666 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4
Pertz et al.
from THF/Et2O to give a white powder: yield 0.96 g (68%);
mp 218-219 °C dec; 1H NMR (pyridine-d5) δ 1.10-1.20 (m, 3
H), 1.48-1.62 (m, 5 H), 1.94 (m, 2 H), 2.37 (m, 1 H), 2.56 (quasi
t, J ) 12.0 Hz, 1 H, H-7â), 2.67 (s, 3 H, NCH3), 3.00 (quasi t,
J ) 14.5 Hz, 1 H, H-4R), 3.38-3.45 (m, 2 H, H-8, H-7R), 3.49
(m, 1 H, H-5), 3.67 (dd, J ) 14.5, 5.5 Hz, 1 H, H-4â), 4.29 (d,
J ) 6.0 Hz, 2 H, H-17â, H-17R), 6.60 (br s, 1 H, H-9), 6.66 (s,
2 H, maleate CHdCH), 7.25 (s, 1 H, H-2), 7.33 (t, J ) 7.5 Hz,
1 H, H-13), 7.41 (d, J ) 7.5 Hz, 1 H, H-12 or H-14), 7.44 (d, J
) 8.0 Hz, 1 H, H-12 or H-14), 11.77 (br s, 1 H, NH); MS (m/z)
364 (M+•, 100). Anal. (C27H32N2O6) Calcd: C, 67.5; H, 6.7; N,
5.8; Found: C, 67.3; H, 6.8; N, 5.8.
of contractile responses.43 Each preparation was mounted in
a 20-mL organ bath containing modified Krebs-Henseleit
solution of the following composition (mM): NaCl 118, KCl
4.7, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25, and
D-glucose 10. The solution was continuously gassed with 95%
O2/5% CO2 and warmed to a constant temperature of 37 °C.
Preparations were connected to a force displacement trans-
ducer attached to a TSE 4711 transducer coupler and a
Siemens C 1016 compensograph for the continuous recording
of isometric changes in tension. Resting force was adjusted to
5 mN at the beginning of each experiment. During an
equilibration period of 120 min, preparations were stimulated
once (after 60 min) with 5-HT (1 µM).
In experiments with agonists two cumulative concentra-
tion-effect curves were determined on each arterial segment
at an interval of 60 min: the first curve for 5-HT and the
second for the respective agonist in the absence or presence
of ketanserin (3 nM). Ketanserin was incubated 30 min before
the second curve. The shift to the right observed in the
presence of ketanserin was calculated by comparing with the
shift measured for the respective control preparation in the
absence of ketanserin. In additional experiments with antago-
nists (partial agonists) two cumulative concentration effect
curves for 5-HT were determined at an interval of 60 min in
the absence and presence of antagonists (partial agonists).
Antagonists (partial agonists) were incubated 30-60 min
before the second curve. Prazosin (30 nM) and cocaine (6 µM)
were present throughout the experiments to block R1-adreno-
ceptors and neuronal uptake of 5-HT.
Gen er a l P r oced u r e for t h e P r ep a r a t ion of Cyclo-
a lk a n eca r boxylic Ester s Der ived fr om Lyser gol a n d
Dih yd r olyser gol-I w ith a n Isop r op yl Su bstitu en t a t N-1
(Meth od A). 9,10-Did eh yd r o-1-isop r op yl-6-m eth yl-8â-er -
golin ylm eth yl Cycloh exa n eca r boxyla te (9b). To a stirred
solution of 9a -base (0.465 g, 1.28 mmol) in dry THF (20 mL)
were added 18-crown-6 (0.24 g, 0.91 mmol), powdered KOH
(0.965 g, 17.2 mmol), and 2-iodopropane (1.52 mL, 15.2 mmol).
The mixture was stirred for 1 h, diluted with CH2Cl2, and
filtered to remove solids. After the filtrate was evaporated to
dryness, the residue was partitioned between CH2Cl2 and a
saturated solution of NaHCO3. The organic layer was dried
over Na2SO4 and the solvent removed under vacuum. Radial
centrifugal chromatography (Chromatotron instrument, eluent
CH2Cl2/cyclohexane/MeOH, 100/20/2 (v/v/v)) of the residue
afforded a yellow oil. The hydrogen maleate salt of 9b was
precipitated from THF/Et2O to give a white powder: yield 0.43
1
g (65%); mp 190-191 °C dec; H NMR (pyridine-d5) δ 1.09-
Partial agonists were characterized by the equilibrium
dissociation constant KP. In experiments where the compounds
were studied as agonists, KP was estimated according to the
method of Kenakin.27 KP was estimated from the slope m of a
plot which related equieffective concentrations of 5-HT and
the partial agonist P according to the equation: c(5-HT) ) m‚
c(5-HT)/c(P) + b, where b is the ordinate intercept. pKP ) -log
KP was calculated from -log KP ) log m. In experiments where
the compounds were studied as antagonists of the effects of
5-HT, KP was estimated according to the method of Marano
and Kaumann.28 KP was estimated from the slope m of a
weighted plot which related equieffective concentrations of
5-HT in the absence (c(5-HT)) and presence (c(5-HT)*) of the
partial agonist P: c(5-HT) ) m‚c(5-HT)* + b, where b is the
ordinate intercept. pKP ) -log KP was calculated from log-
(1/m - 1) ) log c(P) - log KP. Kaumann-Marano plots were
constructed in those cases where antagonist concentrations
were used over 2 log units.29 After log(1/m - 1) versus log c(P)
was plotted, a regression line was constructed and the
intercept on the log concentration axis provided the estimate
of pKP. For the calculation of pKP values, the slope of the
Kaumann-Marano plot was constrained to unity unless it was
significantly different from unity (P < 0.05). Antagonist
dissociation constants given as apparent pA2 values were
calculated from the equation: pA2 ) -log c(B) + log(CR - 1).30
Antagonist dissociation constants given as full pA2 values were
estimated using the method of Arunlakshana and Schild.31
Schild plots were constructed to estimate the pA2 value and
the slope m of the regression line from each experimental
series, which generally comprised at least three different
antagonist concentrations over 2 log units. For the calculation
of pA2 values from Schild plot, the slope was constrained to
unity unless it was significantly different from unity (P <
0.05).32
1.16 (m, 3 H), 1.36 (2 d, J ) 6.5 Hz, 6 H), 1.48-1.62 (m, 5 H),
1.94 (m, 2 H), 2.38 (m, 1 H), 2.44 (quasi t, J ) 10.0 Hz, 1 H,
H-7â), 2.60 (s, 3 H, NCH3), 2.89 (quasi t, J ) 14.5 Hz, 1 H,
H-4R), 3.20-3.27 (m, 2 H, H-8, H-7R), 3.32 (m, 1 H, H-5), 3.60
(dd, J ) 14.5, 5.5 Hz, 1 H, H-4â), 4.28 (d, J ) 6.0 Hz, 2 H,
H-17â, H-17R), 4.53 (sept, J ) 6.5 Hz, 1 H), 6.57 (br s, 1 H,
H-9), 6.66 (s, 2 H, maleate CHdCH), 7.04 (s, 1 H, H-2), 7.31-
7.41 (m, 3 H, H-12, H-13, H-14); MS (m/z) 406 (M+•, 100). Anal.
(C30H38N2O6) Calcd: C, 68.9; H, 7.3; N, 5.4. Found: C, 69.1;
H, 7.3; N, 5.3.
Gen er al P r ocedu r e for th e P r epar ation of N-1-Isopr op-
yl Der iva tives of Sim p le Cla vin es (Meth od B). 1-Isop r op -
ylelym ocla vin e (3b). To a stirred solution of 3a (1.27 g, 5
mmol) in DMSO (10 mL) were added 18-crown-6 (1.32 g, 5
mmol) and powdered KOH (1.72 g, 30.66 mmol). A solution of
isopropyl tosylate (1.6 g, 7.5 mmol) in DMSO (5 mL) was added
dropwise over 45 min. The mixture was stirred for further 60
min and then poured into a saturated solution of NaHCO3.
The solution was extracted with Et2O (4 × 100 mL). The
combined organic layers were dried over Na2SO4 and filtered,
and the solvent was removed under vacuum. Radial centrifugal
chromatography (Chromatotron instrument, eluent CH2Cl2/
MeOH, 95/5 (v/v)) of the residue afforded a white solid. The
hydrogen maleate salt of 3b was precipitated from THF/Et2O
to give a white powder: yield 1.13 g (55%); mp 162-164 °C;
1H NMR (pyridine-d5) δ 1,37 (2 d, J ) 6.5 Hz, 6 H), 2,91 (s, 3
H, NCH3), 3.17 (dd, J ) 12.5, 11.5 Hz, 1 H, H-4R), 3.24 (ddd,
J ) 11.5, 9.5, 3.0 Hz, 1 H, H-5), 3.52 (dd, J ) 13,5, 3,0 Hz, 1
H, H-4â), 3.83 (d, J ) 16.5 Hz, 1 H, H-7â), 4.13 (d, J ) 16.5
Hz, 1 H, H-7R), 4.21 (br s, 1 H, H-10), 4.44 (s, 2 H, H-17), 4.55
(sept, J ) 6.5 Hz, 1 H), 6.63 (s, 2 H, maleate CHdCH), 6.81
(br s, 1 H, H-9), 7.03 (s, 1 H, H-2), 7.15 (d, J ) 7.0 Hz, 1 H,
H-12 or H-14), 7.27-7.37 (m, 2 H, H-13 and H-12 or H-14);
MS (m/z) 296 (M+•, 100). Anal. (C23H28N2O5) Calcd: C, 67.0;
H, 6.8; N, 6.8. Found: C, 66.8; H, 6.8; N, 6.9.
F u n ction a l 5-HT1B Recep tor Assa y (gu in ea -p ig ilia c
a r ter y). Guinea pigs of either sex, 300-450 g, were stunned
by a blow on the neck and bled. The abdominal aorta and the
right and left common iliac arteries were removed and cleared
of adhering connective tissue. Two or three cylindrical seg-
ments of 1-2-mm length from each iliac artery were horizon-
tally suspended between two L-shaped stainless steel hooks
(diameter 0.15 mm) and isometrically mounted as described
for rat tail artery experiments (see above). The bath fluid
(modified Krebs-Henseleit solution with CaCl2 (1.25 mM) and
D-glucose (11.5 mM)) contained ketanserin (1 µM), mepyramine
P h a r m a cology. F u n ction a l 5-HT2A Recep tor Assa y (r a t
ta il a r ter y). Male Wistar rats (280-350 g) were killed by
cervical dislocation. The ventral caudal artery was quickly
dissected and cleared of adhering connective tissue. A stainless
steel wire (diameter 0.3 mm) was inserted into the artery to
rub off the endothelium. Up to 24 cylindrical segments of 4-5-
mm length were prepared from each artery and horizontally
suspended between two L-shaped stainless steel hooks (diam-
eter 0.15 mm) gently inserted into the lumen for the recording