Receptor with Arylpiperazine Ligands. Development of a New 5-HT3

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Further Studies on the Interaction of the 5-Hydroxytryptamine3 (5-HT3) Receptor with Arylpiperazine Ligands. Development of a New 5-HT3 Receptor Ligand Showing Potent Acetylcholinesterase Inhibitory Properties Andrea Cappelli,*,‡ Andrea Gallelli,‡,# Monica Manini,‡ Maurizio Anzini,‡ Laura Mennuni,§ Francesco Makovec,§ M. Cristina Menziani,| Stefano Alcaro,† Francesco Ortuso,† and Salvatore Vomero‡ Dipartimento Farmaco Chimico Tecnologico and European Research Centre for Drug Discovery and Development, Universita` di Siena, Via A. Moro, 53100 Siena, Italy, Rotta Research Laboratorium S.p.A., Via Valosa di Sopra 7, 20052 Monza, Italy, Dipartimento di Chimica, Universita` degli Studi di Modena e Reggio Emilia, Via Campi 183, 41100 Modena, Italy, and Dipartimento di Scienze Farmacobiologiche, Universita` degli Studi Magna Græcia di Catanzaro, Complesso Ninı` Barbieri, 88021, Roccelletta di Borgia (CZ), Italy Received August 9, 2004

Novel arylpiperazine derivatives bearing lipophilic probes were designed, synthesized, and evaluated for their potential ability to interact with the 5-hydroxytryptamine3 (5-HT3) receptor. Most of the new compounds show subnanomolar 5-HT3 receptor affinity. Ester 6bc showing a picomolar Ki value is one of the most potent 5-HT3 receptor ligands so far synthesized. The structure-affinity relationship study suggests the existence of a certain degree of conformational freedom of the amino acid residues interacting with the substituents in positions 3 and 4 of the quipazine quinoline nucleus. Thus, the tacrine-related heterobivalent ligand 6o was designed in an attempt to capitalize on the evidence of such a steric tolerance. Compound 6o shows a nanomolar potency for both the 5-HT3 receptor and the human AChE and represents the first example of a rationally designed high-affinity 5-HT3 receptor ligand showing nanomolar AChE inhibitory activity. Finally, the computational analysis performed on compound 6o allowed the rationalization of the structure-energy determinants for AChE versus BuChE selectivity and revealed the existence of a subsite at the boundary of the 5-HT3 receptor extracellular domain, which could represent a “peripheral” site similar to that evidenced in the AChE gorge. Introduction The serotonin (5-hydroxytryptamine, 5-HT) 5-HT3 receptor is a ligand-gated ion channel that mediates fast depolarizing responses and is apparently selective for the monovalent cations Na+ and K+ and for the divalent ones Ca2+ and Mg2+.1 The receptor shows the pentameric structure characteristic of the ligand-gated ion channels (LGICs), such as nicotinic acetylcholine (nACh), glycine, and type A γ-aminobutyric acid (GABAA) receptors.2 A single 5-HT3 receptor subunit (5-HT3A) was initially cloned,3 and it was found that its cDNA was found to lead to the expression of functional receptors showing a pharmacological profile similar to that of the native 5-HT3 receptors. Subsequently, a new class of human 5-HT3 receptor subunits (5-HT3B) has been cloned and reported to form a functional 5-HT3 receptor when expressed together with 5-HT3A but not when expressed alone. The hetero-oligomer exhibited distinctive pharmacological and functional characteristics compared with those found in the 5-HT3A homo-oligomer.4 Moreover, evidence has been found for expression of heteromeric 5-HT3 receptors in rodent neurons.5 * To whom correspondence should be addressed. Phone: +39 0577 234320. Fax: +39 0577 234333. E-mail: [email protected]. ‡ Universita ` di Siena. # Present address: Dipartimento di Scienze Farmacobiologiche, Universita` degli Studi Magna Græcia di Catanzaro, Complesso Ninı` Barbieri, 88021, Roccelletta di Borgia (CZ), Italy. § Rotta Research Laboratorium S.p.A. | Universita ` degli Studi di Modena e Reggio Emilia. † Universita ` degli Studi Magna Græcia di Catanzaro.

Electron microscopy images of the purified 5-HT3 receptor are available in the literature,2 but its threedimensional (3D) structure has not yet been resolved at the atomic level. However, the great amount of information about the members of this receptor family obtained from photoaffinity labeling, site-directed mutagenesis studies,6 and electron microscopy data7 was used in a pioneering work to build a 3D receptor model.8 More recently, the crystal structure of a homologous snail acetylcholine binding protein (AChBP)9 has been exploited as a structural template for updated models of the ligand-binding domain of the 5-HT3 receptor.10,11 The medicinal chemistry research on the 5-HT3 receptor has produced a large number of potent and selective antagonists (e.g., ondansetron, 1; granisetron, BRL 43694, 2; and tropisetron, 3; for a comprehensive review, see ref 12), which have shown an excellent efficiency in the control of the emesis induced by anticancer chemotherapy and few adverse side effects, to the point of revolutionizing the treatment of nausea in cancer patients.13 Moreover, preclinical investigations have suggested the potential usefulness of these compounds in a number of situations such as cognitive and psychotic disorders, drug and alcohol addiction, treatment of pain, and irritable bowel syndrome.13 It has been suggested that the stimulation of the 5-HT3 receptor in the central nervous system (CNS) enhances the release of dopamine from rat striatal slices14 and that of cholecystokinin from

10.1021/jm0493461 CCC: $30.25 © 2005 American Chemical Society Published on Web 04/16/2005

Development of 5-HT3 Receptor Ligand

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10

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Chart 1

Chart 2

the cerebral cortex and nucleus accumbens15 and inhibits the release of acetylcholine from the entorhinal cortex.16 Our work, which was for more than a decade concerned with the development of 5-HT3 receptor ligands belonging to the classes of arylpiperazines, tropane, and quinuclidine derivatives, started from the study of arylpiperazine derivatives related to quipazine (4, Chart 1), and a huge amount of structure-affinity relationship (SAFIR) and structure-activity relationship (SAR) data was obtained for the interaction of this class of ligands with their receptor.17 Particularly interesting information was obtained on the pivotal role played by the substituents in positions 3 and 4 on both the affinity and the intrinsic efficacy. In this paper we describe the results of the exploration of the receptor pocket interacting with the c edge (positions 3 and 4) of the quipazine quinoline nucleus by means of a systematic approach based on lipophilic probes (primarily alkyl chains of different length in compounds 5 and 6) and an attempt at capitalization of the information obtained. First, the structure of the cyclohexene-condensed reference compound 7a (Chart 2) was manipulated into the one of pyrrolidone-condensed compounds 5a-e to allow the easy introduction of substituents showing different dimensions. The pyrrolidone moiety was then broken to allow a larger number of substituents to be introduced in positions 3 and 4 of the quinoline nucleus (compounds 6c-l). The approach furnished further information on the dimension of the receptor pocket, and the capitalization of this information was attempted by means of the design of bivalent ligands. In this design, an optimized 5-HT3 receptor ligand was used as an anchor and conjugated (by means of a spacer) to tacrine [9-amino-1,2,3,4-tetrahydroacridine, an acetylcholinesterase (AChE) inhibitor]18 in order to obtain a 5-HT3 receptor ligand (6o) endowed with AChE inhibitory properties. The design of compounds possessing this dual activity stemmed from the observation that 5-HT3

receptors mediate the tonic inhibitory control on ACh release in the cortical tissue and do not seem to be significantly impaired in Alzheimer’s disease (AD).19 The molecules showing the synergistic action at 5-HT3 receptors and AChE potentially possess a pharmacological profile suitable for restoring the normal cholinergic tone in AD patients.20 Chemistry Most of the designed quinoline derivatives were synthesized by means of a multistep procedure beginning with a variant of the Pfitzinger quinoline synthesis (Scheme 1). Isatin 8 was acylated with the suitable anhydride (acetic, propionic, butyric, valeric) and subsequently converted into 4-quinolinecarboxylic acids 9-12. The acids bearing alkyl substituents in position 3 of the quinoline nucleus (10-12) were reacted with phosphorus oxychloride and then with ethanol to give esters 1416 or with the appropriate amines to give amides 17, 19-22, 24-29, 31-35. The preparation of quinoline derivatives bearing a hydrogen substituent in position 3 of the quinoline nucleus slightly differs from that described above because 2-chloro-4-quinolinecarboxylic acid chloride was unstable to the aqueous workup. Thus, acid 9 was reacted with phosphorus oxychloride. After workup it was reactivated with thionyl chloride and then reacted with ethanol to give ester 13 or with the appropriate amines to give amides 18, 23, 30. Iminochlorides 13-35 were promptly converted into the final piperazinylquinoline derivatives 6b,ba-bd,c,d,dadc,e,f,fa-fc,g-j,ja-jc,k,l by reaction with either Nmethylpiperazine or piperazine in ethylene glycol. Ester 1417f was the useful intermediate for the synthesis of pyrroloquinolines 5a-e, lactone derivative 5f, and diol 6m (Scheme 2). Ester 14 was brominated with NBS in the presence of dibenzoyl peroxide and cyclized with the suitable amine to obtain chloropyrroloquinoline derivatives 36-40, which were reacted

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Scheme 1a

Cappelli et al.

Scheme 3a

a

Reagents: (i) C6H6, AlCl3; (ii) N-methylpiperazine.

Scheme 4a

a Reagents: (i) (R CH CO) O; (ii) NaOH, H O; (iii) POCl ; (iv) 3 2 2 2 3 SOCl2 (only for compounds in which R3 ) H); (v) EtOH, TEA; (vi) R1N(R2)H, CH2Cl2, TEA; (vii) N-methylpiperazine (or anhydrous piperazine, ethylene glycol).

Scheme 2a

a

a Reagents: (i) NBS, dibenzoyl peroxide, CCl ; (ii) RNH , EtOH; 4 2 (iii) N-methylpiperazine; (iv) HCl, MeOCH2CH2OH; (v) POCl3; (vi) LAH, THF.

with N-methylpiperazine to obtain the final compounds 5a-e. On the other hand, after bromination, ester 14 was heated at reflux with hydrochloric acid in methoxyethanol and then in phosphorus oxychloride to obtain lactone derivative 41, which was reacted with Nmethylpiperazine. Lithium aluminum hydride reduction of lactone 5f gave diol 6m. Ketone 6a was prepared following the procedure described in Scheme 3 starting from acid chloride 42,

Reagents: (i) TEA, CH2Cl2; (ii) N-methylpiperazine.

which was used in a Friedel-Crafts acylation of benzene in the presence of aluminum trichloride to give ketone 43, which was in turn transformed into the final piperazinyl derivative 6a. Compound 6n was synthesized from ethyl 2-chloro-4-methyl-3-quinolinecarboxylate17f and N-methylpiperazine. The conjugate product between our 5-HT3 ligands and tacrine was prepared as described in Scheme 4. Acid chloride 42 was reacted with amine 44 (see ref 18d) to obtain the chloroderivative 45, which was functionalized with N-methylpiperazine to afford the tacrine-related heterobivalent ligand 6o. Structure-Affinity Relationship Studies The newly synthesized heteroarylpiperazine derivatives 5 and 6 were assayed for their potential ability to displace [3H]granisetron specifically bound to the 5-HT3 receptor in rat cortical membrane, in comparison with reference compounds 7 and granisetron (2), following well-established protocols.21 The results of the binding studies are summarized in Tables 1-3.



Table 1. 5-HT3 Receptor Binding Affinities of Compounds 5a-f and 7a,b: Effects of the Variation of the Ring at the c Edge of the Quinoline Nucleus

compd

X

R

Ki ( SEMa (nM)

5a 5b 5c 5d 5e 5f 7ab 7bc 2

N N N N N O CH2CH2 CH2

CH3 CH2CH3 CH2CH2CH3 CH(CH3)CH2CH3 CH2C6H5

2.1 ( 0.47 3.0 ( 0.06 3.6 ( 0.40 4.2 ( 0.10 6.2 ( 1.5 2.4 ( 0.07 0.23 ( 0.03 0.24 ( 0.03 0.35 ( 0.06

a Each value is the mean ( SEM of three determinations and represents the concentration giving half the maximum inhibition of [3H]2 (final concentration 1 nM) specific binding to rat cortical membranes. b See ref 17d. c See ref 22.

The most striking result obtained consists of the fact that most of the new heteroarylpiperazine derivatives show subnanomolar 5-HT3 receptor affinity, and in particular, ester 6bc showing a picomolar Ki value is one of the most potent 5-HT3 receptor ligands so far synthesized. The analysis of the SAFIRs in this class of arylpiperazine ligands showed interesting features. (a) The replacement of a cyclohexene ring fused to the c edge of the quinoline nucleus of compound 7a (or the cyclopentene one of 7b) with the pyrrolidone ring of compounds 5a-e (or the lactone one in the case of 5f) produces a significant decrease in the receptor affinity (Table 1). In the short series of these tricyclic derivatives 5a-f the variation of the volume of the substituent on the pyrrolidone nucleus appears to have negligible effects on the 5-HT3 receptor affinity. These results suggest that the introduction of a carbonyl dipole in the plane of the tricyclic heteroaromatic system of 7a,b is not favorable for the interaction with 5-HT3 receptors, while bulky substituents are easily accommodated in the receptor pocket interacting with the c edge of the quipazine quinoline nucleus. (b) The opening of the pyrrolidone ring of compound 5b or of the lactonic one of 5f to give amide 6d or ester 6b, respectively, produces an affinity enhancement of about 1 order of magnitude (compare also 5c with 6f and 5e with 6j,ja). This result can be explained in light of both the different orientations of the carbonyl dipole and the higher lipophilicity of the seco analogues 6 with respect to the corresponding tricyclic derivatives 5. (c) The results summarized in Table 2 clearly show that the N,N-dibutylaminocarbonyl substituent is easily accommodated in the receptor pocket, but the progressive bulk increase (from the N,N-dipropylaminocarbonyl to N,N-dihexylaminocarbonyl substituents) decreases the affinity by about 1 order of magnitude per step (compare 6f with 6g, 6h, and 6i). On the other hand, nonsymmetric amides 6j and 6k show very similar subnanomolar affinity despite the difference in the substituent volume, while p-chloroanilide 6l was less potent.

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(d) The interchange of the substituent position in compound 6b leading to 6n or the transformation of the same ester 6b into the ketone 6a has little effect on the receptor affinity. (e) Some of the most interesting compounds included in Table 2 were selected to be submitted to the systematic variation of the volume of the substituent in position 3 of the quinoline nucleus (Table 3). In the ethyl ester 6b subseries, the 5-HT3 receptor affinity increases with increasing length of the alkyl side chain, while a biphasic behavior was observed in the case of the diethylamide 6d series and dipropylamide 6f series. Finally, in the N-methylbenzylamide 6j series, the increase of the side chain length decreases the 5-HT3 receptor affinity. On the whole, these results appear to suggest that the lipophilic pocket can be saturated and that the optimization of the dispersion-repulsion forces of the substituents leads to a maximization of the interaction energy, leading to a Ki value of 80 pM (compound 6bc). (f) The replacement of the methyl substituent on the terminal piperazine nitrogen with a hydrogen atom has little effect on the 5-HT3 receptor affinity (compare 6bd with 6b). On the whole, the most interesting information from the SAFIR analysis is the presence of a very large (although apparently saturable) lipophilic pocket. From another perspective, this result can be interpreted as the evidence of a certain degree of conformational freedom of the amino acid residues interacting with the substituents in positions 3 and 4 of the quipazine quinoline nucleus. The design of the tacrine-related heterobivalent ligand 6o represents a way of capitalizing on (and evaluating further) the evidence of such a steric tolerance. Very interestingly, compound 6o shows a nanomolar affinity (Ki ) 5.6 ( 0.02 nM) for the 5-HT3 receptor, a nanomolar potency in inhibiting the human AChE (IC50 ) 4.1 ( 0.6 nM), and a lower potency in inhibiting butyrylcholinesterase (BuChE, IC50 ) 40 ( 5.0 nM). To our knowledge, this compound represents the first example of a high-affinity 5-HT3 receptor ligand showing nanomolar AChE inhibitory activity and rationally designed to obtain this result. The structure-activity relationship analysis performed on the short series of compounds shown in Chart 3 suggests the following considerations. (a) The 5-HT3 receptor appears to be capable of accommodating bivalent ligands by the formation of an additional binding pocket in the proximity of the main binding domain. This appears to confirm the assumed conformational freedom of the amino acid residues interacting with positions 3 and 4 of the quinoline nucleus of quipazine. (b) The peripheral site of AChE appears to be very tolerant in accepting moieties showing different stereoelectronic characteristics (compare 4618 with 45 and 6o). For example, it is capable of accommodating the heteroarylpiperazine moiety of compound 6o, which shows significant structural differences with respect to tacrine. (c) On the other hand, the heteroarylpiperazine moiety alone does not seem to be able to interact effectively with AChE because 5-HT3 receptor ligands 6b,j were devoid of any AChE inhibitory activity. Thus, the tacrine moiety appears to play a key role in the

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Table 2. 5-HT3 Receptor Binding Affinities of Compounds 6a-n: Effects of the Variation of the Substituents in Position 4 of the Quinoline Nucleus

compd

R

R3

R4

Ki ( SEMa (nM)

6a 6b 6c 6d 6e 6f 6g 6h 6i 6j 6k 6l 6m 6n 2

CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3

CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH2OH COOC2H5

COC6H5 COOCH2CH3 CON(CH2)4 CON(CH2CH3)2 CON(CH(CH3)CH3)2 CON(CH2CH2CH3)2 CON(CH2CH2CH2CH3)2 CON(CH2CH2CH2CH2CH3)2 CON(CH2CH2CH2CH2CH2CH3)2 CON(CH3)CH2C6H5 CON(CH3)CH2CtCH CON(CH3)p-Cl-C6H4 CH2OH CH3

0.84 ( 0.17 0.43 ( 0.02 1.6 ( 0.70 0.37 ( 0.09 2.2 ( 0.55 0.11 ( 0.004 0.78 ( 0.12 19 ( 2.4 188 ( 50 0.55 ( 0.15 0.45 ( 0.12 13 ( 4.1 2.7 ( 0.52 0.17 ( 0.02 0.35 ( 0.06

a Each value is the mean ( SEM of three determinations and represents the concentration giving half the maximum inhibition of [3H]2 (final concentration 1 nM) specific binding to rat cortical membranes.

Table 3. 5-HT3 Receptor Binding Affinities of Compounds 6: Effects of the Variation of the Substituents in Position 3 of the Quinoline Nucleus and on the Piperazine Terminal Nitrogen Atom

compd

R

R3

R4

Ki ( SEMa (nM)

6ba 6b 6bb 6bc 6bd 6da 6d 6db 6dc 6fa 6f 6fb 6fc 6ja 6j 6jb 6jc 2

CH3 CH3 CH3 CH3 H CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3

H CH3 CH2CH3 CH2CH2CH3 CH3 H CH3 CH2CH3 CH2CH2CH3 H CH3 CH2CH3 CH2CH2CH3 H CH3 CH2CH3 CH2CH2CH3

COOCH2CH3 COOCH2CH3 COOCH2CH3 COOCH2CH3 COOCH2CH3 CON(CH2CH3)2 CON(CH2CH3)2 CON(CH2CH3)2 CON(CH2CH3)2 CON(CH2CH2CH3)2 CON(CH2CH2CH3)2 CON(CH2CH2CH3)2 CON(CH2CH2CH3)2 CON(CH3)CH2C6H5 CON(CH3)CH2C6H5 CON(CH3)CH2C6H5 CON(CH3)CH2C6H5

0.85 ( 0.40 0.43 ( 0.02 0.21 ( 0.006 0.080 ( 0.02 0.56 ( 0.24 1.6 ( 0.61 0.37 ( 0.09 0.51 ( 0.05 0.92 ( 0.22 0.68 ( 0.06 0.11 ( 0.004 1.1 ( 0.09 1.2 ( 0.11 0.41 ( 0.10 0.55 ( 0.15 10 ( 1.8 14 ( 0.62 0.35 ( 0.06

a Each value is the mean ( SEM of three determinations and represents the concentration giving half the maximum inhibition of [3H]2 (final concentration 1 nM) specific binding to rat cortical membranes.

interaction of 6o with AChE by acting as an anchor in its usual binding site. Molecular Modeling Studies Interaction of Compound 6o with 5-HT3 Receptor. The binding modalities of several antagonist ligands have been discussed recently by Cappelli et al.17h and Maksay et al.10 on the basis of three-dimensional models of the ligand-receptor complexes. The overall shape of the 5-HT3 receptor models used in these papers differs significantly since the first model was developed before

Chart 3

the publication of the AChBP crystal structure. However, in both cases the proposed binding cleft for antagonist ligands is similar, being composed of a vestibule where residue Glu236(171) (the first number corresponds to the whole sequence from rat 5HT3R; the number in parentheses corresponds to the portion of the sequence modeled in ref 8) lies, whereas the back of the cavity is composed of hydrophobic residues such as Trp90(25) and Tyr234(169). Recent results from sitedirected mutagenesis studies confirmed the outstanding importance of these residues23-26 in the ligand binding interaction, thus supporting the predictive value of the modeling procedures. To verify the receptor capability of accommodating bivalent ligands and possibly to identify the putative additional binding pocket, the 6o ligand was docked into an updated three-dimensional



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Figure 1. Energy-minimized structure of the 6o-5HT3 receptor model. (Bottom-left) Overview of the ligand-receptor complex. Ligand 6o (atom types color) is represented as sticks, and the 5HT3 subunits (pink and light-blue) are represented according to their secondary structure. The two insets provide a detailed view of the principal binding pocket for the 5HT3 receptor pharmacophore (top-right) and the tetrahydroacridine moiety (bottom-right).

model obtained by means of the alignment recently reported by Maksay et al.10 Figure 1 shows the best result (i.e., the lowest interaction energy achieved between the ligand and the receptor) obtained by molecular mechanics minimization of the 6o-5HT3 receptor complexes (see the Computational Methods). The gorge between the two subunits is interrupted by the protrusion of loops B and C from one subunit to the other, reducing the binding cavity to a total length of about 25 Å. Therefore, with the charged head of the piperazine moiety establishing an ionic interaction with Glu236 and a cation-π interaction with Trp183, the heptylene linker perfectly fits a gorge between the two receptor subunits and seems to have an optimal length to allow the tetrahydroacridine moiety to accommodate into a cleft located at the receptor boundary. In fact, the π-charge interaction shown in Figure 1 between the ligand and R246 competes with R246solvent interaction, since this residue is solvent-accessible. Other important interactions are obtained with Phe208 and Trp214. It is worth remarking that the binding pockets identified for the tetrahydroacridine moiety in the 5-HT3 receptor and in the AChE and BuChE are composed of very similar residues (see below). Interaction of Compound 6o with AChE and BuChE. The 6o-cholinesterase binding modes were investigated by means of a docking study carried out using the human crystallographic models 1B41 and 1P01 stored in the Protein Data Bank (PDB).27 In the AChE gorge, the presence of the two “anionic” sites, known as the “internal” (Trp 86) and the “peripheral” (Trp 286) ones, respectively, led to a consideration of an extended conformation of 6o for a concurrent recognition with the distal aromatic moieties. High-

affinity AChE ligands, such as donepezil and others,18f,28 showed this kind of interaction. Starting with a manually docked extended conformation of 6o, the binding modes were explored by a flexible Monte Carlo (MC) docking of this ligand into the enzyme clefts, taking into account the induced reciprocal fit effects by consecutive constrained and full energy minimizations of each generated complex configuration. This computational protocol allowed the ligand to invert the reciprocal positions of the aromatic distal moieties with respect to the starting structures. Actually, some high-energy configurations of the 6o neutral formBuChE complex showed this binding fashion, but after full energy minimization in implicit water environment, these were discarded because they proved to be energetically higher than 50 kJ/mol with respect to the global minimum. In all cases only one configuration was found at 300 K with a consistent Boltzmann population: 100% and 99.9% for the protonated and neutral 6o-AChE complexes, respectively, and 59.13% and 77.9% for the charged and neutral 6o-BuChE ones, respectively (Figure 1). A similar binding mode has been observed for both minimum energy configurations. In particular, the bioactive structures in AChE and BuChE clefts have been compared by computing the rms deviation after superimposition with two substructures was taken into account: the former made by the aminoacridine moiety and heptylene linker, the latter formed by this spacer and the arylpiperazine group. The ligand in charged form showed higher rms values, respectively 2.2 and 2.7 Å, while the neutral conformations proved to be quite similar, 0.60 and 1.38 Å, respectively. The orientation of the ligand bioactive conformation was in both cases characterized by the aminoacridine moiety located in the internal anionic site and the quinoline group in the

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Figure 2. Global minimum energy binding modes of the 6o ionized form in complex with AChE (a) and BuChE (b) and of the 6o neutral form in complex with AChE (c) and BuChE (d). For the sake of clarity, only the ligand (green sticks) and the interacting enzyme residues are displayed. The amino acids establishing hydrogen bonds (dashed lines) are represented as sticks, and those with stacking contacts are in CPK.

6o ligand form

∆Gc b

∆Ei c

∆Gc b

∆Ei c

∆∆Gc d

compound, which appeared to be more selective for the AChE enzyme (Chart 3). A high entropic cost is necessary for the bioactive ligand conformation in all cases, especially for the protonated form.

protonated neutral

-298.4 -327.5

82.1 28.9

-251.3 -316.6

78.0 33.5

-47.1 -10.9

Conclusions

Table 4. Thermodynamic Evaluation of 6o-AChE and 6o-BuChE Complexesa AChE

BuChE

a The energies are expressed in kJ/mol. b Free energy of complexation. c Relative ligand internal energy of the bioactive conformation. d Difference of free energies of complexation.

peripheral one. In particular, in the BuChE, the Trp286 of the AChE site was replaced by the Ala277, which could explain the major conformational differences in the arylpiperazine substructures of the bioactive ligand conformations interacting with AChE and BuChE clefts (Figure 1). A detailed analysis of 6o binding modes was carried out by the measurement module of the graphical user interface of the MacroModel package (Maestro GUI) for charged and neutral forms where the ligand was conveniently divided into eight substructures interacting in a different fashion within the two enzyme gorges (see Supporting Information). The comparison of the energy differences reported in Table 4 revealed a consistent higher stabilization of 6o both as neutral and as charged ligands in the AChE cleft with respect to BuChE one. This observation was in agreement with the inhibition activities (IC50) of this

This paper describes the results of a study focused on the exploration of the 5-HT3 receptor pocket interacting with the c edge (positions 3 and 4) of the quipazine quinoline nucleus. In the first step of the investigation, the structure of the cyclohexene-condensed reference compound 7a was transformed into the pyrrolidone- or furanone-condensed structures of compounds 5. Then these heterocyclic moieties were broken to allow a larger number of substituents (lipophilic probes) to be introduced into positions 3 and 4 of compounds 6. Most of the newly synthesized compounds show subnanomolar 5-HT3 receptor affinity, and in particular, ester 6bc showing a picomolar Ki value is one of the most potent 5-HT3 receptor ligands so far synthesized. The SAFIR analysis suggests the presence of a very large (even if apparently saturable) lipophilic pocket or, from another point of view, the existence of a certain degree of conformational freedom of the amino acid residues interacting with the substituents in positions 3 and 4 of the quipazine quinoline nucleus. In the final step of the work, the tacrine-related heterobivalent ligand 6o



was designed in an attempt to capitalize on (and evaluating further) the evidence of such a steric tolerance. Compound 6o shows a nanomolar potency for both 5-HT3 receptor and human AChE and represents (to our knowledge) the first example of a rationally designed high-affinity 5-HT3 receptor ligand showing nanomolar AChE inhibitory activity. Compounds possessing this dual activity potentially possess a pharmacological profile suitable for restoring the normal cholinergic tone in AD patients. The computational analysis performed on compound 6o appeared capable of rationalizing the structureenergy determinants for AChE versus BuChE selectivity and revealed the existence of similarities between the interaction of this bivalent ligand with 5-HT3, AChE, and BuChE. In fact, the two heteroaromatic moieties of 6o appeared to be accommodated by the receptor or enzyme proteins into suitable pockets located approximately at the same distance (14-17 Å) in the three different proteins. In particular, the tetrahydroacridine moiety of 6o appears to find a subsite located at the boundary of the 5-HT3 receptor and composed of an arginine (R246) and two aromatic residues (Phe208 and Trp214) so that this subsite could represent a “peripheral” site similar to the one highlighted in the AChE gorge.

used in the next step. Among them, 2-chloro-3-methyl-4quinolinecarboxylic acid chloride was characterized.17f A mixture of the suitable acid chlorides (1.0 g) in 25 mL of absolute ethanol and 1.2 mL (8.6 mmol) of triethylamine was heated at reflux for 2 h, concentrated under reduced pressure, and partitioned between CH2Cl2 and water. The organic layer was dried over sodium sulfate and concentrated under reduced pressure. Purification of the residue by flash chromatography with n-hexane-ethyl acetate (8:2) as the eluent gave esters 14-16. Ethyl 2-Chloro-3-propyl-4-quinolinecarboxylate (16). This compound was obtained from acid 12 as a colorless oil (yield 66%). 1H NMR (CDCl3): δ 0.99 (t, J ) 7.3, 3H), 1.42 (t, J ) 7.2, 3H), 1.71 (m, 2H), 2.78 (m, 2H), 4.52 (q, J ) 7.2, 2H), 7.50 (m, 1H), 7.64 (m, 2H), 7.97 (m, 1H). Ethyl 2-Chloro-4-quinolinecarboxylate (13). A mixture of acid 9 (1.7 g, 9.0 mmol) in POCl3 (10 mL) was heated at reflux for 3 h under argon. Then the cooled reaction mixture was poured into ice-water, and the precipitate was extracted with CHCl3. The combined extracts were dried over sodium sulfate and evaporated under reduced pressure. To the residue was added thionyl chloride (10 mL), and the resulting mixture was heated at reflux under argon for 2 h. The thionyl chloride excess was then removed under reduced pressure, and 20 mL of ethanol and 6 mL of triethylamine were added. The resulting reaction mixture was heated at reflux for 30 min, concentrated under reduced pressure, and partitioned between CHCl3 and water. The organic layer was dried over sodium sulfate and concentrated under reduced pressure. Purification of the residue by flash chromatography with n-hexane-ethyl acetate (8:2) as the eluent gave 1.6 g of 13 (yield 75%, mp 6364 °C). 1H NMR (CDCl3): δ 1.47 (t, J ) 7.0, 3H), 4.50 (q, J ) 7.1, 2H), 7.64 (m, 1H), 7.77 (m, 1H), 7.87 (s, 1H), 8.06 (m, 1H), 8.72 (m, 1H). General Procedure for the Preparation of 3-Alkyl-2chloro-4-quinolinecarboxamide Derivatives 17, 19-22, 24-29, 31-35. A mixture of the suitable acids 10-12 (1.0 g) in POCl3 (10 mL) was heated at reflux for 3 h under argon. The cooled reaction mixture was poured into ice-water, and the precipitate was extracted with CHCl3. The combined extracts were dried over sodium sulfate and evaporated under reduced pressure. The residue was purified by flash chromatography with dichloromethane as the eluent to obtain the corresponding 3-alkyl-2-chloro-4-chlorocarbonylquinoline derivatives, which were immediately used in the next step. To a mixture of the suitable acid chlorides (1.0 g) in 25 mL of dichloromethane cooled at 0-5 °C was added the appropriate amine (2-10 equiv, or 1.2 equiv plus 5 equiv of triethylamine), and the resulting mixture was stirred at room temperature for a suitable time (typically 0.5 h) while the reaction progress was monitored by TLC. The reaction mixture was concentrated under reduced pressure and partitioned between CH2Cl2 and water. The organic layer was dried over sodium sulfate and concentrated under reduced pressure, and the residue was used in the next step or purified when necessary. 2-Chloro-N,N,3-tripropylquinoline-4-carboxamide (26). This compound was obtained from acid 12 as a colorless oil that crystallized on standing (yield 76%, mp 81-83 °C). 1H NMR (CDCl3): δ 0.59 (t, J ) 7.4, 3H), 1.04 (m, 6H), 1.241.50 (m, 2H), 1.61-1.91 (m, 4H), 2.51-2.68 (m, 1H), 2.802.94 (m, 3H), 3.37-3.52 (m, 1H), 3.68-3.82 (m, 1H), 7.477.71 (m, 3H), 7.99 (d, J ) 8.3, 1H). General Procedure for the Preparation of 2-Chloro4-quinolinecarboxamide Derivatives 18, 23, 30. A mixture of acid 9 (1.5 g, 7.9 mmol) in POCl3 (10 mL) was heated at reflux for 3 h under argon. Then the cooled reaction mixture was poured into ice-water and the precipitate was extracted with CHCl3. The combined extracts were dried over sodium sulfate and evaporated under reduced pressure. To the residue was added thionyl chloride (10 mL), and the mixture was heated at reflux under argon for 2 h. The thionyl chloride excess was then removed under reduced pressure, and the resulting acid chloride was immediately used without further purification. To a mixture of acid chloride in 25 mL of

Experimental Section All chemicals used were of reagent grade. Yields refer to purified products and are not optimized. Melting points were determined in open capillaries on a Gallenkamp apparatus and are uncorrected. Microanalyses were carried out by means of a Perkin-Elmer 240C or a Perkin-Elmer series II CHNS/O analyzer, model 2400. Merck silica gel 60 (230-400 mesh) was used for column chromatography. Merck TLC plates, silica gel 60 F254, were used for TLC. 1H NMR spectra were recorded with a Bruker AC 200 spectrometer in the indicated solvents (TMS as internal standard). The values of the chemical shifts are expressed in ppm, and the coupling constants (J) are in Hz. Mass spectra were recorded on either a Varian Saturn 3 spectrometer or a ThermoFinnigan LCQ-Deca. General Procedure for the Preparation of 2-Hydroxy4-quinolinecarboxylic Acid Derivatives 9-12. A mixture of 10 g (68 mmol) of isatin 8 and 15-24 mL of the appropriate anhydride was heated under reflux for 3 h. The resulting solution was then allowed to cool to room temperature, and the solid formed was collected by filtration, washed with small amounts of diethyl ether, dried, and suspended in 30 mL of water. Sodium hydroxide pellets (4.3 g, 110 mmol) were added, and the resulting mixture was heated at reflux for 3 h, cooled, and acidified with 3 N HCl. The precipitate was collected by filtration, washed with cold water, and then dried under reduced pressure to constant weight. 2-Hydroxy-3-propyl-4-quinolinecarboxylic Acid (12). The title compound was prepared from 10 g (68 mmol) of isatin 8 and 18 mL (91 mmol) of valeric anhydride to obtain 8.0 g of white solid (yield 60%) melting at 266-268 °C. 1H NMR (DMSO-d6): δ 0.87 (t, J ) 7.3, 3H), 1.46-1.57 (m, 2H), 2.46 (t, J ) 7.0, 2H), 7.14-7.50 (m, 4H), 11.86 (s, 1H), 13.95 (br s, 1H). General Procedure for the Preparation of 3-Alkyl-2chloro-4-quinolinecarboxylic Esters 14-16. A mixture of the suitable acids 10-12 (1.0 g) in POCl3 (10 mL) was heated at reflux for 3 h under argon. Then the cooled reaction mixture was poured into ice-water and the precipitate was extracted with CHCl3. The combined extracts were dried over sodium sulfate and evaporated under reduced pressure. The residue was purified by flash chromatography with dichloromethane as the eluent to obtain the corresponding 3-alkyl-2-chloro-4chlorocarbonylquinoline derivatives, which were immediately

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dichloromethane cooled at 0-5 °C was added the appropriate amine (2 equiv of diethylamine, dipropylamine, or N-methylbenzylamine), and the resulting mixture was stirred at room temperature for a suitable time (typically 0.5 h) while the reaction progress was monitored by TLC. The reaction mixture was concentrated under reduced pressure and partitioned between CH2Cl2 and water. The organic layer was dried over sodium sulfate and concentrated under reduced pressure, and the residue was used in the next step or purified when necessary. N-Benzyl-2-chloro-N-methylquinoline-4-carboxamide (30). This compound was obtained as a pale-yellow oil that crystallized on standing (yield 64%, mp 60-62 °C). Since the amide nitrogen of this compound bears two different substituents, the 1H NMR spectrum of the compound shows the presence of two different rotamers in equilibrium. For the sake of simplification the integral intensities have not been given. 1 H NMR (CDCl3): δ 2.73 (s), 3.16 (s), 4.30 (s), 4.86 (s), 7.16 (m), 7.39 (m), 7.61 (m) 7.80 (m), 8.06 (m). General Procedure for the Preparation of Target 2-(4Methyl-1-piperazinyl)quinoline Derivatives 5 and 6. A mixture of the appropriate 2-chloroquinoline derivatives 1335 (1.0 mmol) with N-methylpiperazine (5 mL) was heated at 130-140 °C under argon for a suitable time (1-7 h), and the reaction progress was monitored by TLC. When the 2-chloroquinoline derivative disappeared from the chromatogram, the reaction mixture was then poured into ice-water (250 mL) and extracted with chloroform. The organic layer was washed with water, dried over sodium sulfate, and evaporated under reduced pressure. Purification of the residue by flash chromatography with ethyl acetate-triethylamine (8:2) as the eluent gave the expected 2-(4-methyl-1-piperazinyl)quinoline derivatives 5 and 6 showing a suitable degree of purity as confirmed by 1H NMR spectroscopy, TLC analysis, and elemental (C, H, N) analysis. In some instances the final compound was recrystallized from the appropriate solvent. 2,3-Dihydro-2-methyl-4-(4-methyl-1-piperazinyl)-1Hpyrrolo[3,4-c]quinolin-1-one (5a). This compound was prepared by applying the general procedure to imidoyl chloride 36 (reaction time, 1 h). Compound 5a was recrystallized from acetone to obtain an off-white solid (yield 58%, mp 193-194 °C). 1H NMR (CDCl3): δ 2.37 (s, 3H), 2.59 (t, J ) 4.9, 4H), 3.26 (s, 3H), 3.71 (t, J ) 4.9, 4H), 4.50 (s, 2H), 7.41 (m, 1H), 7.62 (m, 1H), 7.83 (m, 1H), 8.97 (m, 1H). MS (ESI): m/z 297 (M + H+). Anal. (C17H20N4O‚H2O) C, H, N. 4-(4-Methyl-1-piperazinyl)furo[3,4-c]quinolin-1(3H)one (5f). This compound was prepared by applying the general procedure to imidoyl chloride 41 (reaction time, 1 h). Compound 5f was recrystallized from acetone to obtain a yellow solid (yield 81%, mp 173-175 °C). 1H NMR (CDCl3): δ 2.38 (s, 3H), 2.59 (t, J ) 4.9, 4H), 3.70 (t, J ) 5.0, 4H), 5.43 (s, 2H), 7.43 (m, 1H), 7.65 (m, 1H), 7.85 (d, J ) 8.4, 1H), 8.74 (d, J ) 7.8, 1H). MS (ESI): m/z 284 (M + H+). Anal. (C16H17N3O2) C, H, N. 4-Benzoyl-3-methyl-2-(4-methyl-1-piperazinyl)quinoline (6a). This compound was obtained from compound 43 (reaction time, 7 h) as a pale-yellow oil that crystallized on standing. Recrystallization from ethyl acetate gave an analytical sample melting at 174-175 °C (yield 59%). 1H NMR (CDCl3): δ 2.21 (s, 3H), 2.37 (s, 3H), 2.62 (t, J ) 4.6, 4H), 3.36 (m, 4H), 7.26 (m, 2H), 7.40-7.64 (m, 4H), 7.78 (m, 2H), 7.90 (m, 1H). MS (ESI): m/z 346 (M + H+). Anal. (C22H23N3O) C, H, N. Ethyl 2-(4-Methyl-1-piperazinyl)-4-quinolinecarboxylate (6ba). This compound was obtained from ester 13 (reaction time, 3 h) as a pale-yellow oil that crystallized on standing (yield 55%, mp 74-76 °C). 1H NMR (CDCl3): δ 1.44 (t, J ) 6.9, 3H), 2.34 (s, 3H), 2.53 (t, J ) 4.9, 4H), 3.77 (t, J ) 4.9, 4H), 4.47 (q, J ) 7.2, 2H), 7.27 (m, 1H), 7.48 (s, 1H), 7.54 (m, 1H), 7.72 (m, 1H), 8.37 (m, 1H). MS (EI): m/z 299 (M+, 13). Anal. (C17H21N3O2) C, H, N. Ethyl 2-(4-Methyl-1-piperazinyl)-3-propyl-4-quinolinecarboxylate (6bc). This compound was obtained from ester 16 (reaction time, 3 h) as a colorless oil that crystallized on

standing (yield 52%, mp 86-88 °C). 1H NMR (CDCl3): δ 0.94 (t, J ) 7.3, 3H), 1.43 (t, J ) 6.9, 3H), 1.62 (m, 2H), 2.35 (s, 3H), 2.60 (t, J ) 4.7, 4H), 2.73 (m, 2H), 3.28 (t, J ) 4.7, 4H), 4.51 (q, J ) 7.2, 2H), 7.33 (m, 1H), 7.53 (m, 2H), 7.86 (m, 1H). MS (ESI): m/z 342 (M + H+). Anal. (C20H27N3O2) C, H, N. 2-(4-Methyl-1-piperazinyl)-N,N,3-tripropylquinoline4-carboxamide (6fc). This compound was obtained from amide 26 (reaction time, 2 h) as a pale-yellow oil (yield 97%). 1H NMR (CDCl ): δ 0.58 (t, J ) 7.3, 3H), 0.97 (t, J ) 7.3, 3H), 3 1.05 (t, J ) 7.4, 3H), 1.40 (m, 2H), 1.77 (m, 4H), 2.37 (s, 3H), 2.45-2.78 (m, 6H), 2.93 (m, 2H), 3.29 (m, 4H), 3.42-3.78 (m, 2H), 7.33 (m, 1H), 7.54 (m, 2H), 7.86 (m, 1H). MS (ESI): m/z 397 (M + H+). Anal. (C24H36N4O‚1/2H2O) C, H, N. 3,4-Bis(hydroxymethyl)-2-(4-methyl-1-piperazinyl)quinoline (6m). To a suspension of lithium aluminum hydride (0.13 g, 3.4 mmol) in anhydrous THF (10 mL) cooled at 0-5 °C was added a solution of 5f (0.24 g, 0.85 mmol) in anhydrous THF (10 mL). After the mixture was stirred for 30 min at 0-5 °C and 30 min at room temperature, the hydride was hydrolyzed by addition of water and the inorganic material was filtered off and washed with THF. The filtrate was washed with brine, dried over sodium sulfate, and evaporated under reduced pressure to give 6m as an oil that crystallized spontaneously (0.22 g, yield 90%). An analytical sample recrystallized from ethyl acetate melted at 153-155 °C. 1H NMR (CDCl3): δ 2.34 (s, 3H), 2.58 (t, J ) 4.6, 4H), 3.35 (t, J ) 4.7, 4H), 4.96 (s, 2H), 5.16 (s, 2H), 7.42 (t, J ) 7.1, 1H), 7.61 (t, J ) 7.1, 1H), 7.89 (d, J ) 8.6, 1H), 8.05 (d, J ) 8.6, 1H). MS (EI): m/z 287 (M+, 10). Anal. (C16H21N3O2) C, H, N. General Procedure for the Preparation of 4-Chloropyrrolo[3,4-c]quinoline Derivatives 36-40. A mixture of ester 14 (0.37 g, 1.5 mmol) in 30 mL of CCl4 with Nbromosuccinimide (0.27 g, 1.5 mmol) and dibenzoyl peroxide (0.03 g, 0.12 mmol) was heated at reflux under argon for 3 h. The solvent was then removed under reduced pressure, and the residue was diluted with a small portion of the same solvent. The insoluble succinimide was filtered off, and the solvent was removed under reduced pressure. The residue was dissolved in 15 mL of absolute ethanol (or methanol), and the appropriate amine (methylamine, ethylamine, propylamine, sec-butylamine, or benzylamine) was added. The resulting mixture was stirred at room temperature under argon for a suitable time (from 1.5 h to 4 days), and the reaction progress was monitored by TLC. The resulting precipitate was collected by filtration, washed with water and ethanol, and dried under reduced pressure. 4-Chloro-2,3-dihydro-2-methyl-1H-pyrrolo[3,4-c]quinolin-1-one (36). This compound was prepared following the general procedure with a 8 M solution of methylaminemethanol (2 mL, 16 mmol) and methanol as the solvent (reaction time, 1.5 h). Compound 36 was obtained as a white solid (0.18 g, yield 52%, mp 217-220 °C). 1H NMR (CDCl3): δ 3.31 (s, 3H), 4.51 (s, 2H), 7.78 (m, 2H), 8.12 (m, 1H), 9.05 (m, 1H). Anal. (C12H9ClN2O) C, H, N. 4-Chlorofuro[3,4-c]quinolin-1(3H)-one (41). A mixture of ester 14 (1.0 g, 4.0 mmol) in 50 mL of CCl4 with Nbromosuccinimide (0.71 g, 4.0 mmol) and dibenzoyl peroxide (0.10 g) was heated at reflux under argon for 3 h. The solvent was then removed under reduced pressure, and the residue was diluted with a small portion of the same solvent. The insoluble succinimide was filtered off, and the solvent was removed under reduced pressure. The residue was dissolved in 20 mL of 2-methoxyethanol, and 3 N hydrochloric acid (20 mL) was added. The resulting mixture was heated at reflux for 24 h under argon. The cooled reaction mixture was then diluted with water, and the precipitate was collected by filtration, washed with water, and dried under reduced pressure. A mixture of the solid in POCl3 (10 mL) was heated at reflux for 2.5 h under argon. The cooled reaction mixture was then poured into ice-water, and the precipitate was extracted with CHCl3. The combined extracts were dried over sodium sulfate and evaporated under reduced pressure. Purification of the residue by flash chromatography with n-hexane-ethyl acetate (8:2) as the eluent gave pure 41 as a white solid (0.51



g, yield 58%, mp 154-156 °C). 1H NMR (CDCl3): δ 5.40 (s, 2H), 7.82 (m, 2H), 8.17 (d, J ) 8.6, 1H), 8.80 (m, 1H). 4-Benzoyl-2-chloro-3-methylquinoline (43). To a solution of 2-chloro-3-methyl-4-quinolinecarboxylic acid chloride 42 (see ref 17f) (0.50 g, 2.1 mmol) in 30 mL of dry benzene was added anhydrous aluminum trichloride (1.5 g, 11 mmol), and the resulting mixture was stirred at room temperature for 2.5 h. The cooled reaction mixture was decomposed with 3 N HCl (30 mL) and extracted with chloroform. The combined extracts were dried over sodium sulfate and evaporated under reduced pressure. Purification of the residue by flash chromatography with n-hexane-ethyl acetate (8:2) as the eluent gave pure 43 as a white solid (0.49 g, yield 83%, mp 133-135 °C). 1H NMR (CDCl3): δ 2.34 (s, 3H), 7.45 (m, 4H), 7.67 (m, 2H), 7.80 (m, 2H), 8.06 (m, 1H). Anal. (C17H12ClNO) C, H, N. 2-Chloro-3-methyl-N-[7-((1,2,3,4-tetrahydroacridin-9yl)amino)heptyl]quinoline-4-carboxamide (45). A solution of 2-chloro-3-methyl-4-quinolinecarboxylic acid chloride 42 (see ref 17f) (0.26 g, 1.1 mmol) in 20 mL of dichloromethane was added to a mixture of N-(1,2,3,4-tetrahydroacridin-9-yl)heptane-1,7-diamine 44 (see ref 18d) (0.35 g, 1.1 mmol) with triethylamine (1.4 mL, 10 mmol) in dichloromethane (20 mL), and the resulting mixture was stirred at room temperature for 1 h. The reaction mixture was partitioned between dichloromethane and water, and the organic layer was dried over sodium sulfate and evaporated under reduced pressure. Purification of the residue by flash chromatography with ethyl acetate-triethylamine (8:2) as the eluent gave pure 45 as an off-white solid (0.34 g, yield 55%, mp 90-95 °C). 1H NMR (CDCl3): δ 1.40 (br s, 6H), 1.66 (br s, 4H), 1.89 (m, 4H), 2.45 (s, 3H), 2.68 (br s, 2H), 2.99 (br s, 2H), 3.52 (m, 4H), 3.91 (br s, 1H), 6.20 (br s, 1H), 7.30 (m, 1H), 7.50 (m, 2H), 7.67 (m, 2H), 7.83 (d, J ) 8.1, 1H), 7.92 (d, J ) 8.4, 2H). MS (ESI): m/z 515 (M + H+). Anal. (C31H35ClN4O‚3/4H2O) C, H, N. 3-Methyl-2-(4-methyl-1-piperazinyl)-N-[7-[(1,2,3,4-tetrahydroacridin-9-yl)amino]heptyl]quinoline-4-carboxamide (6o). This compound was prepared in a similar way as compounds 6 starting from 45 (0.20 g, 0.39 mmol) and N-methylpiperazine (5 mL, reaction time, 5 h at 140 °C) and purified by flash chromatography with ethyl acetate-triethylamine (8:2) as the eluent. Compound 6o (0.18 g, yield 80%) was obtained as a pale-yellow oil that crystallized on standing (mp 137-140 °C). 1H NMR (CDCl3): δ 1.40 (br s, 6H), 1.63 (m, 4H), 1.92 (m, 4H), 2.35 (s, 3H), 2.37 (s, 3H), 2.57 (t, J ) 4.2, 4H), 2.70 (br s, 2H), 3.02 (br s, 2H), 3.33 (t, J ) 4.5, 4H), 3.49 (m, 4H), 3.90 (br s, 1H), 5.90 (t, J ) 5.5, 1H), 7.31 (m, 2H), 7.58 (m, 3H), 7.87 (m, 3H). MS (ESI): m/z 579 (M + H+). Anal. (C36H46N6O‚1/4H2O) C, H, N. In Vitro Binding Assays. Binding assays were performed as described in ref 21. Male Wistar rats (Charles River, Calco, Italy) were killed by decapitation. Their brains were rapidly removed at 4 °C, and the cortex and hippocampus were dissected out. Tissues were homogenized (Polytron PTA 10TS) in ice-cold 50 mM Hepes buffer, pH 7.4, and centrifuged according to the procedures indicated in the above-cited reference. The pellet obtained was finally suspended in 50 mM Hepes buffer, pH 7.4, just before the binding assay was performed. [3H]Granisetron (s.a. 81 Ci/mmol; NEN Life Science Products) binding was assayed in final incubation volumes of 1 mL. Tissue and [3H]ligand final concentration were 20 mg of tissue/sample and 0.5 nM, respectively. The specific binding of the tritiated ligand was defined as the difference between the binding in the absence (total binding) and in the presence of 100 mM unlabeled 5-HT (nonspecific binding). It represented an average of 70% of the total binding. Incubation was interrupted by rapid filtration under vacuum through Whatman GF/B glass fiber filters presoaked in 50 mM Hepes buffer, pH 7.4, containing 0.1% polyethyleneimine. Filters were immediately rinsed with 12 mL (3 × 4 mL) of ice-cold buffer by means of a Brandel M-24R cell harvester, dried, and immersed into vials containing 8 mL of Ultima Gold MV (Packard Biosciences) for the measurement of trapped radioactivity with a TRI-CARB 1900TR (Packard Biosciences) liquid scintillation spectrometer at a counting efficiency of

about 60%. Competition experiments were analyzed by the “Allfit” program29 to obtain the concentration of unlabeled drug that caused 50% inhibition of [3H]granisetron specific binding (IC50). Apparent affinity constants (Ki) were derived from the IC50 values according to the Cheng and Prusoff equation.30 The Kd value for [3H]granisetron specific binding calculated from saturation isotherms was found to be 0.6 nM. Measurement of AChE/BuChE Activity. AChE/BuChE activities were measured by a fluorimetric assay using the AmplexRed acetylcholine/acetylcholinesterase assay kit (Molecular Probes Inc.). Acetylcholinesterase (from human erytrocytes) and butyrylcholinesterase (from human serum) were purchased from Sigma. Reaction was started by adding 100 µL of AmplexRed reagent working solution (HRP 1 U/mL, Choline oxidase 0.1 U/mL, Ach 50 µM, AmpexRed reagent 200 µM, final concentrations) to 100 µL of enzyme working solution (200 mU/mL in 50 mM Tris-Cl, pH 8, final concentration) either in the absence (control) or in the presence of increasing concentrations of inhibitors. Microplates were incubated at room temperature for 15 min and protected from light, and then fluorescence was measured in a fluorescence microplate reader (Perkin-Elmer HTS 7000PLUS) using excitation at 530-560 nm and emission detection at 590 nm. Background fluorescence, derived from no-enzyme control, was subtracted from each point. By comparison with 100% activity of the control assay, the percentage of enzyme inhibition for the tested compounds was calculated. IC50 values were calculated as indicated in In Vitro Binding Assays. Computational Methods. Interaction of Compound 6o with 5-HT3 Receptor. The extended conformation of the 6o ligand structure was manually docked in the minimized average structure of the receptor built by the program Modeller31 on the basis of the sequence alignment reported by Maksay et al.10 Once the principal interaction modalities of the piperazine moiety, i.e., the ionic interaction with Glu236, were satisfied, in the binding cleft formed by residues Trp90, N128, Trp183, and Tyr234, several conformations of the heptylene linker were considered in order to find a suitable cleft for the tetrahydroacridine moiety. The complexes were energy-minimized by means of the program CHARMM.32 The minimization procedure consisted of 50 steps of steepest descent, followed by a conjugate gradient minimization until the rms gradient of the potential energy was lower than 0.001 kcal mol-1 Å-1. The united atom force field parameters, a 12 Å nonbonded cutoff and a dielectric constant ) 4r, were used. Interaction of Compound 6o with AChE and BuChE. Conformational Analysis and Preorganization Evaluation of Compound 6o. The conformational search of compound 6o was carried out by the MC method as implemented in the MacroModel package.33 Five-thousand conformations of the molecule were generated and energy-minimized with the AMBER* united atom force field34 and GB/SA water implicit model of solvation.35 The monitoring distance on the MC ensemble was calculated between aminoacridine and quinoline centroids. Preparation of Enzyme Models. The crystallographic models 1B41 for the human AChE and 1P0I for the BuChE required preparation before the docking experiments. Ligands and water molecules of both enzymes were removed by means of the Maestro GUI.36 Covalently bonded non-amino-acid residues were kept in both models. Duplicate residues of 1B41 (Arg11, Arg13, Glu91, Glu166, Arg246, His253) and 1P0I (Leu110, Phe227, Ser466, Met511) were removed. Missing atoms of side chain residues for 1B41 (Arg13, Arg246, His253, Glu268, Gln291, Gln369, Arg522) and 1P0I (Gln486) were fixed using the replace option of Maestro GUI. Considering the neutral form of His residues, in agreement with the number of charged residues for 1B41 (29 Glu, 37 Arg, 23 Asp, and 7 Lys) and for 1P0I (31 Glu, 31 Arg, 17 Asp, and 22 Lys) after these fixing operations, the overall total charge was respectively -8 and +5. To reduce the conformational influence of cocrystallized ligands, both enzyme models were subjected to 3000 ps of

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molecular dynamics simulations at 300 K in vacuo with a dielectric constant equal to 80 and AMBER* united atoms. Thirty structures for each enzyme were sampled and submitted to energy minimization in the GB/SA water environment with the convergence criterion modified to 0.5 kJ mol-1 Å-1. The R carbon atoms were taken into account to remove duplicated structures that were judged to be identical if the internal energy difference was found to be within 4.184 kJ/ mol and the rms deviation was lower than 0.25 Å. In the first 20 kJ/mol above the global minimum energy conformers, only one structure was identified for each enzyme and was compared to the PDB conformation by the backbone rms deviation. The backbone rms deviation versus the crystallographic coordinates after the pretreatment were 1.60 Å (AChE) and 2.03 Å (BuChE). Docking Evaluation of 6o AChE/BuChE Binding Modes. Starting complexes of AChE and BuChE with 6o, modeled as charged and neutral forms, were generated by manual docking of a ligand extended conformation. The aminoacridine moiety was positioned in the same way as the crystallographic position of tacrine in 1ACJ PDB model,37 i.e., in front of Trp86 for AChE and in front of Trp82 for BuChE. The arylpiperazine moiety in both enzymes was located at the entrance of the gorge in front of Trp286 and Ala277, respectively. Both complexes were fully energy-minimized in GB/ SA water using the AMBER* united atoms force field. The flexible docking of 6o within the enzymes was performed by 10 000 steps of MC search of the ligand rotatable bonds combined with rotations around its mass center (maximum 180°) and translations within the cleft (maximum 2 Å) with respect to the starting complex conformation. This stage was performed using the AMBER* united atoms force field with a dielectric constant equal to 80. A constrained energy minimization was combined with the MC search considering all the residues within 10 Å from the original ligand position. With an overall population higher than 99% at 300 K, 5 and 22 poses of the charged form of 6o and 39 and 13 poses of its neutral form were respectively obtained in the AChE and BuChE clefts and submitted to a refinement based on full energy minimization in GB/SA water. Finally, 1 and 4 complex configurations were found for the 6o charged and neutral forms with AChE, and 13 and 2 complex configurations were found with BuChE, respectively. The global minimum docking complexes were analyzed by the measurement module of the Maestro GUI,36 considering for the identification of van der Waals interacting residues those located between 0.89 and 1.30 Å from any ligand atom and for the hydrogen bond a cutoff distance maximum equal to 2.5 Å with a minimum angle of 90° and 60° respectively for the donor-hydrogen-acceptor and the hydrogen-acceptorattached atoms. The free interaction energies were computed onto the fully minimized complex ensembles according to a previously reported approach,38 i.e., as the difference between the total and the single contributions of the isolated interacting molecules recalculated in the same conformations of each complex.

HT3A). J. Physiol. 1998, 507, 653-665. (c) Nichols, R. A.; Mollard, P. Direct Observation of Serotonin 5-HT3 Receptor-Induced Increases in Calcium Levels in Individual Brain Nerve Terminals. J. Neurochem. 1996, 67, 581-592. Boess, F. G.; Martin, I. L. Molecular Biology of 5-HT Receptors. Neuropharmacology 1994, 33, 275-317. Maricq, A. V.; Peterson, A. S.; Brake, A. J.; Myers, R. M.; Julius, D. Primary Structure and Functional Expression of the 5-HT3 Receptor, a Serotonin-Gated Ion Channel. Science 1991, 254, 432-437. (a) Dubin, A. E.; Huvar, R.; D’Andrea, M. R.; Pyati, J.; Zhu, J. Y.; Joy, K. C.; Wilson, S. J.; Galindo, J. E.; Glass, C. A.; Luo, L.; Jackson, M. R.; Lovenberg, T. W.; Erlander, M. G. The Pharmacological and Functional Characteristics of the Serotonin 5-HT3A Receptor Are Specifically Modified by a 5-HT3B Receptor Subunit. J. Biol. Chem. 1999, 274, 30799-30810. (b) Davies, P. A.; Pistis, M.; Hanna, M. C.; Peters, J. A.; Lambert, J. J.; Hales, T. G.; Kirkness, E. F. The 5-HT3B Subunit Is a Major Determinant of Serotonin-Receptor Function. Nature 1999, 397, 359363. Hanna, M. C.; Davies, P. A.; Pistis, M.; Hales, T. G.; Kirkness, E. F. Evidence for Expression of Heteromeric Serotonin 5-HT3 Receptors in Rodents. J. Neurochem. 2000, 75, 240-247. Corringer, P.-J.; Le Novere, N.; Changeux, J.-P. Nicotinic Receptors at the Amino Acid Level. Annu. Rev. Pharmacol. Toxicol. 2000, 40, 431-458. Unwin, N. Nicotinic Acetylcholine Receptor at 9 Å Resolution. J. Mol. Biol. 1993, 229, 1101-1124. Menziani, M. C.; De Rienzo, F.; Cappelli, A.; Anzini, M.; De Benedetti, P. G. A Computational Model of the 5-HT3 Receptor Extracellular Domain: Search for Ligand Binding Sites. Theor. Chem. Acc. 2001, 106, 98-104. Brejc, K.; van Dijk, W. J.; Klaassen, R. V.; Schuurmans, M.; van Der Oost, J.; Smit, A. B.; Sixma, T. K. Crystal Structure of an ACh-Binding Protein Reveals the Ligand-Binding Domain of Nicotinic Receptors. Nature 2001, 411, 269-276. Maksay, G.; Bikadi, Z.; Simonyi, M. Binding Interactions of Antagonists with 5-Hydroxytryptamine3A Receptor Models. J. Recept. Signal Transduction Res. 2003, 23, 255-270. Reeves, D. C.; Sayed, M. F.; Chau, P. L.; Price, K. L.; Lummis, S. C. Prediction of 5-HT3 Receptor Agonist-Binding Residues Using Homology Modeling. Biophys. J. 2003, 84, 2338-2334. Gaster, L. M.; King, F. D. Serotonin 5-HT3 and 5-HT4 Receptor Antagonists. Med. Res. Rev. 1997, 17, 163-214. Silverstone, P. H.; Greenshaw, A. J. 5-HT3 Receptor Antagonists. Expert Opin. Ther. Pat. 1996, 6, 471-481. Blandina, P.; Goldfarb, J.; Green, J. P. Activation of a 5-HT3 Receptor Releases Dopamine from Rat Striatal Slices. Eur. J. Pharmacol. 1988, 155, 349-350. Paudice, P.; Raiteri, M. Cholecystokinin Release Mediated by 5-HT3 Receptors in Rat Cerebral Cortex and Nucleus Accumbens. Br. J. Pharmacol. 1991, 103, 1790-1794. Barnes, J. M.; Barnes, N. M.; Costall, B.; Naylor, R. J.; Tyers, M. B. 5-HT3 Receptors Mediate Inhibition of Acetylcholine Release in Cortical Tissue. Nature 1989, 338, 762-763. (a) Anzini, M.; Cappelli, A.; Vomero, S.; Giorgi, G.; Langer, T.; Hamon, M.; Merahi, N.; Emerit, B. M.; Cagnotto, A.; Skorupska, M.; Mennini, T.; Pinto, J. C. Novel, Potent, and Selective 5-HT3 Receptor Antagonists Based on the Arylpiperazine Skeleton: Synthesis, Structure, Biological Activity, and Comparative Molecular Field Analysis Studies. J. Med. Chem. 1995, 38, 26922704. (b) Cappelli, A.; Donati, A.; Anzini, M.; Vomero, S.; De Benedetti, P. G.; Menziani, M. C.; Langer, T. Molecular Structure and Dynamics of Some Potent 5-HT3 Receptor Antagonists. Insight into the Interaction with the Receptor. Bioorg. Med. Chem. 1996, 4, 1255-1269. (c) Campiani, G.; Cappelli, A.; Nacci, V.; Anzini, M.; Vomero, S.; Hamon, M.; Cagnotto, A.; Fracasso, C.; Uboldi, C.; Caccia, S.; Consolo, S.; Mennini, T. Novel and Highly Potent 5-HT3 Receptor Agonists Based on a Pyrroloquinoxaline Structure. J. Med. Chem. 1997, 40, 3670-3678. (d) Cappelli, A.; Anzini, M.; Vomero, S.; Mennuni, L.; Makovec, F.; Doucet, E.; Hamon, M.; Bruni, G.; Romeo, M. R.; Menziani, M. C.; De Benedetti, P. G.; Langer, T. Novel Potent and Selective Central 5-HT3 Receptor Ligands Provided with Different Intrinsic Efficacy. 1. Mapping the Central 5-HT3 Receptor Binding Site by Arylpiperazine Derivatives. J. Med. Chem. 1998, 41, 728741. (e) Cappelli, A.; Anzini, M.; Vomero, S.; Canullo, L.; Mennuni, L.; Makovec, F.; Doucet, E.; Hamon, M.; Menziani, M. C.; De Benedetti, P. G.; Bruni, G.; Romeo, M. R.; Giorgi, G.; Donati, A. Novel Potent and Selective Central 5-HT3 Receptor Ligands Provided with Different Intrinsic Efficacy. 2. Molecular Basis of the Intrinsic Efficacy of Arylpiperazine Derivatives at the Central 5-HT3 Receptors. J. Med. Chem. 1999, 42, 15561575. (f) Cappelli, A.; Anzini, M.; Vomero, S.; Mennuni, L.; Makovec, F.; Doucet, E.; Hamon, M.; Menziani, M. C.; De Benedetti, P. G.; Giorgi, G.; Ghelardini, C.; Collina, S. Novel Potent 5-HT3 Receptor Ligands Based on the Pyrrolidone Structure: Synthesis, Biological Evaluation, and Computational

Acknowledgment. Thanks are due to Italian MIUR (PRIN) and Consiglio Nazionale delle Ricerche (Agenzia 2000) for financial support. Prof. Stefania D’Agata D’Ottavi’s careful reading of the manuscript is also acknowledged. Supporting Information Available: Detailed analysis of the 6o-AChE/BuChE interaction and full experimental details for the synthesis and the characterization of compounds 5 and 6 and their intermediates (chemistry, NMR, MS). This material is available free of charge via the Internet at http:// pubs.acs.org.

References (1) (a) Jackson, M. B.; Yakel, J. L. The 5-HT3 Receptor Channel. Annu. Rev. Physiol. 1995, 57, 447-468. (b) Brown, A. M.; Hope, A. G.; Lambert, J. J.; Peters, J. A. Ion Permeation and Conduction in a Human Recombinant 5-HT3 Receptor Subunit (h5-

(2) (3)

(4)

(5) (6) (7) (8)

(9)

(10) (11) (12) (13) (14) (15) (16) (17)


(18)

(19) (20)

(21) (22) (23)

(24)

(25) (26)

Rationalization of the Ligand-Receptor Interaction Modalities. Bioorg. Med. Chem. 2002, 10, 779-801. (g) Cappelli, A.; Gallelli, A.; Braile, C.; Anzini, M.; Vomero, S.; Mennuni, L.; Makovec, F.; Menziani, M. C.; De Benedetti, P. G.; Donati, A.; Giorgi, G. Novel Potent 5-HT3 Receptor Ligands Based on the Pyrrolidone Structure. Effects of the Quaternization of the Basic Nitrogen on the Interaction with 5-HT3 Receptor. Bioorg. Med. Chem. 2002, 10, 2681-2691. (h) Cappelli, A.; Anzini, M.; Vomero, S.; Mennuni, L.; Makovec, F.; Hamon, M.; De Benedetti, P. G.; Menziani, M. C. The Interaction of 5-HT3 Receptor with Arylpiperazine, Tropane, and Quinuclidine Ligands. Curr. Top. Med. Chem. 2002, 2, 599-624. (a) Pang, Y.-P.; Quiram, P.; Jelacic, T.; Hong, F.; Brimijoin, S. Highly Potent, Selective, and Low Cost Bis-Tetrahydroaminacrine Inhibitors of Acetylcholinesterase. J. Biol. Chem. 1996, 271, 23646-23649. (b) Carlier, P. R.; Du, D.-M.; Han, Y.; Liu, J.; Pang, Y.-P. Potent, Easily Synthesized Huperzine A-Tacrine Hybrid Acetylcholinesterase Inhibitors. Bioorg. Med. Chem. Lett. 1999, 9, 2335-2338. (c) Han, Y. F.; Li, C. P.-L.; Chow, E.; Wang, H.; Pang, Y.-P.; Carlier, P. R. Dual-Site Binding of Bivalent 4-Aminopyridine- and 4-Aminoquinoline-Based AChE Inhibitors: Contribution of the Hydrophobic Alkylene Tether to Monomer and Dimer Affinities. Bioorg. Med. Chem. 1999, 7, 2569-2575. (d) Carlier, P. R.; Chow, E. S.-H.; Han, Y.; Liu, J.; El Yazal, J.; Pang, Y.-P. Heterodimeric Tacrine-Based Acetylcholinesterase Inhibitors: Investigating Ligand-Peripheral Site Interactions. J. Med. Chem. 1999, 42, 4225-4231. (e) Savini, L.; Campiani, G.; Gaeta, A.; Pellerano, C.; Fattorusso, C.; Chiasserini, L.; Fedorko, J. M.; Saxena, A. Novel and Potent Tacrine-Related Hetero- and Homobivalent Ligands for Acetylcholinesterase and Butyrylcholinesterase. Bioorg. Med. Chem. Lett. 2001, 11, 1779-1782. (f) Savini, L.; Gaeta, A.; Fattorusso, C.; Catalanotti, B.; Campiani, G.; Chiasserini, L.; Pellerano, C.; Novellino, E.; McKissic, D.; Saxena, A. Specific Targeting of Acetylcholinesterase and Butyrylcholinesterase Recognition Sites. Rational Design of Novel, Selective, and Highly Potent Cholinesterase Inhibitors. J. Med. Chem. 2003, 46, 1-4. (g) Lewis, W. G.; Green, L. G.; Grynszpan, F.; Radic´, Z.; Carlier, P. R.; Taylor, P.; Finn, M. G.; Sharpless, K. B. Click Chemistry in Situ: Acetylcholinesterase as a Reaction Vessel for the Selective Assembly of a Fentomolar Inhibitor from an Array of Building Blocks. Angew. Chem., Int. Ed. 2002, 41, 1053-1057. Barnes, N. M.; Costall, B.; Naylor, R. J.; Williams, T. J.; Wischik, C. M. Normal Densities of 5-HT3 Receptor Recognition Sites in Alzheimer’s Disease. NeuroReport 1990, 1, 253-254. (a) Mucke, H. A. M.; Castan˜er, J. T-82. Drugs Future 1998, 23, 1075-1077. (b) Sugimoto, H.; Yamanishi, Y.; Iimura, Y.; Kawakami, Y. Donepezil Hydrochloride (E2020) and Other Acetylcholinesterase Inhibitors. Curr. Med. Chem. 2000, 7, 303-339. Nelson, D. R.; Thomas, D. R. [3H]-BRL 43694 (Granisetron), a Specific Ligand for 5-HT3 Binding Sites in Rat Brain Cortical Membranes. Biochem. Pharmacol. 1989, 38, 1693-1695. Castan, F.; Schambel, P.; Enrici, A.; Rolland, F.; Bigg, D. C. H. New Arylpiperazine Derivatives with High Affinity for 5-HT3 Receptor Sites. Med. Chem. Res. 1996, 6, 81-101. Schreiter, C.; Hovius, R.; Costioli, M.; Pick, H.; Kellenberger, S.; Schild, L.; Vogel, H. Characterization of the Ligand-Binding Site of the Serotonin 5-HT3 Receptor: The Role of Glutamate Residues 97, 224, and 235. J. Biol. Chem. 2003, 278, 2270922716. Venkataraman, P.; Joshi, P.; Venkatachalan, S. P.; Muthalagi, M.; Parihar, H. S.; Kirschbaum, K. S.; Schulte, M. K. Functional Group Interactions of a 5-HT3R Antagonist. BMC Biochem. 2002, 3, 16. Venkataraman, P.; Venkatachalan, S. P.; Joshi, P. R.; Muthalagi, M.; Schulte, M. K. Identification of Critical Residues in Loop E in the 5-HT3ASR Binding Site. BMC Biochem. 2002, 3, 15. Beene, D. L.; Price, K. L.; Lester, H. A.; Dougherty, D. A.; Lummis, S. C. R Tyrosine Residues That Control Binding and Gatin in the 5-Hydroxytryptamine3 Receptor Revealed by Un-


(27)

(28)

(29)

(30)

(31) (32)

(33)

(34) (35) (36) (37)

(38)

3575

natural Amino Acid Mutagenesis. J. Neurosci. 2004, 24, 90979104. (a) PDB code 1B41 (AChE): Kryger, G.; Harel, M.; Giles, K.; Toker, L.; Velan, B.; Lazar, A.; Kronman, C.; Barak, D.; Ariel, N.; Shafferman, A.; Silman, I.; Sussman, J. L. Structures of Recombinant Native and E202Q Mutant Human Acetylcholinesterase Complexed with the Snake-Venom Toxin FasciculinII. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2000, 56, 138594. (b) PDB code 1P0I (BuChE): Nicolet, Y.; Lockridge, O.; Masson, P.; Fontecilla-Camps, J. C.; Nachon, F. Crystal Structure of Human Butyrylcholinesterase and of Its Complexes with Substrate and Products. J. Biol. Chem. 2003, 278, 41141-41147. (a) Kryger, G.; Silman, I.; Sussman, J. L. Structure of Acetylcholinesterase Complexed with E2020 (Aricept): Implications for the Design of New Anti-Alzheimer Drugs. Struct. Fold Des. 1999, 7, 297-307. (b) Wong, D. M.; Greenblatt, H. M.; Dvir, H.; Carlier, P. R.; Han, Y.-F.; Pang, Y.-P.; Silman, I.; Sussman, J. L. Acetylcholinesterase Complexed with Bivalent Ligands Related to Huperzine A: Experimental Evidence for SpeciesDependent Protein-Ligand Complementarity. J. Am. Chem. Soc. 2003, 125, 363-373. De Lean, K. W.; Munson, P. J.; Rodbard, D. Simultaneous Analysis of Families of Sigmoidal Curves: Application to Bioassay, Radioligand Assay and Physiological Dose-Response Curves. Am. J. Physiol. 1978, 235, E97-E102. Cheng, Y.; Prusoff, W. H. Relationship between the Inhibition Constant (Ki) and the Concentration of Inhibitor Which Causes 50 Per Cent Inhibition (IC50) of an Enzymatic Reaction. Biochem. Pharmacol. 1973, 22, 3099-3108. Sali, A.; Blundell, T. L. Comparative protein modeling by satisfaction of spatial restraints. J. Mol. Biol. 1993, 234, 779815. Brooks, B. R.; Bruccoleri, R. E.; Olafson, B. D.; States, D. J.; Swaminathan, S.; Karplus, M. CHARMM: A Program for Macromolecular Energy, Minimization and Dynamics Calculations. J. Comput. Chem. 1983, 4, 187-217. Mohamadi, F.; Richards, N. G. J.; Guida, W.; C.; Liskamp, R.; Lipton, M.; Caufield, C.; Chang, G.; Hendrickson, T.; Still, W. C. MacroModelsAn Integrated Software System for Modeling Organic and Bioorganic Molecules Using Molecular Mechanics. J. Comput. Chem. 1990, 11, 440-467. McDonald, D. Q.; Still, W. C. AMBER* Torsional Parameters for the Peptide Backbone. Tetrahedron Lett. 1992, 33, 7743. Still, W. C.; Tempczyk, A.; Hawley, R. C.; Hendrickson, T. Semianalytical Treatment of Solvation for Molecular Mechanics and Dynamics. J. Am. Chem. Soc. 1990, 112, 6127-6129. MacroModel, version 7.2; Schro¨dinger Inc.: Portland, OR, 19982001. Harel, M.; Schalk, I.; Ehret-Sabatier, L.; Bouet, F.; Goeldner, M.; Hirth, C.; Axelsen, P. H.; Silman, I.; Sussman, J. L. Quaternary Ligand Binding to Aromatic Residues in the ActiveSite Gorge of Acetylcholinesterase. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 9031-9035. (a) Alcaro, S.; Gasparrini, F.; Incani, O.; Mecucci, S.; Misiti, D.; Pierini, M.; Villani, C. A. Quasi-Flexible Automatic Docking Processing for Studying Stereoselective Recognition Mechanisms. Part I. Protocol Validation. J. Comput. Chem. 2000, 21, 515-530. (b) Alcaro, S.; Scipione, L.; Ortuso, F.; Posca, S.; Rispoli, V.; Rotiroti, D. Molecular Modeling and Enzymatic Studies of the Interaction of a Choline Analogue and Acetylcholinesterase. Bioorg. Med. Chem. Lett. 2002, 12, 2899-2905. (c) Alcaro, S.; Ortuso, F.; Battaglia, D.; Anzini, M.; Cappelli, A.; Gallelli, A.; Pericot Mohr, G.; Vomero, S.; Galli, A.; Costagli, C.; Makovec, F. Enzymatic and Molecular Modeling Studies of 5-HT3 Receptor Ligands Based on Pyrroloquinoline Structure and Provided with Acetylcholinesterase Inhibitory Activity. Med. Chem. Res. 2003, 12, 147-160.

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Receptor with Arylpiperazine Ligands. Development of a New 5-HT3

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