Synthesis of 13C6-labeled Reyataz (BMS-232632)

Article abstract of DOI:10.1002/jlcr.905

An efficient synthesis of ¹³C₆-labeled Reyataz (BMS-232632) is described. ¹³C₆-labeled Reyataz was synthesized in eight steps from commercially available [ring- ¹³C₆]-L-phenylalanine. The o

Full text of DOI:10.1002/jlcr.905

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2005; 48: 123–130.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.905
Research Article
13
Synthesis of C6-labeled ReyatazTM (BMS-232632)
Richard C. Burrell1,*, Michael W. Lago2, George M. Luke2 and
J. Kent Rinehart3
1Department of Chemical Synthesis, The Bristol-Myers Squibb Pharmaceutical
Research Institute, 5 Research Parkway, Wallingford, CT 06492, USA
2Department of Chemical Synthesis, The Bristol-Myers Squibb Pharmaceutical
Research Institute, 1 Squibb Drive, New Brunswick, NJ 08901, USA
3Department of Chemical Synthesis, The Bristol-Myers Squibb Pharmaceutical
Research Institute, Route 206 and Province Line Road, Princeton, NJ 08540, USA
Summary
An efficient synthesis of C6-labeled ReyatazTM (BMS-232632) is described. 13C6-
13
labeled ReyatazTM was synthesized in eight steps from commercially available
[ring-13C6]-l-phenylalanine. The overall yield of the synthesis was 8% and the purity
of the final product was greater than 98%. Copyright # 2005 John Wiley & Sons,
Ltd.
Key Words: ReyatazTM; Atazanavir; HIV protease inhibitor; carbon-13
Introduction
ReyatazTM (BMS-232632) is a potent human immunodeficiency virus (HIV)
protease inhibitor with a half-life that is suitable for once-daily dosing.1
ReyatazTM has been approved by the FDA to treat patients with HIV. An
important step in the development of ReyatazTM was the validation of rapid,
accurate, and selective assays to quantify the concentration of the drug in
blood samples taken from subjects in clinical studies.2 These assays use LC-
MS-MS to measure the concentration of ReyatazTM relative to an internal
13
standard. The internal standard chosen was C6-labeled ReyatazTM (1).
Isotopically labeled ReyatazTM is an excellent internal standard because its
properties and chromatography are identical to the unlabeled compound. The
only difference is that ReyatazTM has a molecular weight of 704.86 g/mol while
13C6-labeled ReyatazTM (1) has a molecular weight of 710.86 g/mol. This
*Correspondence to: R. C. Burrell, Bristol-Myers Squibb Pharmaceutical Research Institute, 5 Research
Parkway, Department of Chemical Synthesis, MailStop 4BC-377, Wallingford, CT 06492, USA. E-mail:
richard.burrell@bms.com
Copyright # 2005 John Wiley & Sons, Ltd.
Received 7 October 2004
Accepted 12 October 2004

124
R. C. BURRELL ET AL.
N
O
O
OH
O
H
H
N
N
N
OCH3
CH3O
N
N
H
H
O
CH2Ph
*
1
* indicates 13C6
13
Figure 1. Structure of C6-labeled ReyatazTM (1)
difference in molecular weight allows the two compounds to be easily
distinguished by LC–MS–MS. In this paper we describe an efficient synthesis
13
of C6-labeled ReyatazTM (1) (Figure 1).
Results and discussion
The route used to synthesize 13C6-labeled ReyatazTM ([ring-13C6]-1-[4-
(Pyridin-2-yl)phenyl]-5(S)-2,5-bis{[N-(methoxy-carbonyl)-l-tert-leucinyl]ami-
no}-4(S)-hydroxy-6-phenyl-2-azahexane (1)) took advantage of some existing
methodologies developed by Bristol–Myers Squibb’s Discovery Chemistry
department and Process Research & Development department.3,4 The key to
the synthesis was the use of [ring-13C6]-l-phenylalanine as the starting
material. This 13C6-labeled compound is commercially available with high
enantiomeric and isotopic purity.
The synthesis of epoxide 7 was conducted as outlined in Scheme 1. The
amino acid [ring-13C6]-l-phenylalanine (2) was converted to the corresponding
HCl
H2N
O
H
N
O
O
H2N
EtOH, HCl
heat
(t-BuOCO)2O
DIPEA, EtOH
LDA, ClCH2I
THF, -78 °C
OH
Boc
OEt
Boc
OEt
CH2Ph
CH2Ph
CH2Ph
*
2
*
3
*
4
H
H
N
O
H
OH
O
N
Cl
N
Cl
KOH
EtOH
K-Selectride
THF, -78 °C
Boc
Boc
CH2Ph
CH2Ph
CH2Ph
*
*
*
5
6
7
* indicates13 C6
Scheme 1.
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 123–130

SYNTHESIS OF
125
ethyl ester (3) and then Boc-protected to produce 4.5 Treatment of 4 with
ClCH2I and LDA gave the chloromethyl ketone 5.3 Reduction of chloro-
methyl ketone 5 with K-selectride yielded an 8:1 mixture of diastereomeric
alcohols. The major diastereomer (6) was isolated by column chromatography.
Alcohol 6 was treated with KOH to give epoxide 7.
13C6-labeled ReyatazTM (1) was synthesized from epoxide 7 as outlined in
Scheme 2. Epoxide 7 was opened with hydrazine derivative 8 to produce the
chiral alcohol 9.4 Deprotection of 9 with aqueous HCl gave diamine 10.4
Diamine 10 was coupled with carboxylic acid 11 to yield 13C6-labeled
ReyatazTM (1).4 13C6-labeled ReyatazTM (1) was synthesized in an 8% overall
yield with a purity greater than 98%.
N
N
H
N
H
N
OH
O
HN
N
isopropyl alcohol
heat
Boc
+
NHBoc
Boc
NHBoc
CH2Ph
CH2Ph
*
*
7
8
9
N
OH
O
.
3 HCl
H2N
N
HOBT, WSC
DIPEA, CH2Cl2
HCl, THF
heat
NH2
+
CH3O
N
H
CO2H
CH2Ph
*
10
11
N
O
OH
O
H
H
N
N
N
OCH3
CH3O
N
N
H
H
O
CH2Ph
*
O
1
* indicates 13C6
Scheme 2.
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 123–130

126
R. C. BURRELL ET AL.
Experimental procedure
All reagents were obtained from Aldrich Chemical Company and used without
further purification unless otherwise stated. Tetrahydrofuran ultra low water
was obtained from J. T. Baker and used without further purification.
[ring-13C6]-l-Phenylalanine (2) was obtained from Cambridge Isotope
Laboratories with a chemical and enantiomeric purity of greater than 98%.
The hydrazine derivative 8 and carboxylic acid 11 were obtained from Bristol–
Myers Squibb’s Process Research & Development Department.
All experimental procedures were optimized using unlabeled materials. All
glassware was dried and purged with nitrogen or argon before use. All
reactions were monitored by HPLC using the following conditions: YMC
Pack Pro column s-3 mm 4.6 ꢀ 150 mm. Solvent: A=water with 0.05% TFA;
B=acetonitrile with 0.05% TFA. Gradient: 100% A 0–10 min, 100–0% A
10–30 min, 0% A 30–35 min, 0–100% A 35–40 min. Flow: 1 ml/min,
Wavelength 215 nm). HPLC analyses were performed using a Rainin
Dynamax system (model SD-200) equipped with a Varian ProStar PDA
1
detector (model 330). All H NMR spectra were recorded on a Bruker
300 MHz spectrometer using CDCl3 as the solvent.
[ring-13C6]-l-Phenylalanine ethyl ester hydrochloride (3)
Absolute ethanol (110 ml) and [ring-13C6]-l-phenylalanine (2, 3.01 g,
17.59 mmol) were added to a round-bottomed flask.5 Anhydrous hydrochloric
acid was bubbled in for 20 min and the solution was heated to 608C. The
mixture was stirred at 608C under nitrogen for 15.5 h. The reaction mixture
was cooled to room temperature and anhydrous hydrochloric acid was
bubbled in for 5 min. The solution was heated to 608C and stirred for 5 h. At
that time the reaction mixture was cooled to room temperature and
concentrated under reduced pressure. The concentrate was dried under
vacuum for 16 h to give 3.98 g (96% yield) of [ring-13C6]-l-phenylalanine ethyl
ester hydrochloride (3) as a white solid. The white solid coeluted with an
authentic sample of l-phenylalanine ethyl ester hydrochloride when it was
analyzed by HPLC (98% pure).
[ring-13C6]-(tert-Butyloxycarbonyl)-l-phenylalanine ethyl ester (4)
Absolute ethanol (50 ml), [ring-13C6]-l-phenylalanine ethyl ester hydrochloride
(3, 3.98 g, 16.87 mmol), di-tert-butyl dicarbonate (3.68 g, 16.87 mmol) and
diisopropylethylamine (6.47 ml, 4.80 g, 37.11 mmol, 2.2 eq) were added to a
round-bottomed flask. The solution was stirred for 16 h at room temperature
under argon. The reaction mixture was concentrated and the concentrate was
dissolved in EtOAc (60 ml). The solution was washed with H2O (25 ml), 10%
KHSO4 (25 ml) and brine (25 ml). The organic layer was dried (MgSO4),
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SYNTHESIS OF
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filtered and concentrated to obtain a viscous oil. The crude material was
purified by column chromatography (silica, 10% EtOAc/hexanes, TLC Rf ¼
0:53 (ninhydrin) using 30% EtOAc/hexanes) to give 4.59 g (91% yield) of 4 as
1
a white solid. The white solid was analyzed by HPLC (98% pure) and H
NMR: d 7.66–6.77 (m, 5 H), 5.04–4.89 (m, 1 H), 4.62–4.49 (m, 1 H), 4.16 (q,
J¼ 7:2 Hz, 2 H), 3.14–3.02 (m, 2 H), 1.41 (s, 9 H), 1.22 (t, J¼ 7:2 Hz, 3 H).
[ring-13C6]-(1(S)-Benzyl-3-chloro-2-oxo-propyl)-carbamic acid tert-butyl
ester (5)
Anhydrous THF (80 ml), chloroiodomethane (10.81 g, 61.28 mmol) and 4
(4.59 g, 15.32 mmol) were added to a round-bottomed flask.3 The solution was
cooled to ꢁ788C and stirred under argon. An LDA solution (freshly prepared
from n-butyllithium (30.6 ml, 76.59 mmol, 2.5 M solution in hexanes) and
diisopropylamine (11.8 ml, 8.53 g, 84.25 mmol)) at ꢁ788C was added dropwise
to the reaction flask over 40 min via a cannula. As LDA was added, the
reaction mixture changed from a bright yellow suspension to an orange
solution to a brown suspension. The reaction mixture was stirred at ꢁ788C for
an additional 15 min after the addition of LDA was complete. The reaction
was quenched at ꢁ788C by dropwise addition of AcOH:THF (1:2, 75 ml) over
30 min. The reaction mixture was stirred for 10 min at ꢁ788C and then allowed
to warm to room temperature. Brine (50 ml) was added and the mixture was
partially concentrated under reduced pressure. The concentrate was diluted
with EtOAc (200 ml) and the aqueous layer was removed. The organic layer
was washed with saturated NaHCO3 (2 ꢀ 75 ml), 10% NaHSO3 (2 ꢀ 75 ml)
and brine (75 ml). The organic solution was dried (MgSO4), filtered and
concentrated to yield a brown solid. The crude brown solid was partially
purified by column chromatography (silica, 10% EtOAc/hexanes, TLC Rf ¼
0:49 (ninhydrin) using 30% EtOAc/hexanes) to give 3.31 g of 5 as a yellow
solid. The chloroketone 5 coeluted with an authentic sample when it was
analyzed by HPLC (71+% pure).
[ring-13C6]-(1(S)-Benzyl-3-chloro-2(R)-hydroxy-propyl)-carbamic acid tert-
butyl ester (6)
Anhydrous THF (30 ml) and 5 (3.31 g, 10.89 mmol) were added to a round-
bottomed flask and cooled to ꢁ788C. K-Selectride (21.8 ml, 21.8 mmol, 1.0 M
solution in THF) was added to a second flame-dried flask and cooled to
ꢁ788C. The K-selectride solution was added dropwise over 10 min to the
solution of 5 using a cannula. The reaction mixture was stirred under argon for
30 min at ꢁ788C. At that time the mixture was quenched by the addition of
10% KHSO4 (50 ml). The aqueous solution was extracted with EtOAc
(4 ꢀ 50 ml). The combined organic layers were washed with brine (20 ml), dried
(MgSO4), filtered and concentrated to obtain 6.57 g of crude product (ratio of
Copyright # 2005 John Wiley & Sons, Ltd.
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R. C. BURRELL ET AL.
‘‘R’’ to ‘‘S’’ alcohol by HPLC, 8:1). The crude material was purified by column
chromatography (silica, 10% EtOAc/hexanes, TLC Rf ¼ 0:37 (ninhydrin)
using 30% EtOAc/hexanes) to give 1.38 g of 6 as a yellow oil (32% yield for
the two steps based on recovered 4). The yellow oil was analyzed by HPLC
(94% pure) and 1H NMR: d 7.69–6.86 (m, 5 H), 4.93–4.81 (m, 1 H), 3.91–3.69
(m, 2 H), 3.60–3.47 (m, 2 H), 3.10–2.88 (m, 2 H), 1.41 (s, 9 H).
[ring-13C6]-N-(tert-Butyloxycarbonyl)-2(S)-amino-1-phenyl-3(R)-3,4-epoxy-
butane (7)
Absolute ethanol (10 ml), 6 (1.38 g, 4.52 mmol) and KOH (0.30 g, 5.42 mmol,
dissolved in absolute ethanol (20 ml)) were added to a round-bottomed flask.3
The solution was stirred at room temperature under argon for 1 h. At that time
the reaction mixture was concentrated and the concentrate was diluted with
water (50 ml). The aqueous solution was extracted with EtOAc (3 ꢀ 25 ml).
The combined organic layers were washed with brine (25 ml), then dried
(MgSO4), filtered and concentrated under reduced pressure to yield 1.12 g of
crude 7 as an orange oil. The epoxide 7 coeluted with an authentic sample
when it was analyzed by TLC (Rf ¼ 0:41 (ninhydrin) using 30% EtOAc/
hexanes) and HPLC (90% pure).
[ring-13C6]-1-[4-(Pyridin-2-yl)phenyl]-5(S)-2,5-bis[(tert-butyloxy-carbonyl)-
amino]-4(S)-hydroxy-6-phenyl-2-azahexane (9)
Anhydrous isopropyl alcohol (15 ml), 7 (1.12 g, 4.17 mmol) and 8 (1.25 g,
4.17 mmol) were added to a round-bottomed flask.4 The mixture was heated to
758C and stirred for 12 h under argon. At that time the solution was cooled to
room temperature and concentrated under reduced pressure to give a yellow
wax. Column chromatography (silica, 30% EtOAc/hexanes, TLC Rf ¼ 0:11
(ninhydrin) using 30% EtOAc/hexanes) gave 9 contaminated with a small
amount of 8. The product was further purified by washing with TBME (20 ml)
to give 1.10 g of 9 as a white solid (43% for the two steps). The white solid was
analyzed by HPLC (95% pure) and 1H NMR: d 8.85 (brs, 1 H), 8.17–7.83 (m,
4 H), 7.64–7.39 (m, 5.5 H), 7.06–6.83 (m, 2.5 H), 5.39–4.93 (m, 2 H), 4.16–3.83
(m, 2 H), 3.76–3.54 (m, 2 H), 3.04–2.77 (m, 3 H), 2.61–2.44 (m, 1 H), 1.39
(s, 9 H), 1.33 (s, 9 H).
[ring-13C6]-1-[4-(Pyridin-2-yl)phenyl]-5(S)-2,5-bis{[N-(methoxy-carbonyl)-
l-tert-leucinyl]amino}-4(S)-hydroxy-6-phenyl-2-azahexane (1)
Tetrahydrofuran (7 ml), 9 (1.10 g, 1.94 mmol) and aqueous HCl (0.77 ml
9.20 mmol, 12 N solution) were added to a round-bottomed flask.4 The
solution was heated to 608C and stirred for 3 h. Gas evolution was observed
during the first 30 min. The reaction mixture was cooled to room temperature
Copyright # 2005 John Wiley & Sons, Ltd.
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SYNTHESIS OF
129
and concentrated to give crude 10 as a yellow oil. The diamine 10 coeluted
with an authentic sample, when it was analyzed by HPLC (92% pure).
Anhydrous CH2Cl2 (5 ml), 11 (0.92 g, 4.84 mmol), 1-hydroxybenzotriazole
hydrate (0.68 g, 5.04 mmol, 2.6 eq) and 1-[3-(dimethylamino)-propyl]-3-ethyl-
carbodiimide hydrochloride (0.97 g, 5.04 mmol) were added to a round-
bottomed flask.4 Crude 10 (assume 1.94 mmol) and diisopropylethylamine
(1.55 g, 2.10 ml, 12.03 mmol) were dissolved in anhydrous CH2Cl2 (5 ml) and
added to the solution prepared above. The mixture was stirred at room
temperature under argon for 17 h. At that time the reaction mixture was
diluted CH2Cl2 (75 ml) and then washed with water (25 ml), saturated
NaHCO3 (25 ml), and brine (25 ml). The organic solution was concentrated
under reduced pressure to give crude 1 as a yellow foam. The crude material
was partially purified by column chromatography (silica, 10% hexanes/
EtOAc, TLC Rf ¼ 0:31 (UV short wave) using 100% EtOAc, 90% pure by
HPLC). A second purification by column chromatography (silica, 25%
hexanes/EtOAc) gave 0.899 g of 1 as a white solid (65% yield the two steps).
1
The white solid was analyzed by HPLC (98.5% pure) and H NMR: d
8.74–8.67 (m, 1 H), 8.01–7.92 (m, 2 H), 7.85–7.67 (m, 2 H), 7.43–6.84 (m, 8 H),
6.56–6.33 (m, 2 H), 5.37–5.12 (m, 2 H), 4.81 (brs, 1 H), 4.15–3.89 (m, 3 H),
3.83–3.72 (m, 1 H), 3.67 (s, 3 H), 3.63 (s, 3 H), 3.65–3.49 (m, 1 H), 3.02–2.77
(m, 3 H), 2.59–2.48 (m, 1 H), 0.87 (s, 9 H), 0.79 (s, 9 H). The material was also
13C6 H53 N6 O7 (M+H)+:
12
analyzed by HRMS (ES) calculated for
C
32
711.4177, found 711.4183. The isotopic distribution was determined to be:
M+6 (91.4%), M+5 (7.0%), M+4 (1.7%), M+3 (50.1%), M+2 (50.1%),
M+1 (50.1%), M+0 (50.1%).
Conclusion
13C6-Labeled ReyatazTM was synthesized in eight steps from commercially
available [ring-13C6]-l-phenylalanine with an overall yield of 8%. This 13C6-
labeled material was used successfully as an internal standard in an assay
designed to quantify the concentration of ReyatazTM in blood samples taken
from subjects in clinical studies. The validation of this assay was an important
step in the development of ReyatazTM
.
Acknowledgements
We thank Samuel Bonacorsi Jr for helpful conversations and Richard
Gedamke for performing the high resolution MS analysis.
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