It should be fairly evident from the foregoing discussion that each of the above general approaches has as its basis the requirement that a certain amount of information be already in hand. What is often required to achieve selectivity is some sort of structural template that can be exploited. Frequently, other than for the structure of 5-HT itself, such a lead structure might be unavailable; in such cases, a structural template must be identified. Our work with serotonin dates back more than 30 yr to the early 1970s; put in proper perspective, these might be considered the doldrum-days of serotonin research. Although serotonin had been seemingly implicated as being involved in various psychiatric and cardiovascular disorders, proper tools (assay methods, chemical entities) were not readily available. In fact, some investigators still considered serotonin a "putative neurotransmitter." As a consequence, little work was being conducted with serotonin. At that time, it was recognized that there existed at least two types of peripheral serotonin receptor (the so-called D- and M-type 5-HT receptors) (reviewed in ref. 9), but far less was known about central 5-HT receptors. What follows is a discussion of the identification of certain phenylalkylamine derivatives as 5-HT2 ligands (some of this work has been previously reviewed in refs. 10 and 11); this is an example of where selective ligands pre-existed in the primordial pool but were unrecognized as such because of the lack of appropriate pharmacological methodologies.
Our earliest foray into the world of serotonin was associated with studies aimed at elucidation of the mechanism of action of hallucinogenic agents. Certain classical tryptaminergic hallucinogens, such as Y,Y-dimethyltryptamine (DMT; 2 in Fig. 1), its 4-hydroxy and 5-methoxy analogs (psilocin and 5-OMe DMT [3 and 4 in Fig. 1], respectively), and (+)lysergic acid diethylamide (LSD; 5), because of their obvious structural similarity to serotonin (1) were thought to act via a serotonergic mechanism (12). Despite this supposition, whether hallucinogenic agents behaved as serotonin agonists or antagonists was to remain controversial for the next several decades (13).
Other hallucinogenic agents, such as the phenylalkylamines 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM; 6) and mescaline (7), reportedly produced somewhat similar effects in humans, but were structurally distinct in that they did not possess a tryptaminergic nucleus. Actually, because of their structural similarity to the catecholamine neurotransmitters, they were speculated by some investigators to act via either an adrenergic or dopaminergic mechanism (see discussion in ref. 13). Two of the first questions we set out to answer were as follows: (1) Do phenylalkylamine hallucinogens bind at serotonin receptors? and (2) Can examples of tryptamine and phenylalkylamine hallucinogens produce similar behavioral effects? Because of its popularity at the time and because radi-oligand-binding assays for serotonin receptors had not yet been developed, we initially utilized an isolated peripheral tissue preparation—rat fundus tissue— that was thought to be activated by serotonin via a single type of serotonin receptor (i.e., a D-type serotonin receptor). Serotonin, even at very low concentrations, contracts fundus tissue in a robust manner that can be reliably and accurately measured. Using this technique, it was demonstrated that examples of both classes of hallucinogen (tryptamine and phenylalkylamine hallucinogens) bind at serotonin receptors (reviewed in ref. 10). Using a drug discrimination paradigm with rats trained to discriminate either 5-OMe DMT, LSD, DOM or mescaline from the vehicle, we showed that stimulus generalization occurred among this group of agents independent of which was used as the training drug. This was the first indication that these structurally different agents were capable of producing similar stimulus effects in rats. (The drug discrimination paradigm is a behavioral procedure where animals, typically rats, are trained to make one of several possible responses when administered a training drug; in tests of stimulus generalization, test agents are administered to the same animals to determine if they produce an effect similar to that of the training drug [i.e., to determine if substitution occurs]. Where stimulus generalization or substitution occurs, the test drug is considered to be producing stimulus effects similar to those of the training drug.) Next, using rats trained to discriminate DOM from vehicle, we examined a large number of arylalkylamines (i.e., tryptamines and phenylalkylamines), formulated structure-activity relationships, and found that the stimulus potencies of these agents in the rat behavioral paradigm paralleled their human hallucinogenic potencies (in those cases where human data were available in the literature). With evidence from the isolated tissue assays that these compounds might be acting as serotonergic agents, we reasoned that there might exist a relationship between their stimulus or hallucinogenic potency and their affinity for serotonin receptors. Indeed, this was demonstrated to be the case (10). Both the hallucinogenic potency and stimulus potency of various hallucinogens were significantly correlated with their affinity for the serotonin receptors of the rat fundus preparation. It was tentatively concluded that the tryptamine and phenylalkylamine hallucinogens were acting as serotonin agonists. Well and good, but how can the action of centrally acting hallucinogenic substances be adequately, and satisfy-ingly, explained by their affinity for rat gut serotonin receptors? Apart from coincidence, the only logical conclusion was that the serotonin receptors present in the fundus preparation must be similar to those found in the brain. However, brain serotonin receptors had yet to be investigated in detail.
At about this same time, two novel serotonergic agonists were reported: 1-(3-triflurormethyl-phenyl)piperazine (TFMPP; 8) by Fuller and colleagues at Eli Lilly (14) and 8-hydroxy-2-(N,N-di-n-propylaminotetralin) (8-OH DPAT; 9) by Hacksell and co-investigators in Sweden (15). We obtained samples of these two agents and administered them to the DOM-trained animals in tests of stimulus generalization. If hallucinogens are acting as serotonin agonists, animals trained to discriminate a hallucinogen should recognize these new serotonin agonists. This did not happen. Substitution (stimulus generalization) normally occurs when two agents produce similar stimulus effects in animals. What the results told us was that even though each of these agents might be serotonin agonists, they produce dissimilar stimulus effects in animals. We eventually trained separate groups of rats to discriminate TFMPP and 8-OH DPAT from the vehicle. Not only did substitution fail to occur when hallucinogens were administered to these animals, but the TFMPP-trained animals failed to recognize 8-OH DPAT, and 8-OH DPAT-trained animals failed to recognize TFMPP. Evidently, the hallucinogens, TFMPP, and 8-OH
DPAT—although all being purported serotonin agonists—produced different stimulus effects in animals (reviewed in ref. 16).
We examined more carefully the stimulus effects of DOM to determine if serotonin receptors were actually involved in mediating its stimulus actions; stimulus antagonism studies were conducted with various neurotransmitter antagonists, including serotonin antagonists. In tests of stimulus antagonism, pretreatment of the animals with an antagonist that specifically blocks the neu-rotransmitter mechanism underlying the actions of an agonist will cause the animals to make a different response than that typically seen following administration of an agonist. The only antagonists that consistently attenuated the DOM stimulus were serotonin antagonists. However, not all serotonin antagonists were equally effective. Certain serotonin antagonists had little to no effect on the DOM stimulus. Similar studies were conducted with the TFMPP- and 8-OH DPAT-trained animals. Clearly, there were notable differences. It was apparent that these serotonin agonists were producing distinct stimulus effects in animals; because stimulus effects are centrally mediated, the only conclusion that could be reached, other than that these agents activated yet to be identified receptor types, was that there must exist more than one type of serotonin receptor in the brain.
Using radioligand-binding methodology, two populations of brain 5-HT receptors were proposed by Peroutka and Snyder in the fall of 1979: 5-HTj receptors and 5-HT2 receptors (17). A third type of peripheral receptor, the M-type serotonin receptor, was eventually found in the brain and named 5-HT3 receptors (reviewed in refs. 9 and 18). The possibility existed, then, that the three types of agents we had been examining (i.e., DOM, TFMPP, 8-OH DPAT) might be representative ligands for these three receptor types. Although this was not to be the case, we had no way of knowing it at the time. We attempted to sort out the actions of these agents.
Several landmark studies were reported in the early 1980s that help to clarify the situation. Pedigo et al. (19) found that 5-HTj receptors could be further sub-categorized as 5-HT1A and 5-HT1B receptors, ketanserin and pirenpirone were identified as the first useful 5-HT2-selective antagonists (20), and it was proposed that 8-OH DPAT was a 5-HT1A agonist (reviewed in ref. 21). Tritiated ketanserin and tritiated 8-OH DPAT were subsequently introduced as radioligands to label brain 5-HT2 and 5-HT1A receptors, respectively, and it became possible to measure the affinity of hallucinogens at 5-HT2 and 5-HT1A receptors. Although certain of the tryptaminergic hallucinogens displayed affinity both for 5-HT1A and 5-HT2 receptors, the phenylalkylamine hallucinogens such as DOM displayed high affinity for 5-HT2 receptors and lacked affinity for 5-HT1A receptors. In addition, we found that the stimulus actions of DOM were potently antagonized by the then novel 5-HT2 antagonists ketanserin and pirenpirone, indicating that DOM was acting as a 5-HT2 agonist (22). We further demonstrated that the rat brain 5-HT2 receptor affinity of hallucinogens was significantly correlated with their stimulus potency using rats trained to discriminate DOM from the vehicle (23), and we later found a similar correlation between human 5-HT2 receptor affinity and human hallucinogenic potency (24). It seemed that we had identified the first 5-HT2-selective agonist: DOM (6).
In the same series of studies, we had provided mechanistic insight to the actions of hallucinogens and had identified what appeared to be a novel 5-HT2 receptor structural template. The phenylalkylamine template was used to investigate structure-activity and structure-affinity relationships. The availability of these compounds also permitted pharmacophoric investigations and application of QSAR methods (25,26). The bromo and iodo counterparts of DOM (i.e., DOB and DOI [10 and 11], respectively) were identified as possessing structural features deemed optimal for 5-HT2 action. Interestingly, these latter two agents were the most potent phenylalkylamine agonists in contracting rat fundus tissue. The low nanomolar affinities of these latter two ligands in radioligand-binding studies using brain homogenates led to the introduction of [3H]DOB and [125I]DOI as 5-HT2 receptor agonist radioligands for use in binding and autoradiographic investigations (27,28). Subsequently, [123I]DOI and R(-)[123I]DOI were developed for SPECT imaging studies and R(-)[76Br]DOB was examined for autoradiographic studies (29,30).
Our enthusiasm was seriously dampened following several very disconcerting literature reports. A third population of 5-HT1 receptors (termed 5-HT1C receptors at the time) was reported (31), and shortly thereafter we found that DOM and related phenylalkylamine hallucinogens bind with high affinity at this new receptor population. It was also reported that brain 5-HT2 receptors were not identical with rat fundus receptors (20). This latter finding questioned the significance of our earlier correlation between stimulus potency of hallucinogens and receptor affinity for rat fundus serotonin receptors. Subsequently (and fortunately for us), on the basis of additional pharmacological investigation, 5-HT1C receptors were identified as being a member of the 5-HT2 family; the original 5-HT2 receptors were renamed 5-HT2A receptors, and 5-HT1C receptors were renamed 5-HT2C receptors (1). Rat fundus receptors, also later found to belong to the 5-HT2 family, were initially termed 5-HT2F receptors (32) and have been since renamed 5-HT2B receptors (1). So, nearly 20 yr after our initial studies, the circle had been closed. Agents such as DOM, DOB, and DOI are now recognized as being rather selective 5-HT2 agonists, but they show almost no selectivity for the three 5-HT2 subpopulations; that is, these agents bind with nearly comparable affinity at 5-HT2A, 5-HT2B, and 5-HT2C receptors (33). Our initial correlation between fundus (now 5-HT2B) serotonin receptor affinity and hallucinogenic potency might have been fortuitous, but now could be satisfactorily explained; for the series of agents we had examined, 5-HT2B receptor affinity correlated significantly with brain 5-HT2A receptor affinity (33). Phenyl-alkylamines are widely used today as 5-HT2 agonists; actually, depending on the assay system being used, the agents are high-efficacy partial agonists with the R(-) isomers being more effective than their S(+) enantiomers. These compounds have also served as the basis for further ligand development; for example, with the proper aryl substituents appended, DOM-related structures have been shown to display 5-HT2 antagonist action (see Section 2.2).
In effect, a structural template, the aryl-substituted phenylalkylamines, was identified where no structural template had previously existed. Some of the agents, such as DOM, were known but it remained for them to be identified as being selective until the necessary pharmacological techniques were available— and, indeed, for several specific receptor populations to be identified and classified. However, normally, this is not the manner in which selective agents are developed.
DOM (6), DOB (10), DOI (11), and certain related agents are hallucinogenic in humans. Our investigations led to the "5-HT2A hypothesis of hallucinogenic drug action" for the classical hallucinogens (11,23). Could this information be put to use for the development of agents with possible therapeutic benefit? Recently, it was demonstrated that activation of 5-HT2A serotonin receptors represents a novel approach to lowering intraocular pressure—an approach that might be useful in the treatment of glaucoma (34). A local ocular site of action seems to be sufficient for achieving decreased intraocular pressure in a primate model of ocular hypertension. Because 5-HT2A agonists might also produce undesirable central effects should sufficient quantities enter the brain, attempts were made to identify 5-HT2 agonists with reduced propensity to penetrate the blood-brain barrier. In this manner, a 5-HT2A agonist that does not readily penetrate the blood-brain barrier should be effective following local ocular application, and central side effects might be minimized.
Two general strategies are typically employed to reduce the ability of a compound to enter the brain: quaternization of an amine and introduction of polar substituents. Because structure-affinity studies had already shown that quaternization essentially abolishes the affinity of phenylalkylamines for
5-HT2 receptors (35), quaternization was not a viable approach. Limited to the introduction of a polar substituent, it was again necessary to consult the structure-affinity results to determine what and where a polar substituent might be tolerated. 1-(4-Bromo-2,5-dimethoxyphenyl)-2-aminopropan-1-ol (12), an analog of the 5-HT2 agonist DOB (10) bearing a benzylic hydroxyl group, was identified as a candidate structure; the hydroxyl group might sufficiently lower lipophilicity to impede penetration of the blood-brain barrier relative to DOB (36). However, the effect on 5-HT2A affinity of such a structural modification was unknown. Because of the presence of two chiral centers, four optical isomers are possible; all four were prepared and evaluated. Of the four optical isomers, the 1^,2^-isomer 12 (5-HT2A, K = 1.3 nM) was found to bind at 5-HT2A receptors with an affinity similar to that of tf(-)DOB (K =0.5 nM). Like tf(-)DOB, 12 behaved as a partial agonist. In an in vivo test of central action (i.e., stimulus generalization with rats as subjects), 12 was >15 times less potent than ^(-)DOB, suggesting that it might not as readily enter the brain (36). In collaboration with Alcon Laboratories, intraocular administration of 12 to ocular hypertensive monkeys was shown to effectively reduce intraocular pressure (36). Given the route of administration (i.e., topical) and concentrations necessary to reduce intraocular pressure, compounds such as 12 should demonstrate minimal central effects at potentially useful therapeutic doses and offer useful leads for further development. Interestingly, O-methylation of 12 (i.e., 13 in Fig. 2; 5-HT2A, K = 0.7 nM) resulted in an agent that behaved as a full 5-HT2A agonist, indicating that certain oxygen functions at the benzylic position of phenylalkylamines are not only tolerated, but that they can also influence efficacy (36).
The dopaminergic agent spiperone (14 in Fig. 3) was used to initially identify and distinguish 5-HTj from 5-HT2 receptors (17). It was the first recognized 5-HT2 antagonist, and [3H]spiperone was used for several years to label 5-HT2 receptors in radioligand-binding studies. Later, it was demonstrated that spiperone binds at a subpopulation of 5-HTj receptors (i.e., 5-HT1A receptors) (19). One of the most significant early advances in 5-HT2 research
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