6
TRAN ET AL.
4.3.3
(2H)‐one, 7
|
[13C6]5‐(4‐Chlorophenyl)‐4‐(pyridin‐4‐yl)pyridazin‐3
4.3.6 |
[13C6]7‐(4‐Chlorophenyl)‐8‐(pyridin‐4‐yl)‐2‐((6‐
(trifluoromethyl)pyridin‐3‐yl)methyl)‐[1,2,4]triazolo[4,3‐b]
pyridazin‐3(2H)‐one, [13C6]BMS‐725519
In a similar way as [14C]BMS‐725519 was prepared,
compound 9 (201 mg, 0.61 mmol) was reacted with 5‐
[13C6]2‐(4‐Chlorophenyl)‐2‐oxoacetaldehyde (6, 455 mg,
2.6 mmol), 2‐(pyridin‐4‐yl)acetohydrazide (4, 394 mg,
2.6 mmol), and 1‐butanol (13.0 mL) were heated to 123°C
for 2.5 hours. The reaction was monitored by TLC (5%
MeOH/EtOAc, product Rf = 0.5) and by HPLC (product
retention time = 6.30 minutes). The solution was evaporated
to dryness and the residue dissolved in DCM (30 mL),
washed with water (7 mL), brine (5 mL), dried over MgSO4,
filtered, and concentrated to dryness. The crude product was
purified by silica gel flash chromatography eluting with 0%
to 5% MeOH/DCM to afford 494 mg of a white solid (56%
yield). HPLC analysis at 254 nm showed the product to be
(chloromethyl)‐2‐(trifluoromethyl)pyridine
(142.9
mg,
0.73 mmol) to obtain 100 mg of [13C6]BMS‐725519 as a light
yellow solid (40% yield). HPLC purity analysis at 254 nm
showed the product to be 98.9% chemically pure with a reten-
tion time of 7.18 minutes. LC‐MS [M + H]+ = 489/491. Mass
isotopic distribution, [M + 6] = 98%, [M + 2] = 2%. 1H NMR
(500 MHz, DMSO‐d6) δ 8.73 (s, 1H), 8.63 to 8.57 (m, 2H),
8.50 (d, J = 1.6 Hz, 1H), 7.99 (d, J = 8.2 Hz, 1H), 7.90 (d,
J = 8.2 Hz, 1H), 7.61 (br. s, 1H), 7.43 (br. s, 1H), 7.34 to
7.24 (m, 3H), 7.13 (d, J = 8.8 Hz, 1H), and 5.37 (s, 2H).
13C NMR (126 MHz, DMSO‐d6) δ 133.18 to 130.58 (m)
and 128.98 to 128.03 (m).
1
85% pure. H NMR (500 MHz, DMSO‐d6) δ 13.43 (s, 1H),
8.58 to 8.39 (m, 2H), 8.02 (d, J = 1.6 Hz, 1H), 7.56 (d,
J = 3.3 Hz, 1H), 7.39 (d, J = 8.2 Hz, 1H), 7.23 (d,
J = 4.9 Hz, 1H), 7.19 to 7.13 (m, 2H), and 7.11 to 7.03 (m,
1H).
4.4 | Synthesis of [14C]BMS‐811064
4.4.1
tetrahydro‐[1,2,4]triazolo[4,3‐b]pyridazin‐8‐yl)benzonitrile, 11
|
[14C]4‐(7‐(4‐Chlorophenyl)‐5‐ethyl‐3,6‐dioxo‐2,3,5,6‐
4.3.4
pyridazine, 8
|
[13C6]3‐Chloro‐5‐(4‐chlorophenyl)‐4‐(pyridin‐4‐yl)
To
4‐(5‐(4‐chlorophenyl)‐1‐ethyl‐3‐hydrazinyl‐6‐oxo‐1,6‐
To a dry flask was added [13C6]5‐(4‐chlorophenyl)‐4‐
(pyridin‐4‐yl)pyridazin‐3(2H)‐one, 7 (306 mg, 1.1 mmol),
and POCl3 (1.80 mL, 19.3 mmol). The mixture was heated
to 80°C for 4.5 hours while monitoring by HPLC (product
retention time = 6.58 minutes) and TLC (5% MeOH/EtOAc,
product Rf = 0.7). The solution was evaporated to dryness.
The residue was dissolved in EtOAc (30 mL), washed with
saturated NaHCO3 (20 mL), and 1 N NaOH (2.5 mL). The
layers were separated and the organic layer was washed with
brine (7 mL), dried over MgSO4, filtered, and concentrated to
dryness to give an 85% yield of crude product 8. The crude
product was used in the next step without purification.
dihydropyridazin‐4‐yl)benzonitrile (10, 404 mg, 1.1 mmol)
in THF (4.6 mL) was added [14C]CDI (269 mg, 0.95 mmol,
50 mCi, 57% chemically pure, remainder imidazole and/or
imidazole HCl, specific activity = 186 μCi/mg). The
resulting suspension was heated at 65°C for 2 hours. The
reaction was monitored by TLC (60% EtOAc/hexane, starting
material Rf = 0.10, product Rf = 0.20) and by HPLC (starting
material retention time = 7.20 minutes, product retention
time = 9.97 minutes). The solution was evaporated to dry-
ness. The residue was washed with water (8 mL) and
extracted with DCM (3 × 12 mL). The combined organic
extracts were washed with brine (8 mL), dried over MgSO4,
filtered, and concentrated to dryness. The crude product
was purified by flash chromatography eluting with 0% to
5% MeOH/DCM to afford 285 mg of a light yellow solid
(80% yield). The product was used in the next reaction with-
out further purification.
4.3.5
|
[13C6]7‐(4‐Chlorophenyl)‐8‐(pyridin‐4‐yl)‐[1,2,4]
triazolo[4,3‐b]pyridazin‐3(2H)‐one, 9
To [13C6]3‐chloro‐5‐(4‐chlorophenyl)‐4‐(pyridin‐4‐yl)pyridazine
(8, 297 mg, 0.96 mmol) and hydrazinecarboxamide HCl
(183 mg, 1.64 mmol) was added 75% EtOH (6.8 mL) and
concentrated HCl (33 μL). The mixture was heated to 86°C
for 19 hours. The reaction was monitored by TLC (80%
EtOAc/hexane, product Rf = 0.80) and by HPLC (product
retention time = 5.97 minutes). The solution was evaporated
to dryness. The residue was dissolved in EtOAc (60 mL), was
washed with saturated NaHCO3 (15 mL), 1 N NaOH
(1.0 mL), brine (15 mL), dried over MgSO4, filtered, and
was concentrated to dryness. The crude product was purified
by trituration with EtOAc to give 201 mg of a white solid
4.4.2
|
[14C]4‐(7‐(4‐Chlorophenyl)‐5‐ethyl‐2‐(4‐(isoxazol‐5‐yl)
benzyl)‐3,6‐dioxo‐2,3,5,6‐tetrahydro‐[1,2,4]triazolo[4,3‐b]
pyridazin‐8‐yl)benzonitrile, [14C]BMS‐811064
To a dry flask was added compound (11, 140 mg,
0.36 mmol), 5‐(4‐(bromomethyl)phenyl)isoxazole (94 mg,
0.39 mmol), potassium carbonate (99 mg, 0.71 mmol), and
DMF (1.2 mL). The yellow solution was heated to 65°C
and stirred for 0.5 hour under nitrogen. The reaction was
cooled to room temperature and diluted with water
(10 mL). The aqueous solution was extracted with EtOAc
(3 × 12 mL). The combined EtOAc extracts were concen-
trated to give a dark brown oil. The oil was vacuum dried
for 16 hours to give a dark brown solid. The crude solid
was purified by silica gel flash chromatography eluting with
1
(63% yield). H NMR (500 MHz, DMSO‐d6) δ 12.92 (s,
1H), 8.69 (d, J = 5.5 Hz, 2H), 8.44 (s, 1H), 7.62 (br. s,
1H), 7.53 to 7.42 (m, 3H), 7.28 (br. s, 1H), and 7.20 to
7.09 (m, 1H).