**1-(3-***Tert***-Butylphenyl)-2,2,2-Trifluoroethanone as a Potent Transition-State Analogue Slow-Binding Inhibitor of Human Acetylcholinesterase: Kinetic, MD and QM**/**MM Studies**

#### **Irina V. Zueva <sup>1</sup> , Sofya V. Lushchekina <sup>2</sup> , Ian R. Pottie 3,4 , Sultan Darvesh 3,5 and Patrick Masson 6,\***


Received: 23 October 2020; Accepted: 24 November 2020; Published: 27 November 2020 -

**Abstract:** Kinetic studies and molecular modeling of human acetylcholinesterase (AChE) inhibition by a fluorinated acetophenone derivative, 1-(3-tert-butylphenyl)-2,2,2-trifluoroethanone (TFK), were performed. Fast reversible inhibition of AChE by TFK is of competitive type with *K<sup>i</sup>* = 5.15 nM. However, steady state of inhibition is reached slowly. Kinetic analysis showed that TFK is a slow-binding inhibitor (SBI) of type B with *Ki*\* = 0.53 nM. Reversible binding of TFK provides a long residence time, τ = 20 min, on AChE. After binding, TFK acylates the active serine, forming an hemiketal. Then, disruption of hemiketal (deacylation) is slow. AChE recovers full activity in approximately 40 min. Molecular docking and MD simulations depicted the different steps. It was shown that TFK binds first to the peripheral anionic site. Then, subsequent slow induced-fit step enlarged the gorge, allowing tight adjustment into the catalytic active site. Modeling of interactions between TFK and AChE active site by QM/MM showed that the "isomerization" step of enzyme-inhibitor complex leads to a complex similar to substrate tetrahedral intermediate, a so-called "transition state analog", followed by a labile covalent intermediate. SBIs of AChE show prolonged pharmacological efficacy. Thus, this fluoroalkylketone intended for neuroimaging, could be of interest in palliative therapy of Alzheimer's disease and protection of central AChE against organophosphorus compounds.

**Keywords:** acetylcholinesterase; slow-binding inhibition; transition state analog; organophosphorus

#### **1. Introduction**

Fluoroalkylketones (FAK) are potent inhibitors of acetyl cholinesterase (AChE, ES.3.1.1.7) and butyrylcholinesterase (BChE, EC.3.1.1.8) [1–4]. A characteristic of inhibition by these compounds is the slow establishment of equilibrium between enzyme and inhibitor. This process is called slow-binding inhibition (SBI). Unlike classical reversible inhibitors for which equilibrium establishes within microseconds, in SBI, establishment of equilibrium may take seconds, minutes or hours. Three types of SBI have been described: (1) type A is characterized by a single step mechanism with slow *kon* and *ko*ff; (2) type B is a two-step mechanism: after rapid formation of a first enzyme-inhibitor complex, a slow induced-fit step occurs; (3) type C results from the existence of several enzyme forms in slow equilibrium that determine a slow conformational selection for inhibition [5].

Kinetic analysis of AChE inhibition by 1-[*3*-(trimethylamino)phenyl]-2,2,2-trifluoro-1-ethanone (TMTFA) (Figure 1A) showed that this compound is a slow-binding inhibitor of type A for *Torpedo californica* AChE (*K<sup>i</sup>* = 15 × 10−<sup>15</sup> M; *t* diss <sup>1</sup>/<sup>2</sup> = 2.8 h) [3] whereas it a slow-binding inhibitor of type B for electric eel (*Electrophorus electricus*) AChE (*K<sup>i</sup>* = 1.3 × 10−<sup>15</sup> M; *t* diss <sup>1</sup>/<sup>2</sup> = 19 h) [3,4]. Later, the X-ray structure of *Torpedo californica* AChE-TMTFA complex (PDB ID 1AMN [6]) showed that the tight interactions between the enzyme and inhibitor are similar to interactions that take place in the enzyme active center with acetylcholine in the transition state [6]. Thus, FAK, first considered as quasi-substrate inhibitors, are in fact transition state analogues.

**Figure 1.** Chemical structure of related trifluoromethylketone molecules: (**A**): 1-[*3*-(trimethylamino) phenyl]-2,2,2-trifluoro-1-ethanone (TMTFA); (**B**): 2,2,2-trifluoro-1-[*m*-(trimethylsilyl)phenyl]-1-ethanone (Zifrosilone); (**C**): 1-(3-*tert*-butylphenyl)-2,2,2-trifluoroethanone (TFK).

In early 1990s, when AChE inhibitors started to be developed for the palliative treatment of Alzheimer disease (AD), a related silyl compound, Zifrosilone (Figure 1B) [7] was considered as a promising anti-AD symptomatic drug. Though pharmacokinetic/pharmacodynamic (PK/PD) studies were advanced, human clinical trials were discontinued in mid-90s [8–10]. Yet, incorporation of a silicone atom does not induce additional toxicity to molecules of pharmacological interest [11]. After a first report on inhibition of human cholinesterases (hChEs) by a family of <sup>18</sup>F-acetophenones developed for PET neuroimaging [12], it was of interest to investigate thoroughly the carbon analogue of Zifrosilone (Figure 1B), 1-(3-*tert*-butylphenyl)-2,2,2-trifluoroethanone (TFK) (Figure 1C), to describe the mechanism of inhibition of hAChE by kinetics and molecular modeling approaches. Moreover, possible modulation and/or protection of AChE by TFK against OP phosphylation was explored.

#### **2. Materials and Methods**

#### *2.1. Chemicals*

TFK was synthesized as described in [12]. Solution of TFK (0.1 M) was made in acetonitrile. Echothiophate iodide was from Biobasal AG (Basel, Switzerland). Stock solution of echothiophate (0.1 M) was made in water. Paraoxon was purchased from Sigma-Aldrich (Saint Louis, MO, USA). Stock solution of paraoxon (0.1 M) was made in EtOH. Cresylsaligenyl phosphate (CSP) was a gift from Prof O. Lockridge (UNMC, Omaha, NE, USA). Solution of CSP (0.1 M) was made in acetonitrile. Acetylthiocholine iodide was from Sigma-Aldrich (Saint Louis, MO, USA). Stock solution of ATC (0.1 M) was made in water. Stock solutions of substrate and inhibitors were stored at −20 ◦C.

Dithiobisnitrobenzoic acid (DTNB) was from Sigma-Aldrich (Saint Louis, MO, USA). 50 mM solution of DTNB was prepared as described in Ellman [13]. Calbiochem Probe IV (3-(7-Hydroxy-2-oxo-2H-chromen-3-ylcarbamoyl) acrylic acid methylester) was from Merck Millipore (Darmstadt, Germany). Stock solution of Probe IV (1 mM) was in DMSO and stored at −20 ◦C. All other chemicals were of biochemical grade.

#### *2.2. Enzymes*

Recombinant human AChE monomer (MW = 70,000 Da) was expressed in CHO-K1 (Chinese-hamster ovary, ATCC) cells [14]. AChE purification was carried out as described [15] The enzyme was concentrated to 14.7 mg/mL using a Vivaspin 6 (30,000 MW cutoff, Sartorius) and dialyzed at 4 ◦C against 10 mM HEPES pH 7.5 buffer containing 10 mM NaCl. The active site concentration of AChE was determined by the method of residual activity according to Leuzinger [16], using echothiophate, as titrating agent. The residual activity after complete phosphorylation by each concentration of echothiophate was measured by the Ellman's method [13] in 0.1 M sodium phosphate, pH 8.0 at 25 ◦C with 1 mM ATC as the substrate. The active site concentration of the pure rhuAChE monomer was 2.1 × 10−<sup>4</sup> M. The working enzyme was diluted 20,000 time in 0.1 M phosphate buffer containing 1 mg/mL BSA ([E]<sup>w</sup> = 1 × 10−<sup>8</sup> M).

#### *2.3. Kinetic Study of Inhibition*

Kinetic studies of human recombinant AChE inhibition by TFK were performed in 0.1 M sodium phosphate buffer pH 8.0 at 25 ◦C, using the Ellman's method [13] with acetylthiocholine (ATC) as the substrate. Three different concentrations of ATC were used (0.1, 0.5 and 1 mM): Assays were initiated by addition of the enzyme. The final enzyme concentration, E, was 1 × 10−<sup>10</sup> M.

After rapid mixing of solutions, absorbance change 412 nm was recorded for 60 min using a spectrophotometer PerkinElmer λ25 with photodiode detector (PerkinElmer Inc., Waltham, MA, USA). The inhibition process was characterized by a slow onset before reaching steady state. This process is described by Equation (1):

$$[P]\_t = v\_{\rm ss}t + \frac{(v\_i - v\_{\rm ss})(1 - \exp(-k\_{\rm obs}t))}{k\_{\rm obs}}\tag{1}$$

where *v<sup>i</sup>* is the initial velocity, *vss* the steady-state velocity and *kobs*, the first-order rate constant associated with establishment of the steady state. The reciprocal of *kobs* is the lag time before steady state. This is characteristic of slow binding inhibition [17–19].

The initial reversible inhibition step was analyzed from the tangents of progress curves. The general case for linear reversible inhibition is described by following rate Equation (2):

$$v\_{i} = \frac{V\_{\text{max}}[\text{S}]}{K\_{m}[1 + ([I]/K\_{ci})] + [\text{S}](1 + ([I]/K\_{ui}))} \tag{2}$$

where *Vmax* is the maximum velocity, [*S*] the substrate concentration, *K<sup>m</sup>* the Michaelis-Menten constant, [*I*], the inhibitor concentration, *Kci*, the competitive inhibition constant, and *Kui*, the uncompetitive inhibition constant. Rearranging Equation (2) as 1/*v<sup>i</sup>* or [*S*]/*v<sup>i</sup>* as a function of [*I*] provides two complementary plots as decribed by Cornish-Bowden [20]. In the limiting case of competitive inhibition, *Kui*→∝, while for uncompetitive inhibition, *Kci*→∝. Non-competitive inhibition gives *Kci* = *Kui* and mixed-type inhibition gives *Kci* , *Kui*. Therefore, inhibition constant and type of reversible inhibition of the fast step were determined according to the graphical methods of Cornish-Bowden [20] by building Dixon plots (1/*v<sup>i</sup>* vs. [TFK]) and Cornish-Bowden plots ([ATC]/*v<sup>i</sup>* vs. [TFK]) at 3 different ATC concentrations [*S*] = 0.1; 0.5 and 1 mM).

Diagnosis of type of SBI and binding kinetic parameters and inhibition constant corresponding to final reversible step where determined from secondary plots, *kobs* vs. [TFK] at 3 different ATC concentration [5].

For the study of transient enzyme acylation by 2 × 10−<sup>8</sup> M TFK and slow reactivation, the enzyme activity was monitored by the Ellman assay [13]. However, for analyzing slow reactivation after 10- and 1000-fold dilution of the system, because the enzyme concentration became very low (10−<sup>10</sup> and 10−<sup>12</sup> M), the recovery of activity was monitored at 25 ◦C under the same buffer conditions by high sensitivity spectrofluorimetric assay, using the Calbiochem thiol Probe IV [21] instead of DTNB. The Calbiochem probe IV is a coumarinyl derivative that reacts with thiol-chemicals to form a highly fluorescent conjugate. It was found to be the fastest and the most sensitive thiol reagent. Assay of ChEs using this thiol reagent instead of DTNB is more than 2 orders more sensitive than the classical Ellman assay, allowing measurement of activity in media containing <10−<sup>11</sup> M ChE. Kinetic and molecular modeling studies showed that Probe IV does not interfere with activity measurements so that ATC-based assays using DTNB and Probe IV are correlated. The experimental conditions of assay in a Peltier thermostated spectrofluorimeter F-7100 (Hitachi Ltd., Tokyo, Japan) were previously decribed [21,22].

#### *2.4. Modulation of AChE Phosphorylation Following Enzyme Preincubation in the Presence of TFK*

Two OPs were selected, CSP and paraoxon. Inhibition of hAChE by CSP (0.21 µM) was performed in 0.1 M phosphate buffer, pH 8.0 after different pre-incubation times ranging from 5 to 120 min in the presence of various concentrations of TFK (0.1–10 nM). We investigated the effect of pre-incubation by TFK (1–10 nM) on inhibition of huAChE by 50 nM paraoxon under the same conditions.

#### *2.5. Molecular Modeling*

#### 2.5.1. Molecular Docking

Molecular docking was performed using as targets several structures of hAChE co-crystallized with different non-covalent inhibitors (PDB ID 4EY4-4EY8 [15]) and one covalently bound to an organophosphorus adduct (human AChE phosphonylated by sarin, PDB ID 5FPQ, [23]). These structures are missing peripheral loop fragments 259–264 and 495–497. These were inserted from another hAChE X-ray structure (PDB ID 4BDT [24]). Inhibitors, ions and water molecules were removed prior docking. Hydrogen atoms were added with respect of hydrogen bonding network by Reduce software [25]. Molecular docking with a Lamarckian Genetic Algorithm (LGA) [26], was performed with Autodock 4.2.6 [27] software. Grid box for docking of 30 Å × 30 Å × 30 Å included the whole gorge from the mouth to the active site, including PAS. In spite of low number of torsion degrees of freedom of the inhibitor, number of docking runs was increased to analyze cauterization (2 Å tolerance). The main of selected LGA parameters were as follows: 2000 runs, 25 × 10<sup>6</sup> evaluations, 27 × 10<sup>4</sup> generations and population size 300.

#### 2.5.2. Molecular Dynamics

For the preparation of the model systems and further analysis of MD trajectories, VMD software [28] was used.

*Ligand parameterization*: For TFK and TMTFA molecules parameters were taken from Charmm General Force Field [29] through CGenFF interface (https://cgenff.umaryland.edu/), missing parameters were adjusted with the help of ffTK plugin of VMD [30,31].

*System setup*: For MD simulations, complexes of hAChE with the inhibitors (TFK and TMTFA) obtained from molecular docking (X-ray structure PDB ID 4EY7 as a target, binding pose with inhibitors in the active site) were chosen. TIP3P water box was added with boundaries in at least 10 Å from protein and ligand atoms. Sodium and chloride ions were added to final concentration of 0.15 M. Final size of both systems was 67,804 atoms, 84.6 Å × 85.8 Å × 95.4 Å.

For all MD simulations, NAMD 2.11 software [32] with CHARMM36 force field [33] was used in NPT ensemble (298 K, 1 atm) with periodical boundary conditions. MD simulations were run at the Lomonosov Moscow State University supercomputer [34].

For pre-optimization, coordinates of all atoms present in X-ray structure were fixed. Positions of all added atoms were minimized in 5000 steps and subjected 5 ns MD simulation to optimize water box and added loops. Then, all atoms were minimized in 5000 steps. This structure was used as reference and starting point for targeted molecular dynamics (TMD) and QM/MM calculations.

To create starting points for umbrella-sampling free energy calculations, pathways of pulling the inhibitors inside hAChE active site gorge and outside the protein, were obtained by means of targeted molecular dynamics with initial structure as a reference (see Supplementary Materials for details).

*Umbrella sampling (US)*: Pathways obtained by TMD were divided into 240 windows separated by 0.25 Å. State in each window was sampled during 1 ns simulations with harmonic restraining force 50 kcal/mol·Å<sup>2</sup> , with RMSD of trifluoroacetophenone part of the inhibitors as a collective variable. These parameters were adjusted in series of test runs and provided good overlapping of histograms. To construct PMF weighted histogram analysis method (WHAM) [35,36] in A. Grossfield implementation, v. 2.0.9 (http://membrane.urmc.rochester.edu/content/wham) was used.

*Replica exchange molecular dynamics (REMD-US)*: To ensure better sampling, after analysis of PMF obtained by US, 168 of US windows were used as initial coordinates of REMD-US replicas [37,38] with the same other parameters. All replicas were simulated concurrently having Hamiltonians with different biasing potentials. Every 100 steps replicas of the system underwent exchange performed using the Metropolis Monte Carlo criterion. Simulations were performed until full convergence during 10 ns for each replica.

#### 2.5.3. QM/MM Calculations

Initial systems of hAChE-TFK/TMTFA complexes were taken afterMD optimization. First solvation shell (water molecules within 2 Å from the protein) was kept. QM/MM calculations were performed with the NwChem 6.6 software [39]. The density functional theory approach with Grimme empirical dispersion correction [40] PBE0-D3/cc-pvdz was used in QM part. The MM subsystem was modeled with the AMBER force field [41]. Quantum sub-system includes the whole inhibitor molecule and active site residues: the catalytic triad Ser203, His447, Glu334; oxyanion hole Gly121, Gly122, Ala204; other principal residues forming hydrogen-bonding network around the catalytic residues, Glu202, Ser229, Glu450 and three water molecules between them, 129 atoms total, including link atoms. Total size of the system was ~11,500 atoms. For the reactivation process, distance between the inhibitor carbonyl atom and Ser203 O<sup>γ</sup> atoms was increased using harmonic constraints. Unconstrained optimization was performed for obtained reactivation pathway points.

#### **3. Results and Discussion**

#### *3.1. Slow-Binding Inhibition Kinetics of rhAChE by TFK*

Inhibition kinetics of hAChE by TFK ranging from 0.1 to 50 nM was recorded for 60 min in the presence of ATC (0.1, 0.5 and 1 mM). Kinetic analysis of inhibition progress curves showed that there is a slow onset of inhibition before reaching the equilibrium in less than 4 min (Figure 2). This is in agreement with what was previously reported [12]. The steady state was established in less than 35 min. The dependence of the first order rate constant (*kobs*), the reciprocal of the lag time, of the pre-steady state phase on TFK concentration was analyzed according to the formalism of slow binding inhibition (SBI) [5].

**Figure 2.** Typical progress curves (without treatment for smoothing noise) for slow-binding inhibition of AChE by TFK. [E] = 0.08 nM, [ATC] = 0.1 mM and [TFK] ranged from 0.1 to 50 nM. ∆OD<sup>412</sup> is the concentration of ATC hydrolysis product (in Equation (1), [P] = thiocholine = thionitrobenzoate), expressed as the absorbance increase at 412 nm. The reciprocal of *kobs* is the lag time (vertical dotted line at *t* = 1.75 min for 50 nM TFK) before steady state. Progress curves fit to Equation (1).

Initial fast inhibition type and inhibition constant (*K<sup>i</sup>* = *k*−3/*k*+3) for the first binding step (formation of EI) were determined from tangents to progress curves *kobs* vs. time (Figure 2), Dixon and Cornish-Bowden plots (Figure 3). Three different concentrations of ATC were used (0.1; 0.5 and 1 mM). Panels A and B in Figure 3 provide evidence that fast reversible inhibition step is competitive (Dixon plots are intersecting at-[TFK] = *Kci* and Cornish-Bowden plots are parallel) with *Kci* = *K<sup>i</sup>* = 5.15 ± 0.36 nM.

**Figure 3.** Dixon plot (**A**) and Cornish-Bowden plot (**B**) for determination of fast reversible inhibition constant.

The plots of *kobs* vs. [TFK] were built for each ATC concentration (Figure 4). This diagnosis plot established the type of SBI.

**Figure 4.** Typical dependance of *kobs* as a function of TFK concentration for inhibition in the presence of 0.1 mM ATC. The *kobs* values were determined from nonlinear fitting of progress curves in Figure 2 at three substrate concentrations. Data were fitted to Equation (3): ordinate is *k*−<sup>4</sup> and asymptote is *k*−<sup>4</sup> + *k*+4 in Scheme of Figure 5.

Plots of *kobs* vs. [TFK] are upward hyperboles (Figure 4). This is characteristic of SBI of type B (Figure 5). Equation (3) describes the dependence of *kobs* on inhibitor concentration, [*I*], in SBI of type B.

$$k\_{\rm obs} = k\_{-4} + \frac{k\_{+4}[I]}{K\_I(1 + [S]/K\_M) + [I]} \tag{3}$$

Accordingly, after rapid formation of a first complex EI, characterized by an inhibition constant *Ki* , the enzyme undergoes a slow «isomerization», leading to a second complex EI\* characterized by a stronger ligand binding affinity (*Ki*\* < *K<sup>i</sup>* ).

$$\begin{aligned} \mathsf{E} & \xleftarrow{\mathsf{k}\_{+1}[\mathsf{S}]} \mathsf{ES} \xrightarrow{\mathsf{k}\_{+2}} \mathsf{ES} \xrightarrow{\mathsf{k}\_{+2}} \mathsf{E} + \mathsf{E} \\ \mathsf{k}\_{+3}[\mathsf{I}] \xrightarrow{\mathsf{k}\_{-1}} \mathsf{E} & \xleftarrow{\mathsf{k}\_{+3}} \\ \mathsf{E} & \xleftarrow{\mathsf{k}\_{+4}} \mathsf{E}\_{\cdot 4} \end{aligned}$$

**Figure 5.** Slow-binding inhibition model of type B.

The binding kinetic parameters were determined from non-linear curve fitting of secondary plots *kobs* vs. [TFK] (Figure 4) obtained for inhibition in the presence of 0.1, 0.5 and 1 mM ATC. In Figure 4 the ordinate intercept is *k*−*<sup>4</sup>* and the asymptote is *k*−<sup>4</sup> + *k*+4. The constants *k*−<sup>4</sup> and *k*+<sup>4</sup> were found to be independent on ATC concentration, as expected for SBI of type B. Thus, their averages values are *k*−<sup>4</sup> = 0.054 ± 0.006 min−<sup>1</sup> and *k*+<sup>4</sup> = 0.456 ± 0.095 min−<sup>1</sup> . Then, the inhibition constant corresponding to formation of EI\*, *K* \* *<sup>i</sup>* = 0.53 nM, was determined as follows: *K* \* *<sup>i</sup>* = *K<sup>i</sup> k*−4/(*k*−<sup>4</sup> + *k*+4) = 0.53 ± 0.07 nM. This value is in agreement with values previously determined by two different methods (0.4 and 0.56 nM) in the presence of 5 mM ATC in 0.1 phosphate buffer pH 7.4 after 70 min incubation at 23 ◦C [12].

*Biomolecules* **2020**, *10*, 1608

Other important binding kinetic parameters are the residence time on AChE (the reciprocal of the overall *ko*ff rate constant, τ = 1/koff) and the fractional occupancy of AChE (*FOt*) [42]. These parameters were derived from elementary kinetic constants (Equations (4) and (5)):

$$
\tau = \frac{(k\_{-3} + k\_{+4} + k\_{-4})}{k\_{-3}k\_{-4}} \tag{4}
$$

$$FO\_t = \frac{[I]\_t}{[I]\_t + (k\_{-3}/(k\_{+3} + (k\_{+3}k\_{+4})/k\_{-4})} \tag{5}$$

However, as seen in Equations (4) and (5), estimation of τ and *FO<sup>t</sup>* implies knowledge of *k*−3, and *kon*, the second-order rate constant for the initial binding step to hAChE. This constant is not known for TFK in hAChE. However, apparent values of *kon* of several trifluoroketones for binding to different AChE were reported. These constants were determined assuming a single binding step process. For binding of neutral aliphatic trifluoroketones to electric eel AChE, *kon* = 1–5 × 10<sup>9</sup> M−<sup>1</sup> min−<sup>1</sup> [2]. For binding of TFK to mouse AChE, *k*on was similar, 3 × 10<sup>9</sup> M−<sup>1</sup> min−<sup>1</sup> [43]. On the other hand, for the charged counterpart of TFK, TMTFA (Figure 1), an SBI of type A (a single slow step corresponding to formation of EI), *kon* is slower, 6 × 10<sup>6</sup> M−<sup>1</sup> min−<sup>1</sup> for electric eel AChE [3], while for the neutral silylated homologue, Zifrosilone (Figure 1), *kon* = 6 × 10<sup>6</sup> M−<sup>1</sup> min−<sup>1</sup> for electric eel AChE [7]. The *kon* values for binding of neutral trifluoroketones, are of the order of the values reported for binding of small drugs to biological targets, 6 × 10<sup>7</sup> to 6 × 10<sup>9</sup> M−<sup>1</sup> min−<sup>1</sup> [44]. Thus, it may be assumed that the second order association rate constant of TFK in hAChE (*k*+3) is close to the *kon* value for mouse AChE. Thus, taking *k*+<sup>3</sup> ≈ 3 × 10<sup>9</sup> M−<sup>1</sup> min−<sup>1</sup> , with h*Ki*i = 5.15 nM, it follows that *k*−<sup>3</sup> ≈ 15.45 min−<sup>1</sup> . This leads to an overall *ko*ff ≈ 0.052 min−<sup>1</sup> and τ = 19 ± 2 min. The half-time for dissociation of reversibly bound TFK is t1/<sup>2</sup> = ln2/*ko*ff ≈ 13 min. The silylated homologue displays a much longer residence time of about 70 h for rat brain AChE, although its affinity is close to that of TFK (*K<sup>i</sup>* = 0.26 nM) [7].

Because both the concentration of drug in the target compartment and the residence time on physiological target as a function of time, determine the duration of action of a drug in the body, the pharmacological efficacy of a drug depends on the fractional occupancy of the enzyme as a function of time (*FOt*) [42,44]. Then, *FO<sup>t</sup>* depends on both the pharmacokinetic profile of considered drug, i.e., its concentration in the central compartment (the blood circulation) as a function of time, and the binding kinetic parameters on target(s). *FO<sup>t</sup>* change with time is therefore a useful theoretical parameter for estimating the potential pharmacological interest of drugs with long residence times on the target. For TFK, as a SBI of type B, *FO<sup>t</sup>* , for different [I]<sup>t</sup> can be calculated from Eqation (5). Assuming that the initial concentration of TFK in blood is 4.3 µM at time 0 (e.g., after intravenous injection to mice of a dose of 1 mg/kg), it follows that *FOt*<sup>0</sup> = 99.9%. After a certain time (*t*') when concentration in blood has dropped to 4.3 nM, *FOt*<sup>0</sup> = 88.7%. If after time *t*", the concentration has decreased by another order of magnitude (4.3 pM), then *FOt*<sup>0</sup> = 7.3%. Thus, it is clear that with a residence time on AChE of about 20 min, the potential pharmacological efficacy of TFK could be maintained at a high level even though the drug concentration in blood has decreased to a very low value.

#### *3.2. Transient Acylation of AChE by TFK and Subsequent Enzyme Reactivation*

Binding of a transition state analogue, like acetophenone, to ChEs, the formation of a transient hemiketal conjugate mimicking the acetyl tetrahedral intermediate was observed or hypothesized in some cases [6,43]. Thus experiments were performed to study enzyme spontaneous reactivation.

The reaction process was performed under pseudo-first order conditions to reach complete inhibition of enzyme by 2 × 10−<sup>8</sup> M TFK. Then, the enzyme activity was monitored up to 180 min, far beyond establishment of reversible steady-state equilibrium. During this second step, the recovery of enzyme activity was monitored without dilution or after dilution in order to drop TFK concentration in the medium. As seen in Figure 6 red curve, after formation of the second reversible complex EI\*, the enzyme activity progressively started to increase. This suggested, that after formation

of EI\* the enzyme was transiently acylated, and then slowly deacylated. However, during the putative deacylation process, the enzyme could have been re-inhibited by the excess of TFK present in the medium. Thus, after 20 min inhibition, the system was diluted 10- and 1000-fold to drop TFK concentration, and the enzyme activity was monitored as a function of time. Because after dilution, the enzyme activity became very low for accurate monitoring of activity vs. time, instead of the classical Ellman assay, the activity was monitored using a new method with the fluorescent thiol probe (Calbiochem Probe IV) instead of DTNB [21,22]. Results showed that dilution of TFK speeded up the deacylation process (blue and green curves in Figure 6, showing faster recovery of enzyme activity). In the presence of non-inhibitory TFK concentration (2 × 10−<sup>11</sup> M), enzyme reactivation was completed in 40 min with *kreact* ≈ 0.05 min−<sup>1</sup> ; t1/<sup>2</sup> ≈ 14 min (Figure 6, green curve) instead of more than 3 h in the presence of 2 × 10−<sup>8</sup> M TFK (Figure 6, red curve).

The slow enzyme reactivation process was interpreted as spontaneous slow deacylation of bound TFK from AChE active center. Molecular modeling (see next section) confirmed the occurrence of transient acylation of AChE active site serine after TFK binding and shed light on molecular interactions involved in these acylation and deacylation steps. Then, considering that the residence time (τ) on AChE calculated from reversible SBI is 20 min, the subsequent acylation and deacylation processes provide an overall residence time of about 60 min.

**Figure 6.** SBI and subsequent acylation of 2 × 10−<sup>9</sup> M AChE by 2 × 10−<sup>8</sup> M TFK, followed by slow deacylation. Activity was assayed with 1 mM ATC with DTNB as the thiol probe (black, blue, red curves) and with Probe IV as the thiol probe (green curve). The black curve is enzyme activity (control) in the presence of 2% acetonitrile; the red curve is enzyme activity monitored up to 180 min in the presence of 2 × 10−<sup>8</sup> M TFK. Blue curve: after steady-state SBI, the system was diluted 10 times, so that TFK dropped to 2 × 10−<sup>9</sup> M after *t* = 20 min. Green curve: after steady-state SBI, the system was diluted 1000 times, then TFK final concentration dropped to 2 × 10−<sup>11</sup> M after *t* = 20 min. The first-order rate constant of deacylation (*kreac*) was estimated from the slope of Ln increase in activity vs. time.

The slow recovery of enzyme activity depends on the TFK concentration present in the medium. In the presence of remaining 0.02 nM TFK (green curve), it takes less than 40 min to regain an activity similar to that of control enzyme at *t* = 60 min.

#### *3.3. Molecular Modeling of Interaction between TFK and AChE*

Molecular modeling was used to depict in terms of molecular events the different steps of TFK interactions with huAChE. Preliminary results about interaction between TFK and AChE were recently reported [45].

In majority of docking results, TFK was found in the peripheral anionic site (PAS) or bottleneck area (Figure S1). The dominating poses (red clusters in Figure S1) were stabilized by C-Hal . . . π interactions of –CF<sup>3</sup> group with Trp286 ring (Figure S2A). In another pose within cluster making up the red structures, the trifluoro moiety produces hydrogen bonds with the peptide backbone of Phe295, Arg296 (Figure S2B) in a way, similar to interactions in the oxyanion hole. In the next less populated clusters (orange and yellow structures) there were other poses with the same binding pattern (Figure S2C), showing interactions with Ser293 peptide backbone and possible hydrogen bond between the trifluoro moiety and side chain hydroxyl group (Figure S2D). In general in PAS, trifluoroketo-group interacts with the acyl-binding loop as with a huge oxyanion hole, trapping it near entrance to the gorge. Among complexes of AChEs with inhibitors, containing a trifluoro moiety, the majority of them are structures of non-hydrolysable substrate analogues covalently bound to the catalytic serine, and will be discussed below. However, one X-ray structure shows a non-covalent inhibitor, N-(2-diethylamino-ethyl)-3-trifluoromethyl-benzenesulfonamide (PDB ID: 4B84, [46]) with the trifluoro moiety bound in the PAS in the similar fashion (Figure S3), oriented by interactions with acyl-loop main chain NH group.

Binding poses of TFK suitable for covalent interactions with the catalytic triad were found only for X-ray structures PDB ID: 4EY7 and PDB ID: 5FPQ as targets in minor clusters. 4EY7 co-crystallized with donepezil has a wider gorge than the other X-ray structures due to rotation of Tyr337. In many cases this allows to better accommodate bulky inhibitors [47]. In this case, rotation of the catalytic serine side chain, forming hydrogen bond with Glu202, not His447 as usual, gives space for TFK to enter in the active site. Though this is not enough, carbonyl oxygen atom is slightly displaced from the oxyanion hole in these cases, indicating that Trp86 side chain has to move a bit for better fit. Following QM/MM optimization provided necessary expansion of the active site and full accommodation of the inhibitor. X-ray structure 5FPQ is the non-aged covalent conjugate of AChE with sarin. The methyl isopropoxy phosphonyl adduct expands the active site. In particular, Trp86 side chain is moved a bit (Figure S4). These displacements provide enough space for TFK, though between two possible poses, one has *tert*-butyl moiety outside cation-binding site Trp86 and Glu202 (Figure S5A, PDB ID: 4EY7 as a target), and in the other pose, the phenyl ring is out of plane of the trifluoroketo-group (Figure S5B, PDB ID: 5FPQ as a target).

The TFK pose at the bottom of the gorge but outside the active site (Figure S5C) is interesting. Indeed, such a pose was observed only in docking with X-ray structure PDB ID: 4EY8 (complex between AChE and Fasciculin-2) as a target. This provides an additional evidence for Fasciculin-induced conformational changes of AChE, widely discussed previously [48,49].

Generalizing docking results, 3 major binding poses can be outlined: in the PAS, at the area of bottleneck and in the active site (Figure 7A). For comparison, docking results for TMTFA were less diverse, and binding at the area of bottleneck was dominating, while binding to the PAS was negligible. Binding to the active site is similar to TFK, with the trimethyl ammonium group slightly closer to Glu202 due to electrostatic attraction (Figure 7B).

Potential of mean force (PMF) profiles for binding of TFK and TMTFA to hAChE calculated using REMD-US method are in agreement with docking results (Figure 7C). For TFK, two close minima at level 9 and 12 Å, corresponding to binding in the PAS and bottleneck are seen, while for TMTFA only one global minimum at level 9 Å, corresponding to binding in the area of the bottleneck was observed. Transition from these favorable positions in the middle of the gorge to the active site is associated with energy barriers. For TMTFA of 15 kcal/mol, and for TFK it is much higher, more than 20 kcal/mol.

**Figure 7.** Binding of TFK to AChE: (**A**) main docked poses in the gorge: in the PAS (most populated cluster, carbon atoms colored red), at the gorge rim partly blocking the bottleneck (carbon atoms orange), and in the active site ready to react with the catalytic serine (least populated cluster, carbon atoms are colored white) compared to the main binding poses of TMTFA (**B**). Docking results with X-ray structure PDB ID: 4EY7 as a target are shown. (**C**) Free energy (PMF) profile of the binding process calculated with REMD-US method. Process coordinate ξ is the distance between the TFK/TMTFA trifluoroketone group and the active site, oxyanion hole and catalytic serine hydroxyl-group.

In addition to the two distinctive positions inside the gorge, PMF for TFK allows to assume non-specific interactions on the protein surface (local minima valleys in area 17–18 Å and 33–34 Å from the gorge bottom, Figure S6), additionally slowing down the inhibition process. In contrast, TMTFA slides directly into the gorge. This is an illustration of the effect of the positive charge and the role of AChE as a macro-dipole [50,51]. This electrostatic effect acts as a driving force for trafficking of the inhibitor down the gorge and its incorporation in the active site, as it was previously discussed [51–54].

QM/MM optimization of non-covalent complex of hAChE with TFK in the active site obtained by molecular docking leads to size expansion of the active site necessary to accommodate the inhibitor and lower energy barrier for formation of covalent adduct. Traditionally, resulting hemiketals are called "transition state analogs", though practically they are rather analogs of tetrahedral intermediate of AChE-catalyzed ester hydrolysis [55,56]. Hemiketal configuration is very close for TFK and TMTFA, with the only difference that trimethyl ammonium group of TMTFA is 0.6 Å closer to Glu202 than *tert*-butyl moiety of TMTFA. Accordingly, it is similar to configuration X-ray structure of TMTFA bound to *Mus musculus* (PDB ID: 2H9Y [53]) and *Torpedo californica* (PDB ID: 1AMN [6]) AChE (Figure 8A).

**Figure 8.** (**A**) Tetrahedral adducts for reaction between TFK (carbon atoms shown in yellow) and TMTFA (carbon atoms shown in green) and hAChE obtained by QM/MM calculations overlaid with X-ray structures of conjugates of TMTFA with *Mus musculus* (PDB ID: 2H9Y [53], carbon atoms shown in blue) and *Torpedo californica* (PDB ID: 1AMN [6], carbon atoms shown in pink). Double hAChE/TcAChE numbering is provided; (**B**) Non-covalent complex between TFK and hAChE, stable product resulting from the tetrahedral adduct reactivation process.

Energy scan for reactivation process shows that while inhibitor is inside the active site, i.e., carbonyl oxygen atom is within the oxyanion hole, formation of the tetrahedral adduct occurs spontaneously. Stable noncovalent complex was obtained only when the TFK group left the oxyanion hole (Figure 8B).

Though reaction itself occurs barrier-less, it has associated energy barrier for incorporation of the TFK group into the oxyanion hole, estimated with QM/MM method as 19.3 kcal/mol for TFK and 13.0 kcal/mol for TMTFA. Energy barrier for TMTFA estimated with QM/MM method corresponds to values obtained with REMD-US (Figure 7C, free energy profile, 15 kcal/mol energy barrier between position in the gorge at 9 Å and local minima at 1 Å, corresponding to position in the active site), while for TFK, barrier calculated with classical methods was overestimated, probably due to lack of polarizability in classical molecular dynamics.

Molecular modeling results suggest that binding of TFK to hAChE is slow due to non-specific interactions on the protein surface, multiple binding poses inside the gorge (EI\*), and high energy barrier associated with induced-fit entrance of the inhibitor into the active site. Absence of positive charge additionally slows down this process compared to positively charged analogs.

#### *3.4. Modulation of AChE Phosphylation by Pre-Incubation with TFK*

Then, we investigated the possible modulating or protecting effect of TFK on AChE phosphylation by two different OPs. The first OP was cresylsaligenyl phosphate (CSP), the active metabolite of tricresylphosphate. CSP is a strong phosphorylating agent of ChEs [57,58]. Inhibition of ChEs by CSP under first order conditions is biphasic. This atypical phosphylation process has been reported with other OPs and carbamates [45], and was interpreted it in terms of SBI of type C. Accordingly, the enzyme exists as two forms, E and E' in slow equilibrium, each form reacting at different rates with the agent I (Equations (6)–(9) and Figure 9) [57,58].

$$\begin{bmatrix} \mathbf{E}\_{\text{tot}} \end{bmatrix} = \begin{bmatrix} \mathbf{E}\_0 \end{bmatrix} + \begin{bmatrix} \mathbf{E}'\_0 \end{bmatrix} \tag{6}$$

$$\mathbf{E}\left[\mathbf{E}\_{l}\right] = \left[\mathbf{E}\_{0}\right] \exp(-k\_{\rm obs}t) + \left[\mathbf{E}\_{0}^{\prime}\right] \exp(-k\_{\rm obs}^{\prime}t) \tag{7}$$

$$k\_{\rm obs} = \frac{k\_{\rm P}[\rm I]}{K\_{\rm I} + [\rm I]} \tag{8}$$

$$k'\_{\rm obs} = \frac{k'\_{\rm P}[\rm I]}{K'\_{\rm I} + [\rm I]} \tag{9}$$

**Figure 9.** Slow-binding inhibition of type C with a subsequent covalent step, enzyme phosphorylation (*kp*) in the present case.

This type of SBI followed by a covalent step, i.e., phosphylation, is related to enzyme hysteresis [59]. These mechanisms have been thoroughly investigated [60,61]. Thus, we hypothesized that TFK could affect the phosphorylation process by preferential binding to one or both enzyme forms.

Inhibition of hAChE by CSP (0.21 µM) performed after different pre-incubation times in the presence of various concentrations of TFK (0.1–1 nM) showed that the biphasic progressive inhibition process of the enzyme is affected by TFK. The biphasic progressive inhibition was analyzed as a function of pre-incubation time and concentration in TFK.

It was found that pre-incubation of AChE with TFK increased the observed rate of the fast process of phosphorylation but has no significant effect on the slow process (Figure 10). The effects of pre-incubation duration and TFK concentration on the rate of inhibition was directly quantified from the slope of *Ln* (residual Activity) vs time of the fast phosphorylation process and pre-incubation duration the residual activity increased as pre-incubation duration and TFK concentration increased. The deleterious effect of TFK was maximum for 10 nM TFK (Figure 11) and reached a plateau after 120 min pre-incubation (Figure 12).

**Figure 10.** Modulation of progressive biphasic inhibition of hAChE by CBDP CSP (0.21 µM) after pre-incubation of the enzyme for 120 min in the presence of TFK (0.1–10 nM).

**Figure 11.** Dependence of *k*obs,max (fast process, see Equations (7) and (8)) of hAChE phosphorylation by CSP as a function of TFK concentration (after 120 min pre-incubation).

**Figure 12.** Observed first process (fast process) phosphorylation rate constant of hAChE by 0.21 µM CSP as a function of enzyme pre-incubation time in the presence of 10 mM TFK.

Because of the hysteretic behavior of AChE with CSP as a phosphorylating agent, the modulating effect of enzyme pre-incubation in the presence of TFK, makes the enzyme form E more susceptible to phosphorylation by CSP, and accelerates the overall phosphorylation rate of the enzyme system.

Then, we investigated the effect of TFK (1–10 nM) on AChE phosphorylation by 50 nM paraoxon. Unlike progressive inhibition of AChE by CSP, first order inhibition of the enzyme by paraoxon is linear as with most OPs. It was found that pre-incubation of AChE with TFK decreased the observed rate of phosphorylation. Thus, the effects of pre-incubation duration and TFK concentration on the rate of inhibition was directly quantified from the slope of *Ln* (residual Activity) vs time and pre-incubation duration the residual activity increased as pre-incubation duration and TFK concentration increased. The protective effect was maximum for 10 nM TFK (Figure 13) and reached a plateau after 40 min pre-incubation (Figure 14).

A recent in vivo work provided evidence that the slow-binding inhibitor C-547 has a long protective action, up to 3 days, on peripheral AChE against inhibition by paraoxon with no side effects [62]. This suggests that C-547 could be used in pre-treatment of OP-poisoning for long-term protection of the peripheral cholinergic system. However, in the case of TFK, the opposite effect on phosphorylation observed between CSP and paraoxon, makes it difficult to predict whether all SBIs can be protectant or anti-protectant of ChEs against all types of OPs. However, it may be hypothesized that the anti-protective effect on AChE reflects the enzyme hysteretic behavior with certain OPs, and results from preferential phosphorylation of the E form that kept memory of SBI binding. A conclusive answer needs further investigation with different SBIs and OPs.

**Figure 13.** First-order inhibition of AChE by 50 nM paraoxon after pre-incubation of the enzyme in the presence of 10 nM TFK up to 90 min, pH 8.0, 25 ◦C.

**Figure 14.** Observed phosphorylation rate constant of hAChE by 50 nM paraoxon as a function of enzyme pre-incubation time (from 0 to 90 min) in the presence of 10 nM TFK.

#### **4. Conclusions**

Kinetic analysis and molecular modeling of hAChE inhibition by TFK showed that this ligand is a SBI of type B. Moreover, after formation of the final complex, a transiently stabilized tetrahedral conjugate is formed, and then slowly dissociates. The existence of covalent and stable acyl tetrahedral intermediates in ChEs [6,63,64] is not completely understood. It is, however, one of the puzzling features of the catalytic power of these enzymes that deserves further studies.

TFK as an SBI of type B capable of binding to human AChE with high affinity, could be of pharmacological interest. It is already the subject of clinical investigations for neuroimaging of neurodegenerative diseases [12]. The related silyl compound, Zifrosilone, a slow tight binding inhibitor of type A with a long residence time τ = 70 h and *K<sup>i</sup>* = 0.26 nM for rat brain AChE [7] was promising for symptomatic treatment of AD [7,8,10]. However, human clinical trials were discontinued likely because of its longer residence time on AChE target. Thus, TFK with a much shorter residence time (τ ≈ 20 min for reversible binding and overall residence time of about 1 h for full recovery of activity after transient acylation and deacylation of huAChE active serine) appears to be more suitable for further research as an effective and safe pharmacological drug for palliative treatment of AD.

Other possible pharmacological applications may be considered. In particular, research of new molecules for protection of ChEs against phosphylation by OPs is an active field. At the moment, the currently used molecules for pre-treatment of OP poisoning have limited and short protective actions and may induce behavioral and locomotor side effects [65]. Certain SBIs, e.g., huperzine A, galantamine, donepezil have been successfully tested (review in [45]). Novel SBIs may be of interest to expand the duration of AChE protection prior and after exposure to OPs. For example, a recent work from our group provided evidence that the AChE slow-binding inhibitor of type B, C-547, a bulky methyluracil derivative (1,3-bis[5-(diethyl-*o*-nitrobenzylammonium)pentyl]-6-methyluracil dibromide) [45,66] has a long protective action, up to 3 days, on peripheral AChE against its phosphorylation by paraoxon with no side effects [62]. Thus, the low toxicity of TFK in rodents and its protective action on central and peripheral AChEs against toxicity of paraoxon make this compound also of interest for protection of both central and peripheral AChE against OP poisoning (Zueva et al., unpublished).

**Supplementary Materials:** Supplementary Materials: The following are available online at http://www.mdpi.com/ 2218-273X/10/12/1608/s1, Supplementary materials to the Methods section and supplementary figures. Figure S1. Major binding poses found by molecular docking with structures 4EY4-4EY7, 5FPQ as target, clustered together. Representatives of the clusters are colored according to their population from red (most populated) to white (least populated). Figure S2. Individual binding poses of TFK in the PAS from the most populated clusters. Yellow dashes show ordinary hydrogen bonds, cyan dashes show halogen interactions (hydrogen bonds and C-Hal . . . π interactions), and orange dashes show π-π interactions. Figure S3. Overlay of binding pose of TFK in the PAS shown in details in Figure S2-B (here, TFK molecule is highlightd in magenta) with X-ray structures of mAChE in complex with N-(2-Diethylamino- ethyl)-3-trifluoromethyl-benzenesulfonamide (PDB ID: 4B84, carbon atoms are shown green). Figure S4. Overlay of X-ray structures of hAChE in apo-state (PDB ID: 4EY4, carbon atoms are shown blue) and covalent conjugate with sarin (PDB ID: 5FPQ, carbon atoms are shown green, Ser203-sarin conjugate atoms are shown as spheres). Figure S5. Individual binding poses of TFK in the catalytic active site. Based on docking results with X-ray structures PDB ID 4EY7 (A), 5FPQ (B) and 4EY8 (C) used as a target. Figure S6. Binding of TFK to AChE surface, corresponding to local minima valleys in area 17-18 Å (colored green) and 33-34 Å (colored magenta) from the gorge bottom (see Figure 7, C, blue line for TFK).

**Author Contributions:** I.V.Z. performed kinetic studies; S.V.L. performed molecular modeling studies; I.R.P. and S.D. synthesized and developed TFK as a neuroimaging agent; P.M. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was funded by Russian Science Foundation grant # 20-14-00155 to P.M.

**Acknowledgments:** The authors are grateful to J. Dias (IRBA, Brétoche, France) for providing rhuAChE, and Oksana Lockridge (UNMC, Omaha, NE, USA) for the gift of CSP. Computer modeling was carried out using equipment from the shared research facilities of the HPC computing resources at Lomonosov Moscow State University. We acknowledge the Joint Supercomputer Center of the Russian Academy of Sciences for provision of computational time.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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## *Article* **Pursuing the Complexity of Alzheimer's Disease: Discovery of Fluoren-9-Amines as Selective Butyrylcholinesterase Inhibitors and** *N***-Methyl-D-Aspartate Receptor Antagonists**

**Jan Konecny 1,2,†, Anna Misiachna 3,4,5,†, Martina Hrabinova 1,2 , Lenka Pulkrabkova 1,2, Marketa Benkova <sup>2</sup> , Lukas Prchal <sup>2</sup> , Tomas Kucera 1,2 , Tereza Kobrlova <sup>2</sup> , Vladimir Finger 2,6, Marharyta Kolcheva 3,4 , Stepan Kortus 3,4, Daniel Jun 1,2 , Marian Valko <sup>7</sup> , Martin Horak 3,4, Ondrej Soukup 1,2,\* and Jan Korabecny 1,2,\***


**Abstract:** Alzheimer's disease (AD) is a complex disorder with unknown etiology. Currently, only symptomatic therapy of AD is available, comprising cholinesterase inhibitors and *N*-methyl-Daspartate (NMDA) receptor antagonists. Drugs targeting only one pathological condition have generated only limited efficacy. Thus, combining two or more therapeutic interventions into one molecule is believed to provide higher benefit for the treatment of AD. In the presented study, we designed, synthesized, and biologically evaluated 15 novel fluoren-9-amine derivatives. The in silico prediction suggested both the oral availability and permeation through the blood–brain barrier (BBB). An initial assessment of the biological profile included determination of the cholinesterase inhibition and NMDA receptor antagonism at the GluN1/GluN2A and GluN1/GluN2B subunits, along with a low cytotoxicity profile in the CHO-K1 cell line. Interestingly, compounds revealed a selective butyrylcholinesterase (BChE) inhibition pattern with antagonistic activity on the NMDARs. Their interaction with butyrylcholinesterase was elucidated by studying enzyme kinetics for compound **3c** in tandem with the in silico docking simulation. The docking study showed the interaction of the tricyclic core of new derivatives with Trp82 within the anionic site of the enzyme in a similar way as the template drug tacrine. From the kinetic analysis, it is apparent that **3c** is a competitive inhibitor of BChE.

**Keywords:** acetylcholinesterase; Alzheimer's disease; butyrylcholinesterase; fluorene; in vitro; in silico; multi-target directed ligands; *N*-methyl-D-aspartate receptor

**Citation:** Konecny, J.; Misiachna, A.; Hrabinova, M.; Pulkrabkova, L.; Benkova, M.; Prchal, L.; Kucera, T.; Kobrlova, T.; Finger, V.; Kolcheva, M.; Kortus, S.; et al. Pursuing the Complexity of Alzheimer's Disease: Discovery of Fluoren-9-Amines as Selective Butyrylcholinesterase Inhibitors and *N*-Methyl-D-Aspartate Receptor Antagonists. *Biomolecules* **2021**, *11*, 3. https://dx.doi.org/ 10.3390/biom11010003

Received: 25 November 2020 Accepted: 18 December 2020 Published: 22 December 2020

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/ licenses/by/4.0/).

#### **1. Introduction**

Alzheimer's disease (AD) is a debilitating neurodegenerative disorder that manifests as progressive memory loss leading to dementia [1,2]. AD not only represents a serious health burden, but it also imposes social and economic issues [3–6]. Worldwide, nearly 50 million people have developed AD, and it is expected that the number will double within the next 20 years [7]. An effective cure able to halt or slow down the disease progression still does not exist, mainly because of our limited knowledge about AD pathophysiology. However, mechanisms resulting in the clinical symptoms of AD are well-established [8,9]. Among them, amyloid-β (Aβ) and tau proteins are considered as major contributors to AD, forming extracellular aggregates [10,11] and intracellular neurofibrillary tangles, respectively [12–14]. So far, the therapy is only palliative, either enhancing the cholinergic neurotransmission or modulating the synaptic excitotoxicity via *N*-methyl-D-aspartate (NMDA) receptors [15–18]. Oxidative stress [19,20], metal ion imbalance [21], or neuroinflammation [22] are other crucial players in AD pathophysiology.

Currently, AD is treated by acetylcholinesterase (AChE, E.C. 3.1.1.7) inhibitors to restore the physiological levels of acetylcholine (ACh) and the NMDA receptor antagonist, which reduce the excitotoxicity by mitigating the excessive glutamate stimulation of the receptors [23,24]. Donepezil, galantamine, and rivastigmine represent the marketed drugs from the group of AChE inhibitors; memantine acts as a noncompetitive NMDA receptor antagonist, blocking the overstimulation of the respective receptors. Since AChE inhibitors are administered in mild-to-moderate stages of AD, memantine is indicated for severe stages of the disease [25]. It is worth mentioning that the combination of donepezil and memantine into one capsule, known as Namzaric, was approved in 2014. Such a combination offers an improved efficacy compared to single-agent administration with a good pharmacokinetic profile, safety, and tolerability [26,27].

Given the complexity of AD and the high failure rate of single-targeted drug candidates from clinical trials in the last few years, the idea of multitarget directed ligands (MTDLs) has emerged as a new approach to tackle the disease [28–31]. Accordingly, the design of these molecules is oriented towards the modulation of different pathological pathways simultaneously [29,32]. Recently, we have critically pointed out that not all the combined effects are suitable to be amalgamated into a single molecule to achieve the highest therapeutic effect [29]. Indeed, AD is a long-term condition with progressive brain changes occurring 20 years before the AD outbreak into the symptomatic phase [33]. With respect to the combination of therapeutic targets of interest and to achieve maximum synergy, the ideal drugs should be designed by following the timescale for specific pathological cascades. Within this study, we have focused on the symptomatic stage of AD, in which Aβ plaques and neurofibrillary tangles of paired helical filaments are ubiquitous, and their clearance has no effect on improving cognitive functions. This is well-documented, for instance, by the insufficient efficacy of β-secretase inhibitors in phase III clinical trials [34]. With this in mind, we turned our attention to impaired neurotransmission, which is the most critically affected at the later stages of AD, thus pursuing the two most pronounced systems, namely cholinergic and glutamatergic ones [35,36].

Tacrine (THA), an AChE inhibitor, was the first drug approved for AD treatment in 1993. Hepatotoxicity and gastrointestinal discomfort are the culprits responsible for its withdrawal in 2013 [37]. THA acts not only as AChE inhibitor but, also, via the antagonism of NMDA receptors, which might contribute to its pharmacological effects [38]. Indeed, THA inhibits NMDA receptor responses with high specificity in a concentration-dependent manner with an IC<sup>50</sup> = 20 µM at −60 mV and much higher values at positive membrane potentials [39]. Mechanistically, the THA action at NMDA receptors can be classified as reversible, blocking the channel's open state with binding in the proximity of the channel entrance [40,41]. Likewise, other THA derivatives emerged as interesting drugs with a potential applicability for AD treatment due to the pharmacological behavior of both cholinesterases and the NMDA receptor [39,42]. Based on this, our goal was to design

a series of novel multipotent agents, which, in one molecule, shows a representative cholinesterase inhibition together with NMDA antagonist activity.

In a recent work, we developed novel molecules building on a fluorene moiety. The compounds are structurally related to other tricyclic congeners like THA or carbazole, known for their cholinesterase inhibition and/or neuroprotective properties (Figure 1) [43–45]. We took advantage of the above-mentioned and designed novel molecules substituted at the C9 position by various primary or secondary amines, generating 15 novel compounds (**3a**–**o**; Figure 1) as potential cholinesterase inhibitors and NMDA receptor antagonists. Importantly, since several techniques on how to construct MTDLs exist, we applied the so-called merging approach in this study. This approach best corresponds to the drug-likeness of the final molecule compared to the linking or fusing strategies [28,29,32]. Furthermore, the design of the novel family was envisaged in parallel with their physicochemical properties, presuming both oral and central bioavailability. The biological profile includes the assessment of AChE, butyrylcholinesterase (BChE; E.C. 3.1.1.8), and NMDA receptor affinities, with the cytotoxicity and BBB permeation being evaluated as well.

**Figure 1.** Design of a novel 9-aminofluorene overlapping the tacrine (THA) and carbazole moieties. NMDA: *N*-methyl-D-aspartate.

#### **2. Materials and Methods**

#### *2.1. Chemistry*

All chemical solvents and reagents were used in the highest available purity without further purification, and they were purchased from Sigma-Aldrich (Prague, Czech Republic) or FluoroChem (Hadfield, UK). The reactions were monitored by thin-layer chromatography (TLC) on silica gel plates (60 F254, Merck, Prague, Czech Republic), and the spots were visualized by ultraviolet light (254 nm). Purification of crude products was carried out using a PuriFlash Gen5 column, 5.250 (Interchim, Montluçon, France) (silica gel 100, 60 Å, 230–400-mesh ASTM, Sigma-Aldrich, Prague, Czech Republic). NMR spectra were recorded in deuterated chloroform (CDCl3) and deuterated dimethyl sulfoxide (DMSO-d6) on a Bruker Avance NEO 500 MHz spectrometer (499.87 MHz for 1H-NMR and 125.71 MHz for 13C-NMR; Vienna, Austria). Chemical shifts are reported in parts per millions (ppm), and spin multiplicities are given as broad singlet (bs), doublet (d), doublet

of doublet (dd), triplet (t), doublet of triplet (dt), quartet (q), pentet (p), or multiplet (m). Coupling constants (J) are reported in Hz. Recorded NMR spectra are available in the Supplementary Materials. Melting points were measured using an automated melting point recorder M-565 (Büchi, Flawil, Switzerland). The synthesized compounds were analyzed by an LC-MS system consisting of UHLPC Dionex Ultimate 3000 RS coupled with a Q Exactive Plus orbitrap mass spectrometer to obtain high-resolution mass spectra (Thermo Fisher Scientific, Bremen, Germany) (see Supplementary Materials). The samples were dissolved in DMSO/methanol 50/50 (*v*/*v*). Reverse-phase C18 column Kinetex EVO (Phenomenex, Torrance, CA, USA) was used as a stationary phase, and purified water with 0.1% formic acid (mobile phase A) and LC-MS grade acetonitrile with 0.1% formic acid (mobile phase B) were used as the mobile phases. Gradient elution was used to determine purities and mass spectra. Method started with 5% B for 0.3 min, then the gradient rose to 100% B in the third min and was at 100% B for 0.7 min and then went back to 5% B and was equilibrated for 3.5 min. Total run time of the method was 7.5 min. Column was tempered to 27 ◦C, the flow of the mobile phase was 0.5 mL/min, and the injection volume was 1 µL. Gradient LC analysis with UV detection (254 nm) confirmed a >97% purity. High-resolution mass spectra were collected from the total ion current in the scan range 105–1000 *m/z*, with the resolution set to 140,000.

#### *2.2. General Procedure for the Preparation of 9-Bromofluorene hydrochlorides (***3a–o***)*

Appropriate primary or secondary amine (Scheme 1) was dissolved in 10 mL of dry MeCN under argon atmosphere and stirred vigorously at 40 ◦C for 15 min. 9-Bromofluorene (Scheme 1) was dissolved in 5 mL of dry MeCN and added dropwise to the reaction mixture. The reaction was maintained with stirring at 40 ◦C for 3 h. After cooling, the solvent was evaporated under reduced pressure, and the crude material was purified on a FlashChrom column (eluent dichloromethane/methanol (DCM/MeOH) with 1% of NH3, gradient 95:5 → 90:10) to get the appropriate products as a free base. These were converted into hydrochloride salts by the treatment with 1 mL of concentrated aqueous solution of HCl (35%) in 5 mL of MeOH, starting from 0 ◦C to room temperature for 1 h. The solvent was removed in vacuo, and the residual water was distilled via azeotropic distillation with absolute EtOH three times. The solid product was washed by ice-cold acetone, resulting in hydrochloride salt as a white solid.

**Scheme 1.** Synthetic procedure for the preparation of substituted fluoren-9-amines **3a**–**o**. Reaction conditions: (i) MeCN, 40 ◦C, 3 h and (ii) HCl (aq.), MeOH, RT, 1 h.

#### 2.2.1. *N*-Propan-2-yl-9*H*-fluoren-9-amine hydrochloride (**3a**)

Yield 43% as a white solid. Melting point (m.p.): decomposed at 238.5 ◦C. <sup>1</sup>H-NMR (500 MHz, DMSO-*d*6) δ 10.17 (bs, *J* = 5.6 Hz, 2H), 8.12 (dd, *J* = 7.6, 1.0 Hz, 2H), 7.95 (dt, *J* = 7.6, 1.0 Hz, 2H), 7.54 (td, *J* = 7.6, 1.0 Hz, 2H), 7.42 (td, *J* = 7.6, 1.0 Hz, 2H), 5.61–5.57 (m, 1H), 3.27–3.17 (m, 1H), 1.18 (d, *J* = 6.5 Hz, 6H). <sup>13</sup>C-NMR (126 MHz, dmso) δ 141.41, 138.60, 130.43, 128.33, 127.35, 121.21, 58.25, 48.44, 20.18. HRMS (ESI<sup>+</sup> ): [M + H]<sup>+</sup> : calculated for C16H18N<sup>+</sup> (*m*/*z*): 224.14392; found: 224.14343. LC-MS purity 99%.

#### 2.2.2. *N*-Methyl-9*H*-fluoren-9-amine hydrochloride (**3b**)

Yield 13% as a white solid. M.p.: decomposed at 205.9 ◦C. <sup>1</sup>H-NMR (500 MHz, DMSO*d*6) δ 10.52 (bs, 2H), 8.07 (dd, *J* = 7.6, 1.0 Hz, 2H), 7.94 (dd, *J* = 7.6, 1.0 Hz, 2H), 7.53 (td, *J* = 7.6, 1.0 Hz, 2H), 7.42 (td, *J* = 7.6, 1.0 Hz, 2H), 5.64 (s, 1H), 2.12 (s, 3H). <sup>13</sup>C-NMR (126 MHz, dmso) δ 141.42, 137.56, 130.25, 128.20, 126.62, 120.94, 59.75, 27.06. HRMS (ESI<sup>+</sup> ): [M + H]<sup>+</sup> : calculated for C14H14N<sup>+</sup> (*m*/*z*): 196.11262; found: 196.11229. LC-MS purity 100%.

#### 2.2.3. *N*-Cyclohexyl-9*H*-fluoren-9-amine hydrochloride (**3c**)

Yield 43% as a white solid. M.p. 267.3–269.6 ◦C. <sup>1</sup>H-NMR (500 MHz, DMSO-*d*6) <sup>δ</sup> 10.17 (bs, 2H), 8.10 (dd, *J* = 7.6, 1.0 Hz, 2H), 7.95 (dd, *J* = 7.6, 1.0 Hz, 2H), 7.54 (td, *J* = 7.6, 1.0 Hz, 2H), 7.42 (td, *J* = 7.6, 1.0 Hz, 2H), 5.60 (s, 1H), 2.84 (t, *J* = 11.5 Hz, 1H), 1.81 – 1.71 (m, 2H), 1.68–1.59 (m, 2H), 1.50 (m, *J* = 15.0, 5.3, 4.4 Hz, 3H), 1.13–1.00 (m, 3H). <sup>13</sup>C-NMR (126 MHz, dmso) δ 141.09, 138.37, 130.15, 128.09, 127.00, 120.94, 57.68, 54.59, 29.72, 24.68, 23.99. HRMS (ESI<sup>+</sup> ): [M + H]<sup>+</sup> : calculated for C19H22N<sup>+</sup> (*m*/*z*): 264.17522; found: 264.17465. LC-MS purity 99%.

#### 2.2.4. *N*-Cyclopropyl-9*H*-fluoren-9-amine hydrochloride (**3d**)

Yield 57% as a white solid. M.p.: decomposed at 233.1 ◦C. <sup>1</sup>H-NMR (500 MHz, DMSO*d*6) δ 10.63 (bs, 2H), 8.13 (dd, *J* = 7.6, 1.0 Hz, 2H), 7.94 (dd, *J* = 7.6, 1.0 Hz, 2H), 7.53 (td, *J* = 7.6, 1.0 Hz, 2H), 7.41 (td, *J* = 7.6, 1.0 Hz, 2H), 5.63 (s, 1H), 2.25–2.15 (m, 1H), 0.85–0.76 (m, 2H), 0.55–0.46 (m, 2H). <sup>13</sup>C-NMR (126 MHz, dmso) δ 141.38, 138.07, 130.14, 127.95, 127.08, 120.82, 60.17, 26.24, 3.50. HRMS (ESI<sup>+</sup> ): [M + H]<sup>+</sup> : calculated for C16H16N<sup>+</sup> (*m*/*z*): 222.12827; found: 222.12776. LC-MS purity 99%.

#### 2.2.5. *N*-Cyclobutyl-9*H*-fluoren-9-amine hydrochloride (**3e**)

Yield 51% as a white solid. M.p.: decomposed at 271.6 ◦C. <sup>1</sup>H-NMR (500 MHz, DMSO*d*6) δ 10.77 (bs, 2H), 8.10 (dd, *J* = 7.6, 1.0 Hz, 2H), 7.92 (dd, *J* = 7.6, 1.0 Hz, 2H), 7.52 (td, *J* = 7.6, 1.0 Hz, 2H), 7.40 (td, *J* = 7.6, 1.0 Hz, 2H), 5.57 (s, 1H), 3.12 (p, *J* = 9.1, 8.6 Hz, 1H), 2.22–2.10 (m, 2H), 1.62–1.41 (m, 4H). <sup>13</sup>C-NMR (126 MHz, dmso) δ 141.23, 137.87, 130.16, 127.97, 126.96, 120.84, 58.50, 47.72, 27.67, 15.64. HRMS (ESI<sup>+</sup> ): [M + H]<sup>+</sup> : calculated for C17H18N<sup>+</sup> (*m*/*z*): 236.14392; found: 236.14333. LC-MS purity 99%.

#### 2.2.6. 1-(9*H*-fluoren-9-yl)piperidine hydrochloride (**3f**)

Yield 45% as a white solid. M.p.: decomposed at 251.4 ◦C. <sup>1</sup>H-NMR (500 MHz, DMSO*d*6) δ 11.79 (bs, 1H), 8.24 (dd, *J* = 7.6, 1.0 Hz, 2H), 7.94 (dd, *J* = 7.6, 1.0 Hz, 2H), 7.55 (td, *J* = 7.6, 1.0 Hz, 2H), 7.42 (td, *J* = 7.6, 1.0 Hz, 2H), 5.72 (s, 1H), 3.23 (d, *J* = 11.7 Hz, 2H), 2.79 (q, *J* = 11.7 Hz, 2H), 2.07 (q, *J* = 12.6, 11.7 Hz, 2H), 1.67 (d, *J* = 12.6 Hz, 3H), 1.34–1.20 (m, 1H). <sup>13</sup>C-NMR (126 MHz, dmso) δ 141.83, 135.94, 130.66, 128.29, 128.17, 120.91, 67.23, 49.46, 22.29, 21.67. HRMS (ESI<sup>+</sup> ): [M + H]<sup>+</sup> : calculated for C18H20N<sup>+</sup> (*m*/*z*): 250.15957; found: 250.15875. LC-MS purity 98%.

#### 2.2.7. *N*-(2-methoxyethyl)-9*H*-fluoren-9-amine hydrochloride (**3g**)

Yield 11% as a white solid. M.p.: 183.4–185.1 ◦C. <sup>1</sup>H-NMR (500 MHz, DMSO-*d*6) <sup>δ</sup> 10.60 (bs, 2H), 8.15 (dd, *J* = 7.6, 1.0 Hz, 2H), 7.93 (dd, *J* = 7.6, 1.0 Hz, 2H), 7.53 (td, *J* = 7.6, 1.0 Hz, 2H), 7.42 (td, *J* = 7.6, 1.0 Hz, 2H), 5.59 (s, 1H), 3.47 (t, *J* = 5.2 Hz, 2H), 3.19 (s, 3H), 2.47–2.40 (m, 2H). <sup>13</sup>C-NMR (126 MHz, dmso) δ 141.42, 137.56, 130.27, 128.26, 126.69, 120.93, 67.03, 59.32, 58.16, 40.87. HRMS (ESI<sup>+</sup> ): [M + H]<sup>+</sup> : calculated for C16H18NO<sup>+</sup> (*m*/*z*): 240.13884; found: 240.13831. LC-MS purity 99%.

#### 2.2.8. *N*-Ethyl-9*H*-fluoren-9-amine hydrochloride (**3h**)

Yield 36% as a white solid. M.p.: decomposed at 264.8 ◦C. <sup>1</sup>H-NMR (500 MHz, DMSO*d*6) δ 10.46 (bs, *J* = 9.3 Hz, 2H), 8.12 (dd, *J* = 7.6, 1.0 Hz, 2H), 7.94 (dd, *J* = 7.6, 1.0 Hz, 2H), 7.53 (td, *J* = 7.6, 1.0 Hz, 2H), 7.42 (td, *J* = 7.6, 1.0 Hz, 2H), 5.61 (d, *J* = 3.5 Hz, 1H), 2.51–2.48 (m, 2H), 1.14 (t, *J* = 7.2 Hz, 3H). <sup>13</sup>C-NMR (126 MHz, dmso) δ 141.29, 137.89, 130.19, 128.18, 126.72, 120.91, 59.17, 37.35, 11.53. HRMS (ESI<sup>+</sup> ): [M + H]<sup>+</sup> : calculated for C15H16N<sup>+</sup> (*m*/*z*): 210.12827; found: 210.12784. LC-MS purity 99%.

#### 2.2.9. *N*,*N*-Diethyl-9*H*-fluoren-9-amine hydrochloride (**3i**)

Yield 7% as a white solid. M.p.: 166.2–167.9 ◦C. <sup>1</sup>H-NMR (500 MHz, DMSO-*d*6) <sup>δ</sup> 11.66 (bs, 1H), 8.15 (dd, *J* = 7.6, 1.0 Hz, 2H), 7.98 (dd, *J* = 7.6, 1.0 Hz, 2H), 7.57 (td, *J* = 7.6, 1.0 Hz, 2H), 7.44 (td, *J* = 7.6, 1.0 Hz, 2H), 5.85 (s, 1H), 3.12–3.02 (m, 1H), 3.02–2.94 (m, 1H), 1.30 (t, *J* = 7.2 Hz, 6H). <sup>13</sup>C-NMR (126 MHz, dmso) δ 141.62, 136.45, 130.54, 128.37, 127.49, 121.07, 63.15, 46.29, 10.32. HRMS (ESI<sup>+</sup> ): [M + H]<sup>+</sup> : calculated for C17H20N<sup>+</sup> (*m*/*z*): 238.15957; found: 238.15903. LC-MS purity 99%.

#### 2.2.10. 1-(9*H*-fluorene-9-yl)-4-methylpiperazine dihydrochloride (**3j**)

Yield 7% as a white solid. M.p.: decomposed at 206.4 ◦C. <sup>1</sup>H-NMR (500 MHz, DMSO*d*6) δ 10.80 (bs, 1H), 7.85 (dd, *J* = 7.6, 1.0 Hz, 2H), 7.59 (dd, *J* = 7.6, 1.0 Hz, 2H), 7.42 (td, *J* = 7.6, 1.0 Hz, 2H), 7.34 (td, *J* = 7.6, 1.0 Hz, 2H), 5.03 (s, 1H), 3.31–2.71 (m, 8H), 2.68 (s, 3H). <sup>13</sup>C-NMR (126 MHz, dmso) δ 143.00, 140.64, 128.64, 127.51, 125.88, 120.36, 68.65, 53.41, 45.50, 42.34. HRMS (ESI<sup>+</sup> ): [M + H]<sup>+</sup> : calculated for C18H21N<sup>2</sup> + (*m*/*z*): 265.17047; found: 265.16974. LC-MS purity 99%.

#### 2.2.11. 1-(9*H*-fluorene-9-yl)-4-ethylpiperazine dihydrochloride (**3k**)

Yield 25% as a white solid. M.p.: decomposed at 233.6 ◦C. <sup>1</sup>H-NMR (500 MHz, DMSO*d*6) δ 10.75 (bs, 1H), 7.85 (dd, *J* = 7.6, 1.0 Hz, 2H), 7.59 (dd, *J* = 7.6, 1.0 Hz, 2H), 7.42 (td, *J* = 7.6, 1.0 Hz, 2H), 7.33 (td, *J* = 7.6, 1.0 Hz, 2H), 5.03 (s, 1H), 3.02 (q, *J* = 7.3 Hz, 2H), 2.99–2.61 (m, 8H), 1.19 (t, *J* = 7.3 Hz, 3H). <sup>13</sup>C-NMR (126 MHz, dmso) δ 143.05, 140.63, 128.62, 127.49, 125.90, 120.34, 68.67, 51.28, 50.79, 45.45, 9.01. HRMS (ESI<sup>+</sup> ): [M + H]<sup>+</sup> : calculated for C19H24N<sup>2</sup> + (m/z): 279.18612; found: 279.18555. LC-MS purity 99%.

#### 2.2.12. 4-(9*H*-fluorene-9-yl)morpholine hydrochloride (**3l**)

Yield 50% as a white solid. M.p.: decomposed at 239.8 ◦C. <sup>1</sup>H-NMR (500 MHz, DMSO*d*6) δ 12.66 (bs, 1H), 8.21 (dd, *J* = 7.6, 1.0 Hz, 2H), 7.96 (dd, *J* = 7.6, 1.0 Hz, 2H), 7.58 (td, *J* = 7.6, 1.0 Hz, 2H), 7.45 (td, *J* = 7.6, 1.0 Hz, 2H), 5.81 (s, 1H), 4.18–3.93 (m, 2H), 3.94–3.75 (m, 2H), 3.31–3.09 (m, 2H), 3.09–2.87 (m, 2H). <sup>13</sup>C-NMR (126 MHz, DMSO) δ 142.19, 131.09, 128.60, 128.46, 121.30, 67.38, 63.41, 48.58. HRMS (ESI<sup>+</sup> ): [M + H]<sup>+</sup> : calculated for C17H18NO<sup>+</sup> (*m*/*z*): 252.13884; found: 252.13837. LC-MS purity 99%.

#### 2.2.13. *N*-Butyl-9*H*-fluoren-9-amine hydrochloride (**3m**)

Yield 12% as a white solid. M.p.: decomposed at 201.3 ◦C. <sup>1</sup>H-NMR (500 MHz, DMSO*d*6) δ 10.13 (bs, 2H), 8.10 (dd, *J* = 7.6, 1.0 Hz, 2H), 7.93 (dd, *J* = 7.6, 1.0 Hz, 2H), 7.52 (td, *J* = 7.6, 1.0 Hz, 2H), 7.42 (td, *J* = 7.6, 1.0 Hz, 2H), 5.54 (s, 1H), 2.33 (t, 2H), 1.58–1.47 (m, 2H), 1.25–1.14 (m, 2H), 0.74 (t, *J* = 7.3 Hz, 3H). <sup>13</sup>C-NMR (126 MHz, DMSO) δ 141.52, 130.26, 128.40, 126.78, 121.12, 59.98, 41.80, 28.70, 19.70, 13.86. HRMS (ESI<sup>+</sup> ): [M + H]<sup>+</sup> : calculated for C17H20N<sup>+</sup> (*m*/*z*): 238.15957; found: 238.15912. LC-MS purity 97%.

#### 2.2.14. *N*-(2-methylpropyl)-9*H*-fluoren-9-amine hydrochloride (**3n**)

Yield 34% as a white solid. M.p.: 254.8–256.1 ◦C. <sup>1</sup>H-NMR (500 MHz, DMSO-*d*6) <sup>δ</sup> 10.52 (bs, 2H), 8.19 (dd, *J* = 7.6, 1.0 Hz, 2H), 7.94 (dd, *J* = 7.6, 1.0 Hz, 2H), 7.54 (td, *J* = 7.6, 1.0 Hz, 2H), 7.44 (td, *J* = 7.6, 1.0 Hz, 2H), 5.62 (s, 1H), 2.13–2.03 (m, 2H), 1.98–1.85 (m, 1H), 0.82 (d, *J* = 6.7 Hz, 6H). <sup>13</sup>C-NMR (126 MHz, DMSO) δ 141.72, 137.87, 130.54, 128.56, 126.89, 121.22, 59.76, 48.58, 26.03, 20.52. HRMS (ESI<sup>+</sup> ): [M + H]<sup>+</sup> : calculated for C17H20N<sup>+</sup> (*m*/*z*): 238.15957; found: 238.15903. LC-MS purity 97%.

#### 2.2.15. 1-(9*H*-fluoren-9-yl)pyrrolidine hydrochloride (**3o**)

Yield 30% as a white solid. M.p.: decomposed at 239.3 ◦C. <sup>1</sup>H-NMR (500 MHz, DMSO*d*6) δ 12.31 (bs, 1H), 8.14 (dd, *J* = 7.6, 1.0 Hz, 2H), 7.96 (dd, *J* = 7.6, 1.0 Hz, 2H), 7.56 (td, *J* = 7.6, 1.0 Hz, 2H), 7.43 (td, *J* = 7.6, 1.0 Hz, 2H), 5.84 (s, 1H), 3.49–3.27 (m, 2H; signal overlapped with residual DMSO), 3.13–2.92 (m, 2H), 1.97–1.72 (m, 4H). <sup>13</sup>C-NMR (126 MHz, DMSO) δ 141.85, 130.84, 128.57, 127.78, 121.22, 64.49, 50.55, 23.96. HRMS (ESI<sup>+</sup> ): [M + H]<sup>+</sup> : calculated for C17H18N<sup>+</sup> (*m*/*z*): 236.14392; detected: 236.14328. LC-MS purity 99%.

#### *2.3. In Vitro Anti-ChE Assay*

The human AChE/human BChE (*h*AChE/*h*BChE) inhibitory activity of the tested derivatives was determined using Ellman's method [46–48] and is expressed as IC50, i.e., a concentration that reduces the cholinesterase activity by 50%. The human recombinant BChE and AChE were prepared at the Department of Toxicology and Military Pharmacy (Faculty of Military Health Sciences, Hradec Kralove, Czech Republic). 5,5′ -dithiobis(2 nitrobenzoic acid) (Ellman's reagent, DTNB), phosphate buffer (PB, pH 7.4), acetylthiocholine (ATC), and butyrylthiocholine (BTC) were purchased from Sigma-Aldrich, Prague, Czech Republic. For measuring purposes, polystyrene Nunc 96-well microplates with a flat bottom shape (Thermo Fisher Scientific, Waltham, MA, USA) were utilized. All the assays were carried out in 0.1-M KH2PO4/K2HPO<sup>4</sup> buffer, pH 7.4. Enzyme solutions were prepared at 2.0 units/mL in 2-mL aliquots. The assay medium (100 µL) consisted of 40 µL of 0.1-M phosphate buffer (pH 7.4), 20 µL of 0.01-M DTNB, 10 µL of the enzyme, and 20 µL of 0.01-M substrate (ATC/BTC iodide solution). Inhibitor solutions in the concentration range 10−3–10−<sup>11</sup> M were prepared, and IC<sup>50</sup> values were calculated. Tested compounds were preincubated for 5 min. The reaction was started by the immediate addition of 20 µL of the substrate. The activity was determined by measuring the increase in absorbance at 412 for *h*AChE/*h*BChE at 37 ◦C at 2 min intervals using a multimode microplate reader Synergy 2 (BioTek Instruments, Inc., Winooski, VT, USA). Each concentration was assayed in triplicate. Software GraphPad Prism 5 (San Diego, CA, USA) was used for the statistical data evaluation.

#### *2.4. Kinetic Study of hBChE Inhibition*

The kinetic study of *h*BChE was performed by using the above-mentioned modified Ellman's method. The values of *V*max and *K*<sup>m</sup> of the Michaelis-Menten kinetics, as well as the value of *K*<sup>i</sup> , were calculated by nonlinear regression from the substrate velocity curves. Linear regression was used for calculation of the Cornish-Bowden plots. All calculations were performed using GraphPad Prism software version 6.07 for Windows (San Diego, CA, USA).

#### *2.5. Antagonist Activity Towards the NMDA receptor*

#### 2.5.1. HEK293 Cell Culture and Transfection

Human embryonic kidney 293 (HEK293) cells were cultured in Opti-MEM I containing 5% fetal bovine serum (FBS; both from Thermo Fisher Scientific) [49]. The cells grown in a 24-well plate were put in Opti-MEM I media containing a mixture of 0.9 µL of *MATra*-A Reagent (*IBA*) and 900 ng of DNA vectors carrying the human versions of the GluN1-1a (GluN1), GluN2A, or GluN2B subunits and green fluorescent protein (GFP; all vectors were added in equal ratio) [39,50,51]. The cells were placed on a strong magnet plate for 30 min and then trypsinized; resuspended in Opti-MEM I containing 1% FBS, 20-mM MgCl2, and 3-mM kynurenic acid (to inhibit the NMDAR-induced excitotoxicity); and plated on

poly-L-lysine-coated glass coverslips. The electrophysiological experiments were executed 24–48 h after transfection [52].

#### 2.5.2. Electrophysiology

Whole-cell patch-clamp recordings were conducted at room temperature on the GFPpositive HEK293 cells using an Axopatch 200B amplifier (Molecular Devices, San Jose, CA, USA); the series resistance compensation was >80% for all cells [39]. The recorded currents were filtered at 2 kHz with an eight-pole low-pass Bessel filter and digitized at 5 kHz with Digidata 1322A and pClamp 10 software (Molecular Devices). The WAS02 application system, which can reach the solution exchange time around the recorded cell of 10–20 ms, was used to apply the extracellular solution (ECS) [53]. The ECS contained (in mM): 160 NaCl, 2.5 KCl, 10 HEPES, 10 glucose, 0.2 EDTA, and 0.7 CaCl<sup>2</sup> (pH adjusted to 7.3 with NaOH) and was supplemented with the saturating concentrations of co-agonists glycine (50 µM) and agonist glutamate (1 mM; both from Merck). The stock solutions of **3a***–***o** (10 mM) were prepared freshly before each experiment in dimethylsulfoxide (DMSO; Merck), and the background concentration of DMSO was kept equal in each ECS. The borosilicate glass pipettes with a tip resistance ~4-7 MΩ were made using a P-1000 micropipette puller (Sutter Instrument Co., Novato, CA, USA) and were filled with an intracellular solution (in mM: 125 gluconic acid, 15 CsCl, 5 EGTA, 10 HEPES, 3 MgCl2, 0.5 CaCl2, and 2 ATP-Mg salt (pH adjusted to 7.2 with CsOH). Data were analyzed using SigmaPlot 14.0 (Systat Software, Inc., Chicago, IL, USA), and the dose-response curves were built using Equation (1): I = 1/(1 + ([compound]/IC50) h ), where IC<sup>50</sup> is the concentration of the compound that produces a 50% inhibition of the glutamate-evoked current, [compound] is the concentration of the studied compound, and h is the apparent Hill coefficient [54].

#### *2.6. In Vitro Cell Viability Assessment*

Standard MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma Aldrich, Prague, Czech Republic) was used according to the manufacturer's protocols on the CHO-K1 (Chinese hamster ovary, ECACC, Salisbury, UK) and hCMEC/D3 (Sigma Aldrich, St. Louis, MO, USA). The cells were cultured according to recommended conditions and seeded in a density of 8000 cells per well, as described previously [55]. Briefly, the tested compounds were dissolved in dimethyl sulfoxide (DMSO; Sigma Aldrich, St. Louis, MO, USA) and subsequently diluted in the nutrient mixture F-12 ham growth medium (Sigma Aldrich, St. Louis, MO, USA), supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (both Sigma Aldrich, St. Louis, MO, USA), so that the final concentration of DMSO did not exceed 0.5% (*v*/*v*). CHO-K1 cells were exposed to serially diluted tested compounds for 24 h. Then, the medium was replaced by a fresh medium containing 0.5 mg/mL of MTT, and the cells were allowed to produce formazan for another approximately 3 h under surveillance. Thereafter, the medium with MTT was removed, and crystals of formazan were dissolved in DMSO (100 µL/well; PENTA s.r.o., Prague, Czech Republic). Cell viability was assessed spectrophotometrically by the amount of formazan produced. The absorbance was measured at 570 nm with a 650-nm reference wavelength on Synergy HT (BioTek, Winooski, VT, USA). The IC<sup>50</sup> value (halfmaximal inhibitory concentration) was calculated from the control-subtracted triplicates using nonlinear regression (four parameters) by GraphPad Prism 7.03 software (GraphPad Software Inc., San Diego, CA, USA) for the CHO-K1 cell line, and for hCMEC/D3 cells, the cell viability was expressed as the % relative to the untreated control. Final IC<sup>50</sup> and SEM (standard error of the mean) values were obtained as a mean of three independent measurements.

#### *2.7. Determination of In Vitro BBB Permeation*

The D3 assay evaluates the ability of compounds to diffuse from the donor compartment through the D3/hCMEC cell membrane into the acceptor compartment. The D3 cells

were seeded on a PET membrane (area 1.12 cm<sup>2</sup> ) with 3-µm pores of the 12-well plates with 12-mm inserts. The tested compounds were dissolved in DMSO and then diluted with Opti-MEM to reach final a concentration of 30 µM for **3m** and 50 µM for others; the concentration of DMSO did not exceed 0.5% (*v*/*v*). The donor solution (750 µL) was added to the donor compartment (insert), and the same volume of Opti-MEM was added into the acceptor. The concentration of the drug in both compartments was measured by UV-VIS spectrophotometry in 5, 15, 30, and 60 min of incubation in triplicates. The apparent permeability coefficient (*Papp*) was calculated from the concentration ratios. Tightness of the D3 monolayer was conducted by the fluorescein isothiocyanate (FITC) test after each experiment. The accepted values of FITC (fluorescein isothiocyanate) in 0.4 mg/mL in the acceptor compartment after 30 min of incubation must not exceed 1.5% of the initial donor concentration.

$$Papp = \left(\frac{\text{dC}}{\text{dt}}\right) \times \frac{\text{V}\_{\text{r}}}{\text{(A} \times \text{C}\_{0})} \tag{1}$$

A: area of the well/cell monolayer,

dC/dt: amount in the receiver compartment in given time,

Vr: volume of the receiver compartment, and

C0: the initial concentration of tested compounds.

#### *2.8. Molecular Modeling Studies*

The model of *h*BChE (Protein Data Bank (PDB) ID: 4BDS, resolution: 2.10 Å) was downloaded from the RCSB Protein Data Bank (https://rcsb.org) and prepared for flexible molecular docking by MGL Tools [56,57]. The preparation of these receptors involved removal of the inhibitor, water molecules and nonbonded co-crystalized compounds, and the addition of polar hydrogens. Default Gasteiger charges were assigned to all atoms. Flexible parts of the enzymes were set based on previous experiences [58,59]. The rotatable bonds in the flexible residues were detected automatically by AutoDock Tools 1.5.4. The flexible receptor parts contained 39 residues. The studied ligands were firstly drawn in HyperChem 8.0, then manually protonated, as suggested by MarvinSketch 18.24.0. software (http://www.chemaxon.com), geometrically optimized by software Avogadro, and stored as pdb files. The structures of the ligands were processed for docking by the AutoDock Tools 1.5.4 program. Molecular docking was carried out by the software AutoDock Vina 1.1.2 utilizing computer resources of the Czech National Grid Infrastructure MetaCentrum. The search algorithm of AutoDock Vina efficiently combines a Markov chain Monte Carlo-like method for the global search and a Broyden-Fletcher-Goldfarb-Shano gradient approach for the local search [60]. It is a type of memetic algorithm based on interleaving stochastic and deterministic calculations [61]. Each docking task was repeated 20 times with the exhaustiveness parameter set to 8, employing 8 CPUs in parallel multithreading. From the obtained results, the solutions reaching the minimum docking score were taken as the top-scoring modes. The graphical representations of the docked poses were shown in PyMOL (The PyMOL Molecular Graphics System, Version 2.0.6. Schrödinger, LLC, New York, NY, USA). 2D diagrams were generated using Maestro 12.3 (Schrödinger Release, Schrödinger, LLC, New York, NY, USA).

#### *2.9. In Silico Pharmacokinetics and Drug-Likeness Prediction*

SwissADME, web tool was used to predict gastrointestinal absorption, BBB permeation and bioavailability [62]. Physicochemical properties (p*K*a, Clog*P*, HBA, HBD, and TPSA) were predicted by MarvinSketch 20.4.0, ChemAxon Ltd. (Budapest, Hungary; see Table S1, Supplementary Materials). BBB scores were calculated by the BBB calculator available at the ACS website [63].

#### **3. Results and Discussion**

#### *3.1. In Silico Prediction of the CNS and Oral Availability*

All the newly designed compounds were initially screened in silico to predict their peroral and CNS availability. Indeed, both of these features are essential in the drug discovery process of small molecules as potential anti-AD therapeutics and should be estimated before synthesis. To this end, the in silico pharmacokinetics; drug-likeness; and ADME (absorption, distribution, metabolism, and excretion) prediction was applied to compounds **3a**–**o** using a web-based tool SwissADME [62,64,65]. THA and memantine were used as references with known CNS statuses and pharmacokinetic profiles [42,66,67]. Data are presented in Figures 2 and 3 and Table 1 and Table S1.

**Table 1.** Summary of the in silico absorption, distribution, metabolism, and excretion (ADME) and drug-likeness of derivatives **3a**–**o** with tacrine (THA) and memantine using various prediction models.


<sup>a</sup> GIA = gastrointestinal absorption, <sup>b</sup> BBB = blood–brain barrier permeation, <sup>c</sup> Reference [63],

<sup>d</sup> Reference [68], and <sup>e</sup> Bio. Score = bioavailability score [69].

The pink middle area (Figures 2 and 3) represents the optimum range for bioavailability. It is a sum of the lipophilicity (LIPO), size, polarity (POLAR), solubility (INSOLU), saturation (INSATU), and flexibility (FLEX). The red lines show the predicted state for the designed compounds [62,64,65]. The calculated parameters for all the compounds, except for **3b** and **3h**, are situated inside the pink area, suggesting their high oral bioavailability [69]. A positive correlation for the bioavailability was also supported by the BOILED-Egg method [70], proposing high gastrointestinal adsorption and penetration through the BBB [70]. The BBB score, a new predictive model for CNS availability, was also calculated for all the derivatives fitting the range between 5 and 6, indicating a high probability to permeate through the BBB (estimated physicochemical parameters are shown in Table S1, Supplementary Materials) [63]. Besides, all compounds also fulfill the drug-likeness rules of pharmacological models defined by pharmaceutical companies like Veber's (GSK) [71], Lipinski's (Pfizer) [72], Egan's (Pharmacopeia) [73], Ghose's (Amgen) [74], and Muegge's (Bayer) [75] (Table S2, Supplementary Materials). In summary, derivatives **3a**–**o** unambiguously show high predictive peroral bioavailability, BBB permeation, and acceptable pharmacokinetic profiles, making the compounds prospective candidates for the treatment of CNS disorders, including AD.

**Figure 2.** The bioavailability radar chart of the designed fluorene-9-amines **3a–o**.

**Figure 3.** The bioavailability radar chart for the standards THA and memantine (please see detailed description in the text).

#### *3.2. Synthesis*

Fluoren-9-amines were prepared in a one-step procedure from commercially available 9-bromofluorene (**1**) with an excess of various primary or secondary amines (**2a**–**o**) in dry acetonitrile (Scheme 1). This reaction afforded the formation of fluoren-9-amines (**3a**–**o**) in high yields (80–95%). The free bases were then converted to the respective hydrochloride salts. The final products were characterized by <sup>1</sup>H, <sup>13</sup>C-NMR spectra, melting points, and HRMS analysis. LC analysis confirmed the purity for all the derivatives > 97% (Supplementary Materials).

#### *3.3. Evaluation of Cholinesterase Inhibitory Activity*

Fluoren-9-amines were tested for their inhibitory potential against human AChE (*h*AChE) and human BChE (*h*BChE) enzymes using the modified spectrophotometric method of Ellman et al. [46,48,76]. THA was used as a reference compound. The IC<sup>50</sup> values of all tested compounds and their selectivity indexes (SI) for *h*BChE are summarized in Table 2. All tested derivatives showed a preferential inhibition of *h*BChE (usually in the single-digit micromolar range), whereas only compounds **3c** and **3m** showed weak inhibitory activity for *h*AChE at 13.98 and 49.91 µM, respectively. The *h*BChE inhibition ranged between 0.47–54.65 µM. According to the *h*BChE inhibition potency, the top-ranked derivative from all the derivatives under the study was **3c** (IC<sup>50</sup> = 0.47 µM), being 20 times less active compared to THA. However, the relatively unique selectivity to BChE is of immense interest. Indeed, the selectivity profile for the ChE drugs potentially useful in AD treatment is extensively discussed [77]. The currently used ChE inhibitors are AChE-selective (donepezil) or possess more or less nonselective patterns of inhibition (rivastigmine and galantamine) [25]. It was proven that the levels of BChE in the brain of AD patients are elevated with age, while the levels of AChE are suppressed. This finding underlines the importance of selective-BChE inhibitors in the treatment of the later stages of AD [77,78].

#### *3.4. Kinetic Study of hBChE Inhibition*

To determine the inhibition pattern of **3c**, a kinetic study was performed to elucidate the interaction mechanism of the compound with *h*BChE. The kinetics of the inhibition were revealed from velocity curves measured at several concentrations of **3c** and the *h*BChE substrate butyrylthiocholine. The type of enzyme inhibition and appropriate affinity parameter (*K*<sup>i</sup> ) were determined using nonlinear regression analysis. The results for each type of model of inhibition (competitive, noncompetitive, uncompetitive, and mixed) were compared by the sum-of-squares *F-*test. A statistical analysis showed a competitive type of inhibition for *h*BChE (*p* < 0.05) that was in line with the Cornish–Bowden plot used for visualization of the obtained data (Figure 4).


**Table 2.** The inhibitory activities of compounds **3a**–**o** for human acetylcholinesterase (*h*AChE) and human butyrylcholinesterase (*h*BChE).

<sup>a</sup> Results are expressed as the mean of at least three experiments ± SEM. n.a. stands for not active at the tested concentration scale. <sup>b</sup> SI = selectivity index; the selectivity for *h*BChE is determined as a ratio of the half-maximal inhibitory concentration (IC50)(*h*AChE)/IC50(*h*BChE). <sup>c</sup> Data taken from Reference [79].

**Figure 4.** Steady-state inhibition of the *h*BChE substrate (human butyrylthiocholine) hydrolysis by compound **3c** at different concentrations. Cornish−Bowden plots of (S)/*v* against the (I) of the initial velocity at increasing substrate concentrations (2.5–20.0 mM) are presented. Lines were derived from the linear regression of the data points. Each point is the average of three determinations.

The parallel lines indicate a competitive inhibition of *h*BChE by **3c**, which means a reversible binding mode to the active site of the enzyme. With the increasing concentration of the inhibitor, the apparent *V*max remained unchanged and *K*<sup>m</sup> increased. A *K*<sup>i</sup> value of 173 ± 25 nM was determined for **3c** on *h*BChE.

#### *3.5. Evaluation of Antagonist Activity towards the NMDA Receptor*

Further, we aimed to examine the effects of all synthesized compounds (**3a**–**o**) at both the GluN1/GluN2A and GluN1/GluN2B receptors. First, we held the transfected cells at a membrane potential of −60 mV, and we assessed the relative inhibitory effect of each compound at a 10-µM concentration upon its application with the (co-)agonists (Figure 4). These experiments showed that all studied compounds exhibited an inhibitory effect ranging from ~7% to ~52% at both studied NMDAR combinations (Table 3). Interestingly, we revealed that **3e** was the most potent compound at the GluN1/GluN2A receptors, while **3m** was at the GluN1/GluN2B receptors. Subsequently, we generated concentration-response curves for **3e** and **3m** (1–300 µM) for both the GluN1/GluN2A and GluN1/GluN2B receptors at the negative (−60 mV) and positive (40 mV) membrane potentials (Figure 5). Our analysis showed that both compounds exhibited a more profound inhibitory effect at the negative membrane potential (IC<sup>50</sup> values ranged from ~9 µM to ~15 µM), while they were much less potent at the positive membrane potential (IC<sup>50</sup> values ranged from ~83 µM to ~221 µM); the results are summarized in Table 4. Together, our data show that both **3e** and **3m** act as potent voltage-dependent inhibitors of the NMDARs, suggesting that they act as open-channel blockers of NMDARs. Interestingly, the IC<sup>50</sup> values for both **3e** and **3m** at the GluN1/GluN2A and GluN1/GluN2B receptors were in a similar range to those previously obtained with THA (Table 4) [39]. However, both **3e** and **3m** were less potent than memantine under the studied conditions (Table 4).

#### *3.6. In Vitro Cell Viability Assessment*

The in vitro cytotoxicity evaluation on mammalian cells served as a preliminary toxicity indicator for the newly developed compounds (Table 5). In our previous work, we observed a correlation between the calculated log*P* (Clog*P*) values (MarvinSketch software, v. 18.24.0; Table 5) and the toxicity; however, our results within the presented study showed no clear relationship [80]. For instance, one of the most lipophilic compounds (**3f**) yielded as the least toxic, whereas other more hydrophilic fluoren-9-amines were regarded as low-toxic (e.g., **3g** as the most hydrophilic compound) agents as well. The least cytotoxic compound can be classified as **3f** with an attached piperidine moiety. At the pole separation, the most cytotoxic agents were **3c**, **3m**, and **3e**. Importantly, some of the new derivatives were less cytotoxic than THA.


**Table 3.** The inhibitory effect of the derivatives **3a**–**o** at the GluN1/GluN2A and GluN1/GluN2B receptors.

<sup>a</sup> The relative inhibition (RI) was calculated as the ratio of the steady-state currents in the presence and absence of 10 µM of the compound (multiplied by 100) at the membrane potential of −60 mV (see text for more details). Data are shown as mean ± SEM; n = number of recorded cells (see Figure 5).


**Table 4.** The activity of derivatives **3e** and **3m** at the *N*-methyl-D-aspartate (NMDA) receptors at different membrane potentials compared to THA and memantine (Mem).

**− −**

**−**

‐ ‐ ‐ ‐

**−**

<sup>a</sup> The experimental data obtained using six concentrations of each compound (1, 3, 10, 30, 100, and 300 µM) were fitted by Equation (1) (see Section 2.5.2); the values of the IC<sup>50</sup> (in µM), Hill coefficient (*h*), and numbers of analyzed cells (n) obtained from the membrane potentials of −60 mV or 40 mV are shown as mean ± SEM. Dose-response relationships of the inhibitory effects of **3e** or **3m** at the GluN1/GluN2A and GluN1/GluN2B receptors (see Figure 6). <sup>b</sup> THA values were published by our group recently [39]. Mem corresponds to memantine. − ‐

‐ ‐ ‐ − **Figure 5.** The examples of the most (**3e** and **3m**) and least (**3l** and **3g**) potent compounds from all tested fluoren-9-amines at the NMDARs. Representative whole-cell currents induced by 1 mM of glutamate (Glu) and 50 µM of glycine at the negative (−60 mV) membrane potential with (**A**) the least potent compounds: **3l** measured at GluN1/GluN2A and **3g** at GluN1/GluN2B NMDARs and (**B**) the most potent compounds: **3e** measured at GluN1/GluN2A and **3m** at GluN1/GluN2B NMDA receptor (see Table 3).

**Figure 6.** The most potent inhibitory compounds **3e** and **3m** acting at the GluN1/GluN2A and GluN1/GluN2B receptors. (**A**) Whole-cell representative currents of the GluN1/GluN2A receptors showing the inhibitory effects of **3e** (1–300 µM) at membrane potentials −60 mV and +40 mV. (**C**) Representative currents of the GluN1/GluN2B receptors inhibited by **3m** (1–300 µM) at membrane potentials −60 mV and +40 mV. (**B**,**D**) Dose-response inhibitory curves at two membrane potentials (−60 and +40 mV) at the GluN1/GluN2A receptors for **3e** (**B**) and at the GluN1/GluN2B receptors for **3m** (**D**). The inhibition dose-response curves were fitted by Equation (1) (see Section 2.5.2); the resulting values are shown in Table 4.


**Table 5.** The effects of the tested compounds on the CHO-K1 cell viability.

<sup>a</sup> Values are expressed as the mean of three independent measurements. <sup>b</sup> Determined at the maximum solubility of the compound. <sup>c</sup> Data taken from Reference [81]. Clog*P*: calculated log*P*.

#### *3.7. In Vitro BBB Permeation*

The top-ranked BChE inhibitors and NMDA receptor antagonists were selected (i.e., **3e**, **3n**, **3c**, and **3m**) to evaluate their ability to cross the BBB. For this reason, we used the human brain microvascular endothelial cell line hCMEC/D3 as a suitable model for the

normal human BBB [82]. Before that, we investigated whether the applied concentration (50 µM) of the tested compound negatively influenced the cell viability in the monolayer. Despite the relatively low toxicity of the CHO-K1 model, we investigated the effects on the cell viability of the hCMEC/D3 line using MTT at 100 and 50 µM (Table S3). We found that all tested compounds at 50 µM did not substantially reduce the cell viability (>90% of viable cells), except **3m**, which reduced the cell viability to approx. 82% at 50 µM. Due to this fact, a concentration of 30 µM was used for the BBB permeation test for **3m**.

To investigate the potential of the BBB permeation, we used a panel of reference drugs for which the BBB permeation in vivo is known and correlated the P*app* values to them. Thus, our measurements predict that the compounds **3c**, **3e**, and **3n** have a low potential to passively pass the BBB. On the other hand, the P*app* value for **3m** corresponds to those of standard drugs with high CNS permeability (Table 6).


**Table 6.** Prediction of the BBB penetration of drugs expressed as the apparent permeability coefficient (P*app*) ± SEM.

<sup>a</sup> CNS+ and CNS− represent the final predictions of the compound capability to cross or not to cross the BBB, respectively. <sup>b</sup> n stands for the number of independent experiments.

#### *3.8. In Silico Studies*

To better understand the inhibition mechanism between *h*BChE with **3c** (Figure 7A,B) and **3n** (Figure 7C,D), respectively, as the top-ranked inhibitors of the respective enzyme, we performed a molecular modeling simulation. The crystal structure of the THA-*h*BChE complex (Figure 7E,F; PDB ID: 4BDS) was used for comparative purposes [56]. In line with the compounds' designs, both of the inspected fluoren-9-amines **3c** and **3n** adopted a similar arrangement to that of THA within the *h*BChE active site. The crucial observation is that the tricyclic cores of both new molecules interact with Trp82 via parallel π-π stacking in the anionic site of the enzyme. The protonated amino group enabled hydrogen bond formation to carboxylic oxygen from Asp70 (**3c** and **3n**) and the hydroxyl group from Tyr332 (**3n**). In the case of THA, this type of interaction is replaced by two water bridges with the primary amino group. From the catalytic triad residues (His438, Glu197, and Ser198), only His438 is involved in hydrophobic contact with **3c** and **3n**, whereas THA is involved in direct hydrogen contact with this residue. The docking results also highlighted the importance of the attached appendages. Both aliphatic parts, either the cyclohexyl (**3c**) or *iso*-butyl (**3n**) moiety, are engaged in hydrophobic contacts with the Tyr332, Phe329, and Ala328 residues. The cyclohexyl moiety of **3c** seems to deliver another hydrophobic contact to Pro285 and Gly78 that presumably mediates a slightly higher inhibition potency. Contrary to THA anchoring, missing cation-π contacts in the **3c**- and **3n**-*h*BChE complexes can be denoted as the culprit responsible for the slightly decreased affinity of these two agents.

**Figure 7.** Top-scored docking poses of **3c** (**A**,**B**), **3n** (**C**,**D**), and the crystal structure of THA (**E**,**F**) within the *h*BChE active site (Protein Data Bank (PDB) ID: 4BDS) [56]. Ligands are displayed in green (**A**), salmon (**C**), and brown (**E**) for **3c**, **3n**, and THA, respectively. Important amino acid residues involved in the enzyme-ligand interaction are shown in dark-blue, and the catalytic triad is rendered in yellow. Distances are colored as dashed lines, and the distance is measured in Å (**A**,**C**,**E**). 3D figures were created by using PyMOL 2.0.6 viewer. 2D diagrams were generated with Maestro 12.3 (Schrödinger Release, Schrödinger, LLC., New York, NY, USA).

#### **4. Conclusions**

Since the cholinergic hypothesis was first postulated [15], five decades of intensive research have generated a few marketed drugs for symptomatic relief only. The proper and in-depth elucidation of the disease biology imposes challenging tasks that remain

to be resolved by delivering the drugs in the right stage of the disorder [29]. So far, the attempts to approve any drugs have continuously failed, with memantine receiving the final approval in 2003. A glimmer of hope emerged in 2019 when sodium oligomannate (GV-971) was approved in China, acting in a completely different way to other marketed drugs. Indeed, GV-971 suppresses gut dysbiosis, mitigates neuroinflammation, and reverses cognition impairment [83]. Designing MTDLs is an interesting approach that has generated several preclinical candidates for the therapy of AD. However, most of these newly developed agents suffer from limitations, as disclosed recently [29]. Our study was inspired by the appealing combination of donepezil and memantine into one capsule that resulted in a higher therapeutic benefit than the single-agent administrations [27]. Herewith, pursuing the symptomatic stage of AD by modulating the cholinergic and glutamatergic systems, we developed a series of 15 molecules with fluorene tricyclic scaffolds substituted with secondary or tertiary amines at C9. Prior to the synthesis, we took the compound's predicted physicochemical parameters and calculated their oral and CNS availability. Indeed, all the compounds adhered to these criteria. Under in vitro conditions, we demonstrated that the compounds can interact with both NMDAR and BChE while having only a marginal effect on AChE. We also showed that they have a relatively low cytotoxic profile, as determined using CHO-K1 cell lines, and for one compound (**3m**) out of the four selected candidates, a BBB permeation ability was confirmed. The obtained data herein warrant future research with this class of compounds; however, we may state that we found low-toxic BChE-selective compounds with antagonistic activity on the NMDARs that potentially permeate the BBB.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/2218-273 X/11/1/3/s1, Table S1. Physiochemical parameters of compounds **3a**–**o**, THA and Memantine.; Table S2. Drug-likeness of derivatives **3a**–**o**, THA and memantine.; Table S3. Effect of tested compounds on the cell viability of the hCMEC/D3 cells.; <sup>1</sup>H and <sup>13</sup>C NMR spectra; HRMS, and HPLC records of compounds.

**Author Contributions:** J.K. (Jan Konecny), chemical synthesis, data interpretation, and manuscript writing; M.H. (Martina Hrabinova), in vitro measurement of cholinesterase activity; L.P. (Lenka Pulkrabkova) and M.B., cytotoxicity evaluation and BBB permeation; L.P. (Lukas Prchal), HRMS analysis; T.K. (Tereza Kobrlova), BBB permeation; T.K. (Tomas Kucera), in silico calculations; V.F., design of the compounds; D.J., enzyme kinetic analysis; A.M., S.K., M.K., and M.H. (Martin Horak), NMDA receptor measurements; M.V.; manuscript writing; O.S., design of the study, BBB permeation, and manuscript writing; and J.K. (Jan Korabecny), design of the study, data interpretation, docking studies, and manuscript writing. All authors have read and agreed to the published version of the manuscript.

**Funding:** This project was supported by the Czech Health Research Council (No. NU20-08-00296), by the faculty of Military Health Sciences, University of Defence (SV project FVZ201803, Long-term development plan), by MH CZ—DRO (University Hospital Hradec Kralove, No. 00179906), by the European Regional Development Fund: Project "PharmaBrain" (no. CZ.02.1.01/0.0/0.0/16\_025/0007444), and by the project "e-Infrastruktura CZ" (e-INFRA LM2018140) provided within the program Projects of Large Research, Development and Innovations Infrastructures. V.F. acknowledges the support of Charles University (SVV 260 547).

**Data Availability Statement:** Data is contained within the article or Supplementary Materials.

**Acknowledgments:** The authors would like to acknowledge the skillful assistance of Barbora Hejtmankova during the BChE enzyme kinetic evaluation.

**Conflicts of Interest:** The authors declare no conflict of interest.

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