domain: sdv chim

text:
Katarzyna Grychowska

Grzegorz Satala

Tomasz Kos

Anna Partyka

Evelina Colacino

Séverine Chaumont-Dubel

Xavier Bantreil

Anna Wesolowska

Maciej Pawlowski

Jean Martinez

Grzegorz Satała

Severine Chaumont-Dubel

Anna Wesołowska

Maciej Pawłowski

Philippe Marin

Gilles Subra

Andrzej J Bojarski

Fredeŕic Lamaty

Piotr Popik

Paweł Zajdel

quinoline Based 5-HT6 Receptor Antagonists with Potential Application for the Treatment of Cognitive Disorders Associated with Alzheimer's Disease

Keywords: 5-HT 6 R antagonist, 5-HT 6 R inverse agonist, pyrrolo[3, 2-c]quinoline, SB-742457, cognition, memory, novel object recognition test, forced swim test, Vogel test, Alzheimer's disease

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A lzheimer's disease (AD), an irreversible neurodegenerative disorder, is the most common form of dementia worldwide. AD is characterized by the progressive deterioration of various cognitive domains, including a decline in memory and thinking. More than 35 million people are suffering from AD. Currently available therapeutic options for the treatment of AD, based on acetylcholinesterase inhibitors and NMDA receptor antagonist, provide modest efficacy and symptomatic treatment without affecting the neurodegeneration process. 1 In recent years, the serotonin 5-HT 6 receptor (5-HT 6 R) has emerged as a promising molecular target for the treatment of cognitive deficits in AD. 2 The 5-HT 6 R belongs to the family of G-protein coupled receptors, which are positively coupled with adenylyl cyclase. Additionally, 5-HT 6 Rs engage extracellular signal-regulated kinase (ERK)1/2 pathway via the Src family tyrosine kinase Fyn, 3 as well as cyclin-dependent kinase (Cdk)5, [START_REF] Duhr | Cdk5 induces constitutive activation of 5-HT 6 receptors to promote neurite growth[END_REF] and mammalian Target Of Rapamycin (mTOR) pathways. [START_REF] Meffre | 5-HT 6 receptor recruitment of mTOR as a mechanism for perturbed cognition in schizophrenia[END_REF] Moreover, nonphysiological mTOR activation in prefrontal cortex, under the control of 5-HT 6 R, underlies deficits in social cognition and episodic memory in two neurodevelopmental models of schizophrenia in the rat, neonatal administration of the NMDA receptor antagonist phencyclidine and rearing in social isolation after the weaning. [START_REF] Meffre | 5-HT 6 receptor recruitment of mTOR as a mechanism for perturbed cognition in schizophrenia[END_REF] 5-HT 6 R displays high constitutive activity at various signaling cascades, including Gs adenylyl cyclase, [START_REF] Jacobshagen | The serotonin 6 receptor controls neuronal migration during corticogenesis via a ligand-independent Cdk5-dependent mechanism[END_REF] and Cdk5 pathways. [START_REF] Duhr | Cdk5 induces constitutive activation of 5-HT 6 receptors to promote neurite growth[END_REF] Accordingly, the deleterious influence of 5-HT 6 R in AD may reflect an alteration in receptor expression as well as a deregulation of its activity. The 5-HT 6 R is almost exclusively expressed in the CNS, with high levels in the prefrontal cortex, hippocampus, and striatum, the brain regions that are predominantly involved in the learning and memory processes. [START_REF] Geŕard | Immuno-localization of serotonin 5-HT 6 receptor-like material in the rat central nervous system[END_REF] It has also been demonstrated that 5-HT 6 R antagonists modify the transmission of acetylcholine, glutamate, dopamine, and norepinephrine. [START_REF] Dawson | The 5-HT 6 receptor antagonist SB-271046 selectively enhances excitatory neurotransmission in the rat frontal cortex and hippocampus[END_REF][START_REF] Riemer | Influence of the 5-HT 6 receptor on acetylcholine release in the cortex: pharmacological characterization of 4-(2-bromo-6-pyrrolidin-1-ylpyridine-4-sulfonyl)phenylamine, a potent and selective 5-HT 6 receptor antagonist[END_REF] The progress in development of 5-HT 6 R modulators has recently been comprehensively reviewed by Holenz et al., [START_REF] Holenz | Medicinal chemistry strategies to 5-HT 6 receptor ligands as potential cognitive enhancers and antiobesity agents[END_REF] Glennon et al., [START_REF] Glennon | The medicinal chemistry of 5-HT 6 receptor ligands with a focus on arylsulfonyltryptamine analogs[END_REF] and Benhamu et al. [START_REF] Benhamu | Serotonin 5-HT 6 receptor antagonists for the treatment of cognitive deficiency in Alzheimer's disease[END_REF] Important research in the field led to the discovery of several classes of indole and indolelike derivatives (e.g., benzofurans, indazoles, pyrazolopyrimidines, indolines) that preferentially bind to the 5-HT 6 R and behave as antagonists at these sites (Figure 1). [START_REF] Ivachtchenko | Small molecule 5-HT 6 R ligands: A comprehensive insight into their selectivity and activity[END_REF] As a consequence, the structural requirements for 5-HT 6 R antagonists have been described, and pharmacophore models for 5-HT 6 R antagonists have been developed. [START_REF] Lopez-Rodriguez | A three-dimensional pharmacophore model for 5-hydroxytryptamine 6 (5-HT 6 ) receptor antagonists[END_REF] It is worth noting that 5-HT 6 R antagonists displayed procognitive effects in several learning and memory in vivo paradigms in rodents, and their efficacy was confirmed in phase II clinical trials when used in combination with donepezil (AChE inhibitor). [START_REF] Wicke | Investigational drugs targeting 5-HT 6 receptors for the tretament of Alzheimer's disease[END_REF][START_REF] Ramirez | 5-HT 6 receptors and Alzheimer's disease[END_REF] Specifically, SB-742457 and idalopirdine (Lu-AE58054) have been evaluated in phase III clinical trials as an adjunctive therapy to donepezil in patients with mild to moderate AD (Figure 2). [START_REF] Galimberti | Idalopirdine as a treatment for Alzheimer's disease[END_REF] Furthermore, 5-HT 6 R antagonists produced procognitive and/or antiamnesic effects in other neuropsychiatric disorders, i.e. schizophrenia, and Parkinson's disease, and might open therapeutic opportunity for autism treatment. [START_REF] Meffre | 5-HT 6 receptor recruitment of mTOR as a mechanism for perturbed cognition in schizophrenia[END_REF][START_REF] Meneses | Serotonin, neural markers, and memory[END_REF] Continuing our efforts to generate selective 5-HT 6 R ligands, we applied a scaffold-hopping approach to fuse a pyrrole ring onto the c edge of the quinoline fragment of 6-nitroquipazine (selective serotonin-reuptake inhibitor) and then replaced the piperazine moiety with its 3-aminopyrrolidine bioisostere. Although the resulting N-4-(pyrrolidin-3-yl-amino)-1H-pyrrolo-[3,2-c]quinoline I, displayed a low affinity for the 5-HT 6 R, further functionalization of I, introducing the phenylsulfonyl moiety on the nitrogen atom of pyrrole (II), fulfilled the structural requirements of a 5-HT 6 R antagonist and significantly increased the compound's affinity for the 5-HT 6 R. This result prompted us to further investigate the substitution pattern of the arylsulfonyl fragment of derivative II to develop potent 5-HT 6 R antagonists (Figure 3). Herein, we report on a small series of arylsulfonyl derivatives of a novel molecular framework that is based on N-4-(pyrrolidin-3yl-amino)-1H-pyrrolo[3,2-c]quinoline as potent and selective 5-HT 6 R antagonists. The most promising derivatives were evaluated for their ability to reverse drug-induced memory deficits and for their procognitive properties in novel object recognition test in rats. Because noncognitive traits are often associated with dementia and Alzheimer's disease, the most promising derivatives were also evaluated in animal models of anxiety and depression.

■ CHEMISTRY

The designed compounds were synthesized according to a multistep procedure (Scheme 1).

The starting nitrobenzaldehyde, tosylamine, and methyl acrylate reacted in the presence of DABCO and titanium isopropoxide, in isopropanol, under the aza-Bayliss-Hillman reaction conditions, to yield the unsaturated β-aminoester 1. [START_REF] Benakki | Microwave-assisted multi-step synthesis of novel pyrrolo-[3,2-c]quinoline derivatives[END_REF] Subsequently, compound 1 was treated with allyl bromide in a biphasic system to give diene 2. The latter was submitted to a ring-closing metathesis reaction, using Grubbs second-generation catalyst, in refluxing dichloromethane for 30 min, to yield pyrroline 3. This process was promoted by microwave irradiation in the open-vessel mode. Next, the tosyl group of 3 was eliminated in the presence of sodium tert-butoxide (NaOt-Bu). [START_REF] Declerck | 2-Trimethylsilylethanesulfonyl (SES) versus tosyl (Ts) protecting group in the preparation of nitrogen-containing five-membered rings. A novel route for the synthesis of substituted pyrrolines and pyrrolidines[END_REF] Such an approach allowed for the simultaneous aromatization of the pyrroline moiety and generation of the pyrrole derivative 4. Compound 4 was then reduced using palladium on activated charcoal, in methanol, under hydrogen atmosphere. Intramolecular cyclization of aromatic amine 5, upon treatment with acetic acid, provided the lactam derivative 6, which was subsequently submitted to oxidative chlorination to yield 4-chloro-1H-pyrrolo[3,2-c]quinoline (7). Nucleophilic substitution with 1-Boc-3-aminopyrrolidine (either a racemic mixture or pure enantiomers) was carried out under microwave-assisted conditions and gave compounds 8a-8c. Coupling 8a-8c with selected arylsulfonyl chlorides in the presence of a strong phosphazene base, P 1 -t-Bu-tris(tetramethylene) (BTPP), provided sulfonyl derivatives of Boc-protected N-4-(pyrrolidin-3-ylamino)-1H-pyrrolo[3,2-c]quinolines. [START_REF] Zajdel | The pipecolic linkeran acid-labile handle for derivatization of secondary amines on a solid-support. Part 3[END_REF] These compounds furnished the final products 9-27, as hydrochloride salts, upon removal of the Boc group in acidic conditions.

■ RESULTS AND DISCUSSION

Pharmacological in Vitro Evaluation and Structure-Activity Relationship Studies. Pyrroloquinolines have been previously explored as privileged structures for the synthesis of biologically active compounds with potential anticancer, antimalarial, antiobesity, and analgesic properties. [START_REF] Ferlin | Design, synthesis, and structure-activity relationships of azolylmethylpyrroloquinolines as nonsteroidal aromatase inhibitors[END_REF][START_REF] Carroll | Aplidiopsamine A, an antiplasmodial alkaloid from the temperate Australian ascidian, Aplidiopsis confluata[END_REF][START_REF] Adams | Pyrrolo(iso)quinoline derivatives as 5-HT 2C receptor agonists[END_REF][START_REF] Furlotti | Discovery and pharmacological profile of new 1H-indazole-3-carboxamide and 2H-pyrrolo[3,4-c]quinoline derivatives as selective serotonin 4 receptor ligands[END_REF] In the present study, the 1H-pyrrolo[3,2-c]quinoline scaffold was originally proposed as a promising core motif for the development of a new class of 5-HT 6 R antagonists.

The synthesized compounds 9-27 displayed high-to-moderate affinity for the 5-HT 6 R (K i = 3-101 nM) and generally did not bind to serotonergic 5-HT 1A , 5-HT 2A , 5-HT 7 , and dopaminergic D 2 receptors (Table 1). The initially identified compound 9, with the phenylsulfonyl group, displayed high affinity for the 5-HT 6 R (K i = 10 nM). First, the lead optimization focused on the arylsulfonyl moiety.

Introduction of electron-withdrawing substituents, such as fluorine, chlorine (10, 13) or the bulky trifluoromethyl group (16), in the meta position of the phenyl ring slightly increased the affinity for the 5-HT 6 R. A shift of the fluorine atom from the meta to the para position of the phenylsulfonyl fragment induced a 2-fold decrease in the affinity (19 with K i = 18 nM vs 10 with K i = 9 nM). Introduction of an additional fluorine atom in the ortho position of the phenyl ring further decreased the affinity, as compared with the monofluorinated analogue (20 vs 10). Interestingly, replacement of the electron-withdrawing substituent in the meta position of the phenyl ring with electron-donating methoxy (21, K i = 7 nM) or methyl (22, K i = 8 nM) groups maintained the high affinity for the 5-HT 6 R.

Conversely, introduction of sterically hindered tert-butyl or isopropyl groups in the para position (23, 24) significantly decreased the affinity, suggesting that bulky substituents at this position were unfavorable for binding to the 5-HT 6 R (23 vs 9, 24 vs 9). Compounds 23 and 24, with bulky t-Bu and i-Pr substituents, were the least potent ligands among the evaluated series, with K i of 101 nM and 99 nM, respectively.

To further reveal the impact of the size of substituents in the arylsulfonyl fragment on 5-HT 6 R affinity, bicyclic aromatic systems were introduced. Replacement of the phenyl moiety with 1-naphthyl (25, K i = 19 nM) slightly reduced affinity for the 5-HT 6 R, whereas the 8-quinolinyl fragment (26, K i = 4 nM) was better accommodated. Furthermore, it was found that the sterically encumbered 5-Me-benzothien-2-yl moiety caused a 10-fold decrease in the affinity for 5-HT 6 sites (27 with K i = 94 nM vs 9 with K i = 10 nM) and, thus, showed the limitations of the binding pocket. Because the stereochemical properties of molecules might influence their binding in the receptor pocket, three pairs of enantiomers were investigated. A slight preference for the S enantiomers (11, 14, 17) over their R counterparts (12, 15, 18) was observed with respect to their 5-HT 6 receptor affinity and selectivity. Compound 14, an (S)-enantiomer bearing a chloro substituent in the meta position of the phenyl ring, displayed the highest affinity for the 5-HT 6 R (K i = 3 nM).

The most potent compounds (K i < 20 nM) were further evaluated for their functional activity at the 5-HT 6 R in cAMP cellular assays. In the screening procedure carried out in Cerep, compounds were initially classified as 5-HT 6 R antagonists (Table 1). Subsequently, two pairs of enantiomers, 11, 12, 14, and 15, displaying the highest affinity for the 5-HT 6 R and the highest receptor-antagonist activity (% inhibition of control agonist), were evaluated in the cellular functional assay to inhibit 5-CT-induced cAMP production (Table 2). Compounds bearing a chlorine atom in the meta position of the arylsulfonyl displayed stronger antagonist properties than their fluoro counterparts. Among the tested enantiomers, the S isomers (11, 14) were the most potent antagonists and showed a greater antagonistic effect at the 5-HT 6 sites than those exhibited by SB-742457 with K b values 0.83 nM and 0.41 nM for 11 and 14, respectively.

Furthermore, to look for potential inverse agonist activity, two 5-HT 6 R antagonists, 14 and 15, and model 5-HT 6 R antagonist SB-742457, were evaluated in an NG108-15 neuroblastoma cell line that was transiently expressing the 5-HT 6 R. The antagonists' effects on the receptor's constitutive activity at Gs signaling was assessed by BRET using the cAMP sensor CAMYEL. As previously observed, [START_REF] Jacobshagen | The serotonin 6 receptor controls neuronal migration during corticogenesis via a ligand-independent Cdk5-dependent mechanism[END_REF] expressing the 5-HT 6 R strongly activates cAMP production in NG108-15 cells, and the level of constitutive activity represented approximately 60% of the activity of a maximally effective concentration of the full agonist WAY181187 (Figure 4A). The constitutive activity was reduced in a concentration-dependent manner by the 5-HT 6 R antagonist SB-742457, which behaved as an inverse agonist in this model, with pEC 50 of 7.67 (Figure 4A and4B). In contrast, neither 14 nor 15 significantly affected receptor-elicited cAMP production (Figure 4B), indicating that these compounds behaved as neutral antagonists. It was further demonstrated that the selected compounds 14 and 15 did not bind to adrenergic α 1A (<18% at 1 μM), histaminergic H 1 (<17% at 1 μM), muscarinic M 1 (<9% at 1 μM), and serotonin 5-HT 2C and 5-HT 2B receptors (<21% at 1 μM), and they did not show affinity for the serotonin transporter (SERT) (<12% at 1 μM). Of note, this distinguishes the reported compounds 14 and 15 from the well-known 5-HT 6 R agents; for example, SB-742457 displays a high affinity for 5-HT 2A (99% at 1 μM) and 5-HT 2B R (99% at 1 μM), [START_REF] Ivachtchenko | Small molecule 5-HT 6 R ligands: A comprehensive insight into their selectivity and activity[END_REF] and idalopirdine binds at α 1A R (K i = 21 nM) and displays moderate affinity for the 5-HT 2A R (K i = 83 nM). [START_REF] Arnt | Lu AE58054, a 5-HT 6 antagonist, reverses cognitive impairment induced by subchronic phencyclidine in a novel object recognition test in rats[END_REF] Pharmacological in Vivo Evaluation. Several preclinical studies have shown that 5-HT 6 R antagonists reverse drug-induced memory decline or time-dependent natural forgetting in rats in a variety of preclinical behavioral paradigms (e.g., NOR, ASST, 5CCT). [START_REF] Arnt | Lu AE58054, a 5-HT 6 antagonist, reverses cognitive impairment induced by subchronic phencyclidine in a novel object recognition test in rats[END_REF][START_REF] De Bruin | Two novel 5-HT 6 receptor antagonists ameliorate scopolamineinduced memory deficits in the object recognition and object location tasks in Wistar rats[END_REF][START_REF] Hatcher | 5-HT 6 receptor antagonists improve performance in an attentional set shifting task in rats[END_REF][START_REF] Perez-Garcia | Oral administration of the 5-HT 6 receptor antagonists SB-357134 and SB-399885 improves memory formation in an autoshaping learning task[END_REF] The precise impact of 5-HT 6 R antagonists on learning and memory depends on the age of animals and the task used. [START_REF] Silva | Selective 5-HT 6 receptor blockade improves spatial recognition memory and reverses age-related deficits in spatial recognition memory in the mouse[END_REF][START_REF] Foley | The 5-HT 6 receptor antagonist SB-271046 reverses scopolamine-disrupted consolidation of a passive avoidance task and ameliorates spatial task deficits in aged rats[END_REF][START_REF] Woods | 5-HT 6 receptor agonists and antagonists enhance learning and memory in a conditioned emotion response paradigm by modulation of cholinergic and glutamatergic mechanisms[END_REF][START_REF] Meneses | Memory formation and memory alterations: 5-HT 6 and 5-HT 7 receptors, novel alternative[END_REF] Based on rodents' natural preference for novelty, the NOR test is regarded as an etiologically relevant paradigm for studying visual episodic memory. [START_REF] Ennaceur | A new one-trial test for neurobiological studies of memory in rats. 1: Behavioral data[END_REF] This paradigm is based on the spontaneous exploration of novel and familiar objects, and the NOR test has been used for investigating drug-induced (e.g., phencyclidine, scopolamine) cognitive deficits in psychiatric disorders, including schizophrenia and Alzheimer's disease. [START_REF] Grayson | Assessment of disease-related cognitive impairments using the novel object recognition (NOR) task in rodents[END_REF][START_REF] Lyon | Spontaneous object recognition and its relevance to schizophrenia: a review of findings from pharmacological, genetic, lesion and developmental rodent models[END_REF] Furthermore, as the use of long intertrial intervals (24 h) in cognitively unimpaired animals abolishes object discrimination, the NOR test is also a screening method for evaluating potential cognitive enhancers. In such conditions requiring longer retention of information, animals display poor memory that closely resembles natural forgetting. This test allows for determination of the procognitive properties of the evaluated compounds.

The procognitive effects of compounds 14 and 15 were assessed in the NOR task in rats. Following a single administration, 14 and 15 significantly reversed phencyclidine-induced episodic memory decline at doses of 1-3 mg/kg (ip) (Figure 5). The effects were comparable to that produced by SB-742457, which was used as an active comparator. It is worth noting that 15, which is the R enantiomer, produced a slightly stronger effect than its S congener.

The same compounds were further evaluated in the NOR test to determine their memory-enhancing effects 24 h after their administration in cognitively unimpaired rats (Figure 6). In contrast to PCP-impaired conditions, compounds 14 and SB-742457 (3 mg/kg, ip) strongly affected the ability of rats to distinguish between novel and familiar objects, indicating distinct procognitive effects.

Because anxiety and depression are common comorbidities of AD, the improvement of these behavioral symptoms may be considered a valuable therapeutic attribute in the treatment of patients with AD. Thus, to further extend the pharmacological evaluation of the newly designed compounds, their anxiolytic and antidepressant properties were evaluated in Wistar rats in commonly used screening procedures: the Vogel conflict drinking test [START_REF] Vogel | A simple and reliable conflict procedure for testing anti-anxiety agents[END_REF] and the modified forced swim test. [START_REF] Porsolt | Behavioural despair in rats: a new model sensitive to antidepressant treatments[END_REF][START_REF] Detke | Active behaviors in the rat forced swimming test differentially produced by serotonergic and noradrenergic antidepressants[END_REF] SB-742457 was used as a reference. Compound 14, given at a dose of 3 mg/kg (ip), demonstrated anticonflict activity in Vogel test by significantly increasing the number of shocks that were accepted by rats. This effect was almost 2-fold higher than that observed for SB-742457 (340% vs 178%) (Figure 7). Moreover, compounds 14 and 15

were both active at doses of 10 mg/kg (ip), a dose of this level increased the number of shocks that rats accepted by 787% and 729%, respectively.

In the forced swim test, the derivatives 14 (10 mg/kg, ip) and 15 (3 mg/kg, ip) and SB-742457 (3 mg/kg, ip) exerted significant antidepressant-like properties, with similar potencies; the effects were assessed according to the decreased immobility time and increased climbing time of the animals (Figure 8).

The results suggest that the mechanism of antidepressant action of the tested compounds is associated with noradrenergic, rather than serotonergic, neurotransmission, [START_REF] Detke | Active behaviors in the rat forced swimming test differentially produced by serotonergic and noradrenergic antidepressants[END_REF][START_REF] Reneric | Antidepressant behavioral effects by dual inhibition of monoamine reuptake in the rat forced swimming test[END_REF] which is in agreement with literature data for 5-HT 6 receptor antagonists. [START_REF] Lacroix | 5-HT 6 receptor antagonist SB-271046 enhances extracellular levels of monoamines in the rat medial prefrontal cortex[END_REF] The antidepressant-like activity of compounds 14, 15, and SB-742457 seems to be specific because they did not affect the rats' locomotor activity in the open-field paradigm (data not shown).

■ CONCLUSION

As part of ongoing efforts to develop novel procognitive therapies, we demonstrated that the scaffold-hopping approach could be efficiently applied to identify a structurally novel class of 5-HT 6 R antagonists in a group of 1H-pyrrolo[3,2-c]quinolines. Initial lead optimization around the arylsulfonyl fragment afforded compound 14, a more selective and potent 5-HT 6 R antagonist than the reference compound SB-742457. Further evaluation of the 5-HT 6 R's constitutive activity showed that 14 was classified as a neutral antagonist, while SB-742457 behaved as an inverse agonist. The high potency and different functional properties of 14 and SB-742457 that were demonstrated in in vitro tests translated well into in vivo procognitive properties either in PCP-induced memory decline (MED = 3 mg/kg) or in the natural forgetting model in NOR tasks (MED = 3 mg/kg). Compound 14 has additionally demonstrated a higher anxiolytic effect than SB-742457 in Vogel test (MED for 14 = 3 mg/kg) and showed similar antidepressant-like properties in 3-fold higher dose than that observed for SB-742457 in the forced swim test. The study evidenced comparable behavioral effects of 14 (5-HT 6 R antagonist) and SB-742457 (5-HT 6 R inverse agonist). These results support the therapeutic potential of 5-HT 6 R antagonists and/or inverse agonists for the treatment of cognitive decline and other symptoms associated with Alzheimer's disease. It seems that more detailed biochemical studies would provide further information about the action of 5-HT 6 R ligands. Likewise, their therapeutic potential in other neuropsychiatric disorders like autism or Parkinson's disease certainly warrants further exploration.

■ METHODS General Methods. The synthesis was carried out at ambient temperature, unless indicated otherwise. Organic solvents (from Aldrich and Chempur) were of reagent grade and were used without purification. The reagents were purchased from Sigma-Aldrich, Apeiron Synthesis, Chembridge, and Fluorochem. 1 H NMR and [START_REF] Ivachtchenko | Small molecule 5-HT 6 R ligands: A comprehensive insight into their selectivity and activity[END_REF] C NMR spectra were obtained in a Varian BB 200 spectrometer and were recorded at 300 and 75 MHz, respectively; J values are in hertz (Hz), and splitting patterns are designated as follows: s (singlet), d (doublet), t (triplet), and m (multiplet).

UPLC/MS were carried out on a system consisting of a Waters Acquity UPLC, coupled to a Waters TQD mass spectrometer. All the analyses were carried out using an Acquity UPLC BEH C18, 100 × 2.1 mm column, at 40 °C. A flow rate of 0.3 mL/min and a gradient of (0-100)% B over 10 min was used. Eluent A, water/0.1% HCO2H; eluent B, acetonitrile/0.1% HCO2H. Retention times t R were given in minutes. The UPLC/MS purity of all the test compounds and key intermediates was determined to be >99%.

HRMS analyses were performed at The Laboratory for Physical Measurements of the Universitéde Montpellier, on a Synapt G2-S (Waters) with ESI ionization mode.

Melting points were determined with a Buchi apparatus and are uncorrected.

Optical rotation was measured on a Jasco polarimeter (model P-2000, Eastan) using a sodium lamp, emitting light at a wavelength of 589 nm and 10 cm polarimetric tube. Measurements were performed in thermostatic conditions at 20 °C. Elemental analyses for C, H, and N were carried out using the elemental Vario EI III elemental analyzer (Hanau, Germany). Elemental analyses were found within ±0.4% of the theoretical values.

Synthetic Procedures. Methyl 2-[(2-Nitrophenyl)(p-toluenesulfonylamino)-methyl]acrylate (1). In a dried flask, p-toluenesulfonamide (18 g, 105 mmol, 1 equiv) and 1,4-diazabicyclo[2.2.2]octane (DABCO) (1.78 g, 15.7 mmol, 0.15 equiv) were mixed together with the previously activated molecular sieves (4 Å, 21 g). The mixture was suspended in isopropanol (300 mL), followed by addition of nitrobenzaldehyde (15.8 g, 105 mmol, 1 equiv) and methyl acrylate (10.7 mL, 115 mmol, 1.1 equiv). Subsequently, titanium isopropoxide (Ti(iOPr) 4 ) was added as a freshly prepared solution in isopropanol (0.6 mL, 2.1 mmol, 0.02 equiv). The flask was filled with nitrogen and the mixture was stirred at room temperature overnight. Then, a mixture was filtered through Celite, which was rinsed with CH 2 Cl 2 . The solvent was evaporated, and the remaining crude was dissolved in AcOEt (400 mL), washed three times with 1 M KHSO 4 , once with saturated NaHCO 3 , water, and brine, and dried over Na 2 SO 4 . Evaporation of the solvent gave a yellow oil which was subsequently dissolved in AcOEt (300 mL) and precipitated upon portionwise addition of n-hexane (300 mL). The appearing white precipitate was filtered, rinsed with diethyl ether and dried under vacuum.

White solid, 63% yield, t R = 6. Methyl 2-[(N-Allyl-N-tosylamino)(2-nitrophenyl)-methyl]acrylate (2). β-Aminoester 1 (10 g, 25.6 mmol, 1 equiv) was dissolved in DMF (100 mL), followed by addition of K 2 CO 3 (10.5 g, 76.5 mmol, 3 equiv). Subsequently, allyl bromide (4.42 mL, 51.2 mmol, 2 equiv) was added dropwise. The reaction was stirred at room temperature for 6 h. The mixture was diluted with ethyl acetate (300 mL) and washed 5× with water and brine, and dried over Na 2 SO 4 . The organic phase was filtered and hexane was added to precipitate a white solid, which was filtered and dried under vacuum.

White Methyl 2-(2-Aminophenyl)-1H-pyrrole-3-carboxylate (5). The nitro derivative 4 (2.5 g, 10.2 mmol, 1 equiv) was dissolved in methanol, and 10% Pd/C (180 mg, 7 weigh%) and acetic acid (0.26 mL, 4.6 mmol, 0.45 equiv) were subsequently added. The mixture was stirred under a hydrogen atmosphere at room temperature for 2 h. Then, the mixture was filtered though Celite, which was rinsed with methanol. The subsequent evaporation yielded an orange oil.

Orange 4-Chloro-1H-pyrrolo[3,2-c]quinoline (7). 80 mL of POCl 3 was added to the lactam 6 (2 g, 10.8 mmol, 1 equiv), and the mixture was heated at 105 °C for 4 h. Then, the mixture was evaporated under vacuum and 50 g of ice was added. After melting, the solution was neutralized with ammonia solution. The product was extracted three times with CH 2 Cl 2 . The organic layer was dried over Na 2 SO 4 , filtered, and evaporated, yielding a white solid.

White solid, 85% General Procedure for Preparation of Compounds 8a-8c. Compound 7 (0.35 g, 1.7 mmol, 1 equiv) was suspended in 12 mL of MeCN followed by addition of (R,S)-1-Boc-3-aminopyrrolidine (8a), (R)-1-Boc-3-aminopyrrolidine (8b) or (S)-1-Boc-3-aminopyrrolidine (8c) (1.3 g, 6.9 mmol, 4 equiv). The reaction was performed in a Biotage Microwave and stirred at 140 °C for 5h. The solvent was subsequently evaporated and the mixture was purified on silica gel with CH 2 Cl 2 / MeOH 9/1.5 as a developing solvent.

(R,S)-4-(1-tert-Butoxycarbonyl-pyrrolidine-3-yl-amino)-1Hpyrrolo [3,2-c] General Procedure for Preparation of Final Compounds 9-27. Compounds 8a-8c (100 mg, 0.28 mmol, 1 equiv) were dissolved in CH 2 Cl 2 (5 mL), and BTPP (171 μL, 0.56 mmol, 2 equiv) was added. The mixture was placed in an ice-bath, sulfonyl chloride (1.8 equiv) was added, and the reaction mixture was stirred for 3 h. Subsequently, the mixture was evaporated and the remaining yellow crude product was purified on silica gel. The Boc-protected derivatives were treated with 4 N HCl solution in dioxane to give the final products as HCl salts of secondary amines. (bs, 1H), 8.63 (dd, J = 8.50, 1.17 Hz, 1H), 9.52 (bs, 2H). [START_REF] Ivachtchenko | Small molecule 5-HT 6 R ligands: A comprehensive insight into their selectivity and activity[END_REF] obtained with 300 nM serotonin. Compounds were tested at a 10 -6 M concentration. Serotonin was used as a reference compound for determination of the agonist effect and methiothepin for the antagonistic effect. Experimental conditions are described online at www.cerep.fr.

Functional cAMP Assay Protocol. The antagonistic properties of compounds at the 5-HT 6 R were evaluated, as their ability to inhibit cAMP production induced by the agonist 5-CT (100 nM) in HEK293 cells overexpressing 5 HT 6 R. Each compound was tested in triplicate at 8 concentrations (10 -11 -10 -4 M). Cells (prepared with the use of Lipofectamine 2000) were maintained at 37 °C in a humidified atmosphere with 5% CO 2 and were grown in Dulbeco's modified Eagle's medium containing 10% dialyzed fetal bovine serum and 500 mg/mL G418 sulfate. For functional experiments, cells were subcultured in 25 cm 2 diameter dishes, grown to 90% confluence, washed twice with prewarmed (37 °C) phosphate buffered saline (PBS) and were centrifuged for 5 min (160g). The supernatant was aspirated, and the cell pellet was resuspended in stimulation buffer (1 × HBSS, 5 mM HEPES, 0.5 mM IBMX, 0.1% BSA). Total cAMP was measured using the LANCE cAMP detection kit (PerkinElmer), according to the manufacturer instructions. For cAMP levels quantification, cells (5 μL) were incubated with compounds (5 μL) for 30 min at room temperature, in a 384-well white opaque microtiter plate. After incubation, the reaction was stopped and cells were lysed by addition of 10 μL working solution (5 μL Eu-cAMP and 5 μL ULight-anti-cAMP). The assay plate was incubated for 1 h at room temperature. Time-resolved fluorescence resonance energy transfer (TR-FRET) was detected by an Infinite M1000 Pro (Tecan) using instrument settings from LANCE cAMP detection kit manual. K b values were calculated from 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] specific for the analysis of functional inhibition curves: K b = IC 50 /(1 + A/EC 50 ) where A is the agonist concentration, IC 50 is the antagonist concentration producing a 50% reduction in the response to agonist, and EC 50 is the agonist concentration, which causes a half of the maximal response.

Determination of cAMP Production as 5-HT 6 R Constitutive Activity. cAMP measurement was performed in NG108-15 cells transiently expressing 5-HT 6 receptor using the bioluminescence resonance energy transfer (BRET) sensor for cAMP, CAMYEL (cAMP sensor using YFP-Epac-RLuc). [START_REF] Jiang | Use of a cAMP BRET sensor to characterize a novel regulation of cAMP by the sphingosine 1-phosphate/G13 pathway[END_REF] NG108-15 cells were cotransfected in suspension with 5-HT 6 receptor and CAMYEL constructs, using Lipofectamine 2000, according to the manufacturer protocol, and plated in white 96-well plates (Greiner), at a density of 80 000 cells per well. At 24 h after transfection, cells were washed with PBS containing calcium and magnesium. Coelanterazine H (Molecular Probes) was added at a final concentration of 5 μM, and left at room temperature for 5 min. BRET was measured using a Mithras LB 940 plate reader (Berthold Technologies).

In Vivo Pharmacology. Novel Object Recognition Protocol. Procedures are based on earlier studies of Popik et al. [START_REF] Popik | 1-aminocyclopropanecarboxylic acid (ACPC) produces procognitive but not antipsychotic-like effects in rats[END_REF] The experiments were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Ethics Committee for Animal Experiments, Institute of Pharmacology. Male Sprague-Dawley rats (Charles River, Germany) weighing ∼250 g at the arrival were housed in the standard laboratory cages, under standard colony A/C controlled conditions: room temperature 21 ± 2 °C, humidity (40-50%), 12 h light/dark cycle (lights on at 06:00) with ad libitum access to food and water. Rats were allowed to acclimatize for at least 7 days before the start of the experimental procedure. During this week animals were handled for at least 3 times. Behavioral testing was carried out during the light phase of the light/dark cycle. At least 1 h before the start of the experiment, rats were transferred to the experimental room for acclimation. Rats were tested in a dimly lit (25 lx) "open field" apparatus made of a dull gray plastic (66 × 56 × 30 cm 3 ). After each measurement, the floor was cleaned and dried. Procedure consisted of habituation to the arena (without any objects) for 5 min, 24 h before the test and test session comprised of two trials separated by an intertrial interval (ITI). For PCP-induced memory impairment paradigm, 1 h ITI was chosen. For memory improvement paradigm, 24 h ITI was chosen. During the first trial (familiarization, T1) two identical objects (A1 and A2) were presented in opposite corners, approximately 10 cm from the walls of the open field. In the second trial (recognition, T2) one of the objects was replaced by a novel one (A = familiar and B = novel). Both trials lasted 3 min, and animals were returned to their home cage after T1. The objects used were the glass beakers filled with the gravel and the plastic bottles filled with the sand. The heights of the objects were comparable (∼12 cm) and the objects were heavy enough not to be displaced by the animals. The sequence of presentations and the location of the objects was randomly assigned to each rat. The animals explored the objects by looking, licking, sniffing or touching the object while sniffing, but not when leaning against, standing or sitting on the object. Any rat spending less than 5 s exploring the two objects within 3 min of T1 or T2 was eliminated from the study. Exploration time of the objects and the distance traveled were measured using the Any-maze video tracking system. Based on exploration time (E) of two objects during T2, discrimination index (DI) was calculated according to the formula: DI = (EB -EA)/(EA + AB). Phencyclidine (PCP), used to attenuate learning, was administered at the dose of 5 mg/kg (ip) 45 min before familiarization phase (T1). The compounds 14 or 15 were administrated ip 30 min before PCP injection. In nondisturbed paradigm, memory improvement, compounds were administrated ip 30 min before familiarization phase (T1).

Vogel Conflict Drinking Test. The testing procedure was based on a method of Vogel et al. [START_REF] Vogel | A simple and reliable conflict procedure for testing anti-anxiety agents[END_REF] as a conditional model where a noxious stimulus is applied. Anxiety Monitoring System Vogel test produced by TSE Systems (Germany) was used. It was consisted of polycarbonate cages (dimensions 26.5 × 15 × 42 cm 3 ), equipped with a grid floor made from stainless steel bars and drinking bottles containing tap water. Experimental chambers were connected to PC software by control chassis and electric shocks' generator. On the first day of the experiment, the rats were adapted to the test chambers and drink water from the bottle spout for 10 min. Afterward, the rats were returned to their home cages and were given 30 min free access to water followed by a 24 h water deprivation period. The adaptation session and water deprivation protocols were repeated on the second day of the experiment. On the third day, the rats were placed again in the test chambers 60 min after the compounds administration and were given free access to the drinking tube. Recording data started immediately after the first lick and rats were punished with an electric shock (0.5 mA, lasting 1 s) delivered to the metal drinking tube every 20 licks. The number of licks and the number of shocks received during a 5 min experimental session were recorded automatically.

The experiments were performed on male Wistar rats (230-260 g). The animals were housed in polycarbonate Makrolon type 3 cages (dimensions 26.5 × 15 × 42 cm 3 ) in an environmentally controlled room (ambient temperature 21 ± 2 °C; relative humidity 50-60%; 12:12 light/dark cycle, lights on at 08:00), in groups of four rats. Standard laboratory food (LSM-B) and filtered water were freely available. Animals were assigned randomly to treatment groups. Each experimental group consisted of 6-8 animals/dose, and the animals were used only once in each test. All the experiments were performed by three observers unaware of the treatment applied between 9:00 and 14:00 on separate groups of animals. All the experimental procedures were approved by the First Local Ethical Committee on Animal Testing at the Jagiellonian University in Krakoẃ. Compounds 14, 15, and SB-722457 were dissolved in distilled water and injected intraperitoneally (ip) at a volume of 2 mL/kg 60 min before the test procedures. Control animals received a vehicle injection according to the same schedule.

Modified Forced Swim Test in Rats. The experiment was carried out according to the method of Porsolt et al. modified by Detke et al. [START_REF] Porsolt | Behavioural despair in rats: a new model sensitive to antidepressant treatments[END_REF][START_REF] Detke | Active behaviors in the rat forced swimming test differentially produced by serotonergic and noradrenergic antidepressants[END_REF] On the first day of an experiment, the animals were gently individually placed in Plexiglas cylinders (50 cm high, 18 cm in diameter) containing 30 cm of water maintained at 23-25 °C for 15 min. On removal from water, the rats were placed for 30 min in a Plexiglas box under a 60-W bulb to dry. On the following day (24 h later), the rats were replaced in the cylinder and the total duration of immobility, swimming, and climbing was recorded during the whole 5 min test period. The swimming behavior entailed active swimming motions, for example, moving horizontally around in the cylinder. Climbing activity consisted of upward directed movements of the forepaws along the side of the swim chamber, and immobility was assigned when no additional activity was observed other than that necessary to keep the rat's head above the water. [START_REF] Lacroix | 5-HT 6 receptor antagonist SB-271046 enhances extracellular levels of monoamines in the rat medial prefrontal cortex[END_REF] Fresh water was used for each animal. Compounds 14, 15, and SB-742457 were dissolved in distilled water and injected intraperitoneally (ip) 60 min before the test.

Open field test in rats was performed using Motor Monitor System (Campden Instruments, Ltd., UK) consisting of two Smart Frame Open Field (OF) stations (40 × 40 × 38 cm 3 ) with 16 × 16 beams, located in sound attenuating chambers and connected to PC software by control chassis. Individual vehicle-or drug-injected animals were gently placed in the center of the station. An automated Motor Monitor System the total distance covered by a rat for 5 min.

The experiments were performed on male Wistar rats (230-260 g). The animals were housed in polycarbonate Makrolon type 3 cages (dimensions 26.5 × 15 × 42 cm 3 ) in an environmentally controlled room (ambient temperature 21 ± 2 °C; relative humidity 50-60%; 12:12 light/dark cycle, lights on at 08:00), in groups of four rats. Standard laboratory food (LSM-B) and filtered water were freely available. Animals were assigned randomly to treatment groups. Each experimental group consisted of 6-8 animals/dose, and the animals were used only once in each test. All the experiments were performed by three observers unaware of the treatment applied between 9:00 and 14:00 on separate groups of animals. All the experimental procedures were approved by the First Local Ethical Committee on Animal Testing at the Jagiellonian University in Krakoẃ.

■ ASSOCIATED CONTENT

* S Supporting Information

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschemneuro.6b00090. 1 H NMR and [START_REF] Ivachtchenko | Small molecule 5-HT 6 R ligands: A comprehensive insight into their selectivity and activity[END_REF] C NMR spectra for representative compounds (PDF)

■ AUTHOR INFORMATION

Corresponding Author *E-mail: pawel.zajdel@uj.edu.pl. Tel.: +48 126205450. Fax: +48 126205405.

Figure 1 .

 1 Figure 1. Representatives of 5-HT 6 R antagonists from various chemotypes.

Figure 2 .

 2 Figure 2. Structures of 5-HT 6 R antagonists evaluated in phase 3 clinical trials.

Figure 3 .

 3 Figure 3. Strategy to develop a new group of 5-HT 6 R antagonists.

a

  Scheme 1 a

a

  Mean K i values, based on three independent binding experiments. b Mean K i values (SEM ≤ 36%), based on at least two independent binding experiments. c Percent activity of agonist control response at 10 -6 M; performed at Eurofins Cerep. d Percent inhibition of control agonist response at 10 -6 M; performed at Eurofins Cerep e NT: not tested.

Figure 4 .

 4 Figure 4. Influence of compounds 14, 15, and SB-742457 on the 5-HT 6 R constitutive activity at Gs signaling in NG108-15 cells. (A) The 5-HT 6 receptor constitutively activates Gs-cAMP signaling in NG108-15 cells. Cells were transfected with an empty vector (control) or the plasmid encoding the 5-HT 6 receptor and were treated with vehicle (5-HT 6 ), WAY181187, or SB-742457 (1 μM each) for 5 min. (B) The NG108-15 cells expressing the 5-HT 6 receptor were exposed to incremental concentrations of either SB-742457 or 14 or 15 for 5 min. Cyclic AMP levels were estimated by BRET using the cAMP sensor CAMYEL. Data are the means ± SEM of the values obtained in three independent experiments that were performed in quadruplicate using different sets of cultured cells. ***p < 0.001 vs vehicle (ANOVA followed by Student-Newman-Keuls test).

Figure 5 .

 5 Figure 5. Effects of the selected compounds, 14, 15, and SB-742457, in the novel-object recognition test in rats performed in PCP-impaired conditions. Data are presented as the mean ± standard error of the mean of N = 7-10 animals per group. (A) T2, **p < 0.01 vs familiar, ***p < 0.001 vs familiar (paired Student's t test). (B) Discrimination index, *p < 0.05 vs vehicle, **p < 0.01 vs vehicle, ***p < 0.001 vs vehicle, ### p < 0.001 vs PCP (one-way ANOVA followed by Newman-Keuls post hoc test).

Figure 6 .

 6 Figure 6. Effects of the selected compounds 14 (3 mg/kg, ip), 15 (3 mg/kg, ip), and SB-742457 (1 mg/kg and 3 mg/kg, ip) in the novelobject recognition test performed in rats in cognitively unimpaired conditions. Data are presented as the mean ± standard error of the mean of N = 9-10 animals per group. (A) T2, **p < 0.01 vs familiar, ***p < 0.001 vs familiar (paired Student's t test). (B) Discrimination index, *p < 0.05 vs vehicle, ***p < 0.001 vs vehicle (one-way ANOVA followed by Dunnett's post hoc test).

Figure 7 .

 7 Figure 7. Anxiolytic-like effects of 14, 15, and SB-742457 in the Vogel conflict drinking test in rats. Values represent the mean ± standard error of the mean. *p < 0.05 vs vehicle, **p < 0.01 vs vehicle, ****p < 0.0001 vs vehicle (one-way ANOVA followed by Bonferroni's post hoc test); N = 7-8.

Figure 8 .

 8 Figure 8. Antidepressant-like properties of 14, 15 and SB-742457 in the modified forced swim test in rats. Values represent the mean ± standard error of the mean. *p < 0.05 vs vehicle, **p < 0.01 vs vehicle (ANOVA is followed by the Bonferroni's post hoc test); N = 7-8.

  (R,S)-1-(Phenylsulfonyl)-N-(pyrrolidin-3-yl)-1H-pyrrolo[3,2-c]quinolin-4-amine dihydrochloride (9). White solid, 80% yield, t R = 4.78, Mp 220-222 °C, Anal. (C 21 H 22 Cl 2 N 4 O 2 S•H 2 O) C, H, N. MW 483.41. 1 H NMR (300 MHz, DMSO-d 6 ) δ (ppm) 2.21 (d, J = 4.98 Hz, 1H), 2.31-2.45 (m, 1H), 3.56 (d, J = 4.98 Hz, 5H), 5.28 (bs, 1H), 7.44 (d, J = 7.04 Hz, 1H), 7.55-7.65 (m, 3H), 7.68-7.77 (m, 1H), 7.92 (d, J = 7.62 Hz, 2H), 8.19 (d, J = 3.52 Hz, 1H), 8.65 (d, J = 7.92 Hz, 1H), 9.50 (bs, 2H). Monoisotopic mass: 392.13, [M + H] + 393.1. HRMS calcd for C 21 H 21 N 4 O 2 S, 393.1385, found, 393.1389. (R,S)-1-[(3-Fluorophenyl)sulfonyl]-4-(pyrrolidine-3-yl-amino)-1Hpyrrolo[3,2-c]quinoline dihydrochloride (10). White solid, 87% yield, t R = 3.96, Mp 207-209 °C, Anal. (C 21 H 21 Cl 2 FN 4 O 2 S•H 2 O) C, H, N. MW 501.40. 1 H NMR (300 MHz, DMSO-d 6 ) δ (ppm) 2.15-2.26 (m, 1 H), 2.25-2.40 (m, 1H), 3.12-3.18 (m, 1H), 3.20-3.35 (m, 1H), 3.45-3.70 (m, 3H), 5.29 (bs, 1H), 7.41-7.46 (m, 1H), 7.58-7.71 (m, 3H), 7.78 (d, J = 7.06 Hz, 1H), 7.93 (d, J = 7.92 Hz, 1H), 8.06 (bs, 1H), 8.19 (d, J = 3.52 Hz, 1H), 8.39 (bs, 1H), 8.63 (d, J = 8.21 Hz, 1 H), 9.52 (bs, 2H). Monoisotopic mass 410.12, [M + H] + 411.3. HRMS calcd for C 21 H 20 FN 4 O 2 S, 411.1291; found, 411.1291. (S)-1-[(3-Fluorophenyl)sulfonyl]-4-(pyrrolidine-3-yl-amino)-1Hpyrrolo[3,2-c]quinoline dihydrochloride (11). White solid, 85% yield, t R = 3.95, Mp 197-199 °C, Anal. (C 21 H 21 Cl 2 FN 4 O 2 S•H 2 O) C, H, N, MW 501.40. 1 H NMR (300 MHz, DMSO-d 6 ) δ (ppm) 2.21 (d, J = 4.69 Hz, 1H), 2.37-2.42 (m, 1H), 3.23-3.34 (m, 2 H), 3.52-3.59 (m, 3H), 5.28 (bs, 1H), 7.43-7.47 (m, 1H), 7.58-7.69 (m, 2H), 7.77 (d, J = 7.04 Hz, 1H), 7.94 (d, J = 7.92 Hz, 1H), 8.05 (bs, 1H), 8.18 (d, J = 3.52 Hz, 1H), 8.39 (bs, 1H), 8.63 (d, J = 8.21 Hz, 1 H), 9.51 (bs, 2H).

  FN 4 O 2 S, 411.1291; found, 411.1292. (R)-1-[(3-Fluorophenyl)sulfonyl]-4-(pyrrolidine-3-yl-amino)-1Hpyrrolo[3,2-c]quinoline dihydrochloride (12). White solid, 90% yield, t R = 3.76, Mp 220-221 °C, Anal. (C 21 H 21 Cl 2 FN 4 O 2 S•H 2 O) C, H, N. MW 501.40. 1 H NMR (300 MHz, DMSO-d 6 ) δ (ppm) 2.14-2.27 (m, 1H), 2.33-2.45 (m, 1H), 3.20-3.35 (m, 2H), 3.50-3.67 (m, 3H), 5.30 (bs, 1H), 7.42-7.46 (m, 1H), 7.56-7.70 (m, 3H), 7.73-7.80 (m, 1H), 7.94 (d, J = 7.92 Hz, 1H), 8.07 (bs, 1H), 8.19 (d, J = 3.81 Hz, 1H), 8.40

  0998. (R,S)-1-{[3-(Trifluoromethyl)phenyl]sulfonyl}-4-(pyrrolidine-3-ylamino)-1H-pyrrolo[3,2-c]quinoline dihydrochloride (16). White solid, 90% yield, t R = 4.68, Mp 223-225 °C, Anal. (C 22 H 21 Cl 2 F 3 N 4 O 2 S•H 2 O) C, H, N. MW 551.41. 1 H NMR (300 MHz, CDCl 3 /methanol-d 4 ) δ (ppm) 2.22-2.44 (m, 1H), 2.53-2.74 (m, 1H), 3.32 (s, 2H), 3.66 (d, J = 10.18 Hz, 3H), 5.50-5.71 (m, 1H), 7.43 (d, J = 7.70 Hz, 1H), 7.51-7.68 (m, 2H), 7.81 (d, J = 7.70 Hz, 2H), 7.90-8.03 (m, 2H), 8.12 (s, 1H), 8.49 (d, J = 8.25 Hz, 1H), 8.79 (dd, J = 8.53, J = 0.83 Hz, 1H). Monoisotopic mass 460.12, [M + H] + 461.2. HRMS calcd for C 22 H 20 N 4 OF 3 S, 461.1259, found, 461.1259. (S)-1-{[3-(Trifluoromethyl)phenyl]sulfonyl}-4-(pyrrolidine-3-ylamino)-1H-pyrrolo[3,2-c]quinoline dihydrochloride (17

Table 2 .

 2 Antagonist Activity of the Selected Compounds 11, 12, 14, 15, and SB-742457 for the 5-HT 6 R Mean K b values (SEM ≤ 22%), based on three independent binding experiments. c For selectivity panel see ref 13.

	5-HT 6

a Mean K i values, based on three independent binding experiments. b

Table 1 .

 1 Binding Data of Compounds 9-27 for the 5-HT 6 R and Their Functional Activity for 5-HT 6 R, and Binding Data for 5-HT 1A , 5-HT 2A , 5-HT 7 , and D 2 Rs for the Selected Compounds

	K i [nM] a

  30, Mp 109-111 °C, C 18 H 18 N 2 O 6 S, MW 390.41. 1 H NMR (300 MHz, CDCl 3 ) δ (ppm) 2.40 (s, 3H), 3.57 (s, 3H), 5.68 (d, J = 0.77 Hz, 1H), 5.84 (d, J = 8.72 Hz, 1H), 6.08 (d, J = 8.46 Hz, 1H), 6.21 (s, 1H), 7.20-7.26 (m, 2H), 7.40 (m, J = 7.76, 1.41 Hz, 1H), 7.54 (m, J = 7.69, 1.28 Hz, 1H), 7.66-7.71 (m, 3H), 7.82 (dd, J = 8.08, 1.41 Hz, 1H). 13 C NMR (75 MHz, CDCl 3 ) δ (ppm) 21.1, 52.1, 54.4, 124.5, 127.2, 128.8, 129.1, 129.6, 130.1, 133.1, 133.4, 137.1, 137.4, 143.6, 148.0, 165.4. Monoisotopic mass 390.09, [M + H] + 391.4.

  Cl 2(30 mL) in a round-bottom flask, and the Grubbs II catalyst (297 mg, 0.35 mmol, 3 mol %) was added. The reaction was stirred at 36 °C for 30 min under microwave irradiation in the open vessel mode. DMSO (1.22 mL, 17.4 mmol, 1.5 equiv) was added and the mixture stirred at room temperature for 20 h. After this time, silica gel was added into the solution and it was stirred for 5 min. The mixture was diluted with CH 2 Cl 2 and filtered through a layer of silica gel. The filtrate was evaporated and the obtained residue was treated with diethyl ether giving a white precipitate which was filtered and dried under vacuum. To a solution of 2,5-dihydropyrrole 3 (2.5 g, 6.2 mmol, 1 equiv) in DMF (40 mL), sodium tert-butoxide (2.09 g, 18.7 mmol, 3 equiv) was added. The reaction was stirred for 2 h and monitored by TLC. The mixture was diluted with ethyl acetate, neutralized with 1 M KHSO 4 , and washed with a saturated solution of NaHCO 3 , three times with water, and once with brine. The organic layer was dried over Na 2 SO 4 and evaporated. The obtained crude product was purified on silica gel with AcOEt/Hex (4/6) as a developing solvent.Yellow oil, 85% yield, t R = 5.72, C 12 H 10 N 2 O 4 , MW 246.22. 1 H NMR (300 MHz, CDCl 3 ) δ (ppm) 3.62 (s, 3H), 6.64 (t, J = 2.80 Hz, 1H),6.72

	(t, J = 2.95 Hz, 1H) 6.82-6.85 (m, 1H) 7.44-7.48 (m, 1H) 7.51-7.58
	(m, 1H) 7.60-7.66 (m, 1H) 8.02 (dd, J = 8.21, 1.28 Hz, 1H). 13 C NMR
	(75 MHz, CDCl 3 ) δ (ppm) 51.02, 111.45, 113.60, 118.61, 124.37,
	127.29, 129.49, 132.54, 149.57, 164.67. Monoisotopic mass 246.06,
	[M + H] + 247.3.
	solid, 95% yield, t R = 7.48, Mp 83-85 °C, C 21 H 22 N 2 O 6 S, MW 430.47. 1 H NMR (300 MHz, CDCl 3 ) δ (ppm) 2.41 (s, 3H), 3.49-3.55
	(m, 3H), 3.92-4.01 (m, 1H), 4.15-4.25 (m, 1H), 4.92-5.03 (m, 2H),
	5.43-5.59 (m, 2H), 6.44 (s, 1H), 6.79 (s, 1H), 7.23-7.27 (m, 2H),
	7.42-7.49 (m, 1H), 7.61-7.68 (m, 3H), 7.78 (d, J = 7.69 Hz, 1H), 7.93
	(dd, J = 8.21 Hz, 1.28 Hz, 1H). Monoisotopic mass 430.12, [M + H] +
	430.8. HRMS calcd for C 21 H 23 N 2 O 6 S, 431.1277; found, 431.1277.
	Methyl 2,5-Dihydro-2-(2-nitrophenyl)-1-tosyl-1H-pyrrole-3-
	carboxylate (3). The β-aminoester 2 (5 g, 11.6 mmol, 1 equiv) was
	dissolved in CH 2 White solid, 80% yield, Mp 125-127 °C, t R = 6.83, C 19 H 18 N 2 O 6 S, MW 402.42. 1 H NMR (300 MHz, CDCl 3 ) δ (ppm) 2.36 (s, 3H), 3.45 (s,
	3H), 4.29 (m, 1H), 4.54 (m, 1H), 6.61-6.64 (m, 1H), 6.68 (t, J = 2.0 Hz,
	1H), 7.27 (d, J = 8.27 Hz, 2H), 7.31-7.36 (m, 1H), 7.48-7.50 (m, 2H),
	7.74 (d, J = 8.27 Hz, 2H), 7.83 (d, J = 8.22 Hz 1H). 13 C NMR (75 MHz,
	CDCl 3 ) δ (ppm) 21.56, 52.03, 55.73, 62.29, 124.54, 127.75, 128.62,
	129.64, 130.04, 133.16, 133.38, 134.91, 135.68, 136.76, 144.25, 148.70,
	161.57. Monoisotopic mass 402.09, [M + H] + 403.3. HRMS calcd for
	C 19 H 19 N 2 O 6 S, 403.0964; found, 403.0967.

Methyl 2-(2-Nitrophenyl)-1H-pyrrole-3-carboxylate (4).

  oil, 95% yield, t R = 4.84, C 12 H 12 N 2 O 2 , MW 216.24. Compound 5 (2.5 g, 11.6 mmol, 1 equiv) was suspended in sec-butanol (50 mL), and acetic acid (0.5 mL, 8.7 mmol, 0.75 equiv) was added. The reaction mixture was stirred at 70 °C for 3 h. The solvent was evaporated under vacuum giving an orange oil. Orange oil, 99% yield, t R = 2.97, C 11 H 8 N 2 O, MW 184.19. 1 H NMR (300 MHz, methanol-d 4 ) δ (ppm) 6.81 (d, J = 3.08 Hz, 1H), 7.19 (d, J = 3.08 Hz, 1H), 7.23-7.30 (m, 1H), 7.36-7.45 (m, 2H), 7.97 (d, J = 7.69 Hz, 1H). 13 C NMR (75 MHz, DMSO-d 6 ) δ (ppm) 105.53, 113.45, 115.20, 116.25, 121.81, 123.02, 127.51, 134.83, 136.53, 160.20, 172.54. Monoisotopic mass 184.06, [M + H] + 185.0. HRMS calcd for C 11 H 9 N 2 O ,185.0715; found, 185.0714.

	1 H NMR
	(300 MHz, CDCl 3 /methanol-d 4 ) δ (ppm) 3.60-3.62 (m, 3H) 6.59
	(d, J = 3.08 Hz, 1H) 6.67-6.70 (m, 2H) 6.71-6.73 (m, 1H) 7.04-7.11
	(m, 2H). Monoisotopic mass 216.09, [M + H] + 217.0.
	1H-Pyrrolo[3,2-c]quinolin-4(5H)-one (6).

  J = 7.31 Hz, 1H), 7.71-7.81 (d, J = 8.46 Hz, 1H), 7.86 (dd, J = 7.95, 1.28 Hz, 1H).[START_REF] Ivachtchenko | Small molecule 5-HT 6 R ligands: A comprehensive insight into their selectivity and activity[END_REF] C NMR (75 MHz, DMSO-d 6 ) δ (ppm) 28.65, 44.59, 44.75, 50.56, 52.05, 78.65, 102.14, 111.10, 115.93, 120.63, 121.24, 122.72, 126.41, 134.96, 144.33, 152.01, 154.11. Monoisotopic mass 352.19, [M + H] + 353.2. HRMS calcd for C 20 H 25 N 4 O 2 , 353.1978; found, 353.1978. Orange oil, 62% yield, t R = 4.38, C 20 H 24 N 4 O 2 , MW 352.43. 1 H NMR (300 MHz, DMSO-d 6 ) δ (ppm) 1.37-1.50 (m, 9H), 2.00 (bs, 1H), 2.30 (bs, 1H), 3.28-3.56 (m, 4H), 3.70-3.85 (m, 1H), 4.92 (bs, 1H), 6.50-5.58 (d, J = 3.08 Hz, 1H), 7.10-7.15 (d, J = 3.08 Hz, 1H), 7.17-7.24 (m, 1H), 7.25-7.28 (m, 1H), 7.34-7.43 (t, J = 7.18 Hz, 1H), 7.72-7.82 (d, J = 7.95 Hz, 1H), 7.83-7.90 (dd, J = 7.95, J = 1.03 Hz, 1H). Monoisotopic mass 352.19, [M + H] + 353.2. HRMS calcd for C 20 H 25 N 4 O 2 , 353.1978; found, 353.1978.

	(S)-4-(1-tert-Butoxycarbonyl-pyrrolidine-3-yl-amino)-1H-pyrrolo-
	[3,2-c]quinoline (8c). Orange oil, 60% yield, t R = 4.38, C 20 H 24 N 4 O 2 , MW 352.43. 1 H NMR (300 MHz, DMSO-d 6 ) δ (ppm) 1.35-1.49 (m,
	9H) 1.98 (bs, 1H) 2.30 (bs, 1H) 3.27-3.56 (m, 4H) 3.71-3.81 (m, 1H)
	4.93 (bs, 1H) 6.55 (d, J = 3.08 Hz, 1H) 7.13 (d, J = 3.08 Hz, 1H)
	7.16-7.23 (m, 1H) 7.25-7.27 (m, 1H) 7.33-7.34 (t, J = 7.31 Hz, 1H,)
	7.71-7.81 (d, J = 8.46 Hz, 1H) 7.86 (dd, J = 7.95, 1.28 Hz, 1H).
	Monoisotopic mass 352.19, [M + H] + 353.2. HRMS calcd for
	C 20 H 25 N 4 O 2 , 353.1978, found, 353.1978.
	quinoline (8a). Orange oil, 60% yield, t R = 4.38, C 20 H 24 N 4 O 2 , MW 352.43. 1 H NMR (300 MHz, DMSO-d 6 ) δ (ppm)
	1.40 (s, 9H), 1.98 (bs, 1H), 2.30 (bs, 1 H), 3.27-3.56 (m, 4H),
	3.71-3.81 (m, 1 H), 4.93 (bs, 1H), 6.55 (d, J = 3.08 Hz, 1H), 7.13 (d,
	J = 3.08 Hz, 1H), 7.16-7.23 (m, 1H), 7.25-7.27 (m, 1H), 7.33-7.34 (t,

(R)-4-(1-tert-Butoxycarbonyl-pyrrolidine-3-yl-amino)-1H-pyrrolo-[3,2-c]quinoline (8b).

  [START_REF] Ivachtchenko | Small molecule 5-HT 6 R ligands: A comprehensive insight into their selectivity and activity[END_REF] C NMR (75 MHz, DMSO-d 6 ) δ (ppm) 31.07, 44.22, 49.18, 52.49, 108.61, 113.01, 114.85, 115.20, 115.89, 120.49, 123.73, 124.02, 125.46, 130.41, 130.58, 133.41, 134.47, 138.65, 148.62, 160.56, 163.88. Monoisotopic mass 410.12, [M + H] + 411.3. HRMS calcd for C 21H 20

  C NMR (75 MHz, DMSO-d 6 ) δ ppm 31.18, 44.19, 49.10, 52.73, 108.80, 112.94, 114.86 115.20, 115.89, 120.26, 123.73, 124.03, 125.56, 130.16, 130.63, 133.42, 134.44, 138.63, 148.47, 160.53, 163.86. Monoisotopic mass 410.12, [M + H] + 411.3. HRMS calcd for C 21 H 20 FN 4 O 2 S, 411.1291; found, 411.1291. (R,S)-1-[(3-Chlorophenyl)sulfonyl]-4-(pyrrolidine-3-yl-amino)-1Hpyrrolo[3,2-c]quinoline dihydrochloride (13). White solid, 90% yield, t R = 4.13, Mp 220-221 °C, Anal. (C 21 H 21 Cl 3 N 4 O 2 S•H 2 O) C, H, N. MW 517.85. 1 H NMR (300 MHz, DMSO-d 6 ) δ (ppm) 2.20-2.25 (m, 1H), 2.36-2.43 (m, 1H), 3.25-3.36 (m, 1H), 3.49-3.57 (m, 4H), 5.24 (s, 1H), 7.36-7.46 (m, 1H), 7.63 (t, J = 7.96 Hz, 2H), 7.81-7.83 (m, 1H), 7.88-8.08 (m, 2H), 8.19-8.20 (m, 1H), 8.30-8.33 (m, 1H), 8.63-8.66 (m, 1H), 9.46 (s, 2H). Monoisotopic mass 426.09, [M + H] + 427.2. HRMS calcd for C 21 H 20 ClN 4 O 2 S, 427.0995; found, 427.0996. J = 8.59, 1.15 Hz, 1H). 13 C NMR (75 MHz, DMSO-d 6 ) δ ppm 31.16, 44.21, 49.11, 52.70, 108.85, 112.93, 115.91, 120.30, 123.69, 125.56, 126.41, 127.18, 130.19, 130.67, 132.77, 134.42, 135.29, 136.14, 138.49, 148.45. [α] D 20 = +2.93°(0.40, EtOH/H 2 O 9/1). Monoisotopic mass 426.09, [M + H] + 427.2. HRMS calcd for C 21 H 20 ClN 4 O 2 S, 427.0995; found, 427.0992. (R)-1-[(3-Chlorophenyl)sulfonyl]-4-(pyrrolidine-3-yl-amino)-1Hpyrrolo[3,2-c]quinoline dihydrochloride (15). White solid, 91% yield, t R = 4.18, Mp 221-223 °C, Anal. (C 21 H 21 Cl 3 N 4 O 2 S•H 2 O) C, H, N. MW 517.85. 1 H NMR (300 MHz, CDCl 3 /methanol-d 4 ) δ (ppm) 2.24-2.40 (m, 1H), 2.52-2.69 (m, 1H), 3.27-3.44 (m, 2H), 3.56-3.79 (m, 3H), 5.46-5.68 (m, 1H), 7.35-7.46 (m, 1H), 7.52-7.65 (m, 2H), 7.75-7.85 (m, 2H), 7.92-8.02 (m, 2H), 8.05-8.20 (bs, 1H), 8.48 (d, J = 8.25 Hz, 1H), 8.76 (dd, J = 8.53, J = 1.10 Hz, 1H). 13 C NMR (75 MHz, DMSO-d 6 ) δ ppm 31.16, 44.21, 49.11, 52.72, 108.85, 112.92, 115.90, 120.33, 123.69, 125.57, 126.41, 127.18, 130.20, 130.67, 132.77, 134.42, 135.29, 136.14, 138.48, 148.44. [α] D 20 = -5.65°(0.48, EtOH/ H 2 O 9/1). Monoisotopic mass 426.09, [M + H] + 427.2. HRMS calcd for C 21 H 20 ClN 4 O 2 S, 427.0995; found, 427.

	(S)-1-[(3-Chlorophenyl)sulfonyl]-4-(pyrrolidine-3-yl-amino)-1H-
	pyrrolo[3,2-c]quinoline dihydrochloride (14). White solid, 92% yield,
	t R = 4.18, Mp 219-221 °C, Anal. (C 21 H 21 Cl 3 N 4 O 2 S•H 2 O) C, H, N. MW 517.85. 1 H NMR (300 MHz, CDCl 3 /methanol-d 4 ) δ (ppm) 2.25-
	2.38 (m, 1H), 2.54-2.68 (m, 1H), 3.23-3.35 (m, 1H), 3.57-3.76 (m,
	4H), 5.50-5.64 (m, 1H), 7.35-7.45 (m, 2H), 7.48-7.61 (m, 3H),
	7.68-7.72 (m, 1H), 7.84-7.95 (m, 2H), 8.46 (d, J = 8.46 Hz, 1H), 8.75
	(dd,

  ). White solid, 91% yield, t R = 4.67, Mp 219-221 °C, Anal. (C 22 H 21 Cl 2 F 3 N 4 O 2 S•H 2 O) C, H, N, MW 551.41. 1 H NMR (300 MHz, DMSO-d 6 ) δ (ppm) 2.21 (d, C NMR (75 MHz, DMSO-d 6 ) δ ppm 31.18, 44.19, 49.10, 52.70, 108.80, 112.94, 114.86, 115.20, 115.89, 120.26, 123.35, 123.73, 124.02, 125.56, 130.16, 130.63, 133.42, 134.44, 138.61, 148.47, 160.53, 163.86.

	J = 5.28 Hz, 1H), 2.33-2.44 (m, 1H), 3.48 (dd, J = 11.58, 5.72 Hz, 2H),
	3.62-3.68 (m, 3H), 5.29 (bs, 1H), 7.46 (t, J = 7.48 Hz, 1H), 7.64 (t, J =
	7.77 Hz, 1H), 7.79-7.89 (m, 1H), 8.01-8.20 (m, 2H), 8.25 (d, J =
	3.52 Hz, 1H), 8.39 (bs, 2H), 8.67 (d, J = 7.92 Hz, 1H), 9.51 (bs, 2H).
	13 C NMR (75 MHz, DMSO-d 6 ) δ ppm 31.08, 44.22, 49.17, 52.47,
	108.66, 113.03, 114.85, 115.20, 115.91, 120.49, 123.34, 123.73, 124.01,
	125.46, 130.14, 130.58, 133.41, 134.47, 138.66, 148.62, 160.54, 163.87.
	[α] D 20 = +3.50°(0.49, EtOH/H 2 O 9/1). Monoisotopic mass 460.12, [M + H] + 461.2. HRMS calcd for C 22 H 20 N 4 OF 3 S, 461.1259, found,
	461.1258.
	(R)-1-{[3-(Trifluoromethyl)phenyl]sulfonyl}-4-(pyrrolidine-3-yl-
	amino)-1H-pyrrolo[3,2-c]quinoline dihydrochloride (18). White solid,
	88% yield, t R = 4.67, Mp 230-232 °C, Anal. (C 22 H 21 Cl 2 F 3 N 4 O 2 S•H 2 O) C, H, N. MW 551.41. 1 H NMR (300 MHz, DMSO-d 6 ) δ (ppm) 2.11-
	2.27 (m, 1H), 2.30-2.45 (m, 1H), 3.20-3.35 (m, 1H), 3.40-3.52 (m,
	1H), 3.60-3.82 (m, 3H), 5.27 (bs, 1H), 7.42-7.46 (m, 1H), 7.63 (t,
	J = 7.04 Hz, 1H), 7.79-7.87 (m, 1H), 8.01-8.20 (m, 2H), 8.25 (d, J =
	2.93 Hz, 1H), 8.39 (bs, 2H), 8.67 (d, J = 8.50 Hz, 1H), 9.47 (bs, 2H).

[START_REF] Ivachtchenko | Small molecule 5-HT 6 R ligands: A comprehensive insight into their selectivity and activity[END_REF]

DOI: 10.1021/acschemneuro.6b00090 ACS Chem. Neurosci. 2016, 7, 972-983

Funding

The study was financially supported by the Polish Ministry of Science and Higher Education (MNiSW), Grant No. N N405 671540, the project "Prokog", UDA-POIG.01.03.01-12-063/09-00, cofinanced by the European Union from the European Fund of Regional Development (EFRD), and by the University Montpellier, CNRS. F.L. and P.M. were supported by a grant from Institut Carnot Chimie Balard and Pole BioSantéRabelais. K.G. thanks the French Embassy in Warsaw for the cotutelle fellowship.

Notes

The authors declare no competing financial interest.