Conformation, action, and mechanism of action of neuromuscular blocking muscle relaxants

Pharmacology & Therapeutics 98 (2003) 143 – 169 www.elsevier.com/locate/pharmthera

Conformation, action, and mechanism of action of neuromuscular blocking muscle relaxants$ Chingmuh Lee* Department of Anesthesiology, Harbor-UCLA Medical Center Campus of UCLA School of Medicine, 1000 West Carson Street, Torrance, CA 90274, USA

Abstract Since curare was introduced into clinical anaesthesia in 1942, efforts to create better neuromuscular blocking (NMB) muscle relaxants have continued. Today, muscle relaxation remains a mainstay of modern anaesthesia and intensive care. Through manipulation of the traditional structure-action relationships, many new and improved muscle relaxants have been created, and several have been brought to clinical use. However, structure-action relationship is inconsistent and has its limits. Using computer-aided molecular conformational analyses, the conformation-action relationships of NMB agents of various chemical classes have been explored. Conformation, no less than structure, of the NMB agents has shed new light on their mechanisms of action. By reflection, the conformations also suggest new details of the topology of the receptive sites of the nicotinic acetylcholine receptor modeled for the motor endplate of the skeletal muscle. D 2003 Elsevier Science Inc. All rights reserved. Keywords: Structure-action relationship, muscle relaxants; Conformation-action relationship, muscle relaxants; Mechanism of action, muscle relaxants; Nicotinic acetylcholine receptor, motor endplate; Receptive site topology, nicotinic acetylcholine receptor Abbreviations: ACh, acetylcholine; AChR, acetylcholine receptor; C10, decamethonium; CAR, conformation-action relationship; dTc, d-tubocurarine, (+)tubocurarine; GA, genetic algorithm; mTc, metocurine; NMB, neuromuscular blocking; N-N distance, distance between two quaternary N atoms; N-Ovdw distance, distance from the centre of an N atom to the van der Waals extension of an O atom; SAR, structure-action relationship; SDC, succinyldicholine, succinylcholine, suxamethonium; TAAC3, bis[N-(3,4-diacetoxybenzyl)tropanium-3a-yl] glutarate dibromide; TEA, tetraethylammonium; vdw, van der Waals.

Contents 1. 2. 3.

4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The nicotinic acetylcholine receptor model for neuromuscular transmission . . . . . . . . . Historical highlights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Acetylcholine, (+)-tubocurarine, and metocurine . . . . . . . . . . . . . . . . . . . 3.2. Leptocurare and pachycurare . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Pioneering work on the conformation and action of acetylcholine and neuromuscular blocking agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Molecular conformations for nicotinic and muscarinic actions . . . . . . . . . . . . 3.5. Conformation of acetylcholine bound to nicotinic acetylcholine receptor . . . . . . . 3.6. Polymethylene bismethonium congeners . . . . . . . . . . . . . . . . . . . . . . . 3.7. Succinylcholine: its past, present, and future . . . . . . . . . . . . . . . . . . . . . ˚ rules. . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8. The 10-atom and the 14-A 3.9. Prototype bis-tetrahydroisoquinolinium muscle relaxants . . . . . . . . . . . . . . . 3.10. Pancuronium and vecuronium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Recent trends in structure-action relationship exercises and drug development . . . . . . . . 4.1. In search of a non-depolarizing succinylcholine . . . . . . . . . . . . . . . . . . . .

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144 145 146 146 147

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147 147 148 148 149 149 150 150 150 150

$ Disclosure: The author, his associates, and his institutions of association have financial interest in TAAC3 and other bis-tropinium compounds periodically mentioned in the article. * Tel.: 310-222-3863; fax: 310-791-7321. E-mail address: [email protected] (C. Lee).

0163-7258/03/$ – see front matter D 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0163-7258(03)00030-5

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4.2. Non-enzymatic breakdown for shorter duration of action . . . . . . . . . . . . . . . . . 151 4.3. Chiral-specific benzylisoquinolinium muscle relaxants . . . . . . . . . . . . . . . . . . 151 4.4. Molecular asymmetry in novel diester neuromuscular blocking agents . . . . . . . . . . 151 4.5. Rapacuronium as the last mono-quaternary muscle relaxant in clinical anaesthesia? . . . 152 5. Computer analysis of the molecular conformation of neuromuscular blocking agents . . . . . . 152 5.1. Molecular shape of low-energy conformers . . . . . . . . . . . . . . . . . . . . . . . . 152 5.2. Quantitative measurement of molecular flexibility of neuromuscular blocking agents . . 152 5.3. Local and global minimizations of energy of muscle relaxant molecules . . . . . . . . . 152 5.4. Caveats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153 6. Structure, conformation, and action of various classes of neuromuscular blocking agents . . . . 153 6.1. Decamethonium measures the inter-site distance of the endplate acetylcholine receptor . 153 6.2. Energy cost to conform decreases potency as quantitatively illustrated by decamethonium congeners . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154 6.3. Methyl and acetyl as optimal quaternizing and H bond acceptor groups . . . . . . . . . 154 6.4. Inter-methonium connecting chains of larger leptocurares and channel block . . . . . . . 154 6.5. Succinylcholine: its conformation and mechanism of action . . . . . . . . . . . . . . . 154 6.6. Succinylmonocholine not fitting the receptive site . . . . . . . . . . . . . . . . . . . . 156 6.7. Conformation, conformational energy, and pharmacodynamics . . . . . . . . . . . . . . 156 6.8. The neuromuscular blocking conformation and action of gallamine and derivatives . . . 156 6.9. The muscarinic blocking conformation and action of gallamine. . . . . . . . . . . . . . 156 6.10. The twisted A-ring of pancuronium . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156 6.11. The A-ring muscarinic blocking acetylcholine moiety of pancuronium . . . . . . . . . . 157 6.12. The D-ring acetylcholine moiety of pancuronium and vecuronium as neuromuscular blocking pharmacophore. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157 6.13. De-acylated metabolites of potent 3,17-diacyl aminosteroid neuromuscular blocking agents 157 6.14. Need of two functional groups on aminosteroid neuromuscular blocking agents . . . . . 158 6.15. Molecular conformation of pipecuronium explains its high neuromuscular blocking potency and specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158 6.16. Hydrophilic surface of neuromuscular blocking relaxant molecule faces the receptive sites 158 6.17. Planar geometry of vecuronium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158 6.18. N-N distance, ganglionic block, and neuromuscular block . . . . . . . . . . . . . . . . 159 6.19. Optimal molecular length of potent one-bulk neuromuscular blocking agents . . . . . . 159 6.20. Methoxylation improves tetrahydroisoquinolinium neuromuscular blocking relaxants . . 159 6.21. Conformation-action relationship of long-chain tetrahydroisoquinolinium neuromuscular blocking agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160 6.22. Carbonyl group and N-N distance of TAAC3 and derivatives . . . . . . . . . . . . . . 160 6.23. Potency-onset inverse relationship and a new explanation . . . . . . . . . . . . . . . . 160 6.24. Potency-side effect inverse relationship . . . . . . . . . . . . . . . . . . . . . . . . . . 161 6.25. Protonation of d-tubocurarine and vecuronium . . . . . . . . . . . . . . . . . . . . . . 162 7. Receptive site topology remodeled in the light of the conformation-action relationship of the neuromuscular blocking agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162 7.1. Inter-site space available to neuromuscular blocking agents . . . . . . . . . . . . . . . . 162 7.2. Orientation and dimension of acetylcholine-binding subsites . . . . . . . . . . . . . . . 163 7.3. Inter-site topology when two molecules of acetylcholine bind the receptive sites . . . . . 163 8. Conformational mechanism of action of various classes of neuromuscular blocking agents . . . 164 9. Comments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165 9.1. Preference for one receptive site to the other . . . . . . . . . . . . . . . . . . . . . . . 165 9.2. Other conformation-action relationship questions . . . . . . . . . . . . . . . . . . . . . 165 9.3. Conformation-action relationship of channel block . . . . . . . . . . . . . . . . . . . . 165 10. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166

1. Introduction Traditionally, we understand the neuromuscular blocking (NMB) muscle relaxants by their chemical formula and structure. Imperfect as it is, structure-action relationship (SAR) has contributed to the development of new and better muscle relaxants over the decades. However, it has

been pointed out that examination of the muscle relaxants by their molecular conformation, beyond their chemical structure, offers a better understanding of how they may block neuromuscular transmission and cause side effects (Lee, 2001c, 2002c). Furthermore, the conformation of rigid NMB agents must reflect the topology of the receptive site that they bind.

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This review stresses the conformation-action relationship (CAR) of the common NMB agents. It is written from the perspective of an anaesthesiologist, biased towards drugs of clinical relevance. Aspects of structure, conformation, and action of the NMB muscle relaxants recently covered have been updated (Lee, 2001c, 2002c). While SAR is twodimensional ‘‘key-in-lock,’’ CAR is three-dimensional ‘‘hand-in-glove.’’ Readers interested in further details of the traditional SAR of the NMB agents are referred elsewhere (Bovet, 1951; Stenlake, 1963; Kharkevich, 1990; Ducharme & Donati, 1993; Belmont et al., 1993; Hill et al., 1994; Savarese et al., 2000; Moore & Hunter, 2001; Lee, 2001c). Throughout this review, the action of an NMB agent refers mainly to its potency. For quick reference, Table 1 compiles the potency data of selected compounds from several organized sources. It is understood that potency data obtained in vivo customarily refer to the salt, injected intravenously. The tibialis anterior preparation of the cat is a popular model, although it is

Table 1 Potency data of NMB agents Compound

Human ED95 (mg/kg)

Cat ED50 (mg/kg)

dTc Chondocurine mTc Atracurium GW280340A Cisatracurium Mivacurium Doxacurium Rapacuronium 3-OH Rapacuronium Rocuronium Pancuronium Vecuronium Pipecuronium ANQ 9040 Org 7931 HS-342 HS-310 (chandonium) Gallamine Alcuronium SDC AH8165 TAAC3 C10 Undecamethonium (C11) Octadecamethonium (C18)

0.481

0.202 Potency = 2 times dTc3 Potency = 2 – 9 times dTc3

1

0.301 0.211 0.194 0.055 0.0671 0.0241 1.1 (ED90)5 0.42 (ED90)5 0.311 0.0671 0.0431 0.0421 1.36

2.821 0.221 0.269

Savarese et al. (2000). Marshall et al. (1973b). 3 Hill et al. (1994). 4 Belmont et al. (1999). 5 Official package insert. 6 Munday et al. (1994). 7 Bowman et al. (1988b). 8 Gandiha et al. (1974). 9 Kopman et al. (1999). 10 Gyermek et al. (2002b). 11 Paton and Zaimis (1949). 2

0.0132

0.11 (ED90 – 99)7 0.292,8 0.078 0.62 0.052 0.802 0.1810 0.03 (ED95)11 0.06 (ED95)11 1.5 (ED95)11

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particularly sensitive to depolarizing agents. The dose requirement in that model may differ from that of the anaesthesized human, which is usually determined by the thumb twitch evoked by stimulation of the ulnar nerve. Muscle types and many other factors also affect sensitivity.

2. The nicotinic acetylcholine receptor model for neuromuscular transmission A recent synopsis describes the nicotinic acetylcholine receptor (AChR) as being composed of five subunits arranged in the order of aeadb, clockwise looking from the synaptic side down the channel (Prince & Sine, 1998). It is unlikely for the b-subunit to be between the two asubunits (Pedersen & Cohen, 1990). Each subunit has a positive face and a negative face arranged so that the positive face always couples with the negative face of the neighbouring subunit. Thus, the negative face of the esubunit couples with the positive face of the a-subunit to make the ae receptive site for ACh. According to this model, the positively charged methonium head of ACh is attracted to the negative face of the e-subunit, although ACh itself is basically bound to the a-subunit. The ad-subunit constitutes the other receptive site (Pedersen & Cohen, 1990; Sine & Claudio, 1991; Prince & Sine, 1998). When ACh binds both the ae and the ad receptive sites, the AChR channel opens below. Occupation of either receptive site by an antagonist prevents activation of the AChR. In the fetal form, ag exists instead of ae. In general, non-depolarizing NMB agents have greater affinity for the fetal form, although there are exceptions (Fletcher & Steinbach, 1996; Savarese et al., 2000; Paul et al., 2002). In both forms, (+)-tubocurarine (dTc, d-tubocurarine) strongly prefers the ae/ag receptive site, although various NMB agents have different site preferences (Pedersen & Cohen, 1990; Fletcher & Steinbach, 1996). For brevity, throughout this review, ae will be used to mean the receptive site in either adult or fetal forms when differences in site or form are not the issue. Obviously, bis-quaternary NMB agents presumably bind both receptive sites. Readers are referred to a recent review on the neurobiology of the nicotinic AChR (Naguib et al., 2002). The nicotinic AChR channel is generally said to have a funnel-shaped lumen. The extracellular part is the cone of the funnel. The transmembrane part is a gated tube, which is closed at rest. When both receptive sites are ligand-bound, the tube opens below and becomes permeable. The lumen of the open tube is large enough for tetraethylammonium (TEA) and Tris to pass, but not cations that are much larger (Villarroel, 1998; Ortells et al., 1998). The receptive sites are located towards the upper part of the channel. Between ˚ apart; the the receptive sites, the outer borders are
50 A ˚ apart (Taylor et al., 1991). The inner borders are
20 –30 A ˚ lumen of the channel at this level has been put at 20 A across (Egebjerg, 1996). Stabilizing the infrastructure (the

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receptive sites and the channel opening and closing mechanisms below) is a large 5-subunit macromolecule of 250 ˚ , of which 60 A ˚ is kDa, with an overall length of 120 A ˚ ˚ extracellular, 40 A transmembrane, and 20 A intracellular (Taylor et al., 1991). The maximum width of the nicotinic ˚. AChR is
80 A The above model of a nicotinic AChR is adopted for this review as what the AChR at the motor endplate of the skeletal muscle would be like and what an NMB relaxant molecule is supposed to fit when blocking neuromuscular transmission (Lee, 2002b) (Fig. 1). It is understood that the relaxant molecule and the receptive site may change conformation when they interact. The state of the receptor, such as desensitization in vitro and in vivo, also changes the receptor geometry (Martyn et al., 2000). The term ‘‘fit’’ is based on the CAR, and implies only that the agonist or antagonist suits the receptive site at one point to effectuate a neuromuscular block. No docking experiments were performed for this review. Prejunctional receptors play important roles in neuromuscular transmission and block (Bow-

man, 1980; Bowman et al., 1988a; Marshall, 1991). They may have different SARs and CARs. However, they are beyond the scope of this review. All conclusions are only presumed to apply to the endplate nicotinic AChR.

3. Historical highlights 3.1. Acetylcholine, (+)-tubocurarine, and metocurine The classic experiments of Claude Bernard (1856) led to the recognition of the anatomical gap between the motor nerve and the skeletal muscle. Dale and colleagues identified ACh as the transmitter at the neuromuscular junction (Brown et al., 1936; Dale et al., 1936). In 1942, curare was introduced into anaesthesia practice in Canada (Griffith & Johnson, 1942). Originally, dTc was thought to be bisquaternary, but in 1970, its structure was revised to correctly indicate that it is mono-quaternary (King, 1935; Everett et al., 1970). With the tertiary N atom protonated (Pauling &

Fig. 1. Diagram of the nicotinic AChR modeled for the endplate of the mammalian skeletal muscle. Left panel: longitudinal section. The rectangles are the ˚ , large enough to pass TEA, but not cations receptive sites. Upper: resting receptor. Lower: activated receptor. The open trans-membrane channel measures 8 A much larger. Right panel: Cross-section at the level of the receptive sites, viewed from the synaptic side down the channel. The clockwise aeadß sequence of the subunits and their interfaces are after Prince and Sine (1998). Upper: the resting receptor with trans-membrane channel closed. The anionic centres () are at the ae and the ad interfaces. Lower: the open channel. Open trans-membrane tube is shown as dotted circle. N + (shaded) indicates a depolarizing methonium head, such as that of ACh.

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Petcher, 1973), dTc nevertheless may function as a bisquaternary NMB agent. Corresponding to the revision, metocurine (mTc) becomes O,O0,N-trimethyltubocurarine (Sobell et al., 1972), instead of O,O0-dimethyltubocurarine, as it was originally called (Fig. 2). It has two permanent quaternary onium heads. The real bis-quaternized dTc is called chondocurine. 3.2. Leptocurare and pachycurare Bovet (1951) divided NMB agents into ‘‘leptocurare’’ and ‘‘pachycurare’’ types. In general, leptocurares, such as succinylcholine (SDC, succinyldicholine, suxamethonium) and decamethonium (C10), have small onium heads and flexible interonium connecting chains. They are depolarizing. The pachycurares have bulky onium heads that are often bicyclic or polycyclic complexes. Their interonium structure can be a slim flexible chain that allows the onium heads conformational freedom, or a rigid bulky complex that makes one bulk out of the entire molecule. They are non-depolarizing, and may have great potency. 3.3. Pioneering work on the conformation and action of acetylcholine and neuromuscular blocking agents Pauling and associates (Chothia & Pauling, 1968, 1970; Chothia, 1970), as well as Spivak and associates (1986, 1989) and others (Sheridan et al., 1986), have examined the molecular conformation of ACh and its derivatives, and attempted to match their physiological functions to molecu-

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lar conformations. Pauling and Petcher (1973) have analyzed the crystal structure and correlated the three-dimensional geometry of several depolarizing and non-depolarizing NMB agents with their physiological activity. Bovet (1972) drew C10, the cyclo-octadecane derivative of C10, and laudexium with the interonium chain(s) laid in a bent configuration, similar to the fixed configuration of dTc. Citing d’Arcy and Taylor (1962a, 1962b), he attempted to explain their pharmacological similarities with their conformational likeness. 3.4. Molecular conformations for nicotinic and muscarinic actions Various cholinergic receptors and cholinesterases have different conformational requirements or preferences of their agonists (or substrates in the case of cholinesterase) and antagonists. Of the cholinergic agonists and antagonists, a 1970 report (Beers & Reich, 1970) proposed that the distance from the centre of the quaternary N to the van der Waals (vdw) extension of the respective O atom or an equivalent H bond acceptor (N-Ovdw distance) is important in determining whether a compound will be nicotinic or ˚ will impart muscarinic. An N-Ovdw distance of 4.4 A ˚ will impart muscarinic action, while a distance of 5.9 A nicotinic action (Fig. 3). The H bond acceptor is usually an ether O (i.e., -O-) in the former, but a carbonyl O (i.e., = CO) in the latter. For brevity, these two rules will be referred to in ˚ for muscathis review as Beers and Reich’s rule of 4.4 A ˚ rinic action and rule of 5.9 A for nicotinic action. The rule of

Fig. 2. dTc, mTc, atracurium, and laudanosine. Atracurium is drawn with the two benzyltetrahydroisoquinolinium heads in similar layout as in others to illustrate their structural similarities. No conformational precision is intended. See Fig. 4 for an usual presentation of atracurium. Removal of the quaternizing interonium connecting chain of atracurium by Hofmann elimination yields two molecules of laudanosine. Along one path or another, 10 heavy atoms (C, O) can be counted between the two N atoms of dTc and mTc. However, their N-N distances are fixed by the ring complex and bear no similarity to that of C10. ˚ rule are not compatible in this example. Reproduced from Lee (2001c), with permission of the copyright holder, Oxford The 10-atom rule and the 14-A University Press, Oxford.

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˚ for nicotinic action illustrated in the D-ring ACh moiety of vecuronium. The same conformer of the molecule is shown Fig. 3. Beers and Reich’s rule of 5.9 A in two views. The lower view is a side view, slightly rotated to the left for clarity. N is purple, O is red, C is gray. H atoms are hidden. All C atoms of the 16piperidinium on the D-ring are hidden so that the 16-N alone is seen unobstructed. The 17-acetyloxy carbonyl O is marked CO; the unmarked O is the ester O. ˚ ) show the N-Ovdw distance of the carbonyl Thin sticks from the O atoms show the direction and length of the dotted vdw extensions. The long arrows (5.92 A ˚ ) show the N-Ovdw distance of the ester O, in fulfillment of neither rule O, in fulfillment of Beers and Reich’s rule for nicotinic action. The short arrows (2.68 A ˚ for nicotinic nor rule of 4.4 A ˚ for muscarinic action. The carbonyl O protrudes downward and sidewise unshielded. of 5.9 A

˚ is necessary, but an insufficient condition for nicotinic 5.9 A action (Spivak et al., 1986, 1989). In neuromuscular pharmacology, it would be necessary only when the muscle relaxant depends on an intact ACh-like moiety to act by a mono-quaternary mechanism of action, not when it blocks by using two onium heads. Examples are vecuronium for the former and C10 for the latter (Lee, 2001c). Although well recognized (Goldstein et al., 1974; Spivak et al., 1986, 1989; Taylor & Insel, 1990) and quoted in the chapter on autonomic nervous system pharmacology in each edition of the authoritative textbook Anesthesia (Moss & Renz, 2000), these rules have not been validated for the NMB relaxants nor mentioned in the literature on neuromuscular pharmacology until recently (Lee, 2001c). A different version of the conformational requirement of a nicotinic pharmacophore is ˚ from the quaternary N to the carbonyl O, a distance of 4.8 A centre to centre (Sheridan et al., 1986; Abramson et al., 1991; Prince & Sine, 1998). Molecular ACh can fulfill both rules readily. The larger N-Ovdw distance for nicotinic action ˚ ) indicates that the nicotinic AChR has larger (5.9 vs. 4.4 A receptive sites, in which the anionic centre and the H bond donor subsites are farther apart. The large scale of the receptive site topology is further endorsed by the requirement of two ACh molecules acting simultaneously to open one channel. At the endplate, rapid influx of depolarizing cations is required to generate a large endplate potential rapidly.

3.5. Conformation of acetylcholine bound to nicotinic acetylcholine receptor A CAR study concluded that ACh bound to the nicotinic receptor ‘‘must be’’ in the gauche configuration (Spivak et al., 1986, 1989). A conformation of ACh bound to a Torpedo californica nicotinic AChR has been published (Behling et al., 1988). It has a bent conformation, with the two O atoms close to each other to allow the methyl groups to form an uninterrupted lipophilic shield on the other side. The gauche conformation of ACh and related compounds in aqueous solution depends on the presence of the onium group and the partial negative charge of the ß O atom (Partington et al., 1972). 3.6. Polymethylene bismethonium congeners Of the polymethylene bismethonium series of compounds, C5 –C12 and C18; namely, (H3C)3N + (CH2)nN + (CH3)3, where n = 5 –12 or 18, the classic work of Paton and Zaimis (1949) has established that C10 is optimal for neuromuscular block. In a biphasic manner, other congeners, longer and shorter alike, lose NMB potency. Instead of neuromuscular block, C5 and C6 are ganglion blockers. Congeners higher than C11 lose NMB potency, but very long congeners such as C18 show a trend toward regaining potency, while also changing toward a ‘‘non-depolarizing’’

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mechanism of action (Paton & Zaimis, 1949; Hill et al., 1994). Chemically, each congener differs from its neighbour only by one methylene group in the polymethylene chain that connects the two terminal methonium heads. Besides molecular length and a slight increment in lipophilicity with each additional methylene group, nothing structural explains their differences in pharmacological profile (Lee & Jones, 2002). 3.7. Succinylcholine: its past, present, and future SDC was first studied in 1906 as a vasopressor in already curarized animals, and was noted to slow the heart and to raise the blood pressure in a manner ‘‘similar to that of the valeryl compound’’ (Hunt & Traveau, 1906). Its NMB effect was not discovered until 1949, but was applied to clinical anaesthesia soon afterwards, in 1951 in Europe and in 1952 in the United States (Foldes et al., 1952; Lee, 1985, 1994). Although it exhibits an encyclopaedia of complications, some of them life-threatening, it still has clinical utility today (Lee, 1985, 1994). It will continue to be used in anaesthesia until replaced by a ‘‘non-depolarizing SDC.’’ Structurally, SDC is two molecules of ACh joined end on end at the acetyl side. As is in the case of ACh, it takes two molecules of SDC to open one AChR ionic channel (Marshall et al., 1990; Dilger, 1998). The requirement of two SDC to open one AChR channel was explained recently by its bent conformation (Lee, 2001c). 3.8. The 10-atom and the 14-A˚ rules The superior potency of C10 to its congeners established ˚ rule’’ for bis-quaternary NMB a ‘‘10-atom rule’’ and a ‘‘14-A agents. According to the rules, having 10 heavy (C, O) atoms

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between the quaternary N atoms is optimal for NMB block. The rule was re-enforced by the potent non-depolarizing bulky bisonium compounds, such as decamethylene bisatropinium, g-oxalolaudonium, and laudexium (Fig. 4), in which the two bulky heads are connected by a 10-atom chain (Kimura & Unna, 1950; Taylor & Collier, 1951; Brittain et al., 1961; Bovet, 1972; Kharkevich, 1990; Hill et al., 1994; Kimura et al., 1994; Lee, 2001c). Even in the one-bulk pachycurares, such as dTc, mTc (Fig. 2), and pancuronium, one can count 10 heavy atoms between the 2 N atoms along one path or another (Lee, 2001c). It should be obvious that the ‘‘10-atom rule’’ and the ˚ rule’’ are not interchangeable unless the interonium ‘‘14-A structure is straight. Diester chains of the bis-quaternary NMB agents are rarely straight because the chain is bent by the attraction between the carbonyl O and the quaternary N. When the distance between the two N atoms (N-N distance) ˚ , the ‘‘10-atom rule’’ is meaningless. In the oneis not 14 A bulk bis-quaternary NMB agents, such as mTc and pancuronium, the two onium heads are fused and the two rules are not compatible. Other bis-quaternary pachycurares with flexible connecting chains of historical interest include diplacine and DF596 (Bovet, 1972; Hill et al., 1994). For clear distinction, the term ‘‘N-N distance’’ refers to the distance between two quaternary N atoms, centre to centre, whereas the term ‘‘interonium distance’’ refers to the separation between the bulks of two onium heads. As used in this review, the former is a computed quantity (based on the lowest energy conformer available); the latter is not (Lee & Jones, 2002). At one point in rather recent history, anaesthesiology trainees were told that ‘‘nicotinic receptors’’ were separated by units of 5– 6 C atom lengths and that bisonium compounds with one, two, and three times this interonium

Fig. 4. Laudexium, atracurium, mivacurium, doxacurium, and GW280430A illustrating five decades of development of tetrahydroisoquinolinium muscle relaxants. Atoms C1 and N2 are chiral. In GW280430A, one of several possible stereoisomers, the chlorofumarate is asymmetrical and the 1-group is benzyl on the chlorine side, but phenyl on the other side.

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distance would block various nicotinic transmissions by spanning two adjacent, every other, or every third such ‘‘receptors’’ (Stenlake, 1963; Lee, 2001c). This theory was supposed to also explain why long congeners of C10 regain potency and why gallamine, with three onium heads, is advantageous over some bis-quaternary derivatives. Obviously, the ‘‘recurrent receptors’’ theory is of historical interest only. It is not compatible with modern morphology of the nicotinic AChR or the CAR of the NMB Agents. 3.9. Prototype bis-tetrahydroisoquinolinium muscle relaxants As early as 1950 and 1951, researchers published the curarizing effects of synthetic tetrahydroisoquinoline and benzyltetrahydroisoquinoline (including laudanosine) derivatives quaternized by an interonium a,w-decamethylene connection (Taylor & Collier, 1950, 1951). They pointed out great similarity between these NMB agents and that of mTc, and noted that methoxy, di-methoxy, and tri-methoxy compounds have increasing potency in that order. This SAR rule of methoxylation still applies to the modern tetrahydroisoquinolinium NMB agents (Fig. 4). These prototype compounds are reminders that progress toward better NMB agents along a pedigree takes decades.

3.10. Pancuronium and vecuronium Pancuronium was created under the belief that to be potent and non-depolarizing, an NMB agent must be rigid, bulky, and bis-quaternary (Marshall et al., 1980; Savage, 1983; Lee, 2001c). However, although the androstane ring is rather rigid, the A-ring of pancuronium has considerable flexibility (Fielding, 1998) and conformational freedom (details in Section 6.10). It is said that development of vecuronium was delayed in favour of pancuronium, supposedly because it is a ‘‘NOR’’ pancuronium; namely, not bis-quaternary (Fig. 5). Vecuronium turns out to be about equipotent to pancuronium, and nearly devoid of the vagolytic tachycardic side effect (Durant et al., 1979). It established the possibility for mono-quaternary compounds to be potent NMB agents (Marshall et al., 1980; Savage, 1983; Lee, 2001c).

4. Recent trends in structure-action relationship exercises and drug development 4.1. In search of a non-depolarizing succinylcholine In spite of its encyclopaedia of side effects and complications, SDC remains clinically useful, as the only ultrafast- and ultra-short-acting NMB relaxant available clin-

Fig. 5. Some aminosteroid NMB agents. The b (up) side is crowded by, clockwise, 2-R1, 10-CH3, 13-CH3, 17-R4, and 16-R3. Between the 2-N (R1) and the 16N (R3), 10 carbon atoms can be counted along one path or another, but the N-N distance is not equivalent to the length of a straight 10-C chain. The 10-atom ˚ rule are not compatible. In pipecuronium, the N + atoms are farthest out and the R1-R2 and the R3-R4 complexes are not ACh-like in structure rule and the 14-A or shape.

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ically (Hartley & Fidler, 1977; Lee, 1985, 1994). Most of the disadvantages of SDC are attributed to its depolarizing mechanism of action (Lee, 1985, 1994). To avoid these disadvantages, all modern NMB relaxants are non-depolarizing. An editorial by Savarese and Kitz (1975) summed up the prevailing belief that clinical anaesthesiology needs clean NMB relaxants of various durations of action. Since then, a ‘‘non-depolarizing SDC’’ has become recognized by many anaesthesiologists as the ‘‘ideal’’ muscle relaxant. Foldes and colleagues had expressed their feelings in their 1952 debut article on SDC that its duration and speed of change in degree of block are about ideal (Foldes et al., 1952; Lee, 1985). 4.2. Non-enzymatic breakdown for shorter duration of action Mivacurium and doxacurium have cholinester linkages, but while mivacurium is subject to plasma cholinesterase, doxacurium is not. It appears that mivacurium has exhausted the potential of the plasma cholinesterase for exploitation towards creation of shorter-acting non-depolarizing NMB relaxants. In fact, low plasma cholinesterase activity was already recognized as a potential source of difficulty with SDC by Foldes et al. (1952). If the original goal of using the succinate connecting chain was to create an ultra-short-acting NMB agent, then doxacurium would have been a disappointment. Fortunately, doxacurium succeeds as the most potent and clean NMB agent known, matching or exceeding pipecuronium in both aspects (Basta et al., 1988; Foldes et al., 1990; Ducharme & Donati, 1993; Lee, 2001c). In 1851, Hofmann described the elimination of an a-b carbon radical from the quaternary N at high pH and temperature (100C), as described by Stenlake et al. (1983). This is generally called ‘‘Hofmann elimination.’’ In atracurium, the connecting diester is reversed, so that atracurium is a dialcohol ester of two quaternary acids instead of a diacid ester with two quaternary alcohols (cholines) (Fig. 4). This reversed ester arrangement was designed for the acyl group to break away from the laudanosinium under physiological pH and temperature by Hofmann elimination (Stenlake et al., 1983). Atracurium is additionally subject to non-specific ester hydrolysis (Fisher et al., 1986; Hill et al., 1994; Savarese et al., 2000). Rapacuronium is rapidly hydrolyzed to 3-OH rapacuronium, spontaneously or catalyzed ‘‘by esterases of unknown identity and site’’ (quoted from official package insert of Raplon1, by Organon, Inc., West Orange, NJ, USA), not in the liver. As a result, its NMB action is not significantly altered in hepatic failure (Szenohradszky et al., 1999; Duvaldestin et al., 1999). It is short acting, although its metabolite is active and potent, and, theoretically, may lead to cumulative action. GW280430A and bis[N-(3,4diacetoxybenzyl)tropanium-3a-yl] glutarate dibromide (TAAC3) derivatives represent the two new series of diester

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compounds that are candidates for the so-called ‘‘ideal relaxant’’ (Savarese & Kitz, 1975; Boros et al., 1999; Belmont et al., 1999; Moore & Hunter, 2001; Lien, 2002; Gyermek et al., 2002b). Both have demonstrated good pharmacological profiles, and are independent of plasma cholinesterase for breakdown. GW280430A undergoes rapid degradation by chemical mechanisms, including cysteine adduction and ester hydrolysis (Lien, 2002). TAAC3 likely undergoes a spontaneous breakdown that removes the N-benzyl quaternizing group from the tropinium head (Gyermek et al., 2002b), facilitated by the 4-substitution on the benzyl group. In general, spontaneous or enzymatic breakdown in the plasma assures spontaneous recovery of neuromuscular transmission in the presence of renal or hepatic failure. It also reduces the need for pharmacological reversal of the relaxant at the end of surgery (Hunter et al., 1982; Szenohradszky et al., 1992, 1999; Hunter, 1994, 1995, 1996a; Moore & Hunter, 2001). These advantages may be offset by a reduced shelf life, which, in an extreme case, can render a superb product of SAR or CAR exercises clinically useless. 4.3. Chiral-specific benzylisoquinolinium muscle relaxants Receptors are chiral-sensitive, chiral-selective, or chiralspecific. Accordingly, dTc is more potent than l-tubocurarine, and cisatracurium is more potent than atracurium by several fold (Eastwood et al., 1995; Savarese et al., 2000; Moore & Hunter, 2001). Supposedly, their onium heads fit the receptive site(s) better. Following atracurium, doxacurium and mivacurium were synthesized as chiral-selected preparations (Hill et al., 1994; Head-Rapson et al., 1995; Savarese et al., 2000). In the future, all NMB agents probably will have to be marketed as chiral-specific or chiral-selected preparations, unless the use of a nonselective mixture of stereoisomers can be justified (Calvey, 1995). 4.4. Molecular asymmetry in novel diester neuromuscular blocking agents Although bis-quaternary pachycurares of one structural bulk, such as dTc (protonated), mTc, pancuronium, pipecuronium, and some bis-quaternary HS-aminosteroid compounds, have structural asymmetry (Gandiha et al., 1974; Savage, 1983; Kharkevich, 1990; Ducharme & Donati, 1993; Savarese et al., 2000; Lee, 2001c), traditional longchain bis-quaternary NMB agents have symmetrical connecting chains and identical onium heads. However, GW280430A, the latest tetrahydroisoquinolinium NMB agent, has an asymmetrical connecting chain and mixed benzyl/phenyl onium heads (Boros et al., 1999; Belmont et al., 1999; Savarese et al., 2000; Lien, 2002) (Fig. 4). This is interesting considering that the ae and the ad receptive sites of the nicotinic AChR are asymmetric.

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4.5. Rapacuronium as the last mono-quaternary muscle relaxant in clinical anaesthesia?

odds in favour of the straight conformer (Streitwieser et al., 1992b).

Following the discovery of vecuronium, pursuit of shorter-acting aminosteroid NMB agents have followed the line of mono-quaternary compounds to rocuronium, which achieved great improvement in onset (Savage, 1983; Buzello et al., 1996; England et al., 1996; Savarese et al., 2000). Rapacuronium follows the same trend, achieving both rapid onset and short duration of action. Unfortunately, the improvement came with a large reduction in potency and specificity, resulting in side effects, such as bronchospasm, histamine release, tachycardia, and cumulativeness (Hunter, 1996b; Goulden & Hunter, 1999; Bevan, 2000). Bronchospasm has forced the withdrawal of rapacuronium shortly after its clinical introduction in the United States. After rapacuronium, one doubts if any new muscle relaxant in clinical anaesthesia will ever be mono-quaternary again. Considering that most potent NMB agents are bisquaternary, new NMB agents in the future will likely be bisquaternary, because high potency means room to trade for desirable onset and offset. In the past, bronchoconstriction following administration of NMB agents generally was attributed to histamine release. With rapacuronium, bronchoconstriction is disproportionately severe, suggesting an additional more important muscarinic mechanism of action (Levy et al., 1999; Naguib, 2001).

5.2. Quantitative measurement of molecular flexibility of neuromuscular blocking agents

5. Computer analysis of the molecular conformation of neuromuscular blocking agents

To find low-energy conformations of an NMB agent, computer-based local and global minimizations are required (Lee & Jones, 2002). An energy minimization is a local minimization that goes one-way. At every step, the total energy of the molecule must come down, although the components of energy may go up. Allowing both the component energy and the total energy to go up will make the minimization job open-ended, and, therefore, endless and fruitless. Consequentially, an energy minimizer will produce only what is possible downhill, from its starting geometry to its local valley. To find what is possible beyond the limit of the starting geometry, a global minimizer can be used to search a wide conformational space for a collection of conformers down the other valleys ‘‘over the hill.’’ Each of these conformers will be a local minimum when fully energy minimized. The lowest of these local minima is the global minimum. It most credibly represents the molecule because it is the most populated conformer. Simple molecules, such as ACh, SDC, and C10, can have the global minimum and several local minima discovered with ease and certainty. For example, the global minimum of C10 is clearly the straight-chain conformer (Lee & Jones, 2002). In contrast, complex molecules with many rotatable bonds can have their minima only approximated or qualified. For example, the global minimum of mivacurium is hard to ascertain. Nevertheless, one can be sure that it cannot be the

5.1. Molecular shape of low-energy conformers Computer chemistry is a rapidly expanding theoretical branch of chemistry, widely utilized by theoretical chemists, medicinal chemists, and pharmacologists. With cost-effective and user-friendly hardware and software becoming available, researchers in applied sciences may find computer modeling useful in their understanding of the SAR and CAR of drugs used in their specialties. The traditional stereomodels give only imprecise, non-quantitative snapshots. Most bulky and rigid NMB relaxants are physically impossible to assemble with stereomodels (Lee & Jones, 2002). Molecular shape is a fundamental concept of organic chemistry (Streitwieser et al., 1992a). Only low-energy conformers may represent drug molecules in CAR exercises. For illustration, when the configuration of butane is changed from anti to gauche, by a twist of one bond, the four-carbon chain bends and the energy of the molecule increases by
0.9 kcal/mol. Meanwhile, as molecules stabilize at low energy, a 1.4 kcal/mol difference in energy would mean a 10-to-1 odds for the lower-energy conformer to be preferentially populated (Taylor & Insel, 1990). For butane, the energy barrier of 0.9 kcal/mol means a 72-to-28

The traditional division between rigid and non-rigid muscle relaxants is at best qualitative. To add precision, energy required for conformational changes may give a quantitative indication of the rigidity of the molecule or parts of molecule (Lee, 2001c; Lee & Jones, 2002). For example, energy cost to reduce the N-N distance of a long congener of C10 may quantify its ability to bend to fit its onium heads between the two receptive sites. For gallamine, the pertinent measurement may be the energy cost to change the N-N distance of the two outside N atoms, if one assumes that these two ethonium heads are the ones that bind the receptive sites. For vecuronium, the energy cost to change the position of the N relative to the carbonyl O atom, both of the D-ring ACh moiety, may be pertinent. While the N-N distance of mivacurium may be easy to change, the N-N distance of pipecuronium would be hard to change without a major energy penalty. Conceptually, a pachycurare may have ‘‘pachy molecule’’ or ‘‘pachy onium heads’’ with ‘‘lepto connection.’’ Likewise, a leptocurare may have some thicker, less rigid components in the molecule. 5.3. Local and global minimizations of energy of muscle relaxant molecules

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conformer with a straight N-N connecting chain, because many conformers with lower energy and bent connecting chain are easily discovered (unpublished data). Simulated annealing, genetic algorithm (GA) search, and random search are three basic techniques useful in searching for the low-energy conformers of the NMB agents. Simulated annealing is a global minimizer. It thermodynamically heats up a molecule and then cools it down slowly. As in annealing, simulated annealing allows the molecule to resettle at lower energy. At each cycle, the heating gives the conformer the necessary energy to climb over a new hill, and the cooling allows the conformer to cool down a new path to a new valley. The GA search is another global minimizer. It is particularly efficient for molecules with many rotatable bonds (Judson, 1997; Lee & Jones, 2002). It creates a population of individual conformers that undergo a specified number of generations of evolution in which the individuals are crossbred, mutated, evaluated for fitness, and selected according to the principles of evolution. Generation after generation, lower-energy conformers are kept while higher-energy conformers are eliminated. At the end of the evolution, a collection of discrete conformers is harvested. The products of both simulated annealing and GA search must be refined by full minimization of energy because they were selected only based on partial minimization during the search. Random search randomly twists the bonds and fully minimizes the new conformer immediately. Iteration after iteration, new conformers meeting preset criteria are added to the collection. In actual application, simulated annealing is the method capable of producing a conformer of pancuronium with a twisted A-ring that lowers the energy. This is crucial because a modeler would normally model the androstane nucleus of pancuronium without a twisted ring and would not designate bonds inside the androstane nucleus as rotatable. GA search is most efficient for searching molecules with many rotatable bonds (Judson, 1997). Random search is inefficient for molecules with many rotatable bonds because it spends time fully minimizing intermediate products. For small molecules, it is efficient because it completes the local and the global minimizations in one operation. The searches can be combined. For example, one can use simulated annealing to select a geometry for the rigid portion of a molecule and then use GA search to search the rotatable bonds. Upon wide search and thorough minimization of the search products, a credible working global minimum fit for CAR exercises of the NMB agents can often be found. Details of the GA search and the subsequent energy minimization applied to the congeners of C10 have been described (Lee & Jones, 2002). 5.4. Caveats Conformational study is relatively new to the pharmacology of NMB agents. Caveats in its application and interpretation have been proposed (Lee, 2001c; Lee & Jones,

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2002). Inadequate search results can be misleading. Excessive extrapolation should be avoided. All examples used in this review are results of repeated conformational searches; however, they remain preliminary until confirmed by others. While traditional SAR does not specify an environment in which it applies, computer-based conformational searches assume that the molecules are in vacuo. Solvation is a modeling technique that takes into consideration the effect of the solvent on the conformation of the molecule. However, no system of solvation deals with the complex electrolyte composition of the body fluid, and simplistic solvation could compound the errors. Docking requires additional presumptions on the AChR macromolecule (Judson et al., 1995). X-ray crystallography requires special equipment and techniques, and molecules of NMB agents may crystallize in a conformation that differs from how they are in vivo. Advanced techniques, separately or in combination, may improve the precision of the SAR or CAR of the NMB agents in the future, but this is untested.

6. Structure, conformation, and action of various classes of neuromuscular blocking agents 6.1. Decamethonium measures the inter-site distance of the endplate acetylcholine receptor Thermodynamically, C10 congeners prefer straight-chain conformations, with large energy penalty for their molecules to bend, and Lee and Jones have argued that these congeners act as NMB agents in straight conformation (Lee, 2001c; Lee & Jones, 2002). For lack of other plausible explanations, lower congeners may use two molecules, one at each receptive site, as does ACh and SDC, to cause a depolarizing block. C10 and higher congeners likely span the two receptive sites with one molecule. They fit into the same inter-site space, by bending if necessary, and they block by the same methonium heads. The greater the energy penalty required to so conform, the lower the potency. The high congeners, such as C18, have a tendency to regain potency and to exhibit a component of the so-called ‘‘non-depolarizing’’ block (Paton & Zaimis, 1949; Lee, 2001c; Lee & Jones, 2002). From the viewpoint of CAR, the extra length of the C18 polymethylene chain must fold into the inter-site space. Otherwise, it will take two molecules of C18 to bind the two receptive sites. In either case, the non-binding parts of the molecule(s) will be in a position to hinder the flow of the depolarizing cations. Whether this is channel block or non-depolarizing block is discussed in Sections 8 and 9.3. As a corollary, space available to all NMB agents between the two receptive sites of the AChR should be ˚ ; namely, the molecular length of C10 close to 20 A including the vdw extensions of the terminal H atoms (Lee & Jones, 2002; Lee, 2002c). Supporting the hypothesis that C10 is a credible molecular yardstick to size the free intersite space is the observation that gallamine and other classes

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of potent pachycurares also tend to have the same molecular length as optimum (see Sections 6.8 and 6.19) (Lee, 2001c). 6.2. Energy cost to conform decreases potency as quantitatively illustrated by decamethonium congeners One interesting question of importance regarding CAR is how much a conformer may deviate from the lowest-energy conformation before it becomes pharmacologically inferior or irrelevant. For practical purposes, the C10 congeners give a clue (Lee, 2001c; Lee & Jones, 2002), if C11 is presumed to block by conforming to C10. In other words, C11 is being considered a poor conformer of C10, which requires energy to bend to conform to the global minimum of C10 before it works. On revisiting the energy-potency regression line of the polymethylene bismethonium congeners (Lee & Jones, 2002), it was found that it costs C11 3.3 kcal/mol to conform to C10 and that C11 has half the potency of C10 (Paton & Zaimis, 1949). This gives an example of the relationship between the conformational energy and potency of the NMB agents. Before making this snapshot a generalization to other NMB agents, however, one must realize that the energy-potency relationship of NMB agents is complicated and composed of the relationships between energy and conformation, between conformation and receptor binding, and between receptor binding and neuromuscular block, all of which may be non-linear. Besides, the ideal molecular length may be somewhere between that of C10 and one of its neighbours. Furthermore, some molecules of C10 congeners may bind only one receptive site at a time (Lee & Jones, 2002). The chances of using two molecules simultaneously to bind an AChR, one at each receptive site, should increase with the degree of imperfect fit. In any case, conformers of any NMB agent exceeding its global minimum in energy by much more than 3 – 5 kcal/mol should be considered poor conformers and treated with caution in CAR exercises. One realizes that the conformational energy on the receptor side must also affect the binding. 6.3. Methyl and acetyl as optimal quaternizing and H bond acceptor groups A quaternizing group larger than methyl and, to make a complete ACh moiety, an acyl group larger than acetyl generally reduce potency, presumably by distancing the charged N and the carbonyl O from the structures that they bind on the receptive site(s). For example, gallamine, rocuronium, and rapacuronium are relatively weak NMB agents. Hill et al. (1994) has offered a similar explanation for the lower potency of alcuronium versus toxiferine I. 6.4. Inter-methonium connecting chains of larger leptocurares and channel block While keeping the onium heads small (with two or three methyl groups), many compounds with somewhat

larger interonium connecting chains have been synthesized and studied. The common theme is to keep the N-N distance at 10 heavy (C or O) atoms. The connecting chain may be double-bonded, phenyl, or cyclohexyl, or may be cyclo-octadecane (Bovet, 1972; Hill et al., 1994). The last example is C10, with the middle 8 methylene groups duplicated to form a cyclic double strand. In general, any increase in the bulk or rigidity of the connecting chain will shift the nature of the block toward non-depolarizing. This author speculates that if these modified C10 compounds block neuromuscular transmission by binding the same two receptive sites with both onium heads, their redundant lipophilic connecting chain likewise may cause a non-depolarizing component of neuromuscular block by impeding the flow of the depolarizing cations through the channel. From the viewpoint of CAR, it is also noted that these C10-mimicking compounds do not really conform to C10 because their molecular lengths could easily resemble C9 or C11 more than C10. Both factors affect potency. 6.5. Succinylcholine: its conformation and mechanism of action Inadequately appreciated is the report that it takes two molecules of SDC, as it does ACh, to open one nicotinic AChR ionic channel (Marshall et al., 1990; Dilger, 1998). A conformational explanation for this has been offered (Lee, 2001c). In their low energy conformations, each ACh moiety of SDC prefers a bent (cis, gauche) geometry (Fig. 6), as does molecular ACh. The attraction between the N atom and the O atoms in its vicinity (Partington et al., 1972) is greater than the repulsion between the two onium heads at the ends of the molecule. As a result, SDC will be shorter than C10, i.e., short of ideal and too short to span the two receptive sites with one molecule (Lee, 2001b, 2001c, 2002c). Furthermore, one or both ACh moieties of SDC readily validate Beers ˚ for nicotinic action with little and Reich’s rule of 5.9 A energy penalty. In other words, each ACh moiety of SDC can bind a receptive site at both subsites independently, as does ACh. Although SDC is flexible, binding at any two points will tend to point the rest of the molecule in a direction away from the other receptive site. As a result, the remaining ACh moiety will become even less likely to reach the other receptive site, not to mention apposing it in correct orientation. Furthermore, the di-ACh structure of SDC is well known. Less appreciated is that conformationally, as long as the two ACh moieties are joined end on end, they cannot be both clockwise as the pentameric AChR subunits are. In other words, SDC cannot bind both receptive sites by an ACh-like mechanism of action at both moieties. Besides neuromuscular block, the AChlike conformation of the ACh moiety of SDC also explains its ACh-like side effects (Lee, 2001b, 2001c, 2002c).

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Fig. 6. SDC and succinylmonocholine. N is purple, O is red, C is gray, and H is green. Thin sticks show the direction and length of the dotted vdw extensions of the O atoms. Arrows point from the centres of the N atoms to the vdw extensions of the O atoms. In succinylmonocholine, upper, the N-Ovdw distances are ˚ to O1 (proximal ester O), 4.74 A ˚ to CO1 (proximal carbonyl O), 3.43 A ˚ to CO2 (distal carbonyl O), and 7.69 A ˚ to O2 (distal ester O). Both carbonyl O 3.61 A ˚ for nicotinic action. In SDC, lower, the arrow shows an N-Ovdw atoms are pulled close to the solo N atom. Neither CO1 nor CO2 fulfills the rule of 5.9 A ˚ from the centre of the N to the vdw extension of the complementary carbonyl O, fulfilling Beers and Reich’s rule of 5.9 A ˚ for nicotinic action. distance of 6.0 A

Fig. 7. Gallamine, upper, and C10, lower. N is purple, O is red, and C is gray. H atoms are hidden. Thin sticks show the direction and length of the dotted vdw ˚ . All three side chains fulfill Beers and Reich’s rule of 4.4 A ˚ for extensions of the O atoms. Arrow from the centre of N1 illustrates an N-Ovdw distance of 4.27 A ˚ , N2 – N3 = 8.5 A ˚ , N1 – N3 = 8.5 A ˚ ), not equilateral. The two outside ethonium heads muscarinic action. The N atoms form an isosceles triangle (N1 – N2 = 11.9 A ˚ , close to the molecular length of C10, which is a straight molecule of 20 A ˚ , including the vdw extensions of the terminal H atoms. The N1-N2 span 19 A distance of gallamine is shorter than that of C10 by 2 C atoms because the onium heads are triethyl instead of trimethyl. The N-trimethyl derivative of gallamine would be even shorter, by two C atoms, and would have both the N-N distance and the molecular length suboptimal for NMB action.

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6.6. Succinylmonocholine not fitting the receptive site If succinylmonocholine has one intact ACh moiety, and if SDC uses only one of its two ACh moieties at a time, why shouldn’t succinylmonocholine be equipotent with SDC? It is instead only one-twentieth as potent as an NMB agent. Conformational examination shows that low-energy conformers of succinylmonocholine do not readily fulfill Beers and Reich’s rule for nicotinic action (Fig. 6). Missing the second methonium head, the attraction between the lone methonium head of succinylmonocholine and the four O atoms is even stronger than that in SDC. As a result, succinylmonocholine assumes a rather inactive curled conformation. It is interesting that, in SDC, each ACh moiety depends on the other to conform to Beers and Reich’s rule for nicotinic action, but prevents each other from binding simultaneously. 6.7. Conformation, conformational energy, and pharmacodynamics Conceivably, a flexible compound has greater chances of somehow fitting the receptive site(s) with one conformer or another, but the best fitting conformer may not necessarily be of the lowest energy, or most populated. There will be numerous conformers with small energy differences that do not have the best fit, but still will fit. This may be one reason why flexible molecules are generally weak, with flat doseresponse curves. They lack ‘‘conformational density.’’ In contrast, a rigid molecule tends to fit well, or not at all. Conformational rigidity with stiff energy penalty means great concentration of conformers around the global minimum. If the global minimum fits, a rigid muscle relaxant will have high potency. If it does not, few molecules can. As a result, a potent muscle relaxant will have a steep doseresponse curve. Although commonly associated, being ‘‘lepto’’ (i.e., thin) and depolarizing is not synonymous with being flexible and weak. Isoarecolone is thin, but rigid (Spivak et al., 1986, 1989), and C10 has high-energy penalty to change N-N distance (Lee & Jones, 2002). Both are very potent for their size. In general terms, while molecular rigidity contributes significantly to potency, molecular size determines whether an NMB agent will be depolarizing or non-depolarizing. Even if rigid and potent, small molecules cannot fully occupy the cross-sectional area between the receptive sites. Depolarizing cations will flow around them. Large molecules may shut the lumen at the level of the receptive sites, whether the channel is open or closed below (Lee, 2001c, 2002c). Both SDC and C10 are small and depolarizing. However, SDC is much more flexible. The energy cost to change the ˚ is 1.00 kcal/mol for SDC (unpublished N-N distance by 1 A data) vs. 2.83 kcal/mol for C10 (Lee, 2001b; Lee & Jones, 2002). As a result, C10 is nearly 10 times as potent in human. Similarly, gallamine is flexible and weak, as discussed in the following section.

6.8. The neuromuscular blocking conformation and action of gallamine and derivatives Gallamine is three TEA heads attached through ether O linkages to the 1,2,3 positions of a phenyl ring. The middle onium head of gallamine is said to help stabilize the two outside heads, so that the three onium heads would form an equilateral triangle (Stenlake, 1963; Hill et al., 1994). However, a third onium head generally does not increase NMB potency (Marshall, 1968) because the AChR has anionic centres for only two onium heads. Unless positioned to facilitate the other two, a third onium head may even interfere unfavourably (Lee, 2001c, 2002c). Like all multi-quaternary compounds (Marshall, 1968), gallamine is a weak NMB agent. Conformational searches have found that the middle onium head of gallamine helps position the two outside ones ˚ , close to the optimal molecular length so that they span 19 A ˚ of 20 A. Furthermore, the triangle can only be isosceles, not equilateral (Fig. 7). Only the two outside onium heads can span the receptive sites (Lee, 2000a, 2001c). In spite of the third onium head, gallamine is still rather flexible. Measured by the energy penalty required to change the N-N distance between the two outside onium heads by 1 ˚ , it is even more flexible than C10, 1.14 (unpublished data) A vs. 2.83 kcal/mol (Lee & Jones, 2002). Conformationally, gallamine can be considered a ‘‘soft pachycurare’’ of low potency, acting with a bis-quaternary mechanism of action. Lack of a third receptive site per AChR prevents a ‘‘trisquaternary mechanism of action.’’ In contrast, C10 is a ‘‘rigid leptocurare.’’ The trimethyl derivatives of gallamine are weaker than gallamine, in contrast to the general rule that methyl quaternization is optimal for potency (Hill et al., 1994; Lee, 2001c, 2002c). Upon inspection, one finds that the methyl substitution of gallamine would shorten the molecule by two C atoms (Fig. 7), thereby adding suboptimal molecular length to suboptimal N-N distance (Lee, 2000a, 2001c). 6.9. The muscarinic blocking conformation and action of gallamine All low-energy conformers of gallamine have at least one ˚ (4.24 – 4.29 A ˚ ), practically N-Ovdw distance close to 4.27 A ˚ fulfilling Beers and Reich’s rule of 4.4 A for muscarinic action (Lee, 2000a) (Fig. 7). In fact, gallamine is a more potent cardiovagolytic agent than an NMB agent at the lowdose range (Hughes & Chapple, 1976; Savarese et al., 2000). 6.10. The twisted A-ring of pancuronium The 4-ring androstane structure, which is the backbone of the aminosteroid NMB agents (Fig. 5), is reasonably rigid. In

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agreement with Fielding (1998), however, conformational searches using simulated annealing and energy minimization have found that low-energy conformers of pancuronium have the A-ring in the twisted form. Specifically, the C2 of the androstane nucleus is pushed toward the a face. Interestingly, the twisting of the A-ring is not shared by the lowestenergy conformers of pipecuronium and vecuronium (Fig. 8). The charged 2-N onium head of pancuronium probably twists the A-ring to which it is attached. Another source of flexibility of pancuronium is that the 6-member rings on the 2- and 16-positions can attach themselves to the androstane nucleus with their axial or equatorial bonds. 6.11. The A-ring muscarinic blocking acetylcholine moiety of pancuronium While the A-ring ACh moiety of pancuronium will not readily assume a conformation for nicotinic action, its Dring ACh moiety, as well as that of vecuronium (Fig. 3), will not readily assume a conformation for muscarinic action. In the A-ring ACh moiety, however, it is the carbonyl O, not ˚ the ether O, which fulfills Beers and Reich’s rule of 4.4 A (unpublished data). Whereas Beers and Reich did not specify the subtypes of muscarinic action, the muscarinic action of gallamine and pancuronium is mainly m2 in nature

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(Hou et al., 1998). Further work on the conformational requirements of various muscarinic receptor subtypes is indicated. 6.12. The D-ring acetylcholine moiety of pancuronium and vecuronium as neuromuscular blocking pharmacophore By having the same D-ring ACh moiety, pancuronium and vecuronium have similar NMB potency and duration of action, although in some animal species, pancuronium is somewhat more potent. Because vecuronium has the greatest potency and specificity of all mono-quaternary NMB agents, its D-ring ACh moiety must be an excellent NMB pharmacophore. This ACh moiety is cis, in agreement with the statement that the AChR-bound molecular ACh ‘‘must be’’ in the gauche configuration (Spivak et al., 1986, 1989). It ˚ with great precision validates Beers and Reich’s rule of 5.9 A and consistency (Lee, 2000b). Also interesting is that the carbonyl O protrudes out of the molecule, unhindered to appose the H bond donor of the receptive site (Fig. 3). 6.13. De-acylated metabolites of potent 3,17-diacyl aminosteroid neuromuscular blocking agents The 3-OH de-acylated metabolites of potent 3,17-diacyl aminosteroid agents retain significant NMB potency (Booij

Fig. 8. Vecuronium, pipecuronium, and pancuronium in side view. N is purple, O is red, and C is gray. H atoms are hidden. Acetyl groups (CH3CO-) are removed to unobstruct the view of the ester O on the 3- and the 17-positions. N + is quaternary, N is tertiary. The lipophilic methyl groups (CH3) on the 10position (10-CH3) and the 13-position (13-CH3) are marked. They point perpendicularly to the b (up) side. However, the 16-N and the 17-O bonds (both b), especially the former, protrude laterad along the zigzag side view line of the androstane nucleus, which is flat or slightly convex towards the b side. In pancuronium, the A-ring is twisted so that C2 is pushed towards the a side below C3. Pipecuronium has the longest molecular length, with both charged N + atoms extended. Its onium heads are dimethyl and unshielded.

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et al., 1981; Savage, 1983; Caldwell et al., 1994; Schiere et al., 1999), and accumulation of 3-desacetylvecuronium has been believed to cause prolonged paralysis in critically ill patients (Segredo et al., 1992; Ducharme & Donati, 1993). As long as their D-ring ACh moiety is unaltered, their NMB action will remain significant. In the case of rapacuronium, the 3-OH metabolite is even more potent than rapacuronium itself (Schiere et al., 1997, 1999; Goulden & Hunter, 1999; Bevan, 2000). In contrast, the 17-OH de-acylated monoquaternary aminosteroids in general are weak (Savage, 1983). As long as their A-ring ACh moiety is unaltered, their muscarinic action will remain (Norman et al., 1970; Norman & Katz, 1971; Baden, 1976; Savage, 1983). Together, the examples reaffirm the D-ring ACh moiety as the NMB pharmacophore, while the A-ring ACh moiety is muscarinic. 6.14. Need of two functional groups on aminosteroid neuromuscular blocking agents Aminosteroid NMB agents need two functional groups for high potency, presumably to enable them to bind the AChR at two points. The most advantageous two-point arrangement is A-ring and D-ring bis-quaternary, or a complete D-ring ACh-like moiety. Once the requirement has been met, a third functional group has variable effects. Those 16-mono-quaternary compounds without 17-substitutions benefit from 2-quaternization more than from 3hydroxylation or acetylation, as shown by Org 7931 versus ANQ 9040, and some HS-compounds (Gandiha et al., 1974, 1975; Bowman et al., 1988b; Munday et al., 1994; Marshall et al., 1995). Org 7931, the 3,17-unsubstituted, 2,16-bisquaternary pancuronium, has 20 –25% the potency of pancuronium, far more than that of ANQ 9040 (Table 1). Of those already with A-ring and D-ring bis-quaternizations, including Org 7931, the 3- and 17-additional substitutions contribute positively to the NMB effect. Complete removal of these additional substitutions from pancuronium, vecuronium, and HS-compounds reduces potency (Savage, 1983; Bowman et al., 1988b; Marshall et al., 1995; Savarese et al., 2000) (Fig. 5). 6.15. Molecular conformation of pipecuronium explains its high neuromuscular blocking potency and specificity The quaternary and acetyl groups on the A-ring and the D-ring of pipecuronium are far from being ACh-like in structure and geometry (Figs. 5 and 8). Their N-Ovdw distances are too large to fulfill Beers and Reich’s rule for nicotinic or muscarinic action (Beers & Reich, 1970; Lee, 2001c). Such non-ACh-like structure and conformation prevent pipecuronium from binding two points at the same receptive site (intra-site), obliging it instead to bind as bisquaternary with the inter-site mechanism of action (Lee, 2001c). They also free pipecuronium from nicotinic or muscarinic side effects based on ACh-like moiety, and

protect pipecuronium from hydrolysis by cholinesterases. The latter explains its dependence on the kidney for excretion. Pipecuronium is also resistant to hepatic uptake and metabolism, or biliary excretion, because its four methyl groups, two on each quaternary N, make it less lipophilic than most other aminosteroids. ˚ molecular For maximal neuromuscular block, the 21 A length of pipecuronium is close to ideal. Being rigid and slightly longer than C10, pipecuronium may bind both receptive sites from above. For minimal ganglion block, ˚ N-N distance of pipecuronium is farthest away the 16 A from what is favoured by ganglionic blockers. Besides, pipecuronium has ‘‘conformational density.’’ Its A-ring is not twisted. Its onium heads are well exposed, on the far end of the 2- and 16-substitutions. Both the equatorial and axial groups on both quaternary N atoms are methyl, well suited to closely appose the onium heads to the anionic centres of the AChR, with no chirality issue (Fig. 8). The above conformational features working together make pipecuronium the most potent and clean one-bulk bis-quaternary NMB agent known (Boros et al., 1983; Agoston & Richardson, 1985; Foldes et al., 1990; Naguib et al., 1992; Denman et al., 1996; Savarese et al., 2000; Lee, 2001c). More so than any other NMB agents, pipecuronium theoretically fits one, and only one, AChR well, and does so by one (inter-site) mechanism of action. 6.16. Hydrophilic surface of neuromuscular blocking relaxant molecule faces the receptive sites An NMB relaxant molecule presumably faces the AChR with its hydrophilic side, where its charged functional groups are (Lee, 2001c). ACh bound to the nicotinic AChR exposes a continuous lipophilic surface of insulating methyl groups towards the extracellular fluid (Behling et al., 1988). The molecule of mTc has a hydrophilic side that is supposed to interact with the receptor (Sobell et al., 1972). The steroid structure of HS-342 shows crowding with lipophilic methyl groups on the b (up) side. It likely interacts with the receptor on the less hindered, a side (Marshall et al., 1973b; Gandiha et al., 1974). The lipophilic 10-CH3 and the 13-CH3 groups on the androstane nucleus point to and crowd the b side (Figs. 5 and 8). 6.17. Planar geometry of vecuronium From the above examples, one would think that ideally the 16-piperidinium and 17-acetyl groups of the D-ring ACh moiety of vecuronium should preferably be on the a side so that these functional groups would approach the AChR below, while the methyl groups on the b side would provide a lipophilic shield above. In reality, however, they are on the b side. Examination of the conformation of vecuronium shows that the D-ring is particularly crowded on the b side, with its 13-CH3, 16-N, and 17-O bonds all b to the ring.

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Perhaps as a result, the 16-N and the 17-O bonds of vecuronium protrude laterad out of the steroid nucleus. Instead of being perpendicular to the surface of the androstane nucleus, they are planar to the surface along the zigzag side view line of the nucleus. This laterad protrusion allows the b side to remain dominated by the lipophilic methyl groups. It also maximizes the surface area and the molecular length (Figs. 3 and 8). One is tempted to theorize that in this conformation and orientation, vecuronium points its pharmacophore sidewise to bind the receptive site, impedes the inter-site depolarizing cationic flow with the wide surface of its 4-ring androstane nucleus, and shields off ACh and the depolarizing cations from above with its lipophilic b face toward the synapse. No other orientation could fit the pieces of the puzzle and explain its superior NMB action equally well. While pipecuronium is the most potent and clean bisquaternary aminosteroid NMB agent, vecuronium is the most potent and clean mono-quaternary NMB agent known. Both have advantageous conformational features to explain their high potency and specificity. The abovehypothesized planar orientation of vecuronium coincides with how its is customarily drawn on paper, clockwise from the 16-acetyl to the 17-onium group, cis between the functional groups, and with the lipophilic 10-CH3 and 13CH3 groups on the b side perpendicular to the androstane nucleus. Fig. 8 shows the conformational similarities and dissimilarities of vecuronium, pipecuronium, and pancuronium. 6.18. N-N distance, ganglionic block, and neuromuscular block As reviewed recently, the N-N distances of AH8165 (7.5 ˚ ), pentamethonium (C5, 7.7 A ˚ ), HS-342 (8.0 A ˚ ), hexamA ˚ ˚ ), ethonium (C6, 9.0 A), HS-310 (chandonium, 10.2 A ˚ ˚ ), protonated dTc and mTc (10.8 A), pancuronium (11.4 A ˚ ˚ maloue`tine (12.2 A), C10 (14.0 A), and pipecuronium (16.0 ˚ ) illustrate a long-recognized trend that a short N-N A distance imparts ganglion block, while a long N-N distance favours neuromuscular block (Sobell et al., 1972; Brittain & Tyers, 1973; Marshall et al., 1973a; Savarese et al., 2000; Lee, 2001c). 6.19. Optimal molecular length of potent one-bulk neuromuscular blocking agents mTc is among the most rigid NMB agents. It meas˚ in total length; so does dTc. Pancuronium ures
18 A ˚ . Pipecuronium measures and vecuronium measure 19 A ˚ 21 A. The length of these potent rigid one-bulk NMB agents follows the same rank order of their potency. One hypothesizes that their molecules occupy the inter-site space of the AChR with the same rank order of completeness. These pachycurares are the most potent of their respective chemical classes. In agreement, the leptocur-

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ares prefer a similar molecular length. C10 is the most ˚. potent among its congeners, and it measures 20 A Gallamine has low-intermediate bulk and rigidity. It is, nevertheless, the most potent among its chemical class, ˚ (Lee, 2001c; Lee & Jones, 2002) and it measures 19 A (Fig. 7). These molecular yardsticks strongly suggest that while C10 is optimal for binding the receptive sites from in between, pipecuronium is optimal for binding them from above. If C11 is slightly too long and C10 and pipecuronium are ideal, the ideal molecular length for NMB agents, depolarizing and non-depolarizing alike, ˚. should be 20 –21 A 6.20. Methoxylation improves tetrahydroisoquinolinium neuromuscular blocking relaxants dTc has 2 methoxy groups, while mTc has 4. However, both dTc and mTc have 3 O atoms on each onium head. Among the long-chain benzylisoquinolinium compounds, atracurium has 4, mivacurium has 5, and doxacurium has 6 methoxy groups on each onium head (Figs. 2 and 4). As first observed by Taylor and Collier in 1950, and subsequently by others (Bowman et al., 1976; Hill et al., 1994; Savarese et al., 2000; Lee, 2001c), addition of methoxy groups, among other factors, increases potency. GW280340A, with 5 methoxy groups on each onium head, follows the same trend (Boros et al., 1999; Belmont et al., 1999; Savarese et al., 2000; Lien, 2002). How methoxylation increases NMB potency is unclear. It may alter the density and distribution of the charge of the onium head. The O atom of the methoxy group might also serve as the H bond acceptor and bind the H bond donor of the receptive site. However, an intra-site mechanism of action is unlikely, especially for dTc and mTc, because they have their onium heads bound in one bulk in the interior of the bulk, leaving any ACh-mimicking moiety hard to orient itself to maximally appose the receptive site at more than one point. Histamine release is a common concern when using the benzylisoquinolinium NMB agents. The reduction of histamine release from dTc to mTc, as well as from atracurium and mivacurium to cisatracurium, doxacurium, and GW280430A, generally parallels the increase in potency and decrease in dose requirement (Savarese, 1979; Savarese et al., 2000; Lee, 2001c, 2002c) (Table 1). Specific conformational or structural explanations for the differences in their histamine-releasing propensity are not apparent. Laudanosine is a convulsant that is produced by Hofmann elimination of the quaternizing group of atracurium and cisatracurium. Although laudanosine rarely accumulates to a level of clinical concern, anaesthesiologists generally recognize the reduced production of laudanosine as an advantage of cisatracurium. The reduced dose requirement of these relaxants accounts for most of the reduction in histamine release and laudanosine production.

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6.21. Conformation-action relationship of long-chain tetrahydroisoquinolinium neuromuscular blocking agents

decamethylene connecting chain tends to be straight, as it is in C10.

The SAR of atracurium, cisatracurium, mivacurium, doxacurium, GW280430A, and other long-chain bis-quaternary tetrahydroisoquinolinium NMB agents is well publicized. Their CAR, however, is little known because their bulky onium heads and large number of functional groups and rotatable bonds make their molecular conformation hard to determine. Nevertheless, some preliminary conformational features are certain. First, the charges of the N and the O atoms interact (Partington et al., 1972). As in all diester bis-quaternary NMB agents, including SDC (Lee, 2001b), these diacid connecting chains are flexible, except at the double bond, and they bend. Second, the methoxylated 1-phenyl or benzyl group interacts with the rest of molecule in a complex manner to position itself at low energy. Third, the molecule must conform to the same receptive site topology that accommodates all NMB agents. In other words, these NMB agents do not exist in extended conformation as usually diagrammed on paper, and they may not fit between the receptive sites without bending (Lee, 2001c; Lee & Jones, 2002). If extended, the N-N ˚ and the molecular distance of mivacurium would be 21 A ˚ length would be close to 36 A, too large to fit into the space between the receptive sites. Interestingly, the bent connecting chain and the folded onium heads of the long-chain benzylisoquinolinium compounds diagrammed in Fig. 2 are not new concepts. Structurally, dTc and mTc are two benzylisoquinolinium heads folded and joined with two ether linkages. Structural and pharmacological similarities between the one-bulk and the long-chain benzylisoquinolinium compounds were pointed out in 1951 (Bovet, 1951; Taylor & Collier, 1951) and 1962 (d’Arcy & Taylor, 1962a, 1962b). Bovet (1972) drew laudexium with the decamethylene connecting chain bent around, and positioned the onium heads to overlap those of chondocurine. Bowman (personal communication) promoted the concept with a similar illustration in his lectures on atracurium in the 1980s. Realizing that the space between the receptive sites cannot accommodate a molecule of atracurium in fully extended conformation, Lee (2001c, 2002c) adopted a similar version to stress the uncertain interonium distances and molecular lengths of these long-chain diacid relaxants. Preliminary conformational searches have repeatedly uncovered lowenergy conformers of doxacurium also in folded geometry (unpublished data). Both the diacid chain and the 1-benzyl group fold. Even in bent conformation, however, mivacurium, atracurium, and doxacurium tend to have greater N-N distance and molecular length than those of dTc and mTc. Their flexible connections should permit their onium heads freedom to position and orient themselves advantageously. The theory of bent N-N connection probably does not apply to laudexium and decamethylene bisatropinium, whose simple

6.22. Carbonyl group and N-N distance of TAAC3 and derivatives In CAR, all better TAAC3 derivatives have an acyloxy (usually acetoxy) group on the position 4 of the benzyl group that quaternizes the tropine. The carbonyl O atom of this acyl group points toward the quaternary N atom to assume a conformation that satisfies Beers and Reich’s ˚ for nicotinic action (Fig. 9). In SAR, rule of 5.9 A tropine itself is a muscarinic agent, and atropine is a potent vagolytic agent. Quaternization of the N, maximization of the bulk, twinning of the tropinium heads, and optimization of the N-N distance together converted G-1-64 to a nicotinic antagonist with promising NMB potency (Gyermek et al., 1999). Finally, acylation of the quaternizing group further improves the TAAC3 derivatives as NMB agent (Gyermek et al., 2002a, 2002b). Overall, the TAAC3 derivatives are less bulky than the benzylisoquinolinium compounds. Accordingly, they are less potent (Table 1) and their vagolytic side effect tends to remain. The N-N distance of these bistropinium compounds (Fig. 9) is important to their NMB potency, and a glutarate or succinate connecting chain is close to optimum (Lee, 2001a; Gyermek et al., 2002a). When the diester connection of TAAC3 is replaced by the phthalate, the iso-phthalate, or the tere-phthalate, the tere-phthalyl derivative has an N-N ˚ ) closest to that of C10 (14.1 A ˚ ) and distance (14.9 A ˚ TAAC3 (14.3 A), and it has the highest NMB potency. ˚ ) follows in both aspects as a close The iso-phthalate (12.9 A ˚) second (unpublished data). The phthlate derivative (10.3 A is a distant third. 6.23. Potency-onset inverse relationship and a new explanation Little is known independently about the structure-onset or the conformation-onset relationships of the NMB action, except that onset is inversely related to potency (Bowman et al., 1988b; Wierda & Proost, 1995; Nigrovic et al., 1997; Kopman et al., 1999; Savarese et al., 2000). Bowman et al. (1998b) made this observation on the aminosteroid compounds, as did Wierda and Proost (1995) and Nigrovic et al. (1997). Kopman and colleagues (1999) expanded this observation to include NMB agents of all ranges of duration of action, including SDC. The relationship holds even when the NMB agents are applied iontophoretically, in vitro (Min et al., 1992). The new aminosteroids ANQ 9040 and rapacuronium are no exceptions (Munday et al., 1994; Wierda & Proost, 1995; Lee, 2001c). The rapid onset of rocuronium appears to qualify it as an outlier (England et al., 1996), but not really an exception to the rule, considering its relatively low potency

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Fig. 9. TAAC3, the phthalate derivative (TAA-phth), and the tere-phthalate derivative (TAA-t-phth) of TAAC3. N is purple, O is red, and C is gray. H atoms are hidden. Dotted hemispheres are the vdw extensions of the carbonyl O atoms on position 4 of the quaternizing benzyl groups on the right halves of the molecules. Thin sticks show the direction and length of the dotted vdw extensions of the carbonyl O atoms. Arrows point from the centres of the tropinium N ˚ on both halves of the molecule. The N-N distances are 14.3, atoms to the vdw extensions. In all three compounds, the subject N-Ovdw distances are 5.8 – 6.0 A ˚ for TAAC3, TAA-t-phth, and TAA-phth, respectively. 14.9, and 10.3 A

(Mellinghoff et al., 1994; Lee, 2001c). In general, monoquaternary aminosteroids are faster than bis-quaternary compounds (Hill et al., 1994) because they are weaker. Vecuronium is potent and slow, as expected. Compared with pancuronium, however, it is faster for the same potency, qualifying it as another outlier. GW280430A and TAAC3 also appear fast for the potency (Boros et al., 1999; Belmont et al., 1999; Savarese et al., 2000; Gyermek et al., 2001; Lien, 2002). As has been pointed out, significant breakthroughs following initial development along a lead of SAR are often exceptions to the rule or outliers (Lee, 2001c). A plausible explanation for the inverse relationship has been proposed (Donati & Meistelman, 1991). According to Donati and Meistelman (1991), weaker NMB agents are given in larger quantities and, therefore, have more molecules in the central compartment to diffuse into the effect compartment. Once in the effect compartment, all molecules act promptly. This explanation is plausible because drug delivery and receptor binding are of a different order of time scale. If the onset of the NMB agents is solely determined by the kinetics in the central compartment, where does the effect compartment begin and why do not the kinetics and dynamics in the effect compartment matter? Conceptually, kinetic factors, whether injected intravenously or applied iontophoretically (Min et al., 1992), control the distribution of the drugs all the way through the motor endplate. In the

submicro cul de sac environment of the AChR after that, the distance of travel from the mouth of the AChR to the receptive sites, the speed of approach by diffusion or by electrostatic forces, and the time of drug-receptor interaction are on a molecular scale, too small to be perceived by the clinical observers, not to mention discerning a difference among NMB agents. Based on CAR, it is hypothesized that the so-called ‘‘effect compartment’’ starts from the mouth of the receptor where the ‘‘delivery compartment’’ ends (Figs. 1 and 10). The former is a compartment of kinetics and drug delivery; the latter, of dynamics and molecular interaction. 6.24. Potency-side effect inverse relationship From the therapeutic point of view, the inverse potency-onset relationship is regrettable because low potency often means low specificity and a narrow margin of safety from side effects (Lee, 2001c). Low potency also increases the amount of drug that will be given, which, in turn, increases the amount of undesirable metabolites (Boyd et al., 1995; Eastwood et al., 1995). It is a dear price to pay for fast onset. Unfortunately, while low potency usually accelerates onset, it does not assure fast onset. For example, gallamine is weak and slow. Where rapid onset of paralysis is clinically imperative, SDC and rocuronium are at present the two relaxants of best compromise.

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Fig. 10. The nicotinic AChR modeled for the endplate of the skeletal muscle (Fig. 1) remodeled according to the CAR of the NMB agents reviewed. Left ˚ . Lower, activated panel: longitudinal section. Upper, resting receptor. Between the receptive sites, space available to NMB agent inter-site measures 20 A receptor with open trans-membrane channel. ACh molecules bound to the receptive sites are in clockwise orientation. Right panel: Cross-section at the level of the receptive sites, viewed from the synaptic side down the channel. Upper, the resting topology, with the trans-membrane channel below closed. In each site, ˚ . Lower, AChR channel the anionic center () is clockwise to the H bond donor (H) subsite. Space between the two anionic centres should measure 20 A opened by two molecules of ACh. The ACh molecules are clockwise from the carbonyl O to the methonium head. They conform to Beers and Reich’s rule of ˚ for nicotinic action, and match the complementary subsite topology. The ACh molecules and the receptive sites appear large relative to the inter-site 5.9 A space. The trans-membrane channel below, shown as a dotted circular passage, has room to pass cations as large as TEA, but nothing much larger. This is matched by the open passage at the level of the receptive sites. Modified from Lee (2002c).

6.25. Protonation of d-tubocurarine and vecuronium The tertiary N atom of dTc and that of vecuronium are protonated (Pauling & Petcher, 1973; Hill et al., 1994). The protonation has different implications on their CAR and mechanism of action. From the viewpoint of conformation, dTc lacks a carbonyl O to make a good mono-quaternary agent. It depends instead on the protonation to function as bis-quaternary. This ‘‘bis-quaternization by protonation’’ is probably helpful, considering that a permanent dimethyl bis-quaternization, which converts dTc to chondocurine, more than doubles the potency (Hill et al., 1994). In contrast, vecuronium has a superb D-ring ACh moiety to function as a potent mono-quaternary NMB agent. The 2-N protonation has unknown value because even a permanent methyl quaternization there (resulting in pancuronium) adds little to NMB potency. The presumed protonation of vecuronium does not restore the vagolytic action of pancuronium either. According to CAR, bis-quaternization by

protonation is not why vecuronium is as potent as pancuronium.

7. Receptive site topology remodeled in the light of the conformation-action relationship of the neuromuscular blocking agents The inter-site topology of the nicotinic AChR can be better modeled for the motor endplate of the mammalian skeletal muscle by considering the CAR of the NMB agents, as discussed in the following sections. 7.1. Inter-site space available to neuromuscular blocking agents The inner borders between the receptive sites have been ˚ (Egebjerg, 1996) or 20– 30 A ˚ apart estimated to be 20 A (Taylor et al., 1991; Egebjerg, 1996; Dilger, 1998). The

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helical arrangement of the amino acid residues that line the channel with side chains protruding to variable distances into the lumen and the possible existence of intervening water molecules may account for the unavailability of part of this space. Measured by muscle relaxant molecules placed inter-site as molecular yardstick, the space actually available to NMB agents is probably 20 – ˚ (Fig. 10). 21 A 7.2. Orientation and dimension of acetylcholine-binding subsites The methonium head of ACh is attracted to the ae interface, but ACh is largely bound to the a-subunit. On the receptive site, therefore, the H bond donor subsite is probably on the a-subunit. In other words, it is clockwise from the H bond donor (on the a) to the anionic centre (at the ae interface) because the pentameric structure is aeadß clockwise. Likewise, the receptor-bound molecular ACh and the D-ring ACh moiety of vecuronium should also be clockwise from the carbonyl O to the quaternary N (Lee, 2002a, 2002b). As elaborated above, only this hypothetical receptive site orientation allows AChR-bound vecuronium to use its lipophilic b surface to shield off the extracellular fluid from above. Within each receptive site, the two subsites should relate to each other in a manner that complements the D-ring ACh moiety of vecuronium in ˚ (Figs. 10 fulfillment of Beers and Reich’s rule of 5.9 A and 11).

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7.3. Inter-site topology when two molecules of acetylcholine bind the receptive sites Subtracting space taken by two molecules of receptorbound ACh, the remaining space between the receptive sites ˚ ). Otherwise, there would be should at least pass TEA (8 A an inter-site bottleneck, considering that even the crossmembrane portion of the receptor channel below passes TEA. For structural stability, energy efficiency, and ease of return of molecular ACh back into the extracellular fluid, one assumes that receptor-bound ACh molecules should remain superficial and protrude into the lumen. The unoccupied cross-sectional area could then be like a dumbbell in ˚ the space for two shape (Fig. 10). Subtracting from 20 A ˚ ˚ should methonium heads of ACh (6 A each), at least 8 A ˚ indeed remain inter-site to pass TEA. Could more than 8 A remain? If the onium heads of the two ACh molecules can be equated to the two onium heads of C10, then little, if any, ˚ should remain because C10 has a fixed more than 8 A length. Subsequent to the binding, the inter-site distance might change, but this is beyond the scope of the present review. So are changes below the level of the receptive sites. In Fig. 10, the molecule of ACh and the receptive sites appear large relative to the cross-sectional area. This is ˚ . It is appropriate because a methonium head measures 6 A important to recognize that the above view of the topology of the endplate AChR is based on the CAR of the NMB agents only. The dimensions are only approximate. For example, TEA may change to a ‘‘Nordic cross’’ conforma-

Fig. 11. Hypothesized mechanisms of action of ACh, SDC, C10, gallamine, vecuronium, and mivacurium (left upper to right lower), based on their CAR. Vecuronium is shown with its D-ring ACh moiety on the ae receptive site without conformational reason to specify that it prefers this site. Shaded areas in ACh and the interonium structures of C10, gallamine, and vecuronium are intended to show lipophilic regions of the molecules. In vecuronium (see also Figs. 3 and 8), the lipophilic regions, focusing on the 10- and 13-methyl groups, are on the b side facing the synapse. In gallamine and C10, binding at the two anionic centres does not prevent the molecule from rotating along this axis.

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tion with little difference in energy (Wait & Powell, 1958; Stenlake, 1963). In that conformation, its molecular size will be somewhat smaller.

8. Conformational mechanism of action of various classes of neuromuscular blocking agents The conformational features of the NMB agents and the topological features of the AChR must be accommodated in proposing how muscle relaxants initially work. A ‘‘twopoint theory’’ has been proposed (Lee, 2001c). Basically, an NMB agent blocks by binding to two of the four subsites of the AChR, either ‘‘inter-site’’ or ‘‘intra-site.’’ To strengthen binding at the anionic subsite, a small (methyl) quaternizing group permits close apposition, correct chirality facilitates three-dimensional fitting, high charge density strengthens electrostatic attraction, and large onium heads hinder competition from ACh. To strengthen the hold, the onium head should be rigid. To facilitate binding at the H bond donor subsite, the carbonyl O atom should protrude unshielded and the acyl group should be small. An ether O is inadequate. The onium head and the H bond acceptor together should fulfill Beers and Reich’s rule ˚ for nicotinic action. To best occupy the inter-site of 5.9 A ˚ across) and space, the molecule should be bulky (20 – 21 A rigid, and should have a lipophilic surface that can be pointed outward toward the synapse. The interonium structure should put the binding groups at the correct place with correct orientation, or permit them to do so on their own. In the former case, rigidity is advantageous. In the latter case, flexibility is. Because the attraction is strongest between the charged onium head of the relaxant molecule and the anionic centre of the receptive site, good inter-site binding by the bisquaternary mechanism of action generally produces superior NMB potency. Potent mono-quaternary compounds must have a complementary H bond acceptor in the vicinity of the N to make a good ACh-like moiety to bind intra-site. An onium head alone, un-complemented by an H bond acceptor, is insufficient. An un-complemented onium head plus an H bond acceptor at a distance may improve the potency, but such an inter-site mono-quaternary mechanism rarely makes a potent NMB agent. Two H bond acceptors by themselves do not make potent NMB agents. A bis-quaternary compound with a suitable flexible interonium chain connecting two complete ACh-like pharmacophores theoretically may block by binding all four subsites. However, few muscle relaxants demonstrate an important contribution of multi-point binding. Depolarizing NMB agents bind the same receptive sites as do the non-depolarizing NMB agents. They share the same optimal molecular length and they both depend on conformational rigidity for potency. Transitional compounds galore and their mix of mechanisms of action vary accordingly. Subsequent to the binding of the receptive sites, what

happens to the receptor and what distinguishes depolarizing versus non-depolarizing blocks are complex issues. For the endplate to depolarize, in any case, the depolarizing cations presumably must flow past the receptive sites and then through the trans-membrane tube portion of the channel. Molecular size is the main distinction between depolarizing and non-depolarizing mechanisms of action, and the distinction is not absolute. The contribution of this ‘‘channel block’’ varies among relaxants and depends on their molecular size and shape. During neuromuscular block, C10, SDC, C18, gallamine, dTc, vecuronium, pancuronium, pipecuronium, and doxacurium, approximately in that ranking order, may leave room for depolarizing cations to flow through the channel around them. To add to the complexity of composition of mechanisms of action, mTc and atracurium have partial agonistic action and may enhance the response of fetal AChR to ACh (Fletcher & Steinbach, 1996). Existing potent NMB agents (Table 1) fit the above general plan. The mechanisms of action of individual muscle relaxants are proposed below (Fig. 11). Whatever the mechanism or combination of mechanisms, ‘‘conformational density’’ gives rise to steep dose-response curves. Among the aminosteroids, vecuronium best illustrates an intra-site mono-quaternary mechanism of action. Rocuronium and rapacuronium act similarly, although their larger acyl and quaternizing groups reduce their potency. Pipecuronium best illustrates the inter-site bis-quaternary mechanism of action. Pancuronium blocks more like a monoquaternary than a bis-quaternary, because 17-desacetylation reduces its potency, while 2-N de-quaternization does not. The latter only converts it to vecuronium. It has optimal ACh-like moiety, but only suboptimal N-N distance. Unlike most mono-quaternary aminosteroids, which bind intra-site, ANQ 9040 and some HS-compounds rely on the H bond acceptor on the distant part of the molecule to complete the required two-point binding inter-site. While both C10 and SDC have a depolarizing mechanism of action, only C10 keeps a straight conformation with significant rigidity. As a result, only C10 can bind inter-site as bis-quaternary. Possibility exists that some C10 molecules bind only one receptive site. In that case, C10 is similar to SDC, requiring one molecule at each receptive site to open the AChR channel. In CAR, the mechanism of action of SDC is better described as mono-quaternary. Two molecules of SDC act concomitantly, one on each receptive site, binding both subsites with one ACh moiety. The second ACh moiety is required for the molecule to maintain an NMB conformation at one end or the other, or both. It does not bind simultaneously. All C10 congeners have the same methonium heads. Among them, C10 is best suited to have a bis-quaternary mechanism of action. Shorter congeners may not reach, while longer congeners may have to bend to do so. Chances of needing two molecules at the same time also vary accordingly. So does the so-called ‘‘non-depolarizing’’ com-

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ponent of block. Any redundant lipophilic chain folding into the lumen of the AChR channel may cause one form of channel block. Unless bound at both ends, these congeners are more likely than other NMB agents to align themselves lengthwise along the passage of the channel and travel through it because they are straight and thin molecules. This explains why C10 is the most recognized NMB agent that ends up intracellularly (Martyn et al., 2000). Bismethonium derivatives with non-lineal interonium connections bulkier than C10, such as cyclo-octadecane and biscyclohexylethane, will likewise have redundant lipophilic structures to impart some ‘‘non-depolarizing’’ or channel block. Except for the presence of a third ethonium head, gallamine is similar to these compounds in CAR, with two binding sites and a lipophilic centre. Its mediocre bulk, rigidity, and molecular length make it a ‘‘soft pachycurare’’ of low potency. The tetrahydroisoquinolinium compounds bind inter-site as bis-quaternary. Protonation of dTc enables it to bind ‘‘inter-site’’ as an inferior mTc. Those with long-chain interonium structures fold their diester chain and their 1benzyl (phenyl in one onium head of GW280340A) substitutions into the inter-site space to impede the flow of the depolarizing cations. Derivatives of TAAC3 also act as bisquaternary, inter-site, according to similar CAR rules. The carbonyl O on the quaternizing benzyl group fulfills Beers and Reich’s rule for nicotinic action. The contribution of this additional mechanism of action is clear.

9. Comments 9.1. Preference for one receptive site to the other NMB agents may have a different preference for the ae or the ad receptive site. While mTc and gallamine prefer one site, pancuronium and atracurium prefer the other (Fletcher & Steinbach, 1996). Unfortunately, SAR or CAR of the NMB agents has not shed light on such preferences. Between the adult (ae) and the fetal (ag) form of the ae receptive site, the fetal form is generally more sensitive, except to dTc and gallamine (Paul et al., 2002). Excluding this exception, NMB agents rank similarly in sensitivity in both forms (Fletcher & Steinbach, 1996; Paul et al., 2002). Possibility of tailoring NMB agents to specific receptor forms is remote, although conceivable (Naguib et al., 2002; Lee, 2002b). 9.2. Other conformation-action relationship questions Several molecular changes may shed new light on the SAR and CAR of the NMB agents: vecuronium or pancuronium derivatives with the a or b orientation of the functional groups on the D-ring and A-ring ACh moieties systematically altered, benzylisoquinolinium compounds with acetoxy replacing methoxy groups, or two vecuronium

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joined by flexible links to allow the two D-ring ACh moieties to seek a four-point fit. It is not known why bisquaternization of dihydro-b-erythroidine reduces, instead of increases, its potency (Hill et al., 1994). The CAR of chirality is also interesting. 9.3. Conformation-action relationship of channel block The CAR of the NMB agents suggests that blockage of the AChR channel may occur at various levels. The transmembrane portion should admit SDC and C10, especially C10 because it is straight and the thinnest (Lee & Jones, 2002). Depending on the size, charge, and conformation of the molecule, pachycurares too large to enter the tube may cause channel block at various levels of the cone. aBungarotoxin likely fills the entire cone above the binding sites. This concept of nicotinic AChR channel block is inferred from the SAR and CAR of the NMB agents only, and may differ from what is actually measured by crossmembrane events (Colquhoun, 1980; Ogden & Colquhoun, 1985; Albuquerque et al., 1988; Charnet et al., 1990; Zhorov et al., 1991; Zhorov & Brovtsyna, 1993; Marshall et al., 1994; Martyn et al., 2000).

10. Conclusions In conclusion, SAR has historically guided the development of NMB relaxants. However, the utility of SAR is limited. Meanwhile, the nicotinic AChR has become the most studied receptor, and the gap between receptorology and clinical neuromuscular pharmacology has become ever wider. Preliminary as it is, CAR has added to our understanding of how muscle relaxants might work. It explains most potency-related observations in neuromuscular pharmacology, offers new insight into the receptive site topology of the endplate AChR, and suggests specific conformational mechanisms of action of NMB agents of various chemical classes. To have high potency, an NMB agent must fit two points well, either two anionic centres across the channel (inter-site) or an anionic centre along with a complementary H bond donor subsite cis to each other (intra-site). Having more than two binding groups may be counterproductive. Depolarizing or non-depolariz˚ ing, muscle relaxant molecules fit into the same 20– 21 A inter-site space. Molecular rigidity and high concentration of conformers that fit the receptive site topology well will increase potency. The entire molecule may be bulky and rigid, and configured to fit. Alternatively, thin and flexible connecting chains may permit rigid and bulky onium heads to seek their own best fit. In any case, a large lipophilic bulk across the channel may help by impeding the flow of the depolarizing cations. Overall, bis-quaternary compounds have greater potency. Naturally, much of the CAR reviewed will have to be updated as more is elucidated about the nicotinic AChR, the NMB agents, and how

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they interact with each other in vacuo or eventually in the physiologic milieu.

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