Design and synthesis of deuterated boceprevir analogs with enhanced pharmacokinetic properties

Article abstract of DOI:10.1002/jlcr.1905

As part of an ongoing effort to apply the Deuterated Chemical Entity Platform (DCE Platforma) to clinically validated drugs, the synthesis of deuterated analogs of the hepatitis C virus protease inhibitor boceprevir was carried out. The devised synthetic routes allowed for site-selective deuterium incorporation with high levels of isotopic purity. Application of the DCE Platforma to boceprevir enabled the identification of several deuterated analogs that display marked levels of in vitro metabolic stabilization. Most notably, analog 1g exhibits a near doubling of in vitro half-life in human liver microsomal assays. The details of the convergent synthetic route to the boceprevir isotopologs and the results of the metabolic stability assays are described herein.

Full text of DOI:10.1002/jlcr.1905

Research Article
Received 11 February 2011,
Revised 12 April 2011,
Accepted 11 May 2011
Published online 19 July 2011 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.1905
Design and synthesis of deuterated
boceprevir analogs with enhanced
pharmacokinetic properties
Adam J. Morgan, Sophia Nguyen, Vinita Uttamsingh, Gary Bridson,
*
Scott Harbeson, Roger Tung, and Craig E. Masse
As part of an ongoing effort to apply the Deuterated Chemical Entity Platform (DCE Platform™) to clinically validated drugs,
the synthesis of deuterated analogs of the hepatitis C virus protease inhibitor boceprevir was carried out. The devised
synthetic routes allowed for site‐selective deuterium incorporation with high levels of isotopic purity. Application of the
DCE Platform™ to boceprevir enabled the identification of several deuterated analogs that display marked levels of in vitro
metabolic stabilization. Most notably, analog 1g exhibits a near doubling of in vitro half‐life in human liver microsomal
assays. The details of the convergent synthetic route to the boceprevir isotopologs and the results of the metabolic stability
assays are described herein.
Keywords: deuteration; DCE Platform; boceprevir; HCV NS3 protease; KIE
larger percentage of unmetabolized drug reaching the systemic
Introduction
circulation, whereas the overall rate of systemic clearance remains
The deuterium kinetic isotope effect (KIE) has the potential to
unchanged. Deuterated drugs showing this effect may have
alter the biological fate of many drug molecules that are
reduced dosing requirements and produce lower metabolite loads.
metabolized through pathways involving carbon–hydrogen
Because, in many instances, gastrointestinal irritation has been
bond cleavage.1 Within biological systems, numerous compet-
shown to be a dose‐dependent rather than a plasma concentration‐
ing effects can mask the deuterium KIE such that the observed
dependent phenomenon, this effect could allow for enhanced
magnitude and even direction of the KIEs are unpredictable and
tolerability and/or the ability to achieve a higher maximum
depend on a compound’s molecular skeleton, substituents,
tolerated dose. The third panel in Figure 1 illustrates metabolic
shunting effects in cases where a drug is metabolized to form both
specific substitution pattern, and the enzymes responsible for
metabolism.2 As a consequence of the KIE, site‐selective
active and toxic metabolite species. In such a case, the deuterium
deuteration of compounds can result in altered metabolism
KIE can result in the reduced formation of a toxic or reactive
patterns including reduced metabolic rates and metabolic
metabolite as well as the increased formation of desirable active
shunting effects.3 Our research has shown that careful applica-
metabolites. Each of the above cases highlights precision deuterium
tion of deuterium medicinal chemistry as part of the Deuterated
incorporation as a powerful medicinal chemistry tool capable of
Chemical Entity Platform (DCE Platform™) has the unique
benefit, in select cases, of allowing for metabolic optimization
generating deuterated chemical entities (DCEs) with superior
therapeutic properties.
of a compound while preserving the biochemical potency and
Hepatitis C virus (HCV) infection is the major cause of chronic
selectivity of the agent.
Some important pharmacological effects of deuteration,
observed in certain cases, can be classified as illustrated in
Figure 1. The first panel illustrates the case where the major
effect of deuteration is to reduce the rate of systemic clearance.
This results in an increase of the biological half‐life of the
compound. Potential drug benefits could include a reduction in
dosage and the ability to maintain similar systemic exposures
liver disease, leading to cirrhosis, hepatocellular carcinoma, or
liver failure.4 It is estimated that at least 170 million people
worldwide are infected by this virus.4 Currently, the most
effective therapeutic regimen involves treatment with pegylated
α‐interferon in combination with the antiviral agent ribavarin.5
However, even this combination treatment is effective in only
40% of patients with genotype 1 HCV, the most prevalent
genotype in the United States, Japan, and Europe.6
with decreased peak levels (Cmax) and enhance trough levels.
This could result in a lower incidence of Cmax‐associated side
effects and enhanced efficacy, depending on the particular
Concert Pharmaceuticals, Lexington, MA 02421, USA
drug’s pharmacokinetic–pharmacodynanic relationship. The
second panel in Figure 1 illustrates a largely pre‐systemic (or
first‐pass) effect of deuteration. In such a case, reduced rates of
*Correspondence to: Craig E. Masse, Ph.D., 25 First Street, Suite 303, Cambridge,
MA 02141, USA.
E‐mail: cmasse@nimbusdiscovery.com
oxidative metabolism in the gut wall and/or liver result in a
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A. J. Morgan et al.
Figure 1. (A) Reduced systemic clearance resulting in increased half‐life. (B) Decreased pre‐systemic metabolism resulting in higher bioavailability of unmetabolized
drug. (C) Metabolic shunting resulting in reduced exposure to undesirable metabolites or increased exposure to desired active metabolites.
Boceprevir (1) contains an α‐ketoamide electrophilic warhead
Preparation of deuterated P2 synthon 13a14 is outlined in
that reversibly binds to the NS3 serine protease, which plays a Scheme 3. Diphenylethylsulfonium tetrafluoroborate‐d3 (8) was
vital role in the replication of the HCV virus.7 Boceprevir has obtained via treatment of diphenylsulfide with 2,2,2‐iodoethane‐
shown dramatic viral responses in the clinic when administered d3 in the presence of silver tetrafluoroborate.15 Deprotonation of 8
with pegylated α‐interferon and ribavarin, and is currently under with LDA followed by the addition of iodomethane‐d3 allowed for
expedited review in the United States and European Union as a the in situ generation of the diphenylisopropylsulfonium‐d6 salt.
new treatment option for patients living with chronic hepatitis An additional equivalent of LDA served to generate the sulfonium
C.8 The present work is aimed at producing a DCE with ylide, which added to the less hindered exo face of enamide 9 to
enhanced pharmacokinetic properties that may improve the provide dimethylcyclopropane‐d6 10 as a single diastereomer.16,17
antiviral efficacy of boceprevir by raising the trough levels of the It is important to note that when diphenylethylsulfonium
drug. Clinical studies have demonstrated that viral load tetrafluoroborate‐d5 (prepared from iodoethane‐d5) was em-
reductions are positively correlated with boceprevir trough ployed in the cyclopropanation reaction, significantly lower yields
concentrations.9 An enhanced pharmacokinetic profile may also were observed. This is believed to result from a dramatic KIE during
allow for a more convenient dosing regimen over boceprevir’s one or both of the LDA deprotonation events.
three‐times‐daily commercial dosing schedule.10
With dimethylcyclopropane‐d6 10 in hand, removal of the
benzylidene N,O‐acetal was accomplished with LiAlH4, affording
N‐benzyl aminoalcohol‐d6 11 in high yield.18 Palladium‐catalyzed
transfer hydrogenolysis followed by treatment with di‐tert‐butyl
dicarbonate provided the N‐Boc‐aminoalcohol‐d6 12. Oxidation
of the primary alcohol directly to the carboxylic acid under
Results and discussion
Chemistry
The retrosynthetic analysis, outlined in Scheme 1, depicts the key Sharpless conditions19 followed by esterification and subse-
bond‐forming reactions and required synthons for the prepara- quent N‐Boc deprotection ultimately afforded the deuterated P2
tion of deuterated analogs 1a–1g.11 As illustrated, individual synthon 13a.
target compounds arise from a series of peptide coupling
Scheme 4 illustrates the preparation of the N‐Boc‐cyclobutylmethyl
reactions involving selectively deuterated intermediates. The hydroxyamide synthons 25a and 25b from 1,3‐dibromopropane‐
syntheses culminate with the coupling of hydroxyamide synthons d6.14 Cyclobutane dicarboxylic acid‐d6 (14) was obtained in a
25a–25c with appropriately deuterated tert‐butylurea intermedi- two‐step sequence involving alkylation and intramolecular
ates 28a–28d followed by subsequent oxidation to afford cyclization20 of diethyl malonate with 1,3‐dibromopropane‐d6
ketoamides 1a–1g.
followed by saponification of the resulting diester. Decarboxyl-
As shown in Scheme 1, the proposed route relies heavily upon the ation in D2O at 160 °C in a sealed tube generated cyclobutane
use of site‐selectively deuterated synthons, each requiring individual carboxylic acid‐d7 (15), which was subsequently reduced with
preparation. The synthesis of the deuterated P3 subunit 7a (7b LiAlH4, affording cyclobutylmethanol‐d7 (16a), or with LiAlD4,
purchased from Chem Impex Intl.), is outlined in Scheme 2, and affording cyclobutylmethanol‐d9 (16b) (The remainder of the
begins with the formation of trimethylacetaldehyde‐d9 (2) from synthesis was completed in both the d7(a) and d9(b) series to
tert‐butyl chloride‐d9.12 An asymmetric Strecker with aldehyde 2 obtain hydroxyamides 25a and 25b, respectively.) Conversion to
using an (R)‐phenylglycine amide auxiliary in the presence of sodium tosylate 17 followed by subsequent treatment with lithium
cyanide and acetic acid afforded aminonitrile 3 as a single diaste- bromide provided (bromomethyl)cyclobutane 18. Alkylation of
reomer.13 Subsequent exposure of aminonitrile 3 to sulfuric acid 19 with bromide 18 afforded rac‐cyclobutylalanine derivative 20.
afforded bisamide 4, which was then converted to α‐aminoamide Subsequent imine hydrolysis21 and Boc protection delivered rac‐
5 under palladium‐catalyzed hydrogenolysis conditions. Exposure N‐Boc‐cyclobutylalanine t‐butyl ester 21. Reduction of t‐butyl
of 5 to 6 N HCl provided amino acid 6 as its hydrochloric acid salt, ester 21 with diisobutylaluminum hydride22 afforded aldehyde 22,
which was subsequently protected with di‐tert‐butyl dicarbonate, which, upon treatment with acetone cyanohydrin, was converted
to afford N‐Boc‐tert‐leucine‐d9 (7a) in six overall steps.
to cyanohydrin 23. The cyclobutylmethyl hydroxyamide synthon
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A. J. Morgan et al.
Scheme 1. General retrosynthetic strategy to deuterated chemical entities.
25 was then obtained via treatment of 23 with lithium hydroxide hydroxyamide intermediate 24b to afford keto‐amide 29. Sub-
and hydrogen peroxide, affording N‐Boc‐amide 24, followed by sequent coupling with carboxylic acid fragments 28a and 28d
HCl‐mediated Boc deprotection.
afforded final compounds 1b and 1g, respectively. Similar yields
Final compounds 1a–1g were ultimately assembled following have been observed for both strategies, and both methodologies
the general route depicted in Scheme 5, employing the provide final compounds 1 in high chemical and isotopic purities.
appropriately deuterated synthons as needed.11 Coupling of N‐
Boc‐tert‐leucine 7a or 7b with dimethylcyclopropyl proline 13a or
13b was accomplished with BOP reagent to afford Boc‐protected
In vitro metabolic stability
dipeptides 26a–26d.14 Deprotection of the tert‐butyl carbamate In an effort to compare the in vitro metabolic stability of 1a–1g
followed by exposure to tert‐butyl isocyanate 27a23 or 27b with that of boceprevir, the compounds were incubated in human
provided tert‐butyl ureas 28a–28d.14 Ester hydrolysis followed by liver microsomes for a period of 30 min. Monitoring the percent
EDC‐mediated coupling with hydroxyamide 25a–25c and sub- parent remaining for each compound at various time points
sequent oxidation under modified Moffatt conditions24 afforded allows for the determination of in vitro t1/2 and, ultimately, the
the deuterated boceprevir analogs 1a–1g.
average percent change in t1/2 (%Δt1/2) relative to boceprevir
An alternative strategy for incorporation of the keto‐amide (Figure 2). As illustrated in Figure 2 and Table 1, deuteration of the
subunit into the final compounds is shown in Scheme 6.25 This P2 subunit alone seems to have little effect on in vitro metabolic
approach involved oxidation and subsequent Boc deprotection of stability, as compound 1d exhibits a t1/2 similar to that of
Scheme 2. Synthesis of P3 fragment (7a).
J. Label Compd. Radiopharm 2011, 54 613–624
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A. J. Morgan et al.
Scheme 3. Synthesis of P2 fragment (13a).
boceprevir. This may result from a masking of the KIE as a result of Labeling chemistry
metabolic switching away from the P2 site on the molecule.2,3
Trimethylacetaldehyde‐d9 (2)
However, deuteration elsewhere within the molecule seems to
play a more crucial role, and certain metabolic “hot spots” can be
theorized. For instance, the analogs containing deuterium solely
within the P1 subunit (1a and 1b) displayed significant stability
enhancement, with %Δt1/2 = 40% and 51%, respectively. Addi-
tional metabolic stabilization was achieved by site‐selective
deuteration of multiple subunits as highlighted by analog 1g
exhibiting a t1/2 nearly double that of boceprevir. These results
may be indicative of diffuse metabolism at multiple sites within
the parent scaffold. In such cases, the full potential of deuterium‐
mediated stabilization may only be realized through iterative
labeling of several moieties within the compound of interest.
In a 3‐L 4‐necked round bottom flask fitted with mechanical
stirrer, reflux condenser, dropping funnel, and thermometer was
placed a few small crystals of iodine and then magnesium
turnings (24.7 g, 1.03 mol). The bottom of the flask was heated
with a heat gun until the iodine began to vaporize and was then
allowed to cool, and a solution of t‐butyl chloride‐d9 (100.0 g,
1.03 mol, Cambridge Isotope Labs, 98 atom %D) in anhydrous
ether was placed in the dropping funnel. A small amount of the
solution of t‐butyl chloride‐d9 in ether (3–5 mL) was added
directly to the dry magnesium. A large amount of anhydrous ether
(1 L) and a few small crystals of iodine were added, and the
resulting mixture was heated for 0.5 h to initiate the reaction. The
rest of the solution of t‐butyl chloride‐d9 in ether was added with
stirring at a rate not faster than one drop per second. The mixture
was allowed to reflux during the addition, and no external cooling
was applied. The reaction mixture was then heated at reflux for
several hours until almost all of the magnesium disappeared. The
mixture was then cooled to −20 °C, and a solution of anhydrous
DMF (73.0 g, 1.00 mol) in ether (100 mL) was added over a 35‐min
period at such a rate that the temperature of the reaction did not
exceed −15 °C. A second solution of anhydrous DMF (73.0 g,
1.00 mol) was then quickly added at −8 °C. After an additional
5 min, hydroquinone (0.5 g) was added, stirring was stopped, the
cooling bath was removed, and the mixture was left standing
overnight at ambient temperature under nitrogen. The mixture
was then cooled to 5 °C, and aqueous 4 M HCl solution (600 mL)
was added in portions to quench the reaction. The mixture was
diluted with water (400 mL), and the layers were separated. The
aqueous layer was extracted with ether (3 × 200 mL), and the
combined organic layers were dried with MgSO4 and filtered.
The filtrate was subjected to fractional distillation under an atmo-
sphere of nitrogen to remove most of the ether. The residue was
transferred to a small flask, and fractional distillation was
continued to collect aldehyde 2 at 65–75 °C (39.5 g, 40% yield)
as a colorless oil. Compound 2 was stored under nitrogen in the
freezer and used without further purification.
Experimental
General
Nuclear magnetic resonance (NMR) spectra were recorded on a
Bruker Avance 400 spectrometer, operating at 400 MHz for 1H and
100 MHz for 13C and with DMSO‐d6 as solvent unless otherwise
stated. Chemical shifts were reported in parts per million relative
to tetramethylsilane, and J values were reported in hertz.
Multiplicities were reported using the following abbreviations: s,
singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad.
Low‐resolution mass spectra were collected on an Agilent 1100
Series LC/MSD (column: 20 mm C18‐RP 5–95% acetonitrile
(ACN) + 0.1% HCO2H/H2O + 0.1% HCO2H in 7 min with a 2‐min
hold at 95% acetonitrile (ACN) + 0.1% HCO2H/H2O + 0.1% HCO2H;
MSD: single quadrupole LC‐MS (Agilent 6120) mass spectrometer
using ESI in positive or negative mode). Optical rotation data were
collected on a Perkin Elmer model 343 polarimeter at normal
aperture and ambient temperature with a 100 mm path length cell.
All reagents were purchased from commercial sources (Aldrich,
Acros, Chem Impex Intl., Enamine Ltd., Alfa Aesar, CDN Isotopes,
and Cambridge Isotope Labs) unless otherwise specified. The
isotopic purity of commercial deuterated reagents has been noted
in each experimental procedure. Maintenance of isotopic integrity
was monitored using 1H NMR and MS techniques throughout the
synthetic sequences to the final compounds (1a–1g). Column
chromatography was carried out on an ISCO CombiFlash® Rf system
employing ISCO silica gel column cartridges. Human liver
microsomes (20 mg/mL) were obtained from Xenotech, LLC
(Lenexa, KS). β‐Nicotinamide adenine dinucleotide phosphate,
reduced form (NADPH), magnesium chloride (MgCl2), and dimethyl
sulfoxide (DMSO) were purchased from Sigma‐Aldrich.
(R)‐2‐(((S)‐1‐Cyano‐2,2‐dimethylpropyl)amino)‐2‐phenylacetamide‐
d9 (3)
To a stirred suspension of (R)‐phenylglycine amide (60.7 g,
400 mmol) in water (400 mL) was added compound 2 (39.5 g,
415 mmol) at room temperature. Simultaneously, 30% aqueous
NaCN solution (68.8 g, 420 mmol) and glacial acetic acid (25.4 g,
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A. J. Morgan et al.
Scheme 4. Synthesis of P1 fragment (25).
423 mmol) were added in 30 min, whereby the temperature of 15–20 °C through an addition funnel. The resulting mixture was
the reaction increased to 34 °C. The mixture was stirred for 2 h at stirred at room temperature for 1 h. The mixture was then poured
30 °C, followed by stirring at 70 °C for 20 h. After cooling to 30 °C, on ice and carefully neutralized by NH4OH solution to pH 9. The
the product was isolated by filtration. The solid was washed with mixture was extracted with dichloromethane, and the combined
water (500 mL) and dried in vacuo at 50 °C to afford aminonitrile organic layers were washed with water, dried with Na2SO4,
3 (90.0 g, 88% yield) as a tan solid with [α]D = −298° (c = 1.0, filtered, and concentrated in vacuo to afford bisamide 4 (55.0 g,
CHCl3). MS (ESI) 255.3 [(M + H)+].
80% yield) as a yellow foam with [α]D = −140° (c = 1.0, CHCl3). MS
(ESI) 273.3 [(M + H)+].
(S)‐2‐(((R)‐2‐Amino‐2‐oxo‐1‐phenylethyl)amino)‐3,
3‐dimethylbutanamide‐d9 (4)
(S)‐2‐Amino‐3,3‐dimethylbutanamide‐d9 (5)
A solution of compound 3 (64.2 g, 252 mmol) in dichloromethane A mixture of compound 4 (77.0 g, 283 mmol), 10% Pd/C (~50%
(500 mL) at 0 °C was added to conc. sulfuric acid (96%, 350 mL) at water, 20 g), and acetic acid (50 mL) in ethanol (1.2 L) was
Scheme 5. Fragment assembly to afford isotopologs 1a–1g.
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A. J. Morgan et al.
Scheme 6. Alternative fragment coupling approach.
subjected to hydrogenation at 30 psi at room temperature for the crude material from EtOH afforded 8 as a white powder
several days until LC‐MS showed that the reaction was complete. (3.30 g, 86% yield). MS (ESI) 218.2 [(M)+].
The mixture was filtered through Celite® and washed with EtOAc.
(3R,5αS,6αS,6βS)‐3‐Phenyl‐6,6‐bis(trideuteromethyl)tetrahydro‐1H‐
After the filtrate was concentrated in vacuo, the residue was
cyclopropa[3,4]pyrrolo[1,2‐c]oxazol‐5(3H)‐one (10)
diluted with water (1L) and basified with 1 M NaOH solution to
To a solution of freshly prepared LDA (0.94 M) in THF (13.0 mL,
12.4 mmol) at −70 °C was added a solution of 8 (3.64 g,
11.9 mmol) in DME (24 mL). The solution was stirred at −60 °C
for a period of 30 min, followed by the addition of neat
iodomethane‐d3 (0.740 mL, 11.9 mmol, Aldrich, 99.5 atom %D).
The mixture was then warmed slowly to −50°C and stirred at −50 °C
for a period of 2 h. The reaction mixture was then re‐cooled
to −70 °C, and a solution of LDA (0.94 M) in THF (13.8 mL,
13.1 mmol) was added; the mixture was stirred at −70 °C for a
period of 1 h. To the stirred solution at −70 °C was then added
a solution of commercially available (3R,7αS)‐3‐phenyl‐1,7α‐
pH9. The mixture was extracted with dichloromethane, and the
aqueous layer was concentrated in vacuo to half volume, saturated
with solid NaCl, and extracted with THF. The combined extracts
were dried with Na2SO4, filtered, and concentrated in vacuo. The
resulting residue was diluted with toluene and subsequently
evaporated to remove the remaining water, followed by
trituration with dichloromethane to afford the aminoamide 5
(38.0 g, 96% yield) as a white solid, which was used without
further purification.
(S)‐2‐Amino‐3,3‐dimethylbutanoic acid‐d9 hydrochloride (6)
A mixture of compound 5 (31.0 g, 223 mmol) in 6 M aqueous HCl dihydropyrrolo[1,2‐c]oxazol‐5(3H)‐one (9; 0.960 g, 4.77 mmol,
solution (1.5 L) was heated at reflux for 24 h. The mixture was Enamine Ltd.) in DME (9 mL). The reaction mixture was stirred
concentrated in vacuo to give the crude product. The solid was for 1 h at −70 °C, followed by warming to −30 °C and stirring
redissolved in water (500 mL) and washed with EtOAc for an additional 2 h, at which point the mixture was diluted
(2 × 200 mL) to remove impurities from previous steps. The with saturated aqueous NaHCO3 (30 mL) and warmed to
aqueous layer was then concentrated in vacuo, diluted with ambient temperature. The mixture was extracted with Et2O
toluene, evaporated, and dried in vacuo at 50 °C to afford the HCl (3 × 25 mL) and the organic layers were dried with Na2SO4,
salt of the desired compound (S)‐2‐amino‐3,3‐dimethylbutanoic filtered, and concentrated in vacuo. Purification by column
acid‐d9 hydrochloride (6; 33.6 g, 85% yield) as a white solid, which chromatography (SiO2, 20–40% EtOAc/heptane) afforded 10
was used without further purification.
as a white solid (980 mg, 82% yield, 33% with respect to CD3I).
MS (ESI) 250.1 [(M + H)+].
N‐Boc‐tert‐leucine‐d9 (7a)
((1R,2S,5S)‐3‐Benzyl‐6,6‐bis(trideuteromethyl)‐3‐azabicyclo[3.1.0]
hexan‐2‐yl)methanol (11)
To a solution of compound 6 (1.00 g, 5.66 mmol) in a mixture of
dioxane (10 mL) and water (10 mL) was added triethylamine
(3.16 mL, 22.6 mmol) followed by di‐tert‐butyl dicarbonate To a solution of lactam‐d6 10 (376 mg, 1.51 mmol) in THF (3 mL)
(1.48 g, 6.79 mmol). The resulting mixture, stirred at room at 0 °C was added LiAlH4 (2 M in THF, 1.51 mL, 3.02 mmol). The
temperature for 6 h, was then washed with heptane reaction was heated to reflux for 3 h, then cooled to 0 °C, and
(2 × 20 mL). The aqueous fraction was cooled, with the pH quenched by dropwise addition of 10% aqueous KHSO4. The
adjusted to 2 with 1 M HCl, and extracted with ethyl acetate resulting slurry was diluted with ethyl acetate and filtered
(3 × 50 mL). The combined extracts were dried with Na2SO4, (washing the filter cake with ethyl acetate 2 × 10 mL). The
filtered, and concentrated in vacuo to afford Boc‐tert‐leucine‐d9 resulting solution was diluted with water (20 mL) and extracted
(7a, 1.10 g, 81% yield) as a yellow oil. MS (ESI) 239.2 [(M − H)−].
Diphenyl(2,2,2‐trideuteroethyl)sulfonium tetrafluoroborate (8)
with ethyl acetate (3 × 30 mL). The combined organic layers
were washed with brine, dried with MgSO4, and concentrated
in vacuo to afford alcohol‐d6 11 (350 mg, 98% yield). (ESI) 238.30
[(M + H)+].
To a flask containing 2,2,2‐iodoethane‐d3 (10.0 g, 62.9 mmol, CDN
Isotopes, 99 atom %D) was added diphenyl sulfide (2.32 mL,
13.8 mmol) and CH2Cl2 (15 mL). The mixture was cooled to 0 °C
and stirred vigorously. To the cooled mixture was added silver
(1R,2S,5S)‐tert‐Butyl‐2‐(hydroxymethyl)‐6,6‐bis(trideuteromethyl)‐3‐
azabicyclo[3.1.0]‐hexane‐3‐carboxylate (12)
tetrafluoroborate (2.45 g, 12.6 mmol) in one portion, and the To a solution of alcohol‐d6 11 (350 mg, 1.48 mmol) in methanol
mixture was warmed to ambient temperature over a period of (15 mL) was added ammonium formate (571 mg, 9.06 mmol)
24 h. The reaction mixture was then diluted with CH2Cl2 (100 mL) followed by 10% palladium on carbon (70 mg, 20 wt.%). The
and passed through a pad of Celite® to remove the precipitated reaction was then heated to reflux, taking precautions to limit
silver iodide. The filtrate was concentrated in vacuo, and the crude ammonium formate sublimation inside the condenser. After
oil triturated with Et2O to afford a white solid. Recrystallization of stirring at reflux for 2 h, the reaction was cooled to room
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A. J. Morgan et al.
120
100
80
60
Boceprevir (1)
1a
1b
1c
1d
1e
1f
40
20
1g
0
5
10
15
20
25
30
Time (min)
Figure 2. Metabolic stability of boceprevir and 1a–1g in human liver microsomes.
temperature and filtered through Celite®. The Celite® pad was ruthenium trichloride monohydrate (5.50 mg, 0.0252 mmol) and
washed with methanol (2× 10 mL) and then with dichloromethane sodium periodate (2.16 g, 10.0 mmol) in water (15 mL). After
(2 × 20 mL). The resulting solution was then concentrated in vacuo stirring at room temperature for 1 h, the reaction was filtered
to afford the desired amino alcohol‐d6. This material (~1.48 mmol) through Celite®. The Celite® pad was then washed with ethyl
was dissolved in dichloromethane (5mL), and triethylamine acetate (3 × 5 mL), and the resulting solution was concentrated to
(273 μL, 1.96mmol) was added followedbydi‐tert‐butyl dicarbonate dryness. The resulting residue was diluted with 1 M HCl (10 mL)
(428 mg, 1.96 mmol). The reaction was stirred at room temperature and extracted with ethyl acetate (3 × 20 mL). The organic layers
for 15 h, diluted with 1 M HCl (15 mL), and then extracted with were combined, washed with 1 M HCl, dried with Na2SO4, filtered,
dichloromethane (3 × 30 mL). The combined organic layers were and concentrated to afford a dark tan solid. The crude acid‐d6
washed with brine, dried with Na2SO4, filtered, and concentrated. (313 mg, 1.20 mmol) was dissolved in a mixture of benzene
The resulting residue was purified by column chromatography (5.0 mL) and methanol (0.50 mL), and
a 2 M solution of
(SiO2, 0–20% EtOAc/heptane) to afford alcohol‐d6 12 (311 mg, 83%, trimethylsilyl diazomethane in hexanes (780 μL, 1.56 mmol) was
2 step yield). (ESI) 270.2 [(M + Na)+].
added dropwise. The yellow solution was stirred at room
temperature for 15 h and was subsequently quenched by
dropwise addition of acetic acid until effervescence terminated.
The reaction was then concentrated in vacuo with several
repeated heptane dilutions/concentrations to remove excess
acetic acid. The resulting residue was then purified by column
chromatography (SiO2, 0–30% EtOAc/heptane) to afford pure
methyl ester‐d6 (154 mg). To this material was added a 4 M
solution of HCl in dioxane (5.0 mL), and the resulting solution
was stirred at room temperature for 2 h. The reaction was
then concentrated in vacuo to afford pure amine hydro-
chloride‐d6 13a (128 mg, 41%, 3 step yield) as a colorless
solid. (ESI) 176.2 [(M + H)+].
(1R,2S,5S)‐Methyl‐6,6‐bis(trideuteromethyl)‐3‐azabicyclo[3.1.0]
hexane‐2‐carboxylate hydrochloride (13a)
To a solution of alcohol‐d6 12 (311 mg, 1.26 mmol) in ethyl
acetate (10 mL) and ACN (10 mL) was added a solution of
Table 1. Metabolic stability of DCEs in human liver
microsomes
Ave t1/2 (min)a
Ave %Δt1/2
b
Compound
Boceprevir (1)
19.0 0.87
26.6 1.70
28.7 2.19
31.4 2.36
19.1 1.19
23.1 1.31
25.0 0.68
37.3 3.12
—
40
51
65
<10
22
1a
1b
1c
1d
1e
1f
2,2,3,3,4,4‐Hexadeuterocyclobutane‐1,1‐dicarboxylic acid (14)
A 21 wt% solution of sodium ethoxide in ethanol (24.1 mL,
64.2 mmol) was added dropwise to a stirred solution of 1,3‐
dibromopropane‐d6 (3.54 mL, 33.7 mmol, CDN Isotopes, 99 atom
%D) and diethylmalonate (4.87 mL, 32.1 mmol) in ethanol
(35 mL), maintaining a temperature of 60–65 °C. Upon comple-
tion of the addition, the reaction was cooled to ~50 °C and was
subsequently stirred at 100 °C until an aliquot added to water
was neutral on pH paper. The reaction was then cooled to room
temperature, diluted with water, and concentrated in vacuo to
remove ethanol. The resulting aqueous solution was extracted
with EtOAc (3 × 100 mL), dried with Na2SO4, filtered, and
concentrated. The resulting residue was purified by column
32
97
1g
aValues are average of four experiments performed on
separate days.
b%Δt1/2 was calculated according to the following equation:
%Δt1/2 = [(t1/2 DCE) − (t1/2 boceprevir)/(t1/2
boceprevir)] × 100%.
J. Label Compd. Radiopharm 2011, 54 613–624
Copyright © 2011 John Wiley & Sons, Ltd.
www.jlcr.org

A. J. Morgan et al.
chromatography (SiO2, 0–20% EtOAc/heptane) to afford the and filtered. Diethylether was then removed via careful
diethyl ester derivative of 14 (4.00 g, 60% yield). 1H NMR (CDCl3, distillation to afford bromide 18a as a clear oil containing
400 MHz) δ 4.20 (q, 2 H, J = 7.1 Hz), 1.25 (t, 3 H, J = 7.1 Hz). To a diethyl ether (2.02 g total, ~ 1.29 g of 11, ~64% by 1H NMR).
solution of this diester (4.00 g, 19.4 mmol) in ethanol (10 mL) was This material was carried forward without further purification
added 5 M aqueous sodium hydroxide (9.00 mL, 45.0 mmol). into the next step.
The reaction was then heated to reflux and stirred for 2 h.
tert‐Butyl‐2‐((diphenylmethylene)amino)‐3‐(perdeuterocyclobutyl)
propanoate (20a)
Upon cooling to room temperature, the reaction was
concentrated in vacuo and diluted with 6 N HCl (100 mL).
The resulting aqueous solution was extracted with diethyl
ether (3 × 100 mL), and the combined organic layers were
dried with Na2SO4, filtered, and concentrated. The resulting
residue was then recrystallized from EtOAc/heptane to afford
dicarboxylic acid 14 as white crystals (2.21 g, 76%). This
material was carried forward into the next step without
further purification.
To a solution of 19 (2.93 g, 9.92 mmol, purchased from Alfa
Aesar) in THF (30 mL) at −78 °C was added a 1 M solution of
potassium tert‐butoxide in THF (9.92 mL, 9.92 mmol). The
resulting solution was stirred at 0 °C for 1 h, at which time a
solution of bromide 18a (1.29 g, 8.27 mmol) in THF (2 mL) was
added. The reaction was then stirred at room temperature for
15 h. The THF was then removed in vacuo, and the resulting
residue was diluted with water (100 mL). This solution was
then extracted with CH2Cl2 (3 × 50 mL), and the aqueous layer
1,2,2,3,3,4,4‐Heptadeuterocyclobutanecarboxylic acid (15)
A solution of dicarboxylic acid 14 (2.21 g, 14.7 mmol) in D2O was acidified with 1 M HCl and re‐extracted with CH2Cl2
(30 mL, Cambridge Isotope Labs, 99.8 atom %D) in a sealed (3 × 50 mL). All organic layers were combined, dried with
pressure tube was heated to 160 °C for 15 h. After cooling to Na2SO4, filtered, and concentrated to afford a red oil, which
room temperature, the reaction was diluted with excess 1 M was purified by column chromatography (SiO2, 0–20% EtOAc/
HCl and extracted with EtOAc (3 × 100 mL). The combined heptane) to afford imine 20a (1.93 g, 63%). MS (ESI) 371.3
organic layers were dried with Na2SO4, filtered, and concen- [(M + H)+].
trated to afford carboxylic acid 15 (1.57 g, 100%) as a clear oil,
tert‐Butyl‐2‐((tert‐butoxycarbonyl)amino)‐3‐(perdeuterocyclobutyl)
propanoate (21a)
which formed colorless crystals upon cooling in a −20 °C
freezer. This material was carried forward crude into the next
step.
To a solution of 20a (1.93 g, 5.21 mmol) in a mixture of THF and
water (1:1, 24 mL) was added acetic acid (12 mL). The reaction
then stirred at room temperature for 6 h and then quenched with
(Perdeuterocyclobutyl)methyl 4‐methylbenzenesulfonate (17a)
To a solution of 15 (1.00 g, 9.35 mmol) in THF (50 mL) at 0 °C sat. NaHCO3. The resulting solution was then extracted with
was added 2 M solution of LiAlH4 in THF (5.61 mL, EtOAc (3 × 50 mL). The organic layers were then combined, dried
a
11.2 mmol). The reaction was stirred at room temperature with Na2SO4, filtered, and concentrated to afford a 1:1 mixture
for 4 h and then cooled to 0 °C. Upon reaching 0 °C, the of cyclobutylalanine t‐butyl ester‐d7 and benzophenone as
reaction was quenched by dropwise addition of 10% KHSO4 observed by 1H NMR. This mixture was then dissolved in
until a solid white cake formed and further addition of KHSO4 CH2Cl2 (20 mL), and triethylamine (1.09 mL, 7.82 mmol) and di‐
was unreactive. The cake was then broken up, filtered tert‐butyl dicarbonate (1.36 g, 6.25 mmol) were added. The
through Celite®, and washed with diethyl ether repeatedly. reaction was stirred at room temperature for 15 h, diluted
The resulting filtrate was concentrated in vacuo, dissolved in with 1 M HCl (15 mL), and then extracted with CH2Cl2
dichloromethane (100 mL), dried with Na2SO4, filtered, and (3 × 30 mL). The combined organic layers were washed with
concentrated. The resulting alcohol (16a) was dissolved in brine, dried with Na2SO4, filtered, and concentrated. The
DCM (10 mL) and cooled to 0 °C, and pyridine (2.27 mL, resulting residue was purified by column chromatography
2.81 mmol) was added followed by p‐toluenesulfonyl chloride (SiO2, 0–20% EtOAc/heptane) to afford 21a (1.26 g, 80%) as a
(1.78 g, 9.35 mmol). The reaction was stirred at room clear oil. 1H NMR (CDCl3, 400 MHz) δ 4.92 (br d, 1H), 4.08 (q,
temperature for 15 h and then diluted with diethyl ether J = 5.6 Hz, 1H), 1.83 (dd, J = 5.3, 13.6 Hz, 1H), 1.65 (dd, J = 7.6,
(200 mL). The resulting solution was then washed with water 13.6 Hz, 1H), 1.46 (s, 9H), 1.43 (s, 9H).
(100 mL), 1 M HCl (3 × 50 mL), water (100 mL), and brine
(100 mL). The organic layer was then dried with MgSO4,
tert‐Butyl‐(1‐oxo‐3‐(perdeuterocyclobutyl)propan‐2‐yl)carbamate
(22a)
filtered, and concentrated to afford tosylate 17a as a clear oil
1
(1.84 g, 80%), which was used without further purification. H
To a solution of 14 (1.26 g, 4.11 mmol) in toluene (20 mL) cooled
to −78 °C was added a 1 M solution of diisobutylaluminum
hydride in CH2Cl2 (2.12 mL, 2.12 mmol). The reaction was stirred
at −78 °C for 1 h and then quenched with methanol (20 mL).
After stirring at −78 °C for an additional 15 min, the reaction was
NMR (CDCl3, 400 MHz) δ 7.79 (d, J = 8.3Hz, 2H), 7.34 (d, J = 8.1 Hz,
2H), 3.97 (s, 2H), 2.45 (s, 3H).
1‐(Bromomethyl)‐1,2,2,3,3,4,4‐heptadeuterocyclobutane (18a)
A solution of tosylate 17a (3.21 g, 13.0 mmol) and lithium poured into a flask containing saturated Rochelle’s salt (40 mL)
bromide (1.81 g, 20.8 mmol) in acetone (50 mL) was heated to and subsequently stirred at room temperature for 3.5 h. The
reflux and stirred for 3 h. The reaction was then cooled to room organic layer was separated, and the remaining aqueous layer
temperature, diluted with diethyl ether, and filtered through was extracted with EtOAc (3 × 100 mL). The combined organic
Celite® to remove lithium tosylate. The acetone and ether were layers were dried with Na2SO4, filtered, concentrated in vacuo,
then removed via careful distillation (bromomethylcyclobutane, and purified by column chromatography (SiO2, 0–15% EtOAc/
bp = 123 °C). The resulting residue was then diluted with ether heptane) to afford aldehyde 22a (583 mg, 61%). MS (ESI) 135.2
(50 mL), washed with water (2 × 50 mL), dried with MgSO4, [(M‐Boc + H)+].
www.jlcr.org
Copyright © 2011 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2011, 54 613–624

A. J. Morgan et al.
tert‐Butyl‐(1‐cyano‐1‐hydroxy‐3‐(perdeuterocyclobutyl)propan‐2‐
yl)carbamate (23a)
tert‐Butyl‐(1,1‐dideutero‐3‐oxo‐1‐(perdeuterocyclobutyl)propan‐2‐
yl)carbamate (22b)
Acetone cyanohydrin (471 μL, 5.15 mmol) was added to a The procedure was the same as that for 22a, employing 21b
solution of aldehyde 22a (583 mg, 2.49 mmol) and triethyla- (715 mg, 2.32 mmol) to provide 22b (372 mg, 68%). MS (ESI)
mine (420 μL, 3.01 mmol) in CH2Cl2 (4 mL). The reaction was 137.3 [(M‐Boc + H)+].
stirred at room temperature for 15 h and then concentrated
tert‐Butyl‐(1,1‐dideutero‐3‐cyano‐3‐hydroxy‐1‐(perdeuterocyclobutyl)
in vacuo. The resulting residue was diluted with 1 M HCl
propan‐2‐yl)carbamate (23b)
(10 mL) and extracted with CH2Cl2 (3 × 100 mL). The organic
The procedure was the same as that for 23a, employing 22b
(372 mg, 1.57 mmol) to provide 23b (414 mg, 100%). MS (ESI)
286.2 [(M + Na)+].
layers were combined, washed with water and then brine,
dried with MgSO4, filtered, and concentrated in vacuo to
afford cyanohydrin 23a (651 mg, 99%). MS (ESI) 284.2
[(M + Na)+].
tert‐Butyl‐(4‐amino‐1,1‐dideutero‐3‐hydroxy‐4‐oxo‐1‐
(perdeuterocyclobutyl)butan‐2‐yl)carbamate (24b)
tert‐Butyl‐(4‐amino‐3‐hydroxy‐4‐oxo‐1‐(perdeuterocyclobutyl)
butan‐2‐yl)carbamate (24a)
The procedure was the same as that for 24a, employing 23b
(414 mg, 1.57 mmol) to provide 24b (291 mg, 66%). MS (ESI)
304.3 [(M + Na)+].
To a solution of cyanohydrin 23a (651 mg, 2.49 mmol) in
methanol (10 mL) at 0 °C was added 30% H2O2 (1.40 mL,
13.7 mmol) followed by lithium hydroxide (72.0 mg,
2.99 mmol). The reaction was stirred at 0 °C for 3 h and
subsequently quenched at 0 °C via careful addition of
excess sat. NaHSO3. The resulting solution was then diluted
with water (30 mL) and extracted with CH2Cl2 (3 × 100 mL).
The combined organic layers were dried with Na2SO4,
filtered, and concentrated in vacuo to afford hydroxyamide
3‐Amino‐4,4‐dideutero‐2‐hydroxy‐4‐(perdeuterocyclobutyl)
butanamide (25b)
The procedure was the same as that for 25a, employing 24b
(272 mg, 0.984 mmol) to provide 25b (208 mg, 97%). MS (ESI)
182.2 [(M + H)+].
3‐Amino‐4‐cyclobutyl‐2‐oxobutanamide‐d9 (29)
24a (469 mg, 67%) as
a white solid. MS (ESI) 302.3
To a solution of amide 24b (141 mg, 0.501 mmol) in 1:1 toluene/
DMSO (10 mL) at 0 °C was added EDC (960 mg, 5.01 mmol)
followed by dichloroacetic acid (207 μL, 2.50 mmol). The reaction
was stirred at room temperature for 4 h, diluted with 1 M HCl
(50 mL), and then extracted with DCM (3 × 50 mL). The combined
organic layers were washed with 1 M HCl (50 mL), sat. NaHCO3
(50 mL), and brine (50 mL); dried with Na2SO4; filtered; and
concentrated in vacuo. The resulting residue was purified by
column chromatography (SiO2, 0–40% EtOAc/heptane) to afford
the Boc‐protected ketoamide (117 mg, 84%) as an off‐white solid.
MS (ESI) 302.2 [(M + Na)+], 180.2 [M − Boc + H]. The resulting
ketoamide (117 mg, 0.419 mmol) was stirred in 4 M HCl in dioxane
(7 mL) for 3 h. The reaction was then concentrated in vacuo to
afford amine hydrochloride 29 (90 mg, 99%). This material was
carried forward without purification.
[(M + Na)+].
3‐Amino‐2‐hydroxy‐4‐(perdeuterocyclobutyl)butanamide (25a)
A solution of hydroxyamide 24a (469 mg, 1.68 mmol) was stirred
in 4 M HCl in dioxane (5 mL) for 3 h. The reaction was then
concentrated in vacuo to afford hydroxyamide hydrochloride
salt‐d7 25a (337 mg, 93%). MS (ESI) 180.2 [(M + H)+].
Dideutero(perdeuterocyclobutyl)methyl 4‐methylbenzenesulfonate
(17b)
The procedure was the same as that for 17a, employing 15
(3.75 g, 35.0 mmol) and LiAlD4 (Cambridge Isotope Laboratories,
98 atom %D) to provide 17b (5.88 g, 67%). MS (ESI) 272.1
[(M + Na)+].
(1R,2S,5S)‐Methyl‐3‐((S)‐2‐((tert‐butoxycarbonyl)amino)‐4,4,
4‐trideutero‐3,3‐bis(trideuteromethyl)butanoyl)‐6,
6‐bis(trideuteromethyl)‐3‐azabicyclo[3.1.0]hexane‐2‐carboxylate
(26a)
1‐(Bromodideuteromethyl)‐1,2,2,3,3,4,4‐heptadeuterocyclobutane
(18b)
The procedure was the same as that for 18a, employing 17b
(5.88 g, 23.6 mmol) to provide 18b (2.86 g, 76%), which was
carried forward without further purification.
To a solution of 7a (121 mg, 0.506 mmol) and 13a (128 mg,
0.607 mmol) in DCM/DMF (1:1, 3 mL) at 0 °C was added 4‐methyl
morpholine (167 μL, 1.52 mmol) and BOP reagent (269 mg,
0.607 mmol). The reaction was stirred at room temperature for
15 h, diluted with 1 M HCl, and extracted with DCM (3 × 30 mL).
The combined organic layers were washed with 1 M HCl, sat.
NaHCO3, and brine; dried with MgSO4; filtered; and concentrated
in vacuo. The resulting residue was purified by column
chromatography (SiO2, 0–20% acetone/heptane) to afford
methyl ester 26a (112 mg, 56% yield). MS (ESI) 398.3 [(M + H)+].
tert‐Butyl‐3,3‐dideutero‐2‐((diphenylmethylene)amino)‐3‐
(perdeuterocyclobutyl)‐propanoate (20b)
The procedure was the same as that for 20a, employing 18b
(2.86 g, 18.1 mmol) to provide 20b (4.47 g, 66%). MS (ESI) 373.3
[(M + H)+].
(1R,2S,5S)‐Methyl‐3‐((S)‐2‐((tert‐butoxycarbonyl)amino)‐4,4,
4‐trideutero‐3,3‐bis(trideuteromethyl)butanoyl)‐6,6‐dimethyl‐3‐
azabicyclo[3.1.0]hexane‐2‐carboxylate (26b)
tert‐Butyl‐2‐((tert‐butoxycarbonyl)amino)‐3,3‐dideutero‐3‐
(perdeuterocyclobutyl)‐propanoate (21b)
The procedure was the same as that for 21a, employing 20b
(1.49 g, 3.99 mmol) to provide 21b (973 mg, 79%). 1H NMR
(CDCl3, 400 MHz) δ 4.92 (br d, 1H), 4.06 (d, J = 8.3 Hz, 1H), 1.46 (s,
9H), 1.43 (s, 9H).
The procedure was the same as that for 26a, employing amine
13b (693 mg, 3.37 mmol) to afford 26b (465 mg, 35%). MS (ESI)
392.4 [(M + H)+].
J. Label Compd. Radiopharm 2011, 54 613–624
Copyright © 2011 John Wiley & Sons, Ltd.
www.jlcr.org

A. J. Morgan et al.
(1R,2S,5S)‐Methyl‐3‐((S)‐2‐((tert‐butoxycarbonyl)amino)‐3,3‐
dimethylbutanoyl)‐6,6‐bis(trideuteromethyl)‐3‐azabicyclo[3.1.0]
hexane‐2‐carboxylate (26c)
dichloroacetic acid (78.0μL, 0.946 mmol). The reaction was stirred
at room temperature for 4 h, diluted with sat. NaHCO3, and then
extracted with CH2Cl2 (3× 30 mL). The combined organic layers
were washed successively with sat. NaHCO3, 1 M HCl, and brine;
dried with MgSO4; filtered; and concentrated in vacuo. The
resulting residue was purified by column chromatography (SiO2,
0–30% acetone/heptane) to afford 1a (66.0mg, 66%) as a white
solid and as a mixture of diastereomers. 1H NMR (DMSO‐d6,
400 MHz) δ 8.28 (d, J = 7.3Hz, 0.6H), 8.18 (d, J = 7.8Hz, 0.4H), 8.03 (s,
0.6H), 7.99 (s, 0.4H), 7.77 (br s, 1H), 5.97 (s, 1H), 5.87–5.79 (m, 1H),
4.94 (d, J = 7.3Hz, 0.6H), 4.82 (d, J = 7.3Hz, 0.4H), 4.28 (br s, 1H),
4.16–4.06 (m, 1H), 4.00–3.90 (m, 1H), 3.80–3.69 (m, 1H), 1.78 (d,
J = 3.8 Hz, 0.4H), 1.74 (d, J = 3.5 Hz, 0.6H), 1.65–1.50 (m, 1H),
1.46–1.39 (m, 1H), 1.27 (d, J = 4.0 Hz, 0.6H), 1.25 (d, J = 4.0 Hz,
0.4H), 1.17 (br s, 9H), 1.03–0.97 (m, 3H), 0.92–0.86 (m, 9H),
0.86–0.79 (m, 3H); MS (ESI) 527.5 [(M + H)+].
The procedure was the same as that for 26a, employing acid 7a
(305 mg, 1.44 mmol) to afford 26c (358 mg, 77%). MS (ESI) 389.4
[(M + H)+].
(1R,2S,5S)‐3‐((S)‐4,4,4‐Trideutero‐2‐(3‐(1,1,1,3,3,3‐hexadeutero‐2‐
(trideuteromethyl)propan‐2‐yl)ureido)‐3,3‐bis(trideuteromethyl)
butanoyl)‐6,6‐bis(trideuteromethyl)‐3‐azabicyclo[3.1.0]hexane‐2‐
carboxylic acid (28a)
A solution of 26a (545 mg, 1.37 mmol) in 4 M HCl in dioxane (15 mL)
was stirred at room temperature for 3 h and then concentrated in
vacuo to afford the amine hydrochloride salt. This material was
dissolved in dioxane (3.00 mL), and triethylamine (1.15 mL,
8.22mmol) was added. The mixture was subsequently cooled to
−78 °C, and tert‐butyl isocyanate‐d9 (27a; 740mg, 6.85 mmol) was
added. The reaction was stirred at room temperature for 15h,
concentrated in vacuo, diluted with 1 M HCl, and extracted with
DCM (3× 50 mL). The combined organic layers were washed with
1 M HCl, sat. NaHCO3, and brine; dried with MgSO4; filtered; and
concentrated in vacuo. The resulting residue was purified by
column chromatography (SiO2, 10–20% acetone/heptane) to
afford d24‐urea methyl ester as a white crunchy foam. MS (ESI)
428.5 [(M + Na)+]. The resulting urea methyl ester was dissolved in a
mixture of THF/H2O (1:1, 10mL), and lithium hydroxide (35.0mg,
1.45mmol) was added. The reaction was stirred for 3 h, quenched
with 1 M HCl, and concentrated to remove THF. The resulting
aqueous solution was extracted with EtOAc (3× 10mL), and the
combined organic layers were dried with Na2SO4, filtered, and
concentrated to afford carboxylic acid 28a (360mg, 67%), which
was used without further purification.
Boceprevir‐d9 (1b) (method 2: keto‐amide coupling strategy)
To a solution of carboxylic acid 28d (59.0 mg, 0.160 mmol) and
amine hydrochloride 29 (45.0 mg, 0.209 mmol) in ACN (1.5 mL)
at 0 °C was added EDC (46.0 mg, 0.240 mmol), HOBt (6.00 mg,
0.0480 mmol), and N‐methyl morpholine (19.0 μL, 0.176 mmol).
The reaction was stirred at room temperature for 15 h,
concentrated in vacuo, diluted with 1 M HCl, and extracted with
EtOAc (3 × 20 mL). The combined organic layers were washed
with 1 M HCl, sat. NaHCO3, and brine; dried with MgSO4; filtered;
and concentrated in vacuo. The resulting residue was purified by
column chromatography (SiO2, 10–40% acetone/heptane) to
afford 1b (21 mg, 25%) as a white solid and as a mixture of
diastereomers. 1H NMR (CD3OD, 400 MHz) δ 7.55 (br s, 0.3H), 6.07
(br s, 0.6H), 5.95–5.76 (m, 1.3H), 4.38 (s, 0.6H), 4.35–4.20 (m, 2H),
4.13–3.86 (m, 3H), 3.25–3.20 (m, 1H), 1.62–1.27 (m, 2H), 1.29–1.22
(m, 9H), 1.08–1.03 (m, 3H), 1.03–0.96 (m, 9H), 0.95–0.87 (m, 3H).
MS (ESI) 529.5 [(M + H)+].
(1R,2S,5S)‐6,6‐Dimethyl‐3‐((S)‐4,4,4‐trideutero‐2‐(3‐(1,1,1,3,3,
3‐hexadeutero‐2‐(trideuteromethyl)propan‐2‐yl)ureido)‐3,3‐bis
(trideuteromethyl)butanoyl)‐3‐azabicyclo[3.1.0]hexane‐2‐
carboxylic acid (28b)
Boceprevir‐d15 (1c)
The procedure was the same as that for 1a, employing
carboxylic acid 28c to afford 1c in 47% yield and as a mixture
of diastereomers. NMR (DMSO‐d6, 400 MHz) δ 8.28 (d, J = 7.3 Hz,
0.6H), 8.18 (d, J = 7.8 Hz, 0.4H), 8.03 (s, 0.6H), 7.99 (s, 0.4H), 7.78
(br s, 1H), 5.94 (s, 1H), 5.87–5.79 (m, 1H), 4.94 (d, J = 7.3 Hz, 0.6H),
4.82 (d, J = 7.3 Hz, 0.4H), 4.28 (br s, 1H), 4.16–4.06 (m, 1H),
4.00–3.90 (m, 1H), 3.80–3.69 (m, 1H), 1.45–1.39 (m, 1H), 1.31–1.18
(m, 1H), 1.17 (br s, 9H), 0.89 (br s, 9H); MS (ESI) 535.5 [(M + H)+].
The procedure was the same as that for 28a, employing methyl
ester 26b (465 mg, 1.19 mmol) to afford 28b (158 mg, 34%). Urea
methyl ester intermediate: MS (ESI) 422.4 [(M + Na)+].
(1R,2S,5S)‐3‐((S)‐2‐(3‐(tert‐Butyl)ureido)‐3,3‐dimethylbutanoyl)‐
6,6‐bis(trideuteromethyl)‐3‐azabicyclo[3.1.0]hexane‐2‐carboxylic
acid (28c)
Boceprevir‐d6 (1d)
The procedure was the same as that for 28a, employing methyl
ester 26c (358 mg, 0.921 mmol) and tert‐butyl isocyanate (27b,
216 μL, 1.84 mmol) to afford 28c (340 mg, 99%). Urea methyl
ester intermediate: MS (ESI) 388.3 [(M + H)+].
The procedure was the same as that for 1a, employing
carboxylic acid 28c and hydroxyamide 25c to afford 1d in
41% yield and as a mixture of diastereomers. 1H NMR (DMSO‐d6,
400 MHz) δ 8.28 (d, J = 7.3 Hz, 0.7H), 8.21 (d, J = 7.8 Hz, 0.3H), 8.03
(s, 0.7H), 7.99 (s, 0.3H), 7.76 (br s, 1H), 5.94 (s, 1H), 5.87–5.79 (m,
1H), 5.00–4.90 (m, 0.7H), 4.90–4.81 (m, 0.3H), 4.28 (s, 1H),
4.16–4.06 (m, 1H), 4.00–3.90 (m, 1H), 3.80–3.69 (m, 1H), 2.57–2.42
(m, 0.7H), 2.40–2.29 (m, 0.3H), 2.04–1.89 (m, 2H), 1.82–1.68 (m,
3H), 1.68–1.52 (m, 3H), 1.45–1.39 (m, 1H), 1.31–1.18 (m, 1H), 1.17
(br s, 9H), 0.89 (br s, 9H); MS (ESI) 526.5 [(M + H)+].
Boceprevir‐d7 (1a) (method 1: hydroxyamide coupling strategy)
To a solution of 28d (71.0mg, 0.193 mmol) dissolved in a mixture of
CH2Cl2/DMF (1:1, 2.5mL) at 0 °C was added amine hydrochloride
salt 25a (50.0 mg, 0.232 mmol), EDC (56.0mg, 0.290 mmol), HOBt
(39.0mg, 0.290 mmol), and 4‐methyl morpholine (85.0μL,
0.772 mmol). The reaction was stirred at room temperature for
15h and then concentrated in vacuo. The resulting residue was
diluted with 1 M HCl and extracted with EtOAc (3× 30mL). The
combined organic layers were washed successively with 1 M HCl,
sat. NaHCO3, and brine; dried with MgSO4; filtered; and concen-
trated. To a solution of this material in a mixture of toluene/DMSO
(1:1, 4 mL) at 0 °C was added EDC (362 mg, 1.89 mmol) and
Boceprevir‐d18 (1e)
The procedure was the same as that for 1a, employing
carboxylic acid 28b and hydroxyamide 25c to afford 1e in
37% yield and as a mixture of diastereomers. 1H NMR (DMSO‐d6,
400 MHz) δ 8.27 (d, 0.8 H, J = 7.3 Hz), 8.17 (d, 0.2 H, J = 7.8 Hz),
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Copyright © 2011 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2011, 54 613–624

A. J. Morgan et al.
8.02 (s, 0.8 H), 7.97 (s, 0.2 H), 7.76 (br s, 1.0 H), 7.34 (s, 0.5 H), 6.90 using Microsoft Excel Software; in vitro t1/2 = 0.693/k; k = −[slope of
(s, 0.4 H), 5.94 (s, 0.8H), 5.93 (s, 0.2H), 5.87–5.79 (m, 1H), 5.00–4.90 linear regression of % parent remaining(ln) vs incubation time].
(m, 0.8H), 4.90–4.81 (m, 0.2H), 4.28 (s, 0.8H), 4.27 (s, 0.2H),
4.16–4.06 (m, 1H), 4.00–3.90 (m, 1H), 3.80–3.69 (m, 1H), 2.57–2.42
(m, 0.8H), 2.40–2.29 (m, 0.2H), 2.04–1.89 (m, 2H), 1.82–1.68 (m,
Conclusions
3H), 1.68–1.52 (m, 3H), 1.45–1.39 (m, 1H), 1.31–1.18(m, 1H),
In summary, several DCEs derived from the boceprevir scaffold
have been prepared via synthetic routes that allow for highly
site‐selective deuterium incorporation with high levels of
isotopic purity. Our studies demonstrate that judicious applica-
tion of deuterium medicinal chemistry to the boceprevir scaffold
can result in the identification of DCEs that display marked levels
of in vitro metabolic stabilization in human liver microsomes.
The highest degree of metabolic stabilization was observed with
DCE 1g, which exhibited a half‐life approximately double that of
boceprevir. On the basis of previous work in our laboratories, we
anticipate that these metabolically stabilized DCEs will retain the
biochemical potency and selectivity of boceprevir. Further
studies aimed at evaluating the in vivo pharmacokinetics as
well as confirming the preserved pharmacological activity of
these DCEs will be reported in due course. This work highlights
the utility of the DCE Platform™ as a medicinal chemistry tool
capable of modifying the metabolic fate of a drug to potentially
alter its therapeutic profile.
1.03–0.97 (m, 3H), 0.89–0.80 (m, 3H); MS (ESI) 538.5 [(M + H)+].
Boceprevir‐d24 (1f)
The procedure was the same as that for 1a, employing carboxylic
acid 28a and hydroxyamide 25c to afford 1f in 23% yield and as
1
a mixture of diastereomers. H NMR (DMSO‐d6, 400 MHz) δ 8.27
(d, 0.7 H, J = 7.3 Hz), 8.17 (d, 0.3 H, J = 7.8 Hz), 8.02 (s, 0.7 H), 7.97
(s, 0.3 H), 7.76 (br s, 1.0 H), 7.34 (s, 0.6 H), 6.90 (s, 0.6 H), 5.94
(s, 0.7H), 5.93 (s, 0.3H), 5.87–5.79 (m, 1H), 5.00–4.90 (m, 0.7H),
4.90–4.81 (m, 0.3H), 4.28 (s, 0.7H), 4.27 (s, 0.3H), 4.16–4.06 (m, 1H),
4.00–3.90 (m, 1H), 3.80–3.69 (m, 1H), 2.57–2.42 (m, 0.7H), 2.40–2.29
(m, 0.3H), 2.04–1.89 (m, 2H), 1.82–1.68 (m, 3H), 1.68–1.52 (m, 3H),
1.45–1.39 (m, 1H), 1.31–1.18(m, 1H); MS (ESI) 544.5 [(M + H)+].
Boceprevir‐d33 (1g)
The procedure was the same as that for 1b, employing
carboxylic acid 28a to afford 1g in 23% yield and as a mixture
of diastereomers (28 mg, 36%) as a white solid. 1H NMR (CD3OD,
400 MHz) δ 7.55 (br s, 0.3H), 6.07 (br s, 1H), 5.95–5.76 (m, 1H),
4.35–4.20 (m, 2H), 4.20–3.87 (m, 3H), 3.22 (s, 1H), 1.62–1.27
(m, 2H). MS (ESI) 553.5 [(M + H)+].
Acknowledgements
The authors would like to thank Andrew Cottone, James V. Hay,
and Guanglin Bao of Adesis, Inc., for the large‐scale preparation
of the d9‐tert‐leucine intermediate.
Determination of metabolic stability
Stock solutions (7.5 mM) of test compounds were prepared in
DMSO. The 7.5 mM stock solutions were diluted to 12.5 μM in
ACN. The 20 mg/mL human liver microsomes were diluted to
0.625 mg/mL in 0.1 M potassium phosphate buffer (pH 7.4)
containing 3 mM MgCl2. The diluted microsomes were added to
wells of a 96‐well deep‐well polypropylene plate in triplicate.
Ten microliters of the 12.5 μM test compound was added to the
microsomes, and the mixture was pre‐warmed for 10 min.
Reactions were initiated by the addition of pre‐warmed NADPH
solution. The final reaction volume was 0.5 mL and contained
0.5 mg/mL human liver microsomes, 0.25 μM test compound,
and 2 mM NADPH in 0.1 M potassium phosphate buffer (pH 7.4)
and 3 mM MgCl2. The reaction mixtures were incubated at 37 °C,
and 50‐μL aliquots were removed at 0, 5, 10, 20, and 30 min and
then added to shallow 96‐well plates, which contained 50 μL of
ice‐cold ACN with internal standard to stop the reactions. The
plates were stored at 4 °C for 20 min, after which 100 μL of water
was added to the wells of the plate before centrifugation to pellet
the precipitated proteins. Supernatants were transferred to an-
other 96‐well plate and analyzed for amounts of parent remaining
by LC‐MS/MS using an Applied Bio‐systems API 4000 mass
spectrometer. Quantitative analysis by LC‐MS/MS was performed
using an Applied Bio‐systems API 4000 mass spectrometer and
utilized an APCI (Atmospheric Pressure Chemical Ionization) source
operated in positive ion MRM (multiple reaction monitoring) mode.
7‐Ethoxycoumarin (1μM) was used as a positive control.
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