Lobeline: structure-affinity investigation of nicotinic acetylcholinergic receptor binding.

(-)Lobeline (1) and (-)nicotine (2) bind at neuronal nicotinic cholinergic (nACh) receptors with high affinity (K(i) = 4 and 2 nM, respectively). Previous attempts to determine whether lobeline fits the currently accepted nicotinic pharmacophore model have led to suggestions that the carbonyl function, rather than the hydroxyl group, is a major contributor to binding. Interestingly, however, it has never been empirically demonstrated that either oxygen function is actually required for interaction with the receptor. In the present investigation we systematically examined a number of abbreviated analogues of lobeline and found that removal of either one or both oxygen functions reduces the affinity of lobeline by at least 25-fold; furthermore, oxidation of the (-)lobeline hydroxyl group (to afford lobelanine) or reduction of the carbonyl group (to afford lobelanidine) also resulted in decreased affinity. Although it is likely that both oxygen functions contribute to the high affinity of (-)lobeline at nACh receptors, it is concluded that the presence of both oxygen functions is not a requirement for binding; that is, replacement of the (-)lobeline hydroxyl group with a chloro group had no effect on affinity. Another finding of the present investigation is that removal of either one or both oxygen functions of lobeline results in compounds that retain the analgesic activity and potency of (-)lobeline, indicating that there is no direct relationship between neuronal nicotinic cholinergic (primarily alpha(4)beta(2) type) receptor affinity and spinal analgesia as measured in the tail-flick assay.

Influence of amine substituents on 5-HT2A versus 5-HT2C binding of phenylalkyl- and indolylalkylamines.

The effect of 15 different amine substituents on 5-HT2A and 5-HT2C serotonin receptor binding was investigated for two series of compounds (i.e., phenylalkylamine and indolylalkylamine derivatives). In general, amine substitution decreases receptor affinity; however, N-(4-bromobenzyl) substitution results in compounds that bind at 5-HT2A receptors with high affinity (Ki < 1 nM) and with > 100-fold selectivity. Although parallel structural modification in the two series result in parallel shifts in 5-HT2C binding, these same modifications alter 5-HT2A binding in a less consistent manner.

Structure-activity relationships for the binding of arylpiperazines and arylbiguanides at 5-HT3 serotonin receptors.

Arylpiperazines are nonselective agents that bind at 5-HT3 serotonin receptors with moderate to high affinity, whereas 1-phenylbiguanide is a low-affinity but more selective 5-HT3 agonist. In an attempt to enhance the affinity of the latter agent, and working with the assumption that similarities might exist between the binding of the two types of agents, we formulated structure-activity relationships for the binding of the arylpiperazines and then incorporated those substituents, leading to high affinity for the arylpiperazines, into 1-phenylbiguanide. A subsequent investigation examined the structure-activity relationships of the arylbiguanides and identified arylguanidines as a novel class of 5-HT3 ligands. Although curious similarities exist between the structure-activity relationships of the arylpiperazines, arylbiguanides, and arylguanidines, it cannot be concluded that all three series of compounds are binding in the same manner. Furthermore, upon investigating pairs of compounds in the three series, the arylpiperazines behaved as 5-HT3 antagonists (von Bezold-Jarisch assay) whereas the arylbiguanides and arylguanidines acted as 5-HT3 agonists.

2-Substituted tryptamines: agents with selectivity for 5-HT(6) serotonin receptors.

Several 2-alkyl-5-methoxytryptamine analogues were designed and prepared as potential 5-HT(6) serotonin agonists. It was found that 5-HT(6) receptors accommodate small alkyl substituents at the indole 2-position and that the resulting compounds can bind with affinities comparable to that of serotonin. In particular, 2-ethyl-5-methoxy-N, N-dimethyltryptamine (8) binds with high affinity at human 5-HT(6) receptors (K(i) = 16 nM) relative to 5-HT (K(i) = 75 nM) and was a full agonist, at least as potent (8: K(act) = 3.6 nM) as serotonin (K(act) = 5.0 nM), in activating adenylate cyclase. Compound 8 displays modest affinity for several other populations of 5-HT receptors, notably h5-HT(1A) (K(i) = 170 nM), h5-HT(1D) (K(i) = 290 nM), and h5-HT(7) (K(i) = 300 nM) receptors, but is otherwise quite selective. Compound 8 represents the first and most selective 5-HT(6) agonist reported to date. Replacing the 2-ethyl substituent with a phenyl group results in a compound that retains 5-HT(6) receptor affinity (i.e., 10: K(i) = 20 nM) but lacks agonist character. 2-Substituted tryptamines, then, might allow entry to a novel class of 5-HT(6) agonists and antagonists.

Homoazanicotine: a structure-affinity study for nicotinic acetylcholine (nACh) receptor binding.

We have recently identified 3-[(1-methyl)-4,5-dihydro-1H-imidazol-2-yl)methyl]pyridine (homoazanicotine, 8) as a novel nicotinic acetylcholinergic (nACh) receptor ligand. In the present investigation, after we determined that 8 binds selectively at nicotinic (K(i) = 7.8 nM) vs muscarinic (K(i) > 10,000 nM) acetylcholinergic receptors, we examined its structure-affinity relationships for nACh receptor binding. The features investigated included the influence of (i) the composition of connector that separates the two rings, (ii) the N-methyl group, (iii) the ring opening of the imidazoline ring, (iv) the pyridine nitrogen atom, and (v) the aromatization of the imidazoline ring on nACh receptor affinity. As with nicotine, the parent structure seems optimal and most structural changes reduce nACh receptor affinity. Also, as with nicotine analogues, alteration of the spacer group influences affinity in a manner that is somewhat different than that seen with the parent structure.

Benzylimidazolines as h5-HT1B/1D serotonin receptor ligands: a structure-affinity investigation.

Benzylimidazolines may represent a class of 5-HT1D ligands that has yet to be exploited. On the basis of a previous report that the 2-(substituted-benzyl)imidazoline alpha-adrenergic agonist oxymetazoline (8) binds with high affinity at calf brain 5-HT1D receptors, we explored the structure-affinity relationships of a series of related derivatives. Each of the aromatic substituents was removed and then reinstated in a systematic manner to determine the influence of the individual substituents on binding. It was found that all of the aromatic substituents of 8 act in concert to impart high affinity. However, although the 3-hydroxy group could be removed without significantly reducing affinity for h5-HT1D (i.e., human 5-HT1Dalpha) receptors, this modification reduced h5-HT1B (i.e., human 5-HT1Dbeta) receptor affinity by nearly 50-fold. The 2, 6-dimethyl groups also contribute to binding but seem to play a greater role for h5-HT1B binding than h5-HT1D binding. With the appropriate structural modifications, several compounds were identified that display 20- to >100-fold selectivity for h5-HT1D versus h5-HT1B receptors. Preliminary functional data suggest that these compounds behave as agonists. Given that 5-HT1D agonists are currently being explored for their antimigraine action and that activation of h5-HT1B receptors might be associated with cardiovascular side effects, h5-HT1D-selective agents may offer a new lead for the development of therapeutically efficacious agents.

Spiperone: influence of spiro ring substituents on 5-HT2A serotonin receptor binding.

Spiperone (1) is a widely used pharmacological tool that acts as a potent dopamine D2, serotonin 5-HT1A, and serotonin 5-HT2A antagonist. Although spiperone also binds at 5-HT2C receptors, it is one of the very few agents that display some (ca. 1000-fold) binding selectivity for 5-HT2A versus 5-HT2C receptors and, hence, might serve as a useful template for the development of novel 5-HT2A antagonists if the impact of its various substituent groups on binding was known. In the present investigation we focused on the 1, 3,8-triazaspiro[4.5]decanone portion of spiperone and found that replacement of the N1-phenyl group with a methyl group only slightly decreased affinity for cloned rat 5-HT2A receptors. However, N1-methyl derivatives displayed significantly reduced affinity for 5-HT1A, 5-HT2C, and dopamine D2 receptors. Several representative examples were shown to behave as 5-HT2 antagonists. As such, N1-alkyl analogues of spiperone may afford entry into a novel series of 5-HT2A-selective antagonists.

1-[2-methoxy-5-(3-phenylpropyl)]-2-aminopropane unexpectedly shows 5-HT(2A) serotonin receptor affinity and antagonist character.

Certain phenylethylamines, such as 1-(4-bromo-2,5-dimethoxyphenyl)-2-aminopropane (DOB; 1a), are high-affinity 5-HT(2) agonists. Previous structure-affinity studies have concluded that both the 2,5-dimethoxy substitution pattern and the nature of substituents at the 4-position are important determinants of high affinity. We recently demonstrated that replacement of the bromo group of DOB with a 3-(phenyl)propyl substituent results in retention of affinity and that, counter to established structure-affinity relationships, the 2,5-dimethoxy substitution pattern is no longer a requirement for the binding. The present investigation extends these findings by examining a series of analogues, 3, lacking a 5-methoxy group. It was additionally found that shifting the phenylalkyl substituent from the 4- to the 5-position (e.g., 4i) also results in retention of affinity. For example, 1-(2-methoxy-5-(3-phenylpropyl)-2-aminopropane (6; the alpha-methyl derivative of 4i) binds at 5-HT(2A) receptors with high affinity (K(i) = 13 nM) and possesses 5-HT(2A) antagonist character. Thus, not only is the 2,5-dimethoxy substitution pattern not a requirement for the binding of certain phenylethylamines at 5-HT(2A) receptors, the presence of a 4-position substituent (previously thought to serve as a modulator of affinity of DOB-like agents) is also not required. Striking differences in the 5-HT(2A) binding requirements of the present compounds as compared to DOB-like agents suggest multiple substituent-dependent modes of binding.

2-(1-Naphthyloxy)ethylamines with enhanced affinity for human 5-HT1D beta (h5-HT1B) serotonin receptors.

Although the beta-adrenergic antagonist propranolol (1) binds at rodent 5-HT1B serotonin receptors, it displays low affinity (Ki > 10,000 nM) for its species homologue 5-HT1D beta (i.e., h5-HT1B) receptors. The structure of propranolol was systematically modified in an attempt to enhance its affinity for the latter population of receptors. Removal of the alkyl hydroxyl group, shortening of the O-alkyl chain from three to two methylene groups, and variation of the terminal amine substituent resulted in compounds, such as N-monomethyl-2-(1-naphthyloxy)-ethylamine (11; Ki = 26 nM), that display significantly higher h5-HT1B affinity than propranolol. Compound 11 was shown to bind equally well at human 5-HT1D alpha (h5-HT1D) receptors (Ki = 34 nM) and was further demonstrated to possess h5-HT1B agonist character in an adenylate cyclase assay. It would appear that such (aryloxy)alkylamines may represent a novel class of 5-HT1D receptor agonists.

Binding of O-alkyl derivatives of serotonin at human 5-HT1D beta receptors.

In humans, 5-HT1D serotonin receptors represent terminal autoreceptors, and there is some evidence that 5-HT1D ligands may be useful in the treatment of migraine. The most widely used 5-HT1D agonist is sumatriptan; however, this agent reportedly displays little selectivity for 5-HT1D versus 5-HT1A receptors. To identify novel serotonergic agents with enhanced 5-HT1D versus 5-HT1A selectivity, we attempted to take advantage of possible differences in the regions of bulk tolerance associated with the 5-position of the 5-HT binding sites for these two populations of receptors. Examination of a series of 5-(alkyloxy)tryptamine derivatives demonstrated that compounds with unbranched alkyl groups of up to eight carbon atoms bind with high affinity at human 5-HT1D beta receptors (Ki < 5 nM) but demonstrate less than 50-fold selectivity relative to 5-HT1A receptors. Alkyl groups longer than eight carbon atoms impart reduced affinity for 5-HT1A receptors whereas groups longer than nine carbon atoms lead to compounds with reduced affinity at 5-HT1D beta receptors. 5-(Nonyloxy)tryptamine (10) represents a compound with optimal 5-HT1D beta affinity (Ki = 1 nM) and selectivity (> 300-fold). Branching of the alkyl chain, to 5-[(7,7-dimethylheptyl)oxy]tryptamine (15), results in an agent with somewhat lower affinity (5-HT1D beta Ki = 2.3 nM) but with greater (i.e, 400-fold) 5-HT1D versus 5-HT1A selectivity. Replacement of the oxygen atom of 10 with a methylene group (i.e., 20), replacement of the O-proximate methylene with a carbonyl group (i.e., ester 26), or cyclization of the aminoethyl moiety to a carbazole (e.g., 34, 36) or beta-carboline (i.e., 37), result in reduced affinity and/or selectivity. None of the compounds examined displayed significant selectivity for 5-HT1D beta versus 5-HT1D alpha sites; nevertheless, compounds 10 (recently shown to have as a 5-HT1D agonist) and 15 represent the most 5-HT1D versus 5-HT1A selective agents reported to date.

Donitriptan (Pierre Fabre).

Pierre Fabre is developing donitriptan, a piperazide 5-HT1D agonist, as a potential treatment for migraine [175854]. In January 2001, donitriptan had completed phase I trials for migraine and was scheduled to enter phase II development [396027]. This compound has an increased potency and, importantly, markedly higher intrinsic activity in comparison to the well described tryptamine derivatives, naratriptan, zolmitriptan and sumatriptan [295769].

The 5-HT3 agent N-(3-chlorophenyl)guanidine (MD-354) serves as a discriminative stimulus in rats and displays partial agonist character in a shrew emesis assay.

RATIONALE: There is some, albeit conflicting, evidence that 5-HT3 receptors might be involved in the actions of abused stimulants. Most studies have focussed on examinations of 5-HT3 antagonists; this might be due to a lack of high-affinity 5-HT3 agonists that readily penetrate the blood-brain barrier. OBJECTIVES: N-(3-Chlorophenyl) guanidine (MD-354) is a member of a novel class of 5-HT3 ligands developed in our laboratories. We have previously demonstrated that MD-354 can exert agonist effects and now further explore this action. METHODS: Rats (n=9) were trained to discriminate 2 mg/kg MD-354 from saline vehicle in a two-lever drug discrimination task (VI-15 s schedule of reinforcement). The actions of agents with 5-HT3 character were evaluated. The emetic and antiemetic actions of MD-354 were also examined using the shrew as test subject. RESULTS: Various agents with demonstrated 5-HT3 agonist properties substituted for the MD-354 stimulus (MD-354 ED50=0.5 mg/kg): quipazine (ED50=0.2 mg/kg), meta-chlorophenylbiguanide (mCPBG, ED50=1.4 mg/kg), 2-methyl 5-HT (ED50=4.5 mg/kg), 1-(2-naphthyl)biguanide (2-NBG, ED50=1.9 mg/kg), and N-(2-naphthyl)guanidine (2-NG, ED50=0.7 mg/kg). Administration of the training dose of MD-354 in combination with the 5-HT3 antagonists zacopride and tropisetron resulted in stimulus antagonism (AD50=0.02 mg/kg); administered alone, however, zacopride engendered 81% MD-354-appropriate responding (ED50=0.03 mg/kg). MD-354 was shown to produce an emetic effect in the shrew at very high doses (i.e., 40 mg/kg); however, when administered in combination with cisplatin, MD-354 behaved as an antiemetic agent at 10 mg/kg. CONCLUSION: Taken together, the results indicate that MD-354 is a 5-HT3 agonist and that it might be an agent with partial agonist activity.

Pharmacological effects of methamphetamine and other stimulants via inhalation exposure.

The effects of methamphetamine-HCl, methcathinone-HCl, cocaine and ephedrine on locomotor stimulation were compared between inhalation exposure and i.v. injection in mice. Methamphetamine-HCl was readily volatilized upon heating at 300 degrees C in a glass pipe with only trace amounts of amphetamine being produced. The ED50 dose (9.4 and 6.5 mumol/kg for inhalation exposure and i.v. injections, respectively) and biodisposition of methamphetamine-HCl were similar for both routes of administration. Methcathinone-HCl and cocaine were readily volatilized. Their dose response profiles also appeared similar for both routes of administration. Ephedrine did not appear to be easily volatilized and was only effective in stimulating locomotor activity after i.v. administration. These findings indicate that inhalation exposure to methamphetamine-HCl, cocaine and methcathinone possess similar pharmacological characteristics as the i.v. route of administration. In particular, this model may have implications in predicting the pharmacological activity of various stimulants via the inhalation route of administration.

Binding of beta-carbolines and related agents at serotonin (5-HT(2) and 5-HT(1A)), dopamine (D(2)) and benzodiazepine receptors.

A large series of beta-carbolines was examined for their ability to bind at [3H]agonist-labeled 5-HT(2A) serotonin receptors. Selected beta-carbolines were also examined at 5-HT(2C) serotonin receptors, 5-HT(1A) serotonin receptors, dopamine D(2) receptors, and benzodiazepine receptors. Indolealkylamines and phenylisopropylamines were also evaluated in some of these binding assays. The beta-carbolines were found to bind with modest affinity at 5-HT(2A) receptors, and affinity was highly dependent upon the presence of ring substituents and ring saturation. The beta-carbolines displayed little to no affinity for 5-HT(1A) serotonin receptors, dopamine D(2) receptors and, with the exception of beta-CCM, for benzodiazepine receptors. Examples of beta-carbolines, indolealkylamines (i.e. N,N-dimethyltryptamine analogs), and phenylisopropylamines have been previously shown to produce common stimulus effects in animals trained to discriminate the phenylisopropylamine hallucinogen DOM (i.e. 1-(2, 5-dimethoxy-4-methylphenyl)-2-aminopropane) from vehicle. Although the only common receptor population that might account for this action is 5-HT(2A), on the basis of a lack of enhanced affinity for agonist-labeled 5-HT(2A) receptors, as well as on their lack of agonist action in the PI hydrolysis assay, it is difficult to conclude that the beta-carbolines behave in a manner consistent with that of other classical hallucinogens.

Investigation of hallucinogenic and related beta-carbolines.

Certain beta-carbolines are known to be hallucinogenic in humans, and several produce stimulus effects in animals similar to those of the classical hallucinogen 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM). Classical hallucinogens bind at 5-HT2 serotonin receptors and these receptors are thought to play a role in their mechanism of action. In the present study, we examined the binding of 15 beta-carbolines at rat 5-HT2A and 5-HT2C receptors. Affinities (Ki values) of the beta-carbolines ranged from about 100 nM to greater than 10,000 nM depending upon the degree of saturation of the pyridyl ring, and upon the presence and location of methoxy substituents in the benzenoid ring. In a further study, six rats were trained to discriminate the hallucinogenic beta-carboline harmaline (3.0 mg/kg, i.p.) from vehicle using a VI-15s schedule of reinforcement. This represents the first time a hallucinogenic beta-carboline has been used as a training drug in a drug discrimination study. Administration of DOM to the harmaline-trained animals resulted in 76% harmaline-appropriate responding at 1.25 mg/kg DOM and disruption of behavior at a higher dose. Taken together, the results of the present investigation demonstrate that: (a) certain beta-carbolines bind at 5-HT2 receptors; (b) that harmaline serves as a training drug at 3.0 mg/kg in drug discrimination studies with rats as subjects; and that (c) there is some similarity between the stimulus effects produced by harmaline and DOM.

Serotonin receptors and their ligands: a lack of selective agents.

Four major families of serotonin (5-hydroxytryptamine; 5-HT) receptors have been identified: 5-HT1, 5-HT2, 5-HT3 and 5-HT4. At this time, there is a general consensus that the 5-HT1 family can be further subdivided into 5-HT1A, 5-HT1B, 5-HT1C, 5-HT1D, and 5-HT1P subpopulations. In addition, there are several other populations of less well-defined 5-HT receptors. The purpose of this presentation is to discuss 5-HT receptor nomenclature and the agents that are commonly used to investigate each receptor population in as much as it will serve to provide background for the remainder of the symposium. There is presently available an abundance of serotonergic agents; however, these agents are only semiselective, and none can be considered truly selective for a particular population of 5-HT receptors. As useful as these agents have been for the identification and characterization of 5-HT receptors, there remains a need for the development of new, more selective ligands.

5-HT3 agonist 2-methylserotonin as a training drug in discrimination studies.

Using a standard two-lever operant procedure, rats were trained to discriminate 5 mg/kg of the 5-HT3 agonist 2-methylserotonin (2-Me 5-HT; ED50 = 2.6 mg/kg) from saline using a VI 15-s schedule of reinforcement. The 2-Me 5-HT stimulus did not generalize to the 5-HT1/5-HT2 agonist 5-methoxy-N,N-dimethyltryptamine, but did generalize to the new 5-HT3 agonist 1-(m-chlorophenyl)biguanide (ED50 = 1.6 mg kg). The 5-HT3 antagonist ICS 205-930 potently antagonized the 2-Me 5-HT stimulus (ID50 = 0.001 mg/kg), whereas its quaternary amine analog, which does not readily penetrate the blood-brain barrier, failed to completely antagonize the 2-Me 5-HT stimulus at a 10,000-fold higher dose. The results of the present investigation show that 2-Me 5-HT serves as a discriminative stimulus in rats when paired with saline and suggest that its stimulus properties are likely mediated via a central 5-HT3 mechanism. As such, this is the first demonstration that a 5-HT3 agonist can be used as a training drug in drug discrimination studies.

Initial characterization of PMMA as a discriminative stimulus.

The phenylisopropylamine PMMA or N-methyl-1-(4-methoxyphenyl)-2-aminopropane, a structural hybrid of paramethoxyamphetamine (PMA) and methamphetamine, has been previously shown to unexpectedly lack amphetamine-like or hallucinogen-like stimulus properties in animals. For example, in tests of stimulus generalization, neither a (+)amphetamine stimulus nor a DOM stimulus generalized to PMMA. It has also been shown, however, that stimulus generalization does occur in animals trained to discriminate the designer drug MDMA ("Ecstasy" or N-methyl-1-(3,4-methylenedioxyphenyl)-2-aminopropane) from vehicle. In order to further characterize this unique agent, we trained a group of six Sprague-Dawley rats to discriminate 1.25 mg/kg of PMMA (ED50 = 0.44 mg/kg) from saline vehicle. The PMMA stimulus failed to generalize to the phenylisopropylamine stimulant (+)amphetamine, or to the phenylisopropylamine hallucinogen DOM. Stimulus generalization occurred to (+/-)MDMA (ED50 = 1.32 mg/kg) and S(+)MDMA (ED50 = 0.48 mg/kg). Partial generalization occurred with R(+)MDMA, PMA, 3.4-DMA, and fenfluramine. The PMMA stimulus also generalized to the alpha-ethyl homolog of PMMA (EH/PMMA, ED50 = 1.29 mg/kg). Taken together, the results of these studies suggest that PMMA is an MDMA-like agent that lacks the amphetamine-like stimulant character of MDMA. These findings support our previous suggestion that PMMA be considered the structural parent of the MDMA-like family of designer drugs.

Stimulus properties of PMMA: effect of optical isomers and conformational restriction.

para-Methoxymethamphetamine (PMMA), a structural hybrid of two central stimulants, lacks stimulant properties but behaves in a manner similar to that of MDMA [N-methyl-1-(3,4-methylenedioxyphenyl)-2-aminopropane]. PMMA has been established as a training drug in drug discrimination studies, and in the present investigation we sought to determine which optical isomer of PMMA is primarily responsible for its stimulus effects. Because PMMA is a conformationally flexible molecule, it was also of interest to determine what conformation is most important for its actions. Accordingly, we prepared and examined S(+)PMMA, R(-)PMMA, and conformationally restricted forms of PMMA: PMMA-AT, TIQ-1, and TIQ-2. S(+)PMMA (ED50 = 0.32 mg/kg) was found to be at least as potent as PMMA (ED50 = 0.41 mg/kg), whereas R(-)PMMA failed to result in complete stimulus generalization. An aminotetralin-like conformation, as found in PMMA-AT (ED50 = 0.29 mg/kg), seems to better account for the actions of PMMA than a tetrahydroisoquinoline-like conformation because TIQ-1 and TIQ-2 failed to result in stimulus generalization. The results of the present study further support the concept that PMMA and MDMA share considerable similarity with respect to their stimulus properties in animals except that PMMA lacks the amphetaminergic stimulant component of action associated with MDMA.

Structure-activity relationship studies of CNS agents. Part 1: The Free-Wilson analysis of acute toxicity and sedative effect of some 1,2,3,4-tetrahydro-beta-carbolines.

It has been found that 3-alkoxycarbonyl-beta-carbolines are almost non-toxic and showed a sedative effect. The quantitative relationship between the structure of some 1,2,3,4-tetrahydro-beta-carbolines and their acute toxicity and sedative effect was found using the Free-Wilson approach.