Tacrine-resveratrol fused hybrids as multi-target-directed ligands against Alzheimer's disease

Accepted Manuscript Tacrine-resveratrol fused hybrids as multi-target-directed ligands against Alzheimer's disease Jakub Jeřábek, Elisa Uliassi, Laura Guidotti, Jan Korabecny, Ondřej Soukup, Vendula Sepsova, Martina Hrabinova, Kamil Kuca, Manuela Bartolini, Luis Emiliano Peña Altamira, Sabrina Petralla, Barbara Monti, Marinella Roberti, Maria Laura Bolognesi PII:

S0223-5234(16)31054-6

DOI:

10.1016/j.ejmech.2016.12.048

Reference:

EJMECH 9139

To appear in:

European Journal of Medicinal Chemistry

Received Date: 28 October 2016 Revised Date:

22 December 2016

Accepted Date: 23 December 2016

Please cite this article as: J. Jeřábek, E. Uliassi, L. Guidotti, J. Korabecny, O. Soukup, V. Sepsova, M. Hrabinova, K. Kuca, M. Bartolini, L.E. Peña Altamira, S. Petralla, B. Monti, M. Roberti, M.L. Bolognesi, Tacrine-resveratrol fused hybrids as multi-target-directed ligands against Alzheimer's disease, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2016.12.048. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT Tacrine-resveratrol fused hybrids as multi-target-directed ligands against Alzheimer’s disease Jakub Jeřábek,a, b Elisa Uliassi,a Laura Guidotti,a Jan Korabecny,c, d Ondřej Soukup,c, d Vendula Sepsova,e Martina Hrabinova,e Kamil Kuca, c, d Manuela Bartolini,a Luis Emiliano Peña Altamira,a

a

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Sabrina Petralla,a Barbara Monti,a Marinella Roberti*a and Maria Laura Bolognesi*a Department of Pharmacy and Biotechnology, University of Bologna, Via Belmeloro 6/Selmi 3,

40126 Bologna, Italy b

Department of Pharmaceutical Chemistry and Drug Control, Faculty of Pharmacy in Hradec

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Kralove, Charles University in Prague, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech

c

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Republic

Biomedical Research Centre, University Hospital Hradec Kralove, Sokolska 581, 500 05 Hradec

Kralove, Czech Republic d

National Institute of Mental Health, Topolova 748, 250 67 Klecany, Czech Republic

e

Department of Toxicology and Military Pharmacy, Faculty of Military Health Sciences, University

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of Defence, Trebesska 1575, 500 01 Hradec Kralove, Czech Republic

* *

Corresponding author: e-mail, [email protected] Corresponding author: e-mail, [email protected]

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ACCEPTED MANUSCRIPT Abstract Multi-target drug discovery is one of the most followed approaches in the active CNS therapeutic area, especially in the search for new drugs against Alzheimer's disease (AD). This is because innovative multi-target-directed ligands (MTDLs) could more adequately address the complexity of

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this pathological condition. In a continuation of our efforts aimed at a new series of anti-AD MTDLs, we combined the structural features of the cholinesterase inhibitor drug tacrine with that of resveratrol, which is known for its purported antioxidant and anti-neuroinflammatory activities. The

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most interesting hybrid compounds (5, 8, 9 and 12) inhibited human acetylcholinesterase at micromolar concentrations and effectively modulated Aβ self-aggregation in vitro. In addition, 12

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showed intriguing anti-inflammatory and immuno-modulatory properties in neuronal and glial AD cell models. Importantly, the MTDL profile is accompanied by high-predicted blood-brain barrier permeability, and low cytotoxicity on primary neurons.

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neuroinflammation.

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Key words: Alzheimer’s disease, amyloid, acetylcholinesterase, multitarget compounds,

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ACCEPTED MANUSCRIPT 1. Introduction Multi-target-directed ligand (MTDL) drug discovery [1], one of the most innovative concepts to emerge in the AD area in over a decade, is currently being experimented by an ever-increasing

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number of medicinal chemists. Indeed, rationally designed MTDLs have the potential to promote a number of goals that are fundamental to cross the current “Valley of Death” [2] i.e. the muchdeplored inability to translate basic research findings into new effective medications. These goals include (i) an inherently higher adequateness to confront the complex AD pathogenesis; (ii) a larger

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therapeutic window; and (iii) a simplified therapeutic regimen [3].

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There is ample evidence in support of these claims, and many studies in this area are convincing, well designed, innovative, and supported by in vivo proof of principle [4]. However, despite several promising anti-AD hit and lead compounds have been very recently reported [5, 6], no one has progressed beyond the preclinical phase to enter clinical development [4]. Thus, there is still a need

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for novel MTDLs with a genuine translational potential as anti-AD drug candidates. Molecular hybridization has been the most widely applied design strategy towards new MTDLs for AD [7]. To note, marketed acetylcholinesterase (AChE) inhibitor tacrine (1 in Fig. 1) has been

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successfully used as starting scaffold to obtain molecular hybrids with improved and enlarged

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biological profile, beyond the ability to inhibit AChE enzyme [8]. This, in spite of 1’s hepatotoxicity, which led to its withdrawal from the market [9]. Thus, the more potent AChE inhibitor (AChEI) 6-chlorotacrine 2 [10] and the less hepatotoxic 7-methoxytacrine 3 [11], have provided useful back-up scaffolds to generate new hybrid molecules (recently reviewed in [7]). A survey of the literature reveals that a wide set of proposed tacrine hybrids incorporated a fragment derived or inspired by a natural product [12]. This is because natural products have intrinsic multi-target profile, being evolutionarily selected and biologically pre-validated. They are therefore appropriate starting points for the development of more effective MTDLs against AD 3

ACCEPTED MANUSCRIPT [13]. Among the natural products with therapeutic potential in the field of neurodegenerative diseases, we focused our attention on resveratrol (4 in Fig. 1). This naturally occurring polyphenol, mainly

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found in grapes and red wine, has shown beneficial effects against neurodegeneration thanks to its antioxidant and anti-inflammatory properties [14]. These effects have constituted the rationale for a recent clinical trial aimed to evaluate the impact of resveratrol treatment in patients with mild to

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moderate AD [15, 16].

We were particularly attracted by the fact that 4 has been demonstrated to be neuroprotective in

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various neurodegenerative disease models by acting on microglial activation [17]. Indeed, targeting microglial cells by modulating their activity, rather than simply trying to counteract their inflammatory neurotoxicity, might represent a valid therapeutic approach for neurodegenerative diseases [17].

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On these bases, we developed new hybrids 5-12 by fusing the structure of tacrines 1-3 with that of 4 (Fig. 1). We reasoned that this structural hybridization might have positive consequences in terms of both neuroinflammation and safety. From toxicity point of view, resveratrol is known to be safe

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in humans [16], and, more importantly, it has demonstrated beneficial effects in animal models of

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hepatic insult [18]. In addition, the notion that 4 might directly halt amyloid aggregation [19] potentially expands the therapeutic profile of derivatives 5-12. Anticipating our results, we find that derivatives 5-12 display an interesting multi-target profile against multiple target proteins involved in AD pathogenesis. Encouragingly, for compound 12, the favorable in vitro properties translate into effective immunomodulation in an AD cell model.

2. Design rationale

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ACCEPTED MANUSCRIPT The classical strategy of molecular hybridization has been elegantly reevaluated from a MTDL standpoint by Richard Morphy [20]. He defined the framework combination as the approach aimed at combining the frameworks of different single-target molecules to generate a new hybrid [21]. He dubbed the resulting hybrid as linked, fused, and merged depending upon the degree of integration

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of the starting frameworks [22]. Intuitively, by increasing the degree of overlap, a decrease of both molecular weight and structural complexity is achieved. Hence, in principle, a merged hybrid should possess improved drug-like features compared to a linked one [22]. This latter aspect is of

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crucial importance especially when designing hybrids directed to the central nervous system (CNS), which must first permeate the blood-brain barrier (BBB) to exert their effects [23]. Because of more

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stringent physicochemical requirements requested for CNS-acting drugs compared to peripherally acting compounds [23], the development of fused molecules hold particularly high potential. With this in mind, and aiming at combining the anticholinesterase features of 1-3 with the antioxidant, immunomodulatory and neuroprotective framework of resveratrol 4, we developed

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fused derivatives 5-12 (Fig. 1). In particular, toward the goal of a maximal structural overlap, we turned our attention to the 4’-amino derivative of resveratrol 13, which has been previously reported to exert anti-aggregating, antioxidant and neuroprotective effects in AD cellular models [24]. This

the 4’-NH2 of 13).

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allowed us to fuse the two frameworks through the common amino group (i.e. the 9-NH2 of 1 and

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Notably, despite the wealth of anti-AD tacrine- [7-9, 25] and resveratrol-based [26-28] hybrids in the literature, to the best of our knowledge, there is no example of fusing the amino group of 1-3 with that of a resveratrol derivative. However, it should be mentioned that tacrine-resveratrol linked hybrids (featuring an alkoxy linker) have been already patented as useful agents against neurodegenerative diseases [29]. Figure 1 should be put here.

3. Chemistry 5

ACCEPTED MANUSCRIPT Tacrine-resveratrol hybrid compounds 5-12 were obtained as describe in Scheme 1. The key step consisted of a coupling between the amino-stilbene 16 or its corresponding dihydro derivative 15 and the appropriate 9-chlorotacrines 17-19. The reaction, carried out in the presence of phenol under microwave irradiation and solvent free conditions, afforded the desired hybrid methoxylated

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compounds 5-7 and 9-11 in moderate to good yields (32-52%). The dihydroxy derivatives 8 and 12 were obtained, in 93% and 31% yield respectively, by demethylation of the corresponding dimethoxy hybrids 5 and 9 in the presence of boron tribromide at −78 °C. Amino derivatives 15-16

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were prepared starting from the common intermediate trans-nitro-stilbene derivative 14 previously reported [30, 31]. Amino-stilbene 16 [30, 31] was obtained by selective reduction of the nitro group

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with Tin (II) chloride, whereas the corresponding dihydro derivative 15 [32] was obtained by catalytic hydrogenation in continuous-flow reactor that allowed simultaneous reductions of the nitro group and the ethylene bridge. The 9-chlorotacrine derivatives 17-19 has been synthesized as previously described [33, 34].

4. Results and discussion

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Scheme 1 should be put here.

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4.1 hAChE and hBChE activity

To determine the potential interest of the newly synthesized hybrids as MTDLs for the treatment of

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AD, we first assessed their cholinesterase inhibition profile by Ellman’s method [35]. The results, expressed as IC50 values, i.e. the inhibitor concentration that reduces the cholinesterase activity by 50%, are reported in Table 1. Compounds were initially screened at a single concentration, namely 10 µM, and those showing significant inhibition were further evaluated and the IC50 values were determined. Among the synthesized compounds, only 5-6, 8 and 12 showed significant inhibitory activity when tested at 10 µM concentration, giving IC50 values spanning from 0.8 to 14.2 µM. The general lack of significant activity of compounds 9-11 may be ascribed to the fact that this subset of hybrids carries the double bond of the stilbene moiety, thus showing a high degree of structural 6

ACCEPTED MANUSCRIPT rigidity, which may hamper the proper fitting within the enzyme’s gorge. This hypothesis seems confirmed by the activity of the more flexible saturated derivatives 5-6 and 8. Particularly, 5 is the most potent inhibitor of the current series, displaying an IC50 of 0.8 µM, which is comparable to that of reference drug 1 (IC50 = 0.5 µM). Interestingly, the unsaturated analogue 12, with an IC50

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value of 8.8 µM, seems an exception to the observed trend. However, it should be noted that 12, together with 5 and 8, bears a 6-chlorotacrine fragment, which is a more potent AChEI than tacrine itself. Indeed, it is well-known that the chlorine atom in position 6 establishes van der Waals

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contacts with hydrophobic residues within the AChE active site and, simultaneously, decreases the electron density on the tacrine aromatic ring, favoring π-electron interactions with nearby residues

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[36, 37]. A role, albeit minor, could be attributed to the substituent on the terminal aromatic ring of 4. The 2,4-dimethoxy phenyl substituent seems to act as a more suitable fragment for AChE recognition with respect to the 2,4-dihydroxy phenyl one (compare 5 with 8). However, among the unsaturated stilbene derivatives 9-12, the best profile is showed by hybrid 12, which carries the 6-

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chlorotacrine fragment and a catechol ring. Thus, the collected results reinforce the notion that in 1based hybrids, the interaction of tacrine fragment within the AChE binding site provides the major driving force. It is to be understood that the rest of the molecule should allow the proper positioning

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within the enzyme active site.

To better rationalize the interaction patterns of the hybrids to the AChE gorge, we also tried to solve

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the crystal structure of 5 in complex with Torpedo californica AChE. However, no positive results were obtained (data not shown). Since interaction with the peripheral anionic site (PAS) of AChE has been reported to be involved in the induction of amyloid aggregation by AChE [38] leading to the formation of cytotoxic complexes which induce neurite dystrophia and apoptosis as well as alteration of calcium homeostasis [39], the ability of 5 to displace propidium, a well-known PAS binder, was evaluated. Titration experiments showed that 5 was able to weakly interact with AChE’ PAS as demonstrated

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ACCEPTED MANUSCRIPT by its ability to displace propidium. Kd value was 7.31 ± 0.62 µM, a value which is about ten-time higher than the Kd value of propidium [40]. In addition to AChE, we also evaluated the inhibitory profile towards human BChE. Unexpectedly, when tested at 10 µM, all the hybrids of the series, displayed no inhibition. This is apparently in

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contrast with the notion that BChE, due to its wider binding site compared to AChE, should allow a more favorable compound fit. However, the resulting high selectivity for AChE may contribute to the clinically favorable tolerability profile, as observed for the selective AChE inhibitor donepezil

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[41].

4.2 Inhibition of Aβ42 Self-aggregation

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Table 1 should be put here.

Notwithstanding 1-3 were previously shown to be inactive as amyloid inhibitors [37, 42], the potential of tacrine-based hybrids to act as effective inhibitors of Aβ42 aggregation [8, 9] has been

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widely recognized, as well as the antiaggregating properties of 4 [19]. Therefore, to further investigate the anti-AD properties of 5-12, their ability to inhibit Aβ42 self-aggregation was assessed through a thioflavin T-based fluorometric assay [43, 44], which allows quantifying Aβ fibril

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formation in the presence and absence of inhibitor. Searching for agents with multiple activities, the

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compounds 5, 8-9, and 12, endowed with anticholinesterase activity, were selected to be assayed. The anti-aggregating properties of the selected compounds were evaluated at equimolar concentration (50 µM) with respect to Aβ. Notably, 8 and 12 turned out to be the most effective inhibitors of Aβ aggregation within the series, displaying percentages of inhibition of 37.3% and 31.2%, respectively. Intriguingly, all the active compounds possess the 6-chlorotacrine fragment. A clear-cut correlation between a rigid framework and optimal anti-amyloid properties could not be detected. In fact, both saturated and unsaturated hybrids 8 and 12 effectively blocked amyloid aggregation. On the other hand, the presence of a catechol ring seemed to be critical, possibly through establishing hydrogen-bonding interactions. Indeed, 5 and 9, carrying a 2,4-dimethoxy 8

ACCEPTED MANUSCRIPT substituent on the phenyl ring, exhibited a lower inhibitory activity on Aβ self-aggregation (17.6% and 21.6%, respectively) compared to 8 and 12. To confirm the importance of the cathecol moiety, the 2,4-dimethoxy derivative of 4 (trans-3,5-dimethoxy-4’-hydroxystilbene) previously synthesized [30] was assayed (data not shown). The lack of any anti-aggregating activity confirmed the

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importance of having the cathecol unit, as already highlighted by recent works [45]. Derivatives 8 and 12 show a better Aβ-aggregation inhibitory profile with respect to the starting tacrine fragments

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1-3, and similar inhibitory activity with respect to 4 (Table 1).

4.3 Blood–Brain Barrier (BBB) Prediction

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The BBB permeation properties of the current series were preliminary analyzed by exploiting a BBB on-line predictor server [46], belonging to the chemogenomics knowledgebase AlzPlatform [47]. As reported in Table 1, all the compounds displayed high-predicted BBB permeability with scores ranging from 0.068 to 0.105. Importantly, we were pleased to verify that the BBB scores of

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5-12 were higher than that of 4, which showed the lowest BBB score (0.041) and were comparable with those of 1-3 (0.120, 0.111, and 0.102, respectively).

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4.4 Antioxidant activity

4 has been reported to be protective against neurodegeneration by exerting antioxidant activity

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through free radicals scavenging [48]. The potential antiradical activity of compounds 5, 8 and 12 with respect to 4, was assessed in a chemical model, i.e. the 2,2-diphenyl-1-picrylhydrazyl (DPPH) antioxidant assay [49]. For that purpose, various concentrations (10-3 - 10-6 M) of the test compounds were incubated for 30 min in a solution containing the stable free radical and the activities are shown in Table 2, expressed as EC50 values (µM), i.e. the concentration that causes 50% decrease in the DPPH activity. The obtained data were also compared with those of wellknown antioxidants, i.e. Trolox (a polar analogue of vitamin E) and N-acetyl-cysteine. A wide variation between the obtained EC50 values was observed. In contrast with the basic notion of 9

ACCEPTED MANUSCRIPT phenols and polyphenols acting as optimal antioxidants, we found that the presence of free hydroxyl groups on the phenyl ring of 8 and 12 was detrimental to the free radical scavenging efficacy. Conversely, derivative 5, bearing two methoxy groups, showed reasonable antioxidant activity (EC50 = 155 µM), albeit lower than that of 4 (EC50 = 25.6 µM). Overall, the collected data indicate

endowed with an ameliorated antioxidant profile.

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Table 2 should be put here.

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that the combination of tacrines 1-3 with the well-known antioxidant 4 did not lead to hybrids

4.5 Neuroprotection

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Next, on the basis of the well-known neuroprotective profile of 4, we aimed to evaluate the antiinflammatory properties of 5-12 in a neuronal cellular context for which we previously determined their neurotoxic effects on primary rat cerebellar granule neurons (CGNs, Fig. 2). Primary cultures of CGNs were established a few decades ago and since then have become one of the most useful in

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vitro models to study pathological neurobiology [50]. When assessing CGNs viability after 24 h treatment with 5-12, a clear cytotoxic effect was evident for compounds 5-7, 9, and 11, even at 5 µM concentration. Encouragingly, 8 and 10 resulted neurotoxic only at the highest tested

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concentrations (25 and 50 µM). Notably, 12 showed no clear neurotoxicity at all tested concentrations. This profile resembles that of 4, which has been reported to be highly effective in

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maintaining CGNs viability at concentrations ranging between 1-50 µM [51]. Figure 2 should be put here.

On the basis of the neurotoxicity results, only compound 12 was further profiled for its antiinflammatory and immunomodulatory properties against microglial activation. Considering that reactive astrocytes and microglia are associated with amyloid plaques and that the neuronal loss in AD brain involve increased levels of markers of oxidative damage, such as inducible nitric oxide synthase (iNOS), produced by these cells [17], we assessed the potential neuroprotective activity of 12 in primary cultures of astrocytes and microglia, by evaluating iNOS expression and nitrite 10

ACCEPTED MANUSCRIPT production. In fact, neuroprotective compounds aimed at preserving neuronal structure and functionality against toxic stimuli, and at diminishing neuronal loss and degeneration, have been already demonstrated valuable as AD-modifying drugs [17]. To this end, primary cultured glial cells were co-incubated for 24 h with 12 (10-50 µM) and lipopolysaccharide (LPS) (10 µg/mL), a

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potent cytotoxic inducer of inflammation and of a cascade of intracellular events underlying neuronal death (Fig. 3). In LPS-treated cells, we observed a massive induction of nitrite production, which was significantly reduced by treatment with 12 in a dose-dependent manner. To note, 12 was

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remarkably more effective on astrocytes than on microglia, because of significantly decreasing nitrite production to levels comparable to the basal ones at 50 µM (Fig. 3). Altogether these results

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suggest that 12 is a highly promising anti-inflammatory and neuroprotective agent. Figure 3 should put here.

Motivated by these encouraging data, we deeply evaluated its ability to modulate the glial phenotypic switch from the pro-inflammatory M1 to the anti-inflammatory M2 type, by following

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Triggering Receptor Expressed on Myeloid cells 2 (TREM2) and Mannose Receptor type C 1 (MRC1) expression profiles [52, 53]. In fact, M1 activation is associated with the expression of proinflammatory molecules, such as iNOS, whereas M2 activation state is related to the production of

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anti-inflammatory cytokines and markers of phagocytosis, characterized by the expression of TREM2 and MRC1 [52, 53]. Thus, compounds able to modulate the switch from the M1 to M2

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phenotype would determine a decrease of neuroinflammation and an increase of neuronal protection and recovery [17].

On this basis, we investigated the protein extracts of glial cells incubated with 12 (10-50 µM), after treatment with LPS (Fig. 4). In addition to a positive decrease of iNOS expression as marker of M1 phenotype (Fig. 3), the expression of MRC1 was only slightly attenuated by 12 at 10 µM (Fig. 4A and 4C). Even higher concentrations of 12 (25 and 50 µM) determined a marginal diminution of MRC1 expression profile. However, 12 caused the concomitant reduction of the M2 marker TREM2 in a dose-dependent manner (Fig. 4A and 4B). The collected data showed a peculiar 11

ACCEPTED MANUSCRIPT modulation of M2 microglial transcription by 12 in primary cultures, being 12 simultaneously able to slightly modify MRC1 expression, while diminishing iNOS and TREM2 mRNA. In this respect, we have recently showed that the ability of modulating inflammation by interfering with the microglial M1/M2 switch, rather than totally repressing microglia reactivity, might be an important

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feature of novel MTDLs for treating AD [54, 55]. This lent support to the neuroprotective potential of 12, which deserves further investigations in the modulation of microglia activation.

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Figure 4 should be put here.

4.6 Hepatotoxicity

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As previously highlighted, since the serious hepatotoxicity of 1 limited its clinical use [9], the assessment of hepatotoxicity is of critical importance for evaluating the drug-likeness of the newly synthesized hybrids. We attempted to prove a lower hepatotoxicity for 1-12 on the basis of the hepatoprotective properties of 4 [56]. We anticipated that the presence of the resveratrol scaffold in

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the new hybrids could compensate the hepatotoxicity of 1. However, we were well aware that when two fragments are combined together, the resulting hybrid is a completely new chemotype that could possess a completely different cytotoxicity profile. To experimentally test this, MTT assay of

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compounds 1-12 at 0−50 µM for 24 h on liver hepatocellular cells (HepG2) was performed (Fig. 5). The obtained data were compared to those of the starting fragments 1 and 4. As shown in Fig. 5A, 1

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resulted hepatotoxic even at 5 µM concentration determining a decrease in the cell viability of around 20%, whereas at 50 µM a significant reduction of 50% was observed. On the other hand, 4 displayed no significant toxicity in the selected range of concentrations (Fig. 5B). Contrary to our expectations, when tested in the same cellular conditions, 1-12 displayed significant hepatotoxic effects (Fig. 5C). Indeed, compounds 1-8 and 10-12 caused a non-concentration-dependent decrease in the cell viability. Only for derivative 9 a cell viability of 70% is preserved at all the tested concentrations. Collectively, we can conclude that the hybridization strategy did not lead to tacrinebased hybrids endowed with an improved hepatotoxicity profile. 12

ACCEPTED MANUSCRIPT Figure 5 should be put here.

5. Conclusions By a rational design approach, we have identified a new series of fused tacrine-resveratrol hybrids

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5-12 acting as effective MTDL against AD. Indeed, 5-6, 8 and 12 had IC50 values against AChE spanning from the submicromolar to the micromolar range, with 12 also effectively displacing propidium from PAS. 8 and 12 were both more potent anti-aggregating compounds than the parent

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resveratrol 4. A promising antioxidant activity has been registered for 5.

However, only compound 12, thanks to a low neurotoxicity, could be tested in a cell model of AD

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neuroinflammation, where it showed to effectively modulate the M1/M2 switch by positively decreasing iNOS and slightly attenuating MRC1 expressions. In addition, a general hepatotoxicity is evident for all these derivatives, probably due to the presence of the hepatotoxic tacrine fragment. Indeed, despite several non-hepatotoxic 1-based hybrids have been developed [57], the intrinsic

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hepatotoxicity of 1 should be taken into full consideration when embarking in new project aimed to develop novel 1-based MTDLs against AD. Indeed, researchers in the field should carefully monitor that the advantages derived from the synthetic accessibility of 1 would not overbalance the

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disadvantages of its possible toxicity. Then, as we cannot a priori know if the new hybrids (completely new chemotypes) will be toxic or not, toxicity issues should be addressed very early in

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the discovery phase.

Collectively, these findings add new layers of information to multi-target drug discovery and may inform new approaches aimed to the development of novel and safer resveratrol-based hybrids.

6. Experimental section 6.1 Chemistry 6.1.1. General chemical methods

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ACCEPTED MANUSCRIPT Reaction progress was monitored by TLC on pre-coated silica gel plates (Kieselgel 60 F254, Merck) and visualized by UV254 light. Flash column chromatography was performed on silica gel (particle size 40-63 µM, Merck). If required, solvents were distilled prior to use. All reagents were obtained from commercial sources and used without further purification. When stated, reactions

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were carried out under an inert atmosphere. Reactions involving microwave irradiation were performed using a microwave synthesis system (CEM Discover® SP, 2.45 GHz, maximum power 300 W), equipped with infrared temperature measurement. Catalytic hydrogenation was performed

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on H-Cube® Continuous-flow Hydrogenation Reactor (H-Cube, ThalesNano Nanotechnology, Budapest, Hungary). Compounds were named relying on the naming algorithm developed by

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CambridgeSoft Corporation and used in Chem-BioDraw Ultra 12.0. All of the new compounds were analyzed for C, H and N and agreed with the proposed structures within ±0.4% of the theoretical values. Unless state otherwise, 1H-NMR and 13C-NMR spectra were recorded on Varian Gemini at 400 MHz and 100 MHz respectively. Chemical shifts (δ) are reported relative to TMS as

apparatus.

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internal standard. Low resolution mass spectra ESI-MS were recorded on a Waters ZQ 4000

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6.1.2. General procedure for coupling reactions to obtain hybrids 5-7 Tacrine derivatives 17-19 (1 equiv), the amine 15 (1-1.5 equiv) and phenol (4-9 equiv) were

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charged in a pressure tight microwave tube containing a stirring bar. The reaction mixture was submitted to microwave irradiation at 120 °C with an irradiation power of 150W for 1h. The mixture was dissolved in DCM and washed with sodium hydroxide 10%, brine and water, dried over sodium sulfate and evaporated. Crude product was purified by flash chromatography (petroleum ether/ EtOAc, elution gradient 90:10 to 80:20).

6.1.2.1.

6-chloro-N-(4-(3,5-dimethoxyphenethyl)phenyl)-1,2,3,4-tetrahydroacridin-9-amine

(5).

Coupling of compound 15 (0.270 g, 1.05 mmol) and 17 (0.264 g, 1.05 mmol) with phenol (0.889 g, 14

ACCEPTED MANUSCRIPT 9 equiv) was performed according to the general procedure described above. Brown solid, 0.260 g, 0.55 mmol, 52% yield. 1H NMR (CDCl3, 400 MHz) δ: 1.84-1.90 (m, 2H), 1.92-1.98 (m, 2H), 2.72 (t, J= 6.4 Hz, 2H), 2.84 (s, 4H), 3.12 (t, J= 6.4 Hz, 2H), 3.77 (s, 6H), 5.81 (br, 1H, -NH-), 6.30-6.34 (m, 3H, aromatic), 6.65 (d, J= 8.4 Hz, 2H, aromatic), 7.03 (d, J= 8.4 Hz, 2H, aromatic), 7.21-7.24

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(dd, J= 9.0, 2.0, 1H, aromatic), 7.65 (d, J= 9.0 Hz, 1H, aromatic), 7.97 (d, J= 2.0 Hz, 1H, aromatic), 13

C NMR (CDCl3, 100 MHz) δ: 22.8, 22.9, 25.3, 34.2, 37.0, 38.4, 55.4, 98.0, 106.8, 115.4, 117.5,

121.0, 122.5, 125.1, 125.7, 127.8, 129.36, 129.44, 134.5, 134.9, 142.4, 144.0, 144.3, 148.0, 160.9,

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161.2; MS (ESI+): m/z: 473 [M + H]+. Anal. (C29H29ClN2O2) calcd: C 76.64, H 6.18, N 5.92;

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found: C 76.54, H 6.10, N 5.85.

6.1.2.2. N-(4-(3,5-dimethoxyphenethyl)phenyl)-1,2,3,4-tetrahydroacridin-9-amine (6). Coupling of compound 15 (0.170 g, 0.66 mmol) and 18 (0.144 g, 0.66 mmol) with phenol (0.560 g, 9 equiv) was performed according to the general procedure described above. Yellow solid, 0.15 g, 0.34 mmol, 1

H NMR (CDCl3, 400 MHz) δ: 1.83-1.89 (m, 2H), 1.93-1.99 (m, 2H), 2.73 (t, J= 6.4

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52% yield.

Hz, 2H), 2.83 (s, 4H); 3.15 (t, J= 6.4 Hz, 2H), 3.77 (s, 6H), 5.82 (br, 1H, -NH-), 6.31-6.34 (m, 3H, aromatic), 6.64 (d, J= 8.2 Hz, 2H, aromatic), 7.02 (d, J= 8.2 Hz, 2H, aromatic), 7.32 (t, J= 7.6 Hz,

Hz, 1H, aromatic).

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1H, aromatic), 7.59 (t, J= 7.6 Hz, 1H, aromatic), 7.76 (d, J= 8.4 Hz, 1H, aromatic), 7.98 (d, J= 8.4 C NMR (CDCl3, 100 MHz) δ: 22.9, 23.0, 25.5, 34.3, 37.0, 38.5, 55.4, 98.0,

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106.7, 117.1, 122.9, 123.0, 123.3, 124.9, 128.7, 129.0, 129.3, 134.3, 142.7, 143.6, 144.4, 147.6, 160.0, 160.9; MS (ESI+): m/z: 439 [M + H]+. Anal. (C29H30N2O2) calcd: C 79.42, H 6.90, N 6.39; found: C 79.35, H 6.70, N 6.30.

6.1.2.3. N-(4-(3,5-dimethoxyphenethyl)phenyl)-7-methoxy-1,2,3,4-tetrahydroacridin-9-amine (7). Coupling of compound 15 (0.100 g, 0.39 mmol) and 19 (0.096 g, 0.39 mmol) with phenol (0.328 g, 9 equiv) was performed according to the general procedure described above. Brown solid, 0.096 g, 0.13 mmol, 33% yield. 1H NMR (CDCl3, 400 MHz) δ: 1.83-1.88 (m, 2H), 1.92-1.98 (m, 2H), 2.74 15

ACCEPTED MANUSCRIPT (t, J= 6.4 Hz, 2H), 2.83 (s, 4H), 3.11 (t, J= 6.4 Hz, 2H), 3.66 (s, 3H), 3.77 (s, 6H), 5.70 (br, 1H, NH-), 6.31 (t, J= 2.2, 1H, aromatic), 6.34 (d, J= 2.2 Hz, 2H, aromatic), 6.64 (d, J= 8.3 Hz, 2H, aromatic), 6.98 (d, J= 2,8 Hz, 1H, aromatic), 7.03 (d, J= 8.3 Hz, 2H, aromatic), 7.23 (d, J= 2.8 Hz, 1H, aromatic), 7.89 (d, J= 9.2 Hz, 1H, aromatic). 13C NMR (CDCl3, 100 MHz) δ: 22.9, 23.1, 25.5,

RI PT

33.9, 37.0, 38.5, 55.38, 55.44, 98.0, 101.8, 106.7, 117.0, 121.3, 123.4, 123.8, 129.3, 130.4, 134.1, 142.6, 142.7, 143.6, 144.3, 156.8, 157.2, 160.9; MS (ESI+): m/z: 469 [M + H]+. Anal.

SC

(C30H32N2O3) calcd: C 76.90, H 6.88, N 5.98; found: C 76.75, H 6.78, N 5.92.

6.1.3. General procedure for coupling reactions to obtain hybrids 9-11

M AN U

Tacrine derivatives 17-19 (1 equiv), the amine 16 (1-1.5 equiv) and phenol (4-9 equiv) were charged in a pressure tight microwave tube containing a stirring bar. The reaction mixture was submitted to microwave irradiation at 120 °C with an irradiation power of 150W for 2h. The mixture was dissolved in DCM and washed with sodium hydroxide 10%, brine and water, dried

TE D

over sodium sulfate and evaporated. Crude product was purified by flash chromatography (petroleum ether/ EtOAc, elution gradient 90:10 to 80:20).

EP

6.1.3.1. N-(4-(3,5-dimethoxyphenethyl)phenyl)-6-methoxy-1,2,3,4-tetrahydroacridin-9-amine (9). Coupling of compound 16 (0.170 g, 0.66 mmol) and 17 (0.168 g, 0.66 mmol) with phenol (0.248 g,

AC C

4 equiv) was performed according to the general procedure described above. Yellow solid, 0.140 g, 0.29 mmol, 45% yield. 1H NMR (CDCl3, 400 MHz) δ: 1.86-1.90 (m, 2H), 1.94-1.98 (m, 2H), 2.75 (t, J= 6.5 Hz, 2H), 3.14 (t, J= 6.5 Hz, 2H), 3.82 (s, 6H), 5.87 (br, 1H, -NH-), 6.37 (t, J= 2.2 Hz, 1H, aromatic), 6.64 (d, J= 2.2 Hz, 2H aromatic), 6.67 (d, J= 8.5, 2H, aromatic), 6.89 (d, J= 16.3 Hz, 1H), 7.01 (d, J= 16.3 Hz, 1H), 7.28 (d, J= 2.0, 1H, aromatic), 7.37 (d, J= 8.5 Hz, 2H, aromatic), 7.70 (m, 1H, aromatic), 7.99 (d, J= 2.0 Hz, 1H, aromatic).

13

C NMR (CDCl3, 100 MHz) δ: 22.7,

22.8, 25.4, 34.2, 55.5, 99.8, 104.6, 117.0, 121.4, 123.6, 124.9, 126.1, 126.8, 127.9, 128.7, 130.4,

16

ACCEPTED MANUSCRIPT 134.8, 139.8, 143.4, 144.0, 147.9, 161.15, 161.22; MS (ESI+): m/z: 471 [M + H]+. Anal. (C29H27ClN2O2) calcd: C 73.95, H 5.78, N 5.95; found: C 73.90, H 5.65, N 5.85.

6.1.3.2. (E)-N-(4-(3,5-dimethoxystyryl)phenyl)-1,2,3,4-tetrahydroacridin-9-amine (10). Coupling of

RI PT

compound 16 (0.176 g, 0.69 mmol) and 18 (0.100 g, 0.46 mmol) with phenol (0.390 g, 9 equiv) was performed according to the general procedure described above. Orange solid, 0.093 g, 0.21 mmol, 46% yield. 1H NMR (CDCl3, 400 MHz) δ: 1.84-1.90 (m, 2H), 1.94-1.99 (m, 2H), 2.76 (t, J= 6.4 Hz,

SC

2H), 3.17 (t, J= 6.4 Hz, 2H), 3.82 (s, 6H), 5.93 (br, 1H, -NH-), 6.37 (s, 1H, aromatic), 6.63-6.64 (m, 2H, aromatic), 6.68 (d, J= 8.3, 2H aromatic), 6.89 (d, J= 16.2 Hz, 1H), 7.01 (d, J= 16.2 Hz, 1H),

8.5 Hz, 1H, aromatic).

13

M AN U

7.35-7.37 (m, 3H, aromatic), 7.61 (m, 1H, aromatic), 7.79 (d, J= 8.5 Hz, 1H, aromatic), 8.02 (d, J= C NMR (CDCl3, 100 MHz) δ: 22.8, 23.0, 25.6, 34.1, 55.5, 99.8, 104.5,

110.2, 116.8, 123.2, 125.3, 126.5, 127.8, 128.9, 129.0, 130.1, 139.9, 144.3, 144.5, 160.2, 161.2; MS (ESI+): m/z: 437 [M + H]+. Anal. (C29H28N2O2) calcd: C 79.79, H 6.47, N 6.42; found: C 79.80, H

TE D

6.35, N 6.40.

6.1.3.3. (E)-N-(4-(3,5-dimethoxystyryl)phenyl)-7-methoxy-1,2,3,4-tetrahydroacridin-9-amine (11).

EP

Coupling of compound 16 (0.268 g, 0.69 mmol) and 19 (0.174 g, 1.05 mmol) with phenol (0.590 g, 9 equiv) was performed according to the general procedure described above. Brown solid, 0.106 g,

AC C

0.23 mmol, 32% yield. 1H NMR (CDCl3, 400 MHz) δ: 1.83-1.89 (m, 2H), 1.93-1.99 (m, 2H), 2.77 (t, J= 6.4 Hz, 2H), 3.13 (t, J= 6.4 Hz, 2H), 3.70 (s, 3H), 3.82 (s, 6H), 5.78 (br, 1H, -NH-), 6.37 (m, 1H, aromatic), 6.63-6.68 (m, 4H), 6.88 (d, J= 16.2 Hz, 1H), 7.01 (m, 1H, aromatic), 7.01 (d, J= 16.2 Hz, 1H), 7.28-7.29 (m, 1H, aromatic), 7.36 (d, J= 8.5 Hz, 2H, aromatic), 7.91 (d, J= 9.2 Hz, 1H, aromatic).

13

C NMR (CDCl3, 100 MHz) δ: 22.9, 23.0, 25.6, 33.8, 55.5, 55.6, 99.8, 101.7, 104.5,

106.86, 106.89, 116.6, 121.5, 124.1, 126.3, 127.8, 128.9, 129.66, 129.71, 130.25, 130.32, 139.9, 144.3, 157.1, 161.2; MS (ESI+): m/z: 467 [M + H]+. Anal. (C30H30N2O2) calcd: C 77.23, H 6.48, N 6.00; found: C 77.20, H 6.32, N 5.88. 17

ACCEPTED MANUSCRIPT

6.1.4.

5-(4-(6-chloro-1,2,3,4-tetrahydroacridin-9-ylamino)phenethyl)benzene-1,3-diol

(8).

Compound 5 (0.500 g, 1.06 mmol) was dissolved in anhydrous DCM (12 mL). The mixture was cooled to -78 °C and a 1M DCM solution of BBr3 (4.2 mL, 4.2 mmol, 4 eq) was added dropwise

RI PT

under N2 atmosphere. The reaction mixture was stirred at the same temperature for 0.5 h and then at rt for 20h. Ice and then aqueous NaOH 10% were added and the mixture was extracted with DCM (1x10 mL) to remove impurities. The alkaline aqueous phase was acidified with HCl 6N, and a

SC

yellow precipitate was formed. The precipitate was filtered and washed with water and diethyl ether and then purified by flash chromatography (DCM/MeOH 98:2). Yellow solid, 0.440 g, 0.98 mmol,

M AN U

93% yield. 1H NMR (CD3OD, 400 MHz) δ 1.91-1.97 (m, 4H), 2.58 (t, J = 6.0 Hz, 2H), 2.80 (t, J = 7.3 Hz, 2H), 2.94 (t, J = 7.3 Hz, 2H), 3.09 (t, J = 6.0 Hz, 2H), 6.09 (s, 3H, aromatic), 7.11 (d, J = 8.3 Hz, 2H, aromatic), 7.21 (d, J = 8.3 Hz, 2H, aromatic), 7.39 (dd, J= 9.3, 2.0 Hz, 1H, aromatic), 7.67 (d, J = 9.3 Hz, 1H, aromatic), 7.80 (d, J = 2.0 Hz, 1H, aromatic).

13

C NMR (CD3OD, 100 MHz): δ:

TE D

23.1, 23.4, 26.4, 33.0, 38.0, 39.1, 101.2, 108.1, 120.7, 120.9, 121.5, 124.7, 126.5, 127.4, 130.4, 136.9, 137.8, 142.5, 145.2, 146.1, 159.3, 160.0; MS (ESI+): m/z: 445 [M + H]+. Anal.

6.1.5.

EP

(C27H25ClN2O2) calcd: C 72.88, H 5.66, N 6.30; found: C 72.72, H 5.52, N 6.23.

(E)-5-(4-((6-chloro-1,2,3,4-tetrahydroacridin-9-yl)amino)styryl)benzene-1,3-diol

(12).

AC C

Compound 9 (0.400 g, 0.85 mmol) was dissolved in anhydrous DCM (12 mL). The mixture was cooled to -78 °C and a 1M DCM solution of BBr3 (3.39 mL, 3.39 mmol, 4 eq) was added dropwise under N2 atmosphere. The reaction mixture was stirred at the same temperature for 0.5 h and then at rt for 20 h. Aqueous NaOH 10% was added and mixture was extracted with EtOAc (3 x 20 mL). Combined organic phases were dried over sodium sulfate and evaporated. Crude product was purified by flash chromatography (petroleum ether/EtOAc 50:50). Yellow solid, 0.090 g, 0.20 mmol, 31% yield. 1H NMR (CD3OD, 400 MHz) δ: 1.82-1.88 (m, 2H), 1.93-1.99 (m, 2H), 2.73 (t, J= 6.5 Hz, 2H), 3.09 (t, J= 6.5 Hz, 2H), 6.15 (t, J= 2.1 Hz, 1H, aromatic), 6.44 (d, J= 2.1, 2H, 18

ACCEPTED MANUSCRIPT aromatic), 6.70 (d, J= 8.6 Hz, 2H, aromatic), 6.83 (d, J= 16.4 Hz, 1H), 6.96 (d, J= 16.4 Hz, 1H), 7.33-7.37 (m, 3H, aromatic), 7.87-7.89 (m, 2H, aromatic).

13

C NMR (CD3OD, 100 MHz) δ: 23.6,

23.7, 26.7, 34.5, 102.8, 105.9, 118.1, 122.9, 125.1, 126.6, 126.9, 127.1, 127.5, 128.5, 129.2, 131.4, 135.9, 141.2, 145.6, 146.4, 148.5, 159.7, 162.6; MS (ESI+): m/z: 443 [M + H]+. Anal.

6.1.6.

4-(3,5-dimethoxyphenethyl)aniline

(15).

A

solution

RI PT

(C27H23ClN2O2) calcd: C 73.21, H 5.23, N 6.32; found: C 73.10, H 5.18, N 6.15.

of

(E)-1,3-dimethoxy-5-(4-

SC

nitrostyryl)benzene 14 (0.2 g, 0.70 mmol) in EtOAc (20 mL) was reduced on an H-Cube® flow hydrogenator using a Palladium catalyst cartridge 10% Pd/C, with the following conditions: full H2

M AN U

mode, T= rt; P(H2)= 1 bar; flow rate: 1.0 mL/min. Volatile components were evaporated in vacuum to give the crude 4-(3,5-dimethoxyphenethyl)aniline as brown oil. 15 used in the following step without further purification. 0.17 g, 0.65 mmol, 93% yield. 1H-NMR (CDCl3, 400 MHz) δ: 2.89 (s, 4H), 3.77 (s, 6H), 6.31 (t, J= 2.4 Hz, 1H, aromatic), 6.35 (d, J= 2.4 Hz, 2H, aromatic), 6.62-6.64 (m,

6.2. Biological evaluation

EP

6.2.1. Cholinesterase assays

TE D

2H, aromatic), 6.99 (d, J= 8.0 Hz, 2H, aromatic).

The AChE and BChE inhibitory activity of the tested drugs was determined using Ellman´s method

AC C

[35] and is expressed as IC50, i.e. concentration that reduces the cholinesterase activity by 50%. Human red blood cell acetylcholinesterase (hAChE; EC 3.1.1.7), and butyrylcholinesterase (hBChE; EC 3.1.1.8) from human plasma were used as sources of enzymes. 5,5´-dithiobis(2nitrobenzoic acid) (Ellman´s reagent, DTNB), phosphate buffer (PB, pH 7.4), acetylthiocholine (ATC), and butyrylthiocholine (BTC), were purchased (Sigma-Aldrich, Czech Republic). Polystyrene Nunc 96-well microplates with flat bottom (ThermoFisher Scientific, USA) were utilized. The assay was carried out in 0.1 M KH2PO4/K2HPO4 buffer, pH 7.4. The assay mixture (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 19

ACCEPTED MANUSCRIPT enzyme (2.0 units/mL), and 20 µL of 0.01 M substrate (ATC/BTC iodide solution). Assay solutions with were preincubated with inhibitor (10-3 – 10-9 M) for 5 min. The reaction was started by the addition the substrate. The activity was determined by measuring the increase in absorbance at 412 nm at 37 °C at 2 min intervals by using a Multi-mode microplate reader Synergy 2 (Biotek,

Diego, USA) was used for the statistical evaluation.

SC

6.2.2.Propidium displacement studies

RI PT

Vermont, USA). Each concentration was assayed in triplicate. Software GraphPad Prism 5 (San

The affinity of 5 for the peripheral binding site of EeAChE (type VI-S, Sigma-Aldrich) was tested

M AN U

using propidium iodide (P) (Sigma-Aldrich), a known PAS-specific ligand. A shift in the excitation wavelength follows the complexation of propidium iodide and AChE [58]. Fluorescence intensity was monitored by a Jasco 6200 spectrofluorometer (Jasco Europe, Italy) using a 0.5 mL quartz cuvette at room temperature. EeAChE (2 µM) was first incubated with 8 µM propidium iodide in

TE D

1.0 mM Tris-HCl, pH 8.0. A 4.0 mM stock solution of 5 was prepared in methanol. In the back titration experiments of the propidium-AChE complex by the tested inhibitor, aliquots of 5 were sequentially added (final concentration from 8 to 56 µM), and fluorescence emission was monitored

EP

at 635 nm upon excitation at 535 nm. Blanks containing propidium alone, inhibitor plus propidium and EeAChE alone were prepared and fluorescence emission determined. Raw data were processed

AC C

following the method of Taylor and Lappi [59] to estimate KD values, assuming a dissociation constant value for propidium for EeAChE equals to 0.7 µM [40]. GraphPad Prism 4 software (San Diego, USA) was used for the data processing.

6.2.3. Inhibition of Aβ42 Self-Aggregation Aβ42 samples (Bachem AG, Switzerland) were pretreated and resolubilized as previously reported [43, 44] in order to have a stable stock solution ([Aβ42] = 500 µM). Experiments were performed by incubating the peptide in 10 mM phosphate buffer (pH = 8.0) containing 10 mM NaCl, at 30 °C for 20

ACCEPTED MANUSCRIPT 24 h (final Aβ concentration = 50 µM) with and without inhibitor (50 µM). Stock solutions of tested inhibitors were prepared in methanol and dilutions were made in the assay buffer. Blanks containing tested inhibitors and thioflavin T (ThT) were also prepared and evaluated to account for quenching and fluorescence properties. After incubation, samples were diluted to a final volume of

RI PT

2.0 mL with 50 mM glycine-NaOH buffer (pH = 8.5) containing 1.5 µM ThT. A 300-seconds-time scan of fluorescence intensity was carried out (λexc = 446 nm; λem = 490 nm), and values at plateau were averaged after subtracting the background fluorescence of 1.5 µM ThT solution. The

M AN U

SC

fluorescence intensities were compared and the % inhibition was calculated.

6.2.4. Antioxidant assay

Diphenyl-1-picrylhydrazyl stabile free radical assay (DPPH) is a simple method to determine antioxidant activity and is expressed as EC50, i.e. concentration of compound that causes 50% decrease in the DPPH activity [49]. DPPH, methanol, N-acetyl cysteine and trolox (as reference

TE D

standards) were purchased from Sigma-Aldrich (Czech Republic). Polystyrene Nunc 96-well microplates with flat bottom (ThermoFisher Scientific, USA) were used. The assay was carried out in methanol. DPPH solution was prepared at 0.2 mM concentration. The assay medium (200 µL)

EP

consisted of 100 µL of DPPH solution and 100 µL of tested compound (10-3–10-6 M). The

AC C

antioxidant activity was determined after 30 min of incubation by measuring the increase in absorbance at 517 nm at laboratory temperature using Multi-mode microplate reader Synergy 2 (Biotek, Vermont USA). Each concentration was tested in triplicate. Software GraphPad Prism version 5 for Windows (GraphPad Software, San Diego, CA, USA) was used for statistical analysis.

6.2.5. Cell cultures Primary cultures of cerebellar granule neurons (CGNs) were prepared from 7 day-old Wistar rat pups (Rattus norvegicus), as previously described [60]. All animal experiments were authorized by 21

ACCEPTED MANUSCRIPT the University of Bologna bioethical committee (Protocol n° 17-72-1212) and performed according to Italian and European Community laws on animal use for experimental purposes. Briefly, cells were dissociated from cerebella and plated on 96-well plates, previously coated with 10 mg/L polyL-lysine at the density of 0.12x106cells/well in BME with 10% heat-inactivated FBS (Life

RI PT

Technologies), 2mM glutamine, 100µM gentamicin sulfate and 25mM KCl (Sigma-Aldrich). After incubating for 16 h at 37°C in 95% air/5% (vol/vol) CO2 atmosphere, 10 µmol/L cytosine arabinofuranoside was added to reduce the proliferation of non-neuronal cells. After 7 days in vitro

SC

(DIV), differentiated neurons were shifted to serum-free medium for compound testing. Mixed glial cell cultures were prepared from cerebral cortex of newborn Wistar rats, as previously described

M AN U

[43]. Briefly, brain tissue was cleaned from meninges, trypsinized for 15 min at 37°C and, after mechanical dissociation, the cell suspension was washed and plated on poly-L-lysine (SigmaAldrich, St. Louis, MO, USA, 10 µg/mL) coated flasks (75 cm2). Mixed glial cells were cultured for 10-13 days in Basal Medium Eagle (BME, Life technologies Ltd, Paisley, UK) supplemented with

TE D

100 mL/L heat-inactivated fetal bovine serum (FBS, Life technologies), 2 mmol/L glutamine (Sigma-Aldrich) and 100 µmol/L gentamicin sulphate (Sigma-Aldrich). Microglial cells were harvested from mixed glial cells cultures by mechanical shaking, resuspended in fresh medium

EP

without serum and plated on uncoated 35 mm Ø dishes at a density of 1.5 x 106 cells/1.5 mL medium/well for western blot analysis. Cells were allowed to adhere for 30 min and then washed to

AC C

remove non-adhering cells. For the preparation of purified astrocyte cultures, the remaining adherent cells after microglial shaking were detached with trypsin (0.25%)/EDTA (Life technologies). Cells were collected, centrifuged and resuspended in fresh BME medium without serum (Life technologies) and reseeded on poly-L-lysine-coated (Sigma-Aldrich) 35 mm Ø dishes at a density of 1.5 x 106 cells/1.5 mL medium/well for western blot analysis. Afterwards, different treatments were performed. HepG2 liver hepatocellular cells were cultured at 37 °C in a 95% air/5% (vol/vol) CO2 humidifed atmosphere in high glucose Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma Aldrich) 22

ACCEPTED MANUSCRIPT supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2mM L-glutamine, 50 U of penicillin G/mL and 50 µg of streptomycin sulfate/mL. All reagents were from Sigma Aldrich, except for FBS (Life Technologies). Cells were plated on 96-well plates at a density of 8 x 103 cells

viability was measured by trypan blue exclusion.

6.2.6. MTT assay

RI PT

/well. Cell stocks were cryopreserved by standard methods and stored in liquid nitrogen and cell

SC

To test the toxicity of compounds, CGNs or HepG2 cells were exposed to increasing concentration of the compounds, from 0 (controls) to 50 µM, for 24h in serum-free medium. Cell survival was

M AN U

analyzed by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [61]. For MTT assay, thiazolyl blue was added to culture medium at a final concentration of 0.1 mg/mL. After a 20 min incubation at 37 °C in the dark, the MTT precipitate was dissolved in 0.1M Tris-HCl buffer containing 5% Triton X-100 (all from Sigma-Aldrich) and absorbance was read at 570 nm in

6.2.7. Western blot analysis

TE D

a Multiplate Spectrophotometric Reader (Bio-Rad Laboratories S.r.l., Segrate, MI, Italy).

EP

Microglial and astrocytic cells exposed to LPS (10 ng/mL) in presence or absence of different concentrations of compound 12 (from 0 to 50µM) for 24h were directly lysed in ice-cold lysis

AC C

buffer (Tris 50mM, SDS 1 %, 1mM EDTA, protease inhibitor cocktail 0.05%) and protein content was determined by using the Lowry method [62]. 20 µg of protein extract were resuspended in 20 µL of Loading Buffer (0.05M Tris-HCl pH 6.8; 40 g/L sodium dodecyl sulfate; 20 mL/L glycerol; 2 g/L bromophenol blue and 0.02 M dithiothreitol; all chemicals were from Sigma-Aldrich) and loaded into 10% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE; Bio-Rad Laboratories SrL, Segrate, MI, IT). After electrophoresis and transfer on nitrocellulose membranes (GE Healthcare Europe GmbH, Milano, MI, IT), membranes were blocked for 1 h in 5% non-fat milk (Bio-Rad)/0.1% Tween-20 in PBS (Sigma-Aldrich), pH 7.4, and incubated overnight (ON) at 4 °C 23

ACCEPTED MANUSCRIPT with primary antibodies (rabbit polyclonal anti-iNOS or anti-TREM2 or goat polyclonal antiMRC11:1000, or mouse monoclonal anti-GAPDH, 1:2000, all from Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) in 0.1% Tween-20/PBS. Membranes were then incubated with an anti-rabbit or anti-mouse secondary antibody conjugated to horseradish peroxidase (1:2000; Santa Cruz), for

RI PT

90 min at rt in 0.1% Tween-20/PBS. Labeled proteins were detected by using the enhanced chemiluminescence method (ECL; Santa cruz) and detected using the software Image Lab with a ChemiDoc™ MP imaging system (both from Biorad). Densitometric analysis was performed by

SC

using ImageJ software from NIH.

M AN U

6.2.8. NO measurements

Accumulation of nitrite in conditioned media of transfected microglia and astrocytes, was measured by a colorimetric assay based on the Griess reaction. A nitrate standard curve was performed with NaNO2 at known concentrations in order to quantify nitrite concentration in the medium.

TE D

Sulfanilamide 5 mM (Sigma-Aldrich) which was added to the culture medium and the standard curve, reacts with nitrite under acid conditions to form a diazonium cation, which subsequently couples to N-1-naphthyl-ethylenediamine dihydrochloride (NEDA 40 mM; Sigma-Aldrich) to

EP

produce a colored azo dye. After 15 minutes of incubation at room temperature in the dark,

AC C

absorbance was read at 540 nm in a Multiplate Spectrophotometric Reader (Bio-Rad Laboratories).

Acknowledgments

This work was supported by University of Bologna (RFO 2014), MEYS (project Nr. LO1611 under the NPU I program), Ministry of Defense (Long Term Organization Plan-1011) and Ministry of Health of the Czech Republic (grant Nr. 15-30954A). J.J. acknowledges an Erasmus fellowship. The authors would like to acknowledge the contribution of the COST Action CA15135.

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[2] S. Finkbeiner, Bridging the Valley of Death of therapeutics for neurodegeneration, Nat. Med.,

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[6] S. Yahiaoui, K. Hamidouche, C. Ballandonne, A. Davis, J.S. de Oliveira Santos, T. Freret, M. Boulouard, C. Rochais, P. Dallemagne, Design, synthesis, and pharmacological evaluation of multitarget-directed ligands with both serotonergic subtype 4 receptor (5-HT4R) partial agonist and

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EP

5-HT6R antagonist activities, as potential treatment of Alzheimer's disease, Eur. J. Med. Chem.,

AC C

[7] M. Singh, M. Kaur, N. Chadha, O. Silakari, Hybrids: a new paradigm to treat Alzheimer's disease, Mol. Divers., 20 (2016) 271-297. [8] A. Minarini, A. Milelli, E. Simoni, M. Rosini, M.L. Bolognesi, C. Marchetti, V. Tumiatti, Multifunctional tacrine derivatives in Alzheimer's disease, Curr. Top. Med. Chem., 13 (2013) 17711786. [9] A. Romero, R. Cacabelos, M.J. Oset-Gasque, A. Samadi, J. Marco-Contelles, Novel tacrinerelated drugs as potential candidates for the treatment of Alzheimer's disease, Bioorg. Med. Chem. Lett., 23 (2013) 1916-1922. 25

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[11] O. Soukup, D. Jun, J. Zdarova-Karasova, J. Patocka, K. Musilek, J. Korabecny, J. Krusek, M. Kaniakova, V. Sepsova, J. Mandikova, F. Trejtnar, M. Pohanka, L. Drtinova, M. Pavlik, G. Tobin, K. Kuca, A resurrection of 7-MEOTA: a comparison with tacrine, Curr. Alzheimer Res., 10 (2013)

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ACCEPTED MANUSCRIPT Captions

Fig. 1. Design strategy to hybrids 5-12.

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Fig. 2. Neurotoxicity of 5-12 on primary rat cerebellar granule neurons (CGNs) after 24 h treatment. Results are expressed as percentage of controls and are the mean ± SE of four different experiments run at least in quadruplicate. *p<0.05, **p<0.01 compared to its own control at 0 µM

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(Bonferroni’s test after ANOVA).

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Fig. 3. Primary rat microglia and astrocyte cells were treated with LPS (100 ng/mL) in the presence or absence of 12. Immunomodulatory activity in glial cells was evaluated through Western blot analysis and relative densitometry of iNOS expression, using GAPDH as loading control, as well as the indirect extent of released NO trough nitrite measurement in the medium. Densitometric results

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are expressed as percentage of LPS only and are the mean ± SE of 3 different experiments. Nitrite measurement results are the mean ± SE of 3 different experiments run at least in quadruplicate. ###

p<0.001 compared to control; *p<0.05, **p<0.01 and ***p<0.001 compared to LPS treated cells

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Fig. 4. Immunomodulatory activity of 12 in glial cells was evaluated through Western blot analysis (A) and relative densitometries of TREM2 (B) and MRC1 (C) expression as M2 microglial phenotype markers, using GAPDH as loading control. Densitometric results are expressed as percentage of LPS only and are the mean ± SE of 3 different experiments. *p<0.05, **p<0.01 and ***p<0.001 compared to LPS treated cells (Bonferroni’s test after ANOVA).

Fig. 5. Hepatotoxicity of (A) reference compound 1, (B) reference compound 4 and (C) 5-12 on liver hepatocellular cells (HepG2) after 24 h treatment. Results are expressed as percentage of 33

ACCEPTED MANUSCRIPT controls and are the mean ± SE of four different experiments run at least in quadruplicate. *p<0.05

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and **p<0.01 and compared to LPS treated cells (Bonferroni’s test after ANOVA).

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ACCEPTED MANUSCRIPT Table 1. hAChE and hBChE activities, inhibition of Aβ self-aggregation and predicted BBB permeation of 5-12 and reference compounds 1-4.

Compound

IC50 ± SEM (µM) for hAChE

IC50 ± SEM (µM) for hBChE

Aß42 selfaggregation % Inhibition [I] = 50 µM

5

0.80 ± 0.1

n.a.

17.6 ± 1.6

BBB+ (0.101)

6

14.2 ± 3.0

n.a.

n.d.

BBB+ (0.105)

7

n.a.

n.a.

n.d.

8

1.3 ± 0.1

n.a.

9

n.a.

n.a.

10

n.a.

11

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BBB permeation (+/-) and BBB scorea

BBB+ (0.092)

BBB+ (0.068)

21.6 ± 4.9

BBB+ (0.101)

n.a.

n.d.

BBB+ (0.105)

n.a.

n.a.

n.d.

BBB+ (0.092)

12

8.8 ± 0.4

n.a.

31.2 ± 9.0

BBB+ (0.068)

1

0.5 ± 0.1b

0.023 ± 0.003b

<5

BBB+ (0.120)

2

0.007 ± 0.0002b

0.85 ± 0.031b

<5

BBB+ (0.111)

3

10.5 ± 2.0b

21 ± 3.0b

<5

BBB+ (0.102)

4

n.a.c

n.a.c

30.0 ± 8.7

BBB+ (0.041)

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37.3 ± 4.1

The BBB permeability was predicted using the online BBB prediction server (http://www.cbligand.org/BBB/);

b

Data taken from ref. [37];

c

No inhibition observed in tested concentration up to 100 µM

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n.a. – not active, no enzyme inhibition at compound’s concentration of 10 µM; n.d. – not determined.

ACCEPTED MANUSCRIPT Table 2. Antioxidant activity, expressed as EC50 (µM), of 5 and 12 against DPPH, compared to reference compounds 2, Trolox and N-acetyl-cysteine.

8

˃ 5000

12

˃ 5000

4

25.6 ± 1.9

Trolox

16.2 ± 0.4

N-acetyl-cysteine

28.0 ± 1.8

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5

Antioxidant activity EC50 ± SEM (µM) 155 ± 4

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Highlights Novel multitarget ligands against Alzheimer’s disease have been developed

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The new hybrids have been obtained by fusing tacrines with resveratrol

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The most interesting hybrids inhibited hAChE and modulated Aβ self-aggregation

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Hybrid 12 showed a low neurotoxicity and neuroprotective effect on neuroinflammation

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1