A route to the γ-secretase inhibitor [2,3,4-3H]BMS299897 via an α-phenylselenide derivative

Article abstract of DOI:10.1002/jlcr.1719

A method for the preparation of [2,3,4-³H]BMS299897 has been developed. The methyl ester of BMS299897 was oxidized to its double bond derivative, via a phenyl selenide. The resulting double bond was reduced with tritium using Wilkinson's catalyst. The tritiated BMS299897 was isolated, after basic hydrolysis, with a specific activity of 1.6 TBq/mmol. Copyright

Full text of DOI:10.1002/jlcr.1719

Research Article
Received 1 September 2009,
Revised 19 October 2009,
Accepted 1 November 2009
Published online 10 December 2009 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1719
A route to the c-secretase inhibitor [2,3,4-
3H]BMS299897 via an a-phenylselenide
derivative
Ã
¨
Jonas Malmquist, Alexandra Bernlind, and Peter Strom
A method for the preparation of [2,3,4-3H]BMS299897 has been developed. The methyl ester of BMS299897 was oxidized to
its double bond derivative, via a phenyl selenide. The resulting double bond was reduced with tritium using Wilkinson’s
catalyst. The tritiated BMS299897 was isolated, after basic hydrolysis, with a specific activity of 1.6 TBq/mmol.
3
Keywords: [3H]BMS299897; H NMR; Wilkinson’s catalyst; secretase
Reactions were typically run under an inert atmosphere of
nitrogen or argon. Tritium gas was handled in a tritium gas
Introduction
Alzheimer’s disease (AD) is a devastating neurodegenerative
disorder and is the most common form of dementia. Pathological
lesions and plaques, consisting of the amyloid b-peptide (Ab), are
found in the brains of the AD patients. Ab is heterogeneously
produced by the sequential cleavages of amyloid precursor
protein (APP) by the two aspartic proteases, b- and g-secretase.
In this work, a method for tritiation of the g-secretase inhibitor
BMS2998971 (Scheme 1) has been developed for the evaluation
in autoradiographic studies. Initial trials showed that halogena-
tion of BMS299897 was difficult to accomplish using multiple
reaction conditions. The working method for the preparation of
[2,3,4-3H]BMS299897 1 was via reduction of a newly created
double bond in the aliphatic chain2.
mannifold system from RC TRITEC AG, Teufen.
1
3H and H spectra were recorded on a Bruker DRX600 NMR
Spectrometer, operating at 640 MHz for tritium and at 600 MHz
3
for proton, equipped with a 5 mm H/1 H SEX probehead with
Z-gradients. 1H decoupled 3H spectra were recorded on samples
dissolved in CD3OD. For 3H-NMR spectra referencing, a ghost
reference frequency was used, as calculated by multiplying the
frequency of internal TMS in a 1H spectrum with the Larmor
3
1
frequency ratio between H and H (1.06663975), according to
the description by Al-Rawi et al. 3 1H spectra were referenced to
TMS that was set to 0 ppm.
Mass spectra were recorded on a Waters LCMS consisting of
an Waters 1525 micro (LC), Waters PDA 2996, and ELS detector
(Sedex 75) and a ZMD single quadrupole mass spectrometer.
HPLC analyses were performed on a Agilent 1100, HPLC-
system with a binary pump, auto-injector, DAD and column
oven, coupled in series with a Packard Radiomatic Flow
Scintillator 525TR, equipped with a solid scintillator (SolarScint)
cell with a volume of 33 mL. A Zorbax SB (C18, 2.1 Â 100 mm,
3.5 mm); the column temperature was set to 401C and the flow
rate to 0.5 ml/min. A linear gradient was applied, starting at
100% 10 mM ammonium acetate in 5% acetonitrile and ending
at 70% acetonitrile, in 13.5 min.
Preparative chromatography was run on a Gilson 305 with a
Gilson UV/VIS-151 and a RAYTEST Ramona equipped with a
Kromasil column (C18, 5 mm, 10 Â 250 mm or C8, 7 mm,
10 Â 250 mM) using acetonitrile/50 mM ammonium acetate in
MilliQ Water.
Results and discussions
BMS299897 was esterified with trimethylsilyldiazomethane in
methanol and toluene at slightly elevated temperature to give
the methyl ester 2 (Scheme 1) in nearly quantitative yield. The
a-carbon was deprotonated with LHMDS and alkylated with
phenylselenyl chloride at a low temperature and the selenide 3
was isolated in a moderate yield. Oxidation of the selenium with
peroxide, with subsequent elimination, created the a,b-unsatu-
rated ester 4 in a moderate yield. Saturation of the double bond
was performed using Wilkinson’s catalyst and tritium-gas to give
ester 5 in a good yield. The final deprotection of 5 was performed
by hydrolysis using lithium hydroxide. The target compound 1
was isolated in good yield with a specific activity of 1.6 TBq/mmol.
Analysis with 1H/3H-NMR of the labeled material shows incor-
poration in all CH2 positions. The proportions of 3H incorporation
were about 1:3:1, in a-, b- and g-position, respectively (Figure 1).
Liquid scintillation analysis was performed on a PACKARD TRI-
CARB 2900TR. Thin layer chromatography (TLC) was performed
on Merck TLC-plates (Silica gel 60 F254) and UV-light (254 nm)
Experimental
¨
AstraZeneca R&D Sodertalje, Medicinal Chemistry, S-151 85 Sodertalje, Sweden
¨
¨
¨
General methods
*Correspondence to: Jonas Malmquist, AstraZeneca R&D So¨derta¨lje, Medicinal
Chemistry, S-151 85 So¨derta¨lje, Sweden.
E-mail: jonas.malmquist@astrazeneca.com
All solvents used were analytical grade and commercially
available. Anhydrous solvents were routinely used for reactions.
J. Label Compd. Radiopharm 2010, 53 44–46
Copyright r 2009 John Wiley & Sons, Ltd.

J. Malmquist et al.
O H
O
F
F
F
F
Se
O
O
O
F
F
i
ii
O
O
O
N
N
S
S
O
O
F
O
F
N
S
Cl
Cl
O
F
Cl
BM S299897
2
3
iii
T
T
T
O H
T
O
O
F
F
O
T
O
F
F
F
O
T
S
v
iv
O
N
S
O
N
O
N
F
O
F
S
Cl
O
F
O
F
Cl
Cl
1
5
4
Scheme 1. (i) TMS-CH2N2 (ii) LHMDS, PhSe–Cl, À781C (iii) KHCO3, H2O2 (iv) 3H2, Wilkinson’s catalyst, (v) LiOH.
(m, 1 H) 6.90–7.01 (m, 1 H) 7.02–7.09 (m, 1 H) 7.09–7.19 (m, 2 H) 7.53
(d, J=7.57Hz, 1H) 7.58 (d, J= 8.35 Hz, 2 H) 7.62–7.68 (m, 1 H)
7.70–7.77 (m, 2 H). LC-MS m/z 543 ([M1Na]1).
(R)-Methyl 4-(2-(1-(4-Chloro-N-(2,5-difluorophenyl)phenyl-
sulfonamido)ethyl)-5-fluorophenyl)-2-(phenylselanyl)-
butanoate (3)
(R)-Methyl 4-(2-(1-(4-Chloro-N-(2,5-difluorophenyl)phenylsulfo-
namido)ethyl)-5-fluorophenyl)butanoate (52 mg, 0.10 mmol)
was dissolved in tetrahydrofuran (1 mL) under argon-atmo-
sphere. The mixture was cooled to À781C. Lithium bis(trimethyl-
silyl)amide (0.13 mL, 0.13 mmol) was added. After 10 min
phenylselenyl chloride (18.93 mg, 0.10 mmol) dissolved in
tetrahydrofuran (0.3 mL) was added. Room temperature was
reached in 15 min. The reaction was quenched with ammonium
chloride (sat., 1 mL), extracted with ethyl acetate (3 Â 1 mL),
dried over sodium sulfate, filtered and concentrated. The crude
product was chromatographed on silica (12 g) using a gradient
of petroleum ether to petroleum ether/diethyl ether 4:1 to give
3 (40 mg, 0.059 mmol) in 59% yield. 1H NMR (CDCl3) d ppm 1.49
(d, 3 H) 1.91–2.06 (m, 1 H) 2.17–2.30 (m, 1 H) 2.63–2.93 (m, 2 H)
3.00–3.26 (m, 2 H) 3.73–3.80 (m, 1 H) 3.82 (s, 1 H) 3.86 (s, 3 H)
3.87–3.94 (m, 1 H) 5.76–5.96 (m, 1 H) 6.53–6.60 (m, 2 H) 6.66–6.81
(m, 1 H) 6.85–6.95 (m, 2 H) 6.95–7.05 (m, 2 H) 7.40–7.47 (m, 3 H)
7.54–7.75 (m, 4 H). LC-MS m/z 704 ([M1Na]1).
Figure 1. 3H-NMR on 30 MBq [2,3,4-3H]-BMS299897.
visualized the spots. Flash column chromatography was
performed on a RedisepTM prepacked column.
(R)-Methyl 4-(2-(1-(4-Chloro-N-(2,5-difluorophenyl)phenyl-
sulfonamido)ethyl)-5-fluorophenyl)butanoate (2)
(R)-4-(2-(1-(4-Chloro-N-(2,5-difluorophenyl)phenylsulfonamido)ethyl)
-5-fluorophenyl)butanoic acid, BMS299897, (49.7 mg, 0.10 mmol)
was dissolved in methanol (0.2 mL) and toluene (1 mL). Trimethyl-
silyldiazomethane (0.2 mL, 0.40 mmol) was added. The mixture was
heated to 501C under N2-atmosphere for 45 min. The solvents were
evaporated and the residue was dissolved in ethyl acetate (2 mL),
washed with sodium carbonate (sat., 2 Â 1 mL), dried over sodium
sulfate, filtered and concentrated to give 2 in almost quantitative
yield. 1H NMR d ppm 1.29–1.34 (m, 1 H) 1.49 (d, J= 5.67 Hz, 1 H) 1.54
(d, J= 6.94 Hz, 2 H) 1.89–2.00 (m, 3 H) 2.48 (t, J=6.86Hz, 3H)
(R,E)-Methyl 4-(2-(1-(4-Chloro-N-(2,5-difluorophenyl)phenyl-
sulfonamido)ethyl)-5-fluorophenyl)but-2-enoate (4)
2.64–2.75 (m, 1 H) 3.08–3.16 (m, 1 H) 3.73 (s, 4 H) 5.83–5.90 (m, 1 H) (R)-Methyl 4-(2-(1-(4-Chloro-N-(2,5-difluorophenyl)phenylsulfo-
6.32–6.41 (m, 1 H) 6.53–6.60 (m, 1 H) 6.61–6.69 (m, 1 H) 6.86–6.87 namido)ethyl)-5-fluorophenyl)-2-(phenylselanyl)butanoate,
3,
J. Label Compd. Radiopharm 2010, 53 44–46
Copyright r 2009 John Wiley & Sons, Ltd.
www.jlcr.org

J. Malmquist et al.
(61.3 mg, 0.09 mmol) was dissolved in ethyl acetate (1 mL) and containing product were pooled and concentrated to give
tetrahydrofuran (0.5 mL). The mixture was cooled to 01C. ꢀ500 MBq of 5. LC-MS m/z centered around 552 ([M1Na]1).
Potassium hydrogen carbonate (50 mg, 0.50 mmol) and hydro-
gen peroxide (100 mL, 0.98 mmol) was added. The mixture was (R)-[2,3,4-3H]-4-(2-(1-(4-Chloro-N-(2,5-difluorophenyl)phe-
nylsulfonamido)ethyl)-5-fluorophenyl)butanoic acid (1)
stirred overnight at 211C. Ethyl acetate (1 mL) was added and
the organic phase was washed with sodium bisulfite (1 M,
2 Â 1 mL), sodium bicarbonate (sat.) and was then dried over
sodium sulfate. The organic phase was filtered and concen-
trated. The crude product was chromatographed on silica (4 g)
using a gradient of dichloromethane to dichloromethane/
methanol 50:1. Final purification was performed by preparative
HPLC using a gradient of 65–85% acetonitrile. 4 (4 mg,
7.63 mmol) was isolated in 8% yield. 1H NMR d ppm 1.45
(d, J = 5.20 Hz, 1 H) 1.51 (d, J = 6.78 Hz, 1 H) 3.63 (dd, J = 17.10,
4.97 Hz, 1 H) 4.06–4.14 (m, 1 H) 5.65–5.91 (m, 1 H) 6.35–6.47
(m, 1 H) 6.65 (t, J = 8.12 Hz, 1 H) 6.69–6.78 (m, 1 H) 6.82–6.94 (m,
1 H) 6.98 (d, J = 9.30 Hz, 1 H) 7.01–7.06 (m, 1 H) 7.14 (ddd,
J = 12.77, 3.70, 3.39 Hz, 1 H) 7.53 (d, J = 8.04 Hz, 1 H) 7.59
(d, J = 8.35 Hz, 1 H) 7.65 (d, J = 7.88 Hz, 1 H) 7.74 (d, J = 8.51 Hz,
1 H). 13C NMR d ppm 19.45, 35.92, 52.27, 55.82, 111.20, 117.73,
117.88, 121.37, 123.35, 130.54, 130.65, 130.73, 130.76, 130.79,
132.74, 136.57, 138.79, 142.03, 145.92, 148.76, 154.33, 154.54,
158.31, 163.33, 164.99, 166.09. LC-MS m/z 546 ([M1Na]1).
(R)-[2,3,4-3H]-Methyl 4-(2-(1-(4-Chloro-N-(2,5-difluorophenyl)phenyl-
sulfonamido)ethyl)-5-fluorophenyl)butanoate (0.809 mg, 1.53 mmol)
was dissolved in acetonitrile (0.7 mL) and water (0.2 mL).
Lithium hydroxide (4 mg, 0.17 mmol) was added. The solution
was stirred at 211C overnight. The mixture was acidified with
hydrochloric acid (2 M, 4 drops) and was then concentrated. The
residue was dissolved in dimethyl sulfoxide (250 mL) and was
purified on preparative HPLC using 62% acetonitrile. An amount
of 354 MBq was isolated in a specific activity of 1.6 TBq/mmol
and a 99.9% purity. The product was stored in absolute ethanol
at 44 MBq/mL. 3H NMR (200 MBq/mL) d ppm 1.87 (m, CHT), 2.34
(m, –CHT–C = O), 3.08 (m, CHT-Ar). LC-MS m/z centered around
514 ([MÀH]À).
Conclusions
A working method for the preparation of [2,3,4-3H]-BMS299897
has been developed with good specific activity.
(R)-[2,3,4-3H]-Methyl 4-(2-(1-(4-Chloro-N-(2,5-difluorophenyl)-
phenylsulfonamido)ethyl)-5-fluorophenyl)butanoate (5)
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J. Label Compd. Radiopharm 2010, 53 44–46