Neuropathic pain-alleviating activity of novel 5-HT6 receptor inverse agonists derived from 2-aryl-1H-pyrrole-3-carboxamide

signaling pathways engaged by serotonin type 6 receptor (5-HT 6 R) together with its high constitutive activity suggests different types of pharmacological interventions for the treatment of CNS disorders. Non-physiological activation of mTOR kinase by constitutively active 5-HT 6 R under neuropathic pain conditions focused our attention on the possible repurposing of 5-HT 6 R inverse agonists as a strategy to treat painful symptoms associated with neuropathies of different etiologies. Herein, we report the identification of compound 33 derived from the library of 2-aryl-1 H -pyrrole-3-carboxamides as a potential analgesic agent. Compound 33 behaves as a potent 5-HT 6 R inverse agonist at Gs, Cdk5, and mTOR signaling. Preliminary ADME/Tox studies revealed preferential distribution of 33 to the CNS and placed it in the low-risk safety space. Finally, compound 33 dose-dependently reduced tactile allodynia in spinal nerve ligation (SNL)-induced neuropathic rats.


Introduction
Neuropathic pain is a significant public health concern worldwide.According to the International Association for the Study of Pain, 5-10% of adults are affected by chronic pain of neuropathic origin [1,2], resulting from a lesion or a disease affecting the central or peripheral somatosensory nervous system [3][4][5].First-line treatments such as serotonin-noradrenaline reuptake inhibitors (SNRIs, duloxetine and venlafaxine), gabapentin, tricyclic antidepressants, and topical agents (for peripheral neuropathic pain) are only partially effective as revealed by their NNT (number of patients who needed to be treated to obtain a 50% pain relief in one patient) values ranging from 3.6 to 7.7 [6,7].An increasing body of evidence supports both pronociceptive and antinociceptive effects of serotonin and proposes serotonin type 6 receptor (5-HT 6 R) as a potential target for new analgesic agents [8][9][10].
5-HT 6 R belongs to the family of G-protein-coupled receptors (GPCRs).In addition to the canonical Gs-adenylyl cyclase pathway, it recruits several cellular signaling cascades such as extracellular signalregulated kinase (ERK)1/2, cyclin-dependent kinase 5 (Cdk5), and mechanistic target of rapamycin (mTOR) pathways [11].Of note, 5-HT 6 R shows a high level of ligand-independent constitutive activity for both recombinant receptors in cell lines [12] and native receptors in primary cultured neurons [13].
The mechanisms underlying the antinociceptive effects of 5-HT 6 R antagonists remain unclear.We have recently demonstrated that these effects might result from blockade of mTOR activation by constitutively active spinal 5-HT 6 Rs [10].These observations are consistent with studies that identified mTOR kinase as a crucial regulator of central and peripheral pain sensitization [23,24] and demonstrated that its inhibition by rapamycin produces analgesic effects in a wide range of painrelated paradigms in rodents [25][26][27].
In the present study, we describe a systematic structure-activity relationship (SAR) to provide a small library of 5-HT 6 R inverse agonists in a group of 2-aryl-1H-pyrrole-3-carboxamide to obtain 5-HT 6 R inverse agonists that exhibit analgesic effects in neuropathic pain.Structural diversity originates from variation of the alicyclic amines in the 3-carboxamide fragment (R 3 ), functionalizing a phenyl moiety (R 2 ) linked with N 1 -pyrrole by a sulfonyl group or a methylene bridge (X = SO 2 or CH 2 ), and introducing substituents at the 2-phenyl ring of the pyrrole fragment or its replacement with naphth-2-yl or heteroaryl moieties (Ar) (Fig. 1).
Furthermore, we show that compound 33 inhibits agonistindependent 5-HT 6 R-operated activation of Gs, Cdk5, and mTOR signaling, and that it reduces tactile allodynia in spinal nerve ligation (SNL)-induced neuropathic rats.

Chemistry
The designed compounds were synthesized starting from 2-aryl-1Hpyrrole-3-carboxylic acids 7a-k (Scheme 1).The key β-aminoester (1a-i) and diene intermediates (4a-i) bearing substituted phenyl or naphth-2-yl moieties in Ar were synthesized by a two-step approach involving an aza-Baylis-Hillman reaction and N-allylation as previously reported [28].An alternative synthetic route was required to prepare dienes bearing heteroaryl fragments, namely pyridin-2-yl (4j) and thien-2-yl (4k), as the corresponding aza-Baylis-Hillman reaction failed.Thus, a three-step synthesis was envisioned, consisting of Baylis-Hillman reaction of methylacrylate and heteroaryl aldehydes to provide alcohols 2j-k, which were subsequently converted into their corresponding acetates 3j-k.The final nucleophilic substitution performed directly with N-allyl-tosylamine yielded an undesired isomer of 4j-k, originating from a S N 2 ′ reaction of the amine on the allylic acetate.Hence, a first S N 2 ′ with DABCO, followed by a second S N 2 ′ with N-allyltosylamine was necessary to yield the desired dienes 4j-k, as reported in the literature with other tosylamines [29].Next, continuous flow ringclosing metathesis (RCM) was performed in the presence of ruthenium catalyst M2 according to our previously reported procedure [30].Of note, the N-containing heterocyclic compounds, especially pyridines, are generally more difficult substrates in RCM, because the nitrogen atom might coordinate with the ruthenium centre and poison the catalyst.Thus, HCl was used for the flow RCM of 4j to protonate the pyridine and obtain 5j.Then, removal of the tosyl protection group in the presence of sodium tert-butoxide (NaOtBu) with simultaneous aromatization yielded the 2-substituted-1H-pyrrole-3-methylcarboxylates 6a-k.They were subsequently hydrolyzed to their corresponding carboxylic acids (7a-k) in a refluxing aqueous solution of NaOH.
In accordance with our previous studies indicating that substitution of the phenylsulfonyl fragment in the C 3 position with small-size halogens stabilizes the ligand-receptor complex through the formation of a halogen bond (chlorine) or dipole-dipole, van der Waals interaction (fluorine) [28,33,34], we only introduced these atoms (alone or in combinations) in R 2 .As expected, the functionalization of the N 1 -phenylsulfonyl ring in the C 3 position improved the affinity of compounds, with the highest affinity for 5-HT 6 R observed for the chlorinesubstituted compound (18, K i = 28 nM).In contrast, the introduction of two electron-withdrawing substituents (20)(21)(22)(23)(24) was not beneficial for the interaction with 5-HT 6 R as compared to monohalo-substituted derivatives (17)(18) or even an unsubstituted derivative (13) (Table 2).
Inasmuch a structural functionalization of the 2-phenyl ring at the pyrrole moiety might affect the affinity for 5-HT 6 R, selected  substituents, namely methoxy group, chlorine, and fluorine, were introduced (Table 3).Regardless of the position in the 2-phenyl ring, the introduction of an electron-donating methoxy group decreased the affinity for 5-HT 6 R (13 vs. 25, 27, 29) up to 8-fold.Among the electronwithdrawing atoms, chlorine in C 3 and C 4 position slightly increased the affinity of compounds for the receptor (13 vs. 28, 30).In line with published results [28], a fluorine atom in C 4 position was also beneficial for the interaction with 5-HT 6 R (13 vs. 31, 17 vs.32, 18 vs.33).Furthermore, the replacement of the 2-phenyl ring at the pyrrole moiety with naphthyl (18 vs. 34) or heteroaryl moieties, i.e., pyridine-2-yl, thien-2-yl (18 vs. 35, 36), decreased the affinity for 5-HT 6 R. The SAR studies revealed the same trend when the sulfonyl group in N 1 position was replaced by a methylene bridge (33 vs. 37) (Table 3).

Effect of compound 33 on 5-HT 6 R-elicited signaling pathways
Considering the ability of 5-HT 6 R to adopt unique conformations engaging different signaling pathways, we next examined the effect of compound 33 on the recruitment of the canonical Gs-adenylyl cyclase pathway and noncanonical pathways, i.e., Cdk5 and mTOR kinases.
The high level of 5-HT 6 R constitutive activity, corresponding to spontaneous activity of the receptor in the absence of an agonist occupancy, provides the pharmacological distinction between inverse agonists and neutral antagonists [35].The effect of 33 on the agonistindependent 5-HT 6 R-elicited Gs signaling pathway was tested in NG108-15 cells transiently expressing the receptors.Compound strongly decreased the cAMP level in a concentration-dependent Scheme 1. Synthetic route for the preparation of 2-aryl-1H-pyrrole-3-carboxylic acids (7a-k).Scheme 2. General synthetic route for the preparation of final compounds 9-37 obtained as hydrochloride salts.manner; this was reminiscent of the effect of the reference 5-HT 6 R inverse agonist SB-271046 (Fig. 2), and the compound was defined as an inverse agonist.Strikingly, compound 33 produced a stronger inhibition of basal cAMP production than SB-271046 (75.52% ± 3.5% inhibition vs. 82.73%± 6.2% inhibition measured at the maximally effective concentrations of SB-271046 and compound 33, respectively).

5-HT 6 R-operated Cdk5 signaling
5-HT 6 R is also capable of activating Cdk5 signaling in an agonistindependent manner in NG108-15 cells, a process that promotes neurite growth and NG108-15 cell differentiation.Exposure of NG108-15 cells to compound 33 or SB-271046 significantly reduced neurite length, thus indicating that both compounds also behave as inverse agonists of 5-HT 6 R at Cdk5 signaling (Fig. 3).

5-HT 6 R-operated mTOR signaling
5-HT 6 R recruits mTORC1 and stimulates mTOR through a dual mechanism involving the canonical PI3K/Akt/Tsc1,2/Rheb pathway and a physical interaction between the receptor's C-terminal domain and mTOR [11].mTOR is a serine/threonine kinase that plays a crucial role regulating protein synthesis and thus controls many basic physiological functions, including pain modulation and transmission [23,[36][37][38].In addition, mTOR and its downstream effectors were found in the central regions involved in the nociception process, such as the dorsal root ganglion and spinal cord dorsal horn [39].Further body of evidence confirmed that the mTOR inhibitor rapamycin produces analgesic effects in traumatic [26], chemically (bortezomib) [27], and diabetes (streptozotocin)-induced [40] neuropathy in rats.
Our recent investigations demonstrated that the basal level of mTOR phosphorylation at Ser2448 in HEK-293 cells expressing 5-HT 6 Rs was significantly reduced by 5-HT 6 R inverse agonists, i.e., SB-258585, but Table 1 Binding data of synthesized compounds 9-16 and references A, B for 5-HT 6 R. b Data taken from [28] where A is encoded as 7, and B is encoded as 8.

Table 2
Binding data of synthesized compounds 17-24 for 5-HT 6 R. not by neutral antagonists i.e., IIQ, CPPQ [10].Further studies showed that inhibiting mTOR activation by constitutively active 5-HT 6 R alleviates painful symptoms in neuropathic pain models of different etiologies.

No
In line with these observations, we also investigated the effect of compound 33 administration to rats on mTOR signaling in the brain by immunohistochemistry using antibodies against ribosomal protein S6 phosphorylated on Ser 240/244 , a downstream target of mTOR.The administration of compound 33 strongly decreased the level of phosphorylated S6 in the prefrontal cortex, a brain structure involved in the modulation of cognitive processes by 5-HT 6 R (Fig. 4).These observations suggest that compound 33 might be a promising treatment agent for neuropathic pain.

Preliminary ADME/Tox and pharmacokinetics characterization
Because the assessment of in vitro ADME/Tox properties of novel bioactive compounds is a key step in the preclinical lead optimization process, we conducted biotransformation studies of compound 33 by using rat liver microsomes (RLM).Compound 33 exhibited a low value of intrinsic clearance (8.5 μL/min/mg), indicating its high metabolic stability.Additionally, compound 33 exhibited high solubility and chemical stability in a wide pH range (Table 5).
Compound 33 was tested in the human neuroblastoma (SH-SY5Y) and human hepatocellular carcinoma (HepG2) cellular models to exclude potential cytotoxic effects.We investigated the metabolic activity of cells in the MTT test by using doxorubicin (DOX) as a positive control.Compound 33 did not induce neurotoxicity, nor hepatotoxicity.Compound 33 also did not cause significant changes in the number of micronuclei, dicentric bridges as well as nuclear buds, thus indicating the absence of genotoxicity (more details in Supplementary Information; SI : 11, 12).
The PK profile of compound 33 was determined in male Wistar rats after single intragastric gavage (i.g.) at the dose of 10 mg/kg.Compound 33 was slowly absorbed from the gastrointestinal tract with t max = 60 min; it achieved a concentration of 216.6 ng/mL in plasma but crucially crossed the blood-brain barrier, reaching the C max (436.6 ng/mL) in the brain after 30 min.Compound 33 displayed high distribution to the brain, with the brain/plasma ratio of 2.18 (Table 6), and it was eliminated very slowly from the brain, showing a long half-life time of 29 h.

Neuropathic pain alleviating properties of compound 33
Since we have previously shown that 5-HT 6 R inverse agonists

Table 4
The property of compound 33 in 1321N1 cells and its functional activity at 5-HT 6 R-dependent Gs signaling in NG108-5 cells.induced anti-allodynic effect in a preclinical model of traumatic neuropathy of high translational value, i.e., SNL model [10], we chose to evaluate the in vivo activity of compound 33 in SNL rats.Sprague-Dawley male rats were subject to unilateral spinal nerve (L5) ligation.Two weeks later, the paw ipsilateral to nerve injury was tested with von Frey monofilaments to assess tactile allodynia.The allodynic rats (i.e., those presenting a reduction of paw withdrawal threshold) were intraperitoneally (i.p.) injected with either compound 33 (5 and 25 μmol/kg), PZ-1388 (25 µmol/kg) [10], or vehicle (water for injection, 5 mL/kg) and submitted again to von Frey hair application.Compound 33 (25 µmol/kg) significantly increased the 50% threshold at 30 to 90 min after administration and reached the maximal effect at 30-60 min after injection resulting, like PZ-1388 (25 µmol/kg), in a total abolition of SNLinduced tactile allodynia (Fig. 5A).The area under the curves (AUCs) of the 50% threshold variations confirmed the similar anti-allodynic effect of PZ-1388 and compound 33 (Fig. 5B).

Conclusions
In line with our objective to reposition 5-HT 6 R inverse agonists for     Considering the limitations of the currently available therapies, the ability of 5-HT 6 R inverse agonists to alleviate painful symptoms in different models of traumatic neuropathy as well as neuropathies induced by metabolic disorders (e.g., diabetes) and chemotherapies (e. g., anticancer drugs) certainly warrants further investigation.

General methods
Chemicals and solvents were purchased from commercial suppliers (Sigma-Aldrich, Fluorochem, Across, TCI chemicals or Chempur) and used as received.Continuous flow experiments were conducted using a Uniqsis Flowsyn Multi-X equipment.Column chromatography was performed using silica gel Merck 60 (70-230 mesh ASTM) or Biotage Isolera flash chromatography system using Biotage SNAP HP-Sil cartridges.
Mass spectra were recorded on a UPLC-MS/MS system consisted of a Waters ACQUITY UPLC coupled to a Waters TQD mass spectrometer or LC-MS with ESI using Waters Alliance 2695 as LC, coupled to a Waters ZQ mass spectrometer with electrospray source, a simple quadrupole analyzer and a UV Waters 2489 detector.The UPLC/MS purity of all synthesized compounds was determined to be > 96%.Retention times (t R ) were provided in minutes.HRMS analyses were conducted using an UPLC Acquity H-Class from Waters hyphenated to a Synapt G2-S mass spectrometer with a dual ESI source from Waters.The synthesis of starting 2-substituted-1H-pyrrole-3-carboxylic acids was performed according to the previously described procedures [28,30,41], except for 7j-k, for which the detailed synthetic procedure is placed in Supplementary Information.The characterization of selected final compounds 13, 14, 18, 20, 27, 30, 32, 33, 36, 37 is presented below, while the spectroscopic data of all intermediates as well as final compounds excluded from the main manuscript are reported in the Supplementary Information.

General procedure for amidation (8a-r)
The carboxylic acid 7a-k (1 equiv.),1-hydroxybenzotriazole (HOBt) (1.2 equiv) and benzotriazole-1-yl-oxy-tris-(dimethylamino)phosphonium hexafluorophosphate (BOP) (1.2 equiv.)were dissolved in DMF, followed by addition of triethylamine (3 equiv.).The resulting mixture was stirred for 30 min.Next, the appropriate amine (1.2 equiv.) was added and left to react overnight.The mixture was diluted with EtOAc and washed three times with water and brine, dried over Na 2 SO 4 and concentrated under vacuum.The crude product was purified on silica gel (the eluting system is indicated for each compound together with characterisation data).

Determination of functional activity at Gs signaling 4.2.2.1. Impact of compound 33 on cAMP production in 1321N1 cells.
The ability of compound 33 to inhibit 5-CT-induced production of cAMP was evaluated using 1321N1 cells expressing the human 5-HT 6 R (Per-kinElmer).Compound was tested in triplicate at 8 concentrations (10 - 11 -10 -4 M).Total cAMP was measured using the LANCE cAMP detection kit (PerkinElmer), according to the manufacturer's protocol.Timeresolved 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 [45].Detailed description is reported in the Supplementary Information (SI: 7.1).

Impact of compound 33 on cAMP production in NG108-15 cells.
We used NG108-15 cells transiently transfected with 5-HT 6 R and with the cAMP sensor CAMYEL (cAMP sensor using YFP-Epac-RLuc) [19,46].Constitutive activity of the receptor was assessed through bioluminescence resonance energy transfer (BRET) measurement using a Mithras LB 940 plate reader (Berthold Technologies).cAMP production induces a conformational change of the probe, resulting in the decrease of the BRET signal emitted by the CAMYEL probe in cells expressing the receptor when compared to cells expressing the probe alone.This decrease was subsequently used as an index of 5-HT 6 R constitutive activity at Gs signaling.The inverse agonist properties of compound 33 and SB-271046 were tested by measuring BRET signal after a 24 h treatment, as their capacity to restore a BRET signal equivalent to the one measured in cells expressing the probe alone.Detailed description is reported in the Supplementary Information (SI: 7.2).

Impact of compound 33 on Cdk5-dependent neurite growth
The impact of a 24 h treatment with DMSO (control), compound 33 or SB-271046 (10 -8 M) was assessed in NG108-15 cells expressing either cytosolic GFP or a GFP-tagged 5-HT 6 R. Inhibition of Cdk5-5-HT 6 R induced neurite growth was assessed on cells imaged using an AxioI-magerZ1 microscope equipped with epifluorescence (Zeiss).Neurite length was measured using the Neuron J plugin of the ImageJ software (NIH).Detailed description is reported in the Supplementary Information (SI: 8).

Impact of compound 33 on 5-HT 6 R-operated mTOR pathway
Rat were injected with vehicle or compound 33 (6 mg/kg, p.o.).Two hours after injection, rats were deeply anesthetized with ketamine (80 mg/kg) and xylazine (20 mg/kg) and perfused transcardially with mL NaCl solution (0.9%; 32 C) until all remaining blood was removed.Next, the animals were perfused with 500 mL 4% PFA in 0.1 M phosphate-buffered saline (PBS).Brains were post-fixed overnight in the same solution and stored at 4 • C. Fifty μm-thick sections were cut with a vibratome (Leica) and stored at 4 • C in 0.1 M sodium phosphate buffer (PBS), permeabilized with 0.1% Triton X-100 for 20 min and incubated for 48 h at 4 • C with anti-phospho-Ser 240/244 S6 antibody (1:500, Cell Signaling Technology) in PBS containing 0.025% Triton X-100 and 20% goat serum.Sections were then incubated for 1 h with goat Cy3conjugated anti-mouse antibody (1:500, Jackson Laboratory) in PBS containing 20% goat serum.Immunofluorescent staining was observed with a Zeiss Axiophot2 microscope equipped with epifluorescence and quantification of phospho-Ser 240/244 -S6 positive cells was performed on 224 μm × 168 μm images.

In vitro metabolic stability studies
Metabolic stability of compound 33 (10 µM) was analysed in rat liver microsomes (RLMs), using previously reported procedures [28].Samples were analysed using UPLC/MS (Waters Corporation, Milford, MA).All experiments were run in duplicates.Half-life time was evaluated using linear regression model using Graph Pad Prism software and intrinsic clearance was calculated from the equation Cl int = (volume of incubation [µL]/protein in the incubation [mg]) 0.693/t 1/2 [47].Detailed description is reported in the Supplementary Information (SI: 9).

In vitro genotoxicity studies
Micronucleus assay.For the test, CHO-K1 cells (1 × 10 6 ) were seeded in 25 mm 2 culture flasks and allowed to grow for 24 h (37 • C, 5% CO 2 ), then treated with different concentrations of compound 33, mitomycin C (MMC) (Sigma Aldrich) (0.5 µg/mL, positive control) or water (vehicle control).After 24 h of treatment, medium containing cytochalasin B (CytB) (Sigma Aldrich, Darmstadt, Germany) (4.5 µg/mL) was added and incubation was continued for the next 24 h.After trypsinization, the cells were centrifuged and the supernatant was discarded.Then, CHO-K1 cells were briefly exposed to 1% ice-cold sodium citrate (Chempur) and fixed with methanol:acetic acid (3:1; v/v), together with 4 drops of formaldehyde.The cell suspension was centrifuged (5 min, 1000 rpm), the supernatant was discarded, and the pellet was fixed in fixative mixture for two more times, without the addition of formaldehyde.Following fixation, the slides were prepared and dyed with Giemsa dye (1:20 in phosphate buffer) for 5 min [48][49][50].Cells were analyzed using a microscope with a magnification of 40 × .The following parameters were analyzed: number of micronuclei (MN), dicentric bridges (DB) and nuclear buds (NB) per 1000 examined binuclear cells, and the nuclear division index (NDI).NDI was calculated using the formula: [(1 × MOC) + (2 × BC) + (3 × MUC)]/N, where MOC is the number of mononuclear cells, BC is the number of binuclear cells, MUC represents the number of multinuclear cells, and N is the total number of scored cells [49,50].Experiment was conducted two times in two repetition for each condition.

In vivo pharmacokinetic studies
General conditions for pharmacokinetic studies is reported in the Supplementary Information (SI: 10).To determine the concentration of compound 33 in plasma and brain, an original method was developed using a high-performance liquid chromatography coupled to tandem mass spectrometry (LC-ESI/MS/MS).The method was validated according to FDA and EMA guidelines for bioanalytical method validation, in terms of method linearity, precision, accuracy, recovery and matrix effect [51].

Sample preparation.
Prior to chromatographic separation, plasma samples and brain homogenates were purified by protein precipitation with acetonitrile cooled to 4 • C. Whole brains were used to prepare the brain tissue homogenates, which were carefully weighed beforehand, and homogenised using an electric homogeniser after the addition of phosphate buffer (pH 7.4) at a ratio of 1:2.5.For the determination of the concentration of compound 33, a 100 µL of rat plasma or brain homogenate was transferred to 2 mL Eppendorf tubes, and a 5 μL of the internal standard (IS, PH002437, Merck, Darmstadt, Germany) at a concentration of 5 μg/mL was added, thoroughly vortex-mixed for 10 s, whereupon 200 uL of acetonitrile was used to precipitate the proteins.The sample thus prepared was shaken for 20 min, then centrifuged (28,672 × g) for 10 min at 4 • C.After centrifugation, 200 uL of supernatants were collected from the upper layer, and transferred to a chromatography vials in which the inserts had previously been placed.Thereafter 20 uL of the prepared sample was injected on the chromatography column.4.3.4.2.Animals.Evaluation of the pharmacokinetic profile of compound 33 was performed in Wistar rats (16 male, 8-week-old, weighing between 200 and 220 g each), purchased from the Animal House at the Faculty of Pharmacy, Jagiellonian University Medical College, Krakow (Poland).Rats were housed in standard polycarbonate cages (four animals per cage) with maintaining constant environmental conditions, in terms of relative humidity 50%-60%, temperature 22 ± 2 • C, normal 12h light-dark cycle (7 a.m. to 7p.m. light).The animals were given standard rodent chow and water ad libitum.Compound 33 dissolved in PBS saline (pH 7.4) was given intragastrically to each animal at a dose of 6 mg/kg, whereupon the animals were sacrificed by decapitation in deep anasthesia after i.p. injections of 50 mg/kg ketamine plus 8 mg/kg xylazine at specific time-points: 30 min (n = 4), 60 min (n = 4), 90 min (n = 4) and 120 min (n = 4).The blood samples were drawn from each rat into heparinized tubes, and centrifuged at 3000 × g for 10 min to obtain plasma.In the final step, following the animals' euthanasia, the whole brain from each individuals was collected.Both plasma and brain samples were protected from degradation by freezing at temp. at − 80 • C. All experimental procedures were carried out in accordance with EU Directive 2010/63/EU and approved by the I Local Ethics Committee for Experiments on Animals of the Jagiellonian University in Krakow, Poland (No 83/2018).

In vivo assessment of neuropathic pain-alleviating activity
Male Sprague-Dawley rats weighting 150-175 g were purchased from Janvier Labs (Le Genest-Saint-Isle, France).Animals were housed four per cage under standard laboratory conditions and maintained on a 12 h:12 h light/dark cycle in specific pathogen free area.Water and food were available ad libidum.The experiments involving animals were conducted in accordance with the NC3R ARRIVE rules [52].All prodecures were approved by local Ethics Committee of Auvergne (C2EA, France) and by the French Ministry of Higher Education and Innovation (authorization N • 2018111617333273 V5).
Unilateral traumatic neuropathy was induced by spinal nerve ligation according to the method described by Chung [53].Briefly, rats were anesthetized with xylazine (10 mg/kg, intraperitoneal) and ketamine (75 mg/kg, i.p.).Under aseptic conditions, the left L5 spinal nerve was exposed and ligated with a nonabsorbable 5-0 braided silk thread.The muscle and skin were then sutured and the rats received a s.c.injection of meloxicam (non-steroidal anti-inflammatory drug) to reduce the postsurgery pain and inflammation.The animals were allowed to recover for the next 14 days and were monitored daily to insure good health.
Tactile allodynia was assessed using the von Frey hair test [54].The experimenter got the animals habituated to the testing environment 1 h before baseline.Each rat was confined in clear plexiglas compartment placed on an elevated metal mesh floor.A series of 8 Von Frey monofilaments were applied perpendicularly to the central plantar surface of the ipsilateral (side of surgery) hind paw for 5 s in ascending order of force (1.4 to 26 g).Paw withdrawal or licking was considered as a positive response and the next weaker filament was applied.In case of no paw withdrawal or licking, the next stronger filament was applied.This paradigm continued until four measurements have been obtained after an initial change of behavior, or until four consecutive negative responses or five consecutive positive responses.The 50 % response threshold was calculated using the Up-Down method and Dixon's formulae [55].
Drugs were blindly administrated according to the method of blocks and using a randomization procedure.Different animals were used in each experiment.At the end of the experiments, the animals were euthanized by progressive carbon dioxide inhalation (10-30%/min).

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Fig. 2 .
Fig. 2. Inhibition of basal cAMP production in NG108-15 cells transiently transfected with 5-HT 6 Rs by compound 33 and SB-271046.Each point represents the mean ± SEM of the BRET values obtained from quadruplicates measurements in three independent experiments made in different sets of cultured cells.
the treatment of neuropathic pain, we performed structural functionalization of the 2-phenyl-1H-pyrrole-3-carboxamide framework to provide a small library of 5-HT 6 R ligands.Among the series of phenylsulfonyl derivatives of 2-aryl-1H-pyrrole-3-carboxamides, 2-(4fluorophenyl)-1-[(3-chlorophenyl)sulfonyl]-N-(piperidin-4-yl)-1H-pyrrole-3-carboxamide(33) showed high affinity for 5-HT 6 R (K i = 23 nM) and high selectivity over off-target receptors.The in vitro evaluation of functional activity of 33 revealed the inverse agonism effect at Gs signaling and Cdk5-dependent neurite growth.Importantly, compound 33 also inhibited mTOR kinase under the control of constitutively active 5-HT 6 R.This compound showed no cytotoxicity, had high metabolic stability, and easily penetrated into the brain.Finally, compound 33 exhibited rapid and potent in vivo anti-allodynic effect compatible with its pharmacokinetics in SNL-induced neuropathy in rats, an experimental model of traumatic neuropathic pain of high translational value.
a Mean K i values ± SEM are reported for three independent experiments in HEK293 cells.
a Determined at a protein concentration of 0.4 mg/ml in RLM assay.bDetermined in aqueous solution of HCl (pH = 1) and NaOH (pH = 13) by HPLC analysis.cAssessed in DPBS (pH = 7.4) at 20 • C in thermodynamic solubility f Assessed in the micronucleus assay using CHO-K1 cellular model.

Table 6
Pharmacokinetic parameters of compound 33.
a Measured after i.g.gavage of dose 10 mg/kg; t 0.5 -terminal half-life; AUC 0→t -area under the curve from zero to last sampling time; MRTmean residence time; C max -maximum concentration; t max -time to reach the maximum concentration; Vd/Fapparent volume of distribution.bConcentration in brain; number of animals = 16.