A. J. Bojarski et al. / Bioorg. Med. Chem. 13 (2005) 2293–2303
2301
6.1.19. trans-1-(3-Chlorophenyl)-4-(4-succinimidocyclo-
hexyl)piperazine (22). The title compound was prepared
by the general procedure in 36% yield as colorless crys-
tals: mp 218–220 ꢁC, Rf = 0.74 (SiO2, CHCl3/
MeOH = 19/1); 1H NMR (300 MHz) d 7.19 (dd,
J = 8.2, 8.0, 1H, aryl H-5), 6.90 (dd, J = 2.2, 1.9, 1H, aryl
H-2), 6.84–6.80 (m, 2H, aryl H-4 and H-6), 4.05 (tt,
J = 12.4, 4.0, 1H, cyclohexane axial H-4), 3.22 (app br
t, 4H, piperazine 2CH2), 2.74 (app br t, 4H, piperazine
2CH2), 2.69 (s, 4H, CH2CH2 in succinimide), 2.49 (tt,
J = 11.7, 3.3, 1H, cyclohexane axial H-1), 2.31 (qd,
J = 12.9, 3.3, 2H, cyclohexane axial Hꢀs), 2.04 (app br
d, 2H, cyclohexane equatorial Hꢀs), 1.72 (app br d, 2H,
cyclohexane equatorial Hꢀs), 1.41 (qd, J = 12.9, 3.3,
2H, cyclohexane axial Hꢀs). 22Æ2HClÆH2O: mp 252–
254 ꢁC. Anal. (C20H26ClN3O2Æ2HCl H2O) C, H, N.
MeOH = 19/1); 1H NMR (300 MHz) d 7.20 (dd,
J = 8.2, 8.0, 1H, aryl H-5), 6.58 (dd, J = 8.2, 2.2, 1H,
aryl H-6), 6.50 (t, J = 2.2, 1H, aryl H-2), 6.44 (dd,
J = 8.0, 1.9, 1H, aryl H-4), 4.02 (dddd, J = 12.4, 12.1,
4.1, 3.8, 1H, cyclohexane axial H-4), 3.82 (s, 3H,
OCH3), 3.23 (app br t, 4H, piperazine 2CH2), 2.75
(app br t, 4H, piperazine 2CH2), 2.68 (s, 4H, CH2CH2
in succinimide), 2.48 (tt, J = 11.6, 3.3, 1H, cyclohexane
axial H-1), 2.31 (dddd, J = 12.9, 12.6, 3.6, 3.3, 2H, cyclo-
hexane axial Hꢀs), 2.04 (app br d, 2H, cyclohexane equa-
torial Hꢀs), 1.72 (app br d, 2H, cyclohexane equatorial
Hꢀs), 1.42 (dddd, J = 12.9, 12.6, 3.6, 3.3, 2H, cyclohex-
ane axial Hꢀs). 25Æ2HCl: mp 284–286 ꢁC. Anal.
(C21H29N3O3Æ2HCl) C, H, N.
6.1.24.
trans-1-(4-Methoxyphenyl)-4-(4-succinimido-
cyclohexyl)piperazine (26). The title compound was
prepared by the general procedure in 67% yield as color-
less crystals: mp 237–234 ꢁC, Rf = 0.70 (SiO2, CHCl3/
MeOH = 19/1); 1H NMR (300 MHz) d 6.90 (dddd,
J = 9.4, 9.1, 3.0, 2.5, 4H, aryl), 4.02 (dddd, J = 12.4,
12.1, 4.1, 3.8, 1H, cyclohexane axial H-4), 3.80 (s, 3H,
OCH3), 3.13 (app br t, 4H, piperazine 2CH2), 2.77
(app br t, 4H, piperazine 2CH2), 2.69 (s, 4H, CH2CH2,
in succinimide), 2.49 (tt, J = 11.6, 3.3, 1H, cyclohexane
axial H-1), 2.31 (dddd, J = 12.9, 12.6, 3.3, 3.0, 2H, cyclo-
hexane axial Hꢀs), 2.06 (app br d, 2H, cyclohexane equa-
torial Hꢀs), 1.72 (app br d, 2H, cyclohexane equatorial
Hꢀs), 1.42 (dddd, J = 12.9, 12.7, 3.3, 3.0, 2H, cyclohex-
ane axial Hꢀs). 26Æ2HClÆ0.5H2O: mp 264–266 ꢁC. Anal.
(C21H29N3O3Æ2HClÆ0.5H2O) C, H, N.
6.1.20. 4-(4-Succinimidobutyl)-1-(3-trifluoromethylphen-
yl)piperazine (23). The title compound was prepared by
the general procedure in 75% yield as an oil, Rf = 0.35
(SiO2, CHCl3/MeOH = 19/1); 1H NMR (60 MHz) d
7.6–6.8 (m, 4H), 3.8–3.0 (m, 6H), 2.9–2.2 (m, 6H), 2.7
(s, 4H), 1.9–1.4 (m, 4H). 23Æ2HCl: mp 167–168 ꢁC. Anal.
(C19H24F3N3O2Æ2HCl) C, H, N.
6.1.21. trans-4-(4-Succinimidocyclohexyl)-1-(3-trifluoro-
methylphenyl)piperazine (24). The title compound was
prepared by the general procedure in 50% yield as color-
less crystals: mp 181–183 ꢁC, Rf = 0.60 (SiO2, CHCl3/
MeOH = 19/1); 1H NMR (300 MHz) d 7.37 (dd,
J = 8.0, 7.7, 1H, aryl H-5), 7.14–7.07 (m, 3H, aryl H-2,
H-4 and H-6), 4.02 (dddd, J = 12.4, 12.1, 4.1, 3.8, 1H,
cyclohexane axial H-4), 3.28 (app br t, 4H, piperazine
2CH2), 2.77 (app br t, 4H, piperazine 2CH2), 2.69 (s,
4H, CH2CH2 in succinimide), 2.51 (app br t, 1H, cyclo-
hexane axial H-1), 2.32 (dddd, J = 12.9, 12.6, 3.3, 3.0,
2H, cyclohexane axial Hꢀs), 2.06 (app br d, 2H, cyclo-
hexane equatorial Hꢀs), 1.72 (app br d, 2H, cyclohexane
equatorial Hꢀs), 1.42 (qd, J = 12.9, 3.0, 2H, cyclohexane
axial Hꢀs). 24Æ2HCl: mp 255–256 ꢁC. Anal.
(C21H26F3N3O2Æ2HCl) C, H, N.
6.2. In vitro radioligand binding assays
All the assays were carried out on rat brain tissues; inhi-
bition constants (Ki) were determined from at least three
separate experiments in which 8–10 drug concentrations,
run in triplicate, were used. The binding reaction was
terminated by rapid filtration through Whatman GF/B
filters followed by three 4-mL washes with ice-cold incu-
bation buffer.
6.1.22. General procedure for the preparation of com-
pounds 25 and 26. Equimolar amounts (2 mmol) of 14 or
15 and succinic anhydride were refluxed in xylene
(20 mL) for 5 h. The resulting precipitate of noncyclic
amidoacid was filtered off and then was heated in acetic
anhydride (20 mL) in the presence of anhydrous sodium
acetate (30% excess) for 5 h. After cooling the reaction
mixture was poured into ice-water, neutralized with
10% NaOH and extracted with CHCl3 (3 · 30 mL).
The combined extracts were dried (K2CO3) and evapo-
rated, to give the oily residue, which was purified by sil-
ica gel column chromatography. Free bases were then
converted into the hydrochloride salts in CHCl3/MeOH
solution by the treatment with excess of Et2O saturated
with gaseous HCl.
The radioactivity retained on the filters was measured by
liquid scintillation counting (Beckman LS 6500 appara-
tus) in 4 mL scintillation fluid (Akwascynt, BioCare).
Binding isotherms of the tested compounds were ana-
lyzed by nonlinear regression (Prism, GrafPad Software
Inc., San Diego, USA), using the Cheng–Prusoff equa-
tion36 to calculate Ki values.
6.2.1. Serotonin 5-HT1A, 5-HT2A, and a1-adrenergic
binding assays. Radioligand studies with native 5-
HT1A, 5-HT2A, and a1-adrenergic receptors were con-
ducted according to the methods previously described
by us. Briefly: 5-HT1A assays used rat hippocampal
membranes, [3H]-8-OH-DPAT (170 Ci/mmol, NEN
Chemicals) and 5-HT for nonspecific binding; 5-HT2A
assays used rat cortical membranes, [3H]-ketanserin
(88.0 Ci/mmol, NEN Chemicals) and methysergide for
nonspecific binding; a1 assays used rat cortical mem-
branes, [3H]-prazosine (25.0 Ci/mmol, Amersham) and
phentolamine for nonspecific binding.
6.1.23.
trans-1-(3-Methoxyphenyl)-4-(4-succinimido-
cyclohexyl)piperazine (25). The title compound was pre-
pared by the general procedure in 60% yield as colorless
crystals: mp 162–164 ꢁC, Rf = 0.41 (SiO2, CHCl3/