Synthesis and pharmacological evaluation of multifunctional tacrine derivatives against several disease pathways of AD

Article abstract of DOI:10.1016/j.bmcl.2014.12.084

A novel series of tacrine derivatives were designed and synthesized by combining caffeic acid (CA), ferulic acid (FA) and lipoic acid (LA) with tacrine. The antioxidant study revealed that all the hybrids have much more antioxidant capacities compared to

Full text of DOI:10.1016/j.bmcl.2014.12.084

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and pharmacological evaluation of multifunctional tacrine
derivatives against several disease pathways of AD
Maria Digiacomo a, , Ziwei Chen b,c, , Shengnan Wang b,c, Annalina Lapucci a, Marco Macchia a,
Xiaohong Yang b,c, Jiaqi Chu b,c, Yifan Han c,d, Rongbiao Pi b,c, , Simona Rapposelli a,
⇑
⇑
a Dipartimento di Farmacia, Università di Pisa, Via Bonanno, 656126 Pisa, Italy
b Department of Pharmacology & Toxicology, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou 510006, China
c International Joint Laboratory (SYSU-PolyU HK) of Novel Anti-Dementia Drugs of Guangdong, Guangzhou 510006, China
d Department of Applied Biology and Chemical Technology, Institute of Modern Chinese Medicine, The Hong Kong Polytechnic University, Hung Hom, Hong Kong, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of tacrine derivatives were designed and synthesized by combining caffeic acid (CA), ferulic
acid (FA) and lipoic acid (LA) with tacrine. The antioxidant study revealed that all the hybrids have much
more antioxidant capacities compared to CA. Among these compounds, 1b possessed a good ability to
inhibit the b-amyloid protein (Ab) self-aggregation, sub-micromole acetylcholinesterase (AChE)/butyr-
ylcholinesterase (BuChE) inhibitory, modest BACE1 inhibitory. Moreover, compound 1b also was a DPPH
radical scavenger and copper chelatory as well as had potent neuroprotective effects against glutamate-
induced cell death with low toxicity in HT22 cells. Our findings suggest that the compound 1b might be a
promising lead multi-targeted ligand and worthy of further developing for the therapy of Alzheimer’s
disease.
Received 1 December 2014
Revised 23 December 2014
Accepted 24 December 2014
Available online xxxx
Keywords:
Tacrine
Alzheimer’s disease
BACE1
Amyloid protein
Copper
Ó 2015 Published by Elsevier Ltd.
Multi-targeted ligands
Caffeic acid
Alzheimer’s disease (AD) is the most common form of irrevers-
ible dementia in the elderly. It is a multifactorial disorder in which
several factors contribute both to its etiology and pathogenesis. In
particular, decreased levels of acetylcholine (ACh), the formation of
Ab deposits and neurofibrillary tangles, mitochondrial dysfunction
and extensive oxidative stress constitute the principle hallmarks of
AD.1 In the last decade, the design of compounds potentially useful
for the treatment of AD, focused on the development of new
ligands able to interact with different type of targets involved in
(BuChE), and induces oxidative stress, due to reactive oxygen spe-
cies (ROS) production stimulation and glutathione depletion, as
demonstrated by in vitro studies on human liver cell line HepG2.4
Consequently, the simultaneous administration of free radical
scavenger could counteract the tacrine-induced oxidative stress.4
Moreover, it is well known that also bio-metals such as Cu, Zn
and Fe, play a central role in the production of ROS and in the onset
of oxidative stress5 thus contributing to the aggregation of Ab and
progression of AD.
On this basis, new tacrine-like compounds endowed of both
AChEI activity and antioxidant properties have been widely inves-
tigated.6–9 This strategy falls into the new approach in medicinal
chemistry of multi-target design ligands (MTDL), according to
which a single compound able to modulate multiple targets
simultaneously, could result a more effective and safety strategy
than the ‘one drug-one target’ approach.10
AD (i.e., BACE inhibitors,
c
-secretase modulators),2,3 but to date,
the acetylcholinesterase (AChE) inhibitors, such as tacrine, donepe-
zil, rivastigmine and galantamine, still remain the only drugs
currently employed in therapy. The effectiveness of these AChE
inhibitors (AChEI) is limited to the reduction of the symptoms of
AD. Tacrine (TA), the first AChEI approved by FDA for AD, is the
most effective prototype of this class of drugs, nevertheless AChEIs
exert limited disease modification and only attenuate the demen-
tia symptom. TA inhibits both AChE and butyrylcholinesterase
In a previous work, we described a series of tacrine–ferulic acid
(TAnFA)11,12 and tacrine–caffeic acid hybrids (TAnCA).13,14 These
hybrids showed a good multiple biological profile, displaying AChE
inhibition properties and anti-oxidant activity.
As a further development of our work on the design and synthe-
sis of new TA and antioxidant hybrid compounds with a better
⇑
Corresponding authors.
E-mail addresses: pirb@mail.sysu.edu.cn (R. Pi), simona.rapposelli@farm.unipi.it
(S. Rapposelli).
pharmacological profile, we here describe
a new class of
Contributed equally to this work.
http://dx.doi.org/10.1016/j.bmcl.2014.12.084
0960-894X/Ó 2015 Published by Elsevier Ltd.
Please cite this article in press as: Digiacomo, M.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2014.12.084

2
M. Digiacomo et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
hybrid-molecules (1a,b–3a,b) as multi-target derivatives for the
treatment of AD (Fig. 1).
according to the reported procedure,20,21 were subjected to a reac-
tion with 1,3-diamino-2-propanol at 160 °C. The subsequent con-
densation of derivatives 6a,b with the appropriate acids in
presence of DCC and DMAP afforded the products 1a,b and 2a,b.
For the preparation of compound 3a and 3b, the lipoic acid was
converted to the corresponding acid chloride and treated with
the amine-derivative 6a,b.
Cholinergic neurotransmission damage is believed to be one of
the major causes of memory impairment associated with AD. AChE
inhibitor could maintain the Ach in the synaptic cleft by inhibiting
acetylcholinesterase (AChE), and this is the only known means of
treating AD.22 The synthesized compounds (1a,b–3a,b) were firstly
tested for their inhibitory activity of AChE and BuChE following the
experimental protocol previously described.11
In particular, in this Letter we evaluated the possibility to widen
the pharmacological profile of the compounds TAnFA and TAnCA,
through the insertion of 1,3-diamino-2-propanol (Fig. 1A). Actually
this core has been applied successfully to a number of aspartyl pro-
teases, including b-secretase (BACE1).15–18 (Fig. 1B) Moreover, the
presence of both nitrogen and oxygen atoms within the linker
could facilitate the interaction of these ligands with free metals.19
The compounds synthesized (1a,b–3a,b) were evaluated for
their antioxidant and both AChE and BACE1 inhibitor activities.
Furthermore the protective effects on glutamate-induced cell
death in mouse hippocampal neuronal HT22 cells were investi-
gated. The candidate with the best multi-target profile was also
evaluated for its ability to chelate copper.
The IC50s for AChE and BuChE and the ratios of BuChE to AChE,
are summarized in Table 1. Tacrine was taken as reference com-
pound. Compounds 2a, 2b and 3b, were found to be more active
Final compounds 1a,b–3a,b were synthesized following the
procedures described in Scheme 1. Intermediates 5a,b, synthesized
R
OH
R
OH
OH
S
NH
NH
OR1
NH
NH
S
N
O
O
N
1a: R=R1= H
3a: R=H
3b: R=Cl
1b: R=Cl, R1=H
2a: R=H, R1=Me
2b: R=Cl, R1=Me
Figure 1. Multifunctional tacrine-derivatives 1a,b–3a,b.
O
Cl
COOH
NH2
O
a
b
R
N
H
R
N
R
R=H
R=H
5a R=H
5b R=Cl
4
c
R=Cl
d
R
OH
NH2
NH
OH
R
N
OH
e
NH
NH
OR1
O
N
6a R=H
6b R=Cl
1a R=R1 = H
1b R= Cl, R1 = H
2a R= H, R1 = Me
2b R= Cl, R1 = Me
f
R
OH
S
NH
O
NH
S
N
3a R=H
3b R=Cl
Scheme 1. General synthesis of compounds 1a,b–3a,b. Reagents and conditions: (a) cyclohexanone, toluene, reflux, 4 h; (b) POCl3, 120 °C, 2 h; (c) cyclohexanone, POCl3,
reflux, 2 h; (d) 1,3-diamino-2-propanol, 160 °C, 3 h; (e) caffeic acid or ferulic acid, CH2Cl2/DMF, DCC, DMAP, rt, 12 h; (f) (i) lipoic acid, SOCl2, CH2Cl2, 0 °C, 4 h; (ii) Et3N, CH2Cl2/
DMF, 0 °C, 4 h.
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M. Digiacomo et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
Table 1
The inhibition of AChE and BuChE activities (IC50
,
l
M) and DPPH radical scavenging activities (10 and 30
l
M) of new compounds and references
Compounds
IC50
(lM)
Ratio of
BuChE/AChE
IC50
DPPH radical
scavenging rate (%)
DPPH radical
scavenging rate (%)
(30 lM)
AChE
BuChE
(10
lM)
1a
1b
2a
2b
3a
3b
0.70
0.15
0.03
0.02
0.10
0.04
0.10
na
1.01
0.36
0.31
0.71
1.18
1.03
0.04
na
1.45
2.40
10.33
34.53
11.80
25.75
0.40
41.50 3.60
60.87 2.7
10.30 2.6
17.11 1.9
4.23 0.96
0.98 0.55
>1000
89.6 0.57
90.36 1.4
25.0 3.1
22.68 4.1
8.47 2.91
1.57 0.79
>1000
Tacrine
Caffeic acid
na
44.10 4.40
90.27 1.50
na.: not available.
Table 2
Table 3
The BACE1 inhibitory activity of compounds
Inhibition of Ab1-42 aggregation
Compounds
Inhibition of Ab
Compounds
Inhibition of Ab
Compounds (10
lM)
Inhibition rate % SEM
(5
lM)
aggregation (%)
(50
l
M)
aggregation (%)
1a
1b
2a
2b
3a
3b
10.51 2.55
13.07 5.81
14.97 1.25
2.57 7.29
24.77 5.46
46.78 3.12
1a
1b
2a
2b
3a
3b
12.8
9.2
8.4
13.5
na
na
1a
1b
2a
2b
3a
3b
49.8
53.2
46.3
48.9
na
na
Congo red
76.6
Congo red
93.2
na.: not available
than reference drug in inhibiting AChE. The presence of chlorine
atom in 6 position of the tetrahydroacridine fragment (1b, 2b
and 3b) improves the AChE-inhibitory activity and selectivity
toward AChE over BuChE, with respect to the no-substituted deriv-
atives (1a, 2a and 3a). It is also reported that BuChE is involved in
the metabolic degradation of ACh. BuChE activity increases as AD
progresses. Therefore, the concurrent inhibition of both AChE and
BuChE should provide additional benefits in the treatment of
AD.23,24 As list in Table 1, compounds 1b and 2a have equal inhib-
itory activity against BuChE. The best results, in terms of affinity
and selectivity, were obtained for compounds 2b and 3b, that are
ferulic and lipoic chlorine-substituted derivatives, respectively.
Compounds (1a,b–3a,b) were also tested for their radical scav-
enging activities by using 1,1-diphenyl-2-picryl-hydrazyl (DPPH)
assay. The compounds were tested to 10 and 30 lM and the data
are summarized in Table 1. The only derivatives endowed of radi-
cal scavenging activity, comparable to that of caffeic acid, were 1a
and 1b.
In order to evaluate the influence of the tested compounds on
amyloid fibrils, a Th-T fluorescence assay was performed. The com-
pounds were tested to 50
reported in Table 2. Compounds 1a,b–2a,b, at concentration of
50 M, induced a slight significant inhibition of Ab1–42 self-aggre-
lM and 5 lM and the results are
l
gation with percentage values ranging from 46% (2a) to 53% (1b).
On the contrary, the tacrine–lipoic acid hybrids 3a and 3b did
not affect the Ab1–42 self-aggregation.
Hybrids (1a,b–3a,b) were then tested for their BACE1 inhibitory
activity by using a commercially available kit (Sigma–Aldrich, MO,
USA). As showed in Table 3, lipoic acid derivative 3b was able to
Figure 2. The effects of compounds on cell viability in HT22 with or without
glutamate. (A) Cell toxicity of test compounds on HT22 cells. Cell viability was
determined using the MTT assay (n = 6). Cells were treated with indicated
concentration of test compounds for 24 h. ⁄⁄P <0.01 versus control. (B) Compounds
protect HT22 cells against glutamate-induced cytotoxicity. Cells were pretreated
with different concentration of compounds for 30 min and then incubated with/
without 2 mM glutamate for 24 h. Cell viability was determined using the MTT
assay (n = 6). ##P <0.01 versus control; ⁄⁄P <0.01 versus glutamate-treated group.
induce a slight significant inhibition of BACE1 at 10 lM with a per-
centage of about 47% .On the contrary, the compounds 1a,b, 2a,
and 3a exhibited only a modest BACE1 inhibitory activity.
The new synthesized compounds (1a,b–3a,b) were further
investigated for their cell toxicity in HT22 cell, at different concen-
trations (10, 30 and 100 lM) (Fig. 2A). No cytotoxic effects were
observed for caffeic acid-derivative 1a and the ferulic acid-deriva-
tive 2a at various concentrations whereas the 6-chloro analogues
1b, and notably 2b, appeared to be cytotoxic only at the highest
concentrations (100
toxic both at 30 and 100
showed an appreciable cytotoxicity already 10
l
M). Derivatives 3a and 3b showed to be cyto-
M, and in particular compound 3b
M.
l
l
Please cite this article in press as: Digiacomo, M.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2014.12.084

4
M. Digiacomo et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
pharmacokinetic and pharmacodynamic of a multi-targeted com-
pound, when compared with a specific highly selective single-tar-
get molecule.
Acknowledgments
This study was supported by Guangdong Provincial Interna-
tional Cooperation Project of Science
& Technology (No.
2012B050300015, No. 2013B051000038), National Natural Science
Foundation of China (No. 31371070), and Scientific and Technolog-
ical Cooperation between the Italian Republic and the People’s
Republic of China for Year 2013–2015 (No. MAE-M00705/
CN13MO9) to R. Pi and M. Macchia.
Supplementary data
Figure 3. UV–vis (250–500 nm) absorption spectra of 1b (40 lM) with 40, 80 or
120 lM CuCl2 in ethanol.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.12.
084.
In order to evaluate the neuroprotective effects in HT22 cell
against glutamate-induced neuronal death, the synthesized com-
pounds were tested at different concentrations (0.3, 1, 3 and
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.
In conclusion, this study focused on the identification of new
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treatment of AD. In particular we evaluated the AChE, BuChE and
BACE1 inhibitory activities and the ability of the compounds to
inhibit the Ab aggregation. Moreover a determination of the anti-
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carried out.
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possessed a good ability to inhibit the Ab aggregation, even if it dis-
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