Azidoneonicotinoid Photoaffinity Probes for nAChR
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 26 5007
J 2 ) 2 Hz, 1H), 7.31 (d, J ) 7.5 Hz, 1H), 3.71 (m, 4H), 3.49 (s,
CH2), 3.09 (s, 3H), 2.54 (s, 3H). 13C NMR (CDCl3) δ 157.7,
144.2, 142.7, 135.6, 131.1, 127.9, 70.4, 67.6, 48.6, 40.0, 35.7.
IR ν 2123. FAB-LRMS (2-nitrobenzyl alcohol, m/z) 340 (MH+),
307, 289. FAB-HRMS for C11H14ClN9O2, calcd 340.1036 (MH+),
found 340.1041. Anal. (C11H14ClN9O2) C, H, N, Cl.
Method B: To a suspension of 13 (55 mg, 0.32 mmol) in
DMF (0.1 mL) and THF (1.0 mL) was added NaH (15 mg of
60% oil dispersion, 0.38 mmol) at 0 °C. After the mixture was
stirred for 20 min, 3 (64 mg, 0.32 mmol) was added, and the
mixture was stirred a further 12 h at room temperature and
the subsequent 5 h at 50-60 °C. Preparative TLC using
EtOAc/MeOH 15:1 as eluent gave 28 mg of pure product 14
as colorless needles from ether in 26% yield.
2H), 2.45 (m, 4H). Anal. (C10H13ClN2O) C, H, N, Cl.
N-(5-Azido-6-ch lor opyr idin -3-ylm eth yl)m or ph olin e (10).
Method A: A solution of freshly distilled diisopropylamine
(15.5 g, 153 mmol) in dry THF (200 mL) was cooled to -30 °C
and treated with n-butyllithium (91 mL of a 1.6 M solution in
hexane, 146 mmol) over a 15 min period. Then the reaction
mixture was cooled to -78 °C, and a solution of 9 (15.5 g, 73
mmol) in THF (50 mL) was added dropwise over a period of
10 min. After being stirred for 5 min at -78 °C, the reaction
mixture was cooled to -100 °C, and a solution of tosyl azide40
(28.8 g, 146 mmol) in THF (50 mL) was added within 1 min.
During this time the reaction temperature rose to -60 °C. The
cooling bath was then removed, and the reaction mixture was
allowed to warm to 0 °C over a 45 min period. The mixture
was poured into saturated NH4Cl (200 mL) and extracted with
EtOAc. The organic layer was washed with brine, dried with
Na2SO4, and evaporated. The residue was purified by chro-
matography on silica gel using EtOAc as eluent to yield 8.3 g
(45%) of pure product as slightly yellow oil. Rf ) 0.65 in EtOAc.
1H NMR (CDCl3) δ 8.09 (d, J ) 2 Hz, 1H), 7.52 (d, J ) 2 Hz,
1H), 3.72 (m, 4H), 3.49 (s, 2H), 2.47 (m, 4H). Electrospray-MS
(m/z, rel int.) 254 (MH+, 50%). EI-HRMS for C10H12ClN5O,
calcd 253.0730, found 253.0728. Anal. (C10H12ClN5O) C, H, N,
Cl.
N-(5-Azid o-6-ch lor op yr id in -3-ylm eth yl)-N′-m eth yl-N′′-
n itr ogu a n id in e (2). To a solution of 14 (500 mg, 1.47 mmol)
in MeOH (2 mL) was added 1 N HCl (5 mL), and the resulting
reaction mixture was stirred at 80 °C for 90 min. After
evaporation, the residue was purified by chromatography on
silica gel using EtOAc/MeOH 9:1 as eluent to yield 246 mg
(59%) of 2 as colorless crystals. Rf ) 0.70 in EtOAc/MeOH 9:1.
1
Mp ) 170 °C (dec). H NMR (CD3OD) δ 8.14 (d, J ) 2.1 Hz,
1H), 7.78 (d, J ) 2.1 Hz, 1H), 4.53 (s, 2H, py-CH2), 2.95 (s,
3H). 13C NMR (CDCl3) δ 159.6, 145.4, 141.9, 136.3, 136.1,
129.5, 42.5, 28.7. IR ν 2120. UV (EtOH) λmax 260 (log ꢀ 4.10),
272 (shoulder, log ꢀ 4.05), 300 (shoulder, log ꢀ 3.67). FAB-
LRMS (glycerol, m/z) 285 (MH+). FAB-HRMS for C8H9ClN8O2,
calcd 285.0608 (MH+), found 285.0619. Anal. (C8H9ClN8O2) C,
H, N, Cl.
Method B: The conditions were varied by using 1/10th of
the scale in method A and adding the tosyl azide at -78 °C
over a 5 min instead of a 1 min period. Workup as above gave
410 mg (22%) of 10 and 130 mg (6%) of N-(4,5-diazido-6-
chloropyridin-3-ylmethyl)morpholine (11). 11: Rf ) 0.73 in
1-(5-Azid o-6-ch lor op yr id in -3-ylm eth yl)-5-m eth yl-2-n i-
tr oim in oh exa h yd r o-1,3,5-tr ia zin e (16). To a suspension of
NaH (30 mg of 60% oil slurry, 0.75 mmol) in DMF (1 mL) was
added 5-methyl-2-nitroiminohexahydro-1,3,5-triazine (15) (100
mg, 0.63 mmol) at 0 °C. After the mixture was stirred for 30
min, 3 (100 mg, 0.50 mmol) was added, and the resulting
mixture was stirred a further 20 h. NH4Cl (100 mg) was added
to the reaction mixture, and DMF was removed under reduced
pressure at below 40 °C. The residue was purified by prepara-
tive TLC using EtOAc/CHCl3 1:9 as eluent to give 66 mg of
pure product as colorless crystals in 33% yield. Rf ) 0.65 in
1
EtOAc. H NMR (CDCl3) δ 8.08 (s, 1H), 3.71 (m, 4H), 3.48 (s,
2H), 2.49 (m, 4H). Electrospray-MS (m/z, rel int.) 295 (MH+,
100%).
Con ver sion of 10 t o 5-Azid o-6-ch lor op yr id in -3-yl-
m eth yl ch lor id e (3). A solution of 10 (7.8 g, 30.7 mmol) in
THF (100 mL) was treated with ethyl chloroformate (5.9 mL,
61.5 mmol), then stirred at 60 °C for 6 h and evaporated. The
residue was purified by chromatography on silica gel using
EtOAc/hexane 1:4 as eluent to yield 5.46 g (88%) of 3.
1
EtOAc/CHCl3 1:9. Mp ) 156 °C (dec). H NMR (DMSO-d6) δ
1-(5-Azid o-6-ch lor op yr id in -3-ylm et h yl)-2-n it r oim in o-
im id a zolid in e (1). Method A: A mixture of 3 (90 mg, 0.39
mmol) and 12 (58 mg, 0.45 mmol) in acetonitrile (10 mL) was
stirred with K2CO3 (73 mg, 0.54 mmol) at 70 °C for 7 h. After
filtration from inorganic salts, which were rinsed with aceto-
nitrile, the filtrate was concentrated, and the residual solid
was filtered, rinsed with cold water, and dried. The solid was
purified by preparative TLC using EtOAc as eluent to give 50
mg of pure product as colorless crystals in 43% yield. Rf )
0.65 in EtOAc. Mp ) 170 °C (dec). 1H NMR (CDCl3) δ 8.19
(bs, 1H, NH), 8.09 (d., J ) 1.8 Hz, 1H), 7.51 (d, J ) 1.8 Hz,
1H), 4.56 (s, 2H, py-CH2), 3.82 (m, 2H), 3.54 (m, 2H). 13C NMR
(DMSO-d6) δ 160.3, 144.4, 139.7, 134.0, 132.8, 128.9, 45.1, 44.3,
41.5. IR ν 2127. FAB-LRMS (2-nitrobenzyl alcohol, m/z) 297
(MH+), 165, 137. FAB-HRMS for C9H9ClN8O2, calcd 297.0615
(MH+), found 297.0637. Anal. (C9H9ClN8O2) C, H, N, Cl. UV
(EtOH) λmax 262 (log ꢀ 4.25), 268 (shoulder, log ꢀ 4.20), 300
(shoulder, log ꢀ 3.80).
9.45 (bs, 1H, NH), 8.20 (d, J ) 1.8 Hz, 1H), 7.86 (d, J ) 1.8
Hz, 1H), 4.57 (s, 2H, py-CH2), 4.39 (bs, 2H, CH2), 4.29 (bs, 2H,
CH2), 2.52 (s, 3H). 13C NMR (DMSO-d6) δ 154.6, 144.6, 139.5,
134.0, 133.7, 128.8, 67.7, 61.6, 46.0, 38.6. IR ν 2124. FAB-
LRMS (2-nitrobenzyl alcohol, m/z) 326 (MH+). FAB-HRMS for
C
10H12ClN9O2, calcd 326.0880 (MH+), found 326.0873. Anal.
(C10H12ClN9O2) C, H, N.
Con ver sion of 16 to 1-(5-Azid o-6-ch lor op yr id in -3-yl-
m eth yl)-3,5-d im eth yl-2-n itr oim in oh exa h yd r o-1,3,5-tr ia z-
in e (14). To a suspension of dried oil-free NaH (4.8 mg, 0.2
mmol) in dry acetonitrile (3 mL) with
a dried nitrogen
atmosphere was added 18-crown-6 (3 mg, 0.01 mmol) and 16
(32.5 mg, 0.1 mmol). After the mixture was stirred at room
temperature for 30 min, a solution of dry methyl iodide (14.2
mg, 0.1 mmol) in acetonitrile (0.5 mL) was added, and the
resulting mixture was stirred for 1 h. The reaction mixture
was subjected without working-up to preparative TLC using
EtOAc/MeOH 15:1 as eluent. From the band of Rf 0.2, 28.5
mg of the product was isolated in 84% yield.
Bin d in g Site Assa ys. Ra t Br a in Mem br a n es a n d Re-
com bin a n t r4â2 n ACh R.25,26 The standard assay involved
incubation of 5 nM [3H](-)-nicotine (81 Ci/mmol; NEN Life
Science Products, Boston, MA) and various concentrations of
inhibitor with 400 µg of protein from rat brain membranes or
100 µg of protein from recombinant rat R4â2 nAChR in 0.25
mL of buffer for 30 min at 22 °C. The binding buffer consisted
of 20 mM Tris-HCl, pH 7.4, 118 mM NaCl, 4.8 mM KCl, 2.5
mM CaCl2, 1.2 mM MgSO4, and 1.0 mM EDTA. Reactions were
terminated by addition of 5 mL of ice-cold 0.9% NaCl followed
by rapid vacuum filtration with a cell harvester (Brandel
Model MLR-48, Gaithersburg, MD) through GF/B glass fiber
filters and two 5 mL washes on the filter. Filters were
presoaked at least 60 min in 0.1% w/v polyethylenimine (fresh
solution) to reduce nonspecific binding. Specific binding was
Method B: A mixture of 3 (500 mg, 2.46 mmol), 12 (320
mg, 2.46 mmol), and K2CO3 (850 mg, 6.16 mmol) in DMF (10
mL) was stirred at 50 °C for 8 h. After filtration, the filtrate
was concentrated and the residue was purified by chromatog-
raphy on silica gel using EtOAc/MeOH 9:1 as eluent to yield
320 mg (44%) of 1.
1-(5-Azid o-6-ch lor op yr id in -3-ylm eth yl)-3,5-d im eth yl-2-
n itr oim in oh exa h yd r o-1,3,5-tr ia zin e (14). Method A:
A
mixture of 2-nitroiminohexahydro-1,3,5-triazine (13) (1.02 g,
5.9 mmol), 3 (1.20 g, 5.9 mmol), and K2CO3 (2.04 g, 14.8 mmol)
in DMF (20 mL) was stirred at 50 °C for 18 h. After filtration,
the filtrate was concentrated under vacuum, and the residue
was purified by chromatography on silica gel using CH2Cl2/
MeOH 19:1 as eluent to yield 1.66 g (83%) of 14 as colorless
crystals. Rf ) 0.20 in EtOAc/MeOH 9:1. Mp ) 146 °C (dec).
1H NMR (CDCl3) δ 8.08 (d, J ) 1.8 Hz, 1H), 7.64 (d, J ) 1.8
Hz, 1H), 4.70 (s, 2H, py-CH2), 4.34 (bs, 2H, CH2), 4.28 (bs, 2H,