Synthesis of JTT-501 and its metabolite JTP-20604 labelled with 13C

Article abstract of DOI:10.1002/jlcr.700

JTT-501 specifically labelled with ¹³C was obtained via a four-step synthesis at an isotopic enrichment level of 99% and in 14% overall chemical yield starting from 4-hydroxy-[ring-U-¹³C₆]benzaldehyde (3). The hydrogenatio

Full text of DOI:10.1002/jlcr.700

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2003; 46: 605–611.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.700
Research Article
Synthesis of JTT-501 and its metabolite
JTP-20604 labelled with C
1
3
A. Pignatti*, D. Giribone, C. Felicini and E. Fontana
Global Drug Metabolism, Pharmacia, viale Pasteur 10, 20014 Nerviano (MI),
Italy
Summary
1
JTT-501 specifically labelled with C was obtained via a four-step synthesis at
3
an isotopic enrichment level of 99% and in 14% overall chemical yield starting
1
from 4-hydroxy-[ring-U- C ]benzaldehyde (3). The hydrogenation of
3
6
1
3
13
C ]JTT-501 over Pd/C gave [ C ]JTP-20604 in 90% chemical yield.
6 6
[
Copyright # 2003 John Wiley & Sons, Ltd.
Key Words: JTT-501; JTP-20604; oxazolidinedione; insulin-sensitizing agent;
Carbon-13
Introduction
JTT-501 (( Æ )-(RS)-4-[4-[-2-(5-Methyl-2-phenyl-oxazol-4-ethoxy]ben-
zyl] isoxazolidine-3,5-dione) is a novel insulin-sensitizing agent belong-
ing to the isoxalidinedione class, that is currently being evaluated for its
1
efficacy in the treatment of Type 2 diabetes mellitus. Since an LC/MS
method was developed to determine the unchanged drug and its
metabolite JTP-20604 in biological matrices, a non-radioactive iso-
topically labelled version of JTT-501 and JTP-20604, with a molecular
weight of at least three mass units higher than that of the compounds
under investigation, was required for use as internal standard. A
*
2
Correspondence to: A. Pignatti, Global Drug Metabolism Dept., Pharmacia, viale Pasteur 10,
0014 Nerviano (MI), Italy. E-mail: alberto.pignatti@pharmacia.com
Copyright # 2003 John Wiley & Sons, Ltd.
Received 13 January 2003
Accepted 6 February 2003

6
06
A. PIGNATTI ET AL.
method to prepare JTT-501 is reported in the literature that foresees the
2
use of 4-hydroxybenzaldehyde as starting material. This synthetic
approach seemed at first the most convenient way to prepare a suitable
stable labelled version of JTT-501 and its metabolite due to the
13
availability of 4-hydroxy-[ring-U- C]benzaldehyde as well as of the
non-labelled intermediates. In the present paper, the preparation of
1
3
13
C]JTT-501 and [ C]JTP-20604 is reported.
[
Discussion and results
1
3
13
C]JTT-501 and its metabolite [ C]JTP-20604 were prepared as
[
outlined in Scheme 1. The methanesulfonyl derivative 2 was prepared
immediately prior to use by treatment of the commercially available
2
-(5-methyl-2-phenyl-4-oxazolyl)ethanol 1 with methanesulfonyl chlor-
ide in anhydrous pyridine. The reaction of 2 with the commercially
O
N
O
N
a
O
S
OH
O
O
O
2
1
C
C
C
C
C
H
HO
C
b
3
O
O
O
C
C
C
O
N
O
N
C
C
C
C
O
c
C
C
C
C
H
O
d
C
O
O
5
6
4
O
O
O
C
C
C
C
O
N
e
O
N
C
C
C
C
O
C
C
C
C
NH
O
O
O
O
O
[13
C]JTT-501
Reagents and conditions:
a. Methanesulfonyl chloride, pyridine, -10˚C for 3h;
b. NaH, DMF, 60˚C for 16h c. Dimethylmalonate,
f
O
O
toluene, acetic acid, piperidine ,reflux for 5h; d. H
% Pd/C, dioxane:CH OH 1:1, t.a. for 17h; e.
Hydroxylamine hydrochloride, CH ONa, CH
0˚C for 3h; f. H , 5% Pd/C, THF: CH OH 2:1, t.a.
2
,
C
C
5
3
O
N
C
C
C
C
NH2
3
3
OH,
O
HO
6
2
3
for 4h.
[13
C]JTP-20604
Scheme 1.
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 605–611

SYNTHESIS OF JTT-501
607
13
available 4-hydroxy-[ring-U- C]benzaldehyde 3 (1:1 molar ratio) in the
presence of sodium hydride in anhydrous dimethylformamide (DMF) at
6
08C for 16 h, afforded the intermediate 4. The treatment of 4 with a
slight excess of dimethyl malonate (1: 1,1 molar ratio) in the presence of
piperidine and acetic acid in dry toluene at reflux (1108C) for 5 h gave
the benzylidene 5. The hydrogenation over 5% Pd/C in dioxane:
methanol (1:1 v/v) at room temperature for about 17 h of 5 yielded the
corresponding dimethyl malonate 6. The reaction of 6 with hydro-
xylamine hydrochloride and sodium methoxide in anhydrous methanol
1
3
afforded the crude
preparative high performance liquid chromatography (HPLC),
C labelled JTT-501. After purification by
1
3
[
C]JTT-501 was obtained in >97% chemically pure form and an
isotopic enrichment of 99%. The overall chemical yield was approxi-
13
mately 14% from 3. The C labelled metabolite JTP-20604 was
13
accomplished via hydrogenation of [ C]JTT-501 over 5% Pd/C in
tetrahydrofuran: methanol (2:1 v/v) at room temperature for about 4 h.
1
3
[
C] JTP-20604 was obtained in a >99% chemically pure form with a
13
chemical yield of 89% from [ C]JTT-501. These compounds were
suitable for use as internal standards in the LC-MS bioanalytical assay.
Experimental
General methods
1
3
4
-hydroxy-[ring-U- C ]benzaldehyde (3) (99% isotopic enrichment)
6
was purchased from Cambridge Isotope Labs. All solvents and reagents
were of analytical grade and were used without purification unless
otherwise indicated. Chemical purities were determined by HPLC using
a series-200 pump (Perkin-Elmer) equipped with a series 200 solvent
degasser (Perkin-Elmer), series AS-950 autosampler (Jasco) and a LC-
2
95 UV/VIS or a LC-235 UV diode array detector (Perkin-Elmer) and
Turbochrom 4.0 software (Perkin-Elmer). Preparative HPLC was
performed using a PrepStar SD-1 pump (Varian) equipped with a
ProStar 320 UV/Vis detector (Varian), a ProStar 701 fraction collector
(Varian) and PrepStar 4.2 software (Varian).
2
-(5-Methyl-2-phenyl-4-oxazolyl)ethoxymethanesulfonyl (2)
2
-(5-Methyl-2-phenyl-4-oxazoly)ethanol 1 (1 g; 4.92 mmol) was dis-
solved in anhydrous pyridine (52.6 ml) and the solution was cooled to
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 605–611

6
08
A. PIGNATTI ET AL.
–
108C. After 5 min stirring at –108C, methanesulfonyl chloride
0.775 ml; 9.99 mmol ) was added dropwise and the solution was stirred
(
3
for 3 h at –108C. At the end of reaction (as determined by HPLC )
methylene chloride (160 ml) was added, the solution was transferred
into a 500 ml separating funnel and washed with water (3 Â 150 ml),
0
.01N HCl (2 Â 20 ml) and water (2 Â 20 ml). The organic layer was
dried (Na SO ) and, after solvent evaporation, the intermediate 2 was
2
4
3
obtained (1.166 g; 4.14 mmol), 97% chemically pure (by HPLC ;
Rt=18.6 min). The yield of this step was 84%.
1
-[2-(5-Methyl-2-phenyl-4-oxazolyl)ethoxy]-[ring-U- C] benzaldehyde
3
4
(
4)
1
3
A
solution of 4-hydroxy-[ring-U- C]benzaldehyde
3
(300 mg;
2
.34 mmol) in anhydrous DMF (2.5 ml) was added to sodium hydride
(74 mg; 3.04 mmol) in anhydrous DMF (2.5 ml) at 08C. The resulting
suspension was added dropwise to a solution of 2 (990 mg; 3.51 mmol)
in anhydrous DMF (1.5 ml) at 08C. The reaction mixture was then
stirred at 608C for 16 h under nitrogen atmosphere. At the end of the
3
reaction (as determined by HPLC ) the solution was added with water
(15 ml), the mixture transferred into a 100 ml separating funnel and
extracted with diethyl ether (3 Â 20 ml). The organic layer was washed
with brine (20 ml), dried (Na SO ) and after solvent evaporation, the
2
4
crude compound 4 was obtained. The compound was purified by flash-
chromatography on a silica gel column (15 Â 5 cm ID) using a mixture
of hexane:ethyl acetate 2:1 by volume as eluting solvent system. The
fractions containing the compound 4 were combined and after solvent
evaporation to dryness, the intermediate 4 was obtained (943 mg,
3
.83 mmol), 93% chemically pure (by HPLC ; Rt=24.2 min). The yield
2
of this step was 85%.
3
Dimethyl[4-[2-(5-methyl-2-phenyl-4-oxazolyl)ethoxy]-[ring-U- C]ben-
zylidenemalonate (5)
The intermediate 4 (943 mg, 2.83 mmol) was dissolved in anhydrous
toluene (36 ml), the solution was transferred into a round-bottomed
flask equipped with a reflux condenser and a Dean-Stark trap.
Dimethylmalonate (0.39 ml; 3.4 mmol), acetic acid (0.11 ml) and
piperidine (0.11 ml) were then added and the solution refluxed for 5 h.
3
At the end of reaction (as determined by HPLC ) the reaction mixture
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 605–611

SYNTHESIS OF JTT-501
609
was cooled to room temperature, water (30 ml) was added and the
solution, then transferred into a 100 ml separating funnel and extracted
with diethyl ether (3 Â 10 ml). The organic layer was washed with 1 N
HCl aqueous solution (10 ml), a 0.1% NaHCO aqueous solution
3
(10 ml), brine (10 ml) and dried (Na SO ). After solvent evaporation, the
2 4
crude compound 5 was obtained which was purified by flash-
chromatography on a silica gel column (20 Â 3 cm ID) using a mixture
of hexane:ethyl acetate 7:2 by volume as eluting solvent system. After
combining the fractions containing the compound 5 and solvent
evaporation to dryness, the intermediate 5 was recovered (919.4 mg,
3
.18 mmol), 97.7% chemically pure (by HPLC ; Rt=27.2 min). The
2
yield of this step was 77%.
1
4-[2-(5-methyl-2-phenyl-4-oxazolyl)ethoxy]-[ring-U- C]
3
Dimethyl
benzylmalonate (6)
The intemediate 5 (919.4 mg, 2.18 mmol) was dissolved in a mixture
dioxane:methanol 1:1 by volume (18 ml), then 5% Pd/C (103 mg) was
added. The reaction mixture was stirred under hydrogen gas at room
temperature at atmospheric pressure until the reaction was complete
(about 17 h; determined by TLC on silica gel F254 plates (20 Â 5 cm,
0
.25 mm thick; Merck) eluting with ethyl acetate 7:3 by volume). After
catalyst removal by filtration through a D4 sintered-glass filter, the
filtrate was evaporated to dryness. The residue was washed with n-
hexane (2 Â 5 ml) and the compound 6 was obtained (731 mg,
3
.73 mmol) 88% chemically pure (by HPLC ; Rt=27.2 min). The yield
1
of this step was 79%.
( Æ )-(RS)-4-[4-[-2-(5-Methyl-2-phenyl-oxazol-4-yl)ethoxy]-[ring-
13 13
U- C]benzyl] isoxazolidine-3,5-dione ([ C]JTT-501)
Sodium methoxide (281.2 mg, 5.2 mmol) in anhydrous methanol (4 ml)
was added to a solution of hydroxylamine hydrochloride (182 mg,
2.61 mmol) in anhydrous methanol (4 ml) at 08C. The obtained solution
was added dropwise to a solution of 6 (731 mg, 1.73 mmol) in anhydrous
methanol (8 ml) then stirred at 608C for 3 h. At the end of reaction (as
determined by HPLC ), water (10 ml) was added to the mixture which
4
was then washed with diethyl ether (2 Â 25 ml). The aqueous layer was
transferred into a 100 ml separating funnel, adjusted to pH 2 with a 1 N
HCl aqueous solution. A white precipitate was formed which was
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 605–611

6
10
A. PIGNATTI ET AL.
extracted with ethyl acetate (3 Â 25 ml) .The organic layer was
washed with brine (25 ml), dried (Na SO ) and after solvent evapora-
2
4
13
tion, the crude [ C]JTT-501 (326 mg) was obtained. The compound
5
was purified by preparative HPLC. The fractions containing the
compound of interest were collected under nitrogen in the dark, cooled
immediately to about 48C. After pooling, sodium chloride was added to
saturation, pH was adjusted to 2 with hydrochloric acid and the
resulting aqueous phase was extracted with cold ethyl acetate and
toluene (3 Â 25 ml). The organic layers were pooled and dried (Na SO ).
2
4
1
3
After filtration and solvent evaporation to dryness, [ C]JTT-501 was
4
obtained (182.8 mg, 0.46 mmol) 99% chemically pure (by HPLC ;
Rt=19.7 min). The yield of this step was approximately 27%. Mass
+
spectrum (ESI-MS/MS): m/z 399 ([MH] ). H NMR (DMSO d6;
1
5
00 MHz) 2.35 d (singlet, 15 CH ); 2.89 d ( multiplet, 12 CH ); 3.0–3.1 d
3
2
(
multiplet, 4 CH ); 4.14 d, (multiplet, 11 CH ); 6.74 d, (multiplet, 7
2
2
CH+9 CH); 7.07 d (multiplet, 6 CH+10 CH); 7.45 d (multiplet, 19
CH+20 CH+21 CH); 7.91 d (multiplet, 18 CH+22 CH,); 8.45 d
(singlet, CONH-O).
O
1
*
0
1
5
9
4
2
1
22
18
5
1
4
3
O
N
1
*
1
6
*
*
2
0
NH
1
1
1
7
*
8
O
2
1
3
O
6
O
1
9
*
1
2
7
*=denotes position of carbon-13
1
-{4-[2-(5-Methyl-2-phenyl-1,3-oxazol-4-yl)ethoxy]-[ring-U- C]ben-
3
2
zyl}-3-oxo-beta-alanine ([ C]JTP-20604)
13
1
3
[
C] JTT-501 (75.8 mg, 0.19 mmol) was dissolved in a mixture of
tetrahydrofuran: methanol 2:1 by volume (1.5 ml), after which 5% Pd/C
8.3 mg) was added. The reaction mixture was stirred under hydrogen
(
gas at room temperature at atmospheric pressure until the reaction was
4
complete (about 4 h) (determined by HPLC ). After catalyst removal by
filtration through a Uniprep PTFE 0.2 mm filter (Wathman), the filtrate
3
1
was evaporated to dryness. After solvent evaporation, [ C] JTP-20604
was obtained (68 mg, 0.169 mmol) in 99% chemically pure form (by
4
HPLC ; Rt=12.6 min). The overall yield was about 89%. Mass
+
spectrum (ESI-MS/MS): m/z 401 ([MH] ). H NMR (DMSO d6;
1
5
3
6
00 MHz); 2.34 d (singlet, 15 CH ); 2.90 d ( multiplet, 4 CH +12 CH );
.39 d (triplet of doublets, 2 CH); 4.16 d, (triplet of doublets, 11 CH2);
.81 d (multiplet, 7 CH+9 CH); 7.08 d (multiplet, 6 CH+10 CH); 6.95
3
2
2
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 605–611

SYNTHESIS OF JTT-501
611
and 7.40 d (two singlets CONH ); 7.48 d (multiplet, 19 CH+20 CH+21
2
CH); 7.90 d (multiplet, 18 CH+22 CH); 12.39 d (broad signal, COOH).
O
6
*
1
5
4
2
1
22
18
7
O
N
2
*
*
2
0
NH2
1
1
*
* HO
10
O
O
1
9
*
1
2
9
*=denotes position of carbon-13
Acknowledgements
The authors thank Daniela Borghi and Emanuele Arlandini,
Pharmacia PS Predevelopment Analysis- Nerviano (Milan), for NMR
and MS spectra.
References
1
2
3
. Shibata T, Matsui K, Nagao K, et al. Eur J Pharmacol 1999; 364: 211–219.
. Shinkai H, Onogi S, Tanaka M, et al. J Med Chem 1998; 41:1927–33.
. Symmetry C18 column (mm 150 Â 4.6 ID, 5 mm, by Waters) eluting with
CH CN (A) and 25 mM K HPO at pH 2.5 with H PO (B) mixtures:
3
2
4
3
4
1
0 min at 30%A; from 30%A to 50%A in 10 min; 10 min at 50% A. Flow
rate: 1 ml/min. Column temperature: 408C. Analytical wavelength: 254 nm.
. Eclipse XDB C18 column (mm 150 Â 4.6 ID, 5 mm, by Zorbax) eluting with
4
2
0 mM K HPO , 5mM tetrabutylammonium hydroxide at pH 6.5: THF 5:1
2 4
(v/v) (A) and 20 mM K HPO , 5 mM tetrabutylammonium hydroxide at pH
2 4
6
.5: THF 4:6 (v/v) (B) mixtures: 40 min at 100% A; from 100% A to 0% A
in 15 min; 4 min at 100% B. Flow rate: 0.8 ml/min. Column temperature:
08C. Analytical wavelength: 280 nm.
4
5
. Combi HT XDB C18 column (mm 150 Â 21.2 ID, 5 mm, by Zorbax) eluting
with 20 mM K HPO , 5 mM tetrabutylammonium hydroxide at pH 6.5:
2
4
THF 5:1 (v/v) (A) and 20 mM K HPO , 5 mM tetrabutylammonium
2
4
hydroxide at pH 6.5: THF 4:6 (v/v) (B) mixtures: 25 min at 90% A; from
0% A to 0% A in 15 min; 5 min at 100% B. Flow rate: 24 ml/min. Column
temperature: ambient. Analytical wavelength: 280 nm.
9
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 605–611