Journal of Medicinal Chemistry
Article
3
3
7
.00 (s, 3H), 3.05 (s, 6 H), 3.15 (br, 2H), 3.25 (br, 2H), 3.32 (m, 2H),
.53 (ddd, J = J = 9.4, 5.8, 7.2 Hz, 1H), 3.65 (m, 2H), 3.81 (dd, J = 5.9,
.2 Hz, 1H), 3.85 (br, 2H), 3.99 (ddd, J = 9.4, 5.8, 5.9 Hz, 1H), 5.01
(d, J = 3.7 Hz, 1H), 3.78−3.80 (m, 1H), 3.94−3.98 (m, 1H), 4.01 (d, J
= 3.5 Hz, 1H), 4.23 (dd, J = 2.2, 17.4 Hz, 2H), 5.04 (t, J = 5.9 Hz,
13
1H), 5.11 (t, J = 5.11 Hz, 1H), 7.28−7.34 (m, 5H). C NMR (101
(
t, J = 5.8 Hz, 1H), 5.09 (t, J = 5.1 Hz, 1H), 7.33 (m, 5H), 7.89 (m,
MHz, DMSO-d ): δ 21.3, 21.7, 23.9, 25.3, 28.1, 28.3, 28.6, 32.1, 43.5,
6
4
2
6
H). 13C NMR (101 MHz, DMSO-d ): δ 21.3, 21.4, 24.1, 24.3, 25.2,
52.1, 54.1, 62.7, 62.8, 62.9, 64.9, 127.3, 128.1, 128.4, 136.1, 170.8. IR
6
−1
5.3, 31.5, 34.5, 34.6, 49.9, 54.1, 59.8, 59.9, 60.7, 60.8, 62.9, 63.4, 64.7,
4.9, 123.1, 127.4, 128.1, 128.6, 131.7, 134.4, 136.1, 167.7, 170.2. IR
[cm ]: 811, 1043, 1386, 1627, 2927
NOESY NMR Experiments. NOESY studies were acquired using
the noesytp program on a Bruker Avance 400 MHz spectrometer, with
a mixing time of 1 s. The number of scans as well as the number of
dummy scans was 16. The relaxation delay was 3 s, time domain size
−1
[
cm ]: 703, 776, 1354, 1535, 1589, 1651, 1701, 2925, 3418.
8
-[6-(N-(3-(1,3-Dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)-2,2-di-
methylpropyl)-N,N-dimethylammonio)hexyl]-3-[(3-hydroxy-2-
phenylpropanoyl)oxy]-8-methyl-8-azabicyclo[3.2.1]octan-8-ium
Dibromide (6). Yield 60% (yellow solid); mp 169 °C. H NMR (400
2
4
048, acquisition time 0.250725 s, the transmitter frequency offset
.603 ppm, spectral width 102091 ppm, frequency offset of the second
1
MHz, DMSO-d ): δ 1.24 (s, 6H), 1.34 (br, 4H), 1.63−1.90 (m, 7H),
6
nucleus 4.603 ppm, spectral width (F1) 102091 ppm.
2
3
.08−2.29 (m, 3H), 2.52−2.62 (m, 2H), 3.08 (s, 3H), 3.20 (br, 8H),
.33 (s, 2H), 3.45 (br, 2H), 3.50 (s, 2H), 3.69 (ddd, J = 9.7, 5.3, 5.4
Pharmacological Experiments. Cell Culture and Membrane
Preparation. Flp-In-Chinese hamster ovary cells (Flp-In-CHO) stably
expressing the hM2 receptor (CHO-hM2 cells), or the hM2 mutant
Hz, 1H), 3.81 (dd, J = 5.3, 9.1 Hz, 1H), 3.98 (br, 2H), 3.98 (ddd, J =
9
.7, 5.4, 9.1 Hz, 1H), 5.02 (t, J = 5.4 Hz, 1H), 5.00 (t, J = 5.1 Hz, 1H),
3.33
EL2
receptors (CHO-hM2 Y104 A cells; CHO-hM2 Y177 A; CHO-
13
7
.25−7.40 (m, 5H), 7.90 (t, J = 7.7 Hz, 2H), 8.47−8.55 (m, 4H).
C
EL2
EL2
7.36
hM2 Y177 Q; CHO-hM2 Y177 Q, T423 H; CHO-hM2
W422 A) and CHO-K1 cells stably expressing the hM5 receptor
NMR (101 MHz, DMSO-d ): δ 21.4, 21.8, 24.1, 24.3, 25.3, 25.5, 31.6,
7.35
6
4
1
0.2, 48.8, 52.0, 54.1, 60.2, 63.0, 63.5, 64.6, 64.9, 67.0, 71.6, 122.2,
27.3, 127.4, 128.1, 128.6, 130.9, 131.3, 134.3, 136.1, 164.6, 171.2. IR
(
CHO-K1-hM5 cells) were cultured in Ham’s nutrient mixture F-12
−1
supplemented with 10% (v/v) fetal calf serum (FCS), 100 μg mL
streptomycin, 100 U ml penicillin, and 2 mM L-glutamine.
−1
[
2
cm ]: 720, 743, 783, 930, 1028, 1163, 1234, 1339, 1588, 1658, 1702,
947, 3055, 3388.
-[6-(N-(3-(1, 3-Dioxoisoindolin-2-yl)propyl)-N, N-
−1
Radioligand Binding Experiments. Equilibrium binding and
kinetic experiments using membranes of CHO-hM cells were
performed in 96-well microtiter plates (ABgene, Germany) by
applying a rapid filtration procedure essentially as described
9
dimethylammonio)hexyl]-7-[(3-hydroxy-2-phenylpropanoyl)oxy]-9-
2
,4
methyl-3-oxa-9-azatricyclo[3.3.1.0 ]nonan-9-ium Dibromide (7).
1
Yield 20% (light-yellow solid); mp 179 °C. H NMR (400 MHz,
DMSO-d ): δ 1.25 (br, 4H), 1.65 (br, 4H), 1.78 (d, J = 17.2 Hz, 1H),
1
3
3
3
24,29
3
previously.
[ H]NMS binding assays were carried out in a 10
6
mM HEPES, 10 mM MgCl , 100 mM NaCl, 10 μM GDP, pH 7.4 at
2
.93 (d, J = 17.2 Hz, 1H), 2.05 (br, 2H), 2.63 (br, 2H), 2.99 (s, 6H),
.05 (s, 3H), 3.25 (br, 2H), 3.33 (br, 2H), 3.53 (m, 2H), 3.66 (m,
H), 3.76 (d, J = 3.4 Hz, 1H), 3.79 (m, 1H), 3.95 (m, 1H), 4.01 (d, J =
.4 Hz, 1H), 4.19 (d, J = 2.2 Hz, 1H), 4.23 (d, J = 2.2 Hz, 1H), 5.04 (t,
3
3
0 °C. [ H]NMS-binding was characterized by receptor densities of
−6 pmol/mg membrane protein. Incubation times necessary to attain
1
3
[
H]NMS binding equilibrium in the presence of allosteric agents were
29
calculated as described previously. As an exception, Iper-6-naph (15)
13
J = 5.9 Hz, 1H), 5.11 (br, 1H), 7.33 (m, 5H), 7.89 (m, 4H). C NMR
101 MHz, DMSO-d ): δ 21.5, 21.6, 25.1, 25.3, 28.2, 31.8, 34.6, 44.4,
equilibrium data, collected with the receptor mutation M Y104A,
2
(
3
6
used, due to a low [ H]NMS affinity, centrifugation and not rapid
filtration to separate [ H]NMS receptor complexes at the end of the
5
1
1
0.0, 53.5, 53.7, 54.1, 60.8, 62.1, 62.8, 62.9, 63.0, 67.6, 123.1, 127.4,
3
−1
28.2, 128.5, 131.7, 134.4, 136.1, 168.0, 171.2. IR [cm ]: 720, 1038,
396, 1534, 1652, 1710, 1769, 2988.
respective incubation period. This separation step was carried out in
silanized 1.5 mL reaction tubes at 21000g (15.300 rpm) and 15 °C for
9
-[6-(N-(3-(1,3-Dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)-2,2-di-
2
0 min using a table centrifuge (Beckman Mikrofuge 365627, Rotor
methylpropyl)-N,N-dimethyl-ammonio)hexyl]-7-[(3-hydroxy-2-
phenylpropanoyl)oxy]-9-methyl-3-oxa-9-azatricyclo[3.3.1.02,4]-
F241.5, Beckman Instruments, Palo Alto, CA). After removing the
supernatant, the surface of the remaining pellet was washed once with
100 μL of incubation buffer (see above). Finally, the pellet was
resuspended in 80 μL of incubation buffer and a 50 μL aliquot of the
suspension dispensed into a 20 mL scintillation vial. The radioactivity
contained was determined by liquid scintillation counting as described
nonan-9-ium Dibromide (8). Yield 50% (light-yellow solid); mp 212
1
°
C. H NMR (400 MHz, DMSO-d ): δ 1.30 (br, 4H), 1.36 (s, 6H),
6
1
2
3
4
.73 (br, 4H), 1.79 (d, J = 16.9 Hz, 1H), 1.94 (d, J = 16.9 Hz, 1H),
.65 (br, 2H), 3.09 (s, 3H), 3.17 (s, 6H), 3.39 (br, 2H), 3.55 (br, 2H),
.64 (s, 2H), 3.69 (dd, J = 7.7, 8.8 Hz, 1H), 3.77 (d, J = 3.3 Hz, 1H),
.01 (d, J = 3.3 Hz, 1H), 3.80 (ddd, J = 9.9, 5.4, 7.7 Hz, 1H), 3.98
1
5
earlier. Dissociation binding experiments were performed as two-
3
46
point [ H]NMS dissociation experiments.
Dynamic Mass Redistribution Measurements (DMR). DMR
(
4
1
ddd, J = 9.9, 5.4, 8.8 Hz, 1H), 4.15 (s, 2H), 4.20 (d, J = 3.3 Hz, 1H),
.24 (d, J = 3.3 Hz, 1H), 5.04 (t, J = 5.9 Hz, 1H), 5.11 (t, J = 5.4 Hz,
1
5,47
H), 7.35 (m, 5H), 7.91 (t, J = 7.5 Hz, 2H), 8.52 (m, 4H). 13C NMR
assays were carried out as described earlier
using the beta version
of the Epic device (Corning, NY, USA). Flp-In-CHO cells, Hanks’
Balanced Salt Solution (HBSS), and HEPES buffer solution were
obtained from Invitrogen (Carlsbad, CA, USA). In short, Flp-In-CHO
cells, empty or stably transfected with the respective hM gene, were
seeded in a density of 12500 cells per well in 384-well Epic microplates
with 40 μL of growth medium (Ham’s F-12 medium, 10% FBS, 2 mM
L-glutamine, and 1% penicillin/streptomycin) and incubated for 20 h
at 37 °C in an atmosphere of 5% CO until confluence was attained.
After removing the cell culture medium and washing the cells twice
with 50 μL of assay buffer per well (HBSS with 20 mM HEPES, pH
7.0), cells were allowed to rest in 30 μL of assay buffer in the Epic
reader for 2 h at 28 °C. Following the addition of 10 μL of test
compound dissolved in assay buffer, DMR responses were monitored
for 1 h.
(
101 MHz, DMSO-d ): δ 21.7, 21.9, 25.2, 25.3, 25.5, 28.2, 44.4, 48.8,
6
5
1
2.0, 53.6, 53.7, 54.1, 62.1, 62.8, 62.9, 63.0, 67.0, 67.6, 71.6, 122.3,
27.3, 127.4, 127.5, 128.2, 128.5, 130.9, 131.3, 136.1, 164.7, 170.9. IR
−1
[
cm ]: 777, 1236, 1335, 1589, 1649, 1700, 2943, 3410.
3
-[(Hydroxy-2-phenylpropanoyl)oxy]-8-methyl-8-[6-(N,N,N-
trimethylammonio)hexyl]-8-azabicyclo[3.2.1]octan-8-ium Dibro-
mide (9). Yield 20% (brown solid); mp 107 °C. H NMR (400
MHz, DMSO-d ): δ 1.32 (br, 4H), 1.70 (b, 3H), 1.83 (m, 2H), 2.06−
1
6
2
3
5
5
.28 (m, 3H), 2.56 (br, 2H), 3.03 (s, 3H), 3,08 (s, 9H), 3.19 (br, 2H),
.37 (br, 2H), 3.65−3.72 (ddd, J = 9.8, 5.3, 9.2 Hz, 1H), 3.81 (dd, J=
.2, 9.2 Hz, 1H), 3.85 (br, 2H), 3.98 (ddd, J = 9.8, 5.2, 5.3 Hz, 1H),
.00 (t, J = 5.3 Hz, 1H), 5.10 (t, J = 5.3 Hz, 1H), 7.25−7.35 (m, 5H).
1
3
C NMR (101 MHz, DMSO-d ): δ 21.3, 21.7, 24.1, 24.3, 25.2, 31.6,
6
4
1
2
0.1, 52.1, 54.1, 60.2, 62.9, 63.5, 64.6, 64.9, 65.0, 127.4, 128.0, 128.6,
−1
36.1, 171.2. IR [cm ]: 700, 760, 974, 1346, 1480, 1628, 1716, 2868,
953, 3400.
Data Analysis. Equilibrium binding experiments applying
[ H]NMS were analyzed using a four-parameter logistic function
3
7
-[(3-Hydroxy-2-phenylpropanoyl)oxy]-9-methyl-9-[6-(N,N,N-
yielding the p(= −log) IC50 and the slope factor n of the curve. If the
H
2
,4
trimethylammonio]hexyl)-3-oxa-9-azatricyclo[3.3.1.0 ]nonan-9-
observed slope factors and, successively, the bottom plateau did not
ium Dibromide (10). Yield 31% (light-yellow solid); mp 216−217 °C.
differ significantly from unity and zero (F test, P < 0.05), respectively,
1
H NMR (400 MHz, DMSO-d ): δ 1.28 (br, 4H), 1.70 (br, 4H), 1.78
n was constrained to −1 and the bottom plateau to 0.
6
H
(
d, J = 17.3, 1H), 1.93 (d, J = 17.1 Hz, 1H), 2.58−2.68 (m, 2H), 3.07
Homologous competition experiments were analyzed according to
3
(
s, 12 H), 3.30 (b, 2H), 3.52−3.56 (m, 2H), 3.65−3.71 (m, 1H), 3.76
ref 48. For the sake of a semiquantitative comparison of [ H]NMS
6
747
dx.doi.org/10.1021/jm500790x | J. Med. Chem. 2014, 57, 6739−6750