New tetracyclic systems integrated thienopyridine scaffold as an anti-dementia lead: in silico study and biological screening

Alzheimer’s disease (AD) is a multifactorial incurable neurodegenerative disorder. To date, cholinesterase inhibitors (ChEI) are the mainstay line of treatment to ameliorate the symptoms of AD. Tacrine and donepezil are considered two important cornerstones of anti-dementia drugs. Accordingly, novel series of hexahydrobenzothienocyclopentapyridines, octahydrobenzo-thienoquinolines, hexahydrocyclopenta(thienoquinoline/thienodipyridine), and octahydropyrido-thienoquinolines were efficiently synthesized from readily available reagent, e.g. cyclohexanones, cyclopentanone, and 1-methyl-piperidin-4-one to afford 14 new compounds. All new compounds were screened against their acetylcholinesterase, butyrylcholinesterase, and β-amyloid protein inhibition. In AChE inhibition assay, compound 3,7-dimethyl-1,2,3,4,7,8,9,10-octahydrobenzo[4,5]thieno[2,3-b]quinolin-11-amine (2h) showed IC50 value 9.24 ± 0.01 μM × 10−2 excelling tacrine. Compound 1,7-dimethyl-1,2,3,4,7,8,9,10-octahydrobenzo[4,5]thieno[2,3-b]quinolin-11-amine (2e) possess excellent IC50 values 0.58 ± 0.02 μM × 10−2 and 0.51 ± 0.001 μM × 10−4 for both butyrylcholinesterase and β-amyloid protein inhibition assays, sequentially. In silico ADME studies were investigated for the promising members (octahydrobenzo-thienoquinolines 2c, 2d, 2e, 2h, 2i, and octahydropyrido-thienoquinolines 4e) and all the results were illustrated. A comparative docking study was conducted between the promising members and both tacrine and donepezil in both acetyl and butyryl choline active sites. The results revealed extra binding patterns and good agreement with the biological results.


Introduction
Alzheimer's disease (AD) is an irreversible progressive neurodegenerative multi-hitting disease. It affects mostly elderly people leading to a gradual loss of memory, impairment of cognitive abilities, speech difficulties, severe behavioral disturbance, and ultimately death . It is estimated that over 50 million people worldwide are living with dementia with the expected increase in the number up to 152 million by 2050 [1]. The consequent high social and economic complications are demonstrated in Morbidity and Mortality 2022 Report [2]. Consequently, the current attention was directed towards the development of new members able to treat or at least slow down the progression of such a multifactorial disease [3]. Nevertheless, the uncertainty of the definite cause of (AD) entangles the scientists' mission [4,5]. For this reason, several hypotheses including the cholinergic hypothesis, amyloid cascade hypothesis, tau protein hypothesis, oxidative stress hypothesis, bio-metals dyshomeostasis hypothesis, innate immune hypothesis, Osaka mutation, and others have been proposed in an attempt to understand and explain the disease real pathogenesis [6][7][8]. From the cholinergic hypothesis point of view [9,10], elevating the essential neurotransmitter acetylcholine (ACh) levels through simultaneous inhibition of both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) has great benefits in dealing with the disease [11,12]. The AChE and BuChE are two hydrolytic enzymes responsible for the termination of ACh action at the synapse by cleaving Ach to choline and acetate moiety. The two enzymes are very similar in structure and showing 65 identities in amino acids sequence [13]. AChE is found in many types of conducting tissue: nerve and muscle, central and peripheral tissues, motor and sensory fibers. Meanwhile, BuChE, also known as plasma cholinesterase, is found primarily in the liver. Different from AChE, BuChE hydrolyzes butyrylcholine more quickly than ACh [14]. The activity of AChE is higher in motor neurons than in sensory neurons. Accordingly, acetylcholinesterase inhibitors (AChEI) were the pioneer hitting tools for the management of AD by inhibiting AChE. Thus, an increase in ACh bioavailability at neurons synapse causes an improvement in the neuronal transmission in the brain and terminally enhancing memory functions of AD patients [15]. On the other hand, the amyloid hypothesis deals with the abnormal aggregation and deposition of amyloid-β-proteins (AβP) in the brain as the causative agent of AD pathogenesis [7,16]. The genetic mutations in the membrane amyloid precursor protein (APP) gene results in a variation of the APP sequence cleavage producing neurotoxic insoluble amyloid β peptides [17][18][19]. These insoluble peptides aggregate to form polymorphic oligomers and ultimately macroscopic plaques that deposit at neurons causing inflammation, neuron injury, disruption of Ca 2+ homeostasis, disturbances of synapse signaling, neuron loss, and terminal neuron death [20]. Accordingly, a series of anti-Aβ immunotherapies have been conducted to clear the cerebral Aβ deposition, including active Aβ immunization with target antigens and passive immunization via direct administration of Aβ-specific antibodies [21]. More recently, in 2021, the FDA approved Aduhelm ® (Aducanumab), the first putative disease-modifying therapy (DMT) approved for AD. Aducanumab is an amyloid β directed human monoclonal antibody able to reduce brain Aβ plaques as measured by PET-imaging studies [22]. Up to the present moment, no effective treatment for AD disease for curing and arresting the disease progression [23,24]. Tacrine or 9-amino-1,2,3,4tetrahydroaminoacridine (THA) was the first AChEI approved by the Food and Drug Administration (FDA) in 1993 and was launched on the market under the brand name of cognex ® [25]. THA is a centrally acting non-selective competitive inhibitor of both AChE and BChE and also modulates nicotinic receptors [4,26]. Unfortunately, it was withdrawn in 2013 shortly after its release due to its hepatotoxicity [27,28]. In 2006, Donepezil (Aricept ® ) has been licensed by the US Food and Drug Administration (FDA) for the symptomatic treatment of AD. It is worth mentioning that donepezil (Aricept®) is sighted as a potent and selective piperidine AChEI derivative possessing a dual-acting anti-Alzheimer mode with safer margins [29][30][31].
Although relying only on the cholinergic hypothesis is not logical [29], both tacrine and donepezil are still considered cornerstones in the development of new, effective and multi-targeting members [32][33][34][35][36]. Accordingly, many tacrine analogs have been introduced (Fig. 1). The 7-methoxy tacrine I (7-MEOTA) exhibited a good AChE inhibitory profile with limited hepatotoxicity due to its different metabolic clearance pathway compared to THA [37]. Furthermore, introducing different substituents in the 6 and/or 7 positions as in the case of 6-chlorotacrine II improved both the AChE inhibitory profile and selectivity [38]. Meanwhile, replacing the benzene ring in tacrine structure with different heterocyclic fused ring systems to afford tetracyclic structure, III [39], IV, V [40], and VI [41] has proven to be a promising direction. In continuation to modify and uncover new tacrine analogs, our team sketched this piece of work. More specifically, the thiophene nucleus is considered an important pharmacophore in variant bioactive molecules where thiophene derivatives show a wide range of biological activities, such as anti-Alzheimer, anti-inflammatory, anti-fungal, anti-depressant, anti-malarial, anti-cancer, anti-hyperglycemic, and more [42]. Depending on the above-mentioned facts, the sight was drawn toward discovering new thiophene derivatives as anti-Alzheimer agents. Taking into our consideration that thiophene is bioisosteric of benzene; we planned to synthesize tacrine analogs where the benzene ring in tacrine structure (ring A) is replaced by different cycloalkyl thiophenes while keeping intact the central key-binding core "4amino pyridine". Besides, the cyclohexane fused ring in tacrine structure (ring B) is preserved or replaced either by five-membered or six-membered cycloalkanes (Fig. 2). The newly designed compounds were successfully synthesized. Their acetylcholinesterase, butyrylcholinesterase and β-amyloid protein IC 50 inhibition assays were also investigated and compared with both tacrine and donepezil as reference standards.

Results and discussion
Chemistry Novel series of hexahydrobenzothienocyclopentapyridines, octahydrobenzothienoquinolines, hexahydrocyclopenta(thie noquinoline/thienodipyridine), and octahydropyridothieno quinolines were efficiently synthesized and illustrated in Schemes 1 and 2. The synthesis of target tacrine derivatives 2a-i and 4a-e was prepared in two steps. Initially, preparation of substituted thiophene intermediates 1a-c, 3a, and 3b via one-pot Gewald's reaction of starting cycloalkanone a-e, with malononitrile, morpholine, and sulfur in absolute ethanol as a solvent. The target structures 2a-i and 4a-e were prepared through Lewis's acid-catalyzed Friedlander reaction between the obtained thiophene derivatives 1a-c or 3a,b, and different cyclic ketones. It is worth mentioning that anhydrous zinc chloride was used as a Lewis acid [43]. Friedlander reaction [44] provides high yield and rapid access to synthesize the target tacrine products via Lewis acid-mediated cyclodehydration of various aminocyano derivatives with various cyclic ketones [45].
The proposed mechanism [46,47] for the Friedlander reaction is illustrated in Fig. 3. The reaction mechanism for compound 2d synthesis is taken as a good example where the thiophene intermediate (1b) reacted with the cyclohexanone (1) in the presence of ZnCl 2 as a Lewis acid This mechanism involves three steps: (i) The initial addition of the amino group of compound (1b) to the carbonyl carbon of cyclohexanone (1) forming the amino alcohol intermediate A. (ii) Water loss and formation of the imine intermediate B, which tautomerizes to the enamino form C. (iii) The intramolecular aldol-like cyclo condensation with the cyano group forming compound 2d through intermediate D. Accordingly, the large Friedlander annulation is the simplest rapid access method for the synthesis of target tacrine analogs. Meanwhile, the changes in the physical  properties of new compounds from the start were considered a good sign of the success of the reaction. This includes melting points, altered behavior towards crystallization solvents, and physical appearance. Although the required reaction time to synthesize all new target compounds was 8-10 h, their yield varied from 60% to 84% according to the position of substituents and ring size. For example, octahydrobenzo-thienoquinolines as in compounds 2g, 2h, and 2i achieved a higher yield than that of octahydrobenzothienoquinolines as in compounds 2c, 2d, 2e, and 2f. So, the yield was improved by the presence of methyl substituent in the ring (A) at position 3 than that presented at position 1. Also, hexahydrobenzothieno cyclopentapyridines as in compound 2a achieved the highest yield (84%) may be due to the small ring size of cyclopentanone. Finally, octahydropyrido-thienoquinolines as in compounds 4c, 4d, and 4e achieved a high yield may be due to the presence of N-CH 3 substituent in the ring (A). Meanwhile, the IR spectrum of the newly synthesized compounds revealed the absence of the cyano group absorption band at 2188-2195 cm −1 . Regarding the 1 H NMR spectra, both series 2 and 4 showed a singlet signal of (NH 2 ) protons with accurate integration and D 2 O-exchangeable behavior in their spectra. Moreover, the presence of seven peaks in the aromatic region of their 13 C NMR spectra confirms the formation of thienopyridine Scheme 1 Preparation of series 2a-i Scheme 2 Preparation of series 4a-e core. Regarding the 1 H NMR spectra of compounds 2a, 2b , 2d, and 2g, they all exhibited significant doublet peaks at the range of δ 1.03-1.21 ppm corresponding to the 3 protons of (CH 3 ) group. On the other hand, compounds 2e, 2f, 2h, and 2i showed a two doublet/or multiplet peaks equivalent to two (CH 3 ) groups at the range of δ 1.04-1.27 ppm. Nevertheless, the change in the environment around the protons easily affects the 1 H NMR shifts. Compounds 4b, 4c, 4d, and 4e revealed the presence of a singlet peak corresponding to the (N-CH 3 ) protons at δ 2.89, 2.36, and 2.35 ppm, respectively. Both 4d and 4e revealed the presence of an additional doublet peak of (CH 3 ) protons at δ 1.25 and 1.24 ppm, sequentially.

Structure-activity relationship (SAR)
As illustrated (Fig. 4), Compounds 2c, 2d, 2e, 2h, 2i, and 4e excel tacrine and donepezil against all the activities. The close analysis of the structures of these potent compounds revealed that all potent compounds possessing cyclohexyl rings in both rings A and B (Fig. 2). So, the six-membered ring in both rings A and B is the optimum ring size. Moreover, the replacement of either benzene ring or cyclohexyl moieties of tacrine by a smaller size ring (cyclopentyl ring) led to less inhibitory activity in all assays as in compounds 2a, 2b, 4a, and 4b. Besides, the replacement of benzene ring with thiophene one is not only maintaining the activity but also improving the IC 50 values. Regarding the substituents effect, it was found that substitution on both rings A and B was also beneficial to the activity regardless of their position as in compounds 2e, 2h, 2i, and 4e. On the other hand, the monosubstitution on either one of the rings did not show better selective activity as in case of 2a, 2b, 4a, 4b, and 4c. For more evidence on our previously mentioned SAR, Saeedi et al. reported the synthesis of similar tacrine analogs and concluded that cycloalkyl substituents

Physicochemical in silico ADME study and pharmacokinetic properties' prediction
The results from the Swiss ADME predictor showed that compounds (2c, 2d, 2e, 2h, 2i, and 4e) exhibited an anticipated log P value of 4.00, 4.00, 4.28, 4.25, 4.18, and 3.06, respectively. Moreover, they all exhibited moderate water solubility, and high GIT absorption with expected high oral bioavailability. The good values of blood-brain barrier (BBB) permeability assign their efficient ability to reach and bind to their central receptor active site which is quite essential for their biological efficacy. In accordance, the Swiss ADME web tool simply predicts the drugs bioavailability through the bioavailability radar graph where six physicochemical properties are taken into consideration: lipophilicity (LIPO), size (SIZE), polarity (POLAR), solubility (INSOLU), flexibility (FLEX), and saturation (INSATU). The pink colored zone indicates the suitable physicochemical space for oral bioavailability and in which the radar plot of the drug must fall entirely to be considered a bioavailable drug. All six compounds demonstrated a promising bioavailability radar where each compound radar plot entirely falls in the pinkcolored zone that indicates good bioavailability. Furthermore, the BOILED-Egg model produces a rapid way to predict two crucial pharmacokinetic parameters namely human gastrointestinal absorption (HIA) and blood-brain barrier (BBB) permeability as a function of the position of the small molecules in the WLOGP (lipophilicity)-versus-TPSA (polarity) graph. The Boiled-Egg model demonstrates two regions, the white region is the space of the molecules with a greater extent of GIT absorption and the yellow region (yolk) is the space for highly probable BBB permeation. Yellow and white regions are not mutually exclusive. It is worthy to note that all six compounds investigated were classified in the physiochemical area closely like that of the tacrine reference drug (Fig. 5). They showed high gastrointestinal absorption and easily permeated blood-brain barrier. All compounds were found to be a PGP substrate means there would be an issue in the excretion of the drug. The bioavailability radar graph and boiled egg model for six compounds are illustrated in the supplementary data file (Figs. Bio. S1-Bio. S4). On the other hand, the druglikeness generated in accordance with the major pharmaceutical companies; Lipinski (Pfizer) [48], Ghose's (Amgen) [49], Veber's (GSK) [50], Egan's (Pharmacia) [51], and Muegge's The collected boiled egg plot for compounds 2c, 2d, 2e, 2h, 2i, and 4e compared to tacrine (Bayer) [52] filters support their promising bioavailability. Briefly, the six potent compounds exhibited a promising biological efficiency with hopeful pharmacokinetic properties towards their computational results of the physicochemical and pharmacokinetic properties study. The results obtained from the Swiss ADME tool are all summarized in Table 2.

Molecular docking study
In the aim of unwrapping the various ligand interactions with the specific enzymes' active sites and to identify the molecular features responsible for the recorded biological activity, a brief in silico investigation was conducted. It is worth mentioning that the key interactions reproduced by the cocrystallized ligand tacrine with both AChE and BChE active sites are illustrated in the supplementary data file (refer to Fig.  Bio.S5). Accordingly, the interaction between the ligand tacrine with the active-site gorge of AChE showed π-π interaction of its phenyl and pyridine rings with the phenyl ring of Phe-330. Also, both rings showed interaction with the five-membered ring of indole of Trp-84. Furthermore, the amino group of tacrine displayed a water-mediated H-bond with Asp-72, Ser-81, and Ser-122. On the other hand, the interaction between ligand tacrine with the active site of BChE showed π-π interaction of its phenyl and pyridine rings with Trp-A82. The amino group of tacrine displayed a watermediated H-bond with Asp-A70, Ser-A79, and Thr-A120. Meanwhile, a molecular docking study using tacrine as a reference ligand was performed for the most potent newly synthesized compounds (2c, 2d, 2e, 2h, 2i, and 4e) to explore their possible binding modes with both targets (AChE and BChE) and to justify their biological potency. Default docking parameters were set and 13 different docking conformations were carried out for each compound/target.

AChE and BuChE molecular docking simulation study adopting tacrine as a reference ligand
AChE molecular docking findings Finally, the docking energy score for compound 2i was −12.4504 kcal/mol with a similar binding interactions pattern with AChE active site gorge as the previously mentioned docked compounds. On the other hand, the molecular docking study of 4e exhibited an extra different cation-π interaction between the protonated nitrogen of its tetrahydrothienopyridine ring and the indole ring of Trp-432 in the AChE active site gorge with a docking energy score −12.8568 kcal/mol. To sum up, the main common feature is the interaction of the aromatic rings of tacrine and the newly synthesized compounds with the phenyl ring of Phe-330 and the indole ring of Tyr-84 of the AChE active site. The amino pyridine core is an important pharmacophore since the amino group forms water-mediated H-bond with AChE active site. The absence of this amino group leads to a loss of biological activity. The replacement of the benzene ring by the tetrahydrothienopyridine ring as in compound 4e is a promising direction due to its ability to form an extra cation-π interaction with AChE active site that agreed with the biological results. Finally, molecular-docking simulation of the six compounds in the AChE active site showed good agreement with the obtained pharmaco-biological results. In accordance, the superimposition of both compounds 2e and 4e with the ligand tacrine were demonstrated in Fig. 6 as an illustrative example of the mode of docking explained above.  −11.0447, and −12.1545 kcal/mol, respectively in comparison to the tacrine binding score of −10.5068 kcal/mol. The similarity of the docking conformation of compounds 2c, 2d, 2e, and 2i with tacrine was seen in the π-π interactions of their phenyl and pyridine ring with Trp-A82. The amino groups of 2c, 2e, and 2i displayed water-mediated Hbond with Asp-A70, Ser-A79, and Thr-A120 while the amino group of 2d displayed water-mediated H-bond with only Asp-A70, Ser-A79 residues. The conformational superposition of tacrine with compounds 2c, 2d, 2e, and 2i showed comparable overlapping with orientation and position like that seen from the x-ray crystal structure of the tacrine-BuChE complex. Regarding compounds 2h and 4e, their docking energy score was −11.7471 and −15.0700 kcal/mol. The interaction of both compounds in the binding site of BuChE showed a binding pattern that differs from the other docked compounds. The amino group of 2h displayed a water-mediated H-bond with only Thr-A120. Besides, its phenyl and pyridine rings showed cation-π interaction with His-A438. Regarding compound 4e, its amino group showed interaction with His-A438 while its phenyl and pyridine rings showed π-π interaction with Trp-A82. Furthermore, compound 4e showed an extra different interaction between protonated nitrogen of thienopyridine ring and Glu-A197 The interaction of compound 2i is taken as a good example (Fig. 7)   ethyl acetate, and toluene with the ratio 1:2:3. The respectively used ketones (a-e) were commercially available from Acros Organic Company.

General procedure for preparation of compounds 2a-i
The respective thiophenes 1a-c (0.01 mol) were mixed with the appropriate ketones (10 mL) and anhydrous zinc chloride (0.01 mol). The mixture was refluxed for 8-10 h. The resulting solid was filtered, washed with water, and crystallized from the appropriate solvent [40].
General procedure for preparation of compounds 4a-e The respective thiophenes 3a, b (0.01 mol) were mixed with the appropriate ketones (10 mL) and anhydrous zinc chloride (0.01 mol). The mixture was refluxed for 8-10 h. The formed solid was filtered, washed with water, and crystallized from the appropriate solvent [40].

In silico molecular studies
The physicochemical in silico ADME and pharmacokinetic properties' prediction studies were conducted using the Swiss ADME predictor [57]. The Swiss Institute of Bioinformatics (SIB) invented such a free web tool to predict the pharmacokinetics, physicochemical properties, and druglikeness of newly investigated leads. Accordingly, this study was applied to the most potent newly synthesized compounds (2c, 2d, 2e, 2h, 2i, and 4e). The compound structures were imported, and the structures' smiley was entered to run the Swiss ADME study. All the docking studies were performed using Molecular Operating Environment (MOE-2008) software. The X-ray crystal structure of both the tacrine-AChE complex (PDB ID: 1ACJ) [58] and the tacrine-BuChE complex (PDB ID: 4bds) [59] were downloaded from Protein Data Bank (PDB). All minimizations were carried out until an RMSD gradient of 0.05 kcal mol −1 Å −1 with Merk Molecular Force Field (MMFF94x). Docking of the most stable conformers was carried out using Triangle Matcher Replacement and London dG scoring function. Moreover, to ensure the accuracy of the docking study, validation was carried out by re-docking the co-crystallized ligand (tacrine) into the AChE active site resulting in a very close alignment with the original ligand with an RMSD and an energy score of 0.3398 and −10.8740 kcal/mol, sequentially. Furthermore, the re-docking validation of the co-crystallized ligand (tacrine) into the BuChE active site was successfully carried out and produced a binding pattern with an RMSD and an energy score of 0.2398 and −10.5068 kcal/mol, respectively.

Conclusion
Two tacrine analogs series 2a-i and 4a-e were synthesized and eventually structurally elucidated. The new members were screened against their AChE and BuChE enzymes in addition to the Aβ protein inhibition. Both tacrine and donepezil were considered reference drugs in the assays. All compounds showed better inhibitory effects on both AChE and BuChE enzymes than tacrine. On other hand, All Compounds except 2a, 2b, 2f, 2g, 4a, 4b, and 4c displayed an inhibitory effect on both AChE and BuChE enzymes better than donepezil. Finally, compounds 2c, 2d, 2e, 2h, 2i, and 4e resulted in more decrement of Aβ compared to both tacrine and donepezil. The six common promising members in all the assays, 2c, 2d, 2e, 2h, 2i, and 4e, were subjected to molecular modeling and in silico Swiss ADME investigations. All the predicted pharmacokinetic properties revealed moderate water solubility, high human intestinal absorption, and passing blood-brain barrier with a bioavailability score of 0.55. Moreover, the molecular docking results showed extra watermediated H-bonding with the Asp72 residue in the AChE active gorge. More specifically, 4e possessed cation-π interaction between its protonated tetrahydrothienopyridine nitrogen and Trp432. Thus, the tetracyclicthienopyridine fused system has proven to be a promising start for the discovery of new anti-Alzheimer multi-targeting leads.

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