Current Medicinal Chemistry 2001, 8, 999-1034 999 Serotonin Receptor and Transporter Ligands – Current Status

Seung Jun Oh1, Hyun-Joon Ha2,*, Dae Yoon Chi3 and Hee Kyung Lee1

1Department of Nuclear Medicine, Asan Medical Center, University of Ulsan, College of Medicine, 388-1 Pungnap-dong, Songpa-gu, Seoul 138-736, Korea

2Department of Chemistry, Hankuk University of Foreign Studies,Yongin, Kyunggi-Do 449- 791, Korea

3Department of Chemistry, Inha University, Younghun-dong, Nam-gu, Inchon, 402-751, Korea

Abstract: The serotonin (5-HT) receptor system has 14 different subtypes classified by pharmacology and function. Many ligands are widely used for therapeutic and diagnostic purposes in some severe human diseases. Most of the ligands that are specific for each 5-HT receptor have distinctive chemical structures with regard to pharmacophore elements including 4-arylpiperazine, benzimidazole, benzamide, chroman, aminopyridazine, tetralin, and polycycles. However, their affinity and selectivity for 5-HT, dopamine and a 1 receptors depend on their substituents and linker spacers. 5-HT transporter inhibitors have also been developed as potential antidepressants. In contrast to classical tricyclic compounds, newly developed secondary amine derivatives such as paroxetine and tetralin show high binding affinity and selectivity. Radioisotope-labeled ligands have 11 11 18 also been developed, including [carbonyl- C]WAY 100635 for 5-HT1A receptor, [ C or F]ketanserine 125 123 derivatives for 5-HT2 receptor, [ I]DAIZAC for 5-HT3 receptor, and [ I]IDAM for 5-HT transporter, and these are accumulated in brain regions that are rich in the respective receptors. This review summarizes the recent development of 5-HT receptor- and transporter-specific ligands and their pharmacological properties on the basis of their chemical structures.

INTRODUCTION studied with many high-affinity ligands, and the results have been well documented in early reviews [3,4]. Although The diverse pharmacological actions of serotonin (5- many ligands with subnanomolar affinity for serotonin hydroxytryptamine, 5-HT) have been the subject of intense receptors and serotonin transporter have been developed, study since its identification in 1936. These pharmacological more selective ligands with high affinity are required to actions include activation or inhibition of smooth muscle discriminate among structurally or pharmacologically similar movement and cardiac muscle movement, and activation or receptors for clinical applications. Thorough studies have led inhibition of exocrine and endocrine glands, cells of the to new or modified chemical structures to improve the hematopoietic and immune systems, and central and properties of known ligands. These selective ligands have peripheral neurons [1]. The actions of 5-HT have led to the distinctive chemical structures that depend on receptor identification of at least 14 different 5-HT receptors on the subtypes with different binding profiles. basis of operational (function, antagonism, location), transductional (G-protein, ion channel), and structural (gene In addition to serotonin receptors, serotonin transporter is sequence, chromosomal location) criteria. 5-HT receptors are localized on presynaptic axon terminals on serotonin neurons classified into 5 types, namely 5-HT1, 5-HT2, 5-HT3, 5- [5]. This site is a target for antidepressant drugs which are HT4 and 5-HT5 receptors, which are further divided into 14 serotonin transporter inhibitors based on the fact that different subtypes as shown in Fig. (1) [2]. Of these 14 depression is due to a deficiency of monoamines such as classes of 5-HT receptor, the 5-HT3 receptor has a unique noradrenaline or 5-HT at postsynaptic receptors [4]. place in pharmacological investigation not only because it is Therefore, more selective serotonin reuptake site inhibitors the only 5-HT receptor which is coupled to an ion channel, should be good therapeutic agents for depression. but also because its localization in the central nervous system (CNS) is distinct from those of other classes of 5-HT Many compounds with high in vitro affinity and receptors. Although 14 serotonin receptors have been selectivity for specific serotonin receptors sometimes show classified, their distributions and molecular structures are not different pharmacological profiles in vivo due to the yet clear. Among these receptors, 5-HT1-like, 5-TH2, 5-TH3, instability of these compounds against various enzymes and 5-HT4, and serotonin transporter have been extensively their impaired ability to pass through the blood-brain barrier. Therefore, in vivo testing is required for the development of ligands for therapeutic purposes. The use of a radiotracer *Address correspondence to this author at the Department of Chemistry, based on ligands with high affinity and selectivity may be Hankuk University of Foreign Studies, Yongin, Kyunggi-Do, 449-791, helpful for obtaining biological, pharmacological, and Korea; Telephone: 82-335-330-4369; Fax: 82-335-330-4639; E-mail: [email protected] pharmacokinetic information as well as for imaging the in

0929-8673/01 $28.00+.00 © 2001 Bentham Science Publishers Ltd. 1000 Current Medicinal Chemistry, 2001, Vol. 8, No. 9 Ha et al.

5-HT 5-HT4 5-HT 5-HT 5-HT 5-HT 5-HT 5-HT 5-HT 3 6 5A 5B 7 5-HT1A 5-HT1F 1E 5-HT1D 5-HT1B 5-HT2B 2C 2A

Fig. (1). vivo serotonin receptor distribution in human and animal brains for 5-HT1A, 5-HT2, and 5-HT3 receptors. N In this review, we discuss the recent development of 1 4 ligands including radiotracers targeted to the specific N N (CH2)4 R serotonin receptors for 5-HT1A, 5-HT1B/1D, 5-HT2, 5-HT3, N 5-HT4 and 5-HT transporter along with their chemical structures and activities. O O

N R= N 5-HT1A RECEPTOR LIGANDS O The existence of multiple serotonin receptor subsites in O mammalian brain tissue has stimulated research to identify 1; Buspirone 2; Gepirone selective agents as pharmacological tools to evaluate the role of these receptors in various pathological conditions [6]. The OCH3 5-HT1A receptor has been the subject of several studies since 1 4 it was shown to be involved in various physiological N N CH R functions, such as sleep, appetite, and sexual behavior, and 2 pathological status, such as anxiety and depression [7,8]. The 5-HT1A receptor has been cloned, and has been shown to have 421 amino acids with the transmembrane part O arranged in seven helices [9]. R= (CH2)3 N CH 1-Arylpiperazines are one of the most important classes of O N 5-HT1A receptor ligand. Structure-activity relationship O H (SAR) studies have focused on the highly active 4-(w- substituted alkyl)-1-arylpiperazines ( Ki values of 10-8 to 10- 3; NAN-190 4; WAY 100135 10 M ), where an amide or imide function is present at the w position of the alkyl chain. Examples include buspirone (1), gepirone (2), NAN-190 (3), WAY 100135 (4), WAY CH2 100635 (5) and flesinoxan (6), as shown in Fig. (2). N N

Extensive SAR studies regarding these compounds have O shown that 5-HT1A affinity is influenced by the nature of the aryl group at N1 of the piperazine ring and the length of the alkyl chain at the N4 position. With regard to the role of the 5; WAY 100635 amide or imide moiety, some authors have indicated that the presence of the terminal amide fragment plays an important F role in stabilizing the 5-HT1A receptor-ligand complex by p- p or dipole interactions [10-14]. Others have suggested that H N the amide function is not required for binding with the N N receptor [15,16]. In fact, the exact physiochemical requirements of the amide or imide-like region have not been O fully explained. Furthermore, their selectivity between O O dopaminergic D2 and adrenergic a 1 receptors is also not yet 6; Flesinoxan clear because these receptors have a high degree of similarity, OH

Fig. (2). 5-HT Receptor and Transporter Ligands Current Medicinal Chemistry, 2001, Vol. 8, No. 9 1001

R O

N R n N N R n N O OCH3 7 8 9 n = 1 or 2

OH

O O O O N R N R R O O

10 11 12 Fig. (3). with up to 45% homology in their amino acid sequences methoxy derivatives (14-16) at C-6, C-7, or C-8 [14,18,19]. [17]. This indicates that the distance between the methoxy group and the alkyl chain is important. Indeed, when the alkyl Several aromatic analogues (7-12) based on buspirone, chain is at C-1 of the tetralin nucleus, the ideal position for WAY series, and flesinoxan [Fig. (3)], have also been the methoxy group is C-5. When the alkyl chain is at C-2, a studied, and the results have shown that a tetralin moiety methoxy group at C-6 or C-7 (17,18) gives the highest gives the highest selectivity. With regard to the effect of the affinity for the 5-HT1A receptor [Fig. (4)]. Thus, the spatial position of the methoxy group on the tetralin nucleus, the 5- relationship between the methoxy group and the alkyl chain position (13) gave a higher affinity than the corresponding on the tetralin nucleus is very important [14,18,19].

The function of an alkyl chain as a spacer between the tetralin moiety and the terminal group in aryl piperazine N derivatives has also been studied. The highest affinity for 5- HT1A receptor was found in a compound (19) with a three- N carbon chain, while chain lengths of two (20) or four (21) 8 gave less affinity [Fig. (5)] [18,19]. 7 H3CO 13-16 6 CH3O 5

N N N n N H3CO 17 compd n IC50 (nM) H3CO N 19 1 0.5 20 0 140 N 21 2 15 18 Fig. (5). compd position IC50 (nM) Replacing the a -methylene group in the alkyl chain 13 5 0.5 spacer with a secondary amine (22-23) or amide (26) group 14 6 5.4 led to decreased affinity for the 5-HT1A receptor. However, 15 7 9.2 16 8 56 the insertion of an amine in the spacer led to an increase in 17 3.7 selectivity toward 5-HT1A versus dopamine D2 receptor, as 18 3.6 shown in Fig. (6) [18]. Fig. (4). 1002 Current Medicinal Chemistry, 2001, Vol. 8, No. 9 Ha et al.

N N O N

N OCH3 N OCH3 N HN n HN n

OCH3 OCH3 OCH3 22, 23 24, 25 26

n a a compd 5-HT1A 5-HT2 D2 1 2

22 1 7.7 2580 380 220 280 23 2 10 3300 1060 881 458 24 1 0.77 330 18 6.5 17 25 2 1.73 NT 15.4 78.3 NT 26 5310 >10000 >10000 5980 >10000

NT: not tested

Fig. (6).

The chirality of the tetralin moiety at C-1 has a greater flesinoxan, which has a similar chemical structure, is more influence on the selectivity than on the affinity toward 5- selective for 5-HT1A receptors than for dopamine D2 HT1A receptors. As shown in Fig. (7), there were no receptors. Three parts of the structure of flesinoxan have been remarkable differences in affinity for the 5-HT1A receptor been modified, as shown in Fig. (8) [21]. between racemates and (+)- or (-)-enantiomers [20]. When the phenyl ring in part A was replaced by a methyl group, as in compound 30 in Fig. (9) with 1-

N OCH3 N N N R

O O IC (nM) R= 50 R= N N CH3 H OCH3 a H compd 5-HT1A D2 1 31 30 27 (+/-) 0.50 110 43 O O 61 H 28 (R)-(-) 4.3 880 O N 29 (S)-(+) 2.4 610 950 R= N R= N H H Fig. (7). 32 33 O O Although many aryl piperazine derivatives are known to R= N have considerable affinity for dopamine D2 receptors, R= N N H H N N 34 35 F Ki (nM) H O N 6; Flesinoxan compd 5-HT D N N R= 1A 2 A N O H 30 800 1100 O O B 31 1.3 13 36 32 18 73 33 16 64 C OH 34 18 230 35 15 250 36 0.8 23 Fig. (8). Fig. (9). 5-HT Receptor and Transporter Ligands Current Medicinal Chemistry, 2001, Vol. 8, No. 9 1003 methoxyphenylpiperazine, the affinities for 5-HT1A and A study involving structural variation in part C showed dopamine D2 receptors were decreased. Replacement of the that the presence of a 2-methoxy group gives greater affinity phenyl ring with more polar aromatic rings, such as 2-furan for D2 receptor than for 5-HT1A receptor. On the other hand, (32), 2-pyrrole (33), 4-pyridyl (34), and 2,4-pyrimidyl (35), the incorporation of a backbone similar to a 2-methoxy also reduces the affinity for the 5-HT1A receptor. However, group as in benzofuran (42) significantly improves 5-HT1A saturation of the phenyl ring to cyclohexane (36) has little receptor affinity while the affinity for D2 receptors stays the effect on the affinity for either 5-HT1A or D2 receptors. The same. Although several variations were examined, flesinoxan high affinity of 36 for 5-HT1A and D2 receptors indicates that (6) showed the highest selectivity between 5-HT1A and D2 the lipophilicity rather than the aromatic character of the receptors. Thus, the selectivity of 6 for 5-HT1A versus D2 substituent contributes to receptor affinity. receptors seems to be dependent on the arylpiperazine aromatic ring system rather than the N4-[(p- OCH3 fluorobenzoyl)amino]-ethyl substituent [Fig. (11)] [21].

N N R Molecules bearing hydantoin (44-47) [Fig. (12)] based on buspirone (1) and NAN-190 (3) were synthesized and evaluated as 5-HT1A receptor ligands. O O N O H N R N R n N n N 37 F 38 F O n = 1 or 2 45; II Ki (nM) 44; I

O compd 5-HT1A D2

39 37 1.0 5.0 F 38 22 10 O O 39 3.4 3.3 Fig. (10). N R N R Affinity for both receptors was decreased by introducing O O an ether oxygen as in 38, replacing the amide linkage in part 46; III 47; IV B of compound 37. This result suggests that an electronegative atom, such as oxygen, at this position is Fig. (12). unfavorable. Shifting this oxygen atom by one position toward the phenyl ring, as in compound 39, restores affinity Unlike other compounds with amide, imide, or tetralin toward both 5-HT1A and D2 receptors [Fig. (10)]. Although moieties, the selectivity of hydantoin derivatives (48-62) for an amide linkage showed the highest affinity, its role is still 5-HT1A receptor and a 1 receptor depends on the chain controversial [10-14]. length of the alkyl spacer between hydantoin and piperazine. Maximum affinity for both 5-HT1A and a 1 receptors was O observed with a three- or four-methylene spacer, while a F three-methylene spacer was the best for tetralin compounds NH O Ar N N

N n N Ar = N OCH O O O 3 O

N OCH3

40 41 42 43

HO Ki (nM) Ki (nM) compd 5-HT1A D2 O O compd n 5-HT a 1 D 40 4.9 92 1A 2 41 1.0 5.0 48 1 31.7 >1000 >10000 42 0.15 5.3 49 2 45.5 131 246 43 0.30 14 50 3 4.1 9.9 >1000 6 6 1.7 140 51 4 8.8 8.6 >1000 Fig. (11). Fig. (13). 1004 Current Medicinal Chemistry, 2001, Vol. 8, No. 9 Ha et al.

H CO O 3 H3CO

N N N N N N N N O Ki (nM) 52 53 a compd 5-HT1A 1

52 4.4 3.1 53 12.2 323 Fig. (14).

[16,18]. Reduction of the hydrocarbon chain by one or two When the alkyl chain is only one methylene carbon from carbon atoms, as in 48 or 49 in Fig. (13), causes a piperazine most of the tested compounds showed poor Ki significant decrease in affinity for both 5-HT1A and a 1 values, as shown in Fig. (15), regardless of the size or shape receptors [16]. of the imide substructure on the hydantoins.

Loss of a carbonyl group in the hydantoin moiety 53 in However, the affinities for 5-HT1A and a 1 receptors were fig. (14) causes a decrease in affinity for 5-HT1A and a 1 affected by the size and overall ring shapes of hydantoins [ I- receptors by 1.5- and 27-fold compared to their respective IV in Fig. (12) ] bearing three or four methylene carbons as a parent molecules (52). However, the selectivity between a 1 spacer from piperazine (57-62) [Fig. (16)] . Ligands with

O O O

N N N N N N N O O O N OCH3 N N OCH3 OCH3

54 55 56

Ki (nM) a compd 5-HT1A 1 D2

54 34.9 500 >1000 55 93 >1000 >1000 56 61 >1000 >10000 Fig. (15). and 5-HT1A was improved more than 30-fold by this change three-carbon chains as a spacer showed a lower affinity at 5- [22]. HT1A receptor for ring types III and IV (59, 61), while the

H CO 3 H3CO O O N N N N N N N n n O H CO O 57,58 3 59,60 O N Ki (nM) n a N N compd 5-HT1A 1 D2 n 57 1 4.4 3.1 >1000 O 2 61,62 58 5.5 8.3 107 59 1 665 8.1 156 60 2 13 14 >1000 61 1 37 161 >1000 62 2 14 60 397 Fig. (16). 5-HT Receptor and Transporter Ligands Current Medicinal Chemistry, 2001, Vol. 8, No. 9 1005 bicyclohydantoin with a three-carbon spacer (57) showed a Various analogues obtained by modifying the structure of higher affinity at 5-HT1A receptor than those 58 with a four- 63 have been shown to have different binding affinities, carbon spacer [23]. selectivities, and antagonist activities for the 5-HT1A receptor. Introduction of an oxo or hydroxy moiety to the A series of chroman derivatives with potent affinity and a chroman C-4 position, such as in 64 and (-)-65 in Fig. (18), wide range of antagonism for 5-HT1A receptor have also been improved 5-HT1A receptor selectivity. Modification of the introduced. In particular, a 6-fluorochroman derivative (63) methoxy group gave compounds 66-69 with good binding [Fig. (17)] has shown extremely potent affinity (Ki = 0.22 profiles while N-alkylation of the middle amine caused a nM) and in vitro antagonism, though its selectivity for 5- decrease in affinity [25]. HT1A versus a 1-adrenergic and D2-dopamanergic receptors is not satisfactory [24]. Many aporphines can activate dopamine receptors. The racemic N-methyl-substituted aporphine analogue (±)-70 is OCH3 not only a dopamine receptor ligand, but is also a weak partial 5-HT1A receptor agonist [26]. Its (R)-isomer (70) is F O N the most active, and is used as a backbone for modification. H Addition of a methyl group at C-10 (71) improved its O affinity toward 5-HT1A, but a larger substituent such as an 63 ethyl group (72) decreased this affinity by 20-fold [27]. The Ki = 0.221 nM for 5-HT1A receptor effects of various C10-substituents on 5-HT1A receptor binding affinity support the tentative presence of a lipophilic Fig. (17). methyl pocket in the 5-HT1A receptor binding site [Fig. (19)]. Unlike C10-position derivatives, the C11-ethyl and Structural modification of the isochroman ring of 63 to C11-phenyl derivatives 73 and 74 had high affinities for 5- mimic the indole ring of serotonin may change its selectivity HT1A receptors and moderate to low affinities for dopamine

Ki (nM) R R a 2 compd R1 2 R3 5-HT1A 1 D2 H F O 63 C=O H H 0.221 2.71 0.259 N R3 64 OH OCH3 H 3.07 203 62.3 H 65 C=O OCH3 0.310 5.86 4.11 O 66 C=O -OCH2CH2-R3 4.19 169 49.6 67 C=O -OCH2O-R3 2.67 209 42.1 68 C=O -OCH2CH2O-R3 H 2.60 303 25.1 R1 69 C=O OCH3 52.7 1350 ND

ND = not detected Fig. (18). toward various receptors including 5-HT1A, a 1, and D2. D2 receptors. Removal of the N-methyl group decreased the According to a report by Yasunaga et al. [24], a fluoro affinity for 5-HT1A receptors by 7-fold and significantly moiety at the chroman C-6 position is essential for binding reduced the affinities for dopamine D1 and D2 receptors. and potent antagonism. Therefore, the aliphatic part of the Aporphine derivatives showed as high affinity and selectivity chroman ring was modified. for 5-HT1A receptors as aryl piperazine derivatives but

OH OH OH

H3C

N N N N CH CH CH CH 70 3 71 3 72 3 73 3

Ki (nM) OH compd 5-HT D D H3C 1A 1 2 70 9.6 20.4 58.5 N 71 0.45 382 1070 N H 72 9.2 782 2050 73 4.5 270 79.2 CH 75 74 3 74 1.8 3630 233 75 3.2 23800 >10000 Fig. (19). 1006 Current Medicinal Chemistry, 2001, Vol. 8, No. 9 Ha et al.

N N N

* N N * N * H3CO N C H3CO N C OH N C N O N O N O

76; [O-methyl-11C] WAY 100635 77; [carbonyl-11C] WAY 100635 78; [11C] Desmethyl-WAY 100635

F 18 N F O N N * N H3CO N C H3CO N C N O * N O O NH2

79; [11C] CPC-222 80; [18F] MPPF 81; [11C] NAD-299

Fig. (20). required more reaction steps in their synthesis; their binding Unlike 76, [carbonyl-11C]WAY 100635 (77) synthesized 11 mode to 5-HT1A receptors is different from that of aryl from [ C]cyclohexylcarbonyl is readily taken up by the piperazine derivatives [28,29]. brain. Radioactivity is retained in regions rich in 5-HT1A receptors [38], such as the occipital cortex, temporal cortex, The radiolabeled ligands in Fig. (20) were developed to and raphe nuclei, but is rapidly cleared from the cerebellum, 11 image 5-HT1A receptors: [O-methyl- C]WAY 100365 (76) which is almost devoid of 5-HT1A receptors. In monkey and [30,31], [carbonyl-11C]WAY 100635 (77) [32-34], human studies, [carbonyl-11C]WAY 100635 (77) provides [18F]MPPF (80) [35], [11C]CPC-222 (79) [36], and about 3- and 10-fold greater signal contrast (receptor-specific [11C]NAD-299 (81) [37]. to nonspecific binding) than [O-methyl-11C]WAY 100635 (76). In human studies, [11C]cyclohexanecarbonylic acid has WAY 100635 was labeled with 11C at the O-methyl been identified as a significant radioactive metabolite from 77 group of the phenyl ring or the carbonyl carbon of the [32-34]. cyclohexanecarbonyl group. Although [O-methyl-11C]WAY 100635 (76) was labeled in high radiochemical yield (25 %) Following the intravenous injection of p-[18F]MPPF with a specific activity of greater than 38.9 GBq/mmol, its (80), a high accumulation of radioactivity was observed in hepatic metabolism resulted in [O-methyl-11C]WAY 100634 the hippocampus and cerebral cortex in a cat model. Low (82) [Fig. (21)] as a metabolite that penetrates the blood- levels of radioactivity were observed in the cerebellum. At brain-barrier more readily than the parent compound, to 30 min post-injection, the mean hippocampus/cerebellum contribute to nonspecific binding. This problem had not and cortex/cerebellum ratios were 5 and 3.8, respectively been observed in previous rodent studies because this hepatic [35]. Unfortunately, the initial human studies with p- metabolic pathway is not significant in rats [30,31]. [123I]MPPI did not reveal the same specific localization to 5- HT1A receptors as in non-human primates [39]. Another radiolabeled ligand [11C]CPC-222 (79) showed N good brain penetration (1.0 –2.5 %/L) and the ratio of radioactivity in receptor-rich regions to that in the * NH H3CO N cerebellum reached a plateau of 2.5-4.0 by 45 min after N injection in human studies [36]. 99m 5-HT1A receptor ligands with Tc showed low affinity and selectivity due to the large molecular size of 99mTc, and different in vivo activity compared to in vitro properties. 82; [O-methyl-11C] WAY 100635 as metabolite However, the recent successful synthesis of a 99mTc-labeled 5-HT1A receptor ligand with a water- and air-stable 99m + Fig. (21). organometallic aqua complex [ Tc(OH2)3(CO)3] suggests the possibility of building a small molecule bearing 99mTc 5-HT Receptor and Transporter Ligands Current Medicinal Chemistry, 2001, Vol. 8, No. 9 1007

OCH3 OCH3

NH2 N N N N N N 83 84

OCH3

N N N Cl N Tc OC CO 85 CO

Fig. (22). with the bidentate ligand N-CH2-CH2-N consisting of the receptor. However, it fails to discriminate between these two parent ligand for 5-HT1A [Fig. (22)]. receptor subtypes. Many ligands for 5-HT1B receptor antagonists have been discovered. However, most of them The affinity of this compound 85 for the 5-HT1A receptor have shown very poor selectivity between 5-HT1B and 5- (IC50 value) was 5 nM, with an IC50 value of > 1mM for 5- HT1D receptor subtypes. These include benzamide HT2A receptor, dopamine D2 receptor, 5-HT transporter, and derivatives such as GR-127035, naphthyl piperazine, dopamine transporter [40,41]. morpholine, and indole carboxylate [57-59].

O N 5-HT1B AND 5-HT1D RECEPTOR LIGANDS N Molecular biological studies have demonstrated that the CH3 human 5-HT1D receptor site is encoded by a family of two N H distinct genes that were originally called 5-HT1Da and 5- N N HT1Db [42]. Binding studies have shown that the human 5- HT1Db and rat 5-HT1B receptors are pharmacologically O different. For example, propanolol bound to the rodent 5- 86, GR-127935; OCH3 HT1B receptor at a nanomolar concentration, while its IC50 for binding to the 5-HT receptor was > 1000 nM [43]. 5-HT1D: Ki = 0.74 nM 1Db 5-HT : Ki = 0.14 nM This difference in the pharmacological profile appears to be 1B 5-HT1A: Ki = 71.7 nM due to the modification of a single amino acid [44,45]. Since Fig. (23). the amino acid sequence, function, and regional distribution of the rodent 5-HT1B receptor and the human 5-HT1Db Structurally modified compounds bearing alkylamine in receptor are nearly identical, these are considered to be fig. (24) (87, 88, and 89) show a lower binding affinity than species homologues. The Serotonin Club Nomenclature their parent molecule GR-127935 (86). However, selectivity Committee [46] recently proposed a revised nomenclature for for the h5-HT1B receptor subtype versus the h5-HT1A and the 5-HT , 5-HT a , and 5-HT b receptor subtypes. 5- 1B 1D 1D h5-HT1D receptor subtypes is improved by 50- to 70-fold. HT1Da receptors are now called 5-HT1D, while 5-HT1Db receptors are now called 5-HT1B, with a distinction made R between human (h5-HT1B) and rat (r5-HT1B) receptors, considering their different pharmacological properties. X H In contrast to 5-HT1B receptor ligands, 5-HT1D receptors N control neurotransmitter release and may modulate vascular tone [47,48]. There is some evidence, or at least speculation, O that 5-HT1D receptors may be involved in migraine, depression, anxiety, and aggression [48-50]. Two distinct N subtypes of human 5-HT1D receptor have been identified: 5- compd X R HT1Da and 5-HT1Db [51-56]. Most agents that bind rat 5- 87 OH (CH2)3N(CH3)2 HT1B (r5-HT1B) receptor also show affinity for human 5- 88 OCH3 (CH2)3N(CH3)2 HT1D in brain homogenate and/or cloned h5-HT1B receptors 89 OCH3 CCCH2N(CH3)2 [47-50]. Ki (nM)

2’-Methyl-4’-(5-methyl[1,2,4]oxadiazol-3-yl)biphenyl-4- compd h5-HT1A h5-HT1D h5-HT1B carboxylic acid [4-methoxy-3-(4-methyl piperazin-1-yl)- 87 842 700 12.1 phenyl]amide in fig. (23) (86, GR-127935) has been shown 88 >1000 540 70.1 to be the most potent and selective antagonist for 5-HT 89 >1000 1200 16.2 1B/1D Fig. (24). 1008 Current Medicinal Chemistry, 2001, Vol. 8, No. 9 Ha et al.

This suggests that it may be possible to construct ligands r5-HT1B (Ki = 47 nM) than for bovine 5-HT1B (Ki = 630 with better affinity and selectivity by further modifying this nM) receptors. In contrast to (R)-91, (S)-92 showed poor structure. binding affinity and selectivity for r5-HT1B receptors ; Ki = 275 nM [63,64]. An alkyl amine substituent and an ether substituent increase selectivity. However, the large size of the molecule Many agents that bind at 5-HT1D receptors with high leads to a low affinity for h5-HT1B receptor. According to a affinity are tryptamine derivatives of 5-HT (93). For previous report [60], 5-HT1B/1D receptor subtypes possess a example, 5-carbamoyltryptamine (94) is a widely used but deep pocket in the binding domain that recognizes the nonselective 5-HT1D agonist [48]. Sumatriptan (95), which substituent at the 5-O-position of the serotonin residue. In is currently used to treat migraine, is perhaps the most particular, this region of bulk tolerance seems to differentiate commonly used 5-HT1D agonist [fig. (26)] [50]. between 5-HT1B/1D and 5-HT1A receptor subtypes [61]. Naphthyl piperazine derivative 90 [Fig. (25)] was Sumatriptan has led to the discovery of several related synthesized as a new h5-HT1B receptor antagonist, but it compounds such as naratriptan (96) [65], zolitriptan (97) showed only moderate affinity and low selectivity between 5- [66], rizatriptan (98) [67], and eletriptan (99) [68], which are HT1B and 5-HT1D receptors [62]. now available commercially or in late-phase clinical trials. Although their mechanisms of action are still unclear [69], CH3 their pharmacological actions include a direct vasoconstrictor N effect on excessively dilated intracranial, extracerebral arteries, inhibition of vasoactive neuropeptide release from perivascular trigeminal sensory neurons, and the prevention O N of neurogenic vasodilatation [70]. To identify better and O N CH N 3 N

CH3 90, 5-HT1A: Ki = 49.5 nM O O 5-HT : Ki = 0.30 nM S 1B HN 5-HT1D: Ki = 0.69 nM O N H O 96; nazatriptan N O Ki (nM) H N H3C (R)-91 (S)-92 N CH3 CH3SO3H H2O r5-TH1B 47 275 N Fig. (25). N N N Regarding the (R)- and (S)-stereochemisty of the H morphine derivatives of Fig. (25), 2-[[[3- 98; rizatriptan (morpholinomethyl)-2H-chromen-8-yl]oxy]methyl]morpho- Ki = 11 and 41 nM line methanesulfonate showed a 13-fold higher preference for (h5-HTlD and h5-HTlB)

NH NH H3C 2 2 CH3 O N HO HN H N N N O H H O N 93 94 H 97; zolmitriptan NH2 H N S O O N O O S N CH3 H N 95 H h5-HT1B: Ki = 10 nM h5-HT1D: Ki = 6.8 nM 99; eletriptan Fig. (26). Fig. (27). 5-HT Receptor and Transporter Ligands Current Medicinal Chemistry, 2001, Vol. 8, No. 9 1009 more specific ligands for 5-HT1D receptor, the benzene ring essential for the 5-HT1D binding affinity. Extending the in the original serotonin backbone was modified with ethylene chain of 102 to the propyl analogue 103 improved substituents to replace the aminoethyl at C-3 and the both h5-HT1D receptor affinity and binding selectivity over 35 substituents at C-5. h5-HT1B. With a full agonist in the [ S]GTPgS binding assay, further extension of the linker between the indole and Among the compounds shown in Fig. (27), rizatriptan piperazine linker to the butyl homologue 104 gave a lower showed a relatively high binding affinity, but did not show affinity for 5-HT1D receptor [fig. (30)] [72]. any difference in affinity for 5-HT1B and 5-HT1D receptor subtypes. This result led to the development of new h5- There have been some attempts to increase selectivity by HT1D receptor agonists by structural modification, as shown substituting the morpholine (105) or piperidine (106) in Fig. (28). Substitution of dimethylamine for the analogues of propylpiperazine. Although these trials pyrrolidine ring, L-747,201 (101) in Fig. (29), increased increased affinity for these receptors, they did not increase affinity and selectivity by 3- and 7-fold, respectively [71]. binding selectivity so as to be able to discriminate h5-HT1D from h5-HT1B [fig. (31)]. R

O N n N N N N n N N Ki (nM) N compd h5-HT h5-HT N 1D 1B H N H 105 8.8 7.4 106 24 15 100 105 Fig. (28). N N N N N N N N N H

N 106 H

101; L-747, 201 Fig. (31). Receptor Subtype Ki (nM) In addition to extending the alkyl chain, while mono- or di-fluorination of the alkyl chain (107, 108, and 109) had a h5-HT1D 3.1 h5-HT1B 28 very slight effect on 5-HT1D affinity, the selectivity between 5-HT1D and 5-HT1B remained the same. This suggested that Fig. (29). placing these small substituents at the 2-position of the propyl linker had little effect on the conformation of the Based on the structure of L-747,201, replacement of the ligands and thus no effect on their binding to the receptors. pyrrolydine ring with N-methylpiperazine (102) was

R3 CH 3 N N N

N R2 F N N N N R1 N n N N H Ki (nM) N H compd R1 R2 R3 5-HT1D 5-HT1B IC50 (nM) n 107 H H 0.5 72 compound h5-HT1D h5-HT1B Selectivity H 108 H F H 6.8 930 102 1 134 304 2 109 F F H 56 5800 103 2 33 309 9 110 H F CH3 1.0 310 104 3 210 5100 24 111 F F CH3 1.8 1000 Fig. (30). Fig. (32). 1010 Current Medicinal Chemistry, 2001, Vol. 8, No. 9 Ha et al.

Compounds (110, 111) bearing an a -methyl substituent of Although almost all of the tryptamine derivatives piperazine with fluorine at the propyl linker have described above showed subnanomolar binding affinity for 5- significantly greater affinity and selectivity for the 5-HT1D HT1D receptor, little selectivity was usually observed receptor subtype [fig. (32)] [73,74]. between 5-HT1D and 5-HT1B receptors [79-81]. Substituted isochroman-arylpiperazine series have been shown to exhibit A study was conducted to maximize both of the affinity high binding affinity for dopamine D4 receptor [82]. An and selectivity with compounds 112,113, and 114, and the isochroman-arylpiperazine derivative, PNU-109291 in fig. results suggested that propyl and ethyl linkers should be (35) (121), showed more than 5000-fold greater selectivity used to connect the indole and phenyl rings to piperazine, for 5-HT1D receptor than for 5-HT1B receptor. This respectively [fig. (33)]. The position of the substituent for compound does not have the vasoconstrictive activity that fluorine or other small molecules such as CN and Cl on the was observed with Sumatriptan. Although PNU-109291 phenyl ring did not affect on this biological activity [72, 75- (121) has a noble structural framework with isochroman and 77]. arylpiperazine rings, further SAR studies are needed to identify more selective ligands for the 5-HT1D receptor [83]. N n2 O N N N n1 H3C OCH3 N N X H O N N H N Ki (nM) n n (S)-(-)-121 compd 1 2 X 5-HT1d 5-HT1B compd D 5-HT1D 5-HT1B 5-HT1A 5-HT2A D2 4 112 4 1 H 0.1 11 (S)-(-)-121 0.9 5775 1092 168 241 >3704 113 4 2 H 1.4 166 Fig. (35). 114 4 3 H 4.9 85 115 3 2 ortho-F 0.6 100 5-HT2 RECEPTOR LIGANDS 116 3 2 meta-F 0.6 75 The 5-HT2 family of receptors consists of three subtypes, 117 3 2 para-F 0.8 120 5-HT2A, 5-HT2B, and 5-HT2C, which are grouped together Fig. (33). on the basis of molecular sequence, second-messenger system, and operational profile. In general, many compounds Substitution at C-5 of the indole ring in Fig. (34) with that are known to be selective for 5-HT2A receptor also bind sulfonamide (119) and oxazolidone (120) was tolerated. with high affinity to the 5-HT2B and 5-HT2C systems. These substitutions increased the affinity and selectivity Unlike many phenyl piperazine ligands for other serotonin between 5-HT1D and 5-HT1B receptors compared to their receptors, many compounds that are selective for 5-HT2 triazole counterpart 118 [78]. receptors are tri- or pentacycles.

N For 5-HT2A receptor agonists, compounds represented by the general structure 122 in Fig. (36) based on LSD (d- N H lysergic acid N,N-diethylamide) are a good starting point for an SAR study to probe the topography of the serotonin receptor agonist binding sites. Structural requirements for R optimal activity are 1) the primary amine functionality is N separated from the phenyl ring by two carbon atoms, 2) H Ki (nM) alkoxy oxygen (X) is present at C-2 or C-3 of the main aromatic ring, and 3) a hydrophobic substituent such as compd R 5-HT1D 5-HT1B alkyl halo, alkylthio, trifluoromethyl, etc., is present at C-4 (Y). The presence of the methyl group in an a -position N N 118 3.2 260 relative to the amine in compound 122 increased the in vivo N potency and duration of action of the molecule [84,85].

119 1.1 62 X MeHNO2S n NH2 H 120 0.3 49 n= 1-3 O Y X= O NH n Y= halogen X O 122 Fig. (34). Fig. (36). 5-HT Receptor and Transporter Ligands Current Medicinal Chemistry, 2001, Vol. 8, No. 9 1011

O O O O O NH NH2 NH2 NH2 NH2 2

Br Br Br O O O O 123 124 125 126 OCH3 127

compd 5-HT2A sites (Ki, nM) 5-HT1A sites (Ki, nM)

123 2010 29 124 18 4.3 125 2300 5.2 126 34 0.80

Fig. (37).

Compared to compounds 123 and 125, 124 and 126 with than 124. However, this compound did not have selectivity a bromine substituent in the ring showed nanomolar affinity versus other 5-HT2 receptors [87]. for 5-HT2A receptor and high selectivity versus 5-HT1A receptor [fig. (37)]. A bromine substituent in the phenyl ring O seems to play a key role in affinity in this structure. The NH2 tetrahydrobenzofuran ring in 124 and 126 is in an optimal Receptor Affinity (Ki, nM) orientation for good affinity toward 5-HT receptor. The 2A Br lone-pair electrons of the oxygen atom at C-2 in the phenyl 5-HT2A 0.04 ring face the alkyl amine group with a syn orientation. In the O 5-HT2B 0.19 5-HT2C 0.02 benzoxepin 127, the lone-pair electrons of the oxygen atom 129 at C-5 of the phenyl ring face the alkyl amine group in an anti fashion, and these compounds are inactive relative to Fig. (39). analogues with a syn orientation. Consideration of all of these findings gives insight into the topography of the 5- Other compounds that have high affinity for 5-HT2A HT2 receptor binding site for this series of compounds. If the receptor are trazodone,2-[3-[4-(3-chlorphenyl)-1-piperazinyl] - protonated amine undergoes electrostatic binding interaction propyl]-1,2,4-triazolo[4,3-a]pyridin-3-one (130), and its with the conserved aspartate residue in 5-HT2 receptors, as analogues in fig. (40) (131 – 133). Trazodone has been used in Fig. (38), the direction of the approach of the putative H- for human therapy and has two distinct basic nitrogen atoms bond donors that may interact with O2 and/or O5 of the in fragment A, including anilinic (N4) and aliphatic (N1) tetrahydrofuran ring can now be established with respect to nitrogens, similar to 5-HT1A receptor ligands. While the this aspartate-amine interaction site. At O2, the H-bond triazolopyridine and 3-chlorophenyl moieties are fixed within donor likely approaches from a syn direction relative to the the trazodone backbone, the structure was modified to alkyl amine side chain, and at O5, the donor approaches increase its affinity and selectivity as follows (a) an open from an anti direction relative to the alkylamine side chain. chain replaced the piperazine ring (131); (b) a small alkyl A hydrophobic para substituent such as bromine is essential group was introduced on the carbon (132) atom used for the for activating the 5-HT2 receptor [86]. linker to connect the triazolopyridine and piperazine of the A fragment; and (c) an oxo group was introduced into the piperazine ring (133). H-bond donor H These modified compounds showed a lack of affinity for O aspartrate all receptors, including 5-HT2A, a 1, and D2 receptors [88]. + - NH3 OOC residue H Spiperone (134), a high-affinity D2 dopaminergic region of antagonist, was one of the first agents to be able to hydrophobic Br region of distinguish between the original 5-HT1 and 5-HT2 receptor region O steric populations. According to previous reports [89,90], the H occasion binding affinity and selectivity for 5-HT2 could be improved by substituting the N-1 nitrogen with alkyl groups rather than a phenyl group. Replacing of the N1-phenyl group with H-bond donor a cyclohexyl group (135) results in a retention of affinity. 128 Replacement by a methyl group (136) decreases the affinity for 5-HT2A by 10-fold with a lack of affinity for 5-HT2C Fig. (38). receptors. Consistent with an earlier suggestion [91] regarding a region of bulk tolerance, N3-substituents seem to The benzodifuran analogue 129 in fig. (39) showed have little impact on 5-HT2A binding observed with higher affinity for 5-HT2A receptor than tetrahydrobenzofuran compound 137. On the other hand, the lactam carbonyl analogues. It has an approximately 70-fold higher affinity 1012 Current Medicinal Chemistry, 2001, Vol. 8, No. 9 Ha et al.

N N N N N N

N Cl N Cl N CH3 Cl O O O

N1 N4 NH HN N N

A 131 132 130

Percent of inhibition at 10-7M N N a N compd 5-HT2A 1 D2 norepinephirne O 130 78 49 12 0 N N 131 20 7 0 0 132 4 8 3 0 133 5 5 0 0 133 O

Fig. (40). oxygen atom may be important for 5-HT2A binding, as limbic cortical areas and the basal ganglia. Little is known determined by comparison of the compounds 136 and 138. about its functional significance due to the lack of selective Removal of the carbonyl oxygen atom reduced the affinity by 5-HT2C receptor ligands that can distinguish it from the 5- more than 1000-fold [fig. (41)] [92,93]. HT2A receptor [94].

Among the 5-HT2 receptor family, including 5-HT2A, 5- The main structural backbone of selective ligands for 5- HT2B and 5-HT2C receptor subtypes, 5-HT2A and 5-HT2C HT2B and 5-HT2C receptors features polycycles in fig. (42) are present in human tissue. In the central nervous system, (139 –140), which are found on many 5-HT2A receptor there is a high density of postsynaptic 5-HT2A receptors ligands [95-99]. throughout the neocortex and to a lesser extent in the limbic cortex and basal ganglia. The 5-HT2C subtype is highly Structural modification was performed starting from SB- concentrated in the choroid plexus, but is also found in 206553 in fig. (42) (141) as a selective 5-HT2C/2B receptor

O O N O N O

NH NH F N F N

135 134

O O O O N N

NH N F N F N H3C H3C 136 137

O compd 5-HT2A 5-HT2C 5-HT1A D2 N 134 1.8 1600 135 0.7 677 58 N CH3 N 136 23 >10000 0.5 F 137 22 1405 >10000 H3C 138 2400 >10000 265 220 138 Fig. (41). 5-HT Receptor and Transporter Ligands Current Medicinal Chemistry, 2001, Vol. 8, No. 9 1013

H N O N H H H H N N N N N N H C O 3 S O N N N N N CH3 N CH3

CH3 139; SDZ SER-082 140; SB 200646A 141; SB 206553 142; SB 204741 -logK = 8.48 -logK = 7.95 -logKB = 7.34 -logKB = 7.40 B B

Fig. (42). antagonist that shows 100-fold selectivity over the closely After the linear [2,3-f] isomer 141 was found to be the related 5-HT2A receptor. most potent and most selective, further SAR study was carried out with this specific isomeric series with respect to Angular indole isomers in fig. (43) (143-146) were substitution on the pyrrole ring. Removal of the N-methyl designed to investigate the tolerance of steric bulkiness at group in 141 to give 147 [Fig. (44)] led to a significant loss

O O O CH3 HN HN HN N N N N

N CH3 N N N N CH3 141 143 144

O O HN HN N CH3 N N

N N N 145 146 H3C

compd pKi, 5-HT2A pA2, 5-HT2B pKi, 5-HT2C 141 6.3 8.5 8.3 143 5.7 7.8 7.6 144 5.9 145 6.7 146 5.5 7.2

Fig. (43). various positions around the rigid aromatic ring system and of affinity for 5-HT2C and 5-HT2B receptors, suggesting that to determine the effects of altering the orientation of the these receptors had a lipophilic pocket to accommodate the electrostatic potential around the indole ring. The [3,2-e], N-methyl substituent. The presence of the polar indole NH [2,3-g], and [2,3-e] isomers (143, 145, and 146) possess was detrimental to binding. The N-ethyl, N-propyl, and N- weaker affinity for the 5-HT2C receptor, with pKi values 0.7- isopropyl analogues 148, 149, and 150 in fig. (44) all 1.6 log units less than that of the [2,3-f] isomer 141. The retained a 5-HT2C receptor affinity similar to that of 141, linear [3,2-f] isomer 144 is over 100-fold less potent at the with 5-HT2C/5-HT2A selectivity tending to increase with 5-HT2C receptor than 141. size. However, the N-benzyl analogue 151 showed a decrease

compd X X Z pKi, 5-HT2A pA2, 5-HT2B pKi, 5-HT2C

141 H H CH3 6.3 8.5 8.3 O X 147 H H H 5.8 7.5 6.9 HN 148 H H CH2CH3 5.9 8.0 8.1 149 H H CH CH CH 5.5 8.1 7.9 N Y 2 2 3 150 H H CH(CH3)2 5.4 8.2 8.0 N 151 H H CH2Ph 5.2 6.7 7.3 N Z 152 H CH3 CH3 5.5 7.7 7.4 153 CH3 H CH3 5.5 8.2 6.9 Fig. (44). 1014 Current Medicinal Chemistry, 2001, Vol. 8, No. 9 Ha et al.

O HN N A

N

compd A pKi, 5-HT2A pA2, 5-HT2B pKi, 5-HT2C

154 5.3 7.9 7.2 N

CH3

155 6.6 8.0 8.1 S

O

156 6.4 8.9 8.6

CH3 Fig. (45). in affinity for the 5-HT2C and 5-HT2A receptors, and a more possibly due to the presence of the more polar indole significant decrease for the 5-HT2B receptor. This implies a nitrogen. The benzothiophene 155 and benzofuran 156 in fig. limitation to the size of these binding pockets. Other 5,6- (45) were more interesting. They retained high affinity for 5- and 5,7-dimethyl analogues (152 and 153) showed a slight HT2C and 5-HT2B receptors, but unfortunately also showed decrease in 5-HT2C receptor affinity and selectivity. increased affinity at the 5-HT2A receptor, to give only 30- However, it is interesting to note that the 5HT2B receptor and 160-fold increases in 5-HT2C/5-HT2A selectivity, affinity of 153 is relatively unaffected by this variation. respectively. This confirms an earlier observation that steric Clearly, these results indicate subtle differences between the bulkiness around the indole nitrogen in 141 is important for 5-HT2C and 5-HT2B receptors. enhancing 5-HT2C/5-HT2A selectivity [100].

The reduced indole 154 in fig. (45) was found to be less Radioligands for imaging the serotonin 5-HT2 receptor potent at the 5-HT2C receptor than 141 by a factor of 10, family have been developed mainly for the 5-HT2A receptor.

F

O O H H N O N S H3C

N N 18F N N 18F CH3 N O O

157; [18F]Ketanserin 158; [18F]Altanserin 159; [11C]Lu 29-024 N 11 CH3

O O OCH3 O NH N HN N N NH2 F C O CH3 123I F

123 123 160; [ I]IBSP O O 161; [ I]R91150 123 NH I N N X Y 11 11 162; X=F, Y= CH3I; [ C]MSP 123 123 163; X= I, Y= CH3; [ I]MSP Fig. (46). 5-HT Receptor and Transporter Ligands Current Medicinal Chemistry, 2001, Vol. 8, No. 9 1015 [18F]Altanserin (158) [101-103], [18F]-(157) or [11C]-labeled years of age [113]. Although these compounds have a high 11 11 ketanserin [104,105], [ C]MDL 100907 [106], [ C]methyl binding affinity for 5-HT2A receptor and have favorable spiperone (162) [107,108], [11C]Lu 29-024 (159) [109] and characteristics for imaging, there has been considerable 18 [ F]setoperone [110] have been developed to image 5-HT2 interest in developing 5-HT2 receptor binding agents bearing receptor by PET. For SPECT imaging, [123I]IBSP 99mTc due to the desirable characteristics of 99mTc for (iodobenzoyl spiperone, 160) [111], [123I]MSP (163) [112], scintigraphic imaging. These compounds were prepared from [123I]R91150 (161) [113] and several 99mTc-labeled ketanserin by altering the quinazoline-dione portion or the ketanserin analogues have been used [fig. (46)]. (4-fluorobenzoyl)piperidine part, as in 164-169 [fig. (47)], which have nanomolar affinity for the 5-HT2A receptor [114- An animal experiment using rat brain with 116]. [18F]altanserin showed that the radioactivity in the frontal cortex decreased slowly with time from 1.14 %ID/g of tissue Compound 165 in fig. (47) is predominantly at 5 min after injection to 1.00 %ID/g at 60 min, and accumulated in the frontal cortex, anterior olfactory nucleus, remained relatively constant thereafter. The striatum and and caudate putamen which are known to have a high thalamus showed lower uptake values of 0.39 and 0.18 density of 5-HT2A receptors. However, this compound also %ID/g, respectively, beginning 1 hour after injection. The showed unlikely high uptake in the thalamus and white activity in the cerebellum was low and also relatively stable matter region. Accumulation of this compound in these from 1 to 4 hr after injection: 0.12 and 0.09 %ID/g of tissue, lipid-rich areas may be due to its considerable lipophilicity respectively. The frontal cortex-to-cerebellum ratio increased [114-117]. strongly with time and reached a plateau of 10.8 at 2 hr after injection. The frontal cortex-to-cerebellum ratio was dramatically influenced by the specific activity of the 5-HT3 RECEPTOR LIGANDS radiolabeled compound, and decreased with the addition of carrier. Ratios of 12.2. and 3.1 were found for [18F]altanserin While most 5-HT receptors belong to a G-protein-paired injection, with specific activities of 1Ci/mmol and receptor family, 5-HT3 receptors are coupled directly to a 2mCi/mmol respectively [102]. cation channel and are present within the central and peripheral nervous systems [118]. Most agents that bind to 123 A study with [ I]5-iodo-R91150 showed that the 5- other 5-HT receptors display low affinity for 5-HT3 HT2 receptor distribution varied with age and gender in receptors. Consequently, many agents that bind to 5-HT3 healthy subjects. In this study, 5-HT2A binding decreased receptors have fairly unique structures. with age. ligand binding decreased by 42 % from 30 to 70

O O S S CH3 Tc N F O N S S S S F Tc N S 164 165 H3C

O O S S CH3 S S CH3 Tc N Tc N F S S S S

166 167

H3C O S S N S O S Tc Tc N S S S S

168 169 Ki (nM)

compd 5-HT1A 5-HT2A 5-HTT D2

164 142 0.44 1783 12.9 165 19 166 64 167 67 168 7 169 24 Fig. (47). 1016 Current Medicinal Chemistry, 2001, Vol. 8, No. 9 Ha et al.

O O N N CH Cl N N N H 3 N N H NH N H2N CH3 170; Quipazine, Ki=1.4 nM 171; BRL 43694, Ki=0.6 nM 172; Zacopride, Ki=32 nM

O N O O Cl O O Cl N N CH3 H N O H2N OCH3 Cl N H

173; MDL 72222, Ki=54 nM 174; MDL 73147, pA2=9.8 175; Metoclopramide, Ki=348nM

CH O 3 N O N N N N

N N H3C H 176; GR 38032 (Ondansetron), Ki=3.3nM 177; Indolyloxadiazole, IC50 = 1.4 nM Fig. (48).

The development of selective antagonists for the 5-HT3 (170) [123], metoclopramide (175) [124] and their receptor subtype has attracted considerable attention in recent derivatives, such as 174 and 177, are used to treat the emesis years. For example, 5-HT3 receptor antagonists in fig. (48), that is a frequently severe and sometimes incapacitating such as ondansetron (176) [119], granisetron (171) [120], syndrome associated with cancer chemotherapy and MDL 72222 (173) [121], zacopride (172) [122], quipazine anesthesia. Although the only well-established therapeutic

O O O O N Cl N Cl N Cl N N N N N H H H H

N N O2N N H2N N HN HN HN HN 178 179 180 181 Ki = 3.7 Ki = 0.29 Ki = 5.2 Ki = 0.24

O O O N N NH Br N Br N Br N N N N N H H H

N O2N N H2N N HN HN HN 182 183 184 185 Ki = 0.29 Ki = 8.8 Ki = 0.42 pKi = 9.2

N N N N N NH N NH N NH N NH N N N Cl HO F N H3CO

186 187 188 189 pKi = 9.5 pKi = 9.5 pKi = 9.5 pKi = 7.2 Fig. (49). 5-HT Receptor and Transporter Ligands Current Medicinal Chemistry, 2001, Vol. 8, No. 9 1017

N N N N N NH N NH N NH N NH N N N N

CH3

190 191 192 193 pKi = 7.3 pKi = 7.1 pKi = 8.0 pKi = 8.8

N N O O N NH N NH OCH3 N N N N N N N N H

Cl Cl 194 195 196 197 pKi = 9.2 pKi = 7.5 Ki = 40 nM Ki = 210 nM

O O O Cl N N N N N N N N N Cl H3C N H3C N

198 199 200 Ki = 8 nM Ki = 19 nM Ki = 290 nM Fig. (50). indication of 5-HT3 receptor antagonists is as antiemetics, Some substituted benzimidazoles in fig. (50) (190-194) some evidence suggests that 5-HT3 receptor antagonists may at N-1 showed higher affinities than the parent compound also be useful for the treatment of central nervous system (195). Compounds with more voluminous substituents, disorders such as anxiety, schizophrenia, drug abuse and such as cyclopropyl and phenylmethyl groups, are more withdrawal, and age-associated memory impairments [125]. active, with Ki values of 8.8 and 9.2, respectively. In contrast to a benzimidazole moiety, such as in 197, The known 5-HT3 receptor ligands benzimidazolone, substitution at the 2 and/or position by chlorine or methoxy phenylimidazolidin, aminopyridazine, phenylpiperazine, and phenylimidazolidin-2-one derivatives (196, 197, 199 and 1-aryl biguanide have similar structures, with three common 200) decreased 5-HT3 receptor antagonist activity compared pharmacophore elements including a carbonyl group between to that in 198 [fig. (50)] [126]. an aromatic ring and a basic side chain. Many 5-HT3 receptor antagonists with high affinity bear With respect to the aromatic moiety, the introduction of carboxamide or carboxylate linkers. Direct comparison of a chlorine, bromine, nitro, methoxy or amine to the these two forms in 201 and 202 in fig. (51) showed that the benzimidazole ring in fig. (49) is tolerable for binding to 5- carboxamide linker was more potent [126]. HT3 receptors. Regarding 5-HT3/5-HT4 selectivity, unsubstituted compounds (178) and derivatives with Cl or Br at C-6 or NO2 at C-7 (180,183) show high selectivity (Ki O O N N = >1000 nM for 5-HT4 receptor). However, most derivatives N O of benzimidazole with Cl or Br at C-6 and H or NH2 at C-7 H (179, 181, 182 and 184) are moderately active toward 5-HT4 receptors, with Ki values for 5-HT3 and 5-HT4 of (6.1, N N >1000), (0.24, 187), (0.29, 168), and (0.41, 227) [126]. HN HN

Chlorine, fluorine and hydroxy substituents at C-5 on the 201 202 benzimidazole ring, as in compounds 186, 187, and 188, Ki = 3.7 nM Ki = 185 nM improved affinity to the same extent, while this was decreased with a methoxy group (189) [fig. (49)] [127]. Fig. (51). 1018 Current Medicinal Chemistry, 2001, Vol. 8, No. 9 Ha et al.

N N N N N N N N N N N CH3 CH3 N CH3 CH3 203 204 205 206 Ki = 4.2 nM Ki = 16 nM Ki = 46 nM Ki = 29 nM

S S

N N N N N N N N N CH3 CH3 CH3

207 208 209 Ki = 2300 nM Ki = 19 nM Ki = 2700 nM Fig. (52).

In the heteroaryl piperazine series, deletion of the fused with 10-fold higher affinity than a tertiary amine with a benzene ring of the quinoline nucleus seems to have little monomethyl group. However, N,N,-dimethyl quaternary effect, based on a comparison of 204 and 206 with 203 and amine analogues with phenyl piperazine, such as 214 and 205. However, saturation resulted in a dramatic decrease in 215 in Fig. (53), showed decreased affinity [128,129]. the 5-HT3 receptor binding affinity based on the pairs of compounds (203, 207) and (208, 209) [fig. (52)] [128]. 5-HT3 receptor agonist may control gastroenteric motility without completely blocking 5-HT3-sensitized The effect of modification of the piperazine or piperidine nerves. This would be useful for the treatment of moiety was also examined. The replacement of any one of gastroenteric disorders exemplified by irritable bowel the two protonable piperazine nitrogens with an oxygen or syndrome. The agonist activities of 2-methylserotonin (2- carbon atom, as in 211 from 210 and 213 from 212, leads to Me-5-HT, 216) and quipazine in fig. (54) (170) are well a dramatic decrease in affinity. In previous studies [119], a documented, but unlike 2-Me-5-HT, quipazine is able to quaternary amine analogue of 5-HT3 has been shown to bind cross the blood-brain-barrier [130]. Therefore, the general

N NH NH

N N N N N O CH3

210 211 212 213 Ki = 29 nM Ki = 4900 nM Ki = 3000 nM Ki = >10000

CH3 N N + CH3 N N + CH3 CH3 F3C 214 Cl 215 Ki = >10000 Ki = 1450

Fig. (53). 5-HT Receptor and Transporter Ligands Current Medicinal Chemistry, 2001, Vol. 8, No. 9 1019

NH2 with benzyl and allyl substituents, most of them showed poor affinity for 5-HT3 receptors, which suggests that the HO N N affinity of ligands depends mainly on the p-electron density NH rather than on simple steric bulkiness [131,132]. CH3 N H Dimethyl substitution (227) on the thiophene ring in fig. (57) gave a lower affinity than its nonsubstituted analog 216 170 (225) [131,132]. 2-Me-5-HT Quipazine Fig. (54). The introduction of a lipophilic pyrrole moiety (229) on the c-edge of the quinolone system of the quipazine in fig. structure of 5-HT3 receptor agonists was developed on the (58) (170) led to a more potent 5-HT3 receptor ligand that basis of quipazine, and the general pharmacophore of 5-HT3 had the same selectivity ratios versus the other 5-HT receptor agonist is shown in Fig (55). receptor subtypes and showed an improved selectivity versus 5-HT1B and serotonin uptake sites. However, the simple Z addition of a nitrogen atom to the quinolone ring as in R = H or alkyl derivatives quinoxaline in fig. (58) (228) significantly diminished the Y X = N, or C receptor affinity. These findings demonstrated that there was X N Y, Z = 5-or 6-membered ring a favorable lipophilic interaction between the pyrrole portion and the corresponding area in the receptor protein [133]. N R Compared to quipazine (170) with a free nitrogen at N-4 of 217 piperazine, methyl substitution at N-4 of the piperazine ring Fig. (55). improved 5-HT3 receptor affinity, although this was impaired by most alkyl [134]. This structures includes an aromatic ring and an N4- piperazine substituent. A series of compounds (218-227) in Instead of pyrrole, the addition of a benzene ring (230), which the aromatic portion was expanded by a thiophene cyclohexane ring (231), or a saturated bicyclic system in fig. ring as Y in Fig. (56) were evaluated by making (59) (232) to the c-edge of the quinolone nucleus increased substitutions on N-4 of the piperazine ring. Except for those affinity, but saturation of the benzene ring (233) of the

S N

N R

R =

N N N N N N O

218 219 220 221 222

N N N N CH3 N + N O N N

O 223 224 225 226

-log (IC50)

compd 5-HT1A 5-HT1B 5-HT1D 5-HT2A 5-HT2C 5-HT3

218 7.03 5.96 6.87 5.97 6.46 7.22 219 4.49 <4 4.03 4.40 4.38 4.68 220 4.19 <4 <4 <4 4.24 4.14 221 3.34 ND <4 3.95 <4 <4 222 <4 <4 <4 <4 <4 <4 223 5.11 4.39 <4 4.76 4.81 6.90 224 4.47 ND 3.29 3.91 <4 3.69 225 6.40 5.99 4.85 5.36 5.73 8.85 226 7.17 6.38 3.28 5.63 6.12 9.04 Fig. (56). 1020 Current Medicinal Chemistry, 2001, Vol. 8, No. 9 Ha et al. quinolone nucleus significant decreased 5-HT3 affinity. receptor, which is mostly located in the amygdala, Benzofusion at the the h-edge of the quinolone nucleus in entorhinal cortex, and hippocampus. fig. (59) (234,235) also significantly decreased the affinity for 11 5-HT3 receptors. This suggests a limited tolerance to large On the basis of unlabeled compound KF 17643, [ C]KF substituents in the area of the receptor protein surrounding 17643 (236) has 0.32 nM of affinity, which is higher than the h-edge of the quinolone [135]. those of selective 5-HT3 antagonists such as ondasetron (Ki = 7.6 nM) and granisetron (Ki = 2.1 nM). [11C]-labeled KF 17643 showed high non-specific binding in vivo with high S N brain uptake; 2.09 %ID/g at 30 min after injection. This is not suitable for use as a PET ligand for mapping 5-HT H C 3 3 receptor in the central nervous system (CNS) [136]. N N [11C]S21007 (237) showed results similar to those with H3C N [11C]KF 17643. An autoradiographic study with [11C]S21007 failed to reveal in vivo specificity and selectivity for areas rich in 5-HT3 receptors. PET studies in baboon showed consistent results, in that there was no compd 5-HT1A 5-HT1B 5-HT1D 5-HT2A 5-HT2C 5-HT3 selective accumulation of [11C]S21007 in the area postrema 227 3.90 <4 6.33 3.76 3.81 5.44 or hippocampus. Neither displacement nor presaturation with cold S21007 resulted in a significant change in the tissue 11 Fig. (57). distribution or kinetics of [ C]S21007 [137,138]. 125 Receptor distribution studies of 5-HT with radioisotope- The zacopride analogues [ I]MIZAC (238) (KD = 1.5 3 125 125 labeled ligands have been performed with [11C]KF 17643 nM), [ I]LIZAC (239) (KD = 0.3 nM), and [ I] DAIZAC (236) [136], [11C] S21007 (237) [137,138], and (240) (KD = 0.15 nM) were synthesized and studied. Among these compounds, [125I]MIZAC (238) and [125I]LIZAC

H N N

N N N N N CH3 N 228 229 CH3

compd IC50 (nM)

228 22 229 0.37

Fig. (58).

[125I]MIZAC (238) [139-142] and its derivatives (239, 240) (239) appear to bind specifically to an uncharacterized benzac 125 [fig. (60)]. Although many radioligands have been studied site in addition to 5-HT3 receptors. However, [ I] for imaging of 5-HT3 receptors, most of these ligands DAIZAC (240) has significant selectivity and high affinity showed high non-specific binding and low affinity for 5-HT3 only for 5-HT3 receptors [139-142]. In autoradiography for receptor due to the relatively low abundance of 5-HT3 rat brain, the highest binding affinity was observed in the

compd Ki (nM) N N N N N N 230 1.9 N N N 231 0.23 CH CH CH 230 3 231 3 232 3 232 0.83 233 95 234 200 235 200

N N N N N N N N N CH3 CH3 CH3 233 234 235 Fig. (59). 5-HT Receptor and Transporter Ligands Current Medicinal Chemistry, 2001, Vol. 8, No. 9 1021

11CH O N 3 N S 125 N O O I

N N OCH3 N * OCH3 N O 238 237 236 [125I]MIZAC [11C]S21007 [11C]KF17643

O O Cl N Cl N

OCH3 OCH3 O 125 I 125I 240 239 [125I]DAIZAC [125I]LIZAC Fig. (60). temporalimbic cortex and hippocampus, while binding was higher than that for 5-HT4. Both SDZ 205,557 (249) levels were low in the striatum and cerebellum. Although [150] and LY297524 (250) [151], which are related to substituted benzamide may have affinity for 5-HT4 receptors metoclopramide, have been reported to be 5-HT4 receptor and the dopaminergic D2 receptor family, DAIZAC is antagonists, but they also have considerable 5-HT3 receptor suitable as a 5-HT3 ligand without an amino group at C-4 of antagonist activity. GR113808 (243) [152] and SB204070 the benzene ring, which can enhance affinity for 5-HT4 (247) [153] are the first 5-HT4 receptor antagonists with high receptor relative to 5-HT3 [141]. affinity and selectivity for the 5-HT4 receptor. However, neither of these compounds is clinically suitably because the ester linkage in both molecules is readily cleaved in vivo 5-HT4 RECEPTOR LIGANDS [154]. The recently reported 5-HT4 receptor antagonists SB207266 (244) [155] and RS100235 (246) [156] in Fig. 5-HT as a neurotransmitter modulates the activity of the (61) should have better pharmacodynamics than 242 and 245 central nervous system and peripheral tissues by its action at because their amide linkages are more stable than the several receptor subtypes [143]. Due to the wide range of corresponding esters. physiologic and pathophysiologic actions of 5-HT, considerable effort has been made to identify agents which act A structural framework of 5-HT4 receptor antagonists was selectively at each of these receptor subtypes. One of these extracted as 251 [Fig. (62)], which has a piperidine ring receptor subtypes is 5-HT4, which was identified in 1988 bearing a basic nitrogen and an aromatic acyl group [144, 145] and recently cloned [146]. Based on the location connected to N-4 through either an ester or amide linkage. of the 5-HT4 receptor in gastrointestinal tissue, atrial tissue, urinary bladder tissue, and the central nervous system, Replacement of the ester linkage in 241 with an amide in several therapeutic indications have been proposed for both 252 of fig. (63) helps to improve the in vivo stability, but agonists and antagonists for this receptor. Proposed with a significant decrease in 5-HT4 receptor affinity. Alkylation of N-1 in the indole-3-carboxamide ring, like therapeutic applications for agents that block the 5-HT4 receptor include the treatment of irritable bowel syndrome, with an ethyl group as in 253 of fig. (63), improved the atrial arrhythmias, urinary incontinence, and various diseases activity compared to that of non-alkylated compounds. of the central nervous system. Although several compounds The 1-naphthalenecarboxamide 254 and have been identified as 5-HT4 receptors, most do not have the requisite selectivity or pharmacodynamic properties for dihydrobenzofuran derivatives 255 in fig. (64) are two ideal pharmacologic tools or therapeutic agents. Antagonists bicyclic aromatic amides which displayed only weak 5-HT4 receptor antagonism at the concentration tested [157]. While for 5-HT4 receptor can be classified on the basis of their chemical backbone including indolecarboxylate, benzoate, the ring systems are sterically similar to the indazole and aryl ketones, imidazolopyridines, and benzimidazolones benzimidazole rings, they cannot form a similar rigid [147, 148]. On the other hand, there are six distinct classes intramolecular hydrogen bond. This hydrogen-bonding interaction makes the aromatic ring coplanar with the atoms of 5-HT4 agonists consisting of indoles, benzamides, benzoates, arylketones, 8-naphthalimides, and of the amide linkage. The importance of analogous benzimidazolones [147, 148]. intramolecular hydrogen bonding interactions has been demonstrated with a series of dopamine D2 receptor antagonists and serotonin 5-HT receptor antagonists [158, Tropsetron (241) was one of the first 5-HT4 receptor 3 159]. antagonists [149]. However, its affinity for 5-HT3 receptor 1022 Current Medicinal Chemistry, 2001, Vol. 8, No. 9 Ha et al.

CH3 N O H O N N O O S O O CH3

241 N 242 N Tropsetron CH H 3 GR 113808

O O NHSO2CH3 N N H O N N O N 244 243 SB 207266 CH3 GR 113808

O N Cl OCH CH3 3 O O N H CO H N O OCH 3 N 2 3 O O 246 N 245 RS 100235 H DAU 6285

N N O O O O

O O

247 248 O Cl SB 204070 O I SB 207710

NH2 NH2

O O Cl Cl N O N O

H2N OCH3 H2N OCH3 249 250 SDZ 205557 LY 297524 Fig. (61).

CH O N 3

O N O H N N O (CH2)4

249 243 N Tropisetron SB 207266 H

O X X=O, N N R Ar 251 Fig. (62). 5-HT Receptor and Transporter Ligands Current Medicinal Chemistry, 2001, Vol. 8, No. 9 1023

O H O H N N

N CH3 N N N H CH3

252 253

5-HT4 receptor antagonist activity (% relaxation to 10-6M 5-HT) antagonist concentration

10-6M 105M

Tropsetron 33.9 26.4 252 60.0 253 34.3

Fig. (63).

O H O H 5-HT4 receptor antagonist activity N N (% relaxation to 10-6M 5-HT) O antagonist concentration

N N 10-6M 105M CH3 Cl CH3 254 5.4 2.8 255 37.0 22.2 254 255

Fig. (64).

When the rigid tropane ring was replaced by piperidine, N-methyl substitution on the basic nitrogen of piperidine 5-HT4 receptor antagonist activity and selectivity for 5-HT4 as in 259 increased 5-HT4 receptor antagonist activity by receptor versus 5-HT3 was increased. In a previous report more than 10-fold compared to the unsubstituted amine 258. [160], basic nitrogen within a polycyclic framework and the Likewise, incorporation of a benzyl, phenylethyl, conformational lability of compounds increase affinity for the phenylpropyl, or phenylbutyl group improved 5-HT4 5-HT4 receptor rather than the 5-HT3 receptor. Indeed, the receptor antagonist activity (259-262) [fig. (66)] [158]. piperidine 257 was a better 5-HT4 receptor ligand [Fig. (65)], and had 10-fold lower 5-HT3 receptor affinity than its Several compounds (263 – 268) with high affinity for 5- tropane analogue 256 [158]. HT4 receptor have been developed [Fig. (67)].

O O H N NH

N N N N N N CH3 O

256 257

-6 5-HT4 receptor antagonist activity (% relaxation to 10 M 5-HT)antagonist concentration F

-8 3´ 10 M 10-8M 10-7M 10-6M 256 43.2 9.0 8.0 257 37.3 Fig. (65). 1024 Current Medicinal Chemistry, 2001, Vol. 8, No. 9 Ha et al.

O H N piperidine ring and the other is 2,3-dihydro-2-oxo-1H- benzimidazole-1-carboxamide with a piperazine ring. A new family of ester compounds derived from 2-(1-piperidinyl)-4- N N amino-5-chloro-2-methoxybenzoate such as ML 10302 (269) N R and RS 23597-190 (271) [161] were synthesized and their pharmacological characteristics were examined [162]. All of these were potent agonists for this receptor. In particular, the 5-HT receptor antagonist activity (% relaxation introduction of two methyl groups, as in a 3,5- 4 dimethylpiperidine moiety, to the basic chain made to 10-6M 5-HT) antagonist concentration compound 270 a more potent 5-HT4 receptor antagonist R 10-8M 3X10-8M 10-7M 10-6M [160]. 32.0 258 H Carbamate derivatives like 272 in fig. (69) were 38.5 23.3 24.5 3.85 259 CH2Ph synthesized to overcome problems associated with the ester 260 (CH2)2Ph 1.3 functionality, such as the short half-life of ML 10302 (269) 261 (CH ) Ph 2 3 3.7 262 (CH2)4Ph in vivo. However, these were not good ligands for 5-HT4 4.0 receptors, and had about 40-fold lower affinity than the corresponding ester [163]. Methylation of the piperidine ring Fig. (66). as in 273-275 of fig. (69) lowered the affinity for 5-HT4 receptor depending on the substitution site. Another common pharmacophore framework like the bond in Fig. (68) was established on the basis of the Regarding the benzene ring in a benzamide series derived compounds described in Fig. (67), which have two from 4-amino-5-chloro-2-methoxybenzoic acid in ML10302 distinctive backbones applicable to 5-HT4 receptor (269), no clear structure-activity relationships have been antagonists. One is a 4-amino-5-chloro-2-methoxybenzoate proposed to account for the effect of aromatic substituents on

O O Cl Cl N N O F N N H H O H N O H N O F 2 H CO 2 3 264 H3C 263 H3C Cisapride Mosapride N N CH3 CH3 S O O NH N N NH N N N O O N N 265 O O 266 267 VB 20B7 BIMU 1 BIMU 8

O Cl N H N H2N O

CH3 268 SC 53116

Fig. (67).

CH O O 3 Cl Cl N O O N O Cl N O CH3 H2N O H2N O CH CH 3 3 H2N O CH 269; ML 10302 3 Ki = 1.07 +/-0.5 nM 270; Ki = 0.26 +/-0.06 nM 271; RS 23597-190 Fig. (68). 5-HT Receptor and Transporter Ligands Current Medicinal Chemistry, 2001, Vol. 8, No. 9 1025

H O X4 N O Y3 N Cl N O O X3 X1 Y2 X Y H2N OCH3 2 1

269; ML 10302 Ki = 1.07 nM X1 X2 X2 X4 Y1 Y2 Y3 Ki (nM)

272 OCH3 H NH2 Cl H H H 39 273 OCH3 H NH2 Cl CH3 H H 10.2 274 OCH3 H NH2 Cl H CH3 H 25.1 275 OCH3 H NH2 Cl CH3 H CH3 2.4 276 Cl H H H H CH3 H >1000 277 NH2 H H H H CH3 H >1000 278 OCH3 H H H H CH3 H 2.3 279 H H H H H H H 31.7 280 OCH3 H H H H H H 8.9 281 OCH2CH3 H H H H H H 2.6 282 Cl H H H H H H >1000 283 H Cl H H H H H 60.8 284 H H Cl H H H 72.8 Fig. (69). pharmacological activity. A study with several mono- or di- HT4 receptor versus 5-HT3, with an IC50 > 1 mM compared substituted derivatives including 276-278 and 280-284 in to BIMU 8 (266). This improvement in selectivity was

H compd R Ki (nM) O N N BIMU 8 63.9 N N 285 ethyl 75.4 CH O 3 286 isopropyl 91.1 287 cyclopropyl 10.2 N R

Fig. (70). fig. (69) showed widely different affinities, and only methoxy expected from replacement of the rigid tropane ring (BIMU and ethoxy substitutions at the ortho position had a 8) by a flexible piperazine moiety. A larger substituent, such favorable effect [161, 162]. as an ethyl (288) or propyl (289) group, led to an increased affinity for the 5-HT4 receptor. However, an isopropyl (290) 2,3-Dihydro-2-oxo-1H-benzimidazole-1-carboxamide or butyl (291) group was too large to improve affinity [fig. linked to 4-methylpiperazine through a two-methylene spacer (71)]. These results suggest the existence of steric can be derived from BIMU 8 (267) by breakdown of the rigid limitations around N-4 of the piperazine moiety for 5-HT4 tropane ring. The structure of this compound was modified agonism. [148, 160, 164]. by substitution at C-3 of the benzimidazolone ring and at N- 4 of the piperazine ring and by changing the length of the A two-methylene alkyl spacer (294) between the amide linker between the amide and the piperazine ring. Any and the piperazine ring seems to be better for 5-HT4 receptor substituents at C-3 of the benzimidazolone ring in fig. (70), affinity compared to a shorter or longer alkyl spacer, as in except for ethyl, isopropyl, and cyclopropyl substituents, led 293 and 295, respectively [fig. (72)] [148, 160]. to weaker affinity for 5-HT4 receptor (285-287) [148]. [125I]Iodine-labeled SB 207710 in fig. (73) (296) with an Compounds with substitution at N-4 of the piperazine ester functionality was prepared and injected into rats, and ring showed a remarkable improvement in selectivity for 5- showed substantial binding in the caudate nucleus, putamen

H compd R Ki (nM) O N N 5-HT4 5-HT3 BIMU 8 63.9 0.5 N N 288 ethyl 17.9 >1000 R O 289 propyl 16.9 >1000 290 isopropyl N 25.2 >1000 291 butyl 34.1 >1000

Fig. (71). 1026 Current Medicinal Chemistry, 2001, Vol. 8, No. 9 Ha et al.

CH3 N Selective agents for the 5-HT transporter have been H developed for therapeutic and diagnostic purposes. Inhibition O N N of the neuronal transporter for serotonin has long been n established as one of the mechanisms of action of numerous N antidepressants. When a 5-HT transporter inhibitor binds to O the transporter, serotonin accumulates in the synapse, since N its release is no longer accompanied by presynaptic transport back into the neuron. These molecular events occur immediately after administration of a 5-HT transporter inhibitor. First-generation antidepressants have a tricyclic compd n Ki (nM) structure and are used as antidepressants and monoamine oxidase inhibitors [167]. 293 1 49.4 294 2 10.2 295 3 39.7 Imipramine (298) [Fig. (74)] and other tertiary amine- containing congeners are potent inhibitors of serotonin Fig. (72). uptake and norepinephrine uptake in the brain. These tricyclic drugs with a tertiary amine are metabolized in vivo and N-desmethylated to the corresponding secondary amines, and globus pallidum of the basal ganglia by brain such as despramine (297). Consequently, the inhibition of autoradiography in regions known to be 5-HT4 receptor-rich norepinephrine uptake in rat brain was also observed when with [3H]GR 311808 [165]. tertiary amine drugs were administered in vivo [168].

N In addition to tricyclic compounds, 5-HT transporter inhibitors with quite different structures have also been O O shown to have high affinity and selectivity. These compounds are fluoxetine (300), fluvoxamine (301), O citalopram (302), paroxetine (303), sertraline (304), and McN-5652 (305), whose structures are shown in Fig. (75) O I* [168].

NH2 296 125 Imaging of 5-HT transporter in the human brain should [ I] SB 207710 help us to understand how changes in this system are related to depressive illness and other psychiatric disorders. In Fig. (73). addition, saturation of 5-HT transporter and its clinical consequences can be evaluated by the direct measurement of binding of 5-HT transporter in patients undergoing various 5-HT TRANSPORTER LIGANDS drug treatments that target these sites. In the past few years, various cocaine analogues and some compounds based on 5-HT transporter is localized on presynaptic axon tricyclic antidepressants have been developed. The most terminals and cell bodies of serotonin neurons. The well- successful of these is [11C]McN-5652 (trans-1,2,3,5,6,10b- known physiological function of this transporter comes into hexahydro-6-[4-(methylthio)phenyl]pyrrolo[2,1-a ]isoquino- play when serotonin is released. This transporter is used in lone) [169], which strongly inhibits 5-HT transporter in rat the mechanism to remove serotonin from the synapse, which brain synaptosomes (Ki = 0.40 nM) and shows moderate has the effect of terminating the action of serotonin in the selectivity toward other monoamine transporters (dopamine synaptic cleft and allowing captured serotonin to be stored and norepinephrine transporters: Ki = 23.5 nM and 1.82 for subsequent reuse [166]. nM, respectively). Studies in the late 1980s showed that

N N N

Cl

HN N N

297; 298; 299; Desipramine Imipramine Clomipramine

Inhibition of Uptake Desipramine Imipramine Clomipramine Serotonin (IC50, nM) 5000 500 100 Norepinephrine (Ki, nM) 1 20 24 Fig. (74). 5-HT Receptor and Transporter Ligands Current Medicinal Chemistry, 2001, Vol. 8, No. 9 1027

O F N O F3C F O NH N O

300; 301; 302; NH2 Fluoxetine Fluvoxamine NC Citalopram

NH S

F

O N O H N Cl O Cl 303; 304; 305; Paroxetine Sertraline McN-5652

IC50 values (nM)

Norepinephrine Serotonin

Fluoxetine 200 15 Fluvoxamine 500 5 Paroxetine 70 1 Sertraline 4 4

Fig. (75). cocaine inhibited the uptake of 5-HT and dopamine as well after 10-20 min. This compound was also accumulated in as norepinephrine. In fact, the potency of the inhibition of regions rich in 5-HT transporter, such as the thalamus and dopamine uptake was greater than that of the inhibition of 5- the brainstem [179]. HT and norepinephrine uptake. As a result of these studies, many cocaine analogues and GBR derivatives were Removing the N-substituent on the tertiary amine of the developed for the 5-HT transporter, but they did not tropane ring, as in the change from 311 to 310, increased the discriminate between dopamine and 5-HT transporter [170- affinity for both the dopamine transporter and 5-HT 174]. transporter. However, the increased affinity for 5-HT transporter was the greater effect [176, 178]. The addition of Some cocaine analogues that may have changed the larger substituents such as ethyl or iodine to the phenyl ring electron density in the tertiary amine of the tropane ring as shown in 306 - 309 improved the affinity and selectivity showed high binding affinity and selectivity. A tertiary for the 5-HT transporter versus dopamine and norepinephrine amino nitrogen is usually basic enough to be protonated at transporters [175-177]. physiological pH. Since the interaction between cocaine and monoamine reuptake may involve electrostatic or hydrogen- Several different radiolabeled ligands have also been bonding interactions, their 5-HT transporter binding developed on the basis of the known backbones. 403U76 in properties should be affected by an N-substituent that fig. (77) (314) was reported as an inhibitor of 5-HT changes the electron density at the nitrogen atom and the transporter and norepinephrine uptake in rat brain lipophilicity of phenyltropanes to improve the selectivity for synaptosomes, with Ki values of 2.1 nM and 55 nM, specific transporters [175]. The secondary amine derivatives respectively [180]. [123I]Iodine-labeled IDAM [5-iodo-2-((2- in Fig. (76) showed better binding affinity and selectivity ((dimethylamino)methyl)-phenyl)thio)benzyl alcohol] (315) than the tertiary amine derivatives. Compounds 306 [175], based on 314 showed very high binding affinity and 307 [176], 308 [177], and 309 [178] showed a high binding selectivity for 5-HT transporter, with Ki = 0.097 nM. A affinity and selectivity for 5-HT transporter. preliminary imaging study of [123I]IDAM in the brain of a baboon by SPECT at 60-120 min after injection showed Compound 309 labeled with 11C at the ester was excellent contrast in the midbrain area where 5-HT evaluated for use as a 5-HT transporter imaging agent. Eight transporter is found in a high density. The lack of an percent of the injected radioactivity was accumulated in the asymmetric center in these agents makes it possible for them brain, and rapid transient equilibrium in vivo was observed to be synthesized easier than other cocaine derivatives [fig. (77)] [181, 182]. 1028 Current Medicinal Chemistry, 2001, Vol. 8, No. 9 Ha et al.

H O H O O N N O O O N O I

306 307 308

O O O H H H3C N I N I N I O O O Cl Cl

309 310 311 O O H3C H3C N O H N O I

312 313

compd 5-HT Dopamine Norepineprine transporter transporter transporter (Ki; nM) (IC50; nM) Ki; nM

306 0.11 16 94 307 0.6 23 144 308 0.81 11.9 29.1 309 0.69 329 148 310 5.45 0.62 4.13 311 44.5 1.12 37 312 2000 23 920 313 38.8 26 1590 Fig. (76).

5-Iodo-2-((2-((dimethylamino)methyl)-phenoxy)benzyl receptor subtype and 5-HT transporter using recently alcohol [ODAM] (316) derived by replacement of the sulfur developed ligands. 5-HT1A receptor ligands have an aryl in IDAM with oxygen has properties very similar to those of piperazine group as an essential element for high binding IDAM with regard to the 5-HT transporter. Specific binding affinity and selectivity. As an aromatic portion, tetralin was observed in the midbrain at 120 min after injection, derivatives showed the highest binding affinity, and their with an equilibrium midbrain-to-cerebellum ratio of 1.50 : stereochemistry and lipophilicity affect the selectivity versus 0.08, which was slightly lower than that for [123I]IDAM dopamine and a 1 receptor. [11C]-and 99mTc-labeled WAY (1.80 : 0.13). Compared to [123I]IDAM, [123I]ODAM 100635 series have revealed the distribution and function of seemed to be better for 5-HT transporter imaging, with about 5-HT1A receptor in vivo. 5-HT1B and 5-HT1D receptors are 30 % higher brain uptake and slower kinetics and similar with regard to their pharmacology, function and metabolism [183, 184]. regional distribution. Tryptamine derivatives based on sumatriptan were studied as ligands for these receptors. SAR studies have suggested that triazolone substitution in the SUMMARY AND PROSPECTS indole group, a two- or three-methylene carbon spacer and a piperazine group are essential pharmacophore elements. In In this review, we have summarized the pharmacophore addition to tryptamine derivatives, isochroman and elements and pharmacological properties of each 5-HT piperazine derivatives also showed high selectivity with

HO N HO N HO N

S S O

Cl I I 314; 315; 316; 403U76 IDAM ODAM Fig. (77). 5-HT Receptor and Transporter Ligands Current Medicinal Chemistry, 2001, Vol. 8, No. 9 1029

good binding affinity. For 5-HT2 receptor, tricyclic [5] Stahl, S.M. J. Affect. Disord., 1998, 51, 215-235. compounds and trazodone derivatives based on a ketanserin moiety show high binding affinity. Separation of the [6] Peroutka, S.J. J. Neurochem., 1993, 60, 408-416. substituted primary amine functionality from the phenyl ring [7] Barrett, J.E.; Vanover, K.E. Psychopharmacology, 1993, by two carbon atoms is needed along with alkoxy oxygens 112, 1-12. at C-2 or C-3 and a hydrophobic substituent at C-4, such as an alkyl halo, alkylthio, or trifluoromethyl, in the main [8] Romero, A.G.; McCall, R.B. Rep. Med. Chem., 1992, 27, aromatic ring. Although these compounds have high binding 21-30. affinities, selectivity versus other receptors was not [9] Fargin, A.; Raymond, J.R.; Lohse, M.J.; Kobilka, B.K.; satisfactory. Radioisotope-labeled ketanserin derivatives Caron, M.G.; Lefkowitz, R.J. Nature, 1988, 335, 358- showed high uptake in the brain with imaging of receptor 360. distribution in humans. Unlike other serotonin receptors, 5- [10] Ishizumi, K.; Kojima, A.; Antoku, F. Chem. Pharm. HT3 receptor is coupled directly to a cation channel with a different chemical structure from most 5-HT receptor ligands. Bull., 1991, 39, 2288-2300. Using the structure of quipazine as a starting point, many [11] El-Bermawy, M.; Raghupathi, R.; Ingher, S.P.; Teitler, modifications were performed, with the result that the loss of M.; Maayani, S.; Glennon, R.A. Med. Chem. Res., 1992, aromaticity in the quinolone ring leads to a loss of binding 2, 88-95. affinity. 5-HT4 receptor ligands have a similar [12] Perrone, R.; Berardi, F.; Colabufo, N.A.; Leopoldo, M.; pharmacophore as the 5-HT1A receptor system with aryl piperazine, which was used for the development of more Tortorella, V.; Fiorentini, F.; Olgiati, V.; Ghiglieri, A.; specific ligands with azaindole, 4-amine-5-chloro-2-methoxy Govoni, S. J. Med. Chem., 1995, 38, 942-949. benzoate and their analogues. For the 5-HT transporter [13] Perrone, R.; Berardi, F.; Leopoldo, M.; Tortorella, V.; system, which plays a role in the development of Fornaretto, M.G.; Caccia, C.; McArthur, R.A. J. Med. antidepressant drugs, secondary amine derivatives such as Chem., 1996, 39, 3195-3202. paroxetine, fluoxetine and setraline with high binding affinity have been used. Cocaine analogues have also been studied [14] Perrone, R.; Berardi, F.; Colabufo, N.A.; Leopoldo, M.; for use as in vivo imaging agents. Unlike dopamine Tortorella, V.; Fornaretto, M.G.; Caccia, C.; McArthur, R.A. J. Med. Chem., 1996, 39, 4928-4934. transporter ligands, removing the N-substituent on the tertiary amine of the tropane ring increases binding affinity to [15] Lopez-Rodriguez, M.L.; Morcillo, M.J.; Rosado, M.L.; 5-HT transporter. IDAM, which is based on a Benhamu, B.; Sanz, A.M. Bioorg. Med. Chem. Lett., 1996, phenylthiobenzyl analogue of 403U76, showed a high 6, 689-694. binding affinity without any asymmetric center, which makes it easier to synthesize. [16] Lopez-Rodriguez, M.L.; Rosado, M.L.; Benhamu, B.; Morcillo, M.J.; Sanz, A.M.; Orensanz, L.; Eneytez, M.E.; Fuentes, J.A.; Manzanares, J. J. Med. Chem., 1996, 39, Although many ligands have been developed for each 5- 4439-4450. HT receptor subtype and 5-HT transporter, few of them have been successfully applied as pharmaceuticals for therapeutic [17] Trumpp-Kallmeyer, S.; Hoflack, J.; Bruinvels, A.; Hibert, and diagnostic purposes in human diseases. Therefore, more M. J. Med. Chem., 1992, 35, 3448-3462. specific and more selective ligands are required for the treatment of such diseases. [18] Perrone, R.; Berardi, F.; Leopoldo, M.; Tortorella, V. J. Med. Chem., 1996, 39, 3195-3202. [19] Gaillard, P.; Carrupt, P.-A.; Testa, B.; Schambel, P. J. ACKNOWLEDGEMENT Med. Chem., 1996, 39, 126-134.

This work was supported by a grant from the Korea [20] Perrone, R. Berardi, F.; Colabufo, N.A.; Leopoldo, M.; Tortorella, V. J. Med. Chem., 1999, 42, 490-496. Research Foundation (KRF-99-042-000079-03004). [21] Kuipers, W.; Kruse, C.G.; van Wijngaarden, I.; Standaar, P.J.; Tulp. M.; Veldman, N.; Spek, A.L.; Ijzerman, A.P. J. REFERENCES Med. Chem., 1997, 40, 300-312. [22] Lopez-Rodriguez, M.L.; Morcillo, M.J.; Fernandez, E.; [1] Wong, E.H.F.; Bonhaus, D.W.; Eglen, R.M. Adv. Exp. Porras, E.; Murcia, M.; Sanz, A.M.; Orensanz, L. J. Med. Med. Biol., 1995, 363, 97-108. Chem., 1997, 40, 2653-2656. [2] Barnes, N.M.; Sharp, T. Neuropharmacology, 1999, 38, [23] Lopez-Rodriguez, M.L.; Morcillo, M.J.; Rovat, T.K.; 1083-1152. Fernandez, E.; Vicente, B.; Sanz, A.M.; Hernandez, M.; [3] (a) Goodnick, P. J., Benitez, A. Expert Opin. Invest. Orenanz, L. J. Med. Chem., 1999, 42, 36-49. Drug 1995, 4, 935-943. (b) Moller, H. J.; Volz, H. P. [24] Yasunaga, T.; Naito, R.; Kontani, T.; Tsukamoto, S. Drugs 1996, 52, 625-638. . Nomura, T.; Yamaguchi, T.; Mase, T. J. Med. Chem., 1997, [4] (a) Hoyer, D.; Schoeffter, P. J. Recept. Res., 1991, 11, 40, 1252-1257. 197-214. (b) Barens, J.M.; Barens, N.M.; Cooper, S.J. [25] Yasunaga, T.; Kimura, T.; Naito, R.; Kontani, T.; Neurosci. Biobehav. Rev., 1992, 16, 107-113. (c) Hartig, Wanibuchi, F.; Yamashita, H.; Nomura, T.; Tsukamoto, P. R. Experimentia Suppl. 1994, 71P, 93-102. 1030 Current Medicinal Chemistry, 2001, Vol. 8, No. 9 Ha et al.

S.; Yamaguchi, T.; Mase, T. J. Med. Chem., 1998, 41, [45] Metcalf, M.A.; Mc Guffin, R.W.; Hamblin, M.W. 2765-2778. Biochem. Pharamcol., 1992., 44, 1917-1920. [26] Neumeyer, J.L.; Grurchelli, F.E.; Fuxe, K.; Ungerstedt, [46] Hartig, P.R.; Hoyer, D.; Humphrey, P.P.A.; Martin, G.R. U.; Corrodi, H. J. Med. Chem., 1974, 17, 1090-1095. Trends. Pharmacol. Sci. 1996, 17, 103-105. [27] Hedberg, M.H.; Johansson, A.M.; Nordvall, G.; [47] Sandou, F.; Hen, R.; Med. Chem. Res., 1994, 4, 16-84. Yliniemela, A.; Li, B.H.; Martin, A.R.; Hjorth, S.; Unelius, L.; Sundell, S.; Hacksell, U. J. Med. Chem., [48] Zifam E.; Fillion, G. Pharmacol. Rev., 1992, 44, 401- 1995, 38, 647-658. 458. [28] Hedberg, M.H.; Jansen, J.M.; Nordvall, G.; Hjorth, S.; [49] Hoyer, D.; Clarke, D.E.; Fozrd, J.R.; Hartig, P.R.; Martin, Unelius, L.; Johansson, A.M. J. Med. Chem., 1996, 39, G.R.; Mylecharane, E.J.; Saxena, P.R.; Humphrey, P.P.A. 3503-3513. Pharmacol. Rev., 1994, 46, 157-203. [29] Hedberg, M.H.; Linnanen, T.; Jansen, J.M.; Nordvall, G.; [50] Glennon, R.A.; Dukat, M. Glennon, R.A.; Westkaemper, Hjorth, S.; Unelius, L.; Johansson, A.M. J. Med. Chem., R.B. Drug News Perspect., 1993, 6, 390-405. 1996, 39, 3503-3513. [51] Adham, N.; Romanienko, P.; Hartig, P.; Weinshank, [30] Osman, S.; Lundkvist, C.; Pike, V.W.; Halldin, C.; R.L.; Bramcheck, T. Mol. Pharmacol., 1992, 41, 1-7. McCarron, J.A.; Swahn, C.-G.; Ginovart, N.; Luthra, S.K.; Bench, C.J.; Grasby, P.M.; Wilkstrom, H.; Barf, T.; [52] Hamblin, M.W.; Metcalf, M.A. Mol. Pharmacol., 1991, Cliff, I.A.; Fletcher, A.; Farde, L. Nucl. Med. Biol., 1996, 40, 143-148. 23, 627-634. [53] Demchyshyn, L.; Sunahara, R.K.; Miller, K.; Teitler, M.; [31] Wilson, A.A.; Inaba, T.; Fischer, N.; Dixon, L.M.; Hoffman, B.J.; Kennedy, J.L.; Seenman, P.; van Tol, Nobrega, J.; DaSilva, J.N.; Houle, S. Nucl. Med. Biol., H.H.M.; Ninzik, H.B.A. Proc. Natl. Acad. Sci. U.S.A., 1998, 25, 769-776. 1992, 89, 5522-5526. [32] Osman, S.; Lundkvist, C.; Pike, V.W.; Halldin, C.; [54] Jin, H.; Oksenberg, D.; Ashkenazi, A.; Peroutka, S.J.; McCarron, J.A.; Swahn, C.-G.; Farde, L. Ginovart, N.; Duncan, A.M.V.; Rozmahel, R.; Yang, Y.; Mengod, G.; Luthra, S.K.; Bench, C.J.; Grasby, P.M. Nucl. Med. Biol., Palacios, J.M.; O’Dowd, B.F. J. Biol. Chem., 1992, 267, 1998, 25, 215-223. 5735-5738. [33] Ito, H.; Halldin, C.; Farde, L. J. Nucl. Med., 1999, 40, [55] Levy, F.O.; Gudermann, T.; Perez-Reyes, E.; Birnbauman, 102-109. M.; Kaumann, A.J.; Birnbaumann, L. J. Biol. Chem., 1992, 267, 7553-7562. [34] Farde, L.; Ito, H.; Swahn, C.-G.; Pike, V.W.; Halldin, C. J. Med. Chem., 1998, 39, 1965-1971. [56] Hartig, P.R.; Hoyer, D.; Humphrey, P.P.A.; Martin, G. Trends Pharmacol. Sci., 1996, 17, 103-105. [35] Le Bars, D.; Lemaire, C.; Ginovart, N.; Plenevaux, J.; Aerts, J.; Brihaye, C.; Hassoun, W.; Leviel, V.; [57] Pauwels, P.J. CNS Drugs Rev., 1996, 2, 415-428. Mekhsian, P.; Weissmann, D.; Pujol, J.F.; Luxen, A.; [58] Moloney, G.P.; Martin, G.R.; Mathews, N.; Milne, A.; Comar, D. Nucl. Med. Biol., 1998, 25, 343-350. Hobbs, H.; Dodsworth, S.; Sang, P.Y.; Knight, C.; [36] Houle, S.; Wilson, A.A.; Inaba, T.; Fisher, N.; DaSilva, Williams, M.; Maxwell, M.; Glen, R.C. J. Med. Chem., J.N. Nucl. Med. Comm., 1997, 18, 1130-1134. 1999, 42, 2504-2526. [37] Sandell, J.; Halldin, C.; Hall, H.; Thorberg, S.-O.; [59] Halazy, S.; Perez, M.; Fourrier, C.; Pallard, I.; Pauwels, Werner, T.; Sohn, D.; Sedvall, G.; Farde, L. Nucl. Med. P.J.; Palmier, C.; John, G.W.; Valentin, J.-P.; Bonnafous, Biol., 1999, 26, 159-164. R.; Martinez, J. J. Med. Chem., 1996, 39, 4920-4927. [38] Hartig, P.R.; Branckeck, T.A.; Weinshank, R.L. Trends [60] Briley, M.; Moret, C. Clin. Neuropharmacol., 1993, 16, Pharmacol. Sci. 1992, 13, 152-159. 387-420. [39] Kung, H.F.; Frederick, D.; Kim, H.J. Synapse, 1996, 24, [61] Lamothe, M.; Pauwels, P.J.; Belliard, K.; Belliard, K.; 273-281. Schambel, P.; Halazy, S. J. Med. Chem., 1997, 40, 3542- 3550. [40] Alberto, R.; Schibli, R.; Egli, A.; Schubiger, A.P. J. Am. Chem. Soc., 1998, 120, 7987-7988. [62] Jonard-Lebrun, C.; Pauwels, P.J.; Palmier, C.; Moret, C.; Chopin, P.; Perez, M.; Marien, M.; Halazy, S. J. Med. [41] Alberto, R.; Schibli, R.; Schubiger, A.P. J. Am. Chem. Chem., 1997, 40, 3974-3978. Soc., 1999, 121, 6076-6077. [63] Berg, S.; Larsson, L.-G.; Renyl, L.; Ross, B.S.; Thorberg, [42] Weinshank, R.L.; Zgombick, J.M.; Macchi, M.J.; S.-O.; Thorell-Svamtesson, G. J. Med. Chem., 1998, 41, Brancheck, T.A.; Hartig, P.R. Proc. Natl. Acad. 1934-1942. Sci.U.S.A. 1992, 89, 3630-3634. [64] Ismaiel, A.M.; Dukat, M.; Law, H.; Kamboj, R.; Fan, E.; [43] Glennon, R.A. Mol. Pharmacol., 1996, 49, 198-206. Lee, K.H.; Mazzocco, L.; Buekschkens, D.; Teitler, M.; Pierson, M.E.; Glennon, R.A. J. Med. Chem,. 1997, 40, [44] Oksenberg, D.A. Nature, 1992, 360, 161-163. 4415-4419. 5-HT Receptor and Transporter Ligands Current Medicinal Chemistry, 2001, Vol. 8, No. 9 1031

[65] Connor, H.E., Fenuik, W.; Beattie, D.T.; North, P.C.; [82] Tenbrink, R.E.; Bergh, C.L.; Duncan, J.C.; Harris, D.W.; Oxford, A.W.; Saynor, D.A.; Humphrey, P.P.A. Huff, R.M.; Lahti, R.A.; Lawson, C.F.; Lutzke, B.S.; Cephalagia, 1997, 17, 145-152. Martin, I.J.; Rees, S.A. J. Med. Chem., 1996, 39, 2435- 2437. [66] Glen, R.C.; Martin, G.R.; Hill, A.P.; Hyde, R.M.; Woodland, P.M.; Salmon, J.A.; Buckingham, J.; [83] Ennis, M.D.; Ghazal, N.B.; Hoffman, R.L.; Smith, M.W.; Robertson, A.D. J. Med. Chem., 1995, 38, 3566-3580. Schlachter, S.K.; Lawson, C.F.; Im, W.B.; Pregenzer, J.F.; Svensson, K.A.; Lewis, R.A.; Hall, E.D.; Sutter, D.M.; [67] Street, L.J.; Baker, R.; Davey, W.B.; Guilblib, A.R.; Jelly, Harris, L.T.; McCall, R.B. J. Med. Chem., 1998, 41, 2180- R.A.; Reeve, A.J.; Routledge, H.; Sternfeld, F.; Watt, 2183. A.P.; Beer, M.S.; Middlemiss, D.N.; Noble, A.J.; Stanton, J.A.; Scholey, K.; Hargreaves, R.J.; Sohal, B.; Graham, [84] Glennon, R.A. Psychopharmacology, 1980, 68, 155- M.I.; Matassa, V.G. J. Med. Chem., 1995, 38, 1799-1810. 158. [68] Ngo, J.; Rabasseda, X.; Castaner, J. Drugs of the Future, [85] Colpaert, F.C.; Niemegeers, C.J.E.; Janssen, P.A.J. J. 1997, 22, 221-224. Pharamcol. Exp. Ther., 1982, 16, 206-214. [69] Goadsby, P.J. CNS Drugs, 1998, 10, 271-286. [86] Aaron, P.M.; Danuta, M.-L.; Matthew, A.P.; David, B.W.; David, L.N.; David, E.N. J. Med. Chem., 1996, 39, [70] Cumberbatch, M.J.; Hill, R.; Hargreaves, R.J. Eur. J. 2953-2961. Pharmacol., 1997, 328, 37-40. [87] Matthew, A.P.; Danuta, M.L.; Virginia, L.L.; David, [71] Castro, J.L.; Street, L.J.; Guiblin, A.R.; Jelly, R.A.; L.N.; David, E.N. J. Med. Chem., 1998, 41, 5148-5149. Russell, M.G.; Sternfeld, F.; Beer, M.S.; Stanton, J.A.; Matassa, V.G. J. Med. Chem., 1997, 40, 3497-3500. [88] Giannangeli, M.; Cazzolla, N.; Luparini, M.R.; Magnani, M.; Mabilia, M.; Picconi, G.; Tomaselli, M.; Baiocchi, L. [72] Chambers, M.S.; Street, L/J.; Goodacre, S.; Hobbs, S.C.; J. Med. Chem., 1999, 42, 336-345. Hunt, P.; Jelly, R.A.; Matassa, V.G.; Reeve, A.J.; Sterfeld, F.; Beer, M.S.; Stanton, J.A.; Rathbone, D.; Watt, A.P.; [89] Martin, G.R.; Humphrey, P.P.A. Neuropharmacology, MacLeod, A.M. J. Med. Chem., 1999, 42, 691-705. 1994, 33, 261-273 . [73] Niel, M.B.; Collins, I.; Beer, M.S.; Broughton, H.B.; [90] Boess, F.G.; Martin, I.L. Neuropharmacology, 1994, 33, Cheng, S.K.F.; Goodacre, S.C.; Heald, A.; Locker, K.L.; 275-371. Macleod, A.M.; Morrison, D.; Moyes, C.R.; O’Connor, D.; Pike, A.; Roweley, M.; Russel, M.G.N.; Sohal, B.; [91] Metwally, K.A.; Dukat, M.; Egan, C.T.; Smith, C.; DuPre, Stanton, J.A.; Thomas, S.; Verrier, H.; Watt, A.P.; Castro, A.; Ganthier, C.B.; Teitler, M.; Glennon, R.A. J. Med. J.L. J. Med. Chem., 1999, 42, 2087-2104. Chem., 1998, 41, 5084-5093. [74] Castro, J.; Collins, I.; Russell, M.G.N.; Watt, A.P.; Sohal, [92] Pertz, H.; Milhahn, H.-C.; Eich, E. J. Med. Chem., 1999, B.; Rathbone, D.; Beer, M.S.; Stanton, J.A. J. Med. Chem., 42, 659-668. 1998, 41, 2667-2670. [93] Mach, R.; Jackson, J.R.; Leudtke, R.R.; Ivins, K.J.; [75] MacLeod, A.M.; Street, L.J.; Reeve, A.J.; Jelly, R.A.; Molinolf, P.B.; Ehrenkaufer, R.L. J. Med. Chem., 1992, Sternfeld, F.; Beer, M.S.; Stanton, J.A.; Watt, A.P.; 35, 423-430. Rathbone, D.; Matassa, V.G. J. Med. Chem., 1997, 40, 3501-3503. [94] Hoyer, D.; Clarke, D.E.; Fozard, J.R.; Hartig, P.R.; Martin, G.R.; Mylecharane, E.J.; Saxena, P.R.; Humphrey, [76] Russel, M.G.; Matassa, V.G.; Pengilley, R.P.; Niel, M.B.; P.P. Pharmacol. Rev., 1994, 46, 157-203. Sohal, B.; Watt, A.P.; Hitzel, L.; Beer, M.S.; Stanton, J.A.; Castro, J.L. J. Med. Chem., 1999, 42, 4981-5001. [95] Forbes, I.T.; Ham, P.; Booth, D,H.; Martin, R.T.; Thompson, M.; Baxter, G.T.; Blackburn, T.P.; Glen, A.; [77] Castro, J.L; Street, L.J.; Guiblin, A.R.; Jelly, R.A.; Kennett, G.A.; Wood, M.D. J. Med. Chem., 1995, 38, Russell, M.G.N.; Sternfeld, F.; Beer, M.S.; Stanton, J.A.; 2524-2530. Matassa, V.G. J. Med. Chem., 1997, 40, 3497-3500. [96] Nozulak, J.; Kalkman, H.O.; Floerscheim, P.; Hoyer, D.; [78] Sternfeld, F.; Guiblin, A.R.; Jelly, R.A.; Matassa, V.G.; Schoeffer, P.; Buerki, H.R. J. Med. Chem., 1995, 38, 28- Reeve, A.J.; hunt, P.A.; Beer, M.S.; Heald, A.; Stanton, 33. J.A.; Sohal, B.; Watt, A.P.; Street, L.J. J. Med. Chem., 1999, 42, 677-690. [97] Forbes, I.T.; Jones, G.E.; Murphy, O.E.; Holland, V.; Baxter, G.S. J. Med. Chem., 1995, 38, 855-857. [79] Glennon, R.A.; Hong, S.S.; Bondarev, M.; Law, H.; Dukat, M.; Rakhit, S.; Power, P.; Fan, E.; Kinneau, D.; [98] Fludzinski, P.; Wittenauer, L.A.; Schenck, K.W. J. Med. Kamboj, R.; Teitler, M.; Herrick-Davis, K.; Smith, C. J. Chem., 1986, 29, 2415-2418. Med. Chem., 1996, 39, 314-322. [99] Audia, J.E.; Evrard, D.A.; Murdoch, G.R.; Droste, J.J.; [80] Barf, T.J.; Boer, P.; Wikstrom, H. J. Med. Chem., 1996, 39, Nissen, J.S.; Schenck, K.W.; Fludzinski, P.; Lucaites, 4717-4726. V.L.; Nelson, D.L.; Cohen, M.L. J. Med. Chem., 1996, 39, 2773-2780. [81] Xu, Y.; Schaus, J.M.; Walker, C.; Krushinski, J.; Adham, N.; Zgombick, J.M.; Liang, S.X.; Kohlman, D.T.; Audia, [100] Forbes, I.T.; Dubbs, S.; Duckworth, D.M.; Ham, P.; J.E. J. Med. Chem., 1999, 42, 526-531. Jones, G.E.; King, F.D.; Saunders, D.V.; Blaney, F.E.; 1032 Current Medicinal Chemistry, 2001, Vol. 8, No. 9 Ha et al.

Naylor, C.B.; Baxter, G.S.; Blackburn, T.P.; Kennett, [121] Dukat M.; Abdel-Rahman A.A.; Ismaiel A.M.; Ingher S.; G.A.; Wood, M.D. J. Med. Chem., 1996, 39, 4966-4977. Teitler M.; Gyermek L.; Glenmon R.A. J. Med. Chem., 1996, 39, 4017-4021. [101] Tan, P.-Z.; Baldwin, R.M.; Dyck, C.H.; Al-Tikriti, M.; Roth, B.; Khan, N.; Charney, D.S.; Innis, R.B. Nucl. Med. [122] Smith W.W.; Sancilio L.F.; Owera-Atepo J.B.; Naylor Biol., 1997, 26, 601-608. R.J.; Lamberg L. J. Pharm. Pharmacology, 1988, 40, 301-302. [102] Lemaire, C.; Cantineau, R.; Guillaume, M.; Plenevaux, A,; Christiaens, L. J. Med. Chem., 1991, 32, 2266-2272. [123] Ireland S.J.; Tyers M.B. Br. J. Pharmacol., 1987, 90, 229- 238. [103] Biver, F.; Lotstra, F.; Monclus, M.; Dethy, S.; Damhaut, P.; Wikler, D.; Luxen, A.; Goldman, S. Nucl. Med. Biol., [124] King F.D.; Sanger G.J. Drugs of the Future, 1989, 14, 1997, 24, 357-360. 875. [104] Baron, J.C.; Samson, Y.; Comar, D.; Crouzel, C.; Deniker, [125] Heidempergher F.; Pillan A.; Pincirol V.; Vaghi F.; P.; Agid, Y. Rev. Neurol., 1985, 141, 537-545. Arrigoni C.; Varasi M. J. Med. Chem., 1997, 40, 3369- 3380. [105] Berridge, M.; Comar, D.; Crouzel, C.; Baron, J.C. J. Labelled Compd. Radiopharm., 1983, 20, 73-78. [126] Lpez-Radriguez M.L.; Benhaum B.; Morcillo M.J.; Tejade I.D.; Orensanz L.; Alfaro M.J.; Martin M.I. J. Med. Chem., [106] Ito, H.; Nyberg, S.; Halldin, C.; Lundkvist, C.; Farde, L. 1999, 42, 5020-5028. J. Nucl. Med., 1998, 39, 208-214. [127] Orjales A.; Mosquera R.; Labeaga L.; Rodes R. J. Med. [107] Burns, H.D.; Dannals, R.F.; Langstrom, B. J. Nucl. Med., Chem., 1997, 40, 586-593. 1984, 25, 1222-1227. [128] Cappelli A.; Anzimi M.; Vomero S.; Canullo L.; [108] Wagner, H.N.; Burns, H.D.; Dannals, R.F. Science, 1983, Mennuni L.; Makovec F.; Doucet E.; Hamon M.; 221, 1264-1266. Menziani C.; Bruni G.; Donati A. J. Med. Chem., 1999, 42, 1556-1575. [109] Amokhtari, M.; Anderson, K.; Ibazizene, M.; Dhilly, M.; Dauphin, F.; Darre, L. Nucl. Med. Biol., 1998, 25,517- [129] Dukat M.; Miller K.; Teitler M.; Glemmon R.A. Med. 522. Chem. Res., 1999, 11, 271-276. [110] Petit-Taboue, M.-C.; Landeau, B.; Barre, L.; Onfroy, M.- [130] Kilpatrick, G.J.; Bunce, K.T.; Tyers, M.B. Med. Res. Rev., C.; Noel, M.-N.; Baron, J.C. J. Nucl. Med., 1999, 40, 25- 1990, 10, 441-475. 32. [131] Rault, S.; Lancelot, J.-C.; Prunier, H.; Robba, M.; Renard, [111] Samnick, S.; Brandau, W.; Nolken, G.; Gerhards, H.J.; P.; Delagrange, D.; Pfeitter, B.; Caignard, D.-H.; Hamon, Schober, O. Nucl. Med. Biol., 1997, 24, 295-303. M. J. Med. Chem., 1996, 39, 2068-2080. [112] Samnick, S.; Remy, N.; Ametamey, S.; Bader, J.B.; [132] Prunier, H.; Rault, S.; Lancelot, J.C.; Robba, M.; Renard, Brandau, W.; Kirsch, C.-M. Life Sci., 1998, 22, 2001- P.; Delagrange, P.; Pfeiffer, B.; Caingnard, D.H.; Misslin, 2013. R.; Hamon, M. J. Med. Chem., 1997, 40, 1808-1819. [113] Baeken, C.; D’haener, H.; Flamen, D.; Mertens, J.; [133] Camiani, G.; Cappelli, A.; Nacci, V.; Arnzini, M.; Terriere, D.; Chavatte, K.; Boumon, R.; Bossuyt, A. Eur. Vomero, S.; Hamon, M.; Cagnotto, A.; Fracasso, C.; J. Nucl. Med., 1998, 25, 1617-1622. Uboldi, C.; Caccia, S.; Consolo, S.; Mennini, T. J. Med. Chem., 1999, 40, 3670-3678. [114] Johannsen, B.; Pietzsch, H.-J.; Scheunemann, M.; Kretzschmar, M.; Seifert, S.; Brust, P.; Syhre, R.; Spies, H. [134] Camiani, G.; Morelli, E.; Gemma, S.; Nacci, V.; Butini, S.; J. Labelled Compd. Radiopharm., 1999, 42 (suppl 1), Hamon, M.; Novellino, E.; Greco, G.; Cagnotto, A.; S345-S347. Goegom, M.; Cerro, L.; Valle, F.D.; Fracasso, C.; Caccia, S.; Mennini, T. J. Med. Chem., 1999, 42, 4362-4379. [115] Johannsen, B.; Scheunemann, M.; Spies, H.; Brust, P.; Wober, J.; Syhre, R.; Pietxsch, H.-J. Nucl. Med. Biol., [135] Cappelli, A.; Arnzini, M.; Vomero, S.; Mennuni, L.; 1996, 23, 429-438. Makovec, F.; Doucet, E.; Hamon, M.; Bruni, G.; Romeo, M.R.; Menziani, M.C.; Langer, T. J. Med. Chem., 1998, [116] Pietxsch, H.-J.; Scheunemann, M.; Kretzschmar, M.; Elx, 41, 728-741. S.; Pertz, H.H.; Seifert, S.; Brust, P.; Spies, H.; Syhre, R.; Johannsen, B. Nucl. Med. Biol., 1999, 26, 865-875. [136] Ishiwata, K.; Saito, N.; Yanagawa, K.; Furuta, R.; Ishii, S.-I.; Kiyosawa, M.; Homma, Y.; Ishii, K.; Suzuki, F.; [117] Seifert, S.; Pietzsch, H.-J.; Scheunemann, M.; Spies, H.; Senda, M. Nucl. Med. Biol., 1996, 23, 285-290. Syhre, R.; Johannsen, B. Appl. Radiat. Isot., 1998, 49, 5- 11. [137] Delagrange, P.; Emerit, M.B.; Merahi, N.; Abraham, C.; Morain, P.; Rault, S.; Renard, P.; Pfeiffer, B.; Guardiola- [118] Derkach V.; Suprenant A.; North R.A. Nature, 1989, Lemaitre, B.; Hamon, M. Eur. J. Pharmacol., 1996, 316, 339, 706-709. 195-203. [119] Butler A.; Hill J.M.; Ireland S.J.; Jordan C.C.; Tyers M.B. [138] Besret, L.; Dauphin, F.; Guillouet, S.; Dhilly, M.; Br. J. Pharmacol., 1988, 94, 397-412. Gourand, F.; Blaizot, X.; Young, A.R.; Petit-Taboue, [120] Sanget G.J.; Nelson G.R. Eur J. Pharmacol., 1989, 159, M.C.; Mickala, P.; Becquerel, A.; Rault, S.; Barre, L.; 113-124. Baron, J.C. Life Science, 1998, 62, 115-129. 5-HT Receptor and Transporter Ligands Current Medicinal Chemistry, 2001, Vol. 8, No. 9 1033

[139] Paulis, T.; Hwelwtt, W.A.; Schmidt, D.E.; Mason, N.S.; [159] Yang, D.; Soulier, J.H.; Sicsic, S.; Mathe-Allainment, M.; Trivedi, B.L.; Ebert, M.H. Eur. J. Med. Chem., 1997, 32, Bremont, B.; Croci, T.; Caramone, R.; Langlois, M. J. 385-396. Med. Chem., 1997, 40, 608-621. [140] Hewlett, W.A.; Trivedi, B.L.; Zhang, Z.-J.; Paulis, T.; [160] Cohen, M.C.; Bloomquist, W.; Gidda, J.S.; Lacefield, W. Schmidt, D.E.; Lovinger, D.M.; Ansari, M.S.; Ebert, M.H. J. Pharmacol. Exp. Ther., 1990, 254, 350. J. Pharmacol. Exp. Ther., 1999, 288, 221-231. [161] Hoh, K.; Sato, N.; Murozono, T. Drugs of the Future, [141] Hewlett, W.A.; Fridman, S.; Paulis, T.; Ebert, M.H. Prog. 1999, 24, 155-157. Neuro-Psychopharmacol. Biol. Psychiat., 1998, 22, 397-410. [162] Croci, T.; Langlois, M.; Mennini, T.; Landi, M.; Manara, L. Br. J. Pharmacol., 1995, 114, 3829. [142] Hewlett, W.A.; Schmidt, D.E.; Mason, N.S.; Trivedi, B.L.; Ebert, M.H.; Paulis, T. Nucl. Med. Biol., 1998, 25, [163] Soulier, J.L.; Yang, D.; Bremont, B.; Guzzi, U.; Langlois, 141-153. M. J. Med. Chem., 1997, 40, 1755-1761. 143] Hoyer, D.; Clarke, D.E.; Fozard, J.R.; Hartig, P.R.; [164] Mulligan, R.S.; Halldin, C.; Hall, H.; Pilowsky, L.; Ell, Martin, G.R.; Mylecharane, E.J.; Saxena, P.R.; Humphrey, P.J.; Farde, L.; Pike, V.W. J. Labelled Compd. P.P.A. Pharmacol. Rev., 1994, 46, 157-203. Radiopharm., 1999, 42, Suppl. 1. S417-419. [144] Ford, A.P.D.W.; Clark, D.E. Med. Res. Rev., 1993, 13, [165] Waeber, C.; Sebben, M.; Grossman, C.; Javoy-Agid, F.; 633-662. Bockaet, J.; Dumuis, A. Neuroreceptor, 1993, 4, 1239. [145] Dumuis, A.; Bouheld, R.; Sebben, M.; Cory, R.; Bockaert, [166] Stahi, S.M. J. Affect Disord., 1998, 51, 215-235. J.A. Mol. Pharmacol., 1988, 34, 880. [167] Frazer, A. J. Clin. Psychiat., 1997, 58, suppl 6, 9-25. [146] Ullmer, C.; Schumuck, K.; Kalkman, J.O.; Lubbert, H. FEBS Lett., 1995, 370, 215-221. [168] Wong, D.T.; Bymaster, F.P. Neurochemistry in Clinical Application, 1995, 77-95. [147] Laramie, M.; Gaster, F.D. Med. Res. Rev., 1997, 17, 163- 214. [169] Suehiro, M.; Musachio, J.L.; Dannals, R.F.; Mathews, W.B.; Ravert, H.T.; Scheffel, U.; Wagner Jr, H.N. Nucl. [148] Tapia, I.; Alonso-Cires, L.; Mosquera, R.; Labeaga, L.; Med. Biol., 1995, 22, 543-545. Innerarity, A.; Orjales, A. J. Med. Chem., 1999, 42, 2870- 2880. [170] Meltzer, P.C.; Liang, A.Y.; Blundell, P.; Gonzalez, M.D.; Chen, Z.; George, C.; Madras, B.K. J. Med. Chem., 1997, [149] Fagni, L.; Dumuis, A.; Sebben, M.; Bockaert, J. Br. J. 40, 2661-2673. Pharmacol., 1992, 105, 973-979. [171] Kozikowski, A.P.; Araldi, G.L.; Prakash, K.R.C.; Zhang, [150] Egler, R.M.; Alvarez, R.; Johnson, L.G.; Wong, E.H.F. M.; Johnson, K.M. J. Med. Chem., 1998, 41, 4973-4982. Br. J. Pharmacol., 1993, 108, 376-382. [172] Keverline-Frantz, K.I.; Boja, J.W.; Kuhar, M.J.; Abraham, [151] Cohen, M.L.; Susemichel, A.D.; Bloomquist, W.; P.; Burgess, J.P.; Lewin, A.H.; Carroll, F.I. J. Med. Robertson, D.W. Gen. Pharmacol., 1994, 25, 1143- Chem., 1998, 41, 247-257. 1148. [173] Dutta, A.K.; Xu, C.; Reith, M.E. J. Med. Chem., 1996, 39, [152] Gale, J.D.; Grossman, C.J.; Whitehead, J.W.F.; Oxford, 749-756. A.W.; Bunce, K.T.; Humphrey, P.P.A. Br. J. Pharmacol., 1994, 111, 332-338. [174] Kotian, P.; Mascarella, S.W.; Abraham, P.; Lwein, A.H.; Boja, J.W.; Kuhar, M.J.; Carroll, J.W. J. Med. Chem., [153] Wardel, K.A.; Ellis, E.S.; Boxter, G.S.; Kennett, G.A.; 1996, 39, 2753-2763. Gaster, L.M.; Sanger, G.J. Br. J. Pharmacol., 1994, 112, 789-794. [175] Davies, H.M.L.; Kuhn, L.A.; Thornley, C.; Matasi, J.J.; Sexton, T.; Childers, S.R. J. Med. Chem., 1996, 39, 2554- [154] Clark, R.D.; Jahangir, A.; Langston, J.A.; Weinhardt, 2558. K.K.; Miller, A.B.; Leung, E.; Egler, R.M. Bioorg. Med. Chem. Lett., 1994, 20, 2477. [176] Blough, B.E.; Abraham, P.; Lewin, A.H.; Kuhar, M.J.; Boja, J.W.; Carroll, F.I. J. Med. Chem., 1996, 39, 4027- [155] Wardle, K.A.; Bingham, S.; Ellis, E.S.; Gaster, L.M.; 4035. Rushart, B.; Smith, M.I.; Sanger, G.J. Br. J. Pharmacol., 1996, 118, 665-670. [177] Neumeyer, J.L.; Tamagnan, G.; Wang, S.; Gao, Y.; Milius, R.A.; Kula, N.S.; Baldssarini, R.J. J. Med. Chem., 1996, [156] Clark, R.D.; Jahangir, A.; Flippen, L.A.; Miller, A.B.; 39, 543-548. Leung, R.M.; Bonhaus, D.W.; Wong, E.H.F.; Johnson, L.G.; Eglen, R.M. Bioorg. Med. Chem. Lett., 1995, 5, [178] Blough, B.E.; Abraham, P.; Mills, A.C.; Lewin, A.H.; 2119. Boja, J.W.; Scheffel, U.; Kuhar, M.J.; Carroll, F.I. J. Med. Chem., 1997, 40, 3861-3864. [157] Schaus, J.M.; Thompson, D.C.; Bloomquist, W.E.; Susenichel, A.D.; Cohen, M.L. J. Med. Chem., 1998, 41, [179] Sandell, J.; Helfenbein, J.; Halldin, C.; Chou, Y.-W.; 1943-1955. Emond, P.; Guilloteau, D.; Farde, L. J. Labelled Compd. Radiopharm., 1999, 42, Suppl. 1. S51-S53. [158] King, F.; Dabbs, S.; Bermudez, J.; Sanger, G. J. Med. Chem., 1990, 33, 2942. 1034 Current Medicinal Chemistry, 2001, Vol. 8, No. 9 Ha et al.

[180] Ferris, R.M.; Brieaddy, L.; Metha, N.; Hollingsworth, [183] Zhuang, Z-P., Choi, S.-R.; Hou, C.; Kung, H.F. J. M.E.; Rigdon, G.; Wang, C.; Soroko, F.; Wastila, W.; Labelled Compd. Radiopharm., 1999, 42, Suppl. 1. Cooper, B. J. Pharm. Pharmacol., 1995, 47, 775-781. S357-S359. [181] Oya, S.; Kung, M.-P.; Hou, C.; Acton, P.D.; Mu, M.; [184] Zhuang, Z.-P., Choi, S.-R.; Hou, C.; Kung, H.F. Eur. J. Kung, H.F. J. Labelled Compd. Radiopharm., 1999, 42, Nucl. Med., 1999, 26, 1359-1362. Suppl. 1. S57-S59. [182] Oya, S.; Kung, M.-P.; Hou, C.; Acton, P.D.; Mu, M.; Kung, H.F. J. Med. Chem., 1999, 42, 333-335.