Studies on non-thiazolidinedione antidiabetic agents. 3. Preparation and biological activity of the metabolites of TAK-559

Article abstract of DOI:10.1248/cpb.52.120

Preparation and biological activity of the metabolites of the potent antihyperglycemic and antihyperlipidemic agent, (E)-4-{4-[(5-methyl-2-phenyl-1, 3-oxazol-4-yl)methoxy]benzyloxyimino}-4-phenylbutyric acid (TAK-559) (1), were investigated. Metabolites M

Full text of DOI:10.1248/cpb.52.120

120
Chem. Pharm. Bull. 52(1) 120—124 (2004)
Vol. 52, No. 1
Studies on Non-Thiazolidinedione Antidiabetic Agents. 3.1,2) Preparation
and Biological Activity of the Metabolites of TAK-559
Hiroshi IMOTO,* Mitsuharu MATSUMOTO, Hiroyuki ODAKA, Junichi SAKAMOTO, Hiroyuki KIMURA,
Masami NONAKA, Yutaka KIYOTA, and Yu MOMOSE
Takeda Chemical Industries, Ltd. Pharmaceutical Research Division; 2–17–85 Juso-Honmachi, Yodogawa-ku, Osaka
532–8686, Japan. Received September 19, 2003; accepted November 1, 2003
Preparation and biological activity of the metabolites of the potent antihyperglycemic and antihyperlipi-
demic agent, (E)-4-{4-[(5-methyl-2-phenyl-1,3-oxazol-4-yl)methoxy]benzyloxyimino}-4-phenylbutyric acid (TAK-
559) (1), were investigated. Metabolites M-I (2), M-II (3), M-III (4) and M-IV (5) were synthesized and their bio-
logical activities were evaluated by in vitro and in vivo experiments. Compounds 2—4 activate human peroxisome
proliferator-activated receptor gamma one (hPPARg1) and hPPARa, but their activities are weaker than those of
TAK-559 (1). Compound 5 only activates hPPARg1 weakly. TAK-559 (1) showed potent in vivo plasma glucose
and triglyceride lowering activities in Wistar fatty rats after intraperitoneal administration, while its metabolites
(2—5) showed comparatively weak activities.
Key words antidiabetic agent; oxyiminoalkanoic acid; type 2 diabetes; peroxisome proliferator-activated receptor; TAK-559
Peroxisome proliferator-activated receptor gamma (PPAR- of isolated metabolites (M-I, M-II, M-III, M-IV) and synthe-
g) is one of a subfamily of PPARs encoded by independent sized compounds (2—5) were shown to be identical respec-
genes. Three human PPARs, designated PPARa, PPARg, and tively (data not shown). Transactivation activities of com-
PPARd, have been identified.3—5) Recent studies suggest that pounds 1—5 for human PPAR subtypes were examined
PPARg ligands have the ability to lower plasma glucose and using in vitro experiments, and the glucose and lipid lower-
triglyceride levels in insulin-resistant animal models.6) Some ing effects of the compounds were evaluated in Wistar fatty
glucose and lipid lowering agents, which have transcriptional rats using in vivo experiments. The results are described
activity at PPARg, are currently used for clinical treatment of below.
type 2 diabetes.7—9)
In a previous paper, we showed (E)-4-{4-[(5-methyl-2- Chemistry
phenyl-1,3-oxazol-4-yl)methoxy]benzyloxyimino}-4-phenyl-
Compounds 2 and 3 were synthesized from commercially
butyric acid (TAK-559) (1), a novel oxyiminoalkanoic acid available 6a and 6b, respectively (Chart 2). Friedel-Crafts
derivative, had strong functional activity at PPARg (Chart acylation of 6a—b gave aryl ketones 7a—b, of which the
1).2) TAK-559 also exhibits marked glucose and lipid lower- methoxy groups were converted into hydroxy groups using
ing activities in insulin-resistant animal models, KKAy mice boron tribromide to yield 8a—b. Reactions of 8a—b with 92)
and Wistar fatty rats,2,10—13) and is under clinical trials.
Metabolites M-I, M-II, M-III and M-IV were found during
gave aldoximes, which were hydrolyzed to afford 2 and 3.
Compound 4 was prepared following the procedure de-
preclinical and clinical studies on TAK-559 (1), and their scribed in Chart 3. The hydroxy group of compound 1014)
proposed structures are shown in Chart 1. To confirm the was protected using chloromethyl methyl ether to give 11.
structures and to study the biological properties of the Lithiation at the 5-position of the oxazole 11 with n-butyl-
metabolites, compounds 2—5 (Charts 2—4) were synthe- lithium, followed by treatment with N,N-dimethylformamide,
sized and their biological activities were evaluated by both in yielded aldehyde 12. Reduction of 12 gave the corresponding
vitro and in vivo experiments. Analytical high-performance alcohol, which was protected using tert-butylchlorodiphenyl-
liquid chromatography (HPLC) and mass spectroscopy (MS) silane to yield 13. Selective removal of the methoxymethyl
group of 13 was achieved using bromotrimethylsilane to give
alcohol 14. Compound 14 was reacted with methanesulfonyl
chloride, then coupled with 152) to provide 16. Removal of
the tert-butyldiphenylsilyl group of 16 and subsequent hy-
drolysis of the ester group gave the desired compound 4.
Chart 4 shows the synthesis of 5. Chloromethyloxazole
1715) was reacted with methyl 4-hydroxybenzoate under basic
conditions to yield 18. Hydrolysis of 18 gave 5 in good yield.
Results and Discussion
To investigate the effect of TAK-559 (1) and its metabo-
lites (2—5) on the full-length PPAR subtypes, COS-1 cells
were transiently transfected with PPAR expression plasmid,
human retinoid X receptor alpha (hRXRa) expression plas-
mid and reporter construct containing four copies of the rat
Chart 1
acetyl-CoA oxidase peroxisome proliferator response ele-
To whom correspondence should be addressed. e-mail: Imoto_Hiroshi@takeda.co.jp
© 2004 Pharmaceutical Society of Japan

January 2004
121
Chart 2
Chart 3
Table 1. Transactivation Activities of TAK-559 and Its Metabolites for
hPPARg1 and hPPARaa)
EC50 (nM)
Compounds
hPPARg1
hPPARa
1 (TAK-559)
31b)
130
210
200
970
67b)
240
540
Chart 4
2
3
4
5
ment (PPRE), and then transfected cells were treated with
TAK-559 and its metabolites.
580
NDc)
TAK-559 (1) and its metabolites (2—5) activated
hPPARg1 in a dose-dependent manner (data not shown). The
EC50 values for 1, 2, 3, 4 and 5 were 31 nM,16) 130 nM,
210 nM, 200 nM and 970 nM, respectively (Table 1). Thus,
TAK-559 is the most potent activator of hPPARg1 among
these compounds.
a) Full-length hPPARg1 (or hPPARa)– hRXRa expression plasmids were cotrans-
fected into COS-1 cells with PPAR responsive luciferase reporter plasmid, and cells
were cultured in the presence of 1, 3, 10, 30, 100, 300, 1000, 3000, and 10000 nM of
each compound or vehicle (0.1% DMSO) alone for 48 h. The cell extracts were then as-
sayed for luciferase activity. Transactivation activity is represented as EC50. Data are the
mean (nϭ5). b) From reference 16. c) Not determined because of poor activity.
In a transient cotransfection assay for hPPARa, TAK-559 5) activate hPPARd, we performed a transient cotransfection
(1) and its metabolites (2—4) activated hPPARa in a dose- assay with or without hPPARd expression plasmid and a
dependent manner (data not shown), while compound 5 high concentration (10 mM) of these compounds. Although
failed to activate it. The EC50 values for 1, 2, 3, and 4 were the hPPARd activation by TAK-559 was significantly depen-
67 nM,16) 240 nM, 540 nM and 580 nM, respectively (Table 1), dent on the exogenous expression of hPPARd in COS-1
indicating that TAK-559 (1) is significantly more potent than cells, its metabolites did not activate the exogenous hPPARd
its metabolites as an activator of hPPARa.
(data not shown).
The in vitro results described above indicate that TAK-559
To examine whether TAK-559 (1) and its metabolites (2—

122
Vol. 52, No. 1
of test compounds, and then cultured in 5% CO2 at 37 °C for 48 h. After re-
moving the medium, 40 ml of PICAGENE-LT7.5 (Wako Pure Chemical Ind.,
Ltd., Japan) was added. After stirring, luciferase activities were determined
in a microplate-based luminescence reader (Amersham Pharmacia, U.K.).
(b) In Vivo Male Wistar fatty rats11) were bred in Takeda Chemical In-
dustries, Ltd. and used at the age of 27-weeks. Throughout the study, they
were housed in metal mesh cages and fed a commercial diet CE-2 (Clea,
Japan) and water ad libitum. They were divided into 16 groups (5 rats in
each group) based on plasma glucose and triglyceride, and they were in-
traperitoneally injected with TAK-559 (1) and its metabolites (2—5) for 7 d.
Compounds (1—5) were suspended in 0.5% methylcellulose saline solution.
TAK-559 was injected into 3 groups of rats at the doses of 0.1, 0.3 and
1.0 mg/kg/d, and each metabolite was injected into 3 groups of rats at the
doses of 0.3, 1.0 and 3.0 mg/kg/d. Before and after the 7-d treatment, blood
was withdrawn from the tail vein, and the plasma glucose and triglyceride
levels were enzymatically measured using an Autoanalyzer 7070 (Hitachi,
Japan). The change (%) from the initial level of plasma glucose and triglyc-
eride after the 7-d treatment was calculated in each rat. The degrees of varia-
tion (% control value) in the plasma parameters after treatment were also es-
timated from the relative ratio of the change (%) in each rat to the average of
the change (%) in the control group, and the ED25 values for the glucose and
triglyceride lowering activities of TAK-559 and its metabolites were calcu-
lated by least-squares linear regression analysis using % control values.
Chemical Methods Melting points were recorded on a Yanagimoto
micro melting point apparatus and are uncorrected. Elemental analyses (C,
H, N) were carried out in the Takeda Analytical Research Laboratories, and
all values are within Ϯ0.4% of calculated values, unless otherwise noted. IR
Table 2. Effects of TAK-559 and Its Metabolites on Plasma Glucose and
Triglyceride Levels in Wistar Fatty Ratsa)
ED25 (mg/kg/d)
Compounds
Plasma glucose
Plasma triglyceride
1 (TAK-559)
0.13 (0.062—0.19)
0.079b)
2
3
4
5
Ͼ3
2.4 (0.94—ND)
Ͼ3
Ͼ3
Ͼ3
Ͼ3
Ͼ3
2.6 (1.0—ND)
a) TAK-559 and its metabolites were intraperitoneally injected into male Wistar
fatty rats (27-week-old) for 7 d. Values in the parentheses show the 95% confidence in-
terval of the ED25 values. The ED25 values greater than 3.0 mg/kg/day are shown as
“Ͼ3”. ND: not determined. b) This value was obtained by extrapolation below the
lowest dose.
plays the key role for in vivo glucose and lipid lowering ac-
tivities, rather than its metabolites (2—5). However, it is well
known that the in vivo activities of compounds can be signifi-
cantly different from their in vitro activities. So, to under-
stand the role of metabolites (2—5) on glucose and lipid
lowering activities, the in vivo activities of these metabolites
were measured in Wistar fatty rats after intraperitoneal ad-
ministration.
1
spectra were recorded on a JASCO IR-810. H-NMR spectra were recorded
on a Varian Gemini-200 spectrometer in CDCl3 or DMSO-d6 using tetra-
methylsilane as an internal standard. Chemical shifts are expressed as d
Table 2 shows the effects of TAK-559 (1) and its metabo-
lites (2—5) on the ED25 values of plasma glucose and (ppm) values for protons relative to the internal standard. All compounds ex-
1
hibited H-NMR spectra and analytical data consistent with their proposed
triglyceride lowering activities in Wistar fatty rats. TAK-559
structures. Column chromatography was performed using Merck Silica Gel
(1) showed potent plasma glucose and triglyceride lowering
activities, decreasing the plasma triglyceride level by 30%
60 (0.063—0.200 mm). The following abbreviations are used: sϭsinglet,
dϭdoublet, tϭtriplet, qϭquartet, quinϭquintet, sextϭsextet, septϭseptet,
even at the lowest dose (0.1 mg/kg). Hence, it was necessary
to estimate the ED25 value for the plasma triglyceride lower-
ing effect of TAK-559 (1) by extrapolation. Compounds 2
and 4 had less potent effects on plasma glucose and triglyc-
eride levels than TAK-559 (1). The plasma glucose levels in
3-injected rats and the plasma triglyceride levels in 5-injected
rats showed relatively weak effects compared with those in 1-
injected rats.
In conclusion, TAK-559 (1) is a more potent activator of
both hPPARg1 and hPPARa than its metabolites (2—5).
TAK-559 (1) also showed potent plasma glucose and triglyc-
eride lowering activities, while its metabolites (2—5) showed
comparatively weak activities, in Wistar fatty rats after in-
traperitoneal administration. These results indicate that TAK-
559 itself plays a key role for treatment of diabetes, rather
than its metabolites (2—5).
mϭmultiplet, brϭbroad, dec.ϭdecomposed.
Ethyl 4-(4-Methoxyphenyl)-4-oxobutanoate (7a) Ethyl succinyl chlo-
ride (48.8 g, 297 mmol) was added dropwise to a suspension of aluminum
chloride (43.6 g, 327 mmol) in dichloromethane (300 ml) at 0 °C, and the
mixture was stirred at 0 °C for 15 min. To this was added anisole (32.2 ml,
297 mmol) dropwise at 0 °C. The reaction mixture was stirred at room tem-
perature for 15 h, then poured onto ice (500 g). After stirring at room tem-
perature for 1 h, the organic layer was separated, washed with brine, dried
over magnesium sulfate, and concentrated in vacuo. The residue was chro-
matographed on silica gel with ethyl acetate–hexane (1 : 5, v/v) and recrys-
tallized from ethyl acetate–hexane to give 7a (47.1 g, 67%) as colorless crys-
1
tals. mp 49—50 °C. H-NMR (CDCl3) d: 1.27 (3H, t, Jϭ7.1 Hz), 2.74 (2H,
t, Jϭ6.8 Hz), 3.27 (2H, t, Jϭ6.8 Hz), 3.87 (3H, s), 4.16 (2H, q, Jϭ7.1 Hz),
6.94 (2H, d, Jϭ8.8 Hz), 7.97 (2H, d, Jϭ8.8 Hz). IR (KBr) cmϪ1: 2980, 1732,
1678, 1601, 1260, 1171, 1030, 833. Anal. Calcd for C13H16O4: C, 66.09; H,
6.83. Found: C, 66.08; H, 6.58.
Ethyl 4-(3,4-Dimethoxyphenyl)-4-oxobutanoate (7b) Using the proce-
dure for preparation of 7a, 7b (81% yield) was prepared from veratrole (6b)
1
as a colorless oil. H-NMR (CDCl3) d: 1.27 (3H, t, Jϭ7.1 Hz), 2.75 (2H, t,
Jϭ6.8 Hz), 3.29 (2H, t, Jϭ6.8 Hz), 3.93 (3H, s), 3.95 (3H, s), 4.17 (2H, q,
Jϭ7.1 Hz), 6.90 (1H, d Jϭ8.4 Hz), 7.54 (1H, d, Jϭ2.0 Hz), 7.64 (1H, dd,
Experimental
Biological Procedures. (a) In Vitro Transient Cotransfection Assay Jϭ2.0, 8.4 Hz). IR (KBr) cmϪ1: 2980, 1732, 1678, 1516, 1267, 1157, 1024,
COS-1 cells were seeded at 5ϫ106 cells in a 150 cm2 tissue culture flask, 858, 766.
and cultured in 5% CO2 at 37 °C overnight. Transfections were performed
Ethyl 4-(4-Hydroxyphenyl)-4-oxobutanoate (8a) Boron tribromide
with LipofectAMINE (GIBCO BRL, U.S.A.) according to the instructions (2.40 ml, 25.4 mmol) was added to a stirred mixture of ethyl 4-(4-
of the manufacturer. Briefly, the transfection mixture contained 125 ml of methoxyphenyl)-4-oxobutanoate (7a, 2.00 g, 8.46 mmol) in dichloromethane
LipofectAMINE, 100 ml of LipofectAMINE Plus, 2.5 mg of each expression (15 ml) at 0 °C, and the mixture was stirred at 0 °C for 1 h and at room tem-
plasmid pMCMVneo-hPPARg (pMCMVneo-hPPARd or pMCMVneo-hP- perature for an additional 15 h. The reaction was quenched by addition of
PARa), pMCMVneo-hRXRa, 5 mg of reporter plasmid pGL3-PPREϫ4-tk- water and extracted with ethyl acetate. The extract was washed with brine,
luc-neo and 5 mg of pRL-tk (Promega, U.S.A.). Cells were incubated in dried over magnesium sulfate, and concentrated in vacuo to leave an oil. The
25 ml of transfection mixture for 3 h in 5% CO2 at 37 °C. After adding 25 ml oil was dissolved in ethanol (20 ml), then sulfuric acid (0.2 ml) was added.
of Dulbecco’s modified eagle medium (DMEM, Nikken Bio Medical Lab., The mixture was refluxed for 2.5 h. After cooling to room temperature, it
Japan) containing 0.1% fatty acid-free bovine serum albumin (BSA), the was diluted with ethyl acetate, washed with water, brine, dried over magne-
cells were then incubated for 24 h in 5% CO2 at 37 °C. After transfection, sium sulfate, and concentrated in vacuo. The residue was chromatographed
cells were detached by treating with trypsin-EDTA (GIBCO BRL, U.S.A.) on silica gel with ethyl acetate–hexane (1 : 2, v/v) to give 8a (1.16 g, 62%) as
centrifuged and then suspended in DMEM medium containing 0.1% fatty crystals. Recrystallization of 8a from ethyl acetate–hexane gave colorless
acid free-BSA. The suspended cells were added in an OPAQUE PLATE crystals. mp 111—112 °C. 1H-NMR (CDCl3) d: 1.28 (3H, t, Jϭ7.1 Hz), 2.76
(white 96 well plate, COSTAR, U.S.A.) at the density of 8.8ϫ103 cells/well (2H, t, Jϭ6.6 Hz), 3.25 (2H, t, Jϭ6.6 Hz), 4.18 (2H, q, Jϭ7.1 Hz), 6.06 (1H,
in 80 ml of DMEM medium containing 0.1% fatty acid free-BSA and 20 ml
br s), 6.85 (2H, d, Jϭ8.8 Hz), 7.86 (2H, d, Jϭ8.8 Hz). IR (KBr) cmϪ1: 3335,

January 2004
123
1732, 1603, 1225, 1169, 839. Anal. Calcd for C12H14O4: C, 64.85; H, 6.35. phenyl-1,3-oxazole-5-carbaldehyde (12, 50.9 g, 206 mmol) in tetrahydrofu-
Found: C, 64.96; H, 6.59. ran (300 ml) and methanol (30 ml). The mixture was stirred at 0 °C for 0.5 h,
Ethyl 4-(3,4-Dihydroxyphenyl)-4-oxobutanoate (8b) Using the proce- then poured into water and extracted with ethyl acetate. The organic extract
dure for preparation of 8a, 8b (45% yield) was prepared from ethyl 4-(3,4- was washed with brine, dried over magnesium sulfate, and concentrated in
dimethoxyphenyl)-4-oxobutanoate (7b) as colorless crystals. mp 118— vacuo to leave an oil. The oil was dissolved in N,N-dimethylformamide
119 °C (ethyl acetate–hexane). 1H-NMR (CDCl3) d: 1.28 (3H, t, Jϭ7.1 Hz), (500 ml). The solution was cooled to 0 °C, then imidazole (29.5 g,
2.75 (2H, t, Jϭ6.6 Hz), 3.26 (2H, t, Jϭ6.6 Hz), 4.17 (2H, q, Jϭ7.1 Hz), 433 mmol) was added, followed by addition of tert-butylchlorodiphenylsi-
5.95—6.40 (2H, br), 6.90 (1H, d, Jϭ8.0 Hz), 7.51 (1H, dd, Jϭ2.2, 8.0 Hz), lane (67.9 g, 247 mmol) dropwise. The reaction mixture was stirred at room
7.59 (1H, d, Jϭ2.2 Hz). IR (KBr) cmϪ1: 3250, 1715, 1669, 1595, 1292,
1169, 912, 743. Anal. Calcd for C12H14O5: C, 60.50; H, 5.92. Found: C,
60.50; H, 5.81.
temperature for 1 h. The solvent was evaporated in vacuo. The residue was
diluted with ethyl acetate, washed with 1 M hydrochloric acid, water, brine,
dried over magnesium sulfate, and concentrated in vacuo. The residue was
(E)-4-[4-(5-Methyl-2-phenyl-1,3-oxazol-4-ylmethoxy)benzyloxyimino]- chromatographed on silica gel with ethyl acetate–hexane (1 : 8, v/v) to give
1
4-(4-hydroxyphenyl)butanoic Acid (2) A mixture of 4-[(5-methyl-2- 13 (80.1 g, 66%) as a colorless oil. H-NMR (CDCl3) d: 1.06 (9H, s), 3.34
phenyl-1,3-oxazol-4-yl)methoxy]benzyloxyamine2) (9, 1.54 g, 4.95 mmol), (3H, s), 4.37 (2H, s), 4.64 (2H, s), 4.80 (2H, s), 7.34—7.49 (9H, m), 7.69—
ethyl 4-(4-hydroxyphenyl)-4-oxobutanoate (8a, 1.00 g, 4.50 mmol), acetic 7.75 (4H, m), 7.97—8.03 (2H, m). IR (KBr) cmϪ1: 2932, 1555, 1427, 1150,
acid (0.773 ml, 13.5 mmol), sodium acetate (738 mg, 9.00 mmol), and 1111, 1042, 704.
ethanol (30 ml) was refluxed for 48 h, then diluted with ethyl acetate, washed
[5-(tert-Butyldiphenylsilyloxymethyl)-2-phenyl-1,3-oxazol-4-yl]-
with water, brine, dried over magnesium sulfate, and concentrated in vacuo. methanol (14) Bromotrimethylsilane (21.5 ml, 163 mmol) was added
The residue was chromatographed on silica gel with ethyl acetate–hexane dropwise to a cold (Ϫ40 °C) stirred solution of 5-(tert-butyldiphenylsilyl-
(1 : 2, v/v) to leave an oil. The oil was dissolved in tetrahydrofuran (20 ml), oxymethyl)-4-(methoxymethoxymethyl)-2-phenyl-1,3-oxazole (13, 20.0 g,
water (10 ml) and ethanol (10 ml), then lithium hydroxide monohydrate 41.0 mmol) in chloroform (250 ml). The mixture was stirred at Ϫ40 °C for
(755 mg, 18.0 mmol) was added. The reaction mixture was stirred at room 0.5 h and at 0 °C for an additional 1 h. The reaction mixture was poured into
temperature for 4 h, then made acidic by addition of 1 M hydrochloric acid
saturated aqueous sodium hydrogen carbonate and extracted with chloro-
(18.5 ml) and extracted with ethyl acetate. The extract was washed with form. The extract was dried over magnesium sulfate, and concentrated
brine, dried over magnesium sulfate, and concentrated in vacuo. The residue in vacuo. The residue was chromatographed on silica gel with ethyl
was recrystallized from ethyl acetate–hexane to give 2 (1.82 g, 83%) as col- acetate–hexane (1 : 2, v/v) and crystallized from ethyl acetate–hexane to give
1
1
orless crystals. mp 178—179 °C. H-NMR (DMSO-d6) d: 2.30—2.40 (2H, 14 (10.2 g, 56%) as colorless crystals. mp 95—96 °C. H-NMR (CDCl3) d:
m), 2.45 (3H, s), 2.82—2.92 (2H, m), 5.00 (2H, s), 5.08 (2H, s), 6.77 (2H, d, 1.06 (9H, s), 2.15 (1H, t, Jϭ6.2 Hz), 4.44 (2H, d, Jϭ6.2 Hz), 4.80 (2H, s),
Jϭ8.8 Hz), 7.04 (2H, d, Jϭ8.8 Hz), 7.35 (2H, d, Jϭ8.8 Hz), 7.44—7.54 (5H,
7.34—7.49 (9H, m), 7.67—7.74 (4H, m), 7.95—8.01 (2H, m). IR (KBr)
m), 7.90—7.98 (2H, m), 9.72 (1H, s). IR (KBr) cmϪ1: 3397, 1690, 1514,
cmϪ1: 2932, 1553, 1427, 1113, 912, 743, 702. Anal. Calcd for C27H29NO3Si:
1285, 1240, 1019, 943, 912, 808. Anal. Calcd for C28H26N2O6: C, 69.12; H, C, 73.10; H, 6.59; N, 3.16. Found: C, 72.82; H, 6.87; N, 3.08.
5.39; N, 5.76. Found: C, 68.96; H, 5.47; N, 5.64. Methyl (E)-4-(4-{[5-(tert-Butyldiphenylsilyloxymethyl)-2-phenyl-1,3-
(E)-4-[4-(5-Methyl-2-phenyl-1,3-oxazol-4-ylmethoxy)benzyloxyimino]- oxazol-4-yl]methoxy}benzyloxyimino)-4-phenylbutanoate (16) Meth-
4-(3,4-dihydroxyphenyl)butanoic Acid (3) Using the procedure for anesulfonyl chloride (0.617 ml, 7.98 mmol) was added dropwise to a cold
preparation of 2, 3 (77% yield) was prepared from ethyl 4-(3,4-dihydroxy- (0 °C) stirred solution of [5-(tert-butyldiphenylsilyloxymethyl)-2-phenyl-
phenyl)-4-oxobutanoate (8b) as pale brown crystals. mp 168—169 °C (ethyl 1,3-oxazol-4-yl]methanol (14, 3.00 g, 6.70 mmol) in ethyl acetate (30 ml).
acetate). 1H-NMR (CDCl3) d: 2.30—2.39 (2H, m), 2.45 (3H, s), 2.79—2.88 The mixture was stirred at 0 °C for 2 h, then poured into water and extracted
(2H, m), 5.00 (2H, s), 5.07 (2H, s), 6.73 (1H, d, Jϭ8.0 Hz), 6.92 (1H, dd,
with ethyl acetate. The extract was washed with brine, dried over magnesium
Jϭ2.0, 8.0 Hz), 7.01—7.10 (3H, m), 7.35 (2H, d, Jϭ8.4 Hz), 7.47—7.54 sulfate, and concentrated in vacuo to leave an oil. The oil was dissolved in
(3H, m), 7.91—7.98 (2H, m), 9.08 (1H, d, Jϭ2.0 Hz), 9.23 (1H, d,
Jϭ2.0 Hz), 12.23 (1H, s). IR (KBr) cmϪ1: 3245, 2930, 1696, 1514, 1238,
1026, 781, 720, 691. Anal. Calcd for C28H26N2O7·1/4H2O: C, 66.33; H,
5.27; N, 5.53. Found: C, 66.18; H, 5.21; N, 5.32.
N,N-dimethylformamide (15 ml). Methyl (E)-4-(4-hydroxybenzyloxyimino)-
4-phenylbutanoate2) (15, 2.00 g, 6.38 mmol) was added to the solution, fol-
lowed by addition of potassium carbonate (1.76 g, 12.8 mmol). The reaction
mixture was stirred at 50 °C for 1 h, then poured into water and extracted
4-(Methoxymethoxymethyl)-2-phenyl-1,3-oxazole (11) Sodium hy- with ethyl acetate. The organic extract was washed with brine, dried over
dride (60% in oil, 12.5 g, 314 mmol) was added to a cold (0 °C) stirred solu-
magnesium sulfate, and concentrated in vacuo. The residue was chro-
tion of (2-phenyl-1,3-oxazol-4-yl)methanol14) (10, 50.0 g, 285 mmol) in
matographed on silica gel with ethyl acetate–hexane (1 : 6, v/v) to give 16
1
tetrahydrofuran (700 ml), and the mixture was stirred at 0 °C for 10 min and (3.43 g, 73%) as a colorless oil. H-NMR (CDCl3) d: 1.05 (9H, s), 2.50—
at room temperature for an additional 1 h. The mixture was cooled to 0 °C, 2.59 (2H, m), 3.02—3.11 (2H, m), 3.61 (3H, s), 4.78 (2H, s), 4.84 (2H, s),
then chloromethyl methyl ether (26.0 ml, 342 mmol) was added dropwise. 5.15 (2H, s), 6.87 (2H, d, Jϭ8.4 Hz), 7.25—7.48 (14H, m), 7.60—7.72 (6H,
The reaction mixture was stirred at 0 °C for 10 min and at room temperature m), 7.98—8.04 (2H, m). IR (KBr) cmϪ1: 2932, 1736, 1512, 1238, 1113,
for an additional 3 h. The mixture was poured into water and extracted with 1071, 1024, 824, 704.
ethyl acetate. The extract was washed with brine, dried over magnesium sul-
fate, and concentrated in vacuo. The residue was chromatographed on silica zyloxyimino)-4-phenylbutanoic Acid (4) Tetrabutylammonium fluoride
gel with diethyl ether–hexane (1 : 1, v/v) to give 11 (61.4 g, 98%) as a pale trihydrate (2.93 g, 9.28 mmol) was added to a stirred solution of methyl (E)-
(E)-4-(4-{[5-(Hydroxymethyl)-2-phenyl-1,3-oxazol-4-yl]methoxy}ben-
yellow oil. 1H-NMR (CDCl3) d: 3.44 (3H, s), 4.61 (2H, s), 4.77 (2H, s), 4-(4-{[5-(tert-butyldiphenylsilyloxymethyl)-2-phenyl-1,3-oxazol-4-
7.42—7.49 (3H, m), 7.68 (1H, s), 8.03—8.09 (2H, m). IR (KBr) cmϪ1
2934, 1555, 1449, 1150, 1055, 716.
:
yl]methoxy}benzyloxyimino)-4-phenylbutanoate (16, 3.43 g, 4.64 mmol) in
tetrahydrofuran (30 ml). The mixture was stirred at room temperature for
4-(Methoxymethoxymethyl)-2-phenyl-1,3-oxazole-5-carbaldehyde (12) 1 h. The reaction mixture was poured into water and extracted with ethyl ac-
n-Butyllithium (1.6 M in hexane, 193 ml, 308 mmol) was added dropwise to etate. The organic extract was washed with brine, dried over magnesium sul-
a cold (Ϫ78 °C) stirred solution of 4-(methoxymethoxymethyl)-2-phenyl- fate, and concentrated in vacuo to leave an oil. The oil was dissolved in
1,3-oxazole (11, 61.4 g, 280 mmol) in diethyl ether (1000 ml), and the mix- tetrahydrofuran (20 ml), water (10 ml) and methanol (10 ml), then lithium
ture was stirred at Ϫ78 °C for 1 h. N,N-Dimethylformamide (65.0 ml, hydroxide monohydrate (584 mg, 13.9 mmol) was added. The reaction mix-
840 mmol) was added dropwise to the mixture, then the resultant mixture ture was stirred at room temperature for 1 h, then made acidic by addition of
was allowed to warm to room temperature and stirred at that temperature for 1 M hydrochloric acid (14.0 ml) and extracted with ethyl acetate. The extract
1 h. The mixture was poured into water and extracted with diethyl ether. The was washed with brine, dried over magnesium sulfate, and concentrated in
organic extract was washed with brine, dried over magnesium sulfate, and vacuo. The residue was crystallized from ethyl acetate–hexane to give 4
1
concentrated in vacuo. The residue was chromatographed on silica gel with (1.89 g, 84%) as colorless crystals. mp 139—141 °C. H-NMR (CDCl3) d:
diethyl ether–hexane (1 : 1, v/v) to give 12 (55.9 g, 81%) as a pale yellow oil. 2.53—2.62 (2H, m), 3.01—3.10 (2H, m), 4.77 (2H, s), 5.11 (2H, s), 5.16
1H-NMR (CDCl3) d: 3.45 (3H, s), 4.82 (2H, s), 4.90 (2H, s), 7.45—7.58
(2H, s), 6.99 (2H, d, Jϭ8.8 Hz), 7.30—7.50 (8H, m), 7.58—7.65 (2H, m),
(3H, m), 8.15—8.21 (2H, m), 10.03 (1H, s). IR (KBr) cmϪ1: 2944, 1682, 8.00—8.06 (2H, m). IR (KBr) cmϪ1: 2938, 1717, 1512, 1236, 1020, 912,
1541, 1451, 1152, 1046, 914, 743. 739, 693. Anal. Calcd for C28H26N2O6: C, 69.12; H, 5.39; N, 5.76. Found: C,
5-(tert-Butyldiphenylsilyloxymethyl)-4-(methoxymethoxymethyl)-2- 69.10; H, 5.24; N, 5.63.
phenyl-1,3-oxazole (13) Sodium borohydride (3.90 g, 103 mmol) was
Methyl 4-[(5-Methyl-2-phenyl-1,3-oxazol-4-yl)methoxy]benzoate (18)
added to a cold (0 °C) stirred solution of 4-(methoxymethoxymethyl)-2- A mixture of 4-chloromethyl-5-methyl-2-phenyl-1,3-oxazole15) (17, 6.00 g,

124
Vol. 52, No. 1
28.9 mmol), methyl 4-hydroxybenzoate (4.84 g, 31.8 mmol), potassium car-
bonate (4.80 g, 34.7 mmol) and N,N-dimethylformamide (50 ml) was stirred
at 70 °C for 3 h. The reaction mixture was poured into water and extracted
with ethyl acetate. The organic extract was washed with water, brine, dried
over magnesium sulfate, and concentrated in vacuo. The residue was chro-
matographed on silica gel with ethyl acetate–hexane (1 : 3, v/v) to give 18
(8.09 g, 87%) as crystals. Recrystallization of 18 from ethyl acetate–diethyl
ether gave colorless needles. mp 104—105 °C. 1H-NMR (CDCl3) d: 2.45
(3H, s), 3.89 (3H, s), 5.05 (2H, s), 7.05 (2H, d, Jϭ9.0 Hz), 7.40—7.53 (3H,
m), 7.95—8.08 (4H, m). IR (KBr) cmϪ1: 1708, 1600, 1275, 1240, 1167,
1100, 993, 765. Anal. Calcd for C19H17NO4: C, 70.58; H, 5.30; N, 4.33.
Found: C, 70.50; H, 5.22; N, 4.30.
Bull., 51, 138—151 (2003).
3) Isseman I., Green S., Nature (London), 347, 645—650 (1990).
4) Schmidt A., Endo N., Rutledge S. J., Vogel R., Shinar D., Rodan G. A.,
Mol. Endocrinol., 6, 1634—1641 (1992).
5) Kliewer S. A., Forman B. M., Blumberg B., Ong E. S., Borgmeyer U.,
Manglesdorf D. J., Umesono K., Evans R. M., Proc. Natl. Acad. Sci.
U.S.A., 91, 7355—7359 (1994).
6) Lehmann J. M., Moore L. B., Smith-Oliver T. A., Wilkison W. O.,
Willson T. M., Kliewer S. A., J. Biol. Chem., 270, 12953—12956
(1995).
7) Troglitazone: Yoshioka T., Fujita T., Kanai T., Aizawa Y., Kurumada
T., Hasegawa K., Horikoshi H., J. Med. Chem., 32, 421—428 (1989).
Although launched first in the market, troglitazone had been with-
drawn because of liver toxicity and related deaths associated with the
drug.
4-[(5-Methyl-2-phenyl-1,3-oxazol-4-yl)methoxy]benzoic Acid (5)
A
mixture of methyl 4-[(5-methyl-2-phenyl-1,3-oxazol-4-yl)methoxy]benzoate
(18, 2.00 g, 6.19 mmol), 1 M sodium hydroxide solution (18.6 ml), methanol
(20 ml) and tetrahydrofuran (30 ml) was stirred at 60 °C for 1 h. The reaction
mixture was made acidic by addition of 1 M hydrochloric acid and extracted
with ethyl acetate. The organic extract was washed with brine, dried over
magnesium sulfate, and concentrated in vacuo to give 5 (1.73 g, 90%) as
8) Pioglitazone: Sohda T., Momose Y., Meguro K., Kawamatsu Y.,
Sugiyama Y., Ikeda H., Arzneim.-Forsch., 40, 37—42 (1990).
9) Rosiglitazone: Cantello B. C., Cawthorne 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).
crystals. Recrystallization of 5 from ethyl acetate–diisopropyl ether gave
1
colorless needles. mp 185—186 °C. H-NMR (CDCl3) d: 2.46 (3H, s), 5.07 10) Iwatsuka H., Shino A., Suzuoki Z., Endocrinol. Jpn., 17, 23—35
(2H, s), 7.06 (2H, d, Jϭ9.0 Hz), 7.39—7.50 (3H, m), 7.96—8.11 (4H, m).
(1970).
IR (KBr) cmϪ1: 1670, 1600, 1302, 1252, 1215. Anal. Calcd for C18H15NO4: 11) Ikeda H., Shino A., Matsuo T., Iwatsuka H., Suzuoki Z., Diabetes, 30,
C, 69.89; H, 4.89; N, 4.53. Found: C, 69.60; H, 5.01; N, 4.20.
1045—1050 (1981).
12) Suzuki M., Odaka H., Matsumoto M., Suzuki N., Imoto H., Momose
Y., Kimura H., Sugiyama Y., Diabetologia, submitted for publication.
Acknowledgements We thank Kenji Yamashita, Norio Tada, and Mi-
dori Ono for isolation and structural determination of the TAK-559 metabo- 13) Tsuji Y., Odaka H., Suzuki M., Itasaka M., Sugiyama Y.,
lites (2—5); Shinji Moriyama for in vitro testing; Masami Suzuki and
Osamu Kataoka for in vivo testing.
Diabetologia, submitted for publication.
14) Yoo S. K., Tetrahedron Lett., 33, 2159—2162 (1992).
15) Goto Y., Yamazaki M., Hamana M., Chem. Pharm. Bull., 19, 2050—
2057 (1971).
References and Notes
1) Part 1: Imoto H., Imamiya E., Momose Y., Sugiyama Y., Kimura H.,
Sohda T., Chem. Pharm. Bull., 50, 1349—1357 (2002).
2) Part 2: Imoto H., Sugiyama Y., Kimura H., Momose Y., Chem. Pharm.
16) Sakamoto J., Kimura H., Moriyama S., Imoto H, Momose Y., Odaka
H., Sawada H., Eur. J. Pharmacol., submitted for publication.