Cycloalkanecarboxylic esters derived from lysergol, dihydrolysergol-I, and elymoclavine as partial agonists and antagonists at rat 5-HT(2A) receptors: Pharmacological evidence that the indolo[4,3-fg]quinoline system of the ergolines is responsible for high 5-HT(2A) receptor affinity

Article abstract of DOI:10.1021/jm981092u

Three series of cycloalkanecarboxylic esters derived from the naturally occurring clavine alkaloids lysergol, dihydrolysergol-I, and elymoclavine were synthesized to study their interaction with 5-HT(2A) receptors and α₁- adrenoceptors in rat tail artery and aorta, respectively. Especially cycloalkanecarboxylic esters derived from lysergol showed complex behavior as partial agonists and antagonists of the contractile effect of 5-HT. Within this group, partial 5-HT(2A) receptor agonist activity was most potent for cyclopropanecarboxylic ester 6a (pK(p) = 7.67, α = 0.21) and decreased as the volume requirement of the alicyclic ring increased. This tendency was echoed in experiments where the compounds were used as antagonists of the contractile effect of 5-HT. From the structure-activity study, the N-1- isopropyl homologue of 6a, compound 6b, emerged as the ligand with the highest affinity for rat 5-HT(2A) receptors (pA₂ = 8.74). For cycloalkanecarboxylic esters derived from dihydrolysergol-I and elymoclavine, no clear structure-affinity relationship could be deduced, although those compounds that had smaller cycloalkyl rings in the acyl portion and an isopropyl substituent at N-1 showed the highest 5-HT(2A) receptor affinity. On the other hand, cycloalkanecarboxylic esters derived from lysergol, dihydrolysergol-I, and elymoclavine displayed low or marginal affinity at α₁-adrenoceptors. A further aim of the study was to examine to what extent the complete removal of the acyl portion of the esters would affect 5-HT(2A) receptor affinity. The parent alcohols of the three series of N-1-isopropyl homologues, 1-isopropyllysergol (1b), 1-isopropyldihydrolysergol-I (2b), and 1-isopropylelymoclavine (3b), displayed higher affinity for 5-HT(2A) receptors (pA₂ = 9.15, 8.50, 9.14) than the corresponding esters. Compounds 1b-3b had no contractile effects by themselves and displayed low affinity at guinea-pig 5-HT(1B) receptors and rat α₁-adrenoceptors. The high affinity for rat 5-HT(2A) receptors was retained when clavines even more simple in structure than 1b-3b, compounds 4b and 5b, were examined as 5-HT(2A) receptor antagonists. The nanomolar antagonist activity of simple clavines (1b-5b) in the rat suggests that the indolo-[4,3-fg]quinoline system of the ergolines is the molecular fragment that is responsible for 5-HT(2A) receptor affinity, and not the substituent at position C-8.

Full text of DOI:10.1021/jm981092u

J . Med. Chem. 1999, 42, 659-668
659
Cycloa lk a n eca r boxylic Ester s Der ived fr om Lyser gol, Dih yd r olyser gol-I, a n d
Elym ocla vin e a s P a r tia l Agon ists a n d An ta gon ists a t Ra t 5-HT2A Recep tor s:
P h a r m a cologica l Evid en ce th a t th e In d olo[4,3-fg]qu in olin e System of th e
Er golin es Is Resp on sible for High 5-HT2A Recep tor Affin ity
Heinz H. Pertz,* H.-Christian Milhahn, and Eckart Eich
Fachbereich Pharmazie, Freie Universita¨t Berlin, Ko¨nigin-Luise-Strasse 2 + 4, D-14195 Berlin (Dahlem), Germany
Received October 1, 1998
Three series of cycloalkanecarboxylic esters derived from the naturally occurring clavine
alkaloids lysergol, dihydrolysergol-I, and elymoclavine were synthesized to study their
interaction with 5-HT2A receptors and R1-adrenoceptors in rat tail artery and aorta, respectively.
Especially cycloalkanecarboxylic esters derived from lysergol showed complex behavior as partial
agonists and antagonists of the contractile effect of 5-HT. Within this group, partial 5-HT2A
receptor agonist activity was most potent for cyclopropanecarboxylic ester 6a (pKP ) 7.67, R )
0.21) and decreased as the volume requirement of the alicyclic ring increased. This tendency
was echoed in experiments where the compounds were used as antagonists of the contractile
effect of 5-HT. From the structure-activity study, the N-1-isopropyl homologue of 6a , compound
6b, emerged as the ligand with the highest affinity for rat 5-HT2A receptors (pA2 ) 8.74). For
cycloalkanecarboxylic esters derived from dihydrolysergol-I and elymoclavine, no clear
structure-affinity relationship could be deduced, although those compounds that had smaller
cycloalkyl rings in the acyl portion and an isopropyl substituent at N-1 showed the highest
5-HT2A receptor affinity. On the other hand, cycloalkanecarboxylic esters derived from lysergol,
dihydrolysergol-I, and elymoclavine displayed low or marginal affinity at R1-adrenoceptors. A
further aim of the study was to examine to what extent the complete removal of the acyl portion
of the esters would affect 5-HT2A receptor affinity. The parent alcohols of the three series of
N-1-isopropyl homologues, 1-isopropyllysergol (1b), 1-isopropyldihydrolysergol-I (2b), and
1-isopropylelymoclavine (3b), displayed higher affinity for 5-HT2A receptors (pA2 ) 9.15, 8.50,
9.14) than the corresponding esters. Compounds 1b-3b had no contractile effects by themselves
and displayed low affinity at guinea-pig 5-HT1B receptors and rat R1-adrenoceptors. The high
affinity for rat 5-HT2A receptors was retained when clavines even more simple in structure
than 1b-3b, compounds 4b and 5b, were examined as 5-HT2A receptor antagonists. The
nanomolar antagonist activity of simple clavines (1b-5b) in the rat suggests that the indolo-
[4,3-fg]quinoline system of the ergolines is the molecular fragment that is responsible for 5-HT2A
receptor affinity, and not the substituent at position C-8.
Ch a r t 1
In tr od u ction
Despite their low selectivity, ergolines are important
ligands for serotonin 5-HT2A receptors, where they
display complex behavior as partial agonists or antago-
nists.1 For example, it has been suggested that the
classical hallucinogenic agent lysergic acid diethylamide
(LSD) may exert its psychotomimetic effect predomi-
nantly by acting as a partial agonist at 5-HT2A recep-
tors.2,3 Furthermore, there is evidence that the powerful
contractions produced by ergometrine and methylergo-
metrine on uterine smooth muscle involve partial ago-
nism of these agents at 5-HT2A receptors, since the
effects can be blocked by the 5-HT2A receptor antago-
nists methysergide and ICI169,369, respectively.4 On
the other hand, ergoline-based 5-HT2A receptor antago-
nists such as the widely used LY53857 and structurally
related compounds such as sergolexole, amesergide, and
LY215840 are known to inhibit 5-HT-induced platelet
aggregation and to block the direct contractile effect of
platelet-released 5-HT on vascular smooth muscle.5-9
These agents may be useful in the treatment of ischemic
heart disease and other vascular disorders.10 From the
chemical standpoint, sergolexole is a 6-methylergoline-
8-carboxylic acid cycloalkyl ester, while amesergide and
LY215840 are 6-methylergoline-8-carboxylic acid cy-
cloalkylamides. The compounds share the structural
property to be substituted with an isopropyl group at
the indole nitrogen (Chart 1).
* To whom correspondence should be addressed. Phone: (49) 30-
8383135. Fax: (49) 30-8383729. E-mail: sieker@schunet.pharmazie.fu-
berlin-de.
Recently, we have found that ester formation of the
naturally occurring clavines lysergol (9,10-didehydro-
10.1021/jm981092u CCC: $18.00 © 1999 American Chemical Society
Published on Web 02/04/1999

660 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4
Pertz et al.
Ch a r t 2
Sch em e 1a
a
Reagents and experimental conditions: (i) pyridine, 4-DMAP,
addition of the respective acid chloride at 0 °C within 160 min,
then 12 h stirring at rt. Chemical structures: for details, see Chart
2.
in the vasculature not only via interaction with 5-HT2A
receptors but also with 5-HT1B (formerly 5-HT1D-like)
receptors16-18 and R1-adrenoceptors, it was of special
interest to examine whether selected compounds would
display selectivity for vascular 5-HT2A receptors over
these other sites. A preliminary report of some of these
data has been published.19
8â-hydroxymethyl-6-methylergoline) and dihydrolyser-
gol-I (8â-hydroxymethyl-6-methylergoline) with suitable
aliphatic branched-chain carboxylic acids and N-1-
isopropyl substitution afforded compounds with en-
hanced antagonist activity at vascular 5-HT2A recep-
tors.11 As an extension of our previous efforts, we now
report the conversion of lysergol (1a ), dihydrolysergol-I
(2a ), and elymoclavine (3a ) into three series of cyclo-
alkanecarboxylic esters (6a ,b-21a ,b) (Chart 2) and the
interaction of the new ergolines with 5-HT2A receptors
in rat tail artery and R1-adrenoceptors in rat aorta. It
was hypothesized that alicyclic carboxylic esters derived
from lysergol, dihydrolysergol-I, and elymoclavine could
ultimately have a similar affinity profile at 5-HT2A
receptors as 6-methylergoline-8-carboxylic acid cy-
cloalkyl esters such as sergolexole.
It has recently been demonstrated that N-1-unsub-
stituted ergolines have higher affinity for the human
versus the rat 5-HT2A receptor, whereas the correspond-
ing ergolines with an alkyl (methyl, isopropyl) substitu-
ent at N-1 have higher affinity for the rat versus the
human 5-HT2A receptor.12,13 A single amino acid differ-
ence, Ser242 in the human receptor and Ala242 in the
rat receptor, is responsible for the differential affini-
ties.14 In addition, it has been shown that mutation of
Ala242 f Ser242 in the rat 5-HT2A receptor converts
the operational characteristics of the rat receptor to that
of the human receptor.15 It has been speculated that
serine in the human 5-HT2A receptor serves as a
hydrogen bond acceptor for the indole NH of N-1-
unsubstituted ergolines, whereas alanine in the rat
5-HT2A receptor allows aliphatic stacking with the alkyl
group of N-1-alkylated ergolines.15 Hence, a further aim
of the present study was to investigate the pharmaco-
logical profile of the parent alcohols of the N-1-isopropyl
homologues (1-isopropyllysergol (1b), 1-isopropyldihy-
drolysergol-I (2b), 1-isopropylelymoclavine (3b)) in order
to estimate their possible contribution to 5-HT2A recep-
tor antagonist activity in the rat. Affinity estimates of
even more simple ergolines such as 1-isopropylagrocla-
vine (4b) and 1-isopropylfestuclavine (5b) should elu-
cidate the structural requirements of the 5-HT2A recep-
tor with regard to the ergoline molecule. 4b and 5b have
a methyl group instead of a hydroxymethyl group at C-8,
and 5b has a C-8-C-9 single bond (Chart 2). Since
ergolines generally exert their pharmacological effects
Ch em istr y
Sergolexole is the most prominent 6-methylergoline-
8-carboxylic acid cycloalkyl ester that shows potent
5-HT2A receptor antagonist activity (pA2 of 9.05 in rat
jugular vein) without appreciably binding to 5-HT1,
dopamine D1, dopamine D2, histamine H1, muscariner-
gic, R1-adrenergic, and â-adrenergic receptors.6,7 We
intended to synthesize structurally related cycloalkane-
carboxylic esters derived from 9,10-didehydro-8â-hy-
droxymethyl-6-methylergoline (lysergol), 8â-hydroxy-
methyl-6-methylergoline (dihydrolysergol-I), and 8,9-
didehydro-8-hydroxymethyl-6-methylergoline (elymo-
clavine), so-called ergoline “reverse esters” (reverse in
comparison with lysergic acid esters), which have an
alicyclic ring in the acyl portion and an isopropyl
substituent at the indole nitrogen. Lysergol (1a ), dihy-
drolysergol-I (2a ), and elymoclavine (3a ) served as
starting points for the preparation of the esters. Ely-
moclavine (3a ) was available from submerged cultures
of Claviceps strain SD-58 which can be readily adapted
to large-scale production.20,21 Lysergol (1a ) was prepared
from elymoclavine (3a ) by heating on activated Al2O3
in pyridine22 and dihydrolysergol-I (2) from lysergol (1a )
by catalytic hydrogenation with 10% Pd/C in DMF/
pyridine (100:2, v/v) at 5 bar.23 Agroclavine (4a ) was
isolated from Claviceps strain SD-58, while festuclavine
(5a ) was synthesized from 4a by catalytic transfer
hydrogenation using Raney nickel in EtOH.24 O-Acyla-
tion of 1a -3a was achieved by Einhorn reaction using
the respective alicyclic acid chloride in pyridine in the
presence of 4-DMAP (Scheme 1).
Introduction of the isopropyl substituent at the N-1
position of the esters derived from 1a and 2a was
achieved by addition of isopropyl iodide to a solution of
the respective O-acylated lysergol or dihydrolysergol-I
derivative in THF using as base powdered KOH in the
presence of 18-crown-6 (method A). Simple clavines such
as 1a -5a were alkylated by using isopropyl tosylate as
alkylating agent in DMSO (method B) (Scheme 2).25
O-Acylated derivatives of 3a with an isopropyl substitu-
ent at the N-1 position were prepared from 3b because

Role of the Indoloquinoline System of Ergolines
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4 661
Sch em e 2a
a
Reagents and experimental conditions: (i) dry THF, 18-crown-
6, powdered KOH, isopropyl iodide, 1 h stirring at rt (method A);
(ii) DMSO, powdered KOH, isopropyl tosylate, 2 h stirring at rt
(method B). Chemical structures: for details, see Chart 2.
the direct alkylation of the esters led to hydrolysis. After
workup, all compounds were purified and separated by
radial chromatography under an atmosphere of nitrogen
or argon.
F igu r e 1. Antagonism of 5-HT-induced contractions by the
partial agonist 6a and the silent antagonist 1b in rat tail
artery. Shown are concentration-effect curves for 5-HT in the
absence (O, 17-42) and presence of 6a and 1b, respectively.
A. Compound 6a at 15.8 nM (b, n ) 8), 39.8 nM (2, n ) 8),
100 nM (1, n ) 8), 300 nM (9, n ) 8), and 1000 nM ([, n )
10). Contractions elicited by 15.8, 39.8, 100, 300, and 1000 nM
6a were 3 ( 1, 9 ( 2, 9 ( 2, 11 ( 2, and 14 ( 3% (not shown)
and partially faded to 3 ( 1, 8 ( 2, 9 ( 2, 10 ( 2, and 13 ( 2%
within 30 min. Contractions of 6a after 30 min are shown in
the left segment of the abscissa. Inset: Kaumann-Marano plot
for the calculation of partial agonist affinity (pKP).29 pKP was
8.40 ( 0.04 (slope m ) 1.11 ( 0.02, significantly different from
unity (P < 0.05)). B. Compound 1b at 3 nM (b, n ) 10), 30
nM (2, n ) 9), and 300 nM (1, n ) 6). Inset: Schild analysis
of antagonist-induced curve displacements. pA2 was 9.15 (
0.04 (slope m ) 1.05 ( 0.03, not significantly different from
unity (P < 0.05)). All values are means ( SEM.
Biology
The interaction of the compounds with 5-HT2A and
5-HT1B receptors and R1-adrenoceptors was studied in
various functional in vitro assays. Partial agonism at
5-HT2A receptors was studied in the absence and pres-
ence of ketanserin (3 nM) in cylindrical segments of the
isolated rat tail artery. The effectiveness of the com-
pounds in blocking 5-HT2A receptor-mediated contrac-
tions by 5-HT was determined in the same tissue. The
ability of the compounds to block R1-adrenoceptor-
mediated contractions by (R)-phenylephrine was studied
in cylindrical segments of the isolated rat thoracic aorta.
The interaction of selected compounds (1b-3b) with
5-HT1B receptors was examined in cylindrical segments
of the isolated guinea-pig iliac artery, moderately pre-
contracted by PGF2R (50-500 nM).26
I, and elymoclavine were tested for their ability to
inhibit 5-HT-induced contractions of rat tail artery and
(R)-phenylephrine-induced contractions of rat aorta. All
compounds studied caused a rightward shift of the
concentration-response curves to 5-HT and (R)-phen-
ylephrine, respectively, with little or no effect on
maximum responses (Tables 1-3). Cyclopropanecar-
boxylic acid ester 6a (pKP ) 8.40, Figure 1A) and its
N-1-isopropyl homologue 6b (pA2 ) 8.74) showed the
highest affinity for 5-HT2A receptors. Within the series
of O-acylated derivatives of lysergol (6a,b-10a,b) 5-HT2A
receptor affinity continuously decreased as the volume
requirement of the alicyclic ring increased. Increasing
the size of the alicyclic ring resulted in a decrease in
antagonist activity for N-1-unsubstituted homologues
(6a -10a ) that paralleled the potency in experiments for
5-HT2A receptor agonist activity (see below). It should
be mentioned that the pKP values for the partial
agonist-5-HT2A receptor complex calculated from an-
tagonism by 6a -10a of the contractile response to 5-HT
were slightly higher than the pKP values calculated from
the contractile response to 6a -10a . This suggests that
Antagonism by cycloalkanecarboxylic esters derived
from lysergol (6a ,b-10a ,b), dihydrolysergol-I (11a ,b-
15a ,b), and elymoclavine (16a ,b-21a ,b) of the contrac-
tile effect of 5-HT via 5-HT2A receptors and (R)-
phenylephrine via R1-adrenoceptors is summarized in
Tables 1-3. The contractile effects of O-acylated lysergol
derivatives 6a -10a on 5-HT2A receptors are sum-
marized in Table 4. The interaction of selected clavine
derivatives 1b-5b with 5-HT2A receptors, 5-HT1B recep-
tors, and R1-adrenoceptors is summarized in Table 5.
Partial agonists were characterized by estimation of the
equilibrium dissociation constant KP (given as pKP).27-29
Antagonist dissociation constants are given as apparent
or full pA2 values.30-32 For details, see the Experimental
Section.
Resu lts a n d Discu ssion
Cycloa lk a n eca r boxylic Ester s Der ived fr om Ly-
ser gol, Dih yd r olyser gol-I, a n d Elym ocla vin e a s
An ta gon ists a t 5-HT2A Recep tor s a n d R1-Ad r en o-
cep tor s. “Reverse esters” of lysergol, dihydrolysergol-

662 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4
Pertz et al.
Ta ble 1. Physical Properties and Pharmacological Effects of Cycloalkanecarboxylic Esters Derived from Lysergol on 5-HT-Induced
Contractions of Rat Tail Artery and (R)-Phenylephrine-Induced Contractions of Rat Aorta
a
All compounds exhibit 1H NMR spectral data in agreement with assigned structures. All compounds were characterized and tested
as their hydrogen maleate salts. Salt 6b was hydrated with 0.5 mol of water. All salts were crystallized from a THF/Et2O mixture.
b
Elemental analyses were within (0.4% of the theoretical values for C, H, N. Values are expressed as mean ( SEM from n individual
vascular segments of at least two animals (number n of independent experiments in parentheses). c These values are apparent pA2 values
d
which were estimated according to ref 30. 5-HT2A/R1 is the ratio of KB values. e This value is the pKP value which was estimated from
a Kaumann-Marano plot according to ref 29. f Slope 1.11 ( 0.02 of the Kaumann-Marano plot, significantly different from unity (P <
g
h
0.05). This value is the full pA2 value which was estimated from the Schild plot according to ref 31. Slope 1.05 ( 0.06 of the Schild
i
j
k
plot. Slope 1.00 ( 0.12 of the Kaumann-Marano plot. Slope 1.10 ( 0.07 of the Schild plot. This value is the pKP value which was
estimated according to ref 28.
6a -10a and 5-HT may bind in two slightly different
orientations at the 5-HT2A receptor. In the series of
O-acylated derivatives of dihydrolysergol-I (11a ,b-
15a ,b), increasing the size of the alicyclic ring resulted
in a decrease in 5-HT2A receptor affinity only for N-1-
isopropyl homologues. For unsubstituted homologues no
clear structure-affinity relationship could be deduced.
Moreover, 5-HT2A receptor affinity was only slightly
affected by the structure of the cycloalkyl ring in the
acyl portion of the esters. This finding was echoed in
the series of O-acylated derivatives of elymoclavine
(16a ,b-21a ,b), where 5-HT2A receptor affinities for
unsubstituted and N-1-isopropyl homologues showed
differences of only 0.86 and 0.36 log unit between the
most potent compound and the weakest compound. It
should be mentioned that the introduction of a methoxy
group in the 4-position of the cyclohexyl ring (19a ,b and
20a ,b) failed to enhance 5-HT2A receptor affinity. This
was in contrast to the observation of Garbrecht et al.
who synthesized substituted 6-methylergoline-8-car-
boxylic acid cyclohexyl esters of which an oxygen in the
4-position of the ester cyclohexyl ring was of crucial
importance for 5-HT2A receptor affinity.5 Moreover, the
findings in our laboratory suggest that the stereochem-
ical orientation of the substituent in the 4-position
seems to be of minor importance for 5-HT2A receptor
affinity. Compounds 20a ,b with a cyclohexyl ring in the
acyl portion characterized by a 4-methoxy group trans
to the carboxyl group showed nearly equal antagonist
activity compared to 19a ,b with a 4-methoxy-substitut-
ed cyclohexyl ring existing as a cis/trans diastereomeric
mixture.
Comparing the 5-HT2A receptor affinity of N-1-un-
substituted derivatives with that of compounds with an
N-1-isopropyl group revealed the importance of a sub-
stituent at N-1. “Reverse esters” derived from lysergol
and elymoclavine, respectively, with an N-1-isopropyl
group showed higher affinity at rat 5-HT2A receptors
than their unsubstituted homologues with the exception
of one case (compound 21b). Similar findings have
previously been reported with 8â-6-methylergoline-8-
carboxylic acid cycloalkyl esters and amides.12,13 With
O-acylated derivatives of dihydrolysergol-I, no clear
structure-affinity relationship could be deduced. N-1-
Alkylation resulted in higher 5-HT2A receptor affinity
only for those derivatives that were characterized by
smaller cycloalkyl rings (cyclopropyl derivative 11b,
cyclobutyl derivative 12b).
The examination of the pharmacological activity of
lysergol, dihydrolysergol-I, and elymoclavine “reverse
esters” at R1-adrenoceptors revealed low affinity at these
sites for all compounds tested. This was especially true
for ergolines with an N-1-isopropyl substituent. Er-
golines with an isopropyl group at N-1 seem to be highly
specific antagonists at 5-HT2A receptors (see ratio
5-HT2A/R1 of Tables 1-3).

Role of the Indoloquinoline System of Ergolines
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4 663
Ta ble 2. Physical Properties and Pharmacological Effects of Cycloalkanecarboxylic Esters Derived from Dihydrolysergol-I on
5-HT-Induced Contractions of Rat Tail Artery and (R)-Phenylephrine-Induced Contractions of Rat Aorta
a
All compounds exhibit 1H NMR spectral data in agreement with assigned structures. All compounds were characterized and tested
as their hydrogen maleate salts. All salts were crystallized from a THF/Et2O mixture. Elemental analyses were within (0.4% of the
b
theoretical values for C, H, N. Values are expressed as mean ( SEM from n individual vascular segments of at least two animals
(number n of independent experiments in parentheses). c These values are apparent pA2 values which were estimated according to ref
d
30. 5-HT2A/R1 is the ratio of KB values. e This value is the pKP value which was estimated according to ref 28. f This value is the full pA2
g
h
value which was estimated from the Schild plot according to ref 31. Slope 0.92 ( 0.05 of the Schild plot. Slope 0.84 ( 0.05 of the Schild
plot, significantly different from unity (P < 0.05).
Cycloa lk a n eca r boxylic Ester s Der ived fr om Ly-
ser gol a s P a r tia l Agon ists a t 5-HT2A Recep tor s.
Within the series of O-acylated lysergol derivatives, N-1-
unsubstituted compounds (6a -10a ) contracted rat tail
arteries with pKP values of 6.35-7.67 and intrinsic
activities R of 0.11-0.21 with respect to 5-HT (Table
4). Partial agonist effects of the compounds were sur-
mountably antagonized by ketanserin (3 nM). The
similarity of blocking potency of ketanserin (pA2 of
9.10-9.18) is consistent with an interaction of the
compounds with the same receptor class (5-HT2A) (Table
4). Compounds 6a -10a are therefore further ergolines
with 5-HT2A receptor agonist activity. In agreement with
the affinities obtained from antagonist experiments (see
above), agonist activity was most potent for cyclopro-
panecarboxylic acid ester 6a (pKP ) 7.67) and continu-
ously decreased in this series of homologues to reveal
the lowest activity for cycloheptanecarboxylic acid ester
10a (pKP ) 6.35). Especially compounds 6a , 7a , and 8a
were more potent than 5-HT and lysergol (1), respec-
tively (see relative potencies of Table 4). On the other
hand, O-acylated dihydrolysergol-I derivatives produced
only marginal contractile responses in rat tail artery
(R of 0.02-0.05). Therefore pKP values for the agonist
effects of these compounds were not calculated. Com-
pound 11a was the only outlier in this series. The
compound showed appreciable agonist potency and
intrinsic activity (pKP ) 6.10, R ) 0.35), but the
contractile effect was resistant to blockade by ketanserin
(3 nM). It should be mentioned that there was no
compound within the series of O-acylated elymoclavine
derivatives that produced any measurable contractile
effect by itself at vascular 5-HT2A receptors. Further-
more, when O-acylated derivatives of lysergol and
dihydrolysergol-I had an N-1-isopropyl substituent, no
agonist activity was observed. The finding that partial
5-HT2A receptor agonism of the ergolines examined is
restricted to N-1-unsubstituted compounds with a double
bond in the 9,10-position is consistent with previously
reported findings on several ergolines which show the
same structural pattern. The most prominent represen-
tative in this connection is LSD which has been reported
to act as a partial agonist at 5-HT2A receptors of rabbit
aorta (R ) 0.14) and calf coronary artery (R ≈ 0.2).34,35
In addition LSD has been reported to act as a partial
agonist (R ) 0.25) at 5-HT2A receptors coupled to the
phosphatidylinositol (PI) second-messenger system.36
Further ergoline-based compounds with partial agonist
activity at 5-HT2A receptors are lisuride, and ergo-
metrine.37,38,4 Although it has been found that N-1-
unsubstituted ergolines such as LSD, lisuride and
ergometrine display selectivity for human versus rat
5-HT2A receptors (see above) and thus are reliable
probes for unmasking species differences,12,13,39 it should
be emphasized that these ergolines have a different
quality of action (partial agonism) compared to ergolines
for which silent 5-HT2A receptor antagonism can be
demonstrated.
N-1-Su b st it u t ed Cla vin es a s An t a gon ist s a t
5-HT2A Recep tor s. The observation that ergoline “re-

664 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4
Pertz et al.
Ta ble 3. Physical Properties and Pharmacological Effects of Cycloalkanecarboxylic Esters Derived from Elymoclavine on
5-HT-Induced Contractions of Rat Tail Artery and (R)-Phenylephrine-Induced Contractions of Rat Aorta
a
All compounds exhibit 1H NMR spectral data in agreement with assigned structures. All compounds were characterized and tested
as their hydrogen maleate salts with the exception of compounds 16b and 17b which were characterized and tested as their hydrochloride
salts. All salts with the exception of compounds 16b and 17b were crystallized from a THF/Et2O mixture. Compounds 16b and 17b were
crystallized from a MeOH/Et2O mixture. The 4-methoxy-substituted cyclohexyl ring of 19a ,b exists as cis/trans diastereomeric mixture
b
(1:3). Elemental analyses were within (0.4% of the theoretical values for C, H, N. Values are expressed as mean ( SEM from n individual
vascular segments of at least two animals (number n of independent experiments in parentheses). c These values are apparent pA2 values
d
which were estimated according to ref 30. 5-HT2A/R1 is the ratio of KB values.
Ta ble 4. Cycloalkanecarboxylic Esters Derived from Lysergol
as Agonists at 5-HT2A Receptors of Rat Tail Artery
artery than the corresponding esters (Table 5). Com-
pounds 1b, 2b, and 3b produced potent antagonism of
the effects of 5-HT with pA2 values of 9.15 (Figure 1B),
8.50, and 9.14, respectively. In contrast, 1b, 2b, and 3b
showed lower affinity for vascular 5-HT1B receptors and
vascular R1-adrenoceptors (Table 5). It is worth men-
tioning that 1b, 2b, and 3b had no contractile effects
by themselves at 5-HT2A receptors, 5-HT1B receptors,
and R1-adrenoceptors. The high affinity for rat 5-HT2A
receptors was retained when analogues even more
simple in structure than 1b, 2b, and 3b were examined
as antagonists of 5-HT-induced contractions of rat tail
artery. Simple clavines such as compounds 4b and 5b,
in which a methyl group instead of a hydroxymethyl
group is attached to C-8, produced competitive antago-
nism of the effects of 5-HT with pA2 values of 8.84 and
8.50, respectively. Compared to the high antagonist
activity of 4b and 5b at 5-HT2A receptors, antagonist
activity of the compounds at R1-adrenoceptors of rat
aorta was low (Table 5).
a
b
Number
n of independent experiments was 11-12. The
concentration of ketanserin was 3-10 nM (n ) 5-6). c pKP values
d
were estimated according to ref 27. Apparent pA2 values were
estimated according to ref 30. e Full pA2 value from the Schild plot
according to ref 31. f Data are from ref 33.
verse esters” with a small alicyclic ring in the acyl
portion and an isopropyl substituent at the indole
nitrogen were potent 5-HT2A receptor antagonists led
us to examine to what extent the complete removal of
the acyl portion would affect 5-HT2A receptor affinity.
Most surprisingly, the parent alcohols of the isopropyl
homologues (1-isopropyllysergol (1b), 1-isopropyldihy-
drolysergol-I (2b), and 1-isopropylelymoclavine (3b))
showed higher affinity for 5-HT2A receptors of rat tail
Perhaps most informative is the comparison between
the antagonist activities of the N-1-alkylated esters and
their corresponding alcohols 1b-3b. O-Acylated deriva-
tives of lysergol, dihydrolysergol-I, and elymoclavine
which are characterized by an isopropyl substituent at
N-1 have 2.5-25-, 2-28-, and 10-23-fold decreased

Role of the Indoloquinoline System of Ergolines
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4 665
Ta ble 5. Physical Properties and Pharmacological Effects of N-1-Substituted Clavine Alkaloids
a
All compounds exhibit 1H NMR spectral data in agreement with assigned structures. All compounds were characterized and tested
as their hydrogen maleate salts. Salt 1b was hydrated with 0.5 mol of water. All salts were crystallized from a THF/Et2O mixture.
b
Elemental analyses were within (0.4% of the theoretical values for C, H, N. Full pA2 values were estimated from the Schild plot according
d
to ref 31. c Apparent pA2 values were estimated according to ref 30 (concentration of antagonists 3-30 µM). 5-HT2A/5-HT1B and 5-HT2A
/
R1 are the ratios of KB values. e Slope 1.05 ( 0.03 of the Schild plot. f Slope 1.03 ( 0.03 of the Schild plot. Slope 0.89 ( 0.04 of the Schild
plot, significantly different from unity (P < 0.02). Slope 1.06 ( 0.08 of the Schild plot. Slope 1.00 ( 0.07 of the Schild plot. Number n
g
h
i
of independent experiments in parentheses.
antagonist activity at 5-HT2A receptors compared to the
corresponding parent alcohols 1b-3b. It could be shown
that a number of variations at the 8-position may be
tolerated without a major effect on antagonist potency.
Moreover, the nanomolar antagonist activity of the
parent drugs demonstrates that the derivatization of the
C-8 substituent is not crucial for 5-HT2A receptor
affinity. The observation is consistent with the nano-
molar antagonist activity of compounds 4b and 5b of
which the pharmacophore is structurally reduced to the
crude 8,9-didehydro-6,8-dimethylergoline or 6,8-di-
methylergoline skeleton due to the lack of the -OH
group at C-17 and the double bond in the D-ring,
respectively. Thus, the indolo[4,3-fg]quinoline system of
the ergolines is, in fact, the molecular fragment that is
somehow ultimately responsible for 5-HT2A receptor
affinity, and not the substituent at position C-8. The
present study of structure-activity relationships shows
that the importance of the tetracyclic structure of the
ergolines can be demonstrated by means of simple
ergolines, of which the indolo[4,3-fg]quinoline system
represents more or less the complete molecule. The
crucial role of aromatic groups of many 5-HT receptor
ligands including ergolines for 5-HT receptor interaction
has recently been demonstrated by molecular modeling
studies.40-42 In addition, site-directed mutagenesis tech-
niques have shown that within the 5-HT2A receptor
protein it is the highly conserved aromatic residue
phenylalanine at position 340 which is essential for
ergoline binding. It has been suggested that the phenyl
moiety of phenylalanine may allow a specific aromatic
interaction (e.g., π or hydrophobic) with the aromatic
ring of the ergoline nucleus of compounds such as
LY53857 and amesergide.42
The complete removal of the acyl portion in the series
of N-1-isopropyl homologues yields compounds which
are even more potent 5-HT2A receptor antagonists than
their corresponding esters and display low affinity at
5-HT1B receptors and R1-adrenoceptors. Therefore, it is
suggested that the indolo[4,3-fg]quinoline system of the
ergolines is the structural fragment that is decisive for
5-HT2A receptor affinity, and not the substituent at
position C-8.
Exp er im en ta l Section
Ch em istr y. 1H NMR spectra were recorded on a Bruker
AC 300 or AC 400 spectrometer. Chemical shifts are given in
ppm (δ) downfield from TMS. EIMS were obtained using a
MAT-711 spectrometer operating at 70 eV. Elemental analyses
(C, H, N) for novel compounds were determined with a Perkin-
Elmer 240C instrument. Melting points were taken on a Bu¨chi
530 melting point apparatus and are uncorrected. Chemical
purifications on a preparative scale were performed by radial
centrifugal chromatography with a Chromatotron 7924 (Har-
rison Research, Palo Alto, CA) using glass rotors with 1-, 2-,
or 4-mm layers of silica gel 60 PF254 containing gypsum (Merck
art. no. 7749) and appropriate eluents. The Chromatotron
chamber was continuously purged with dry nitrogen and
protected from light. All experiments were monitored by thin-
layer chromatography (TLC) using aluminum sheets coated
with a 0.2-mm layer of silica gel 60 F254 (Merck art. no. 5554)
and appropriate eluents. Detection of compounds on TLC was
additionally achieved with van Urk’s reagent. All reactions
were carried out in the dark under an inert atmosphere of dry
nitrogen or argon using glassware that had been carefully
cleaned and dried overnight in a 120 °C oven.
Gen er a l P r oced u r e for th e P r ep a r a tion of Cycloa l-
k a n eca r boxylic Ester s Der ived fr om Lyser gol, Dih y-
d r olyser gol-I, a n d Elym ocla vin e. 9,10-Did eh yd r o-6-m e-
th yler golin -8â-ylm eth yl Cycloh exa n eca r boxyla te (9a ).
To a cooled (0 °C) and stirred solution of 1a (0.75 g, 2.96 mmol)
in dry pyridine (45 mL) was added dropwise, and in the
presence of catalytic amounts of 4-DMAP, a solution of freshly
distilled cyclohexanecarbonyl chloride (0.86 g, 5.85 mmol) in
CHCl3 (12 mL) over 160 min. After the addition was finished,
the reaction mixture was allowed to stand at room temperature
overnight. The resulting solution was evaporated to dryness
and the residue partitioned between CHCl3 and a saturated
solution of NaHCO3. The organic layer was dried over Na2-
SO4 and the solvent removed under vacuum. Radial centrifugal
chromatography (Chromatotron instrument, eluent CH2Cl2/
cyclohexane/MeOH, 100/10/2 (v/v/v)) of the residue afforded a
yellow oil. The hydrogen maleate salt of 9a was precipitated
In conclusion, the present findings demonstrate that
cycloalkanecarboxylic esters derived from lysergol, di-
hydrolysergol-I, and elymoclavine exhibit complex be-
havior as partial agonists and antagonists at rat 5-HT2A
receptors. Those compounds that have smaller cycoalkyl
rings in the acyl portion and an isopropyl substituent
at the indole nitrogen emerged as silent 5-HT2A receptor
antagonists of high potency. Partial 5-HT2A receptor
agonist activity is restricted to N-1-unsubstituted de-
rivatives of lysergol (double bond in the 9,10-position).

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

Role of the Indoloquinoline System of Ergolines
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4 667
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Wainscott, D. B. Species Differences in the Pharmacology of the
5-Hydroxytryptamine2 Receptor: Structurally Specific Differ-
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(13) J ohnson, M. P.; Audia, J . E.; Nissen, J . S.; Nelson, D. L. N(1)-
Substituted Ergolines and Tryptamines Show Species Differ-
ences for the Agonist-Labeled 5-HT2 Receptor. Eur. J . Pharma-
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(14) Kao, H.-T.; Adham, N.; Olsen, M. A.; Weinshank, R. L.; Branchek,
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(16) Hartig, P. R.; Hoyer, D.; Humphrey, P. P. A.; Martin, G. R.
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(0.3 µM), cimetidine (30 µM), and cocaine (30 µM) to block
5-HT2A receptors, R1-adrenoceptors, histamine H1 receptors,
histamine H2 receptors, and neuronal uptake of 5-HT. The
applied resting force was 5 mN. During an equilibration period
of 4.5 h, the organs were stimulated after 100 min with
prostaglandin F2R (PGF2R; 30 µM). Relaxation was achieved
by subsequent addition of carbachol (10 µM). After 175 min
the organs were moderately precontracted with an EC10-EC20
(50-500 nM) of PGF2R and subsequently stimulated with 5-HT
(0.3 µM). Two cumulative concentration-effect curves for 5-HT
were determined at an interval of 80 min in the absence and
presence of 1b-3b, on organs precontracted with an EC10
-
EC20 of PGF2R as above. Compounds 1b-3b were incubated for
45 min.
F u n ction a l r1-Ad r en ocep tor Assa y (r a t a or ta ). The
thoracic aorta was removed from rats used for studies at
5-HT2A receptors in rat tail artery (see above). When cleared
of connective tissue the aorta was cut into 6-12 rings of 4-6-
mm length. Each cylindrical segment was rolled with a pair
of tweezers to destroy the endothelium. The segments were
horizontally suspended between two L-shaped stainless steel
holders (diameter 0.3 mm).44 The organs were isometrically
mounted as described for rat tail artery experiments (see
above). The bath fluid (modified Krebs-Henseleit solution of
the above composition at 37 °C, gassed with 95% O2/5% CO2)
contained (R,S)-propranolol (1 µM) to block â-adrenoceptors.
The applied resting force was 20 mN. During an equilibration
period of 140 min the organs were stimulated twice with (R)-
phenylephrine (100 nM). Two cumulative concentration-
response curves for the contractile effect of (R)-phenylephrine
were determined in the absence and presence of antagonist.
Antagonists were incubated 30 min before the second curve.
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(23) Polgar, I.; Fo¨ldesi, J .; Kiss, J .; Major, P.; Molnar, K.; Sugar, A.;
Szen, T.; Balogh, K. (Gideon Richter Vegyeszeti Gyar) Prepara-
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Lysergol. FR 2,648,815, 1990.
Ack n ow led gm en t. The authors wish to thank Dr.
W. Schunack for financial support of the studies and
Dr. S. Elz for fruitful discussions.
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