Novel tacrine–benzofuran hybrids as potential multi-target drug candidates for the treatment of Alzheimer’s Disease

Journal of Enzyme Inhibition and Medicinal Chemistry

ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20

Novel tacrine–benzofuran hybrids as potential

multi-target drug candidates for the treatment of

Alzheimer’s Disease

Gaia Fancellu, Karam Chand, Daniel Tomás, Elisabetta Orlandini, Luca

Piemontese, Diana F. Silva, Sandra M. Cardoso, Sílvia Chaves & M. Amélia

Santos

To cite this article: Gaia Fancellu, Karam Chand, Daniel Tomás, Elisabetta Orlandini, Luca

Piemontese, Diana F. Silva, Sandra M. Cardoso, Sílvia Chaves & M. Amélia Santos (2020)

Novel tacrine–benzofuran hybrids as potential multi-target drug candidates for the treatment of

Alzheimer’s Disease, Journal of Enzyme Inhibition and Medicinal Chemistry, 35:1, 211-226, DOI:

10.1080/14756366.2019.1689237

To link to this article: https://doi.org/10.1080/14756366.2019.1689237

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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

2020, VOL. 35, NO. 1, 211–226

https://doi.org/10.1080/14756366.2019.1689237

RESEARCH PAPER

Novel tacrine–benzofuran hybrids as potential multi-target drug candidates for

the treatment of Alzheimer’s Disease

a

Gaia Fancellu^a,b

, Karam Chand

, Elisabetta Orlandini^b, Luca Piemontese^c, Diana F.

ꢀ

, Daniel Tomas

Silva^d, Sandra M. Cardoso^d,e, Sılvia Chaves

and M. Amelia Santos

a

ꢀ

a

b

ꢀ

Centro de Quımica Estrutural, Instituto Superior Tecnico, Universidade de Lisboa, Lisboa, Portugal; Department of Earth Sciences, University of

c

d

ꢁ

Pisa, Pisa, Italy; Dipartimento di Farmacia–Scienze del Farmaco, Universita degli Studi di Bari “Aldo Moro”, Bari, Italy; CNC–Center for

Neuroscience and Cell Biology, Universidade de Coimbra, Coimbra, Portugal; Institute of Molecular and Cell Biology, Faculty of Medicine,

e

Universidade de Coimbra, Coimbra, Portugal

ABSTRACT

ARTICLE HISTORY

Received 27 August 2019

Revised 28 October 2019

Accepted 29 October 2019

Pursuing the widespread interest on multi-target drugs to combat Alzheimerꢀs disease (AD), a new series of

hybrids was designed and developed based on the repositioning of the well-known acetylcholinesterase

(AChE) inhibitor, tacrine (TAC), by its coupling to benzofuran (BF) derivatives. The BF framework aims to

endow the conjugate molecules with ability for inhibition of AChE (bimodal way) and of amyloid-beta pep-

tide aggregation, besides providing metal (Fe, Cu) chelating ability and concomitant extra anti-oxidant activ-

ity, for the hybrids with hydroxyl substitution. The new TAC-BF conjugates showed very good activity for

AChE inhibition (sub-micromolar range) and good capacity for the inhibition of self- and Cu-mediated Ab

aggregation, with dependence on the linker size and substituent groups of each main moiety.

Neuroprotective effects were also found for the compounds through viability assays of neuroblastoma cells,

after Ab_1-42induced toxicity. Structure-activity relationship analysis provides insights on the best structural

parameters, to take in consideration for future studies in view of potential applications in AD therapy.

KEYWORDS

Alzheimer’s disease; multi-

target drugs; tacrine-

benzofuran hybrids; AChE

inhibitors; metal chelators

GRAPHICAL ABSTRACT

Design, synthesis and evaluation of new tacrine-benzofuran hybrids as multitargeting anti- Alzheimer’s

disease agents; high AChE inhibition associated with other relevant properties; structure–activity rela-

tionship analysis

ꢀ

Centro de Quımica Estrutural, Instituto Superior Tecnico, Universidade de Lisboa, Av, Rovisco Pais 1,

CONTACT M. Amelia Santos

masantos@tecnico.ulisboa.pt

1049-001, Lisboa, Portugal

ꢀ

G.F. and K.C. contributed equally to this work.

ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

212

G. FANCELLU ET AL.

1. Introduction

2. Results and Discussion

Alzheimer’s Disease (AD) is one of the main causes of dementia 2.1. Molecular design

among elderly people, whose prevalence by 2050 is expected to

The study of the new multifunctional hybrid compounds (Figure 1)

be 14 and 130 million people in Europe and worldwide, respect-

ively^1,2. It is characterised by progressive impairment of memory

cognitive functions due to the degeneration of synapses and to

the death of neurons, especially in hippocampus³. The main

pathological hallmarks of AD are the presence of thick extracellu-

lar b-amyloid plaques (Ab) and intra-neuronal neurofibrillary tan-

gles (NFT)⁴, which compromise brain function due to the death of

many neurons. Other relevant characteristics of the patient brains

are the deficit of choline neurotransmitters, mostly acetylcholine

due to its hydrolysis by acetylcholinesterase (AChE) in neuronal

synapses, and also the metal dyshomeostasis associated with

advanced oxidative processes. In spite of the huge amount of

research aimed at understanding and to combat this pathology,

there is no cure so far. Along the past two decades, several strat-

egies have been followed bringing important contributions to AD

drug design and therapy⁵. The inhibition of AChE appeared as the

first approach and four AChE inhibitor drugs (tacrine, donepezil,

galantamine and rivastigmine) were approved by the US FDA,

although they can only lead to temporary symptom amelioration⁶.

Despite enormous efforts in the recent past years from academia

and pharmaceutical companies, several new molecules failed on

clinical trials⁷.

involved a preliminary molecular design approach. Therefore, two

main molecular moieties were selected, namely tacrine (TAC) and

benzofuran (BF), to guarantee the hitting of at least two main

pathophysiological targets of AD. TAC could assure the AChE inhib-

ition; BF was selected to mimic the indanone role in donepezil

(DNP), enabling a bimodal enzyme interaction for AChE, and also to

inhibit the self-aggregation of Ab peptide; the hydroxyl BF substitu-

ent (b-positioned to the oxygen atom of BF) can also endow the

hybrids with metal (Fe, Cu) chelating capacity and concomitant

extra anti-oxidant activity. Besides the selection of the main scaf-

folds and corresponding linkers, substituent groups were also intro-

duced in each moiety, namely: at C6 of TAC, the chlorine atom,

aimed at improving the AChE inhibitory capacity; at C7 of BF, the

methoxyl and hydroxyl groups, with the goal of mimicking DNP

indanone substituent groups as well as to provide metal chelation

capacity, respectively. To achieve the bimodal interaction of the

hybrids with AChE, through the binding to both the catalytic

anionic site (CAS) and the peripheral anionic site (PAS), the size of

the linker chain, connecting both main moieties, is of expected rele-

vance. Based on our previous experience about TAC hybrids with

dual-binding site¹⁶, alkyl linkers were selected with 3–4 methylene

carbons (n ¼ 1, 2), meaning a total spacer length of 5–6 atoms.

Therefore, to get some insight on the effects of these structural var-

iations on the binding pattern and binding affinity between the lig-

and and the receptor, molecular docking simulation appears as an

important tool.

The model structure for the active site of AChE was obtained

from the RCSB Protein Data Bank (PDB, entry 1ODC)²⁰, particularly

from the X-Ray structure of Torpedo Californica AChE (TcAChE)

complexed with the inhibitor (N-4⁰-quinolyl-N⁰-9⁰⁰-(1⁰⁰,2⁰⁰,3⁰⁰,4⁰⁰-tetra-

hydroacridinyl)-1,8-diaminooctane), hereinafter named as the ori-

ginal ligand, which has structural similarities with our hybrids.

Notwithstanding some recognised differences between the

respective inhibitor-enzyme complexes, reported for human

acetylcholine esterase, hAChE, and also of the electric ray

(Torpedo californica) homologue, TcAChE, both enzymes are fairly

conservative in terms of the main aminoacid residues that coat

the active site gorge²¹, and so the herein described modelling

study was performed with TcAChE.

Due to the multifactorial nature of this disease, the perspective

of AD treatment with a single-target molecule has been recently

changed with the emergence of multi-target anti-AD drug candi-

dates, aimed at targeting the disease pathogenesis and get dis-

ease– modifying effects. This type of compounds can act on

several disease targets, such as amyloid beta peptide (Ab) aggre-

gation, inhibition of AChE, modulation of metal dyshomeostasis

and inhibition of MAO involved in the production of reactive oxy-

gen species (ROS)^8–11. Therefore, the world of hybrid molecules

has been discovered and deepened, namely by repositioning and

extra-functionalization of already approved drugs such as acetyl-

cholinesterase inhibitors (AChEi). Tacrine (TAC) is the first single

drug developed for AD treatment as AChE inhibitor. Although it

was withdrawn from the market due to its hepatic toxicity at

therapeutic doses, it has been, by far, the mostly used AChE

inhibitory moiety in the development of multi-target anti-

AD drugs^11–13

.

Continuing our recent efforts to discover novel multi-target

directed ligands (MTDLs) for the treatment of AD, especially based

on templates of already known AChEi drugs^14–16, the current

study is focused on a new set of tacrine-benzofuran (TAC-BF)

hybrids, which have been developed and evaluated for their mul-

tiple properties as potential multi-target anti-AD agents. The nat-

urally inspired benzofuran (BF) scaffold can be considered as a

mimic of the indanone moiety of donepezil, and it has been

recently applied in many natural-based compounds with import-

ant biological properties, even as anti-neurodegeneratives^15,17–19

.

Particularly in this study, the new hybrids were designed to decide

on the most adequate spacer length between both main molecu-

lar units, with aid from molecular docking simulations. Afterwards,

the selected compounds were synthesised and tested for their

physico-chemical properties, as anti-oxidant and metal chelation

capacity, and biological properties (AChE inhibition, anti-Ab aggre-

gation); the effects of the compounds on cell viability and neuro-

protection were also evaluated in neuroblastoma cells after Ab_1-42

induced toxicity.

Figure 1. General structure of the tacrine-benzofuran (TAC-BF) hybrids

under study.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

213

Analysis of the outcomes of docking results (Figure 2) shows a

Regarding the effect of substituent groups, the chloro substi-

perfect insertion of the TAC moiety into the CAS, lined with the resi- tution at C6 of TAC is supposed to account for a better activity

dues Trp84, Ph330, Glu199, with a good superimposition of the TAC than the non-substituted analogues, due to the good fitting of

of the hybrids and that of the original ligand. This is mainly due to the chlorine atom inside a hydrophobic pocket of TcAChE formed

their ability to establish p-p stacking binding interactions with the by Trp432, Met436 and Ile439²³, otherwise identically reported

aromatic rings of Trp84 and Phe330 (see Figure 2), which can block for the human AChE (hAChE)²⁴, although, in Figure 2, only the

the entrance of the substrate (ACh) and therefore avoid its hydroly- first residue (Trp432) can be seen in a close distance from the

sis by the catalytic triad (Ser200, His440, Glu327)²². On the other chlorine atom. Regarding the BF moiety, the C7 substitution by

hand, the BF moiety, linked to TAC by the alkylamide spacers, seems methoxyl/hydroxyl groups aimed at enabling eventual new

also to be able to establish p-p interactions with PAS through Tyr70 potential interactions into the PAS. Indeed, it is interesting to

and Trp279 residues. Both the alkyl-amidic chain linkers of the con- observe that the BF moiety is well accommodated into the PAS,

jugates, with three or four methylene groups (n ¼ 1, 2) in the alkyl where -OCH₃/-OH seems to create H-bond interactions with the

chains, appear to be well accommodated along the hydrophobic Tyr70, although the presence of the substituents may also cause

channel, providing the adequate lengths for dual mode interaction a slight rearrangement of the whole structure inside this site, as

inside the AChE gorge and also H-bond interactions between the compared with the non-substituted compounds. Overall, the

amide-NH and the phenolic group of Tyr121, in accordance with molecular modelling studies gave some support to the predicted

previously reported results for other TAC hybrids^14,16. Remarkably, ability of the compounds for a bimodal interaction at CAS and

the docking results show only small differences in the enzyme inter- PAS binding sites of AChE. Furthermore, BF is expected to bring

action with an apparent better interaction with shorter spacer (5 other important contributions to the whole multifunctional activ-

atoms in the tether chain; n ¼ 1) than with the corresponding longer ity of the studied hybrids (antioxidant activity, anti-Ab aggrega-

homologue (6 atoms in the tether chain; n ¼ 2), (cf. 23 and 24, tion and metal chelation), which will be object of study in the

Figure 2).

respective sections.

Figure 2. Molecular modelling of four representative compounds inside the binding site of AChE, superimposed with the original ligand (N-4⁰-quinolyl-N⁰-9⁰⁰-(1⁰⁰,2⁰⁰,3⁰⁰,4⁰⁰-

tetrahydroacridinyl)-1,8-diaminooctane), green Coloured. These compounds have the same TAC substituent (R¼ Cl), but different BF substituent (R₁) and spacer length: 15

(turquoise Coloured; n ¼ 1; R₁¼ H), 19 (dark blue Coloured; n ¼ 1; R₁¼ -OCH₃), 23 (yellow Coloured; n ¼ 1; R₁¼ -OH), 24 (orange Coloured; n ¼ 2; R₁¼ H).

214

G. FANCELLU ET AL.

It should be mentioned that after the design and synthesis of reaction involved a standard acidic hydrolysis under very mild

the herein studied series of hybrids, a publication appeared also conditions, namely with a mixture of the Lewis acid boron tri-

with TAC-BF hybrids²⁵, although they have different linkers and chloride (BCl₃) and the catalyst tetra-n-butylammonium iodide

do not include the same substituent groups, in particular the

hydroxyl group of the benzofuran ring which is able to provide

metal chelating capacity and additional anti-oxidant activity.

Comparison of the present docking simulations with those

recently reported,²⁵for two other Tacrine-benzofuran hybrid ana-

logues, indicates quite identical interaction within the CAS binding

site, although interaction with PAS at the entrance of the gorge

involves different aminoacid residues, attributable to the differ-

ence in the used crystal structure of the inhibitor-bound TcAChE

complexes and also to the much longer length of the linker for

the two most closed analogues (from 3 to 6 carbon atom chain,

in the present and the reported studies).

(TBAI), in dry CH₂Cl₂, under N₂atmosphere and low temperature

(ꢁ78 ^ꢂC), following a protocol previously reported^27,28

.

2.3. Physico-chemical studies

2.3.1. Radical scavenging activity

A selection of the newly synthesised hybrids was tested for their

radical scavenging activity (anti-oxidant activity, EC₅₀), based on

their interaction with the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free

radical, as previously described²⁹.

Analysis of the obtained results indicate for compound 20 a

weak activity (EC₅₀>10³mM), otherwise similar to that found for

the reference compound, TAC. However, the introduction of the

hydroxyl substituent in the BF moiety resulted in a considerable

increase of the radical scavenging activity, though with still mod-

erate EC₅₀values (434–480 mM, for 24 and 21). Overall, the pres-

ence of the hydroxyl group increases the hybrid anti-oxidant

activity, as previously anticipated, which can be attributed to their

capacity for proton donation from the OH group.

2.2. Chemistry

The series of eight TAC-BF hybrids was prepared by following the

synthetic strategy outlined in Scheme 1. It involved two independ-

ent sequential reaction steps leading to the preparation of the

two main molecular moieties (TAC and BF), which were then

coupled to each other. The TAC starting fragments, 9-chloro-

1,2,3,4-tetrahydroacridines (3/4), were prepared from the coupling

the commercially available anthranilic acid (1) or 2-amino-4-chlor-

obenzoic acid (2) with cyclohexanone in the presence of POCl₃, as

previously reported²⁶. The synthesis of the BF derivatives involved

2.3.2. Metal complexation

It has been largely accepted that chelators interfere both in

metal-induced Ab aggregation/neurotoxicity and on metal homeo-

stasis of AD brains. Among the hybrids with BF hydroxyl substitu-

ent, compound 21 was chosen as a representative model to

analyse the concomitant chelating capacity towards the redox-

active (Fe(III), Cu(II)) and Ab-binding (Cu(II)) metal ions. Due to

solubility reasons, these studies were performed in a mixed 25%

(w/w) DMSO/water medium, In fact, DMSO was chosen because it

is a widely used and well tolerated solvent in biological and cellu-

a first condensation of the salicylaldehyde derivatives (5/6) (R₁

¼

H/OMe) with ethylbromoacetate in dry DMF under reflux and

basic conditions for substitution at the phenol group to obtain

the intermediate esters, ethyl 2-phenoxyacetates (5⁰/6⁰), which

were subsequently cyclized by further heating at high tempera-

ture conditions to afford the corresponding intermediate BF-

esters (7/8). Reaction of the diaminoalcane spacers with the

benzofuran-2-ethylcarboxylates in methanol gave the correspond- lar studies, the amounts of ligand (<7 mM) and DMSO (<1%)

ing N-(aminoalkyl)benzofuran-2-carboxamides (9–12). These employed in culture media being quite low with concomitant no

aminoalkyl-benzofuran derivatives were finally attached at the alterations observed in cells³⁰.

The protonation constants of compound 21, corresponding to

the phenolic hydroxyl (log K₁¼ 9.01) and to the tacrine N-amine

9-position of the TAC derivatives (3/4), by their reaction in the

presence of phenol and a catalytic amount of potassium iodide,

affording the final compounds (13–20). The metoxy-containing (log K₂¼ 7.99) groups, were determined by ultraviolet-visible (UV-

compounds (R₁¼ -OCH₃, 18–20) were further demethylated to vis) spectrophotometric titration (see Figure 3 and Table 1). Figure 3

obtain the corresponding derivatives with R₁¼ -OH (21–24). This shows that the absorvance maxima at 339 and 351 nm correspond

Scheme 1. Synthesis of TAC-BF hybrids. Reagents and conditions: (a) cyclohexanone, POCl₃, 180 ^ꢂC, 3 h; (b) DMF, K₂CO₃(1.2 eq), ethylbromoacetate (1.05 eq), 135-

140 ^ꢂC, 5–6 h; (c) diaminoalkane (3.0 eq), dry MeOH, overnight; (d) phenol, KI, 165–170 ^ꢂC 35–60 min; (e) BCl₃, TBAI, DCM, under N₂(ꢁ78 ^ꢂC), 2 h.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

215

to both species (H₂L^þand HL), while those at 295 and 321 nm are the global formation constants of the complexes, obtained by

attributed to the deprotonated (L^ꢁ) form.

treatment of the UV-vis spectrophotometric data (see Table 1).

From analysis of Table 1 it is possible to conclude that the

obtained log K_ivalues are in accordance with the values found, in

50% w/w DMSO/water medium, for the protonation of the phen-

From Figure 4(a)) it is possible to observe that, under the work-

ing conditions, FeHL and FeHL₂are the predominant species up to

pH ca 8 and above that pH FeL₃and FeL₂(OH)₂are subsequently

formed. Compound 21 is a strong chelator for iron (pFe ¼ 22.2 at

pH 7.4, C_L/C_M¼ 10, C_M¼ 10^ꢁ6M), that can be attributed to the hard

donor atom (O,O) chelation core of the BF moiety and the formation

of stable five-membered metal chelate rings.

olic oxygen (log K₁¼ 9.02 and the tacrine N-amine (log K₂

¼

8.18) of TAC-BIM1³¹.

The chelating ability of compound 21 towards the two metal

ions under study (Fe(III) and Cu(II)) was evaluated on the basis of

Concerning copper chelation, Figure 4(b)) presents CuHL as the

dominant species below pH 6.7 while above it, CuL₂is the predomin-

ant species. Compound 21 is a good copper chelator (pCu ¼ 11.9),

similarly to already found for the hydroxyphenyl-benzimidazole (BIM)

derivatives (pCu ¼ 10.7–11.1)³¹.

As a conclusion, the hydroxyl-containing derivatives of these

TAC-BF compounds showed to be effective chelators of Fe(III) and

Cu(II), well-known redox-active metal ions associated to AD.

2.4. Biological activity

2.4.1. AChE inhibition

Inhibition of TcAChE by the new set of hybrids (13–24) was evaluated

by using a previously described method²⁹, based on an adaptation of

the Ellman’s test³². The standard reference compound selected for

these assays was tacrine (IC₅₀¼ 0.35 mM)¹⁴, which was also evaluated

under our experimental conditions. The IC₅₀values obtained for

AChE inhibition (AChEi) are depicted in Table 2. All the studied

Figure 3. Species distribution curves for compound 21 along with molar extinc-

tion coefficients at the maximum absorption wavelengths (C_L¼ 4.0ꢃ 1 0 ^ꢁ5M).

Table 1. Stepwise protonation constants for the ligand 21 and global formation

constants^afor the corresponding Fe(III) and Cu(II)) complexes (T ¼ 25.0 0.1 ^ꢂC,

I ¼ 0.1 M KCl, 25% w/w DMSO/water) as well as pM^bvalues.

log

K_i

log

b_Fe

log

b_Cu

Compound

M_mH_hL_l

_mH_hL_l

(011)

(021)

(101)

(111)

(102)

(112)

(103)

(1–11)

(1–22)

pM

9.01(1)

7.99(3)

–

10.87(6)

19.02(2)

20.68(8)

–

22.90(6)

–

38.37(6)

36.95(7)

–

12.21(8)

22.2

–

2.25(3)

-

11.9

21

(011)

(021)

(031)

(111)

(122)

(102)

pM

9.02(3)

8.18(5)

3.32(6)

–

17.27(5)

34.38(6)

20.14(8)

11.1

TAC-BIM1^c

^ab_M

¼ [M_mH_hL_l]/[M]^m[H]^h[L]^l.

^bpM^m^H¼^h^L^lꢁlog[M] at pH 7.4 (C_L/C_M¼ 10, C_M¼ 1 0 ^ꢁ6M).

Figure 4. Species distribution curves for the systems (a) Fe(III)/21 1:3 and

(b) Cu(II)/21 1:2 (C_L¼ 4.0ꢃ 1 0 ^ꢁ5M).

^cIn 50/50 w/w DMSO/water³¹

.

216

G. FANCELLU ET AL.

compounds showed good inhibitory activity, with IC₅₀values in sub-

Graph A illustrates the effect of the linker length on the enzyme

micromolar range, close or even inferior to that of the standard refer- inhibitory capacity. Remarkably, the compounds with propylic

ence. A brief structure-activity relationship analysis was performed, to chains evidenced higher AChE inhibitory activity than the butylic

aid the comparison and understanding of the contribution of differ- analogues, although the molecular modelling only indicated

ent structural variations in the inhibitory activity, namely the length a subtle better accommodation of the shorter length inhibitors

of the spacer, as well as the presence of substituent groups in tacrine inside the enzyme active site. The compounds with longer linker

(H, Cl) and in benzofuran (-H, -OCH₃, -OH), as illustrated on the graph- may be forced to some structural distortions, namely in their inter-

ics (A, B, C) of Figure 5.

actions inside the lipophilic channel between CAS and PAS and

Table 2. Summary of results for the biological assays of TAC-BF hybrids

Comp.

Ab aggregation

inhibition^b,c(%)

AChE

inhibition^a

Self-Ab

Cu-ind

n

R

R₁

IC₅₀(mM)

aggr

Ab aggr

13

14

15

16

17

18

19

20

21

22

23

24

TAC

1

2

1

2

1

2

1

2

1

2

1

2

–

H

Cl

H

Cl

H

Cl

–

H

OCH₃

OH

–

0.58

0.72

0.12

0.34

0.36

0.76

0.13

0.67

0.81

0.98

0.13

0.61

0.35

60.6

51.5

51.0

43.0

57.5

51.9

57.8

64.4

44.0

30.4

31.5

43.4

20

–

50.1

54.0

52.5

53.4

–

^aThe values are mean of five independent experiments SD; AChE from Electric eel.

^bInhibition of self-mediated Ab₄₂aggregation (%) with or without copper (40 mM). The thioflavin-T fluorescence method was used, and the

measurements were carried out in the presence of an inhibitor (80 mM).

^cThe values are the mean of two independent measurements in duplicate (SEM < 10%).

1.2

1

1.2

1

(A)

(B)

0.8

0.6

0.4

0.2

0

0.8

0.6

0.4

0.2

0

17 19

13 15

18 20

14 16

21 23

22 24

17 18

13 14

19 20

15 16

21 22

23 24

R = H R = Cl

Propylic Chain

Butylic Chain

(C)

(D)

70

1.2

1

20

17

19

60

50

40

30

20

10

0

22

18

24

21

23

21

24

0.8

0.6

0.4

0.2

0

23

22

1

2

R=Cl, n=1

3

4

R=H, n=1

R=H, n=2

aggregation; R₁= OCH₃

aggregation; R = OH

R=Cl, n=2

13 17 21

R₁= - H

14 18 22

R₁= -OCH₃

15 19 23

16 20 24

self-A

β

R₁= -OH

Cu-induced aggregation; R = OH

Figure 5. Graphical summary of the effect of different structural parameters on the inhibition of AChE (AChEi, IC₅₀) and on the inhibition of Ab aggregation (%): (A)

size of alkylchain linker in AChEi: n ¼ 1 (propylic chain, n ¼ 2 (butylic chain); (B) substituents at C6 of TAC (R ¼ H, Cl) in AChEi; (C) substituents at C7 of BF (R₁¼ H,

-OCH₃, -OH) in AChEi; (D) inhibition of Ab aggregation (self- and Cu-induced).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

217

even in the stacking interaction of BF with PAS. Interestingly, identi- studied compounds. The results, expressed as percentage of aggre-

cal chain length effects were also found in other tacrine hybrids¹⁶

gation inhibition, are summarised in Table 2 and graphically pre-

Graph B illustrates the effect of chlorine substitution in TAC, which sented in Figure 5(D), including the values obtained for Tacrine

.

(20% of inhibition) as standard reference for comparison purpose.

They showed that all compounds can inhibit the Ab aggregation

with good-to-moderate activity and improved as compared with

TAC. Their activity and their dependence on ligand structural features

may be rationalised by different intercalation into the fibrils, as

already postulated and reported for other inhibitors^14,37. Among the

compounds in study, 20 exhibited the highest inhibitory activity in

both kinds of induced Ab aggregation, while others (ex. 17, 18, 19)

present also very good activity for self-mediated Ab aggregation, but

they are expected to have moderate activity for Cu^2þ-induced Ab

aggregation due to absence of the OH group. Interestingly, the

hydroxyl substituent (21–24) seems to lead to a general inhibitory

activity decrease of the self-mediated Ab aggregation (as compared

with the non-substituted or the metoxy-substituted analogues), while

the inhibitory capacity towards the Cu^2þ-induced Ab aggregation

increased, as compared with conditions of metal absence. This may

be rationalised on the basis of their copper chelating capacity, as pre-

led to improvement of the inhibitory activity of the hybrids, consist-

ently with other results previously described^14,22, although the

major effect was shown for those with the propylic chain and OH

substituent (cf. 21 and 23). Graph C indicates that the BF substitu-

ents (-OCH₃or –OH) do not lead to activity improvement. Overall,

the best AChEi activity was achieved for hybrids 15, 19 and 23 with

propylic chain linkers and, except 19, as well as chloro substitution

on TAC and no substitution on the BF moiety.

Although not measured under the framework of this paper,

the inhibition of butylcholinesterase (BuChE) and the selectivity of

both cholinesterases could be also of interest to be pursued in

future evaluations.

2.4.2. Inhibition of self- and Cu^2þ-induced Ab_1-42aggregation

The Ab amyloid plaques are one of the main hallmarks of AD and

they result from over production and aggregation of b-amyloid pep-

tide, which can accumulate outside the neurons, by self-mediated

or Cu^2þ-induced process, interfering with the synaptic impairment,

neurotransmission and memory³³. Therefore, anti-AD drugs have

been designed aimed to disrupt and disaggregate the Ab’s fibrils

into monomers, obtaining non-toxic forms of b-secretase’s prod-

ucts³⁴. To assess the effect of these new hybrids in the inhibition of

amyloid peptide aggregation, a selection of the compounds was

in vitro assayed, based on the thioflavin T (ThT) method³⁵. Indeed

ThT is a histochemical dye able to create ionic or hydrophobic inter-

actions with the peptide b–sheets, the predominant secondary

structure of the amyloid fibrils in aqueous solutions³⁶. This binding

interactions can be monitored by fluorimetry since the presence of

ThT-fibrils increases the absorbance and the emission of the ThT

dye, and induces red shifts on the absorbance (from 385 to 446 nm)

viously shown by other metal chelating compounds^14,38

.

Furthermore, despite some exceptions (e.g. 20 and 24), the increase

in linker length and the chloro TAC substitution induce some small

negative effects in the inhibition of Ab self-aggregation.

Nevertheless, since quantitative analysis based on fluorescence

of copper(II) containing solutions may result in somehow errone-

ous conclusions due to some potential emission quenching by

this paramagnetic ion, a fluorescence-independent study by trans-

mission electron microscopy (TEM) was also performed for com-

pound 21. The TEM images depicted in Figure 6 show the

presence of heavily intertwined aggregates, for Ab₄₂alone, while

in the case of its co-incubation with 21 the aggregates became

sparser and more elongated, thus suggesting that for this TAC-BF

hybrid the Ab self-induced aggregation maybe be mainly attrib-

and emission peaks (from 445 to 485 nm)¹⁴. All the measurements uted to the ligand intercalation between b-sheets of Ab fibrils.

were performed after incubation of the self-mediated and Cu^2þ

The presence of copper induced changes in the morphology of

induced Ab aggregates in the presence/absence of the herein the Ab aggregates, both in the absence and in the presence of

-

Metal-free

CuCl₂

Ab

Ab+21

Figure 6. TEM images of Ab aggregation inhibition performed with samples incubated (37^ꢂC) for 24 h. Experimental conditions: [Ab_1–42] ¼ [CuCl₂] ¼ 25lM;

[21] ¼ 50 lM; pH ¼ 6.6.

218

G. FANCELLU ET AL.

21, although in this last case there is a considerable change, calculated for clog P are close to the limit imposed by the used

apparently with the formation of homogenous low molecular range, warning for possible negative consequences on the absorp-

aggregates. Overall, TEM confirms the role of the ligand in the tion through the BBB due to their lipophilic components (pres-

inhibition of Ab aggregation.

ence of longer chain or absence of polar hydroxyl groups).

Furthermore, all the compounds evidenced good capacity to cross

the intestinal cell barrier for a good GUT absorption, though the

last four compounds have lower values, apparently due to the

presence of that hydrophilic group. Lastly, but not less important,

the predicted CNS activity, related to toxicological effects, indi-

cates they should be safe and without anticipated side effects.

2.4.3. Pharmacokinetic parameters

To assess the drug-likeness of these new drug candidates, some

relevant pharmacokinetic parameters were in silico calculated to

identify possible peripheral or central toxicity and to evaluate their

ability to cross membranes, namely the blood-brain barrier (BBB),

in case of drugs which main goal is the CNS. Thus, all the studied

compounds have been evaluated by QikProp V. 2.5 program³⁹, to

2.4.4. Cell viability and neuroprotection

gain prediction on some pharmacokinetic parameters, such as: the The neuroprotective effect of these new multi-target drugs

lipophilic character, (the octanol–water partition coefficient, clog designed for AD was evaluated using SH-SY5Y cells treated with

P), the capacity to cross the BBB (log BB), the velocity of intestinal Ab_1-42peptides or ascorbate/iron. For each compound we per-

absorption (caco-2 cell permeability), the activity in the CNS and formed a dose-response curve to select a non-toxic concentration

also the verification of Lipinski’s rule of five, to give an idea of (Figure 7).

potential future oral formulation as anti-AD agents (see Table 3).

AD is characterised by the extracellular accumulation of senile

Analysis of the results depicted in Table 3 shows that import- plaques composed of aggregated amyloid-beta peptide (Ab). Ab

ant parameters, such as molecular weight, clog P and log BB, are peptides are synthesised inside cells where they oligomerize and

perfectly inside the range determined by Lipinski’s rule to act in may react with active metal-ions such as copper, iron or zinc. The

the CNS, though it has been noticed that one parameter has been production of ROS conjugated with oligomeric Ab peptide is

violated except for 21, 22 and 23. In fact, most of the values involved in the neurodegenerative process of AD⁴⁰. We observed

Table 3. Summary of calculated pharmacokinetic molecular descriptors for the new hybrids by QikProp v.2.540³⁹

Caco-2

Permeability^d

(nm/sec)

Violations

of Lipinski’s

rule of 5^e

Comp.

Ref.

(n,R,R1)

(1,H,H)

(2,H,H)

(1,Cl,H)

(2,Cl,H)

(1,H,OCH₃)

(2,H,OCH₃)

(1,Cl,OCH₃)

(2,Cl,OCH₃)

(1,H,OH)

(2,Cl,OH)

(1,Cl,OH)

(2,Cl,OH)

MW^a

clog P^b

log BB^c

CNS activity^f

13

14

15

16

17

18

19

20

21

22

23

24

399.491

413.518

433.936

447.963

429.518

443.544

463.963

477.989

415.491

429.518

449.936

463.963

5.143

5.106

5.685

6.039

5.327

5.745

5.852

5.907

4.567

4.755

4.764

5.208

ꢁ0.690

ꢁ0.488

ꢁ0.396

ꢁ0.559

ꢁ0.573

ꢁ0.802

ꢁ0.431

ꢁ0.597

ꢁ1.522

ꢁ1.466

ꢁ1.261

ꢁ1.324

1764

2173

2149

1893

2423

1867

2324

1890

460

1

0

1

þ/ꢁ

–

þ/ꢁ

–

520

469

493

–

^aMW < 500.

b

ꢁ2 < clog P < ꢁ6.5.

c

ꢁ3.0 < log BB < 1.2.

^d<25 poor and >500 great.

^eMaximum is 4.

^f(–) inactive toxicologically – (þþ) very active toxicologically³⁹

.

Figure 7. Dose-response screening to select non-toxic concentrations of TAC-BF hybrids. Cells were treated with varying concentration of TAC-BF conjugates (from

10 lM to 40 lM) for 25 h and cell viability was determined using the MTT reduction assay. Results are expressed relatively to SH-SY5Y untreated cells, with the

ꢀ

ꢀꢀ

mean SEM derived from three different experiments. p < 0.05; p < 0.01, significantly different when compared with SH-SY5Y untreated cells.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

219

Figure 8. Neuroprotective effect of TAC-BF hybrids from Ab₄₂-induced toxicity on

SH-SY5Y cells. Cells were treated with Ab₄₂peptide (2.5 lM) for 24 h in the

absence or in the presence of the compounds (1 h pre-incubation þ 24 h co-incu-

bation). Evaluation of cell viability was performed using the MTT reduction assay.

Results are expressed relatively to SH-SY5Y untreated cells, with the mean SEM

Figure 9. Neuroprotective effect of TAC-BF conjugates against L-Ascorbic Acid

(AscH(-))/Ferrous Sulphate (Fe) toxicity on SH-SY5Y cells. Cells were treated with

Asc/Fe (5 mM/500 lM, respectively) for 24 h, after treatment for 1 h in the

absence or in the presence of the compounds. Evaluation of cell viability was

performed by using MTT reduction assay. Results are expressed relatively to SH-

SY5Y untreated cells, with the mean SEM derived from 4 different experiments.

ꢀ

derived from 4 different experiments. p < 0.05, significantly different when com-

pared with SH-SY5Y untreated cells; #p < 0.05, significantly different when com-

pared with Ab₄₂treated SH-SY5Y cells. (compounds: 18, 19, 20 and 23 – 20 mM;

21, 24 – 35 mM).

ꢀꢀ

p < 0.01, significantly different when compared with SH-SY5Y untreated cells.

#p < 0.05, significantly different when compared with Asc/Fe treated SH-SY5Y

cells. (compounds: 18, 19, 20 and 23 – 20mM; 21, 24 – 35 mM).

that Ab₄₂decreased cellular viability and, interestingly, the

TAC–BF conjugates 19 and 23 prevented that Ab-induced cell tox-

icity (Figure 8).

intercalating ability of the compound inside the fibrils. The best

capacity for inhibition of Ab self-aggregation was presented by 20

followed by compounds 17, 18, 19. The OH-containing com-

pounds present a general decrease in the self-aggregation inhib-

ition although, in the presence of Cu^2þ, the inhibition is generally

increased attributed to metal chelating effect. Furthermore com-

pounds 19 and 23 presented the best achievements in terms of

cell neuroprotective effects. Overall, a set of TAC-BF hybrids pre-

sented very good properties for potential drugs against AD and

they deserve to be further explored for the challenging discovery

of new AD therapies.

Compound 19 showed ability to inhibit Ab-self-aggregation,

thus being expected to decrease the formation of oligomers and

ROS-dependent production. In addition, oxidative stress is an early

event in the course of AD neurodegenerative process and it is

associated with increased oxidation of proteins, lipids and nucleic

acids in AD hippocampus and cortex. These data correlates with

the elevated levels of Ab₄₀and Ab₄₂in the same brain regions⁴⁰.

To induce oxidative stress in our cellular model we used the pair

Asc/Fe, which showed a decrease in the viability of SHSY-5Y cells

(Figure 9). Nevertheless, among the new TAC-BF hybrids tested,

compound 19 appeared to best achieve cell protection from Asc/

Fe-induce oxidative stress, thus presenting the best radical scav-

enging properties in cell environment. Therefore, although in solu-

tion conditions the hydroxyl-containing compounds (21–24)

revealed higher radical scavenging capacity than the tested non-

hydroxyl compound (20), that feature was not paralleled in cell

environment. This may be due to the higher hydrophilic character

of these compounds (see Table 3). In fact, compound 21 evi-

denced the worst values calculated for BBB and Caco permeability

and presented also the lowest capacity for in cell rad-

ical scavenging.

4. Experimental part

4.1. Materials and equipment

Analytical grade reagents were purchased from Sigma-Aldrich and

Acros, and were used as supplied. Solvents were dried according

to standard methods⁴¹. The chemical reactions were monitored by

Thin Layer Chromatography (TLC) using alumina plates coated

with silica gel 60 F254 (Macherey-Nagel). Column chromatography

purifications were performed on silica gel (Merck 230–400 mesh

(Geduran Si 60). The melting points (mp) were measured with a

Leica Galen III hot stage apparatus and are uncorrected. The

Proton and Carbon-13 NMR were recorded either on Bruker

AVANCE III-300 (300 MHz and 75.5 MHz) or Bruker AVANCE III-400

(400 MHz and 100.5 MHz) NMR spectrometers, at 25 ^ꢂC. Chemical

shifts (d) are reported in ppm from the standard internal reference

tetramethylsilane (TMS) and the coupling constant values (J) are

3. Conclusion

A new set of tacrine-benzofuran hybrids was designed, synthes-

ised and studied through a multidisciplinary approach. These new

compounds evidenced very good capacity for inhibition of AChE

(submicromolar range), with the best activity found for com-

pounds 15, 19, 23, with IC₅₀values much lower than that of tac-

rine (TAC). Therefore, according to the design aims, the extra-

functional benzofuran (BF) moiety enabled the hybrids with dual-

binding capacity in their interaction with the active binding sites

(CAS and PAS) of the enzyme. Structure activity relationships, indi-

cate that the best activity corresponds to compounds with a

shorter linker (propylic chain) and chloride substituent at C6 pos-

ition of TAC; the substituents at the C7 of BF did not evidence

considerably systematic effects in the inhibitory activity, but the

–OH causes a general small decrease. Regarding the inhibition of

1

determined in Hertz. In H NMR description, the following abbrevi-

ations are used: s ¼ singlet, d ¼ doublet, t ¼ triplet, m ¼ multiplet,

dt ¼ double triplet, dd ¼ double doublet, td ¼ triple doublet, bs

¼ broad singlet, bd ¼ broad doublet. Mass spectra (ESI-MS) were

performed on a 500 MS LC Ion Trap mass spectrometer (Varian

Inc., Palo Alto, CA, USA) equipped with an ESI ion source, oper-

ated in the positive ion mode. High-resolution mass spectra

(HRMS) were obtained on a Bruker Impact II quadrupole mass

spectrometer (Bruker Daltoniks).

For the metal complexation studies, the aqueous iron (FeCl₃,

Ab aggregation, the activity seems to be mainly related to the 0.0177 M) and copper (CuCl₂, 0.015 M) stock solutions were

220

G. FANCELLU ET AL.

prepared from 1000 ppm standards (Titrisol) and their metal con- 4.2.4. Ethyl 7-methoxybenzofuran-2-carboxylate (8)

1

Yield 75%, mp 56 ꢁ 57 ^ꢂC; H NMR (CDCl₃, 300 MHz) d: 7.51 (s, 1H,

tent was evaluated by atomic absorption. To prevent hydrolysis,

the iron stock solution was prepared in excess of acid chloride

and its exact concentration in HCl determined by the usual stand-

ard-addition method using 0.1 M HCl (Titrisol). The 0.1 M HCl solu-

H-3), 7.18 ꢁ 7.25 (m, 2H, H-4 and H-5), 7.10 (d, 1H, J ¼ 7.8 Hz, H-6),

4.34 (q, 2H, J ¼ 7.2 Hz, CH₂CH₃), 3.94 (s, 3H, OCH₃), 1.32 (t, 3H,

J ¼ 7.2 Hz, CH₃CH₂); ¹³C NMR (CDCl₃, 100 MHz) d: 159.34, 145.95,

145.93, 145.35, 128.57, 124.37, 114.52, 114.00, 108.86, 61.38, 56.00,

tion, used in spectrophotometric titrations, was prepared from a

Titrisol ampoule while the titrant was from carbonate free com-

mercial concentrate (Titrisol, KOH 0.1 M ampoules). The KOH solu-

tion was standardised by titration with a solution of potassium

hydrogen phthalate and was discarded whenever the percentage

of carbonate, determined by Gran’s method⁴², was greater than

0.5% of the total amount of base.

The electronic spectra and the AChE inhibition assays were per-

formed in a 1-cm path length quartz cell with a Perkin Elmer

Lambda 35 spectrophotometer equipped with a temperature pro-

grammer PTP1 þ 1 Peltier System (T ¼ 25.0 0.1 ^ꢂC). The enzyme

AChE 500 U, extracted from Electrophorus electricus (electric eel),

was purchased from Sigma-Aldrich.

For the study of the b-amyloid (Ab) aggregation, the ThT fluor-

escence assay was performed using a Varian Cary Eclipse fluorim-

eter at the following wavelengths: excitation (446 nm) and

emission (485 nm). Amyloid b-peptide (1–42), Ab_1-42, was pur-

chased from GeneCust as a lyophilised powder stored at ꢁ20 ^ꢂC.

TEM assays were performed with a Hitachi H8100 transmission

electron microscope with a LaB6 filament (200 kV, 10000–20000 ꢃ

magnification) at MicroLab/IST.

14.30; m/z (ESI-MS): 243.08 [M þ Na]^þ.

4.2.5. General procedure for synthesis of N-(aminoalkyl)benzo-

furan-2-carboxamide (9–12)

To the mixture of diaminoalkane (4 eq) in methanol, 1 M solution

of ethyl benzofuran-2-carboxylate (7, 8) in methanol was added

dropwise over a period of 5 h at room temperature. The reaction

mixture was stirred overnight and completion of reaction was

monitored on TLC. On completion, the reaction mixture was con-

centrated under reduced pressure and the residue so obtained

was diluted 4 M HCl solution (100 mL) under ice cold condition

and washed with DCM (3 ꢃ 25 mL). Finally the aqueous layer was

basified with saturated sodium hydroxide solution to 11–12 pH,

extracted with ethyl acetate and dried on sodium sulphate. The

evaporation of ethyl acetate under reduced pressure gave the

desired pure N-(aminoalkyl)benzofuran-2-carboxamides (9–12) as

light yellow solids in 64 ꢁ 71% yield.

4.2.6. N-(3-aminopropyl)benzofuran-2-carboxamide (9)

The title compound was synthesised from reaction of compound

8 with 1,3-diaminopropane. Yield 64%; mp 120–121 ^ꢂC. ¹H NMR

(400 MHz, MeOD-d₄) d: 7.64 (d, 1H, J ¼ 8.0 Hz, H-4), 7.52 (d, 1H,

J ¼ 8.0 Hz, H-7), 7.44 (s, 1H, H-3), 7.39 (t, 1H, J ¼ 8.0 Hz, H-6), 7.25

(t, 1H, J ¼ 8.0 Hz, H-5), 3.48 (t, 2H, J ¼ 8.0 Hz, CH₂NHCO), 2.72 (t, 2H,

J ¼ 8.0 Hz, CH₂NH₂), 1.75–1.82 (m, 2H, CH₂CH₂CH₂); ¹³CNMR

(100 MHz, MeOD-d₄) d: 159.89, 154.93, 148.67, 127.39, 126.74,

123.47, 122.34, 111.38, 109.76, 38.42, 36.39, 32.09; m/z (ESI-MS):

219.26 [M þ H]^þ.

4.2. Synthesis of the compounds

The schematic representation and the synthetic steps involved in

the preparation of all TAC-BF hybrids and their intermediates are

shown in Scheme 1.

4.2.1. General procedure for synthesis of 9-chloro-1,2,3,4-tetrahy-

droacridine (3 ꢁ 4)

The title compounds, 9-chloro-1,2,3,4-tetrahydroacridine (3) and

6,9-dichloro-1,2,3,4-tetrahydroacridine (4), were prepared from

commercially available anthranilic acids according to our previous

4.2.7. N-(4-aminobutyl)benzofuran-2-carboxamide (10)

The title compound was synthesised from reaction of compound

8 with 1,4-diaminobutane. Yield 69%; mp 94–95 ^ꢂC. ¹H NMR

(400 MHz, MeOD-d₄) d: 7.70 (d, 1H, J ¼ 8.0 Hz, H-4), 7.57 (d, 1H,

J ¼ 8.0 Hz, H-7), 7.42 ꢁ 7.47 (m, 2H, H-3 & H-6), 7.30 (t, 1H,

J ¼ 8.0 Hz, H-5), 3.44 (t, 2H, J ¼ 8.0 Hz, CH₂NHCO), 2.73 (t, 2H,

J ¼ 8.0 Hz, CH₂NH₂), 1.55–1.71 (m, 4H, CH₂CH₂CH₂CH₂); ¹³CNMR

(100 MHz, MeOD-d₄) d: 159.81, 154.98, 148.74, 127.42, 126.73,

123.48, 122.33, 111.36, 109.70, 40.52, 38.71, 28.92, 26.48; m/z (ESI-

MS): 233.37 [M þ H]^þ.

reported procedure^14,16

.

4.2.2. General procedure for synthesis of ethyl benzofuran-2-carb-

oxylate (7 ꢁ 8)

To a solution of substituted salicyaldehydes (72 mmol) and potas-

sium carbonate (10 g, 72 mmol) in DMF (100 mL) was added ethyl-

bromoacetate (12 g, 72 mmol). The reaction was refluxed in oil

bath for 12 h and after completion of reaction, it was poured in

cold water and the product was extracted with ethyl acetate¹⁹.

The crude product was purified by column chromatography using

1% ethyl acetate-petroleum ether as eluent to yield the desired

product (70 ꢁ 75%).

4.2.8. N-(3-aminopropyl)-7-methoxybenzofuran-2-carboxamide (11)

The title compound was synthesised from reaction of compound

8 with 1,3-diaminopropane. Yield 66%; mp 116 ꢁ 117 ^ꢂC. ¹H NMR

(400 MHz, MeOD-d₄) d: 7.43 (s, 1H, H-3), 7.17 ꢁ 7.24 (m, 2H, H-4 &

H-5), 6.96 (d, 1H, J ¼ 8.0 Hz, H-6), 3.98 (s, 1H, OCH₃) 3.50 (t, 2H,

J ¼ 8.0 Hz, CH₂NHCO), 2.75 (t, 2H, J ¼ 8.0 Hz, CH₂NH₂), 1.77 ꢁ 1.84

(m, 2H, CH₂CH₂CH₂); ¹³CNMR (100 MHz, MeOD-d₄) d: 159.82,

148.60, 145.66, 144.40, 128.93, 124.24, 114.09, 110.06, 108.33,

55.10, 38.34, 36.33, 31.96; m/z (ESI-MS): 249.45 [M þ H]^þ.

4.2.3. Ethyl benzofuran-2-carboxylate (7)

1

Yield 70%, mp 48 ꢁ 50 ^ꢂC; H NMR (CDCl₃, 300 MHz) d: 7.69 (d, 1H,

J ¼ 2.0 & 7.8 Hz, H-4), 7.60 (d, 1H, J ¼ 8.4 Hz, H-7), 7.54 (s, 1H, H-3),

7.43–7.48 (m, 1H, H-6), 7.26–7.33 (m, 1H, H-5), 4.45 (q, 2H,

J ¼ 7.2 Hz, CH₂CH₃), 1.44 (t, 3H, J ¼ 6.9 Hz, CH₃CH₂); ¹³C NMR

(CDCl₃, 100 MHz) d: 159.59, 155.67, 145.69, 127.53, 126.94, 123.73,

122.76, 113.76, 112.35, 61.50, 14.32; m/z (ESI-MS): 190.54 [M]^þ.

4.2.9. N-(4-aminobutyl)-7-methoxybenzofuran-2-carboxamide (12)

The title compound was synthesised from reaction of compound

8 with 1,4-diaminobutane. Yield 71%; mp 89 ꢁ 90 ^ꢂC. ¹H NMR

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

221

(400 MHz, MeOD-d₄) d: 7.44 (s, 1H, H-3), 7.18 ꢁ 7.25 (m, 2H, H-4 & 6⁰), 7.39 (s, 1H, H-3⁰), 7.27 ꢁ 7.32 (m, 2H, H-5⁰& H-7), 3.86 (t, 2H,

H-5), 6.97 (d, 1H, J ¼ 8.0 Hz, H-6), 3.98 (s, 1H, OCH₃) 3.43 (t, 2H, J ¼ 8.0 Hz, CH₂NH), 3.55 (t, 2H, J ¼ 8.0 Hz, CH₂NHCO), 2.87 (brs, 2H,

J ¼ 8.0 Hz, CH₂NHCO), 2.70 (t, 2H, J ¼ 8.0 Hz, CH₂NH₂), 1.54–1.69 (m, H-4), 2.66 (brs, 2H, H-1), 2.06–2.09 (m, 2H, CH₂CH₂CH₂)

2H, CH₂CH₂CH₂CH₂); ¹³CNMR (100 MHz, MeOD-d₄) d: 159.68, 1.85–1.86(m, 4H, H-2, H-3); ¹³CNMR (100 MHz, MeOD-d₄) d: 160.07,

148.70, 145.66, 144.39, 128.95, 124.26, 114.11, 110.03, 108.32, 154.90, 154.61, 153.49, 148.32, 141.69, 136.88, 127.26, 126.91,

55.12, 40.75, 38.82, 29.50, 26.51;; m/z (ESI-MS): 263.44 [M þ H]^þ.

126.24, 124.68, 123.56, 122.36, 120.28, 115.27, 113.20, 111.34,

109.89, 45.05, 36.15, 30.08, 29.61, 23.89, 21.78, 20.87; HRMS (ESI)

calcd for C₂₅H₂₅ClN₃O₂[M þ H]^þ, m/z 434.6482, found 434.6449.

4.2.10. General procedure for the synthesis of N-(4-((1,2,3,4-tetra-

hydroacridin-9-yl)amino)alkyl)benzofuran-2-carboxamide deriva-

tives (13 ꢁ 20)

4.2.14. N-(4-((6-chloro-1,2,3,4-tetrahydroacridin-9-yl)amino)butyl)-

benzofuran-2-carboxamide (16)

The mixture of 9-chloro-1,2,3,4-tetrahydroacridine (3, 4) (1 eq) and

N-(aminoalkyl)benzofuran-2-carboxamide (9 ꢁ 12) (1 eq) was

heated in phenol (0.5 eq) with the catalytic amount of potassium

iodide at 155 ꢁ 160 ^ꢂC for 35 ꢁ 60 min. The completion of reaction

was monitored on TLC. On completion, the dense oily reaction

mixture was sonicated with ethyl acetate. The precipitates so

obtained were filtered and washed with water and hexane to give

light yellow coloured N-(4-((1,2,3,4-tetrahydroacridin-9-yl)amino)-

butyl)benzofuran-2-carboxamide derivatives (13–20); the com-

pounds were purified either through crystallisation in

ethylacetate-hexane or through column chromatography over sil-

ica (0.5 ꢁ 1.0% MeOH-DCM) in 67 ꢁ 79% yield.

The title compound was synthesised from reaction of compounds

4 and 10. Yield 70%; mp 127 ꢁ 129 ^ꢂC. ¹H NMR (400 MHz, MeOD-

d₄) d: 8.32 (d, 1H, J ¼ 8.0 Hz, H-8), 7.72 (d, 1H, J ¼ 8.0 Hz, H-4⁰), 7.60

(s, 1H, H-5) 7.53 (d, 1H, J ¼ 8.0 Hz, H-7⁰), 7.45 ꢁ 7.49 (m, 2H, H-6⁰&

H-7), 7.39 (s, 1H, H-3⁰), 7.34 (t, 1H, J ¼ 8.0 Hz, H-5⁰), 3.95 (t, 2H,

J ¼ 8.0 Hz, CH₂NH), 3.45 (t, 2H, J ¼ 8.0 Hz, CH₂NHCO), 2.93 (brs, 2H,

H-4), 2.69 (brs, 2H, H-1), 1.90–1.93 (m, 6H, H-2, H-3 & NHCH₂CH₂)

1.77–1.81(m, 2H, CH₂CH₂CONH); ¹³CNMR (100 MHz, MeOD-d₄) d:

159.75, 155.41, 154.91, 152.37, 148.52, 140.66, 137.71, 127.32,

126.84, 125.03, 123.56, 122.35, 119.21, 114.92, 112.88, 111.31,

109.67, 38.47, 28.93, 27.32, 25.87, 23.52, 21.66, 20.67; HRMS (ESI)

calcd for C₂₆H₂₇ClN₃O₂[M þ H]^þ, m/z 448.6636, found 448.6677.

4.2.11.

N-(3-((1,2,3,4-tetrahydroacridin-9-yl)amino)propyl)benzo-

4.2.15. 7-Methoxy-N-(3-((1,2,3,4-tetrahydroacridin-9-yl)amino)pro-

pyl)benzofuran-2-carboxamide (17)

furan-2-carboxamide (13)

The title compound was synthesised from reaction of compounds

1

3 and 9. Yield 67%; mp 145 ꢁ 146 ^ꢂC. H NMR (400 MHz, MeOD-d₄)

The title compound was synthesised from the reaction of com-

1

pounds 3 and 11. Yield 75%; mp 137 ꢁ 138 ^ꢂC. H NMR (400 MHz,

d: 8.39 (d, 1H, J ¼ 8.0 Hz, H-8), 7.71 ꢁ 7.77 (m, 3H, H-5, H-6 & H-4⁰),

7.48ꢁ.57 (m, 3H, H-7, H-7⁰& H-6⁰⁰), 7.42 (s, 1H, H-3⁰), 7.34 ꢁ 7.36

(m, 1H, H-5⁰), 4.09 (brs, 2H, CH₂NH), 3.59 (brs, 2H, CH₂NHCO), 2.94

(brs, 2H, H-4), 2.75 (brs, 2H, H-1), 2.17 (brs, 2H, CH₂CH₂CH₂), 1.92

(brs, 4H, H-2 & H-3); ¹³CNMR (100 MHz, MeOD-d₄) d: 160.16,

156.69, 154.98, 150.31, 148.32, 138.33, 132.55, 127.32, 126.94,

124.89, 123.60, 122.39, 118.64, 115.66, 111.63, 111.37, 109.92,

44.98, 36.05, 29.94, 27.87, 23.55, 21.54, 20.34; HRMS (ESI) calcd for

C₂₅H₂₆N₃O₂[M þ H]^þ, m/z 400.2020, found 400.2034.

MeOD-d₄) d: 8.28 (d, 1H, J ¼ 8.0 Hz, H-8), 7.62 ꢁ 7.70 (m, 2H, H-5 &

H-6), 7.45 (t, 1H, J ¼ 8.0 Hz, H-7), 7.33 (s, 1H, H-3⁰) 7.19 ꢁ 7.20 (m,

2H, H-4⁰& H-5⁰), 6.96 ꢁ 6.97 (m, 1H, H-6⁰), 3.98 (t, 2H, J ¼ 8.0 Hz,

CH₂NH), 3.94 (s, 3H, OCH₃), 3.52–3.53 (m, 2H, CH₂NHCO), 2.85 (brs,

2H, H-4), 2.66 (brs, 2H, H-1), 2.08–2.13 (m, 2H, NHCH₂CH₂) 1.84

(brs, 4H, H-2 & H-3); ¹³C NMR (100 MHz, MeOD-d₄) d: 159.98,

156.02, 150.85, 148.32, 145.67, 144.40, 138.99, 132.08, 128.84,

124.72, 124.37, 119.34, 115.94, 114.08, 111.84, 110.17, 108.43,

55.07, 45.00, 36.15, 29.95, 28.27, 23.64, 21.60, 20.47; HRMS (ESI)

calcd for C₂₆H₂₈N₃O₃[M þ H]^þ, m/z 430.2135, found 430.2142.

4.2.12. N-(4-((1,2,3,4-tetrahydroacridin-9-yl)amino)butyl)benzofuran-

2-carboxamide (14)

4.2.16. 7-Methoxy-N-(4-((1,2,3,4-tetrahydroacridin-9-yl)amino)butyl)-

benzofuran-2-carboxamide (18)

The title compound was synthesised from reaction of compounds

3 and 10. Yield 72%; mp 117 ꢁ 118 ^ꢂC. ¹H NMR (400 MHz, MeOD-

d₄) d: 8.30 (d, 1H, J ¼ 8.0 Hz, H-8), 7.69 (t, 1H, J ¼ 8.0 Hz, H-6),

7.60 ꢁ 7.63 (m, 2H, H-5 & H-4⁰), 7.45 ꢁ 7.49 (m, 2H, H-7 & H-7⁰),

7.39 (t, 1H, J ¼ 8.0 Hz, H-6⁰), 7.32 (s, 1H, H-3⁰), 7.25 (t, 1H, J ¼ 8.0 Hz,

H-5⁰), 3.96 (t, 2H, J ¼ 8.0 Hz, CH₂NH), 3.42 (t, 2H, J ¼ 8.0 Hz,

CH₂NHCO), 2.89 (brs, 2H, H-4), 2.62 (brs, 2H, H-1), 1.87 (brs, 6H, H-

2, H-3 & NHCH₂CH₂) 1.73 ꢁ 1.77(m, 2H, CH₂CH₂CONH); ¹³CNMR

(100 MHz, MeOD-d₄) d: 159.73, 156.28, 154.86, 150.30, 148.52,

138.37, 132.43, 127.29, 126.79, 125.00, 124.88, 123.52, 122.30,

118.79, 115.72, 111.60, 111.34, 109.65, 38.48, 27.98, 27.42, 25.99,

The title compound was synthesised from the reaction com-

1

pounds 3 and 12. Yield 79%; mp 111 ꢁ 113 ^ꢂC. H NMR (400 MHz,

MeOD-d₄) d: 8.23 (d, 1H, J ¼ 8.0 Hz, H-8), 7.62 ꢁ 7.68 (m, 2H, H-5 &

H-6), 7.43 (t, 1H, J ¼ 8.0 Hz, H-7), 7.37 (s, 1H, H-3⁰) 7.24 ꢁ 7.25 (m,

2H, H-4⁰& H-5⁰), 7.02 (d, 1H, J ¼ 8.0 Hz, H-6⁰), 3.99 (s, 3H, OCH₃),

3.80 (t, 2H, J ¼ 8.0 Hz, CH₂NH), 3.43 (t, 2H, J ¼ 8.0 Hz, CH₂NHCO),

2.93 (brs, 2H, H-4), 2.71 (brs, 2H, H-1), 1.90 (brs, 4H, H-2 & H-3),

1.73–1.86 (m, 4H, NHCH₂CH₂CH₂); ¹³C NMR (100 MHz, MeOD-d₄) d:

159.66, 154.03, 148.57, 145.71, 144.42, 142.40, 130.36, 128.93,

23.47, 21.57, 20.40; HRMS (ESI) calcd for C₂₆H₂₈N₃O₂[M þ H]^þ, m/z 124.30, 124.12, 123.95, 117.86, 114.09, 113.62, 109.99, 108.37,

414.2176, found 414.2147.

55.05, 38.53, 30.33, 27.77, 26.15, 24.06, 22.10, 21.30; HRMS (ESI)

calcd for C₂₇H₃₀N₃O₃[M þ H]^þ, m/z 444.2282, found 444.2260.

þ

4.2.13. N-(3-((6-chloro-1,2,3,4-tetrahydroacridin-9-yl)amino)propyl)-

benzofuran-2-carboxamide (15)

4.2.17. N-(3-((6-chloro-1,2,3,4-tetrahydroacridin-9-yl)amino)propyl)-

The title compound was synthesised from reaction of compounds 7-methoxybenzofuran-2-carboxamide (19)

4 and 9). Yield 73%; mp 124 ꢁ 125 ^ꢂC. ¹H NMR (400 MHz, MeOD- The title compound was synthesised from the reaction of com-

1

d₄) d: 8.17 (d, 1H, J ¼ 8.0 Hz, H-8), 7.67 (d, 1H, J ¼ 8.0 Hz, H-4⁰), 7.60 pounds 4 and 11. Yield 70%; mp 157 ꢁ 158 ^ꢂC. H NMR (400 MHz,

(s, 1H, H-5) 7.52 (d, 1H, J ¼ 8.0 Hz, H-7⁰), 7.43 (t, 1H, J ¼ 8.0 Hz, H- MeOD-d₄) d: 8.35 (d, 1H, J ¼ 8.0 Hz, H-8), 7.62 (s, 1H, H-5), 7.48 (d,

222

G. FANCELLU ET AL.

1H, J ¼ 8.0 Hz, H-4⁰), 7.35 (s, 1H, H-3⁰), 7.27 (brs, 2H, H-7 & H-5⁰), H-6⁰), 3.80 (t, 2H, J ¼ 8.0 Hz, CH₂NH), 3.43 (t, 2H, J ¼ 8.0 Hz,

7.05 (m, 1H, H-6⁰), 4.08 (brs, 2H, CH₂NH), 4.01 (s, 3H, OCH₃), 3.58 CH₂NHCO), 2.93 (brs, 2H, H-4), 2.71 (brs, 2H, H-1), 1.90 (brs, 4H, H-

2 & H-3), 1.73–1.86 (m, 4H, NHCH₂CH₂CH₂); ¹³C NMR (100 MHz,

(brs, 2H, CH₂NHCO), 2.87 (brs, 2H, H-4), 2.71 (brs, 2H, H-1), 2.17 (m,

2H, NHCH₂CH₂) 1.90 (brs, 4H, H-2 & H-3); ¹³CNMR (100 MHz,

MeOD-d₄) d: 159.66, 154.03, 148.57, 145.71, 144.42, 142.40, 130.36,

128.93, 124.30, 124.12, 123.95, 117.86, 114.09, 113.62, 109.99,

55.05, 38.53, 30.33, 27.77, 26.15, 24.06, 22.10, 21.30. HRMS (ESI)

calcd for C₂₆H₂₇N₃O₃[M þ H]^þ, m/z 429.2058, found 429.2083.

MeOD-d₄) d: 159.87, 156.56, 150.68, 148.24, 145.68, 144.39, 139.00,

138.55, 128.81, 127.11, 125.28, 124.44, 117.61, 114.11, 113.99,

112.09, 110.18, 108.52, 55.06, 45.18, 36.02, 29.52, 27.84, 23.40,

21.38, 20.22; HRMS (ESI) calcd for C₂₆H₂₇ClN₃O₃[M þ H]^þ, m/z

464.6577, found 464.6565.

4.2.22. N-(3-((6-chloro-1,2,3,4-tetrahydroacridin-9-yl)amino)propyl)-

7-hydroxybenzofuran-2-carboxamide (23)

4.2.18. N-(4-((6-chloro-1,2,3,4-tetrahydroacridin-9-yl)amino)butyl)-

7-methoxybenzofuran-2-carboxamide (20)

The title compound was synthesised from compound 19. Yield

50%; mp 85 ꢁ 87 ^ꢂC. ¹H NMR (400 MHz, MeOD-d₄) d: 8.35 (d, 1H,

J ¼ 8.0 Hz, H-8), 7.62 (s, 1H, H-5), 7.48 (d, 1H, J ¼ 8.0 Hz, H-4⁰), 7.35

(s, 1H, H-3⁰), 7.27 (brs, 2H, H-7 & H-5⁰), 7.05 (m, 1H, H-6⁰), 4.08 (brs,

2H, CH₂NH), 4.01 (s, 3H, OCH₃), 3.58 (brs, 2H, CH₂NHCO), 2.87 (brs,

2H, H-4), 2.71 (brs, 2H, H-1), 2.17 (m, 2H, NHCH₂CH₂) 1.90 (brs, 4H,

H-2 & H-3); ¹³C NMR (100 MHz, MeOD-d₄) d: 159.87, 156.56, 150.68,

148.24, 145.68, 144.39, 139.00, 138.55, 128.81, 127.11, 125.28,

124.44, 117.61, 114.11, 113.99, 112.09, 110.18, 55.06, 45.18, 36.02,

29.52, 27.84, 23.40, 21.38, 20.22. HRMS (ESI) calcd for C₂₅H₂₅ClN₃O₃

[M þ H]^þ, m/z 450.6442, found 450.6450.

The title compound was synthesised from the reaction of com-

1

pounds 4 and 12. Yield 79%; mp 245 ꢁ 247 ^ꢂC. H NMR (400 MHz,

MeOD-d₄) d: 8.39 (d, 1H, J ¼ 8.0 Hz, H-8), 7.51 ꢁ 7.52 (m, 2H, H-5 &

H-4⁰), 7.33 (s, 1H, H-3⁰), 7.28 ꢁ 7.29 (m, 2H, H-7 & H-5⁰), 7.05 ꢁ 7.07

(m, 1H, H-6⁰), 4.05 (t, 2H, J ¼ 8.0 Hz, CH₂NH), 4.01 (s, 3H, OCH₃),

3.45 (t, 2H, J ¼ 8.0 Hz, CH₂NHCO), 2.91 (brs, 2H, H-4), 2.69 (brs, 2H,

H-1), 1.96–1.97 (m, 6H, NHCH₂CH₂, H-2

& H-3), (m, 2H,

CH₂CH₂NHCO); ¹³CNMR (100 MHz, MeOD-d₄) d: 159.55, 156.17,

150.66, 148.45, 145.66, 144.32, 140.16, 138.43, 128.84, 127.27,

125.30, 124.40, 117.85, 114.08, 112.39, 109.93, 108.42, 55.04, 38.49,

28.11, 27.09, 25.45, 23.24, 21.45, 20.35; HRMS (ESI) calcd for

C₂₇H₂₉ClN₃O₃[M þ H]^þ, m/z 478.6745, found 478.6734.

4.2.23. N-(4-((6-chloro-1,2,3,4-tetrahydroacridin-9-yl)amino)butyl)-

7-hydroxybenzofuran-2-carboxamide (24)

The title compound was synthesised from compound 20. Yield

1

66%; mp 100 ꢁ 101 ^ꢂC; H NMR (400 MHz, MeOD-d₄) d: 8.39 (d, 1H,

4.2.19. General procedure for the deprotection of the hydroxy

group (21–24)

J ¼ 8.0 Hz, H-8), 7.51 ꢁ 7.52 (m, 2H, H-5 & H-4⁰), 7.33 (s, 1H, H-3⁰),

7.28 ꢁ 7.29 (m, 2H, H-7 & H-5⁰), 7.05 ꢁ 7.07 (m, 1H, H-6⁰), 4.05 (t,

2H, J ¼ 8.0 Hz, CH₂NH), 3.45 (t, 2H, J ¼ 8.0 Hz, CH₂NHCO), 2.91 (brs,

2H, H-4), 2.69 (brs, 2H, H-1), 1.96–1.97 (m, 6H, NHCH₂CH₂, H-2 & H-

3), (m, 2H, CH₂CH₂NHCO); ¹³C NMR (100 MHz, MeOD-d₄) d: 159.55,

156.17, 150.66, 148.45, 145.66, 144.32, 140.16, 138.43, 128.84,

127.27, 125.30, 124.40, 117.85, 114.08, 112.39, 109.93, 55.04, 38.49,

28.11, 27.09, 25.45, 23.24, 21.45, 20.35. HRMS (ESI) calcd for

C₂₆H₂₆ClN₃O₃[M þ H]^þ, m/z, 464.6511, found 463.6498.

A mixture of 7-methoxy-N-(4-((1,2,3,4-tetrahydroacridin-9-yl)ami-

no)alkyl)-benzofuran-2-carboxamide (1 eq.) and n-Bu₄NI (3 eq.) in

dry CH₂Cl₂(15 mL) was stirred under N₂atmosphere and cooled

to ꢁ78 ^ꢂC. To this solution was dropwise added an excess of a

solution 1 M BCl₃in CH₂Cl₂(5 eq.). After ca 5 min, the solution

could warm to room temperature and was stirred for 2 h. The

reaction was quenched with ice water and it was left to stir for

30 min. A precipitate was formed and the product was recrystal-

lized from CH₃CN/Et₂O to afford the pure final compounds as a

pale yellow solids. Yield: 40 ꢁ 66%.

4.3. Molecular modelling

The interactions between ligands and proteins are of fundamental

importance in the modern structure–based drug design, and so

docking simulations with GOLD v. 5.1 program⁴³were performed.

The inhibitor models were docked inside the protein model of

Torpedo californica AChE (TcAChE), which was obtained from the

X-ray structure of its complex with an AChE inhibitor (N-4’-qui-

nolyl-N’-9’’-(1’’,2’’,3’’,4’’-tetrahydroacridinyl)-1,8-diaminooctane, herein

4.2.20. 7-Hydroxy-N-(3-((1,2,3,4-tetrahydroacridin-9-yl)amino)pro-

pyl)benzofuran-2-carboxamide (21)

The title compound was synthesised from compound 17. Yield

40%; mp 93 ꢁ 95 ^ꢂC. ¹H NMR (400 MHz, MeOD-d₄) d: 8.28 (d, 1H,

J ¼ 8.0 Hz, H-8), 7.62 ꢁ 7.70 (m, 2H, H-5 & H-6), 7.45 (t, 1H,

J ¼ 8.0 Hz, H-7), 7.33 (s, 1H, H-3⁰) 7.19–7.20 (m, 2H, H-4⁰& H-5⁰),

6.96–6.97 (m, 1H, H-6⁰), 3.98 (t, 2H, J ¼ 8.0 Hz, CH₂NH), 3.52–3.53 called original ligand), retrieved from the RCSB Protein Data Bank

(m, 2H, CH₂NHCO), 2.85 (brs, 2H, H-4), 2.66 (brs, 2H, H-1), (PDB entry 1ODC)²⁰. This structure was chosen due to the similar-

2.08–2.13 (m, 2H, NHCH₂CH₂) 1.84 (brs, 4H, H-2 & H-3); ¹³C NMR ity between this ligand and the herein studied ligands, namely

the TAC and the aromatic moiety linked by an alkylic chain, inter-

acting respectively with the catalytic active site (CAS) and the per-

ipheral anionic site (PAS). Structures from Homo sapiens were not

selected because they are complexed with smaller inhibitors. To

get the protein model, the original complex model was treated

with the programme MAESTRO v. 9.3⁴⁴by removal of the original

ligand, solvent and co-crystalized molecules, as well as by addition

of hydrogen atoms. This programme was also used to design the

ligand structures, which afterwards were optimised with software

GHEMICAL v. 2.0⁴⁵, through a random conformational search of

100 cycles and 2500 optimisation steps. To complete these simula-

(100 MHz, MeOD-d₄) d: 159.98, 156.02, 150.85, 148.32, 145.67,

144.40, 138.99, 132.08, 128.84, 124.72, 124.37, 119.34, 115.94,

114.08, 111.84, 110.17, 55.07, 45.00, 36.15, 29.95, 28.27, 23.64,

21.60, 20.47. HRMS (ESI) calcd for C₂₅H₂₅N₃O₃^þ[M þ H]^þ, m/z

415.1904, found 415.1951.

4.2.21. 7-Hydroxy-N-(4-((1,2,3,4-tetrahydroacridin-9-yl)amino)bu-

tyl)benzofuran-2-carboxamide (22)

The title compound was synthesised from 18. Yield 50%; mp

79 ꢁ 81 ^ꢂC. ¹H NMR (400 MHz, MeOD-d₄) d: 8.23 (d, 1H, J ¼ 8.0 Hz,

H-8), 7.62–7.68 (m, 2H, H-5 & H-6), 7.43 (t, 1H, J ¼ 8.0 Hz, H-7), 7.37 tions, the ligand models were docked inside the active site of the

(s, 1H, H-3⁰), 7.24–7.25 (m, 2H, H-4⁰& H-5⁰), 7.02 (d, 1H, J ¼ 8.0 Hz, AChE structure through the programme GOLD v. 5.1, using its

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

223

standard parameters and the Astex Statistical Potential (ASP) scor- were included in the fitting of experimental data towards the

ing function. The zone of interest of the docking was defined as

the residues within 10 Å from the original position of the ligand in

the crystal structure. The docking protocol used herein was vali-

dated, trough the re-docking of the co-crystallized ligand (original

ligand)²⁰into the respective AChE model, leading to a structure

with very good superimposition to that of reported X-ray crystal

structure, as recently reported.¹⁶

equilibrium models related to the Fe(III)/L and Cu(II)/L systems:

log b_FeH-2¼ -6.78, log b_FeH-3¼ -10.78; logb_{Cu H}¼ -9.94. The spe-

2

ꢁ2

cies distribution curves were obtained with the Hyss program⁴⁷.

4.5. Acetylcholinesterase inhibition

The enzymatic activity of AChE was determined using an adapta-

tion of the Ellman’s method, as described previously^29,32. Firstly, a

stock solution of AChE was prepared by dissolving enzyme 500 U

in 10 mL of tris(hydroxymethyl)aminomethane (TRIS) buffer

(50 mM, pH 8). Then, 4–(2-hydroxyethyl)-1-piperazineethanesul-

fonic acid (HEPES) buffer was used for further dilution of the

enzyme solution therefore obtaining the final working solution of

AChE. The assay solution consisted of 374 mL of HEPES buffer

(50 mM and pH 8.0), a variable volume (10–50 mL) of the com-

pound’s stock solution (1 mg/mL of MeOH), 476 mL of 3 mM bis(3-

carboxy-4-nitrophenyl) disulphide (DTNB), 25 mL of AChE stock

solution and the necessary amount of methanol to attain the

same volume of sample mixture in a 1 mL cuvette. After mixing,

samples were left to incubate for 15 min and then 75 mL of 16 mM

acetylthiocholine iodide (AChI) solution was added and immedi-

ately the reaction was monitored at 405 nm for 5 min. Firstly,

enzyme activity was determined by measuring the rate of enzym-

atic reaction in solution by recording absorbance (with or without

enzyme) at 405 nm. To calculate the percent inhibition of AChE,

the enzyme activity in the presence of increasing concentrations

of the test compound, as well as for a blank reaction (methanol

without compounds), were measured at 405 nm.

4.3.1. Prediction of pharmacokinetic properties

To get an insight on drug-likeness properties of these new

hybrids, in silico calculations of some pharmacokinetic descriptors

were performed by the QikProp program³⁹provided by MAESTRO.

Parameters such as the lipo-hydrophilic character (clog P), blood-

brain barrier partition coefficient (log BB), ability to be absorbed

through the intestinal tract (Caco-2 cell permeability) and CNS

activity were calculated. These predictions are for orally delivered

drugs and assume non active transport.

4.4. Physico-chemical properties

4.4.1. Anti-oxidant activity

The anti-oxidant capacity or, more precisely, radical scavenging

activity based on the electron and proton donation ability, was

evaluated by the DPPH method previously described^29,46. To a

2.5 mL solution of DPPH (0.002%) in methanol, solutions of each

compound were added in different volumes to obtain different

concentrations in a 3.5 mL final volume. The samples were incu-

bated for 30 min at room temperature. The absorbance was meas-

ured at 517 nm against the corresponding blank (methanol). The

antioxidant activity was calculated by Eq. (1)²⁹.

The obtained results were expressed in percentage of inhib-

ition through the Equation (2):

%1 ¼ 100ꢁðv₁=v₀ꢃ 100Þ

(2)

%AA ¼ ððA_DPPHꢁA_sampleÞ=A_DPPHÞ ꢃ 100

(1)

Where v₁is the initial reaction rate in the presence of the

inhibitor and v₀is the initial rate of the control reaction. The

The tests were carried out in triplicate. The compound concen-

tration providing 50% of antioxidant activity (EC₅₀) was obtained

by plotting the antioxidant activity against the compound curves of inhibition were obtained using a plot of percentage of

concentration.

enzymatic inhibition versus inhibitor concentration, and a calibra-

tion curve was obtained from which the linear regression parame-

ters were obtained.

4.4.2. Metal chelation studies

The metal-complexation studies of compound 21 were performed

by UV-Vis spectroscopic titration. Due to solubility reasons, these

equilibrium solution studies were accomplished in a mixed 25%

(w/w) DMSO/water medium, at T ¼ 25.0 0.1 ^ꢂC and ionic strength

(I) 0.1 M KCl. The glass and Ag/AgCl reference electrodes were pre-

viously conditioned in different DMSO/H₂O mixtures of increasing

DMSO % composition and the response of the glass electrode

was checked by strong acid – strong base (HCl/KOH) calibrations

and analysis of the respective Nernst parameters by Gran’s

method⁴⁷. For the spectrophotometric titrations (total volume

30 mL), the ligand concentration (C_L) was 4.0 ꢃ 1 0 ^ꢁ5M and C_M/C_L

ratios of 0:1, 1:1 (M ¼ Cu, Fe), 1:2 (M ¼ Cu) and 1:3 (M ¼ Fe) were

used. The spectrophotometric measurements were carried out in a

280–450 nm wavelength range at pH ca 2.4–10. The value deter-

mined for the water ionisation constant (pK_w) was 14.40. The step-

wise protonation constants of the ligand, K_i¼ [H_iL]/[H_i-1L][H]

(i ¼ 1–2), and the overall metal–complex stability constants,

4.6. Inhibition of self-mediated and Cu²¹induced Ab_1-42

aggregation

Among all the methods to measure the aggregation of Ab_1-42, the

assay based on Thioflavin T (ThT) is the standard procedure based

on the fluorescence of this dye^49,50. This test is performed with

Ab_1-42peptide previously prepared by dissolving it in

1,1,1,3,3,3–hexafluoro-2-propanol (HFIP), an organic solvent useful

to solubilise and monomerize the b–sheet protein aggregates and

reserve them in the fridge. Films are re-dissolved in an Eppendorf

tube with a fresh solution of a mixture of CH₃CN/Na₂CO₃/NaOH,

to have a stable stock solution, and afterwards, added to an opti-

mised fibrillation phosphate buffer¹⁴. The samples were prepared

using MeOH (1 mg/mL) as solvent, followed by incubation in a

water bath for 24 h at 37 ^ꢂC with gentle shaking. After incubation,

the samples were added to a 96-well plate (BD Falcon) with

180 mL of 5 mM ThT in 50 mM glycine-NaOH (pH 8.5) buffer. Blank

samples were prepared for each concentration in a similar way,

devoid of peptide. After 5-min incubation with the dye, the ThT

fluorescence was measured at 446 nm (excitation) and

b_M

¼ [M_mH_hL_l]/[M]^m[H]^h[L]^l, were calculated by fitting the spec-

_mH_hL_l

trophotometric data with PSEQUAD program⁴⁸. The Fe(III) and

Cu(II) hydrolysis models were determined under the defined

experimental conditions (I ¼ 0.1 M KCl, 25% w/w DMSO/H₂O,

T ¼ 25.0 0.1 ^ꢂC) and the following values of stability constants 485 nm (emission).

224

G. FANCELLU ET AL.

4.7. Transmission electron microscopy (TEM)

incubated at 37 ^ꢂC for 1 h 30 min protected from light. At the end

of the incubation period, the formazan precipitates were solubi-

lised with 0.5 mL of acidic isopropanol (0.04 M HCl/isopropanol).

The absorbance was measured at 570 nm⁵¹. Cell reduction ability

was expressed as a percentage of untreated control cells.

All data were expressed as mean SEM of at least three inde-

pendent experiments performed in duplicates. Statistical analyses

were performed using GraphPad Prism 5 (GraphPad Software,

San Diego, CA, USA). Differences between two datasets were eval-

uated by two-tailed unpaired Student’s t test A p-value <0.05 was

considered statistically significant.

The Transmission Electron Microscopy (TEM) is a high resolution

technique used to study biological or inorganic samples at

2–3 nm level, through black and white high-quality images. The

compound 21 was selected among those with better anti-Ab

aggregation assay. Imaging acquisition was performed with sam-

ples of the ligand mixed with Ab, in the absence or presence of

copper, prepared by following the single–droplet method in the

negative staining protocol. The Ab stock solutions were prepared

by dissolving the lyophilised peptide in a mixture of 48 mL of

CH₃CN, 10 mL of NH₄OH (2%) and 48 mL of NaCl (300 mM), followed

by addition of a buffered solution containing 4–(2-hydroxyethyl)-

1-piperazine-ethanesulfonic acid (HEPES, 50 mM, pH ¼ 6.6) to

obtain a total concentration of 50 mM. The ligands were treated

with amyloid-b peptide and prepared previously to be studied in

absence or in presence of copper chloride (25 mM) and everything

was followed by incubation for 24 h at 37 ^ꢂC. Formvar/Carbon

200-mesh Cu grids (Ted Pella) were treated with Ab peptide

aggregated samples (10 mL) for 2 min at rt. Excess sample solution

was removed using filter paper followed by washing twice with

deionised water. Each grid incubated with uranyl acetate (1%,

10 mL, 1 min) was stained and dried for 15 min at rt. The support

device used to hold the sample inside TEM was a copolymer of

polyvinyl acetate from polyvnyl alcohol and formaldehyde called

Formvar stabilised with a thin layer of carbon, using copper 200 –

mesh grids as a main support (purchased to Ted Pella).

Acknowledgements

~

^

The authors thank the Portuguese Fundac¸ao para a Ciencia e

Tecnologia (FCT), for the financial support of the projects UID/QUI/

00100/2013, UID/QUI/00100/2019, the postdoctoral fellowship

(KC), and also thank the Erasmus^þprogramme (GF).

Acknowledgements are also due to the Portuguese NMR (IST-UL

Center) and Mass Spectrometry Networks (Grant LISBOA-01–0145-

FEDER-022125).

Disclosure statement

No potential conflict of interest was reported by the authors.

ORCID

4.8. Cell viability and neuroprotection

Gaia Fancellu

Karam Chand

http://orcid.org/0000-0002-9705-3779

http://orcid.org/0000-0001-7691-4392

http://orcid.org/0000-0002-9000-0535

http://orcid.org/0000-0003-3398-2787

http://orcid.org/0000-0002-7980-5818

http://orcid.org/0000-0002-0612-2645

http://orcid.org/0000-0002-2199-0555

http://orcid.org/0000-0002-8554-4992

http://orcid.org/0000-0002-4069-9368

SH-SY5Y human neuroblastoma cell line (ATCC-CRL-2266) is grown

in Dulbecco’s modified Eagle’s medium (DMEM), and were

obtained from Gibco-Invitrogen (Life Technologies Ltd, UK) with

10% heat inactivated foetal calf serum, containing 50 U/mL penicil-

lin and 50 mg/mL streptomycin, under a humidified atmosphere of

95% air- 5% CO₂at 37 ^ꢂC. Cells were plated at 0.12 ꢃ 10⁶cells/mL

for cell viability assay. The tested compounds (18, 19, 20, 21, 23

and 24) were dissolved in DMSO at a concentration of 50 mM and

aliquots were stored at ꢁ20 ^ꢂC. We performed a dose-response

screening (from 10 mM to 40 mM) in order to choose the highest

non-toxic concentration. As a result we selected: 20 mM final con-

centrations for compounds 18, 19, 20 and 23; 20 mM final concen-

tration for compound 19; 35 mM final concentration for

compounds 21 and 24. The final concentration of DMSO in cul-

ture media did not exceed 0.05% (v/v) and no alterations on cells

were observed. The compounds were added to the cell media 1 h

before the incubation with Ab_1-42or L-ascorbic acid/ferrous sul-

phate. Ab_1-42or L-ascorbic acid/ferrous sulphate where incubated

alone or with the compound for an additional 24 h. Ab_1-42was

prepared as 276.9 mM stock in sterile water and added to the

medium at 2.5 mM final concentration. Ferrous sulphate was

freshly prepared as 0.36 M stock in water and added to the

medium at 500 mM final concentration. L-Ascorbic Acid was freshly

prepared as 80 mM stock in water and added to the medium at

5 mM final concentration. Ab_1-42was purchased from Bachem

(Torrance, CA, USA) and ferrous sulphate and L-ascorbic acid from

Sigma Chemical Co (St. Louis, MO, USA).

ꢀ

Daniel Tomas

Elisabetta Orlandini

Luca Piemontese

Diana F. Silva

Sandra M. Cardoso

ꢀ

Sılvia Chaves

ꢀ

M. Amelia Santos

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Article Doi

Novel tacrine–benzofuran hybrids as potential multi-target drug candidates for the treatment of Alzheimer’s Disease

DOI: 10.1080/14756366.2019.1689237

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Article abstract of DOI:10.1080/14756366.2019.1689237

Full text of DOI:10.1080/14756366.2019.1689237

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