Targeting the aryl hydrocarbon receptor with a novel set of triarylmethanes

European Journal of Medicinal Chemistry 207 (2020) 112777

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry

journal homepage: http://www.elsevier.com/locate/ejmech

Targeting the aryl hydrocarbon receptor with a novel set of

*

triarylmethanes

Elizabeth Goya-Jorge ^a^,^b, Celine Rampal , Nicolas Loones , Stephen J. Barigye ,

c

b

c

c, *

Laureano E. Carpio , Rafael Gozalbes , Clotilde Ferroud , Mait eꢀ Sylla-Iyarreta Veitía

,

a, **

Rosa M. Giner

a

Departament de Farmacologia, Facultat de Farm aꢁ cia, Universitat de Val ꢁe ncia. Av. Vicente Andr ꢀe s Estell ꢀe s, S/n, 46100, Burjassot, Valencia, Spain

ProtoQSAR SL, CEEI (Centro Europeo de Empresas Innovadoras), Parque Tecnol oꢀ gico de Valencia, Av. Benjamin Franklin 12, 46980, Paterna, Valencia, Spain

Equipe de Chimie Mol ꢀe culaire Du Laboratoire G ꢀe nomique, Bioinformatique et Chimie Mol ꢀe culaire (EA 7528), Conservatoire National des Arts et M ꢀe tiers

b

c

(

Cnam), 2 Rue Cont ꢀe , 75003, HESAM Universit ꢀe , Paris, France

a r t i c l e i n f o

a b s t r a c t

Article history:

Received 17 March 2020

Received in revised form

The aryl hydrocarbon receptor (AhR) is a chemical sensor upregulating the transcription of responsive

genes associated with endocrine homeostasis, oxidative balance and diverse metabolic, immunological

and inﬂammatory processes, which have raised the pharmacological interest on its modulation. Herein, a

novel set of 32 unsymmetrical triarylmethane (TAM) class of structures has been synthesized, charac-

terized and their AhR transcriptional activity evaluated using a cell-based assay. Eight of the assayed TAM

compounds (14, 15, 18, 19, 21, 22, 25, 28) exhibited AhR agonism but none of them showed antagonist

effects. TAMs bearing benzotriﬂuoride, naphthol or heteroaromatic (indole, quinoline or thiophene) rings

seem to be prone to AhR activation unlike phenyl substituted or benzotriazole derivatives. A molecular

docking analysis with the AhR ligand binding domain (LBD) showed similarities in the binding mode and

in the interactions of the most potent TAM identiﬁed 4-(pyridin-2-yl (thiophen-2-yl)methyl)phenol (22)

compared to the endogenous AhR agonist 5,11-dihydroindolo[3,2-b]carbazole-12-carbaldehyde (FICZ).

Finally, in silico predictions of physicochemical and biopharmaceutical properties for the most potent

agonistic compounds were performed and these exhibited acceptable druglikeness and good ADME

proﬁles. To our knowledge, this is the ﬁrst study assessing the AhR modulatory effects of unsymmetrical

TAM class of compounds.

20 August 2020

Accepted 21 August 2020

Available online 2 September 2020

Keywords:

Triarylmethane

Ah receptor

Agonistic activity

CYP1A1

Transcription factor

©

1

. Introduction

in cells of epithelial origin in the liver, kidney, lung and spleen [2].

The ligand binding domain (LBD) of AhR is allocated in the PAS-B

[(PER)/AhR nuclear translocator (ARNT)/single-minded (SIM)]

domain of the receptor. Once ligands arrive at the cytosolic loca-

tions of the receptor, they induce or inhibit the conformational

modiﬁcations needed to prompt its nuclear translocation. If AhR is

activated, the chaperone proteins are dissociated and its HLH

domain forms a heterodimer with the nuclear translocator ARNT.

The differential recognition of speciﬁc sequences in the promoter of

downstream genes is determined by the recruitment of coac-

tivators and corepressors, modulating thereby AhR expression.

Such sequences of recognition are known as xenobiotic response

The widely expressed and multifunctional aryl hydrocarbon

receptor (AhR) protein is ligand-activated, evolutionarily

a

conserved and pleiotropic transcription factor. It is classiﬁed as a

member of the basic helixeloopehelix (bHLH) family of receptors.

The cytosolic and resting state of AhR is found in association with

the chaperones heat shock protein 90 (Hsp 90), the immunophilin-

like protein XAP2 (ARA9 or AIP) and p23 [1]. Although AhR is

present in most tissues, its highest level of transcriptional activity is

0

elements (XRE) and they are identiﬁed by the core sequence 5 -

*

(

M.S.-I. Veitía and RM. Giner equally contributed as the last authors).

0

GCGTG-3 of the DNA [3]. Some XRE-independent mechanisms of

*

Corresponding author.

AhR activation have been suggested on inﬂammatory and auto-

** Corresponding author.

E-mail addresses: maite.sylla@lecnam.net (M. Sylla-Iyarreta Veitía), rosa.m.

giner@uv.es (R.M. Giner).

immune conditions, particularly in selective hormone-sensitive

https://doi.org/10.1016/j.ejmech.2020.112777

E. Goya-Jorge, C. Rampal, N. Loones et al.

European Journal of Medicinal Chemistry 207 (2020) 112777

Abbreviations

IC50

LRMS

half Inhibitory Concentration

low-resolution mass spectra

ADME

AhR

AhR-HepG2 AhR-Lucia

ANOVA

bHLH

BSD

absorption, distribution, metabolism, and excretion

Aryl hydrocarbon Receptor

human liver carcinoma HepG2

(one-way) analysis of variance

basic helixeloopehelix

bisacodyl

m-CPBA m-chloroperbenzoic acid

MEM

MTT

Minimum Essential Medium

3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium

bromide

™

NEAA

NMR

OECD

non-essential amino acids

Nuclear Magnetic Resonance

Organisation for Economic Co-operation and

Development

calcd

calculated

CH223191 2-methyl-2H-pyrazole-3-carboxylic acid

Cy cyclohexane

CYP1A1 cytochrome P450 family 1 subfamily A polypeptide 1

PAS

PER, ARNT (AhR-nuclear translocator), Single-

minded SIM

DCM

DCE

DMSO

EC50

ER

FBS

FCC

FICZ

dichloromethane

dichloroethane

dimethyl sulfoxide

half effective concentration

estrogen receptor

PBS

PC

Phosphate Buffer Saline

Positive Control

p-toluenesulfonic acid

triarylmethane

PTSA

TAM

THF

TLC

RPCmax

rt

SAR

SEM

SERM

XRE

tetrahydrofuran

fetal bovine serum

Thin Layer Chromatography

maximum response relative to the positive control

room temperature

Structure-Activity Relationship

standard error of the mean

selective Estrogen Receptor modulators

xenobiotic response elements

Flash Column Chromatography

5,11-dihydroindolo[3,2-b]carbazole-12-

carbaldehyde

Gas Chromatography-Mass Spectrometry

High Performance Liquid Chromatography

High Resolution Mass Spectra

GC-MS

HPLC

HRMS

cancer [4]. However, the main outcome of AhR expression is its

canonical XRE-mediated signaling linked to the induction of

xenobiotic metabolizing enzyme of the cytochrome P450 (CYP), in

particular CYP1A1 from family 1, subfamily A, polypeptide 1 [5].

Several ligands have been identiﬁed as modulators of AhR

including endogenous metabolites such 5,11-dihydroindolo[3,2-b]

carbazole-12-carbaldehyde (FICZ) and indoxyl sulfate [6] as well as

extensively used drugs such as omeprazole and leﬂunomide [7,8],

and dietary phytocompounds such as quercetin [9]. While exact

interaction patterns of different ligands upon binding with AhR still

lack a completed crystallized structure of the receptor, important

contributions are available for the LBD [10]. Moreover, vast studies

of the toxic ligand/agonist of AhR known as 2,3,7,8-

tetrachlorodibenzo-p-dioxin (TCDD) has shed light on the activa-

tion mechanism as well as on the signaling patterns of the receptor

modulated through AhR activation and particularly those affecting

gut and intestinal tissues [31e33]. Hence, promising drug candi-

dates such as NPD-0414-2 and NPD-0414-24 have been recently

suggested in the pharmacotherapy of colitis [34]. AhR-mediated

transcription converge with various nuclear receptor signaling

pathway, mainly with the estrogen receptor (ER) [35]. Indeed, se-

lective ER modulators (SERM) have been also identiﬁed as AhR li-

gands, which probably contributes to their therapeutical effects in

postmenopausal osteoporosis and breast cancers [36]. Some SERMs

identiﬁed hold the triarylmethane (TAM) skeleton [37]. Moreover,

the symmetric TAM compound tris-indolyl methane was evaluated

in a recent publication as a dual modulator of AhR and Pregnane X

receptor (PXR) [38]. However, to the best of our knowledge, un-

symmetrical TAM compounds have never been addressed as po-

tential modulators of AhR.

[

11,12]. The functional activity of AhR has proved to be determined

The TAMs are privileged structures in medicinal chemistry [39].

Numerous TAM derivatives have found applicability in neurode-

generative diseases and vascular disorders and as anti-

inﬂammatory, antitumoral and anti-infective agents against

tuberculosis, human immunodeﬁciency virus and respiratory syn-

cytial virus [40e42]. Notable examples are the TAM drug bisacodyl

(BSD) and its analogs pointed out as anti-inﬂammatory, antimi-

crobial and antiproliferative agents [43,44], and the well-known

antimycotic drug clotrimazole suggested in the antiproliferative

and antiangiogenic pharmacotherapy [44,45].

Considering the aforementioned evidence that endows TAMs as

an interesting scaffold in medicinal chemistry, added to the phar-

macological relevance of targeting AhR [21], led to the hypothesis

pursued herein. That is, TAM class of compounds could modulate

AhR activation with potential therapeutic applicability in malig-

nancies, immunological and inﬂammatory processes. Hence, novel

TAMs were synthetized and their AhR-mediated transcriptional

activity in AhR-HepG2 cells was assayed in vitro. The differential

effects displayed by the set of compounds allowed to suggest

theoretical contributions of the substituents in the AhR modulatory

effects. ADME properties were predicted and the binding afﬁnity

preliminarily studied using computational methods for the most

by each speciﬁc ligand that binds to the LBD and that ultimately

leads to dissimilar ligand- and AhR-dependent biological responses

[

are crucial structural determinants in all kinds of AhR modulators

suggested to date [14e18].

AhR ligands are associated with key physiological processes

such as proper development and metabolism, cell cycle regulation

and immune defense [19]. Hence, while earlier perspectives

focused on the function of AhR as xenobiotic sensor of toxicants like

dioxins and polyaromatic hydrocarbons, recent suggestions placed

AhR as an attractive pharmacological target [20e22]. Among the

potential therapeutical uses of AhR modulation are included lung

and vascular tissues health [23,24], treatment of liver and cystic

ﬁbrosis [25,26], control of the antioxidant response [27] and

regulation of neural functions in both vertebrates and invertebrates

13]. In general, aromatic or heteroaromatic hydrocarbon moieties

[

28]. Moreover, probably the most signiﬁcant pharmacological

applications of targeting AhR are in the treatment of several cancer

types, in which the prodrug Phortress (NSC 710305) has been rec-

ommended as anticancerogenic and tumor suppressor chemo-

therapy for CYP1A1-positive tumors [4,29,30]. In addition,

important inﬂammatory and immunological conditions could be

2

E. Goya-Jorge, C. Rampal, N. Loones et al.

European Journal of Medicinal Chemistry 207 (2020) 112777

signiﬁcant AhR activators identiﬁed.

optimized yields in a range of 38%e88%.

The syntheses of the benzotriazolyl triarylmethanes 10e12 were

carried out following the synthetic pathways represented in

Scheme 2.

2

. Results and discussion

First, the synthesis of the (4-methoxyphenyl) (pyridin-2-yl)

methanol 9 was performed by a bromine-lithium exchange as

previously described for compounds 3 from 2-bromopyridine 1 and

2

.1. Synthesis of triarylmethanes

The syntheses of triarylmethanes derivatives are shown in

p-anisaldehyde

8 in anhydrous tetrahydrofuran. Pyridylaryl-

Schemes 1e4. Details about the synthetic protocol and chemical

characterization of all intermediates are given in the Supplemen-

tary Information (SI-1).

benzotriazol 10 was prepared from benzotriazole and the corre-

sponding diarylmethanol 9 in the presence of a catalytic amount of

p-toluenesulfonic acid in perﬂuorooctane (C F18). In these condi-

8

tions, the desired regioisomer 10 was obtained in 50% yield in high

purity (HPLC, 95%). The regioisomer 10a was also isolated and its

characterization is described in the SI-1. An optimization of this

procedure could probably improve the obtained yield.

The syntheses of the p,p-N-oxides 6a-e and o,p-diary-

lmethylpyridines 7a-e were carried out following the synthetic

pathways represented in Scheme 1. First, synthesis of the corre-

sponding carbinols 3a-e was performed from 2-bromopyridine 1

and the corresponding aromatic aldehydes 2a-e, by a bromine-

lithium exchange following the procedure of Seto et al., 2004 [46]

or by a bromine-magnesium exchange using isopropylmagnesium

chloride in tetrahydrofuran at room temperature [47].

The dimethoxylated compound 11 was synthesized by reaction

with boron tribromide in dichloromethane. The reaction was

ꢀ

conveniently carried out by mixing the reagents at 0 C in an inert

solvent and then allowing the mixture to warm up to room tem-

perature during 6 h. Under these conditions the 4-((1H-benzo [d]

The key step to obtain the desired TAMs involved a regiose-

lective Friedel-Crafts hydroxyalkylation of the corresponding

carbinol 3a-e with phenol in nitrobenzene under acidic activation

[

5

1e3]triazol-1-yl) (pyridin-2-yl)methyl)phenol 11 was obtained in

0% yield. The corresponding acetate derivative 12 was obtained by

[

48]. The p,p regioisomers 4a-e were obtained with 4 equivalents of

ꢀ

treating 11 with acetic anhydride in the presence of sodium hy-

droxide at room temperature. After workup and puriﬁcation by

FCC, the desired compound 12 was isolated in 52% yield.

sulfuric acid at 80 C in a range of 33%e72% yield. The o,p com-

pounds 7a-e were obtained with 20 equivalents of catalyst at 0 C in

a range of 23%e98% yield. Acetates 5a-e were obtained by treating

the corresponding triarylmethanes derivatives with acetic anhy-

dride in the presence of sodium hydroxide at room temperature.

After workup, the desired compounds 5a-e were isolated in a range

ꢀ

The syntheses of the TAMs bearing heteroaromatic rings

(naphthol, indole, quinoline or thiophene) are outlined in Scheme

3

. Unsymmetrical naphthol (14, 15), pyridylaryl indoles (18,19),

and thiophene (21, 22) were synthesized under acid conditions by

condensation of the corresponding heterocycle with (4-

methoxyphenyl) (pyridin-2-yl)methanol 9 previously obtained by

a lithium-bromine exchange as described in Scheme 2. On the other

hand, TAM 25 bearing a quinoline fragment was prepared from the

corresponding aryl ketone 23 previously synthesized from the

carbinol 9 in excellent yield (98%) via a base-promoted aerobic

of 76%e98% yield and pure enough to be used in the next step

1

without any supplementary puriﬁcation as suggested by the

H

NMR analysis. N-oxide derivatives 6a-e were prepared from the

corresponding acetates by oxidation with m-chloroperbenzoic acid

in dichloromethane at room temperature. After 2e3 h of reaction,

N-oxide derivatives 6a-e were isolated with prior puriﬁcation by

ﬂash column chromatography (FCC) on silica gel with non-

ꢀ

Scheme 1. Synthesis of p,p- and o,p-triarylmethanes. (i) i-PrMgCl (1 M) in 2-Me-THF, anh THF, 2 h, rt, Ar; or n-BuLi, anh THF, ꢁ78 C/rt, Ar. (ii) phenol, H

2 4

SO (4 eq.), nitrobenzene,

ꢀ

O, NaOH, ꢂ15 h at 20 ^ꢀC or 40 C (iv) m-CPBA, anh DCM, 2 h at 20 C. (v) phenol, H

ꢀ

method A or B (A: 5 min at 80 C, then at rt. B: from 0 C to rt), Ar (iii) Ac

2

2 4

SO (20 eq.),

ꢀ

nitrobenzene at 80 C, Ar, 5 min..

3

E. Goya-Jorge, C. Rampal, N. Loones et al.

European Journal of Medicinal Chemistry 207 (2020) 112777

ꢀ

Scheme 2. Synthesis of benzotriazolyl triarylmethanes. (i) n-BuLi, anh THF, ꢁ78 C/rt. (ii) benzotriazole, PTSA monohydrate, C

8

F

18, 104 C, 24 h (iii) BBr

3

, DCM, 6 h, from 0 C to rt.,

ꢀ

Ar (iv) Ac

2

O, NaOH, 24 h, from 0 C to rt.

ꢀ

Scheme 3. Synthesis of naphthol, indole, thiophene and quinoline triarylmethanes. (i) NH

2

SO

3

H, DCE, 20 h, at 85 C, Ar (ii) HI, AcOH, 5.5 h at 100 C, Ar (iii) Ac

2

O, NaOH, 24 h, from

ꢀ

0

C to rt. (iv) BBr

3

, DCM, 19 h, from 0 C to rt (v) 18a, Ac

2

O, NaOH, 3.5 h, from 0 C to rt. (vi) CH

3

SO

3

H, DCE, microwave irradiation 2 h at 80 C (vii) BBr

, NaOH, toluene at 110 C (ix) prior mix of n-BuLi and 24 in anh THF, 1.5 h at ꢁ78 C, Ar, then 23 in dry THF, 17 h at rt.

3

, DCM, 19 h, from 0 C to rt., Ar

ꢀ

(

viii) O

2

oxidation using air as a free and clean oxidant [49]. The other

desired TAMs (16, 19) were prepared using sequence series of

including methoxy group deprotection followed by acylation as

indicated conditions in Scheme 3.

The synthesis of the TAMs bearing triﬂuoromethyl group was

carried out following the synthetic pathways represented in

Scheme 4. The synthesis of the pyridin-2-yl (4-(triﬂuoromethyl)

phenyl)methanol 26 and the corresponding arylketone 27 was

performed following the same procedure described in Scheme 1.

Then, TAM 28 was obtained by a halogen-metal exchange from 4-

bromoanisole in 67% yield. Demethoxylation was conveniently

carried out with hydroiodic acid in acetic acid at reﬂux. Under these

conditions the 4-(pyridin-2-yl (4-(triﬂuoromethyl)phenyl)methyl)

phenol 29 was obtained in 82% yield. The acetate derivative 30 was

obtained by treating 29 with acetic anhydride in presence of so-

dium hydroxide at room temperature. After workup and puriﬁca-

tion by FCC, the desired compound 4-(pyridin-2-yl (4-

(triﬂuoromethyl)phenyl) methylphenyl acetate 30 was isolated in

87% yield.

All compounds biologically evaluated were obtained in high

purity (HPLC or NMR, generally > 95%).

4

E. Goya-Jorge, C. Rampal, N. Loones et al.

European Journal of Medicinal Chemistry 207 (2020) 112777

ꢀ

Scheme 4. Synthesis of TAMs bearing triﬂuoromethyl group. (i) prior mix of n-BuLi and 2-bromopyridine in anh THF at ꢁ78 C, Ar, then 4-(triﬂuoromethyl)benzaldehyde, 17 h at rt.

, toluene, reﬂux, 24 h (iii) prior mix of n-BuLi and 4-bromoanisole in anh THF at ꢁ78 ^ꢀC, Ar and then 27 (iv) HI 57%, AcOH, reﬂux, Ar (v) Ac

O, NaOH, 4 h, from 0 C to rt.

ꢀ

(

ii) NaOH, O

2

.2. Biological evaluation

viability percentages obtained for 32 novel TAMs and the drug BSD

are shown in Fig. 1. The adopted criterium considered as cytotoxic

was a reduction of cell viability above 15% upon treatment with

TAMs.

.2.1. Cell viability

The effects on cell viability caused by the synthetized TAMs on

AhR-HepG2 cell line were determined by the 3-(4,5-

dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT)

assay, which constitutes a valuable method to study cell prolifera-

tion, cytotoxicity and chemosensitivity in vitro [50]. The cell

The eight TAMs 14, 15, 18, 19, 21, 22, 25 and 28 were studied in

more detail due to their AhR agonist effects as will be described

later. For these compounds, no cytotoxic effect was observed at the

six assayed concentrations. The same conditions but longer

Fig. 1. Viability percentages of cells exposed to the TAMs by MTT assay. a) Compounds 4 a-e, 10, 12, 16, b) Compounds 5 a-e and 6 a-c, c) Compounds 6d, 6e, 7 a-e and 30, d)

Compounds 14, 15, 18, 19, 21, 22, 25 and 28. All compounds were assayed at 0.1 M, 1.0 M, 5.0 M and 10.0 M, and compounds in d) were also tested at 2.5 M and 7.5 M. Each

m

chart represents the mean percentage ± SEM from at least three independent experiments (n ¼ 3). Cell viability lower than 85% was considered cytotoxic. *p < 0.05 signiﬁcantly

different from vehicle control (using one-way ANOVA followed by Dunnett’s post-test).

5

E. Goya-Jorge, C. Rampal, N. Loones et al.

European Journal of Medicinal Chemistry 207 (2020) 112777

exposure times (48 h and 72 h) in the MTT test revealed similar

results (data not shown) than those obtained after 24 h of treat-

ment (Fig. 1d).

remarkable high induction during the co-exposure was registered

for compounds 25 (RPCmax ¼ 272.13%) and 28 (RPCmax ¼ 242.95%)

despite that they were not by themselves among the strongest

activators of AhR. Similarly, a notable induction of AhR activation in

presence of FICZ during the antagonist assay was showed by the

inactive compounds 5b, 4d and 7a (RPCmax 203.94%, 200.45% and

196.65%, respectively).

The TAMs 4a, 5a, 6a and 30 were found to be cytotoxic over

5

mM. The strongest cytotoxicity was caused by compound 4a,

whose reduction of cell viability was above 90%, while comparable

reductions (~50%) of cell viability were observed for compounds 5a,

6

a and 30. The concentration limits established in the AhR tran-

scriptional activity bioassay only considered no cytotoxic concen-

trations of the tested TAMs.

2.3. SAR considerations

2

.2.2. AhR transcriptional activity

Standard Positive Controls. During the validation and optimi-

The AhR-mediated transactivation induced by the TAMs

allowed a comprehensive structure-activity relationship (SAR)

analysis. The key structural features of the active TAMs and their

AhR agonist effects expressed as fold responses are shown in

Fig. 2.

zation of the in vitro method, sigmoidal dose-response curves of the

endogenous AhR agonist FICZ were obtained with concentrations

from 0.01 to 18.0

tions of the AhR-HepG2 cell line provider (See SI-2). Similarly, the

antagonist bioassay was validated with the inhibition curve of the

2

m

M (R ¼ 0.99), according to the recommenda-

1

X1, X2 substitution. Regarding X , the presence of a pyridine ring

(14, 15, 18, 19, 21, 22, 25, 28) was important for AhR agonist activity

similarly to some other compounds reported in vivo as CYP1A1

inducers [54]. The N-oxidation had no inﬂuence on AhR activation,

noticeable when comparing the TAMs 5a-e vs. the corresponding

N-oxides 6a-e. None of the substituted phenyls at Ar showed any

effect on AhR except for 28 in which the introduction of a hydroxyl

AhR

antagonist

2-methyl-2H-pyrazole-3-carboxylic

acid

(

CH223191), obtained by co-exposure the EC50 of FICZ and con-

centrations from 1 to 30

where [51].

The maximum induction of AhR transcription caused by FICZ

was up to 30 folds at the maximum concentration tested (18 M).

From the dose-response curve of AhR agonist, the estimated EC50 of

FICZ in the cell model was 9.06 M. Meanwhile, in presence of FICZ

mM of the CH223191 as described else-

m

2

group at X position turned it moderately active. Compound 25

bearing a quinoline and a hydroxyl group at X

niﬁcant AhR agonist induction.

2

also exhibited sig-

m

concentration at the EC50, the antagonist compound CH223191

reduced to half the transcriptional activity of AhR at the maximum

concentration tested (30 mM). From the dose-response curve of AhR

R substitution. The presence of different oxygenated functional

group at R does not appear to determine the agonist effects on AhR

of the synthetized TAMs as it has been reported for other aromatic

compounds in the literature [55]. Most of the active compounds in

this study carry a hydroxyl or methoxy group at R. The methoxy

substitution in most cases was a better feature to exhibit AhR

agonist activity (14 vs. 15 and 18 vs. 19). However, the free hydroxyl

group in 22 resulted in a 30-fold increase of the AhR agonism when

compared with the methoxylated derivative 21.

Ar substitution. The AhR-agonist activity was crucially inﬂu-

enced by the third aromatic or heteroaromatic system occupying

Ar. Thus, the most potent AhR agonism was exhibited by TAMs

with heteroaromatic moiety such as thiophene (22), indole (18,

19) and quinoline (25). Otherwise, derivatives with a naphthol

substituent (14, 15) displayed some AhR agonist ability although

considerably weaker than the rest of heteroaromatic derivatives

(except for 21) as shown in Fig. 2. Curiously, compounds bearing a

benzotriazole moiety (10, 12) were found to be inactive. It should

be notice that even though 15 and 21 were considered agonists

according to the RPCmax threshold (Table 1), their induced fold

response was not signiﬁcant compared to the vehicle control as

shown in Fig. 2.

inhibition, the estimated IC50 of CH223191 was 2.43

with data reported in the literature [52].

mM, consistent

TAM compounds. The results of AhR agonist and AhR antago-

nist assays are presented in Table 1 for all the studied compounds,

that include the 32 novel TAMs synthetized, the commercial TAM

drug BSD and the positive controls. Dose response-curves are

provided as SI-2.

In the AhR reporter gene assay, the maximum effect observed

corresponded to the maximum concentration tested for all TAMs

except for 15 and 18. Compounds 4a, 5a, 6a and 30 were assayed at

non-cytotoxic concentrations (up to 1 mM) and they were unable to

induce or blockage AhR transcriptional activity in this cell model.

AhR agonist assay. The eight TAMs 14, 15, 18, 19, 21, 22, 25 and

2

8 were identiﬁed as agonists of AhR (RPCmax> 10%) while the rest

of them and the BSD were classiﬁed as inactive AhR agonists

Table 1). The agonist effectiveness of the active compounds

(

compared to FICZ followed the order: 18 z 22 > 19 > 25 > 14 > 28

z 15 > 21. Compounds 18 and 22 were more active as agonist than

FICZ at a comparable exposure concentration showing an RPCmax of

1

14.2% and 111.0%, respectively.

On the other hand, the introduction of heteroaromatic substit-

uent at Ar were in no case harmful to cells according to the cell

viability study (Fig. 1). Most of the substituted phenyl derivatives at

Ar that caused cytotoxicity at the highest concentrations tested (4a,

5a, 6a) bear a tertbutyl functional group.

Consistent to the above results, phloroglucinol TAMs have

shown better safety index and anti-HIV effects when bearing a

heteroaromatic moiety [42]. Additionally, indole-containing

chemicals have long been recognized as AhR ligands from endog-

enous and dietary sources, sustaining the strong agonism displayed

by 18 and 19 [19,56]. Although the thiophene ring in TAM-class of

compounds has been suggested as an attractive moiety for anti-

mycobacterial activity [57], it is not commonly found in either

classical or nonclassical AhR modulators identiﬁed to date [58].

Thus, to our knowledge, the agonist effects on AhR transcriptional

activity of thiophene derivatives are suggested herein for the ﬁrst

time.

In cases where a dose-response curve of agonism was achieved,

the half effective concentration (EC50) was estimated as a measure

of the potency of active compounds. As reported in Table 1, com-

pound 22 (EC50 ¼ 13.16

agonist. Compounds 25 and 18 showed comparable half effective

concentrations of 19.88 M and 21.72 M, respectively, while

M). The EC50 esti-

M and 52.03 M,

mM) was suggested as the most potent AhR

m

compound 19 was less potent (EC50 ¼ 27.86

m

mated for compounds 14 and 28 were 53.62

m

respectively. Lastly, it was not possible to obtain a dose-response

curve for compounds 15 nor 21.

AhR antagonist assay. None of the tested compounds showed

antagonist effects on AhR activation. However, additive effects and

probably synergism in presence of FICZ were observed for most of

the agonist compounds. Interestingly, the level of AhR transcrip-

tional response in presence of FICZ was not proportional to the

effectiveness of compounds individually tested as agonists. Thus, a

6

E. Goya-Jorge, C. Rampal, N. Loones et al.

European Journal of Medicinal Chemistry 207 (2020) 112777

Table 1

AhR-mediated transcriptional activity of the 32 rationally designed TAMs 4e7 [a-e], 10, 12, 14e16, 18, 19, 21, 22, 25, 28, 30, the drug bisacodyl and the agonist (FICZ) and

antagonist (CH223191) controls.

AhR-HepG2 transcriptional activity

ID

Ar

R

X

1

X

2

[

m

M]^a

Agonist

EC50

±SEM

(

m

M)

Antagonist

Activity

Criteria^d

RPC (%) ±SEM^b

c

RPC (%) ±SEM^b

4

5

6

7

1

2

3

a

b

c

d

e

a

b

c

d

e

a

b

c

d

e

a

b

c

d

e

0

2

4

5

6

8

9

1

2

5

8

0

Phe (4-C(CH

3

)

3

)

OH

N

H

OH

H

1.0

6.89 ± 0.29

7.09 ± 0.35

7.71 ± 0.56

6.59 ± 0.37

7.79 ± 0.52

5.32 ± 0.29

9.42 ± 0.46

6.32 ± 0.13

6.26 ± 0.10

8.77 ± 0.33

6.10 ± 0.23

6.28 ± 0.18

8.80 ± 0.52

7.78 ± 0.28

6.51 ± 0.31

6.81 ± 0.19

6.93 ± 0.15

7.99 ± 0.17

7.18 ± 0.16

6.89 ± 0.14

8.86 ± 0.26

9.23 ± 0.24

39.70 ± 0.76

28.57 ± 0.51

5.17 ± 0.25

114.20 ± 0.80

76.95 ± 0.63

10.54 ± 0.41

111.01 ± 0.86

53.78 ± 0.37

26.60 ± 0.89

7.71 ± 0.22

6.58 ± 0.19

100%

ND

113.44 ± 3.16

138.98 ± 4.72

168.82 ± 7.81

200.45 ± 7.41

150.92 ± 7.29

112.56 ± 2.01

203.94 ± 4.05

143.15 ± 6.44

128.16 ± 5.01

118.36 ± 5.34

120.3 ± 2.55

93.35 ± 1.09

106.22 ± 3.50

78.81 ± 3.89

92.41 ± 2.75

196.65 ± 7.59

114.43 ± 6.55

117.10 ± 3.45

114.57 ± 5.72

111.59 ± 3.21

163.53 ± 7.29

141.94 ± 6.44

142.31 ± 2.78

168.86 ± 5.74

143.80 ± 1.99

182.99 ± 5.12

226.23 ± 7.45

136.57 ± 2.70

202.27 ± 4.42

272.13 ± 5.51

242.95 ± 5.42

117.24 ± 3.81

107.20 ± 3.03

e

Inactive

Agonist

Inactive

Agonist

Inactive

Agonist

Antagonist

Phe (4-CH

3

)

10.0

1.0

10.0

1.0

10.0

5.0

10.0

1.0

>100

>400

ND

Phe (4-Br)

Phe (4-Cl)

Phe (4-F)

OH

Phe (4-C(CH

Phe (4-CH

OCOCH

3

)

>100

Phe (4-Br)

Phe (4-Cl)

Phe (4-F)

>1000

>100

ND

þ

ꢁ

Phe (4-C(CH

Phe (4-CH

N O

3

)

>200

>100

>200

>1000

>100

>1000

>100

53.62 ± 0.22

>50

>200

21.72 ± 0.32

27.86 ± 0.15

>50

13.16 ± 0.08

19.88 ± 0.08

52.03 ± 0.29

ND

Phe (4-Br)

Phe (4-Cl)

Phe (4-F)

Phe (2-OH)

1H-benzotriazole

1-Naph (2-OH)

1-Naph (2-OCOCH

3-indole

þ

N O

(CH

3

)

3

C

N

CH

Br

Cl

F

3

OCH

OCOCH

3

OCH

OH

OCOCH

OCH

OCOCH

OCH

OH

OCH

3

)

3

3-indole

2-thiophene

2-quinoline

3

Phe(4-CF

3

)

3

OCOCH

e

3

BSD

Control

Phe(4-OCOCH

FICZ

CH223191

3

)

10.0

18.0

30.0

>1000

9.06 ± 0.02

ðIC50Þ2.43 ± 0.18

e

54.50 ± 0.79

a

Maximum concentration tested in the absence of limitations due to cytotoxicity or insolubility.

Average of the percentages of the maximum response relative to the positive control (RPCmax) ± SEM of AhR agonist/antagonist activity from at least three independent

b

experiments (n ¼ 3).

c

Estimated half effective concentration (EC50) for agonists or half inhibitory concentration (IC50) for antagonist ± SEM, all extrapolated from the dose-response curve. ND:

non-determined.

d

Activity criteria for AhR agonist (>10%) or antagonist (<70%) compounds [53].

2

.4. Computational studies

GLU 320 were also identiﬁed for 22, FICZ and TCDD, respectively as

represented in Fig. 3. The obtained results suggested differences in

the predictive binding for these three ligands that could ultimately

lead to distinct biological responses [13]. A comparison between

the binding energies and interactions modes of compounds 22 and

21 did not provided plausible rationalization for the remarkable

activity differences in vitro identiﬁed (see Section 2, SI-3).

On the other hand, AhR ligands often modulate other tran-

scription factors, particularly nuclear receptors [59,60]. Therefore,

as a preliminary off-targeting screening, the binding capacity of

compound 22 in ER, Androgen Receptor (AR), Progesterone Re-

ceptor (PR) and Pregnane X Receptor (PXR) was analyzed by mo-

lecular docking. A comparison between 22 and well-known ligands

of each receptor did not reveal any apparent binding resemblances.

Although the binding energies of 22 were in most cases similar to

those exhibited by the speciﬁc ligand for each receptor, the pocket

and binding sites were different in all cases (details are provided in

Section 4, SI-3).

.4.1. Molecular docking

The binding to AhR of the strongest TAM agonist identiﬁed (22)

was compared by means of molecular docking analysis with the

known ligand/agonist compounds FICZ and TCDD. In the absence of

a crystalized structure of AhR-LBD, the structurally related PAS-B

domain of HIF2a was used for molecular docking analysis (details

are provided as SI-3). The best poses obtained for the three ligands

during the docking simulations are represented in Fig. 3.

The three docked ligands (22, FICZ and TCDD) seem to concurr

in the internal cavity of the crystallized PAS-B heterodimer as ex-

pected [10]. While 22 and FICZ were predicted to bound with AhR-

LBD in a similar region of the cavity, the best pose estimated for

TCDD binding seems to lie in a different region as shown in Fig. 3.

Details on the residues involved in the molecular docking for each

ligand are provided in Supplementary Information (SI-3). Further-

more, the hydrophobic interactions in the predicted protein/ligand

complex for 22 as well as for the endogenous agonist FICZ shared

PHE 254 and ALA 277 residues.

hydrogen bond with TYR 281 and THR 321, and halogen bond with

P-Cation interactions with HIS 248,

2

.4.2. Druglikeness and ADME proﬁle

Characterizing the druglikeness as well as the bioavailability are

7

E. Goya-Jorge, C. Rampal, N. Loones et al.

European Journal of Medicinal Chemistry 207 (2020) 112777

Figure 2. Maximum AhR agonist activity induced in cells by compounds 14, 15, 18, 19, 21 22, 25 and 28. General structure and agonist compounds are represented at the left. Data

are expressed as fold responses, as compared to non-induced cells (i.e. vehicle control (c)). The bar chart at the right shows mean fold response ± SEM (n ¼ 4) as: ꢃ25 folds (red),

between 10 and 20 folds (orange), ꢂ10 folds and signiﬁcant (light orange), <10 folds and not signiﬁcant (white). The levels of signiﬁcance were determined using one-way ANOVA,

followed by Dunnett’s post-test when compared to vehicle control (***p < 0.001) or by Bonferroni post-test when compared between pairs of structural analogous (##p < 0.01,

###p < 0.001).

Fig. 3. Representation of molecular docking and the molecular interactions between HIF2

a (PBD ID: 3F1O) and 22 (A), FICZ (B) and TCDD (C). The structure of the protein is

represented as transparent lilac ribbons and the best pose obtained for 22, FICZ and TCDD are displayed as sticks. The residues involved in hydrophobic interactions are labelled.

P

-

Cation (orange), hydrogen bond (blue) and halogen bond (green) interactions are represented in dot lines. (color online only). (For interpretation of the references to color in this

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

important steps toward the prioritization in drug discovery [61].

Therefore, physicochemical properties (Table 2) were predicted in

silico for the six novel synthesized TAMs with the most signiﬁcant

AhR agonist activity. Additional properties are provided in SI-3.

The presence of reactive functional groups (#rtvFG) has been

related to decomposition, reactivity and toxicity in vivo. Therefore,

this molecular descriptor serves as an alert system of structural

groups such as azo, diazo, carbonate, aluminum or silicon (full list

8

E. Goya-Jorge, C. Rampal, N. Loones et al.

European Journal of Medicinal Chemistry 207 (2020) 112777

Table 2

Physicochemical properties and druglikeness criteria of the AhR-agonist TAMs and the drug bisacodyl.

ID

#rtvFG^a

MW

g/mol]^b

Dipole^c

SASA

[Å ]

Volume

[Å ]

TPSA^f

Donor

HB

Accpt

HB

Polrz

[Å ]

logP

o/w^j

logS

[S: mol/dm ]

Lipinski’s

2

d

3

e

g

h

3

i

3

k

rule of ﬁve^l

[

1

2

4

8

9

2

5

8

0

1

0

2

341.41

314.39

342.40

267.35

342.40

359.35

361.40

0.68

1.75

3.53

6.52

7.33

7.66

599.04

578.44

625.36

503.96

615.79

606.32

658.56

1081.50

1034.32

1111.64

869.78

1090.79

1064.55

1169.98

42.35

37.91

54.98

50.36

61.36

42.35

65.49

1

0

2.50

1.75

3.50

1.75

3.50

2.50

6.00

38.39

36.98

40.02

29.76

38.90

36.68

40.99

5.32

5.36

4.82

4.09

4.95

5.57

3.66

ꢁ5.46

ꢁ5.51

ꢁ5.74

ꢁ4.32

ꢁ5.41

ꢁ6.12

ꢁ4.74

1

0

1

0

BSD

a

#

rtvFG: Number of reactive functional groups in the structure of the molecule (listed in Experimental Section). Recommended values: 0e2.

b

c

d

e

f

MW: Molecular weight of the molecule. Recommended values: 130e725 g/mol.

Dipole: Computed dipole moment of the molecule. Recommended values: 1.0e12.5.

2

SASA: Total solvent accessible surface area. Recommended values: 300e1000 Å using a probe with a 1.4 Å radius.

Volume: Total solvent-accessible volume. Recommended values: 500e2000 Å³using a probe with a 1.4 Å radius.

TPSA: Topological polar surface area.

g

h

i

Donor HB: Estimated number of hydrogen bonds that would be donated by the solute to water molecules in an aqueous solution. Recommended values: 0e6.

Accpt HB: Estimated number of hydrogen bonds that would be accepted by the solute from water molecules in an aqueous solution. Recommended values: 2-20.

3

Polrz: Predicted polarizability. Recommended values: 13e70 Å .

j

logP o/w: Predicted logarithm of octanol/water partition coefﬁcient. Recommended values: ꢁ2.0 to 6.5.

k

l

3

logS: Predicted logarithm of solubility (S expressed in mol/dm ). Recommended values: ꢁ6.5 to 0.5.

Lipinski’s rule of ﬁve (druglikeness): Number of violations of Lipinski’s rule of ﬁve: MW < 500, logP o/w < 5, donor HB ꢂ 5, accpt HB ꢂ 10. Maximum: 4 violations.

in SI-3). All the agonist TAMs were consistent the recommended

criterium for drug-like compounds. They all were free from

potentially reactive functional groups except for 19. Interestingly,

the TAM drug bisacodyl possessed two. The molecular weights, the

computed dipole moments, the topological polar surface area

AhR-agonist TAMs synthetized probably have good permeability

to cross the gut-blood barrier, a high human oral absorption and

only 19 and 28 violated the solubility criterium of Jorgensen’s rule.

Adequate binding to human serum albumin (logKhsa) as well as

an appropriate number of metabolic reactions (#Metab) were

predicted for all the TAMs. Lastly, the apparent capacity to cross

the brain/blood barrier (logBB) was predicted as good for all the

studied TAMs while the predicted skin permeability (logKp) was

in acceptable limit only for the strongest AhR-agonist 22 and BSD.

(

TPSA) and the total solvent accessible surface area (SASA) and

volume, the polarizability as well as the estimated number of

hydrogen bonds (HB) that may be donated or accepted in an

aqueous solution were predicted within the recommended values

for all the studied TAMs.

The octanol/water partition coefﬁcient (logP o/w) directly in-

ﬂuences the effects of chemical entities on biological systems,

particularly their pharmacokinetics and pharmacodynamics [62].

Similarly, the aqueous solubility (logS) of a compound inﬂuences its

ability to reach the site of action and produce any kind of effect.

According to the predicted logP o/w and logS, all the agonist TAMs

met the recommended range for druglikeness. Finally, Table 2

shows the Lipinski’s rule of ﬁve as a global criterium of druglike-

ness that suggests the limits of some physicochemical properties as

follow: MW < 500, logP o/w < 5, donor HB ꢂ 5, accpt HB ꢂ 10.

Hence, none of the agonist TAMs showed more than one violation

of such rule and only the predicted logP o/w for the TAMs 14,18 and

3. Conclusions

TAM compounds were straightforwardly synthetized and char-

acterized by means of efﬁcient synthetic strategies in this work. The

effects of 32 newly TAM derivatives as potential modulators of the

emerging pharmacological target AhR were determined in vitro

using a novel secreted luciferase assay system. The bioassays

revealed an exclusive agonism of eight derivatives and a lack of

antagonist activity on AhR activation across the TAM set. Hetero-

aromatic or naphthol moieties crucially determined the occurrence

of AhR agonism and the thiophene derivative 22 was the most

potent agonist compound on AhR-mediated transcription yielding

over 30-fold response, comparable to the endogenous metabolite

FICZ. The structural adequacy, absence of cytotoxicity as well as

druglikeness and favorable ADME proﬁle, allow to suggest 22 as a

new lead compound in the study of AhR-mediated transcription. In

general, these results could provide valuable insights to design new

potent AhR modulators based on the TAM scaffold.

2

8 was slightly higher than 5.

In order to analyze the six agonist compounds in the context of

rapidly metabolized AhR ligands (RMAhRLs) or Selective AhR

modulators (SAhRMs), a comparative analysis was performed

based on their physicochemical proﬁles, as proposed by Dolciami D.

et al. [63]. The mean values ± SD of the molecular descriptors

suggested for RMAhRLs are MW (335 ± 91), log P o/w (3.46 ± 1.10)

and TPSA (65.1 ± 24.8), while those for SAhRMs are MW (307 ± 77),

log P o/w (4.24 ± 2.24) and TPSA (43.7 ± 34) [63]. Comparisons

performed using one-way ANOVA (p < 0.05) followed by Dunnett’s

post-test of the TAMs within the RMAhRLs and SAhRMs context,

revealed no signiﬁcant differences, as represented in Fig. 4 a) and

b), respectively. Therefore, the studied TAMs cannot be classiﬁed as

SAhRMs or RMAhRLs according to this criterion.

4. Experimental Section

4.1. Chemistry

4.1.1. Materials and methods

All reagents were obtained from commercial sources unless

otherwise noted and used as received. Heated experiments were

conducted using thermostatically controlled oil baths. Reaction

requiring anhydrous conditions were performed under an atmo-

sphere oxygen-free in oven-dried glassware. Drying of the products

On the other hand, predictive results of some properties

contributing to the ADME proﬁle of the AhR-agonist TAMs, are

shown in Table 3.

Considering the predicted parameters collectively related to

oral bioavailability (Caco-2 permeability, Human Oral Absorption

was carried out under reduced pressure using a vacuum pump and/

ꢀ

or a desiccant heated to 40 C in the presence of P

2

O

5

. All reactions

(

HOA) and Jorgensens rule of three), it can be concluded that the

were monitored by analytical thin layer chromatography (TLC) or

9

E. Goya-Jorge, C. Rampal, N. Loones et al.

European Journal of Medicinal Chemistry 207 (2020) 112777

Fig. 4. Mean values of the molecular descriptors MW, logP and TPSA for compounds 14, 18, 19, 22, 25 and 28 suggested to classify rapidly metabolized AhR ligands (RMAhRLs) and

Selective AhR modulators (SAhRMs) [63]. One-way ANOVA (p < 0.05) followed by Dunnett’s post-test did not revealed signiﬁcant differences between TAMs and RMAhRLs or

SAhRMs, respectively.

Table 3

Prediction of ADME descriptors for the AhR-agonist TAMs and the drug bisacodyl.

ID

Caco-2

nm/sec]^a

MDCK

[nm/sec]

logBB^c

logKp

[nm/sec]

logKhsa

Jm

ꢁ2 ꢁ1

]^f

g cm h

HOA^g

#Metab^h

Jorgensen’s rule of threeⁱ

b

d

[nm/sec]^e

[

m

1

2

4

8

9

2

5

8

4561.02

5349.67

2083.59

2157.45

4130.44

5013.57

846.79

2551.05

3031.00

1093.83

1872.28

2291.76

ꢁ0.07

0.07

ꢁ0.37

ꢁ0.19

ꢁ0.12

0.26

ꢁ0.05

ꢁ0.07

ꢁ0.88

ꢁ1.07

ꢁ0.08

ꢁ0.34

ꢁ1.81

0.87

0.93

0.80

0.47

0.70

0.82

0.20

1.06

0.83

0.08

1.11

1.12

0.12

0.10

3

5

4

3

5

4

3

0

1

0

1

0

10000.00

413.32

BSD

ꢁ0.87

a

Caco-2 (model for the gut-blood barrier): Predicted apparent Caco-2 cell permeability (non-active transport) [nm/sec]. Criteria: <25 poor permeability,>500 great

permeability.

b

MDCK (mimic for the blood-brain barrier): Predicted apparent MDCK cell permeability (non-active transport) [nm/sec]. Criteria: <25 poor permeability,>500 great

permeability.

c

logBB: Predicted brain/blood partition coefﬁcient (model for orally delivery drugs). Recommended values: from ꢁ3.0 to 1.2.

d

e

f

logKp: Predicted skin permeability. Recommended values: from ꢁ8.0 to ꢁ1.0.

logKhsa: Predicted binding to human serum albumin. Recommended values: from ꢁ1.5 to 1.5.

ꢁ2 ꢁ1

mg cm .h ).

Jm: Predicted maximum transdermal transport rate obtain from: Kp ꢄ MW ꢄ S (

g

h

i

HOA (Human Oral Absorption): Predicted qualitative human oral absorption: low (1), medium (2), high (3).

Metab: Number of likely metabolic reactions (listed in SI-3). Recommended values: 1e8.

Jorgensen’s rule of three (oral availability): Fewer (and preferably no) violations of the follow: logS > ꢁ5.7, Caco-2 > 22 nm/s, #PrimaryMetabolites< 7.

Gas chromatography-Mass spectrometry (GC-MS). TLC was per-

formed on aluminium sheets, silica gel coated with ﬂuorescent

indicator F254, Merck. TLC plates were visualized using irradiation

with light at 254 nm or in an iodine chamber as appropriate. FCC

was carried out when necessary using silica gel 60 (particle size

performed with an Agilent 6890 N instrument equipped with a

12 m ꢄ 0.20 mm dimethyl polysiloxane capillary column and an

Agilent 5973

N MS detector-column temperature gradient

ꢀ

80e300 C (method 160): 160 C (1 min), 180 Ce260 C (10 C/

ꢀ

min), 260 C (4 min); (method 180): 180 C (1 min), 180 Ce300 C

ꢀ

0

.040e0.063 mm, Merck). The eluent mixture is speciﬁed for each

(10 C/min), 300 C (2 min), gradient 200e300 C (method 200):

ꢀ

puriﬁcation.

200 C (1 min), 200 Ce300 C (10 C/min), 300 C (4 min). Low-

resolution mass spectra (LRMS) result from ionization by elec-

tronic impact. Infrared spectra were recorded over the 400-

4.1.2. Physical measurements

ꢁ

1

Melting points (Mp) were determined on a Leica VMHB system

Koﬂer apparatus. The structure of the products prepared by

4000 cm range with an Agilent Technologies Cary 630 FTIR/ATR/

ZnSe spectrometer. High-resolution mass spectra (HRMS) analyses

were acquired on a Thermo Scientiﬁc LTQ Orbitrap mass spec-

trometer. The HPLC analyses were carried out on a normal phase

column Hypersil Si (length: 150 mm, diameter: 4.6 mm, stationary

different methods was checked by comparison of their NMR, IR and

1

13

MS data and by the TLC behavior. H and C NMR spectra were

acquired on a Bruker BioSpin GmbH spectrometer 400 MHz, at

room temperature. Chemical shifts are reported in

d

units, parts per

phase: 5

(length: 150 mm, diameter: 4.60 mm, stationary phase: 5

a Water 2998 Photodiode Array Detector (260e370 nm) and an

isocratic system of elution. The retention time (R ) is expressed in

min in the decimal system. HPLC purity was determined on the

Hypersil Si column, using n-heptane/ethyl acetate 7/3 with a ﬂow

m

m) and a reverse phase column Hypersil ODS C18

million (ppm). Coupling constants (J) are measured in hertz (Hz).

Splitting patterns are designed as follows: s, singlet; d, doublet; dd,

doublet of doublets; dm, doublet of multiplets, ddd, doublet of

doublets of doublets; m, multiplet; br, broad. Various 2D tech-

niques and DEPT experiments were used to establish the structures

and to assign the signals. For the assignments of the NMR signals,

we use the convention presented in Fig. 5. GC-MS analyses were

mm) using

t

rate of 0.8 mL per min and UV detection at

otherwise notiﬁed.

l

¼ 262e264 nm, unless

10

E. Goya-Jorge, C. Rampal, N. Loones et al.

European Journal of Medicinal Chemistry 207 (2020) 112777

Fig. 5. Convention adopted to assign signals of ¹H and ¹³C NMR spectra. Only the 32 TAM compounds evaluated in vitro are described herein. Intermediates and other TAMs

obtained are detailed as SI-1.

4.1.3. General procedure for the preparation of p,p-triarylmethanes

9 3 7

123.37 (C ), 124.87 (C15, C17),128.60 (C14, C18),129.88 (C , C ), 133.31

Method A: To a solution of the corresponding carbinol (1 eq.)

(C

2

),136.56 (C10),140.59 (C13),148.22 (C16), 149.02 (C12),155.58 (C

5

),

þ.

and phenol (1.2 eq.) in nitrobenzene (0.4 M) was added dropwise

163.15 (C

302 [M - CH

8

); GC-MS method 180, R

t

¼ 7.82 min, m/z 317 [M ] (100),

ꢀ

þ.

concentrated sulfuric acid (4 eq.) at 0 C. The reaction progress was

3

] (30), 286 [M - 2 CH

3

] (5), 260 [M - C(CH

3

)

3

] (19),

þ

monitored by GC-MS and TLC (eluent DCM/MeOH 90/10). After

239 [OHPhCHt-BuPh] (23), 224 [OHPhCHt-BuPh - CH

3

] (8); IR

ꢀ

ꢁ1

5

min at 80 C the reaction was cooled to room temperature and

(ATR) (cm ) 3058, 3020 (

1510 (

C ¼ C), 1167 (

o-disubst); HPLC purity: 96%, (Hypersyl Si, n-heptane/EtOAc 30/

70, ﬂow rate 0.80 mL/min, ¼ 2.84 min).

max ¼ 264 nm, R

nCsp2-H), 2957 (

n

Csp3-H), 1615, 1600,

neutralized with a saturated solution of NaHCO

extracted with ethyl acetate three times. The combined organic

phases were dried over anhydrous Na SO , ﬁltered and concen-

3

(pH 7e8), then

n

nC-O), 810 (dCsp2-H p-disubst), 755(dCsp2-H

2

4

l

t

trated. The crude residue was puriﬁed by FCC on silica gel (eluent

gradient DCM, DCM/MeOH 98/2, DCM/MeOH 90/10) to afford the

corresponding p,p-triarylmethane: Yields: 4a (72%), 4b (63%) and

4

0

2

.1.3.2. 4-(pyridin-2-yl (p-tolyl)methyl)phenol (4b). Yield: 73 mg,

.26 mmol, beige solid, 63%. 18 mg (0.07 mmol, 15%) of 2-(pyridin-

-yl (p-tolyl)methyl)phenol are also isolated, (Method A).

4

c (39%).

Method B: To a solution of the corresponding carbinol (1 eq.)

ꢀ

Mp ¼ 156e158 C. TLC CyHex/EtOAc 50/50, Rf ¼ 0.44, DCM/MeOH

and phenol (1.2 eq.) in nitrobenzene (0.4 M) was added dropwise

1

9

H

J

7

0/10, Rf ¼ 0.53; H NMR (DMSO-d

19), 5.50 (s, 1H, H

), 6.69 (d, 2H, Jortho ¼ 8.5 Hz, H

ortho ¼ 8.5 Hz, H , H ), 7.03e7.11 (m, 4H, H14, H15,H18, H17),

.16e7.24 (m, 2H, H

6

, 400 mHz) (

d

ppm) 2.24 (s, 3H,

ꢀ

concentrated sulfuric acid (4 eq.) at 0 C. The reaction progress was

1

4

6

, H ), 6.99 (d, 2H,

monitored by GC-MS and TLC (eluent CyHex/EtOAc 50/50). After

7

3

ꢀ

stirring at 0 C the reaction was cooled to room temperature and

9

, H11), 7.70 (ddd, 1H, J10-9 ¼ J10-11 ¼ 7.7 Hz, J10-

3

neutralized with a saturated solution of NaHCO (formation of a

gum which solubilizes once pH 7e8 is reached), then extracted

with ethyl acetate four times. The combined organic phases were

12

¼ 1.80 Hz, H10), 8.51 (ddd, 1H, J12-11 ¼ 5 Hz, J12-10 ¼ 2.8 Hz, J12-

13

9

¼ 0.8 Hz, H12), 9.24 (s, 1H, OH); C NMR (DMSO-d

6

, 100 mHz) (

d

),

ppm) 20.56 (C19), 57.12 (C

28.78 (C14, C15, C17, C18), 129.91 (C

36.57 (C10), 140.63 (C13), 149.05 (C12), 155.70 (C

1

), 114.98 (C

6

, C

), 133.40 (C16), 135.05 (C

), 163.21 (C

4

), 121.40 (C11), 123.38 (C

9

washed with brine, dried over anhydrous Na

concentrated. The crude residue was puriﬁed by FCC on silica gel

eluent gradient DCM, DCM/MeOH 98/2, DCM/MeOH 90/10) to

afford the corresponding p,p-triarylmethane: Yields: 4d (33%) and

2 4

SO , ﬁltered and

1

3

, C

7

2

5

8

); GC-

(

þ.

MS method 180, R

t

¼ 7.22 min, m/z 274 [M ] (100), 259 [M - CH

3

]

þ

3

(

12),197 [OHPhCHPhCH

3

] (34),181 [OHPhCHPhCH

- OH] (24),167

Csp2-H),

C-O), 754 ( Csp2-

H o-disubst); HPLC purity: 98%, (Hypersyl Si, n-heptane/EtOAc 30/

0, ﬂow rate 0.80 mL/min, ¼ 3.00 min).

max ¼ 264 nm, R

4

e (47%).

þ

ꢁ1

[PhCHPy] , (8), 78 [Py] (4); IR (ATR) (cm ) 3018, 3005 (

n

2

915 (

n

Csp3-H), 1614, 1592, 1508 (

n

C ¼ C), 1233 (

n

d

4

.1.3.1. 4-((4-(tert-butyl)phenyl) (pyridin-2-yl)methyl)phenol (4a).

Yield: 96 mg, 0.30 mmol, white solid, 72%. 13 mg (0.04 mmol, 9.6%)

of 2-((4-(tert-butyl)phenyl) (pyridin-2-yl)methyl)phenol are also

7

l

t

ꢀ

isolated, (Method A). Mp ¼ 154e156 C. TLC CyHex/EtOAc 50/50,

4.1.3.3. 4-((4-bromophenyl)

Yield: 51 mg, 0.15 mmol, oil, 39%, (Method A). TLC DCM/MeOH 90/

(pyridin-2-yl)methyl)phenol

(4c).

1

Rf ¼ 0.54, DCM/MeOH 90/10, Rf ¼ 0.80; H NMR (DMSO-d

6

,

1

4

00 mHz) (

ortho ¼ 8 Hz, H

ortho ¼ 8 Hz, H14, H18), 7.18e7.24 (m, 2H, H

ortho ¼ 8 Hz, H15, H17), 7.71 (ddd, 1H, J10-9 ¼ J10-11 ¼ 8 Hz, J10-

¼ 4 Hz, H10), 8.52 (ddd, 1H, J12-11 ¼ 4.7 Hz, J12-10 ¼ 1.8 Hz, J12-

d

ppm) 1.24 (s, 9H, H20), 5.49 (s, 1H, H

, H , H

), 7.01 (d, 2H, Jortho ¼ 8 Hz, H

, H11), 7.30 (d, 2H,

1

), 6.69 (d, 2H,

10, Rf ¼ 0.60; H NMR (DMSO-d

6

, 400 mHz) (

, H

), 7.00 (d, 2H, Jortho ¼ 8.5 Hz,

), 7.14 (d, 2H, Jortho ¼ 8.2 Hz, H14, H18), 7.20e7.28 (m, 2H, H

d ppm) 5.55 (s, 1H,

J

4

6

7

3

), 7.11 (d, 2H,

H

1

), 6.69 (d, 2H, Jortho ¼ 8.70 Hz, H

4

6

9

7

, H

3

9

,

11), 7.46 (d, 2H, Jortho ¼ 8.3 Hz, H15, H17), 7.73 (ddd, 1H, J10-9 ¼ J10-

12

11 ¼ 7.7 Hz, J10-12 ¼ 1.8 Hz, H10), 8.53 (dd, 1H, J12-11 ¼ 5.3 Hz, J12-

13

9

¼ 0.95 Hz, H12), 9.27 (s, 1H, OH); C NMR (DMSO-d

6

, 100 mHz) (

d

10 ¼ 1.9 Hz, H12), 9.32 (s, 1H, OH); C NMR (DMSO-d

6

, 100 mHz) (d

ppm) 31.12 (C19), 34.03 (C20), 57.06 (C ), 114.96 (C , C

1

6

4

), 121.38 (C11),

1 6 4

ppm) 56.56 (C ), 115.15 (C , C ), 119.32 (C16), 121.68 (C11), 123.58

11

E. Goya-Jorge, C. Rampal, N. Loones et al.

European Journal of Medicinal Chemistry 207 (2020) 112777

1

(

1

C

9

), 129.94 (C

3

, C

7

), 131.02 (C15, C17), 131.23 (C14, C18), 132.79 (C

2

),

50/50, Rf ¼ 0.73, DCM/MeOH 90/10, Rf ¼ 0.20; H NMR (DMSO-d

6

,

1

),

,

36.83 (C10), 143.16 (C13), 149.21 (C12), 159.91 (C

7

), 162.44 (C ); GC-

8

400 mHz) (

d

ppm) 1.25 (s, 9H, H19), 2.25 (s, 3H, H21), 5.65 (s, 1H, H

, H

), 7.17 (d, 2H, Jortho ¼ 8.3 Hz, H14

, H

, H11), 7.32 (d, 2H, Jortho ¼ 8.4 Hz,

15, H17), 7.74 (ddd, 1H, J10-9 ¼ J10-11 ¼ 7.6 Hz, J10-12 ¼ 2 Hz, H10),

þ.

MS method 180, R

t

¼ 8.81 min, m/z 340 [M ] (100), 324 [M - OH]

7.04 (d, 2H, Jortho ¼ 8.6 Hz, H

4

6

þ.

þ

(

[

3), 259 [M

-

Br] (52), 181 [OHPhCH

2

Ph] (85), 167

H

18), 7.23e7.29 (m, 4H, H

3

, H

7

9

þ

ꢁ1

PhCH

2

Py] (21), 78 [Py] (15); IR (ATR) (cm ) 3055, 3018 (

n

Csp2-

C-O); HPLC

purity: 95%, (Hypersyl Si, n-heptane/EtOAc 30/70, ﬂow rate

H

13

H), 2924 (

n

Csp3-H), 1593, 1511, 1486 (

n

C ¼ C), 1168 (

¼ 3.00 min).

(pyridin-2-yl)methyl)phenol

n

8.51e8.56 (m, 1H, H12); C NMR (DMSO-d

20.79 (C21), 31.09 (C19), 34.06 (C20), 56.91 (C ),121.48 (C

(C11), 123.58 (C ), 125.06 (C15, C17), 128.58 (C14, C18), 129.91 (C

36.79 (C10), 139.88 (C ), 140.61 (C13), 148.55 (C16), 148.77 (C

149.17 (C12), 162.33 (C

6

, 100 mHz) (

d ppm)

1

4

6

, C ),121.65

0

.80 mL/min,

l

max ¼ 263 nm, R

t

9

3

, C

7

5

),

1

2

4

.1.3.4. 4-((4-chlorophenyl)

(4d).

8

), 169.23 (C22); GC-MS method 180,

þ

Yield: 865 mg, 2.94 mmol, white solid, 33%, (Method B).

R

CH

t

¼ 9.38 min, m/z 359 [M ] (99), 344 [M ꢁ CH

3

] (11), 316 [M ꢁ 2

ꢀ

þ

Mp ¼ 170e171 C. TLC CyHex/EtOAc 50/50, Rf ¼ 0.49, DCM/MeOH

3

] (100), 302 [M ꢁ C(CH

3

)

3

] (33), 239 [OHPhCHt-BuPh] (35),

1

þ

9

H

0/10, Rf ¼ 0.70; H NMR (DMSO-d

6

, 400 mHz) (

d

ppm) 5.57 (s, 1H,

224 [OHPhCHt-BuPh - CH

3

](14), 209 [OHPhCHt-BuPh - 2 CH

] (9), 167 [PhCHPy] (23); IR

Csp2-H), 2960, 2905, 2868 (

3

]

þ

1

), 6.70 (d, 2H, Jortho ¼ 8.6 Hz, H

, H

4 6

), 7 (d, 2H, Jortho ¼ 8.6 Hz, H

7

,

(25), 193 [OHPhCHt-BuPh - C(CH

3

)

3

ꢁ

1

3

), 7.19e7.25 (d þ m, 4H, Jortho ¼ 8.5 Hz, H

9

, H11, H14, H18), 7.34 (d,

(ATR) (cm ) 3053 (

C ¼ O), 1587, 1504, 1467 (

disubst).

n

Csp3-H), 1759

Csp2-H p-

2

H, Jortho ¼ 8.5 Hz, H15, H17), 7.73 (ddd, 1H, J10-9 ¼ J10-11 ¼7.6 Hz, J10-

¼ 1.9 Hz, H10), 8.53 (dd, 1H, J12-11 ¼ 5.6 Hz, J12-10 ¼ 2,2 Hz, H12),

(n

n

C ¼ C), 1192 ( C-O), 749 (

n

d

1

9

2

13

6 1

.31 (s, 1H, OH); C NMR (DMSO-d , 100 mHz) (d ppm) 56.48 (C ),

1

C

15.12 (C

4

, C

6

), 121.64 (C11), 123.55 (C

9

), 128.07 (C15, C17), 129.91 (C3,

), 136.80 (C10), 142.70

); GC-MS method 180,

4.1.4.2. 4-(pyridin-2-yl (p-tolyl)methyl)phenyl acetate (5b). The re-

action was performed during 2 h 20 min at 20 C. Yield: 542 mg,

ꢀ

7

), 130.77 (C14, C18), 130.80 (C16), 132.85 (C

2

(

C

13), 149.19 (C12), 155.90 (C

5

), 162.50 (C

8

1.51 mmol, brown oil, 96%. TLC CyHex/EtOAc 50/50, Rf ¼ 0.74, DCM/

þ.

þ

1

R

t

¼ 8.04 min, m/z 295 [M ] (100), 280 [M -OH] (2), 259 [M ꢁ Cl]

MeOH 90/10, Rf ¼ 0.80; H NMR (DMSO-d

6

, 400 mHz) (

d

ppm) 2.25

, H ), 7.21

, H11), 7.31

), 7.51 (d, 2H, Jortho ¼ 8.4 Hz, H15

17), 7.76 (ddd, 1H, J10-9 ¼ J10-11 ¼ 7.6 Hz, J10-12 ¼ 1.80 Hz, H10), 8.56

þ

(

[

(

25), 217 [OHPhCHPhCl] (36), 201 [OHPhCHPhCl -OH] (9), 181

OHPhCHPhCl - Cl] (35), 167 [PhCHPy] (14), 78 [Py] (7); IR (ATR)

cm ) 3054 (

510 (

C ¼ C), 1235 (

o-disubst), 624(

EtOAc 30/70, ﬂow rate 0.80 mL/min,

(s, 3H, H20), 5.72 (s, 1H, H

(d, 2H, J ortho ¼ 8.4 Hz, H14, H18), 7.24e7.26 (m, 3H, H

(dd, 1H, J9-10 ¼ 8 Hz, J9-11 ¼ 2 Hz, H

1

), 7.06 (d, 2H, Jortho ¼ 8.6 Hz, H

4

6

þ

3

, H

7

ꢁ1

n

O-H),

n

3019 (

n

Csp2-H), 2928 (

nCsp3-H), 1615, 1592,

9

,

1

n

C-O), 806 (

dCsp2-H p-disubst), 756 (

dCsp2-H

H

13

n

C-Cl); HPLC purity: 95%, (Hypersyl Si, n-heptane/

max ¼ 263 nm, R ¼ 3.01 min).

(ddd, 1H, J12-11 ¼7.6 Hz, J12-10 ¼ 2.7 Hz, J12-9 ¼ 0.9 Hz, H12); C NMR

(DMSO-d , 100 mHz) ( ppm) 20.83 (C20), 56.40 (C ), 119.64 (C16),

21.68 (C , C ), 121.90 (C11), 123.76 (C ), 129.94 (C , C ), 131.19 (C14

18), 131.24 (C15, C17), 137.02 (C10), 140.08 (C ), 142.39 (C13), 148.97

l

t

6

d

1

4

6

9

3

7

,

4.1.3.5. 4-((4-ﬂuorophenyl)

(pyridin-2-yl)methyl)phenol (4e).

C

2

Yield: 89 mg, 0.32 mmol, yellow oil, 47%, (Method B). TLC CyHex/

(C

5

), 149.35 (C12), 161.66 (C

8

), 169.24 (C19); GC-MS method 180, m/z

1

þ

EtOAc 50/50, Rf ¼ 0.44, DCM/MeOH 90/10, Rf ¼ 0.70; H NMR

R

t

¼ 9.46 min, 382 [M ] (61), 340 [M ꢁ COCH

3

] (100), 261

þ

(

DMSO-d

6

, 400 mHz) (

d

ppm) 5.58 (s, 1H, H

1

), 6.73 (d, 2H,

[M ꢁ CleCOCH

3

] (38), 184 [OPhCH

2

Ph] (46), 167 [PhCH

Csp2-H), 1754 (

C-O), 763 ( Csp2-H p-disubst); HPLC

purity: 98%, (Hypersyl Si, n-heptane/EtOAc 30/70, ﬂow rate

0.80 mL/min, ¼ 2.79 min).

max ¼ 262 nm, R

2

Ph] (24);

ꢁ

1

J

(

ortho ¼ 8.5 Hz, H

m, 2H, H , H11), 7.17e7.27 (m, 4H, H14, H15, H17, H18), 7.70 (ddd, 1H,

10-9 ¼ J10-11 ¼7.8 Hz, J10-12 ¼ 2 Hz, H10), 8.53 (dd, 1H, J12-11 ¼ 4.8 Hz,

4

, H

6

), 7.02 (d, 2H, Jortho ¼ 8.5 Hz, H

7

, H

3

), 7.05e7.14

IR (ATR) (cm ) 3061, 3029, 3009 (

1504, 1487 (

C ¼ C), 1202 (

n

C ¼ O), 1585,

9

n

d

J

(

1

3

12-10 ¼ 1.7 Hz, H12); C NMR (DMSO-d

), 114.81 (d, 2C, JC-F ¼ 21 Hz, C15, C17), 115.24 (C

23.59 (C ), 130.02 (C , C

), 130.83 (d, 2C, JC-F ¼ 8 Hz, C14, C18),

33.31 (C

), 136.85 (C10), 139.70 (d, 1C, JC-F ¼ 3 Hz, C13), 149.25 (C12),

55.95 (C ); GC-MS

), 160.65 (d, 1C, JC-F ¼ 240 Hz, C16), 162.89 (C

method 180, R

6

, 100 mHz) (

d

ppm) 56.64

l

t

C

1

4

6

, C ), 121.68 (C11),

9

3

7

4.1.4.3. 4-((4-bromophenyl) (pyridin-2-yl)methyl)phenyl acetate

(5c). The reaction was performed during 1 h at 20 C. Yield:

344 mg, 0.90 mmol, brown oil, 77%. TLC CyHex/EtOAc 50/50,

ꢀ

2

1

5

8

þ.

1

t

¼ 6.37 min, m/z 278 [M ] (100), 261 [M -OH] (30),

Rf ¼ 0.82; H NMR (DMSO-d

6

, 400 mHz) (

d

ppm) 2.25 (s, 3H, H20),

, H ), 7.24e7.29 (m, 5H,

),

þ

2

01 [OHPhCHPhF] (42), 183 [OHPhCHPhCl -F] (23), 78 [Py] (4);

5.74 (s, 1H, H

, H

1

), 7.07 (d, 2H, Jortho ¼ 8.6 Hz, H

4

6

ꢁ1

IR (ATR) (cm ) 3056, 3005 (

C-O), 811 (

n

Csp2-H), 1592, 1506 (

n

C ¼ C), 1221

H

3

7

, H11, H14, H18), 7.31 (dd, 1H, J9-10 ¼ 7.8 Hz, J9-11 ¼1.20 Hz, H

9

(

n

d

Csp2-H p-disubst), 757 (

d

Csp2-H o-disubst); HPLC

7.38 (d, 2H, Jortho ¼ 8.5 Hz, H15, H17), 7.76 (dd, 1H, J10-11 ¼ 7.8 Hz, J10-

purity: 100%, (Hypersyl Si, n-heptane/EtOAc 30/70, ﬂow rate

12 ¼ 1.9 Hz, H10), 8.56 (ddd, 1H, J12-11 ¼ 7.5 Hz, J12-10 ¼ 2.7 Hz, J12-

13

0

.80 mL/min,

lmax ¼ 264 nm, R

t

¼ 3.07 min).

9

5

¼ 0.9 Hz, H12); C NMR (DMSO-d

6

, 100 mHz) (

), 121.90 (C11), 123.75 (C

7

), 130.85 (C14, C18), 131.11 (C16), 137.70 (C10), 140.16

d

ppm) 20.83 (C20),

6.36 (C

1

), 121.68 (C

, C

), 141.95 (C13), 148.97 (C

4

, C

6

9

), 128.27 (C15, C17),

4.1.4. General procedure for the preparation of triarylmethane

129.94 (C

(C

GC-MS method 180, R

[M ꢁ COCH

3

acetates

To a solution of the corresponding compound (1 eq.) in acetic

anhydride (110 eq.) was added an aqueous solution of sodium hy-

2

5

), 149.35 (C12), 161.73 (C

8

), 169.24 (C19);

þ

t

¼ 8.67 min m/z 338 [M ] (54), 294

þ

3

] (100), 259 [M ꢁ Py] (17), 217 [OPhCH

2

PhCl] (28),

ꢀ

þ

droxide 1 M (1.30 eq.) at 20 C or 40 C. The reaction progress was

monitored by GC-MS and TLC (eluent CyHex/EtOAc 90/10). After

stirring at room temperature, the reaction mixture was concen-

trated, and ethyl acetate was added. The solution was washed with

202 [PyCH

IR (ATR) (cm ) 3055, 3007 (

C ¼ O), 1585, 1505, 1466 ( C ¼ C), 1202 (

disubst), 763 ( Csp2-H p-disubst); HPLC purity: 95%, (Hypersyl Si,

n-heptane/EtOAc 30/70, ﬂow rate 0.80 mL/min,

max ¼ 262 nm,

¼ 2.78 min).

2

PhCl] (7), 184 [OPhCH

2

Ph] (18), 167 [PhCH

Csp2-H), 2917 (

C-O), 817 (

2

Ph] (13);

ꢁ

1

n

Csp3-H), 1754

Csp2-H o-

(n

n

d

water, an aqueous solution of NaHCO

Then the organic phases were dried over anhydrous Na

3

then with brine and water.

SO

l

2

4

,

R

t

ﬁltered and concentrated. The expected compounds were isolated,

and purity was checked by NMR. Yields: 5a (90%), 5b (96%), 5c

4.1.4.4. 4-((4-chlorophenyl) (pyridin-2-yl)methyl)phenyl acetate

(5d). The reaction was performed during 1 h at 20 C. Yield:

ꢀ

(

77%), 5d (76%), and 5e (78%).

3

49 mg, 1.03 mmol, orange oil, 76%. TLC CyHex/EtOAc 50/50,

1

4

.1.4.1. 4-((4-(tert-butyl)phenyl) (pyridin-2-yl)methyl)phenyl acetate

Rf ¼ 0.74, CyHex/EtOAc 50/50, Rf ¼ 0.74; H NMR (DMSO-d

400 mHz) ( ppm) 2.25 (s, 3H, H20), 5.73 (s, 1H, H ), 7.05 (d, 2H, J4-

¼ 8.6 Hz, H , H ), 7.15 (dd, 2H, Jortho ¼ JH-F ¼ 8.9 Hz, H15, H17),

6

,

ꢀ

(

5a). The reaction was performed during 4 h at 20 C then 15 h at

d

1

ꢀ

4

0 C. Yield: 524 mg, 0.35 mmol, brown oil, 90%. TLC CyHex/EtOAc

6

4

6

12

E. Goya-Jorge, C. Rampal, N. Loones et al.

European Journal of Medicinal Chemistry 207 (2020) 112777

7

.23e7.31 (m, 6H, H

¼ 7.7 Hz, J10-12 ¼ 1.9 Hz, H10), 8.55e8.57 (ddd, 1H, J12-9 ¼ 0.80 Hz,

3

, H

7

, H

9

, H11, H14, H18), 7.76 (ddd, 1H, J10-9 ¼ J10-

C18, MeOH/H

2

O 90/10, ﬂow rate 0.80 mL/min,

l

max ¼ 264 nm,

1

R

t

¼ 2.47 min).

13

J

12-10 ¼ 1.8 Hz, J11-12 ¼ 4.8 Hz, H12); C NMR (DMSO-d

ppm) 20.83 (C20), 56.28 (C

), 115.04 (d, 2C, JC-F ¼ 20.90 Hz, C15, C17),

21.63 (C , C ), 121.84 (C11), 123.69 (C ), 129.90 (C , C ), 130.82 (d,

C, JC-F ¼ 7,9 Hz, C14, C18), 136.98 (C10), 139.09 (d, 2C, JC-F ¼ 3.20 Hz,

13), 140.49 (C ), 148.91 (C ), 149.33 (C12), 160.82 (d, 1C, JC-

6

,100 mHz) (d

1

4.1.5.2. 2-((4-acetoxyphenyl) (p-tolyl)methyl)pyridine 1-oxide (6b).

The reaction was performed during 2 h at 20 C. Yield: 92 mg,

ꢀ

1

2

C

4

6

9

3

7

ꢀ

0.28 mmol, white solid, 88%. Mp ¼ 164e166 C. TLC DCM/MeOH 97/

1

2

5

3, Rf ¼ 0.30; H NMR (DMSO-d

6

, 400 mHz) (d ppm) 2.26 (s, 3H,

F

¼ 239.60 Hz, C16), 169.25 (C

8

), 172.06 (C19); GC-MS method 180,

H21), 2.29 (s, 3H, H19), 6.12 (s, 1H, H

1

), 6.96e6.98 (m, 3H, H11, H14,

þ

R

t

¼ 7.18 min, m/z, 321 [M ] (6), 278 [M ꢁ CH

3

CO] (100),183)

Csp2-H), 2927

C ¼ C), 1192 ( C-O),

Csp2-H p-disubst);

HPLC purity: 97%, (Hypersyl Si, n-heptane/EtOAc 30/70, ﬂow rate

H18), 7.08 (s, 4H, H , H , H , H

3

4

6

7

), 7.15 (d, 2H, Jortho ¼ 7.8 Hz, H15, H17),

þ

ꢁ1

[

PyCHPhO] (15); IR (ATR) (cm ) 3051, 3006 (

Csp3-H), 1754 (

C ¼ O), 1571, 1588, 1503 (

160 ( C-F), 819 ( Csp2-H o-disubst), 750 (

n

7.29 (ddd,1H, J10-9 ¼ 7.7 Hz, J10-11 ¼7.8 Hz, J10-12 ¼ 1.30 Hz, H10), 7.35

(ddd, 1H, J9-10 ¼ 7 Hz, J9-11 ¼ 6.4, J9-12 ¼ 2.2 Hz, H ), 8.27 (dd, 1H, J12-

10 ¼ 6.4 Hz, J12-9 ¼ 1.04 Hz, H12); C NMR (DMSO-d , 100 mHz) (

ppm) 20.61 (C21), 20.86 (C19), 48.80 (C ), 121.87 (C , C ), 124.65 (C

10), 126.55 (C11), 128.90 (C14,C18), 129.24 (C , C ), 129.85 (C15, C17),

(

n

9

13

1

n

d

6

d

1

4

6

9

,

0

.80 mL/min,

lmax ¼ 262 nm, R

t

¼ 2.84 min).

C

3

7

136.05 (C16), 137.47 (C

2

), 138.16 (C13), 139.21 (C12), 149.15 (C

8

),

þ

4.1.4.5. 4-((4-ﬂuorophenyl) (pyridin-2-yl)methyl)phenyl acetate (5e).

152.59 (C

5

), 169.21 (C20); LRMS (ESI, CV ¼ 30) 356 [Mþ23] (100),

ꢀ

þ

ꢁ1

The reaction was performed during 3 h at 40 C. Yield: 470 mg,

357 [M þ Hþ23] (15), 689 [2 M þ 23] (12); IR (ATR) (cm ) 3071,

3049 ( Csp2-H), 2921 ( Csp3-H), 1756 (

C ¼ C), 1250 ( N-O), 1202 ( C-O), 838 (

Csp2-H p-disubst); HPLC purity: 97%, (Hypersil ODS C18, MeOH/

O 90/10, ﬂow rate 0.80 mL/min, ¼ 2.38 min).

max ¼ 263 nm, R

1

.46 mmol, brown oil, 78%. TLC CyHex/EtOAc 50/50, Rf ¼ 0.60; H

NMR (DMSO-d , 400 mHz) ( ppm) 2.25 (s, 3H, H20), 5.73 (s, 1H,

), 7.05 (d, 2H, J4-6 ¼ 8.6 Hz, H , H ), 7.15 (dd, 2H, Jortho ¼ JH-

¼ 8.9 Hz, H15, H17), 7.23e7.31 (m, 6H, H , H , H , H11, H14, H18), 7.76

n

C ¼ O), 1607, 1501, 1488

6

d

(n

n

d

Csp2-H o-disubst), 769

H

1

4

6

(d

F

3

7

9

H

2

l

t

(

ddd, 1H, J10-9 ¼ J10-11 ¼7.7 Hz, J10-12 ¼ 1.9 Hz, H10), 8.55e8.57 (ddd,

13

1

H, J12-9 ¼ 0.80 Hz, J12-10 ¼ 1.8 Hz, J11-12 ¼ 4.8 Hz, H12); C NMR

, 100 mHz) ( ppm) 20.83 (C20), 56.28 (C ), 115.04 (d, 2C,

C-F ¼ 20.90 Hz, C15, C17), 121.63 (C , C ), 121.84 (C11), 123.69 (C ),

29.90 (C , C

), 130.82 (d, 2C, JC-F ¼ 7,9 Hz, C14, C18), 136.98 (C10),

39.09 (d, 2C, JC-F ¼ 3.20 Hz, C13), 140.49 (C ), 148.91 (C ), 149.33

4.1.5.3. 2-((4-acetoxyphenyl) (4-bromophenyl)methyl)pyridine 1-

(

J

1

DMSO-d

6

d

1

oxide (6c). The reaction was performed during 2 h 30 min at

ꢀ

4

6

9

20 C. After puriﬁcation by FCC, the product was solubilized in

3

7

dichloromethane and then washed three times with NaHCO

Na CO aqueous solution (1:1) to remove the m-chloroperbenzoic

acid residue. Yield: 78 mg, 0.20 mmol, yellow oil, 66%. TLC DCM/

3

/

2

5

2

3

(

C

12), 160.82 (d, 1C, JC-F ¼ 239.60 Hz, C16), 169.25 (C

8

), 172.06 (C19);

þ

1

GC-MS method 180, R

t

¼ 7.18 min, m/z, 321 [M ] (6), 278

MeOH 97/3, Rf ¼ 0.36; H NMR (DMSO-d

(s, 3H, H20), 6.14 (s, 1H, H

), 7.10 (dd, 1H, J9-10 ¼ 7.8 Hz, J9-11 ¼ 2.1 Hz,

, H , H , H ),

6

, 400 mHz) (d ppm) 2.27

þ

ꢁ1

[M

ꢁ CH

Csp2-H), 2927 (

C ¼ C), 1192 ( C-O), 1160 (

Csp2-H p-disubst); HPLC purity: 97%, (Hypersyl Si, n-heptane/

max ¼ 262 nm, R ¼ 2.84 min).

3

CO] (100),183) [PyCHPhO] (15); IR (ATR) (cm ) 3051,

Csp3-H), 1754 (

C ¼ O), 1571, 1588, 1503

C-F), 819 ( Csp2-H o-disubst), 750

1

3

006 (

n

H

9

), 7.06 (d, 2H, Jortho ¼ 8.4 Hz, H14, H18), 7.11 (s, 4H, H

3

4

6

7

(

n

d

n

d

7.31 (ddd, 1H, J10-9 ¼ 7.8 Hz, J10-11 ¼ 7.7 Hz, J10-12 ¼ 1.3 Hz, H10), 7.38

(ddd, 1H, J11-10 ¼ 7.7 Hz, J11-12 ¼ 7.4 Hz, J11-9 ¼ 2.1 Hz, H11), 7.54 (d,

2H, Jortho ¼ 8.5 Hz, H14, H18), 8.30 (ddd, 1H, J12-11 ¼ 7.4 Hz, J12-

EtOAc 30/70, ﬂow rate 0.80 mL/min,

l

t

13

1

0

¼ 2.8 Hz, J12-9 ¼ 1 Hz, H12); C NMR (DMSO-d

6

, 100 mHz) (

), 120.07 (C16), 122.02 (C , C ),

124.81(C11), 124.88 (C10), 126.55 (C ), 129.99 (C , C ), 131.10 (C14

d

4.1.5. General procedure for the preparation of triarylmethane

ppm) 20.85 (C20), 48.74 (C

1

4

6

acetate N-oxides

9

3

7

,

To a solution of the corresponding triarylmethane acetate (1 eq.)

in anhydrous dichloromethane (0.22 M), was added in one portion

m-chloroperbenzoic acid (3 eq.). The suspension was stirred at

room temperature. The reaction medium became a clear solution

and then pale yellow milky one. The reaction progress was moni-

tored by GC-MS and TLC (eluent CyHex/EtOAc 60/40). At the end of

the reaction, the reaction mixture was neutralized by a 40%

aqueous solution of KOH (pH 7e8) then diluted with distilled water

and extracted with dichloromethane four times. The combined

C

18), 131.52 (C15, C17), 137.45 (C

2

), 139.26 (C12), 139.92 (C13), 149.34

þ

(C ), 151.91 (C

8

5

), 169.17 (C19); LRMS (ESI, CV ¼ 30) 420 [Mþ23]

þ

ꢁ1

(100), 689 [2 M þ 23] (12); IR (ATR) (cm ) 3083 (

n

Csp2-H), 1754

N-O), 1201 ( C-O), 850

Csp2-H p-disubst); HPLC purity: 96%,

O 90/1, ﬂow rate 0,8 mL/min,

(n

C ¼ O), 1505, 1484, 1428 (

n

C ¼ C), 1248 (

n

(d

Csp2-H o-disubst), 771 (

d

(Hypersil ODS C18, MeOH/H

max ¼ 264 nm, R ¼ 2.42 min).

2

l

t

4.1.5.4. 2-((4-acetoxyphenyl) (4-chlorophenyl)methyl)pyridine 1-

organic phases were dried over anhydrous Na

concentrated. The crude product was puriﬁed by FCC on silica gel

eluent DCM/MeOH 97/3) to afford the corresponding N-oxides:

Yields: 6a (43%), 6b (88%), 6c (66%), 6d (38%), and 6e (80%).

2

SO

4

, ﬁltered and

oxide (6d). The reaction was performed during 2 h 30 min at

20 C. After puriﬁcation by FCC, the product was solubilized in

ꢀ

(

dichloromethane and then washed three times with NaHCO

Na CO aqueous solution (1:1) to remove the m-chloroperbenzoic

acid residue. Yield: 40 mg, 0.11 mmol, yellow oil, 38%. TLC DCM/

3

/

2

3

1

4

1

4

.1.5.1. 2-((4-acetoxyphenyl) (4-(tert-butyl)phenyl)methyl)pyridine

-oxide (6a). The reaction was performed during 2 h at 20 C. Yield:

5 mg, 0.12 mmol, light yellow oil, 43%. TLC DCM/MeOH 97/3,

MeOH 97/3, Rf ¼ 0.38; H NMR (DMSO-d

(s, 3H, H20), 6.15 (s, 1H, H

), 6.97 (dd,1H, J9-10 ¼ 7.8 Hz, J9-11 ¼ 2.1 Hz,

, H , H , H ),

6

, 400 mHz) (d ppm) 2.27

ꢀ

1

H

9

), 7.11 (d, 2H, Jortho ¼ 8.4 Hz, H14, H18), 7.11 (s, 4H, H

3

4

6

7

1

Rf ¼ 0.20; H NMR (DMSO-d

6

, 400 mHz) (

), 6.98e7.02 (m, 3H, H11, H14, H18),

, H ), 7.29e7.38 (m, 4H, H , H10, H15

d

ppm) 1.27 (s, 9H, H20),

7.32 (ddd, 1H, J10-9 ¼ 7.8 Hz, J10-11 ¼ 7.7 Hz, J10-12 ¼ 1.3 Hz, H10),

2

.26 (s, 3H, H22), 6.13 (s, 1H, H

, H , H

1

7.36e7.42 (m, 1H, H11), 7.31 (d, 2H, Jortho ¼ 8.5 Hz, H14, H18), 8.29

13

7.07e7.12 (m, 4H, H

3

4

6

7

9

,

(ddd, 1H, J12-11 ¼ 7.3 Hz, J12-10 ¼ 2.8 Hz, J12-9 ¼ 1 Hz, H12); C NMR

13

H

17), 8.27 (dd, 1H, J12-11 ¼ 5.4 Hz, J12-9 ¼ 0.8 Hz, H12); C NMR

(DMSO-d

124.81 (C11), 124.87 (C10), 126.55 (C

), 130.74 (C14, C18), 131.54 (C16), 137.52 (C

(C13), 149.34 (C ), 151.98 (C ), 169.17 (C19); IR (ATR) (cm ) 3080,

3050 ( Csp2-H), 1755( C ¼ C), 1251 ( N-

C ¼ O), 1506, 1487, 1430 (

O), 1200 ( C-O), 857 ( Csp2-H o-disubst), 765 ( Csp2-H p-disubst);

HPLC purity: 96%, (Hypersil ODS C18, MeOH/H O 90/10, ﬂow rate

0.80 mL/min, ¼ 2.38 min).

max ¼ 264 nm, R

6

,100 mHz) (

d

ppm) 20.84 (C20), 48.67 (C

), 128.60 (C15, C17), 129.99 (C

), 139.27 (C12), 139.49

1 4 6

),122.02 (C , C ),

(

DMSO-d

6

, 100 mHz) (

d

ppm) 20.84 (C22), 31.12 (C19), 34.17 (C20),

), 124.63 (C ,C10), 125.42 (C15,C17), 126.51

, C ), 137.43 (C ), 138.11 (C13),

, C16), 152.55 (C ), 169.19 (C21); IR (ATR)

Csp3-H), 1759 (

C ¼ O), 1506,

C-O), 852 ( Csp2-H o-

Csp2-H p-disubst); HPLC purity: 99%, (Hypersil ODS

9

3

,

4

8.71 (C

1

), 121.87 (C

4

, C

6

9

C

7

2

ꢁ1

(

C

11), 128.66 (C14, C18), 129.86 (C

3

7

2

8

5

1

39.20 (C12), 149.13 (C

8

5

n

ꢁ1

(

cm ) 3054 (

486, 1431 (

disubst), 762 (

n

Csp2-H), 2962, 2954 (

n

d

1

n

C ¼ C), 1250 (

n

N-O), 1183 (

n

d

2

d

l

t

13

E. Goya-Jorge, C. Rampal, N. Loones et al.

European Journal of Medicinal Chemistry 207 (2020) 112777

4

.1.5.5. 2-((4-acetoxyphenyl) (4-ﬂuorophenyl)methyl)pyridine 1-

H), 1590, 1509, 1474 (

n

C ¼ C), 1166 (

nC-O), 830 (dCsp2-H p-disubst).

oxide (6e). The reaction was performed during 4 h 30 min at

ꢀ

2

4

0

C. Yield: 220 mg, 0.65 mmol, yellow solid, 80%. Mp ¼ 226-

4.1.6.3. 2-((4-bromophenyl)

(pyridin-2-yl)methyl)phenol

(7c).

ꢀ

1

24 C. TLC DCM/MeOH 97/3, Rf ¼ 0.20; H NMR (DMSO-d

00 mHz) ( ppm) 2.26 (s, 3H, H20), 6.17 (s, 1H, H ), 6.98 (dd, 1H, J9-

¼ 2 Hz, J9-10 ¼ 7.8 Hz, H ), 7.10 (s, 4H, H , H , H15, H17), 7.12e7.20

m, 4H, H , H

, H14, H18), 7.31 (ddd, 1H, J10-9 ¼ J10-11 ¼ 7.7 Hz, J10-

¼ 1.2 Hz, H10), 7.37 (ddd, 1H, J11-10 ¼ J11-12 ¼ 7.7 Hz, J11-9 ¼ 2.1 Hz,

6

,

Yield: 31 mg, 0.09 mmol, orange oil, 23%. TLC DCM/MeOH 90/10,

d

1

Rf ¼ 0.10; H NMR (DMSO-d

6 1

, 400 mHz) (d ppm) 5.61 (s, 1H, H ),

1

9

4

6

.73 (d, 1H, Jortho ¼ 8.4 Hz, H

¼ 2.4 Hz, H ), 7.14e7.20 (m, 3H, H14, H18, H

11), 7.32 (d, 1H, J3-5 ¼ 2.30 Hz, H ), 7.49 (d, 2H, Jortho ¼ 8.5 Hz, H15

6

), 7.06 (ddd, 1H, J4-5 ¼ J4-3 ¼ 8,4 Hz, J4-

(

3

7

6

4

5

), 7.22e7.29 (m, 2H, H

9

,

1

2

H

3

13

H

11), 8.28 (dd, 1H, J12-11 ¼ 6.4 Hz, J12-9 ¼ 1.0 Hz, H12); C NMR

, 100 mHz) ( ppm) 20.85 (C20), 48.49 (C ), 115.42 (d, 2C,

C-F ¼ 20.60 Hz, C15, C17),121.98 (C4, ),124.79 (C10, C11),126.53 (C ),

29.90 (C , C

), 130.85 (d, 2C, JC-F ¼ 8.30 Hz, C14, C18), 136.59 (d, 1C,

), 139.26 (C12), 149.28 (C ), 152.29 (C ),

61.08 (d,1C, JC-F ¼ 241.90 Hz, C16),169.19 (C19); LRMS (ESI, CV ¼ 30)

17), 7.75 (ddd, 1H, J10-9 ¼ J10-11 ¼ 7.7 Hz, J10-12 ¼ 1.9 Hz, H10), 8.54

(

DMSO-d

6

d

1

(

1

ddd, 1H, J12-11 ¼ 4.8 Hz, J12-10 ¼ 1.8 Hz, J12-9 ¼ 0.9 Hz, H12), 10.40 (s,

J

1

C

6

9

13

H, OH); C NMR (DMSO-d

6

, 100 mHz) (

), 119.44 (C16), 121.77 (C11), 123.64 (C

), 131 (C15, C17), 131.08 (C14, C18), 131.24 (C

), 162.07 (C ); IR (ATR) (cm ) 3403

Csp2-H), 2922 ( Csp3-H), 1590, 1486

d

ppm) 56.26 (C

), 127.33 (C , C

), 136.93 (C10), 142.77

1

), 116.40

), 130.53

3

7

(

C

n

6

9

3

5

J

C-F ¼ 2.40 Hz, C13), 137.86 (C

2

5

8

2

4

1

3

ꢁ

1

13), 149.22 (C12), 151.94 (C

O-H), 3068, 2926 (

C ¼ C), 1164 ( C-O).

7

8

þ

ꢁ1

60 (100) [Mþ23] , 361 360 (100) [M þ Hþ23] ; IR (ATR) (cm

066 ( Csp2-H), 2922 ( Csp3-H), 1753 (

C ¼ O), 1603, 1504, 1427

N-O), 1193 ( C-O), 1160 ( C-F), 843 ( Csp2-H o-

Csp2-H p-disubst); HPLC purity: 97%, (Hypersil ODS

O 90/10, ﬂow rate 0.80 mL/min,

max ¼ 264 nm,

¼ 2.25 min).

)

n

(

n

C ¼ C), 1275 (

n

d

disubst), 765 (

C18, MeOH/H

R

d

2

l

4.1.6.4. 2-((4-chlorophenyl)

(pyridin-2-yl)methyl)phenol

(7d).

ꢀ

t

Yield: 106 mg, 0.36 mmol, beige solid, 80%, Mp ¼ 200e202 C. TLC

1

DCM/MeOH 90/10, Rf ¼ 0.21; H NMR (DMSO-d

ppm) 5.65 (s, 1H, H

), 6.73 (d, 1H, Jortho ¼ 8.4 Hz, H

ortho ¼ 8.6 Hz, J4-6 ¼ 2.5 Hz, H ), 7.23 (d, 2H, Jortho ¼ 8.4 Hz, H14, H18),

7.26e7.32 (m, 4H, H , H11, H , H

), 7.37 (d, 2H, Jortho ¼ 8.5 Hz, H15

6

, 400 mHz) (

d

4

.1.6. General procedure for the preparation of o,p-triarylmethanes

To a solution of the corresponding carbinol (1 eq.) and phenol

1

6

), 7.06 (dd, 1H,

J

4

(

1.2 eq.) in nitrobenzene (0.40 M) was added dropwise concen-

9

3

5

,

ꢀ

trated sulfuric acid (20 eq.) at 0 C. The reaction progress was

monitored by GC-MS and TLC (eluent DCM/MeOH 90/10). After

5

neutralized with a saturated solution of NaHCO

extracted with ethyl acetate three times. The combined organic

phases were dried over anhydrous Na SO , ﬁltered and concen-

trated. The crude residue was puriﬁed by FCC on silica gel (eluent

gradient DCM, DCM/MeOH 95/5, DCM/MeOH 90/10) to afford the

corresponding o,p-triarylmethane: Yields: 7a (98%), 7b (45%), 7c

H

17), 7.80 (ddd, 1H, J10-9 ¼ J10-11 ¼ 7.7 Hz, J10-12 ¼ 1.8 Hz, H10), 8.57

13

(dd, 1H, J12-11 ¼ 5.5 Hz, J12-10 ¼ 1.8 Hz, H12), 10.41 (s, 1H, OH);

C

ꢀ

min at 80 C the reaction was cooled to room temperature and

NMR (DMSO-d

6

, 100 mHz) (

, C

4

), 132.55 (C16), 137.47 (C10), 142.13 (C13), 148.96 (C12),

d

ppm) 56 (C

1

), 116.46 (C

6

), 121.98 (C11),

3

(pH 7e8), then

123.83 (C

9

), 127.37 (C

3

5

), 128.22 (C15, C17), 130.56 (C

2

), 130.86 (C14

,

C

18), 131.47 (C

ꢁ

1

2

4

152 (C

2957 (

H p-disubst), 623 (

7

n

), 161.85 (C

Csp3-H), 1591, 1488, 1450 (

C-Cl).

8

); IR (ATR) (cm ) 3422 (

n

O-H), 3064 (

nCsp2-H),

n

C ¼ C), 1163 (

n

C-O), 818 (dCsp2-

n

(

23%), 7d (80%), and 7e (64%).

4

.1.6.5. 2-((4-ﬂuorophenyl)

(pyridin-2-yl)methyl)phenol

(7e).

ꢀ

Yield: 78 mg, 0.28 mmol, light yellow solid, 64%, Mp ¼ 222e224 C.

4

.1.6.1. 2-((4-(tert-butyl)phenyl) (pyridin-2-yl)methyl)phenol (7a).

TLC DCM/MeOH 90/10, Rf ¼ 0.39; 1H NMR (DMSO-d

ppm) 5.62 (s, 1H, H ), 7.06 (dd,1H, J3-

), 6.71 (d,1H, Jortho ¼ 8.5 Hz, H

¼ J4-5 ¼ 8.5 Hz, J4-6 ¼ 2.2 Hz, H ), 7.09e7.16 (m, 2H, H14, H18),

, H11, H15, H17), 7.31 (d, 2H, Jortho ¼ 2.1 Hz, H

), 7.75 (ddd, 1H, J10-9 ¼ J10-11 ¼ 7.7 Hz, J10-12 ¼ 1.9 Hz, H10), 8.54

6

, 400 mHz) (d

ꢀ

Yield: 134 mg, 0.42 mmol, beige solid, 98%, Mp ¼ 202e204 C. TLC

1

6

DCM/MeOH 90/10, Rf ¼ 0.16; H NMR (DMSO-d

ppm) 1.25 (s, 9H, H19), 5.54 (s, 1H, H

), 6.71 (d, 1H, Jortho ¼ 8.4 Hz,

), 7.07 (dd, 1H, J4-6 ¼ 2.3 Hz, Jortho ¼ 8.50 Hz, H ), 7.13 (d, 2H,

ortho ¼ 8.2 Hz, H14, H18), 7.20e7.27 (m, 2H, H , H11), 7.29e7.36 (m,

H, H15, H17, H , H

), 7.74 (ddd, 1H, J10-9 ¼ J10-11 ¼ 7.7 Hz, J10-

¼ 2 Hz, H10), 8.54 (ddd, 1H, J12-11 ¼ 4.7 Hz, J12-10 ¼ 1,8 Hz, J12-

6

, 400 mHz) (d

4

1

7.22e7.28 (m, 4H, H

9

3

,

H

J

4

6

4

H

5

9

13

(

1

dd, 1H, J12-11 ¼ 4.8 Hz, J12-10 ¼ 1.9 Hz, H12); C NMR (DMSO-d

00 mHz) ( ppm) 56.18 (C ), 114.83 (C15, C17), 115.04 (C ), 121.70

11),123.57 (C , C ),127.30 (C ),130.52 (C ),130.83 (C14, C18),131.43

), 136.88 (C10), 139.47 (C13), 149.27 (C12), 157.33 (C ), 159.54 (C16),

Csp2-H), 1595,

Csp2-H p-disubst), 750

6

,

5

3

d

1

6

1

2

(

C

5

3

9

2

1

3

9

¼ 0.9 Hz, H12), 10.38 (s, 1H, OH); C NMR (DMSO-d

), 116.17 (C ), 121.50 (C11),

, C ), 128.58 (C14, C18), 130.37

6

, 100 mHz) (d

4

7

ppm) 31.13 (C19), 34.05 (C20), 56.84 (C

23.42 (C ), 124.97 (C15, C17), 127.24 (C

), 131.42 (C

51.73 (C

1

6

ꢁ

1

(

62.46 (C

506, 1432 (

dCsp2-H o-disubst).

8

); IR (ATR) (cm ) 3424 (

nO-H), 3064 (n

1

(

1

9

3

5

nC ¼ C), 1156 ( C-O), 809 (

n

d

C

2

4

), 136.68 (C10), 140.19 (C13), 148.34 (C16), 149.11 (C12),

þ

7

), 162.77 (C

8

); LRMS (ESI, CV ¼ 30) 318.18 (50) [MþH] ; IR

ꢁ

1

(

ATR) (cm ) 3454 (

n

O-H), 3057 (

n

Csp2-H),

C-O).

n 2959, 2904 (nCsp3-H),

1

489, 1592, 1473 (

nC ¼ C), 1168 (

4.1.7. Preparation of benzotriazole-triarylmethanes

A mixture of benzotriazole (5 mmol, 1 eq.), (4-methoxyphenyl)

(pyridin-2-yl)methanol 9 (4.6 mmol, 1 eq.) and p-toluenesulfonic

acid monohydrate (13 mmol, 2.8 eq.) was stirred and reﬂuxed

4

0

1

d

.1.6.2. 2-(pyridin-2-yl (p-tolyl)methyl)phenol (7b). Yield: 53 mg,

ꢀ

.21 mmol, beige solid, 45%, Mp ¼ 210e212 C. TLC DCM/MeOH 90/

1

ꢀ

0, Rf ¼ 0.20, DCM/MeOH 90/10, Rf ¼ 0.10e0.20; H NMR (DMSO-

overnight in perﬂuorooctane (20 mL, bp 104 C) under argon. The

6

, 400 mHz) (

d

ppm) 2.26 (s, 3H, H19), 5.54 (s, 1H, H

), 7.03 (dd, 1H, J4-3 ¼ J4-5 ¼ 8.5 Hz, J4-6 ¼ 2.3 Hz,

), 7.05e7.13 (m, 5H, H14, H15, H18, H17, H ), 7.19e7.25 (m, 2H, H

11), 7.29 (d, 1H, J9-10 ¼ 2.3 Hz, H ), 7.73 (ddd, 1H, J10-9 ¼ J10-

¼ 7.7 Hz, J10-12 ¼ 1.9 Hz, H10), 8.53 (ddd, 1H, J12-11 ¼ 7.5 Hz, J12-

1

), 6.70 (d, 1H,

perﬂuorocarbon ﬂuid was removed on cooling. A solution of

methanol saturated with KOH (20 mL) was added to the remaining

solid and sonication was applied until solubilization was complete.

Methanol was removed under vacuum and water (20 mL) was

added. The mixture was extracted with ethyl acetate four times.

J

H

ortho ¼ 8.4 Hz, H

6

4

3

5

,

9

11

13

1

0

¼ 4.8 Hz, J12-9 ¼ 0.9 Hz, H12), 10.38 (s, 1H, OH); C NMR (DMSO-

, 100 mHz) ( ppm) 20.61 (C19), 56.91 (C ), 116.28 (C ), 121.54 (C ),

23.46 (C ), 127.30 (C ), 128.83 (C ), 128.89 (C14, C15, C17, C18, C11),

30.40 (C16), 131.51 (C ), 135.22 (C13), 136.71 (C10), 140.27 (C ),

49.16 (C12), 162.87 (C

); LRMS (ESI, CV ¼ 30) 298 (100) [Mþ23] ;

O-H), 3052, 3007 ( Csp2-H), 2922 ( Csp3-

2 4

The combined organic phases were dried over Na SO , ﬁltered and

d

1

6

d

1

6

9

concentrated. The crude product was puriﬁed by FCC on silica gel

(eluent CyHex/EtOAc, 60/40) and afforded the desired compound

10 as a beige solid. The regioisomer 2-((4-methoxyphenyl) (pyr-

idin-2-yl)methyl)-2,3-dihydro-1H-benzo [d] [1e3]triazole 10a was

also isolated and characterized (see SI-1).

5

3

2

4

7

þ

8

n

ꢁ1

IR (ATR) (cm ) 3418 (

n

14

E. Goya-Jorge, C. Rampal, N. Loones et al.

European Journal of Medicinal Chemistry 207 (2020) 112777

1

4

.1.7.1. 1-((4-methoxyphenyl)

(pyridin-2-yl)methyl)-1H-benzo[d]

40, R

6.48 (s, 1H, H

ortho ¼ 8.7 Hz, H

f

0.2; H NMR (DMSO-d

), 6.79 (d, 2H, Jortho ¼ 8.7 Hz, H

, H

6

, 400 mHz) (

d

ppm)

d

3.67 (s, 3H, CH

3

),

ꢀ

[

1e3] triazole (10). Yield: 730 mg (50%). Mp ¼ 170e172 C. TLC

1

4

, H ), 7.43 (d, 2H,

6

1

CyHex/EtOAc 60/40, R

f

¼ 0.20; H NMR (DMSO-d

), 7.00 (d, 2H, Jortho ¼ 8.8 Hz, H , H

), 7.39 (ddd,1H, J11-10 ¼ 7.6 Hz, J11-12 ¼ 4.5 Hz, J11-9 ¼ 0.9 Hz,

11), 7.42 (m,1H, HBt3), 7.50 (ddd,1H, JBt2-Bt1 ¼8.3 Hz, JBt2-Bt3 ¼ 6.9 Hz,

),

6

, 400 mHz) (

d

ppm)

J

3

7

), 7.15 (d, 1H, JN3-N4 ¼ 8.8 Hz, HN3), 7.25 (ddd,

3

H

.78 (s, 3H, CH

3

4

6

), 7.32e7.38 (m, 3H,

1H, JN7-N6 ¼ 7.9 Hz, JN7-N8 ¼ 6.8 Hz, JN7-N9 ¼ 0.85 Hz, HN7), 7.29 (ddd,

1H, J11-10 ¼ 7.5, J11-12 ¼ 4.9 Hz, J11-9 ¼ 1.1 Hz, H11), 7.36 (ddd, 1H, JN8-

N9 ¼ 8.5, JN9-N8 ¼ 7.9 Hz, JN8-N6 ¼ 1.3 Hz, HN8), 7.51 (d, 1H, J9-

3 7 9

, H , H

J

Bt2-Bt4 ¼1 Hz, HBt2), 7.61 (dt,1H, JBt4-Bt3 ¼ 8.4 Hz, HBt4), 7.69 (s,1H, H

1

10 ¼ 7.9 Hz, H ), 7.74 (d, 1H, JN3-N4 ¼ 7.9 Hz, NN4), 7.80 (d, 1H, JN6-

9

7

1

d

C

.88(ddd,1H, J10-9 ¼7.8 Hz, J10-11 ¼7.7 Hz, J10-12 ¼1.8 Hz, H10), 8.09(dd,

N7 ¼ 7.9 Hz, HN6), 7.78e7.83 (m, 1H, H10), 8.18 (d, 1H, JN9-

H, JBt1-Bt2 ¼ JBt4-Bt3 ¼ 8.3Hz,JBt1-Bt3 ¼ JBt4-Bt2 ¼1.8Hz,HBt1), 8.56(ddd,

N8 ¼ 7.9 Hz, HN9), 8.52 (ddd, 1H, J12-11 ¼ 4.9 Hz, J12-10 ¼ 1.8 Hz,

13

H, J12-11 ¼4.8 Hz, J12-10 ¼1.8 Hz, J12-9 ¼ 0.8 Hz, H12); C NMR (DMSO-

J

12-9 ¼ 0.7 Hz, H12), 11.13 (s, 1H, OH); C NMR (DMSO-d

6

, 100 mHz)

6

, 100 mHz) (

),119.20 (C Bt1),122.71 (C11), 123.19 (C

andC ),133.08(C

), 159.14 (C ); IR (ATR) (cm ) 3054, 3005

Csp3-H), 2837( OMe), 1609, 1587, 1510, 1463

C - O); GC-MS method 200, R

¼ 8.83 min m/z: 316

] (100), 79[PyH] (10); HPLC: purity: 95%,

m, A165, Isooctane/EtOAc 70/30, ﬂow rate 1.20 mL/

max ¼ 260 nm, R ¼ 12.3 min).

d

ppm) 55.12 (C13), 66.10 (C

),123.97 (CBt2), 127.30 (C Bt3),

),137.30(C10),145.23(CBt6),

1

), 111.23 (C Bt4), 114.02 (C

6

,

(d

ppm) 48.08 (C

(CN1), 121.89 (CN9), 122.27 (C

(CN8), 128.50 (CN6), 128.57 (CN5), 128.90 (CN4), 129.24 (C

133.37 (CN10), 133.69 (C ), 137.59 (C10), 148.10 (C12), 153.63 (CN2),

1

), 54.92 (CH

3

), 113.39 (C

), 123.20 (CN7), 124.07 (C11), 126.16

and C ),

6 4

, C ), 119.91 (CN3), 120.34

4

9

1

29.12 (C Bt5),130.15 (C

49.37 (C12), 157.44 (C

Csp2-H), 2933,2905(

7

3

2

7

3

ꢁ

1

5

n

8

2

(

[

(

n

157.41 (C

5

), 163.00 (C

8

); GC-MS: method 180, R

t

¼ 11.64 min m/z:

þ

C ¼ C), 1243 (

n

t

341 [M] (100), 324 [M ꢁ OH] (90), 393 [M ꢁ 47] (15); IR (ATR)

þ

ꢁ1

M] (5),198[PyCHPhOCH

Hypersil 250, 5

3

(cm ) 3031 (

n

Csp2-H), 2952, 2929 (

n

Csp3-H), 2834 (

n

OMe), 1618,

m

1597, 1507 (

nC ¼ C), 1243 (

nC-O); HRMS: calcd. for C23H19NO

2

min,

l

t

[MþH]^þ(342.1489), found (342.1489).

4

.1.7.2. 4-((1H-benzo[d] [1e3]triazol-1-yl) (pyridin-2-yl)methyl)

4.1.8.2. 1-((4-hydroxyphenyl) (pyridin-2-yl)methyl)naphthalen-2-ol

(15). To a solution of 14 (0.6 mmol, 1 eq.) in glacial acetic acid

(2.5 mL) under argon, was added 0.6 mL of stabilized hydriodic acid

(d ¼ 1.701, 57%, 4.5 mmol, 7.5 eq.). The mixture was reﬂuxed

phenyl acetate (12). To a previously synthesized solution of 4-((1H-

benzo [d] [1e3]triazol-1-yl) (pyridin-2-yl)methyl)phenol 11

(

0.19 mmol, 1 eq.) (see SI-1) in acetic anhydride (40 mmol, 210 eq.)

ꢀ

at 0 C was slowly added a solution of NaOH 1 M (0.6 mmol, 3.1 eq.).

The mixture was stirred for 24 h at room temperature, concentrated

under vacuum and water was added (10 mL). The mixture was

extracted with ethyl acetate three times then the organic phases

were neutralized with saturated NaHCO

washed with brine and then dried over Na

(T ¼ 100 C) for 5 h 30 then neutralized with a saturated solution of

3

NaHCO (pH z 8). Then the mixture was extracted with ethyl ac-

etate (3 ꢄ 20 mL), dried over Na

2 4

SO and ﬁltrated. The ﬁltrate was

concentrated under vacuum and puriﬁed by FCC on silica gel

(CyHex/EtOAc, 60/40) to provide 15 as a yellow solid.

3

solution (pH z 8),

SO . The product was

ꢀ

2

4

Yield: 70.10 mg (35%). Mp ¼ 218e220 C. TLC CyHex/EtOAc 60/

1

puriﬁed by FCC (eluent DCM/MeOH, 95/5) to afford 12 as a white

40, R

f

0.45; H NMR (DMSO-d

6

, 400 mHz) (

d

ppm) 3.67 (s, 3H, CH

3

),

solid.

6.48 (s, 1H, H

1

), 6.62 (d, 2H, Jortho ¼ 8.5 Hz, H

, H

), 7.14 (d, 1H, JN3-N4 ¼ 7.1 Hz, HN3), 7.23e7.31

),

4 6

, H ), 6.83 (d, 2H,

ꢀ

Yield: 33.6 mg (52%). Mp ¼ 172e174 C. TLC DCM/MeOH 95/5,

J

ortho ¼ 8.5 Hz, H

3

7

1

R

7

1

H

f

¼ 0.2; H NMR (DMSO-d

6

, 400 mHz) (

d

ppm) 2.26 (s, 3H, CH

3

),

(m, 2H, HN7 and H11), 7.37 m,1H, HN8), 7.53 (d,1H, J9-10 ¼ 7.8 Hz, H

9

.17 (d, 2H, Jortho ¼ 8.6 Hz, H

4

, H

6

), 7.31 (d, 1H, J11-10 ¼ 7.7 Hz, H

9

7.73 (d, 1H, JN3-N4 ¼ 7.1 Hz, NN4), 7.77e7.83 (m, 2H, HN6 and H10),

.36 (ddd, 1H, J11-10 ¼ 4.8, J11-12 ¼ 4.7 Hz, J11-9 ¼ 1.1 Hz, H11),

.39e7.42 (m, 1H, HBt2), 7.43 (d, 2H, Jortho ¼ 8.6 Hz, H , H ),

.47e7.51 (m, 1H, HBt3), 7.64 (dt, 1H, JBt4-Bt3 ¼ 8.4 Hz, HBt4), 7.75 (s,

H, H

), 7.86 (ddd, 1H, J10-9 ¼ 7.7 Hz, J10-11 ¼ 7.7 Hz, J10-12 ¼ 1.8 Hz,

10), 8.09 (dd, 1H, JBt1-Bt2 ¼ JBt4-Bt3 ¼ 8.3 Hz, JBt1-Bt3 ¼ JBt4-

8.19 (d, 1H, JN9-N8 ¼ 8.6 Hz, HN9), 8.50e8.54 (m, 1H, H12), 9.19 (s, 1H,

13

3

7

6

OH), 11.20 (s, 1H, OH); C NMR (DMSO-d , 100 mHz) (d ppm) 48.12

(C

1

), 114.77 (C

), 123.20 (CN7), 124.09 (C11), 126.12 (CN8), 128.48 (CN6), 128.56

, C ), 131.92 (CN10), 133.42 (C ), 137.59

6 4

, C ), 119.99 (CN3), 120.50 (CN1), 121.85 (CN9), 122.23

1

9

(CN5), 128.80 (CN4), 129.14 (C

7

3

2

Bt2 ¼ 1.6 Hz, HBt1), 8.56 (ddd, 1H, J12-11 ¼ 4.8 Hz, J12-10 ¼ 1.8 Hz,

(C10), 148.03 (C12), 153.67 (CN2), 155.44 (C

5

), 163.21 (C

8

); GC-MS:

1

3

þ

J

12-9 ¼ 0.85 Hz, H12); C NMR (DMSO-d

6

, 100 mHz) (

, C ), 119.20 (C Bt1), 122.84

and C ),

), 134.72 (C Bt5), 137.44 (C10), 145.21 (C Bt6), 149.46 (C12),

d

ppm) 20.80

method 200, R

t

¼ 9.12 min m/z: 327 [M] (90), 310 [M ꢁ OH] (100);

ꢁ

1

(

CH

3

), 65.85 (C

1

), 111.10 (C Bt4), 122.06 (C

6

4

IR (ATR) (cm ) 3339 (

n

O-H), 3077, 3016 (

CeO), 1227( C-O), 801 (

Csp2-H o-disubst); HRMS: calcd. for

nCsp2-H),1616,1591,1511

C

9

), 123.37 (C11), 124.12 (CBt2), 127.18 (C Bt3), 130.02 (C

33.09 (C

50.31 (C

7

3

(n

C ¼ C), 1410 (

d

n

dCsp2-H p-disubst), 740

1

2

(d

C

22

H17NO

2

[MþH]^þ

ꢁ1

5

), 157.00 (C

Csp2-H), 2923, 2904 (

C - O), 835 (

8

), 169.09 (C]O); IR (ATR) (cm ) 3054, 3005

Csp3 - H), 1750 ( C-O), 1610, 1588, 1509

Csp2-H p-disubst), 772 (

(328.1332), found (328.1332).

(

n

C ¼ C), 1245 (

n

d

Csp2-H o-

4.1.8.3. 4-((2-Acetoxynaphthalen-1-yl) (pyridin-2-yl)methyl)phenyl

acetate (16). To a stirred solution of 15 (0.12 mmol, 1 eq.) in acetic

anhydride (13 mmol, 110 eq.) at 0 C, was slowly added a solution of

NaOH 1 M (0.3 mmol, 2.6 eq.). The mixture was stirred for 24 h at

room temperature, concentrated under vacuum and dissolved in

10 mL of water. The reaction mixture was extracted with ethyl ac-

þ

disubst); GC-MS: method 200, R

t

¼ 9.92 min m/z: 344 [M] (12),

þ

ꢀ

2

26 [M-Bt] (25), 184 [PyCHNH-Ph] (100); HRMS: calcd. for

þ

C

20

H

16

N

4

O

2

[MþNa] (367.1165), found (367.1165).

4

.1.8. Preparation of naphthol triarylmethanes

.1.8.1. 1-((4-methoxyphenyl) (pyridin-2-yl)methyl)naphthalen-2-ol

etate three times, neutralized with saturated NaHCO

(pH z 8). The combined organic phases were washed with brine

dried over Na SO , ﬁltered and concentrated. FCC on silica gel

(CyHex/EtOAc, 50/50) afforded 16 as a transparent oil.

Yield: 47.70 mg (97%). TLC CyHex/EtOAc 70/30, R 0.60; H NMR

(DMSO-d , 400 mHz) ( ppm) 1.84 (s, 3H, CH ), 2.24 (s, 3H, CH ),

3.67, 6.54 (s, 1H, H , H ), 7.09 (d, 1H,

), 7.79 (d, 2H, Jortho ¼ 8.6 Hz, H

N3-N4 ¼ 7.9 Hz, HN3), 7.22 (d, 2H, Jortho ¼ 8.6 Hz, H , H ), 7.20e7.24

(m, 1H, H11), 7.30 (d, 1H, J9-10 ¼ 8.9 Hz, H ), 7.43e7.51 (m, 2H, HN7

N8), 7.72 (ddd, 1H, J10-9 ¼ 7.7, J10-11 ¼ 7.8 Hz, J10-12 ¼ 1.2 Hz, H10),

7.92 (d, 1H, JN6-N7 ¼ 8.9 Hz, HN6), 7.94e7.97 (m, 1H, NN4), 8.16e8.21

(m, 1H, H

), 8.47 (ddd, 1H, J12-11 ¼ 4.9 Hz, J12-10 ¼ 1.9 Hz, J12-

3

solution

(

14). 2-Naphthol 13 (2.70 mmol, 1.2 eq.) was mixed with (4-

methoxyphenyl) (pyridin-2-yl)methanol (2.30 mmol 1.5 eq.) in

dichloroethane (2.5 mL) and sulfamic acid (3.45 mmol, 1.5 eq.)

under argon. The mixture was heated at 85 C for 20 h. After re-

2

4

ꢀ

1

f

action completion, the reaction mixture was cooled at room tem-

6

d

3

perature, neutralized with a saturated solution of NaHCO

and extracted with dichloromethane three times. The organic

layers were dried over MgSO , ﬁltered and concentrated under

vacuum. The product was puriﬁed by FCC on silica gel (DCM 100%)

to afford 14 as a whitish solid.

3

(pH z 8)

1

4

6

J

3

7

4

9

,

H

ꢀ

Yield: 38.5 mg (49%). Mp ¼ 158e160 C. TLC CyHex/EtOAc 60/

9

15

E. Goya-Jorge, C. Rampal, N. Loones et al.

European Journal of Medicinal Chemistry 207 (2020) 112777

¼ 0.9 Hz, H12); ¹³C NMR (DMSO-d

, 100 mHz) (

d

ppm) 25.66 and

),

NMR (DMSO-d

(CIn4), 116.56 (CIn2), 118.42 (C11), 118.72 (C

), 121.56 (CIn5), 122.91 (CIn1), 123.94 (CIn6), 126.42 (CIn3), 129.56

, C ), 136.40 (C ), 136.70 (C10), 140.81 (CIn8), 148.70 (C ), 149.06

(C12), 162.89 (C ), 169.24 (C]O); GC-MS: method 180,

¼ 11.76 min m/z: 342 [M] (90), 300 [M ꢁ COCH

, 100 mHz) (

d

ppm) 20.80 (CH

9

2

1

C

(

1

[

(

6

3 1

), 49.82 (C ), 111.49

6.05 (CH

3

), 55.24 (C

1

), 126.73 (C

6

, C ), 126.93 (CN3),128.32 (C

4

9

), 121.09 (CIn7), 121.36 (C

6

,

30.19 (CN9),130.56 (CN7),131.80 (CN8),133.76 (CN5),133.85 (CN4 and

N6), 129.14 (C , C ), 137.01 (CN1), 137.58 (CN10), 141.94 (C10), 143.90

), 152.00 (CN2), 154.05 (C ), 154.15 (C12), 167.21 (C ), 173.90 and

74.46 (C]O); GC-MS: method 180, R

C

4

7

3

(C

7

3

2

5

C

2

5

8

þ

t

¼ 12.39 min m/z: 411

R

t

3

]

(100), 264 [M-

PyH] (15), 222 [PyCHIn] (100); IR (ATR) (cm ) 3408 ( N-H). 3060

C ¼ O), 1589, 1570, 1505

Csp2-H o-disubst); HRMS: calcd. for

þ

ꢁ

þ

ꢁ1

M] (1), 369 [M ꢁ Ac] (3), 252 [PyCHPhNapth] (100); IR (ATR)

n

1

cm ) 3059 (

469 (

C ¼ C), 1180 (

for C26

[MþNa] (434.1363), found (434.1363).

n

Csp2-H). 2926 (

n

Csp3-H), 1749 (

n

C ¼ O), 1572, 1503,

(

n

Csp2 - H), 2989 (nCsp3 - H), 1749 (n

1

n

C - O), 811 (

dCsp2-H p-disubst). HRMS: calcd.

C ¼ C), 1223 (

n

C - O), 743 (

d

þ

H21NO

4

C

H

22 18

N

2

O

2

[MþNa] (365.1260), found (365.1260).

4

1

.1.9. Preparation of indole triarylmethanes

.1.9.1. 3-((4-methoxyphenyl) (pyridin-2-yl)methyl)-3a,7a-dihydro-

H-indole (18). Indole 17 (4.7 mmol, 2 eq.) was mixed with (4-

4.1.10. Preparation of thiophen triarylmethanes

4.1.10.1. 2-((4-methoxyphenyl) (thiophen-2-yl)methyl)pyridine (21).

A

mixture of (4-methoxyphenyl) (pyridin-2-yl)methanol

9

methoxyphenyl) (pyridin-2-yl)methanol 9 (2.3 mmol, 1 eq.),

(1.3 mmol, 1 eq.), thiophene 20 (13 mmol, 10 eq.) and meth-

anesulfonic acid (0.7 mL, d ¼ 1,48,10 mmol, 8 eq.) in dichloroethane

(6 mL) was placed in a vial. The vial was sealed, and the mixture was

stirred and submitted to microwave irradiation for 2 h (300 W

dichloroethane (2.5 mL) and sulfamic acid (2.3 mmol, 1 eq.) under

ꢀ

argon. The mixture was heated at 85 C during 20 h. After reaction,

the mixture was cooled to room temperature. A solution of meth-

anol saturated with NaOH (20 mL) was added to the remaining

solid and sonication was applied until complete solubilization.

Methanol was removed under vacuum and water (20 mL) was

added. The mixture was extracted with dichloromethane three

ꢀ

power, T ¼ 80 C). The mixture was neutralized with saturated

3

NaHCO solution (pH z 8), extracted with dichloromethane

(4 ꢄ 30 mL) and dried MgSO

SiO (10 mL) and puriﬁcation on FCC (CyHex/EtOAc, 70/30) afforded

21 as greenish oil.

Yield: 80 mg (25%). TLC CyHex/EtOAc 70/30, R

(DMSO-d , 400 mHz) ( ppm) 3.71 (s, 3H, CH ), 5.80 (s, 1H, H

(dt, 1H, JT2-T3 ¼ 3.5 Hz, JT2-T4 ¼ 2.4 Hz, HT2), 6.86 (d, 2H,

ortho ¼ 8.8 Hz, H , H ), 6.93 (dd, 1H, JT3-T2 ¼ 3.5 Hz, JT3-T4 ¼ 5.1 Hz,

T3), 7.23e7.27 (m, 1H, HT4), 7.25 (d, 2H, Jortho ¼ 8.8 Hz, H , H ), 7.34

(d, 1H, J11-10 ¼ 7.8 Hz, H11), 7.38 (m, 1H, H ), 7.74 (ddd, 1H, J10-

¼ 7.7 Hz, J10-11 ¼ 7.7 Hz, J10-12 ¼ 1.9 Hz, H10), 8.51 (ddd, 1H, J12-

4

. Concentration under vacuum over

2

2 4

times. The combined organic phases were dried over Na SO ,

ﬁltered and concentrated. FCC on silica gel (CyHex/EtOAc, 50/50)

afforded 18 as a brown solid.

f

0.4; ¹H NMR

), 6.80

6

d

3

1

ꢀ

Yield: 59 mg (81%). Mp ¼ 136e138 C. TLC: CyHex/EtOAc 50/50,

1

R

f

0.4; H NMR (DMSO-d

6

, 400 mHz) (

d

ppm) 3.70 (s, 3H, CH

3

), 5.69

J

4

6

(

H

s, 1H, H

In1, HIn6), 7.0 (ddd, 1H, JIn7- In6 ¼ 8.0 Hz, JIn7- In5 ¼ 7.0 Hz, J In7

In4 ¼ 1.1 Hz, HIn7), 7.14 (d, 1H, J11-10 ¼ 7.7 Hz, H11), 7.20 (ddd, 1H, J9-

¼ 7.8 Hz, J9-11 ¼ 4.9 Hz, J9-12 ¼ 1.1 Hz, H ), 7.23 (d, 2H,

ortho ¼ 8.6 Hz, H , H ), 7.29 (dt, 1H, JIn4- In5 ¼ 8.9 Hz, HIn4), 7.35 (dt,

H, NIn5, J In5

In4 ¼ 8.9 Hz, HIn5), 7.68 (ddd, 1H, J10-9 ¼ 7.7 Hz, J10-

¼ 7.7 Hz, J10-12 ¼ 1.8 Hz, H10), 8.51 (ddd, 1H, J12-11 ¼ 4.9 Hz, J12-

1

), 6.84 (d, 2H, Jortho ¼ 8.7 Hz, H

4

, H

6

), 6.82e6.88 (m, 2H,

H

3

7

e

9

1

3

1

J

1

0

9

11 ¼ 4.8 Hz, J12-10 ¼ 1.8 Hz, J12-9 ¼ 0.8 Hz, H12); C NMR (DMSO-d

100 mHz) ( ppm) 52.53 (C ), 54.99 (CH ), 113.64 (C , C ), 121.10

(C11), 122.18 (C ), 125.10 (CT4), 125.10 (CT2), 126.41 (CT3), 129.47 (C

), 135.09 (C

6

,

3

7

d

1

3

4

6

e

9

3

,

11

C

7

2

), 136.93 (C10), 146.68 (CT1), 149.12 (C12), 157.90 (C

5

),

13

þ

1

0

¼ 1.9 Hz, J12-9 ¼ 0.8 Hz, H12), 10.9 (s, 1H, NH); C NMR (DMSO-d

00 mHz) ( ppm) 49.08 (C ), 55.92 (CH ),111.49 (CIn4),114.56 (CIn2),

18.50 (C11), 119.72 (C ), 120.09 (CIn7), 120.36 (C , C ), 122.56 (CIn5),

22.90 (CIn1), 122.94 (CIn6), 128.42 (CIn3), 130.56 (C , C ), 135.40 (C ),

); GC-

6

,

162.89 (C

8

); GC-MS: method 160, R

t

¼ 8.06 min m/z: 281 [M] (100),

þ

ꢁ1

d

1

3

266 [M ꢁ CH

3

] (25), 203 [ThCHPhOCH

3

] (100); IR (ATR) (cm

Csp2-H), 2962, 2932 ( Csp3-H), 2837 ( OMe),

C ¼ C),1242( C-O), 808 (

Csp2-H o-disubst); HRMS: calcd. for

(282.0947), found (282.0946).

)

9

6

4

3081, 3048 3005 (

1604, 1584,1509 (

n

7

3

2

n

dCsp2-H p-disubst), 702

1

36.80 (C10), 139.81 (CIn8), 148.80 (C

5

), 149.08 (C12), 160.89 (C

8

(d

C

17

H

15NOS [MþH]^þ

þ

MS: method 160, R

t

¼ 13.14 min m/z: 314 [M] (90), 299

þ

ꢁ1

[

M ꢁ 15] (10), 236 [InCHPhOCH

3

] (100); IR (ATR) (cm ) 3412

Csp2-H), 2920 ( Csp3-H), 2857 ( OMe), 1592,

C ¼ C), 1246 ( C - O), 743 ( Csp2-H o-disubst); HRMS:

O [MþH] (315.1492), found (315.1492).

(

n

N-H). 3052, 3009 (

507, 1456 (

calcd. for C21

n

4.1.10.2. 4-(Pyridin-2-yl(thiophen-2-yl)methyl)phenol (22). To

a

1

n

H

n

d

stirred solution of 21 (0.5 mmol, 1 eq.) in anhydrous dichloro-

þ

ꢀ

18

N

2

methane (7 mL) under argon at 0 C, was added dropwise a 1 M

solution of BBr

room temperature, MeOH (10 mL) was added. The solution was

dried over Na SO , ﬁltrated and concentrated under vacuum over

SiO (10 mL). The dry SiO powder was loaded onto a silica gel

3

in dichloromethane (2.4 mmol, 5 eq.). After 19 h at

4.1.9.2. 4-((3a,7a-dihydro-1H-indol-3-yl) (pyridin-2-yl)methyl)

phenyl acetate (19). To a stirred solution of 18a (1.6 mmol, 1 eq.) in

2

4

ꢀ

acetic anhydride (183 mmol, 115 eq.) at 0 C, was slowly added a

2

solution of NaOH 1 M (2.15 mmol, 1.3 eq.). The mixture was stirred

for 3 h 30 at room temperature and concentrated under vacuum

and dissolved in 10 mL of water. The reaction mixture was extracted

with ethyl acetate three times and neutralized with saturated

column and eluted (DCM/MeOH, 95/5). Concentration under vac-

uum afforded 22 as a black oil.

Yield: 80 mg (60%). TLC DCM/MeOH 95/5, R

(DMSO-d , 400 mHz) ( ppm) 5.72 (s, 1H, H ), 6.67 (d, 2H,

, H

), 6.80 (dt, 1H, JT2-T3 ¼ 3.4 Hz, JT2-T4 ¼ 2.1 Hz,

T2), 6.93 (dd, 1H, JT3-T2 ¼ 3.5 Hz, JT3-T4 ¼ 5.1 Hz, HT3), 7.11 (d, 2H,

, H

), 7.24 (dd, 1H, JT4-T3 ¼ 7.5 Hz, JT4-T2 ¼ 2.1 Hz,

), 7.73 (ddd, 1H,

0.3; ¹H NMR

f

6

d

1

NaHCO

3

solution (pH z 8). The combined organic phases were

SO , ﬁltered and concentrated.

J

H

ortho ¼ 8.6 Hz, H

4

6

washed with brine, drying over Na

2

4

FCC on silica gel (CyHex/EtOAc, 30/70) afforded 19 as a light brown

J

H

ortho ¼ 8.6 Hz, H

3

7

solid.

T4), 7.32 (d, 1H, J11-10 ¼ 7.9 Hz, H11), 7.36 (m, 1H, H

9

ꢀ

Yield: 39 mg (68%). Mp ¼ 152e154 C. TLC CyHex/EtOAc 30/70,

J

10-9 ¼ 7.7 Hz, J10-11 ¼ 7.7 Hz, J10-12 ¼ 1.9 Hz, H10), 8.53 (ddd, 1H, J12-

0.60; ¹H NMR (DMSO-d

, 400 mHz) (

d

ppm) 2.23 (s, 3H, CH

),

11 ¼ 4.9 Hz, J12-10 ¼ 1.8 Hz, J12-9 ¼ 0.9 Hz, H12); C NMR (DMSO-d

13

R

f

6

3

6

,

5

7

.78 (s, 1H, H

.02 (d, 2H, Jortho ¼ 8.7 Hz, H

In5 ¼ 7.0 Hz, J In7 In4 ¼ 1.1 Hz, HIn7), 7.14 (d, 1H, J11-10 ¼ 7.9 Hz, H11),

.22 (ddd,1H, J9-10 ¼ 7.5 Hz, J9-11 ¼4.8 Hz, J9-12 ¼ 1.1 Hz, H ), 7.32 (m,

), 7.71 (ddd, 1H, J10-

¼ 8.6 Hz, J10-11 ¼ 7.6 Hz, J10-12 ¼ 1.9 Hz, H10), 8.51 (ddd, 1H, J12-

1

), 6.85e6.87 (m, 1H, HIn1), 6.88e6.89 (m, 1H, HIn6),

100 mHz) (

122.20 (C ), 125.24 (CT4), 125.36 (CT2), 126.40 (CT3), 130.47 (C

135.45 (C

162.40 (C

IR (ATR) (cm ) 3400 (

1249( C-O), 816 (

HRMS: calcd. for C16

1 4 6

d ppm) (d ppm) 50.50 (C ), 114.68 (C , C ), 120.10 (C11),

4

, H

6

), 7.03 (dd,1H, JIn7- In6 ¼ 8.0 Hz, JIn7-

9

3

, C

7

),

e

2

), 136.83 (C10), 146.74 (CT1), 149.10 (C12), 157.76 (C

5

þ

7

9

8

); GC-MS: method 180, R

t

¼ 6.5 min m/z: 267 [M] (100);

ꢁ

1

2

H, HIn4, HIn5), 7.35 (d, 2H, Jortho ¼ 8.7 Hz, H

3

, H

7

n

C-O), 3001 (

n

Csp2-H), 1590, 1511 (

n

C ¼ C),

9

n

dCsp2-H p-disubst), 751(dCsp2-H o-disubst);

13

13NOS [MþH]^þ(268.0791), found (268.0790).

11

¼ 4.8 Hz, J12-10 ¼ 1.8 Hz, J12-9 ¼ 0.8 Hz, H12), 10.94 (s, 1H, NH);

C

H

16

E. Goya-Jorge, C. Rampal, N. Loones et al.

European Journal of Medicinal Chemistry 207 (2020) 112777

4

.1.11. Preparation of quinoline triarylmethane

.1.11.1. (4-methoxyphenyl) (pyridin-2-yl) (quinolin-2-yl)methanol

4 h at room temperature then neutralized with a saturated solution

of NaHCO (pH z 8). Acetone was added (10 mL) and the mixture

3

(

25). n-Butyllithium in hexane (1.6 M, 2.5 mmol, 1.3 eq.) was added

was stirred for another 30 min (a white precipitate of sodium ac-

etate formed). The mixture was extracted with ethyl acetate three

dropwise to a stirred solution of 2-bromoquinoline 24 (2.2 mmol,

1

ꢀ

.1 eq.) in anhydrous tetrahydrofuran (2 mL) at ꢁ78 C under argon.

2 4

times, dried with Na SO , ﬁltrated and concentrated under vacuum.

After 1 h 30, (4-methoxyphenyl) (pyridin-2-yl)methanone 23

1.9 mmol, 1 eq.) in dry tetrahydrofuran (3 mL) was added drop-

FCC on silica gel (CyHex/EtOAc, 50/50) afforded 30 as a yellow oil.

1

(

Yield: 130 mg (87%). TLC: CyHex/EtOAc 50/50, R

(DMSO-d , 400 mHz) ( ppm) 2.30 (s, 3H, CH ), 6.45 (s, 1H, C

(d, 2H, Jortho ¼ 8.6 Hz, H16, H18), 7.24e7.30 (m, 3H, H11, H15, H19), 7.33

(d, 1H, J9-10 ¼ 7.9 Hz, H ), 7.47 (d, 2H, Jortho ¼ 8.4 Hz, H , H ), 7.67 (d,

2H, Jortho ¼ 8.2 Hz, H , H ), 7.76 (ddd, 1H, J10-9 ¼ 8.6 Hz, J10-

11 ¼ 7.6 Hz, J10-12 ¼ 1.9 Hz, H10), 8.57 (ddd, 1H, J12-11 ¼ 4.8 Hz, J12-

f

0.45; H NMR

wise. After 17 h at room temperature, water (10 mL) was added. The

reaction mixture was extracted with ethyl acetate three times,

6

d

3

1

), 7.07

dried over Na

0/20) afforded 25 as a brown oil.

Yield: 53 mg (81%). TLC CyHex/EtOAc 80/20, R

DMSO-d , 400 mHz) ( ppm) 3.70 (s, 3H, CH ), 6.85 (d, 2H,

, H

), 7.06 (s, 1H, OH), 7.24 (d, 2H, Jortho ¼ 8.9 Hz,

), 7.29 (ddd, 1H, J11-10 ¼ 7.7 Hz, J11-12 ¼ 4.8 Hz, J11-9 ¼ 1.1 Hz,

11), 7.59 (ddd, 1H, JQ6-Q5 ¼ 8 Hz, JQ6-Q7 ¼ 6.9 Hz, JQ6-Q8 ¼ 1.1 Hz,

Q6), 7.72e7.77 (m, 3H, H

, HQ2, HQ5), 7.82 (ddd, 1H, J10-9 ¼ 7.8 Hz,

2

SO

4

, ﬁltered and concentrated. FCC (CyHex/EtOAc,

9

3

7

8

4

6

0.3; ¹H NMR

f

1

3

(

6

d

3

10 ¼ 1.9 Hz, J12-9 ¼ 0.9 Hz, H12); C NMR (DMSO-d

ppm) 20.78 (CH ), 56.65 (C ), 121.72 (C16, C18), 121.95 (C11), 122.93

(C ), 123.83 (C , C ), 129.73 (C , C ), 129.79 (C15, C19), 129.96 (CF ),

136.83 (C10), 139.70 (C ), 147.66 (C14), 149.04 (C ), 149.36 (C12),

161.31 (C17), 169.16 (C

6

, 100 mHz) (d

J

ortho ¼ 8.9 Hz, H

4

6

3

1

H

3

, H

7

9

4

6

3

7

3

5

2

9

8

), 170.29 (C]O); GC-MS: method 180,

þ

J

(

10-11 ¼7.4 Hz, J10-12 ¼ 1.9 Hz, H10), 7.94e7.97 (m, 2H, HQ7, HQ8), 8.32

d, 1H, JQ3-Q2 ¼ 8.0 Hz, HQ3), 8.49 (ddd, 1H, J12-11 ¼ 4.8 Hz, J12-

R

t

¼ 6.96 min m/z 371 [M ] (50), 328 [M ꢁ Ac] (100); IR (ATR)

ꢁ

1

(cm ) 3051 (

n

Csp2 eH), 2931 (

n

Csp3-H), 1755 (

n

C]O), 1618, 1588,

13

1

0

¼ 1.8 Hz, J12-9 ¼ 0.9 Hz, H12); C NMR (DMSO-d

ppm) 54.98 (CH ), 90.63 (C ), 113.01(C , C ), 121.23 (C11), 122.24 and

22.28 (CQ2, CQ5), 126.67 (CQ8), 126.79 (CQ4), 127.69 (CQ7), 128.49

11), 128.77 (C and C ), 129.70 (C ), 136.15 (C10), 136.74 (CQ3),

37.86 (C ), 145.16 (CQ9), 147.49 (C12), 158.15 (C

63.99 (C

6

, 100 mHz) (

d

1505 (

n

C ¼ C), 1323 (

n

C-O), 1108 (

n

H

C-O-C), 1066 (

n

C-F), 823 (

d

Csp2-

þ

3

1

4

6

H p-disubst); HRMS: calcd. for C21

found (394.1026).

16

F

3

O

2

Na [Mþ23] (394.1025),

1

(

1

3

C

3

7

9

2

8

), 163.81 (CQ1),

4.2. AhR transcriptional activity

4.2.1. Materials

Unless otherwise speciﬁed, chemical reagents and biological

products for in vitro assays were obtained from Gibco, Life Tech-

nologies (Thermo Fisher Scientiﬁc Inc), MilliporeSigma (Merck

KGaA) or InvivoGen. Fungible material was provided by Falcon®,

Corning ® or Eppendorf ®.

þ

5

); GC-MS: method 180, R

t

¼ 10.71 min m/z: 342 [M] (75),

þ

ꢁ1

25 [M ꢁ OH] (75), 128 [C

9

H

6

N] (75); IR (ATR) (cm ) 3408 (

nO-

H). 3031, 3025 (

587, 1509 (

C ¼ C), 1220(

calcd. for C22

n

Csp2H), 2992 (

n

Csp3 eH), 2851 (

n

OMe), 1580,

1

n

H

n

C-O), 742 (

d

Csp2-H o-disubst); HRMS:

þ

18

N

2

O

2

[MþH] (343.1368), found (343.1367).

4

.1.12. Preparation of triarylmethanes bearing triﬂuoromethyl

.1.12.1. (4-methoxyphenyl) (pyridin-2-yl) (4-(triﬂuoromethyl)

phenyl)methanol (28). n-Butyllithium in hexane (1.6 M, 2.8 mmol,

4.2.2. Cell culture

1

.6 eq.) was added dropwise to a stirred solution of 4-bromoanisole

Cell-Line and luciferase assay system. AhR-Lucia™ Human liver

carcinoma HepG2 (AhR-HepG2) reporter cells were obtained from

InvivoGen engineered to detect endogenous AhR expression. This

cell line is stably transfected with a pSELECT-zeo-Lucia plasmid that

ꢀ

(

2.5 mmol, 1.4 eq.) in anhydrous tetrahydrofuran (2 mL) at ꢁ78 C

under argon. After 1½ h, pyridin-2-yl (4-(triﬂuoromethyl)phenyl)

methanone 27 (1.7 mmol, 1 eq.) dissolved in dry tetrahydrofuran

(

1 mL) was added dropwise. After 17 h at room temperature, water

30 mL) was added. Extraction with ethyl acetate (4 ꢄ 30 mL),

contains the resistance marker to the antibiotic Zeocin™. EF-1a/

HTLV composite promoter is combined to the elongation factor 1

alpha core promoter and the 5’ untranslated region of the Human T-

cell Leukemia Virus. The secreted luciferase Lucia™ is expressed by

2 4

drying over Na SO , concentration under vacuum and column

chromatography (CyHex/EtOAc, 70/30) afforded 28 as a white solid.

ꢀ

Yield: 39 mg (67%). Mp ¼ 98e100 C. TLC CyHex/EtOAc 70/30, R

f

a synthetic reporter gene codon optimized for prolonged

1

0

1

.5; H NMR (DMSO-d

6

, 400 mHz) (

d

ppm) 3.55 (s, 3H, CH

3

), 6.45 (s,

mammalian cell expression. The promoter is coupled with the

human Cyp1a1 gene entire regulatory sequence, that contains six

XREs. The secreted coelenterazine Lucia™ is a novel luciferase re-

porter technology that does not involve cells lysis to measure the

bioluminescence.

Quality and sterility. Quality control of the reporter activity and

guaranteed of mycoplasma-free contamination was provided by

the cell line suppliers. Additional routine inspections were con-

ducted as standard quality control procedures to avoid myco-

plasma, fungi, yeast and/or viruses’ contamination. All the

experiments were steered with less than 20 passages after thawing

as recommended. The cell culture facilities from the Central Service

for Experimental Research (SCSIE) at the University of Valencia

where the experiments were conducted have certiﬁed proﬁciency

to maintain, subculturing and guarantee aseptic conditions.

Maintenance. AhR-HepG2 cells were handled and cultured ac-

cording to supplier’s information under strict sterility conditions in

H, OH), 6.70 (d, 2H, Jortho ¼ 9.0 Hz, H16, H18), 6.90 (d, 2H,

J

ortho ¼ 9.0 Hz, H15, H19), 7.15 (ddd, 1H, J11-10 ¼ 7.6 Hz, J11-12 ¼ 4.8 Hz,

11-9 ¼ 1.1 Hz, H11), 7.30 (d, 2H, Jortho ¼ 8.2 Hz, H , H ), 7.50 (d, 2H,

ortho ¼ 8.2 Hz, H , H ), 7.48 (d, 1H, J9-10 ¼ 8.1 Hz, H ), 7.60 (ddd, 1H,

10-9 ¼ 7.9 Hz, J10-11 ¼ 7.5 Hz, J10-12 ¼ 1.9 Hz, H10), 8.31 (ddd, 1H, J12-

3

7

4

6

9

13

1

¼ 4.7 Hz, J12-10 ¼ 1. Hz, J12-9 ¼ 0.84 Hz, H12); C NMR (DMSO-d

00 mHz) ( ppm) 54.98 (CH ), 80.15 (C ), 112.94 (C16, C18), 121.30

), 122.04 (C11), 124.21 and 124.25 (C , C ), 126.60 (C ,C ), 128.94

15, C19), 129.10 (CF ), 136.75 (C10), 138.60 (C ), 147.91 (C12), 151.90

6

,

1

d

3

1

(

C

9

4

6

3

7

3

5

2

), 137.86 (C

5

), 158.09 (C17), 164.59 (C

8

); GC-MS: method 160,

þ

R

t

¼ 8.59 min m/z 359 [M ](100), 281 [HOPhCOHPhCF

3

]

(43), 252

þ

ꢁ1

[

3

1

CH

423 (

590, 1525 (

3

OPhCH

2

PhCF

3

]

(20), 173 [OCPhCF

Csp2-H), 2934 ( Csp3-H), 2854 (

C-O), 1109 (

3

]

(65); IR (ATR) (cm

)

n

OH), 3012 (

n

OMe), 1612,

n

C ¼ C), 1324 (

n

C-O-C), 1065 (

n

C-F), 831

(

d

Csp2-H p-disubst); HRMS: calcd. for C20

H

16

F

3

NO

2

[MþH]^þ

360.1206), found (360.1206).

T-75 culture ﬂasks under an aqueous saturated atmosphere with 5%

ꢀ

4.1.12.2. 4-(hydroxy(pyridin-2-yl)

(4-(triﬂuoromethyl)phenyl)

CO

2

at 37 C. The growth medium was prepared as follow: to

methyl)phenyl acetate (30). To a stirred solution of 4-(pyridin-2-yl

4-(triﬂuoromethyl)phenyl)methyl)phenol 29 (0.4 mmol, 1 eq.) in

Minimum Essential Medium (MEM) containing non-essential

ꢀ

(

amino acids (NEAA) was added 10% (v/v) of 56

C heat-

acetic anhydride (44 mmol, 110 eq.) was slowly added a solution of

NaOH 1 M (0.5 mmol, 1.3 eq.) at 0 C. The mixture was stirred for

inactivated fetal bovine serum (FBS) and a mixture of Penicillin-

Streptomycin (100 U/mL-100 g/mL). The antimicrobial formula

ꢀ

m

17

E. Goya-Jorge, C. Rampal, N. Loones et al.

European Journal of Medicinal Chemistry 207 (2020) 112777

Normocin™ (0.1 mg/mL) and, after the third passage, the selective

antibiotic Zeocin™ (0.2 mg/mL) were supplemented to prevent

mycoplasma, bacterial and fungal contamination.

coelenterazine substrate for the luciferase reaction was prepared

according to suppliers by pouring the lyophilized powder protected

from light in sterile water. Finally, 50 mL/well of the QUANTI-Luc

Subculturing. Once the cells reached 85% conﬂuency in the

culture ﬂasks, they were rinsed twice with 10 mL of PBS and later

detached through the incubation with 3e5 mL of 0.25% trypsin-

solution was added and the light signal produced was immedi-

ately measured in a microplate reader (VICTORx3, PerkinElmer Inc.,

USA).

ꢀ

EDTA during 6 min at 37 C. After inactivation, the cell suspen-

sion was centrifugated at 1200 rpm during 5 min, the supernatant

was removed, and the pellet resuspended in fresh medium. To

dissociate the clumps during the passages, sterile 10 mL syringes

with 18-gauge (18G) needles were used. This last step also guar-

4.2.4. Activity results

Activity criteria. The capacity of the synthetized TAMs to activate

(i.e. act as agonists) and to suppress (i.e. act as antagonists) AhR-

mediated transcription was analyzed in terms of magnitude of

the effects and based on the concentration at which such effects

occurs. Therefore, the fold response induced (compared with

vehicle control) was used to inform the magnitude of the effect

while half effective/inhibitory concentrations (EC50 or IC50) were

estimated from dose-response curves of agonist or antagonist ac-

tivity respectively. In the absence of a regulatory guidance for AhR,

the threshold used to identify active from inactive compounds in

both agonist and antagonist assays followed the OECD guidelines

for ER transactivation, where the induced response is compared

with a positive control (PC) [53]. Thus, the maximum response

relative to the positive control (RPCmax) in this work represented

the maximum fold response induced by each TAM (x) compared to

the positive control FICZ.

anteed the accuracy of the cell counting performed by mixing 10

of cell suspension with 10 L of 0.4% Trypan Blue Solution in a

chamber slide read in cell counters on the Countess™ II instrument

Invitrogen™, Thermo Fisher Scientiﬁc).

mL

m

(

4.2.3. Bioassays

Assay medium and seeding. The assay medium used was the

growth medium without Normocin™ nor Zeocin™. A volume of

00 L of cells/well was seeded into 96-wells microplates at a

2

m

density of 2.0 ꢄ 105 cells/mL and incubated overnight prior to

treatment.

Treatment. The 32 synthetized TAMs and the commercial TAM-

drug bisacodyl were dissolved in DMSO (0.5% ﬁnal maximum

concentration/well). The cells were exposed during 24 h to at least

In the AhR-agonist assay, the RPCmax was calculated as per-

centage of the maximum AhR induction of FICZ (in relative light

units of the luciferase gene expression), that is expressed as

Fold response PC_M_a_xðFICZÞ in Equation (1). Meanwhile, in the

antagonist assay, the RPCmax was calculated as percentage of the

effect induced by the EC₅₀of FICZ that is expressed as

Fold response EC₅₀ðFICZÞ in Equation (2).

4

different concentration of the TAMs (0.1e10

their cytotoxicity and/or solubility. In the agonist assay, cells were

exposed to 10 L/well of the tested compounds while in the

antagonist assay, cells were treated with 10 L/well of the tested

compounds plus 10 L/well of the EC50 of FICZ.

mM), depending on

m

Cell viability assay. Cell viability was assessed by the 3-(4,5-

dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT)

assay, which is a colorimetric method based on the reduction of the

yellowish solution of the MTT tetrazolium salt to form the formazan

precipitate, as an identiﬁcation of the redox potential in metabol-

ically actives cells [50]. After seeding, cells were treated with

Compounds showing RPCmax ꢃ 10 % were considered active in

the agonist bioassay while compounds with RPCmax ꢂ 70 % were

considered active in the antagonist bioassay.

Agonist RPCmax ð%Þ ¼ Fold response ðxÞ₌_F_o_l_d_r_e_s_p_o_n_s_e_P_C_M_a_x

ðFICZÞ

ꢄ 100

(1)

(2)

Antagonistic RPCmax ð%Þ ¼ Fold response ðxÞ₌_F_o_l_d_r_e_s_p_o_n_s_e_E_C₅₀

ꢄ 100

ðFICZÞ

different concentrations of the tested compounds and incubated

during 24e72 h. Then, the medium was discarded and 100 L/well

On the other hand, when possible, sigmoidal curves of x [log

(concentration)] vs. y (Fold response) were designed constraining

the Hill Slope value to 1.0 and using the Top and Bottom plateaus in

the units of the yaxis. The concentration of agonist required to

provoke a response halfway between the baseline and maximum

responses (EC50 or IC50) was estimated from Equation (3).

m

of 0.5 mg/mL of MTT solution was added. Plates were incubated at

ꢀ

3

7 C allowing the transformation, the supernatant was removed,

and the formazan crystals were dissolved adding 100 mL/well of

DMSO. Finally, the optical density was determined by reading the

absorbance at 490 nm using a microplate reader (VICTORx3, Per-

kinElmer Inc., USA).

AhR-transactivation assay. The induction of AhR-mediated

transcriptional activity was measured by transferring 20 mL of the

ꢀ

ꢁ

Top ꢁ Bottom

y ¼ Bottom þ

(3)

ðlogEC50ꢁxÞ*Hill Slope

1

þ 10

Statistical analysis. All data informed represent means obtained

from at least three independent experiments (n ¼ 3) and sextu-

plicate in all cases. The precision of results was reported through

supernatant of stimulated cells to white sterile and ﬂat-bottom 96-

wells microplates. The QUANTI-Luc™ assay reagent containing the

18

E. Goya-Jorge, C. Rampal, N. Loones et al.

European Journal of Medicinal Chemistry 207 (2020) 112777

the standard error of the mean (SEM). All active compounds were

re-tested at least once again (n ¼ 4) and a greater number of con-

centrations was evaluated (ꢃ5). Statistical signiﬁcance was deter-

mined with a one-way analysis of variance (ANOVA) following by

Dunnett’s post-test for comparison with controls or Bonferroni

post-test to compare the studied compounds.

References

[1] C.A. Brad ﬁ eld, E. Glover, A. Poland, Puri ﬁ cation and N-terminal amino acid

sequence of the Ah receptor from the C57BL/6J mouse, Mol. Pharmacol. 39

(1991) 13e19.

[2] K.W. Bock, Aryl hydrocarbon receptor ( AHR ): from selected human target

genes and crosstalk with transcription factors to multiple AHR functions,

Biochem. Pharmacol. 168 (2019) 65e70, https://doi.org/10.1016/

j.bcp.2019.06.015.

4

.3. Computational Studies

[3] S.-H. Seok, W. Lee, L. Jiang, K. Molugu, A. Zheng, Y. Li, S. Park, C.A. Bradﬁeld,

Y. Xing, Structural hierarchy controlling dimerization and target DNA recog-

nition in the AHR transcriptional complex, Proc. Natl. Acad. Sci. U. S. A 114

.3.1. Molecular docking simulations

(2017) 5431e5436, https://doi.org/10.1073/pnas.1617035114.

Molecular docking analysis was performed with Autodock Vina

[4] I.A. Murray, A.D. Patterson, G.H. Perdew, Aryl hydrocarbon receptor ligands in

cancer: friend and foe, Nat. Rev. Canc. 14 (2014) 801e814, https://doi.org/

[

64] as implemented in YASARA [65]. The crystalized protein

structure of HIF2 was used for docking analysis (PDB ID 3F1O). The

sequence of the PAS-B domain of HIF2 shows the highest level of

10.1038/nrc3846.

a

[

5] E.J. Wright, K.P. De Castro, A.D. Joshi, C.J. Elferink, Canonical and non-canonical

a

aryl hydrocarbon receptor signaling pathways Toxicology, Curr. Opin. Toxicol.

2

(2017) 87e92, https://doi.org/10.1016/j.cotox.2017.01.001.

identity and similarity with AhR among all the PAS identiﬁed to

date [66]. Hence, it is commonly used as template structure in

molecular modeling of AhR ligand binding domain [67]. An addi-

tional evaluation of the aforementioned sequence identity and

similarity is provided in Section 3, SI-3. The analyzed ligands were

the strongest agonist identiﬁed 22, the structurally related com-

pound 21, and the known AhR ligand/agonist compounds FICZ and

TCCD. Besides, a preliminary off-targeting docking analysis of

compound 22 with four nuclear receptors (ER, AR, PR and PXR) was

performed.

All simulations were performed for the entire target structure

making the protein ridig and the ligand compounds totally ﬂexible.

Protein Ligand Interaction Proﬁler server [68] was used to predict

the interactions of the best protein/ligand complex for each ligand

and molecular graphics and analyses were performed with UCSF

Chimera [69].

6] L. Stejskalova, Z. Dvorak, P. Pavek, Endogenous and exogenous ligands of aryl

hydrocarbon receptor: current state of art, Curr. Drug Metabol. 12 (2011)

198 e 212, https://doi.org/10.2174/138920011795016818 .

7] L.C. Quattrochi, R.H. Tukey, Nuclear uptake of the Ah (dioxin) receptor in

response to omeprazole: transcriptional activation of the human CYP1A1

gene, Mol. Pharmacol. 43 (1993) 504e508.

[8] E.F. O’Donnell, K.S. Saili, D.C. Koch, P.R. Kopparapu, D. Farrer, W.H. Bisson,

L.K. Mathew, S. Sengupta, N.I. Kerkvliet, R.L. Tanguay, S.K. Kolluri, The anti-

inﬂammatory drug leﬂunomide is an agonist of the aryl hydrocarbon recep-

tor, PLoS One 5 (2010), https://doi.org/10.1371/journal.pone.0013128.

[9] H.P. Ciolino, P.J. Daschner, G.C. Yeh, Dietary ﬂavonols quercetin and kaemp-

ferol are ligands of the aryl hydrocarbon receptor that affect CYP1A1 tran-

scription differentially, Biochem. J. 340 (1999) 715e722, https://doi.org/

1

0.1042/0264-6021:3400715.

10] T.H. Scheuermann, D.R. Tomchick, M. Machius, Y. Guo, R.K. Bruick,

K.H. Gardner, Artiﬁcial ligand binding within the HIF2 PAS-B domain of the

[

a

HIF2 transcription factor, Proc. Natl. Acad. Sci. U. S. A 106 (2009) 450e455,

https://doi.org/10.1073/pnas.0808092106.

11] M.B. Kumar, P. Ramadoss, R.K. Reen, J.P. Vanden Heuvel, G.H. Perdew, The Q-

rich subdomain of the human ah receptor transactivation domain is required

for dioxin-mediated transcriptional activity, J. Biol. Chem. 276 (2001)

4

2302e42310, https://doi.org/10.1074/jbc.M104798200 .

4

.3.2. Druglikeness and ADME proﬁle

The physicochemical parameters and ADME descriptors were

[12] F.J. Quintana, A.S. Basso, A.H. Iglesias, T. Korn, M.F. Farez, E. Bettelli,

M. Caccamo, M. Oukka, H.L. Weiner, Control of Treg and TH17 cell differen-

tiation by the aryl hydrocarbon receptor, Nature 453 (2008) 65e71, https://

doi.org/10.1038/nature06880 .

13] A.A. Soshilov, M.S. Denison, Ligand promiscuity of aryl hydrocarbon receptor

agonists and antagonists revealed by site-directed mutagenesis, Mol. Cell Biol.

predicted using the QikProp v5.9 Panel from Schr o€ dinger software

v11.9. Conformational averages from OPLS-AA force ﬁeld were used

for calculations.

[

34 (2014) 1707e1719, https://doi.org/10.1128/mcb.01183-13 .

[

14] Y. Xing, M. Nukaya, K.A. Satyshur, L. Jiang, V. Stanevich, E.N. Korkmaz,

L. Burdette, G.D. Kennedy, Q. Cui, C.A. Bradﬁeld, Identiﬁcation of the Ah-

receptor structural determinants for ligand preferences, Toxicol. Sci. 129

Author contribution

(

2012) 86e97, https://doi.org/10.1093/toxsci/kfs194 .

The manuscript was written through contributions of all au-

thors. All authors have given approval to the ﬁnal version of the

manuscript.

[

15] D. Dolciami, M. Gargaro, B. Cerra, G. Scalisi, L. Bagnoli, G. Servillo, M.A. Della

Fazia, P. Puccetti, F.J. Quintana, F. Fallarino, A. Macchiarulo, Binding mode and

structureeactivity relationships of ITE as an aryl hydrocarbon receptor (AhR)

agonist, ChemMedChem 13 (2018) 270e279, https://doi.org/10.1002/

cmdc.201700669 .

Declaration of competing interest

[16] K.N. Chitrala, X. Yang, P. Nagarkatti, M. Nagarkatti, Comparative analysis of

interactions between aryl hydrocarbon receptor ligand binding domain with

its ligands: a computational study, BMC Struct. Biol. 18 (2018), https://doi.org/

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.

10.1186/s12900-018-0095-2 .

[17] E. Goya-Jorge, T.Q. Doan, M.L. Scippo, M. Muller, R.M. Giner, S.J. Barigye,

R. Gozalbes, Elucidating the aryl hydrocarbon receptor antagonism from a

chemical-structural perspective, SAR QSAR Environ. Res. 31 (2020) 209e226,

https://doi.org/10.1080/1062936X.2019.1708460 .

Acknowledgments

[18] J. Chen, C.A. Haller, F.E. Jernigan, S.K. Koerner, D.J. Wong, Y. Wang, J.E. Cheong,

R. Kosaraju, J. Kwan, D.D. Park, B. Thomas, S. Bhasin, R.C. de la Rosa,

A.M. Premji, L. Liu, E. Park, A.C. Moss, A. Emili, M. Bhasin, L. Sun, E.L. Chaikof,

Modulation of lymphocyte-mediated tissue repair by rational design of het-

erocyclic aryl hydrocarbon receptor agonists, Sci. Adv. 6 (2020) 1e16, https://

doi.org/10.1126/sciadv.aay8230 .

This project has received funding from the European Union’s

Horizon 2020 research and innovation programme under the Marie

Skłodowska-Curie grant agreement No. 722634. The Early Stage

Researcher Goya-Jorge E. of this Innovative Training Network

named ‘PROTECTED’ (http://protected.eu.com/) gratefully thanks

for her PhD scholarship. Authors also thank the FONGECIF for the

ﬁnancial support offered to C eꢀ line Rampal and Nicolas Loones

[19] M. Mescher, T. Haarmann-Stemmann, Modulation of CYP1A1 metabolism:

from adverse health effects to chemoprevention and therapeutic options,

Pharmacol.

Ther.

187

(2018)

71e87,

https://doi.org/10.1016/

j.pharmthera.2018.02.012 .

[

20] K.W. Bock, From TCDD-mediated toxicity to searches of physiologic AHR

functions, Biochem. Pharmacol. 155 (2018) 419e424, https://doi.org/10.1016/

j.bcp.2018.07.032 .

21] C. Esser, B.P. Lawrence, D.H. Sherr, G.H. Perdew, A. Puga, R. Barouki,

X. Coumoul, Old receptor, new tricksdthe ever-expanding universe of aryl

hydrocarbon receptor functions. Report from the 4th AHR meeting, 29 e 31

August 2018 in Paris, France, Int. J. Mol. Sci. 19 (2018), https://doi.org/

during their engineering internship at Cnam.

Appendix A. Supplementary data

1

0.3390/ijms19113603 .

Supplementary data to this article can be found online at

https://doi.org/10.1016/j.ejmech.2020.112777 .

[

22] S. Zhang, P. Lei, X. Liu, X. Li, K. Walker, L. Kotha, C. Rowlands, S. Safe, The aryl

19

E. Goya-Jorge, C. Rampal, N. Loones et al.

European Journal of Medicinal Chemistry 207 (2020) 112777

hydrocarbon receptor as a target for estrogen receptor-negative breast cancer

chemotherapy, Endocr. Relat. Canc. 16 (2009) 835e844, https://doi.org/

triarylmethanes: synthesis, antimicrobial activity, anti-proliferative behavior

and in silico studies, Bioorg. Chem. 96 (2020), https://doi.org/10.1016/j.bio-

org.2020.103591 , 103591.

10.1677/erc-09-0054 .

[

23] K.W. Bock, Human AHR functions in vascular tissue: pro- and anti-

inﬂammatory responses of AHR agonists in atherosclerosis, Biochem. Phar-

macol. 159 (2019) 116e120, https://doi.org/10.1016/j.bcp.2018.11.021 .

24] N. Guerrina, H. Traboulsi, D.H. Eidelman, C.J. Baglole, The aryl hydrocarbon

receptor and the maintenance of lung health, Int. J. Mol. Sci. 19 (2018), https://

doi.org/10.3390/ijms19123882 .

[44] M.S.-I. Veítia, D. Siverio Mota, V. Lerari, M. Marín, R.M. Giner, L. Vicet Muro,

Y.R. Guerra, F. Dumas, C. Ferroud, P.A.M. De Witte, A.D. Crawford, V.J. Ar aꢀ n,

Y.M. Ponce, Fishing anti-inﬂammatories from known drugs: in silico repur-

posing, design, synthesis and biological evaluation of bisacodyl analogues,

Curr. Top. Med. Chem. 17 (2017) 2866e2887, https://doi.org/10.2174/

1568026617666170817161953 .

25] C. Duval, E. Blanc, X. Coumoul, Aryl hydrocarbon receptor and liver ﬁ brosis,

[45] R.A. Al-Qawasmeh, Y. Lee, M.Y. Cao, X. Gu, A. Vassilakos, J.A. Wright, A. Young,

Triaryl methane derivatives as antiproliferative agents, Bioorg. Med. Chem.

Lett 14 (2004) 347e350, https://doi.org/10.1016/j.bmcl.2003.11.004 .

[46] M. Seto, Y. Aramaki, H. Imoto, K. Aikawa, T. Oda, N. Kanzaki, Y. Iizawa, M. Baba,

M. Shiraishi, Orally active CCR5 antagonists as anti-HIV-1 agents 2: synthesis

and biological activities of anilide derivatives containing a pyridine N-oxide

moiety, Chem. Pharm. Bull. 52 (2004) 818e829, https://doi.org/10.1248/

8

(2018)

8e13,

https://doi.org/10.1016/

j.cotox.2017.11.010 .

26] M. Puccetti, G. Paolicelli, V. Oikonomou, A. De Luca, G. Renga, M. Borghi,

M. Pariano, C. Stincardini, L. Scaringi, S. Giovagnoli, M. Ricci, L. Romani,

T. Zelante, Towards targeting the aryl hydrocarbon receptor in cystic ﬁ brosis,

Mediat. Inﬂamm. 2018 (2018), 1601486, https://doi.org/10.1155/2018/

1601486 , 7 pages.

[

27] C. Dietrich, Antioxidant functions of the aryl hydrocarbon receptor, Stem Cell.

Int. 2016 (2016), 7943495, https://doi.org/10.1155/2016/7943495 , 10 pages.

28] L. Juricek, X. Coumoul, The aryl hydrocarbon receptor and the nervous system,

Int. J. Mol. Sci. 19 (2018) 2504, https://doi.org/10.3390/ijms19092504 .

29] T. Bradshaw, A. Westwell, The development of the antitumour benzothiazole

prodrug, phortress, as a clinical candidate, Curr. Med. Chem. 11 (2005)

[47] F. Tr eꢀ court, G. Breton, V. Bonnet, F. Mongin, F. Marsais, G. Qu eꢀ guiner, New

syntheses of substituted pyridines via bromine-magnesium exchange, Tetra-

hedron 56 (2001) 1349e1360, https://doi.org/10.1016/S0040-4020(00)

00027-2 .

[48] M. Sylla-Iyarreta Veitía, C. Rampal, C. Ferroud, An ef ﬁ cient access to unsym-

metrical triarylmethanes by regioselective Friedel-Crafts hydroxyalkylation,

Trends Org. Chem. 20 (2019) 1 e 13 .

1009e1021, https://doi.org/10.2174/0929867043455530 .

[

30] S. Safe, Y. Cheng, U.H. Jin, The aryl hydrocarbon receptor (AhR) as a drug target

[49] M. G o€ rmen, M.S.I. Veitía, F. Trigui, M. El Arbi, C. Ferroud, Ferrocenyl analogues

for cancer chemotherapy, Curr. Opin. Toxicol. 1 (2017) 24e29, https://doi.org/

of bisacodyl: synthesis and antimicrobial activity, J. Organomet. Chem. 794

(2015) 274e281, https://doi.org/10.1016/j.jorganchem.2015.07.016 .

[50] J.C. Stockert, R.W. Horobin, L.L. Colombo, A. Bl aꢀ zquez-Castro, Tetrazolium salts

10.1016/j.cotox.2017.01.012 .

31] B. Lamas, J.M. Natividad, H. Sokol, Aryl hydrocarbon receptor and intestinal

immunity review-article, Mucosal Immunol. 11 (2018) 1024e1038, https://

doi.org/10.1038/s41385-018-0019-2 .

32] J. Gao, K. Xu, H. Liu, G. Liu, M. Bai, C. Peng, T. Li, Y. Yin, Impact of the gut

microbiota on intestinal immunity mediated by tryptophan metabolism,

and formazan products in Cell Biology: viability assessment, ﬂuorescence

imaging, and labeling perspectives, Acta Histochem. 120 (2018) 159e167,

https://doi.org/10.1016/j.acthis.2018.02.005 .

[51] E. Goya-Jorge, F. Abdmouleh, L.E. Carpio, R.M. Giner, M.Sylla-I Veitía, Dis-

covery of 2-aryl and 2-pyridinylbenzothiazoles endowed with antimicrobial

and aryl hydrocarbon receptor agonistic activities, Eur. J. Pharmaceut. Sci. 151

(2020) 105386, https://doi.org/10.1016/j.ejps.2020.105386 .

[52] A. Mohammadi-Bardbori, M. Omidi, M.R. Arabnezhad, Impact of CH223191-

induced mitochondrial dysfunction on its aryl hydrocarbon receptor

agonistic and antagonistic activities, Chem. Res. Toxicol. 32 (2019) 691e697,

https://doi.org/10.1021/acs.chemrestox.8b00371 .

[53] OECD, Test No. 455: Performance-Based Test Guideline for Stably Transfected

Transactivation in Vitro Assays to Detect Estrogen Receptor Agonists and

Antagonists, 2016, https://doi.org/10.1787/9789264265295-en .

[54] H. Kim, S. Reddy, R.F. Novak, 3-Methylcholanthrene and pyridine effects on

CYP1A1 and CYP1A2 expression in rat renal tissue, Drug Metab. Dispos. 23

Front. Cell Infect. Microbiol.

fcimb.2018.00013 .

8 (2018) 1e22, https://doi.org/10.3389/

[

33] C. Esser, A. Rannug, B. Stockinger, The aryl hydrocarbon receptor in immunity,

Trends Immunol. 30 (2009) 447e454, https://doi.org/10.1016/

j.it.2009.06.005 .

34] I. Maraﬁni, D. Di Fusco, V. Dinallo, E. Franz eꢁ , C. Stolﬁ, G. Sica, G. Monteleone,

I. Monteleone, NPD-0414-2 and NPD-0414-24, two chemical entities designed

as aryl hydrocarbon receptor (AHR) ligands, inhibit gut in ﬂ ammatory signals,

Front. Pharmacol. 10 (2019) 1e9, https://doi.org/10.3389/fphar.2019.00380 .

35] P. Tarnow, T. Tralau, A. Luch, Chemical activation of estrogen and aryl hy-

drocarbon receptor signaling pathways and their interaction in toxicology and

metabolism, Expet Opin. Drug Metabol. Toxicol. 15 (2019) 219e229, https://

doi.org/10.1080/17425255.2019.1569627 .

36] E.F. O’Donnell, D.C. Koch, W.H. Bisson, H.S. Jang, S.K. Kolluri, The aryl hydro-

carbon receptor mediates raloxifene-induced apoptosis in estrogen receptor-

negative hepatoma and breast cancer cells, Cell Death Dis. 5 (2014) 1e12,

https://doi.org/10.1038/cddis.2013.549.

[

(1995) 818. LP

abstract .

e

824, http://dmd.aspetjournals.org/content/23/8/818.

[55] P. De Medina, R. Casper, J.F. Savouret, M. Poirot, Synthesis and biological

properties of new stilbene derivatives of resveratrol as new selective aryl

hydrocarbon modulators, J. Med. Chem. 48 (2005) 287e291, https://doi.org/

10.1021/jm0498194 .

[56] M.S. Denison, A. Pandini, S.R. Nagy, E.P. Baldwin, L. Bonati, Ligand binding and

activation of the Ah receptor, Chem. Biol. Interact. 141 (2002) 3e24, https://

doi.org/10.1016/S0009-2797(02)00063-7 .

[57] M. Nambo, Z.T. Ariki, D. Canseco-Gonzalez, D.D. Beattie, C.M. Crudden, Ary-

lative desulfonation of diarylmethyl phenyl sulfone with arenes catalyzed by

scandium tri ﬂ ate, Org. Lett. 18 (2016) 2339e2342, https://doi.org/10.1021/

acs.orglett.6b00744 .

ꢀ

~

37] G. Guedes, A. Amesty, R. Jimenez-Monzon, J. Marrero-Alonso, M. Díaz,

0

L. Fern aꢀ ndez-P eꢀ rez, A. Est eꢀ vez-Braun, Synthesis of 4,4 -diaminotriphenyl-

methanes with potential selective estrogen receptor modulator (SERM)-like

activity, ChemMedChem 10 (2015) 1403e1412, https://doi.org/10.1002/

cmdc.201500148 .

[

38] Z. Dvo ꢂr ꢀa k, F. Kopp, C.M. Costello, J.S. Kemp, H. Li, A. Vrzalov ꢀa , M. Stepankova,

ꢂ ꢂ ꢀ

ꢀ

I. Barto nꢂ kov ꢀa , E. Jiskrov aꢀ , K. Poulíkov ꢀa , B. Vyhlídalov aꢀ , L.U. Nordstroem, C. V

Karunaratne, H.S. Ranhotra, K.S. Mun, A.P. Naren, I.A. Murray, G.H. Perdew,

J. Brtko, L. Toporova, A. Sch o€ n, W.G. Wallace, W.G. Walton, M.R. Redinbo,

K. Sun, A. Beck, S. Kortagere, M.C. Neary, A. Chandran, S. Vishveshwara,

M.M. Cavalluzzi, G. Lentini, J.Y. Cui, H. Gu, J.C. March, S. Chatterjee, A. Matson,

D. Wright, K.L. Flannigan, S.A. Hirota, R.B. Sartor, S. Mani, Targeting the

pregnane X receptor using microbial metabolite mimicry, EMBO Mol. Med. 12

[58] R. Pohjanvirta, The AH Receptor in Biology and Toxicology, John Wiley and

Sons, 2011, https://doi.org/10.1002/9781118140574 .

ꢀ

[59] A.C. Roman, J.M. Carvajal-Gonzalez, J.M. Merino, S. Mulero-Navarro,

P.M. Fern aꢀ ndez-Salguero, The aryl hydrocarbon receptor in the crossroad of

signalling networks with therapeutic value, Pharmacol. Ther. 185 (2018)

50e63, https://doi.org/10.1016/j.pharmthera.2017.12.003 .

(

2020) 1e19, https://doi.org/10.15252/emmm.201911621.

[

39] S. Mondal, G. Panda, Synthetic methodologies of achiral diarylmethanols,

diaryl and triarylmethanes (TRAMs) and medicinal properties of diaryl and

[60] S. Safe, M. Wormke, Inhibitory aryl hydrocarbon ReceptorꢁEstrogen receptor

a

cross-talk and mechanisms of action, Chem. Res. Toxicol. 16 (2003)

triarylmethanes-an overview, RSC Adv.

doi.org/10.1039/c4ra01341g .

4

(2014) 28317e28358, https://

807e816, https://doi.org/10.1021/tx034036r .

[61] A.J. Lucas, J.L. Sproston, P. Barton, R.J. Riley, Estimating human ADME prop-

erties, pharmacokinetic parameters and likely clinical dose in drug discovery,

Expet Opin. Drug Discov. 14 (2019) 1313e1327, https://doi.org/10.1080/

17460441.2019.1660642 .

[62] A. Leo, C. Hansch, D. Elkins, Partition coef ﬁ cients and their uses, Chem. Rev. 71

(1971) 525e616, https://doi.org/10.1021/cr60274a001 .

[63] D. Dolciami, M. Ballarotto, M. Gargaro, L.C. L oꢀ pez-Cara, F. Fallarino,

40] J.L. Douglas, M.L. Panis, E. Ho, K.-Y. Lin, S.H. Krawczyk, D.M. Grant, R. Cai,

S. Swaminathan, T. Cihlar, Inhibition of respiratory syncytial virus fusion by

the small molecule VP-14637 via speci ﬁ c interactions with F protein, J. Virol.

77 (2003) 5054e5064, https://doi.org/10.1128/jvi.77.9.5054-5064.2003 .

41] G. Panda, Shagufta, A.K. Srivastava, S. Sinha, Synthesis and antitubercular

activity of 2-hydroxy-aminoalkyl derivatives of diaryloxy methano phenan-

threnes, Bioorg. Med. Chem. Lett 15 (2005) 5222e5225, https://doi.org/

A. Macchiarulo, Targeting Aryl hydrocarbon receptor for next-generation

immunotherapies: selective modulators (SAhRMs) versus rapidly metabo-

lized ligands (RMAhRLs), Eur. J. Med. Chem. 185 (2020), https://doi.org/

10.1016/j.ejmech.2019.111842 .

10.1016/j.bmcl.2005.08.045 .

42] S.K. Chauthe, S.B. Bharate, S. Sabde, D. Mitra, K.K. Bhutani, I.P. Singh, Bio-

mimetic synthesis and anti-HIV activity of dimeric phloroglucinols, Bioorg.

Med.

Chem.

18

(2010)

2029e2036,

https://doi.org/10.1016/

[64] O. Trott, A. Olson, Software news and update. AutoDock Vina improving the

speed and accuracy of docking with a new scoring function, efﬁcient opti-

mization, and multithreading, J. Comput. Chem. 31 (2009) 455e461, https://

doi.org/10.1002/jcc.21334 .

j.bmc.2010.01.023 .

43] C. Ricco, F. Abdmouleh, C. Riccobono, L. Guenineche, F. Martin, E. Goya-Jorge,

N. Lagarde, B. Liagre, M. Ben Ali, C. Ferroud, M. El Arbi, M.S.I. Veitía, Pegylated

20

E. Goya-Jorge, C. Rampal, N. Loones et al.

European Journal of Medicinal Chemistry 207 (2020) 112777

doi.org/10.1016/j.cotox.2017.01.011.

[68] S. Salentin, S. Schreiber, V.J. Haupt, M.F. Adasme, M. Schroeder, PLIP: fully

automated protein-ligand interaction pro ﬁ ler, Nucleic Acids Res. 43 (2015)

W443eW447, https://doi.org/10.1093/nar/gkv315 .

[69] E.F. Pettersen, T.D. Goddard, C.C. Huang, G.S. Couch, D.M. Greenblatt,

E.C. Meng, T.E. Ferrin, UCSF Chimerada visualization system for exploratory

research and analysis, J. Comput. Chem. 25 (2004) 1605e1612, https://

doi.org/10.1002/jcc.20084 .

[

65] E. Krieger, G. Vriend, YASARA View - molecular graphics for all devices - from

smartphones to workstations, Bioinformatics 30 (2014) 2981e2982, https://

doi.org/10.1093/bioinformatics/btu426.

66] S. Giani Tagliabue, S.C. Faber, S. Motta, M.S. Denison, L. Bonati, Modeling the

binding of diverse ligands within the Ah receptor ligand binding domain, Sci.

Rep. 9 (2019) 1e14, https://doi.org/10.1038/s41598-019-47138-z .

67] L. Bonati, D. Corrada, S. Giani Tagliabue, S. Motta, Molecular modeling of the

AhR structure and interactions can shed light on ligand-dependent activation

and transformation mechanisms, Curr. Opin. Toxicol. 1 (2017) 42e49, https://

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