M. Plesescu et al.
[14C]-[(2,5-Dichloro-N-[2-({(1R)-3-methyl-1-[(3aS,4S,6S,7aR)-3a,5,5-
trimethylhexahydro-4,6-methano-1,3,2-benzodioaxaborol-2-yl]
butyl}amino)-2-oxoethyl}benzamide (17).
Upon adding heptanes (3mL), a white solid was formed. After filtration, it
was dried over night under high vacuum to obtain the title compound
(400 mg, 58%).
1H-NMR (DMSO): d 8.93–9.02 (1H, br), 8.68–8.80 (1H, br), 7.75–8.00
(1.5H, m), 7.20–7.50 (1.5H, m), 4.20–4.30 (1H, t), 3.75–3.95 (1H, t), 2.60–2.70
(1H, m), 1.57–1.70 (1H, m), 1.15–1.40 (2H, m), 0.70–0.85 (6H, m). MS: m/z
352 (M+ 1-H2O).
A mixture of [14C]-[(2,5-dichlorobenzoyl)amino]acetic acid (16, 91 mg,
0.36 mmol), (1S,2S,3R,5S)-pinanediol leucine boronate trifluoroacetate
salt (8, 140 mg, 0.37 mmol, 1 eq) and TBTU (130 mg, 0.4 mmol, 1.1 eq)
was stirred in anhydrous DMF (2 mL) at room temperature for 15 min
under an atmosphere of nitrogen. This mixture was cooled in ice bath,
and N,N-diisopropylethylamine (0.2 mL, 1 mmol, 3 eq) was added
dropwise at a rate of 1 drop/30 s. The reaction was allowed to proceed
at 0 ꢀC for 1 h and quenched with water (5 mL). A chunky white solid
was formed, and after stirring at room temperature for 30 min, it was
filtered and dried under high vacuum (165 mg, 90%).
[13C9]-(R)-2,20-(2-(1-(2-(2,5-Dichlorobenzamido)acetamido)-3-
methylbutyl)-5-oxo-1,3,2-dioxaborolane-4,4-diyl)diacetic acid (11)
A solution of citric acid (128 mg, 0.6mmol, 1.1eq) in ethyl acetate (2.5mL)
was prepared by heating at 60ꢀC under an atmosphere of nitrogen.
Separately, a similar solution of [13C9]-(R)-(1-(2-(2,5-dichlorobenzamido)
acetamido)-3-methylbutyl)boronic acid (9, 222mg, 0.6 mmol) was prepared
in ethyl acetate (0.5 mL). It was added dropwise to the hot citric acid
solution. After stirring at 60 ꢀC for 15 min, the solution was cooled slowly
to room temperature, when a cloudy precipitate was formed. Further
cooling in ice bath and filtering followed by drying under high vacuum
at 40 ꢀC provided the desired compound (287 mg, 91%).
[14C]-(R)-(1-(2-(2,5-Dichlorobenzamido)acetamido)-3-methylbutyl)
boronic acid (18)
A
mixture of [
14C]-[(2,5-dichloro-N-[2-({(1R)-3-methyl-1-[(3aS,4S,6S,7aR)-
3a,5,5-trimethylhexahydro-4,6-methano-1,3,2-benzodioaxaborol-2-yl]butyl}
amino)-2-oxoethyl}benzamide (17, 165 mg, 0.33mmol), (2-methylpropyl)
boronic acid (80mg, 0.8mmol, 2 eq) in methanol (10 mL) and hexane
(10 mL) with 1 N HCl (1mL, 1 mmol, 3eq) was stirred at room temperature
for 4 h. Upon completion of reaction by HPLC, the two layers were
separated, and the methanol (bottom) layer was washed with additional
hexane (10 mL). The methanol layer was concentrated, redissolved in
2 mL of 2 N NaOH and washed with dichloromethane (3ꢂ 5 mL). The
aqueous layer was made acidic (pH 5) by adding 1 N HCl dropwise, then
the product was extracted into dichloromethane (5ꢂ 10mL). The
combined organic extracts were dried over MgSO4, filtered and
concentrated down. The residue was redissolved in dichloromethane, and
upon addition of hexane (1.5mL), a white solid formed. Solvent was
removed under a stream of nitrogen to obtain the title compound
(79 mg, 91% radiochemical purity). The impure product was redissolved in
1H-NMR (DMSO): d 12.10–12.40 (1H, br), 10.65–10.90 (1H, br), 9.10–9.30
(1H, br), 8.99–7.75 (1.5H, m), 7.20–7.50 (1.5H, m), 4.45–4.60 (1H, t),
3.95–4.15 (1H, t), 2.85–2.95 (1H, t), 2.60–2.80 (4H, m), 1.57–1.70 (1H,
m), 1.15–1.40 (2H, m), 0.70–0.85 (6H, m).
[14C]-2,5-Dichlorobenzoic acid (14)
To a solution of 1-bromo-2,5-dichlorobenzene (12, 580 mg, 2.5 mmol)
in ether (6 mL) at ꢁ78 ꢀC and under an atmosphere of nitrogen was
added 1.6-M n-butyl lithium in hexanes (1.6 mL, 1 eq) dropwise. The
resulting yellow solution was stirred at low temperature for 30 min.
This solution containing lithiated species 13 was treated at ꢁ78 ꢀC
with a freshly prepared stream of 14CO2 (generated from 100 mCi of
Ba14CO3, 58.4 mCi/mmol, and concentrated H2SO4) for 4 h. The
reaction was quenched by adding water slowly (6 mL) and 5% aq.
Na2CO3 (1 mL). The layers were separated, and the organic layer was
further washed with 5% aq. Na2CO3 (4 mL). The combined basic
extracts were neutralized with concentrated HCl (1 mL), and the
product was extracted into dichloromethane (3ꢂ 8 mL). After drying
over MgSO4 and filtering, the organic solution was concentrated to a
white solid, which was dried under high vacuum to give the title
compound (300 mg, 62%).
a
mixture of water, and acetonitrile and chromatographed on a
Phenomenex Luna C8(2) column (10ꢂ 250mm, 5mm, flow rate: 4 mL/
min, 234 nm, linear gradient elution from15% B to 45% B in 40 min, ramp
up to 45% B in 5 min, then 95% B in 5 min). Radiochemical purity of the
combined fractions was 99% (60mg, 50%).
MS: m/z 345 (M + 1-H2O).
[14C]-(R)-2,20-(2-(1-(2-(2,5-Dichlorobenzamido)acetamido)-3-
methylbutyl)-5-oxo-1,3,2-dioxaborolane-4,4-diyl)diacetic acid (19)
[14C]-Methyl-[(2,5-dichlorobenzoyl)amino]acetate (15)
A solution of citric acid (33mg, 0.17mmol, 1eq) in ethyl acetate (1.5mL) was
prepared by heating at 60ꢀC under an atmosphere of nitrogen. Separately
was prepared a similar solution of [14C]-(R)-(1-(2-(2,5-dichlorobenzamido)
acetamido)-3-methylbutyl)boronic acid, (18, 60mg, 0.17mmol) in ethyl
acetate (0.5mL). It was added dropwise to the hot citric acid solution. After
stirring at 60ꢀC for 10 min, the solution was cooled slowly to room
temperature. It became cloudy as a precipitate formed and further cooled
in ice bath, then filtered and dried under high vacuum at 40 ꢀC to obtain
the desired compound (79mg, 7.3mCi, 90%, 108.8mCi/mg).
A mixture of [14C]-2,5-dichlorobenzoic acid (14, 300 mg, 1.57 mmol),
glycine methyl ester (197 mg, 1.5 mmol, 1 eq), 1-hydroxybenzotriazole
(210 mg, 1.6 mmol, 1 eq) and N,N-diisopropylethylamine (0.55 mL,
3.2 mmol, 2 eq) was dissolved in tetrahydrofuran (7 mL) under an
atmosphere of nitrogen. The solution was cooled in ice bath and finally
added N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride
(300 mg, 1.5 mmol, 1 eq). The resulting yellow solution was stirred at
room temperature overnight. The reaction was quenched by adding
saturated aq. NaHCO3 solution (8mL), and the product was extracted with
dichloromethane (3ꢂ 10 mL). The organic extracts were concentrated to
an orange oil and used as it is in the next step (410 mg, 87%).
1H-NMR (DMSO): d 12.0–12.20 (1H, br), 10.63–10.85 (1H, br), 9.10–9.25
(1H, br), 7.79–7.75 (1.H, s), 7.70–7.58 (2H, s), 4.43–4.22 (1H, t), 3.95–4.15
(1H, t), 2.85–3.2.95 (1H, t), 2.60–2.80 (4H, m), 1.84–1.67 (1H, m), 1.45–1.20
(2H, m), 0.78–1.00 (6H, m).
[14C]-[(2,5-Dichlorobenzoyl)amino]acetic acid (16)
To a solution of [14C]-methyl-[(2,5-dichlorobenzoyl)amino]acetate (15,
410 mg, 1.55 mmol) in methanol (5 mL) was added lithium hydroxide
Acknowledgements
(130 mg, 3.1 mmol, 2 eq) and water (1.5 mL). The reaction was stirred at The authors would like to thank the Medicinal Chemistry and
room temperature overnight. It was acidified with 6 N HCl (1 mL), and Process Chemistry groups at Millennium Pharmaceuticals for
the organic solvent was removed by rotary evaporation. The remaining assistance with this project. We would also like to acknowledge
mixture was partitioned between water (5 mL) and ethyl acetate the analytical group for assistance with the planned good
(10 mL). After the layers were separated, the aqueous extract was further manufacturing practices synthesis of [14C]-MLN9708.
washed with ethyl acetate (2ꢂ 10 mL). The combined organic extracts
were concentrated down to a yellow oil, which was recrystallized from
hot water (5 mL). After cooling to room temperature, a white solid of high
Conflict of interest
radiochemical purity was isolated by filtration (92 mg, 23%).
The authors did not report any conflict of interest.
J. Label Compd. Radiopharm 2013, 56 464–470
Copyright © 2013 John Wiley & Sons, Ltd.
Plesescu, Mihaela
