Multi-Targeting Tacrine Conjugates with Cholinesterase and Amyloid-Beta Inhibitory Activities: New Anti-Alzheimer's Agents

Article abstract of DOI:10.1002/cbdv.202000083

Alzheimer's disease (AD) is a severe age dependent and chronic problem with no cure so far. The available treatments are temporary, acting over short period of time. The main pathological hallmark of the disease includes cholinergic dysfunction, oxidative stress, accumulation of Aβ fibrils and tau tangles. In context with the multi-factorial nature of this disease, two different series of molecules were developed to hit the multifactorial disease targets. Mainly, the molecules were designed to inhibit the AChE and aggregation of Aβ, and also oxidative damage. Two novel series of TAC-fenbufen/menbutone conjugated molecules were designed, synthesized and bio-assayed. All compounds showed inhibition capacity towards AChE, Aβ aggregation and moderate to good radical scavenging capacity. Particularly, five TAC-menbutone molecules showed improved AChE and Aβ aggregation inhibition capacity compared to TAC-fenbufen conjugated molecules. Overall, these novel series of molecules may be potential drug lead molecules in the treatment of AD.

Full text of DOI:10.1002/cbdv.202000083

DOI: 10.1002/cbdv.202000083
FULL PAPER
Multi-Targeting Tacrine Conjugates with Cholinesterase and
Amyloid-Beta Inhibitory Activities: New Anti-Alzheimer’s Agents
Asha Hiremathad,a, b Sílvia Chaves,b and Rangappa S. Keri*a
a
Organic and Medicinal Chemistry Laboratory, Center for Nano and Material Sciences, Jain University, Jain
Global Campus, Jakkasandra Post, Kanakapura Road, Ramanagara District, Karnataka-562112, India,
e-mail: keriphd@gmail.com; sk.rangappa@jainuniversity.ac.in
Centro de Química Estrutural and Departamento de Engenharia Química, Instituto Superior Técnico,
b
Universidade de Lisboa, Av. Rovisco Pais 1, 1049-001 Lisboa, Portugal
Alzheimer’s disease (AD) is a severe age dependent and chronic problem with no cure so far. The available
treatments are temporary, acting over short period of time. The main pathological hallmark of the disease
includes cholinergic dysfunction, oxidative stress, accumulation of Aβ fibrils and tau tangles. In context with the
multi-factorial nature of this disease, two different series of molecules were developed to hit the multifactorial
disease targets. Mainly, the molecules were designed to inhibit the AChE and aggregation of Aβ, and also
oxidative damage. Two novel series of TAC-fenbufen/menbutone conjugated molecules were designed,
synthesized and bio-assayed. All compounds showed inhibition capacity towards AChE, Aβ aggregation and
moderate to good radical scavenging capacity. Particularly, five TAC-menbutone molecules showed improved
AChE and Aβ aggregation inhibition capacity compared to TAC-fenbufen conjugated molecules. Overall, these
novel series of molecules may be potential drug lead molecules in the treatment of AD.
Keywords: Alzheimer’s disease, tacrine, acetylcholinesterase inhibitors, Aβ self-aggregation inhibitors.
effective drugs to tackle this disease. Unfortunately,
there are frequent failures due to the unclear patho-
Introduction
Alzheimer’s disease (AD) is the most common form of genesis and multi-factorial nature of AD.[3] Some
dementia and chronic, progressive age-related neuro- important characteristics of AD are extracellular depo-
degenerative disorder. The characteristics of the dis- sition of misfolded amyloid-β (Aβ),[4,5] and intercellular
ease are impaired cognition, behavioral disturbances, accumulation of neurofibrillary tangles (NFT)[6] due to
and memory loss, resulting in difficulties to perform hyperphosphorylation of the tau protein which are
regular activities. This has become a devastating toxic materials to the neurons, leading to neuronal
problem to the society, being estimated that more death. Cholinergic dysfunction also occurs,[7,8] due to
than 13.2 million people will be affected from AD by the rapid hydrolysis of acetylcholine (ACh) by acetyl-
2050, and also this illness has no cure so far. It is cholinesterase (AChE). Other substantial causes for the
expected that the treatment for AD and other disease growth are oxidative damage[9] and variation
dementia will cost over $259 billion, which could even in the homeostasis of metal ions which enhances Aβ
raise up to $1.1 trillion by 2050.[1] Most of the available aggregation leading to AD development.[10–12] It has
drugs are AChE inhibitors,[2] which are symptomatic been hypothesized that the cholinergic dysfunctions,
and work over a short period of time. Thus, many probably induced by formation of extracellular Aβ
researchers across the globe attempted to discover plaques and tau tangles, may be formed in the
hippocampus and cerebral cortical areas. Similarly, it is
also hypothesized that neurodegeneration may occur
at the cholinergic terminal due to Aβ plaques.[13,14]
However, it remains unclear whether cholinergic
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Chem. Biodiversity 2021, 18, e2000083
dysfunctions or Aβ plaques formation are primary Results and Discussion
events causing neuronal losses in the brain. Therefore,
to tackle this disease, it is very important to focus on
Chemistry
more than one target of AD.
The designed tacrine-fenbufen (TAC-fenbufen) and
In this framework, the extended research of our tacrine-menbutone (TAC-menbutone) molecules were
group has made an attempt to find out novel prepared by the synthetic route shown in Scheme 2.
derivatives to treat AD.[15] Herein, we present hybrids The 9-chloro-1,2,3,4-tetrahydroacridine (3) was pre-
of tacrine (TAC)-fenbufen/menbutone by using an pared from commercially available anthranilic acid/
amide linker to couple TAC with two main scaffolds, substituted anthranilic acid (1), with cyclohexanone
biphenyl acid and methoxy naphthalene acid, which (2).[13] Further, compound 3 was treated with alkyl-
are pharmacophore entities. The TAC was selected to diamines 4a–4c, using a catalytic amount of KI and
endeavor the hybrids with acetylcholine inhibition phenol, to give the TAC amines 5a–5f.[13] Simulta-
(AChEI),[15,16] while the other two main moieties were neously, synthesis of two other main entities (8, 11)
introduced to improve the inhibition of Aβ self- were carried out by substitution of alkyl acids to
aggregation (Scheme 1). Inclusion of fenbufen and biphenyl (6) and 1-methoxynaphthalene (10) with
menbutone moieties with adequate chain length will succinic anhydride (7) correspondingly to yield fenbu-
allow interaction with peripheral anionic site (PAS) of fen (8) and menbutone (11). Finally, TAC amine
AChE enzyme which is at the entrance side. Eventually, precursors 5a–5f were coupled with acids (8, 11),
these new series of compounds will improve the respectively, by using T3P�50 wt.% in ethyl acetate as
capacity of AChE inhibition and may delay the self- a coupling reagent at inert conditions for about 12 h
aggregation of Aβ protein. Therefore, molecular dock- at room temperature (r.t.) resulting in the novel TAC-
ing simulation studies were performed to select the fenbufen 9a–9d and TAC-menbutone 12a–12f con-
appropriate chain length and it is evidenced a good jugated molecules.
interaction of the molecules with the targeted AChE
enzyme and superimposition with the original ligand
(tacrine dimer). Furthermore, the synthesized com-
Molecular Modeling and Docking Simulations
pounds were assayed for their biological properties, The strategy herein adopted proposed to develop
such as inhibition of AChE, Aβ aggregation and radical active molecules to inhibit the targeted enzyme. The
scavenging activity.
two main scaffolds fenbufen and menbutone mole-
Scheme 1. Design of TAC-fenbufen and TAC-menbutone conjugates.
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Chem. Biodiversity 2021, 18, e2000083
°
Scheme 2. Synthesis of TAC-fenbufen/menbutone conjugated molecules. Reagents and conditions: i) POCl3, 180 C, reflux, 4 h; ii)
°
phenol, KI, 180 C, reflux, 35 min; iii) EDC, AlCl3, r.t. 4 h; iv) T3P, NMM, DCM, N2 atm, r.t., 12 h.
cules are pharmacophore entities, coupled with TAC. action with the targeted enzyme in CAS and PAS
The TAC moiety was selected to assure AChE inhib- showing that these designed molecules could inhibit
ition, while the other two main moieties were the enzyme (Figure 1). The TAC moiety in the CAS side
conjugated simultaneously to improve the inhibition showed good πÀ π stacking with Trp84 and blocks of
of Aβ self-aggregation. Additionally, the coupling of other amino acids (Ser 200, His440 and Glu327). In PAS
these two moieties with an adequate linker size could binding site, the selected biphenyl ring is in πÀ π
improve the targeted enzyme inhibition. Therefore, stacking with the two methylene linker (n=2) 9a and
the molecular simulation studies herein performed 9d, while the molecule with three methylene linker
allowed in selecting the appropriate chain length. The (n=3) chain length is quite longer than the original
structure obtained for this hybrid-enzyme complex is ligand and slightly out of PAS, even though it interacts
similar to the known crystal structure of TcAChE with amino acids namely, Trp279 and Tyr70. Interest-
complexed with (N-4’-quinolyl-N’-9“-(1”,2“,3”,4“-tetra- ingly, most of the hybrids showed H-bonding with
hydroacridinyl)-1,8-diaminooctane;
PDB
entry Tyr121 mainly, compounds 9a and 9b with shortest
1ODC).[16] Hence, the interaction of the designed bonding distance (2.612 Å and 2.180 Å, respectively)
molecules with AChE was studied and compared with (Figure 1). In vitro studies revealed that among all,
the original ligand 1ODC.
compound 9b (n=3) exhibited good inhibitory ca-
In the case of TAC and fenbufen hybrids, the pacity towards AChE, maybe explained by the fact that
methylene linkers n=2, 3 correspond to adequate the extended biphenyl ring is interacting well with the
chain lengths and the compound can be exactly fitted amino acid residue Tyr279. Overall, novel TAC-fenbu-
into the enzyme gorge and along that the active site; fen conjugated molecules 9a–9d are able to moder-
compounds 9a–9d exhibited πÀ π stacking and inter- ately inhibit the AChE.
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Chem. Biodiversity 2021, 18, e2000083
Figure 1. Docking results for the TAC-fenbufen with TcAChE: (A) superimposition of 9a (yellow) with original ligand (N-4’-quinolyl-
N’-9“-(1”,2“,3”,4“-tetrahydroacridinyl)-1,8-diaminooctane; PDB entry 1ODC); (B) superimposition of 9b (green) with original ligand.
The molecular simulations of TAC-menbutone parent drug tacrine. Particularly, compounds 12b and
hybrids showed that they exhibit good superimposi- 12e presented the best AChE inhibition and they also
tion between the original ligand (PDB entry 1ODC) exhibited good inhibition capacity towards Aβ self-
and the selected pharmacophore (menbutone), which aggregation (Figure 2B,C). In general, the docking
means that the designed molecules could act as AChE studies provided good information between the
inhibitors. Here, the chain length was modulated in designed drug and the targeted enzyme, which
accordance with the superimposition of the designed showed that the designed molecules could be able to
hybrids with the original ligand and the enzyme inhibit AChE.
gorge. The docking studies showed that the designed
hybrids with chain linkers (n=2–4) form good πÀ π
Pharmacokinetic Properties
stacking interaction with the targeted enzyme. Re-
markably, the hybrids with chain linkers (n=3, 4) To endeavor the drug-likeness of the novel com-
showed good interaction and adequate linker size to pounds and their potential to penetrate important
insert into enzyme gorge. For the molecule with the membranes such as the blood-brain barrier (BBB),
shortest linker (n=2, 12a) an H-bond between some indicators of their pharmacokinetic profiles were
carbonyl oxygen of menbutone and À OH of Tyr121 predicted using QikProp program, v. 2.5.[17] Parameters
(2.056 Å) is formed. Although compounds which such as the calculated octanol-water partition coef-
establish H-bonding are having usually good AChE ficient (clog P), the ability to cross the blood-brain-
inhibition capacity, in fact these molecules displayed barrier BBB (log BB), the capacity to be absorbed
low inhibition capacity in in vitro measurements (Fig- through the intestinal tract to the blood (Caco-2 cell
ure 2A). For the compounds with longer linker (n=3) permeability) and the verification of Lipinski’s rule of
12b and 12e, very good AChE inhibition capacity was five, may give an idea of their drug-likeness for being
exhibited, almost two fold higher than that for the orally active as anti-AD agents.[18]
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Chem. Biodiversity 2021, 18, e2000083
Figure 2. Docking results for the TAC-menbutone hybrids with TcAChE: (A) superimposition of 12a (green) with original ligand (N-
4’-quinolyl-N’-9“-(1”,2“,3”,4“-tetrahydroacridinyl)-1,8-diaminooctane; PDB entry 1ODC); (B) superimposition of 12b (cyan) with
original ligand; (C) superimposition of 12e (yellow) with original ligand.
Newly designed tacrine-fenbufen and tacrine-men- Caco-2. This indicates that these compounds can pass
butone conjugated hybrids were studied for drug through blood brain barrier and have good ability to
likeliness in silico by using QikProp v. 2.5.[17] The be absorbed through the intestinal tract to the blood.
compounds have zero to two violations of Lipinski Values of Caco-2 permeability >500 are considered
rule.[18] Namely, compound 12a has zero violations good values for cell permeability, and the herein
and most of the compounds have one to two studied compounds have shown values in the range
violations due to the higher value of molecular weight 591 to 1046 nm/s (Table 1). As a conclusion, it can be
(�500 g/mol) and lipo-hydrophilic character (>5) stated that these designed molecules have shown
according to the Lipinski rule.[18] Interestingly, the promising results for drug likeliness and therefore, it
designed molecules have good value of logBB and can be predicted that they are orally active agents.
Table 1. Summary of the calculated pharmacokinetic descriptors.
Compound
MW
clogP[a][b]
log BB[a][c]
Caco-2 Permeability
(nm/s)[a]
CNS[a] activity
Violations of
Lipinski rule[a]
9a
9b
9c
477.60
491.63
512.05
526.07
481.59
516.03
509.64
516.03
530.06
544.09
5.299
5.875
4.949
6.374
4.181
5.684
5.263
5.684
4.755
5.548
À 0.949
À 1.353
À 0.540
À 0.784
À 0.870
À 1.116
À 1.216
À 1.116
À 0.532
À 0.889
833
649
911
1015
854
661
591
661
1010
1046
À
1
1
1
2
0
2
2
2
1
2
À
+/À
À
9d
12a
12b
12c
12d
12e
12f
À
À
À
À
+/À
À
[a]Predicted values using program Qikprop v.2.5. [b]Calculated octanol/water partition coefficient. [c]Brain/blood partition coefficient.
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Chem. Biodiversity 2021, 18, e2000083
Radical Scavenging Capacity
ylene linker (n=3) showed improved inhibition ca-
pacity than those with the smaller chain linker (n=2),
Two series of molecules were screened for their radical which may be due to the better accommodation in
scavenging (RS) capacity by DPPH method.[19] This the enzyme active site and extended biphenyl ring
method was based on the radical scavenging capacity interaction with amino acid residue Tyr279 in PAS
of molecules and interaction with DPPH free radical. (Figure 1).
Firstly, TAC-fenbufen hybrids 9a–9d results revealed a
Further, TAC-menbutone hybrids 12a–12f showed
moderate scavenging capacity (EC50 =400 to 745 μM). two fold more inhibitory capacity than TAC. In fact,
However, compound 9d showed the highest RS compounds 12b (IC50 =0.12 μM) and 12e (IC50 =
capacity (EC50 =400 μM). Furthermore, TAC-menbu- 0.18 μM) showed the highest inhibitory capacity
tone hybrids 12a–12f were tested for radical scaveng- among these two series of hybrids. These two
ing capacity and better results were obtained for RS compounds 12b and 12e contain a three methylene
capacity (EC50 =220 to 452 μM). Mainly, compound linker (n=3) and showed the best AChE inhibition
12b exhibited the best RS capacity (EC50 =220 μM). which was evident from the molecular docking
The methoxy substitution improved the RS capacity of simulations, previewing a dual mode interaction (CAS
the molecules. Overall, these two series of newly and PAS). Specifically, the menbutone hybrids dis-
studied molecules displayed moderate to good radical played better AChE inhibition, being well inserted into
scavenging capacity by DPPH method.
the enzyme gorge, and establishing interactions with
amino acid residues and πÀ π stacking, the methoxy
substitution aiding in the enhancement of AChE
inhibition. Compounds with a longer chain linker (n=
Biological Studies
AChE inhibition. Newly synthesized two series of 4) exhibited less inhibitory capacity towards AChE.
molecules 9a–9d and 12a–12f are evaluated for However, all these newly developed hybrids showed
inhibition of TcAChE by a previously described improved AChE inhibition compared to the parent
method.[20,21] The IC50 values for inhibition of AChE are drug tacrine.
presented in Table 2. TAC-fenbufen hybrids 9a–9d,
showed good AChE inhibition, although few showed
better inhibitory capacity than the parent drug TAC.
Inhibition of Self-Mediated Aβ42 Aggregation
Compound 9b showed the highest inhibition (IC50 = To study the anti-amyloidogenic capacity of these
0.34 μM). Especially, compounds with a longer meth- novel TAC-fenbufen and TAC-menbutone hybrid mole-
Table 2. In vitro activities of the TAC-fenbufen hybrids 9a–9d and TAC-menbutone hybrids 12a–12f: Radical scavenging capacity
(DPPH), inhibition of AChE and Aβ42 self-aggregation.
Compound
n
R
RS Capacity[a]
EC50 μM
AChE Inhib[b]
IC50 (μM) � SD
Aβ42 self-aggreg.[c]
Inhibition (%)
9a
9b
9c
9d
12a
12b
12c
12d
12e
12f
Tacrine
Fenbufen
Menbutone
Trolox
2
3
2
3
2
3
4
2
3
4
À
H
H
Cl
Cl
H
H
H
Cl
Cl
Cl
745�0.14
450�0.08
542�0.14
440�0.10
242�0.16
220�0.20
452�0.12
316�0.32
248�0.20
324�0.18
>1000
0.42�0.08
0.34�0.05
0.57�0.06
0.45�0.09
0.28�0.07
0.12�0.03
0.32�0.08
0.22�0.06
0.18�0.1
0.28�0.08
0.38�0.02
0.97�0.07
0.85�0.08
À
33.5�1.2
28.2�2.2
46.2�1.4
42.6�2.8
45.6�0.8
70.5�1.2
55.0�1.6
76.2�1.8
64.5�2.4
34.5�0.8
22�1.6
Nd
>1000
>1000
Nd
Nd
15.6�0.2
[a]EC50 values for the DPPH assay. [b]The values are mean of five independent experiments � SD. [c]Inhibition of self-mediated Aβ42
aggregation (in %). The Thioflavin-T fluorescence method was used, and the measurements were carried out in the presence of an
inhibitor (80 μM). The values are the mean of two independent measurements in duplicate (SEM <10%). Nd – Not determined.
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Chem. Biodiversity 2021, 18, e2000083
cules, the in vitro ThT assay was performed by a Aβ42 inhibition capacity improved with chloro substitu-
previously described method.[22,23] The studies were tion (R=Cl) and biphenyl moiety which is interacting well
performed after incubation of Aβ42 with or without at PAS. Further, these exhibited moderate-good radical
ligands. The results are displayed in Table 2 and all the scavenging capacity (9d; EC50 =440 μM) (Figure 3).
compounds were able to reduce ThT fluorescence
associated with Aβ fibril binding, which indicates the menbutone molecules, two to three methylene chain
inhibition of the Aβ42 self-aggregation process. lengths are adequate to insert into the AChE enzyme
It was found that in the case of the TAC-
TAC-fenbufen hybrids 9a–9d exhibited a moderate gorge. However, molecules with three methylene
inhibition capacity towards Aβ42 aggregation. Mainly, chain length showed significant inhibition capacity
compounds with shorter methylene linker (n=2) and towards AChE and Aβ aggregation. The TAC moiety
chloro substitution on TAC moiety 9c and 9d showed coupled with resulted in an enhanced AChE and Aβ42
the best inhibition (up to 46% and 42%) than com- inhibition capacity upon coupling with menbutone.
pounds with a longer methylene linker (n=3). Further- Additionally, improved radical scavenging capacity
more, TAC-menbutone conjugated molecules were also was observed for the menbutone series of molecules
assayed for anti-Aβ aggregation capacity by ThT assay that could be due the methoxy group (12b; EC50 =
method. Interestingly, compounds 12a–12f presented 220 μM). In general, these series of molecules 12a–12f
good inhibitory capacity towards Aβ42 self-aggregation exhibited improved inhibition capacity when com-
(45 to 76%). Compound 12d displayed the highest pared to the TAC-fenbufen hybrids 9a–9d.
inhibition of Aβ42 aggregation (76%) and 12b also
exhibited a very good inhibition (70%). In fact, an
improved percentage of inhibition of Aβ42 aggregation
was observed for the molecules which also interact well
Conclusions
with PAS in the AChE active site. Additionally, com- Two novel series of TAC-fenbufen 9a–9d and TAC-
pounds 12c and 12e of the same series presented menbutone 12a–12f hybrids were developed and bio-
moderate to good inhibition capacity (55 and 64%, assayed such as inhibition of AChE and Aβ aggregation
respectively). Besides, no noticeable changes are ob- and radical scavenging capacity against Alzheimer’s
served with variations in chain length and chloro disease. Firstly, TAC-fenbufen hybrids exhibited good
substitution. Overall, among these two novel series, TAC- AChE activity as reference drug tacrine. Mainly, com-
menbutone hybrids displayed better inhibitory capacity pound 9b showed the highest AChE inhibition (IC50 =
for Aβ42 aggregation than TAC-fenbufen compounds.
0.34 μM), RS capacity was improved for these newly
conjugated molecules 9b–9d (EC50 =450 μM and
440 μM, respectively). Additionally, these displayed mod-
erate capacity to inhibit the aggregates of Aβ (9c; 46%).
Secondly, TAC-menbutone hybrids showed the best
Possible Structure-Activity Relationship (SAR) Studies of
TAC-Fenbufen/Menbutone Hybrids
Based on biological studies, herein it is predicted the inhibitory capacity of AChE and Aβ aggregation. This
possible SAR of the TAC-fenbufen hybrids: two methylene may be due to the menbutone moiety and methoxy
linker length (n=2) was appropriate and the AChE and substitution is interacting well at PAS. Especially, com-
Figure 3. Possible SAR study of TAC-fenbufen/menbutone hybrids.
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Chem. Biodiversity 2021, 18, e2000083
pounds 12b and 12e showed the best AChE inhibition were recorded on Bruker AVANCE III spectrometers at
(IC50 =0.12 and 0.18 μM, respectively) which is twofold 300 and 400 MHz, respectively. Chemical shifts (δ) are
more active than the parent drug tacrine. Additionally, reported in ppm from the standard internal reference
compounds 12b and 12d displayed very good inhibition tetramethylsilane (TMS). Mass spectra (ESI-MS) were
of Aβ (70% and 76%, respectively). Among all, com- performed on a 500 MS LC Ion Trap (Varian Inc., Palo
pound 12b showed the best RS capacity (EC50 = Alto, CA, USA) mass spectrometer equipped with an
220 μM). However, among these two series of molecules ESI ion source, operated in the positive ion mode. The
TAC-menbutone hybrids showed higher activity than electronic spectra were recorded with a PerkinElmer
TAC-fenbufen hybrids. Generally, the newly synthesized Lambda 35 spectrophotometer, using thermostatic
exhibited moderate to good capacity of inhibition 1 cm path length cells.
towards targeted important pathological targets and
interestingly, few of the molecules 12b, 12d, and 12e
presented promising multi-functional activity. Hence,
Molecular Modeling and Docking Simulations
further study of these would leads to development of To perform our docking studies the X-ray crystallo-
potential drugs to treat the AD.
graphic structure of Torpedo californica-AChE
(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
(N-4’-quinolyl-N’-9“-(1”,2“,3”,4“-tetrahydroacridinyl)-
Experimental Section
Abbreviations
AChE: acetylcholinesterase; AChEI: acetylcholinesterase 1,8-diaminooctane, PDB entry 1ODC) and our ligands:
inhibitors; AD: Alzheimer’s disease; Aβ: amyloid-β; CAS: all have a tacrine moiety and an aromatic group
cationic active site; DCM: dichloromethane; DPPH: di- linked through an alkyl chain. The original structure
(phenyl)-(2,4,6-trinitrophenyl)iminoazanium; DTNB: 5,5- was treated using Maestro v. 9.3 by removing the
dithiobis(2-nitrobenzoicacid); HFIP: 1,1,1,3,3,3-hexa- original ligand, solvent, and co-crystallization mole-
fluoropropan-2-ol; HEPES: 2-[4-(2-hydroxy ethyl)piper- cules, and then, adding the hydrogen atoms.[25] The
azin-1-yl]ethane sulfonic acid; NFT: neurofibrillary ligands were built using Maestro, and then, using
tangles; PAS: peripheral anionic site; SAR: structure- Ghemical v. 2.0,[26] they were submitted to random
activity relationship; TAC: tacrine; ThT: thioflavin T; T3P: conformational search (RCS) of 1000 cycles, and
propylphosphonic anhydride solution.
2500 optimization steps using Tripos 5.2 force
field.[27] The minimized ligands were docked into the
AChE structure with GOLD software v. 5.1,[28] and the
zone of interest was defined as the residues within
General Methods and Materials
Analytical grade reagents were purchased from Sigma- 10 Å from the original position of the ligand in the
Aldrich, Fluka, Alfa Aesar and Acros and were used as crystal structure. The ‘allow early termination’ option
supplied. Solvents were dried according to standard was deactivated, and the remaining default parame-
methods.[24] The chemical reactions were monitored ters of Gold were used. The ligands were subjected to
by TLC using alumina plates coated with silica gel 60 100 genetic algorithm steps using ASP as fitness
F254 (Merck). Column chromatography separations function.
were performed on silica gel Merck 230–400 mesh
(Geduran Si 60). The melting points (m.p.) were
Pharmacokinetics Properties
measured with a Leica Galen III hot stage apparatus
1
and are uncorrected.1 The H- and 13C-NMR spectra To analyze the drug likeliness of novel conjugated
molecules as new anti-AD drugs, a brief prediction on
pharmacokinetic proprieties was performed in silico.
Parameters such as the lipo-hydrophilic character
(clogP), blood-brain barrier partition coefficient (log
BB), the ability to be absorbed through the intestinal
tract (Caco-2 cell permeability) and CNS activity were
calculated.
1 IR characterization of the samples was not performed as
the FT-IR instrument was out of service and its main-
tenance support was not received due the lockdown
implemented during COVID-19 pandemic. However, oth-
er characterization which includes mass and NMR was
performed, which confirmed the structure of the mole-
cules.
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The ligands were built and minimized as previously calculated as well as the enzyme activity. A control
mentioned for the docking studies. The structures reaction was carried out using the sample solvent
were submitted to the calculation of these relevant (methanol) in the absence of any tested compound,
pharmacokinetic proprieties and descriptors using and it was considered as 100% activity. The percent-
QikProp v. 2.5.[17] These predictions are for orally age inhibition of the enzyme activity due to the
delivered drugs and assume non-active transport.
presence of increasing test compound concentration
was calculated by the following Eqn. 3.
�
�
Radical Scavenging Capacity
Vi
V0
%I ¼ 100 À
� 100
(3)
The radical scavenging activity was evaluated by the
DPPH method previously described.[19,29] To a 2.5 mL
solution of DPPH (0.002%) in methanol, four samples where vi is the initial reaction rate in the presence of
of each compound in solution were added in different inhibitor and v0 is the initial rate of the control
volumes to obtain different concentrations in a 3.5 mL reaction. The inhibition curves were obtained by
final volume. The samples were incubated for 30 min plotting the percentage of enzymatic inhibition versus
at room temperature. The absorbance was measured inhibitor concentration, and a calibration curve was
at 517 nm against the corresponding blank (metha- obtained from which the linear regression parameters
nol). The antioxidant activity was calculated by Eqn. were obtained.
1.[29]
�
�
50 À b
IC50
¼
(4)
ADPPH À Asample
m
%AA ¼
� 100
(1)
ADPPH
Where b is the interception in the y axis and m is the
The tests were carried out in triplicate. The compound slope. The statistical analysis was performed in Micro-
concentrations providing 50% of antioxidant activity soft Office Excel®.
(EC50) were obtained by plotting the antioxidant
activity against the compound concentration.
Inhibition of Self-Mediated Aβ42 Aggregation
�
�
50 À b
Amyloid-β peptide (1–42) (Aβ42) was purchased from
Sigma-Aldrich as a lyophilized powder and stored at
EC50
¼
(2)
m
°
À 20 C. The samples were treated with 1,1,1,3,3,3-
Where b is the interception in the y axis and m is the hexafluoropropan-2-ol (HFIP) to avoid self-aggregation
slope. The parameters for statistical analysis were and reserved. HFIP pretreated Aβ42 samples were re-
calculated with Microsoft Office Excel®.
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
Biological Studies
AChE inhibition. The enzymatic activity TcAChE was (0.215 M, pH 8.0) to obtain a 40 μM solution. Due to
measured using an adaptation of the method pre- the hydrophobic nature of the compounds under
viously described.[20,21] The assay solution contained study, they were first dissolved in methanol (1 mg/mL)
374 μL of HEPES buffer (50 mM and pH 8.0), a variable and then, further diluted in phosphate buffer to a final
volume (10–50 μL) of the stock solution of each concentration of 80 μM.
compound in methanol (1 mg/mL), 25 μL of AChE
stock solution, and the necessary amount of methanol
To study the Aβ42 aggregation inhibition, a re-
to attain 0.925 mL of the sample mixture in a 1 mL ported method, based on the fluorescence emission of
cuvette. The samples were left to incubate for 15 min, thioflavin T (ThT), was followed.[22,23,30] Firstly, Aβ42
and then, 75 μL of acetylthiocholine iodide (AChI) (10 μL) samples and the tested compounds (10 μL)
solution (16 mM) and 476 μL of DTNB (3 mM) were were diluted with the phosphate buffer to a final
added. The reaction was monitored for 5 min at concentration of 40 μM (Aβ) and 80 μM (compounds),
°
40 nm. Assays were run with a blank containing all the and then, they were incubated for 24 h at 37 C,
components except AChE, which was replaced by without stirring. As for the control, a sample of the
HEPES buffer. The velocities of the reaction were peptide was incubated under identical conditions but
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Chem. Biodiversity 2021, 18, e2000083
[4] R. Jakob-Roetne, H. Jacobsen, ‘Alzheimer’s disease: From
without the inhibitor. After incubation, the samples
were added to a 96-well plate (BD Falcon) with 180 μL
of 5 μM 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 using a Spectramax Gemini EM (Molecular
Devices) at the following wavelengths: 446 nm (ex-
citation) and 485 nm (emission). The percent inhibition
of the self-induced aggregation due to the presence of
the test compound was calculated by the Eqn. 5. In
which IFi and IF0 corresponded to the fluorescence
intensities, in the presence and the absence of the test
compound, respectively, minus the fluorescence in-
tensities due to the respective blanks.
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�
�
IFi
IF0
% I ¼ 100 À
� 100
(5)
The reported values were obtained as the mean �
SEM of duplicate of two different experiments.
Acknowledgements
Authors acknowledge Jain University for financial
support and A.H. is thankful to Erasmus NAMASTE
consortium (unique grant number: NAMASTE_-
20140147) for the award of her doctoral exchange
fellowship and also the Portuguese NMR (IST-UL
Center) and Mass Spectrometry Networks (Node IST-
CTN) for providing access to their facilities.
[15] A. Hiremathad, R. S. Keri, A. R. Esteves, S. M. Cardoso, S.
Chaves, M. A. Santos, ‘Tacrine-Hydroxyphenylbenzimida-
zole hybrids as potential multitarget drug candidates for
Alzheimer’s disease’, Eur. J. Med. Chem. 2018, 148, 255–
267.
Author Contribution Statement
Dr. Rangappa S. Keri: Work design and written. Dr.
Asha Hiremathad: Experiment carried and written. Dr.
Silvia Chaves: Modeling and biological studies.
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Received February 4, 2020
Accepted December 21, 2020
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