Copyright © 2002 by Institute of Pharmacology Polish Journal of Pharmacology Polish Academy of Sciences Pol. J. Pharmacol., 2002, 54, 327–341 ISSN 1230-6002

REVIEW

IN THE SEARCH FOR SELECTIVE LIGANDS OF 5-HT5, 5-HT6 AND 5-HT7 RECEPTORS

Anna Weso³owska Department of New Drug Research, Institute of Pharmacology, Polish Academy of Sciences, Smêtna 12, PL 31-343 Kraków, Poland

In the search for selective ligands of 5-HT#, 5-HT$ and 5-HT% serotonin receptors. A. WESO£OWSKA. Pol. J. Pharmacol., 2002, 54, 327–341.

In recent years much attention has been focused on the functional impor- tance of 5-HT#, 5-HT$ and 5-HT% receptors in the pathogenesis of neuropsy- chiatric and other diseases. In this connection, intensive studies with ligands of these receptors are currently in progress. Recognition of the structural characteristics responsible for the binding of a ligand molecule to an appro- priate receptor, and development of an active complex have reached an ad- vanced stage in the search for selective compounds. This review was under- taken to summarize the results of structure-activity relationship studies with ligands of 5-HT#, 5-HT$ and 5-HT% receptors. Additionally, some data on lo- calization, pharmacological properties and the functional role of those recep- tors were reported.

Key words: structure-activity relationship, 5-HT# ligands, 5-HT$ li- gands, 5-HT% ligands A. Weso³owska

Receptors through which serotonin (5-HT) pro- to demonstrate effects on signal transduction sys- duces its physiological and pathological effects tems such as adenylate cyclase (AC) or phospholi- have been the subject of thorough investigation, pase C [38, 44, 63]. Although no second messenger initially using both in vitro and in vivo pharmaco- coupling could be detected for mouse 5-HT5A re- logical methods, and later on by means of radio- ceptor, Francken et al. [18] and Hurley et al. [29] ligand binding. However, a large body of new data reported functional coupling of the human 5-HT5A were accumulated in the past years, also thanks to receptor to G proteins and receptor-mediated inhi- molecular techniques including in situ hybridiza- bition of AC activity in HEK293 cells expressing tion, which makes modification of the above classi- human 5-HT5A receptors. fication justifiable. The Serotonin Club Receptor The nothern blot analysis, quantitative polyme- Nomenclature Committee has recently proposed rase chain reaction (PCR) and in situ hybridization new classification of 5-HT receptors, which requir- experiments showed the presence of 5-HT5A mes- es three fundamental properties of a receptor to be senger ribonucleic acid (mRNA) in human brain described to ensure a comprehensive classification: cortex, hippocampus (dentate gyrus, CA1,CA2 and its operational (drug-related), transductional (re- CA3), hypothalamic area, amygdala and cerebel- ceptor-coupling) and structural (primary amino lum. The 5-HT5A receptor mRNA is expressed in acid sequence) characteristics. When applied to the many regions of the rat brain, the highest levels currently recognized 5-HT receptors, the above cri- being found in the hippocampus and hypothalamus, teria indicate the existence of up to seven receptor with lower concentrations in the cortex, thalamus classes in the central nervous system (CNS) [4, 12]. and striatum. The presence of 5-HT5A mRNA in To date, the least time has been spent on 5-HT5, the mouse brain was shown in the cerebral cortex, 5-HT6 and 5-HT7 receptor studies, mostly due to hippocampus, cerebellum and olfactory bulb, but the lack of selective ligands of these receptors. This not in the kidney, liver, spleen, lung or heart [4, 41, review is focused on structure-activity relationship 42, 44, 45]. investigations with 5-HT5, 5-HT6 and 5-HT7 recep- The physiological function of 5-HT5A receptors tor ligands. Additionally, some data on localization, is still unclear. On the basis of their localization it is pharmacological properties and functional role of proposed that 5-HT5A receptors may be involved in 5-HT5, 5-HT6 and 5-HT7 receptors as well as their multiple functions of forebrain 5-HT, such as regu- potential contribution to neuropsychiatric and other lation of affective states, cognition, anxiety (and diseases have been presented. related behaviors), sensory perception and neuro- endocrine functions [41]. Limbic distribution of 5-HT receptors and their ligands 5-HT5A mRNA seems to suggest its role in learn- 5 ing, memory and emotional behavior [42, 44]. Ex- Two 5-HT5 receptor subtypes, i.e. 5-HT5A and pression of 5-HT5A immunoreactive cells in the 5-HT5B, have been recognized. Human and rodent substantia nigra points to involvement of this re- 5-HT5A complementary deoxyribonucleic acid ceptor in the regulation of nigrostriatal dopaminer- (cDNA) encodes a protein of 357 amino acid resi- gic transmission and sensorimotor integration. The dues with seven hydrophobic domains, two puta- above assumption is evidenced by an increased lo- tive N-linked glycosylation sites and several poten- comotor and exploratory behavior in 5-HT5A re- tial phosphorylation sites for protein kinase C and ceptor knockout mice [23]. Interestingly, 5-HT5A 3’,5’-cyclic adenosine monophosphate (cAMP)-de- immunoreactive cells are in abundance in the su- pendent protein kinase. Murine 5-HT5B receptor prachiasmatic nucleus, which suggests its potential cDNA codes for a protein of 370 amino acids with involvement in circadian rhythms [41]. The results one putative site for N-linked glycosylation and of genetic studies seem to testify to a dysfunction consensus sites for phosphorylation by protein ki- of the 5-HT system in complex psychiatric disor- nase C (PKC) and cAMP-dependent protein kinase. ders, since Birkett et al. [3] found that an allelic 5-HT5A and 5-HT5B receptors display a 68% amino variation in human 5-HT5A receptor gene may play acid identity [4, 38, 45]. a role in the development of schizophrenia and af- The expression of murine and human 5-HT5 re- fective disorder. ceptors has already been shown in various cell sys- 5-HT5B receptor mRNA was present exclusive- tems, however, most of the described studies failed ly in the CA1 field of the hippocampus, the habe-

328 Pol. J. Pharmacol., 2002, 54, 327–341 5-HT#, 5-HT$ AND 5-HT% LIGANDS nula and dorsal raphe in murine brain, but not in sumption is supported by the high affinity of ergo- peripheral organs such as, e.g. the kidney, heart, lines, such as LSD (Ki = 0.9 nM), which contain an lung or liver [4, 38]. The recent findings of Grailhe embedded conformationally-constrained trypta- et al. [22] showed that, in contrast to mouse 5-HT5B mine moiety. , which represents a different gene, human 5-HT5B gene does not encode a func- conformation of the moiety, does not tional protein because its coding sequence is inter- bind at 5-HT5A receptors (Ki > 10000 nM); how- rupted by stop codons. Hence, the latter authors ever, its lack of affinity may be associated with suggested that the 5-HT5B receptor was lost during a bulk of the bridged ring (Fig. 1). evolution, after rodents and primates have di- Successive structure-5-HT5A receptor affinity verged. The 5-HT5B receptor is the first example of investigations consisted in modifications of a mole- a brain-specific protein not found in humans. cule of dimethyltryptamine (DMT) which showed COS-7 and NIH-3T3 cells expressing murine no significant affinity for 5-HT5A receptors (Ki = 5-HT5A or 5-HT5B receptors displayed high affinity 2815 nM) [54]. N1-methylation, transposition of binding of [125J]-2-iodo-lysergic acid diethylamide the nitrogen atom within the pyrrole moiety or its (LSD), that could be displaced by various non-se- removal, reduction of the pyrrole moiety as well as lective substances including 2-bromo- homologation of a side chain of DMT did not yield -LSD, , and methiothepin substances showing affinity for 5-HT5A receptors (Tab. 1) [4, 22, 44]. (Ki = 3000–25000 nM). Only introduction of a 5-me- thoxy group, but not a 4- or 6-methoxy one, into DMT enhanced about threefold the affinity (Ki = Table 1. The affinity of some compounds for 5-HT5A and 5-HT5B receptors [4] 850 nM for 5-methoxyDMT). Introduction of more bulky substituents to position 5 of DMT (e.g. for pKi 5-benzyloxy-DMT Ki = 2660 nM), as well as to the Compound amine group (Ki = 5075–10330 nM) yielded sub- 5-HT5A 5-HT5B stances with a reduced affinity for 5-HT5A recep- (mouse COS-7) (mouse COS-7) tors. In contrast, arylpiperazines showed a diverse 2-Bromo-LSD 8.7 8.5 affinity for 5-HT5A receptors (Fig. 2). In particular, Ergotamine 8.4 7.4 1-(1-naphthyl) represent high-affinity 5-Carboxyamidotryptamine 7.8 6.9 5-HT5A ligands (Ki = 40 and 3 nM for 1-(1-naph- thyl) (1-NP) and 7-hydroxy-1-NP, re- Methysergide 7.2 7.8 spectively). Although insufficient information is Methiothepin 7.0 6.6 available to permit a conclusion that the naphthyl 5-HT 6.6 6.4 ring of 1-NP binds to 5-HT5A receptors in the same RU24969 6.5 6.4

NH2 Up to the present, only Teitler et al. [54] have conducted preliminary structure-5-HT5A receptor HO affinity relationship studies. Beginning with the N structure of 5-HT, which binds only with modest H affinity to 5-HT receptors (K = 170 nM), minor 5A i 5-HT Ki = 170 nM [54] structural modifications were examined in a step- wise fashion. Methylation of 5-HT in position 2, O homologation of its side chain, dimethylation of its CH3 (C2H5)2N N N H amine group or removal of the pyrrole moiety H3CO yielded substances with a reduced affinity for N CH CH 5-HT5A receptors (Ki = 1290–10000 nM). Only H 2 3 methylation of 5-HT in position 5 improved almost N H twofold the affinity of 5-methoxy-5-HT (Ki =98 nM) in relation to 5-HT itself [54]. 5-HT likely (+)-LSD Ki = 0.9 nM [54] Ibogaine Ki > 10000 nM [54] binds at 5-HT5A receptors with its side chain being Fig. 1. Chemical structures of 5-HT, (+)-LSD and ibogaine and in a fully extended conformation. The above as- their affinity for 5-HT#) receptors

ISSN 1230-6002 329 A. Weso³owska

H H H N N N

N N N

H3CO

Cl

1-Phenylpiperazine 1-(3-Chlorophenyl)piperazine 1-(2-Methoxyphenyl)piperazine

Ki > 10000 nM [54] Ki = 8700 nM [54] Ki = 480 nM [54]

H H H N N N

N N N HO HO

1-(2-Hydroxyphenyl)piperazine 1-(1-Naphthyl)piperazine 7-Hydroxy-1-(1-naphthyl)piperazine

Ki = 215 nM [54] Ki = 40 nM [54] Ki = 3 nM [54]

Fig. 2. Chemical structures of arylpiperazine derivatives and their affinity for 5-HT#) receptors

manner as does the indole nucleus of 5-HT, the Human 5-HT6 cDNA encodes a 440-amino-acid high affinity of these agents supports the notion polypeptide which sequence significantly diverges that an indole ring is not essential for 5-HT5A bind- from that described for rat 5-HT6 receptor [32]. ing. Naphthylpiperazines, such as 7-methoxy-1-NP, 5-HT6 receptors are positively linked to AC via were previously reported to bind to multiple popu- GS= protein, since 5-HT and 5-HT6 in- lations of 5-HT receptors; for example, 7-methoxy- crease cAMP levels in cells which stably expressed -1-NP binds to 5-HT1A receptors with Ki = 3.2 nM 5-HT6 receptors [32, 39, 49, 50]. [21] and thus cannot be considered selective for The presence of 5-HT6 mRNA was showed in 5-HT5A receptors. However, due to their high affi- several human and rat brain regions, most distinctly nity, 1-NPs may serve as a suitable non-indolic in the striatum, low levels were found in the cortex, template for the prospective development of agents nucleus accumbens, olfactory tubercle, hippocam- with a greater 5-HT5A selectivity. pus, hypothalamus, amygdala and cerebellum, while no signal was detected in peripheral organs [32, 49, 5-HT receptors and their ligands 62]. 6 Bourson et al. [7] attempted to determine the Monsma et al. [39] isolated the first cDNA cod- physiological role of 5-HT6 receptors using oligo- ing for a 5-HT6 receptor from rat striatum. The nucleotide antisenses (AOs) to selectively prevent cDNA obtained, by this approach encodes a protein translation of the 5-HT6 receptor in vivo and to of 437 amino acid residues with seven hydrophobic measure various behavioral parameters such as food regions, one potential N-linked glycosylation site intake, body weight, body temperature, locomotor and several potential sites for phosphorylation by activity, nociception and free behavior. Repeated cAMP-dependent PKC in the predicted third cyto- intracerebroventricular treatment with AOs, but not plasmatic loop and the carboxyl-terminal tail. Within with a scrambled form of the antisense sequence, the transmembrane regions, this receptor showed gave rise to a specific behavioral syndrome of homologies of 36–41% to other 5-HT receptors. yawning, stretching and chewing. Some well-known

330 Pol. J. Pharmacol., 2002, 54, 327–341 5-HT#, 5-HT$ AND 5-HT% LIGANDS stereotypic effects are induced, first of all, by dopa- rotonergic substances, including 2-bromo-LSD, di- mine D2 receptor agonists, yet in this case the spe- hydroergotamine, methysergide and methiothepin, cific behavioral syndrome did not seem to be typical and drugs (e.g. chlor- caused by modulation of dopaminergic transmis- prothixene, ), as well as some antidepres- sion, since no changes in tissue levels of either do- sants (e.g. , ) showed rela- pamine or its metabolites 3,4-dihydroxyphenylace- tively high affinities for those receptors (Tab. 2). tic acid and homovanillic acid were observed nor Tryptamine and derivatives are functional did (a D2 receptor antagonist) reduce agonists, while antipsychotic and the number of yawns or stretches. On the other drugs are regarded as antagonists of 5-HT6 recep- hand, atropine (a muscarine receptor antagonist) tors [32, 47, 50]. It should be stressed here that the dose-dependently antagonized the behavioral syn- abovementioned drugs and substances have a di- drome induced by AOs [7] or Ro 04-6790 (a selec- versified chemical structure. tive 5-HT6 receptor antagonist) [53]. Ro 04-6790 5-HT6 receptors have a characteristic pharma- also inhibited the rotational behavior of unilaterally cological profile that distinguishes them from other 6-hydroxydopamine lesioned rats, induced by the 5-HT receptors. Boess et al. [5] attempted to iden- muscarinic antagonists, scopolamine and atropine tify specific interactions between 5-HT6 receptors [6]. Hence, one of the functions of 5-HT6 receptors and nonselective ligands. They showed that 5-HT appears to be the control of cholinergic neurotrans- and N1-unsubstituted (e.g. LSD, ergota- mission which is increased by 5-HT6 antagonists. mine) bind to threonine 196 (Thr196) in the trans- Therefore, it is interesting to speculate about a pos- membrane region V, having created a hydrogen sible clinical role of selective 5-HT6 antagonists as bond between the hydroxyl group of threonine and potential drugs to be used in pathological states the indole nitrogen of such ligands. In contrast, re- when a change of cholinergic neurotransmission is moval of a potential hydrogen bond-forming site in indicated. This would include depression, maybe transmembrane helix of five of the 5-HT6 receptors memory deficits, and anxiety. The results obtained by changing Thr196 to alanine, selectively reduced by Yoshioka et al. [65] indicated that 5-HT6 recep- the affinity of the natural N1-unsubstituted indole- tors may be involved in certain states of anxiety. amine and ergolines without affecting or increasing The latter authors used conditioned fear stress (CFS) to induce both an increase in 5-HT release Table 2. The affinity of some compounds for 5-HT6 receptors from the prefrontal cortex and freezing behavior in [4, 47] rats. Treatment with AOs for 5-HT6 receptor pKi Compound mRNA suppressed the CFS-induced 5-HT release, 5-HT6 (rat COS-7) but not the freezing behavior. After infusion with Agonists: AOs, Hamon et al. [25] observed a decrease in 7.9 5-HT6 receptor-like immunostaining of the nucleus 2-Bromo-LSD 7.8 accumbens and an anxiogenic behavior of rats in social interaction and elevated plus maze tests. 5-Methoxytryptamine 7.4–7.7 The evidence obtained from positron emission 5-HT 6.8–7.3 tomography indicates that the limbic prefrontal 5-Carboxyamidotryptamine 6.7 cortex containing cells which make connections to Antagonists: the nucleus accumbens may be involved in the etio- Methiothepin 8.7–9.4 logy of schizophrenia itself [13]. Demonstration of 8.5 the presence of 5-HT6 receptors in the nucleus ac- 8.4 cumbens, prefrontal cortex and other limbic areas Clozapine 7.9–8.4 [62] and high affinity of some neuroleptics for 6.4 them [47] give support to the concept that 5-HT 6 Amoxypine 7.5 receptors may play an important role in mediating Mianserin 7.4 the effects of some antipsychotic agents. 7.2 COS-7 cells, transiently expressing rat 5-HT6 receptors, displayed high affinity binding sites for Clomipramine 7.3 [125J]LSD and [3H]5-HT. Various non-selective se- 6.7

ISSN 1230-6002 331 A. Weso³owska the affinity of N1-methylated compounds (e.g. me- 5-HT6 AOs, which consisted in yawning, stretching tergoline, methysergide and ). The and chewing [53]. mentioned increased affinity of N1-methylated The next described 5-HT6 antagonist was com- ligands may be the result of elimination of an un- pound 1, a benzenesulphonamide derivative (Fig. 3). favorable steric interaction between the methyl It showed an excellent affinity for 5-HT6 receptors group in the N1 position of these ligands and (pKi = 8.3) and more than fiftyfold higher selecti- Thr196 of 5-HT6 receptors. vity for more than 50 receptors, enzymes or ion The abovementioned 5-HT6 receptor antagonist channels tested so far [8]. After examination of the Ro 04-6790 and its analog Ro 63-0563 are ben- whole series of compound 1 analogs (whose ben- zenesulfonamide derivatives with high affinities for zene moiety was enlarged), it turned out that com- 5-HT6 receptors (pKi = 7.3 and 7.9, respectively, pound 2 with a benzothiophene ring had the highest [53]) (Fig. 3). Both these compounds have an over affinity for 5-HT6 receptors (pKi = 9.2) (Fig. 3). hundredfold higher selectivity for 5-HT6 receptors However, a pharmacokinetic study indicated that with respect to the other 23 binding sites studied, compound 2 was rapidly metabolically N-dealkyl- however, Ro 63-0563 is characterized by some af- ated in rats and its CNS penetration was poor [8]. finity for 5-HT2A and 5-HT2C receptors (pKi = 5.3 Consequently, the N-demethylated derivative was and 5.7, respectively) [23]. When they were tested synthesized (compound SB-271046) (Fig. 3) and by functional assays, neither compound had any was found to be a high-affinity (pKi = 8.9), selec- significant effect on basal cAMP accumulation, tive (over 200 times more selective for the 5-HT6 which suggests that they are neither agonists nor receptor versus 55 other receptors, binding sites or inverse agonists. At the same time, both Ro 04- ion channels) and orally active 5-HT6 receptor an- 6790 and Ro 63-0563, behaved like competitive tagonist [48]. SB-271046 produced a potent and antagonists in the HeLa cells expressing human long-lasting anticonvulsant activity in a rat maxi- 5-HT6 receptors, since they inhibited the 5-HT-sti- mal electroshock seizure threshold test. However, mulated AC activity. Ro 04-6790 given intraperito- the magnitude of those antiseizure effects was mo- neally (3–30 mg/kg) produced a dose-dependent dest in comparison with that of some well-known behavioral syndrome similar to that produced by antiepileptic drugs, e.g. carbamazepine. Such a low

SO2NH N NHCH3 SO2NH NHCH3 N CH3 SO NH N N N 2

NH2 NH2 NHCH NHCH3 OCH 3 Br 3

Ro 04-6790 pKi = 7.3 [53] Ro 63-0563 pKi = 7.9 [53] 1 pKi = 8.3 [8]

CH3 CH3 F Cl Cl Br N CH3 NH N SO2NH N SO2NH N H S S NHSO2 N Br OCH3 OCH3 OCH3 2 pKi = 9.2 [8] SB-271046 pKi = 8.8 [53] SB-357134 pKi = 8.5 [9]

N N N N N N Ar = phenyl lub 1-naphthyl SO2 N N SO2Ar n=1lub2 SO2Ar (n) (n)

3 Ki = 0.2-4.9 nM [30] 4 Ki = 0.2-1.3 nM [30] 3a Ki = 0.2 nM [30]

Fig. 3. Chemical structures of sulfonamide derivatives and their affinity for 5-HT$ receptors

332 Pol. J. Pharmacol., 2002, 54, 327–341 5-HT#, 5-HT$ AND 5-HT% LIGANDS level of the anticonvulsant efficacy, associated with were synthesized in the hope that replacement of 5-HT6 receptor blockade, probably contributes to polar sulfonamide NH would increase their CNS the apparent absence of dose-dependence of SB- penetration. A number of these compounds main- -271046 in the latter test. Therefore, a possible tained an excellent 5-HT6 receptor affinity (pKi = clinical use of SB-271046 as a drug in the treat- 8.4–9.5) and diversified selectivity. Generally in ment of epilepsy is still controversial at this stage that series, attempts to increase the brain penetra- of experimentation. In separate behavioral [46] and tion by augmenting lipophilicity also led to an en- neurochemical [15] studies, SB-271046 produced hanced in vivo clearance in the rat [9]. indications of improvement in cognitive function. Isaac et al. [30] described a new series of 6-bi- At present, it is in the first phase of clinical trials cyclopiperazinyl-1-arylsulfonylindole (3) and 6-bi- for the treatment of cognitive disorders [9]. cyclopiperidinyl-1-arylsulfonylindole (4) derivati- The aim of successive studies was to further ex- ves (Fig. 3) as new 5-HT6 antagonists. In general, plore the scope of activity of SB-271046-related all the derivatives were found to be very potent structures. Therefore, a number of 4-piperazinyl qui- ligands of 5-HT6 receptors, whose Kis were lower noline derivatives connected with the benzothio- than 10 nM. Compound 3a with the 1-naphthyl phene moiety, showing excellent 5-HT6 binding af- group and n = 1 had the highest 5-HT6 receptor af- finities (pKi = 7.6-9.2) and > 100 higher selectivity finity (Ki = 0.2 nM) and a good binding selectivity in comparison with a range of other receptors, were compared to other key receptors. In a functional identified. It was shown that the selectivity of AC assay, it was found to be an antagonist, since it 2-substituted benzothiophene derivatives depended decreased AC activity measured in HEK293 cells on the size of 3-substituent, and the preferred point expressing human 5-HT6 receptors. Those findings of attachment of the piperazine ring to quinoline make it a promising candidate for the possible was at position 4. In a functional model, the tested treatment of schizophrenia, depression and memo- compounds were found to reverse the 5-HT-stimu- ry dysfunctions. Compound 3a is currently being lated AC activity and were classified as 5-HT6 re- further evaluated for its therapeutic potential [30]. ceptor antagonists. These compounds are under The possibility that the 5-HT6 receptor popula- further investigation for their potential utility in the tion may play a role in neuropsychiatric disorders treatment of CNS disorders [10]. has attracted considerable attention, but to date re- In the course of consecutive studies with sul- latively little is known about structural require- fonamide derivatives into the structure-activity re- ments for the binding at 5-HT6 receptors and lationship, compound SB-357134 (Fig. 3) was syn- ligands which may possibly be selective for this thesized and examined. It was found to be a high population of receptors. Glennon et al. [19] attemp- affinity (pKi = 8.5 [9]) and selective 5-HT6 receptor ted to determine structural characteristics of trypta- antagonist with good oral bioavailability in rats. All mine derivatives, which would decide the affinity its analogs (with different halogen substituens of these compounds for 5-HT6 receptors. In the within the benzene moiety) also demonstrated an first phase of their studies with tryptamine (Ki = excellent 5-HT6 affinity (pKi = 7.5–9.3 [9]), but 180 nM for 5-HT6 receptors), as a starting point diverse selectivities. In contrast to 2,4-, 2,5- or they investigated the role of aminoethyl side chain 3,5-disubstituted derivatives of SB-357134, mono- and substitution mode of the indole fragment. substituted compounds were very rapidly cleared in However, methylation in position 1 or a, methoxy- vivo and their brain to blood ratios were very low. lation in position 4, 6 or 7, transposition of the ni- In an effort to combine the metabolic stability with trogen atom or its removal resulted in a dramatic enhanced CNS penetration, a series of 2,3,5-tri- reduction of the affinity for 5-HT6 receptors (Ki = substituted analogs of SB-357134 was prepared. 350–20000 nM), whereas introduction of the me- Most of those compounds had good 5-HT6 affinity thyl group to position 2 (Ki = 46 nM for 2-methyl- (pKi = 6.8–8.7 [9]) and an exceptional selectivity, 5-HT) or of the hydroxyl group to position 5 of but their pharmacokinetic profile was similar to tryptamine molecule, as well as dimethylation of that of 3,5-disubstituted derivatives. In an attempt the amine group (Ki = 30 nM for N,N-dimethyl- to further increase brain penetration of these com- tryptamine) yielded derivatives with significantly pounds, conformationally restricted indoline, tetra- higher 5-HT6 affinities. Those studies indicated hydroquinoline and tetrahydroisoquinoline analogs that the presence of a lateral substituent in position

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5 of dimethyltryptamine was an optimum arrange- as a novel 5-HT6 antagonist, since it inhibited the ment (Ki = 16 nM for 5-methoxy-N,N-dimethyl- 5-HT-stimulated AC measured in HEK293 cells tryptamine, and Ki = 11 nM for 5-methylthio-N,N- expressing human 5-HT6 receptors [20]. While dis- dimethyltryptamine). Those findings also gave cussing these analogs, it is also noteworthy that support to an earlier, advanced hypothesis [28] that even small changes in the molecule structure may hydrogen bond formation by the 5-OH group is not completely alter the intrinsic activity. important for the 5-HT6 binding. Methylthio de- To continue the structure-activity relationship rivatives tend to form weaker hydrogen bonds with studies of this class of ligands, a series of N1-(aryl- 5-HT6 receptors, and in in vitro studies 5-methyl- sulfonyl)-N,N-dimethyltryptamine derivatives was thio-N,N,-dimethyltryptamine (Ki = 11 nM) showed synthesized and investigated [58]. While changing a higher affinity for 5-HT6 receptors than its 5-hy- aromatic substituents at the suflonyl group and the droxy derivative (Ki = 95 nM) [19]. position of the methoxy group within the indole On the grounds of the abovementioned results, moiety, it was found that compound 5 (Fig. 4) with Glennon et al. [20] synthesized several 2-alkyl-5-me- an unsubstituent phenyl ring bound with a high af- thoxytryptamines with affinities at least comparable finity (Ki = 2.3 nM) to 5-HT6 receptors, but also to that of 5-HT itself (Ki = 75 nM). In particular, showed a significant affinity for 5-HT2A (Ki = 130 2-ethyl-5-methoxy-N,N-dimethyltryptamine (EMDT) nM) and 5-HT2C (Ki = 23 nM) receptors. The struc- (Fig. 4) showed a high 5-HT6 receptor affinity (Ki ture of compound 5 was modelled using SYBYL, = 16 nM) and a reasonable selectivity for 5-HT6 and three families of low-energy conformations versus other 5-HT subtype receptors. In functional were identified. The results of those molecular studies, EMDT was demonstrated to behave like modeling investigations indicated that members of a 5-HT6 (it stimulated AC activity) with the three conformational families of 5 were super- a potency at least equal to that of 5-HT. A 2-phenyl imposed with the corresponding members of fami- derivative (PMDT) (Ki = 20 nM) (Fig. 4), which lies of the selective 5-HT6 receptor antagonist Ro has a binding profile slightly different from that of 63-0563. Functional studies brought support to that EMDT, also seems of interest. Compound PMDT observation. Compound 5 produced inhibition of lacks an agonist activity and may thus be regarded the 5-HT-stimulated AC activity in a dose-depen- dent manner and was lacking an agonist character CH CH 3CH3 3 CH3 [58]. Other modifications of the phenyl ring or its N N replacement with 1- or 2-naphthalene, as well as a simultaneous change of the methoxy group posi- H3CO CH3 H3CO tion in the indole moiety yielded novel 5-HT6 re- N N ceptor ligands with a high, but diversified, affinity H H (Ki = 0.9–93 nM) [58]. Although the obtained com- pounds (e.g. 5a (Fig. 4)) showed a higher affinity EMDT Ki = 16 nM [21] PMDT Ki = 20 nM [21] for 5-HT6 receptors than did compound 5, they were not subjected to further functional in vitro or

CH3 CH3 in vivo studies; therefore, it is not possible to con- CH3CH3 N clude what was the impact of the introduced struc- N tural modifications on the functional activity of these ligands. H3CO H3CO The role of the sulfonyl group in N1-benzene- N N sulfonyl has also been investigated. SO2 The obtained results indicate that, as a rule, the sul- SO2 fonyl group enhances the 5-HT6 receptor affinity of tryptamines and although the presence of the sul- foxide moiety is optimal, it is unnecessary for bind- ing [34]. The lack of functional and pharmacoki- 5 K = 2.3 nM [58] 5a K = 0.9 nM [58] i i netic studies with these ligands limits evaluation of Fig. 4. Chemical structures of tryptamine derivatives and their the effect of the sulfonyl group on the intrinsic ac- affinity for 5-HT$ receptors tivity and metabolism of compounds of this group.

334 Pol. J. Pharmacol., 2002, 54, 327–341 5-HT#, 5-HT$ AND 5-HT% LIGANDS

5-HT7 receptors and their ligands generalized low levels of the expression of 5-HT7(c) (hindbrain, cerebellum, spleen) and 5-HT7(d) (spleen, caudate nucleus) isoforms (Tab. 3) [27, 59]. The Heidmann et al. [26] reported that alternative small amount of 5-HT7(c) and 5-HT7(d) suggests that splicing in human and rat tissues produced several these isoforms result from a “leaky” transcription, 5-HT7 receptor isoforms which differed in amino and that they have no physiological relevance [27]. acid sequences of their carboxyl terminal tails. In rat tissues, three 5-HT7 isoforms, called 5-HT7(a), Table 3. The number of amino acids and regional distribution of 5-HT7 receptor isoforms [4, 33, 59] 5-HT7(b), and 5-HT7(c) in line with the NC-IUPHAR nomenclature [60], were found. Rat 5-HT7(a) (448- Receptor Number of Regional distribution -amino acid) and 5-HT7(b) (435-amino acid) forms amino acids arise from alternative splice donor sites. A third new isoform found in the rat, 5-HT7(c) (470-amino acid), 5-HT7a 445 (human) thalamus, hypothalamus, results from a retained exon cassette. Three 5-HT7 448 (rat) hippocampus, brain mRNA isoforms were also identified in human tis- 448 (mouse) stem, cortex, striatum, sues. Two human isoforms represent 5-HT (a long 446 (gwinea pig) olfactory bulb, olfactory 7(a) tubercle, spleen, kidney, form) and 5-HT(7b) (a short form) forms (445- and heart, coronary artery 432-amino acid, respectively), but the third one does not correspond to rat 5-HT7(c). Instead, it consti- 5-HT7b 432 (human) caudate nucleus, tutes a distinct isoform, 5-HT7(d) (479-amino acid), 435 (rat) hippocampus, spleen resulting from retention of a separate exon cassette. 5-HT7(d) transcripts are not present in the rat, be- 5-HT7c (rat) 470 (rat) cerebellum, hindbrain, spleen cause a 5-HT7(d)-specifying exon is absent from rat 5-HT7d (human) 479 (human) caudate nucleus, spleen 5-HT7 gene [26, 27]. 5-HT7 receptor protein is unique, with a low (< 40%) overall homology with other 5-HT receptors [16]. It has seven hydropho- On the grounds of 5-HT7 receptor localization bic domains, two putative sites for N-linked glyco- in the suprachiasmatic nuclei (SCN) of the hypo- sylation in the amino-terminal region, and two cys- thalamus [27, 33], it has been postulated that 5-HT7 teine residues which may form a structurally im- receptors may play an important role in circadian portant disulfide bond. One putative recognition rhythms. Hence, selective 5-HT7 receptor com- site for PKC, and another one for both cAMP-de- pounds may be useful in the treatment of jet lag and pendent protein kinase and multifunctional calmo- sleep disorders of a circadian nature. It is also be- dulin-dependent protein kinase II are conserved in lieved that a disregulated circadian rhythm may the carboxyl-terminal domain of murine 5-HT7 re- lead to mental fatigue and depression. Recent re- ceptors [4]. 5-HT7 receptors preferentially activate sults show that administration of antidepressant AC, supposedly via coupling to GSa [1, 2, 43]. drugs according to a profile consistent with the ac- 5-HT and nonselective agonists of these receptors tivity of the 5-HT7 receptor ( pKi = 6.0, increase the cAMP level measured in different cells amitriptyline pKi = 6.2) induces the immediate expressing human, rat and mouse 5-HT7 receptors early gene Fos in the SCN, which is indicative of [27, 33, 59]. neuronal activation. That effect was diminished A northen blot analysis of various mammalian upon chronic exposure which, in turn, was corre- tissues has shown the highest levels of 5-HT7(a) lated with down-regulation of 5-HT7 receptors mRNA in the hypothalamus and thalamus, its high [40]. Thus, one of the consequences of antidepres- amount in the brainstem and hippocampus, and sant treatment may be modulation of a possible lower levels in the cerebral cortex, striatum, olfac- dysrhythmic circadian function in depression, in tory bulb and olfactory tubercle. The 5-HT7(a) iso- which 5-HT7 receptors might be one of the key fac- form also predominates in the spleen, kidney, heart tors. Further evidence for the role of 5-HT7 recep- and coronary artery [4, 16, 27, 59]. The isoform of tors in depression comes from the fact that they are the 5-HT7(b) receptor comprises 31–45% of 5-HT7 under inhibitory regulation exerted by adrenal ster- receptor mRNA in the caudate, hippocampus and oids [14, 52, 64] whose concentration also under- spleen [59]. In situ hybridization studies indicate goes day-night variations. Both the hypothalamic-

ISSN 1230-6002 335 A. Weso³owska pituitary-adrenocortical axis and 5-HT are impli- Table 4. The affinity of some compounds for 5-HT7 receptors cated in depressive illnesses [11], therefore, further [4, 16, 47] studies with selective 5-HT7 ligands are necessary to elucidate possible involvement of the receptor in Compound pKi 5-HT7 (rat HEK293) depression and antidepressant therapeutic response. The expression of mRNA for 5-HT7 receptors in Agonists: thalamic and limbic structures points to their role in 5-Methoxytryptamine 8.3–8.8 affective behavior. The fact that such antipsychot- 5-HT 8.1–8.7 ics as risperidone (pKi = 8.9) and clozapine (pKi = 5-Carboxyamidotryptamine 9.0–9.5 8.2) show a high affinity for 5-HT7 receptors and 8-OH-DPAT 6.3–7.4 have features of antagonists [47] has led to an as- sumption that these receptors may be important for Antagonists: mediating the unique actions of certain antipsy- Methiothepin 8.4–9.0 chotic drugs. The role of 5-HT7 receptors in schizo- Mesulergine 7.7–8.2 phrenia is corroborated by the observation that 9.3 a decrease in 5-HT7 mRNA is observed in the pre- Chlorprothixene 8.3 frontal cortex of schizophrenic patients [59]. Phar- Chlorpromazine 7.6 macological data show that 5-HT7 receptors media- Risperidone 8.9 te the spasmolytic action of 5-HT on the smooth Clozapine 7.9–8.2 muscle of cerebral and peripheral vessels [55, 61]. Mianserin 6.9–7.3 Thus, ligands of these receptors could be used in Amitriptyline 6.2 migraine prophylaxis. The presence of these recep- Fluoxetine 6.0 tors in the periphery may suggest their involvement in immunological reactions, inflamatory processes, hypertension and peripheral vessels diseases [59]. The results of structure-activity relationship At this stage of knowledge, it may only be studies with benzenesulfonamide derivatives, 5-HT7 stated that further studies with selective ligands of receptor ligands [17], showed that the chirality cen- ter a (e.g. in the isomer (R,R)6,pKi = 7.2 [17]) 5-HT7 receptors are necessary to elucidate involve- ment of these receptors in the pathogenesis and (Fig. 5) was essential for the binding to 5-HT7 re- treatment of schizophrenia and other emotional and ceptors, whereas the chirality center b was appar- psychic disturbances, as well as vascular disorders. ently less important. Additionally, it was found that High affinity binding sites for [3H]5-HT were moving the methyl group to position 4 of piperi- dine, as well as replacement of the bulk aromatic found in COS-7 cells expressing mouse 5-HT re- 7 moiety in the sulfonamide group (compound 6) ceptors or rat and human homologs [4, 16, 47]. Re- with a mono- or disubstituted phenyl or thienyl cently, a number of substances with a differentiated moiety resulted in improvement of the affinity for chemical structure have been reported to show high 5-HT7 receptors. Following the abovementioned affinity for 5-HT7 receptors. Among them there are suggestions, compound SB-258719 (pKi = 7.5 for 5-carboxyamidotryptamine, 5-methoxytryptamine 5-HT7 receptors) (Fig. 5) was designed and synthe- and 8-hydroxy-2-(di-n-propylamino)tetraline (8-OH- sized. SB-258719 inhibited the 5-carboxyamido- -DPAT, a 5-HT1A ligand), all of them being ago- tryptamine-stimulated activity of AC in HEK293 nists of 5-HT receptors. Methiothepin, mesuler- 7 cells expressing human 5-HT7 receptors, and was gine, a few typical (e.g. chlorprothixene) and atypi- classified as a 5-HT7 antagonist [17, 57]. A confor- cal (e.g. clozapine) antipsychotic drugs and some mational analysis of compound SB-258719 using (e.g. mianserin, amitriptyline) are MACROMODEL revealed that all bonds were antagonists of these receptors (Tab. 4). All the relatively free to rotate, except for the S-N and abovementioned substances are nonselective, since N(Me)-C(Me) bonds. The Ramachandran plot they also have an excellent affinity for other 5-HT showing rotation around these two bonds in SB- and non-5-HT receptors [4, 16, 47, 51], therefore, 258719 molecule reveals an energy minimum when studies are in progress at present to develop new, the two methyl groups (one in the chirality center a, selective ligands of these receptors. and the another in nitrogen atom of the sulfona-

336 Pol. J. Pharmacol., 2002, 54, 327–341 5-HT#, 5-HT$ AND 5-HT% LIGANDS

CH3 CH3 CH3 b SO a N CH 2 N SO2 N N SO2 N 3 CH3 N CH3 CH3

CH3

(R,R)6 pKi = 7.2 [17] SB-258719 pKi = 7.5 [17] (R)7 pKi = 7.8 [37]

(S)7 pKi = 6.4 [37]

R SO2 N SO2 N 3,4-dichloro N N 3-bromo 3-methyl R CH3 CH3 3-methoxy (R)8 pKi = 8.0 [37] 9 pKi = 8.0-8.9 [37] 3-hydroxy

(S)8 pKi = 6.4 [37]

CN SO2 N H3C N N

OH O N N H CH3 CH3

SB-269970-A pKi = 8.9 [37] DR 4004 pKi = 8.7 [31] (6aR,aR)10 Ki =20.8nM[35]

Fig. 5. Chemical structures of benzenesulfonamide, tetrahydrobenzindole and derivatives and their affinity for 5-HT% receptors mide group) adopt a gauche orientation (60°). This 269970-A showed good CNS penetration, how- suggested the synthesis of analogs in which both ever, was rapidly cleared from the blood in the rat. methyl groups have been tied together into a pi- In in vivo experiments, SB-269970-A dose-depen- peridine (7) or pyrrolidine (8) ring (Fig. 5). The R dently inhibited the 5-carboxyamidotryptamine-in- enantiomers of both these derivatives are potent duced hypothermia in guinea pigs and paradoxical 5-HT7 ligands [37]. Using an optimized R-pyrroli- sleep in rats without effects on other sleep stages. dinylethyl side chain, further investigations were These data suggest that 5-HT7 receptors play a role carried out into the effect of the 3-substituted in sleep control. These observations support the hy- phenyl ring (i.e. 9 derivative) (Fig. 5) on 5-HT7 re- pothesis that 5-HT7 receptor antagonists have po- ceptor affinity and selectivity. Suprisingly, intro- tential utility for the treatment of depression (in duction of a polar 3-hydroxy group afforded SB- which disturbances of sleep are present) and/or cir- 269970-A (Fig. 5) with the highest 5-HT7 receptor cadian rhythm disturbances [24, 56]. affinity (pKi = 8.9) and an excellent selectivity pro- Of the tetrahydrobenzindole derivatives, com- file (> 100 times) compared with a total of 50 other pound DR 4004 is a highly potent ligand of 5-HT7 receptors, enzymes, or ion channels, except for receptors (pKi = 8.7 [31]) (Fig. 5), with at least 47- 5-HT5A receptors (50 times). In a functional model, fold higher selectivity over the 5-HT2 (pKi = 7.0 that compound showed features of an antagonist, [31]) and other receptors. DR 4004 inhibits the since it inhibited 5-HT- or 5-carboxyamidotrypt- 5-HT-induced stimulation of cAMP accumulation amine-stimulated AC activity in HEK293 cells sta- in COS-7 cells transfected with human 5-HT7 re- bly expressing human 5-HT7 receptors [17, 24, 37]. ceptors, hence, it is regarded as a 5-HT7 receptor Pharmacokinetic studies demonstrated that SB- antagonist [31].

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Hydrophobic region However, it is should be stressed here that the above-described studies bore fruit, having devel- oped 5-HT7 receptor ligands which are character- 4.9–5.9 Å ized by a significant affinity, distinct selectivity and 11.6–12.6 Å activity typical of antagonists. On the other hand, no selective agonist of these receptors has been available as yet. Active charged center As it has been mentioned elsewhere in this pa- per, intensive studies with ligands of serotonin re- 7.1–8.1 Å ceptors of the 5-HT5, 5-HT6 and 5-HT7 type are 7.1–8.1 Å currently in progress, and they are concerned with the structure-affinity and intrinsic activity relation- Acceptor ships. Recognition of the structural features respon- sible for the binding of a ligand molecule to an ap- Aromatic ring 4.1–5.1 Å propriate receptor and the creation of an active Fig. 6. Proposed pharmacophore for 5-HT% antagonists accor- complex, in which information is encoded that is ding to López-Rodríguez et al. [36] later passed into higher organizational levels of an organism, is an important stage in the search for se- lective compounds. Such selective ligands, espe- Of the series of atropisomeric biaryl derivatives cially agonists of 5-HT5, 5-HT6 and 5-HT7 recep- of (R)-aporphine, Linnanen et al. [35] synthesized tors, would certainly be helpful in determining their compounds which interacted in a stereoselective functional importance and involvement in the manner with 5-HT7, 5-HT1A, and D2 receptors. The pathogenesis of diseases, not exclusively of the novel derivative, compound (6aR,aR)10 (Fig. 5), CNS. However, it should be kept in mind that al- showed preference for the 5-HT7 receptor subtype tough selective receptor ligands are an important (Ki = 20.8 nM) and – in contrast to its analogs – and indispensable research tool, they rarely happen a significantly lower affinity for 5-HT1A (Ki = 778 in practice to be drugs. nM) and D2 (Ki = 2470 nM) receptors. (6aR,aR)10 turned out to be 5-HT7 receptor antagonist, since it REFERENCES inhibited the 5-HT-stimulated cAMP production in 1. Adham N., Zgombick J.M., Bard J.A., Branchek T.A.: CHO cells transfected with rat 5-HT7 receptors [35]. Using the software package Catalyst, López- Functional characterization of the recombinant human 5-hydroxytryptamine7(a) receptor isoform coupled to -Rodríguez et al. [36], performed a study with a set adenylate cyclase stimulation. J. Pharmacol. Exp. of thirty 5-HT7 antagonists, structurally different Ther., 1998, 287, 508–514. from a chemical feature standpoint, which was se- 2. Bard J.A., Zgombick J.M., Adham N., Vaysse P., lected from the reported data as the target training Branchek T.A., Weinshank R.L.: Cloning of a novel set for Catalyst analysis. The results of their study human serotonin receptor (5-HT7) positively linked to adenylate cyclase. J. Biol. Chem., 1993, 268, 23422– indicated that the minimal structural requirements 23426. for 5-HT7 antagonism consist of an aromatic ring, 3. Birkett J.T., Arranz M.J., Munro J., Osbourn S., Ker- a basic nitrogen atom (a positive ionizable center), win R.W., Collier D.A.: Association analysis of the a H-bonding acceptor group and a hydrophobic re- 5-HT5A gene in depression, psychosis and antipsy- gion at 4.9-5.9 Å away from the basic center (Fig. 6). chotic response. NeuroReport, 2000, 11, 2017–2020. 4. Boess F.G., Martin I.L.: Molecular biology of 5-HT It is very likely that compounds synthesized ac- receptors. Neuropharmacology, 1994, 33, 275–317. cording to these principles will show an antagonis- 5. Boess F.G., Monsma F.J., Mayer V., Zwingelstin C., tic action towards 5-HT7 receptors. This pharma- Sleight A.J.: Interaction of tryptamine and ergoline cophore model for 5-HT7 antagonists represents the compounds with threonine 196 in the ligand binding first contribution to the rational design of agents site of the 5-hydroxytryptamine6 receptor. Mol. Phar- acting on this receptor type. It offers a structural in- macol., 1997, 52, 515–523. 6. Bourson A., Boess F.G., Bös M., Sleight A.J.: In- sight to aid the development of novel 5-HT li- 7 volvement of 5-HT6 receptors in nigro-striatal func- gands which are esesential for the knowledge of the tion in rodents. Brit. J. Pharmacol., 1998, 125, 1562– (patho)physiological role of 5-HT7 receptors. 1566.

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