Synthesis of 3H, 14C and 13C6 labelled Sch 58235

Article abstract of DOI:10.1002/jlcr.539

³H-Sch 58235 was prepared at a specific activity of 29.1 Ci/mmol by Ir(COD)(Cy₃P)PyPF₆ catalysed exchange with tritium gas. ¹⁴C-Sch 58235 was prepared in three steps from p-hydroxy[ring-U-¹⁴C]benzaldehyde with an overall radiochemical yield of 21%. ¹³C₆-Sch 58235 was similarly prepared in three steps from p-hydroxy[ring-U-¹³C₆]benzaldehyde in an overall yield of 41%. Copyright

Full text of DOI:10.1002/jlcr.539

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2002; 45: 145–155.
DOI: 10.1002/jlcr.539
Research Article
3
14
13
Synthesis of H, C and C6 labelled
Sch 58235
D. Hesk*, G. Bignan, J. Lee, J. Yang, K. Voronin, C. Magatti,
P. McNamara, D. Koharski, S. Hendershot, S. Saluja and S. Wang
Schering Plough Research Institute, 2015 Galloping Hill Road,
Kenilworth NJ 07033, USA
Summary
3H-Sch 58235 was prepared at a specific activity of 29.1 Ci/mmol by
Ir(COD)(Cy3P)PyPF6 catalysed exchange with tritium gas. 14C-Sch 58235
was prepared in three steps from p-hydroxy[ring-U-14C]benzaldehyde with an
overall radiochemical yield of 21%. 13C6-Sch 58235 was similarly prepared in
three steps from p-hydroxy[ring-U-13C6]benzaldehyde in an overall yield of
41%. Copyright # 2002 John Wiley & Sons, Ltd.
Key Words: Sch 58235; tritium; carbon-13; carbon-14; synthesis
Introduction
Sch 582351,2 inhibits the absorption of dietary cholesterol and is
currently in Phase II clinical trials as a successor to the earlier candidate
Sch 484613. The preparation of labelled Sch 48461 compound has
already been reported.4
As is typical during the progression of a compound from the
discovery to development phase, 3H-Sch 58235 was initially requested to
conduct preliminary AME studies. Once the compound had progressed
to the development phase, 14C-Sch 58235 and SIL-Sch 58235 were
prepared. This paper thus describes the synthesis of each labelled form.
*Correspondence to. D. Hesk, Schering Plough Research Institute, 2015 Galloping Hill Road,
Kenilworth, NJ 07033, USA.
Copyright # 2002 John Wiley & Sons, Ltd.
Received 4 October 2001
Accepted 24 October 2001

146
D. HESK ET AL.
Results and discussion
3H-Sch 58235 was synthesized by Ir(COD)(Cy3P)PyPF6 (Crabtree’s
Catalyst)5 catalysed exchange with tritium gas.
3H NMR analysis showed tritium was exclusively located in the
fluoroaniline ring and ortho to the b-lactam.
320 MHz proton decoupled 3H NMR of 3H-Sch 58235 in d6-DMSO.
Such a finding is consistent with earlier deuterium labelling results in
2
model acetanilides and H-Sch 48461, suggesting the formation of a
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SYNTHESIS OF 3H, 14C AND 13C6
147
6-membered cyclometallated species with the ortho position and the
carbonyl in the b-lactam6,7.
The first proposed route to 14C-Sch 58235 is shown below:
The label was to be introduced from p-bromo-[ring-U-14C]-fluor-
obenzene 3 by firstly making the Grignard reagent and converting this
to the zinc complex 4. The freshly prepared 14C-zincate would be then
reacted with the acid chloride 5 in the presence of Pd(0). Although this
method was viable using unlabelled commercially available Grignard
reagent, the reaction failed when 14C-p-bromofluorobenzene was the
starting material. Lack of formation of the desired 14C Grignard and
hence the 14C-zincate was the most likely cause. This route was
abandoned in favour of an alternative route shown below.
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D. HESK ET AL.
In this route, p-fluoro-[ring-U-14C]-acetophenone 6 was converted
to the TMS enol ether 7 and reacted with the freshly prepared
chiral aldehyde 8. The alcohol 9 was obtained in 27% yield, with the low
yield likely due to competing enolisation of the aldehyde. Dehydration
with PTSA proceeded in low yield and attempts to reduce the small
amount of product with Wilkinson’s catalyst gave none of the desired
ketone 10. This route was abandoned in favour of the route shown
below.
This route, which has become the working Chemical Development
synthesis, used p-hydroxy-[ring-U-14C]-benzaldehyde as the starting
material. This route was also used for the preparation of 13C6-Sch
58235:
The initial steps involved preparation of S alcohol 12 via CBS
reduction of the ketone 11 supplied by Chemical Development and
preparation of the 14C-imine 13 in 91% yield by heating p-fluoroaniline
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SYNTHESIS OF 3H, 14C AND 13C6
149
with p-hydroxy-[ring-U-14C]-benzaldehyde in isopropanol. The S
alcohol 12 and 1.3 equivalents of the 14C-Schiff base 13 were silylated
with TMS chloride and then treated with titanium tetrachloride to
yield intermediate 14 in 39% radiochemical yield with the desired
R, S configuration. Attempts to use 14C-Schiff base 13 stoi-
chiometrically led to incomplete reaction and still lower yields.
Intermediate 14 was then re-silylated with N,O-bis-(trimethylsilyl)
acetamide (BSA) and then cyclized with catalytic tetrabutyl ammonium
fluoride to yield crude 14C-Sch 58235. Recrystallisation from isopropa-
nol and wateryielded 19 mCi of 14C-Sch 58235 in 21% overall
radiochemical yield.
Forthe 13C-synthesis, the imine was prepared in 97% yield from
13C6-p-hydroxybenzaldehyde and p-fluoroaniline. The chiral aldol
proceeded in 40% yield after recrystallization to generate 14. Finally,
cyclization and removal of the chiral auxillary resulted in 13C6-Sch
58235 16 in 90% yield after recrystallization.
Initially it was attempted to prepare SIL-Sch 58235 with the
use of deuterium and Crabtree’s catalyst, as shown in the following
scheme:
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D. HESK ET AL.
Starting from intermediate 17, exchange of the protons ortho to the
ketone and ortho in the aromatic attached to the lactam nitrogen, was
attempted with Crabtree’s catalyst. Although the protons ortho to the
ketone were totally exchanged, three successive exchange reactions
afforded a M+3 and M+4 cluster 18. After chiral reduction to alcohol
19, attempts at further exchange were also unsuccessful. Finally after
de-benzylation to form 2H-Sch 58235 20 a final exchange was
attempted, with no further deuterium enrichment seen. Hence as a
SIL standard with clean enrichment was desired, this approach was
abandoned in favourof 13C6-Sch 58325.
Experimental
Materials
p-Hydroxy[ring-U-14C]benzaldehyde was purchased from Amersham
plc. Compound 11 and unlabelled Sch 58235 1 were obtained
from Schering Plough Research Institute, Chemical Develop-
ment. 13C6-p-hydroxybenzaldehyde was obtained from Cambridge
Isotope Laboratories. (Tricyclohexylphosphine) (1-5-cyclooctadiene)
(pyridine) iridium (I) hexafluorophosphate was purchased from the
Aldrich Chemical Company. All remaining reagents and solvents were
purchased from Aldrich or Acros Organics and were used as received.
All 14C and 13C-synthetic steps were carried out under an atmosphere of
argon.
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SYNTHESIS OF 3H, 14C AND 13C6
151
Liquid scintillation counting
Quantitation of radioactivity was performed using a Packard 2200CA
liquid scintillation analyser, with Scintiverse BD cocktail used
throughout.
Thin layer chromatography
Thin layer chromatography was performed using Whatman
LK6DF (silica gel 60) 5 Â 20 cm, 0.25 mm plates. The plates were
scanned on a Bioscan 1000 linearanalyse.r The following systems
were used:
1. Ethyl acetate:hexane (1:1)
2. Ethyl acetate:hexane (6:4)
High performance liquid chromatography
3H and 14C-Sch 58235 were analysed by hplc for radiochemical,
chemical and chiral purity. 13C6-Sch 58235 was assayed forchemical
purity alone. A Waters 600E system controller was used with a Waters
712 WISP auto injector. Chemical purity was determined using a
Waters 486 single channel UV detector and radiochemical purity using a
Radiomatic Flow 1 radioflow detector with Radiomatic Flo-Scint III
liquid scintillation cocktail. The following systems were used:
1. Phase-Separations Spherisorb S5 ODS2, 10 cm  4.6 mm ID,
240 nm, methanol:water(1:1) at 1.5 ml/min forabout 15 min followed
by a gradient to acetonitrile.
2. Keystone scientific PVA-silica, 15 cm  4.6 mm ID, 240 nm,
hexane: ethanol (90:10) at 1.5 ml/min followed by a gradient to 100%
ethanol.
3. Supelco ABZ, 15 cm  4.6 mm ID, 248 nm, acetonitrile:water
(35:65) to (58:42) over20 min to (95:5) over10 min at 1.5 ml/min.
4. Daicel Chiralcel OD-R, 25 cm  4.6 mm ID, 248 nm, acetonitrile:-
water(45:55) at 0.4 ml/min.
5. Daicel Chiralcel OD-H, 25 cm  4.6 mm ID, 254 nm, hexane:etha-
nol (70:30) at 1 ml/min.
3
Synthesis of H-Sch 58235 (2)
Sch 58235 (12 mg) and Ir(COD)(Cy3P)(Py)PF6 (1.2 mg) were dissolved
in dry methylene chloride (2 ml) and stirred with tritium gas (20 Ci) at
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D. HESK ET AL.
room temperature for 16 h. At the completion of the reaction, the
contents were diluted in ethanol and evaporated to dryness. The
product was redissolved and the evaporation repeated twice more. A
total of 1.3 Ci was isolated with a radiochemical purity as determined in
tlc system 1 of 80%.
40 mCi of the crude product was purified by hplc system 2 to yield
3
10.4 mCi of H-Sch 58235 at a specific activity of 29.1 Ci/mmol. The
radiochemical purity as determined by hplc systems 1 and 2 was >99%.
3
3H NMR of H-Sch 58235, d7.25 ppm.
Synthesis of 14C-Sch 58235 (15)
3À[5À(p-fluorophenyl)-5-hydroxy-1-oxopentyl]-4-phenyl-2-oxazolidi-
none 12. RÀ(+)-a,a-Diphenyl-2-prolinol (759 mg, 3 mmol) and tri-
methylboroxine (510 ml, 3.66 mmol) were dissolved in toluene (100 ml)
and distilled. Upon completion of the distillation, a further 100 ml of
toluene was added and the distillation repeated. The formed catalyst
was dissolved in anhydrous methylene chloride (10 ml), borane dimethyl
sulphide (1 ml, 10.54 mmol) was added and the solution was cooled to
08C. A solution of 3-[5-(1, 5-Dioxo-5-(p-fluorophenyl) pentyl]-4-phenyl-
2-oxazolidinone (3.56 g, 10 mmol) in methylene chloride (10 ml) was
then added dropwise over 40 min and the reaction continued for 2 h at
08C.
Afterchecking forcomplete reaction by tlc system 1, methanol (3 ml)
was added and the reaction stirred at ice temperature for 10 min before
1.5 ml of 30% hydrogen peroxide was added followed by 15 ml of water
after a further 10 min. The organic layer was separated and washed with
5% sodium sulphite solution, 1 M sulphuric acid and brine. After drying
over anhydrous sodium sulphate, filtration and drying, a quantitative
yield of 12 was obtained. Analysis in hplc system 5 showed >99% ee of
the desired SS product.
4À[[(p-fluorophenol) imino] methyl] [ring-U-14C] phenol 13. p-Hydro-
xy[ring-U-14C]benzaldehyde (90 mCi, 93 mg, 0.73 mmol) and p-hydroxy-
benzaldehyde (185 mg, 1.51 mmol) were mixed and suspended in
isopropanol (1 ml). The suspension was heated until complete solution
was obtained and p-fluoroaniline (220 ml, 2.36 mmol) was added dropwise.
The reaction was heated at 50–608C for1 h duirng which a yellow
precipitate formed. The reaction was diluted with isopropanol (300 ml) and
stirred at room temperature for a further 30 min before the product was
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SYNTHESIS OF 3H, 14C AND 13C6
153
collected by filtration and dried under vacuum. A yield of 82 mCi, 441 mg
(91%) of 13 was obtained which was used dir ectly in the next step.
3À[5À(4Àfluorophenyl)-2-[[4-fluorophenyl) amino] [4-[(trimethylsi-
lyl) - oxy] [ring-U-14C] phenyl] methyl- 1- oxo- 5- [trimethylsilyl) oxy]
pentyl-2-oxzaolidinone 14. A solution of 12 (566 mg, 1.58 mmol) in
methylene chloride (11 ml) was transferred via cannula into a flame
dried flask containing 13 (82 mCi, 441 mg, 2.05 mmol). Diisopropy-
lethylamine (1.65 ml, 9.46 mmol) was added and the solution cooled to
0–58C. Trimethylsilyl chloride (660 ml, 5.23 mmol) was added and the
reaction stirred for 75 min at this temperature before it was cooled to
between À35 and À308C.
Titanium tetrachloride (180 ml, 1.66 mmol) was added dropwise and
the reaction was stirred for 4 h at this temperature before it was
quenched by the addition of acetic acid (450 ml) while maintaining the
temperature below À258C. The reaction was stirred for 15 min and
poured into 5% aqueous tartaric solution (11.6 ml) at 08C. Stirring was
continued at 08C for 15 min and at room temperature for a further hour.
Sodium bisulphite (566 mg) was then added. The organic layer was split
off and the aqueous layer extracted with methylene chloride (5 ml). The
combined organic layers were washed with water (12 ml) and
evaporated to dryness.
The solid was dissolved in methylene chloride (3.5 ml), N,O-Bis-
(trimethylsilyl)acetamide (BSA) (460 ml) was added and the solution
refluxed for 30 min. The reaction was evaporated to dryness and the
resulting solid residue recrystallized from ethyl acetate and hexane to
yield 32 mCi, 576 mg, of 14 (39%). The radiochemical purity as
determined by tlc system 2 was 89%.
1À(4Àfluorophenyl-3-[3-(4-fluorophenyl-3-hydroxypropyl-4-([ring-
U-14C])-4Àhydroxy phenyl-2-azetidinone, 14C-Sch 58235 15. BSA
(440 ml, 1.75 mmol) was added to a solution of 14 (32 mCi, 576 mg,
0.8 mmol) in anhydrous methylene chloride (3.8 ml) under argon. After
stirring for 15 min at room temperature, the reaction was cooled to 08C
and tetrabutyl ammonium fluoride trihydrate (31.4 mg, 0.10 mmol) added.
The reaction was stirred at 08C for2 h and then evaporated to an oil.
The oil was dissolved in isopropanol (4.2 ml) and sulphuric acid (1 M,
1.2 ml) was added. After stirring for 1 h, water was added dropwise until
a precipitate was observed. The mixture was cooled to 08C and the
precipitate collected by filtration. The crude product was recrystallized
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D. HESK ET AL.
twice from isopropanol:water to yield 19.1 mCi, 196 mg (60%) of 14C-
Sch 58235 at a specific activity of 40.5 mCi/mmol. The radiochemical
purity determined by reverse phase hplc (hplc system 3) and chiral hplc
(hplc system 4) was in excess of 99%.
Synthesis of 13C6-Sch 58235 (16)
4À[[(p-fluorophenol) imino] methyl] [13C6] phenol 13. p-Hydro-
xy[13C6]benzaldehyde (989 mg, 7.71 mmol)) was suspended in isopropa-
nol (4 ml) and heated until complete solution was obtained.
p-Fluoroaniline (760 ml, 8.1 mmol) was then added dropwise and
reaction was heated at 50–608C for 1 h during which a yellow precipitate
formed. The product was collected by filtration and dried under
vacuum. A yield of 1650 mg (97%) of 13 was obtained.
3À[5À(4Àfluorophenyl)-2-[[4-fluorophenyl) amino] [4-[(trimethylsi-
lyl)-oxy] [13C6] phenyl] methyl- 1- oxo- 5- [trimethylsilyl) oxy] pentyl-
2-oxzaolidinone 14. A solution of 12 (1.03 g, 2.89 mmol) in methylene
chloride (5 ml) was transferred via cannula into a flame dried flask
containing 13 (795 mg, 3.6 mmol) in methylene chloride (18 ml).
Diisopropylethylamine (3.2 ml, 15.2 mmol) was added and the solution
cooled to 0–58C. Trimethylsilyl chloride (1.2 ml, 9.6 mmol) was added
and the reaction stirred for 60 min at this temperature before it was
cooled to between À35 and À308C.
Titanium tetrachloride (332 ml, 3.03 mmol) was added dropwise and
the reaction was stirred for 3 h at this temperature before it was
quenched by the addition of acetic acid (800 ml) while maintaining the
temperature below À258C. The reaction was stirred for 15 min and
poured into 5% aqueous tartaric solution (25 ml) at 08C. Stirring was
continued at 08C for 15 min and at room temperature for a further hour.
Sodium bisulphite (1 g) was then added. The organic layer was split off
and the aqueous layer extracted with methylene chloride (3 Â 20 ml).
The combined organic layers were washed with water (12 ml) and
evaporated to dryness.
The solid was dissolved in methylene chloride (4.5 ml), N,O-Bis-
(trimethylsilyl)acetamide (BSA) (800 ml) was added and the solution
refluxed for 30 min. The reaction was evaporated to dryness and the
resulting solid residue recrystallized from ethyl acetate and hexane to
yield 995 mg, of 14 (47%).
Copyright # 2002 John Wiley & Sons, Ltd.
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SYNTHESIS OF 3H, 14C AND 13C6
155
1À(4Àfluorophenyl)-3-[3-(4-fluorophenyl)-3-hydroxypropyl]-4-([13C6]-
4-hydroxyphenyl) -2-azetidinone, 13C6-Sch 58235 16. BSA (480 ml,
1.75 mmol) was added to a solution of 14 (490 mg, 0.77 mmol) plus
(an additional 148 mg , 0.2 mmol of 14 from an earlier synthesis) in
anhydrous methylene chloride (3.5 ml) under argon. After stirring for
15 min at room temperature, the reaction was cooled to 08C and
tetrabutyl ammonium fluoride trihydrate (35 mg, 0.11 mmol) added.
The reaction was stirred at 08C for90 min and then evaporated to an oil.
The oil was dissolved in isopropanol (2.2 ml) and sulphuric acid (1 M,
0.32 ml) was added. After stirring for 1 h, water was added dropwise
until a precipitate was observed. The mixture was cooled to 08C and the
precipitate collected by filtration. The product was vacuum dried to
yield 330 mg (90%) of 13C6-Sch 58235 The chemical purity as
determined by reverse phase hplc (hplc system 3) and chiral hplc (hplc
system 4) was in excess of 98%.
Acknowledgements
The authors would like to thank Professor J. R. Jones, Mrs. L. Carroll
and Mr. J. P. Bloxsidge of the University of Surrey for the tritium nmr
work. Thanks are also due to Dr. M. J. Morley from Amersham
Corporation for the 3H-Sch 58235 synthesis and to Dr. T. K.
Thiruvengadam Dr. G. Wu, Mr. Y. Wong and Mr. Z. Ding from
SPRI Chemical Development for intermediates, procedures and helpful
discussions during the 14C and 13C synthesis.
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Copyright # 2002 John Wiley & Sons, Ltd.
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