In vitro and in vivo isotope effects with hepatitis C protease inhibitors: Enhanced plasma exposure of deuterated telaprevir versus telaprevir in rats

Article abstract of DOI:10.1021/jm901023f

Telaprevir 2 (VX-950), an inhibitor of the hepatitis C virus (HCV ^a) NS3-4A protease, is in phase 3 clinical trials. One of the major metabolites of 2 is its P1-(R)-diastereoisomer, 3 (VRT-394), containing an inversion at the chiral center next

Full text of DOI:10.1021/jm901023f

J. Med. Chem. 2009, 52, 7993–8001 7993
DOI: 10.1021/jm901023f
In Vitro and In Vivo Isotope Effects with Hepatitis C Protease Inhibitors: Enhanced Plasma
Exposure of Deuterated Telaprevir versus Telaprevir in Rats
Franc-ois Maltais, Young Chun Jung, Minzhang Chen, Jerry Tanoury, Robert B. Perni,† Nagraj Mani, Leena Laitinen,
Hui Huang, Shengkai Liao, Hongying Gao,‡ Hong Tsao, Eric Block, Chien Ma, Rebecca S. Shawgo, Christopher Town,
Christopher L. Brummel, David Howe, S. Pazhanisamy, Scott Raybuck, Mark Namchuk, and Youssef L. Bennani*
Vertex Pharmaceuticals Incorporated, 130 Waverly Street, Cambridge, Massachusetts 02139. †Sirtris, a GlaxoSmithKline company,
200 Technology Square, Cambridge, MA 02139. ‡Pfizer Inc., 558 Eastern Point Rd, Groton, CT 06340-5196.
Received July 10, 2009
Telaprevir 2 (VX-950), an inhibitor of the hepatitis C virus (HCVa) NS3-4A protease, is in phase 3
clinical trials. One of the major metabolites of 2 is its P1-(R)-diastereoisomer, 3 (VRT-394), containing
an inversion at the chiral center next to the R-ketoamide on exchange of a proton with solvent.
Compound 3 is approximately 30-fold less active against HCV protease. In an attempt to suppress the
epimerization of 2 without losing activity against the HCV protease, the proton at that chiral site was
replaced with deuterium (d). The compound 1 (d-telaprevir) is as efficacious as 2 in in vitro inhibition of
protease activity and viral replication (replicon) assays. The kinetics of in vitro stability of 1 and 2 in
buffered pH solutions and plasma samples, including human plasma, suggest that 1 is significantly more
stable than 2. Oral administration (10 mg/kg) in rats resulted in a ∼13% increase of AUC for 1.
Introduction
In early stage clinical trials, the NS3-4A inhibitor 2, dra-
matically lowered plasma levels of HCV RNA when given as a
single agent, although there was evidence of viral break through
with selection of resistant viral variants.11 When given in
combination with PEG-IFN-R, or PEG-IFN-R and ribavirin,
the selection of viral variants was minimized.12,13 Compound 2
was well tolerated in these short viral kinetic studies and in the
follow-on clinical trials. Presently, compound 2 is being tested
Hepatitis C viral infection (HCVa) is a serious health crisis
affecting over 180 million people worldwide.1 The current
approved therapy for chronic HCV is a combination of
weekly injection of PEG-IFN-R with a twice-a-day oral
administration of ribavirin.2,3 Development of new therapies
for hepatitis C is focusing on STAT-C compounds to directly
block the viral life cycle. Most new compounds target the
HCV NS5B polymerase or the NS3-4A protease,4 an enzyme
needed for the release of nonstructural proteins from the
initially translated HCV polypeptide. Several polymerase
and NS3-4A protease inhibitors have been advanced to
clinical testing with varying degrees of success.5-16 The
HCV polymerase has multiple binding pockets and lends itself
to inhibition by competitive inhibitors (NIs) and allosteric
inhibitors (NNIs). Clinical trials of NS5B inhibitors are
numerous; many failed after advancing to phase II trials due
to adverse effects or viral rebound due to resistant strains. At
present, Abbott (NNI), Anadys (NNI), and GSK (NNI) are
in phase I; Gilead (NNI), Pfizer (NNI), and Pharmasset/
Roche (NI) have advanced to phase II clinical trials.5-8 The
active site of HCV protease is shallow requiring multiple inter-
actions from the inhibitor to have potency. Protease inhibitors
are very efficacious in lowering the viral load to undetectable
levels. A number of compounds from Boehringer-Ingelheim,
InterMune/Roche, and Merck areinphase I; Medivir/Tibotec
in phase II; Schering-Plough and Vertex are in phase III
clinical trials.8,9
in many late stage clinical trials in treatment naıve as well as
¨
PEG-IFN-R/ribavirin treatment-failure patients.
Compound2 is a peptidomimetic inhibitor, whose structure
is shownin Figure1. Thechiral center nexttothe R-ketoamide
(P1) in 2 (S-configuration) is stable at acidicpH but is proneto
epimerization through proton exchange with solvent water at
alkaline pH via an enol tautomer as depicted in Figure 1. The
resulting P1-(R)-diastereoisomer, 3 (VRT-394; Figure 1),
has shown approximately 30-fold lower inhibitory activity
(Ki > 1 μM).10 Epimer 3 is one of the primary metabolites of 2
in vivo (the concentrations of 3 can reach up to 40% of the
concentration of 2 at some time points). Because of the
significant observed concentrations of 3, understanding of
the kinetics for the interconversion of 2 to 3 (in vitro and in
vivo) is important to the characterization of 2.
In an effort to increase in vivo exposure of the biologically
active drug 2, we substituted the P1-proton with a deuterium and
examined the net effect on in vitro stabilityandinvivoexposurein
rats. This strategy of specific deuteration, which has long been in
use in the investigation of organic and enzymatic reaction mec-
hanisms and biosynthetic pathways analysis,17-20 was viewed as
the least structurally disruptive because of the SAR sensitivity to
substitution at the R-keto amide position. Isotopic substitution
can result in a substantial reduction of the reaction rate (or
metabolism) if the covalent bond with the substituted atom is
partially broken in the rate-limiting transition state.21,22 This
primary kinetic isotope effect, which is expressed as the fold
*To whom correspondence should be addressed. Phone: 1-617-444-
6616. Fax: 1-617-444-7822. E-mail: youssef_bennani@vrtx.com.
aAbbreviations: HCV, hepatitis C virus; AUC, area under curve;
PEG-IFN-R, pegylated interferon alpha; STAT-C compounds, specifi-
cally targeted antiviral therapies for hepatitis C; NIs, nucleoside inhibi-
tors; NNIs, non-nucleoside inhibitors; RNA, ribonucleic acid; IV,
intravenous; PO, oral administration.
r
2009 American Chemical Society
Published on Web 11/06/2009
pubs.acs.org/jmc

7994 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 24
Maltais et al.
presence of palladium on carbon, gave the racemic mixture
of R-hydroxy-β-amino amide 11 in good overall yield (70%).
At this stage, the mass spectral analysis revealed the deuter-
ium content to be unchanged (∼99%). Chiral resolution of
amide 11 with deoxycholic acid, followed by hydrolysis with
hydrochloric acid, afforded enantiomerically pure key inter-
mediate 13 in high deuterium content (∼99%), as assessed by
chiral HPLC and mass spectroscopy, respectively. Amine 13,
the synthesis of which was reported earlier for 2,24 underwent
amide-coupling with carboxylic acid 1424 to give the alcohol 15.
Tempo oxidation of 15 gave the desired final compound, 1. The
combined yield for the amide coupling and oxidation steps was
80%. The mass spectral analysis confirmed the deuterium
content of the final product, 1, at 97%. Experimental details
of the synthesis of 1 are given in the Materials and Methods
section.
In Vitro Characterization of 1. The antiviral characteristics
of 1 and 2 were assessed side-by-side in biochemical assays.
In the enzyme inhibition assay with full-length NS3-4A
protease, 1 had a Ki value of 20 nM, which is similar to
the value of 44 nM for 2.10 In the viral replication assay
(replicon assay) 1 and 2 had IC99 values of 4.0 and 3.3 μM,
respectively. Therefore, the in vitro antiviral properties
of 2 are not significantly altered upon substitution with
deuterium in 1.
Compounds 1 and 2 were also subjected to buffer stability
studies in solutions of varying pH from 1 to 9. Aliquots were
withdrawn as a function of time and analyzed by LC-MS for
the content of 1, 2, and the biologically inactive diastereo-
isomer 3. The LC-MS analysis is described in the General
Experimental Details. Below pH 7, the epimerization rate
was insignificant over a 3 day period (data not shown). The
percentage of 3 formed at pHs 7 and 9 were plotted as a
function of time in Figure 2. These data were fitted to eq 2,
where B and Beq are % of 3 at time t and tµ, respectively, and
kobs (the sum of rate constants for the forward and reverse
reactions as shown in eq 1) is the rate constant for the
approach to equilibrium. The kobs for 2 was not dependent
on the phosphate concentration at pH 7 (Table 1) and the
epimerization half-life at pH 7 was ∼35 h. The rate-constant
for epimerization of 2 at pH 9 was 19-fold higher than that at
pH 7 indicating that specific base-catalyzed (HO-) compo-
nent of epimerization reaction dominated at alkaline pH.
The equilibrium concentration of 3, Beq, was determined to
be between 35 and 40% of the total amount of 2 and 3.
At pH 7 and 9, compound 1 was more stable than 2, and
the observed kinetic isotope effect on the initial rate of 3
formation was approximately 5-fold. Deuterium substitu-
tion, therefore, significantly suppresses the inactivation of 2
via epimerization. Kinetic isotope effects of such magnitude
for the substitution of hydrogen with deuterium have been
observed previously in a number of reactions where a C-H
bond was being modified.18
Figure 1. Reaction scheme for 2and its (R)-epimer (3) interconversion.
decrease in rate constant for the deuterium substitution,
kH/kD, can be as high as 7-10-fold. Kinetic isotope effect studies
have long been employed to identify metabolic fate of drug
substances, to determine rate-limiting steps of organic and
enzymatic reactions, and to infer the rate-limiting transition-state
structures.23
In this study, it was investigated whether the in vitro rate
of epimerization of 1 (d-telaprevir) was slower than that of
2 in rat, dog, and human plasma and whether higher
S-diastereoisomer/R-diasteroisomer ratios could be obtained
in rat plasma after administration of 1 as compared to
administration of 2.
Herein we report the synthesis of 1, its kinetic stability
compared to 2 as a function of buffer pH, plasma stability,
and pharmacokinetic profiles in rats after intravenous and
oral administration.
Synthesis of 1. The synthesis of 1, described in Scheme 1,
started with 2-hexyne-1-ol 4, which was reduced with Red-
Al, and the subsequent intermediate was quenched with D2O
to deliver 3-deutero-allylic alcohol 5 in high isotope content
(99%) and high reaction yield (83%). Oxidation of the
alcohol 5 with MnO2 to the corresponding aldehyde 6 was
achieved in excellent yield (90%). Compound 6 was further
oxidized to the carboxylic acid 7 using standard methods. At
this stage, the deuterium incorporation was not determined
because of nonionization. Amide formation of compound 8
with cyclopropylamine proceeded in 65% yield using iso-
butyl chloroformate in the presence of N-methyl morpho-
line. The mass spectral analysis showed the preservation of
the deuterium content at 99%. Epoxidation of 8 was
achieved with urea hydrogen peroxide in trifluoroacetic
anhydride to yield 9. The crude reaction mixture from above
was used in the next step without further purification.
Epoxide 9 was regiospecifically ring-opened with sodium
azide to intermediate 10, the reduction of which, in the
kf
F
2
3
(1Þ
(2Þ
R
kr
B ¼ Beqð1 - eð -k
Þ
obsÞt
The stability of 1 and 2 was also tested in plasma at 1 and
10 μM concentrations. Because the stability profile was
similar for 1 and 10 μM concentrations, only 1 μM data is
shown in Figure 3. The relative initial rates and the asso-
ciated kinetic isotope effects are presented in Table 2. It is

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 24 7995
Scheme 1. Synthesis of 1
obvious that human plasma catalyzed the epimerization by
a factor of about 10 compared to buffer (used as a control),
while the rat or dog plasma showed less effect on the
epimerization rates (Table 2). The concentration of 3 reached
a steady state level of approximately 40% of the concentra-
tion of the initial 2 in human plasma, which is similar to what
was observed in phosphate buffer. The compound 1 was
more stable in all plasma samples when compared to 2. The
kinetic isotope effect on the initial rate of epimerization was
in the range of 4-7 for dog, rat, or human plasma-catalyzed
samples. The kobs value for the approach to equilibrium in
human plasma-catalyzed epimerization of 2, obtained by
fitting the progress curve data to eq 2, was 0.038 min-1
yielding a half-life of ∼20 min. This has the effect of reducing
the concentration of 2 by 35% (by conversion to 3) over the
course of approximately an hour. In that period only 10% of
the deuterated drug was converted to 3 by epimerization
(Figure 3). Epimerization of 1 also results in the production
of 3 because the abstracted deuterium will be exchanged with
a proton in the solvent water. Compounds 1 and 2 go
through the same enol tautomeric intermediate (Figure 1
and eq 3), therefore, they have the same probability to
generate 3 versus 2, depending on which side of the enol
intermediate receives the proton. We followed the kinetics of
the appearance of 3 as well as the formation of 2 from 1.
As is clear from the data for compound 1 (Figure 4), the
overall production of 2 is 1.4-fold faster than the formation
of the inactive epimer 3. Therefore, the rate constant leading
to the formation of 2 (kv) is 1.4-fold larger than that leading
to the formation of the inactive epimer (ke). This observation
is in line with previous work, indicating that the equilibrium
favors 2 over 3 by a 60:40 ratio.
In all in vitro conditions studied, 1 was significantly more
stable than 2 with respect to epimerization to form 3. The
next challenge was to determine if the additional in vitro
stability of 1 would translate to increased exposure in vivo.
As a first approximation, both 1 and 2 should have similar
chemical and metabolic reactivity at all other sites on the
molecule except for the isotopic center. Therefore, the ad-
vantage bestowed by the isotopic substitution depends on
what the elimination mechanisms are with respect to epimeri-
zation. Short of testing its metabolic stability in man, the
pharmacokinetics of 1 in rats was obtained.
In Vivo Characterization. Both 1 and 2 were administered
to rats at 1 mg/kg IV and 10 mg/kg PO. In all experiments the
concentrations of the active drug (1 and 2) and 3 in plasma
were monitored as a function of time.
When 1 and 2 were dosed IV separately, the PK profiles
were virtually identical (Figure 5A). However, when 1 and 2
were dosed PO, significant differences were observed in the
PK profile (Figure 5B): (a) higher levels of 1 were observed at
several time points, (b) 2 was observed over time, albeit at a
low level (<4%), in 1 administered rats, and (c) 3 levels were

7996 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 24
Maltais et al.
Figure 3. Stability of 2 (panel A) and 1 (panel B) in plasma samples.
Compound 2 or 1 dissolved in DMSO was added either to pH 7.4
buffer (0.1 M phosphate; ꢀ), rat plasma (0), dog plasma (9), or
human plasma (O) to a final concentration of 1 μM and 10 μM (data
not shown). The final DMSO concentration was 1%. Aliquots
withdrawn at various intervals were processed and analyzed as in
General Experimental Details.
Figure 2. Epimerization rate for 2 (A) and 1 (B) in buffer. The
initial concentration of 2 and 1 were 0.25 mg/mL in all experiments
(0: pH 7 water; 9: pH 7 phosphate buffer (0.05 M); O: pH 9 borate
buffer (0.1 M)). Aliquots were drawn at various intervals, processed,
and analyzed for the content of 3, 2, and 1, as described in General
Experimental Details.
Table 1. Epimerization Rate Constants for 2
medium
kobs (hr-1 a
)
rel. rate
pH 7 (H2O)
pH 7 (0.05 M phosphate)
pH 9 (0.1 M borate)
0.020 ( 0.002
0.023 ( 0.004
0.375 ( 0.019
1
1
19
a kobs is the sum of forward (kf) and reverse (kr) rate constants for the
epimerization of 2, as given in eq 1.
Table 2. Effect of Plasma on the Epimerization of 2 and 1
kinetic isotope effectb (kH/kD)
medium
relative ratea
@ 1 μM
@ 10 μM
buffer (pH 7.4)
rat plasma
dog plasma
1
5
6
1.0 - 1.5
1.4 - 3.4
>8
7
7
4
>5
6
>5
Figure 4. Appearance of 3 and 2 from 1 in human plasma. The
DMSO solution of 1 was added to human plasma to a final
concentration of 10 μM. Aliquots were drawn at various intervals,
processed, and analyzed for the content of 2 (O) and 3 (9) as in
General Experimental Details.
human plasma
a Initial rate of epimerization of 2. b Standard error of (1.
less in 1 dosed animals. These observations are qualitatively
in agreement with the expected effect of deuterium in 1. Each
time point represents an average concentration observed
in three rats. Because the animal-to-animal variability was
high, it was not possible to confidently conclude if the AUC
increase for 1 was statistically significant. To this end, the PO
studies were repeated with a 50:50 mixture of 1 and 2 and the
number of rats in the study were increased to six. By formu-
lating the mixture of 1 and 2, any animal-to-animal vari-
ability is removed because each animal is exposed to both 1
and 2 at the same time. Instead of plotting the absolute
concentration of 1 and 2 in the PO samples, we plotted the
mean ratio of 1 over 2, as shown in Figure 6, as this gives a
direct estimate of any increase in 1 due to its increased
stability. The actual concentrations of 1 and 2 are presented
as supplement data. The peak concentration of the active
drug (1 or 2) was about 200 ng/mL. A cutoff of <10 ng/mL
was used to exclude data points that may be unreliable.
As the isotopic content of 1 is 97%, the 50:50 mixture
sample will produce a ratio of 1 over 2 of 0.94, as indicated by
the dashed line in Figure 6. It is obvious from the PO data in
Figure 6 that most of the time points are above this reference
line. The ratio peaks at about 2 to 3 h after dosing to a net
increase of 12-15%. This is consistent with the observations
in Figure 5B. Interestingly, a similar effect was not observed
in the IV-dosed animals (Figure 5A); the lack of a significant
difference may be due to variability in the data. The increase
in the AUC for PO-dosed 1 is still small (13%), but ob-
servable. This analysis ignores the fact that a small portion of
2 was indeed generated from 1, putting 13% increase in the
AUC as a lower limit. Because the epimerization half-life is
long (>4 h) in rat plasma compared to the terminal half-life
(∼1.5 h; Figure 5B) in vivo, the observed small increase in the
AUC for 1 is expected.
Summary
It has been well documented in the literature that isotopic
substitution can alter physical properties of a compound.17-25
However, the property changes will be subtle for atomic
substitution compared to appending a chemical group.

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Journal of Medicinal Chemistry, 2009, Vol. 52, No. 24 7997
administration of deutero-rofecoxib in rats26 resulted in 1.53-
and 1.60-fold increases in the AUC and Cmax, respectively.
Other examples of deuterated drugs exhibiting superior pro-
perties over their counterparts in preclinical studies include
30-d2-butethal, showingover a 2-fold increase in sleeptime27 in
mice, and deuterated sildenafil, with higher selectivity for
phosphodiesterases.28 Herein, we showed that specific deu-
teration of 2 led to increased pH and plasma stability in vitro.
In rats, 1 exhibited a small but measurable increase in AUC
and the clearance of 2 is not dominated by epimerization to 3.
Based on the epimerization of 1 to 3 in vitro, it is possible that
the AUC in humans will be more significantly impacted than
the minimal effect observed in rats, however, to determine this
it would be necessary to dose 1 in human subjects.
Materials and Methods
General Experimental Details. All commercial reagents and
anhydrous solvents were obtained from commercial sources and
were used without further purification, unless otherwise speci-
fied. Mass samples were analyzed on a Micro Mass ZQ, ZMD,
Quattro LC, or Quatro II mass spectrometer operated in a single
MS mode with electrospray ionization. Samples were intro-
duced into the mass spectrometer using flow injection (FIA)
or chromatography. The mobile phase for all mass analysis
consisted of acetonitrile-water mixtures with either 0.2% for-
mic acid or ammonium formate. The high-resolution mass
spectrum was measured using a 9.4T APEX III FTMS Bruker
Daltonics instrument. The following HPLC methods were used
to obtain the reported retention times. (i) Method A: YMC
ProC18 column, 2.0 ꢀ 50 mm; linear gradient from 5 to 95%
CH3CN in H2O over 15 min (0.1% trifluoroacetic acid); flow
rate 1 mL/min; 214/254 nM. (ii) Method B: Daicel Chiralpak
AD-H, 4.6ꢀ 50 mm; isocratic gradient 92% heptane, 8%
ethanol for 40 min (0.1% methanesulfonic acid); flow rate
1.0 mL/min at 5 °C; detection diode array. (iii) Method C:
YMC-Pack PVA-Sil NP, 4.6 ꢀ 50 mm; linear gradient from 2 to
90% heptane in THF over 45 min (0.1% AcOH, 0.001% TFA);
flow rate 1.5 mL/min; detection diode array. 1H NMR spectra
(δ, ppm) was recorded either using a Bruker DRX-500 (500
MHz) or a Bruker Avance II-300 (300 MHz) instrument.
Column chromatography was performed using Merck silica
gel 60 (0.040-0.063 mm). The purity of all intermediates and
final product showed a purity of g95%, determined by HPLC
and HRMS.
Figure 5. IV and PO dose-response curves for 2 and 1 dosed
separately in rats. (A) 2 or 1 was given at 1 mg/kg dose to rat by
injection into a preimplanted jugular cannula. Blood was with-
drawn at specified intervals, the drug substance was extracted as in
Materials and Methods and analyzed by LC/MS. The (0) and (O)
represent 2 and 1 concentrations, respectively. (B) 1 and 2 were given
at 10 mg/kg dose to rat by gavage, blood samples withdrawn at
specified times, extracted as in Materials and Methods, and ana-
lyzed by LC/MS. The (0) and (4) represent the parent drug from 2
and 1, respectively. The (b) and (]) represent 3, the metabolite from
2 and 1, respectively. The (O) represents 2 formed from 1.
(E)-Hex-3-deutero-2-en-1-ol (5). To a three-neck 250 mL
round-bottom flask equipped with mechanical stirrer and reflux
condenser was charged 2-hexyn-1-ol 4 (10 g, 0.1 mol) and THF
(100 mL, 10 vol). The resulting mixture was cooled down to 0 (
5 °C and then Red-Al (65% in toluene, 32 mL, 1.6 equiv) was
added slowly under a nitrogen atmosphere between 0 and
20 °C. The resulting mixture was allowed to warm up to 25 °C
and stirred for 5 h. The reaction mixture was cooled down to
-5 ( 5 °C, and D2O (8.2 g, 4 equiv) was added dropwise between
0 and 15 °C. To the resulting mixture was charged isopropyl
acetate (50 mL, 5 vol) and saturated NH4Cl solution (50 mL,
5 vol). After stirring the mixture for 10 min, the white solid
formed was filtered out. The organic layer from the filtrate was
separated and the aqueous layer was extracted with isopropyl
acetate (30 mL, 3 vol). The organic layers were combined and
washed with water (30 mL, 3 vol), dried over MgSO4, and
concentrated to afford compound 5 (9.8 g, 95%) as a colorless
oil. The crude product was used for the next step without fur-
ther purification. HPLC (Method A) tR = 9.128 min. For the
Figure 6. PO dosing of 50:50 mixture of 1 and 2. A 50:50 mixture of
1 (97% isotopic purity) and 2 was formulated as given in the General
Experimental Details and orally administered at 10 mg/kg to six
male Sprague-Dawley rats. Blood samples were collected at speci-
fied times, extracted, and analyzed by MS. The ratio of 1 to 2 is
plotted as a function of time. The dashed line corresponds to the
initial ratio of 1 to 2 of 0.94. The actual concentrations of 2 are
provided in Table 1 of the Supporting Information.
Therefore, isotopic substitution is an attractive way to boost
the concentration of the active drug by slowing down
either the oxidative metabolism or the epimerization rate, as
exemplified in the case of 2, without significantly altering the
pharmacological properties of the drug. Currently we do not
know of any deuterated drug on the market. However, a
number of approved drugs were deuterated either with the
intention to improve their stability or to understand the
metabolic fate.18 Recently, one publication reported that oral
1
compound: H NMR (500 MHz, CDCl3) δ 5.66 (t, 1H, J =
5.0 Hz), 4.12 (d, 2H, J = 5.0 Hz), 2.04 (t, 2H, J = 5.0 Hz),
1.38-1.46 (m, 2H), 0.93 (t, 3H, J = 5.0 Hz); 13C NMR (125
MHz, CDCl3) δ 132.95 (t, J = 23.8 Hz), 128.90, 63.76, 34.17,

7998 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 24
Maltais et al.
22.25, and 13.66. Deuterium content by LCMS/MS = 99%.
HRMS =no signal in positive or negative ion modes.
urea hydrogen peroxide (25 g, 4 equiv), and p-TsOH (12.3 g,
1 equiv) in CH2Cl2 (100 mL, 10 vol) at 0 °C was added trifluoro-
acetic anhydride (40.9 g, 3 equiv) in CH2Cl2 (50 mL, 5 vol) over
30 min. The reaction mixture was heated to 40 ( 5 °C and stirred
for 3 h. After cooling down the reaction solution to 0 °C, the
reaction was quenched by adding 6 N NaOH (100 mL, 10 vol)
slowly and stirring for 30 min. The organic layer was separated,
and the organic layer was washed with brine (5 vol) and water
(5 vol). The resulting organic layer was dried over MgSO4 and
evaporated to give the epoxide 9 (9.7 g, 86%) as pale yellow oil.
The crude product was used for the next step without further
purification. HPLC (Method A) tR = 7.86 min. For the com-
pound: 1H NMR (500 MHz, DMSO) δ 8.01 (s, 1H), 3.09 (s, 1H),
2.63-2.65 (m, 1H), 1.39-1.54 (m, 4H), 0.91 (t, 3H, J=5.0 Hz),
0.60 (t, 2H, J = 3.0 Hz), 0.45 (t, 2H, J = 3.0 Hz); 13C NMR
(125 MHz, CDCl3) δ 168.67, 56.73 (t, J=26.3 Hz), 54.08, 32.69,
22.06, 18.59, 13.56, 5.38, and 5.37. HRMS calcd for C6H15-
DNO2, 171.12383; found, 171.12383. Deuterium content by
LCMS/MS =no ionization.
3-Azido-3-deutero-N-cyclopropyl-2-hydroxyhexanamide (10).
To a three-necked 250 mL round-bottom flask equipped with
mechanical stirrer and reflux condenser containing the epoxide 9
(10 g, 0.06 mol) and anhydrous magnesium sulfate (14.1 g,
2.0 equiv) in MeOH (100 mL, 10 vol) was added sodium azide
(15.3 g, 4.0 equiv) in one portion. The resulting mixture was
heated to 65 ( 5 °C and stirred for 5 h. The reaction solution
was cooled down to room temperature and isopropyl acetate
(100 mL, 10 vol) was added and stirred for 10 min. The mixture
was filtered through a pad of Celite to get rid of insoluble salts
and the resulting clear solution was concentrated to 3 vol. To the
resulting solution was added isopropyl acetate (170 mL, 17 vol)
and the mixture was stirred for 10 min. Again, the solution was
filtered through a pad of Celite to afford the product 10 as a clear
solution in isopropyl acetate (about 200 mL) for the next step
without further purification. HPLC (Method A) tR =8.88 min.
For the compound: 1H NMR (500 MHz, DMSO) δ 7.91 (s, 1H),
6.00 (d, 1H, J=5.0 Hz), 4.03 (d, 1H, J=5.0 Hz), 2.66-2.67 (m,
1H), 1.30-1.58 (m, 4H), 0.88 (t, 3H, J=5.0 Hz), 0.60 (t, 2H, J=
3.0 Hz), 0.48 (t, 2H, J=3.0 Hz); 13C NMR (125 MHz, CDCl3) δ
171.93, 73.43, 62.90 (t, J=23.8 Hz), 29.25, 22.11, 18.89, 13.55,
and 5.42. HRMS calcd for C9H16DN4O2, 214.14088; found,
214.14076. Deuterium content by LCMS/MS =99%.
(E)-Hex-3-deutero-2-enal (6). To a three-neck 250 mL round-
bottom flask equipped with mechanical stirrer containing the
alcohol 5 (10 g, 0.1 mol) in CH2Cl2 (150 mL, 15 vol) was charged
activated MnO2 (87 g, 10 equiv) at room temperature. After
vigorous stirring for 1 h, another portion of MnO2 (16 g, 2 equiv)
was added and the shaking was continued for 4 h. The reaction
solution was filtered through a pad of Celite. The solvent was
removed in vacuo (25 °C, 100 mmHg) to give the crude aldehyde
6 as a pale yellowish oil (8.8 g, 90%). The crude product was
used for the next step without further purification. HPLC
1
(Method A) tR = 10.141 min. For the compound: H NMR
(500 MHz, CDCl3) δ 9.54 (d, 1H, J=10.0 Hz), 6.14 (s, 1H), 2.34
(m, 2H), 1.55-1.60 (m, 2H), 1.00 (t, 3H, J=5.0 Hz); 13C NMR
(125 MHz, CDCl3) δ 194.06, 158.32 (t, J = 23.8 Hz), 133.00,
34.53, 21.07, and 13.59. Deuterium content by LCMS/MS=no
ionization. HRMS=no signal in positive or negative ion modes.
(E)-Hex-3-deutero-2-enoic acid (7). To a three-neck 500 mL
round-bottom flask equipped with mechanical stirrer and reflux
condenser was charged the aldehyde 6 (10 g, 0.1 mol), tert-
BuOH (90 mL, 9 vol), and 2-methyl-2-butene (30 mL, 3 vol). The
resulting solution was added with freshly prepared aqueous
NaClO2 (27.4 g, 3 equiv) and NaH2PO4 (62.9 g, 4 equiv) in
water (200 mL) over 30 min. The reaction mixture was stirred at
room temperature for 2 h. The reaction solution was cooled
down to 0 °C and was added with saturated Na2SO3 aqueous
solution until the reaction became colorless. The stirring was
stopped, the organic layer was separated, and the aqueous layer
was extracted with EtOAc (3 vol ꢀ 3). The organic layers were
combined and concentrated in vacuo until the total volume
became 3 vol. The resulting solution was extracted with 1 N
NaOH (3 vol ꢀ 3), and the remaining organic layer was
discarded. The combined aqueous solution was acidified with
6 N HCl to the pH 1.0. The solution was extracted with CH2Cl2
(3 vol ꢀ 5). The combined organic layer were dried over MgSO4
and concentrated to give the acid 7 (8.7 g, 75%) as a white solid.
HPLC (Method A) tR=9.02 min. For the compound: 1H NMR
(500 MHz, CDCl3) δ 5.84 (s, 1H), 2.23 (t, 2H, J = 5.0 Hz),
1.51-1.55 (m, 2H), 0.98 (t, 3H, J = 5.0 Hz); 13C NMR (125
MHz, CDCl3) δ 172.33, 151.90 (t, J = 23.8 Hz), 120.71, 34.17,
21.12, and 13.63. HRMS calcd for C6H9DO2, 116.08163; found,
116.08163. Deuterium content by LCMS/MS= no ionization.
(E)-N-Cyclopropylhex-3-deutero-2-enamide (8). A three-neck
250 mL round-bottom flask equipped with mechanical stirrer
and reflux condenser was charged with the acid 7 (10 g, 0.09 mol)
and isobutyl chloroformate (13 g, 1.1 equiv) in CH2Cl2 (100 mL,
10 vol). The resulting solution was cooled down to 0 °C, and
N-methyl morpholine (13.2 g, 1.5 equiv) was added slowly while
controlling the temperature between 0 and 20 °C. Then, the
mixture was allowed to warm up to room temperature and
stirred for 1 h. To the resulting solution was added cyclopropyl
amine (5.9 g, 1.2 equiv), and the solution was stirred for 2 h.
The reaction mixture was washed with 1 N NaOH (3 vol ꢀ 2),
1 N HCl (3 vol ꢀ 2), brine solution (3 vol), and water (3 vol).
The organic layer was dried over MgSO4 and concentrated to
afford the crude product as an oil. The crude product was
dissolved with heptane (5 vol) and cooled down to -78 °C with
stirring. The precipitated solid was filtered and dried to give the
amide 8 (8.7 g, 65%) as a white solid. HPLC (Method A) tR =
3-Amino-3-deutero-N-cyclopropyl-2-hydroxyhexanamide (11).
A 500 mL autoclave hydrogenation reactor equipped with
mechanical stirrer containing the azido compound 10 (200 mL,
0.05 mol) in isopropyl acetate (obtained in the previous step) was
charged with Pd/C (10% Pd, water 50%, 0.8 g). The solution was
charged with nitrogen (1.0atm) andreleased three times andthen
charged with hydrogen (3.0 atm) and released three times. The
resulting solution was charged with hydrogen (3 atm) and stirred
for 5 h. After releasing the hydrogen gas, the solution was purged
with nitrogen for 5 min. To the resulting solution was added
MeOH (30 mL, 3 vol), and the reaction mixture was heated to
50 ( 5 °C. The reaction mixture was filtered through a pad of
Celite to afford a clear solution. The product was isolated by
concentrating the solution at 20 ( 5 °C until 3 vol of the solution
remained. The solid was collected by filtration, washed (iso-
propyl acetate, 3 vol), and dried to give the amine 11 (7.7 g, 70%,
two steps) as a white crystalline solid. HPLC (Method A) tR =
1
4.51 min. For the compound: H NMR (500 MHz, DMSO) δ
1
8.51 min. For the compound: H NMR (500 MHz, DMSO) δ
7.70 (s, 1H), 5.31 (s, 2H), 3.68 (s, 1H), 2.64-2.66 (m, 1H),
1.10-1.50 (m, 4H), 0.82 (t, 3H, J = 5.0 Hz), 0.59 (t, 3H, J =
3.0 Hz), 0.45 (t, 3H, J=3.0 Hz); 13C NMR (125 MHz, CDCl3) δ
173.61, 75.38, 52.93 (t, J = 20.0 Hz), 33.40, 21.93, 18.78, 14.01,
5.49, and 5.47. HRMS calcd for C9H18DN2O2, 188.15038;
found, 188.15028. Deuterium content by LCMS/MS=99%.
(2S,3S)-3-Amino-3-deutero-N-cyclopropyl-2-hydroxyhexan-
amide Deoxycholic Acid Salt (12). To a three-neck 250 mL
round-bottom flask equipped with mechanical stirrer and
containing the racemic amine 11 (10 g, 0.05 mol) in THF
7.92 (s, 1H), 5.78 (s, 1H), 2.66-2.68 (m, 1H), 2.08 (t, 2H, J =
5.0 Hz), 1.38-1.42 (m, 2H), 0.87 (t, 3H, J=5.0 Hz), 0.63 (t, 2H,
J = 3.0 Hz), 0.40 (t, 2H, J = 3.0 Hz); 13C NMR (125 MHz,
CDCl3) δ 165.88, 141.52 (t, J = 23.8 Hz), 124.25, 33.02, 22.15,
20.09, 13.44, and 5.65. HRMS calcd for C9H15DNO, 155.12892;
found, 155.12873. Deuterium content by LCMS/MS =99%.
3-Deutero-N-cyclopropyl-3-propyloxirane-2-carboxamide (9).
To a three-neck 250 mL round-bottom flask equipped with
mechanical stirrer and containing the amide 8 (10 g, 0.06 mol),

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 24 7999
(100 mL, 10 vol) was charged deoxycholic acid (15.7 g, 0.75
equiv). The reaction mixture was heated to 65 ( 5 °C and stirred
for 1 h at this temperature. The resulting homogeneous mixture
was cooled down to 23 ( 2 °C over 1 h, and was maintained at
the same temperature range for 1 h. The precipitated solids were
collected by filtration, washed with THF (50 mL, 5 vol), and dried
to give the resolved amine 12 (12.4 g, 41%, enantiomeric ratio
(ER)=2:98) as a white solid. HPLC (Method B) tR=27.11 min.
HRMS calcd for C9H18DN2O2, 188.15038; found, 188.15060.
Deuterium content by LCMS/MS= 98%.
to afford 1 (9.2 g, 80%) as a white solid. HPLC (Method C) tR =
18.0 min. For the compound: 1H NMR (500 MHz, DMSO) δ 9.21
(s, 1H), 8.91 (d, 1H, J=2.0 Hz), 8.77 (t, 1H, J=1.0 Hz), 8.72 (d,
1H, J=5.0 Hz), 8.51 (d, 1H, J=9.0 Hz), 8.25 (s, 1H), 8.22 (d, 1H,
J=9.0 Hz), 4.70 (m, 1H), 4.54 (m, 1H), 4.28 (d, 1H, J=3.5 Hz),
3.75 (ABX, 1H, JAB = 8.0 Hz, JAX = 8.0 Hz), 3.64 (ABX, 1H,
JAB =10.5 Hz, JAX =3.0 Hz), 2.75 (m, 1H), 2.64 (m, 1H), 1.83-
1.30 (m, 16H), 1.19-1.00 (m, 6H), 0.94 (s, 9H), 0.89 (t, 3H, J =
7.0 Hz), 0.66 (m, 2H), 0.58 (m, 2H); 13C NMR (125 MHz, CDCl3)
δ 196.91, 171.74, 170.33, 169.19, 168.93, 161.99, 161.84, 147.78,
143.96, 143.37, 143.34, 64.64, 56.31, 56.23, 54.08, 47.12, 42.18,
41.21, 35.63, 34.33, 32.01, 31.70, 31.48, 28.99, 27.92, 26.28, 25.63,
25.57, 24.56, 22.39, 22.33, 18.64, 13.66, 13.41, 5.33, and 5.27.
HRMS calcd for C36H53DN7O6, 681.41929; found, 681.41946.
Deuterium content as determined by LCMS/MS was 97%.
Stability Kinetics in Buffer. Stability kinetics were performed
in four different buffers from Fisher Scientific: pH 1 (potassium
chloride, SB140-500), pH 3 (potassium bipthalate, SB97-500),
pH 5 (potassium bipthalate, SB102-500), pH 7 (potassium
phosphate monobasic, SB108-500), and pH 9 (boric acid-
potassium chloride, SB114-500). Simulated gastric fluid was
prepared as specified in the U.S. Pharmacopeia (USP), giving a
sodium chloride concentration of 2 mg/mL, a pepsin concentra-
tion of 3.2 mg/mL, and a final pH of 1.2 adjusted by 12 N HCl.
Simulated intestinal fluid was prepared as a 6.8 mg/mL mono-
basic potassium phosphate solution and the pH was adjusted
to 6.8 by 0.2 N sodium hydroxide. Due to the low aqueous
solubility of 2 and 1, a 1:1 (v/v) mixture of aqueous buffer
(above) and acetonitrile was prepared to test the stability of
compounds at different pHs. The analytes were incubated at
RT or 37 °C with buffers/ACN mixture at final concentrations
of 0.25 mg/mL. Liquid chromatography (HPLC) was used to
quantitate the concentrations of the inactive 3 and 2 or 1 in the
buffer at various time points from time 0 to 3 days.
(2S,3S)-3-Amino-3-deutero-N-cyclopropyl-2-hydroxyhexan-
amide Hydrochloric Acid Salt (13). To a three-neck 250 mL
round-bottom flask equipped with mechanical stirrer was
charged with compound 12 and 2-propanol (62 mL, 5 vol).
The solution was heated to 75 ( 5 °C and 5-6 N HCl solution in
2-propanol (12 mL, 3 equiv) was added slowly with vigorous
stirring. The resulting solution was stirred at the same tempera-
ture for 1 h and was cooled down to 23 ( 2 °C. The reaction
mixture was maintained at the same temperature for 1 h.
The precipitated solids were collected by filtration, washed with
2-propanol (36 mL, 3 vol), and dried to give the enantiomeri-
cally pure amine 13 (3.0 g, 75%, ER = 0:100) as a white solid.
HPLC (Method B) tR = 24.86 min. For the compound: 1H
NMR (500 MHz, DMSO) δ 8.07 (s, 1H), 7.97 (s, 3H), 6.25 (d,
1H, J = 5.0 Hz), 4.16 (d, 1H, J = 5.0 Hz), 2.67-2.70 (m, 1H),
1.33-1.46 (m, 4H), 0.84 (t, 3H, J=5.0 Hz), 0.61 (t, 3H, J=3.0
Hz), 0.53 (t, 3H, J = 3.0 Hz); 13C NMR (125 MHz, CDCl3) δ
171.70, 70.54, 52.37 (t, J=23.8 Hz), 29.07, 22.17, 18.04, 13.69,
5.34, and 5.30. HRMS calcd for C9H18DN2O2, 188.15038;
found, 188.15048. Deuterium content by LCMS/MS =99%.
(1S,3aR,6aS)-N-((R)-3-Deutero-1-(cyclopropylamino)-1,2-di-
oxohexan-3-yl)-2-((S)-2-((S)-2-cyclohexyl-2-(pyrazine-2-carbox-
amido)acetamido)-3,3-dimethylbutanoyl) Octahydrocyclopenta-
[c]pyrrole-1-carboxamide, 1. CH2Cl2 (6 vol) was charged into a
300 mL, three-neck flask and cooled to 0-5 °C. (1S,3aR,6aS)-
2-((S)-2-((S)-2-cyclohexyl-2-(pyrazine-2-carboxamido)acetamido)-3,
3-dimethylbutanoyl)octahydrocyclopenta[c]pyrrole-1-carboxylic
acid 1429 (10.0 g, 1.0 equiv), HOBt hydrate (2.85 g, 1.1 equiv),
EDCI (3.53 g, 1.1 equiv), and the chiral amine 13 (4.12 g,
1.1 equiv) were charged to the same flask in that order. NMM
(3.68 mL, 2.0 equiv) was added over 10 min while maintaining the
reaction temperature below 5 °C. The reaction mixture was
warmed up 20-25 °C over 30 min and stirred for an additional
6 h. The reaction completion of compound 15 was monitored
by HPLC. The reaction mixture was washed with water (5 vol), 1
N HCl (5 vol), and 5 wt % aq NaHCO3 (5 vol) to yield a solution
of (1S,3aR,6aS)-N-((R)-3-deutero-1-(cyclopropylamino)-2-hydroxy-
1-oxohexan-3-yl)-2-((S)-2-((S)-2-cyclohexyl-2-(pyrazine-2-carbo-
xamido)acetamido)-3,3-dimethylbutanoyl) octahydrocyclopenta[c]-
pyrrole-1-carboxamide 15 in CH2Cl2.
TEMPO (0.25 g, 0.06 equiv) was added to the above alcohol 15
solution in CH2Cl2 and stirred at 20-25 °C until all TEMPO was
dissolved. To this solution a solution of NaHCO3 (1.5 equiv) in
water (4 vol) was added. The resulting biphasic mixture was cooled
to 0-5 °C. While maintaining the reaction temperature at 0-5 °C,
a 10-13 wt % NaOCl solution (20.0 g, 1.1 equiv) was added over
30 min and stirred for an additional 1 h. The layers were separated
and the organic layer was washed with H2O (5 vol), 1% Na2SO3
(5 vol), and H2O (5 vol) at 0-5 °C. Glacial acetic acid (0.12 equiv)
was added to the solution of 1 in CH2Cl2. The organic layer is
reduced to 3 vol by vacuum distillation at less than 20 °C. After
distillation, the solution was brought up to 40 °C. EtOAc (0.8 vol)
was added, followed by the addition of 1. The resulting mixture
was stirred for 15 min at 40 °C. EtOAc (8 vol) was added to the
mixture while maintaining a temperature at 40 °C. The total
volume of the slurry was then reduced to 4 vol by distillation at
30-40 °C. The resulting slurry was then cooled down to 25 °C over
1 h and stirred for an additional 1 h. The slurry was filtered. The
filter cake was washed with EtOAc (3 vol), dried under vacuum
with nitrogen bleed at 45 °C for 10 h, and cooled down to 20-25 °C
Stability Kinetics in Plasma. The analytes (1 or 2) were
incubated at 37 °C with rat, dog, and human plasma at final
concentrations of 1 or 10 μM. The concentrations of analytes in
plasma were monitored at various time points over time.
Specifically, 495 μL aliquots of rat, dog, and human plasma
were preincubated in cluster tubes in a shaking 37 °C water bath.
The 5 μL volumes of 0.1 and 1.0 mM stock solutions of 1 and 2
were added to the aliquots. The final concentrations were 1 and
10 μΜ of 1 and 2. All incubations were conducted in triplicates
in a shaking 37 °C water bath. Control incubations were con-
ducted in 0.1 M KPO4 buffer. Incubation times were 0, 30, 60,
and 120 min for rat and dog, and 0, 30, 60, 120, and 180 min for
buffer and human plasma. At the end of the incubation, 100 μL
volumes were transferred to cluster plates placed on dry ice and
flash frozen. The plates were sealed and stored in -80 °C freezer
until extraction. This procedure was followed for all concentra-
tions of 1 and 2 (1 and 10 μM) investigated using buffer and
plasma samples from all species investigated.
Sample Extraction and Preparation for LC-MS Analysis.
Prior to thawing the samples, 5 μL formic acid and 10 μL of
internal standard solution were added to all the samples, followed
by 1200 μL ethyl acetate, using a Quadra 3 multichannel pipetter
(TomTec, Hamden CT). The plate was then covered tightly and
vortexed for 20 min and centrifuged at 3000 rpm for 10 min. After
centrifugation, 900 μL of supernantant was transferred to a V-
bottomed 96 deep-well plate. The solution was then dried under
nitrogen gas at flow rate of 60 L/min at 25 °C for about 30 min.
The contents of the plate were then reconstituted with 100 μL of
ethyl acetate. The reconstituted solution was transferred into a
96-well plate containing glass inserts. The plate was covered and
20 μL was injected into LCMS/MS.
Instrumentation
All sample analysis was carried out on an API 4000 mass
spectrometer from Applied Biosystems/MDS Sciex (Applied

8000 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 24
Maltais et al.
Biosystems, Foster City, CA) equipped with Turbo V sources
and TurboIonspray interface. The HPLC system consisted of
an Agilent series 1100 binary pump (Agilent Technologies,
Palo Alto, CA). The auto sampler was a CTC Analytics PAL
autosampler (CTC Analytics AG, Zwingen, Switzerland). All
instrumentations were controlled and synchronized by Ana-
lyst software (version 1.4.1) from Applied Biosystems/MDS
Sciex.
Liquid Chromatographic and Mass Spectrometric Meth-
ods. Chiral separation was accomplished on a ChiralPak
AD column (Chiral Technologies, Inc., West Chester, PA).
The mobile phase consisted of two solvents, solvent A
(2-propanol) and solvent B (n-heptane). The gradient was
maintained at 10% A for 0.2 min, followed by a linear
increase to 50% A in 5 min, and kept at 50% B for 6 min,
and then the gradient was decreased to 10% A in 0.01 min.
The column was then equilibrated at 10% A for 9.99 min.
The total run time for each injection was 20 min. The flow
rate was 0.7 mL/min. The injection volume was 20 μL. To
improve the signal, a makeup solvent consisting of 0.2%
formic acid in water was added at a flow rate of 0.1 mL/min
post column.
The MRM parameters were optimized by infusing the
standard solutions of test compound and internal standard
in the mixture of 0.1% formic acid in 50% acetonitrile with
water. The MRM transitions for 2 was 680.36 > 322.30; 1
was 681.36 > 323.30; and the internal standard of isotope
labeled 2 with 11 deuterium atoms was 691.36 > 322.30. The
dwell time of each transition was 150 ms.
Pharmacokinetics in Rat. Male Sprague-Dawley rats (six/
dose group; weight range 275-300 g) from Charles River
Laboratories with preimplanted jugular and carotid can-
nulas were dosed with 10 mg/kg of 2 or 10 mg/kg of 1 by the
oral route. The dosing vehicle contained 1 mg/mL of 1 or 2 in
a vehicle made from a rotovapped solid dispersion contain-
ing 49.5% 2, 49.5% HPMC-AS, and 1% SLS, which was
dissolved in 1% HPMC-AS and 10% vitamin E TPGS. The
dosing volume was 10 mL/kg.
blood by centrifugation (Eppendorf model 5415D centri-
fuge, 2 min at 16100 rpm) within 30 min after sample
collection. A total of 100 μL of the resulting plasma was
combined with 5 μL of 10% formic acid (Fluka, Germany).
The acidified plasma sample was frozen at approximately
-60 °C until analysis with the LC/MS/MS.
Pharmacokinetic parameters were determined using plas-
ma concentration-time profiles and tissue concentration-
time profiles of 1 and 2 in rats at scheduled (nominal)
sampling times with non compartmental pharmacokinetic
methods using WinNonlin Professional Edition software,
version 5.1.1 (Pharsight Corporation, Mountain View, CA).
Acknowledgment. We would like to thank Nigel Ewing
and Wojciech Dworakowski for help in generating the sup-
porting data, and Dr. Franc-oise Berlioz-Seux, Dr. Lindsay
ꢀ
McNair, Dr. Valerie Philippon, and Sarah Cowherd from
Vertex Pharmaceuticals for their input and editorial help in
preparing this manuscript.
Supporting Information Available: Analytical data of com-
pounds 1 and 5-13. This material is available free of charge via
the Internet at http://pubs.acs.org.
References
(1) (a) Memon, M. I.; Memon, M. A. Hepatitis C: An epidemiological
review. J. Virol. Hepat. 2002, 9, 84–100.(b) also see: http://www.who.
int/vaccine_research/diseases/viral_cancers/en/index2.html.
(2) Fried, M. W.; Shiffman, M. L.; Reddy, K. R.; Smith, C.; Marinos,
G.; Goncales, F. L., Jr.; Haussinger, D.; Diago, M.; Carosi, G.;
Dhumeaux, D.; Craxi, A.; Lin, A.; Hoffman, J.; Yu, J. Peginter-
feron R-2a plus Ribavirin for chronic hepatitis C virus infection.
N. Engl. J. Med. 2002, 347, 975–982.
(3) Strader, D. B.; Wright, T.; Thomas, D. L.; Seeff, L. B. Diagnosis,
management, and treatment of hepatitis C. Hepatology 2004, 39,
1147–1171.
(4) Kolykhalov, A. A.; Mihalik, A. K.; Feinstone, S. M.; Rice, C. M.
Hepatitis C virus encoded enzymatic activities and conserved RNA
elements in the 30 nontranslated region are essential for virus
replication in vivo. J. Virol. 2000, 74, 2046–2051.
(5) Burton, J. R., Jr.; Everson, G. T. HCV NS5B poymerase inhibitors.
Two additional groups of male Sprague-Dawley Rats
(six/dose group; weight range 230-270 g) from Charles
River Laboratories with preimplanted jugular and carotid
cannulas were dosed with 1 mg/kg of 2 or 1 mg/kg of 1 by
the intravenous route. The dosing vehicle contained 1 mg/
mL of 1 or 2, which was dissolved in 10% dimethyl iso-
sorbide, 15% ethanol, 40% propylene glycol, and 35% D5W
(dextrose 5% in water). The intravenous formulation was
filtered using a Millex-LG filter (Millipore Corporation,
Bedford, MA) and injected through the preimplanted jugu-
lar cannula.
A fifth group of six male Sprague-Dawley rats (weight
range 250-275 g) from Charles River Laboratories with
preimplanted jugular and carotid cannulas were dosed with
10 mg/kg of 1 and 2 in a 50/50 mixture by the oral route. The
dosing vehicle contained 0.5 mg/mL of 2 and 0.5 mg/mL 1 in
a vehicle made from a rotovapped solid dispersion contain-
ing 49.5% 2, 49.5% HPMC-AS, and 1% SLS, which was
dissolved in 1% HPMC-AS and 10% vitamin E TPGS. The
dosing volume was 10 mL/kg.
Clin. Liver Dis. 2009, 13, 453–465.
(6) Kronenberger, B.; Zeuzem, S. Current and future treatment op-
tions for HCV. Ann. Hepatol. 2009, 8, 103–112.
(7) Beaulieu, P. L. Recent advances in the development of NS5B
polymerase inhibitors for the treatment of hepatitis C virus infec-
tion. Expert Opin. Ther. Pat. 2009, 19, 145–164.
(8) Reiser, M.; Timm, J. Serine protease inhibitors as anti-hepatitis C
virus agents. Expert Rev. Anti-Infect. Ther. 2009, 7, 537–547.
(9) Chen, K. X.; Njoroge, F. G. A review of HCV protease inhibitors.
Curr. Opin. Invest. Drugs 2009, 10, 821–837.
(10) Perni, R. B.;Almquist, S. J.;Byrn, R. A.;Chandorkar, G.; Chaturvedi,
P. R.; Courtney, L. F.; Decker, C J.; Dinehart, K.; Gates, C. A.;
Harbeson, S. L.; Heiser, A.; Kalkeri, G.; Kolaczkowski, E.; Lin, K.;
Luong, Y.-P.; Rao, B. G.; Taylor, W. P.; Thomson, J. A.; Tung, R. d.;
Wei, Y.; Kwong, A. D.; Lin, C. Preclinical profile of telaprevir,
a potent, selective, and orally bioavailable inhibitor of hepatitis C
virus NS3-4A serine protease. Antimicrob. Agents Chemother. 2006, 50,
899–909.
(11) Reesink, H. W.; Zeuzem, S.; Weegink, C. J.; Forestier, N.;
Van Vliet, A.; De RooiJ, J. J. V. D. W.; McNair, L.; Purdy, S.;
Kauffman, R.; Alam, J.; Jansen, P. L. M. Rapid decline of viral
RNA in hepatitis C patients treated with telaprevir: A phase Ib,
placebo-controlled, randomized study. Gastroenterology 2006, 131,
997–1002.
(12) Forestier, N.; Reesink, H. W.; Weegink, C. J.; et al. Antiviral
activity of telaprevir and peginterferon R-2a in patients with
hepatitis C. Hepatology 2007, 46, 640–648.
(13) Lawitz, E.; Rodriguez-Torres, M.; Muir, A. J.; et al. Antiviral
effects and safety of telaprevir, peginterferon R-2a, and riba-
virin for 28 days in hepatitis C patients. J. Hepatol. 2008, 49,
163–169.
Blood samples were collected from each rat at 0 (predose),
0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 12, and 24 h after oral dosing. Blood
samples (0.250 mL) were collected using the Instech Auto-
mated Blood Samples (Model ABS11, Instech Laboratories,
Plymouth Meeting, PA) into EDTA containing tubes that
were maintained at 4 °C. The plasma was isolated from the
(14) Kieffer, T. L.; Sarrazin, C.; Miller, J. S.; Welker, M. W.; Forestier,
N.; Reesink, H. W.; Kwong, A. D.; Zeuzem, S. Telaprevir and

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 24 8001
pegylated interferon-R-2a inhibit wild-type and resistant genotype
isotope effects, and rate-limiting steps. J. Biol. Chem. 2005, 280,
19496–19506.
(22) Cleland, W. W. The use of kinetic isotope effects to determine
enzyme mechanism. Arch. Biochem. Biophys. 2005, 433, 2–12.
(23) Klinman, J. P. Primary kinetic isotope effects. In Transition states
of biochemical processes; Gandour, R. D., Schowen, R. L., Eds.;
Plenum Press: New York, NY, 1978.
(24) Tanoury, G. J.; Chen, M.; Jung, Y. C.; Forslund, R. E. Process for
preparation of R-amino-β-hydroxycarbonyl compounds. PCT Int.
Appl. WO 2007109023 A1, 2007.
(25) Cleland, W. W.; O’Leary, M. H.; Northrop, D. B.; Eds. In Iso-
tope effects on enzyme-catalyzed reactions; University Park Press:
Baltimore, MD, 1977.
(26) Schneider, F.; Hillgenberg, M.; Koytchev, R.; Alken, R.-G.
Enhanced plasma concentration by selective deuteration of rofe-
coxib in rats. Arzneim. Forsch./Drug Res. 2007, 56, 295–300.
(27) Tanabe, M.; Yasuda, D.; LeValley, S.; Mitoma, C. The pharma-
cologic effect of deuterium substitution on 5-n-butyl-5-ethylbutyric
acid. Life Sci. 1969, 8, 1123–1128.
1 hepatitis C virus replication in patients. Hepatology 2007, 46,
631–639.
(15) Sarrazin, C.; Kieffer, T. L.; Bartels, D.; Hanzelka, B.; Muh, U.;
Welker, M.; Wincheringer, D.; Zhou, Y.; Chu, H.-M.; Lin, C.;
Weegink, C.; Reesink, H.; Zeuzem, S.; Kwong, A. D. Dynamic
hepatitis C virus genotypic changes in patients treated with the
protease inhibitor Telaprevir. Gastroenterology 2007, 132, 1767–
1777.
(16) Poordad, F.; Shiffman, M.; Sherman, K.; Smith, J.; Yao, M.;
George, S.; Adda, N.; McHutchison, J. A study of teleprevir
with peginterferon R-2A (P) and ribavirin (R) in subjects
with well-documented prior P/R null response, non-response
or relapse: preliminary results. J. Hepatol. 2008, 48, Supplement 2,
S374-S375.
(17) Melander, L.; Saunders, W. H. Reaction Rates of Isotopic Mole-
cules; Wiley: New York, NY, 1980.
(18) Blake, M. I.; Crespi, H. L.; Katz, J. J. Studies with deuterated
drugs. J. Pharm. Sci. 1975, 64, 367–391.
(19) Hall, L. H.; Hanslik, R. P. Kinetic deuterium isotope effects on the
N-demethylation of tertiary amides by cytochrome P-450. J. Biol.
Chem. 1990, 265, 12349–12355.
(20) Nelson, S. D.; Tragger, W. F. The use of deuterium isotope effects
to probe the active site properties, mechanism of cytochrome
P-450-catalyzed reactions, and mechanisms of metabolically de-
pendent toxicity. Drug Metab. Dispos. 2003, 31, 1481–1498.
(21) Krauser, J. A.; Guengerich, F. P. Cytochrome P450 3A4-catalyzed
testaterone 6b-hydroxylation stereochemistry, kinetic deuterium
(28) Scheider, F.; Mattern-Dogru, E.; Hillgenberg, M.; Alen, R.-G.
Changed phsophodiesterase selectivity and enhanced in vivo
efficacy by selective deuteration of sildenafil. Arzneim. Forsch./
Drug Res. 2007, 57, 293–298.
(29) Synthesis: Yip, Y.; Victor, F.; Lamar, J.; Johnson, R.; Wang,
Q. M.; Glass, J. I.; Yumibe, N.; Wakulchik, M.; Munroe, J.; Chen,
S.-H. P4 and P10 optimization of bicycloproline P2 bearing tetra-
peptidyl a-ketoamides as HCV protease inhibitors. Bioorg. Med.
Chem. Lett. 2004, 14, 5007-5011.