Facile synthesis of stable isotope-labeled antibacterial agent RWJ-416457 and its metabolite

Article abstract of DOI:10.1002/jlcr.2936

Synthesis of multiple stable isotope-labeled antibacterial agent RWJ-416457, (N-{3-[3-fluoro-4-(2-methyl-2,6-dihydro-4H-pyrrolo[3,4-c]pyrazol-5- yl)-phenyl]-2-oxo-oxazolidin-5-ylmethyl}-acetamide), and its major metabolite, N-{3-[4-(2,6-dihydro-4H-pyrrolo[3,4-c]pyrazol-5-yl)-3-fluoro-phenyl] -2-oxo-oxazolidin-5-ylmethyl}-acetamide, is described. The stable isotope-labeled [¹³CD₃]RWJ-416457 was prepared readily by acetylation of the precursor amine, 5-aminomethyl-3-[3-fluoro-4-(2-methyl-2,6- dihydro-4H-pyrrolo[3,4-c]pyrazol-5-yl)-phenyl]-oxazolidin-2-one with CD ₃¹³COCl in pyridine. Synthesis of the stable isotope-labeled metabolite involved a construction of multiple isotope-labeled pyrazole ring. N,N-dimethyl(formyl-¹³C,D)amide dimethyl acetal was first prepared by treating N,N-dimethyl(formyl-¹³C,D)amide with dimethyl sulfate, followed by sodium methoxide. Then, N-{3-[3-fluoro-4-(3-oxo- pyrrolidin-1-yl)-phenyl]-2-oxo-oxazolidin-5-ylmethyl}-acetamide was condensed with N,N-dimethyl(formyl-¹³C,D)amide dimethyl acetal, and the resultant β-ketoenamine intermediate underwent pyrazole ring formation with hydrazine-¹⁵N₂, to give the [¹³C ¹⁵N₂D]-labeled metabolite. Copyright

Full text of DOI:10.1002/jlcr.2936

Research Article
Received 17 April 2012,
Revised 30 April 2012,
Accepted 14 May 2012
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.2936
Facile synthesis of stable isotope-labeled
antibacterial agent RWJ-416457 and
its metabolite
*
Ronghui Lin and Larry E. Weaner
Synthesis of multiple stable isotope-labeled antibacterial agent RWJ-416457, (N-{3-[3-fluoro-4-(2-methyl-2,6-dihydro-4H-pyrrolo
[3,4-c]pyrazol-5-yl)-phenyl]-2-oxo-oxazolidin-5-ylmethyl}-acetamide), and its major metabolite, N-{3-[4-(2,6-dihydro-4H-pyrrolo
[3,4-c]pyrazol-5-yl)-3-fluoro-phenyl]-2-oxo-oxazolidin-5-ylmethyl}-acetamide, is described. The stable isotope-labeled [13CD3]
RWJ-416457 was prepared readily by acetylation of the precursor amine, 5-aminomethyl-3-[3-fluoro-4-(2-methyl-2,6-dihydro-
4H-pyrrolo[3,4-c]pyrazol-5-yl)-phenyl]-oxazolidin-2-one with CD133COCl in pyridine. Synthesis of the stable isotope-labeled
metabolite involved a construction of multiple isotope-labeled pyrazole ring. N,N-dimethyl(formyl-13C,D)amide dimethyl acetal
was first prepared by treating N,N-dimethyl(formyl-13C,D)amide with dimethyl sulfate, followed by sodium methoxide. Then,
N-{3-[3-fluoro-4-(3-oxo-pyrrolidin-1-yl)-phenyl]-2-oxo-oxazolidin-5-ylmethyl}-acetamide was condensed with N,N-dimethyl
(formyl-13C,D)amide dimethyl acetal, and the resultant b-ketoenamine intermediate underwent pyrazole ring formation with
hydrazine-15N2, to give the [13C15N2D]-labeled metabolite.
Keywords: isotope label; RWJ-416457; antibacterial agent; metabolite; pyrazole construction
difference to nullify the effect of naturally abundant heavy
isotopes in the drug molecule. The mass difference depends on
Introduction
RWJ-416457, N-{3-[3-fluoro-4-(2-methyl-2,6-dihydro-4H-pyrrolo
the molecular weight and elements of the analyte. The stable
[3,4-c]pyrazol-5-yl)-phenyl]-2-oxo-oxazolidin-5-ylmethyl}-acetamide
isotopes should be labeled at non-exchangeable positions. The
(1) is an orally active antibacterial agent under investigation for
isotopically labeled compounds should be chemically pure, free
or less than 0.1% of unlabeled compound, and usually possess a
difference of at least 3 amu’s compared with the unlabelled parent
molecules.
Pyrazole is a versatile and important building block for many
biologically active and pharmaceutical compounds. For example,
the treatment of infections caused by clinically important
gram-positive bacteria.1–4 This pyrrolopyrazolyl-substituted
oxazolidinone shows a high selectivity against gram-positive
bacteria, as well as it demonstrates a potent in vivo efficacy.
In comparison with (Food and Drug Administration) FDA-approved
antibiotic Linezolid (marketed as ZyvoxW), RWJ-416457 inhibited
the growth of Linezolid-susceptible staphylococci, enterococci, and
streptococci at concentrations of ≤4mg/mL, generally exhibiting
twofold to fourfold greater potency than that of Linezolid.2a
The des-methyl analog on the pyrazole ring of the parent
drug, that is, N-{3-[4-(2,6-dihydro-4H-pyrrolo[3,4-c]pyrazol-5-yl)-
3-fluoro-phenyl]-2-oxo-oxazolidin-5-ylmethyl}-acetamide (2), was
identified as the major metabolite. To support drug development
efforts, stable isotope-labeled RWJ-416457, as well as its metabo-
lites were required.
it was built into antibacterial agents,1–4,6,7 and other therapeutic
agents such as those for treating cognitive disorder such as
Alzheimer’s disease.8 There have been some literature reports
for the synthesis of isotope-labeled pyrazole-containing com-
pounds such as 14C-labeled stanozolo9 but none for the con-
struction of multiple stable isotope-labeled pyrazole ring. Herein,
we report a rapid and efficient synthesis of stable isotope-
labeled antibacterial agent RWJ-416457 (3, Figure 1), as well
as its pyrazole-containing metabolite (4), which involves con-
struction of multiple stable isotope-labeled pyrazole ring.
Stable isotope-labeled compounds are commonly used as
internal standards for liquid chromatography tandem mass
spectrometry (LC–MS–MS) quantitative bioanalysis of clinical and
forensic samples.5 In order to better evaluate biological behavior
of drug candidates, accurate analysis is important to establish the
exposure of the drug compounds in biological subjects. LC–MS
has been generally used to quantify low levels of drug compounds
in biological samples such as blood, plasma, serum, or urine. The
nature of the internal standard is an important aspect of any
quantitative analytical procedure. The optimum internal
standard is a pure, stable, and isotopically labeled analog of
the drug compound with a sufficiently large enough mass
Results and discussion
The synthesis of RWJ-416457 (1) and its N-de-methylated analog
(2) has been reported.3,4 The synthesis of stable isotope-labeled
Janssen Research and Development LLC, Janssen Pharmaceutical Companies of
Johnson & Johnson, 1000 Route 202, Raritan, New Jersey 08869, USA
*Correspondence to: Ronghui Lin, Janssen Research and Development LLC,
Janssen Pharmaceutical Companies of Johnson & Johnson, Raritan, NJ 08869, USA.
E-mail: Ronghui.Lin@yahoo.com
J. Label Compd. Radiopharm 2012
Copyright © 2012 John Wiley & Sons, Ltd.

R. Lin and L. E. Weaner
O
labeled hydrazine-15N2 was generated in situ from commer-
cially available hydrazine sulfate-15N2 15NH125NH2.H2SO4) and
(
O
N
N
Me
N
N
potassium t-butoxide solution in THF. Similar condensation
reaction with hydrazine sulfate-15N2 without prior treatment
with potassium t-butoxide failed to produce the desired pro-
duct. Also, similar reaction with prior treatment of hydrazine sul-
fate-15N2 with two equivalents of diethyl isopropyl amine,
instead of potassium t-butoxide, was unsuccessful.
NHAc
N
F
1
O
O
N
N
HN
NHAc
An alternative synthesis for the stable isotope-labeled
RWJ-416457 could be achieved by methylation of the stable
isotope-labeled metabolite N-{3-[4-(2,6-dihydro-4H-pyrrolo[3,4-c]
pyrazol-5-yl)-3-fluoro-phenyl]-2-oxo-oxazolidin-5-ylmethyl}-
acetamide (4) with methyl iodide following a previously reported
method.4 However, because of the known poor regioselectivity
of the methylation on N1 and N2 of the pyrazole ring, as well
as the difficult separation of the two methylated regioisomers
this approach was not pursued.
F
2
O
O
H
N
N
N
Me
N
CD3
13C
N
F
3
O
O
D
13C
15N
O
N
N
H
15N
NHAc
General experimental
F
4
Stable isotope-labeled reagents acetyl chloride-1-13C,D3 (CD313COCl),
Figure 1. RWJ-416457 (1), its major metabolite (2), and their corresponding
designed isotope-labeled compounds (3 and 4).
N,N-dimethyl(formyl-13C,D)amide and hydrazine sulfate-15N2
(
15NH125NH2.H2SO4) were purchased from Isotec (a division of
Sigma-Aldrich, St. Louis, MO). Dimethyl sulfate, potassium t-butoxide
in THF solution (1M), and deuterated methanol (CD3OD) were
purchased from Sigma-Aldrich and were used as received. Other
reagents and solvents were obtained from VWR International.
Nuclear magnetic resonance spectra were acquired on a Bruker
300-Avance (300 MHz) spectrometer with Tetramethylsilane
(TMS) as an internal standard. Chemical shifts are expressed in
parts per million (ppm, d scale). LC–MS analysis was performed
on an Agilent 1100 series LC/MSD (Mass spectrometric detector)
with an Agilent ZorbaxW SB C18 column (3 mm, 2.1 Â 50 mm),
gradient 10–100% CH3CN-H2O, 0.05% TFA or 0.05% NH4OAc over
3.5 min, hold at 100% CH3CN for 2.5 min, flow rate at 0.5 mL/min,
detection at 214 and 254 nm, and mass scan range 120–1500 amu.
Flash chromatography was performed using a Teledyne Isco
CombiFlash Companion system and a RediSepW silica gel (Teledyne
Isco, Lincoln, Nebraska, USA) column unless otherwise specified.
Reverse-phase preparative HPLC purifications were performed
using a Gilson system equipped with a Phenomenex Gemini C18
column (5 mm, 21.2 Â 250 mm, 110 Å) eluted at 15 mL/min with
UV detection at 254 nm with a 20-min gradient from 10–90% CH3CN
in H2O in the presence of 0.05% TFA.
RWJ-416457 (3) is outlined in Scheme 1. Preparation of the
amine precursor (5) was first attempted by selective hydrolysis
of the acetamide group of RWJ-416457 (1) but failed. Instead,
the amine precursor (5) was prepared according to general pro-
cedures described in PCT publication WO01/42242.4 Then,
[13CD3]RWJ-416457 was conveniently prepared by acetylation
of the amine precursor, 5-aminomethyl-3-[3-fluoro-4-(2-methyl-
2,6-dihydro-4H-pyrrolo[3,4-c]pyrazol-5-yl)-phenyl]-oxazolidin-2-
one (3) with CD133COCl in pyridine in 92% isolated yield.
The synthesis of the stable isotope labeled metabolite (4) is
shown in Scheme 2. N,N-dimethyl(formyl-13C,D)amide dimethyl
acetal (8) was first prepared in 65% yield by treating commercially
available N,N-dimethyl(formyl-13C,D)amide (6) first with dimethyl
sulfate, followed by with sodium methoxide in deuterated metha-
nol in reference to a modified literature procedure.10 When unlabeled
methanol was used as the reaction solvent in the treatment
with sodium methoxide, the deuterium of the desired product
(8, Me2N13CD(OMe)2) could be substantially exchanged with
proton to give Me2N13CH(OMe)2 as the major product based
on proton nuclear magnetic resonance (NMR) and 13C-NMR
analysis. Thus, deuterated methanol was employed as the
solvent to avoid the Hydrogen/Deuterium (H/D) exchange.
Next, N-{3-[3-fluoro-4-(3-oxo-pyrrolidin-1-yl)-phenyl]-2-oxo-
oxazolidin-5-ylmethyl}-acetamide (9) was prepared according
to known literature procedures.11 Its condensation with N,N-
dimethyl(formyl-13C,D)amide dimethyl acetal (8) produced
5-Aminomethyl-3-[3-fluoro-4-(2-methyl-2,6-dihydro-4H-
pyrrolo[3,4-c]pyrazol-5-yl)-phenyl]-oxazolidin-2-one (5)
The amine precursor (5) was prepared according to general
procedures described in PCT publication WO01/42242.4 MS
m/z 332 (M + H+). 1H NMR ((CD3)2SO) d 7.53 (1H, s), 7.46 (1H,
dd, J = 15, 3 Hz), 7.17 (1H, dd, J = 9, 3 Hz), 6.86 (1H, t, J = 9 Hz),
4.57 (1H, m), 4.46 (4H, s), 4.01 (1H, t, J = 9 Hz), 3.84–3.79 (4H, m),
2.81 (2H, m), 1.59 (2H, s).
[
13CD]-labeled b-ketoenamine intermediate (10) in 54% yield.
The resultant b-ketoenamine intermediate (10) underwent pyra-
zole ring formation with 15N2-labeled hydrazine (15NH215NH2) to
give the [13C15N2D]-labeled metabolite (4) in 55% yield. The
O
O
13C
O
O
H
N
N
O
N
Me
D3C
Cl
N
N
N
Me
N
CD3
N
13C
NH2
N
F
Pyridine
O
F
3
5
Scheme 1. Synthesis of stable isotope [13CD3]-labeled RWJ-416457, N-{3-[3-fluoro-4-(2-methyl-2,6-dihydro-4H-pyrrolo[3,4-c]pyrazol-5-yl)-phenyl]-2-oxo-oxazolidin-5-
ylmethyl}-acetamide (3).
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Copyright © 2012 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2012

R. Lin and L. E. Weaner
D
Me2N13CD(OMe)2
(MeO)2SO2
Me2N13CDO
MeONa
CD3OD
Me2N+ 13C
-
MeOSO3
OMe
8
6
7
NMe2
13C
O
N
O
D
O
D
NH NH . H SO
4
13C
15
15
O
2
2
2
8
O
O
15N
N
N
N
H
N
N
NHAc
NHAc
t-BuOK, DMF-d6
DMF
NHAc
15N
O
O
F
F
F
4
9
10
Scheme 2. Synthesis of the stable isotope [13CD15N2]-labeled metabolite, N-{3-[4-(2,6-dihydro-4H-pyrrolo[3,4-c] pyrazol-5-yl)-3-fluoro-phenyl]-2-oxo-oxazolidin-5-ylmethyl}-
acetamide (4).
[
13CD3]RWJ-416457 (3)
evaporated and the oily residue (7, quantitative yield) was used
as it was in the next step.
(2) N,N-Dimethyl(formyl-13C,D)amide dimethyl acetal (8)
5-Aminomethyl-3-[3-fluoro-4-(2-methyl-2,6-dihydro-4H-pyrrolo
[3,4-c]pyrazol-5-yl)-phenyl]-oxazolidin-2-one (5, 820 mg) was
dissolved completely in anhydrous pyridine (15 mL) at 60ꢀC
with sonicating. The resultant solution was then cooled at
room temperature and maintained as a clear solution. To this
solution, a solution of CD133COCl (218 uL) in anhydrous dichlor-
omethane (2 mL) was added. The reaction mixture was stirred
at room temperature for 2 h. Crude product as a pale brown
solid formed was collected by using vacuum filtration and
rinsed with water to give the first batch of crude product.
The filtrate was diluted with the addition of 75 mL of water to
give more of crude product. The combined crude product
was dissolved in a minimal amount of DMSO (~2 mL). The resul-
tant DMSO solution was added dropwise to distilled water (15 mL)
under vigorous stirring. The formed precipitate was collected by
using suction filtration, rinsed with water, and dried in vacuo in
the absence of light to give the desired product [13CD3]RWJ-
416457 (3) as light brown solid (857 mg, 92%). MS m/z 378
(M+ H+). 1H NMR ((CD3)2SO) d 8.2t (1H, m), 7.53 (1H, s), 7.45
(1H, dd, J = 15, 3 Hz), 7.15 (1H, dd, J = 9, 3 Hz), 6.86 (1H, t,
J = 9 Hz), 4.70 (1H, m), 4.48 (4H, s), 4.08 (1H, t, J = 9 Hz), 3.85
(3H, s), 3.70 (1H, dd, J= 9, 6 Hz), 3.40 (2H, m). The stable isotope-
labeled compound was confirmed additionally by using HPLC
analysis with co-injection of unlabeled authentic RWJ-416457.
Isotope analysis indicated that the labeled compound was
free of unlabeled compound. The stable isotope-labeled com-
pound was found to have a purity of 98.7 area percent by HPLC
analysis using an Agilent 1200 series HPLC system with a UV
detector set at 240nm and a Waters XTerra RP18 column
(Waters Corporation, MA, USA) (4.6 Â 150 mm, 3.5 mM) and a gra-
dient of acetonitrile in water in the presence of 0.05% TFA by
volume at flow rate 1 mL/min and column temperature at 25ꢀC.
To a dried round-bottom flask under nitrogen, sodium
methoxide (3.6 g, 66.67 mmol, 1 eq) and 21 mL of anhydrous
deuterated methanol (CD3OD) was added. This solution was
cooled down to À10ꢀC and at À10ꢀC under nitrogen, was
added dropwise the aforementioned complex (7) in a period
of 1.5 h. When the addition was complete, stirring was contin-
ued for another 2 h. The mixture was then distilled slowly at
atmospheric pressure in an oil bath gradually heated from
70 to 90ꢀC and various fractions were collected into a
round-bottom flask containing magnesium sulfate. Once the
residue solid was dry, the liquid-containing product was then
slowly distilled using a vigreux long distillation column in
order to eliminate methanol solvent. Fractions containing
the products (b.p. 105–106ꢀC/760 mmHg) were collected
(5.62 g, 65%). 1H NMR (CDCl3) d 3.34 and 3.32 (each 3H, s),
2.29 and 2.28 (each 3H, s).
N-{3-[3-Fluoro-4-(3-oxo-pyrrolidin-1-yl)-phenyl]-2-oxo-
oxazolidin-5-ylmethyl}-acetamide (9)
This intermediate was prepared according to the procedures
described on the literature.11 MS m/z 336 (M + H+).
[
13CD]N-{3-[4-(3-Dimethylaminomethylene-4-oxo-pyrrolidin-
1-yl)-3-fluoro-phenyl]-2-oxo-oxazolidin-5-ylmethyl}-
acetamide (10)
To a round-bottom flask was charged with the aforementioned
intermediate ketone11 (9, 761 mg, 2.28 mmol) and anhydrous
DMF (2.3 mL). The mixture was heated in an oil bath at 60ꢀC with
agitation to achieve a clear dissolution. To this solution, the
aforementioned prepared N,N-dimethyl(formyl-13 C,D)amide
dimethyl acetal (8, 1.9 mL, 6.4 eq) under nitrogen atmosphere
was added. The mixture was heated in an oil bath at 60ꢀC and
agitated for 1.5 h. The reaction was cooled at room temperature,
and then heptane (5 mL) was added with fast agitation for 5 min.
N,N-Dimethyl(formyl-13C,D)amide dimethyl acetal (8)
Synthesis of the labeled DMF dimethyl acetal was referred to a
modified literature procedure.10
(1) Dimethyl N,N-dimethyl(formyl-13C,D)amide-sulfate (7)
In a round-bottom flask (500 mL) under nitrogen, anhydrous The brown solid was filtered, washed with heptane (5 mL Â 2),
N,N-dimethyl(formyl-13C,D)amide (6, 5.0 g, 66.67 mmol) and and dried by air suction. The resulting solid was dried at 40ꢀC
dimethyl sulfate (8.41 g, 6.31 ml, 1 eq) were added. The mixture under vacuum overnight. The intermediate was obtained as a
was then heated at 80ꢀC for 3 h. Then, after cooling at room brown powder (482 mg, ~54%), which was used in the next reac-
temperature, it was added to the reaction mixture of an equal tion without further purification. MS analysis confirmed the iden-
volume of anhydrous diethyl ether (~12 mL). The solvent was tity of the compound (MS m/z 393 (M + H+)).
J. Label Compd. Radiopharm 2012
Copyright © 2012 John Wiley & Sons, Ltd.
www.jlcr.org

R. Lin and L. E. Weaner
[
13C15N2D]N-{3-[4-(2,6-Dihydro-4H-pyrrolo[3,4-c]pyrazol-5-yl)- of the stable isotope-labeled metabolite (4) involved the
3-fluoro-phenyl]-2-oxo-oxazolidin-5-ylmethyl}-acetamide (4)
condensation of N-{3-[3-fluoro-4-(3-oxo-pyrrolidin-1-yl)-phenyl]-
2-oxo-oxazolidin-5-ylmethyl}-acetamide (9) with N,N-dimethyl
(formyl-13C,D)amide dimethyl acetal (8), which was prepared by treat-
ing N,N-dimethyl(formyl-13C,D)amide (6) with dimethyl sulfate, fol-
lowed by sodium methoxide to give b-ketoenamine intermediate
(10) and subsequent pyrazole ring formation with 15N-labeled
hydrazine 15NH215NH2. The synthesis of the pyrazole-containing
metabolite, involved the construction of multiple stable
isotope-labeled pyrazole ring from ketone compound, provides a
general and convenient synthetic method for multiple stable
isotope labeling synthesis of biologically and pharmaceutically
important pyrazole compounds.
The aforementioned labeled enamine intermediate (10) (435 mg,
1.11mmol) was dissolved in DMF-d6 (2.2 mL) in a round-bottom
flask heated in an oil bath at 90ꢀC and with agitation. Hydrazine
sulfate-15N2 (15NH215NH2.H2SO4) (179.2 mg, 1.35 mmol) in another
round-bottom flask was dissolved in 1.1 mL of D2O in a warm
bath with sonication. To this solution, 1.34 mL of 1M potassium
t-butoxide in THF solution was added. The resultant solution
was then added to the aforementioned enamine solution. This
reaction mixture was then heated in an oil bath at 90ꢀC and
stirred under nitrogen for 6 h until TLC analysis indicated the
completion of the reaction. The reaction mixture was cooled at
room temperature and added dropwise into distilled water
(20 mL) with vigorous stirring. The suspension mixture was
stirred at room temperature for 20 min and was allowed to stand
for 20 min and the solid was isolated by using filtration. This solid
was dried by using suction filtration and then placed in a vacuum
oven at room temperature overnight to give the crude product as
a light brown solid.
Acknowledgements
We are grateful to Dr. Xun Li for providing intermediate
N-{3-[3-fluoro-4-(3-oxo-pyrrolidin-1-yl)-phenyl]-2-oxo-oxazolidin-5-
ylmethyl}-acetamide (9) and to Dr. Hartmut Zinser for providing
intermediate 5-aminomethyl-3-[3-fluoro-4-(2-methyl-2,6-dihydro-
4H-pyrrolo[3,4-c]pyrazol-5-yl)-phenyl]-oxazolidin-2-one (5). We
also like to thank Drs. Mark Macielag, Gilles Bignan, and Daksha
Desai-Krieger for the helpful discussions.
The crude product was dissolved in a minimal amount of
DMSO (~1.5 mL), and filtrated to give a clear solution. The resultant
DMSO solution was added dropwise to distilled water (15 mL)
under vigorous stirring. The formed precipitate was collected by
using suction filtration, rinsed with water, and dried in vacuo in
the absence of light. The precipitation procedure was repeated
second time to give the desired product as a light brown solid
Conflict of Interest
The authors did not report any conflict of interest.
1
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(222 mg, 55%). MS m/z 364 (M + H+). H NMR ((CD3)2SO) \ 12.66
(1H, d, br, J= 108 Hz, H15N), 8.25 (1H, t), 7.48 (1H, dd, J=15, 3 Hz),
7.15 (1H, dd, J= 9, 3 Hz), 6.88 (1H, t, J= 9 Hz), 4.70 (1H, m), 4.48
(4H, s), 4.10 (1H, t), 3.70 (1H, t), 3.40 (2H, t), 1.85 (3H, s). The stable
isotope-labeled compound was confirmed additionally by HPLC
analysis with co-injection of unlabeled authentic compound.
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was free of unlabeled compound. HPLC analysis showed the
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antibacterial agent RWJ-416457, (3, N-{3-[3-fluoro-4-(2-methyl-2,
6-dihydro-4H-pyrrolo[3,4-c]pyrazol-5-yl)-phenyl]-2-oxo-oxazolidin-
5-ylmethyl}-acetamide), and its major metabolite, N-{3-[4-(2,
6-dihydro-4H-pyrrolo[3,4-c]pyrazol-5-yl)-3-fluoro-phenyl]-2-
oxo-oxazolidin-5-ylmethyl}-acetamide (4), is described. [13CD3]-
labeled RWJ-416457 was conveniently prepared by acetylation of
the precursor amine, 5-aminomethyl-3-[3-fluoro-4-(2-methyl-
2,6 - dihydro-4H-pyrrolo[3,4-c]pyrazol-5-yl)-phenyl]-oxazolidin-
2-one (5) with CD133COCl in pyridine. Key steps for the synthesis
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