Synthesis and evaluation of 2-nonylaminopyridine derivatives as PPAR ligands

Article abstract of DOI:10.1248/cpb.55.1053

To find novel PPAR ligands, we prepared several 3-{3 or 4-[2-(nonylpyridin-2-ylamino)ethoxy]phenyl}-propanoic acid derivatives which were designed based on the structure of our previous PPARγ ligand 1. In PPAR binding affinity assays, compound 4, which ha

Full text of DOI:10.1248/cpb.55.1053

July 2007
Chem. Pharm. Bull. 55(7) 1053—1059 (2007)
1053
Synthesis and Evaluation of 2-Nonylaminopyridine Derivatives as PPAR
Ligands
Shinya USUI,a Hiroki FUJIEDA,a Takayoshi SUZUKI,a Naoaki YOSHIDA,a Hidehiko NAKAGAWA,a
b
b
,a
*
Michitaka OGURA, Makoto MAKISHIMA, and Naoki MIYATA
a Graduate School of Pharmaceutical Sciences, Nagoya City University; 3–1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi
467–8603, Japan: and b Nihon University School of Medicine; 30–1 Oyaguchi-kamicho, Itabashi-ku, Tokyo 173–8610,
Japan. Received March 26, 2007; accepted May 7, 2007; published online May 9, 2007
To find novel PPAR ligands, we prepared several 3-{3 or 4-[2-(nonylpyridin-2-ylamino)ethoxy]phenyl}-
propanoic acid derivatives which were designed based on the structure of our previous PPARg ligand 1. In PPAR
binding affinity assays, compound 4, which had an ethoxy group at the C-2 position of the propanoic acid of 1,
showed selective binding affinity for PPARg. Compound 3, with an ethyl group at the C-2 position, was found to
be a PPARa/g dual ligand. Compound 6, the meta isomer of 1, has been shown to be a PPARa ligand. The intro-
duction of methyl (7) and ethyl (8) groups to the C-2 position of the propanoic acid of 6 further improved
PPARa-binding potency. In cell-based transactivation assay, compounds 3 and 4 showed dual-agonist activity to-
ward PPARa and PPARg. Compound 6 was found to be a triple agonist and compound 8 proved to be a selective
PPARa agonist. In the human hypodermic preadipocyte differentiation test, it was demonstrated that the maxi-
mal activity of compounds 3 and 4 was higher than that of rosiglitazone.
Key words peroxisome proliferator-activated receptor (PPAR); agonist; selectivity
The peroxisome proliferator-activated receptors (PPARa, ever, the use of TZDs has been limited because of their seri-
PPARg and PPARd) are a set of ligand-activated transcrip- ous side effects such as hepatic toxicity, weight gain and
tion factors in the nuclear hormone receptor superfamily.1—4) edema. Meanwhile, PPARa is highly expressed in metaboli-
These receptors regulate the expression of a large number of cally active tissues such as the liver, heart and muscle, and
genes involved in lipid metabolism and energy balance by regulates lipid homeostasis. PPARa agonists such as clofi-
binding to a DNA sequence termed PPAR response brate (Fig. 1) have demonstrated the ability to reduce serum
elements.5) The PPARg is predominantly expressed in adi- triglyceride and increase HDL cholesterol levels,9) and are
pose tissues, and plays a pivotal role in adipose differentia- being utilized as hypolipidemic agents. In addition, recent
tion, and the regulation of glucose and lipid homeostasis. studies revealed that dual agonists of PPARa/g decrease the
The clinically useful thiazolidinedione (TZD) class of insulin free triglyceride plasma concentration and increase plasma
sensitizers such as rosiglitazone6) and pioglitazone7) (Fig. 1) HDL concentration in an insulin-resistant animal model.10,11)
are potent PPARg agonists used in the treatment of Type 2 Thus, many groups have ongoing research programs to iden-
diabetes. TZDs are known to improve insulin resistance, tify more potent and less toxic PPARa agonists, PPARg ago-
which is a key underlying feature of Type 2 diabetes8); how- nists and PPARa/g dual agonists.
We previously reported compound 1 (Fig. 1), which was
designed based on the structure of rosiglitazone and 15d-
PGJ2,12,13) as a potent PPARg ligand.14) To find more potent
PPARg agonists and novel PPARa agonists, we chose com-
pound 1 as the lead structure, because recent reports indi-
cated that PPARg affinity can be increased by the introduc-
tion of substituents into the C-2 position of propanoic
acid,15—19) and minor structural modifications can convert
PPAR subtype selectivity.20—22) In this article, we report the
synthesis, binding affinity and biological activity of PPAR
ligands based on the structure of compound 1.
Chemistry The compounds prepared for this study are
shown in Fig. 2, and the routes used for synthesis are shown
in Charts 1—3. Chart 1 shows the preparation of compounds
1, 3, 4, 6—10, 12 and 13. The 2-nonylaminopyridine 18 was
prepared by the method of Buchwald23): treatment of n-nonyl-
amine 17 with 2-bromopyridine, Pd2(DBA)3, BINAP, and t-
BuONa in toluene at 105 °C. p-Hydroxybenzaldehyde 19a,
m-hydroxybenzaldehyde 19b and isovaniline 19c were al-
lowed to react with 1,2-dibromoethane to give ethers 20a—c.
The Horner–Wadsworth–Emmons reaction24) was applied to
Fig. 1. Structures of Rosiglitazone, Pioglitazone, 15d-PGJ2, Clofibrate,
the conversion of 20a—c into acrylic acid ethyl esters 21a—
GW7647, GW501516 and Compound 1
∗ To whom correspondence should be addressed. e-mail: miyata-n@phar.nagoya-cu.ac.jp
© 2007 Pharmaceutical Society of Japan

1054
Vol. 55, No. 7
j. The double bonds of 21a—j were hydrogenated to yield (2-pyridinyl)-N-nonylpropanoic acid ethyl esters 23a—j. The
compounds 22a—j. Coupling between 2-nonylaminopyri- subsequent hydrolysis of 23a—j gave the desired carboxylic
dine 18 and propanoic acid ethyl esters 22a—j afforded N- acids 1, 3, 4, 6—10, 12 and 13.
The preparation of compounds 2, 5, 11 and 14, which have
one or two methyl groups at the C-2 position of propanoic
acid, is outlined in Chart 2. Aldehydes 20a and 20c were re-
duced by NaBH4 and allowed to react with acetic anhydride
to give 25a and 25b. Compounds 25a and 25b were treated
with 1-methoxy-1-trimethylsilyloxypropene or dimethyl-
ketene methyltrimethylsilyl acetal in the presence of magne-
sium perchlorate in anhydrous CH2Cl2 to give esters 26a—
d.25) Coupling between 2-nonylaminopyridine 18 and
propanoic acid methyl esters 26a—d afforded N-(2-
pyridinyl)-N-nonyl compounds 27a—d and subsequent hy-
drolysis gave carboxylic acids 2, 5, 11 and 14.
Preparation of the acrylic acid derivatives 15 and 16 is
shown in Chart 3. Acrylic acid ethyl esters 21a and 21h were
allowed to react with 2-nonylaminopyridine 18 to give com-
pounds 28a and 28b. Treatment of 28a and 28b with aqueous
NaOH gave N-(2-pyridinyl)-N-nonyl acrylic acids 15 and 16.
Fig. 2. Structures of Compounds 1—16
(a) 2-bromopyridine, Pd2(DBA)3, BINAP, t-BuOH, toluene, 105 °C, 70%; (b) 1,2-dibromoethane, Cs2CO3, THF, 65 °C, 33—57%; (c) (EtO)2P(O)CH(R)CO2Et, NaH, anhydrous
THF, 0 °C to rt, 47—95%; (d) H2, Pd/C, EtOH, 79—97%; (e) 18, Et3N, KI, 105 °C, 9—17%; (f) 2 N aq. NaOH, EtOH, THF, rt, 82—100%.
Chart 1

July 2007
1055
Fig. 3. Binding Affinity for PPARg of Compounds 1—16 at 0.1, 1.0 and
10 mM
Values are the means of at least three experiments.
(a) NaBH4, EtOH, rt, 81—95%; (b) Ac2O, DMAP, rt, 96—97%; (c) 1-methoxy-1-
trimethylsilyloxypropene, or dimethylketene methyltrimethylsilyl acetal, Mg(ClO4)2, rt,
90—94%; (d) 18, Et3N, KI, 105 °C, 5—13%; (e) 2 N aq. NaOH, EtOH, rt, 75—97%.
Chart 2
Fig. 4. Binding Affinity for PPARa of Compounds 1—16 at 0.1, 1.0 and
10 mM
Values are the means of at least three experiments.
and 5, respectively. GW764727) (PPARa), rosiglitazone6)
(PPARg) and GW50151628) (PPARd) were used as reference
compounds (Fig. 1).
The lead compound 1 showed relatively high affinity for
PPARg and little affinity for PPARa and PPARd (Fig. 3—5).
We initially examined the effect of substituents at the C-2 po-
sition of the propanoic acid of 1, because it has been reported
that the introduction of an alkyl or an alkoxy group at this
position increases the activity of PPARg and PPARa.15—19)
Among compound 1, methyl 2, ethyl 3, ethoxy 4, and di-
methyl 5, compound 4 showed the strongest affinity for
PPARg, so compound 4 was founded to be a potent and se-
(a) 18, Et3N, KI, 105 °C, 9—13%; (b) 2 N aq. NaOH, EtOH, THF, rt, 82—99%.
Chart 3
Results and Discussion
The binding affinity of the compounds for PPARs was lective PPARg ligand (Figs. 3—5). In addition, the PPARg
evaluated with the CoA-BAP System (Microsystems).26) In affinity of compound 3 was comparable to that of compound
this system, alkaline phosphatase (AP) activity is directly 1, whereas compound 3 displayed strong affinity for PPARa
proportional to the affinity of the ligands for PPARs. The as compared with 1 (Figs. 3, 4).
abilities of compounds 1—16 to bind PPARa, PPARg and
Next, we investigated the PPARs affinity of compound 6,
PPARd were evaluated and the results are shown in Figs. 3, 4 the meta isomer of compound 1.29) Compound 6 showed

1056
Vol. 55, No. 7
Table 1. In Vitro Functional PPAR Transactivation Activity of Compounds
EC50a) (mM)
Compound
R
Position
PPARg
PPARd
PPARa
1
3
4
6
7
8
H
Et
OEt
H
Me
Et
p
p
p
m
m
m
>10
2.62
0.32
7.76
4.72
>8
>10
>10
>10
6.42
>10
>10
>10
3.68
0.74
3.20
2.65
1.81
a) Compounds were screened for agonist activity on PPAR-GAL4 chimeric receptors
in transiently transfected HEK-293 cells as described. EC50 value is the molar concen-
tration of the test compound that affords 50% of maximal reporter activity.33)
Fig. 5. Binding Affinity for PPARd of Compounds 1—16 at 0.1, 1.0 and
10 mM
Values are the means of at least three experiments.
much higher affinity for PPARa than 1 (Fig. 4). Further-
more, the affinity for PPARg of 6 was lower than that of 1
(Fig. 3), and compound 6 exhibited little affinity for PPARd
(Fig. 5). To find more potent PPARa ligands, we prepared
and tested compounds 7, 8 and 9 in which a methyl, ethyl
and ethoxy group was substituted at the C-2 position of the
propanoic acid of 6, respectively. Compounds 7 and 8
showed strong affinity for PPARa and compound 8 displayed
slightly weak affinity for PPARg as compared with the parent
compound 6. The effect of the introduction of a methoxy
group at the C-4 position of the benzene ring of 6 was also
investigated; however, compounds 10—14 showed no pro-
nounced affinity for PPARs as compared to compound 6
(Figs. 3—5).
Fig. 6. Accumulation of Fatty Acid in Human Preadipocytes by Com-
pounds 3, 4 and Rosiglitazone
Values are the means of at least three experiments.
The acrylic acid derivatives 15 and 16 did not show no- of PPARg enhances adipocyte differentiation31) and increases
table affinity for PPARs (Figs. 3—5). insulin sensitivity, compounds 3 and 4 were subjected to a
Based on the findings in the binding assay, we next investi- human hypodermic preadipocyte differentiation test.32) The
gated the PPAR transactivation activity of compounds 1, 3, 4, accumulation of triglycerides in the cells was observed after
6, 7, and 8 by reporter gene assay30) (Table 1). We initially the administration of compounds 3 and 4 at concentrations of
tested the activity of compound 1, which did not show signif- 0, 0.613, 1.25, 2.5 and 5 mM, and the activity of compounds 3
icant transcriptional activity for PPARa, d, and g. We then and 4 was found to be higher than that of rosiglitazone at
examined the activity of compounds 3 and 4, which have an 1 mM and higher concentrations (Fig. 6).
ethyl or ethoxy group at the C-2 position of the propanoic
acid of 1. As expected from the binding assay, compound 3 Conclusion
was a PPARa/g dual agonist. Although the binding assay in-
To find novel PPAR ligands, we prepared several 2-nonyl-
dicated that compound 4 is a selective PPARg ligand, it aminopyridine derivatives which were designed based on the
showed potent dual-agonist activity toward PPARa and structure of a PPARg ligand 1. In PPAR binding affinity as-
PPARg. Compound 6, the meta isomer of compound 1, was says, compound 4, which had an ethoxy group at the C-2 po-
found to be a triple agonist. Compound 7, which had an in- sition of the propanoic acid of 1, showed high binding affin-
troduced methyl group at the C-2 position of the propanoic ity for PPARg and little affinity for PPARa and PPARd.
acid moiety of 6, improved the EC50 values and selectivity Compound 3, which had an ethyl group at the C-2 position of
for PPARa and PPARg, and the introduction of ethyl group propanoic acid, was found to be a PPARa/g dual ligand.
(compound 8) increased the activity and selectivity for Compound 6, the meta isomer of 1, has been shown to be a
PPARa. Among these compounds, compound 8 showed the PPARa ligand. The introduction of methyl (7) and ethyl (8)
highest selectivity for PPARa.
groups at the C-2 position of the propanoic acid of 6 further
As compounds 3 and 4 were found to have relatively high improved PPARa-binding potency. In cell-based transactiva-
PPARg agonist activity in our study, we used them for fur- tion assay, compounds 3 and 4 showed dual-agonist activity
ther evaluation. Since it has been reported that the activation toward PPARa and PPARg. Compound 6 was found to be a

July 2007
1057
triple agonist and compound 8 proved to be a selective (toluene : CHCl3 : AcOEtꢀ45 : 5 : 3) gave 55 mg (11%) of 23a as a colorless
oil: 1H-NMR (CDCl3, 500 MHz, d: ppm) 8.13 (1H, ddd, Jꢀ4.8, 1.8, 0.9 Hz),
PPARa agonist. In the human hypodermic preadipocyte dif-
7.40 (1H, ddd, Jꢀ8.8, 7.0, 1.8 Hz), 7.09 (2H, d, Jꢀ8.5 Hz), 6.83 (2H, dt,
ferentiation test, it was demonstrated that the activity of com-
Jꢀ8.5, 2.7 Hz), 6.50 (2H, m), 4.14 (2H, t, Jꢀ6.1 Hz), 4.11 (2H, q,
Jꢀ7.3 Hz), 3.91 (2H, t, Jꢀ6.1 Hz), 3.47 (2H, t, Jꢀ7.9 Hz), 2.87 (2H, t,
pounds 3 and 4 was higher than that of rosiglitazone. The
findings of this study will help provide an effective agent for
Type 2 diabetes and hyperlipidemia.
Jꢀ7.6 Hz), 2.57 (2H, t, Jꢀ7.9 Hz), 1.66—1.59 (2H, m), 1.36—1.20 (12H,
m), 1.23 (3H, t, Jꢀ7.0 Hz), 0.88 (3H, t, Jꢀ6.7 Hz); HR-MS Calcd for
C27H40N2O3 440.3039, Found 440.3036.
Experimental
3-{4-[2-(Nonylpyridin-2-ylamino)ethoxy]phenyl}propanoic Acid (1)
(Chart 1) To a solution of 23a (49 mg, 0.119 mmol) in 1.0 ml of THF and
1.0 ml of EtOH was added a solution of 2 N aqueous NaOH (0.60 ml,
1.19 mmol). The reaction mixture was stirred for 1 d at room temperature,
and neutralized to pH 7 with 2 N aqueous HCl. The mixture was subjected to
silica gel flash chromatography (CHCl3/MeOHꢀ19/1) to give 44 mg (90%)
Melting points were determined using a Yanagimoto micro melting point
apparatus or a Büchi 545 melting point apparatus and were left uncorrected.
Proton nuclear magnetic resonance spectra (1H-NMR) were recorded on a
JEOL JNM-LA500 spectrometer in solvent as indicated. Chemical shifts (d)
were reported in parts per million relative to the internal standard tetra-
methylsilane. High-resolution mass spectra (HR-MS) were recorded on a
JEOL JMS-SX102A mass spectrometer. Reagents and solvents were pur-
chased from Aldrich, Tokyo Kasei Kogyo, Wako Pure Chemical Industries,
and Kanto Kagaku, and used without purification. Flash column chromatog-
raphy was performed using Silica Gel 60 (particle size 0.046—0.063 mm)
supplied by Merck.
1
of 1 as a colorless oil: H-NMR (CDCl3, 500 MHz, d: ppm) 8.14 (1H, dd
Jꢀ4.9, 1.2 Hz), 7.45 (1H, t, Jꢀ7.1 Hz), 7.10 (2H, d, Jꢀ8.5 Hz), 6.81 (2H, d,
Jꢀ8.5 Hz), 6.54 (2H, m), 4.12 (2H, t, Jꢀ5.6 Hz), 3.93 (2H, t, Jꢀ5.6 Hz),
3.50 (2H, t, Jꢀ7.9 Hz), 2.88 (2H, t, Jꢀ7.8 Hz), 2.61 (2H, t, Jꢀ7.8 Hz),
1.66—1.62 (2H, m), 1.35—1.25 (12H, m), 0.88 (3H, t, Jꢀ6.8 Hz); MS (EI)
m/z: 412 (Mꢁ); HR-MS Calcd for C25H36N2O3 412.2727, Found 412.2726.
2-Nonylaminopyridine (18) To a solution of 2-bromopyridine (0.9 ml,
9.20 mmol) in 10 ml of anhydrous toluene were added n-nonylamine (17,
10.0 ml, 55.2 mmol), Pd2(DBA)3 (0.169 g, 0.18 mmol), rac-BINAP (0.229 g,
0.37 mmol) and sodium tert-butoxide (1.24 g, 12.9 mmol). The reaction mix-
ture was stirred at 105 °C for 3 d under Ar, and poured into water. The whole
was extracted with AcOEt. The AcOEt layer was separated, washed with
water and brine, and dried over Na2SO4. The solvent was removed by evapo-
ration in vacuo. Purification by silica gel flash chromatography (AcOEt/n-
2-{4-[2-(Nonylpyridin-2-ylamino)ethoxy]benzyl}butyric
Acid
(3)
Compound 3 was prepared from 19a using the procedure described for 1 in
2.5% yield. In the step of the synthesis of 21b, 2-(diethoxyphosphoryl)bu-
tyric acid ethyl ester was used instead of ethyl diethylphosphono acetate:
colorless oil; 1H-NMR (CDCl3, 500 MHz, d: ppm) 8.11 (1H, ddd, Jꢀ4.9,
1.8, 0.9 Hz), 7.41 (1H, ddd, Jꢀ8.8, 7.0, 1.8 Hz), 7.08 (2H, d, Jꢀ8.5 Hz),
6.79 (2H, d, Jꢀ8.5 Hz), 6.50 (1H, dd, Jꢀ7.0, 4.8 Hz), 6.48 (1H, d,
Jꢀ7.9 Hz), 4.05 (1H, m), 3.99 (1H, m), 3.85 (2H, m), 3.46 (2H, t,
Jꢀ7.9 Hz), 2.88 (1H, dd, Jꢀ13.7, 8.5 Hz), 2.70 (1H, dd, Jꢀ13.7, 6.4 Hz),
2.58 (1H, m), 1.68—1.55 (4H, m), 1.31—1.21 (12H, m), 0.96 (3H, t,
Jꢀ7.6 Hz), 0.88 (3H, t, Jꢀ6.7 Hz); HR-MS Calcd for C27H40N2O3 440.3039,
Found 440.3029.
1
hexaneꢀ1/5) gave 1.43 g (70%) of 18 as a yellow solid: H-NMR (CDCl3,
500 MHz, d: ppm) 8.07 (1H, dd, Jꢀ4.9, 1.5 Hz), 7.41 (1H, ddd, Jꢀ8.5, 6.7,
1.8 Hz), 6.55 (1H, dd, Jꢀ6.7, 5.5 Hz), 6.37 (1H, d, Jꢀ8.2 Hz), 4.45 (1H, m),
3.24 (2H, m), 1.65—1.59 (2H, m), 1.41—1.27 (12H, m), 0.88 (3H, t,
Jꢀ6.9 Hz).
2-Ethoxy-3-{4-[2-(nonylpyridin-2-ylamino)ethoxy]phenyl}propanoic
Acid (4) Compound 4 was prepared from 19a using the procedure de-
scribed for 1 in 2.1% yield. In the step of the synthesis of 21c, 2-(di-
ethoxyphosphoryl)ethoxyacetic acid ethyl ether was used instead of ethyl di-
4-(2-Bromoethoxy)benzaldehyde (20a) To a solution of p-hydroxy-
benzaldehyde (19a, 1.25 g, 10.0 mmol) in 13 ml of THF were added Cs2CO3
(4.28 g, 13.0 mmol) and 1,2-dibromoethane (1.76 ml, 20.0 mmol). The mix-
ture was stirred at 65 °C for 14 h. The mixture was poured into 2 N aqueous
NaOH. The mixture was extracted with AcOEt. The AcOEt layer was sepa-
rated, washed with water and brine, and dried over Na2SO4. The solvent was
removed by evaporation in vacuo. Purification by silica gel flash chromatog-
raphy (AcOEt/n-hexaneꢀ1/3) gave 0.758 g (33%) of 21a as a light yellow
solid: 1H-NMR (CDCl3, 500 MHz, d: ppm) 9.90 (1H, s), 7.85 (2H, d,
Jꢀ8.5 Hz), 7.02 (2H, d, Jꢀ8.8 Hz), 4.38 (2H, t, Jꢀ6.1 Hz), 3.67 (2H, t,
Jꢀ6.1 Hz).
1
ethylphosphono acetate: colorless oil; H-NMR (CDCl3, 500 MHz, d: ppm)
8.12 (1H, dd, Jꢀ4.9, 1.2 Hz), 7.43 (1H, ddd, Jꢀ8.8, 7.0, 1.8 Hz), 7.28 (2H,
d, Jꢀ8.5 Hz), 6.80 (2H, d, Jꢀ8.5 Hz), 6.51 (2H, m), 4.05 (2H, m), 3.90 (2H,
m), 3.60 (1H, m), 3.47 (2H, t, Jꢀ7.6 Hz), 3.05 (1H, dd, Jꢀ14.0, 4.5 Hz),
2.94 (1H, dd, 14.3, 7.6 Hz), 1.65—1.59 (2H, m), 1.35—1.20 (12H, m), 1.17
(3H, t, Jꢀ7.0 Hz), 1.17 (3H, t, Jꢀ7.0 Hz), 0.88 (3H, t, Jꢀ7.0 Hz); HR-MS
Calcd for C27H40N2O4 456.2988, Found 456.2999.
3-{3-[2-(Nonylpyridin-2-ylamino)ethoxy]phenyl}propanoic Acid (6)
Compound 6 was prepared from 19b using the procedure described for 1 in
7.7% yield: colorless oil; 1H-NMR (CDCl3, 500 MHz, d: ppm) 8.14 (1H,
ddd, Jꢀ4.9, 2.0, 0.8 Hz), 7.42 (1H, ddd, Jꢀ8.5, 6.7, 2.1 Hz), 7.18 (1H, t,
Jꢀ7.9 Hz), 6.75 (3H, m), 6.5 (2H, m), 4.15 (2H, t, Jꢀ6.1 Hz), 3.91 (2H, t,
Jꢀ5.8 Hz), 3.47 (2H, t, Jꢀ7.6 Hz), 2.91 (2H, t, Jꢀ7.6 Hz), 2.65 (2H, t,
Jꢀ7.9 Hz), 1.65—1.61 (2H, m), 1.35—1.25 (12H, m), 0.88 (3H, t,
Jꢀ6.7 Hz); HR-MS Calcd for C25H36N2O3 412.2726, Found 412.2729.
2-Methyl-3-{3-[2-(nonylpyridin-2-ylamino)ethoxy]phenyl}propanoic
Acid (7) Compound 7 was prepared from 19b using the procedure de-
scribed for 1 in 3.5% yield. In the step of the synthesis of 21e, 2-(di-
ethoxyphosphoryl)propanoic acid ethyl ester was used instead of ethyl di-
ethylphosphono acetate: light yellow oil; 1H-NMR (CDCl3, 500 MHz, d:
ppm) 8.14 (1H, ddd, Jꢀ4.8, 1.8, 0.9 Hz), 7.42 (1H, ddd, Jꢀ8.8, 6.7, 2.1 Hz),
7.17 (1H, t, Jꢀ7.9 Hz), 6.74—6.78 (3H, m), 6.52 (1H, m), 6.50 (1H, d,
Jꢀ8.8 Hz), 4.14 (2H, t, Jꢀ6.1 Hz), 3.90 (2H, m), 3.46 (3H, t, Jꢀ7.6 Hz),
3.02 (1H, dd, Jꢀ13.4, 6.7 Hz), 2.75 (1H, m), 2.64 (1H, dd, Jꢀ13.4, 7.6 Hz),
1.66—1.58 (2H, m), 1.35—1.25 (12H, m), 1.17 (3H, d, Jꢀ7.0 Hz), 0.88
(3H, t, Jꢀ7.3 Hz); HR-MS Calcd for C26H38N2O3 426.2882, Found; Mꢁ
426.2877.
3-[4-(2-Bromoethoxy)phenyl]acrylic Acid Ethyl Ester (21a) To a sus-
pension of NaH (191 mg, 4.78 mmol) in 4 ml of anhydrous THF was added
dropwise a solution of ethyl diethylphosphono acetate (1.07 ml, 5.21 mmol)
in 5 ml of anhydrous THF at 0 °C under Ar. The mixture was stirred for 1 h
at 0 °C. To the mixture was added dropwise a solution of 20a (916 mg,
4.00 mmol) in 8 ml of anhydrous THF. The reaction mixture was stirred for
16 h at 0 °C. The reaction mixture was poured into ice-water and extracted
with AcOEt. The AcOEt layer was separated, washed with brine, and dried
over Na2SO4. The solvent was removed by evaporation in vacuo. Purification
by silica gel flash chromatography (AcOEt/n-hexaneꢀ1/5) gave 901 mg
(76%) of 20a as a white solid: 1H-NMR (CDCl3, 500 MHz, d: ppm) 7.64
(1H, d, Jꢀ15.8 Hz), 7.48 (2H, d, Jꢀ8.8 Hz), 6.92 (2H, d, Jꢀ8.5 Hz), 6.32
(1H, d, Jꢀ15.8 Hz), 4.32 (2H, t, Jꢀ6.1 Hz), 4.26 (2H, q, Jꢀ7.3 Hz), 3.65
(2H, t, Jꢀ6.1 Hz), 1.34 (3H, t, Jꢀ7.0 Hz).
3-[4-(2-Bromoethoxy)phenyl]propanoic Acid Ethyl Ester (22a) To a
solution of 21a (445 mg, 1.49 mmol) in 5 ml of EtOH was added 64 mg of
7% Pd/C. The reaction mixture was stirred for 1 d under H2 at room temper-
ature. The catalyst was removed by filtration, and the solvent was removed
by evaporation in vacuo. 359 mg (80%) of 22a was obtained as a light yel-
1
low oil: H-NMR (CDCl3, 500 MHz, d: ppm) 7.12 (2H, dt, Jꢀ8.8 , 3.0 Hz),
2-{3-[2-(Nonylpyridin-2-ylamino)ethoxy]benzyl}butyric
Acid
(8)
6.83 (2H, dt, Jꢀ8.5, 3.0 Hz), 4.26 (2H, t, Jꢀ6.1 Hz), 4.12 (2H, q,
Jꢀ7.0 Hz), 3.62 (2H, t, Jꢀ6.1 Hz), 2.89 (2H, t, Jꢀ7.6 Hz), 2.58 (2H, t,
Jꢀ8.2 Hz), 1.23 (3H, t, Jꢀ7.0 Hz).
Compound 8 was prepared from 19b using the procedure described for 1 in
4.9% yield. In the step of the synthesis of 21f, 2-(diethoxyphosphoryl)bu-
tyric acid ethyl ester was used instead of ethyl diethylphosphono acetate:
yellow oil; 1H-NMR (CDCl3, 500 MHz, d: ppm) 8.13 (1H, ddd, J ꢀ5.2,
1.2 Hz), 7.42 (1H, ddd, Jꢀ8.8, 6.7, 2.1 Hz), 7.15 (1H, t, Jꢀ7.9 Hz), 6.71—
6.79 (3H, m), 6.52 (1H, dd, Jꢀ6.7, 5.2 Hz), 6.50 (1H, d, Jꢀ8.8 Hz), 4.12
(2H, t, Jꢀ5.5 Hz), 3.83—3.94 (2H, m), 3.46 (3H, t, Jꢀ7.6 Hz), 2.93 (1H, dd,
Jꢀ13.7, 8.2 Hz), 2.72 (1H, dd, Jꢀ13.7, 6.7 Hz), 2.60 (1H, m), 1.69—1.55
(4H, m), 1.35—1.25 (12H, m), 0.96 (3H, t, Jꢀ7.3 Hz), 0.82 (3H, t,
Jꢀ7.0 Hz); HR-MS Calcd for C27H40N2O3 440.3039, Found 440.3043.
3-{4-[2-(Nonyl-pyridin-2-ylamino)ethoxy]phenyl}propanoic
Acid
Ethyl Ester (23a) To a solution of 22a (0.343 g, 1.14 mmol) in 1 ml of
THF were added 2-nonylaminopyridine (1.01 g, 4.56 mmol), KI (0.189 g,
1.14 mmol) and Et3N (0.45 ml, 2.30 mmol). The reaction mixture was stirred
for 28 h at 105 °C. The reaction mixture was poured into water and extracted
with AcOEt. The AcOEt layer was separated, washed with water and brine,
and dried over Na2SO4. Purification by silica gel flash chromatography

1058
2-Ethoxy-3-{3-[2-(nonylpyridin-2-ylamino)ethoxy]phenyl}propanoic
Vol. 55, No. 7
Jꢀ6.1 Hz), 2.95 (1H, dd, Jꢀ13.4, 7.1 Hz), 2.69 (1H, m), 2.61 (1H, dd,
Jꢀ13.4, 7.6 Hz), 1.14 (3H, d, Jꢀ7.0 Hz).
3-{4-[2-(Nonylpyridin-2-ylamino)ethoxy]phenyl}-2,2-dimethyl-
propanoic Acid Methyl Ester (27a) Compound 27a was prepared from
Acid (9) Compound 9 was prepared from 19b using the procedure de-
scribed for 1 in 2.5% yield. In the step of the synthesis of 21g, 2-(di-
ethoxyphosphoryl)ethoxyacetic acid ethyl ether was used instead of ethyl di-
ethylphosphono acetate: yellow oil; 1H-NMR (CDCl3, 500 MHz, d: ppm)
8.13 (1H, dd, Jꢀ4.9, 1.2 Hz), 7.44 (1H, ddd, Jꢀ8.8, 7.0, 1.8 Hz), 7.17 (1H, t,
Jꢀ7.6 Hz), 6.83 (2H, m), 6.77 (1H, dd, Jꢀ7.9, 1.8 Hz), 6.53 (2H, m), 4.15
(2H, t, Jꢀ5.5 Hz), 4.07 (1H, dd, Jꢀ7.3, 4.9 Hz), 3.90 (2H, m), 3.59 (1H, m),
3.47 (2H, t, Jꢀ7.9 Hz), 3.43 (1H, m), 3.09 (1H, dd, Jꢀ13.7, 4.6 Hz), 2.98
(1H, dd, Jꢀ13.7, 7.3 Hz), 1.67—1.59 (2H, m), 1.36—1.23 (12H, m), 1.16
(3H, t, Jꢀ7.0 Hz), 0.88 (3H, t, Jꢀ6.7 Hz); HR-MS Calcd for C27H40N2O4
456.2988, Found 456.2985.
1
26a using the procedure described for 23a in 11% yield: H-NMR (CDCl3,
500 MHz, d: ppm) 8.13 (1H, ddd, Jꢀ4.9, 1.8, 0.9 Hz), 7.41 (1H, ddd, Jꢀ8.8,
7.0, 2.1 Hz), 7.04 (2H, d, Jꢀ8.5 Hz), 6.82 (2H, dt, Jꢀ8.5, 1.8 Hz), 6.5 (2H,
m), 4.14 (2H, t, Jꢀ6.1 Hz), 3.91 (2H, t, Jꢀ5.8 Hz), 3.63 (3H, s), 3.47 (2H, t,
Jꢀ7.9 Hz), 2.94 (1H, dd, Jꢀ13.4, 6.7 Hz), 2.68 (1H, m), 2.59 (1H, dd,
Jꢀ13.4, 7.6 Hz), 1.64—1.57 (2H, m), 1.35—1.25 (12H, m), 1.13 (3H, d,
Jꢀ7.0 Hz), 0.88 (3H, t, Jꢀ7.2 Hz); HR-MS Calcd for C27H40N2O3 440.3039,
Found 440.3046.
3-{4-Methoxy-3-[2-(nonylpyridin-2-ylamino)ethoxy]phenyl}propanoic
Acid (10) Compound 10 was prepared from 19c using the procedure de-
scribed for 1 in 2.7% yield: yellow oil; 1H-NMR (CDCl3, 500 MHz, d: ppm)
8.16 (1H, ddd, Jꢀ4.9, 1.8, 0.9 Hz), 7.43 (1H, ddd, Jꢀ8.8, 7.0, 1.8 Hz), 6.93
(1H, d, Jꢀ1.5 Hz), 6.79 (1H, d, Jꢀ8.2 Hz), 6.74 (1H, dd, Jꢀ8.2, 1.8 Hz),
6.53 (1H, dd, Jꢀ7.0, 5.2 Hz), 6.51 (1H, d, Jꢀ8.8 Hz), 4.24 (2H, t,
Jꢀ6.7 Hz), 3.94 (2H, t, Jꢀ6.4 Hz), 3.83 (3H, s), 3.45 (2H, t, Jꢀ7.6 Hz),
2.88 (2H, t, Jꢀ7.6 Hz), 2.61 (2H, t, Jꢀ7.6 Hz), 1.64—1.58 (2H, m), 1.31—
1.25 (12H, m), 0.88 (3H, t, Jꢀ7.0 Hz); HR-MS Calcd for C26H38N2O4
442.2832, Found 442.2846.
3-{4-[2-(Nonylpyridin-2-ylamino)ethoxy]phenyl}-2-methylpropanoic
Acid (2) Compound 2 was prepared from 27a using the procedure de-
scribed for 1 in 97% yield: colorless oil; 1H-NMR (CDCl3, 500 MHz, d:
ppm) 8.13 (2H, d, Jꢀ4.8 Hz), 7.41 (1H, ddd, Jꢀ8.8, 6.7, 1.8 Hz), 7.08 (2H,
d, Jꢀ8.5 Hz), 6.82 (2H, d, Jꢀ8.5 Hz), 6.5 (2H, m), 4.12 (2H, m), 3.90 (2H, t,
Jꢀ5.8 Hz), 3.47 (2H, t, Jꢀ7.9 Hz), 2.98 (1H, dd, Jꢀ13.4, 7.0 Hz), 2.73 (1H,
m), 2.63 (1H, dd, Jꢀ13.4, 7.6 Hz), 1.65—1.58 (2H, br), 1.35—1.25 (12H,
m), 1.17 (3H, d, Jꢀ6.7 Hz), 0.88 (3H, t, Jꢀ7.0 Hz); HR-MS Calcd for
C26H38N2O3 426.2882, Found 426.2889.
3-{4-[2-(Nonylpyridin-2-ylamino)ethoxy]phenyl}-2,2-dimethyl-
propanoic Acid (5) Compound 5 was prepared from 20a using the proce-
dure described for 2 in 4.2% yield. In the step of the synthesis of 26b, di-
methylketene methyltrimethylsilyl acetal was used instead of 1-methoxy-1-
trimethylsilyloxypropene: light yellow oil; 1H-NMR (CDCl3, 500 MHz, d:
ppm) 8.14 (2H, dd, Jꢀ4.9, 1.5 Hz), 7.41 (1H, ddd, Jꢀ8.8, 6.7, 1.8 Hz), 7.05
(2H, d, Jꢀ8.5 Hz), 6.80 (2H, d, Jꢀ8.5 Hz), 6.50 (2H, m), 4.12 (2H, t,
Jꢀ5.8 Hz), 3.90 (2H, t, Jꢀ5.8 Hz), 3.47 (2H, t, Jꢀ7.6 Hz), 2.81 (2H, s),
1.65—1.59 (2H, m), 1.35—1.25 (12H, m), 1.17 (6H, s), 0.88 (3H, t,
Jꢀ7.0 Hz); HR-MS Calcd for C27H40N2O3 440.3039, Found 440.3040.
3-{4-Methoxy-3-[2-(Nonylpyridin-2-ylamino)ethoxy]phenyl}-2-
methylpropanoic Acid (11) Compound 11 was prepared from 20c using
the procedure described for 2 in 3.3% yield: colorless oil; 1H-NMR (CDCl3,
500 MHz, d: ppm) 8.16 (1H, ddd, Jꢀ4.9, 1.8, 0.9 Hz), 7.44 (1H, ddd, Jꢀ8.8,
6.7, 1.8 Hz), 6.95 (1H, d, Jꢀ1.8 Hz), 6.78 (1H, d, Jꢀ7.9 Hz), 6.71 (1H, dd,
Jꢀ8.2, 1.8 Hz), 6.54 (1H, dd, Jꢀ7.0, 5.1 Hz), 6.50 (1H, d, Jꢀ8.8 Hz), 4.25
(2H, m), 3.92 (2H, m), 3.83 (3H, s), 3.43 (2H, t, Jꢀ7.3 Hz), 2.92 (1H, m),
2.70 (2H, m), 1.64—1.54 (2H, m), 1.31—1.20 (12H, m), 1.16 (3H, d,
Jꢀ6.7 Hz), 0.88 (3H, t, Jꢀ6.7 Hz); HR-MS Calcd for C27H40N2O4 456.2988,
Found 456.3007.
3-{4-Methoxy-3-[2-(Nonylpyridin-2-ylamino)ethoxy]phenyl}-2,2-di-
methylpropanoic Acid (14) Compound 14 was prepared from 20c using
the procedure described for 2 in 8.4% yield. In the step of the synthesis of
26d, dimethylketene methyltrimethylsilyl acetal was used instead of 1-
methoxy-1-trimethylsilyloxypropene: colorless oil; 1H-NMR (CDCl3,
500 MHz, d: ppm) 8.16 (1H, ddd, Jꢀ5.1, 1.8, 0.6 Hz), 7.45 (1H, ddd, Jꢀ8.8,
7.0, 1.8 Hz), 7.00 (1H, d, Jꢀ2.1 Hz), 6.76 (1H, d, Jꢀ8.2 Hz), 6.68 (1H, dd,
Jꢀ8.2, 1.8 Hz), 6.55 (1H, dd, Jꢀ7.0, 5.2 Hz), 6.50 (1H, d, Jꢀ8.8 Hz), 4.27
(2H, t, Jꢀ7.0 Hz), 3.90 (2H, t, Jꢀ7.3 Hz), 3.84 (3H, s), 3.40 (2H, t,
Jꢀ7.6 Hz), 2.79 (2H, s), 1.63—1.57 (2H, m), 1.35—1.25 (12H, m), 1.20
(6H, s), 0.88 (3H, t, Jꢀ7.0 Hz); HR-MS Calcd for C28H42N2O4 470.3145,
Found 470.3144.
2-{4-Methoxy-3-[2-(nonylpyridin-2-ylamino)ethoxy]benzyl}butyric
Acid (12) Compound 12 was prepared from 19c using the procedure de-
scribed for 1 in 3.0% yield. In the step of the synthesis of 21i, 2-(di-
ethoxyphosphoryl)butyric acid ethyl ester was used instead of ethyl di-
ethylphosphono acetate: yellow oil; 1H-NMR (CDCl3, 500 MHz, d: ppm)
8.16 (1H, ddd, Jꢀ4.8, 1.8, 0.6 Hz), 7.43 (1H, ddd, Jꢀ9.1, 7.3, 2.1 Hz), 6.91
(1H, d, Jꢀ1.8 Hz), 6.76 (1H, d, Jꢀ8.2 Hz), 6.71 (1H, dd, Jꢀ8.2, 2.1 Hz),
6.53 (1H, dd, Jꢀ6.4, 5.1 Hz), 6.50 (1H, d, Jꢀ8.8 Hz), 4.23 (2H, m), 3.98
(2H, m), 3.86 (1H, m), 3.83 (3H, s), 3.43 (2H, m), 2.87 (1H, dd, Jꢀ13.7,
8.5 Hz), 2.71 (1H, dd, Jꢀ13.6, 6.1 Hz), 2.56—2.50 (1H, m), 1.70—1.51
(4H, m), 1.34—1.23 (12H, m), 0.95 (3H, t, Jꢀ7.0 Hz), 0.88 (3H, t,
Jꢀ7.0 Hz); HR-MS Calcd for C28H42N2O4 470.3145, Found 470.3142.
2-Ethoxy-3-{4-methoxy-3-[2-(nonylpyridin-2-ylamino)ethoxy]phenyl-
propanoic Acid (13) Compound 13 was prepared from 19c using the pro-
cedure described for 1 in 3.8% yield. In the step of the synthesis of 21j, 2-
(diethoxyphosphoryl)ethoxyacetic acid ethyl ether was used instead of ethyl
1
diethylphosphono acetate: yellow oil; H-NMR (CDCl3, 500 MHz, d: ppm)
8.13 (1H, dd, Jꢀ4.9, 1.8 Hz), 7.45 (1H, ddd, Jꢀ8.8, 7.0, 1.8 Hz), 6.96 (1H,
s), 6.78 (2H, m), 6.54 (1H, dd, Jꢀ6.7, 5.5 Hz), 6.52 (1H, d, Jꢀ8.5 Hz), 4.25
(2H, m), 4.04 (1H, m), 3.93 (2H, m), 3.83 (3H, s), 3.59 (1H, m), 3.44 (2H, t,
Jꢀ7.6 Hz), 3.05 (1H, dd, Jꢀ13.7, 4.9 Hz), 2.97 (1H, dd, Jꢀ13.7, 6.4 Hz),
1.65—1.58 (2H, m), 1.35—1.21 (12H, br), 1.17 (3H, t, Jꢀ7.0 Hz), 0.88 (3H,
t, Jꢀ6.7 Hz); HR-MS Calcd for C28H42N2O5 486.3094, Found 486.3111.
{4-(2-Bromoethoxy)phenyl}methanol (24a) To
a solution of 20a
(890 mg, 3.89 mmol) in 10 ml of EtOH was added NaBH4 (163 mg,
3.89 mmol). The reaction mixture was stirred for 6 h at room temperature.
The reaction mixture was poured into water, and extracted with AcOEt. The
AcOEt layer was separated, washed with water and brine, and dried over
Na2SO4. The solvent was removed by evaporation in vacuo. 763 mg (81%)
1
of 24a was obtained as a white solid: H-NMR (CDCl3, 500 MHz, d: ppm)
7.30 (2H, d, Jꢀ8.5 Hz), 6.91 (2H, d, Jꢀ8.8 Hz), 4.62 (2H, d, Jꢀ5.1 Hz),
4.29 (2H, t, Jꢀ6.1 Hz), 3.64 (2H, t, Jꢀ6.4 Hz), 1.58 (1H, t, Jꢀ5.5 Hz).
Acetic Acid 4-(2-Bromoethoxy)benzyl Ester (25a) To a solution of
24a (2.31 g, 10.0 mmol) in 20 ml of CH2Cl2 were added Ac2O (3.10 ml,
30.0 mmol) and a catalytic amount of DMAP. The reaction mixture was
stirred for 1 d at room temperature and diluted with AcOEt. The AcOEt so-
lution was washed with water and brine, and dried over Na2SO4. Purification
by silica gel flash chromatography (AcOEt/n-hexane ꢀ1/5) gave 2.62 g
(96%) of 25a as a light yellow oil: 1H-NMR (CDCl3, 500 MHz, d: ppm)
7.29 (2H, d, Jꢀ8.8 Hz), 6.90 (2H, d, Jꢀ8.8 Hz), 5.04 (2H, s), 4.28 (2H, t,
Jꢀ6.1 Hz), 3.63 (2H, t, Jꢀ6.1 Hz), 2.07 (3H, s).
3-{4-[3-(Nonylpyridin-2-ylamino)propyl]phenyl}acrylic Acid Ethyl
Ester (28a) (Chart 3) Compound 28a was prepared from 21a using the
1
procedure described for 23a in 13% yield: H-NMR (CDCl3, 500 MHz, d:
ppm) 8.14 (1H, ddd, Jꢀ4.8, 2.1, 0.9 Hz), 7.63 (1H, d, Jꢀ16.2 Hz), 7.45 (2H,
d, Jꢀ8.8 Hz), 7.42 (1H, ddd, Jꢀ8.8, 7.0, 1.8 Hz), 6.91 (2H, d, Jꢀ8.5 Hz),
6.52 (1H, dd, Jꢀ7.0, 5.5 Hz), 6.50 (1H, d, Jꢀ8.8 Hz), 6.30 (1H, d,
Jꢀ15.8 Hz), 4.25 (2H, q, Jꢀ7.0 Hz), 4.21 (2H, t, Jꢀ5.8 Hz), 3.94 (2H, t,
Jꢀ5.8 Hz), 3.46 (2H, t, Jꢀ7.9 Hz), 1.64—1.60 (2H, m), 1.36—1.30 (3H, t,
Jꢀ7.0 Hz), 1.33—1.23 (12H, br), 0.88 (3H, t, Jꢀ7.0 Hz); HR-MS Calcd for
C27H38N2O3 438.2882, Found 438.2855.
3-{4-(2-Bromoethoxy)phenyl}-2-methylpropionic Acid Methyl Ester
(26a) To a solution of 25a (0.948 g, 3.47 mmol) and 1-methoxy-1-
trimethylsilyloxypropene (1.00 g, 6.24 mmol) in 35 ml of anhydrous CH2Cl2
was added magnesium perchlorate (0.129 g, 0.694 mmol) under Ar. The re-
action mixture was stirred for 1 d at room temperature. The reaction mixture
was diluted with CH2Cl2. The CH2Cl2 solution was washed with water and
brine, and dried over Na2SO4. The solvent was removed by evaporation in
vacuo. Purification by silica gel flash chromatography (AcOEt/n-
hexaneꢀ1/10) gave 0.935 g (90%) of 26a as a colorless oil: 1H-NMR
(CDCl3, 500 MHz, d: ppm) 7.07 (2H, dt, Jꢀ8.5, 3.0 Hz), 6.82 (2H, dt,
Jꢀ8.5, 3.0 Hz), 4.26 (2H, t, Jꢀ6.4 Hz ), 3.63 (3H, s), 3.62 (2H, t,
3-{4-[3-(Nonylpyridin-2-ylamino)propyl]phenyl}acrylic Acid (15) To
a solution of 28a (84 mg, 0.192 mmol) in 2 ml of THF and 2 ml of EtOH was
added a solution of 2 N aqueous NaOH (0.29 ml, 0.580 mmol). The reaction
mixture was stirred for 18 h at room temperature, and neutralized to pH 7
with aqueous 2 N HCl. Purification by silica gel flash chromatography
(CHCl3/MeOHꢀ19/1) gave 78 mg (99%) of white solid. The 41 mg of the
white solid was recrystallized from AcOEt–n-hexane and collected by filtra-
tion to give 22 mg (52%) of 15 as a white solid: mp 142—143 °C; 1H-NMR
(CDCl3, 500 MHz, d: ppm) 8.15 (1H, dd, Jꢀ4.9, 1.8 Hz), 7.70 (1H, d,
Jꢀ15.9 Hz), 7.47 (2H, d, Jꢀ8.8 Hz), 7.43 (1H, ddd, Jꢀ8.8, 7.0, 1.8 Hz),
6.93 (1H, d, Jꢀ8.8 Hz), 6.53 (1H, dd, Jꢀ6.4, 5.5 Hz), 6.50 (1H, d,

July 2007
1059
Chem. Pharm. Bull., 39, 1440—1445 (1991).
8) Hauner H., Diabetes Metab. Res. Rev., 18, S10—S15 (2002).
9) Francis G. A., Annicotte J. S., Auwerx J., Curr. Opin. Parmacol., 3,
186—191 (2003).
Jꢀ8.5 Hz), 6.29 (1H, d, Jꢀ16.1 Hz), 4.22 (2H, t, Jꢀ5.8 Hz), 3.95 (2H, t,
Jꢀ5.8 Hz), 3.46 (2H, t, Jꢀ8.2 Hz), 1.65—1.61 (2H, m), 1.34—1.24 (12H,
m), 0.88 (3H, t, Jꢀ7.0 Hz); Anal. Calcd for C25H34N2O3: C, 73.14; H, 8.35;
N, 6.82. Found: C, 73.23; H, 8.46; N, 6.89.
10) Lohray B. B., Lohray V. B., Bajji A. C., Kalchar S., Poondra R. R.,
Padakanti S., Chakrabarti R., Vikramadithyan R. K., Misra P., Juluri
S., Mamidi N. V., Rajagopalan R., J. Med. Chem., 44, 2675—2678
(2001).
11) Duran-Sandoval D., Thomas A. C., Bailleul B., Fruchart J. C., Staels
B., Med. Sci., 19, 819—825 (2003).
12) Forman B. M., Tontonoz P., Chen J., Brun R. P., Spiegelman B. M.,
Evans R. M., Cell, 83, 803—812 (1995).
13) Kliewer S. A., Lenhard J. M., Willson T. M., Patel I., Morris D. C.,
Lehmann J. M., Cell, 83, 813—819 (1995).
3-{4-Methoxy-3-[2-(nonylpyridin-2-ylamino)ethoxy]phenyl}acrylic
Acid (16) Compound 16 was prepared from 21h using the procedure de-
scribed for 15 in 7.4% yield: colorless oil; H-NMR (CDCl3, 500 MHz, d:
ppm) 8.22 (1H, dd, Jꢀ4.9, 1.8 Hz), 7.69 (1H, d, Jꢀ15.8 Hz), 7.44 (1H, ddd,
Jꢀ8.8, 7.0, 1.8 Hz), 7.31 (1H, d, Jꢀ1.5 Hz), 7.09 (1H, dd, Jꢀ8.2, 1.8 Hz),
6.86 (1H, d, Jꢀ8.2 Hz), 6.56 (1H, dd, Jꢀ7.0, 5.2 Hz), 6.51 (1H, d,
Jꢀ8.5 Hz), 6.31 (1H, d, Jꢀ15.8 Hz), 4.31 (2H, t, Jꢀ6.4 Hz), 3.99 (2H, t,
Jꢀ6.1 Hz), 3.90 (3H, s), 3.45 (2H, t, Jꢀ7.6 Hz), 1.65—1.61 (2H, m), 1.32—
1.22 (12H, m), 0.88 (3H, t, Jꢀ7.0 Hz); HR-MS Calcd for C26H36N2O4
440.2675, Found 440.2716.
1
14) Usui S., Suzuki T., Hattori Y., Etoh K., Fujieda H., Nishizuka M., Ima-
gawa M., Nakagawa H., Kohda K., Miyata N., Bioorg. Med. Chem.
Lett., 15, 1547—1551 (2005).
15) Buckle D. R., Cantello B. C. C., Cawthorne M. A., Coyle P. J., Dean D.
K., Faller A., Haigh D., Hindley R. M., Jefcott L. J., Lister C. A., Pinto
I. L., Rami H. K., Smith D. G., Smith S. A., Bioorg. Med. Chem. Lett.,
6, 2121—2126 (1996).
16) Buckle D. R., Cantello B. C. C., Cawthorne M. A., Coyle P. J., Dean D.
K., Faller A., Haigh D., Hindley R. M., Jefcott L. J., Lister C. A., Pinto
I. L., Rami H. K., Smith D. G., Smith S. A., Bioorg. Med. Chem. Lett.,
6, 2127—2130 (1996).
17) Hulin B., Newton L. S., Lewis D. M., Genereux P. E., Gibbs E. M.,
Clark D. A., J. Med. Chem., 39, 3897—3907 (1996).
18) Sauerberg P., Pettersson I., Jeppesen L., Bury P. S., Mogensen J. P.,
Wassermann K., Brand C. L., Sturis J., Woldike H. F., Fleckner J.,
Andersen A. S., Mortensen S. B., Svensson L. A., Rasmussen H. B.,
Lehmann S. V., Polivka Z., Sindelar K., Panajotova V., Ynddal L.,
Wulff E. M., J. Med. Chem., 45, 789—804 (2002).
19) Cronet P., Petersen J. F., Folmer R., Blomberg N., Sjoblom K., Karls-
son U., Lindstedt E. L., Bamberg K., Structure, 9, 699—706 (2001).
20) Nomura M., Tanase T., Ide T., Tsunoda M., Suzuki M., Uchiki H., Mu-
rakami K., Miyachi H., J. Med. Chem., 46, 3581—3599 (2003).
21) Weigand S., Bischoff H., Dittrich-Wengenroth E., Heckroth H., Lang
D., Vaupel A., Woltering M., Bioorg. Med. Chem. Lett., 15, 4619—
4623 (2005).
Binding Assay The assay for PPAR binding activity was performed
using a CoA-BAP System kit (NuLigand series, Microsystems). Briefly, glu-
tathione-S-transferase-human nuclear receptor ligand binding domain (GST-
hNR LBD) fusion proteins were incubated in a glutathione-fixed micro-well
plate at 4 °C overnight. After excessive proteins were removed, human tran-
scriptional intermediary factor 2 (hTIF2)-bacterial alkaline phosphatase
(BAP) fusion proteins were added to the well with test chemicals. After 1 h
incubation at 4 °C, excessive proteins were removed carefully. The enzyme
reaction was allowed to start with the addition of p-nitrophenylphosphoric
acid (NPP) as a substrate, and incubated at 30 °C. After 3 h, the reaction was
stopped by the addition of 0.5 N aqueous NaOH. The product was measured
by reading absorbance at 405 nm with a 1420 ARVOTM multilabel counter
(ParkinElmer, Boston, MA, U.S.A.). AP activity was determined by sub-
tracting background absorption from the reading at 405 nm.
Cell-Based Transactivation Assay30) Human embryonic kidney (HEK)
293 cells were cultured in DMEM containing 5% fetal bovine serum at
37 °C in a humidified atmosphere of 5% CO2 in air. Transfections of PPAR
and reporter gene constructs were performed by calcium phosphate copre-
cipitation. Eight hours after transfection, ligands were added. Cells were har-
vested 12—16 h after treatment, and luciferase and b-galactosidase activities
were assayed using a 1420 ARVOTM MX multilabel counter (ParkinElmer,
Boston, MA, U.S.A.). DNA cotransfection experiments included 58 ng of re-
porter plasmid, 12 ng of CMX-b-galactosidase, and 18 ng of each receptor
expression plasmid per well in a 96-well plate. Luciferase data were normal-
ized to an internal b-galactosidase control and reported values are the means
of triplicate assays.
Preadipocyte Differentiation Test Human preadipocytes from hypo-
dermic tissues, a preadipocyte growth medium and a preadipocyte differenti-
ation medium were purchased from TOYOBO. Co., Ltd (Osaka, Japan).
Human preadipocytes were cultured for 8 d in preadipocyte growth medium
in a humidified incubator at 37 °C and 5% CO2. The medium was renewed
every other day. When the preadipocytes reached confluence, the cells were
treated with preadipocyte differentiation medium containing compounds 3,
4, or rosiglitazone. The cells were cultured for a further 7 d with the differ-
entiation medium renewed every 3 d. The accumulation of triglycerides was
evaluated by measuring the absorbance at 570 nm with a 1420 ARVOTM
multilabel counter (ParkinElmer, Boston, MA, U.S.A.) after staining with
Lipidos Liquid® (TOYOBO Co., Ltd, Osaka, Japan).
22) Kasuga J., Makishima M., Hashimoto Y., Miyachi H., Bioorg. Med.
Chem. Lett., 16, 554—558 (2006).
23) Wagaw S., Buchwald S. L., J. Org. Chem., 61, 7240—7241 (1996).
24) Maryanoff B. E., Reitz A. B., Chem. Rev., 89, 863—927 (1989).
25) Grieco P. A., Handy S. T., Tetrahedron Lett., 38, 2645—2648 (1997).
26) Kanayama T., Mamiya S., Nishihara T., Nishikawa J., J. Biochem.
(Tokyo), 133, 791—797 (2003).
27) Brown P. J., Stuart L. W., Hurley K. P., Lewis M. C., Winegar D. A.,
Wilson J. G., Wilkison W. O., Ittoop O. R., Willson T. M., Bioorg.
Med. Chem. Lett., 11, 1225—1227 (2001).
28) Sznaidman M. L., Haffner C. D., Maloney P. R., Fivush A., Chao E.,
Goreham D., Sierra M. L., LeGrumelec C., Xu H. E., Montana V. G.,
Lambert M. H., Willson T. M., Oliver W. R., Jr., Sternbach D. D.,
Bioorg. Med. Chem. Lett., 13, 1517—1521 (2003).
29) Usui S., Fujieda H., Suzuki T., Yoshida N., Nakagawa H., Miyata N.,
Bioorg. Med. Chem. Lett., 16, 3249—3254 (2006).
30) Fukuen S., Iwaki M., Yasui A., Makishima M., Matsuda M., Shimo-
mura I., J. Biol. Chem, 280, 23653—23659 (2005).
31) Henke B. R., J. Med. Chem., 47, 4118—4127 (2004).
32) Lagace D. C., Nachtigal M. W., J. Biol. Chem., 279, 18851—18860
(2004).
References and Notes
1) Nuclear Receptors Nomenclature Committee. Cell, 97, 161—163
(1999).
2) Willson T. M., Brown P. J., Sternbach D. D., Henke B. R., J. Med.
Chem., 43, 527—550 (2000).
4) Kersten S., Desvergne B., Wahli W., Nature (London), 405, 421—424
(2000).
33) Relative transactivation activity at 10 mM: compound 4, 163% of
WY14643 for a-activity, and 88% of rosiglitazone for g-activity, com-
pound 8, 275% of WY14643 for a-activity.
6) Cantello B. C. C., Cawthorone M. A., Cottam G. P., Duff P. T., Haigh
D., Hindley R. M., Lister C. A., Smith S. A., Thurlby P. L., J. Med.
Chem., 37, 3977—3985 (1994).
7) Momose Y., Meguro K., Ikeda H., Hatanaka C., Oi S., Sohda T.,