New Hybrids of 4-Amino-2,3-polymethylene-quinoline and p-Tolylsulfonamide as Dual Inhibitors of Acetyl- and Butyrylcholinesterase and Potential Multifunctional Agents for Alzheimer’s Disease Treatment

New hybrid compounds of 4-amino-2,3-polymethylene-quinoline containing different sizes of the aliphatic ring and linked to p-tolylsulfonamide with alkylene spacers of increasing length were synthesized as potential drugs for treatment of Alzheimer’s disease (AD). All compounds were potent inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with selectivity toward BChE. The lead compound 4-methyl-N-(5-(1,2,3,4-tetrahydro-acridin-9-ylamino)-pentyl)-benzenesulfonamide (7h) exhibited an IC50 (AChE) = 0.131 ± 0.01 µM (five times more potent than tacrine), IC50(BChE) = 0.0680 ± 0.0014 µM, and 17.5 ± 1.5% propidium displacement at 20 µM. The compounds possessed low activity against carboxylesterase, indicating a likely absence of unwanted drug-drug interactions in clinical use. Kinetics studies were consistent with mixed-type reversible inhibition of both cholinesterases. Molecular docking demonstrated dual binding sites of the conjugates in AChE and clarified the differences in the structure-activity relationships for AChE and BChE inhibition. The conjugates could bind to the AChE peripheral anionic site and displace propidium, indicating their potential to block AChE-induced β-amyloid aggregation, thereby exerting a disease-modifying effect. All compounds demonstrated low antioxidant activity. Computational ADMET profiles predicted that all compounds would have good intestinal absorption, medium blood-brain barrier permeability, and medium cardiac toxicity risk. Overall, the results indicate that the novel conjugates show promise for further development and optimization as multitarget anti-AD agents.

Cholinesterases are still the most promising targets for ameliorating cognitive dysfunctions in AD. Accordingly, in the design of multi-target drugs, a cholinesterase inhibitor is selected as one of the pharmacophores. A second pharmacophore is then attached via a spacer to the cholinesterase inhibitor [40][41][42][43][44]. Such hybrid molecules expand the range of pharmacological activities of a parent anticholinesterase compound by adding antiaggregant, antioxidant, and other desirable properties [45][46][47][48][49][50].
Tacrine is the first drug approved for AD treatment. It has a high affinity for the AChE CAS and is widely employed as a pharmacophore to create multifunctional cholinesterase inhibitors possessing additional neuroprotective and disease-modifying properties [46,[51][52][53][54][55][56][57].
In the present work, we used 4-amino-2,3-polymethylene-quinoline with different sizes of the aliphatic ring as the main anticholinesterase pharmacophore ( Figure 1A); the homologue with n = 2 is tacrine. The aromatic p-tolylsulfonamide moiety ( Figure 1B) was used as our second pharmacophore. Sulfonamides are widely utilized in various pharmaceutical applications due to their therapeutic versatility that encompasses antibacterial, antiviral, antimalarial, antifungal, anticancer, antidepressant, and other properties [58,59]. They are also among the most effective and highly investigated derivatives in the field of carbonic anhydrases inhibition and related clinical applications [60][61][62]. Recently, publications have appeared on the presence of inhibitory activity against AChE and BChE in various sulfonamide derivatives [63][64][65][66][67]. Sulfonamide-containing selective and reversible human BChE inhibitors with nanomolar potency were developed that improved learning and memory functions in mouse models [21]. Moreover, antioxidant activity has been shown for certain sulfonamide derivatives [65,66,68,69]. Several sulfonamide-containing compounds exhibited promising Aβ self-assembly and cholinesterase inhibition, and in parallel showed good free radical scavenging properties [63].
The incorporation of a sulfonamide fragment into hybrid structures for anti-AD drugs has likewise been demonstrated. For example, a series of tacrine derivatives containing a sulfonamide group was synthesized; the compounds exhibited potent inhibitory activity on both cholinesterases and were shown to have antioxidant properties [69]. In addition, quinoline-sulfonamide hybrids have been developed as potential multifunctional therapeutic agents for AD with high anti-AChE and anti-BChE activity; the lead compound in this series was shown to improve hippocampal-dependent working memory in a rat model [70].
Herein, we have synthesized new hybrid compounds of 4-amino-2,3-polymethylene-quinoline containing different sizes of the aliphatic ring and linked to p-tolylsulfonamide using alkylene spacers of increasing length. We investigated the action of the synthesized compounds on enzyme targets of the cholinergic nervous system using as surrogates human erythrocyte acetylcholinesterase (EC 3.1.1.7, AChE) and equine serum butyrylcholinesterase (EC 3.1.1.8, BChE), along with a structurally related enzyme, porcine liver carboxylesterase (EC 3.1.1.1, CES). We also studied the ability of our novel conjugates to bind to the PAS of AChE from Electrophorus electricus (EeAChE) and competitively displace propidium iodide from this site as a measure of their potential ability to block AChE-induced aggregation of β-amyloid. Enzyme kinetics studies were used to ascertain the mechanism of inhibition, and molecular docking was employed to provide insights into the binding modes of conjugates in the AChE and BChE active sites. We also measured the The aromatic p-tolylsulfonamide moiety ( Figure 1B) was used as our second pharmacophore. Sulfonamides are widely utilized in various pharmaceutical applications due to their therapeutic versatility that encompasses antibacterial, antiviral, antimalarial, antifungal, anticancer, antidepressant, and other properties [58,59]. They are also among the most effective and highly investigated derivatives in the field of carbonic anhydrases inhibition and related clinical applications [60][61][62]. Recently, publications have appeared on the presence of inhibitory activity against AChE and BChE in various sulfonamide derivatives [63][64][65][66][67]. Sulfonamide-containing selective and reversible human BChE inhibitors with nanomolar potency were developed that improved learning and memory functions in mouse models [21]. Moreover, antioxidant activity has been shown for certain sulfonamide derivatives [65,66,68,69]. Several sulfonamide-containing compounds exhibited promising Aβ self-assembly and cholinesterase inhibition, and in parallel showed good free radical scavenging properties [63].
The incorporation of a sulfonamide fragment into hybrid structures for anti-AD drugs has likewise been demonstrated. For example, a series of tacrine derivatives containing a sulfonamide group was synthesized; the compounds exhibited potent inhibitory activity on both cholinesterases and were shown to have antioxidant properties [69]. In addition, quinoline-sulfonamide hybrids have been developed as potential multifunctional therapeutic agents for AD with high anti-AChE and anti-BChE activity; the lead compound in this series was shown to improve hippocampal-dependent working memory in a rat model [70].
Herein, we have synthesized new hybrid compounds of 4-amino-2,3-polymethylene-quinoline containing different sizes of the aliphatic ring and linked to p-tolylsulfonamide using alkylene spacers of increasing length. We investigated the action of the synthesized compounds on enzyme targets of the cholinergic nervous system using as surrogates human erythrocyte acetylcholinesterase (EC 3.1.1.7, AChE) and equine serum butyrylcholinesterase (EC 3.1.1.8, BChE), along with a structurally related enzyme, porcine liver carboxylesterase (EC 3.1.1.1, CES). We also studied the ability of our novel conjugates to bind to the PAS of AChE from Electrophorus electricus (EeAChE) and competitively displace propidium iodide from this site as a measure of their potential ability to block AChE-induced aggregation of β-amyloid. Enzyme kinetics studies were used to ascertain the mechanism of inhibition, and molecular docking was employed to provide insights into the binding modes of conjugates in the AChE and BChE active sites. We also measured the radical-scavenging activity of the conjugates in the ABTS assay. Finally, to assess the potential pharmacokinetic properties of the new compounds, we computationally estimated their profiles for absorption, distribution, metabolism, excretion, and toxicity (ADMET). Some preliminary results have been published earlier [71].

Chemistry
We have developed a method for the synthesis of hybrid multi-target compounds 7a-k based on 4-amino-2,3-polymethylene-quinolines as the first pharmacophore and p-tolylsulfonamide as the second pharmacophore. Previously, we synthesized such compounds 7b,f,i,k in which the quinoline and sulfonamide pharmacophores were combined into one molecule with a diaminopropane linker, in the form of hydrochlorides [71]. Here, we devised a procedure for the synthesis of target conjugates in the form of free bases. As shown in Figure 2, these compounds were obtained by the reaction of aminoquinolines 5 with tosyl chloride 6 in the presence of triethylamine. The aminoquinolines 5 needed for this were synthesized by the following method. First, by boiling anthranilic acid (2) with ketones 1 (cyclopentanone, cyclohexanone, cycloheptanone or cyclooctanone) in phosphorus oxychloride, chlorine derivatives 3 were obtained. Then, derivatives 3 were condensed with diamines 4 according to a published procedure [72] to afford the necessary aminoquinolines 5.

. Chemistry
We have developed a method for the synthesis of hybrid multi-target compounds 7a-k based 4-amino-2,3-polymethylene-quinolines as the first pharmacophore and p-tolylsulfonamide as the ond pharmacophore. Previously, we synthesized such compounds 7b,f,i,k in which the quinoline sulfonamide pharmacophores were combined into one molecule with a diaminopropane linker, the form of hydrochlorides [71]. Here, we devised a procedure for the synthesis of target jugates in the form of free bases. As shown in Figure 2, these compounds were obtained by the ction of aminoquinolines 5 with tosyl chloride 6 in the presence of triethylamine. The inoquinolines 5 needed for this were synthesized by the following method. First, by boiling hranilic acid (2) with ketones 1 (cyclopentanone, cyclohexanone, cycloheptanone or looctanone) in phosphorus oxychloride, chlorine derivatives 3 were obtained. Then, derivatives 3 re condensed with diamines 4 according to a published procedure [72] to afford the necessary inoquinolines 5.
Thus, conjugates 7 with different sizes of the aliphatic ring of mino-2,3-polymethylene-quinolines and various lengths of the alkyl chain linking the two rmacophores have been synthesized. The size of the aliphatic ring and the length of the alkyl cer for each compound 7 are shown in Table 1.

. Inhibition Studies of AChE, BChE and CES. Structure-Activity Relationships
For all conjugates 7, we determined their esterase profile, i.e., the relative ability to inhibit eral related serine esterases -AChE, BChE, and CES. This approach makes it possible to estimate h the primary pharmacological effects of the tested compounds (the inhibition of AChE and hE) and their possible unwanted side effects (the inhibition of CES that hydrolyzes numerous er-containing drugs) [73][74][75][76][77]. AChE from human erythrocytes was used along with two enzymes non-human origin: equine serum BChE and porcine liver CES. These sources of BChE and CES Thus, conjugates 7 with different sizes of the aliphatic ring of 4-amino-2,3-polymethylenequinolines and various lengths of the alkyl chain linking the two pharmacophores have been synthesized. The size of the aliphatic ring and the length of the alkyl spacer for each compound 7 are shown in Table 1.

Inhibition Studies of AChE, BChE and CES. Structure-Activity Relationships
For all conjugates 7, we determined their esterase profile, i.e., the relative ability to inhibit several related serine esterases -AChE, BChE, and CES. This approach makes it possible to estimate both the primary pharmacological effects of the tested compounds (the inhibition of AChE and BChE) and their possible unwanted side effects (the inhibition of CES that hydrolyzes numerous ester-containing drugs) [73][74][75][76][77]. AChE from human erythrocytes was used along with two enzymes of non-human origin: equine serum BChE and porcine liver CES. These sources of BChE and CES were used because of their relatively low cost, high sequence identity to human enzymes [73,74,76], and the exploratory character of this work.
The inhibitory activities of our conjugates against the esterases were characterized as IC 50 values, i.e., the inhibitor concentrations required to decrease a given enzyme activity by 50%. Tacrine, an effective AChE and BChE inhibitor, and bis-4-nitrophenyl phosphate (BNPP), a selective CES inhibitor, were used as positive controls. The results are shown in Table 1.
As can be seen from Table 1, conjugates 7 generally inhibited both cholinesterases effectively, exhibiting, like tacrine, a higher activity toward BChE. The activities of compounds 7b,f,i,k obtained here coincided with the previously determined results [71]. Conjugates 7 practically did not inhibit CES, which suggests that potential unwanted drug-drug interactions would be lacking if these compounds were used clinically for AD treatment.
The degree and selectivity of enzyme inhibition was found to be determined by the structure of the synthesized conjugates; namely, the aliphatic ring size of the 4-amino-2,3-polymethylene-quinoline fragment (i.e., the tacrine moiety) and the length of the spacer linking the pharmacophores. The effect of the aliphatic ring size was demonstrated by compounds 7b, 7f, 7i, 7k with a spacer length m = 3 (Table 1). The most active compound toward AChE was conjugate 7f with an unmodified six-membered tacrine ring (C-6, n = 2), whose inhibitory activity was only three times lower than that of tacrine. The conjugate 7i with a seven-membered tacrine ring (C-7, n = 3) exhibited maximum activity toward BChE: it inhibited the enzyme in the nanomolar range with an IC 50 = 0.044 µM, close to that of tacrine. An increase in the size of the aliphatic ring to 8 (C-8, 7k) reduced the anti-AChE activity ten-fold and decreased anti-BChE activity six-fold. Thus, the six-membered aliphatic ring of tacrine was optimal for the anti-AChE activity of conjugates 7, whereas a 7-membered ring was best for their anti-BChE potency.
An increase in the spacer length (m) for conjugates with the same ring size in series 7a-7e (C-5, n = 1), 7f-7h (C-6, n = 2), and 7i-7j (C-7, n = 3) led to an increase in the inhibitory activity of compounds toward AChE. The most active among compounds with a five-membered ring were compounds 7c and 7d containing four and five methylene groups, respectively. In a series of compounds with a six-membered tacrine aliphatic ring 7f-7h (C-6, n = 2), the inhibitory activity toward AChE increased more than 14 times with an increase in the length of the spacer from three to five CH 2 groups. Compound 7h exhibited the maximum anti-AChE activity, being five times more potent than tacrine.
The results are consistent with the known data that, in contrast to extremely powerful bis-tacrine inhibitors [78], tacrine-based hybrid compounds linking tacrine with an uncharged aromatic group had anti-AChE activities comparable to that of tacrine [79,80].
In the case of anti-BChE activity, it was not possible to reveal a clear pattern of the dependence of enzyme inhibition on the spacer length of the conjugate. The maximum anti-BChE activity, close to that of tacrine, was found for compounds 7g and 7h with a six-membered tacrine ring and a spacer length of four and five CH 2 groups, respectively, as well as for compounds 7i and 7j with a seven-membered tacrine aliphatic ring and a spacer length of three and four CH 2 groups, respectively.

Kinetic Studies of AChE and BChE Inhibition
The mechanism of inhibitory action of conjugates 7 towards AChE and BChE was studied using compound 7g as an example. The graphical analysis of the kinetic data on AChE ( Figure 3A) and BChE ( Figure 3B) inhibition by 7g in the Lineweaver-Burk double-reciprocal plot demonstrates the changes in both K m and V max that attests to a mixed type of inhibition. The inhibition constants were K i = 0.390 ± 0.008 µM (competitive component) and

Molecular Docking Studies
The differential interaction of bulky inhibitors with AChE and BChE is highly dependent on the difference in structure of the active site gorge that leads from the protein surface to the active site [49,50,[81][82][83][84]. AChE has a narrow gorge divided by the so-called bottleneck into the PAS and CAS [85,86]. The AChE gorge is lined with aromatic residues that favor binding of positively charged substrates/inhibitors, and the dynamically changing radius of the bottleneck determines

Molecular Docking Studies
The differential interaction of bulky inhibitors with AChE and BChE is highly dependent on the difference in structure of the active site gorge that leads from the protein surface to the active site [49,50,[81][82][83][84]. AChE has a narrow gorge divided by the so-called bottleneck into the PAS and CAS [85,86]. The AChE gorge is lined with aromatic residues that favor binding of positively charged substrates/inhibitors, and the dynamically changing radius of the bottleneck determines the kinetics of binding; in particular, slow-binding inhibition [87,88]. BChE has a wider gorge than AChE without a pronounced narrowing to separate the PAS and CAS, and most of the BChE gorge residues that are aromatic in AChE are replaced in BChE with aliphatic ones [86]. This makes the BChE gorge more permissive to uncharged or even negatively charged substrates and inhibitors [89], and less prone to slow-binding inhibition than is the case with AChE [90,91].
Molecular docking of conjugates 7 into AChE demonstrated that they are dual binding-site inhibitors. This confirms our previous finding [49,71]. The tacrine fragment binds in the CAS and the p-tolylsulfonamide moiety binds to the PAS. Comparison of docking results of the compounds to different X-ray structures of human AChE showed that the best binding poses were obtained with the X-ray structure of AChE co-crystallized with donepezil (PDB: 4EY7). This structure has conformational changes of CAS and PAS amino acids necessary to accommodate bulky inhibitors [92].
Molecular docking of compounds 7b, 7f, 7i, 7k with the spacer m = 3 explains the observed effect of the aliphatic ring size on the inhibitory activity toward AChE. The position of the 4-amino-2,3-polymethylene-quinoline (tacrine) ring inside the AChE active site is similar for compounds 7b, 7f, 7i, but not for compound 7k with the largest C-8 aliphatic ring ( Figure 4A). The increased size of the aliphatic ring does not allow it to fit in the same region below the bottleneck; the displaced C-8 tacrine ring leads to the loss of a hydrogen bond between the protonated amino group of the tacrine fragment and the Tyr124 side chain. This results in the experimentally observed reduced binding affinity.
In the case of binding to BChE, the influence of the aliphatic ring size is not as pronounced as with AChE; all tacrine fragments are located inside the BChE gorge in a uniform way ( Figure 4B), with the sulfonamide group binding to the active site and forming hydrogen bonds with catalytic residues and the oxyanion hole. The protonated amino group in the tacrine fragment forms ionic interactions with Asp70 in the PAS. However, for compound 7k (C-8), the large aliphatic ring size impairs ionic interactions of the positively charged external amino group of the tacrine fragment with the Asp70 anionic side chain. Otherwise, compound 7i with a 7-membered ring did not have more specific interactions than compounds 7b and 7f, but it appeared to fit better than the others in the active site gorge cavity.
As was observed earlier for other dual binding site AChE inhibitors with lengthy spacers [49,50,[81][82][83][84], they are located inside the AChE gorge in an elongated conformation whereas they are bent inside the wider BChE gorge. Consequently, an increase in the spacer length for conjugates with the same ring size leads to better occupation of the AChE PAS by the p-tolylsulfonamide fragment. Figure 4C shows binding poses of compounds 7a-7e (C-5) as an example, while for compounds 7f-7h (C-6) the same trend was observed. Although for the compounds with a shorter linker (7a, 7b) hydrogen bonding between the sulfo-group and the Tyr124 side chain is possible while no specific interactions of the conjugate molecules in the PAS were observed, more favorable occupancy of the PAS by compounds with a longer linker leads to better propidium displacement and increased inhibitory activity due to hydrophobic interactions. In contrast, the position of the compounds inside the BChE gorge was almost unaffected by spacer length (Figure 4D), in accordance with the weak influence of the spacer length on experimentally measured inhibitory activities.
Thus, the molecular docking results show that the differences in the structure of the active sites of AChE and BChE give rise to different structure-inhibitory activity relationships for inhibition of these enzymes by these hybrid inhibitors. Thus, the molecular docking results show that the differences in the structure of the active sites of AChE and BChE give rise to different structure-inhibitory activity relationships for inhibition of these enzymes by these hybrid inhibitors.

Displacement of Propidium Iodide from the PAS of EeAChE
Propidium is a selective ligand for the PAS of AChE responsible for the Aβ binding [24,29,30,93], which showed a significant decrease in AChE-induced Aβ aggregation (82% at 100 µM) [26]. This observation has served as the basis for the fluorescent evaluation of competitive propidium displacement from the PAS of AChE as a screening method to assess the potential ability of compounds to bind PAS and thus block the pro-aggregation activity of AChE [28]. Propidium iodide exhibits a fluorescence increase upon binding to AChE. A decrease in propidium fluorescence in the presence of test compounds indicates that they are able to displace propidium and bind to the PAS of AChE, suggesting that they would thereby block the AChE-mediated aggregation of β-amyloid [24,27,29,33].
Here, donepezil was used as a reference compound. Donepezil is a mixed-type AChE inhibitor, for which the ability to block AChE-PAS-induced Aβ aggregation has been demonstrated [26]. The results are presented in Table 1.
It can be seen in Table 1 that the sulfonamide-containing conjugates 7 at a concentration of 20 µM reduced the fluorescence intensity by 9-17% and more effectively displaced propidium from the PAS of AChE compared to the reference compound donepezil. For compounds 7f, i, there were no differences with the results obtained earlier [71]. For all 4-amino-2,3-polymethylene-quinoline-sulfonamide heterodimers with five, six, or seven-membered aliphatic rings, the degree of propidium displacement increased with elongation of the spacer length, which agrees with the results of molecular docking.
Taken together, the results from propidium displacement, kinetics, and molecular docking indicate that the conjugates 7 are dual-site AChE inhibitors occupying both CAS and PAS pockets. Thanks to binding to the PAS of AChE, they can potentially block AChE-induced aggregation of β-amyloid, thus having an additional positive disease modification effect.

Antioxidant Activity
The presence of antioxidant activity in a number of sulfonamide derivatives [66,68,69]

Predicted ADMET Profiles and PAINS Analysis
The results of the computational estimation of a number of ADMET and physicochemical properties for the compounds 7a-k are shown in Table 2. As can be seen, all of the compounds had high predicted values for intestinal absorption, enabling their oral administration. Thanks to moderate predicted blood-brain barrier permeability (brain concentration is about 10-25% of the plasma concentration), sufficient CNS activity can be expected. Both parameters of the cardiac toxicity risk (pK i and pIC 50 ) for all analyzed compounds (4.7-6.5 log units) are in the lower or medium part of their possible range (3-9 log units). Although these values are acceptable, additional studies and structure optimization are desirable for a few compounds in order to ensure maximal safety. The predicted lipophilicities and aqueous solubilities of the compounds are also good for potential drug compounds. The integral quantitative estimates of drug-likeness (QED) are above 0.3 for all the compounds and above 0.5 for most of them. The Pan Assay INterference compoundS (PAINS) filter check for the compounds listed in Table 2 did not identify any structural alerts. Thus, the predicted ADMET, physicochemical, and PAINS properties of the compounds 7a-k are quite acceptable for potential lead compounds at the early drug development stages.

Chemistry
All solvents, chemicals, and reagents were obtained commercially and used without purification. 1 H-NMR (200 MHz) spectra were recorded on a DPX-200 NMR spectrometer (Bruker, Karlsruhe, Germany) using tetramethylsilane as an internal standard. Chemical shifts, δ, are given in parts per million (ppm), and spin multiplicities are given as s (singlet), br s (broad singlet), d (doublet), t (triplet), q (quartet) or m (multiplet). Coupling constants, J, are expressed in hertz (Hz). Melting points were recorded on a SMP10 Melting Point Apparatus (Stuart, Staffordshire, UK) and are uncorrected. Yields refer to isolated pure products and were not maximized. CHN analysis was performed on the ER-20 analyzer (Carlo-Erba, Val-de-Reuil, France). All compounds exhibited analytical and spectroscopic data that strongly agreed with their expected structures.

Synthesis of Compounds
The synthesis and characteristics of compounds are described below. The original NMR spectra are presented in the Supplementary Materials.

General Procedure for the Preparation of Derivatives 7a-k
A solution of tosyl chloride (190.65 mg, 1 mmol) in methylene chloride (5 mL) was added to a solution of NEt 3 (2 mmol) and aminoquinoline 5 (1 mmol) in methylene chloride (4 mL). The reaction mixture was stirred for 1 h at room temperature, diluted with 10 mL of water, and extracted with ethyl acetate (3 × 10 mL). The combined extracts were washed with brine, dried over anhydrous Na 2 SO 4, and evaporated under vacuum to give the required crude product that was recrystallized from isopropanol.
AChE and BChE activities were measured by the colorimetric method of Ellman et al. [94]. The assay solution consisted of 0.1 M K/Na phosphate buffer pH 7.5, 25 • C, 0.33 mM DTNB, 0.02 unit/mL AChE or BChE, and 1 mM substrate (ATCh or BTCh, respectively). Reagent blanks consisted of reaction mixtures without substrates.
The activity of CES was determined spectrophotometrically at 405 nm to monitor the release of 4-nitrophenol [95] in 0.1 M K/Na phosphate buffer pH 8.0, 25 • C. Final enzyme and substrate (4-nitrophenyl acetate) concentrations were 0.02 unit/mL and 1 mM, respectively. Assays were carried out with a blank containing all constituents except porcine CES to assess non-enzymatic hydrolysis.
Test compounds were dissolved in DMSO; reaction mixtures contained 2% (v/v) of the solvent, a concentration shown not to affect the activity of the enzymes on its own (data not shown). Tacrine, donepezil, and bis-4-nitrophenyl phosphate (BNPP), a selective CES inhibitor, were used as positive controls.
An initial evaluation of inhibitory activity of compounds was carried out by determination of the AChE, BChE and CES inhibition at a compound concentration of 20 µM. For this, a sample of the corresponding enzyme was incubated with the test compound for 5 min at 25 • C for temperature equilibration; then the enzyme residual activity was determined. Each experiment was performed in triplicate.
For the most active compounds, the IC 50 values (the concentration of inhibitor required to decrease the enzyme activity by 50%) were determined. Eight different concentrations of the test compounds in the range 10 −12 -10 −4 M were selected in order to obtain inhibition of esterase activity between 20% and 80%. The test compounds were added to the assay solution and incubated with the enzyme at 25 • C for 5 min (for temperature equilibration) followed by the addition of substrate. A parallel control was made for the assay solution with no inhibitor.
Measurements were performed in the FLUOStar OPTIMA microplate spectrophotometer (BMG Labtech, Ortenberg, Germany). Each experiment for the IC 50 assay was performed in triplicate. The results were expressed as the mean ± SEM. The reaction rates in the presence and absence of inhibitor were compared, and the percent residual enzyme activity due to the presence of test compounds was calculated. IC 50 values were determined graphically from inhibition curves (log inhibitor concentration vs. percent residual enzyme activity) using Origin 6.1 software (OriginLab, Northampton, MA, USA).

• Kinetic Study of AChE and BChE Inhibition. Determination of Steady-State Inhibition Constants
Mechanisms of AChE and BChE inhibition were assessed via a thorough analysis of enzyme kinetics. Residual activity was measured following 5 min incubation at 25 • C (for temperature equilibration) with three increasing concentrations of inhibitor and six decreasing substrate concentrations. Substrate was added immediately after the 5 min incubation with inhibitor and the rates of absorption were monitored at 412 nm using the FLUOStar OPTIMA (BMG Labtech) microplate spectrophotometer. Inhibition constants K i (competitive component) and αK i (noncompetitive component) were determined by linear regression of 1/V versus 1/[S] double-reciprocal (Lineweaver-Burk) plots using Origin 6.1 software. Data were expressed as mean ± SEM (n = 3 experiments).

Propidium Displacement Studies
The ability of the test compounds to competitively displace propidium, a selective ligand of the PAS of AChE, was evaluated by the fluorescence method [93,96]. EeAChE was used owing to its high degree of purification, high activity, and lower cost than human AChE (hAChE). In addition, we performed 3D alignment of the crystal structures of EeAChE (PDB: 1C2O) and hAChE (PDB: 4EY7) using YASARA-Structure 18.4.24 for Windows, which showed that the two structures were essentially congruent with an RMSD of 0.623 Å over 527 aligned residues and 88.6% sequence identity. The fluorescence intensity of propidium iodide bound with AChE increases several times; decreasing fluorescence intensity of the bound propidium in the presence of the test compounds shows their ability to bind to the PAS of AChE [24,27].
To determine the degree of displacement (% displacement) of propidium from the PAS of AChE, EeAChE (final concentration, 7 µM) was incubated with the test compound at a concentration of 20 µM in 1 mM Tris-HCl buffer pH 8.0, 25 • C, for 15 min. Then, propidium iodide solution (final concentration, 8 µM) was added, the samples were incubated for 15 min and the fluorescence spectrum (530 nm (excitation) and 600 nm (emission)) was taken. Donepezil and tacrine were used as reference compounds. The blank contained propidium iodide of the same concentration in 1 mM Tris-HCl buffer pH 8.0. The measurements were carried out in triplicate on the FLUOStar Optima microplate reader (BMG Labtech), and the results were calculated by the following formula: (1) where IF AChE + Propidium is the fluorescence intensity of the propidium associated with AChE in the absence of the test compound (taken as 100%), and IF AChE + Propidium + inhibitor is the fluorescence intensity of the propidium associated with AChE in the presence of the test compound.
The solution of cation radical ABTS •+ was produced by incubation of ABTS with potassium persulfate in deionized water for 12-16 h at room temperature in the dark. Radical scavenging capacity of the compounds was analyzed by mixing 10 µL of compound with 240 µL of ABTS •+ working solution in ethanol (100 µM final concentration). After 1 h of incubation, the reduction in absorbance was measured spectrophotometrically at 734 nm using the xMark microplate UV/VIS microplate spectrophotometer (Bio-Rad, Hercules, CA, USA). Ethanol blanks were run in each assay. Values were obtained from three replicates of each sample and three independent experiments. Trolox was used as the antioxidant standard.
The antioxidant activity is reported as Trolox equivalent antioxidant capacity (TEAC values) by comparing (A 0 − A test ) of the test antioxidant with (A 0 − A Trolox ) of the Trolox standard at a concentration of 20 µM after a reaction time of 1 h. A 0 is the absorbance of a control lacking any radical scavenger and A test/Trolox is the absorbance of the remaining ABTS •+ in the presence of the test compound or Trolox, respectively:

Prediction of ADMET Profiles and PAINS Analysis
Human intestinal absorption (HIA) [105], blood-brain barrier permeability (LogBB) [106], and hERG-mediated cardiac toxicity risk (channel affinity pK i and inhibitory activity pIC 50 ) [107] were estimated using the integrated online service for ADMET properties prediction (ADMET Prediction Service) [108]. It implements the predictive QSAR models based on accurate and representative training sets, fragmental descriptors, and artificial neural networks. The lipophilicity (LogPow) and aqueous solubility (pS) were estimated by the ALogPS 3.0 neural network model implemented in the OCHEM platform [109]. The quantitative estimate of drug-likeness (QED) values [110] were calculated and the Pan Assay INterference compoundS (PAINS) alerts were checked using the RDKit version 2019.03.4 software [111].

Statistical Analyses
Results are presented as mean ± SEM calculated using GraphPad Prism version 6.05 (San Diego, CA, USA) for Windows. Plots, linear regressions, and IC 50 values were determined using Origin 6.1 (Northampton, MA, USA) for Windows.

Conclusions
New hybrids of 4-amino-2,3-polymethylene-quinoline and p-tolylsulfonamide with different aliphatic ring sizes and alkylene spacers of increasing length were synthesized and their structures established. All synthesized compounds exhibited high dual anticholinesterase activity and greater selectivity toward BChE. They possessed low anti-CES activity, suggesting the absence of potential unwanted drug-drug interactions in prospective clinical use.
Kinetics studies were consistent with mixed-type reversible inhibition. Molecular docking into AChE and BChE provided detailed insights into the binding modes demonstrating dual-site binding of the conjugates in AChE and clarifying differences in the structure-activity relationships for AChE and BChE inhibition.
The conjugates had the ability to bind to the AChE PAS and displace propidium, indicating their potential to block AChE-induced β-amyloid aggregation, thereby potentially exerting disease-modifying capability. All compounds demonstrated low antioxidant activity.
Computational ADMET profiles predicted that all the conjugates should have good intestinal absorption, medium blood-brain barrier permeability, and medium cardiac toxicity risks. Moreover, the PAINS filter check for the compounds did not identify any alerts.
Overall, the developed hybrid compounds demonstrated potent dual anticholinesterase activity along with the predicted propensity to block AChE-induced aggregation of β-amyloid. Thus, they have the potential for treating symptoms as well as exerting disease-modifying effects and show promise for further development and optimization as multitarget anti-AD agents. In particular, the lead compound 4-methyl-N-(5-(1,2,3,4-tetrahydro-acridin-9-ylamino)-pentyl)benzenesulfonamide (7h), with an unmodified six-membered tacrine ring and m = 5, exhibited maximum anti-AChE activity with IC 50 = 0.131 ± 0.01 µM (five times more potent than tacrine) and high anti-BChE activity. It is a mixed-type AChE inhibitor with the highest percentage of propidium displacement, very good intestinal absorption and medium predicted blood-brain barrier permeability. Funding: This research was partly supported by grant #19-53-26016_a of the Russian Foundation for Basic Research and by the IPAC RAS State Targets Project # 0090-2019-0005. Support for RJR's contributions was provided by the University of Michigan. The funding sources had no role in the study design; collection, analysis and interpretation of data; writing of the manuscript; or the decision to submit the article for publication.