1-(Arylsulfonyl-isoindol-2-yl)piperazines as 5-HT6R Antagonists: Mechanochemical Synthesis, In Vitro Pharmacological Properties and Glioprotective Activity

In addition to the canonical Gs adenylyl cyclase pathway, the serotonin type 6 receptor (5-HT6R) recruits additional signaling pathways that control cognitive function, brain development, and synaptic plasticity in an agonist-dependent and independent manner. Considering that aberrant constitutive and agonist-induced active states are involved in various pathological mechanisms, the development of biased ligands with different functional profiles at specific 5-HT6R-elicited signaling pathways may provide a novel therapeutic perspective in the field of neurodegenerative and psychiatric diseases. Based on the structure of SB-258585, an inverse agonist at 5-HT6R-operated Gs and Cdk5 signaling, we designed a series of 1-(arylsulfonyl-isoindol-2-yl)piperazine derivatives and synthesized them using a sustainable mechanochemical method. We identified the safe and metabolically stable biased ligand 3g, which behaves as a neutral antagonist at the 5-HT6R-operated Gs signaling and displays inverse agonist activity at the Cdk5 pathway. Inversion of the sulfonamide bond combined with its incorporation into the isoindoline scaffold switched the functional profile of 3g at Gs signaling with no impact at the Cdk5 pathway. Compound 3g reduced the cytotoxicity of 6-OHDA and produced a glioprotective effect against rotenone-induced toxicity in C8-D1A astrocyte cell cultures. In view of these findings, compound 3g can be considered a promising biased ligand to investigate the role of the 5-HT6R-elicited Gs and Cdk5 signaling pathways in neurodegenerative diseases.


Introduction
The serotonin type 6 receptor (5-HT 6 R) is a Gs-coupled receptor widely expressed in brain regions involved in cognitive functions [1] and has been considered an important target to alleviate cognitive symptoms related to neurodegenerative and psychiatric disorders [2][3][4][5][6]. Several lines of evidence indicate that modulation of cognition by 5-HT 6 R ligands might be independent of its coupling to Gs, suggesting the engagement of alternative signaling mechanisms. Indeed, interactomic studies revealed that the 5-HT 6 R is linked to different GIPs (GPCR interacting proteins) that underlie its modulation of cognitive (mechanistic target of rapamycin (mTOR)) and neuro-developmental (cyclin-dependent brain regions involved in cognitive functions [1] and has been considered an important target to alleviate cognitive symptoms related to neurodegenerative and psychiatric disorders [2][3][4][5][6]. Several lines of evidence indicate that modulation of cognition by 5-HT6R ligands might be independent of its coupling to Gs, suggesting the engagement of alternative signaling mechanisms. Indeed, interactomic studies revealed that the 5-HT6R is linked to different GIPs (GPCR interacting proteins) that underlie its modulation of cognitive (mechanistic target of rapamycin (mTOR)) and neuro-developmental (cyclin-dependent kinase 5 (Cdk5) and G protein-regulated inducer of neurite outgrowth 1 (GPR1N1)) processes [7,8]. Recently, a non-canonical 5-HT6R-elicited Gαq/11-RhoA pathway, which modulates nuclear actin and increases histone acetylation and chromatin accessibility, has been identified in hippocampal neurons [9]. An important feature of the 5-HT6R is its high level of ligand-independent constitutive activity, which corresponds to the ability of a receptor to be active even in the absence of an agonist [10]. The role of 5-HT6R constitutive activity at canonical Gs signaling and non-canonical signaling has been described in various pathophysiological conditions [11][12][13][14][15]. Consistent with those findings, the development of 5-HT6R ligands acting as inverse agonists and/or neutral antagonists at the different 5-HT6R-operated signaling pathways is of utmost interest to decipher the cellular mechanism(s) under the control of the various 5-HT6R signal transduction mechanisms and of constitutive vs. agonist-dependent receptor activation.
These observations prompted us to investigate further the role of the arylsulfonamide fragment in the development of active 5-HT 6 R ligands. Starting from SB-258585, an inverse agonist at 5-HT 6 R-operated Gs and Cdk5 signaling [20,21], we designed a set of 1-(arylsulfonyl-isoindol-2-yl)piperazines. We focused on the inversion of the sulfonamide bond combined with its incorporation into differently substituted isoindoline scaffold and flipping of the piperazine moiety in the 2-, 3-, or 4-position at the phenyl ring ( Figure 2). These observations prompted us to investigate further the role of the arylsulfonamide fragment in the development of active 5-HT6R ligands. Starting from SB-258585, an inverse agonist at 5-HT6R-operated Gs and Cdk5 signaling [20,21], we designed a set of 1-(arylsulfonyl-isoindol-2-yl)piperazines. We focused on the inversion of the sulfonamide bond combined with its incorporation into differently substituted isoindoline scaffold and flipping of the piperazine moiety in the 2-, 3-, or 4-position at the phenyl ring ( Figure 2). . Figure 2. Design of a 1-(arylsulfonyl-isoindol-2-yl)piperazines library.
The impact of compounds synthesized using a mechanochemical approach on 5-HT6R-operated Gs and Cdk5 signaling pathways was examined. The glioprotective properties of the selected compound with the highest potency at Gs and Cdk5 signaling and favorable preliminary ADMET profile were investigated in C8-D1A astrocytes exposed to the gliotoxic treatments using 6-hydroxydopamine (6-OHDA) and rotenone (ROT).

General Chemical Methods
All commercially available reagents were of the highest purity from Fluorochem, Across Organic. The milling treatments were carried out in a vibratory ball-mill Retsch MM400 operated at 30 Hz. The milling load was defined as the sum of the mass of the reactants per free volume in the jar and was equal to 10 mg/mL. All reactions using the vibratory ball mill were performed under air.

General Procedure A for Sulfonylation of Isoindoline Derivatives to Obtain Intermediates 2a-l
Intermediates 1a-d (1 eq), different fluoro-substituted benzenesulfonyl chloride (1.1 eq), and NaOH (3 eq) were introduced in a 35 mL SS jar (milling load 10 mg/mL) with one SS ball (ϕball = 1.5 cm). The reaction was carried out for 5-7 min at rt. The mixture was The impact of compounds synthesized using a mechanochemical approach on 5-HT 6 Roperated Gs and Cdk5 signaling pathways was examined. The glioprotective properties of the selected compound with the highest potency at Gs and Cdk5 signaling and favorable preliminary ADMET profile were investigated in C8-D1A astrocytes exposed to the gliotoxic treatments using 6-hydroxydopamine (6-OHDA) and rotenone (ROT).

General Chemical Methods
All commercially available reagents were of the highest purity from Fluorochem, Across Organic. The milling treatments were carried out in a vibratory ball-mill Retsch MM400 operated at 30 Hz. The milling load was defined as the sum of the mass of the reactants per free volume in the jar and was equal to 10 mg/mL. All reactions using the vibratory ball mill were performed under air.

General Procedure A for Sulfonylation of Isoindoline Derivatives to Obtain Intermediates 2a-l
Intermediates 1a-d (1 eq), different fluoro-substituted benzenesulfonyl chloride (1.1 eq), and NaOH (3 eq) were introduced in a 35 mL SS jar (milling load 10 mg/mL) with one SS ball (φ ball = 1.5 cm). The reaction was carried out for 5-7 min at rt. The mixture was transferred into a filtration funnel, washed with distilled water, and dried under reduced pressure yielding intermediate 2a-l in high yields (85-98%).

General Procedure B for Aromatic Substitution to Obtain Final Compounds 3a-l
Intermediates 2a-l (1 eq) and anhydrous piperazine (3 eq) were introduced in a 10 mL SS jar (milling load 10 mg/mL) with one stainless steel ball (φ ball = 1.5 cm) followed by the addition of MeCN (34 µL, η = 0.4 µL mg −1 ) as a liquid assistant. The reaction was carried out for 1.5 h at 50 • C. In the case of intermediates 2e, 2f, 2g, and 2h, DMSO (42 µL, η = 0.4 µL mg −1 ) were used as a liquid additive, and the reactions were milled for 1.5 h at 80 • C. After the addition of cold water (5 mL), the resulting mixture was transferred into a filtration funnel, washed with distilled water, and dried under reduced pressure. The products were purified via crystallization in ethanol, yielding final compounds 3a-l as a white powder (yields 75-88%).
2.1.4. General Procedure C for One-Pot Two-Step Reaction for Obtaining Compounds 3e, 3f, and 3g Isoindoline derivative (1 eq), sodium hydroxide (3 eq), and 3-fluorobenzenesulfonyl chloride (1.1 eq) were introduced in a 35 mL SS jar (milling load 10 mg/mL) with one stainless steel ball (φ ball = 1.5 cm). After 10 min of milling at rt, anhydrous piperazine (3 eq) and DMSO (140 µL, η = 0.4 µL mg −1 ) were added into the jar, and the reaction was carried out for an additional 60 min at 80 • C. The resulting mixture was transferred into a filtration funnel, washed with distilled water, and dried under reduced pressure. The products were purified via crystallization in ethanol, yielding final compounds 3e, 3f, and 3g as white powders (yields 85-88%).

Impact of Evaluated Compounds on cAMP Production in 1321N1 Cells
The ability of compounds 3e, 3f, and 3g to inhibit 5-CT-induced production of cAMP was assessed using 1321N1 cells expressing the human 5-HT 6 R (PerkinElmer) using previously described procedures [15,17]. The level of cAMP was measured using the LANCE cAMP detection kit (PerkinElmer) according to the manufacturer's protocol. TR-FRET (Time Resolved Fluorescence Resonance Energy Transfer) was detected by an Infinite M1000 Pro (Tecan) using instrument settings from the LANCE cAMP detection kit manual. Tested compounds were evaluated at 8 concentrations (10 −11 -10 −4 M) in triplicate. K b values were calculated from the Cheng-Prusoff equation [25]. A detailed description is reported in the Supporting Information.

Impact of Evaluated Compounds on cAMP Production Elicited by Constitutively Active 5-HT 6 R in NG108-15 Cells
The functional properties at Gs signaling of 3e, 3f, 3g, and the prototypic inverse agonist SB-258585 were evaluated using NG108-15 cells transiently expressing 5-HT 6 R and the cAMP sensor CAMYEL (cAMP sensor using YFP-Epac-RLuc) [26]. We previously demonstrated that the 5-HT 6 R displays a high level of constitutive activity in this model. Changes in cyclic AMP levels upon exposure to increasing concentrations of the tested compounds were assessed by BRET (Bioluminescence Resonance Energy Transfer) measurement using a Mithras LB 940 plate reader (Berthold Technologies), as previously described [17]. A detailed description is reported in the Supporting Information.

Impact of Tested Compounds on Cdk5-Dependent Neurite Growth
The inverse agonist properties at Cdk5 signaling of 3e, 3f, 3g, and the prototypic inverse agonist SB-258585 were evaluated in NG108-15 cells transiently expressing 5-HT 6 R by measuring neurite outgrowth, as previously reported. NG108-15 cells were transfected with plasmids encoding either cytosolic GFP or a GFP-tagged 5-HT 6 R and grown on glass coverslips for 6 h. Then, cells were treated with either vehicle or the evaluated compounds. Neurite growth was measured on cells imaged using an AxioImagerZ1 microscope equipped with epifluorescence (Zeiss) using the Neuron J plugin of the ImageJ software (NIH). A detailed description is reported in the Supporting Information.

In Vitro Assessment of Metabolic Stability
Metabolic stability studies were performed according to reported protocols [27,28]. Evaluated compounds 3e, 3f, and 3g at the final concentration of 20 µM were preincubated in phosphate buffer (pH = 7.4), containing rat liver microsomes (microsome from rat male liver, pooled; 0.5 mg/mL; Merck/Millipore Sigma, Darmstadt, Germany, St. for 10 min at 37 • C. The reaction was initiated by adding the NADPH-regenerating system. After 0, 30, and 60 min, reactions were quenched with ice-cold methanol containing internal standard (pentoxifylline @ 100 nM). Next, samples were centrifuged, and the supernatants were analyzed by UPLC/MS. T 1/2 was determined from the slope of the line on Ln (%remaining of parent compounds) vs. time plots. Cl int was calculated from the equation: Cl int = [volume of incubation (µL)/amount of protein (mg) × 0.693]/t 1/2 . All samples were analyzed in duplicate. The assay performance was confirmed by using extensive or low metabolized drugs as references (Imipramine and Donepezil, respectively).

In Vitro Cytotoxicity Evaluation
Hepatotoxicity, neurotoxicity, and glial cytotoxicity were investigated using the following cellular models: human hepatocellular carcinoma (HepG2), human neuroblastoma (SHSY-5Y), and mouse astrocytes (C8-D1A), all from ATCC (American Type Culture Collection, Manassas, VA, USA). Cells were cultured in appropriate culture media recommended by ATCC supplemented with 10% fetal bovine serum (FBS; Gibco, Life Technologies, Carlsbad, CA, USA) and 1% antibiotic mixture (Gibco, Life Technologies, Carlsbad, CA, USA). Cells were cultured at standard conditions (5% CO 2 , 95% humidity, 37 • C). For cytotoxicity experiments, cells were seeded into 96 well plates at an initial density of 2000 cells/well. After overnight incubation, cells were treated with 3e, 3f, and 3g in a concentration range of 0.78-50 µM for 48 h; then, an MTT assay was performed as described previously [17,29]. Briefly, following cell exposure, 10 µL of MTT reagent was added to each well. After the next 4 h, the medium was aspirated, and formazan produced in cells appeared as dark crystals in the bottom of the wells. Next, 100 µL DMSO was added to each well. Then, the optical density of the solution (OD) at 570 nm was determined on a plate reader (Spectra Max iD3, Molecular Devices, San Jose, CA, USA). The experiments were run three times in triplicates.

In Vitro Assessment of Glioprotective Properties
Mouse astrocytes (C8-D1A cell line, ATCC) were cultured in DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10% FBS (FBS; Gibco, Life Technologies, Carlsbad, CA, USA) and 1% antibiotic mixture (Gibco, Life Technologies, Carlsbad, CA, USA) at standard condition until the cells reached 80-90% confluence. Cells were harvested and seeded in 96-well plates at a density of 5000 cells/well. All experiments were conducted with a reduced amount of serum equal to 2.5%. The optimal amount of serum was determined experimentally on the basis of preliminary studies. After 24 h, cells were pre-incubated in the presence of compound 3g or references CPPQ, SB-258585, and WAY-181187 (all at 0.25 µM). Then 6-OHDA (20 µM) or rotenone (0.5 µM) were added and co-incubated for the next 24 h. The ability of tested compounds to prevent 6-OHDA-induced or rotenoneinduced cytotoxicity was assessed by MTT assay following the protocol described above. The experiments were run three times in triplicates.

Mechanochemical Synthesis of Compounds 3a-3l
A medicinal mechanochemical approach [28,[30][31][32] was employed for the synthesis of a novel group of 1-(arylsulfonyl-isoindol-2-yl)piperazines 3a-3l (Scheme 1). The optimization of the synthetic process started with the sulfonylation of unsubstituted isoindoline with the 4-fluorobenzenesulfonyl chloride (1.1 eq) (Table S1-SI). The reaction was initially performed in a 10 mL stainless-steel (SS) jar with a 1.5 cm diameter ball of the same material by using a vibratory ball mill (vbm) operated at 30 Hz. Potassium carbonate and sodium hydroxide guaranteed high conversions of reagents with a milling time of only 5 min. Although the use of potassium carbonate provided a better conversion rate, sodium hydroxide was chosen for further optimization due to its lower molecular weight. Increasing the amount of base from 2 to 3 equivalents resulted in full conversion to intermediate 2a (isolated yield 98%). The reaction was then scaled up using a 35 mL SS jar without affecting the conversion or the yield (Table S1- for sulfonylation on a larger scale were then applied for the generation of intermediates 2b-2l. Regardless of the substituent at the isoindoline scaffold and the kind of arylsulfonyl chloride, all intermediates 2a-2l were obtained after a short milling time, ranging from 5 to 7 min. Of note, products 2a-2l were isolated after filtration of the inorganic base, followed by washing the filtrate with water to provide desired compounds in high yields (85-98%). and sodium hydroxide guaranteed high conversions of reagents with a milling time only 5 min. Although the use of potassium carbonate provided a better conversion rat sodium hydroxide was chosen for further optimization due to its lower molecular weigh Increasing the amount of base from 2 to 3 equivalents resulted in full conversion to inte mediate 2a (isolated yield 98%). The reaction was then scaled up using a 35 mL SS j without affecting the conversion or the yield (Table S1-SI). The optimized reaction co ditions for sulfonylation on a larger scale were then applied for the generation of interm diates 2b-2l. Regardless of the substituent at the isoindoline scaffold and the kind of ary sulfonyl chloride, all intermediates 2a-2l were obtained after a short milling time, rangin from 5 to 7 min. Of note, products 2a-2l were isolated after filtration of the inorganic bas followed by washing the filtrate with water to provide desired compounds in high yield (85-98%). Scheme 1. Mechanochemical synthesis of final compounds 3a-l using vbm operating at 30 Hz. R agents and conditions: (i) ϕball = 1.5 cm, 35 mL SS jar, the total mass of reagents = 350 mg, millin load = 10 mg/mL; differently substituted isoindolines (1 eq), arylsulfonyl chloride (1.1 eq), NaOH eq), 5-7 min, isolated yields (85-98%); (ii) ϕball = 1.5 cm, 10 mL SS jar, the total mass of reagents = mg, milling load = 10 mg/mL; sulfonylated intermediates 2a-l (1 eq), anhydrous piperazine (3 eq MeCN or DMSO (η = 0.4 μL/mg), 50 or 80 °C, 1.5 h, isolated yields (75-88%).
Next, a mechanochemical procedure was applied to perform a nucleophilic aromat substitution (SNAr) between fluorinated derivatives 2a-2l and anhydrous piperazine obtain final compounds 3a-3l. The advantages of using temperature-controlled mechan chemical procedures have been recently demonstrated for different organic reactions [33 36]. In line with those findings, heating of a 10 mL SS jar containing intermediate 2a (1 e and anhydrous piperazine (1 eq) at 50 °C for 1.5 h by using a temperature-controlled he gun pre-set at 90 °C enabled the formation of the product 3a (55% of conversion). N product was observed after milling of reagents at room temperature (Table S2-SI). B cause the conversion rate was still not satisfactory, MeCN, a widely used aprotic pol solvent for SNAr reactions in solution, was evaluated as a non-toxic liquid additive to en hance the overall mixing and reaction kinetics. Employing liquid-assisted grinding (LAG in tandem with the increase in the amount of amine (3 eq of anhydrous piperazine), dra tically improved the conversion rates up to 90% to furnish the desired product 3a in 85 yield (Table S2-SI). Regardless of the kind of substituent at the isoindoline core, simil results were obtained with different 4-fluoro and 2-fluoro-containing substrates (isolate yields 80-88%). Despite the low reactivity of the fluorine atom in the 3-position with r spect to sulfonamide moiety, optimization of reaction conditions was required. Th Scheme 1. Mechanochemical synthesis of final compounds 3a-l using vbm operating at 30 Hz. Reagents and conditions: (i) φ ball = 1.5 cm, 35 mL SS jar, the total mass of reagents = 350 mg, milling load = 10 mg/mL; differently substituted isoindolines (1 eq), arylsulfonyl chloride (1.1 eq), NaOH (3 eq), 5-7 min, isolated yields (85-98%); (ii) φ ball = 1.5 cm, 10 mL SS jar, the total mass of reagents = 85 mg, milling load = 10 mg/mL; sulfonylated intermediates 2a-l (1 eq), anhydrous piperazine (3 eq), MeCN or DMSO (η = 0.4 µL/mg), 50 or 80 • C, 1.5 h, isolated yields (75-88%).
Next, a mechanochemical procedure was applied to perform a nucleophilic aromatic substitution (S N Ar) between fluorinated derivatives 2a-2l and anhydrous piperazine to obtain final compounds 3a-3l. The advantages of using temperature-controlled mechanochemical procedures have been recently demonstrated for different organic reactions [33][34][35][36]. In line with those findings, heating of a 10 mL SS jar containing intermediate 2a (1 eq) and anhydrous piperazine (1 eq) at 50 • C for 1.5 h by using a temperaturecontrolled heat gun pre-set at 90 • C enabled the formation of the product 3a (55% of conversion). No product was observed after milling of reagents at room temperature (Table S2-SI). Because the conversion rate was still not satisfactory, MeCN, a widely used aprotic polar solvent for S N Ar reactions in solution, was evaluated as a non-toxic liquid additive to enhance the overall mixing and reaction kinetics. Employing liquid-assisted grinding (LAG), in tandem with the increase in the amount of amine (3 eq of anhydrous piperazine), drastically improved the conversion rates up to 90% to furnish the desired product 3a in 85% yield (Table S2-SI). Regardless of the kind of substituent at the isoindoline core, similar results were obtained with different 4-fluoro and 2-fluoro-containing substrates (isolated yields 80-88%). Despite the low reactivity of the fluorine atom in the 3-position with respect to sulfonamide moiety, optimization of reaction conditions was required. The replacement of MeCN with DMSO as a liquid additive enabled to increase the milling temperature of the jar to 80 • C (heat gun pre-set at 120 • C), providing final compounds 3e, 3f, 3g, and 3h in acceptable yields (75-78%). All the final compounds were isolated after simple precipitation from water followed by filtration to remove the excess of piperazine and crystallization in ethanol (Table S2-SI).

Radioligand Binding Assays in HEK-293 Cells and SAR Studies
All synthesized compounds were evaluated in 3 [H]-LSD competition binding experiments for their affinity for 5-HT 6 R expressed in HEK-293 cells (Table 1). Embedding the sulfonamide moiety into an unsubstituted isoindoline scaffold was sufficient to provide potent compounds 3a and 3e. Compounds bearing the chlorine or bromine atom at the 4or 5-position of isoindoline scaffold were equipotent (3b and 3c vs. 3a) or showed lower affinity than their unsubstituted analogs (3d vs. 3a and 3l vs. 3j). Regardless of the kind of halogen at the isoindoline core, 3-piperazinyl derivatives 3e, 3f, 3g, and 3h were the most potent compounds (K i < 5 nM). A shift of the piperazine moiety from 3-to 4-position at the phenyl ring slightly decreased the affinity for the 5-HT 6 R but still provided high-affinity ligands 3a, 3b, and 3c. In contrast, the introduction of piperazine moiety at the 2-position at the phenyl ring was not beneficial for the interaction with the 5-HT 6 R, as compounds 3i, 3j and 3l were not active (K i > 900 nM). An exception, compound 3k bearing the 4-bromo substituent at the isoindoline scaffold, displayed average affinity at the 5-HT 6 R. This effect might result from the favorable localization of the bromine atom at the isoindoline fragment, which enabled compound 3k to adopt an optimal position in the receptor binding pocket via the formation of hydrophobic/steric interactions. Table 1. The affinity of synthesized compounds 3a-l and reference SB-258585 at the 5-HT 6 R. potent compounds 3a and 3e. Compounds bearing the chlorine or bromine atom at the 4or 5-position of isoindoline scaffold were equipotent (3b and 3c vs. 3a) or showed lower affinity than their unsubstituted analogs (3d vs. 3a and 3l vs. 3j). Regardless of the kind of halogen at the isoindoline core, 3-piperazinyl derivatives 3e, 3f, 3g, and 3h were the most potent compounds (Ki < 5 nM). A shift of the piperazine moiety from 3-to 4-position at the phenyl ring slightly decreased the affinity for the 5-HT6R but still provided high-affinity ligands 3a, 3b, and 3c. In contrast, the introduction of piperazine moiety at the 2position at the phenyl ring was not beneficial for the interaction with the 5-HT6R, as compounds 3i, 3j and 3l were not active (Ki > 900 nM). An exception, compound 3k bearing the 4-bromo substituent at the isoindoline scaffold, displayed average affinity at the 5-HT6R. This effect might result from the favorable localization of the bromine atom at the isoindoline fragment, which enabled compound 3k to adopt an optimal position in the receptor binding pocket via the formation of hydrophobic/steric interactions. The most potent 5-HT6R ligands 3e, 3f, and 3g bearing the piperazine moiety localized at position-3 at the sulfonamide fragment were then tested for their activity at serotonin 5-HT1A, 5-HT2A, 5-HT7, and dopaminergic D2 receptors ( Table 2). These compounds were highly selective over the off-target 5-HT7R and D2R (Ki > 2 μM). They also displayed The most potent 5-HT 6 R ligands 3e, 3f, and 3g bearing the piperazine moiety localized at position-3 at the sulfonamide fragment were then tested for their activity at serotonin 5-HT 1A , 5-HT 2A , 5-HT 7 , and dopaminergic D 2 receptors (Table 2). These compounds were highly selective over the off-target 5-HT 7 R and D 2 R (K i > 2 µM). They also displayed good selectivity toward the 5-HT 1A R subtype (up to 680-folds). The selectivity index over the 5-HT 2A R (K i < 200 nM) was still satisfactory. Table 2. The antagonist properties of selected compounds 3e, 3f, and 3g, their impact on 5-HT 6 R constitutive activity, and their K i values for 5-HT 6, 5-HT 1A , 5-HT 2A , 5-HT 7 , and D 2 Rs.

One-Pot Two-
Step Mechanochemical Synthesis of Compounds 3e, 3f, and 3g To optimize the developed mechanochemical procedure, a one-pot, two-step protocol was employed for the synthesis of the most potent derivatives 3e, 3f, and 3g (Scheme 2).

One-Pot Two-Step Mechanochemical Synthesis of Compounds 3e, 3f, and 3g
To optimize the developed mechanochemical procedure, a one-pot, two-step protocol was employed for the synthesis of the most potent derivatives 3e, 3f, and 3g (Scheme 2).

8.9
NT Inverse agonist 645 1023 3388 3802 a Mean Ki values ± SEMs from three independent binding experiments; b Mean Kb values ± SEMs from three independent experiments in 1321N1 cells; c Data were obtained from four independent. transfection experiments in NG108-15 cells and measured in triplicate; d Data taken from reference [20]; NT = not tested.
According to the newly elaborated protocol, intermediates generated upon milling the isoindolines 1a-c (1 eq.) with 3-fluorobenzenesulfonyl chloride (1.1 eq) and NaOH (3 eq) were directly submitted to SNAr by the addition of anhydrous piperazine (3 eq) and DMSO (η = 0.4 μL/mg) in the 35 mL SS jar (Scheme 2). The simplified one-pot two-step synthetic procedure enabled to obtain final compounds in higher overall yields (85-88%) when compared to the sequential two-step protocol (75-78%) by improving the conversion rates of the SNAr reaction. A possible explanation could be found in the role of NaOH in neutralizing the generated in situ HF, maintaining a favorable basic environment for the substitution reaction.
According to the newly elaborated protocol, intermediates generated upon milling the isoindolines 1a-c (1 eq.) with 3-fluorobenzenesulfonyl chloride (1.1 eq) and NaOH (3 eq) were directly submitted to S N Ar by the addition of anhydrous piperazine (3 eq) and DMSO (η = 0.4 µL/mg) in the 35 mL SS jar (Scheme 2). The simplified one-pot two-step synthetic procedure enabled to obtain final compounds in higher overall yields (85-88%) when compared to the sequential two-step protocol (75-78%) by improving the conversion rates of the S N Ar reaction. A possible explanation could be found in the role of NaOH in neutralizing the generated in situ HF, maintaining a favorable basic environment for the substitution reaction.

Antagonist Properties of Selected Compounds at 5-HT 6 R-Operated Gs Signaling
The antagonist properties of compounds 3e, 3f, and 3g at 5-HT 6 R-operated Gs signaling were assessed in comparison with the reference ligand intepirdine and using 5-CT as an agonist in 1321N1 cells over-expressing the 5-HT 6 R ( Figure 3A). Likewise, intepirdine (K b = 1 ± 0.4 nM, Figure 3A), all compounds inhibited the 5-CT-stimulated cAMP accumulation and, thus, were classified as receptor antagonists (K b = 0.7−6.9 nM, Table 2).
Subsequently, the ability of compounds 3e, 3f, and 3g to inhibit 5-HT 6 R constitutive activity were further investigated in neuroblastoma NG108-15 cells, in line with our previous observation indicating that transiently expressed receptors exhibit a high level of constitutive activity at Gs signaling in this cell population. Neither of the evaluated compounds significantly affected basal cAMP production in NG108-15 cells, indicating that they behave as neutral antagonists (Table 2, Figure 3B). In contrast and consistent with previous findings, the reference compound SB-258585 reduced the basal level of cAMP, showing inverse agonist properties [14].
In light of these findings and considering structure-functional activity relationships, it seems that the more flexible primary sulfonamide of SB-258585 provides a particular orientation of the aromatic fragment that allows inhibition of the spontaneously active conformation of 5-HT 6 R [20], while inversion of the sulfonamide and its concomitant incorporation into isoindoline scaffold confers neutral antagonist properties.
an agonist in 1321N1 cells over-expressing the 5-HT6R ( Figure 3A). Likewise, intepirdine (Kb = 1 ± 0.4 nM, Figure 3A), all compounds inhibited the 5-CT-stimulated cAMP accumulation and, thus, were classified as receptor antagonists (Kb = 0.7−6.9 nM, Table 2). Subsequently, the ability of compounds 3e, 3f, and 3g to inhibit 5-HT6R constitutive activity were further investigated in neuroblastoma NG108-15 cells, in line with our previous observation indicating that transiently expressed receptors exhibit a high level of constitutive activity at Gs signaling in this cell population. Neither of the evaluated compounds significantly affected basal cAMP production in NG108-15 cells, indicating that they behave as neutral antagonists (Table 2, Figure 3B). In contrast and consistent with previous findings, the reference compound SB-258585 reduced the basal level of cAMP, showing inverse agonist properties [14].
In light of these findings and considering structure-functional activity relationships, it seems that the more flexible primary sulfonamide of SB-258585 provides a particular orientation of the aromatic fragment that allows inhibition of the spontaneously active conformation of 5-HT6R [20], while inversion of the sulfonamide and its concomitant incorporation into isoindoline scaffold confers neutral antagonist properties.
Beyond the ability to adopt different conformational states able to recruit the canonical Gs-adenylyl cyclase pathway in the presence and absence of an agonist, 5-HT6R also activates Cdk5 signaling in an agonist-independent manner [21]. We previously showed that neurite growth in NG108-15 cells transiently expressing the 5-HT6R provides an in vitro model to assess agonist-independent activation of Cdk5 signaling, enabling pharmacological distinction between inverse agonists and neutral antagonists at Cdk5 signaling [21,37]. Beyond the ability to adopt different conformational states able to recruit the canonical Gs-adenylyl cyclase pathway in the presence and absence of an agonist, 5-HT 6 R also activates Cdk5 signaling in an agonist-independent manner [21]. We previously showed that neurite growth in NG108-15 cells transiently expressing the 5-HT 6 R provides an in vitro model to assess agonist-independent activation of Cdk5 signaling, enabling pharmacological distinction between inverse agonists and neutral antagonists at Cdk5 signaling [21,37].
As previously observed, transient expression of the 5-HT 6 R in NG108-15 cells markedly increased neurite growth compared with cells expressing GFP alone. Treatment of NG108-15 cells expressing the receptor with compounds 3e, 3f, and 3g for 24 h significantly reduced neurite length (11.81 ± 2.61 µm, n = 112 neurites, 10.92 ± 0.89 µm n = 182 neurites and 8.08 ± 0.84 µm, n = 105 neurites, respectively vs. 16.95 ± 1.5 µm, n = 228 neurites in vehicle-treated cells, Figure 4). Of note, compound 3g was the most effective compound providing a 50% reduction in neurite growth slightly higher than that measured in cells treated with the reference SB-258585 (10.3± 0.77 µm, n = 182 neurites). In this case, reducing the flexibility of the sulfonamide fragment of SB-258585 by its inversion and incorporation into isoindoline moiety did not impact the functional profile of tested compounds at the 5-HT 6 R-elicited Cdk5 pathway. To our knowledge, compound 3g represents a unique example of a 5-HT 6 R ligand that behaves as a neutral antagonist at Gs signaling and displays potent inverse agonist properties at a non-canonical pathway engaged by the receptor.
in cells treated with the reference SB-258585 (10.3± 0.77 μm, n = 182 neurites). In this case, reducing the flexibility of the sulfonamide fragment of SB-258585 by its inversion and incorporation into isoindoline moiety did not impact the functional profile of tested compounds at the 5-HT6R-elicited Cdk5 pathway. To our knowledge, compound 3g represents a unique example of a 5-HT6R ligand that behaves as a neutral antagonist at Gs signaling and displays potent inverse agonist properties at a non-canonical pathway engaged by the receptor. Figure 4. Impact of compounds 3e, 3f, 3g, and SB-258585 on Cdk5 signaling pathway. NG108-15 cells were transfected with a plasmid encoding a GFP-tagged 5-HT6R or GFP alone. Cells expressing the receptor were exposed to vehicle (DMSO), compounds 3e, 3f, 3g, and reference SB-2758585 for 48 h. The left panel shows representative images of cells in each condition (scale bar 20 μm). The histogram shows the mean ± SEM of values obtained from at least three independent experiments. *** p < 0.001, ** p < 0.02 vs. cells expressing GFP. All groups were compared using one-way ANOVA followed by Tukey's multiple comparison test.

In Vitro Metabolic Stability and Preliminary Safety Assessment for 3e, 3f, and 3g
Preliminary ADME/Tox properties of compounds 3e, 3f, and 3g were screened using in vitro methods. First, biotransformation studies using rat liver microsomes (RLM) revealed that all tested compounds were metabolically stable (Table 3). Unsubstituted derivative 3e displayed a lower clearance value (Clint = 3.78 μL/min/mg) than its halogenated analogs 3f (4-Cl) and 3g (4-Br), similar to that of the reference drug donepezil (Clint = 3.65 μL/min/mg). Imipramine, an extensively metabolized drug used as a positive control, displayed much higher clearance (Clint = 115.65 μL/min/mg). Table 3. Preliminary metabolic stability and safety profile for compounds 3e, 3f, and 3g.

ID
Metabolic Stability a Cytotoxicity b Figure 4. Impact of compounds 3e, 3f, 3g, and SB-258585 on Cdk5 signaling pathway. NG108-15 cells were transfected with a plasmid encoding a GFP-tagged 5-HT 6 R or GFP alone. Cells expressing the receptor were exposed to vehicle (DMSO), compounds 3e, 3f, 3g, and reference SB-2758585 for 48 h. The left panel shows representative images of cells in each condition (scale bar 20 µm). The histogram shows the mean ± SEM of values obtained from at least three independent experiments. *** p < 0.001, ** p < 0.02 vs. cells expressing GFP. All groups were compared using one-way ANOVA followed by Tukey's multiple comparison test.

Glioprotective Properties of Compound 3g
Considering the pivotal physiological role of astrocytes in contrasting oxidative stress and promoting tissue repair [38,39], supporting their neuroprotective function might be regarded as a tangible strategy to improve the current therapeutic needs in the treatment of neurodegenerative disorders. We next investigated the ability of compound 3g to protect astrocytes against 6-OHDA-and ROT-induced cytotoxicity in C8-D1A cells, in line with our previous findings indicating that blocking of 5-HT 6 R results in glioprotective effect against toxic insults [17,40]. In comparison, we assessed the effect of WAY-181187, a 5-HT 6 R agonist, SB-258585, an inverse agonist, and CPPQ, a neutral antagonist [18]. For this purpose, cells were co-incubated with the evaluated compounds (at the non-toxic concentration of 0.25 µM) and 6-OHDA or ROT (20 and 0.5 µM, respectively). After 24 h of incubation, the viability of astrocytes was determined using the MTT assay.
Both compound 3g and CPPQ, which behave as neutral antagonists at the 5-HT 6 Roperated Gs signaling pathway, decreased the cytotoxicity of 6-OHDA and ROT in C8-D1A astrocytes ( Figure 5A,B), whereas the inverse agonist SB-258585 did not evoke any protective effect. Likewise, treatment of C8-D1A cells with WAY-181187, a 5-HT 6 R agonist, slightly but not significantly enhanced the gliotoxic effects of 6-OHDA and ROT. These findings suggest that 5-HT 6 R activation by 5-HT contained in the serum from the cellgrowing medium contributes to the toxicity of 6-OHDA and rotenone in C8-D1A astrocytes.

Conclusions
Structural modifications around the sulfonamide moiety of SB-258585 led to the design of 1-(arylsulfonyl-isoindol-2-yl)piperazines as a new framework for developing biased 5-HT6R ligands. An application of a one-pot two-step mechanochemical procedure enabled the synthesis of selected derivatives in a fast and efficient manner (isolated yields 85-88%). Tested compounds displayed high metabolic stability in RLM assays and no toxic effect toward human hepatocellular carcinoma and neuroblastoma or a mouse astrocyte cell line. In vitro functional evaluation identified compounds that behave as potent neutral antagonists at 5-HT6R-elicited Gs signaling and simultaneously display inverse agonist properties at the Cdk5 pathway. These findings underline the important role of the sulfonamide moiety, in particular its inversion and rigidification, for the design of

Conclusions
Structural modifications around the sulfonamide moiety of SB-258585 led to the design of 1-(arylsulfonyl-isoindol-2-yl)piperazines as a new framework for developing biased 5-HT 6 R ligands. An application of a one-pot two-step mechanochemical procedure enabled the synthesis of selected derivatives in a fast and efficient manner (isolated yields 85-88%). Tested compounds displayed high metabolic stability in RLM assays and no toxic effect toward human hepatocellular carcinoma and neuroblastoma or a mouse astrocyte cell line. In vitro functional evaluation identified compounds that behave as potent neutral antagonists at 5-HT 6 R-elicited Gs signaling and simultaneously display inverse agonist properties at the Cdk5 pathway. These findings underline the important role of the sulfonamide moiety, in particular its inversion and rigidification, for the design of biased ligands at 5-HT 6 R-operated Gs and Cdk5 signaling pathways. Moreover, the neutral antagonists 3g and CPPQ exerted glioprotective properties against 6-OHDA-and ROT-induced toxicity in the C8-D1A astrocyte cell line. In contrast, no protective effect and even a slight increase in cytotoxicity were observed upon treatment with the inverse agonist SB-258585 or the agonist WAY-181187, respectively. The identified biased ligand 3g might be considered as a molecular probe to explore the role of 5-HT 6 R in neurodegenerative diseases further.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/biom13010012/s1. Characterization of all intermediates; UPLC-MS, 1 H-NMR, and 13 C-NMR of all intermediates and final compounds; Table S1-SI: Optimization of milling conditions for sulfonylation of isoindolines 1a-d; Table S2-SI: Optimization of milling conditions for S N Ar of intermediates 2a-l; Figure S1-SI: The effect of compound 3g on cellular viability.