selective agonist 5, these [1,2,4]thiadia-
zole derivatives appear as a new series of potent and
selective PPARa/d dual agonists.
Table 5. Activities in human PPAR co-transfection assays
O
R2
O
HO
R1
N
To determine the activity of these compounds on the
endogenous human receptor, we employed real time
PCR to measure the expression levels of a panel of genes
involved in lipid metabolism and energy expenditure.
The cell types utilized in these experiments were primary
human skeletal muscle cells. To date, 40 induced several
target genes in a dose-dependent manner (Fig. 2). For
example, a robust induction of ATP-binding cassette
transporter A1 (ABC-A1)23 by 40 was detected in skele-
tal muscles (1.5-fold increase at 1 nM to 4.8-fold in-
crease at 1000 nM). Since ABC-A1 is involved in lipid
and protein metabolism, the induction of this gene by
40 suggests PPARa/d dual agonists may play an impor-
tant role in this function. Meanwhile, a significant
induction of carnitine palmitoyltransferase type 1
(CPT-1)24 by 40 (3-fold at 1 nM to 7.8-fold at
1000 nM) suggests 40 may be involved in fatty acid oxi-
O
S
N
Compound R1
R2
a
EC50 (nM)
PPARa
PPARd
PPARc
(% resp.) (% resp.) (% resp.)
76
77
79
80
81
82
83
CF3
OCF3
CF3
H
H
F
94 (71)
94 (80)
86 (65)
97 (101)
504 (83)
3 (79)
12 (83)
3 (76)
>3000
>3000
>3000
>3000
>3000
>3000
>3000
OCF3 Cl
Cl
13 (79)
114 (75)
534 (60)
148 (85)
Cl
CH3
CH3
CH3 643 (58)
Cl 297 (75)
a EC50 is the concentration of test compounds needed to induce 50% of
the maximum luciferase activity. The EC50 value is the average of
more than two separate tests.

3328
L. Shen et al. / Bioorg. Med. Chem. 16 (2008) 3321–3341
Table 6. Activities in human PPAR co-transfection assays
O
R3
R2
O
R4
HO
R4
R1
N
S
S
N
R1
R2
H
R3
H
R4
a
EC50 (nM)
Compound
PPARa (% resp.)
PPARd (% resp.)
PPARc (% resp.)
86
CF3
CH3
155 (31)
20 (70)
>3000
87
89
OCF3
Cl
H
CH3
CH3
736 (31)
>3000
>1000
>1000
>3000
>3000
Cl
a EC50 is the concentration of test compounds needed to induce 50% of the maximum luciferase activity. The EC50 value is the average of more than
two separate tests.
ABC-A1
CPT-1
11
10
9
7
6
5
4
3
2
1
0
1nM
10nM
100nM
1000nM
1nM
10nM
100nM
1000nM
8
7
6
5
4
3
2
1
0
Pa9
20
15
10
5
1nM
10nM
100nM
1000nM
0
Figure 2. Induction of selected target genes by 40 in primary, human skeletal muscle cells. Genes include: ABCA1, ATP binding cassette transporter
A1; CPT-1, Carnitine palmitoyl transferase type I; and Pa9, PPARa in-house target gene. Fold increase over vehicle was expressed as mean SD
(standard error mean) of triplicate samples.
dation. In addition, a strong induction of a new PPARa
target gene, Pa9,25 was also observed (3.8-fold at 1 nM
to 15-fold at 1000 nM). While the monosubstituted 4-
OCF3-analog 23 was found to have P450 induction issue
at 10 lM concentration, we were pleased to find that the
disubstituted 3,4-dichloro-analog 40 displayed only
modest P450 induction at the same concentration. How-
ever, the reason for the different degree of P450 induc-
tion caused by the subtle change in chemical structure
is unclear at this point. Meanwhile, in human liver
microsome cytochrome P450 inhibition assay, 40 dis-
played IC50 > 30 lM for all subtypes tested. Therefore,
it should have low risk for potential drug–drug
interactions.
terolemic rat model. While the high-cholesterol diet fed
hypercholesterolemic rat displayed about 2.8-fold lower
HDL-C level (17.7 mg/dL, Fig. 3) than the standard
chow diet fed rat (49 mg/dL), compound 40 increased
the serum HDL-C level (from 17.7 to 45 mg/dL) in a
dose-related manner after these rats were dosed orally
for 8 days. In addition, while this hypercholesterolemic
rat displayed about 8.7-fold elevated LDL-C level
(96 mg/dL, Fig. 3) than that of the standard chow diet
fed rat (11 mg/dL), 40 attenuated the high LDL-C level
(from 96 to 70 mg/dL) in a dose-related manner. This
LDL-C lowering effect of 40 seems more related to
PPARa agonist rather than PPARd agonist property.
When dosed from 0.1 to 1 mg/kg/d, compound 40
dose-dependently decreased the total cholesterol from
423 to 263 mg/dL. Similar to PPARa agonists, there
was also a dose-related decrease in triglycerides ob-
served when dosed with 40. A pharmacokinetic (PK)
arm of this study is shown in Table 7. The Cmax and
3.2. In vivo studies
The ability of compound 40 to affect lipid/cholesterol
homeostasis in vivo was first examined in a hypercholes-

L. Shen et al. / Bioorg. Med. Chem. 16 (2008) 3321–3341
3329
HDL-C
LDL-C
55
50
45
40
35
30
25
20
15
10
5
**
130
120
110
100
90
**
Lean Vehicle
HF Vehicle
0.1 (mg/kg)
0.3 (mg/kg)
1 (mg/kg)
Lean Vehicle
HF Vehicle
0.1 (mg/kg)
0.3 (mg/kg)
1 (mg/kg)
**
**
**
**
80
**
70
60
50
3 (mg/kg)
3 (mg/kg)
40
30
20
**
10
0
0
Total Cholesterol
Triglycerides
500
400
300
200
100
0
300
200
100
0
Lean Vehicle
HF Vehicle
0.1 (mg/kg)
0.3 (mg/kg)
1 (mg/kg)
Lean Vehicle
HF Vehicle
0.1 (mg/kg)
0.3 (mg/kg)
1 (mg/kg)
*
*
*
*
3 (mg/kg)
3 (mg/kg)
**
Figure 3. Effects of 40 on HDL-C, LDL-C, total cholesterol, and triglyceride levels in hypercholesterolemic rats (8 days oral dosing). Bars represent
means SD (n = 8 rats per group; *p < 0.05, **p < 0.01 relative to vehicle HF).
AUC increased proportionally with dose. High plasma
drug exposure (e.g., AUC = 13,900 ng h/mL at 1 mg/
kg) and long plasma duration (t1/2 = 5.7 h at 1 mg/kg)
was observed, no unexpected accumulation occurred
after multiple dosing.
The capability of 40 to affect lipid homeostasis was next
examined in a human apoA1 transgenic mouse model.
Since apoA1 is the primary protein component of
HDL-C, a human apoA1 transgenic mouse model has
proven useful in the testing of PPARa and PPARd hyp-
olipidemic compounds.26 Male hApoA1 transgenic mice
were dosed orally with 40 for 7 days (Fig. 4). Compound
40 dose-dependently (from 0.3 to 3 mg/kg) increased ser-
um HDL-C levels, which paralleled the increases in ser-
um hApoA1 levels. This result suggests that the
compound increased a PPARa target ApoA1 protein,
which may contribute to the increase in HDL-C levels.
Table 7. PK parameters in Sprague–Dawley rats following either
multiple (8 days of treatment) or a single dose of 40
Dose (mg/kg) Interval Cmax (ng/mL) AUC (ng h/mL) t1/2
0.1
0.3
1
Multiple
Multiple
82 18
231 16
738
1
5.5
5.1
5.7
7.1
2401 284
13,900 4051
27,124 4284
Multiple 1361 542
Multiple 2004 425
3
3
Single
2521 1204
24,241 11,415 7.2
Data are expressed as means SD, N = 2–3 per group. For the single
dose group, the animals were treated with vehicle for 7 days and
administered a single dose of 40 on day 8.
hAPO A1
550
HDL
300
200
100
0
**
**
*
*
Vehicle
Vehicle
500
450
400
350
300
250
200
150
100
50
*
0.3 (mg/kg)
1 (mg/kg)
3 (mg/kg)
0.3 (mg/kg)
1 (mg/kg)
3 (mg/kg)
0
Triglycerides
**
NEFA
120
110
100
90
80
70
60
50
40
30
20
10
0
1.25
1.00
0.75
0.50
0.25
0.00
Vehicle
Vehicle
*
0.3 (mg/kg)
1 (mg/kg)
3 (mg/kg)
0.3 (mg/kg)
1 (mg/kg)
3 (mg/kg)
**
**
**
Figure 4. Effects of 40 on hApoA1, high-density lipoprotein, triglyceride, and non-esterified free fatty acid levels in male human Apo A1 transgenic
mice (7 days oral dosing). Bars represent means SD (n = 8 rats per group; *p < 0.05, **p < 0.01 relative to vehicle HF).

3330
L. Shen et al. / Bioorg. Med. Chem. 16 (2008) 3321–3341
Table 8. PK parameters in ob/ob mice following either multiple (11
days of treatment) or a single dose of 40
Compound 40 also significantly reduced triglycerides
and decreased free fatty acids.
Dose (mg/kg) Interval Cmax (ng/mL) AUC (ng h/mL) t1/2
The ob/ob mice, a genetic leptin deficient model, are ob-
ese and insulin resistant with hypertriglyceridemia,
hyperglycemia and has been used as a rodent model of
obesity-induced insulin resistance. Seven-week-old fe-
male ob/ob mice were dosed orally (from 0.1 to 3 mg/
kg) with 40 for 11 days (Fig. 5). Compound 40 signifi-
cantly reduced serum glucose (52% decrease, from 779
to 375 mg/dL), insulin (50% decrease, from 40 to
20 ng/mL), and triglycerides (83% decrease, from 530
to 92 mg/dL). Unlike PPARc agonists in this model,
compound 40 improved insulin sensitivity but reduced
the body weight at the same time. However, liver
weights increased (65% increase, from 2.6 to 4.3 g) sim-
ilar to the effect observed with PPARa agonists. A PK
arm of this study is shown in Table 8. The Cmax and
AUC increased proportionally up to 1 mg/kg dose.
From 1 to 3 mg/kg dose, a minor unexpected accumula-
tion occurred. High plasma drug exposure (e.g.,
AUC = 47,600 ng h/mL at 1 mg/kg) and long plasma
duration (t1/2 = 5.2 h at 1 mg/kg) were observed in this
ob/ob mice model.
0.1
0.3
1
multiple
multiple
multiple
480 108
1836 346
6830 922
2785 553
10,595 578
47,600 891
5.1
5.0
5.2
3
multiple 26,337 6603 248,545 25,829 6.7
single 19,343 7914 114,823 2370 5.1
3
Data are expressed as means SD, N = 2–3 per group. For the single
dose group, the animals were treated with vehicle for 10 days and
administered a single dose of 40 on day 11.
(Fig. 6). From 0.3 to 3 mg/kg doses, there was no change
in serum level of liver enzyme at AST (or ALT, data not
shown). However, at 10 mg/kg, there was a significant
increase (1.7-fold) at AST level. PK arm data are shown
in Table 9. The Cmax and AUC increased proportionally
with dose, and no unexpected accumulation occurred
after multiple dosing. High plasma drug exposure
(e.g., AUC = 24,120 ng h/mL at 3 mg/kg) and long plas-
ma duration (t1/2 = 6 h at 3 mg/kg) were also observed in
this female Beagle dog model.
3.3. Molecular modeling
The in vivo antihyperlipidemic activity of 40 was also
evaluated in obese, mildly hypertriglyceridemic female
Beagle dogs. Animals were dosed with 40 (0.3–10 mg/
kg) by oral gavage for 7 days, approximately 3 h before
feeding. There was a dose-dependent reduction in serum
triglycerides, total cholesterol, and free fatty acids
Receptor–ligand docking studies were carried out to
help gain insight into the binding mode of compound
21 (Table 1) and ligand subtype selectivity. Figure 7
illustrates a low energy binding mode of 21 in PPARd
as determined by Glide4.5.27,28 The binding mode is sim-
Triglycerides
Glucose
900
800
600
Vehicle
Vehicle
500
400
300
200
100
0
0.1 (mg/kg)
0.3 (mg/kg)
1 (mg/kg)
0.1 (mg/kg)
0.3 (mg/kg)
1 (mg/kg)
3 (mg/kg)
700
600
*
**
500
**
3 (mg/kg)
400
300
200
100
0
**
**
**
Insulin
BW Change
45
40
35
30
25
20
15
10
5
7
6
5
4
3
2
1
0
Vehicle
Vehicle
0.1 (mg/kg)
0.3 (mg/kg)
1 (mg/kg)
3 (mg/kg)
0.1 (mg/kg)
0.3 (mg/kg)
1 (mg/kg)
3 (mg/kg)
**
**
**
**
0
Liver Wt
5
4
3
2
1
0
Vehicle
**
0.1 (mg/kg)
0.3 (mg/kg)
1 (mg/kg)
3 (mg/kg)
**
Figure 5. Effects of 40 on triglyceride, glucose, insulin levels, body weight change, and liver weight change in female ob/ob mice (11 days oral dosing).
Bars represent means SD (n = 8 rats per group; *p < 0.05, **p < 0.01 relative to vehicle HF).

L. Shen et al. / Bioorg. Med. Chem. 16 (2008) 3321–3341
3331
Triglycerides
Total Cholesterol
300
200
100
0
400
Vehicle
0.3 (mg/kg)
1 (mg/kg)
3 (mg/kg)
Vehicle
0.3 (mg/kg)
1 (mg/kg)
3 (mg/kg)
10 (mg/kg)
300
**
**
200
*
10 (mg/kg)
*
**
*
100
0
NEFA
AST
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
70
60
50
40
30
20
10
0
**
Vehicle
Vehicle
0.3 (mg/kg)
1 (mg/kg)
3 (mg/kg)
10 (mg/kg)
0.3 (mg/kg)
1 (mg/kg)
3 (mg/kg)
10 (mg/kg)
**
**
**
**
Figure 6. Effects of 40 on triglyceride, total cholesterol, non-esterified fatty acids, and liver enzyme activity levels in female Beagle dogs (7 days oral
dosing). Bars represent means SD (n = 8 rats per group; *p < 0.05, **p < 0.01 relative to vehicle HF).
Table 9. PK parameters in female Beagle dogs following either
multiple (7 days of treatment) or a single dose of 40
ilar to many PPAR/ligand structures in that an acidic,
ligand ‘head’ group forms hydrogen bonds with residues
at the terminus of the so-called Arm I pocket.29 In our
case, the fibrate-like head group of 21 forms a network
Dose (mg/kg) Interval Cmax (ng/mL) AUC (ng h/mL) t1/2
0.3
1
Multiple
Multiple
Multiple
398 121 2649 1049
1529 84 6505 1984
6711 1288 24,120 4631
6.5
4.7
6.0
of hydrogen bonds with H323, Y473, and H449. At
the mid-section of Arm I, the ortho substituted methyl
group of the phenol occupies a small hydrophobic pock-
et formed by F282, C285, and I363.30 In addition, the
para-trifluoro phenyl extends deep into hydrophobic
Arm II. In an attempt to help rationalize PPARa/d iso-
type selectivity, the compounds in Table 1 were docked
3
10
10
Multiple 10,068 2215 61,563 24,563 6.2
Single 10,138 5637 57,238 35,201 5.8
Data are expressed as means SD, N = 2–3 per group. For the single
dose group, the animals were treated with vehicle for 6 days and
administered a single dose of 40 on day 7.
Figure 7. Predicted binding mode of 21 in PPARd. The carboxylate head group of 21 (blue) forms multiple hydrogen bonds with Y473, H323, and H449
in Arm I. The phenolic ortho-methyl projects into a small hydrophobic pocket outlined by F282, I363, and C285. The para trifluoromethyl phenyl side
of the molecule occupies Arm II of the PPARd ligand binding domain. PPARd is rendered semi-transparent for ease of view, 21 lies behind H3.

3332
L. Shen et al. / Bioorg. Med. Chem. 16 (2008) 3321–3341
into both PPAR isotypes. Docking experiments, with
and without hydrogen bonding constraints, were car-
ried out. Unfortunately, docking energies and binding
modes in all experiments revealed little about ligand/
PPAR isotype selectivity. These results concur with
previous studies whose authors find that PPAR isotype
selectivity remains beyond the limits of virtual screen-
ing technology and is therefore poorly represented by
docking experiments.31 The barrier to ligand/isotype
selectivity in structure-based modeling is primarily
due to the following reasons: high conservation of ac-
tive site residues between the three PPAR isotypes.32
Approximately 34 residues make up the ligand-binding
cavities of the PPARs with at least 80% of these con-
served32; very large ligand binding cavities in all the
PPARs. PPAR receptors have some of the largest
5.1.2. 4-(4-Trifluoromethyl-phenyl)-but-3-yn-2-one (2).
To a solution of 1 (1.25 g, 7.36 mmol) in THF (10 mL)
at room temperature was added 1.0 M EtMgBr
(8.8 mL, 8.8 mmol) in tert-butyl methyl ether. After
heating at 35 ꢁC for 2 h, the solution was cooled to room
temperature and then transferred via a cannula to an-
other flask containing acetic anhydride (1.4 mL,
14.9 mmol) and THF (5 mL) at 4 ꢁC. The solution was
stirred at 4 ꢁC and warmed to room temperature over-
night. Saturated NH4Cl was added, and the organic
phase was separated and the aqueous layer was ex-
tracted with CH2Cl2. The combined organic layers were
dried, concentrated, and the product was purified by
column chromatography (EtOAc/hexane) to give
1
1.46 g (94%) of 2 as a yellow oil: H NMR (300 MHz,
CDCl3) d 7.69 (d, J = 8.8 Hz, 2H), 7.65 (d, J = 8.9 Hz,
2H), 2.48 (s, 3H).
3
˚
(ꢁ1400 A ) binding pockets identified in nuclear hor-
mone receptor (NHR) ligand binding domains.29 Final-
ly, the ligand binding domains are very flexible. PPAR
receptors have been shown to undergo induced fit upon
ligand binding and can bind different conformations of
the same ligand.33
5.1.3. 3-Methyl-5-(4-trifluoromethyl-phenyl)-isothiazole
(3). A mixture of 2 (435 mg, 2.05 mmol) and hydroxyl-
amine-O-sulfonic acid (245 mg, 2.17 mmol) in water
(0.8 mL) was stirred at room temperature for 30 min.
Solid NaHCO3 (183 mg, 2.18 mmol) and water
(0.5 mL) were added and the mixture was stirred for
15 min. NaSH (1.0 M) (2.3 mL, 2.3 mmol) in water
was added and the mixture was stirred overnight and ex-
tracted with CH2Cl2. The extracts were dried, concen-
trated, and the product was purified by column
chromatography to give 347 mg (70%) of 3 as a yellow
4. Conclusion
In an attempt to rationalize the unexpected potent and
selective PPARa/d dual agonist activity, 21 was docked
into all three PPAR isotypes. Unfortunately, docking
energies and binding mode experiments revealed little
about SAR selectivity. Nonetheless, compound 40 and
its close analogs represent a new series of PPARa/d dual
agonist. The high potency, high selectivity, significant
gene induction, excellent PK profile, low P450 inhibition
or induction, and good in vivo efficacy in four animal
models support 40 being selected as a pre-clinical study
candidate, and may render 40 as valuable pharmacolog-
ical tool in elucidating the complex roles of PPARa/d
dual agonists, and the potential usage for the treatment
of metabolic syndrome.
1
solid: H NMR (300 MHz, CDCl3) d 7.69 (s, 4H), 7.24
(s, 1H), 2.55 (s, 3H); MS (ES) m/z: 244 (M+H+).
5.1.4. 3-Bromomethyl-5-(4-trifluoromethyl-phenyl)-iso-
thiazole (4). A mixture of 3 (110 mg, 0.453 mmol), N-
bromosuccinimide (88 mg, 0.49 mmol), and benzoyl per-
oxide (11 mg, 0.045 mmol) in CCl4 (4 mL) was refluxed
for 18 h, and then diluted with CH2Cl2 and water. The
organic layer was separated and the aqueous layer was
extracted with CH2Cl2. The combined organic layers
were dried, concentrated, and column chromatographed
(CH2Cl2/hexane) to provide 61 mg (42%) of 4 as a white
1
solid: H NMR (300 MHz, CDCl3) d 7.71 (s, 4H), 7.53
(s, 1H), 4.58 (s, 2H).
5. Experimental
5.1. Chemical synthesis
5.1.5. {2-Methyl-4-[5-(4-trifluoromethyl-phenyl)-isothiazol-
3-ylmethylsulfanyl]-phenoxy}-acetic acid (6). A mixture
of 4 (60 mg, 0.18 mmol) and (4-mercapto-2-methyl-
phenoxy)acetic acid ethyl ester 5 (63 mg, 0.28 mmol) in
CH3CN (3 mL) was degassed under N2 for about
15 min. After addition of Cs2CO3 (91 mg, 0.28 mmol),
the mixture was stirred overnight under N2, concen-
trated, and purified by column chromatography
(EtOAc/hexane) to give 67 mg (77%) of 6-ethyl ester as
5.1.1. General remarks. 1H NMR spectra were measured
on a Bruker AC-300 (300 MHz) spectrometer using tet-
ramethylsilane as an internal standard. Elemental anal-
yses were obtained by Quantitative Technologies Inc.
(Whitehouse, New Jersey), and the results were within
0.4% of the calculated values unless otherwise men-
tioned. Melting points were determined in open capil-
lary tubes with a Thomas–Hoover apparatus and were
uncorrected. Electrospray mass spectra (MS-ES) were
recorded on a Hewlett Packard 59987A spectrometer.
High resolution mass spectra (HRMS) were obtained
on a Micromass Autospec. E. spectrometer. The term
‘DMAP’ refers to dimethylamino-pyridine, ‘TFA’ refers
to trifluoroacetic acid, ‘NMP’ refers to 1-methyl-2-pyr-
rolidinone, ‘DPPA’ refers to diphenylphosphoryl azide.
The syntheses of compounds 21, 22, 23, 24, 56, 57, 76,
77, and 87 were described in preliminary report.17
1
white crystals: H NMR (300 MHz, CDCl3) d 7.69 (d,
J = 8.5 Hz, 2H), 7.65 (d, J = 8.6 Hz, 2H), 7.31 (s, 1H),
7.19 (d, J = 1.8 Hz, 1H), 7.14 (dd, J = 8.4, 2.2 Hz, 1H),
6.60 (d, J = 8.4 Hz, 1H), 4.61 (s, 2H), 4.25 (q,
J = 7.1 Hz, 2H), 4.16 (s, 2H), 2.23 (s, 3H), 1.28 (t,
J = 7.1 Hz, 3H); MS (ES) m/z: 468 (M+H+). Anal. calcd
for C22H20F3NO3S2: C, 56.52; H, 4.31; N, 3.00. Found:
C, 56.54; H, 3.86; N, 3.00. A mixture of 6-ethyl ester
(54 mg, 0.12 mmol) and 2 M NaOH (0.2 mL, 0.4 mmol)
in MeOH (1.5 mL) was stirred under N2 for 2 h and

L. Shen et al. / Bioorg. Med. Chem. 16 (2008) 3321–3341
3333
1
concentrated. CH2Cl2 and water were added, and the
mixture was acidified with concentrated HCl. The or-
ganic phase was separated and the aqueous phase was
extracted with CH2Cl2. The combined organic layers
were dried, concentrated, and column chromatographed
(0.3% AcOH in EtOAc) to give 49 mg (97%) of 6 as a
white solid: 1H NMR (300 MHz, CDCl3) d 7.69 (d,
J = 8.7 Hz, 2H), 7.65 (d, J = 8.6 Hz, 2H), 7.35 (s, 1H),
7.18 (s, 1H), 7.16 (m, 1H), 6.64 (d, J = 8.2 Hz, 1H),
4.66 (s, 2H), 4.17 (s, 2H), 2.22 (s, 3H); MS (ES) m/z:
440 (M+H+). Anal. calcd for C20H16F3NO3S2Æ0.6H2O:
C, 53.35; H, 3.85; N, 3.11. Found: C, 53.20; H, 3.51;
N, 2.91.
(41%, 3 steps) of 10-ethyl ester as a clear oil: H NMR
(300 MHz, CDCl3) d 7.98 (d, J = 8.2 Hz, 2H), 7.68 (d,
J = 8.3 Hz, 2H), 7.30 (s, 1H), 7.25 (d, J = 2.1 Hz, 1H),
7.17 (dd, J = 8.4, 2.2 Hz, 1H), 6.61 (d, J = 8.4 Hz, 1H),
4.62 (s, 2H), 4.25 (s, 2H), 4.24 (q, J = 7.1 Hz, 2H), 2.24
(s, 3H), 1.27 (t, J = 7.1 Hz, 3H); MS (ES) m/z: 490
(M+Na+).
A mixture of 10-ethyl ester (56 mg,
0.12 mmol) and 2 M NaOH (0.15 mL, 0.30 mmol) in
MeOH (2.5 mL) was stirred under N2 for 2 h and concen-
trated. CH2Cl2 and water were added, and the mixture
was acidified with concentrated HCl. The organic phase
was separated and the aqueous phase was extracted with
CH2Cl2. The combined organic layers were dried, con-
centrated, and column chromatographed (0.3% AcOH
in EtOAc) to give 50 mg (95%) of 10 as a yellow oil which
solidified upon standing over time: 1H NMR (300 MHz,
CDCl3) d 7.96 (d, J = 8.1 Hz, 2H), 7.67 (d, J = 8.2 Hz,
2H), 7.32 (s, 1H), 7.24 (d, J = 1.7 Hz, 1H), 7.19 (dd,
J = 8.4, 2.2 Hz, 1H), 6.63 (d, J = 8.4 Hz, 1H), 4.67 (s,
2H), 4.26 (s, 2H), 2.23 (s, 3H); MS (ES) m/z: 440
(M+H+) Anal. calcd for C20H16F3NO3S2: C, 54.66; H,
3.67; N, 3.19. Found: C, 54.30; H, 3.52; N, 3.06.
5.1.6. 3-(4-Trifluoromethyl-phenyl)-isothiazole-5-carbox-
ylic acid ethyl ester (8). The reaction mixture of 7
(608 mg, 2.46 mmol) and ethyl propiolate (726 mg,
7.41 mmol) in chlorobenzene (10 mL) was heated at
135 ꢁC for 20 h. TLC showed some of the starting mate-
rial 7 still remained. More ethyl propiolate (726 mg,
7.41 mmol) and 1,2-dichlorobenzene (10 mL) were
added and the solution was heated at 160 ꢁC for 7 h.
After cooling to room temperature, the reaction mixture
was concentrated and the product was purified by col-
umn chromatography to give 264 mg (36%) of 11 as
an off-white solid and 229 mg (31%) of 8 as a white solid:
1H NMR (300 MHz, CDCl3) d 8.15 (s, 1H), 8.09 (d,
J = 8.1 Hz, 2H), 7.73 (d, J = 8.2 Hz, 2H), 4.44 (q,
J = 7.1 Hz, 2H), 1.43 (t, J = 7.1 Hz, 3H); MS (ES) m/z:
302 (M+H+).
5.1.9. 3-(4-Trifluoromethyl-phenyl)-isothiazole-4-carbox-
ylic acid ethyl ester (11). From the same reaction that
provided 8 as shown above, 264 mg (36%) of 11 was also
isolated as an off-white solid: 1H NMR (300 MHz,
CDCl3) d 9.39 (s, 1H), 7.76 (d, J = 8.3 Hz, 2H), 7.70
(d, J = 8.4 Hz, 2H), 4.28 (q, J = 7.1 Hz, 2H), 1.27 (t,
J = 7.1 Hz, 3H); MS (ES) m/z: 302 (M+H+).
5.1.7. [3-(4-Trifluoromethyl-phenyl)-isothiazol-5-yl]-meth-
anol (9). To the solution of 8 (104 mg, 0.345 mmol) in
THF (2 mL) at ꢀ78 ꢁC was added 1.0 M LiAlH4
(0.21 mL, 0.21 mmol) in THF. After stirring at ꢀ78 ꢁC
for 30 min, water was slowly added and the mixture
was allowed to warm up to room temperature. The pre-
cipitated solid was filtered and rinsed with CH2Cl2. The
filtrate was washed with saturated NH4Cl, and the aque-
ous solution was back extracted with CH2Cl2. The com-
bined organic phases were dried and concentrated to
5.1.10. [3-(4-Trifluoromethyl-phenyl)-isothiazol-4-yl]-meth-
anol (12). To the solution of 11 (96 mg, 0.32 mmol) in
EtOH (4 mL) at room temperature was added NaBH4
(30 mg, 0.79 mmol). After overnight, more NaBH4
(154 mg, 4.05 mmol) and EtOH (4 mL) were added
and stirring was continued for 24 h. The solvent was
evaporated, and the residue was partitioned between
CH2Cl2 and water. The organic phase was dried and
concentrated to provide 77 mg (93%) of 12 as a clear
1
oil: H NMR (300 MHz, CDCl3) d 8.76 (s, 1H), 7.90
(d, J = 8.1 Hz, 2H), 7.73 (d, J = 8.2 Hz, 2H), 4.81 (s,
1
give 98 mg of 9 as a yellow solid: H NMR (300 MHz,
CDCl3) d 8.04 (d, J = 8.3 Hz, 2H), 7.70 (d, J = 8.4 Hz,
2H), 7.51 (s, 1H), 5.06 (s, 2H); MS (ES) m/z: 260
(M+H+).
2H); MS (ES) m/z: 260 (M+H+).
5.1.11.
{2-Methyl-4-[3-(4-trifluoromethyl-phenyl)-isot-
hiazol-4-ylmethylsulfanyl]-phenoxy}-acetic acid (13).
The mixture of 12 (77 mg, 0.30 mmol), methanesulfonyl
chloride (53 mg, 0.47 mmol), and triethylamine
(0.083 mL, 0.60 mmol) in CH2Cl2 (3 mL) was stirred
at room temperature overnight. The solution was
washed with water and the aqueous phase was back ex-
tracted with CH2Cl2. The combined organic layers were
dried and concentrated to provide 107 mg of the crude
mesylate as an off-white solid. A mixture of the crude
mesylate (107 mg) and (4-mercapto-2-methyl-phen-
oxy)acetic acid ethyl ester 5 (90 mg, 0.40 mmol) in
CH3CN (2 mL) was degassed under N2 for about
15 min. After addition of Cs2CO3 (167 mg, 0.512 mmol),
the mixture was stirred for 4 h under N2, concentrated,
and purified by column chromatography (EtOAc/hex-
ane) to give 114 mg (77%, 2 steps) of 13-ethyl ester as
a yellow oil: 1H NMR (300 MHz, CDCl3) d 8.30 (s,
1H), 7.80 (d, J = 8.2 Hz, 2H), 7.72 (d, J = 8.3 Hz, 2H),
5.1.8. {2-Methyl-4-[3-(4-trifluoromethyl-phenyl)-isothiazol-
5-ylmethylsulfanyl]-phenoxy}-acetic acid (10). To a solu-
tion of 9 (98 mg, 0.38 mmol) in CH2Cl2 (3 mL) were
added methanesulfonyl chloride (59 mg, 0.52 mmol)
and triethylamine (0.096 mL, 0.69 mmol). The solution
was stirred at room temperature overnight and diluted
with CH2Cl2 and water. The organic layer was separated
and the aqueous layer was extracted with CH2Cl2. The
combined organic phases were dried and concentrated
to provide 95 mg of the crude mesylate as a yellow solid.
A mixture of the crude mesylate (95 mg) and (4-mer-
capto-2-methyl-phenoxy)acetic acid ethyl ester
5
(115 mg, 0.51 mmol) in CH3CN (2 mL) was degassed un-
der N2 for about 15 min. After addition of Cs2CO3
(220 mg, 0.67 mmol), the mixture was stirred for 4 h un-
der N2, concentrated, and the product was purified by
column chromatography (EtOAc/hexane) to give 66 mg

3334
L. Shen et al. / Bioorg. Med. Chem. 16 (2008) 3321–3341
7.11 (d, J = 1.7 Hz, 1H), 7.05 (dd, J = 8.4, 2.2 Hz, 1H),
6.58 (d, J = 8.4 Hz, 1H), 4.63 (s, 2H), 4.27 (q,
J = 7.1 Hz, 2H), 4.04 (s, 2H), 2.23 (s, 3H), 1.30 (t,
J = 7.1 Hz, 3H); MS (ES) m/z: 490 (M+Na+). A mixture
of 13-ethyl ester (103 mg, 0.221 mmol) and 2 M NaOH
(0.30 mL, 0.60 mmol) in MeOH (2.5 mL) was stirred un-
der N2 for 2 h and concentrated. CH2Cl2 and water were
added, and the mixture was acidified with concentrated
HCl. The organic phase was separated and the aqueous
phase was extracted with CH2Cl2. The combined organ-
ic layers were dried, concentrated, and column chro-
matographed (0.3% AcOH in EtOAc) to give 90 mg
(93%) of 13 as a white solid: 1H NMR (300 MHz,
MeOH-d4) d 8.56 (s, 1H), 7.75 (s, 4H), 7.01 (s, 1H),
6.99 (m, 1H), 6.66 (d, J = 8.2 Hz, 1H), 4.67 (s, 2H),
4.10 (s, 2H), 2.14 (s, 3H); MS (ES) m/z: 440 (M+H+).
Anal. calcd for C20H16F3NO3S2: C, 54.66; H, 3.67; N,
3.19. Found: C, 54.54; H, 3.53; N, 3.01.
5.1.14. 5-(4-Trifluoromethyl-phenyl)-isoxazole-3-carbox-
ylic acid ethyl ester (18). To the solution of 17 (23.0 g,
79.9 mmol) in EtOH (400 mL) was added hydroxyl-
amine hydrochloride (16.65 g, 240 mmol). After stirring
at room temperature for 2 h, the reaction mixture was
refluxed for 5 h. The cooled reaction mixture was con-
centrated under reduced pressure, and the residue was
partitioned between CH2Cl2 and water. The organic
layer was dried, concentrated, and column chromato-
graphed to provide 20.8 g (91%) of 18 as a yellow solid:
1H NMR (300 MHz, CDCl3) d 7.94 (d, J = 8.2 Hz, 2H),
7.77 (d, J = 8.3 Hz, 2H), 7.04 (s, 1H), 4.49 (q,
J = 7.1 Hz, 2H), 1.45 (t, J = 7.1 Hz, 3H); MS (ES) m/z:
286 (M+H+). Anal. calcd for C13H10F3NO3: C, 54.74;
H, 3.53; N, 4.91. Found: C, 54.89; H, 3.20; N, 4.55.
5.1.15. [5-(4-Trifluoromethyl-phenyl)-isoxazol-3-yl]-meth-
anol (19). To a solution of 18 (19.4 g, 68.1 mmol) in
THF (420 mL) at ꢀ78 ꢁC was added 1.0 M LiAlH4
(41.0 mL, 41.0 mmol) in THF. After stirring at ꢀ78 ꢁC
for 1 h, water (4 mL) was slowly added and the mixture
was allowed to warm to room temperature. After the
addition of brine, the precipitated solid was filtered,
rinsed with CH2Cl2, refluxed in THF (200 mL) two
times, and filtered again. The combined filtrates were
separated and the aqueous phase was extracted with
CH2Cl2. The combined organic phases were dried, con-
centrated, and column chromatographed (EtOAc/hex-
5.1.12. 5-Chloromethyl-3-(4-trifluoromethyl-phenyl)-isox-
azole (15). To a mixture of 14 (500 mg, 2.24 mmol) and
propargyl chloride (235 mg, 3.15 mmol) in CH2Cl2
(12 mL) at 4 ꢁC was added Et3N (0.38 mL, 2.73 mmol).
After removing of the cooling bath, the reaction mixture
was stirred at room temperature for 5 h and then
washed with water. The aqueous phase was back ex-
tracted with CH2Cl2, and the combined organic layers
were dried, concentrated and column chromatographed
1
1
to give 373 mg (64%) of 15 as a white solid: H NMR
ane) to give 7.45 g (45%) of 19 as a yellow solid: H
(300 MHz, CDCl3) d 7.93 (d, J = 8.2 Hz, 2H), 7.73 (d,
J = 8.3 Hz, 2H), 6.68 (s, 1H), 4.68 (s, 2H); MS (ES)
m/z: 262 (M+H+).
NMR (300 MHz, CDCl3) d 7.88 (d, J = 8.2 Hz, 2H),
7.72 (d, J = 8.5 Hz, 2H), 6.69 (s, 1H), 4.83 (s, 2H); MS
(ES) m/z: 266 (M+Na+). Anal. calcd for C11H8F3NO2:
C, 54.33; H, 3.32; N, 5.76. Found: C, 54.38; H, 3.08;
N, 5.77.
5.1.13. {2-Methyl-4-[3-(4-trifluoromethyl-phenyl)-isoxazol-
5-ylmethylsulfanyl]-phenoxy}-acetic acid (16). A mixture
of 15 (262 mg, 1.00 mmol) and 5 (340 mg, 1.50 mmol) in
CH3CN (8 mL) was degassed under N2 for about
15 min. After addition of Cs2CO3 (490 mg, 1.50 mmol),
the reaction mixture was stirred for 4 h under N2, then
concentrated, and purified by column chromatography
(EtOAc/hexane) to give 410 mg (91%) of 16-ethyl ester
5.1.16. {2-Methyl-4-[5-(4-trifluoromethyl-phenyl)-isoxazol-
3-ylmethylsulfanyl]-phenoxy}-acetic acid (20). To a solu-
tion of 19 (7.07 g, 29.1 mmol) in CH2Cl2 (320 mL) at
4 ꢁC were added methanesulfonyl chloride (4.44 g,
38.8 mmol) and triethylamine (6.0 mL, 43.1 mmol).
The cooling bath was removed and the solution was stir-
red at room temperature for 3 h. After removal of about
two-thirds of CH2Cl2 under reduced pressure, Et2O was
added. The precipitated solid was filtered and rinsed
with Et2O. The filtrate was washed with water, and the
aqueous solution was back extracted with CH2Cl2.
The combined organic layers were dried and concen-
trated to provide 9.19 g of the crude mesylate as a brown
solid. A mixture of the crude mesylate (9.19 g) and 5
(8.50 g, 37.6 mmol) in CH3CN (200 mL) was degassed
under N2 for about 15 min. After addition of Cs2CO3
(14.0 g, 42.9 mmol), the mixture was stirred for 2.5 days
under N2 and concentrated. The residue was diluted
with CH2Cl2 and filtered. The filtrate was concentrated
and purified by column chromatography (EtOAc/hex-
ane) to give 10.41 g (79%, 2 steps) of 20-ethyl ester as
1
as a clear oil which solidified upon standing: H NMR
(300 MHz, CDCl3) d 7.87 (d, J = 8.1 Hz, 2H), 7.70 (d,
J = 8.3 Hz, 2H), 7.24 (d, J = 2.7 Hz, 1H), 7.18 (dd,
J = 8.4, 2.4 Hz, 1H), 6.61 (d, J = 8.4 Hz, 1H), 6.30 (s,
1H), 4.62 (s, 2H), 4.24 (q, J = 7.1 Hz, 2H), 4.08 (s,
2H), 2.25 (s, 3H), 1.28 (t, J = 7.1 Hz, 3H); MS (ES)
m/z: 474 (M+Na+). Anal. calcd for C22H20F3NO4S: C,
58.53; H, 4.47; N, 3.10. Found: C, 58.51; H, 4.08; N,
2.94. A mixture of 16-ethyl ester (348 mg, 0.772 mmol)
and 2 M NaOH (0.4 mL, 0.8 mmol) in water (0.4 mL)
and MeOH (11 mL) was stirred under N2 for 3 h and
concentrated. CH2Cl2 and water were added, and the
mixture was acidified with concentrated HCl. The or-
ganic phase was separated and the aqueous phase was
extracted with CH2Cl2. The combined organic layers
were dried and concentrated to give 334 mg (100%) of
16 as a white solid: 1H NMR (300 MHz, CDCl3) d
7.87 (d, J = 8.1 Hz, 2H), 7.70 (d, J = 8.0 Hz, 2H), 7.25
(s, 1H), 7.20 (dd, J = 8.3, 1.9 Hz, 1H), 6.65 (d,
J = 8.3 Hz, 1H), 6.34 (s, 1H), 4.68 (s, 2H), 4.10 (s,
2H), 2.24 (s, 3H); MS (ES) m/z: 446 (M+Na+). HRMS
calcd for C20H16F3NO4S: 423.0752. Found: 423.0763.
1
a yellow solid: H NMR (300 MHz, CDCl3) d 7.85 (d,
J = 8.2 Hz, 2H), 7.71 (d, J = 8.3 Hz, 2H), 7.23
(d, J = 1.8 Hz, 1H), 7.17 (dd, J = 8.4, 2.2 Hz, 1H), 6.61
(d, J = 8.4 Hz, 1H), 6.53 (s, 1H), 4.61 (s, 2H), 4.25 (q,
J = 7.1 Hz, 2H), 4.05 (s, 2H), 2.24 (s, 3H), 1.28 (t,
J = 7.1 Hz, 3H); MS (ES) m/z: 474 (M+Na+). Anal.
calcd for C22H20F3NO4S: C, 58.53; H, 4.47; N, 3.10.

L. Shen et al. / Bioorg. Med. Chem. 16 (2008) 3321–3341
3335
Found: C, 58.46; H, 4.30; N, 3.01. A mixture of 20-ethyl
ester (5.66 g, 12.5 mmol) and 2 M NaOH (6.6 mL,
13.2 mmol) in water (10 mL) and MeOH (160 mL) was
stirred under N2 for 2 h and concentrated. CH2Cl2 and
water were added, and the mixture was acidified with
concentrated HCl (1.2 mL). The organic phase was sep-
arated and the aqueous phase was extracted with
CH2Cl2. The combined organic layers were dried, con-
centrated, and column chromatographed (0.3% AcOH
in EtOAc) to give 5.24 g (99%) of 20 as an off-white so-
5.1.18. General procedure for the synthesis of 27, 33, 38,
43, 48, and 53.
5.1.18.1. 3-(3-Fluoro-4-trifluoromethyl-phenyl)-[1,2,4]-
thiadiazole-5-carboxylic acid ethyl ester (27). A reaction
mixture of 26 (480 mg, 1.81 mmol) and ethyl cyanofor-
mate (722 mg, 7.29 mmol) in 1,2-dichlorobenzene
(7 mL) was heated at 160 ꢁC for 20 h. After cooling
down to room temperature, the reaction mixture was
purified by column chromatography to give 400 mg
(69%) of 27 as a brown solid: 1H NMR (400 MHz,
1
lid: H NMR (300 MHz, CDCl3) d 7.84 (d, J = 8.2 Hz,
CDCl3)
d
8.26 (d, J = 8.2 Hz, 1H), 8.22 (d,
2H), 7.71 (d, J = 8.3 Hz, 2H), 7.22 (d, J = 1.6 Hz, 1H),
7.17 (dd, J = 8.4, 2.1 Hz, 1H), 6.63 (d, J = 8.4 Hz, 1H),
6.56 (s, 1H), 4.65 (s, 2H), 4.05 (s, 2H), 2.22 (s, 3H);
MS (ES) m/z: 446 (M+Na+). Anal. calcd for
C20H16F3NO4S: C, 56.73; H, 3.81; N, 3.31. Found: C,
56.76; H, 3.46; N, 3.20.
J = 11.2 Hz, 1H), 7.74 (t, J = 7.6 Hz, 1H), 4.57 (q,
J = 7.1 Hz, 2H), 1.50 (t, J = 7.1 Hz, 3H); MS (ES) m/z:
321 (M+H+).
5.1.18.2. 3-(3-Chloro-4-trifluoromethoxy-phenyl)-[1,2,4]-
thiadiazole-5-carboxylic acid ethyl ester (33). Using 32
and following the procedure as in the preparation of
27 gave 33 (36%) as a white solid: 1H NMR
(300 MHz, CDCl3) d 8.52 (d, J = 2.1 Hz, 1H), 8.31
(dd, J = 8.6, 2.1 Hz, 1H), 7.44 (dd, J = 8.6, 1.4 Hz,
1H), 4.56 (q, J = 7.1 Hz, 2H), 1.49 (t, J = 7.1 Hz, 3H);
MS (ES) m/z: 375 (M+Na+).
5.1.17. General procedure for the synthesis of 26, 32, 37,
42, 47, and 52
5.1.17.1. 5-(3-Fluoro-4-trifluoromethyl-phenyl)-[1,3,4]oxa-
thiazol-2-one (26). The reaction mixture of 3-fluoro-
4-trifluoromethylbenzamide 25 (2.82 g, 13.6 mmol),
chlorocarbonylsulfenyl chloride (3.57 g, 27.2 mmol) in
toluene (35 mL) was heated at 60 ꢁC for 15 h and con-
centrated. CH2Cl2 was added and the mixture was fil-
tered. The filtrate was concentrated and the product
was purified by column chromatography (EtOAc/hex-
ane) to provide 3.46 g (96%) of 26 as off-white solids:
1H NMR (400 MHz, CDCl3) d 7.87 (d, J = 8.2 Hz,
1H), 7.83 (d, J = 10.6 Hz, 1H), 7.77 (m, 1H).
5.1.18.3. 3-(3,4-Dichloro-phenyl)-[1,2,4]thiadiazole-5-
carboxylic acid ethyl ester (38). Using 37 and following
the procedure as in the preparation of 27 gave 38
1
(91%) as a yellow solid: H NMR (300 MHz, CDCl3)
d 8.48 (d, J = 2.0 Hz, 1H), 8.20 (dd, J = 8.4, 2.0 Hz,
1H), 7.57 (d, J = 8.4 Hz, 1H), 4.56 (q, J = 7.1 Hz, 2H),
1.49 (t, J = 7.1 Hz, 3H); MS (ES) m/z: 303 (M+H+).
5.1.17.2. 5-(3-Chloro-4-trifluoromethoxy-phenyl)-[1,3,4]-
oxathiazol-2-one (32). Using 31 and following the proce-
dure as in the preparation of 26 gave 32 (88%) as a white
solid: 1H NMR (300 MHz, CDCl3) d 8.12 (d, J = 2.0 Hz,
1H), 7.92 (dd, J = 8.7, 2.0 Hz, 1H), 7.45 (dd, J = 8.6, 1.1,
1H).
5.1.18.4. 3-(2,4-Dichloro-phenyl)-[1,2,4]thiadiazole-5-
carboxylic acid ethyl ester (43). Using 42 and following
the procedure as in the preparation of 27 gave 43
1
(55%) as a beige solid: H NMR (300 MHz, CDCl3) d
7.99 (d, J = 8.4 Hz, 1H), 7.56 (d, J = 2.0 Hz, 1H), 7.38
(dd, J = 8.4, 2.0 Hz, 1H), 4.56 (q, J = 7.1 Hz, 2H), 1.48
(t, J = 7.1 Hz, 3H); MS (ES) m/z: 303 (M+H+).
5.1.17.3. 5-(3,4-Dichloro-phenyl)-[1,3,4]oxathiazol-2-
one (37). Using 36 and following the procedure as in
the preparation of 26 gave 37 (94%) as an off-white solid:
1H NMR (300 MHz, CDCl3) d 8.07 (d, J = 2.0 Hz, 1H),
7.80 (dd, J = 8.4, 2.0 Hz, 1H), 7.58 (d, J = 8.4 Hz, 1H).
5.1.18.5. 3-(3,4-Dimethyl-phenyl)-[1,2,4]thiadiazole-5-
carboxylic acid ethyl ester (48). Using 47 and following
the procedure as in the preparation of 27 gave 48
1
(47%) as a brown solid: H NMR (300 MHz, CDCl3)
d 8.15 (s, 1H), 8.09 (d, J = 7.9 Hz, 1H), 7.24 (m, 1H),
4.55 (q, J = 7.1 Hz, 2H), 2.35 (s, 3H), 2.33 (s, 3H),
1.48 (t, J = 7.1 Hz, 3H); MS (ES) m/z: 263 (M+H+).
5.1.17.4. 5-(2,4-Dichloro-phenyl)-[1,3,4]oxathiazol-2-
one (42). Using 41 and following the procedure as in
the preparation of 26 gave 42 (54%) as an off-white so-
lid: H NMR (300 MHz, CDCl3) d 7.81 (d, J = 8.5 Hz,
1H), 7.56 (d, J = 2.1 Hz, 1H), 7.39 (m, 1H).
1
5.1.18.6. 3-(3-Chloro-4-methyl-phenyl)-[1,2,4]thiadia-
zole-5-carboxylic acid ethyl ester (53). Using 52 and fol-
lowing the procedure as in the preparation of 27 gave 53
1
5.1.17.5. 5-(3,4-Dimethyl-phenyl)-[1,3,4]oxathiazol-2-
one (47). Using 46 and following the procedure as in
the preparation of 26 gave 47 (47%) as a white solid:1H
NMR (400 MHz, CDCl3) d 7.74 (s, 1H), 7.69 (d,
J = 7.9 Hz, 1H), 7.24 (d, J = 8.2 Hz, 1H), 2.33 (s, 6H).
(87%) as a white solid: H NMR (300 MHz, CDCl3) d
8.37 (d, J = 1.7 Hz, 1H), 8.15 (dd, J = 7.9, 1.7 Hz, 1H),
7.35 (d, J = 7.9 Hz, 1H), 4.55 (q, J = 7.1 Hz, 2H), 2.44
(s, 3H), 1.49 (t, J = 7.1 Hz, 3H); MS (ES) m/z: 283
(M+H+).
5.1.17.6. 5-(3-Chloro-4-methyl-phenyl)-[1,3,4]oxathi-
azol-2-one (52). Using 51 and following the procedure
as in the preparation of 26 gave 52 (57%) as a light
brown solid: H NMR (300 MHz, CDCl3) d 7.96 (d,
J = 1.7 Hz, 1H), 7.75 (dd, J = 8.0, 1.8 Hz, 1H), 7.35 (d,
J = 8.0 Hz, 1H).
5.1.19. General procedure for the synthesis of 28, 34, 39,
44, 49, and 54.
5.1.19.1. [3-(3-Fluoro-4-trifluoromethyl-phenyl)-[1,2,4]-
thiadiazol-5-yl]-methanol (28). To a solution of 27
(212 mg, 0.662 mmol) in EtOH (10 mL) at room temper-
ature was added NaBH4 (64 mg, 1.7 mmol). After
1

3336
L. Shen et al. / Bioorg. Med. Chem. 16 (2008) 3321–3341
stirring for 2 h, a few drops of water were added to
quench excess of hydride. EtOH was evaporated, and
the residue was partitioned between CH2Cl2 and water.
The organic phase was dried and concentrated to pro-
vide 162 mg (88%) of 28 as a yellow oil: 1H NMR
(300 MHz, CDCl3) d 8.17 (d, J = 8.5 Hz, 1H), 8.13 (d,
J = 11.6 Hz, 1H), 7.71 (m, 1H), 5.19 (s, 2H); MS (ES)
m/z: 279 (M+H+).
DMF (0.1 mL) was added Cs2CO3 (100 mg, 0.31 mmol).
After stirring at room temperature for 15 min, the mix-
ture was concentrated. The residue was diluted with
EtOAc, washed with water and brine, dried, concen-
trated, and column chromatographed to give 85 mg
(79%) of 30-ethyl ester as a clear oil: 1H NMR
(300 MHz, CDCl3) d 8.11 (d, J = 8.4 Hz, 1H), 8.07 (d,
J = 11.3 Hz, 1H), 7.69 (t, J = 7.7 Hz, 1H), 7.26 (m,
1H), 7.14 (dd, J = 8.5, 2.5 Hz, 1H), 6.57 (d, J = 8.5 Hz,
1H), 4.40 (s, 2H), 4.20 (q, J = 7.1 Hz, 2H), 2.19 (s,
3H), 1.58 (s, 6H), 1.20 (t, J = 7.1 Hz, 3H); MS (ES)
m/z: 515 (M+H+). A solution of 30-ethyl ester (82 mg,
0.16 mmol) in MeOH (1.0 mL) and THF (1.0 mL) was
treated with 2 N NaOH (1.0 mL, 2.0 mmol) for 4 h
and concentrated. The residue was diluted with EtOAc
and water, and acidified with concentrated HCl. The or-
ganic layer was separated and the aqueous layer was ex-
tracted with EtOAc. The combined organic phases were
washed with brine, dried, concentrated, and column
chromatographed to provide 77 mg (99%) of 30 as a yel-
low oil: 1H NMR (300 MHz, CDCl3) d 8.11 (d,
J = 8.1 Hz, 1H), 8.06 (d, J = 11.3 Hz, 1H), 7.69 (t,
J = 7.6 Hz, 1H), 7.28 (m, 1H), 7.17 (dd, J = 8.6,
2.5 Hz, 1H), 6.72 (d, J = 8.5 Hz, 1H), 4.42 (s, 2H),
2.20 (s, 3H), 1.61 (s, 6H); MS (ES) m/z: 487 (M+H+).
HRMS calcd for C21H18F4N2O3S2: 486.0695. Found:
486.0712.
5.1.19.2. [3-(3-Chloro-4-trifluoromethoxy-phenyl)-[1,2,4]-
thiadiazol-5-yl]-methanol (34). Using 33 and following
the procedure as in the preparation of 28 gave 34
1
(93%) as a beige solid: H NMR (300 MHz, CDCl3) d
8.42 (d, J = 2.1 Hz, 1H), 8.21 (dd, J = 8.6, 2.1 Hz, 1H),
7.42 (dd, J = 8.6, 1.4 Hz, 1H), 5.18 (s, 2H).
5.1.19.3. [3-(3,4-Dichloro-phenyl)-[1,2,4]thiadiazol-5-
yl]-methanol (39). Using 38 and following the procedure
as in the preparation of 28 gave 39 (86%) as a yellow so-
lid: 1H NMR (400 MHz, DMSO-d6)
d 8.33 (d,
J = 1.9 Hz, 1H), 8.15 (m, 1H), 7.82 (d, J = 8.4, 1H),
4.99 (s, 2H).
5.1.19.4. [3-(2,4-Dichloro-phenyl)-[1,2,4]thiadiazol-5-
yl]-methanol (44). Using 43 and following the procedure
as in the preparation of 28 gave 44 (56%) as a white so-
1
lid: H NMR (300 MHz, CDCl3) d 7.91 (d, J = 8.4 Hz,
1H), 7.55 (d, J = 2.1 Hz, 1H), 7.36 (dd, J = 8.4, 2.1 Hz,
1H), 5.20 (s, 2H); MS (ES) m/z: 261 (M+H+).
5.1.20.2.
2-{4-[3-(3-Chloro-4-trifluoromethoxy-phe-
nyl)-[1,2,4]thiadiazol-5-ylmethylsulfanyl]-2-methyl-phen-
oxy}-2-methyl-propionic acid (35). Using 34 and
following the procedure as in the preparation of 30 gave
35 (20%) as a light yellow oily solid: 1H NMR
(300 MHz, CDCl3) d 8.37 (d, J = 2.1 Hz, 1H), 8.16
(dd, J = 8.6, 2.1 Hz, 1H), 7.40 (dd, J = 8.6, 1.5 Hz,
1H), 7.28 (d, J = 2.0 Hz, 1H), 7.17 (dd, J = 8.4, 2.3 Hz,
1H), 6.72 (d, J = 8.5 Hz, 1H), 4.42 (s, 2H), 2.20 (s,
3H), 1.61 (s, 6H); MS (ES) m/z: 519 (M+H+). HRMS
calcd for C21H18F3ClN2O4S2: 518.0349. Found:
518.0365.
5.1.19.5. [3-(3,4-Dimethyl-phenyl)-[1,2,4]thiadiazol-5-
yl]-methanol (49). Using 48 and following the procedure
as in the preparation of 28 gave 49 (85%) as a white so-
lid: 1H NMR (300 MHz, CDCl3) d 8.06 (s, 1H), 8.00 (d,
J = 7.8 Hz, 1H), 7.24 (d, J = 7.9 Hz, 1H), 5.17 (s, 2H),
2.62 (brs, 1H), 2.34 (s, 3H), 2.32 (s, 3H); MS (ES) m/z:
221 (M+H+).
5.1.19.6.
[3-(3-Chloro-4-methyl-phenyl)-[1,2,4]thia-
diazol-5-yl]-methanol (54). Using 53 and following the
procedure as in the preparation of 28 gave 54 (90%) as
1
a white solid: H NMR (400 MHz, CDCl3) d 8.28 (d,
5.1.20.3. 2-{4-[3-(3,4-Dichloro-phenyl)-[1,2,4]thiadiazol-
5-ylmethylsulfanyl]-2-methyl-phenoxy}-2-methyl-propionic
acid (40). Using 39 and following the procedure as in the
preparation of 30 gave 40 (46%) as a light yellow solid:
1H NMR (300 MHz, CDCl3) d 8.34 (d, J = 2.0 Hz, 1H),
8.06 (dd, J = 8.4, 2.0 Hz, 1H), 7.52 (d, J = 8.4 Hz, 1H),
7.28 (d, J = 2.1 Hz, 1H), 7.17 (dd, J = 8.4, 2.4 Hz, 1H),
6.73 (d, J = 8.5 Hz, 1H), 4.41 (s, 2H), 2.20 (s, 3H),
1.61 (s, 6H); MS (ES) m/z: 469 (M+H+). Anal. calcd
for C20H18Cl2N2O3S:20.10H2O: C, 50.98; H, 3.89; N,
5.94. Found: C, 50.60; H, 3.53; N, 5.71.
J = 1.7 Hz, 1H), 8.06 (dd, J = 7.9, 1.7 Hz, 1H), 7.33 (d,
J = 8.0 Hz, 1H), 5.17 (s, 2H), 2.44 (s, 3H); MS (ES) m/
z: 241 (M+H+).
5.1.20. General procedure for the synthesis of 30, 35, 40,
45, 50, and 55.
5.1.20.1. 2-{4-[3-(3-Fluoro-4-trifluoromethyl-phenyl)-
[1,2,4]thiadiazol-5-ylmethylsulfanyl]-2-methyl-phenoxy}-
2-methyl-propionic acid (30). To a solution of 28
(684 mg, 2.46 mmol) in CH2Cl2 (10 mL) were added car-
bon tetrabromide (896 mg, 2.70 mmol) and triphenyl-
phosphine (707 mg, 2.70 mmol). The mixture was
stirred at 0 ꢁC for 1 h and room temperature for 1 h,
concentrated, and purified by column chromatography
to give 554 mg (66%) of the bromide as a white solid:
1H NMR (300 MHz, CDCl3) d 8.17 (d, J = 8.4 Hz,
1H), 8.12 (d, J = 11.5 Hz, 1H), 7.72 (t, J = 7.6 Hz,
1H), 4.82 (s, 2H); MS (ES) m/z: 343 (M+H+). To a mix-
ture of the bromide (75 mg, 0.22 mmol) and 2-(4-mer-
capto-2-methyl-phenoxy)-2-methyl-propionic acid ethyl
ester 29 (52 mg, 0.21 mmol) in CH3CN (1.5 mL) and
5.1.20.4. 2-{4-[3-(2,4-Dichloro-phenyl)-[1,2,4]thiadi-
azol-5-ylmethylsulfanyl]-2-methyl-phenoxy}-2-methyl-pro-
pionic acid (45). Using 44 and following the procedure
as in the preparation of 30 gave 45 (62%) as a light
1
yellow solid: H NMR (300 MHz, CDCl3) d 7.85 (d,
J = 8.4 Hz, 1H), 7.52 (d, J = 2.0 Hz, 1H), 7.33 (dd,
J = 8.4, 2.1 Hz, 1H), 7.28 (d, J = 2.2 Hz, 1H), 7.19
(dd, J = 8.4, 2.4 Hz, 1H), 6.73 (d, J = 8.5 Hz, 1H),
4.44 (s, 2H), 2.20 (s, 3H), 1.61 (s, 6H); MS (ES) m/
z: 469 (M+H+). Anal. calcd for C20H18Cl2N2O3S2:

L. Shen et al. / Bioorg. Med. Chem. 16 (2008) 3321–3341
3337
C, 51.17; H, 3.87; N, 5.97. Found: C, 50.80; H, 3.53;
N, 5.72.
5.1.23. General procedure for the synthesis of 61 and 65.
5.1.23.1. 3-Bromomethyl-5-(4-chloro-phenyl)-[1,2,4]thia-
diazole (61). To a solution of 60 (558 mg, 2.46 mmol) in
CH2Cl2 (10 mL) were added carbon tetrabromide
(896 mg, 2.70 mmol) and triphenylphosphine (707 mg,
2.70 mmol). The reaction mixture was stirred at 0 ꢁC
for 1 h and room temperature for 1 h, concentrated,
and purified by column chromatography to give
5.1.20.5. 2-{4-[3-(3,4-Dimethyl-phenyl)-[1,2,4]thiadi-
azol-5-ylmethylsulfanyl]-2-methyl-phenoxy}-2-methyl-pro-
pionic acid (50). Using 49 and following the procedure as
in the preparation of 30 gave 50 (67%) as a light yellow
1
solid: H NMR (400 MHz, CDCl3) d 8.00 (s, 1H), 7.94
1
(dd, J = 7.8, 1.5 Hz, 1H), 7.28 (d, J = 1.9 Hz, 1H), 7.21
(d, J = 7.8 Hz, 1H), 7.17 (dd, J = 8.5, 2.2 Hz, 1H), 6.71
(d, J = 8.5 Hz, 1H), 4.42 (s, 2H), 2.32 (s, 3H), 2.31 (s,
3H), 2.19 (s, 3H), 1.59 (s, 6H); MS (ES) m/z: 429
(M+H+). HRMS calcd for C22H24N2O3S2: 428.1228.
Found: 428.1215.
599 mg (84%) of the bromide 61 as a white solid: H
NMR (300 MHz, CDCl3) d 7.92 (d, J = 8.6 Hz, 2H),
7.49 (d, J = 8.6 Hz, 2H), 4.68 (s, 2H); MS (ES) m/z:
289 (M+H+).
5.1.23.2. 3-Bromomethyl-5-(3,4-dichloro-phenyl)-[1,2,4]-
thiadiazole (65). Using 64 and following the procedure
as in the preparation of 61 gave 65 (83%) as a white
5.1.20.6. 2-{4-[3-(3-Chloro-4-methyl-phenyl)-[1,2,4]thia-
diazol-5-ylmethylsulfanyl]-2-methyl-phenoxy}-2-methyl-pro-
pionic acid (55). Using 54 and following the procedure as
in the preparation of 30 gave 55 (55%) as a light yellow
solid: 1H NMR (300 MHz, CDCl3) d 8.21 (d, J = 1.7 Hz,
1H), 8.01 (dd, J = 8.0, 1.7 Hz, 1H), 7.30 (d, J = 8.2 Hz,
1H), 7.27 (s, 1H), 7.17 (dd, J = 8.5, 2.2 Hz, 1H), 6.72
(d, J = 8.5 Hz, 1H), 4.41 (s, 2H), 2.42 (s, 3H), 2.19 (s,
3H), 1.59 (s, 6H). HRMS calcd for C21H21ClN2O3S2:
448.0682. Found: 448.0713.
solid: 1H NMR (300 MHz, CDCl3)
d 8.03 (d,
J = 2.1 Hz, 1H), 7.71 (dd, J = 8.3, 2.1 Hz, 1H), 7.52 (d,
J = 8.3 Hz, 1H), 4.61 (s, 2H); MS (ES) m/z: 349
(M+Na+).
5.1.24. General procedure for the synthesis of 62 and 66.
5.1.24.1. 2-{4-[5-(4-Chloro-phenyl)-[1,2,4]thiadiazol-3-
ylmethylsulfanyl]-2-methyl-phenoxy}-2-methyl-propionic
acid (62). Using 61 and following the procedure as in
the preparation of 30 gave 62 (46%) as an oily solid:
1H NMR (300 MHz, CDCl3) d 7.87 (m, 2H), 7.47 (m,
2H), 7.27 (m, 1H), 7.18 (dd, J = 8.4, 2.3 Hz, 1H),
6.73 (d, J = 8.5 Hz, 1H), 4.34 (s, 2H), 2.19 (s, 3H),
1.60 (s, 6H); MS (ES) m/z: 435 (M+H+). HRMS
calcd for C20H19ClN2O3S2: 434.0526. Found:
434.0618.
5.1.21. General procedure for the synthesis of 59 and 63.
5.1.21.1. 5-(4-Chlorophenyl)-[1,2,4]thiadiazole-3-car-
boxylic acid ethyl ester (59). A mixture of 58 (1.77 g,
10.1 mmol) and 4-chlorobenzonitrile (6.97 g, 50.5
mmol) in 1,2-dichlorobenzene (10 mL) was heated at
160 ꢁC for 4 days. After cooling down to room tem-
perature, the reaction mixture was concentrated and
the product was purified by column chromatography
to provide 353 mg (13%) of 59 as light brown crystals:
1H NMR (400 MHz, CDCl3) d 7.99 (d, J = 8.6 Hz,
2H), 7.51 (d, J = 8.6 Hz, 2H), 4.55 (q, J = 7.1 Hz,
2H), 1.49 (t, J = 7.1 Hz, 3H); MS (ES) m/z: 269
(M+H+).
5.1.24.2. 2-{4-[5-(3,4-Dichloro-phenyl)-[1,2,4]thiadiazol-
3-ylmethylsulfanyl]-2-methyl-phenoxy}-2-methyl-propionic
acid (66). Using 65 and following the procedure as in the
1
preparation of 30 gave 66 (68%) as an oily solid: H
NMR (400 MHz, CDCl3) d 8.06 (d, J = 2.0 Hz, 1H),
7.73 (dd, J = 8.3, 2.0 Hz, 1H), 7.57 (d, J = 8.3 Hz, 1H),
7.26 (m, 1H), 7.16 (m, 1H), 6.72 (d, J = 8.4 Hz, 1H),
4.34 (s, 2H), 2.20 (s, 3H), 1.60 (s, 6H); MS (ES) m/z:
469 (M+H+). Anal. calcd for C20H18Cl2N2O3S2: C,
51.17; H, 3.87; N, 5.97. Found: C, 51.06; H, 3.68; N,
5.63.
5.1.21.2. 5-(3,4-Dichloro-phenyl)-[1,2,4]thiadiazole-3-
carboxylic acid ethyl ester (63). Using 3,4-dic-
hlorobenzonitrile and following the procedure as in
the preparation of 59 gave 63 (31%) as an off-white so-
1
lid: H NMR (300 MHz, CDCl3) d 8.19 (d, J = 2.0 Hz,
1H), 7.85 (dd, J = 8.3, 2.1 Hz, 1H), 7.61 (d, J = 8.4 Hz,
1H), 4.55 (q, J = 7.1 Hz, 2H), 1.49 (t, J = 7.1 Hz, 3H);
MS (ES) m/z: 305 (M+H+).
5.1.25. General procedure for the synthesis of 67 and 72.
5.1.25.1. [3-(4-Trifluoromethyl-phenyl)-[1,2,4]thiadiazol-
5-yl]-acetic acid ethyl ester (67). A reaction mixture of 7
(0.995 g, 4.03 mmol) and ethyl cyanoacetate (1.8 g,
16.1 mmol) in 1,2-dichlorobenzene (18 mL) was heated
at 160 ꢁC for 20 h. After cooling down to room temper-
ature, the reaction mixture was purified by column chro-
matography to give 153 mg (12%) of 67 as a white solid:
1H NMR (300 MHz, CDCl3) d 8.41 (d, J = 8.1 Hz, 2H),
7.73 (d, J = 8.2 Hz, 2H), 4.34 (q, J = 7.2 Hz, 2H), 4.27
(s, 2H), 1.37 (t, J = 7.1 Hz, 3H); MS (ES) m/z: 317
(M+H+).
5.1.22. General procedure for the synthesis of 60 and 64.
5.1.22.1. [5-(4-Chloro-phenyl)-[1,2,4]thiadiazol-3-yl]-
methanol (60). Using 59 and following the procedure
as in the preparation of 28 gave 60 (97%) as a yellow so-
1
lid: H NMR (400 MHz, CDCl3) d 7.91 (d, J = 8.6 Hz,
2H), 7.49 (d, J = 8.6 Hz, 2H), 4.98 (s, 2H); MS (ES)
m/z: 227 (M+H+).
5.1.22.2. [5-(3,4-Dichloro-phenyl)-[1,2,4]thiadiazol-3-
yl]-methanol (64). Using 63 and following the procedure
as in the preparation of 28 gave 64 (84%) as a white so-
5.1.25.2. [3-(3,4-Dichloro-phenyl)-[1,2,4]thiadiazol-5-
yl]-acetic acid ethyl ester (72). Using 71 and following
the procedure as in the preparation of 67 gave 72
(14%) as a white solid: H NMR (300 MHz, CDCl3) d
8.39 (d, J = 1.9 Hz, 1H), 8.11 (dd, J = 8.4 Hz, 2.0 Hz,
1
lid: H NMR (300 MHz, CDCl3) d 8.09 (d, J = 2.1 Hz,
1H), 7.78 (dd, J = 8.4, 2.1 Hz, 1H), 7.59 (d, J = 8.4 Hz,
1
1H), 4.99 (s, 2H); MS (ES) m/z: 261 (M+H+).

3338
L. Shen et al. / Bioorg. Med. Chem. 16 (2008) 3321–3341
1H), 7.53 (d, J = 8.4 Hz, 1H), 4.33 (q, J = 7.1 Hz, 2H),
4.25 (s, 2H), 1.36 (t, J = 7.1 Hz, 3H); MS (ES) m/z:
317 (M+H+).
5.1.29. General procedure for the synthesis of 79, 80, 81,
82, and 83.
5.1.29.1. 2-{4-[3-(3-Fluoro-4-trifluoromethyl-phenyl)-
[1,2,4]thiadiazol-5-ylmethoxy]-2-methyl-phenoxy}-2-meth-
ylpropionic acid (79). Using 28 and 78, and following the
procedure as in the preparation of 30 gave 79 (40%) as a
5.1.26. General procedure for the synthesis of 68 and 73.
5.1.26.1. 2-[3-(4-Trifluoromethyl-phenyl)-[1,2,4]thia-
diazol-5-yl]-ethanol (68). To a solution of 67 (180 mg,
0.57 mmol) in EtOH-THF (8–2 mL) at room tempera-
ture was added NaBH4 (136 mg, 3.6 mmol). After stir-
ring for 20 h, a few drops of water were added to
quench excess of hydride. The solvent was evaporated,
and the residue was partitioned between CH2Cl2 and
water. The organic phase was dried and concentrated
to give the crude alcohol 68: MS (ES) m/z: 275
(M+H+).
1
colorless oil: H NMR (400 MHz, DMSO-d6) d 8.18–
8.26 (m, 2H), 7.99–8.03 (t, J = 7.9 Hz, 1H), 7.00 (d,
J = 3.0 Hz, 1H), 6.87–6.90 (m, 1H), 6.74 (d,
J = 8.9 Hz, 1H), 5.66 (s, 2H), 2.17 (s, 3H), 1.46 (s,
6H); MS (ES) m/z: 471 (M+H+); HRMS calcd for
C21H18F4N2O4S: 470.0923. Found: 470.0914.
5.1.29.2. 2-Methyl-2-{2-methyl-4-[3-(4-trifluorometh-
oxy-phenyl)-[1,2,4]thiadiazol-5-ylmethoxy]-phenoxy}-pro-
pionic acid (80). Using 34 and 78, and following the
procedure as in the preparation of 30 gave 80 (51%) as
5.1.26.2. 2-[3-(3,4-Dichloro-phenyl)-[1,2,4]thiadiazol-
5-yl]-ethanol (73). Using 72 and following the procedure
as in the preparation of 68 gave 73 (64%) as a white so-
1
a white solid: H NMR (300 MHz, CD3OD) d 8.47 (d,
J = 2.0 Hz, 1H), 8.32 (dd, J = 8.6, 2.1 Hz, 1H), 7.58
(dd, J = 8.6, 1.4 Hz, 1H), 6.94 (s, 1H), 6.83–6.82 (m,
2H), 5.55 (s, 2H), 2.23 (s, 3H), 1.53 (s, 6H); MS (ES)
m/z: 503 (M+H+). Anal. calcd for C21H18ClF3N2O5S:
C, 50.16; H, 3.61; N, 5.57. Found: C, 50.16; H, 3.33;
N, 5.43.
1
lid: H NMR (300 MHz, CDCl3) d 8.39 (d, J = 2.0 Hz,
1H), 8.11 (dd, J = 8.4 Hz, 2.0 Hz, 1H), 4.11 (t,
J = 5.6 Hz, 2H), 3.41 (t, J = 5.6 Hz, 2H).
5.1.27. General procedure for the synthesis of 69 and 74.
5.1.27.1. 5-(2-Bromo-ethyl)-3-(4-trifluoromethyl-phe-
nyl)-[1,2,4]thiadiazole (69). Using 68 and following the
procedure as in the preparation of 61 gave 69 (44%) as
5.1.29.3. 2-{4-[3-(3,4-Dichloro-phenyl)-[1,2,4]thiadi-
azol-5-ylmethoxy]-2-methyl-phenoxy}-2-methyl-propionic
acid (81). Using 39 and 78, and following the procedure
as in the preparation of 30 gave 81 (15%) as a white so-
1
a white solid: H NMR (300 MHz, CDCl3) d 8.42 (d,
J = 8.1 Hz, 2H), 7.74 (d, J = 8.2 Hz, 2H), 3.84–3.80
(m, 2H), 3.76–3.71 (m, 2H).
1
lid: H NMR (300 MHz, CD3OD) d 8.40 (s, 1H), 8.19
(d, J = 8.4 Hz, 1H), 7.66 (d, J = 8.4 Hz, 1H), 6.92 (d,
J = 2.6 Hz, 1H), 6.87 (d, J = 8.5 Hz, 1H), 6.79 (d,
J = 8.4 Hz, 1H), 5.53 (s, 2H), 2.23 (s, 3H), 1.52 (s,
6H); MS (ES) m/z: 453 (M+H+). HRMS calcd for
C20H18Cl2N2O4S: 452.0364. Found: 452.0385.
5.1.27.2. 5-(2-Bromo-ethyl)-3-(3,4-dichloro-phenyl)-
[1,2,4]thiadiazole (74). Using 73 and following the proce-
dure as in the preparation of 61 gave 74 (86%) as a white
solid: 1H NMR (300 MHz, CDCl3) d 8.39 (d, J = 2.0 Hz,
1H), 8.11 (dd, J = 8.4 Hz, 2.0 Hz, 1H), 3.83–3.78 (m,
2H), 3.73–3.69 (m, 2H).
5.1.29.4. 2-{4-[3-(3,4-Dimethyl-phenyl)-[1,2,4]thiadi-
azol-5-ylmethoxy]-2-methyl-phenoxy}-2-methyl-propionic
acid (82). Using 49 and 78, and following the procedure
as in the preparation of 30 gave 82 (17%) as a white so-
lid: 1H NMR (300 MHz, CDCl3) d 8.07 (s, 1H), 8.02 (d,
J = 7.7 Hz, 1H), 7.23 (s, 1H), 6.85–6.91 (m, 2H), 6.75–
6.79 (dd, J = 8.7, 3.2 Hz, 1H), 5.48 (s, 2H), 2.34 (d,
J = 7.0 Hz, 6H), 2.26 (s, 3H), 1.57 (s, 6H); MS (ES)
m/z: 413 (M+H+); HRMS calcd for C22H24N2O4S:
413.1535. Found: 413.1533.
5.1.28. General procedure for the synthesis of 70 and 75.
2-Methyl-2-(2-methyl-4-{2-[3-(4-trifluoro-
5.1.28.1.
methyl-phenyl)-[1,2,4]thiadiazol-5-yl]-ethylsulfanyl}-phen-
oxy)-propionic acid (70). Using 69 and following the
procedure as in the preparation of 30 gave 70 (40%) as
1
a white solid: H NMR (300 MHz, CD3OD) d 8.42 (d,
J = 8.1 Hz, 2H), 7.78 (d, J = 8.2 Hz, 2H), 7.23 (d,
J = 1.8 Hz, 1H), 7.17 (d, J = 8.3 Hz, 1H), 6.76 (d,
J = 8.4 Hz, 1H), 3.43 (t, J = 6.1 Hz, 2H), 3.30 (m, 2H),
2.16 (s, 3H), 1.54 (s, 6H); MS (ES) m/z: 481 (MꢀH+).
HRMS calcd for C21H20Cl2N2O3S2: 482.0946. Found:
482.0952.
5.1.29.5. 2-{4-[3-(3-Chloro-4-methyl-phenyl)-[1,2,4]thia-
diazol-5-ylmethoxy]-2-methyl-phenoxy}-2-methyl-propi-
onic acid (83). Using 54 and 78, and following the
procedure as in the preparation of 30 gave 83 (26%) as
1
5.1.28.2. 2-(4-{2-[3-(3,4-Dichloro-phenyl)-[1,2,4]thia-
diazol-5-yl]-ethylsulfanyl}-2-methyl phenoxy)-2-methyl-
propionic acid (75). Using 74 and following the proce-
dure as in the preparation of 30 gave 75 (35%) as a
white solid: 1H NMR (300 MHz, CD3OD) d 8.29
(d, J = 1.9 Hz, 1H), 8.07 (dd, J = 8.4, 1.9 Hz, 1H),
7.58 (d, J = 8.4 Hz, 1H), 7.23 (d, J = 1.8 Hz, 1H),
7.15 (dd, J = 8.3, 1.9 Hz, 1H), 6.70 (d, J = 8.4 Hz,
1H), 3.38 (t, J = 6.0 Hz, 2H), 3.31 (m, 2H), 2.15 (s,
3H), 1.56 (s, 6H); MS (ES) m/z: 483 (M+H+). HRMS
calcd for C21H20Cl2N2O3S2: 482.0292. Found:
482.0302.
a white solid: H NMR (400 MHz, CD3OD) d 6.72 (s,
1H), 6.57 (d, J = 7.9 Hz, 1H), 5.89 (d, J = 7.7 Hz, 1H),
5.40 (s, 1H), 5.26–5.33 (m, 2H), 4.0 (d, J = 1.5 Hz,
2H), 1.77 (s, 6H), 0.90 (s, 3H), 0.70 (s, 3H); MS (ES)
m/z: 433 (M+H+). Anal. calcd for C21H21ClN2O4S: C,
58.26; H, 4.89; N, 6.47. Found: C, 57.85; H, 4.86; N,
6.46.
5.1.30. 4-[3-(4-Trifluoromethyl-phenyl)-[1,2,4]thiadiazol-
5-ylmethylsulfanyl]-phenol (85). To a solution of 84
(795 mg, 3.06 mmol) in CH2Cl2 (30 mL) at 0 ꢁC were
added methanesulfonyl chloride (518 mg, 4.52 mmol)

L. Shen et al. / Bioorg. Med. Chem. 16 (2008) 3321–3341
3339
and triethylamine (617 mg, 6.11 mmol). The mixture
was stirred at room temperature for 1 h and then parti-
tioned between water and CH2Cl2 (80 mL). The organic
layer was washed with brine, dried, concentrated, and
column chromatographed (EtOAc/hexane) to provide
859 mg (83%) of the mesylate as a white solid. A mixture
of the mesylate (210 mg, 0.621 mmol) and 4-mercap-
tophenol (113 mg, 0.897 mmol) in CH3CN (8 mL) was
degassed under N2 for about 10 min. After the addition
of Cs2CO3 (242 mg, 0.742 mmol), the mixture was stir-
red at room temperature for 40 min, concentrated, and
purified by column chromatography (EtOAc/hexane)
to give 228 mg (100%) of 85 as a white solid: 1H
NMR (300 MHz, CDCl3) d 8.35 (d, J = 8.1 Hz, 2H),
7.71 (d, J = 8.3 Hz, 2H), 7.36 (d, J = 8.7 Hz, 2H), 6.78
(d, J = 8.7 Hz, 2H), 4.97 (s, 1H), 4.40 (s, 2H); MS (ES)
m/z: 369 (M+H+).
d 8.34 (d, J = 2.0 Hz, 1H), 8.06 (dd, J = 8.4, 2.0 Hz,
1H), 7.52 (d, J = 8.4 Hz, 1H), 7.28–7.26 (m, 1H), 7.17
(dd, J = 8.4, 2.3 Hz, 1H), 6.50 (d, J = 8.5 Hz, 1H), 4.39
(s, 2H), 2.36–2.27 (m, 2H), 2.21–2.15 (m, 2H), 2.17 (s,
3H), 1.82–1.79 (m, 4H); MS (ES) m/z: 495 (M+H+).
HRMS calcd for C22H20Cl2N2O3S2: 494.0292. Found:
494.0311.
5.2. Biology
5.2.1. PPAR assays. All cell culture and co-transfection
reagents were purchased from Invitrogen (Carlsbad,
CA) with the exception of the Charcoal-treated FBS
(Hyclone; Logan, UT). HEK293 cells were grown in
DMEM/F-12 medium supplemented with 10%FBS and
glutamine. The Steady-Glo Luciferase Assay Kit (Pro-
mega; Madison, WI) was used for measuring luciferase
reporter activity. For a 175 cm2 tissue culture flask,
100 · 105 of HEK293 cells were seeded in the growth
medium and incubated at 37 ꢁC in 5% CO2 incubator
until the cells were 80% confluent. The cells were then
co-transfected with DNA constructs containing the li-
gand-binding domains of either PPARa, c, or d-Gal4
chimeric receptors and Gal4-luciferase reporter using
DMRIE-C transfection reagent and OPTI-MEM re-
duced serum medium according to the manufacturer’s
instructions. On the following day, the DNA-containing
medium was replaced with 30 mL of 5% Charcoal-trea-
ted FBS growth medium. After 6 h, the cells were re-pla-
ted at a density of 50,000 cells/well in 96-well plates and
incubated overnight at 37 ꢁC in a 5% CO2 incubator.
Cells were treated with 5 lL of compound or vehicle
(0.1% DMSO) solution and incubated for 24 h at
37 ꢁC in 5% CO2 incubator. The next day, luciferase
activity was measured with the Steady-Glo Luciferase
Assay kit according to the manufacturer’s instructions.
5.1.31. 2-Methyl-2-{4-[3-(4-trifluoromethyl-phenyl)-[1,2,4]-
thiadiazol-5-ylmethylsulfanyl]-phenoxy}-propionic acid
(86). To a three-necked flask containing NaH (36 mg,
0.90 mmol; 60% in mineral oil) was added a solution
of 85 (220 mg, 0.598 mmol) in THF. To the mixture at
40 ꢁC was added tert-butyl 2-bromoisobutyrate
(287 mg, 1.29 mmol). After heating at 70 ꢁC for 2 h,
more
tert-butyl
2-bromoisobutyrate
(215 mg,
0.970 mmol) was added and the heating was continued
overnight. The reaction mixture was quenched with
water (0.1 mL), concentrated, and chromatographed
(EtOAc/hexane) to give 81 mg (27%) of 86-tert-butyl es-
ter:1H NMR (300 MHz, CDCl3) d 8.35 (d, J = 8.2 Hz,
2H), 7.71 (d, J = 8.2 Hz, 2H), 7.34 (d, J = 8.8 Hz, 2H),
6.78 (d, J = 8.7 Hz, 2H), 4.42 (s, 2H), 1.55 (s, 6H),
1.38 (s, 9H); MS (ES) m/z: 511 (M+H+). The mixture
of 86-tert-butyl ester (80 mg, 0.16 mmol) in CH2Cl2
(1.5 mL) and trifluoroacetic acid (0.5 mL) was stirred
at room temperature for 1.5 h, concentrated, and col-
umn chromatographed (EtOAc/hexane, CH2Cl2/
MeOH) to give 40 mg (56%) of 86 as a light yellow solid:
1H NMR (300 MHz, CDCl3) d 8.34 (d, J = 8.0 Hz, 2H),
7.71 (d, J = 8.2 Hz, 2H), 7.37 (d, J = 8.8 Hz, 2H), 6.87
(d, J = 8.8 Hz, 2H), 4.45 (s, 2H), 1.59 (s, 6H); MS (ES)
m/z: 453 (MꢀH+). HRMS calcd for C20H17F3N2O3S2:
454.0633. Found: 454.0658.
5.2.2. Induction of selected genes. Human peripheral
blood lymphocytes were freshly prepared (ABS Inc.,
Wilmington, DE) and total RNA was isolated from
the cells following manufacturer’s instruction (Trizon
method, Invitrogen Inc., CA). PCR primers and fluores-
cent probes (TaqMan probe) were designed using the
software PrimerDesign (Applied Biosystems, Foster
City, Ca). The sequences of the primers and TaqMan
probe for PDHK4 (GenBank Accession No.: U54617)
are 50-TGCATTTTTGCGACAAGAATTG (forward),
50-TTGGGTCGGGAGGATATCAA (reverse), and
5-FAM-CTGTGAGACTCGCCAACATTCTGAAGG
A-TAMRA-3. The sequences of the primers and Taq-
Man probe for CPT1 (GenBank Accession No.:
NM_001876) are 50-CATTCCTTCC CATTCGTAGC
(forward), 50-AGCTGCACAA AGGCGTCTG (re-
verse), and 5-FAM-AGGAATCATC AAGAAATGTC
GCACGAGC-TAMRA-3 (TaqMan). They were syn-
thesized by Keystone Labs (Camarillo, CA). Reverse
transcription (RT) was conducted following manufac-
turer’s instruction using TaqMan Reverse Transcription
Reagents (Applied BioSystems, CA). The RT reaction
mixture was 20 lL containing 1· RT buffer, 5.5 mM
MgCl2, 0.5 mM dNTP, 2.5 lM of random hexamer,
1 U of RNase Inhibitor, 1 U of MultisScribe RT En-
zyme, and 0.5 lg of total RNA. The reaction was carried
5.1.32. 1-{4-[3-(3,4-Dichloro-phenyl)-[1,2,4]thiadiazol-5-
ylmethylsulfanyl]-2-methyl-phenoxy}-cyclopentanecarb-
oxylic acid (89). To a mixture of the bromide of 39 (39A)
(58 mg, 0.18 mmol) and 88 (76 mg, 0.18 mmol) in THF
(0.5 mL) at 0 ꢁC was added 1.0 M tetrabutylammonium
fluoride (0.18 mL, 0.18 mmol) in THF dropwise. After
stirring at the same temperature for 15 min, the mixture
was concentrated and the residue was purified by col-
umn chromatography to afford 88 mg (93%) of 89-
methyl ester as a yellow oil: 1H NMR (300 MHz,
CDCl3) d 8.35 (d, J = 2.0 Hz, 1H), 8.07 (dd, J = 8.4,
2.0 Hz, 1H), 7.53 (d, J = 8.4 Hz, 1H), 7.26 (s, 1H),
7.13 (dd, J = 8.5, 2.4 Hz, 1H), 6.37 (d, J = 8.5 Hz, 1H),
4.38 (s, 2H), 3.70 (s, 3H), 2.28–2.12 (m, 4H), 2.18 (s,
3H), 1.81–1.76 (m, 4H); MS (ES) m/z: 509 (M+H+).
Using 89-methyl ester and following the same base
hydrolysis procedure as in the preparation of 30 gave
1
89 (35%) as a yellow oil: H NMR (400 MHz, CDCl3)

3340
L. Shen et al. / Bioorg. Med. Chem. 16 (2008) 3321–3341
out at 48 ꢁC for 30 min, then 95 ꢁC for 5 min and finally
at 4 ꢁC. The reaction volume was extended into 62.5 lL
and 5 lL of the RT (equivalent to 40 ng of total RNA)
was used for PCR amplification. Each reaction was car-
ried out in a total volume of 25 lL containing 5 lL of
RT, 1· Master Mix (reaction buffer, dNTP, Polymerase,
Applied Biosystems), 0.2 lM of forward, reverse prim-
ers, and TaqMan probe. The reaction was performed
in a DNA Sequence Detector (Model 7900, Applied Bio-
Systems) with the condition of 50 ꢁC for 2 min, 94 ꢁC for
10 min, followed by 40 cycles of 94 ꢁC for 15 s and 59 ꢁC
for 1 min. For real time PCR quantitation, the measure-
ment of human 18S ribosomal RNA was used as an
internal control for normalization of measuring and
loading errors. PCR data were collected by Ct value
(the cycle number at which logarithmic PCR plots cross
a calculated threshold line) according to the manufac-
turer’s guidelines and the value was used to determine
the DCt (Ct of the target gene minus the Ct of 18S ribo-
somal RNA controls). Relative mRNA level was calcu-
Serum levels of triglycerides, total cholesterol, free fatty
acids, AST, and ALT were measured by COBAS Mira
Plus blood chemistry analyzer (Roche Diagnostic Sys-
tems, Indianapolis, IN).
5.2.6. Pharmacokinetic assay. Rats are normally dosed
intravenously (iv) at levels of 1–3 mg/kg and by oral ga-
vage at a level of 3–10 mg/kg with drug candidates.
Drug compound is typically formulated for iv dosing
as a solution in 10% w/v Solutol in 5% dextrose in sterile
water vehicle (D5W). Drug compound is typically for-
mulated for oral dosing as a uniform suspension in
0.5% methylcellulose vehicle. Blood samples (0.5 mL)
are collected into heparinized tubes post-dose via orbital
sinus puncture. Blood samples are centrifuged for cell
removal, and precisely 200 lL of plasma supernatant
is then transferred to a clean vial, placed on dry ice,
and subsequently stored in a ꢀ70 ꢁC freezer prior to
analysis. Plasma samples are normally prepared as fol-
lows. Four hundred microliters of acetonitrile contain-
ing internal standard is added to 200 lL of plasma to
precipitate proteins. Samples are centrifuged at 5000g
for 3 min and supernatant removed for analysis by
LC–MS–MS. Calibration standards are prepared by
adding appropriate volumes of stock solution directly
into plasma and treated identically to collected plasma
samples. Calibration standards are typically prepared
in the range of 0.1–10 mM for quantitation. LC–MS–
MS analysis was performed using either multiple reac-
tion or selected ion monitoring for detection of charac-
teristic ions for each drug candidate and internal
standard used is Propranolol.
lated using the equation 2ꢀDDCt
.
5.2.3. In vivo rat model. Male Sprague–Dawley rats
weighing between 275 and 325 g at the start of treatment
were used (ACE Animals, Inc., Boyertown, PA). All but
six rats were fed an atherogenic (high cholesterol) diet
(C13002, Research Diets, New Brunswick, NJ) for 6
days before starting treatment. The remaining six rats
received normal chow (Purina 5001) and were also oral-
ly dosed with vehicle during the treatment period. The
diets continued during the treatment period. The com-
pound was formulated in 0.5% MethocelTM, which was
also the vehicle for the control animals and the animals
were orally dosed at a volume of 10 mL/kg once daily
for 8 days. On day 9, the animals were anesthetized with
70% CO2/30% O2 and blood was obtained from the ret-
ro-orbital sinus. Serum was prepared and the lipid
parameters were measured using a COBAS analyzer
(Roche Diagnostics). The animals were then sacrificed,
the livers were removed (from six animals per group)
and weighed.
5.3. Computational details
PPARa/az242 (PDB Accession 1I7G)33 and PPARd/
gw2433 (PDB Accession 1GWX)34 were used as protein
coordinates in the Glide docking studies. All ligand ini-
tial 3D coordinates were generated with an in-house
implementation of a Stochastic Proximity Embedding
(SPE) algorithm,35 followed by minimization using the
MMFF94s force-field.36–39 Explicit hydrogens were
added to the proteins using the ‘all-atom with no lone
pair treatment’ followed by restrained minimization, as
implemented in PPREP utility of Glide4.5.27,28 Various
Coulombic and van der Waals grids were generated
with, and without, hydrogen bond constraints to
Y473d and Y464a. Compound 21 was then allowed to
flexibly dock into all receptors using standard precision
(SP) or extra precision (XP) Glide. GlideScore, the de-
fault internal docking scoring function in Glide, evalu-
ated the interaction energies of ligand with the
Coulombic and van der Waals grids. Then the best scor-
ing poses are minimized using the OPLS-AA force-
field40 on precomputed van der Waals and electrostatic
grids.
5.2.4. In vivo hApoA1 and ob/ob mouse models. Male
hAPOA1 transgenic mice and Female ob/ob mice
(C57BL/6J-Lepob) were purchased from Jackson Labs
(Bar Harbor, ME). Compound 40 in 0.5% methylcellu-
lose suspension was dosed orally for 7 or 11 days,
respectively. Serum levels of HDL-C, triglyceride, free
fatty acids, and/or glucose were collected under fed con-
ditions measured using a COBAS Mira Plus blood
chemistry analyzer (Roche Diagnostic Systems, India-
napolis, IN). Statistical analysis was performed using
the program Prism (Graphpad, Monrovia, CA) and
with one-way analysis of variance and Dunnett’s multi-
ple comparison test.
5.2.5. Obese Beagle dog model. Female obese Beagle
dogs (retired breeders) (Marshall Bioresources, North
Rose, NY) were maintained on a regular canine diet
and food was supplied once daily. Compound 40 in a
dose range of 0.3–10 mg/kg was orally administered dai-
ly for continuous 7 days, approximately 3 h before feed-
ing. Blood samples were collected 6 h after the last dose.
Acknowledgment
We thank Chemical and Biological Support Team of
J&JPRD for the pharmacokinetic study of compound
40.

L. Shen et al. / Bioorg. Med. Chem. 16 (2008) 3321–3341
3341
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