Copyright © 2003 by Institute of Pharmacology Polish Journal of Pharmacology Polish Academy of Sciences Pol. J. Pharmacol., 2003, 55, 543–552 ISSN 1230-6002

NOVEL 4-ALKYL-1-ARYLPIPERAZINES AND 1,2,3,4-TETRAHYDROISOQUINOLINES CONTAINING DIPHENYLMETHYLAMINO OR DIPHENYLMETHOXY FRAGMENT WITH DIFFERENTIATED 5-HT1A/5-HT2A/D2 RECEPTOR ACTIVITY

Maria H. Paluchowska1,#, Maria J. Mokrosz 1, Sijka Charakchieva-Minol1, Beata Duszyñska1, Aneta Kozio³1, Anna Weso³owska2, Katarzyna Stachowicz2, Ewa Chojnacka-Wójcik2 Department of Medicinal Chemistry, Department of New Drugs Research, Institute of Pharmacology, Polish Academy of Sciences, Smêtna 12, PL 31-343 Kraków, Poland

Novel 4-alkyl-1-arylpiperazines and 1,2,3,4-tetrahydroisoquinolines containing diphenylmethylamino or diphenylmethoxy fragment with dif- ferentiated 5-HT )/5-HT )/D receptor activity. M.H. PALUCHOWSKA, M.J. MOKROSZ , S. CHARAKCHIEVA-MINOL, B. DUSZYÑSKA, A. KOZIO£, A. WESO£OWSKA, K. STACHOWICZ, E. CHOJNACKA- WÓJCIK. Pol. J. Pharmacol., 2003, 55, 543–552.

Two series of 4-alkyl-1-arylpiperazines (1–4) and 1,2,3,4-tetrahydroiso- quinolines (5, 6) with diphenylmethylamino (series a) or diphenylmethoxy (series b) fragment were synthesized in order to obtain potential ligands of 5-HT ) and/or 5-HT ) and dopamine D receptors. Four new arylpiperazi- nes (1a, 3a, 1b, 3b) were found to demonstrate high 5-HT ) receptor affinity (KE = 1.5–35 nM); among them, 3a exhibited satisfactory 5-HT ) receptor affinity (KE = 74 nM). Only compounds 1b and 2b showed moderate affinity for D receptor sites (KE = 123 and 128 nM, respectively). Compounds 1a, 3a, 1b and 3b were investigated in vivo to determine their functional activity at 5-HT ) receptors; additionally, 3a was tested for 5-HT ) receptor activity. Derivatives 1a, 1b and 3b produced effects characteristic of antagonists of postsynaptic 5-HT ) receptors. Moreover, 1b exhibited features of an ago- nist of presynaptic 5-HT ) receptors, while 3a behaved like a partial agonist of postsynaptic 5-HT ) sites. The latter derivative may also be classified as a 5-HT ) receptor antagonist. Thus, novel potent 5-HT ) receptor ligands were successfully obtained, and the most promising compound 3a showed mixed 5-HT )/5-HT ) receptor activity in in vitro and in vivo tests.

Key words: new arylpiperazines, 5-HT )/5-HT )/D receptor affinity, 5-HT )/5-HT ) receptor functional activity

 correspondence M.H. Paluchowska, M.J. Mokrosz , S. Charakchieva-Minol, B. Duszyñska, A. Kozio³, A. Weso³owska, K. Stachowicz, E. Chojnacka-Wójcik

INTRODUCTION The latter has recently been used for studying li- gand-5-HT1A receptor interactions, since its basic Structures containing 1-arylpiperazine or 1,2,3,4- nitrogen atom mimics that of 1-arylpiperazines at tetrahydroisoquinoline (THIQ) fragment constitute 5-HT1A receptor sites [15, 19]. Diphenylmethyl- a numerous group of serotonin (5-HT)1A receptor amino (series a) or diphenylmethoxy (series b) ligands including agonists, partial agonist and an- fragment was selected as a second pharmacophoric tagonists [8, 15, 16, 19]. It is noteworthy that some component of those molecules. The diphenylmeth- of them show anxiolytic/antidepressant-like activi- ane system is part of the structure of such 5-HT2A ty [4, 8, 27]. Recently, some interest has focused on receptor antagonists as amperozide or ritanserin compounds acting at both 5-HT1A and 5-HT2 re- [7]. Both those pharmacophoric fragments in the ceptors as potential therapeutic agents. Compounds designed compounds were connected to an ethyl- with dual activity at these receptor systems are pre- ene or a trimethylene aliphatic chain. dicted to be more efficacious than those acting at In the present study, we have described the syn- either subtype exclusively. This happens to be the thesis of 12 new derivatives and their pharmacol- case with the arylpiperazine derivatives flibanserin ogical characteristics at 5-HT1A, 5-HT2A and D2 re- and adatanserin, 5-HT1A receptor agonists with ceptors. 5-HT2A receptor antagonistic activity, which show potent activity in preclinical models of anxiety and MATERIALS and METHODS depression [28]. Moreover, atypical antipsychotic drugs exhibit high affinity not only for dopamine D2 receptors, but also for 5-HT1A and/or 5-HT2A CHEMICAL PART ones [12, 14, 26]. Based on our previous experiences with search- The methods used for the synthesis of the de- ing for compounds with mixed 5-HT1A/5-HT2A re- sired compounds are shown in Figure 1. Melting ceptor activity [17, 18, 20–22] we designed structu- points were determined on a Boetius apparatus and res containing 1-(2-methoxyphenyl)piperazine, 1-(3- are uncorrected. 1H NMR spectra were taken with chlorophenyl)piperazine or THIQ moieties (Tab. 1). a Varian EM-360 L (60 MHz) spectrometer in deu-

Table 1. Structure of the investigated compounds and their affinities for the 5-HT ), 5-HT ) and D receptors

CH X (CH2)n Het

KE ± SEM (nM)

Comp. Het n series a, X = NH series b,X=O

5-HT ) 5-HT ) D 5-HT ) 5-HT ) D

OCH 1 3 2 1.5 ± 0.1 125 ± 6 434 ± 13 27 ± 1 113 ± 9 123 ± 4 2 N N 3 77 ± 14 850 ± 25 2030 ± 210 64 ± 12 1180 ± 9 128 ± 9

Cl 3 2 14 ± 2 74 ± 3 980 ± 30 35 ± 2 186 ± 2 2960 ± 330 4 NN 3 330 ± 20 320 ± 20 NT 298 ± 4 330 ± 6 NT

5 2 350 ± 20 2260 ± 40 NT 1230 ± 20 600 ± 20 NT N 6 3 380 ± 5 6290 ± 20 NT 250 ± 50 470 ± 80 NT

544 Pol. J. Pharmacol., 2003, 55, 543–552 5-HT )/5-HT )/D RECEPTOR ACTIVITY OF NEW ARYLPIPERAZINES

a CH NH2 + Cl (CH2)n Het CH NH (CH2)n Het

1a-6a

b CH O (CH2)n X + HHet CH O (CH2)n Het

1b-6b

n = 2, 3

OCH3 Cl

Het: N N ,,NN N

Fig. 1. Methods of preparation of compounds 1a–6a and 1b–6b.(a) NaHCO!, n-BuOH, reflux; (b)Et!N, CH!CN, reflux

teriochloroform solutions with tetramethylsilane as 4.0 (s, 3H, OCH3); 4.9 (s, 1H, CH); 7.0 (s, 4H, an internal standard. The spectral data for amines C6H4); 7.1–7.7 (m, 10H, 2C6H5). 1 refer to their free bases. Chemical shifts are ex- 2a: H NMR d: 1.4–2.0 (m, 2H CH2CH2CH2); 2.2 pressed as d (ppm), and the coupling constants J in (s, 1H, NH); 2.3–2.9 (m, 8H, CH2CH2CH2N(CH2)2); hertz (Hz). Column chromatography separations 2.9–3.3 (m, 4H, N(CH2)2); 3.8 (s, 3H, OCH3); 4.8 were carried out on a Merck gel 60 or aluminum (s, 1H, CH); 7.0 (s, 4H, C6H4); 7.1–7.7 (m, 10H, oxide 90, neutral (70–230 mesh). The stationary 2C6H5). phases and eluents are shown in Table 2. For bio- 3a: 1H NMR d: 2.1 (s, 1H, NH); 2.3–2.9 (m, 8H, logical assays, the obtained free bases were con- NHCH2CH2N(CH2)2); 3.0–3.3 (m, 4H, N(CH2)2); verted into hydrochloride or fumarate salts. The 4.8 (s, 1H, CH); 6.6–7.6 (cluster, 14H, aromat). 1 physicochemical properties of the new compounds 4a: H NMR d: 1.4–2.0 (m, 2H, CH2CH2CH2); 2.0 are presented in Table 2. The elemental analyses of (s, 1H, NH); 2.3–2.9 (m, 8H, CH2CH2CH2N(CH2)2); the salts were within ± 0.4% of the theoretical values. 3.0–3.4 (m, 4H, N(CH2)2); 4.8 (s, 1H, CH); 6.7–7.7 (cluster, 14H, aromat). 1 General procedure for the preparation of com- 5a: H NMR d: 2.2 (s, 1H, NH); 2.4–3.0 (m, 8H, pounds 1a–6a NHCH2CH2NCH2CH2); 3.6 (s, 2H, NCH2); 4.8 (s, 1H, CH); 6.8–7.6 (cluster, 14H, aromat). 1 Equimolar amounts (2 mmol) of diphenylme- 6a: H NMR d: 1.5–2.1 (m, 2H, CH2CH2CH2); 2.2 thylamine and the appropriate 1-aryl-4-(w-chloro- (s, 1H, NH); 2.4–3.0 (m, 8H, NHCH2CH2CH2NCH2CH2); alkyl)-1,2,3,4-tetrahydroisoquinoline were refluxed 3.6 (s, 2H, NCH2); 4.8 (s, 1H, CH); 6.9–7.6 (clus- in n-butanol (20 ml) in the presence of a 10% ex- ter, 14H, aromat). cess of NaHCO3 for 6–21 h. The solvent was re- General procedure for the preparation of com- moved under reduced pressure, and the residue was pounds 1b–6b treated with water (50 ml), extracted with CHCl3, and dried over anhydrous K2CO3. Crude products Equimolar amounts (2 mmol) of the appropriate were purified by column chromatography (Tab. 2). arylpiperazine or THIQ and 2-(diphenylmethoxy)- 1a: 1H NMR d: 2.2 (s, 1H, NH); 2.4–2.9 (m, 8H, ethyl bromide or 3-(diphenylmethoxy)propyl chlo- NHCH2CH2N(CH2)2); 2.9–3.3 (m, 4H, N(CH2)2); ride were refluxed in acetonitrile (30 ml) in the

ISSN 1230-6002 545 M.H. Paluchowska, M.J. Mokrosz , S. Charakchieva-Minol, B. Duszyñska, A. Kozio³, A. Weso³owska, K. Stachowicz, E. Chojnacka-Wójcik

Table 2. Physicochemical data of new compounds

Comp. Reaction time Yield= M.p. (°C) Conditions Molecular formula (h) (%) Cryst. solvent> of column (mol. weight)@ chromatography?

1a 16 55 230–232 AC $H! N!O · 3HCl · 1.5H O acetone-ethanol (538.0)

1b 21 72 114–115 BC $H!N O ·C"H"O" ·2H O acetone (554.6)

2a 19 38 259–261 AC %H!!N!O · 3HCl · H O acetone-ethanol (503.0)

2b 30 29 145–147 CC %H! N O ·C"H"O" acetone (532.6)

3a 6 43 254–256 DC #H &N!Cl · 3HCl acetone (515.4)

3b 21 25 163–165 AC #H %N OCl·C"H"O" ·3H O acetone (577.1)

4a 6 77 159–161 EC $H!N!Cl·3HCl·2H O acetone-ethanol (565.4)

4b 25 22 150–152 FC $H 'N OCl · 2HCl · 0.8H O acetone (508.3)

5a 13 37 256–258 CC "H $N · 2HCl acetone (415.4)

5b 17 41 128–130 CC "H #NO · C"H"O" ·2H O acetone (495.6)

6a 21 68 186–188 CC #H &N · 2HCl ethanol-hexane (429.4)

6b 31 45 139–140 CC #H %NO · C"H"O" acetone (473.6)

= > ? for free bases; for salts; A–SiO , chloroform : methanol (49:1, v/v), B – SiO , chloroform : ethyl acetate (2:1, v/v); C – SiO , chloroform : methanol (19:1, v/v), D – Al O!, ethyl acetate : n-hexane (1:4, v/v), E – SiO , ethyl acetate : n-hexane (1:1, v/v), @ F – SiO , chloroform; calculated from elemental analysis

1 presence of a 10% excess of triethylamine for 3b: H NMR d: 2.3–2.9 (m, 6H, CH2N(CH2)2); 17–31 h. After evaporation of the solvent, the resi- 2.9–3.3 (m, 4H, N(CH2)2); 3.6 (t, J = 6 Hz, 2H, due was chromatographed on the column with sili- CH2O); 5.4 (s, 1H, CH); 6.6–7.6 (cluster, 14H, aro- ca gel (Tab. 2). mat). 1 1b: H NMR d: 2.5–2.9 (m, 6H, CH2N(CH2)2); 4b: 1.5–2.2 (m, 2H, CH2CH2CH2); 2.3–2.8 (m, 2.9–3.3 (m, 4H, N(CH2)2); 3.6 (t, J = 6 Hz, 2H, 6H, CH2N(CH2)2); 2.9–3.3 (m, 4H, N(CH2)2); 3.5 CH2O); 3.8 (s, 3H, OCH3); 5.4 (s, 1H, CH); 7.0 (s, (t, J = 6 Hz, 2H, CH2O); 5.4 (s, 1H, CH); 6.7–7.6 4H, C6H4); 7.2–7.6 (m, 10H, 2C6H5). (cluster, 14H, aromat). 1 2b: H NMR d: 1.6–2.2 (m, 2H, CH2CH2CH2); 5b: 2.6–3.1 (m, 6H, OCH2CH2NCH2CH2); 2.3–2.8 (m, 6H, CH2N(CH2)2); 2.8–3.3 (m, 4H, 3.6–3.9 (m, 4H, OCH2CH2NCH2); 5.4 (s, 1H, CH); N(CH2)2); 3.6 (t, J = 6 Hz, 2H, CH2O); 3.8 (s, 3H, 6.9–7.6 (cluster, 14H, aromat). 1 OCH3); 5.4 (s, 1H, CH); 7.0 (s, 4H, C6H4); 7.2–7.6 6b: H NMR d: 1.6–2.2 (m, 2H, CH2CH2CH2); (m, 10H, 2C6H5). 2.3–3.1 (m, 6H, OCH2CH2NCH2CH2); 3.3–3.8 (m,

546 Pol. J. Pharmacol., 2003, 55, 543–552 5-HT )/5-HT )/D RECEPTOR ACTIVITY OF NEW ARYLPIPERAZINES

4H, OCH2CH2NCH2); 5.4 (s, 1H, CH); 6.9–7.6 (clu- In vivo experiments ster, 14H, aromat). The experiments were performed on male Wis- tar rats (250–300 g) or male Albino Swiss mice PHARMACOLOGICAL PART (24–28 g). The animals were kept at a room tem- perature (20 ± 1oC) on a natural day-night cycle (November–December), and were housed under In vitro experiments standard laboratory conditions. They had free ac- cess to food and tap water before the experiment. 5-HT ) and 5-HT ) binding assays Each experimental group consisted of 6–8 ani- mals/dose, and all the animals were used only once. The affinity of the investigated compounds for (±)-1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopro- 5-HT1A and 5-HT2A receptors was evaluated on the pane hydrochloride [(±)DOI, Research Biochemi- basis of their ability to displace [3H]-8-OH-DPAT cal Inc.], 8-hydroxy-2-(di-n-propylamino)tetralin (222 Ci/mmol, Amersham) and [3H]-ketanserin hydrobromide (8-OH-DPAT, Research Biochemi- (66.4 Ci/mmol, NEN Chemicals), respectively. Ra- cal Inc.), reserpine (Ciba, ampoules) and N-{2-[4- dioligand binding experiments were performed on (2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-py- rat brain using the following structures: the hippo- ridinyl)cyclohexanecarboxamide trihydrochloride campus for 5-HT1A, and the cortex for 5-HT2A re- (WAY 100635, synthesized by Dr. J. Boksa, Institu- ceptors, according to published procedures [3]. te of Pharmacology, Polish Academy of Sciences, Kraków, Poland) were used as aqueous solutions. D binding assays Compounds 1a, 1b, 3a and 3b were suspended in a 1% aqueous solution of Tween 80. 8-OH-DPAT, The preparation of rat striatal membranes was reserpine and WAY 100635 were injected subcuta- conducted as described previously [24]. The final neously (sc), (±)DOI, 1a, 1b, 3a and 3b were given tissue concentration for D2 receptor binding was 3 intraperitoneally (ip) in a volume of 2 ml/kg (rats) –1 mg of original wet weight ml . All the assays were and 10 ml/kg (mice). The obtained data were ana- carried out in a 50 mM potassium phosphate buffer lyzed by Dunnett’s test (when only one drug was 3 (pH 7.4). The radioligand used was [ H]-spiperone given), or by the Newman-Keuls test (when two (15.70 Ci/mmol, NEN Chemicals), applied in the drugs were administered). presence of 50 nM ketanserin (ketanserin tartrate salt, Sigma-RBI) to prevent the radioligand binding Body temperature in mice to 5-HT2A receptors. Displacement experiments were preformed in a total volume of 1.2 ml. Assay The effects of the tested compounds given tubes (in triplicate) containing: 0.1 ml of 1 nM alone on the rectal body temperature in mice [3H]-spiperone, 0.1 ml of a competing drug or (measured with an Ellab thermometer) were re- 0.1 ml of the vehicle (total binding), and 1 ml of the corded 30, 60, 90 and 120 min after their admini- tissue were incubated at 37°C for 30 min. Non- stration. In an independent experiment, the effect of specific binding was determined using 0.1 ml of WAY 100635 (0.1 mg/kg) on the hypothermia 5 mM butaclamol [(+)-butaclamol HCl, Sigma- induced by compounds 1a, 1b or 3b was tested. RBI]. The binding reaction was terminated by rapid WAY 100635 was administered 15 min before 1a, filtration through Whatman GF/B filters, followed 1b or 3b, and rectal body temperature was recorded by three 4 ml washes with an ice-cold incubation 30 and 60 min after injection of the tested com- buffer. The radioactivity retained on the filters was pounds. In another experiment, the effect of 3a measured by a liquid scintillation counting (Beck- (which did not change body temperature in mice) man LS 6500 apparatus) in 4 ml of the scintillation on the 8-OH-DPAT (5 mg/kg)-induced hypother- fluid (Akwascynt). mia was tested. Compound 3a was administered Ki values were determined from at least three 45 min before 8-OH-DPAT, and rectal body tem- competition binding experiments in which 10 drug perature was measured 15, 30, 45 and 60 min after concentrations were used. The Cheng and Prusoff injection of 8-OH-DPAT. The results were ex- equation [5] was used for Ki calculations. pressed as a change in body temperature (Dt) with

ISSN 1230-6002 547 M.H. Paluchowska, M.J. Mokrosz , S. Charakchieva-Minol, B. Duszyñska, A. Kozio³, A. Weso³owska, K. Stachowicz, E. Chojnacka-Wójcik respect to the basal body temperature, measured at RESULTS and DISCUSSION the beginning of the experiment. The two designed series of compounds (a and b) Lower lip retraction (LLR) in rats were synthesized via the general routes shown in Figure 1. The compounds were tested for their in LLR was assessed according to the method de- vitro activity towards 5-HT1A, 5-HT2A and D2 re- scribed by Berendsen et al. [2]. The rats were indi- ceptors; binding data are summarized in Table 1. In vidually placed in cages (30 × 25 × 25 cm) and series a, the highest 5-HT1A receptor affinity (Ki = were scored three times (at 15, 30 and 45 min after 1.5 nM) was demonstrated by compound 1a with administration of the tested compounds) as fol- 1-(2-methoxyphenyl)piperazine fragment and the lows: 0 = lower incisors not visible, 0.5 = partly ethylene chain connecting two basic nitrogen atoms visible, 1 = completely visible. The total maximum in the molecule. The replacement of 2-methoxy score amounted to 3/rat. In a separate experiment, group in piperazine moiety with 3-chloro substitu- the effect of the studied compounds on LLR ent caused a 10-fold decrease in 5-HT1A receptor induced by 8-OH-DPAT (1 mg/kg) was tested. affinity, notwithstanding compound 3a was still The compounds were administered 45 min before a very potent ligand of those receptors (Ki =14nM). 8-OH-DPAT, and the animals were scored 15, 30 The elongation of the aliphatic chain to three me- and 45 min after 8-OH-DPAT administration. thylene groups in both the arylpiperazines resulted in a decrease in 5-HT1A receptor affinity (cf. com- Behavioral syndrome in reserpinized rats pounds 1a vs 2a and 3a vs 4a). When the arylpi- perazine system was substituted with THIQ moiety Reserpine (1 mg/kg) was administered 18 h be- 5a 6a fore the test. The rats were individually placed in for the analogous compounds and , significant experimental cages (30 × 25 × 25 cm) 5 min before diminution of 5-HT1A receptor affinity was ob- injection of the tested compounds. Observation ses- served. In the case of arylpiperazine derivatives of series b, a tendency similar to that stated in the sions, lasting 45 s each, began 3 min after the injec- a tion and were repeated every 3 min. Reciprocal amine series was noted, i.e. the shorter the spacer, the higher the 5-HT1A receptor affinity. Thus, aryl- forepaw treading (FT) and flat body posture (FBP) 1b 3b were scored using a ranked intensity scale, where piperazines and with ethylene spacer bound 0 = absent, 1 = equivocal, 2 = present, and 3 = in- very well to 5-HT1A receptor sites (Ki = 27 and 35 nM, respectively), whereas in the case of their tense. The total maximum score of five observation 2b 4b periods amounted to 15/for each symptom/animal trimethylene analogs and the respective Ki values were 64 and 298 nM. In that series, THIQs [29]. The effect of the tested compounds on the 5b 6b behavioral syndrome induced by 8-OH-DPAT and also exhibited very weak 5-HT1A recep- (5 mg/kg) in reserpinized rats was estimated in an tor affinity (Ki = 1230 and 250 nM, respectively). independent experiment. The investigated com- Satisfactory 5-HT2A receptor affinity was shown only by two arylpiperazines from series a, both pounds were administered 60 min before 8-OH- 1a 3a DPAT. Observations began 3 min after 8-OH-DPAT with ethylene spacer, i.e. compounds and (Ki administration and were repeated every 3 min for = 125 and 74 nM, respectively). As to the remain- a period of 15 min. der of the amines of that series, their binding con- stant values ranged between 320–6290 nM. Ethers b Head twitches in mice of series demonstrated low 5-HT2A receptor af- finity (Ki = 330–1180 nM), except for compound In order to habituate mice to the experimental 1b whose binding constant equalled 113 nM. The environment, each animal was randomly trans- in vitro experiments discussed above showed that ferred to a 12 cm (diameter) × 20 cm (height) glass in the group of the studied arylpiperazines replace- cage, lined with sawdust, 30 min prior to treatment. ment of the nitrogen atom in the spacer with oxy- Head twitches were induced by (±)DOI (2.5 mg/kg) gen was not a profitable structural modification; in in mice [6]. Immediately after treatment with general, ethers showed lower 5-HT1A receptor af- (±)DOI, the head twitches were counted for 20 min. finity than their amine analogs. On the other hand, The tested compounds were administered 60 min the distance between the heteroatom (N or O) and before (±)DOI. the basic N-4 piperazine atom in both the aryl-

548 Pol. J. Pharmacol., 2003, 55, 543–552 5-HT )/5-HT )/D RECEPTOR ACTIVITY OF NEW ARYLPIPERAZINES piperazine series (a and b) affected not only 5-HT1A effect claimed to be mediated through 5-HT1A so- but also 5-HT2A receptor affinities and was optimal matodendritic receptor [11, 13]. This hypothermia in the case of two methylene groups in a spacer. In is abolished by such antagonists of 5-HT1A recep- the case of six compounds (1–3a and 1–3b) with tors as, e.g. WAY 100635 [10], WAY 100135 [9, the most interesting 5-HT1A/5-HT2A receptor pro- 25] or MP 3022 [23]. Compounds 1a, 1b and 3b, file, their D2 receptor affinity was determined. Only like 8-OH-DPAT, induced hypothermia in mice, ethers 1b and 2b showed moderate affinity for that whereas 3a, like WAY 100635, did not change receptor type, furthermore, the length of the ali- body temperature in mice (Tab. 3). However, in phatic spacer did not influence that affinity (Ki = contrast to 1a and 3b, only the hypothermia in- 123 and 128 nM, respectively). To sum up this part duced by compound 1b (10 mg/kg) was attenuated of the study, we found four new arylpiperazine (by ca. 50%, p < 0.05) by WAY 100635 (0.1 mg/kg) 5-HT1A receptor ligands (1a, 3a, 1b and 3b; Ki = (data not shown). Therefore, the decrease in mouse 1.5–35 nM), among which compound 3a demon- body temperature, produced by 1b, but not by 1a or strated satisfactory 5-HT2A receptor affinity (Ki = 3b, can be regarded as a measure of its presynaptic 74 nM). 5-HT1A receptor agonistic activity. It seems that Finally, compounds 1a, 1b, 3a and 3b were tested these receptors are not involved in the 1a-or3b-in- in in vivo models commonly used for evaluating duced hypothermias. Compound 3a (20 mg/kg) did functional 5-HT1A or 5-HT2A receptor activity. As not change the hypothermia induced by 8-OH- has already been established, 8-OH-DPAT, a 5-HT1A DPAT (5 mg/kg) in mice, while WAY 100635 receptor agonist, induces hypothermia in mice, an (0.1 mg/kg) completely abolished the latter effect

Table 3. The effect of the tested compounds on body temperature in mice

Dt ± SEM (°C) Dose Compound (mg/kg) 30 min 60 min 90 min 120 min

Vehicle – –0.1 ± 0.1 –0.1 ± 0.1 –0.2 ± 0.1 0.1 ± 0.1 1a 10 –0.6 ± 0.1= –0.3 ± 0.1 –0.4 ± 0.1 –0.2 ± 0.1 20 –0.7 ± 0.1> –0.5 ± 0.1= –0.3 ± 0.1 –0.1 ± 0.1

Vehicle – 0.0 ± 0.1 0.0 ± 0.1 –0.2 ± 0.1 –0.2 ± 0.1 1b 5 –0.8 ± 0.2> –0.6 ± 0.2 –0.5 ± 0.2 –0.4 ± 0.2 10 –1.5 ± 0.1> –1.0 ± 0.1> –1.0 ± 0.1> –0.7 ± 0.2

Vehicle – 0.0 ± 0.1 –0.1 ± 0.1 –0.1 ± 0.1 –0.2 ± 0.1 3a 10 –0.3 ± 0.1 0.0 ± 0.1 –0.1 ± 0.1 –0.1 ± 0.1 20 –0.4 ± 0.1 –0.2 ± 0.1 –0.2 ± 0.1 –0.2 ± 0.1

Vehicle – –0.1 ± 0.1 –0.1 ± 0.1 0.0 ± 0.1 0.0 ± 0.1 3b 10 –1.0 ± 0.1> –0.9 ± 0.1> –0.8 ± 0.1> –0.6 ± 0.1= 20 –1.2 ± 0.2> –1.0 ± 0.2> –1.1 ± 0.2> –0.9 ± 0.3>

Vehicle – 0.0 ± 0.1 0.1 ± 0.1 0.1 ± 0.1 0.0 ± 0.1 8-OH-DPAT 5 –1.2 ± 0.1> –0.1 ± 0.1> –0.7 ± 0.1> –0.2 ± 0.1 WAY 100635 0.1 0.1 ± 0.1 0.2 ± 0.1 0.1 ± 0.1 0.2 ± 0.2

The tested compounds were administered 30 min before the test. The absolute mean initial body temperatures were within a range of 36±0.5°C;n=8mice per group, = p<0.05,> p < 0.01 vs. respective vehicle

ISSN 1230-6002 549 M.H. Paluchowska, M.J. Mokrosz , S. Charakchieva-Minol, B. Duszyñska, A. Kozio³, A. Weso³owska, K. Stachowicz, E. Chojnacka-Wójcik of 8-OH-DPAT (data not shown); therefore, it LLR in rats (with poor intensity, Tab. 4); at the seems that the presynaptic activity of 3a is negligi- same time, it inhibited the effect induced by 8-OH- ble in this model. In order to determine postsynap- DPAT. In reserpinized rats, 3a did not mimic the ef- tic 5-HT1A receptor agonistic effects of the tested fect of 8-OH-DPAT (data not shown), but reduced compounds (1a, 1b, 3a and 3b), their ability to in- the FBP and FT evoked by that 5-HT1A receptor duce LLR and a behavioral syndrome (FBP, and agonist (Tab. 4). The remaining compounds (1a, 1b FT) in reserpinized rats was tested. The 8-OH- and 3b) showed features of weak antagonists of DPAT-induced LLR and behavioral syndrome in postsynaptic 5-HT1A receptors, since – when used rats depend on stimulation of postsynaptic 5-HT1A at high doses – they inhibited the behavioral effects receptors [1, 2, 29]. Moreover, evidence has been of 8-OH-DPAT (Tab. 4); at the same time – when presented that those symptoms are sensitive to given alone – they evoked neither LLR (Tab. 4), 5-HT1A receptor antagonists [10, 23, 25, 29]. nor FBP, nor FT in rats (data not shown). It is note- Hence, the ability of the tested compounds to in- worthy that compound 3a also demonstrated 5-HT2A hibit the 8-OH-DPAT-induced LLR, FBP and FT receptor antagonistic properties; it dose-dependent- was regarded as a measure of their postsynaptic ly inhibited the (±)DOI-induced head twitches in 5-HT1A receptor antagonistic activity. In those be- mice (ED50 = 12.5 (8.9–17.5) mg/kg), this effect havioral experiments only compound 3a behaved being connected with activation of 5-HT2A recep- like a weak partial agonist of 5-HT1A postsynaptic tors [6]. The results of the in vivo studies indicate receptors, since its high dose (20 mg/kg) induced that despite high affinity of 1a, 1b, 3a and 3b for

Table 4. Induction of lower lip retraction (LLR) (A) by the tested compounds, and their effect on the 8-OH-DPAT-induced LLR (B) in rats and on 8-OH-DPAT-induced behavioral syndrome in reserpinized rats (C)

Mean ± SEM behavioral score Dose Compound AB C (mg/kg) LLR FBP FT

Vehicle – 0.1 ± 0.1 2.8 ± 0.1 12.9 ± 1.0 12.4 ± 0.7 1a 10 0.4 ± 0.2 1.6 ± 0.1> 10.0 ± 0.7 4.6 ± 0.8> 20 0.5 ± 0.2 1.0 ± 0.2> 8.4 ± 0.7= 3.8 ± 0.7>

Vehicle – 0.0 2.8 ± 0.1 13.8 ± 0.5 13.4 ± 0.5 1b 10 0.0 1.8 ± 0.1> 11.6 ± 1.0 7.8 ± 1.1> 20 0.5 ± 0.2 1.1 ± 0.2> 8.3 ± 0.4> 4.3 ± 0.9>

Vehicle – 0.1 ± 0.1 2.8 ± 0.1 14.0 ± 0.6 13.8 ± 0.7 3a 10 0.2 ± 0.1 1.5 ± 0.2> 11.8 ± 0.8 9.7 ± 0.9 20 1.3 ± 0.2> 1.3 ± 0.2> 9.8 ± 0.7= 7.7 ± 1.1=

Vehicle – 0.0 2.8 ± 0.1 14.6 ± 0.2 13.0 ± 0.6 3b 10 0.0 1.8 ± 0.3> 12.0 ± 1.3 9.2 ± 1.0> 20 0.2 ± 0.1 1.6 ± 0.3> 10.6 ± 0.8= 7.7 ± 0.7>

WAY100635 0.1 0.1 ± 0.1 0.3 ± 0.2> 0.8 ± 0.4> 1.2 ± 0.7>

(A) The tested compounds were administered 15 min before the test; (B) the tested compounds were administered 45 min before 8-OH-DPAT (1 mg/kg); (C) reserpine (1 mg/kg) and the tested compounds were administered 18 h and 60 min, respectively, before 8-OH-DPAT (5 mg/kg); FBP – flat body posture, FT – forepaw treading; n = 6 rats per group; = p<0.05,> p < 0.01 vs. respective vehicle

550 Pol. J. Pharmacol., 2003, 55, 543–552 5-HT )/5-HT )/D RECEPTOR ACTIVITY OF NEW ARYLPIPERAZINES

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