Synthesis of [14C]boceprevir, [13C 3]boceprevir, and [D9]boceprevir, a hepatitis C virus protease inhibitor

Article abstract of DOI:10.1002/jlcr.1960

Boceprevir is a hepatitis C virus (HCV) NS3 protease inhibitor for HCV treatment. [¹⁴C]Boceprevir (SCH 503034, trade name Victrelis) was synthesized from K¹⁴CN in 11 steps with an overall yield of 16.4%. [¹³C₃]Boceprevir was synthesized in 16 steps with a 2.5% overall yield. The carbon-13 in the molecule was distributed along the peptide chain. [D₉]Boceprevir was synthesized from [D₉]-t- butylamine in four steps with an overall yield of 69%. Copyright 2012 John Wiley & Sons, Ltd. Boceprevir is an HCV NS3 protease inhibitor for HCV treatment. [14C]Boceprevir (SCH 503034, trade name Victrelis) was synthesized from K14CN in 11 steps with an overall yield of 16.4%. [13C3] Boceprevir was synthesized in 16 steps with a 2.5% overall yield. The carbon-13 in the molecule was distributed along the peptide chain. [D9]Boceprevir was synthesized from [D9]-t-butylamine in 4 steps with an overall yield of 69%.

Full text of DOI:10.1002/jlcr.1960

Research Article
Received 17 November 2011,
Revised 05 December 2011,
Accepted 12 December 2011
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.1960
Synthesis of [14C]boceprevir, [13C3]boceprevir,
and [D9]boceprevir, a hepatitis C virus
protease inhibitor
*
Sumei Ren, Pernilla Royster, Carolee Lavey, David Hesk, Paul McNamara,
David Koharski, Van Truong, and Scott Borges
Boceprevir is a hepatitis C virus (HCV) NS3 protease inhibitor for HCV treatment. [14C]Boceprevir (SCH 503034,
trade name Victrelis) was synthesized from K14CN in 11 steps with an overall yield of 16.4%. [13C3]Boceprevir was
synthesized in 16 steps with a 2.5% overall yield. The carbon-13 in the molecule was distributed along the peptide
chain. [D9]Boceprevir was synthesized from [D9]-t-butylamine in four steps with an overall yield of 69%.
Keywords: boceprevir; Victrelis; SCH 503034; HCV protease inhibitor; carbon-13; carbon-14; synthesis
transformation, and (R,S) amino nitrile precipitated out in
Introduction
79% yield. Hydrolysis of 2 with H2SO4 gave diamide 3 in
Chronic hepatitis C virus (HCV) infection is a major global
82% yield. Catalytic hydrogenolysis to remove the chiral
auxiliary afforded (S)-t-[14C]leucine amide 4 in 94% yield.
public health problem, affecting more than 170 million
people worldwide. Boceprevir, a novel, potent and orally
bioavailable NS3 protease inhibitor, has been approved recently
Hydrolysis of amide 4 with HCl gave (S)-t-[14C]leucine 5 in
quantitative yield. Reaction of 5 with (Boc)2O in the presence
of Et3N gave Boc-(S)-t-[14C]leucine 6 in 68% yield.
by the FDA to be used in combination with Peginterferon
and ribavirin to treat hepatitis C genotype 1 infection.1–5
The rest of the synthesis of [14C]boceprevir is as shown in
Scheme 2.
[
14C]Boceprevir was synthesized to support ADME studies
(S)-t-[14C]Leucine was coupled with amine
7 to give
and environmental assessment study. [D9]Boceprevir was
initially requested as an internal standard in the development
of bioanalytical LC/MS/MS methods. [13C3]Boceprevir was
requested for the development of LC-MS methods for profil-
ing and structural characterization of metabolites in various
biological matrices. After oral administration of a mixture
containing unlabeled boceprevir, [14C]boceprevir, and [13C3]
boceprevir to male rats, the drug-derived material obtained
from various matrices was collected and analyzed. The
isotopic pattern of [13C3]SCH 503034 facilitated the detection
of drug-derived material in the presence of endogenous ions.
compound 8 in 74% yield. The Boc group was removed with
HCl to generate compound 9 in quantitative yield. Treatment
of amine 9 with tert-butyl isocyanate gave ester 10 in 84%
yield. Hydrolysis of ester 10 gave acid 11 in quantitative
yield. Coupling of acid 11 with amine 12 using EDCI/HOBt
chemistry gave compound 13 as an approximately equal
mixture of four diastereomers in 98% yield. Finally, oxidation
of hydroxyamide 13 using Dess–Martin reagent gave [14C]
boceprevir as an approximately equal mixture of two
diastereomers in 65% yield. The diastereomer ratio is same
as that of the unlabeled boceprevir.
Results and discussion
Synthesis of [14C]boceprevir
Synthesis of [13C3]boceprevir
Boceprevir is a linear, peptide-like molecule. It was required that
the molecule was labeled in multiple sites along the tri-peptide
[
14C]Boceprevir was synthesized from K14CN as shown in
Schemes 1 and 2. (S)-t-[14C]Leucine (5) was synthesized by a
diastereoselective Strecker reaction using (R)-phenylglycine
amide 1 as a chiral auxiliary, as shown in Scheme 1.6
(R)-(À)-2-Phenylglycine amide 1, synthesized from (R)-(À)-2-
phenylglycine methyl ester hydrochloride by a modified Merck Research Laboratories, 126 E. Lincoln Avenue, PO Box 2000, Rahway, NJ
literature procedure,7 reacted with pivaldehyde to form the
07065, USA
imine in situ, which was reacted with K14CN in aqueous HOAc
*Correspondence to: Sumei Ren, Labeled Compound Synthesis, Merck Research
Laboratories, 126 E. Lincoln Avenue, PO Box 2000, Rahway, NJ 07065, USA.
to give amino nitrile 2. The mixture of (R,S) and (R,R)
amino nitrile underwent a crystallization-induced asymmetric E-mail: sumei.ren@merck.com
J. Label Compd. Radiopharm 2012, 55 108–114
Copyright © 2012 John Wiley & Sons, Ltd.

S. Ren et al.
O
H
K14CN, HOAc, H2O
RT,2 h then 70oC 24 h
79%
H2SO4 Conc., CH2Cl2
+
NH2
NH2
HN
HN
NH2
20~40oC, 2 h
82%
H2N
O
O
14C NH2
O
O
14CN
1
2
3
O
14C
O
14C
O
14C
H2/Pd/C, 2atm
(Boc)2O, Et3N, DMF
6N HCl
100oC, 24 h
OH
NH2
OH
50oC,4 h
68%
NHBoc
EtOH, HOAc, 20 h
94%
NH2.HOAc
NH2.HCl
6
4
5
Scheme 1. Synthesis of Boc-(S)-t-[14C]leucine.
O
14C
COOMe
COOMe
N
14C
COOMe
7
N
14C
N
OH
HCl/CH3OH
H.HCl
O
NHBoc
O
-20oC to RT, 20 h
HATU, DIPEA,CH2Cl2
-20oC to -10oC, 20 h
NH2.HCl
NHBoc
6
9
8
74%
NH2 NH2.HCl
N
COOMe
O
N
14C
COOH
H
H
N
14C
OH
N
N
H
N
H
12
LiOH/THF/H2O
O
O
N
O
NMM, CH2Cl2
-78oC to 40oC,16 h
84%
EDCI, NMM, HOBt
-20oC to RT, 22 h
98%
O
RT, 7 h
100%
O
10
11
O
O
H2N
H2N
H
N
H
OH
O
N
Dess-Martin Reagent
N
14C
N
14C
H
H
H
N
H
N
O
O
N
N
RT, 1 h
65%
O
O
O
O
13
[
14C]Boceprevir
Scheme 2. Synthesis of [14C]boceprevir.
backbone. The retrosynthetic analysis is shown in Scheme 3.
None of the labeled synthetic intermediates were commercially acid. The carbamic acid solution was treated with the
available. They were synthesized as shown in Schemes 4 and 5. pre-formed Mitsunobu zwitterion to give compound 14 in
13C]Compound 15 was synthesized from K13CN in seven 39% yield after distillation.
[
13C]CO2 was reacted with t-butyl amine to give carbamic
[
steps with 35% overall yield in the same manner as that of
Compound 16 was synthesized in four steps in 79% overall
yield as outlined in Scheme 5.
SCH 503034D was oxidized with Dess–Martin periodinane
[
14C]compound 9 (Schemes 1 and 2).
tert-Butyl [13C]isocyanate (14; Scheme 4) was synthesized
from tert-butyl amine and [13C]CO2 in the presence of to generate aldehyde 17 in quantitative yield. Treatment of
Mitsunobu zwitterions according to the literature procedure8 aldehyde 17 with K13CN and NaHSO3 gave 18 as a diastereo-
for the unlabeled compound, as shown in Scheme 4.
isomeric mixture in 96% yield. Cyanohydrin 18 was converted
N
*
C
O
H2N
O
*
14
H
O
N
HO
N
H
H
CO2Me
NH2
HCl.H2N
O
N
*
N
N
*
*
O
O
*
O
O
NH2.HCl
15
*=13
C
13C3]Boceprevir
16
[
Scheme 3. Retrosynthesis of [13C3]boceprevir.
J. Label Compd. Radiopharm 2012, 55 108–114
Copyright © 2012 John Wiley & Sons, Ltd.
www.jlcr.org

S. Ren et al.
Ph P (CH ) CHOOCN=NCOOCH(CH ) (DIAD)
,
N
3
3 2
3 2
*
*
C
NH2
+ CO2
O
CH2Cl2 , -78oC, then RT, 16 h
39%
14
Scheme 4. Synthesis of tert-butyl [13C]isocyanate.
H
O
O
N
H
OH
K*CN, NaHSO3
O
N
Dess-Martin Reagent
H
O
THF/H2O, 5oC, 5 h
96%
O
CH2Cl2, RT, 2 h
100%
SCH 503034D
17
OH
H
OH
OH
O
N
H
*
NH2
HCl.H2N
NH2
CN
H2O2, K2CO3
O
N
*
*
HCl, Dioxane
DMSO, 5oC to RT, 4 h
94%
O
O
O
O
RT, 4 h
88%
18
19
16
Scheme 5. Synthesis of [13C]compound 16.
to hydroxyamide 19 in 94% yield by reacting with H2O2/
K2CO3/DMSO. Finally, removal of the Boc group with HCl
gave compound 16 in 88% yield.
Experimental
General
The final compound [13C3]boceprevir as an approximately
equal mixture of two diastereomers was synthesized with
these labeled intermediates in four steps in 23% overall
yield (Scheme 6) in a similar manner to the synthesis of
[
13C]CO2 was purchased from Isotec. K13CN was purchased
from Cambridge Isotope Laboratories, Inc. [2H9]-t-Butylamine
was purchased from C/D/N Isotopes, Inc.
14C]Potassium
[
cyanide was obtained from Amersham. Compounds 7, 12,
SCH 503034D and unlabeled boceprevir were from Merck
Research Laboratories, Process Chemistry. All remaining
reagents, authentic standards, and solvents were purchased
from standard commercial sources (Aldrich, Fluka, Fisher,
Acros) and were used without further purification.
[
14C]boceprevir. In the last step of the synthesis, DMSO/
dichloroacetic acid/EDCI was used as oxidizing reagent, which
gave a comparable yield with that using Dess–Martin reagent.
Synthesis of [D9]boceprevir
LC-MS: Waters Micromass with Waters 2695 Separation
[D9]Boceprevir was synthesized from [D9]-t-butylamine in Module operating in the ES+ ionization mode. YMC-Pack
four steps with an overall yield of 69%. Compound 23 was ProC18 column, 150 mm  4.6 mm, 230 nm, isocratic, pH 9.5,
synthesized from (S)-t-leucine and amine 7 in the same way 0.04 M NH4OAc/H2O:CH3OH (35:65), 1.0 mL/min.
as [14C]compound 8. Amine 23 was reacted with triphosgene
Analytical HPLC system 1: Cyclobond 2000I, 150 Â 4.6 mm,
to generate the isocyanate, which was reacted with [D9]-t- 220 nm, CH3CN/water 99:1, 1.0 mL/min, LS Cocktail 2.4 mL/
butylamine to give urea 24 in 93% yield. The rest of the min, isocratic.
synthesis was completed in the same manner as that of
[
Analytical HPLC system 2: Waters 600 Multisolvent Delivery
System with Waters 2487 Absorbance Detector: YMC-Pack Pro
13C3]boceprevir (Scheme 7).
N
C
*
14
CO2H
CO2Me
CO2Me
N
O
N
*
N
LiOH
H
N
H
H
H
N
N
*
N
*
THF/H2O
RT, 4 h
100%
*
NMM, CH2Cl2
-78oC to -46oC, 4 h
*
O
O
O
O
O
NH2.HCl
15
20
then RT, 16 h
44%
21
OH
*
O
H2N
O
*
NH2
HCl.H2N
*
H2N
H
N
OH
H
N
O
O
toluene/DMSO
EDCI, Cl2CHCO2H
N
16
H
N
H
N
N
O
*
H
N
H
N
0oC, 20 h
73%
O
*
O
*
EDCI, NMM, HOBt
-20oC, 2 h, then RT 20 h
*
O
O
22
O
71%
[
13C3]Boceprevir
Scheme 6. Synthesis of [13C3]boceprevir.
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Copyright © 2012 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2012, 55 108–114

S. Ren et al.
COOMe
LiOH, THF/H2O
COOMe
1, Triphosgen, NaHCO3, CH2Cl2
0oC, 30min
N
N
H
N
H
D3C
D3C
N
O
O
RT, 7 h
100%
2. D9-t-butylamine, 42oC, 2.75 h
CD3
O
NH2.HCl
23
93%
24
NH2 NH2.HCl
O
H2N
O
COOH
H
N
N
OH
H
N
H
OH
12
D3C
D3C
N
N
O
H
H
O
D3C
D3C
N
N
CD3
O
EDCI, NMM, HOBt
O
-20oC to RT, 20 h
86%
O
CD3
25
26
O
H2N
H
O
N
EDCI, Cl2CO2H, toluene/DMSO
N
H
H
N
O
0oC, 20 h
87%
D3C
D3C
N
O
CD3
O
[D9]Boceprevir
Scheme 7. Synthesis of [D9]boceprevir.
C18 column, 150 mm  4.6 mm, 230 nm, 30ꢀC, 0.05 M pH 7.5, Synthesis of [14C]diamide 3
K2HPO4/H2O:CH3OH (35:65), 1.0 mL/min, isocratic.
To a solution of concentrated H2SO4 (5.4 mL) at 10ꢀC, [14C]
amino nitrile 2 (196.8 mCi, 3.58 mmol) in CH2Cl2 (7.0 mL) was
added dropwise over 30 min. The flask containing the [14C]
amino nitrile 2 was washed with CH2Cl2 (2 Â 1 mL), and the
mixture was added to the reaction over 10 min. The reaction
was stirred at 20ꢀC for 30 min, warmed to 40ꢀC, and stirred
for an additional 2 h. After being cooled to RT, the mixture
was poured on ice, neutralized with 25% NH4OH to pH ~ 9,
and extracted with EtOAc (7 Â 35 mL). The combined organic
phase was washed with brine (50 mL), dried over Na2SO4,
filtered, and concentrated to give 178 mCi of crude com-
pound 3. The crude product was purified on a silica column
(elution with 0–10% CH3OH/CH2Cl2) to give 162 mCi (82%)
of (S),(R)- [14C]diamide 3 with 95.0% RCP by analytical HPLC
system 1. A small amount of (R),(R)- [14C]diamide 3 diastereo-
mer was removed during the purification.
Analytical HPLC system 3: Restek Allure Biphenyl 150 mm
3.2 mm, 5 mm, 220 nm, 40ꢀC. MeOH/0.05 M TEAP pH 6 (65:35)
for 20 min followed by a step gradient to CH3CN for 20
min, 0.5 mL/min.
Semi-preparative HPLC: Waters 600 Multisolvent Delivery
System with Waters 2487 Absorbance Detector: Zorbox SB
Phenyl C18, 250 mm  9.4 mm, 220 nm, 0.01 M pH 7.5,
K2HPO4/H2O:CH3OH (30:70), 4.0 mL/min, isocratic.
1H NMR (400 or 600 MHz) spectra were obtained on a Var-
ian spectrometer.
Synthesis of [14C]boceprevir
Synthesis of [14C]amino nitrile 2
To a stirred suspension of (R)-(À)-2-phenylglycine amide 1
(0.682 g, 4.54 mmol) in H2O (3.0 mL) was added pivaldehyde
(0.50 mL, 4.54 mmol) at room temperature (RT). K14CN
(250 mCi, 0.305 g, 4.54 mmol, 55.0 mCi/mmol) was added.
The vial containing K14CN was washed with H2O
(2 Â 0.5 mL), and the mixture was added to the reaction flask.
Finally, glacial acetic acid (0.28 mL, 4.77 mmol) was added
over 10 min. The mixture was stirred at RT under N2 for 2 h,
warmed to 70ꢀC and stirred for 24 h. A big chunk of white
solid formed in the reaction and was broken to small pieces
periodically. After being cooled to RT, the product was
collected by filtration and washed with H2O (2 Â 3 mL). The
solid, after being concentrated and dried, was dissolved in
CH2Cl2 (12 mL) and counted to give 196.8 mCi (79%) of [14C]
amino nitrile 2 as a light yellow solid. TLC of the final product
indicated 99% radiochemical purity (RCP) (SiO2, ethyl acetate/
hexanes 60:40, Rf = 0.54).
Synthesis of (S)-[14C]tert-leucine amide 4
To a solution of [14C]diamide 3 (162 mCi, 2.94 mmol) in EtOH
(30 mL, 200 proof), Pd/C (10% wt on activated carbon,
150 mg) and glacial HOAc (3.0 mL) were added. The mixture
was shaken under H2 (55 psi) for 20 h and filtered through a
Celite pad. The Celite was washed with EtOH (3 Â 20 mL),
and the combined organic phase was concentrated (this
sometimes did not go to completion; in such case, the
mixture was re-submitted to the hydrogenation condition to
push the reaction to completion). The crude product was
purified by flash chromatography on silica gel (elution with
0–10% CH3OH/CH2Cl2) to give 152 mCi (94% yield) of
(S)-[14C]tert-leucine amide 4 with 98.8% RCP by radio-TLC
(10% CH3OH/CH2Cl2, Rf = 0.29).
J. Label Compd. Radiopharm 2012, 55 108–114
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S. Ren et al.
Synthesis of (S)-[14C]tert-leucine 5
(S)-[14C]tert-Leucine amide
refluxed in aqueous HCl (60 mL, 6 N) for 24 h and then cooled
to RT. The solvent was removed under reduced pressure to
give the crude product 5 in 79.8% RCP by radio-TLC (10%
CH3OH/CH2Cl2, Rf = 0.028). The crude product was used
directly in the next step.
CH2Cl2 (3 Â 30 mL). The combined organic phase was dried
(Na2SO4), filtered, concentrated, and purified by silica gel
chromatography (0–40% EtOAc/hexanes) to give 26.1 mCi of
10 (84% yield) with 100% RCP by radio-TLC (20% EtOAc in
hexanes, Rf = 0.13).
4
(152 mCi, 2.76 mmol) was
Synthesis of [14C]compound 11
[
14C]Compound 10 (26.1 mCi, 0.474 mmol) was dissolved in
Synthesis of (S)-N-Boc-[14C]tert-leucine 6
THF (3.0 mL) at RT, and LiOH (50.0 mg in 2.0 mL of H2O) was
added. The reaction was stirred at RT under N2 for 7 h and
concentrated. The residue was diluted with EtOAc (20 mL)
and acidified with HCl (0.3 N, 20 mL), extracted with EtOAc
(3 Â 30 mL), washed with brine (30 mL), dried (Na2SO4),
filtered, and concentrated to give 26.1 mCi of 11 with 100%
RCP by radio-TLC (20% EtOAc in hexanes, Rf =0.07) in quanti-
tative yield.
To a solution of 5 (152 mCi, 2.76 mmol) in anhydrous DMF
(5.0 mL), di-t-butyl-dicarbonate (1.50 g, 6.87 mmol) and Et3N
(0.96 mL, 6.88 mmol) were added. The reaction was stirred at
50ꢀC under N2 for 2 h, and an additional Et3N (0.5 mL) was
added. The heavy suspension was stirred at 50ꢀC for 2 h,
then at RT over the weekend. After removing the solvent
under vacuum at 50ꢀC, the residue was dissolved in HCl
(30 mL, 1 N, pre-cooled on ice) and extracted with EtOAc
(4 Â 40 mL). The combined organic phase was dried (Na2SO4),
filtered, and concentrated. The crude product was purified by
flash chromatography on silica gel (elution with 1% CH3OH/
0.5% HOAc/CH2Cl2) to give 104 mCi of 6 with 95.3% RCP by
radio-TLC as a white solid in 68% yield. The product co-eluted
with authentic N-Boc-t-leucine (Fluka) on TLC (Rf = 0.18, 10%
CH3OH/0.5% HOAc/CH2Cl2,).
Synthesis of [14C]compound 13
A suspension of [14C]compound 11 (26.1 mCi, 0.474 mmol),
compound 12 (119 mg, 0.569 mmol), HOBt (64 mg,
0.474 mmol), and EDCI (136 mg, 0.712 mmol) in anhydrous
THF/DMF (4 mL/2.0 mL, anhydrous) was treated with
N-methylmorpholine (0.24 mL, 2.14 mmol) at À20ꢀC under
N2. The reaction was gradually warmed to RT during 2 h
and stirred at RT for 20 h. The reaction was diluted with
EtOAc (20 mL) and washed with HCl (1 N, 30 mL). The
aqueous phase was extracted with EtOAc (3 Â 40 mL), and
the combined organic phase was washed with saturated
NaHCO3 (30 mL) and brine (30 mL), dried (Na2SO4), filtered,
concentrated, and purified by silica gel chromatography
(gradient, 10–50% acetone/hexanes) to give 25.5 mCi of
Synthesis of [14C]compound 8
The amine HCl salt 7 (204 mg, 0.993 mmol) and DIPEA
(0.53 mL, 2.97 mmol) were dissolved in CH2Cl2 (anhydrous,
3.0 mL) at À20ꢀC, and added to the flask containing [14C]
compound 6 (42.0 mCi, 0.764 mmol). The reaction mixture was
cooled to À20ꢀC under N2, and HATU (377 mg, 0.993 mmol)
was added. The reaction mixture was gradually warmed to
À10 ꢀC and stirred at À10 ꢀC under N2 for 20 h. The reaction
was diluted with CH2Cl2 (50 mL) and washed with NaHCO3
(saturated, 20 mL) and brine (20 mL). The organic phase was
dried (Na2SO4), filtered, and concentrated. The crude product
was purified by flash chromatography on silica gel (50 g silica,
elution with EtOAc/hexanes) to give 31.0 mCi with 97.3% RCP
by radio-TLC (20% EtOAc in hexanes, Rf = 0.22) of compound
8 as a light yellow solid in 74% yield.
[
14C]compound 13 as an approximately equal mixture of four
diastereomers (98% yield) with 99.6% RCP by analytical HPLC
system 2. All the four diastereomers were separated in this
HPLC system; the retention times of the peaks were identical
with the authentic unlabeled standard.
Synthesis of [14C]boceprevir
To a solution of [14C]compound 13 (2.0 mCi, 0.036 mmol) in
CH2Cl2 (0.6 mL, anhydrous), Dess–Martin periodinane (15 mg,
0.036 mmol) was added. The reaction was stirred under N2
at RT for 1 h. The reaction was diluted with diethyl ether
(20 mL) and then washed with saturated NaHCO3/brine
Synthesis of [14C]compound 9
[
14C]Compound 8 (31.0 mCi, 0.563 mmol) was dissolved in (20 mL, 1:1) and water (20 mL). The aqueous phase was
CH3OH (2.0 mL) and cooled to À20 ꢀC. To the solution, HCl extracted with diethyl ether (2 Â 10 mL). The combined
in CH3OH (2.0 mL, prepared fresh by bubbling HCl gas into organic phase was dried (Na2SO4), filtered, and concentrated.
cold CH3OH for 20 min) was added. The reaction was The crude product was initially purified by silica gel chroma-
gradually warmed to RT and stirred under N2 for 20 h. The tography (gradient, 10–40% acetone/hexanes) and then by
reaction mixture was concentrated in vacuo to give [14C] semi-preparative HPLC to give 1.3 mCi of [14C]boceprevir as
amine hydrochloride salt with 96.7% RCP by radio-TLC an approximately equal mixture of two diastereomers in
(5% CH3OH (7 N NH3)/CH2Cl2, Rf = 0.56) in quantitative yield. 65% yield with 98.5% RCP by analytical HPLC system 3. The
The compound was used directly in the next step.
retention times of the peaks were identical with the authen-
tic unlabeled standard. The specific activity of the compound
was 55.7 mCi/mmol.
Synthesis of [14C]compound 10
[
14C]Compound 9 (31.0 mCi, 0.563 mmol) was dissolved in
Synthesis of [13C3]boceprevir
CH2Cl2 (anhydrous, 6.0 mL) and cooled to À78 ꢀC under N2.
To the solution, N-methylmorpholine (0.12 mL, 1.13 mmol)
was added dropwise, followed by the addition of t-butyl
isocyanate (0.13 mL, 1.13 mmol). The reaction was gradually
Synthesis of tert-butyl [13C]isocyanate 14
[
13C]CO2 (3 L) was bubbled through a solution of t-butylamine
warmed to 40ꢀC and stirred at 40ꢀC under N2 16 h. The (1.85 mL, 17.6 mmol) in CH2Cl2 (40 mL) at À10ꢀC for 10 min
reaction was diluted with CH2Cl2 (40 mL) and washed with and then cooled to À78ꢀC to form the carbamate salt
HCl (1 N, 30 mL). The aqueous phase was extracted with solution. In a separate flask, diisopropyl azodicarboxylate
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J. Label Compd. Radiopharm 2012, 55 108–114

S. Ren et al.
(4.2 mL, 21.3 mmol) was added dropwise to a solution of PPh3 Synthesis of [13C]compound 16
(5.54 g, 21.1 mmol) in CH2Cl2 (60 mL) under N2 at À20ꢀC. The
HCl (11 mL, 4.0 M in dioxane) was added dropwise to a
resulting light yellow solution was cooled to À78ꢀC, then
transferred via cannula to the above carbamate salt solution.
Additional [13C]CO2 (1 L) was bubbled into the reaction
for 5 min. The reaction was warmed to RT and stirred in the
sealed flask overnight. Most of the CH2Cl2 was removed
by fractional distillation at 60ꢀC. The rest of the CH2Cl2
and tert-butyl [13C]isocyanate 1 was separated from the
byproducts PPh3O and DIADH2 by vacuum distillation. A final
fractional distillation to remove the small amount of CH2Cl2
gave 693 mg of tert-butyl [13C]isocyanate (14) in 39% yield
as a colorless oil. 1H NMR (400 MHz, CDCl3): d 1.37 (s, 9H).
LC-MS (EI+) m/z 133 (M + CH3OH + H)+.
solution of compound 19 (2.01 g, 7.35 mmol) in dioxane
(11 mL). The reaction mixture was stirred at RT for 4 h, and
the product was collected by filtration. The solid was washed
with Et2O (2 Â 5 mL) and dried to give 1.36 g of 16 in 88%
yield. The product was used directly in the next step. LC-MS
(EI+) m/z 196 (M + Na)+, 174 (M + H)+, 130 (M-CONH2 + H)+.
The rest of the synthesis of [13C3]boceprevir was conducted
in the same manner as that of [14C]boceprevir. In the last
step of the synthesis, DMSO/dichloroacetic acid/EDCI was
used as oxidizing reagent, which gave a comparable yield
with that using Dess–Martin reagent. The detailed procedure
is as follows:
Synthesis of [13C3]boceprevir
Synthesis of compound 17
To a solution of compound 17 (455 mg, 0.867 mmol) in DMSO
(2.5 mL) and toluene (5.0 mL) at 0ꢀC, EDCI (1.60 g, 8.346 mmol)
was added. To the resulting suspension, dichloroacetic acid
(0.40 mL, 4.86 mmol) was added dropwise, and the mixture
was stirred under N2 at 0ꢀC for 20 h. The reaction was diluted
with EtOAc (40 mL) and then washed with HCl (1 N, 40 mL),
saturated NaHCO3 (40 mL) and brine (40 mL). The organic
solution was dried (Na2SO4), filtered, concentrated, and
purified by silica gel chromatography (gradient, 30–40%
To a solution of SCH 503034D (1.0 g, 4.36 mmol) in CH2Cl2
(15 mL), Dess–Martin periodinane (15.0 g, 15 wt/wt% solution
in CH2Cl2, 5.30 mmol) was added dropwise at RT. After being
stirred for 2 h, the reaction mixture was diluted with Et2O
(50 mL) and then washed with NaOH (25 mL, 1 N), H2O
(25 mL), and brine (25 mL). The organic phase was dried
(Na2SO4), filtered, and concentrated to give 0.99 g of crude
product 17 in quantitative yield. Compound 17 was used
directly in the next step without further purification. 1H
NMR (400 MHz, CD3OD): d 9.45 (s, 1H), 6.90 (s, 1H), 3.82–3.80
(m, 1H), 2.40–2.19 (m, 2H), 2.06–1.44 (m, 7H), 1.42 (s, 9H).
LC-MS (EI+) m/z 479 (2M + Na + H)+.
acetone/hexanes) to give 332 mg of
white solid in 73% yield. This batch was combined
[
13C3]boceprevir as
a
with another batch of product (total, 830 mg) and repurified
by silica gel chromatography (gradient, 30–40% acetone/
hexanes) to give a final batch of 722 mg of [13C3]boceprevir
as an approximately equal mixture of two diastereomers.
The chemical purity was 95.2% by HPLC system 3. 1H NMR
(400 MHz, DMSO-d6): d 8.20 (dd, J = 41.70 Hz, J = 7.32, 1H, NH),
8.00–7.73 (m, 2H, NH2), 5.95 (s, 1H, NH), 5.84 (t, J = 10.25, 1H,
NH), 4.99–4.81 (m, 1H), 4.28 (s, 1H), 4.15–4.08 (m, 1H),
3.99–3.91 (m, 1H), 3.79–3.71 (m, 1H), 2.51–2.32 (m, 1H),
1.99–1.90 (m, 2H), 1.81–1.71 (m, 3H), 1.66–1.54 (m, 3H),
1.45–1.40 (m, 1H), 1.29–1.24 (m, 1H), 1.15 (s, 9H), 1.10–0.80 (m,
15H). 13C NMR (DMSO-d6, d): 197.8, 170.9, 170.8, 162.8, 157.3,
59.1, 56.6, 51.7, 48.9, 47.4, 36.7, 34.0, 32.1, 30.6, 29.1, 27.7, 27.3,
27.0, 26.4, 26.1, 18.5, 17.6, 12.6. LC-MS (EI+) m/z 523 (M + H)+,
423 (M-tBuNHCO + H)+, 309 (M-tBuNHCONHCHtBuCO + H)+. HRMS-
Synthesis of [13C]compound 18
To a solution of 17 (2.0 g, 8.8 mmol) and K13CN (684 mg,
10.4 mmol) in THF/H2O (10 mL, 1:1) at 5ꢀC, a saturated
aqueous solution of sodium bisulfate (8.0 mL) was added
slowly over 2 h, and the mixture was stirred for a further 5 h
at 5ꢀC. The reaction was diluted with H2O (40 mL) and
extracted with CH2Cl2 (5 Â 100 mL). The combined organic
phase was washed with brine, dried (Na2SO4), filtered, and
concentrated. The crude product was purified by flash
chromatography on silica gel (50 g silica, elution with
10–30% EtOAc/hexanes) to give 2.17 g of diastereomer
mixture of 18 as a colorless liquid in 96% yield. 1H NMR
(400 MHz, CDCl3): d 5.35 (bs, 0.5H, NH), 4.85–4.76 (m, 1H), 4.46
(bs, 1.5H, NH, OH), 3.81–3.48 (m, 1H), 2.41–1.57 (m, 9H), 1.45(s,
9H). LC-MS (EI+) m/z 256 (M + H)+, 200 (M-CHCNOH + H)+.
12 13
FAB (m/z): [M + Na]+calcd for C24C3H45N5O5Na, 545.34190; found
545.34430.
Synthesis of [D9]boceprevir
Synthesis of [13C]compound 19
Synthesis of [D9]compound 24
To a solution of 18 (2.00 g, 7.84 mmol) in DMSO (13 mL) at
5ꢀC, K2CO3 (886 mg, 6.41 mmol) was added. H2O2 (3.5 mL, Into a 250-mL round-bottomed flask containing compound
30.9 mmol) was then added dropwise during 30 min. The 23 (which was prepared in the same manner as that of
reaction mixture was warmed to RT, stirred for 4 h, diluted compound 8, 1.05 g, 3.29 mmol) was added CH2Cl2 (14 mL)
with H2O (12 mL), and stirred for an additional 10 min. The and saturated NaHCO3 (14 mL). The mixture was stirred at
resulting white precipitate was filtered, washed with H2O 0 ꢀC, and triphosgene (0.322 g, 1.09 mmol) was added. The
(3 Â 5 mL), and dried under vacuum to give 1.21 g of reaction mixture was stirred for 30 min at 0 ꢀC and then
compound 19. The filtrate was extracted with EtOAc extracted with CH2Cl2 (3 Â 30 mL). The combined organic
(3 Â 50 mL); the combined organic phase was washed with extracts were dried (Na2SO4) and then concentrated by rotary
H2O (2 Â 50 mL), dried (Na2SO4), filtered, and concentrated evaporation to give a yellow oil (isocyanate) that was resus-
to give an additional 0.80 g of compound 19 for a total pended in anhydrous CH2Cl2 (32 mL), and the mixture was
94% yield. Compound 19 was used directly in the next step. stirred under argon. [2H9]-t-Butylamine (0.431 mL, 3.62 mmol)
LC-MS (EI+) m/z 296 (M + Na)+, 274 (M + H)+, 218 (M-t-Bu + H)+, was added, and the reaction was heated in a 42 ꢀC oil
174 (M-Boc + H)+.
bath for 2.75 h. The reaction was concentrated by rotary
J. Label Compd. Radiopharm 2012, 55 108–114
Copyright © 2012 John Wiley & Sons, Ltd.
www.jlcr.org

S. Ren et al.
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evaporation to give a light yellow oil, which was purified on a
silica gel column eluting with 2:8 EtOAc/hexanes to give [D9]
compound 24 as a white solid (1.19 g, 93%).
The rest of the synthesis of [D9]boceprevir as an approxi-
mately equal mixture of two diastereomers was conducted
in the same manner as that of [13C3]boceprevir.
In summary, [14C]boceprevir, [13C3]boceprevir, and [D9]
boceprevir were synthesized to support the drug develop-
ment at different stages.
Acknowledgements
The authors would like to thank Ms. Rebecca Osterman and Dr. T.
M Chan for the NMR analysis, and Mr. Abrahim Daaro and Dr.
Pradip R. Das for the MS analysis.
Conflict of Interest
The authors did not report any conflict of interest.
[6] W. H. J. Boesten, J. P. G. Seerden, B. D. Lange, H. G. A. Dielemans, H. L.
M. Elsenberg, B. Kaptein, H. M. Moody, R. M. Kellogg, Q. B. Broxterman,
Org. Lett. 2001, 3, 1121–1124.
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www.jlcr.org
Copyright © 2012 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2012, 55 108–114