Synthesis of [2H, 13C] and [14C] labeled vasopressin V1a/V2 receptor antagonist RWJ-676070 and its stable labeled N-des-benzoyl metabolite

Article abstract of DOI:10.1002/jlcr.1515

Stable isotope-labeled ([¹³C₂, D₃]) and carbon 14-labeled vasopressin receptor antagonist RWJ-676070, a spirobenzazepine amide, were prepared in separate syntheses for use in drug metabolism studies. The stable isotopically labeled sample was prepared starting from [ ¹³C₂, D₆]dimethyl sulfate and [ ¹³C]copper (I) cyanide in nine steps with a 14% overall isotopic yield. The ¹⁴C label was introduced in five steps starting from [¹⁴C]potassium cyanide to provide material having a specific activity of 58 mCi/mmol (2.15 GBq/mmol). Selective hydrolysis of the metabolically labile amide bond in [¹³C₂, D₃]RWJ-676070 gave the corresponding labeled metabolite in one step. Copyright

Full text of DOI:10.1002/jlcr.1515

Research Article
Received 12 December 2007,
Revised 29 February 2008,
Accepted 12 March 2008
Published online in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1515
13
Synthesis of [2H, C] and [14C] labeled
vasopressin V1a/V2 receptor antagonist RWJ-
676070 and its stable labeled N-des-benzoyl
metabolite
Ã
Yong Gong,a David C. Hoerr,b Larry E. Weaner,b and Ronghui Lina
Stable isotope-labeled ([13C2, D3]) and carbon 14-labeled vasopressin receptor antagonist RWJ-676070, a spirobenzazepine
amide, were prepared in separate syntheses for use in drug metabolism studies. The stable isotopically labeled sample was
prepared starting from [13C2, D6]dimethyl sulfate and [13C]copper (I) cyanide in nine steps with a 14% overall isotopic yield.
The 14C label was introduced in five steps starting from [14C]potassium cyanide to provide material having a specific activity
of 58 mCi/mmol (2.15 GBq/mmol). Selective hydrolysis of the metabolically labile amide bond in [13C2, D3]RWJ-676070 gave
the corresponding labeled metabolite in one step.
Keywords: isotope label; vasopressin receptor antagonist; spirobenzazepine; metabolite
fluoro-2-iodobenzene (4) with [13C]copper (I) cyanide intro-
duced the fifth label to provide [13C]nitrile 5, which was
Introduction
Vasopressin V1a/V2 receptor antagonists are promising in
treating certain cardiovascular-related syndromes.1 A series of
nonpeptide-substituted spirobenzazepines was discovered to
be potent and orally active vasopressin antagonists.2–5 One of
the representative compounds, RWJ-676070, was selected for
clinical studies.6 To support drug development efforts, both
stable-labeled and radio-labeled RWJ-676070 were required. The
stable-labeled compound was designed and synthesized as an
internal standard for LC-MS-MS quantitative analysis.7 Following
completion of the synthesis, a request for next-day delivery of a
labeled major metabolite of RWJ-676070 was received to
support an on-going study. The metabolite, formed by in vivo
cleavage of an amide bond with subsequent loss of the chloro-
fluorobenzoyl moiety, was facilely prepared to meet the request
by direct hydrolysis of [13C2, D3]RWJ-676070 to provide stable-
labeled metabolite 12. The carbon-14-labeled sample was
prepared using chemistry similar to that used for preparation
of the stable-labeled compound, but with the label in a known,
metabolically stable position.
hydrolyzed to the corresponding acid 6 followed by conversion
to acid chloride 7. Coupling of 7 with aniline 3 generated amido
ester 8 with the required five isotopic labels [13C2, D3] in 22%
isotopic yield for six steps. Amido acid 9 from hydrolysis of ester
8 was converted to acid chloride 10 and coupled with
spirobenzazepine 11. The resulting [13C2, D3]RWJ-676070 was
obtained in 14% overall isotopic yield after high-performance
liquid chromatography (HPLC) purification.
For the synthesis of [13C, D3]N-des-benzoyl metabolite 12, a
multi-step synthetic route similar to, but shorter than, the above
procedure to [13C2, D3]RWJ-676070 was obviously the normal
choice. However, the urgent request for next-day delivery of a
small amount of material prompted us to look for an alternative
synthesis. The desired-labeled metabolite was prepared in one
step by selective hydrolysis of the metabolically labile amide
bond in [13C2, D3]RWJ-676070. The reaction was completed at an
elevated temperature (up to 1501C) with a reduced reaction
time (10 min) using 1 N sodium hydroxide. The [13C2, D3]RWJ-
676070 was cleanly hydrolyzed to the target [13C, D3]aniline
metabolite 12, along with [13C]2-chloro-5-fluorobenzoic acid (6)
as the co-product (Scheme 2). The resulting [13C, D3]metabolite
was purified by HPLC.
Results and discussion
The synthesis of stable-labeled [13C2, D3]RWJ-676070 is outlined
in Scheme 1. The five stable labels were incorporated into two
different functional groups, –O13CD3 and –13CN, in intermedi-
ates 2 and 5, respectively. The methoxy-13CD3 group was
introduced through O-methylation of methyl 3-hydroxy-4-
nitrobenzoate (1) with [13C2, D6]dimethyl sulfate and the
resulting [13C, D3]-labeled nitro compound 2 was hydrogenated
to provide aniline intermediate 3. Both steps proceeded in
nearly quantitative chemical yields. Cyanation of 1-chloro-4-
aGlobal Preclinical Development, Johnson & Johnson Pharmaceutical Research
& Development, Raritan, NJ 08869, USA
bGlobal Preclinical Development, Johnson & Johnson Pharmaceutical Research
& Development, Spring House, PA 19477, USA
*Correspondence to: Yong Gong, Global Preclinical Development, Johnson &
Johnson Pharmaceutical Research & Development, Raritan, NJ 08869, USA.
E-mail: ygong@prdus.jnj.com
J. Label Compd. Radiopharm 2008, 51 268–272
Copyright r 2008 John Wiley & Sons, Ltd.

Y. Gong et al.
CO2CH3
CO2CH3
CO2CH3
(
13CD3)2SO4
H2, Pd/C
EtOAc
O13CD3
O13CD3
K2CO3/acetone
OH
NO2
NO2
NH2
1
2
3
Cl
Cl
Cl
F
Cl
O
13C
1) KOH
EtOH/H2O
13CN
I
13CO2H
Cu13CN
DMF
SOCl2
DCM
Cl
2) IN HCl
NEt3
CH2Cl2
F
F
F
6
4
5
7
OH
OCH3
1) LiOH
H2O/THF
SOCl2
toluene
Cl
O
O
O
Cl
F
O
13C
13C
N
N
2) IN HCl
13CD3O
13CD3O
H
H
F
8
9
CO2H
CO2H
Cl
N
N
O
O
Cl
F
11
H
13C
O
N
TMSCl, Et3N, toluene
13CD3O
O
13C
H
Cl
N
10
13CD3O
H
[
13C2, D3]RWJ-676070
F
Scheme 1. Synthesis of [13C2, D3]RWJ-676070
CO2H
Cl
13CO2H
NaOH, H2O
N
[
13C2, D3]RWJ-676070
+
O
F
6
NH2
13CD3O
12
Scheme 2. Synthesis of [13C, D3]N-des-benzoyl metabolite (12)
The synthesis of [14C]RWJ-676070 is outlined in Scheme 3.
Cyanation of 4-bromo-2-methoxyphenylamine (13) with
K14CN (400 mCi) and CuI yielded nitrile 14 in 65% yield.
Hydrolysis of 14 under acidic conditions provided aminoben-
Experimental
General
zoic acid 15, which was purified by HPLC, treated with [13C2, D6]Dimethyl sulfate (99.81 at% D, 99.57 at% 13C) and
chlorotrimethylsilane and acylated with 2-chloro-5-fluoroben- [13C]copper (I) cyanide (99 at% 13C) were purchased from Isotec
zoyl chloride 16 to provide amido acid 17. The latter was (Division of Sigma-Aldrich, St. Louis, MO). [14C]Potassium
converted to the acid chloride by treatment with thionyl cyanide was obtained from PerkinElmer (Boston, MA, 54.8 mCi/
chloride and coupled with spirobenzazepine 11. The resulting mmol). Methyl 3-hydroxy-4-nitrobenzate (1) was obtained from
[
14C]RWJ-676070 (61.4 mCi, 15.3% radiochemical yield) was Lancaster Synthesis (Windham, NH) and 1-chloro-4-fluoro-2-
obtained with a specific activity of 58 mCi/mmol after HPLC iodobenzene (4) from Oakwood Product Inc (West Columbia,
purification.
SC). 4-Bromo-2-methoxy-phenylamine (13) was purchased from
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Y. Gong et al.
14CN
14CO2H
Br
K14CN, CuI
NMP
48% HBr
AcOH
CH3O
CH3O
14
CH3O
NH2
NH2
NH2
15
13
CO2H
14CO2H
1) SOCl2, toluene
2) TMSCl, pyridine
N
C14
TMSCl, pyridine
toluene
CH3O
O
HN
O
COCl
Cl
11
O
Cl
Cl
N
H
CH3O
F
F
14C]RWJ-676070
16
17
F
[
Scheme 3. Synthesis of [14C]RWJ-676070
AstaTech (Bristol, PA). Other reagents and solvents were column temperature was 551C, and UV detection was at 275 nm.
obtained from Sigma-Aldrich, VWR International and other The sample was injected in 10 mL of a solvent mixture of
suppliers, and used as received.
CH3CN–H2O (70:30, v/v). Radioactivity in the HPLC effluent
1H and 13C NMR spectra were acquired on a Bruker 300- stream was measured by mixing the HPLC flow stream with
Avance (300 MHz) spectrometer with TMS as an internal EcoScint scintillation cocktail at 2 mL/min in a radioactive flow
standard. Chemical shifts are expressed in parts per million detector equipped with a 0.5 mL flow cell. The sample size for
(ppm, d scale). LC-MS was performed on an Agilent 1100 series the radioactive measurements was 1 mL.
LC/MSD with an Agilent Zorbax SB C18 column (3 mm,
2.1 Â 50 mm), gradient 10–100% CH3CN–H2O, 0.05% TFA or [Methoxy-13C, D3]methyl 3-methoxy-4-nitrobenzoate (2)
0.05% NH4OAc over 3.5 min, held at 100% CH3CN for 2.5 min,
Methyl 3-hydroxy-4-nitrobenzoate (1, 0.98 g, 5.0 mmol) was
stirred with [13C2, D6]dimethyl sulfate (1.0 g, 7.5 mmol) in
flow rate 0.5 mL/min, detection at 214 and 254 nm, mass scan
range 120–1500 amu. Flash chromatography was performed
acetone (20 mL) in the presence of K2CO3 (1.38 g, 10 mmol) at
using a Teledyne Isco CombiFlash Companion system and a
ambient temperature for 17 h. The mixture was concentrated to
RediSeps silica gel column. Reverse-phase preparative HPLC
dryness. The crude solid was partitioned between H2O (12 mL)
purifications were performed using a Gilson system equipped
and EtOAc (12 mL). The organic layer was separated and the
aqueous layer was extracted with EtOAc (4 mL). The combined
with a Phenomenex Gemini C18 column (5 mm, 21.2 Â 250 mm,
˚
110 A) eluted at 20 mL/min with UV detection at 214 and
EtOAc extracts were washed with H2O (4 mL), dried over
anhydrous Na2SO4 and concentrated under reduced pressure
at 371C to give 2 as a yellow solid (1.06 g, 99% chemical yield
and 33% isotopic yield). 1H NMR (CDCl3) d 7.85 (d, 1H, J = 8.3 Hz),
7.76 (d, 1H, J = 1.5 Hz), 7.70 (dd, 1H, J = 8.3, 1.5 Hz), 3.97 (s, 3H).
MS m/z 216.0 (MH)1.
254 nm, with a 20-min gradient from 10 to 90% CH3CN in H2O,
0.05% TFA. Analytical HPLC was performed with an Agilent
Zorbax SB C8 column (5 mm, 4.6 Â 250 mm) eluted with solvent
B: CH3CN–TFA (0.1%) and solvent A: H2O–TFA (0.1%) starting
from 10% B to 50% B at 20 min then increased to 90% B at
30 min. The flow rate was 1 mL/min, column temperature was
351C and UV detection was at 254 nm. The sample was injected
in a mixture of CH3CN–H2O–TFA (1:1:0.1%). HPLC with radio-
active monitoring of the effluent was performed with an Agilent
Zorbax Eclipse XDB C18 column (1.8 mm, 4.6 Â 100 mm), eluted
with a gradient from 10% solvent D (10 mM NH4OAc in CH3CN,
15:85, v/v) and 90% solvent C (10 mM NH4OAc in CH3CN, 95:5, v/
v) to 100% solvent D at 8 min then held at this composition for
4 min before returning to 90% solvent C and 10% solvent D for
1 min. Run time was 13 min, the flow rate was 1 mL/min, column
temperature was 551C and UV detection was at 275 nm. Sample
was injected in 5 mL of a CH3CN–H2O (2:1, v/v) solvent mixture
for analysis with the UV detector. The final 14C-labeled sample
was analyzed with a Waters Xterra RP18 column (3.5 mm,
4.6 Â 150 mm) using a 35-min solvent gradient from 10% B to
100% B, where solvent A contained 5% CH3CN in 10 mM
aqueous NH4OAc buffer and solvent B contained 85% CH3CN in
10 mM aqueous NH4OAc buffer. The flow rate was 1 mL/min,
[Methoxy-13C, D3]methyl 4-amino-3-methoxybenzoate (3)
Nitrobenzoate 2 (1.06 g, 4.9 mmol) was hydrogenated in EtOAc
(10 mL) in the presence of 10% Pd/C (56 mg) at ambient
temperature for 16 h. The Pd/C catalyst was then filtered and
washed with EtOAc (3 Â 2 mL). Removal of the solvent under
reduced pressure at 371C gave 3 as an off-white crystalline solid
(0.90 g, 99%). 1H NMR (CDCl3) d 7.55 (dd, 1H, J = 8.1, 1.8 Hz), 7.45
(d, 1H, J = 1.7 Hz), 6.67 (d, 1H, J = 8.2 Hz), 4.23 (br, 2H), 3.87 (s, 3H).
MS m/z 186.1 (MH)1.
[Nitrile-13C]2-chloro-5-fluorobenzonitrile (5)
A
mixture of 1-chloro-4-fluoro-2-iodobenzene (4, 2.30 g,
9.0 mmol) and Cu13CN (0.72 g, 8.0 mmol) in dimethyl formamide
(DMF) (6 mL) was heated at 1201C oil bath for 24 h, then at
1401C for 4 h. The reaction mixture was cooled to ambient
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Y. Gong et al.
temperature and filtered. The filter cake was washed with CH2Cl2 The mixture was heated at 971C in an oil bath for 100 min and
(2 Â 2 mL). The filtrate was diluted with CH2Cl2 (10 mL), washed then concentrated under reduced pressure at 371C to give 10 as
with H2O (6 Â 10 mL), and concentrated under reduced pressure a white solid, which was dissolved in CH2Cl2 (4 mL) and used
at ambient temperature to give 5 as an off-white crystalline solid directly in the next step reaction.
(1.12 g, 89%) after air drying (Caution: sublimes). 1H NMR (CDCl3)
d 7.48–7.57 (m, 1H), 7.37–7.43 (m, 1H), 7.25–7.32 (m, 1H).
[Methoxy-13C, D3, amido-13C]RWJ-676070
To an ice–water bath cooled solution of compound 11 (0.523 g,
1.1 mmol, as a camphorsulfonic acid salt) in toluene (4 mL)
[Carboxyl-13C]2-chloro-5-fluorobenzoic acid (6)
To a suspension of benzonitrile 5 (1.12 g, 7.15 mmol) in EtOH containing NEt3 (0.83 mL, 6.0 mmol) was added chlorotrimethyl-
(6 mL) was added KOH (1.22 g, 21.8 mmol) in H2O (6 mL). The silane (0.46 mL, 3.6 mmol) dropwise and stirred at 0–41C for 1 h.
mixture was heated in a sealed tube at 1401C in an oil bath for To the resulting slurry was added benzoyl chloride 10 in CH2Cl2
100 min and then concentrated under reduced pressure at 371C. prepared above, followed by a CH2Cl2 rinse (2 mL) in a dropwise
The resulting slurry was acidified with 1 N HCl (22 mL) and manner. The cooling bath was removed and the slurry was
filtered. The filter cake was washed with H2O (3 Â 10 mL) and stirred at ambient temperature for an additional 17 h. The
dried in vacuo at ambient temperature to give 6 as an off-white reaction was quenched with 1 N HCl (4 mL) and stirred for
1
solid (1.09 g, 87%). H NMR (DMSO-d6) d 13.71 (s, 1H), 7.58–7.66 20 min. The crude mixture was concentrated under reduced
(m, 2H), 7.41–7.47 (m, 1H). MS m/z 176.1 (MH)1.
pressure at 371C and the remaining crude product was dissolved
in CH3CN (6.5 mL) and purified by preparative HPLC as described
above. The combined product fractions were concentrated and
the product was obtained as a white solid (0.37 g, 61% in two
[Carbonyl-13C]2-chloro-5-fluorobenzoyl chloride (7)
To a suspension of benzoic acid 6 (0.392 g, 2.23 mmol) in CH2Cl2
(8 mL) was added SOCl2 (1 mL, 14 mmol). The mixture was
heated to 601C and refluxed for 4 h. The resulting solution was
concentrated under reduced pressure at 371C to give crude 7,
which was dissolved in CH2Cl2 (3 mL) and used directly in the
next step.
1
steps). H NMR (DMSO-d6) d 12.34 (br, 1H), 9.78 (s, 1H), 7.84 (d,
1H, J = 8.8 Hz), 7.56 (m, 1H), 7.45 (m, 1H), 7.10–7.38 (m, 3H), 7.02
(m, 1H), 6.68–6.81 (m, 3.6 H), 6.24 (s, 0.4 H), 4.70 (m, 1H), 3.38 (m,
1H, partial overlap with H2O in DMSO), 2.50–3.02 (m, 4H, partial
overlap with DMSO), 1.95 (m, 2H), 1.58 (m, 2H). 13C NMR (DMSO-
d6) d 163.7, 54.8. MS m/z 554.1 (MH)1.
[Methoxy-13C, D3, amido-13C]methyl 4-(2-chloro-5-fluoro-
benzoylamino)-3-methoxybenzoate (8)
[Methoxy-13C, D3]metabolite 12
A mixture of [methoxy-13C, D3, amido-13C]RWJ-676070 (10 mg,
18 mmol) and 1.0 N NaOH (0.30 mL) in a sealed tube was
immersed in an oil bath heated at 1301C and the temperature
was then increased to 1501C. The mixture was stirred at 1501C
for 10 min, cooled to ambient temperature and acidified with
1 N HCl (0.30 mL). The resulting white slurry was concentrated to
dryness, treated with MeOH (1.4 mL) and filtered. The filtrate was
purified by preparative HPLC as described above. The product
fraction was concentrated to give [methoxy-13C, D3] metabolite
12 as an off-white solid (7 mg as TFA salt, 76%). 1H NMR (CD3CN)
d 6.98–7.26 (m, 3H), 6.61–6.72 (m, 3.6 H), 6.46 (d, 1H, J = 8.0 Hz),
6.33 (br, 0.4 H), 4.76 (m, 1H), 3.35 (m, 1H), 2.59–3.04 (m, 4H),
1.58–1.96 (m, 4H, partial overlap with CD3CN). MS m/z 397.1
(MH)1.
The solution of benzoyl chloride 7 (r2.23 mmol) in CH2Cl2
prepared in the previous step was added slowly to a solution of
aminobenzoate 3 (0.414 g, 2.23 mmol) in CH2Cl2 (7 mL) contain-
ing NEt3 (0.93 mL, 6.7 mmol) while cooling in an ice bath. The
mixture was stirred at ambient temperature for 2 h and then
quenched with 1 N HCl (10 mL). The organic layer was separated,
washed with saturated NaHCO3 (10 mL) and concentrated under
reduced pressure at 371C. The crude product was purified by
flash chromatography on silica gel, eluted with 0–30% EtOAc in
heptane, to yield 8 as a white solid (0.663 g, 86% in two steps).
1H NMR (CDCl3) d 8.85 (s, 1H), 8.61 (d, 1H, J = 8.4 Hz), 7.75 (d, 1H,
J = 8.4 Hz), 7.52–7.60 (m, 2H), 7.43–7.48 (m, 1H), 7.12–7.19 (m,
1H), 3.93 (s, 3H). MS m/z 343.0 (MH)1.
[Methoxy-13C, D3, amido-13C]4-(2-chloro-5-fluorobenzoyla-
mino)-3-methoxybenzoic acid (9)
[Nitrile-14C]4-cyano-2-methoxyphenylamine (14)
A mixture of 4-bromo-2-methoxyphenylamine (13, 1.475 g,
7.3 mmol), K14CN (490 mg, 7.3 mmol, 400 mCi, 54.8 mCi/mmol)
and CuI (685 mg, 3.6 mmol) in 1-methyl-2-pyrrolidinone (10 mL)
was heated at 2201C for 7 h. The reaction was cooled to ambient
temperature and EtOAc (2 mL) and NaCN (2 g) in H2O (6 mL)
were added. This mixture was stirred for 20 min and extracted
with EtOAc. The organic extracts were combined, washed with
H2O (4 Â 10 mL) and dried over anhydrous MgSO4. Filtration and
removal of the solvent on a rotary evaporator gave an oil (14,
262 mCi, 54.8 mCi/mmol, 65.5% radiochemical yield), which was
used directly in the next reaction step.
To a solution of methyl benzoate 8 (0.40 g, 1.17 mmol) in THF
(4 mL) was added 1.0 N LiOH (3.5 mL, 3.5 mmol). The mixture was
stirred at ambient temperature for 5 h and the solvent was
removed under reduced pressure at 371C. The remaining
mixture was acidified with 1 N HCl (4 mL) and filtered. The filter
cake was washed with H2O (3 Â 2 mL) and dried in vacuo for 18 h
at ambient temperature to give product 9 as a white solid
(0.38 g, 100%). H NMR (DMSO-d6) d 12.96 (s, 1H), 10.03 (s, 1H),
8.22 (d, 1H, J = 8.4 Hz), 7.50–7.62 (m, 4H), 7.36–7.42 (m, 1H). MS
m/z 329.1 (MH)1.
1
[Methoxy-13C, D3, amido-13C]4-(2-chloro-5-fluorobenzoyla-
mino)-3-methoxybenzoyl chloride (10)
[Carboxyl-14C]4-amino-3-methoxybenzoic acid (15)
To the crude nitrile 14 (4.85 mmol) obtained from above was
added glacial AcOH (2.9 mL) and 48% HBr (4.85 mL). This mixture
To a suspension of benzoic acid 9 (0.38 g, 1.1 mmol) in toluene
(4 mL) was added SOCl2 (0.15 mL, 2.0 mmol) and DMF (2 drops).
J. Label Compd. Radiopharm 2008, 51 268–272
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Y. Gong et al.
was stirred and heated at 1301C for 7 h. The reaction was cooled removal of the solvent gave a yellow solid (862 mg) that was
to ice-bath temperature and EtOAc and 50% NaOH were added dissolved in a mixture of CH3CN–H2O (10:2.2, v/v) and purified
to pH 6. The solvent was removed to dryness and the product by preparative HPLC on a Supelcosil LC-ABZ1PLUS column
was extracted with a 95:5 (v/v) mixture of EtOAc–CH3OH. (5 mm, 2.1 Â 25 cm, Supelco). The product was eluted from the
Filtration and removal of the solvent in vacuo gave a dark brown column using a 40-min linear gradient from 20 to 85% CH3CN in
solid. This was dissolved in CH3CN–H2O (3:2, v/v, 18 mL) and H2O with 0.05% TFA present at a flow rate of 10 mL/min. After a
injected in 2 mL portions onto a 2.14 Â 25 cm Dynamax C18 10-min hold at the end of the gradient at 85% CH3CN in
preparative HPLC column (Varian). The compound was eluted H2O–TFA, the column was re-equilibrated to the initial condi-
using a linear gradient solvent mixture from 10 to 22% tions. The product eluted from the column with a 34-min
CH3CN–H2O containing 0.05% TFA over 30 min. The solvent retention time. The center portion of the fractions containing
flow rate was 10 mL/min, column temperature was at ambient product were collected and the solvent was immediately
temperature and UV detection was at a wavelength of 254 nm. removed on a rotary evaporator at ambient temperature. The
The product eluted from the column between 16 and 22 min product having a radiochemical purity of 99.6% was recovered
and was collected and combined. The solvent was removed on a as a white solid (583 mg, 61.4 mCi, 58 mCi/mmol), dissolved in
rotary evaporator at 301C. Product 15 was dried in vacuo to an EtOH–H2O (90:10, v/v) and stored at À201C.
off-white solid (as the TFA salt, 639 mg, 123.7 mCi, 54.8 mCi/
mmol, 47% yield) that was 98% radiochemically pure.
Conclusion
An efficient synthesis of stable-labeled [methoxy-13C, D3,
amido-13C]RWJ-676070 is described. The nine-step procedure
achieved five isotopic labels in a 14% overall isotopic yield. A
novel one-step route to the [13C, D3]-labeled N-des-benzoyl
metabolite by selective hydrolysis of the metabolically labile
amide bond in [methoxy-13C, D3, amido-13C]RWJ-676070 was
revealed. This successful approach enabled us to meet a next-
day delivery challenge. Finally, a carbon-14 label was introduced
in the metabolically stable carboxyl group of RWJ-676070 via a
five-step sequence that provided material having a specific
activity of 58 mCi/mmol.
[Carboxyl-14C]4-(2-chloro-5-fluorobenzoylamino)-3-methox-
ybenzoic acid (17)
Under a nitrogen gas atmosphere was placed aminobenzoic
acid TFA salt 15 (639 mg, 2.25 mmol), toluene (5.6 mL) and dry
pyridine (0.80 mL 9.89 mmol). This mixture was cooled in an ice
bath and chlorotrimethylsilane (0.70 mL, 5.52 mmol) was slowly
added over 10 min. The resulting mixture was stirred for 30 min
at 01C and a solution of 2-chloro-5-fluorobenzoyl chloride (16,
463 mg, 2.4 mmol) in toluene (1.65 mL) was slowly added and
the reaction was stirred at 01C for 3 h. The reaction was
quenched by the addition of a mixture of concentrated HCl
(0.4 mL), H2O (1.5 mL), and EtOH (1.9 mL). After stirring for
15 min, the mixture was heated to 801C for 30 min then cooled
to room temperature. The resulting suspension was filtered and
the filter cake was washed with H2O and toluene. Product 17
was obtained as a white solid (593 mg, 1.82 mmol, 99.9 mCi)
after drying in vacuo.
Acknowledgement
We are grateful to the Endocrine and Metabolic teams from
Research and Early Development, and the Chemical Develop-
ment group of Johnson & Johnson Pharmaceutical Research and
Development for their earlier synthetic efforts and procedures;
to Cilag AG for the supply of chemical intermediates used in this
effort.
[Amido-14C]RWJ-676070
To a solution of benzoic acid 17 (593 mg, 1.82 mmol) in toluene
(3.9 mL) under a nitrogen gas atmosphere was added SOCl2
(0.4 mL) and a catalytic amount of DMF (5 mL). This mixture was
heated at 951C for 1 h then at 1001C for an additional hour.
Toluene and excess SOCl2 were removed by vacuum distillation
and the acid chloride residue was dissolved in CH2Cl2 (6 mL).
Into a second reaction vessel was placed compound 11 (0.856 g,
1.80 mmol, as a camphorsulfonic acid salt), pyridine (0.832 mL,
10.9 mmol) and toluene (2.8 mL) under a nitrogen gas atmo-
sphere. Chlorotrimethylsilane (0.716 mL, 5.65 mmol) was added
at 01C and the mixture was stirred for 1 h. At this temperature
was added the above solution of the acid chloride to the
reaction mixture and this was stirred for 5 h at ambient
temperature. The reaction was quenched by the addition of
18% HCl (1.3 mL) and the solvent was removed on a rotary
evaporator. The remaining solid was dissolved in EtOAc (30 mL)
and this solution was washed with H2O. The aqueous phase was
back extracted with EtOAc (4 Â 10 mL) and the organic extracts
were washed with H2O and dried over MgSO4. Filtration and
References
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[7] For application of stable isotopically labeled internal standard for
´
´
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www.jlcr.org
Copyright r 2008 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2008, 51 268–272