Subscriber access provided by University of Pennsylvania Libraries Article AVN-211, Novel and Highly Selective 5-HT6 Receptor Small Molecule Antagonist, for the Treatment of Alzheimer's Disease Alexandre V. Ivachtchenko, Yan Lavrovsky, and Yan A. Ivanenkov Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00830 • Publication Date (Web): 17 Feb 2016 Downloaded from http://pubs.acs.org on February 22, 2016

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1 2 AVN211, Novel and Highly Selective 5HT 6 Receptor Small Molecule 3 4 5 Antagonist, for the Treatment of Alzheimer's Disease 6 7 8 9 Alexandre V. Ivachtchenko, a,b Yan Lavrovsky,d and Yan A. Ivanenkov c,e* 10 11 a Alla Chem LLC, 1835 E. Hallandale Beach Blvd # 442, Hallandale Beach, Fl 33009 USA, 12 13 14 tel: + 18584423727, email: [email protected] 15 b 16 Avineuro Pharmaceuticals, Inc., 1835 E. Hallandale Beach Blvd # 442, Hallandale Beach, Fl 17 18 33009 USA, tel: + 18584423727, email: [email protected] 19 20 с Moscow Institute of Physics and Technology (State University), 9 Institutskiy lane, 21 22 23 Dolgoprudny city, Moscow Region, 141700, Russian Federation, fax: +74954087277, tel: 24 25 +74987446916 26 27 d RPharm Overseas, Inc., 12526 High Bluff Drive, Suite # 300, San Diego, CA 92130 USA, tel: 28 29 +16179013890, email: lavrovsky@rpharm.com 30 31 32 e Moscow State University, Chemistry Dept., 119991, Moscow, Leninskie gory, building 1/3, 33 34 GSP1, tel: +74959394020, email: [email protected] 35 36 37 * Corresponding author, email: [email protected] 38 39 40 41 42 ABSTRACT 43 44 Within the past decade several novel targets have been indicated as key players in 45 46 Alzheimertype dementia and associated conditions, including a 'frightening' memory loss as 47 48 well as severe cognitive impairments. These proteins are deeply implicated in crucial cell 49 50 processes e.g. autophagy, growth and progression, apoptosis and metabolic equilibrium. Since 51 52 53 recently, 5HT 6R has been considered as one of the most prominent biological targets in AD 54 55 drug therapy. Therefore, we investigated the potential procognitive and neuroprotective effects 56 57 of our novel selective 5HT 6R antagonist, AVN211. During an extensive preclinical evaluation 58 59 60 1 ACS Paragon Plus Environment Molecular Pharmaceutics Page 2 of 55

1 2 the lead compound demonstrated a relatively high therapeutic potential and improved selectivity 3 4 towards 5HT 6R as compared to reference drug candidates. It was thoroughly examined in 5 6 different in vivo behavioral models directly related to AD and showed evident improvements in 7 8 cognition and learning. In many cases, the observed effect was considerably greater than that 9 10 11 determined for the reported drugs and drug candidates, including memantine, SB742457 and Lu 12 13 AE58054, evaluated under the same conditions. In addition, AVN211 showed a similar or better 14 15 anxiolytic efficacy than fenobam, rufinamide, lorazepam and buspirone in an elevated plusmaze 16 17 model, elevated platform and open field tests. The compound demonstrated low toxicity and no 18 19 20 side effects in vivo , an appropriate pharmacokinetic profile as well as stability. In conclusion, 21 22 AVN211 significantly delayed or partially halted the progressive decline in memory function 23 24 associated with AD that makes it an interesting drug candidate for the treatment of 25 26 neurodegenerative and psychiatric disorders. The advanced clinical trials is currently under 27 28 active discussion and in high priority. 29 30 31 32 33 KEYWORDS: 5HT 6R, antagonists, Alzheimer’s disease, selectivity, preclinical study, 34 35 pharmacokinetics 36 37 38 39 40 TABLE OF CONTENTS 41 42 43 44 45 AVN-211 46 47 O S 48 O 49 N 50 51 S N 52 N 53 54 IC = 2.34 nM (5-HT R) 55 50 6 56 57 58 59 60 2 ACS Paragon Plus Environment Page 3 of 55 Molecular Pharmaceutics

1 2 3 4 INTRODUCTION 5 6 Alzheimer's disease (AD) is, undoubtedly, the most common and progressive form of 7 8 dementia (up to 60–70% of cases) diagnosed particularly among the elderly, and 60% of these 9 10 11 people live in Western, low and middleincome countries. Currently, according to World Health 12 1 13 Organization, more than 35 million alzheimer's report numbers exist. In Western hemisphere, 14 15 the prevalence of AD is roughly estimated around 1% among the subjects aged 60‒65, however 16 17 this number increases up to 35% in people aged over 85. As a result, above 5.5 million people 18 19 20 are currently diagnosed with AD in United States. Generally, the disease is accompanied by 21 22 severe memory loss and disorientation, social detachment, significant loss of independence, and 23 24 ultimately leads to lethal outcome within 3−9 years since the diagnosis has been confirmed and 25 26 this frightening statistics is actually expected to get much worse. Therefore, without radical 27 28 breakthroughs in the field of innovative treatment and care of AD the associated costs are indeed 29 30 31 expected to grow dramatically. During the past decade, considerable advances have been made 32 33 towards the understanding of tangled nature of this pathology and neurodegeneration associated 34 35 with this disease. However, despite the outstanding achievements in this field, an effective cure 36 37 strategy is still wanting. With regard to drug treatment, many painstaking attempts have been 38 39 40 made resulting in several smallmolecule compounds with promising activity against AD (Table 41 42 1). 43 44 45 46 Table 1 . Representative examples of molecules targeted against AD 47 48 49 Mechanism of 50 Drug Name Originator Highest Phase Ref 51 action 52 53 DMXB nicotinic α7 partial 54 CoMentis Phase II 2 55 Anabaseine agonist 56 57 58 Ladostigil butyrylcholinesterase Avraham Phase II 3 59 60 3 ACS Paragon Plus Environment Molecular Pharmaceutics Page 4 of 55

1 2 inhibitor, MAOB/A 3 4 inhibitor 5 6 VX 745 MAPK p38 inhibitor EIP Pharma Phase II 4 7 8 9 Ttype calcium Sonexa 10 ZSET1446 Phase II 5 11 channel activator Therapeutics 12 13 Latrepirdine multi targeted Pfizer Phase III 6 14 15 Lu AE58054 5HT 6R antagonist Lundbeck Phase III 7,8 16 17 18 SB 742457 5HT 6R antagonist GSK Phase II 9 19 20 FORUM 21 Encenicline α7 agonist Phase III 10 22 Pharmaceuticals 23 24 AC 1202 analogue 25 26 27 AC1204 (Dietary Accera Phase II/III 11 28 29 Supplement) 30 31 Verubecestat BACE1 inhibitor Merck & Co. Phase III 12 32 33 AZD3293 BACE1 inhibitor AstraZeneca Phase II/III 13 34 35 36 MAO inhibitor, Tau 37 38 aggregation TauRx 39 LeucoMTx Phase III 14 40 inhibitor, NO Therapeutics 41 42 production inhibitor 43 44 Monoamine oxidases (MAO); p38 mitogenactivated protein kinases (MAPK p38); 5hydroxytryptamine (serotonin) 45 46 receptor 6 (5HT 6R); Betasecretase 1 (BACE1) 47 48 49 50 Particularly, it has been proposed that Latrepirdine (Dimebon) may modulate several 51 52 53 targets including voltagegated calcium channels, mitochondrial permeability pore transition, or 54 15–21 55 several neurotransmitter receptors. However, this type of therapy is specifically targeted on 56 57 symptomatology (BPSD) and improvements in the quality of life thereby providing per se 58 59 60 4 ACS Paragon Plus Environment Page 5 of 55 Molecular Pharmaceutics

1 2 temporary effect. Recent findings have indicated several novel prominent targets deeply 3 4 implicated in AD neuropathology, including GLP1R (AD is increasingly regarded as type 3 5 6 diabetes), 22,23 proteins that regulates autophagy (e.g. PINK1|2,24 mTOR,25 TLRs,26 SUMO1,27 7 8 Wnt/βcatenin,28 ACAT1,29 TREM2)30 and prosurvival intracellular pathways 9 10 31,32 11 (repair/regenerative processes) as well as γ|β secretase, growth factors and serotonin 12 33,34 13 receptors (improvements in memory and/or cognition). It should be noted that 5HT 6R was 14 15 tentatively suggested to be involved in autophagy regulation as well via mTOR signaling 16 17 pathway.25,35,36 18 19 20 In recent years the important role of 5HT 6R and 5HT 6R antagonists in AD 21 34,37–41 22 neuropathology has been abundantly highlighted [for review see: ]. Despite several 23 24 “unsuccessful” stories with 5HT 6R antagonists in clinics, including the one with Dimebon, 25 26 novel smallmolecule compounds possessing high activity against this receptor subtype are 27 28 29 currently undergoing clinical evaluation related to AD neuropathology. For instance, Lu 30 42 31 AE58054 ( vide supra ), PF05212377 (Phase II, Pfizer), SUVN502 (Phase II, concomitant 32 33 therapy, Suven Life Sciences),43 as well as our parent compounds, including AVN10144 and 34 35 AVN322 8 (Phase I/II, Alla Chem.). Interestingly, some leading pharmaceutical companies have 36 37 made the decision to reprofile their antischizophrenia molecules towards AD trials, since 38 39 40 selective 5HT6R antagonists have recently been considered “behindthescenes” as prospective 41 42 drug candidates against AD. 43 44 Here we describe the pharmacokinetic features, activity, efficiency as well as toxicity 45 46 profile of AVN211. We clearly demonstrated that this compound is a novel highly effective 47 48 49 (single digits nM) 5HT 6R antagonist with dramatically improved selectivity as compared to 50 51 other reported molecules targeted against the title receptor, including the most advanced in 52 53 clinics LuAE58054 and SB742457. AVN211 (4 and 8 mg, 26 weeks) has recently been 54 55 evaluated in Phase II clinical trial at Avineuro for the treatment of schizophrenia. Based on the 56 57 obtained results, we made a decision to test the compound within an extensive clinical trial 58 59 60 5 ACS Paragon Plus Environment Molecular Pharmaceutics Page 6 of 55

1 2 following the parent indication as well as reprofile it against AD by analogy to other 5HT 6R 3 4 antagonists LuAE58054 and SB742457. The results of a comparative preclinical trial disclosed 5 6 herein have revealed AVN211 as a promising drug candidate, which has demonstrated an 7 8 improved PK profile and efficiency in different in vivo ADrelated models as compared to other 9 10 11 therapeutics including selective 5HT 6R antagonists, Memantine (NMDAR antagonist) and 12 13 Tacrine (AChE inhibitor). Considering a prominent role of 5HT 6R and selective 5HT 6R 14 15 antagonists in Alzheimer's neuropathology, AVN211 can be reasonably regarded as novel drug 16 17 candidate with an outstanding therapeutic potency against AD. 18 19 20 21 22 MATERIAL AND METHODS 23 24 Due to severe limitations in the available paper space, the detailed experimental 25 26 protocols, procedures as well as lab. tools are properly discussed in supplementary materials 27 28 (SM) . Below, we provide only a brief description. 29 30 31 32 33 Screening 34 35 HTS and follow up studies were performed using a recombinant human 5HT 6R stably 36 37 expressed in HEK293 cells in a TRex TM System, a tetracycline regulated mammalian expression 38 39 40 assay kit. All the reagents used were obtained from Sigma Chemical Company (St. Louis, MO) 41 42 unless otherwise stated. Radiochemicals were purchased from Amersham Radiochemicals (GE 43 44 Healthcare Life Sciences, Pittsburgh, PA). Membrane preparations from cells either transfected 45 46 with or endogenously expressed receptors and enzymes were obtained from MDS Pharma 47 48 Services and Eurofins Sc. (Taipei, Taiwan). SKNSH cells were from American Type Culture 49 50 51 Collection (Manassas, VA). Human recombinant tetracyclineinducible HEK293 5HT 6RTRex 52 53 cells were obtained from Etogen Scientific (San Diego, CA). Competitive radioligand binding 54 55 assay for AVN211 towards a broad panel of therapeutically significant targets has been 56 57 58 59 60 6 ACS Paragon Plus Environment Page 7 of 55 Molecular Pharmaceutics

1 2 performed in MDS Pharma Services and Eurofins Sc. following the procedure described in the 3 4 paper by Okun et al. 47 5 6 7 8 Stability in human liver s9 fraction 9 10 Analysis of AVN211 stability in the presence of human liver s9 fraction (CellzDirect, 11 12 USA) was performed in according to the procedure reported in manufacturer guidelines.48 13 14 15 16 17 Permeability 18 19 Permeability of AVN211 was determined using Caco2 cells (ATCC cat № HTB37). 20 21 Assay was performed according to Caco2 plate manufacturer guidelines.49 22 23 24 25 26 Interaction with P-gp 27 28 29 Two different approaches, luminescent ATPase assay with PgpGlo System and Caco2 30 31 32 monolayer efflux assay, were used to study Pgp/AVN211 interaction. The first assay has been 33 34 applied to detect the effects of compounds on recombinant human Pgp in a cell membrane 35 36 fraction. The assay relies on the ATP dependence of the lightgenerating reaction of firefly 37 38 luciferase. MultiScreen Caco2 Assay System (Millipore Corp., USA) was used for the 39 40 monolayer efflux studies. Caco2 cells are morphologically and functionally very similar to 41 42 43 intestinal barrier cells and are frequently utilized as in vitro permeability model. The cells also 44 45 express a variety of transporters including Pgp, thus may be used in Pgpmediated transport 46 47 studies. Particularly, the effect of AVN211 on Rhodamine 123 transport has been estimated. 48 49 50 51 52 Interaction with CYPs 53 54 The interaction of AVN211 with CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and 55 56 57 CYP3A4 (Invitrogen Vivid® CYP450 Screening Kits, Cat. no. P2863, P2859, P2864, P2972, 58 59 60 7 ACS Paragon Plus Environment Molecular Pharmaceutics Page 8 of 55

1 2 P3021, P2856) and CYP2B6 (BD Biosciences P450 Inhibition Kit, Cat. no. 459220) was studied 3 4 in full accordance with the manufacturer’s protocols.50 5 6 7 8 9 10 Animals 11 12 13 Mice (1733 g, 89 wk old) and rats (150320 g, 1314 wk old) were obtained from the 14 15 State Scientific Center (GU NC) of Biomedical Technologies of the Russian Academy of 16 17 Medical Sciences (RAMS) “Andreevka” (Moscow reg.). Studies with Rhesus Macaques ( M. 18 19 mulatta ) were performed at the Research Institute of Medical Primatology (Adler, Russia). 20 21 Monkeys were recommended by the regulatory body as nonrodent species for a number of 22 23 24 reasons: data on CNS drug candidates have usually better translation from primates to humans; it 25 26 is assumed that primates have more "humanlike" pattern of serotonin receptors expression. 27 28 29 30 31 32 In vivo PK study 33 34 35 Pharmacokinetic properties of the compound were evaluated using 24 male rats of WAQ 36 37 strain. Oral bioavailability of AVN211 was calculated based on the obtained results as a ratio of 38 39 AUQ 0→t (p.o.)/AUQ 0→t (i.v.). We also investigated the PK features of AVN211 in rat brain. The 40 41 studies were performed on male Wistar rats. Preliminary escalating dose study was conducted in 42 43 male Wistar rats (300–330 g). Animals were kept in standard laboratory conditions. Animals 44 45 46 were divided into 6 separate groups (3 animals per each group). Three groups were administered 47 48 with AVN211 per os in 6 doses: 1, 3, 10, 20, 50 and 100 mg/kg. The remaining groups were 49 50 administered with AVN211 intravenously in 3 doses: 1, 3 and 10 mg/kg. After the injection, 51 52 animals were allowed to rest for 60 minutes. Then, animals were sacrificed and plasma samples 53 54 55 were collected and subsequently analyzed for AVN211 concentration using LCMS/MS 56 57 API2000. SHK mice were also used to estimate PK features of AVN211. The BBB permeability 58 59 of AVN211 was estimated in rats (10 mg/kg, p.o.). Five rhesus monkeys were used for PK 60 8 ACS Paragon Plus Environment Page 9 of 55 Molecular Pharmaceutics

1 2 evaluation. Animals were not recruited in other studies (e.g. as a placebocontrol group) for at 3 4 least 2,5 months prior the PK trials. Animals were kept in cages, feeding was performed with 5 6 usual balanced fodders, eggs, vegetables and fruits. Clinical status of animals and their behavior 7 8 were normal. Animal IDs, weight, birthday as well as the route of administration and doses of 9 10 11 the tested compounds are presented in SM. Biodistribution of the studied compound was 12 3 13 examined in rats using [H ]AVN211 (10 mg/kg). In this experiment, 18 male rats (180–200 g, 14 15 WAQ strain) were used. The animals were kept in standard laboratory conditions. Maintenance 16 17 and care were in accordance with the Guide for the Care and Use of Laboratory Animals. 51 18 19 20 21 22 23 In vivo efficacy 24 25 26 The rats ( Wistar and SpragueDawley , 160–300 g, 13–14 wk old) and mice (BALB/c and 27 28 SHK, 18–32 g, 8–9 wk old). Before the studies, the animals were quarantined for 7 days. During 29 30 this period, the animals were monitored for the presence of possible diseases. They were housed 31 32 33 in standard plastic cages under controlled conditions: temperature 19–25 °C, humidity 50–70%, 34 35 12:12 h lightdark cycle, concentration of carbon dioxide <0.15% (vol.), concentration of 36 37 ammonia <0.001 mg/L. The animals were monitored at least once daily (or 1 h after subacute 38 39 injections). The results of monitoring were traced in a laboratory register. The specificity of the 40 41 subjects used are listed in Table 9 (see below ). 42 43 44 Most of the reference compounds, including commercially available Sibutramine, 45 46 47 Haloperidole, Lorazepam, Fenobam, Rufinamide, Buspirone, Memantine and Tacrine were used 48 49 in consistent doses and the doses were chosen based on the literature and tests performed at the 50 51 lab. Particularly, SB742457 and PRX07034 were tested in different doses (titrated) in the same 52 53 fashion as it was done for AVN211. It is important to emphasize that the most efficacious dose 54 55 of each compound varied in different species of rodents (for example there was clear difference 56 57 58 between efficacy in SHK and BALB/C mice) and also it varied depending on the challenge given 59 60 9 ACS Paragon Plus Environment Molecular Pharmaceutics Page 10 of 55

1 2 to animals (MK801 vs. scopolamine). The most efficacious dose of SB742457 or PRX07034 3 4 is cited in the manuscript ( vide infra ). The details of titration and corresponding dose range are 5 6 presented in SI. 7 8 9 10 11 12 hERG channels inhibition 13 14 15 Potassium current was measured using the patch clamp technique in Molecular Devices’ 16 17 Patch Express 7000A following the procedure described in SM. 18 19 20 21 22 23 Toxicity and side-effects 24 25 26 LD 50 , MTD and 14days toxicity of AVN211 were investigated in SHK strain male mice 27 28 (20–25 g). We estimate 29 and 30days toxicity of AVN211 in 80 Wistar rats and rabbits, 29 30 respectively. Longterm 180days toxicological study was also performed in 80 Wistar rats (8 31 32 weeks old). In addition, six healthy Rhesus Macaques (Macaca Mulatta, 3 males and 3 females) 33 34 35 were used to determine 30days toxicity of AVN211. Pathomorphological examination, 36 37 including histopathology and macroscopic study, was performed in mice (10 males and 10 38 39 females) after AVN211 administration per os in the dose of 3 mg/kg. Rabbits and Wistar rats 40 41 were also used under the same conditions. In vitro mutagenicity was estimated using Ames MPF 42 43 Test, while mutagenicity in vivo was analyzed in C57BL/6 mice (18–20 g, 8–12 weeks old). All 44 45 46 the details of the procedures mentioned above are conveniently described in SM with 47 48 corresponding tables and figures. 49 50 51 52 53 54 Statistical analysis 55 56 All data were fed into Excel 2003 spread sheets. Subsequent statistical analyses were 57 58 59 performed using Statistica 6.0 software. The obtained results are presented as mean ± standard 60 10 ACS Paragon Plus Environment Page 11 of 55 Molecular Pharmaceutics

1 2 error of the mean (S.E.M.), unless otherwise stated. A pvalue less than 0.05 was considered 3 4 statistically significant. LD 50 was calculated from the mortality rate table according to the 5 6 method of Finny using BioStat 2006 program. 7 8 9 10 11 12 RESULTS 13 14 Molecule 15 16 AVN211 (5,7dimethyl2(methylthio)3(phenylsulfonyl)pyrazolo[1,5a]pyrimidine, 17 18 b 19 Figure 1) is a highly selective 5HT 6R antagonist, with K i=1.09 nM (competitive binding assay), 20 f 45 21 K i=0.83‒1.97 nM (functional assay) obtained by a convenient 3step synthetic route in high 22 23 yield (up to 90%) using common chemical reactions. 46 24 25 26 27 MW=333.44 28 LogD=2.37 (pH=7.4) O S 29 O LogP=1.21 (pH=4) H3C N LogP=2.37 (pH=9.5) 30 logSw=-6.12 S 31 N HBA=4 N CH 32 3 HBD=0 33 CH RBN=1 3 PSA= 98.0 34 AVN-211 35 36 Figure 1 . Structure of AVN211 and key features. 37 38 39 40 41 Screening and selectivity 42 43 Historically, more than 100K compounds available within ChemDiv proprietary library 44 45 have been screened on a panel of GPCRs reportedly involved in pathophysiology of AD. 46 47 Disclosed hits were then tested in in vitro and in vivo behavioral models and further optimized 48 49 on a smaller panel of GPCRs. Targetbased HTS and profiling have revealed AVN211 as a 50 51 b 52 highly potent and selective 5HT 6R antagonist (Figure 2): IC 50 =2.34 nM, K i=1.09 nM and EC 50 53 f 54 = 7.1‒16.0 nM, K i = 0.83‒1.97 nM. 55 56 57 58 59 60 11 ACS Paragon Plus Environment Molecular Pharmaceutics Page 12 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13

14 15 a b 16 17 3 18 Figure 2. Activity of AVN211: ( a) competitive displacement of radiolabeled [ H]LSD 19 20 from 5HT 6R expressed in the cell membrane fraction; ( b) dosedependent inhibition of cAMP 21 22 production induced by serotonin in HEK293 cells transfected with functional human 5HT 6R. 23 24 25 26 27 AVN211 specificity profile was accurately determined using a wide panel of various 28 29 proteins, including GPCR family, ion channels, and transporters (Figure 3, Supplementary Table 30 31 1). As clearly shown in Figure 3 (and Table S1 in SI), AVN211 exhibited 5Kfold higher 5 32

33 HT 6R selectivity over other 65 receptors, enzymes, and ion channels except 5HT 2B R subtype 34 35 (IC f =0.196 µM, EC >10 µM). Within the 5HTRs group the compound showed about 100 36 50 50 37 52 38 fold 5HT 6R selectivity. 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 12 ACS Paragon Plus Environment Page 13 of 55 Molecular Pharmaceutics

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

25 26 Figure 3. Selectivity profile of AVN211 (at 1 and 10 M) towards 66 targets determined in the 27 28 29 radioligand binding assay (the detailed data is presented in SM, Supplementary Table 1). 30 31 32 33 One of the most crucial point of AVN211 is an outstanding selectivity exclusively 34 35 53 towards 5HT 6R as compared to known antagonists which has recently been reported. For 36 37 38 instance, the most advanced drug candidates LuAE58054 and SB742457 demonstrated rather 39 40 moderate 5HT 6R selectivity that was respectively more than 100 and 50fold lower than the 41 42 selectivity of AVN211 keeping a comparative target affinity. Indeed, the selectivity profile of 43

44 LuAE58054 (Ki=0.83 nM, 5HT 6R) determined in more than 80 binding as well as in 20 45 46 enzymatic assays indicated >50 fold selectivity except for the adrenergic α (K =21 nM) and α 47 1A i 1B 48 49 (Ki=22 nM) receptors. LuAE58054 also showed nanomolar activity towards 5HT 2A R (Ki=83 50 54 51 nM) and 5HT 2C R (Ki=250 nM). With regard to SB742457 (p Ki=9.63, 5HT 6R, binding 52 53 assay), it showed >100fold increase in the target activity over 85 other proteins of different 54 55 types.55 However, in a radioligand binding assay, at the concentration of 1 µM, it demonstrated 56 57 58 a multitargeted profile, including the core 5HT 6R (101% inhibition) as well as 5HT 1A R (73%), 59 60 13 ACS Paragon Plus Environment Molecular Pharmaceutics Page 14 of 55

1 2 5HT 1B R (102%), 5HT 2A R (99%), 5HT 2B R (99%), 5HT 2C R (95%), 5HT 7R (84%) receptor 3 4 subtypes. Moreover, we have demonstrated that SB742457 was quite active against adrenergic 5 6 α1B (50%), α 2A (51%), β 1 (90%) and β 2 (83%), dopamine D 2L (76%) and D 2S (76%), imidazoline 7 8 I central (60%) and σ (57%) receptors, calcium Ltype benzothiazepine (66%) and 9 2 1 10 11 dihydropyridine (55%) and sodium site 2 (55%) channels, as well as NET (78%) and DAT 12 56 13 (85%) transporters. 14 15 16 17 Affinity to benzodiazepine receptor 18 19 20 In vivo evaluation revealed AVN211 as a potent anxiolytic ( vide infra ). In order to assess 21 22 the mechanism of anxiolytic action, an additional radioligand displacement assay was performed 23 24 with peripheral benzodiazepine receptor. As clearly shown in Figure 3, AVN211 did not interact 25 26 with benzodiazepine receptor, thus it’s anxiolytic effects observed in 3 different in vivo models 27 28 were not likely to be related to peripheral benzodiazepine receptors. 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 Figure 3. Radioligand displacement assay with peripheral benzodiazepine receptor 44 45 46 47 Interaction with 5-HT R 48 2B 49 50 In some cases, the 5HT 2B R agonism has been shown to be implicated in a cardiac 51 57 52 impairment. Our data, showing that AVN211 binds to the receptor, has made it necessary to 53 54 study its effect on 5HT 2B R in more details to address its potential liability. Thus, in a 55 56 radioligand competitive binding assay, AVN211 showed Ki values of 0.125 M (Figure 4a), 57 58 which was 60fold higher than that observed for 5HT R. AVN211 was tested in a functional 59 6 60 14 ACS Paragon Plus Environment Page 15 of 55 Molecular Pharmaceutics

1 2 tissue assay, rat fundus contraction, both as an agonist (induction of fundus contractions) and 3 4 antagonist (inhibition of αmethylserotonininduced contractions). The data depicted in Figure 5 6 4b shows that AVN211 is a partial agonist of the Rat receptor with EC around 13 M, which 7 50 8 9 is about 100fold higher than its antagonistic activity against the same receptor, IC 50 =163 nM 10 11 (cellbased functional assay). Additional functional aequorin kinetic assays (Euroscreen) were 12 13 performed to measure possible AVN211 agonism of human 5HT 2B R. AVN211 was used in 14 15 two concentrations – 10 M and 20 M (Figure 4c) and did not show any agonism at either 16 17 18 concentration. Dosedependence curve of AVN211 was also obtained using aequorin functional 19 20 assay (Figure 5). 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 a b 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 c 56 57 58 59 60 15 ACS Paragon Plus Environment Molecular Pharmaceutics Page 16 of 55

1 3 2 Figure 4. ( a) Dosedependent displacement of [ H]LSD from 5HT 2B receptor with AVN211; 3 4 (b) dosedependence agonist/antagonist curve of AVN211; ( c) intracellular calcium dynamics in 5 6 AVN211treated cells. AVN211 was used in two concentrations 10 M ( left ) and 20 M 7 8 (right ) 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Figure 5. Activation of h5HT 2B R by AVN211 30 31 32 33 The aforementioned data clearly indicates that AVN211 is a highly selective and specific 34 35 36 antagonist of 5HT 6R with very good safety window. 37 38 39 40 In vitro ADME features 41 42 Analysis of AVN211 (17 M) stability in the presence of human liver s9 fraction (Figure 43 44 6) showed t value of more than 2 h. The compound reached the maximal degradation of only 45 1/2 46 47 5% of the initial concentration. Thus, AVN211 was quite stable in the presence of s9 human 48 49 liver fraction. 50 51 52 53 54 55 56 57 58 59 60 16 ACS Paragon Plus Environment Page 17 of 55 Molecular Pharmaceutics

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Figure 6. Dynamics of AVN211 concentration during incubation with human s9 fraction 17 18 19 20 21 Analysis of AVN211 (500 ng/mL) stability in the human plasma showed only 16% 22 23 reduction in the original compound concentration during the first 30 min of the incubation. 24 25 AVN211 binding to human plasma proteins was assessed using the Pierce rapid equilibrium 26 27 device (RED) system. According to the obtained data, 87.8% of AVN211 was bound to human 28 29 plasma proteins. The same binding was observed in rat plasma (PPB=88.2%). Permeability of 30 31 32 AVN211 was estimated in Caco2 cells ( for details, see SM ). It was demonstrated that AVN 33 6 34 211 has a relatively high permeability in Caco2 assay conditions (Papp(10 sm/s) = 41.4; 35 36 LC/MS/MS). 37 38 Pglycoprotein is an ATPdependent efflux transporter that affects the absorption, 39 40 41 distribution and clearance of a variety of molecules. The modulation of Pgp function through 42 43 inhibition or stimulation can lead to drugdrug interactions and alter pharmacokinetics, efficacy, 44 45 safety and target organ specificity. Thus, the in vitro Pgp assays are used in drug development 46 47 to identify the potential interaction of drug candidates with Pgp. Compounds could be classified 48 49 as Pgp substrates, inhibitors, inducers or have no effect on Pgp function. It is well known that a 50 51 52 single assay format is unable to provide a complete and definite information about Pgprelated 53 54 effects of compounds. Therefore, we used two different approaches ( vide supra ). Thus, during 55 56 the first assay AVN211 stimulated PgpATPase activity in a concentrationdependent manner 57 58 at concentrations above 10 M, while at 45 M the compound showed the same effect as 59 60 17 ACS Paragon Plus Environment Molecular Pharmaceutics Page 18 of 55

1 2 verapamil, a Pgpinhibitor used as a positive control (see SI). Stimulation effect was only seen in 3 4 the concentrations exceeding 10 M, which were much higher than therapeutic concentrations. 5 6 Therefore, it can be concluded that AVN211 would not affect PgpATPase activity in 7 8 therapeutic conditions. 9 10 11 AVN211 showed good passive permeability in MultiScreen Caco2 assay system. There 12 13 was no difference between transport rates in apical to basolateral and basolateral to apical 14 15 directions. The Papp BA/AB ratio was about 1.0 at the whole concentration range. This finding 16 17 suggests that AVN211 does not undergo efflux and is not transported by Pgp. However, there 18 19 20 is an opinion that the monolayer efflux assays tend to be insensitive to highly permeable

21 6 58 22 compounds (Papp AB > 30×10 cm/s), thereby yielding false negative results. The compound 23 24 may not be effluxed by Pgp, but could affect the transport of other Pgp substrates due to the 25 26 complexity of the possible molecule–protein interactions. Thus, we used Rhodamine123 27 28 (Rh123), a wellcharacterized and easily detectable Pgp substrate to estimate this effect under 29 30 31 the analogue conditions. According to the results obtained in both tests: AVN211 was negative 32 33 in Caco2 monolayer efflux assay, but stimulated Pgp ATPase activity at concentrations above 34 35 10 M. In addition, a weak decrease in Rhodamine123 efflux was observed in the presence of 36 37 20 M AVN211. Thus, AVN211 can interact with Pgp at micromolar concentration. It is 38 39 40 unlikely to be transported by Pgp or its efflux was not detected due to its very high 41 42 permeability. 43 44 The effect of AVN211 on CYP450 family was investigated following the procedure 45 46 described above. The obtained results are presented in Table 2 and Figure 7. As clearly shown 47 48 below, AVN211 interacts with CYP2B6, CYP2C9 and CYP2C19. The PK profile of AVN211 49 50 51 and its metabolites have been estimated in humans at different oral doses and schedule. Thus, we 52 53 revealed several key metabolically active points, including the sulphur atom ( Soxidation), 54 55 methyl groups attached to pyrimidine moiety (monooxygenation) and phenyl ring ( pposition, 56 57 58 59 60 18 ACS Paragon Plus Environment Page 19 of 55 Molecular Pharmaceutics

1 2 monooxygenation). The details of this study will be discussed properly within the follow up 3 4 paper. 5 6 7 8 Table 2. Results of the CYP450 inhibition assays 9 10 11 CYP isoform Max% inh SD IC 50 , pM Hillslope Conclusion 12 13 1A2 not active 14 15 2B6 95.5 1.6 0.76 0.93 active 16 17 2C9 43.7 4.7 2.68 2.515 active 18 19 20 2C19 36.26 0.83 8.08 1.742 active 21 22 2D6 not active 23 24 2E1 not active 25 26 27 3A4 not active 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 a b 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 19 ACS Paragon Plus Environment Molecular Pharmaceutics Page 20 of 55

1 2 c 3 4 Figure 7. CYP inhibition curves for AVN211: IC 50 : 0.75, 2.68 and 8.08 M (partial inhibition), 5 6 respectively 7 8 9 10 11 In vivo PK profile 12 13 Figure 8 shows concentrations of AVN211 (ng/mL) in rat blood plasma after a single 14 15 intravenous injection (i.v.) in the dose of 5 mg/kg and 10 mg/kg (p.o.). Pharmacokinetic 16 17 parameters are listed in Table 3. 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 a 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 b 53 54 55 56 57 58 59 60 20 ACS Paragon Plus Environment Page 21 of 55 Molecular Pharmaceutics

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 c 16 17 Figure 8. Pharmacokinetics curve for AVN211: a) 5 mg/kg, i.v.; b) 10 mg/kg, p.o.; c) 18 19 pharmacokinetics curve, AVN211, brain rats (10 mg/kg, p.o.) 20 21 22 23 24 Table 3. Main pharmacokinetic parameters of AVN211 in rats 25 26 Tmax Cmax AUQ 0→t AUC 0→∞ T1/2 Kel Cl 27 28 min ng/m L ng ×min/m L ng ×min/m L min min 1 mL/min/kg 29 30 31 5 (i.v.) 959 .7 61766 .4 64921 .6 50 .86 0.0136 32 33 60 (p.o.) 204.0 29995.8 44627.8 131.15 0.0053 34 35 60 (p.o.) 5589. 5 36 49.9 7793 153.1 1283.2 37 rats, brain (AUQ ) 38 0→240 39 40 41 42 43 44 Figure 8c shows the concentrations of AVN211 (ng/mL) in rat brain after a single 45 46 administration per os at the dose of 10 mg/kg. The obtained PKplots are presented in SM. Oral 47 48 49 bioavailability of AVN211 was 24% in WAQ strain rats. Despite the average solubility (22 50 51 g/mL), plasma concentrations of AVN211 showed a linear dosedependence up to 20 mg/kg 52 53 dose given per os . In the 20–100 mg/kg (p.o.) range a plateau was observed. The PK features of 54 55 AVN211 were also determined in SHK mice upon intraperitoneal administration (i.p.) in doses 56 57 58 of 1, 0.2 and 0.05 mg/kg (Table 4). The corresponding PK plots are presented in SM. 59 60 21 ACS Paragon Plus Environment Molecular Pharmaceutics Page 22 of 55

1 2 3 4 Table 4. PK parameters for AVN211 upon i.p. administration 5 6 PK Parameters 7 8 9 Dose Tmax Cmax AUC 0→t AUC 0→∞ t1/2 Kel Vd Cl 10 11 (mg/kg) min ng/ml ng×min/ml ng×min/ml min min 1 mL/kg mL/min/kg 12 13 1 5.0 158 3632 3726 21 0.0337 7962 268 14 15 16 0.2 5 67 1205 1259 23 0.0295 5381 159 17 18 0.05 5 13.70 197.18 230.59 9.88 0.0701 3091.39 216.83 19 20 21 22 One of the most crucial features of CNSactive compounds is the permeability across the 23 24 25 blood brain barrier (BBB). For instance, among the reported bisaryl sulfonamides the following 26 59 27 B/P ratio was reported: 0.1 (SB271046), 0.9 (indolebased analogue by Suven Life 28 29 Sciences),60 0.19 (SB357134),61 0.24 (Ro 660074),62 0.01 (Ro046790 and Ro630563) 59 , 1.0 30 31 (4,5dihydro1Hpyrazolecontaining analogue).63 For other chemotypes including indoles, their 32 33 isosteric modifications and nonsulfonyl compounds, B/P ratio is within the range of 0.5–23.64–69 34 35 36 However, other PK parameters significantly influence the therapeutic efficiency of such 37 38 molecules, e.g. t 1/2 (brain), Cl (brain), bioavailability, etc. Certainly, all the CNSrelated PK 39 40 features should be considered cumulatively to get the success. Based on the parameters above, 41 42 B/P ratio for AVN211 was calculated using different methods as following: 43 44 45 BrAUC 0→240/PlAUC 0→240 =18.6; BrAUC 0→∞ /PlAUC 0→∞ =17.5; BrC max /PlC max =24.4. AVN211 46 47 was found in the rat brain after oral administration providing very good B/P ratio close to 25% 48 49 based on C max values. CSF/plasma ratio was also determined in rats. AVN211 was administered 50 51 per os in the dose of 20 mg/kg to the 6 male Wistar rats. After 60 minutes animals were 52 53 54 anaesthetized and samples of CSF, brain and plasma were taken. CSF samples were carefully 55 56 monitored for the absence of blood contaminations. AVN211 was extracted from CSF, brain 57 58 and plasma with acetonitrile and the concentrations were detected using API2000 LCMS/MS. 59 60 22 ACS Paragon Plus Environment Page 23 of 55 Molecular Pharmaceutics

1 2 The obtained results are depicted in Figure 9. Summarizing, an average concentration of AVN 3 4 211 in rat CSF after oral administration in the dose of 20 mg/kg is 44.2 ng/mL, CSF/plasma ratio 5 6 is 21.66%, B/P ratio is 19.5%. 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Figure 9. Relative concentrations of AVN211 in CSF and plasma 24 25 26 27 28 Pharmacokinetics in Monkeys 29 30 AVN211 (2 mg/kg, 9.4, 11.0 and 11.6 mg per animal) diluted in saline was injected into 31 32 the right elbow vein in 3 mL or administered per os (10 mg/kg, 44 and 45 mg per animal) in 3,5 33 34 35 mL. No signs of an anxiety, vomiting or unusual behavior were observed. Blood was taken from 36 37 the left elbow vein. The obtained results are summarized in Tables 5 and 6. The corresponding 38 39 PK plots are presented in SM. Oral bioavailability of AVN211 was calculated as a ratio of 40 41 AUC 0→t ,(p.o.)/AUC 0→t ,(i.v.) and equals to 10% (F abs ,%). 42 43

44 45 46 Table 5. Main PK parameters of AVN211 upon i.v. administration in monkeys 47 48 Route of PK parameters 49 Dose 50 * Tmax Cmax C0 AUC 0→t AUC 0→∞ T1/2 Kel 51 Monkey administra 52 (mg/kg) 53 tion min ng/m L ng/m L ng×min/m L ng×min/m L min min 1 54 55 56 1 i.v. 2 1 5732 7878.6 57286.3 59571.9 29.34 0.0236 57 58 59 60 23 ACS Paragon Plus Environment Molecular Pharmaceutics Page 24 of 55

1 2 2 3962 5404.9 44220.4 49402.9 43.28 0.016 3 4 5 3 4312 5888.3 57221.7 61673.8 35.47 0.0195 6 7 8 n 3 3 3 3 3 3 3 9 10 11 M 1 4669 6391 52909 56883 36.03 0.0197 12 13 SD 937 1311 7525 6563 6.99 0.0038 14 15 16 SEM 541 757 4345 3789 4.03 0.0022 17 18 19 CV, % 20.1 20.5 14.2 11.54 19.39 19.31 20 21

22 * Kel elimination rate constant 23 24 25 26 Table 6. Main PK parameters of AVN211 upon p.o. administration in monkeys 27 28 PK parameters 29 Route of 30 Dose T C AUC AUC T K 31 Monkey administ max max 0→t 0→∞ 1/2 el 32 (mg/kg) 33 1 34 ration min ng/m L ng×min/m L ng×min/m L min min 35 36 37 4 15 143.0 12233 14383 212.98 0.0033 38 p.o. 10 39 5 15 377.0 40245 41793 107.29 0.0065 40 41 n 2 2 2 2 2 2 42 43 M 15.00 260 26239 28088 160.14 0.0049 44 45 46 SD 165 19808 19381 74.73 0.0023 47 48 SEM 117 14006 13705 52.84 0.0016 49 50 CV, % 63.6 75.5 69.0 46.67 46.67 51 52 53 54 55 Biodistribution of the compound was examined in rats. The resulting PK curve and the 56 57 main PK parameters are shown in Table 7 and Figure 10, respectively. Homogenates of liver, 58 59 60 24 ACS Paragon Plus Environment Page 25 of 55 Molecular Pharmaceutics

1 2 heart, lungs, spleen, kidneys, brain, small and large intestines and pancreas were collected at 480 3 4 min time point and analyzed in the betacounter. The obtained results are presented in Table 8. 5 6 As clearly shown in Table 8, after oral administration at the dose of 10 mg/kg AVN211 was 7 8 found in all the studied organs. A preferential accumulation was found in large and small 9 10 11 intestines and liver. 12 13 14 15 Table 7. PK parameters of [H 3]AVN211 observed in WAQ strain rats (p.o. administration) 16 17 Parameter Units Estimate 18 19 20 Kel 1/min 0.0006 21 22 t1/2 min 1142 23 24 Tmax min 120 25 26 C ng/mL 38.5 27 max 28 29 AUC (0→480) min×ng/mL 15610 30 31 AUC (0→∞) min×ng/mL 65735 32 33 Vd/F L/kg 251 34 35 Cl/F (mL/min)/kg 152 36 37 38 MRT min 1678 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 25 ACS Paragon Plus Environment Molecular Pharmaceutics Page 26 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Figure 10 . PK curve of AVN211 in plasma after p.o. administration in rats 21 22 23 24 Table 8. AVN211 concentrations in various organs and tissues 25 26 Large Small 27 28 480 min Brain Pancreas Liver Lungs Heart Spleen Kidneys Blood 29 intestine intestine 30 31 13.1 31.9 91.3 66.4 57.0 33.7 20.6 13.1 41.6 32.4 32 33 Concentration 34 11.4 37.2 85.2 83.7 76.9 48.2 21.0 18.0 24.5 29.6 35 36 ng/g 37 16.3 39.4 115.7 65.7 64.5 30.8 19.0 18.6 34.5 29.3 38 39 40 Mean 13.6 36.2 97.4 71.9 66.1 37.6 20.2 16.6 33.5 30.4 41 42 43 STD 2.5 3.9 16.1 10.2 10.1 9.3 1.1 3.0 8.6 1.7 44 45 46 In vivo efficacy 47 48 AVN211 was evaluated in different in vivo models directly associated with cognitive 49 50 and memory impairments, including passive avoidance test 70,71 and Morris water maze test 51 52 72,73 53 (MWM). The obtained results are briefly summarized in Table 9, all the related procedures, 54 55 experimental protocols and plots are presented in SM. As clearly shown in Table 9, AVN211 56 57 can be reasonably regarded as novel promising drug candidate for the treatment of AD as well as 58 59 60 26 ACS Paragon Plus Environment Page 27 of 55 Molecular Pharmaceutics

1 2 schizophrenia. In many cases, the observed effects were considerably higher than that 3 4 demonstrated for PRX07034 and SB742457 or comparative to Donepezil, Memantine, 5 6 Buspirone, Lorazepam, Fenobam, Rufinamide, Piracetam, Haloperidol, and Apomorphine . 7 8 9 10 * 11 Table 9. In vivo efficacy of AVN211 12 13 Description Study Subject Brief summary 14 15 AVN211 (0.05 1 mg/kg, i.p. or 0.2 16 17 mg/kg, p.o.) was more effective than 18 19 20 Memantine (5 mg/kg, i.p.) or Tacrine (10 21 Passive Avoidance Test : 22 mg/kg, i.p.), while PRX07034 (10 mg/kg, 23 24 i.p.) was not active in the test. SB742457 25 scopolamine was BALB/c mice 26 (1 mg/kg, i.p.) was slightly active affecting 27 administered i.p. at the dose (24–25 g) 28 29 time spent in the light chamber of the 30 of 0.3 mg/kg, 30 min prior 31 passive avoidance apparatus. The most 32 training 33 pronounced effect of AVN211 was 34 Scopolamine 35 observed at 0.05 mg/kg (i.p.) and 0.2 mg/kg 36 Based Models of 37 (p.o.) doses 38 39 Amnesia 40 10wk old Given either per os or i.p. AVN211 (0.25 41 Passive Avoidance Test : 42 male and mg/kg, 30 min before the test) significantly 43 scopolamine (2.5 mg/kg, 44 female Wistar restored memory in scopolaminetreated 45 i.p.) 20 min before the 46 rats rats. The control group animals were 47 test 48 49 (160–185 g) injected with physiological solution 50 51 Morris Water Maze Test: Scopolamine was administered 30 min prior 52 53 (a) scopolamine (1.5 BALB/c mice training, Tacrine and Donepezil – 60 min 54 55 mg/kg, i.p.) or ( b) (24–25 g) before training, while AVN211 5 min 56 57 scopolamine (1.5 mg/kg, prior training. The control group animals 58 59 60 27 ACS Paragon Plus Environment Molecular Pharmaceutics Page 28 of 55

1 2 i.p.) combined with Tacrine were injected with physiological solution. 3 4 (3 mg/kg, i.p.), Donepezil (3 AVN211 showed antiamnestic activity 5 6 mg/kg, i.p.) or comparable to that of AChE in hibitor 7 8 AVN211 (0.05, 0.2 or 1 Donepezil (Aricept) 74 9 10 11 mg/kg, i.p.) 12 13 Scopolamine was administered 30 min 14 Morris Water Maze Test: 15 before training, while AVN211 5 min 16 (a) scopolamine (4.5 17 Wistar rats prior training. AVN211 was active 18 19 mg/kg, i.p.) or ( b) 20 (160–185 g, effectively restoring time in target zone 21 scopolamine (4.5 mg/kg, 22 13–14 wk old) almost to a placebo level . The control group 23 i.p.) combined with AVN 24 animals were injec ted with physiological 25 211 (0.2 mg/kg, i.p.) 26 solution 27 28 29 The scopolamineinduced deficit in novel 30 31 object recognition was prevented by 32 33 administration of Memant ine (10 mg/kg, 34 Novel Object Recognition 35 i.p.) 60 min prior the training, AVN211 36 Test : the test was 37 (0.05 and 0.2 mg/kg, i.p.) 5 minutes before 38 39 abolished by scopolamine SHK mice 40 the training, or SB742457 (0.2 mg/kg, i.p.) 41 administered in the dose of 1 (24–30 g) 42 15 min before the training but not PRX 43 mg/kg i.p. 30 minutes before 44 07034 (10 mg/kg, i.p.), 15 minutes before 45 the training. 46 the test. AVN211 showed an antiamnesic 47 48 49 effect exceeding that of SB742457 and 50 51 Memantine 52 53 MK801 Based Passive Avoidance Test : on Independent groups of mice were treated 54 BALB/c mice 55 Models of day of training, mice were additionally with one of the following 56 (24–25 g) 57 Amnesia treated with proamnesic drugs: Tacrine, 10 mg/kg, i.p., 60 min 58 59 60 28 ACS Paragon Plus Environment Page 29 of 55 Molecular Pharmaceutics

1 2 agent MK801 (0.1 mg/kg, before the training, Memantine, 10 mg/kg, 3 4 i.p.) 30 minutes prior the i.p., 60 min prior the training, SB742457, 1 5 6 training. MK801 pre mg/kg, i.p., 15 min before the training, 7 8 treatment significantly PRX07034 , 10 mg/kg, i.p., 30 min before 9 10 11 reduced effect of the training the training, or with AVN211 (0.020.2 12 13 producing socalled mg/kg, i.p.), which was administered 5 min 14 15 anterograde amnesia before the test. The control group animals 16 17 were injected with physiological solution. 18 19 20 AVN211 was more effective compared to 21 22 SB742457, while Memantine, Tacrine and 23 24 PRX07034 were not active in the test 25 26 Morris Water Maze Test : 27 28 29 mice were treated with ( a) 30 31 MK801 (0.2 mg/kg, i.p.) or AVN211 was administered 5 min prior to 32 33 (b) MK801 (0.2 mg/kg, i.p.) the training. The control group animals 34 35 combined with Tacrine (3 demonstrated successful learning, since 36 37 mg/kg, i.p.), Donepezil (1 they spent more time in the quadrant of the 38 39 rd 40 mg/kg, i.p.), Memantine (5 platform location on the 3 day. MK801 at 41 BALB/c mice 42 mg/kg, i.p.), or AVN211 the dose of 0.2 mg/kg disrupted training 43 (24–25 g) 44 (0.05 or 0.2 mg/kg, i.p.). process under the described conditions. At 45 46 MK801 was administered the same time, coadmi nistration of 47 48 49 30 min prior the test. Memantine or AVN211 (0.05 mg/kg) 50 51 Tacrine, Donepezil and significantly improved the learning phase of 52 53 Memantine were spatial navigation 54 55 administered 60 min before 56 57 the training 58 59 60 29 ACS Paragon Plus Environment Molecular Pharmaceutics Page 30 of 55

1 2 Morris Water Maze Test : 3 4 the experiment was 5 6 performed as described male and 7 8 above with only one female Wistar AVN211 (0.2 mg/kg, i.p.) was active in the 9 10 11 modification – MK801 (0.2 rats test effectively restoring time in target zone 12 13 mg/kg, i.p.) was used instead (160–185 g, to a placebo level 14 15 of scopolamine. MK801 10 wk old) 16 17 was injected 30 min before 18 19 20 the training 21 22 Novel Object Recognition 23 24 Test : MK801 was 25 26 administered in the dose of 27 28 29 0.2 mg/kg i.p. 30 minutes 30 31 before the training. The MK 32 AVN211 as well as all the tested reference 33 801induced deficit in novel 34 SHK mice molecules did not reveal an antiamnesic 35 object recognition was not 36 (22–32 g) effect within the test under the applied 37 prevented by co 38 39 conditions 40 administration of Tacrine (3 41 42 mg/kg, i.p.) 60 min prior the 43 44 training or AVN211 (0.051 45 46 mg/kg, i.p.) 5 minutes before 47 48 49 the training 50 51 Elevated Plus Maze : mice Buspirone and Lorazepam were 52 53 Anxiolytic were treated with either BALB/c mice administered at the maximal dose which did 54 55 Activity placebo, or Buspirone (5 (24 g) not produce sedative sideeffects seen in the 56 57 mg/kg, i.p., 30 min prior the test as a decrease in a general exploratory 58 59 60 30 ACS Paragon Plus Environment Page 31 of 55 Molecular Pharmaceutics

1 2 training), or Lorazepam (0.05 activity. Buspirone, Lorazepam, Fenobam, 3 4 mg/kg, i.p., 60 min before the Rufinamide and AVN211 produced a clear 5 6 training), or Fenobam (5 anxiolytic effect in the elevated plus maze 7 8 mg/kg, i.p., 60 min before the test: they significantly increased the number 9 10 training), or Rufinamide (15 11 of visits into the open arms of the maze, 12 mg/kg, i.p., 60 min prior the 13 time spent in the open arms and decreased 14 test) or with AVN211 (0.05, 15 the number of defecations. AVN211, 16 0.2, or 1 mg/kg, i.p., 5 min 17 Lorazepam, Buspirone, Fenobam and 18 19 before the training) 20 Rufinamide did not affect locomotor 21 22 activity, thus their anxiolytic activity was 23 24 not related to a sedative effect. The most 25 26 prominent anxiolytic effect was seen with 27 28 AVN211 injected i.p. at the doses of 0.01– 29 30 31 0.2 mg/kg, Lorazepam injected i.p. at the 32 33 dose of 0.05 mg/kg and Fenobam injected 34 35 i.p. in the dose of 5 mg/kg 36 37 Both lorazepam (0.05 mg/kg, i.p.), 60 38 39 Male 40 minutes before the test, and AVN211 (0.05 41 Sprague 42 Elevated Platform Test mg/kg, i.p.), 5 minutes before the test, 43 Dawley rats 44 decreased the total time of freezing during 45 (250–300 g) 46 the first minutes in the test 47 48 49 Open Field Test : Lorazepam 50 51 (0.05 mg/kg, i.p.) was AVN211 and Lorazepam increased the 52 SHK mice 53 administered 30 minutes prior number of entries and the total time spent in 54 (20–25 g) 55 the testing and AVN211 the central zone 56 57 (0.05 and 0.2 mg/kg, i.p.) was 58 59 60 31 ACS Paragon Plus Environment Molecular Pharmaceutics Page 32 of 55

1 2 administered 5 minutes prior 3 4 to the testing 5 6 Prepulse Inhibition of 7 8 Acoustic Startle : 9 10 11 apomorphine was dissolved 12 13 in 0.1% ascorbic acid in 14 The obtained results demonstrated about 15 sterilized water. Haloperidol 16 53% prepulse inhibition in the placebo 17 was dissolved in Tween 80. 18 group. Propsychotic agent apomorphine (3 19 AVN211 was dissolved in 20 21 mg/kg) reduced this variable showing 22 sterile water with 0.1% 23 deterioration of ability for filtration of 24 Tween 80. The appropriate 25 sensory signals. AVN211 (0.05 and 0. 2 26 vehicle solution was sterile 27 mg/kg) and Haloperidol (1 mg/kg) 28 water with 0.1% Tween 80. Naive male 29 Antipsychotic prevented disruptive effect of apomorphine 30 31 Haloperidol was SHK (24–30 32 Activity on the startle prepulse inhibition. AVN 33 administered 60 min prior to g), 34 211 at doses of 0.05 and 0.2 mg/kg revealed 35 the testing (volume of 36 antipsychoticlike properties. Prepulse 37 injection was 10 ml/kg). 38 inhibition (% to pulse alone) for 39 Apomorphine was 40 41 apomorphine+placebo was 29 %, for 42 administered s.c. 20 min 43 Haloperidol+apomorphine – 55%, for 44 before the testing (volume of 45 AVN211+apomorphine – 46% (0.05 46 injection was 1 ml/kg.). 47 mg/kg) and 42% (0.2 mg/kg) 48 AVN211 was administered 49 50 51 i.p. 5 min before the testing 52 53 (volume of injection was 10 54 55 ml/kg) 56 57 Nootropic Cognitive Deficit in SHK Mice The test revealed that AVN211 and 58 59 60 32 ACS Paragon Plus Environment Page 33 of 55 Molecular Pharmaceutics

1 2 Activity Exploratory Low : on days (22–32 g) piracetam produce a marked decrease in 3 4 47, the mice o.d. received number of visits during the 1 st patrolling 5 6 i.p. injection of placebo, demonstrating a clear cognitionenhancing 7 8 piracetam (400 mg/kg, i.p.) effect . Number of arm visits: placebo (EH 9 10 11 or AVN211 (0.1 mg/kg, mice) – av. 5.5, placebo (EL mice) – av. 12 13 i.p.). The second crossmaze 5.7, Piracetam (400 mg/kg, EL mice) – av. 14 15 test was performed on Day 7 5.1, AVN211 (0.1 mg/kg, EL mice) – av. 16 17 an hour after the last 5.6. EH (exploratory high) or EL 18 19 20 injection (exploratory low) animal subgroups 21 22 * ref. compounds were evaluated in doses published previously in scientific literature. In some cases, dose was 23 24 dependent on exp. conditions and trial specificity. AVN211, SB742457 and PRX07034 were evaluated in 25 different doses ( titrated ). 26 27 28 29 30 Safety pharmacology and sideeffects 31 32 hERG channel inhibition 33 34 Drugs belonging to different classes have been shown to be associated with QT 35 36 prolongation leading in some cases to serious ventricular arrhythmias. The most common 37 38 39 mechanism for these adverse events is the inhibition of one or more cardiac potassium channels, 40 41 in particular hERG channel. Potassium currents controlled by this channel are important for re 42 43 polarization of cardiac myocytes and unintended blockage of these channels by some drugs 44 45 causes prolonged myocardial QT interval with potentially lethal consequences. AVN211 was 46 47 tested for its ability to block hERG channel using HEK cells stably expressing the hERG 48 49 50 potassium channel ( for details, see SM ). Thus, AVN211 exhibited a potency to block hERG 51 52 channel two orders of magnitude lower than that of blocking serotonininduced cell response 53 54 mediated through 5HT 6R (Figure 4). In the patch clamp experiments (ion current inhibition) the 55 56 compound showed an IC 50 value of 18.0 M. Taking into account such a low potency, we expect 57 58 59 that AVN211 will not affect QT interval in vivo . 60 33 ACS Paragon Plus Environment Molecular Pharmaceutics Page 34 of 55

1 2 3 4 Assessment of LD 50 5 6 AVN211 was injected in mice i.p. 10 ml/kg in different doses ranged from 500 to 20000 7 8 mg/kg (Figure 11). As shown in Figure 11, AVN211 should be assigned to nontoxic organic 9 10 11 compounds and demonstrates the LD 50 value of 13684±2370 mg/kg. 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Figure 11 . Mortality among SHK mice upon AVN211 treatment in different doses 27 28 29 30 31 AVN211 was injected i.p. daily during 5 days in 10, 30, 100, 200 and 400 mg/kg doses 32 33 (only 200 and 400 mg/kg groups data presented). Each experimental group consisted of 8 mice. 34 35 Animal weight and mortality were measured. Injection of AVN211 did not cause statistically 36 37 significant reduction in body weight in comparison to the control group (Figure 12). The data 38 39 40 suggests that MTD of AVN211 is not less than 400 mg/kg. 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 Figure 12 . Determination of MTD for AVN211 57 58 59 60 34 ACS Paragon Plus Environment Page 35 of 55 Molecular Pharmaceutics

1 2 14Days toxicity study was performed in 20 male adult SHK mice (2025 g). During the 3 4 trial no statistically significant change in the enzymatic activity of alanine aminotransferase 5 6 (ALT) and aspartate aminotransferase (AST) as well as in the level of glucose and urea in 7 8 plasma was observed after 14days treatment with AVN211 (i.p., o.d. 20 mg/kg), however a 9 10 11 trend in a body weight decrease was detected and might be attributed to a mechanismbased 12 13 action as described above. 14 15 Morphology of internal organs was examined visually and microscopically (paraffin 16 17 slices 4–5 m, hematoxylineosin staining). Mass coefficients of internal organs were 18 19 20 determined by weighing respective organs: thymus, heart, lungs, kidneys, spleen, brain and 21 22 cerebellum. Necropsy of the mice was performed after 14 days of AVN211 i.p. administration 23 24 in the dose of 20 mg/kg. Five males and five females were studied. Animals were sacrificed by 25 26 decapitation. Study was performed within an hour after decapitation. Samples of heart, liver, 27 28 29 kidneys, brain and cerebellum were collected for histological analysis. Clinical observation 30 31 revealed no signs of toxicity: no hair loss, glossy and shiny hair, skin is elastic, subepidermal 32 33 cellular tissue moderately expressed (Figure 13, Table 10). 34 35 Appearance and condition of internal organs in control and AVN211treated mice were 36 37 similar (Table 10): lungs – lobes correctly shaped, pale pink colored, foamy liquid is seen on the 38 39 40 slice; liver – regular size, trabecules (no enlargement), dark brown, plethoric, intralobular 41 42 capillary network was developed, hepatocytes and Kupfer cells were regularly shaped; spleen – 43 44 elongated, purple, moderately solid; kidneys – light brown, fibrous capsule was shiny and thin, 45 46 easily removable, capillaries of glomerules of cortical substance were plethoric, no alterations in 47 48 49 epithelium of proximal and distal tubules, tubular glimpses were open; brain regular size, brain 50 51 tunics were uniformly colored without visible hemorrhages, cortex and subcortical formations 52 53 were of normal shape and appearance, ventricles of brain were of a normal size (not enlarged); 54 55 cerebellum – regular size, no alterations in composition, no visible hemorrhages, all structural 56 57 elements were without alterations; heart – was seen on the slices as longitudinal and transverse 58 59 60 35 ACS Paragon Plus Environment Molecular Pharmaceutics Page 36 of 55

1 2 muscle fibers, coronary vessels of a regular appearance; Purkinje cells were regularly shaped, 3 4 blood vessels were not enlarged, study of mass coefficient of internal organs did not reveal 5 6 statistically significant difference between control and AVN211treated animals. Organs of both 7 8 groups (control and AVN211 20 mg/kg i.p.) were observed using microscope and located 9 10 11 regularly. Based on the obtained results, it can be concluded that AVN211 did not show any 12 13 signs of toxicity during the study performed. Similar results were obtained in rats (see SM). 14 15 16 17 18 19 20 21 22 23 24 a b c 25 26 27 28 29 30 31 32 33 34 35 36 37 d e 38 39 Figure 13. AVN211 treated mice: ( a) cardiac wall, endocardium, no alterations observed 40 41 (hematoxylin, eosin 40x10); ( b) liver, no alterations, hematoxylin and eosin, 20x10; ( c) kidneys, 42 43 44 no alterations, hematoxylin and eosin, 20x10; (d) hippocampal formation, pyramidal neurons 45 46 and granular cells without alterations (hematoxylin and eosin, 40x10); ( e) cerebellum, structural 47 48 elements and Purkinje cells without alterations (hematoxylin, eosin, 20x10). 49 50 51 52 53 54 55 Table 10 . Mass coefficients of internal organs after 14 days of the study 56 57 Group Organ mass coefficient (g/kg body weight) 58 59 60 36 ACS Paragon Plus Environment Page 37 of 55 Molecular Pharmaceutics

1 2 Thymus Lungs Heart Liver Spleen Kidneys Brain Cerebellum 3 4 Control 2.8±0.2 8.9±0.4 5.2±0.1 59.4±3.6 6.3±1.3 15.4±0.6 9.7±0.3 3.8±0.1 5 6 AVN211 7 3.0±0.3 9.5±0.6 5.5±0.3 52.0±1.7 5.9±1.0 15.4±0.9 10.5±0.5 3.6±0.3 8 9 20 mg/kg 10 11 12 13 Morphology of internal organs, body weight dynamics, general behavior (open field test 14 15 and grid test), plasma biochemistry as well as hematology were evaluated by analogy to 16 17 aforementioned approaches during the 29 and 30days trial. During the first study, the 18 19 20 compound was tested in doses of 1, 3 and 10 mg/kg and no toxicity was revealed. This 21 22 observation was supported by lack of a body weight change in all animal groups. General 23 24 behavior tests did not reveal any signs of toxicity. There were no changes related to AVN211 25 26 administration in microstructure of studied organs, while some changes in mass coefficient 27 28 29 (liver, lungs, testicles) were not related to AVN211 administration. 30 31 In the second trial, AVN211 administered in the dose of 3 mg/kg orally was not toxic as 32 33 well. No changes in the body weight, parameters of plasma biochemistry markers, macro and 34 35 microscopic observations were detected. Observed alterations in the lung parenchyma could be a 36 37 consequence of distress syndrome. It should be noted that the estimation of 30days toxicity of 38 39 40 AVN211 (10 mL/kg, p.o.) in monkeys revealed no toxicity except minor alterations in blood 41 42 plasma biochemistry that could be explained by ketamine tranquilization. 43 44 The signs of adverse effects were revealed during the longterm 180days toxicological 45 46 study. Thus, statistically significant body weight reduction was observed in male and female rats 47 48 49 in 1 mg/kg group. This can be explained either by moderate toxicity caused by AVN211 50 51 administration or by AVN211 action as a 5HT 6R antagonist. However, no dose dependent 52 53 weight reduction was found in AVN211treated female rats suggesting rather 5HT 6Rrelated 54 55 than toxicological explanation of body weight reduction in female rats. With respect to plasma 56 57 biochemistry, the increase in bilirubin level was observed at the highest dose in males which can 58 59 60 37 ACS Paragon Plus Environment Molecular Pharmaceutics Page 38 of 55

1 2 be associated with liver damage. However, such outcome was not observed in 3 4 pathomorphological experiments. Macroscopic study of the organs did not reveal any difference 5 6 between placebo and AVN211treated animals. Thus, as a result of conducted 7 8 pathomorphological investigation, no toxic action of AVN211 administered orally during 180 9 10 11 days was observed (. The noted special features and changes in the histological structure of the 12 13 internal organs of experimental animals did not bear the signs of pathology, they did not have 14 15 noticeable dose dependence and, often, they were manifested by similar means in animals of 16 17 placebo group as well. 18 19 20 AVN211 in concentrations up to 200 M was not mutagenic according to the results 21 22 obtained in Ames test. Results of two independent experiments support this conclusion. No 23 24 significant chromosomal aberrations were found in AVN211treated mice, suggesting absence 25 26 of AVN211induced mutagenic activity in vivo . According to FLIGRLOinduced paw edema 27 28 29 test, AVN211 did not reveal any allergenic properties in the doses of 1, 3 and 10 mg/kg. 30 31 We also demonstrated that scopolamine, in gastrointestinal transit test performed in mice, 32 33 produced marked increase in time of first defecation after charcoal administration showing 34 35 decrease in GI transit velocity, whereas AVN211 did not produce any significant effect on the 36 37 transit. 38 39 75 40 Catalepsy was evaluated in rats by method adopted from studies by Bourson and 41 42 Bristow. 76 It was revealed that AVN211 did not produce cataleptic effect in either of the tested 43 44 doses (0.2 and 1 mg/kg) while haloperidol caused a pronounced cataleptic effect. 45 46 Considering the aforementioned results, AVN211 can be reasonably regarded as 47 48 49 generally nontoxic organic substance with promising ADME features and activity for further 50 51 clinical evaluation against AD. 52 53 54 55 DISCUSSION 56 57 58 59 60 38 ACS Paragon Plus Environment Page 39 of 55 Molecular Pharmaceutics

1 2 The results above clearly show that AVN211 leads to considerable improvements in 3 4 cognition and learning investigated in different behavioral models of AD. In vivo efficacy of 5 6 AVN211 was compared to that of Memantine (a fixeddose combination of memantine 7 8 hydrochloride and donepezil hydrochloride was launched in 2015, U.S., by Actavis against AD 9 10 11 type dementia), Tacrine (launched in 1993 by Shionogi), SB742457 (Phase II, GSK) and PRX 12 13 07034 (Phase II, Epix Pharmaceuticals). During all the behavioral tests performed, AVN211 14 15 significantly restored both scopolamine and MK801induced cognitive dysfunction. In some 16 17 cases, the cognition restorative effect of the lead molecule was higher as compared with that of 18 19 20 the control compounds. Additionally, in an elevated plusmaze model, elevated platform and 21 22 open field tests AVN211 has shown a similar or better anxiolytic efficacy than fenobam, 23 24 rufinamide, lorazepam and buspirone. Additionally, AVN211 has a clear IP and easily scalable 25 26 chemistry, allowing us to consider the molecule as a new promising drug candidate for the 27 28 treatment of neurodegenerative and psychiatric disorders. 29 30 31 Among the current trends matured in AD drug treatment the reprofiling of selective 5 32 33 HT 6R ligands, especially antagonists, can be highlighted. Thus, Lu AE58054 was evaluated in 34 35 Phase II clinical trial in Lundbeck against schizophrenia. However, in 2010, in spite of some 36 37 initial success,77 the company discontinued the development of the compound for the treatment 38 39 40 of cognitive impairment associated with schizophrenia (CIAS) based on efficacy data which did 41 78 42 not support further development for this indication. Intriguingly, Lundbeck has recently 43 44 initiated the extensive clinical trials of Lu AE58054 as adjuvant therapy against AD, including 45 46 four Phase III,79,80–82 since Phase II evaluation was successfully completed in 2012.83–85 In 2013, 47 48 the compound was licensed by Lundbeck to Otsuka Pharmaceutical for codevelopment and co 49 50 51 commercialization in Canada, the U.S., certain East Asian countries, including Japan, major 52 53 European countries and Nordic countries for the treatment of ADtype dementia. 54 55 SB742457, developed by GSK, was initially focused against schizophrenia, depression, 56 57 anxiety and AD.85–90 The company successfully completed preclinical evaluation of the 58 59 60 39 ACS Paragon Plus Environment Molecular Pharmaceutics Page 40 of 55

1 2 compound mainly focusing on common cognitive disorders then made the decision to advance 3 4 this molecule in clinics exclusively against AD. As a result, Phase II clinical trial was completed 5 6 demonstrating that SB742457 was generally safe and well tolerated and may be efficacious in 7 8 AD.91 In last quarter of 2014, Roivant Neurosciences has entered into an agreement with GSK 9 10 11 for the acquisition of SB742457. GSK conducted 13 clinical studies of an excellent safety and a 12 13 some involving over 1,250 healthy subjects and 357 patients with AD. The results of these 14 15 studies provide some evidence that SB742457 holds promise for patients with mild to moderate 16 17 AD, however, no relevant proofs were obtained following this indication.92 18 19 20 Roivant intends to meet with the FDA in the first half of 2015 to confirm the regulatory 21 22 requirements for the continued development of the product for adjuvant therapy of patients with 23 24 mild to moderate AD. Phase III clinical evaluation has recently been launched for RVT101 (re 25 26 named SB742457) against AD.93 The company may also explore the clinical development of 27 28 SB742457 for other neurological disorders for which there are limited or no therapeutic options. 29 30 31 Avineuro Pharmaceuticals, Inc. has conducted Phase I and Phase IIa clinical trials with 32 33 AVN211 as adjuvant therapy in patients with schizophrenia in a condition of partial remission 34 35 receiving stable antipsychotic therapy. The results of this a multicenter doubleblind randomized 36 37 placebocontrolled study supported good safety and tolerability profiles of AVN211, as well as 38 39 40 gave an encouraging perspective in this adjuvant therapy. We plan to continue clinical trials 41 94,95 42 AVN211 as adjuvant therapy in patients with schizophrenia in partial remission. 43 44 Summarizing the results above, selective 5HT 6R antagonists have recently emerged as a 45 46 prominent class of smallmolecule compounds with an enormous therapeutic potential against 47 48 AD. The comprehensive preclinical evaluation presented here allows us to make a decision 49 50 51 towards the initiation of a separate clinical program against AD, since AVN211 showed high 52 53 efficiency and low toxicity in a range of in vivo animal tests specific for this neuropathology. 54 55 Clinical advances of Lu AE58054 and SB742457 in the adjuvant therapy of AD obtained in 56 57 recent years firmly confirm the validity of the approach.95 However, despite a range of great 58 59 60 40 ACS Paragon Plus Environment Page 41 of 55 Molecular Pharmaceutics

1 2 achievements, several preclinical studies have indicated rather controversial results obtained for 3 96 4 the reported 5HT 6R antagonists. For instance, the beneficial effects of 5HT 6R antagonists 5 6 disclosed in early animal studies during the MWM test could not be replicated.9799 Moreover, 7 8 SB271046 demonstrated a relatively poor or not detectable efficiency at all towards the 9 10 100 11 scopolamineinduced learning deficit in the MWM test and contextual fear conditioning 12 98 13 (CFC) paradigms. The compound also did not show clozapinelike effects in models for 14 15 positive and negative symptoms of schizophrenia.101 In addition, during a typical object 16 17 recognition test Ro4368554 had no impact on the delayinduced recognition deficit. 102 SB 18 19 20 258585 (3–30 mg/kg, i.p.) and Ro4368554 (1–10 mg/kg, i.p.) have recently been tested in 21 22 different models for schizophrenia, including apomorphineinduced deficit in PPI of acoustic 23 24 startle and amphetamineevoked locomotor activity in rats. In addition, the therapeutic effects of 25 26 both compounds on learning and memory deficits in rats produced by natural forgetting and by 27 28 MK801 or scopolamine, in the contextual fear conditioning, MWM and spontaneous alternation 29 30 96 31 paradigms has assessed. As a results, it was revealed that Ro4368554 increased activity at 1 32 33 mg/kg but not at higher doses, whereas SB258585 had no effect on the spontaneous locomotor 34 35 activity. Ro4368554 and SB258585 enhanced memory, as evidenced by an increase in the 36 37 exploration of the new object versus the old object. Ro046790 and SB271046 prevented 38 39 103,104 40 scopolamineinduced amnesia in the PA test. However, in a model of cholinergic 41 42 dysfunction, that is, scopolamineinduced memory impairment (ADrelated model), Ro4368554 43 44 and SB258585 were unable to prevent the impairment induced by scopolamine in CFC and 45 46 passive avoidance tests, as it was demonstrated previously for Ro 046790 and SB271046. 102 In 47 48 MWM model, SB258585 showed a beneficial effect on memory in contrast to Ro4368554. 96,105 49 50 51 Neither Ro4368554 or SB258585 reversed the effect of amphetamine in amphetamineinduced 52 53 hyperlocomotion in the open field and apomorphineinduced impairment of PPI (two models for 54 55 psychoticlike symptoms).96 However, SB258585 significantly enhanced amphetamineinduced 56 57 hyperlocomotion at the highest tested dose of 30 mg/kg, while Ro4368554 was able to reverse 58 59 60 41 ACS Paragon Plus Environment Molecular Pharmaceutics Page 42 of 55

1 2 apomorphineinduced deficits in PPI at a low prepulse intensity. The analogues comparative in 3 4 vivo tests have been performed for AVN211 and the corresponding results are summarized in 5 6 Table 9 ( vide supra ) indicating the enhanced therapeutic effect in contrast to the reference 7 8 compounds. Therefore, good clinical perspectives can be imagined for AVN211 keeping in 9 10 11 mind the upper selectivity and efficiency over the reported drug candidates revealed during an 12 13 extensive preclinical study disclosed herein. Indeed, a substantial enhancement of both 14 15 chemicallyimpaired and nonimpaired cognition in mice and rats given at low doses makes AD 16 17 as a primary therapeutic indication for AVN211. Additionally, AVN211 has shown a 18 19 20 considerable anxiolytic effect that allows us to consider the molecule for alternative therapeutic 21 22 indication. This compound has the best selectivity profile among the reported 5HT 6R 23 24 antagonists and appropriate ADME features reliable for oral administration. The results of 25 26 subsequent clinical evaluation of AVN211 against AD will be disclosed in a follow up 27 28 communication. 29 30 31 32 33 ACKNOWLEDGMENTS 34 35 We would like to thank D.V. Kravchenko, (Chemical Diversity Research Institute, 36 37 Russia), R.N. Karapetian (Chemical Diversity Research Institute, Russia), I.M. Okun (ChemDiv, 38 39 40 Inc., USA), V.I. Kazey (Exactelabs, Russia) and R.M. Salimov (Incuron LLC, Russia) for their 41 42 help with experimental work, interpretation of the results and manuscript preparation. The 43 44 authors would like to acknowledge Mark S. Veselov (Moscow Institute of Physics and 45 46 Technology, Russia) for the preparation of the manuscript and valuable expert opinion. The 47 48 authors declare that there are no conflicts of interest. 49 50 51 52 53 SUPPORTING INFORMATION 54 55 The supplementary material is available in the electronic version of this article. 56 57 58 59 60 42 ACS Paragon Plus Environment Page 43 of 55 Molecular Pharmaceutics

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