Elsevier

Bioorganic & Medicinal Chemistry

Volume 18, Issue 21, 1 November 2010, Pages 7548-7564
Bioorganic & Medicinal Chemistry

Design, synthesis, and subtype selectivity of 3,6-disubstituted β-carbolines at Bz/GABA(A)ergic receptors. SAR and studies directed toward agents for treatment of alcohol abuse

https://doi.org/10.1016/j.bmc.2010.08.049Get rights and content

Abstract

A series of 3,6-disubstituted β-carbolines was synthesized and evaluated for their in vitro affinities at αxβ3γ2 GABAA/benzodiazepine receptor subtypes by radioligand binding assays in search of α1 subtype selective ligands to treat alcohol abuse. Analogues of β-carboline-3-carboxylate-t-butyl ester (βCCt, 1) were synthesized via a CDI-mediated process and the related 6-substituted β-carboline-3-carboxylates 6 including WYS8 (7) were synthesized via a Sonogashira or Stille coupling processes from 6-iodo-βCCt (5). The bivalent ligands of βCCt (32 and 33) were also designed and prepared via a palladium-catalyzed homocoupling process to expand the structure–activity relationships (SAR) to larger ligands. Based on the pharmacophore/receptor model, a preliminary SAR study on 34 analogues illustrated that large substituents at position-6 of the β-carbolines were well tolerated. As expected, these groups are proposed to project into the extracellular domain (LDi region) of GABAA/Bz receptors (see 32 and 33). Moreover, substituents located at position-3 of the β-carboline nucleus exhibited a conserved stereo interaction in lipophilic pocket L1, while N(2) presumably underwent a hydrogen bonding interaction with H1. Three novel β-carboline ligands (βCCt, 3PBC and WYS8), which preferentially bound to α1 BzR subtypes permitted a comparison of the pharmacological efficacies with a range of classical BzR antagonists (flumazenil, ZK93426) from several different structural groups and indicated these β-carbolines were ‘near GABA neutral antagonists’. Based on the SAR, the most potent (in vitro) α1 selective ligand was the 6-substituted acetylenyl βCCt (WYS8, 7). Earlier both βCCt and 3PBC had been shown to reduce alcohol self-administration in alcohol preferring (P) and high alcohol drinking (HAD) rats but had little or no effect on sucrose self-administration.1, 2, 3 Moreover, these two β-carbolines were orally active, and in addition, were anxiolytic in P rats but were only weakly anxiolytic in rodents. These data prompted the synthesis of the β-carbolines presented here.

Graphical abstract

A series of 3,6-disubstituted β-carbolines was synthesized and evaluated for their in vitro affinities at αxβ3γ2 GABAA/benzodiazepine receptor subtypes by radioligand binding assays in search of α1 subtype selective ligands to treat alcohol abuse. Analogues of β-carboline-3-carboxylate-t-butyl ester (βCCt, 1) were synthesized via a CDI-mediated process and the related 6-substituted β-carboline-3-carboxylates 6 including WYS8 (7) were synthesized via a Sonogashira or Stille coupling processes from 6-iodo-βCCt (5). The bivalent ligands of βCCt (32 and 33) were also designed and prepared via a palladium-catalyzed homocoupling process to expand the structure–activity relationships (SAR) to larger ligands. Based on the pharmacophore/receptor model, a preliminary SAR study on 34 analogues illustrated that large substituents at position-6 of the β-carbolines were well tolerated. As expected, these groups are proposed to project into the extracellular domain (LDi region) of GABAA/Bz receptors (see 32 and 33). Moreover, substituents located at position-3 of the β-carboline nucleus exhibited a conserved stereo interaction in lipophilic pocket L1, while N(2) presumably underwent a hydrogen bonding interaction with H1. Three novel β-carboline ligands (βCCt, 3PBC and WYS8), which preferentially bound to α1 BzR subtypes permitted a comparison of the pharmacological efficacies with a range of classical BzR antagonists (flumazenil, ZK93426) from several different structural groups and indicated these β-carbolines were ‘near GABA neutral antagonists’. Based on the SAR, the most potent (in vitro) α1 selective ligand was the 6-substituted acetylenyl βCCt (WYS8, 7). Earlier both βCCt and 3PBC had been shown to reduce alcohol self-administration in alcohol preferring (P) and high alcohol drinking (HAD) rats but had little or no effect on sucrose self-administration.1, 2, 3 Moreover, these two β-carbolines were orally active, and in addition, were anxiolytic in P rats but were only weakly anxiolytic in rodents. These data prompted the synthesis of the β-carbolines presented here.

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Introduction

Alcohol addiction and dependence remain significant public health concerns, impacting physical and mental well-being, family structure and occupational stability.4 While advances have been made in the development of novel therapies to treat alcoholism,5, 6, 7, 8 alcohol-dependent individuals represent a heterogeneous group,9, 10, 11 and it is unlikely that a single pharmacological treatment will be effective for all alcoholics. Hence, a better understanding of the neuromechanisms which regulate alcohol seeking behaviors and the design of clinically safe and effective drugs that reduce alcohol addiction and dependence remain a high priority.7, 12 While the precise neuromechanisms regulating alcohol-seeking behaviors remain unknown, there is now compelling evidence that the GABAA receptors within the striatopallidal and extended amygdala system are involved in the ‘acute’ reinforcing actions of alcohol.13, 14, 15, 16, 17, 18 Among the potential GABAA receptor isoforms within the VP regulating alcohol-seeking behaviors, GABA receptors containing the α1 receptor subtype (GABA α1) appear preeminent. Thus, Criswell et al. observed that acute alcohol administration selectively enhanced the effects of iontophoretically applied GABA in the ventral pallidum (VP).19, 20 However, no effects were seen in the septum, ventral tegmental area (VTA), and CA1 hippocampus. These data suggest the α1 Bz/Gaba(A)ergic receptor plays an important role in alcohol-motivated behaviors. Research on the neuroanatomical basis of alcohol reward has shown that the NACC, VTA, VP, central amygdala (CeA), and hippocampus are all involved in GABAergic regulation of ethanol (EtOH) reinforcement.2, 21, 22, 23, 24 Other investigators have identified a dense reciprocal projection from the VP to the NACC,25, 26, 27 and many of these have been found to be GABAergic neurons.28, 29, 30 The NACC is now well established as a substrate that regulates the reinforcing properties of abused drugs.13 Finally, immunohistochemical31, 32 and in situ hybridization studies33, 34, 35 have demonstrated that the VP contains one of the highest concentrations of mRNA encoding the α1 subunit in the CNS. These findings, together with pharmacological studies suggesting the VP plays a role in reward-mediated behaviors of psychostimulants and opiates,28, 36, 37, 38, 39 suggest a possible role of the VP-α1 receptors in the euphoric properties of alcohol. Findings of previous studies concluded that inhibition of VP-α1 receptors by the α1-preferring antagonist 3-PBC produced marked reductions on alcohol-maintained responding.9, 15, 40, 41 The α1-mediated suppression at the VP level by 3-PBC showed a high degree of neuroanatomical specificity. Specifically, the α1-mediated suppression was not observed with the more dorsal placements in the NACC or caudate putamen. The failure of 3-PBC to alter alcohol self-administration in the NACC/striatum is in agreement with previous research which has consistently reported a lack of expression of the α1 transcript in the NACC and caudate putamen.31, 32, 33, 34, 35

An understanding of the neuromechanisms that regulate alcohol drinking is key in the development of drugs to treat alcohol addiction and dependence in humans.2 In recent years, much evidence has accumulated in favor of the GABA system;22, 23, 42, 43 however much remains unknown about the role of specific GABAA receptor subtypes in regulating ethanol reinforcement. This is due to both a lack of high affinity and selective ligands capable of discriminating among the GABAA receptor subunits and the heterogeneity of various subunits within the known alcohol reward circuitry.31, 34 Of the potential GABAA receptors involved in the reinforcing properties of alcohol, evidence suggests the α1 subtype within the VP may play an important role in regulating alcohol-seeking behaviors, as mentioned above. The VP contains one of the highest distributions of α1 subunits in the mesolimbic system.32, 33, 34, 35 Finally, acute ethanol administration has been reported to selectively enhance the effects of iontophoretically applied GABA in the VP. These effects correlate highly with [3H] zolpidem binding (an α1 subtype selective agonist).19, 20

The GABAA receptor is the major inhibitory neurotransmitter receptor of the central nervous system (CNS) and the site of action of a variety of pharmacologically and clinically important drugs, such as benzodiazepines, barbiturates, neuroactive steroids, anesthetics, and convulsants.44 It is now clear that these receptors regulate the excitability of the brain, anxiety, muscle tone, circadian rhythms, sleep, vigilance, memory, and learning.44 There are several disease states thought to be associated with the improper functioning of this system, including anxiety, epilepsy,45 insomnia,46 depression and bipolar disorder,47, 48 schizophrenia,49 as well as mild cognitive impairment and Alzheimer’s disease.50 A role of GABAA receptors in drug and alcohol abuse has also been reported.51, 52, 53 Briefly, GABAA receptors are composed of five subunits that form a central chloride channel and can belong to different subunit classes. A total of 19 subunits (6α, 3β, 3γ, 1δ, 1ε, 1π, 1θ, 3ρ) of the GABAA receptor have been cloned and sequenced from the mammalian nervous system.54, 55, 56, 57, 58, 59 All these polypeptides possess an approximate molecular mass of ∼50 kD and are structurally related.

To evaluate the role of the α1 receptor in regulating alcohol reinforcement, 3-propoxy-β-carboline hydrochloride (3-PBC), a mixed benzodiazepine (BDZ) agonist–antagonist with binding selectivity at the α1 receptor was developed.2 Compared with the prototypical BDZ agonist zolpidem, 3-PBC exhibited a slightly higher binding selectivity for the α1 receptor.60, 61 Preliminary behavioral studies in several species (e.g., rats, mice, and primates) show that 3-PBC is a BDZ antagonist, exhibiting competitive binding-site interactions with BDZ agonists at low to moderate doses (2.5–15 mg/kg).60, 62, 63 At higher doses (15–60 mg/kg), 3-PBC produces anxiolytic effects in the plus maze that are comparable with those of chlordiazepoxide in alcohol preferring (P) rats.62, 63, 64, 65 Thus given the proposed subunit composition of the GABA receptors within the CeA,31, 32, 34, 66 pharmacological compounds capable of exploiting the α1, α2, and α3 subunit-containing GABAA receptors represent optimal tools to evaluate the role of GABAA receptors in alcohol reinforcement and better understand neurobehavior and ethanol responding.

The β-carboline-3-carboxylate-t-butyl ester (βCCt) is a mixed benzodiazepine agonist–antagonist ligand with binding selectivity at α1 receptors;2, 62, 67 βCCt also exhibits some affinity (albeit lower) for both α2 and α3 receptors. Behavioral studies in several species (e.g., rats, mice, primates) show that βCCt is a BDZ antagonist exhibiting competitive binding-site interactions with BDZ agonists over a broad range of doses.60, 62, 68, 69, 70, 71, 72 Other studies show that βCCt produces anxiolytic effects in rodents62 and potentiates the anticonflict response induced by α1 subtype agonists in primates.70 Thus, βCCt displays a weak agonist or antagonist profile depending on the behavioral task, species, and dose employed.

In studies involving the α1 subtype, βCCt, and 3-PBC were observed to selectively reduce alcohol-motivated behaviors in a variety of experiments.2, 73 However, unlike the α5 selective inverse agonist RY-23, both the β-carboline antagonists βCCt and 3-PBC displayed mixed weak agonist–antagonist profiles in vivo in alcohol P and HAD rats. Therefore, in addition to being able to study the molecular basis of alcohol reinforcement, α1 Bz β-carboline ligands which display mixed agonist–antagonist pharmacology in alcohol P and HAD rats may be capable of reducing alcohol intake while eliminating or greatly reducing the anxiety associated with habitual alcohol, abstinence or detoxification. Thus, these types of ligands may be ideal clinical agents for the treatment of alcohol dependent individuals.2, 73

Consequently, several series of structurally different compounds have been synthesized which possess some α1 subtype selectivity.67, 74, 75, 76, 77 The discovery of high affinity, saturable, and stereospecific ligands for the BzR has been coupled with the demonstration that β-carbolines exhibited an affinity for the BzR.78, 79, 80, 81, 82, 83, 84, 85 Some of these agents act on the BzR to induce effects that are functionally opposite (inverse agonists/antagonists) to those of classical BDZs. Consequently, the affinities of a wide variety of β-carbolines have been reported on synaptosomal membranes from this laboratory,60, 72, 78, 80, 81, 86, 87, 88, 89, 90 and the laboratories of others,91, 92, 93, 94, 95, 96 and this prompted the study of the binding affinities of a series of β-carbolines67 at five recombinant GABAA/BzR subtypes (α1β3γ2, α2β3γ2, α3β3γ2, α5β3γ2 and α6β3γ2) expressed from recombinant human cell lines.91, 97 In general, this series of β-carboline ligands exhibited some selectivity at α1 receptor subtypes including βCCt (1) and 3-PBC (2).2, 3 These two ligands displayed a 20-fold and 10-fold selectivity, respectively, for the α1 subtype over the α2 and α3 receptors, as well as over 150-fold selectivity for the α1 site over the α5 subtype.2, 3 βCCt (1) was more selective at the α1 subtype in vitro than the classical α1 selective agonists zolpidem (3) and CL 218872 (4) (Fig. 1).60, 98, 99 A number of in vitro and in vivo studies employing α1 (e.g., zolpidem, CL 218872,68 βCCt, and 3-PBC3) selective ligands suggest the α1-containing GABAA/Bz receptors of the ventral pallidum (VP) play an important role in regulating alcohol’s neurobehavioral effects; particularly alcohol’s reinforcing properties as mentioned above.2, 3, 19, 20, 35

Section snippets

Structure–activity relationships

A predictive 3-D QSAR pharmacophore/receptor model for inverse agonist/antagonist β-carbolines was initially developed via Comparative Molecular Field Analysis (CoMFA) and later refined.100, 101 Affinities of ligands from 15 different structural classes have been evaluated.61 Based on this CoMFA study of a series of β-carbolines, Huang et al. reported that β-carbolines bind to all diazepam sensitive (DS) sites of the BzR with some selectivity at the α1-containing receptor isoform and this was

Chemistry

The synthesis of the ligands under study is outlined in Scheme 1, Scheme 2, Scheme 3. The important precursor β-carboline-3-carboxylate ethyl ester (βCCE, 8) and its corresponding acid (9) were the intermediates required for large-scale synthesis of βCCt (1), as well as an intermediate required for the synthesis of the new β-carbolines. As outlined in Scheme 1, d,l-tryptophan 10 was converted into tetrahydro-β-carboline (11) via a Pictet–Spengler reaction on kilogram scale. Fischer

βCCt bivalent ligands

The in vitro biological protocols employed in the present study follow the published procedure119, 120 and are detailed in Section 6. Although the α1β3γ2 BzR/GABAergic subtype is very similar in structure to the α2 and α3 subtypes, there are slight differences.61, 121 One major difference is in region LDi, which appears larger in the α1 subtype than in either the α2, or α3 or α5 subtypes. This is located near position-6 of βCCt (1) (Fig. 3).60, 110, 122

The western region of the

Conclusion

Ethanol allosterically modulates the GABA receptor complex to open the chloride channel and hyperpolarize cells. At the pharmacological level, the effects of ethanol can be antagonized with GABA antagonists.109 Unfortunately, the paucity of high affinity subtype selective ligands capable of discriminating among the various GABAA receptor subtypes has, thus far, precluded study of the precise role GABAA subunits play in mediating EtOH-maintained responding.

A series of β-carboline ligands

Biological and pharmacological testing

Methods for in vitro receptor binding and efficacy in oocytes follow previous work.129, 130 Competition binding assays were performed in a total volume of 0.5 mL at 4 °C for 1 h using [3H] flunitrazepam as the radiolabel. For these binding assays, 20–50 μg of membrane protein harvested with hypotonic buffer (50 mM Tris–acetate pH 7.4 at 4 °C) was incubated with the radiolabel as previously described.119 Nonspecific binding was defined as radioactivity bound in the presence of 100 μM diazepam and

Acknowledgments

This work was supported in part by NIMH 046851 (J.M.C.) as well as NIAAA AA016179 (DMP) and NCRR RR00168 (DMP). We acknowledge support of this work by the Research Growth Initiative of the University of Wisconsin-Milwaukee and the Lynde and Harry Bradley Foundation.

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