Tacrine-allyl/propargylcysteine-benzothiazole trihybrids as potential anti-Alzheimer's drug candidates

Article abstract of DOI:10.1039/c6ra03455a

On continuing our research into new drug candidates for Alzheimer's disease (AD), we have designed, synthesized and evaluated a series of multifunctional trihybrid agents. The design strategy was based on the incorporation of a benzothiazole (BTA) moiety on a series of very recently reported bihybrids, resulting from the conjugation of a tacrine (TAC) with natural based moieties, namely S-allylcysteine (SAC) (garlic constituent) and S-propargylcysteine (SPRC). Thus, in addition to the acetylcholinesterase inhibition (AChEI) and anti-ROS capacity of the bihybrids (TAC-SAC/SPRC), the new trihybrids (TAC-SAC/SPRC-BTA) appeared endowed with a 5-fold capacity for inhibition of amyloid beta-peptide (Aβ) aggregation. The BTA moiety led also to considerable enhancement of the AChEI on the trihybrids, which molecular modeling suggested as being due to simultaneous binding to the catalytic active site and peripheral anionic site of AChE. The trihybrids were also assessed for MAO inhibition, but resulted in lower activity than expected, ascribed to the low accessibility of the propargyl groups to the enzyme active site. Finally, the effects of the compounds on the viability of neuroblastome cells stressed with Aβ₄₂ and H₂O₂ showed moderate cell protection. Overall, the performed studies illustrate the importance (and limitations) of enclosing several molecular scaffolds in one molecular entity to allow the modulation of multiple AD targets.

Full text of DOI:10.1039/c6ra03455a

View Article Online
View Journal
RSC Advances
This article can be cited before page numbers have been issued, to do this please use: A. Santos, A.
Hiremathad, K. Chand, A. R. Esteves, S. Cardoso, R. R. Ramsay, S. Chaves and R. S. Keri, RSC Adv., 2016,
DOI: 10.1039/C6RA03455A.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. This Accepted Manuscript will be replaced by the edited,
formatted and paginated article as soon as this is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/advances

Page 1 of 35
RSC Advances
View Article Online
DOI: 10.1039/C6RA03455A
Tacrineꢀallyl/propargylcysteineꢀbenzothiazole trihybrids as potential
antiꢀAlzheimer´s drug candidates
Asha Hiremathad,a,b Karam Chand,a A. Raquel Esteves,c Sandra M. Cardoso,c,d
Rona R. Ramsay,e Sílvia Chavesa, Rangappa S. Keri,b,* M. Amélia Santosa,*
a Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, Av.
Rovisco Pais 1, 1049ꢀ001 Lisboa, Portugal.
b Centre for Nano and Material Sciences, Jain University, Jain Global Campus,
Bangalore, Karnataka, 562112, India.
c
CNBC – Centro de Neurociências e Biologia Celular, Universidade de Coimbra,
Coimbra, Portugal.
d Faculdade de Medicina, Universidade de Coimbra, 3030 Coimbra, Portugal.
e
Biomedical Sciences Research Complex, University of St Andrews, Biomolecular
Sciences Building, North Haugh, St Andrews KY16 9ST, UK.
Keywords: Alzheimer´s disease; Trihybrid drugs; Tacrine; AntiꢀAβ aggregation; MAOꢀ
B; Antiꢀneurodegeneration
*
Corresponding Authors:
*For M.A.S.: phone, +351ꢀ218419273; email, masantos@ist.utl.pt
*For K.S.R.: phone, +91ꢀ8027577199; email, keriphd@gmail.com
1

RSC Advances
Page 2 of 35
View Article Online
DOI: 10.1039/C6RA03455A
Abstract
On continuing our research on new drug candidates for Alzheimer´s disease (AD), we
have designed, synthesized and evaluated a series of multifunctional trihybrid agents. The
design strategy was based on the incorporation of a benzothiazole (BTA) moiety on a
series of very recently reported bihybrids, resulting from the conjugation of a tacrine
(TAC) with natural based moieties, namely Sꢀallylcysteine (SAC) (garlic constituent) and
Sꢀpropargylcysteine (SPRC). Thus, in addition to the acetylcholinesterase inhibition
(AChEI) and antiꢀROS capacity of the bihybrids (TACꢀSAC/SPRC), the new trihybrids
(TACꢀSAC/SPRCꢀBTA) appeared endowed with 5ꢀfold capacity for inhibition of the
amyloid betaꢀpeptide (Aβ) aggregation. The BTA moiety led also to considerable
enhancement of the AChEI on the trihybrids, which molecular modeling suggested as
being due to the simultaneous binding to the catalytic active site and peripheral anionic
site of AChE. The trihybrids were also assessed for the MAO inhibition, but resulted in
lower activity than expected, ascribed to the low accessibility of the propargyl groups to
the enzyme active site. Finally, the effects of the compounds on the viability of
neuroblastome cells stressed with Aβ42 and H2O2 showed moderate cell protection.
Overall, the performed studies illustrate the importance (and limitations) of enclosing
several molecular scaffolds in one molecular entity to allow the modulation of multiple
AD targets.
1. Introduction
Alzheimer´s disease (AD) is one of the most devastating ageꢀrelated neurodegenerative
disorders with no cure so far, characterized by progressive memory loss and other
cognitive impairements. The increase of life expectancy of humans and the exponential
rise in the number of cases of AD support the need for developing an effective treatment
for this frontline health problem.1 Although its etiology has not been yet well understood,
the patient brains are characterized by βꢀamyloid (Aβ) deposits (plaques) around neurons
and neurofibrillary tangles (NFT) inside neurons,2 increased oxidative stress and
disruption of metal homeostasis.3 Other prominent features of AD are disturbances in
neurotransmitters, especially acetylcholine (cholinergic) deficit due to its hydrolysis by
2

Page 3 of 35
RSC Advances
View Article Online
DOI: 10.1039/C6RA03455A
acetylcholinesterase (AChE). Since around two decades ago, AD patients have been
mainly treated with inhibitors of AChE (AChEI), but with limited therapeutic success. In
fact they can provide amelioration of symptoms (improving patient memory) for a limited
time window and for mild situations, but cannot alter the course of the disease.4 The
multifactorial nature and the complexity of AD is believed to be the main reason for the
absence of effective drugs, and has lead to the emergence of multitargeting drugs. Under
this approach, one molecular entity is conveniently polyfunctionalized to enable its
simultaneous binding interaction with several of the most important targets, but mostly
focused on inhibition of AChE and Aβꢀaggregation and/or on the control of the brain
oxidative stress.5 Monoamine oxidase (MAO) has also been considered an important
target for the treatment of AD,6 since it catalyzes the oxidative deamination of the
monoamine based neurotransmitters, such as serotonine and dopamine, with the
concomitant production of H2O2 that can promote the Fenton reaction and formation of
reactive oxygen species (ROS).7
In this context, the development of multitargeting drugs for treatment of AD has been a
challenge for many research groups.8,9,10 Most of the multitarget drug design strategies
have been driven by the recognized importance of maintaining the cholinergic functions
(cholinergic hypothesis). Thus, they include a main molecular framework based on
approved antiꢀAD drugs (AChE inhibitors), which is modified (hybridized) with other
functional units to hit other disease targets. Among the USA FDA antiꢀAD drugs that are
AChEI (e.g. tacrine, donepezil, rivastigmine, and galanthamine),11 tacrine was the first
and most potent AChEI (discontinued due to side effects such as hepatotoxicity),12 with
superiority in the structural modification, thus making it a priviliged structure for
incorporation into the overall molecular skeletons of the multiꢀtargetꢀdirected drugs for
the treatment of AD.13 In fact, we can find tacrine hybrids containing fragments of
donepezil or galanthamine, while also a number of hybrids containing a structural
fragment derived from natural based products such as alkaloids, flavonoids or others.
An important part of our current research has also been a focus on the development of
acetylcholine esterase inhibitors functionalized for the inhibition/modulation of other AD
targets, namely Aβ aggregation, oxidative stress and redoxꢀactive metal ions.14,15,16 Very
recently, the conjugation of tacrine (TAC) with Sꢀallylcysteine (SAC) and Sꢀ
3

RSC Advances
Page 4 of 35
View Article Online
DOI: 10.1039/C6RA03455A
propargylcysteine (SPRC), two natural based products with neuroprotective and
antioxidant roles (e.g. garlic derivatives),17,18 resulted in a set of bihybrids (TACꢀSAC
and TACꢀSPRC) with important neuroprotecting properties which, besides the expected
AChEI and antiꢀoxidant properties, presented also inhibitory activity for the selfꢀ and Cuꢀ
induced Aβ aggregation.19,20 Interestingly, Nꢀpropargylamine is also an important
pharmacophore used in the design of MAO inhibitors.8,21 In this work we report the
design, synthesis and biological evaluation of a new set of trihybrids (see Fig. 1) which,
Insert here Fig 1
in addition to the functional groups presented in the above bihybids, include also a
benzothiazole (BTA) moiety to enhance the inhibition of Aβ aggregation, since BTA is a
derivative of thioflavine T (ThT), a well known Aβ dye widely used in the design of AD
drugs.14,22 Furthermore this new set of eight hybrids was designed by selection of suitable
linkers to enable a dual mode binding, via the tacrine and BTA moieties, with both active
binding sites of AChE. Besides the description of the design and synthetic procedures, we
include herein the results and discussion of the bioassays, namely the inhibition of AChE,
Aβ aggregation, MAOs and antiꢀoxidant properties, as well as their effect on the neuronal
cell viability upon being subjected to model stressors of AD.
2. Results and Discussions
2.1 Molecular design and modeling
The design of the new trihybrids, to be subsequently prepared and studied (Fig. 2),
involved a preliminary selection of the three molecular scaffolds, to address for main
biological symptoms associated with AD, and then docking simulations were carried out
to set the optimal length of the linker between the main frameworks.
Insert here Fig 2
The selection of the main molecular moieties was based on our previous works,
specifically: tacrine (TAC) and the natural based Sꢀallylcysteine (SAC) or Sꢀ
propargylcysteine (SPRC) to endow the hybrids with antiꢀAChE and antiꢀoxidant
properties;19 benzothiazole (BTA) to promote the interaction with Aβ and antiꢀAβ
aggregation properties; on the other hand, an adequate linkage of TAC with BTA to
warrant a dual binding mode within the active site of AChE and increase the enzymatic
4

Page 5 of 35
RSC Advances
View Article Online
DOI: 10.1039/C6RA03455A
inhibition.14 Based on previous studies, an alkylꢀamide linker could avoid linker strain
and the length was selected based on docking simulation of the ligand into the enzyme,
TcAChE. These studies also explained the interaction modes of the trihybrids with AChE.
Preliminairy docking results showed that three to five methylenes could provide the
linker with adequate length to enable the dual mode interaction with the two molecular
fragments (TAC, BTA) within the AChE gorge. A more detailed analysis of the
simulation results shows that the trihybrids could simultaneously bind to both the
catalytic anionic site (CAS) and the peripherical anionic site (PAS) (see representative
results of the docking studies in Fig 3).
Insert here Fig 3
The tacrine appears inserted in the botton of the enzyme gorge, adopting a parallel πꢀπ
stacking interaction with Trp84 and Phe330 of CAS, and blocking the accessibility of
substract and water to the active site (formed by the “catalytic triad” Ser200, His440 and
Glu327);23 The benzothiazole moiety occupies the PAS having a πꢀπ interaction with
Trp279 and Tyr70. The structure found for these hybridꢀenzyme complex models is quite
similar to the reported crystal structure of TcAChE complexed with a tacrineꢀquinoline
inhibitor (Nꢀ4'ꢀquinolylꢀN'ꢀ9"ꢀ(1",2",3",4"ꢀtetrahydroacridinyl)ꢀ1,8ꢀdiaminooctane; PDB
entry 1ODC),24 as shown for 10g (Fig. 3A). These simulations also indicated the capacity
of the linker amide group to establish Hꢀbond interactions between its NH and the
phenolic group of Tyr121. Interestingly, in opposition to the observed previously for the
corresponding bihybrids (TACꢀSAC and TACꢀSPRC),19 all the trihybrids could
accommodate the allyl and propargyl groups outwards the lipophylic part of PAS, which
is left free for the stronger interaction with the BTA moiety. Furthermore, these allyl and
propargyl groups do not seem to result in considerable differences on the ligandꢀenzyme
interaction (see Fig.3B for 10c and 10g). These simulations also illustrate that compounds
with longer chains (n = 5), as 10b, have the BTA moiety slightly shifted outwards the
PAS aromatic residues, which may result in somehow lower πꢀπ interaction with this site
than the shorter compounds (n = 3), as 10a (see Fig. 3C). Lastly, although 6ꢀchoroꢀtacrine
is known for its higher activity, as compared with the corresponding tacrine derivatives,
this was not clearly evidenced from the docking results, namely superimposition of 10c
and 10a (see Fig. 3D). Notably, this chloro atom (green coloured) appears to be well
5

RSC Advances
Page 6 of 35
View Article Online
DOI: 10.1039/C6RA03455A
accommodated in close distance of Hist440, which is thought to be the rationale for that
enhancement on inhibitory activity, namely due to an Hꢀbond interaction between its
carbonyl oxygen and the tacrine pyridinium group.25
Overall, the modeling studies give some support to the expected ability of the trihybrids
to interact with both the CAS and PAS of AChE, thus explaining some of the stronger
calculated inhibitory activities, as compared with the corresponding bihybrids. However,
since the binding modes found for these compounds were so similar to each other, their
relative potency as inhibitors would be difficult to predict with confidence.
2.2 Chemistry
The designed tacrineꢀSꢀallylcysteineꢀbenzothiazole (TACꢀSACꢀBTA) and tacrineꢀSꢀ
propargylcysteineꢀbenzothiazole (TACꢀSPRCꢀBTA) hybrids were synthesized according
to a convergent synthetic approach shown in Scheme 1. The 9ꢀchloroꢀ1,2,3,4ꢀ
tetrahydroacridine (3) was prepared from commercially available anthranilic acid as
previously reported.26,27 Amino alkyl acids were attached to the 3a and 3b, using a
catalytic amount of KI and phenol to obtain the intermediates 2ꢀ(1,2,3,4ꢀ
tetrahydroacridineꢀ9ꢀylamino) acids 4(aꢀd). The synthesis of the BTAꢀcysteine
intermediates 9(a,b) involved four steps: firstly, Lꢀcysteine (5) was Sꢀalkylated with allyl
bromide and propargyl bromide in the presence of NH4OH to yield the 3ꢀallylsulfanylꢀ2ꢀ
aminoꢀpropionic acid (6a) and 2ꢀaminoꢀ3ꢀpropꢀynylsulfanylꢀpropionic acid (6b),
respectively; their amino group was Bocꢀprotected by reaction with diꢀtertꢀbutyl
dicarbonate (Boc) to yield compounds 7(a,b); these compounds were coupled with a
BTA unit, through the condensation of the benzothiazoꢀ2ꢀamine (8) to their carboxylic
group (T3Pꢀactivated), and subsequently the Boc group was released from the amine
group with TFA to obtain the intermediates 9(a,b). Lastly the condensation of both
intermediate compounds 4(aꢀd) and 9(a,b) in the presence of T3P and NMM afforded the
ultimate novel hybrids 10(aꢀh).
Insert here Scheme 1
2.3. Biological activity
2.3.1. AChE inhibition
6

Page 7 of 35
RSC Advances
View Article Online
DOI: 10.1039/C6RA03455A
TcAChE enzymatic inhibition by the newly synthesized compounds 10(aꢀh) was
evaluated by adaptation of a method previously described.15 The IC50 values for AChE
inhibition are depicted in Table 1 (see also data plots in Fig. S9). These series of
compounds showed inhibitory activity in submicromolar range, even slightly better than
the standard drug (tacrine). Among those, as we expected, the compounds with the
chloroꢀsubstituted tacrine moiety showed higher inhibition (IC50 = 0.25ꢀ0.28 µM), with
no considerable difference between the propargyl/allyl cysteine substituents. Overall, the
compound with chlorotacrine and Sꢀpropargylcysteine groups (10g) showed highest
AChE inhibition with IC50 value of 0.25 µM, while the compound with allyl substitution
10d exhibited inhibition with IC50 value of 0.28 µM. Generally, the size of the linker
tested (n = 3, 5) seems to have a small effect on the inhibitory capacity of the compounds.
Overall, comparison of the AChE inhibitory activity obtained for this series of trihybrids
(TACꢀSAC/SPRCꢀBTA) with that obtained previously for the corresponding bihybrid
analogues (TACꢀSAC/SPRC, see Table S1 in Supplementary Data),19 showed that the
inclusion of the third moiety (BTA) resulted in a considerable enhancement (up to one
order of magnitude, with IC50 in subꢀꢁM range), attributable to its stronger dualꢀbinding
mode with both the CAS and PAS of AChE, especifically due to the strong BTAꢀPAS πꢀ
π interaction.
2.3.2. Inhibition of selfꢀmediated and Cu(II)ꢀinduced Aβ42 aggregation
To test the antiꢀamyloidogenic activity of these novel trihybrid compounds, our
experimental approach relied on in vitro assays with thioflavine T (ThT) to monitor and
quantify the aggregation of Aβ42 synthetic peptide into fibrils. ThT is a histochemical dye
that is known to bind to the peptide βꢀsheet conformation, which is the predominant
secondary structure of amyloidꢀbeta fibrils. The presence of fibrils can be monitored by
the fluorescence emission of ThT, with the absorption peak at 446 nm and the emission
peak at 485 nm.28,29,30
The inhibition studies were carried out upon incubating Aβ42 in the presence and absence
of the ligands. The results, summarized in Table 1, showed that all the compounds
induced a decrease of the ThT fluorescence associated with the Aβ fibril binding, thus
suggesting an inhibition of the Aβ42 selfꢀaggregation process. In particular, all of them
7

RSC Advances
Page 8 of 35
View Article Online
DOI: 10.1039/C6RA03455A
exhibited very goodꢀtoꢀmoderate aggregation inhibitoiry capacity. Compounds 10b and
10e showed very good inhibitory capacity (78.2% and 77.2%, respectively), while other
sets of compounds (10a, 10c, 10f) and (10d, 10g, 10h) exhibited, respectively, good and
moderate activities. Comparison between the antiꢀAβ aggregation capacities found for
these trihybrids (TACꢀSACꢀBTA and TACꢀSPRCꢀBTA) and the previously reported
values for corresponding bihybrids (TACꢀSAC and TACꢀSPRC, see Table S1)19 or
tacrine itself, showed a general increase of up to 5ꢀfold the percentage of inhibition for
the same experimental conditions, which is clearly attributed to the further inclusion of
the BTA moiety in these hybrids. Among the set of compounds studied herein, the chloro
substitution resulted in the decrease of the interaction with Aβ, with apparent slightly
stronger effect for compounds with longer chain size. In general, the allylic derivatives
led to a somewhat better inhibition than the corresponding propargylic ones, which may
be due to higher rigidity and increased lipophilicity (some water solubility limitations).
A survey on the effect of compounds on the inhibition of copperꢀinduced Aβ aggregation
was carried out for 10c and 10g. It was observed a higher inhibitory activity for the
incubation of Aβ42 in the coꢀpresence of copper (56.6, 37.6%) than that in its absence
(31.6, 24.1%), thus suggesting that the ligands may modulate the metalꢀinduced
aggregation of Aβ42, similarly to previously reported for the analogues tacrineꢀSAC and
TACꢀSPRC.20 This can be rationalized by the metalꢀchelating properties of compounds
which may compete with the amyloid peptide for the copper and decrease their
aggregation ability, thus resulting in higher inhibition percentage. However, the
enhancement of Aβ42 inhibitory activity due to the coꢀpresence of copper observed for the
trihybrids is lower than that previously observed for dihybrids (ca 3.8 fold, see Table
S1),20 which may be attributed to the substitution of one N-amine by N-amide donor
groups.
Insert here Table1
2.3.3. Antioxidant activity
The newly invented trihybrid compounds of TACꢀSACꢀBTA and TACꢀSPRCꢀBTA were
screened for their antioxidant activities, based on their interaction with the 2,2ꢀdiphenylꢀ
1ꢀpicrylhydrazyl (DPPH) free radical.31 Analysis of the results evidenced low antiꢀ
8

Page 9 of 35
RSC Advances
View Article Online
DOI: 10.1039/C6RA03455A
oxidant activity (EC50 > 1000 µM, see Fig. S10), similarly to tacrine and lower than the
values previously found for the bihybrid analogues (TACꢀSAC and TACꢀSPRC, see
Table S1),19 and so the particular values are not included herein (see representative plot
in Fig. S9). These series of trihybrid compounds presented antioxidant capacity similar
to tacrine, which is in contrast with the expectation due to the inclusion of the naturalꢀ
based SAC and SPRC neuroprotectors.32 The recognized in vitro neuroprotective activity
provided by these groups may be associated with an increased antiꢀoxidant activity inside
the cells due to some enzymatic deprotection of the sulfhydryl group.33
2.3.4. Inhibition of MAO
Four of the new compounds 10(a,c,g,h) were investigated for their inhibitory activity
against MAOs using a coupled fluorescent method.34 Compounds neither quenched the
fluorescence of resorufin nor compromised the activity of the coupling enzyme,
horseradish peroxidase. The IC50 values are given in Table 1. All compounds were poor
inhibitors of MAO B (IC50 >50 ꢁM), but were slightly more effective against MAO A
that has a wider active site to accommodate bulky molecules.
Without preincubation, the inhibition of MAO A was less, giving IC50 values as follows:
10a, 48.4 ꢁM; 10c, 25.9 ꢁM; 10g, 42.1 ꢁM; 10h, 87.5 ꢁM. Thus, preincubation decreased
the IC50 only slightly (< 3ꢀfold) in contrast to the standard MAO A inhibitor, clorgyline,
where the IC50 decreases 100ꢀfold with 30 min preincubation.35 The very small difference
in IC50 after 30 min preincubation was the same for both the allyl and propargyl
compounds suggesting that the new propargyl derivatives do not inactivate MAO to any
great extent. The high IC50 values obtained when inhibitor and substrate are added at the
same time indicate low affinity binding, so it is likely that the propargyl group is not
close enough to the N5 of the FAD in the active site of MAO to undergo enzymeꢀ
catalyzed activation.
2.3.5. Cell viability and neuroprotection
To evaluate the potential therapeutic effect of TACꢀSACꢀBTA and TACꢀSPRCꢀBTA
compounds 10(a–h), SHꢀSY5Y cells were treated with Aβ42 peptide or H2O2 in the
presence or absence of the tacrine hybrids. Aβ peptides that accumulate in AD relevant
9

RSC Advances
Page 10 of 35
View Article Online
DOI: 10.1039/C6RA03455A
neurons are widely used to induce cell toxicity and model AD. Indeed, Aβ oligomers
induced a decrease in cell viability (Fig.4A). Despite our previous results showed that
similar tacrine hybrids prevented from Aβꢀinduced cell toxicity.19 the addition of a new
benzothiazol to potentiate the interaction with Aβ eliminated any potential protective
role. We may emphasize that an increased interaction between Aβ peptides and our new
tacrine hybrids 10(c,e,h) prevented Aβ aggregation but still allowed soluble oligomers to
interact with organelles, such as mitochondria and elicit its toxic effect. Nevertheless,
compound 10d showed the lowest inhibition of Aβ aggregation (14%) very similar to our
data obtained with previous compounds that were able to prevent Aβꢀinduced cellular
toxicity.19 Regarding compound 10f, its highest molecular weight and lipophilicity (see
below and Table 1S), as well as concomitant limited solubilization, may have also
contributed for the decreasing in its cellular internalization and the lowest recovering
effect of the amyloid stressor..
Additionally, it is generally accepted that ADꢀrelated neurodegenerative processes
involve oxidative stress that may be induced by H2O2. Indeed, H2O2 (100 ꢀM, 24 h)
induced a decrease in cell viability in SHSYꢀ5Y cells (see Fig. 4B). Among the tacrine
hybrids tested 10 (b,g,h) were the most effective in preventing H2O2ꢀmediated toxicity.
These results are similar to those previously obtained with other tacrine hybrid
compounds.19
Insert here Fig 4
2.3.6. Pharmacokinetic properties
In an effort to establish the drugꢀlikeness of the novel compounds and their potential to
penetrate important membranes such as the blood–brain barrier (BBB), some indicators
of their pharmacokinetic profiles were predicted using QikProp program, v. 2.5.36
Parameters such as the calculated octanol–water partition coefficient (clogP), the ability
to cross the BBB (log BB), the capacity to be absorbed through the intestinal tract to the
blood (Cacoꢀ2 cell permeability), and the verification of Lipinski’s rule of five, may give
an idea of their drugꢀlikeness for being orally active as antiꢀAD agents.
All the compounds presented clogP values greater than five (Table 1S), thus with higher
lipophilic character than recommended by Lipinski’s rule.37 The compounds also have
10

Page 11 of 35
RSC Advances
View Article Online
DOI: 10.1039/C6RA03455A
molecular weights higher than 500, which accounts for two violations of this rule. The
high lipophilic character and the low BBB permeability (log BB) indicate that the
compounds are not eligible as drug candidates for oral administration. However, they
exhibited very good results in Cacoꢀ2 permeability, ranging from ca. 600 to 1400 nm/s
(higher than 500 nm/s is considered good)36 with compounds 10e and 10g having lower
cell permeabilities (according to the standard value). The other compounds presented
greater values, indicating that the absorption through the intestinal tract to the blood is
possible.
3. Conclusion
The conjugation of several functional moieties in one molecular entity has recently
emerged as an important drug development strategy to combat the complex multifactorial
Alzheimer`s disease (AD). Following this strategy in this work, a series of eight novel
trihybrids has been designed, prepared and bioassayed aimed at gathering information
about potential antiꢀAD drug candidates. They incorporate three main moieties, tacrine
(TAC), Sꢀallylꢀ or Sꢀpropargylꢀcysteine (SAC/SPRC) and benzothiazole (BTA), for
inhibition/modulation of three main important AD targets: acetylcholinesterase
inhibition, antiꢀoxidant activity and inhibition of amyloid β (Aβ) aggregation, with
additional possible inhibition of the MAO enzyme. Analysis of the biological properties
shown for these trihybrids was made in comparison with those recently reported for
identical chemical library of bihybrids, lacking the BTA moiety. As hypothesized, the
inclusion of the third moiety resulted in a considerable enhancement of the AChE
inhibitory activity (up to one order of magnitude, with IC50 in subꢀꢁM range, below that
of the tacrine drug), attributed to its stronger dualꢀbinding mode with both the CAS and
PAS of AChE, specifically due to the strong BTAꢀPAS πꢀπ interaction. Also, insertion of
the BTA moiety on the trihybrids led to an enhancement of up to ca 5ꢀfold the percentage
of selfꢀAβ aggregation inhibition, attributed to the wellꢀknown affinity of BTA
derivatives for Aβ. Nevertheless, antioxidant activity of the trihybrids is lower than the
biꢀhybrids and similar to that of TAC. In addition, a preliminary study with some of these
new trihybrids revealed only weak capacity for MAO inhibition, another known drug
target for AD.
11

RSC Advances
Page 12 of 35
View Article Online
DOI: 10.1039/C6RA03455A
Overall, a comparative analysis of some relevant antiꢀAD properties of the present
trihybrids and those of the previous bihybrid analogues shows that conjugation of these
three structural scaffolds in a molecular unit can provide a basis for the development of
potential multifunctional drug candidates for Alzheimer´s disease.
4. Experimental part
4.1. Chemistry
General methods and materials
Analytical grade reagents were purchased from SigmaꢀAldrich, Fluka and Acros and
were used as supplied. Solvents were dried according to standard methods38. The
chemical reactions were monitored by TLC using alumina plates coated with silica gel 60
F254 (Merck). Column chromatography separations were performed on silica gel Merck
230ꢀ400 mesh (Geduran Si 60). The melting points (m.p.) were measured with a Leica
1
13
Galen III hot stage apparatus and are uncorrected. The H and C NMR spectra were
recorded on Bruker AVANCE III spectrometers at 300 and 400 MHz, respectively.
Chemical shifts (δ) are reported in ppm from the standard internal reference
tetramethylsilane (TMS). The following abbreviations are used: s = singlet, d = doublet, t
= triplet, m = multiplet. Mass spectra (ESIꢀMS) were performed on a 500 MS LC Ion
Trap (Varian Inc., Palo Alto, CA, USA) mass spectrometer equipped with an ESI ion
source, operated in the positive ion mode. For the target compounds, the elemental
analyses were performed on a Fisons EA1108 CHNS/O instrument and were within the
limit of ± 0.4%. The electronic spectra were recorded with a Perkin Elmer Lambda 35
spectrophotometer, using thermostatic 1 cm path length cells.
4.1.1. General procedure for the preparation of 2ꢀ(1,2,3,4ꢀtetrahydroacridineꢀ9ꢀ
ylamino) acid 4(aꢀd)
To a mixture of 3 (1 eq) and different aliphatic acids (3ꢀaminopropanoic, 4ꢀ
aminobuytric and 6ꢀamino hexanoic acid (2 eq) was added to phenol and catalytic
o
amount of KI. The mixture is heated at 180 C for 4 h and then cooled to room
temperature. The mixture is dissolved in 5% NaOH, washed with diethyl ether (3ꢀ4
times). The collected aqueous layer was adjusted to pH 8 with HCl, and then washed with
diethyl ether (3ꢀ4 times). The aqueous layer was acidified with HCl up to pH 2 and then
12

Page 13 of 35
RSC Advances
View Article Online
DOI: 10.1039/C6RA03455A
extracted with dichloromethane. The organic layer was dried over anhydrous Na2SO4 and
concentrated under reduced pressure to give the crude product. Purification was
performed by column chromatography, eluent: CH2Cl2/MeOH (6/1) v/v, plus 10 mL 25%
ammonia aqueous, for 1L total solution.
4.1.2.1. 4ꢀ(1,2,3,4ꢀtetrahydroacridinꢀ9ꢀylamino)butanoic acid (4a)
9ꢀChloroꢀ1,2,3,4ꢀtetrahydroacridine (3a) and 4ꢀaminobuytric acid afforded the
o
1
pure title product as a light yellow colour solid; yield 62%; m.p. 157ꢀ160 C; H NMR
(MeOD) δ: 8.45 (d, J = 8.6 Hz, 1H, Hꢀ8), 7.85ꢀ7.73 (m, 2H,Hꢀ5 & Hꢀ6), 7.59ꢀ7.53 (m,
1H, Hꢀ7), 4.00 (t, J = 7.0 Hz, 2H, Hꢀ1′), 3.00 (brs, 2H, Hꢀ4), 2.69 (brs, 2H, Hꢀ1), 2.50 (t,
J = 6.7 Hz, 2H, Hꢀ3′), 2.16ꢀ2.07 (m, 2H, Hꢀ2′), 1.96ꢀ1.93 (m, 4H, Hꢀ2 & Hꢀ3); MSꢀESI
(m/z): 285 (M+1)+, 286 (M+2)+.
4.1.2.2. 6ꢀ(1,2,3,4ꢀtetrahydroacridinꢀ9ꢀylamino)hexanoic acid (4b)
9ꢀChloroꢀ1,2,3,4ꢀtetrahydroacridine (3a) and 6ꢀaminohexanoic acid afforded the
o
1
pure title product as a light yellow colour solid; yield 67%; m.p. 181ꢀ183 C; H NMR
(300 MHz, MeOD) δ 8.19 (d, J = 8.5 Hz, 1H, Hꢀ8), 8.04 (d, J = 8.4 Hz, 1H, Hꢀ5), 7.68ꢀ
7.62 (m, 1H, Hꢀ6), 7.44ꢀ7.39 (m, 1H, Hꢀ7), 3.85 (t, J = 7.0 Hz, 2H, Hꢀ1′), 3.00 (brs, 2H,
Hꢀ4), 2.59 (brs, 2H, Hꢀ1), 2.24 (t, J = 7.0 Hz, 2H, Hꢀ5′), 1.85ꢀ1.79 (m, 6H, Hꢀ2, Hꢀ3, &
Hꢀ2′), 1.61ꢀ1.59 (m, 2H, Hꢀ4′), 1.42ꢀ1.39 (m, 2H, Hꢀ3′); MSꢀESI (m/z): 313 (M+1)+ )+,
314 (M+2)+.
4.1.2.3. 4ꢀ(6ꢀchloroꢀ1,2,3,4ꢀtetrahydroacridinꢀ9ꢀylamino)butanoic acid (4c)
6,9ꢀChloroꢀ1,2,3,4ꢀtetrahydroacridine (3b) and 4ꢀaminobuytric acid afforded the
o
1
pure title product as a light yellow colour solid; yield 59%; m.p. 230ꢀ232 C; H NMR
(MeOD) δ: 8.45 (d, J = 8.7 Hz, 1H, Hꢀ8), 7.76 (d, J = 1.9 Hz, 1H, Hꢀ5), 7.52 (dd, J = 7.2
&1.9 Hz, 1H, Hꢀ7), 4.00 (t, J = 6.9 Hz, 2H, Hꢀ1′), 3.00 (brs, 2H, Hꢀ4), 2.71 (brs, 2H, Hꢀ
1), 2.50 (t, J = 8.1 Hz, 2H, Hꢀ3′), 2.20ꢀ2.13 (m, 2H, Hꢀ2′), 1.99ꢀ1.96 (m, 4H, Hꢀ2 & Hꢀ3);
MSꢀESI (m/z): 318 (M)+, 319 (M+1)+.
4.1.2.4. 6ꢀ(6ꢀchloroꢀ1,2,3,4ꢀtetrahydroacridinꢀ9ꢀylamino)hexanoic acid (4d)
6,9ꢀChloroꢀ1,2,3,4ꢀtetrahydroacridine (3b) and 6ꢀaminohexanoic acid afforded the
o
1
pure title product as a light yellow colour solid; yield 69%; m.p. 155ꢀ157 C; H NMR
13

RSC Advances
Page 14 of 35
View Article Online
DOI: 10.1039/C6RA03455A
(300 MHz, MeOD) δ 8.39 (d, J = 8.9 Hz, Hꢀ8), 7.82 (d, J = 2.1 Hz, 1H, Hꢀ5), 7.55 (dd,
1H, J = 7.0 & 2.1 Hz, Hꢀ7), 3.98 (t, J = 7.2 Hz, 2H, Hꢀ1′), 3.00 (br, 2H, Hꢀ4), 2.72 (br,
2H, Hꢀ3), 2.35 (t, J = 7.2 Hz, 2H, Hꢀ5′), 2.00ꢀ1.98 (m, 4H, Hꢀ2 & Hꢀ3); 1.94ꢀ1.86 (m, 2H,
Hꢀ2′), 1.73ꢀ1.65 (m, 2H, Hꢀ4′), 1.55ꢀ1.49 (m, 2H, Hꢀ3′); MSꢀESI (m/z): 346 (M+), 347
(M+1)+.
4.1.4. Procedure for the preparation of 3ꢀallylsulfanylꢀ2ꢀaminoꢀpropionic
acid (6a) and 2ꢀaminoꢀ3ꢀpropꢀynylsulfanylꢀpropionic acid (6b)
Lꢀcysteine (1 eq) were dissolved in NH4OH solution (2M, 45 mL), and the
o
reaction mixture was stirred at 0 C around 30 min. Afterwards, allyl bromide/propargyl
bromide (1.5 eq) was dropwise added and reaction mixture was stirred for another 3 h,
concentrated in vaccuo and filtered; the filtrate was washed with ethanol and
recrystallized from waterꢀethanol affording the pure product as white shiny particles.
4.1.3.1. (R)ꢀ3ꢀallylsulfanylꢀ2ꢀaminoꢀpropionic acid (6a)
Lꢀcysteine (5) and allyl bromide afforded the pure title product as a white powder;
o
1
yield 75%; m.p. 220ꢀ221 C; H NMR (300 MHz, D2O) δ: 5.96ꢀ5.79 (m, 1H, HC=C),
5.29ꢀ5.19 (m, 2H, CH2=CH), 3.94ꢀ3.82 (m, 1H, CHNH2), 3.21 (d, J = 7.2 Hz, 2H, CH2ꢀ
allyl), 3.00ꢀ2.94 (m, 2H, CH2CHN); MSꢀESI (m/z): 160(Mꢀ1)+, 162(M+1)+.
4.1.3.2. (R)ꢀ2ꢀaminoꢀ3ꢀpropꢀynylsulfanylꢀpropionic acid (6b)
Lꢀcysteine (5) and propargyl bromide afforded the pure title product as a white
o
1
shiny solids; yield 72%; m.p. 294ꢀ296 C; H NMR (300 MHz, D2O) δ: 3.83ꢀ3.90 (m,
1H, CHNH2), 3.32ꢀ3.30 (m, 2H, CH2ꢀpropargyl), 3.24ꢀ3.01 (m, 2H, CH2CHN), 2.61 (t, J
= 2.5 Hz,1H, HCC); MSꢀESI (m/z): 160 (M+1)+.
4.1.4.
Procedure
for
the
preparation
of
(S)ꢀ3ꢀ(allylthio)ꢀ2ꢀ(tertꢀ
butoxycarbonylamino)propionic acid (7a) and (S)ꢀ2ꢀ(tertꢀbutoxycarbonylamino)ꢀ3ꢀ
(propꢀ2ꢀynylthio)propanoic acid (7b)
Compound 6(a, b) (1 eq) and Boc anhydride (1 eq) were both dissolved and
stirred in THF (25 mL), saturated with sodium bicarbonate solution (60 mL) and left
stirring for 5 h at room temperature. The reaction mixture was diluted with H2O (15 mL)
and EtOAc (15 mL), the pH was adjusted to about 2ꢀ2.5, and then separate the organic
14

Page 15 of 35
RSC Advances
View Article Online
DOI: 10.1039/C6RA03455A
extracts were washed with brine and dried over Na2SO4. The solvent was removed under
vacuum and the crude material was purified by silica gel chromatography to give 7(a,b).
4.1.4.1. (S)ꢀ3ꢀ(allylthio)ꢀ2ꢀ(tertꢀbutoxycarbonylamino)propanoic acid (7a)
3ꢀAllylsulfanylꢀ2ꢀaminoꢀpropionic acid (6a) was treated with Boc anhydride to
afford the pure title product as semi solid; yield 90%; 1H NMR (300 MHz, DMSOꢀd6) δ:
7.1 (d, J = 8.3 Hz, 1H, HNCH), 5.79ꢀ5.69 (m, 1H, HC=C), 5.14ꢀ5.06 (m, 2H, CH2=HC),
4.06ꢀ4.01(m, 1H, CHNH), 3.15 (d, J = 7.1 Hz, 2H, CH2ꢀallyl), 2.82ꢀ2.61 (m, 2H,
CH2CHN), 1.38 (s. 9H, tBuH); MSꢀESI (m/z): 261 (M+).
4.1.4.2. (S)ꢀ2ꢀ(tertꢀbutoxycarbonylamino)ꢀ3ꢀ(propꢀ2ꢀynylthio)propanoic acid (7b)
2ꢀAminoꢀ3ꢀpropꢀynylsulfanylꢀpropionic acid (6b) and Boc anhydride afforded the
1
pure title product as semi solid; yield 83%; H NMR (300 MHz, DMSO) δ: 7.15 (d, J =
8.4 Hz, 1H, HNCH), 4.15ꢀ4.08 (1H, m, HCNH), 3.41ꢀ3.39 (m, 2H, CH2ꢀpropargyl), 3.08ꢀ
3.81 (m, 2H, CH2CHN), 2.67 (t, J = 2.5 Hz,1H, HCC), 1.41 (s. 9H, tBuH); MSꢀESI (m/z):
259 (M+).
4.1.5. Procedure for the preparation of tertꢀbutyl 3ꢀ(allylthio)ꢀ1ꢀ(benzo[d]thiazolꢀ2ꢀ
ylamino)ꢀ1ꢀoxopropanꢀ2ꢀylꢀcarbamate (8a) and tert-butyl 1ꢀ(benzo[d]thiazolꢀ2ꢀ
ylamino)ꢀ1ꢀoxoꢀ3ꢀ(propꢀ2ꢀynylthio)propanꢀ2ꢀylꢀcarbamate (8b)
T3P (1.5 eq) was added dropwise to a stirred suspension of 2ꢀamino benzothiazole
(1 eq) (8), NꢀBoc protected SAC/SPRC (1 eq) 7(a,b) and NMM (2.5 eq) in DCM under
nitrogen and the mixture was stirred at room temperature for 3 h; after completing the
reaction, it was diluted with water and extracted in ethyl acetate (3×20 mL). The
combined extracts were washed with HCl (0.1 N, 3×50 mL) and NaOH (1 N, 3×50 mL)
and then with water and brine. The combined organic phases were dried over Na2SO4 and
solvent was removed to afford the substitled compounds 8(a,b).
4.1.5.1.
carbamate (8a)
2ꢀAminobenzothiazole (8) and NꢀBoc protected SAC (7a) afforded the pure title
Tertꢀbutylꢀ3ꢀ(allylthio)ꢀ1ꢀ(benzo[d]thiazolꢀ2ꢀylamino)ꢀ1ꢀoxopropanꢀ2ꢀylꢀ
product as an colorless solid with 78% yield; m.p. 83ꢀ85 oC; 1H NMR (300 MHz, CDCl3)
δ: 7.82ꢀ7.83 (m, 2H, Hꢀ4 & Hꢀ7), 7.43 (t, J = 8.0 Hz, 1H, Hꢀ5 or Hꢀ6), 7.31 (t, J = 8.0 Hz,
1H, Hꢀ6 or Hꢀ5), 5.73ꢀ5.65 (m, 1H, HC=C), 5.11ꢀ5.07 (m, 2H, H2C=CH), 4.66ꢀ4.55 (m,
15

RSC Advances
Page 16 of 35
View Article Online
DOI: 10.1039/C6RA03455A
1H, HCNH), 3.08 (d, J = 7.1 Hz, 2H, CH2ꢀallyl), 2.98ꢀ2.95 (m, 2H, CH2CHN), 1.46 (s.
9H, tBuH); MSꢀESI (m/z): 393 (M+).
4.1.5.2. Tertꢀbutylꢀ1ꢀ(benzo[d]thiazolꢀ2ꢀylamino)ꢀ1ꢀoxoꢀ3ꢀ(propꢀ2ꢀynylthio)propanꢀ
2ꢀylꢀcarbamate (8b)
2ꢀAminobenzothiazole (8) and NꢀBoc protected SPRC (7b) afforded the pure title
o
1
product as an colourless solid; yield 83%; m.p.133ꢀ135 C; H NMR (300 MHz, CDCl3)
δ: 7.82ꢀ7.85 (m, 2H, Hꢀ4 & Hꢀ7), 7.45 (t, J = 8.0 Hz, 1H, Hꢀ5 or Hꢀ6), 7.31 (t, J = 8.0 Hz,
1H, Hꢀ6 or Hꢀ5), 4.82ꢀ4.80 (1H, m, HCNH), 3.26ꢀ3.22 (m, 4H, CH2ꢀpropargyl, &
CH2CHN), 2.26 (s, 1H, HCC), 1.41 (s, 9H, tBuH); MSꢀESI (m/z): 391 (M+).
4.1.6. Procedure for the preparation of 3ꢀ(allylthio)ꢀ2ꢀaminoꢀNꢀ(benzo[d]thiazolꢀ2ꢀ
yl)propanamide
(9a)
and
2ꢀaminoꢀNꢀ(benzo[d]thiazolꢀ2ꢀyl)ꢀ3ꢀ(propꢀ2ꢀ
ynylthio)propanamide (9b)
Compounds 8(a,b) and trifluoroacetic acid (TFA, 1 mL) were dissolved DCM (2
mL) and stirred at room temperature for 2 h. After completing the reaction, the solvent
was evaporated and afforded the crude product and washed with diluted ammonia
solution (10%). Purification was performed by column chromatography, eluent
CH2Cl2/MeOH (6/1), affording pure compounds 9(a,b).
4.1.6.1. 3ꢀ(allylthio)ꢀ2ꢀaminoꢀNꢀ(benzo[d]thiazolꢀ2ꢀyl)propanamide (9a)
Tertꢀbutylꢀ3ꢀ(allylthio)ꢀ1ꢀ(benzo[d]thiazolꢀ2ꢀylamino)ꢀ1ꢀoxopropanꢀ2ꢀylꢀ
carbamate (8a) and TFA afforded the pure title product as a colourless solid; yield 86%;
m.p.105ꢀ107 oC; 1H NMR (CDCl3) δ: 7.78ꢀ7.82 (m, 2H, Hꢀ4 & Hꢀ7), 7.44 (t, J = 8.0 Hz,
1H, Hꢀ5 or Hꢀ6), 7.29 (t, J = 8.0 Hz, 1H, Hꢀ6 or Hꢀ5), 5.71ꢀ5.22 (m, 1H, HC=C), 5.13ꢀ
5.20 (m, 2H, H2C=CH), 3.79 (brs, 1H, HCNH), 3.08ꢀ3.15 (m, 3H, H2Cꢀallyl &
H2CCHN), 3.09ꢀ2.80 (m, 1H, H2CCHN); MSꢀESI (m/z): 293 (M+).
4.1.6.2. 2ꢀaminoꢀNꢀ(benzo[d]thiazolꢀ2ꢀyl)ꢀ3ꢀ(propꢀ2ꢀynylthio)propanamide (9b)
Tertꢀbutylꢀ1ꢀ(benzo[d]thiazolꢀ2ꢀylamino)ꢀ1ꢀoxoꢀ3ꢀ(propꢀ2ꢀynylthio)propanꢀ2ꢀylꢀ
carbamate (8b) and TFA afforded the pure title product as a colourless solid; yield 89%;
o
m.p.112ꢀ114 C; 1H NMR (DMSO) δ: 7.98 (d, J = 7.8 Hz, 1H, Hꢀ4) 7.75 (d, J = 7.8 Hz,
1H, Hꢀ7), 7.45 (t, J = 8.0 Hz, 1H, Hꢀ5 or Hꢀ6), 7.32 (t, 1H, J = 8.0 Hz, Hꢀ6 or Hꢀ5), 3.76
16

Page 17 of 35
RSC Advances
View Article Online
DOI: 10.1039/C6RA03455A
(t, J = 6.7 Hz, 1H, HCNH), 3.19 (s, J = 6.7 Hz, 2H, CH2ꢀpropargyl), 2.97ꢀ2.86 (m, 3H,ꢀ
CH2CHN & HCC); MSꢀESI (m/z): 291 (M+).
4.1.7. General procedure for the preparation of 10(aꢀh)
T3P (1.5 eq) was added dropwise to a stirred suspension of BTAꢀSAC/BTAꢀ
SPRC (1 eq) 9(a,b), 2ꢀ(1,2,3,4ꢀtetrahydroacridineꢀ9ꢀylamino) acid (1 eq) 4(aꢀd) and
NMM (2.5 eq) in DCM under nitrogen and the mixture was stirred at room temperature
for 3 h; after completing the reaction, it was diluted with water and extracted in ethyl
acetate (3×20 mL). The combined extracts were washed with HCl (0.1N, 3×50 mL) and
NaOH (1N, 3×50 mL) and then with water and brine. The combined organic phases were
dried over Na2SO4 and solvent was removed to afford the substitled compounds 10(aꢀh).
The final product was purified by column chromatography, eluent: CH2Cl2/MeOH (6/1)
v/v, plus 10 mL 25% ammonia aqueous, for 1L total solution and some of the compounds
recrystalized by ethanol and ethyl ether.
4.1.7.1. Nꢀ(3ꢀ(Allylthio)ꢀ1ꢀ(benzo[d]thiazolꢀ2ꢀylamino)ꢀ1ꢀoxopropanꢀ2ꢀyl)ꢀ4ꢀ(1,2,3,4ꢀ
tetrahydroacridinꢀ9ꢀylamino)butanamide (10a)
3ꢀ(Allylthio)ꢀ2ꢀaminoꢀNꢀ(benzo[d]thiazolꢀ2ꢀyl)propanamide (9a) and 4ꢀ(1,2,3,4ꢀ
tetrahydroacridinꢀ9ꢀylamino)butanoic acid (4a) afforded the pure title product as a light
o
1
yellow color solid; yield 65%; m.p. 95ꢀ102 C; H NMR (400 MHz, MeODꢀd4) δ: 8.33ꢀ
8.34 (m, 1H, Hꢀ8), 7.75ꢀ7.78 (m, 2H, Hꢀ4′′′ & Hꢀ7′′′), 7.60ꢀ7.64 (m, 2H, Hꢀ5 & Hꢀ6),
7.47ꢀ7.52 (m, 1H, Hꢀ5′′′ or Hꢀ6′′′), 7.40ꢀ7.41 (m, 1H, Hꢀ6′′′ or Hꢀ5′′′), 7.29ꢀ7.30 (m, 1H,
Hꢀ7), 5.70ꢀ5.80 (m, 1H, CHꢀallyl), 5.14 (d, 1H, J = 16.0 Hz, CH2ꢀallyl), 5.08 (d, 1H, J =
8.0 Hz, CH2ꢀallyl), 4.72 (t, J = 8.0 Hz, 1H, Hꢀ2′′), 3.88ꢀ3.90 (m, 2H, Hꢀ1′), 3.11ꢀ3.18 (m,
3H, ꢀSCH2ꢀallyl & Hꢀ3′′), 2.94ꢀ2.2.99 (m, 3H, Hꢀ4 & Hꢀ3′′), 2.74 (brs, 2H, Hꢀ1), 2.55 (t,
J = 8.0 Hz, 2H, Hꢀ3′), 2.10ꢀ2.15 (m, 2H, Hꢀ2′), 1.85 (brs, 4H, Hꢀ2 & Hꢀ3); 13C NMR
(100.5 MHz, DMSOꢀd6) δ: 172.8, 171.0, 158.1, 157.3, 151.6, 148.9, 145.9, 134.6, 131.9,
129.0, 126.6, 124.1, 123.9, 123.8, 122.2, 121.0, 120.0, 117.9, 115.7, 53.0, 47.9, 34.4,
34.2, 33.1, 32.8, 31.9, 31.7, 26.8, 25.3, 22.9, 22.5; MSꢀESI (m/z): 558 (Mꢀ1)+, 559 (M+),
560 (M+1)+; Analysis calc. for C30H33N5O2S2.0.52 EtOH: C 62.83, H 6.06, N 12.01, S
11.00 %; found: C 63.05, H 6.21, N 11.61 %.
17

RSC Advances
Page 18 of 35
View Article Online
DOI: 10.1039/C6RA03455A
4.1.7.2. Nꢀ(3ꢀ(Allylthio)ꢀ1ꢀ(benzo[d]thiazolꢀ2ꢀylamino)ꢀ1ꢀoxopropanꢀ2ꢀyl)ꢀ6ꢀ(1,2,3,4ꢀ
tetrahydroacridinꢀ9ꢀylamino)hexanamide (10b)
3ꢀ(Allylthio)ꢀ2ꢀaminoꢀNꢀ(benzo[d]thiazolꢀ2ꢀyl)propanamide (9a) and 6ꢀ(1,2,3,4ꢀ
tetrahydroacridinꢀ9ꢀylamino)hexanoic acid (4b) afforded the pure title product as a light
o
yellow color solid; yield 71 %; m.p. 96ꢀ100 C ; 1H NMR (400 MHz, MeODꢀd4) δ: 8.13
(d, J = 8.0 Hz, 1H, Hꢀ8), 7.67ꢀ7.75(m, 2H, Hꢀ4′′′ & Hꢀ7′′′), 7.59 (t, J = 8.0 Hz, 1H, Hꢀ5),
7.39 (t, J = 8.0 Hz, 1H, Hꢀ6), 7.36ꢀ7.37 (m, 2H, Hꢀ5′′′ & Hꢀ6′′′), 7.25 (t, J = 8.0 Hz, 1H,
Hꢀ7), 5.50ꢀ5.78 (m, 1H, CHꢀallyl), 5.13 (d, 1H, J = 20.0 Hz, =CH2ꢀallyl), 5.06 (d, 1H, J =
8.0 Hz, =CH2ꢀallyl), 4.75 (t, 1H, J = 8.0 Hz, Hꢀ2′′), 3.61 (t, 2H, J = 8.0 Hz, Hꢀ1′), 3.15
(d, 2H, J = 8.0 Hz, CH2ꢀallyl), 2.93ꢀ2.96 (m, 3H, Hꢀ4 & Hꢀ3′′), 2.86ꢀ2.88 (m, 1H, Hꢀ3′′),
2.68 (brs, 2H, Hꢀ1), 2.30 (t, J = 8.0 Hz, 2H, Hꢀ5′), 1.87 (brs, 4H, Hꢀ2 & Hꢀ3), 1.66ꢀ1.73
13
(m, 4H, Hꢀ2′ & Hꢀ4′), 1.42ꢀ1.45 (m, 2H, Hꢀ3′); C NMR (100.5 MHz, MeODꢀd4) δ:
174.7, 170.9, 159.1, 155.5, 153.1, 148.4, 144.1, 133.9, 131.9, 129.5, 125.8, 124.3, 123.7,
123.6, 123.4, 120.8, 120.2, 116.6, 53.1, 51.8, 35.0, 34.2, 31.6, 31.4, 30.3, 29.3, 25.9, 24.9,
24.3, 22.4, 21.7; MSꢀESI (m/z): 588(M+), 589 (M+1)+, 590 (M+2)+; Analysis calc. for
C32H37N5O2S2.0.20 EtOH·0.5 H2O: C 63.42, H 6.65, N 11.56%; found: C 63.01, H 6.62,
N 11.37%.
4.1.7.3.
Nꢀ(3ꢀ(Allylthio)ꢀ1ꢀ(benzo[d]thiazolꢀ2ꢀylamino)ꢀ1ꢀoxopropanꢀ2ꢀyl)ꢀ4ꢀ(6ꢀ
chloroꢀ1,2,3,4ꢀtetrahydroacridinꢀ9ꢀylamino)butanamide (10c)
3ꢀ(Allylthio)ꢀ2ꢀaminoꢀNꢀ(benzo[d]thiazolꢀ2ꢀyl)propanamide (9a) and 4ꢀ(6ꢀchloroꢀ
1,2,3,4ꢀtetrahydroacridinꢀ9ꢀylamino)butanoic acid (4c) afforded the pure title product as
o
1
a light yellow color solid; yield 61%; m.p. 195ꢀ198 C; H NMR (400 MHz, MeODꢀd4)
δ: 8.15 (d, J = 8.0 Hz, 1H, Hꢀ8), 7.80 (d, J = 8.0 Hz, 1H, Hꢀ5) 7.62ꢀ7.67 (m, 2H, Hꢀ4′′′ &
Hꢀ7′′′), 7.40 (t, J = 8.0 Hz, 1H, Hꢀ7), 7.27ꢀ7.33 (m, 2H, Hꢀ5′′′ & Hꢀ6′′′), 5.70ꢀ5.80 (m,
1H, CHꢀallyl), 5.13 (d, 1H, J = 20.0 Hz, CH2ꢀallyl), 5.07 (d, 1H, J = 8.0 Hz, CH2ꢀallyl),
4.73 (t, J = 8.0 Hz, 1H, Hꢀ2′′), 3.68 (t, 2H, J = 8.0 Hz, Hꢀ1′), 3.14 (d, 2H, J = 8.0 Hz, ꢀ
SCH2ꢀallyl), 2.91ꢀ294 (m, 1H, Hꢀ3′′), 2.83ꢀ2.86 (m, 3H, Hꢀ4 & Hꢀ3′′), 2.67 (brs, 2H, Hꢀ
1), 2.47 (t, J = 8.0 Hz, 2H, Hꢀ3′), 2.05ꢀ2.06 (m, 2H, Hꢀ2′), 1.84 (brs, 4H, Hꢀ2 & Hꢀ3); 13C
NMR (100.5 MHz, DMSOꢀd6) δ: 172.8, 171.0, 158.6, 158.0, 151.6, 148.9, 147.6, 134.6,
133.5, 131.9, 126.7, 126.3, 124.2 124.1, 122.2 121.0, 117.9, 115.9, 53.0, 47.9, 34.2,
33.2, 32.8, 31.9, 26.8, 25.3, 22.8, 22.4; MSꢀESI (m/z): 594 (M+), 595 (M+1)+, 596
18

Page 19 of 35
RSC Advances
View Article Online
DOI: 10.1039/C6RA03455A
(M+2)+; Analysis calc. for C30H32N5O2S2Cl. 0.70 H2O: C 59.38, H 5.55, N 11.54 %;
found : C 59.07, H 5.55, N 11.36 %.
4.1.7.4.Nꢀ(3ꢀ(Allylthio)ꢀ1ꢀ(benzo[d]thiazolꢀ2ꢀylamino)ꢀ1ꢀoxopropanꢀ2ꢀyl)ꢀ6ꢀ(6ꢀ
chloroꢀ1,2,3,4ꢀtetrahydroacridinꢀ9ꢀylamino)hexanamide (10d)
3ꢀ(Allylthio)ꢀ2ꢀaminoꢀNꢀ(benzo[d]thiazolꢀ2ꢀyl)propanamide (9a) and 6ꢀ(6ꢀchloroꢀ
1,2,3,4ꢀtetrahydroacridinꢀ9ꢀylamino)hexanoic acid (4d) afforded the pure title product as
o
a light yellow color solid; yield 68 %; m.p. 94ꢀ98 C; 1H NMR (400 MHz, MeODꢀd4) δ:
8.11 (d, J = 8.0 Hz, 1H, Hꢀ8), 7.76 (d, J = 8.0 Hz, 1H, Hꢀ4′′′) 7.69ꢀ7.71 (m, 2H, Hꢀ5 &
Hꢀ7′′′), 7.40 (t, J = 8.0 Hz, 1H, Hꢀ5′′′ or Hꢀ6′′′), 7.32ꢀ7.35 (m, 1H, Hꢀ7), 7.27 (t, J = 8.0
Hz, 1H, Hꢀ6′′′ or Hꢀ5′′′), 5.72ꢀ5.79 (m, 1H, CHꢀallyl), 5.14 (d, 1H, J = 20.0 Hz, =CH2ꢀ
allyl), 5.08 (d, 1H, J = 12.0 Hz, =CH2ꢀallyl), 4.75 (t, J = 8.0 Hz, 1H, Hꢀ2′′), 3.59 (t, 2H, J
= 8.0 Hz, Hꢀ1′), 3.15 (d, 1H, J = 4.0 Hz, CH2ꢀallyl), 2.92ꢀ2.98 (m, 3H, Hꢀ4 & Hꢀ3′′),
2.80ꢀ2.83 (m, 1H, Hꢀ3′′), 2.67 (brs, 2H, Hꢀ1), 2.30 (t, J = 8.0 Hz, 2H, Hꢀ3′), 1.88 (brs,
4H, Hꢀ2 & Hꢀ3), 1.66ꢀ1.73 (m, 4H, Hꢀ2′ & Hꢀ4′), 1.40ꢀ1.48 (m, 2H, Hꢀ3′); 13C NMR
(100.5 MHz, DMSOꢀd6) δ: 172.9, 171.2, 159.7, 158.3, 150.9, 148.9, 148.0, 134.6, 132.9,
131.9, 127.1, 126.5, 125.9, 123.8, 121.0, 119.0, 117.9, 116.3, 53.0, 48.3, 35.3, 34.2, 33.9,
32.0, 30.7, 26.3, 25.4, 25.3, 23.0, 22.7; MSꢀESI (m/z): 622 (M+), 623 (M+1)+, 624
(M+2)+; Analysis calc. for C32H36N5O2S2Cl.·0.79 EtOH: C 61.24, H 6.23, N 10.63 %;
found: C 61.08, H 6.38, N 10.30 %.
4.1.7.5. Nꢀ(1ꢀ(Benzo[d]thiazolꢀ2ꢀylamino)ꢀ1ꢀoxoꢀ3ꢀ(propꢀ2ꢀynylthio)propanꢀ2ꢀyl)ꢀ4ꢀ
(1,2,3,4ꢀtetrahydroacridinꢀ9ꢀylamino)butanamide (10e)
2ꢀAminoꢀNꢀ(benzo[d]thiazolꢀ2ꢀyl)ꢀ3ꢀ(propꢀ2ꢀynylthio)propanamide (9b) and 4ꢀ(1,2,3,4ꢀ
tetrahydroacridinꢀ9ꢀylamino)butanoic acid (4a) afforded the pure title product as a light
o
yellow color solid; yield 63%; m.p. 105ꢀ110 C; 1H NMR (400 MHz, MeODꢀd4) δ: 8.17
(d, J = 8.0 Hz, 1H, Hꢀ8), 7.78 (d, J = 8.0 Hz, Hꢀ4′′′) 7.64ꢀ7.70 (m, 2H, Hꢀ5 & Hꢀ7′′′),
7.56 (t, J = 8.0 Hz, 1H, Hꢀ6), 7.39 (t, 2H, J = 8.0 Hz, Hꢀ5′′′ & Hꢀ6′′′), 7.27 (t, J = 8.0 Hz,
1H, Hꢀ7), 4.82 (t, 1H, J = 8.0 Hz, Hꢀ2′′), 3.69 (t, 2H, J = 8.0 Hz, Hꢀ1′), 3.35 (s, 2H, ꢀ
SCH2ꢀpropargyl), 3.21ꢀ3.26 (m, 1H, Hꢀ3′′), 3.01ꢀ3.06 (m, 1H, Hꢀ3′′), 2.90 (brs, 2H, Hꢀ4),
2.72 (brs, 2H, Hꢀ1), 2.60 (s, 1H, ꢀCHꢀpropargyl), 2.48 (t, J = 8.0 Hz, 2H, Hꢀ3′), 2.04ꢀ2.09
(m, 2H, Hꢀ3′), 1.86 (brs, 4H, Hꢀ2 & Hꢀ3); 13C NMR (100.5 MHz, DMSOꢀd6) δ: 172.8,
19

RSC Advances
Page 20 of 35
View Article Online
DOI: 10.1039/C6RA03455A
171.0, 158.2, 157.7, 151.1, 148.9, 146.6, 131.9, 128.6, 127.7, 126.6, 124.1, 123.8, 123.6,
122.19, 121.0, 120.3, 116.1, 80.6, 74.4, 52.8, 48.0, 33.4, 32.8, 26.9, 25.4, 23.1, 22.7,
19.1; MSꢀESI (m/z): 556(Mꢀ1)+, 557(M+), 558 (M+1)+; Analysis calc. for
C30H31N5O2S2·0.23 EtOH: C 63.00, H 5.66, N 12.32, S 11.28%; found: C 64.02, H 5.79,
N 12.18%.
4.1.7.6. Nꢀ(1ꢀ(Benzo[d]thiazolꢀ2ꢀylamino)ꢀ1ꢀoxoꢀ3ꢀ(propꢀ2ꢀynylthio)propanꢀ2ꢀyl)ꢀ6ꢀ
(1,2,3,4ꢀtetrahydroacridinꢀ9ꢀylamino)hexanamide (10f)
2ꢀAminoꢀNꢀ(benzo[d]thiazolꢀ2ꢀyl)ꢀ3ꢀ(propꢀ2ꢀynylthio)propanamide (9b) and 6ꢀ(1,2,3,4ꢀ
tetrahydroacridinꢀ9ꢀylamino)hexanoic acid (4b) afforded the pure title product as a light
o
1
yellow color solid; yield 75%; m.p. 96ꢀ100 C; H NMR (400 MHz, MeODꢀd4) δ: 8.30
(d, J = 8.0 Hz, 1H, Hꢀ8), 7.78 (m, 2H, Hꢀ4′′′ & Hꢀ7′′′), 7.50ꢀ7.54 (m, 1H, Hꢀ5), 7.39 (t,
2H, J = 8.0 Hz, Hꢀ6), 7.26 (t, J = 8.0 Hz, 1H, Hꢀ5′′′ or Hꢀ6′′′), 7.15 (t, J = 8.0 Hz, 1H, Hꢀ
6′′′ or Hꢀ5′′′), 6.75ꢀ6.80 (m, 1H, Hꢀ7), 4.82 (t, 1H, J = 8.0 Hz, Hꢀ2′′), 3.86 (t, 2H, J = 8.0
Hz, Hꢀ1′), 3.37 (s, 2H, ꢀSCH2ꢀpropargyl), 3.18ꢀ3.23 (m, 1H, Hꢀ3′′), 3.01ꢀ3.07 (m, 1H, Hꢀ
3′′), 2.93 (brs, 2H, Hꢀ4), 2.63ꢀ2.64 (m, 3H, Hꢀ1 & ꢀCHꢀpropargyl), 2.35 (t, J = 8.0 Hz,
2H, Hꢀ5′), 1.91 (brs, 4H, Hꢀ2 & Hꢀ3), 1.79ꢀ187 (m, 2H, Hꢀ2′), 1.69ꢀ1.73 (m, 2H, Hꢀ4′),
1.48ꢀ1.53 (m, 2H, Hꢀ3′); 13C NMR (100.5 MHz, DMSOꢀd6) δ: 173.0, 171.1, 158.3, 158.0,
151.0, 148.9, 146.9, 131.9, 128.5, 128.3, 126.6, 124.0, 123.7, 123.6, 122.1, 121.0, 120.4,
116.0, 80.6, 74.3, 52.8, 48.3, 35.3, 33.7, 32.9, 30.8, 26.3, 25.4, 25.3, 23.1, 22.8, 19.1;
MSꢀESI (m/z): 584(Mꢀ1)+, 585(M+), 586 (M+1)+; Analysis calc. for C30H31N5O2S2·0.51
EtOH: C 64.06, H 6.13, N 11.49, S 10.52%; found: C 64.01, H 5.85, N 11.20%.
4.1.7.7. Nꢀ(1ꢀ(Benzo[d]thiazolꢀ2ꢀylamino)ꢀ1ꢀoxoꢀ3ꢀ(propꢀ2ꢀynylthio)propanꢀ2ꢀyl)ꢀ4ꢀ
(6ꢀchloroꢀ1,2,3,4ꢀtetrahydroacridinꢀ9ꢀylamino)butanamide (10g)
2ꢀAminoꢀNꢀ(benzo[d]thiazolꢀ2ꢀyl)ꢀ3ꢀ(propꢀ2ꢀynylthio)propanamide (9b) and 4ꢀ(6ꢀ
chloroꢀ1,2,3,4ꢀtetrahydroacridinꢀ9ꢀylamino)butanoic acid (4c) afforded the pure title
o
product as a light yellow color solid; yield 63%; m.p.119ꢀ122 C; 1H NMR (400 MHz,
MeODꢀd4) δ: 8.19 (d, J = 8.0 Hz, 1H, Hꢀ8), 7.80 (d, J = 8.0 Hz, 1H, Hꢀ4′′′), 7.61ꢀ7.65 (m,
2H, Hꢀ5 & Hꢀ7′′′), 7.41 (t, J = 8.0 Hz, 1H, Hꢀ5′′′ or Hꢀ6′′′), 7.35 (d, 1H, J = 8.0 Hz, Hꢀ7),
7.30 (t, J = 8.0 Hz, 1H, Hꢀ6′′′ or Hꢀ5′′′), 4.80 (t, 1H, J = 8.0 Hz, Hꢀ2′′), 3.73 (t, 2H, J =
8.0 Hz, Hꢀ1′), 3.36 (s, 2H, ꢀSCH2ꢀpropargyl), 3.18ꢀ3.23 (m, 1H, Hꢀ3′′), 3.03ꢀ3.07 (m,
20

Page 21 of 35
RSC Advances
View Article Online
DOI: 10.1039/C6RA03455A
1H, Hꢀ3′′), 2.84ꢀ2.86 (m, 2H, Hꢀ4), 2.67 (brs, 2H, Hꢀ1), 2.62 (s, 1H, ꢀCHꢀpropargyl), 2.49
13
(t, J = 8.0 Hz, 2H, Hꢀ3′), 2.07ꢀ2.10 (m, 2H, Hꢀ3′), 1.85 (brs, 4H, Hꢀ2 & Hꢀ3); C NMR
(100.5 MHz, MeODꢀd4) δ: 174.4, 170.3, 158.1, 155.7, 153.4, 148.3, 144.1, 135.7, 131.9,
126.0, 125.8, 124.3, 123.6, 122.3, 120.8, 120.3, 116.6, 114.2, 78.9, 71.6, 53.0, 49.3, 32.6,
32.1, 30.9, 29.2, 25.8, 24.1, 22.1, 18.5; MSꢀESI (m/z): 592 (M+), 593(M+1)+, 594
(M+2)+; Analysis calc. for C30H30N5O2S2Cl·0.98H2O·0.9EtOH: C 58.64, H 5.78, N
10.75%; found: C 58.06, H 5.34, N 11.07%.
4.1.7.8. Nꢀ(1ꢀ(Benzo[d]thiazolꢀ2ꢀylamino)ꢀ1ꢀoxoꢀ3ꢀ(propꢀ2ꢀynylthio)propanꢀ2ꢀyl)ꢀ6ꢀ
(6ꢀchloroꢀ1,2,3,4ꢀtetrahydroacridinꢀ9ꢀylamino)hexanamide (10h)
2ꢀAminoꢀNꢀ(benzo[d]thiazolꢀ2ꢀyl)ꢀ3ꢀ(propꢀ2ꢀynylthio)propanamide (9b) and 6ꢀ(6ꢀ
chloroꢀ1,2,3,4ꢀtetrahydroacridinꢀ9ꢀylamino)hexanoic acid (4d) afforded the pure title
o
1
product as a light yellow color solid; yield 70%; m.p. 98ꢀ106 C; H NMR (400 MHz,
MeODꢀd4) δ: 8.10 (d, J = 8.0 Hz, 1H, Hꢀ8), 7.75 (d, J = 8.0 Hz, 1H, Hꢀ4′′′), 7.68ꢀ7.70 (m,
2H, Hꢀ5 & Hꢀ7′′′), 7.39 (t, J = 8.0 Hz, 1H, Hꢀ5′′′ or Hꢀ6′′′), 7.33 (d, 1H, J = 8.0 Hz, Hꢀ7),
7.27 (t, J = 8.0 Hz, 1H, Hꢀ6′′′ or Hꢀ5′′′), 4.83 (t, 1H, J = 8.0 Hz, Hꢀ2′′), 3.59 (t, 2H, J =
8.0 Hz, Hꢀ1′), 3.36 (s, 2H, ꢀSCH2ꢀpropargyl), 3.18ꢀ3.23 (m, 1H, Hꢀ3′′), 3.03ꢀ3.06 (m, 1H,
Hꢀ3′′), 2.92 (brs, 2H, Hꢀ4), 2.66 (brs, 2H, Hꢀ1), 2.62 (m, 1H, ꢀCHꢀpropargyl), 2.32 (t, J =
8.0 Hz, 2H, Hꢀ5′), 1.88 (brs, 4H, Hꢀ2 & Hꢀ3), 1.67ꢀ1.73 (m, 4H, Hꢀ2′ & Hꢀ4′), 1.42ꢀ1.48
(m, 2H, Hꢀ3′); 13C NMR (100.5 MHz, DMSOꢀd6) δ: 173.0, 170.9, 158.2, 158.0, 151.8,
148.8, 146.4, 133.7, 131.9, 126.6, 126.3, 125.5, 124.1, 122.1, 121.0, 118.2, 115.5, 80.6,
74.3, 52.8, 48.1, 35.2, 32.9, 30.6, 26.3, 25.3, 25.2, 22.8, 22.3, 19.1; MSꢀESI (m/z): 620
(M+), 620 (M+1)+, 621 (M+2)+; Analysis calc. for C32H34N5O2S2Cl·1.1 EtOH: C 61.23, H
6.10, N 10.44%; found: C 61.57, H 6.15, N 9.87%.
4.2. Molecular Modeling
To perform our docking studies the Xꢀray crystallographic structure of Torpedo
californicaꢀAChE (TcAChE) complexed with an inhibitor was taken from RCSB Protein
Data Bank (PDB entry 1ODC), in order to be used as receptor. This structure was chosen
because of the similarity between its original inhibitor ((Nꢀ4'ꢀquinolylꢀN'ꢀ9"ꢀ(1",2",3",4"ꢀ
tetrahydroacridinyl)ꢀ1,8ꢀdiaminooctane) and our ligands: all have a tacrine moiety and an
aromatic group linked through an alkyl chain.39 The original structure was treated using
21

RSC Advances
Page 22 of 35
View Article Online
DOI: 10.1039/C6RA03455A
Maestro v. 9.340 by removing the original ligand, solvent, and cocrystallization
molecules, and then adding the hydrogen atoms. The ligands were built using Maestro,
and then, using Ghemical v. 2.041 they were submitted to random conformational search
(RCS) of 1000 cycles, and 2500 optimization steps using Tripos 5.2 force field.42 The
minimized ligands were docked into the AChE structure with GOLD software v. 5.1,43
and the zone of interest was defined as the residues within 10 Å from the original position
of the ligand in the crystal structure. The ‘allow early termination’ option was
deactivated, and the remaining default parameters of Gold were used. The ligands were
subjected to 100 genetic algorithm steps using ASP as fitness function.
4.3. Prediction of pharmacokinetic properties
To analyze the potential of the new compounds as new antiꢀAD drugs, a brief prediction
on pharmacokinetic proprieties was performed in silico. Parameters such as the lipoꢀ
hydrophilic character (clogP), blood–brain barrier partition coefficient (log BB), the
ability to be absorbed through the intestinal tract (Cacoꢀ2 cell permeability) and CNS
activity were calculated.
The ligands were built and minimized as previously mentioned for the docking studies.
The structures were submitted to the calculation of these relevant pharmacokinetic
proprieties and descriptors using QikProp v. 2.5.36 These predictions are for orally
delivered drugs and assume nonactive transport
4.4. Antioxidant activity:
The antioxidant activity was evaluated by the DPPH method previously described.31 To a
2.5ꢀmL solution of DPPH (0.002%) in methanol, four samples of each compound in
solution were added in different volumes to obtain different concentrations in a 3.5ꢀmL
final volume. The samples were incubated for 30 min at room temperature.
The absorbance was measured at 517 nm against the corresponding blank (methanol).
The antioxidant activity was calculated by an Eq. (3).44
(3)
22

Page 23 of 35
RSC Advances
View Article Online
DOI: 10.1039/C6RA03455A
The tests were carried out in triplicate. The compound concentrations providing 50% of
antioxidant activity (EC50) were obtained by plotting the antioxidant activity against the
compound concentration.
4.5. Acetylcholinesterase activity
The enzymatic activity TcACHE was measured using an adaptation of the method
previously described.15 The assay solution contained 374 µL of Hepes buffer (50 mM and
pH8.0), a variable volume (10–50 µL) of the stock solution of each compound in
methanol (1 mg/mL), 25 µL of AChE stock solution, and the necessary amount of
methanol to attain 0.925 mL of the sample mixture in a 1ꢀmL cuvette. The samples were
left to incubate for 15 min, and then, 75 µL of acetylthiocholine iodide (AChI) solution
(16 mM) and 476 µL of DTNB (3 mM), were added. The reaction was monitored for 5
min at 405 nm. Assays were run with a blank containing all the components except
AChE, which was replaced by Hepes buffer. The velocities of the reaction were
calculated as well as the enzyme activity. A control reaction was carried out using the
sample solvent (methanol) in the absence of any tested compound, and it was considered
as 100% activity. The percentage inhibition of the enzyme activity due to the presence of
increasing test compound concentration was calculated by the following Eq. (1)
%I = 100ꢀ(vi /vo × 100)
(1)
in which vi is the initial reaction rate in the presence of inhibitor and vo is the initial rate
of the control reaction. The inhibition curves were obtained by plotting the percentage of
enzymatic inhibition versus inhibitor concentration, and a calibration curve was obtained
from which the linear regression parameters were obtained.
4.6. Inhibition of selfꢀmediated and Cu(II)ꢀinduced Aβ (1–42) aggregation
Amyloidꢀβ peptide (1–42) (Aβ42) was purchased from Aldrich as a lyophilized powder
and stored at ꢀ20 °C. The samples were treated with 1,1,1,3,3,3ꢀhexafluoropropanꢀ2ꢀol
(HFIP) to avoid selfꢀaggregation and reserved. HFIP pretreated Aβ42 samples were reꢀ
solubilized with a CH3CN/Na2CO3 (300 µM)/NaOH (250 µM) (48.3:48.3:4.3,v/v/v)
solvent mixture in order to have a stable stock solution. This Aβ42 alkaline solution (500
µM) was diluted in phosphate buffer (0.215 M, pH 8.0) to obtain a 40 µM solution. Due
to the hydrophobic nature of the compounds under study, they were first dissolved in
23

RSC Advances
Page 24 of 35
View Article Online
DOI: 10.1039/C6RA03455A
methanol (1 mg/mL) and then further diluted in phosphate buffer to a final concentration
of 480 µM.
To study the Aβ42 aggregation inhibition, a reported method, based on the fluorescence
emission of thioflavin T (ThT), was followed.16,28,29 It is based on the assumption that
reduction of the ThT fluorescence, due to competition of the compounds for the peptide
interaction, imply reduced Aβ aggregation. To study the effects of the compounds on
copperꢀinduced Aβ1ꢀ42 aggregation, solutions of Cu(II) (200 ꢁM) in phosphate buffer (0.2
M, pH 8.0) were prepared from a CuCl2 stock solution (0.015 M). The assay consisted on
the incubation of Aβ1ꢀ42 (40 ꢁM) with or without Cu(II) (40 ꢁM) in phosphate buffer with
or without the ligands (80 ꢁM). The incubation was performed at 37º C, for 24 h, and for
the control, a sample of the peptide was incubated under identical conditions but without
the inhibitor. After incubation, the samples were added to a 96ꢀwell plate (BD Falcon)
with 180 µL of 5 µM ThT in 50 mM glycineꢀNaOH (pH 8.5) buffer. Blank samples were
prepared for each concentration in a similar way, devoid of peptide. After 5ꢀmin
incubation with the dye, the ThT fluorescence was measured using a Spectramax Gemini
EM (Molecular Devices) at the following wavelengths: 446 nm (excitation) and 485 nm
(emission). The percent inhibition of aggregation due to the presence of the test
compound was calculated by Eq. (2), in which IFi and IF0 corresponded to the
fluorescence intensities, in the presence and the absence of the test
I% = 100ꢀ(IFi/IF0 ×100)
(2)
compound, respectively, minus the fluorescence intensities due to the respective blanks.
The reported values were obtained as the mean ± SEM of duplicate of two different
experiments.
4.7. MAOs Inhibition
MAO activity was measured in 96ꢀwell plates (triplicate wells) by coupling the
production of hydrogen peroxide is via horseradish peroxidase to a dye producing the
fluorescent resorufin.35,45 Membraneꢀbound human MAO A and MAO B, horseradish
peroxidase, and Ampliflu Red were obtained from SigmaꢀAldrich (UK). For reversible
inhibition, MAO was added last to the assay mixture containing (at final concentrations)
20 µM Ampliflu Red, 1 U/mL horseradish peroxidase, 1 mM tyramine. To see whether
there was irreversible inhibition; MAO and inhibitor were preincubated in 100 µL
24

Page 25 of 35
RSC Advances
View Article Online
DOI: 10.1039/C6RA03455A
phosphate buffer pH 7.5 at 30 °C for 30 min before adding 100 µL containing the rest of
the assay components to start the reaction. Solubility limited the maximum concentration
of inhibitor to 100 µM. The data were analyzed in Graphpad PRISM 4 using the
sigmoidal equation constrained to a bottom value of zero.
4.8. Cells and treatments
Human Neuroblastoma SHꢀSY5Y cell line was purchased from ATCCꢀCRLꢀ2266.
Briefly, the cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) obtained
from GibcoꢀInvitrogen (Life Technologies Ltd, UK) with 10% heat inactivated fetal calf
serum, containing 50 U/mL penicillin, and 50 ꢁg/mL streptomycin, under a humidified
atmosphere of 95% airꢀ 5% CO2 at 37 ºC. Cells were plated at 0.1 × 106 cells/mL for
MTT cell proliferation assay. Medium was changed after 24 h and immediately before
treatments.
The tested compounds (10a, 10b, 10c, 10d, 10e, 10f, 10g, 10h) were dissolved in DMSO
at a concentration of 25 mM and aliquots were stored at ꢀ20 ˚C. The compounds 10a, 10d
and 10h were added to the medium at 2.5 ꢁM final concentration; the compounds 10b
10c, 10e, 10f, 10g were added to the medium at 1 ꢁM final concentration; KC1 was
added to the medium at 10 nM final concentration. The final concentration of DMSO in
culture media did not exceed 0.05% (v/v) and no alterations on cells were observed. Cells
were preꢀincubated for 1 h with the compounds and then incubated with Aβ42 or H2O2 for
another 24 h. Aβ42 was prepared as 276.9 ꢁM stocks in sterile water and added to the
medium to 1 ꢁM final concentration. H2O2 was freshly prepared as 5 mM stocks in water
and added to the medium to 100 ꢁM final concentrations. Aβ42 was purchased from
American Peptide (Sunnyvale, CA, USA) and H2O2 from Sigma Chemical Co (St. Louis,
MO, USA). For all conditions tested, control experiments were performed in which the
compounds tested, Aβ42 or H2O2 were not added.
MTT cell proliferation assay
Cell proliferation was determined by the colorimetric MTT (3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ
2,5ꢀdiphenyltetrazolium bromide) assay.46 In viable cells, MTT is metabolized into a
formazan that absorbs light at 570 nm. Following the cell treatment protocol the medium
was aspirated and 0.5 mL MTT (0.5 mg/ml) was added to each well. The plate was then
25

RSC Advances
Page 26 of 35
View Article Online
DOI: 10.1039/C6RA03455A
incubated at 37 °C for 3 h. At the end of the incubation period the formazan precipitates
were solubilized with 0.5 mL of acidic isopropanol (0.04 M HCl/isopropanol). The
absorbance was measured at 570 nm. Cell reduction ability was expressed as a percentage
of the untreated cells.
All data result from the analysis of duplicates per experimental condition in at least three
independent experiments and are expressed as the mean±SEM. Statistical analyses were
performed using GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA).
Statistical tests between multiple data sets and conditions were carried out using a oneꢀ
way analysis of variance (ANOVA) with pairwise multiple comparison procedures using
the post hoc Bonferroni’s test to determine statistical significance, as appropriate. A p
value <0.05 was considered statistically significant.
Acknowledgements. The authors acknowledge the Portuguese Fundação para a Ciência
e Tecnologia (FCT) for the project UID/QUI/00100/2013, postdoctoral fellowships (RSK
and KC), and also the Portuguese NMR (ISTꢀUL Center) and Mass Spectrometry
Networks (Node ISTꢀCTN) for providing access to their facilities. We also thank the
doctoral fellowship from Erasmus Namaste program (AH) and PꢀL. Joffrin for his
contribution to the MAO bioassays at the University of St Andrews.
References
1 Alzheimer’s Association, Alzheimer´s Dement., 2014, 10, 47.
2
S. Oddo, A. Caccamo, J. D. Shepherd, M. P. Murphy, T. E. Golde, R. Kayed, R. Metherate, M. P.
Mattson, Y. Akbari and F. M. LaFerla, Neuron., 2003, 39, 409.
3 S. Ayton, P. Lei and A. I. Bush, Free Radical Biol. Med., 2013, 62, 76.
4 J. L. Cummings, M. Travis and K. Zhong, Alzheimer´s Res. Ther., 2014, 6, 37.
5
A. Cavalli, M. L. Bolognesi, A. Minarini, M. Rosini, V. Tumiatti, M. Recanatini and C. Melchiorre, J.
Med. Chem., 2008, 51, 347.
6
H. Zheng, T. Amit, O. BarꢀAm, M. Fridkin, M. B. H. Youdim and S. A. Mandel, J. Alzheimer’s Dis.
2012, 30, 1.
7 A. Mattevi, C. Binda, M. Li, F. Hubálek, Curr. Med. Chem., 2004, 11, 1983.
26

Page 27 of 35
RSC Advances
View Article Online
DOI: 10.1039/C6RA03455A
8 M. B. Youdim, and J. J. Buccafusco, Trends Pharmacol. Sci., 2005, 26, 27.
9
E. Nepovimova, E. Uliassi, J. Korabecny, L. E. PeñaꢀAltamira, S. Samez, A. Pesaresi, G. E. Garcia, M.
Bartolini, V. Andrisano, C. Bergamini, R. Fato, D. Lamba, M. Roberti, K. Kuca, B. Monti and M. L.
Bolognesi, J. Med. Chem., 2014, 57, 8576.
10 R. Leon, A.G. Garcia and J. MarcoꢀContelles, Med. Res. Rev., 2013, 33, 139.
11 G. Small and R. Bullock, Alzheimer´s Dement., 2011, 7, 177.
12 M. L. Crismon, Ann. Pharmacother., 1994, 28, 744.
13
A. Romero, R. Cacabelos, M. J. OsetꢀGasque, A. Samadi and J. MarcoꢀContelles, Bioorg. Med. Chem.
Lett., 2013, 23, 1916.
14 R. S. Keri, C. Quintanova, S. M. Marques. A. R. Esteves, S. M. Cardoso and M. A. Santos, Bioorg. Med.
Chem., 2013, 21, 4559.
15
A. Nunes, S. M. Marques, C. Quintanova, D. F. Silva, S. M. Cardoso, S. Chaves, M. A. Santos, Dalton
Trans., 2013, 42, 6058.
16
C. Quintanova, R. S. Keri, S. M. Marques, M. GꢀFernandes, S. M. Cardoso, M. L. Serralheiro, M. A.
Santos, Med. Chem. Comm., 2015, 6, 1969.
17 A. Dairam, R. Fogel, S. Daya and J. L. Limson, J. Agric. Food Chem., 2008, 56, 3350.
18 J. M. Kim H. J. Chang, W. K. Kim, N. Chang and H. S. Chun, J. Agric. Food Chem., 2006, 54, 6547.
19
R. S. Keri, C. Quintanova, S. Chaves, D. F. Silva, S. M. Cardoso and M. A. Santos, Chem. Biol. Drug
Des. 2016, 87, 101.
20 C. Quintanova, R. S. Keri, S. Chaves and M. A. Santos, J. Inorg. Biochem., 2015, 151, 58.
21 C. Lu, Q. Zhou, J. Yan, Z. Du, L. Huang and X. Li, Eur. J. Med. Chem., 2013, 62, 745.
22 S. Noel, S. Cadet, E. Gras and C. Hureau, Chem. Soc. Rev., 2013, 42, 7747.
23 H. Dvir, I. Silman, M. Harel, T. L. Rosenberry and J. L. Sussman, ChemꢀBiol. Interact. 2010, 187, 10.
24 PDB, entry 1ODC. http://www.rcsb.org/pdb/explore/explore.do?structureId=1ODC.
25 E. H. Rydberg, B. Brumshtein, H. M. Greenblatt, D. M. Wong, D. Shaya, L. D. Williams, P. R. Carlier,
Y. Pang, I. Silman and J. L. Sussman, J. Med. Chem., 2006, 49, 5491.
26 L. J. Sargent and L. J. Small, Org. Chem., 1946, 11, 359.
27 M. K. Hu and C. F. Lu, Tetrahedron Lett., 2000, 41, 1815.
28
M. Bartolini, C. Bertucci, M. L. Bolognesi, A. Cavalli, C. Melchiorre and V. Andrisano, Chem.
BioChem., 2007, 8, 2152.
27

RSC Advances
Page 28 of 35
View Article Online
DOI: 10.1039/C6RA03455A
29 X. Chao, X. He, Y. Yang, X. Zhou, M. Jin, S. Liu, Z. Cheng, P. Liu, Y. Wang, J. Yu, Y. Tan, Y. Huang,
J. Qin and S. Rapposelli, Bioorg. Med. Chem. Lett., 2012, 22, 6498.
30 A. Hawe, M. Sutter, W. Jiskoot, Pharm. Res., 2007, 25, 1487.
31 B. Tepe, D. Daferera, A. Sokmen, M. Sokmen and M. Polissiou, J. Agric. Food. Chem., 2005, 90, 333.
32 Q. H. Gong, Q. Wang, L. L. Pan, X. H. Liu, H. Xin and Y. Z. Zhu, Brain Behav. Immun., 2011, 25, 110.
33 S. M. Denecke, Curr. Top Cell Regul., 2000, 36, 151.
34 M. J. Zhou, Z. J. Diwu, N. PanchukVoloshina and R. P. Haugland, Anal. Biochem., 1997, 253, 162.
35
G. Esteban, J. Allan, A. Samadi, A. Mattevi, M. Unzeta, J. MarcoꢀContelles, Claudia Binda, Rona R.
Ramsay, Biochim. Biophys. Acta, Proteins Proteomics, 2014, 1844, 1104.
36 QikProp, version 2.5, Schrödinger, LLC, New York, NY, 2005.
37 C. A. Lipinski, F. Lombardo, B. W. Dominy and P. J. Feeney, Adv. Drug Deliv. Rev., 1997, 23, 3.
38
W. L. F. Armarego, D. D. Perrin, Purification of Laboratory Chemicals, 4th ed.; Butterworthꢀ
Heinemann: Oxford, 1999.
39
E. H. Rydberg, B. Brumshtein, H. M. Greenblatt, D. M. Wong, D. Shaya, L. D. Williams, P. R. Carlier
Y. Pang, I. Silman and J. L. Sussman, J. Med. Chem., 2006, 49, 5491.
40 Maestro, version 9.3. Schrödinger Inc.: Portland, OR, 2012.
41 T. Hassinen and M. J. Peräkylä, Comput. Chem., 2001, 22, 1229.
42 M. Clark, R. D. Cramer III and N. Van Opdenbosch, J. Comput. Chem., 2004, 10, 982.
43 G. Jones, P. Willett, R. C. Glen, A. R. Leach and R. Taylor, J. Mol. Biol., 1997, 67, 727.
44
J. Sebestík, S. M. Marques, P. L. Falé, S. Santos, D. M. Arduíno, S. M. Cardoso, C. R. Oliveira, M. L.
Serralheiro and M. A. Santos, J. Enzyme Inhib. Med. Chem., 2011, 26, 485.
45 R. R. Ramsay, A. Olivieri and A. Holt, J. Neural Transm., 2011, 118, 1003.
46 T. Mosmann, J. Immunol. Methods, 1983, 65, 55.
28

Page 29 of 35
RSC Advances
View Article Online
DOI: 10.1039/C6RA03455A
Scheme, Figs and tables
Scheme 1. Reagents and conditions (i) POCl3, 180 oC, reflux, 3 h; (ii) 2 equiv H2N(CH2)nCO2H,
phenol, KI, reflux, 4 h; (iii) allyl bromide (iiiꢀ1) or propargyl bromide (iiiꢀ2), NH4OH (2M), r.t.,
3 h; (iv) diꢀtertꢀbutyl dicarbonate and Na2CO3, THF, r.t., 5 h; (v) 1.5 equiv T3P, 2.5 equiv NMM,
CH2Cl2, rt, 3 h; (vi) TFA, CH2Cl2, rt, 2 h; (vii) 1.5 equiv T3P, 2.5 equiv NMM, CH2Cl2, r.t., 3 h;