Spiro[pyrrolidine-3,3′-oxindoles] and Their Indoline Analogues as New 5-HT6 Receptor Chemotypes

Synthetic derivatives of spiro[pyrrolidinyl-3,3′-oxindole] alkaloids (coerulescine analogues) were investigated as new ligands for aminergic G-protein coupled receptors (GPCRs). The chemical starting point 2′-phenylspiro[indoline-3,3′-pyrrolidin]-2-one scaffold was identified by virtual fragment screening utilizing ligand- and structure based methods. As a part of the hit-to-lead optimization a structure-activity relationship analysis was performed to explore the differently substituted 2′-phenyl-derivatives, introducing the phenylsulphonyl pharmacophore and examining the corresponding reduced spiro[pyrrolidine-3,3′-indoline] scaffold. The optimization process led to ligands with submicromolar affinities towards the 5-HT6 receptor that might serve as viable leads for further optimization.

The 5-HT 6 R is a member of the Class A G-protein coupled receptors (aminergic family) considered to be a current and promising drug target for the treatment of several central nervous system related indications, such as: cognitive, learning and memory deficits related to Alzheimer's disease [6], Parkinson's disease [7] and schizophrenia [8]. Chemical similarity to the endogenous agonist serotonin explains the most frequent heteroaromatic ring systems routinely used in 5-HT 6 R ligands that include indoles, indolines, indazoles, pyrrolo [2,3-b]pyridines, pyrazolo [1,5-a]pyridines, [1,2,3]-triazolo [1,5-a]pyrimidines and further 5 + 6 condensed N-heterocycles. Pharmacophore based approaches on 5-HT 6 R antagonists are typically focused to the canonical pharmacophore model [9] ( Figure 2), which is defined as having two hydrophobic rings/ring systems connected by a hydrogen bond acceptor (e.g., sulfonyl, sulfonamide linker). Optionally, a positively ionizable residue is included in one of the hydrophobic sites of the ligands, typically offering an interacting moiety with the D 3.32   The tryptamine derived scaffold of 6 and its alignment with the published 5-HT6R pharmacophore [9] prompted us to explore the structure-activity relationship around the spiropyrrolidinyl-oxindole core. Here we report the results of our hit-to-lead program, which led to an identification of promising 5-HT6R ligands of this chemotype.

Identification and Early Structure-Activity Data on Spiro[pyrrolidine-3,3′-oxindole] Derivatives
Fragment libraries containing a couple hundreds or even thousands of small polar molecules are routinely used for hit identification at the early stage of drug discovery. Fragment screening provides diverse chemotypes and significant operational freedom for the further optimization of promising hits. Inspired by these advantages in a recent study we developed a strategy for aminergic focused fragment libraries using a sequential filtering methodology applying ligand-and structure-based scoring functions [4,5]. The prospective validation was performed on our in-house library of 1183 fragments. A physicochemical-property based scoring, followed by docking the fragments into an ensemble of carefully selected aminergic GPCR X-ray structures (PDB ID: 3PBL, 3RZE, 4IB4, 4IAQ, A further pharmacophore feature might contain an additional intramolecular hydrogen bond donor moiety further stabilizing the binding conformation of the ligands [11] (see Figure 2). Selectivity among other aminergic GPCR's was shown [12] to be accessible through omitting the positively ionizable group in the 5-HT6R antagonists. Bis(hetero)arylsulphonyl-and sulfonamide substituents also contribute to 5-HT6R affinity and selectivity.  The tryptamine derived scaffold of 6 and its alignment with the published 5-HT6R pharmacophore [9] prompted us to explore the structure-activity relationship around the spiropyrrolidinyl-oxindole core. Here we report the results of our hit-to-lead program, which led to an identification of promising 5-HT6R ligands of this chemotype.

Identification and Early Structure-Activity Data on Spiro[pyrrolidine-3,3′-oxindole] Derivatives
Fragment libraries containing a couple hundreds or even thousands of small polar molecules are routinely used for hit identification at the early stage of drug discovery. Fragment screening provides diverse chemotypes and significant operational freedom for the further optimization of promising hits. Inspired by these advantages in a recent study we developed a strategy for aminergic focused fragment libraries using a sequential filtering methodology applying ligand-and structure-based scoring functions [4,5]. The prospective validation was performed on our in-house library of 1183 fragments. A physicochemical-property based scoring, followed by docking the fragments into an ensemble of carefully selected aminergic GPCR X-ray structures (PDB ID: 3PBL, 3RZE, 4IB4, 4IAQ, The tryptamine derived scaffold of 6 and its alignment with the published 5-HT 6 R pharmacophore [9] prompted us to explore the structure-activity relationship around the spiropyrrolidinyl-oxindole core. Here we report the results of our hit-to-lead program, which led to an identification of promising 5-HT 6 R ligands of this chemotype.

Identification and Early Structure-Activity Data on Spiro[pyrrolidine-3,3 -oxindole] Derivatives
Fragment libraries containing a couple hundreds or even thousands of small polar molecules are routinely used for hit identification at the early stage of drug discovery. Fragment screening provides diverse chemotypes and significant operational freedom for the further optimization of promising hits. Inspired by these advantages in a recent study we developed a strategy for aminergic focused fragment libraries using a sequential filtering methodology applying ligand- and structure-based scoring functions [4,5]. The prospective validation was performed on our in-house library of 1183 fragments. A physicochemical-property based scoring, followed by docking the fragments into an ensemble of carefully selected aminergic GPCR X-ray structures (PDB ID:  3PBL, 3RZE, 4IB4, 4IAQ, 3UON, 4MQT, 4LDE, 2RH1, 3NY9). This resulted in a set of 36 top ranked hit molecules which were measured on an aminergic target not being included in the original docking ensemble, namely 5-HT 6 R. We demonstrate the usefulness of the method for comprehensive aminergic focused screenings. Out of the four hits with low micromolar inhibitory results, the structurally novel 2 -(3-fluorophenyl)spiro[indoline-3,3 -pyrrolidin]-2-one (6) possessing the spiro[pyrrolidine-3,3 -oxindole] scaffold (Table 1), was selected for further optimization. As the next step of exploring the structure-activity relationship substructure search in the MCULE purchasable database [13] of 5 million compounds resulted in further 887 spiro[pyrrolidine-3,3′-oxindole] derivatives. The molecules were prepared by Schrödinger's LigPrep [14] creating possible conformers, tautomers and protonation states by default settings. The ligands were then projected to single precision molecular docking analysis on a nine membered ensemble of molecular dynamics frames of an 5-HT6R homology model [15] (built using 5-HT2BR X-ray crystal structure [16] in complex with ergotamine as template (PDB ID: 4IB4)). The docking poses were filtered in a post-processing step keeping only binding modes forming hydrogen bonds towards Asp106 3.32 , Asn288 6.55 and optionally Ser193 5.43 , occupying a primary hydrophobic cleft defined by Trp281 6.48 , Phe284 6.51 and Phe285 6.52 . A secondary hydrophobic subpocket was defined by the pose filtering criteria as following: Val107 3.33 , Ala157 4.56 , Leu160 4.59 , Pro161 4.60 , Leu164 4.63 . The satisfactory poses of the molecules were scored to have the best ranking in possibly all of the 9 frames using consensus ranking [5]. Altogether ten compounds (7)(8)(9)(10)(11)(12)(13)(14)(15)(16) were purchased and tested in our serotonergic panel ( Table 2). As the next step of exploring the structure-activity relationship substructure search in the MCULE purchasable database [13] of 5 million compounds resulted in further 887 spiro[pyrrolidine-3,3 -oxindole] derivatives. The molecules were prepared by Schrödinger's LigPrep [14] creating possible conformers, tautomers and protonation states by default settings. The ligands were then projected to single precision molecular docking analysis on a nine membered ensemble of molecular dynamics frames of an 5-HT 6 R homology model [15] (built using 5-HT 2B R X-ray crystal structure [16] in complex with ergotamine as template (PDB ID: 4IB4)). The docking poses were filtered in a post-processing step keeping only binding modes forming hydrogen bonds towards Asp106 3.32 , Asn288 6.55 and optionally Ser193 5.43 , occupying a primary hydrophobic cleft defined by Trp281 6.48 , Phe284 6.51 and Phe285 6.52 . A secondary hydrophobic subpocket was defined by the pose filtering criteria as following: Val107 3.33 , Ala157 4.56 , Leu160 4.59 , Pro161 4.60 , Leu164 4.63 . The satisfactory poses of the molecules were scored to have the best ranking in possibly all of the 9 frames using consensus ranking [5]. Altogether ten compounds (7)(8)(9)(10)(11)(12)(13)(14)(15)(16) were purchased and tested in our serotonergic panel (Table 2).
In spite of the structural diversity, all virtual screening hits were lacking substitution at the oxindole scaffold. However, compounds 7, 9 and 10 having oxygen-containing substituents in the 3,4-positions of the 2 -phenyl ring showed somewhat improved affinity compared to the initial compound 6. The moderate improvement in affinity and the lack of selectivity amongst the closely related serotonergic targets have driven us to interpret the results in the context of the known 5-HT 6 R pharmacophore patterns [17] (Figure 3). This analysis suggests that introducing a phenylsulfonyl moiety either to the 1-nitrogen at the oxindole ring or to the 1 -nitrogen at the pyrrolidine ring would be beneficial for both the affinity and selectivity of this chemotype. post-processing step keeping only binding modes forming hydrogen bonds towards Asp106 , Asn288 6.55 and optionally Ser193 5.43 , occupying a primary hydrophobic cleft defined by Trp281 6.48 , Phe284 6.51 and Phe285 6.52 . A secondary hydrophobic subpocket was defined by the pose filtering criteria as following: Val107 3.33 , Ala157 4.56 , Leu160 4.59 , Pro161 4.60 , Leu164 4.63 . The satisfactory poses of the molecules were scored to have the best ranking in possibly all of the 9 frames using consensus ranking [5]. Altogether ten compounds (7)(8)(9)(10)(11)(12)(13)(14)(15)(16) were purchased and tested in our serotonergic panel (Table 2). post-processing step keeping only binding modes forming hydrogen bonds towards Asp106 3.32 , Asn288 6.55 and optionally Ser193 5.43 , occupying a primary hydrophobic cleft defined by Trp281 6.48 , Phe284 6.51 and Phe285 6.52 . A secondary hydrophobic subpocket was defined by the pose filtering criteria as following: Val107 3.33 , Ala157 4.56 , Leu160 4.59 , Pro161 4.60 , Leu164 4.63 . The satisfactory poses of the molecules were scored to have the best ranking in possibly all of the 9 frames using consensus ranking [5]. Altogether ten compounds (7)(8)(9)(10)(11)(12)(13)(14)(15)(16) were purchased and tested in our serotonergic panel ( Table 2). post-processing step keeping only binding modes forming hydrogen bonds towards Asp106 3.32 , Asn288 6.55 and optionally Ser193 5.43 , occupying a primary hydrophobic cleft defined by Trp281 6.48 , Phe284 6.51 and Phe285 6.52 . A secondary hydrophobic subpocket was defined by the pose filtering criteria as following: Val107 3.33 , Ala157 4.56 , Leu160 4.59 , Pro161 4.60 , Leu164 4.63 . The satisfactory poses of the molecules were scored to have the best ranking in possibly all of the 9 frames using consensus ranking [5]. Altogether ten compounds (7)(8)(9)(10)(11)(12)(13)(14)(15)(16) were purchased and tested in our serotonergic panel ( Table 2). post-processing step keeping only binding modes forming hydrogen bonds towards Asp106 3.32 , Asn288 6.55 and optionally Ser193 5.43 , occupying a primary hydrophobic cleft defined by Trp281 6.48 , Phe284 6.51 and Phe285 6.52 . A secondary hydrophobic subpocket was defined by the pose filtering criteria as following: Val107 3.33 , Ala157 4.56 , Leu160 4.59 , Pro161 4.60 , Leu164 4.63 . The satisfactory poses of the molecules were scored to have the best ranking in possibly all of the 9 frames using consensus ranking [5]. Altogether ten compounds (7)(8)(9)(10)(11)(12)(13)(14)(15)(16) were purchased and tested in our serotonergic panel ( Table 2). In spite of the structural diversity, all virtual screening hits were lacking substitution at the oxindole scaffold. However, compounds 7, 9 and 10 having oxygen-containing substituents in the 3,4-positions of the 2′-phenyl ring showed somewhat improved affinity compared to the initial compound 6. The moderate improvement in affinity and the lack of selectivity amongst the closely related serotonergic targets have driven us to interpret the results in the context of the known 5-HT6R pharmacophore patterns [17] (Figure 3). This analysis suggests that introducing a phenylsulfonyl moiety either to the 1-nitrogen at the oxindole ring or to the 1′-nitrogen at the pyrrolidine ring would be beneficial for both the affinity and selectivity of this chemotype. 49 In spite of the structural diversity, all virtual screening hits were lacking substitution at the oxindole scaffold. However, compounds 7, 9 and 10 having oxygen-containing substituents in the 3,4-positions of the 2′-phenyl ring showed somewhat improved affinity compared to the initial compound 6. The moderate improvement in affinity and the lack of selectivity amongst the closely related serotonergic targets have driven us to interpret the results in the context of the known 5-HT6R pharmacophore patterns [17] (Figure 3). This analysis suggests that introducing a phenylsulfonyl moiety either to the 1-nitrogen at the oxindole ring or to the 1′-nitrogen at the pyrrolidine ring would be beneficial for both the affinity and selectivity of this chemotype. 45 In spite of the structural diversity, all virtual screening hits were lacking substitution at the oxindole scaffold. However, compounds 7, 9 and 10 having oxygen-containing substituents in the 3,4-positions of the 2′-phenyl ring showed somewhat improved affinity compared to the initial compound 6. The moderate improvement in affinity and the lack of selectivity amongst the closely related serotonergic targets have driven us to interpret the results in the context of the known 5-HT6R pharmacophore patterns [17] (Figure 3). This analysis suggests that introducing a phenylsulfonyl moiety either to the 1-nitrogen at the oxindole ring or to the 1′-nitrogen at the pyrrolidine ring would be beneficial for both the affinity and selectivity of this chemotype. 7 In spite of the structural diversity, all virtual screening hits were lacking substitution at the oxindole scaffold. However, compounds 7, 9 and 10 having oxygen-containing substituents in the 3,4-positions of the 2′-phenyl ring showed somewhat improved affinity compared to the initial compound 6. The moderate improvement in affinity and the lack of selectivity amongst the closely related serotonergic targets have driven us to interpret the results in the context of the known 5-HT6R pharmacophore patterns [17] (Figure 3). This analysis suggests that introducing a phenylsulfonyl moiety either to the 1-nitrogen at the oxindole ring or to the 1′-nitrogen at the pyrrolidine ring would be beneficial for both the affinity and selectivity of this chemotype. 7 In spite of the structural diversity, all virtual screening hits were lacking substitution at the oxindole scaffold. However, compounds 7, 9 and 10 having oxygen-containing substituents in the 3,4-positions of the 2′-phenyl ring showed somewhat improved affinity compared to the initial compound 6. The moderate improvement in affinity and the lack of selectivity amongst the closely related serotonergic targets have driven us to interpret the results in the context of the known 5-HT6R pharmacophore patterns [17] (Figure 3). This analysis suggests that introducing a phenylsulfonyl moiety either to the 1-nitrogen at the oxindole ring or to the 1′-nitrogen at the pyrrolidine ring would be beneficial for both the affinity and selectivity of this chemotype. 40 In spite of the structural diversity, all virtual screening hits were lacking substitution at the oxindole scaffold. However, compounds 7, 9 and 10 having oxygen-containing substituents in the 3,4-positions of the 2′-phenyl ring showed somewhat improved affinity compared to the initial compound 6. The moderate improvement in affinity and the lack of selectivity amongst the closely related serotonergic targets have driven us to interpret the results in the context of the known 5-HT6R pharmacophore patterns [17] (Figure 3). This analysis suggests that introducing a phenylsulfonyl moiety either to the 1-nitrogen at the oxindole ring or to the 1′-nitrogen at the pyrrolidine ring would be beneficial for both the affinity and selectivity of this chemotype. 3,4-positions of the 2′-phenyl ring showed somewhat improved affinity compared to the initial compound 6. The moderate improvement in affinity and the lack of selectivity amongst the closely related serotonergic targets have driven us to interpret the results in the context of the known 5-HT6R pharmacophore patterns [17] (Figure 3). This analysis suggests that introducing a phenylsulfonyl moiety either to the 1-nitrogen at the oxindole ring or to the 1′-nitrogen at the pyrrolidine ring would be beneficial for both the affinity and selectivity of this chemotype.

Hit-to-Lead Optimization of Spiro[pyrrolidine-3,3 -oxindoles]
Detailed elaboration of the spiro[pyrrolidine-3,3 -oxindole] scaffold required a viable synthesis strategy for the preparation of designed analogues. The first, conventional approach is based on an intramolecular Mannich-reaction used in case of several alkaloids including (±)-horsfiline (4) [18] and Spirotryprostatin B [19]. An alternative approach is the oxidative reaction of tryptolines induced by tert-butyl hypochlorite, N-bromosuccinimide, N-chlorosuccinimide, sodium tungstate, lead tetraacetate, or osmium tetroxide [20,21]. The reaction is completed by the subsequent elimination of water that finally results in the reorganization of the ring system to spiro[pyrrolidine-3,3 -oxindoles] (Scheme 1).
For the effective exploration of the structure-activity relationship we need a feasible and universal approach with acceptable functional group tolerance and high variability around the spirooxindole core. Considering the requirements of the early stage optimization program we aimed a synthesis strategy that Further, sophisticated approaches such as [1,3]-dipolar cycloaddition reactions of azomethine ylides [22], radical cyclization by AIBN [23], intramolecular Heck reaction [24] and asymmetric nitroolefination of oxindoles [25] are also available, however, used less frequently.
For the effective exploration of the structure-activity relationship we need a feasible and universal approach with acceptable functional group tolerance and high variability around the spiro-oxindole core. Considering the requirements of the early stage optimization program we aimed a synthesis strategy that • uses readily accessible, simple starting materials • applies well-documented, readily available reagents • has key intermediates to access a wide variety of derivatives • is not necessarily stereoselective at this stage of the optimization The oxidative spiro-rearrangement reactions offer a wide variety of oxidative reagents [20,21,[26][27][28][29][30][31][32][33] and the corresponding starting materials are readily accessible through Pictet-Spengler reaction of tryptamines [34] (Scheme 2). oxindole core. Considering the requirements of the early stage optimization program we aimed a synthesis strategy that Following this synthesis strategy, tryptamine (23) was used as a starting material for the synthesis of the common intermediate tryptoline (25). The Pictet-Spengler condensation reaction was performed by glyoxylic acid-monohydrate in aqueous medium [35]. The crude acid intermediate 24 was decarboxylated in refluxing concentrated hydrochloric acid affording the tetrahydro-β-carboline. The first synthetic route depicted in Scheme 3 starts with the N-acylation of the tryptoline (25). The application of Cbz (carboxybenzyl) [36] protecting group was necessary for two reasons: 1. achieve the sulfonylation at the oxindol-nitrogen, 2. the spiro-rearrangement reaction is not occurring in case of unsubstituted pyrido-nitrogen. N-chlorosuccinimide [37] was used in the first experiments as halogenating reagent, providing the Cbz-protected spiro[pyrrolidine-3,3′-oxindole]. The Following this synthesis strategy, tryptamine (23) was used as a starting material for the synthesis of the common intermediate tryptoline (25). The Pictet-Spengler condensation reaction was performed by glyoxylic acid-monohydrate in aqueous medium [35]. The crude acid intermediate 24 was decarboxylated in refluxing concentrated hydrochloric acid affording the tetrahydro-β-carboline. The first synthetic route depicted in Scheme 3 starts with the N-acylation of the tryptoline (25). The application of Cbz (carboxybenzyl) [36] protecting group was necessary for two reasons: 1. achieve the sulfonylation at the oxindol-nitrogen, 2. the spiro-rearrangement reaction is not occurring in case of unsubstituted pyrido-nitrogen. N-chlorosuccinimide [37] was used in the first experiments as halogenating reagent, providing the Cbz-protected spiro[pyrrolidine-3,3 -oxindole]. The phenylsulfonylation was performed either by using lithium-hexamethyl disilazane [38] and sodium hydride [39] as deprotonating agents and acid scavengers. Finally, the deprotection of the Cbz-group by hydrogenation [40] resulted in the desired 1-(phenylsulfonyl)spiro[indoline-3,3 -pyrrolidin]-2-one (17) compound. Following an alternative way, the more basic pyrido-nitrogen of tryptoline 29 might be first sulfonylated [41] followed by the N-bromosuccinimide assisted spiro-cyclization to 18 [42]. phenylsulfonylation was performed either by using lithium-hexamethyl disilazane [38] and sodium hydride [39] as deprotonating agents and acid scavengers. Finally, the deprotection of the Cbz-group by hydrogenation [40] resulted in the desired 1-(phenylsulfonyl)spiro[indoline-3,3′-pyrrolidin]-2-one (17) compound. Following an alternative way, the more basic pyrido-nitrogen of tryptoline 29 might be first sulfonylated [41] followed by the N-bromosuccinimide assisted spiro-cyclization to 18 [42]. Compounds 17 and 18 showed improved selectivity towards the 5-HT6R (Table 3) as compared to the non-sulfonylated 2′-phenyl derivatives, investigated in the first batch (compounds 15-24), however, no improvement in the binding affinity was detected.  Compounds 17 and 18 showed improved selectivity towards the 5-HT 6 R (Table 3) as compared to the non-sulfonylated 2 -phenyl derivatives, investigated in the first batch (compounds 15-24), however, no improvement in the binding affinity was detected. Compounds 17 and 18 showed improved selectivity towards the 5-HT6R (Table 3) as compared to the non-sulfonylated 2′-phenyl derivatives, investigated in the first batch (compounds 15-24), however, no improvement in the binding affinity was detected. Facilitating the potency optimization, we followed the classical 5-HT6R pharmacophore pattern and tried to remove the polar interacting hydrogen bond acceptor feature in the oxindole ring of 17.
For better understanding of the indole, indoline and oxindole-related chemical space of known 5-HT6R ligands we collected all compounds, with at least 0.1 µM activity towards h5-HT6R from the ChEMBL database.
Substructure filtering by the query of 30 ( Figure 4) revealed that altogether 3 examples were found for oxindoles, as 5-HT6R ligands. All hits belong to the 3′-phenylspiro[indoline-3,2′thiazolidine]-2,4′-dione scaffold 31 that was identified by Hostetler et al. [43]. Searching for the 1- Compounds 17 and 18 showed improved selectivity towards the 5-HT6R (Table 3) as compared to the non-sulfonylated 2′-phenyl derivatives, investigated in the first batch (compounds 15-24), however, no improvement in the binding affinity was detected. Facilitating the potency optimization, we followed the classical 5-HT6R pharmacophore pattern and tried to remove the polar interacting hydrogen bond acceptor feature in the oxindole ring of 17.
For better understanding of the indole, indoline and oxindole-related chemical space of known 5-HT6R ligands we collected all compounds, with at least 0.1 µM activity towards h5-HT6R from the ChEMBL database.
Substructure filtering by the query of 30 ( Figure 4) revealed that altogether 3 examples were found for oxindoles, as 5-HT6R ligands. All hits belong to the 3′-phenylspiro[indoline-3,2′thiazolidine]-2,4′-dione scaffold 31 that was identified by Hostetler et al. [43]. Searching for the 1- To conclude, we have aimed to synthesize different 1-(phenylsulfonyl)indolines (scaffold 32), being substituted in the 1 -pyrrolidine nitrogen with either basic-nitrogen containing, or lipophilic groups, presented in Scheme 4-compounds 39a-b. Based on these considerations, first we benzylated [44] the tryptoline (25) to afford 36a, further converting it to the corresponding spiro-derivative [45] 37a. The reduction of the oxindole oxo-group was performed by applying borane-tetrahydrofuran complex in refluxing absolutized tetrahydrofuran [46]. It was an important observation, that the reduction step has to precede the sulfonylation, to avoid the formation of side products when treating the sulfonamide with the boronic reagent. The phenyl-sulfonylated product 39a was afforded either by applying LiHMDS [38] or trietylamine/dimethylaminopyridine [47] reagents. The pyridine-4-ylmethyl derivative (39b) was synthesized based on the same route, however, as a first step, the tryptoline (25) was treated with 4-chloromethylpyridine [48,49].  To conclude, we have aimed to synthesize different 1-(phenylsulfonyl)indolines (scaffold 32), being substituted in the 1′-pyrrolidine nitrogen with either basic-nitrogen containing, or lipophilic groups, presented in Scheme 4-compounds 39a-b. Based on these considerations, first we benzylated [44] the tryptoline (25) to afford 36a, further converting it to the corresponding spiro-derivative [45] 37a. The reduction of the oxindole oxo-group was performed by applying borane-tetrahydrofuran complex in refluxing absolutized tetrahydrofuran [46]. It was an important observation, that the reduction step has to precede the sulfonylation, to avoid the formation of side products when treating the sulfonamide with the boronic reagent. The phenyl-sulfonylated product 39a was afforded either by applying LiHMDS [38] or trietylamine/dimethylaminopyridine [47] reagents. The pyridine-4ylmethyl derivative (39b) was synthesized based on the same route, however, as a first step, the tryptoline (25) was treated with 4-chloromethylpyridine [48,49].
We also intended to examine the effect of only transforming our initial test compound 17 to the corresponding reduced indoline, however the debenzylation step of 39a afforded unexpected products (Scheme 5, 40a and 40b). In case, when methanol was applied as solvent for the debenzylation by catalytic hydrogenation, a methylated product 40a has formed and respectively, the use of ethanol afforded an ethyl-alkylated derivative 40b. However, both the alkylated derivatives showed some improvement of binding affinity to reach the low micromolar range, the compound possessing the biggest lipophilic, bulky group 43a has produced the best, submicromolar binding affinity, with both selectivity against the other closely related serotonergic targets (Table 4). We also intended to examine the effect of only transforming our initial test compound 17 to the corresponding reduced indoline, however the debenzylation step of 39a afforded unexpected products (Scheme 5, 40a and 40b). In case, when methanol was applied as solvent for the debenzylation by catalytic hydrogenation, a methylated product 40a has formed and respectively, the use of ethanol afforded an ethyl-alkylated derivative 40b. However, both the alkylated derivatives showed some improvement of binding affinity to reach the low micromolar range, the compound possessing the biggest lipophilic, bulky group 43a has produced the best, submicromolar binding affinity, with both selectivity against the other closely related serotonergic targets (Table 4). However, both the alkylated derivatives showed some improvement of binding affinity to reach the low micromolar range, the compound possessing the biggest lipophilic, bulky group 43a has produced the best, submicromolar binding affinity, with both selectivity against the other closely related serotonergic targets (Table 4). However, both the alkylated derivatives showed some improvement of binding affinity to reach the low micromolar range, the compound possessing the biggest lipophilic, bulky group 43a has produced the best, submicromolar binding affinity, with both selectivity against the other closely related serotonergic targets (Table 4). However, both the alkylated derivatives showed some improvement of binding affinity to reach the low micromolar range, the compound possessing the biggest lipophilic, bulky group 43a has produced the best, submicromolar binding affinity, with both selectivity against the other closely related serotonergic targets (Table 4). The submicromolar affinity of 39a prompted us investigating the substituent vectors at both the benzylic and the phenylsulfonyl rings by walking fluorine substituents around. This methodology, often referred as fluoro-scan has been used effectively for the identification of sites tolerant to functionalization. In particular, fluoro-scan explores the impact of enhanced lipophilicity, H-bonding and/or filling a small pocket [50]. In case of the N′-benzylic substitution [13] pattern, 2-, 3-and 4-fluorobenzylchlorides were used as alkylating agents in order to synthesize the corresponding fluorinated indolines (Scheme 6, 44a-c The submicromolar affinity of 39a prompted us investigating the substituent vectors at both the benzylic and the phenylsulfonyl rings by walking fluorine substituents around. This methodology, often referred as fluoro-scan has been used effectively for the identification of sites tolerant to functionalization. In particular, fluoro-scan explores the impact of enhanced lipophilicity, H-bonding and/or filling a small pocket [50]. In case of the N′-benzylic substitution [13] pattern, 2-, 3-and 4-fluorobenzylchlorides were used as alkylating agents in order to synthesize the corresponding fluorinated indolines (Scheme 6, 44a-c). The submicromolar affinity of 39a prompted us investigating the substituent vectors at both the benzylic and the phenylsulfonyl rings by walking fluorine substituents around. This methodology, often referred as fluoro-scan has been used effectively for the identification of sites tolerant to functionalization. In particular, fluoro-scan explores the impact of enhanced lipophilicity, H-bonding and/or filling a small pocket [50]. In case of the N -benzylic substitution [13] pattern, 2-, 3-and 4-fluorobenzylchlorides were used as alkylating agents in order to synthesize the corresponding fluorinated indolines (Scheme 6, 44a-c).
The submicromolar affinity of 39a prompted us investigating the substituent vectors at both the benzylic and the phenylsulfonyl rings by walking fluorine substituents around. This methodology, often referred as fluoro-scan has been used effectively for the identification of sites tolerant to functionalization. In particular, fluoro-scan explores the impact of enhanced lipophilicity, H-bonding and/or filling a small pocket [50]. In case of the N′-benzylic substitution [13] pattern, 2-, 3-and 4-fluorobenzylchlorides were used as alkylating agents in order to synthesize the corresponding fluorinated indolines (Scheme 6, 44a-c). The substitution of benzylic ring with fluorine caused a minor decrease in affinity, thus has shown, that growing towards this direction is not beneficial ( Table 5). On the other hand, however, some improvement in the selectivity was observed.
The alternative growing direction towards the phenylsulfonyl ring (compounds 46a-c) showed improvement in the affinities (see Table 6), compared to the results of 39a, also retaining the good selectivity against the 5-HT7 subtype. These data suggest, that position 2 of the phenylsulfonyl group of 46a is beneficial and therefore it is worth to explore this vector during the forthcoming lead optimization program, Scheme 6. Fluoro-scan of the N -benzyl direction.
The substitution of benzylic ring with fluorine caused a minor decrease in affinity, thus has shown, that growing towards this direction is not beneficial ( Table 5). On the other hand, however, some improvement in the selectivity was observed.     The alternative growing direction towards the phenylsulfonyl ring (compounds 46a-c) showed improvement in the affinities (see Table 6), compared to the results of 39a, also retaining the good selectivity against the 5-HT 7 subtype. These data suggest, that position 2 of the phenylsulfonyl group of 46a is beneficial and therefore it is worth to explore this vector during the forthcoming lead optimization program.

Binding Mode Analysis of the Optimized 5-HT6R Ligand
We have performed a docking analysis of the 1′-benzyl-1-((2-fluorophenyl)sulfonyl)-spiro [indoline-3,3′-pyrrolidine] (46a) 5-HT6R antagonist using the receptor model reported earlier [15]. Docking of 46a by Schrödinger-Glide either with, or without applying any constraints (requiring hydrogen bonding with both Asn288 6.55 and/or Ser193 5.43 ) resulted in consistent docking poses inside the orthosteric binding pocket ( Figure 5). The indoline core occupies the primary hydrophobic cavity of the binding pocket (formed by Trp281 6.48 , Phe284 6.51 , Phe285 6.52 ), while the protonated quaternary pyrrolidine-nitrogen is oriented towards the conserved Asp106 3.32 , forming a well-established hydrogen bond. The phenyl-sulfonyl moiety of 46a is sitting at a secondary hydrophobic cleft defined by Val107 3.33 , Ala157 4.56 , Leu160 4.59 , Pro160 4.60 and Leu164 4.63 , positioning the sulfonyl-linker as hydrogen-bond acceptor against Ser193 5.43 and Asn288 6.55 . Interestingly, the fluorine atom as hydrogen bond acceptor is offering a possible polar interaction with the Asn288 6.55 residue, underlining the preference of the ortho-substitution at the phenyl-sulfonyl ring. The indoline core occupies the primary hydrophobic cavity of the binding pocket (formed by Trp281 6.48 , Phe284 6.51 , Phe285 6.52 ), while the protonated quaternary pyrrolidine-nitrogen is oriented towards the conserved Asp106 3.32 , forming a well-established hydrogen bond. The phenyl-sulfonyl moiety of 46a is sitting at a secondary hydrophobic cleft defined by Val107 3.33 , Ala157 4.56 , Leu160 4.59 , Pro160 4.60 and Leu164 4.63 , positioning the sulfonyl-linker as hydrogen-bond acceptor against Ser193 5.43 and Asn288 6.55 . Interestingly, the fluorine atom as hydrogen bond acceptor is offering a possible polar interaction with the Asn288 6.55 residue, underlining the preference of the ortho-substitution at the phenyl-sulfonyl ring.

Conclusions
The structure-activity relationship around the original spiropyrrolidinyl-oxindole hit (6) was first investigated using the "SAR-by-catalog" approach that resulted in some moderate improvement in affinity and a low level of selectivity. Therefore, we decided to explore the novel 5-HT 6 R chemotype by a more conventional medicinal chemistry strategy. A synthetic tree (summarized in Scheme 8) of the synthesized oxindoles and indoles was elaborated starting from the core tryptoline intermediate (25).

Materials and Methods
All chemical reagents used were purchased from commercial chemical suppliers. The NMR experiments were performed at 500 MHz ( 1 H) on a Varian VNMR SYSTEM spectrometer. Chemical shifts are referenced to the residual solvent signals, 2.50 ppm for 1 H in DMSO-d6 and 7.28 ppm for 1 H in CDCl3. The MS measurements were performed on Shimadzu LCMS2020 LC/MS system. Flash

Materials and Methods
All chemical reagents used were purchased from commercial chemical suppliers. The NMR experiments were performed at 500 MHz ( 1 H) on a Varian VNMR SYSTEM spectrometer. Chemical shifts are referenced to the residual solvent signals, 2.50 ppm for 1 H in DMSO-d 6 and 7.28 ppm for 1 H in CDCl 3 . The MS measurements were performed on Shimadzu LCMS2020 LC/MS system. Flash chromatography was performed using Teledyne ISCO CombiFlash Lumen+ Rf. Purifications by preparative-HPLC were performed with Hanbon NS4205 Binary high pressure semi-preparative HPLC. Thin-layer chromatography was performed on TLC Silica Gel 60 F254. High resolution mass spectrometric measurements were performed using a Q-TOF Premier mass spectrometer (Waters Corporation, Milford, MA, USA) in positive electrospray ionization mode. Compounds 7-16 were purchased from Mcule Inc.

Radioligand Binding Assays
Cell pellets were thawed and homogenized in 10 volumes of assay buffer using an Ultra Turrax tissue homogenizer and centrifuged twice at 35,000 g for 20 min at 4 • C, with incubation for 15 min at 37 • C in between. The composition of the assay buffers was as follows: for 5-HT 1A R: 50 mM Tris-HCl, 0.1 mM EDTA, 4 mM MgCl 2 , 10 µM pargyline and 0.1% ascorbate; for 5-HT 2A R: 50 mM Tris-HCl, 0.1 mM EDTA, 4 mM MgCl 2 and 0.1% ascorbate; for 5-HT 6 R: 50 mM Tris-HCl, 0.5 mM EDTA and 4 mM MgCl 2 , for 5-HT 7b R: 50 mM Tris-HCl, 4 mM MgCl 2 , 10 µM pargyline and 0.1% ascorbate. All assays were incubated in a total volume of 200 µL in 96-well microtiter plates for 1 h at 37 • C, except for 5-HT 1A R and 5-HT 2A R which were incubated at room temperature for 1 h and 1.5 h respectively. The process of equilibration is terminated by rapid filtration through Unifilter plates with a 96-well cell harvester and radioactivity retained on the filters was quantified on a Microbeta plate reader (PerkinElmer).
For displacement studies the assay samples contained as radioligands: 2. Non-specific binding is defined with 10 µM of 5-HT in 5-HT 1A R and 5-HT 7 R binding experiments, whereas 10 µM of chlorpromazine or 10 µM of methiothepine were used in 5-HT 2A R and 5-HT 6 R assays, respectively. Each compound was tested in triplicate at 7-8 concentrations (10 −11 -10 −4 M). The inhibition constants (K i ) were calculated from the Cheng-Prusoff equation [47]. Results were expressed as means of at least three separate experiments (SD ≤ 24%).

Docking Procedure
The homology model of h-5HT 6 R [15] was prepared for docking experiment my Schrödinger's Protein Preparation Wizard by default settings for protomer-state optimization and restrained minimization by OPLS_2005 force field. Single precision docking was performed with Schrödinger's Glide by default settings. The ligand (46a) was prepared for docking by Schrödinger's LigPrep, generating possible ionization states (at pH range of 7.0 ± 2.0), tautomers and stereoisomers. Constrained docking was set to two required interactions (at least 1 match): hydrogen bond formed with Asn288 6.55 and/or hydrogen bond formed with Ser193 5. 43 .