Synthesis and pharmacokinetic profile of highly deuterated brecanavir analogs

Article abstract of DOI:10.1016/j.ejmech.2013.02.001

Several highly deuterated analogs of the HIV-1 protease inhibitor brecanavir have been prepared to study the effect of deuterium upon metabolic stability. The sites for deuterium incorporation were initially chosen to maximize the potential for a kinetic isotope effect; locations where C-H bond breaking is the rate limiting step. The analogs have been profiled in both in vitro and in vivo pharmacokinetic studies and the result will be described herein.

Full text of DOI:10.1016/j.ejmech.2013.02.001

European Journal of Medicinal Chemistry 63 (2013) 202e212
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and pharmacokinetic profile of highly deuterated
brecanavir analogs
*
Emile J. Velthuisen , Todd M. Baughman, Brian A. Johns, David P. Temelkoff,
Jason G. Weatherhead
GlaxoSmithKline Research & Development, Infectious Diseases Therapeutic Area Unit, Five Moore Drive, Research Triangle Park, NC 27709, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Several highly deuterated analogs of the HIV-1 protease inhibitor brecanavir have been prepared to study
the effect of deuterium upon metabolic stability. The sites for deuterium incorporation were initially
chosen to maximize the potential for a kinetic isotope effect; locations where CeH bond breaking is the
rate limiting step. The analogs have been profiled in both in vitro and in vivo pharmacokinetic studies and
the result will be described herein.
Received 8 November 2012
Received in revised form
30 January 2013
Accepted 1 February 2013
Available online 14 February 2013
Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords:
HIV
AIDS
Protease inhibitor
Ritonavir
Deuterium
Kinetic isotope effect
1. Introduction
In 2006, GlaxoSmithKline disclosed the discovery of brecanavir
(Fig. 1), a tyrosyl peptidomimetic that demonstrated low nano-
HIV continues to be one of the leading causes of death world-
wide. In 2009, there were an estimated 33.3 million people living
with HIV/AIDS with 2.6 million new infections and 1.8 million
deaths [1]. The overall rates of morbidity and mortality have been
decreasing in part due to the advent of highly active antiretroviral
therapy (HAART), but despite this progress the treatment of HIV
remains problematic due to drug resistance, tolerance, and toxicity.
Protease represents one of three essential enzymes required for
replication of HIV and is an important target for antiretroviral
therapy. However, the current generation of protease inhibitors
(PIs) is susceptible to drug resistance [2,3] and require co-
administration with a pharmacokinetic booster to inhibit the
metabolism of the parent drug by cytochrome P450-3A4 [4,5].
molar activity against both wild-type and PI-resistant HIV [6,7]. In
clinical studies the drug was safe and well tolerated but poor sol-
ubility and first pass metabolism were challenges uncovered during
the studies. The metabolic profile was significantly improved by co-
administration with the pharmacoenhancer ritonavir. However,
due to potential side effects and drugedrug interactions it would be
favorable to design brecanavir analogs that had a superior meta-
bolic profile without the aid of a pharmacoenhancer.
One strategy to improve the ADME properties of brecanavir is to
incorporate deuterium in place of hydrogen where CeH bond
breaking is the rate limiting step during metabolism. Although the
use of stable isotope labeled compounds has been important for the
study of drug metabolism and organ toxicity [8,9], only recently has
deuterium been incorporated into molecules with the specific aim
of treating disease [10,11].
Abbreviations: ADME, absorption, distribution, metabolism, excretion; AIBN,
azobisisobutylnitrile; AIDS, acquired immunodeficiency syndrome; DIPEA, diiso-
propyl ethyl amine; DMP, DesseMartin periodinane; DMPK, drug metabolism and
pharmacokinetics; DNAUC, dose-normalized area under the curve; HAART, highly
active antiretroviral therapy; HIV, human immunodeficiency virus; IPA, isopropyl
alcohol; KIE, kinetic isotope effect; LDA, lithium diisopropylamide; NBS, N-bro-
mosuccinimide; PI, protease inhibitor; PNP, p-nitrophenyl; TBDMSCl, t-butyldime-
thylsilyl chloride; TFA, trifluoroacetic acid; TMEDA, tetramethylethylenediamine.
* Corresponding author.
Deuterium is a naturally occurring, non-radioactive, and stable
isotope of hydrogen. Whereas hydrogen contains a single proton
and electron, deuterium also contains an additional neutron in its
nucleus effectively doubling the atomic mass of the atom. Because
deuterium is isotopic to hydrogen there is no difference in the
pharmacological activity of the molecule. However, due to the
increased mass, a significant kinetic isotope effect (KIE) is observed
between these two isotopes ranging from one to seven [12].
E-mail address: emile.j.velthuisen@gsk.com (E.J. Velthuisen).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.02.001

E.J. Velthuisen et al. / European Journal of Medicinal Chemistry 63 (2013) 202e212
203
Although several reports suggest a considerable ADME advantage
was gained by the incorporation of deuterium into a drug-like
molecule it was unclear from the onset of this investigation if the
KIE would be enough to have a noticeable impact upon the in vivo
pharmacokinetics [11].
In designing brecanavir analogs it would be important to un-
derstand where metabolism occurs so that deuterium can be
strategically incorporated to maximize the primary isotope effect.
Characterization of the major metabolites of brecanavir in human
and rat hepatocytes indicated that glucuronidation, O-dealkylation,
and mono-oxidation were predominant (Fig. 1) [13]. Because the
primary isotope effect would likely have little influence upon glu-
curonidation the sites for mono-oxidation were initially targeted
for deuterium incorporation.
Due to the extensive amount of oxidation in brecanavir it
seemed beneficial to initially incorporate the maximum amount of
deuterium into an analog and then profile the in vivo pharmaco-
kinetics to observe any potential benefit. Due to the modular nature
of the synthesis, it would then be feasible to pinpoint any metabolic
soft spots by selectively reverting deuterium back to hydrogen. To
avoid ambiguous or partial deuterium incorporation, readily
exchangeable hydrogen atoms would be avoided.
benzodioxole-5-sulfonyl chloride 11 was accomplished by alkyl-
ation of catechol 9 with chloroiodomethane-d2 and subsequent
chlorosulfonylation. With these three components synthesized it
was now possible to construct the key amino alcohol-d12 core of
brecanavir. Epoxide 5 was stereoselectively opened with isobutyl-
amine-d9 8 and the resulting amine was treated with deuterated
1,3-benzodioxole 11 give sulfonamide 12. Following removal of the
tert-butyloxycarbonyl protecting group under acidic conditions our
attention turned to the synthesis of the remaining thiazole and
bisfuran fragments.
Synthesis of the thiazole-d2 fragment of brecanavir was accomplished
by treatment of 2-methylthiazole 14 with lithium aluminum deuteride
followed by chlorination with phosphorus oxychloride (Scheme 2). Unfor-
tunately, the synthesis of a more highly deuterated thiazole fragment
proved problematic. Upon treatment of known thioamide-d3 [16,17] with
ethyl bromopyruvate in ethanol, thiazole-d318 wasisolatedin60%yieldbut
with only 33% deuterium incorporation based upon integration of the 1H
NMR spectrum. To better understand the source of deuteriumehydrogen
exchange the same reaction was performed using methanol-d4 as sol-
vent. In this second experiment, the percent deuterium incorporation was
held constant following formation of the thiazole but deuteriumehydrogen
exchange of the methyl group was observed once again following reduction
of the ester with lithium aluminum deuteride. Unfortunately, the facile
nature of deuteriumehydrogen exchange of 2-methyl thiazole precluded
this site for the incorporation of deuterium.
2. Results and discussion
Synthesis of the bisfuran-d5 started with a lithium aluminum
deuteride reduction of dimethyl-D-tartrate acetonide (Scheme 3)
2.1. Chemistry
[17]. Deprotection of the acetonide with Amberlyst-15 afforded
symmetrical tetraol 22 in which the primary alcohols were selec-
tively protected as tert-butyl dimethyl silyl ethers. Subsequent
cleavage of diol 23 with sodium periodate revealed acetaldehyde-d2
Synthesis of the d1-tyrosine core of brecanavir started with a
Kowalski ester homologation [14] of tyrosine 2 [15] to afford
chloromethyl ketone 3 (Scheme 1). A stereoselective sodium bor-
odeuteride reduction, followed by ethanolic KOH mediated cycli-
24.
A
lithium aluminum deuteride reduction of 4-
zation provided the desired oxirane
5 in high yield. The
benzyloxybutanoate 25 followed by orthogonal silyl protection of
the resulting alcohol provided 27. Hydrogenolysis of the O-benzyl
ether and oxidation of the resulting alcohol gave aldehyde 28. The
isobutylamine-d9 coupling partner was then prepared by formation
of the mixed anhydride of commercially available isobutyric acid-
d7 6, aminolysis with ammonia gas and subsequent reduction with
lithium aluminum deuteride. Synthesis of deuterated 1,3-
penultimate precursor to the bisfuranwas assembled byan L-proline
catalyzed mixed aldol of acetaldehyde-d2 24 and 4-siloxybutanal 29
where synproduct 31 was isolated as the major diastereomer in a 7:1
ratio [18]. Following an acid catalyzed silyl deprotection/cyclization
cascade, bisfuran 32 was isolated in 30% yield. To incorporate an
additional deuterium into the bisfuran unit, the secondary alcohol
was first oxidized to ketone 33 using DesseMartin periodinane and
subsequently reduced with lithium aluminum deuteride. Approach
of the deuteride from the convex face of the molecule afforded a
single alcohol isomer that was enzymatically resolved and then
activated as the p-nitrophenyl carbonate 35.
In addition to the bisfuran-d5 moiety, a complementary bis-
furan-d2 unit was constructed that contained a deuterium at the
bridgehead acetal carbon. Synthesis of this fragment commenced
with a directed lithiation of dihydrofuran 36 and quenching with
D2O (Scheme 4). The dihyrdofuran was then brominated with NBS
and the resulting oxocarbenium ion trapped with propargyl alcohol
to provide furan 39. A radical mediated 5-exo-dig cyclization of the
alkynlfuran followed by ozonolysis of the resulting methylene
afforded ketone 40. The second deuterium was then introduced by
lithium aluminum deuteride reduction of ketone 41 followed by
acetylation of the resulting secondary alcohol. The racemic bis-
furan-d2 was then enzymatically resolved using Chiro-Clec PC to
give chiral bisfuran 43 as a single isomer. Following removal of the
acetyl group and activation of the chiral secondary alcohol as a p-
nitrophenyl carbonate, the bisfuran-d2 was ready for incorporation
into the brecanavir-d12 core.
The bisfuran subunits were introduced by acylation of amino
alcohol 13 with the respective PNP-carbonate derivative of alcohols
35 and 44. To avoid any competitive alkylation of the secondary
Fig. 1. Structure of the protease inhibitor brecanavir and the major sites of metabolism
in human and rat hepatocytes.

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Reagents and Conditions: (i) ICH2Cl, LDA, THF, -78º C, (80%); (ii) NaBD4, EtOH, (63%); (iii) KOH, EtOH, (99%); (iv) EtCO2Cl,
Et3N, DCM; NH3 (g), (99%); (v) LiAlD4, (99%); (vi) chloroiodomethane-d2, Cs2CO3, DMF, 110 °C, (96%); (vii) SO3·DMF;
(COCl)2, DMF, (50%); (viii) IPA, 8; (ix) 11, DIPEA, (65% two steps); (x) TFA, DCM, (99%).
Scheme 1. Synthesis of the brecanavir-d12 core.
alcohol in the brecanavir core it was protected as an acetonide
using dimethoxypropane and TsOH in DCM. Hydrogenolysis of the
O-benzyl ether afforded the phenol that was subsequently alky-
lated with thiazole-d2 16. Following deprotection of the acetonide
under acidic conditions, brecanavir-d19 52 and brecanavir-d16 53
were isolated (Scheme 5).
When dosed intravenously in rats, brecanavir was a high
clearance compound with a short half-life and low exposure
(Table 1). Upon co-dosing with the pharmacokinetic enhancer ri-
tonavir (RTV), the in vivo profile was significantly improved.
Clearance was reduce by 12-fold, and both the t1/2 and exposure
was increased by approximately 11-fold. Unfortunately both the
brecanavir-d19 and -d16 analogs had nearly identical intravenous
in vivo profiles as unboosted 1.
2.2. In vivo DMPK
To help reconcile any metabolic differences between brecanavir-
d16 53 and brecanavir 1 the major metabolites were characterized
following incubation with human and rat hepatocytes (Fig. 2).
Interestingly, the overall metabolic profile appears to have changed
with the incorporation of deuterium but the net result of high
in vivo clearance remains the same. In rats, carbamate bond
cleavage of the brecanavir-d16 53 carbonate is now apparent as is
To better understand if there was any metabolic benefit to
incorporating deuterium into brecanavir, compounds 52 and 53
were dosed intravenously in rats. We decided to forego the oral arm
of the in vivo experiment because the metabolic advantage should
manifest in a lower overall clearance for the analog if deuterium
had any affect.
Reagents and Conditions: (i) LiAlD4, THF, (66%); (ii) POCl3, THF, (97%); (iii) Ethyl bromopyruvate, solvent, (60%); (iv) LiAlD4,
THF (99%)
Scheme 2. Synthesis of the thiazole-d2 fragment 16.

E.J. Velthuisen et al. / European Journal of Medicinal Chemistry 63 (2013) 202e212
205
Reagents and conditions: (i) LiAlD4, THF, (45%); (ii) Amberlyst-15, DCM, (84%); (iii) TBSCl, imidazole, DMF, (47%); (iv)
NaIO4, (50%); (v) LiAlD4, THF (85%); (vi) TBSCl, imidazole, DMF (97%); (vii) H2, Pd/C, EtOAc (91%); (viii) (COCl)2, DMSO,
Et3N, DCM (99%); (ix) L-Proline, (cat.); (x) HCl, THF, (30% over 2-steps); (xi) Dess-Martin, NaHCO3, DCM, (78%); (xii) LiAlD4,
THF; (xiii) Ac2O, DMAP, Na2CO3, THF/H2O; (xiv) ChiroCLEC PC; (xv) K2CO3, MeOH; (xvi) PNPCOCl, Pyridine, DCM (42%
from 33).
Scheme 3. Synthesis of the bisfuran-d5 35.
dehydrogenation of the core and glucuronidation of the O-deal-
kylated benzodioxane. In addition, mono-oxidation of the fully
deuterated isobutyl amine-d9 side chain was observed in both rats
and humans as was replacement of a single deuterium with a hy-
droxyl group in the bisfuran unit. Surprisingly, these latter products
suggest the KIE of deuterium was not sufficient to overcome the
bond breaking step involved with oxidation. In comparison,
humans only had a single unique metabolite not found in rats; bond
scission of the benzodioxane. Other common metabolites include
glucuronidation of the secondary alcohol and oxidation of the
phenoxymethyl thiazole side chain.
Although further deuterated analogs could be made, no signif-
icant KIE was observed at the key metabolic sites identified in
human and rat hepatocytes. It’s possible that the in vivo rat
Reagents and conditions: (i) a) nBuLi/TMEDA, b) D2O; (ii) NBS, DCM, 0 °C; (iii) Bu3SnH, AIBN (26% three steps); (iv) O3,
DCM/MeOH (99%); (v) LiAlD4; (vi) Ac2O, Na2CO3, DMAP, (54% over 2 steps); (vii) ChiroCLEC PC, (39%); (viii) K2CO3, MeOH;
(ix) PNPCOCl, Pyr, DCM, (73% two steps).
Scheme 4. Synthesis of bisfuran-d2 44.

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E.J. Velthuisen et al. / European Journal of Medicinal Chemistry 63 (2013) 202e212
Reagents and conditions: (i) Hunig's Base, DCM, (46 = 78%, 47 = 61%); (ii) dimethoxypropane, TsOH, DCM, (48 = 81%, 49 =
68%); (iii) Pd/C, H2, EtOAc; (iv) 16, Cs2CO3, (v) HCl (52 = 66% three steps; 53 = 35% three steps).
Scheme 5. Final assembly of brecanavir-d19 52 and -d16 53.
pharmacokinetic profile is not reflective of humans. There were
several key differences in the metabolite profiles between the two
species and perhaps a higher species would more closely resemble
that of humans. However, until a better model system exists, or we
better understand the human metabolism of brecanavir, it becomes
difficult to justify further work on deuterated brecanavir analogs
despite the potential benefit this would offer patients.
established by analytical HPLC (C-18 column, 5.0 micron, 0 / 100%
CH3CN (or MeOH)/water with 0.05e0.1% HCOOH (or TFA)) and UV
detection with or without mass spectrometer detection. All test
compounds showed >95% purity (AUCs by UV detection).
4.1.1. (S)-tert-butyl (1-(4-(benzyloxy)phenyl)-4-chloro-3-
oxobutan-2-yl)carbamate (3)
LDA (169 mL, 163 mmol) was added dropwise at ꢀ78 ꢁC through
a cannula to a mixture of iodochloromethane (9.45 mL, 130 mmol)
and ethyl N-{[(1,1-dimethylethyl)oxy]carbonyl}-O-(phenylmethyl)-
3. Conclusion
L
-tyrosinate (prepared as described in J. Med. Chem. 1990, 33, 1620)
In conclusion, several highly deuterated analogs of the protease
inhibitor brecanavir have been synthesized and evaluated for im-
provements in metabolic stability. However, when dosed intrave-
nously in rats they had a nearly identical in vivo profile as unboosted
1. Interestingly, the metabolic profile appears to have shifted with
the incorporation of deuterium but the net result of high clearance
remains the same. Also worthy of note is that several locations that
contained deuterium were metabolized suggesting the KIE was not
sufficient to overcome oxidation under all circumstances.
also at ꢀ78 ꢁC. The resulting dark solution was stirred for one hour
at ꢀ78 ꢁC, then 50 mL of a 1:1 acetic acid:tetrahydrofuran solution
was added slowly and the reaction was warmed to ambient tem-
perature, poured onto a mixture of ice water and ethyl acetate, and
the aqueous solution was extracted with ethyl acetate, the organic
layer was washed with brine, dried over sodium sulfate, filtered
and concentrated under reduced pressure. The brown solid was
suspended in cold ether and filtered to give the title compound
(3.17 g). The filtrate was concentrated under reduced pressure to
give an additional 7.4 g of slightly impure title compound (10.57 g,
4. Experimental section
80% yield). 1H NMR (400 MHz, CDCl3)
d 1.40 (s, 9H), 2.80e3.21 (m,
2H), 3.87e4.25 (m, 2H), 4.42e4.72 (m, 1H), 5.03 (s, 2H), 6.81e7.17
4.1. Chemistry
(m, 4H), 7.29e7.57 (m, 5H); LC/MS (m/z) ESþ ¼ 427 (M þ 23).
All commercially available reagents were used without further
purification. Column chromatography was carried out on silica gel
(70e230 mesh). TLC was conducted on silica gel 250 micron, F254
plates. 1H NMR spectra were recorded at 300 or 400 MHz. Melting
points are uncorrected. Purities of test compounds were
4.1.2. tert-butyl ((2S,3S)-1-(4-(benzyloxy)phenyl)-4-chloro-3-
hydroxybutan-2-yl)carbamate-d1 (4)
Sodium borodeuteride (1.64 g, 39 mmol) was added in portions
to a suspension of 1,1-dimethylethyl [(1S)-3-chloro-2-oxo-1-({4-
[(phenylmethyl) oxy]phenyl}methyl)propyl]carbamate (3.16 g,
7.82 mmol) in 60 mL of a 4:1 ethanol:tetrahydrofuran solution
at ꢀ78 ꢁC. The mixture was warmed to 0 ꢁC and stirred 1 h. The
suspension was poured onto ice, placed in a 0 ꢁC bath, acidified
with 1 N HCl to a pH w2 and filtered to collect a brown solid. The
filtrate was extracted with ethyl acetate, dried over sodium sulfate
and combined with the isolated solid. The brown solid was sus-
pended in ethyl acetate (10 vol.), heated to reflux and stirred for
15 min. The hot solution was filtered and allowed to cool to ambient
temperature and aged overnight. The mixture was filtered and the
Table 1
Rat in vivo pharmacokinetic screening results [19].
Cmpd
Number of
deuterium
Clp
(mL/min/kg)
t1/2
(h)
i.v. DNAUC
(ng h/mL/mg)
1
0
0
19
16
65
5.4
59
64
0.2
2.2
0.2
0.2
66
774
59
1 þ RTV
52
53
64

E.J. Velthuisen et al. / European Journal of Medicinal Chemistry 63 (2013) 202e212
207
Fig. 2. Major metabolites of brecanavir-d16 53 in rat and human hepatocytes. Bonds/atoms in red designate possible sites of oxidation. A ¼ metabolite identified in rats,
B ¼ metabolite identified in humans. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
filter cake was washed with cold ethyl acetate. A second crop was
(50 mL) was treated with ethyl chloroformate (1.053 mL,
10.96 mmol). After stirring for one hour a white suspension had
formed. Ammonia gas was bubbled through the mixture for 45 min
to give a thick suspension. The mixture was poured into water and
the layers were partitioned and separated. Solid NaCl was added
and the mixture was extracted with CHCl3/IPA (3:1). The combined
extracts were dried over Na2SO4, filtered and concentrated to give
the crude compound 7 as a white solid. The crude product was
dissolved in THF (20 mL) and cooled to 0 ꢁC. Lithium aluminum
deuteride (14.95 mL,14.95 mmol,1 M in THF) was slowly added and
the mixture was heated at 50 ꢁC for 2 h. The mixture was cooled to
ambient temperature and then to 0 ꢁC. To the mixture was slowly
isolated in a similar manner to give the title compound as a white
solid in >10:1 d.r. (2 g, 63%). 1H NMR (400 MHz, CDCl3)
d 1.36 (s,
9H), 2.71e3.16 (m, 3H), 3.43e3.71 (m, 2H), 3.74e3.94 (m, 1H),
4.40e4.80 (m, 1H), 5.03 (s, 2H), 6.91 (d, J ¼ 8.6 Hz, 2H), 7.12 (d,
J ¼ 8.5 Hz, 2H), 7.27e7.49 (m, 5H); LC/MS (m/z) ESþ ¼ 429 (M þ 23).
4.1.3. tert-butyl ((S)-2-(4-(benzyloxy)phenyl)-1-((S)-oxiran-2-yl)
ethyl)carbamate-d1 (5)
Potassium hydroxide (0.82 mL, 1.5 eq, 1 N) was added to a sus-
pension of 1,1-dimethylethyl [(1S,2S)-3-chloro-2-hydroxy-1-({4-
[(phenylmethyl)oxy]phenyl}methyl)propyl]-d1-carbamate (221 mg,
0.543 mmol) in ethanol (8 mL) and the suspension was stirred for 1 h
at ambient temperature. The yellow solution was concentrated un-
der reduced pressure, diluted with water, the aqueous solution was
extracted with dichloromethane and the organics were dried over
sodium sulfate, filtered and concentrated under reduced pressure to
give the title compound as a light yellow solid (200 mg, 99%).1H NMR
added water (630
mL) and then stirred for 5 min. The mixture was
then treated with 15% NaOH (630
mL) and stirred for 5 min. Lastly,
water (1.9 mL) was added, the mixture was diluted with diethyl
ether and then stirred at ambient temperature for 30 min. The
suspension was filtered over Celite. The filtrate was treated with
HCl (g) for 5 min and the mixture was concentrated. The crude
residue was azeotroped with toluene and MeOH to give 2-methyl-
1-propanamine-d9 as a white solid (1.19 g, quantitative yield). 2H
(400 MHz, CDCl3)
d 1.38 (s, 9H), 2.67e2.99 (m, 4H), 3.39e3.74
(m, 2H), 4.41e4.59 (m, 1H), 4.95e5.11 (m, 2H), 6.86e6.98 (m, 2H),
7.06e7.17 (m, 2H), 7.20e7.51 (m, 5H); LC/MS (m/z) ESþ ¼ 393
(M þ 23).
NMR (61 MHz, CDCl3)
d ppm 0.69e1.15 (m, 6D), 1.88e2.19 (m, 1D),
2.66e3.00 (m, 2D); Rf ¼ 0.15 (80:19:1 DCM/MeOH/NH4OH).
4.1.4. 2-methyl-1-propanamine-d9 (8)
4.1.5. 1,3-benzodioxole-d2 (10)
An ice cold solution of 2-methylpropanoic acid-d7 (1.0 mL,
9.96 mmol) and triethylamine (1.666 mL, 11.96 mmol) in DCM
A suspension of catechol (5.0 g, 45.4 mmol) and Cs2CO3
(22.19 g, 68.1 mmol) in N,N-dimethylformamide (DMF) (108 mL)

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E.J. Velthuisen et al. / European Journal of Medicinal Chemistry 63 (2013) 202e212
was treated with chloroiodomethane-d2 (4.96 mL, 68.1 mmol). The
resulting suspension was heated to 110 ꢁC. After 1 h, the reaction
mixture was cooled to ambient temperature and filtered through a
pad of Celite and rinsed with diethyl ether. The filtrate was diluted
with water and extracted with diethyl ether. The combined
organic layers were washed with water, brine, dried (MgSO4),
filtered and concentrated in vacuo. The residue was then diluted
with Et2O and the solids were filtered off. The filtrate was
concentrated to afford 5.39 g (96%) as a brown oil: 1H NMR
stirring and then 1.2 mL 15% NaOH, followed by 10 min stirring and
finally 3.6 mL of H2O. The resulting suspensionwas filtered through a
pad of Celite and concentrated to afford (2-methyl-1,3-thiazol-4-yl)
methanol-d2 (5.05 g, 38.5 mmol, 66% yield) as a yellow oil: 1H NMR
(400 MHz, CDCl3)
d
¼ 7.03 (s, 1H), 2.72 (s, 3H).
4.1.10. 4-(chloromethyl)-2-methyl-1,3-thiazole hydrochloride-d2
(16)
A solution of (2-methyl-1,3-thiazol-4-yl)methanol-d2 (6.0 g,
45.7 mmol) in dichloromethane (229 mL) was treated with thionyl
chloride (9.98 mL, 137 mmol). After 30 min, the reaction mixture
was concentrated in vacuo to afford 4-(chloromethyl)-2-methyl-1,3-
thiazole hydrochloride-d2 (8.26 g, 44.4 mmol, 97% yield) as a brown
(400 MHz, CDCl3)
d
¼ 6.84 (s, 5H).
4.1.6. 1,3-benzodioxole-5-sulfonyl chloride-d2 (11)
A suspension of SO3eDMF complex (7.51 g, 49.1 mmol) in 1,2-
dichloroethane (DCE) (43.4 mL) was treated dropwise with a so-
lution of 1,3-benzodioxole-d2 (5.39 g, 43.4 mmol) in 1,2-
dichloroethane (43.4 mL) and heated to 75 ꢁC. After 18 h, the re-
action mixture was cooled to room temperature and treated
dropwise with oxalyl chloride (4.15 mL, 49.1 mmol) and heated to
65 ꢁC. After 2 h, the reaction mixture was quenched by the slow
addition of water (5 mL). The layers were partitioned and the
combined organics were washed with water, brine, dried (Na2SO4),
filtered and concentrated in vacuo. The residue was purified by ISCO
(0e30% EtOAcehexanes) to afford 1,3-benzodioxole-5-sulfonyl
chloride-d2 (4.86 g, 21.83 mmol, 50% yield) as a white solid: 1H
solid: 1H NMR (400 MHz, DMSO-d6)
d
¼ 7.57 (s, 1H), 2.65 (s, 3H).
4.1.11. (4R,5R)-dimethyl 2,2-dimethyl-1,3-dioxolane-4,5-
dicarboxylate (20)
(2R,3R)-dimethyl 2,3-dihydroxysuccinate (8.0 g, 44.9 mmol) and
p-toluene sulfonic acid (100 mg, 0.526 mmol) were dissolved in 1,1-
dimethoxypropane (35 mL, 285 mmol) and the mixture was heated
at 85 ꢁC for 5 h. Additional p-toluene sulfonic acid (134 mg) was
added and the reaction was heated at 95 ꢁC overnight. The reaction
was allowed to cool to room temperature, diluted with a solution of
sodium bicarbonate, and the aqueous layer was extracted with
diethyl ether. The organic layer was dried over sodium sulfate and
concentrated under reduced pressure to a red oil. High vacuum
distillation gave (4R,5R)-dimethyl 2,2-dimethyl-1,3-dioxolane-4,5-
NMR (400 MHz, CDCl3)
6.96 (d, J ¼ 8.4 Hz, 1H); ES ꢀ MS: 203 ((M ꢀ Cl) þ OH).
d
¼ 7.63 (dd, 1H), 7.42 (d, J ¼ 2.0 Hz, 1H),
4.1.7. tert-butyl ((2S,3R)-1-(4-(benzyloxy)phenyl)-3-hydroxy-4-(N-
isobutylbenzo[d][1,3]dioxole-5-sulfonamido)butan-2-yl)carbamate-
d12 (12)
dicarboxylate (8.34 g, 85%) as
(400 MHz, CDCl3) (ppm) 4.79 (s, 2H), 3.81 (s, 6H), 1.47 (s, 6H).
a
clear yellow oil. 1H NMR
d
2-methyl-1-propanamine-d9, (15.55 mL, 6.84 mmol, 0.44 M in
THF) was added to 1,1-dimethylethyl ((1S)-1-[(2S)-2-oxiranyl]-2-
4.1.12. ((4S,5S)-2,2-dimethyl-1,3-dioxolane-4,5-diyl)dimethanol-d4
(21)
{4-[(phenylmethyl)oxy]phenyl}ethyl)carbamate-d1
(507
mg,
(4R,5R)-dimethyl 2,2-dimethyl-1,3-dioxolane-4,5-dicarboxylate
(4.35 g, 19.94 mmol) was dissolved in diethyl ether and cooled to
0
diethyl ether) was added dropwise to form a thick white suspen-
sion. The reaction was heated to reflux for 2 h and then cooled in an
ice water bath. Water (0.7 mL), 1 N NaOH (0.7 mL) and water
(2.3 mL) were successively added and the mixture was stirred
15 min, filtered through Celite and the filtrate was carefully
concentrated to give (4S,5S)-2,2-dimethyl-1,3-dioxolane-4,5-diyl)
dimethanol-d4 (1.47 g, 45%) as a pale yellow oil. 1H NMR (400 MHz,
1.369 mmol) and heated to 50 ꢁC for 48 h and then concentrated.
The light brown solid was dissolved in dichloromethane (20 mL)
and triethylamine (0.382 mL, 2.74 mmol) was added. The mixture
was cooled to 0 ꢁC and 1,3-benzodioxole-5-sulfonyl chloride-d2
(366 mg, 1.642 mmol) was added. The mixture was stirred at
ambient temperature for 50 min. Water was added and the mixture
was extracted with DCM. The extracts were washed with saturated
NaHCO3, dried over Na2SO4, filtered and concentrated. The residue
was purified by ISCO (0e50% EtOAc/hex) to give the title compound
ꢁC. Lithium aluminum deuteride (20.33 mL, 20.33 mmol, 1 M in
as a white foam. 1H NMR (400 MHz, CDCl3)
d
ppm 1.37 (s, 9H), 2.83e
CDCl3) d (ppm) 3.98 (s, 2H), 1.98 (s, 2H, 1.41 (s, 6H).
2.97 (m, 2H), 3.01e3.10 (m, 2H), 3.67e3.75 (m, 1H), 3.87 (br. s., 1H),
4.61e4.67 (m, 1H), 5.05 (s, 2H), 6.83e6.97 (m, 3H), 7.11e7.21 (m,
3H), 7.30e7.48 (m, 6H); ES þ MS: 629 (M þ 1).
4.1.13. (2S,3S)-butane-1,2,3,4-tetraol-d4 (22)
((4S,5S)-2,2-dimethyl-1,3-dioxolane-4,5-diyl)dimethanol
(2.96 g, 17.81 mmol) was dissolved in a 10% methanol/water solu-
tion (37 mL) and Amberlyst-15 (59 mg) was added and the reaction
was heated at 60 ꢁC overnight. The mixture was cooled to room
temperature, filtered and the filtrate was concentrated to give
(2S,3S)-butane-1,2,3,4-tetraol-d4 (1.89 g, 84%) as a colorless res-
4.1.8. N-((2R,3S)-3-amino-4-(4-(benzyloxy)phenyl)-2-
hydroxybutyl)-N-isobutylbenzo[d][1,3]dioxole-5-sulfonamide-d12
(13)
A solution of tert-butyl ((2S,3R)-1-(4-(benzyloxy)phenyl)-3-
hydroxy-4-(N-isobutylbenzo[d][1,3]dioxole-5-sulfonamido)butan-
2-yl)carbamate-d12 (0.08 g, 0.13 mmol) was dissolved in DCM
(2 mL) and treated with TFA (0.5 mL). After 30 min, the reaction
mixture was concentrated in vacuo to afford the title compound
(0.07 g, 100%) that was used immediately without further purifi-
cation. ES þ MS: 539.32.
idue. 1H NMR (400 MHz, CDCl3)
d (ppm) 4.63 (s, 2H).
4.1.14. (6S,7S)-2,2,3,3,10,10,11,11-octamethyl-4,9-dioxa-3,10-
disiladodecane-6,7-diol-d4 (23)
(2S,3S)-butane-1,2,3,4-tetraol-d4 (148 mg, 1.17 mmol) and
imidazole (176 mg, 2.58 mmol) were dissolved in DMF (2 mL) and
the mixture was cooled to 0 ꢁC. TBDMSCl (354 mg, 2.35 mmol) was
added dropwise as a solution in DMF (5 mL). When the addition
was complete, the ice bath was removed and the reaction was
stirred at room temperature overnight. The mixture was diluted
with water, the aqueous layer was extracted with diethyl ether, and
the organic layer was washed with water. The organic layer was
dried over sodium sulfate and concentrated to give (6S,7S)-
2,2,3,3,10,10,11,11-octamethyl-4,9-dioxa-3,10-disiladodecane-6,7-
4.1.9. (2-methyl-1,3-thiazol-4-yl)methanol-d2 (15)
To a ꢀ78 ꢁC solution of lithium aluminum deuteride (33.3 mL,
33.3 mmol) in THF (39.7 mL) was slowly added ethyl 2-methyl-1,3-
thiazole-4-carboxylate (10 g, 58.4 mmol) in THF (43.7 mL).
Following the addition, the mixture was allowed to warm to ambient
temperature over a 2 h period. The reaction mixture was then
quenched by the slow addition of 1.2 mL H2O followed by 10 min of

E.J. Velthuisen et al. / European Journal of Medicinal Chemistry 63 (2013) 202e212
209
diol-d4 (195 mg, 47%). 1H NMR (400 MHz, CDCl3)
2H), 2.82 (m, 2H), 0.87 (s, 18H), 0.05 (s, 12H).
d
(ppm) 3.65 (m,
brine, dried over sodium sulfate and concentrated under reduced
pressure to give 4-{[(1,1-dimethylethyl)(dimethyl)silyl]oxy}butanal-
d2 (1.49 g, 100%) as a yellow oil. 1H NMR (400 MHz, CDCl3)
d 0.01 (s,
4.1.15. 2-((tert-Butyldimethylsilyl)oxy)acetaldehyde-d2 (24)
6H), 0.85 (s, 9H), 1.76e1.87 (m, 2H), 2.41e2.53 (m, 2H), 9.68e9.85
Sodium periodate (7.08 g, 33.1 mmol) was added to a mixture of
(6S,7S)-2,2,3,3,10,10,11,11-octamethyl-4,9-dioxa-3,10-
(m, 1H).
disiladodecane-6,7-diol-d4 (5.87 g, 16.55 mmol) in DCM (41 mL),
THF (16 mL) and water (41 mL). After stirring at room temperature
overnight, additional sodium periodate (7.2 g) was added until the
reaction was complete by TLC (9:1 hexanes:ethyl acetate). The
mixture was diluted with pentane, extracted aqueous layer with a
9:1 pentane:DCM solution, and washed organic layer with water
and brine. The combined organic layer was dried over sodium
sulfate and carefully concentrated under reduced pressure to afford
2-((tert-Butyldimethylsilyl)oxy)acetaldehyde-d2 (4.21 g, 50%) as a
4.1.20. (3R,3S)-hexahydrofuro[2,3-b]furan-3-ol-d4 (32)
A solution of 4-{[(1,1-dimethylethyl)(dimethyl)silyl]oxy}butanal-
d2 (1.21 g, 5.92 mmol) in tetrahydrofuran (5 mL) was cooled to 0 ꢁC
and treated with and L-proline (0.136 g, 1.182 mmol) and 2-((tert-
Butyldimethylsilyl)oxy)acetaldehyde-d2 (4.2 g, 23 mmol). The reac-
tion was stirred at room temperature for 12 h. Hydrochloric acid
(1.4mL,1 N)wasadded, andthe mixturewasstirredfor48 h. Pyridine
(0.23 mL), toluene (6.4 vol.), and water (3.4 mL) were added and the
mixture was stirred 10 min and then filtered through a pad of Celite.
The organic layer was extracted with water, the aqueous layer was
washed with toluene, and the aqueous layer was concentrated under
reduce pressure to give a yellow oil. Purification by silica gel chro-
matography (20e100% ethyl acetate/hexanes) gave (3R,3S)-hexahy-
drofuro[2,3-b]furan-3-ol-d4 (234 mg, 30% for two steps) as a
colorless oil after distillation. 1H NMR (400 MHz, CDCl3)
9.69 (s, 1H), 0.92 (s, 9H), 0.09 (s, 6H).
d (ppm)
4.1.16. 4-[(phenylmethyl)oxy]-1-butanol-d2 (26)
Ethyl 4-[(phenylmethyl)oxy]butanoate (8.23 g, 45.7 mmol) was
dissolved in THF, cooled to 0 ꢁC and a solution of lithium aluminum
deuteride (1 M diethyl ether, 22.84 mL) was added dropwise. After
stirring 20 min, water (0.84 mL), 1 N sodium hydroxide (0.84 mL),
and water (2.5 mL) were successively added and the mixture was
vigorously stirred for 10-min and filtered through a pad of Celite.
The filtrate was concentrated under reduced pressure to give 4-
[(phenylmethyl)oxy]-1-butanol-d2 (6 g, 85%) as a colorless oil. 1H
colorless oil.1H NMR (400 MHz, CDCl3)
d 1.71e1.80 (m,1H),1.81e1.91
(m, 1H), 2.28 (m, 1H), 2.83 (m, 1H), 4.43 (m, 1H), 5.68 (m, 1H).
4.1.21. (3R)-tetrahydrofuro[2,3-b]furan-3(2H)-one-d4 (33)
DMP (506 mg, 1.19 mmol) was added to a suspension of
(3R,3aS)-hexahydrofuro[2,3-b]furan-3-ol-d4 (100 mg, 0.745 mmol)
and sodium bicarbonate (315 mg, 3.75 mmol) in DCM (3 mL) at 0 ꢁC.
The mixture was stirred at room temperature for 2 h. The reaction
was diluted with a solution of sodium thiosulfate and a sodium
bicarbonate solution was added. The aqueous layer was extracted
with DCM. The combined organics were washed with sodium
thiosulfate solution and sodium bicarbonate solution and then
dried over sodium sulfate and concentrated to a white solid. Puri-
fication by silica gel chromatography afforded (3R)-tetrahydrofuro
[2,3-b]furan-3(2H)-one-d4 (77 mg, 78%) as a white solid.
NMR (400 MHz, CDCl3)
d 1.67 (m, 6 H), 2.11 (s, 1H), 3.50 (m, 2H),
4.50 (s, 2H), 7.00e7.50 (s, 5H).
4.1.17. (4-(benzyloxy)butoxy)(tert-butyl)dimethylsilane-d2 (27)
TBDMSCl (5.39 g, 35.7 mmol) was added to a mixture of 4-
[(phenylmethyl)oxy]-1-butanol-d2 (5 g, 27.5 mmol), imidazole
(2.43 g, 35.7 mmol), and N,N-dimethylformamide (20 mL) and the
mixture was stirred 2 h at room temperature. Water was added, the
aqueous layer was extracted with diethyl ether, and the combined
organics were washed with brine, dried over sodium sulfate and
concentrated under reduced pressure to give (4-(benzyloxy)
butoxy)(tert-butyl)dimethylsilane-d2 (7.94 g, 97%) as a colorless oil.
4.1.22. (3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl acetate (34)
(3R)-tetrahydrofuro[2,3-b]furan-3(2H)-one-d4 (58 mg, 0.439 mmol)
was dissolved in DCM (5 mL) and cooled to 0 ꢁC. Lithium aluminum
deuteride (0.198 mmol, 0.2 mL) was added dropwise and the
reaction was stirred 10 min. Water (0.01 mL), 1 N NaOH (0.01 mL)
and water (0.03 mL) were successively added and the mixture
was filtered through Celite and the filtrate was concentrated
under reduced pressure. The residue was dissolved in THF (5 mL)
and cooled to 0 ꢁC. Sodium carbonate (116 mg, 1.1 mmol), and
DMAP (0.5 mg) were added followed by dropwise addition of
acetic anhydride (0.06 mL, 0.658 mmol). The reaction was stirred at
room temperature for 2 h, filtered, and the filtrate was diluted
with 1 N HCl. The aqueous layer was extracted with DCM and
the combined organics were dried over sodium sulfate and
concentrated to a give (3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl
acetate (69 mg, 89%) as a colorless residue. 1H NMR (400 MHz,
1H NMR (400 MHz, CDCl3)
d 0.03 (s, 6H), 0.88 (s, 9H), 1.50e1.80 (m,
4H), 3.40e3.54 (m, 2H), 4.49 (s, 2H), 7.00e7.50 (m, 5H).
4.1.18. 4-{[(1,1-dimethylethyl)(dimethyl)silyl]oxy}-1-butanol-d2
(28)
A mixture of (1,1-dimethylethyl)(dimethyl)({4-[(phenylmethyl)
oxy]butyl}oxy)silane-d2 (7.94 g, 26.8 mmol), Pd/C (812 mg) and
methanol (10 mL) was stirred under an atmosphere of H2 (40 psi) for
12 h. Acetic acid (4 mL) was added and the mixture was stirredunder
an atmosphere of H2 (50 psi) for 12 h. The suspension was filtered
through Celite and the filtrate was concentrated under reduced
pressure to give 4-{[(1,1-dimethylethyl)(dimethyl)silyl]oxy}-1-
butanol-d2 (5.03 g, 91%) as a colorless oil. 1H NMR (400 MHz,
CDCl3)
d
0.05 (s, 6H), 0.88 (s, 9H), 1.51e1.73 (m, 4H), 3.63 (s, 2H).
CDCl3)
d 5.74 (m, 1H), 3.05 (m, 1H), 2.11 (s, 3H), 1.99 (m, 1H), 1.88
(m, 1H).
4.1.19. 4-{[(1,1-dimethylethyl)(dimethyl)silyl]oxy}butanal-d2 (29)
A ꢀ78 ꢁC solution of oxalyl chloride (0.964 mL, 11.01 mmol)
in dichloromethane (30 mL) was treated slowly with DMSO
(1.56 mL, 22 mmol) and stirred for 10 min. A solution of 4-{[(1,1-
dimethylethyl)(dimethyl)silyl]oxy}-1-butanol-d2 (1.5 g, 7.34 mmol)
in dichloromethane (20 mL) was added slowly and the mix was
stirred 15 min at ꢀ78 ꢁC. Triethylamine (3.07 mL, 22 mmol) was
added slowly and the mix was stirred 10 min at ꢀ78 ꢁC and allowed
to warm to room temperature and stirred 30 min. Saturated sodium
bicarbonate solution and brine were added, the aqueous layer was
extracted with dichloromethane, the organics were washed with
4.1.23. (3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl acetate
(155 mg, 0.875 mmol) was dissolved in a sodium hypo-
phosphate solution (pHw 4) (1 mL) and 1 N NaOH (0.5 mL) was
added to adjust the pHw5. Added two pipet drops of Altus Bi-
ologics Chiro-Clec PC, (0.05 g/mL dry weight suspension) and the
mixture was heated to 40 ꢁC overnight. The pH was adjusted to
w5 with 1 N NaOH and the mixture was heated at 40 ꢁC for
another 7 h at 40 ꢁC. The reaction was cooled to room tempera-
ture, filtered and the filtrate was extracted with DCM. The organic
layer was washed with water several times and the combined

210
E.J. Velthuisen et al. / European Journal of Medicinal Chemistry 63 (2013) 202e212
organic layer was dried over sodium sulfate and concentrated to
give 111 mg of a colorless residue. This material was dissolved in
MeOH (5 mL) and potassium carbonate (84 mg, 0.608 mmol) was
added and the mixture was stirred at room temperature overnight.
The reaction was diluted with ethyl acetate and filtered. The
filtrate was concentrated to give (3R,3aS)-hexahydrofuro[2,3-b]
furan-3-ol-d5 (85 mg) which was used directly in the next step
bubbled through the reaction mixture to evacuate the remaining
ozone. Dimethyl sulfide (10.92 mL, 148 mmol) was cooled to 0 ꢁC
and slowly transferred to the reaction flask which was held
at ꢀ78 ꢁC. The mixture was warmed to ambient temperature and
then concentrated to give tetrahydrofuro[2,3-b]furan-3(2H)-one-
d1 as a pale yellow oil. 1H NMR (400 MHz, CDCl3)
d ppm 2.15e
2.34 (m, 2H), 2.92e3.08 (m, 1H), 3.69e3.85 (m, 1H), 3.97e4.11
(m, 1H), 4.15 (s, 2H).
without purification. 1H NMR (400 MHz, CDCl3)
d
5.61 (m, 1H),
2.77 (m, 1H), 2.24 (m, 1H), 1.77 (m, 1H).
4.1.27. hexahydrofuro[2,3-b]furan-3-yl acetate-d2 (42)
4.1.24. (3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl (4-
nitrophenyl) carbonate-d5 (35)
To a ꢀ78 ꢁC solution of tetrahydrofuro[2,3-b]furan-3(2H)-one-d1
(3178 mg, 24.61 mmol) in THF (15 mL) was added lithium aluminum
deuteride (18 mL, 18.0 mmol, 1 M in THF). The mixture was stirred
for 20 min at ꢀ78 ꢁC and then warmed to 0 ꢁC for 30 min. At 0 ꢁC,
(3R,3aS)-hexahydrofuro[2,3-b]furan-3-ol-d5 (216 mg, 1.6 mmol)
was suspended in DCM (5 mL) and cooled to 0 ꢁC. Pyridine (0.39 mL,
4.79 mmol) and 4-nitrophenylchloroformate (338 mg, 1.68 mmol)
were added and the mixture was stirred 2 h letting ice bath expire.
The mixture was diluted with water and the aqueous layer was
extracted with DCM. The combined organics were washed with a
sodium bicarbonate solution, 1 N HCl, and sodium bicarbonate so-
lution successively and then dried over sodium sulfate. Purification
by silica gel chromatography afforded (3R,3aS,6aR)-hexahydrofuro
[2,3-b]furan-3-yl (4-nitrophenyl) carbonate (46% over 2 steps from
(3R,3aS,6aR)-Hexahydrofuro[2,3-b]furan-3-yl acetate). Chiral SFC
(15% MeOH on an OJH column, at 140 bar, with a temperature of
40 ꢁC and a flow rate of 2 mL/min) of a with racemic sample show
>99% purity (see Supporting information for chiral HPLC trace). 1H
water (760
mL) was added and the mixture was stirred for 5 min. The
mixture was then treated with 15% NaOH (760
mL) and stirred for
5 min. Lastly, water (2.3 mL) was added, the mixture was diluted
with ether and stirred at ambient temperature for 30 min. The
mixture was filtered over Celite and the filtrate was concentrated to
a pale yellow oil. The crude alcohol intermediate was dissolved in
THF (25 mL) and cooled to 0 ꢁC. Sodium carbonate (7825 mg,
73.8 mmol) and DMAP (50.0 mg, 0.409 mmol) were added, followed
by dropwise addition of acetic anhydride (3.48 mL, 36.9 mmol). The
mixturewas stirred at ambient temperature for 2 h and then filtered.
DCM was added and the organic phase was washed with 1 N HCl,
then dried over Na2SO4, filtered and concentrated. The residue was
purified on silica gel (0e50% EtOAc/hexanes gradient) to give hex-
ahydrofuro[2,3-b]furan-3-yl acetate-d2 as a colorless oil. 1H NMR
NMR (400 MHz, CDCl3) d ppm 1.90e2.26 (m, 2H), 3.07e3.25 (m,1H),
5.68e5.95 (m, 1H), 7.39 (d, J ¼ 9.2 Hz, 2H), 8.30 (d, J ¼ 9.2 Hz, 2H).
(400 MHz, CDCl3) d ppm 1.82e1.98 (m,1H),1.98e2.06 (m,1H), 2.08e
4.1.25. 3-methylidenehexahydrofuro[2,3-b]furan-d1 (40)
2.14 (m, 3H), 3.06 (d, J ¼ 9.4 Hz,1H), 3.77 (d, J ¼ 9.6 Hz,1H), 3.87e3.96
An ice cold solution of 2,3-dihydrofuran (5.0 mL, 66.1 mmol) in
TMEDA (2.0 mL,13.25 mmol) was treated by dropwise addition of n-
butyllithium (30.9 mL, 77 mmol, 2.5 M solution in hexanes). The
mixture was stirred for 2 h, then cooled to ꢀ78 ꢁC and treated
dropwise with deuterium oxide (3 mL, 166 mmol). The cooling bath
was allowed to expire and the mixture was stirred overnight. The
pale yellow solution was filtered over Celite to remove the pre-
cipitates, dried over sodium sulfate, filtered and the solution was
used crude in the next step. A mixture of n-bromosuccinimide
(12.95 g, 72.7 mmol) in DCM (40 mL) was cooled to 0 ꢁC and treated
with the crude solution of 2,3-dihydrofuran-d1 and propargyl
alcohol (5.76 mL, 98.9 mmol). The ice bath was allowed to expire and
the mixture was stirred for 2 h at ambient temperature. The mixture
was diluted with DCM and then washed with saturated sodium
thiosulfate and saturated sodium bicarbonate. The extracts were
dried over sodium sulfate, filtered and concentrated to give a pale
yellow oil that was used without further purification. A mixture of
tributyltin hydride (21.91 mL, 83 mmol) and AIBN (0.109 g,
0.661 mmol) in toluene (10 mL) was heated to 105 ꢁC and a solution
of the crude 3-bromo-2-(2-propyn-1-yloxy)tetrahydrofuran-d1 in
toluene (10 mL) was slowlyadded. The mixture was heated at 105 ꢁC
for 4 h and then stirred at room temperature overnight. The mixture
was concentrated and then azeotroped with acetonitrile. The
resultant yellow oil was purified on silica gel (0e30% ethyl acetate/
hexanes gradient) to give the desired product as a pale yellow oil
(m, 1H), 3.96e4.04 (m, 1H), 4.08 (d, J ¼ 9.8 Hz, 1H).
4.1.28. (3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl acetate-d2
(43)
Hexahydrofuro[2,3-b]furan-3-yl acetate-d2 (2.30 g, 13.2 mmol)
was dissolved in 0.1 M NaH2PO4 (7 mL) and the mixture was
adjusted to pH w5 by adding 1.0 N NaOH. Altus Biologics Chiro-Clec
PC, 0.05 g/mL dry weight suspension (13.20 mmol) (2 drops) was
added and the mixture was heated to 40 ꢁC for 36 h with periodic
adjustments to pH 5 by addition of 1 N NaOH. The mixture was
cooled and filtered. The filtrate was extracted with DCM and the
extracts were washed with water until TLC (1:1 EtOAc/hexanes)
indicated no alcohol present in the organic layer. The organic phase
was dried over Na2SO4, filtered and concentrated. The residue was
purified on silica gel (0e50% EtOAc/hexanes gradient) to give
(3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl acetate-d2 as
a
colorless oil. 1H NMR (400 MHz, CDCl3)
d
ppm 1.80e1.97 (m, 1H),
1.97e2.06 (m, 1H), 2.11 (s, 3H), 3.06 (d, J ¼ 9.4 Hz, 1H), 3.76 (d,
J ¼ 9.6 Hz, 1H), 3.91 (ddd, J ¼ 10.2, 8.6, 6.1 Hz, 1H), 3.96e4.03 (m,
1H), 4.08 (d, J ¼ 9.6 Hz, 1H).
4.1.29. (3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl (4-
nitrophenyl) carbonate-d2 (44)
A solution of (3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl ace-
tate-d2 (904 mg, 5.19 mmol) in MeOH (15 mL) was treated with
potassium carbonate (861 mg, 6.23 mmol) and stirred until the
reaction was judged complete by TLC. The mixture was concen-
trated and the residue was triturated with EtOAc and DCM, filtered
and concentrated. 1H NMR indicated reversion to the starting ma-
terial. The residue was dissolved in MeOH (15 mL), treated with
potassium carbonate (10 mg) and then stirred overnight at ambient
temperature. The mixture was concentrated to give a pale yellow
oil which was used crude in next step. 1H NMR (400 MHz, CHLO-
(2.17 g, 26%). 1H NMR (400 MHz, CDCl3)
d
ppm 1.94 (m, J ¼ 12.3, 5.3,
1.6, 1.6 Hz, 1H), 2.19 (tt, J ¼ 11.8, 8.2 Hz, 1H), 3.30 (d, J ¼ 8.4 Hz, 1H),
3.81 (ddd, J ¼ 11.1, 8.6, 5.3 Hz,1H), 3.96 (td, J ¼ 8.2,1.6 Hz,1H), 4.35e
4.46 (m, 1H), 4.46e4.57 (m, 1H), 5.06 (dq, J ¼ 12.4, 2.2 Hz, 2H).
4.1.26. tetrahydrofuro[2,3-b]furan-3(2H)-one-d1 (41)
A
solution of 3-methylidenehexahydrofuro[2,3-b]furan-d1
(3.13 g, 24.61 mmol) in DCM (15 mL) and methanol (15 mL)
at ꢀ78 ꢁC was treated with a steady stream of ozone until
starting material was consumed as monitored by TLC. O2 gas was
ROFORM-d)
d
ppm 1.89 (m, J ¼ 12.9, 9.9, 9.9, 8.5 Hz, 1H), 2.22e2.39
(m, 1H), 2.86 (d, J ¼ 9.8 Hz, 1H), 3.65 (d, J ¼ 9.2 Hz, 1H), 3.91 (td,

E.J. Velthuisen et al. / European Journal of Medicinal Chemistry 63 (2013) 202e212
211
J ¼ 9.3, 6.4 Hz, 1H), 4.00 (m, J ¼ 8.6, 5.6, 2.7, 2.7 Hz, 2H). To an ice
cold solution of the crude alcohol in DCM (15 mL) was added pyr-
idine (0.839 mL, 10.38 mmol) followed by 4-nitrophenyl chlor-
oformate (2092 mg, 10.38 mmol). The mixture was stirred at
ambient temperature until complete by TLC (several hours). The
mixture was diluted with DCM and partitioned with water. The
organic phase was washed with 1 N HCl, then saturated NaHCO3,
dried over Na2SO4, filtered and concentrated. The residue was pu-
rified twice on silica gel (0e50% EtOAc/hexanes gradient) to give
2-hydroxy-1-({4-[(phenylmethyl)oxy]phenyl}methyl)propyl]carba-
mate-d14 (32 mg, 0.046 mmol) and dimethoxypropane (0.080 mL,
0.65 mmol) in DCM (1 mL) was treated with p-toluenesulfonic acid
monohydrate (31 mg, 0.16 mmol) and then heated to 40 ꢁC for
90 min. The mixture was cooled to ambient temperature, saturated
NaHCO3 was added and the mixture was extracted with DCM. The
extracts were dried over Na2SO4, filtered and concentrated and
then purified on silica gel (0e50% EtOAc/hexanes gradient) to give
(6aR)-hexahydrofuro[2,3-b]furan-3-yl (4S,5R)-5-{[(1,3-benzodi-
oxol-5-ylsulfonyl)(2-methylpropyl)amino]methyl}-2,2-dimethyl-4-
({4-[(phenylmethyl)oxy]phenyl}methyl)-1,3-oxazolidine-3-carboxy-
(3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl
carbonate-d2 as a white solid (1.13 g, 73%). 1H NMR (400 MHz,
CDCl3)
(4-nitrophenyl)
d
ppm 2.01 (m, J ¼ 13.2, 9.9, 9.9, 8.2 Hz, 1H), 2.13e2.25 (m,
late-d14 as
(400 MHz,CDCl3)
a
pale yellow residue (23 mg, 68%). 1H NMR
1H), 3.15 (d, J ¼ 9.6 Hz, 1H), 3.85e4.11 (m, 3H), 4.17 (d, J ¼ 10.0 Hz,
1H), 7.35e7.45 (m, 2H), 8.26e8.35 (m, 2H).
d
¼ 7.48e7.37 (m, 4H), 7.37e7.32 (m, 1H), 7.21e
7.00 (m, 4H), 6.92 (d, J ¼ 8.6 Hz, 2H), 6.88e6.75 (m, 1H), 5.06 (s,
2H), 4.42e4.06 (m, 1H), 4.03e3.93 (m, 1H), 3.93e3.71 (m, 2H),
3.51e3.23 (m, 2H), 3.09e2.64 (m, 4H), 1.99e1.84 (m, 2H), 1.61e1.54
(m, 3H), 1.53e1.40 (m, 3H); ES þ MS: 737 (M þ 1).
4.1.30. (3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl ((2S,3R)-1-(4-
(benzyloxy)phenyl)-3-hydroxy-4-(N-isobutylbenzo[d][1,3]dioxole-
5-sulfonamido)butan-2-yl)carbamate-d17 (46)
Compound 45 (63 mg, 117 mmol) was dissolved in MeCN (5 mL),
DIPEA (0.06 mL, 0.34 mmol) was added followed by compound 35
(35 mg, 0.117 mmol) and the mixture was heated at 76 ꢁC overnight.
The reaction was concentrated, diluted with sodium bicarbonate
solution, and the aqueous layer was extracted with ethyl acetate.
The organic layer was dried over sodium sulfate, concentrated and
purified by silica gel chromatography to afford the title compound
4.1.33. (3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl (4S,5R)-5-
{[(1,3-benzodioxol-5-ylsulfonyl)(2-methylpropyl)amino]methyl}-4-
[(4-hydroxyphenyl)methyl]-2,2-dimethyl-1,3-oxazolidine-3-
carboxylate-d14 (51)
A
solution of (3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl
(4S,5R)-5-{[(1,3-benzodioxol-5-ylsulfonyl)(2-methylpropyl)amino]
methyl}-2,2-dimethyl-4-({4-[(phenylmethyl)oxy]phenyl}methyl)-
1,3-oxazolidine-3-carboxylate-d14 (23 mg, 0.031 mmol) in EtOAc
(5 mL) was treated with Pd/C, Degussa type (33 mg, 0.031 mmol)
and then stirred under a balloon of hydrogen gas for 2 h. The
mixture was filtered over Celite and the filtrate was concentrated to
give (3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl (4S,5R)-5-{[(1,3-
benzodioxol-5-ylsulfonyl)(2-methylpropyl)amino]methyl}-4-[(4-hy-
droxyphenyl)methyl]-2,2-dimethyl-1,3-oxazolidine-3-carboxylate-
d14 as an off-white solid (19 mg, 92%) which was used crude
without further purification.
(64 mg, 78%) as a white solid. 1H NMR (400 MHz, CDCl3)
d 7.29e7.47
(m, 6H), 7.05e7.20 (m, 3H), 6.81e6.99 (m, 3H), 5.59e5.74 (m, 1H),
5.03 (m, 2H), 4.89 (m,1H), 3.82 (m,1H), 3. 56 (m, 1H), 2.66e3.25 (m,
5H); ES þ MS: 700.34.
4.1.31. (3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl [(1S,2R)-3-
[(1,3-benzodioxol-5-ylsulfonyl)(2-methylpropyl)amino]-2-hydroxy-
1-({4-[(phenylmethyl)oxy]phenyl}methyl)propyl]carbamate-d14
(47)
A solution of 1,1-dimethylethyl [(1S,2R)-3-[(1,3-benzodioxol-5-
ylsulfonyl)(2-methylpropyl)amino]-2-hydroxy-1-({4-[(phenylmethyl)
oxy]phenyl}methyl)propyl]carbamate-d12 (114 mg, 0.178 mmol) in
DCM (2 mL) was treated with TFA (0.4 mL). The pink solution was
stirred for one hour and then concentrated. The residue was parti-
tioned between EtOAc and saturated NaHCO3. The aqueous phase was
extracted with EtOAc. The combined extracts were dried over Na2SO4,
filtered and concentrated. The crude amine was dissolved in MeCN
(2 mL), treated with DIPEA (0.125 mL, 0.714 mmol) and (3R,3aS,6aR)-
hexahydrofuro[2,3-b]furan-3-yl 4-nitrophenyl carbonate-d2 (85 mg,
0.286 mmol) and the mixture was heated to 45 ꢁC for 2 h. The mixture
was cooled to ambient temperature and then concentrated. The res-
idue was dissolved in EtOAc, washed with saturated NaHCO3 (ꢂ3),
dried over Na2SO4, filtered and concentrated. The residue was purified
on silica gel (0e50% EtOAc/hexanes gradient) to give (3R,3aS,6aR)-
hexahydrofuro[2,3-b]furan-3-yl [(1S,2R)-3-[(1,3-benzodioxol-5-ylsul
fonyl)(2-methylpropyl)amino]-2-hydroxy-1-({4-[(phenylmethyl)
oxy]phenyl}methyl)propyl]carbamate-d14 as a white foam (76 mg,
4.1.34. (3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl {(1S,2R)-3-
[(1,3-benzodioxol-5-ylsulfonyl)(2-methylpropyl)amino]-2-hydroxy-
1-[(4-{[(2-methyl-1,3-thiazol-4-yl)methyl]oxy}phenyl)methyl]
propyl}carbamate-d19 (52)
The title compound was prepared using the procedures
described for compound 53 to give the desired product from
compound 13 (54 mg, 0.073 mmol) and thiazole 19 (11 mg,
0.073 mmol) in 66% yield over three steps after purification by
reverse phase HPLC. 1H NMR (400 MHz, CDCl3)
d 1.25e1.75 (m, 3H),
2.60e3.23 (m, 7H), 3.50e4.00 (m, 1H), 4.89e5.03 (m, 1H), 5.64 (m,
1H), 6.81e6.95 (m, 3H), 7.07e7.18 (m, 3H), 7.22 (s, 1H), 7.28e7.36
(m, 1H); HRMS cacld for 723.3504 (M þ H); found 723.3506.
4.1.35. (3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl {(1S,2R)-3-
[(1,3-benzodioxol-5-ylsulfonyl)(2-methylpropyl)amino]-2-hydroxy-
1-[(4-{[(2-methyl-1,3-thiazol-4-yl)methyl]oxy}phenyl)methyl]
propyl}carbamate-d16 (53)
61%). 1H NMR (400 MHz, CDCl3)
d
ppm 1.42e1.58 (m, 1H), 1.60e1.75
A suspension of (3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl
(4S,5R)-5-{[(1,3-benzodioxol-5-ylsulfonyl)(2-methylpropyl)amino]
methyl}-4-[(4-hydroxyphenyl)methyl]-2,2-dimethyl-1,3-oxazolidine-
3-carboxylate-d14 (18.5 mg, 0.029 mmol) and cesium carbonate
(37.3 mg, 0.114 mmol) in DMF (1.5 mL) was treated with 4-(chlor-
omethyl)-2-methyl-1,3-thiazole-d2 (14.37 mg, 0.077 mmol) and
then heated to 70 ꢁC for 4 h. The mixture was cooled to ambient
temperature, water was added and the mixture was diluted with
ethyl acetate. The phases were separated and the organic phase was
washed with water, dried over Na2SO4, filtered and concentrated.
The residue was dissolved in 1,4-Dioxane (0.5 mL) and then treated
with 4 M HCl/dioxane (1 mL, 4.00 mmol) and water (0.2 mL). The
mixture was stirred for 4 h and then concentrated. The residue was
(m, 1H), 2.77 (dd, J ¼ 14.1, 9.4 Hz, 1H), 2.85e3.07 (m, 3H), 3.08e3.21
(m, 1H), 3.58 (s, 1H), 3.66e3.78 (m, 2H), 3.79e3.92 (m, 2H), 3.98 (d,
J ¼ 9.8 Hz, 1H), 4.92 (d, J ¼ 9.2 Hz, 1H), 5.03 (s, 2H), 6.90 (dd, J ¼ 8.5,
1.7 Hz, 3H), 7.09e7.22 (m, 3H), 7.29e7.37 (m, 2H), 7.37e7.46 (m, 4H);
ES þ MS: 697 (M þ 1).
4.1.32. (3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl (4S,5R)-5-
{[(1,3-benzodioxol-5-ylsulfonyl)(2-methylpropyl)amino]methyl}-
2,2-dimethyl-4-({4-[(phenylmethyl)oxy]phenyl}methyl)-1,3-
oxazolidine-3-carboxylate-d14 (49)
A
solution of (3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl
[(1S,2R)-3-[(1,3-benzodioxol-5-ylsulfonyl)(2-methylpropyl)amino]-

212
E.J. Velthuisen et al. / European Journal of Medicinal Chemistry 63 (2013) 202e212
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purified by RP-HPLC to give (3R,3aS,6aR)-hexahydrofuro[2,3-b]
furan-3-yl {(1S,2R)-3-[(1,3-benzodioxol-5-ylsulfonyl)(2-methylpropyl)
amino]-2-hydroxy-1-[(4-{[(2-methyl-1,3-thiazol-4-yl)methyl]oxy}
phenyl)methyl]propyl}carbamate-d16 as a white solid (7.9 mg,
38%). 1H NMR (400 MHz, CDCl3)
d ppm 1.55e1.75 (m, 2 H), 2.76e
2.82 (m, 4H), 2.92e3.03 (m, 3H), 3.12e3.15 (m, 1H), 3.70e3.77
(m, 2H), 3.81e3.91 (m, 2H), 3.99 (d, J ¼ 9.6 Hz, 1H), 4.94 (d,
J ¼ 9.2 Hz, 1H), 6.90e6.93 (m, 3H), 7.14e7.18 (m, 3H), 7.23 (s, 1H),
7.33e7.36 (m, 1H); ES þ MS: 720 (M þ 1).
Conflicts of interest
None.
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Appendix A. Supporting information
Supporting information related to this article can be found at
http://dx.doi.org/10.1016/j.ejmech.2013.02.001.
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