N. Tschammer et al.
MED
with CH2Cl2 (10 mL). The combined organic layers were dried over
MgSO4 and concentrated under reduced pressure. The residue was
purified by flash chromatography (3% MeOH in CH2Cl2), yielding
product 3 as a yellow solid (71 mg, 72%). Additionally, 12 mg of 3
were recovered by flash chromatography (17%). The product was
used in the next step without NMR or IR characterization: HR-EIMS
([M+H]+, m/z): 711.3042, calcd: 711.3044; RP-HPLC (254 nm): tR =
21.5 min, purity >95%.
2.3 Hz, 1H), 8.64 (dd, J=7.9, 1.9 Hz, rotamer), 8.58 (dd, J=7.6,
1.9 Hz, 1H), 7.54 (dd, J=7.6, 4.5 Hz, 1H), 7.53 (dd, J=6.8, 3.0 Hz,
rotamer, signal is overlapped with previous multiplet), 7.43 (dd, J=
7.9, 4.9 Hz, 1H), 7.37–7.41 (m, rotamer), 7.33 (dd, J=6.8, 2.6 Hz,
1H), 7.21–7.25 (m, rotamer), 7.12–7.19 (m, overlap of 2 rotamers),
7.11 (d, J=9.4 Hz, 1H), 7.05–7.12 (m, overlap of 2 rotamers), 6.99–
7.05 (m, overlap of 2 rotamers), 5.21 (q, J=7.2 Hz, 1H), 5.03 (q, J=
6.4 Hz, rotamer), 4.07 (qd, J=7.2, 2.3 Hz, overlap of 2 rotamers),
3.79 (d, J=15.5 Hz, 1H), 3.66 (d, J=15.9 Hz, 1H), 3.60 (dd, J=16.2,
7.6 Hz, 1H), 3.54 (dd, J=16.2, 6.8 Hz, 1H), 3.38 (dd, J=13.2, 5.7 Hz,
rotamer), 3.12 (dd, J=14.0, 6.8 Hz, rotamer), 2.90 (m, overlap of 2
rotamers), 2.74 (d, J=11.3 Hz, rotamer), 2.59 (d, J=11.0 Hz, rota-
mer), 2.31 (d, J=16.6 Hz, rotamer), 2.27 (s, 3H), 2.14 (s, rotamer),
1.91 (t, J=11.7 Hz, overlap of 2 rotamers), 1.85 (t, J=11.3 Hz, over-
lap of 2 rotamers), 1.70 ꢀ1.81 (m, overlap of 2 rotamers), 1.52–1.67
(m, overlap of 2 rotamers), 1.47 (d, J=7.2 Hz, 3H), 1.45 (t, J=
6.8 Hz, overlap of 2 rotamers), 1.28–1.40 (m, overlap of 2 rotamers),
1.00 ppm (qd, J=12.1, 3.8 Hz, rotamer); 13C NMR (91 MHz, 300 K,
CDCl3, mixture of rotamers): d=171.7, 170.4, 162.9, 162.6, 160.3,
159.8, 159.6, 157.9, 157.3, 156.9, 156.7, 156.5, 156.2, 136.9, 135.0,
134.8, 131.1, 130.7, 130.6, 129.7, 129.2, 129.0, 129.0, 128.2, 128.1,
127.9, 127.7, 127.7, 123.4, 123.4, 122.4, 121.6, 118.3, 118.0, 117.0,
116.8, 116.5, 116.4, 116.2, 115.7, 115.3, 64.2, 63.8, 60.4, 55.5, 55.3,
54.2, 53.4, 53.3, 51.1, 47.9, 46.2, 39.4, 38.1, 37.4, 35.4, 35.4, 30.9,
30.7, 30.3, 30.2, 29.7, 29.7, 17.8, 16.7, 14.8, 14.6 ppm; IR: n˜ =
2929.34, 2852.20, 2783.74, 1696.09, 1590.02, 1508.06, 1435.74,
1326.79, 1241.93, 1115.62, 1056.80 cmꢀ1. HRMS (ESI, [M+H]+, m/z):
calcd: 626.2749, found: 626.2753; RP-HPLC (254 nm): tR =17.5 min,
purity >98%.
N-{1-[3-(4-Ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimi-
din-2-yl]ethyl}-2-[4-fluoro-3-(trifluoromethyl)phenyl]-N-(piperi-
din-4-ylmethyl)acetamide (4): A solution of 3 (66 mg, 0.093 mmol)
and TFA (0.35 mL, 4.54 mmol) in CH2Cl2 (3 mL) was stirred for 4 h
at room temperature. The reaction mixture was then diluted with
CH2Cl2 (10 mL) and washed with concentrated NaHCO3 and brine.
The organic layer was dried over MgSO4, filtered, and concentrated
under reduced pressure. The residue was purified by flash chroma-
tography (9% MeOH and 0.3% conc. NH3(aq) in CH2Cl2) to give the
1
product as a white foam (48 mg, 85%): H NMR (600 MHz, CDCl3,
mixture of rotamers in 1:0.45 ratio): d=9.06 (dd, J=4.7, 2.1 Hz,
rotamer), 8.94 (dd, J=4.5, 1.9 Hz, 1H), 8.64 (dd, J=7.9, 2.3 Hz, rota-
mer), 8.57 (dd, J=7.9, 1.9 Hz, 1H), 7.60 (dd, J=8.7, 2.6 Hz, 1H),
7.54 (dd, J=7.7, 4.7 Hz, rotamer), 7.41 (dd, J=7.9, 4.5 Hz, 1H),
7.33–7.39 (m, 2H), 7.23–7.26 (m, rotamer), 7.06–7.18 (m, overlap of
2 rotamers), 7.05 (dd, J=8.7, 2.6 Hz, 1H), 7.01 (dd, J=8.7, 3.0 Hz,
1H), 5.12 (q, J=7.2 Hz, 1H), 5.03 (q, J=6.8 Hz, rotamer), 4.07 (q,
J=7.2 Hz, overlap of 2 rotamers), 3.76 (d, J=15.9 Hz, 1H), 3.71 (d,
J=15.9 Hz, 1H), 3.57 (dd, J=15.9, 8.3 Hz, 1H), 3.50 (dd, J=15.9,
6.0 Hz, 1H), 3.37–3.47 (m, 2H), 3.20–3.28 (brd, rotamer), 3.03–3.16
(m, overlap of 2 rotamers), 2.93 (d, J=16.2 Hz, rotamer), 2.80 (brt,
J=12.1 Hz, rotamer), 2.62–2.72 (brt, rotamer), 2.49 (brt, J=13.4 Hz,
rotamer), 2.35 (d, J=16.2 Hz, rotamer), 2.17–2.31 (m, 1.87H, over-
lap of 2 rotamers), 1.84 (brd, J=12.5 Hz, 1H), 1.73 (brd, J=
12.1 Hz, rotamer), 1.62–1.69 (m, 1H), 1.61 (d, J=6.8 Hz, rotamer),
1.49–1.58 (m, 1H), 1.45 (t, J=6.8 Hz, overlap of 2 rotamers), 1.18–
1.34 (m, 1H), 0.81–0.91 ppm (m, rotamer); 13C NMR (151 MHz,
300 K, CDCl3, mixture of rotamers): d=171.4, 170.6, 162.9, 162.6,
162.5, 160.4, 159.8, 159.6, 157.9, 157.3, 156.8, 156.8, 156.4, 156.2,
137.0, 136.8, 135.0, 134.9, 134.9, 130.8, 130.4, 130.4, 129.9, 129.3,
129.0, 129.0, 128.2, 128.0, 127.9, 127.7, 127.5, 123.6, 123.4, 122.4,
121.6, 118.4, 118.3, 118.1, 118.1, 117.1, 116.9, 116.9, 116.8, 116.5,
116.4, 116.2, 115.7, 115.3, 64.2, 63.8, 54.3, 53.9, 50.4, 39.5, 38.1, 36.9,
29.7, 28.7, 28.1, 18.2, 16.4, 14.8, 14.6 ppm; IR: n˜ =3402.78, 3049.87,
2982.37, 2930.31, 2853.17, 2726.85, 1687.41, 1653.66, 1625.70,
1607.38, 1590.99, 1509.03, 1435.74, 1409.71, 1325.82, 1252.54,
1163.83, 1128.15, 1056.80, 1043.30, 827.31 cmꢀ1; HRMS (ESI, [M+
H]+, m/z): calcd: 612.2592, found: 612.2599; RP-HPLC (254 nm):
tR =17.0 min, purity >94%.
General conditions for radiosynthesis: Commercial reagents and
solvents were purchased from Acros Organics (Geel, Belgium), Alfa
Aesar (Karlsruhe, Germany), or Merck KGaA (Darmstadt, Germany)
and were used without further purification unless otherwise stated.
All solvents were of analytical grade, and DMSO was distilled prior
to use and stored over 4 ꢂ molecular sieves. [3H]Methyl iodide dis-
solved in toluene (as =3.15 TBqmmolꢀ1
,
85 Cimmolꢀ1
;
av =
37 GBqmLꢀ1 1 CimLꢀ1
,
)
was from Hartmann Analytic (Braun-
schweig, Germany). Scintillation cocktail Rotiscint Eco Plus was
from Carl Roth (Karlsruhe, Germany). Analytical HPLC was per-
formed on a system from Waters (Eschborn, Germany), equipped
with a pump control module, a 510 HPLC pump, a 486 UV/VIS de-
tector, and a Flo-One beta series A-500 radiodetector (Packard,
Meriden, USA). The stationary phase was a Synergi Hydro-RP (250ꢁ
4.6 mm, 4 mm) column equipped with a Luna C18 (4ꢁ3.0 mm)
column guard (Phenomenex, Aschaffenburg, Germany). Linear gra-
dients of CH3CN/TFA 0.05% (v/v) and H2O/TFA 0.05% (v/v) were
used as mobile phases at a flow rate of 0.8 mLminꢀ1. Absorbance
was detected at 220 nm, and radioactivity was measured with the
radiodetector by liquid scintillation counting (liquid scintillator: Ro-
tiscint Eco Plus, flow rate: 3.8 mLminꢀ1).
N-{1-[3-(4-Ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimi-
din-2-yl]ethyl}-2-[4-fluoro-3-(trifluoromethyl)phenyl]-N-[(1-meth-
[3H]N-{1-[3-(4-Ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyri-
midin-2-yl]ethyl}-2-[4-fluoro-3-(trifluoromethyl)phenyl]-N-[(1-
methylpiperidin-4-yl)methyl]acetamide (RAMX3, [3H]5): Com-
pound 4 (402.7 mg, 0.6588 mmol, 11.2 equiv), dissolved in CH2Cl2
(50 mL), and DIPEA (123.9 mg, 0.9588 mmol, 16.3 equiv) in CH2Cl2
(50 mL) were added to a solution of [3H]methyl iodide (8.875 mg,
0.0588 mmol, 1 equiv, 5 mCi) in toluene (5 mL), and the reaction mix-
ture was diluted with CH2Cl2 (100 mL) and stirred for 20 h at room
temperature.
ylpiperidin-4-yl)methyl]acetamide (5): Compound
4 (20 mg,
0.033 mmol) was dissolved in aqueous formaldehyde (37%)
(0.8 mL, 10.65 mmol). The solution was stirred for 20 min at ambi-
ent temperature, then sodium triacetoxyborohydride (22 mg,
0.104 mmol) was added, and stirring was continued for 3 h. After
completion of the reaction, the solution was diluted with saturated
NaHCO3 to 25 mL and extracted with CH2Cl2 (3ꢁ10 mL). The com-
bined organic layers were dried over MgSO4, filtered, and concen-
trated under reduced pressure. The crude product was purified by
flash chromatography (5% MeOH and 0.5% conc. NH3(aq) in CH2Cl2),
yielding the product as a white foam after trituration with n-
hexane: 1H NMR (600 MHz, CDCl3, mixture of rotamers in ratio
1:0.45): d=9.06 (dd, J=4.5, 1.9 Hz, rotamer), 8.98 (dd, J=4.9,
The reaction was monitored by HPLC. Reaction mixture (1 mL) was
added to 104 mL of unlabeled compound 5 solution (100 mm), the
“cold” version of RAMX3, to allow for UV detection in addition to
&8
&
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ChemMedChem 0000, 00, 1 – 10
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