2216 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Muth et al.
precipitates were collected by filtration and washed several
times with small amounts of acetonitrile and with pentane and
dried in vacuo. 3: yield 49%; mp 242 °C; 1H NMR (δ(ppm),
DMSO-d6) 1.07 (CHCH3, d, J 6.6), 1.27 (N+(CH2)2CH2, br), 1.66
KI and K2CO3 (1:1) was added. The reaction solution was
refluxed for 3-7 days. After the reaction was completed
(TLC monitoring: silica gel, eluent CH3OH:0.2 M NH4NO3
solution (aq) ) 3:2) about one-half of the solvent was evapo-
rated and the solution was cooled to 4 °C for several days. The
obtained white precipitates were collected by filtration and
washed several times with small amounts of acetonitrile and
with pentane and dried in vacuo. 4b: yield 34%; mp 186 °C;
1H NMR (δ(ppm), DMSO-d6) 0.95 (CH2CH3, d, J 7.3), 1.30
(N+(CH2)2CH2, br), 1.34 (CHCH2, br), 1.68 (N+CH2CH2,
br), 2.33 (NPhthCH2CH, br), 3.09 (N+(CH3)2, s), 3.31 (N+CH2,
br), 3.38 (CH2N+, br), 3.55-3.68 (NPhthCH2, m), 7.88 (CHar,
m). 4c: yield 51%; mp 71 °C; 1H NMR (δ(ppm), DMSO-d6)
(N+CH2CH2,
br),
2.05
(NPhthCH2CH2,
br),
2.50
(NPhthCH2CH, br), 3.01, 3.03 (N+(CH3)2, s), 3.27 (N+CH2, br),
3.37 (CH2N+, br), 3.49-3.62 (NPhthCH2, m), 3.66 (NPhthCH2, t,
J 6.2), 7.87 (Har, m). 7b: yield 44%; mp 221 °C; 1H NMR
(δ(ppm), DMSO-d6) 1.06 (CHCH3, d, J 6.6), 1.30 (N+(CH2)2CH2,
br), 1.68 (N+CH2CH2, br), 2.06 (NPhthCH2CH2, br), 2.50
(NPhthCH2CH, br), 2.51 (phth-CH3, s), 3.04, 3.11 (N+(CH3)2, s),
3.30 (N+CH2, br), 3.38 (CH2N+, br), 3.48-3.62 (NPhthCH2, m),
3.66 (NPhthCH2, t, J 6.2), 7.67 (Har, d, J 7.6), 7.71 (Har, s), 7.77
(Har, d, J 7.6), 7.89 (Har, m).
0.85 (CH2CH3, t,
J
6.7), 1.29 (N+(CH2)2CH2, br), 1.40
(CH2CH2CH3, br), 1.48 (CHCH2, br), 1.72 (N+CH2CH2, br), 2.35
(NPhthCH2CH, br), 3.10, 3.11 (N+(CH3)2, s), 3.32 (N+CH2, br),
3.39 (CH2N+, br), 3.56-3.67 (NPhthCH2, m), 7.88 (CHar, m).
Synthesis of 2-{3-[1-(6-{1,1-Dimethyl-1-[3-(1,3-dioxo-
1,3-dihydroisoindol-2-yl)-2-methylpropyl]ammonio}-
hexyl)-1,1-dimethylammonio]-2-methylpropyl}isoindoline-
1,3-dione Dibromide (4a). Two equivalents of the phthal-
imidopropylamine 9a (2 mmol) and 1 equiv of 1,6-dibromo-
hexane (1 mmol) were dissolved in acetonitrile (50 mL), and a
catalytic amount of KI and K2CO3 (1:1) was added. The
reaction solution was refluxed for 4 d. After the reaction was
completed (TLC monitoring: silica gel, eluent CH3OH:0.2 M
NH4NO3 solution (aq) ) 3:2), the solution was allowed to cool
to room temperature and a white precipitate (yield 65%) was
collected by filtration. It was washed several times with
1
4d: yield 65%; mp 77 °C; H NMR (δ(ppm), DMSO-d6) 0.82,
0.91 (CH2CH(CH3)2, d,
J 6.5), 1.25 (CHCH2, br), 1.27
(N+(CH2)2CH2, br), 1.69 (N+CH2CH2, br), 1.75 (CH2CH(CH3)2,
m), 2.35 (NPhthCH2CH, br), 3.08, 3.09 (N+(CH3)2, s), 3.31
(N+CH2, br), 3.40 (CH2N+, br), 3.61 (NPhthCH2, br), 7.88
(CHar, m).
Determination of the Lipophilicity. The test and refer-
ence substances (2-phenylethylamine, 2-phenylethanol, ben-
zene, N,N′-dimethylaniline, chlorobenzene, toluene, ethylben-
zene, cumene, biphenyl, and anthracene), respectively, were
1
acetonitrile and pentane and dried in vacuo: mp 227 °C; H
dissolved in methanol (4 µg/mL). Using
a RP column
NMR (δ(ppm), DMSO-d6) 1.06 (CHCH3, d, J 6.6), 1.26
(N+(CH2)2CH2, br), 1.68 (N+CH2CH2, br), 2.51 (NPhthCH2CH,
br), 3.08 (N+(CH3)2, s), 3.28 (N+CH2, br), 3.33 (CH2N+, br),
3.49-3.62 (NPhthCH2, m), 7.87 (CHar, m).
(LiChroCart 125-4 HPLC cartridge; LiChrospher 100, RP 18,
5 µm, end-capped, Merck) and a mobile phase composed of
methanol/phosphate buffer pH 7.4 ) 70:30 (0.02% N,N-
dimethylamine added), the retention times were determined
and converted to k′ values. k′ ) (tR-t0)/t0 with tR ) retention
time of the test substance and t0 ) hold-up time. The log k′
values of the reference substances were correlated with the
log P values reported in ref 17. The calibration curve was
established (lipophilicity: y ) 2.4103x + 1.4865; R2 ) 0.9778)
and the log P values of the test substances calculated. The log
P values are summarized in Table 3 (Supporting Information).
General Procedure for the Synthesis of 2-(2-(Bromo-
methyl)butyl)isoindoline-1,3-dione (10), 2-(2-(Bromo-
methyl)pentyl)isoindoline-1,3-dione (11), and 2-(2-(Bro-
momethyl)-4-methylpentyl)isoindoline-1,3-dione (12). A
mixture of potassium phthalimide (6 mmol) and the corre-
sponding alkylated 1,3-dibromopropane derivative (18 mmol)
in 30 mL of N,N-dimethylformamide was heated at 80 °C for
7 h. After the reaction was completed, the precipitated
potassium bromide was removed from the solution and the
solvent was evaporated under reduced pressure. The liquid
residues were dissolved in diethyl ether and extracted with
water. The organic layers were dried over Na2SO4, and the
solvent was evaporated. The resulting liquids were purified
by means of column chromatography (silica gel, eluent
CH2Cl2). Colorless oils were obtained that crystallized after
several days at 4 °C. 10: yield 41%; mp 42 °C; 1H NMR
(δ(ppm), CDCl3) 0.98 (CH3, t, J 7.5), 1.42-1.58 (CHCH2CH3,
m), 2.17 (CH2CHCH2, br), 3.44, 3.49 (CH2Br, dd, J 4.8, 10.6),
3.70 (NCH2, dd, J 6.6, 13.9), 3.79 (NCH2, dd, J 7.3, 13.9), 7.74,
7.84 (CHar, m). 11: yield 46%; mp 53 °C; 1H NMR (δ(ppm),
CDCl3) 0.93 (CH3, t, J 7.5), 1.32-1.57 (CH2CH2CH3, m), 2.24
(CH2CHCH2, br), 3.42, 3.47 (CH2Br, dd, J 4.7, 10.5), 3.69
(NCH2, dd, J 6.4, 13.9), 3.75 (NCH2, dd, J 7.1, 13.9), 7.74,
7.84 (CHar, m). 12: yield 46%; mp 61 °C; 1H NMR (δ(ppm),
CDCl3) 0.93 (CH3, dd, J 6.5, 25.5), 1.15-1.22 (CHCH2CH, m),
1.42-1.49 (CHCH2CH, m), 1.68-1.78 (CH(CH3)2, m), 2.31
(CH2CHCH2, br), 3.40, 3.45 (CH2Br, dd, J 4.7, 10.5), 3.67
(NCH2, dd, J 5.9, 13.9), 3.75 (NCH2, dd, J 8.0, 13.9), 7.74, 7.84
(CHar, m).
General Procedure for the Synthesis of 2-{3-[1-(6-
{1,1-Dimethyl-1-[3-(1,3-dioxo-1,3-dihydroisoindol-2-yl)-2-
ethylpropyl]ammonio}hexyl)-1,1-dimethylammonio]-2-
ethylpropyl}isoindoline-1,3-dione Dibromide (4b), 2-{3-
[1-(6-{1,1-Dimethyl-1-[3-(1,3-dioxo-1,3-dihydroisoindol-2-
yl)-2-propylpropyl]ammonio}hexyl)-1,1-dimethylammo-
nio]-2-propylpropyl}isoindoline-1,3-dione Dibromide (4c),
and 2-{3-[1-(6-{1,1-Dimethyl-1-[3-(1,3-dioxo-1,3-dihydro-
isoindol-2-yl)-2-isobutylpropyl]ammonio}hexyl)-1,1-di-
methylammonio]-2-isobutylpropyl}isoindoline-1, 3-di-
one Dibromide (4d). Two equivalents of the corresponding
N-(3-bromopropyl)phthtalimides 10-12 (2 mmol) and 1 equiv
of N,N,N′,N′-tetramethyl-1,6-hexanediamine (1 mmol) were
dissolved in acetonitrile (50 mL), and a catalytic amount of
Determination of Stability in Buffer Solution. For the
determination of the stability the compounds were dissolved
in Mg/Tris buffer, pH 7.3 (20 µg/mL). By means of UV
spectroscopy the absorption maxima of the test compounds
were determined. The absorption values in the maximum
wavelengths were observed over a period of 24 h. For com-
pounds that exhibited an exponential decrease of absorption,
the half-life periods (t1/2) were determined and are summarized
in Table 3 (Supporting Information).
Pharmacology. Preparation of porcine heart homogenates
has been described previously.18,19 Experiments were carried
out at 37 °C using a buffer consisting of 3 mM MgHPO4 and
50 mM Tris-HCl at pH 7.3. [3H]-N-methylscopolamine
([3H]NMS, PerkinElmer Life Sciences, Boston, MA) with a
specific activity of 81.0-83.5 Ci/mmol was used as radioligand
at a concentration of 0.2 nM. To determine nonspecific binding,
1 µM atropine was added. [3H]NMS binding affinity under
control conditions amounted to pKD ) 9.37 (9.33, 9.41; two
homogenates, three experiments each). Rapid vacuum filtra-
tion (glass filters No. 6; Schleicher and Schu¨ll, Dassel,
Germany) was used to separate the membranes. After washing
the filters twice with 5 mL of distilled water of 0 °C, 5 mL of
scintillation fluid (Ready protein, Beckman, Palo Alto, CA) was
added to measure radioactivity (Beckman LS 6000 counter).
To measure the effects of the test compounds on the
dissociation of [3H]NMS, “two-point kinetic experiments” were
carried out (cf. ref 20). Therefore, membranes were preincu-
bated for 30 min with [3H]NMS to attain equilibrium binding.
[3H]NMS-dissociation was revealed after addition of 1 µM
atropine either alone or in combination with the test com-
pound. Specific [3H]NMS binding at t ) 0 and 10 was measured
to characterize the monoexponential time course of [3H]NMS
dissociation. The obtained apparent rate constant of dissocia-
tion k-1 relative to the rate constant of dissociation of the
control served to achieve concentration-effect curves for the