1230
M. J. Mokrosz et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1229–1231
into hydrochloride or fumarate salts for biological
assays.
of Z isomers showed a strong influence of arylidene
substituents, especially two of them with p-methoxy-
phenyl (2c) and 2-thienyl (2d) groups, in which the pre-
sence of additional heteroatoms rendered the respective
derivatives practically inactive at 5-HT1A binding sites.
Among Z and E isomers, the former were generally
better tolerated by those receptors.
Pharmacology
Radioligand binding studies were carried out in the
hippocampus of rats for 5-HT1A receptors, and in the
cortex for 5-HT2A receptors according to the previously
published procedures.10 The radioligands used were
[3H]-8-OH-DPAT (190 Ci/mmol, Amersham) and [3H]-
ketanserin (60 Ci/mmol, NEN Chemicals) for 5-HT1A
and 5-HT2A receptors, respectively. Ki values were
determined on the basis of at least three competition
binding experiments in which the tested compounds
were used at concentrations of 10À10–10À3 M, run in
triplicate.
On the other hand, the 5-HT2A affinity remained at the
same high level (Ki=200–64 nM) for the majority of the
tested compounds except for the (Z)-thiophene deriva-
tive 2d, which is a less active compound (Ki=138 nM),
and the more active (E)-3-pyridyl one 3e (Ki=7 nM).
Moreover, a clear difference between the 5-HT2A bind-
ing constants of the two isomers was observed for those
two compounds only, which suggests that the E isomer
is more active than Z. For the rest of the compounds, Ki
values seem to be independent of the structure and
geometry of the 3-substituted indolinone core.
To determine the postsynaptic 5-HT1A agonistic effect
of 2a and 2e, their ability to induce a lower lip retraction
(LLR) in rats was tested. The ability of those com-
pounds to inhibit that symptom produced by 8-OH-
DPAT, a 5-HT1A receptor agonist, was regarded as a
postsynaptic 5-HT1A antagonistic activity.11,12 The
blocking effect of the tested compounds on the head
twitches induced by the 5-HT2A agonist (Æ)DOI in
mice13 was a measure of their antagonistic activity at
those receptors.
The next step of our investigation was concentrated on
the in vivo effects induced by some selected compounds.
As already mentioned, all of the presented compounds
(except 2d) demonstrated a high affinity for 5-HT2A
receptors and only two of them (2a and 2e) for 5-HT1A
ones. For that reason those 5-HT1A and/or 5-HT2A
ligands were studied in vivo in tests commonly used for
evaluation of the 5-HT1A or 5-HT2A functional
activity.11À13 In those models, compounds 2a and 2e—
like (S)-WAY-100135, a well-known 5-HT1A antago-
nist—did not evoke LLRin rats, but, when used in a
dose of 20 mg/kg, strongly inhibited the LLRinduced
by 8-OH-DPAT (Table 2). The above results show that
derivatives 2a and 2e act as 5-HT1A receptor antago-
nists. The ability of 2a–c, 2e, 3a–e and ketanserin, a
well-known 5-HT2A receptor antagonist, to antagonize
head twitches in mice, observed after administration of
(Æ)DOI, a 5-HT2A agonist,13 was used to evaluate 5-
HT2A receptor antagonistic properties. The results pre-
sented in Table 1 indicate that all the investigated com-
pounds, as well as the reference compounds ketanserin and
1 effectively inhibited the (Æ)DOI-induced head twitches
in mice; ED50 values of 2a–3e derivatives ranged between
9.5–28.5 mg/kg, hence they may be classified as 5-HT2A
antagonists. Interestingly, the tested derivatives—despite
Results and Discussion
As seen in Table 1, the 3-arylidene substituted com-
pounds have much diversified affinities at 5-HT1A
receptors. Of those compounds, only two Z isomers,
which possess phenyl and 3-pyridyl substituents (2a and
2e, respectively) have a relatively high affinity (Ki
ffi50 nM) at these sites; however, the values obtained in
the present study were twice as low as those for the
parent compound 1 (Ki=29 nM).14 The other tested
compounds showed a low or a very low 5-HT1A recep-
tor affinity. A comparison of the 5-HT1A binding data
Table 1. In vitro and in vivo activity of compounds 2 and 3
No.
Ar
Isomer
Ki [nM]
ED50c(mg/kg), ip
5-HT1A
5-HT2A
Table 2. Induction of a lower lip retraction (LLR) by 2a and 2e and
the effect of the investigated compounds on the 8-OH-DPAT-induced
LLRin rats (1 mg/kg, sc)
1a
—
Phenyl
Phenyl
p-Cl-C6H4
p-Cl-C6H4
—
Z
E
Z
E
Z
E
Z
E
Z
E
29Æ4
54Æ13
25Æ3
56Æ3
38Æ6
36Æ4
32Æ2
36Æ1
20Æ4
6.5 (4.5–9.4)
27.5 (20.8–36.3)d
28.5 (22.3–36.5)
15.0 (10.0–22.5)
20.0 (14.3–28.0)
16.0 (12.3–20.8)
28.5 (18.4–44.2)
—
2ab
3a
399Æ11
204Æ13
1610Æ4
7930Æ18
538Æ3
2bb
3b
Treatment
Dose (mg/kg)
Inductiona
Inhibitionb
MeanÆSEM
2cb p-OCH3-C6H4
3c p-OCH3-C6H4
Vehicle
2a
—
10
20
—
10
20
10
0.1Æ0.1
0.1Æ0.1
0.2Æ0.1
0.1Æ0.1
0.1Æ0.2
0.1Æ0.1
0.0Æ0.0
2.8Æ0.2
1.9Æ0.4
0.8Æ0.1c
2.7Æ0.1
2.3Æ0.3
0.9Æ0.3c
0.8Æ0.3c
2d
3d
2e
3e
2-Thienyl
2-Thienyl
3-Pyridyl
3-Pyridyl
Ketanserin
20,500Æ100 138Æ21
>50,000
57Æ2
64Æ15
20Æ4
7Æ1
16.0 (9.4–27.2)
27.0 (21.6–33.8)
9.5 (7.0–12.8)
Vehicle
2e
101Æ5
1933Æ219e 1.5Æ0.2e 0.12 (0.07–0.2)
(S)-WAY-100135
aRef 14.
bRef 15.
aThe investigated compounds were administered ip 15 min before the
test.
cED50 — the dose inhibiting the (Æ)DOI-induced (2.5 mg/kg, ip) head
twitches in mice by 50%.
bThe investigated compounds were administered ip 45 min before 8-
OH-DPAT.
dConfidence limits (95%) given in parentheses.
eData from ref 16.
cP<0.01 versus vehicle + 8-OH-DPAT.
Mokrosz, Maria J.
