Tania De

La Fuente
email: tfuentem@quim.ucm.es

Mar Martín-Fontecha

Jessica Sallander

Bellinda Benhamú
email: bellinda.benhamu@quim.ucm.es

Mercedes Campillo
email: mercedes.campillo@uab.es

Rocío A Medina
email: rociomedina@quim.ucm.es

Lucie P Pellissier
email: lucie.pellissier@igf.cnrs.fr

Sylvie Claeysen
email: sylvie.claeysen@igf.cnrs.fr

Aline Dumuis
email: aline.dumuis@igf.cnrs.fr

Leonardo Pardo
email: leonardo.pardo@uab.es.

Tania De La Fuente

Lucie P Pellisier

María L López-Rodríguez

Receptor Antagonists. Molecular Mechanisms of Receptor Inactivation

Keywords: GPCR, G protein-coupled receptor, CNS, central nervous system, h5-HT 6 positive ionizable atom, HBA, H-bonding acceptor group, HYD, hydrophobic, TM, transmembrane helix, EL, extracellular loop, SEM, 2-

Introduction

Serotonin (5-hydroxytryptamine, 5-HT) research is now more than 50 years old, [1][2][START_REF] Adayev | Transmembrane signaling in the brain by serotonin, a key regulator of physiology and emotion[END_REF] but still represents one of the most attractive areas in medicinal chemistry. 4 Fourteen serotonin receptor subtypes have been identified so far, that are classified into seven families (5-HT 1- 7 ) [START_REF] Uphouse | Multiple serotonin receptors: too many, not enough, or just the right number?[END_REF][START_REF] Hoyer | 5-HT receptor classification and nomenclature: towards a harmonization with the human genome[END_REF] based on amino acid sequences, pharmacology, and intracellular mechanisms. With the exception of the ionotropic 5-HT 3 receptor, all of the 5-HT subtypes are G protein-coupled receptors (GPCRs a ). [START_REF] Bartfai | The state of GPCR research in 2004[END_REF][START_REF] Filizola | The structure and dynamics of GPCR oligomers: a new focus in models of cell-signaling mechanisms and drug design[END_REF][9][START_REF] Rosenbaum | The structure and function of Gprotein-coupled receptors[END_REF] To date many serotoninergic agents acting at 5-HT 1-4 receptors are clinically used 11 for the treatment of certain central nervous system (CNS) illnesses. One of the most recent additions to the family of serotonin receptors is the human 5-HT 6 receptor (h5-HT 6 R), that was cloned in 1996 12,[START_REF] Kohen | Cloning, characterization, and chromosomal localization of a human 5-HT 6 serotonin receptor[END_REF] as a gene codifying a polypeptide chain of 440 amino acids [START_REF] Kohen | Cloning, characterization, and chromosomal localization of a human 5-HT 6 serotonin receptor[END_REF][START_REF] Kohen | Cloning of the mouse 5-HT 6 serotonin receptor and mutagenesis studies of the third cytoplasmic loop[END_REF] that is positively coupled to the adenylate cyclase [START_REF] Plassat | Molecular cloning of a mammalian serotonin receptor that activates adenylate cyclase[END_REF][START_REF] Sebben | 5-HT 6 receptors positively coupled to adenylyl cyclase in striatal neurones in culture[END_REF][17] cascade via the G s protein.

The high affinity of several antipsychotic and antidepressant agents [START_REF] Kohen | Cloning, characterization, and chromosomal localization of a human 5-HT 6 serotonin receptor[END_REF][START_REF] Monsma | Cloning and expression of a novel serotonin receptor with high affinity for tricyclic psychotropic drugs[END_REF][START_REF] Davies | Drug discovery targets: 5-HT 6 receptor[END_REF] boosted the first studies exploring the 5-HT 6 R potential for the treatment of schizophrenia and bipolar affective disorder. However, contradictory results [START_REF] Davies | Drug discovery targets: 5-HT 6 receptor[END_REF] were obtained about the role of the receptor in these therapeutic indications. Later on, 5-HT 6 R function was associated with the control of cholinergic neurotransmission, [START_REF] Bourson | Determination of the role of the 5-ht 6 receptor in the rat brain: a study using antisense oligonucleotides[END_REF]21 which prompted much interest into the possible implication of the receptor in cognitive impairment [START_REF] Davies | Drug discovery targets: 5-HT 6 receptor[END_REF]22,[START_REF] Mitchell | 5-HT 6 receptors: a novel target for cognitive enhancement[END_REF] (memory and learning) related to neurological diseases such as Alzheimer's. In the last five years it has been shown that selective 5-HT 6 R ligands are able to reduce food intake, [START_REF] Svartengren | The selective serotonin 5-HT 6 receptor antagonist BVT5182 decreases food intake and body weight in both rats and mice. 34 th[END_REF]25 though this effect had been unnoticed in early studies. Thus, the 5-HT 6 R has emerged as a promising target for the treatment of obesity and related metabolic syndrome, [START_REF] Davies | Drug discovery targets: 5-HT 6 receptor[END_REF]22,[START_REF] Heal | Selective 5-HT 6 receptor ligands: progress in the development of a novel pharmacological approach to the treatment of obesity and related metabolic disorders[END_REF] a disease with an increasing global prevalence and high unmet clinical need. Clearly, there is much evidence that the h5-HT 6 R is involved in the pathogenesis of CNS diseases 4,[START_REF] Davies | Drug discovery targets: 5-HT 6 receptor[END_REF]22,[START_REF] Mitchell | 5-HT 6 receptors: a novel target for cognitive enhancement[END_REF][START_REF] Heal | Selective 5-HT 6 receptor ligands: progress in the development of a novel pharmacological approach to the treatment of obesity and related metabolic disorders[END_REF][START_REF] Glennon | Higher-end serotonin receptors: 5-HT(5), 5-HT(6), and 5-HT(7)[END_REF] related to cognitive or eating disorders, so it appears to be an attractive therapeutic target that should be exploited for drug development. Since it is known to be expressed almost exclusively in the CNS [START_REF] Monsma | Cloning and expression of a novel serotonin receptor with high affinity for tricyclic psychotropic drugs[END_REF][START_REF] Ruat | A novel rat serotonin (5-HT 6 ) receptor: molecular cloning, localization and stimulation of cAMP accumulation[END_REF][START_REF] Gerard | Immuno-localization of serotonin 5-HT 6 receptorlike material in the rat central nervous system[END_REF][START_REF] Sleight | The 5hydroxytryptamine(6) receptor: localisation and function[END_REF][START_REF] Hirst | Differences in the central nervous system distribution and pharmacology of the mouse 5-hydroxytryptamine-6 receptor compared with rat and human receptors investigated by radioligand binding, site-directed mutagenesis, and molecular modeling[END_REF] and mainly in the olfactory tubercle, striatum, nucleus accumbens, and hippocampus, it is possible that new therapeutic agents targeting this receptor might have relatively few peripheral side effects. The first 5-HT 6 R antagonists Ro 04-6790, [START_REF] Sleight | Characterization of Ro 04-6790 and Ro 63-0563: potent and selective antagonists at human and rat 5-HT 6 receptors[END_REF] Ro 63-0563 [START_REF] Sleight | Characterization of Ro 04-6790 and Ro 63-0563: potent and selective antagonists at human and rat 5-HT 6 receptors[END_REF] and SB-258585 [START_REF] Bromidge | 5-Chloro-N-(4-methoxy-3-piperazin-1-yl-phenyl)-3-methyl-2benzothiophenesulfonamide (SB-271046): a potent, selective, and orally bioavailable 5-HT 6 receptor antagonist[END_REF] (Chart 1) were identified in the late 1990s by high-throughput screening at Roche and GlaxoSmithKline. Soon afterward, Glennon et al., [START_REF] Glennon | 5-HT 6 serotonin receptor binding of indolealkylamines: A preliminary structure-affinity investigation[END_REF][START_REF] Glennon | 2-Substituted tryptamines: agents with selectivity for 5-HT(6) serotonin receptors[END_REF][START_REF] Tsai | N 1 -(Benzenesulfonyl)tryptamines as novel 5-HT 6 antagonists[END_REF][START_REF] Lee | 5-HT 6 serotonin receptor binding affinities of N 1 -benzenesulfonyl and related tryptamines[END_REF] pioneers in the synthesis of 5-HT 6 R ligands, reported the first selective agonist EMDT [START_REF] Glennon | 2-Substituted tryptamines: agents with selectivity for 5-HT(6) serotonin receptors[END_REF] and antagonist MS-245 [START_REF] Tsai | N 1 -(Benzenesulfonyl)tryptamines as novel 5-HT 6 antagonists[END_REF] (Chart 1), concurrently discovered by Merck Sharp & Dohme. [START_REF] Russell | N-Arylsulfonylindole derivatives as serotonin 5-HT(6) receptor ligands[END_REF] Since then, the number of patent applications and scientific publications concerning the 5-HT 6 R experienced a dizzying growth, mostly in the last few years. At present, several research groups from both academia and the pharmaceutical industry have contributed to the development of structurally different 5-HT 6 R agonists and antagonists 4,[START_REF] Davies | Drug discovery targets: 5-HT 6 receptor[END_REF]22,[START_REF] Heal | Selective 5-HT 6 receptor ligands: progress in the development of a novel pharmacological approach to the treatment of obesity and related metabolic disorders[END_REF][START_REF] Glennon | Higher-end serotonin receptors: 5-HT(5), 5-HT(6), and 5-HT(7)[END_REF] (such as WAY-181187, E-6801 and SB-357134, Chart 1), that have been used as pharmacological tools to study the receptor. To our knowledge [START_REF] Heal | Selective 5-HT 6 receptor ligands: progress in the development of a novel pharmacological approach to the treatment of obesity and related metabolic disorders[END_REF] eight of these compounds have entered clinical trials, and five of them are still under active development in Phase I or II for the treatment of cognitive dysfunction associated with Alzheimer's disease or schizophrenia, as well as for the treatment of obesity.

However, no 5-HT 6 R ligand has been marketed so far, and the search for new compounds acting at this receptor remains an area of high interest for medicinal chemists, aiming at either highly selective agents or compounds combining 5-HT 6 R activity with other serotonin activities thought to be beneficial for the treatment of CNS disorders.

In this context, we are involved in a project aimed at the development of new serotonin 5-HT 6 R ligands using a rational approach. As a starting point, using Catalyst software, [START_REF]Catalyst, version 4.9[END_REF] we generated a ligand-based pharmacophore model for 5-HT 6 R antagonism [START_REF] Lopez-Rodriguez | A three-dimensional pharmacophore model for 5-hydroxytryptamine 6 (5-HT 6 ) receptor antagonists[END_REF] from a training set of 45 structurally diverse compounds previously described. Based on this model, here we have designed new benzimidazole derivatives containing a piperazine ring, either a carbonyl (-CO-) or a methylene (-CH 2 -) group, and an aromatic ring (benzene or naphthalene) (Figure 1). We report the synthesis of new compounds 1-20 and their binding affinity for the 5-HT 6 R (Table 1). The identified ligands have been characterized as antagonists in COS-7 cells transfected with the h5-HT 6 R, representing a new family of 5-HT 6 R antagonists. Site-directed mutagenesis and a β 2 -adrenoceptor-based homology model of the 5-HT 6 R were used to predict the mode of binding of these ligands. This combined experimental and computational study has permitted us to propose the molecular mechanisms by which the new benzimidazole derivatives act as 5-HT 6 R antagonists.

Results and Discussion

Pharmacophore-based Design of New 5-HT 6 R Ligands. Our previously reported 3-D pharmacophore model for 5-HT 6 R antagonists showed four key pharmacophore elements: a positive ionizable atom (PI), an aromatic ring-hydrophobic site (AR), a hydrogen bond acceptor group (HBA), and a hydrophobic site (HYD). [START_REF] Lopez-Rodriguez | A three-dimensional pharmacophore model for 5-hydroxytryptamine 6 (5-HT 6 ) receptor antagonists[END_REF] Based on these structural requirements, we have designed a series of new compounds (Figure 1A) that contain a piperazine ring as PI; the AR feature is a benzimidazole system; the HBA feature is a carbonyl group (X = CO); and a benzene or naphthalene ring as HYD. Hence, the designed derivatives could represent a new structural class of 5-HT 6 R ligands, containing a benzimidazole scaffold in the central core. In the new compounds the piperazine ring is attached to the benzimidazole system at either position 4 or position 5, providing two different orientations of the AR, HBA, and HYD moieties relative to the receptor-anchoring PI group. Figure 1B shows designed compound 3: Y = 4-(piperazin-1-yl), X = CO, Ar = 1naphthyl mapped onto the 3-D model. Additionally, compounds lacking the HBA feature or the HYD region were studied in order to assess the influence of these pharmacophore elements on the binding to the receptor. Therefore, we also synthesized analogues (Figure 1A) where the carbonyl group (X = CO) was replaced by a methylene (X = CH 2 ) with no hydrogen bond accepting capability, or where the Ar moiety was missing (X = H).

Synthesis.

The synthesis of new benzimidazole derivatives 1-20 (Figure 1A, Table 1) was performed by using two different strategies, depending on the nature of X moiety (X = CH 2 or X = CO, see Schemes 1-3). Compounds 10-20 (X = CH 2 ) were obtained by nucleophilic substitution of 3-chloro-or 5-chloro-2-nitroaniline with piperazine or 1-methylpiperazine, followed by catalytic hydrogenation of the resulting piperazinyl-2-nitroanilines 21-24, and subsequent oxidative cyclization of the obtained o-phenylenediamines 25-28 with the appropriate readily available arylacetaldehydes (Scheme 1). On the other hand, those benzimidazole derivatives having a carbonyl group (X = CO, 1-9) were synthesized by butyllithium-mediated acylation at the 2-position of N-protected derivatives followed by removal of 2-(trimethylsilyl)ethoxymethyl (SEM) or tert-butoxycarbonyl (BOC) groups (Schemes 2 and 3). Thus, the reduction of commercially available 3-chloro-2-nitroaniline, Phillips cyclization, and SEM protection of the obtained 4-chlorobenzimidazole (29) [START_REF] Montanari | Ricerche sui benzimidazoli[END_REF][START_REF] Dandegaonker | 4(7)-Halobenzimidazoles[END_REF] afforded a non-separable mixture of N-protected regioisomers 30, in a 1:1 ratio (Scheme 2).

Subsequent Pd(0)-catalyzed coupling [START_REF] Lopez-Rodriguez | Pd(0) Amination of benzimidazoles as an efficient method towards new (benzimidazolyl)piperazines with high affinity for the 5-HT(1A) receptor[END_REF] of 30 with 1-BOC-or 1-methylpiperazine yielded only 4-substituted intermediates 31,32. Finally, derivatives 31,32 were acylated via butyllithium deprotonation, and SEM or BOC groups were subsequently removed to obtain desired compounds 1-5 (X = CO). Similarly, the cyclization of o-phenylenediamines 27,28 [START_REF] Sanchez-Alonso | Piperazine derivatives of benzimidazole as potential anthelmintics. Part 1: Synthesis and activity of methyl-5-(4-substituted piperazin-1-yl)benzimidazole-2-carbamates[END_REF] followed by the protection of 5-substituted benzimidazoles 38,43 and the 2-acylation of Nprotected derivatives 39,44 afforded final compounds 6-9 (X = CO), after deprotection of intermediates 40-42,45 (Scheme 3).

Binding Affinities and Structure-Affinity Relationships. Target compounds 1-20 were assessed for in vitro affinity at the h5-HT 6 R by radioligand binding assays, using [ 3 H]LSD in transfected HEK-293 cells (see Experimental Section for details). The competitive inhibition assays were first performed at a fixed dose of 10 -6 M, and the complete dose-response curve, at six different concentrations of the ligand, was determined for those compounds that presented a displacement of the radioligand over 50%. The inhibition constant K i was calculated from the IC 50 value using the Cheng-Prusoff equation, [START_REF] Cheng | Relationship between the inhibition constant (K i ) and the concentration of inhibitor which causes 50% inhibition (I 50 ) of an enzymatic reaction[END_REF] and the values in Table 1 are the mean of two to four experiments. Compounds binding the 5-HT 6 R were tested for selectivity over the 5-HT 7 receptor, using [ 3 H]LSD in transfected HEK-293 cells (see Experimental Section for details), and found to be inactive (K i > 500 nM) at the 5-HT 7 R. The following structure-affinity relationships can be drawn from the 5-HT 6 R binding affinity data presented in Table 1. Most of the 4-substituted benzimidazole derivatives show high affinity (K i = 13-66.8 nM) whereas 5-substituted analogues are inactive (K i > 1000 nM). These experimental data show that 4-substituted benzimidazoles display a favorable orientation of the AR, HBA, and HYD moieties relative to the receptor-anchoring PI group. In 4-substituted derivatives containing a carbonyl group as HBA (1-5: X = CO) only compounds 3 and 4, both containing 1-naphthyl ring as HYD moiety, show 5-HT 6 R affinity [K i (3) = 13 nM; K i (4) = 34 nM]. In contrast, phenyl [K i (1) > 1000 nM vs K i (3) = 13 nM, K i (2) > 1000 nM vs K i (4) = 34 nM)] is insufficient for high-affinity binding, and 2-naphthyl [K i (5) > 1000 nM vs K i (4) = 34 nM] aromatic system is not tolerated at the HYD region. However, when the carbonyl group is replaced by a methylene moiety all 4-substituted compounds (10-14: X = CH 2 ) display affinity for the 5-HT 6 R (K i = 16-66.8 nM). These results show that the hydrogen bonding capability proposed for HBA pharmacophore element is not essential for the binding of this new family of ligands to the 5-HT 6 R. Also, comparison of the affinity data obtained for compounds 4 (X = CO, K i = 34 nM) vs 13 (X = CH 2 , K i = 36.22 nM) and 3 (X = CO, K i = 13 nM) vs 12 (X = CH 2 , K i = 16 nM) suggest that the aromatic ring (Ar) is accommodated at different parts of the receptor for compounds with X = CO and X = CH 2 (see Computer-based Analysis of the Structure-affinity Relationships). The inactivity of 4-(4-methylpiperazin-1-yl)-1H-benzimidazole (K i > 1000 nM, data not shown), lacking the aromatic ring Ar, supports the hypothesis that the presence of HYD region is important for the receptor binding.

Functional Activity of 5-HT 6 R Ligands. Compounds displaying affinity for the 5-HT 6 R were evaluated for their functional profile, by determining the adenylate cyclase activity in COS-7 cells expressing the human receptor. Cells were treated with the ligand for 10 min at 37 ºC, in the absence (agonist effect) or the presence (antagonist effect) of serotonin (100 μM), and the cAMP content was then quantified by homogeneous time-resolved fluorescence (HTRF®) (see Experimental Section for details). Adenylate cyclase activity is expressed as the percentage of the maximal effect obtained with serotonin alone. All assayed compounds show a dose-dependent effect in the presence of 5-HT, and no effect in the absence of 5-HT, indicating their antagonist profile. The K i values in Table 2 are the mean of three experiments, using eight different concentrations of the tested compound. These data (K i = 5-31 nM) show that 4-substituted benzimidazoles represent a new family of potent 5-HT 6 R antagonists.

Defining the Mode of Binding of Ligand 4 by Site-directed Mutagenesis and

Computational Modeling. Figure 2 shows the sequence alignment of transmembrane helices (TMs) 3, 5, 6, and 7 and extracellular loop (EL) 2 for β 1 -and β 2 -adrenergic receptors, selected serotonin receptors, and the 5-HT 6 R. These domains form the ligand binding site in the crystal structures of β 1 -46 and β 2 - [START_REF] Cherezov | Highresolution crystal structure of an engineered human β 2 -adrenergic G protein-coupled receptor[END_REF][START_REF] Rosenbaum | GPCR engineering yields high-resolution structural insights into β 2 -adrenergic receptor function[END_REF] adrenergic receptors, as they were previously identified by site-directed mutagenesis. [START_REF] Shi | The binding site of aminergic G protein-coupled receptors: the transmembrane segments and second extracellular loop[END_REF] In order to characterize the amino acid residues of the 5-HT 6 R involved in the binding of ligand 4, mutagenesis assays were performed with Ala substitution at C3.36, S5.43, W6.48, F6.52, and N6.55 sites. Receptor expression levels at the plasma membrane relative to the expression of wild-type (WT) receptor were determined by an ELISA assay. These mutant receptors are expressed at the 80-110% range with the exception of W6.48A (30%). Figure 3A In order to understand these observed effects at the molecular level, we constructed a computer 3-D model of the complex between compound 4 and a β 2 -adrenoceptor-based model of the 5-HT 6 R (see Experimental Section). In this model (Figure 3B) D3.32 anchors the protonated piperazine ring; the benzimidazole ring expands toward TM 6, parallel to EL 2, to hydrogen bond O δ of the key N6.55; the coplanar carbonyl group also hydrogen bonds the N δ -H moiety of N6.55; and the aromatic naphthalene ring enters deeply into a small cavity between TMs 3 and 6, to form edge-to-face interactions with the key W6.48 and F6.52 side chains in TM 6. This computational model explains the experimentally determined importance of W6.48, F6.52 and N6.55 of the 5-HT 6 R in the binding of ligand 4 (Figure 3A).

The finding that the S5.43A mutation enhances the activity of 4 (Figure 3A) is attributed to the fact that S5.43 hydrogen bonds N6.55 (Figure 3B); thus, the mutation at this site increases the flexibility of N6.55 favoring its interaction with 4. Other significant interactions between compound 4 and the 5-HT 6 R, as revealed by the computational model, are: the interaction between the highly polar methyl group attached to the protonated piperazine ring and W3.28; the interaction of the aromatic naphthalene ring with V3.33 in TM 3; and the interaction between the aromatic benzimidazole ring and F6.51 in TM 6, F7.35 in TM 7, and L182 in EL 2 (Figures 3B and3C).

Computer-based Analysis of the Structure-affinity Relationships. In an attempt to get a better understanding of the above described structure-affinity relationships, we have performed additional computational simulations of the complexes between 4-substituted benzimidazole derivatives and the 5-HT 6 R. Regarding HBA, replacement of the sp 2 carbonyl group (X = CO) by the sp 3 methylene unit (X = CH 2 ) allows the benzimidazole ring and the aromatic systems (Ar = phenyl, 1-naphthyl, 2-naphthyl) in the HYD region to form an angle of approximately 90°. Thus, in compounds with X = CH 2 the HYD moiety is located between TMs 3 and 5, to interact with the hydrophobic side chains of V3.33 and A5.42 (Figure 4A). In contrast, the Ar aromatic moiety in compounds containing X = CO is located between TMs 3 and 6, interacting with V3.33, W6.48 and F6.52 side chains (Figure 4B). The different location of the HYD feature in the receptor explains the observed different structure-affinity relationships at this pharmacophore element in both types of compounds (X = CH 2 or X = CO). Clearly, all tested aromatic rings phenyl, 1-naphthyl, and 2-naphthyl are permitted in the cavity between TMs 3 and 5 (X = CH 2 , Figure 4A), whereas phenyl and 1-naphthyl are permitted in the cavity between TMs 3 and 6 (X = CO, Figure 4B). In the cavity between TMs 3 and 6 the smaller phenyl ring, relative to 1-naphthyl, does not optimally interact with F6.52 (Figure 4B), resulting in the corresponding loss of affinity [K i (1) > 1000 nM, K i (2) > 1000 nM]. The presence of the 2-naphthyl system is not permitted because it clashes with TM 3 (see orange ring in Figure 4B), leading to an inactive analogue [K i (5) > 1000 nM].

Comparison of the binding of SB-258585 with the binding of benzimidazole derivatives 4 and 13 to the 5-HT 6 R. SB-258585 [START_REF] Bromidge | 5-Chloro-N-(4-methoxy-3-piperazin-1-yl-phenyl)-3-methyl-2benzothiophenesulfonamide (SB-271046): a potent, selective, and orally bioavailable 5-HT 6 receptor antagonist[END_REF] (Chart 1) is a commercially available potent 5-HT 6 R antagonist that contains the common piperazine ring as the PI pharmacophore element, a methoxyphenyl system as AR, a sulfonamide group as HBA, and a iodophenyl ring as HYD. [START_REF] Lopez-Rodriguez | A three-dimensional pharmacophore model for 5-hydroxytryptamine 6 (5-HT 6 ) receptor antagonists[END_REF] In order to compare the binding mode of this 5-HT 6 R antagonist with the binding mode of benzimidazole derivatives we evaluated the antagonist activity of SB-258585 in the C3.36A, S5.43A, W6.48A, F6.52A, and N6.55A mutant receptors (Figure 5A). Clearly, with the only exception of W6.48A, all these mutations decrease the antagonist activity of SB-258585: 72-fold, relative to WT, in the C3.36A mutant receptor; 8-fold in S5.43A; 152-fold in F6.52A; and 36-fold in N6.55A (Figure 5A). These mutational data are compatible with a mode of binding of SB-258585 to the 5-HT 6 R as depicted in Figure 5B: D3.32 anchors the protonated piperazine ring, the methoxyphenyl system interacts with F6.52, the methoxy group hydrogen bonds C3.36, the sulfonamide group hydrogen bonds both S5.43 and N6.55, and the substituted benzene interacts with the hydrophobic side chains of V3.33 and A5.42.

Previously reported rhodopsin-based models have suggested that arylsulfonyltryptamines bind between TMs 1, 6 and 7; 50 the sulfonyl oxygens of MS-245 (Chart 1) bind S3.37 and T5.46; 51 or N6.55 in SB-357134 (Chart 1) and related sulfonamide derivatives. [START_REF] Hirst | Differences in the central nervous system distribution and pharmacology of the mouse 5-hydroxytryptamine-6 receptor compared with rat and human receptors investigated by radioligand binding, site-directed mutagenesis, and molecular modeling[END_REF][START_REF] Lopez-Rodriguez | A three-dimensional pharmacophore model for 5-hydroxytryptamine 6 (5-HT 6 ) receptor antagonists[END_REF] Figure 5C shows SB-258585 (white) and benzimidazole derivatives 4 (green) and 13 (orange) bound to the 5-HT 6 R for comparison purposes. With respect to the HBA feature, the large sulfonamide moiety of SB-258585 is capable to hydrogen bond both S5.43 and N6.55 while the carbonyl group of compound 4 hydrogen bonds exclusively N6.55, which also binds the N-H of the benzimidazole ring (AR feature) of compounds 4 and 13. Clearly, the piperazine-benzimidazole rings of 4 and 13 are placed near the extracellular side, parallel to EL 2, while the piperazine-methoxyphenyl rings of SB-258585 are located deeper in the TM bundle. Notably, the methoxyphenyl system of SB-258585 (AR feature) is placed at similar position than the naphthalene ring (HYD feature) of compound 4. On the other hand, the position of the HYD feature of SB-258585 (iodophenyl ring) is similar to the HYD feature of compound 13 (naphthalene ring). Thus, the different location of the AR features in the binding site suggests that compounds 4 and 13 elicit their antagonist function by a different mechanism than ligand SB-258585 (see below).

Molecular Mechanisms of Receptor Inactivation.

The understanding of the molecular mechanisms by which these benzimidazole derivatives act as 5-HT 6 R antagonists, and GPCR function in general, is a key subject in medicinal chemistry. The recent knowledge of the molecular steps of agonist-induced GPCR activation (see Smit et al. [START_REF] Smit | Pharmacogenomic and structural analysis of constitutive G protein-coupled receptor activity[END_REF] and Nygaard et al. [START_REF] Nygaard | Ligand binding and micro-switches in 7TM receptor structures[END_REF] for recent reviews) facilitates this task. It has been proposed, from engineered GPCRs with metal ion binding sites, that TM 6 performs an inward movement of the extracellular part toward TM 3. [START_REF] Schwartz | Molecular mechanism of 7TM receptor activation-a global toggle switch model[END_REF] Thus, in the new family of 5-HT 6 R antagonists the role of the benzimidazole ring may be to maintain, by forming a hydrogen bond interaction with N6.55, TM 6 away from the center of the bundle (Figure 6A). In addition, L182 in EL 2 forms favorable C-H•••π interactions [START_REF] Ozawa | The importance of CH/π hydrogen bonds in rational drug design: An ab initio fragment molecular orbital study to leukocyte-specific protein tyrosine (LCK) kinase[END_REF] with the benzimidazole ring, which may serve to immobilize both EL 2 and TM 6 in the inactive conformation (Figure 6A). Similar interactions are observed between the antagonist cyanopindolol and the partial inverse agonist carazolol with the homologous F201 [START_REF] Warne | Structure of a β 1 -adrenergic Gprotein-coupled receptor[END_REF] and F193 [START_REF] Rosenbaum | GPCR engineering yields high-resolution structural insights into β 2 -adrenergic receptor function[END_REF] of the β 1 -and β 2 -adrenergic receptors, respectively. Fluorescence spectroscopy in the β 2 -adrenergic receptor provides evidence that agonist binding induces or stabilizes relocation of the extracellular side of TM 5, to facilitate binding of the catechol hydroxyls of the adrenergic agonists. [START_REF] Swaminath | Sequential binding of agonists to the β 2 adrenoceptor. Kinetic evidence for intermediate conformational states[END_REF] The Ar system in CH 2 -containing compounds is located between TMs 3 and 5, interacting with V3.33 and A5.42, and probably blocking these helices in the inactive conformation (Figure 6B). In addition to these activation pathways, agonist binding might also trigger the rotamer toggle switch of W6.48 from pointing toward TM 7 to pointing toward the binding site crevice, [START_REF] Shi | The binding site of aminergic G protein-coupled receptors: the transmembrane segments and second extracellular loop[END_REF][START_REF] Crocker | Location of Trp265 in metarhodopsin II: implications for the activation mechanism of the visual receptor rhodopsin[END_REF][START_REF] Ruprecht | Electron crystallography reveals the structure of metarhodopsin I[END_REF] through the formation of specific hydrogen bonds. [START_REF] Lopez-Rodriguez | Synthesis and structure-activity relationships of a new model of arylpiperazines. 8. Computational simulation of ligand-receptor interaction of 5-HT(1A)R agonists with selectivity over α 1adrenoceptors[END_REF] The 1-naphthyl ring in CO-containing compounds occupies a small cavity between TMs 3 and 6 (Figure 6C), and might act by blocking the proposed conformational transition of W6.48 and the subsequent steps in the activation process. [START_REF] Smit | Pharmacogenomic and structural analysis of constitutive G protein-coupled receptor activity[END_REF] We have also shown that the conformational transition of W6.48 is accompanied, in a concerted manner, by the conformational transition of the side chain at position 3.36 from pointing toward TM 6 to pointing toward TM 7. [START_REF] Pellisier | Conformational toggle switches implicated in basal constitutive and agonist-induced activated states of 5-HT 4 receptors[END_REF] Thus, the methoxy group of SB-258585 might block receptor activation by hydrogen bonding C3.36 in the inactive conformation (Figure 6D). Accordingly, the C3.36A mutation has a significant effect in the antagonist activity of SB-258585 (the effect for compound 4 is less significant), while the W6.48A mutation impairs the antagonist activity of compound 4 (the effect for SB-258585 is minor).

Conclusions

Based on our previously described pharmacophore model for 5-HT 6 R antagonists, [START_REF] Lopez-Rodriguez | A three-dimensional pharmacophore model for 5-hydroxytryptamine 6 (5-HT 6 ) receptor antagonists[END_REF] we have identified benzimidazole derivatives as a new structural class of serotonin 5-HT 6 R ligands. Computational modeling and site-directed mutagenesis have allowed us to characterize the amino acid residues of 5-HT 6 R involved in ligand binding (Figure 3). The distinctive positively charged piperazine ring anchors the ligand to D3.32 in TM 3, whereas the benzimidazole ring is situated parallel to EL 2, to hydrogen bond N6.55 in TM 6 (Figures 3 and4). An important feature of these molecules is the linker between the benzimidazole ring and the terminal Ar system (phenyl, 1-naphthyl, 2-naphthyl). The sp 2 carbonyl group (-CO-) enforces both aromatic domains to be coplanar, whereas the sp 3 methylene unit (-CH 2 -) allows the rings to form an angle of approximately 90°. Thus, in CH 2 -containing compounds the Ar system is located between TMs 3 and 5, to interact with V3.33 and A5.42 (Figure 4A), whereas in CO-containing compounds the Ar aromatic moiety is located between TMs 3 and 6, interacting with V3.33, W6.48 and F6.52 (Figure 4B). Both types of compounds display high binding affinity for the 5-HT 6 R as shown for ligands 12 [K i (12) = 16 nM] and 13 [K i (13) = 36 nM], having CH 2 as a linker; and ligands 3 [K i (3) = 13 nM] and 4 [K i (4) = 34 nM] containing CO (Table 1). The new compounds were pharmacologically characterized as potent 5-HT 6 R antagonists (K i = 5-31 nM).

This combined experimental and computational study has permitted to understand how ligand binding blocks receptor activation (Figure 6), which may contribute to the rational design of new compounds acting at the serotonin 5-HT 6 R. As some of the residues involved in ligand binding and receptor inactivation are strongly conserved, these findings can likely be extended to other members of the rhodopsin-like family of GPCRs.

Experimental Section

Chemistry. Melting points (uncorrected) were determined on a Stuart Scientific electrothermal apparatus. Infrared (IR) spectra were measured on a Shimadzu-8300 or Bruker Tensor 27 instrument equipped with a Specac ATR accessory of 5200-650 cm -1 transmission range; frequencies (ν) are expressed in cm -1 . Nuclear Magnetic Resonance (NMR) spectra were recorded on a Bruker Avance 500 ( 1 H, 500 MHz; [START_REF] Kohen | Cloning, characterization, and chromosomal localization of a human 5-HT 6 serotonin receptor[END_REF] C, 125 MHz), Bruker Avance 300-AM ( 1 H, 300 MHz; [START_REF] Kohen | Cloning, characterization, and chromosomal localization of a human 5-HT 6 serotonin receptor[END_REF] C, 75 MHz) or Bruker 200-AC spectrometer ( 1 H, 200 MHz; [START_REF] Kohen | Cloning, characterization, and chromosomal localization of a human 5-HT 6 serotonin receptor[END_REF] C, 50 MHz) at the UCM's NMR facilities. Chemical shifts (δ) are expressed in parts per million relative to internal tetramethylsilane; coupling constants (J) are in hertz (Hz). The following abbreviations are used to describe peak patterns when appropriate: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad), app (apparent). Mass spectrometry (MS) was carried out on a Bruker LC-Esquire in electrospray mode (ESI) or a HP 5989 A in electron impact mode (EI, 70 eV). Elemental analyses (C, H, N) were obtained on a LECO CHNS-932 apparatus at the UCM's analysis services and were within 0.5% of the theoretical values, confirming a purity of at least 95% for all tested compounds. Analytical thin-layer chromatography (TLC) was run on Merck silica gel plates (Kieselgel 60 F-254) with detection by UV light (254 nm), ninhydrin solution, or 10% phosphomolybdic acid solution in ethanol. Flash chromatography was performed on glass column using silica gel type 60 (Merck, particle 230-400 mesh) or on a Supelco VersaFlash station using silica gel cartridges (Supelco, particle size 20-45 μm). Unless stated otherwise, starting materials, reagents and solvents were purchased as high-grade commercial products from Sigma-Aldrich, Lancaster, Scharlab or Panreac, and were used without further purification. THF was distilled from sodium benzophenone ketyl and used immediately. Dichloromethane was distilled from calcium hydride.

The following compounds were synthesized according to described procedures: 23, [START_REF] Abuzar | Synthesis of 2-carbalkoxyamino-5(6)-(1-substituted piperazin-4yl/piperazin-4-ylcarbonyl)benzimidazoles and related compounds as potential anthelmintics[END_REF] 24, [START_REF] Sanchez-Alonso | Piperazine derivatives of benzimidazole as potential anthelmintics. Part 1: Synthesis and activity of methyl-5-(4-substituted piperazin-1-yl)benzimidazole-2-carbamates[END_REF] 28, [START_REF] Kelly | DNA Binding compounds. V Synthesis and characterization of boron-containing bibenzimidazoles related to the DNA minor groove binder, Hoechst 332585[END_REF] and 29, [START_REF] Dandegaonker | 4(7)-Halobenzimidazoles[END_REF] and their spectroscopic data are in agreement with those previously described. Spectroscopic data of all described compounds were consistent with the proposed structures. For series 1-8, 10-20 we include the data of compounds 5, 6, 8, 11, 12, 15, and 19.

For intermediates 25-28, 33-37, 40-42 the data of compounds 26, 35 and 42 are described.

General Procedure for the Synthesis of 2-Nitro-ω-piperazin-1-ylanilines (21-24).

To a solution of 3-or 5-chloro-2-nitroaniline (5.12 g, 29.5 mmol) in anhydrous DMF (70 mL), piperazine or 1-methylpiperazine (78 mmol), Cs 2 CO 3 or K 2 CO 3 (78 mmol) were added, respectively. The reaction was heated at 120 ºC for 24 h. Upon cooling, the mixture was poured into ice-cold water and the solid was filtered, washed with cold water and dried under vacuum. The solid was purified by column chromatography using the appropriate eluent, to afford pure 21-24.

2-Nitro-3-piperazin-1-ylaniline (21

2-Nitro-5-piperazin-1-ylaniline (23).

Obtained from 5-chloro-2-nitroaniline and piperazine, in quantitative yield. Chromatography: CH 2 Cl 2 /EtOH, 9:1; mp 164-165 ºC (lit. [START_REF] Abuzar | Synthesis of 2-carbalkoxyamino-5(6)-(1-substituted piperazin-4yl/piperazin-4-ylcarbonyl)benzimidazoles and related compounds as potential anthelmintics[END_REF] 170 ºC).

5-(4-Methylpiperazin-1-yl)-2-nitroaniline (24).

Obtained from 5-chloro-2-nitroaniline and 1-methylpiperazine, in 89% yield. Chromatography: CH 2 Cl 2 /EtOH, 98:2; mp 153-154 ºC (lit. [START_REF] Sanchez-Alonso | Piperazine derivatives of benzimidazole as potential anthelmintics. Part 1: Synthesis and activity of methyl-5-(4-substituted piperazin-1-yl)benzimidazole-2-carbamates[END_REF] 156-157 ºC).

General Procedure for the Synthesis of ω-Piperazin-1-ylbenzene-1,2-diamines (25-28).

To a suspension of 21-24 (1.3 mmol) in absolute MeOH (10 mL), 10% Pd(C) (25 mg) was added and the reaction was hydrogenated at room temperature for 24 h, with an initial hydrogen pressure of 50 p.s.i. The mixture was filtered over celite and the solvent was evaporated to dryness to give 25-28, which were used in the next step without further purification.

3-Piperazin-1-ylbenzene-1,2-diamine (25).

Obtained from 21 in quantitative yield.

3-(4-Methylpiperazin-1-yl)benzene-1,2-diamine (26).

Obtained from 22 in quantitative yield. IR (CHCl 3 ): 3325, 1600, 1478, 1455. 1 H NMR (CDCl 3 ) δ 2.31 (s, 3H), 2.60 (m, 4H), 2.94 (br t, J = 4.7, 4H), 6.54 (dd, J = 6.9, 1.4, 1H), 6.63-6.72 (m, 2H). [START_REF] Kohen | Cloning, characterization, and chromosomal localization of a human 5-HT 6 serotonin receptor[END_REF]

4-Piperazin-1-ylbenzene-1,2-diamine (27).

Obtained from 23 in quantitative yield.

4-(4-Methylpiperazin-1-yl)benzene-1,2-diamine (28).

Obtained from 24 in 89% yield; mp 101-103 ºC (lit. [START_REF] Kelly | DNA Binding compounds. V Synthesis and characterization of boron-containing bibenzimidazoles related to the DNA minor groove binder, Hoechst 332585[END_REF] 101-102 ºC).

General Procedure for the Synthesis of Compounds 29, 38, 43. To a suspension of 3chlorobenzene-1,2-diamine, [START_REF] Montanari | Ricerche sui benzimidazoli[END_REF] 27 or 28 (9.8 mmol) in water (14 mL), formic acid (1.2 mL, 29 mmol) was added and the mixture was heated at 100 ºC for 3 h. After the complete disappearance of the starting material (tlc), the reaction was poured on ice and 1 M KOH was added until pH 9-10. The resulting solid was filtrated, dried and recrystallized; otherwise, the aqueous solution was extracted with CH 2 Cl 2 (3 x 50 mL), the organic layers were dried (Na 2 SO 4 ), and evaporated to dryness. The residue was purified by column chromatography using the appropriate eluent, to afford pure benzimidazoles 29, 38, 43.

4-Chloro-1H-benzimidazole (29).

Obtained from 3-chlorobenzene-1,2-diamine in quantitative yield; mp 167-169 ºC (lit. [START_REF] Dandegaonker | 4(7)-Halobenzimidazoles[END_REF] 170-171 ºC) (EtOAc).

5-(4-Methylpiperazin-1-yl)-1H-benzimidazole (38)

. Obtained from 28 in 60% yield.

Chromatography: CHCl 3 /MeOH, 95:5; mp 81-83 ºC (lit. [START_REF] Lopez-Rodriguez | Pd(0) Amination of benzimidazoles as an efficient method towards new (benzimidazolyl)piperazines with high affinity for the 5-HT(1A) receptor[END_REF] 82-84 ºC). Spectroscopic data are in agreement with those previously described.

5-Piperazin-1-yl-1H-benzimidazole (43).

Obtained from 27 in 87% yield.

Chromatography CHCl 3 /MeOH/NH 3 6:4:0.1; mp 123-125 ºC (lit. [START_REF] Lopez-Rodriguez | Pd(0) Amination of benzimidazoles as an efficient method towards new (benzimidazolyl)piperazines with high affinity for the 5-HT(1A) receptor[END_REF] 126-128 ºC). Spectroscopic data are in agreement with those previously described.

General Procedure for the Synthesis of Compounds 30 and 39. To a suspension of K 2 CO 3 (2.86 g, 20.7 mmol) or NaH (444 mg, 11.1 mmol, 60% mineral oil) in anhydrous DMF (10 mL), 29 or 38 (6.9 mmol) was added portionwise, respectively, under an argon atmosphere. The mixture was stirred at room temperature for 1 h, then 2-(trimethylsilyl)ethoxymethyl chloride (SEMCl) (2.1 mL, 11.1 mmol) was added dropwise, and the reaction was stirred for 2 h. The reaction was quenched with water and extracted with CH 2 Cl 2 (30) or EtOAc (39). The organic layers were dried (Na 2 SO 4 ) and evaporated under vacuum, and the residue was purified by column chromatography, using the appropriate eluent, to provide 30 or 39.

4-and 7-Chloro-1-[(2-(trimethylsilyl)ethoxy)methyl]-1H-benzimidazoles (30

6-(4-Methylpiperazin-1-yl)-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-benzimidazole (39).

Obtained from 38 in 44% yield, after the separation from its 5-substituted regioisomer* (38% yield) by column chromatography (CH 2 Cl 2 /EtOH, 9:1). IR (CHCl 3 ) 1625, 1581, 1502, 1490, 1454. 1 H NMR (CDCl 3 ) δ -0.03 (s, 9H), 0.91 (app t, J = 8.2, 2H), 2.39 (s, 3H), 2.64 (br t, J = 4.8, 4H), 3.26 (br t, J = 4.8, 4H), 3.51 (app t, J = 8.2, 2H), 5.49 (s, 2H), 7.00 (d, J = 2.1, 1H), 7.05 (dd, J = 9.0, 2.3, 1H), 7.69 (d, J = 8.8, 1H), 7.84 (s, 1H). [START_REF] Kohen | Cloning, characterization, and chromosomal localization of a human 5-HT 6 serotonin receptor[END_REF] C NMR (CDCl 3 ) δ -1.5, 17.6, 46.0, 50.5, 55.1, 66.2, 74.0, 96.9, 114.5, 120.4, 134.9, 137.9, 141.9, 149.5. MS (EI) m/z (%) 346 (M, 100), 331 (21), 229 (21), 199 (9), 73 (46), 43 (40).

*Spectroscopic data of 5-(4-Methylpiperazin-1-yl)-1-{[2-(trimethylsilyl)ethoxy]methyl}-

1H-benzimidazole: IR (CHCl 3 ) 1624, 1586, 1491, 1454. 1 H NMR (CDCl 3 ) δ -0.05 (s, 9H), 0.90 (app t, J = 8.2, 2H), 2.39 (s, 3H), 2.65 (br t, J = 4.9, 4H), 3.24 (br t, J = 4.9, 4H), 3.50 (t, J = 8.2, 2H), 5.49 (s, 2H), 7.10 (dd, J = 8.8, 2.4, 1H), 7.34 (d, J = 2.1, 1H), 7.43 (d, J = 9.0, 1H), 7.90 (s, 1H). [START_REF] Kohen | Cloning, characterization, and chromosomal localization of a human 5-HT 6 serotonin receptor[END_REF]

Synthesis of Tert-butyl 5-and 6-[4-tert-butoxycarbonyl)piperazin-1-yl]-1Hbenzimidazole-1-carboxylates (44

, 290 (100), 246 (50), 216 (23), 57 (45), 41 (37).

General

General Procedure for the Synthesis of Benzimidazole Derivatives 10-20 (X = CH 2 ).

To a solution of previously synthesized 25-28 (1.5 mmol) and the appropriate aldehyde (2.3 mmol) in freshly distilled CH 2 Cl 2 (8 mL), ytterbium triflate (93 mg, 0.2 mmol) was added and the reaction was stirred at room temperature overnight. The mixture was filtered over celite, the solvent was evaporated under reduced pressure, and the residue was purified by column chromatography using the appropriate eluent, to afford pure 10-20.

2-Benzyl-4-piperazin-1-yl-1H-benzimidazole (10).

Obtained from 25 and phenylacetaldehyde, in 45% yield. Chromatography: CH 2 Cl 2 /EtOH, 9:1; mp 126-128 ºC. Cell Surface ELISA. COS-7 cells were transfected with HA-tagged-5-HT 6 R, WT or mutants cDNAs. Cells were grown on 96-well plates in DMEM medium with 10% dFCS and fixed as described previously. [START_REF] Barthet | Uncoupling and endocytosis of 5-hydroxytryptamine 4 receptors. Distinct molecular events with different GRK2 requirements[END_REF] Cells were then incubated with anti-HA antibody at 0.6 μg/mL for 60 min in the same buffer and incubated with anti-rabbit/HRP conjugate [START_REF] Pellisier | Conformational toggle switches implicated in basal constitutive and agonist-induced activated states of 5-HT 4 receptors[END_REF] The color code of the helices is: TM 3 (red), 5 (green), and 6 (blue).

2-Benzyl-4-(4-methylpiperazin-1-yl)-1H-benzimidazole (11

  shows the antagonist activity of 4 relative to 5-HT-induced activation in the five mutant receptors and in the WT 5-HT 6 R. Notably, the substitution of W6.48, F6.52, or N6.55 by Ala fully impedes compound 4 to block 5-HT-induced activation. The C3.36A mutation decreases the antagonist activity of the ligand by 7-fold [K i (WT) = 5 nM vs K i (C3.36A) = 34 nM]. Surprisingly, the S5.43A mutation substantially increases the antagonist activity of 4 by 98-fold [K i (WT) = 5 nM vs K i (S5.43A) = 0.051 nM].

[ 4 -( 4 -

 44 Procedure for the Synthesis of Compounds 33-37, 40-42, and 45. To a solution of SEM-protected or BOC-protected benzimidazole 31, 32, 39 or 44 (1.3 mmol) in anhydrous THF (5.2 mL), BuLi (1.8 mmol, 1.6 M in hexane) was added under an argon atmosphere at -78 ºC. The mixture was stirred at this temperature for 1 h, then corresponding alkyl benzoate or naphthoate (1.4 mmol) was added slowly and the reaction mixture was allowed to reach room temperature and stirred overnight. The reaction was quenched with a saturated aqueous solution of NH 4 Cl, then extracted with CH 2 Cl 2 , and the organic layers were washed with water and brine, dried (Na 2 SO 4 ), and evaporated to dryness. The residue was purified by column chromatography using the appropriate eluent, to afford pure 33-37, 40-42, 45. Obtained from 31 and ethyl benzoate, in 21% yield. Chromatography: hexane/EtOAc, 8:2. Methylpiperazin-1-yl)-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-benzimidazol-2yl](phenyl)methanone (34

  Nonspecific binding was determined by radioligand binding in the presence of a saturating concentration of 500 μM serotonin, and represented less than 10% of total binding.5-HT 7Receptor. Cell paste (6.8 mg/mL) was homogenized in 200 volumes of assay buffer (50 mM Tris-HCl, 10 mM MgSO 4 , 0.5 mM EDTA, pH 7.4 at 25 ºC). Fractions of 500 μL of the membranes suspension were incubated at 27 ºC for 120 min with 3 nM [ 3 H]LSD, in the presence or absence of the competing drug, in a final volume of 540 μL of assay buffer.Nonspecific binding was determined by radioligand binding in the presence of a saturating concentration of 25 μM clozapine, and represented less than 15% of total binding. For all binding assays, competing drug, nonspecific, total, and radioligand bindings were defined in triplicate. Incubation was terminated by rapid vacuum filtration through Wallac Filtermat A filters, presoaked in poly(ethylenimine) (0.5% for the 5-HT 6 R and 0.3% for the 5-HT 7 R), using a FilterMate Unifilter 96-Harvester. The filters were then washed 5-9 times with 500 μL of ice-cold 50 mM Tris-HCl buffer (pH 7.4 at 25 ºC), and heated at 80 ºC. The radioactivity bound to the filters was measured by scintillation spectrometry, using a Microbeta TopCount instrument. The data were analyzed by an iterative curve-fitting procedure using GraphPad Prism, which provided IC 50 , K i , and r 2 values for test compounds, K i values being calculated from the Cheng-Prusoff equation.[START_REF] Cheng | Relationship between the inhibition constant (K i ) and the concentration of inhibitor which causes 50% inhibition (I 50 ) of an enzymatic reaction[END_REF] Plasmid Construct. HA-tagged-5-HT 66 receptor cDNAs in pRK5 were generated by fusing the sequence of the HA epitope (YPYDVPDYA) to a cleavable signal peptide (MVLLLILSVLLLKEDVRGSAQS) derived from the metabotropic glutamate receptor 5 (mGlu5R). This sequence was inserted into the pRK5 vector using Xba I and BsrG I restriction sites. Then, full-length mouse 5-HT 6 R cDNA was subcloned in frame using BsrG I and Hind III. HA-5-HT 6 -C3.36A, HA-5-HT 6 -S5.43A, HA-5-HT 6 -W6.48A, HA-5-HT 6 -F6.52A, HA-5-HT 6 -N6.55A receptor mutants were generated from HA-5-HT 6 R, cloned in pRK5, with the Quick Change Site-Directed Mutagenesis Kit (Stratagene, Amsterdam, Netherlands).

Figure 1 .

 1 Figure 1. A previously reported 3-D pharmacophore model for 5-HT 6 R antagonists showed

Figure 2 .

 2 Figure 2. Sequence alignment of TMs 3, 5-7 and EL2 of β 1 -and β 2 -adrenergic receptors, and

Figure 3 .

 3 Figure 3. (A) The antagonist activity of compound 4 was tested relative to 5-HT-induced

Figure 4 .

 4 Figure 4. Computational models of the complexes between ligand 13 (A) and 4 (B) and the 5-

Figure 5 .

 5 Figure 5. (A) The antagonist activity of SB-258585 was tested relative to 5-HT-induced

HT 6 R

 6 . In this model D3.32 anchors the protonated piperazine ring, the methoxyphenyl system interacts with F6.52, the methoxy group hydrogen bonds C3.36, the sulfonamide moiety hydrogen bonds both S5.43 and N6.55, and the iodophenyl ring interacts with the hydrophobic side chains of V3.33 and A5.42. (C) Comparison of the binding modes of SB-258585 (white) and benzimidazole derivatives 4 (green) and 13 (orange) to the 5-HT 6 R. The color code of the helices is: TM 3 (red), 5 (green), 6 (blue), and 7 (brown).

Figure 6 .

 6 Figure 6. Molecular mechanisms of receptor inactivation. (A) The benzimidazole ring (in

a

  Scheme 1 a

  ). Obtained from 3-chloro-2-nitroaniline and piperazine, in 40% yield. Chromatography: CH 2 Cl 2 /EtOH, 8:2; mp 212-213 ºC. IR (KBr): 3308, 1514, 1443. 1 H NMR (CDCl 3 , δ): 1.90 (br s, 1H), 3.01 (m, 8H), 4.95 (br s, 2H), 6.57 (d, J = 8.1, 2H), 7.14 (t, J = 8.1, 1H). 13 C NMR (CDCl 3 , δ): 46.2, 53.0, 109.3, 111.2, 122.0, 132.9, 142.9, 148.0. MS (ESI): 223.0 (M+H).

	3-(4-Methylpiperazin-1-yl)-2-nitroaniline (22). Obtained from 3-chloro-2-nitroaniline
	and 1-methylpiperazine, in quantitative yield. Chromatography: CH 2 Cl 2 /EtOH, 9:1. IR
	(CHCl 3 ): 3383, 1605, 1573, 1511, 1459. 1 H NMR (CDCl 3 ) δ 2.32 (s, 3H), 2.53 (br t, J = 4.7,
	4H), 3.01 (br t, J = 4.7, 4H), 4.80 (br s, 2H), 6.38 (d, J = 8.1, 2H), 7.11 (t, J = 8.1, 1H). 13 C
	NMR (CDCl 3 ) δ 46.1, 51.7, 55.1, 109.1, 111.2, 121.3, 132.9, 142.6, 147.6. MS (ESI): 258.9
	(M+Na).

  C NMR (CDCl 3 ) δ 46.1, 51.3, 55.9, 111.6, 112.7, 118.9, 130.1, 134.4, 140.2. MS (ESI) 207.0 (M+H), 228.9

	(M+Na).

  ). Obtained from 29 in quantitative yield, as a non-separable mixture of regioisomers in a 1:1 ratio. Chromatography: hexane/EtOAc, 7:3. IR (CHCl 3 ) 1613, 1576, 1499, 1428. 1 H NMR (CDCl 3 ) δ 0.03 (s, 9H), 0.93, 0.99 (br t, J = 4.5, 2H), 3.55, 3.62 (app t, J = 8.0, 2H), 5.59, 5.88 (s, 2H), 7.20-7.36 (m, 2H), 7.46, 7.72 (dd, J = 5.4, 1.0, 1H), 7.99, 8.03 (s, 1H). 13 C NMR (CDCl 3 ) δ -1.4, -1.3, 17.8, 17.9, 66.3, 66.8, 74.6, 75.1, 109.2, 116.8, 119.4, 122.8, 123.4, 124.2, 125.0, 125.2, 130.1, 133.4, 143.4, 145.1, 146.3. MS (ESI) 283.0 (M+H).

Procedure for the Synthesis of Compounds 31 and 32. To

  C NMR (CDCl 3 ) δ -1.6, 17.6, 46.0, 50.9, 55.2, 66.2, 74.2, 107.2, 110.2, 116.0, 128.3, 142.9, 144.9, 148.3. MS (EI) m/z (%) 346 (M, 100), 331(21), 229(21), 199(9), 73(46), 43(40).

	General a solution of 30 (537
	mg, 1.9 mmol) in o-xylene (17 mL) under an argon atmosphere, 1-BOC-or 1-
	methylpiperazine	(2.7	mmol),	NaOt-Bu	(250	mg,	2.7	mmol),
	tris(dibenzylideneacetone)dipalladium(0) (190 mg, 0.19 mmol) and tri-t-butylphosphine (150
	mg, 0.8 mmol) were added. The mixture was heated at 110 ºC for 24 h. Upon cooling to room
	temperature, the reaction was filtered over celite, the solvent was removed under reduced
	pressure, and the residue was purified by column chromatography using the appropriate
	eluent, to afford pure 31 or 32.
								01 (s,
	9H), 1.08 (app t, J = 8.6, 2H), 1.46 (s, 9H), 2.72 (br t, J = 4.8, 4H), 3.40-3.50 (m, 4H), 3.73
	(app t, J = 8.3, 2H), 5.70 (s, 2H), 6.77 (dd, J = 8.0, 1.2, 1H), 7.26 (t, J = 8.0, 1H), 7.99 (dd, J
	= 8.4, 1.1, 1H), 8.04 (s, 1H). 13 C NMR (CDCl 3 , δ): -1.7, 18.1, 28.2, 51.8, 67.5, 75.3, 79.7,
	114.7, 116.0, 125.0, 129.5, 138.0, 144.3, 149.9, 154.5.
	4-(

Tert-butyl 4-(1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-benzimidazol-4-yl)piperazine-1carboxylate (31).

Obtained from 30 and 1-BOC-piperazine in 49% yield. Chromatography: hexane/EtOAc, 8:2. IR (CHCl 3 ) 1697, 1603, 1543, 1477, 1450.

1

H NMR (CDCl 3 ) δ -0.

4-Methylpiperazin-1-yl)-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-benzimidazole

	(32). Obtained from 30 and 1-methylpiperazine in 43% yield. Chromatography:
	CH 2 Cl 2 /EtOH, 98:2. IR (CHCl 3 ) 1499, 1453. 1 H NMR (CDCl 3 ) δ -0.04 (s, 9H), 0.90 (app t, J
	= 8.4, 2H), 2.44 (s, 3H), 2.73 (br t, J = 4.8, 4H), 3.50 (app t, J = 8.1, 2H), 3.61 (m, 4H), 5.52
	(s, 2H), 6.69 (d, J = 8.3, 1H), 7.12 (d, J = 8.3, 1H), 7.24 (t, J = 8.1, 1H), 7.90 (s, 1H). 13 C
	NMR (CDCl 3 , δ): -1.5, 17.8, 46.0, 52.8, 55.5, 65.9, 74.8, 115.3, 116.7, 122.8, 128.1, 139.9,

144.3, 145.4. MS (ESI) 347.0 (M+H).

  Cl 2 (6 mL), di-tert-butyl dicarbonate (648 mg, 3 mmol), triethylamine (0.4 mL, 3 mmol) and 4-(dimethylamino)pyridine (37 mg, 0.3 mmol) were added, and the mixture was stirred at room temperature overnight. The reaction solution was washed with 1 M NaOH and the organic layer was dried (Na 2 SO 4 ) and evaporated at reduced pressure. The resulting crude

	was purified by column chromatography to give 44 in 69% yield, as a non-separable mixture
	of regioisomers in a 1:1 ratio. IR (KBr) 1755, 1740, 1694, 1624, 1487, 1451. 1 H NMR
	(CDCl 3 ) δ 1.50, 1.70 (s, 18H), 3.14-3.21, 3.62-3.66 (m, 8H), 7.05, 7.12 (dd, J = 8.9, 2.3, 1H),
	7.31, 7.59 (br s, 1H), 7.66, 7.87 (d, J = 10.3, 1H), 8.30, 8.39 (s, 1H). 13 C NMR (CDCl 3 ) δ
	28.3, 28.6, 50.7, 51.1, 80.0, 85.4, 102.2, 108.2, 114.7, 115.9, 117.3, 120.9, 132.8, 133.0,
	138.4, 140.2, 142.4, 147.6, 149.1, 149.7, 154.7. MS (EI) m/z (%) 402 (M, 18), 346 (44), 302

). To a solution of 43 (200 mg, 1 mmol) in anhydrous CH 2

  .9, 121.6, 125.8, 126.9, 127.7, 127.9, 128.8, 129.9, 131.9, 132.9, 133.3, 135.0, 135.6, 137.6, 147.2, 150.3, 182.5. MS (ESI) 371.0 (M+H). Anal. (C 23 H 22 N 4 O) C, H, N. The reaction mixture was stirred at room temperature for 5 h, then evaporated under reduced pressure. The resulting crude was resuspended in CH 2 Cl 2 and washed with 1 M NaOH. The organic layer was dried (Na 2 SO 4 ) and evaporated, and the obtained solid was purified by recrystallization from Et 2 O, to afford pure 9 in quantitative yield; mp 155-157 ºC. IR (KBr) 3442, 1626, 1600, 1516, 1461. 1 H NMR (CD 3 OD) δ 3.22-

	Synthesis of 2-naphthyl-(5-piperazin-1-yl-1H-benzimidazol-2-yl)methanone (9). To a
	solution of 45 (69 mg, 0.15 mmol) in anhydrous CH 2 Cl 2 (1 mL), trifluoroacetic acid (0.25
	mL, 3 mmol) was added dropwise. 3.39 (m, 8H), 7.17 (br s, 1H), 7.23 (dd, J = 9.0, 2.2, 1H), 7.54-7.69 (m, 2H), 7.70 (d, J = 9.0,
	1H), 7.95-8.09 (m, 3H), 8.29 (dd, J = 8.7, 1.7, 1H), 9.20 (s, 1H).

). Obtained from 32 and ethyl benzoate, in 47% yield. Chromatography: CH 2 Cl 2 /EtOH, 95:5. 11513 C NMR (CD 3 OD) δ 44.5, 49.2, 93.4, 111.7, 118.2, 125.6, 127.1, 127.9, 128.3, 129.0, 130.0, 132.0, 132.1, 132.8, 133.6, 133.8, 136.2, 148.2, 150.3, 183.9. MS (ESI) 357.0. Anal. (C 22 H 20 N 4 O) C, H, N.

  5H).[START_REF] Kohen | Cloning, characterization, and chromosomal localization of a human 5-HT 6 serotonin receptor[END_REF] C NMR (CDCl 3 ) δ 36.3, 45.7, 49.6, 55.2, 105.6, 110.0, 123.6, 127.7, 129.4, 129.5, 135.1, 136.5, 137.0, 141.5, 151.3. MS (ESI) 307.0 (M+H). Anal. (C 19 H 22 N 4 ) C, H, N. Cell paste (6.0 mg/mL) was homogenized in 200 volumes of assay buffer (50 mM Tris-HCl, 10 mM MgCl 2 , 0.5 mM EDTA, pH 7.4 at 25 ºC). Fractions of 20 μL of the membranes suspension were incubated at 37 ºC for 60 min with 2.6 nM [ 3 H]LSD, in the presence or absence of the competing drug, in a final volume of 200 μL of assay buffer.

	5-(4-Methylpiperazin-1-yl)-2-(1-naphthylmethyl)-1H-benzimidazole (18). Obtained
	from 28 and 1-naphthylacetaldehyde, in 75% yield. Chromatography: CH 2 Cl 2 /EtOH, 9:1; mp
	). Obtained from 26 and phenylacetaldehyde, in 42% yield. Chromatography: CH 2 Cl 2 /EtOH, 8:2; mp 120-123 ºC. IR naphthylacetaldehyde, in 45% yield. Chromatography: CH 2 Cl 2 /EtOH, 9:1; mp 163-165 ºC. IR (KBr) 3454, 1617, 1597, 1531, 1455. 1 H NMR (CD 3 OD) δ 3.47-3.52 (m, 8H), 4.74 (s, 2H), 6.73-6.76 (m, 1H), 7.11-7.14 (m, 2H), 7.35 (d, J = 6.8, 1H), 7.42-7.50 (m, 3H), 7.83 (d, J = 8.2, 1H), 7.88-7.91 (m, 1H), 8.10-8.14 (m, 1H). 13 C NMR (CD 3 OD) δ 34.0, 45.3, 106.2, 108.7, 124.3, 124.7, 126.8, 127.1, 127.5, 128.3, 129.2, 129.9, 132.2, 134.2, 135.6, 136.2, 136.9, 142.2, 151.9. MS (ESI) 343.2 (M+H). Anal. (C 22 H 22 N 4 ) C, H, N. 4-(4-Methylpiperazin-1-yl)-2-(1-naphthylmethyl)-1H-benzimidazole (13). Obtained from 26 and 1-naphthylacetaldehyde, in 48% yield. Chromatography: CH 2 Cl 2 /EtOH, 9:1; mp 95-96 ºC. 4-(4-Methylpiperazin-1-yl)-2-(2-naphthylmethyl)-1H-benzimidazole (14). Obtained from 26 and 2-naphthylacetaldehyde, in 51% yield. Chromatography: CH 2 Cl 2 /EtOH, 8:2. 2-Benzyl-5-piperazin-1-yl-1H-benzimidazole (15). Obtained from 27 and phenylacetaldehyde, in 67% yield. Chromatography: CHCl 3 /MeOH/NH 3 , 9:1:0.1; mp 173-175 ºC. IR (KBr) 3450, 1633, 1493, 1455. 1 H NMR (Acetone-d 6 ) δ 2.78-2.92 (m, 8H), 2.97 (br s, 1H), 4.05 (s, 2H), 6.76 (dd, J = 8.7, 2.3, 1H), 6.84 (d, J = 2.1, 1H), 7.07-7.25 (m, 6H), 129.2, 133.9, 136.1, 138.4, 149.3, 152.8. MS (ESI) 293.0 (M+H). Anal. (C 18 H 20 N 4 ) C, H, N. 2-Benzyl-5-(4-methylpiperazin-1-yl)-1H-benzimidazole (16). Obtained from 28 and phenylacetaldehyde, in 53% yield. Chromatography: CH 2 Cl 2 /EtOH, 97:3; mp 108-109 ºC. 2-(1-Naphthylmethyl)-5-piperazin-1-yl-1H-benzimidazole (17). Obtained from 27 and 1-naphthylacetaldehyde, in 44% yield. Chromatography: CH 2 Cl 2 /EtOH, 9:1; mp 112-114 ºC. 121-123 ºC. 2-(2-Naphthylmethyl)-5-piperazin-1-yl-1H-benzimidazole (19). Obtained from 27 and 2-naphthylacetaldehyde, in 42% yield. Chromatography: CH 2 Cl 2 /EtOH 9:1; mp 157-159 ºC. IR (KBr) 3443, 1632, 1490, 1456. 1 H NMR (CD 3 OD) δ 3.25-3.32 (m, 8H), 4.27 (s, 2H), 6.92 (dd, J = 8.8, 2.3, 1H), 7.02 (d, J = 2.0, 1H), 7.29-7.39 (m, 4H), 7.67-7.73 (m, 4H). 13 C NMR (CD 3 OD) δ 36.3, 45.3, 50.1, 103.9, 116.5, 116.7, 127.0, 127.5, 128.0, 128.5, 128.8, 129.7, 134.1, 135.3, 135.5, 135.8, 140.0, 148.6, 155.3. MS (ESI) 343.0 (M+H). Anal. (C 22 H 22 N 4 ) C, H, N. 5-(4-Methylpiperazin-1-yl)-2-(2-naphthylmethyl)-1H-benzimidazole (20). Obtained from 28 and 2-naphthylacetaldehyde, in 59% yield. Chromatography: CH 2 Cl 2 /EtOH, 95:5; mp 149-152 ºC. Pharmacology. Radioligand Binding Assays. Membranes from HEK-293 cells expressing human 5-HT 6 or 5-HT 7 receptors, were purchased from Perkin-Elmer and conserved at -80 ºC in packaging buffer for subsequent use. Specific radioligand [ 3 H]LSD (79.2 Ci/mmol) was from NEN (Perkin-Elmer). Competitive inhibition assays were performed according to standard procedures, briefly detailed below. (KBr) 3356, 1593, 1533, 1494, 1456. 2-(1-Naphthylmethyl)-4-piperazin-1-yl-1H-benzimidazole (12). Obtained from 25 and 1-7.90 (br s, 1H). 13 C NMR (Acetone-d 6 ) δ 35.7, 46.6, 52.6, 102.0, 114.3, 114.4, 126.9, 128.8, 5-HT 6 Receptor.

1

H NMR (CDCl 3 ) δ 2.44 (s, 3H), 2.79 (m, 4H), 3.62 (m, 4H), 4.30 (s, 2H), 6.66 (d, J = 8.0, 1H), 7.09 (m, 1H), 7.12 (t, J = 8.0, 1H), 7.

30-7.38 (m,

Table 1 .

 1 Binding Affinity of Synthesized Compounds 1-20 at the h5-HT 6 R.

	compd	positio n	R	X	Ar	K i ± SEM (nM) a
	1	4	H	CO	phenyl	> 1000
	2	4	CH 3 CO	phenyl	>1000
	3	4	H	CO 1-naphthyl	13 ± 1
	4	4	CH 3 CO 1-naphthyl	34 ± 2
	5	4	CH 3 CO 2-naphthyl	> 1000
	6	5	CH 3 CO	phenyl	> 1000
	7	5	CH 3 CO 1-naphthyl	> 1000
	8	5	CH 3 CO 2-naphthyl	> 1000
	9	5	H	CO 2-naphthyl	> 1000
	10	4	H CH 2	phenyl	58 ± 3
	11	4	CH 3 CH 2	phenyl	66.8 ± 0.7
	12	4	H CH 2 1-naphthyl	16 ± 2
	13	4	CH 3 CH 2 1-naphthyl	36.22 ± 0.08
	14	4	CH 3 CH 2 2-naphthyl	49 ± 3
	15	5	H CH 2	phenyl	> 1000
	16	5	CH 3 CH 2	phenyl	> 1000
	17	5	H CH 2 1-naphthyl	> 1000
	18	5	CH 3 CH 2 1-naphthyl	> 1000
	19	5	H CH 2 2-naphthyl	> 1000
	20	5	CH 3 CH 2 2-naphthyl	> 1000

a Values are the mean of two to four experiments performed in triplicate.

Table 2 .

 2 Potency of New 5-HT 6 R Antagonists.

	compd R	X	Ar	K i ± SEM (nM) a
	3	H	CO 1-naphthyl	15 ± 2
	4	CH 3 CO 1-naphthyl	5 ± 2
	10	H CH 2	phenyl	31 ± 4
	11	CH 3 CH 2	phenyl	16 ± 5
	12	H CH 2 1-naphthyl	16 ± 3
	13	CH 3 CH 2 1-naphthyl	27 ± 3
	14	CH 3 CH 2 2-naphthyl	17 ± 4

a Values are the mean of three experiments performed in triplicate.

Acknowledgment. This work has been supported by grants from the Spanish Ministerio de Educación (MEC, SAF-2007/67008), Comunidad Autónoma de Madrid (CAM, S-SAL-249-2006), and Instituto de Salud Carlos III (RD07/0067/0008). The authors thank Fundación Ramón Areces and MEC for predoctoral grants to T.F., J.S, and R.A.M.

Chart 1. Structures of 5-HT 6 R agonists EMDT, WAY-181187 and E-6801, and antagonists MS-245, SB-258585, Ro 04-6790 and SB-357134.

(Amersham Pharmacia Biotech) at 1 μg/mL for 60 min. Chromogenic substrate was added (Supersignal® ELISA femto-maximum sensitivity, Pierce, Perbio-Brebières, France).

Chemiluminescence was detected and quantified by a Wallac Victor2 luminescence counter.

Determination of cAMP Production in Transfected Cells. COS-7 cells were transfected with the appropriate cDNA and seeded into 24-well plates (100 000 cells/well). 24 hours post-transfection, a 10 min-stimulation with the appropriate concentrations of each tested compound in absence or presence of 100 μM 5-HT was performed, as previously described. [START_REF] Abuzar | Synthesis of 2-carbalkoxyamino-5(6)-(1-substituted piperazin-4yl/piperazin-4-ylcarbonyl)benzimidazoles and related compounds as potential anthelmintics[END_REF] Quantification of cAMP production was performed by homogenous time resolved fluorescence (HTRF®) using the cAMP Dynamic Kit (Cisbio International, Bagnols-sur-Cèze, France), according to the manufacturer's instructions.

Data Analysis. The dose-response curves were fitted using GraphPad Prism and the following equation for monophasic dose-response curves: y = (y max -y min ) / 1 + [(x / IC 50 ) n H ] + y min , where IC 50 is the concentration of the antagonist necessary to reduce 50% of the maximal effect induced by 5-HT and n H is the Hill coefficient. All data represented correspond to the mean ± SEM of three independent experiments performed in triplicate.

Computational Methods. Models of the Ligand-Receptor Complexes. Modeller v9.5 [START_REF] Marti-Renom | Comparative protein structure modeling of genes and genomes[END_REF] was used to build a homology model of the human 5-HT 6 R using the crystal structure of the β 2 -adrenergic receptor (PDB code 2RH1) [START_REF] Cherezov | Highresolution crystal structure of an engineered human β 2 -adrenergic G protein-coupled receptor[END_REF][START_REF] Rasmussen | Crystal structure of the human β 2 adrenergic G-protein-coupled receptor[END_REF] as template. Internal water molecules 506, 519, 528, 529, 532, 534, 537, 543, 546, and 548 that mediate a number of interhelical interactions, [START_REF] Rosenbaum | GPCR engineering yields high-resolution structural insights into β 2 -adrenergic receptor function[END_REF] and seem conserved in the rhodopsin-like family of GPCRs, [START_REF] Pardo | The role of internal water molecules in the structure and function of the rhodopsin family of G proteincoupled receptors[END_REF] were also included in the model. The Duan et al. (2003) force field [START_REF] Duan | A point-charge force field for molecular mechanics simulations of proteins based on condensed-phase quantum mechanical calculations[END_REF] was used for receptors and the general Amber force field (GAFF) [START_REF] Wang | Development and testing of a general amber force field[END_REF] and HF/6-31G*-derived RESP atomic charges were used for the ligands. Molecular dynamics simulations of the ligand-receptor complexes were performed with the Sander module of AMBER 9 69 using the protocol previously described. 70 This material is available free of charge via the Internet at http://pubs.acs.org.

Supporting Information


Baz Ihab

Champoussin Lance Michel
email: michel.lance@ec-lyon.fr

Investigation of two-phase flows generated by cavitation inside high-pressure injection nozzles

In this paper, we develop experimental techniques based on shadowgraphy and tomography techniques, associated with LIF, to visualize the two-phase flow structure into a typical nozzle geometry. The experiments were performed using a Common Rail system for generating periodic injection conditions. A transparent tip with sac and axial spray hole, drilled into quartz to allow optical access, is mounted over a standard type nozzle. The hole diameters of the nozzles vary in the rang of 0.3 to 0.4 mm with a length/diameter ratio of 4. The experiments show that cavitation starts at the inlet of the orifice and develops to reach the exit. The local position and range of cavitation layers, lying between the flow and the nozzle wall and within the flow itself, are observed at different instants of the injection process. At high cavitation number (K>5) and low Reynolds number (Re<12000) the cavitation region spreads to the middle of the flow.

However, when increasing the Reynolds number, the cavitation appears as a thin layer near the wall of the orifice. It is shown that small surface disturbances as well as the nozzle geometry have a strong influence on the flow inside the spray hole. Moreover, an histeresis is observed when pressure released.

Introduction

It is well known today that spray atomization in direct injection engines depends mainly on the cavitation of the flow within the nozzle. The challenge is to characterize high speed two-phase flows ( > 200 m/s), at high pressure (> 70 Mpa), in very tiny channels (300-400 micrometers in diameter), in very unsteady conditions. In this paper, we develop experimental techniques based on shadowgraphy and tomography techniques, associated with LIF, to visualize the flow structure into a typical nozzle geometry. The first evidence of the role of cavitation in the atomization process stems to Bergwerk's paper (1959). Since this pioneering work, many experiments have been carried out to characterize thz structure of the two-phase flow inside the injector channel, and its influence on the atomization of the liquid jet [START_REF] Chaves | Experimental study of cavitation in the nozzle hole of diesel injectors using transparent nozzles[END_REF][START_REF] Arcoumanis | Cavitation in Real-Size Multi-Hole Diesel Injector Nozzles[END_REF], Baddock, 1999[START_REF] Roosen | Investigation of cavitation phenomena inside fuel injector nozzles[END_REF]. As a matter of fact, the onset of the hydrodynamic instability of the liquid cylindrical jet, which is responsible for its break-up, is now recognized to originate from disturbances in growing upstream in the injection channel. The latter may come from strong turbulence fluctuations and/or the presence of gas bubbles in the liquid associated with cavitation. In the range of very high pressure found in Diesel injectors, the occurrence of cavitation is unavoidable due to strong acceleration of the liquid at the inlet of the nozzle. The presence of sharp edges in the injection device, which triggers detachment of the flow, still contributes to amplify this effect. Such a phenomenon is in principle well known at moderate pressures, and can be described with classical cavitation models. The situation is less clear for the present range of pressure and liquid velocity, for which non equilibrium effects may be expected. Moreover, the development of the flow inside the injection channel is conditioned by the existence of extremely strong shear (say 10 6 s -1 ), which has no equivalent in usual situations. On the other end, the time scale of the flow is also very small, since the typical convection time in the nozzle is 10 -5 s. These time scales have to be compared to the duration of the fuel injection, a few milliseconds. This suggests that a quasisteady approximation could be adopted to describe the flow in the nozzle. This must be checked experimentally. As mentioned above, several studies have already been devoted to this problem. However, due to the experimental difficulties, the previous experiments were restricted to larger nozzle sizes and/or smaller pressures. The aim of the present study is to contribute to fill the gap between the experiments and the real flow conditions.

Experimental setup

Due the special pressure conditions encountered in the Diesel injection device, it proved necessary to use adapt a commercial injection device. Therefore, a standard type nozzle has been used. In order to perform optical measurements, the nozzle tip is replaced by an optically polished quartz, with the exact shape and dimensions of the sac and hole spray (fig. 1). The cylindrical orifice has a diameter of 0.4 mm and an aspect ratio of 4. The experiments were conducted using a Bosch Common-Rail injection system providing real unsteady Diesel injection conditions.

Fig. 1 Transparent Diesel nozzle

The main advantage of this system lies in the possibility to adjust the injection time and pressure, as well as the number of injection sequences. Since high rail pressures decrease the lifetime of the quartz nozzle tip, all our experiments are carried out at rail pressures smaller than 50 MPa and with energizing time of 2.5 ms. The fluid used is an oil with physical properties identical to Diesel fuel. In particular, the values of density, viscosity and surface tension are respected. An advantage of this test oil for optical measurements is the good matching of the refractive index n to that of quartz (n oil =n quartz =1.46). Thus, reflection and refraction, when the light passes the internal surface of the quartz nozzle, is minimized. The oil is injected into a constant volume spray chamber capable of carrying high pressures (8 MPa). The optical system consists of a long-distance microscope and an intensified CCD camera (CCD-chip 1280x1024 pixels, exposure time down to 3ns, delay between two images down to 500 ns).

To reduce the blurring effects in the pictures caused by high flow velocities higher than 150 m/s, an exposure time of 20 ns must be used. As a light source, a xenon arc lamp is used (fig. 2) for shadowgraphy, and Argon-ion Laser is used for tomography with an optical setup to produce a light sheet thickness of less than 30 µm (fig. 3). In this work, a direct measurement of the sac pressure is made by using an AVL pressure transducer mounted on an injector tip drilled into steel and having the same dimensions as the transparent one.

Results and discussion

Due to the very small time scale of the flow, it proves impossible to resolve in time the whole sequence of the injection device. Therefore, phase synchronisation with periodic injection has been used. This supposes that the flow structure is also periodic and reproducible for each realization, which is obviously very unlikely. The visualizations presented here are only intended to shed a qualitative view of the flow development. In order to bring more quantitative information it is necessary to account for some cyclic variations with phase averaging. So for each parameters setting, a sequence of about 50 pictures is recorded.

Figure 4 shows the time sequence of the injection, for a typical rail pressure of 250 bar. It is observed that this pressure decreases only very slightly with time, so that it can be assumed constant during the process. The flow rate is controlled by the lift of the needle, which can be adjusted to get different delays. The pressure in small volume before the injection channel (the sac) is an important parameter since it determines the actual liquid flow rate. It is worth noting the huge pressure drop associated with the path of the flow in the needle clearance.

Fig. 4 Needle lift, sac pressure and rail pressure during the injection cycle. P R = 30 MPa, t e = 2.5 ms

In figure 5 a reconstruction of the two-phase flow development is given. In the first image, the injector is filled up with liquid and a gas bubble is sucked from the chamber downstream when the needle lifts up. The next image shows clearly the onset of cavitation at the inlet of the injection channel. The development of a two-phase flow is observed in the next three pictures. The gas bubbles generated at the inlet are entrained by the high speed liquid flow and produce an annularlike structure, with a layer of gas close to the walls. At full needle lift the flow rate is maximum and the cavitation layer stretches till the end of the channel. Unfortunately, it was not possible with this device to observe the shape of the resulting spray. Other experiments however, in different conditions, have shown a reduced penetration length of the liquid jet in the chamber at this period of the injection cycle.

The mean velocity in the channel has been estimated from the displacement of gas bubbles measured between two images separated by 500 ns, assuming that the velocity slip is negligible. The resulting instantaneous Reynolds number is plotted in figure 6 for cycle duration. where P s is the nozzle sac pressure upstream to the flow orifice, P v is the vapor pressure of the fuel and P b is the downstream pressure. The evolution of this quantity, shown in figure 7, points out the high values reached by this parameter in this experiment. It is difficult to get quantitative data for the cavitation from the shadowgraphy pictures, since the light intensity is integrated along the chords of the cylindrical pipe. Laser tomography provides a better estimation of the sectional area occupied by the vapor phase. We define a cavitation rate as this area to the total area of the plane enlighten by the laser sheet. This quantity is shown in figure 8. The onset of cavitation is as expected advanced when pressure increases. Now, except for the beginning of the cycle, the influence of the pressure seems to be limited, and the cavitation rate reaches an asymptotic value of 20%. The accuracy of the measurements does not allow to discuss safely the apparent overshoot for the lower pressure. Interestingly, cavitation re-increases when the needle is closing, despite of the decay of the cavitation number. It must noticed that the high pressure drop for small needle lifts cause cavitation to occur near the needle passage, and therefore a higher void fraction may be observed in the injector. This effect is not very significant in practice for the corresponding flow rate is small. Figure 9 shows visualizations obtained with laser tomography coupled with PLIF. A fluorescent dye added to the test oil used for the experiments makes it possible to better distinguish between the liquid fluorescent phase and the vapor phase. It must be stressed out that the strong light absorption associated with the vapor layer and the dye permits to observe only one half of the flow (right side in the figures). The vapor layer is well delimited, the resolution however does not show its fine structure, likely a layer of small bubbles. An interesting effect is the histeresis observed when the needle is closing. As a matter of fact, under quasi-steady conditions, it should be expected that the flow structure would be the same for identical cavitation numbers, or in other words, that the opening and closing phase would be similar (except for the very beginning and the very end, as discussed above). This is clearly not the case here, which perhaps indicates that the role of activated cavitation sites on the wall could be significant.

Conclusion

It has been possible in this work to visualize the two-phase flow developing in a small fuel injection channel, for very severe flow conditions. The onset and the development of cavitation have been characterized. Further efforts are needed to still improve the accuracy of the data, and to couple the structure of the flow and that of the atomizing jet in the chamber, as well as the size distribution of droplets in the spray.

Fig. 2

 2 Fig. 2 Experimental setup for shadowgraphy

Fig. 5 Fig. 6

 56 Fig. 5 Cavitation inside the spray hole : Rail pressure = 30 MPa, Back pressure = 0.1 MPa, Energizing time of the injector = 2.5 ms (Shadowgraphy images taken at different instants in the injection cycle)

Fig. 7 Fig. 8

 78 Fig.7 Evolution of the cavitation number during the injection cycle.

Fig. 9

 9 Fig.9 Visualization of the flow in the injector during the injection cycle using PLIF

  Closing phase5th International Conference on Multiphase Flow, ICMF'04Yokohama, Japan, May 30-June 4, 2004 Paper No. 488

	Opening phase
	t

a = 700 µs t a = 2200 µs t a = 2600 µs t a = 3100 µs Quasi-steady phase t a = 650 µs t a = 3200 µs

Acknowledgements

This work is sponsored by the CNRS, PSA PEUGEOT-CITROËN and RENAULT. The authors would like to thank Mr. Rémy Point and Mr. Michel Gaud for their technical assistance.