Synthesis and identification of [1,2,4]thiadiazole derivatives as a new series of potent and orally active dual agonists of peroxisome proliferator-activated receptors α and δ

Article abstract of DOI:10.1021/jm070511x

Cardiovascular disease is the most common cause of morbidity and mortality in developed nations. To effectively target dyslipidemia to reduce the risk of cardiovascular disease, it may be beneficial to activate the peroxisome proliferator-activated recept

Full text of DOI:10.1021/jm070511x

3954
J. Med. Chem. 2007, 50, 3954-3963
Synthesis and Identification of [1,2,4]Thiadiazole Derivatives as a New Series of Potent and
Orally Active Dual Agonists of Peroxisome Proliferator-Activated Receptors r and δ
Lan Shen, Yan Zhang, Aihua Wang, Ellen Sieber-McMaster, Xiaoli Chen, Patricia Pelton, Jun 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, New Jersey 08512
ReceiVed May 2, 2007
Cardiovascular disease is the most common cause of morbidity and mortality in developed nations. To
effectively target dyslipidemia to reduce the risk of cardiovascular disease, it may be beneficial to activate
the peroxisome proliferator-activated receptors (PPARs) PPARR and PPARδ simultaneously through a single
molecule. Replacement of the methylthiazole of 5 (the PPARδ selective agonist) with [1,2,4]thiadiazole
gave compound 13, which unexpectedly displayed submicromolar potency as a partial agonist at PPARR in
addition to the high potency at PPARδ. Optimization of 13 led to the identification of 24 as a potent and
selective PPARR/δ dual agonist. Compound 24 and its close analogs represent a new series of PPARR/δ
dual agonists. The high potency, significant gene induction, excellent PK profiles, and good in vivo efficacies
in three animal models may render compound 24 as a valuable pharmacological tool in elucidating the
complex roles of PPARR/δ dual agonists and as a potential treatment of the metabolic syndrome.
Introduction
regulator for insulin sensitivity, and two PPARγ agonists,
rosiglitazone 3 and pioglitazone 4, have been used for the
clinical treatment of type 2 diabetes since 1999.10 In contrast,
the biological role of PPARδ and the potential clinical utility
of its ligand were unclear, in part, due to its broad tissue
expression11 and lack of good chemical tools to study its
pharmacology. Recently, a potent and selective PPARδ agonist,
5 (GW501516, phase II clinical trial), was developed and shown
to increase plasma HDL-C levels together with decreasing
LDL-C and triglycerides in obese and dyslipidemic rhesus
monkeys.12 In addition, while one study supported an athero-
protective effect of PPARδ agonists in LDLR-/- mice,13 other
data suggested that PPARδ activation may attenuate the
metabolic syndrome, including obesity.14 To effectively target
dyslipidemia to reduce the risk of cardiovascular diseases, it
may be beneficial to activate PPARR and PPARδ simulta-
neously through a single molecule. To date, there are only two
series of PPARR/δ dual agonists [6 (GW2433)15 and 716] with
in vitro data reported. This paper describes our finding of [1,2,4]-
thiadiazole derivatives as a new series of potent and orally active
PPARR/δ dual agonists.
Cardiovascular disease (CVDa) is the most common cause
of morbidity and mortality in developed nations.1 Atherogenic
dyslipidemia, characterized by an abnormal circulating lipid
profile, including low levels of high-density lipoprotein cho-
lesterol (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 accumulate
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
returning it to the liver.6
The peroxisome proliferator-activated receptors (PPARs)
belong to the nuclear hormone receptor superfamily and consist
of three members, PPARR, PPARγ, and PPARδ. PPARR is
expressed mainly in tissues involved in lipid oxidation such as
liver, kidney, adrenal glands, cardiac muscle, and skeletal
muscles. PPARR regulates the expression of genes involved in
lipid metabolism.7 The fibrate drugs, such as fenofibrate 1 and
ciprofibrate 2 (Figure 1), have been used for the clinical
treatment of dyslipidemia by lowering serum triglycerides and
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
PPARR.9 PPARγ is expressed in adipose tissue, macrophages,
and vascular smooth muscles.7 PPARγ was identified as a key
Chemistry
The synthesis of the first target molecule 13 is shown in
Scheme 1. Cyclocondensation of 4-trifluoromethylbenzamide
8 with chlorocarbonylsulfenyl chloride at 60 °C gave the [1,3,4]-
oxathiazol-2-one 9 intermediate. Reaction of 9 with ethyl
cyanoformate at 160 °C with the extrusion of CO2 provided
the desired [1,2,4]thiadiazole-5-carboxylic acid ethyl ester 10.
Reduction of the ethyl ester with NaBH4 resulted in the
formation of primary alcohol 11. Conversion of 11 to its
mesylate, followed by S-alkylation with thiophenoxide 1217 and
base hydrolysis of the ethyl ester, gave the desired target
molecule 13.
* To whom correspondence should be addressed. Tel: 609-655-6967.
Fax: 609-655-6930. E-mail: gkuo@prdus.jnj.com.
a Abbreviations: CVD, cardiovascular disease; HDL-C, high-density
lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; PPARs,
peroxisome proliferator-activated receptors; FFA, free fatty acid; TG,
triglycerides; FXR, farnesoid X receptor; LXR, liver X receptor; RXR,
retinoic acid X receptor; GR, glucocorticoid receptor; RAR, retinoic acid
receptor; ABC-A1, ATP-binding cassette transporter A1; CPT-1, carnitine
palmitoyltransferase type1; DMAP, (dimethylamino)pyridine; TFA, trif-
luoroacetic acid; NMP, 1-methyl-2-pyrrolidinone; DPPA, diphenylphos-
phoryl azide.
The synthesis of its geminal dimethyl analog 15 is shown in
Scheme 2. Conversion of 11 to the mesylate, followed by
S-alkylation with 4-mercapto-2-methylphenol, provided phenol
14. O-Alkylation of 14 with 2-bromoisobutyric acid gave the
10.1021/jm070511x CCC: $37.00 © 2007 American Chemical Society
Published on Web 07/04/2007

[1,2,4]Thiadiazole DeriVatiVes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 16 3955
Figure 1. Structures of representative PPAR agonists: PPARR (1, 2), PPARγ (3, 4), PPARδ (5), and PPARR/δ (6, 7).
Scheme 1a
Scheme 3a
a (i) EtO2CC(Me)2Br, Cs2CO3, dioxane, 100 °C, 68%; (ii) ClSO3H,
0-20 °C, 47%; (iii) SnCl4, HCl, EtOH, reflux, 100%.
Scheme 4a
a (i) Chlorocarbonylsulfenyl chloride, toluene, 60 °C, 39-70%; (ii) ethyl
cyanoformate, 1,2-dichlorobenzene, 160 °C, 81-97%; (iii) NaBH4, EtOH,
70-100%; (iv) MsCl, Et3N, CH2Cl2; (v) 12, CH3CN, Cs2CO3, 64% (two
steps); (vi) NaOH, MeOH-H2O, 97%.
Scheme 2a
a (i) PPh3, CBr4, CH2Cl2, 0-20 °C, 55-81%; (ii) 19, CH3CN, Cs2CO3;
(iii) NaOH, MeOH-H2O, 47-74% (two steps).
intermediate 22 needed for the synthesis of the OCF3 analog
24 was prepared in the same method as that of 11 (Scheme 1).
Once the alcohol 22 was obtained, reaction of 22 with PPh3/
CBr4 afforded the primary bromide 23 smoothly (Scheme 4).
S-Alkylation of 23 with thiophenoxide 19, followed by base
hydrolysis, generated the OCF3 analog 24. The synthesis of Cl
analog 30 and t-Bu analog 36 is identical to that of 24.
The synthesis of reverse-thiadiazole analog 41 (or 45) is
shown in Scheme 5. Reaction of 2-oxo[1,3,4]oxathiazole-5-
carboxylic acid ethyl ester 3718 with 4-trifluoromethylbenzoni-
trile at 160 °C provided [1,2,4]thiadiazole-3-carboxylic acid
ethyl ester 38. Reduction of 38 with NaBH4 generated the
alcohol 39. Reaction of 39 with PPh3/CBr4 afforded the bromide
40, followed by S-alkylation with 19, and base hydrolysis
provided desired target 41.
a (i) MsCl, Et3N, CH2Cl2; (ii) 4-mercapto-2-methylphenol, CH3CN,
Cs2CO3, 95% (two steps); (iii) NaH, THF, 60 °C, 2-bromoisobutyric acid,
21%.
desired compound 15. The synthesis of the corresponding OCF3
analog 24, Cl analog 30, or t-Bu analog 36 could also be
accomplished in a slightly different manner when compared to
that of CF3 analog 15. Therefore, we first prepared the
thiophenoxide 19, followed by S-alkylation of the right-hand
biheteroaryl moiety to give 24, 30, or 36. The synthesis of 19
is shown in Scheme 3. O-Alkylation of 2-methylphenol 16 with
2-bromo-2-methylpropionic acid ethyl ester generated 17. Treat-
ment of 17 with excess ClSO3H at 0 °C, with the temperature
being allowed to warm to 20 °C slowly, gave the chlorosulfonyl
derivative 18. Reduction of 18 with SnCl4 provided the
thiophenoxide 19 in quantitative yield. The required key
The synthesis of oxygen-link target 50 (or 51) is shown in
Scheme 6. O-Alkylation of phenol 46 with 2-bromo-2-methyl-
propionic acid ethyl ester gave 47. Oxidation of 47 with
m-CPBA, followed by base hydrolysis, provided phenol 48.
Alkylation of 48 with the bromide 49 (or 23), followed by base
hydrolysis, afforded target 50 (or 51).

3956 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 16
Shen et al.
Scheme 5a
a (i) 4-Trifluoromethylbenzonitrile, 1,2-dichlorobenzene, 160 °C, 4-34%;
(ii) NaBH4, EtOH, 93-94%; (iii) PPh3, CBr4, CH2Cl2, 0-20 °C, 72-87%;
(iv) 19, CH3CN, Cs2CO3. (v) NaOH, MeOH-H2O, 50-74% (two steps).
Figure 2. Effects of 24 on serum HDL-C and LDL-C levels in
hypercholesterolemic rat treated via oral gavage (0.1-3 mg/kg) for 8
days. Bars represent means ( SEM (n ) 8 rat per group; *p < 0.05,
**p < 0.01 relative to vehicle HF).
Scheme 6a
followed by bromination with NBS, afforded bromide 55.
Reaction of 55 with triisopropylsilanethiol in the presence of
Pd(PPh3)4 gave the thio analog 56. S-Alkylation of 56 with
bromide 23 in the presence of TBAF, followed by base
hydrolysis, resulted in the target 57.
Results and Discussion
Lead Generation. During this study, HEK293 cells were
grown in DMEM/F-12 medium supplemented with 10% FBS
and glutamine. The cells were cotransfected with DNA con-
structs containing the ligand-binding domains of either PPARR,
PPARγ, or δ-Gal4 chimeric receptors. The steady-Glo luciferase
assay kit was used for measuring luciferase reporter activity.
Percent efficacies for PPARR, PPARδ, and PPARγ activation
were reported relative to PPARR agonist (compound 2.1),19
PPARδ agonist (compound f),20 and rosiglitazone, respectively.
A primary goal of our PPARs research programs has been
the identification of a functionally potent and selective PPARδ
agonist with optimal in vivo efficacy and maximum safety
margin. Since 5 is the first reported potent and selective PPARδ
agonist, we were interested in conducting structure-activity
relationship (SAR) studies with respect to 5 as the lead molecule.
It seems that the methyl-substituted thiazole of 5 may contribute
uniquely to its high functional potency and selectivity at PPARδ;
therefore, we first screened various five-membered or six-
membered heteroaryls or heterocycles in all three human PPARs
cotransfection assays to see if we could uncover any new series
that may meet our program goal. In summary, replacement of
the methylthiazole of 5 with [1,2,4]thiadiazole gave compound
13. Unexpectedly, 13 displayed submicromolar potency as a
partial agonist at PPARR (EC50 ) 468 nM, 42%, Table 1) in
addition to the retained high potency and high activity at PPARδ
(EC50 ) 10 nM, 79%) and no activity at PPARγ (EC50 >3000
nM). Therefore, while maintaining the similar shape and size
of the entire molecule, a simple replacement of the methylthi-
azole to the thiadiazole resulted in the conversion of a highly
potent and selective PPARδ agonist (∼1000-fold selectivity as
shown in 5, Table 1) to a PPARR/δ dual agonist (only ∼47-
fold selectivity for δ over R as observed in 13). Since geminal
dimethyl substitution of the acidic head moiety appears to be
a (i) 2-Bromo-2-methylpropionic acid ethyl ester, Cs2CO3, dioxane,
100 °C, 57%; (ii) mCPBA, TsOH, CH2Cl2, 38 °C, 82%; (iii) NaOEt, EtOH-
THF, 81%; (iv) 49 (or 23), CH3CN, Cs2CO3; (v) NaOH, MeOH-H2O, 71-
72% (two steps).
Scheme 7a
a (i) ClSO3H, Br2, 1,2-dichloroethane, reflux; MeOH, 86%; (ii) 2-me-
thylphenol, CH3CN, Cs2CO3,70 °C, 22%; (iii) NBS, CH3CN, 95%; (iv)
NaH, toluene, triisopropylsilanethiol, Pd(PPh3)4, THF, 90 °C, 82%; (v) 23,
TBAF, THF, 0 °C, 94%; (vi) NaOH, MeOH-H2O, 68%.
The synthesis of cyclopentyl analog 57 is shown in Scheme
7. Treatment of cyclopentanecarboxylic acid 52 with ClSO3H
and Br2 gave bromide 53. Alkylation of 53 with 2-methylphenol,

[1,2,4]Thiadiazole DeriVatiVes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 16 3957
Table 1. Activities of Compounds in Human PPAR Co-Transfection Assays
EC50 ( SEM,a nM (% resp)
compd
R2
Z
Y
X
R1
PPARRb
PPARδc
PPARγd
>3000
407 ( 31 (25)
>3000
>3000
>3000
>3000
>3000
>3000
>3000
>3000
13
15
24
30
36
41
45
50
51
57
H
S
S
S
S
S
S
S
O
O
S
S
S
S
S
S
N
N
S
S
S
N
N
N
N
N
S
CF3
CF3
OCF3
Cl
t-Bu
CF3
OCF3
CF3
OCF3
OCF3
468 ( 10 (42)
79 ( 9 (51)
33 ( 1 (44)
186 ( 10 (38)
64 ( 13 (64)
425 ( 12 (38)
656 ( 13 (47)
94 ( 2 (71)
94 ( 7 (80)
736 ( 16 (31)
10 ( 2 (79)
6 ( 2 (76)
3 ( 1 (73)
18 ( 2 (72)
93 ( 7 (48)
26 ( 3 (67)
58 ( 3 (55)
3 ( 1 (79)
12 ( 2 (83)
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
S
N
N
N
>1000
5
1100e
1e
850e
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. SEM: standard error of the mean. b The internal PPARR agonist (compound 2.1)19 was used as the standard. c The internal PPARδ agonist
(compound f)20 was used as the standard. d Rosiglitazone was used as the standard. e The data was reported in ref 17c.
Table 2. Induction of Selected Target Genes by 24 in Primary Human
four examples (15, 24, 30, and 36), it seems the high potency
Skeletal Muscle Cells
observed at both PPARR and PPARδ of 24 may not derive from
concn, nM
ABC-A1
CPT-1
Pa9
the steric bulkiness at the 4-position of the phenyl group.
We turned our interest to examine the reverse-[1,2,4]-
thiadiazole series to see if the specific orientation of the five-
membered thiadiazole ring is critical to the potency or selectivity
observed at PPARs. The representative CF3 analog 41 and OCF3
analog 45 of reverse-[1,2,4]thiadiazole were thus synthesized.
Compound 41 displayed 5-fold reduced potency with slightly
lower activity at PPARR (EC50 ) 425 nM, 38%) and 4-fold
reduced potency at PPARδ (EC50 ) 26 nM, 67%) compared to
that of 15. Even with the assumed more favorable OCF3-
substituent, 45 displayed 8-fold reduced potency at PPARR
(EC50 ) 656 nM, 47%) and 10-fold reduced potency at PPARδ
(EC50 ) 58 nM, 55%) compared to that of 15. It appears that
the reverse-[1,2,4]thiadiazole analogs may not have the favorable
interactions with the ligand-binding pockets of PPARR and
PPARδ as the [1,2,4]thiadiazole analogs have.
Since the oxygen atom may behave as the bioisostere of the
sulfur atom in some cases, we decided to synthesize the oxygen-
link analogs of 15 and 24. Compound 50, the oxygen-link CF3
analog, exhibited slightly lower potency but higher activity at
PPARR (EC50 ) 94 nM, 71%) and ∼2-fold higher potency at
PPARδ (EC50 ) 3 nM, 79%) compared to that of the sulfur-
link CF3 analog 15. Therefore, the replacement of a sulfur atom
with an oxygen atom led to an interesting potent PPARR/δ dual
agonist 50, although it is not as potent as 24 at PPARR.
Replacing the CF3 substituent with OCF3 in the oxygen-link
series resulted in the formation of 51. Compound 51 exhibited
1
10
100
1000
1.9 ( 0.3a
2 ( 0.1
4.7 ( 1.3
5.4 ( 0.2
3 ( 0.9
2.6 ( 0.5
5.9 ( 0.7
11.4 ( 3.2
4.1 ( 1
6.5 ( 0.2
9.9 ( 1.2
22.4 ( 5.4
a Fold increase over vehicle were expressed as mean ( SEM (standard
error mean) of triplicate samples.
beneficial for many PPARR agonists, including 1, 2, and 6, we
decided to explore PPARs activities of the geminal dimethyl
analog of 13. Indeed, 15 displayed 6-fold improved potency at
PPARR (EC50 ) 79 nM, 51%) and even slightly higher potency
at PPARδ (EC50 ) 6 nM, 76%) compared to that of 13.
Meanwhile, 15 also exhibited submicromolar potency but with
low activity at PPARγ (EC50 ) 407 nM, 25%). Overall, with
the incorporation of the additional geminal dimethyl group to
the [1,2,4]thiadiazole series, 15 displayed better potency at both
PPARR and PPARδ than the corresponding CH2 analog 13.
This observation is also consistent with another PPARδ agonist
series reported recently.21
We then examined the impact of other phenyl ring substit-
uents on PPARs activities. Replacing the CF3 substituent with
OCF3 gave 24. Interestingly, OCF3 analog 24 displayed ∼2-
fold higher potency at PPARR (EC50 ) 33 nM, 44%) and ∼2-
fold higher potency at PPARδ (EC50 ) 3 nM, 73%), but with
abolished activity at PPARγ (EC50 > 3000 nM) compared to
CF3 analog 15. Up to this point, 24 behaves as a quite potent
and selective PPARR/δ dual agonist. It is unclear if the high
potency of 24 observed at both PPARR and PPARδ may derive
from the increasing steric bulk of OCF3 or from the H-bond-
acceptor capability of the oxygen atom. We thus prepared
sterically less bulky Cl analog 30 and sterically more bulky t-Bu
analog 36 to get more insights. Compound 30 exhibited >2-
fold reduced potency at PPARR (EC50 ) 186 nM, 38%) and
3-fold reduced potency at PPARδ (EC50 ) 18 nM, 72%) and
maintained no activity at PPARγ (EC50 > 3000 nM) compared
to that of CF3 analog 15. Meanwhile, 36 displayed about equal
potency with higher activity at PPARR (EC50 ) 64 nM, 64%)
and 16-fold reduced potency with lower activity at PPARδ (EC50
) 93 nM, 48%) compared to that of CF3 analog 15. From these
slightly lower potency but higher activity at PPARR (EC50
94 nM, 80%) and ∼2-fold reduced potency at PPARδ (EC50
)
)
12 nM, 83%) compared to that of 15. Overall, in the oxygen-
link series, the OCF3 analog 51 does not seem to have more
favorable interaction with the ligand-binding pockets of PPARR
and PPARδ than that of the CF3 analog 50. This observation is
not consistent with the previous result observed between CF3
analog 15 and OCF3 analog 24 in the sulfur-link series. The
nonparallel SAR suggests that the oxygen atom is not an
equivalent bioisostere to the sulfur atom in this series.
Since we already demonstrated that the incorporation of the
geminal dimethyl group in the acidic head moiety improved

3958 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 16
Shen et al.
Figure 3. Effects of 24 on HDL-C, TG, and FFA levels in male hApoA1 transgenic mice dosed orally (3 mg/kg) for 7 days. Bars represent means
( SEM (n ) 8 mice per group; *p < 0.05, **p < 0.01 relative to vehicle control).
the potencies at both PPARR and PPARδ (15 versus 13), we
would like to see if we could further enhance the potency by
introducing a sterically bulkier group. Unfortunately, installation
of a cyclopentyl group as in 57 not only lowered the potency
and the activity at PPARR (EC50 ) 736 nM, 31%) but also
abolished the activities at both PPARδ (EC50 > 1000 nM) and
PPARγ (EC50 > 3000 nM). In summary, as compared to 5,
these [1,2,4]thiadiazole derivatives appear as a new series of
potent and selective PPARR/δ dual agonists. In addition, there
was no cross-activity of 24 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 Vivo Studies. The ability of compound 24 to affect lipid/
cholesterol homeostasis in vivo was first examined in a
hypercholesterolemic rat model. The high-cholesterol diet fed
hypercholesterolemic rat displayed about a 3.7-fold lower
HDL-C level (13 mg/dL, Figure 2) than the rat fed standard
chow (48 mg/dL). Compound 24 dose-dependently (from 0.1
to 3 mg/kg/d) increased serum HDL-C levels (from 35 to 74
mg/dL) after these rats were dosed orally for 8 days. With this
hypercholesterolemic rat model, a PPARδ selective agonist will
generally normalized HDL-C levels to that of vehicle lean rats
(data not shown), but the current PPARR/δ dual agonist 24 is
able to increase the HDL-C levels even 50% higher than that
of vehicle lean rats. In addition, while the hypercholesterolemic
rat displayed about 11-fold elevated LDL-C levels (113 mg/
dL, Figure 2) than the rat fed standard chow (10 mg/dL), 24
attenuated the high LDL-C levels (from 82 to 49 mg/dL) in a
dose-related manner. On the other hand, 24 exhibited no effect
on serum triglycerides and no effect on liver weight except at
the 3 mg/kg high dose (∼50% increase), which may be
attributed to its PPARR activity.25
The capability of 24 to affect lipid homeostasis was next
examined in the 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 PPARR and PPARδ hypolipidemic compounds.26 Male
hApoA1 mice were dosed orally with 3 mg/kg of 24 for 7 days.
Compound 24 caused significant increase in HDL-C (74%
increase, from 287 to 499 mg/dL, Figure 3) and reductions in
triglycerides (85% decrease, from 230 to 34 mg/dL) and in free
fatty acids (31% decrease, from 1.84 to 1.27 mequiv/L)
compared to that of vehicle control group.
To determine the activity of these compounds on the 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 muscles cells. To date, 24 induced
several target genes in a dose-dependent manner (Table 2). For
example, a robust induction of ATP-binding cassette transporter
A1 (ABC-A1)22 by 24 was detected in skeletal muscles (1.9-
fold increase at 1 nM to 5.4-fold increase at 1000 nM). Since
ABC-A1 is involved in lipid and protein metabolism, the
induction of this gene by 24 suggests PPARR/δ dual agonists
may play an important role in this function. Meanwhile, a
significant induction of carnitine palmitoyltransferase type 1
(CPT-1)23 by 24 (3-fold at 1 nM to 11.4-fold at 1000 nM)
suggests that 24 may be involved in fatty acid oxidation. In
addition, a strong induction of a new PPARR target gene, Pa9,24
was also observed (4.1-fold at 1 nM to 22.4-fold at 1000 nM).
Most of the compounds displayed very good pharmacokinetic
profiles in rat. For example, using 10% Solutol in D5W for
intravenous formulation and 0.5% methylcellulose for oral
gavage formulation, when dosing at 3 mg/kg, 24 displayed high
maximum plasma concentration (Cmax ) 6675 ( 779 ng/mL),
rapid oral absorption (Tmax ) 0.8 h), high plasma drug exposure
The ob/ob mice are obese, insulin resistant with hypertrig-
lyceridemia and hyperglycemia, and have been used as a rodent
model of obesity-induced insulin resistance. Seven-week-old
female ob/ob mice were dosed orally with 24 for 11 days.
Compound 24 significantly reduced serum TG (80% decrease,
from 492.9 to 98.9 mg/dL, Table 3), serum glucose (51%
decrease, from 630.9 to 308.7 mg/dL), and serum FFA (58%
decrease, from 1.2 to 0.5 mequiv/L). Meanwhile, liver weights
(AUC ) 22033 ( 2375 ng h/mL), long plasma duration (t1/2
)
5.3 ( 2.3 h), low systemic clearance (CL ) 2.2 ( 0.2 mL/
min.kg), and high oral bioavailability (F ) 96%).

[1,2,4]Thiadiazole DeriVatiVes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 16 3959
Table 3. Effect of 24 on Serum TG, Glucose, FFA, and Liver Weight
J ) 8.2 Hz, 2 H), 4.57 (q, J ) 7.1 Hz, 2 H), 1.49 (t, J ) 7.1 Hz,
3 H); MS (ES) m/z 303 (M + H+).
in ob/ob Micea
TG,b
mg/dL
gluc,b
mg/dL
FFA,b
mequiv/L
liver wt,b
g
3-(4-Trifluoromethoxyphenyl)[1,2,4]thiadiazole-5-carboxyl-
ic Acid Ethyl Ester (21). Using 20 and following the procedure
as in the preparation of 9 and 10 gave 21 (97%) as a colorless oil:
1H NMR (300 MHz, CDCl3) δ 8.42 (m, 2 H), 7.33 (dd, J ) 8.9,
0.8 Hz, 2 H), 4.56 (q, J ) 7.1 Hz, 2 H), 1.49 (t, J ) 7.1 Hz, 3 H).
3-(4-Chlorophenyl)[1,2,4]thiadiazole-5-carboxylic Acid Ethyl
Ester (27). Using 26 and following the procedure as in the
preparation of 10 gave 27 (94%) as a white solid: 1H NMR (300
MHz, CDCl3) δ 8.31 (m, 2 H), 7.47 (m, 2 H), 4.55 (q, J ) 7.1 Hz,
2 H), 1.48 (t, J ) 7.1 Hz, 3 H); MS (ES) m/z 269 (M + H+).
3-(4-tert-Butylphenyl)[1,2,4]thiadiazole-5-carboxylic Acid Eth-
yl Ester (33). Using 32 and following the procedure as in the
preparation of 10 gave 33 (81%) as a yellowish crystal: 1H NMR
(300 MHz, CDCl3) δ 8.29 (m, 2 H), 7.51 (m, 2 H), 4.55 (q, J )
7.1 Hz, 2 H), 1.48 (t, J ) 7.1 Hz, 3 H), 1.37 (s, 9 H); MS (ES) m/z
291 (M + H+).
dose,
mg/kg
vehicle
1
492.9 ( 40.4
98.9 ( 5.4
630.9 ( 23.4
308.7 ( 24.7
1.2 ( 0.1
0.5 ( 0.1
3.0 ( 0.2
4.5 ( 0.1
a Seven-week-old female ob/ob mice dosed orally with 24 for 11 days.
b Mean value ( SEM (n ) 8), p < 0.01 versus vehicle control.
increased (50% increase, from 3 to 4.5 g) similar to the effect
observed with PPARR agonists. The good in vivo efficacy of
24 observed in the mouse model is consistent with the good
potency displayed in the mouse PPARR HD assay (EC50 ) 127
nM).
Conclusion
In an attempt to rationalize the unexpected potent and
selective PPARR/δ dual agonist activity, 24 was docked into
all three PPAR isotypes. Unfortunately, docking energies and
binding modes experiments revealed little about SAR selectivity.
Nonetheless, compound 24 and its close analogs represent a
new series of PPARR/δ dual agonists. The high potency, high
selectivity, significant gene induction, excellent PK profiles, and
good in vivo efficacies in three animal models may render
compound 24 as a valuable pharmacological tool in elucidating
the complex roles of PPARR/δ dual agonists and as a potential
treatment of the metabolic syndrome.
General Procedure for the Synthesis of 11, 22, 28, and 34.
[3-(4-Trifluoromethylphenyl)[1,2,4]thiadiazol-5-yl]methanol (11).
To a solution of 10 (200 mg, 0.662 mmol) in EtOH (10 mL) at
room temperature was added NaBH4 (64 mg, 1.7 mmol). After
stirring for 2 h, a few drops of water were added to quench the
excess of hydride. EtOH was evaporated, and the residue was
partitioned between CH2Cl2 and water. The organic phase was dried
and concentrated to provide 167 mg (97%) of 11 as off-white
crystals: 1H NMR (300 MHz, CDCl3) δ 8.40 (d, J ) 8.1 Hz, 2 H),
7.74 (d, J ) 8.2 Hz, 2 H), 5.20 (s, 2 H), 2.65 (brs, 1 H); MS (ES)
m/z 261 (M + H+).
[3-(4-Trifluoromethoxyphenyl)[1,2,4]thiadiazol-5-yl]metha-
nol (22). Using 21 and following the procedure as in the preparation
of 11 gave 22 (70%) as a white solid: 1H NMR (300 MHz, CDCl3)
δ 8.32 (m, 2 H), 7.31 (d, J ) 8.1 Hz, 2 H), 5.17 (d, J ) 5.8 Hz,
2 H), 1.26 (t, J ) 7.1 Hz, 1 H); MS (ES) m/z 277 (M + H+).
[3-(4-Chlorophenyl)[1,2,4]thiadiazol-5-yl]methanol (28). Using
27 and following the procedure as in the preparation of 11 gave 28
(87%) as an off-white solid: 1H NMR (300 MHz, CDCl3) δ 8.22
(m, 2 H), 7.45 (m, 2 H), 5.17 (s, 2 H); MS (ES) m/z 227 (M +
H+).
[3-(4-tert-Butylphenyl)[1,2,4]thiadiazol-5-yl]methanol (34). Us-
ing 33 and following the procedure as in the preparation of 11 gave
34 (100%) as an off-white solid: 1H NMR (300 MHz, CDCl3) δ
8.19 (m, 2 H), 7.50 (m, 2 H), 5.16 (s, 2 H), 1.36 (s, 9 H); MS (ES)
m/z 249 (M + H+).
{2-Methyl-4-[3-(4-trifluoromethylphenyl)[1,2,4]thiadiazol-5-
ylmethylsulfanyl]phenoxy}acetic Acid (13). A mixture of 11 (88
mg, 0.34 mmol), methanesulfonyl chloride (58 mg, 0.51 mmol),
and triethylamine (70 mg, 0.69 mmol) in CH2Cl2 (3 mL) was stirred
at room temperature for 1.5 h. The mixture was washed with water
and the aqueous phase was back-extracted with CH2Cl2. The
combined organic layers were dried and concentrated to provide
111 mg of the mesylate as a yellow solid.
A mixture of the crude mesylate (111 mg) and (4-mercapto-2-
methylphenoxy)acetic acid ethyl ester 12 (111 mg, 0.491 mmol)
in CH3CN (4 mL) was degassed under N2 for about 15 min. After
the addition of Cs2CO3 (214 mg, 0.656 mmol), the mixture was
stirred overnight under N2, concentrated, and purified by column
chromatography (EtOAc/hexane) to give 102 mg (64%, 2 steps)
of the ethyl ester as a white solid: 1H NMR (300 MHz, CDCl3) δ
8.35 (d, J ) 8.1 Hz, 2 H), 7.71 (d, J ) 8.2 Hz, 2 H), 7.30 (d, J )
1.7 Hz, 1 H), 7.23 (dd, J ) 8.6, 2.2 Hz, 1 H), 6.62 (d, J ) 8.5 Hz,
1 H), 4.61 (s, 2 H), 4.41 (s, 2 H), 4.24 (q, J ) 7.1 Hz, 2 H), 2.25
(s, 3 H), 1.27 (t, J ) 7.1 Hz, 3 H); MS (ES) m/z 491 (M + Na+).
Anal. (C19H15F3N2O3S2·0.5H2O) C, H, N.
A mixture of ethyl ester (77 mg, 0.16 mmol) and 2 M NaOH
(0.30 mL, 0.60 mmol) in MeOH (4 mL) was stirred under N2 for
30 min and concentrated. EtOAc and water were added, and the
mixture was acidified with concentrated HCl. The organic phase
was separated and the aqueous phase was extracted with EtOAc.
The combined organic layers were dried and concentrated to give
70 mg (97%) of 13 as an off-white solid: 1H NMR (300 MHz,
Experimental Section
1
Chemistry. H NMR spectra were measured on a Bruker AC-
300 (300 MHz) spectrometer using tetramethylsilane as an internal
standard. Elemental analyses were obtained by Quantitative Tech-
nologies Inc. (Whitehouse, NJ), and the results were within 0.4%
of the calculated values unless otherwise mentioned. Melting points
were determined in open capillary 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 Micro-
mass Autospec. E. spectrometer.
General Procedure for the Synthesis of 9, 26, and 32. 5-(4-
Trifluoromethylphenyl)[1,3,4]oxathiazol-2-one (9). The reaction
mixture of 4-trifluoromethylbenzamide 8 (2.57 g, 13.6 mmol) and
chlorocarbonylsulfenyl chloride (3.57 g, 27.2 mmol) in toluene (35
mL) was heated at 60 °C for 15 h and concentrated. CH2Cl2 was
added and the mixture was filtered. The white solid was washed
with CH2Cl2 and dried under high vacuum to give 922 mg (36%)
of 4-trifluoromethylbenzamide 8 as recovered starting material. The
filtrate was concentrated and column chromatographed (EtOAc/
hexane) to provide 1.31 g (39%) of 9 as white crystals: 1H NMR
(300 MHz, CDCl3) δ 8.11 (d, J ) 8.2 Hz, 2 H), 7.77 (d, J ) 8.3
Hz, 2 H).
5-(4-Chlorophenyl)[1,3,4]oxathiazol-2-one (26). Using 25 and
following the procedure as in the preparation of 9 gave 26 (63%)
as a colorless oil: 1H NMR (300 MHz, CDCl3) δ 7.91 (d, J ) 8.6
Hz, 2 H), 7.48 (d, J ) 8.6 Hz, 2 H).
5-(4-tert-Butylphenyl)[1,3,4]oxathiazol-2-one (32). Using 31
and following the procedure as in the preparation of 9 gave 32
(70%) as a colorless oil: 1H NMR (300 MHz, CDCl3) δ 7.90 (m,
2 H), 7.50 (m, 2 H), 1.35 (s, 9 H).
General Procedure for the Synthesis of 10, 21, 27, and 33.
3-(4-Trifluoromethylphenyl)[1,2,4]thiadiazole-5-carboxylic Acid
Ethyl Ester (10). A reaction mixture of 9 (448 mg, 1.81 mmol)
and ethyl cyanoformate (722 mg, 7.29 mmol) in 1,2-dichloroben-
zene (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 505 mg (92%) of 10 as a yellow solid:
1H NMR (300 MHz, CDCl3) δ 8.50 (d, J ) 8.1 Hz, 2 H), 7.76 (d,

3960 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 16
Shen et al.
MeOH-d4) δ 8.37 (d, J ) 8.1 Hz, 2 H), 7.78 (d, J ) 8.3 Hz, 2 H),
7.28 (s, 1 H), 7.26 (m, 1 H), 6.76 (d, J ) 8.3 Hz, 1 H), 4.66 (s, 2
H), 4.53 (s, 2 H), 2.20 (s, 3 H); MS (ES) m/z 441 (M + H+); FAB-
HRMS (M+) calcd 440.0476, found 440.0465.
2-Methyl-4-[3-(4-trifluoromethylphenyl)[1,2,4]thiadiazol-5-yl-
methylsulfanyl]phenol (14). To a solution of 11 (795 mg, 3.06
mmol) in CH2Cl2 (30 mL) at 0 °C were added methanesulfonyl
chloride (518 mg, 4.52 mmol) and triethylamine (617 mg, 6.11
mmol). The mixture was stirred at room temperature for 1 h and
then partitioned 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-mer-
capto-2methylphenol (126 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 stirred at room temperature
for 40 min, concentrated, and purified by column chromatography
(EtOAc/hexane) to give 228 mg (95%) of 14 as a white crystalline
solid: 1H NMR (300 MHz, CDCl3) δ 8.35 (d, J ) 8.0 Hz, 2 H),
7.71 (d, J ) 8.2 Hz, 2 H), 7.26 (s, 1 H), 7.20 (dd, J ) 8.0, 2.1 Hz,
1 H), 6.71 (d, J ) 8.2 Hz, 1 H), 4.83 (s, 1 H), 4.40 (s, 2 H), 2.21
(s, 3 H); MS (ES) m/z 383 (M + H+).
2-Methyl-2-{2-methyl-4-[3-(4-trifluoromethylphenyl)[1,2,4]-
thiadiazol-5-ylmethylsulfanyl]phenoxy}propionic Acid (15). To
a solution of 14 (39 mg, 0.10 mmol) in THF (1 mL) was added
NaH (20 mg, 0.50 mmol; 60% in mineral oil) and the mixture was
heated at 60 °C for 30 min, during which the solution changed to
blue and then brown. 2-Bromoisobutyric acid (34 mg, 0.20 mmol)
was added, and the mixture was heated at the same temperature
for 1 h, acidified with 1 N HCl, and diluted with CH2Cl2. The
organic phase was separated, washed with brine, dried, concentrated,
and column chromatographed (EtOAc/hexane) to isolate 10 mg
(21%) of 15 as a yellow oil: 1H NMR (300 MHz, CDCl3) δ 8.35
(d, J ) 8.0 Hz, 2 H), 7.71 (d, J ) 8.2 Hz, 2 H), 7.28 (m, 1 H),
7.18 (dd, J ) 8.4, 2.4 Hz, 1 H), 6.72 (d, J ) 8.5 Hz, 1 H), 4.43 (s,
2 H), 2.20 (s, 3 H), 1.61 (s, 6 H); MS (ES) m/z 469 (M + H+).
Anal. (C21H19F3N2O3S2·0.3 H2O) C, H. N.
2-Methyl-2-o-tolyloxypropionic Acid Ethyl Ester (17). To a
mixture of 2-bromo-2-methylpropionic acid ethyl ester (8.27 mL,
64.0 mmol) and 2-methylphenol (7.60 g, 70.2 mmol) in dioxane
(100 mL) was added Cs2CO3 (31.3 g, 96.0 mmol). After the mixture
was refluxed at 100 °C for 4 h and allowed to cool to room
temperature, the solvent was evaporated under reduced pressure.
The residue was dissolved in Et2O, washed with 1 N NaOH, dried,
and concentrated to give 9.69 g (68%) of 17: 1H NMR (300 MHz,
CDCl3) δ 7.13 (d, J ) 7.3 Hz, 1 H), 7.03 (t, J ) 7.6 Hz, 1 H), 6.87
(t, J ) 7.3 Hz, 1 H), 6.66 (d, J ) 8.2 Hz, 1 H), 4.24 (q, J ) 7.1
Hz, 2 H), 2.23 (s, 3 H), 1.59 (s, 6 H), 1.25 (t, J ) 7.1 Hz).
2-(4-Chlorosulfonyl-2-methylphenoxy)-2-methylpropionic Acid
Ethyl Ester (18). To a flask containing 17 (11.3 g, 0.051 mol) at
0 °C was slowly added ClSO3H (15.2 mL, 0.229 mol). The
temperature was allowed to warm to room temperature and the
solution was stirred for 1 h. Upon stirring, the reaction mixture
was poured into ice. The solid was filtered, washed with water,
and vacuum-dried to give 7.7 g (47%) of 18: 1H NMR (300 MHz,
CDCl3) δ 7.82 (d, J ) 2.5 Hz, 1 H), 7.75 (dd, J ) 8.9, 2.5 Hz, 1
H), 6.67 (d, J ) 8.8 Hz, 1 H), 4.23 (q, J ) 7.1 Hz, 2 H), 2.31 (s,
3 H), 1.70 (s, 6 H), 1.22 (t, J ) 7.1 Hz); MS (ES) m/z 343 (M +
Na+).
2-(4-Mercapto-2-methylphenoxy)-2-methylpropionic Acid Eth-
yl Ester (19). To a solution of 18 (2.00 g, 6.25 mmol) in EtOH
(7.8 mL) were added HCl in dioxane (4.0 M, 7.8 mL, 31 mmol)
and tin powder (3.70 g, 31.2 mmol). The mixture was refluxed for
3 h, poured into ice, and extracted with CH2Cl2 (50 mL × 3). The
organic layers were combined and dried over Na2SO4. After
filtration, the filtrate was concentrated to give 3.37 g (∼100%) of
19: 1H NMR (300 MHz, CDCl3) δ 7.12 (d, J ) 2.0 Hz, 1 H), 7.00
(dd, J ) 8.4, 2.4 Hz, 1 H), 6.56 (d, J ) 8.4 Hz, 1 H), 4.23 (q, J )
7.1 Hz, 2 H), 3.31 (s, 1 H), 2.18 (s, 3 H), 1.57 (s, 6 H), 1.25 (t, J
) 7.1 Hz); MS (ES) m/z 255 (M + H+).
General Procedure for the Synthesis of 23, 29, and 35.
5-Bromomethyl-3-(4-trifluoromethoxyphenyl)[1,2,4]-
thiadiazole (23). To a solution of 22 (679 mg, 2.46 mmol) in CH2-
Cl2 (10 mL) were added carbon tetrabromide (896 mg, 2.70 mmol)
and triphenylphosphine (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 678 mg (81%) of
23 as a white solid: 1H NMR (300 MHz, CDCl3) δ 8.32 (m, 2 H),
7.31 (dd, J ) 8.9, 0.8 Hz, 2 H), 4.82 (s, 2 H); MS (ES) m/z 339
(M + H+).
5-Bromomethyl-3-(4-chlorophenyl)[1,2,4]thiadiazole (29). Us-
ing 28 and following the procedure as in the preparation of 23 gave
29 (65%) as an off-white solid: 1H NMR (300 MHz, CDCl3) δ
8.22 (m, 2 H), 7.45 (m, 2 H), 4.81 (s, 2 H).
5-Bromomethyl-3-(4-tert-butylphenyl)[1,2,4]thiadiazole (35).
Using 34 and following the procedure as in the preparation of 23
gave 35 (55%) as a white solid: 1H NMR (300 MHz, CDCl3) δ
8.19 (m, 2 H), 7.49 (m, 2 H), 4.82 (s, 2 H), 1.36 (s, 9 H); MS (ES)
m/z 313 (M + H+).
General Procedure for the Synthesis of 24, 30, and 36.
2-Methyl-2-{2-methyl-4-[3-(4-trifluoromethoxyphenyl)[1,2,4]-
thiadiazol-5-ylmethylsulfanyl]phenoxy}propionic Acid (24). To
a mixture of 23 (73 mg, 0.22 mmol) and 2-(4-mercapto-2-methyl-
phenoxy)-2-methyl-propionic acid ethyl ester 19 (52 mg, 0.21
mmol) in CH3CN (1.5 mL) and DMF (0.1 mL) was added Cs2CO3
(100 mg, 0.31 mmol). After stirring at room temperature for 15
min, the mixture was concentrated. The residue was diluted with
EtOAc, washed with water and brine, dried, concentrated, and
column chromatographed to give 82 mg (78%) of 24-ethyl ester:
1H NMR (300 MHz, CDCl3) δ 8.27 (d, J ) 8.9 Hz, 2 H), 7.29 (dd,
J ) 8.9, 0.9 Hz, 1 H), 7.26 (m, 2 H), 7.13 (dd, J ) 8.5, 2.4 Hz, 1
H), 6.56 (d, J ) 8.5 Hz, 1 H), 4.40 (s, 2 H), 4.20 (q, J ) 7.1 Hz,
2 H), 2.18 (s, 3 H), 1.58 (s, 6 H), 1.20 (t, J ) 7.1 Hz, 3 H); MS
(ES) m/z 513 (M + H+). Anal. (C23H23F3N2O4S2) C, H, N.
A solution of 24-ethyl ester (80 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 organic
layer was separated and the aqueous layer was extracted with
EtOAc. The combined organic phases were washed with brine,
dried, concentrated, and column chromatographed to provide 71
mg (95%) of 24: 1H NMR (400 MHz, CDCl3) δ 8.27 (d, J ) 8.8
Hz, 2 H), 7.30-7.23 (m, 3 H), 7.17 (d, J ) 8.4 Hz, 1 H), 6.72 (d,
J ) 8.4 Hz, 1 H), 4.42 (s, 2 H), 2.20 (s, 3 H), 1.60 (s, 6 H); MS
(ES) m/z 507 (M + Na+). Anal. (C21H19F3N2O4S2) C, H, N.
2-{4-[3-(4-Chlorophenyl)[1,2,4]thiadiazol-5-ylmethylsulfanyl]-
2-methylphenoxy}-2-methylpropionic Acid (30). Using 29 and
following the procedure as in the preparation of 24 gave 30 (64%)
as a light yellow solid: 1H NMR (400 MHz, CDCl3) δ 8.17 (m, 2
H), 7.42 (m, 2 H), 7.28 (d, J ) 2.2 Hz, 1 H), 7.18 (dd, J ) 8.5, 2.4
Hz, 1 H), 6.72 (d, J ) 8.5 Hz, 1 H), 4.42 (s, 2 H), 2.19 (s, 3 H),
1.61 (s, 6 H); MS (ES) m/z 435 (M + H+). Anal. (C20H19ClN2O3S2)
C, H, N.
2-{4-[3-(4-tert-Butylphenyl)[1,2,4]thiadiazol-5-ylmethylsulfa-
nyl]-2-methylphenoxy}-2-methylpropionic Acid (36). Using 35
and following the procedure as in the preparation of 24 gave 36
(47%) as a yellow gummy solid: 1H NMR (400 MHz, CDCl3) δ
8.14 (m, 2 H), 7.47 (m, 2 H), 7.28 (d, J ) 2.3 Hz, 1 H), 7.17 (dd,
J ) 8.5, 2.4 Hz, 1 H), 6.72 (d, J ) 8.5 Hz, 1 H), 4.43 (s, 2 H),
2.19 (s, 3 H), 1.60 (s, 6 H), 1.35 (s, 9 H); MS (ES) m/z 457 (M +
H+). Anal. (C24H28N2O3S2) C, H, N.
General Procedure for the Synthesis of 38 and 42. 5-(4-
Trifluoromethylphenyl)[1,2,4]thiadiazole-3-carboxylic Acid Eth-
yl Ester (38). A mixture of 37 (1.77 g, 10.1 mmol) and
4-trifluoromethylbenzonitrile (8.63 g, 50.5 mmol) in 1,2-dichlo-
robenzene (10 mL) was heated at 160 °C for 4 days. After cooling
down to room temperature, the mixture was purified by column
chromatography to provide 120 mg (3.9%) of 38 as light brown
crystals: 1H NMR (300 MHz, CDCl3) δ 8.17 (d, J ) 8.1 Hz, 2 H),
7.80 (d, J ) 8.2 Hz, 2 H), 4.56 (q, J ) 7.1 Hz, 2 H), 1.50 (t, J )
7.1 Hz, 3 H); MS (ES) m/z 303 (M + H+).

[1,2,4]Thiadiazole DeriVatiVes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 16 3961
5-(4-Trifluoromethoxyphenyl)[1,2,4]thiadiazole-3-carboxyl-
ic Acid Ethyl Ester (42). Using 4-trifluoromethoxybenzonitrile and
following the procedure as in the preparation of 38 gave 42 (34%)
as a yellow gummy solid: 1H NMR (300 MHz, CDCl3) δ 8.10 (m,
2 H), 7.37 (d, J ) 8.0 Hz, 2 H), 4.55 (q, J ) 7.1 Hz, 2 H), 1.49 (t,
J ) 7.1 Hz, 3 H); MS (ES) m/z 319 (M + H+).
General Procedure for the Synthesis of 39 and 43. [5-(4-
Trifluoromethylphenyl)[1,2,4]thiadiazol-3-yl]methanol (39). Us-
ing 38 and following the procedure as in the preparation of 11 gave
39 (93%) as a yellow solid: 1H NMR (300 MHz, CDCl3) δ 8.09
(d, J ) 8.1 Hz, 2 H), 7.78 (d, J ) 8.2 Hz, 2 H), 5.01 (s, 2 H); MS
(ES) m/z 261 (M + H+).
[5-(4-Trifluoromethoxyphenyl)[1,2,4]thiadiazol-3-yl]metha-
nol (43). Using 42 and following the procedure as in the preparation
of 39 gave 43 (94%) as a white solid: 1H NMR (400 MHz, CDCl3)
δ 8.01 (m, 2 H), 7.35 (d, J ) 8.2 Hz, 2 H), 4.99 (s, 2 H), 2.70 (brs,
1 H).
General Procedure for the Synthesis of 40 and 44. 3-Bro-
momethyl-5-(4-trifluoromethylphenyl)[1,2,4]thiadiazole (40). Us-
ing 39 and following the same procedure as in the preparation of
23 gave 40 (72%) as a light yellow oil: 1H NMR (400 MHz,
CDCl3) δ 8.10 (d, J ) 8.2 Hz, 2 H), 7.78 (d, J ) 8.2 Hz, 2 H),
4.71 (s, 2 H); MS (ES) m/z 323 (M + H+).
3-Bromomethyl-5-(4-trifluoromethoxyphenyl)[1,2,4]-
thiadiazole (44). Using 43 and following the same procedure as
in the preparation of 40 gave 44 (87%) as a clear oil: 1H NMR
(300 MHz, CDCl3) δ 8.02 (m, 2 H), 7.35 (d, J ) 8.1 Hz, 2 H),
4.69 (s, 2 H); MS (ES) m/z 341 (M + H+).
General Procedure for the Synthesis of 41 and 45. 2-Methyl-
2-{2-methyl-4-[5-(4-trifluoromethylphenyl)[1,2,4]thiadiazol-3-
ylmethylsulfanyl]phenoxy}propionic Acid (41). Using 40 and
following the same procedure as in the preparation of 24 gave 41
(50%): 1H NMR (300 MHz, CDCl3) δ 8.05 (d, J ) 8.1 Hz, 2 H),
7.76 (d, J ) 8.2 Hz, 2 H), 7.28 (d, J ) 2.3 Hz, 1 H), 7.18 (dd, J
) 8.5, 2.3 Hz, 1 H), 6.73 (d, J ) 8.5 Hz, 1 H), 4.37 (s, 2 H), 2.19
(s, 3 H), 1.60 (s, 6 H); MS (ES) m/z 469 (M + H+). Anal.
(C21H19F3N2O3S2) C, H, N.
The acetate (1.858 g, 6.63 mmol) was dissolved in THF (10 mL).
NaOEt in EtOH (2.68 M, 6.63 mmol) was added. The mixture was
stirred at 20 °C for 2 h and concentrated. The residue was
partitioned between EtOAc and H2O. The organic phase was dried
and concentrated. Purification by column chromatography (eluting
with EtOAc/hexane) provided compound 48 (1.28 g, 81%) as a
light yellow oil: 1HNMR (300 MHz, CDCl3) δ 6.62-6.65 (m, 2H),
6.48-6.52 (dd, J ) 8.7, 3.0 Hz, 1H), 4.51 (s, 1H), 4.22-4.29 (q,
J ) 7.1 Hz, 2H), 2.19 (s, 3H), 1.53 (s, 6H), 1.26-1.30 (t, J ) 7.1
Hz, 3H); MS (ES) m/z 261 (M + Na).
General Procedure for the Synthesis of 50 and 51. 2-Methyl-
2-{2-methyl-4-[3-(4-trifluoromethylphenyl)[1,2,4]thiadiazol-5-
ylmethoxy]phenoxy}propionic Acid (50). O-Alkylation of 48 with
the bromide 49 (CF3 analog of 23) and following the same
procedure as in the preparation of 24 gave 50 (71%) as a white
solid: 1H NMR (300 MHz, MeOH-d4) δ 8.48 (d, J ) 8.2 Hz, 2
H), 7.81 (d, J ) 8.3 Hz, 2 H), 6.95 (s, 1 H), 6.83 (s, 2 H), 5.56 (s,
2 H), 2.23 (s, 3 H), 1.54 (s, 6 H); MS (ES) m/z 453 (M + H+).
Anal. (C21H19F3N2O4S) C, H, N.
2-Methyl-2-{2-methyl-4-[3-(4-trifluoromethoxyphenyl)[1,2,4]-
thiadiazol-5-ylmethoxy]phenoxy}propionic Acid (51). O-Alky-
lation of 48 with the bromide 23 and following the same procedure
as in the preparation of 50 gave 51 (72%) as a white solid: 1H
NMR (300 MHz, CD3OD) δ 8.39 (d, J ) 8.8 Hz, 2 H), 7.41 (d, J
) 8.4 Hz, 2 H), 6.94 (d, J ) 2.5 Hz, 1 H), 6.83 (m, 2 H), 5.54 (s,
2 H), 2.23 (s, 3 H), 1.53 (s, 6 H); MS (ES) m/z 469 (M + H+).
Anal. (C21H19F3N2O5S) C, H, N.
1-Bromocyclopentanecarboxylic Acid Methyl Ester 53. To a
solution of cyclopentanecarboxylic acid (1.14 g, 10.0 mmol) in 1,2-
dichloroethane (50 mL) were added bromine (1.60 g, 10.0 mmol)
and chlorosulfonic acid (1.17 g, 10.0 mmol). The reaction mixture
was refluxed for 2 h and concentrated. The residue was dissolved
in MeOH (30 mL) and the mixture was refluxed overnight. After
removal of the solvent, the residue was diluted with Et2O, washed
with water (×2) and brine, dried over Na2SO4, filtered, and
concentrated to give 1.79 g (86%) of 53: 1H NMR (300 MHz,
CDCl3) δ 3.80 (s, 3 H), 2.33-2.28 (m, 4 H), 2.03-1.95 (m, 2 H),
1.85-1.75 (m, 2 H).
2-Methyl-2-{2-methyl-4-[5-(4-trifluoromethoxyphenyl)[1,2,4]-
thiadiazol-3-ylmethylsulfanyl]phenoxy}propionic Acid (45). Us-
ing 44 and following the same procedure as in the preparation of
41 gave 45 (74%) as a white gummy solid: 1H NMR (300 MHz,
CDCl3) δ 7.97 (m, 2 H), 7.33 (d, J ) 8.1 Hz, 2 H), 7.27 (s, 1 H),
7.17 (dd, J ) 8.5, 2.4 Hz, 1 H), 6.73 (d, J ) 8.5 Hz, 1 H), 4.35 (s,
2 H), 2.19 (s, 3 H), 1.60 (s, 6 H); MS (ES) m/z 485 (M + H+).
Anal. (C21H19F3N2O4S2) C, H, N.
2-(4-Acetyl-2-methylphenoxy)-2-methylpropionic Acid Ethyl
Ester (47). A mixture of 2-bromo-2-methylpropionic acid ethyl ester
(7.93 g, 40.65 mmol), 46 (6.10 g, 40.65 mmol), and cesium
carbonate (13.2 g, 40.65 mmol) was refluxed in dioxane (100 mL)
for 18 h. After cooling, the mixture was partitioned between ethyl
acetate and water. The organic layers were dried and concentrated.
Purification by column chromatography (EtOAc/hexane) gave
compound 47 (6.169 g, 57%) as a colorless oil: 1H NMR (300
MHz, CDCl3) δ 7.78 (d, J ) 1.5 Hz, 1H), 7.67-7.70 (dd, J ) 2.1,
8.6 Hz, 1H), 6.60-6.63 (d, J ) 8.6 Hz, 1H), 4.19-4.26 (q, J )
7.1 Hz, 2H), 2.53 (s, 3H), 2.27 (s, 3H), 1.66 (s, 6H), 1.24 (t, J )
7.1 Hz, 3H); MS (ES) m/z 265 (M + H+).
2-(4-Hydroxy-2-methylphenoxy)-2-methylpropionic Acid Eth-
yl Ester (48). Compound 47 (2.14 g, 8.10 mmol) was dissolved in
CH2Cl2 (25 mL). mCPBA (77 wt %, 3.18 g, 14.17 mmol) and TsOH
monohydrate (154 mg, 0.81 mmol) were added. The mixture was
stirred at refluxing for 4 h and cooled. Solid Na2S2O3 was added,
followed by saturated aqueous Na2S2O3, NaHCO3, and brine. The
organic phase was dried and evaporated. Purification by column
chromatography (eluting with EtOAc/hexane) gave acetate (1.858
g, 82%) as a light yellow oil: 1HNMR (300 MHz, CDCl3) δ 6.88
(d, J ) 2.8 Hz, 1H), 6.74-6.78 (dd, J ) 2.8, 8.8 Hz, 1H), 6.66 (d,
J ) 8.8 Hz, 1H), 4.20-4.27 (q, J ) 7.1 Hz, 2H), 2.26 (s, 3H),
2.22 (s, 3H), 1.58 (s, 6H), 1.23-1.27 (t, J ) 7.1 Hz, 3H); MS
(ES) m/z 298 (M + H+).
1-o-Tolyloxycyclopentanecarboxylic Acid Methyl Ester 54.
To a solution of 2-methylphenol (90 mg, 0.83 mmol) in CH3CN
(2 mL) was added Cs2CO3 (678 mg, 2.08 mmol), followed by 53
(207 mg, 1.0 mmol). The mixture was heated at 70 °C for 4.5 h,
diluted with water, and extracted with Et2O. The extracts were dried,
concentrated, and column chromatographed (EtOAc/hexane 1/12)
to give 52 mg (22%) of 54: 1H NMR (300 MHz, CDCl3) δ 7.13
(d, J ) 7.3 Hz, 1 H), 7.03 (m, 1 H), 6.83 (td, J ) 7.4, 0.7 Hz, 1
H), 6.45 (d, J ) 8.1 Hz, 1 H), 3.73 (s, 3 H), 2.29-2.17 (m, 4 H),
2.23 (s, 3 H), 1.84-1.76 (m, 4 H); MS (ES) m/z 235 (M + H+).
1-(4-Bromo-2-methylphenoxy)cyclopentanecarboxylic Acid
Methyl Ester 55. To a solution of 54 (76 mg, 0.32 mmol) in CH3-
CN (1.5 mL) was added N-bromosuccinimide (63 mg, 0.35 mmol).
After stirring at room temperature for 1.5 h, additional N-
bromosuccinimide (29 mg, 0.16 mmol) was added and the mixture
was stirred for another 1 h. After removal of the solvent under
reduced pressure, the residue was purified by column chromatog-
raphy (CH2Cl2/hexane 1/1) to provide 95 mg (95%) of 55: 1H NMR
(300 MHz, CDCl3) δ 7.25 (d, J ) 0.5 Hz, 1 H), 7.13 (dd, J ) 8.6,
2.4 Hz, 1 H), 6.32 (d, J ) 8.7 Hz, 1 H), 3.73 (s, 3 H), 2.28-2.12
(m, 4 H), 2.19 (s, 3 H), 1.82-1.77 (m, 4 H); MS (ES) m/z 314 (M
+ H+).
1-(2-Methyl-4-triisopropylsilanylsulfanylphenoxy)-
cyclopentanecarboxylic Acid Methyl Ester 56. To a suspension
of NaH (17 mg, 0.43 mmol; 60% in mineral oil) in toluene (1.5
mL) was added triisopropylsilanethiol (82 mg, 0.43 mmol). After
stirring at room temperature for 30 min, to the mixture were added
a solution of 55 (122 mg, 0.390 mmol) in THF (1 mL) and tetrakis-
(triphenylphosphine) palladium (45 mg, 0.039 mmol), and the
mixture was degassed under N2. After heating at 90 °C for 4 h, the
solvents were evaporated and the residue was purified by column
chromatography (EtOAc/hexane 1/8) to afford 135 mg (82%) of
56: 1H NMR (300 MHz, CDCl3) δ 7.25 (d, J ) 1.8 Hz, 1 H), 7.13

3962 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 16
Shen et al.
(dd, J ) 8.5, 2.3 Hz, 1 H), 6.32 (d, J ) 8.5 Hz, 1 H), 3.70 (s, 3 H),
2.25-2.13 (m, 4 H), 2.16 (s, 3 H), 1.82-1.77 (m, 4 H), 1.27-
1.19 (m, 3 H), 1.07 (s, 9 H), 1.04 (s, 9 H); MS (ES) m/z 423 (M
+ H+).
reverse primers, and TaqMan probe. The reaction was performed
in a DNA sequence detector (Model 7900, Applied BioSystems)
with the conditions of 50 °C for 2 min and 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 quatitation, the measurement 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 manufacturer’s guidelines
and the value was used to determine the ∆Ct (Ct of the target gene
1-{2-Methyl-4-[3-(4-trifluoromethoxyphenyl)[1,2,4]thiadiazol-
5-ylmethylsulfanyl]phenoxy}cyclopentanecarboxylic Acid 57. To
a mixture of 23 (53 mg, 0.18 mmol) and 56 (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 column chromatography to afford
88 mg (94%) of 57-methyl ester as a clear oil: 1H NMR (300
MHz, CDCl3) δ 8.27 (d, J ) 8.9 Hz, 2 H), 7.30-7.26 (m, 3 H),
7.14 (dd, J ) 8.5, 2.2 Hz, 1 H), 6.37 (d, J ) 8.5 Hz, 1 H), 4.39 (s,
2 H), 3.70 (s, 3 H), 2.27-2.12 (m, 4 H), 2.18 (s, 3 H), 1.80-1.76
(m, 4 H); MS (ES) m/z 525 (M + H+).
minus the Ct of 18S ribosomal RNA controls). Relative mRNA
Ct
level was calculated using the equation 2-∆∆
.
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 orally
dosed with vehicle during the treatment period. The diets continued
during the treatment period. The compound was formulated in 0.5%
Methocel, 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 retro-orbital sinus. Serum
was prepared and the lipid parameters were measured using a
COBAS analyzer (Roche Diagnostics). The animals were then
sacrificed, and the livers were removed (from six animals per group)
and weighed.
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 24 in
0.5% methylcellulose 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 conditions and measured
using a COBAS Mira Plus blood chemistry analyzer (Roche
Diagnostic Systems, Indianapolis, IN). Statistical analysis was
performed using the program Prism (Graphpad, Monrovia, CA) and
with one-way analysis of variance and Dunnett’s multiple com-
parison test.
Pharmacokinetic Assay. Rats were dosed intravenously (iv) at
levels of 1-3 mg/kg and by oral gavage at a level of 3-10 mg/kg
with drug candidates. Drug compound was formulated for iv dosing
as a solution in 10% w/v Solutol in 5% dextrose in sterile water
vehicle (D5W). Drug compound was formulated for oral dosing as
a uniform suspension in 0.5% methylcellulose vehicle. Blood
samples (0.5 mL) were collected into heparinized tubes post dose
via orbital sinus puncture. Blood samples were centrifuged for cell
removal, and precisely 200 µL of plasma supernatant was then
transferred to a clean vial, placed on dry ice, and subsequently stored
in a -70 °C freezer prior to analysis. Plasma samples were prepared
as follows. Four hundred microliters of acetonitrile containing
internal standard was added to 200 µL of plasma to precipitate
proteins. Samples were centrifuged at 5000g for 3 min, and
supernatant was removed for analysis by LC-MS-MS. Calibration
standards were prepared by adding appropriate volumes of stock
solution directly into plasma and treated identically to collected
plasma samples. Calibration standards were prepared in the range
from 0.1 to 10 mM for quantitation. LC-MS-MS analysis was
performed using either multiple reaction or selected ion monitoring
for detection of characteristic ions for each drug candidate, and
the internal standard used was Propranolol.
Using 57-methyl ester and follow the same base hydrolysis
procedure as in the preparation of 50 gave 57 (68%) as a colorless
oil: 1H NMR (400 MHz, CDCl3) δ 8.27 (m, 2 H), 7.30-7.27 (m,
3 H), 7.17 (dd, J ) 8.5, 2.3 Hz, 1 H), 6.50 (d, J ) 8.5 Hz, 1 H),
4.40 (s, 2 H), 2.36-2.27 (m, 2 H), 2.21-2.15 (m, 2 H), 2.18 (s, 3
H), 1.82-1.78 (m, 4 H). Anal. (C23H21F3N2O4S2) C, H, N.
Biology. PPARs Assays. All cell culture and cotransfection
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 (Promega; 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.
Then the cells were cotransfected with DNA constructs containing
the ligand-binding domains of either PPARR, PPARγ, or δ-Gal4
chimeric receptors and Gal4-luciferase reporter using DMRIE-C
transfection reagent and OPTI-MEM reduced serum medium
according to the manufacturer’s instructions. On the following day,
the DNA-containing medium was replaced with 30 mL of 5%
charcoal-treated FBS growth medium. After 6 h, the cells were
replated 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 µL 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 Lu-
ciferase Assay kit according to the manufacturer’s instructions.
Induction of Selected Genes. Human peripheral blood lym-
phocytes were freshly prepared (ABS Inc., Wilmington, DE) and
total RNA was isolated from the cells following manufacturer’s
instruction (Trizon method, Invitrogen Inc.). PCR primers and
fluorescent probes (TagMan probe) were designed using the
software PrimerDesign (Applied Biosystems, Foster City, CA). The
sequences of the primers and TaqMan probe for PDHK4 (GanBank
accession number U54617) are 5′-TGCATTTTTGCGACAAGAAT-
TG (forward), 5′-TTGGGTCGGGAGGATATCAA (reverse), and
5-FAM-CTGTGAGACTCGCCAACATTCTGAAGGA-TAMRA-
3. The sequences of the primers and TaqMan probe for CPT1
(GanBank accession number NM_001876) are 5′-CATTCCTTC-
CCATTCGTAGC (forward), 5′-AGCTGCACAAAGGCGTCTG
(reverse), and 5-FAM-AGGAATCATCAAGAAATGTCGCAC-
GAGC-TAMRA-3 (TaqMan). They were synthesized by Keystone
Labs (Camarillo, CA). Reverse transcription (RT) was conducted
following manufacturer’s instruction using TaqMan reverse tran-
scription reagents (Applied BioSystems). The RT reaction was 20
µL and contained 1× RT buffer, 5.5 mM MgCl2, 0.5 mM dNTP,
2.5 µM of random hexamer, 1 unit of RNase Inhibitor, 1 unit of
MultisScribe RT enzyme, and 0.5 µg of total RNA. The reaction
was carried out at 48 °C for 30 min, and then 95 °C for 5 min, and
finally 4 °C. The reaction volume was extended into 62.5 µL, and
5 µL of the RT (equivalent to 40 ng of total RNA) was used for
PCR amplification. Each reaction was carried out a total volume
of 25 µL containing 5 µL of RT, 1× Master Mix (reaction buffer,
dNTP, polymerase, Applied Biosystems), 0.2 µM of forward,
Acknowledgment. We thank the Chemical and Biological
Support Team at Raritan of J&JPRD for the pharmacokinetic
study of compound 24.
Supporting Information Available: Elemental analysis data
for target molecules, PPARR agonist (compound 2.1), and PPARδ
agonist (compound f) are included. This material is available free
of charge via the Internet at http://pubs.acs.org.

[1,2,4]Thiadiazole DeriVatiVes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 16 3963
derivatives as human peroxisome proliferator-activated receptor
(PPAR) R/δ dual agonists for the treatment of metabolic syndrome.
Bioorg. Med. Chem. 2006, 14, 8405-8414.
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