Synthesis, Spectral Characterization and Molecular Docking Studies of Novel Benzidin-bis-tetrahydroacridine Analogues

Alzheimer’s is a progresive neurodegenerative disease that interferes with human cognitive ability, memory and behavior. The inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes are major therapeutic routes for the treatment of Alzheimer disease. In the study, nevel bis-polymethylenquinoline-bis-carboxamides (3a-f) and bis-polymethylenquinoline-bis-carboxylic acids (5a-b) having as precursor benzidine, were obtained in good yields by Pfitzinger condensation reactions of bis-isatines with corresponding cyclanones. The compounds were characterized by elemental analysis, FT-IR, NMR and mass spectrometry. Furthermore, the compounds were subjected to molecular docking dynamics simulations to ascertain their potentials as inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Molecular docking simulations showed varied binding activities towards the two binding sites of acetylcholinesterase: 4EY7 and 1OCD, and human butyrylcholinesterase: 1P0I. Compounds 3e and 5b demostrated strong binding affinities with 1P0I, 1OCD and 4EY7 biotargets similar to the binding modes of donepezil and tacrine (co-crystallized inhibitors of acetylcholinesterase) and butyrate (co-crystallized inhibitors of butyrylcholinesterase).

Previously, we described the synthesis of other tetrahydroacridine derivatives [25,26] obtained by Pfitzinger reaction of isatines with cyclanones and their AChE inhibitory activity was predicted using the structure-based QSAR model recently developed in our lab [27]. The QSAR model indicated a correlation between the higher number of methylene groups present in the target molecule and the higher inhibitory AChE activity. In the present work, novel eight bis tetrahydroacridines compounds were synthesized and subjected to molecular docking studies and their binding energies, Drug-likeness and Pharmacokinetics evaluations were carried out.

Materials and methods
All chemicals were purchased from commercial suppliers and used without any further purification. The IR spectra were recorded using a FT-IR Bruker Vertex 70 equipped with a diamond ATR. 1 H and 13 C-NMR spectra were recorded using a Gemini 300BB spectrometer operating at 300MHz for 1 H and 75MHz for 13 C in DMSO-d6, using TMS as internal reference. Mass spectra were recorded on Varian 1200 LC/MS/MS mass spectrometer with electrospray interface ESI, both in positive and negative mode. Substance dissolved in methanol (1% formic acid) to 1 ppm was direct introduced ESI interface using a Prostar 240 SDM pump at 50 µl flow rate. ESI needle was set at 5000 V both positive and negative mode, drying and nebulizing gas was nitrogen from Domnick Hunter generator. For negative mode methanol had 0.5 % ammonium carbonate instead of formic acid. Fragmentations were performed in second quadrupole using argon as collision gas at 1.5 mTorr. Fragments were scanned by the third quadrupole. Chloral hydrate (0.22mol, 32.5g) was dissolved in 1000 mL water with Na2SO4 (1.60 mol, 230g). The solution was warmed at 60 0 C before the solution of hydroxylamine hydrochloride (0.22 mol in 100 mL water) was added. In a separate flask, slurry of benzidine (0.1 mol, 18.4g) in water (120mL) with concentrated HCl (17.3mL) was prepared. The two reaction mixtures were combined and the reaction temperature was increased to 90-95 0 C for 20 min. Heating was then stopped and the mixture was cooled in an ice bath for 15 min. The solid precipitates were collected by filtration, recrystalized in C2H5OH, filtrated off, and poured into a cold dilute ammonia solution. The resultant yellowish precipitates ( Figure  2) were obtained by filtration, washed with water (30mL) and C2H5OH, and dried [28].   (2-(hydroxyimino)acetamide derivative (1a) (0.03 mol, 9.78g) is added in small portions, over 8-10 minutes, to a solution of sulphuric acid (0.72 mol, 44.14mL, 88.3%, d=1.81) at 70-72 0 C, under vigorous stirring. The temperature is maintained at 87-95 0 C for 30 min to complete the reaction. The mixture is cooled at room temperature and poured into a mixture of ice and water. The crude product is stirred for 30 min, filtered off and washed several times with water to neutral pH. Deep-purple powder was obtained ( Figure 3). The bis-isatines (2a) were purified by dissolving it in an aqueous alkali 5%, neutralising with dilute acetic acid, filtering off the dark material separated, and acidifying the solution with hydrochloric acid [29]. The solution is then cooled rapidly, and the bis-isatin which separates is filtered off. Yield

General procedure for synthesis of corresponding dicarboxylic acids (5a-b):
Target compounds (5a-b) were obtained according the reaction oulined in scheme 1, by cyclocondensation of bis-indoline-tetraones with a cyclanone in etanolic solution of KOH: 0.1 mol of benzidinbisisatin (2a) was dissolved in 0.3 mol of KOH (30% solution) in 100 mL of EtOH. At the end, 0.3 mol of cyclanone is added with stirring. The reaction was stirred at reflux for 12 hours. Then the half of the solution is concentratad by vacuum distilation and the precipitate formed is filtered by vacuum and washed several times with acetone and ethyl ether. After cooling 0.34-0.4 moles of 50% CH3COOH were added. The resulting yellow solid was insoluble in common organic solvents and therefore could not be purified. The compounds decompose above 240 0 C.
Following the general procedure, 0.

Drug-likeness and Pharmacokinetics Studies
The physicochemical properties to determine the drug-likeness of the synthesized compounds were evaluated in silico. The properties evaluated include molecular weight (MW); hydrogen bond acceptor (HBA); hydrogen bond donor (HBD); total polar surface area (TPSA); number of rotatable bond (nRB); number of acid (nAc); molar refractivity (MR); number of atoms (nA). Also, the pharmacokinetic parameters were generated using Pre ADMET online free software (www.preadmet.bmdrc.kr). The pharmacokinetic parameters evaluated include: cell membrane permeability (Caco2), human intestinal absorption (HIA), CYP 2C19 inhibition, blood-brain barrier (BBB) and plasma protein binding (PPB).

Chemistry
The general synthetic procedure for the new heterodimers is shown in Scheme 1, and consists of three steps: Scheme 1. Synthesis of tetrahydroacridines analogous a. CCl3-CHO, NH2OH, Na2SO4, t = 95-100 0 C; b. H2SO4, t = 70 0 C; c. NH3, Cyclanones; d. KOH alcoholic solution, Cyclanones (used cyclanones were: cyclohexanone, 2-methyl-cyclohexanone, 4-methylcyclohexanone, cycloheptanone, cyclooctanone, tetralone, indanone) The bis-isonitrosoacetanilides, 1a were prepared in low yield (30%) by Sandmayer reaction of dianiline with chloral and hydroxylamine. In the second step of the synthesis, compound (1a) were heated at 87-95 0 C in H2SO4 for twenty minutes to obtain the corresponding bisindoline-tetraones (2a), in good yields, by cyclocondensation. In the third step, carboxamides are obtained by Pfitzinger condensation of bis-isatine (2a) with cyclanones in aqueous ammonia on heating (130-150 0 C), in a closed vessel. If the Pfitzinger condensation occurred in solution of KOH in ethanol, the corresponding acids are obtained. The final compounds have little solubility in organic solvents and melting points above 240 0 C with decomposition [28,29]. The new compounds were boiled several times in chloroform in order to remove the soluble impurities as no purification of the di-amides (3a-f) and di-acides (5a-b), was achieved by recristalization, and/or classic chromatography.
Finally semipreparative HPLC Chromatography is done, with a reversed phase Zorbax C18 column (250 mm 21.2 mm i.d.; particle size 8 µm) (DuPont, USA). Mobile phase consisted in 20/40/40 water (pH 8.2 with ammonium carbonate)/ methanol (0.5 % ammonium carbonate)/ acetonitrile ( 0.5 % ammonium carbonate). Flow rate was established at 10 mL/min, using a Prostar 240 SDM pump (Varian). For chromatography it was used 'heart cut' strategy with a Prostar 330 UV-VIS DAD. Sample dissolved in DMF (1% formic acid) at 100 mg/mL, was manually injected into system at portion of 1 ml, using an Reodyne 7725i . Chromatography was ten times repeated. Solvents (methanol, acetonitrile) were removed in vacuum, from water a grey-beige deposit was collected, filtered off and dried. The method was applied to all synthesized compounds (Figure 4 and 5) [31].

NMR Spectral data
The 1 H NMR spectra of isonitrosoacetanilides, 1a shows for the aromatic protons the AA'BB 'systems with coupling constants of J = 8.8 Hz at δ = 7.63; 7.7 ppm. The azomethynic protons occur at about δ = 7.68 ppm. The signals for the amide group proton -NH-CO-appears at δ = 10.3 ppm and for the hydroxylamine at δ = 12 ppm.
The 1 H NMR spectra of bis-isatine, 2a (obtained by acid cyclization) show three distinct signals for each proton in part. The most shielded is the proton H-7 which appears at δ = 6.9 ppm as a doublet with a coupling constant of J = 8.2 Hz. Proton at H-6 gives a doublet of doublets that occurs at δ = 7.89 ppm. The proton H-4 appears at δ = 7.75 ppm, with a meta coupling constant of J = 1.9 Hz.
By cyclocondensation of bis-isatin, 2a with cyclanones to bis-tacrincarboxamides / bis-tacrinacids, aromatic protons retain their multiplicity, but their chemical shifts are significantly modified in the tetrahydroacridinic system. A deshield of about δ = 0.8 -1ppm is observed for the proton H-7 (doublet) positioned in the ortho position towards the quinolinic nitrogen atom, and to a lesser extent of about δ = 0.2 -0.4 ppm for the proton H-4, of the benzidine. The amide group as well as the carboxyl group, induces a deshielding through space over the H-4 proton and the methylene groups of the saturated cycles which occurs in the range of δ =3.01-3.32ppm.
The 13 C NMR spectra of the bistacrines (resulting from the condensation of the bisisatine (2a) with different cyclanones) showed signals for the amide group at δ = 168.5 -168.0 ppm and for the acids at δ = 166.5 -168.0 ppm. The carbon atom in the positions: , C-9 and C-9 'show signals at δ = 157 -162 ppm. Compared to bisisatins, tertiary carbon atoms do not suffer significant displacements. The methylene groups of the saturated cycles of bistacrinamides, present signals at δ = 25 -30ppm, the most distorted ones being those placed nearest by the amide and the carboxyl groups.

Mass Spectra
The basicity of tetrahydroacridines is comparable or even greater than that of the quinoline. Anyway, in the molecule of tetrahydroacridinic carboxamides, the quinolinic nitrogen has a higher basicity comparing to the amidic nitrogen, due to the presence of the saturated cycle adjacent to the quinolinic N-heterocycle, which increases both the density of electrons and the steric hindrance.
Therefore, electrospray ionization, in the positive ion mode, will produce protonated pseudomolecular ions by the placement of a proton on the nitrogen of the quinolinic heterocycle. When an acidified solvent (e.g. with formic acid) is used for dissolution, the electrospray ionization of the diamides and di-acids, in the positive ion mode, take place with the formation of the parent molecular ion as a protonated molecule [M + H] + . Instead, when a basic mobile phase (water with 0.5% ammonium carbonate with a pH of 8.2) is used, the retention is better for amides and the shape of the chromatographic peaks is significantly improved. Unfortunately, the acids didn't produce good retention neither in acidic nor basic eluents.
Ionization of the amides, Figure 6, occurs with the formation of double ions [M + 2H] 2+ having m/z values corresponding to half; the abundance of these ions is significantly much lower than that of the molecular ions [M + H] + .
Their fragmentation occurs with the successive loss of the amidic groups. In the positive mode, amides are broken down as follows: loss of an ammonia molecule from an amide group, loss of another ammonia molecule from the second amide group, loss of a carbon monoxide molecule (CO), loss of another molecule of carbon monoxide (CO) from the second amide group. Amides without bridge are difficult fragmented.
In the positive mode, amides are broken down as follows: loss of an ammonia molecule from an amide group, loss of another ammonia molecule from the second amide group, loss of a carbon monoxide molecule (CO), loss of another molecule of carbon monoxide (CO) from the second amide group. Amides without bridge are difficult fragmented: The acids are ionized also in negative ion mode in the electrospray interface, when a solvent slightly basic is used, such as methanol / water with 0,5 % ammonium carbonate.
The di-acids are strongly ionized in the positive ion mode and fragmentation occurs with difficulty at high collision energies and higher argon pressures (2.5 mTorr). The first fragmentation occurs with the loss of formic acid and the second by the loss of another molecule of CO2:  Table 1 shows the binding free energy of our compounds, as compared with the co-crystallized inhibitors (CCI) and donepezil. The compounds, especially 3e and 5b showed higher binding affinities than the standard drug. 5b showed highest binding affinities of -13.88 and -15.60 kcal/mol with 1OCD and 4EY7 respectively while 3e had the highest binding affinity with 1POI (-13.82 kcal/mol). These compounds seem to have better binding interactions with the drug targets when compared to CCI and donepezil. Consequently, compounds 3e and 5b were used to further illustrate their binding pose and interactions with the targets as shown in figures 8-10. Docking studies of the synthesized compounds into AChE and BChEI revealed several interactions between the compounds and the amino acid residues in the binding sites of the drug targets. Figures 6 A and B show the binding pose of compound 3e and donepezil respectively in the binding cavity of BChE. Unlike donepezil which had a few significant interactions at the binding cavity of BChE, 3e established so many significant interactions with the amino acid residues in the BChE binding site (Figure 8). The amino group of 3e interacted with the oxygen atom of PRO 285 through H-bonding (3.62 Å). There were three different H-pi interactions observed with 3e in the binding cavity of BChE. Firstly, the 5-membered ring of TRP 82 interacted with C-26 of the cyclooct-1-enyl of the 3e. Secondly, the 6-membered ring of TYR 332 interacted with the N-atom of the amino group of 3e. Thirdly, 6-membered ring of PHE 329 interacted with the same N-atom as TYR 332 did. CA PRO 285, CE2 PHE 329 and CE2 PHE 329, through pi-H bonding, interacted with two aromatic 6-membered rings and one non-aromatic 6-membered ring of 3e respectively. In Figure 7, compound 3e, donezepil and the co-crystallized ligand (butyrate) were concurrently overlaid in the binding cavity of 1POI. These various significant interactions could explain the high binding affinity 3e had with BChE (-13.82 kcal/mol) when compared to the binding affinity of donepezil (-11.41 kcal/mol) and the crystallized ligand (-8.02 https://doi.org /10.37358/Rev. Chim.1949 Rev. Chim., 71(5), 2020, 163-181 175 kcal/mol) ( Table 1). The few interactions of the crystallized ligand with BChE could possibly account for its weaker BChE inhibitory activity.  In Figure 8A, O-33 and O-34 of 5b interacted with N ARG 296 and N PHE 295 through H-acceptor interactions. There was H-π interaction between the C-16 of 5b and 6-membered ring of TRP 86 through an intermolecular distance of 3.75Å. Also, two 6-membered rings of 5b combined with 6-membered rings of TYR 286 and TYR 341 respectively through π-π interactions. AChE contains two binding site domains namely: catalytic anionic site (CAS) and peripheral anionic site (PAS). The major amino acid residues that constitute PAS include TYR124, TRP286, PHE295, ARG296 and PHE297. It is therefore clear that 5b bound to the PAS of the binding site domain of AChE. PAS, is located at the entrance of the AChE gorge, serves as the binding targets for enzyme inhibitors [32].

Drug-likeness and pharmacokinetics
Physicochemical parameters have been used to predict the drug-likeness of a chemical moeity. These parameters influences the pharmacodynamics and pharmacokinetic properties of these chemical compounds. For example, lipophilicity is a property that plays vital role on solubility, absorption, distribution, metabolism, and excretion properties as well as pharmacological activity. When a molecule is highly lipophilic, it tends to go into the inner membrane which is mainly lipoidal and is retained. Octanolwater partition coefficient (log P) is a parameter that measures the degree of solubility of drugs both in aqueous and lipid phases. The higher the log P, the higher the chances of the drug molecule to have low solubility. Conversely, when log P is lower than the lower limit, the drug will find it difficulty to penetrate the lipid membranes [33]. As a result of higher or lower log P outside the normal range, the drug molecule will have poor bioavailability when administered through oral route. This will directly affect the pharmacological activity of such drug. There are other physicochemical parameters which affect bioavailability of drug molecules and they are shown in Table 2.  Lipinski predicted that a drug candidate with good oral bioavailability should have MW ≤ 500 Da, log P ≤ 5, HBD ≤ 5 and HBA ≤ 10. Reckitt has reported the modified Lipinski's rule of 5 (ro5). He stated that a likely drug molecule should have an log P between -0.4 and 5.6, MR between 40 and 130, nA between 20 and 70, HBD ≤ 5, HBA ≤ 10 and MW ≤ 500 Da. Any violation of more than one of these physicochemical parameters will be attributed to poor bioavailability and the compound may not be qualified to be a drug candidate. Our synthesized compounds namely 3a-d and 5a obeyed both the Lipinski's ro5 and the modified Lipinski's ro5 reported by Reckitt. These imply that these compounds can be orally bioavailable and hence are drug candidates. Compounds 5b obeyed only Lipinski's ro5. However, 3e-f disobeyed both rules. However, it should be noted that though some compounds violate these rules, they are still orally bioavailable since they possess groups which act as substrates for biological transporters. They can have up to 4 violations [34]. Furthermore, other surrogate properties have been explored to check whether a compound will be orally bioavailable. A study by [35] has reported that the number of rotatable bond (NoRB) influences bioavailability in rats and recommended that a cut off of NoRB ≤ 10 show good oral bioavailability. All the synthesized compounds showed NoRB ≤ 10 indicating a good oral availability. Total polar surface area (TPSA) is another surrogate marker for cell permeability. A molecule with TPSA ≤ 140 Å 2 would be able to permeate the cell. Again, all the synthesized compounds agreed with this cut off and hence, have the ability to permeate the cell membranes. We further calculated the percentage solubility represented as % ABS from % ABS = 109 -0.345 x TPSA [36]. All the synthesized had good solubility at 70.37% and above which is an indication for good oral bioavailability AD is a neurological disorder. For any drug to be considered as an effective treatment for AD, it must essentially cross the BBB to gain access to the brain cells. This is of primary consideration in the design of drugs for neurological intervention. Besides crossing the BBB, a potential neurotherapeutic drug candidate would need to stay in the brain tissue for a reasonable time in order to elicit the expected pharmacological action [37,38]. Andreas Reichel has modified the Lipinski's ro5 for neurotherapeutics to 'CNS likeness' [39]. He stated that a neurotherapeutic must have MW < 450 Da, Log P < 3, HBD < 4 and HBA < 8. However, Reichel failed to take into consideration other essential drug properties such as polar surface area, number of rotatable bonds, pKa, flexibility, and log D at pH 7.4 (logD7.4) which are desirable for BBB permeation. These drug properties are shown in Table 3 and are modifications from [40,41]. Apart from the violation of the TPSA which is inherent in all the compounds, most of the synthesized compounds met the requirement/cut-off for BBB penetrability. This implies that they can cross the blood brain barrier and might be useful to treat AD.

Pharmacokinetic assessment
Only unbounded drug will be available for diffusion and transport across cell membranes. This will ultimately be available to interact with the pharmacological targets. Therefore, the extent of plasma protein binding of drug molecules will determine its therapeutic outcome, distribution and duration in the system. It has been found out that drug molecules with PPB of more than 90% are strongly bound to the plasma protein and those with less than 90% are weakly bound. From table 4, it can be seen that all the synthesized compounds are strongly bound to plasma protein except 3d. Drugs that are highly bound to plasma protein will be less readily available in the systemic circulation. However, they can be formulated as sustained release drugs. Caco-2 model and Human Intestinal Absorption (HIA) are useful in assessing the cell permeability and intestinal absorption of drug molecule. Caco-2 values being less than 4 imply low permeability; 4 -70 implies middle permeability and greater than 70 implies high permeability [44]. Yee [43] collaborated this finding in 1999, by showing that compounds with HIA (0 -20%) indicates poor absorption; 20 -70% are moderately absorbed and (70 -100%) are well absorbed. Our compounds in table 4 show moderate permeability and they can be well absorbed in the intestine.
CYP2C19 is a member of the cytochrome P450 superfamily of drug-metabolizing enzymes involved in the metabolism of the majority of currently prescribed drugs and new chemical entities. It is a polymorphic enzyme that accounts for less than 5% of hepatic P450 [44] and 2 to 3% of intestinal P450 content [45]. All the synthesized compounds are inhibitors of CYP2C19. That implies that drugs which are metabolized by this enzyme should not be co-administered with these compounds if formulated as drugs. Example: proton pump inhibitors such as omeprazole and lansoprazole, psychotropic drugs including diazepam and imipramine, and anticonvulsants such as phenobarbital and mephenytoin are metabolized by CYP2C19.

4.Conclusions
The Bis-polymethylenquinoline-bis-carboxamides can be considered dual-binding site heterodimers in which the two tetrahydroacridine units are connected directly through the benzenic nucleus of tetrahydroacridinic skeleton. In this present work, a series of di-polymethylencarboxamides and the corresponding diacids were designed to simultaneously interact with the active and peripheral binding sites of human AChE. The compounds where obtained by Pizinger condensation reaction of precursors 5,5'-5,5'-biindoline-2,2',3,3'-tetraone ,2a with substituted cyclanones. The carboxamides are obtained with a yield of 40-50% .Opposed to the mono tetrahydroacridines, the new dimers had very high melting points (mostly decomposed above 240 o C) and are insoluble in common organic solvents. The pharmacological potentials of the compounds, in terms of binding modes, as inhibitors of human acetylcholinesterase (hAChE) and butyrylcholinesterase (hBChE) were evaluated by molecular docking studies with the bio-target sites of AChE: 4EY7, 1OCD; BChE: 1P0I. The compounds had good interactions with the biotargets. However 3e and 5d showed the best binding (greater than the cocrystallized inhibitors and donezepil). These compounds could become lead in the treatment of Alzheimer's disease.