Discovery of Novel Indolealkylpiperazine Derivatives As Potent 5-HT1A Receptor Agonists for the Potential Future Treatment of Depression

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Article Discovery of Novel Indolealkylpiperazine Derivatives as Potent 5-HT1A Receptor Agonists for the Potential Future Treatment of Depression

Chen Zhu, Xinwei Li, Weiqing Peng and Wei Fu *

Department of Medicinal Chemistry, School of Pharmacy, Fudan University, 826 Zhangheng Road, Pudong New District, Shanghai 201203, China; [email protected] (C.Z.); [email protected] (X.L.); [email protected] (W.P.) * Correspondence: [email protected]

Received: 26 August 2020; Accepted: 1 October 2020; Published: 1 November 2020

Abstract: Depression is a severe psychiatric disorder that aﬀects over 100 million people worldwide. 5-HT1A receptor agonists have been implicated in the treatment of a variety of central nervous system diseases, especially depression. In this study, based on FW01, a selective potent 5-HT1AR agonist discovered via dynamic pharmacophore-based virtual screening, a series of indolealkylpiperazine derivatives with a benzamide moiety were designed and synthesized by the modiﬁcation of the amide tail group as well as indole head group of FW01. Among all tested compounds, 13m displayed potent agonistic activity towards 5-HT1AR with an EC50 value of 1.01 nM. Molecular docking studies were performed to disclose the mechanism of its potent agonistic activity and high selectivity. Finally, the activation model of 5-HT1AR induced by 13m was proposed.

Keywords: 5-HT1A receptor agonist; depression; drug design; molecular dynamics

1. Introduction Depression is a severe psychiatric disorder that affects over 100 million people worldwide [1]. The main symptoms include low mood, lack of energy, sadness, insomnia, etc. [2]. Patients also suffer a high risk of suicide [3,4]. Though the etiology of depression is not clear, it is certain that genetic and psychological factors as well as environmental factors are all involved in the pathogenesis [5,6]. Selective serotonin reuptake inhibitors (SSRIs) and serotonin norepinephrine reuptake inhibitors (SNRIs) are common antidepressants clinically [7–9]. Though SSRIs are first-line drugs for the treatment of depression, they still have some drawbacks including delayed onset and limited efficacy [10,11]. Thus, drug discovery for new antidepressants with rapid onset and good efficacy is still urgent [12,13]. Serotonin (5-hydroxytryptamine, 5-HT), as an important neurotransmitter, is involved in multiple physiological and behavioral processes [14,15]. 5-HT mediates its physiological effects through at least 14 different receptor subtypes [16]. Among them, 5-HT1A receptor (5-HT1AR) plays a significant role in mood regulation and has been a hot target for various central nervous system disorders, especially depression [17–20]. Medicines like gepirone, tandospirone and vilazodone acting as 5-HT1AR partial agonists are representative antidepressants clinically [21]. Meanwhile, the combination of 5-HT1AR agonists and SSRIs not only accelerate the onset of SSRIs but also improve the therapeutic effect [10]. Therefore, based on our previous research, we aimed at discovering selective 5-HT1AR agonists with novel scaffold with the eventual aim to treat depression.

Molecules 2020, 25, 5078; doi:10.3390/molecules25215078 www.mdpi.com/journal/molecules Molecules 2020, 25, x FOR PEER REVIEW 2 of 14

2. Results and Discussion

2.1. Compound Design

In our previous study, a series of lead compounds as 5-HT1AR agonists with novel scaffolds were obtained through the combination of pharmacophore-based virtual screening and biological assay [22]. Among them, FW01 (Figure 1) possessed high activity for 5-HT1AR (Ki = 51.9 ± 16.4 nM), medium activity for 5-HT2A receptor (5-HT2AR) (Ki = 206.71 ± 7.46 nM) and low activity for dopamine D2 receptor (D2R) (Ki = 2161.35 ± 25.55 nM). FW01 was bound to 5-HT1AR via multiple interaction forces [22]. The docking predicted binding mode of FW01 with 5-HT1AR (Figure 1) showed that the indole head group was inserted into the pocket formed by transmembrane helix 3 (TM3), TM5, TM6 and TM7 of 5-HT1AR. The NH group of the indole head formed hydrogen bonding with T3.37. The protonated nitrogen of piperazine participated in the salt bridge with D3.32. As for the amide tail Moleculesgroup, the2020 ,phenyl25, 5078 ring was inserted into the pocket formed by residues F3.28, Y2.64, and A6.21.2 of 13 However, there was an empty pocket formed by TM6 and TM7 which was not reached by FW01. Due 2.to Resultsthe presence and Discussion of the upper pocket and the lower pocket formed by TM6 and TM7, the amide tail moiety of FW01 was replaced with different 2,5-substituted benzamide moieties. As for the indole 2.1.moiety, Compound the development Design of marketed antidepressant drug vilazodone showed that a 5-CN indole moiety could enhance the 5-HT1AR affinity, which was also proved in our previous study [23,24]. In our previous study, a series of lead compounds as 5-HT R agonists with novel scaﬀolds Therefore, the 5-F indole moiety of FW01 was replaced by 5-CN indole1A moiety to raise the agonistic were obtained through the combination of pharmacophore-based virtual screening and biological activity of designed compounds towards 5-HT1AR. assay [22]. Among them, FW01 (Figure1) possessed high activity for 5-HT R(Ki = 51.9 16.4 nM), Finally, a series of indolealkylpiperazine derivatives with a benzamide1A moiety were± designed medium activity for 5-HT receptor (5-HT R) (Ki = 206.71 7.46 nM) and low activity for dopamine and synthesized. Their 2Acellular function2A activities on dopamine± and serotonin receptors were D receptor (D R) (Ki = 2161.35 25.55 nM). FW01 was bound to 5-HT R via multiple interaction evaluated2 by Ultra2 Lance cAMP ±assay and Fluorometric Imaging Plate Reader1A (FLIPR) assay. forces [22].

Figure 1. Predicted binding mode of FW01 (green sticks) with 5-HT R (cartoon) and the design of Figure 1. Predicted binding mode of FW01 (green sticks) with 5-HT1A1AR (cartoon) and the design of indolealkylpiperazine derivatives.derivatives. Ligands Ligands are are shown shown as greenas green sticks. sticks. The bindingThe binding residues residues of 5-HT of1A 5-R are shown in gray. Hydrogen bond interactions are represented as dotted red lines. 5-HT R was HT1AR are shown in gray. Hydrogen bond interactions are represented as dotted red lines. 1A5-HT1AR obtained by homology modeling from the X-ray structure of 5-HT R (PDB code: 6G79). was obtained by homology modeling from the X-ray structure of 5-HT1B 1BR (PDB code: 6G79). The docking predicted binding mode of FW01 with 5-HT R (Figure1) showed that the indole 2.2. Chemical Synthesis 1A head group was inserted into the pocket formed by transmembrane helix 3 (TM3), TM5, TM6 and TM7 of 5-HTSynthesis1AR. The of NHintermediate group of the5 was indole shown head in formedScheme hydrogen 1. Commercially bonding available with T3.37. 5-cyanoindole The protonated was nitrogenfirst acylated of piperazine with 3-chloropropionyl participated in the chloride salt bridge via withFriedel-Crafts D3.32. As foracylation the amide to tailobtain group, 2, which the phenyl was ringreduced was by inserted NaBH4 into/CF3COOH the pocket system formed to give by compound residues F3.28, 3. Compound Y2.64, and 4,A6.21. which was However, obtained there via was the anreaction empty of pocket 3 and N-Boc-piperazine, formed by TM6 and was TM7 deprotected which was to not give reached 5. by FW01. Due to the presence of the upper pocket and the lower pocket formed by TM6 and TM7, the amide tail moiety of FW01 was replaced with diﬀerent 2,5-substituted benzamide moieties. As for the indole moiety, the development of marketed antidepressant drug vilazodone showed that a 5-CN indole moiety could enhance the 5-HT1AR aﬃnity, which was also proved in our previous study [23,24]. Therefore, the 5-F indole moiety of FW01 was replaced by 5-CN indole moiety to raise the agonistic activity of designed compounds towards 5-HT1AR. Finally, a series of indolealkylpiperazine derivatives with a benzamide moiety were designed and synthesized. Their cellular function activities on dopamine and serotonin receptors were evaluated by Ultra Lance cAMP assay and Fluorometric Imaging Plate Reader (FLIPR) assay.

2.2. Chemical Synthesis Synthesis of intermediate 5 was shown in Scheme1. Commercially available 5-cyanoindole was ﬁrst acylated with 3-chloropropionyl chloride via Friedel-Crafts acylation to obtain 2, which was Molecules 2020, 25, 5078 3 of 13

reduced by NaBH4/CF3COOH system to give compound 3. Compound 4, which was obtained via the reactionMoleculesMolecules 20202020 of 3,, 2525and,, xx FORFOR N-Boc-piperazine, PEERPEER REVIEWREVIEW was deprotected to give 5. 33 ofof 1414

Scheme 1. Synthesis of intermediate 5. Reagents and conditions: (aa) 3-chloropropionyl chloride, AlCl3, Scheme 1. Synthesis of intermediate 5.. Reagents Reagents and conditions: ( (a) 3-chloropropionyl 3-chloropropionyl chloride,chloride, AlClAlCl33, anhydrous DCM, r.t., overnight;overnight; ((bb)) NaBHNaBH4, CF 3COOH,COOH, anhydrous anhydrous THF, 0 °C,C, 1 h; h; ( (cc)) Boc-piperazine, Boc-piperazine, anhydrous DCM, r.t., overnight; (b) NaBH44, CF33COOH, anhydrous THF, 0 ◦°C, 1 h; (c) Boc-piperazine, CH3CN, K 2COCO3, ,75 75 °C,C, 36 36 h; h; (d (d) )CF CF3COOH,COOH, DCM, DCM, r.t., r.t., 4 4h. h. CH33CN, K2CO33, 75 °C,◦ 36 h; (d) CF33COOH, DCM, r.t., 4 h.

Synthesis of the 5-sulfonyl benzoic acid was outlined in Scheme2. Compound 7a was obtained SynthesisSynthesis ofof thethe 5-sulfonyl5-sulfonyl benzoicbenzoic acidacid waswas outlinedoutlined inin SchemeScheme 2.2. CompoundCompound 7a7a waswas obtainedobtained by the sulfonation reaction of 6a and chlorosulfonic acid. 7a reacted with sodium sulﬁte and then byby thethe sulfonationsulfonation reactionreaction ofof 6a6a andand chlorosulfonicchlorosulfonic acid.acid. 7a7a reactedreacted withwith sodiumsodium sulfitesulfite andand thenthen alkylated by iodomethane to give 8a. 8a was deprotected by lithium hydroxide to give 10a. Synthesis alkylatedalkylated byby iodomethaneiodomethane toto givegive 8a8a.. 8a8a waswas deprotecteddeprotected byby lithiumlithium hydroxidehydroxide toto givegive 10a10a.. SynthesisSynthesis of 10b–10d was accomplished by reaction of 8a and corresponding reagents and then deprotection by ofof 10b10b––10d10d waswas accomplishedaccomplished byby reactionreaction ofof 8a8a andand correspondingcorresponding reagentsreagents andand thenthen deprotectiondeprotection lithium hydroxide. For 8b–8d, the starting material was methyl salicylate and the synthetic route was byby lithiumlithium hydroxide.hydroxide. ForFor 8b8b––8d8d,, thethe startingstarting materialmaterial waswas methylmethyl salicylatesalicylate andand thethe syntheticsynthetic routeroute similar to 8a. Compounds 11a–11m were obtained by Mitsunobu reaction of 8b–8d and corresponding waswas similarsimilar toto 8a8a.. CompoundsCompounds 11a11a––11m11m werewere obtainedobtained byby MiMitsunobutsunobu reactionreaction ofof 8b8b––8d8d andand alcohol or alkylation of 8b–8d and 3-iodo-oxetane, which were then deprotected by lithium hydroxide correspondingcorresponding alcoholalcohol oror alkylationalkylation ofof 8b8b––8d8d andand 3-iodo-oxetane,3-iodo-oxetane, whichwhich werewere thenthen deprotecteddeprotected byby to give compounds 12a–12m. lithiumlithium hydroxidehydroxide toto givegive compoundscompounds 12a12a––12m12m..

O F O F HO HO O F O F i O F O F i O F O O F efHO O S O efHO O O S HO O HO O O S O S O h 10a O S Cl O S h 10a O Cl O O R1 O R1 6a 7a 8a O R1 O R1 6a 7a 8a O i HO O i HO

O O S O S O O S O S O O 9a~9c9a~9c 10b~10d 10b~10d

R3 R3 O OH O OH O O R3 O O R3 O OH O OH O O O O O OH j i O OH e O f/g O j O i HO e O f/g O O HO O O O O O O S O S O S O S O S S S O Cl O S R2 O R2 O R2 O Cl O R2 O R2 O R2 6b6b 7b 7b 8b~8d 8b~8d 11a~11m11a~11m 12a~12m 12a~12m

O O CF O O CF3 R1=,N 3 ,O R1=,N ,O

H H H H R2=,N ,N R2=,N ,N

O O RR3=,,,,,=,,,,, 3 O O

Scheme 2. SynthesisSynthesis of ofbenzoic benzoic acid acid intermediates. intermediates.Reagents Reagents and and conditions: conditions: (e)( eHClSO) HClSO3, 40, 40°C, 30C, Scheme 2. Synthesis of benzoic acid intermediates. Reagents and conditions: (e) HClSO3, 403 °C,◦ 30 30min; min; (f) (i.f )Na i. Na2SO3SO, H2,HSO4,SO H2O,,H r.t.,O, 2 r.t., h; 2ii. h; iodide ii. iodide alkanes, alkanes, K2CO K3,CO DMF,, DMF, r.t., 3 r.t., h; ( 3g h;) amine, (g) amine, DCM, DCM, r.t., min; (f) i. Na2SO2 3, H32SO24, H42O, r.t.,2 2 h; ii. iodide alkanes, K2CO23, DMF,3 r.t., 3 h; (g) amine, DCM, r.t., r.t.,2 h; 2(h h;) (correspondingh) corresponding reagent, reagent, K2CO K CO3, DMF,, DMF, 150 150 °C, ◦18C, h; 18 ( h;i) LiOH·H (i) LiOH2O,H THF:HO, THF:H2O =O 4:1,= 4:1,r.t., r.t.,5 h; 5 ( h;j) 2 h; (h) corresponding reagent, K2CO2 3, DMF,3 150 °C, 18 h; (i) LiOH·H2·O,2 THF:H2O 2= 4:1, r.t., 5 h; (j) (alcohol,j) alcohol, PPh PPh3, DBAD,, DBAD, THF, THF, r.t., r.t., 6 h 6 or h or3-iodo-oxetane, 3-iodo-oxetane, Cs Cs2COCO3, DMF,, DMF, 80 80°C,◦ 10C, h. 10 h. alcohol, PPh3, DBAD,3 THF, r.t., 6 h or 3-iodo-oxetane, Cs2CO2 3, DMF,3 80 °C, 10 h.

As shownshown inin SchemeScheme3 ,3, compounds compounds13a 13a–13q–13qwere were obtained obtained by condensationby condensation of 5 ofand 5 and benzoic benzoic acid As shown in Scheme 3, compounds 13a–13q were obtained by condensation1 of 5 and13 benzoic intermediate. Structure of all compounds were conﬁrmed by ESI-MS, HRMS, H NMR1 and C NMR. 13 acidacid intermediate.intermediate. StructureStructure ofof allall compoundscompounds werewere confirmedconfirmed byby ESI-MS,ESI-MS, HRMS,HRMS, 1HH NMRNMR andand 13CC NMR.NMR.

Molecules 2020, 25, 5078 4 of 13 Molecules 2020, 25, x FOR PEER REVIEW 4 of 14

Scheme 3. Synthesis of compounds 13a–13q.. Reagents Reagents and and conditions: conditions: ( (kk)) EDC·HCl, EDC HCl, HOBT, HOBT, DIEA, DIEA, · DCM, r.t., 8 h.

2.3. In In Vitro Vitro Biological Biological Activity Evaluation After these these new new derivatives derivatives were were synthesized, synthesized, they they were were all all subjected subjected to toin vitroin vitro evaluationevaluation of functionalof functional activities activities towards towards 5-HT 5-HT1A,1A 5-HT, 5-HT2A 2Aandand D2 D 2receptors.receptors. As Asshown shown in in Table Table 11,, indolealkylpiperazine derivatives derivatives displayed displayed varied selectivity forfor 5-HT5-HT1A1ARR over D2RR and 5-HT2AR.R. When R 2 waswas methyl methyl group, group, R R11 waswas subjected subjected to to substituents substituents of of different different size size to to examine examine the the SAR. SAR. Compound 13a with an F atom displayed reduced agonistic activity for 5-HT1A1AR.R. The The introduction introduction of morpholine group ( 13b) resulted in drastically drastically decreased decreased activi activityty towards towards three three receptors, receptors, indicating indicating that large size group of RR11 position was not compatible with these receptors. Replacing morpholine group with with smaller smaller size size 1,1,1-trifluoro-2-pr 1,1,1-trifluoro-2-propylopyl or orcyclopropylmethoxy cyclopropylmethoxy group group afforded afforded 13c and13c 13dand, respectively.13d, respectively. By comparing By comparing 13b, 13bthey, theyshowed showed increased increased activity activity for 5-HT for 5-HT1AR 1AandR and5-HT 5-HT2AR. 2AWeR. postulatedWe postulated that that the thecyclopropylmethoxy cyclopropylmethoxy group group extended extended into into the the hydrophobic hydrophobic pocket pocket where where the phenyl ring of FW01 located, thus stabilizing the binding conformation of of 13d in the active pockets of 5-HT1ARR and and 5-HT 5-HT2A2AR.R. Then, Then, the the ethyl, ethyl, methylamino methylamino and and cyclopropylmethylamino cyclopropylmethylamino groups groups were employed to replace methyl of 13d andand compoundscompounds 13e13e––13g13g werewere designed.designed. When adopting the sulfonamide substituents ( (13f13f and 13g), the activityactivity forfor 5-HT5-HT1A1ARR showed a remarkable elevation in comparison to compound 13a–13e, presumably due to the the less less flexible flexible nitrogen nitrogen chain chain posing posing a a limited limited conformation to form a more stablestable hydrophobic interaction.

MoleculesMolecules 2020 2020, 25, ,25 x ,FOR x FOR PEER PEER REVIEW REVIEW 5 of5 of15 15 MoleculesMolecules 2020 2020, ,25 25, ,x x FOR FOR PEER PEER REVIEW REVIEW 55 ofof 1515 MoleculesTableTable 2020 1., 25 1.Functional, xFunctional FOR PEER activities REVIEWactivities of ofcompounds compounds 13a 13a–13g–13g for for dopamine dopamine receptor receptor (D (D2R)2R) and and serotonin serotonin5 of 15 receptorsTablereceptors 1. (5-HT Functional (5-HT1AR1A Rand and activities 5-HT 5-HT2AR).2A ofR). compounds 13a–13g for dopamine receptor (D2R) and serotonin MoleculesTable 2020 ,1. 25 Functional, x FOR PEER activities REVIEW of compounds 13a–13g for dopamine receptor (D2R) and serotonin5 of 15 receptors (5-HT1AR and 5-HT2AR). Molecules 2020Tablereceptors, 25, 50781. Functional (5-HT1AR and activities 5-HT2A ofR). compounds 13a–13g for dopamine receptor (D2R) and serotonin 5 of 13 MoleculesMolecules 2020, 252020, x, FOR25, x FORPEER PEER REVIEW REVIEW 5 of 155 of 15 Tablereceptors 1. Functional(5-HT1AR and activities 5-HT2A R).of compounds 13a–13g for dopamine receptor (D2R) and serotonin Molecules 2020, 25, x FOR PEER REVIEW 5 of 15 receptors (5-HT1AR and 5-HT2AR). Table 1. Functional activities of compounds 13a–13g for dopamine receptor (D2R) and serotonin Table 1. Functional activities of compounds 13a–13g for dopamine receptor (D2R) and serotonin Table 1. Functional activities of compounds 13a–13g for dopamine receptor (D2R) and serotonin Tablereceptors 1. Functional(5-HT1AR and activities 5-HT2A R).of compounds 13a–13g for dopamine receptor (D2R) and serotonin receptors (5-HT1AR and 5-HT2AR). receptors (5-HT R and 5-HT R). receptors (5-HT1A 1AR and 5-HT2A2AR).

D2DR2 R 5-HT5-HT1AR1A R 5-HT5-HT2AR2A R CompoundCompound R1R 1 R2R 2 ICIC50D D(nM)502 R2(nM)R EC5-HT EC5-HT50 (nM)50 1A(nM)1ARR IC5-HT5-HT IC50 (nM)50 2A(nM)2ARR 1 2 CompoundCompound RR1 RR2 a a a a FW01 - - 2161.3550 ± 25.55 50 7 206.7150 ± 7.46 FW01 - - 2161.35ICICD50 2±(nM) R(nM) 25.55 EC5-HT EC50 7 (nM) 1A(nM)R 206.715-HT IC IC50 ±(nM) 2A(nM)7.46R Compound R1 R2 13aFW0113a -F -F- -CH-CH -3 3 2161.35NANA ± 25.55 a a 167.7 167.7 7 206.71>5000>5000 ± 7.46 a a FW01 - - 2161.35IC50D (nM)2 R± 25.55 EC5-HT50 (nM) 71A R 206.71 IC5-HT50 (nM) ±2A 7.46R 1 2 Compound13a R -F R-CH 33 NA a 167.7 >5000 a FW0113a -F- -CH - 2161.35IC50NA (nM)± 25.55 EC 50 167.7 7(nM) 206.71 IC50>5000 (nM)± 7.46 D2R 5-HT1AR 5-HT2AR 1 2 3 a a CompoundFW0113a R-F- -CHR - 2161.35NA D± 225.55 R 5-HT 167.7 7 1A R 206.71>5000 5-HT ± 7.46 2AR Compound13b13b R -CH-CHR3 3 IC50NA (nM)NA EC989.750 989.7(nM) IC50NA (nM)NA 1 2 D2R 5-HT1AR 5-HT2AR Compound13a R-F1 -CHR2 3 ICNAD2R (nM) a EC 167.75-HT(nM) 1AR >5000 IC5-HT(nM) 2Aa R CompoundFW0113b13b R1 - R-CH-CH2 - 3 3 2161.35IC50NA 50NA (nM)± 25.55 EC50989.7989.750 7(nM) 206.71 IC50NA NA50(nM)± 7.46 50 50 50 13a -F -CH3 ICNA (nM) a EC 167.7 (nM) >5000 IC (nM)a FW0113b - -CH - 3 2161.352161.35NA ± 25.55 25.55 989.7 7 206.71NA ± 7.46 a FW01 -- a 7 206.71 7.46 a FW01 - - 2161.35 ±a ±25.55 7 206.71 ± 7.46 13b13a -F -CH3 NA 989.7 167.7 >5000NA ±

13a 13c13a13c -F -F -CH-CH -CH-CH3 3 33 NANANA 197.2 167.7 197.2 167.7 1144>11445000>5000 13b -CH3 NA 989.7 NA 13c13c -CH-CH3 3 NANA 197.2 197.2 11441144

13b13b -CH-CH3 3 NANA 989.7 989.7 NA NA 13c -CH3 NA 197.2 1144

13b -CH3 NA 989.7 NA 13c -CH3 NA 197.2 1144 13d13d -CH-CH3 3 NANA 88.0 88.0 887.4887.4

13c13c -CH-CH3 3 NANA 197.2 197.2 1144 1144 13d13d -CH-CH3 3 NANA 88.0 88.0 887.4887.4

13c -CH3 NA 197.2 1144 13d -CH3 NA 88.0 887.4

13c 13d13d -CH-CH-CH3 3 3 NANANA 88.0 88.0 197.2 887.4 887.41144 13e13e -CH-CH2CH2CH3 3 >5000>5000 49.2 49.2 1292 1292

13d13e13e -CH-CH-CH2CH2CH3 3 3 >5000NA>5000 88.0 49.2 49.2 887.4 1292 1292

13d13e13e -CH-CH-CH2CH23CH 3 3 >5000NA>5000 49.288.0 49.2 887.4 1292 1292

13e -CH2CH3 >5000 49.2 1292 13f13f NANA 23.623.6 10991099 13d -CH 3 NA 88.0 887.4 13e13f13f -CH2CH3 >5000NANANA 49.223.623.6 129210991099 1099

13e13f -CH2CH3 >5000NA 23.6 49.2 1099 1292

13f NA 23.6 1099 13g13g13g 43114311 5.35 5.355.35 22772277 2277

13e 13f13g13g -CH2CH3 >5000NA43114311 23.65.355.35 49.2 109922772277 1292 Risperidone - - 2.6 - 3.2

RisperidoneRisperidone13g13f - - - - 4311NA 2.6 2.6 5.3523.6 - - 227710993.23.2 8-OH-DPAT - - - 7.3 - 8-OH-DPATRisperidoneRisperidone8-OH-DPAT13g ------4311 2.6 - 2.6 - 5.35 7.3 - 7.3 - 22773.2-3.2 -

Risperidone8-OH-DPAT8-OH-DPAT - - - a:a: a:Ki -K - value; -i value; NA:NA: NA: nono no activity.activity. 2.6activity. - - 7.3 - 7.3 3.2- - 13f 13g 4311NA 5.3523.6 22771099 8-OH-DPAT - a:a: K K-i ivalue; value; NA: NA: no no activity. activity. - 7.3 - RisperidoneEncouragedEncouraged13g by by the the results- results above, above, we- we next next exte extendednded4311 2.6 our our efforts efforts to 5.35to -investigate investigate the the22773.2 effect effect of of Encouraged8-OH-DPAT by the results - above, a: K wei- value; next NA: extended no activity. - our eﬀorts 7.3 to investigate-the eﬀect of substituentssubstituentsRisperidoneEncouragedEncouraged of of position position byby thethe 2 -resultsof2 results of the the phenyl above, above,phenyl ring -we wering onnextnext on compounds compoundsexteextendednded 2.6 our13four 13f and efforts effortsand 13g 13g to. toOverall,. - investigateOverall,investigate compounds compounds thethe3.2 effect effect 13f 13f of–of – a: Ki value; NA: no activity. substituents13qsubstituentssubstituents13q 8-OH-DPATRisperidoneshowedEncouraged showed of position selective ofof selective positionposition by 2the ofagonistic agonistic 2theresults2 - ofof phenyl thethe activity above, phenylphenylactivity ring forwe ring- ring onfor 5-HTnext compoundson5-HTon compounds1AcompoundsexteR.1AR. Amongnded Among 2.6 - 13f our 13f13ftheseand these efforts andand 13gcompounds, 13gcompounds,13g .to. Overall, 7.3. Overall, -investigateOverall, 13g compounds13g compoundscompounds and theand 3.2 13m-effect 13m– 13q13f13f13f– of13q– –13q showedbearingsubstituents13q13qbearing13g selective8-OH-DPAT Encouraged showedshowed cyclopropylmethylamino cyclopropylmethylamino of agonistic selectiveselective position by the agonistic agonisticactivity2 results-of the forphenylactivityabove,activity moiety 5-HTmoietya: K ringwe ifor- for 1Avalue;displayed R. displayednext5-HTon5-HT Among NA:compounds 1Aexte1AR. noR. more nded Amongactivity.Amongmore4311 these - potentour 13fpotent compounds,thesethese effortsand activity compounds,activity13gcompounds, to. 7.3Overall, investigate5.35for 13gfor 5-HT and 5-HT13g 13gcompounds1A 13m andR1Atheand Rthan -– 13mthaneffect 13m13q2277 their13f– –13q bearingtheir13q of–

bearingcorresponding cyclopropylmethylamino compounds 13f moietyand 13h displayed–13l1A bearing more methylaminopotent activity moiety, for 5-HT suggesting1A1AR than theirthat cyclopropylmethylaminocorrespondingsubstituents13qbearing showedEncouraged cyclopropylmethylamino ofselective positioncompounds by the moietyagonistic 2 resultsof the13f displayed activityphenyl above, andmoietya: K 13hringforwei morevalue; displayed– 5-HT next13lon potentNA:compounds bearingexteR. no Amongndedmore activity. activity methylamino ourpotent13f these forandefforts 5-HTactivitycompounds, 13g to.1A moiety,Overall, investigate Rfor than 5-HT 13g compoundssuggesting their and theR corresponding than 13meffect –13ftheirthat13q of– cyclopropylmethylaminobearingcorrespondingcorrespondingcyclopropylmethylamino cyclopropylmethylamino compoundscompounds moiety moiety 13f13f was moietyandandwas more 13hmore13h displayed ––favorabl13l 13lfavorabl 1Abearingbearing emore ethan than methylamino potentmethylaminomethylamino methylamino activity moiety, moiety,moietyfor moiety 5-HT atsuggesting suggesting1A atpositionR position than their 2 that that2as as compoundsRisperidone13qsubstituents showedEncouraged13f and ofselective position 13hby –the 13lagonistic- 2 resultsbearingof the activityphenylabove, methylamino -ringfor we 5-HT nexton compounds exte moiety,R. Amongnded 2.6 suggesting our 13f these effortsand compounds, 13g thatto. Overall, investigate cyclopropylmethylamino - 13g compounds and the 13meffect3.2 –13f13q of– showncorrespondingbearingcyclopropylmethylaminocyclopropylmethylaminoshown in cyclopropylmethylaminoin Table Table 2.compounds 2.Compounds Compounds moietymoiety 13f 13h 13h andwasmoietyandwas and 13mmore13hmore 13m displayed– bearing 13l favorablbearingfavorabl bearing the more ethee thanisobutylthan isobutylmethylamino potent methylaminomethylamino moiety moietyactivity were moiety,were moietyformoiety found found5-HT suggesting attoat1A to positionRdemonstrateposition demonstratethan their that22 asas moiety8-OH-DPATsubstituents13q was showed more of favorableselective position agonistic- than2 of the methylamino activityphenyl -ring for moiety5-HT on compounds1AR. at Among position - 13f these 2 and as showncompounds, 13g. Overall, in 7.3 Table 13g compounds2. and Compounds 13m -–13f 13q– 13h thecorrespondingcyclopropylmethylaminoshownshownthe most most in in potent Table Tablepotent compounds2. agonistic2. Compoundsagonistic Compounds moietyactivity activity13f 13h 13h wasandfor and andfor 5-HTmore 13h13m 5-HT13m1A– 13lbearing favorablR1Abearing Ramong bearingamong the ethe methylaminethan isobutyl methylamineisobutylmethylamino methylamino moiety moiety and and were weremoiety,cyclopropylmethylamino moietycyclopropylmethylamino found found suggestingat to toposition demonstrate demonstrate 2that as and 13m13qbearing showedbearing cyclopropylmethylamino selective the isobutyl agonistic moiety activity moietya: were Ki forvalue; founddisplayed 5-HT NA:1A toR. no demonstrate moreAmong activity. potent these theactivitycompounds, most for potent 5-HT 13g 1A agonisticandR than 13m –their13q activity moietycyclopropylmethylaminoshownthethemoiety mostmost inpossessing possessingTable potentpotent 2. agonisticCompoundsagonistic compounds, compounds, moiety activityactivity 13h respectively. wasrespectively. and forfor more 5-HT13m5-HT 1Abearing1Afavorabl RCompoundR Compound amongamong thee than methylamineisobutylmethylamine 13j 13jmethylamino with withmoiety isopropyl isopropyl andand were cyclopropylmethylaminomoietycyclopropylmethylamino foundgroup group at to wasposition demonstratewas found found 2 asto to for 5-HTcorrespondingbearing1AR cyclopropylmethylamino among compounds methylamine 13f andandmoiety cyclopropylmethylamino13h displayed–13l bearing more methylamino potent activity moiety moiety, for possessing 5-HT suggesting1AR than compounds, theirthat moietycompletely possessing lose activity compounds, for 5-HT 1Arespectively.1AR, which1A wasCompound due to the 13j isopropyl with isopropyl moiety groupof 13j wasforming found steric to Encouragedcompletelyshownthecyclopropylmethylaminomoiety most in possessing potentTable lose by 2. activity agonistic theCompounds compounds, results for moietyactivity 5-HT above,13h respectively.was forR,and which 5-HT morewe13m next bearingwasfavorablR Compound among dueexte the etonded thanmethylamine isobutylthe 13j isopropylourmethylamino with moietyefforts isopropyl and moiety were tocyclopropylmethylamino moiety investigatefound ofgroup 13j at to formingwasposition demonstrate thefound steric 2effect asto of respectively.corresponding Compound compounds13j with 13f isopropyl and 13h group–13l wasbearing found methylamino to completely moiety, lose activitysuggesting for 5-HTthat 1AR, substituentshindrancethemoietycompletelycompletelyhindrance most possessingof potent withposition losewithlose 5-HT agonisticactivityactivity5-HT compounds, 1A2 R.1Aof R. for Asfor the activityAs 5-HTfor5-HT phenylfor 13orespectively. 1A 13o1AforR, R,which which5-HTringwhich showed 1Aon showedwasRwasCompound amongcompounds duedue potent potent totomethylamine the the 13jactivity activity isopropyl 13fisopropylwith and forisopropyl forand 5-HT 13g moiety moiety5-HT cyclopropylmethylamino. 1AOverall,R, 1A group ofR, ofthe 13jthe13j presence was formingpresencecompoundsforming found of stericstericof the tothe 13f – whichcyclopropylmethylaminoshown was due in Table to the 2. isopropylCompounds moiety moiety 13h was and of more 13j13mforming bearingfavorabl stericthee than isobutyl hindrance methylamino moiety with were moiety 5-HT found atR. to position Asdemonstrate for 13o 2 aswhich N-(cyclopropylmethyl)sulfamoylcompletelymoietyhindrancehindranceN-(cyclopropylmethyl)sulfamoyl possessing withlosewith activity 5-HT5-HT compounds,1A1AR. R.for As As 5-HT forfor respectively. 13omoiety1A13o R,moiety whichwhich limited limited showedshowedwas Compound the due the conformationpotent potent conformationto the 13j activity activityisopropyl with of forofisopropylthefor the moiety5-HT 5-HTcompound. compound.1A1A R,groupofR, the1A13jthe By presenceforming presencewasBy contrast, contrast, found steric ofof the thetothe the 13q showedshownthe most in selective potentTable 2. agonistic Compounds agonistic activity activity 13h forand 5-HT for13m 5-HT1A bearingR among1AR. the Among methylamine isobutyl these moiety andcompounds, were cyclopropylmethylamino found to13g demonstrate and 13m– 13q showedintroductioncompletelyhindranceN-(cyclopropylmethyl)sulfamoylN-(cyclopropylmethyl)sulfamoylintroduction potent with lose activity of of a 5-HTactivity polara polar1A for R.atom foratom 5-HTAs 5-HT tofor to 1Athe 13othe1A R,moiety moietyRR, 3Rthewhich groupwhich3 group presencelimited limited showed resultedwas resulted the duethe ofpotent conformationin conformation toin thea thedecreaseda decreased N-(cyclopropylmethyl)sulfamoylactivity isopropyl of forofactivity the activitythe 5-HTmoiety compound. compound. for1A forR, of5-HT the 5-HT13j 1A presence By formingByR1A Rbycontrast, contrast, by an an ofordersteric orderthe the moietythe bearingthemoiety cyclopropylmethylaminomost possessing potent agonistic compounds, activity respectively.moiety for 5-HT displayed1A RCompound among more methylamine 13j potent with isopropyl activityand cyclopropylmethylamino forgroup 5-HT was1A Rfound than to their limitedhindranceN-(cyclopropylmethyl)sulfamoylintroductionintroduction the conformation with of of 5-HTa a polar polar1AR. ofatom atom As the forto to compound. the the13o moiety R Rwhich3 3group group limited showedBy resulted resulted contrast, the potentconformation in in a a decreased thedecreased activity introduction of for activity theactivity 5-HT compound. 1Afor for ofR, 5-HT a5-HTthe polar Bypresence1A1AR Rcontrast, atombyby an an of order toorder the the R3 corresponding completelymoiety possessing losecompounds activity compounds, for 13f 5-HT andrespectively.1AR, which13h–13l was Compound bearingdue to the 13jmethylamino isopropyl with isopropyl moiety moiety, groupof 13j formingwassuggesting found steric to that groupN-(cyclopropylmethyl)sulfamoylintroduction resulted in of a a decreased polar atom activity to the moiety R3 for group 5-HTlimited resulted1A Rthe by conformation in an a decreased order of ofmagnitude, activity the compound. for 5-HT indicating 1AByR contrast, by an that order the a polar cyclopropylmethylaminocompletelyhindrance with lose 5-HTactivity1A R.moiety for As 5-HT for was13o1AR, which morewhich showed favorablwas due potent eto than the activity isopropyl methylamino for 5-HTmoiety1A R,moiety of the 13j presence forming at position ofsteric the 2 as groupintroduction was unfavorable of a polar to atom the binding to the R3 a groupﬃnity resulted towards in5-HT a decreasedR(13i activity, 13l, 13nfor 5-HT, 13q1A).R by an order shown hindranceN-(cyclopropylmethyl)sulfamoyl in Table with 2. Compounds 5-HT1AR. As 13hfor 13o andmoiety which 13m limited bearingshowed the potentconformationthe isobutyl activity1A ofmoiety for the 5-HT compound. were1AR, thefound Bypresence contrast,to demonstrate of the N-(cyclopropylmethyl)sulfamoylintroduction of a polar atom to the moiety R3 group limited resulted the conformation in a decreased of activity the compound. for 5-HT 1AByR contrast, by an order the the most potent agonistic activity for 5-HT1AR among methylamine and cyclopropylmethylamino introduction of a polar atom to the R3 group resulted in a decreased activity for 5-HT1AR by an order moiety possessing compounds, respectively. Compound 13j with isopropyl group was found to completely lose activity for 5-HT1AR, which was due to the isopropyl moiety of 13j forming steric hindrance with 5-HT1AR. As for 13o which showed potent activity for 5-HT1AR, the presence of the N-(cyclopropylmethyl)sulfamoyl moiety limited the conformation of the compound. By contrast, the introduction of a polar atom to the R3 group resulted in a decreased activity for 5-HT1AR by an order

MoleculesMolecules 2020 2020, ,25 25, ,x x FOR FOR PEER PEER REVIEW REVIEW 77 ofof 1515 Molecules 20202020,, 2525,, xx FORFOR PEERPEER REVIEWREVIEW 77 ofof 1515 Table 2. Functional activities of compounds 13f–13q for dopamine receptor (D2R) and serotonin MoleculesTable 2020 2.,, 25Functional,, xx FORFOR PEERPEER activities REVIEWREVIEW of compounds 13f–13q for dopamine receptor (D2R) and serotonin7 of 15 Tablereceptors 2. Functional (5-HT1AR andactivities 5-HT2A ofR). compounds 13f–13q for dopamine receptor (D22R) and serotonin MoleculesMoleculesreceptorsTable 2020 2020 2., ,25 25 Functional(5-HT, ,x x FOR FOR1A PEERR PEER and activities REVIEW REVIEW5-HT2A R).of compounds 13f–13q for dopamine receptor (D R) and serotonin77 ofof 1515 receptorsreceptors (5-HT(5-HT1A1AR and 5-HT2A2AR). MoleculesMoleculesTable 2020 2020 2., ,25 25 Functional, ,x x FOR FOR PEER PEER activities REVIEW REVIEW of compounds 13f–13q for dopamine receptor (D2R) and serotonin77 ofof 1515 receptorsTable 2. Functional(5-HT1AR and activities 5-HT2A R).of compounds 13f–13q for dopamine receptor (D22R) and serotonin MoleculesMoleculesTablereceptors 2020 2020 2., ,25 25Functional (5-HT, ,x x FOR FOR PEER RPEER and activities REVIEW REVIEW 5-HT ofR). compounds 13f–13q for dopamine receptor (D R) and serotonin77 ofof 1515 TablereceptorsTablereceptors 2.2. Functional(5-HT Functional(5-HT1A1ARR and and activitiesactivities 5-HT 5-HT2A2A ofR).ofR). compounds compounds 13f13f––13q13q forfor dopaminedopamine receptorreceptor (D(D22R)R) andand serotoninserotonin MoleculesMolecules 2020 2020, 25, 25, x, xFOR FOR PEER PEER REVIEW REVIEW 7 7of of 15 15 receptorsTablereceptorsTable 2.2. Functional (5-HTFunctional(5-HT1A1ARR andand activitiesactivities 5-HT5-HT2A2A R).ofR).of compounds compounds 13f13f––13q13q forfor dopaminedopamine receptorreceptor (D(D22R)R) andand serotoninserotonin Molecules 2020, 25, x FOR PEER REVIEW 7 of 15 Moleculesreceptors 2020, 25 (5-HT, x FOR1A RPEER and REVIEW5-HT2AR). 7 of 15 TablereceptorsTable 2. 2. Functional Functional(5-HT1AR andactivities activities 5-HT 2Aof R).of compounds compounds 13f 13f–13q–13q for for dopamine dopamine receptor receptor (D (D2R)2R) and and serotonin serotonin

MoleculesMoleculesreceptorsreceptors 2020 2020, ,25 25(5-HT, (5-HT,x x FOR FOR1A1A PEERR PEERR and and REVIEW REVIEW5-HT 5-HT2A2AR). R). 77 of of 15 15 TableTable 2.2. FunctionalFunctional activitiesactivities ofof compoundscompounds 13f13f––13q13q forfor dopaminedopamine receptorreceptor (D(D2R)2R) andand serotoninserotonin MoleculesMolecules 2020 2020, ,25 25, ,x x FOR FOR PEER PEER REVIEW REVIEW 77 of of 15 15 receptorsreceptors (5-HT (5-HT1A1ARR and and 5-HT 5-HT2A2AR).R). TableTable 2.2. FunctionalFunctional activitiesactivities ofof compoundscompounds 13f13f––13q13q forfor dopaminedopamine receptorreceptor (D(D2R)2R) andand serotoninserotonin MoleculesMolecules 2020 2020, 25, 25, x, xFOR FOR PEER PEER REVIEW REVIEW 7 7of of 15 15 receptorsreceptorsTable 2. (5-HT Functional(5-HT1A1ARR and and activities 5-HT 5-HT2A2AR). R).of compounds 13f –13q for dopamine receptor (D2 2R) and serotonin MoleculesMoleculesTable 2020 2020 2., ,25 25Functional, ,x x FOR FOR PEER PEER activities REVIEW REVIEW of compounds 13f–13q for dopamine receptor (D R) and serotonin77 ofof 1515 Molecules 20202020,, 2525,, 5078x FOR PEER REVIEW D22R 5-HT1A1AR 5-HT2A2AR 76 of 1315 1A1A 2A2A D R 5-HT R 2 5-HT R TablereceptorsCompoundreceptorsTableCompound 2. 2. Functional (5-HT Functional(5-HT RR and andactivities activitiesR R5-HT 25-HT2 R).of R).of compounds compoundsRR33 13f 13f––13q13q for for dopamine dopamine receptor receptor (D (DR)2R) and and serotonin serotonin IC50 (nM) EC50 (nM) IC50 (nM) IC50D (nM)22R EC5-HT50 (nM)1A1AR IC5-HT50 (nM)2A2AR receptorsTableTableCompoundreceptors 2.2. Functional(5-HT Functional(5-HT 1A1ARR and and activitiesactivitiesR 5-HT 25-HT2 2A 2A R).ofofR). compoundscompounds RR33 13f13f––13q13q forfor dopaminedopamine receptorreceptor (D(D2R)2R) andand serotoninserotonin Table 2. Functional activities of compounds 13f–13qIC for5050 (nM) dopamine ECreceptor5050 (nM) (D2R) ICand5050 (nM) serotonin ICD (nM)2R EC5-HT (nM)1AR IC5-HT (nM)2AR receptorsreceptorsCompound (5-HT (5-HT 1A1ARR and and R5-HT 5-HT2 2A2AR).R). R3 TableCompound 2. Functional activitiesR of compoundsR 13f–13q for dopamine receptor (D2R) and serotonin receptors 13f(5-HT13f 1AR and 5-HT2AR). ICD50NADNA 2(nM)2RR EC5-HT5-HT23.65023.6 (nM)1A1A R R IC5-HT5-HT1099501099 (nM)2A2A R R CompoundCompound RR22 RR33 receptors (5-HT1AR and 5-HT 2AR). IC5050 (nM) EC5050 (nM) IC5050 (nM) 13f13f ICDNAD 2(nM)2RR EC5-HT5-HT23.6 (nM)1A1A RR IC5-HT5-HT1099 (nM)2A2A RR CompoundCompound RR22 RR33 IC5050 2(nM) EC5050 (nM)1A IC5050 (nM)2A 13f IC DDNA (nM)2RR EC5-HT5-HT23.6 (nM)1A RR IC5-HT5-HT1099 (nM)2A RR CompoundCompound RR22 RR33 IC5050 (nM) EC5050 (nM) IC5050 (nM) 13f13h13f ICNADNA2 (nM)2R EC5-HT23.623.65.67 1A(nM)1A R IC5-HT1099567.41099 (nM)2A2A R 13h DNAR 5-HT5.67 R 5-HT567.4R CompoundCompound RR2 2 RR3 3 13h13f13f ICIC50NA50 NA(nM) (nM) EC EC5023.65.6723.650 (nM) (nM) IC IC50567.41099109950 (nM) (nM) 13h DNA2R 5-HT5.671A R 5-HT567.42AR D2R 5-HT1AR 5-HT2AR CompoundCompound13f RR 2 2 RR3 3 NA 23.6 1099 13h13f 50NA 505.6723.6 50567.41099 13h ICIC50NA (nM) (nM) EC EC5.6750 (nM) (nM) IC IC567.450 (nM) (nM) D2R2 5-HT1A1AR 5-HT2A2AR 13f DNAR 5-HT23.6 R 5-HT1099 R CompoundCompound13f13h13h13i RR 2 2 RR3 3 NANANA 23.65.67400.55.67 1099567.4567.44186 13i IC5050NA (nM) EC50400.550 (nM) IC50418650 (nM) ICDD2 R(nM)2R 5-HT EC5-HT (nM)1A1ARR 5-HT IC5-HT (nM)2A2ARR CompoundCompound13h13h RR2 2 RR3 3 NANA 5.675.67 567.4567.4 13f13i13i13f NANANA 400.523.623.6 109941861099 ICIC50D50D (nM)2 R(nM)2R EC5-HT EC 5-HT5050 (nM) (nM)1A1ARR IC5-HT IC5-HT5050 (nM) (nM)2A2ARR Compound13h R2 2 R3 3 NA 5.67 567.4 Compound13h13i R R NA 400.55.67 567.44186 13f ICIC50NA50 (nM) 2(nM) EC EC5023.650 (nM) (nM)1A IC IC50109950 (nM) (nM)2A 13f DDNA2DRR2 R5-HT5-HT 5-HT23.61AR R1A R5-HT5-HT1099 5-HT2AR R 2A R CompoundCompound13h13i13h13i RR 2 2 2 RRR3 33 NANANA 400.55.67400.55.67 567.44186567.44186 13f13j13f13j ICIC50NA50NA NANA(nM) (nM) EC EC5023.650NA23.6 NA(nM) (nM) IC IC501099501099NA NA(nM) (nM) IC50 (nM) EC50 (nM) IC50 (nM) 13i13i NADNA2R 400.55-HT400.5 1AR 418641865-HT 2AR 13h13f13j13h13j13f NANANA 23.65.67NA5.6723.6 567.41099567.41099NA Compound R 2 R3 13i13i IC50NANA (nM) EC400.5400.550 (nM) 41864186 IC50 (nM) 13f13f13j NANA 23.623.6NA 10991099NA 13h13h NANANA 5.675.67 23.6 567.4567.4 1099

13i13j13k13j13i NANANA 400.5400.5NA77.5NA 4186NA41861778NA 13h13k13h NANANA 5.6777.55.67 567.41778567.4

13f 13i13k13j13k13j NANANA 400.5NA77.5NA23.6 41861778NANA 1099 13h13h13i NANANA 5.67400.55.67 5.67 567.4567.44186 567.4

13j13j NANA NANA NANA 13h13i13h13k NANA 400.55.675.6777.5 567.44186567.41778 13i NA 400.5 4186 13j13k13j NANA NA77.5NA NA1778NA 13k13l13l NANA 523.977.5523.9 1778NANA 13i13i NANANA 400.5400.5 400.5 41864186 4186 13k NA 77.5 1778 13i13k13j13l13l13j13i NANANA 400.5523.9NA77.5400.5NA 41861778NA4186NANA 13h NA 5.67 567.4 13k NA 77.5 1778 13k13l NA 523.977.5 1778NA 13j13i13i13j NANANA 400.5NA400.5NA NA 4186NA4186NA NA

13k13l13k13l NANANA 523.977.5523.977.5 1778NA1778NA 13j13g13j13g NA43114311NA NA5.35NA5.35 NA22772277NA

13k13l13l NANA 523.977.5523.9 1778NANA 13k13j13g13g13k13j NA43114311NANA NA5.355.3577.5NA 77.5 NA227722771778NA 1778 13i NA 400.5 4186 13l13l NANA 523.9523.9 NANA 13k13j13g13j NA4311NA 77.5NA5.35NA 1778NA2277NA 13k NA 77.5 1778 13l13g13l NA4311NANA 523.9523.95.35 523.9 NA2277NA NA 13m13g13m >50004311>5000 5.351.011.01 2277 1528 1528 13k13k NANA 77.577.5 17781778 13g13g 43114311 5.355.35 22772277 13k13m13l13m13k13l >5000>5000NANA 523.977.5523.91.011.0177.5 1778NA 1528 15281778NA

13g13g13g 431143114311 5.355.35 5.35 22772277 2277 13j 13k13m13k >5000NANA 77.577.51.01NA 17781778 1528 NA 13l13l NANA 523.9523.9 NANA

13g13m13m13g13n >50004311>50004311NA 5.351.015.351.0131.6 2277 1528>50002277 1528 13l13n13l NANANA 523.9523.931.6 >5000NANA

13m13m13g13n13m >50004311>5000NA>5000 5.351.0131.61.01 1.01 >50002277 1528 1528 1528 13l13n13g13l NA4311NA 523.9523.931.65.35 >5000NA2277NA

13m13m >5000>5000 1.011.01 1528 1528 13k 13g13l13n13l 4311NANA 523.95.35523.931.677.5 2277>5000NANA 1778 13g 4311 5.35 2277 13m13m13n >5000>5000NA 1.011.0131.6 1528>5000 1528 13n13n13o13g13o 4311NANANA 31.622.85.3522.8 31.6 >50002277NANA> 5000 13g 4311 5.35 2277 13n13n NANA 31.631.6 >5000>5000 13m13g13m13o13o13g >50004311>50004311NA 5.351.0122.81.015.35 2277 1528 15282277NA

13n13n NANA 31.631.6 >5000>5000 13o13g13g13o 43114311NANA 5.355.3522.8 22.8 22772277NA NA 13m13m >5000>5000 1.011.01 1528 1528 13l NA 523.9 NA 13n13o13n13o13p NANANA 31.622.822.831.648.2 >5000>5000NA4730NA 13m13p13m >5000>5000NA 1.0148.21.01 15284730 1528

13p13n13o13p13p13o NANANA 31.622.848.222.8 >50004730NANA 13m13m13n >5000>5000NANA 1.0131.61.01 48.2 1528>5000 1528 4730

13o13p13o NANA 22.848.222.8 4730NANA 13m13n13m13p13n >5000>5000NANA 1.0131.61.0148.231.6 >5000 1528>5000 15284730

13g 13o13p13p13o NA4311NANA 22.848.222.848.25.35 NA47304730NA 2277 13q13n13q13n13q NANANANANA 31.6174.7174.731.6 174.7 >5000>5000NANA NA

13p13p NANA 48.248.2 47304730 13n13o13q13q13o13n NANANA 31.6174.722.822.831.6 >5000>5000NANANA

Risperidone13p13p - - NANA 2.6 48.248.2 -47304730 3.2 13n13n13q NANA 31.6174.731.6 >5000>5000NA 8-OH-DPAT13o13o - -NANA - 22.822.8 7.3 NANA -

13p13q13q13p NANANA 174.748.2174.748.2 4730NA4730NA 13m 13o13o >5000NANA 22.822.81.01 NANA 1528

13o13p13q13p13q13o NA: no activity.NANANA 174.722.848.2174.748.222.8 4730NANA4730NA

13q NA 174.7 NA 13q NA 174.7 NA 13p13o13o13p NANA 22.848.222.848.2 4730NA4730NA

13q13q NANA 174.7174.7 NANA 2.4. Binding Mode13p13p NANA 48.248.2 47304730 13n NA 31.6 >5000 13p13q13q13p NANA 174.748.2174.748.2 4730NA4730NA

We next studied the binding mode of 13m with agonistic conformation of 5-HT1AR. We used

13q13p13p13q NANA 174.748.2174.748.2 4730NA4730NA the GOLD 5.1 program to dock 13m into the binding site of the 5-HT1AR which was obtained by

13q13q NANA 174.7174.7 NANA homology13o modeling from the X-ray structure of 5-HT1B receptorNA (PDB code:22.8 6G79) [25].NA The results 13q13q NANA 174.7174.7 NANA of molecular modeling were shown in Figure 2 . The protonated nitrogen atom of 13m formed a 13q13q NANA 174.7174.7 NANA strong salt bridge with D3.32 in 5HT R. The NH group of the indole ring formed hydrogen bonding 1A with T3.37. The N-(cyclopropylmethyl)sulfamoyl moiety interacted with A2.61, Y2.64 and F3.28 13p NA 48.2 4730 through hydrophobic packing, which was in accordance with the binding mode of FW01 with 5-HT1AR. The isobutoxy group was inserted into another hydrophobic pocket formed by F6.51, V6.54 and I7.38.

13q NA 174.7 NA

Molecules 2020,, 25,, 5078x FOR PEER REVIEW 7 of 1314

Figure 2. Binding mode of 13m with 5-HT1AR. The ligand is shown as green sticks. 5-HT1AR and the Figure 2. Binding mode of 13m with 5-HT1AR. The ligand is shown as green sticks. 5-HT1AR and the surrounding residues are in gray. Hydrogen bond interactions are represented as dotted red lines. surrounding residues are in gray. Hydrogen bond interactions are represented as dotted red lines.

To explain the high selectivity of 13m toward 5-HT1AR, the sequences of 5-HT1AR, 5-HT2AR and To explain the high selectivity of 13m toward 5-HT1AR, the sequences of 5-HT1AR, 5-HT2AR and D2R at the binding site were aligned. As shown in Figure3A, the residues of the active site in three receptorsD2R at the di bindingffered greatly site were and aligned. thus the As conformations shown in Figure of the 3A, three the residues receptors of at the the active binding site site in three were receptors differed greatly and thus the conformations of the three receptors at the binding site were different. The 3D structures of 13m-bounded 5-HT1AR and 5-HT2AR were superimposed to explain different. The 3D structures of 13m-bounded 5-HT1AR and 5-HT2AR were superimposed to explain the selectivity of 13m for 5-HT1AR and 5-HT2AR. The PDB code of 5-HT2AR was 6A93 [26]. As shown the selectivity of 13m for 5-HT1AR and 5-HT2AR. The PDB code of 5-HT2AR was 6A93 [26]. As shown in Figure3B, compared with 5-HT 1AR, the amino acid residues in the hydrophobic pocket of 5-HT2AR stretchedin Figure 3B, out compared further. Thus with the 5-HT N-(cyclopropylmethyl)sulfamoyl1AR, the amino acid residues in ofthe13m hydrophobiccollapsed pocket with W3.28 of 5-HT in the2AR stretched out further. Thus the N-(cyclopropylmethyl)sulfamoyl of 13m collapsed with W3.28 in the active pocket of 5-HT2AR, which is obvious to be observed in Figure3C. It accounted for the reduced active pocket of 5-HT2AR, which is obvious to be observed in Figure 3C. It accounted for the reduced affinity of 13m for 5-HT2AR. The same phenomenon was found in the case of D2R when superposing affinity of 13m for 5-HT2AR. The same phenomenon was found in the case of D2R when superposing 5-HT1AR and D2R (Figure3D). The PDB code of D 2R was 6CM4 [27]. The amino acid residues in 5-HT1AR and D2R (Figure 3D). The PDB code of D2R was 6CM4 [27]. The amino acid residues in the the active pocket of D2R stretched out further than those in the active pocket of 5HT1AR. Thus the indoleactive pocket head and of D the2R N-(cyclopropylmethyl)sulfamoylstretched out further than those in ofthe13m activecollapsed pocket withof 5HT S5.421AR. andThus L2.64 the indole in the head and the N-(cyclopropylmethyl)sulfamoyl of 13m collapsed with S5.42 and L2.64 in the active active pocket of D2R. This explains why 13m showed high selectivity toward 5-HT1AR over 5-HT2AR pocket of D2R. This explains why 13m showed high selectivity toward 5-HT1AR over 5-HT2AR and and D2R. D2R. 2.5. 13m Induced Activation Mechanism of 5-HT1AR

Molecular dynamic simulations were carried out to explore the activation process of 5-HT1AR by 13m. Two systems, 13m bound to the active-state 5-HT1AR and the inactive-state 5-HT1AR without ligand (apo), were performed for 200 ns simulation, respectively. The inactive-state 5-HT1AR was modelled from the X-ray structure of 5-HT1BR (PDB code: 5V54). The Gromos conformational cluster analysis was used to obtain the representative structures of two systems in the course of simulation. In 13m-5-HT1AR system, a conserved hydrogen bond was formed between the NH group of the indole ring in 13m and the oxygen atom of hydroxyl in T3.37 during the simulation (Figure4A,D). At the same time, the protonated nitrogen atom in 13m maintained a stable hydrogen bond with the carbonyl oxygen of D3.32 and a relatively weak interaction with the hydroxyl oxygen of Y7.43 (Figure4B). Therefore, the interaction between D3.32 and Y7.43 (also called 3-7 lock) stayed unbroken in 13m-5-HT1AR system but experienced big ﬂuctuation in apo system (Figure4C). This phenomenon was in agreement with the previous study of our group [28]. The oxygen atom on the sulfonyl group

Molecules 2020, 25, 5078 8 of 13 of 13m formed a hydrogen bond with Q2.65 initially, but it ﬂuctuates greatly due to the swing of the Moleculesmolecular 2020 tail, 25, after x FOR 80 PEER ns (FigureREVIEW4 E). 8 of 14

Figure 3. ((A)) Sequences of 5-HT5-HT1A1AR,R, 5-HT 2AR,R, and and D D22RR at at the the binding binding site. site. ( (BB)) The The superimposition superimposition of 5-HT2ARR with with 13m13m-bound-bound 5-HT 5-HT1AR.R. ( (CC)) Electrostatic Electrostatic potential potential energy diagram ofof 13m13m andand 5-HT5-HT2A2AR at

the binding site. ( D) The superimposition ofof DD22R with 13m-bound 5-HT1A1AR.R. ( (EE)) Electrostatic Electrostatic potential potential

energy diagram ofof 13m13m andand DD22R at the binding site.site. The ligand is shown asas greengreen sticks.sticks. 5-HT5-HT1A1AR and

the surrounding residuesresidues areare inin gray.gray. 5-HT5-HT2A2AR and the surrounding residues areare inin pink.pink. D22R and the surrounding residuesresidues are inin blue.blue.

The strong interaction between 13m and 5-HT R induced the movement of TM3, TM5 and 2.5. 13m Induced Activation Mechanism of 5-HT1AR 1A TM6. The triplets P5.50, I3.40 and F6.44 were located under the binding pockets of small Molecular dynamic simulations were carried out to explore the activation process of 5-HT1AR by molecules, their arrangement was important for stabilizing the antagonistic conformation of 5-HT1AR. The13m. interaction Two systems, of 13m 13mwith bound D3.32 to andthe T3.37active-state adjusted 5-HT the1A sideR and chain the ofinactive-state I3.40 to ﬂip up,5-HT subsequently1AR without ligandleading (apo), to the were relocation performed of P5.50 for and 200 F6.44 ns simu (Figurelation,5A). Theirrespectively. rearrangement The inactive-state was closely related5-HT1AR to was the modelledmovement from of the the helices. X-ray Atstructure the same of 5-HT time,1B W6.48R (PDB moved code: outward5V54). The due Grom to theos steric conformati restraintsonal between cluster 13manalysisand was residue used W6.48. to obtain As the a rotamer representative toggle switch,structures W6.48 of two further systems pushed in the the course intracellular of simulation. end of the TM6In 13m to move-5-HT inward1AR system, and a the conserved external endhydrogen of the TM6bond towas move formed outward between (Figure the5 NHB). Finally, group of TM3, the indole ring in 13m and the oxygen atom of hydroxyl in T3.37 during the simulation (Figure 4A,D). TM5 and TM6 underwent large movement to create space for the binding of Gα/s protein (Figure5C). AtIt was the worthsame time, noting the that protonated the TM6 nitrogen moved outward atom in 11.513m Å. maintained a stable hydrogen bond with the carbonyl oxygen of D3.32 and a relatively weak interaction with the hydroxyl oxygen of Y7.43 (Figure 4B). Therefore, the interaction between D3.32 and Y7.43 (also called 3-7 lock) stayed unbroken in 13m- 5-HT1AR system but experienced big fluctuation in apo system (Figure 4C). This phenomenon was in agreement with the previous study of our group [28]. The oxygen atom on the sulfonyl group of 13m formed a hydrogen bond with Q2.65 initially, but it fluctuates greatly due to the swing of the molecular tail after 80 ns (Figure 4E).

Molecules 2020, 25, 5078 9 of 13 Molecules 2020, 25, x FOR PEER REVIEW 9 of 14

Figure 4. (A) The interaction between 13m and D3.32, Y7.43, Q2.65 and T3.37. The ligand is shown Figure 4. (A) The interaction between 13m and D3.32, Y7.43, Q2.65 and T3.37. The ligand is shown as as green sticks. 5-HT1AR and the surrounding residues are in pink. Hydrogen bond interactions are green sticks. 5-HT1AR and the surrounding residues are in pink. Hydrogen bond interactions are B C representedrepresented as as dotted dotted red red lines. lines. ( (B)) Distance Distance ofof the ligand with with D3.32 D3.32 and and Y7.43. Y7.43. (C () Distance) Distance between between D3.32D3.32 and and Y7.43. Y7.43. (D (D) Distance) Distance between between ligandligand andand T3.37. ( (EE)) The The interaction interaction between between ligand ligand and and Q2.65 Q2.65 in 13m-5-HT R system. Moleculesin 2020 13m, 25-5-HT, x1A FOR1AR PEERsystem. REVIEW 10 of 14

The strong interaction between 13m and 5-HT1AR induced the movement of TM3, TM5 and TM6. The triplets P5.50, I3.40 and F6.44 were located under the binding pockets of small molecules, their arrangement was important for stabilizing the antagonistic conformation of 5-HT1AR. The interaction of 13m with D3.32 and T3.37 adjusted the side chain of I3.40 to flip up, subsequently leading to the relocation of P5.50 and F6.44 (Figure 5A). Their rearrangement was closely related to the movement of the helices. At the same time, W6.48 moved outward due to the steric restraints between 13m and residue W6.48. As a rotamer toggle switch, W6.48 further pushed the intracellular end of the TM6 to move inward and the external end of the TM6 to move outward (Figure 5B). Finally, TM3, TM5 and TM6 underwent large movement to create space for the binding of Gα/s protein (Figure 5C). It was worth noting that the TM6 moved outward 11.5 Å.

Figure 5. (A) Superimposed structures of apo and 13m-5-HT1AR systems and the rearrangements of Figure 5. (A) Superimposed structures of apo and 13m-5-HT1AR systems and the rearrangements of I3.40, P5.50 and F6.44 when 13m was bound to 5-HT1AR. (B) the movement of rotamer toggle switch I3.40, P5.50 and F6.44 when 13m was bound to 5-HT1AR. (B) the movement of rotamer toggle switch W6.48 in 13m-5-HT1AR system contributed to the movement of the TM6. (C) Superimposed structures of W6.48 in 13m-5-HT1AR system contributed to the movement of the TM6. (C) Superimposed structures apo and 13m-5-HT1AR systems and the rearrangements of I3.40, P5.50, and F6.44 when 13m was bound of apo and 13m-5-HT1AR systems and the rearrangements of I3.40, P5.50, and F6.44 when 13m was to 5-HT1AR. The apo system and surrounding residues are represented as grey. The 13m-5-HT1AR bound to 5-HT1AR. The apo system and surrounding residues are represented as grey. The 13m-5- system and surrounding residues are represented as pink. HT1AR system and surrounding residues are represented as pink.

On the basis of simulation trajectory and analysis results, a possible activation process of 5-HT1AR On the basis of simulation trajectory and analysis results, a possible activation process of 5- could be proposed (Figure6). Typically, unbound 5-HT 1AR maintained in an inactive state or a HT1AR could be proposed (Figure 6). Typically, unbound 5-HT1AR maintained in an inactive state or a basic active state. Owing to the intramolecular interaction and inherent flexibility, the molecular thermodynamic motion would lead to the dynamic equilibrium of several energy minima conformations of 5-HT1AR. When 13m got close to the binding pocket of 5-HT1AR, the electrostatic effect between them promoted the ligand recognition and binding process. Then, 13m stably stayed in the pocket enclosed by TM3, TM5, TM6 and TM7 through several anchoring interactions with the residues D3.32, Q2.65 and T3.37. These residues played the role of molecular triggers, propagating the conformational changes to the inner-middle part of the seven helices. The conformational changes induced the rearrangement of P5.50, I3.40 and F6.44 as well as the adjustment of W6.48, ultimately leading to the outward movement of TM3 and TM6, along with TM5 getting closer to TM6. The synergic helix movement of TM3, TM5, and TM6 created space for the binding of Gα/s protein. Then, 5-HT1AR was fully activated, subsequently leading to the downstream signal transduction.

Molecules 2020, 25, 5078 10 of 13 basic active state. Owing to the intramolecular interaction and inherent ﬂexibility, the molecular thermodynamic motion would lead to the dynamic equilibrium of several energy minima conformations of 5-HT1AR. When 13m got close to the binding pocket of 5-HT1AR, the electrostatic eﬀect between them promoted the ligand recognition and binding process. Then, 13m stably stayed in the pocket enclosed by TM3, TM5, TM6 and TM7 through several anchoring interactions with the residues D3.32, Q2.65 and T3.37. These residues played the role of molecular triggers, propagating the conformational changes to the inner-middle part of the seven helices. The conformational changes induced the rearrangement of P5.50, I3.40 and F6.44 as well as the adjustment of W6.48, ultimately leading to the outward movement Moleculesof TM3 2020 and, TM6,25, x FOR along PEER with REVIEW TM5 getting closer to TM6. 11 of 14

Figure 6. ProposedProposed activation process of 5-HT1AR.R. The The apo apo system system and surrounding residues are represented as as grey. grey. The The 13m13m-5-HT-5-HT1A1AR systemR system and and surrounding surrounding residues residues are represented are represented as pink. as pink. The

GTheα/s protein Gα/s protein is represented is represented as blue. as blue.

3. ExperimentThe synergic Protocols helix movement of TM3, TM5, and TM6 created space for the binding of Gα/s protein. Then, 5-HT1AR was fully activated, subsequently leading to the downstream signal transduction. 3.1. Chemistry and Biological Evaluation 3. Experiment Protocols The compound synthesis methods, NMR data and biological evaluation methods were provided in3.1. the Chemistry supplementary and Biological material. Evaluation The compound synthesis methods, NMR data and biological evaluation methods were provided 3.2.in the Molecular Supplementary Modeling Materials.

3.2.1.3.2. Molecular Homology Modeling Modeling

The amino acid sequence of 5-HT1AR were downloaded from the UniProtKB database (Entry 3.2.1. Homology Modeling code: P08908), and sequence similarity search was performed using NCBI BLAST server (Bethesda, MD, TheUSA) amino [29]. acidThe sequencestructure of (PDB 5-HT code:1AR were 6G79 downloaded and 5V54) fromof 5-HT the1B UniProtKB receptor databasewere selected (Entry as code: the templatesP08908), and to construct sequence similaritythe agonistic search and was antagonistic performed conformation using NCBI BLASTof 5-HT server1A receptor. (Bethesda, Sequence MD, alignmentUSA) [29]. of The 5-HT structure1B receptor (PDB and code: 5-HT 6G791AR and was 5V54) carried of 5-HT out1B usingreceptor Discovery were selected Studio as2016 the (hereafter templates abbreviatedto construct to the DS) agonistic (Waltham, and antagonisticMA, USA). Homology conformation modeling of 5-HT was1A receptor.performed Sequence with DS. alignment Ten models of were5-HT 1Bgeneratedreceptor after and loop 5-HT refinement1AR was carried and the out one using with Discovery the lowest Studio Discrete 2016 Optimized (hereafter Protein abbreviated Energy to (DOPE)DS) (Waltham, score was MA, submitted USA). Homology to energy modeling minimizati was performedon (100 steps with steepest DS. Ten descent models werewith generatedbackbone constrained).after loop reﬁnement The PROCHECK and the one program with the (Cambridge, lowest Discrete UK) Optimizedwas used to Protein evaluate Energy the stereochemical (DOPE) score quality of 5-HT1AR [30].

3.2.2. Molecular Docking Operations Molecular docking was carried out using GOLD 5.0.1 (Cambridge, UK). The binding site was defined to include all residues within a 15.0 Å radius of the conserved D3.32 Cγ carbon atom of 5- HT1AR. Ten conformations were produced for each ligand, and Gold-Score was used as scoring function. Other parameters were set as standard default. High-scoring complexes were inspected visually to identify the most reasonable solution.

Molecules 2020, 25, 5078 11 of 13 was submitted to energy minimization (100 steps steepest descent with backbone constrained). The PROCHECK program (Cambridge, UK) was used to evaluate the stereochemical quality of 5-HT1AR[30].

3.2.2. Molecular Docking Operations Molecular docking was carried out using GOLD 5.0.1 (Cambridge, UK). The binding site was deﬁned to include all residues within a 15.0 Å radius of the conserved D3.32 Cγ carbon atom of 5-HT1AR. Ten conformations were produced for each ligand, and Gold-Score was used as scoring function. Other parameters were set as standard default. High-scoring complexes were inspected visually to identify the most reasonable solution.

3.3. Molecular Dynamic Simulations The molecular dynamic (MD) simulations were performed using the GROMACS 5.1.2 package (Uppsala, Sweden) [31]. Two systems, 13m bound to active-state 5-HT1AR and inactive-state 5-HT1AR without ligand (apo), were embedded in the hydrated, equilibrated palmitoyloleoylphosphatidylcholine (POPC) bilayer via the CHARMM-GUI server. The TIP3P water model was used for the MD simulation. Sodium and chloride ions were added to neutralize two systems to an ionic concentration of 0.15 mol/L. Both systems were minimized and gradually equilibrated in an NPT ensemble at 310 K and 1 bar. Periodic boundary conditions were applied to these simulation systems. Bonds connected to hydrogen atoms were restrained with the LINCS algorithm to allow an integration time step of 2 fs. Electrostatic interactions were calculated using the particle mesh Ewald (PME) method. Finally, the MD simulations for both systems were performed for 200 ns. All analysis of MD trajectories was performed using tools implemented in the GROMACS 5.1.2 package (Uppsala, Sweden). All the structural graphics were processed with PyMOL software (San Carlos, CA, USA) [32].

4. Conclusions In summary, a series of indolealkylpiperazine derivatives with a benzamide moiety were designed and synthesized. Their activities on dopamine and serotonin receptors were evaluated and these compounds were found to be selective 5-HT1AR agonists. Among all tested compounds, 13m displayed the most potent agonistic activity over 5-HT1AR with an EC50 value of 1.01 nM. The investigation of the binding mode uncovered the mechanism of 13m’s potent agonistic activity and high selectivity. In addition, molecular dynamic simulations were carried out to propose 13m induced activation model of 5-HT1AR. The electrostatic interaction between the negatively charged active site of the 5-HT1AR and the positively protonated charged 13m is the initial driving force for the ligand recognition. 13m formed anchoring interactions with residues D3.32, Q2.65, T3.37 and induced conformation changes of 5-HT1AR. This leads to the rearrangement of P5.50, I3.40 and F6.44 as well as the adjustment of W6.48, causing the outward movement of TM3 and TM6, along with TM5 getting closer to TM6. Then, 5-HT1AR was ready to bind Gα/s protein, leading itself to a fully activated state. This model would further promote structure-based agonist design of 5-HT1AR for the treatment of depression.

Supplementary Materials: The following are available online, the compound synthesis methods, NMR data, biological evaluation methods. Author Contributions: C.Z., X.L. and W.P. contributed equally in this work. Compound design, C.Z. and W.F.; compound synthesis, C.Z., X.L. and W.P.; writing—original draft preparation, C.Z. and X.L.; writing—review and editing, W.F.; funding acquisition, W.F. All authors have read and agreed to the published version of the manuscript. Funding: This work is supported by grants from National Natural Science Foundation of China (NO. 81773635, NO. 82073765) and Shanghai Science and Technology Development Funds (14431900500, 20S11902400). Conﬂicts of Interest: The authors declare no conﬂict of interest. Molecules 2020, 25, 5078 12 of 13

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