Isotope labeled 'HEA Moiety' in the synthesis of labeled HIV-protease inhibitors - Part 1

Article abstract of DOI:10.1002/jlcr.870

[(S)-1′-((N-tert-Butyloxycarbonyl)amino)-2S-[²H ₅]phenyl-ethyl]oxirane 11, made from [²H ₅]-bromobenzene, was transformed into the HIV-protease inhibitors [2H5J-DPH 153893 and [²H₅]-DPH 140

Full text of DOI:10.1002/jlcr.870

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2004; 47: 821–835.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.870
Research Article
Isotope labeled ‘HEA Moiety’ in the synthesis
of labeled HIV-protease inhibitors – Part 1
I. Victor Ekhato1,*, Yuan Liao1,2 and Mihaela Plesescu1,3
1 Bristol-Myers Squibb Company, Pharmaceutical Research Institute, 5 Research
Parkway, Wallingford, CT 06492, USA
2 Pfizer Global Research and Development, Ann Arbor, MI 48105, USA
3 Millennium Pharmaceuticals, Cambridge, MA 023139, USA
Summary
[(S)-10-((N-tert-Butyloxycarbonyl)amino)-2S-[2H5]phenyl-ethyl]oxirane 11, made from
[2H5]-bromobenzene, was transformed into the HIV-protease inhibitors [2H5]-DPH
153893 and [2H5]-DPH 140662. Both compounds are members of the hydroxyethy-
lamine class of protease inhibitors (HIV-PIs). The method of synthesis is applicable to
members of this class and the HEE group of HIV-PIs. Copyright # 2004 John Wiley
& Sons, Ltd.
Key Words: protease inhibitor; anti-HIV; AIDS; [2H5]-HEA isostere
Introduction
DPH 153893 and DPH 140662, like amprenavir1 and saquinavir,2 are
members of the hydroxyethylamine (HEA) isostere class of HIV protease
inhibitors (HIV-PIs).3 The two compounds are highly potent HIV-aspartyl
protease inhibitors with an average IC90 of 4–8 nM against wild-type viruses.
Both were selected for pre-clinical evaluation as potential new generation anti-
HIV drugs.4
Aspects of the studies involved LC-MS analysis and the stable isotope-
labeled forms of DPH 153893 and DPH 140662 were needed as internal
standards. We wanted to make the penta-deuterated DPH 153893 and DPH
140662 molecules in which the [2H5] is situated at the core as the [phenyl
ring-2H5]-HEA. We preferred to make the penta-deuterated analog based on
the calculation that the molecular weight is sufficiently increased, such that
overlap with other ions due to isotopic mass distribution is avoided.
*Correspondence to: I. V. Ekhato, Bristol-Myers Squibb Company, Pharmaceutical Research Institute,
5 Research Parkway, Wallingford, CT 06492, USA. E-mail: ihoezo.ekhato@bms.com
Copyright # 2004 John Wiley & Sons, Ltd.
Received 15 July 2003
Revised 25 September 2003
Accepted 17 June 2004

822
I. V. EKHATO ET AL.
Since both compounds possess the HEA core structure, we planned to
prepare DPH 153893 and DPH 140662 from a common labeled precursor
bearing the elements of the [phenyl ring-2H5]-HEA moiety. We believe that the
phenyl group of the HEA sub-unit is metabolically stable, since metabolites
have not been reported.5 Similarly, these metabolites are absent from our
internal study6 of DPH 153893 or DPH 140662. As a result, we were
encouraged to explore the current synthesis with the prospect that a protease
inhibitor containing the [phenyl ring-2H5]-HEA moiety might, in addition to
our initial reason for synthesis, be suitable for in vivo investigation. By routine
reactions we made the required [2H5]-intermediate from [2H5]-bromobenzene
and transformed this compound into [2H5]-DPH 153893 and [2H5]-DPH
140662.
Results and discussion
In the synthetic literature on anti-HIV protease inhibitors, unlabeled (1S, 2S)-
(1-oxiranyl-2-[2H5]phenylethyl)-carbamic acid tert-butyl ester 11 is extensively
used as an intermediate3,7 for synthesis of aspartyl protease inhibitors. We
considered 11 to be an attractive key intermediate from which to make [2H5]-
protease inhibitors. Compound 11, Figure 1, has the epoxide functionality from
which to build [2H5]-DPH 153893 and [2H5]-DPH 140662. In addition, 11
provides the [phenyl ring-2H5]-HEA isostere core of both inhibitors. Unlike
previous work,7 the present approach does not draw on any chiral pools of
reagents, such as optically pure amino acids or derivatives, to construct 11.
Therefore, constructing the [2H5]-labeled carbon skeleton from [2H5]-bromo-
benzene 1 with the desired 1S, 2S-configuration in 11 was a challenging task.
To address this point, we implemented a facially selective syn-aldol
condensation reaction with S-4-benzyl-(3-[2H5]phenylpropionyl)-oxazolidin-
2-one 5. [2H5]-Bromobenzene was used to prepare compound 5, and the
Figure 1. Structures of [2H5]-epoxide intermediate, [2H5]-DPH 153893 and
[2H5]-DPH 140662
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 821–835

SYNTHESIS OF LABELED HIV-PROTEASE INHIBITORS
823
sequence of routine reactions that was employed is shown in Scheme 1. Thus,
following a lithium-halogen exchange reaction with [2H5]-bromobenzene 1,
DMF was added and acid workup provided the aldehyde, 2. A Wittig-type
homologation protocol8 with compound 2 furnished 3-[2H5]phenylacrylic
acid ethyl ester, 3. The carboxylic acid made by the hydrolysis of 3 was
activated as the mixed pivaloyl anhydride, and reacted with (S)-(-)-4-benzyl-
N-lithio-2-oxazolidinone, according to a literature procedure,9 to form (S)-4-
benzyl-3-(3-[2H5]phenylacryloyl)-oxazolidin-2-one, 4. Compound
4
was
saturated by subjecting it to Pd/C catalyzed hydrogenation to furnish (S)-4-
benzyl-3-(3-[2H5]phenylpropionyl)-oxazolidin-2-one, 5.
2H
2H
2H
2H
2H
2H
2H
CHO
2H
2H
Br
(i) BuLi, DMF
-78oC
(ii) 0.5 N HCl
NaH, DMF
(EtO)2P(O)CH2CO2Et
CO2Et
2H
2H
2H
92 %
2H
1
2H
2
2H
3
O
O
(i)LiOH, MeOH; dil. HCl
(ii) NMM, Me2CCOCl
O
2H
O
O
N
2H
O
2H
2H
O
N
(1) 10 % Pd/C, H2
THF:EtOAc (1:1)
Bn
2H
2H
O
NLi
Bn
(iii)
Bn
2H
84 %
2H
2H
5
2H
4
Scheme 1. Conversion of [2H5]-bromobenzene into the acyl oxazolidinone 5 to
enable the asymmetric synthesis of 11
To complete the required carbon skeleton and simultaneously achieve the
desired (S, S)-configuration, compound 5 was condensed with (benzyloxy)
acetaldehyde,10 as shown in Scheme 2. The combination of dibutylboron
triflate and diisopropylethylamine reagent resulted in the highly diastereofa-
cially selective syn-aldol condensation reaction yielding 6. In the manner
described in the literature,11 lithium peroxide was used to cleave the amide
bond in compound 6 to obtain (2S,3S)-[(2-[2H5]benzyl-4-(benzyloxy)-3-
hydroxybutyric acid, 7, in 84% yield. Compound 7 was refluxed in a solution
of toluene containing diphenylphosphoryl azide and triethylamine to produce
8, an oxazolidinone. Formation of compound 8 is explained by the intra-
molecular cyclization reaction of the intermediate isocyanate with the
participation of the neighboring hydroxy group, as described in the
literature.10 The deprotection of 8 and the formation of (1S,2S)-1-[N-tert-
butyloxycarbonyl)amino]-1-[25H5]benzyl-3-(benzyloxy)propan-2-ol, 9, was
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 821–835

824
I. V. EKHATO ET AL.
Scheme 2. Sequence of reactions to create desired (S,S)-configuration
achieved in a one-pot procedure. Compound 5 was subsequently hydrogenated
under Pd/C catalysis to yield 10 (see Scheme 2).
The diol grouping in 10 was converted to the epoxide ring by a modified
literature protocol12 to give 11 (Scheme 3). Specifically, compound 10 was
treated with 1-(bromocarbonyl)-1-methylethyl acetate in ethanol-free chloro-
form at 08C, converting it to an intermediate bromohydrin. The bromohydrin
was cyclized by treatment with a solution of sodium methoxide in THF to
provide (1S, 2S)-(1-oxiranyl-2-[2H5]phenyl-ethyl)-carbamic acid tert-butyl
ester, 11, in 73% yield.
From intermediate 11 we proceeded to make [2H5]-DPH 153893 and [2H5]-
DPH 140662. At the second step in the reaction sequence isobutylamine was
reacted with 11 in hot isopropyl alcohol to give 12. Compound 12 is the last
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 821–835

SYNTHESIS OF LABELED HIV-PROTEASE INHIBITORS
825
Scheme 3. Elaboration of [2H6]-epoxide 11 into 14 via compound 12, a common
intermediate to [2H6]-DPH 153893 and [2H5]-DPH 140662
intermediate common to both synthetic targets, i.e. the reaction step after 12 is
the point of divergence of the reaction sequences to DPH 140662 and DPH
153893.
In order to make [2H5]-DPH 140662, compound 12 was treated with 4-nitro-
benzenesulfonyl chloride in the presence of potassium carbonate as acid
scavenger. After removal of the Boc group, 13 was obtained as the
methanesulfonic acid salt in 80% overall yield over the two steps. By a
standard coupling method with EDC.HCl, 4-methylmorpholine and HOBt13
in DMF, compound 13 and Boc-l-tert-leucine formed the Boc protected 14.
We found that the stepwise sequence of coupling Boc-l-tert-leucine to make
the Boc protected 14, the removal of Boc to yield 14 and the coupling reaction
of compound 14 with IS066 (IS066 was generously supplied by the
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 821–835

826
I. V. EKHATO ET AL.
Department of Chemical Process, DuPont Pharmaceuticals Company,
Wilmington, DE) to give 15, as shown in Scheme 4, was the best sequence
for making [2H5]-DPH 140662. An alternative single coupling reaction to
DPH 140662 from 13 was complicated by intra-molecular cyclization of the
dipeptide reagent under our reaction conditions. Chromatographic purifica-
tion was required to obtain 15, and then only in poor yield. The single
coupling approach was therefore abandoned.
Scheme 4. Conversion of 14 into [2H5]-DPH 140662
A standard coupling protocol in DMF containing EDC.HCl, HOBt and
4-methylmorpholine as acid scavenger converted 14 to compound 15. After
Pd/C catalyzed hydrogenation of 15 to the 4-aminobenzenesulfonamide, the
Boc group was removed with TFA-Et3SiH and the reaction mixture was
neutralized with NaHCO3 to provide [2H5]-DPH 140662 in 61% yield.
By substituting 3-nitrobenzenesulfonyl chloride for 4-nitrobenzenesulfonyl
chloride in Scheme 3, and following a sequence of reactions analogous to
Schemes 3 and 4, we accomplished the synthesis of [2H5]-DPH 153893 in
comparable yield.
Conclusion
Two HIV protease inhibitors were specifically labeled with [2H5] at the HEA
sub-unit by transforming [2H5]-bromobenzene into a common intermediate for
these agents. The intermediate was made using established reactions that
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 821–835

SYNTHESIS OF LABELED HIV-PROTEASE INHIBITORS
827
included an asymmetric reaction step. The intermediate was used to create the
[phenyl ring-2H5] labeled HEA core structure of DPH 140662 and DPH
153893. Very high deuterium incorporation was achieved by the sequence we
have described and the synthetic approach may be applicable, with some
modifications, to other HIV-protease inhibitors.
Experimental
All reactions were carried out under an atmosphere of argon unless otherwise
specified. Solvents were commercial grade and used without purification or
drying. Column chromatography was carried out on Merck Kiesegel 60
(230 m) silica gel. Flash chromatographic separations were on a Biotage Flash
System using a pre-packed silica gel cartridge. TLC visualization reagents
included (10% iodine plus 10% AcOH) in 40% aqueous KI. 1H NMR spectra
were recorded at 300, 400 or 500 MHz. Chemical shifts are given in ppm
relative to tetramethylsilane (TMS). Only the important IR absorptions
(cmꢀ1) and the molecular ion and/or base peaks in MS are given. HPLC
columns used include Zorbax Rx C18, Discovery Amide C16, and Waters
Symmetry C18 columns.
4S-Benzyl-3-(3-[2H5]phenyl-propionyl)-oxazolidin-2-one, 5
%
To a stirred solution of [2H5]-phenylacrylic acid (10.1 g, 65.07 mmol, see
Scheme 1 for preparation) and 4-methylmorpholine (7.15 ml, 65.07 mmol) in
dry THF (120 ml) maintained at ꢀ788C with a dry ice-acetone bath was added
trimethylacetyl chloride (8.01 ml, 65.07 mmol). After 30 min the cold bath was
replaced with an ice-water bath and the reaction was stirred for another
45 min. The precipitate was removed by filtration, and the filtrate was
transferred to a solution of N-lithio-oxazolidinone that was pre-generated as
follows. (S)-(-)-4-Benzyl-2-oxazolidinone (11.53 g, 65.07 mmol) in dry THF
(120 ml) was stirred under argon atmosphere at ꢀ788C while BuLi (1.6 M
solution in hexanes, 40.6 ml, 65.07 mmol) was added dropwise. The solution
was stirred at ꢀ788C for 1.0 h and the above filtrate, a solution of mixed
anhydride in THF, was added in one portion. The reaction was quenched
after 1 h with saturated NH4Cl solution (100 ml) and the organic phase
was separated. The aqueous portion was further extracted with ethyl acetate
(3 ꢁ 200 ml), the combined organic extract was dried and evaporated
to dryness to give (S)-4-benzyl-3-(3-[2H5]phenylacryloyl)-oxazolidin-2-one
4. Compound 4 was dissolved in ethyl acetate (150 ml) and a suspension of
10% Pd/C (1.2 g) in ethyl acetate (20 ml) was added. After the reaction mixture
was degassed, it was stirred under a hydrogen atmosphere for 3 h and filtered
through a pad of Celite to remove the spent catalyst. The filtrate was
evaporated to a product which crystallized from EtOAc-hexane to give 5
1
(17.10 g, 84%). H NMR (400 MHz) d 2.74 (dd, 1H), 3.03 (m, 2H), 3.28 (m,
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 821–835

828
I. V. EKHATO ET AL.
3H), 4.16 (m, 2H), 4.65 (m, 1H), 7.17 (d, 2H) and 7.32 (m, 3H). 13C NMR
(75 MHz) d 30.30, 37.12, 37.94, 55.17, 66.26, 127.4, 128.0, 128.2, 129.0, 129.5,
140.4, 153.4 and 172.5. IR nmax (CHCl3) 2276, 1785, 1699, 1490, 1478, 1390,
1372, and 1202 cmꢀ1
.
4S-Benzyl-3-[(2S-[2H5]benzyl-4-(benzyloxy)-3S-hydroxy-butyryl)]-oxazoli-
din-2-one, 6
%
To a stirred solution 4S-benzyl-3-(3-[2H5]phenylpropionyl)-oxazolidin-2-one 5
(23.68 g, 75.3 mmol) in CH2Cl2 (140 ml) at 08C was added dibutylboron triflate
(1.0 M solution in dichloromethane, 98.0 ml, 98 mmol) followed by diisopro-
pylethylamine (19.67 ml, 112.97 mmol). After stirring the mixture for 1 h at
room temperature it was cooled to ꢀ788C and a solution of (benzyloxy)ace-
taldehyde (20 g, 133.17 mmol) in CH2Cl2 (30 ml) was added. The reaction
mixture was stirred for 30 min at ꢀ788C and warmed to room temperature for
2 h. A phosphate buffer of pH 7.3 (40 ml) was added and while the reaction
was cooled to 08C, methanol (140 ml) was added followed by a mixture of (2:1)
MeOH and 30% H2O2 (180 ml). The resulting mixture was stirred for 1 h,
concentrated to a small volume and extracted with EtOAc (2 ꢁ 200 ml). The
organic portions were combined and washed with saturated NaHCO3
(200 ml), brine (200 ml) and dried over anhydrous magnesium sulfate.
Evaporation of the solvent gave the crude product which was purified by
chromatography on silica gel. The column was eluted with 10–25% ethyl in
hexane to give 6 (25 g, 71.46%). 1H NMR (500 MHz) d 2.10 (dd, Hz, 1H), 2.64
(brs, 1H), 2.83 (dd, 1H), 3.08 (q, Hz, 1H), 3.17 (q, 1H), 3.59 (m, 2H), 3.78 (tr,
1H), 3.86 (q, 1H), 4.18 (q, 1H), 4.42 (m, 1H), 4.50 (s, 2H), 4.64 (m, 1H), 6.93
(dd, 2H), 7.20–7.343 (m, 10H). IR nmax (CHCl3) 2274, 1778, 1694, 1496, 1386,
1210, and 1104 cmꢀ1
.
S-2-[2H5]Benzyl-4-(benzyloxy)-3S-hydroxy-butyric acid, 7
%
To a stirred solution of 4S-benzyl-3-[(2S-[2H5]benzyl-4-(benzyloxy)-3S-hydro-
xy-butyryl)]-oxazolidin-2-one 6 (23.1 g, 49.72 mmol) in 50% aqueous THF
(105 ml) at 08C were added 30% H2O2 (25 ml, 220 mmol) and LiOH.H2O
(4.25 g, 101.2 mmol) in succession. The reaction mixture was stirred in an ice
bath and monitored by TLC until acyloxazolidinone was consumed in a
calculated 6 h. Saturated sodium metabisulfite (100 ml) was added and the
mixture was extracted with EtOAc (3 ꢁ 200 ml). After acidification of the
aqueous portion to pH 2 with dilute HCl, it was extracted with EtOAC
(2 ꢁ 120 ml). The organic portions were combined, dried over anhydrous
Na2SO4, filtered and evaporated to an oil. TLC on silica gel (ether: pet. ether:
acetic acid, 60/40/1 v/v/v) showed fast running desired product and slow
running oxazolidinone. The mixture was applied to a column of silica gel and
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 821–835

SYNTHESIS OF LABELED HIV-PROTEASE INHIBITORS
829
eluted with (ether: pet. ether: acetic acid 60/40/1,v/v/v/) to afford crystalline
1
(CH2Cl2-hexane) 7 (12.98 g, 84%). H NMR (500 MHz) d 2.95 (m, 1H), 3.10
(2H), 3.49 (dd, 1H), 3.59 (dd, 1H), 4.03 (m, 1H), 4.54 (s, 2H), 7.31–7.36 (m,
5H). 13C NMR (75 MHz) d 33.67, 50.09, 70.38, 71.64, 73.57, 127.9, 128.0,
128.2, 128.3, 128.6, 137.6, 138.6, and 177.9. IR nmax (CHCl3) 3254, 2275, 1728,
1454 and 1169 cmꢀ1
.
S-4-[2H5]Benzyl-5S-((benzyloxymethyl)-oxazolidin-2-one, 8
%
A mixture of S-(2-[2H5]benzyl-4-(benzyloxy)-3S-hydroxy-butyric acid 7
(11.0 g, 36.0 mmol), diphenylphosphoryl azide (9.84 ml, 45.66 mmol) and
triethylamine (6.68 ml, 47.92 mmol) in toluene (78 ml) was refluxed for 4 h
and cooled to room temperature. Ethyl acetate (250 ml) was added and the
solution was washed with saturated NaHCO3 (100 ml), dried and evaporated
to give a residue that was chromatographed on silica gel. The fast running
impurities were first eluted with 25% EtOAc in hexane, and 35% EtOAc in
1
hexane was used to elute the desired product 8 (11.7g, 98%). H NMR
(300 MHz) d 2.75 (dd, 1H), 2.92 (dd, 1H), 3.797 (d, 2H), 4.12 (m, 1H), 5.62 (s,
2H), 5.8 (m, 2H) and 7.40 (m, 5H).
(S)-1-[N-tert-Butyloxycarbonyl)amino]-1-[2H5]benzyl-3-(benzyloxy)propan-
2S-ol, 9
%
S-4-[2H5]Benzyl-5S-(benzyloxymethyl)-oxazolidin-2-one 8 (11.7 g, 38.74 mmol)
in 50% aqueous ethanol (40 ml) containing KOH (9.0 g, 160.4 mmol) was
heated at 708C for 3 h and then cooled to room temperature. The solution was
adjusted to pH 7 with 1.0 M HCl and concentrated under reduced pressure.
Di-tert-butyl dicarbonate (16.99 g, 77.84 mmol) in CH2Cl2 (60 ml) was added
at 08C and the reaction was stirred for 2 h at room temperature. Saturated
NH4Cl solution (40 ml) was added. The mixture was extracted with CH2Cl2
(2 ꢁ 50 ml), the combined organic portions were dried and evaporated to yield
a solid residue. The crude product was applied to a column of silica gel and the
excess di-tert-dibutyl dicarbonate was eluted with 10% EtOAc in hexane.
Further elution with 35% EtOAc in hexane gave a solid which crystallized
from hexane to afford 9 (15.2g, 94%). 1H NMR (300 MHz) d 1.35 (s, 9H), 2.62
(brs), 2.88 (d, 2H), 3.56 (m, 2H), 3.73 (brs, 1H), 3.93 (brs, 1H), 4.51 (s, 2H),
4.72 (d, 1H), 7.33 (m, C6H5). 13C NMR (75 MHz) d 28.34, 36.36, 54.20, 71.72,
71.82, 73.68, 79.53, 125.9(tr) 127.8, 127.9, 128.1, 128.5, 129.1(tr), 137.8, 137.9
and 155.9. IR nmax (CHCl3) 1685, 2273, 1526, and 1172 cmꢀ1
.
S-1-[N-tert-Butyloxycarbonyl)amino]-1-[2H5]benzyl-propan-2S,3-diol, 10
To a solution of S-1-[N-tert-butyloxycarbonyl)amino]-1-[2H5]benzyl-3-(benzy-
loxy)-propan-2S-ol 9 (14.0 g, 37.18 mmol) in EtOAc (140 ml) was added a
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 821–835

830
I. V. EKHATO ET AL.
suspension of 5% Pd/C (1.40 g) in ethyl acetate (10 ml). The stirred reaction
mixture was purged to remove air and stirred under hydrogen atmosphere for
18 h. The spent catalyst was filtered off and the filtrate was concentrated to a
white solid that crystallized from hexane-ethyl acetate to give 10 (10.48 g,
1
ꢂ 100%). H NMR (500 MHz) d 1.37 (s, 9H), 1.59 (brs, 1H), 2.75 (d, 1H),
2.91 (dd, 1H), 3.09 (dd, 1H), 3.33 (brs, 2H), 3.65 (m, 2H), 3.82 (m, 1H), 4.53
(d, 1H). 13C NMR (75 MHz) d 28.30, 36.55, 52.59, 63.06, 73.27, 80.41,
126.2(tr), 128.2 (tr), 129.0 (tr), 137.3, and 157.1. IR nmax (CHCl3) 3356, 2273,
1685, 1525, 1445, 1250, and 1171 cmꢀ1
.
[(S)-10-((N-tert-Butyloxycarbonyl)amino)-2S-[2H5]phenyl-ethyl]oxirane, 11
1-Bromocarbonyl-1-methylethyl acetate (2.0 ml, 13.63 mmol) was added to a
stirred suspension of (S)-1-[N-tert-butyloxycarbonyl)amino]-1-[2H5]benzyl-
propan-2S,3-diol 10 (3.0 g, 10.48 mmol) in dry, ethanol free, chloroform
(30 ml) maintained at 08C in an ice water bath. After 3 h at room temperature
the reaction was quenched by the addition of saturated NaHCO3 solution
(20 ml) and extracted with EtOAc (3 ꢁ 30 ml). The organic extract was washed
with brine (30 ml), dried over anhydrous Na2SO4 and evaporated to give a
solid. The solid was stirred as a solution in dry THF (40 ml), cooled to 08C in
an ice bath and NaOCH3 (0.92 g, 17.03 mmol) was added. After stirring the
reaction mixture at room temperature for 4 h, saturated ammonium chloride
solution (30 ml) was added and the mixture extracted with EtOAc (3 ꢁ 30 ml).
The organic extract was dried over anhydrous Na2SO4, filtered, and the filtrate
was evaporated to a small volume. The material was applied to a column of
silica gel and the compound was eluted with 22% EtOAc in hexane to give a
1
white solid 11 (2.08 g, 73%). H NMR (300 MHz) d 1.75 (d, J=7.0 Hz, 3H),
2.45 (s, 3H), 4.97 (q, J=7.0 Hz, 1H), 7.10–7.27 (m, 3H), 7.32–7.43 (m, 2H),
7.51 (d, J=8.2 Hz, 2H) and 7.75 (d, J=7.3 Hz, 1H). 13C NMR (75 MHz) d
28.34, 37.65, 41.61, 46.81, 53.3, 79.71, 126.2 (tr), 128.1(tr), 129.1(tr), 136.7 and
155.3. IR nmax (CHCl3) 2275, 1679, 1523 and 1168 cmꢀ1
.
3S-[N-(tert-Butyloxycarbonyl)amino-1-(2-methylpropyl)amino-4-[2H5]phe-
nylbutan-2R-ol, 12
To a solution of 2S-[10-(S)-((N-tert-butyloxycarbonyl)amino)-2-[2H5]phenyl-
ethyl]-oxirane 11 (2.06 g, 7.20 mmol) in isopropanol (6 ml) was added
isobutylamine (6 ml, 111.5 mmol) and heated at 868C for 4 h under argon
atmosphere. The solvent was evaporated after cooling to room temperature.
The white solid obtained was crystallized from ether-petroleum ether to give
1
12 (2.32 g, ꢂ 100%). H NMR (500 MHz) d 0.89 (dd, 6H), 1.35 (s, 9H), 1.70
(m, 1H), 2.39 (d, 3H), 2.68 (d, 2H), 2.86 (dd, 1H), 2.98 (dd, 1H), 3.44 (m, 1H),
3.79 (brs, 1H), 4.67 (d, 1H). IR nmax (CHCl3) 3365, 1648, 1521, and 1173 cmꢀ1
.
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 821–835

SYNTHESIS OF LABELED HIV-PROTEASE INHIBITORS
831
N-(3S-Amino-2R-hydroxy-4-[2H5]phenyl-butyl)-N-isobutyl-4-nitro-benzene-
sulfonamide, 13
A solution of K2CO3 (430 mg, 3.11 mmol) in water (3 ml) was added to a
solution of 3S-[N-(tert-butyloxycarbonyl)amino-1-(2-methylpropyl)amino-4-
[2H5]phenylbutan-2R-ol 12 (575.19 mg, 1.68 mmol) in isopropyl acetate (3 ml).
The mixture was stirred at 568C to attain solution. 4-Nitro-benzenesulfonyl
chloride (360 mg, 1.62 mmol) in isopropyl acetate (3 ml) was added and stirring
continued for 30 min after the addition was completed. A solid product
separated upon cooling the reaction to room temperature, and it was collected
by filtration. This material (Boc protected amine) (885.8 mg, 1.681 mmol) was
stirred in isopropyl acetate (18 ml) and the temperature was brought to 858C
under argon atmosphere. Methanesulfonic acid (130.9 ml, 2.0 mmol) in
isopropyl acetate (5 ml) was added slowly and the reaction mixture was
stirred for another 30 min. Upon cooling to room temperature a crystalline
product separated and it was collected by filtration to afford 13 (722.6 mg,
80%). 1H NMR (300 MHz) (DMSO-d6) d 0.89 (dd, 6H), 1.80 (m, 1H), 2.36 (s,
3H), 2.85 (m, 2H), 3.10 (m, 3H), 3.49 (dd, 1H), 3.95 (m, 1H), 5.58 (d, 1H), 7.80
(brs, 2H), 8.15 (d, 1H), 8.40 (d, 1H).
2-Amino-N-{1-[2H5]benzyl-2-hydroxy-3-[isobutyl-(4-nitro-benzenesulfonyl)-
amino]-propyl-3,3-dimethylbutyramide, 14
N-tert-Butyloxycarbonyl tert-butylglycine (Boc-l-tert-leucine) (360.80 mg,
1.56 mmol), 1-hydroxybenzotriazole hydrate (210.8 mg, 1.56 mmol) in anhy-
drous DMF (10 ml) was treated with 4-methylmorpholine (343 ml, 3.12 mmol)
followed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
(298.8 mg, 1.56 mmol). Compound 13 (680 mg, 1.302 mmol) was added and
the reaction mixture was stirred under argon atmosphere overnight. The
reaction mixture was concentrated to a small volume, partitioned between
ethyl acetate (20 ml) and water (15 ml), and the aqueous portion was
separated. The aqueous portion was further extracted with ethyl acetate
(3 ꢁ 20 ml) and the combined organic portions were washed sequentially with
saturated NH4Cl solution (20 ml), saturated K2CO3 solution (20 ml), brine
(20 ml) and dried. The filtrate was evaporated to a residue (843 mg), and then
dissolved in isopropyl acetate (10 ml). While the stirred solution was
maintained at 858C, methanesulfonic acid (102 ml, 1.85 mmol) in isopropyl
acetate (3 ml) was added dropwise. The reaction mixture was cooled to room
temperature after 30 min, and concentrated to a small volume. Upon the
addition of ether, a crystalline solid precipitated, and the material was
collected by filtration to afford 14 (708 mg, 84%). HPLC analysis on a Zorbax
Rx C18 column 4.6 ꢁ 250 mm at a flow rate of 1.0 ml/min under isocratic
conditions using 40% water in acetonitrile containing 0.05% TFA gave a
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 821–835

832
I. V. EKHATO ET AL.
retention time of 3.3 min.1H NMR (300 MHz) (DMSO-d6) d 0.75 (dd, 6H),
0.96 (s, 9H), 1.89 (m, 1H), 2.28 (s, 3H), 2.65 (dd, 1H), 2.82 (m, 1H), 2.96 (dd,
1H), 3.08 (dd, 1H), 3.40 (m, 2H), 3.65 (m, 1H), 3.85 (brs), 4.10 (m, 1H), 7.85
(brs, 2H), 8.10 (d, 1H), 8.38 (d, 1H). IR nmax (CHCl3) 2276, 1691, 1530, 1352,
1350, and 1150 cmꢀ1
.
[{1-{1-[2H5]Benzyl-2-hydroxy-3-[isobutyl-{4-nitro-benzenesulfonyl)-amino]-
propylcarbamoyl}-2,2-dimethyl-propylcarbamoyl}-methyl](3-fluoro-benzyl)-
carbamic acid tert-butyl ester, 15
To a solution of N-[(tert-butyloxycarbonyl)-(3-fluorobenzyl)]glycine (IS066)
(373.95 mg, 1.32 mmol), 1-hydroxybenzotriazole hydrate (HOBT), 178.38 mg,
1.32 mmol) in anhydrous DMF (5 ml) was added 4-methylmorpholine (290 ml,
2.64 mmol) followed by EDC.HCl (252.56 mg, 1.32 mmol). While stirring
under an argon atmosphere, 14 (700 mg, 1.1 mmol) was added. After stirring
overnight, the mixture was concentrated under high vacuum to a small volume
and partitioned between EtOAc (20 ml) and water (15 ml). The organic phase
was separated and the aqueous portion was further extracted with ethyl
acetate (2 ꢁ 20 ml). The combined organic portions were washed with water
(20 ml), saturated NH4Cl solution (20 ml), saturated K2CO3 (20 ml), brine
(20 ml) and dried over magnesium sulfate. The solvent was evaporated to give
a foamy solid 15 (880 mg, 91%). HPLC analysis on a Zorbax Rx C18 column
4.6 ꢁ 250 mm at a flow rate of 1.0 ml/min under isocratic conditions using 40%
water in acetonitrile containing 0.05% TFA gave a retention time of
11.12 min.
N-{3-[(4-Amino-benezenesulfonyl)-isobutyl-amino]-1-[2H5]benzyl-2-hydroxy-
propyl}-2-[2-(3-fluorobenzylamino)-acetylamino]-3,3-dimethyl-butyramide,
[2H5]-DPH 140662
To a solution of 15 (700 mg, 0.869 mmol) in methanol (25 ml) was added a
suspension of 10% Pd/C (224 mg) in methanol (8 ml). The reaction mixture
was stirred at room temperature in an atmosphere of hydrogen for 3 h and
then filtered. The solvent was evaporated to yield a foamy solid. Trifluor-
oacetic acid (2.616 ml) and triethylsilane (Et3SiH) (0.4 ml) were added
successively to a solution of the solid in methylene chloride (18 ml). The
mixture was stirred at room temperature and the reaction was monitored by
TLC (3% MeOH in dichloromethane). After 2 h the reaction mixture was
evaporated to a residue and, EtOAc (20 ml), water (15 ml), and solid NaHCO3
(1.3 g) were added. The mixture was stirred for 20 min and the organic portion
was separated. The aqueous portion was further extracted with ethyl acetate
(2 ꢁ 20 ml) and the combined organic extract was dried over MgSO4.
The product was purified by chromatography on silica gel column eluted
with 8% EtOH in toluene to give an oil. Trituration of the oil with ether gave
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 821–835

SYNTHESIS OF LABELED HIV-PROTEASE INHIBITORS
833
[2H5]-DPH 140662 (385 mg, 61%; D5 98.9%, D4, 1.1%). [a]2D0 – 15.648
(c=0.41% in MeOH). The product was analyzed by HPLC on a Discovery
Amide C16 Supelco 250 ꢁ 4.6 mm, 5 mm column. The solvent combination of A
(10 mmol ammonium formate) and B (acetonitrile) was used under gradient
conditions of 20–30% B in 0–5 min, 30–50% B in 5–18 min and 20–30% B in
18–20 min at a flow rate of ml/min. The product peak was detected by UV at a
wavelength of 254 nm and gave >98% chemical purity at a retention time of
1
14.13 min. IR nmax (CHCl3) 1314, 1285, and 1143 cmꢀ1. H NMR (300 MHz,
d6-DMSO) d 0.85 (m, 15H), 1.82 (m, 1H), 2.52 (dd, 1H), 2.68 (m, 1H), 2.85 (m,
2H), 2.95 (dd, 1H), 3.05 (s), 3.22 (dd, 1H), 3.30 (s, 3H), 3.50 (s), 3.60 (m, 1H),
3.89 (m, 1H), 4.18 (d, 1H), 4.89 (d, 1H), 5.98 (brs, 2H), 6.60 (d, 2H), 7.12 (m,
3H), 7.42 (m, 3H), 7.78 (d, 1H), 7.98 (d, 1H). HRMS (TOF MS ES+; M+1);
calcd. 675.3762, found 675.3766. ESI full ms gave z at 675 (M+1, 100%) and
397 (5%), 372 (15%) and 354 (2%).
N-{1-[2H5]Benzyl-2-hydroxy-3-[isobutyl-(3-nitro-benzenesulfonyl)-amino]-
propyl}-2-[2-(3-fluorobenzylamino)-acetylamino]-3,3-dimethyl-butyramide,
[2H5]-DPH 153893
N-[(tert-butyloxycarbonyl)-(3-fluorobenzyl)]glycine (356.0 mg, 1.256 mmol)
was reacted with 2-amino-N-{1-[2H5]benzyl-2-hydroxy-3-[isobutyl-(3-nitro-
benzenesulfonyl–amino]-propyl-3,3-dimethyl-butyramide
methanesulfonic
acid salt 17 (799 mg, 1.256 mmol) to make [(1{1-[2H5]benzyl-2-hydroxy-
3-[isobutyl-(3-nitro-benzenesulfonyl)-amino]-propyl-carbamoyl}-2,2-dimethyl-
propylcarbamoyl)-methyl]–(3-fluorobenzylcarbamic acid tert-butyl ester
(902 mg, 89%) as described under experiment 15. Sequential hydrogenolysis
and boc deprotection with methanesulfonic acid in isopropyl acetate gave
N-{1-[2H5]benzyl-2-hydroxy-3-[isobutyl-(3-nitro-benzenesulfonyl)-amino]pro-
pyl}-2-[2-(3-fluorobenzylamino)-acetylamino]-3,3-dimethyl-butyramide [2H5]-
DPH 153893 (947 mg, 1.09 mmol, 98%; D5, 98.9%, D4, 1.1%) as the
methanesulfonic acid salt. [a]2D0 – 17.848 (c=0.43% in MeOH). HPLC analysis
on Discovery Amide C16 Supelco 250 ꢁ 4.6 mm, 5 mm column with elution
solvent combination of A (10 mmol ammonium formate) and B (acetonitrile),
under the gradient conditions of 20–30% B in 0–5 min, 30–50% B in 5–18 min
and 20–30% B in 18–20 min and a flow rate of 1.0 ml/min. The product peak
was detected by UV at a wavelength of 254 nm, and gave >99.6% chemical
purity at a retention time of 10.7 min. HRMS (TOF MS ES+; M+1); calcd.
1
675.3762, found 675.3765. H NMR (300 MHz, d6-DMSO) d 0.85 (dd, and s,
overlapped, 15 H), 1.92 (m, 1 H), 2.50 (dd, 1 H overlapped with DMSO), 2.80
(m, 2H), 2.90 (m, 2H), 3.03 (s), 3.50 (s), 3.60 (m, 1H), 3.95 (m, 1H), 4.20 (d,
1H), 4.90 (d, 1H), 5.51 (brs), 6.70 (d, 1H), 6.80 (d, 1H), 6.90 (s, 1H), 7.10 (m,
3H), 7.30 (m, 1H), 6.65 (d, 1H), 7.90 (d, 1H). ESI full MS gave z 675 (M+1,
100%), 397 (2%), 372 (18%), and 354 (5%).
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 821–835

834
I. V. EKHATO ET AL.
Acknowledgements
The authors are grateful to Dr Gerald Miwa and Shimoga Prakash
(Millennium Pharmaceuticals) for their support. IVE gratefully acknowledges
D. R. Schroeder and S. Huang (DAS, BMS Wallingford, CT) for NMR
support; Julie Nielsen (DAS, BMS, Wallingford, CT) for the determination of
Optical Rotations of Protease Inhibitors and Richard E. Gedamke (ARD,
BMS, Lawrenceville, NJ) for the determination of at% D.
References
1. (a) Kim EE, Baker CT, Dwyer MD, Murcko MA, Rao BG, Tung RD, Navia
MA. J Am Chem Soc 1995; 117: 1181–1182. (b) Tung RD, Murcko MA, Bhissetti
GR. Vertex Pharmaceuticals Inc., 1994; WO94/05639.
2. (a) Roberts NA, Martin JA, Kinchington D, Broadhurst AV, Craig JC, Duncan
IB, Gaplin SA, Handa BA, Kay J, Kroehn A, Lambert RW, Merret JH,
Mills JS, Parkes KEB, Redshaw S, Ritchie AJ, Taylor DL, Thomas GJ,
Machin PJ. Science 1990; 248: 358. (b) Skinner C, Sedwick A, Bragman K,
Pinching AJ, Weber J. Abstracts at the 9th International Conference on
AIDS, Berlin, 1993. (c) Parkes KEB, Bushnell DJ, Crackett PH,
Dunsdon SJ, Freeman AC, Gunn MP, Hopkins RA, Lambert RW, Martin JA,
Merrett JH, Redshaw S, Spurden WC, Thomas GJ. J Org Chem 1994; 59:
3656–3664.
3. (a) Rich DH, Green J, Toth MV, Marshall GR, Kent SBH. J Med Chem 1990;
33: 1285–1287. (b) Rich DH, Sun CQ, Vara Prasad JVN, Pathiasseril A,
Toth MV, Marshall GR, Clara M, Mueller RA, Houseman KJ. J Med Chem
1991; 34: 1222–1225. (c) Beaulieu PL, Wernic D. J Org Chem 1996; 61:
3635–3645.
4. Kaltenbach III RF, Trainor D, Getman G, Harris S, Diamond M, Saye JJ,
Erickson-Viitanen S. Antimicrobial Agents Chemother 2001; 45: 3021–3028.
5. Treluyer JM, Bowers G, Cazali N, Sonnier M, Rey E, Pons G, Cresteil T. Drug
Metab Dispos 2003; 31: 275–281.
6. Solon EG, Yang TD. Dept of Drug Metabolism and Pharmacokinetics, Dupont
Pharmaceutical Company, Newark, DE 19714; Personal Communication.
7. Albeck A, Persky R. Tetrahedron 1994; 50: 6333–6346. (b) Thottahthil JK,
Polniaszek RP. Synlett 2000; 6: 902–904. (c) Rotella DP. Tetrahedron Lett 1995;
35: 5453–5456. (d) Beaulieu PL, Wernic D, Duceppe J-S, Guindon Y. Tetrahedron
Lett 1995; 36: 3317–3319. (e) Beier C, Schaumann E, Adiwidjaja G. Synlett 1998;
1: 42–44.
8. (a) Lombardo L, Taylor RJK. Synthesis 1978; 2: 131–132. (b) Coutrot P, Snoussi
M, Savignac P. Synthesis 1978; 2: 133–134. (c) Monache GD, Di Giovanni MC,
Maggio F, Misiti D, Zappia G. Synthesis 1995; 9: 1155–1158.
9. Dobarro A, Velasco D. Tetrahedron 1996; 52: 13525–13530.
10. (a) Ghosh AK, Hussain KA, Fidanze S. J Org Chem 1997; 62: 6080–6082.
(b) Ghosh AK, Fidanze S. J Org Chem 1998; 63: 6146–6152.
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 821–835

SYNTHESIS OF LABELED HIV-PROTEASE INHIBITORS
835
11. Hoekstra MS, Sobieray DM, Schwindt MA, Mulhern TA, Groae TM, Huckabee
BK, Henderickson VS, Franklin LC, Granger EJ, Karrick GL. Org Process Res
Dev 1997; 1: 26–38.
12. (a) Bazin J, Chattopadhyaya J. Synthesis 1985; 12: 1108–1111. (b) Hansske F,
Robins MJ. Tetrahedron Lett 1985; 26: 4295–4298. (c) Verheyden JPH, Moffatt
JG. J Org Chem 1972; 37: 2289–2299. (d) Greenberg S, Moffatt JG. J Am Chem
Soc 1973; 95: 4016–4025. (e) Greenberg S, Moffatt JG. J Am Chem Soc 1973; 95:
4025–4030.
13. Ekhato IV, Huang Y. J Label Compd Radiopharm 1997; 39: 1019–1038.
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 821–835