Heterocycles as physiological ligands for the benzodiazepine receptor and for other binding sites

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HETEROCYCLES AS PHYSIOLOGICAL LIGANDS FOR THE BENZODIAZEPINE RECEPTOR AND FOR OTHER BINDING SITES JOHANNES WILDMANN Institut für Biochemie, Abteilung Enzymchemie der Georg-August- Universität, D- 3400 Göttingen Received in final form 14 April 1989

SUMMARY Recently pharmacologically active benzodiazepines, including diazepam, have been identified in common foodstuffs, e .g. wheat and potato . The chronical intake by way of plant food may explain the existence of benzodiazepines in the brain and in other tissues of various mammalians and man . Hitherto these alkaloid-like compounds were considered to be merely products of industrial synthesis . All the benzodiazepines used in therapy show a similar chemical structure . However, depending on particular substituents, agonistic benzodiazepines can he subdivided into groups of different pharmacological potency . Besides benzodiazepines, in the past years other alkaloid drugs, e .g . derivatives of morphine, norharmane and tetrahydronorharmane, have been isolated from animals . Some of these substances have been discussed as physiological ligands of specific neuronal binding sites . Indications have been provided that at least part of these compounds or their precursors may be of plant origin too . The presence of these compounds in plants used for food may suggest complex interactions between plant and animal, exceeding the nutritional aspect . Kt:v woRios :

heterocycles, benzodiazepine structure, physiological ligand . BENZODIAZEPINES AND THEIR BINDING SITES

Since the introduction of chlordiazepoxide (Librium, 1960) and diazepam (Valium, 1961) benzodiazepine tranquillizers have become the remedy of choice for the treatment of anxiety and insomnia . In addition they have been used as antiepileptics because of their anticonvulsant properties . However, evidence was not provided until the mid-1970s that all the main benzodiazepine effects were due to their impact on the GABA ergic system [ 1, 2], which represents the most important neuronal inhibitory network in mammalian brain . When specific neuronal benzodiazepine binding sites were detected [3, 4], the site of benzodiazepine action was elucidated . The benzodiazepine receptor was found to be part of a complicated molecular complex comprising the Cl - ionophore, the GABA binding sites and additional 1043-6618/89/060673-10/$03 .00/0

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drug recognition sites . The receptor complex has been proposed to be a tetramer consisting of two sets of a- and #-subunits [5]. It shows striking structural similarities to other chemically gated ion channel receptors . An exceptional situation was found for the benzodiazepine receptor : besides agonistic ligands (e .g . benzodiazepines), which facilitate GABA induced opening of the Cl - ionophore, inverse agonistic ligands were detected, which show effects exactly opposite to those of the benzodiazepines . Some /3-carboline derivatives are potent inverse agonists at the benzodiazepine receptor [6] . Benzodiazepine receptor antagonists are also known [7] . Pure antagonists like flumazenil exhibit no intrinsic efficacy when occupying benzodiazepine receptors . Agonists, inverse agonists and antagonists are thought to bind to special-possibly overlapping-domains of the same benzodiazepine receptor site . The GABA/benzodiazepine receptor complex might only be modulated effectively by a bidirectional impact of agonists and inverse agonists . The existence of the receptor complex in the brain of all vertebrates leads to the suggestion that endogenous ligands may exist for these binding sites . Conservation during evolution indicates a function in signal transmission . During the past 10 years considerable efforts have been made to elucidate natural agonists of the benzodiazepine receptor. A variety of candidates were suggested but no definite compound emerged [8] .

STRUCTURAL CHARACTERIZATION OF BENZODIAZEPINE RECEPTOR AGONISTS Generally, drug effects, both endogenous and exogenous, may be initiated by molecular interactions with respective recognition sites . In the case of potent drugs, which, like the benzodiazepines, already act in nM concentrations, such interactions must be highly specific . Drug action is defined by the affinity to the recognition site and the intrinsic efficacy and may be affected by kinetic and metabolic parameters . When endogenous and artificial ligands occupy the same recognition site, prompting the same physiological effects, this could be due to structural similarities of both kinds of ligands . Therefore the structural analysis of a drug, known to compete specifically for a receptor site, may be helpful to develop an idea about the conformation of prospective endogenous ligands. Figure 1 gives an overview over the variety of the benzodiazepine structures, which were used in therapy up to 1985 . Generally it is believed that all agonistic benzodiazepines show the same mode of action . Some compounds are not pharmacologically active per se, but require molecular modification to become effective and therefore can be considered as prodrugs . The active metabolite of a number of benzodiazepines acting as prodrugs is nordazepam (N-desmethyldiazepam) . Examples are chlordiazepoxide, chlorazepate, halazepam, oxazolam, pinazepam or prazepam. Medazolam and ketazolam are activated by transformation to diazepam. Diazepam itself becomes desmethylated to nordazepam, emphasizing the central role which nordazepam plays among the benzodiazepines .

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N-Desmethylation and C-3-hydroxylation are major steps in benzodiazepine metabolization followed by glucuronidation which terminates pharmacological activity and allows urinary excretion . All benzodiazepines mentioned so far are substituted at position C-7 by residues of strong electronegativity, such as the halogens Cl, F, Br or a NO, group . Electronegative substitution of C-7 dramatically increases the affinity of a respective benzodiazepine to the benzodiazepine receptor . The affinity of diazepam (IC 5t) 8 . 5 nM) exceeds about 40 times the affinity of its non-chlorinated equivalent Ro 5-3464 (ICS, 350 nM) . Introduction of a second electronegative substituent in position C-2' further increases the receptor affinity by a factor 4-5 . Delormetazepam, the respective 2'chlorinated derivative of diazepam, exhibits an IC 51 value of 1 . 8 nM . Methylation at position N-1 or hydroxylation at position C-3 do not significantly change receptor affinity, but may have pharmacokinetic implications . Substitutions at other positions clearly are disadvantageous and therefore are not to be found among the collection of therapeutically used benzodiazepines . From benzodiazepines of the nordazepam type benzodiazepine structures analogous to the dichlorinated compound delorazepam can be discerned, which are generally endowed by a higher pharmacological potency . This group comprises prodrugs (e .g. cloxazolam) and molecular variations due to the exchange of haloxazolam, flurazepam, electronegative substituents (ethyl loflazepate, clonazepam, phenazepam) and/or by N-1 methylation (flunitrazepam, fludiazepam) or C-3 hydroxylation (lorazepam, lormetazepam) . 1,2-Annelated benzodiazepines, such as the imidazo and triazolo derivatives midazolam and triazolam, are thought to be transformed to the respective ketones at C-2 [9] . In conclusion the total collection of benzodiazepine tranquillizers, which were therapeutically available by 1985 may be divided into merely two groups, the 7-mono- and 7,2'-disubstituted compounds, which in their turn can be derived from nordazepam and delorazepam . The mono- and dichlorinated benzodiazepines, however, are metabolically not interrelated as transformation and degradation follow separate pathways and have no metabolites in common . N N

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Actually a third metabolically independent group can be constituted : the 2'monosubstituted benzodiazepines . Although such compounds are generally endowed by a high receptor affinity [10], no representative of this group is used in therapy so far . Interestingly, nordazepam shows in part a peptide formation (Fig . 2) . Excepting the chlorine residue and replacing the C-5 phenyl substituent by a carbonyl group results in a 1,4-benzodiazepine, which can be considered as a cyclic dipeptide, formed by the ß-amino acid anthranilic acid and glycine (Fig . 2) . Glycine can be substituted for other a-amino acids . The exchange of glycine by sarcosine leads to a compound structurally resembling the benzodiazepine antagonist flumazenil (Fig . 2) .

IDENTIFICATION OF ENDOGENOUS LIGANDS These considerations lead us to search for endogenous benzodiazepine receptor ligands as compounds, structurally closely related to synthetic benzodiazepines of the nordazepam or delorazepam type [11] . This idea eventually resulted in the isolation of eight different benzodiazepines (Fig . 3) including nordazeparn and delorazepam itself from plants commonly utilized for human consumption, i .e. wheat and potato [12] . Among the benzodiazepines identified from biological sources are non-chlorinated, 7-chlorinated, 7,2'-dichlorinated and also 2'chlorinated compounds . All of them are closely related structurally . Of special interest is 7-deschloro-2'-chlorodiazepam, which exhibits higher receptor affinity (IC, 3 .8 nM) than its isomer diazepam. The compound shows a moderate GABA shift in vitro and is not sedative in mice up to 5 mg/kg [13], indicating properties of a partial agonist. While the natural 7-chlorinated and 7,2'-dichlorinated benzodiazepines are full agonists, the 2'-monochlorinated compound isodiazepam shows a lower intrinsic efficacy. It may be of interest to examine whether this is a general property of benzodiazepine structures bearing only one electronegative substituent at C-2' .

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The chronic intake of traces of benzodiazepines as food constituents may provide a basis to explain the presence of nordazepam and diazepam in the brain and other organs of rats [14] not treated with benzodiazepines . Interestingly nordazepam was first independently isolated as an endogenous ligand of the benzodiazepine receptor from mammalian brains using a completely different method [15] but which is to a certain extent also based on structural prerequisites . In this case,-monoclonal antibodies, directed against the benzodiazepine compound 3-hemisuccinyloxyclonazepam, were used in immunoaffinity chromatography . Success would be attained only if the ligand to be traced showed a close structural relationship to the antigen used for the production of the monoclonal antibodies . In brains of drug free rats and cattle amounts of 5-10 ug/kg [(2-4) X 10 - ' M] of the benzodiazepines diazepam and/or nordazepam have been found [14-16] . After the administration of pharmacologically effective doses, the concentration of free benzodiazepines in the brain is thought to be in the range of 10 - s-10 - ' M [10] . Therefore the amounts of natural benzodiazepines found in brain may be considered to be physiologically relevant . Pharmacologically active benzodiazepines contained in the diet (wheat, potato, etc .) may amount a few ug per day, which is far below a single therapeutic dose (i .e . 5-10 mg diazepam) . However, it has to be kept in mind, that the chronic intake of trace amounts of benzodiazepines from foodstuffs could lead to their accumulation in the brain . Pharmacologically active natural benzodiazepines belong to the chlorinated compounds . In many cases their representatives are highly effective drugs . Chlorine and other halogens containing compounds are frequently found in marine algae and in lower fungi, and in higher plants more than 130 chlorine bearing compounds have been identified [17] . Benzodiazepines can be considered as alkaloid-like structures . Alkaloids are secondary plant metabolites originating from amino-acid metabolic pathways, comprising at least one cyclically integrated N atom . Many alkaloids show profound physiological effects in humans and animals . Since neuronal mechanisms are concerned, these drugs are in many cases psychoactive . Plants with such constituents have been known since antiquity and were used for cultic purposes . Some of the effective entities have been isolated and identified in the last or in the first half of the present century . Examples are : morphine, atropine, nicotine, cocaine and psilocybin . Several substances have become important tools in experimental pharmacology . Some are therapeutically useful or serve as lead substances in the development of synthetic drugs . The actions of many natural or synthetic alkaloid-like compounds are due to their affinity to specific binding sites localized on the outer cell membrane . For some specific binding sites, such as the imipramine recognition site, no corresponding endogenous ligands are known .

HETEROCYCLIC COMPOUNDS In the last decade peptide structures have been found to be of greatest interest as candidates of endogenous ligands . For example enkephalin and /endorphin were identified as endogenous ligands for the morphine receptors . However, there are



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hints that in animals neuroregulation might not exclusively he confined to peptides , but that heterocyclic compounds of an alkaloid-like structure may also serve . Recently it was reported that morphine and other opiate alkaloids are not only present at a concentration of grams per 100 g dry weight in distinct preparations of Papaver somniferum but are also detectable in common plants, including those used for food, but at concentrations several orders of magnitude less . Evidence could be provided for the occurrence of morphine in ppb amounts in grass and other fodder for the cow [18] . The presence of morphine and other opiate alkaloids in the mammalian brain [19, 20] and of morphine in human and bovine milk may thus originate from food [18] . According to Hazum [ 18] morphine could represent the physiological ligand of the u-opiate receptor to which enkephalins exhibit only moderate affinity . Other heterocyclic compounds of recent interest which are known to be plant constituents [21] are the ß-carbolines (harmane alkaloids) and the tetrahydro-ß-carbolines (Fig. 4) . Some representatives of the ß-carbolines are potent pharmacologically active ligands of the benzodiazepine receptor . Ethyl-ßcarboline-3-carboxylate, like most compounds of this structure, act as inverse agonists and some of them have been reported to be anxiogenic in man [22] . Recently, butyl-ß-carboline-3-carboxylate was isolated from bovine cortex and was suggested to function as a natural ligand for the benzodiazepine receptor . However, it has not yet been clarified whether this compound has agonistic or inverse agonistic properties [23] . The tetrahydro-ß-carboline derivative 5-methoxy-tryptoline and other tryptolines have been discussed as endogenous ligands of the imipramine recognition sites . They occur naturally in mammalian brain and adrenals and have an appropriate pharmacological profile and a reasonable affinity for these binding sites [24, 25 ) . The tricyclic dibenzoazepine compound imipramine has been used as an antidepressant drug for over 30 years . Its antipsychotic effect is thought to he due to its affinity to specific neuronal binding sites, which control the re-uptake mechanism of serotonin and noradrenaline . COOCH 'CH 3 N

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Reserpine, a constituent of the plant Rauwolfia serpentina, represents another member of the tetrahydro-ß-carboline family which exhibits psychoactive properties . It interferes with the neuronal storage of catecholamines and was used as a neuroleptic in the pre-chlorpromazine era .

PERSPECTIVES AND DISCUSSION Further research will be required to reveal whether traces of psychoactive substances, especially distinct heterocycles of an alkaloid-like structure, such as #carbolines, opiates, benzodiazepines or their precursors, occur frequently in food plants . Some of these substances could modulate neuronal processes in humans and animals . The hypothetical existence of 'psychamines' of plant origin could have escaped detection so far, because an alternative to the plant based nutritiondirectly or indirectly-essentially does not exist . Moreover deficiencies may not have been observed, because alkaloid-like structures-different from several vitamins-are rather inert against environmental interference and unlike the peptide modulators they are orally active . In addition, in some cases the amounts of these compounds contained in the plants may be only in the ppb range and remain near to the limit of detection . Alkaloid-like heterocycles of plant origin, occurring widely in the plant kingdom, can be taken into consideration as effectors of neuronal processes when the following criteria are fulfilled : 1. interaction with neuronal high affinity binding sites ; 2. compartmentalization (storage, neuronal release/uptake) ; 3. modulation of signal transformation (i .e . cyclase or ionophore) . Interestingly modified 1,4-benzodiazepine structures have been synthesized or isolated from microbiotic sources, interacting with binding sites different from the benzodiazepine receptors (Fig . 5) . Tifluadom is a synthetic benzodiazepine

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derivative found to bind specifically to K-opiate receptors . Among the compounds structurally related to benzodiazepine tranquillizers are also the exceptional neuroleptic clozapine, representing a chlorine bearing dibenzodiazepine, the antidepressant drug dibenzepine and the natural benzodiazepine derivative asperlicin and its synthetic variations, which are potent specific antagonists of the peptide hormone cholecystokinin (CCK) [26] . The phenomenon that structurally closely related compounds, belonging to the benzodiazepine or the harmane family, exhibit affinities for binding sites mediating completely different physiological effects, gives rise to the question of whether these structures may represent molecular principles favourable to serve in ligand/ receptor interactions . The detection of natural benzodiazepine receptor ligands and other mood modulating substances in animals and in plant nutritives may open a new field in biochemical-neuropharmacological investigation . First, it will be necessary to establish the incidence of the corresponding natural psychoactive compounds . Second, functional aspects in the animals will become a matter of interest . In the future this area might become as important as the peptide endogenous effectors .

REFERENCES I . Costa E, Guidotti A, Mao CC . Evidence for involvement of GABA in the action of benzodiazepines: studies on rat cerebellum . Adv Biochem Psychopharmacol 1975 : 14 : 113-30 . 2 . Haefely W, Kulesar A, Mähler H, Pieri L, Polc P, Schaffner R . Possible involvement of GABA in the central actions of benzodiazepines. Adv Biochem Psychopharmacol '975 : 14 : 131-51 . 3 . Squires RF, Braestrup C . Benzodiazepine receptors in the rat brain . Nature 1977 : 266 : 732-4 . 4 . Mähler H, Okada T. Benzodiazepine receptor : demonstration in the central nervous system . Science 1977 ; 198 : 849-51 . 5 . Schofield PR, Darlison MG, Fujita N, et al. Sequence and functional expression of the GABA receptor shows a ligand-gated receptor super-family. Nature 1987 ; 328 : 221-7 . 6 . Braestrup C, Nielsen M, Honore T. Benzodiazepine receptor ligands with positive and negative efficacy . In: Mandel P, De Feudis FV, eds . CNS receptors : from molecular pharmacology to behavior . New York : Raven Press, 1982 : 237-45 . 7 . Hunkeler W, Mähler H, Pieri L, et al. Selective antagonists of benzodiazepines . Nature 1981 ; 290 : 514 . 8 . Haefely W. Endogenous ligands of the benzodiazepine receptor . Pharmacopsychiat 1988 ;21 :43-6 . 9 . Schröder E, Rufer C, Schmiechen R . Tranquilizer . In : Arzneimittelchemie . Stuttgart : Thieme Verlag, 1976 ; 1 : 308-23 . 10 . Haefely W, Kyburz E, Gerecke M, Mähler H . Recent advances in molecular pharmacology of benzodiazepine receptors and in the structure-activity relationship of their agonists and antagonists . Adv Drug Res 1985 ; 14 : 165-322 . 11 . Wildmann J, Niemann J, Matthaei H . Endogenous benzodiazepine receptor agonist in human and mammalian plasma . JNeural Transm 1986 ; 66 :151-60 . 12 . Wildmann J, Vetter W, Ranalder U, Schmidt K, Maurer R, Mähler H . Occurrence of pharmacologically active benzodiazepines in trace amounts in wheat and potato . Biochem Pharmacol 1988 ; 37: 3549-59 . 13 . Wildmann J . The natural benzodiazepine isodiazepam is a partial agonist at the benzodiazepine receptor . Biol Chem Hoppe Segler 1988 ; 369 : 1217 .



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14. Wildmann J, Möhler H, Vetter W, Ranalder U, Schmidt K, Maurer R. Diazepam and Ndesmethyldiazepam are found in rat brain and adrenal and may be of plant origin . J Neural Transm 1987 ; 70 : 383-98 . 15 . Sangameswaran L, Fales HM, Friedrich P, De Blas AL . Purification of a benzodiazepine from bovine brain and detection of benzodiazepine-like immunoreactivity in human brain . Proc Natl Acad Sci 1986 ; 83 : 92 36-40 . 16 . Medina JH, Pena C, Piva M, Paladini AC, De Robertis E . Presence of benzodiazepinelike molecules in mammalian brain and milk . Biochem Biophys Res Commun 1989; 152 : 534-9 . 17 . Engvild KC. Chlorine-containing natural compounds in higher plants . Phytochemistry 1986 ; 25 : 781-91 . 18 . Hazum E, Sabatka JJ, Chang K-J, Brent DA, Findley JW, Cuatrecasas P . Morphine in cow and human milk : could dietary morphine constitute a ligand for specific morphine (lc) receptors? Science 1981 ; 213 : 1010-12 . 19. Weitz CJ, Lowney LI, Faull KF, Feistner G, Goldstein A . Morphine and codeine from mammalian brain. Proc Natl Acad Sci 1986 ; 83 :9784-8 . 20 . Donnerer J, Oka K, Rice KC, Spector S . Presence and formation of codeine and morphine in the rat . Proc Natl Acad Sci 1986 ; 83 : 4566-7 . 21 . Holmstedt B. Betacarbolines and tetrahydroisoquinolines : historical and ethnopharmacological background . Progr Clin Biol Res 1982 ; 90 : 3-13 . 22 . Dorow R, Horowski R, Paschelke G, Amin M, Braestrup C . Severe anxiety induced by FG 7142, a ß-carboline ligand for benzodiazepine receptors . Lancet 1983 ; ii : 98-9 . 23 . Medina JH, Novas ML, De Robertis E, Pena C, Paladini AC . Identification of a potent endogenous benzodiazepine binding inhibitor from bovine cerebral cortex . In: Racagni G, Donoso AO, eds . GABA and endocrine function. New York : Raven Press, 1986 . 24 . Barbaccia ML, Melloni P, Pozzi O, Costa E . [ 3 H]Imipramine displacement and 5HT uptake inhibition by tryptoline derivatives : in rat brain 5-methoxytryptoline is not the autocoid for [3H]imipramine recognition sites . EurJ Pharmacol 1986 ; 123 : 45-52 . 25 . Langer SZ, Lee CR, Schoemaker H, Segonzac A, Esnaud H . 5-Methoxytryptoline and close analogs as candidates for the endogenous ligand of the 3 H-imipramine recognition site. In : Lal H, ed . Endocoids. New York: Alan Liss, 1985 :441-55 . 26. Evans BE, Bock MG, Rittle KE, et al. Design of potent, orally effective, nonpeptidal antagonists of the peptide hormone cholecystokinin . Proc Natl Acad Sci 1986 ; 83 : 4918-22 .