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Allosteric Interactions of Quaternary Strychnine and Brucine Derivatives with Muscarinic Acetylcholine Receptors Parviz Gharagozloo,*,† Sebastian Lazareno,† Angela Popham,† and Nigel J. M. Birdsall‡ Medical Research Council Collaborative Centre, 1-3 Burtonhole Lane, Mill Hill, London, NW7 1AD, U.K., Division of Physical Biochemistry, National Institute for Medical Research, Mill Hill, London, NW7 1AA, U.K. Received November 24, 1997
The affinity and allosteric properties of 22 quaternary derivatives of strychnine and brucine at the m1-m4 subtypes of muscarinic receptors have been analyzed and compared. The subtype selectivity, in terms of affinity, was in general m2 > m4 > m1 > m3. The highest affinities were found for N-benzyl, N-2-naphthylmethyl, and N-4-biphenylylmethyl strychnine (13, 14, and 18, respectively). All the strychnine and brucine derivatives were positively cooperative with the antagonist, N-methylscopolamine, at m2 receptors and, in the case of the strychnine analogues, were positively cooperative with N-methylscopolamine at least at one other subtype. The strychnine analogues were negatively cooperative with the neurotransmitter, acetylcholine, at all subtypes whereas brucine and five of the six derivatives examined were positively cooperative with acetylcholine at one or more subtypes (m1-m5) and exhibited different patterns of subtype selectivity. The ability to generate subtype-selective allosteric enhancers of acetylcholine binding and function may be of use in the development of drugs for the treatment of Alzheimer’s disease. Introduction Muscarinic receptors have a widespread distribution in both the central and peripheral nervous systems where they mediate the actions of released acetylcholine (ACh). These actions include the stimulation of smooth muscle contraction, glandular secretion, modulation of cardiac function, motor control, temperature regulation, and memory.1 There are five subtypes of muscarinic receptors (m1-m5), each with a characteristic regional distribution.2,3 In addition, different biochemical and electrophysiological responses are associated with the different subtypes.1-3 Potential targets for subtypeselective agents include disorders of intestinal motility, cardiac and urinary bladder function, asthma, analgesia, and Parkinson’s disease.2
Major efforts are also being made to develop agents which act to alleviate the effects on cognition of the cholinergic deficit present in the early stages of Alzheimer’s disease. Such agents include cholinesterase inhibitors4,5 (to prolong the actions of ACh) and muscarinic agonists6-9 (as a replacement therapy). However, cholinesterase inhibition results in prolonged and nonselective stimulation of all cholinergic receptors, both * To whom correspondence should be sent. Phone: 44 (0) 181-906 3811. Fax: 44 (0) 181-906 1395. E-mail:
[email protected]. † Medical Research Council Collaborative Centre. ‡ National Institute for Medical Research.
Figure 1. Ternary complex model of allosteric action of a ligand L with an allosteric agent X at a receptor R.
nicotinic and muscarinic. Equally there are no muscarinic agonists of substantial selectivity,2,10 at least based on “affinity”. We have been investigating an alternative approach, one which aims to increase the efficiency of the binding interaction between ACh and its receptors in a subtype-selective manner. Muscarinic receptors have a second (allosteric) binding site in addition to the site which binds acetylcholine and competitive antagonists.11-15 Occupancy of the allosteric site modulates but does not abolish the actions of ligands binding to the primary site. The interactions follow the allosteric ternary complex model (Figure 1), where KX and KL are the affinity constants for the binding of the allosteric ligand, X, and the primary ligand, L, respectively.12,13,15 The value of R defines the nature of the cooperative interaction. For R < 1 there is negative cooperativity, i.e., X and L inhibit the binding of each other, whereas if R > 1 there is positive cooperativity, i.e., X and L increase the binding of each other. In situations where R ) 1, there is neutral
10.1021/jm970799y CCC: $18.00 © 1999 American Chemical Society Published on Web 01/27/1999
Quaternary Strychnine and Brucine Derivatives
cooperativity, i.e., X and L have no effect on the equilibrium binding of each other to the receptor. The allosteric interactions with muscarinic receptors were first quantitated using gallamine as the allosteric ligand.12,13 A number of other ligands were later shown to interact allosterically,14 but like gallamine, all these ligands were negatively cooperative with agonists and antagonists. More recently the neuromuscular blocker alcuronium 1 has been reported to enhance the binding of the antagonist N-methylscopolamine (NMS) to m2 receptors.16 Subsequently we15 and others17 also discovered that the alkaloid strychnine 2 exhibited allosteric properties similar to alcuronium at muscarinic receptors.
Strychnine is the prerequisite starting material for the synthesis of alcuronium and its analogues and, in simplest terms, may be viewed as a monomer of the functionalized dimer, alcuronium. Although strychnine is much smaller than alcuronium, it binds with similar affinities to the unliganded and NMS-liganded m1, m3, and m4 receptors.15 The major difference in the binding of the two agents is at the unliganded and NMSliganded m2 receptors where alcuronium exhibits a greater than 10-fold higher affinity than strychnine. There are also differences in the cooperativities of the two compounds with an antagonist (NMS) and agonist (ACh). For example, with NMS as antagonist, strychnine manifests neutral cooperativity at m1 receptors and positive cooperativity at m4 receptors,15 whereas alcuronium inhibits NMS binding to m1 receptors and is neutrally cooperative at m4 receptors.19 In addition, alcuronium is inhibitory against ACh at all muscarinic subtypes18,19 whereas strychnine approaches neutral cooperativity with ACh at m1 and m4 receptors.15 The identification of strychnine as a muscarinic allosteric agent prompted us to search for other closely related structures. This work led to our discovery that another well-known alkaloid, brucine 3 (10,11-dimethoxy strychnine), is an allosteric muscarinic ligand,20 a result which has now been confirmed.18,21 Brucine binds with about 2-10-fold lower affinity to the unliganded and NMS-liganded m1-m4 receptors than strychnine.20,21 Despite its low affinity for muscarinic receptors, brucine is of special interest since it is the only allosteric agent reported so far to increase both the affinity and functional potency of ACh at m1 receptors.20 The binding data for strychnine and brucine indicate that small changes in ligand structure (i.e., the two methoxy groups in brucine) result in different allosteric properties and hence different pharmacological properties. This is as one expects, since the effect of chemical modification of an allosteric ligand on its binding to the unliganded and liganded receptors is unlikely to be
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Scheme 1. Quaternization Reactions of Strychnine and Brucine
equal. There is therefore the potential for discovering related allosteric agents which exhibit positive, neutral, or negative cooperativities with ligands which occupy the primary binding site (including, and especially, the endogenous agonist). Since the size and nature of the cooperativity of an allosteric ligand depends on its affinity for both the unliganded and liganded state of a receptor, it follows that the analysis of both sets of structure-activity relationships (SARs) is necessary for the design of such agents. The complex structures of these alkaloids somewhat limit their usefulness as versatile chemical templates for the development of SAR series. Quaternization of the basic tertiary nitrogen atom appeared as the most appropriate synthetic strategy to generate a wide range of analogues or derivatives, taking into consideration that alcuronium itself contains quaternary ammonium centers. In this paper, we examine the effect of quaternization of both alkaloids on the affinity and cooperativity with an antagonist (NMS) and agonist (ACh) at the m1-m4 subtypes. A preliminary report of some of the findings has been published.22 Chemistry The quaternization of strychnine and brucine with a variety of alkyl halides were carried out using either method A or B according to Scheme 1. In general the reactions proceeded in good yields but there were a number of reactions which failed, presumably in part due to the presence of electron-withdrawing groups on the R-carbon atom of the alkyl halide. Strychnine and brucine did not undergo quaternization with the following alkyl halides: bromomethyl methyl ether, 2-bromoethyl methyl ether, 2-iodo-1,1,1-trifluoroethane, 2-iodoethanol, 2-bromomethyl-1,3-dioxolane, and 2-(bromomethyl)tetrahydropyran. Strychnine N-oxide 11 was prepared from the reaction of strychnine and hydrogen peroxide.23 N-Amination of brucine and strychnine to give 12 and 22, respectively, was achieved with omesitylenesulphonylhydroxylamine, following a literature procedure described for N-amination of tertiary amines.24,25 N-Chloromethyl brucine20 was prepared following a procedure similar to that reported for N-chloromethyl strychnine.26
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Table 1. Estimated log Affinities (log M-1) of the Quaternary Derivatives of Strychnine at the Free and NMS Liganded Muscarinic (m1-m4) Receptorsa
m1 compd 2 4 5 6 7 8 9 10 11 12 13 14 15 16 17
R H CH3 CH2CH3 CH2CH2CH3 C6H11CH2 CH2CHdCH2 CH2CtCH CH2CN ONH2 PhCH2 PhCH2CH2 m-NO2PhCH2 p-NO2PhCH2
18 1
alcuronium
m2
m3
m4
free
NMS
free
NMS
free
NMS
free
NMS
4.9
