Docking of 6-chloropyridazin-3-yl derivatives active on nicotinic acetylcholine receptors into molluscan Acetylcholine Binding Protein (AChBP)

Il Farmaco 60 (2005) 313–320 http://france.elsevier.com/direct/FARMAC/

Docking of 6-chloropyridazin-3-yl derivatives active on nicotinic acetylcholine receptors into molluscan Acetylcholine Binding Protein (AChBP) Roberto Artali *, Gabriella Bombieri, Fiorella Meneghetti Istituto di Chimica Farmaceutica e Tossicologica, Università di Milano, Viale Abruzzi 42, 20131 Milano, Italy Received 26 October 2004; received in revised form 5 January 2005; accepted 8 January 2005 Available online 05 March 2005

Abstract The crystal structure of Acetylcholine Binding Protein (AChBP), homolog of the ligand binding domain of nAChR, has been used as model for computational investigations on the ligand–receptor interactions of derivatives of 6-chloropyridazine substituted at C3 with 3,8diazabicyclo[3.2.1]octane, 2,5-diazabicyclo[2.2.1]heptane and with piperazine and homopiperazine, substituted or not at N4. The ligand– receptor complexes have been analyzed by docking techniques using the binding site of HEPES complexed with AChBP as template. The good relationship between the observed binding affinity and the calculated docking energy confirms that this model provides a good starting point for understanding the binding domain of neuronal nicotinic receptors. An analysis of the possible factors significant for the ligand recognition has evidenced, besides the cation–p interaction, the distance between the chlorine atom of the pyridazinyl group and the carbonylic oxygen of Leu B112 as an important parameter in the modulation of the binding energy. © 2005 Elsevier SAS. All rights reserved. Keywords: Docking; 6-Chloropyridazin-3-yl derivatives; AChBP

1. Introduction Nervous systems are intended to transmit their signals rapidly and vital components of this process are post-synaptic receptors, termed ligand-gated ion channels (LGICs) able to convert from chemical to electrical signals in less than one millisecond [1]. LGICs respond to ligand binding generating an electrical signal that depends on both selectivity of the ion channel and electrochemical gradients across the membrane, as the Central Nicotinic Acetylcholine (nACh). The recent advances in genomic provide a wide heterogeneity of nicotinic receptors, which led to design new selective ligands possibly devoid of nicotinic side effects [2,3]. nAChRs are implicated in neurodegenerative disorders such as Parkinson’s and Alzaheimer’s disease [4], where the nicotinic receptors are less than those present in the brain of healthy individuals [5]. The neuronal types, which are either hetero- or homomeric,

* Corresponding author. Tel.:+39 02 503 175 52; fax:+39 02 503 175 65. E-mail address: [email protected] (R. Artali). 0014-827X/$ - see front matter © 2005 Elsevier SAS. All rights reserved. doi:10.1016/j.farmac.2005.01.004

located on both pre- and post-synaptic nerve terminals, mediate the positive effects of nicotine on cognition, memory and attention [6]. In nAChRs, the ligand-binding site is located at the interface between two subunits [7], and acetylcholine (ACh) is expected to bind through cation–p interactions, where the cationic nitrogen of ACh interacts with electron-rich aromatic side chains [8]. For AchRs, the most recent electron microscopy data at 4.6 Å resolution identified ACh binding-pockets surrounded by seven-stranded b-sheet structures [9]. During an investigation on the glia role in synaptic transmission, Smit et al. [10] isolated a soluble Acetylcholine Binding Protein (AChBP), secreted by glial cells to modulate the cholinergic transmission at synapse. The structure of this molluscan acetylcholine binding protein, solved by X-ray crystallography at 2.7 Å resolution [11], shares 18% sequence identity with GABAA receptor and up to 24% sequence identity with the extracellular portion of the nACh receptor. On these basis, several homology models [12–15] were created using AChBP structure that contains the aminoacid residues needed for the ligand binding.

314

R. Artali et al. / Il Farmaco 60 (2005) 313–320

Fig. 1. (a) Schematic representation of the homopentamer.1 (b) HEPES (in CPK) in the ligand binding site at the interface between two protomers.

The structural studies point out that AChBP is a radial symmetric homopentamer, where the only subunit contacts are dimer interfaces (Fig. 1a), with dimensions similar to those defined by electron microscopy of the extracellular domain of the nACh receptor [9]. The ligand binding sites are at each of the five subunit interfaces where the molecules of HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonate acid), coming from the buffer used in the crystallization process, are located. This weak ligand has a positive charged nitrogen similar to ACh. The orientation of HEPES that was not well defined in the 2.7 Å data has been clarified in the recent 2.1 Å resolution structure [16]. The HEPES reorientation does not affect the position of the charged ammonium group that remains unchanged (Fig. 1b). The piperazinium moiety lies between two subunits having residual identical to those deduced by affinity labeling and of mutagenesis for the ACh binding site in the ACh receptors [12,17]. Celie et al. [16] reported also the structures of the AChBP complexed with nicotine or carbamylcholine that permit to identify the classical geometrical features expected in the nicotinoid pharmacophore for maximal binding affinity [18]. These agonists are buried in the same binding site and the characteristics of the ligands binding are deduced comparing the structures of nicotine and carbamylcholine with the high resolution structure of HEPES [16] that could represent the model for the interaction with the charged quaternary group present in the nicotinic ligands. In fact, the superposition of these ligands bound to AChBP shows their charged nitrogens at about the same position comparable to that of HEPES (Fig. 2). HEPES stacks with its quaternary ammonium onto Trp A143 making cation–p interaction: the face of the aromatic ring provides a region of negative electrostatic potential that binds the cation with considerable strength [19]. This is supported by quantum mechanical calculations of interaction energies between unnatural Trp analogs and a cation [20] and by theoretical studies that clearly indicate, among the natural aminoacids, Trp has the most potent cation–p binding site

Fig. 2. Superposition of HEPES (ball and stick), Carbamylcholine (light gray) and Nicotine (dark gray) as found at the ligand binding site [16].

[21], making van der Waals contact with the six membered ring of the indole moiety and the charged nitrogen. The synthesis and analgesic activity of a series of 3-(6chloro-3-pyridazinyl)-3,8-diazabicyclo[3.2.1]octane derivatives, as possible analogs of the nAChR modulator epibatidine, has been previously reported [22] and more recently [23] the effects of the quaternization of the basic nitrogen of these ligands were investigated (Table 1). Since the structure of the cholinergic receptor (AChR) is not known at atomic level, the structure–activity relationships of these ligands and the docking experiments could contribute to a better characterization of the binding site of the nicotinic drugs. The examined compounds were 3,8diazabicyclo[3.2.1]octane (1), 2,5-diazabicyclo[2.2.1]heptane (2), piperazine (3), and homopiperazine (4), substituted at one nitrogen atom with the 6-chloro-3-pyridazinyl group while the other nitrogen atom was either unsubstituted (1a–4a), or mono- (1b–4b), or di-methylated (1c–4c). The present paper reports the results of docking experiments of these ligands with AChBP with the ultimate purpose to provide a possible relationship between their affinity and the docking energy.

R. Artali et al. / Il Farmaco 60 (2005) 313–320

315

Table 1 Binding properties [23] of the docked compounds

Comp. 1a

Log(Ki × 109) 0.61

Comp. 1b

Log(Ki × 109) 1.59

Comp. 1c

Log(Ki × 109) 0.36

2a

0.94

2b

1.81

2c

0.63

3a

1.50

3b

1.15

3b

–0.35

4a

0.17

4b

2.34

4b

2.07

2. Experimental procedures All calculations were done on a dual-Pentium III workstation, running SGI-SMP version of Linux RedHat 7.3 Professional running the Molecular Operating Environment (MOE) ver. 2002.03 [24], GROMACS [25] and MOPAC [26] ver. 7.0 programs. The AChBP has been used as macromolecular receptor target. Representative models of the binding site of the protein were constructed from the crystal structure coordinates [11] by including only the relevant subunits A and B (Fig. 1b). The edited crystal structure (water molecules removed) was imported into MOE and all hydrogen atoms were added to the structure with their standard geometry. The MMFF94 [27] forcefield and partial atomic charges were assigned to the protein. The resulting structure was then equilibrated for 100 ps to remove bad contacts between hydrogen atoms, using a positional restrained molecular dynamics protocol (GROMACS) in which part of the system (in this case all non-hydrogen atoms) is not allowed to move from its initial position. For the ligands 3c and 4c, the crystallographic coordinates have been used [28] and those of 3c were employed as template to build the starting models for 1a–c, 2a–c, 3a–b, 4a–b in their protonated form. The geometry of these latter was optimized with the semiempirical PM3 method, as implemented in the MOPAC 7.0 program (convergence criteria to 0.0005 kcal mol–1), using partial charges from electrostatic potential (ESP) calculations, fitted to a PM3 wave function. MOE-Dock [24] was used for docking purpose and the ligands were positioned in the binding pocket by using a flexible fit to the crystallographic HEPES molecule position, to minimize any bad steric interactions and to ensure starting conditions congruent with the pharmacophoric model of interaction. A Monte Carlo simulated annealing process was used for docking the substrate into the active site of the AChBP, search-

ing six spatial degrees of freedom. A random perturbation of the ligand was applied at each step and the interaction energy (IE) evaluated for every new state. The resulting structure is then accepted or rejected based on a probabilistic test. In the present case, a docking box of 50 × 50 × 60 points with a grid spacing of 0.375 Å was created, centered at the center of mass of the ligand and oriented with the long side along its main direction, allowing the ligand search in the entire active site of AChBP and in a significant part of the binding pocket environment. Each docking run begins with the compounds in a random conformation and for each ligand 35.000 iterations were used, while the number of cycles and runs was set to 30, producing a molecular database with 30 docked configurations for each calculation. After each simulation, the resulting database was analyzed and the five configurations with the lowest interaction energies (IEs) were chosen as the “best structures”. A further energy minimization was used to refine the orientation of the substrate in the binding site of AChBP. Using this protocol, both the ligands and the side chains of the binding site were relaxed, taking into account the aminoacids mobility within a 10.0 Å radius around the ligands. For each examined compound only the most stable conformation was chosen as representative of the ligand/AChBP complex with an energy difference higher than 10 kcal/mol between the most stable conformation and the next one. 3. Results and discussion The chemical structure and the nicotinic inhibitory activities (Ki) [22] of the 12 analyzed compounds (1a–c, 2a–c, 3a–c, 4a–c) are shown in Table 1. The crystal structure of AChBP is the established model for the extracellular domain of the pentameric LGICs, and

316

R. Artali et al. / Il Farmaco 60 (2005) 313–320

differences in the contacts with the centroids of the aromatic rings as reported in Table 2. A superposition of the examined compounds at the binding site is shown in Fig. 3. In particular, the docking site of the 3c molecule is shown in Fig. 4a while a detail of the environment of its charged nitrogen at the center of an “aromatic” cluster p is in Fig. 4b. The analyses of the aminoacidic environment around the ligands show some differences when their contacts in a region of 4 Å radius are considered, as summarized in Table 3. At the ligand binding site, only Trp A143 and Tyr A192 make contacts with all docked compounds, while the

was then used as the receptor model [11]. We performed 30 docking procedures for each ligand and the best configuration of each of the 12 ligand–receptor complexes was selected on energetic grounds. Their geometrical features were correlated to the different activities of the ligands with their morphology and the aminoacidic environment in the macromolecular cavity. The ligands lay in the zone defined by two subunits, with the charged nitrogen in contact with the Tyr and Trp residues of subunit A. The docked ligands form, mainly with Trp A143, a cation–p interaction [29] as expected for nicotinic agonists. This arrangement is common to all docked ligands with small

Table 2 Environment of the charged nitrogen for 1a–c, 2a–c, 3a–c, 4a–c: distances (Å) are calculated between charged nitrogen and the centroids of the aminoacids aromatic moieties. For comparison are reported the distances of HEPES, Carbamylcholine and Nicotine [16] SubunitA Tyr A89 Trp A143 Tyr A185 Tyr A192 SubunitB Trp B53 Tyr B164

Carbamyl choline 5.72 4.60 4.94 4.83

Nicotine

HEPES 1a

1b

1c

2a

2b

2c

3a

3b

3c

4a

4b

4c

5.76 4.18 5.55 4.92

4.10 5.17 4.32 6.71

4.37 4.30 5.88 5.32

5.08 5.38 4.16 5.24

5.20 4.70 5.43 4.67

4.79 5.55 4.11 5.02

4.98 4.54 4.96 5.65

5.69 5.31 4.39 5.12

4.81 5.40 4.18 5.20

5.55 5.70 4.04 4.77

5.47 4.96 4.79 4.98

4.56 5.18 4.30 5.72

5.48 4.92 4.63 5.66

5.40 5.29 4.37 5.02

5.37 7.77

6.23 9.17

4.75 6.66

6.37 9.07

5.37 7.25

6.19 8.62

5.83 7.64

5.02 7.47

5.18 7.11

5.39 7.47

5.66 7.34

5.45 7.64

5.20 7.23

4.71 6.91

5.41 7.36

Fig. 3. Superposition of the ligands inside the binding site (for sake of clarity only Leu112B and Tyr113B of AChBP are rendered in ball and stick).

R. Artali et al. / Il Farmaco 60 (2005) 313–320

317

Fig. 4. (a) Docking site of 3c: the protein is represented in solid ribbon, the binding site is contoured by the solid SAS (solvent accessible surface) and the ligand is in CPK. (b) Environment of 3c charged nitrogen.

missed ones of the other aromatic residues appear randomly related to the binding affinity and the docking energy. The unique contact common to the examined ligands and to HEPES, Carbamylcholine and Nicotine [16] (Table 3) is with Trp A143 confirming its important role. The charged nitrogen positions for 1a–c, 2a–c and 3a–c are in an interval of about 1.3 Å for the homopiperazine compounds, the dimethylated 4c is 2.2 Å apart, the monomethylated 4b 1.4 Å, and the non-methylated 4a 1.7 Å, with respect to the reference compound 3c. As reported in Table 3, only the ligands 2b, 4b, and 4c with the lowest activity are close to Leu B112, with a short distance between their chlorine atom and the carbonyl oxygen of the aminoacidic residue. This contact appears as a destabilizing factor for the binding and it can be considered important for activity discrimination. The respective position of the most active compound 3c and the less active compound 4b are compared in Fig. 5. The different position of the chlorine atom with respect to Leu B112 is clearly evidenced. This result could be confirmed by the distances between the carbonyl oxygen of Leu B112 and the closest atoms of Carbamylcholine and Nicotine [16], greater than 4 Å in both cases. A MOPAC/PM3 analysis of the examined compounds shows their dipolar moment in the direction of the chlorine atom, which, in 2b, 4b, and 4c, is oriented toward the Leu B112 carbonyl oxygen, causing an unfavorable ligand–

receptor dipole–dipole interaction. This feature, considering its steric and electrostatic components, destabilizes the complexes in our computational models and could justify their reduced biological activity. The role of the chlorine atom in nicotinic ligands has been largely debated. Following the observation that epibatidine has an affinity for nACh receptors enhanced over nicotine, a significant positive contribution of the chloro substituent to affinity has been suggested [30]. This seems to clash with the observation that deschloroepibatidine has a similar affinity as epibatidine [31]. However, the presence of the added chloro substituent in 6-chloronicotine doubles the affinity for the nACh receptors. In general, it has been observed that the introduction of a chlorine a to the pyridine nitrogen has variable effects in different class of compounds, diminishing the affinity in some cases [18] and enhancing in others [32]. In the examined compounds, the short distance of chlorine atom from Leu B112 appears a detrimental factor for affinity. In order to give quantitative weight to the previous observations, we verified the existence of any correlation between the nicotinic binding affinity of 1–4 compounds and the calculated docking energies or the geometrical features of the complexes. The binding models obtained with MOE-Dock [24] and the relative interaction energies of the ligands (Edock) have been correlated to the experimental values of Ki [23] (Fig. 6). The regression coefficient (r = 0.9123) supports the statistical significance of this model for the observed variations in binding energies.

Table 3 Contacts in a sphere of 4 Å radius around the ligands (the presence or absence of contact is represented with + or –). HEPES, Carbamylcholine and Nicotine [16] are reported for comparison

Tyr A89 Trp A143 Thr A144 Tyr A185 Tyr A192 Leu B112

Carbamyl choline – + – – – –

Nicotine

HEPES

1a

1b

1c

2a

2b

2c

3a

3b

3c

4a

4b

4c

– + – – – –

+ + – – – –

+ + + + + –

+ + + + + –

– + + + + –

+ + + + + –

– + + + + +

– + – + + –

+ + – + + –

+ + – + + –

+ + – + + –

– + + – + –

+ + – – + +

+ + – + + +

318

R. Artali et al. / Il Farmaco 60 (2005) 313–320

Fig. 5. Particular of the protein binding site represented as Connolly surface (CPK): the zone of the common cationic interaction is represented by a black enclosure, in light gray is the 3c chlorine and in dark gray 4b chlorine.

Fig. 6. Plot of the interaction energy (kcal/mol) vs Log(Ki × 109) for 1a–c, 2a–c, 3a–c, 4a–c complexes with AChBP.

Fig. 7. Plot of the distance (Å) Cl-OLeuB112 vs Log(Ki × 109) for 1a–c, 2a–c, 3a–c, 4a–c complexes.

4. Conclusions Considering the geometrical features of the complexes, a good correlation could be evidenced between Log Ki and the distance Cl-OLeu112B (Fig. 7). The parameters are more correlated (r = 0.9521) than the previous ones, and a link between the measured activity and the distance can be observed.

The docking results of 12 6-chloro-pyridazin-3-yl derivatives show that they bind to AChBP with orientation and position very close to that resulting from the crystallographic analysis in the AChBP complexes with HEPES, Nicotine and

R. Artali et al. / Il Farmaco 60 (2005) 313–320

Carbamylcholine [16]. The stability of the ligand–protein complexes is controlled by electrostatic and steric interactions, the main effect being the cation–p interaction of the ligand charged ammonium group with the aromatic cluster at the receptor binding site, underlining the important role of this interaction for the activity. The analysis of the electronic properties of the ligands at the binding site shows an unfavorable interaction of the chlorine atom at the sixth position of the pyridazine ring with the Leu B112 oxygen, that is modulated by the geometric characteristics of the ligands: their shape and bulk may produce steric strain pushing the chlorine more or less close to Leu B112, in particular in 2b, 4b, and 4c compounds, that are very close to this residue and present an increased binding energy. In our docking experiments, a good correlation of the binding affinity (Log Ki) with the calculated interaction energies (Edock) and with the chlorine-OLeu B112 distance was found, evidencing the significant role of chlorine in the interaction with the receptor. However, although the generalized fold and the characteristics of the nAChRs binding site are present in AChBP, any conclusion drawn from AChBP and from its homology models needs to be validated in the real pentameric ligand-gated receptor.

Acknowledgements Thanks are due to BRACCO Imaging for a grant to R.A.

References [1]

[2]

[3]

[4]

[5]

[6] [7]

[8] [9]

B.A. Cromer, C.J. Morton, M.W. Parker, Anxiety over GABAA receptor structure relieved by AchBP, Trends Biochem. Sci. 27 (2002) 280–287. A.O. Koren, A.G. Horti, A.G. Mukhin, D. Gündisch, A.S. Kimes, R.F. Dannals, E.D. London, 2-, 5-, and 6-Halo-3-(2(S)-azetidinylmethoxy)pyridines: synthesis, affinity for nicotinic acetylcholine receptors, and molecular modeling, J. Med. Chem. 41 (1998) 3690– 3698. M.A. Abreo, N.H. Lin, D.S. Garvey, D.E. Gunn, A.M. Hettinger, J.T. Wasicak, P.A. Pavlik, Y.C. Martin, D.L. Donnelly-Roberts, D.J. Anderson, J.P. Sullivan, M. Williams, S.P. Arneric, M.W. Holladay, 3-Pyridyl ethers with subnanomolar affinity for central neuronal nicotinic acetylcholine receptors, J. Med. Chem. 39 (1996) 817–825. P.A. Newhouse, A. Potter, E.D. Levin, Nicotinic system involvement in Alzheimer’s and Parkinson’s diseases. Implications for therapeutics, Drug Aging 11 (1997) 206–228. M.W. Holladay, M.J. Dart, J.K. Lynch, Neuronal nicotinic acetylcholine receptors as targets for drug discovery, J. Med. Chem. 40 (1997) 4169–4194. D. Paterson, A. Nordberg, Neuronal nicotinic receptors in the human brain, Prog. Neurobiol. 61 (2000) 75–111. H.R. Aria, Localization of agonist and competitive antagonist binding sites on nicotinic acetylcholine receptors, Neurochem. Int. 36 (2000) 595–645. D.A. Dougherty, Cation–p interactions in chemistry and biology. A new view of benzene, Phe, Tyr, and Trp, Science 271 (1996) 163–168. A. Miyazawa, Y. Fujiyoshi, M. Stowell, N. Unwin, Nicotinic acetylcholine receptor at 4.6 Å resolution: transverse tunnels in the channel wall, J. Mol. Biol. 288 (1999) 765–786.

319

[10] A.B. Smit, N.I. Syed, D. Schaap, J. van Minnen, J.A. Klumperman, A glia-derived acethylcholine-binding protein that modulates synaptic transmission, Nature 411 (2001) 261–268. [11] K. Brejc, W.J. Van Dijk, R.V. Klaassen, M. Schuurmans, J. Van der Oost, A.B. Smit, T.K. Sixma, Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors, Nature 411 (2001) 269–276. [12] N. Le Novere, T. Grutter, J.P. Changeux, Models of the extracellular domain of the nicotinic receptors and of agonist- and Ca2+-binding sites, Proc. Natl. Acad. Sci. USA 99 (2002) 3210–3215. [13] B.E. Molles, I. Tsigelny, P.D. Nguyen, S.X. Gao, S.M. Sine, P. Taylor, Residues in the epsilon subunit of the nicotinic acetylcholine receptor interact to confer selectivity of waglerin-1 for the alpha-epsilon subunit interface site, Biochemistry 41 (2002) 7895–7906. [14] M. Schapira, R. Abagyan, M. Totrov, Structural model of nicotinic acetylcholine receptor isotypes bound to acetylcholine and nicotine, BMC Struct. Biol. 2 (2002) 1. [15] S.M. Sine, H.L. Wang, N.D. Bren, Lysine scanning mutagenesis delineates structural model of the nicotinic receptor ligand binding domain, J. Biol. Chem. 277 (2002) 29210–29223. [16] P.H.N. Celie, S.E. Van Rossum-Fikkert, W.J. Van Dijk, K. Brejc, A.B. Smit, T.K. Sixma, Nicotine and carbamylcholine binding to nicotinic acetylcholine receptors as studied in AChBP crystal structure, Neuron 41 (2004) 907–914. [17] A. Karlin, Emerging structure of the nicotinic acetylcholine receptors, Nat. Rev. Neurosci. 3 (2002) 102–114. [18] R.A. Glennon, M. Dukat, Central nicotinic receptor ligands and pharmacophores, Pharm. Helv. Acta 74 (2000) 103–114. [19] D.A. Dougherty, D.A. Stauffer, Acetylcholine binding by a synthetic receptor: implications for biological recognition, Science 250 (1990) 1558–6015. [20] W. Zhong, J.P. Gallivan,Y. Zhang, L. Li, H.A. Lester, D.A. Dougherty, From ab initio quantum mechanics to molecular neurobiology: a cation–p binding site in the nicotinic receptor, Proc. Natl. Acad. Sci. USA 95 (1998) 12088–12093. [21] S. Mecozzi, A.P. West Jr., D.A. Dougherty, Cation–p interactions in aromatics of biological and medicinal interest: electrostatic potential surfaces as a useful qualitative guide, Proc. Natl. Acad. Sci. USA 93 (1996) 10566–10571. [22] D. Barlocco, G. Cignarella, D. Tondi, P. Vianello, S. Villa, A. Bartolini, C. Ghelardini, N. Galeotti, D.J. Anderson, T.A. Kuntzweiler, D. Colombo, L. Toma, Mono- and disubstituted-3,8-diazabicyclo[3.2.1]octane derivatives as analgesics structurally related to epibatidine: synthesis, activity and modeling, J. Med. Chem. 41 (1998) 674–681. [23] L. Toma, P. Quadrelli, W.H. Bunnelle, D.J. Anderson, M.D. Meyer, G. Cignarella, et al., 6-Chloropyridazin-3-yl derivatives active as nicotinic agents: synthesis, binding, and modeling studies, J. Med. Chem. 45 (2002) 4011–4017. [24] MOE (The Molecular Operating Environment) Version 2002.03, software available from Chemical Computing Group Inc., 1010 Sherbrooke Street West, Suite 910, Montreal, Canada H3A 2R7. http: //www.chemcomp.com. [25] T.A. Halgren, Merck Molecular Force Field, I. Basis, form, scope, parameterization and performance of MMFF94, J. Comp. Chem. 17 (1996) 490–519. [26] D. Van der Spoel, R. Van Drunen, H.J.C. Berendsen, GRoningen MAchine for Chemical Simulations; Department of Biophysical Chemistry, BIOSON Research Institute: Nijenborgh 4-9717 AG, Groningen, The Netherlands, 1994. E-mail: [email protected]. (b) D. van der Spoel, A. R. van Buuren, E. Apol, P. J. Meulenhoff, D. P. Tieleman, A. L. T. M. Sijbers, R. van Drunen, H. J. C. Berendsen, Gromacs User Manual Version 1.3; Department of Biophysical Chemistry, BIOSON Research Institute: Nijenborgh 4 NL-9717 AG, Groningen, The Netherlands, 1996. Internet: http: //rugmd0.chem.rug.nl/gm × 1996. (c) W. F van Gunsteren, S. R.

320

R. Artali et al. / Il Farmaco 60 (2005) 313–320

Billeter, A. A. Eising, P. H. Hunenberger, P. Kruger, A. E. Mark, W. R. P. Scott, I. G. Tironi, Biomolecular Simulation: The GROMOS96 Manual and User Guide; Hochschulverlag AG an der ETH Zurich: Zurich, 1996. [27] J.J.P. Stewart, Optimization of parameters for semi-empirical methods i-method, J. Comp. Chem. 10 (1989) 209–220. [28] R. Artali, G. Bombieri, F. Meneghetti, Structural and conformational studies of 3-(6-chloropyridazin-3-yl)-N,N-dimethyl-homopiperazinium iodide with central nicotinic action, Heterocycles 60 (2003) 2085– 2093.

[29] N. Zacharias, D.A. Dougherty, Cation–p interactions in ligand recognition and catalysis trends, Pharmacol. Sci. 23 (2002) 281–287. [30] M. Dukat, M.I. Damaj, W. Glassco, D. Dumas, E.L. May, B.R. Martin, R.A. Glennon, Epibatidine: a very high affinity nicotinic receptor ligand, Med. Chem. Res. 4 (1994) 131–139. [31] F.I. Carrol, F. Liang, H.A. Navarro, L.E. Brieaddy, M.I. Damaj, B.R. Martin, J. Med. Chem. 44 (2001) 2229–2237. [32] G. Balboni, M. Marastoni, S. Meriggi, P.A. Borea, R. Tomatis, Synthesis and activity of 3-pyridylamine ligands at central nicotinic receptors, J. Med. Chem. 35 (2000) 979–988.