3-methyl-4-nitrobenzoic acid synthesis
Based on the topology of the active site of AChE we hypothesized that the combination of tacrine with trolox or tryptoline through linkers of varied chain lengths would lead to multifunctional hybrid compounds with the ability to modulate a number of important drug targets in the neurodegenerative cascade (Fig. 4 ). It is expected that the tacrine moiety will interact with conserved aromatic residues in the catalytic anionic site (CAS) of AChE necessary for AChE inhibition while trolox or tryptoline will interact with conserved aromatic residues in the peripheral anionic site (PAS) necessary for the possible prevention of AChE-induced Aβ aggregation [ 19 - 22 ]. Varying the linker chain length explores the appropriate binding of the pharmacophores in the active site of AChE. The aim of these hybrids is to provide additive or synergistic therapeutic effects that might help overcome the limitation of current AD drugs and to aid in the search for novel AD lead compounds.
Tacrine is an attractive lead compound that has successfully been used in the multitarget design strategy [ 9 , 10 ]. It has ChE inhibitory activity and additional biological activity such as monoamine oxidase (MAO) inhibition and ion channel modulating ability [ 12 ] that may be of value in the treatment of AD has been described for it. Recent studies have demonstrated that lead optimization of tacrine in the design of novel AD drugs can improve its biological profile and alleviate its hepatotoxicity [ 13 ]. Based on these studies, tacrine appears to be a suitable lead compound for the design of multitarget agents. Trolox is an analog of Vitamin E (α-tocopherol) with well documented antioxidant capacity which may neutralize free radical species believed to be linked to aging or diseases such as AD [ 14 ]. Trolox also has the ability to prevent neurotoxicity induced by Aβ and hydrogen peroxide [ 15 ]. Another source revealed that trolox could inhibit glycogen synthase kinase 3β (GSK 3β) whose hyperactivity causes neurofibrillary tangle formation [ 16 ]. These different biological activities of trolox consolidate its neuroprotective capacity and make it an excellent lead for the design of multifunctional drugs for treating AD. Tryptoline is a β-carboline derivative and derivatives thereof have been shown to inhibit ChE, MAO, NMDAR and other targets involved in the proposed pathogenesis of AD [ 17 , 18 ].
A more appropriate strategy was proposed by developing multitarget directed ligands (MTDL) with the ability to modulate multiple targets involved in the pathogenesis of AD simultaneously [ 8 , 9 ]. Numerous examples (Fig. 2 ) of hybrid compounds with multifunctional activities have already been developed and published, including bis-7-tacrine dimers [ 10 ], galantamine-memantine hybrids [ 8 ], phenylthiazole-tacrine hybrids [ 9 ] and bivalent β-carboline hybrids [ 11 ] with pharmacological profiles offering promise of slowing or stopping neurodegenerative disease progression. These positive findings in the development of multifunctional hybrid compounds have prompted us to develop new hybrid compounds using tacrine, trolox and tryptoline as lead compounds (Fig. 3 ).
Alzheimer’s disease (AD) is a chronic, multifactorial disease of the central nervous system [ 1 ]. Its characteristic symptoms include short-term memory impairment that worsens as the disease progresses and eventually leads to severe cognitive and physical disability [ 2 ]. AD represents the main cause of dementia and no curative drug is available. To date, it is estimated that 36.6 million people worldwide are suffering from the disease. This number is expected to double or triple by 2030 and 2050 [ 3 ]. The sequence of molecular events that underlies the occurrence of AD is still not well understood [ 4 ]. Current hypotheses suggest a decrease in the level of the neurotransmitter acetylcholine (ACh) in the brain regions involved in learning and memory, β-amyloid plaque formation (Aβ), oxidative stress, excitotoxicity, neuroinflammation and aggregation of tau proteins (τ-proteins) [ 5 , 6 ]. The current treatment of AD is limited to acetylcholinesterase inhibitor drugs (donepezil, galantamine and rivastigmine) and a N-methyl-D-aspartate receptor (NMDAR) antagonist (memantine, Fig. 1 ). These drugs act through the “one-molecule-one target” paradigm, offer only symptomatic treatment and do not stop the progression of AD [ 7 ].
The synthesis of the tacrine-trolox hybrids (8a – 8d) required intermediate 9-chloro-1,2,3,4-tetrahydroacridine (4), which was obtained from two sequential reactions; condensation of anthranilic acid (1) and cyclohexanone (2), followed by a reaction of the Spiro intermediate (3) with phosphoryl chloride. The intermediate (4) was then aminated in the microwave with appropriate alkyl diamine linkers followed by conjugation to trolox through an amide bond using the activation agent N,N-carbonyldiimidazole to give compounds 8a - 8d (Scheme 1). The tacrine-tryptoline hybrid (14) was synthesized by a two-step reaction including amination of 1,7-dibromoheptane with 9-amino-1,2,3,4-tetrahydroacridine to produce intermediate 12, followed by conjugation to tryptoline through a further amination reaction. The tryptoline dimer (16) was obtained as a side-product from the reaction of tryptoline with an excess amount of 1,2-dibromoethane (Scheme 2) in a failed attempt to synthesize the tryptoline-bromoethane monomer. Compound 16 was added to the series of test compounds as a serendipitously synthesized compound that could show some biological activities because of possible fit inside the ChE active site cavity.
Scheme 1. Reagents and conditions: (a) anthranilic acid (1), cyclohexanone (2), toluene, reflux, 4 hrs; (b) spiro[2H-3,1-benzoxazine-2,1-cyclohexane]-4(1H)-one (3), POCl3, alkaline workup (KOH), 2 hrs; (c) 9-chloro-1,2,3,4-tetrahydroacridine (4), alkylenediamines in excess (5n), DCM, NaHCO3, microwave irradiation at 200 0C, 250 W and maximum pressure for 30 minutes; (d) N1-(1,2,3,4-tetrahydroacridin-9-yl)-alkane-1,n-diamine (6n), Trolox (7), N,N-carbonyldiimidazole, THF, rt, overnight, giving the tacrine-trolox hybrids (8a – 8d).The selective loss of cholinergic neurons in AD results in a deficit of acetylcholine (ACh) in specific regions of the brain that mediate learning and memory functions [23]. Consequently, AD patients have been treated with AChE inhibitors [24-26] but unfortunately with limited therapeutic success, mainly because of the multifactorial nature of AD. Recent studies have also shown that compounds that are able to inhibit butyrylcholinesterase (BuChE) may be of value in the treatment of AD [27]. In AD, the ratio of BuChE/AChE gradually increases as the disease progresses, partially as a consequence of the progressive loss of the cholinergic synapses where AChE enzymes are located [28]. A compound able to inhibit both AChE and BuChE may thus be of more therapeutic value in AD.
Scheme 2. Reagents and conditions: (e) tacrine hydrochloride (9), H2O, K2CO3, 1 hr; (f) tacrine (10), 1,7-dibromoheptane (11), CH3CN, KOH, rt, overnight; (g) N-(7-bromoheptyl)-1,2,3,4-tetrahydroacridin-9-amine (12), tryptoline (13), DMF, K2CO3, KI, microwave 160 0C, 250 PSI, 200 W, 1h, giving the tacrine-tryptoline hybrid (14); (h), tryptoline (13), 1,2-dibromoethane (15), KOH, CH3CN, microwave 100 0C, 250 PSI, 130 W, 1 hr, giving the bistryptoline dimer (16).The inhibitory activity of the target compounds 8a – 8d, 14 and 16 against TcAChE (from electric eel) and eqBuChE (from equine serum) were measured according to the method of Ellman using tacrine and donepezil as reference compounds [29]. The IC50 values of the test compounds and reference compounds are given in Table 1. The tacrine-trolox hybrid compounds (8a – 8d) showed moderate to high AChE inhibitory activities (IC50: 49.31 – 2200 nM) comparable to or better than the reference compounds donepezil, tacrine and previously reported β-carbolines [30]. Compound 16 displayed no inhibitory activity against AChE and though tryptoline is known to have AChE inhibitory activity, it is possible that the two carbon linker chain length limited the flexibility of the tryptoline moieties and prevented the dimer from adopting a stable conformation for effective activity. This scenario is slightly different for 8a where the 2 carbon linker chain length is extended by the amide bond conferring moderate AChE inhibitory activity. Compound 8d (IC50 = 49.31 nM) with a 6 carbon linker displayed higher activities than 8a, 8b and 8c with 2, 3 and 4 carbon linker chain lengths respectively. Compound 14 with the 7 carbon linker exhibited the highest activity (IC50 = 17.37 nM) and this correlates to the modelling study that clearly illustrate the interaction of the two pharmacophore moieties (tacrine and tryptoline) with Trp84 and Trp279, which are crucial for AChE activity (Fig. 5). In this series, it is clear that hybrids with longer linker chain lengths show increased AChE inhibitory activities compared to the shorter ones. This correlates well with other studies described in the literature [8-11]. The idea of varying linker chain length seeks to assign to the two linked pharmacophore units, the ability to span the PAS and CAS of AChE, and obtain the appropriate orientation that would enable them to interact with crucial amino acids in the active site, generating better activities. As confirmed by these results, shorter linker chain lengths (less than 4 carbons) are less favorable for optimal activity.
Table 1.In vitro IC50 values of test compounds 8a – 8d, 14 and 16 for TcAChE, eqBuChE and DPPH.
aBuChE selectivity index = IC50(TcAChE)/IC50(eqBuChE). n.d. = not determined.
The test compounds showed moderate to high BuChE inhibitory activities comparable or higher than their reference compound tacrine (Table 1) and previously reported β-carbolines [30]. Compound 8d (IC50 = 4.74 nM) and 14 (IC50 = 3.16 nM) with 6 and 7 carbon linker chain lengths respectively displayed higher activities than 8a, 8b, and 8c with 2, 3 and 4 carbons linker chain lengths. It can be deduced from this observation that hybrids with longer linker chain lengths also seem to exhibit increased BuChE inhibitory activities compared to the shorter ones. This similarity of interaction of synthesized compounds and target proteins (AChE / BuChE) is consistent with the similarity of the topology of their active sites. Compounds 8d and 14 exhibited BuChE inhibitor activity more than 3 fold higher than tacrine. All the synthesized compounds showed higher inhibitory activity for BuChE than for AChE. It has been demonstrated that the two proteins share 65% amino acid sequence similarity and the 35% difference in their amino sequence confer to BuChE the ability to accommodate bulkier substrates than AChE [31]. The similarity in amino acid sequence of AChE and BuChE may prove to be a challenge in efforts to design a 100% non-selective cholinesterase inhibitor. The goal of this study was however to discover new dual inhibitors of cholinesterase since both proteins are believed to contribute, to different degrees, to the depletion of ACh levels in AD brain [24-28]. Based on this observation, selective inhibitors could be less effective as therapeutic options.
To provide insight into the binding mode of the synthesized compounds docking studies were carried out on TcAChE and eqBuChE. The protein structure of TcAChE co-crystallized with tacrine (PDB code: 2CMF) was retrieved from the Brookhaven Protein Data Bank (www.rcsb.org/pdb) and docking was carried out using the Dock application of Molecular Operating Environment (MOE 2015.10) software.
The BuChE molecular modeling experiments were conducted using a homology model because of the absence of a X-ray structure of eqBuChE. The automated homology-modeling program, SWISS-MODEL, was used to model the putative three-dimensional structure of eqBuChE based on the crystal structure of hBuChE (PDB: 2PM8) which shows an 89% sequence similarity [32]. The docking of the compounds into this homology model of eqBuChE was carried out with Autodock Vina software [33]. The docking results generated were directly loaded into and analyzed with MOE.
Fig. (4). Compounds 8d (top) and 14 (bottom) docked into the active site of TcAChE (left) and possible ligands interactions with conserved aromatic residues (right). Compounds 8d and 14 below are shown in white. In pink and cyan are Trp84 (CAS) and Trp 279 (PAS), crucial amino acids regularly involved in inhibition of AChE.The docking revealed that the tacrine-trolox hybrids 8a – 8d, the tacrine-tryptoline hybrid (14) and the tryptoline dimer (16) show similar binding patterns. All the compounds spanned the CAS and PAS of TcAChE but with different energy scores. Compounds with the short linker chain lengths tend to have higher energy scores of -8.10 kcal/mol (16) and -9.84 kcal/mol (8a) while 8b, 8c, 8d and 14 have relatively lower energy scores of -10.20 kcal/mol, -10.30 kcal/mol, -11.43 kcal/mol and -10.40 kcal/mol, respectively. The difference in energy scores indicate a disparity in how strong / fast / stable the synthesized compounds bind to the active site. Based on this concept, compounds with longer linker chain lengths should display the highest activity. This is in line with the biological results where the same trend was observed. In all docking experiments, tacrine occupied the CAS and trolox / tryptoline occupied the PAS, which correlated well with our hypothesis and also demonstrated the high selectivity of the tacrine moiety for the CAS as supported in the literature [31]. Fig. (4) shows the docking and binding interactions of compounds 8d and 14 in TcAChE. It is clear from the images that the quinoline ring of tacrine form π-π interactions with Trp84 in both compounds 8d and 14 with the trolox and tryptoline moieties reaching the PAS region of the enzyme. An H-π interaction is also observed with compound (14) between 9-H of the tryptoline moiety and Trp279 in the PAS. These results confirm that the hybrid compounds are able to span both the CAS and PAS of AChE which may account for the promising AChE inhibitory activity observed. The molecular modeling experiments were also done with the test compounds in their protonated state. The only additional interaction observed was an ionic interaction between the protonated acridine nitrogen and HIS440. The binding energies and orientation of the molecules did not show any significant differences when compared to their protonated counterparts.
To explore the binding mode in eqBuChE, compounds 8d and 14 were used as representative compounds and were docked into the active site of the eqBuChE homology build model (Fig. 5). The docking results of 8d and 14 showed that the tacrine moiety binds in the CAS region, establishing π-π stacking interactions with Trp82. In both these compounds the trolox and tryptoline moieties spanned the active site gorge and were stabilized within the PAS region of eqBuChE. The binding energies of 8d and 14 were -11.83 kcal/mol and -12.23 kcal/mol, respectively. The low binding energies and favourable positions of 8d and 14 within the active site of eqBuChE may account for the high affinity and slight selectivity observed for BuChE over AChE. As observed for the AChE docking experiments, when the molecules were redocked in their protonated state, no significant difference was observed in their binding orientations or binding energies. An additional ionic interaction between the protonated acridine amine and HIS438 was the only additional binding interaction predicted.
Fig. (5). Complex of compound 8d (top) and 14 (bottom) with the eqBuChE homology model (left) and the interaction maps (right). Compounds 8d and 14 below are shown in white and Trp82 in the CAS site is shown in pink.Reactive oxygen and nitrogen species contribute to the pathophysiology of neurodegenerative disorders [34]. Antioxidants are compounds capable of scavenging free radicals and antioxidant therapy is therefore considered as one of the options in neuroprotection [14]. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay was used to assess the free radical scavenging or antioxidant effect of the test compounds. The synthetic nitrogen-centred DPPH˙+ is not biologically relevant, but is used as indicator compound in testing hydrogen transfer capacity related to antioxidant activity [35]. The results of the ability of synthesized compounds to scavenge the DPPH free radical are included in Table 1. Trolox has well documented antioxidant properties and was used as reference compound in this assay. The conjugation of trolox to tacrine through varying linker chain lengths did not affect its ability to scavenge free radicals. The tacrine-trolox compounds (8b – 8d, IC50: 11.48 – 14.38 µM) exhibited activity with IC50 values in the same range as that of trolox (IC50 = 17.57 µM). The exception is compound 8a (IC50: 46.23 µM) which showed free radical scavenging activity of about half of that of trolox. Compounds 14 and 16 exhibited lower free radical scavenging activity with IC50 values of 129.41 µM and 125.24 µM. This was expected as the trolox moiety is replaced by the less potent free radical scavenging tryptoline moiety.