Design, Synthesis, and Evaluation of Orally Bioavailable Quinoline-Indole Derivatives as Innovative Multitarget-Directed Ligands: Promotion of Cell Proliferation in the Adult Murine Hippocampus for the Treatment of Alzheimer's Disease

Article abstract of DOI:10.1021/acs.jmedchem.7b01417

A novel series of quinoline-indole derivatives were synthesized and evaluated as multitarget-directed ligands for the treatment of Alzheimer's disease (AD). Biological evaluation revealed that the derivatives had multifunctional profiles including antioxidant effects, blood-brain barrier (BBB) penetration, biometal chelation, Aβ aggregation modulation, neurotrophic and neuroprotective properties. Moreover, several representative target derivatives demonstrated hippocampal cell proliferation in living adult mice by intracerebroventricular (icv) injection or oral administration. Further drug-like property analysis demonstrated that the optimized compound, 8d (WI-1758), had liver microsomal metabolic stability, was well tolerated (>2000 mg/kg), and had a rational pharmacokinetic profile, as well as an oral bioavailability of 14.1% and a positive log BB (-0.19) to cross the BBB in vivo. Pharmacodynamics studies demonstrated that chronic oral administration of 8d·HCl substantially ameliorated the cognitive and spatial memory deficits in APP/PS1 AD mice and noticeably reduced overall cerebral β-amyloid deposits.

Full text of DOI:10.1021/acs.jmedchem.7b01417

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Article
Design, Synthesis and Evaluation of Orally Bioavailable
Quinoline-Indole Derivatives as Innovative Multitarget-
Directed Ligands: Promotion of Cell Proliferation in the Adult
Murine Hippocampus for the Treatment of Alzheimer’s Disease
Zhiren Wang, Jinhui Hu, Xiaoping Yang, Xing Feng, Xingshu Li, Ling Huang, and Albert S. C. Chan
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01417 • Publication Date (Web): 08 Feb 2018
Downloaded from http://pubs.acs.org on February 8, 2018
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Design, Synthesis and Evaluation of Orally Bioavailable
QuinolineꢀIndole Derivatives as Innovative MultiꢀTargetꢀDirected
Ligands: Promotion of Cell Proliferation in the Adult Murine
Hippocampus for the Treatment of Alzheimer’s Disease
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†,‡
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,†
,†
Zhiren Wang, Jinhui Hu, Xiaoping Yang, Xing Feng, Xingshu Li,* Ling Huang,* and
†
Albert S. C. Chan
†
School of Pharmaceutical Sciences, Sun Yatꢀsen University, Guangzhou, 510006, China
‡
Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, School of Medicine,
Hunan Normal University, Changsha, 410013, China
Abstract
A novel series of quinolineꢀindole derivatives were synthesized and evaluated as
multiꢀtargetꢀdirected ligands for the treatment of Alzheimer’s disease (AD).
Biological evaluation revealed that the derivatives had multifunctional profiles
including antioxidant effects, bloodꢀbrain barrier (BBB) penetration, biometal
chelation, Aβ aggregation modulation and neurotrophic and neuroprotective
properties. Moreover, several representative target derivatives demonstrated
hippocampal cell proliferation in living adult mice by intracerebroventricular (ICV)
injection or oral administration. Further drugꢀlike property analysis demonstrated that
the optimized compound, 8d (WIꢀ1758), had liver microsomal metabolic stability,
was well tolerated (>2000 mg/kg), and had a rational pharmacokinetic profile, as well
as an oral bioavailability of 14.1% and a positive log BB (ꢀ0.19) to cross the BBB in
vivo. Pharmacodynamics studies demonstrated that chronic oral administration of
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d•HCl substantially ameliorated the cognitive and spatial memory deficits in
APP/PS1 AD mice and noticeably reduced overall cerebral βꢀamyloid deposits.
Introduction
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Alzheimer’s disease (AD), the most prevalent and chronic type of
1
neurodegenerative disease, affects approximately 47 million patients worldwide. The
pathologic changes of AD are convoluted and multilayered; these changes include the
aberrant accumulation of amyloidꢀβ (Aβ) plaques, loss of synapses, neurofibrillary
tangles within neurons, oxidative stress, dyshomeostasis of biometals in the brain,
2
etc. Presently, the primary therapeutic options for AD treatment are
acetylcholinesterase inhibitors (AChEIs), such as donepezil, rivastigmine, and
galantamine, as well as the NꢀmethylꢀDꢀaspartate (NMDA) receptor (NMDAR)
antagonist, memantine, which can improve memory and cognitive function to a
certain extent but cannot definitively cure this disease. Due to its complex
pathogenesis, the multiꢀtargetꢀdirected ligands (MTDLs) approach for AD treatment
3
ꢀ6
has been developed in both symptomatic and diseaseꢀmodifying efficiencies .
Among the numerous neuropathological biomarkers of AD, the extracellular Aβ
plaque deposits between neurons and intracellular neurofibrillary tangles within the
7
brains of patient are primary markers. The selfꢀassembled and hydrophobic
aggregations of Aβ peptides are associated with complex pathways, soluble oligomers,
8
paranuclei, and protofibrils, and they ultimately generate insoluble filaments. The
dyshomeostasis of biometals is another biomarker, and through comparison with the
ageꢀmatched healthy parenchyma within the brains of AD patients, 3ꢀ5ꢀfold higher
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2
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concentrations of metal ions in Aβ plaque deposits, such as Cu (0.4 mM, 5.7 times),
2
+
2+/3+
Zn (1.0 mM, 2.8 times) and Fe
(0.9 mM, 2.9 times), were found. These
biometals have a comparatively greater affinity towards the Nꢀterminus of the Aβ
peptide, especially at residues His6, His13, and His14, which aggravated Aβ
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aggregation and exacerbated their noxious properties. Furthermore, the
dyshomeostasis redox active metal ions, including those unbonded and bonded with
Aβ peptides, could exacerbate the overproduction of reactive oxygen species (ROS)
that jeopardizes biological tissue and finally results in neuronal damage or apoptosis
1
0
in the central nervous system (CNS) of AD patients. Consequently, the use of
appropriate metal chelating agents to inhibit the production or accumulation of Aβ
plaques and restore biometal homeostasis in the brains of AD patients has been
11
developed as a potent therapeutic strategy for AD treatment. Clioquinol (CQ, Figure
1) and its analogs, with 8ꢀhydroxy quinoline as a crucial scaffold, have a moderate
affinity for metal ions and can extract metals from extracellular Aβ aggregates and
shift them to copperꢀcarrier proteins in the parenchymal cells without perturbing the
overall essential metal biochemistry in the brain, demonstrating safety, noticeably
strengthened cognition and substantially decreased cerebrospinal fluid (CSF) levels of
1
2, 13
Aβ in recent phase II clinical trials.
The more recently developed preclinical
multiꢀtargetꢀdirected
metalꢀchelating
agents
(Mꢀ30),
and
5ꢀ((methyl(propꢀ2ꢀynꢀ1ꢀyl)amino)methyl)quinolinꢀ8ꢀol
1ꢀ(5ꢀchloroꢀ8ꢀmethoxyquinolinꢀ2ꢀyl)undecanꢀ1ꢀol
1
(J2326),
(L2ꢀb)
1
1
4
N ,N ꢀdimethylꢀN ꢀ(pyridinꢀ2ꢀylmethyl)benzeneꢀ1,4ꢀdiamine
revealed
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noticeable efficacies in multiple accessible murine models of AD (Figure 1).
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Figure 1. Structure of CQ, multiꢀtargetꢀdirected metalꢀchelating agents, melatonin, melatoninꢀNꢀbenzylamine
hybrids and several small molecules reported to manipulate neuronal cells in vitro and in vivo.
All adult mammalian species have reservoirs of neuronal stem cells in the
hippocampal dentate gyrus (DG), which integrates new neurons into the hippocampus
1
7
throughout life. This adult neurogenesis provides an indispensable mechanism by
which lost or damaged neural cells from neurodegeneration can be replaced with
functioning normal ones, which facilitates longꢀterm hippocampalꢀdependent learning,
1
8, 19
memory and neuronal plasticity.
Nevertheless, with aging or neurodegeneration in
rodents and elderly humans, the rate of regeneration of new neurons from the neural
2
0
stem cells within the hippocampus decreases exponentially. Furthermore, before the
new neural precursor cells are fully matured into functional neurons and migrate into
the adult hippocampus, which is generally over approximately 30 days for mice, the
vast majority of neural precursor cells undergo normal apoptosis during the first week
17, 18, 21
with no explicit reason that has been currently identified.
Therefore, the
promotion of adult hippocampal neurogenesis with small drugꢀlike molecules was
identified as an effective therapeutic strategy to address longꢀterm neurodegeneration,
19
which has significant unmet regenerative medical needs, particularly for AD.
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Allopregnanolone
(6S,9aS)ꢀNꢀbenzylꢀ6ꢀ(4ꢀhydroxybenzyl)ꢀ8ꢀ(naphthalenꢀ1ꢀylmethyl)ꢀ4,7ꢀdioxohexahyd
roꢀ2Hꢀpyrazino[1,2ꢀa]pyrimidineꢀ1(6H)ꢀcarboxamide (ICGꢀ001, Figure 1)
(Figure
1)
and
the
compound
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significantly strengthened hippocampal neurogenesis and markedly reduced the
22, 23
pathological burden in a preclinical in vivo model of AD.
Through enhancing
remyelination, benztropine (Figure 1) exhibited a striking mitigation of disease
24
severity in a murine model of relapsingꢀremitting multiple sclerosis. Recently, the
compound Nꢀ(3ꢀ(3,6ꢀdibromoꢀ9Hꢀcarbazolꢀ9ꢀyl)ꢀ2ꢀfluoropropyl)ꢀ3ꢀmethoxyaniline
(P7C3A20,
Figure
1)
and
its
further
analog
Nꢀ(3ꢀ(3,6ꢀdibromoꢀ9Hꢀcarbazolꢀ9ꢀyl)ꢀ2ꢀfluoropropyl)ꢀ4ꢀ(4ꢀ(propꢀ2ꢀynꢀ1ꢀyloxy)benzo
yl)benzamide (P7C3ꢀS326, Figure 1), which has a preeminent ability to promote adult
hippocampal neurogenesis, demonstrated significant efficacies in rodent models of
Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), traumatic brain injury
17,
18,
25ꢀ29
(TBI) and ageꢀrelated cognitive decline.
Interestingly, the
melatoninꢀNꢀbenzylamine hybrids (Figure 1), which have an indole framework, can
6,
promote the effective development of neural stem cells into the neuronal phenotype.
3
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Taking into account neuronal loss, deficits in adult neurogenesis and the
formation of plaques and tangles associated with AD, in this work, we merged the
skeleton of indole and CQ to generate a series of quinolineꢀindole scaffolds to study
their ability to modulate adult hippocampal neurogenesis in vivo, biometal
dyshomeostasis and antiꢀamyloid aggregation (Figure 2). Through structureꢀactivity
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relationship (SAR) studies of antioxidant ability, BBB permeability, modulation of Aβ
aggregation and neuronal cell proliferation activity, the potential compounds with
neurotrophic function were further evaluated for their neuroprotective capacities,
metabolic stability, acute toxicity and hippocampal cell proliferation in living adult
mice. Ultimately, the pharmacokinetic profile and pharmacodynamics evaluation of
the optimal compound was performed.
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Figure 2. Multiꢀtargetꢀdirected design strategy by introducing an indole fragment into the CQ framework.
Results and Discussion
Chemistry
The synthetic routes of the target compounds (7aꢀ7g and 8aꢀ8g) are depicted in
Scheme 1. The commercially available 8ꢀhydroxyquinaldine or one of its analogs (1a
or 2a) was reacted with the corresponding oꢀnitrobenzaldehyde derivative to produce
3aꢀ3g and 4aꢀ4g, respectively, followed by reductive cyclization of the
oꢀnitrostyrenes derivatives (3aꢀ3g and 4aꢀ4g) catalyzed by Pd(OAc) under CO, thus
2
leading to the key indole intermediates 5aꢀ5g and 6aꢀ6g. Finally, the removal of the
3 2 3
acetyl group by interesterification in the presence of CH OH and K CO (for 5aꢀ5g)
or NaOCH (for 6aꢀ6g) led to the target compounds 7aꢀ7g and 8aꢀ8g with good yields.
3
In addition, by treating the pure target compounds 7g, 8c, 8d and 8g with HCl (g) in
an ethyl acetate solvent, the corresponding hydrochloride compounds were obtained
with a high yield. The synthesis of the target compounds 9a, 9d and 10d is described
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in Scheme 2. Compounds 2d and 2e, which were prepared according to a previous
31
method, were reacted with chloro(methoxy)methane to give 2f and 2g. The
subsequent reactions of 2f and 2g with oꢀnitrobenzaldehyde or its analogs in the
presence of NaH gave 3i, 3j and 4j, respectively. Finally, target compounds 9a, 9d
and 10d were produced by reductive cyclization of the oꢀnitrostyrene intermediates
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(3i, 3j or 4j) with CO, followed by deprotection of the methoxymethane groups under
acidic conditions.
Scheme 1. Synthesis of 7aꢀ7g (X = H), 8aꢀ8g (X = Cl), 7g•HCl, 8c•HCl, 8d•HCl and 8g•HCl. Reagents and
2 2 3 2 3
conditions: a. acetic anhydride, 150 °C, 12 h; b. DMF, phen•H O, Pd(OAc) , CO, 80 °C, 24 h; c. CH OH, K CO
(
3
for 5aꢀ5g) or NaOCH (for 6aꢀ6g), rt, 1 h, d. AcOEt, HCl (g), rt, 12 h.
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Scheme 2. Synthesis of 9a, 9d and 10d. Reagents and conditions: a. HCHO, HCl (g), rt, 72 h; b. P(OEt) , 110 °C,
2 2 2 2
12 h; c. H O, KOH, NaClO, rt, 2 h; d. CH Cl , DIPEA, ClMOM, rt, 5 h; e. DMF, NaH, rt, 4 h; f. DMF, phen•H O,
2 3
Pd(OAc) , CO, 80 °C, 24 h; g. CH OH, HCl (37%), rt, 5 h.
Free Oxygen Radical Scavenging Ability
Oxidative stress is one of the earliest events in AD pathogenesis and oxidative
damage is present within the brain of AD patients. Considering that 8ꢀhydroxy
quinoline derivatives generally have good metal ion complexing ability, we first
determined the antioxidant ability of the quinolineꢀindole compounds, performed the
free oxygen radical absorbance capacity (ORAC) assay using fluorescein (ORACꢀFL)
method with a vitamin E analog, Trolox, as the internal standard. Table 1 revealed that
all of the target compounds exhibited significantly higher antioxidant efficacies
compared with clioquinol (0.5), melatonin (2.4) and clioquinol + melatonin (2.9). The
results also showed that the radical scavenging efficacies of the target compounds
were principally derived from the indole moiety. An SAR study demonstrated that the
phenolic hydroxyl groups on the indole moieties were pivotal for the antioxidant
activity. Compounds 7f (5.4) and 8f (4.9), which have phenolic hydroxyl groups at the
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ꢀposition of the indole moieties, provided considerably better antioxidant efficacies
compared with the analogs that were methylated of the hydroxyl group (7e, 3.5; 8e,
.4). Notably, compound 9d (6.6), which has an indole moiety on the 5ꢀposition of the
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quinoline, demonstrated the greatest radical scavenging efficacy. The presence of the
chlorine on the oxine framework indicated a slight detrimental effect on the
antioxidant activity. Compounds 7d, 7f, and 9d, lacking a chlorine atom in the oxine
scaffold, exhibited ORAC values of 5.9, 5.4 and 6.6, respectively, compared with 8d,
8
f, and 10d (ORAC values 5.0, 4.9 and 5.3), which have a chlorine in the oxine
scaffold.
ꢀ6
ꢀ1
e
Table 1. Oxygen Radical Absorbance Capacity (ORAC, Trolox Equivalents), Permeability (P × 10 cm s )
Determined by the PAMPAꢀBBB Assay for Target Compounds, Predicted Penetration of the CNS and Inhibition of
Aβ Selfꢀaggregation.
Aβ1ꢀ42 selfꢀinduced
P
e
1
2
a
Comd.
R
R
X
ORAC
Pred.
aggregation
ꢀ
6
ꢀ1 b
(
×10 cm s )
c
(
%Inhib.)
7
7
a
H
F
H
H
H
H
H
H
H
H
4.5 ± 0.1
4.2 ± 0.3
4.6 ± 0.2
5.9 ± 0.3
8.0 ± 1.2
10.6 ± 2.3
9.8 ± 1.6
14.8 ± 0.7
CNS+
CNS+
CNS+
CNS+
6.1 ± 1.3
34.2 ± 3.8
25.1 ± 3.9
51.2 ± 9.1
b
7
c
Cl
OH
7
d
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e
OMe OMe
OH OMe
H
H
H
3.5 ± 0.2
5.4 ± 0.1
4.3 ± 0.2
5.2 ± 1.3
12.1 ± 2.1
5.9 ± 0.3
6.6 ± 1.4
5.3 ± 0.4
5.6 ± 0.8
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
21.5 ± 7.4
42.3 ± 5.8
19.4 ± 5.0
13.6 ± 4.2
42.9 ± 5.1
35.9 ± 1.9
7f
7
g
ꢀ
ꢀ
0
1
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8
9
0
1
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9
0
1
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8
9
0
1
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8
9
0
1
2
3
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5
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7
8
9
0
8a
H
F
H
H
H
Cl 3.1 ± 0.2
Cl 3.2 ± 0.1
Cl 3.0 ± 0.1
8
b
8
c
Cl
8d
OH
H
Cl 5.0 ± 0.2
10.2 ± 0.1
CNS+
66.8 ± 8.2
(
WIꢀ1758)
8
e
OMe OMe Cl 3.4 ± 0.2
OH OMe Cl 4.9 ± 0.1
3.9 ± 1.6
8.4 ± 1.5
4.9 ± 0.2
12.8 ± 1.4
9.1 ± 0.6
6.8 ± 0.4
CNS±
CNS+
CNS+
CNS+
CNS+
CNS+
32.8 ± 7.1
50.3 ± 8.3
30.8 ± 5.6
43.1 ± 7.8
57.2 ± 4.8
68.7 ± 7.9
1.9 ± 1.2
8
f
8g
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Cl 3.2 ± 0.1
9
a
H
H
H
2.1 ± 0.1
6.6 ± 0.1
9d
0d
OH
OH
1
Cl 5.3 ± 0.2
d
d
clioquinol
melatonin
clioquinol +
melatonin
curcumin
ꢀ
ꢀ
0.5 ± 0.2
2.4 ± 0.1
ꢀ
ꢀ
d
d
ꢀ
ꢀ
19.3 ± 3.4
d
d
ꢀ
2.9 ± 0.2
ꢀ
ꢀ
22.5 ± 5.3
d
d
d
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
36.7 ± 6.6
42.1 ± 5.8
d
d
d
resveratrol
ꢀ
ꢀ
ꢀ
d
d
chlorpromazine
hydrocortisone
ꢀ
5.9 ± 0.4
1.1 ± 0.3
CNS+
CNS–
ꢀ
d
d
ꢀ
ꢀ
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a
The means ± SD of three independent experiments. Data are expressed as ꢁmol of trolox equivalent/ꢁmol of
b
ꢀ1
tested compound. Compounds were dissolved in DMSO at 5 mg mL and diluted with PBS/EtOH (70:30).
Values are expressed as the means ± SD at least three independent experiments, compounds with permeabilities
1
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0
ꢀ6
ꢀ1
c
e
values P > 4.7 × 10 cm s could cross the bloodꢀbrain barrier by passive diffusion. The Aβ1ꢀ42/inhibitor ratio
was equal to 5/1, with inhibitor at 5 ꢁM and Aβ1ꢀ42 at 25 ꢁM. Values are the mean of three independent
d
experiments in triplicate. n.t. = not tested.
BloodꢀBrain Barrier Permeability Assay
Penetration of the bloodꢀbrain barrier (BBB) is a critical factor for successful
CNS drugs. To determine the BBB penetration of the target compounds, we used the
parallel artificial membrane permeability (PAMPA) assay, which was initially
established by Di et al and has been successfully applied to a multitude of diverse
compounds to predict passive BBB permeation. The results presented in Table 1
indicated that most of the target compounds can significantly permeate the
bloodꢀbrain barrier by passive diffusion. The SAR study indicated that compounds 7d
e
(P = 14.8) and 7f (12.1), which contain a phenolic hydroxyl group on the indole
moiety, demonstrated significantly higher BBB permeability compared with the
analogs that replaced the phenolic hydroxyl groups with hydrogen (7a, 8.0), halogens
(
7b, 10.6; 7c, 9.8), dioxoles (7g, 5.9) or the methylation of the phenolic hydroxyl
groups (7e, 5.2). In contrast, for compounds with the indole scaffold on the 5ꢀposition
of the quinoline, such as 9a (12.8) and 9d (9.1), the phenolic hydroxyl groups on the
indole moiety demonstrated slight unfavorable effects on BBB permeability. Likewise,
the effect of chlorine on the oxine moiety displayed a slight undesirable effect on
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brain penetration. The chloroꢀsubstituted compounds 8d (10.2) and 10d (6.8)
exhibited slightly lower BBB permeabilities compared with the hydrogenꢀsubstituted
counterparts 7d (14.8) and 9d (9.1).
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Inhibition of Aβ SelfꢀAggregation
To determine the inhibitory activities of the quinolineꢀindole derivatives on Aβ1–
42 selfꢀinduced aggregation, the thioflavin T (ThT) fluorescence assay was first
performed with curcumin and resveratrol as reference compounds (Table 1). Among
the four series of compounds, 7 (X = H), 8 (X = Cl), 9 (X = H) and 10 (X = Cl),
compounds 7d (inhibition: 51.2%) and 8d (inhibition: 66.8%), in which a phenolic
1
hydroxyl group was on the indole moiety at the R position, had more potent effects
than clioquinol (1.9%), melatonin (19.3%), clioquinol + melatonin (22.5%) and the
2
other analogs. Compounds 7f and 8f, with a methoxy group on the ortho (R ) position
of the hydroxyl group, exhibited slightly weaker activities (42.3%, and 50.3%,
respectively) than the parent compounds. The methylation of the hydroxyl group or
cyclization with the methoxy group seemed unfavorable for the activities, and
compounds 7e (21.5%), 8e (32.8%), 7g (19.4%) and 8g (30.8%) showed dramatic
decreases in inhibiting Aβ1–42 aggregation, which demonstrated that the phenolic
hydroxyl group on the indole moiety was preferable for the inhibitory activity.
Replacement of the phenolic hydroxyl group with an H, F or Cl, as in compounds
7aꢀ7c (6.1 – 34.2%), 8aꢀ8c (13.6 – 42.9%), and 9a (43.1%), also provided much less
potent activities than compounds 7d, 8d and 9d, respectively. Compounds 9d (57.2%)
and 10d (68.7%), with the indole moiety on the 5ꢀposition of the quinoline, had
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activities comparable with 7d and 8d. The results also demonstrated that the presence
of a chlorine atom on the oxine moiety plays a pivotal role in the inhibitory activity,
and compounds 8d (66.8%) and 10d (68.7%) exhibited more potent activities than
their hydrogenꢀsubstituted counterparts 7d (51.2%) and 9d (57.2%), respectively.
MetalꢀChelating Property Studies
1
1
1
1
1
1
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Considering compound 8d showed excellent antioxidant ability, bloodꢀbrain
barrier (BBB) permeability and inhibition of Aβ1–42 selfꢀinduced aggregation, and
structurally, the quinolineꢀindole derivatives should also have good metalꢀchelating
properties, we chose 8d as an typical for studying the metalꢀchelating ability of the
31
quinolineꢀindole derivatives. The results in Figure 3 demonstrate that substantial
new characteristic absorptions were detected at 338, 309, 324 and 309 nm after
4 2 4 3
titrating 8d (314 nm) with CuSO , ZnCl , FeSO and FeCl , respectively, which
2
+
2+
2+
3+
indicated that compound 8d was capable of chelating Cu , Zn , Fe , and Fe and
2+
generating corresponding complexes. The stoichiometry of the Cu ꢀ8d complexes in
buffer was initially determined using the inflexion point analysis, which measures the
2
+
absorption at 338 nm by titrating 8d with Cu . As shown in Figure 3B, an
2+
2+
isoabsorptive point (at 0.5) revealed a unique Cu ꢀ8d complex via a 1:2 Cu /ligand
molar ratio. Moreover, highꢀresolution mass spectrometry was also performed to
further probe the explicit components of the chelation complex, and the primary
2+
molecular ion peak of [(8d)
2
Cu] (calculated: 375.0269, detected: 375.0277, Figure
2+
3
C) illustrated a molar ratio of 1:2 Cu /ligand for the complex.
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4
Figure 3. (A) UV absorbance spectrum of compound 8d (50 ꢁM) alone or in the presence of CuSO (50 ꢁM),
ZnCl (50 ꢁM) FeSO (50 ꢁM) or FeCl (50 ꢁM) in buffer (20 mM HEPES, 150 mM NaCl, pH = 7.4). (B) UVꢀvis
2
4
3
2
+
titration of compound 8d (50 ꢁM) with Cu in buffer (20 mM HEPES, 150 mM NaCl, pH = 7.4) at room
2
+
temperature. (C) Highꢀresolution mass spectrometry of the Cu ꢀ8d complex in solution.
Inhibition of Selfꢀ and MetalꢀInduced Aβ Aggregation and Disaggregation of Aβ
Aggregates by Quinolineꢀindole Derivatives
To further elucidate the inhibitory effects of quinolineꢀindole derivatives on selfꢀ
or metalꢀinduced Aβ aggregation, translating to less toxic or nonꢀtoxic offꢀpathway
amorphous Aβ aggregates, transmission electron microscopy (TEM) and the ThT
assay were performed for both the inhibition of Aβ aggregates and disaggregation of
Aβ aggregation fibrils in the presence of quinolineꢀindole derivatives. As indicated by
the ThT assay (Figures 4), compounds 8d and 8g provided 71.6%ꢀ48.6% inhibition of
selfꢀinduced Aβ1ꢀ42 aggregation and 72.7%ꢀ48.55% disaggregation of the preformed
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Aβ1ꢀ42 aggregate fibrils (Figures 4a and 4b), especially 8d, which were higher than the
inhibition values of CQ (19.6% and 13.0%, respectively, p < 0.01 and 0.001) and
melatonin (49.1% and 44.3%, respectively, p < 0.05 and 0.01). The morphological
assessment of compound 8d by TEM (Figure 4c, sequence 5) showed less dense and
thinner fibrils as well as fewer unstructured Aβ1ꢀ42 aggregates in the 8dꢀtreated sample
compared with the control (Figure 4c, sequence 2), CQ (Figure 4c, sequence 3) or
melatonin (Figure 4c, sequence 4). Alternatively, in the disaggregation assay, 8d
exhibited narrower and shorter aggregates (Figure 4d, sequence 5) than CQ (Figure 4d,
sequence 3) and melatonin (Figure 4d, sequence 4). These results validated that 8d
could effectively modulate selfꢀinduced Aβ1ꢀ42 aggregation by its overall structure
rather than its structural components.
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9
0
Figure 4. Experiments of inhibition selfꢀinduced Aβ1–42 aggregation and disaggregation of selfꢀinduced Aβ1–42
aggregates in the presence of 8d. (a and b) Top: Scheme for the inhibition or disaggregation experiments. Bottom:
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The fluorescence intensity of the ThT binding assay, data are expressed as the mean ± SD at least three
independent experiments. Statistical significance was analyzed by ANOVA:
∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.
(
c and d) TEM image analysis of the inhibition of Aβ1–42 aggregation and disaggregation of selfꢀinduced Aβ1–42
0
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aggregates. Serial number: (1) fresh Aβ1–42, (2) Aβ1–42 alone, (3) Aβ1–42 + CQ, (4) Aβ1–42 + melatonin, (5) Aβ1–42
+
8
d, (6) Aβ1–42 + 8g. Experimental conditions: Aβ1–42 (25 ꢁM); compound/Aβ1ꢀ42 = 1/1; PBS (50 mM); pH 7.4;
3
7 °C.
2
+
The abilities of 8d to inhibit Cu ꢀinduced Aβ aggregation and induce the
disaggregation of Aβ aggregation fibrils were investigated (Figure 5). The ThT
fluorescence assay demonstrated that 8d had a rate of 85.8% inhibition for Aβ
2+
aggregation and 83.3% for disaggregation of Cu ꢀassociated Aβ aggregation fibrils,
which were distinctly better than the values found for CQ (65.4% and 36.9%,
respectively, p < 0.01 and 0.01) and melatonin (47.8% and 24.0%, respectively, p <
0.001 and 0.01). These results were further supported by TEM images of Aβ samples,
which revealed the smallerꢀsized nonꢀfibrillar amorphous Aβ species in the presence
2
+
of Cu and 8d (Figure 5c and 5d, sequence 5). In contrast, larger Aβ aggregates were
observed with CQ (Figure 5c and 5d, sequence 4), melatonin (Figures 5c and 5d,
sequence 6) and the control sample (Figures 5c and 5d, sequence 2). Because CQ
2
+
exhibited a weaker ability to inhibit Cu ꢀinduced Aβ aggregation and to disaggregate
the wellꢀformed Aβ fibrils and melatonin provided almost no ability for either, these
observations suggest that both the indole and 8ꢀhydroxy quinoline moieties in 8d
could be crucial for redirecting and interacting with metalꢀAβ. The above results also
demonstrated that substituents on the indole moiety of 8d, especially the hydroxyl
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group, could influence the regulation of Aβ aggregation. These similar scenarios often
appeared in the literature describing the development of specific chemical ligands for
15, 32ꢀ34
antiꢀamyloidogenic activity against metalꢀAβ complexes.
Taken together, all of
1
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the results suggest that 8d has a strong ability to modulate selfꢀ and metalꢀinduced Aβ
aggregation.
2
+
2+
Figure 5. Experiments of inhibition Cu ꢀinduced Aβ
aggregation and disaggregation of Cu ꢀinduced Aβ
1–42
1
–42
aggregates in the presence of 8d. (a and b) Top: Scheme for the inhibition or disaggregation experiments. Bottom:
The fluorescence intensity of the ThT binding assay, data are expressed as the mean ± SD at least three
independent experiments. Statistical significance was analyzed by ANOVA: ∗ p < 0.01, ∗∗ p < 0.01, ∗∗∗ p < 0.001.
2+
(
c and d) TEM images analysis of the inhibition of Cu ꢀinduced Aβ1–42 aggregation and disaggregation of
2
+
2+
Cu ꢀinduced Aβ
aggregates. Serial number: (1) fresh Aβ1–42, (2) Aβ1–42 alone, (3) Aβ1–42 + Cu , (4) Aβ
+
1–42
1–42
2+
2+
2+
Cu + CQ, (5) Aβ1–42 + Cu + 8d, (6) Aβ1–42 + Cu + melatonin. Experimental conditions: Aβ1–42 (25 ꢁM);
2+
chelator/Aβ1ꢀ42/Cu = 2/1/1; HEPES (20 ꢁM) and NaCl (150 ꢁM); pH 6.6; 37 °C.
Effects of the QuinolineꢀIndole Derivatives on Neuronal Proliferation of PC12
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Cells
Inspired by the melatoninꢀNꢀbenzylamine hybrids can promote the development
of neural stem cells into the neuronal phenotype, we assessed the efficacies of the
0
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target compounds on neuronal proliferation with undifferentiated PC12 neuronal cells
16, 35
according to a wellꢀestablished method.
After the cells were cultured for 72 h in
the presence of the tested compounds (at concentrations ranging from 0.5 ꢁM to 50
M) or nerve growth factor (NGF, 50 ng/ml, as the positive control), the cells were
surveyed under a digital microscope. As the results indicate in Figure 6A, compounds
g, 8c, 8d and 8g did not show any neurotoxicity and revealed significant abilities to
ꢁ
7
increase the number of cells. The SAR investigation indicated that replacement of the
1
phenolic hydroxyl group on the indole scaffold of 8d (X = Cl, R = OH) with chlorine
1
(8c, X = Cl, R = Cl) generated a slightly decreased neurotrophic activity. However,
1
1
replacement with hydrogen (8a, X = Cl, R = H) and fluorine (8b, X = Cl, R = F) or
1
2
the introduction of a methoxy group (8f, X = Cl, R = OH, R = OMe) resulted in the
complete loss of the increased proliferation, and further methylation of the hydroxyl
1
2
group (8e, X = Cl, R = R = OMe) displayed increased neurotoxicity. Similar trends
were also observed for the compounds containing chlorine on the quinoline moiety,
1
1
such as compounds 7c (X = H, R = Cl) and 7d (X = H, R = OH), and when the
chlorine was removed from 8c or 8d, they exhibited a rather poor neurotrophic effect.
In addition, shifting the indole moiety to the benzene ring of the quinoline, for
example in compounds 9a, 9d and 10d, led to considerable neurotoxicity compared
with the vehicle. Compounds 7g and 8g, which have a dioxole moiety on the indole,
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demonstrated striking neurotrophic efficacies. The literature has reported that two
mitogenꢀactivated protein kinases (MAPKs), extracellular signalꢀregulated kinase
16, 36
(ERK) and p38, play crucial roles in neurotrophic actions.
Therefore, we used the
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ERK inhibitor U0126 (CAS: 109511ꢀ58ꢀ2) and the p38 inhibitor SB203580 (CAS:
52121ꢀ47ꢀ6) to study the effects of neuronal proliferation by quinolineꢀindole
derivatives using the 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyl tetrazolium bromide
MTT, 570 nm) assay. With the addition of the ERK inhibitor U0126 (Figure 6B), the
dꢀtriggered proliferation of neuronal cells was completely abolished, and the
1
(
8
addition of the p38 inhibitor SB203580 significantly decreased the proliferation effect
of 8d. These preliminary results suggested that quinolineꢀindole derivatives might
promote neuronal proliferation in a MAPKꢀdependent mechanism. Additionally, the
morphological studies in Figure 7 shows that compounds 7g, 8c, 8d and 8g exhibited
neurotrophic effects on PC12 cells, in contrast to the lack of increase in neurons using
CQ and melatonin.
Figure 6. (A) The quantification of neuron number using imageꢀbased analysis. Statistics were expressed as the
percentage relative to control and as the mean ± SD of at least three independent experiments. NGF was tested at a
constant concentration of 50 ng/ml. (B) The effects of 8d, 8d + U0126 (ERK inhibitor, 1 ꢁM) and 8d + SB203580
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(
p38 inhibitor, 1 ꢁM) on neuronal survival measured by MTT assay. Statistical significance was analyzed by
ANOVA: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 by compared with the control group (0 ꢁM quinolineꢀindole
derivatives).
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Figure 7. Images from representative experiments showing the quinolineꢀindole derivatives in undifferentiated
PC12 cells: cells were treated with 50 ng/ml NGF or 10 µM of melatonin, CQ, 7g, 8c, 8d, or 8g, respectively.
Neuroprotective Capacity against Intracellular Oxidative Stress
Cellular damage in the central nervous system by oxidative stress is a typical
feature in AD, PD, Huntington’s disease (HD), and ALS, especially intracellular
oxidative stress, such as mitochondrial ROS, which is closely involved in the most
17
crucial intrinsic pathway leading to apoptosis emanating from the mitochondria. To
explore the therapeutic potentials for compounds 7g, 8c, 8d and 8g as close as
possible to the physiological conditions of the onset of neurodegenerative disease, an
assay to investigate their neuroprotective capacities against intracellular oxidative
stress was performed using the human neuroblastoma cell line SHꢀSY5Y (which is
usually used in the determination of ROS) with tertꢀbutyl hydroperoxide (tꢀBuOOH)
as the ROS stimulant along with the fluorescence probe dichlorofluorescein diacetate
(
DCFHꢀDA), which was used to evaluate the intracellular ROS production. In the trial,
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all compounds were tested at 1ꢀ0.01 ꢁM, a concentration close to their concentrations
for promoting neurite outgrowth. As shown in Figure 8, after tꢀBuOOH was
introduced into the cells, the production of intracellular ROS units increased from
37.5 ± 5.2 (vehicle) to 415.5 ± 39.4 (control). In contrast, significantly less ROS
production was detected in 7gꢀ, 8cꢀ, 8dꢀ and 8gꢀincubated cells. The EC50 values
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(0.26, 0.82, 0.10 and 0.33 ꢁM, respectively, for 7g, 8c, 8d and 8g) in Table 2
demonstrated that these compounds have the greatest capacities against intracellular
oxidative stress compared with melatonin (EC50 > 1.0 ꢁM) and the parent compound
CQ (EC50 > 1.0 ꢁM).
Figure 8. Inhibition of tꢀBuOOHꢀinduced ROS production in the presence of CQ, melatonin, 7g, 8c, 8d and 8g
determined by DCFHꢀDA. Data are the mean ± SD of at least three independent experiments performed in
sextuplicate and expressed as fluorescence unit. Statistical comparisons with control (only tꢀBOOH) were
performed by oneꢀway ANOVA. (levels of significance * p < 0.05;** p < 0.01;*** p < 0.001, ns p > 0.05).
Table 2. The EC50 Values of Neuroprotective Capacity against Intracellular Oxidative Stress in SHꢀSY5Y Cell.
a
a
Compound
EC50 (ꢁM)
Compound
EC50 (ꢁM)
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melatonin
> 1.0
clioquinol
> 1.0
7g
0.26 ± 0.07
0.10 ± 0.03
8c
8g
0.82 ± 0.12
0.33 ± 0.05
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a
Data are expressed as the mean ± SD at least three independent experiments.
Metabolic Stability in Liver Microsomes of SD Rats
Proper liver microsomal stability of the optimal compounds is beneficial for the
stability in metabolism. To evaluate this property, the metabolic stabilities of
compounds 7g•HCl, 8c•HCl, 8d•HCl and 8g•HCl in the liver microsomes of SD rats
were examined according the procedure described in a previous study with CQ and
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donepezil as the reference antiꢀAD agents and testosterone as a positive control.
According to the results in Table 3, the hydrochlorides of 8c, 8d and 8g exhibited T1/2
values in liver microsomes of 195.8, 116.8 and 389.3 min, respectively, which
demonstrated better stabilities compared with CQ (T1/2 = 34.4 min) and donepezil
(T1/2 = 77.9 min). Compound 7g•HCl displayed a somewhat weaker stability and had
a T1/2 of 58.6 min toward liver enzymes, which may be attributed to the lack of
chlorine substituent to block the metabolic position of the quinoline scaffold. Overall,
these results revealed that the hydrochlorides of 7g, 8c, 8d and 8g had considerable in
37, 38
vitro metabolic stabilities and thus deserve further detailed studies in vivo
.
Table 3. Metabolic Stability of 7g•HCl, 8c•HCl, 8d•HCl and 8g•HCl in Liver Microsomes of SD Rats
ꢀ1
a
Compound
testosterone
k (min )
T1/2 (min)
b
0.31323 ± 0.04206
0.00889 ± 0.00041
2.2 ± 0.4
77.9 ± 5.1
c
donepezil
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CQ
0.02012 ± 0.00156
0.01183 ± 0.00026
0.00354 ± 0.00038
0.00593 ± 0.00020
0.00178 ± 0.00005
34.4 ± 3.8
58.6 ± 1.8
7g•HCl
8
c•HCl
8d•HCl
g•HCl
195.8 ± 23.1
116.8 ± 5.6
389.3 ± 15.5
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Results are expressed as the mean ± SD of at least three independent experiments performed in triplicate. The
positive control (testosterone) and the compound donepezil exhibited metabolic stability that was consistent with
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the literature and internal validation data.
Acute Toxicity Studies
The acute toxicity profiles of the hydrochlorides of 7g, 8c, 8d and 8g were
evaluated in adult C57BL/6 mice (12 weeks old). Each of the test compounds was
given in single doses of 0, 677, 1333, or 2000 mg/kg by intragastric infusion (n = 6
for each dosage, three male and three female). After the administration of the
compounds, any abnormal behavior and mortality changes were observed
continuously during the first 4 h, intermittently for the following 24 h, and
occasionally thereafter for 14 consecutive days for any delayed effects. During the
observation period, no acute toxicity phenomena, including mortality, significant
abnormal behaviors, drastically altered consumption of water or food and marked
weight loss or gain, were observed. Additionally, no macroscopic abnormities of the
stomach, intestines, heart, lung, liver, spleen, kidneys and other organs were detected
after all of the mice were sacrificed on the 14th day. Furthermore, no significant
histological abnormal changes were detected in the liver, heart, lung and brain of the
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mice at the maximum tested dosage 2000 mg/kg in the histopathological studies by
hematoxylin and eosin (HE) staining (Figure 9). Overall, these results revealed that
7g•HCl, 8c•HCl, 8d•HCl and 8g•HCl were well tolerated and exhibited no acute
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toxicity at a dose up to 2000 mg/kg.
Figure 9. Histological analysis of liver, heart, lung and brain for the acute toxicity studies of hydrochlorides of 7g,
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c, 8d and 8g at dosage of 2000 mg/kg in mice; 1000 ꢁm and 100 ꢁm indicate the scale bar of images (HE staining,
n = 6). Representative images of HEꢀstained liver, heart, lung and brain for each group are shown.
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Selective Promotion of Cell Proliferation in the Hippocampus of Living Adult
Mice
To further determine the efficacies of the target quinolineꢀindole compounds on
cell proliferation in the hippocampus of living adult mice, an unbiased in vivo
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screening technique was performed. Each compound (hydrochlorides of 7g, 8c, 8d
and 8g, at a concentration of 10 ꢁM) was administered by intracerebroventricular
(ICV) injection at a constant rate (0.5 ꢁL/h) over 7 days into the left lateral ventricle
of living adult wild type C57BL/6 mice (12 weeks old, n = 6 for each compound) via
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subcutaneously implanted osmotic minipumps. According to the previous studies,
the attainable intracerebroventricular concentrations of the compounds were in a
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lowꢀmicromolar to midꢀnanomolar concentration range. During the infusion of the
compound, animals received a daily intraperitoneal (IP) injection with the thymidine
analog bromodeoxyuridine (BrdU, 50 mg/kg/day) to mark new cells and the survival
of proliferating hippocampal neural precursor cells. Apart from the ICV injection
method, the administration of hydrochlorides of 7g, 8c, 8d and 8g (PO group, 30
mg/kg/day) by oral gavage to evaluate the efficacy of cell proliferation in the
hippocampus of adult C57BL/6 mice was also performed (n = 6 for each group). To
ensure a low baseline level of cell proliferation, mice were housed individually
without access to running wheels seven days prior to the assay, as social interaction
and voluntary exercise stimulate hippocampal neurogenesis. After one week of the
administration of the compounds (including the ICV and PO groups), all animals were
sacrificed and transcardially perfused. BrdU immunohistochemistry was used to
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quantify cell proliferation by counting the number of BrdU+ cells in the brain
hemisphere opposite to the side of infusion to avoid complications arising from the
surgical implantation of the pump. The number of BrdU+ cells was normalized
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against the volume of the hippocampus to obtain consistent data across six animals.
It is worth noting that the immunohistochemical staining of BrdU cells in the brains of
mice illustrated that the quinolineꢀindole compounds could specifically incorporate
BrdU in the hippocampal regions, including the DG, cornus ammonis (CA) 1, CA2
and CA3 regions, rather than the regions near the hippocampus, such as the cortex and
striatum (Figure 10). This valid evidence further demonstrated that the target
compounds had a preeminent ability to promote cell proliferation in the hippocampus
of living adult mice rather than universally throughout the entire brain. This
characteristic is very important for the treatment of AD, as all adult mammals form
new neurons within the hippocampus, which is indispensable for learning, memory
and neuronal plasticity.
Figure 10. The administration of quinolineꢀindole compounds by ICV indicated selective BrdU incorporation in
the hippocampus of living adult mice, including the dentate gyrus (DG), cornus ammonis (CA) 1, CA2 and CA3
regions, rather than cortex and striatum (immunohistochemical staining × 40, n = 6).
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The cell proliferation efficacies displayed in Figures 11 and 12 demonstrated a
substantial difference in the number of BrdU+ cells in the subgranular zone (SGZ) of
DG regions between the quinolineꢀindole compoundꢀtreated mice, CQꢀ or
melatoninꢀtreated mice and the vehicle group. After the living mice were treated with
the hydrochlorides of 7g, 8c, 8d and 8g over the 7 day period, the
immunohistochemical results indicated that the rates of BrdUꢀincorporated new and
surviving hippocampal neurons in the DG regions of the hippocampus were 43.6% (p
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<
0.001), 28.5% (p < 0.01), 41.3% (p < 0.001) and 31.2% (p < 0.01), respectively,
which revealed strikingly greater results than for the vehicle (7.8%) and CQ (6.2%)
groups. Melatonin displayed moderate effects (27.4%) on the hippocampal neurons,
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which was consistent with previously reported results. Consequently, the results
indicated that the quinolineꢀindole compounds could promote cell proliferation from
the reservoirs of neuronal stem cells in the subgranular zone (SGZ) of the
hippocampal DG. Furthermore, the ICV injection of the hydrochlorides of 7g, 8c, 8d
and 8g noticeably induced the cell proliferation of neural precursor cells in the CA1,
CA2 and CA3 regions of the hippocampus (7g: 61.9%, 56.1% and 49.3%, p < 0.001;
8c: 15.3%, 18.1% and 20.3%, p < 0.01; 8d: 50.1%, 39.6% and 36.5%, p < 0.001; 8g:
49.8%, 38.0% and 37.6%, p < 0.001), which demonstrated significantly greater
effectiveness compared with the vehicle (3.2%, 2.4% and 1.4%), CQ (2.7%, 2.1% and
1.5%) or melatonin (2.7%, 2.1% and 1.2%) groups (Figure 11 and 12). These results
further suggested that compounds 7g•HCl, 8c•HCl, 8d•HCl and 8g•HCl can induce
cell proliferation in the hippocampus of living adult mice.
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Figure 11. Immunohistochemically visualized BrdU incorporation in the new hippocampal neurons of living adult
C57BL/6 mice (immunohistochemical staining × 200, n = 6). The compounds 7g•HCl, 8c•HCl, 8d•HCl and
8g•HCl were administered ICV at the indicated concentration (0.5 ꢁL/h) for 7 days, during which mice were dosed
IP daily with BrdU (50 mg/kg/day) to label new hippocampal neurons. CA1, CA2, CA3 and DG indicate the
cornus ammonis (CA) 1, CA2, CA3 and dentate gyrus (DG) regions of the hippocampus, respectively.
Representative 200 × magnification images of an immunohistochemically stained hippocampus for each group
were shown.
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Figure 12. Efficacy of 7g•HCl, 8c•HCl, 8d•HCl and 8g•HCl in the living adult mouse model of cell proliferation
within the hippocampus. The compounds were administered ICV at the indicated concentration (0.5 ꢁL/h) for 7
days, during which time the mice were dosed IP daily with BrdU (50 mg/kg/day) to label new hippocampal
neurons. CA1, CA2, CA3 and DG indicate the cornus ammonis (CA) 1, CA2, CA3 and dentate gyrus (DG) regions
of the hippocampus, respectively. The results were expressed as the means ± SD (n = 6), and statistical
significance was analyzed by twoꢀway ANOVA: ns p > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 versus the
vehicle, CQ and melatonin groups.
Alternatively, after oral gavage administration of compounds at the dose of 30
mg/kg/day for 7 days, the new hippocampal neurons in the DG, CA1, CA2 and CA3
regions of the hippocampus for quinolineꢀindoleꢀcompound treated groups (7g: 7.6%,
8
.0%, 7.5% and 3.9%; 8c: 4.4%, 4.8%, 5.1% and 4.7%; 8d: 30.6%, 21.1%, 24.3% and
3.9%, p < 0.01; 8g:17.0%, 14.6%, 16.3% and 17.1%, p < 0.05) were higher than the
2
vehicle (2.2%, 2.3%, 2.4% and 1.5%), CQ (2.0%, 2.4%, 2.3% and 1.4%) or melatonin
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