Spiro[Pyrrolidine-3,3'-Oxindoles] and Their Indoline Analogues As New 5

molecules

Article Spiro[pyrrolidine-3,30-oxindoles] and Their Indoline Analogues as New 5-HT6 Receptor Chemotypes

Ádám A. Kelemen 1, Grzegorz Satala 2, Andrzej J. Bojarski 2 and György M. Keser ˝u 1,* 1 Medicinal Chemistry Research Group, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok körútja 2, H1117 Budapest, Hungary; [email protected] 2 Department of Medicinal Chemistry, Institute of Pharmacology, Polish Academy of Sciences, 12 Sm˛etnaStreet, 31-343 Krakow, Poland; [email protected] (G.S.); [email protected] (A.J.B.) * Correspondence: [email protected] or [email protected]; Tel.: +36-1-382-6821

Received: 1 November 2017; Accepted: 12 December 2017; Published: 14 December 2017

Abstract: Synthetic derivatives of spiro[pyrrolidinyl-3,30-oxindole] alkaloids (coerulescine analogues) were investigated as new ligands for aminergic G-protein coupled receptors (GPCRs). The chemical starting point 20-phenylspiro[indoline-3,30-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 20-phenyl-derivatives, introducing the phenylsulphonyl pharmacophore and examining the corresponding reduced spiro[pyrrolidine-3,30-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.

Keywords: oxindole; indoline; coerulescine; 5-HT6R; G-protein coupled receptor

1. Introduction The recent isolation of naturally occurring and biologically active spiropyrrolidinyl-oxindole alkaloids initiated a significant research on synthetic derivatives [1], particularly compounds with the spiro[indoline-3,30-pyrrolidine]-2-one ring system. Spiropyrrolidinyl-oxindole alkaloids were isolated from different species including Gelsemium sempervirens, Aspidosperma, Mitragyna, Ourouparia, Rauwolfia, Vinca species [2]. The most prominent examples are rynchophylline (1), formosanine (2), coerulescine (3), horsfiline (4) and elacomine (5) that show diverse biological activity (Figure1). Although these compounds have a common tryptamine derived motif, a ChEMBL-analysis [3] revealed that no representatives have been ever tested against serotonergic targets. Developing ligand-(FrAGs) [4] and structure-based (FrACS) [5] methods for the identification of aminergic receptor ligands we identified 6 as a micromolar 5-hydroxytryptamine receptor 6 (5-HT6R) ligand. The 5-HT6R 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-HT6R 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-HT6R antagonists are typically focused to the canonical pharmacophore model [9] (Figure2), 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 D3.32

Molecules 2017, 22, 2221; doi:10.3390/molecules22122221 www.mdpi.com/journal/molecules Molecules 2017, 22, 2221 2 of 25

Molecules 2017, 22, x 2 of 24 aspartate residue of aminergic GPCR’s as a key factor of aminergic 7TM-receptor activation [10]. A further pharmacophore feature might contain an additional intramolecular hydrogen bond donor moiety further stabilizing the binding conformation of the ligandsligands [[11]11] (see(see FigureFigure2 ).2). SelectivitySelectivity among other aminergic GPCR’s was shown [12] [12] to be accessible through omitting the positively ionizable group inin thethe 5-HT5-HT66R antagonists. Bis(hetero)arylsulphonyl-Bis(hetero)arylsulphonyl- and sulfonamide substituents also contribute to 5-HT6R affinity afﬁnity and selectivity.

Figure 1. 1. RepresentativeRepresentative natural natural spiropyr spiropyrrolidinyl-oxindolerolidinyl-oxindole alkaloids. alkaloids. The tryptamine The tryptamine scaffold scaffold is shown is shownas red skeleton. as red skeleton.

Figure 2. GeneralGeneral pharmacophore model of 5-HT66R antagonists. PI: positive ionisable portion; HYD1,21,2:: hydrophobic sites; iHBD: intramolecular hydrogen bond donor site; HBA: hydrogen bond acceptor

feature; HSi,j:: substituents substituents on on th thee hydrophobic hydrophobic sites. sites.

The tryptamine derived scaffold of 6 and its alignment with the published 5-HT6R pharmacophore The tryptamine derived scaffold of 6 and its alignment with the published 5-HT6R [9] prompted us to explore the structure-activity relationship around the spiropyrrolidinyl-oxindole pharmacophore[9] prompted us to [9 explore] prompted the structure-activity us to explore relationship the structure-activity around the spiropyrrolidinyl-oxindole relationship around the core. Here we report the results of our hit-to-lead program, which led to an identification of promising spiropyrrolidinyl-oxindolecore. Here we report the results core. of Here our wehit-to-lead report the program, results ofwhich our hit-to-leadled to an id program,entification which of promising led to an 5-HT6R ligands of this chemotype. identiﬁcation of promising 5-HT6R ligands of this chemotype.

2. Results and Discussion

2.1. Identification Identification and Early Structure-ActivityStructure-Activity Data on Spiro[pyrrolidine-3,30′-oxindole]-oxindole] DerivativesDerivatives Fragment libraries libraries containing containing a acouple couple hundreds hundreds or or even even thousands thousands of small of small polar polar molecules molecules are areroutinely routinely used used for hit for identification hit identification at the atearly the st earlyage of stage drug ofdiscovery. drug discovery. Fragment Fragment screening screening provides providesdiverse chemotypes diverse chemotypes and significant and significantoperational operationalfreedom for freedomthe further for optimization the further optimizationof promising ofhits. promising Inspired by hits. these Inspired advantages by these in a advantagesrecent study in we a developed recent study a strategy we developed for aminergic a strategy focused for aminergicfragment libraries focused using fragment a sequential libraries filtering using a methodology sequential filtering applying methodology ligand- and applying structure-based ligand- 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, Molecules 2017, 22, 2221 3 of 25

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 Moleculesthe fragments2017, 22, x into an ensemble of carefully selected aminergic GPCR X-ray structures (PDB3 of 24 ID: 3PBL, 3RZE, 4IB4, 4IAQ, 3UON, 4MQT, 4LDE, 2RH1, 3NY9). This resulted in a set of 36 top 3UON, 4MQT, 4LDE, 2RH1, 3NY9). This resulted in a set of 36 top ranked hit molecules which were ranked hit molecules which were measured on an aminergic target not being included in the measured on an aminergic target not being included in the original docking ensemble, namely original docking ensemble, namely 5-HT6R. We demonstrate the usefulness of the method for 5-HT6R. We demonstrate the usefulness of the method for comprehensive aminergic focused comprehensive aminergic focused screenings. Out of the four hits with low micromolar inhibitory screenings. Out of the four hits with low micromolar inhibitory results, the structurally novel 2′-(3- results, the structurally novel 20-(3-ﬂuorophenyl)spiro[indoline-3,30-pyrrolidin]-2-one (6) possessing fluorophenyl)spiro[indoline-3,3′-pyrrolidin]-2-one (6) possessing the spiro[pyrrolidine-3,3′-oxindole] the spiro[pyrrolidine-3,30-oxindole] scaffold (Table1), was selected for further optimization. scaffold (Table 1), was selected for further optimization. Table 1. Serotonergic G-protein coupled receptor (GPCR) panel of 6 as measured in binding assays of Table 1. Serotonergic G-protein coupled receptor (GPCR) panel of 6 as measured in binding assays four serotonin receptors (Ki values are in µM). of four serotonin receptors (Ki values are in µM). ID Structure 5-HT 5-HT 5-HT 5-HT ID Structure 5-HT1A 1A 5-HT2A2A 5-HT6 6 5-HT7 7

6 6 16.9116.91 5.05 6.75 6.75 8.488.48

As the next step of exploring the structure-activity relationship substructure search in the As the next step of exploring the structure-activity relationship substructure search in MCULE purchasable database [13] of 5 million compounds resulted in further 887 spiro[pyrrolidine- the MCULE purchasable database [13] of 5 million compounds resulted in further 887 3,3′-oxindole] derivatives. The molecules were prepared by Schrödinger’s LigPrep [14] creating spiro[pyrrolidine-3,30-oxindole] derivatives. The molecules were prepared by Schrödinger’s possible conformers, tautomers and protonation states by default settings. The ligands were then LigPrep [14] creating possible conformers, tautomers and protonation states by default settings. projected to single precision molecular docking analysis on a nine membered ensemble of molecular The ligands were then projected to single precision molecular docking analysis on a nine membered dynamics frames of an 5-HT6R homology model [15] (built using 5-HT2BR X-ray crystal structure [16] ensemble of molecular dynamics frames of an 5-HT R homology model [15] (built using 5-HT R in complex with ergotamine as template (PDB ID: 4IB4)).6 The docking poses were filtered in a2B X-ray crystal structure [16] in complex with ergotamine as template (PDB ID: 4IB4)). The docking post-processing step keeping only binding modes forming hydrogen bonds towards Asp1063.32, poses were filtered in a post-processing step keeping only binding modes forming hydrogen bonds Asn2886.55 and optionally Ser1935.43, occupying a primary hydrophobic cleft defined by Trp2816.48, towards Asp1063.32, Asn2886.55 and optionally Ser1935.43, occupying a primary hydrophobic cleft Phe2846.51 and Phe2856.52. A secondary hydrophobic subpocket was defined by the pose filtering defined by Trp2816.48, Phe2846.51 and Phe2856.52. A secondary hydrophobic subpocket was defined criteria as following: Val1073.33, Ala1574.56, Leu1604.59, Pro1614.60, Leu1644.63. The satisfactory poses of by the pose filtering criteria as following: Val1073.33, Ala1574.56, Leu1604.59, Pro1614.60, Leu1644.63. the molecules were scored to have the best ranking in possibly all of the 9 frames using consensus The satisfactory poses of the molecules were scored to have the best ranking in possibly all of the ranking [5]. Altogether ten compounds (7–16) were purchased and tested in our serotonergic panel 9 frames using consensus ranking [5]. Altogether ten compounds (7–16) were purchased and tested in (Table 2). our serotonergic panel (Table2). TableIn 2. spite Serotonergic of the structural GPCR panel diversity, of derivatives all virtual substituted screening in the 2 hits′-phenyl were moiety lacking as measured substitution in at the oxindole scaffold. However, compounds 7, 9 and 10 having oxygen-containing substituents in binding assays of four serotonin receptors (Ki values are in µM). the 3,4-positions of the 20-phenyl ring showed somewhat improved affinity compared to the initial compoundID6. The moderateStructure improvement in affinity 5-HT and1A the5-HT lack2A of5-HT selectivity6 5-HT7 amongst the closely related serotonergic targets have driven us to interpret the results in the context of the known 5-HT6R pharmacophore patterns [17] (Figure3). This analysis suggests that introducing a phenylsulfonyl 7 7.07 5.45 2.26 2.69 moiety either to the 1-nitrogen at the oxindole ring or to the 10-nitrogen at the pyrrolidine ring would be beneficial for both the affinity and selectivity of this chemotype.

8 1.74 2.28 3.18 1.09

9 9.84 2.69 3.20 8.82

10 12.08 4.33 4.11 12.74 Molecules 2017, 22, x 3 of 24 MoleculesMolecules 2017 2017, 22,, 22x , x 3 of3 24 of 24

3UON, 4MQT, 4LDE, 2RH1, 3NY9). This resulted in a set of 36 top ranked hit molecules which were 3UON,3UON, 4MQT, 4MQT, 4LDE, 4LDE, 2RH1, 2RH1, 3NY9). 3NY9). This This resulted resulted in ina set a set of of36 36top top ranked ranked hit hit molecules molecules which which were were measured on an aminergic target not being included in the original docking ensemble, namely measuredmeasured on on an anaminergic aminergic target target not not being being included included in inthe the original original docking docking ensemble, ensemble, namely namely 5-HT6R.6 We demonstrate the usefulness of the method for comprehensive aminergic focused 5-HT5-HT6R. R.We We demonstrate demonstrate the the usefulness usefulness of ofthe the me methodthod for for comprehensive comprehensive aminergic aminergic focused focused screenings. Out of the four hits with low micromolar inhibitory results, the structurally novel 2′-(3- screenings.screenings. Out Out of ofthe the four four hits hits with with low low micromol micromolar arinhibitory inhibitory results, results, the the structurally structurally novel novel 2′-(3- 2′-(3- fluorophenyl)spiro[indoline-3,3′-pyrrolidin]-2-one (6) possessing the spiro[pyrrolidine-3,3′-oxindole] fluorophenyl)spiro[indoline-3,3fluorophenyl)spiro[indoline-3,3′-pyrrolidin]-2-one′-pyrrolidin]-2-one (6) ( possessing6) possessing the the spiro[pyrrolidine-3,3 spiro[pyrrolidine-3,3′-oxindole]′-oxindole] scaffold (Table 1), was selected for further optimization. scaffoldscaffold (Table (Table 1), 1),was was selected selected for for further further optimization. optimization.

Table 1. Serotonergic G-protein coupled receptor (GPCR) panel of 6 as measured in binding assays TableTable 1. Serotonergic1. Serotonergic G-protein G-protein couple coupled receptord receptor (GPCR) (GPCR) panel panel of 6of as 6 measuredas measured in bindingin binding assays assays of four serotonin receptors (Ki valuesi are in µM). of fourof four serotonin serotonin receptors receptors (K i (valuesK values are are in µM).in µM).

ID Structure 5-HT5-HT1A 1A 5-HT5-HT2A 2A 5-HT5-HT6 6 5-HT7 IDID StructureStructure 5-HT1A 5-HT2A 5-HT6 5-HT5-HT7 7

6 16.91 5.05 6.75 8.48 6 6 16.9116.91 5.055.05 6.75 6.75 8.488.48

As the next step of exploring the structure-activity relationship substructure search in the AsAs the the next next step step of ofexploring exploring the the structure-acti structure-activityvity relationship relationship substr substructureucture search search in inthe the MCULE purchasable database [13] of 5 million compounds resulted in further 887 spiro[pyrrolidine- MCULEMCULE purchasable purchasable database database [13] [13] of 5of million 5 million compounds compounds resulted resulted in furtherin further 887 887 spiro[pyrrolidine- spiro[pyrrolidine- 3,3′-oxindole] derivatives. The molecules were prepared by Schrödinger’s LigPrep [14] creating 3,33,3′-oxindole]′-oxindole] derivatives. derivatives. The The mole moleculescules were were prepared prepared by bySchrödinger’s Schrödinger’s LigPrep LigPrep [14] [14] creating creating possible conformers, tautomers and protonation states by default settings. The ligands were then possiblepossible conformers, conformers, tautomers tautomers and and protonation protonation stat states esby bydefault default settings. settings. The The ligands ligands were were then then projected to single precision molecular docking analysis on a nine membered ensemble of molecular projectedprojected to tosingle single precision precision molecular molecular docking docking analysis analysis on on a nine a nine membered membered ensemble ensemble of ofmolecular molecular dynamics frames of an 5-HT6R 6homology model [15] (built using 5-HT2BR2B X-ray crystal structure [16] dynamicsdynamics frames frames of anof an5-HT 5-HT6R homologyR homology model model [15] [15] (built (built using using 5-HT 5-HT2BR X-rayR X-ray crystal crystal structure structure [16] [16] in complex with ergotamine as template (PDB ID: 4IB4)). The docking poses were filtered in a in incomplex complex with with ergotamine ergotamine as astemplate template (PDB (PDB ID: ID: 4IB4)). 4IB4)). The The docking docking poses poses were were filtered filtered in ina a post-processing step keeping only binding modes forming hydrogen bonds towards Asp1063.32,3.32 post-processingpost-processing step step keeping keeping only only binding binding modes modes forming forming hydrogen hydrogen bonds bonds towards towards Asp106 Asp1063.32, , Asn2886.55 6.55and optionally Ser1935.43,5.43 occupying a primary hydrophobic cleft defined by Trp2816.48,6.48 Asn288Asn2886.55 and and optionally optionally Ser193 Ser1935.43, occupying, occupying a primarya primary hydropho hydrophobicbic cleft cleft defined defined by byTrp281 Trp2816.48, , Phe2846.51 6.51and Phe2856.52.6.52 A secondary hydrophobic subpocket was defined by the pose filtering Phe284Phe2846.51 and and Phe285 Phe2856.52. A. Asecondary secondary hydrophobic hydrophobic subpocket subpocket was was defined defined by bythe the pose pose filtering filtering criteria as following: Val1073.33,3.33 Ala1574.56,4.56 Leu1604.59,4.59 Pro1614.60,4.60 Leu1644.63.4.63 The satisfactory poses of criteriacriteria as asfollowing: following: Val107 Val1073.33, Ala157, Ala1574.56, Leu160, Leu1604.59, Pro161, Pro1614.60, Leu164, Leu1644.63. The. The satisfactory satisfactory poses poses of of the molecules were scored to have the best ranking in possibly all of the 9 frames using consensus thethe molecules molecules were were scored scored to tohave have the the best best rankin ranking ing inpossibly possibly all allof ofthe the 9 frames9 frames using using consensus consensus rankingMolecules [5].2017 Altogether, 22, 2221 ten compounds (7–16) were purchased and tested in our serotonergic panel4 of 25 rankingranking [5]. [5]. Altogether Altogether ten ten compounds compounds (7 –(167–)16 were) were purchased purchased and and tested tested in inour our serotonergic serotonergic panel panel (Table 2). (Table(Table 2). 2). Table 2. Serotonergic GPCR panel of derivatives substituted in the 20-phenyl moiety as measured in Table 2. Serotonergic GPCR panel of derivatives substituted in the 2′-phenyl moiety as measured in TableTablebinding 2. Serotonergic2. Serotonergic assays of fourGPCR GPCR serotonin panel panel of receptors derivativesof derivatives (Ki substitutedvalues substituted arein inµ theinM). the 2′-phenyl 2′-phenyl moiety moiety as measuredas measured in in binding assays of four serotonin receptors (Ki valuesi are in µM). bindingbinding assays assays of fourof four serotonin serotonin receptors receptors (K i (valuesK values are are in µM).in µM). ID Structure 5-HT1A 5-HT2A 5-HT6 5-HT7 ID ID StructureStructure 5-HT 5-HT1A 1A 5-HT5-HT2A 2A5-HT 5-HT6 6 5-HT5-HT7 7 ID Structure 5-HT1A 5-HT2A 5-HT6 5-HT7

7 7.07 5.45 2.26 2.69 77 7 7.077.077.07 5.455.45 5.45 2.262.26 2.262.692.69 2.69

8 1.74 2.28 3.18 1.09 88 8 1.741.741.74 2.282.28 2.28 3.183.18 3.181.091.09 1.09

9 9.84 2.69 3.20 8.82 99 9 9.849.849.84 2.692.69 2.69 3.203.20 3.208.828.82 8.82

10 12.08 4.33 4.11 12.74 1010 10 12.0812.0812.08 4.334.33 4.33 4.114.11 4.1112.7412.74 12.74 Molecules 2017, 22, x 4 of 24 Molecules 2017, 22, x 4 of 24

1111 49.1149.11 8.68 8.686.20 6.2058.96 58.96 11 49.11 8.68 6.20 58.96

12 45.67 1.12 6.63 10.45 1212 45.6745.67 1.12 1.126.63 6.6310.45 10.45

13 7.14 5.77 7.73 7.49 1313 7.147.14 5.77 5.777.73 7.737.49 7.49

14 7.76 4.54 8.62 8.98 1414 7.767.76 4.54 4.548.62 8.628.98 8.98

15 40.50 23.70 9.49 46.72 1515 40.5040.50 23.70 23.709.49 9.4946.72 46.72

16 not active 53.89 16.54 not active 1616 notnot active active 53.89 53.8916.54 not16.54 active not active

In spite of the structural diversity, all virtual screening hits were lacking substitution at the 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 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 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 compound 6. The moderate improvement in affinity and the lack of selectivity amongst the closely 6 related serotonergic targets have driven us to interpret the results in the context of the known 5-HT6R 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 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 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. be beneficial for both the affinity and selectivity of this chemotype.

(a) (b) (a) (b) Figure 3. (a) 1-(phenylsulfonyl)spiro[indoline-3,3′-pyrrolidin]-2-one (17) superimposed onto the Figure 3. (a) 1-(phenylsulfonyl)spiro[indoline-3,3′-pyrrolidin]-2-one (17) superimposed onto the classical pharmacophore; (b) 1′-(phenylsulfonyl)spiro[indoline-3,3′-pyrrolidin]-2-one (18) fitting to classical pharmacophore; (b) 1′-(phenylsulfonyl)spiro[indoline-3,3′-pyrrolidin]-2-one (18) fitting to the pharmacophore pattern lacking a positive ionisable moiety. the pharmacophore pattern lacking a positive ionisable moiety.

Molecules 2017, 22, x 4 of 24

11 49.11 8.68 6.20 58.96

12 45.67 1.12 6.63 10.45

13 7.14 5.77 7.73 7.49

14 7.76 4.54 8.62 8.98

15 40.50 23.70 9.49 46.72

16 not active 53.89 16.54 not active

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 Molecules 2017, 22, 2221 5 of 25 be beneficial for both the affinity and selectivity of this chemotype.

(a) (b)

Figure 3.3. ((aa)) 1-(phenylsulfonyl)spiro[indoline-3,31-(phenylsulfonyl)spiro[indoline-3,3′-pyrrolidin]-2-one0-pyrrolidin]-2-one (17 (17) )superimposed superimposed onto onto thethe classical pharmacophore;pharmacophore; ( b(b)) 1 01-(phenylsulfonyl)spiro[indoline-3,3′-(phenylsulfonyl)spiro[indoline-3,30-pyrrolidin]-2-one′-pyrrolidin]-2-one (18 ()18 fitting) fitting to the to pharmacophorethe pharmacophore pattern pattern lacking lackin a positiveg a positive ionisable ionisable moiety. moiety. Molecules 2017, 22, x 5 of 24 2.2. Hit-to-Lead Optimization of Spiro[pyrrolidine-3,3 0-oxindoles] 2.2. Hit-to-Lead Optimization of Spiro[pyrrolidine-3,3′-oxindoles] Detailed elaboration of the spiro[pyrrolidine-3,30-oxindole] scaffold required a viable synthesis strategyDetailed for the elaboration preparation of ofthe designed spiro[pyrrolidine-3,3 analogues. The′-oxindole] first, conventional scaffold required approach a viable is based synthesis on an intramolecularstrategy for the Mannich-reactionpreparation of designed used in analogues. case of several The first, alkaloids conventional including approach (±)-horsfiline is based (4 on)[18 an] andintramolecular Spirotryprostatin Mannich-reaction B [19]. An used alternative in case of several approach alkaloids is the including oxidative (± reaction)-horsfiline of tryptolines(4) [18] and inducedSpirotryprostatin by tert-butyl B [19]. hypochlorite, An alternativeN-bromosuccinimide, approach is the oxidativeN-chlorosuccinimide, reaction of tryptolines sodium induced tungstate, by leadtert-butyl tetraacetate, hypochlorite, or osmium N-bromosuccinimide, tetroxide [20,21]. N The-chlorosuccinimide, reaction is completed sodium by thetungstate, subsequent lead tetraacetate, elimination ofor waterosmium that tetroxide finally results[20,21]. inThe the reaction reorganization is complete of thed by ring the systemsubsequent to spiro[pyrrolidine-3,3 elimination of water0-oxindoles] that finally (Schemeresults in1 the). reorganization of the ring system to spiro[pyrrolidine-3,3′-oxindoles] (Scheme 1).

Scheme 1. Mechanism of the succinimide assisted oxidativeoxidative spiro-rearrangement of tryptolines.

Further, sophisticated approaches such as [1,3]-dipolar cycloaddition reactions of azomethine 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 ylides [22], radical cyclization by AIBN [23], intramolecular Heck reaction [24] and asymmetric nitroolefination of oxindoles [25] are also available, however, used less frequently. 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 For the effective exploration of the structure-activity relationship we need a feasible and universal universal approach with acceptable functional group tolerance and high variability around the spiro- approach with acceptable functional group tolerance and high variability around the spiro-oxindole oxindole core. Considering the requirements of the early stage optimization program we aimed a core. Considering the requirements of the early stage optimization program we aimed a synthesis synthesis strategy that strategy that • uses readily accessible, simple starting materials • usesapplies readily well-documented, accessible, simple readily starting available materials reagents • applieshas key well-documented,intermediates to access readily a wide available variety reagents of derivatives • hasis not key necessarily intermediates stereoselective to access a at wide this varietystage of of the derivatives optimization • is not necessarily stereoselective at this stage of the optimization The oxidative spiro-rearrangement reactions offer a wide variety of oxidative reagents [20,21,26–33] and the corresponding starting materials are readily accessible through Pictet-Spengler reaction of tryptamines [34] (Scheme 2).

NH2 glyoxylic acid monohydrate conc.HCl/H2O H2O, RT, 2h NH reflux, 2h NH

N N COOH N H H H H 24 25 23 Scheme 2. Synthesis of tryptoline by Pictet-Spengler condensation.

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 Molecules 2017, 22, x 5 of 24

2.2. 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).

Scheme 1. Mechanism of the succinimide assisted oxidative spiro-rearrangement of tryptolines.

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 Molecules• has2017 key, 22 intermediates, 2221 to access a wide variety of derivatives 6 of 25 • is not necessarily stereoselective at this stage of the optimization The oxidative spiro-rearrangement reactionsreactions offeroffer aa widewide varietyvariety ofof oxidativeoxidative reagentsreagents [[20,21,26–33]20,21,26–33] and the correspondingcorresponding starting materials are readilyreadily accessibleaccessible throughthrough Pictet-SpenglerPictet-Spengler reaction of tryptamines [[34]34] (Scheme(Scheme2 2).).

NH2 glyoxylic acid monohydrate conc.HCl/H2O H2O, RT, 2h NH reflux, 2h NH

N N COOH N H H H H 24 25 23 Scheme 2. Synthesis of tryptoline by Pictet-Spengler condensation.

Following this synthesis strategy, tryptaminetryptamine ((2323)) waswas usedused asas aa startingstarting materialmaterial for the synthesis of the common intermediate tryptoline (25). The Pictet-Spengler condensa condensationtion reaction was performed by glyoxylicglyoxylic acid-monohydrateacid-monohydrate in aqueousaqueous mediummedium [[35].35]. The The crude acid intermediate 24 was decarboxylated in in refluxing refluxing concentrated concentrated hy hydrochloricdrochloric acid acid affording affording the thetetrahydro- tetrahydro-β-carboline.β-carboline. The Thefirst synthetic first synthetic route depicted route depicted in Scheme in 3 Schemestarts with3 starts the N- withacylation the ofN the-acylation tryptoline of (25 the). The tryptoline application (25). Theof Cbz application (carboxybenzyl) of Cbz (carboxybenzyl)[36] protecting group [36] protecting was necessary group for was two necessary reasons: for 1. twoachieve reasons: the 1.sulfonylation achieve the at sulfonylationthe oxindol-nitrogen, at the oxindol-nitrogen, 2. the spiro-rearrangement 2. the spiro-rearrangement reaction is not occurring reaction in case is not of unsubstituted pyrido-nitrogen. N-chlorosuccinimide [37] was used in the first experiments Moleculesoccurring 2017 in, 22 case, x of unsubstituted pyrido-nitrogen. N-chlorosuccinimide [37] was used in the6 firstof 24 experimentsas halogenating as halogenating reagent, reagent,providing providing the Cbz-protected the Cbz-protected spiro[pyrrolidine-3,3 spiro[pyrrolidine-3,3′-oxindole].0-oxindole]. The phenylsulfonylation was performed either by usingusing lithium-hexamethyl disilazane [38] [38] and sodium hydride [39] [39] as deprotonating agents and acid scav scavengers.engers. Finally, Finally, the deprotection of the Cbz-group by hydrogenation [40] [40 ]resulted resulted in in the the de desiredsired 1-(phenylsulfony 1-(phenylsulfonyl)spiro[indoline-3,3l)spiro[indoline-3,3′-pyrrolidin]-2-one0-pyrrolidin]-2-one (17) (compound.17) compound. Following Following an alternative an alternative way, way, the themore more basic basic pyrido-nitrogen pyrido-nitrogen of tryptoline of tryptoline 29 might29 might be firstbe first sulfonylated sulfonylated [41] [41 followed] followed by bythe the N-bromosuccinimideN-bromosuccinimide assisted assisted spiro-cyclization spiro-cyclization to 18 to 18[42].[42 ].

Scheme 3. Synthesis of 1- and 1 ′0-phenylsulfonyl-phenylsulfonyl derivatives.

Compounds 17 and 18 showed improved selectivity towards the 5-HT6R (Table 3) as compared Compounds 17 and 18 showed improved selectivity towards the 5-HT R (Table3) as compared to to the non-sulfonylated 2′-phenyl derivatives, investigated in the first batch6 (compounds 15–24), the non-sulfonylated 20-phenyl derivatives, investigated in the ﬁrst batch (compounds 15–24), however, however, no improvement in the binding affinity was detected. no improvement in the binding afﬁnity was detected.

Table 3. Serotonergic GPCR panel of the N-phenylsulfonylated derivatives at N′ and N-positions as

measured in binding assays of four serotonin receptors (Ki values are in µM).

ID Structure 5-HT1A 5-HT2A 5-HT6 5-HT7

17 not active not active 12.30 58.31

18 not measured 87.35 6.86 not active

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- (phenylsulfonyl) indoline scaffold 32, 27 actives were found with the structure 33. Out of the 27 active molecules 4 possess a positive ionizable moiety built upon the phenyl side of the sulfonyl and all of the remaining examples are equipped by the classical tryptamine-type amine feature. Searching by MoleculesMolecules 2017, 201722, x, 22, x 6 of 246 of 24 phenylsulfonylationphenylsulfonylation was wasperformed performed either either by usin by using lithium-hexamethylg lithium-hexamethyl disilazane disilazane [38] [38]and andsodium sodium hydridehydride [39] [39]as deprotonating as deprotonating agents agents and andacid acid scav scavengers.engers. Finally, Finally, the deprotection the deprotection of the of Cbz-group the Cbz-group by hydrogenationby hydrogenation [40] resulted[40] resulted in the in de thesired desired 1-(phenylsulfony 1-(phenylsulfonyl)spiro[indoline-3,3l)spiro[indoline-3,3′-pyrrolidin]-2-one′-pyrrolidin]-2-one (17) (17) compound.compound. Following Following an alternative an alternative way, way, the morethe more basic basic pyrido-nitrogen pyrido-nitrogen of tryptoline of tryptoline 29 might 29 might be be first firstsulfonylated sulfonylated [41] [41]followed followed by the by Nthe-bromosuccinimide N-bromosuccinimide assisted assisted spiro-cyclization spiro-cyclization to 18 to [42]. 18 [42].

SchemeScheme 3. Synthesis 3. Synthesis of 1- ofand 1- 1and′-phenylsulfonyl 1′-phenylsulfonyl derivatives. derivatives.

CompoundsCompounds 17 and 17 and18 showed 18 showed improved improved selectivity selectivity towards towards the 5-HTthe 5-HT6R (Table6R (Table 3) as 3) compared as compared Moleculesto theto non-sulfonylatedthe2017 non-sulfonylated, 22, 2221 2′-phenyl 2′-phenyl derivatives, derivatives, investigated investigated in the in firstthe firstbatch batch (compounds (compounds 157– of 2415 25),– 24), however,however, no improvement no improvement in the in bindingthe binding affinity affinity was wasdetected. detected. Table 3. Serotonergic GPCR panel of the N-phenylsulfonylated derivatives at N0 and N-positions as TableTable 3. Serotonergic 3. Serotonergic GPCR GPCR panel panel of the of N the-phenylsulfonylated N-phenylsulfonylated derivatives derivatives at N ′at and N′ Nand-positions N-positions as as measured in binding assays of four serotonin receptors (Ki values are in µM). measuredmeasured in binding in binding assays assays of four of fourserotonin serotonin receptors receptors (Ki values (Ki values are in are µM). in µM).

IDID ID Structure StructureStructure 5-HT 5-HT1A 5-HT1A 1A 5-HT5-HT5-HT2A2A 2A5-HT 5-HT5-HT6 6 65-HT5-HT5-HT7 7 7

1717 17 not activeactivenot active not not active activenot active 12.30 12.3012.30 58.3158.3158.31

1818 18 not measuredmeasurednot measured 87.35 87.35 87.35 6.866.86 6.86not notactivenot active active

Facilitating the potency optimization, we followed the classical 5-HT6R pharmacophore pattern FacilitatingFacilitating the potencythe potency optimization, optimization, we followed we followed the classicalthe classical 5-HT 5-HTR pharmacophore6R pharmacophore pattern pattern and tried to remove the polar interacting hydrogen bond acceptor feature6 in the oxindole ring of 17. andand tried tried to remove to remove the the polar polar interacting interacting hydrogen hydrogen bond bond acceptor acceptor feature feature in in the the oxindole oxindole ring ring of of 17. For better understanding of the indole, indoline and oxindole-related chemical space of known 17. ForFor betterbetter understanding understanding of of the the indole, indole, indoline indoline and an oxindole-relatedd oxindole-related chemical chemical space space of known of known 5-HT6R ligands we collected all compounds, with at least 0.1 µM activity towards h5-HT6R from the 5-HT5-HTR ligands6R ligands we collectedwe collected all compounds, all compounds, with with at least at least 0.1 µ 0.1M activityµM activity towards towards h5-HT h5-HTR from6R from the the ChEMBL6 database. 6 ChEMBLChEMBL database. database. Substructure filtering by the query of 30 (Figure 4) revealed that altogether 3 examples were SubstructureSubstructure filtering filtering by the by query the of query30 (Figure of 304 )(Figure revealed 4) that revealed altogether that 3 examplesaltogether were 3 examples found for were found for oxindoles, as 5-HT6R ligands. All hits belong to the 3′-phenylspiro[indoline-3,2′- oxindoles,found asfor 5-HT oxindoles,R ligands. as All 5-HT hits6 belongR ligands. to the All 30-phenylspiro[indoline-3,2 hits belong to the 30-thiazolidine]-2,4′-phenylspiro[indoline-3,20-dione ′- thiazolidine]-2,4′-dione6 scaffold 31 that was identified by Hostetler et al. [43]. Searching for the 1- scaffoldthiazolidine]-2,431 that was identified′-dione scaffold by Hostetler 31 that etwas al. [identified43]. Searching by Hostetle for ther 1-(phenylsulfonyl) et al. [43]. Searching indoline for the 1- (phenylsulfonyl) indoline scaffold 32, 27 actives were found with the structure 33. Out of the 27 active scaffold(phenylsulfonyl)32, 27 actives in weredoline found scaffold with 32 the, 27 structureactives were33. found Out of with the 27the active structure molecules 33. Out 4 of possess the 27 active a molecules 4 possess a positive ionizable moiety built upon the phenyl side of the sulfonyl and all of positivemolecules ionizable 4 possess moiety a built positive upon ionizable the phenyl moiety side ofbuilt the upon sulfonyl the andphenyl all of side the of remaining the sulfonyl examples and all of the remaining examples are equipped by the classical tryptamine-type amine feature. Searching by are equippedthe remaining by the examples classical tryptamine-type are equipped by amine the classi feature.cal tryptamine-type Searching by the 1-(phenylsulfonyl)indoleamine feature. Searching by 34 query resulted in 397 examples with the structure 35. The structures were analyzed based on the chain-length distance between the 3-carbon of the indole ring and the positive ionizable basic nitrogen atom, if any. We found 20 examples representing indoles with attached basic nitrogen (all tertiary amines). Only five compounds were identified with a single methylene linker out of which 2 were primary amines and 3 were tertiary amines. The largest part of the with structure 34 contains ethylene linker (201 examples out of 397) of which 123 were tertiary amines, 51 were secondary amines and 19 were primary amines. Much less compounds have 3 atom long linkage, 54 examples were identified, with 21 tertiary amines and 33 secondary amines and no primary amines. No ligands were found with basic nitrogen’s being further than 3 atoms from the indole core. To conclude, we have aimed to synthesize different 1-(phenylsulfonyl)indolines (scaffold 32), being substituted in the 10-pyrrolidine nitrogen with either basic-nitrogen containing, or lipophilic groups, presented in Scheme4—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]. Molecules 2017, 22, x 7 of 24 the 1-(phenylsulfonyl)indole 34 query resulted in 397 examples with the structure 35. The structures were analyzed based on the chain-length distance between the 3-carbon of the indole ring and the positive ionizable basic nitrogen atom, if any. We found 20 examples representing indoles with attached basic nitrogen (all tertiary amines). Only five compounds were identified with a single methylene linker out of which 2 were primary amines and 3 were tertiary amines. The largest part of the with structure 34 contains ethylene linker (201 examples out of 397) of which 123 were tertiary amines, 51 were secondary amines and 19 were primary amines. Much less compounds have 3 atom long linkage, 54 examples were identified, with 21 tertiary amines and 33 secondary amines and no

Moleculesprimary2017 amines., 22, 2221 No ligands were found with basic nitrogen’s being further than 3 atoms from8 ofthe 25 indole core.

O oxindoles R N S 2/3 examples O O N N H H

30 31

R N R

1-(phenylsulfonyl)indolines 23/27 examples

N N O O O S O S

4/27 examples 32 33

N R R

1-(phenylsulfonyl)indoles H/R N (H2C) n H/R 397 examples N N S O O O O S

34 35

Molecules 2017, 22, x Figure 4. Substructure analysis of ChEMBL 5-HT66R active compounds. 8 of 24

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]. 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. Scheme 4. Synthesis of N′0-substituted-substituted and and N-phenylsulfonylated-phenylsulfonylated indolines. indolines.

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 (Scheme5, 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.

Scheme 5. Synthesis of N′-methyl and ethyl derivatives.

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).

Table 4. Serotonergic GPCR panel of N-phenylsulfonyl indolines with different N′-substituents as

measured in binding assays of four serotonin receptors (Ki values are in µM).

ID Structure 5-HT1A 5-HT2A 5-HT6 5-HT7 N

39a N not measured 4.76 0.76 55.76 O O S

N N

39b N not active 26.85 6.60 not active O O S Molecules 2017, 22, x 8 of 24

MoleculesMolecules 2017 2017, ,22 22, ,x x 88 of of 24 24

Molecules 2017, 22, 2221 9 of 25 Scheme 4. Synthesis of N′-substituted and N-phenylsulfonylated indolines. SchemeScheme 4. 4. Synthesis Synthesis of of N N′-substituted′-substituted and and N N-phenylsulfonylated-phenylsulfonylated indolines. indolines.

Scheme 5.5. Synthesis of N0′-methyl and ethyl derivatives. SchemeScheme 5. 5. Synthesis Synthesis of of N N′-methyl′-methyl and and ethyl ethyl derivatives. derivatives. However, both the alkylated derivatives showed some improvement of binding affinity to reach However, both the alkylated derivatives showed some improvement of binding afﬁnity to reach the lowHowever, micromolar both the range, alkylated the compound derivatives po showedssessing some the improvementbiggest lipophilic, of binding bulky affinity group to43a reach has the lowHowever, micromolar both the range, alkylated the compoundderivatives possessingshowed some the improvement biggest lipophilic, of binding bulky affinity group to43a reachhas theproduced low micromolar the best, submicromolar range, the compound binding poaffinity,ssessing with the bothbiggest selectivity lipophilic, against bulky the group other 43a closely has theproduced low micromolar the best, submicromolarrange, the compound binding po afﬁnity,ssessing with the bothbiggest selectivity lipophilic, against bulky the group other 43a closely has producedrelated serotonergic the best, submicromolar targets (Table 4). binding affinity, with both selectivity against the other closely producedrelated serotonergic the best, submicromolar targets (Table4 ).binding affinity, with both selectivity against the other closely relatedrelated serotonergic serotonergic targets targets (Table (Table 4). 4). Table 4. Serotonergic GPCR panel of N-phenylsulfonyl indolines with different N′-substituents as Table 4. Serotonergic GPCR panel of N-phenylsulfonyl indolines with different N0-substituents as TableTablemeasured 4. 4. Serotonergic Serotonergic in binding assaysGPCR GPCR ofpanel panel four of serotoninof N N-phenylsulfonyl-phenylsulfonyl receptors ( Kindolines indolinesi values arewith with in different µM).different N N′-substituents′-substituents as as measured in binding assays of four serotonin receptors (Ki values are in µM). measuredmeasured in in binding binding assays assays of of four four serotonin serotonin receptors receptors ( K(Ki ivalues values are are in in µM). µM). ID Structure 5-HT1A 5-HT2A 5-HT6 5-HT7 ID Structure 5-HT1A 5-HT2A 5-HT6 5-HT7 IDID StructureStructureN 5-HT5-HT1A1A 5-HT5-HT2A2A 5-HT5-HT66 5-HT5-HT7 7 NN 39a N not measured 4.76 0.76 55.76 S O 39a39a39a ONN notnotnot measured measured measured 4.76 4.76 4.76 0.760.76 0.76 55.7655.76 55.76 S OO OO S

N N NN NN 39b N not active 26.85 6.60 not active S O 39b39b39b ONN notnotnot active active active 26.85 26.85 26.85 6.606.60 6.60notnot active activenot active S OO OO S

Molecules 2017, 22, x 9 of 24

40a40a N notnot measured measured 4.52 4.52 1.85 1.85 40.17 40.17 S O O S

40b40b N notnot measured measured 3.82 3.82 2.42 2.42not activenot active S O O S

The submicromolar affinity of 39a prompted us investigating the substituent vectors at both the 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, 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 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 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).

F F

F NH N N N 1.) NBS, AcOH N N THF/H O, BH /THF a:2-, b:3-, c:4-fluorobenzylchloride THF/H2O, BH3/THF K2CO3, DMF 0°C, 1.5 h abs. THF NH K2CO3, DMF NH O RT, overnight 2.) cc. NaHCO N reflux, overnight N RT, overnight 2.) cc. NaHCO3, H reflux, overnight H RT, 0.5 h 43a-c 25 41a-c 42a-c 43a-c

N N F

a: 2-fluoro PhSO2Cl, TEA b: 3-fluoro 2 N b: 3-fluoro c: 4-fluoro S O c: 4-fluoro DMAP, DCM O S RT, overnight

44a-c 44a-c 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 corresponding fluorophenylsulfonyl derivatives 46a–c were synthesized from compound 38a intermediate, previously synthesized (Scheme 7). 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, Molecules 2017, 22, x 9 of 24

40a N not measured 4.52 1.85 40.17 O O S

40b N not measured 3.82 2.42 not active O O S

The submicromolar affinity of 39a prompted us investigating the substituent vectors at both the Moleculesbenzylic2017 and, 22, 2221the phenylsulfonyl rings by walking fluorine substituents around. This methodology,10 of 25 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 0 and/orand/or filling filling a a small small pocketpocket [50[50].]. In case of the N′-benzylic-benzylic substitution substitution [13] [13 pattern,] pattern, 2-, 2-, 3- 3-and and 4-fluorobenzylchlorides4-fluorobenzylchlorides werewere usedused asas alkylatingalkylating agents in in order order to to synthesize synthesize the the corresponding corresponding fluorinatedfluorinated indolines indolines (Scheme (Scheme6 ,6,44a 44a––cc).).

F F

F NH N 1.) NBS, AcOH N N a:2-, b:3-, c:4-fluorobenzylchloride THF/H2O, BH3/THF 0°C, 1.5 h abs. THF NH K2CO3, DMF NH O N N RT, overnight 2.) cc. NaHCO3, H reflux, overnight H RT, 0.5 h 25 41a-c 42a-c 43a-c

N F

a: 2-fluoro PhSO Cl, TEA 2 N b: 3-fluoro O c: 4-fluoro DMAP, DCM O S RT, overnight

44a-c SchemeScheme 6. 6.Fluoro-scan Fluoro-scan of the N′0-benzyl-benzyl direction. direction.

The substitution of benzylic ring with fluorine caused a minor decrease in affinity, thus has shown, 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 thatimprovement growing towards in the selectivity this direction was observed. is not beneficial (Table5). On the other hand, however, some improvement in the selectivity was observed. Molecules The2017, corresponding22, x fluorophenylsulfonyl derivatives 46a–c were synthesized from compound10 of 24 38a Molecules 2017, 22, x 10 of 24 intermediate, previously synthesized (Scheme 7). TableTable 5. 5. SerotonergicSerotonergic GPCR GPCR panel panel of th ofe fluorobenzyl the fluorobenzyl derivatives derivatives as measured as measured in binding in assays binding of assays of TableTableThe 5. 5. alternativeSerotonergic Serotonergic growingGPCR GPCR panel panel direction of of th thee fluorobenzyl fluorobenzyl towards the derivatives derivatives phenylsulfonyl as as measured measured ring in in (compounds binding binding assays assays 46a of of –c) showed three serotonin receptors (Ki values are in µM). µ threethree serotonin serotonin receptors receptors (Ki (valuesKi values are in are µM). in M). improvementthree serotonin in receptors the affinities (Ki values (see are Ta inble µM). 6), compared to the results of 39a, also retaining the good selectivity against theID 5-HT7 subtype.Structure These data suggest, 5-HT1A that 5-HTposition6 5-HT 2 of 7the phenylsulfonyl group ID Structure 5-HT1A 5-HT6 5-HT7 ID StructureStructure 5-HT 5-HT1A1A 5-HT65-HT5-HT6 7 5-HT7 of 46a is beneficial and therefore it is worthF to explore this vector during the forthcoming lead F optimization program, N N

44a not active 4.53 78.93 44a N notnot active active 4.53 4.5378.93 78.93 SN O O S O O S

F F N N

44b not active 1.97 31.49 44b44b N not notactive active 1.97 1.9731.49 31.49 44b SN O not active 1.97 31.49 O S O O S

N N F F 44c44c N not notactive active 1.77 1.7715.74 15.74 44c NS O not active 1.77 15.74 O S O O S

Scheme 7. Fluoro-scan of the N-phenylsulfonyl direction. SchemeScheme 7. 7. Fluoro-scan Fluoro-scan of of the the N N-phenylsulfonyl-phenylsulfonyl direction. direction. Table 6. Serotonergic GPCR panel of the fluorophenylsulfonyl derivatives as measured in binding TableTable 6. 6. Serotonergic Serotonergic GPCR GPCR panel panel of of the the fluorophenyl fluorophenylsulfonylsulfonyl derivatives derivatives as as measured measured in in binding binding assays of three serotonin receptors (Ki values are in µM). assays of three serotonin receptors (Ki values are in µM). assays of three serotonin receptors (Ki values are in µM). ID Structure 5-HT1A 5-HT6 5-HT7 ID Structure 5-HT1A 5-HT6 5-HT7 ID Structure 5-HT1A 5-HT6 5-HT7

N N

46a not active 0.19 42.57 46a N not active 0.19 42.57 46a SN O not active 0.19 42.57 O S O O S F O F Molecules 2017, 22, x 10 of 24

Table 5. Serotonergic GPCR panel of the fluorobenzyl derivatives as measured in binding assays of

three serotonin receptors (Ki values are in µM).

ID Structure 5-HT1A 5-HT6 5-HT7 F

Molecules 2017, 22, x N 10 of 24

Table 5. Serotonergic 44aGPCR panel of the fluorobenzyl derivativesnot active as measured 4.53 in 78.93binding assays of N three serotonin receptors (Ki values are in µM).O O S

ID Structure 5-HT1A 5-HT6 5-HT7 F F N N

44a not active 4.53 78.93 N 44b S NO not active 1.97 31.49 O O O S

F N N F Molecules 2017, 22, 2221 11 of 25 44b44c N N notnot active active 1.971.77 31.4915.74 O O O SO S The corresponding ﬂuorophenylsulfonyl derivatives 46a–c were synthesized from compound 38a intermediate, previously synthesized (Scheme7).

N F

44c N not active 1.77 15.74 O O S

Scheme 7. Fluoro-scan of the N-phenylsulfonyl direction.

Table 6. Serotonergic GPCR panel of the fluorophenylsulfonyl derivatives as measured in binding The alternative growing direction towards the phenylsulfonyl ring (compounds 46a–c) showed assays of three serotonin receptors (Ki values are in µM). improvement in the afﬁnities (see Table6), compared to the results of 39a, also retaining the good selectivity against theID 5-HT 7 subtype.Structure These data suggest,5-HT1A that5-HT position6 5-HT 27 of the phenylsulfonyl group of 46a is beneﬁcial and therefore it is worth to explore this vector during the forthcoming lead N optimization program.Scheme 7. Fluoro-scan of the N-phenylsulfonyl direction.

TableTable 6. Serotonergic 6. Serotonergic 46aGPCR GPCR panel panel ofN the of the fluorophenyl ﬂuorophenylsulfonylnotsulfonyl active derivatives derivatives0.19 as42.57 measured as measured in binding in binding S O µ assaysassays of three of three serotonin serotonin receptors receptorsO (Ki (valuesKi valuesF areare in µM). in M).

IDID StructureStructure 5-HT 5-HT1A1A 5-HT65-HT5-HT6 7 5-HT7

46a 46a N notnot active active 0.19 0.1942.57 42.57 S O O F Molecules 2017, 22, x 11 of 24

46b not active 0.71 76.00 46b 46b N notnot active active 0.71 0.7176.00 76.00 S O O S

46c N not active 2.45 56.05 46c 46c S O notnot active active 2.45 2.4556.05 56.05 O S

6 2.3. 2.3.Binding Binding Mode Mode Analysis Analysis of the of Optimized the Optimized 5-HT 5-HT6R Ligand6R Ligand We Wehave have performed performed a dockingdocking analysis analysis of theof 1the0-benzyl-1-((2-fluorophenyl)sulfonyl)-spiro[indoline-3, 1′-benzyl-1-((2-fluorophenyl)sulfonyl)-spiro 0 6 [indoline-3,33 -pyrrolidine]′-pyrrolidine] (46a) 5-HT (46a6R) antagonist 5-HT6R antagonist using the receptorusing the model receptor reported model earlier reported [15]. Docking earlier of[15].46a by DockingSchrödinger—Glide of 46a by Schrödinger—Glideeither with, orwithout either with, applying or wi anythout constraints applying (requiringany constraints hydrogen (requiring bonding 6.55 5.43 hydrogen bonding with both Asn2886.55 and/or Ser1935.43) resulted in consistent docking poses inside the orthosteric binding pocket (Figure 5).

Figure 5. Binding mode of compound 46a in the orthosteric pocket of 5-HT6R. Figure 5. Binding mode of compound 46a in the orthosteric pocket of 5-HT6R.

The indoline core occupies the primary hydrophobic cavity of the binding pocket (formed by 6.48 6.51 6.52 Trp2816.48, Phe2846.51, Phe2856.52), while the protonated quaternary pyrrolidine-nitrogen is oriented 3.32 towards the conserved Asp1063.32, forming a well-established hydrogen bond. The phenyl-sulfonyl 3.33 4.56 4.59 moiety of 46a is sitting at a secondary hydrophobic cleft defined by Val1073.33, Ala1574.56, Leu1604.59, 4.60 4.63 5.43 Pro1604.60 and Leu1644.63, positioning the sulfonyl-linker as hydrogen-bond acceptor against Ser1935.43 6.55 and Asn2886.55. Interestingly, the fluorine atom as hydrogen bond acceptor is offering a possible polar 6.55 interaction with the Asn2886.55 residue, underlining the preference of the ortho-substitution at the phenyl-sulfonyl ring.

Molecules 2017, 22, x 11 of 24

46b N not active 0.71 76.00 O O S

N 46c O not active 2.45 56.05 O S

2.3. Binding Mode Analysis of the Optimized 5-HT6R Ligand

MoleculesWe2017 have, 22, 2221performed a docking analysis of the 1′-benzyl-1-((2-fluorophenyl)sulfonyl)-spiro12 of 25 [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 6.55 5.43 withhydrogen both Asn288bonding withand/or both Asn288 Ser193 6.55 )and/or resulted Ser193 in consistent5.43) resulted docking in consistent poses inside docking the poses orthosteric inside bindingthe orthosteric pocket binding (Figure5 pocket). (Figure 5).

Figure 5. Binding mode of compound 46a in the orthosteric pocket of 5-HT6R. Figure 5. Binding mode of compound 46a in the orthosteric pocket of 5-HT6R. The indoline core occupies the primary hydrophobic cavity of the binding pocket (formed by The indoline core occupies the primary hydrophobic cavity of the binding pocket (formed by Trp2816.48, Phe2846.51, Phe2856.52), while the protonated quaternary pyrrolidine-nitrogen is oriented Trp2816.48, Phe2846.51, Phe2856.52), while the protonated quaternary pyrrolidine-nitrogen is oriented towards the conserved Asp1063.32, forming a well-established hydrogen bond. The phenyl-sulfonyl 3.32 towardsmoiety of the 46a conserved is sitting Asp106at a secondary, forming hydrophobic a well-established cleft defined hydrogen by Val107 bond.3.33, Ala157 The phenyl-sulfonyl4.56, Leu1604.59, moiety of 46a is sitting at a secondary hydrophobic cleft deﬁned by Val1073.33, Ala1574.56, Leu1604.59, Pro1604.60 and Leu1644.63, positioning the sulfonyl-linker as hydrogen-bond acceptor against Ser1935.43 4.60 4.63 5.43 Pro160and Asn288and6.55. Leu164 Interestingly,, positioning the fluorine the sulfonyl-linkeratom as hydrogen as hydrogen-bond bond acceptor is acceptor offering against a possible Ser193 polar 6.55 andinteraction Asn288 with. Interestingly, the Asn2886.55 the residue, ﬂuorine underlining atom as hydrogen the preference bond acceptor of the is ortho-substitution offering a possible at polar the 6.55 interactionphenyl-sulfonyl with ring. the Asn288 residue, underlining the preference of the ortho-substitution at the phenyl-sulfonyl ring.

3. 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-HT6R chemotype by a more conventional medicinal chemistry strategy. A synthetic tree (summarized in Scheme8) of the synthesized oxindoles and indoles was elaborated starting from the core tryptoline intermediate (25). The removal of the bulky lipophilic site at the 20-position of the pyrrolidine ring and insertion of the classical phenylsulfonyl part resulted in notable selectivity across the serotoninergic GPCR panel of 4 receptors. The next optimization step was analyzing the chemistry of known indole, indoline and oxindole-based 5-HT6R inhibitors in ChEMBL. We have synthesized different 1-(phenylsulfonyl) indolines, being substituted in the 10-pyrrolidine nitrogen with either basic-nitrogen containing, or lipophilic groups. As a result, 10-benzyl-1-(phenylsulfonyl) spiro[indoline-3,30-pyrrolidine] (39a) and its corresponding 2-, 3-substituted analogues 46a–b were identified as 5-HT6R starting points. This opened a new chemical space for the under-represented spiro[pyrrolidine-3,30-indoline] chemotype. Molecules 2017, 22, x 12 of 24

3. 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-HT6R 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). The removal of the bulky lipophilic site at the 2′-position of the pyrrolidine ring and insertion of the classical phenylsulfonyl part resulted in notable selectivity across the serotoninergic GPCR panel of 4 receptors. The next optimization step was analyzing the chemistry of known indole, indoline and oxindole-based 5-HT6R inhibitors in ChEMBL. We have synthesized different 1-(phenylsulfonyl) indolines, being substituted in the 1′-pyrrolidine nitrogen with either basic-nitrogen containing, or lipophilic groups. As a result, 1′-benzyl-1-(phenylsulfonyl) spiro[indoline-3,3′-pyrrolidine] (39a) and itsMolecules corresponding2017, 22, 2221 2-, 3-substituted analogues 46a-b were identified as 5-HT6R starting points.13 This of 25 opened a new chemical space for the under-represented spiro[pyrrolidine-3,3′-indoline] chemotype.

Scheme 8. Spiro[pyrrolidine-3,30′-oxindoles] and spiro[pyrrolidine-3,3 0′-indolines] as 5HT6 ligands.

4. Materials and Methods All chemical reagents used were purchased from commercial chemical suppliers. The NMR All chemical reagents used were purchased from commercial chemical suppliers. The NMR experiments were performed at 500 MHz (1H) on a Varian VNMR SYSTEM spectrometer. Chemical experiments were performed at 500 MHz (1H) on a Varian VNMR SYSTEM spectrometer. Chemical shifts are referenced to the residual solvent signals, 2.50 ppm for 1H in1 DMSO-d6 and 7.28 ppm for 1H shifts are referenced to the residual solvent signals, 2.50 ppm for H in DMSO-d6 and 7.28 ppm in CDCl1 3. The MS measurements were performed on Shimadzu LCMS2020 LC/MS system. Flash for H in CDCl3. 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. Molecules 2017, 22, 2221 14 of 25

4.1. General Procedures for the Synthesis of Spiro[pyrrolidine-3,30-oxindole] and Spiro[pyrrolidine-3,30-indoline] Derivatives

4.1.1. Procedures to Afford 1-(Phenylsulfonyl)spiro[indoline-3,30-pyrrolidin]-2-one (17)

Synthesis of Benzyl 2-Oxospiro[indoline-3,30-pyrrolidine]-10-carboxylate (27) To the solution of benzyl 3,4-dihydro-1H-pyrido[3,4-b]indole-2(9H)-carboxylate (26) (918 mg, 3 mmol) in 10 mL tetrahydrofuran was added 10 mL distilled water and triethylamine (0.436 mL, 3.15 mmol, 1.05 equiv.) at room temperature. Afterwards N-chlorosuccinimide (431 mg, 3.24 mmol, 1.08 equiv.) was added at 0 ◦C in portions. The reaction mixture was stirred for 1.5 h at room temperature. The reaction mixture was diluted with 10 mL brine and was extracted using 2 × 10 mL ethyl acetate. The combined organic phases were dried over sodium-sulfate, filtered and evaporated under reduced pressure. The crude material was purified using flash chromatography (gradient: dichloromethane: methanol 0% to 5% methanol) to give 27 as a yellow solid. Yield: 399 mg (41%). 1 H-NMR (500 MHz, DMSO-d6) δ ppm 7.42–7.28 (m, 8H, ArH-benzyl, ArH-4, ArH-5, ArH-6), 7.14 0 (d, J = 7.8 Hz, 1H, ArH-7), 5.11 (s, 2H, benzyl CH2), 3.70–3.53 (m, 4H, pyrrolidine H-2 , pyrrolidine 0 0 13 H-5 ), 2.19–2.05 (m, 2H, H-4 ). C-NMR (125 MHz, DMSO-d6) δ ppm 176.9 (C-2), 154.8 (C=O), 142.8 (C-3a), 141.5 (C-7a), 135.9 (benzyl C-1), 128.5 (benzyl C-3, benzyl C-5), 127.9 (benzyl C-4, benzyl C-2, 0 benzyl C-6), 123.8 (C-4), 122.1 (C-5), 109.3 (C-7), 67.9 (benzyl CH2), 57.7 (C-3), 45.1 (pyrrolidine C-2 ), 43.3 (pyrrolidine C-50), 34.2 (pyrrolidine C-40). MS (DUIS+) m/z 323 [M + H]+.

Synthesis of Benzyl 2-oxo-1-(phenylsulfonyl)spiro[indoline-3,30-pyrrolidine]-10-carboxylate (28) (1) Procedure Using Sodium-Hydride To the solution of benzyl 2-oxospiro[indoline-3,30-pyrrolidine]-10-carboxylate (27) (175 mg, 0.542 mmol) in 5 mL anhydrous tetrahydrofuran was added sodium-hydride (60%, 28 mg, 0.705 mmol; 1.3 equiv.) at 0 ◦C. After stirring the reaction mixture at room temperature for 0.5 h, benzenesulfonyl chloride (0.076 mL, 0.596 mmol, 1.1 equiv.) was added dropwise at 0 ◦C. The reaction mixture was stirred overnight at room temperature, then it was quenched by 10 mL brine and was extracted using ethyl acetate (2 × 10 mL). The collected organic phases were dried using sodium sulfate, filtered and evaporated under reduced pressure. The crude product was purified using flash chromatography (gradient: hexane: ethyl acetate 0% to 20%) to afford 28 as a colorless solid. Yield: 94 mg (38%). (2) Procedure Using Lithium-Hexamethyl-Disilazane To the solution of benzyl 2-oxospiro[indoline-3,30-pyrrolidine]-10-carboxylate (27) (161 mg, 0.5 mmol) and benzenesulfonyl chloride (0.076 mL, 0.6 mmol, 1.2 equiv.) in 5 mL anhydrous tetrahydrofuran was added lithiumhexamethyl-disilazane (1M in THF, 0.85 mL, 0.85 mmol, 1.7 equiv.) at 0 ◦C and the reaction mixture was stirred at this temperature for 0.5 h. After quenching the reaction by 10 mL brine, the reaction mixture was extracted using ethyl acetate (2 × 10 mL) and the collected organic phases were dried over sodium sulfate, filtered and evaporated under reduced pressure. The crude product was purified using flash chromatography (gradient: hexane: ethyl acetate 0% to 1 20%) to give 28 as a white solid. Yield: 230 mg (99%). H-NMR (500 MHz, DMSO-d6) δ ppm 8.02 (d, J = 7.7 Hz, 2H, ArH-3”, ArH-5”), 7.77 (m, 2H, ArH-4”, ArH-7), 7.67 (m, 2H, phenyl-sulfonyl ArH-2”, phenyl-sulfonyl ArH-6”), 7.43–7.30 (m, 7H, ArH-4, ArH-6, benzyl ArH-2,3,4,5,6), 7.23 (t, J = 7.4 Hz, 1H, 0 0 ArH-5), 5.09 (m, 2H, benzyl CH2), 3.65–3.50 (m, 4H, pyrrolidine H-2 , pyrrolidine H-5 ), 2.25–2.09 (m, 0 13 2H, pyrrolidine H-4 ). C-NMR (125 MHz, DMSO-d6) δ ppm 170.1 (C-2), 154.3 (C=O), 136.2 (benzyl C-1), 135.5 (C-7a), 134.8 (phenyl-sulfonyl C-1”), 133.9 (C-3a), 133.1 (phenyl-sulfonyl C-4”), 129.2 (C-3”, C-5”), 128.5 (benzyl C-3, benzyl C-5), 128.0 (C-6, benzyl C-4), 127.5 (benzyl C-2, benzyl C-6), 127.4 0 0 (C-2”, C-6”), 123.3 (C-4), 121.8 (C-5), 113.8 (C-7), 67.5 (benzyl CH2), 66.1 (C-3), 46.4 (C-2 ), 46.1 (C-5 ), 30.3 (C-40). MS (DUIS+) m/z 463 [M + H]+. Molecules 2017, 22, 2221 15 of 25

Synthesis of 1-(Phenylsulfonyl)spiro[indoline-3,30-pyrrolidin]-2-one (17) To the solution of benzyl 2-oxo-1-(phenylsulfonyl)spiro[indoline-3,30-pyrrolidine]-10-carboxylate (28) (180 mg, 0.389 mmol) in methanol was added palladium (10%) on charcoal (90 mg, 5 w/w %) in an autoclave. The reactor was filled with 5 atm hydrogen gas and was stirred for 1 h at room temperature. The reaction mixture was then filtered on Celite and was evaporated under reduced pressure. The crude material was purified by flash chromatography (gradient: dichloromethane: methanol 0% to 5% methanol) to give 17 as an orange solid. Yield: 108 mg (85%). 1H-NMR (500 MHz, CDCl3) δ ppm 8.10 (d, J = 8.0 Hz, 2H, phenyl-sulfonyl ArH-3”, phenyl-sulfonyl ArH-5”), 7.91 (d, J = 8.4 Hz, 1H, phenyl-sulfonyl ArH-4”), 7.65 (t, J = 7.2 Hz, 1H, ArH-7), 7.53 (t, J = 7.2 Hz, 2H, phenyl-sulfonyl ArH-2”, phenyl-sulfonyl ArH-6”), 7.47 (d, J = 7.2 Hz, 1H, ArH-4), 7.32 (t, J = 8.0 Hz, 1H, ArH-6), 7.19 (t, J = 8.0 Hz, 1H, ArH-5), 3.08 (m, 1H, H-20), 2.93 (m, 1H, pyrrolidine H-20), 2.74–2.69 (m, 2H, pyrrolidine H-50), 2.28–2.23 (m, 1H, pyrrolidine H-40), 2.11–2.04 (m, 1H, pyrrolidine 0 13 H-4 ). C-NMR (125 MHz, DMSO-d6) δ ppm 175.2 (C-2), 166.8 (C-7a), 140.4 (phenyl-sulfonyl C-1”), 135.2 (C-3a), 133.0 (phenyl-sulfonyl C-4”), 129.5 (phenyl-sulfonyl C-3”, phenyl-sulfonyl C-5”), 128.4 (C-6), 127.1 (phenyl-sulfonyl C-2”, phenyl-sulfonyl C-6”), 122.5 (C-4), 121.7 (C-5), 109.6 (C-7), 55.8 (C-3), 48.3 (pyrrolidine C-20), 47.2 (pyrrolidine C-50), 33.0 (pyrrolidine C-40). MS (DUIS+) m/z 329 + + [M + H] . HRMS (ESI+): m/z [M + H] calcd. for C17H17N2O3S: 329.0960; found: 329.0949.

4.1.2. Synthesis of 10-(Phenylsulfonyl)spiro[indoline-3,30-pyrrolidin]-2-one (18) To the solution of 2-(phenylsulfonyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (29) (312 mg, 1 mmol) in tetrahydrofuran: water (10:10 mL) was added 10 mL glacial acetic acid at 0 ◦C. N-bromosuccinimide (178 mg, 1 mmol, 1 equiv.) was added slowly in portions at 0 ◦C. The reaction mixture was stirred for 1.5 h at 0 ◦C. Afterwards the reaction was quenched at 0 ◦C by 10 mL concentrated sodium-carbonate solution and was stirred for 0.5 h, allowing to warm up to room temperature. The mixture was extracted using ethyl acetate (2 × 10 mL). The combined organic phases were washed with concentrated sodium-hydrogen carbonate (2 × 10 mL) and by brine (2 × 10 mL). The organic phases were dried over sodium-sulfate, filtered and evaporated under reduced pressure. The crude product was purified using flash chromatography (gradient: hexane: ethyl acetate 0% to 20% ethyl acetate) to afford 18 as a colorless solid. Yield: 180 mg (55%). 1H-NMR (500 MHz, DMSO-d6) δ ppm 10.47 (s, 1H, NH), 7.87 (d, J = 7.3 Hz, 2H, phenyl-sulfonyl ArH-3”, ArH-5”), 7.76 (t, J = 7.3 Hz, 1H, phenyl-sulfonyl ArH-4”), 7.67 (t, J = 7.3 Hz, 2H, phenyl-sulfonyl ArH-2”, phenyl-sulfonyl ArH-6”), 7.15 (t, J = 7.3 Hz, 1H, ArH-4), 6.82 (t, J = 8.5 Hz, 2H, ArH-5, ArH-6), 6.69 (d, J = 7.3 Hz, 1H, ArH-7), 3.56 (t, J = 6.7 Hz, 1H, pyrrolidine H-20), 3.35 (t, J = 7.3 Hz, 1H, pyrrolidine H-20), 3.35 (d, J = 3.5 Hz, 2H, pyrrolidine H-50), 2.10–2.05 (m, 1H, pyrrolidine H-40), 0 13 1.94–1.88 (m, 1H, pyrrolidine H-4 ). C-NMR (125 MHz, DMSO-d6) δ ppm 181.4 (C-2), 167.0 (C-3a), 141.3 (C-7a), 135.8 (phenyl-sulfonyl C-1”), 133.3 (phenyl-sulfonyl C-4”), 129.5 (phenyl-sulfonyl C-3”, phenyl-sulfonyl C-5”), 128.3 (C-6), 127.4 (phenyl-sulfonyl C-2”, phenyl-sulfonyl C-6”), 122.4 (C-4), 121.8 (C-5), 109.6 (C-7), 55.6 (C-3), 47.4 (pyrrolidine C-20, pyrrolidine C-50), 35.8 (pyrrolidine C-40). + + MS (DUIS+) m/z 329 [M + H] . HRMS (ESI+): m/z [M + H] calcd. for C17H17N2O3S: 329.0960; found: 329.0957.

4.1.3. Procedures to Afford 10-Benzyl-1-(phenylsulfonyl)spiro[indoline-3,30-pyrrolidine] (39a) To the solution of 10-benzylspiro[indoline-3,30-pyrrolidin]-2-one (37a) (500 mg, 1.7985 mmol) in 15 mL absolutized tetrahydrofuran was added borane/tetrahydrofuran (1M) complex solution (4.495 mL, 4.495 mmol, 2.5 equiv.) and the reaction mixture was refluxed overnight. The reaction was allowed to cool down to room temperature and was quenched using 30 mL concentrated ammonium-chloride solution and was stirred for 20 min. Afterwards, 20 mL ethyl acetate was used for extraction. The combined organic phases were dried over sodium-sulfate, filtered and evaporated under reduced pressure to afford 400 mg 38a, used as a crude product in the further Molecules 2017, 22, 2221 16 of 25 steps without purification. To the solution of crude 10-benzylspiro[indoline-3,30-pyrrolidine] (38a) (400 mg 1.513 mmol) (in anhydrous dichloromethane was added triethylamine (0.419 mL, 3.03 mmol, 2 equiv.) and N,N-dimethylpyridin-4-amine (10 mg, 0.076 mmol, 0.5 equiv.) at room temperature. Afterwards benzenesulfonyl chloride (0.203 mL, 1.59 mmol, 1.05 equiv.) was added to the solution at 5 ◦C. The reaction mixture was stirred at room temperature overnight. The reaction was quenched using 10 mL 10% HCl solution and was extracted by 10 mL dichloromethane. The dichloromethane phase was collected and dried over sodium-sulfate. The drying agent was filtered-off and the solution was evaporated under reduced pressure. The crude product was purified by flash chromatography to 1 afford 39a as yellow oil. Yield: 621 mg (85% calculated to 37a). H-NMR (500 MHz, DMSO-d6) δ ppm 7.73 (d, J = 7.6 Hz, 2H, phenyl-sulfonyl H-2”, phenyl-sulfonyl H-6”), 7.58 (t, J = 7.6 Hz, 1H, H-6), 7.46 (t, J = 7.9 Hz, 3H, phenyl-sulfonyl H-3”, phenyl-sulfonyl H-4”, phenyl-sulfonyl H-5”), 7.31 (t, J = 7.6 Hz, 2H, phenyl-sulfonyl H-3”, phenyl-sulfonyl H-5”), 7.27–7.21 (m, 5H, benzyl H-1-5), 7.05 (t, J = 7.6 Hz, 1H, H-5), 3.89 (d, J = 10.9 Hz, 1H, H-2), 3.78 (d, J = 10.9 Hz, 1H, H-2), 3.52 (s, 2H, benzyl CH2), 2.69 (m, 1H, pyrrolidine H-20), 2.57 (m, 1H, pyrrolidine H-20), 2.24 (d, J = 9.1 Hz, 1H, pyrrolidine H-50), 2.15 (d, J = 9.2 Hz, 1H, pyrrolidine H-50), 1.91–1.85 (m, 1H, pyrrolidine H-40), 1.78–1.73 (m, 1H, pyrrolidine 0 13 H-4 ). C-NMR (125 MHz, DMSO-d6) δ ppm 141.0 (C-3a), 139.0 (C-7a), 134.2 (benzyl C-1), 129.8 (phenyl-sulfonyl C-1”), 129.8 (phenyl-sulfonyl C-4”), 128.7 (phenyl-sulfonyl C-3”, phenyl-sulfonyl C-5”), 128.7 (benzyl C-3, benzyl C-5), 127.4 (benzyl C-2, benzyl C-6), 127.4 (C-6), 124.9 (phenyl-sulfonyl C-2”, phenyl-sulfonyl C-6”, benzyl C-4), 124.2 (C-4), 121.8 (C-5), 114.6 (C-7), 66.7 (pyrrolidine C-20), 0 0 + 63.5 (benzyl CH2), 59.3 (C-3), 53.3 (pyrrolidine C-5 ), 49.8 (C-4 ). MS (DUIS+) m/z 405 [M + H] . HRMS + (ESI+): m/z [M + H] calcd. for C24H25N2O2S: 405.1637; found: 405.1620.

4.1.4. Procedures to Afford 1-(Phenylsulfonyl)-10-(pyridin-4-ylmethyl)spiro[indoline-3,30-pyrrolidine] (39b)

Synthesis of 10-(Pyridin-4-ylmethyl)spiro[indoline-3,30-pyrrolidin]-2-one (37b) To the solution of the crude 2-(pyridin-4-ylmethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (36b) (812 mg) in tetrahydrofuran:water (10:10 mL) was added 10 mL glacial acetic acid at 0 ◦C. N-bromosuccinimide (549 mg, 3.0874 mmol) was added slowly in portions at 0 ◦C. The reaction mixture was stirred for 1.5 h at 0 ◦C. Afterwards the reaction was quenched at 0 ◦C by 20 mL concentrated sodium-carbonate solution and was stirred for 0.5 h, allowing to warm up to room temperature. The mixture was extracted using ethyl-acetateethyl acetate (2 × 10 mL). The combined organic phases were washed with concentrated sodium-hydrogen carbonate (2 × 10 mL) and by brine (2 × 10 mL). The organic phases were dried over sodium-sulfate, filtered and evaporated under reduced pressure. The crude product was purified using flash chromatography (gradient: dichloromethane: methanol 0% to 10% methanol) to afford 37b as a yellow solid. Yield: 293 mg (35% 1 calculated to 25). H-NMR (500 MHz, DMSO-d6) δ ppm 11.63 (s, 1H, NH), 8.52 (d, J = 5.6 Hz, 2H, pyridine ArH-2, pyridine ArH-6), 7.59 (d, J = 7.2 Hz, 1H, ArH-4), 7.40 (d, J = 8.0 Hz, 1H, ArH-7), 7.32 (d, J = 5.5 Hz, 2H, pyridine ArH-3, pyridine ArH-5), 7.23 (t, J = 7.6 Hz, 1H, ArH-6), 7.05 (t, J = 7.3 Hz, 0 1H, ArH-5), 4.72 (s, 2H, pyridyl-methyl CH2), 3.65 (t, J = 6.7 Hz, 2H, pyrrolidine H-2 ), 3.31 (m, 2H, 0 0 13 pyrrolidine H-5 ), 3.02 (t, J = 6.7 Hz, 2H, pyrrolidine H-4 ). C-NMR (125 MHz, DMSO-d6) δ ppm 179.3 (C=O), 148.5 (pyridine C-2, pyridine C-6), 147.2 (pyridine C-1), 141.6 (C-7a), 134.7 (C-3a), 128.1 (C-6), 123.5 (C-4), 122.4 (pyridine C-3, pyridine C-5), 109.7 (C-7), 61.2 (pyridyl-methylene CH2), 60.1 (C-3), 54.4 (pyrrolidine C-20), 53.6 (pyrrolidine C-50), 34.7 (pyrrolidine C-40). MS (DUIS+) m/z 280 [M + H]+.

Synthesis of 1-(Phenylsulfonyl)-10-(pyridin-4-ylmethyl)spiro[indoline-3,30-pyrrolidine] (39b) To the solution of 10-(pyridin-4-ylmethyl)spiro[indoline-3,30-pyrrolidin]-2-one (37b) (289 mg, 1.05 mmol) in 10 mL anhydrousdry tetrahydrofuran was added borane/tetrahydrofuran (1 M) complex solution (2.63 mL, 2.63 mmol, 2.5 equiv.) and the reaction mixture was refluxed overnight. The reaction was allowed to cool down to room temperature and was quenched using 15 mL concentrated ammonium-chloride solution and was stirred for 20 min. Afterwards, 10 mL ethyl acetate was used Molecules 2017, 22, 2221 17 of 25 for extraction. The combined organic phases were dried over sodium-sulfate, filtered and evaporated under reduced pressure to afford 292 mg 38b, used as a crude product in the further steps without purification. To the solution of crude 10-(pyridin-4-ylmethyl)spiro[indoline-3,30-pyrrolidine] (38b) (0.292 mg) in 5 mL anhydrous dichloromethane was added triethylamine (0.307 mL, 2.22 mmol) and N,N-dimethylpyridin-4-amine (6.8 mg, 0.0555 mmol) at room temperature. Afterwards benzenesulfonyl chloride (204 mg, 1.161 mmol) was added to the solution at 5 ◦C. The reaction mixture was stirred at room temperature overnight. The reaction was quenched using 10 mL 10% HCl solution and was extracted by 5 mL dichloromethane. The dichloromethane phase was collected and dried over sodium-sulfate. The drying agent was filtered-off and the solution was evaporated under reduced pressure. The crude product was purified by flash chromatography to afford 39b as yellow 1 solid. Yield: 0.003 mg. H-NMR (500 MHz, DMSO-d6) δ ppm 8.50 (d, J = 6.2 Hz, 2H, pyridine ArH-2, pyridine ArH-6), 7.72 (d, J = 7.9 Hz, 2H, phenyl-sulfonyl ArH-2”, phenyl-sulfonyl ArH-6”), 7.58–7.53 (m, 3H, pyridine ArH-3, pyridine ArH-5, phenyl-sulfonyl ArH-4”), 7.49 (d, J = 7.8 Hz, 1H, ArH-7), 7.43 (t, J = 8.0 Hz, 2H, phenyl-sulfonyl ArH-3”, phenyl-sulfonyl ArH-5”), 7.28–7.23 (m, 2H, ArH-4, ArH-6), 7.07 (t, J = 7.1 Hz, 1H, ArH-5), 3.90 (d, J = 11.1 Hz, 1H, pyridyl methyl CH2), 3.83 (d, J = 11.1 Hz, 1H, pyridyl methyl CH2), 3.74 (d, J = 15.2 Hz, 1H, H-2), 3.67 (d, J = 15.2 Hz, 1H, H-2), 2.77 (m, 1H, pyrrolidine H-20), 2.62 (m, 1H, pyrrolidine H-20), 2.17 (m, 2H, pyrrolidine H-50), 1.95–1.89 (m, 1H, 0 0 13 pyrrolidine H-4 ), 1.82–1.78 (m, 1H, pyrrolidine H-4 ). C-NMR (125 MHz, DMSO-d6) δ ppm 148.6 (pyridine C-2, pyridine C-6), 141.0 (pyridine C-1), 143.9 (C-3a), 143.1 (C-7a), 139.5 (phenyl-sulfonyl C-1”), 134.1 (phenyl-sulfonyl C-4”), 130.0 (phenyl-sulfonyl C-3,” phenyl-sulfonyl C-5”), 128.1 (C-6), 127.3 (phenyl-sulfonyl C-2”, phenyl-sulfonyl C-6”), 127.6 (C-4), 124.2 (pyridine C-3, pyridine C-5), 124.8 0 (C-5), 114.1 (C-7), 65.6 (pyrrolidine C-2 ), 61.5 (benzyl CH2), 60.1 (C-2), 53.0 (C-3), 51.3 (pyrrolidine C-50), 32.9 (pyrrolidine C-40). MS (DUIS+) m/z 406 [M + H]+. HRMS (ESI+): m/z [M + H]+ calcd. for C23H24N3O2S: 406.1589; found: 406.1586.

4.1.5. Synthesis of 10-Methyl-1-(phenylsulfonyl)spiro[indoline-3,30-pyrrolidine] (40a) The solution of 10-benzyl-1-(phenylsulfonyl)spiro[indoline-3,30-pyrrolidine] (39a) (2 mg, 0.495 mmol) in 5 mL methanol was filled into an autoclave. Palladium (10%) on charcoal (100 mg, 5 w/w %) was added to the solution and reaction was stirred overnight under 5 bar hydrogen atmosphere at 50 ◦C. The reaction mixture was filtered through a pad of Celite and was evaporated under reduced pressure. The crude residual was purified by flash chromatography (gradient: dichloromethane: methanol 0% to 10% methanol) to afford 40a as a colorless solid. Yield: 37 mg (23%). 1 H-NMR (500 MHz, DMSO-d6) δ ppm 7.79 (d, J = 7.5 Hz, 2H, phenyl-sulfonyl ArH-2”, phenyl-sulfonyl ArH-6”), 7.66 (t, J = 7.2 Hz, 1H, phenyl-sulfonyl ArH-4”), 7.56 (t, J = 7.9 Hz, 2H, phenyl-sulfonyl ArH-3”, phenyl-sulfonyl ArH-5”), 7.48 (d, J = 7.9 Hz, 1H, ArH-7), 7.22 (m, 2H, ArH-4, H-6), 7.03 (t, J = 7.9 Hz, 1H, ArH-5), 3.87 (d, J = 11.8 Hz, 1H, H-2), 3.75 (d, J = 11.8 Hz, 1H, H-2), 2.62 (m, 1H, pyrrolidine H-20), 2.44 (m, 1H, pyrrolidine H-20), 2.24 (d, J = 9.1 Hz, 1H, pyrrolidine H-50), 2.17 0 0 (s, 3H, methyl CH3), 2.15 (d, J = 9.1 Hz, 1H, pyrrolidine H-5 ), 1.85–1.80 (m, 1H, pyrrolidine H-4 ), 0 13 1.74–1.69 (m, 1H, pyrrolidine H-4 ). C-NMR (125 MHz, DMSO-d6) δ ppm 143.1 (C-3a, C-7a), 133.1 (phenyl-sulfonyl C-1”, phenyl-sulfonyl C-4”), 129.2 (phenyl-sulfonyl C-3”, phenyl-sulfonyl C-5”), 127.5 (C-6, phenyl-sulfonyl C-2”, phenyl-sulfonyl C-6”), 124.1 (C-4), 114.5 (C-5), 110.0 (C-7), 69.1 (pyrrolidine C-20), 60.2 (pyrrolidine C-50), 55.5 (C-3), 52.8 (C-2), 42.3 (pyrrolidine N(10)-methyl), 32.5 (pyrrolidine 0 + + C-4 ). MS (DUIS+) m/z 329 [M + H] . HRMS (ESI+): m/z [M + H] calcd. for C18H21N2O2S: 343.1324; found: 343.1320.

4.1.6. Synthesis of 10-Ethyl-1-(phenylsulfonyl)spiro[indoline-3,30-pyrrolidine] (40b) The solution of 10-benzyl-1-(phenylsulfonyl)spiro[indoline-3,30-pyrrolidine] (39a) (100 mg, 0.2475 mmol) in 5 mL methanol was ﬁlled into an autoclave. Palladium (10%) on charcoal (500 mg, 5 w/w %) was added to the solution and reaction was stirred overnight under 5 bar hydrogen atmosphere at 50 ◦C. The reaction mixture was ﬁltered through a pad of Celite and was evaporated under reduced pressure. Molecules 2017, 22, 2221 18 of 25

The crude residual was puriﬁed by ﬂash chromatography (gradient: dichloromethane: methanol 0% to 1 10% methanol) to afford 40b as a colorless solid. Yield: 19 mg (22%). H-NMR (500 MHz, DMSO-d6) δ ppm 7.78 (m, 2H, phenyl-sulfonyl ArH-3”, phenyl-sulfonyl ArH-5”), 7.66 (m, 1H, phenyl-sulfonyl ArH-4”), 7.56 (m, 2H, phenyl-sulfonyl ArH-2”, phenyl-sulfonyl ArH-6”), 7.50–7.47 (m, 1H, ArH-7), 7.24–7.21 (m, 2H, ArH-4, ArH-6), 7.04 (t, J = 7.6 Hz, 1H, ArH-5), 3.86 (d, J = 10.8 Hz, 1H, H-2), 3.75 (d, J = 10.8 Hz, 1H, H-2), 2.68 (m, 1H, pyrrolidine H-20), 2.44 (m, 1H, pyrrolidine H-20), 2.34 (q, J = 6.4 Hz, 0 0 2H, ethyl CH2), 2.24 (m, 1H, pyrrolidine H-5 ), 2.11 (q, J = 8.9 Hz, 1H, pyrrolidine H-5 ), 1.86–180 (m, 0 0 13 1H, pyrrolidine H-4 ), 1.73–167 (m, 1H, pyrrolidine H-4 ), 0.93 (t, J = 7.6 Hz, 3H, ethyl CH3). C-NMR (125 MHz, DMSO-d6) δ ppm 143.3 (C-3a, C-7a), 133.1 (phenyl-sulfonyl C-1”, phenyl-sulfonyl C-4”), 129.5 (phenyl-sulfonyl C-3”, phenyl-sulfonyl C-5”), 127.7 (C-6, phenyl-sulfonyl C-2”, phenyl-sulfonyl C-6”), 124.1 (C-4), 114.1 (C-5), 110.1 (C-7), 69.1 (pyrrolidine C-20), 60.2 (pyrrolidine C-50), 55.3 (C-3), 0 0 0 52.1 (C-2), 50.0 (pyrrolidine N(1 )-ethyl CH2), 32.5 (pyrrolidine C-4 ), 13.6 (pyrrolidine N(1 )-ethyl + + CH3). MS (DUIS+) m/z 343 [M + H] . HRMS (ESI+): m/z [M + H] calcd. for C19H23N2O2S: 343.1480; found: 343.1496.

4.1.7. Procedures to Afford 10-(2-Fluorobenzyl)-1-(phenylsulfonyl)spiro[indoline-3,30-pyrrolidine] (44a)

Synthesis of 10-(2-Fluorobenzyl)spiro[indoline-3,30-pyrrolidin]-2-one (42a) To the solution of 41a (1.54 g, 5.5 mmol) in tetrahydrofuran: water (30:30 mL) was added 30 mL glacial acetic acid at 0 ◦C. N-bromosuccinimide (979 mg, 5.5 mmol, 1 equiv.) was added slowly in portions at 0 ◦C. The reaction mixture was stirred for 1.5 h at 0 ◦C. Afterwards the reaction was quenched at 0 ◦C by 40 mL concentrated sodium-carbonate solution and was stirred for 0.5 h, allowing to warm up to room temperature. The mixture was extracted using ethyl acetate (2 × 20 mL). The combined organic phases were washed with concentrated sodium-hydrogen carbonate (2 × 20 mL) and by brine (2 × 20 mL). The organic phases were dried over sodium-sulfate, filtered and evaporated under reduced pressure. The product 42a was purified by flash chromatography (gradient: 1 dichloromethane: methanol 0% to 10% methanol) Yield: 970 mg (56%). H-NMR (500 MHz, DMSO-d6) δ ppm 10.31 (s, 1H, NH), 8.01 (d, J = 8.8 Hz, 1H, benzyl ArH-6), 7.77 (d, J = 7.9 Hz, 1H, ArH-4), 7.65 (t, J = 7.9 Hz, 1H, benzyl ArH-4), 7.47 (t, J = 6.2 Hz, 1H, benzyl ArH-5), 7.24 (t, J = 8.4 Hz, 1H, ArH-6), 7.16 (m, 1H, benzyl ArH-3), 6.94 (t, J = 7.5 Hz, 1H, ArH-5), 6.80 (d, J = 7.5 Hz, 1H, ArH-7), 3.75 (s, 2H, 0 0 benzyl CH2), 3.06 (m, 1H, pyrrolidine H-2 ), 2.76 (d, J = 8.9 Hz, 1H, pyrrolidine H-5 ), 2.69 (d, J = 8.9 Hz, 1H, pyrrolidine H-50), 2.61 (q, J = 7.9 Hz, 1H, pyrrolidine H-2’), 2.20–2.15 (m, 1H, pyrrolidine H-40), 0 13 1.92–1.87 (m, 1H, pyrrolidine H-4 ). C-NMR (125 MHz, DMSO-d6) δ ppm 181.5 (C=O), 178.0 (benzyl C-2), 141.6 (C-7a), 137.0 (C-3a), 135.5 (benzyl C-6), 130.2 (benzyl C-4), 127.6 (C-6), 124.7 (benzyl C-1), 123.9 (benzyl C-5), 123.3 (C-4), 122.3 (C-5), 115.7 (benzyl C-3), 109.6 (C-7), 64.0 (C-3), 53.86 (benzyl CH2), 52.9 (pyrrolidine C-20), 51.7 (pyrrolidine C-50), 37.1 (pyrrolidine C-40). MS (DUIS+) m/z 297 [M + H]+.

Synthesis of 10-(2-Fluorobenzyl)-1-(phenylsulfonyl)spiro[indoline-3,30-pyrrolidine] (44a) Compound 43a was prepared according to the same procedure as 38a, starting from 42a (100 mg, 0.3378 mmol), using borane-tetrahydrofuran (0.6757 mL, 0.6757 mmol, 2.5 equiv.), to afford 10 mg crude product used in the next step without further puriﬁcation. Compound 44a was prepared according to the same procedure as 39a, starting from crude 43a (10 mg, 0.03545 mmol), using benzylsulfonyl chloride (50 mg, 0.07445 mmol, 1.05 equiv.), triethylamine (0.010 mL, 0.0709 mmol, 2 equiv.) and N,N-dimethylpyridin-4-amine (0.2 mg, 0.0017 mmol, 0.5 equiv.). The product was puriﬁed by preparative-HPLC (gradient: water: acetonitrile 0% to 100% acetonitrile) Yield: 2 mg. 1 H-NMR (500 MHz, CDCl3) δ ppm 8.22 (t, J = 6.7 Hz, 2H, phenyl-sulfonyl ArH-3”, phenyl-sulfonyl ArH-5”), 7.88 (m, 1H, phenyl-sulfonyl ArH-4”), 7.69 (m, 2H, phenyl-sulfonyl ArH-2”, phenyl-sulfonyl ArH-6”), 7.53 (t, J = 6.2 Hz, 1H, benzyl ArH-4), 7.47 (m, 1H, ArH-7), 7.33 (t, J = 7.8 Hz, 1H, benzyl ArH-5), 7.23 (t, J = 7.8 Hz, 1H, benzyl ArH-6), 7.17–7.08 (m, 2H, ArH-5, ArH-6), 6.73 (d, J = 7.0 Hz, 2H, 0 benzyl ArH-3), 4.22 (m, 2H, H-2), 3.98 (m, 2H, benzyl CH2), 3.75 (m, 1H, pyrrolidine H-2 ), 3.40 (m, 1H, Molecules 2017, 22, 2221 19 of 25 pyrrolidine H-20), 3.14 (m, 1H, pyrrolidine H-50), 2.98 (m, 1H, pyrrolidine H-50), 2.28 (m, 1H, pyrrolidine 0 0 13 H-4 ), 1.51 (m, 1H, pyrrolidine H-4 ). C-NMR (125 MHz, CDCl3) δ ppm 159.3 (benzyl C-2), 144.1 (C-3a), 142.5 (C-7a), 137.3 (phenyl-sulfonyl C-1”), 134.2 (phenyl-sulfonyl C-4”), 130.3 (benzyl C-6), 129.5 (phenyl-sulfonyl C-3”, phenyl-sulfonyl C-5”), 129.2 (benzyl C-4), 128.2 (C-6), 127.6 (phenyl-sulfonyl C-2”, phenyl-sulfonyl C-6”), 125.8 (benzyl C-1), 125.3 (benzyl C-5), 125.0 (C-4), 121.6 (C-5), 115.7 0 0 0 (benzyl C-3), 113.0 (C-7), 62.3 (C-2 ), 59.6 (benzyl CH2), 55.8 (C-3), 54.9 (C-5 ), 53.6 (C-2), 33.1 (C-4 ). + + MS (DUIS+) m/z 423 [M + H] . HRMS (ESI+): m/z [M + H] calcd. for C24H24N2O2FS: 423.1543; found: 423.1531.

4.1.8. Procedures to Afford 10-(3-Fluorobenzyl)-1-(phenylsulfonyl)spiro[indoline-3,30-pyrrolidine] (44b)

Synthesis of 10-(3-Fluorobenzyl)spiro[indoline-3,30-pyrrolidin]-2-one (42b) To the solution of 41b (1.681 g, 6 mmol) in tetrahydrofuran: water (30:30 mL) was added 30 mL glacial acetic acid at 0 ◦C. N-bromosuccinimide (1.069 g, 6 mmol, 1 equiv.) was added slowly in portions at 0 ◦C. The reaction mixture was stirred for 1.5 h at 0 ◦C. Afterwards the reaction was quenched at 0 ◦C by 40 mL concentrated sodium-carbonate solution and was stirred for 0.5 h, allowing to warm up to room temperature. The mixture was extracted using ethyl acetate (2 × 20 mL). The combined organic phases were washed with concentrated sodium-hydrogen carbonate (2 × 20 mL) and by brine (2 × 20 mL). The organic phases were dried over sodium-sulfate, filtered and evaporated under reduced pressure. The product 42b was purified by flash chromatography (gradient: dichloromethane: 1 methanol 0% to 10% methanol) Yield: 1.31 g (76% calculated to 29). H-NMR (500 MHz, DMSO-d6) δ ppm 10.31 (s, 1H, NH), 7.38–7.33 (m, 2H, benzyl H-5, benzyl ArH-6), 7.21–7.16 (m, 3H, ArH-4, benzyl H-2, benzyl ArH-4), 7.04 (m, 1H, ArH-6), 6.98 (t, J = 7.0 Hz, 1H, ArH-5), 6.81 (d, J = 8.0 Hz, 1H, ArH-7), 0 0 3.72 (s, 2H, benzyl CH2), 3.07 (m, 1H, pyrrolidine H-2 ), 2.74 (d, J = 8.8 Hz, 1H, pyrrolidine H-5 ), 2.66 (d, J = 8.8 Hz, 1H, pyrrolidine H-20), 2.56 (m, 1H, pyrrolidine H-50), 2.20 (m, 1H, pyrrolidine H-40), 0 13 1.90 (m, 1H, pyrrolidine H-4 ). C-NMR (125 MHz, CDCl3) δ ppm 181.96 (C=O), 161.2 (benzyl C-3), 142.3 (C-7a), 141.4 (benzyl C-1), 130.4 (benzyl C-5), 127.8 (benzyl-C6, C-6), 122.1 (benzyl C-2, benzyl 0 0 C-4), 109.4 (C-7), 64.7 (benzyl CH2), 63.9 (C-3), 53.9 (pyrrolidine C-2 ), 52.7 (C-5 ), 36.8 (pyrrolidine C-40). MS (DUIS+) m/z 297 [M + H]+.

Synthesis of 10-(3-Fluorobenzyl)-1-(phenylsulfonyl)spiro[indoline-3,30-pyrrolidine] (44b) Compound 43b was prepared according to the same procedure as 38a, starting from 42b (385 mg, 1.3 mmol), using borane-tetrahydrofuran (3.250 mL, 3.25 mmolm 2.5 equiv.), to afford 338 mg crude product used in the next time without further puriﬁcation. Compound 44b was prepared according to the same procedure as 39a, starting from crude 43b (338 mg, 1.197 mmol), using benzylsulfonyl chloride (221 mg, 1.2579 mmol, 1.05 equiv.), triethylamine (0.331 mL, 2.396 mmol, 2 equiv.) and N,N-dimethylpyridin-4-amine (7 mg, 0.0599 mmol, 0.5 equiv.). The product was puriﬁed by preparative HPLC (gradient: dichloromethane: methanol 0% to 10% methanol) Yield: 11 mg. 1H-NMR (500 MHz, CDCl3) δ ppm 7.83 (d, J = 8.2 Hz, 1H, ArH-7), 7.77 (d, J = 7.6 Hz, 1H, benzyl ArH-6), 7.73 (d, J = 7.6 Hz, 1H, ArH-4), 7.64 (d, J = 8.2 Hz, 1H, benzyl ArH-4), 7.58–7.53 (m, 1H, benzyl ArH-5), 7.50–7.39 (m, 3H, phenyl-sulfonyl ArH-3”, phenyl-sulfonyl ArH-4”, phenyl-sulfonyl ArH-5”), 7.37–7.27 (m, 2H, phenyl-sulfonyl ArH-2”, phenyl-sulfonyl ArH-6”), 7.16–7.12 (m, 1H, phenyl-sulfonyl ArH-6), 7.09–7.02 (m, 1H, phenyl-sulfonyl ArH-5), 6.99 (m, 1H, benzyl H-2), 4.23 (m, 1H, H-2), 4.11 (m, 1H, H-2), 0 0 3.93–3.71 (m, 2H, benzyl CH2), 3.23 (m, 1H, pyrrolidine H-2 ), 2.94 (m, 1H, pyrrolidine H-2 ), 2.83 (m, 1H, pyrrolidine H-50), 2.69 (m, 1H, pyrrolidine H-50), 2.39–2.25 (m, 1H, pyrrolidine H-40), 2.18 0 13 (m, 1H, pyrrolidine H-5 ). C-NMR (125 MHz, CDCl3) δ ppm 163.0 (benzyl C-3), 143.6 (C-3a), 143.5 (C-7a), 137.6 (phenyl-sulfonyl C-1”), 134.2 (phenyl-sulfonyl C-4”), 130.4 (benzyl C-5), 129.5 (phenyl-sulfonyl C-3”, phenyl-sulfonyl C-5”), 129.1 (C-6), 128.0 (phenyl-sulfonyl C-2”, phenyl-sulfonyl C-6”), 127.7 (benzyl C-6), 125.6 (benzyl C-1), 125.3 (C-4), 124.1 (C-5), 121.8, 115.8 (benzyl C-2), 113.4 0 0 (C-7), 113.3 (benzyl C-4), 61.2 (benzyl CH2), 59.9 (pyrrolidine C-2 ), 55.3 (C-3), 54.8 (pyrrolidine C-5 ), Molecules 2017, 22, 2221 20 of 25

53.6 (C-2), 33.1 (pyrrolidine C-4’). MS (DUIS+) m/z 423 [M + H]+. HRMS (ESI+): m/z [M + H]+ calcd. for C24H24N2O2SF: 423.1543; found: 423.1558.

4.1.9. Procedures to Afford 10-(4-Fluorobenzyl)-1-(phenylsulfonyl)spiro[indoline-3,30-pyrrolidine] (44c)

Synthesis of 10-(4-Fluorobenzyl)spiro[indoline-3,30-pyrrolidin]-2-one (42c) To the solution of 41c (2.361 g, 8.4321 mmol) in tetrahydrofuran: water (30:30 mL) was added 30 mL glacial acetic acid at 0 ◦C. N-bromosuccinimide (1.501 g, 8.4321 mmol, 1 equiv.) was added slowly in portions at 0 ◦C. The reaction mixture was stirred for 1.5 h at 0 ◦C. Afterwards the reaction was quenched at 0 ◦C by 40 mL concentrated sodium-carbonate solution and was stirred for 0.5 h, allowing to warm up to room temperature. The mixture was extracted using ethyl acetate (2 × 20 mL). The combined organic phases were washed with concentrated sodium-hydrogen carbonate (2 × 20 mL) and by brine (2 × 20 mL). The organic phases were dried over sodium-sulfate, filtered and evaporated under reduced pressure. The product 42c was purified by flash chromatography (gradient: dichloromethane: methanol 0% to 10% methanol) Yield: 320 mg (19% calculated to 25). 1H-NMR (500 MHz, DMSO-d6) δ ppm 10.30 (s, 1H, NH), 7.41–7.35 (m, 3H, benzyl H-2, benzyl ArH-6, ArH-4), 7.16 (m, 3H, benzyl ArH-3, benzyl ArH-5, ArH-6), 6.96 (t, J = 7.2 Hz, 1H, ArH-5), 6.81 (d, J = 7.4 Hz, 0 1H, ArH-7), 5.73 (s, 2H, benzyl CH2), 3.03 (m, 1H, pyrrolidine H-5 ), 2.73 (d, J = 9.3 Hz, 1H, pyrrolidine H-20), 2.64 (d, J = 9.3 Hz, 1H, pyrrolidine H-20), 2.58–2.53 (m, 1H, pyrrolidine H-50), 2.21–2.16 (m, 1H, 0 0 13 pyrrolidine H-4 ), 1.95–1.87 (m, 1H, pyrrolidine H-4 ) C-NMR (125 MHz, DMSO-d6) δ ppm 179.1 (C=O), 161.9 (benzyl C-4), 141.6 (C-7a), 138.2 (benzyl C-1), 134.6 (C-3a), 130.7 (benzyl C-2, benzyl C-6), 128.0 (C-6), 123.3 (C-4), 122.3 (C-2), 115.4 (benzyl C-3, benzyl C-5), 109.6 (C-7), 64.0 (benzyl CH2), 58.3 (C-3), 54.0 (pyrrolidine C-20), 52.9 (pyrrolidine C-50), 37.0 (pyrrolidine C-40). MS (DUIS+) m/z 297 [M + H]+.

Synthesis of 10-(4-Fluorobenzyl)-1-(phenylsulfonyl)spiro[indoline-3,30-pyrrolidine] (44c) Compound 43c was prepared according to the same procedure as 38a, starting from 42c (310 mg, 1.0473 mmol), using borane-tetrahydrofuran (2.620 mL, 2.620 mmol, 2.5 equiv.), to afford 461 mg crude product used in the next step without further purification. Compound 44c was prepared according to the same procedure as 39a, starting from crude 43c (100 mg, 0.354 mmol), using benzylsulfonyl chloride (131 mg, 0.745 mmol, 1.05 equiv.), triethylamine (0.099 mL, 0.7092 mmol) and N,N-dimethylpyridin-4-amine (2 mg, 0.01773 mmol). The product 44c was purified by flash chromatography (gradient: dichloromethane: methanol 0% to 10% methanol) Yield: 10 mg. 1H-NMR (500 MHz, CDCl3) δ ppm 7.84–7.77 (m, 2H, phenyl-sulfonyl ArH-3”, phenyl-sulfonyl ArH-5”), 7.74–7.70 (m, 1H, phenyl-sulfonyl ArH-4”), 7.67–7.57 (m, 3H, phenyl-sulfonyl ArH-2”, phenyl-sulfonyl ArH-6”, ArH-7), 7.50–7.45 (m, 2H, benzyl ArH-2, benzyl ArH-6), 7.43–7.40 (m, 1H, ArH-4), 7.24–7.20 (m, 1H, ArH-6), 7.16–7.12 (m, 2H, benzyl ArH-3, benzyl ArH-5), 7.09 (m, 1H, ArH-5), 4.39 (m, 1H, H-2), 4.29 0 (m, 1H, H-2), 4.18 (m, 1H, benzyl CH2), 413.06 (m, 1H, benzyl CH2), 3.90 (m, 1H, pyrrolidine H-2 ), 3.70 (m, 1H, pyrrolidine H-20), 3.06 (m, 1H), 2.68 (m, 1H, pyrrolidine H50), 2.33 (m, 1H, pyrrolidine 0 0 0 13 H-5 ), 2.33 (m, 1H, pyrrolidine H-4 ), 1.75 (m, 1H, pyrrolidine H-4 ). C-NMR (125 MHz, CDCl3) δ ppm 159.8 (benzyl C-4), 144.1 (C-3a), 142.6 (C-7a), 137.5 (benzyl C-1), 134.2 (phenyl-sulfonyl C-1”, phenyl-sulfonyl C-4”), 130.7 (benzyl C-2, benzyl C-6), 129.2 (phenyl-sulfonyl C-3”, phenyl-sulfonyl C-5”), 128.3 (C-6), 127.7 (phenyl-sulfonyl C-2”, phenyl-sulfonyl C-6”), 125.8 (C-4), 125.2 (C-5), 115.5 0 (benzyl C-3, benzyl C-5), 113.1 (C-7), 62.4 (pyrrolidine C-2 ), 59.8 (benzyl CH2), 55.5 (C-3), 55.0 (pyrrolidine C-50), 53.7 (C-2), 33.0 (pyrrolidine C-40). MS (DUIS+) m/z 423 [M + H]+. HRMS (ESI+): + m/z [M + H] calcd. for C24H24N2O2SF: 423.1543; found: 423.1542.

4.1.10. Synthesis of 10-Benzyl-1-((2-fluorophenyl)sulfonyl)spiro[indoline-3,30-pyrrolidine] (46a) Compound 46a was prepared according to the same procedure as 39a, starting from crude 38a (15 mg, 0.0567 mmol), using 2-fluorobenzylsulfonyl chloride (23 mg, 0.11819 mmol), triethylamine (0.015 Molecules 2017, 22, 2221 21 of 25 mL, 0.1188 mmol, 2.1 equiv.) and N,N-dimethylpyridin-4-amine (0.7 mg, 0.006 mmol, 0.1 equiv.). The product was purified by flash chromatography (gradient: dichloromethane: methanol 0% 1 to 10% methanol) Yield: 6.5 mg. H-NMR (500 MHz, CDCl3) δ ppm 8.00 (t, J = 6.7 Hz, 1H, phenyl-sulfonyl ArH-4”), 7.53 (m, 3H, phenyl-sulfonyl ArH-3”, phenyl-sulfonyl ArH-6”, ArH-7), 7.41–7.34 (m, 5H, benzyl ArH-2, benzyl ArH-3, benzyl ArH-5, benzyl ArH-6, ArH-4), 7.27 (t, J = 8.1 Hz, 1H, phenyl-sulfonyl ArH-5”), 7.17 (t, J = 8.1 Hz, 1H, benzyl ArH-4), 7.10 (t, J = 9.4 Hz, 0 1H, ArH-6), 7.04 (t, J = 7.6 Hz, 1H, ArH-5), 4.16–4.07 (m, 5H, benzyl CH2, H-2, pyrrolidine H-2 ), 3.34 (m, 1H, pyrrolidine H-20), 3.17 (m, 1H, pyrrolidine H-50), 3.05 (m, 1H, pyrrolidine H-50), 2.36 0 0 13 (m, 1H, pyrrolidine H-4 ), 2.14 (m, 1H, pyrrolidine H-4 ). C-NMR (125 MHz, DMSO-d6) δ ppm 155.6 (phenyl-sulfonyl C-2”), 149.7 (C-3a), 142.8 (C-7a), 133.2 (benzyl C-1), 131.8 (phenyl-sulfonyl C-4”), 131.0 (phenyl-sulfonyl C-6”), 129.6 (benzyl C-3, benzyl C-5), 128.4 (benzyl C-2, benzyl C-6), 124.2 (phenyl-sulfonyl C-5”), 122.9 (benzyl C-4), 122.5 (C-4), 121.0 (C-5), 120.4 (phenyl-sulfonyl C-1”), 0 116.8 (phenyl-sulfonyl C-3”), 111.3 (C-7), 63.2 (pyrrolidine C-2 ), 62.6 (benzyl CH2), 55.1 (C-3), 52.5 (pyrrolidine C-50), 49.2 (C-2), 37.1 (pyrrolidine C-40). MS (DUIS+) m/z 423 [M + H]+. HRMS (ESI+): + m/z [M + H] calcd. for C24H24N2O2SF: 423.1543; found: 423.1550.

4.1.11. Synthesis of 10-Benzyl-1-((3-fluorophenyl)sulfonyl)spiro[indoline-3,30-pyrrolidine] (46b) Compound 45b was prepared according to the same procedure as 39a, starting from crude 38a (15 mg, 0.0567 mmol), using 3-fluorobenzylsulfonyl chloride (23 mg, 0.1188 mmol, 2.1 equiv.), triethylamine (0.015 mL, 0.1188 mmol, 2.1 equiv.) and N,N-dimethylpyridin-4-amine (0.7 mg, 0.006 mmol, 0.1 equiv.). The product was purified by flash chromatography (gradient: dichloromethane: methanol 0% to 10% 1 methanol) Yield: 5.5 mg. H-NMR (500 MHz, CDCl3) δ ppm 8.25 (m, 1H, phenyl-sulfonyl ArH-2”), 7.65–7.58 (m, 2H, phenyl-sulfonyl ArH-5”, phenyl-sulfonyl ArH-6”), 7.48–7.38 (m, 6H, ArH-7, benzyl ArH-2–6), 7.29–7.25 (m, 2H, ArH-4, ArH-6), 7.21 (m, 1H, phenyl-sulfonyl ArH-4”), 7.08 (t, J = 7.6 Hz, 1H, ArH-5), 4.16 (d, J = 12.7 Hz, 1H, benzyl CH2), 4.09 (d, J = 12.7 Hz, 1H, benzyl CH2), 3.99 (d, J = 10.9 Hz, 1H, H-2), 3.85 (d, J = 10.9 Hz, 1H, H-2), 3.33–3.24 (m, 2H, pyrrolidine H-20), 3.12 (d, J = 11.6 Hz, 1H, pyrrolidine H-50), 3.00 (d, J = 11.6 Hz, 1H, pyrrolidine H-50), 2.26–2.20 (m, 1H, pyrrolidine H-40), 0 13 2.05–2.00 (m, 1H, pyrrolidine H-4 ). C-NMR (125 MHz, CDCl3) δ ppm 158.6 (phenyl-sulfonyl C-3”), 146.8 (C-3a), 145.4 (C-7a), 137.5 (benzyl C-1), 130.5 (phenyl-sulfonyl C-1”), 129.7 (phenyl-sulfonyl C-5”), 129.0 (benzyl C-3, benzyl C-5), 128.7 (benzyl C-2, benzyl C-6), 124.5 (C-6), 122.8 (benzyl C-4), 122.7 (phenyl-sulfonyl C-6”), 121.7 (C-4), 120.2 (C-5), 117.7 (phenyl-sulfonyl C-4”), 114.3 (phenyl-sulfonyl 0 0 C-2”), 109.5 (C-7), 63.0 (pyrrolidine C-2 ), 62.6 (benzyl CH2), 55.4 (C-3), 52.7 (pyrrolidine C-5 ), 49.2 (C-2), 37.9 (pyrrolidine C-40). MS (DUIS+) m/z 423 [M + H]+. HRMS (ESI+): m/z [M + H]+ calcd. for C24H24N2O2SF: 423.1543; found: 423.1547.

4.1.12. Synthesis of 10-Benzyl-1-((4-fluorophenyl)sulfonyl)spiro[indoline-3,30-pyrrolidine] (46c) Compound 46c was prepared according to the same procedure as 39a, starting from crude 38a (15 mg, 0.0567 mmol), using 4-fluorobenzylsulfonyl chloride (23 mg, 0.1182 mmol, 2.1 equiv.), triethylamine (0.015 mL, 0.1188 mmol, 2.1 equiv.) and N,N-dimethylpyridin-4-amine (0.7 mg, 0.006 mmol, 0.1 equiv.). The product was purified by flash chromatography (gradient: dichloromethane: 1 methanol 0% to 10% methanol) Yield: 7.7 mg. H-NMR (500 MHz, CDCl3) δ ppm 7.80 (m, 2H, phenyl-sulfonyl ArH-3”, phenyl-sulfonyl ArH-5”), 7.63 (d, J = 7.9 Hz, 1H, ArH-7), 7.47 (m, 2H, benzyl H-3, benzyl ArH-5), 7.40 (m, 3H, benzyl ArH-2, benzyl ArH-4, benzyl ArH-6), 7.28 (m, 2H, phenyl-sulfonyl ArH-3”, phenyl-sulfonyl ArH-5”), 7.09–7.05 (m, 3H, ArH-4, ArH-5, ArH-6), 4.10 (d, J = 12.3 Hz, 1H, H-2), 4.02–3.96 (m, 2H, benzyl CH2), 3.84 (d, J = 11.3 Hz, 1H, H-2), 3.25 (m, 1H, pyrrolidine H-20), 3.16 (m, 1H, pyrrolidine H-20), 2.94 (m, 1H, pyrrolidine H-50), 2.89 (m, 1H, pyrrolidine H-50), 2.24–2.18 (m, 1H, pyrrolidine H-40), 2.03–1.99 (m, 1H, pyrrolidine H-40). 13C-NMR (125 MHz, CDCl3) δ ppm 163.7 (phenyl-sulfonyl C-4”), 152.7 (C-3a), 150.6 (C-7a), 147.0 (benzyl C-1), 129.6 (phenyl-sulfonyl C-1”), 129.5 (phenyl-sulfonyl C-2”, phenyl-sulfonyl C-6”), 129.2 (benzyl C-3, benzyl C-5), 129.1 (benzyl C-2, benzyl C-6), 128.8 (C-6), 128.6 (benzyl C-4), 125.6 (C-4), 124.4 (C-5), 122.8 Molecules 2017, 22, 2221 22 of 25

0 (phenyl-sulfonyl C-3”, phenyl-sulfonyl C-5”), 116.1 (C-7), 63.1 (pyrrolidine C-2 ), 62.4 (benzyl CH2), 54.1 (C-3), 52.8 (pyrrolidine C-50), 49.1 (pyrrolidine C-20), 36.6 (pyrrolidine C-40). MS (DUIS+) m/z 423 + + [M + H] . HRMS (ESI+): m/z [M + H] calcd. for C24H24N2O2SF: 423.1543; found: 423.1563.

4.2. General Procedure for the Serotonergic Screening Assays

4.2.1. Cell Culture and Preparation of Cell Membranes

HEK293 cells with stable expression of human serotonin 5-HT1A, 5-HT6 or 5-HT7b receptors (all prepared with the use of Lipofectamine 2000) or CHO-K1 cells with plasmid containing the ◦ sequence coding for the human serotonin 5-HT2A receptor (PerkinElmer) were maintained at 37 C in a humidified atmosphere with 5% CO2 and were grown in Dulbeco’s Modifier Eagle Medium containing 10% dialysed fetal bovine serum and 500 µg/mL G418 sulphate. For membranes preparations, cells were subcultured in 150 cm2 flasks, grown to 90% confluence, washed twice with prewarmed to 37 ◦C phosphate buffered saline (PBS) and were pelleted by centrifugation (200 g) in PBS containing 0.1 mM EDTA and 1 mM dithiothreitol. Prior to membrane preparations pellets were stored at −80 ◦C.

4.2.2. 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-HT1AR: 50 mM Tris–HCl, 0.1 mM EDTA, 4 mM MgCl2, 10 µM pargyline and 0.1% ascorbate; for 5-HT2AR: 50 mM Tris–HCl, 0.1 mM EDTA, 4 mM MgCl2 and 0.1% ascorbate; for 5-HT6R: 50 mM Tris–HCl, 0.5 mM EDTA and 4 mM MgCl2, for 5-HT7bR: 50 mM Tris–HCl, 4 mM MgCl2, 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-HT1AR and 5-HT2AR 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: 2.5 nM [3H]-8-OH-DPAT 3 (5002.4 GBq/mmol) for 5-HT1AR; 1 nM [ H]-Ketanserin (1975.8 GBq/mmol) for 5-HT2AR; 2 nM 3 3 [ H]-LSD (3093.2 GBq/mmol) for 5-HT6R or 0.8 nM [ H]-5-CT (1450.4 GBq/mmol) for 5-HT7R. Non-specific binding is defined with 10 µM of 5-HT in 5-HT1AR and 5-HT7R binding experiments, whereas 10 µM of chlorpromazine or 10 µM of methiothepine were used in 5-HT2AR and 5-HT6R assays, respectively. Each compound was tested in triplicate at 7–8 concentrations (10−11–10−4 M). The inhibition constants (Ki) were calculated from the Cheng-Prusoff equation [47]. Results were expressed as means of at least three separate experiments (SD ≤ 24%).

4.2.3. Docking Procedure

The homology model of h-5HT6R[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 Asn2886.55 and/or hydrogen bond formed with Ser1935.43.

Acknowledgments: The authors are grateful to László Drahos and Ágnes Gömöry (RCNS, Hungary) for the exact mass (high-resolution MS) measurements and we are thankful for Aaron Keeley (RCNS, Hungary) for carefully reading the manuscript and for Stefan Mordalski (Institute of Pharmacology, Poland) for helpful discussions. The support of the National Brain Research Program (KTIA_NAP_13-1-2013-0001) is acknowledged. Molecules 2017, 22, 2221 23 of 25

Author Contributions: G.M.K. and A.J.B. conceived the study; Á.A.K. performed the syntheses, G.S. performed the in vitro measurements. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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Sample Availability: Samples of the compounds are not available from the authors.

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