Discovery of a Novel Acetylcholinesterase Inhibitor by Fragment-Based Design and Virtual Screening

Home , Acetylcholinesterase, Enzyme inhibitor

Article Discovery of a Novel Acetylcholinesterase Inhibitor by Fragment-Based Design and Virtual Screening

Georgi Stavrakov 1,2,* , Irena Philipova 2, Atanas Lukarski 1, Mariyana Atanasova 1 , Borislav Georgiev 3, Teodora Atanasova 1, Spiro Konstantinov 1 and Irini Doytchinova 1,*

1 Department of Chemistry, Faculty of Pharmacy, Medical University of Sofia, Sofia 1000, Bulgaria; [email protected]fia.bg (A.L.); [email protected]fia.bg (M.A.); [email protected]fia.bg (T.A.); [email protected]fia.bg (S.K.) 2 Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Sofia 1113, Bulgaria; [email protected] 3 Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, Sofia 1113, Bulgaria; [email protected] * Correspondence: [email protected]fia.bg (G.S.); [email protected]fia.bg (I.D.)

Abstract: Despite extensive and intensive research efforts in recent decades, there is still no effective treatment for neurodegenerative diseases. On this background, the use of drugs inhibiting the enzyme acetylcholinesterase (AChE) remains an eternal evergreen in the symptomatic treatment of mild to moderate cognitive impairments. Even more, the cholinergic hypothesis, somewhat forgotten in recent years due to the shift in focus on amyloid cascade, is back to life, and the search for new, more Citation: Stavrakov, G.; Philipova, I.; effective AChE inhibitors continues. We generated a fragment-based library containing aromatic Lukarski, A.; Atanasova, M.; moieties and linkers originating from a set of novel AChE inhibitors. We used this library to design Georgiev, B.; Atanasova, T.; 1220 galantamine (GAL) derivatives following the model GAL (binding core) - linker (L) - aromatic Konstantinov, S.; Doytchinova, I. fragment (Ar). The newly designed compounds were screened virtually for blood–brain barrier (BBB) Discovery of a Novel permeability and binding to AChE. Among the top 10 best-scored compounds, a representative lead Acetylcholinesterase Inhibitor by molecule was selected and tested for anti-AChE activity and neurotoxicity. It was found that the Fragment-Based Design and Virtual selected compound was a powerful non-toxic AChE inhibitor, 68 times more active than GAL, and Screening. Molecules 2021, 26, 2058. https://doi.org/10.3390/ could serve as a lead molecule for further optimization and development. molecules26072058 Keywords: acetylcholinesterase inhibitor; fragment-based library; molecular docking; BBB perme- Academic Editors: Irena Kostova and ability; galantamine; neurotoxicity; neuro-2A cell line; Ellman’s method Diego Muñoz-Torrero

Received: 8 March 2021 Accepted: 31 March 2021 1. Introduction Published: 3 April 2021 Acetylcholinesterase (AChE) is one of the most active enzymes in the central cholinergic system [1]. At the cholinergic synapses, it catalyzes the degradation of the neuro- Publisher’s Note: MDPI stays neutral transmitter acetylcholine (ACh) to choline and acetyl and terminates its action on the with regard to jurisdictional claims in postsynaptic ACh receptors. An extensive network of cortical pyramidal neurons, rich in published maps and institutional afﬁl- AChE, has been observed by histochemical analysis of human brains [2]. It has been found iations. that these neurons are absent at birth and during childhood, gradually emerge during adolescence, reach a maximum in early adulthood, and then gradually decrease in the course of normal aging. In patients with neurodegenerative disorders, a severe loss of cholinergic neurons in the cortex and hippocampus is observed due to abnormal accumu- Copyright: © 2021 by the authors. lation of misfolded proteins like amyloid-β (Aβ), τ-protein, α-synuclein [3–7]. This severe Licensee MDPI, Basel, Switzerland. loss is one of the factors responsible for cognitive impairments, typical for Alzheimer’s and This article is an open access article Parkinson’s diseases [8,9]. distributed under the terms and In this regard, the use of drugs able to inhibit the enzyme AChE and hence, to increase conditions of the Creative Commons the levels of ACh in the brain and to improve the cognitive functions has a clear, rational Attribution (CC BY) license (https:// beneﬁt in the treatment of neurodegenerative disorders especially Alzheimer’s disease creativecommons.org/licenses/by/ 4.0/). (AD). Currently, the AChE inhibitors like donepezil, galantamine and rivastigmine, are the

Molecules 2021, 26, 2058. https://doi.org/10.3390/molecules26072058 https://www.mdpi.com/journal/molecules Molecules 2021, 26, x 2 of 14

disease (AD). Currently, the AChE inhibitors like donepezil, galantamine and rivastig- Molecules 2021, 26, 2058 mine, are the only FDA- and EMA-approved drugs for the symptomatic treatment2 of 14 of mild to moderate cognitive impairments [10]. The levels of AChE inhibition found in patients treated by current AChE inhibitors are less than the minimum of 50% required for effectiveonly FDA- therapy and EMA-approved of AD [11]. Any drugs attempt for the to symptomatic increase the treatment dose to achieve of mild toa moderatebetter AChE inhibitioncognitive results impairments in an increase [10]. The in levels drug of toxicity AChE and inhibition limits foundthe clinical in patients benefits treated [12]. byThus, thecurrent demand AChE for inhibitors more powerful are less thanand theless minimum toxic AChE of 50% inhibitors required is forstill effective an extremely therapy im- portantof AD [11and]. Anyurgent attempt scientific to increase challenge. the dose to achieve a better AChE inhibition results in anGalantamine increase in drug (GAL) toxicity is one and of the limits most the widely clinical prescribed benefits [12 drug]. Thus,s for the treating demand AD for [13]. Apartmore powerfulfrom being and an less AChE toxic inhibitor, AChE inhibitors GAL is is an still allosteric an extremely modulator important of andnicotinic urgent ACh receptorscientific [14 challenge.], has antioxidant properties [15] and facilitates the clearance of Aβ peptide in rat brainsGalantamine [16]. Due (GAL) to this is onevariety of the of mostactions, widely GAL prescribed can serve drugs as а formultitarget treating ADscaffol [13].d to designApart fromnew derivatives being an AChE with inhibitor, a higher GALAChE is inhibitory an allosteric effect modulator and lower of nicotinictoxicity. AChSteadily inreceptor recent [14 decades], has antioxidant, GAL is the properties focus of [15 many] and labfacilitates projects the around clearance the of worldAβ peptide aiming in to developrat brains safer [16 ].and Due more to this powerful variety anti of actions,-neurodegenerative GAL can serve agents as a multitarget [17–27]. scaffold to design new derivatives with a higher AChE inhibitory effect and lower toxicity. Steadily in In the present study, we use GAL as a core structure and design a series of deriva- recent decades, GAL is the focus of many lab projects around the world aiming to develop tives to improve the binding affinity to AChE. Several well-defined domains exist in the safer and more powerful anti-neurodegenerative agents [17–27]. deep Inand the narrow present binding study, we site use of GAL AChE. as aAmong core structure them of and greatest design importance a series of derivatives is the active catalyticto improve site the (CAS), binding consisting affinity of to the AChE. triad Several Ser203 well-defined, Glu334 and domains His447, existand responsible in the deep for ACh'sand narrow hydrolysis binding [28 site]. Along of AChE. the Among binding them gorge of greatest are situated importance the anionic, is the active acyclic, catalytic and ox- yanionsite (CAS), domains. consisting Near of the the triadgorge Ser203, entranc Glu334e is placed and His447, the peripheral and responsible anionic for site ACh’s (PAS), whichhydrolysis is associated [28]. Along with the the binding initiation gorge of areamyloidogenesis situated the anionic, [29]. The acyclic, X-ray and structure oxyanion of the complexdomains. rh NearAChE the-GAL gorge [28 entrance] (Figure is1) placedshows thethat peripheral GAL binds anionic at the sitebottom (PAS), of whichthe binding is siteassociated where with the CAS the initiation is located of amyloidogenesis and fills only one [29-].third The X-rayof the structure binding of gorge. the complex The GAL ammoniumrhAChE-GAL group [28] (Figure is pointing1) shows towards that GAL the bindsentrance at the of bottomthe binding of the bindinggorge and site is where the only positionthe CAS in is locatedthe GAL and molecule fills only available one-third for of the substitution binding gorge. without The affecting GAL ammonium the binding mode.group is pointing towards the entrance of the binding gorge and is the only position in the GAL molecule available for substitution without affecting the binding mode.

FigureFigure 1. 1. XX-ray-ray structure structure of the complexcomplex galantaminegalantamine (GAL)- (GALrh)AChE-rhAChE (pdb (pdb code: code: 4ey6). 4ey6). Protein Protein is is givengiven in in gr grayay ribbon, GAL—inGAL—in ball-and-stickball-and-stick colored colored by by element. element. Our previous structure—AChE affinity studies on GAL derivatives showed that the Our previous structure—AChE affinity studies on GAL derivatives showed that the substitution of CH3 group at the quaternary N-atom with a linker containing five to seven substitution of CH3 group at the quaternary N-atom with a linker containing five to seven carboncarbon atoms atoms and ending withwith aromaticaromatic moiety moiety increases increases the the affinity affinity to AChEto AChE more more than than 10001000 times times [2 [244]. Here, wewe designeddesigned a a fragment-based fragment-based library library containing containing aromatic aromatic moieties moieties andand linkers linkers originating originating fromfrom aa setset of of novel novel AChE AChE inhibitors inhibitors recently recentl discoveredy discovered in our in our lab lab byby docking docking-based-based virtual virtual screening screening of aof ZINC a ZINC lead-like lead- databaselike database [30]. We[30 used]. We this used library this li- braryto design to design GAL derivativesGAL derivatives following following the model the model GAL-linker GAL (L)-aromatic-linker (L)-aromatic fragment fragment (Ar) (Ar)(Figure (Figure2), screened 2), screened them virtually them virtually for blood–brain for blood barrier–brain (BBB) barri permeabilityer (BBB) permeability and docked and them into rhAChE. Among the best-scored inhibitors, a representative lead molecule was selected and tested for anti-AChE activity and neurotoxicity.

Molecules 2021, 26, x 3 of 14

Molecules 2021, 26, x 3 of 14 docked them into rhAChE. Among the best-scored inhibitors, a representative lead molecule was selected and tested for anti-AChE activity and neurotoxicity.

Molecules 2021, 26, 2058 docked them into rhAChE. Among the best-scored inhibitors,3 ofa 14representative lead molecule was selected and tested for anti-AChE activity and neurotoxicity.

Figure 2. Common structure of the designed galantamine derivatives (GAL-L-Ar).

2. Results FigureFigure2.1. Design 2. Common 2. Common of structureGAL structureDerivatives of the designed of theUsing galantamine designed Fragment derivatives galantamine-Based (GAL-L-Ar). Libraries derivatives (GAL-L-Ar). 2. ResultsThe novel GAL derivatives were designed to contain a core, a linker, and an aro- 2.1.2.matic Results Design group of GAL (Figure Derivatives 2) Using. The Fragment-Based GAL molecule Libraries was used as a core. The linkers were designed followingThe novel our GAL previous derivatives werestructure designed–activity to contain studies a core, a linker,on GAL and an derivatives aromatic [22–27], showing group2.1. Design (Figure2 ).of TheGAL GAL Derivatives molecule was Using used Fragment as a core. The-Based linkers Libraries were designed that a linker containing five to seven carbon atoms is optimal for dual-site binding to followingThe our novel previous GAL structure–activity derivatives studies were on designed GAL derivatives to contain [22–27], showing a core, a linker, and an aro- thatAChE. a linker This containing finding ﬁve towas seven applied carbon atoms to select is optimal linkers for dual-site from binding the AChE to AChE. inhibitors discovered Thismaticrecently ﬁnding group from was applied(Figure the ZINC to 2) select. Thedatabase linkers GAL from mol[30 the].ecule AChEThus inhibitorswas, 18 usedtypes discovered asof alinkers core. recently The were linkers designed were and designed as- fromfollowingsigned the ZINCby L1our database–18 previous (Figure [30]. Thus, 3structure). 18 types– ofactivity linkers were studies designed on and GAL assigned derivatives by [22–27], showing L1–18that (Figure a linker3). containing five to seven carbon atoms is optimal for dual-site binding to AChE. This finding was applied to select linkers from the AChE inhibitors discovered recently from the ZINC database [30]. Thus, 18 types of linkers were designed and assigned by L1–18 (Figure 3).

FigureFigure 3. Linkers 3. Linkers used inused the design in the of eddesign galantamine of ed hybrids.galantamine hybrids.

Sixteen aromatic groups (assigned by Ar1–16) were designed to mimic the aromatic rings of ZINC AChE inhibitors (Figure 4) [30]. Each group consists of derivatives (denoted by letters a–v) of the corresponding aromatic core or a single aromatic moiety Figure(groups 3. Ar12Linkers–16). used The in following the design aromatic of ed galantamine fragments hybrids. were used in the design of the novel set of GAL derivatives: biphenyl (Ar1), pyridine-2(1H)-one (Ar2), 1H-indole (Ar3), Sixteen aromatic groups (assigned by Ar1–16) were designed to mimic the aromatic rings of ZINC AChE inhibitors (Figure 4) [30]. Each group consists of derivatives (denoted by letters a–v) of the corresponding aromatic core or a single aromatic moiety (groups Ar12–16). The following aromatic fragments were used in the design of the novel set of GAL derivatives: biphenyl (Ar1), pyridine-2(1H)-one (Ar2), 1H-indole (Ar3),

Molecules 2021, 26, 2058 4 of 14

Molecules 2021, 26, x 4 of 14

Sixteen aromatic groups (assigned by Ar1–16) were designed to mimic the aromatic rings of ZINC AChE inhibitors (Figure4)[ 30]. Each group consists of derivatives (denoted by letters a–v) of the corresponding aromatic core or a single aromatic moi- ety1H (groups-benzo[ Ar12–16).d]imidazole The (Ar4), following 6-phenylpyridazin aromatic fragments-3(2 wereH)-one used (Ar5), in the 1 design-isopropyl of -1H-indole the(Ar6), novel quinoline set of GAL derivatives: (Ar7, Ar9), biphenyl 1-methyl (Ar1),-3- pyridine-2(1phenyl-1H-Hpyrazole)-one (Ar2), (Ar8), 1H-indole phenyl (Ar10), (Ar3),quinazoline 1H-benzo[-2,4(1d]imidazoleH,3H)-dione (Ar4), 6-phenylpyridazin-3(2 (Ar11, Ar12), H 1,2,3,4)-one (Ar5),-tetrahydroquinoline 1-isopropyl-1H- (Ar13), indole1,2,3,4 (Ar6),-tetrahydroisoquinoline quinoline (Ar7, Ar9), 1-methyl-3-phenyl-1 (Ar14, Ar15) andH-pyrazole isoquinoline (Ar8), phenyl (Ar16). (Ar10), The substituents quinazoline-2,4(1H,3H)-dione (Ar11, Ar12), 1,2,3,4-tetrahydroquinoline (Ar13), 1,2,3,4- vary between F, Cl, Br, NH2, methyl, methoxy, i-propyl, CHF2, SO2NH2, NHSO2CH3, tetrahydroisoquinoline (Ar14, Ar15) and isoquinoline (Ar16). The substituents vary be- NHSO2NH2. Thus, 120 aromatic moieties were designed (16 of Ar1, 22 of Ar2, 9 of Ar3, 15 tween F, Cl, Br, NH2, methyl, methoxy, i-propyl, CHF2, SO2NH2, NHSO2CH3, NHSO2NH2. Thus,of Ar4, 120 2 aromatic of Ar5, moieties Ar6 and were Ar11, designed 3 of Ar7, (16 of 14 Ar1, of 22Ar8, of Ar2,17 of 9 ofAr9, Ar3, 13 15 of of Ar10 Ar4, 2 and of 1 of Ar12– Ar5,16). Ar6 For and each Ar11, aromatic 3 of Ar7, fragment 14 of Ar8,, ten 17 oflinkers Ar9, 13 (L1 of– Ar1010) were and 1 used, of Ar12–16). except For for each Ar13–16, where aromaticseven linkers fragment, (L1 ten–3, linkers L6–8, (L1–10) and were L10) used, were except used. for For Ar13–16, Ar9d, where Ar9e, seven Ar9h linkers and Ar9j, eight (L1–3,linkers L6–8, (L11 and–L18) L10) were used.used For additionally. Ar9d, Ar9e, Ar9hIn total, and 1220 Ar9j, eightnovel linkers GAL (L11–L18)derivatives were de- weresigned used in additionally. the present In total,study: 1220 160 novel with GAL Ar1, derivatives 220 with were Ar2, designed 90 with in theAr3, present 150 with Ar4, 20 study: 160 with Ar1, 220 with Ar2, 90 with Ar3, 150 with Ar4, 20 with Ar5, 20 with Ar6, 30 with Ar5, 20 with Ar6, 30 with Ar7, 140 with Ar8, 202 with Ar9, 130 with Ar10, 20 with with Ar7, 140 with Ar8, 202 with Ar9, 130 with Ar10, 20 with Ar11, 10 with Ar12, 7 with Ar13,Ar11, 7 with10 with Ar14, Ar12, 7 with 7 Ar15,with 7Ar13, with Ar16.7 with The Ar14, full fragment 7 with Ar15, library 7 usedwith in Ar16. the present The full fragment studylibrary is given used in in Table the present S1 from Supplementary study is given Materials. in Table S1 from Supplementary Materials.

FigureFigure 4. Structures4. Structures of the of aromatic the aromatic fragments. fragments. The full fragmentThe full fragment library is given library in Table is given S1. in Table S1.

2.2. Blood–Brain Barrier (BBB) Permeability The 1220 newly designed GAL derivatives were tested for BBB permeability applying the filters implemented in SwissADME [31] and BBB Predictor [32]. SwissADME has one BBB permeability filter, and 381 compounds passed it. Then, these compounds were filtered through the 8 algorithms of the BBB predictor, and 199 passed all of them. Their distribution by groups was as follows: 22 with Ar1, 25 with Ar2, 17 with Ar3, 20 with Ar4, 10 with Ar5, 3 with Ar6, 4 with Ar7, 21 with Ar8, 39 with Ar9, 38 with Ar10, and none with Ar11–16.

Molecules 2021, 26, 2058 5 of 14

2.2. Blood–Brain Barrier (BBB) Permeability The 1220 newly designed GAL derivatives were tested for BBB permeability applying the filters implemented in SwissADME [31] and BBB Predictor [32]. SwissADME has one BBB permeability filter, and 381 compounds passed it. Then, these compounds were filtered through the 8 algorithms of the BBB predictor, and 199 passed all of them. Their distribution by groups was as follows: 22 with Ar1, 25 with Ar2, 17 with Ar3, 20 with Ar4, 10 with Ar5, 3 with Ar6, 4 with Ar7, 21 with Ar8, 39 with Ar9, 38 with Ar10, and none with Ar11–16.

2.3. Virtual Screening by Molecular Docking The 199 molecules that passed the nine BBB permeability ﬁlters were docked to human rhAChE. GAL was used as a referent structure. Eight compounds with RMSD >1.5 Å were excluded. The GoldScores of the remaining 191 derivatives ranged from 115.38 to 77.40, and all of them were higher than the GoldScore of GAL (77.73), except for one (77.40). One hundred sixty-six compounds (87%) had scores below 100, while 25 compounds (13%) showed scores above 100 (Table1).

Table 1. The best-scored GAL derivatives with GoldScore >100. Compound 8 (given in bold) was selected for synthesis.

Compound No. GoldScore Linker Aromatic Fragment 1 115.38 L10 Ar1d 2 114.14 L10 Ar1h 3 111.49 L10 Ar1n 4 111.28 L10 Ar1m 5 110.42 L5 Ar1p 6 109.51 L10 Ar1i 7 108.14 L10 Ar1o 8 105.56 L10 Ar1a 9 105.53 L6 Ar1a 10 103.90 L6 Ar10e 11 103.73 L1 Ar8l 12 103.24 L2 Ar4e 13 102.87 L10 Ar1e 14 102.87 L7 Ar13 15 102.84 L6 Ar1j 16 102.49 L2 Ar8n 17 102.31 L2 Ar8e 18 102.08 L6 Ar1k 19 101.87 L10 Ar6a 20 101.87 L3 Ar10e 21 101.67 L3 Ar10l 22 101.03 L6 Ar1b 23 100.60 L1 Ar9o 24 100.31 L1 Ar3d 25 100.04 L1 Ar15 GAL 77.73

2.4. GAL Derivative Selected for Synthesis Nine of the top 10 best-scored GAL derivatives contained the aromatic fragment Ar1, and seven of them-the linker L10. Among them, compound 8 includes the basic structure of Ar1, i.e., Ar1a, and is a good representative lead of both Ar1 and L10. This compound was selected for further synthesis and tests. The physicochemical properties of compound 8 are given in Table2. It has almost twice the molecular weight of GAL, lower pKa, higher logP and logD7.4, wider polar surface area (PSA), 2 hydrogen-bond donors and 6 acceptors. The HBDs is the enol OH group of GAL core and the linker’s NH group. All heteroatoms in the molecule act as HBAs. Molecules 2021, 26, x 6 of 14

Table 2. Physicochemical properties, neurotoxicity and anti-AChE activity of compound 8. Molecules 2021, 26, 2058 6 of 14 Parameter Compound 8 GAL MW 1 524.65 287.35 pKa 7.10 7.92 Table 2. Physicochemical properties, neurotoxicity and anti-AChE activity of compound 8. logP 4.49 1.75 ParameterlogD7.4 Compound 84.34 GAL 1.12 MWPolar1 surface area (Å) 524.6571.03 287.35 41.93 HydrogenpKa -bond donors (HBD) 7.102 7.92 1 logP 4.49 1.75 HydrogenlogD7.4-bond acceptors (HBA) 4.346 1.12 4 NeurotoxicityPolar surface area on(Å) neuro-2A cells (IC50)71.03 >100 µM 41.93 >50 µM 2 Hydrogen-bond donors (HBD) 2 1 Inhibitory activity on eeAChE (IC50) 27.79  5.49 nM 1.92  0.1 µM Hydrogen-bond acceptors (HBA) 6 4 1 2 NeurotoxicityThe physicochemical on neuro-2A properties cells (IC50 were) calculated >100 byµM ACD/logD v.9.08 >50(ACDµM Inc., Toronto, Cana- 2 da).Inhibitory [24]. activity on eeAChE (IC50) 27.79 ± 5.49 nM 1.92 ± 0.1 µM 1 The physicochemical properties were calculated by ACD/logD v.9.08 (ACD Inc., Toronto, Canada). 2 [24]. 2.5. Synthesis of the Selected GAL Derivative 2.5. SynthesisTwo building of the Selected blocks GAL-an Derivative an acid 27 and an amine 30, were needed for the synthesis of compoundTwo building 8, which blocks-an were an subsequentlyacid 27 and an amine connected30, were via needed an amide for the bond. synthesis Thus, initial ofKnoevenagel compound 8,condensation which were subsequently of biphenyl connected-2-carbaldehyde via an amide with bond.malonic Thus, acid initial afforded the Knoevenagel condensation of biphenyl-2-carbaldehyde with malonic acid afforded the unsaturated acid 26, which was hydrogenated to give 3-(biphenyl-2-yl)propanoic acid 27. unsaturated acid 26, which was hydrogenated to give 3-(biphenyl-2-yl)propanoic acid 27. NucleophilicNucleophilic substitution substitution of tert of-butyl tert-butylN-(2-bromoethyl)carbamate N-(2-bromoethyl)carbamate with norgalantamine with norgalantamine 2828gave gave the the the thetert tert-butylcarbamate-butylcarbamate protected protected diamine diamine29. The29. The latter latter was was deprotected deprotected and andimmediately immediately subjected subjected to to amide amide bond bond formation withwith acid acid27 27to to give give the the selected selected com- compoundpound 8 in8 in a amoderate moderate yield yield (Scheme1 ).1).

SchemeScheme 1. 1.Synthesis Synthesis of the of selectedthe selected lead compoundlead compound8. 8. 2.6. Neurotoxicity of the Selected GAL Derivative 2.6. Neurotoxicity of the Selected GAL Derivative The cytotoxicity of compound 8 was tested on neuro-2A cells as described in the MaterialsThe and cytotoxicityМethods section. of compound No toxicity 8 waswas observed tested on in concentrationsneuro-2A cells up as to described 100 µM in the (TableMaterials2). For and comparison, Мethods the section toxicity. No of GAL toxicity on neuro-2A was observed is >50 µ inM[ concentrations24]. Thus, the newly up to 100 µM designed(Table 2) GAL. For derivative comparison, is practically the toxicity non-toxic. of GAL on neuro-2A is >50 µM [24]. Thus, the newly designed GAL derivative is practically non-toxic.

Molecules 2021, 26, x 7 of 14

2.7. Anti-AChE Activity of the Selected GAL Derivative The inhibitory activity of compound 8 was measured in vitro on the enzyme acetylcholinesterase from electric eel (eeAChE) by Ellman’s method [33] with some modifi- cations [34]. However, the docking simulations were conducted on human AChE (rhA- ChE). The sequence alignment of the two proteins has shown that the 17 residues forming the deep and narrow binding gorge on the enzyme are identical [25]. Hence, the in vitro test on eeAChE assesses adequately the inhibitory activities of compounds generated from molecular docking simulations on rhAChE, as it was proven in several studies [22–27]. The selected compound 8 inhibited the enzyme AChE with IC50 = 27.79 nM (Table 2).

Molecules 2021, 26, 2058 It was 68 times more active than GAL, which showed IC50 = 71.92 of 14 µM in the same test.

2.8. Stability of the Complex AChE-Compound 8 assessed by Molecular Dynamics Simulation 2.7. Anti-AChE Activity of the Selected GAL Derivative The best docking pose of compound 8 into rhAChE was selected as starting coordi- The inhibitory activity of compound 8 was measured in vitro on the enzyme acetyl- cholinesterasenates of the from complex electric eel for (eeAChE) the molecularby Ellman’s method dynamics [33] with (MD) some modiﬁca- simulations. The complex was tionssolvated [34]. However, in saline the docking in an simulationsoctahedral were box, conducted energy on human minimized, AChE (rhAChE). heated to 310 °C, equilibrated The sequence alignment of the two proteins has shown that the 17 residues forming the deepand andsimulated narrow binding for gorge1000 onns the (1µs) enzyme by are AMBER identical [ 25v.]. 18 Hence, (UCSF, the in vitroSantest Francisco, US) [35]. For the ontimeeeAChE of simulation assesses adequately, the thecomplex inhibitory was activities stable of compounds, and the generatedligand fromremained bound in the bind- rh molecularing site. docking simulations on AChE, as it was proven in several studies [22–27]. The selected compound 8 inhibited the enzyme AChE with IC50 = 27.79 nM (Table2). It was 68 times more active than GAL, which showed IC50 = 1.92 µM in the same test.

2.8.3. Discussion Stability of the Complex AChE-Compound 8 assessed by Molecular Dynamics Simulation TheGAL best docking is a safe pose ofbut compound moderate8 into rhinhibitorAChE was selectedof AChE as starting. To coordinatesimprove its inhibitory activity, in of the complex for the molecular dynamics (MD) simulations. The complex was solvated inthe saline present in an octahedral study, box,we energydesigned minimized, a library heated of to 310fragments◦C, equilibrated generated and from compounds dis- simulatedcovered for previously 1000 ns (1µs) by as AMBER AChE v. 18 inhibitors (UCSF, San Francisco, [30]. The US) [ 35library]. For the consisted time of of two sections: aro- simulation,matic fragments the complex and was stable, linkers. and the Sixteen ligand remained aromatic bound fragments in the binding with site. various substituents and 3.18 Discussion linkers were derived and combined with being attached to GAL as a main binding core.GAL Thus, is a safe a butfragment moderate- inhibitorbased oflibrary AChE. To of improve 1220 itsGAL inhibitory derivatives activity, in thewas generated. Initially, the present study, we designed a library of fragments generated from compounds discovered previouslycompounds as AChE were inhibitors screened [30]. The libraryfor the consisted ability of two to sections: cross aromaticthe BBB. fragments One hundred ninety-nine de- andrivatives linkers. Sixteen (16%) aromatic passed fragments the nine with various BBB substituentsfilters of andSwissADME 18 linkers were derivedand BBB Predictor. Next, these and combined with being attached to GAL as a main binding core. Thus, a fragment-based librarycompounds of 1220 GAL were derivatives docked was generated. into the Initially, rh theAChE compounds and were assessed screened for by the the GoldScore function of abilityGOLD to cross v.5.2.2 the BBB. (CCDC, One hundred Cambridge, ninety-nine derivatives UK). Among (16%) passed the the top nine BBB10 of filters the best-scored derivatives, of9 SwissADMEcontained and the BBB aromatic Predictor. Next, fragment these compounds Ar1, and were docked7 contained into the rh AChEthe linker and L10. The fragment Ar1 assessed by the GoldScore function of GOLD v.5.2.2 (CCDC, Cambridge, UK). Among the topand 10 ofthe the linker best-scored L10 derivatives, originate 9 contained from theone aromatic of the fragment most Ar1, active and 7 novel contained AChE inhibitors (Figure 5) thederived linker L10. by The a docking fragment Ar1-based and the screening linker L10 originate of a ZINC from one lead of the-like most activedatabase containing 6,053,287 novel AChE inhibitors (Figure5) derived by a docking-based screening of a ZINC lead-like databasemolecules containing [30]. 6,053,287 molecules [30].

Figure 5. Figure One5. One of the of most the active most novel active AChE novel inhibitors AChE derived inhibitors previously derived by a docking-based previously by a docking-based screening of a ZINC lead-like database with Kd = 0.525 µM, measured by isothermal titration calorimetryscreening [30 of]. a ZINC lead-like database with Kd = 0.525 µM, measured by isothermal titration calo- rimetry [30]. Compound 8 was selected as a representative lead molecule of GAL derivatives with Ar1 and L10. It was synthesized and tested for cytotoxicity and AChE activity. It was found that thisCompo derivativeund is non-toxic 8 was and selected inhibits the as enzyme a representative with IC50 of 28 nM, leadwhich makes molecule of GAL derivatives itwith 68 times Ar1 more and active L10. than It GAL. was The synthesized enzyme–inhibitor and complex tested is stable for forcytotoxicity 1 µs MD and AChE activity. It simulation in saline at 310 K. These initial tests on compound 8 make it a perspective lead ofwas a new found series ofthat congeners this withderivative potential AChE is non inhibitory-toxic activity. and inhibits the enzyme with IC50 of 28 nM, which makes it 68 times more active than GAL. The enzyme–inhibitor complex is stable for 1 µs MD simulation in saline at 310 K. These initial tests on compound 8 make it a perspective lead of a new series of congeners with potential AChE inhibitory activity.

Molecules 2021, 26, x 8 of 14

Molecules 2021, 26, 2058 The best-scored docking pose of compound 8 and its interactions8 of 14 with the enzyme are visualized in Figure 6. The GAL core is located on the bottom of the binding gorge and forms a network of hydrogen bonds: between GAL’s quaternary ammonium and Tyr337,The best-scored between docking OCH3 pose and of Ser203, compound and8 and between its interactions OH and with Glu202. the enzyme A are cation-π interaction visualizedis formed in Figurebetween6. The the GAL positively core is located charged on the ammonium bottom of the bindingatom and gorge Tyr337. and An additional formshydrogen a network bond of hydrogen is formed bonds: between between GAL’s the linker’s quaternary NH ammonium group and and Tyr337, Tyr337. The aromatic between OCH3 and Ser203, and between OH and Glu202. A cation-π interaction is formed betweenfragment the is positively involved charged in a ammoniumπ-π stacking atom with and Tyr337.Tyr72 Anand additional Trp286. hydrogen Similar interactions were bondobserved is formed between between the the linker’s other NHtop- groupscored and GAL Tyr337. derivatives The aromatic (data fragment not is shown). For com- involvedpounds in 2 aandπ-π stacking10, the withlinker’s Tyr72 carbonyl and Trp286. O Similaratom makes interactions a hydrogen were observed bond with Tyr124. In betweenthe complexes the other top-scored of compounds GAL derivatives 3, 4, 6 (data, and not 7 shown)., the aromatic For compounds fragments2 and 10 form, π-π stacking the linker’s carbonyl O atom makes a hydrogen bond with Tyr124. In the complexes of compoundswith Tyr1243, 4, 6instead, and 7, the of aromatic Tyr72, fragments while in form theπ- π complexstacking with of Tyr124compound instead of10, this stacking is Tyr72,formed while with in the Tyr341. complex of compound 10, this stacking is formed with Tyr341.

FigureFigure 6. The6. The best-scored best-scored docking docking pose of compound pose of compound8 into rhAChE. 8Hydrogen into rhAChE. bonds areHyd shownrogen in bonds are shown in yellowyellow dashes, dashes, cation- cationπ interaction-in-π interaction blue line-in and blueπ-π lineinteraction-in and π-π red interaction line. The enzyme-in red residues line. The enzyme resi- aredues given are as given gray sticks. as gr Compounday sticks.8 isCompound colored by element. 8 is colored The structural by element. water molecule The structural within the water molecule bindingwithin site the is binding presented site in ball-and-stick. is presented Interactions in ball- areand visualized-stick. Interactions by YASARA v. are 17.1.28 visualized (IMBM, by YASARA v. Graz,17.1.28 Austria) (IMBM, [36]. Graz, Austria) [36]. 4. Materials and Methods 4.1.4. Materials Materials and Methods Reagents were commercial grade and used without further purification. Thin-layer chromatography4.1. Materials (TLC) was performed on aluminum sheets pre-coated with Merck Kieselgel 60 F254Reagents 0.25 mm (Merck, were Sofia,commercial Bulgaria). grade Flash column and used chromatography without further was carried purification. out Thin-layer usingchromatography Silica Gel 60 230–400 (TLC) mesh was (Fluka performed Labimex, Sofia, on aluminum Bulgaria). Commercially sheets pre available-coated with Merck Kie- solvents were used for reactions, TLC and column chromatography. Melting points were determinedselgel 60 inF254 capillary 0.25 tubesmm on(Merck SRS MPA100, Sofia, OptiMelt Bulgaria (Sunnyvale,). Flash CA, column USA) automated chromatography was car- meltingried out point using system Silica with Gel the heating60 230 rate–400 set mesh at 1◦C/min (Fluka (uncorrected). Labimex, OpticalSofia, rotationBulgaria). Commercially 20 ([availableα] D) was measured solvents on were JASCO usedP-2010 for POLARIMETER reactions, TLC (JASCO and INTERNATIONAL column chromatography. CO. Melting Tokyo,points Japan). were The determined NMR spectra in were capillary recorded tubes on a Bruker on SRS Avance MPA100 II+ 600 OptiMelt (BRUKER (Sunnyvale, CA, BioSpin GmbH, Rheinstetten, Germany) (600.13 for 1H-MHz and 150.92 MHz for 13C-NMR) USA) automated melting point system with the heating rate set at 1°C/min (uncorrected). Optical rotation ([α]20D) was measured on JASCO P-2010 POLARIMETER (JASCO IN- TERNATIONAL CO. Tokyo, Japan). The NMR spectra were recorded on a Bruker Avance II+ 600 (BRUKER BioSpin GmbH, Rheinstetten, Germany) (600.13 for 1H-MHz and 150.92 MHz for 13C-NMR) spectrometer with TMS as internal standards for chemical shifts (δ, ppm). 1H- and 13C-NMR data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, br = broad, m = multiplet), coupling constants (Hz), integration, identification. The assignment of the 1H- and 13C-NMR spectra

Molecules 2021, 26, x 9 of 14

Molecules 2021, 26,were x made based on COSY and HSQC experiments. Elemental analyses were performed 9 of 14 by the Microanalytical service Laboratory of the Institute of Organic Chemistry, Bulgar- ian Academy of Science. were made based on COSY and HSQC experiments. Elemental analyses were performed 4.2. Synthesis and Analytical Data of (E)-3-(biphenyl-2-yl)acrylic acid 26 Molecules 2021, 26, 2058 by the Microanalytical service Laboratory of the Institute9 of 14 of Organic Chemistry, Bulgar- A mixtureian of Academybiphenyl- 2of-carbaldehyde Science. (0.300 g, 1.65 mmol), malonic acid (0.343 g, 3.29 mmol) and a catalytic amount of piperidine was refluxed overnight in 2 mL dry pyridinspectrometere. The mixture with TMS aswas internal cooled standards and for acidified chemical shifts with (δ, ppm).1 N H1H-Cl. and The13C-NMR resulting mixture was data are reported4.2. as Synthesis follows: chemical and Analytical shift, multiplicity Data (s of = singlet, (E)-3- d(biphenyl = doublet, t-2 =- triplet,yl)acrylic acid 26 extracted three times with ether. Then the organic phase was extracted with aq. Na2CO3. q = quartet, br = broad,A mixture m = multiplet), of biphenyl coupling constants-2-carbaldehyde (Hz), integration, (0.300 identification. g, 1.65 mmol), malonic acid (0.343 g, ThenThe aqueous assignment phase of the was1H- andacidified13C-NMR with spectra 1 N were NCl made and based extracted on COSY andwith HSQC ether. The combined experiments. Elemental3.29 mmol) analyses and were a performed catalytic by theamount Microanalytical of piperidine service Laboratory was refluxed overnight in 2 mL dry organic extracts were dried over MgSO4, filtered and concentrated under reduced pres- of the Institutepyridin of Organice. Chemistry,The mixture Bulgarian was Academy cooled of and Science. acidified with 1 N HCl. The resulting mixture was sure. The residue was purified by flash column chromatography (silica gel, petroleum extracted three times with ether. Then the organic phase was extracted with aq. Na2CO3. ether/EtOAc4.2. Synthesis = and1:2) Analytical to give Data 0.352 of (E)-3-(biphenyl-2-yl)acrylic g of 26 as white acidcrystals,26 m.p. 201–202 °C. Yield: 95%. A mixtureThen of biphenyl-2-carbaldehyde aqueous phase was (0.300 acidified g, 1.65 mmol), with malonic1 N NCl acid and (0.343 extracted g, with ether. The combined 1H-NMR (CDCl3, 600 MHz):  = 11.70 (br, 1H, OH), 7.81 (d, J = 15.9 Hz, 1H, 3-H), 7.72 (dt, J 3.29 mmol) andorg aanic catalytic extracts amount were of piperidine dried wasover refluxed MgSO overnight4, filtered in 2 and mL dry concentrated under reduced pres- = 8.3, 0.5 Hz, 1H, arom.), 7.48–7.43 (m, 3H, arom.), 7.42–7.38 (m, 3H, arom.), 7.32–7.30 (m, pyridine. Thesure. mixture The was residue cooled and was acidified purified with 1by N HCl.flash The column resulting mixturechromatography (silica gel, petroleum 2H, arom.),was extracted 6.39 three(d, J times= 15.9 with Hz, ether. 1H, Then 2-H) the ppm. organic 13 phaseC-NMR was extracted(CDCl3, with 150.9 aq. MHz):  = 171.97 Na CO . Thenether/EtOAc aqueous phase was= 1:2) acidified to give with 10.352 N NCl g and of extracted 26 as white with ether. crystals, The m.p. 201–202 °C. Yield: 95%. (CO), 146.142 3 (C-3), 143.20 (C, arom.), 139.67 (C, arom.), 132.15 (C, arom.), 130.57 (CH, combined organic1H-NMR extracts (CDCl were dried3, 600 over MHz): MgSO 4 ,= filtered 11.70 and(br, concentrated1H, OH), 7.81 under (d, J = 15.9 Hz, 1H, 3-H), 7.72 (dt, J arom.),reduced 130.27 pressure. (CH, The arom.), residue 129.80 was purified (2CH, by flasharom.), column 128.33 chromatography (2CH, arom.), (silica gel, 127.69 (CH, arom.), petroleum ether/EtOAc= 8.3, 0.5 Hz, = 1:2) 1H, to give arom.), 0.352 g 7.48 of 26–as7.43 white (m, crystals, 3H, arom.), m.p. 201–202 7.42◦C–7.38. (m, 3H, arom.), 7.32–7.30 (m, 127.97 (CH, arom.),1 126.97 (CH, arom.), 118.08 (C-2) ppm. 13 Yield: 95%. H-NMR2H, arom.), (CDCl3 ,6.39 600 MHz): (d, J δ== 15.9 11.70 Hz,(br, 1H, 1H, OH), 2-H) 7.81 ppm. (d, J = 15.9C Hz,-NMR 1H, (CDCl3, 150.9 MHz):  = 171.97 3-H), 7.72 (dt,(CO),J = 8.3, 146.14 0.5 Hz, 1H, (C- arom.),3), 143.207.48–7.43 (C, (m, arom.), 3H, arom.), 139.67 7.42–7.38 (C, arom.), (m, 3H, 132.15 (C, arom.), 130.57 (CH, arom.), 7.32–7.30 (m, 2H, arom.), 6.39 (d, J = 15.9 Hz, 1H, 2-H) ppm. 13C-NMR (CDCl3, arom.), 130.27 (CH, arom.), 129.80 (2CH, arom.), 128.33 (2CH, arom.), 127.69 (CH, arom.), 150.9 MHz): δ = 171.97 (CO), 146.14 (C-3), 143.20 (C, arom.), 139.67 (C, arom.), 132.15 (C, arom.), 130.57127.97 (CH, (CH, arom.), arom.), 130.27 (CH,126.97 arom.), (CH, 129.80 arom.), (2CH, 11 arom.),8.08 128.33(C-2) (2CH,ppm. arom.), 127.69 (CH, arom.), 127.97 (CH, arom.), 126.97 (CH, arom.), 118.08 (C-2) ppm.

4.3. Synthesis and Analytical Data of 3-(biphenyl-2-yl)propanoic acid 27

To a solution of 26 (0.300 g, 1.34 mmol) in MeOH (10 mL), flashed with Ar and cooled4.3. to Synthesis 0 °C, and10% Analytical Pd/C Datawas of added 3-(biphenyl-2-yl)propanoic at one portion acid, 27and the atmosphere was changed to H2 with theTo a solutionhelp4.3. of ofSynthesis a26 hydrogen(0.300 g, and 1.34 - mmol)Analyticalfilled in balloon. MeOH Data (10 The mL),of 3- flashed(biphenylmixture with -was Ar2- andyl)propanoic monitored cooled acid by 2TLC7 , and ◦ after to2 0h C,at 10% room Pd/C temperature was added at one, portion,no traces and theof atmospherethe starting was changed compound to H2 with were detected. The To a solution of 26 (0.300 g, 1.34 mmol) in MeOH (10 mL), flashed with Ar and mixturethe helpwas of afiltered hydrogen-filled through balloon. a pad The mixture of Celite was monitored and concentrated by TLC, and after to 2give h at 0.300 g of 27 as room temperature,cooled no tracesto 0 °C, of the 10% starting Pd/C compound was added were detected. at one Theportion mixture, and was the atmosphere was changed to 1 whitefiltered crystals, through m.p. a pad 112 of– Celite113 °C. and Yield: concentrated 99%. to H give-NMR 0.300 g(CDCl of 27 as3 white, 600 crystals,MHz):  = 11.77 (br, 1H, H◦ 2 with the1 help of a hydrogen-filled balloon. The mixture was monitored by TLC, and OH), m.p.7.42 112–113–7.40 (m,C. Yield: 2H, 99%. arom.),H-NMR 7.37 (CDCl–7.343, 600 (m, MHz): 1H,δ = arom.), 11.77 (br, 1H,7.31 OH),–7.27 7.42–7.40 (m, 5H, arom.), 7.22– after 2 h at room temperature, no traces of the starting compound were detected. The (m, 2H, arom.), 7.37–7.34 (m, 1H, arom.), 7.31–7.27 (m, 5H, arom.), 7.22–7.21 (m, 1H, arom.), 13 7.21 (m, 1H, arom.), 2.93 (t, J = 7.8 Hz, 1H, 3-H), 2.4513 (t, J = 8.2 Hz, 1H, 2-H) ppm. C-NMR 2.93 (t, J = 7.8 Hz,mixture 1H, 3-H), was 2.45 (t,filteredJ = 8.2 Hz, through 1H, 2-H) ppm. a padC-NMR of Celite (CDCl 3,and 150.9 concentrated MHz): to give 0.300 g of 27 as (CDClδ =3, 178.6150.9 (CO), MHz): 141.99  (C, = 178.6 arom.), (CO), 141.32 (C, 141.99 arom.), (C, 137.46 arom.), (C, arom.), 141.32 130.27 (C, (CH, arom.), arom.), 137.46 (C, arom.), white crystals, m.p. 112–113 °C. Yield: 99%. 1H-NMR (CDCl3, 600 MHz):  = 11.77 (br, 1H, 130.27129.01 (CH, (2CH, arom.), arom.), 129.01 128.91 (CH, (2CH, arom.), arom.), 128.25 (2CH,128.91 arom.), (CH, 127.61 arom.), (CH, arom.), 128.25 127.05 (2CH, arom.), 127.61 (CH, arom.), 126.31OH), (CH, 7.42 arom.),–7.40 34.89 (m, (C-2), 2H, 28.00arom.), (C-3) ppm.7.37–7.34 (m, 1H, arom.), 7.31–7.27 (m, 5H, arom.), 7.22– (CH, arom.), 127.05 (CH, arom.), 126.31 (CH, arom.), 34.89 (C-2), 28.00 (C-3) ppm. 7.21 (m, 1H, arom.), 2.93 (t, J = 7.8 Hz, 1H, 3-H), 2.45 (t, J = 8.2 Hz, 1H, 2-H) ppm. 13C-NMR (CDCl3, 150.9 MHz):  = 178.6 (CO), 141.99 (C, arom.), 141.32 (C, arom.), 137.46 (C, arom.),

130.27 (CH, arom.), 129.01 (2CH, arom.), 128.91 (CH, arom.), 128.25 (2CH, arom.), 127.61

(CH, arom.), 127.05 (CH, arom.), 126.31 (CH, arom.), 34.89 (C-2), 28.00 (C-3) ppm.

4.4. Synthesis and Analytical Data of tert-butyl {2-[(4aS,6R,8aS)-6-hydroxy-3-methoxy-

5,6,9,10-tetrahydro-4aH-[1]benzofuro[3a,3,2-ef][2]benzazepin-11(12H)-yl]ethyl}carbamate 29

To a solution of norgalanthamine 28 (0.100 g, 0.36 mmol) in anhydrous acetonitrile (10 mL) under4.4. argon Synthesis atmosphere and Analytical was Data added of tert tert--butylbutyl {2 N-[(4aS,6R-(2-bromoethyl)carbamate,8aS)-6-hydroxy-3-methoxy - (0.098 g, 0.44 mmol)5,6,9,10 and-tetrahydro anhydrous-4aH K-[1]benzofur2CO3 (0.150o[3 g,a,3,2 1.08- ef][2]benzazepinmmol). After stirring-11(12H) for-yl]ethyl}carbamate 24 h at 29 To a solution of norgalanthamine 28 (0.100 g, 0.36 mmol) in anhydrous acetonitrile (10 mL) under argon atmosphere was added tert-butyl N-(2-bromoethyl)carbamate (0.098 g, 0.44 mmol) and anhydrous K2CO3 (0.150 g, 1.08 mmol). After stirring for 24 h at

Molecules 2021, 26, x 10 of 14 Molecules 2021, 26, 2058 10 of 14

60 °C, the4.4. Synthesis mixture and was Analytical filtered Data of through tert-butyl {2-[(4aS,6R,8aS)-6-hydroxy-3-methoxy- a pad of Celite. The filtrate was concentrated 5,6,9,10-tetrahydro-4aH-[1]benzofuro[3a,3,2-ef][2]benzazepin-11(12H)-yl]ethyl}carbamate 29 under reducedTo a solutionpressure of norgalanthamine and subjected28 (0.100to purification g, 0.36 mmol) inby anhydrous flash column acetonitrile chromatography on silica(10 gel mL) (CH under2Cl argon2/CH atmosphere3OH/NH was4OH added = 25/1/0.02) tert-butyl N-(2-bromoethyl)carbamate to give the desired (0.098 product g, 29 as waxy ◦ solid. Yield:0.44 mmol) 83%. and [α] anhydrousD20 = −64.5 K2 CO(c 0.4205,3 (0.150 g, CHCl 1.08 mmol).3). 1H After-NMR stirring (CDCl for 243, h600 at 60 MHz):C,  = 6.65 (d, J the mixture was filtered through a pad of Celite. The filtrate was concentrated under = 8.2 Hz,reduced 1H, H pressure-2), 6.61 and (d, subjected J = 8.2 to purificationHz, 1H, H by-1), flash 6.06 column (d, chromatographyJ = 10.2 Hz, 1H, on silica H-8), 6.01 (dd, J = 10.2, 4.9gel Hz, (CH 1H,2Cl2 /CHH-7),3OH/NH 5.04 (br,4OH 1H, = 25/1/0.02) NH), 4.60 to give (br, the 1H, desired H product-6), 4.1529 as(d, waxy J = solid.15.6 Hz, 1H, H-12), Yield: 83%. [α] 20 = −64.5 (c 0.4205, CHCl ). 1H-NMR (CDCl , 600 MHz): δ = 6.65 (d, 4.14–4.12 (m, 1H, HD-4a), 3.83 (s, 3H, OCH3 3), 3.78 (d, J = 15.63 Hz, 1H, H-12), 3.40 (t, J = 13.7 J = 8.2 Hz, 1H, H-2), 6.61 (d, J = 8.2 Hz, 1H, H-1), 6.06 (d, J = 10.2 Hz, 1H, H-8), 6.01 (dd, Hz, 1H, JH= 10.2-10),, 4.9 3.20 Hz,– 1H,3.18 H-7), (m, 5.04 2H, (br, H 1H,-1′), NH), 3.14 4.60 (d, (br, J 1H, = H-6),14.5 4.15Hz, (d, 1H,J = 15.6H-10), Hz, 1H,2.70–2.66 (m, 1H, H-5), 2.65H-12),–2.59 4.14–4.12 (m, 2H, (m, 1H,H-2′), H-4a), 2.40 3.83 (br, (s, 3H, 1H, OCH OH),3), 3.78 2.04 (d, J–=1.98 15.6 (m, Hz, 1H,2H, H-12), H-5, 3.40 H (t,-9), 1.54–1.51 (m, J = 13.7 Hz, 1H, H-10), 3.20–3.18 (m, 2H, H-10), 3.14 (d, J = 14.5 Hz, 1H, H-10), 2.70–2.66 (m, 3 3 13 3  1H, H-9),1H, 1.44 H-5), (s, 2.65–2.59 9H, C(CH (m, 2H,) H-2) ppm.0), 2.40 (br,C 1H,-NMR OH), 2.04–1.98(CDCl (m,, 150.9 2H, H-5, MHz) H-9), 1.54–1.51 = 156.05 (CO), 145.83 13 (C-3a), 144.23(m, 1H, H-9),(C-3), 1.44 133.01 (s, 9H, C(CH(C-12b),3)3) ppm. 127.75C-NMR (C-7, (CDCl C-12a),3, 150.9 126.62 MHz) δ =(C 156.05-8), 122.13 (CO), (C-1), 111.10 (C-2), 88.67145.83 (C (C-3a),-6), 79.19 144.23 (C-3),(C(CH3)3), 133.01 (C-12b), 62.00 127.75 (C-4a), (C-7, 56.98 C-12a), (C 126.62-12), (C-8), 55.85 122.13 (OCH3), (C-1), 51.65 (C-10), 111.10 (C-2), 88.67 (C-6), 79.19 (C(CH3)3), 62.00 (C-4a), 56.98 (C-12), 55.85 (OCH3), 51.65 49.60 (C-2′),48.38 (C-08a), 37.30 (C-1′), 32.850 (C-9), 29.87 (C-5), 28.40 (C(CH3)3) ppm. (C-10), 49.60 (C-2 ),48.38 (C-8a), 37.30 (C-1 ), 32.85 (C-9), 29.87 (C-5), 28.40 (C(CH3)3) ppm.

4.5. Synthesis and Analytical Data of 3-(biphenyl-2-yl)-N-{2-[(4aS,6R,8aS)-6-hydroxy- 3-methoxy- 4.5. Synthesis5,6,9,10-tetrahydro-4aH-[1]benzofuro[3a,3,2-ef][2]benzazepin-11(12H)-yl]ethyl}propanamide and Analytical Data of 3-(biphenyl-2-yl)-N-{2-[(4aS,6R,8aS)-6-8hydroxy- 3-methoxy-5,6,9,10To a solution-tetrahydro of 29 (0.100-4aH g,-[1]benzofuro[3a,3,2 0.24 mmol) in dichloromethane-ef][2]benzazepin (5 mL) was-11(12H) added -yl]ethyl}prop ◦ anamide CF8 3CO2H (0.25 mL) dropwise at 0 C. The mixture was stirred for 2 h at r.t. and quenched by dropwise addition of aq NH4OH. The mixture was extracted with CH2Cl2. The organic To layers a solution dried and of concentrated 29 (0.100 to give g, 0.24 0.125 g mmol) of crude in (4a S dichloromethane,6R,8aS)-11-(2-aminoethyl)-3- (5 mL) was added CF3CO2Hmethoxy-5,6,9,10,11,12-hexahydro-4a (0.25 mL) dropwise at 0 °C.H-[1 The]benzofuro[3a,3,2-ef][ mixture was2]benzazepin-6-ol stirred for 2 30h, at which r.t. and quenched was used without further puriﬁcation. Yield: 86%. by dropwise addition of aq NH4OH. The mixture was extracted with CH2Cl2. The organic To a solution of acid 27 (0.052 g, 0.23 mmol) in CH2Cl2 (7 mL) was added EDC (0.044 g, layers 0.23 dried mmol), HOBT and (0.031 g, concentrated 0.23 mmol) and amine to30 (0.065 give g, 0.21 mmol). 0.125 The mixture g of crude (4aS,6R,8awasS stirred)-11-(2 for-aminoethyl) 1 h at r.t. and the-3 product-methoxy formation-5,6,9,10,11,12 was monitored-hexahydro by TLC. The- mixture4aH-[1]benzofuro[3 was concentrated to dry and directly subjected to ﬂash column chromatography on silica a,3,2-ef][2]benzazepingel (CH2Cl2/MeOH/NH-6-ol4 OH30, =which 20/1/0.02) was to giveused 0.058 without g of the desired further product purification. 8 as white Yield: 86%. ◦ 20 1 To amorphousa solution solid, of acid m.p. 131–13327 (0.052C. Yield: g, 0.23 54%. mmol) [α]D = in -56.7 CH (c 0.4115,2Cl2 (7 CHCl mL)3). wasH-NMR added EDC (0.044 (CDCl3, 600 MHz): δ = 7.42–7.40 (m, 2H, arom.), 7.36–7.34 (m, 1H, arom.), 7.32–7.28 (m, 3H, g, 0.23 mmol),arom.), 7.27–7.24 HOBT (m, (0.031 2H, arom.), g, 0.23 7.22–7.21 mmol) (m, 1H,and arom.), amine 6.66 30 (d, J(0.065= 8.2 Hz, g, 1H, 0.21 H-2), mmol). 6.58 The mixture was stirred(d, J =for 8.2 1 Hz, h 1H,at r.t. H-1), and 6.06 (dd,the Jproduct= 10.4, 0.8 formation Hz, 1H, H-8), 6.02was (dd, monitoredJ = 10.2, 4.7 by Hz, TLC. 1H, The mixture was concentratedH-7), 5.78 (br, 1H,to dry NH), and 4.60 (br, directly 1H, H-6), subjected 4.15 (br, 1H, H-4a),to flash 4.09 column (d, J = 15.6 Hz,chromatography 1H, H-12), on silica 3.83 (s, 3H, OCH3), 3.65–3.63 (m, 1H, H-12), 3.63 (t, J = 13.8 Hz, 1H, H-10), 3.24–3.14 (m, gel (CH2Cl2/MeOH/NH4OH = 20/1/0.02) to give 0.058 g of the desired product 8 as white 2H, CH2NHCO), 3.03 (d, J = 14.7 Hz, 1H, H-10), 2.97 (t, J = 7.8 Hz, 2H, COCH2), 2.71–2.68 20 1 amorphous(m, 1H, solid, H-5), 2.56–2.55m.p. 131 (m,–133 1H, NCH°C. 2Yield:), 2.50–2.46 54%. (m, 1H,[α] NCHD =2 ),-56.7 2.25 (t,(cJ =0.4115, 8.0 Hz, 2H,CHCl3). H-NMR COCH CH ), 2.02–1.94 (m, 2H, H-5, H-9), 1.51 (d, J = 12.6 Hz, 1H, H-9) ppm. 13C-NMR (CDCl3, 600 MHz):2 2  = 7.42–7.40 (m, 2H, arom.), 7.36–7.34 (m, 1H, arom.), 7.32–7.28 (m, 3H, arom.), 7.27–7.24 (m, 2H, arom.), 7.22–7.21 (m, 1H, arom.), 6.66 (d, J = 8.2 Hz, 1H, H-2), 6.58 (d, J = 8.2 Hz, 1H, H-1), 6.06 (dd, J = 10.4, 0.8 Hz, 1H, H-8), 6.02 (dd, J = 10.2, 4.7 Hz, 1H, H-7), 5.78 (br, 1H, NH), 4.60 (br, 1H, H-6), 4.15 (br, 1H, H-4a), 4.09 (d, J = 15.6 Hz, 1H, H-12), 3.83 (s, 3H, OCH3), 3.65–3.63 (m, 1H, H-12), 3.63 (t, J = 13.8 Hz, 1H, H-10), 3.24– 3.14 (m, 2H, CH2NHCO), 3.03 (d, J = 14.7 Hz, 1H, H-10), 2.97 (t, J = 7.8 Hz, 2H, COCH2), 2.71–2.68 (m, 1H, H-5), 2.56–2.55 (m, 1H, NCH2), 2.50–2.46 (m, 1H, NCH2), 2.25 (t, J = 8.0 Hz, 2H, COCH2CH2), 2.02–1.94 (m, 2H, H-5, H-9), 1.51 (d, J = 12.6 Hz, 1H, H-9) ppm. 13C-NMR (CDCl3, 150.9 MHz) δ = 172.21 (CO), 145.83 (C-3a), 144.28 (C-3), 141.79 (C, arom.), 141.52 (C, arom.), 138.09 (C, arom.), 132.93 (C-12b), 130.18 (CH, arom.), 129.26 (CH, arom., C-12a), 129.02 (2CH, arom.), 128.23 (2CH, arom.), 127.83 (C-7), 127.53 (CH, arom.), 126.95 (CH, arom.), 126.50 (C-8), 126.20 (CH, arom.), 122.11 (C-1), 111.12 (C-2), 88.64 (C-6), 61.96 (C-4a), 56.81 (C-12), 55.85 (OCH3), 51.54 (C-10), 48.92 (NCH2), 48.34 (C-8a), 37.48 (COCH2CH2), 35.95 (CH2NHCO), 32.79 (C-9), 29.86 (C-5), 29.22 (COCH2) ppm. C33H36N2O4 (524.65): calcd. C 75.55, H 6.92, N 5.34, found C 75.23, H 7.09, N 5.06.

Molecules 2021, 26, 2058 11 of 14

(CDCl3, 150.9 MHz) δ = 172.21 (CO), 145.83 (C-3a), 144.28 (C-3), 141.79 (C, arom.), 141.52 (C, arom.), 138.09 (C, arom.), 132.93 (C-12b), 130.18 (CH, arom.), 129.26 (CH, arom., C-12a), Molecules 2021, 26, x 129.02 (2CH, arom.), 128.23 (2CH, arom.), 127.83 (C-7), 127.53 (CH, arom.), 126.95 (CH, 11 of 14 arom.), 126.50 (C-8), 126.20 (CH, arom.), 122.11 (C-1), 111.12 (C-2), 88.64 (C-6), 61.96 (C-4a), 56.81 (C-12), 55.85 (OCH3), 51.54 (C-10), 48.92 (NCH2), 48.34 (C-8a), 37.48 (COCH2CH2), 35.95 (CH2NHCO), 32.79 (C-9), 29.86 (C-5), 29.22 (COCH2) ppm. C33H36N2O4 (524.65): calcd. C 75.55, H 6.92, N 5.34, found C 75.23, H 7.09, N 5.06.

4.6. Prediction of Blood–Brain Barrier (BBB) Permeability The ability of the GAL derivatives to cross the BBB by passive diffusion was predicted 4.6. Predictionby SwissADME of Blood (http://www.swissadme.ch–Brain Barrier (BBB) Permeability Accessed on: 5 February 2019) [31] and BBB Predictor (https://www.cbligand.org/BBB/predictor.php Accessed on: 5 February 2019) [32]. TheSwissADME ability of predicts the GAL BBB derivatives permeability based to cross on the the lipophilicity BBB by andpassive polarity diffusion of small was predicted bymolecules SwissADME [37]. The ( BBBhttp://www predictor uses.swissadme.ch a support vector Accessed machine (SVM) on: 5.02.2019 and LiCABEDS) [31] and BBB Predictor(ligand (https://www.cbligand.org/BBB/predictor.php classifier of adaptively boosting ensemble decision stumps) Accessed algorithms on: developed 5.02.2019 ) [32]. based on four types of fingerprints for 1593 compounds with known BBB permeability [38]. SwissADME predicts BBB permeability based on the lipophilicity and polarity of small molecules4.7. Virtual[37]. The Screening BBB by predictor MolecularDocking uses a support vector machine (SVM) and LiCABEDS (ligand classifierThe GAL of derivatives adaptively were constructedboosting withensemble Avogadro decision software v.stumps) 1.1.0 (University algorithms of developed basedPittsburgh, on four Pittsburgh, types PA,of fingerprints USA) [39] andminimized for 1593 withcompounds MMFF94s forcewith field known [40], then BBB permea- structures were docked into the X-ray structure of human recombinant acetylcholinesterase bility [38(rh].AChE, pdb ID: 4EY6, R = 2.40 Å) [28]. The docking simulations were performed by GOLD v.5.2.2 (CCDC Ltd., Cambridge, UK) using a protocol previously optimized in 4.7. Virtualterms Screening of scoring function,by Molecular rigid/flexible Docking ligand and binding site, the radius of the binding site, presence/absence of a water molecule (HOH846) within the binding site, number of Thegenetic GAL algorithms derivatives (GA) were runs [ 22constructed–27]. The docking with simulations Avogadro in thesoftware present studyv. 1.1.0 were (University of Pittsburgh,performed US) at the [39 following] and minimized settings: scoring with function MMFF94s GoldScore, force flexible field ligand, [40], flexible then structures were dockedbinding into site, thethe radius X-ray of struc the bindingture of site human 10 Å, a structural recombinant water moleculeacetylcholinesterase within the (rhA- binding sire (HOH 846), 100 GA runs. The residues from the binding site, which are in close ChE, pdbproximity ID: 4EY6, to the R bound = 2.40 ligand, Å ) were[28]. set The as flexible.docking These simulations were Tyr72, Asp74,were Trp86,performed Tyr124, by GOLD v.5.2.2 (CCDCSer125, Trp286, Ltd., Phe297,Cambridge, Tyr337, Phe338,UK) using Tyr341. a GALprotocol from X-raypreviously structure optimized was used as ain terms of scoringreferent function, molecule. rigid/flexible The best-scored ligand compounds and binding were analyzed site, forthe synthetic radius feasibility of the and binding site, presence/absenceprotein–ligand of interactions. a water molecule (HOH846) within the binding site, number of ge- netic algorithm4.8. Neurotoxicitys (GA) Test runs [22–27]. The docking simulations in the present study were performed Toat assessthe following the neurocytotoxicity settings: scoring of the selected function compound, GoldScore, murine flexible neuroblastoma ligand, flexible bindingneuro-2A site, the cells radius (German of the collection binding DSMZ, site Braunschweig, 10 Å , a structural Germany) water were molecule used. They within the were cultivated under standard conditions: complete medium (90% DMEM, 10% heat- binding sire (HOH 846), 100 GA runs. The residues◦ from the binding site, which are in inactivated FBS, and non-essential amino acids); 37 C and 5% CO2 in a fully humidified close proximityatmosphere. to Thethe cell bound line was ligand kept, in were the logarithmic set as flexible growth. These phase bywere splitting Tyr72, 1:4 onceAsp74, Trp86, Tyr124,a Ser125, week using Trp286, trypsin/EDTA. Phe297, AboutTyr337, 30% Phe338, of the cells Tyr341. grew like GAL neuronal from cells. X-ray For structure the was used asexperimental a referent evaluation molecule. of the The cytotoxicity, best-scored the cells compounds were plated in were 96-well analy flat-bottomedzed for synthetic feasibility and protein–ligand interactions.

4.8. Neurotoxicity Test To assess the neurocytotoxicity of the selected compound, murine neuroblastoma neuro-2A cells (German collection DSMZ, Braunschweig, Germany) were used. They were cultivated under standard conditions: complete medium (90% DMEM, 10% heat-inactivated FBS, and non-essential amino acids); 37 °C and 5% CO2 in a fully humidified atmosphere. The cell line was kept in the logarithmic growth phase by splitting 1:4 once a week using trypsin/EDTA. About 30% of the cells grew like neuronal cells. For the experimental evaluation of the cytotoxicity, the cells were plated in 96-well flat-bottomed plates at the recommended density of around 1100 cells/25 cm2. After 24 h, the cells were treated with a series of concentrations (100, 50, 25, 12.5, 6.25 µM) of the tested compound, and after 72 h incubation, an MTT-dye reduction assay was performed [41]. MTT stock solution (10 mg/mL in PBS) was added (0.01 mL/well) at the end of incubation. Plates were further incubated at 37 °C for 4 h. Next, the formazan crystals were dissolved by the addition of 0.110 mL/well 5% formic acid in 2-propanol (v/v). Absorp-

Molecules 2021, 26, 2058 12 of 14

plates at the recommended density of around 1100 cells/25 cm2. After 24 h, the cells were treated with a series of concentrations (100, 50, 25, 12.5, 6.25 µM) of the tested compound, and after 72 h incubation, an MTT-dye reduction assay was performed [41]. MTT stock solution (10 mg/mL in PBS) was added (0.01 mL/well) at the end of incubation. Plates were further incubated at 37 ◦C for 4 h. Next, the formazan crystals were dissolved by the addition of 0.110 mL/well 5% formic acid in 2-propanol (v/v). Absorption was measured at 580 nm wavelength on an automated ELISA reader Labexim LMR1. At least six wells per concentration were used, and data were processed using the software 2.0 (GraphPad, San Diego, CA, USA).

4.9. Assessment of AChE Inhibitory Activity The microplate assay used for measuring AChE inhibitory activity was as described by Ellman [33] with the modiﬁcations added by López [34]. Acetylthiocholine iodide (ATCI) in a solution with 5,50-dithiobis(2-nitrobenzoic acid) (DTNB) was used as a substrate for the acetylcholinesterase from Electrophorus electricus (Steinheim, Sigma-Aldrich, Germany). 50 µL of AChE (0.25 U/mL) dissolved in phosphate buffer (8 mM K2HPO4, 2.3 mM NaH2PO4, 0.15 M NaCl, pH 7.5) and 50 µL of the sample dissolved in the same buffer were added to the wells. The plates were incubated for 30 min at room temperature before the addition of 100 µL of the substrate solution (0.04 M Na2HPO4, 0.2 mM DTNB, 0.24 mM ATCI, pH 7.5). The absorbances were read in a microplate reader (BIOBASE, ELISA-EL10A, Jinan, Shandong, China) at 405 nm after 3 min. Enzyme activity was calculated as an inhibition percentage compared to an assay using a buffer without any inhibitor. GAL was used as a positive control. The AChE inhibitory data were analyzed with the software package Prism 9 (GraphPad Inc., San Diego, USA). The IC50 values were measured in triplicate, and the results are presented as means ± SD.

4.10. Molecular Dynamics Protocol The best-scored pose of the selected GAL derivative in complex with AChE was used as a starting structure for the MD simulation by Amber 18 [35]. The small molecule was parametrized using GAFF2.11 force ﬁeld and AM1-BCC charges, and the complex was solvated in a truncated octahedral box with TIP3P water and 0.15 M NaCl. The system was subjected to energy minimization, heating to 310 K, density equilibration, preproduction equilibration and production dynamics for 1µs (1000 ns). Frames were saved every 1 ns.

5. Conclusions The fragment-based design of 1220 GAL derivatives and their virtual screening for BBB permeability and docking into rhAChE delivered a set of 25 novel prospective AChE inhibitors. One of them was selected as a representative of the most common linker and aromatic fragment among the top 10 best-scored ligands. The in vitro tests for cytotoxicity and AChE inhibitory activity conﬁrmed that the selected compound is a powerful non-toxic AChE inhibitor and can serve as a lead molecule for further optimization and development.

Supplementary Materials: The following are available online, Table S1: Fragment library used in the present study. Author Contributions: Conceptualization, G.S., I.P., A.L., M.A. and I.D.; methodology, G.S., I.P., A.L., M.A., B.G., S.K. and I.D.; investigation, G.S., I.P, A.L., M.A. and B.G.; writing—original draft preparation, G.S., M.A. and I.D.; writing—review and editing, G.S., I.P, A.L., M.A., B.G., S.K. and I.D.; visualization, G.S., M.A. and I.D.; supervision, I.D.; project administration, I.D.; funding acquisition, I.D. All authors have read and agreed to the published version of the manuscript. Funding: This work was funded by the Bulgarian National Science Fund (Grant DN03/9/2016) and Grant No BG05M2OP001-1.001-0003, ﬁnanced by the Science and Education for Smart Growth Operational Program and co-ﬁnanced by the European Union through the European Structural and Investment funds. The APC was funded by the Bulgarian National Roadmap for Research Infrastructure, Grant No. D01-271/2019. Molecules 2021, 26, 2058 13 of 14

Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: Authors would like to thank Strahil Berkov from the Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, for the helpful discussions. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results. Sample Availability: Sample of the compound 8 is available from the authors.

References 1. Geula, C.; Mesulam, M.M. Cholinergic systems in Alzheimer’s disease. In Alzheimer Disease, 2nd ed.; Terry, R.D., Katzman, R., Bick, K.L., Sisodia, S.S., Eds.; Lippincott, Williams & Wilkins: Philadelphia, PA, USA, 1999; pp. 269–292. [CrossRef] 2. Janeczek, M.; Gefen, T.; Samimi, M.; Kim, G.; Weintraub, S.; Bigio, E.; Rogalski, E.; Mesulam, M.-M.; Geula, C. Variations in Acetylcholinesterase Activity within Human Cortical Pyramidal Neurons Across Age and Cognitive Trajectories. Cereb. Cortex 2017, 28, 1329–1337. [CrossRef] 3. Palop, J.J.; Mucke, L. Amyloid-β-induced neuronal dysfunction in Alzheimer’s disease: From synapses toward neural net-works. Nat. Neurosci. 2010, 13, 812–818. [CrossRef] 4. Wozniak, M.A.; Itzhaki, R.F.; Shipley, S.J.; Dobson, C.B. Herpes simplex virus infection causes cellular β-amyloid accumu-lation and secretase upregulation. Neurosci. Lett. 2007, 429, 95–100. [CrossRef] 5. Eimer, W.A.; Kumar, D.K.V.; Shanmugam, N.K.N.; Rodriguez, A.S.; Mitchell, T.; Washicosky, K.J.; György, B.; Breakefield, X.O.; Tanzi, R.E.; Moir, R.D. Alzheimer’s Disease-Associated β-Amyloid Is Rapidly Seeded by Herpesviridae to Protect against Brain Infection. Neuron 2018, 99, 56–63. [CrossRef] 6. Guo, T.; Noble, W.; Hanger, D.P. Roles of tau protein in health and disease. Acta Neuropathol. 2017, 133, 665–704. [CrossRef] 7. Sulzer, D.; Edwards, R.H. The physiological role of α-synuclein and its relationship to Parkinson’s Disease. J. Neurochem. 2019, 150, 475–486. [CrossRef] 8. Hardy, J.A.; Higgins, G.A. Alzheimer’s disease: The amyloid cascade hypothesis. Science 1992, 256, 184–185. [CrossRef][PubMed] 9. Irwin, D.J.; Lee, V.M.-Y.; Trojanowski, J.Q. Parkinson’s disease dementia: Convergence of α-synuclein, tau and amyloid-β pa-thologies. Nat. Rev. Neurosci. 2013, 14, 626–636. [CrossRef][PubMed] 10. Colović,M.B.; Krstić,D.Z.; Lazarević-Pašti,T.D.; Bondžić,A.M.; Vasić,V.M. Acetylcholinesterase inhibitors: Pharmacology and toxicology. Curr. Neuropharmacol. 2013, 11, 315–335. [CrossRef][PubMed] 11. Moss, D.E. Improving Anti-Neurodegenerative Benefits of Acetylcholinesterase Inhibitors in Alzheimer’s Disease: Are Irreversible Inhibitors the Future? Int. J. Mol. Sci. 2020, 21, 3438. [CrossRef][PubMed] 12. Galimberti, D.; Scarpini, E. Old and new acetylcholinesterase inhibitors for Alzheimer’s disease. Expert Opin. Investig. Drugs 2016, 25, 1181–1187. [CrossRef] 13. Han, S.; Sweeney, J.; Bachman, E.; Schweiger, E.; Forloni, G.; Coyle, J.; Davis, B.; Joullie, M. Chemical and pharmacological characterization of galanthamine, an acetylcholinesterase inhibitor, and its derivatives. A potential application in Alzheimer’s disease? Eur. J. Med. Chem. 1992, 27, 673–687. [CrossRef] 14. Dajas-Bailador, F.A.; Heimala, K.; Wonnacott, S. The Allosteric Potentiation of Nicotinic Acetylcholine Receptors by Galantamine Is Transduced into Cellular Responses in Neurons: Ca2+ Signals and Neurotransmitter Release. Mol. Pharmacol. 2003, 64, 1217–1226. [CrossRef] 15. Simeonova, R.; Zheleva, D.; Valkova, I.; Stavrakov, G.; Philipova, I.; Atanasova, M.; Doytchinova, I. A Novel Galantamine- Curcumin Hybrid as a Potential Multi-Target Agent against Neurodegenerative Disorders. Molecules 2021, 26, 1865. [CrossRef] 16. Takata, K.; Kitamura, Y.; Saeki, M.; Terada, M.; Kagitani, S.; Kitamura, R.; Fujikawa, Y.; Maelicke, A.; Tomimoto, H.; Taniguchi, T.; et al. Galantamine-induced Amyloid-β Clearance Mediated via Stimulation of Microglial Nicotinic Acetylcholine Receptors*. J. Biol. Chem. 2010, 285, 40180–40191. [CrossRef] 17. Bores, G.M.; Kosley, R.W. Galanthamine derivatives for the treatment of Alzheimer’s disease. Drugs Future 1996, 21, 621–635. [CrossRef] 18. Mary, A.; Renko, D.Z.; Guillou, C.; Thal, C. Potent acetylcholinesterase inhibitors: Design, synthesis, and structure–Activity relationships of bis-interacting ligands in the galanthamine series. Bioorganic Med. Chem. 1998, 6, 1835–1850. [CrossRef] 19. Guillou, C.; Mary, A.; Renko, D.Z.; Gras, E.; Thal, C. Potent acetylcholinesterase inhibitors: Design, synthesis and structure-activity relationships of alkylene linked bis-galanthamine and galanthamine-galanthaminium salts. Bioorganic Med. Chem. Lett. 2000, 10, 637–639. [CrossRef] 20. Herlem, D.; Martin, M.-T.; Thal, C.; Guillou, C. Synthesis and structure–activity relationships of open D-Ring galanthamine analogues. Bioorganic Med. Chem. Lett. 2003, 13, 2389–2391. [CrossRef] Molecules 2021, 26, 2058 14 of 14

21. Jia, P.; Sheng, R.; Zhang, J.; Fang, L.; He, Q.; Yang, B.; Hu, Y. Design, synthesis and evaluation of galanthamine derivatives as acetylcholinesterase inhibitors. Eur. J. Med. Chem. 2009, 44, 772–784. [CrossRef][PubMed] 22. Atanasova, M.; Yordanov, N.; Dimitrov, I.; Berkov, S.; Doytchinova, I. Molecular Docking Study on Galantamine Derivatives as Cholinesterase Inhibitors. Mol. Inform. 2015, 34, 394–403. [CrossRef] 23. Atanasova, M.; Stavrakov, G.; Philipova, I.; Zheleva, D.; Yordanov, N.; Doytchinova, I. Galantamine derivatives with indole moiety: Docking, design, synthesis and acetylcholinesterase inhibitory activity. Bioorganic Med. Chem. 2015, 23, 5382–5389. [CrossRef] 24. Stavrakov, G.; Philipova, I.; Zheleva, D.; Atanasova, M.; Konstantinov, S.; Doytchinova, I. Docking-based design of galantamine derivatives with dual-site binding to acetylcholinesterase. Mol. Inf. 2016, 35, 278–285. [CrossRef] 25. Stavrakov, G.; Philipova, I.; Zheleva-Dimitrova, D.; Valkova, I.; Salamanova, E.; Konstantinov, S.; Doytchinova, I. Docking-based design and synthesis of galantamine-camphane hybrids as inhibitors of acetylcholinesterase. Chem. Biol. Drug Des. 2017, 90, 709–718. [CrossRef] 26. Stavrakov, G.; Philipova, I.; Lukarski, A.; Valkova, I.; Atanasova, M.; Dimitrov, I.; Konstantinov, S.; Doytchinova, I. Acetyl- cholinesterase inhibitors selected by docking-based screening–proof-of-concept study. Bulg. Chem. Commun. 2018, 50, 40. 27. Stavrakov, G.; Philipova, I.; Lukarski, A.; Atanasova, M.; Zheleva, D.; Zhivkova, Z.D.; Ivanov, S.; Atanasova, T.; Konstantinov, S.; Doytchinova, I. Galantamine-Curcumin Hybrids as Dual-Site Binding Acetylcholinesterase Inhibitors. Molecules 2020, 25, 3341. [CrossRef] 28. Cheung, J.; Rudolph, M.J.; Burshteyn, F.; Cassidy, M.S.; Gary, E.N.; Love, J.; Franklin, M.C.; Height, J.J. Structures of Human Acetylcholinesterase in Complex with Pharmacologically Important Ligands. J. Med. Chem. 2012, 55, 10282–10286. [CrossRef] [PubMed] 29. Inestrosa, N.C.; Dinamarca, M.C.; Alvarez, A. Amyloid-cholinesterase interactions. Implications for Alzheimer’s disease. FEBS J. 2008, 275, 625–632. [CrossRef] 30. Doytchinova, I.; Atanasova, M.; Valkova, I.; Stavrakov, G.; Philipova, I.; Zhivkova, Z.; Zheleva-Dimitrova, D.; Konstantinov, S.; Dimitrov, I. Novel hits for acetylcholinesterase inhibition derived by docking-based screening on ZINC database. J. Enzym. Inhib. Med. Chem. 2018, 33, 768–776. [CrossRef] 31. Daina, A.; Michielin, O.; Zoete, V. SwissADME: A free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. Sci. Rep. 2017, 7, 42717. [CrossRef][PubMed] 32. Liu, H.; Wang, L.; Lv, M.; Pei, R.; Li, P.; Pei, Z.; Wang, Y.; Su, W.; Xie, X.-Q. AlzPlatform: An Alzheimer’s disease do-main-specific chemogenomics knowledgebase for polypharmacology and target identification research. J. Chem. Inf. Model. 2014, 54, 1050–1060. [CrossRef] 33. Ellman, G.L.; Courtney, K.; Andres, V.; Featherstone, R.M. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 1961, 7, 88–95. [CrossRef] 34. López, S.; Bastida, J.; Viladomat, F.; Codina, C. Acetylcholinesterase inhibitory activity of some Amaryllidaceae alkaloids and Narcissus extracts. Life Sci. 2002, 71, 2521–2529. [CrossRef] 35. Case, D.A.; Ben-Shalom, I.Y.; Brozell, S.R.; Cerutti, D.S.; Cheatham, T.E., III; Cruzeiro, V.W.D.; Darden, T.A.; Duke, R.E.; Ghoreishi, D.; Gilson, M.K.; et al. Amber; University of California: San Francisco, CA, USA, 2018; Available online: https://ambermd.org/ doc12/Amber18.pdf (accessed on 8 March 2021). 36. Krieger, E.; Vriend, G. YASARA View—molecular graphics for all devices—from smartphones to workstations. Bioinformatics 2014, 30, 2981–2982. [CrossRef][PubMed] 37. Daina, A.; Zoete, V. A BOILED-Egg To Predict Gastrointestinal Absorption and Brain Penetration of Small Molecules. ChemMed- Chem 2016, 11, 1117–1121. [CrossRef] 38. Ma, C.; Wang, L.; Xie, X.-Q. Ligand Classifier of Adaptively Boosting Ensemble Decision Stumps (LiCABEDS) and Its Application on Modeling Ligand Functionality for 5HT-Subtype GPCR Families. J. Chem. Inf. Model. 2011, 51, 521–531. [CrossRef] 39. Hanwell, M.D.; Curtis, D.E.; Lonie, D.C.; Vandermeersch, T.; Zurek, E.; Hutchison, G.R. Avogadro: An advanced semantic chemical editor, visualization, and analysis platform. J. Cheminform. 2012, 4, 17. [CrossRef][PubMed] 40. Halgren, T.A. Merck molecular force field. I. Basis, form, scope, parameterization, and performance of MMFF94. J. Comput. Chem. 1996, 17, 490–519. [CrossRef] 41. Momekov, G.; Ferdinandov, D.; Bakalova, A.; Zaharieva, M.; Konstantinov, S.; Karaivanova, M. In vitro toxicological evalu-ation of a dinuclear platinum(II) complex with acetate ligands. Arch. Toxicol. 2006, 80, 555–560. [CrossRef][PubMed]