Structural requirements for agonist actions at the benzodiazepine receptor: studies with analogues of 6-(benzyloxy)-4-(methoxymethyl)-beta-carboline-3-carboxylic acid ethyl ester.

The analogues 2, 3, 8a-c, 9, 13, and 14 of the beta-carboline 6-(benzyloxy)-4-(methoxymethyl)-beta-carboline-3-carboxylic acid ethyl ester (ZK-93423, 1) were synthesized in order to determine which functional groups are required for agonist activity exhibited by the parent compound 1 at the benzodiazepine receptor (BzR). The potencies of these compounds were evaluated in vitro and in vivo; the beta-carbolines 2, 8a-c, and 13 were found to exhibit high affinity (0.5-22 nM) for BzR, but in vivo studies demonstrated they were not anticonvulsant while analogues 8a and 8b antagonized the effects of diazepam. These results are in agreement with the model proposed for the pharmacophore of the agonist site (domain) of the BzR, which requires the interaction of two sites of electron density [N(2) and C(4)], a third at E+ (electrostatic or steric), and two of lipophilicity [C(3) and C(6)] of the ligand with the BzR protein (see Figure 1). The pharmacophore for the agonist site is derived from the structure-activity relationships of the benzodiazepines, pyrazoloquinolines, and the beta-carboline 1, and compared to interactions required for ligand inverse agonist activity.

Synthesis and dopamine transporter affinity of the four stereoisomers of (+/-)-2-(methoxycarbonyl)-7-methyl-3-phenyl-7-azabicyclo[2.2.1]heptane.

All four stereoisomers of (+/-)-2-(methoxycarbonyl)-7-methyl-3-phenyl-7-azabicyclo[2.2. 1]heptane were synthesized and evaluated as cocaine binding site ligands at the dopamine transporter. The in vitro binding affinities (Ki) of the 7-azabicyclo[2.2.1]heptane derivatives were measured in rat caudate-putamen tissue and found to be 100-3000-fold less potent (Ki = 5-96 microM) than cocaine and 2beta-(methoxycarbonyl)-3beta-phenyltropane (2, WIN 35,065-2). Surprisingly, the 3alpha-phenyl isomers (6c, 6d) were more potent than the 3beta-phenyl isomers (6a, 6b). Molecular modeling studies revealed that the rigid 7-azabicyclo[2.2.1]heptane derivatives possess molecular topologies which are significantly different than the molecular topologies of the 2beta-(methoxycarbonyl)-3-phenyltropanes.

Synthesis and dopamine transporter affinity of 2-(methoxycarbonyl)-9-methyl-3-phenyl-9-azabicyclo[3.3.1]nonane derivatives.

A series of 9-methyl-3 beta-phenyl-2-substituted-9-azabicyclo[3.3.1]nonane derivatives were synthesized and evaluated as cocaine-binding site ligands at the dopamine transporter (DAT). The conformation of the bicyclic structures and the stereochemistry of the substituents were determined by NMR and X-ray crystallography. The in vitro binding affinity (Ki) of the 9-azabicyclo[3.3.1]nonane derivatives was measured in rat caudate-putamen tissue, and they were found to be 100-fold (Ki = 2-14 microM) less potent than cocaine and other tropane analogs. From these results it is evident that the cocaine-binding site at the DAT is very sensitive to structural modifications of the unsubstituted methylene bridge [C(6)-C(7)] of cocaine and cocaine-like compounds.

Synthesis, structure, dopamine transporter affinity, and dopamine uptake inhibition of 6-alkyl-3-benzyl-2-[(methoxycarbonyl)methyl]tropane derivatives.

A series of 6-alkyl-3 beta-benzyl-2-[(methoxycarbonyl)methyl]tropane analogues were synthesized and evaluated as cocaine binding site ligands at the dopamine transporter (DAT). The in vitro affinity (Ki) for the DAT of the 6-alkyl-3 beta-benzyl-2-[(methoxycarbonyl) methyl]tropane analogues was determined by inhibition of [3H]WIN 35,428 in rat caudate putamen tissue. The inhibition of dopamine uptake (IC50) was also measured for selected compounds which demonstrated moderate affinity for the dopamine transporter. The unsubstituted enantiopure analogues (-)-19a (Ki = 33 nM) and surprisingly (+)-20a (Ki = 60 nM) were found to be almost equipotent with the high-affinity binding components of cocaine and WIN 35,065-2 and exhibited slightly more potent dopamine uptake inhibition than both cocaine and WIN 35,065-2. In general, substitution at the 6-position of racemic 19a and 20a with alkyl groups was found to result in decreased activity relative to increased chain length of the substituent. The 3 beta-benzyl-2 beta-[(methoxycarbonyl)methyl]-6 beta-methyltropane (21b; Ki = 57 nM) was the only 6-alkyl derivative to exhibit moderately potent activity. The 6 beta-isomer 21b was 4-fold more potent than the 6 alpha-isomer 19b (Ki = 211 nM) and was nearly equipotent with (-)-19a and (+)-20a as well as with cocaine and WIN 35,065-2. The results of this study further demonstrate the steric constraints associated with the C(6)-C(7) methylene bridge of the tropane ring system for molecular recognition of cocaine analogues at the cocaine binding site(s) on the DAT.

Synthesis, dopamine transporter affinity, dopamine uptake inhibition, and locomotor stimulant activity of 2-substituted 3 beta-phenyltropane derivatives.

A series of 2 beta-substituted 3 beta-phenyltropanes were synthesized as analogs of cocaine and tested in vitro for their ability to displace bound [3H]WIN 35,428 (2b) and inhibit dopamine uptake in rat caudate-putamen tissue. The analogs bound with high affinity (Ki = 11-22 nM) to the dopamine transporter. Increased lipophilicity at the beta-C(2)-position was found to lead to increased binding affinity and increased dopamine uptake potency. However, a direct correlation between clogP values and binding affinity and potency of uptake inhibition was not observed. The unsaturated ester 7 was found to possess weak dopamine uptake inhibition relative to the high binding affinity (IC50/Ki = 10.2). In vivo measurement of stimulated locomotor activity and drug discrimination against cocaine (10 mg/kg, ip) with selected analogs (4, 6, and 7) demonstrated that the behavioral effects of these drugs were approximately equipotent with those of cocaine. The structure-activity relationships of this series of cocaine analogs supports a pharmacophore model in which lipophilic interactions between the beta-C(2)-position of 3 beta-phenyltropanes and the cocaine binding site on the dopamine transporter lead to enhanced potency while electrostatic interactions have a nonspecific effect.

Synthesis of substituted 7,12-dihydropyrido[3,2-b:5,4-b']diindoles: rigid planar benzodiazepine receptor ligands with inverse agonist/antagonist properties.

A series of 1-, 2-, 3-, 4-, 5-, 6-, 7-, 10-, and 12-substituted pyridodiindoles were synthesized and screened in vitro against [3H]diazepam for activity at the benzodiazepine receptor (BzR). In vitro, the 2-substituted pyridodiindoles were found to be the most potent (IC50 less than 10 nM) of this new class of BzR ligands. In vivo, 2-methoxypyridodiindole 19a (IC50 = 8 nM) was found to be the most potent partial inverse agonist (proconvulsant) of the series. The parent compound 2 (IC50 = 4 nM) was only slightly less potent. In addition, 2-hydroxypyridodiindole 21a (IC50 = 6 nM) was found to exhibit potent proconvulsant activity when administered as a prodrug derivative, pivaloyl ester 22. 2-Chloropyridodiindole 16a (IC50 = 10 nM) was devoid of preconvulsant activity; however, 16a was found to be the most potent antagonist of the anticonvulsant effects of diazepam in this class of BzR ligands. From the in vivo data available, substitution on ring E of 2 with electron-withdrawing groups results in antagonists at BzR, while replacement of hydrogen at C-2 with electron-releasing groups provides enhanced inverse agonist activity. The pyridodiindoles were used as "templates" for the formulation of a model of the inverse agonist/antagonist active site of the BzR. The proposed model consists of a hydrogen bond acceptor site (A1) and a hydrogen bond donor site (D2) disposed 6.0-8.5 A from each other on the receptor protein. The hydrogen-bonding sites are believed to be located at the base of a narrow cleft. A large lipophilic pocket at the mouth of the narrow cleft serves to direct molecules into the binding site, while the presence of a small lipophilic pocket permits substitution only at position 2 of the pyridodiindole nucleus for maximum binding potency.

Synthesis of novel 3-substituted beta-carbolines as benzodiazepine receptor ligands: probing the benzodiazepine receptor pharmacophore.

The 3-substituted beta-carbolines 2-4 and 5-7 were prepared from 3-amino-beta-carboline (8) in one step via diazotization, followed by reaction with the appropriate nucleophile in order to determine their binding affinity for benzodiazepine receptors (BzR). All three of the 3-alkoxy-beta-carbolines 2 (IC50 = 124 nM), 3 (IC50 = 24 nM), and 4 (IC50 = 11 nM) have high affinities for BzR. The beta-carbolines substituted with electron-withdrawing groups including 5 (Cl; IC50 = 45 nM), 6 (NO2; IC50 = 125 nM), and 7 (N = C = S; IC50 = 8 nM) also had high affinities for BzR. The affinities of 5-8 clearly indicate that a carbonyl moiety at position 3 of a beta-carboline is not required for high-affinity binding to BzR. These findings have led to the development of a model for the binding of ligands to an inverse agonist domain at BzR. This model is supported by the recent synthesis of 3-ethoxy-beta-carboline (3), a potent, long-lived partial inverse agonist, and 7, an irreversible BzR ligand.

Synthesis and ligand binding of eta(6)-(2beta-carbomethoxy-3beta-phenyltropane) transition metal complexes.

The transition metal complexes [eta(6)- (2beta-carbomethoxy-3beta-phenyltropane)]tricarbonylchromium (3) and [eta(6)-(2beta-carbomethoxy-3beta-phenyltropane)] [eta(5)-(pentamethylcyclopentadienyl)]ruthenium(II)triflate (4) were synthesized from 2beta-carbomethoxy-3beta-phenyltropane (2, WIN 35,065) to further elucidate the influence of substituents on the 3beta-aryl on the affinity of the ligand for cocaine-binding sites at the dopamine transporter. The compounds were tested for their ability to displace bound [(3)H]WIN 35,428 (5) from rat caudate putamen tissue and for their ability to inhibit [(3)H]dopamine uptake. The binding affinity for 3 was 2-fold greater than those observed for cocaine (1) and 2, while the binding affinity for 4 was found to be 100-fold less than those of 1 and 2. In addition, 3 was equipotent with 1 and 2 in [(3)H]dopamine uptake inhibition studies, while 4 was 10-fold less potent. The potencies of the complexes 3 and 4 correlated well with the structure-activity relationships of other 2beta-carbomethoxy-3beta-aryltropane derivatives. These data further support a pharmacophore model in which the region occupied by the aryl ring is a lipophilic pocket with electropositive character.

Synthetic and computer-assisted analyses of the pharmacophore for the benzodiazepine receptor inverse agonist site.

The structural requirements for ligand binding to the benzodiazepine receptor (BzR) inverse agonist site were probed through the synthesis and in vitro evaluation of 3-substituted beta-carbolines 6, 7, 11, 12, gamma-carboline 13, and diindoles 18-21, 23-25, 27, 28, and 34. On the basis of the apparent binding affinities of these and other analogues, a hydrogen bond acceptor site (A2) on the receptor is proposed to interact with the N(9) hydrogen atom of the beta-carbolines or the N(7) hydrogen nuclei of the diindoles. Likewise, a proposed hydrogen bond donating site (H1) interacts with the N(2) nitrogen atom of the beta-carbolines or the N(5) nitrogen atom of the diindoles. It appears that interaction with both sites is a prerequisite for high affinity since analogues which have either one or both of these positions blocked exhibit substantial reduction in affinity. Moreover, H1 appears to be capable of engaging in a three-centered hydrogen bond with appropriately functionalized ligands, which explains the increase in potency observed in the following series of 3-substituted beta-carbolines: the n-butyl (12, IC50 = 245 nM), n-propoxy (9, IC50 = 11 nM), and propyl ketone (11, IC50 = 2.8 nM) congeners. In addition to H1 and A2, there appears to be a relatively narrow hydrophobic pocket in the binding cleft that can accommodate substituents at the 3-position of the beta-carbolines which have chain lengths less than or equal to C5. There is a 1 order of magnitude decrease in affinity between n-propoxy analogue 9 (IC50 = 11 nM, chain length = 4) and n-butoxy derivative 7 (IC50 = 98 nM, chain length = 5). Furthermore, alpha- and gamma-branching [e.g. ethoxycarbonyl (2), IC50 = 5 nM and tert-butoxycarbonyl (31) IC50 = 10 nM] but not beta- and delta-branching [e.g. isopropoxy (6), IC50 = 500 nM and (neopentyloxy) carbonyl (48), IC50 = 750 nM] at position 3 are tolerated. Occupation of this hydrophobic pocket is clearly important for high affinity as evidenced by the relatively low affinity of 30, a beta-carboline which possesses a hydrogen atom at the 3-position. This same hydrophobic pocket is partially filled by the D and E rings of the diindoles, which accounts for the high affinity of several members of this series. An excluded volume analysis using selected 3-substituted beta-carbolines and ring-E substituted pyridodiindoles is consistent with the presence of this hydrophobic pocket (see Figure 1).(ABSTRACT TRUNCATED AT 400 WORDS)

Molecular yardsticks. Rigid probes to define the spatial dimensions of the benzodiazepine receptor binding site.

A series of rigid planar azadiindoles (8a, 8b, and 8d), benzannelated pyridodiindoles (11a, 11b, and 11d), and indolopyridoimidazoles (11c, 20, and 24) were synthesized from 4-oxo-1,2,3,4-tetrahydro-beta-carboline 5 via the Fischer indole cyclization with the appropriate arylhydrazines. These analogues were employed as probes ("molecular yardsticks") to define the spatial dimensions of the lipophilic regions of the benzodiazepine receptor (BzR) binding cleft. Benzannelated indoles 11a-d and indolopyridoimidazoles 20 and 24 were important in establishing an area of negative interaction (S1, see Figure 6, part b) in the binding cleft common to the interactions of both inverse agonists and agonists. Data from this chemical and computer-assisted analysis of the pharmacophore (see Figure 6) indicates that inverse agonists and agonists bind to the same binding region, but the pharmacophoric descriptors required for the two activities are different, in keeping with previous studies with these planar ligands. However, the hydrogen bond donating site H1 and the lipophilic region L1 in the receptor binding site are common interactions experienced by both series of ligands. The low affinities of both indolo[3,2-c]carbazole (3a) and indolo[3,2-b]isoquinoline (3b) for the BzR are consonant with the requirements of a hydrogen bond acceptor interaction at donor site H1 and a hydrogen bond donor interaction at acceptor site A2 for potent inverse agonist activity in the beta-carboline series. The hydrochloride salts of 1-aza- 8a (IC50 10.6 nM), 2-aza- 8b (IC50 51.5 nM), and 4-azadiindole 8d (IC50 11.2 nM) were found to be much more soluble in water than the corresponding salt of the parent diindole 2. Moreover, aza analogues 8a and 8b were shown to be partial inverse agonists with proconvulsant potencies comparable to that of the parent diindole 2.

Synthesis of n-heteroaryl-7-azabicyclo[2.2.1]heptane derivatives via palladium-bisimidazol-2-ylidene complex catalyzed amination reactions.

[reaction in text] A one-step approach to novel N-heteroaryl-substituted-7-azabicyclo[2.2.1]heptanes from readily available heteroaryl halides and 7-azabicyclo[2.2.1]heptane has been achieved. The cross-coupling amination reaction employs palladium-bisimidazol-2-ylidene complexes as catalysts to give good to moderate yields over a wide variety of substrates.

Synthesis and stereochemical assignment of exo- and endo-7-methyl-7-azabicyclo[2.2.1]heptan-2-ol.

[formula: see text] The syntheses of both exo and endo stereoisomers of 7-methyl-7-azabicyclo[2.2.1]heptan-2-ol were achieved in straightforward fashion. Alternatively, the intramolecular cyclization of syn-4-N-methylaminocyclohexane 1,2-epoxide was found to give exo-7-methyl-7-azabicyclo-[2.2.1]heptan-2-ol as the sole product. The stereochemistry of the exo isomer was unequivocally confirmed by X-ray crystallography.

Synthesis, dopamine and serotonin transporter binding affinities of novel analogues of meperidine.

A series of meperidine analogues was synthesized and the binding affinities for the dopamine and serotonin transporters were determined. The substituents on the phenyl ring greatly influenced the potency and selectivity of these compounds for the transporter binding sites. In general, meperidine (3) and its analogues were more selective for serotonin transporter binding sites and the esters 9 were more potent than the corresponding nitriles 8. The 3,4-dichloro derivative 9e was the most potent ligand of the series for dopamine transporter binding sites while the 2-naphthyl derivative 9g exhibited the most potent binding affinity and was highly selective for serotonin transporter binding sites.