Laboratory Note
Syntheses and evaluation of halogenated cytisine derivatives and of bioisosteric thiocytisine as potent and selective nAChR ligands

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Abstract

We have developed one-step syntheses of halogenated derivatives of (−)-cytisine featuring a halogen substituent at positions 3, 5 or 3 and 5 of the 2-pyridone fragment, and prepared the novel bioisosteric thiocytisine by oxygen–sulphur exchange. The affinities of these pyridone-modified analogs of (−)-cytisine for (α4)2(β2)3 and α7* nAChRs in rat forebrain membranes were determined by competition with (±)-[3H]epibatidine and [3H]MLA, respectively. The 3-halocytisines 7 possess subnanomolar affinities for (α4)2(β2)3 nAChRs, higher than those found for (−)-cytisine as well as for the 5-halocytisines 8 and 3,5-dihalocytisines 6. In contrast to the parent alkaloid the 3-halogenated species display much a higher affinity for the α7* nAChR subtype. The most potent molecule was 3-bromocytisine (7b) with preferential selectivity (200-fold) for the (α4)2(β2)3 subtype [Ki=10 pM (α4β2) and 2.0 nM (α7*)]. Replacement of the lactam with a thiolactam pharmacophore to thiocytisine (12) resulted in a subnanomolar affinity for the (α4)2(β2)3 nAChR subtype (Ki=0.832 nM), but in a drastic decrease of affinity for the α7* subtype; thiocytisine (12) has a Ki value of 4000 nM (α7*), giving a selectivity of 4800-fold for the neuronal (α4)2(β2)3-nAChR and thus displaying the best affinity–selectivity profile in the series under consideration.

Introduction

There is accumulating evidence that ligands acting with high agonistic affinity at nicotinic acetylcholine receptors (nAChRs) may possibly be utilised as therapeutics [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18] in the treatment of various neurological and mental disorders related to a decrease in cholinergic function: these include, e.g. senile dementia of the Alzheimer type, Parkinson's disease, attention deficit hyperactivity disorder, Tourette's syndrome, depression and ulcerative colitis, in addition to nicotine addiction, tardive dyskinesia and schizophrenia. Moreover, worldwide interest in nAChR agonists as potential analgesics has emerged, representing an attractive area of research in pain control [1], [2], [4], [6], [11], [14]. Although several promising ligands for nAChRs have been developed [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18] during the past few years, there is still a need for potent agents that would interact more selectively with the neuronal nicotinic receptors and display no or minimal side-effects as compared with naturally occurring prototypical agonists for nAChRs such as (−)-nicotine (1) or (−)-epibatidine (2) [1], [4], [10], [18], whose therapeutic usefulness is limited by several untoward effects. (see figure 1.)

1,2,3,4,5,6-Hexahydro-1,5-methano-pyrido[1,2-a][1], [5]diazocin-8-one, (−)-cytisine (3) [19], [20] with numbering through the text, easily accessible by extracting seeds from Laburnum anagyroides medicus (Fabaceae) [21], showed a remarkable combination of properties, sharing various physiological effects with (−)-nicotine (1) [22]. It is acutely toxic and its toxicological responses include nausea, convulsions, and death by respiratory failure. In comparison with (−)-nicotine (1), however, (−)-cytisine (3) is a more potent nAChR ligand, displaying higher selectivity toward the (α4)2(β2)3 nAChR subtype combined with subnanomolar affinity [22], [23], [24], [25]. Advantageously the Laburnum-alkaloid shows a longer half-life in vivo than (−)-nicotine [26], crosses the blood–brain-barrier [18] and exhibits comparable efficacy and potency to (−)-nicotine in stimulating dopamine release from striatal synaptosomes [18].

Until now, only a few structure–activity relationship (SAR) studies on (−)-cytisine derivatives have been reported [18], [27]. Thus, in our effort to gain novel nAChR agonists [28], [29] with possibly improved pharmacodynamic profiles and safety over the natural alkaloids 1, 2 and 3, we decided to synthesise a number of structurally modified derivatives of (−)-cytisine. Replacement of a hydrogen atom by a halogen in one or more specific positions of a biologically active lead compound may substantially improve the intrinsic biological activity [15], [30], [31]. Thus, we investigated the influence of halogen substituents such as chlorine, bromine and iodine on the in vitro affinity for (α4)2(β2)3 and α7* nAChRs, in order to gain further insight into the SAR of these species.

In addition we were interested in the divalent bioisosteric replacement [32] of the lactam pharmacophore, the hydrogen bond acceptor functionality of which is required for maintaining biological activity [4], [23]. Since bioisosteric replacement of a lactam by a thiolactam pharmacophore has been used extensively in medicinal chemistry, retaining activity both in vitro and in vivo [32], we anticipated that an oxygen–sulphur exchange in the pharmacophoric lactam moiety might lead to a retention of activity or even enhanced potency associated with higher selectivity. This would reveal whether the size of the substituents and the presence of hydrogen bond acceptors were important factors for the affinity of the nAChR ligand. Such compounds might also possess reduced toxicity.

A recent paper describing the synthesis of analogs of (−)-cytisine for in vivo studies of nAChRs using PET [33] (positron emission tomography) and several patents [18], [34] claiming (−)-cytisine (3) and its derivatives as useful agents for the treatment of neurogenerative diseases and their use in addiction therapy prompted us to report our own independently obtained results.

In this communication we describe one-step preparations of several halogenated cytisine derivatives and the first synthesis of the previously unknown thiocytisine, as well as the in vitro binding affinities of the target molecules for (α4)2(β2)3 and α7* nAChRs, determined by competition with (±)-[3H]epibatidine [35] and [3H]MLA, respectively [36].

Section snippets

Chemistry

(−)-Cytisine (3) is a chiral quinolizidine alkaloid with a tricyclic skeleton, consisting of the A-, B-, and C-rings. It is characterised by a bispidine framework 4 fused to a 2-pyridone moiety. The constitution of 3 has been elucidated by chemical degradation [37] and by syntheses [38], [39], [40], [41], [42]; the absolute configuration of the two chiral centers was established by Okuda et al. to be 7R,9S [37]. The solution and crystal structure of the alkaloid 3 has been studied using NMR

Pharmacology

The cytisine derivatives listed in table I were tested for their in vitro affinity for (α4)2(β2)3 and α7* nAChRs subtypes by radioligand binding assays. To determine the affinities for the (α4)2(β2)3 nAChR subtype a previously described competition assay was used with (±)-[3H]epibatidine and the P2 membrane fraction of Sprague–Dawley rat forebrain. These studies demonstrated that the specific binding of (±)-[3H]epibatidine to crude synaptic membranes of rat forebrain, at concentrations up to

Results and discussion

We have demonstrated that halogenation of the 2-pyridone moiety of (−)-cytisine (3) can be carried out successfully in a one-step approach utilising (−)-cytisinium acetate (5), in situ prepared in aqueous acetic acid (60%) as the starting material. The syntheses of the twofold substituted halocytisines of type 6 were accomplished with yields better than 80%, employing a twofold excess of the halogenating agent mentioned. The same method proved useful in the preparation of the 3-halocytisines 7

Conclusions

In our continuing efforts to develop new ligands with high affinity and better selectivity for the multivarious subtypes of nAChRs, we have prepared and evaluated several analogs of the highly toxic alkaloid (−)-cytisine (3). We found that several of these (−)-cytisine-based compounds with halogen atoms as the substituents at position 3, 5, or 3 and 5 of the 2-pyridone fragment or characterised by a bioisosteric thiolactam pharmacophore instead of the lactam functionality proved to be highly

General chemistry

Standard vacuum techniques were used in the handling of the air-sensitive materials. Melting points were determined on a ‘Leitz-Heiztischmikroskop’ HM-Lux and are uncorrected. Solvents were dried and freshly distilled before use according to literature procedures. IR spectra were recorded on a Perkin–Elmer 257, 398 and a Nicolet FT-IR spectrometer 510-P; liquids were run as films, solids as KBr pellets. 1H NMR and 13C NMR were recorded on a JEOL JNM-GX 400 and LA 500 and δ values are given in

Supplementary material

Crystallographic data (excluding structure factors) for the structure(s) reported in this paper have been deposited with the Cambridge Crystallographic Data Center as supplementary publication no. CCDC-144569. Copies of the data can be obtained free of charge on application to The Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44-1223-336033; e-mail: [email protected]).

Acknowledgements

Financial support from Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie is gratefully acknowledged. We thank Bayer, A.G., Merck, A.G., and Degussa, A.G. for supplying us with valuable chemicals.

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