Alkyl-modified side chain variants of anatoxin-a: A series of potent nicotinic agonists

DRUG DEVELOPMENT RESEARCH 31~147-156 (1994)

Alkyl-Modified Side Chain Variants of Anatoxin-a: A Series of Potent Nicotinic Agonists

Philip Thomas, Paul A. Brough, Timothy Catlagher, and Susan Wonnacott Department of Biochemistry, University of Bath, Bath (P.T., S. W.), and School of Chemistry, University

of Bristol, Bristol (P.A.B., T.C.), England

Strategy, Management and Health Policy

ABSTRACT The potent nicotinic agonist anatoxin-a has a semi-rigid structure amenable to chemical synthesis and modification, making it an attractive candidate for exploring the structure-activity relationships of ligands at nicotinic acetylcholine receptors. Racemic anatoxin-a and a series of three analogues with one or more methine or methylene units added to the acetyl sidechain were synthesised and designated homoanatoxin, propylanatoxin, and isopropylanatoxin. In competition binding assays on two neuronal nlcotinic receptors in rat brain membranes, labelled with 13H]nicotine and [1251]abungarotoxin, the analogues retained or exceeded the potency of the parent structure. K, values for anatoxin-a, homoanatoxin, propylanatoxin, and isopropylanatoxin were 19, 5.5,24 and 7.5 nM, respectively, at the [3H]nicotine site and 900,340,40, and 120 nM, respectively, at the [1251]abungarotoxin site. Thus propylanatoxin appears to show a preference for the latter receptor site. Functional potencies of homoanatoxin and isopropylanatoxin were determined at neuronal a7 nicotinic receptors reconstituted in Xenopus oocytes, by two electrode voltage clamp recording. EC,, values of 0.72 pM and 0.66 pM were determined, similar to that previously published for (+ )anatoxin-a. Molecular modelling of anatoxin-a and its alkyl-modified analogues shows them to have very similar energy profiles, consistent with their similar potencies. In the preferred (low energy) conformation, the enone moiety adopts an s-trans arrangement, although this configuration does not fit the classical pharmacophore model for nicotinic ligands. This series of alkyl-modified analogues based on anatoxin-a provides some novel potent nicotinic agonists and with propylanatoxin, receptor subtype selectivity may be emerging. This rational approach to ligand design also enables us to explore further the requirements for ligand recognition through the application of computational chemistry. o 1994 Wdey-Llss, inc

Key Words: toxins, brain membranes, [3H]nicotine binding, ['Z51]abungarotoxin binding, oocyte expression

INTRODUCTION

Anatoxin-a (AnTx; Fig. 1: 5a) is a potent naturally occurring nicotinic agonist, produced by freshwater cyanobacteria Anabaena f lo s aqua and Oscilla- toria [Carmichael et al., 1979; Skulberg et al., 19921. AnTx is between 3 and 50 times more potent than (-)nicotine as an agonist at several subtypes of neuronal nicotinic acetylcholine receptor [nAChR; Thomas et al., 19931. As a secondary amine, AnTx can cross the blood brain barrier, and produces a partial nicotine-like discriminative stimulus effect in rats

0 1994 Wiley-Liss, Inc.

trained to discriminate nicotine [Stolerman et al., 19921.

AnTx has been synthesised de novo [Koskinen and Rapoport, 1985; Huby et al., 19911. The scope for structural modification, together with the rigid 9-azabicyclo[4.2. llnonene skeleton which favours the application of computational chemistry, has stimu- lated structure-activity relationship (SAR) pro-

Address reprint requests to Susan Wonnacott, Department of Biochemistry, University of Bath, Bath BA2 7AY, UK.

148 THOMAS ET AL.

(1 1 (2a-d)

(4a-d)

(3a-d) CI

(Sa) h T x R= CH3 (Sb) H o m h T x R= CH2CH3 (Sc) PropylAnTx R= CH2CH2CH3 (Sd) IsopropylAnTx R= CH(CH3)2

Figure 1. Synthesis of anatoxin-a and sidechain analogues from ketene dithioacetal (1). Reagents and conditions: (i) n-BuLi, THF; (ii) RI, (=R=CH3; bR= CH,CH,; 'R= CH,CH,CH,; dR=CH(CH,),) (iii) Vinylchloroformate, K,CO,, CH,CI,; (iv) HCI (aq); (v) Boc,O, Et,N; (vi) 1,4-dioxane, HCI (aq). In 4 and 5 the enone moiety i s shown in the s-cis configuration.

grammes based on AnTx. All members of an extensive series of analogues [Sardina et al., 1989; Howard et a]., 19901 were shown to have weaker activities than the parent compound in functional and binding assays for muscle nAChR [Swanson et al., 19911 and in nicotinic binding assays on brain tissue [Wonnacott et al., 19911. This exercise defined the secondary amine and carbonyl functions of AnTx as crucial for potency. Consequently, we have recently focussed on new analogues that conserve these features. Addition of one methylene unit to the acetyl sidechain of AnTx, cre- ating homoanatoxin (HomoAnTx, Fig. 1: 5b) did not diminish potency at neuronal nAChR, as judged by ligand binding assays [Wonnacott et a]., 19921. Sub- sequently, HomoAnTx has been identified as a naturally occurring algal toxin [Skulberg et al., 19921. Here we have extended this synthetic series to in- clude propyl- and isopropyl-analogues (Fig. 1: 5c, d). They have been compared with AnTx and HomoAnTx in binding assays, and a preliminary assessment of functional potency has been undertaken. The results are compared with the predictions arising from the application of computational chemistry to these novel structures.

MATERIALS AND METHODS Materials

( -)[N-methyl-3H]Nicotine (78Ci/mmol) was from DuPont-NEN (Stevenage, UK) and [1251]Na was

purchased from Amersham International (Amersham, UK). aBungarotoxin was obtained from the Sigma Chemical Co. (Poole, UK) and was iodinated to a specific activity of 700 Ci/mmol. Reagents and solvents for synthetic chemistry were purified by standard pro- cedures. The hydrochloride salts of the AnTx analogues were dissolved in aqueous ethanol (AnTx, 19%; homoanatoxin [ HomoAnTx], 50%; propylanatoxin [ PropylAnTx] , 95%; isopropylanatoxin [ Isopropy- IAnTx], 50%) to give stock solutions of 10-'M which were stored in aliquots at -20°C. Molecular models were generated using HYPERCHEM 3.0 (Autodesk Inc., Sansalito, CA).

Methods

Chemistry

PropylAnTx and IsopropylAnTx were synthesised from ketene dithioacetal (Fig. 1: 1) as follows:

2-(2-Propyl-1,3-dithian-2-yl)-9-azabicyclo[4.2.1] non-2-ene (Fig. 1: 2c). To a stirred solution of ketene dithioacetal(1) (0.741 g, 2.9 mmol) in tetrahydrofuran (THF) (22 ml) at -78°C under nitrogen was added n-butyl lithium (1.6 M, 2.53 ml, 4.06 mmol) dropwise, and the solution slowly allowed to warm to room temperature over a period of 2 h. The solution was then recooled to -78°C and n-propyl iodide (0.592g, 3.34 mmol) in THF (5 ml) was added dropwise over a period of 2.5 h using a syringe pump. The reaction mixture was then allowed to reach ambient tempera-

POTENT NICOTINIC AGONIST ANALOGUES OF ANATOXIN-A 149

ture, quenched with water (10 ml), diluted with ethyl acetate and the phases separated. The aqueous phase was extracted with ethyl acetate (1 x 25 ml) and the combined organic phases were dried (Na,SO,), evaporated and the residue was purified by flash chromatography (ethyl acetate, silica gel Merck No. 11695) to give the title compound as a yellow oil (0.588 g, 68%) (Found: 297.1577. C,,H,,NS, requires 297.1585);. omZ (film)/cm-, 1,470, 1,450, 1,435, 1,425; 6, (270 MHz, CDCI,) 0.89 (3 H, t, J 7.3), 1.20-1.60 (3 H, m), 1.60-2.40 (11 H, m), 2.40 (3 H, s, NCH,), 2.56-2.70 (2 H, m), 2.79 (1 H, d d d, J 3.3, 10.6, 13.9 Hz, SCH,CH,) 2.92 (1 H, d d d, J 3.1, 10.6, 13.9 Hz, SCH,CH,), 3.40 (1 H, m), 4.00 (1 H, d, J 9.1 Hz), 6.20 (1 H, d m, J 7.3 Hz); 6, (67.8 MHz, CDC1,) 145.1, 130.1, 64.4, 62.6, 60.1, 41.9, 38.2, 32.1, 27.9,27.6,27.4,25.4,23.9, 17.4, 14.1;m/z(EI) 297 ( M t , 58%).

2-(2-Propyl-1,3-dithianyl-2-yl)-9-vinyloxycar- bonyl-9-azabicyclo[4.2.l]non-2-ene (Fig. 1: 3c). To a stirred solution of the alkylated dithiane (2c) (0.588 g, 1.98 mmol) in dichloromethane (19 ml) was added anhydrous potassium carbonate (0.50 g). This mixture was stirred for 0.5 h then cooled to 0°C. Vinyl chloroformate (0.178 pl, 1.98 mmol, Aldrich, 94%, which was stored over dry sodium sulphate for at least 6 h) was added dropwise and the mixture was then stirred for 18 h under nitrogen at ambient temperature. Fur- ther vinyl chloroformate (18 p1, 0.19 mmol) was added and stirring continued for 2 h. Silica gel was added to the reaction mixture and solvent evaporated in vacuo to give a solid residue which was purified by flash chromatography [ethyl acetate-light petroleum (0:l-1:9)] to give the title compound (0.508 g, 72%) as a colourless solid. An analytical sample was obtained by recrystallization from di-iso-propyl ether, m. p. 113-115°C; (Found: 353.1490. C,,H2,N0,S, requires 353.1483); urnax (CHCl,)/cni-l 1,713, 1,645, 1,418; 6, (270 MHz, CDC1,) (complicated by carbamate resonance) 0.87 (3H, q, J 7 Hz), 1.0-1.5 (3 H, m), 1.5-2.2 (8 H, m), 2.20-2.90 (7 H, m), 4.43 (1 H, m), 4.60-4.82 (2 H, m), 5.08 (1 H, br t, J 9.7 Hz), 6.23 (1 H, m), 7.21 (1 H, d d, J 6.2, 14.1 Hz); m/z (EI) 353 ( M + , 80%).

9-((Tert-butyloxy)carbonyl)-2-(butan- l-oxo- 1-yl) -9-azabicyclo[4.2.l]non-2-ene (Fig. 1: 4c). To a solution of the vinyloxycarbamate (3c) (0.4648, 1.3 mmol) in 1,4-dioxane (20 ml) was added water (5 ml) and concentrated HCl(O.9 ml). The mixture was heated to reflux under a nitrogen atmosphere for 18 h. The majority of the solvents were removed in vaciio and the residue diluted with water (10 ml), cooled to 0°C and then made basic by dropwise addition of 2 M sodium hydroxide. The solution was extracted with

dichloromethane (5 x 10 ml) and the combined organic phases evaporated in vacuo. The residue was taken up in 2:l THF-water (25 ml) and triethylamine (0.45 ml, 3.26 mmol) was added followed by di-tert- butyldicarbonate (0.71 g, 4.59 mmol). After standing at room temperature for 18 h the solvent was removed in vacuo, the residue diluted with water (10 ml) and then extracted with dichloromethane (5 x 10 ml). The combined organic phases were dried (Na,SO,) and evaporated to an oil which was purified by flash chromatography [ethyl acetate-light petroleum (1:4)] to give the title compound (0.250g, 66%) as a colourless oil, (Found: 293.1984. C,,H,,NO, requires 293.1991); amax (film)/cm-' 1,691, 1,670, 1,400; 6,, (270 MHz, CDC1,) 0.94 (3 H, t, J 7.3 Hz), 1.36 and 1.43 (9 H, s, 3 X CH,), 1.50-2.75 (5 H, m), 2.02-2.24 (2 H, m), 2.25-2.48 (3 H, m), 2.52-2.64 (2 H, m), 4.22-2.48(1H,m),5.12(1H,d,J9.3Hz),6.81(1H, t, J 7 Hz); 6, (67.8 MHz, CDCI,), 199.9, 153.0, 149.8, 140.7, 79.1, 55.5, 53.1, 39.0, 32.5, 31.5, 30.4, 28.6, 28.3, 24.1, 18.3, 18.0, 13.9; m / ~ (EI) 293 ( (Mt) ,

2-(Butan-l-oxo-l-yl)-9-azabicyclo[4.2. llnon- 2-ene hydrochloride (Fig. 1: 5c). The N-Boc derivative (4c) (14 mg, 48 pmol) was dissolved in 1,4-dioxane (250 1.1) and hydrochloric acid (2 M, 2.5 ml) was added. The reaction mixture was stirred vigorously for 4 h. The solution was then evaporated in vacuo to give a quantitative yield of the pure hydrochloride salt as a colourless glass. (Found: 193.1462 (Mf) . C,,H1,NO requires 193.1467); omax (CHCl,/cm-l) 1,672, 1,585; 6, (270 MHz, CDCI,) 0.91 (3 H, t, J 7.3 Hz), 1.59 (2 H, sextet, J 7.3 Hz), 1.80-2.03 (3 H, m), 2.08-2.24 (1 H, m), 2.31-2.65 (6 H, m), 4.32 (1 H, br ~ ) ,5 .24 (1H,m) , 7 .16(1H,br t , J6 .0Hz) , 8.62(1H, br s), 9.42 (1 H, br s); 6, (67.8 MHz, CDC1,) 198.7, 144.2, 143.4, 58.3, 52.4, 38.8, 30.4, 27.8, 27.5, 23.6, 17.9, 13.8; m/z 193 ( M + , 61%), 164(93), 150(100), 122(96), 82(71), 69(69).

2- (2-Iso-propyl- 1,3-dithian-2 -yl) -9-azabicyclo [4.2.l]non-2-ene (Fig. 1: 2d). The ketene dithioacetal (1) (0.510 g, 2.0 mmol) was alkylated with iso-propyl iodide (0.24 ml, 2.4 mmol) using the same procedure applied to the propyl analogue above to give the title compound as a yellow oil (0.500 g, 84%) (Found: 297.1569. C,,H,,NS, requires 297.1585); om, (film)/ cm-I 1,470, 1,446, 1,422; 6, (270 MHz, CDCI,) I .01 (3 H, d, J6.8 Hz), 1.10 (3 H, d, J6 .8 Hz), 1.45 (1 H, m), 1.58-2.40 (12 H, m), 2.30 (3 H, s, NCH,), 2.78 (1 H, t , J 12 Hz), 3.01 (1 H, t , J 12 Hz), 3.32 (1 H, m), 3 . 9 2 ( 1 H , d , J 7 . 7 H z ) , 6 . 1 8 ( 1 H , dm,J9 .3Hz) ;6 , (67.8 MHz, CDCI,) 145.4, 131.3, 67.4, 65.8, 64.2, 41.1, 35.4, 31.5, 30.3, 27.7, 27.3, 25.5, 23.7, 17.9, 17.8; m/z (EI) 297 (M', 33%).

3.4%).

150 THOMAS ET AL.

2-(2-Iso-propyl-l,3-dithianyl-2-yl)-9-vinyloxycar- bonyl-9-azabicyclo[4.2.l]non-2-ene (Fig. 1: 3d). The dithiane (2d) (0.490 g, 1.64 mmol) was demethylated using the same procedure as applied to the propyl analogue. Purification by flash chromatography [ethyl acetate-light petroleum (0:l-1:9)] gave the title compound (3d) as a colourless solid. Although spectral data were consistent with the required product, the compound could not be obtained in a sufficiently pure form for a full characterisation and was used crude for the next synthetic step.

9-( (Tert-butyloxy)carbonyl)-2-(2-methylpropan- l-oxo-l-yl)-9-azabicyclo[4.2.l]non-2-ene (Fig. 1: 4d). The impure vinyloxycarbamate (3d) (0.160 g) was hy- drolyzed and protected as its N-Boc derivative using the same procedure as applied to the propyl analogue. Purification by flash chromatography [ethyl acetate- light petroleum (1:4)] gave the title compound as a colourless solid (78 mg, 59%). An analytical sample was recrystallized from di-iso-propyl ether, m. p. 78-81°C; (Found: 293.1990. C17Hz7N0, requires 293.1991); o,, (CHCl,)/cm-l 1,684, 1,410, 1,173, 1,112; 6, (270 MHz, CDCl,) 1.10 (6 H, t, J 6.5 Hz), 1.36 and 1.43 (9 H, s, ~ x C H , ) , 1.5-1.8 (4 H, m), 2.0-2.3 (2 H, m), 2.3-2.5 (2 H, m), 3.25 (1 H, p , J 7 Hz), 4.23-4.48 (1 H, m), 5.12 (1 H, d, J 9.3 Hz), 6.70-6.87 (I H, m); 6, (67.8 MHz, CDCI,) 204.1, 153.1, 148.4, 140.3, 79.2,55.4,53.5, 33.6,31.8, 30.7, 28.8, 28.4, 24.2, 20.2, 19.0; m/z (EI) 293 (Mf, 4%).

2-(2-Methylpropan-l-oxo-l-yl(-9-azabicyclo[4.2. l]non-2-enehydrochloride (Fig. 1: 5d). The N-Boc derivative (4d) (15 mg, 51 pmol) was deprotected using the same procedure as applied to the propyl analogue to give the title compound as a colourless glass in quantitative yield. (Found: 193.1468. C1,H19N0 requires 193.1467); omax (CHCl,)/cm-l 1,671, 1,602, 1,470, 1,243; 6, (270 MHz, CDC1,) 1.06 (3 H, d, J 6.8 Hz), 1.07 (3 H, d, J 6.8 Hz), 1.80-2.03 (3 H, m), 2.13-2.30 (1 H, m), 2.30-2.65 (4 H, m), 3.23 (1 H, septet, J 6.8 Hz), 4.32 (1 H, br s), 5.21 (1 H, m), 7.17 (1 H, br t, J 5.3 Hz), 8.93 (1 H, br s), 9.57 (1 H, br s); 6, (67.8 MHz, CDC1,) 202.9, 144.3, 142.5, 58.5, 52.6, 33.4, 30.4, 27.6, 27.5, 23.5, 19.6, 19.1; m/z 193 ( M t , 71%), 178 (48), 164 (22), 150 (loo), 122 (88).

AnTx and HomoAnTx were synthesised by the same route (Fig. 1: 1-5,a,b).

Radioligand binding assays

The activities of the analogues were measured in competition binding assays at two rat brain nAChR subtypes labelled by [3H](-)nicotine and [ '251]cx-bun- garotoxin (aBgt) exactly as previously described [Wonnacott et al., 19921. Binding data were analysed by iterative curve fitting using the programme Sigma-

Plot 4.1. Data were fitted to the non-linear Hill equation: y = 1/(1+ (IC,~X)"~) , where x = analogue concentration, IC,, = concentration producing 50% inhibition of specific binding and nH = Hill number. Inhibition constants, Ki, were determined from IC,, estimates using the Cheng-Prusoff relationship [Cheng and Prusoff, 19731: Ki = IC,d(l+ Kd/[L]), where [L] = radioligand concentration used and K, = dissociation constant for the radioligand. K, values of 1 nM and 10 nM were assumed for [ '251]aBgt and [,HI( -)nicotine respectively [ MacAl- lan et al., 19881 and these concentrations were used in the binding assays.

Dual electrode voltage clamp electrophysiology

Xenopus oocytes were injected with 2 ng of a 7 cDNA [Couturier et al., 19901. Electrophysiological recording was performed 2 2 days later using a con- ventional dual electrode voltage clamp as previously described [Amar et al., 19931. Cells were clamped at -70 mV and perfused (5 mlimin) with Modified Barth's Solution, (MBS: NaCl 88 mM, KCl 1 mM, HEPES 10 mM, MgSO, 0.82 mM, Ca(NO,), 0.37 mM, CaCl, 0.91 mM, NaHCO, 2.4 mM, pH 7.5), containing 0.5 p M atropine to block endogenous muscarinic receptors. AnTx analogues were applied in perfusion as 3 s pulses. Data were stored on a digital- to-analogue DAT converter and processed using the aquisition and analysis programs AQ and PAT2L [Amar et al., 19911 and SIGMAPLOT 4.1. Dose-response curves were fitted to the non-linear Hill equation: y = l/(l + (EC,dX)"").

Computational chemistry

The molecular modelling package HYPER- CHEM (Autodesk Inc., CA) was used to construct protonated (+) enantiomers of each AnTx analogue in both twist chair and twist boat conformations. Partial charges were calculated using MNDO methods and structures were minimised with the MM + molecular mechanics forcefield. All rotatable bonds were sub- jected to dihedral driver calculation (30" incrementsj to derive intermediate conformations prior to mini- misation. All conformers approximately fitting the Beers-Reich distance for the nicotinic pharmacophore (i. e., 4.3-5.3A between the charged nitrogen and car- bony1 oxygen) and within lOkcal of the lowest energy structure, were assessed as candidates for bioactive conformations.

RESULTS Synthesis of AnTx Analogues

(2)PropylAnTx (5c) and (?)IsopropylAnTx (5d) were prepared from ketene dithioacetal (1) as shown


120

100

80

60

40

20

0

-11-10-9 -8 -7 -6 -5 -4 -3

log [AnTx analogue] (M)

Figure 2. Competition binding assays of AnTx analogues at (a)[3H]nicotine and (b)[’2511aBgt binding sites in rat brain membranes. Serial dilutions of AnTx (o), HomoAnTx (e), Isopropyl- AnTx (O), and PropylAnTx (V) were assayed. Values are the

in Figure 1. Alkylation of the allylic anion derived from (1) with the appropriate alkyl iodide gave the %substituted dithianes (2c,d) which were de-meth- ylated using vinyl chloroformate to give carbamates (3c,d). Acid hydrolysis of both the carbamate and dithiane residues, followed by reaction with Boc,O, gave the N-boc protected toxins (4c,d) which were deprotected with aqueous acid to give (5c,d) as the hydrochloride salts. The HCl salts of (?)AnTx and (2)HomoAnTx were also synthesised from ketene dithioacetal (l), exactly as described for the new analogues (Fig. l). For HomoAnTx this represents a more direct synthetic route than described earlier [Wonnacott et a]., 19921. The purity of these analogues, as judged by ‘H NMR was greater than 99%.

Competition Binding Assays

(+)AnTx inhibited [3H]nicotine and [ 12sII]abungarotoxin (aBgt) binding to rat brain membranes with Ki values of 19 nM and 0.9 p M respectively (Table 1). Taking into account the stereoselectivity of nAChR for the ( + ) enantiomer of AnTx, the K, value determined here for the racemic compound may underestimate that of the active enantiomer by a factor of two. Thus the values for the racemic compound agree with previous data for (+)AnTx (Ki = 3.5 nM and 0.4 pM, respectively [Wonnacott et al., 19911). HomoAnTx, PropylAnTx, and IsopropylAnTx competed for [3H]nicotine binding sites with Ki values of 5.5, 24, and 7.5 nM, respectively (Fig. 2; Table 1). At [1251]aBgt binding sites, Ki values were 0.3, 0.04, and

120 1 100

80

60

40

20

0

T

-10-9 -8 -7 -6 -5 -4 -3 -2

log [AnTx onologue] (M)

means from at least 3 independent assays, with s.e.m. indicated by the vertical bars. Data points were fitted to the non-linear Hill equation (see Materials and Methods); Ki values are presented in Table 1.

0.1 p M , respectively. These estimates for HomoAnTx are in excellent agreement with previous determinations [Wonnacott et al., 19921. Clearly the addition of further methine or methylene units does not diminish binding potency. None of these analogues showed any muscarinic potency as judged by [3H]yuinuclidinyl benzilate binding assays (data not shown).

Functional Assays

A preliminary assessment of the agonist potencies of HomoAnTx and IsopropylAnTx was carried out by two electrode voltage clamp recording from Xeno- pus oocytes expressing a7 nicotinic acetylcholine receptor (nAChR) [Amar et al., 19931. Brief application of agonist resulted in large inward currents of up to 1 pA. At high agonist concentrations, currents dis- played fast onset and rapid desensitisation, characteristic of a7 nAChR (Fig. 3a) [Couturier et a]., 19901. A range of analogue concentrations was tested on a single oocyte, and dose-response curves of the peak currents were averaged from several such experiments (Fig. 3b). The EC,, value for HomoAnTx was 0.72 p M , compared with 0.66 p M for IsopropylAnTx (Ta- ble 1). Comparison of concentrations eliciting maximum responses (HomoAnTx, 10 pM; IsopropylAnTx, 3 p M ) with responses to a maximally effective concentration of (+)AnTx [Amar et al., 19931 were made on the same oocyte and demonstrated that the AnTx analogues were as efficacious as the parent compound. However it was consistently observed that IsopropylAnTx produced a particularly steep dose re-

152 THOMAS ET AL.

~~ ~

TABLE 1. Binding Affinities and Functional Potencies of AnTx Analogues at Neuronal nAChR

Competition binding assays Agonist potency

[EC50 (nM)lb: [Ki (nM)I”

[’2511aBgt site a7 nAChR Ligand [3H]Nicotine site

(_‘)AnTx 1 9 t 3 900 ?: 50 n .d .‘ (_‘)HomoAnTx 5.5 -C 0.5 340 * 80 720 2 550 (t)PropylAnTx 2 4 ” 11 44 r’: 7 n.d. (4)lsopropylAnTx 7.5 t 3.0 120 t 30 660 2 20

aK, values are the mean ? s.e.m. from at least 3 determinations. bEC,, values are the mean t s.e.m. from at least 3 dose response curves. ‘n.d. = not determined

sponse curve and concentrations above 3 p M were less efficacious, resulting in a “bell-shaped” dose-response curve (Fig. 3b). Similar curves for several agonists were obtained from nAChR preparations using biochemical protocols over a longer timecourse [Thomas et al., 19931. This result from the rapid electrophysiological recording suggests that Isopropyl AnTx may produce very rapid desensitisation or receptor inactivation. Current-voltage relationships were determined for HomoAnTx and IsopropylAnTx (Fig. 4). The two analogues behaved similarly, with current responses decreasing as the membrane potential was stepped from - 100 to -30 mV. At more pos- itive potentials, marked inward rectification was observed.

Computational Chemistry

The bridged ring of AnTx can adopt either boat or chair conformations, whereas the enone moiety can exist in either a s-cis or s-trans arrangement. To explore the possible bioactive conformations, protonated (+) enantiomers of AnTx and each analogue were constructed. Four low energy conformations were isolated, corresponding to chair s-trans (CT), chair s-cis (CC), boat s-trans (BT), and boat s-cis (BC). As AnTx would be >99% protonated at physiological pH (pKa=9.36) [Koskinen and Rapoport, 19851 all calculations were performed on protonated structures. Briefly, the s-trans, twist chair (CT) conformation of each of the AnTx structures was the lowest energy form (Table 2). The difference in energy between s-trans chair and boat forms was very small (-0.03-0.2 kcal/mol), so both forms should be well populated. The chair forms of the s-cis conformers were also lower energy than the corresponding boat forms (AE-0.4-0.5kcal/mol), with the apparent ex- ception of AnTx.

The s-trans conformer was some 4.3-5.1 kcal/ mol lower in energy than its corresponding s-cis form in both chair and boat conformations (Table 2). The enone moiety (C = C-C = 0) was calculated to adopt a

planar arrangement (however, the crystal structure of AnTx.HC1 [Koskinen and Rapoport, 19851 predicts distortion from planarity of -IS0). All other conformations explored by dihedral driver calculations of the enone moiety (Fig. 5a) increased the energy of AnTx and all analogues. The presence of the increased hydrophobic bulk adjacent to the enone moiety, as a consequence of the addional carbon units, appears to have no significant influence on the preference for the s-trans configuration: each of the analogues retains the difference in energies between cis and trans configu- rations.

The optimal distance between the ammonium ion and the carbonyl oxygen in nicotinic ligands is regarded to be 4.8 5 0.3 K [Sheridan et al., 19861. s-Cis boat structures best fit this distance (Table 2) whereas in s-trans structures these pharmacophore points are in close proximity (Fig. 5b), which may also favour additional electrostatic attractive forces between the ligand and the receptor molecule.

DISCUSSION

Here we describe the synthesis of novel nicotinic ligands based on the natural algal toxins (+)am- toxin-a and homoanatoxin. For this initial characterisation we have employed the racemic compounds but the nicotinic potency of AnTx resides predominantly (and probably exclusively) in the configuration repre- sented by the (+ ) enantiomer of anatoxin [Spivak et al., 19831; thus the assays may underestimate nicotinic potency by a factor of two.

In competition binding assays, AnTx and the three analogues competed for [3H]( -)nicotine binding sites with Ki values between 5 and 24 nM; the rank order of potency was HomoAnTx>Isopro- pylAnTx>AnTx>PropylAnTx. This high affinity nicotinic agonist binding site is proposed to correspond to the nAChR composed of 014 and 02 subunits [Whit- ing et al., 1987; Flores et al., 19921. Equilibrium binding assays determine the affinity for the desensi-


Figure 3. Activation of a7 nAChR expressed in Xenopus oocytes by AnTx analogues. a: Representative traces of inward currents elicited by 1 JLM HomoAnTx (0) and IsopropylAnTx (V) in an oocyte previously injected with 017 cDNA. b: Dose-response curves for HomoAnTx (0) and IsopropylAnTx (0). Oocytes were clamped at -70 mV and exposed to consecutive 3 s pulses of increasing concentrations of agonist; pulses were separated by

tised state of the receptor, whereas this nAChR subtype is activated by 48 nM (+)AnTx [Thomas et al., 19931, indicating a 14-fold difference between K, and EC,, values.

Nicotinic binding sites characterised by [ lZ51] aBgt binding have been correlated with nAChR subtypes containing the a7 subunit [Anand et al., 19931. This nAChR is generally less sensitive to nicotinic ligands: Ki values between 40 nM and 900 nM were determined in the present study, with the rank order PropylAnTx > IsopropylAnTx > HomoAnTx > AnTx. Thus the parent compound was the least potent of the series, while PropylAnTx appeared to show a preference for this site. HomoAnTx and IsopropylAnTx gave comparable Ki values (0.34 p M and 0.12 pM, respectively). These two analogues also showed comparable potencies in activating a7 nAChR in Xenopus oocytes, with EC,, values of 0.72 p M and 0.66 pM, giving EC,dK, ratios of 2 and 5, respectively. These potencies compare favourably with our published evaluation of enantiomerically pure ( + )AnTx at the a7 nAChR [Amar et al., 19931, which yielded an EC,, value of 0.58 p M and an EC5dKi ratio of 1.5. Six other agonists gave EC,dKi ratios between 0.8 and 8.4 [Amar et al., 19931 so the AnTx analogues fall within this range.

120

100 - 80 -

60 -

-10 -9 -8 -7 -6 -5 -4 -3

log [Analogue] (M) m5min washing to allow full recovery from desensitisation. Re- sponses were normalised to maximum peak height and values from at least 3 oocytes have been averaged, with s.e.m. indicated by the vertical bars. Data points were fitted to the non-linear Hil l equation (see Materials and Methods). EC,, values are presented in Table 1.

Interestingly, the dose-response curve for Iso- propylAnTx was particularly steep and hooked, sug- gestive of rapid desensitisation or inactivation at higher agonist concentrations. Various AnTx analogues have been shown previously to exhibit channel blocking activity [ Swanson et al., 19911, although high micromolar concentrations are typically required. The current-voltage relationships for HomoAnTx and IsopropylAnTx did not reveal any difference between these analogues, and showed the inward rectification characteristic of a7 nAChR [Couturier et al., 19901.

From the ligand binding assays, PropylAnTx appears to be less selective for the [3H]nicotine site: its Ki value at the [1251]aBgt site (44nM) was less than twice that at the [3H]nicotine site (compared with K, ratios for the two sites of 46, 62, and 16 for AnTx, HomoAnTx, and IsopropylAnTx, respectively). A previous series of AnTx analogues [Wonnacott et al., 19911 typically gave Ki ratios for [3H]nicotine:[ 1251] aBgt sites of between 30 and 100; AnTx and Ho- moAnTx clearly fall in this category. It will be of in- terest to see if the apparent selectivity of PropylAnTx for the [lZ51]a-Bgt site is manifested in its agonist potency at a7 nAChR.

While AnTx is a relatively rigid molecule, the enone moiety exists in s-cis and s-trans conformations;

154

Potent ia l (mv)

THOMAS ET AL.

(+)chair conformations

Figure 4. Current-voltage relationships for HomoAnTx and Iso- propylAnTx. The voltage dependency of agonist-activated a7 currents was determined by applications (3 s) of analogue at 25min intervals to oocytes while stepping the holding potential from -100 mV to +30 mV. Analogue concentrations approximated to their EC,, concentrations: 0.6 pM (k)HomoAnTx(a), and 0.6 pM (?)lsopropylAnTx(V). Current responses were normalised with respect to the response observed at -100 mV. Inset: Superim- posed traces of currents evoked by HomoAnTx (0.6 )LM) in a single oocyte.

which of these is the bioactive conformation is still controversial [Gund and Spivak, 19911. The s-cis conformer is consistent with the classical Beers-Reich pharmacophore for nicotinic ligands [Beers and Reich, 1970; Koskinen and Rapoport, 19851. How- ever, an “excluded receptor volume” model, which adequately accounts for the enantiospecificity of AnTx, does not satisfactorily accommodate the s-cis conformer [Hacksell and Mellin, 19891. Conforma- tionally constrained s-cis variants of AnTx are, however, weak nicotinic ligands [Brough et al., 19921 (Brough, Thomas, Wonnacott and Gallagher, unpub- lished) but further work is necessary to establish the viability, or otherwise, of the s-trans arrangement as a bioactive conformation.

Molecular modelling studies of AnTx and its alkyl-modified sidechain analogues (Fig. 5) indicate that the s-trans arrangement of the enone moiety is the lowest energy configuration, in agreement with previous studies [Thompson et al., 19921, and with the crystal structures of AnTx [Koskinen and Rapo- port, 19851 and its N-acetyl analogue [Huber, 19721. It may be that some revision of the classical Beers- Reich model of the nicotinic pharmacophore is needed, as implicated in the analysis of acetylcholine

I n

0 30 60 90 120 150 180210 240 2 7 0 3 0 0 3 3 0 3 6 0

0 30 60 90 120 150 180 210240 270 300330360

Dihedral angle

Figure 5. Comparison of AnTx and alkyl-modified analogues by computational chemistry. Intermediate conformations of ( -t ) enantiomers of the AnTx structures were explored by rotating the enone dihedral angle (see Materials and Methods). Changes to (a) the total energy, and (b) the N - 0 distance were recorded. Graphs represent calculations performed on the lowest energy chair conformations. 0, AnTx; *, HomoAnTx; V, IsopropylAnTx; V, Propyl- AnTx.

bound to the nAChR [Behling e t al., 19881. Although there is a clear energy preference for the s-trans conformation, this does not necessarily represent the bioactive arrangement.

Extension of the alkyl sidechain does not distort the conformation of the AnTx skeleton, and the energy barrier to rotation between s-cis and s-trans ar- rangements remains essentially unchanged. These observations are consistent with the retention of bio- logical activity by these analogues. The nAChR binding site can evidently accommodate the increased hydrocarbon bulk of the alkyl sidechain. Indeed substantial extension of this group for the generation of affinity ligands results in only slight loss of potency [Huby et al., 19911. In contrast, changes or additions to other positions in the AnTx structure are poorly tolerated [Swanson et al., 1991; Wonnacott et a]., 19911.


TABLE 2. Molecular Properties of the Lowest Energy Forms of Anatoxin Structures

a) Total energy (kcalhol) and b) N-0 distance (A) of structures

Boat, trans (BT) Boat, cis (BC) Chair, trans (CT)

a b a b a b a b

Chair, cis (CC)

( + )AnTx 24.30 3.82 29.05 (+ )HornoAnTx 26.74 3.81 31.31 ( +)PropylAnTx 27.30 3.81 31.82 (+ )IsopropylAnTx 31.31 3.79 36.04

These structure-activity studies based on the potent naturally occurring agonist AnTx have defined three new nicotinic agonists at neuronal nAChR that retain or exceed the potency of the parent compound. This comparison demonstrates that the binding pocket of the two neuronal nAChR subtypes studied can accommodate the increasing hydrocarbon bulk of the extended sidechain of these analogues. Indeed, in the case of PropylAnTx some a7 subtype selectivity may be apparent. While molecular modelling con- firms the similarities between these analogues with respect to their energy profiles, the results challenge assumptions regarding the bioactive conformation of nicotinic ligands, since the lowest energy (s-trans) conformation is not the best fit of the current pharmacophore model. Further work in this programme of rational synthesis and evaluation will be needed to address this issue.

A C K N O W L E D G M E N T S

This work was supported by grants from the SERC to T.G. and S.W. P.A.B. is the recipient of an SERC CASE studentship in collaboration with Merck Sharp & Dohme Research Laboratories. We are grateful to Marc Ballivet for providing the a7 cDNA, and to Mark Stephens for carrying out some of the binding assays.

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Alkyl-modified side chain variants of anatoxin-a: A series of potent nicotinic agonists

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