New 9- or 10-arylpiperazinoalkyl substituted pyrimido- or diazepino[2,1- f]purines with partial or full 5-HT(1A) agonistic activity

Article abstract of DOI:10.1016/S0223-5234(99)80050-6

5HT(1A) and 5HT(2A) receptor affinities of several γ-(3'-chloro- or 2'- methoxyphenyl)-piperazinopropyl derivatives of pyrimido [2,1-f]purines or 1,3-diazepino[2,1-f]purines are reported. Some behavioral models demonstrated that 1,3-dimethyl-9-{3-[4-(2'-methoxyphenyl)1-piperazinyl]propyl}-2,4- dioxo-1,3,6,7,8,9-hexahydropyrimido[2,1-f]purine 5b and 1,3-dimethyl-9-{3- [4-(2'-methoxyphenyl)-1-piperazinyl]propyl}-2,4,8-trioxo-1,3-dihydro-9H- pyrimido[2,1-f]purine 8b, may be classified as partial agonist and a full agonist, respectively of pre- and post-synaptic 5HT(1A) receptors.

Full text of DOI:10.1016/S0223-5234(99)80050-6

Eur. J. Med. Chem. 34 (1999) 167−175
© Elsevier, Paris
167
Short communication
New 9- or 10-arylpiperazinoalkyl substituted pyrimido-
or diazepino[2,1-f]purines with partial or full 5-HT1A agonistic activity
Maciej Pawlowskia*, Jacek Katlabia, Anna Drabczynskaa, Beata Duszynskab,
Sijka Charakchieva-Minolb, Anna Deren-Wesolekb, Ewa Tatarczynskab, Ewa Chojnacka-Wójcikb,
Maria J. Mokroszb, Andrzej J. Bojarskib
aDepartment of Pharmaceutical Chemistry, Collegium Medicum of Jagiellonian University, 9 Medyczna St., 30-688 Kraków, Poland
bInstitute of Pharmacology, Polish Academy of Sciences, 12 Smetna St., 31-343 Kraków, Poland
(Received 29 July 1998; accepted 24 September 1998)
Abstract – 5HT1A and 5HT2A receptor affinities of several γ-(3’-chloro- or 2’-methoxyphenyl)-piperazinopropyl derivatives of pyrimido[2,1-
f]purines or 1,3-diazepino[2,1-f]purines are reported. Some behavioral models demonstrated that 1,3-dimethyl-9-{3-[4-(2’-methoxyphenyl)-
1-piperazinyl]propyl}-2,4-dioxo-1,3,6,7,8,9-hexahydropyrimido[2,1-f]purine 5b and 1,3-dimethyl-9-{3-[4-(2’-methoxyphenyl)-1-pipera-
zinyl]propyl}-2,4,8-trioxo-1,3-dihydro-9H-pyrimido[2,1-f]purine 8b, may be classified as partial agonist and a full agonist, respectively of
pre- and post-synaptic 5HT1A receptors. © Elsevier, Paris
1-arylpiperazine / 5-HT1A agonists / pyrimidopurines / diazepinopurines
1. Introduction
5-HT1A affinity and 5-HT1A/5-HT2A selectivity of the
The majority of 1-arylpiperazines is classified as par-
tial agonists of 5-HT1A receptors [1–3], although the
postsynaptic antagonism may also be regarded as their
common pharmacological property [1, 2, 3]. There are,
however, only few examples of either full (pre- and
post-synaptic) agonists [4, 5] 1a,b or antagonists [6–8] 2
and 3. (figure 1).
In the course of searching for the ligands possessing
one of the above pharmacological properties, we tested
1-phenyl and 1-(2-pyrimidyl)piperazines 4 which con-
tained a complex heterocyclic ring system. (figure 2).
These derivatives have been found to be the potent
5-HT1A receptor ligands and behaved either like antago-
nists or partial agonist of the postsynaptic 5-HT1A recep-
tors [9]. In the present study we further examined the
influence of structural modifications pyrimido[2,1-
f]purine and diazepino[2,1-f]purines together with 1-(3-
chlorophenyl)- and 1-(3-methoxyphenyl)piperazine on
Figure 1. Structure of full (pre- and post-synaptic) 5-HT1A
receptor agonists 1a,b and antagonists 2, 3.
*Correspondence and reprints

168
The starting bromo(chloro)propyl derivatives of tricy-
clic pyrimido[2,1-f]theophylline or diazepino[2,1-f]theo-
phylline were obtained in the several-step synthesis
reported previously [10–13].
Melting points, yields and results from elemental and
TLC analyses are given in table I. The final products
1
were characterized by their H-NMR and MS spectral
data, which were in accordance with the proposed struc-
tures (table II and III).
Figure 2. Structure of tricyclic theophylline derivatives, anta-
gonists and partial agonists of postsynaptic 5-HT1A receptors.
3. Pharmacology
The receptor affinities of the investigated compounds
5–9 (hydrochlorides) were determined in competition
ligands and the consequences of these modifications on in
vivo pharmacological properties of the most active de-
rivatives.
experiments
using
8-hydroxy-2-di-n-propylamino-
tetraline, [3H]-8-OH-DPAT (hippocampus) for 5-HT1A
and [3H] ketanserin (cortex) for 5-HT2A receptors. The
results are presented in table IV.
2. Chemistry
The functional profile of 5b and 8b at pre- and
post-synaptic 5-HT1A receptors was demonstrated in
several in vivo models. The 8-OH-DPAT-induced lower
lip retraction (LLR) in rats [14] and behavioral syndrome
The following new compounds 5–9 were synthesized
by condensation of the bromo(chloro)propyl substrate
with corresponding substituted piperazines (figure 3).
Table I. Yields, melting points and analytical data of new compounds.
a
Compound M.p. (oC), recryst. solvent
Yield (%) Formula, molecular weight
Analyses
C, H, N
C, H, N
TLC, Rf solvent
0.79 E
5a, HCl
5b, HCl
6a, HCl
6b, HCl
7a, base
7a, HCl
7b, base
7b, HCl
8a, base
8a, HCl
8b, base
8b, HCl
9a, base
9a, HCl
9b, base
9b, HCl
244–245, EtOH/H2O
88
74
65
82
64
C23H30N7O2Cl HCl, 508.45
C24H33N7O3 H2O HCl, 522.04
C24H32N7O2Cl HCl, 522.47
C25H32N7O3 H2O HCl, 536.01
C23H28N7O3Cl, 486.00
176 decomp., EtOH/H2O
245–247, EtOH/H2O
0.73 E
C, H, N
0.87 E
b
c
190 decomp., EtOH/H2O
190–191, 96° EtOH
C
H, N
0.78 E
b
C , H, N
C, H, N
C, H, N
0.23 A, 0.53 B, 0.76 C
272 decomp., 80° EtOH
230–231, 2-methoxyethanol
258 decomp., 96° EtOH
174–175, 96° EtOH
C23H28N7O3Cl HCl, 522.44
C24H31N7O4, 481.56
50
0.10 A, 0.30 B, 0.55 C
0.31 A, 0.60 B, 0.75 C
0.18 A, 0.39 B, 0.56 C
0.30 A, 0.56 B, 0.75 C
0.15 A, 0.36 B, 0.80 D
b
C24H31N7O4 HCl, 518.02
C23H26N7O3Cl, 483.96
C , H, N
62
–
C, H, N
b
288–290 decomp., 10° EtOH
216–218, 96° EtOH
C23H26N7O3Cl HCl, 520.42
C24H29N7O4, 479.55
C , H, N
68
C, H, N
b
273–275 decomp., 96° EtOH
145–146, 96° EtOH
C24H29N7O4 HCl, 515.99
C24H30N7O3Cl, 500.00
C , H, N
40
–
C, H, N
c
167–169 decomp., anh. EtOH
145–147, 96° EtOH
C24H30N7O3Cl HCl, 536.45
C25H33N7O4, 495.59
C, H, N
c
41
–
C, H, N
b
242–244 decomp., 96° EtOH
C25H33N7O4 HCl, 532.04
C , H, N
a
b
Elemental analysis is within ±0.4% for elements indicated unless otherwise noted; C found (calc.): 6b HCl 56.52 (56.02); 7a base 56.40
(56.84); 7b HCl 55.08 (55.64); 8a HCl 52.62 (53.08); 8b HCl 55.28 (55.86); 9b HCl 55.90 (56.43); c N found (calc.) 6b HCl 18.93 (18.29);
9a HCl 18.70 (18.27); 9b base 19.33 (19.78).

169
(flat body posture and forepaw treading) in reserpinized
rats [15] were mediated by postsynaptic receptors.
However, the 8-OH-DPAT-induced hypothermia in
mice was mediated by presynaptic 5-HT1A receptors [16,
17]. The results of in vivo studies are given in tables V,
VI, VII and VIII.
4. Results and discussion
The radioligand binding data on the 5-HT1A receptor
affinities of 5–9 show large differences and depend on
both, 1-arylpiperazine moiety and the amide fragment.
All the investigated compounds are potent 5-HT1A recep-
tor ligand with Ki within the region 2.8–43 nM for 5b and
7a respectively, except 9a and 9b of which the affinity is
moderate.
The compounds containing o-methoxyphenyl-
piperazine (o-OMePhP, series b) are generally more
active at 5-HT1A receptor sites than their counterparts
with m-chloro-phenylpiperazine (m-CPP, series a) if
comparing 5b vs. 5a (Ki = 2.8 and 25.7 nM) or 8b vs. 8a
(Ki = 10.4 and 32.8 nM). On the other hand their high
affinity makes them more sensitive for any change in the
amide fragment. Enlargement of the ring of the alkyl
substitution causes 6-fold loss of activity for o-methoxy
derivative 6b vs. 5b and retains activity of 6a comparing
to 5a. Addition of the amide function makes both series of
the compounds less afin but again o-methoxy derivatives
are more affected, Ki ratios are 7b/5b = 7.7 and 7a/5a =
1.7. Both modifications, amide function along with en-
largement of the ring, dramatically decrease activity of
9b, 60 times in relation to the most active compound 5b,
while 9a is only slightly, above 2 times less potent than
5a (table IV, figure 4).
The 5-HT2A affinities of the derivatives studied extend
from the moderate, Ki = 126 nM for 5a, to very weak,
5014 nM for 9b. The influence of the structure modifi-
cations on 5-HT2A affinity is comparable in both series of
the investigated compounds but not as strong as in case of
the 5-HT1A affinity however, the direction of these
changes is similar (table IV, figure 4).
The selectivity factor S1,2, for the m-CPP series varies
from 4.2 for 7a to 33.9 for 8a and these derivatives
should be regarded rather non-selective ones. On the
contrary, the o-OMePhP derivatives are highly selective
5-HT1A/5-HT2A receptor ligands as their selectivity fac-
tor is within 30 for 9b up to 373 for 8b, especially that
their 5-HT2A affinities are within the range which can not
be expected to evoke any behavioral effects.
It has been shown that lipophilicity or dipole moment
of the terminal amide or cycloamide fragment of
1-arylpiperazines significantly affects the 5-HT1A and
Figure 3. Synthesis of tricyclic chloro- and methoxy-
arylpiperazinopropyl derivatives.

170
1
Table II. H-NMR data on selected compounds.
No.
δ ppm in CDCl3 (200 MHz)
5a, HCl
2.10–2.29 (m, 2H, CH2CH2CH2); 2.32–2.45 (m, 10H, CH2N-(CH2)4-N); 3.06–3.11 (m, 2H, C7H2); 3.36 (s, 3H, N3CH3); 3.47
(s, 3H, N1CH3); 3.56–3.68 (t, 2H, J = 7 Hz, N9CH2); 4.19–4.25 (t, 2H, J = 6 Hz, N5CH2); 6.75–7.20 (m, 4H, aromat).
5b, HCl
2.17–2.30 (m, 2H, CH2CH2CH2); 2.32–2.45 (m, 10H, CH2N-(CH2)4-N); 3.06–3.15 (m, 2H, C7H2); 3.36 (s, 3H, N3CH3); 3.48
(s, 3H, N1CH3); 3.65–3.72 (t, 2H, J = 7 Hz, N9CH2); 3.86 (s, 3H, OCH3); 4.15–4.25 (t, 2H, J = 6 Hz, N5CH2); 6.80–7.10
(m, 4H, aromat).
6a, HCl
6b, base
7a, base
1.90–2.17 (m, 2H, CH2CH2CH2); 2.32–2.41 (m, 10H, CH2N-(CH2)4-N); 2.90–3.20 (m, 2H, C7H2); 3.37 (s, 3H, N3CH3); 3.48
(s, 3H, N1CH3); 3.60–3.66 (t, 2H, J = 7 Hz, N10CH2); 4.34–4.36 (d, 2H, J = 5 Hz, N5CH2); 6.80–7.20 (m, 4H, aromat).
1.92–2.00 (m, 2H, CH2CH2CH2); 2.30–2.45 (m, 10H, CH2N-(CH2)4-N); 3.10–3.20 (m, 2H, C7H2); 3.36 (s, 3H, N3CH3); 3.50
(s, 3H, N1CH3); 3.90 (s, 3H, OCH3); 4.30–4.40 (d, 2H, J = 5 Hz, N5CH2); 6.90–7.20 (m, 4H, aromat).
1.79–1.96 (m, 2H, CH2CH2CH2); 2.37–2.59 (m, 6H, CH2N-(CH2)2); 2.84–3.02 (t, 2H, C7H2); 3.08–3.20 (m, 4H,
(CH2)2N-C6H4Cl); 3.34 (s, 3H, N3CH3); 3.50 (s, 3H, N1CH3); 3.98–4.14 (t, 2H, N9CH2); 4.50 (t, 2H, N5CH2); 6.67–6.70
(m, 3H, aromat); 7.01–7.02 (d, 1H, 2’-C6H4Cl).
7b, base
8a, base
8b, base
9a, base
9b, base
1.80–2.02 (q, 2H, CH2CH2CH2); 2.39–2.65 (m, 6H, CH2N-(CH2)2); 2.84–3.10 (m, 6H, (CH2)2N-C6H4OCH3 + C7H2); 3.35
(s, 3H, N3CH3); 3.50 (s, 3H, N1CH3); 3.81 (s, 3H, OCH3); 3.97–4.15 (t, 2H, N9CH2); 4.33–4.51 (t, 2H, N5CH2); 6.86–6.97
(m, 4H, aromat).
1.81–1.98 (m, 2H, CH2CH2CH2); 2.43–2.59 (m, 6H, CH2N-(CH2)2); 3.04–3.17 (m, 4H, (CH2)2N-C6H4Cl); 3.38 (s, 3H,
N3CH3); 3.52 (s, 3H, N1CH3); 4.24–4.42 (t, 2H, N9CH2); 6.19–6.29 (d, 1H, 7CH); 6.68–6.78 (m, 3H, aromat); 7.00–7.02 (d,
1H, 2’-C6H4Cl); 8.41–8.51 (d, 1H, C6H).
1.81–2.16 (m, 2H, CH2CH2CH2); 2.45–2.64 (m, 6H, CH2N-(CH2)2); 2.94–3.16 (m, 4H, (CH2)2N-C6H4OCH3); 3.38 (s, 3H,
N3CH3); 3.56 (s, 3H, N1CH3); 3.81 (s, 3H, OCH3); 4.25–4.43 (t, 2H, N9CH2); 6.20–6.29 (d, 1H, C7H); 6.75–7.02 (m, 4H,
aromat); 8.42–8.51 (d, 1H, C6H).
1.74–2.01 (q, 2H, CH2CH2CH2); 2.32–2.57 (m, 10H, CH2N-(CH2)2 + C7H2 + C8H2); 3.06–3.18 (m, 4H, (CH2)2N-C6H4Cl);
3.37 (s, 3H, N3CH3); 3.52 (s, 3H, N1CH3); 3.87–4.05 (t, 2H, N9CH2); 4.35–4.52 (t, 2H, N5CH2); 6.67–6.79 (m, 3H, aromat);
7.01–7.03 (d, 1H, 2’-C6H4Cl).
1.65–2.00 (m, 2H, CH2CH2CH2); 2.32–2.62 (m, 10H, CH2N-(CH2)2 + C7H2 + C8H2); 2.84–3.07 (m, 4H, (CH2)2N-
C6H4OCH3); 3.37 (s, 3H, N3CH3); 3.52 (s, 3H, N1CH3); 3.81 (s, 3H, OCH3); 3.88–4.06 (t, 2H, N9CH2); 4.21–4.51 (t, 2H,
N5CH2); 6.75–7.02 (m, 4H, aromat).
5-HT2A receptor affinity [2, 18, 19]. This is not however,
to be such an obvious case. Although, in both series we
found the tendency that the higher dipole moment in-
creases the affinity at 5-HT1A receptors but this is only a
qualitative relationship between these two properties. A
tentative suggestion may be drawn however, that the
differences in the affinities of the investigated compounds
can be attributed to the subtle stereochemical distortions
within the heterocyclic fragment.
Finally, concerning 5-HT1A receptor affinity and the
selectivity towards 5-HT1A/5-HT2A receptors, we se-
lected two compounds i.e. 5b and 8b, to determine their
functional profile at 5-HT1A receptors. Compounds 5b
and 8b (2.5–5 mg/kg i.p.), given alone induced a dose-
Table III. MS data on selected compounds.
No.
M+
m/z intensity
7a, base
7b, base
8a, base
8b, base
9a, base
9b, base
486 (18)
481 (45)
483 (37)
479 (80)
499 (10)
495 (22)
470 (20); 345 (8); 319 (47); 290 (24); 209 (100); 166 (25); 70 (25); 55 (19).
466 (30); 325 (37); 290 (18); 205 (100); 190 (40); 177 (30); 70 (30); 55 (27).
468 (30); 317 (58); 288 (44); 248 (41); 209 (100); 166 (25); 70 (40); 56 (19).
464 (20); 287 (58); 248 (28); 205 (100); 190 (41); 62 (30); 136 (28); 70 (25); 56 (19).
484 (57); 347 (18); 333 (50); 304 (30); 209 (100); 195 (38); 70 (58); 56 (16).
480 (70); 333 (17); 304 (17); 219 (20); 205 (100); 190 (38); 164 (26); 70 (30); 56 (16).

171
Table IV. 5-HT1A and 5-HT2A receptor affinities of compounds 5–9.
a
Compound
Ki ± SEM [nM]
Selectivity
5-HT1A
5-HT2A
5-HT2A/5-HT1A
5a
5b
6a
6b
7a
7b
8a
8b
9a
9b
25.7 ± 1
126 ± 12
486 ± 31
330 ± 9
562 ± 37
182 ± 14
4219 ± 183
1080 ± 40
3882 ± 418
359 ± 10
5014 ± 6
4.9
173
12.3
32.7
4.2
195
32.9
373
5.9
2.8 ± 0.3
26.7 ± 3.1
17.2 ± 0.5
42.9 ± 4
21.6 ± 0.8
32.8 ± 1.6
10.4 ± 1.2
60.3 ± 10.2
168 ± 1
29.8
a
Mean values from at least three independent experiments/receptors [16, 17].
dependent LLR in rats, the maximum score being 73%
and 77%, respectively, after the highest dose used (ta-
ble V). The same effect was observed after administration
of 8-OH-DPAT (0.25–1 mg/kg s.c.), a full agonist of
5-HT1A receptors; the maximum possible score was
100% after a dose of 1 mg/kg s.c.. Therefore it may be
concluded that, like 8-OH-DPAT, the two investigated
compounds, 5b and 8b, are agonists of postsynaptic
5-HT1A receptors in the behavioral (LLR) model studied.
In reserpinized rats, 5b (5–10 mg/kg) alone did not evoke
a behavioral syndrome (flat body posture and/or forepaw
treading), whereas 8b in the highest dose used (10 mg/kg
i.p.) induced a flat body posture, the maximum possible
score being 50%; however, forepaw treading was not
observed after the latter administration. 8-OH-DPAT
(5 mg/kg i.p.) induced a flat body posture (97–100% of
possible scores) and forepaw treading (88–90% of pos-
sible scores) in reserpinized rats. The 8-OH-DPAT-
induced forepaw treading was dose-dependently attenu-
ated by 5b (5–10 mg/kg i.p.) and blocked by (S)-WAY
100135 (10 mg/kg s.c.), a 5-HT1A receptor antagonist, 8b
did not change that effect of 8-OH-DPAT. At the same
time, 5b and 8b (5–10 mg/kg i.p.) had no influence on the
flat body posture induced by 8-OH-DPAT in reserpinized
rats; the reference drug, (S)-WAY 100135 (5–10 mg/kg
s.c.), slightly reduced (by ca. 40%) that effect of 8-OH-
DPAT, the reduction being equal after either dose used
(table VI). The above results indicate that in this model
(serotonin syndrome) 8b shows features characteristic of
a weak agonist of postsynaptic 5-HT1A receptors,
whereas 5b behaves like antagonist of these receptors.
The results of both these behavioral studies permit a
conclusion that 8b (which induced LLR and flat body
posture) may be classified as an agonist of postsynaptic
5-HT1A receptors, whereas compound 5b behaves like a
partial agonist of these receptors: it induced LLR and, at
the same time, attenuated the forepaw treading induced
by 8-OH-DPAT, a 5-HT1A receptor agonist. In those tests,
Table V. The induction of lower lip retraction (LLR) by 5b, 8b and 8-OH-DPAT in rats.
Compounds
Dose (mg/kg)
LLR score (mean ± SEM)
Vehicle
5b
–
0.1 ± 0.1
1.2 ± 0.2
2.2 ± 0.2
2.5
5.0
–
b
Vehicle
0.1 ± 0.1
a
8b
2.5
5.0
–
0.25
0.5
1.0
1.2 ± 0.2
2.3 ± 0.2
0.1 ± 0.1
1.5 ± 0.1
2.1 ± 0.5
3.0 ± 0.0
b
Vehicle
8-OH-DPAT
a
b
b
a
b
5b and 8b were administered (i.p.) and 8-OH-DPAT (s.c.), 15 min before the test. Each group consisted of 6 rats. p < 0.05, p < 0.01 vs.
respective vehicle group (Newman–Keuls test).

172
Table VI. The induction of serotonin syndrome by 5b, 8b and (S)-WAY 100135 (A) and the effect of compounds 5b, 8b and (S)-WAY 100135
on the 8-OH-DPAT-induced syndrome (B) in reserpinized rats.
Compounds
Dose (mg/kg)
Mean ± SEM behavioral score
A
B
Flat body posture
Forepaw treading
Flat body posture
Forepaw treading
Vehicle
5b
–
0.2 ± 0.1
0.1 ± 0.1
0.1 ± 0.1
0.3 ± 0.1
0.1 ± 0.1
0.2 ± 0.1
0.3 ± 0.1
0.3 ± 0.1
0.2 ± 0.1
0.5 ± 0.1
0.3 ± 0.1
0.2 ± 0.1
0.2 ± 0.1
0.2 ± 0.1
14.5 ± 0.3
13.0 ± 0.7
12.9 ± 1.0
14.5 ± 0.3
15.0 ± 0.0
13.1 ± 1.6
15.0 ± 0.0
13.5 ± 0.2
9.5 ± 1.0
5.9 ± 0.8
13.5 ± 0.2
11.4 ± 0.5
11.9 ± 1.0
13.2 ± 0.9
5.5 ± 0.6
1.0 ± 0.3
a
5.0
10.0
–
5.0
10.0
–
b
Vehicle
8b
b
7.6 ± 1.6
Vehicle
(S)-WAY 100135
0.2 ± 0.1
0.2 ± 0.1
0.3 ± 0.1
b
b
5.0
10.0
9.0 ± 1.0
9.7 ± 1.0
b
b
Reserpine (1 mg/kg, s.c.) was administered 18 h before the test. A: 5b, 8b (i.p.) and (S)-WAY 100135 (s.c.) were administered 3 min before
the test. B: 5b, 8b (i.p.) and (S)-WAY 100135 (s.c.) were administered 45 min before 8-OH-DPAT (5 mg/kg, i.p.). Each group consisted of
a
b
6 rats. p < 0.05, p < 0.01 vs. respective vehicle group (Newman–Keuls test).
the same functional profile, characteristic of 5b, was
described for ipsapirone, a well-known partial ago-
nist [14, 20, 21], and a potent anxiolytic drug. Com-
pounds 5b and 8b (2.5–10 mg/kg), given alone, produced
dose-dependent hypothermic responses in mice; after the
highest dose of 5b and 8b, the decrease in body tempera-
ture mediated by 5-HT1A receptors was back to the
control level within 60 min. 8-OH-DPAT (1.25–5 mg/kg),
used as a reference compound, also evoked hypothermia
in mice, which lasted up to 45 min after its administration
(table VII). The hypothermic effects observed after 5b
(10 mg/kg) and 8b (5 mg/kg) administration were attenu-
ated and abolished (30 min after 8b administration),
respectively, by (S)-WAY-100135 (10 mg/kg). The 8-OH-
DPAT (5 mg/kg)-induced hypothermia in mice was com-
pletely blocked by (S)-WAY 100135 (10 mg/kg) (ta-
ble VIII).This finding indicates that, like 8-OH-DPAT, 5b
and 8b may be agonists of presynaptic 5-HT1A receptors.
In conclusion, it should be mentioned that the previ-
ously described derivatives possessing the same as 5b
and 8b, heteroaromatic terminal fragments and 1-phe-
nylpiperazine moiety were classified as typical postsyn-
aptic 5-HT1A antagonists [9]. The replacement of
1-phenyl by 1-(o-methoxyphenyl)piperazine, which is
usually present in 5-HT1A receptor antagonist i.e. MP-
3022 or WAY-100135, surprisingly resulted in the ligands
with high intrinsic activity, which demonstrated dominat-
ing agonistic properties at 5-HT1A receptors. In fact, the
Table VII. The effect of 5b, 8b and 8-OH-DPAT on the body temperature in mice.
a
Compounds
Dose (mg/kg)
∆t ± SEM (°C)
15 min
30 min
45 min
60 min
Vehicle
5b
–
0.2 ± 0.2
0.4 ± 0.2
–0.1 ± 0.2
0.1 ± 0.1
–0.6 ± 0.2
0.2 ± 0.1
–0.7 ± 0.2
–1.0 ± 0.3
0.3 ± 0.2
–0.6 ± 0.3
–0.6 ± 0.3
–1.2 ± 0.2
0.5 ± 0.2
–0.0 ± 0.1
0.3 ± 0.1
–0.2 ± 0.2
0.3 ± 0.1
–0.5 ± 0.3
–0.6 ± 0.2
0.1 ± 0.1
–0.1 ± 0.1
–0.3 ± 0.2
–0.9 ± 0.2
0.5 ± 0.2
0.3 ± 0.1
0.5 ± 0.1
0.1 ± 0.2
0.2 ± 0.1
–0.3 ± 0.2
–0.2 ± 0.2
0.1 ± 0.2
–0.1 ± 0.2
–0.4 ± 0.2
–0.6 ± 0.2
2.5
5.0
10.0
–
2.5
5.0
–
1.25
2.5
5.0
–0.5 ± 0.2 b
b
–0.4 ± 0.1
–0.7 ± 0.2
–0.1 ± 0.2
–1.3 ± 0.3
c
c
b
Vehicle
8b
c
c
b
–1.6 ± 0.2
Vehicle
8-OH-DPAT
0.1 ± 0.2
–0.3 ± 0.2
–0.3 ± 0.2
b
b
c
c
b
–1.0 ± 0.2
5b, 8b and 8-OH-DPAT (i.p.) were administered 15 min before the test. Each group consisted of 7–8 mice. a The absolute mean initial body
b
c
temperatures were within a range of 36.3 ± 0.5 °C; p < 0.05, p < 0.01 vs. respective vehicle group (Dunnett’s test).

173
Table VIII. The effect of (S)-WAY 100135 on the 5b-, 8b- and 8-OH-DPAT-induced hypothermia in mice.
a
Compounds
Dose (mg/kg)
∆t ± SEM (°C)
15 min
30 min
45 min
60 min
Vehicle
5b
(S)-WAY 100135
Vehicle
8b
(S)-WAY 100135
Vehicle
8-OH-DPAT
(S)-WAY 100135
–
–
10
–
–
10
–
–
–0.3 ± 0.2
–1.7 ± 0.3
–0.7 ± 0.2
–0.3 ± 0.2
–1.4 ± 0.3
–0.8 ± 0.2
0.3 ± 0.1
–1.3 ± 0.3
0.2 ± 0.2
–0.2 ± 0.2
–1.4 ± 0.3
–0.5 ± 0.2
–0.2 ± 0.2
–1.0 ± 0.2
–0.1 ± 0.5
0.2 ± 0.1
0.1 ± 0.2
–0.9 ± 0.2
0.1 ± 0.2
0.1 ± 0.2
–0.2 ± 0.2
–0.1 ± 0.2
0.1 ± 0.2
–1.8 ± 0.4
0.3 ± 0.2
0.1 ± 0.1
–0.3 ± 0.3
0.2 ± 0.1
0.1 ± 0.1
0.3 ± 0.2
0.2 ± 0.2
0.1 ± 0.3
–1.4 ± 0.3
0.1 ± 0.2
c
b
B
A
c
A
c
c
c
c
–1.6 ± 0.2
0.0 ± 0.2
B
B
B
B
10
5b (10 mg/kg, i.p.), 8b (5 mg/kg, i.p.) and 8-OH-DPAT (5 mg/kg, i.p.) were administered 15 min before the test. (S)-WAY 100135 (s.c.) was
a
administered 45 min before the experiment. Each group consisted of 7–8 mice. The absolute mean initial body temperatures were within
a range of 36.3 ± 0.5 °C; b p < 0.05, c p < 0.01 vs. respective vehicle group; A p < 0.05, B p < 0.01 vs. respective 5b, 8b or 8-OH-DPAT group
(Dunnett’s test).
observed in vivo activity of compounds 5b and 8b,
resulting from their high affinity for 5-HT1A receptors,
permits to classify 5b as presynaptic agonist and postsyn-
aptic partial agonist, while 8b as pre- and post-synaptic
agonist of 5-HT1A receptors. It should be stressed also
that different functional activity of these two compounds
can be attributed to the enlargement of the π-system in 8b
vs. 5b and/or diminished flexibility of the heterocyclic
fragment.
5. Experimental protocol
5.1. Chemistry
The m.p.’s were uncorrected. All compounds were
analyzed for C, H, N.
1H-NMR: Varian 200 BB (200 MHz) apparatus using
TMS as internal standard, MS: AMD-604 (70 eV) with
direct inlet. TLC was performed on Merck plates (Kie-
selgel 60 F254), solvent A: benzene + acetone (7 + 3),
solvent B: benzene + acetone (1 + 1), solvent C: methanol
+ CH3COOH (3 + 7), solvent D: chloroform + methanol
+ CH3COOH (7 + 2 + 1). solvent E: benzene + acetone
+ methanol (1 + 1 + 1).
5.2. Synthesis of compounds 5 and 6 (general
procedure)
A solution of 1-(3’-chlorophenyl)piperazine hydro-
chloride or 1-(2’-methoxyphenyl)piperazine hydrochlo-
ride (0.01 mol) in 10 mL of 30% NaOH was extracted
with chloroform (4 × 20 mL). The combined organic
layers were dried over anhydrous K2CO3, filtered off and
evaporated. The oily residue was treated with 30 mL of
Figure 4. A relationship between the pKi values and different
arylpiperazine fragments.

174
anhydrous toluene then 0.0045 mol of anhydrous K2CO3
and 0.0045 mol of γ-bromopropyl substrates were added
in one portion. The reaction mixture was refluxed for 6 h
(compounds 5a,b) or 10 h (compounds 6a,b). The inor-
ganic salts were filtered off from the hot mixture and the
solvent was evaporated to dryness. To the residue 10 mL
H2O was added, the crystalline product (5a,b) was
filtered off washed with water dried and used for the salt
preparation. In the case of compounds 6a,b, oily residues
after treating with H2O were obtained.
Compounds 5a and 6a were obtained previously in the
reaction of γ-bromopropyl substrates (synthesized by
other methods) with piperazine derivatives, using differ-
ent solvent and reaction conditions [23].
were determined on the basis of at least three competition
binding experiments in which 10–14 drug concentrations
(10–10–10–3 M), run in triplicates, were used.
5.5.2. In vivo experiments
The experiments were performed on male Wistar rats
(250–300 g) or male Albino–Swiss mice (22–26 g). The
animals were kept at ambient temperature (20 ± 1 °C) on
a natural day–night cycle (September–January), and were
housed under standard laboratory conditions. They had
free access to food (bacutil pellets) and tap water before
the experiment. Each experimental group consisted of six
to eight animals per dose, and all the animals were used
only once.
8-Hydroxy-2-(di-n-propylamino)tetralin hydrobromide
(8-OH-DPAT•HBr, Research Biochemicals, Inc.), (S)-N-
tert-butyl-3-[4-(2-methoxyphenyl)piperazin-1-yl]-2-
phenylpropanamide ((S)-WAY 100135, synthesized by
Dr. J. Boksa, Institute of Pharmacology, Polish Academy
of Sciences), reserpine (Ciba) and the investigated hydro-
chloride salts of 5b and 8b were used in the form of
freshly prepared aqueous solutions.
Hydrochloride salts were prepared as follows: the
crystalline bases (5a,b) or oily residues (6a,b) were
dissolved in conc. HCl, the solution was evaporated to
dryness then isopropanol was added. The separated crys-
tals of the salts were recrystallized from isopropanol.
5.3. Synthesis of compounds 7–9 (general procedure)
The free bases 1-(3’-chlorophenyl)piperazine and
1-(2’-methoxyphenyl)piperazine were obtained by ex-
traction from their hydrochlorides (0.01 mol) by analogi-
cal manner as for compounds 5 and 6. The oily residue
after evaporation of chloroform was treated with 10 mL
of 2-methoxyethanol then 0.005 mol of γ-chloropropyl
substrate was added and the reaction mixture was re-
fluxed for 9 h. After refrigerating (–15 °C) for 12 h the
precipitated product was filtered off, washed with small
amount of cold anhydrous ethanol and with H2O. The
crude free base was recrystallized.
The obtained in vivo data were analyzed by Dunnett’s
test.
5.5.2.1. Induction of lower lip retraction (LLR) in rats
The LLR was conducted according to the method
described by Berendsen et al. [14]. The animals were
individually placed in cages, having been scored three
times at 15, 30 and 45 min after administration of 5b
(i.p.), 8b (i.p.) and 8-OH-DPAT (s.c.) as follows: 0 =
lower incisors invisible, 0.5 = partly visible, 1 = clearly
visible. The summed up, maximum score was up to 3 for
each rat.
5.4. Hydrochlorides of compounds 7–9
5.5.2.2. Induction of behavioral syndrome in
reserpinized rats
The suspension of the base (1.0 g) in 20 mL of anhy-
drous ethanol was saturated with gaseous HCl. The
mixture was cooled on the water–ice bath. The com-
pounds was dissolved, after few minutes the salt was
precipitated. The product was filtered off, washed with
anhydrous ethanol and recrystallized.
The animals were individually placed in cages 5 min
before injection of 5b (i.p.), 8b (i.p.) and (S)-WAY
100135 (s.c.). Observation sessions, lasting 45 sec each,
began 3 min after administration of the investigated
compounds, and were repeated every 3 min. Flat body
posture and reciprocal forepaw treading were scored
using a ranked intensity scale, where 0 = absent, 1 =
equivocal, 2 = present, and 3 = intense, according to
Tricklebank et al. [15]. The maximum score, summed up
over five observation periods, amounted to 15 for each
symptom per animal. Reserpine (1 mg/kg, s.c.) was
administered 18 h before the test.
5.5. Pharmacology
5.5.1. In vitro experiments
Radioligand binding experiments were conducted in
the hippocampus of the rat brain for 5-HT1A receptors,
and in the cortex for 5-HT2A receptors according to the
published procedures [22]. The following radioligands
were used: [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
In a separate experiment, the effect of 5b, 8b and
(S)-WAY 100135 on the behavioral syndrome induced by
8-OH-DPAT (5 mg/kg, i.p.) was estimated. The tested
compounds were administered 45 min before 8-OH-

175
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DPAT. Observation sessions began 3 min after injection
of 8-OH-DPAT.
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The rectal body temperature of mice (measured with an
Ellab thermometer) was recorded at 15, 30, 45 and
60 min after injection of 5b (i.p.), 8b (i.p.) and 8-OH-
DPAT (i.p.).
In a separate experiment, the effect of (S)-WAY
100135 (10 mg/kg, s.c.) on the hypothermia induced by
5b (10 mg/kg, i.p.), 8b (5 mg/kg, i.p.) or 8-OH-DPAT
(5 mg/kg, i.p.) was investigated. (S)-WAY 100135 was
administered 30 min before the tested compounds, the
rectal body temperature of mice was measured 15, 30, 45
and 60 min after their injection.
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This study was partly supported by the Polish State
Committee for Scientific Research (KBN), grant no.
6-P206-004-07 (1994–1997).
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