Synthesis and biological assessment of 4,1-benzothiazepines with neuroprotective activity on the Ca2+ overload for the treatment of neurodegenerative diseases and stroke

molecules

Article

Synthesis and Biological Assessment of 4,1-Benzothiazepines

with Neuroprotective Activity on the Ca²⁺Overload for the

Treatment of Neurodegenerative Diseases and Stroke

Lucía Viejo ^1,2, Marcos Rubio-Alarcón ¹, Raquel L. Arribas ¹, Manuel Moreno-Castro ¹, Raquel Pérez-Marín ¹,

María Braun-Cornejo ¹, Martín Estrada-Valencia ^{1, ,†}

*

and Cristóbal de los Ríos ^1,2,

*

1

Instituto-Fundación Teóﬁlo Hernando and Departamento de Farmacología y Terapéutica, Universidad

Autónoma de Madrid, C/Arzobispo Morcillo, 4, 28029 Madrid, Spain; lucia.viejo@estudiante.uam.es (L.V.);

marcru02@ucm.es (M.R.-A.); raquel.arribas@urjc.es (R.L.A.); manmoren@ucm.es (M.M.-C.);

raquelpm4@gmail.com (R.P.-M.); m.brauncornejo@gmail.com (M.B.-C.)

Instituto de Investigación Sanitaria, Servicio de Farmacología Clínica, Hospital Universitario de La Princesa,

28006 Madrid, Spain

2

*

†

Correspondence: M.H.Estrada-Valencia@leeds.ac.uk (M.E.-V.); cristobal.delosrios@inv.uam.es (C.d.l.R.)

Present address: Leeds Institute of Cardiovascular and Metabolic Medicine, School of Medicine, University of

Leeds, Leeds LS2 9JT, UK.

ꢀꢁꢂꢀꢃꢄꢅꢆꢇ

ꢀꢁꢂꢃꢄꢅꢆ

Abstract: In excitable cells, mitochondria play a key role in the regulation of the cytosolic Ca²⁺levels.

A dysregulation of the mitochondrial Ca²⁺buffering machinery derives in serious pathologies, where

neurodegenerative diseases highlight. Since the mitochondrial Na⁺/Ca²⁺exchanger (NCLX) is the

principal efﬂux pathway of Ca²⁺to the cytosol, drugs capable of blocking NCLX have been proposed

to act as neuroprotectants in neuronal damage scenarios exacerbated by Ca²⁺overload. In our search

of optimized NCLX blockers with augmented drug-likeness, we herein describe the synthesis and

pharmacological characterization of new benzothiazepines analogues to the ﬁrst-in-class NCLX

blocker CGP37157 and its further derivative ITH12575, synthesized by our research group. As a

result, we found two new compounds with an increased neuroprotective activity, neuronal Ca²⁺

regulatory activity and improved drug-likeness and pharmacokinetic properties, such as clog p or

brain permeability, measured by PAMPA experiments.

Citation: Viejo, L.; Rubio-Alarcón,

M.; Arribas, R.L.; Moreno-Castro, M.;

Pérez-Marín, R.; Braun-Cornejo, M.;

Estrada-Valencia, M.; de los Ríos, C.

Synthesis and Biological Assessment

of 4,1-Benzothiazepines with

Neuroprotective Activity on the Ca²⁺

Overload for the Treatment of

Neurodegenerative Diseases and

Stroke. Molecules 2021, 26, 4473.

https://doi.org/10.3390/

molecules26154473

Keywords: CGP37157; benzothiazepines; neuroprotection; Ca²⁺overload; oxidative stress; mitochondria

Academic Editors: Helen Osborn,

Diego Muñoz-Torrero,

Robert J. Doerksen and Silvia Socorro

Received: 9 June 2021

Accepted: 20 July 2021

Published: 24 July 2021

1. Introduction

Calcium ions act as very versatile intracellular second messengers. Calcium originates

a plethora of cell responses, such as neurotransmitters exocytosis, muscle contraction or

gene transcription activation [1

]. The cytosolic Ca²⁺concentration ([Ca²⁺]_c) is an essential

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parameter that needs to be ﬁnely regulated. Calcium homeostasis depends on the quantity

of Ca²⁺uptake through plasmalemmal transporters and its release from intracellular or-

ganelles [

channels. The ﬁrst ones comprise the N-methyl-D-aspartate-sensitive glutamate receptors

(NMDAR) [ ], the -amino-3-hydroxy-5-methyl-4-isoxazolpropionic acid-sensitive gluta-

mate receptors (AMPAR) [ ], and some subtypes of the acetylcholine nicotinic receptors [ ].

Among the second ones, voltage-gated Ca²⁺channels (VGCC) outstand [ ]. Ca²⁺ions can

be released to cytosol from endoplasmic reticulum (ER) through inositol-1,4,5-triphosphate

receptors (IP3R) or ryanodine receptors [ ], and from mitochondria through both mito-

chondrial Na⁺/Ca²⁺(NCLX) and H⁺/Ca²⁺exchangers [

]. Cells are capable of decreasing

[Ca²⁺]_cwith Ca²⁺-chelating proteins, such as Calbindin-D28K and parvalbumin, or with

plasmalemmal transporters like the Na⁺/Ca²⁺exchanger [

]. Furthermore, ER and mito-

chondria uptake Ca²⁺through the sarcoplasmic/endoplasmic reticulum Ca²⁺ATPase [

and the mitochondrial Ca²⁺uniporter [

]. The malfunction of any of these mechanisms

1

]. The Ca²⁺entry to the cytosol mainly occurs via ligand or voltage-gated ionic

α

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Attribution (CC BY) license (https://

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]

Molecules 2021, 26, 4473. https://doi.org/10.3390/molecules26154473

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Molecules 2021, 26, 4473

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gives rise to cell Ca²⁺overload or deﬁcit, in sensitive areas of excitable cells, which promote

situations of cell morbidity [10].

The impairment of the [Ca²⁺]_cbalance has been widely documented in several central

and vascular diseases. In particular, this is a common pathological event in neurodegen-

erative diseases, where Ca²⁺dyshomeostasis is more exacerbated than the minor Ca²⁺

deregulation observed in a normal aging. In Alzheimer’s disease (AD), the Ca²⁺dyshome-

ostasis hypothesis points out that Ca²⁺dysregulation in intracellular stores, plasma mem-

brane transporters, and buffering mechanisms accelerate the progression of the disease.

Therefore, the Ca²⁺dysregulation evidenced in AD causes functional impairments, leading

to brain damage and cognitive deﬁcit [11]. Moreover, Ca²⁺imbalance has been strongly

correlated with several hallmarks of AD, as amyloid

β

-peptide (A

β

) oligomers can form

pores in the membranes permeable to Ca²⁺

[

12]. In addition, soluble A

β

oligomers induce

Ca²⁺dyshomeostasis through NMDAR overstimulation, driving to synapsis loss in mice

brains [13]. Interestingly, the ﬁrst approved drug for AD within the last 18 years (approved

in 7 June 2021 by FDA), aducanumab [14], reduced Ca²⁺overload in animal models [15].

Besides, the other non-anticholinesterasic drug approved for AD is other Ca²⁺regulator, the

NMDAR blocker memantine [16]. Various Ca²⁺permeable channels such as NMDAR [17],

AMPAR [18], VGCC [19] or ionotropic purinergic receptors 7 [20,21] are also implicated

in the progression of the amyotrophic lateral sclerosis. These Ca²⁺channels participate in

the ampliﬁcation of the neuronal lesion, for instance, triggered by the glutamate released

by activated microglia, and where the mitochondrial dysfunction seems to be the main

target [22,23]. The α-synuclein, chief component of the Lewy bodies of dopaminergic

neurons compromised in Parkinson’s disease, forms small aggregates (protoﬁbrils) able to

generate ionic pores in the lipid membrane [24], and to induce an uncontrolled Ca²⁺entry

to neurons [25]. Additionally, the continuous ﬁring exerted by dopaminergic neurons needs

high ﬂuxes of Ca²⁺, what makes these neurons particularly vulnerable to Ca²⁺dyshome-

ostasis [26]. The polyglutamined huntingtin protein, characteristic of the Huntington’s

disease, binds directly to IP3R, and induces a massive Ca²⁺release from the endoplasmic

reticulum [27]. The pathological huntingtin protein also affects both NMDA and VGCC

permeability to Ca²⁺

[28,29]. As far as stroke, it is considered as an “acute” neurodegener-

ation process, where cerebral ischemic injury shares physiopathological mechanisms of

neuronal death with neurodegenerative diseases. Many of these mechanisms control Ca²⁺

dysregulation as a key event generated by the large exposure to glutamate [30] and the

subsequent recruitment of NMDAR and AMPAR. Thus, compounds studied for ischemic

brain injury, such as melatonin, have featured their neuroprotective actions through the

expression of Ca²⁺-binding proteins [31].

In summary, all these observations reinforce the clinical study of Ca²⁺channels antago-

nists and the search of new drugs capable of regulating [Ca²⁺]_c. These remain a therapeutic

challenge, where innovative approaches focused on interacting with intracellular organelles

buffering Ca²⁺are getting more attention every day. Under this premise, our research

group studies new 4,1-benzothiazepine derivatives analogues to CGP37157 (Figure 1) [32

]

with important neuroprotective actions in several in vitro models of neurodegeneration

and stroke. Their pharmacological activity involves biological targets handling [Ca²⁺]_c, as

VGCC [33], NCLX [34], or the Ca²⁺homeostasis modulator 1 [35]. Hence, these compounds

can be considered as a privileged family of Ca²⁺modulators. Our main goal aimed to

improve their pharmacokinetic properties, increasing their aqueous solubility while keep-

ing their neuroprotective proﬁle. Then, through some isosteric replacements, compounds

with better Lipinsky’s rules of ﬁve parameters were obtained without compromising

pharmacodynamics (Table S1, Supplementary Material) [36].

Molecules 2021, 26, 4473

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Figure 1. Chemical structures of the hit CGP37157, and the leads ITH12575 [34 ,35 ] and ITH12662 [36 ,37 ],

as the best compounds of the previously evaluated 4,1-benzothiazepines synthesized by our re-

search group.

Figure 1 shows lead compounds of previously described families of 4,1-benzothiazepines.

Based on these results, we herein described a newly-synthesized CGP37157 analogues pre-

senting chemical descriptors that afforded the best pharmacological activities in previous

studies, as well as the replacement by pyridine of the pending phenyl ring, in order to supply

less lipophilic drugs. Alternatively, isomers of ITH12575 were also prepared with the goal

of assessing the effect of shifting the position of the isopropyl group on the pharmacological

activity. In addition, the use of dimethylamine as an isoster of chlorine has been probed,

which would also enhance pharmacokinetic profile of derivatives.

2. Results and Discussion

2.1. Chemistry

The preparation of 4,1-benzothiazepine derivatives was achieved through two main

reaction steps, namely o-lithiation (Scheme 1, step a) and S-alkylation (Scheme 1, step b),

following experimental procedures previously reported with slight modiﬁcations [34].

t

◦

78 C, 2 h; (b) methyl

Scheme 1.

(a

) (i) BuLi, THF,

−

78 C to

−

20 C, 2 h; (ii) ArCHO, THF,

−

◦

thioglycolate, TFA, 85 C, 24 h.

Non-commercial isopropylbenzaldehydes, necessary to prepare intermediates

5 and

6

, were synthesized from the corresponding bromoisopropylbenzene by metal–halogen

exchange with ⁿBuLi in presence of DMF as a source of carbonyl (Supplementary Material).

Boc-protected p-chloroaniline [35] suffered NH–deprotonation by ^tBuLi dropwise addition

at

◦

−

78 C. To promote the subsequent ortho-metallation process, temperature was elevated

Molecules 2021, 26, 4473

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up to

−

20 ^◦C. Then, the resulting organolithium intermediates underwent nucleophilic

addition on the selected aromatic aldehydes to produce diarylcarbinol derivatives 5–7

in medium to low yields. The addition of aryllithium species over arylaldehydes was

affected by various obstacles, being the main one product degradation by an intramolecular

nucleophilic attack of the generated alcohol over the carbamate, similarly to what was

previously reported [38]. With the aim of increasing polarity and aqueous solubility

by the incorporation of aromatic nitrogen atoms, some pyridylaldehydes were used as

substrates for the addition reaction, which produced intermediates

8–

11. Next, preparation

of compounds 12

–

18 was executed by treatment with an excess of methyl thioglycolate in

reﬂuxing of triﬂuoroacetic acid (TFA; 85 ^◦C). In this acidic environment, several reactions

proceed in one pot. A SN1 reaction occurs due to removal of H₂O and attack of the thiol

reagent over the stabilized carbocation, along with the release of the Boc-protected amine.

Thus, the newly free amine attacks the pending methyl ester in an intramolecular fashion,

to yield the corresponding benzothiazepines 12–18 (Scheme 1) in moderate to excellent

yields. In previous works, substrates possessing aromatic nitrogen atoms needed the use

of alternative reagents and additional reaction steps [36]. By contrast, the 5-(pyridin-N-

yl)benzothiazepines 15

to those for the synthesis of the 5-arylbenzothiazepines 12

(Supplementary Material), pyridine-bearing derivatives 15

–

18 were obtained in good yields and under similar conditions

14. As shown in Table S1

18 present a better aqueous

–

solubility, according to their calculated partition coefﬁcient logarithm (clog p) value that

are between 2.5 and 3.2.

A second strategy to increase polarity of 4,1-benzothiazepines was explored. It con-

sisted of replacing the chlorine atom at position 7 by a dimethylamino group. The Boc-

protected N,N-dimethyl-p-phenylenediamine 19 (Scheme 2) (Supplementary Material)

was subjected to the same conditions of ortho-lithiation, and the nucleophilic addition

over o-isopropylbenzaldehyde led to the formation of intermediate 20 in moderated yield

(33%). In this case, the ortho-lithiation reaction followed by the nucleophilic addition gave

lower yields with several other tested aromatic aldehydes (e.g., with o-chlorobenzaldehyde,

yielding the corresponding intermediate in a 12% yield), presumably due to an enhanced

intramolecular addition of carbinol to the carbamate, with removal of tert-butanol. Then,

the reaction of 20 with methyl thioglycolate in TFA at reﬂux afforded the corresponding

7-(dimethylamino)-5-(2-(propan-2-yl)phenyl-1,5-dihydro-4,1-benzothiazepin-2(3H)-one 21

with a 38% yield (Scheme 2).

◦

20 C, 2 h; (ii) ortho-(iso-propyl)benzaldehyde, THF,

Scheme 2.

(

a

) Boc₂O, Et₃N, THF, rt, 2 h; (

b

) (i) ^tBuLi, THF,

−

78 C to

−

◦

−78 C, 2 h; (c) methyl thioglycolate, TFA, 90 C, 24 h.

Attempting to increase yields, we replaced the Boc- group with pivaloyl (2,2-dimethyl

propanoyl) as a protecting group, which had offered a reliable alternative for the preparation

of pyridothiazepines [36 ]. Thus, N,N-dimethyl-p-phenylenediamine was protected at the free

amine with pivaloyl chloride using triethylamine as a base in excellent yields. The subsequent

◦

ortho-lithiation reaction of 22 was promoted with ^tBuLi at

−

20 C as commented above, and its

addition to selected aromatic aldehydes furnished the corresponding open intermediates 23–25

Molecules 2021, 26, 4473

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in low to good yields, but with a more straightforward purification than that experimented

with the Boc-protected intermediate 20 and analogues. Formation of the benzothiazepine

scaffold needed higher temperatures, i.e., 110 ^◦C, to avoid the isolation of S-alkylated non-

cycled intermediate. Thus, we obtained in one-pot products 26–28 in low yields (Scheme 3 ).

Nevertheless, this entails a significant optimization since the removal of pivaloyl required in

previous reports a three-step protocol [36 ] (removal of pivaloyl protecting group, S-alkylation,

and finally, a LiHMDS-catalyzed cyclization).

t

◦

t

◦

20 C, 2 h;

Scheme 3.

(

a

) BuCOCl, Et₃N, CH₂Cl₂, 0 C to rt, 3 h; (

b

) (i) BuLi, THF,

−

78 C to

−

◦

(ii) ArCHO, THF, −78 C, 2 h; (c) methyl thioglycolate, TFA, 110 C, 24 h.

The replacement of chlorine by dimethylamine at C7 afforded a slight reduction of

the predicted clog p (Table S1, Supplementary Material). Some other derivatives proposed

to be tested were benzothiazepines unsubstituted at C7. These new analogues were

expected to be more lipophilic than the head CGP37157, but their clog p also lowered,

about one unit. Consequently, we synthesized new derivatives starting from aniline, which

was ﬁrstly protected with pivaloyl chloride, and the subsequent addition of aromatic

aldehydes via ortho-lithiation provided the expected intermediates 30 and 31. With the

goal of probing alternative methodologies, preparation of ﬂuorinated intermediate 30

n

was carried out through ortho-lithiation with BuLi in smooth conditions. Although

yield was not disappointing in this example (43%), the reaction with other aldehydes

were unsuccessful. Moreover, the disturbing presence of the by-product derived from the

addition of n-butyllithium to p-ﬂuorobenzaldehyde in the crude product moved us back

to use ^tBuLi as metallating agent. We achieved addition to the bulky o-anisaldehyde by

inducing ortho-metallation of 29 with ^tBuLi, affording intermediate 31 in low yield. The

treatment with methyl thioglycolate at 100 ^◦C underwent deprotection, S-alkylation, and

cyclization in one pot; however, this treatment produced very low yields (Scheme 4).

Scheme 4.

(

a

) ^tBuCOCl, Et₃N, _◦CH₂Cl₂, rt, 3 h; (

b

) for R = p-F: (i) ⁿBuLi, THF, 0 ^◦C to rt, 4 h; (ii) p-

0

ﬂuorobenzaldehyde, THF,

−

78 C to rt, 2 h; (

c

) for R = o-OCH₃or 3 -pyridylaldehyde: (i) ^tBuLi, THF,

−

78 ^◦C to

100 C, 24 h.

−

10 ^◦C, 2–3 h; (ii) ArCHO, THF,

−

78 ^◦C to

−

20 ^◦C, 2 h; (

d) methyl thioglycolate, TFA,

◦

Molecules 2021, 26, 4473

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The alternative three-independent-steps methodology was carried out as described [36].

Nevertheless, it did not entail an improvement in terms of overall chemical yield compared

with the one-pot preparation, in addition to increasing the time required and expenses.

Finally, we appreciated that 4-aminopyridine could become an eligible starting point

for the synthesis of new pyridothiazepines with enhanced polarity, different to the previ-

ously described pyrido [2,3-e][1,4] thiazepines [36], as they would give rise to interesting

pyrido[4,3-e][

1

,

4

] thiazepine bicycles. Protection of 4-aminopyridine with pivaloyl chloride

◦

was conducted in good yield at lower temperature (

diacylated product, observed at rt. Regular ortho-lithiation conditions and the subse-

quent addition to benzaldehyde, conducted this time at

provided the corresponding intermediate 35 in excellent yield (90%). T_◦hen, the one-pot

reaction using methyl thioglycolate in TFA at high temperature (up to 115 C, including the

use of sealed tube) failed. Alternatively, we recruited the three steps plan. Hence, pivaloyl

group was removed under basic aqueous conditions (KOH in ethanol/H₂O reﬂux) and the

free amino alcohol 36 suffered nucleophilic substitution of methyl thioglycolate aided by

TFA under CH₂Cl₂reﬂux, obtaining 36 in low yield, presumably due to the scarce contribu-

tion of the pyridine ring to the stability of the intermediate carbocation. Lastly, cyclization

−

20 C), to avoid the formation of

−

20 ^◦C unlike previous reactions,

to form the desired pyrido[4,3-e][1,4] thiazepine 38 was accomplished by deprotonation of

the amine with LiHMDS in THF (Scheme 5). The global yield of these three chemical steps

reached a decent 22% (13% from aniline, ﬁve reaction steps). Intriguingly, the incorporation

of an aromatic nitrogen at position 7 gave rise to a pyridothiazepine derivative with a very

good clog p value (1.93, Table S1, Supplementary Material), much better than those of the

previously reported pyrido[2,3-e][1,4] thiazepines [36].

◦

Scheme 5. (a b

) ^tBuCOCl, Et₃N, CH₂Cl₂, rt, 3 h; ( ) for R = p-F: (i) ⁿBuLi, THF, 0 C to rt, 4 h; (ii) p-ﬂuorobenzaldehyde, THF,

−

78 _◦^◦C to rt, 2_◦h; (

c

) for R = o-OCH₃or 3⁰-pyridylaldehyde: (i) ^tBuLi, THF,

−

78 ^◦C to

−

10 ^◦C, 2–3 h; (ii) ArCHO, THF,

◦

−78 C to −20 C, 2 h; (d) methyl thioglycolate, TFA, 100 C, 24 h; (e) LiHMDS, THF, −78 C to rt, 24 h.

2.2. Pharmacology

2.2.1. Assessment of per se Toxicity of the New Derivatives in Cell Cultures

Firstly, we aimed to ensure that the new synthesized compounds had no negative

effect over the viability of our cellular models. Thus, SH-SY5Y neuroblastoma cells and

rat cortical neurons were exposed to compounds at increasing concentrations (1, 3, 10,

and 30

tested compounds evidenced that compound 13, bearing an isopropyl group at meta

position, showed a remarkable cytotoxic proﬁle at 30 M (Figure 2), reason why 13 was

µM) (Figure 2). First, experiments of viability in SH-SY5Y cells in presence of

µ

discontinued from the pharmacological analysis. Despite compound 12 did not produce a

dramatic loss of cell viability, this new molecule was not further evaluated on the grounds

that some solubility problems were appreciated. Indeed, both compounds are highly

lipophilic benzothiazepines analogues to ITH12575, as conﬁrmed by their clog p (Table

S1, Supplementary Material). The other compounds herein described did not affect cell

viability in a statistically signiﬁcant fashion. Regarding cortical neurons, compounds 18

26 27 28, and 33 slightly reduced cell viability in a signiﬁcant manner at 30 M. Thus, we

paid attention to their therapeutic range in experiments using cortical neurons.

,

µ

Molecules 2021, 26, 4473

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Figure 2. Effect of compounds on the cell viability of SH-SY5Y cells and rat cortical neurons, measured by the MTT

method. Data are means SEM of at least three independent experiments, normalized over the maximum cell viability

±

(basal = 100%), obtained from SH-SY5Y cells or rat cortical neurons not exposed to any compound.*** p < 0.001, ** p < 0.01,

* p < 0.05, compared with basal group.

Overall, these experiments helped us to stablish proper testing concentrations. For

instance, considering that the neuroprotective experiments need long incubation period,

the selected concentration was 10 µ

M. In the case of the modulation of neuronal Ca²⁺

oscillations, assays to evaluate IC₅₀in the micromolar range seemed reliable, as they are

acute assays.

2.2.2. Effect of Compounds on the Membrane Depolarization-Induced and

NMDA-Induced Ca²⁺Inﬂux

Subsequently, we assessed the ability of the compounds to minimize altered Ca²⁺

elevations into cells in our two cellular models. On one hand, we subjected SH-SY5Y

neuroblastoma cells to a high extracellular K⁺concentration (70 mM), which evokes cell

depolarization and VGCC opening [33]. This experimental model mimics what occurs in a

plethora of neurodegenerative diseases, where Ca²⁺overload is a common event and mito-

chondria play a valuable role to buffer such Ca²⁺elevations. Should our compounds slow

the rate of Ca²⁺release from mitochondria, they would improve the capacity of neurons to

handle the neurotoxic cytosolic Ca²⁺accumulation. Thus, most of the synthesized com-

pounds, preincubated to SH-SY5Y cells at the concentration of 10

mitigating effect of the depolarization-stimulated [Ca²⁺]_celevation in SH-SY5Y cells by

about 50% (Figure 3). Compounds 18 M in a

21, and 32 decreased Ca²⁺signaling at 10

µM, showed an amenable

,

µ

similar manner or even better than the reference compound nifedipine (10 µM) (Figure 3)

.

This appreciable Ca²⁺antagonist feature allowed us to calculate their IC₅₀, deﬁned as the

concentration of compound capable of halving the 70 mM K⁺-evoked Ca²⁺elevation. For

that purpose, SH-SY5Y cells were incubated with the test compounds at several concentra-

tions from 1 to 30 µ

M and then stimulated with high K⁺to induce depolarization. In this

situation, increasing concentrations of compounds produced a proportional decline of Ca²⁺

uptake, allowing us to draw concentration–response curves (Figure 3). Those compounds

prevailing in the single concentration experiments, 18

IC₅₀of the family, 11.5, 5.4, and 9.2 µM, respectively.

, 21, and 32, presented the lowest

Molecules 2021, 26, 4473

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Figure 3. Representative ﬂuorescent curve of [Ca²⁺]_coscillations for each test compound and the standard nifedipine (Nife),

at the concentration of 10 M. The curves represent the % of ﬂuorescence compared to a control situation, i.e., cells just

stimulated with 70 mM K⁺, and they are the mean

SEM of at least 3 independent experiments by triplicate. Bottom right,

table with the IC₅₀data calculated from experiments using compounds concentrations from 1 to 30 µM.

µ

±

On the other hand, NMDAR is one the principal pathways of Ca²⁺overload reported

in neurodegenerative diseases [39]. Indeed, a unique non-anticholinesterasic drug pre-

scribed for AD is the NMDAR antagonist memantine [16]. For this reason, we considered

of note to assess whether these potential NCLX blockers could also mitigate the altered

Ca²⁺ﬂuctuations derived from NMDAR stimulation. To achieve this aim, we chose the

primary culture of the rat cortical embryonic neurons, as SH-SY5Y neuroblastoma cells

have not operative NMDAR [40]. In this protocol, NMDA was administered at the con-

centration of 10

NMDAR. The subsequent [Ca²⁺]_celevation was slightly reduced by compounds exposed

to neurons at 10 M (Figure 4 ), mostly by about 25%, except 21 and 27, which showed a

noticeable mitigation of the NMDA-elicited Ca²⁺increase, even higher than that exerted

by the reference compound memantine at 10 M. Therefore, only compounds 21 and 27

offered IC₅₀values below 30 µM, being 6.2 and 18.5 µM, respectively.

µ

M to rat cortical embryonic neurons to induce Ca²⁺entry through the

µ

Molecules 2021, 26, 4473

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Figure 4. Representative ﬂuorescent curves of [Ca²⁺]_coscillations for each test compound and the standard memantine

(Mem), at the concentration of 10 M. The curves represent the % of ﬂuorescence compared to a control situation, i.e., cells

just stimulated with NMDA 10 M, and they are the mean SEM of at least 3 independent experiments by triplicate.

µ

±

Bottom right, table with the IC₅₀data calculated from experiments using compounds concentrations from 1 to 30 µM.

2.2.3. Effect of Compounds against Toxic Stimuli Related to Neurodegeneration

The neuroprotective effects of these compounds in in vitro models of neurodegenera-

tion were evaluated against a cocktail of rotenone and oligomycin A (R/O, 30 M/10 M)

and the glutamate exposure (50 M). For the former, SH-SY5Y neuroblastoma cells were

µ

subjected to oxidative stress by administration of the stressor cocktail R/O, which disrupts

the correct function of the mitochondrial electron transport chain by inhibiting complex I

and V, respectively. It is worthwhile mentioning that this oxidative stress model related

to aging produces, in terms of the mitochondrial impairment, neuronal Ca²⁺dishome-

ostasis [41]. Hence, a potential NCLX partial blocker should efﬁciently counteract the

neurotoxicity elicited by R/O. Thus, the effect of the compounds administered at 10

against the R/O damage was evaluated by the MTT reduction method. As it can be ap-

preciated in Figure 5, dimethylamino analogues 21 26 27, and 28 present a clear trend

µM

,

to protect against oxidative stress, as these compounds are able to reduce the cell death

induced by R/O. Beside this family, pyridothiazepine 38 also showed a neuroprotective

proﬁle against oxidative stress.

Molecules 2021, 26, 4473

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Figure 5. Effect of compounds on cell viability compromised by rotenone and oligomycin A (R/O)

in SH-SY5Y cells (

Data are means

A

) or glutamate 50

µM in rat cortical neurons (B) measured by the MTT method.

±

SEM of at least three independent experiments in triplicate, normalized over the

maximum viability (control) obtained from cells not exposed to the toxic stimulus. Melatonin (Mela,

10 nM) or memantine (Mem, 30 nM) were used as standard compounds, respectively. ### p < 0.001,

compared with control group (white bars), *** p < 0.001, ** p < 0.01, * p < 0.05 compared with cells

stimulated with toxic stimulus in absence of compounds (black bars).

Regarding the other neurodegeneration model, the effect against the excitotoxicity

produced by high glutamate concentration was selected, considering that during the pro-

gression of neurodegenerative diseases, neurons lose their ability to control glutamate

concentrations, while this neurotransmitter keeps in the synaptic cleft, producing cell death

by over-activating NDMAR [42]. Similar to the ﬂuorescence experiments of NMDA-evoked

[Ca²⁺]_coscillations, the cell culture used for these assays were rat cortical embryonic neu-

rons, considering their susceptibility to glutamate administration. In these experiments,

compounds elicited a different pattern of neuroprotection. For instance, compounds 21

,

32, and 33 presented a statistically signiﬁcant dissipation of the glutamate-elicited neuro-

toxicity. Again, compound 38 nicely recovered cell viability of the glutamate-challenged

cortical neurons.

2.2.4. Blood–Brain Barrier Crossing Ability

Considering the potential use of these compounds as drugs targeting the central

nervous system (CNS), we evaluated their ability to penetrate the blood–brain barrier

(BBB) using the parallel artiﬁcial membrane permeation assay (PAMPA) prediction method.

The in vitro permeabilities (Pe) of compounds to CNS were obtained by executing these

experiments in the presence of the lipid extract of a porcine brain as a model of human

BBB, as described in the experimental section. Negative and positive reference drugs were

used to assess the correct development of each assay. Thus, most of the benzothiazepine

derivatives described in this work were capable of crossing the BBB by passive diffusion,

showing Pe values higher than 4, a widely accepted value that deﬁnes the limit for passive

diffusion [43]. As it can be appreciated in Figure 6, only compounds 14, 26, and 32 did

not reach this Pe value. In the case of 14, the presence of the highly charged nitro group

presumably hinders its passive diffusion.

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Figure 6. Blood–brain barrier (BBB) permeability evaluated by the parallel artiﬁcial membrane permeation assay (PAMPA)

method. Permeability is represented as means SEM of three independent experiments by triplicate. C (caffeine) and T

±

(testosterone) are negative and positive control, respectively. A value of Pe

≥

4 indicates ability to cross the BBB passively.

3. Materials and Methods

3.1. General

Commercially available reagents for synthesis or biological experiments were pur-

chased from Sigma-Aldrich/Merck (Madrid, Spain). Solvents, with analytical grade, were

obtained from VWR/Avantor (Barcelona, Spain) and freshly distilled when used for reac-

tions. Thin layer chromatography (TLC) under silica gel was used to monitor the evolution

of reactions by UV light exposure at 254 nm. Reactions were carried out under Schlenk

conditions (vacuum/argon purges and argon atmosphere). Further puriﬁcations of com-

pounds were conducted by automatized ﬂash chromatography ISOLERA One (Biotage,

Uppsala, Sweden), using pre-charged “SNAP” columns. Melting points, without correc-

tion, were obtained with a SMP-10 apparatus (Stuart, Stone, Staffordshire, UK). MS spectra

were acquired in a QSTAR ABSciex apparatus (Applied Biosystems, Foster City, CA, USA).

We acquired 1H and 13C NMR spectra in a AVANCE 300 MHz station (Bruker, Billerica,

MA, USA), shown as parts per million (ppm) calibrated by the residual proton signal from

the deuterated solvents. The purity of the ﬁnal compounds was evaluated by elemental

analysis in a CHNS-932 station (Leco, St Joseph, MI, USA), where mass percentages were

within the 0.4% compared with the calculated value, or by HRMS in an ABSciex QSTAR

spectrometer under the high-resolution conﬁguration with electrospray as the ionization

source, where m/z values were within the 5 ppm compared with the calculated value.

3.2. General Procedure for the Synthesis of Intermediates 5–11, 20, 26–28, 31, and 35

General procedure was similar to what has been described, ref. [34] with slight modi-

ﬁcations. To a solution of the Boc- or Piv-protected anilines

mentary Material) (1 equiv) in dry THF (4–8 mL/mmol) at

4

−

[

35], 19

, 22, and 29 (Supple-

78 ^◦C under Ar atmosphere,

^tBuLi (1.7 M in hexanes, 2.7 equiv) was added dropwise. When the addition was complete,

◦

the mixture was stirred for 15 min at

−

78 C, then it was warmed up to

−

20 C and stirred

◦

for 2 h more. After this time, the reaction mixture was cooled down to

−

78 C and a

solution of the corresponding arylaldehyde (1.1 equiv) in THF (5 mL) was slowly added,

stirring the resulting mixture at

78 ^◦C until no longer evolution was observed (2–5 h)

by TLC. Reaction was interrupted by slow addition of H₂O (15 mL) and allowed to reach

rt. Afterwards, it was extracted with diethyl ether (3 30 mL) and combined organic

−

×

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layer was washed with brine (3

evaporated, obtaining an oil that was puriﬁed by automated ﬂash chromatography using

ethyl acetate/n-hexane mixtures as eluent.

×

50 mL), dried over anhydrous MgSO₄, ﬁltered, and

3.2.1. 4-Chloro-2-[hydroxy(4⁰-isopropylphenyl)methyl]-N-tert-butoxycarbonylaniline (5)

Following the General procedure 4.2, reaction of 4-chloro-N-tert-butoxycarbonylaniline

4

[

35] (2.28 mmol, 519 mg) with p-isopropylbenzaldehyde (Supplementary Material) (2.51

1

mmol, 372 mg) [^tBuLi (6.16 mmol)] yielded

5

(341 mg, 40%). H NMR (300 MHz, CDCl₃)

δ

7.81 (d, J = 8.70 Hz, 1H, Ar), 7.66 (s, 1H, NH), 7.30 (m, 6H, Ar), 7.08 (d, J = 2.5 Hz, 1H,

H3), 5.84 (s, 1H, CHOH), 3.60 (bs, 1H, OH), 2.94 [m, J = 6.9 Hz, 1H, CH(CH₃)₂], 1.47 [s, 9H,

C(CH₃)₃], 1.28 [d, J = 6.9 Hz, 6H, CH(CH₃)₂].

3.2.2. 4-Chloro-2-[hydroxy(3⁰-isopropylphenyl)methyl]-N-tert-butoxycarbonylaniline (6)

Following the General procedure 4.2, reaction of 4-chloro-N-tert-butoxycarbonylaniline

4

[

35 ] (0.66 mmol, 150 mg) with m-isopropylbenzaldehyde (Supplementary Material) (0.72

1

mmol, 107 mg) [^tBuLi (1.78 mmol)] yielded

6

(72 mg, 29%). H NMR (300 MHz, CDCl₃)

δ

7.84 (d, J = 8.9 Hz, 1H, Ar), 7.55 (s, 1H, NH), 7.35–7.15 (m, 4H, Ar), 7.10–7.05 (m, 2H, Ar), 5.85

(s, 1H, CHOH), 3.50 (bs, 1H, OH), 2.92 [m, J = 6.8 Hz, 1H, CH(CH₃)₂], 1.45 [s, 9H, C(CH₃)₃],

1.25 [d, J = 6.8 Hz, 6H, CH(CH₃)₂].

3.2.3. 4-Chloro-2-[hydroxy(4⁰-nitrophenyl)methyl]-N-tert-butoxycarbonylaniline (7)

Following the General procedure 4.2, reaction of 4-chloro-N-tert-butoxycarbonylaniline

4 [

35 ] (3.01 mmol, 685 mg) with p-nitrobenzaldehyde (3.31 mmol, 500 mg) [^tBuLi (8.13 mmol)]

yielded

7 δ 8.20 (d, J = 9.0 Hz, 2H, Ar), 7.69

(264 mg, 23%). ¹H NMR (300 MHz, CDCl₃)

(d, J = 8.6 Hz, 1H, Ar), 7.55 (d, J = 9.0 Hz, 2H, Ar), 7.48 (s, 1H. NH), 7.31 (m, 1H, Ar), 7.09 (d,

J = 2.5 Hz, 1H, H3), 5.93 (s, 1H, CHOH), 4.80 (bs, 1H, OH), 1.43 [s, 9H, C(CH₃)₃].

3.2.4. 4-Chloro-2-[hydroxy(pyridin-4-yl)methyl]-N-tert-butoxycarbonylaniline (8)

Following the General procedure 4.2, reaction of 4-chloro-N-tert-butoxycarbonylaniline

4 [

35 ] (4.24 mmol, 965 mg) with isonicotinaldehyde (4.67 mmol, 500 mg) [^tBuLi (11.45 mmol)]

yielded 8 δ 8.29 (d, J = 6.2 Hz, 2H, Ar), 8.04 (s, 1H,

(618 mg, 44%). ¹H NMR (300 MHz, CDCl₃)

NH), 7.77 (d, J = 8.8 Hz, 1H, Ar), 7.34 (d, J = 6.2 Hz, 2H, Ar), 7.28 (dd, J = 8.7, 2.4 Hz, 1H, Ar),

7.10 (d, J = 2.4 Hz, 1H, H3), 5.78 (s, 1H, CHOH), 4.70 (bs, 1H, OH), 1.41 [s, 9H, C(CH₃)₃].

3.2.5. 4-Chloro-2-[hydroxy(pyridin-3-yl)methyl]-N-tert-butoxycarbonylaniline (9)

Following the General procedure 4.2, reaction of 4-chloro-N-tert-butoxycarbonylaniline

4 [

35 ] (4.24 mmol, 965 mg) with nicotinaldehyde (4.67 mmol, 500 mg) [^tBuLi (11.45 mmol)]

yielded 9 δ 8.42 (s, 1H, Ar), 8.30 (m, 1H, Ar), 8.18

(718 mg, 51%). ¹H NMR (300 MHz, CDCl₃)

(s, 1H, NH), 7.75 (m, 2H, Ar), 7.24 (m, 2H, Ar), 7.10 (d, J = 2.3 Hz, 1H, H3), 5.86 (s, 1H, CHOH),

4.90 (bs, 1H, OH), 1.43 [s, 9H, C(CH₃)₃].

3.2.6. 4-Chloro-2-[hydroxy(pyridin-2-yl)methyl]-N-tert-butoxycarbonylaniline (10)

Following the General procedure 4.2, reaction of 4-chloro-N-tert-butoxycarbonylaniline

4 [

35 ] (4.24 mmol, 965 mg) with picolinaldehyde (4.67 mmol, 500 mg) [^tBuLi (11.45 mmol)]

yielded 10 (394 mg, 28%). ¹H NMR (300 MHz, CDCl₃)

δ 8.52 (d, J = 6.0 Hz, 1H, Ar), 8.38 (s,

1H, NH), 7.75 (d, J = 8.6 Hz, 1H, Ar), 7.67 (m, 1H, Ar), 7.24 (m, 4H, Ar), 5.79 (s, 1H, CHOH),

4.93 (bs, 1H, OH), 1.42 [s, 9H, C(CH₃)₃].

3.2.7. 4-Chloro-2-[(3-chloropyridin-4-yl)hydroxymethyl]-N-tert-butoxycarbonylaniline (11)

Following the General procedure 4.2, reaction of 4-chloro-N-tert-butoxycarbonylaniline

4 [

35 ] (1.93 mmol, 439 mg) with 3-isonicotinaldehyde (2.11 mmol, 300 mg) [^tBuLi (5.21 mmol)]

yielded 11 (144 mg, 20%). ¹H NMR (300 MHz, CDCl₃)

1H, Ar), 7.67 (d, J = 5.1 Hz, 1H, Ar), 7.55 (s, 1H, NH), 7.53 (d, J = 8.7 Hz, 1H, Ar), 7.22 (dd,

δ 8.44 (d, J = 5.1 Hz, 1H, Ar), 8.38 (s,

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J = 8.7, 2.5 Hz, 1H, Ar), 6.89 (d, J = 2.5 Hz, 1H, H3), 6.03 (s, 1H, CHOH), 5.48 (bs, 1H, OH),

1.47 [s, 9H, C(CH₃)₃].

3.2.8. 4-Dimethylamino-2-[hydroxy(2⁰-Isopropylphenyl)methyl]-N-tert-butoxycarbonylaniline (20

)

Following the General procedure 4.2, reaction of 4-dimethylamino-N-tert-butoxycarbon

ylaniline 19 (Supplementary Material) (4.23 mmol, 1 g) with o-isopropylbenzaldehyde (4.65

mmol, 690 mg) [^tBuLi (11.42 mmol)] yielded 20 (537 mg, 33%). ¹H NMR (300 MHz, acetone-

d₆) δ 7.83 (bs, 1H, NH), 7.45 (m, 2H, Ar), 7.34–7.15 (m, 3H, Ar), 6.65 (dd, 1H, J = 8.7, 3.0 Hz,

H5), 6.42 (s, 1H, CHOH), 6.22 (d, 1H, J = 3.0 Hz, H3), 4.94 (s, 1H, OH), 3.16 [m, J = 6.0 Hz, 1H,

CH(CH₃)₂], 2.76 [s, 6H, N(CH₃)₂], 1.46 [s, 9H, C(CH₃)₃], 1.20 [d, J = 6.0 Hz, 3H, CH(CH₃)],

0.98 [d, J = 6.0 Hz, 3H, CH(CH₃)].

3.2.9. 2-[(2⁰-Chlorophenyl)hydroxymethyl]-4-dimethylamino-N-tert-butylcarbonylaniline (23)

Following the General procedure 4.2, reaction of N-[4-(dimethylamino)phenyl]-2,2-dimethy

lpropanamide 22 (Supplementary Material) (2.26 mmol, 0.5 g) with o-chlorobenzaldehyde

(0.28 mL, 318 mg, 2.26 mmol) [^tBuLi (6.1 mmol)] yielded 23 (284 mg, 35%). ¹H NMR (300 MHz,

CDCl₃) δ 7.96 (bs, 1H, NH), 7.57 (bd, J = 6.0, 1H, Ar), 7.4 (d, J = 9.0 Hz, 1H, H6), 7.3–7.20 (m, 3H,

Ar), 6.65 (dd, J = 9.0, 3.0 Hz, H5), 6.38 (d, J = 3.0 Hz, 1H, H3), 6.05 (d, J = 2.7 Hz, 1H, CHOH), 4.11

(d, J = 2.7 Hz, 1H, OH), 2.81 [s, 6H, N(CH₃)₂], 1.25 [s, 9H, C(CH₃)₃].

3.2.10. 4-Dimethylamino-2-[hydroxy(2⁰-Methoxyphenyl)methyl]-N-tert-butylcarbonylaniline (24)

Following the General procedure 4.2, reaction of N-[4-(dimethylamino)phenyl]-2,2-dimethy

lpropanamide 22 (Supplementary Material) (3.40 mmol, 750 mg) with o-methoxybenzaldehyde

(0.45 mL, 509 mg, 3.74 mmol) [^tBuLi (9.18 mmol)] yielded 24 (865 mg, 71%). ¹H NMR (300 MHz,

CDCl₃) δ 8.79 (s, 1H, NH), 7.97 (d, 1H, J = 8.4 Hz, H6), 7.30 (m, 1H, Ar), 6.95 (d, J = 8.0 Hz, 1H,

Ar), 6.87 (m, 2H, Ar), 6.74 (bd, J = 8.4 Hz, 1H, H5), 6.51 (d, J = 2.7 Hz, 1H, H3), 6.04 (d, J = 3.6 Hz,

1H, CHOH), 4.04 (d, 1H, J = 3.6 Hz, OH), 3.93 (s, 3H, OCH₃), 2.86 [s, 6H, N(CH₃)₂], 1.06 [s, 9H,

C(CH₃)₃].

3.2.11. 4-Dimethylamino-2-[(4⁰-fluorophenyl)hydroxymethyl]-N-tert-butylcarbonylaniline (25)

Following

the

General

procedure

4.2,

reaction

of

N-[4-(dimethy

lamino)phenyl]-2,2-dimethylpropanamide 22 (Supplementary Material) (2.26 mmol, 0.5 g) with

p-fluorobenzaldehyde (0.27 mL, 309 mg, 2.49 mmol) [^tBuLi (6.10 mmol)] yielded 25 (390 mg,

50%). ¹H NMR (300 MHz, CDCl₃)

δ 8.21 (s, 1H, NH), 7.82 (d, J = 9.0 Hz, 1H, H6), 7.27 (m, 2H,

Ar), 6.98 (m, 2H, Ar), 6.72 (dd, J = 9.0, 3.0 Hz, 1H, H5), 6.50 (d, J = 3.0 Hz, 1H, H3), 5.75 (s, 1H,

CHOH), 3.77 (s, 1H, OH), 2.88 [s, 6H, N(CH₃)₂], 1.04 [s, 9H, C(CH₃)₃].

3.2.12. 2-[Hydroxy(2⁰-methoxyphenyl)methyl]-N-tert-butylcarbonylaniline (31)

Following the General procedure 4.2, reaction of N-phenyl-2,2-dimethylpropanamide

29 (Supplementary Material) (2.80 mmol, 0.5 g) with o-methoxybenzaldehyde (0.88 mL,

1

780 mg, 5.60 mmol) [^tBuLi (7.00 mmol)] yielded 31 (215 mg, 25%). H NMR (300 MHz,

CDCl₃)

δ 9.53 (s, 1H, NH), 8.48 (d, J = 8.4 Hz, 1H, H6), 7.67–7.06 (m, 7H, Ar), 6.31 (d, J =

4.3 Hz, 1H, CHOH), 4.46 (d, 1H, J = 4.3 Hz, OH), 4.17 (s, 3H, OCH₃), 1.35 [s, 9H, C(CH₃)₃].

3.2.13. N-[3-[hydroxy(phenyl)methyl]pyrydin-4-yl]-2,2-dimethylpropanamide (35)

Following the General procedure 4.2, reaction of 2,2-dimethyl-N-(4-pyridinyl)propanamide

34 (Supplementary Material) (4.21 mmol, 750 mg) benzaldehyde (0.47 mL, 491 mg, 4.62 mmol)

[^tBuLi (11.37 mmol)] yielded 35 (1.08 g, 90%). ¹H NMR (300 MHz, CDCl₃)

δ 9.37 (s, 1H, NH),

8.37 (d, J = 5.7 Hz, 1H, Ar), 8.31 (d, J = 5.7 Hz, 1H, Ar), 7.99 (s, 1H, Ar), 7.36–7.30 (m, 5H, Ar),

5.85 (s, 1H, CHOH), 3.72 (s, 1H, OH), 1.07 [s, 9H, C(CH₃)₃].

3.3. Synthesis of 2-[(4⁰-Fluorophenyl)hydroxymethyl]-N-tert-butylcarbonylaniline (30)

Similar to what has been described for the use of ^tBuLi [34], with modiﬁcations. To a

solution of the Piv-protected aniline 29 (Supplementary Material) (2.82 mmol, 0.5 g) in dry

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◦

78 C under Ar atmosphere, ⁿBuLi (1.9 M in hexanes, 2.5 equiv, 7.05 mmol,

THF (12 mL) at

3.7 mL) was added dropwise. When the addition was complete, the mixture was stirred for

15 min at

78 ^◦C, then it was warmed up to 0 ^◦C and stirred for 4 h more. After this time,

the reaction mixture was cooled down to

78 ^◦C and a solution of p-ﬂuorobenzaldehyde

−

(0.62 mL, 0.7 g, 5.64 mmol) in THF (5 mL) was slowly added, and the reaction mixture

was allowed to reach 0 ^◦C for 2 h. Reaction was interrupted by slow addition of HCl 1N

(12 mL). Afterwards, it was extracted with ethyl acetate (3

organic layer was dried over anhydrous Na₂SO₄, ﬁltered, and evaporated, obtaining an

×

20 mL) and the combined

oil that was puriﬁed by automatized ﬂash chromatography using ethyl acetate/n-hexane

1

mixtures as eluent, yielding 30 as a yellow oil (360 mg, 43%). H NMR (300 MHz, CDCl₃)

δ

9.15 (s, 1H, NH), 8.37 (d, J = 8.1 Hz, 1H, Ar), 7.62–7.27 (m, 5H, Ar), 7.23 (m, 2H, Ar), 6.09 (s,

1H, CHOH), 4.22 (s, 1H, OH), 1.31 [s, 9H, C(CH₃)₃].

3.4. General Procedure for the Synthesis of 4,1-Benzothiazepines 12–18, 21, 26–28

Similar to what has been described [34], with slight modiﬁcations. Methyl thioglyco-

late (6 equiv) and triﬂuoroacetic acid (TFA, 14 equiv) were added to the intermediates 5–11

or 20 under Ar. The mixture was stirred at 85–110 ^◦C for 24 h. Then, it was dissolved in

CH₂Cl₂(30 mL) and washed subsequently with brine (30 mL), NaOH (1N, 30 mL), and

brine (30 mL). The organic layer was dried over Na₂SO₄, ﬁltered, and evaporated. The

crude material was puriﬁed by automatized ﬂash chromatography using ethyl acetate/n-

hexane mixtures as eluent to give pure compounds.

3.4.1. 7-Chloro-5-(4⁰-isopropylphenyl)-3,5-dihydro-4,1-benzothiazepin-2-(1H)-one (12)

Following the General procedure 4.4, reaction of

5

(0.66 mmol, 248 mg) [methyl

L)] yielded 12 (140 mg,

64%) as a white solid. Mp: 240–243 C. ¹H NMR (300 MHz, CDCl₃)

8.20 (s, 1H, NH), 7.39

thioglycolate (3.96 mmol, 420 mg), TFA (9.24 mmol, 1.05 g, 707

µ

◦

δ

(d, J = 8.1 Hz, 2H, Ar), 7.27 (m, 3H, Ar), 7.04 (d, J = 8.4 Hz, 1H, H9), 6.98 (d, J = 2.3 Hz, 1H,

H6), 5.64 (s, 1H, H5), 3.34 (d, J = 12.2 Hz, 1H, H3a), 2.99 (d, J = 12.2 Hz, 1H, H3b), 2.94 [m,

J = 6.9 Hz, 1H, CH(CH₃)₂], 1.28 [d, J = 6.9 Hz, 6H, CH(CH₃)₂]. ¹³C NMR (75.4 MHz, CDCl₃)

δ

170.6, 149.4, 136.9, 134.8, 133.4, 133.2, 129.4, 128.9, 128.7, 127.2, 125.4, 47.2, 34.0, 31.7, 24.1,

24.0. MS (API-ES+): m/z 332.09 [(M + H)⁺], 663.18 [(2M + Na)⁺]. Anal. C₁₈H₁₈ClNOS

(C,H,N).

3.4.2. 7-Chloro-5-(3⁰-isopropylphenyl)-3,5-dihydro-4,1-benzothiazepin-2-(1H)-one (13)

Following the General procedure 4.4, reaction of

colate (1.15 mmol, 122 mg), TFA (2.66 mmol, 303 mg, 204

white solid. Mp: 218–220 ^◦C. ¹H NMR (300 MHz, CDCl₃)

6

(0.19 mmol, 71 mg) [methyl thiogly-

L)] yielded 13 (21 mg, 33%) as a

8.01 (s, 1H, NH), 7.30 (m, 2H,

µ

δ

Ar), 7.25 (m, 3H, Ar), 6.98 (d, J = 8.4 Hz, 1H, H9), 6.90 (d, J = 2.3 Hz, 1H, H6), 5.59 (s, 1H,

H5), 3.28 (d, J = 12.2 Hz, 1H, H3a), 2.92 (d, J = 12.2 Hz, 1H, H3b), 2.86 [m, J = 6.9 Hz, 1H,

CH(CH₃)₂], 1.21 [d, J = 6.9 Hz, 3H, CH(CH₃)], 1.19 [d, J = 6.9 Hz, 3H, CH(CH₃)]. ¹³C NMR

(75.4 MHz, CDCl₃) δ 149.8, 136.8, 136.0, 134.8, 133.3, 129.0, 128.9, 128.7, 127.8, 127.0, 126.8,

125.4, 47.6, 34.2, 31.7, 24.2, 24.0. MS (API-ES+): m/z 332.10 [(M + H)⁺], 663.20 [(2M + Na)⁺].

HRMS: m/z (M + H)⁺calcd. for C₁₈H₁₈ClNOS 332.0870, found, 332.0869.

3.4.3. 7-Chloro-5-(4⁰-nitrophenyl)-3,5-dihydro-4,1-benzothiazepin-2-(1H)-one (14)

Following the General procedure 4.4, reaction of

thioglycolate (4.20 mmol, 446 mg), TFA (9.80 mmol, 1.12 g, 750

>99%) as a white solid. Mp: 289–291 ^◦C. ¹H NMR (300 MHz, DMSOd₆)

7

(0.70 mmol, 265 mg) [methyl

L)] yielded 14 (234 mg,

9.52 (s, 1H, NH),

µ

δ

8.25 (d, J = 8.8 Hz, 2H, Ar), 7.74 (d, J = 8.8 Hz, 2H, Ar), 7.45 (dd, J = 8.3, 2.4 Hz, 1H, Ar), 7.29

(d, J = 2.4 Hz, 1H, H6), 7.06 (d, J = 8.4 Hz, 1H, H9), 5.78 (s, 1H, H5), 3.14 (d, J = 12.2 Hz, 1H,

H3a), 3.07 (d, J = 12.2 Hz, 1H, H3b). ¹³C NMR (75.4 MHz, DMSOd₆)

δ 168.4, 146.8, 146.4,

136.6, 135.6, 130.3, 129.8, 128.8, 128.7, 126.5, 123.5, 46.3, 30.5. MS (API-ES+): m/z 335.02

[(M + H)⁺]. Anal. C₁₅H₁₁ClN₂O₃S (C,H,N).

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3.4.4. 7-Chloro-5-(pyridin-4-yl)-3,5-dihydro-4,1-benzothiazepin-2-(1H)-one (15)

Following the General procedure 4.4, reaction of (1.85 mmol, 619 mg) [methyl

thioglycolate (11.10 mmol, 1.18 g), TFA (25.90 mmol, 2.95 g, 1.98 mL)] yielded 15 (369 mg,

69%) as a white solid. Mp: 224–226 ^◦C. ¹H NMR (300 MHz, DMSOd₆)

9.51 (s, 1H, NH),

8

δ

8.58 (m, 2H, Ar), 7.46 (m, 3H, Ar), 7.33 (d, J = 2.4 Hz, 1H, H6), 7.07 (d, J = 8.4 Hz, 1H, H9),

5.64 (s, 1H, H5), 3.15 (d, J = 12.2 Hz, 1H, H3a), 3.04 (d, J = 12.2 Hz, 1H, H3b). ¹³C NMR

(75.4 MHz, DMSOd₆)

δ 168.4, 149.7, 147.8, 136.6, 135.3, 130.3, 128.9, 128.8, 126.5, 123.4, 45.9,

30.3. MS (API-ES+): m/z 291.03 [(M + H)⁺]. Anal. C₁₄H₁₁ClN₂OS (C,H,N).

3.4.5. 7-Chloro-5-(pyridin-3-yl)-3,5-dihydro-4,1-benzothiazepin-2-(1H)-one (16)

Following the General procedure 4.4, reaction of

9 (2.14 mmol, 716 mg) [methyl

thioglycolate (12.84 mmol, 1.36 g), TFA (29.96 mmol, 3.42 g, 2.29 mL)] yielded 16 (476 mg,

76%) as a white solid. Mp: 224–226 ^◦C. ¹H NMR (300 MHz, DMSOd₆)

δ 9.62 (s, 1H, NH),

8.62 (d, J = 2.4 Hz, 1H, H2⁰), 8.55 (m, 1H, Ar), 7.93 (m, 1H, Ar), 7.43 (m, 2H, Ar), 7.13 (d,

J = 2.4 Hz, 1H, H6), 7.07 (d, J = 8.4 Hz, 1H, H9), 5.70 (s, 1H, H5), 3.10 (AA’BB’, J = 12.5 Hz,

2H, H3). ¹³C NMR (75.4 MHz, DMSOd₆)

δ 168.5, 149.7, 149.0, 136.6, 136.5, 135.5, 133.8,

130.3, 128.7, 128.3, 126.3, 123.5, 44.2, 30.7. HRMS: m/z (M + H)⁺calcd. for C₁₄H₁₂ClN₂OS

291.0353, found, 291.0347.

3.4.6. 7-Chloro-5-(pyridin-2-yl)-3,5-dihydro-4,1-benzothiazepin-2-(1H)-one (17)

Following the General procedure 4.4, reaction of 10 (1.18 mmol, 395 mg) [methyl

thioglycolate (7.08 mmol, 751 mg), TFA (16.52 mmol, 1.88 g, 1.26 mL)] yielded 17 (200 mg,

◦

58%) as a white solid. Mp: 215–217 C. ¹H NMR (300 MHz, CDCl₃)

δ 8.65 (m, 1H, Ar), 7.78

(dt, J = 7.7, 1.9 Hz, 1H, Ar), 7.65 (s, 1H, NH), 7.60 (d, J = 8.9 Hz, 1H, H6), 7.27 (m, 2H, Ar),

7.00 (m, 2H, Ar), 5.72 (s, 1H, H5), 3.26 (d, J = 12.5 Hz, 1H, H3a), 3.08 (d, J = 12.5 Hz, 1H,

H3b). ¹³C NMR (75.4 MHz, CDCl₃)

δ 169.9, 156.6, 149.9, 137.3, 136.2, 134.8, 133.4, 129.0,

125.5, 123.9, 123.2, 49.3, 31.2. MS (API-ES+): m/z 291.03 [(M + H)⁺]. HRMS: m/z (M + H)⁺

calcd. for C₁₄H₁₂ClN₂OS 291.0353, found, 291.0342.

3.4.7. 7-Chloro-5-(3⁰-chloropyridin-4-yl)-3,5-dihydro-4,1-benzothiazepin-2-(1H)-one (18)

Following the General procedure 4.4, reaction of 11 (0.39 mmol, 144 mg) [methyl

thioglycolate (2.34 mmol, 248 mg), TFA (5.46 mmol, 1.88 g, 1.26 mL)] yielded 18 (80 mg,

63%) as a white solid. Mp: 226–229 ^◦C. ¹H NMR (300 MHz, CDCl₃)

δ 8.64 (d, J = 6.0 Hz,

1H, H6⁰), 8.61 (s, 1H, H2⁰), 7.83 (d, J = 5.1 Hz, 1H, H5⁰), 7.51 (s, 1H, NH), 7.31 (dd, J = 2.4,

8.4 Hz, 1H, H8), 7.07 (d, J = 8.4 Hz, 1H, H9), 6.78 (d, J = 2.4 Hz, 1H, H6), 5.94 (s, 1H, H5),

3.28 (d, J = 12.3 Hz, 1H, H3a), 3.10 (d, J = 12.3 Hz, 1H, H3b). ¹³C NMR (75.4 MHz, CDCl₃)

δ

169.2, 150.0, 148.2, 148.1, 135.3, 135.0, 133.9, 133.7, 129.6, 128.5, 125.8, 125.4, 43.9, 31.2.

HRMS: m/z (M + H)⁺calcd. for C₁₄H₁₁Cl₂N₂OS 324.9964, found, 324.9953.

3.4.8. 7-Dimethylamino-5-(2⁰-isopropylphenyl)-3,5-dihydro-4,1-benzothiazepin-2-(1H)-one (21)

Following the General procedure 4.4, reaction of 20 (0.26 mmol, 100 mg) [methyl

thioglycolate (1.56 mmol, 165 mg),_◦TFA (3.64 mmol, 415 mg, 279

µ

L)] yielded 21 (29 mg,

38%) as a white solid. Mp: 108–112 C. ¹H NMR (300 MHz, acetone-d₆)

δ

8.49 (s, 1H, NH),

7.78 (m, 1H, Ar), 7.40–7.25 (m, 3H), 7.00 (d, J = 8.4 Hz, 1H, H9), 6.66 (dd, J = 3.0, 8.4 Hz, 1H,

H8), 6.09 (m, 2H, H6, H5), 3.33 (d, J = 12.0 Hz, 1H, H3a), 2.93 [m, J = 6.9 Hz, 1H, CH(CH₃)₂],

2.83 (d, J = 12.0 Hz, 1H, H3b), 2.72 [s, 6H, N(CH₃)₂], 1.19 [d, J = 6.9 Hz, 3H, CH(CH₃)], 0.86

[d, J = 6.9 Hz, 3H, CH(CH₃)]; ¹³C NMR (75.4 MHz, CDCl₃)

δ 169.8, 150.7, 148.0, 136.4, 135.4,

130.6, 129.3, 127.5, 127.0, 126.6, 125.8, 113.0, 112.4, 44.0, 40.6, 32.4, 24.5, 23.7. HRMS: m/z

(M + H)⁺calcd. for C₂₀H₂₅N₂OS 341.1682, found, 341.1671.

3.4.9. 5-(2⁰-Chlorophenyl)-7-dimethylamino-3,5-dihydro-4,1-benzothiazepin-2-(1H)-one (26

Following the General procedure 4.4, reaction of 23 (0.39 mmol, 140 mg) [methyl

thioglycolate (2.33 mmol, 247 mg), TFA (5.43 mmol, 619 mg, 415 L)] yielded 26 (36 mg,

27%) as a white solid. Mp: 178–181 ^◦C. ¹H NMR (300 MHz, DMSOd₆)

9.44 (s, 1H, NH),

)

µ

δ

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7.81 (d, J = 7.5 Hz, 1H, Ar), 7.49 (m, 2H, Ar), 7.45 (m, 1H, Ar), 6.93 (d, J = 8.7 Hz, 1H, Ar),

6.66 (dd, J = 11.4, 5.4 Hz, 1H, H8), 5.95 (m, 2H, H6, H5), 3.16 (d, 1H, H3a), 2.90 (d, 1H, H3b),

2.75 [s, 6H, N(CH₃)₂]. ¹³C NMR (75.4 MHz, DMSOd₆):

δ 168.6, 148.9, 134.9, 132.9, 132.8,

131.0, 129.9, 129.8, 127.3, 126.3, 125.0, 112.0, 110.3, 43.5, 31.1. HRMS: m/z (M + H)⁺calcd.

for C₁₇H₁₈ClN₂OS 333.0822, found, 333.0829. Anal. C₁₇H₁₇ClN₂OS (C,H,N).

3.4.10. 7-Dimethylamino-5-(2⁰-methoxyphenyl)-3,5-dihydro-4,1-benzothiazepin-2-(1H)-one (27)

Following the General procedure 4.4, reaction of 24 (0.70 mmol, 250 mg) [methyl

thioglycolate (4.18 mmol, 444 mg), TFA (9.76 mmol, 1.11 g, 750

µ

L)] yielded 27 (24 mg,

10%) as a white solid. Mp: 233–236 ^◦C. ¹H NMR (300 MHz, CDCl₃)

δ

7.75 (d, J = 9.0 Hz,

1H, Ar), 7.33 (m, 1H, Ar), 7.11 (s, 1H, NH), 7.05 (m, 1H, Ar), 6,90 (m, 2H, Ar), 6.56 (dd,

J = 9.0, 3.0 Hz, 1H, H8), 6.25 (d, J = 3.0 Hz, 1H, H6), 6.17 (s, 1H, H5), 3.70 (s, 3H, OCH₃),

3.35 (d, J = 12.0 Hz, 1H, H3a), 2.95 (d, J = 12.0 Hz, 1H, H3b), 2.79 [s, 6H, N(CH₃)₂]. ¹³C

NMR (75.4 MHz, DMSOd₆):

δ 170.6, 157.0, 150.0, 135.7, 130.8, 129.2, 125.7, 125.2, 125.2

120.4, 111.8, 111.1, 55.7, 40.6, 40.4, 31.7. Anal. C₁₈H₂₁N₂O₂S·H₂O (C,H,N).

0

3.4.11. 7-Dimethylamino-5-(4 -fluorophenyl)-3,5-dihydro-4,1-benzothiazepin-2-(1H)-one (28

)

Following the General procedure 4.4, reaction of 25 (0.43 mmol, 150 mg) [methyl

thioglycolate (2.61 mmol, 277 mg), TFA (6.09 mmol, 694 mg, 466 L)] yielded 28 (25 mg,

18%) as a white solid. Mp: 219–222 ^◦C. ¹H NMR (300 MHz, DMSOd₆)

9.14 (s, 1H, NH),

7.52 (m, 2H, Ar), 7.21 (m, 2H, Ar), 6.87 (d, J = 9.0 Hz, 1H, H9), 6.65 (dd, J = 9.0 Hz, J = 3.0 Hz

1H, H8), 6.33 (d, J = 3.0, 1H, H6), 5.54 (s, 1H, H5), 3.05 (d, J = 12.0 Hz, 1H, H3a), 2.93 (d,

J = 12.0 Hz, 1H, H3b), 2.78 [s, 6H, N(CH₃)₂]. ¹³C NMR (75.4 MHz, DMSOd₆):

168.8, 148.9,

µ

δ

,

δ

134.7, 134.5, 130.8 (JC-F = 8.3 Hz), 126.1, 125.2, 115.2 (JC-F = 21.3 Hz), 111.9, 111.6, 46.7, 40.0,

30.9. HRMS: m/z (M + H)⁺calcd. for C₁₇H₁₈FN₂OS 317.1120, found, 317.1120.

3.4.12. 5-(4⁰-Fluorophenyl)-3,5-dihydro-4,1-benzothiazepin-2-(1H)-one (32)

Following the General procedure 4.4, reaction of 30 (0.36 mmol, 109 mg) [methyl

thioglycolate (2.16 mmol, 229 mg), TFA (5.04 mmol, 575 mg, 388

12%) as a white solid. Mp: 251–254 ^◦C. ¹H NMR (300 MHz, DMSOd₆)

7.46–7.23 (m, 3H, Ar), 7.08 (m, 3H, Ar), 6.98 (m, 1H, Ar), 5.66 (s, 1H, H5), 3.29 (d, J = 12.2 Hz,

1H, H3a), 3.02 (d, J = 12.2 Hz, 1H, H3b). ¹³C NMR (75.4 MHz, CDCl₃):

170.0, 157.6 (d,

µL)] yielded 32 (12 mg,

δ

7.47 (m, 2H, Ar),

δ

J = 245 Hz), 136.2, 134.9, 133.2, 131.1 (d, J = 8.0 Hz), 128.9, 128.8, 127.9, 124.3, 115.8 (d,

J = 21.0 Hz), 47.2, 31.5. HRMS: m/z (M + Na)⁺calcd. for C₁₅H₁₂FNNaOS 296.0621, found,

296.0621.

3.4.13. 5-(2⁰-Methoxyphenyl)-3,5-dihydro-4,1-benzothiazepin-2-(1H)-one (33)

Following the General procedure 4.4, reaction of 31 (0.98 mmol, 308 mg) [methyl

thioglycolate (5.88 mmol, 624 mg)_◦, TFA (13.72 mmol, 1.56 g, 1.06 mL)] yielded 33 (23 mg,

δ 7.76 (d, J = 9.2 Hz, 1H,

8%) as a white solid. Mp: 216–218 C. ¹H NMR (300 MHz, CDCl₃)

Ar), 7.63–6.79 (m, 8H, Ar), 6.20 (s, 1H, H5), 3.68 (s, 3H, OCH₃), 3.33 (d, J = 12.1 Hz, 1H, H3a),

2.99 (d, J = 12.1 Hz, 1H, H3b). ¹³C NMR (75.4 MHz, CDCl₃):

δ

170.3, 156.7, 136.4, 134.9,

130.8, 129.4, 128.4, 128.4, 127.8, 125.4, 124.0, 120.6, 111.1, 55.7, 40.1, 31.6. Anal. C₁₆H₁₅NO₂S

(C,H,N).

3.5. Synthesis of (4-Aminopyridin-3-yl)(phenyl)methanol (36)

Similar to what we have described [36]. To a solution of intermediate 35 (0.63 mmol,

179 mg) in 1,4-dioxane (18 mL), KOH_aq2 M (18 mL) was added, and the mixture was

reﬂuxed for 24 h. Then, reaction mixture was cooled down to rt and extracted with ethyl

acetate (3

×

50 mL). The combined organic layer was dried over anhydrous Na₂SO₄, ﬁltered,

1

and evaporated, obtaining 36 as a yellow solid (132 mg, >99%). H NMR (300 MHz, CDCl₃)

δ

8.00 (d, J = 5.7 Hz, 1H, Ar), 7.86 (s, 1H, Ar), 7.37–7.30 (m, 5H, Ar), 6.42 (d, J = 5.7 Hz, 1H,

Ar), 5.78 (s, 1H, CHOH), 4.76 (s, 3H, OH, NH₂).

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3.6. Synthesis of Methyl 2-[(4-Aminopyridin-3-yl)(phenyl)methylthio]acetate (37)

Similar to what we have described [36]. To a solution of intermediate 36 (445 mg,

2.22 mmol) in dry CH₂Cl₂(25 mL), methyl thioglycolate (13.3 mmol, 1.41 g) and TFA

(10 mL) were added under Ar. The mixture was stirred at 45 ^◦C for 22 h. Then, reaction

mixture was concentrated, dissolved in CH₂Cl₂(50 mL), and washed subsequently with

saturated NaHCO₃(50 mL), NaOH (1N, 50 mL), and brine (50 mL). The organic layer was

dried over Na₂SO₄, ﬁltered, and evaporated. The crude was puriﬁed by automatized ﬂash

chromatography using ethyl acetate/n-hexane mixtures as eluent to give 37 (144 mg, 25%).

¹H NMR (300 MHz, CDCl₃)

δ 8.12 (d, J = 5.7 Hz, 1H, Ar), 7.89 (s, 1H, Ar), 7.51 (m, 2H, Ar),

7.41–7.32 (m, 3H, Ar), 6.56 (d, J = 5.7 Hz, 1H, Ar), 5.35 (s, 1H, CH), 5.08 (s, 2 H, NH₂), 3.74

(s, 3H, OCH₃), 3.22 (d, J = 16.8 Hz, 1H, H2a), 3.14 (d, J = 16.8 Hz, 1H, H2b).

3.7. Synthesis of 5-Phenyl-3,5-dihydropyrido[4,3-e][1,4]thiazepin-2-(1H)-one (38)

Similar to what we have described [36]. To a solution of intermediate 37 (150 mg,

0.52 mmol) in dry THF (23 mL), LiHMDS (1.04 mmol, 1.0 M in hexane, 1.04 mL) was

◦

added dropwise at

−

78 C under Ar and stirred at this temperature for 15 min. Then,

reaction mixture was allowed to warm up to rt for 24 h. Reaction was terminated by

adding a saturated solution of NH₄Cl (50 mL) and extracted with CH₂Cl₂(3 30 mL).

The combined organic layer was washed with brine (3

×

50 mL), dried over anhydrous

◦

Na₂SO₄, ﬁltered and evaporated, to obtain an orange solid (135 mg, 90%). Mp: 138–143 C.

¹H NMR (300 MHz, CDCl₃)

δ

8.52 (d, J = 5.7 Hz, 1H, Ar), 8.30 (s, 1H, Ar), 7.48–7.37 (m,

5H, Ar), 6.93 (d, J = 5.7 Hz, 1H, Ar), 5.63 (s, 1H, H5), 3.30 (d, J = 13.2 Hz, 1H, H3a), 3.15

(d, J = 13.2 Hz, 1H, H3b). ¹³C NMR (75.4 MHz, CDCl₃):

δ 170.4, 151.0, 150.0, 144.0, 136.4,

129.3, 129.0, 128.9, 128.4, 117.4, 46.3, 31.6. HRMS: m/z (M + H)⁺calcd. for C₁₄H₁₃N₂OS

257.0743, found, 257.0742.

3.8. Experimental Use of Animals

Maximum efforts were made to reduce the number of pregnant rats sacriﬁced for

culturing cortical embryonic neurons and their suffering, following the guidelines of the EU

Council Directive. Experiments were approved by the Ethics Committee of the Universidad

Autónoma de Madrid.

3.9. Cell Cultures

The neuroblastoma SH-SY5Y cell line was obtained from the American Type Culture

Collection (ATCC-CRL-2266), maintained and cultured in a 1:1 mixture of MEM and

F12 media with 10% of fetal bovine serum (FBS), according to procedures previously

described [36]. For ﬂuorescence-based [Ca²⁺]_cmeasurements, SH-SY5Y cells were seeded

in clear-bottomed, 96-well black plates at a density of 40,000 cells/well. For viability assays,

SH-SY5Y cells were seeded in 48-well plates at a density of 70,000 cells/well following

the procedure recently reported [44]. Rat cortical embryonic neurons were prepared

from 19-day-old embryos directly seeded in the plate well treated with poly-D-lysine in

neurobasal medium (Gibco 21103, Madrid, Spain) supplemented with 1% of L-glutamine

(Sigma, G6392), 0.5% of penicillin/streptomycin and 0.1% of gentamincine. In ﬂuorescence-

based [Ca²⁺]_cmeasurement, neurons were seed at clear-bottomed, 96-well black plates at a

density of 30,000 cells/well. For viability assays, the rat cortical neurons were also seeded

in 48-well plates at a density of 40,000 cells/well. All the biological assays were conducted

under sterile conditions.

3.10. Per se Toxicity Assay

The per se toxicity of the new synthetized compounds was assessed in both cell

cultures by the method of MTT reduction. After 24 h of seeding, SH-SY5Y cells were

preincubated with compounds at different concentrations (1, 3, 10, and 30 µM) for 24 h. Af-

terwards, MEM/F12 medium was replaced by one fresh with 1% FBS with the compounds

at the same concentration. The viability of cultures was measured 20 h later, following a

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procedure recently described [44]. In the case of rat cortical neurons, 7 days after seeding,

compounds were added to the wells to reach a ﬁnal concentration of 1, 3, 10, and 30 µM.

Forty-eight hours later, neuron viability was measure by the same procedure as the one

described for SH-SY5Y cells.

3.11. Blockade of Cytosolic Ca²⁺Fluctuations Assay

To assess the blocking effect of compounds on the VGCC, we used SH-SY5Y cells

charged with the Ca²⁺-sensitive ﬂuorescent dye Fluo-4/AM at 10

µM in Krebs-HEPES

buffer, following a protocol recently described [44]. Compounds were incubated at the

desired concentrations (1, 3, 10, and 30 µ

M). The Ca²⁺entry was favored by addition of

K⁺70 mM to the extracellular medium. To evaluate the blocking effect of compounds

on NMDA receptors, we have followed a protocol recently reported by our research

group [45]. Rat cortical neurons were charged with Fluo-4/AM at 3

µM in Krebs-HEPES

buffer and compounds incubated at same concentrations than before. To stimulate Ca²⁺

entry through the NMDA-sensitive glutamate receptors, NMDA was applied at 10 µM.

In both cases, ﬂuorescence oscillations as result of the [Ca²⁺]_cincreases were real-time

monitored in a multi-well ﬂuorescence plate reader (FluoStar Optima, BMG, Germany),

with excitation and emission wavelengths of 485 and 520 nm, respectively. Data were

calculated as ﬂuorescence increase (∆F), according to the formula:

(Fx − Fo)

∆F =

(Fmax − Fmin)

where F_xis the maximum ﬂuorescence obtained after Ca²⁺entry stimulation, F₀is the

averaged ﬂuorescence before, F_maxis the maximum ﬂuorescence of the well obtained by

treating cells with 5% Triton X-100, and the F_minis the minimum ﬂuorescence of well

obtained by adding MnCl₂1 M to the previously Triton-lysed cells. The

normalized respect to the control ( F_c). Data are presented as percentage of blockade (100%

F) at the concentration of 10 M, or as IC₅₀, i.e., the concentration of compound capable

∆F data were

∆

µ

of halving the ∆F_c(%∆F = 50).

3.12. Neuroprotection Assays

The effect of compounds on the SH-SY5Y cells and rat cortical neurons viability

compromised of different toxics was also evaluated by method of MTT reduction [46].

Twenty-four hours after seeding, SH-SY5Y cells were preincubated with compounds at

the concentration of 10

one fresh with 1% FBS, compounds at the same concentration, and rotenone/oligomycin

A administered to media at the concentration of 30/10 M, respectively. The cultures’

µM for 24 h. Afterwards, MEM/F12 medium was replaced by

µ

viability was measured 20 h later, following the MTT procedure recently described [44]. As

reference neuroprotective compound, melatonin 10 nM was selected. In the case of the rat

cortical neurons, 7 days after seeding in 48-well plates, neurons were preincubated adding

the compounds at a ﬁnal concentration of 10 µM for 24 h. Then, toxic stimuli of 50 µM

of glutamate was added to the wells. After 20 h, cell viability was measured by the MTT

procedure [44]. Here, the reference protective compound used was memantine (30 nM).

3.13. Blood–Brain Barrier Permeation Assay (PAMPA)

The parallel artiﬁcial membrane permeation assay (PAMPA) was used to predict the

capacity of compounds to cross the blood–brain barrier (BBB) by passive diffusion, in

a similar manner as previously described [43]. Compounds were dissolved in 10 mM

phosphate-buffered saline (PBS) buffer (pH 7.4) to the concentration of 100

membrane of the donor 96-well plate (Multiscreen IP sterile clear plate PDVF membrane,

pore size 0.45 M) was impregnated with 4 L of porcine brain lipid (PBL) (Avanti Polar

Lipids, Inc., Alabaster, AL, USA) in dodecane (20 mg/mL), and after 5 min, 180 L of each

compound solution was added. The acceptor 96-well plate (Multiscreen, Millipore Corp.

Burlington, MA, USA) was ﬁlled with 180 L of PBS and 1% of DMSO. Finally, the donor

µM. The ﬁlter

µ

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ﬁlter plate was carefully put on the acceptor plate to form a “sandwich”, which was left

undisturbed for 4 h at rt. After incubation, an UV plate reader determined the concentration

of compounds in both acceptor and donor wells (150 µL/well) as the maximum absorption

wavelength of each compound. Concentration of the compounds in the donor and acceptor

well, and equilibrium concentration were calculated from the standard curve and expressed

as the permeability (Pe), according to the following formula:

ꢀ

ꢁ

ꢂꢃ

[drug]acceptor

[drug]equilibrium

log Pe = log C × − ln 1 −

Vdonor × Vaceptor

(Vdonor + Vaceptor) × Area × Time)

Results are expressed as mean ± SEM of three different experiments in duplicate.

where C =

3.14. Data Analysis

To calculate the necessary sample size (n) for each experiment, taking a conﬁdence

interval (Z) of 95% and an error margin admitted (e) of 5%, we applied the formula

n = Z²·s²/e², where the averaged standard deviation (s) in our similar previous experiments

using neuroblastoma cells was 0.04. Thus, a sample size of a minimum of three experiments

was necessary. Comparisons between control and test groups were executed by a one-way

analysis of variance (ANOVA), followed by Dunnett or Newman–Keuls post hoc test, using

the GraphPad Prism 5.0 for Mac OSX software, and considered statistical different when

p < 0.05.

4. Conclusions

A series of new CGP37157 derivatives with improved physicochemical properties

were synthesized, and their pharmacological properties were evaluated. Their ability to

buffer the neuronal Ca²⁺by controlling the mitochondrial regulatory machinery, and thus

protecting against neurotoxic stimuli derived from Ca²⁺overload, make them promis-

ing potential neuroprotective drugs. Despite their modest capacity to mitigate neu-

ronal Ca²⁺elevation, the ﬁrst subgroup of new 4,1-benzothiazepines present a signiﬁ-

cant protection against oxidative stress, and a slight capacity to mitigate neuronal Ca²⁺

elevation. Among these derivatives, compound 18 stands out thanks to its ability to

reduce Ca²⁺overload induced by both K⁺-evoked depolarization and NMDA stimula-

tion, as well as its neuroprotective proﬁle against both R/O-induced oxidative stress and

high glutamate-exerted excitotoxicity. Notably, compound 18 is the closest analogue to

CGP37157, with an added aromatic nitrogen in the para position of the pending phenyl

ring. This slight modiﬁcation allowed the compound to overcome the solubility problems

of CGP37157, keeping its pharmacological properties similar. Regarding to the family

of 7-dimethilaminobenzothiazepines, the derivative 21, which possesses an o-isopropyl

substituent at the pending phenyl ring, showed the most promising regulatory capacity

of [Ca²⁺]_clevels, correlating with an interesting reduction of the neuronal death in an

excitotoxicity scenario. Again, compound 21 is the closest analogue to our previous head

compound ITH12575 [35], which is currently under investigation for several pathologies

affecting mitochondrial function, such as acute traumatic cerebral damage. In the case

of the new derivatives lacking substitution at C7, compounds 32 and 33, although they

were not good at regulating Ca²⁺overload, they have shown a signiﬁcant neuroprotection

against glutamate. Finally, the 4-aminopyridine-fused thiazepine 38, has a very interesting

protective proﬁle against the neurotoxic stimuli used, even without presenting good regula-

tory capability of neuronal Ca²⁺. Almost all the new compounds feature a highly probable

passive diffusion through the BBB, which is a crucial fact for any drug targeting the CNS. In

fact, the most promising compounds, 18 and 21, have a Pe value higher than the threshold

value of 40 nm/s (Figure 6). Excluding compounds 13 and 14, which surprisingly were

slightly soluble and toxic, the rest of the new compounds presenting diverse substitution

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have shown interesting neuroprotective proﬁles. Overall, we highlight compounds 18 and

21, as promising optimized benzothiazepine derivatives for further studies to deﬁne their

neuroprotective properties.

Supplementary Materials: The following are available online. Synthesis of precursors and interme-

diates and Table S1, Lipinski’s parameters.

Author Contributions: Conceptualization, L.V. and C.d.l.R.; methodology, L.V., C.d.l.R. and M.E.-V.;

formal analysis, L.V.; investigation, L.V., M.R.-A., R.L.A., M.M.-C., R.P.-M., and M.B.-C.; data curation,

C.d.l.R. and L.V.; writing—original draft preparation, L.V. and C.d.l.R.; writing—review and editing,

C.d.l.R., M.E.-V., R.L.A., and L.V.; visualization, C.d.l.R., M.E.-V., R.L.A., and L.V.; supervision, C.d.l.R.

and M.E.-V.; project administration, C.d.l.R.; funding acquisition, C.d.l.R. All authors have read and

agreed to the published version of the manuscript.

Funding: This research was funded by Acciones Estratégicas en Salud, Proyectos de Investigación en

Salud (ISCIII/FEDER, PI16/01041, PI19/01724).

Institutional Review Board Statement: The study was conducted according to the guidelines of

the Declaration of Helsinki (2010/63/EU; Spanish legislation: Articles 33.1 and 33.8, RD53/2013),

and approved by the Ethics Committee of the Universidad Autónoma de Madrid (registry number

ES-28079-0000097).

Informed Consent Statement: Not applicable.

Data Availability Statement: Data supporting obtained results can be obtained from the authors

upon request.

Acknowledgments: We thank Antonio G. García (UAM) for continuous support and advice. We

thank Fundación Teóﬁlo Hernando for technical support.

Conﬂicts of Interest: The authors declare no conﬂict of interest.

Sample Availability: Samples of the compounds 12–18, 21, 26–28, 32, 33, and 38 are available from

the authors.

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Article Doi

Synthesis and biological assessment of 4,1-benzothiazepines with neuroprotective activity on the Ca2+ overload for the treatment of neurodegenerative diseases and stroke

DOI: 10.3390/molecules26154473

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Article abstract of DOI:10.3390/molecules26154473

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