Tuning melatonin receptor subtype selectivity in oxadiazolone-based analogues: Discovery of QR2 ligands and NRF2 activators with neurogenic properties

European Journal of Medicinal Chemistry 190 (2020) 1 12090

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Research paper

Tuning melatonin receptor subtype selectivity in oxadiazolone-based

analogues: Discovery of QR2 ligands and NRF2 activators with

neurogenic properties

a

ꢀ

Clara Herrera-Arozamena , Martín Estrada-Valencia , Concepcion Perez ,

a

b

,

c

,

d

b, c

ꢀ

Laura Lagartera , Jose A. Morales-García

Juan Felipe Franco-Gonzalez , Patrycja Michalska , Pablo Duarte , Rafael Leon

, Ana Perez-Castillo

,

e

e, f

ꢀ

,

e

g

b, c, h

ꢀ

Manuela G. Lopez , Alberto Mills , Federico Gago , Angel Juan García-Yagüe

b

,

c

,

c

,

, *

ꢀ

Raquel Fernandez-Gines

^h, Antonio Cuadrado ^{b h}, María Isabel Rodríguez-Franco ^a

a

ꢀ

Instituto de Química Medica, Consejo Superior de Investigaciones Cientíﬁcas (IQM-CSIC), C/ Juan de La Cierva 3, E-28006, Madrid, Spain

Instituto de Investigaciones Biomedicas (CSIC-UAM), C/ Arturo Duperier, 4, E-28029, Madrid, Spain

b

c

ꢀ

Centro de Investigacion Biomedica en Red Sobre Enfermedades Neurodegenerativas (CIBERNED), C/ Valderrebollo, 5, E-28031, Madrid, Spain

^dDepartamento de Biología Celular, Facultad de Medicina, Universidad Complutense de Madrid, Avda. Complutense S/n, E-28040, Madrid, Spain

e

ꢀ

Instituto Teoﬁlo Hernando de IþD Del Medicamento, Departamento de Farmacología y Terapeutica, Facultad de Medicina, Universidad Autonoma de

Madrid, E-28029, Madrid, Spain

f

ꢀ

Instituto de Investigacion Sanitaria, Servicio de Farmacología Clínica, Hospital Universitario de La Princesa, E-28006, Madrid, Spain

Area de Farmacología, Departamento de Ciencias Biomedicas & “Unidad Asociada IQM-CSIC”, Facultad de Medicina y CC. Salud, Universidad de Alcala, E-

g

ꢀ

28805, Alcala de Henares, Madrid, Spain

h

ꢀ

Universidad Autonoma de Madrid (UAM), Departamento de Bioquímica, Facultad de Medicina, E-28029, Madrid, Spain

a r t i c l e i n f o

a b s t r a c t

Article history:

New multi-target indole and naphthalene derivatives containing the oxadiazolone scaffold as a bio-

isostere of the melatonin acetamido group have been developed. The novel compounds were charac-

terized at melatonin receptors MT₁R and MT₂R, quinone reductase 2 (QR2), lipoxygenase-5 (LOX-5), and

monoamine oxidases (MAO-A and MAO-B), and also as radical scavengers. We found that selectivity

within the oxadiazolone series can be modulated by modifying the side chain functionality and co-

planarity with the indole or naphthalene ring. In phenotypic assays, several oxadiazolone-based de-

rivatives induced signalling mediated by the transcription factor NRF2 and promoted the maturation of

neural stem-cells into a neuronal phenotype. Activation of NRF2 could be due to the binding of indole

derivatives to KEAP1, as deduced from surface plasmon resonance (SPR) experiments. Molecular

modelling studies using the crystal structures of QR2 and the KEAP1 Kelch-domain, as well as the

Received 27 November 2019

Received in revised form

20 January 2020

Accepted 21 January 2020

Available online 25 January 2020

Keywords:

Indole or naphthalene e oxadiazolones

Melatonin receptors (MT₁R and MT₂R)

Quinone reductase-2 (QR2)

Nuclear erythroid 2-related factor (NRF2)

Kelch-like ECH associated protein 1 (KEAP1)

Abbreviations: AD, Alzheimer’s disease; AREs, antioxidant response elements;

, amyloid- peptide; COSY, homonuclear correlation spectroscopy; GPCR, G-

A

b

protein-coupled receptor; HMBC, heteronuclear multiple bond correlation; HSQC,

heteronuclear single quantum correlation; KEAP1, Kelch-like ECH associated pro-

tein 1; LOX-5, lipoxygenase-5; MAO-A and MAO-B, monoamine oxidases; MAP-2,

microtubule-associated protein 2 marker; MMGBSA, molecular mechanics gener-

alized born surface area approach; MT₁R and MT₂R, GPCRs of melatonin; MTDL,

multi-target directed ligand; MW, microwaves; ND, neurodegenerative disease;

NDGA, nordihydroguaiaretic acid; NRF2, nuclear erythroid 2-related factor; NSC,

neural stem cells; ORAC, oxygen radical absorbance capacity assay; PAMPA-BBB,

parallel artiﬁcial membrane permeability assay for the bloodꢀbrain barrier; PDB,

protein data bank; QR2, quinone reductase 2; ROS and RNS, reactive oxygen and

nitrogen species; SGZ, subgranular zone; SPR, surface plasmon resonance assay;

SVZ, subventricular zone; TuJ-1, b_III-tubulin marker; uMD, unrestrained molecular

dynamics; XFEL, X-ray free-electron laser.

* Corresponding author.

E-mail address: isabelrguez@iqm.csic.es (M.I. Rodríguez-Franco).

https://doi.org/10.1016/j.ejmech.2020.1 12090

2

C. Herrera-Arozamena et al. / European Journal of Medicinal Chemistry 190 (2020) 112090

Monoamine oxidases (MAO-A and MAO-B)

Lipoxygenase-5 (LOX-5)

Neurogenic activity

recently described X-ray free-electron laser (XFEL) structures of chimeric MT₁R and MT₂R, provided a

rationale for the experimental data and afforded valuable insights for future drug design endeavours.

license (http://creativecommons.org/licenses/by/4.0/).

1. Introduction

has been identiﬁed as the ﬂavin adenine dinucleotide (FAD)-

dependent enzyme quinone reductase-2 (QR2, EC 1.10.99.2) [16,17].

Alzheimer’s disease (AD) is the most common age-associated

neurodegenerative disorder (ND), whose main hallmarks are the

Melatonin activates MT₁R and MT₂R at sub-nanomolar concentra-

tions (K_d~ 0.1 nM), whereas its afﬁnity as a QR2 inhibitor is about

100-fold lower [16].

extracellular deposits of the misfolded amyloid-b peptide (Ab), the

intracellular accumulation of the hyperphosphorylated

QR2 catalyses the two-electron reduction of quinones into un-

stable hydroquinones, which are excreted through conjugation or

reconverted to quinones in the presence of oxygen while producing

ROS [18]. Given that QR2 levels are increased in the hippocampus of

AD patients, inhibitors of this enzyme are promising therapeutic

agents to ﬁght neurodegeneration by reducing the oxidative stress

[19e21].

Adult neurogenesis is a matter of intense research, although its

scope and function remain debatable [22,23]. Restricted to small

regions, namely the subventricular zone (SVZ) of the lateral

ventricle and the subgranular zone (SGZ) of the hippocampus, the

adult mammalian brain retains neural stem cells (NSC) niches that

could develop and integrate new cells in the neuronal circuitry.

Therefore, a neurogenic drug would induce the differentiation of

NSCs into mature neurons capable of replacing those lost by neu-

rodegeneration, allowing the brain to recover its own self-renewal

capacity [24].

microtubule-associated tau protein and a massive neuronal loss [1].

AD aetiology is not completely understood, although different

causative factors have been postulated, such as oxidative stress and

neuroinﬂammation [2].

Oxidative stress is the consequence of the progressive failure of

the antioxidant defence systems, particularly noticeable in the

brains of AD-patients, where the increased levels of oxidized bio-

molecules correlate to disease progression [3]. Thereby, the control

of transcription factors involved in the regulation of antioxidant

genes and the inhibition of the main enzymes that produce reactive

oxygen and nitrogen species (ROS and RNS) would be a valuable

strategy to combat AD [4].

The nuclear erythroid 2-related factor (NRF2) is a transcriptional

protein that regulates the expression of antioxidant and anti-

inﬂammatory enzymes [5]. In non-stressed conditions, NRF2 is

mainly located in the cytosol bound to the Kelch-like ECH associ-

ated protein 1 (KEAP1), which favours its proteasome degradation.

Under pathological conditions (oxidative stress, toxic insults, etc.),

KEAP1 changes its conformation and NRF2 is released and trans-

located to the nucleus, where it binds to the DNA antioxidant

response elements (AREs), inducing the expression of antioxidant

enzymes such as NAD(P)H quinone oxidoreductase and heme

oxygenase-1 [6,7].

In recent years, numerous molecular targets and signalling

cascades involved in neurogenesis have been identiﬁed and, as a

consequence, different types of drugs have been evaluated in

neuronal regeneration. For example, neurogenic properties have

been found in MTRs’ ligands, NRF2 activators, antioxidants and

anti-inﬂammatory agents [25].

Melatonin modulates hippocampal neurogenesis by increasing

both cellular precursor proliferation and survival in the hippocampi

of aged mice [26]. Given these outstanding properties, many

research groups (including ours) have devoted great efforts aimed

to the identiﬁcation of new MTR ligands and their molecular

interaction pathways [27e30]. In this context, our melatonin

analogue IQM316 was recently found to be capable of inducing

hippocampal neurogenesis in mice at a healthy and sustainable

rate, thus helping to preserve previous memories [31,32]. We have

also described the bioisosteric replacement of the acetamido group

of melatonin by a series of reversed amides and azoles [29]. Among

these azole derivatives, 5-[2-(5-methoxy-1H-indol-3-yl)ethyl]-

1,3,4-oxadiazol-2(3H)-one (1) was the most potent partial agonist

at the human MTRs, displaying the highest afﬁnity for both human

MTRs (K_i¼ 35 and 4 nM for hMT₁R and hMT₂R, respectively).

Moreover, this compound also showed potent neurogenic proper-

ties in vitro, better than melatonin itself, but it was predicted that it

would not enter the CNS in view of the unfavourable results in

in vitro blood-brain barrier (BBB) permeability assays.

Given the complex nature of AD, the multi-target directed

ligand (MTDL) strategy has emerged as a valid alternative to ﬁght

different pathological cascades involved in this disease. To achieve

maximum efﬁciency, MTDLs should promote endogenous defen-

sive and/or regenerative pathways and be directed towards bio-

molecular targets located upstream in the neurotoxic cascades

[2,33].

With these ideas in mind, in this work we planned to develop

new melatonin-based MTDLs that could improve the CNS perme-

ability of 1 and also incorporate additional activities on targets

Monoamine oxidases (MAO-A and MAO-B) are outer mito-

chondrial membrane-bound ﬂavoproteins, whose levels are

increased in the brain of patients suffering NDs, such as AD and

Parkinson’s disease. Both enzymes catalyse the oxidative deami-

nation of biogenic amines, including several neurotransmitters,

what it produces hydrogen peroxide, ROS, and accordingly oxida-

tive stress [8]. Besides, overexpression of MAO-B increases the

formation of aberrant Ab [9]. In this regard, MAO inhibition could

provide neuroprotection against AD, by regulating neurotransmit-

ters’ levels, ameliorating oxidative pathologies and stopping the

deposition of amyloid plaques.

Neuroinﬂammation is another important player in AD that

generates cytokines and neurotoxins, leading to neuronal damage

and death [10]. The iron-containing enzyme lipoxygenase-5 (LOX-

5, EC 1.13.11.34) catalyses two early steps of the oxidation of the

arachidonic acid, producing ﬁnally pro-inﬂammatory leukotrienes

[11]. LOX-5 is present in the CNS particularly in the hippocampus,

where its levels appear to increase in AD [12]. Several works have

demonstrated that blockade of LOX-5 in transgenic mice di-

minishes cytokines levels and both Ab and tau pathologies [13,14].

Therefore, LOX-5 inhibitors could be valuable therapeutic agents, as

they could reduce neuroinﬂammation and aberrant protein

aggregation.

In mammals, melatonin is secreted in nanomolar concentrations

from the pineal gland to the blood, when it transmits the circadian

rhythm to the peripheral organs by interacting with two G-protein-

coupled receptors (GPCRs), called MT₁and MT₂(MT₁R and MT₂R)

[15]. Melatonin also interacts with a third binding site, MT₃, which

C. Herrera-Arozamena et al. / European Journal of Medicinal Chemistry 190 (2020) 112090

3

related to oxidative stress and neuroinﬂammation. For this pur-

pose, we modiﬁed each part of the starting molecule by: (i) addition

of different substituents to the oxadiazolone nitrogen; (ii)

replacement of the oxadiazolone ring by an oxadiazolamine; (iii)

insertion of a double bond in the linker; and (iv) replacement of the

indole by a dihydronaphthalene or naphthalene ring (Figure 1).

giving the corresponding a,b-unsaturated ester 19 in high yields

(95%). Then, 19 was reduced by catalytic hydrogenation to the

saturated ester 20, followed by its transformation to the corre-

sponding hydrazide and further MW-assisted cyclization in the

presence of 1,1-carbonildiimidazole (CDI) to give the 1,3,4-

oxadiazol-2(3H)-one derivative 21 in 48% yield.

The conjugated double bond in the linker was introduced by the

treatment of 18 with malonic acid in basic media that generate the

a,b-unsaturated acid 22, in almost quantitative yield by a Knoeve-

2. Results and discussion

nagel-Doebner reaction. Activation of 22 with hydroxybenzotriazole

(HOBt) and N-(3-dimethylaminopropyl)-N⁰-ethylcarbodiimide hy-

drochloride (EDC$HCl) as coupling agents, and a catalytic amount of

4-dimethylaminopyridine (DMAP) at rt during 3 h, followed by the

addition of hydrazine hydrate, yielded the corresponding hydrazide

in quantitative yield. It is worth-mentioning that the order of

addition of the reagents is crucial for obtaining high yields. Once

the hydrazide is formed, its MW-assisted cyclization with CDI

afforded the indoleeNH-oxadiazolone derivative 23 in good yield

(85%). The treatment of 23 with propargyl bromide in basic media

(K₂CO₃) and acetone in an MW-oven at 120 ^ꢁC during 10 min

afforded the corresponding N-propargyl-oxadiazolone 24 (Scheme

1). Bioisosteric replacement of the oxadiazolone ring by an amide

2.1. Chemistry. Synthesis of new indole- and naphthalene-based

compounds

The microwave (MW)-assisted reaction (120 ^ꢁC, 10 min) of 1

with the corresponding alkyl halide in basic media (K₂CO₃) using

acetone as solvent gave derivatives 2e15, in moderate to excellent

yields (49e92%) (Scheme 1).

Moreover, the treatment of 1 with methyl- or ethylamine in the

presence of (benzotriazol-1-yloxy)tris(dimethylamino)phospho-

nium hexaﬂuorophosphate (BOP) and triethylamine (TEA) in DMF,

yielded 1,3,4-oxadiazol-2-amines (16 and 17) in low yields (18 and

6%, respectively).

Modiﬁcations in the linker were performed using the

commercially available 5-methoxy-1H-indole-3-carbaldehyde (18)

as starting material. The introduction of a methyl group in the beta-

position with respect to the indole ring was carried out from 18 and

(carbethoxyethylidene)triphenylphosphorane by a Wittig reaction,

group was carried out from the

a,b-unsaturated acid 22 by

condensation with propargyl or allyl amine in acetonitrile at rt

during 3 h, in the presence of EDC$HCl, HOBt, and DMAP, giving the

corresponding propargyl (25) or allyl derivative (26) in moderate to

Scheme 1. Reagents and conditions: (a) RX, K₂CO₃, acetone, MW, 120 ^ꢁC, 10 min; (b) RNH₂, BOP, TEA, DMF, rt, overnight; (c) Ph₃P]CHeCO₂Et, DCM, reﬂux, overnight; (d) H₂/PdeC

(5%), EtOH, rt, overnight; (e) N₂H₄$H₂O, MW, 150 ^ꢁC, 45 min; (f) CDI, DMF, MW, 120 ^ꢁC, 25 min; (g) Malonic acid, piperidine, pyridine, 70 ^ꢁC, overnight; (h) HOBt, EDC$HCl, DMAP,

ACN, rt, 3 h; (i) N₂H₄$H₂O, rt; (j) Propargyl bromide, K₂CO₃, acetone, MW, 120 ^ꢁC, 10 min; (k) Propargylamine or allylamine, rt.

4

C. Herrera-Arozamena et al. / European Journal of Medicinal Chemistry 190 (2020) 112090

good yields (Scheme 1).

modifying the palladium-based catalyst [Pd(OAc)₂þPPh₃or

Pd(PPh₃)₂Cl₂], temperature (100e140 ^ꢁC) and reaction time

(10e40 min). Optimized conditions for the synthesis of 35 resulted

from the use of Pd(PPh₃)₂Cl₂as catalyst and MW irradiation at

105 ^ꢁC for 15 min, obtaining the desired ester derivative in 87%

yield.

Then, the ester 35 was hydrolysed under basic condition with

lithium hydroxide (LiOH) followed by acid treatment with HCl to

obtain the corresponding acid. This acid was not isolated but

transformed into the corresponding hydrazide, followed by cycli-

zation with CDI to afford the desired dihydronaphthalene-based

oxadiazolone 33 in low yields (21%) due to its easy aromatization.

In fact, the aromatic analogue was detected by HPLC-MS, although

it was not isolated (Scheme 2).

Indole scaffold replacement by either a dihydronaphthalene or a

naphthalene ring was carried out from commercial 7-methoxy-1-

tetralone (27) by two different synthetic routes. Reaction of 27

with a solution of methyl magnesium iodide in ether at rt for 3 h

afforded the corresponding alcohol (non-isolated intermediate),

which was treated with HCl to yield the 1-methyl dihydronaph-

thalene 28 in quantitative yield [34]. Oxidation of the above methyl

group with selenium dioxide gave aldehyde 29, in low yield due to

the easy aromatization of the dihydronaphthalene ring. In spite of

the mild oxidative conditions used, a mixture of dihydronaph-

thalene 29 and naphthalene 30 [35] was obtained, in 25% and 20%

yield respectively (Scheme 2).

Then, we planned to transform the dihydronaphthalene deriv-

ative 29 into the unsaturated acid 31 by the treatment with malonic

acid in Knoevenagel-Doebner conditions, obtaining unexpected ¹H

and ¹³C NMR data for the isolated product. A deeper NMR analysis

(COSY, HSQC, and HMBC) demonstrated that this structure corre-

sponded to lactone 32, formed under basic conditions by an

intramolecular cyclization (Scheme 2). Probably, the carboxylate

group attacked the intracyclic double bond forming the interme-

diate enol, which could develop to lactone 32. Nevertheless, given

that in solution this cyclization is an equilibrium, acid activation

was possible after treatment with coupling reagents (EDC$HCl,

HOBt). Further treatment with N₂H₄$H₂O and CDI, led to the

desired dihydronaphthalene-based NH-oxadiazolone 33, although

in low yield (15%), probably due to the easy aromatization that

could also give the corresponding naphthalene (non-isolated).

Aiming to improve the yield of the oxadiazolone derivative with

a dihydronaphthalene core 33, another alternative synthetic route

was explored. 7-Methoxy-1-tetralone (27) was reacted with tri-

ﬂuoromethanesulfonic anhydride (Tf₂O) and 2-chloropyridine as

base to yield the vinyl triﬂate 34 in good yield (78%) [36] (Scheme

2).

Given that the best yields were achieved using the Heck reaction

in triﬂate derivatives, this strategy was applied for obtaining

naphthalene derivatives. The conditions previously used in the

synthesis of the vinyl triﬂate 34 (Scheme 2) were extended to the

dimethoxylated derivative (Scheme 3). However, derivative 37 was

obtained in low yields due to the formation of several by-products,

probably due to the lesser electrophilic character of the carbonyl

group. Aromatization of triﬂates 34 and 37 with 2,3-dichloro-5,6-

dicyano-1,4-benzoquinone (DDQ) at rt, led naphthalenes 38 and

39, respectively (Scheme 3). Then, the ethyl acrylic moiety was

introduced by a Heck reaction, treating the corresponding triﬂate

derivative with ethyl acrylate in the presence of Pd(OAc)₂, 1,10-

phenanthroline and TEA, to yield naphthalene esters 40 and 41 in

high yields (90 and 81%, respectively). These esters were hydro-

lysed under basic conditions, obtaining acids 42 and 43 in quanti-

tative yields. Finally, these acids were transformed into the

corresponding hydrazides, followed by cyclization with CDI to

afford naphthaleneeNH-oxadiazolones 44 and 45 in good yields

(91 and 85%, respectively).

All new compounds were isolated and puriﬁed using ﬂash col-

umn chromatography (automatic IsoleraOne-Biotage equip), pre-

parative thin-layer plates (TLC) or semipreparative HPLC (Waters

Autopuriﬁcation system). Purity (>95%) was evaluated by HPLC-MS

and chemical structure was characterized by their spectroscopic

data (¹H NMR, ¹³C NMR). Complete NMR assignments were made

Then, the introduction of an a,b-unsaturated ester was carried

out by oleﬁnation of vinyl triﬂates under palladium-catalysed

conditions (Heck reaction), using ethyl acrylate as reactant and

TEA as base. Since ﬁrst attempts to obtain 35 in good yields failed,

different experimental conditions in a MW oven were probed,

Scheme 2. Reagents and conditions: (a) CH₃MgI, Et₂O, rt, 3 h; (b) 2 M HCl, 40 ^ꢁC, 3 h; (c) SeO₂, EtOH:H₂O (10:1), reﬂux, overnight; (d) Malonic acid, piperidine, pyridine, 70 ^ꢁC,

overnight; (e) EDC$HCl, HOBt, DMAP, ACN, rt, 3 h; (f) N₂H₄$H₂O, rt; (g) CDI, DMF, MW, 120 ^ꢁC, 25 min; (h) Tf₂O, 2-chloropyridine, DCM, 2 h, rt; (i) Ethyl acrylate, Pd(PPh₃)₂Cl₂, TEA,

MW, 105 ^ꢁC, 15 min; (j) LiOH, THF:H₂O (1:1), rt, overnight.

C. Herrera-Arozamena et al. / European Journal of Medicinal Chemistry 190 (2020) 112090

5

Scheme 3. Reagents and conditions: (a) Tf₂O, 2-chloropyridine, DCM, rt, 2 h; (b) DDQ, DCM, rt, 10 min; (c) Ethyl acrylate, Pd(OAc)₂, 1,10-phenanthroline, DMF, MW, 150 ^ꢁC, 1 h; (d)

LiOH, THF:H₂O 1:1, rt, overnight; (e) 1 M HCl; (f) EDC$HCl, HOBt, DMAP, ACN, rt; (g) N₂H₄$H₂O, rt; (h) CDI, DMF, MW, 130 ^ꢁC, 25 min.

by two-dimensional NMR experiments, mainly COSY (homonuclear

correlation spectroscopy), HSQC (heteronuclear single quantum

correlation) and HMBC (heteronuclear multiple bond correlation).

oxadiazolones were not evaluated in QR2.

The replacement of the oxadiazolone ring by a 1,3,4-oxadiazol-

2-amine to give 16 provided binding constants in the hundred-

nanomolar range for the three melatonin targets (K_i¼ 550, 110,

and 330 nM, for hMT₁R, hMT₂R and QR2, respectively). Thus, this

change maintained the binding constants for hMT₁R and hMT₂R

and improved the afﬁnity for QR2, compared to some N-substituted

oxadiazolone derivatives (e.g., 12 and 15).

The addition of a methyl group in the aliphatic linker of 1 to give

the beta-methyl derivative 21, resulted in the complete loss of af-

ﬁnity for hMT₁R, a similar binding for hMT₂R (K_i¼ 12 nM) and

better afﬁnity for QR2 (K_i¼ 230 nM) than the starting molecule 1.

Introduction of a double bond in the linker of the indole series

provided interesting effects on the behaviour of these unsaturated

oxadiazolones (23 and 24) and amides (25 and 26). These four

derivatives lost their afﬁnities toward hMT₁R and hMT₂R (K_is >

100 nM), while drastically improved their binding to QR2. Unsat-

urated indole-oxadiazolone compounds 23 and 24, and the prop-

argyl amide derivative 25 showed K_is between 3.2 and 9.3 nM in

QR2, one order of magnitude better than melatonin itself

(K_i¼ 77 nM).

2.2. Characterization in melatonin receptors

Experiments on human MT₁R and MT₂R were performed in

Chinese hamster ovary cells, where these receptors were stably

transfected. The QR2 binding assays were carried out using mem-

brane homogenates of hamster brains. In all cases, displacement of

the radioligand 2-[¹²⁵I]iodomelatonin was measured in the absence

or presence of the tested compound and nonspeciﬁc binding was

determined with melatonin, following described protocols

[37e39].

Firstly, radioligand displacements were measured at a ﬁxed

concentration of compound (100 or 10,000 nM) in each receptor

subtype. Then, binding constants (K_i) were calculated only for

compounds with a radioligand displacement above 80%, using a

range of 5 different concentrations of tested compound in 3 inde-

pendent experiments. Melatonin was assayed for comparative

purposes and results are gathered in Table 1.

According to previously published results [41], we also found

that the NH-indole fragment was not indispensable for activity on

melatonin targets. In fact, the 7-methoxy-3,4-dihydronaphthalene

33 showed afﬁnities toward hMT₂R and QR2 in the 10^ꢀ7M range

(K_i¼ 410 and 260 nM, respectively), although resulted poor active

in hMT₁R. Ring aromatization to give the naphthalene counterpart

greatly modiﬁed its proﬁle, as 44 recovered a moderated afﬁnity for

hMT₁R (K_i¼ 260 nM), maintained a similar afﬁnity for QR2

(K_i¼ 270 nM), and interestingly, displayed a potent binding con-

stant in hMT₂R in the low-nanomolar range (K_i¼ 5.1 nM). The

introduction of a second methoxy group in position 6 of the

naphthalene ring was partially detrimental for afﬁnity, since 45 was

inactive in hMT₁R, not as active as 44 in hMT₂R (K_i¼ 69 nM),

although 45 was one order of magnitude more active in QR2

(K_i¼ 35 nM).

The indoleeNH-oxadiazolone 1 showed high binding afﬁnities

for hMT₁R and hMT₂R (K_i¼ 35 and 4 nM, respectively) [29]. In

contrast, it did not show a substantial afﬁnity toward QR2, as it only

displaced 5% of the radioligand at 100 nM (Table 1). The intro-

duction of substituents in the NH group of the oxadiazolone ring to

give derivatives 2e15 caused a great decrease in the afﬁnity for all

melatonin receptors, reaching in some cases the total loss of ac-

tivity. Only compounds with longer chains namely, 7 [R ¼

(CH₂)₅CH₃],

8

[R

¼ (CH₂)₆CH₃], 12 (R ¼ CH₂cyclobutyl), 13

(R ¼ CH₂cyclopentyl), and 15 [R ¼ (CH₂)₃phenyl] showed a slight

selectivity for hMT₂R over hMT₁R, with binding constants around

10^ꢀ7M in the case of hMT₂R. This afﬁnity drop could be due to a

possible hydrogen bond between the NH group of the oxadiazolone

ring of 1 and the receptors, which is lost when it is substituted. The

evaluation of derivative 4 in QR2 gave only a 68% displacement at

5-(2-(7-Methoxynaphthalen-1-yl)vinyl)-1,3,4-oxadiazol-2(3H)-

10

mM, so its K_iwas not calculated and the rest of the indole-

6

C. Herrera-Arozamena et al. / European Journal of Medicinal Chemistry 190 (2020) 112090

Table 1

Binding constants at hMT₁R, hMT₂R and QR2 (K_i, nM), or percentage of radioligand displacement at the speciﬁed concentration (in brackets), antioxidant properties (ORAC,

trolox equiv.) and NRF2 induction capability (CD, M) of new indole and naphthalene derivatives.

m

Compd.

K_i(nM)^a

ORAC (Trolox equiv)^b

NRF2 (CD, m

M)^c

hMT₁R

hMT₂R

QR2

1

4

7

8

12

13

15

16

21

23

24

25

26

33

44

45

35 1^d

4.0 0.5^d

>10⁴

>10²(5%)

~10⁴(68%)

n.d.

2.7 0.3

3.0 0.2

2.1 0.2

2.2 0.1

2.8 0.2

2.7 0.2

1.8 0.2

2.3 0.1

2.4 0.1

1.8 0.1

1.3 0.1

1.8 0.1

0.4 0.04

<0.1

n.d.

>30

n.d.

>60

15.1 0.9

1.76 0.20

27.4 1.5

>30

n.d.

>30

>10⁴

530 40

300 30

230 20

390 30

320 20

110 20

12 0.9

>10⁴

550 40

>10⁴

n.d.

690 60

550 40

>10²(37%)

>10²(8%)

>10²(0%)

>10²(13%)

>10²(34%)

~10⁴(46%)

260 20

~10⁴(63%)

0.27 0.03

n.d.

330 30

220 20

6.6 0.4

3.2 0.2

9.3 0.5

110 10

250 20

270 20

>10²(17%)

>10²(0%)

~10²(58%)

~10²(78%)

260 20

5.1 0.4

69

6

35

77

2

1

<0.1

2.3 0.1

n.d.

>30

Melatonin

Sulforaphane

0.13 0.02

n.d.

0.54 0.07^e

n.d., not determined.

a

Results are the mean SEM of three independent experiments (n ¼ 3).

b

c

Results are the mean SD of three independent experiments (n ¼ 3).

Values are expressed as the mean SEM of four independent experiments in duplicate.

Value from Ref. [29].

Value from Ref. [40].

d

e

one (44), which displayed the highest afﬁnity for hMT₂R, was also

functionally characterized by measuring its effects on cAMP mod-

ulation, according to a described HTRF-detection protocol [38,42].

Compound 44 was found to be a potent agonist in hMT₂R with a

half maximal effective concentration in the low-nanomolar range

(EC₅₀¼ 1.2 0.1 nM) and a maximum activity of 85% with respect

to melatonin.

concentration tested (10 mM). In summary, it can be inferred that in

these compounds the radical absorbance capacity was mainly due

to the presence of the indole ring, rather than other structural

motifs.

2.4. Induction of the transcription factor NRF2 pathway in the

AREc32 cell line

The ability to activate NRF2 was evaluated using an NRF2-

dependent luciferase reporter assay in the AREc32 cell line

[40,48]. Cells were cultured for 24 h and then treated with

increasing concentrations of the corresponding compound (0.3, 3,

2.3. Evaluation of the oxygen radical absorbance capacity (ORAC)

As a measure of antioxidant properties, we tested new indole-

and naphthalene-based compounds in the ORAC assay, following

10 and 30 mM). A selection of new derivatives, covering different

described

protocols

[43e46].

( )-6-Hydroxy-2,5,7,8-

structural features, was tested as inducers of NRF2 signalling and

the results are gathered in Table 1. Sulforaphane was used as

reference compound and data are expressed as the concentration

needed to duplicate the speciﬁc activity of the luciferase reporter

(CD).

tetramethylchromane-2-carboxylic acid (trolox), the aromatic

part of vitamin E responsible for its scavenging properties, was used

as the internal standard with the arbitrary value of ORAC ¼ 1.0.

Results are expressed as trolox equivalents (trolox mmol/tested

compd. mmol) in a comparative scale that indicates if a compound

is a better (ORAC > 1.0 trolox equiv) or a worse oxygen radical

scavenger (ORAC < 1.0 trolox equiv) than vitamin E. Melatonin was

also evaluated for comparative purposes, giving an ORAC value 2.3-

fold higher than trolox. This activity fully agrees with the ORAC

value previously described by Soﬁc et al. (2.0 trolox equiv) [47], thus

supporting the reliability of our experiments.

In general, saturated indole-oxadiazolone derivatives (e.g., 4)

did not show activity at the maximum concentration tested

(30

and its N-propargylic counterpart 24 were able to increase lucif-

erase activity, with CD values of 15.1 and 1.8 M, respectively

mM). On the contrary, the unsaturated indole-oxadiazolone 23

m

(Table 1). Removal of the oxadiazolone ring in 24 to give the

propargylic amide 25 caused a decrease by one order of magnitude

As shown in Table 1, all indole derivatives displayed ORAC values

ranging from 1.3 to 3.0 trolox equiv., independently of the nature of

the substituent attached to position 3 of the heterocycle. Conse-

quently, they could be considered as excellent antioxidant agents.

Surprisingly, the unsaturated derivative 23 did not show a sub-

stantial difference in the ORAC value (2.4 trolox equiv) compared to

its saturated analogue 1 (2.7 trolox equiv), despite the view that an

increase of the conjugation between the indole and oxadiazolone

rings would bring about augmented radical scavenger properties.

Replacement of indole by a dihydronaphthalene or naphthalene

core was detrimental to the antioxidant activity in the ORAC assay.

Dihydronaphthalene 33 displayed a reduced ORAC value (0.4 trolox

equiv) and naphthalenes 44 and 45 were inactive at the maximum

in NRF2 activation (CD ¼ 27.4

mM). When the propargyl moiety was

replaced by an allyl fragment (26) the activity vs. NRF2 was lost.

Dihydronaphthalene and naphthalene analogues were inactive.

The above results indicated the importance of the presence of the

indole core and a double bond in the linker to achieve good in-

duction of NRF2.

2.5. Binding studies by surface plasmon resonance (SPR) assay

As explained in the introduction, activation of NRF2 could be

due to conformational changes in KEAP1 that preclude the union

between both proteins and stimulate the expression of antioxidant

C. Herrera-Arozamena et al. / European Journal of Medicinal Chemistry 190 (2020) 112090

7

enzymes by NRF2. To evaluate the ability of our molecules to

interact with the Kealch domain of KEAP1, a surface plasmon

resonance (SPR) experiment was carried out. The Kelch domain was

obtained from Escherichia coli transformed with pET15b-6xHIS-

Kelch plasmid and induced by adding isopropyl-1-thio-b-D-gal-

actopyranoside (IPTG). 6xHIS-Kelch was puriﬁed using the ProBond

Puriﬁcation System (Invitrogen). The Kelch domain of KEAP1 was

immobilized on a carboxymethyl cellulose sensor chip (CM5) and a

solution of the corresponding molecule (100 mM) was then ﬂuxed

over the sensor chip in order to detect a change in the SPR signal,

resulting from the interaction between the compounds and the

Kelch domain. The well-known KEAP1 binder ML334 was used as

internal reference [49], which produced a change in the SPR signal

of 10 resonance units (Fig. 2).

As shown in Fig. 2, SPR responses of compounds 23e25 were

equal to, or greater than ML334, while the rest of the compounds

tested (4, 21, 26, and 45) gave fewer values. Importantly, the most

active compounds in the phenotypic assay based on AREc32 cells

showed a better binding to KEAP1 than the less active derivatives.

Therefore, we can tentatively think that the activation of NRF2 by

compounds 23e25 in the cellular assay could be due to the binding

of these molecules to KEAP1.

Fig. 2. Binding of selected compounds (4, 21, 23e26 and 45) to immobilized KEAP1

Kelch domain protein in comparison with ML334, determined by SPR spectroscopy.

Mean

**P < 0.05.

SD (n ¼ 3), one-way ANOVA plus Dunnett’s test compared to ML334 (#).

Table 2

Inhibition (IC₅₀, m

M)^aof human monoamine oxidases (hMAO-A and hMAO-B) and

human lipoxygenase-5 (hLOX-5) of indole and naphthalene derivatives^b.

Compd.

hMAO-A

hMAO-B

hLOX-5

2.6. Inhibition of human MAO and LOX-5

12

13

16

23

>50

53.0 3.5

40.6 1.1

68.7 4.2

6.7 0.8

161.4 19.4

n.d.

>50

74.2 2.2

22.5 1.8

60.6 2.6

>100

12.5 0.3

n.d.

0.15 0.03

0.097 0.019

All compounds were evaluated as inhibitors of human MAO-A,

MAO-B and LOX-5 and the results of the most representative

compounds are collected in Table 2. For the sake of clarity, data of

inactive compounds in the three enzymes are omitted. The hMAO-

A/B inhibition was determined by the production of oxygen

peroxide from a common substrate for both isoenzymes (p-tyra-

mine) and quantiﬁed by the Amplex Red MAO assay kit [50]. (R)-

Deprenyl, iproniazid and moclobemide were also tested for

comparative purposes. The inhibition of hLOX-5 was performed

following the ﬂuorescence-based method described by Pufahl et al.

[51], using the two well-known inhibitors zileuton and nordihy-

droguaiaretic acid (NDGA) as internal references.

37.0 0.7

68.3 4.6

35.7 3.3

0.017 0.002

7.5 0.4

>100

33

(R)-Deprenyl

Iproniazid

Moclobemide

(R,S)-Zileuton

NDGA

n.d.

a

Data are the mean

SD of three independent experiments performed in

triplicate.

b

Data of inactive compounds in the three enzymes are not shown.

In general, saturated indoleeoxadiazolone derivatives did not

show any appreciable inhibition of hMAOs or hLOX-5

and 45 were inactive in the three enzymatic assays. Surprisingly,

the introduction of a propargyl substituent in the oxadiazolone ring

of the MAO-A/B inhibitor 23 to give 24 led to a total lack of activity

on these enzymes (data not shown). Contrariwise, the

dihydronaphthalene-oxadiazolone 33 inhibited hMAO-A, hMAO-B

(IC₅₀> 50

modest inhibition of hLOX-5 (IC₅₀¼ 74.2 and 22.5

The 5-methoxyindole-oxadiazolamine 16 inhibited both hMAO-B

M, respectively). Introduction of

mM), with the exception of 12 and 13 which displayed a

mM, respectively).

and hLOX-5 (IC₅₀¼ 37.0 and 60.6

m

and hLOX-5 with IC₅₀values of 40.6, 35.7 and 12.5 mM, respectively

(Table 2).

a double bond in the linker changed this proﬁle, as the 5-

methoxyindole-oxadiazole 23 displayed moderate inhibition of

MAO-A and MAO-B (IC₅₀¼ 53.0 and 68.3

m

M, respectively)

although it was inactive in LOX-5 (IC₅₀> 100

mM). This positive

2.7. In vitro bloodꢀbrain barrier permeation assay (PAMPA-BBB)

effect of the double bond in the linker was not valid for the indole-

amide and the naphthalene-oxadiazole series, because 25, 26, 44,

In order to determine the capability of the new compounds to

Fig. 1. Design strategy for the novel melatonin-based MTDLs.

8

C. Herrera-Arozamena et al. / European Journal of Medicinal Chemistry 190 (2020) 112090

cross the BBB and reach their CNS targets, we evaluated them in the

in vitro parallel artiﬁcial membrane permeability assay for the BBB

(PAMPA-BBB) described by Di et al. [52], and modiﬁed by our group

for testing molecules with limited water solubility [44e46,53,54].

The passive CNS permeation of new compounds through a lipid

extract of porcine brain was measured at room temperature. In each

experiment, 11 commercial drugs of known brain permeability

were also tested, and their permeability values were normalized to

the reported PAMPA-BBB data. As previously established in the

literature [52], compounds with P_e> 4.0$10^ꢀ6cm s^ꢀ1would be able

to cross the BBB (cnsþ), whereas those displaying

P_e< 2.0$10^ꢀ6cm s^ꢀ1would not reach the CNS (cns-). Between these

values, the predicted CNS permeability remains uncertain (cns þ/

ꢀ).

The neurogenic potential of each compound was determined using

ﬂuorescence confocal microscopy, by direct observation of the

expression of two immunoﬂuorescent dyes linked to two well-

known neuronal markers, b_III-tubulin (TuJ-1 clone) shown in

green and microtubule-associated protein 2 (MAP-2) in red. TuJ-1 is

expressed in new born neurons in early stages of their differenti-

ation, whereas the expression of MAP-2 indicates a consolidated

neuronal stage [59]. Vehicle-treated cultures (considered as basal)

showed only a few scattered positive TuJ-1 or MAP-2 cells. In

contrast, treatment with the compounds resulted in an increase in

the number of TuJ-1 and MAP-2-positive cells compared to basal

levels.

In the melatonin-based family several compounds promoted the

expression of TuJ-1 and MAP-2, indicating positive neurogenic ac-

tivities (Fig. 3). Comparing derivatives with a dimethylene liker, the

indoleeoxadiazolone 4 displayed good neurogenic properties,

whereas its indoleeoxadiazolamine counterpart 16 was inactive.

This result points out the importance of the presence of an oxa-

diazolone ring for successful neurogenesis.

In the melatonin-based family, the indoleeNH-oxadiazolone 1

and its beta-methyl analogue 21 (both bearing a saturated linker)

were predicted as not CNS-permeable (P_e< 2$10^ꢀ6cm s^ꢀ1). In

contrast, all indoleeN-substituted oxadiazolone derivatives (2e15),

showed permeability values exceeding 4.0 10^ꢀ6cm s^ꢀ1and thus,

they

were

predicted

to

be

CNS-permeable.

The

Among indole compounds bearing a double bond in the linker,

the indoleeN-propargyloxadiazolone 24 was one of the most active

derivatives, as it greatly increased the expression of both TuJ-1 and

MAP-2. Conversely, its indoleeNH-oxadiazolone analogue 23 was

inactive, suggesting that the N-propargyl group is beneﬁcial for

activity.

Both naphthaleneeNH-oxadiazolone derivatives 44 and 45

showed positive effects in the expression of TuJ-1 and MAP-2. In

contrast, the dihydronaphthalene analogue 33 was inactive, sug-

gesting that the naphthalene core is more useful for biasing the

compounds towards neurogenesis.

indoleeoxadiazolamine 16 displayed negative CNS permeation,

whereas the indoleeNH-oxadiazolone 23, which is the unsaturated

analogue of 1, displayed a permeability value in the ambiguous

range (P_e¼ 3.2$10^ꢀ6cm s^ꢀ1). The attachment of a propargylic

moiety onto the oxadiazolone ring nitrogen of 23 gave 24, which

-as expected-was found to be CNS-permeable. Unsaturated amides

25 and 26 showed negative CNS permeability.

The exchange of the indole heterocycle by a dihydronaph-

thalene (33) or a naphthalene (44 and 45) generated compounds

with good CNS permeability values (P_e

¼

9.0, 9.8 and 4.5

10^ꢀ6cm s^ꢀ1, respectively), even when the NH-unsubstituted oxa-

diazolone ring was retained. From these experiments we can

conclude that, in general, the new indole and naphthalene de-

rivatives with a single NH-unsubstituted motif are expected to be

CNS-penetrating, whereas compounds with two NH groups would

experience more difﬁculties to reach the CNS.

2.10. Theoretical studies. Conformational analysis of indole and

naphthalene derivatives 23 and 44

As explained above, the indoleeNH-oxadiazolone derivative 23

showed marked selectivity towards QR2 (K_i¼ 6.6 nM), in com-

parison with hMT₁R and hMT₂R (K_i> 100 nM). In contrast, substi-

tution of a naphthalene ring for the indole heterocycle gave 44,

which was a potent and selective agonist at hMT₂R (EC₅₀¼ 1.1 nM),

compared to hMT₁R and QR2 (K_i¼ 260 and 270 nM, respectively)

(Table 1).

With the aim of explaining this distinct behaviour in NH-oxa-

diazolone derivatives that only differ in the nature of the fused

heterocycle, we performed a conformational analysis of 23 and 44

using quantum mechanics (Fig. 4).

The conformational analysis revealed higher energy barriers

(~2 kcal mol^ꢀ1) for rotation about the C2eC3 bond and a preferred

ﬂat conformation in the indoleeNH-oxadiazolone 23 (Fig. 4). This

co-planar arrangement of the ring systems is prevented in the

naphthalene derivative 44 due to steric clash as a consequence of

the decreased C1eC2eC3 angle. These results seemed to indicate

that the ﬂat conformation of 23 might favour the stacking inter-

action with the isoalloxazine ring of FAD in QR2, whereas the slight

non-planarity of 44 (dihedral angle of ~40^ꢁ) would be preferred for

its agonist activity on hMT₂R. On the other hand, the energy bar-

riers for rotation about the C4eC5 bond are of similar magnitude in

both molecules.

2.8. Drug-like calculations

To choose the best candidates for neurogenic assays, we studied

the medicinal chemistry alerts of new indole- or naphthalene-

oxadiazolones in two free databases, namely ZINC15 (http://

zinc15.docking.org/) [55] and Aggregator Advisor (http://advisor.

bkslab.org/) [56] (Table S2 in the Supplementary Information).

None of the new compounds was highlighted as a pan assay

interference compound (PAINS), according to the ZINC15 database.

However, the Aggregator Advisor website identiﬁed compounds 13

and 15 with a fairly high calculated LogP in the range reported for

other aggregators. Thus, these compounds were discarded for

further biological assays.

Moreover, we calculated several physicochemical and ADME

properties (log P, solubility, pK_aand Lipinski rule) of selected

compounds (23, 24 and 44) in comparison with melatonin (Table S3

in the Supplementary Information). According to the Swiss Insti-

tute of Bioinformatics (http://www.swissadme.ch/) [57], these

compounds showed a good drug-like proﬁle and thus, they are

good candidates for further pharmacological studies.

2.9. Neurogenic studies

2.11. Molecular dynamics simulations on hQR2, hMT₁R and hMT₂R

We investigated the capacity of a selection of the new com-

pounds, covering different structural features, to promote cell dif-

ferentiation into neurons. To this end, hippocampal-derived

neurospheres adhered to a substrate and cultured under differen-

tiation conditions in the presence of compounds were used [27,58].

To test the previous hypotheses, we undertook some molecular

dynamics studies. In agreement with the previous statement, our

modelled complexes of hQR2 (Fig. 5) and hMT₂R (Fig. 6) with 23

and 44, respectively, show that the proposed stacking of the ﬂat 23

molecule on FAD is reminiscent of that described for resveratrol

C. Herrera-Arozamena et al. / European Journal of Medicinal Chemistry 190 (2020) 112090

9

Fig. 3. Melatonin-based compounds promote neuronal differentiation on SGZ-derived neurospheres. Neurospheres were cultured in the presence of compounds (10

mM) for 7 days

and later adhered for 3 days to allow differentiation in the presence of compounds. Confocal images show the expression of the neuronal markers TuJ clone (early neurogenesis) in

green and MAP-2 (mature neurons) in red. DAPI was used for nuclear staining. Fluorescent representative images from four independent experiments were displayed. Scale

bar ¼ 100

mm (images of inactive compounds are not shown). (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the Web version of this

article.)

[60], with the indole nitrogen establishing a hydrogen bond to the

carbonyl oxygen of Gly174 that is similar to that observed for one of

the resorcinol hydroxyls of this naturally occurring polyphenol. It

can also be seen that the oxadiazolone nitrogen is engaged in

hydrogen bonds to water molecules, which would be displaced

when the attached hydrogen is replaced with a short substituent, as

is the case for the propargyl group present in 24 (Fig. S1, Supporting

Information).

In contrast, for the less coplanar naphthalene derivatives, the

variety of binding orientations we found is commensurate with

that observed in X-ray crystal structures of QR2 in complex with

melatonin and derivatives [61]. Nonetheless, the out-of-plane

conformation of 44 is ideally suited to mimic the bound confor-

mation of melatonin that has been observed in the XFEL structures

of chimeric MT₁R and MT₂R constructs, very recently described by

Stauch et al. [62] and Johansson et al. [63], respectively. At this

location in our modelled MT₂R, binding of 44 is largely stabilized by

the phenyl ring of Phe192^ECL2, additional hydrophobic interactions

with neighbouring residues (including the highly conserved

Trp264^6.48that was replaced by Phe in the experimental con-

structs) and a number of direct and water-mediated hydrogen

bonds with the polar side chains of Gln194^ECL2, Asn162^4.60, and

Asn268^6.52. The most important difference relative to MT₁R is the

orientation of Tyr200^5.38, which is close to the side chains of Val178

^ECL2and Ser180 ^ECL2and whose phenolic group hydrogen bonds to

the carbonyl oxygen of Ala171^4.56. In MT₁R, the equivalent residues

are Ala165 ^ECL2and Thr167 ^ECL2so that the side chain of Tyr187^5.38

points towards the membrane [62,63] and slightly different in-

teractions are observed (Fig. S2, Supporting Information).

2.12. Molecular docking studies and molecular dynamics

simulations on KEAP1

Molecular docking studies of 23 and 24 were performed on four

different crystal structures of the KEAP1 Kelch domain (PDB entries

4XMB, 5CGJ, 5FNU and 4IFL) to consider, at least partially, protein

ﬂexibility. At physiologic pH, compound 23 exists as two different

species, as deduced from its experimental pK_aof 7.45 0.09 (Fig. S3,

Supporting Information). No substantial differences in the average

binding energy values were observed in both species, named 23-

0 and 23-1. On the other hand, the higher afﬁnity score obtained for

24 correlated with the experimental NRF2 induction value in the

AREc32 cell line (Table S1, Supporting Information).

The most stable molecular docking poses found for 23 and 24 on

the four 3D-structures of the KEAP1 Kelch domain (Figs. S4 and S5,

Supporting Information) were used as the starting structures for

uMD simulations, which after 200 ns of production run showed

some drifting of the bound ligand towards a new ligand-protein

interface (Fig. 7). The binding site residues involved in the NRF2-

KEAP1 interaction are very well known and have been reported

by several authors [64]. It has been shown that residues

Arg380,415,483; Ser363,508,555,602; and Tyr525,572,334 on

10

C. Herrera-Arozamena et al. / European Journal of Medicinal Chemistry 190 (2020) 112090

Fig. 4. Global energy conformations of indoleeNH-oxadiazolone 23 (A) and naphthaleneeNH-oxadiazolone 44 representative molecules (B), together with the rotational barriers

(in vacuum, blue; using an implicit solvent model for water, orange) upon rotation about the bonds connecting atoms labelled 2 and 3 (top) and 4 and 5 (bottom). (For interpretation

of the references to color in this ﬁgure legend, the reader is referred to the Web version of this article.)

KEAP1 are essential to interact with NRF2. Our uMD simulations

show that 23 and 24 do interact with some of these residues (Fig. 7)

and also 23 experiences greater ﬂuctuations within the binding

pocket in KEAP1. This difference mostly arises from the fact that 24

interacts with hydrophobic residues Phe577 and Ala556 whereas

23 does not.

Finally, thermodynamic calculations over the last 10 ns of the

production run, using the Molecular Mechanics Generalized Born

Surface Area approach (MMGBSA) [65], revealed a noticeably

higher afﬁnity for 24 relative to 23 that is in consonance with the

C. Herrera-Arozamena et al. / European Journal of Medicinal Chemistry 190 (2020) 112090

11

Fig. 5. Representative models of hQR2 in complex with indole e NH-oxadiazolone derivative 23 (sticks with C atoms coloured in grey) obtained after the simulated annealing

procedure following molecular dynamics simulations. The starting reference structure was PDB entry 1SG0.

Fig. 6. Model of a membrane-embedded hMT₂R (rainbow coloured) in complex with naphthaleneeNH-oxadiazolone derivative 44 (sticks with C atoms coloured in grey). The

enlarged region in the box shows the superposition of eight cooled and energy-minimized snapshots from the last 170e200 ns of unrestrained molecular dynamics (uMD)

simulation. The starting reference structure was PDB entry 6ME8.

12

C. Herrera-Arozamena et al. / European Journal of Medicinal Chemistry 190 (2020) 112090

Fig. 7. Dynamic conformation of KEAP1-ligand complexes. A) KEAP1-23 complex. B) KEAP1-24 complex. Water molecules and ions are not depicted for clarity. KEAP1 is represented

as a brown molecular surface [66]. Residues involved into protein-ligand interaction are represented as sticks by colour type: green are polar residues, blue are positive charged

residues and white are hydrophobic residues. Ligands are represented as sticks with grey carbon atoms, blue nitrogen atoms, red oxygen atoms and white hydrogen atoms. Thinner

white sticks correspond to the ligand conformations during the last 10ns of the MD production. These images were made with VMD software support [67]. The most stable

molecular docking poses found for 23 and 24 on the 3D-structures of the KEAP1 Kelch domain with pdbcode 4XMB were used as the starting reference structure. (For interpretation

of the references to color in this ﬁgure legend, the reader is referred to the Web version of this article.)

experimental ﬁnding that 24 leads to a larger activation and

expression of NRF2 in AREc32 cells (Table 1).

selectivity and afford valuable insight that can aid in future drug

design endeavours.

In the ORAC assays, indole derivatives were found to be as good

radical scavengers as melatonin, whereas naphthalene-based

compounds showed worse ORAC values. Thus, the radical absor-

bance capacity was mainly linked to the indole ring.

3. Conclusions

By structural modiﬁcations of our described hMT₁R and hMT₂R

nanomolar ligand 5-[2-(5-methoxy-1H-indol-3-yl)ethyl]-1,3,4-

oxadiazol-2(3H)-one

In relation to NRF2 induction, best results were obtained with

the indoleeNH-oxadiazolone derivative with a double bond in the

linker 23 and its N-propargylic counterpart 24 (CD ¼ 15.1 and

(1),

new

indolee

and

naphthaleneeoxadiazolone MTDLs have been obtained and eval-

uated on targets related to oxidative stress and neuroinﬂammation.

Introduction of lipophilic substituents on the nitrogen of the oxa-

diazolone ring improved their in vitro CNS permeation but was

detrimental for their afﬁnity for the GPCRs hMT₁R and hMT₂R. The

addition of a methyl group in the aliphatic linker of 1 or the

replacement of the oxadiazolone ring by a 1,3,4-oxadiazol-2-amine

scaffold yielded compounds with K_is in the hundred-nanomolar

range in melatonin targets, but they were predicted as not CNS-

permeable.

Introduction of a double bond in the linker provided interesting

effects on the behaviour of these less ﬂexible indole- and

naphthalene-oxadiazolone compounds. The 5-methoxyindole de-

rivatives 23 and 24 displayed remarkable selectivity towards QR2

(K_i¼ 6.6 and 3.2 nM, respectively) in reference to hMT₁R and

hMT₂R (K_is > 100 nM). In contrast, when the 5-methoxyindole of 23

was replaced by a 7-methoxynaphthalene the selectivity changed

drastically, since 44 was preferably bound to hMT₂R (K_i¼ 5.1 nM)

compared to hMT₁R and QR2 (K_i¼ 260 and 270 nM, respectively).

Moreover, 44 was functionally characterized as a potent hMT₂R

agonist with a maximum activity of 85% with respect to melatonin

and an EC₅₀of 1.1 nM.

1.8 mM, respectively). When comparing with other derivatives, we

can infer the importance of the presence of the indole core and a

double bond in the connector to achieve a good induction of NRF2.

From the SPR experiments using the KEAP1 Kelch domain, we could

infer that the cellular NRF2 induction by compounds 23 and 24

could be due to the binding of these molecules to KEAP1. Moreover,

molecular dynamics simulations on the KEAP1 Kelch domain

showed that 23 and 24 interact with several essential residues

involved in the binding KEAP1-NRF2. Thermodynamic calculations

revealed higher afﬁnity for 24 than for 23, according to the

experimental ﬁnding that 24 leads to a larger activation and

expression of NRF2 in cellular experiments.

Several saturated indoleeoxadiazolone derivatives were modest

hLOX-5 inhibitors and inactive in hMAOs. However, the introduc-

tion of a double bond in the linker again reversed selectivity, as the

indoleeNH-oxadiazolone 23 inhibited hMAOs but it is inactive in

hLOX-5. Only the dihydronaphthalene-oxadiazolone 33 was found

to inhibit the three enzymes, with IC₅₀values in the two-digit

micromolar range.

In neurogenic assays, several of the new melatonin-

oxadiazolone derivatives stimulated the differentiation of NSC to

a consolidated neuronal stage, as they could contribute to the auto-

repair processes of the CNS.

Finally, it can be emphasized that it is possible to modulate the

selectivity of these new oxadiazolone-based MTDLs by modifying

the side chain functionality and co-planarity with the indole or

naphthalene ring.

Conformational analyses using quantum mechanics revealed

that the indole-NH-oxadiazolone 23 presents a co-planar arrange-

ment of the ring systems, whereas the naphthalene-NH-oxadia-

zolone 44 shows

a

dihedral angle of ~40^ꢁbetween both

heterocycles. These differences could explain the observed selec-

tivity of 23 and 44 towards QR2 and hMT₂R, respectively. Molecular

modelling and computer simulations, using the crystal structure of

QR2 and the recently described XFEL structures of chimeric MT₁R

and MT₂R, have provided

a rationale for the experimental

C. Herrera-Arozamena et al. / European Journal of Medicinal Chemistry 190 (2020) 112090

13

4. Experimental section

4.3. Characterization of compounds 2e45

4.1. Chemistry. General methods

4.3.1. 5-[2-(5-Methoxy-1H-indol-3-yl)ethyl]-3-methyl-1,3,4-

oxadiazol-2(3H)-one (2)

Chromatography: DCM to DCM:MeOH 95:5. White solid (66%

yield) of mp 83e86 ^ꢁC. ¹H NMR (500 MHz, CDCl₃)

7.97 (s, 1H, NH),

High-grade reagents and solvents were purchased from com-

mercial suppliers and were used without further puriﬁcation. Re-

actions were followed by thin-layer chromatography (TLC) or high-

performance liquid chromatography coupled to mass spectrometry

(HPLC-MS). TLC was performed using Merck silica gel 60 F254

d

7.25 (d, J ¼ 8.6 Hz, 1H, H₇), 7.00 (d, J ¼ 2.5 Hz, 1H, H₂), 6.99 (d,

J ¼ 2.4 Hz, 1H, H₄), 6.87 (dd, J ¼ 8.7, 2.4 Hz, 1H, H₆), 3.87 (s, 3H,

OCH₃), 3.35 (s, 3H, NCH₃), 3.12 (t, J ¼ 7.7 Hz, 2H, H_a), 2.92 (t,

plates and compounds were detected under UV-light (

l

¼ 254 or

J ¼ 7.7 Hz, 2H, H_b). ¹³C NMR (126 MHz, CDCl₃)

d 155.9 (C_5’), 154.4

365 nm) and/or stained with 10% wt. phosphomolybdic acid,

ninhydrin, or vanillin solutions in EtOH. HPLC-MS data were ac-

quired in an Alliance Watters 2695 coupled to a quadrupole mass

spectrometer (Micromass ZQ). HPLC was equipped with a SunFire

(C_2’), 154.2 (C₅), 131.5 (C_7a), 127.4 (C_3a), 122.5 (C₂), 113.6 (C₃), 112.6

(C₆), 112.2 (C₇), 100.3 (C₄), 56.1 (OCH₃), 32.5 (NCH₃), 27.4 (C_b), 21.5

(C_a). HPLC-MS (15:95- g.t.5 min) ^tR 3.97 min, m/z ¼ 274.20 [MþH]^þ,

calcd. for [C₁₄H₁₅N₃O₃þH]^þ274.29. HRMS [ESI^þ] m/z ¼ 273.11145

[M]^þ, calcd. for [C₁₄H₁₅N₃O₃]^þ273.11134.

C₁₈column (3.5

diode array detector (

m

m, 4.6 mm ꢂ 50 mm) and a UVevisible photo-

l

¼ 190e700 nm). MS were acquired in an

electrospray ionization (ESI) interface working in the positive or

negative-ion mode. Microwave driven reactions were performed in

a Biotage Initiator 2.5 reactor. Unless otherwise stated, products

were puriﬁed by automatized ﬂash column chromatography using

an IsoleraOne (Biotage) equipment, with different cartridges of

4.3.2. 3-Ethyl-5-[2-(5-methoxy-1H-indol-3-yl)ethyl]-1,3,4-

oxadiazol-2(3H)-one (3)

Chromatography: DCM to DCM:MeOH 95:5. White solid (56%

yield) of mp 69e72 ^ꢁC. ¹H NMR (500 MHz, CDCl₃)

d 7.90 (s, 1H, NH),

silica gel Biotage ZIP KP-Sil 50

ative TLC on Merck silica gel 60 F254 plates or semipreparative

HPLC on Waters Autopuriﬁcation system with UVevisible

photodiode array detector (

rupole mass spectrometer (3100 Mass Detector). Purity of all

compounds (ꢃ95%) was determined by HPLC on a Waters 2690

equipment, at a ﬂow rate of 1.0 mL/min, with a UVevisible

m

m. Alternatively, we used prepar-

7.26 (d, J ¼ 8.8 Hz, 1H, H₇), 7.01 (d, J ¼ 2.5 Hz, 1H, H₂), 6.99 (d,

J ¼ 2.3 Hz, 1H, H₄), 6.87 (dd, J ¼ 8.8, 2.4 Hz, 1H, H₆), 3.87 (s, 3H,

OCH₃), 3.71 (q, J ¼ 7.2 Hz, 2H, CH₂CH₃), 3.15e3.11 (m, 2H, H_a),

2.95e2.90 (m, 2H, H_b), 1.28 (t, J ¼ 7.2 Hz, 3H, CH₂CH₃). ¹³C NMR

a

l

¼ 190e700 nm) coupled to a quad-

(126 MHz, CDCl₃) d 155.9 (C_5’), 154.3 (C₅), 154.0 (C_2’), 131.5 (C_7a),

127.5 (C_3a), 122.5 (C₂), 113.8 (C₃), 112.7 (C₆), 112.1 (C₇), 100.3 (C₄),

56.1 (OCH₃), 40.8 (CH₂CH₃), 27.6 (C_b), 21.6 (C_a), 13.5 (CH₂CH₃).

t

photodiode array detector (

l

¼ 190e700 nm), using a SunFire C₁₈

HPLC-MS (15:95- g.t.5 min) R 4.21 min, m/z ¼ 288.15 [MþH]^þ,

column (3.5

mm, 4.6 mm ꢂ 50 mm). The gradient of the mobile

calcd. for [C₁₅H₁₇N₃O₃þH]^þ288.32. HRMS [ESI^þ] m/z ¼ 287.1275

phase consisted of H₂O:ACN with formic acid (0.1%), and the gra-

dients time (g.t.) are indicated for each compound. Melting points

(mp) (uncorrected) were determined in a MP70 apparatus (Mettler

Toledo). Nuclear magnetic resonance (¹H NMR and ¹³C NMR)

spectra were obtained in the following spectrometers: Varian

INOVA-300, Varian INOVA-400, Varian Mercury-400 or Varian

[M]^þ, calcd. for [C₁₅H₁₇N₃O₃]^þ287.12699.

4.3.3. 5-[2-(5-Methoxy-1H-indol-3-yl)ethyl]-3-propyl-1,3,4-

oxadiazol-2(3H)-one (4)

Chromatography: DCM to DCM:MeOH 95:5. White solid (49%

Unity-500. Chemical shifts (

d) are reported in parts per million

yield) of mp 95e98 ^ꢁC. ¹H NMR (500 MHz, DMSO‑d₆)

d 10.67 (s, 1H,

(ppm) relative to internal tetramethylsilane scale, and coupling

constants (J) are expressed in hertz (Hz). 2D NMR experiments

namely, homonuclear correlation spectroscopy (H,HeCOSY), het-

eronuclear multiple quantum correlation (HMQC) and hetero-

nuclear multiple bond correlation (HMBC) were acquired to assign

protons and carbons of new structures. High resolution mass

spectra (HRMS) analyses were carried out by using an Agilent 1200

Series LC system (equipped with a binary pump, an autosampler,

and a column oven) coupled to a 6520 quadrupole-time of ﬂight

(QTOF) mass spectrometer. ACN:H₂O (75:25, v:v) was used as

mobile phase at 0.2 mL/min. The ionization source was an ESI

interface working in the positive-ion mode. The electrospray

voltage was set at 4.5 kV, the fragmentary voltage at 150 V and the

drying gas temperature at 300 ^ꢁC. Nitrogen (99.5% purity) was used

as nebulizer (207 kPa) and drying gas (6 L/min).

NH), 7.21 (dd, J ¼ 8.7, 0.5 Hz, 1H, H₇), 7.11 (d, J ¼ 2.5 Hz, 1H, H₂), 6.97

(d, J ¼ 2.4 Hz, 1H, H₄), 6.70 (dd, J ¼ 8.8, 2.4 Hz, 1H, H₆), 3.75 (s, 3H,

OCH₃), 3.53 (t, J ¼ 6.8 Hz, 2H, NCH₂), 3.00 (t, J ¼ 7.4 Hz, 2H, H_a), 2.90

(t, J ¼ 7.5 Hz, 2H, H_b), 1.57 (h, J ¼ 7.2 Hz, 2H, CH₂CH₃), 0.78 (t,

J ¼ 7.4 Hz, 3H, CH₂CH₃). ¹³C NMR (126 MHz, DMSO‑d₆)

d 155.6 (C_5’),

153.5 (C_2’), 153.0 (C₅), 131.3 (C_7a), 127.1 (C_3a), 123.3 (C₂), 112.1 (C₇),

111.9 (C₃), 111.2 (C₆), 99.7 (C₄), 55.3 (OCH₃), 46.4 (NCH₂), 26.9 (C_b),

21.0 (CH₂CH₃), 20.8 (C_a), 10.6 (CH₂CH₃). HPLC-MS (15:95- g.t.5 min)

^tR 4.48 min, m/z ¼ 302.28 [MþH]^þ, calcd. for [C₁₆H₁₉N₃O₃þH]^þ

302.35. HRMS [ESI^þ] m/z

¼

301.14327 [M]^þ, calcd. for

[C₁₆H₁₉N₃O₃]^þ301.14264.

4.3.4. 3-Butyl-5-[2-(5-methoxy-1H-indol-3-yl)ethyl]-1,3,4-

oxadiazol-2(3H)-one (5)

Chromatography: DCM. White solid (92% yield) of mp 64e65 ^ꢁC.

¹H NMR (500 MHz, DMSO‑d₆)

d

10.67 (s, 1H, NH), 7.21 (d, J ¼ 8.7 Hz,

4.2. General procedure for the synthesis of 2e15

1H, H₇), 7.10 (d, J ¼ 2.4 Hz, 1H, H₂), 6.96 (d, J ¼ 2.5 Hz, 1H, H₄), 6.70

(dd, J ¼ 8.7, 2.4 Hz, 1H, H₆), 3.75 (s, 3H, OCH₃), 3.55 (t, J ¼ 6.9 Hz, 2H,

NCH₂), 3.00 (t, J ¼ 7.4 Hz, 2H, H_a), 2.91 (t, J ¼ 7.5 Hz, 2H, H_b), 1.52 (p,

J ¼ 7.0 Hz, 2H, NCH₂CH₂), 1.17 (h, J ¼ 7.4 Hz, 2H, CH₂CH₃), 0.83 (t,

A mixture of 1 (1 equiv.), K₂CO₃(1.2 equiv.) and the corre-

sponding halide (1.2 equiv) in acetone (7 mL/mmol) was heated

under MW-irradiation at 120 ^ꢁC for 10 min. Solvent was evaporated

under reduced pressure, EtOAc (10 mL/mmol) was added and the

organic layer was washed with H₂O (x3) and brine and then, dried

over MgSO₄, ﬁltered, and evaporated to dryness under reduced

pressure. The crude was puriﬁed by column chromatography using

the appropriate eluent to afford the corresponding alkyl derivative

2e15.

J ¼ 7.4 Hz, 3H, CH₂CH₃). ¹³C NMR (126 MHz, DMSO‑d₆)

d 155.6 (C_5’),

153.4 (C_2’), 153.0 (C₅), 131.3 (C_7a), 127.1 (C_3a), 123.3 (C₂), 112.1 (C₇),

111.9 (C₃), 111.2 (C₆), 99.7 (C₄), 55.3 (OCH₃), 44.4 (NCH₂), 29.5

(NCH₂CH₂), 26.9 (C_b), 20.8 (C_a), 18.8 (CH₂CH₃), 13.3 (CH₂CH₃). HPLC-

MS (15:95- g.t.5 min) ^tR 4.79 min, m/z ¼ 316.11 [MþH]^þ, calcd. for

[C₁₇H₂₁N₃O₃þH]^þ316.37. HRMS [ESI^þ] m/z ¼ 315.15838 [M]^þ, calcd.

for [C₁₇H₂₁N₃O₃]^þ315.15829.

14

C. Herrera-Arozamena et al. / European Journal of Medicinal Chemistry 190 (2020) 112090

4.3.5. 5-[2-(5-Methoxy-1H-indol-3-yl)ethyl]-3-pentyl-1,3,4-

oxadiazol-2(3H)-one (6)

4.3.9. 5-[2-(5-Methoxy-1H-indol-3-yl)ethyl]-3-(2-methylpropyl)-

1,3,4-oxadiazol-2(3H)-one (10)

Chromatography: DCM. Pale yellow solid (90% yield) of mp

Chromatography: DCM. White solid (82% yield) of mp 54e56 ^ꢁC.

48e51 ^ꢁC. ¹H NMR (500 MHz, CDCl₃)

d

7.90 (s, 1H, NH), 7.25 (d,

¹H NMR (500 MHz, DMSO‑d₆)

d

10.67 (s, 1H, NH), 7.21 (d, J ¼ 8.7 Hz,

J ¼ 8.5 Hz, 1H, H₇), 7.01 (d, J ¼ 2.5 Hz, 1H, H₂), 6.99 (d, J ¼ 2.4 Hz, 1H,

H₄), 6.87 (dd, J ¼ 8.8, 2.5 Hz, 1H, H₆), 3.87 (s, 3H, OCH₃), 3.63 (t,

J ¼ 7.2 Hz, 2H, NCH₂), 3.13 (t, J ¼ 7.4 Hz, 2H, H_a), 2.92 (t, J ¼ 7.5 Hz,

2H, H_b), 1.67 (p, J ¼ 7.3 Hz, 2H, NCH₂CH₂), 1.36e1.30 (m, 2H,

CH₂CH₃), 1.30e1.21 (m, 2H, N(CH₂)₂CH₂), 0.89 (t, J ¼ 7.2 Hz, 3H,

1H, H₇), 7.10 (d, J ¼ 2.4 Hz, 1H, H₂), 6.97 (d, J ¼ 2.4 Hz, 1H, H₄), 6.70

(dd, J ¼ 8.7, 2.4 Hz, 1H, H₆), 3.75 (s, 3H, OCH₃), 3.37 (d, J ¼ 7.1 Hz, 2H,

NCH₂), 3.00 (t, J ¼ 7.3 Hz, 2H, H_a), 2.91 (t, J ¼ 7.2 Hz, 2H, H_b),

1.90e1.87 (m, 1H, CH(CH₃)₂), 0.79 (d, J ¼ 6.7 Hz, 6H, CH(CH₃)₂). ¹³

C

NMR (126 MHz, DMSO‑d₆) d 155.6 (C_5’), 153.7 (C_2’), 153.0 (C₅), 131.3

CH₂CH₃). ¹³C NMR (126 MHz, CDCl₃)

d

155.8 (C_5’), 154.3 (C₅), 154.3

(C_7a), 127.1 (C_3a), 123.3 (C₂), 112.1 (C₇), 111.9 (C₃), 111.2 (C₆), 99.7 (C₄),

55.3 (OCH₃), 51.8 (NCH₂), 27.4 (CH(CH₃)₂_t), 26.9 (C_b), 20.8 (C_a), 19.4

(CH(CH₃)₂). HPLC-MS (15:95- g.t.5 min) R 4.77 min, m/z ¼ 316.34

[MþH]^þ, calcd. for [C₁₇H₂₁N₃O₃þH]^þ316.37. HRMS [ESI^þ] m/

z ¼ 315.15958 [M]^þ, calcd. for [C₁₇H₂₁N₃O₃]^þ315.15829.

(C_2’), 131.5 (C_7a), 127.5 (C_3a), 122.5 (C₂), 113.7 (C₃), 112.7 (C₆), 112.1

(C₇), 100.3 (C₄), 56.1 (OCH₃), 45.8 (NCH₂), 28.6 (N(CH₂)₂CH₂), 27.9

(NCH₂CH₂), 27.6 (C_b), 22.3 (CH₂CH₃), 21.6 (C_a), 14.1 (CH₂CH₃). HPLC-

MS (30:95- g.t.5 min) ^tR 4.60 min, (50:95- g.t.5 min) ^tR 3.17 min, m/

z ¼ 330.13 [MþH]^þ, calcd. for [C₁₈H₂₃N₃O₃þH]^þ330.40. HRMS

[ESI^þ] m/z ¼ 329.17515 [M]^þ, calcd. for [C₁₈H₂₃N₃O₃]^þ329.17394.

4.3.10. 3-(Cyclopropylmethyl)-5-[2-(5-methoxy-1H-indol-3-yl)

ethyl]-1,3,4-oxadiazol-2(3H)-one (11)

4.3.6. 3-Hexyl-5-[2-(5-methoxy-1H-indol-3-yl)ethyl]-1,3,4-

oxadiazol-2(3H)-one (7)

Chromatography: DCM. White solid (88% yield) of mp 94e97 ^ꢁC.

¹H NMR (500 MHz, CDCl₃)

d

7.89 (s, 1H, NH), 7.26 (d, J ¼ 8.8 Hz, 1H,

Chromatography: hexane to DCM. Colorless oil (70% yield). ¹H

H₇), 7.02 (d, J ¼ 2.4 Hz, 1H, H₂), 7.00 (d, J ¼ 2.7 Hz, 1H, H₄), 6.87 (dd,

J ¼ 8.8, 2.4 Hz, 1H, H₆), 3.87 (s, 3H, CH₃), 3.52 (d, J ¼ 7.2 Hz, 2H,

NCH₂), 3.13 (q, J ¼ 7.0 Hz, 2H, H_a), 2.96e2.90 (m, 2H, H_b), 1.19e1.09

(m, 1H, CH(CH₂)₂), 0.57e0.51 (m, 2H, CH(CH₂)₂), 0.34 (dt, J ¼ 6.2,

NMR (500 MHz, MeOD)

d

7.20 (d, J ¼ 8.7 Hz, 1H, H₇), 7.03 (bs, 1H,

H₂), 6.95 (d, J ¼ 2.4 Hz, 1H, H₄), 6.74 (dd, J ¼ 8.8, 2.4 Hz, 1H, H₆), 3.82

(s, 3H, OCH₃), 3.57 (t, J ¼ 6.9 Hz, 2H, NCH₂), 3.11 (t, J ¼ 7.2 Hz, 2H,

H_a), 2.92 (t, J ¼ 7.2 Hz, 2H, H_b), 1.56 (p, J ¼ 7.0 Hz, 2H, NCH₂CH₂),

1.30e1.21 (m, 4H, N(CH₂)₃(CH₂)₂CH₃), 1.20e1.10 (m, 2H,

N(CH₂)₂CH₂), 0.88 (t, J ¼ 6.9 Hz, 3H, CH₂CH₃). ¹³C NMR (126 MHz,

4.8 Hz, 2H, CH(CH₂)₂). ¹³C NMR (126 MHz, CDCl₃)

d 155.9 (C_2’), 155.8

(C_5’), 154.3 (C₅), 131.5 (C_7a), 127.5 (C_3a), 122.5 (C₂), 113.8 (C₃), 112.7

(C₆), 112.1 (C₇), 100.3 (C₄), 56.1 (CH₃), 50.5 (NCH₂), 27.5 (C_b), 21.6

(C_a), 10.0 (CH(CH₂)₂), 3.6 (CH(CH₂)₂). HPLC-MS (15:95- g.t.5 min) ^tR

4.57 min, m/z ¼ 314.03 [MþH]^þ, calcd. for [C₁₇H₁₉N₃O₃þH]^þ314.36.

HRMS [ESI^þ] m/z ¼ 313.14324 [M]^þ, calcd. for [C₁₇H₁₉N₃O₃]^þ

313.14264.

MeOD)

d 157.6 (C_5’), 156.0 (C_2’), 155.1 (C₅), 133.3 (C_7a), 128.7 (C_3a),

124.1 (C₂), 113.6 (C₃), 113.0 (C₇), 112.8 (C₆), 100.7 (C₄), 56.2 (OCH₃),

46.4 (NCH₂), 32.3 (N(CH₂)₃CH₂), 28.9 (NCH₂CH₂), 28.7 (C_b), 26.9

(N(CH₂)₂CH₂), 23.5 (CH₂CH₃), 22.4 (C_a), 14.3 (CH₂CH₃). HPLC-MS

t

(50:95- g.t.5 min) R 3.77 min, m/z ¼ 344.38 [MþH]^þ, calcd. for

[C₁₉H₂₅N₃O₃þH]^þ344.43. HRMS [ESI^þ] m/z ¼ 343.18957 [M]^þ,

calcd. for [C₁₉H₂₅N₃O₃]^þ343.18959.

4.3.11. 3-(Cyclobutylmethyl)-5-[2-(5-methoxy-1H-indol-3-yl)

ethyl]-1,3,4-oxadiazol-2(3H)-one (12)

4.3.7. 3-Heptyl-5-[2-(5-methoxy-1H-indol-3-yl)ethyl]-1,3,4-

oxadiazol-2(3H)-one (8)

Chromatography: hexane to DCM. Pale yellow solid (75% yield)

of mp 74e76 ^ꢁC. ¹H NMR (500 MHz, CDCl₃)

d 7.88 (bs, 1H, NH), 7.25

Chromatography: hexane to DCM. Yellow oil (85% yield). ¹H

(d, J ¼ 8.0 Hz, 1H, H₇), 7.00 (d, J ¼ 2.4 Hz, 1H, H₂), 6.99 (d, J ¼ 2.5 Hz,

1H, H₄), 6.87 (dd, J ¼ 8.8, 2.4 Hz, 1H, H₆), 3.87 (s, 3H, CH₃), 3.66 (d,

J ¼ 7.3 Hz, 2H, NCH₂), 3.12 (dd, J ¼ 8.6, 6.7 Hz, 2H, H_a), 2.91 (dd,

J ¼ 8.4, 6.9 Hz, 2H, H_b), 2.68 (p, J ¼ 7.7 Hz, 1H, CH(CH₂)₂), 2.06e1.96

(m, 2H, CH(CH₂)₂), 1.93e1.83 (m, 2H, CH₂(CH₂)₂), 1.81e1.69 (m, 2H,

NMR (300 MHz, CDCl₃)

d

7.95 (s, 1H, NH), 7.25 (d, J ¼ 8.9 Hz, 1H, H₇),

7.02e6.95 (m, 2H, H₂, H₄), 6.86 (dd, J ¼ 8.8, 2.4 Hz, 1H, H₆), 3.87 (s,

3H, OCH₃), 3.63 (t, J ¼ 7.2 Hz, 2H, NCH₂), 3.13 (dd, J ¼ 8.6, 6.3 Hz, 2H,

H_a), 2.92 (dd, J ¼ 8.6, 6.4 Hz, 2H, H_b), 1.67 (p, J ¼ 7.3 Hz, 2H,

NCH₂CH₂), 1.34e1.19 (m, 8H, N(CH₂)₂(CH₂)₄CH₃), 0.88 (t, J ¼ 6.7 Hz,

CH(CH₂)₂). ¹³C NMR (126 MHz, CDCl₃)

d 155.7 (C_5’), 154.5 (C_2’), 154.3

3H, CH₂CH₃). ¹³C NMR (75 MHz, CDCl₃)

d 155.9 (C_5’), 154.3 (C₅),

(C₅), 131.5 (C_7a), 127.5 (C_3a), 122.5 (C₂), 100.3 (C₄), 56.1 (CH₃), 50.6

(NCH₂), 34.3 (CH(CH₂)₂), 27.6 (C_b), 25.7 (CH(CH₂)₂), 21.5 (C_a), 18.3

(CH₂(CH₂)₂). HPLC-MS (50:95- g.t.5 min) ^tR 2.35 min, m/z ¼ 328.37

[MþH]^þ, calcd. for [C₁₈H₂₁N₃O₃þH]^þ328.38. HRMS [ESI^þ] m/

z ¼ 327.15892 [M]^þ, calcd. for [C₁₈H₂₁N₃O₃]^þ327.15829.

154.3 (C_2’), 131.5 (C_7a), 127.5 (C_3a), 122.5 (C₂), 113.7 (C₃), 112.6 (C₆),

112.1 (C₇), 100.3 (C₄), 56.1 (OCH₃), 45.8 (NCH₂), 31.8 (N(CH₂)₄CH₂),

28.9 (N(CH₂)₃CH₂), 28.2 (NCH₂CH₂), 27.6 (C_b), 26.4 (N(CH₂)₂CH₂),

22.7 (CH₂CH₃), 21.6 (C_a), 14.2 (CH₂CH₃). HPLC-MS (70:95- g.t.5 min)

^tR 1.56 min, m/z ¼ 358.25 [MþH]^þ, calcd. for [C₂₀H₂₇N₃O₃þH]^þ

358.45. HRMS [ESI^þ] m/z ¼ 357.2056 [M]^þ, calcd. for [C₂₀H₂₇N₃O₃]^þ

357.20524.

4.3.12. 3-(Cyclopentylmethyl)-5-[2-(5-methoxy-1H-indol-3-yl)

ethyl]-1,3,4-oxadiazol-2(3H)-one (13)

4.3.8. 5-[2-(5-Methoxy-1H-indol-3-yl)ethyl]-3-(propan-2-yl)-

1,3,4-oxadiazol-2(3H)-one (9)

Chromatography: hexane to DCM. White solid (72% yield) of mp

81e84 ^ꢁC. ¹H NMR (500 MHz, CDCl₃)

d 7.88 (bs, 1H, NH), 7.25 (d,

Chromatography: DCM. White solid (81% yield) of mp

J ¼ 8.8 Hz, 1H, H₇), 7.01 (d, J ¼ 2.5 Hz, 1H, H₂), 6.99 (d, J ¼ 2.4 Hz, 1H,

H₄), 6.86 (dd, J ¼ 8.8, 2.4 Hz, 1H, H₆), 3.87 (s, 3H, CH₃), 3.56 (d,

J ¼ 7.4 Hz, 2H, NCH₂), 3.13 (t, J ¼ 7.6 Hz, 2H, H_a), 2.92 (t, J ¼ 7.6 Hz,

2H, H_b), 2.29 (p, J ¼ 7.6 Hz, 1H, CH(CH₂)₂), 1.70e1.59 (m, 4H,

CH(CH₂)₂, H_d, H_c), 1.58e1.50 (m, 2H, CH(CH₂)₂(CH₂)₂), 1.27e1.16 (m,

130e133 ^ꢁC. ¹H NMR (500 MHz, CDCl₃)

d 7.88 (bs, 1H, NH), 7.25 (d,

J ¼ 7.8 Hz, 1H, H₇), 7.01 (d, J ¼ 2.4 Hz, 1H, H₂), 6.99 (d, J ¼ 2.4 Hz, 1H,

H₄), 6.86 (dd, J ¼ 8.8, 2.4 Hz, 1H, H₆), 4.25e4.21 (m, 1H, CH(CH₃)₂),

3.87 (s, 3H, OCH₃), 3.13 (dd, J ¼ 7.6 Hz, 2H, H_a), 2.92 (dd, J ¼ 7.7 Hz,

2H, H_b), 1.30 (d, J ¼ 6.7 Hz, 6H, CH(CH₃)₂). ¹³C NMR (126 MHz,

2H, CH(CH₂)₂). ¹³C NMR (126 MHz, CDCl₃)

d 155.7 (C_5’), 154.5 (C_2’),

CDCl₃)

d

155.8 (C_5’), 154.3 (C₅), 153.6 (C_2’), 131.5 (C_7a), 127.6 (C_3a),

154.3 (C₅), 131.5 (C_7a), 127.5 (C_3a), 122.5 (C₂), 113.7 (C₃), 112.7 (C₆),

112.1 (C₇), 100.3 (C₄), 56.1 (CH₃), 50.5 (NCH₂), 39.0 (CH(CH₂)₂), 30.1

(CH(CH₂)₂), 27.6 (C_b), 25.2 (CH(CH₂)₂(CH₂)₂), 21.6 (C_a). HPLC-MS

122.5 (C₂), 113.9 (C₃), 112.7 (C₆), 112.1 (C₇), 100.3 (C₄), 56.1 (OCH₃),

47.9 (CH(CH₃)₂), 27.7 (C_b), 21.6 (C_a), 20.8 (CH(CH₃)₂). HPLC-MS

t

(15:95- g.t.5 min) R 4.49 min, m/z ¼ 302.35 [MþH]^þ, calcd. for

(50:95- g.t.5 min) R 3.14 min, m/z ¼ 342.20 [MþH]^þ, calcd. for

[C₁₆H₁₉N₃O₃þH]^þ302.35. HRMS [ESI^þ] m/z ¼ 301.14244 [M]^þ,

[C₁₉H₂₃N₃O₃þH]^þ342.41. HRMS [ESI^þ] m/z ¼ 341.17482 [M]^þ, calcd.

calcd. for [C₁₆H₁₉N₃O₃]^þ301.14264.

for [C₁₉H₂₃N₃O₃]^þ341.17394.

C. Herrera-Arozamena et al. / European Journal of Medicinal Chemistry 190 (2020) 112090

15

4.3.13. 3-Benzyl-5-[2-(5-methoxy-1H-indol-3-yl)ethyl]-1,3,4-

oxadiazol-2(3H)-one (14)

H_a, H_b), 1.11 (t, J ¼ 7.2 Hz, 3H, CH₂CH₃). ¹³C NMR (126 MHz,

DMSO‑d₆) d 163.3 (C_2’), 159.3 (C_5’), 153.0 (C₅), 131.3 (C_7a), 127.2 (C_3a),

Chromatography: DCM. White solid (57% yield) of mp

123.2 (C₂), 112.4 (C₃), 112.0 (C₇), 111.2 (C₆), 99.8 (C₄), 55.3 (OCH₃),

37.3 (CH₂CH₃), 25.8 (C_b), 22.0 (C_a), 14.5 (CH₂CH₃). HPLC-MS (15:95-

118e121 ^ꢁC. ¹H NMR (500 MHz, CDCl₃)

d 7.85 (s, 1H, NH), 7.38e7.33

(m, 2H, Ph_o), 7.32e7.30 (m, 1H, Ph_p), 7.30e7.21 (m, 3H, Ph_m, H₇),

6.98 (d, J ¼ 2.4 Hz, 1H, H₄), 6.94 (s, 1H, H₂), 6.87 (dd, J ¼ 8.7, 2.4 Hz,

1H, H₆), 4.81 (s, 2H, NCH₂), 3.86 (s, 3H, CH₃), 3.11 (t, J ¼ 7.6 Hz, 2H,

g.t.5 min) ^tR 3.53 min, m/z

¼

287.11 [MþH]^þ, calcd. for

[C₁₉H₂₅N₃O₃þH]^þ287.34. HRMS [ESI^þ] m/z ¼ 286.14359 [M]^þ,

calcd. for [C₁₅H₁₈N₄O₂]^þ286.14298.

H_a), 2.90 (t, J ¼ 7.6 Hz, 2H, H_b). ¹³C NMR (126 MHz, CDCl₃)

d 156.1

(C_5’), 154.3 (C₅), 154.2 (C_2’), 135.1 (Ph_i), 131.4 (C_7a), 129.0 (Ph_o), 128.4

(Ph_p), 128.3 (Ph_m), 127.4 (C_3a), 122.5 (C₂), 113.6 (C₃), 112.7 (C₆), 112.1

(C₇), 100.2 (C₄), 56.0 (CH₃), 49.5 (NCH₂), 27.6 (C_b), 21.5 (C_a). HPLC-

4.3.17. 5-[1-(5-Methoxy-1H-indol-3-yl)propan-2-yl]-1,3,4-

oxadiazol-2(3H)-one (21)

Entitled compound was prepared in a multi-step synthesis

starting from a mixture of 5-methoxy-1H-indole-3-carbaldehyde

(18, 1.0 g, 5.7 mmol) and (carbethoxyethylidene) triphenylphos-

phorane (1.3 equiv) in DCM (50 mL) that was reﬂuxed overnight.

Solvent was evaporated under reduced pressure to give interme-

diate 19 [68] as a white solid of mp 116e117 ^ꢁC (1.4 g, 95% yield)

after a column chromatography (pure hexane to hexane: EtOAc

t

MS (30:95- g.t.5 min) R 4.23 min, m/z ¼ 350.26 [MþH]^þ, calcd.

for [C₂₀H₁₉N₃O₃þH]^þ350.14. HRMS [ESI^þ] m/z ¼ 349.14257[M]^þ,

calcd. for [C₂₀H₁₉N₃O₃]^þ349.14264.

4.3.14. 5-[2-(5-Methoxy-1H-indol-3-yl)ethyl]-3-(3-phenylpropyl)-

1,3,4-oxadiazol-2(3H)-one (15)

Chromatography: hexane to hexane:DCM 8:2. Pale yellow oil

1:1). Then, the a,b-unsaturated ester 19 (500 mg, 1.93 mmol) in

(92% yield). ¹H NMR (500 MHz, CDCl₃)

d

7.85 (s, 1H, NH), 7.32e7.09

EtOH (20 mL) was treated with a catalytic amount of PdeC (5%)

under N₂atmosphere. N₂was displaced by H₂and the ﬂask was

sealed up with a septum. A balloon with H₂was connected with a

needle to stir at rt overnight. Then, PdeC was removed by ﬁltration

and the solvent was evaporated to dryness under reduced pressure,

obtaining 20 [69] as a white solid (500 mg, > 99% yield), which was

used without further puriﬁcation. A mixture of ester 20 (360 mg,

1.38 mmol) and hydrazine hydrate in EtOH (15 mL) was heated to

150 ^ꢁC for 45 min under MW irradiation. After the solvent was

evaporated, DCM (10 mL) was added to the crude and the organic

layer was washed with saturated aq. solution of NaHCO₃(3x10 mL),

brine (10 mL) and dried over MgSO₄. Solvent was ﬁltered and

evaporated under reduced pressure to obtain the corresponding

hydrazide, which was not isolated but identiﬁed by its HPLC-MS

(m, 6H, H₇, Ph), 6.98 (bs, 2H, H₂, H₄), 6.85 (dd, J ¼ 8.8, 2.5 Hz,1H, H₆),

3.86 (s, 3H, CH₃), 3.68 (t, J ¼ 7.0 Hz, 2H, NCH₂), 3.12 (dd, J ¼ 8.6,

6.4 Hz, 2H, H_a), 2.91 (dd, J ¼ 8.3, 6.4 Hz, 2H, H_b), 2.58 (t, J ¼ 7.6 Hz,

2H, CH₂Ph), 2.00 (p, J ¼ 7.2 Hz, 2H, NCH₂CH₂). ¹³C NMR (125 MHz,

CDCl₃)

d 155.9 (C_5’), 154.3 (C₅), 154.2 (C_2’), 140.9 (Ph_i), 131.5 (C_7a),

128.6 (Ph), 128.5 (Ph), 126.3 (Ph_p), 122.5 (C₂), 113.7 (C₃), 112.7 (C₆),

112.1 (C₇), 100.3 (C₄), 56.1 (CH₃), 45.2 (NCH₂), 32.7 (CH₂Ph), 29.7

(NCH₂CH₂), 27.6 (C_b), 21.5 (C_a). HPLC-MS (50:95- g.t.5 min) ^tR

3.42 min, m/z ¼ 378.31[MþH]^þ, calcd. for [C₂₂H₂₃N₃O₃þH]^þ378.44.

HRMS [ESI^þ] m/z

377.17394.

¼

377.1737[M]^þ, calcd. for [C₂₂H₂₃N₃O₃]^þ

4.3.15. 5-[2-(5-Methoxy-1H-indol-3-yl)ethyl]-N-methyl-1,3,4-

oxadiazol-2-amine (16)

t

data (15:95- g.t.5), R 2.97 min, m/z ¼ 248.17 [MþH]^þ, calcd. for

To a solution of 1 (150 mg, 0.58 mmol) in 5.8 mL of anhydrous

[C₁₃H₁₇N₃O₂þH]^þ248.30. Intermediate hydrazide was reacted with

CDI (1.2 equiv) in anhydrous DMF (10 mL/mmol) under MW-

irradiation at 120 ^ꢁC for 25 min. Solvent was removed and the

residue was dissolved in EtOAc, washed with H₂O (3x10 mL) and

brine (10 mL), dried over MgSO₄, ﬁltered and evaporated to dryness

under reduced pressure. Puriﬁcation was made by column chro-

matography (hexane to hexane:EtOAc 1:1) obtaining 21 (180 mg,

48% yield) as a white solid of mp 90e93 ^ꢁC. ¹H NMR (400 MHz,

DMF, TEA (162

mL,1.16 mmol), BOP (282 mg, 0.64 mmol) and methyl

amine (580 L of a 2M solution in THF) were slowly added. The

m

mixture was stirred at rt overnight and the solvent was removed

under reduced pressure. The crude was extracted with EtOAc (3x10

mL) and washed with H₂O (10 mL) and brine (10 mL). The organic

layer was dried over MgSO₄and the solvent was evaporated. The

residue was puriﬁed by ﬂash chromatography (DCM to DCM:MeOH

9:1) and washed with hexane to afford derivative 16 (29 mg, 18%

yield) as a white solid of mp 145e148 ^ꢁC. ¹H NMR (500 MHz,

CDCl₃)

d 8.40 (bs, 1H, NHCO), 7.91 (bs, 1H, NH_indole), 7.25 (d,

J ¼ 9.0 Hz, 1H, H₇), 6.98 (d, J ¼ 2.4 Hz, 1H, H₂), 6.97 (d, J ¼ 2.4 Hz, 1H,

H₄), 6.86 (dd, J ¼ 8.8, 2.4 Hz, 1H, H₆), 3.87 (s, 3H, OCH₃), 3.19 (dd,

J ¼ 13.7, 6.7 Hz, 1H, H_a), 3.11 (h, J ¼ 6.8 Hz, 1H, H_b), 2.94 (dd, J ¼ 13.8,

7.0 Hz, 1H, H_a), 1.32 (d, J ¼ 6.8 Hz, 3H, CH_bCH₃). ¹³C NMR (101 MHz,

DMSO‑d₆)

d

10.65 (bs, 1H, NH_indole), 7.24 (t, J ¼ 4.9 Hz, 1H, NHCH₃),

7.21 (d, J ¼ 8.9 Hz, 1H, H₇), 7.08 (d, J ¼ 2.5 Hz, 1H, H₂), 6.93 (d,

J ¼ 2.5 Hz, 1H, H₄), 6.70 (dd, J ¼ 8.7, 2.4 Hz, 1H, H₆), 3.75 (s, 3H,

OCH₃), 2.99 (m, 4H, H_a, H_b), 2.75 (d, J ¼ 4.9 Hz, 3H, NHCH₃). ¹³C NMR

CDCl₃)

d 161.4 (C_5’), 155.0 (C_2’), 154.3 (C₅), 131.4 (C_7a), 127.8 (C_3a),

(126 MHz, DMSO‑d₆)

d

164.0 (C_2’), 159.5 (C_5’), 153.0 (C₅), 131.3 (C_7a),

123.4 (C₂), 112.6 (C₆), 112.3 (C₃), 112.1 (C₇), 100.4 (C₄), 56.1 (OCH₃),

t

127.1 (C_3a), 123.2 (C₂), 112.4 (C₃), 112.0 (C₇), 111.2 (C₆), 99.8 (C₄), 55.3

(OCH₃), 29.0 (NHCH₃), 25.8 (C_b), 22.0 (C_a). HPLC-MS (15:95-

33.7 (C_b), 29.4 (C_a), 16.9 (CH_bCH₃). HPLC-MS (40:95- g.t.10 min) R

1.25 min, m/z ¼ 274.22 [MþH]^þ, calcd. for [C₁₄H₁₅N₃O₃þH]^þ274.29.

HRMS [ESI^þ] m/z ¼ 273.11073 [M]^þ, calcd. for [C₁₄H₁₅N₃O₃]^þ

273.11134.

g.t.5 min) ^tR 3.17 min, m/z

¼

273.34 [MþH]^þ, calcd. for

[C₁₄H₁₆N₄O₂þH]^þ273.31. HRMS [ESI^þ] m/z ¼ 272.12816 [M]^þ, calcd.

for [C₁₄H₁₆N₄O₂]^þ272.12733.

4.3.18. 5-[(E)-2-(5-Methoxy-1H-indol-3-yl)ethenyl]-1,3,4-

4.3.16. N-Ethyl-5-[2-(5-methoxy-1H-indol-3-yl)ethyl]-1,3,4-

oxadiazol-2-amine (17)

oxadiazol-2(3H)-one (23)

Derivative 23 was prepared in

a multi-step synthesis as

Ethyl derivative 17 was synthesized from 1 following the same

procedure as for 16, but using ethyl amine. In this case, a column

chromatography (DCM to DCM:MeOH 9:1) and a semi-preparative

HPLC separation (20:80 g t. 60 min, isocratic gradient) were

necessary to puriﬁed 17 (6 mg, 6% yield). ¹H NMR (500 MHz,

explained below. To a solution of the commercial aldehyde 18

(500 mg, 2.8 mmol) in pyridine (20 mL), malonic acid (2 equiv) and

piperidine (13 mL/mmol) were added and the mixture was stirred at

70 ^ꢁC overnight under N₂atmosphere. Solvent was evaporated

under reduced pressure and EtOAc was added. The organic layer

was washed with H₂O (3x10 mL) and 10% citric acid solution

(10 mL), dried over MgSO₄, ﬁltered, and evaporated to dryness, to

give 22 (97% yield) as a white solid of mp 208e211 ^ꢁC (bibl [70].

DMSO‑d₆)

d

10.65 (bs, 1H, NH_indole), 7.32 (t, J ¼ 5.6 Hz, 1H, NHCH₂),

7.21 (dd, J ¼ 8.7, 0.5 Hz, 1H, H₇), 7.09 (d, J ¼ 2.3 Hz, 1H, H₂), 6.94 (d,

J ¼ 2.4 Hz, 1H, H₄), 6.70 (dd, J ¼ 8.7, 2.4 Hz, 1H, H₆), 3.75 (s, 3H,

OCH₃), 3.15 (qd, J ¼ 7.2, 5.6 Hz, 2H, CH₂CH₃), 3.04e2.95 (m, 4H, 4H,

197e200 ^ꢁC). Then, to a suspension of the

a,b-unsaturated acid 22

16

C. Herrera-Arozamena et al. / European Journal of Medicinal Chemistry 190 (2020) 112090

(500 mg, 2.30 mmol) and activated 4 Å molecular sieves in anhy-

drous ACN (40 mL) at rt under N₂atmosphere, HOBt (1.2 equiv),

EDC$HCl (1.2 equiv) and DMAP (0.12 equiv) were orderly added.

The mixture was stirred for 3 h until the complete acid activation

and then, N₂H₄$H₂O (excess) was added. Once ﬁnished the reac-

tion, H₂O was added, extracted with DCM and washed with satu-

rated NaHCO₃(aq). The organic layer was separated and evaporated

under reduced pressure to obtain the corresponding N-acylhy-

drazine, which was not isolated but identiﬁed by HPLC-MS (15:95-

CH₃), 3.16 (t, J ¼ 2.6 Hz, 1H, ≡CH). ¹³C NMR (126 MHz, DMSO‑d₆)

d

166.1 (CO), 154.5 (C₅), 133.9 (C_a), 132.3 (C_7a), 131.0 (C₂), 125.3 (C_3a),

114.7 (C_b), 112.8 (C₇), 111.8 (C₆), 109.5 (C₃), 102.5 (C₄), 81.5 (≡C), 73.1

(≡CH), 55.5 (CH₃), 27.8 (CH₂). HPLC-MS (15:95- g.t.10 min) ^tR

5.80 min, m/z ¼ 255.07 [MþH]^þ, calcd. for [C₁₅H₁₄N₂O₂þH]^þ

255.29. HRMS [ESI^þ] m/z

¼

254.10667 [M]^þ, calcd. for

[C₁₅H₁₄N₂O₂]^þ254.10553.

4.3.21. (2E)-3-(5-Methoxy-1H-indol-3-yl)-N-(prop-2-en-1-yl)

prop-2-enamide (26)

Similarly, to the synthesis of 25 but using allylamine, derivative

26 was obtained (72% yield) as a pale yellow solid of mp

124e126 ^ꢁC. Chromatography: hexane to hexane:EtOAc 3:7. ¹H

g.t.5), ^tR 1.99 min, m/z

¼

232.39 [MþH]^þ, calcd. for

[C₁₂H₁₃N₃O₂þH]^þ232.26. Intermediate hydrazide (525 mg,

2.28 mmol) was reacted with CDI (1.2 equiv) in anhydrous DMF

(20 mL) under MW-irradiation at 120 ^ꢁC for 25 min. Solvent was

removed and the residue was dissolved in EtOAc (10 mL), washed

with H₂O (3x10 mL) and brine (10 mL), dried over MgSO₄, ﬁltered

and evaporated to dryness under reduced pressure. Puriﬁcation

was made by column chromatography (hexane to hexane:EtOAc

1:1) obtaining 23 (502 mg, 1.95 mmol, 85% yield) as a white solid of

NMR (500 MHz, CDCl₃)

d

8.77 (s, 1H, NH_indole), 7.87 (d, J ¼ 15.5 Hz,

1H, H_a), 7.40 (d, J ¼ 2.8 Hz, 1H, H₂), 7.30 (d, J ¼ 2.6 Hz, 1H, H₄), 7.30

(d, J ¼ 8.7 Hz, 1H, H₇), 6.90 (dd, J ¼ 8.9, 2.3 Hz, 1H, H₆), 6.35 (d,

J ¼ 15.6 Hz,1H, H_b), 5.92 (ddt, J ¼ 17.1,10.1, 5.6 Hz,1H, CH ¼ ), 5.72 (t,

J ¼ 6.4 Hz, 1H, CONH), 5.25 (dd, J ¼ 17.2, 1.5 Hz, 1H, CH_2trans¼ ), 5.17

(dd, J ¼ 10.3, 1.5 Hz, 1H, CH_2cis¼ ), 4.05 (tt, J ¼ 5.8, 1.6 Hz, 2H, CH₂),

mp 206e209 ^ꢁC. ¹H NMR (500 MHz, DMSO‑d₆)

d 11.53 (bs, 1H,

NH_indole), 7.85 (d, J ¼ 2.8 Hz, 1H, H₂), 7.49 (d, J ¼ 16.4 Hz, 1H, H_a),

7.34 (d, J ¼ 2.7 Hz, 1H, H₄), 7.34 (d, J ¼ 8.5 Hz, 1H, H₇), 6.82 (dd,

J ¼ 8.8, 2.3 Hz, 1H, H₆), 6.59 (d, J ¼ 16.4 Hz, 1H, H_b), 3.82 (s, 3H, CH₃).

3.87 (s, 3H, CH₃). ¹³C NMR (126 MHz, CDCl₃)

d 167.3 (CO), 155.3 (C₅),

135.1 (C_a), 134.6 (CH ¼ ), 132.2 (C_7a), 128.7 (C₂), 126.2 (C_3a), 116.6

(¼CH₂), 115.2 (C_b), 113.3 (C₃), 112.9 (C₆), 112.6 (C₇), 102.8 (C₄), 56.2

(CH₃), 42.3 (CH₂). HPLC-MS (15:95- g.t.10 min) ^tR 6.12 min, m/

z ¼ 257.23 [MþH]^þ, calcd. for [C₁₅H₁₆N₂O₂þH]^þ257.31. HRMS

[ESI^þ] m/z ¼ 256.12237 [M]^þ, calcd. for [C₁₅H₁₆N₂O₂]^þ256.12118.

¹³C NMR (126 MHz, DMSO‑d₆)

d 155.4 (C_5’), 154.6 (C₅), 154.4 (C_2’),

132.0 (C_7a), 131.1 (C_a), 129.7 (C₂), 125.5 (C_3a), 112.9 (C₇), 112.3 (C₆),

111.9 (C₃), 103.9 (C_b), 101.5 (C₄) 55.5 (CH₃). HPLC-MS (15:95-

g.t.10 min) ^tR 6.04 min, m/z

¼

258.32 [MþH]^þ, calcd. for

[C₁₃H₁₁N₃O₃þH]^þ258.25. HRMS [ESI^þ] m/z ¼ 257.08107 [M]^þ,

4.3.22. 6-Methoxy-4-methyl-1,2-dihydronaphthalene (28)

calcd. for [C₁₃H₁₁N₃O₃]^þ257.08004.

To a solution of 7-methoxy-1-tetralone (200 mg, 1.13 mmol) in

5 mL of Et₂O, CH₃MgI (1.3 equiv) was added dropwise at rt. The

mixture was stirred for 3 h, washed with a saturated solution of

NH₄Cl and extracted with Et₂O (3x10 mL), obtaining the interme-

diate alcohol. Then, a 2 M HCl solution was added dropwise until

pH 5 and the mixture was heated at 40 ^ꢁC for 3 h. The layers were

separated, and the organic layer was washed with a solution of

Na₂S₂O₃, and brine; dried over MgSO₄, ﬁltrated and the solvent was

removed under reduced pressure. The crude was puriﬁed by ﬂash

chromatography in hexane, obtaining 28 [34] (191 mg, 1.10 mmol,

97% yield) as a colorless oil.

4.3.19. 5-[(E)-2-(5-Methoxy-1H-indol-3-yl)ethenyl]-3-(prop-2-yn-

1-yl)-1,3,4-oxadiazol-2(3H)-one (24)

Alkylation of 23 (60 mg, 0.23 mmol) with propargyl bromide

(1.2 equiv) in the presence of K₂CO₃(1.2 equiv) under MW-

irradiation at 120 ^ꢁC for 10 min, using acetone (7 mL/mmol) as

solvent, gave 24 (12 mg, 17% yield) as a white solid of mp

201e203 ^ꢁC. Puriﬁcation by semipreparative HPLC (gradient 35:40-

g.t.30 min). ¹H NMR (400 MHz, DMSO‑d₆)

d 11.59 (s, 1H, NH), 7.88

(d, J ¼ 2.5 Hz, 1H, H₂), 7.56 (d, J ¼ 16.4 Hz, 1H, H_a), 7.37 (d, J ¼ 2.4 Hz,

1H, H₄), 7.35 (d, J ¼ 8.8 Hz, 1H, H₇), 6.83 (dd, J ¼ 8.7, 2.4 Hz, 1H, H₆),

6.63 (d, J ¼ 16.4 Hz, 1H, H_b), 4.58 (d, J ¼ 2.6 Hz, 2H, CH₂), 3.83 (s, 3H,

CH₃), 3.45 (t, J ¼ 2.5 Hz, 1H, ≡CH). ¹³C NMR (101 MHz, DMSO‑d₆)

4.3.23. Oxidation reaction of 28 with selenium oxide

To compound 28 (238 mg, 1.37 mmol) in EtOH (5 mL), a solution

of SeO₂(5 equiv) in EtOH:H₂O (15 mL, 10:1) was added dropwise

(35 min addition). The mixture was heated at reﬂux overnight and

then, it was extracted with EtOAc (3x10 mL) and washed with a

saturated solution of NaHCO₃(10 mL) and brine (10 mL). The

organic layer was dried over MgSO₄, ﬁltered and concentrated

under reduced pressure. The crude was puriﬁed by column chro-

matography (hexane to hexane:EtOAc 6:4) to afford 7-

methoxynaphthalene-1-carbaldehyde (30) [35] as a colorless oil

(8 mg, 20% yield) and 7-methoxy-3,4-dihydronaphthalene-1-

carbaldehyde (29) (64 mg, 25% yield) as a brown oil. ¹H NMR

d

154.7 (C₅), 154.3 (C_5’), 152.1 (C_2’), 132.3 (C_a), 132.1 (C_7a), 130.4 (C₂),

125.4 (C_3a), 113.0 (C₇), 112.5 (C₆), 111.9 (C₃), 102.9 (C_b), 101.6 (C₄),

77.3 (≡C), 76.0 (≡CH), 55.5 (CH₃), 35.2 (CH₂). HPLC-MS (15:95-

g.t.10 min) ^tR 7.63 min, m/z

¼

296.25 [MþH]^þ, calcd. for

[C₁₆H₁₃N₃O₃þH]^þ296.30. HRMS [ESI^þ] m/z ¼ 270.10827 [M]^þ,

calcd. for [C₁₆H₁₃N₃O₃]^þ270.10044.

4.3.20. (2E)-3-(5-Methoxy-1H-indol-3-yl)-N-(prop-2-yn-1-yl)

prop-2-enamide (25)

Under N₂atmosphere, to a solution of the acid 22 (100 mg,

0.46 mmol) in anhydrous ACN (15 mL/mmol) in the presence of

activated 4 Å molecular sieves, HOBt (1.2 equiv), EDC$HCl (1.2

equiv) and DMAP (0.12 equiv) were orderly added at rt. The mixture

was stirred for 3 h and then, propargylamine (1.2 equiv) was added.

Afterward, H₂O was added, extracted with DCM (3x10 mL) and

washed with saturated NaHCO₃aq (10 mL). The organic layer was

separated, dried with MgSO₄and solvent evaporated under

reduced pressure. Residue was puriﬁed by column chromatography

(hexane to hexane:EtOAc 1:1), obtaining 25 as an oil (60 mg,

(400 MHz, CDCl₃)

d

9.68 (s, 1H, COH), 7.87 (d, J ¼ 2.7 Hz,1H, H₈), 7.10

(d, J ¼ 8.5 Hz, 1H, H₅), 7.04 (t, J ¼ 4.7 Hz, 1H, H₂), 6.80 (dd, J ¼ 8.3,

2.7 Hz, 1H, H₆), 3.83 (s, 3H, CH₃), 2.76 (t, J ¼ 7.9 Hz, 2H, H₄),

2.61e2.54 (m, 2H, H₃). ¹³C NMR (101 MHz, CDCl₃)

d 192.7 (CO),

158.4 (C₇), 153.7 (C₂), 138.0 (C₁), 130.3 (C_8a), 128.4 (C₅), 127.8 (C_4a),

114.2 (C₆), 111.4 (C₈), 55.5 (CH₃), 26.3 (C₄), 24.8 (C₃). HPLC-MS

t

(50:95- g.t.10 min) R 2.46 min, m/z ¼ 189.39 [MþH]^þ, calcd. for

[C₁₂H₁₂O₂þH]^þ189.23.

0.23 mmol, 51% yield). ¹H NMR (500 MHz, DMSO‑d₆)

d

11.44 (s, 1H,

4.3.24. 9-Methoxy-2,4a,5,6-tetrahydro-3H-naphtho[2,1-b]pyran-3-

one (32)

NH_indole), 8.29 (t, J ¼ 5.5 Hz, 1H, NH_amide), 7.72 (d, J ¼ 2.9 Hz, 1H, H₂),

7.62 (d, J ¼ 15.8 Hz, 1H, H_a), 7.34 (d, J ¼ 8.8 Hz, 1H, H₇), 7.34 (d,

J ¼ 1.9 Hz, 1H, H₄), 6.85 (dd, J ¼ 8.7, 2.5 Hz, 1H, H₆), 6.53 (d,

J ¼ 15.8 Hz, 1H, H_b), 4.01 (dd, J ¼ 5.5, 2.5 Hz, 2H, CH₂), 3.84 (s, 3H,

To a solution of aldehyde 29 (280 mg, 1.49 mmol) in pyridine

(7 mL/mmol), malonic acid (2 equiv) and piperidine (13

mL/mmol)

were added and the mixture was stirred at 70 ^ꢁC overnight under

C. Herrera-Arozamena et al. / European Journal of Medicinal Chemistry 190 (2020) 112090

17

N₂atmosphere. Solvent was evaporated under reduced pressure

and EtOAc (10 mL) was added. The organic layer was washed with

H₂O (10 mL), 10% citric acid solution (10 mL), dried over MgSO₄,

ﬁltered, and evaporated to dryness, obtaining lactone 32 as a white

(C₁), 135.1 (C₂), 134.8 (C_8a), 129.9 (C_4a), 129.6 (C₅), 120.0 (C_b), 113.3

(C₆), 111.4 (C₈), 61.7 (OCH₂), 55.8 (OCH₃), 27.9 (C₄), 25.0 (C₃), 14.6

t

(CH₂CH₃). HPLC-MS (50:95- g.t.10 min) R 5.38 min, m/z ¼ 259.11

[MþH]^þ, calcd. for [C₁₆H₁₈O₃þH]^þ259.32.

solid of mp 96e98 ^ꢁC (23% yield). ¹H NMR (500 MHz, CDCl₃)

d 7.10

(d, J ¼ 2.7 Hz, 1H, H₁₀), 7.07 (d, J ¼ 8.4 Hz, 1H, H₇), 6.81 (dd, J ¼ 8.4,

2.6 Hz, 1H, H₈), 6.37 (dt, J ¼ 5.6, 2.7 Hz, 1H, H₁), 5.15 (dtd, J ¼ 13.7,

4.4, 1.6 Hz, 1H, H_4a), 3.82 (s, 3H, CH₃), 3.36 (ddd, J ¼ 20.6, 5.9, 1.5 Hz,

1H, H₂), 3.21 (ddd, J ¼ 20.7, 4.4, 2.8 Hz, 1H, H₂), 2.91e2.75 (m, 2H,

H₆), 2.42 (dq, J ¼ 12.4, 4.3 Hz, 1H, H₅), 1.99 (tddd, J ¼ 12.2, 11.4, 5.0,

4.3.27. 6,7-Dimethoxy-3,4-dihydronaphthalen-1-yl

triﬂuoromethanesulfonate (37)

Tf₂O (1.1 equiv) and 2-chloropyridine (1.1 equiv) were added to a

solution of commercial 6,7-dimethoxy-1-tetralone (550 mg,

2.67 mmol) in 10 mL of dry DCM at rt, under inert atmosphere.

After 2 h of reaction, the solvent was evaporated and the crude was

puriﬁed by ﬂash chromatography (hexane to hexane:EtOAc 75:25),

obtaining 301 mg (33% yield) of 37 as a colorless oil [71].

0.6 Hz, 1H, H₅). ¹³C NMR (126 MHz, CDCl₃)

d 170.2 (C₃), 158.5 (C₉),

134.5 (C_1b), 131.8 (C_10a), 130.0 (C₇), 128.7 (C_6a), 114.8 (C₈), 114.7 (C₁),

108.0 (C₁₀), 77.7 (C_4a), 55.5 (CH₃), 31.8 (C₂), 29.6 (C₅), 26.9 (C₆).

t

HPLC-MS (50:95- g.t.10 min) R 1.75 min, m/z ¼ 231.17 [MþH]^þ,

calcd. for [C₁₄H₁₄O₃þH]^þ231.26.

4.3.28. 7-Methoxynaphthalen-1-yl triﬂuoromethanesulfonate (38)

To a solution of 34 (165 mg, 0.53 mmol) in DCM (5 mL/mmol), a

solution of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ, 1.2

equiv) in DCM (5 mL/mmol) was added. The mixture was stirred for

10 min and washed with a saturated solution of NaHCO₃(3x10 mL)

and brine (10 mL). Organic layer was dried over MgSO₄, ﬁltrated

and the solvent was removed under reduced pressure. The crude

was puriﬁed by column chromatography (hexane), giving 38 as a

colorless oil (142 mg, 88% yield) [72].

4.3.25. 5-[(E)-2-(7-Methoxy-3,4-dihydronaphthalen-1-yl)ethenyl]-

1,3,4-oxadiazol-2(3H)-one (33)

Lactone 32 (70 mg, 0.30 mmol) was treated with HOBt (1.2

equiv), EDC$HCl (1.2 equiv) and DMAP (0.12 equiv) in anhydrous

ACN (15 mL) in the presence of activated 4 Å molecular sieves at rt

for 3 h. Then, N₂H₄$H₂O (excess) was added to give the corre-

sponding hydrazide, which was not isolated but identiﬁed by HPLC-

MS. Intermediate hydrazide was treated with CDI (1.2 equiv) in

anhydrous DMF (10 mL) under MW-irradiation at 120 ^ꢁC for 25 min.

Solvent was removed and the residue was dissolved in EtOAc,

washed with H₂O and brine, dried over MgSO₄, ﬁltered and evap-

orated to dryness under reduced pressure. Puriﬁcation was made

by column chromatography (hexane to hexane:EtOAc 3:7) obtain-

ing 33 (15% yield) as a white solid of mp 133e136 ^ꢁC. ¹H NMR

4.3.29. 6,7-Dimethoxynaphthalen-1-yl triﬂuoromethanesulfonate

(39)

Similarly to the synthesis of 38 but using 37 as starting material,

the 6,7-dimethoxynaphthalene derivative 39 [72] was obtained as a

colorless oil (86% yield), after a column chromatography (hexane to

hexane:EtOAc 9:1).

(400 MHz, MeOD)

d

7.11 (d, J ¼ 8.4 Hz, 1H, H₅), 7.09 (dd, J ¼ 16.2,

1.2 Hz, 1H, H_a), 6.91 (d, J ¼ 2.6 Hz, 1H, H₈), 6.75 (dd, J ¼ 8.2, 2.6 Hz,

1H, H₆), 6.50 (d, J ¼ 16.2 Hz, 1H, H_b), 6.45 (t, J ¼ 4.8 Hz, 1H, H₂), 3.78

4.3.30. Ethyl (2E)-3-(7-methoxynaphthalen-1-yl)prop-2-enoate

(40)

(s, 3H, CH₃), 2.66 (t, J ¼ 7.7 Hz, 2H, H₄), 2.35e2.27 (m, 2H, H₃). ¹³

C

To a solution of triﬂate 38 (220 mg, 1.70 mmol) in anhydrous

DMF (5 mL/mmol), 1,10-phenanthroline (5.5% mol), Pd(OAc)₂(5.5%

mol), TEA (1.2 equiv) and ethyl acrylate (5 equiv) were successively

added. The reaction was heated under MW irradiation at 150 ^ꢁC for

1 h. The solvent was evaporated and the product was puriﬁed by

column chromatography (hexane to hexane:EtOAc 95:5), giving

ester 40 [73] (390 mg, 1.52 mmol, 90% yield) as a white solid of mp

76e78 ^ꢁC.

NMR (126 MHz, CDCl₃) d 158.5 (C₇),155.6 (C_5’),153.9 (C_2’),137.5 (C_a),

134.7 (C₁), 133.7 (C_8a), 133.1 (C₂), 128.9 (C_4a), 128.8 (C₅), 112.4 (C₆),

111.3 (C_b), 110.7 (C₈), 55.6 (CH₃), 27.1 (C₄), 24.1 (C₃). HPLC-MS

t

(50:95- g.t.10 min) R 4.72 min, m/z ¼ 271.10 [MþH]^þ, calcd. for

[C₁₅H₁₄N₂O₃þH]^þ271.29. HRMS [ESI^þ] m/z ¼ 270.10827 [M]^þ,

calcd. for [C₁₅H₁₄N₂O₃]^þ270.10044.

4.3.26. Ethyl (2E)-3-(7-Methoxy-3,4-dihydronaphthalen-1-yl)

prop-2-enoate (35)

4.3.31. Ethyl (2E)-3-(6,7-dimethoxynaphthalen-1-yl)prop-2-enoate

(41)

Under inert atmosphere, Tf₂O (0.6 mL, 3.31 mmol) and 2-

chloropyridine (0.3 mL, 3.31 mmol) were added to a solution of

commercial 7-methoxy-1-tetralone (507 mg, 2.88 mmol) in 7 mL of

dry DCM at rt. After 2 h the reaction was ﬁnished and the solvent

was evaporated to dryness. The crude was puriﬁed by ﬂash chro-

matography on hexane, obtaining the triﬂate 34 [36] as colorless oil

(697 mg, 78% yield). Intermediate 34 (120 mg, 0.39 mmol) was

solved in 1 mL of anhydrous DMF under N₂atmosphere and a so-

Similarly to the synthesis of 40, but using the triﬂate derivative

39 as reactant, the

yield as a white solid of mp 89e90 ^ꢁC. Chromatography: hexane to

hexane:EtOAc 85:15. ¹H NMR (400 MHz, CDCl₃)

8.44 (d,

a,b-unsaturated ester 41 was obtained in 81%

d

J ¼ 15.7 Hz, 1H, H_a), 7.74 (d, J ¼ 8.1 Hz, 1H, H₄), 7.62 (d, J ¼ 7.4 Hz, 1H,

H₂), 7.39 (s, 1H, H₈), 7.34 (t, J ¼ 7.7 Hz, 1H, H₃), 7.14 (s, 1H, H₅), 6.52

(d, J ¼ 15.7 Hz, 1H, H_b), 4.32 (q, J ¼ 7.1 Hz, 2H, CH₂), 4.05 (s, 3H,

lution of ethyl acrylate (93

mL, 0.86 mmol, 2.2 equiv), TEA (189

m

L,

CH₃OC₇), 4.01 (s, 3H, CH₃OC₆), 1.37 (t, J ¼ 7.1 Hz, 3H, CH₂CH₃). ¹³

C

1.36 mmol, 3.5 equiv) and Pd(PPh₃)₂Cl₂(6 mg, 2.2 mol %) in 0.5 mL

of DMF was added. Reaction was heated under MW irradiation at

105 ^ꢁC for 15 min. DMF was evaporated to dryness and the residue

was extracted with ether (3x10 mL). Organic layer was washed with

H₂O (3x10 mL), dried over MgSO₄and ﬁltered. Crude was puriﬁed

by preparative TLC in hexane:EtOAc 8:2 to afford unsaturated ester

35 as a white solid of mp: 72e74 ^ꢁC (46 mg, 50% yield). ¹H NMR

NMR (101 MHz, CDCl₃) d 167.3 (CO),150.4 (C₇), 149.7 (C₆), 142.1 (C_a),

130.4 (C₁), 129.9 (C_4a), 129.0 (C₄), 127.6 (C_8a), 124.1 (C₃), 123.6 (C₂),

120.5 (C_b), 107.1 (C₅), 102.4 (C₈), 60.7 (CH₂), 56.2 (CH₃OC₇), 56.0

(CH₃OC₆), 14.5 (CH₂CH₃). HPLC-MS (50:95- g.t.10 min) ^tR 3.50 min,

m/z ¼ 287.27 [MþH]^þ, calcd. for [C₁₇H₁₈O₄þH]^þ287.33.

4.3.32. (2E)-3-(7-Methoxynaphthalen-1-yl)prop-2-enoic acid (42)

LiOH (1.3 equiv) was added to a solution of 40 (100 mg,

0.39 mmol) in THF:H₂O 1:1 (10 mL) and the mixture was stirred at

rt overnight. The solution was adjusted to pH ¼ 1 by addition of 1 M

HCl. The product was precipitated, washed with H₂O until neutral

pH, centrifuging to avoid losing product, and lyophilized to remove

water traces to obtain acid 42 [73] (889 mg, 0.39 mmol, >99% yield)

(400 MHz, MeOD)

d

7.60 (d, J ¼ 16.0 Hz, 1H, H_a), 7.12 (d, J ¼ 8.3 Hz,

1H, H₅), 6.87 (d, J ¼ 2.6 Hz, 1H, H₈), 6.77 (dd, J ¼ 8.2, 2.7 Hz, 1H, H₆),

6.56 (t, J ¼ 5.0 Hz, 1H, H₂), 6.26 (d, J ¼ 15.9 Hz, 1H, H_b), 4.24 (q,

J ¼ 7.2 Hz, 2H, OCH₂), 3.78 (s, 3H, OCH₃), 2.66 (t, J ¼ 7.8 Hz, 2H, H₄),

2.33 (td, J ¼ 7.6, 4.7 Hz, 2H, H₃), 1.32 (t, J ¼ 7.1 Hz, 3H, CH₂CH₃). ¹³

C

NMR (101 MHz, MeOD)

d 168.8 (CO), 159.9 (C₇), 144.7 (C_a), 135.7

18

C. Herrera-Arozamena et al. / European Journal of Medicinal Chemistry 190 (2020) 112090

as a white solid of mp 212e215 ^ꢁC.

using hMT₁R and hMT₂R stably transfected in Chinese hamster

ovary cells (catalog refs. 1538 and 1687, respectively) [37,38]. In the

4.3.33. (2E)-3-(6,7-Dimethoxynaphthalen-1-yl)prop-2-enoic acid

(43)

In the same way to the synthesis of 42, from ester 41 (150 mg,

0.52 mmol) acid 43 was obtained in quantitative yield (134 mg,

0.52 mmol), as a white solid of mp 220e222 ^ꢁC. ¹H NMR (500 MHz,

case of hMT₁R cell membrane homogenates (about 80 mg protein)

are incubated for 240 min at 22 ^ꢁC with 0.01 nM [¹²⁵I]iodomela-

tonin in the absence or presence of the test compound in a buffer

containing 50 mM Tris-HCl (pH 7.4), 5 mM MgCl₂and 0.1% BSA. For

hMT₂R cell membrane homogenates (6 mg protein) are incubated

MeOD)

d

8.45 (d, J ¼ 15.7 Hz,1H, H_a), 7.84 (d, J ¼ 8.1 Hz,1H, H₄), 7.75

for 120 min at 37 ^ꢁC with 0.05 nM [¹²⁵I]iodomelatonin in the

absence or presence of the test compound in a buffer containing

50 mM Tris-HCl (pH 7.4) and 5 mM MgCl₂.

(d, J ¼ 7.0 Hz, 1H, H₂), 7.53 (s, 1H, H₈), 7.38 (t, J ¼ 7.9 Hz, 1H, H₃), 7.36

(s, 1H, H₅), 6.57 (d, J ¼ 15.7 Hz, 1H, H_b), 4.02 (s, 3H, CH₃OC₇), 3.96 (s,

3H, CH₃OC₆). ¹³C NMR (126 MHz, MeOD)

d

167.8 (CO₂H), 151.6 (C₇),

In both cases, nonspeciﬁc binding is determined in the presence

151.0 (C₆), 142.6 (C_a), 131.0 (C₁), 130.9 (C_4a), 129.8 (C₄), 128.3 (C_8a),

124.6 (C₃),124.1 (C₂),121.2 (C_b),108.1 (C₅),103.0 (C₈), 56.0 (CH₃OC₇),

55.9 (CH₃OC₆). HPLC-MS (50:95- g.t.10 min) ^tR 1.62 min, it does not

ionize.

of 1 mM melatonin. Following incubation, the samples are ﬁltered

rapidly under vacuum through glass ﬁbre ﬁlters (GF/B, Packard)

presoaked with 0.3% PEI and rinsed several times with ice-cold

50 mM Tris-HCl using a 96-sample cell harvester (Uniﬁlter, Pack-

ard). The ﬁlters are dried then counted for radioactivity in a scin-

tillation counter (Topcount, Packard) using a scintillation cocktail

(Microscint 0, Packard).

4.3.34. 5-[(E)-2-(7-Methoxynaphthalen-1-yl)ethenyl]-1,3,4-

oxadiazol-2(3H)-one (44)

Acid 42 (230 mg, 1.01 mmol) was transformed into the corre-

sponding hydrazide by treatment with HOBt (1.2 equiv), EDC$HCl

(1.2 equiv) and DMAP (0.12 equiv) in anhydrous ACN (10 mL) at rt

for 3 h and then, with N₂H₄$H₂O. The corresponding acylhydrazine

was not isolated, but identiﬁed by HPLC-MS (50:95- g.t.10 min and

4.4.2. Binding assays in quinone reductase-2

Evaluation of the afﬁnity of compounds for QR2 in the hamster

brain was performed by a radioligand binding assay (Euroﬁns-

CEREP, France, catalog ref. 0088) [39]. Membrane homogenates of

2:30- g.t.10 min), m/z

¼

243.11 [MþH]^þ, calcd. for

brain (750

¹²⁵I]iodomelatonin in the absence or presence of the test com-

pound in a buffer containing 50 mM Tris-HCl (pH 7.4) and 4 mM

CaCl₂. Nonspeciﬁc binding is determined in the presence of 30

m

g protein) are incubated for 60 min at 4 ^ꢁC with 0.1 nM

[C₁₄H₁₄N₂O₂þH]^þ243.28. Intermediate hydrazide was treated with

CDI (1.2 equiv) in anhydrous DMF (10 mL/mmol) under MW-

irradiation at 130 ^ꢁC for 25 min. Solvent was removed and the

residue was dissolved in EtOAc (10 mL), washed with H₂O (3x10

mL) and brine (10 mL), dried over MgSO₄, ﬁltered and evaporated to

dryness under reduced pressure. Puriﬁcation was made by column

chromatography (hexane to hexane:EtOAc 6:4) obtaining 44

(245 mg, 91% yield) as a white solid of mp 207e208 ^ꢁC. ¹H NMR

[

m

M

melatonin. Following incubation, the samples are ﬁltered rapidly

under vacuum through glass ﬁber ﬁlters (Filtermat B, Wallac)

presoaked with 0.5% PEI (pH 7.4) and rinsed several times with an

ice-cold buffer containing 50 mM Tris-HCl and 2 M NaCl using a 48-

sample cell harvester (Mach II, Tomtec). The ﬁlters are dried then

counted for radioactivity in a scintillation counter (Betaplate 1204,

Wallac) using a solid scintillator (Meltilex B/HS, Wallac).

(500 MHz, MeOD)

d

8.10 (d, J ¼ 16.1 Hz, 1H, H_a), 7.84 (d, J ¼ 8.4 Hz,

1H, H₄), 7.82 (d, J ¼ 9.0 Hz, 2H, H₅, H₂), 7.45 (d, J ¼ 2.5 Hz, 1H, H₈),

7.37 (dd, J ¼ 8.0, 7.4 Hz,1H, H₃), 7.20 (dd, J ¼ 9.0, 2.4 Hz,1H, H₆), 6.83

(d, J ¼ 16.1 Hz, 1H, H_b), 3.98 (s, 3H, CH₃). ¹³C NMR (126 MHz, MeOD)

4.4.3. Methodology and expression of the results of binding assays

in hMT₁R, hMT₂R and QR2

d

160.0 (C₇), 156.6 (C_5’), 135.4 (C_a), 133.9 (C_8a), 132.1 (C₁), 131.4 (C₅),

131.0 (C₄), 130.8 (C_4a), 126.1 (C₂), 124.3 (C₃), 119.9 (C₆), 113.5 (C_b),

102.5 (C₈), 55.9 (CH₃). HPLC-MS (50:95- g.t.10 min) ^tR 1.83 min, m/

z ¼ 269.16 [MþH]^þ, calcd. for [C₁₅H₁₂N₂O₃þH]^þ269.27. HRMS

[ESI^þ] m/z ¼ 268.08547 [M]^þ, calcd. for [C₁₅H₁₂N₂O₃]^þ268.08479.

Radioligand displacements were measured at a ﬁxed concen-

tration of compound in each receptor subtype in 3 independent

experiments. Then, compounds displaying a radioligand displace-

ment above 80% were re-evaluated using a range of 5 different

concentrations in 3 independent experiments, to determine the

binding constants (K_i). The standard reference compound was

melatonin, which was tested in each experiment at several con-

centrations to obtain a competition curve from which its IC₅₀was

calculated.

The IC₅₀values (concentration causing a half-maximal inhibi-

tion of control speciﬁc binding) and Hill coefﬁcients (nH) were

determined by non-linear regression analysis of the competition

curves generated with mean replicate values using Hill equation

curve ﬁtting

4.3.35. 5-[(E)-2-(6,7-Dimethoxynaphthalen-1-yl)ethenyl]-1,3,4-

oxadiazol-2(3H)-one (45)

Similarly to the synthesis of 44, acid 43 (100 mg, 0.39 mmol)

was ﬁrst transformed into the corresponding hydrazide that was

identiﬁed by HPLC-MS (2:30- g.t.10 min), m/z ¼ 273.11 [MþH]^þ,

calcd. for [C₁₅H₁₆N₂O₃þH]^þ273.30. This intermediate hydrazide

was then reacted with CDI to obtain the oxadiazolone 45 (99 mg,

85% yield) as a yellow pale solid of mp 232e234 ^ꢁC. Chromatog-

raphy: hexane to hexane:EtOAc 6:4. ¹H NMR (400 MHz, DMSO‑d₆)

d

8.04 (d, J ¼ 16.1 Hz, 1H, H_a), 7.82 (d, J ¼ 8.1 Hz, 1H, H₄), 7.78 (d,

nH

J ¼ 7.3 Hz, 1H, H₂), 7.46 (s, 1H, H₈), 7.41e7.32 (m, 2H, H₅, H₃), 6.93 (d,

Y ¼ D þ [(A-D) / [1þ(C/IC₅₀

)

]

J ¼ 16.1 Hz, 1H, H_b), 3.97 (s, 3H, C₇OCH₃), 3.90 (s, 3H, C₆OCH₃). ¹³

C

NMR (101 MHz, DMSO‑d₆)

d

154.4 (C_5’), 154.2 (C_2’), 149.9 (C₇), 149.3

where Y ¼ speciﬁc binding, A ¼ left asymptote of the curve,

D ¼ right asymptote of the curve, C ¼ compound concentration, and

nH ¼ slope factor.

(C₆), 133.7 (C_a), 130.1 (C₁), 129.4 (C_4a), 128.4 (C₄), 126.6 (C_8a), 123.8

(C₃), 122.7 (C₂), 112.6 (C_b), 107.2 (C₅), 102.2 (C₈), 55.6 (C₇OCH₃), 55.4

t

(C₆OCH₃). HPLC-MS (50:95- g.t.10 min) R 1.22 min, m/z ¼ 299.18

The inhibition constants (K_i) were calculated using the Cheng

Prusoff equation

[MþH]^þ, calcd. for [C₁₆H₁₄N₂O₄þH]^þ299.30. HRMS [ESI^þ] m/

z ¼ 298.09568 [M]^þ, calcd. for [C₁₆H₁₄N₂O₄]^þ298.09536.

K_i¼ IC₅₀/ (1 þ L/K_D)

4.4. Pharmacology

where

L

¼

concentration of radioligand in the assay, and

4.4.1. Assays for human MT₁R and MT₂R subtypes

K_D¼ afﬁnity of the radioligand for the receptor. A scatchart plot is

These experiments were performed at Euroﬁns-CEREP (France),

used to determine the K_D. In all cases, K_is are expressed as the

C. Herrera-Arozamena et al. / European Journal of Medicinal Chemistry 190 (2020) 112090

19

mean SEM of three independent experiments.

area under the ﬂuorescence decay curve (AUC) was calculated. The

net AUC corresponding to a sample was calculated by subtracting

the AUC corresponding to the blank. Regression equations between

net AUC and antioxidant concentration were calculated for all the

samples, measured in three independent experiments. ORAC values

were expressed as trolox equivalents by using the standard curve

calculated for each assay, where the ORAC value of trolox was taken

as 1.0.

4.4.4. Functional assay at human MT₂R

Evaluation of the agonist activity of 44 at hMT₂R expressed in

transfected CHO cells was performed by measuring its effects on

cAMP modulation using the HTRF detection method (Euroﬁns-

CEREP, France, catalog ref. G028-2092) [38]. The cells were sus-

pended in HBSS buffer (Invitrogen) complemented with 20 mM

HEPES (pH 7.4) and 500 mM IBMX, then distributed in microplates

at a density of 7.10³cells/well in the presence of either of the

following: HBSS (basal control), the reference agonist at 10 nM

(stimulated control) or various concentrations (EC₅₀determina-

tion), or the test compound. Thereafter, the adenylyl cyclase acti-

4.4.6. NRF2 induction by luciferase activity in AREc32 cells

AREc32 cells were kindly provided by Prof. Roland Wolf (Uni-

versity of Dundee, UK) [48]. AREc32 cells were cultured in DMEM

with glutamax and high glucose, supplemented with 1% penicillin-

streptomycin (10,000 units), geneticin (0.8 mg mL^ꢀ1) and 10% FBS,

at 37 ^ꢁC in a 5% CO₂air atmosphere. AREc32 cells were sub-cultured

in 96-well white plates at 2 ꢂ 10⁴cells/well density. Cells were

incubated with increasing concentrations of tested compounds in

duplicate for 24 h. Luciferase expression was assayed with the

Luciferase Assay System by luminescence in an Orion II microplate

luminometer (Berthold, Germany). Luciferase activity increase was

normalized to basal conditions considered as 1. CD (concentration

that duplicates the luciferase activity) values are expressed as the

mean SEM of four independent experiments in duplicate.

vator NKH 477 is added at a ﬁnal concentration of 5 mM. Following

10 min incubation at 37 ^ꢁC, the cells are lysed and the ﬂuorescence

acceptor (D2-labelled cAMP) and ﬂuorescence donor (anti-cAMP

antibody labelled with europium cryptate) are added. After

60 min at room temperature, the ﬂuorescence transfer is measured

at

l

ex ¼ 337 nm and em ¼ 620 and 665 nm using a microplate

l

reader (Envison, PerkinElmer). The cAMP concentration is deter-

mined by dividing the signal measured at 665 nm by that measured

at 620 nm (ratio).

The EC₅₀value (concentration producing

a half-maximal

response) was determined by non-linear regression analysis of

the concentration-response curves generated with mean replicate

values of three independent experiments using Hill equation curve

ﬁtting

4.4.7. Expression of KEAP1

4.4.7.1. pET15b-6xHis-Kelch domain construction. The human Kelch

domain fragment from vector pcDNA3.1-KEAP1-HA [74] was

subcloned in pET15b-6xHIis using the following oligonucleotides

that introduce an NdeI restriction site at both ends (Forward: 5⁰-

Y ¼ Dþ{(A-D) / [1þ(C/EC₅₀

)

^nH]}

TCGACATATGCAGGCAGTGCCCTGCCGCCGC-3⁰)

(Reverse: 5⁰-TGACGGATCCTTAGGTGACGGCCACACCCAC-3⁰).

and

BamHI

where Y ¼ response, A ¼ left asymptote of the curve, D ¼ right

asymptote of the curve,

nH ¼ slope factor.

C

¼

compound concentration, and

4.4.7.2. Expression of recombinant 6xHis-Kelch domain. The

pET15b-6xHIis-Kelch expression vector was used to express the

Kelch domain of KEAP1 as a 6xHis-fusion protein in the BL21-DE3

strain of Escherichia coli cultivated overnight at 37 ^ꢁC in LB medium.

Expression was induced at an optical density A₅₅₀¼ 0.4 by adding

The standard reference agonist is melatonin, which was tested

in each experiment at several concentrations to generate a

concentration-response curve from which its EC₅₀value was

calculated.

isopropyl-1-thio-b-D-galactopyranoside (IPTG) to a ﬁnal concen-

4.4.5. Oxygen radical absorbance capacity assay (ORAC)

tration of 0.5 mM. After 3 h at 37 ^ꢁC temperature, cells were lysed,

and 6xHIS-fusion protein was puriﬁed using the ProBond Puriﬁ-

cation System (Invitrogen) as described by the manufacturer.

Antioxidant activities were measured using the ORAC method

[43], in a Polarstar Galaxy plate reader (BMG Labtechnologies

GmbH, Offenburg, Germany) with 485-P excitation and 520-P

emission ﬁlters. The equipment was controlled by the Fluorostar

Galaxy software (version 4.11-0) for ﬂuorescence measurement.

2,2⁰-Azobis-(amidinopropane) dihydrochloride (AAPH), ( )-6-

hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (trolox)

and ﬂuorescein (FL) were purchased from Sigma-Aldrich. The re-

action was carried out in 75 mM phosphate buffer (pH 7.4) and the

4.4.8. Surface plasmon resonance (SPR) assays

SPR experiments were performed at 20 ^ꢁC with a Biacore X-100

apparatus (Biacore, GE) in PBS-T with 1 mM DTT and 2% de DMSO

(running buffer) at 25 ^ꢁC. The KEAP1 Kelch domain protein was

immobilized on a CM5 sensor chip (Biacore, GE) following standard

amine coupling method [75]. The carboxymethyl dextran surface of

the ﬂow cell 2 was activated with a 7-min injection of a 1:1 ratio of

0.4 M EDC and 0.1 M NHS. The protein was coupled to the surface

ﬁnal reaction mixture was 200

(120 L; 70 mM, ﬁnal concentration) solutions were placed in a

black 96-well microplate (96F untreat, Nunc). The mixture was pre-

incubated for 15 min at 37 ^ꢁC and then, AAPH solution (60

L,

mL. Antioxidant (20 mL) and FL

m

with a 7-min injection at 100 mg/mL in 10 mM sodium acetate, pH

12 mM, ﬁnal concentration) was added rapidly using a multi-

channel pipette. The microplate was immediately placed in the

reader and the ﬂuorescence recorded every minute for 80 min. The

microplate was automatically shaken prior each reading. Samples

5.0. The unreacted N-hydroxysuccinimide esters were quenched by

a 7-min injection of 0.1 M ethanolamine-HCl (pH 8.0). The level of

immobilization was 7000 RUs. Flow cell 1 treated as a ﬂow cell 2

(amine coupling procedure) without protein was used as a refer-

ence. Prior to use 10 mM stock solutions of compounds were

were measured at eight different concentrations (0.1e1

blank (FL þ AAPH in phosphate buffer) instead of the sample so-

lution and eight calibration solutions using trolox (1e8 M) were

mM). A

diluted several times until 100

running buffer. Typically, a series of different compounds was

injected onto the sensor chip a ﬂow rate of 45 L/min for a period of

mM ﬁnal concentration in the

m

also carried out in each assay. All the reaction mixtures were pre-

pared in duplicate, and at least three independent assays were

performed for each sample. Raw data were exported from the

Fluostar Galaxy Software to an Excel sheet for further calculations.

Antioxidant curves (ﬂuorescence vs. time) were ﬁrst normalized to

the curve of the blank corresponding to the same assay, and the

m

1 min followed by a dissociation period of 1 min. After the disso-

ciation process an extra-wash treatment was made over the ﬂow

cells with a 50% DMSO solution. No regeneration was needed.

Sensograms data were double-referenced and solvent corrected

using the Biaevaluation X-100 software (Biacore, GE).

20

C. Herrera-Arozamena et al. / European Journal of Medicinal Chemistry 190 (2020) 112090

4.4.9. Inhibition of human monoamine oxidases (hMAO-A and

hMAO-B)

was determined by UVeVis spectroscopy. Every sample is analysed

at ﬁve wavelengths, in four wells and at least in three independent

runs, and the results are given as the mean standard deviation. In

each experiment, 11 quality control standards of known BBB

permeability were included to validate and normalize the analysis

set.

MAO inhibition measurements were evaluated following the

general procedure previously described [76]. Brieﬂy, test drugs and

adequate amounts of recombinant hMAO-A or hMAO-B (Sigma-

Aldrich, Spain) required and adjusted to oxidize 165 pmol of p-

tyramine/min in the control group, were incubated for 15 min at

37 ^ꢁC in a ﬂat-black-bottom 96-well microtest plate (BD Bio-

sciences, Franklin Lakes, NJ) placed in the dark ﬂuorimeter cham-

ber. The reaction was started by adding 200 mM Amplex Red

reagent (Molecular Probes, Inc., Eugene, OR), 1 U/mL horseradish

peroxidase, and 1 mM p-tyramine. Then, the production of resor-

uﬁn was quantiﬁed at 37 ^ꢁC in a multidetection microplate ﬂuo-

rescence reader (FLX800, Bio-Tek Instruments, Inc., Winooski, VT)

based on the ﬂuorescence generated (excitation, 545 nm; emission,

590 nm). The speciﬁc ﬂuorescence emission was calculated after

subtraction of the background activity, which was determined from

wells containing all components except the hMAO isoforms, which

were replaced by a sodium phosphate buffer solution. IC₅₀values

were the mean SD of three independent experiments performed

in triplicate.

4.4.12. Neurogenic assays

Ten three-month-old male C57BL/6 mice were used throughout

this study. All experimental procedures were previously approved

by the Ethics Committee for Animal Experimentation of the CSIC in

accordance with the European Communities Council, directive

2010/63/EEC and National regulations, normative 53/2013. Special

care was taken to minimize animal suffering. Neural stem cells

(NSCs) were isolated from the SGZ of the hippocampal dentate

gyrus and prepared following previously published methods

[58,81]. The isolated NSCs were seeded into 12-well dishes at a

density of ~40,000 cells per cm2 in DMEM/F12 (1:1) containing N2

medium. NSCs were grown as free-ﬂoating cells under proliferative

conditions (10 ng/mL epidermal growth factor þ 10 ng/mL ﬁbro-

blast growth factor) allowing the formation of neural stem cells-

enriched growing spheres also called neurospheres (NS). After 7

days, with all NS having the same stage and size, cultures were

4.4.10. Inhibition of human lipoxygenase-5 (hLOX-5)

The ﬂuorescence-based enzyme method developed by Pufahl

et al. was followed [77], in 96-well microtiter plates. The assay

solution consists of Tris buffer (50 mM, pH 7.5), ethyl-

enediaminetetraacetic acid (EDTA, 2 mM), CaCl₂(2 mM), arach-

treated daily for 7 days with vehicle or 10

m

M of compound 6 (10).

Now, NS were adhered onto 100

m

g mL^ꢀ1poly-

L-lysine-coated

coverslips and treated for 3 additional days under differentiation

conditions (medium containing 1% fetal bovine serum and without

exogenous growth factors) [82]. Once neurospheres were differ-

entiated (72 h), coverslips were processed as previously described⁴¹

and used for immunoﬂuorescence analysis. Then the expression of

neuronal markers was analysed. The following antibodies linked to

idonic

acid

(AA,

3

mM),

ATP

(10

mM),

2⁰,7⁰-

dichlorodihydroﬂuorescein diacetate (H₂DCFDA, 10

mM), hLOX-5

(100 mU/well), bovine glutathione peroxidase (GPx, 25 mU/well)

and reduced glutathione (GSH, 1 mM). Compounds to be tested

were added to the test solution prior to AA and ATP, and pre-

incubated for a period of 10 min at room temperature. Then, the AA

and ATP substrates were added; the enzymatic reaction allowed to

neurogenesis were used:

b-III-tubulin polyclonal antibody (TuJ

clone; Abcam), a protein expressed at early stages of neurogenesis

and a monoclonal microtubule-associated protein type 2 (MAP-2)

antibody, a classical marker of late neuronal maturation. To visu-

alize primary antibodies Alexa-ﬂuor-labelled secondary antibodies

(Molecular probes) were used. Nuclei were stained with DAPI.

Fluorescent representative images from four independent experi-

ments were acquired in a LSM710 laser scanning spectral confocal

microscope (Zeiss). Confocal microscope settings were adjusted to

produce the optimum signal to noise ratio.

progress for 20 min and ended by the addition of 40 mL of aceto-

nitrile. The ﬂuorescence measurements (excitation: 485 nm;

emission: 520 nm) were performed on a FLUOstar OPTIMA (BMG

LABTECH, Offenburg, Germany). IC₅₀is deﬁned as the concentration

of compound that inhibits enzymatic activity by 50% over the

control of untreated enzyme. Data were the mean SD of three

independent experiments performed in triplicate.

4.4.11. In vitro bloodebrain barrier permeation assay (PAMPA-BBB)

Prediction of the brain penetration was evaluated using a par-

allel artiﬁcial membrane permeation assay (PAMPA-BBB), in a

similar manner as previously described [27,29,78e80]. Pipetting

was performed with a semi-automatic pipettor (CyBi®-SELMA) and

UV reading with a microplate spectrophotometer (Multiskan

Spectrum, Thermo Electron Co.). Commercial drugs, phosphate

buffered saline solution at pH 7.4 (PBS), and dodecane were pur-

chased from Sigma, Aldrich, Acros, and Fluka. Millex ﬁlter units

4.4.13. In silico model building of ligands and proteins

The molecular graphics program PyMOL [83] was employed in

the construction of the initial molecular models, trajectory visual-

ization and 3D ﬁgure generation. Ab initio geometry optimization of

the derivatives studied was achieved using a 6-31G* basis set and

the Hartree-Fock method, as implemented in the Gaussian 09

program [84]. The SCRF ¼ (Solvent ¼ Water) option was used to

place the solute in a cavity within the solvent reaction ﬁeld and

mimic hydration effects. For melatonin, 2-iodomelatonin and

resveratrol, the optimized geometries were also written in Crys-

tallographic Information File (CIF) format [85] using eLBOW [86] for

re-reﬁnement of several human quinone reductase II (QR2) X-ray

crystal structures retrieved from the Protein Data Bank (entries

2QWX, 2QX8 [87], and 1SG0 [60]) against structure factor ampli-

tudes using Phenix [88,89] and Coot [90]. Six macrocycles were

used in each reﬁnement round and concerted motions of atomic

groups were taken into account by means of the translation-

libration-screw (TLS) model [91]. PDB entries 6ME3 [62] and

6ME6 [63] containing the XFEL structures of chimeric MT₁and MT₂

receptor constructs, respectively, in complex with 2-

phenylmelatonin were used as templates to model the complexes

of the studied compounds with the wild-type human receptors

with the aid of the Swiss-Model server [92,93]. Consequently, our

(PVDF membrane, diameter 25 mm, pore size 0.45 mm) were ac-

quired from Millipore. The porcine brain lipid (PBL) was obtained

from Avanti Polar Lipids. The donor microplate was a 96-well ﬁlter

plate (PVDF membrane, pore size 0.45

microplate was an indented 96-well plate, both from Millipore. The

acceptor 96-well microplate was ﬁlled with 200 L of PBS: ethanol

(70:30) and the ﬁlter surface of the donor microplate was

impregnated with 5 L of porcine brain lipid (PBL) in dodecane

(20 mg mL^ꢀ1). Compounds were dissolved in PBS: ethanol (70:30)

at 100

g mL^ꢀ1, ﬁltered through a Millex ﬁlter, and then added to

the donor wells (200 L). The donor ﬁlter plate was carefully put on

mm) and the acceptor

m

the acceptor plate to form a sandwich, which was left undisturbed

for 120 min at 25 ^ꢁC. After incubation, the donor plate is carefully

removed and the concentration of compounds in the acceptor wells

C. Herrera-Arozamena et al. / European Journal of Medicinal Chemistry 190 (2020) 112090

21

MT₁and MT₂receptor models (UniProt [94] identiﬁers P48039 and

P49286, respectively) contained the original truncations at both

termini but had all the introduced point mutations replaced by the

natural amino acids and a short added sequence bridging the ICL3

gap (residues 219e227 and 232e240 in MT₁and MT₂receptors,

respectively). The conserved disulﬁde bridge between C100^3.25and

C177^ECL2in MT₁and between C113^3.25and C190^ECL2in MT₂were

explicitly deﬁned.

compounds using our reﬁned structure from PDB entry 1SG0; in

these cases, however, we found it necessary to add distance re-

straints (100 kcal mol^ꢀ1^ꢀ2) to the Zn^2þcoordination sphere and

Å

weak positional restraints (1 kcal mol^ꢀ1Å^ꢀ2) to the Ca trace of the

protein. The highly converged ensemble of 40 low-energy conﬁg-

urations thus obtained for each complex is expected to represent

more accurately the global energy minimum than isolated trajec-

tory frames [104,105].

Atom-centered charges for all ligands were calculated on the 6-

31G*-optimized geometries with the sqm program [95] in Amber-

Tools17 and the default AM1-BCC option [96], so as to reproduce

the molecular electrostatic potential in a way consistent with the

ff14SB force ﬁeld parameter set [97] in AMBER 16 (http://ambermd.

org).

4.4.16. Potentiometric pK_adetermination

Titrations were carried out at 25 ^ꢁC in 0.15 M KCl solution (aq) in

a SiriusT3 equipment (Sirius Analytical Instruments Ltd, East Sus-

sex, Britain) equipped with an Ag/AgCl double junction reference

pH electrode, a Peltier temperature system and a turbidity sensor.

Standardized 0.5 M KOH and 0.5 M HCl were used as titration re-

agents. KOH solution was standardized by potassium phthalate. The

pK_avalue is the mean of 3 titrations SD [106].

4.4.14. Molecular docking

The protein in the X-ray crystal structure of KEAP1 Kelch

Domain was prepared with the Protein Preparation procedure as

implemented in Maestro. All hydrogens were added and the charge

states of titratable amino acids were deﬁned using the PROTKA

procedure, followed by H-bond optimization and constrained

relaxation to 0.2 rmsd tolerance. The resulting structures were used

to generate docking grids and perform docking studies with com-

pounds 23 and 24 using GLIDE [98]. The ligands were prepared

with the LigPrep module using the parameters of OPLS3 Force Field.

Generation of possible tautomers was done with Epik routine in

Declaration of competing interest

The authors declare that they have no known competing

ﬁnancial interests or personal relationships that could have

appeared to inﬂuence the work reported in this paper.

Acknowledgments

Maestro at pH range of 7

Maestro 11.5 Release 2018-1 (Schrodinger LLC).

2. All the modelling was done with

The authors gratefully acknowledge the following ﬁnancial

supports: Spanish Ministry of Science, Innovation and Universities;

Spanish Research Agency; and European Regional Development

Funds (grants RTI2018-093955-B-C21 and SAF2015-64948-C2-1-R

to M.I.R.-F.; RTI2018-095793-B-I00 to M.G.L., SAF2015-64629-C2-

2-R to F.G.), General Council for Research and Innovation of the

Community of Madrid and European Structural Funds (grant

4.4.15. Molecular dynamics simulations

The complexes of 44 with MT₁R and MT₂R were embedded in a

lipid bilayer made up of 240 1,2-dioleoyl-sn-glycero-3-

phosphocholine (DOPC) and 21 cholesterol molecules with the

aid of the web-based CHARMM-GUI Membrane Builder graphical

user interface [99]. The system size along the Z axis was determined

by specifying a 10 Å thickness of 150 mM NaCl aqueous solution on

each side of the membrane. The AMBER ff14SB [97] and lipid14

[100] force ﬁelds were used for protein and lipids, respectively.

For each complex studied, all hydrogens and water molecules

were ﬁrst reoriented in the electric ﬁeld of the solute and then all

protein residues, ligands and waters were relaxed by performing 25

000 steps of steepest descent followed by 25 000 steps of conjugate

gradient energy minimization. The resulting geometry-optimized

coordinate sets were then heated up to 300 K using the Langevin

thermostat; thereafter the temperature was maintained at 300 K

whereas the system’s dimensions and density were equilibrated at

a constant pressure of 1 atm by means of an anisotropic Berendsen

weak-coupling barostat, essentially as described [100]. The uMD

simulations were run for 200 ns using the pmemd.cuda code [101],

as implemented in AMBER16, running on single Nvidia® GPUs. The

coupling constants for the temperature and pressure baths were 1.0

and 0.2 ps, respectively. The application of SHAKE to all bonds

allowed an integration time step of 2 fs to be used. A cutoff distance

of 9 Å was selected for the nonbonded interactions and the list of

nonbonded pairs was updated every 25 steps. Periodic boundary

conditions were applied and electrostatic interactions were rep-

resented using the smooth particle-mesh Ewald method [102] with

a grid spacing of 1 Å. Trajectory snapshots were saved every 0.1 ns

for further analysis. Besides, a simulated annealing procedure [103]

was followed every 5 ns to cool the system down from 300 to 273 K

over a 1-ns period. Thereafter the geometries of these “frozen”

complexes were optimized by carrying out an energy minimization

until the root-mean-square of the Cartesian elements of the

gradient was less than 0.01 kcal mol^ꢀ1Å^ꢀ1. A similar procedure was

used to simulate the fully solvated complexes of human QR2 with

B2017/BMD-3827

e NRF24ADCM), Health Institute Carlos III

(Miguel Servet II Program CP16/00014 and grant PI17/01700 to R.L.).

CH-A and P.M. thank their PhD fellowships from Spanish Ministry

of Education (MEC, PhD grant FPU16/01704 and mobility grant

FPUEST17/00233 to CH-A and FPU13/03737 to P.M.).

Appendix A. Supplementary data

Supplementary data to this article can be found online at

https://doi.org/10.1016/j.ejmech.2020.112090.

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Article Doi

Tuning melatonin receptor subtype selectivity in oxadiazolone-based analogues: Discovery of QR2 ligands and NRF2 activators with neurogenic properties

DOI: 10.1016/j.ejmech.2020.112090

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