Design and synthesis of multipotent 3-aminomethylindoles and 7-azaindoles with enhanced protein phosphatase 2A-activating profile and neuroprotection

Article abstract of DOI:10.1016/j.ejmech.2018.07.030

We report the synthesis and pharmacological evaluation of new 3-aminomethylindoles derivatives with neuroprotective properties designed to present multi-target activity centered on reducing the neuronal Ca²⁺ overload and preventing phosphatase

Full text of DOI:10.1016/j.ejmech.2018.07.030

Accepted Manuscript
Design and synthesis of multipotent 3-aminomethylindoles and 7-azaindoles with
enhanced protein phosphatase 2A-activating profile and neuroprotection
Rocío Lajarín Cuesta, Raquel L. Arribas, Carmen Nanclares, Eva M. García-Frutos,
Luis Gandía, Cristóbal de los Ríos
PII:
S0223-5234(18)30586-5
10.1016/j.ejmech.2018.07.030
EJMECH 10565
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 22 May 2018
Revised Date: 9 July 2018
Accepted Date: 11 July 2018
Please cite this article as: Rocí.Lajarí. Cuesta, R.L. Arribas, C. Nanclares, E.M. García-Frutos, L.
Gandía, Cristó. de los Ríos, Design and synthesis of multipotent 3-aminomethylindoles and 7-azaindoles
with enhanced protein phosphatase 2A-activating profile and neuroprotection, European Journal of
Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.07.030.
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Graphical Abstract
Gramine derivatives
R'
N
R'
X
N
Y
R
X = H, Cl, Br, OMe
Y = N, CH
R = Bn, Bu,CH2C CH
Ca2+
PP2A
Alzheimer's disease
P
P
P
P
P
P
P
P
P
P
Neurofibrilary
Tangles
Tau Kinases
(PLEASE USE COLOR FOR THIS FIGURE)
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Design and synthesis of multipotent 3-aminomethylindoles and 7-azaindoles
with enhanced protein phosphatase 2A-activating profile and neuroprotection
Rocío Lajarín Cuesta a, Raquel L. Arribas a, Carmen Nanclares a, Eva M. García-
Frutos b, Luis Gandía a, and Cristóbal de los Ríos a,c,
*
a Instituto-Fundación Teófilo Hernando and Departamento de Farmacología y
Terapéutica, Universidad Autónoma de Madrid, C/ Arzobispo Morcillo, 4, 28029
Madrid, Spain
b Instituto de Ciencia de Materiales de Madrid, Consejo Superior de Investigaciones
Científicas, C/ Sor Juan Inés de la Cruz, s/n, 28049, Madrid, Spain
c Instituto de Investigación Sanitaria, Hospital Universitario de la Princesa, C/ Diego
de León, 62, 28006, Madrid, Spain
Corresponding author: Cristóbal de los Ríos, e-mail address:
cristobal.delosrios@inv.uam.es
Abbreviations: Aβ, amyloid beta peptides; AD, Alzheimer’s disease; ICa, Ca2+
currents; NFT, neurofibrillary tangles; NMDA, N-methyl-D-aspartate; OA, okadaic
acid; PAD, PP2A-activating drugs; PD, Parkinson’s disease, pNPP, p-nitrophenol
phosphate; PP2A, phosphoprotein phosphatase 2A; pTau, phosphoTau; PSA, polar
surface area; R/O, rotenone plus oligomycin A; VGCC, voltage-gated Ca2+ channels.
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ABSTRACT
We report the synthesis and pharmacological evaluation of new 3-
aminomethylindoles derivatives with neuroprotective properties designed to present
multi-target activity centered on reducing the neuronal Ca2+ overload and preventing
phosphatase 2A (PP2A) inhibition, which are two important early physiophathological
events observed in neurodegenerative scenarios. Chemical syntheses of proposed
compounds were achieved in two straightforward reaction steps with high yields.
Most of the compounds mitigated the okadaic acid-provoked inhibition of PP2A and
protected SH-SY5Y cells against toxic stimuli related to Tau-hyperphosphorylation
and oxidative stress, similarly to the observed in in Alzheimer’s disease (AD). In
addition, some of them mitigated the Ca2+ overload induced by depolarization. The
derivative 1-(1-benzyl-5-chloro-1H-indol-3-yl)-N,N-dimethylmethanamine (19)
outstood by its high recovery of the PP2A activity and blockade of voltage-gated
Ca2+ channels, accompanied by good neuroprotective profile. These findings make
this compound eligible for further preclinical assays with the goal of positioning new
innovative drugs for the treatment of AD.
Keywords: Gramine, multi-target strategy, PP2A, Ca2+ channels, neuroprotection,
Alzheimer’s disease
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1. Introduction
Indole alkaloids are considered as one of the most successful small molecule drugs.
Both traditional medicine and current marketed drugs are full of examples where the
indole constitutes a chief part of the chemical structure, being the 5% of the total
FDA-approved drugs. Interestingly, two third of them present a 3-aminoethylindole
moiety, which is indeed found in paradigmatic drugs families such as the ergot
alkaloids (ergotamine, cabergoline, nicergoline, ergometrine, etc), the triptans
(sumatriptan, zolmitriptan, almotriptan, rizatriptan, etc), reserpine and related vinca
alkaloids (deserpidine, rescinnamine, yohimbine, etc) or the blockbuster tadalafil. In
addition, endogenous substances such as tryptophan, 5-HT or melatonin are also 3-
aminoethylindoles (Fig. 1).
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Fig. 1. Highlighted examples of FDA-approved natural products, synthetic drugs, and
endogenous indoles. All of these examples possess the 3-aminoethyl substructure,
traced in blue. (PLEASE USE COLOR FOR THIS FIGURE)
Conversely, much less interest, but not negligible, has aroused the therapeutic use
of their homologous 3-aminomethylindoles [1-5]. Among all the indole-based FDA-
approved drugs, such moiety can only be appreciated in the highly complex
vinorelbine (Fig. 2). Gramine (1, Fig. 2), extracted from the family plant of the
Poaceae, is the simplest example of 3-aminomethylindole discovered in biota [6].
Gramine has been object of several pharmacological studies, highlighting some of
effect on the serotoninergic system [7] or on mitochondrial bioenergetic [8]. Synthetic
analogues of 1 have shown pharmacological activities such as antioxidant and anti-
inflammatory [9, 10], anti-serotoninergic [11] or cell Ca2+ modulators [12]. Moreover,
1 and its analogues have been used as precursors for the synthesis of a huge
amount of indole derivatives, taking advantage their easy transformation through a
retro-Mannich type reaction in acidic media into intermediates susceptible to
smoothly suffer nucleophilic substitutions [13]. However, this chemical reactivity has
hindered their positioning as investigational drugs. In this context, our research
group has humbly contributed to set up experimental conditions that improve
chemical stability and handling of 1 derivatives [14], facilitating their isolation and
biological assessment. As a result, we have recently reported several 1 derivatives
with interesting neuroprotective properties related to a dual pharmacological profile,
the mitigation of neuronal Ca2+ overload and the reactivation of the Ser/Thr
phosphatases enzymatic activity, among which the derivative ITH12657 (2) (Fig. 2)
outstood by its wide-spectrum neuroprotective actions [14, 15].
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Fig. 2. Selected examples of 3-aminomethylindoles. Vinorelbine, the only drug in
clinic possessing this moiety; gramine (1), as the simplest natural product, and its
synthetic derivative ITH12657 (2) [14], under investigation for central nervous system
diseases. (PLEASE USE COLOR FOR THIS FIGURE)
This multipotent feature makes these compounds of interest for the treatment of
several human diseases where the alteration of both cell Ca2+ homeostasis and
protein phosphorylation occur. Among them, neurodegenerative diseases like
Alzheimer’s (AD) prevail, due to the social and economic issues that entail,
exacerbated by the lack of efficient medicines to face them [16]. In the case of AD,
the alteration of cytosolic Ca2+ levels evidenced in vulnerable neurons is a widely
observed characteristic [17, 18]. In this context, it is worthwhile mentioning that
memantine, which diminishes the Ca2+ influx through the N-methyl-
D-aspartate-
sensitive glutamate (NMDA) receptors [19], is one of the few prescribed drugs for
AD, along three anticholinesterasics (galantamine, donepezil, and rivastigmine) [20].
On the other hand, hyperphosphorylation of Tau protein that drives to the formation
of the so-called neurofibrillary tangles (NFT) is one of the most validated therapeutic
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targets in AD [21]. Although the real causes behind the AD onset remain elusive,
increasing evidences reveal that, after the senile plaques formation by aggregation
of amyloid β (Aβ) peptides, the generation of NFT, formed by hyperphosphorylated
Tau proteins, is an essential event for the progression of the disease [22], in such a
way that the sole presence of senile plaques accompanied by a total absence of
NFT is characteristic of aged patients without dementia symptoms [23, 24].
Otherwise, therapeutic approaches to treat AD, such as those targeting
amyloidogenesis, the use of pro-cholinergic drugs, or several antioxidant-based
strategies, have failed [25, 26]. Not even drugs capable of inhibiting Tau kinases
have afforded the expected crops [27]. In this sense, the activation of the opposite
process, namely Tau dephosphorylation, mostly performed by the Ser/Thr
phosphatase PP2A [28], has raised marginal interest in drug discovery [29], despite
there is a plethora of biomedical literature reporting that PP2A enzymatic activity is
key for the correct phosphorylation state of Tau [30], and more, PP2A down-
regulation has been observed in neurodegenerative diseases like AD [31],
Parkinson’s disease (PD) [32], and amyotrophic lateral sclerosis [33], among others
[34].
By these reasons, a multi-target strategy based on activating Tau dephosphorylation
and mitigating neuronal Ca2+ overload keeps being an interesting therapeutic
alternative for the development of innovative potential drugs for AD. Attempting to
optimize the neuroprotective properties of the lead compound 2, we herein describe
the design, synthesis, and pharmacological assessment of gramine derivatives that
present different substitution at C5, as well as aza-indole analogues, assessing the
effect of these chemical modifications on both PP2A-activating potential and
blockade of voltage-gated Ca2+ channels (VGCC).
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2. Results and Discussion
2.1. Chemistry
The design of new 1 analogues aimed at enhancing their activity as PP2A-activating
drugs (PAD) [35] to favor the dephosphorylation of NFT-aggregating Tau proteins,
while maintaining VGCC blocking properties. With this purpose, we selected as
substituents at the indole nitrogen those present in the best PAD of the previous
family of 1 derivatives, i.e. benzyl, n-butyl, and propargyl. Modifications at C5 of the
indole ring aimed to replace the metabolism-sensitive methyl group present in 2 (Fig.
2) by a more stable group or by its removal. Thus, the isosteric replacement of the
methyl group by a chlorine atom was one of the chemical strategies selected, in
order to improve the metabolic stability without losing biological activity. In addition,
the relevance of substitution at C5 for the pharmacological properties studied was
challenged with the preparation and further evaluation of non-substituted analogues
13–18. Another remarkable modification was replacing the benzene-fused ring by a
pyridine, to access the corresponding 7-azaindole analogues (formally pyrrolo[2,3-
b]pyridines). We selected the easily accessible 5-bromo and 5-methoxy substitutions
to start probing these new aza-indoles. The designed ligands are supposed to
present more druggable properties, according to computational prediction centered
on parameters of drug-likeness (Table S1, supplementary material). Such
computational prediction included the Lipinsky’s rule of 5 (molecular weight, clog P,
and number of hydrogen bonds donors and acceptors) [36], the polar surface area
(PSA), and, since the proposed derivatives possess ionizable groups, both pKa (of
the most basic center) and clog D [37] (Table S1, supplementary material). Most of
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the compounds fulfilled the requirements for druggability, except compound 20 [X =
Cl, R = Bn, R’ = -(CH2)5-, Scheme 1] that showed two alerts (Table S1,
supplementary material). However, realizing that it could be considered as the
closest isoster of the lead compound 2, it was synthesized and biologically
assessed, too. With regard to the 7-azaindole derivatives, they drew a significant
decrease in both clog P and clog D, except for compound 26, which showed a slight
alert in clog D (Table S1, supplementary material).
Scheme 1. Preparation of 1 derivatives. Reagents and conditions: (a) (1) NaH,
DMSO, rt, 1–2 h, (2) alkyl or benzyl halide, rt, 1–2 h; (b) Me2NH (aq) or piperidine,
HCHO (aq), AcOH, 0 ºC to rt, 2–5 h.
Compounds were prepared in two steps, starting with a N-alkylation reaction. 1H-
indoles were deprotonated with NaH at room temperature, for 1 h in the case of the
chlorinated substrate, and 2 h in the case of non-substituted 1H-indole. Afterwards,
the corresponding alkyl or benzyl halide was added, to furnish intermediates 3–8 in
excellent yields (Scheme 1), without further chromatographic purification. Despite
this reaction classically recruits DMF as solvent [1, 38], we alternatively use DMSO
[39, 40] because of its easier removal after the work-up. For the N-alkylation of 7-
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azaindoles, smoother conditions were achieved [41], using KOH under phase-
transfer catalysis. Commercially available 5-bromo-7-azaindole or the 5-methoxy-7-
azaindole, prepared by aromatic nucleophilic substitution of the former with MeONa
[42], gave rise to 9–12 in quantitative yields, except for the propargyl-bearing
compound 11, obtained in medium yield (Scheme 2), which was elevated with the
previous NaH/DMSO conditions. Automatized flash chromatography was required to
isolate intermediates 9–12 from the phase transfer catalyst waste.
Scheme 2. Preparation of 7-azaindole derivatives analogues to 1. Reagents and
conditions: (a) CuBr, MeO-Na+, DMF, MeOH, 175 ºC, 4.5 h; (b) Bu4N+HSO4-, KOH,
alkyl or benzyl halide, acetone, 75 ºC, 2–5 h; (c) HCHO (aq), dimethylamine (aq) or
piperidine in glacial acetic acid, 0 ºC to rt, 2–5 h.
Targeted products 13–32 were synthesized by Mannich-type reaction of
intermediates 3–12 with formaldehyde and dimethylamine or piperidine in acetic
acid, with good yields in most of the cases (Scheme 1 and 2). The addition order of
reagents sensibly influences chemical yield, as the indole enamine moiety should
attack the in situ-formed iminium species. Thus, formaldehyde and the secondary
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amine, either dimethylamine or piperidine are first mixed in AcOH and then the
resulting solution injected to the reaction mixtures. This is presumably one of the
causes of the scattered reactions yields found in literature for the synthesis of N,N-
dimethylaminomethyl-3-indoles and related analogues. The other, in our opinion, is
that some authors conduct Mannich reaction over N1-unsubstituted indoles for then
to promote the N-alkylation [1]. Otherwise, taking into account that such N-alkylation
confers stability versus the possible retro-Mannich reaction of 3-aminomethyl-1H-
indoles, we consider a better election to promote Mannich reaction over N-alkylated
indoles. Few examples in literature report the use of ZnCl2 in ethanol instead acetic
acid to promote Mannich reaction in similar indoles, but describing lower yields and
longer reaction times than those shown herein [43]. As mentioned before, 3-
aminomethyl-1H-indoles suffer a retro-Mannich process leading to the generation of
3-methylen-3H-indoles in acidic aqueous. To avoid such by-reaction, work-ups were
carried out at pH > 10 (NaOH), chlorinated solvents were neutralized from acid
traces with basic alumina, and flash chromatography, when necessary, was
executed with basic alumina as stationary phase. Due to most of the compounds
appear as oils of complicated manipulation and limited aqueous solubility, they were
salinized to their oxalate salts, thus becoming white powdered solids, highly soluble
(10 mM) in aqueous buffers, possessing high chemical stability, confirmed by 1H-
NMR length dissolved in D2O. In contrast, 13C-NMR spectra of oxalates were carried
out using DMSOd6, to get sufficient concentration for adequate acquisition
(Supplementary material).
2.2. Gramine (1) derivatives mitigated the okadaic acid-induced Ser/Thr
phosphatases inhibition
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The rationale behind the development of the 1 derivatives we pursue is that, by
interacting with the catalytic subunit C of PP2A in a binding site that does not
compromise the PP2A-leaded Tau-dephosphorylating enzymatic activity,
compounds could avoid the approach of specific inhibitors to PP2A catalytic center,
and at this way keep or recover the PP2A phosphatase activity, which is found out
depressed in several neurodegeneration scenarios [28], such as that observed in
AD, where it has been described high cytosolic levels of PP2A endogenous
inhibitors. To probe this hypothesis, the PP2A inhibition is modeled by subjecting
SH-SY5Y neuroblastoma cells to okadaic acid (OA), a marine toxin widely used as
experimental model of Tau hyperphosphorylation [44]. Exposure to OA reproduces
morphological and cognitive hallmarks of AD in both neuronal cultures and animal
models as a consequence of the impairment of PP2A [45]. To monitor changes in
the phosphatase activity of SH-SY5Y cell cultures, we selected the colorimetric
method of the p-nitrophenol phosphate (pNPP).[46] Colorless pNPP is a
chromogenic substrate of phosphatase enzymes that hydrolyze it to form yellow-
colored p-nitrophenolate (λabs = 405 nM). The NMDA receptor blocker memantine at
10 nM was used as reference compound in these experiments by its activating effect
of PP2A [14].
Table 1
%Recovery of the okadaic acid (OA)-challenged Ser/Thr phosphatase activity of SH-
SY5Y neuroblastoma cells in presence of compounds 13–32.a
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Compound
Y
-
X
-
R
R’
-
%Recovery of PP2A activity
28 ± 3*
Memantine
13
-
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
H
Bn
CH3
45 ± 9**
14
H
Bn
-(CH2)-
CH3
37 ± 4**
15
H
nBu
12 ± 13ns
30 ± 9*
16
H
nBu
-(CH2)-
CH3
CH2C≡CH
17
H
59 ± 12**
17 ± 11ns
78 ± 11***
27 ± 11ns
39 ± 13*
18
H
CH2C≡CH -(CH2)-
19
Cl
Cl
Cl
Cl
Cl
Cl
Br
Br
Br
Br
Br
Br
OCH3
Bn
CH3
-(CH2)-
CH3
20
Bn
21
nBu
22
nBu
-(CH2)-
CH3
38 ± 15*
23
CH2C≡CH
23 ± 6*
24
CH2C≡CH -(CH2)-
44 ± 13**
21 ± 10ns
31 ± 8**
25
Bn
CH3
-(CH2)-
CH3
26
N
Bn
27
N
nBu
34 ± 9*
28
N
nBu
-(CH2)-
CH3
22 ± 10ns
17 ± 5ns
29
N
CH2C≡CH
CH2C≡CH
Bn
30
N
-(CH2)-
CH3
25 ± 11ns
33 ± 7**
31
N
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32
N
OCH3
Bn
-(CH2)-
47 ± 9***
aSH-SY5Y cells, previously preincubated with test compounds at 0.1 µM for 24 h,
were treated with the selective PP2A inhibitor OA at 15 nM and compounds for 18 h
more. Phosphatase activity was assessed by the pNPP colorimetric method. Data
are mean of five assays ± SEM in triplicates, expressed as recovered phosphatase
activity. ***p < 0.001, **p < 0.01, *p < 0.05, ns = not significant, respect to SH-SY5Y
neuroblastoma cells exposed to OA in absence of compounds.
Following the protocol described in the experimental section, SH-SY5Y cells
subjected to OA 15 nM for 18 h suffered a decrease of 26 ± 2% in the phosphatase
activity, without compromising cell viability (unlike the exposure to OA at 20 nM for
20 h, which decreased SH-SY5Y cells viability in a significant fashion, as shown in
the neuroprotection experiments). Most of the compounds softened the drop of the
phosphatase activity in OA-treated cells in a significant manner (Table 1), recovering
it up to 78% result obtained when compound 19 was tested. Globally, this family of 1
derivatives diminished the effects of OA in a 34 ± 4%. Stressing on structure-activity
relationships, benzyl-substituted 1 analogues recovered PP2A activity by an average
of 40%, whilst those bearing butyl or propargyl groups did it by about a 30%.
Similarly, a recovery average of 40% was also observed in those derivatives
presenting chlorine or methoxy group at C5, which was much better than those
observed in brominated or non-substituted analogues. The replacement of aromatic
fused ring from benzene to pyridine to furnish derivatives 25–32 did not affected
PAD activity dramatically, but an slight drop of the averaged %recovery was
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appreciated. Similarly, the N,N-dimethylaminomethyl moiety at C3 afforded slightly
better recovery than compounds bearing a piperidinylmethyl moiety at C3 analogues.
Thus, the most sensible substitution seems to be that at the indole nitrogen.
Collecting the best options for each chemical substitution proposed, a compound
that presented benzyl at the indole nitrogen, chlorine or methoxy at C5, benzene as
aromatic fused ring, and dimethylamine instead of piperidine, should theoretically be
the best PP2A-activating compound. Indeed, compound 19 (R = Bn; R’ = Me; X = Cl;
Y = CH) presented the best %recovery of the PP2A enzymatic activity (75%, Table
1). Taking into account that OA is about 500-fold more selective for inhibiting PP2A
than for PP1 [47], we firmly state that the reactivation of the phosphatase activity
carried out by our compounds in presence of OA 15 nM (a concentration where PP1
is marginally affected) is directly ascribed to their ability to counteract the OA-
induced PP2A inhibition. We have proved this hypothesis through western-blot
analysis, testing the effect of the best PAD, i.e. 19, in the phosphorylation rate of Tau
(Fig. 3).
2.3. Compound 19 reduced phosphorylation of Tau of SH-SY5Y neuroblastoma cells
exposed to OA
It is important to remark that PP2A is by far the principal phosphatase enzyme of
Tau protein [28]. The quantification of phosphoTau (pTau) levels was achieved with
the primary antibody anti-pTau AT8 that recognizes pSer 202 and pThr 205
phosphoepitopes, extensively found in NFT-forming Tau aggregates [48].
75kDa
p-tau (AT8)
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37kDa
β-actine
basal
OA
OA + Mem OA + 19
Figure 3. Effect of compound 19 on the 15 nM OA-induced Tau phosphorylation in
SH-SY5Y neuroblastoma cells. Top, immunoblot of a representative experiment.
Data are expressed as the mean ± SEM of three independent experiments. #p <
0.05, compared with basal group, *p < 0.05 compared with cells subjected to OA in
absence of compounds. Compound 19 was tested at the concentration of 100 nM.
Memantine (Mem) 10 nM was used as a reference compound.
The 24 h-exposure of the selective PP2A inhibitor OA to SH-SY5Y neuroblastoma
cells augmented almost 2.5-fold the phosphorylation rate of Tau at Ser 202 and Thr
205, which was noticeably prevented by the presence of 19 at 0.1 µM (Fig. 3), the
same concentration used for enzymatic assays shown in Table 1. Hence, we can
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confirm that compound 19 avoids the inhibition of PP2A, which could drive, for
instance, to the NFT-generating Tau hyperphosphorylation observed in AD. With the
goal of finding structural clues for this highlighted pharmacological activity, molecular
docking studies of 19 with PP2A were performed.
2.4. Docking studies on the interaction of 1 derivatives and PP2A
Molecular docking studies were implemented to figure the possible interactions of
PP2A with 1 derivatives, using its 3D structure deposited in the Protein Data Bank
(ID: 2IE4; www.rdcb.org) [49]. We selected derivative 19 to run the docking
experiments (Fig. 4), as it was the best PAD of the family, and compound 32 (Fig. 5),
as the most active 7-azaindole 1 derivatives, which exerted a 47% recovery of PP2A
enzymatic activity (Table 1).
(A)
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(B)
Fig. 4. Molecular docking of 19—PP2A complex. (A) Pose of 19 with the interacting
amino acid residues within a 6 Å radius. (B) Electrostatic map where the charge-
transfer interaction between the indole ring of 19 and the positively-charged His 191
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is observed (high electron density areas are red-colored, while electron-deficient
areas are in blue). (PLEASE USE COLOR FOR THIS FIGURE)
Fig. 4A shows compound 19 in a shape able to establish a strong H-bond between
its dimethylamine group, protonated at physiological pH, and the hydroxyl of Ser 120
(d = 1.75 Å). The benzene-fused ring of 19 seems to interact in planar (π–π)
hydrophobic fashion with Trp 200, while the electron-filled indole-forming pyrrole
finely faces the electron-demanding His 191 (Fig. 4B).
Fig. 5. Molecular docking of 32—PP2A complex. Pose of 32 with the interacting
amino acid residues within a 8 Å radius. (PLEASE USE COLOR FOR THIS
FIGURE)
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As far as the 7-azaindole analogue 32, it can be appreciated a weak H-bond
between Ser 120 and the methoxy group at C5, where the steric hindrance elicited
by methyl would prevent a better fit. Otherwise, 32 nicely poses into the hydrophobic
pocket defined by Ala 216 (to the piperidine substituent; d = 3.59 Å), Ile 123 and Val
126 (to the benzyl group; d = 4.01 and 3.86 Å, respectively)(Fig. 5).
2.5. Effect of 1 derivatives on the increases of neuronal Ca2+ stimulated by
depolarization
SH-SY5Y neuroblastoma is an excitable cell line that responds to extracellular
exposure to 70 mM K+ opening VGCC, and thus taking up ungovernable large
amounts of Ca2+ that trigger neuronal death phenomena, resembling the Ca2+
overload evidenced in several neurodegenerative diseases such as AD [50]. Since
several 1 derivatives described in literature behaved as Ca2+-antagonists, we
wondered whether these newly synthesized 1 derivatives related to head compound
2 could buffer depolarization-induced Ca2+ elevations, too. To respond this question,
96-well-plated SH-SY5Y cells were charged with the Ca2+-sensitive fluorescent dye
fluo-4, which detects cytosolic Ca2+ and its oscillation, and stimulated with an
extracellular solution of 70 mM K+ in presence of the test compounds. The well-
known Ca2+-antagonist nifedipine [51], was used in our experiments as reference
compound. As shown in Table 2, Similarly, some 1 derivatives reduced the K+-
stimulated Ca2+ increase through VGCC in SH-SY5Y cells monitored by fluo-4
(Table 2).
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Table 2
Blockade of the high K+-Induced Ca2+ uptake by compounds 13–32 in SH-SY5Y
neuroblastoma cells.a
Compound
Y
X
-
R
R’
%blockade
40 ± 5***
22 ± 6*
Nifedipine
13
-
-
-
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
H
Bn
Bn
CH3
14
H
-(CH2)-
CH3
11 ± 7ns
7 ± 5ns
15
H
nBu
16
H
nBu
-(CH2)-
CH3
13 ± 8ns
3 ± 4ns
CH2C≡CH
CH2C≡CH
Bn
17
H
18
H
-(CH2)-
CH3
35 ± 5**
40 ± 6***
37 ± 7***
23 ± 7*
19
Cl
Cl
Cl
Cl
Cl
Cl
Br
20
Bn
-(CH2)-
CH3
21
nBu
22
nBu
-(CH2)-
CH3
24 ± 8*
23
CH2C≡CH
CH2C≡CH
Bn
18 ± 11ns
32 ± 8**
46 ± 6***
24
-(CH2)-
CH3
25
20

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26
27
28
29
30
31
32
N
N
N
N
N
N
N
Br
Br
Bn
-(CH2)-
CH3
15 ± 6ns
10 ± 6ns
9 ± 4ns
nBu
Br
nBu
-(CH2)-
CH3
Br
CH2C≡CH
CH2C≡CH
Bn
11 ± 6ns
8 ± 5ns
Br
-(CH2)-
CH3
OCH3
OCH3
10 ± 6ns
19 ± 8ns
Bn
-(CH2)-
aSH-SY5Y cells, previously preincubated with test compounds at 1 µM or nifedipine
at 3 µM for 10 min, were stimulated with a high K+ solution to get a final
concentration of 70 mM in the extracellular medium, monitoring the subsequent
cytosolic Ca2+ elevation with the fluorescent dye fluo-4. Compounds diminished such
increase by the percentage expressed in the table. Data are mean of five assays ±
SEM in triplicates. ***p < 0.001, **p < 0.01, *p < 0.05, and ns = not significant,
respect to SH-SY5Y neuroblastoma control cells exposed to 70 mM K+ in absence of
tested compounds.
Only 8 of 20 compounds blocked the Ca2+ entry exacerbated by depolarization in
SH-SY5Y cells in a statistically significant manner. Interestingly, the presence of a
chlorine atom at C5 favor better VGCC blockade (Table 2), what is consistent with its
nature as bioisoster of the methyl group, present in the head compound 2, which
showed important Ca2+ antagonist properties [14]. Besides, benzyl group outstands
as a privileged substituent over butyl and propargyl at the indole nitrogen. Most of
the 7-aza-analogues 25–32 did not block VGCC but, surprisingly, compound 25
appears as the best blocker of the current family, what underlines the complexity to
21

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infer structure-activity relationships over some biological targets such as ionic
channels. The best VGCC blockers 19 and 25 were further studied with
electrophysiological experiments using a whole-cell patch-clamp configuration, to
directly measure Ca2+ currents (ICa) through VGCC of bovine chromaffin cells.
Fig. 6. Blockade of Ca2+ currents by 19 and 25 in bovine chromaffin cells.
Normalized intensity/voltage (I/V) relationship of ICa before (control, black line, A or
B) and after treatment with compound 19 (grey line, A) or compound 25 (grey line,
22

ACCEPTED MANUSCRIPT
B). Data are means ± SEM of 9 cells from 4 cultures, as indicated in parentheses.
***p < 0.001; **p < 0.01; *p < 0.05, respect to control for each voltage.
To characterize blockade of ICa by 19 or 25, we performed intensity vs voltage (I/V)
curves, applying depolarizing pulses of 5 10-2 s from –60 to +70 mV each 20 s, in
the presence or the absence of compounds. As shown in Fig. 6, both compounds
produced a partial blockade of ICa that was statistically significant when positive
depolarizing pulses were applied, without affecting the amplitude of ICa recorded at
negative potentials. These data would indirectly suggest that the blocking effects of
19 and 25 on ICa are selective for non-L-type VGCC (i.e. N- or P/Q-type channels),
as reported for 2 [14]. The absence of effects on L-type VGCC would elude
cardiovascular-related side effects for these compounds.
Hence, five derivatives (13, 19, 21, 22, and 24) behaved as multipotent ligands,
drawing a blockade of VGCC and maintaining the PP2A activity compromised by
inhibitory actions. Among all the chemical modifications proposed, the substitution of
chlorine at C5 and a benzyl group linked to the indole nitrogen would be maximizing
this dual action. Hence, the pharmacological properties shown by 2 are matched by
24 and amenably improved by 19, which indeed exerted a reduction of the pTau
levels measured by western-blot (Fig. 3), although the use of other Tau antibodies
would be necessary to confirm this attractive feature. In any case, further
experiments were planned to assess whether this dual action achieved
neuroprotection against toxic stimuli related to AD.
2.6. Neuroprotection experiments
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Before appraising the ability of compounds for counteracting toxic stimuli related to
neurodegeneration in SH-SY5Y cells, we estimated their possible intrinsic toxicity in
the same cell model, by per se exposing cell cultures to compounds for 48 h at much
higher concentrations, up to 30 µM, than those used for pharmacological activities
and neuroprotection. Thus, none of the compounds reduced viability of SH-SY5Y
cells measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) method at the concentration of 3 µM, and most of them were harmless at 10
µM (Fig. S1, supplementary material). Hence, the derivatives 13–32 would not
damaging neurons at concentrations where they show PAD, Ca2+-antagonist and
neuroprotective activities.
According to the pharmacological activities of 13–32 shown in Table 1 and 2, we
proposed to run the neuroprotective assays under two experimental models of
neurodegeneration closely related to the progression of AD, i.e. Tau
hyperphosphorylation and oxidative stress, monitoring the viability of SH-SY5Y cells
in presence of compounds by the method of the MTT reduction [52]. Tau
hyperphosphorylation-related damage was simulated by exposing SH-SY5Y cells to
OA. Thus, when cells were subjected to the damage exerted by OA at 20 nM for 20
h, cell viability was reduced by 30 ± 1%. In this situation, cells preincubated and
coincubated with compounds 13–32, relieved the OA-elicited loss of cell viability by
the percentages expressed in Table 3, which was statistically significant for all
compounds. Memantine at 10 nM was used as standard in these experiments for its
protective actions against the impairment of PP2A enzymatic activity [14].
Table 3
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% Protectiona by compounds 13–32 of the cell viability of SH-SY5Y neuroblastoma
cells compromised by okadaic acid (OA)b or the cocktail of rotenone plus oligomycin
A (R/O)c
Compound
Y
X
-
R
R’
OAb
63 ± 2***
-
R/Oc
Memantine
-
-
-
-
-
Melatonin
13
-
-
-
36 ± 5*
34 ± 3***
28 ± 2***
20 ± 6ns
25 ± 5**
32 ± 4***
41 ± 3***
32 ± 3*
24 ± 2**
38 ± 3*
27 ± 5*
26 ± 6*
16 ± 2ns
32 ± 4*
25 ± 3*
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Br
Br
Bn
CH3
67 ± 3***
14
Bn
-(CH2)- 65 ± 4***
CH3 50 ± 3***
-(CH2)- 55 ± 4***
CH3 75 ± 2***
15
nBu
16
nBu
17
CH2C≡CH
18
CH2C≡CH -(CH2)- 58 ± 4***
19
Bn
CH3
-(CH2)- 62 ± 4***
CH3 93 ± 1***
-(CH2)- 98 ± 1***
CH3 43 ± 6**
88 ± 2***
20
Bn
21
nBu
22
nBu
23
CH2C≡CH
24
CH2C≡CH -(CH2)- 55 ± 7*
25
Bn
Bn
CH3
84 ± 2***
26
N
-(CH2)- 60 ± 4***
25

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27
28
29
30
31
32
N
N
N
N
N
N
Br
Br
Br
Br
nBu
CH3
-(CH2)- 98 ± 1***
CH3 63 ± 5**
57 ± 4***
18 ± 3ns
39 ± 3*
17 ± 3ns
13 ± 3ns
29 ± 6*
40 ± 6**
nBu
CH2C≡CH
CH2C≡CH -(CH2)- 68 ± 3***
OCH3 Bn
OCH3 Bn
CH3
51 ± 5***
-(CH2)- 62 ± 2***
aData, assessed by the MTT method, are shown as the recovered %viability by
compounds when the toxic stimulus is present. bSH-SY5Y cells preincubated with
compounds at 0.1 µM for 24 h, and subjected to 20 nM OA and 0.1 µM of tested
compounds for 20 h. cSH-SY5Y cells preincubated with compounds at 10 nM for 24
h, and subjected to R/O 30 and 10 µM in presence of compounds at 10 nM for 24 h
more. Data are mean of five assays ± SEM by triplicates. ***p < 0.001, **p < 0.01, *p
< 0.05, ns = not significant, respect to SH-SY5Y cells exposed to toxic stimulus in
absence of compounds.
Establishing structure-activity relationships from neuroprotective experiments is
always a though challenge, due to neuroprotective compounds could dissipate
neuronal damage through interactions with a plethora of proteins implicated in the
physiopathological cascade triggered by the toxic stimulus. Indeed, when we
analyzed the effect of compounds over the toxicity exerted by OA, no clear
evidences of privileged substituents were found among all the synthetic alternatives
proposed. Nevertheless, we appreciated that the propargyl group at the indole
nitrogen (R = CH2C≡CH) and both methoxy group and hydrogen at C5 (X = OCH3,
H) are the less efficacious chemical modifications to mitigate the OA-driven
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neurotoxicity. In addition, there were five compounds able to recover the viability of
OA-challenged SH-SY5Y cells by more than 80% (Table 3). Three of them (19, 21,
and 22) modulated both PP2A-played phosphatase activity and cell Ca2+ overload
(Table 1 and 2). These compounds are therefore merely considered multitarget-
directed neuroprotectants.
Next, SH-SY5Y neuroblastoma cells were subjected to oxidative stress by
administration of stressor cocktail formed by rotenone plus oligomycin A (R/O),
which blocks complexes I and V of the mitochondrial electron transport chain,
respectively, and therefore impairs ATP synthesis [53]. It is worthwhile mentioning
that several models of oxidative stress related to aging produce PP2A inactivation
[54], as well as neuronal Ca2+ dishomeostasis [55], where VGCC overactivation
plays an essential role [56]. Thus, 24 h of exposure to the R/O cocktail at the
concentration of 30 and 10 µM, respectively, diminished viability of SH-SY5Y
neuroblastoma cells by 38 ± 1%. Previously, we had observed that 1, tested in a
wide range of concentrations, featured a decent neuroprotective profile against R/O
neurotoxicity from concentrations as low as 10 nM (data not shown), in a similar
fashion to the well-known antioxidant and PP2A activator melatonin [57, 58]. This
fact prompted us to test compounds 13–32 at 10 nM in the in vitro model of oxidative
stress elicited by R/O in SH-SY5Y cells, although these compounds were actually
not designed either to scavenge free radicals or bind any redox enzyme. Otherwise,
14 out 20 compounds healed part of the damage elicited by R/O (Table 3). None of
the chemical substitutions outstood, but propargyl group at the indole nitrogen
seems to be the less favored substituent to achieve neuroprotection. Compounds 18,
21, 28, and 32 performed the best antioxidant effects, being better than the standard
melatonin and reaching about 40% of protection (Table 3). Compound 19 also
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deserves to be mentioned herein, as its 32% of protection against the R/O-evoked
toxic signal, added to the best PAD profile (Table 1), the second best depolarization-
stimulated Ca2+ blockade (Table 2), and its great neuroprotective effect against OA
(88%, Table 3), make it the lead compound of the series. In addition, multipotent
compounds 21 and 22 showed important neuroprotective actions, unlike 24, which
did not protect against the R/O toxicity, despite being PAD and VGCC blocker.
Besides, the important Ca2+ antagonist effect of compound 25 (Fig. 6), though
lacking PP2A up-regulatory actions, led to a decent neuroprotective profile in both
models. On the other hand, the good neuroprotective profile of 28 (Table 3) opens
the door to new experiments to figure out its real pharmacological mechanism of
action, as it did not affect phosphatase activity or cell Ca2+ overload. As far as the 7-
azaindole analogues 25–32, they have not globally afforded better results than their
benzene-fused analogues 13–24. Otherwise, they would present beneficial
physicochemical properties to feature acceptable pharmacokinetic parameters, such
as the lower clog P, clogD[7.4], pKa (Table S1, supplementary material) or minor in
vitro toxicity (Figure S1, supplementary material). Finally, the family of chlorinated 1
derivatives evidenced the most promising biological activities. In our opinion, this
should be due in part to the bio-isosteric nature of the chlorine atom as a first-
election, metabolically stable, replacement for methyl groups, present in the head
compound 2. Its most promising derivative seems to be 19 that, although more
western-blot analyses need to be executed to fully characterize its
dephosphorylating effect on Tau, it herein demonstrated that reduces the levels of
pTau measured by AT8 antibody, which recognizes phosphoepitopes at Ser 202 and
Thr 205 sites.
28