Design, stereoselective synthesis, conﬁgurational stability and biological activity of 7-chloro-9-(furan-3-yl)-2,3,3a,4-tetrahydro-1 H - benzo[ e ]pyrrolo[2,1-c ][1,2,4]thiadiazine 5,5-dioxide

4-tetrahydro-1 H -benzo[ e ]pyrrolo[2,1-c ][1,2,4] thiadiazine 5,5-dioxide bearing a pyrrolo moiety coupled with the 5-(furan-3-yl) substituent on benzo-thiadiazine core. A stereoselective synthesis was projected to obtain single enantiomer of the latter compound. Absolute conﬁguration was assigned by X-ray crystal structure. Patch clamp experiments evaluating the activity of single enantiomers as AMPAr positive allosteric modulator showed that R stereoisomer is the active component. Molecular modeling studies were performed to explain biological results. An on-column stopped-ﬂow bidimensional recycling HPLC procedure was applied to obtain on a large scale the active enantiomer with enantiomeric enrichment starting from the racemic mixture of the compound.


Introduction
L-Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system (CNS).2][3][4] On the basis of their sensitivity to selective agonists, iGluRs have been further classified into three subclasses: kainic acid (KA), N-methyl-D-aspartic acid (NMDA) and the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. 5A dysfunction of glutamatergic neurotransmission has been implicated in a number of neurological and psychiatric diseases. 6Many studies have been carried out to develop compounds able to enhance glutamatergic function without causing excitotoxicity.2][3][4][5][6] Compared to direct AMPA receptor agonists, allosteric modulators are anticipated to possess finer tuning to increase glutamatergic function since they have no effects in the absence of the natural ligand in the synapse.These agents, binding to an allosteric site, enhance receptor function by decreasing desensitization and/or deactivation.5][16][17][18][19][20][21] Among benzothiadiazine derivatives, IDRA21, a compound resulting from saturation of the nitrogenAcarbon double bond of the thiadiazide ring of the antihypertensive diazoxide, was demonstrated as a promising clinical candidate.
IDRA21 has attracted particular clinical interest despite its modest in vitro activity since it is effective in increasing learning and memory performances in behavioral tests, representing an important lead compound able to cross the blood-brain barrier (BBB). 22,23Recent crystallographic studies regarding the binding mode of benzothiadiazine derivatives on S1S2 GluA2 subunits of AMPAr suggested that these compounds bind to the dimer interface, leading to inhibition of the receptor desensitization by stabilization of the ligand-binding domain within a dimer interface. 24,25In particular detailed analysis of the crystal structure of IDRA21 in complex with S1S2 GluA2 subunits reveal the presence of an unfilled hydrophilic pocket that could accommodate an aryl or heteroaryl substituent at C5 atom of IDRA21 core. 26Molecular modeling studies have suggested that heteroaromatic substituents, like thiophenyl or furanyl, and aromatic substituents, on C5 position of IDRA21 can fit into the unfilled hydrophilic pocket increasing the affinity of IDRA21derivatives towards AMPA receptor.Therefore a series of 5-arylbenzothiadiazine derivatives were recently designed and synthesized by our research group. 26Among them, 7-chloro-5-(3-furanyl)-3-methyl-3,4-dihydro-2H-1,2, 4-benzothiadiazine 1,1-dioxide (1, Fig. 1) has attracted particular attention because it exhibited an interesting pharmacological activity as AMPAr positive allosteric modulator. 26][38] Further studies have demonstrated that all activity resided in the S enantiomer of 2; moreover, it showed high chemical and configurational stability. 33Starting from this interesting chemical and pharmacological profile, we have designed a new compound bearing the pyrrolo moiety of compound 2 coupled with 5-aryl substituent on benzothiadiazine core following the approach of combining in a single molecule two different pharmacophores.7-Chloro-9-(furan-3-yl)-2,3,3a,4-tetrahydro-1H-benzo[e]pyrrolo[2,1-c][1,2,4]thiadiazine 5,5-dioxide (3) was designed and synthesized in order to enhance configurational and chemical stability preserving the relevant biological activity (Fig. 1).

Configurational and chemical stability of (
Recent studies conducted by dynamic chromatography on compound (±)-1 underlined its configurational and chemical lability. 35ndeed during the chiral separation on a Chiralcel OD-R column at temperatures between 25 and 45 °C employing aqueous mobile phases (water/acetonitrile) at different pH a pronounced plateau was observed between the peaks corresponding to the two enantiomers, indicating a rapid enantiomerization of (±)-1. 35Differently the same experiments applied to (±)-3 evidenced its stereo stability during chromatographic separation in aqueous mobile phases.No enantiomerization and hydrolysis occurred when (±)-3 was chromatographed with similar conditions (see Fig. 2).
In order to evaluate stereo and chemical stability of (±)-3 in absence of chiral stationary phase a recently developed method was employed. 32topped-flow bidimensional recycling HPLC (sf-BD-rHPLC) method was applied employing immobilized artificial membrane (IAM) stationary phase as reactor column.No enantiomerization and/or hydrolysis product peaks appeared in the sf-BD-rHPLC chromatograms performed in IAM column at both pH 2.2 and 7.4 after 90 min at 37.5 °C.A moderate racemization occurs only at pH 1 at 45 °C for 90 min suggesting a great increase of the configurational stability of (±)-3 with respect to compound (±)-1.Moreover the hydrolysis product of (±)-3 was not observed suggesting a complete chemical stability of the latter compound.A similar trend was previously observed for compound 2 confirming the stabilizing effect of the pyrrolo moiety.Since previous studies suggested that chiral benzothiadiazines display a stereoselective pharmacological action, it was important to evaluate the activity of single stereoisomers of (±)-3 as AMPAr modulators. 40,41oreover, the configurational stability observed for (±)-3 prompted us to develop an asymmetric synthesis.The synthetic pathway employed is shown in Scheme 2. Once obtained racemic 3, it was oxidized in presence of potassium permanganate in basic conditions to give 9, which was subjected to asymmetric reduction.
A similar protocol was employed by Desos et al. to obtain S18986 with high optical purity. 41The enantiomeric excess achieved in this reaction was about 70% as calculated by chiral HPLC.Since enantiomeric excess obtained by asymmetric synthesis was insufficient to conduct biological experiments, a semipreparative chromatographic method was developed in order to purify the enantiomer of 3. The enriched enantiomeric mixture was chromatographed on semipreparative Chiralcel OD column with hexane/2-propanol 95:5 (v/v) as mobile phase.The collected fractions containing the single enantiomer show high enantiomeric excess values (ee > 99%).Enantiomeric excess of (+)-3 was calculated employing an OD-R column with water/acetonitrile 50:50 (v/v) as mobile phase.Subsequently, optical rotation values for the enantiomer obtained was calculated and the specific rotation in chloroform was [a] D +32.4°(26 mg/mL; chloroform, 24 °C).In order to assign the absolute configuration, single crystal X-ray diffraction analysis were performed on the isolated dextrorotatory enantiomer (Fig. 3).The data analysis indicated that the enantiomer obtained employing Chirald Ò as chiral auxiliary had S configuration.Thus the reaction proceeds with the same stereochemical outcome observed for S18986. 41n order to obtain (R)-3 the procedure described by Cohen et al. and Deeter et al., was applied to 9. 42-44  They demonstrated that by choosing Chirald Ò or its enantiomer (2R,3S)-(À)-4-dimethylamino-1,2-diphenyl-3-methyl-2-butanol in complex with LiAlH 4 for the reduction of acetylenic ketones it was possible to obtain selectively alcohols with opposite configuration with good enantioselectivity. 43This strategy applied to compound 9 furnished the desired (R)-3 with good enantiomeric excess (73%); further purification on semipreparative Chiralcel OD column gave enantiopure (R)-3 (ee > 99%) with [a] D À33.8°(34 mg/mL; chloroform, 24 °C).

Biological activity
The activity of compound 3 and its enantiomers as allosteric modulators of AMPA/kainate receptors was evaluated by patchclamp technique in primary cultures of cerebellar neurons.Kainate (KA)-evoked current was mainly mediated by AMPAr activation because application of GYKI 53655 (100 lM), an AMPA receptors antagonist, almost completely abolished the current (data not shown).Since IDRA 21 is one of the most relevant benzothiadiazine derivatives reported in literature as AMPAr-positive allosteric modulator, it was selected as reference compound.Data obtained indicate that compound 3 and IDRA21 potentiate by 30 ± 9% (n = 4) and 8 ± 11% (n = 4) KA-evoked currents at 10 lM (Fig. 4).

Docking studies
Several and various binding modes have been observed for benzothiadiazine type compounds active as AMPA positive allosteric modulators. 25,24Thus in order to understand the possible stereospecific interactions of (À)-(R)-3 and (+)-(S)-3 docking studies were performed.Taking advantage of crystal structures of the AMPA GluA2 ligand binding domain co-crystallized with several benzothiadiazines, Molegro Virtual Docker (MVD) software was applied to dock (À)-(R)-3 and (+)-(S)-3 within the binding pocket of the GluA2 dimer interface.The software MVD was previously evaluated on several crystal structure of benzothiadiazine. 26,34he average root mean square distance (RMSD) of the best ranked pose of tested compounds compared to their binding pose in their respective crystal structures was found to be less than 1.0 Å proving that MVD is able to accurately dock this class of compounds.The crystal structure obtained for (+)-(S)-3 was employed as input file to build the ligands with Spartan Wavefunction 08.Among the tested crystal structures, the GluA2 dimer in complex with Cyclothiazide and IDRA21 (PDB codes: 1LBC and 3IL1) were selected for docking studies of both enantiomers 3. 25 The docking protocol template was built using the following chemical properties of Cyclothiazide and IDRA21: ring atoms, hydrogen-bond acceptors, hydrogen-bond donors and steric interactions.Minimization was performed after docking in order to increase the precision of the analysis.
The docking output clearly demonstrated that (À)-(R)-3 and (+)-(S)-3 adopt a binding mode close to that of IDRA21 (Fig. 5).This result is particularly significant since it was obtained for both the crystal structures employed.
The primary polar interaction partners for both the enantiomers of 3 are Pro494 and Gly731 (Fig. 6).The carbonyl oxygen atom of Pro494 forms a polar interaction with the N4 atom of (À)-(R)-3 and (+)-(S)-3, whereas the amidic backbone of Gly731 makes an hydrogen bond with the oxygen of the sulphone moiety since it's located for both the stereoisomer within 3 Å.
However, in contrast to what observed in IDRA21 crystal structure, the N1a hydrogen atom in compound 3 has been substituted with a pyrrole group; therefore, the hydrogen bond to Ser754 is not possible.The lack of this interaction could explain the moderate decrease of activity showed for both the enantiomer of 3 with respect to 1.The main difference observed for (À)-(R)-3 and (+)-(S)-3 is the position of the pyrrole.The S configuration of the C3a atom imposes a spatial orientation on the five membered ring such that favored steric interactions with hydrophobic cleft defined by Val750, Leu751, and Leu259 residues (Fig. 5).As described by Ptak et al. the substituent at C3 and thus the hydrophobic interactions played an important role in the orientation and in the position of the benzothiadiazine rings.In compound (+)-(S)-3 the pyrrole moiety is buried deep inside in the lipophilic cavity previously described preventing the formation of the hydrogen bond between the heteroatom of 3-furanyl fragment and the amidic backbone of Ser497 (Figs. 5 and 6).This interaction, as recently reported, is crucial in conferring high activity as AMPA positive allosteric modulator. 26,44On the other hand, (À)-(R)-3 poorly interact with the hydrophobic region of the ligand binding domain since the pyrrole is located diametrically opposite to Val750, Leu751, and Leu259 residues (Fig. 5).Therefore the furan moiety could be accommodated within the hydrophilic pocket lined by Lys763, Tyr424, Ser729, Phe495 and Ser497 residues interacting via hydrogen bond with the latter amino acid.

Synthesis of (À)-(R)-3 and (+)-(S)-3 by on-line racemization
Notwithstanding the asymmetric synthesis previously described proved to be successful, the modest enantiomeric excess obtained with the reaction, the high costs of the chiral auxiliaries employed and the moderate yields obtained led us to develop a new strategy to gain access to enantiopure (À)-(R)-3.
Our goal was to achieve (À)-(R)-3 on a preparative scale in order to conduct full and detailed pharmacological in vivo experiments.
The good enantioselectivity achieved during the chromatography of (±)-3 on a semipreparative Chiralcel OD column and the moderate racemization observed at pH 1 suggested the possible application of a recently developed enantiomeric enrichment process. 46e stopped-flow bidimensional recycling HPLC method consists in a 3-step experimental protocol.In step 1, the racemate was injected and quantitatively separated on chiral column.Subsequently, one of the two enantiomers was trapped into a reactor achiral column, which was filled with appropriate racemization solvent.Afterwards, the mobile phase flow was reinforced into the achiral column and the two enantiomers were separated in the chiral column.The cycle can be repeated by trapping one of the two eluted enantiomers in the achiral column (Fig. 7).
(±)-3 was injected on a semipreparative Chiralcel OD with hexane/2-propanol 95:5 (v/v) as mobile phase.The first eluted stereoisomer (À)-(R)-3 of the racemic mixture injected was initially collected.The second eluted enantiomer, the undesired (+)-(S)-3, was trapped in the achiral column, that was subsequently filled with 2-propanol added of 1% (v/v) of trifluoroacetic acid as catalyst.The trapped enantiomer was left to racemize for 60 min at 45 °C.Afterwards the original mobile phase (hexane/2-propanol 95:5 (v/v)) was introduced in the achiral column forcing the original enantiomer (+)-(S)-3 and its interconversion product (À)-(R)-3 to the semipreparative Chiralcel OD where they was enantioseparated.Thus by harvesting again the selected stereoisomer it is possible to collect additional 25% of (À)-(R)-3.The racemization/ separation cycle could be repeated several times in order to increase the yield of (À)-(R)-3.200 mg of racemic compound by consecutive injections afforded approximately 170 mg of the desired enantiomer.
This method will assist us to gain access to relevant data on the pharmacokinetic profile of (À)-(R)-3.

Conclusions
Recently, we designed and synthesized compound 1 that has attracted particular attention because it exhibited an interesting pharmacological activity as AMPA receptor positive modulator.Preliminary configurational stability studies suggested a rapid enantiomerization in condition similar to those it will meet in vivo.
In order to enhance stability towards enantiomerization and hydrolysis, preserving AMPAr affinity, 7-chloro-9-(furan-3-yl)-2,3,3a,4-tetrahydro-1H-benzo[e]pyrrolo[2,1-c][1,2,4]thiadiazine 5,5-dioxide (±)-3 was synthesized.Studies on (±)-3 confirmed a great increase in the configuration stability and a complete suppression of the hydrolysis in physiological conditions.A stereoselective synthesis was developed to obtain the single enantiomers of (±)-3.The absolute configuration of the enantiomers of 3 was assigned thanks to X-ray diffraction spectroscopy.Biological studies suggested a stereospecific interaction of (À)-(R)-3 with AMPA receptor.Molecular modeling experiments performed on AMPA GluA2 ligand binding domain identified the probable binding mode of (À)-(R)-3 and the source of the stereospecific recognition with the receptor.The 'on line racemization' technique was applied to synthesize the eutomer (À)-(R)-3 on preparative scale.In vivo pharmacokinetic experiments in rats are in development in order to confirm the stereo-pharmacological activity of 3.

Instrumentation
The chromatographic apparatus was a Shimadzu LC-10AD Pump (Shimadzu Italia, Milan), a Merck Hitachi L-6200A Pump (Merck KGaA, Darmstadt, Germany), a Rheodyne 7725 manual injector equipped with a 20 ll sample loop (Jasco Europe, Italy, Milan).A Merck Hitachi L-7400UV (Merck KGaA, Darmstadt, Germany) was used as detector.Chromatograms were recorded with a Jasco J-700 program (Jasco Europe, Italy, Milan).Two Rheodyne 7000 valves were used to switch the mobile phase flow (Jasco Europe, Italy, Milan).Column temperature regulation was obtained with a Jasco CO-2067 column oven (Jasco Europe, Italy, Milan).
Melting points were determined with an Electrothermal Apparatus and they are uncorrected.
IR spectra were recorded on a PerkinElmer Model 1600 FT-IR spectrometer. 1 H NMR spectra were recorded with a Bruker DPX 400 spectrometer with CDCl 3 as solvent and tetramethylsilane (TMS) as external standard.Chemical shifts (d) are in part per million and coupling constant (J) in hertz.Multiplicities are abbreviated as follows: s, singlet; d, doublet; dd, double doublet; t, triplet; m, multiplet.The electrospray ionization (HR-ESI-MS) experiments were carried out in a hybrid QqTOF mass spectrometer (PE SCIEX-QSTAR) equipped with an ion spray ionization source.MS (+) spectra were acquired by direct infusion (5 mL/min) of a solution containing the appropriate sample (10 pmol/mL), dissolved in a solution 0.1% acetic acid, methanol/water 50:50 at the optimum ion voltage of 4800 V.
All pH measurements were made using Orion Research EA940 pH-meter.

X-ray crystallography
X-ray diffraction experiments were carried out at room temperature (293°K) by a Bruker-Nonius KappaCCD single crystal diffractometer, equipped a with a charge-coupled device (CCD detector), using monochromatized MoK (k radiation = 0.71073 Å).The automatic data collection strategy was defined by the COLLECT software, cell determination and refinement by DIRAX and data reduction by EVAL. 47,48Absorption effects were corrected by SAD-ABS, exploiting a semi-empirical approach.[51]

Chromatography
The separation factor (a) was calculated as k 2 /k 1 and retention factors (k 1 and k 2 ) as k 1 = (t 1 À t 0 )/t 0 where t 1 and t 2 refer to the retention times of the first and second eluted enantiomers.The resolution factor (R s ) was calculated by the formula R s = 2(t 2 À t 1 )/ (w 1 + w 2 ) where w 1 and w 2 are the peak widths at base for the first and second eluted enantiomers.The dead time of the columns (t 0 ) was determined by injection of 1,3,5-tri-tert-butylbenzene.
The injection volume was 500 ll.The detector was set at 254 nm.The collected fractions corresponding to the enantiomers were analyzed by injection on the same column and in the same chromatographic conditions.
Separation of enantiomers of compound 3 was carried out isocratically at 25-45 °C on Chiralcel OD-RH column using water/acetonitrile (50:50, v/v) as mobile phase.The compound was dissolved in ethanol and subsequently diluted 1:10 (v/v) with mobile phase at final concentration of 100 lg/ml.The injection volume was 50 ll.The detector was set at 254 nm.
The sf-BD-rHPLC method was previously described. 24The racemic mixture of 3 was injected on Chiralcel OD.The mobile phase consisted of n-hexane and 2-propanol 95:5 (v/v).The injection volume was 500 ll.The detector was set at 254 nm.The racemization of the trapped enantiomer was conducted employing an IAM column as reactor column and isopropanol added of 1% (v/v) of trifluoroacetic acid as racemization solvent.The reactor column was left 1 h at 45 °C.

Docking studies
The GluA2-S1S2J crystal structures in complex with Cyclothiazide and IDRA21 were retrieved from the Protein Data Bank (PDB codes: 1LBC and 3IL1) and imported into MVD. 25All water molecules and co-factors were deleted.(S)-3 and (R)-3 were built in Spartan employing the crystal structure of (S)-3 as input file.Then they were exported as mol2 files and docked in GluA2 by using MVD.We used the template docking available in MVD and evaluated MolDock score, Rerank score, and protein-ligand interaction score from Mol-Dock and MolDock [GRID] options.Cyclothiazide and IDRA21 were selected as reference compounds for the template.It was used the default settings, including a grid resolution of 0.30 Å, the MolDock optimizer as a search algorithm, and the number of runs was set to 10.A population size of 50, maximum iteration of 2000, scaling factor of 0.50, crossover rate of 0.90.The maximum number of poses to generate was increased to 10 from a default value of 5.

Figure 4 .
Figure 4. (A) Representative whole cell recording showing KA (100 lM)-evoked current recorded from a cerebellar neuron in culture in control conditions (left traces), after application of (R)-3 (10 lM) (middle trace) and after washing (right trace).(B) Histogram showing the potentiation of (R)-3, (S)-3, 3 and IDRA 21 of 5-Will-evoked current and of KA-evoked current.Each data point is the mean ± SEM of at least five cells.Significative differences among (R)-3 and (S)-3 enantiomers were obtained (T-test, p < 0.05).

Figure 6 .
Figure 6.Principal polar interactions of (R)-3 (yellow, panel a and b) and (S)-3 (magenta, panel c and d).Blue dashes indicate H-bond interactions.Red dashes indicate absence of H-bond interactions.