Multifunctional Small Molecules as Potential Anti ...

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Multifunctional Small Molecules as Potential Anti-Alzheimer's Disease Agents

Beatrice Bargagna , 1,2, Lidia Ciccone 3,, Susanna Nencetti 3, M. Am?lia Santos 2,*, S?lvia Chaves 2, Caterina Camodeca 3 and Elisabetta Orlandini 1,4,*

1 Department of Earth Sciences, University of Pisa, Via Santa Maria 53-55, 56100 Pisa, Italy; beatrice.bargagna@dst.unipi.it

2 Centro de Qu?mica Estrutural and Departamento de Engenharia Qu?mica, Instituto Superior T?cnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal; silvia.chaves@tecnico.ulisboa.pt

3 Department of Pharmacy, University of Pisa, Via Bonanno 6, 56126 Pisa, Italy.; lidia.ciccone@unipi.it (L.C.); susanna.nencetti@unipi.it (S.N.); caterina.camodeca@unipi.it (C.C.)

4 Research Center "E. Piaggio", University of Pisa, 56122 Pisa, Italy * Correspondence: masantos@tecnico.ulisboa.pt (M.A.S.); elisabetta.orlandini@unipi.it (E.O.) These authors contributed equally to this work.

Citation: Bargagna, B.; Ciccone, L.; Nencetti, S.; Santos, M.A.; Chaves, S.; Camodeca, C.; Orlandini, E. Multifunctional Small Molecules as Potential Anti-Alzheimer's Disease Agents. Molecules 2021, 26, 6015. molecules26196015

Academic Editors: Valentina Oliveri and Luciana Mosca

Received: 7 July 2021 Accepted: 29 September 2021 Published: 3 October 2021

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Copyright: ? 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( /by/4.0/).

Abstract: Alzheimer's disease (AD) is a severe multifactorial neurodegenerative disorder characterized by a progressive loss of neurons in the brain. Despite research efforts, the pathogenesis and mechanism of AD progression are not yet completely understood. There are only a few symptomatic drugs approved for the treatment of AD. The multifactorial character of AD suggests that it is important to develop molecules able to target the numerous pathological mechanisms associated with the disease. Thus, in the context of the worldwide recognized interest of multifunctional ligand therapy, we report herein the synthesis, characterization, physicochemical and biological evaluation of a set of five (1a?e) new ferulic acid-based hybrid compounds, namely feroyl-benzyloxyamidic derivatives enclosing different substituent groups, as potential antiAlzheimer's disease agents. These hybrids can keep both the radical scavenging activity and metal chelation capacity of the naturally occurring ferulic acid scaffold, presenting also good/mild capacity for inhibition of self-A aggregation and fairly good inhibition of Cu-induced A aggregation. The predicted pharmacokinetic properties point towards good absorption, comparable to known oral drugs.

Keywords: Alzheimer's disease (AD); multifunctional drugs; metal chelation; antioxidant activity; A stabilizers; neurodegeneration; ferulic acid; multitarget drugs

1. Introduction Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder

characterized by a progressive loss of neurons in the brain. Early symptoms are memory decline and language problems, followed by other cognitive serious dysfunctions related to brain atrophy [1]. AD is the most common cause of dementia and, in 2019, it was estimated that 50 million individuals were affected by dementia worldwide. This number is projected to reach 152 million cases by 2050 [2]. Despite research efforts, the pathogenesis and mechanism of AD progression are not yet completely understood. However, it is well-known that a common feature in AD patients is the presence of extracellular amyloid- (A) plaques and intracellular neurofibrillary tangles (NFT) of hyperphosphorylated tau protein, the two major hallmarks in AD [3].

There are only a few symptomatic drugs approved for the treatment of AD. Four of them hamper the pathway that downregulates the neurotransmitter acetylcholine (ACh) acting as acetylcholinesterase inhibitors (AChE)--tacrine, donepezil, rivastigmine and

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galantamine--and the fifth is a N-methyl-D-aspartate (NMDA) receptor antagonist (memantine) [4]. Moreover, on June 7th 2021, the Food and Drug Administration (FDA) approved Aduhelm (aducanumab) for the treatment of AD. Aducanumab is a human IgG1 anti-A monoclonal antibody specific for -amyloid oligomers and fibrils [5].

The multifactorial character of AD suggests that it is important to develop molecules able to target the numerous pathological mechanisms associated with the disease. Studies revealed that A is the most abundant peptide found among the numerous proteins present in AD amyloid plaques [6]. It has been demonstrated that these proteins can interact with A peptides favoring or contrasting AD progression--negative and positive cross-interaction, respectively. Recently, the positive amyloid cross-interaction has been reported as a potential emerging multi-target strategy against AD [7,8] and several proteolysis targeting chimera (PROTAC) constructions have been proposed [9?11].

Dyshomeostasis of physiological metal ions is a common feature of neurological disorders such as AD [12?19]. Studies report that higher levels of metal ions, such as Cu2+, Zn2+ and Fe3+, are found in cerebral amyloid plaques of AD patients compared to the concentrations detected in the brains of non-AD patients [13,20]. Moreover, it has been reported that redox active metal ions, as Cu2+ and Fe2+, interact with A producing reactive oxygen species (ROS) and, finally, inducing neuronal death [21]. Therefore, most chelators included in anti-AD drugs are based on heterocyclic structures of hard or hard-soft ligands, namely containing hexocyclic or hexocyclic pairs of electron donor atoms such as (O-O), (O-N), and (N-N). According to the multifactorial nature of AD, the most recent studies aim to develop molecules that can act simultaneously against different pathological features, at the same time being A stabilizers, antioxidants and metal chelators [22,23].

In the context of multifunctional ligand therapy, marine and terrestrial organisms are a fundamental source for the discovery of new bioactive agents [24?28]. In the last few years, several different compounds isolated from plants and microorganisms have shown good effects for the treatment of AD and several drug candidates are in clinical trials, confirming that the use of natural compounds against AD is an active and interesting area of research [23,29?31].

Ferulic acid (FA), as with other hydroxycinnamic derivatives, is a phenolic compound largely present in the human diet. FA has been considered as a multifunctional antioxidant because, besides the more typical radical scavenging role by electron or hydrogen donation to existing radicals, it can also chelate redox-active metal ions, thus disabling their participation in the Fenton reaction. FA is also well known for its antiinflammatory properties and recent studies have demonstrated its potential role in the treatment of AD, particularly due to its capacity to inhibit A aggregation in vitro and in in vivo AD mouse models protecting the brain from A neurotoxicity [32]. In fact, in the last few years, FA has been largely used as a scaffold to design new multifunctional ligands against AD progression [33].

Given the considerations above, in this study, a new set of FA hybrid derivatives, 1a? e, is presented in which the FA moiety is coupled with benzyloxyamines substituted with methoxyl and trifluoromethyl groups or also chlorine atoms (Figure 1). In particular, the methoxyl and trifluoromethyl groups were chosen for their ability to establish H-bond interactions as acceptors or donors, or only as acceptors, respectively. Concerning the chlorine atom, it increases the lipophilic character of the molecule, thus favoring hydrophobic interactions with A peptides.

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Figure 1. General structures of ferulic acid (FA) and its benzyloxyamidic derivatives.

Herein, we report the synthesis and characterization of a new set of hybrid compounds enclosing the ferulic acid scaffold, followed by the evaluation of their physicochemical and biological properties, envisaging their potential role as anti-AD agents.

2. Results and Discussion 2.1. Chemistry

The (E)-N-(benzyloxy)-3-(4-hydroxy-3-methoxyphenyl)acrylamides compounds 1a? e were obtained following the synthetic procedure reported in Scheme 1.

Scheme 1. (i) Et3N, anhydrous DMF, r.t., 4 h; (ii) NH3 7M in MeOH, r.t., 2 h; (iii) Et2O?HCl, Et2O, T = 0 ?C; (iv) EDCI, HOBt, N-methylmorpholine, anhydrous DMF, r.t., 24 h.

The FA derivatives 1a?e were synthesized coupling the commercially available ferulic acid (6) and hydrochloride benzylhydroxylamines 5a?e variously substituted. The Oarylmethylhydroxylamine hydrochloride 5a?e were synthetized according to the procedure previously described [34?36]. Briefly, the O-arylmethylhydroxylamine hydrochloride 5a?e were obtained by reaction between the suitably substituted benzyl bromide 2a? e and the N-hydroxyphthalimide (3) by Mitsunobu reaction, and successive deprotection of the phthalimido group with ammonia solution 7N in MeOH. Compounds 5a?e were purified by crystallization and isolated as their hydrochloride salts.

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Finally, the coupling reaction of the free amino group of these benzylhydroxylamines with the carboxylic group of FA was carried out in anhydrous DMF and inert argon atmosphere, in the presence of the carboxyl activating agent N-(3-dimethylaminopropyl)N-ethylcarbodiimide hydrochloride (EDCI), hydroxybenzotriazole (HOBt) and Nmethylmorfoline. The reaction mixture was stirred at r.t. for 24 h, followed by the corresponding workup and purification to afford the final compounds 1a?e as pure solids under good yields (57?69%).

2.2. Physicochemical Studies

2.2.1. Antioxidant Activity

Cinnamic acid derivatives, such as FA, have been shown to avoid chain-breaking in the oxidation of low density lipoproteins, related with their hydrogen or electron-donating capacity and to the stability of the formed phenoxyl radicals [37].

Commercial ferulic acid (FA) and all the newly synthesized compounds 1a?e were studied for their radical scavenging activity, following the protocol previously reported [38,39]. The activity of each compound is expressed as EC50 and is related to its interaction with the free stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH?). Analysis of the results contained in Table 1 shows that benzyloxyamidic derivatives 1a?e have good antioxidant activity, similar to the one of ferulic acid and slightly lower than the one of ascorbic acid [40], with an EC50 value in the order of low ?M. The transformation of the carboxylic portion of ferulic acid and the increasing size of the molecule do not affect their radical scavenging capacity, which must be related to the proton donor phenolic group, both in the precursor and in the hybrids.

Table 1. Antioxidant results based on radical scavenging activity using the DPPH method.

O

R

O

NO

H

R

HO

Compound

Radical Scavenging Activity a (EC50, M)

1a

2-OCH3

34 ? 1

1b

3-OCH3

40 ? 2

1c

2-CF3

34 ? 1

1d

3-Cl

33 ? 1

1e

2,4-Cl

32 ? 3

Ferulic acid

-

36 ? 2

Ascorbic acid

-

25 ? 1 [40]

a Mean ? SD of 3 independent experiments for 50% antioxidant activity.

2.2.2. Metal Chelation Studies

Besides antioxidant capacity, FA can also play other roles such as chelation of transition metal ions (e.g., copper, iron), which are catalysts of oxidative stress, and interference in metal-induced A aggregation.

In order to evaluate the chelation capacity of the herein developed FA derivatives, compounds 1a and 1d were selected to be investigated on their acid-base behavior and metal chelating ability towards Cu2+ and Fe3+ ions, by using UV?Vis spectrophotometric titrations. These results were further compared to those of FA, studied by pH-potentiometric titrations.

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Acid-Base Properties

To evaluate the metal complexation capacity of the selected compounds, their protonation constants were determined.

Compounds were obtained in their neutral form, H2L (FA) and HL (1a, 1d), respectively. Due to some water-solubility limitations, especially for the benzyloxyamidic derivatives 1a and d, a mixed (20%, w/w) DMSO/water medium was chosen. The values obtained for the protonation constants are reported in Table 2.

Table 2. Stepwise protonation constants of FA, 1a, and 1d as well as global formation constantsa of their Fe3+, Cu2+ complexes and corresponding pM b values. (T = 25.0 ? 0.1 ?C, I = 0.1 M KCl, 20% w/w DMSO/water).

Compound FA

MmHhLl (mhl) (011) (021) (111) (101) (1-11) (102) (1-12) (1-22) (103) pM

log Ki

log (FemHhLl) a (CumHhLl) a

9.43(2) c 4.83(4) c

12.15(6) c

20.31(6) c

7.81(8) c 26.70(8) c

17.4

13.30(4) c 6.52(8) c

11.14(6) c 1.90(8) c

6.2

(011)

(101)

16.49(3) d

6.27(7) d

(1-21) (102)

8.75(2) d

23.69(4) d

-13.16(7) d -

(1-12)

-

1.12(5) d

1a

pM

17.6

6.3

(011)

(101)

15.42(7) d

7.49(7) d

(102) (1-12)

8.93(3) d

25.43(5) d -

12.14(7) d 2.20(8) d

(1-22)

8.50(5)

-

1d

pM

18.2

7.0

a (MmHhLl) = [MmHhLl]/[M]m[H]h[L]l; b pM = -log [M] at pH 7.4 (CL/CM = 10, CM = 10-6 M); c pH-

potentiometric data; d UV?Vis spectrophotometric data.

The values were obtained by fitting analysis of the experimental pH-potentiometric (FA) and spectrophotometric data (1a, 1d) with an equilibrium model using Hyperquad 2008 [41] and Psequad [42] programs, respectively. Figure 2 includes the potentiometric titration curves obtained for FA, as an example.

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