Design, synthesis and biological evaluation of 2-substituted-6-[(4-substituted-1-piperidyl)methyl]-1H-benzimidazoles as inhibitors of ebola virus infection

European Journal of Medicinal Chemistry 214 (2021) 113211

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry

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

Research paper

Design, synthesis and biological evaluation of 2-substituted-6-[(4-

substituted-1-piperidyl)methyl]-1H-benzimidazoles as inhibitors of

ebola virus infection

Maxime Bessi eꢀ res ^a^,¹, El z_ bieta Plebanek ¹, Payel Chatterjee ^b,

a,

b

c

Punya Shrivastava-Ranjan , Mike Flint , Christina F. Spiropoulou , Dawid Warszycki ,

c

a

,

**

, Luigi A. Agrofoglio ^a

, *

Andrzej J. Bojarski , Vincent Roy

a

Univ. Orl ꢁe ans, CNRS, ICOA, UMR 7311, F-45067, Orl ꢁe ans, France

Viral Special Pathogens Branch, Centers for Disease Control and Prevention, Atlanta, GA, USA

May Institute of Pharmacology Polish Academy of Sciences, Krak oꢁ w, Poland

b

c

a r t i c l e i n f o

a b s t r a c t

Article history:

Received 30 August 2020

Received in revised form

Novel 2-substituted-6-[(4-substituted-1-piperidyl)methyl]-1H-benzimidazoles were designed and syn-

thesized as Ebola virus inhibitors. The proposed structures of the new prepared benzimidazole-

piperidine hybrids were conﬁrmed based on their spectral data and CHN analyses. The target com-

pounds were screened in vitro for their anti-Ebola activity. Among tested molecules, compounds 26a

20 November 2020

Accepted 12 January 2021

Available online 27 January 2021

(

EC50¼0.93

m

M, SI ¼ 10) and 25a (EC50¼0.64

m

M, SI ¼ 20) were as potent as and more selective than

Toremifene reference drug (EC50 ¼ 0.38

m

M, SI ¼ 7) against cell line. Data suggests that the mechanism

by which 25a and 26a block EBOV infection is through the inhibition of viral entry at the level of NPC1.

Furthermore, a docking study revealed that several of the NPC1 amino acids that participate in binding to

GP are involved in the binding of the most active compounds 25a and 26a. Finally, in silico ADME pre-

diction indicates that 26a is an idealy drug-like candidate. Our results could enable the development of

small molecule drug capable of inhibiting Ebola virus, especially at the viral entry step.

Keywords:

Ebola virus

Viral entry

Niemann-pick C1

Benzimidazole-piperidine derivatives

Docking studies

1. Introduction

envelope glycoprotein (GP) of ﬁlovirus mediates binding to cellular

receptors and the subsequent fusion of the virus envelope with the

The family Filoviridae includes ﬁve genera: Cuevavirus, Mar-

burgvirus, Ebolavirus, Striavirus and Thamnovirus [1,2]. Since 1967,

when Marburg virus was ﬁrst isolated, numerous ﬁlovirus hemor-

rhagic fever (HF) outbreaks have occurred, often with high fatality

rates. The most virulent Ebolavirus species is Zaire ebolavirus

host cell membrane [5e8]. Study of the host factors that promote or

restrict Ebola replication is of great interest as it may thus lead to

the development of novel therapeutics. It has been reported that

ﬁlovirus GP interacts with multiple molecules for entry into host

cells [4]; the entry mechanism is complex, involving not only cell-

surface molecules but also intracellular proteins. Several cell-

surface molecules are thought to participate in viral entry

[4,6,9,10] including the cellular lectins DC-SIGN, DC-SIGNR, LSECtin,

ASPGR-1 and hMGL [11e13]. The T-cell immunoglobulin and mucin

domain-containing protein 1 (TIM-1) was also shown to be a factor

with a role in virus entry [14]. Another known host cell factor is the

Tyro3/Axl/Mer (TAM) family of tyrosine kinase receptors [15] .

(

EBOV) with mortality rate of up to 90%, [3]. Filoviruses cause HF,

associated with immune suppression and a systemic inﬂammatory

response in human and non-human primates. Filoviruses are

enveloped, non-segmented, negative-sense, single-stranded RNA

viruses [4]. To begin the infection cycle, the virus must transport its

genetic information through the membrane of a target cell. The

Other molecules involved in ﬁlovirus entry are

a3 and b1 integrins

[

16] as well as cathepsins (L and B) [17]. If promising results with

*

Corresponding author.

antibody therapies have been reported, and very recently one has

been approved for the treatment of EBOV infection [18] one

important application for an anti-EBOV small molecule could be the

*

* Corresponding author.

E-mail addresses: vincent.roy@univ-orleans.fr (V. Roy), luigi.agrofoglio@univ-

oleans.fr (L.A. Agrofoglio).

1

These authors contributed equally to this work.

https://doi.org/10.1016/j.ejmech.2021.113211

M. Bessi ꢀe res, E. Plebanek, P. Chatterjee et al.

European Journal of Medicinal Chemistry 214 (2021) 113211

treatment of persistently-infected patients, as antibodies may be

unable to access the immunologically-privileged sites that harbor

the virus. Thus, some representative antiﬁlovirus small molecules,

belonging to several chemical classes [19,20], are shown in Fig. 1;

they target virus genome replication such as nucleosides BCX4430

and GS-5734 (remdesivir) (two non-obligate RNA chain termi-

nator), or virus entry such as CID23631927 (a cathepsin L inhibitor),

the benzimidazole FGI-103 (mechanism to be established), tor-

emifene and terconazole (two Niemann-Pick C1-dependent in-

hibitors). In 2011, C o^ t eꢁ et al. identiﬁed the benzylpiperazine

adamantane diamide-derived (3.47) as inhibitor of Ebola virus

entry [21]. Compound 3.47, alongside a genetic screening study,

was used to identify Niemann-Pick C1 protein (NPC1) as an

essential receptor required for EBOV entry [21,22]. Compound 3.47

is reported to blocked binding of EBOV-GP to NPC1; however, this

inhibitor has unfavourable properties for in vivo application. In

within the EBOV entry mechanism that might be subject to thera-

peutic intervention.

Several of the virus entry inhibitors are sharing common moi-

eties, such as benzimidazole, piperazine and piperidine. Based on

this ﬁnding, we report herein the synthesis of hitherto unknown 2-

substituted-6-[(4-substituted-1-piperidyl)methyl]-1H-benzimid-

azoles (Fig. 1); those compounds are targeting NPC1 involved in

Ebola virus entry. The main scaffold is formed by a benzimidazole

moiety, which has been an important pharmacophore and privi-

leged structure in medicinal chemistry [31], linked to a piperidine

ether and its isostere 4-methylpiperidine. Modiﬁcations are done at

C2 position on the benzymidazole moiety with substituted phenyl

group having (or not) a triﬂuoromethyl- and triﬂuoromethyl ether

due to its peculiar properties (steric and electronic effects and

enhanced lipophilicity) [32] (blue dashed rectangle). We investi-

gate also the inﬂuence of [(phenoxy-methyl)phenyl group bearing a

enzymatic stable phosphonate diester (green dashed rectangle)

bounded at C4 of the piperidine moiety by either X ¼ O (4-aryloxy-,

2

013, Shoemaker et al. demonstrated the inhibition of EBOV entry

by multiple cationic amphiphiles [23]. The mechanism of this in-

hibition was NPC1-dependent, but different from that described for

the compound 3.47. More recently, Rong et al. reported some 4-

serial A) or X ¼ CH

2

(4-benzyl-, serial B). All compounds were

evaluated for their ability to inhibit EBOV entry.

(

aminomethyl)benzamides (CBS1118) as potent entry inhibitors of

Ebola virus [24]. Toremifene, and other selective estrogen receptor

modulators, were ﬁrst identiﬁed as inhibitors of EBOV infection in

screens of approved drugs, with toremifene conferring a statisti-

cally signiﬁcant survival beneﬁt in the mouse model of infection

2

. Results and discussion

2.1. Chemistry

[

25e27]. Crystallographic and biochemical studies have subse-

2

1

.1.1. Synthesis of 2-substitued-6-[(4-aryloxy-1-piperidyl)methyl]-

H-benzimidazoles (serial A)

The chosen strategy to the desired 2-substituted-6-[(4-

quently shown that toremifene’s mechanism of action is through

binding in a large cavity in the EBOV glycoprotein and destabilizing

its structure [28e30]. Thus, there are multiple steps and targets

substituted-1-piperidyl)methyl]-1H-benzimidazoles was to follow

Fig. 1. Some anti-ebola molecules and general structure of our new inhibitors.

2

M. Bessi ꢀe res, E. Plebanek, P. Chatterjee et al.

European Journal of Medicinal Chemistry 214 (2021) 113211

a convergent synthesis ending with the coupling of a (4-

substituted)-1-piperidine fragment at C6 position of a 6-(chlor-

omethyl)-1H-benzimidazole fragment, under SN conditions. Thus,

the synthesis of the (4-substituted)-1-piperidine fragment started

Thus, the 2-bromophenol 17 was protected to its tetrahydropyranyl

ether 18. The transmetallation of 18 with n-BuLi followed by the

nucleophilic addition on N-Boc-piperidine-4-carboxaldehyde

afforded 19. Unfortunately, the dehydroxylation/phenol depro-

with either the

xylene (1b), which were converted to their phosphonate analogs 2a

para position) and 2b (meta position), respectively, respectively, by

a

,

a

-dibromo-m-xylene (1a) or the

a

,

a

-dibromo-p-

3

tection of 19 with TFA/Et SiH gave the desired 22 in only 21% poor

yield, probably due to some removal of BOC under acidic traces. In

order to circumvent this problem, 17 was protected as benzyl ether

20, which, following a similar pathway, afforded 21 in 65% yield.

Final dehydroxylation/phenol deprotection of 21 by hydrogenation

under pressure (10 bars) during 24 h gave 22 in quantitative yields.

22 was then reacted with 1-(bromomethyl)-4-(diethox-

yphosphorylmethyl)benzene (2a) or its meta analog 2b in DMA at

(

SN of bromine with diethylphosphite. Beside, catechol (3) was

monoprotected to 4. Then, the nucleophilic substitution with 2a

and 2b in the presence of a 4 afforded the compounds 5a and 5b,

ꢀ

respectively, after 20 min at 80 C under microwave irradiation.

Removal of the pyrane protecting group by a treatment with pyr-

idinium p-toluenesulfonate (PPTS) during 3 h in ethanol afforded

ꢀ

140 C under microwave irradiation to afford 23a and 23b,

6

a and 6b, respectively, in excellent yields (Scheme 1).

The protected piperidine-4-one (7) was reduced to 8 and acti-

respectively, (Scheme 5). Subsequent deprotection of the carba-

mate protecting group was realized in acidic conditions and led 24a

and 24b, respectively. Finally, 2-substituted-6-[(4-benzyl-1-

piperidyl)methyl]-1H-benzimidazoles 25a and 26a-e were ob-

tained, respectively, as above described by (1) in situ chlorination of

the benzylic alcohol of 14a-e, in the presence of thionyl chloride

and subsequent substitution with piperidine fragment 24a,b under

basic conditions.

vated as tosyl leaving group analog 9. After a brief optimization of

the solvent, the activation and the substrate, it appears that the

nucleophilic substitution of tosylpiperidine 9 with 6a and 6b,

respectively, runs in optimal conditions with dimethylacetamide

(

DMA) under microwave irradiation (Scheme 2) and gave the

protected piperidines 10a and 10b, respectively, in good yields.

After deprotection of the carbamate group with triﬂuoroacetic acid,

the (4-aryloxy)-1-piperidines 11a (para-position) and 11b (meta-

position) were isolated, respectively.

2.2. Antiviral evaluation

The 6-(chloromethyl)-1H-benzimidazole fragment was syn-

thesized from methyl 3,4-diaminobenzoate and various benzalde-

The anti-EBOV activity and cytotoxicity of our molecules were

assessed using an infectious recombinant reporter EBOV and by cell

viability assay, respectively (Table 1), toremifene being used as

positive control [25].

hydes 12a-e via one-step process using NaHSO

3

40% as oxidant,

(

Scheme 3). The desired compounds 13a-e were isolated in a range

of 76e97% yields. After reduction of 13a-e, the (2-phenyl-3H-ben-

zimidazol-5-yl)methanol derivatives 14a-e were isolated in quan-

titative yields. The ﬁnal coupling was done by (1) in situ

chlorination of the benzylic alcohol of 14a-e, in the presence of

thionyl chloride and subsequent substitution with piperidine

fragment 11a,b under basic conditions. The ﬁnal 2-substitued-6-

As a result, all compounds having our benzimidazole-piperidine

scaffold were found to possess a good potency (EC50

introduction of CF - and CF O- groups (for increased lopophilicity)

didn’t improve the SI. Analogs bearing a piperidine-ether moiety

e.g. 2-substitued-6-[(4-aryloxy-1-piperidyl)methyl]-1H-benz-

mM range). The

3

(

imidazoles - serial A) appear to be less active than the 2-

substituted-6-[(4-benzyl-1-piperidyl)methyl]-1H-benzimidazoles

[(4-aryloxy-1-piperidyl)methyl]-1H-benzimidazoles 15a-e and

16a-e were isolated in moderate yields (over two-steps).

(

Serial B). Compounds 16a (EC50¼1.88

EC50¼0.87 M, SI ¼ 5) displayed good inhibitory activity, and a SI

M;

M and SI ¼ 7). Among those molecules, compound 26a

m

M, SI ¼ 7) and 26b

m

2

1

.1.2. Synthesis of 2-substituted-6-[(4-benzyl-1-piperidyl)methyl]-

H-benzimidazoles (serial B)

comparable or nearly equipotent to toremifene (EC50 ¼ 0.38

CC50 ¼ 2.50

(EC50¼0.93 M, SI ¼ 10) and 25a (EC50¼0.64 M, SI ¼ 20) are of great

m

Facing some difﬁculties to build these 2-substituted-6-[(4-

m

benzyl-1-piperidyl)methyl]-1H-benzimidazoles

through

the

interest, meanwhile compound 25a stood out as the most potent

against EBOV. 25a and 26a are isomers of position and they both

belong to serial B. To test if 25a and 26a inhibited EBOV entry, we

used HIV pseudotype particles bearing the EBOV GP. Both 25a and

26a inhibited the EBOV GP-mediated entry of these particles into

similar above described approach, we considered an alternative

approach based ﬁrst on the synthesis of the 2-(4-piperidylmethyl)

phenol (22), followed then on its selective functionalization of

either the piperidine moiety or the phenol moiety, (Scheme 4).

ꢀ

Scheme 1. Synthesis of compounds 6a and 6b. Reagents and conditions: (a) diethylphosphite, DMF, 150 C, MW, 2 min, 90% (b) DHP, PPTS, DCM, r.t., 3 h, 88e94% (c) NaI, K

DMF, MW, 80 C, 20 min, 72% (d) PPTS, EtOH, 55 C, 3 h, 99%.

2 3

CO ,

ꢀ

3

M. Bessi ꢀe res, E. Plebanek, P. Chatterjee et al.

European Journal of Medicinal Chemistry 214 (2021) 113211

Scheme 2. Synthesis of (4-aryloxy)-1-piperidines 11a and 11b. Reagents and conditions: (a) NaH, MeOH, rt, 20 h, 95% (b) TsCl, Et

MW, 140 C, 30 min, 72% (d) TFA in DCM 1:2, r.t., 1 h.

3

N, DCM, r.t., 20 h, 98% (c) 6a and 6b, K

2 3

CO , DMA,

ꢀ

Scheme 3. Synthesis of 2-substitued-6-[(4-aryloxy-1-piperidyl)methyl]-1H-benzimidazoles 15a-e and 16a-e. Reagents and conditions: (a) NaHSO

3

40%, EtOH, 100 C, 1e4 h,

76e97% b) LiAlH

4

, THF, r.t., 2e4 h, 94e99% (c) 1. SOCl

2

, reﬂux, 2.5 h, 2.11a or 11b, DIPEA, CH

3

CN, r.t., 19 h, 21e40% (over two-steps).

ꢀ

Scheme 4. Synthesis of compound 22. Reagents and conditions: (a) PPTS, DHP, DCM, r.t., 12 h, quant. (b) 1. n-BuLi, THF, ꢂ78 C, 30 min, 2.1-Boc-piperidine-4-carboxaldehyde, THF,

ꢀ

r.t., 3 h, 60e65%. c) TFA, Et

3

SiH, DCM

,

rt, 1 h, (21% from 19) and (65% from 21) (d) BnCl, K

2

CO

3

, DMF, 70 C, 12 h, quant. (e) H

2

, Pd/C, EtOAc, rt, 24 h, 10 bars, quant.

Huh7 cells, with EC50 and CC50 values similar to those seen with

infectious EBOV (Fig. 2A). The EBOV entry receptor is NPC1, which

functions in the cellular trafﬁcking of cholesterol. We looked to see

the effect of 25a and 26a on the subcellular localization of choles-

terol in HeLa cells. Both of these compounds induced a dramatic

accumulation of cholesterol to intracellular vacuoles, similar to that

seen with U18666A, a known inhibitor of EBOV entry that acts

through NPC1 (Fig. 2B). This effect was associated with minimal

cytotoxicity in HeLa cells (Fig. 2C). Together, this data suggests that

the mechanism by which 25a and 26a block EBOV infection is

through the inhibition of viral entry at the level of NPC1.

2.3. Chemoinformatics

In order to explain these data, 25a and 26a were docked in the

active site of induced ﬁt optimized NPC1 crystallized structure

(Fig. 3). The recently crystallized structure of NPC1 (PDB id: 5U73)

[33] was used for the initial docking model generation in Schrӧ-

dinger Suite (Protein Preparation Wizard Tool [34] and Glide [35] at

default settings, except the size of the grid box set as 30x30 ꢁ 30 Å).

Because the model showed highly unsatisfactory efﬁciency in

retrospective virtual screening experiments, cluster-speciﬁc

models were next obtained in an induced-ﬁt docking approach.

All 1637 compounds active against NPC1 fetched from the ChEMBL

4

M. Bessi ꢀe res, E. Plebanek, P. Chatterjee et al.

European Journal of Medicinal Chemistry 214 (2021) 113211

ꢀ

Scheme 5. Synthesis of 2-substituted-6-[(4-benzyl-1-piperidyl)methyl]-1H-benzimidazoles 25a and 26a-e. Reagents and conditions: (a) 2a or 2b, K

b) TFA, r.t., 1 h, 88e94% (c) 1.14a-e, SOCl , reﬂux, 2.5 h, 2. DIPEA, ACN, r.t., 19 h, 18e24%.

2

CO

3

, DMA, MW, 140 C, 30 min,

(

2

[

36] and the PDSP databases [37] (Ki or equivalent less than

3. Conclusions

1000 nM for ChEMBL ligands or marked as an active for PDSP

compounds) were clustered with the Hierarchical Clustering tool in

Canvas [38]. After manual reﬁnements, compounds were split into

We have developed an efﬁcient synthesis for the generation of

functionalized 2-substituted-6-[(4-substituted-1-piperidyl)

2

0 distinct chemical classes. Centroids from each cluster were used

methyl]-1H-benzimidazoles as entry inhibitors of Ebola virus. The

exploration of our scaffolds was focused on the piperidine moiety

as center core, and we also investigated the inﬂuence of various

substitution at the benzimidazole moiety. Our compounds also

exhibited a potent anti-EBOV activity, with four compounds with a

submicromolar activity, and two of them 25a and 26a with an

excellent selectivity index of, respectively, 10 and 20 (2e3 times

more selective than the FDA-approved anti-cancer drug Tor-

emifene). Data suggests that the mechanism by which 25a and 26a

block EBOV infection is through the inhibition of viral entry at the

level of NPC1. Finally, the ADME-Tox proﬁle of our compounds

showed an excellent in silico proﬁle for almost all synthesized

compounds, with good calculated permeability and solubility, low

HERG toxicity, high permeability and good protein plasma inter-

action. Altogether, these compounds deserve to be further opti-

mized and developed as potential antiﬁloviral drugs.

for the induced-ﬁt docking procedure [39] which generated one

model per cluster. Screening efﬁciency (tested as previously, with

the application of all actives and 88k decoys generated with the

application of DUD-E formalism [40]) was signiﬁcantly improved

and the best performing model (AUC increased from 0.501 for the

crystal structure to 0.722 for the best-optimized model) was used

for the docking studies of compounds 25a and 26a.

Analysis of the docking modes showed that several of the NPC1

amino acids that participate in binding to GP [41e43] are involved

in the binding of compounds 25a and 26a. The benzimidazole

moiety of both compounds formed a hydrogen bond with Q421 and

pi-pi stacking interaction with Y423. Both compounds created

additional pi-pi stacking interaction: compound 25a with F504 and

compound 26a with F503. Moreover, compound 26a was

hydrogen-bonded with Y423.

The #stars parameter, of the QikProp ADME prediction program,

can be used to help determine the drug-likeness of a particular

compound. The #stars values of approved drugs are generally

found in the acceptable range of 0e5. The most favorable value

4

. Experimental

4.1. Chemistry

#

star ¼ 0 was found for compound 26a. This value indicated 26a is

an ideally drug-like molecule (Table 2). Investigated compounds

showed few violations of Lipinski’s rule of ﬁve (RuleOfFive) and

Jorgensen’s rule of three (RuleOfThree). However, these guidelines

for deﬁning drug-like properties and oral availability are not always

entirely predictive for an optimal lead molecule. All compounds

showed good predicted human oral absorption. Their low aqueous

solubility (ClQPlogS) and high - predicted octanol/water partition

coefﬁcient (QPlogPo/w) indicated the hydrophobic properties. The

distribution of the compounds was simulated by calculation of

binding afﬁnities to human serum albumin (QPlogKhsa). The esti-

mated values have been found in the acceptable range for almost all

our compounds. To predict the drug cytotoxicity, the estimation of

IC50 values for blockage of HERG Kþ channel was used, and every

compound was found to be in the acceptable range. All molecules

showed excellent predicted Caco-2 cell (model for the gut-blood

barier) permeability and great predicted MDCK cell (good mimic

for the blood-brain barrier) permeability. Thus, these compounds

are predicted to cross the blood/brain barrier (QPlogBB) and may

potentially exhibit some activity in the central nervous system.

Commercially available chemicals were of reagent grade and

used as received. The reactions were monitored by thin layer

chromatography (TLC) analysis using silica gel plates (Kieselgel

6

0F254, E. Merck). Column chromatography was performed on

1

13

Silica Gel 60 M (0.040e0.063 mm, E. Merck). The H and C NMR

spectra were recorded on a Varian InovaUnity 400 spectrometer

(

3

400 MHz) in (d4) methanol, CDCl , shift values in parts per million

relative to SiMe as internal reference. High Resolution Mass

4

spectra were performed on a Bruker maxis mass spectrometer by

the “F eꢁ d eꢁ ration de Recherche”.

4.1.1. General procedure 1 (for the synthesis of 2a and 2b)

The microwave vial was charged with a,a-dibromo-m/p-xylene

(0.40 g, 1.5 mmol, 2 equiv), phosphite derivative (0.75 mmol, 1

ꢀ

equiv) and DMF (0.8 mL). The reaction mixture was heated at 150 C

for 2 min through microwave activation. Then, mixture was poured

into water and product was extracted with EtOAc. Combined

organic phases were washed with water and brine, then dried over

4

MgSO and concentrated under vacuum. Puriﬁcation by column

chromatography on silica gel (PE/EtOAc or DCM/MeOH) gave the

5

M. Bessi ꢀe res, E. Plebanek, P. Chatterjee et al.

European Journal of Medicinal Chemistry 214 (2021) 113211

Table 1

Anti-EboV activity of benzimidazole analogs.

Number

Compound

EC50^a

2.95

(m

M)

CC50^b

14.72

(m

M)

SI^c

5

15a

15b

2.95

14.72

5

1

5c

1.19

4.20

100

4

3

5d

29.67

1

5e

6a

1.73

1.88

3.82

2

7

13.14

16b

2.08

9.56

5

1

6c

1.97

2.49

4.23

4.59

2

6d

0

16e

8.73

15.50

2

5a

6a

0.64

0.93

13.21

9.17

20

10

26b

0.87

4.45

5

6

M. Bessi ꢀe res, E. Plebanek, P. Chatterjee et al.

Table 1 (continued)

European Journal of Medicinal Chemistry 214 (2021) 113211

Number

Compound

EC50^a

1.11

(m

M)

CC50^b

4.20

(m

M)

SI^c

4

26c

26d

26e

1.40

1.57

4.22

4.56

3

27

Toremifene

0.38

2.50

7

a

b

c

Effective concentration required to inhibit virus-expressed reporter ﬂuorescence by 50%.

Cytotoxic concentration required to cause a loss of cell viability by 50%.

Selectivity Index deﬁned as CC50/EC50

.

5

eP), 2.16e1.79 (m, 3H, H²^/³^/⁴^/⁵),

desired product.

H ), 3.15 (d, J ¼ 21.6 Hz, 2H, CH

2

/3/4/5

13

1

.74e1.56 (m, 4H, H

), 1.23 (t, J ¼ 7.0 Hz, 6H, CH

3

). C NMR

quat

(

C

(

101 MHz, CDCl 149.45 (C

3

)

d

),147.16 (C

), 129.83 (d, J ¼ 6.8 Hz, C ), 127.42

Ar Ar

),136.13 (d, J ¼ 3.6 Hz,

4

.1.1.1. Diethyl 4-(bromomethyl)benzylphosphonate (2a). The title

compound was prepared from -dibromo-p-xylene (1a) (0.40 g,

.5 mmol, 2 equiv) and triethyl phosphite (0.13 mL, 0.75 mmol, 1

equiv) following the general procedure 1. Compound 2a (0.21 g,

quat

Ar

), 131.11 (d, J ¼ 9.3 Hz, C

a,a

Ar

d, J ¼ 3.0 Hz, C ), 122.58 (C ), 121.84 (C ), 118.56 (C ), 115.58

1

Ar

Anomeric

5

C ), 97.61 (C

), 71.06 (CH

2

eO), 62.12 (d, J ¼ 6.8 Hz,

2/3/4

1

CH eOeP), 61.99 (C ), 33.55 (d, J ¼ 138.1 Hz, CH eP), 30.39 (C

),

8

d

4

8%) was obtained as a colorless oil. H NMR (400 MHz, CDCl

7.36 (d, J ¼ 8.1 Hz, 2H), 7.32e7.23 (m, 2H), 4.49 (s, 2H, BreCH

.09e3.89 (m, 4H, OeCH eP), 1.26 (t,

), 3.16 (d, J ¼ 21.8 Hz, 2H, CH

).

3

)

),

2

/3/4

2/3/4

31

2

(

5.30 (C

), 18.72 (C

), 16.36 (d, J ¼ 5.9 Hz, CH

3

). P NMR

2

þ

162 MHz, CDCl

3

)

d

26.27. HRMS-ESI (m/z) [MþNa] calcd for

2

C

23

H

31NaO P 457.1756, found 457.1760.

6

J ¼ 7.1 Hz, 6H, CH

3

4

.1.1.2. Diethyl 3-(bromomethyl)benzylphosphonate (2b). The title

compound was prepared from -dibromo-m-xylene (1b) (0.40 g,

.5 mmol, 2 equiv) and triethyl phosphite (0.13 mL, 0.75 mmol, 1

equiv) following the general procedure 1. Compound 2b (0.23 g,

4.1.4. Diethyl ({3-[2-(oxan-2-yloxy)phenoxymethyl]phenyl}methyl)

phosphonate (5b)

a,a

1

Compound 4 (665 mg, 1.1 eq., 3.43 mmol) was dissolved in DMF

(10 mL) in a 10e20 mL microwave vial. To this solution were added

potassium carbonate (868 mg, 2 eq., 6.28 mmol), compound 2b

(1 g, 1eq., 3.14 mmol) and few crystals of sodium iodide (catalytic

1

9

d

4%) was obtained as a colorless oil. H NMR (400 MHz, CDCl

7.38e7.21 (m, 4H), 4.49 (s, 2H, BreCH ), 4.12e3.94 (m, 4H,

), 3.16 (d, J ¼ 21.7 Hz, 2H, CH eP), 1.26 (t, J ¼ 7.1 Hz, 6H, CH ).

3

)

2

ꢀ

OeCH

2

3

amount). The solution was then stirred 20 min at 80 C under

microwave irradiation. After evaporation of all volatiles, the residue

was puriﬁed by silica gel chromatography to afford desired product

4

.1.2. 2-(Oxan-2-yloxy)phenol (4)

Catechol 3 (2 g, 18.2 mmol, 1 equiv) and dihydropyran (1.66 mL,

8.2 mmol, 1 equiv) were added to a solution of pyridinium p-tol-

1

5

b as a colorless oil. (974 mg, 72%). H NMR (250 MHz, CDCl

3

)

d

7.30

eO-

Ar

(

m, 4H, H ), 7.07 (s, 1H, H ) 6.88 (m, 3H, H ), 5.08 (s, 2H, CH

Ar), 3.97 (m, 5H, CH eOeP, OeCHeO), 3.57 (m, 1H, CH

eP), 2.11e1.44 (m, 7H, CH THP), 1.20 (t,

26.21. HRMS-ESI

P 457.1756, found 457.1760.

2

1

2

eO THP)

uenesulfonate (46 mg, 0.182 mmol, 1 mmol%) in DCM (35 mL). The

solution was then stirred at room temperature for 3 h, and the

solvent were removed under reduced pressure. The resulted

mixture was then dissolved in EtOAc, washed twice with NaHCO3,

once with brine, dried over MgSO4 and concentrated to afford pure

compound 4 (3.30 g, 94%) as a yellowish oil, [44]. CAS: 21645-25-0.

2

3

.12 (d, J ¼ 21.7 Hz, 2H, CH

2

31

J ¼ 7.1 Hz, 6H, CH

3 3

). P NMR (162 MHz, CDCl ) d

(

m/z) [MþNa]^þcalcd for C23

H31NaO

6

4.1.5. Diethyl {[4-(2-hydroxyphenoxymethyl)phenyl]methyl}

phosphonate (6a)

4

.1.3. Diethyl ({4-[2-(oxan-2-yloxy)phenoxymethyl]phenyl}methyl)

To a solution of phosphonate 5a (1.16 g, 1 eq., 2.67 mmol) in

ethanol (27 mL) pyridium p-toluenesulfonate (34 mg, 5 mol%,

0.13 mmol) was added. The mixture was then stirred at 55 C for

phosphonate (5a)

ꢀ

In a 10e20 mL microwave vial, compound 4 (1.12 g, 1 eq.,

5

.74 mmol) was dissolved in DMF (18 mL). To this solution, potas-

3 h, followed by the evaporation of all volatiles. The residue was

sium carbonate (1.58 g, 2 eq., 11.48 mmol), compound 2a (2.03 g, 1.1

then puriﬁed by ﬂash chromatography, eluting Petroleum Ether/

eq., 6.32 mmol) and few crystals of sodium iodide (catalytic

amount) were added. The mixture was then stirred 20 min at 80 C

under microwave irradiation. After evaporation of all volatiles, the

EtOAc 65:35 to 1:1, to give unprotected compound 6a as a white

ꢀ

1

solid. (936 mg, quantitative yield). H NMR (400 MHz, CDCl

3

)

d

7.35

eO),

Ar

(m, 4H, H ), 6.89 (m, 4H, H ), 5.68 (s, 1H, OH), 5.08 (s, 2H, CH

4.02 (ddq, J ¼ 10.2, 7.0, 3.0 Hz, 4H, CH eOeP), 3.17 (d, J ¼ 21.7 Hz,

13

2

residue was puriﬁed by silica gel chromatography to afford desired

2

1

product 5a as a colorless oil. (1.42 g, 57%). H NMR (400 MHz, CDCl

3

)

2H, CH

2

eP), 1.25 (t, J ¼ 7.0 Hz, 6H, CH

3 3

). C NMR (101 MHz, CDCl )

Ar

quat

d

(

7.40 (d, J ¼ 7.8 Hz, 2H, H ), 7.30 (dd, J ¼ 8.2, 2.5 Hz, 2H, H ), 7.15

d

145.96 (C

), 145.74 (C

), 135.02 (d, J ¼ 3.8 Hz, C

), 131.95 (d,

Ar

anomeric

quat

Ar

m, 1H, H ), 6.93 (m, 3H, H ), 5.43 (s, 1H, H

), 5.09 (t,

J ¼ 9.3 Hz, C

), 130.10 (d, J ¼ 6.5 Hz, C ), 128.03 (d, J ¼ 3.2 Hz, C ),

Ar

Ar Ar Ar

J ¼ 2.1 Hz, 2H, CH eO), 4.01 (m, 4H, CH eO-Phosph), 3.60 (m, 1H,

2

121.89 (C ), 120.08 (C ), 114.79 (C ), 112.33 (C ), 70.83 (CH eO),

7

M. Bessi ꢀe res, E. Plebanek, P. Chatterjee et al.

European Journal of Medicinal Chemistry 214 (2021) 113211

Fig. 2. Compounds block EBOV GP-mediated entry and alter the subcellular distribution of intracellular cholesterol, consistent with inhibition of NPC1 activity. (A & B) Compounds

block the entry of HIV pseudotype particles bearing the EBOV GP. Data represent the mean ± standard deviation from 3 biological repeats. Blue circles, entry; green, viability for (A)

compound 25a and (B) compound 26a. (C) Compounds alter the distribution of intracellular cholesterol, consistent with an inhibition of NPC1 activity. Immunoﬂuorescence

micrographs of HeLa cells treated with compounds 25a, 26a or their DMSO-vehicle, or U18666A or its water-vehicle, then stained with ﬁlipin to visualize intracellular cholesterol.

Bars are equivalent to 100

mm. (D) Minimal cytotoxicity in HeLa cells treated with these compounds. Cellular viability, relative to the DMSO vehicle-control, as determined by

CellTiter-Glo. Data represent the mean ± standard deviation from 3 biological repeats.

6

2.17 (d, J ¼ 6.9 Hz, CH

2

eOeP), 33.54 (d, J ¼ 138.0 Hz, CH

2

eP), 16.38

26.08. HRMS-ESI

P351.1362, found 351.1353.

CH

2

eP), 1.23 (t, J ¼ 7.1 Hz, 6H, CH

3

). ¹³C NMR (101 MHz, CDCl

)

3

31

quat

(

d, J ¼ 6.0 Hz, CH

3

). P NMR (162 MHz, CDCl

3

)

d

145.99 (C

), 145.76 (C

), 136.77 (d, J ¼ 3.2 Hz, C

), 132.28 (d,

þ

quat

Ar

m/z) [MþH] calcd for C18

H O

24 5

J ¼ 9.0 Hz, C

), 129.79 (d, J ¼ 6.7 Hz, C ), 129.11 (d, J ¼ 6.6 Hz, C ),

Ar

1

28.91 (d, J ¼ 3.0 Hz, C ), 126.26 (d, J ¼ 3.5 Hz, C ), 121.88 (C ),

Ar

120.08 (C ), 114.86 (C ), 112.37 (C ), 70.91 (CH

2

eO), 62.17 (d,

eP), 16.37 (d,

26.05. HRMS-ESI (m/

P 351.1362, found 351.1360.

4.1.6. Diethyl {[3-(2-hydroxyphenoxymethyl)phenyl]methyl}

J ¼ 6.7 Hz, CH

2

eOeP), 33.77 (d, J ¼ 138.2 Hz, CH

2

phosphonate (6b)

31

J ¼ 5.9 Hz, CH

3 3

). P NMR (162 MHz, CDCl ) d

To a solution of phosphonate 5b (1.60 g, 1 eq., 3.6 mmol) in

þ

z): [MþH] calcd for C18

24

H O

5

ethanol (37 mL) pyridium p-toluenesulfonate (45 mg, 5 mol%,

ꢀ

0

3

.18 mmol) was added. The mixture was then stirred at 55 C for

h, followed by the evaporation of all volatiles. The residue was

4.1.7. tert-Butyl 4-hydroxypiperidine-1-carboxylate (8)

tert-Butyl 4-oxopiperidine-1-carboxylate (5 g,

then puriﬁed by ﬂash chromatography, eluting Petroleum Ether/

7

1

eq.,

EtOAc 65:35 to 1:1, to give unprotected compound 6b as a light

25.1 mmol) was dissolved in MeOH (50 mL) under inert atmo-

sphere. This solution was cooled at 0 C and sodium borohydride

was subsequently added portionwise (950 mg, 1 eq., 25.1 mmol).

The mixture was allowed to reach room temperature and stirred for

1

ꢀ

brown oil. (1.28 g, quant.) H NMR (400 MHz, CDCl

3

)

d

7.32 (m, 4H,

eO), 4.01

eOeP), 3.17 (d, J ¼ 21.7 Hz, 2H,

Ar

H ), 6.89 (m, 4H, H ), 5.75 (s, 1H, OH), 5.10 (s, 2H, CH

ddq, J ¼ 8.2, 7.1, 3.2 Hz, 4H, CH

2

(

2

8

M. Bessi ꢀe res, E. Plebanek, P. Chatterjee et al.

European Journal of Medicinal Chemistry 214 (2021) 113211

Fig. 3. The docking binding poses of compound 25a (orange) 26a (purple) and in the active site of NPC1 (PDB ID: 5U73). Residues within a distance of 4 Å from compounds have

been shown. Hydrogen bond and pi-pi stacking interactions between NPC1 and docked compounds are in red and blue, respectively. In the left bottom corner, whole NPC1 protein is

shown, with the binding site highlighted in the red box.

Table 2

ADME proﬁle of synthesized compounds.

#stars QPlogPo/w QPlogKhsa QPlogHERG QPPCaco

QPPMDCK

Percent

Human

Oral

Rule of Five Rule of Three

Absorption

Range or recommended values 0e5 ꢂ2.0e6.5 ꢂ1.5e1.5 Below ꢂ5 <25 poor, >500 great <25 poor, >500 great <25 poor, >80% high Max 4

Max 3

3

1

2

.47 [22]

5a

3

2

4

7

8

3

4

7

4

7

6

2

0

1

5

1

4.54

6.31

7.61

7.83

7.93

7.67

6.90

8.07

7.64

7.88

7.86

6.81

6.32

7.08

7.53

8.07

7.36

0.54

1.04

1.40

1.44

1.55

1.37

1.18

1.49

1.29

1.44

1.27

1.02

1.19

1.36

1.51

1.26

ꢂ5.21

ꢂ8.16

ꢂ7.90

ꢂ8.33

ꢂ7.71

ꢂ8.72

ꢂ8.34

ꢂ7.66

ꢂ8.64

ꢂ8.58

ꢂ6.72

ꢂ6.05

ꢂ5.86

ꢂ6.66

ꢂ7.51

ꢂ6.27

60.06

71.04

72.39

87.82

96.89

100

1

2

1

0

1

0

1

2

1

608.80

736.35

752.54

748.73

321.19

5b

5c

5d

5e

6a

6b

6c

6d

6e

5a

6a

6b

6c

1285.39

1879.53

1490.82

3433.29

625.09

2646.59

3833.36

2744.82

2721.34

584.78

100

95.94

100

95.09

96.08

100

1397.40

1113.77

1033.88

1455.18

1113.92

1111.66

1063.19

1741.80

1676.22

1728.33

1423.42

1731.97

997.06

4173.72

3415.32

3672.55

3660.05

100

6d

6e

ꢀ

2

0h. The solution was carefully quenched at 0 C with 2 N sodium

4

with DCM (2 x 40 mL), dried over MgSO and evaporated. The crude

hydroxide (20 mL), followed by evaporation of all volatiles. The

mixture was then dissolved in EtOAc (40 mL) and water (20 mL).

The aqueous phase was extracted with EtOAc (3 x 40 mL), the

was then puriﬁed by silica gel column chromatography (Petroleum

ether/EtOAc 8:2) to afford desired product 9 as a white crystalline

solid. (8.7 g, 99%), [46]. CAS: 118811-07-7.

organic phases were washed with H

2

O (20 mL), brine (20 mL), dried

over MgSO and concentrated under reduced pressure. Puriﬁcation

4

by silica gel column chromatography (Petroleum Ether/EtOAc 8:2)

afforded desired product 8 as a white solid. (4.8 g, 95%), [45]. CAS:

4.1.9. tert-Butyl-4-{[2-({4-[(diethoxyphosphoryl)methyl]phenyl}

methoxy)phenyl]-methyl }piperidine-1-carboxylate (10a)

In a 10e20 mL microwave vial, phosphonate compound 6a

109384-19-2.

(

512 mg, 1 eq., 1.46 mmol) was dissolved in dimethylacetamide

13 mL). To this solution, potassium carbonate (404 mg, 2 eq.,

4

.1.8. tert-Butyl 4-[(4-methylbenzenesulfonyl)oxy]piperidine-1-

2.92 mmol) and compound 9 (778 mg, 1.5 eq., 2.19 mmol) were

then added and this mixture was stirred at 140 C during 30 min

ꢀ

carboxylate (9)

To a solution of compound 8 (5 g, 1 eq., 24.8 mmol) in DCM

50 mL), triethylamine (13.9 mL, 4 eq., 99.4 mmol) and p-toluene-

under microwave irradiation. The resulting residue was dissolved

in EtOAc (20 mL) and water (25 mL), and the aqueous phase was

extracted thrice with EtOAc (3 x 20 mL). The organic phases were

then washed 5 times with water (5 x 25 mL), once with brine

(25 mL) and dried over magnesium sulfate. The residue was

concentrated under reduced pressure and puriﬁed by silica gel

column chromatography, eluting DCM/MeOH 99:1, to afford the

(

sulfonyl chloride (9.47 g, 2 eq., 49.7 mmol) were added. The

mixture was stirred 20 h at room temperature followed by the

concentration of this solution under reduced pressure. The residue

was then dissolved in DCM (20 mL) and water (20 mL), extracted

with DCM (20 mL), washed with 1 N HCl (40 mL), extracted twice

9

M. Bessi ꢀe res, E. Plebanek, P. Chatterjee et al.

European Journal of Medicinal Chemistry 214 (2021) 113211

intermediate 10a as colorless oil which was directly engaged in the

4.1.11.2. Diethyl

({3-[2-(piperidin-4-ylmethyl)phenoxymethyl]

next step.

phenyl}methyl) phosphonate (11b). Following general procedure 2,

triﬂuoroacetic acid (1.5 mL, 100 eq., 19.7 mmol) was added to a

solution of compound 10b (105 mg, 1 eq., 0.197 mmol) in DCM

4.1.10. tert-Butyl-4-{[2-({3-[(diethoxyphosphoryl)methyl]phenyl}

(3 mL). Pure compound 11b was obtained after ﬂash column

methoxy) phenyl]-methyl}piperidine-1-carboxylate (10b)

chromatography (DCM/MeOH 95:5), affording desired product as

In a 2e5 mL microwave vial, phosphonate compound 6b

1

3

an orange solid. (58 mg, 68%). H NMR (400 MHz, CDCl ) d 7.50 (d,

(

100 mg, 1 eq., 0.29 mmol) was dissolved in dimethylacetamide

DMA, 2.5 mL). To this solution potassium carbonate (81 mg, 2 eq.,

Ar

J ¼ 2.3 Hz, 1H, H ), 7.29 (m, 1H, H ), 7.15 (m, 2H, H ), 6.96 (m, 4H,

Ar

4

H ), 4.98 (s, 2H, CH

6.6, 0.5 Hz, 4H, CH

.12 (d, J ¼ 21.8 Hz, 2H, CH

2

eO), 4.60 (s, 1H, H pip), 3.95 (2x ddq, J ¼ 7.6,

0

.57 mmol) and compound 9 (152 mg, 1.5 eq., 0.43 mmol) were

2

6

ꢀ

2

eOeP), 3.32 (td, J ¼ 12.9, 3.8 Hz, 2H, H , H pip),

afterwards added. This reaction mixture was stirred at 140 C for

2 6

3

2

eP), 3.09 (m, 2H, H , H pip), 2.25 (tdd,

3

0 min under microwave irradiation. The resulting residue was

3

5

3

J ¼ 12.8, 4.7, 2.2 Hz, 2H, H , H pip), 1.96 (dd, J ¼ 13.1,2.9 Hz, 2H, H ,

dissolved in EtOAc (10 mL) and water (5 mL), and the aqueous

phase was extracted thrice with EtOAc (3 x 10 mL). The organic

phases were then washed 5 times with water (5 x 5 mL), once with

brine (5 mL) and dried over magnesium sulfate. The crude was

concentrated under reduced pressure and puriﬁed by silica gel

column chromatography, eluting DCM/MeOH 99:1, to afford

5

13

H pip), 1.22 (td, J ¼ 7.1, 0.5 Hz, 6H, CH

3

). C NMR (101 MHz, CDCl

3

)

quat

d

150.77 (C

J ¼ 9.5 Hz, C

), 146.38 (C

), 137.04 (d, J ¼ 3.4 Hz, C

), 131.41 (d,

quat

Ar

),129.87 (d, J ¼ 6.7 Hz, C ),128.91 (d, J ¼ 6.0 Hz, C ),

Ar

1

26.78 (d, J ¼ 3.2 Hz, C ), 124.51 (d, J ¼ 5.0 Hz, C ), 123.47(C ),

Ar Ar Ar 4

1

21.87(C ), 119.29(C ), 114.14(C ), 71.32(C pip), 70.80 (CH

2

eO),

2

6

1

63.09 (d, J ¼ 6.9 Hz, CH

2

eOeP), 39.44(C , C pip), 32.96 (d,

desired product 10b as colorless oil. (100 mg, 71%). H NMR

3

5

31

Ar

J ¼ 139.5 Hz, CH

2

eP), 26.66(C , C pip), 16.06 (d, J ¼ 6.2 Hz, CH

3

).

P

(

400 MHz, DMSO) d 7.29 (m, 3H, H ), 7.19 (s, 1H, H ), 7.01 (m, 2H,

Ar

4

NMR (162 MHz, CDCl

3

)

d

26.57. HRMS-ESI (m/z) [MþH]^þcalcd for

2

H ), 6.85 (m, 2H, H ), 5.03 (s, 2H, CH eO), 4.41 (m, 1H, H pip),

2

6

23 5

C H33NO P 434.2097, found 434.2094.

3

.87 (ddq, J ¼ 6.9, 6.1, 0.4 Hz, 4H, CH

2

eOeP), 3.56 (m, 2H, H , H

2

eP),3.15 (m, 2HH , H pip),1.79 (s,

Boc), 1.07 (dt,

). C NMR (101 MHz, DMSO) d 154.36 (C]O),

2

6

pip), 3.17 (d, J ¼ 21.5 Hz, 2H, CH

3

5

3

5

4.1.12. General procedure 3 (for benzimidazole ring construction)

Solution of an aldehyde (12a-e, respectively), (1 equiv) in 40%

3

NaHSO (4 mL) was stirred for 1h at room temperature, then so-

2

H, H , H pip), 1.51 (s, 2H, H , H pip), 1.35 (s, 9H, CH

3

1

3

J ¼ 6.9, 0.4 Hz, 6H, CH

3

quat

150.05 (C

), 147.18 (C

), 137.83 (d, J ¼ 3.0 Hz, C

), 132.88 (d,

quat

Ar

lution of methyl 3,4-diaminobenzoate (0.65 g, 3.94 mmol, 1 equiv)

J ¼ 8.7 Hz, C

),129.61 (d, J ¼ 6.8 Hz, C ),129.07 (d, J ¼ 6.8 Hz, C ),

ꢀ

Ar Ar Ar

in EtOH (2 mL) was added. Resulting mixture was stirred at 100 C

1

28.74 (d, J ¼ 2.5 Hz, C ), 126.00 (d, J ¼ 3.7 Hz, C ), 122.57 (C ),

Ar

quat

4

6

5

for 1e4h, then concentrated. Residue was dissolved in water and

extracted with EtOAc. Combined organic phases were dried over

MgSO and concentrated. The crude product was puriﬁed by ﬂash

4

column chromatography (PE/EtOAc or DCM/MeOH) to afford

desired product.

1

21.79 (C ), 118.59 (C ), 115.52 (C ), 79.11 (C

pip), 70.43(CH eOeP), 39.74 (C , C

eO), 61.83 (d, J ¼ 6.5 Hz, CH

pip, under DMSO peak),32.71 (d, J ¼ 134.0 Hz, CH

pip), 28.53 (CH

Boc), 16.64 (d, J ¼ 5.9 Hz, CH

162 MHz, DMSO)

P 534.2621, found 534.2615.

Boc), 74.19 (C

2

3

2

eP), 31.00 (C , C

eOeP). P NMR

26.34. HRMS-ESI (m/z) [MþH] calcd for

31

3

2

þ

(

d

28 7

C H41NO

4

.1.12.1. 5-Methyl

The title compound was prepared from benzaldehyde 12a (0.4 mL,

.94 mmol, 1 equiv) following the general procedure 3. Compound

carboxylate-2-phenyl-benzimidazole

(13a).

4.1.11. General procedure 2 (for BOC removal)

3

Triﬂuoroacetic acid (100 eq.) was added dropwise to a mixture

1

3a (0.97 g, 97%) was obtained as a white solid. H NMR (250 MHz,

CDCl

8.39 (s, 1H), 8.18e8.10 (m, 2H), 8.00 (dd, J ¼ 8.5, 1.5 Hz, 1H),

.66 (d, J ¼ 8.2 Hz,1H), 7.53e7.45 (m, 3H), 3.97 (s, 3H, CH

101 MHz, CDCl 167.97, 130.99, 129.44, 129.38, 127.08, 125.02,

of Boc-compound (1 eq.) in DCM (2:1 DCM/TFA v/v). The reaction

was stirred at room temperature for 2h and then volatiles were

removed under reduced pressure. The crude product was extracted

3

) d

13

7

(

3

). C NMR

3

) d

with EtOAc, washed with NaHCO

3

until pH 7, dried over MgSO

4

,

þ

124.70, 52.42 (CH

3

). HRMS (ESI) m/z [MþH] calcd for C15

13 2 2

H N O

ﬁltrated and concentrated under vacuum. Pure compounds were

obtained after puriﬁcation by ﬂash column chromatography with

DCM/MeOH (95:5) as eluent.

2

53.0972 found 253.0974.

4

.1.12.2. 5-Methyl carboxylate-2-[4-(triﬂuoromethyl)phenyl]-

benzimidazole (13b). The title compound was prepared from 4-

triﬂuoromethyl-benzaldehyde (0.41 mL, 3.00 mmol, 1 equiv)

4.1.11.1. Diethyl

({4-[2-(piperidin-4-yloxy)phenoxymethyl]phenyl}

methyl) phosphonate (11a). Following general procedure 2, tri-

ﬂuoroacetic acid (11.1 mL, 100 eq., 146 mmol) was added to a so-

lution of intermediate 10a in DCM (20 mL). Pure compound 11a was

obtained after ﬂash column chromatography (DCM/MeOH 95:5),

following the general procedure 3. Compound 13b (0.90 g, 93%) was

1

obtained as a white solid. H NMR (400 MHz, DMSO)

d 13.51 (s, 1H,

NH), 8.39 (d, J ¼ 8.3 Hz, 2H), 8.35e7.99 (m, 1H), 7.95 (d, J ¼ 8.3 Hz,

13

2H), 7.92e7.82 (m, 1H), 7.81e7.59 (m, 1H), 3.88 (s, 3H, CH

NMR (101 MHz, DMSO) 166.63, 133.31, 130.33, 127.43, 126.05,

126.01, 125.40, 122.69, 111.82, 52.05 (CH

3

).

C

1

affording desired product as an orange solid. (588 mg, 67%). H

d

Ar

19

NMR (400 MHz, CDCl

3

)

d

7.45 (d, J ¼ 8.0 Hz, 2H, H ), 7.29 (d, J ¼ 8.2,

3

). F NMR (376 MHz,

Ar

þ

2

.5 Hz, 2H, H ), 7.09 (m, 2H, H ), 7.01 (t, J ¼ 7.3 Hz, 1H, H ), 6.94

DMSO)

d

ꢂ61.30. HRMS (ESI) m/z [MþH] calcd for C16

12 3 2 2

H F N O

Ar

4

ꢂ1

(

m, 1H, H ), 5.10 (s, 2H, CH

2

eO), 4.57 (quint., J ¼ 4.3 Hz, 1H, H pip),

eOeP), 3.38 (dt, J ¼ 13.0,

.0 Hz, 2H, H , H pip), 3.26 (d, J ¼ 21.8 Hz, 2H, CH eP), 3.09 (m, 2H,

321.0845 found 321.0848. IR (cm ) 3296, 1688, 1618, 1443, 1319,

1289, 1239, 1225, 1163, 1114, 1100, 1064, 1019, 984, 850, 765, 749,

4

7

.04 (2x ddq, J ¼ 7.7, 6.7, 2.3 Hz, 4H, CH

2

6

ꢀ

2

696, 653. Mp 260 C.

2

6

3

5

H , H pip), 3.13 (tdd, J ¼ 12.8, 5.0 Hz, 2H, H , H pip), 2.03 (dt,

3

5

13

J ¼ 7.1,5.3 Hz, 2H, H , H pip), 1.26 (t, J ¼ 7.0 Hz, 6H, CH

3

). C NMR

4.1.12.3. 5-Methyl carboxylate-2-[3-(triﬂuoromethyl)phenyl]-

benzimidazole (13c). The title compound was prepared from 3-

triﬂuoromethyl-benzaldehyde (0.24 mL, 1.8 mmol, 1 equiv)

quat

Ar

(

101 MHz, CDCl

3

)

d

146.55 (C

), 135.15 (d, J ¼ 3.8 Hz, C

), 129.78

Ar

d, J ¼ 6.6 Hz, C ), 127.65 (d, J ¼ 3.3 Hz, C ), 123.08 (C ), 121.41

Ar

4

C ), 118.82 (C ), 114.95 (C ), 70.80 (C pip), 70.52 (CH

2

eO), 62.35

following the general procedure 3. Compound 13c (0.44 g, 76%) was

2

6

1

d, J ¼ 7.0 Hz, CH

2

eOeP), 40.24(C , C pip), 31.86(d, J ¼ 138.0 Hz,

obtained as a white solid. H NMR (400 MHz, DMSO)

d

13.45 (s, 1H,

3

5

31

CH

2

eP), 27.06(C , C pip), 15.24(d, J ¼ 6.1 Hz, CH

3

). P NMR

NH), 8.53 (s, 1H), 8.49 (d, J ¼ 7.8 Hz, 1H), 8.22 (s, 1H), 7.92e7.80 (m,

þ

13

(

162 MHz, CDCl

3

)

d

27.50. HRMS-ESI (m/z) [MþH] calcd for

3H), 7.75e7.68 (m,1H), 3.88 (s, 3H, CH

3

). C NMR (101 MHz, DMSO)

C

23

H33NO

5

P 434.2097, found 434.2100.

d

166.64, 152.21, 130.60, 130.55, 130.36, 130.03, 129.71, 126.85,

10

M. Bessi ꢀe res, E. Plebanek, P. Chatterjee et al.

European Journal of Medicinal Chemistry 214 (2021) 113211

1

26.82, 125.35, 123.84, 123.66, 123.14, 123.10, 122.64, 52.05 (CH

3

).

4.1.13.3. 5-Hydroxymethyl-2-[3-(triﬂuoromethyl)phenyl]-benzimid-

azole (14c). The title compound was prepared from 13c (0.31 g,

0.96 mmol, 1 equiv) following the general procedure 4. Compound

19

þ

F NMR (376 MHz, DMSO)

for C16

d

ꢂ61.32. HRMS (ESI) m/z [MþH] calcd

ꢂ1

H

12

3

F N

2

O

2

321.0845 found 321.0848. IR (cm ) 3296, 1694,

1

628,1538,1459,1438,1331,1306,1285,1270,1221,1182, 1166,1121,

108, 1098, 1070, 991, 919, 803, 772, 751, 700, 688, 650, 612.Mp

14c (0.27 g, 95%) was obtained as a white solid. H NMR (400 MHz,

MeOD)

7.69e7.55 (m, 2H), 7.31 (dd, J ¼ 8.3, 0.9 Hz, 1H), 4.75 (s, 2H, CH

NMR (101 MHz, MeOD) 151.92, 132.76, 132.44, 132.10, 131.14,

127.66, 127.62, 126.77, 124.41, 124.37, 124.07, 65.55 (CH

d

8.44 (s, 1H), 8.34 (d, J ¼ 7.7 Hz, 1H), 7.85e7.71 (m, 2H),

ꢀ

13

190 C.

2

). C

d

1

9

4

.1.12.4. 5-Methyl carboxylate-2-[4-(triﬂuoromethoxy)phenyl]-

benzimidazole (13d). The title compound was prepared from 4-

triﬂuoromethoxybenzaldehyde (0.75 mL, 5.3 mmol, 1 equiv)

2

). F NMR

þ

(376 MHz, MeOD)

d

ꢂ64.33. HRMS (ESI) m/z [MþH] calcd for

ꢂ1

C

15

H

12

3

F N

2

O 293.0896 found 293.0901. IR (cm ) 3063, 2866, 2824,

following the general procedure 3. Compound 13d (1.38 g, 78%) was

1437, 1399, 1328, 1312, 1281, 1168, 1119, 1072, 1002, 975, 820, 798,

720, 699, 686, 657, 650.Mp 202 C.

1

ꢀ

obtained as a light yellow solid. H NMR (400 MHz, DMSO)

d

8.31 (d,

Ar

J ¼ 8.7 Hz, 2H, H ), 8.21 (s, 1H, H ), 7.87 (dt, J ¼ 8.4 Hz, 1H, H ),

Ar

13

7

.70 (d, J ¼ 8.7 Hz, 1H, H ), 7.59 3.88 (s, 3H, CH

3

). C NMR

4.1.13.4. 5-Hydroxymethyl-2-[4-(triﬂuoromethoxy)phenyl]-benz-

imidazole (14d). The title compound was prepared from 13d (1.38 g,

4.11 mmol, 1 equiv) following the general procedure 4. Compound

quat

(

101 MHz, DMSO)

d

167.15(C]O), 152.98(C

29.31(C ), 129.25(C ), 126.73(C ), 124.11(C ), 123.96(C ),

), 150.12(C

),

Ar

1

Ar

19

1

21.99(C ), 121.77(C ), 52.50 (CH

3

). F NMR (376 MHz, DMSO)

ꢂ56.64. HRMS (ESI) m/z [MþH] calcd for C16 337.0839

found 337.0842.

14d (1.21 g, 95%) was obtained as a white solid. H NMR (400 MHz,

þ

Ar

d

H

12

F

3

N

2

O

2

DMSO)

d

12.94 (s, 1H, NH), 8.29 (d, J ¼ 8.4 Hz, 2H, H ), 7.61 (s, 1H,

Ar

H ), 7.56 (d, J ¼ 8.4 Hz, 2H, H ), 7.51 (s, 1H, H ), 7.18 (s, 1H, H ),

13

5.22 (s, 1H, OH), 4.61 (s, 2H, CH

2

). C NMR (101 MHz, DMSO)

4

.1.12.5. 5-Methyl carboxylate-2-[3-(triﬂuoromethoxy)phenyl]-

d

150.30, 149.62, 144.25, 143.21, 138.14, 136.94, 135.50, 129.98,

Ar

quat

Ar

benzimidazole (13e). The title compound was prepared from 3-

triﬂuoromethoxybenzaldehyde (0.75 mL, 5.3 mmol, 1 equiv)

following the general procedure 3. Compound 13e (1.61 g, 91%) was

128.77 (C ), 124.35, 122.68 (C

(C

), 121.91 (C ), 121.80 (C ), 121.49

quat

Ar

), 119.25, 118.20 (d, J ¼ 157.8 Hz, C ), 116.69, 110.53 (d,

Ar 19

J ¼ 174.2 Hz, C ), 63.85 (CH

2

). F NMR (376 MHz, DMSO)

d

ꢂ56.68.

1

þ

obtained as a light yellow solid. H NMR (400 MHz, DMSO)

d

8.24

HRMS (ESI) m/z [MþH] calcd for C15

12 3 2

H F N O 309.0890 found

Ar

(

7

d, J ¼ 7.6 Hz, 2H, H ), 8.16 (s, 1H, H ), 7.88 (d, J ¼ 8.4 Hz, 1H, H ),

309.0893.

Ar

13

.75 (m, 2H, H ), 7.55 (d, J ¼ 7.6 Hz, 1H, H ), 3.89 (s, 3H, CH

3

).

C

quat

NMR (101 MHz, DMSO)

d

167.12 (C]O), 153.88 (C

), 132.26 (C ), 131.81 (C

), 152.66

4.1.13.5. 5-Hydroxymethyl-2-[3-(triﬂuoromethoxy)phenyl]-benz-

imidazole (14e). The title compound was prepared from 13e (1.61 g,

4.80 mmol, 1 equiv) following the general procedure 4. Compound

14e (1.41 g, 95%) was obtained as a white solid. H NMR (400 MHz,

DMSO)

quat

Ar

quat

Ar

(

C

), 149.31 (C

), 126.20 (C ),

Ar

19

124.27 (C ), 123.31 (C ), 119.39 (C ), 52.53 (CH

3

)

F NMR

þ

1

(

376 MHz, DMSO)

d

ꢂ56.71. HRMS (ESI) m/z [MþH] calcd for

Ar

C

16

H

12

F

3

N

2

O

2

337.0839 found 337.0841.

d

13.05 (bs, 1H, NH), 8.20 (d, J ¼ 8.0 Hz, 1H, H ), 8.13 (s, 1H,

Ar

H ), 7.70 (t, J ¼ 8.0 Hz, 1H, H ), 7.58 (d, J ¼ 8.2 Hz, 1H, H ), 7.56 (s,

Ar

4

.1.13. General procedure 4 (for reduction with AlLiH

4

)

1H, H ), 7.49 (d, J ¼ 8.0 Hz,1H, H ), 7.20 (d, J ¼ 8.2 Hz,1H, H ), 5.21

13

To the mixture of LiAlH

4

(0.03 g, 0.8 mmol, 2 equiv) in dry THF

(bs, 1H, OH), 4.62 (s, 2H, CH

2

). C NMR (101 MHz, DMSO)

d

150.02

ꢀ

quat

Ar

quat

Ar

(

1 mL), cooled at 0 C, solution of methyl 2-substitued-benzimid-

(C

122.54 (C ), 121.86 (C ), 119.31 (C

NMR (376 MHz, DMSO)

O 309.0890 found 309.0892.

), 149.33 (C

), 132.94 (C ), 131.64 (C

), 125.70 (C ),

Ar

quat

19

azole-5 carboxylate (1 equiv) in dry THF (1 mL) was slowly added.

The ice bath was removed, and the reaction mixture was stirred for

2

), 118.93 (C ), 63.80 (CH

2

).

F

þ

d

ꢂ56.69. HRMS (ESI) m/z [MþH] calcd for

e4h. After completion EtOAc and water were added. Then,

15 12 3 2

C H F N

aqueous phase was extracted with EtOAc (3x) and combined

organic phases were dried over MgSO

desired product.

4

and concentrated to give

4.1.14. General procedure 5 (for coupling piperidine fragment with

benzimidazole fragment)

2

To a ﬂask containing benzimidazole derivative (1 eq.), SOCl (29

4

.1.13.1. 5-Hydroxymethyl-2-phenyl-benzimidazole (14a). The title

eq.) was added and the resulted mixture was stirred for 2h30 at

ꢀ

compound was prepared from 13a (0.10 g, 0.4 mmol, 1 equiv)

following the general procedure 4. Compound 14a (0.09 g, quan-

titative) was obtained as a white solid. H NMR (250 MHz, DMSO)

80 C. After cooling to room temperature, SOCl

2

was evaporated

and the obtained solid was diluted with ACN (4 mL) and cooled to

1

ꢀ

0 C. Then, to this solution, piperidine derivative (1 eq.) and dii-

d

7

2

12.86 (s, 1H, NH), 8.16 (dd, J ¼ 8.0, 1.3 Hz, 2H), 7.76e7.36 (m, 5H),

sopropylethylamine (5.3 eq.) were added. The ice bath was

removed and the reaction mixture was allowed to stir for

16e19 h at room temperature. After concentration, the crude

product was puriﬁed by column chromatography (DCM/MeOH) to

give the desired product.

.16 (d, J ¼ 8.1 Hz, 1H), 5.20 (t, J ¼ 5.4 Hz, 1H, OH), 4.60 (d, J ¼ 5.0 Hz,

13

H, CH

2

). C NMR (101 MHz, DMSO)

d

130.24, 129.70, 128.91,

þ

126.31, 63.40 (CH

2

). HRMS (ESI) m/z [MþH] calcd for C14

13 2

H N O

2

25.1022 found 225.1024.

4

.1.13.2. 5-Hydroxymethyl-2-[4-(triﬂuoromethyl)phenyl]-benzimid-

4.1.14.1. Diethyl

({4-[2-({1-[(2-phenyl-1H-1,3-benzodiazol-6-yl)

azole (14b). The title compound was prepared from 13b (0.40 g,

.25 mmol, 1 equiv) following the general procedure 4. Compound

methyl]piperidin-4-yl}oxy)phenoxymethyl]-phenyl}methyl)phospho-

nate (15a). The title compound was obtained following the general

procedure 5 from benzimidazole 14a (31 mg, 1 eq., 0.138 mmol),

1

4b (0.35 g, 94%) was obtained as a white solid. H NMR (400 MHz,

DMSO)

8.37 (d, J ¼ 8.1 Hz, 2H), 7.93 (d, J ¼ 8.3 Hz, 2H), 7.62e7.49

m, 2H), 7.21 (d, J ¼ 8.8 Hz,1H), 5.21 (s,1H, OH), 4.62 (s, 2H, CH

NMR (101 MHz, DMSO) 149.58, 134.01, 129.68, 126.95, 125.96,

25.92, 125.50, 122.79, 63.32 (CH

d

SOCl

2

(293

mL, 29 eq., 4.01 mmol) and then compound 11a (60 mg, 1

1

3

(

2

)$ C

eq., 0.138 mmol), diisopropylethylamine (124 m

L, 5.3 eq.,

d

0.73 mmol) in ACN (2 mL). After puriﬁcation by silica gel column

19

1

d

2

)$ F NMR (376 MHz, DMSO)

chromatography, eluting DCM/MeOH 95:5, the clean compound

þ

1

ꢂ61.16.HRMS (ESI) m/z [MþH] calcd for C15

H

12

F

3

N

2

O 293.0896

15a was obtained as an amorphous creamy solid. (29 mg, 40%) H

ꢂ1

Ar

found 293.0900.IR (cm ) 3131, 2944, 2877, 1621, 1434, 1316, 1281,

NMR (400 MHz, MeOD)

d

8.12 (d, J ¼ 7.0 Hz, 2H, H ), 7.63 (m, 5H,

Ar

1

6

164, 1116, 1065, 1010, 961, 876, 849, 808, 786, 749, 693, 646,

34.Mp 228 C.

H ), 7.37 (m, 5H, H ), 7.04 (m, 2H, H ), 6.91 (dquint, J ¼ 7.3,

ꢀ

A

r

4

2

1.9 Hz,2H, H ), 5.06 (s, 2H, CH eO), 4.48 (s, 1H, H pip), 4.00 (ddq,

11

M. Bessi ꢀe res, E. Plebanek, P. Chatterjee et al.

European Journal of Medicinal Chemistry 214 (2021) 113211

J ¼ 8.8, 7.0, 1.8 Hz, 4H, CH

2

eOeP), 3.93 (s, 2H, CH

J ¼ 21.7 Hz, 2H, CH eP), 3.06 (bs, 2H, H , H pip), 2.76 (bs, 2H, H , H

2

eN), 3.24 (d,

methyl}phosphonate (15d). The title compound was obtained

following the general procedure 5 from benzimidazole 14d (36 mg,

2

6

2

6

2

3

5

13

pip), 1.97 (m, 4H, H , H pip), 1.23 (t, J ¼ 7.0 Hz, 6H, CH

3

). C NMR

1 eq., 0.12 mmol), SOCl

compound 11a (50 mg, 1 eq., 0.12 mmol), diisopropylethylamine

(104 L, 5.3 eq., 0.61 mmol) in ACN (2 mL). After puriﬁcation by

2

(244 mL, 29 eq., 3.35 mmol) and then

quat

(

101 MHz, MeOD)

d

150.15 (C

), 147.01 (C

), 136.24 (d,

quat

Ar

J ¼ 3.9 Hz, C

), 131.11 (d, J ¼ 9.2 Hz, C

), 130.19 (C ), 129.71 (d,

m

Ar

J ¼ 6.6 Hz, C ), 129.42 (C ), 128.79 (C ), 127.59 (d, J ¼ 4.0 Hz, C ),

silica gel column chromatography, eluting DCM/MeOH 95:5, the

clean compound 15d was obtained as an amorphous creamy solid.

Ar

1

26.48 (C ), 122.46 (C ), 121.46 (C ), 118.52 (C ), 115.23 (C ),

1

7

4

0.49 (CH eOeP), 62.09 (CH eN),

2

eO), 62.30 (d, J ¼ 6.9 Hz, CH

2

(17 mg, 23%) H NMR (400 MHz, MeOD)

d

8.17 (d, J ¼ 9.0 Hz, 2H,

2

6

3

5

Ar

8.99 (C , C pip), 32.00 (d, J ¼ 138.2 Hz, CH

2

eP), 29.14 (C , C pip),

). P NMR (162 MHz, MeOD) 27.43 HRMS-

ESI (m/z) [MþH] calcd for C37 P 640.2941, found 640.2935.

H ), 7.61 (m, 2H, H ), 7.46 (d, J ¼ 8.7 Hz, 2H, H ), 7.39 (d, J ¼ 7.8 Hz,

31

Ar Ar Ar Ar

15.23 (d, J ¼ 6.0 Hz, CH

3

d

2H, H ), 7.30 (m, 3H, H ), 6.99 (m, 2H, H ), 6.89 (m, 2H, H ), 5.02

þ

4

H

43

N

3

O

5

(s, 2H, CH

CH eOeP), 3.80 (s, 2H, CH

2.95 (bs, 2H, H , H pip), 2.58 (bs, 2H, H , H pip), 1.92 (m, 4H, H , H

2

eO), 4.40 (s, 1H, H pip), 3.99 (dqd, J ¼ 9.8, 7.0, 1.6 Hz, 4H,

2

eN), 3.21 (d, J ¼ 21.9 Hz, 2H, CH

2

eP),

2

6

2

6

3

5

4

.1.14.2. Diethyl {[4-(2-{[1-({2-[4-(triﬂuoromethyl)phenyl]-1H-1,3-

1

3

benzodiazol-6-yl}methyl)piperidin-4-yl]oxy}phenoxy-methyl)phenyl]

methyl}phosphonate (15b). The title compound was obtained

following the general procedure 5 from benzimidazole 14b

pip), 1.21 (t, J ¼ 7.1 Hz, 6H, CH

3

). C NMR (101 MHz, MeOD)

d

151.27

quat

(C

), 150.42 (C

), 150.08 (C

), 131.00 (d, J ¼ 9.2 Hz, C

), 147.13 (C

), 136.27 (d,

quat

J ¼ 4.0 Hz, C

), 129.68 (d, J ¼ 6.7 Hz,

Ar

(

0.16 mmol), SOCl

2

(340

mL, 29 eq., 4.68 mmol) and then compound

C ), 128.59 (C ), 128.33 (C ), 127.53 (d, J ¼ 2.7 Hz, C ), 125.04

Ar Ar Ar Ar Ar Ar

11a (70 mg, 1 eq., 0.16 mmol), diisopropylethylamine (144

mL, 5.3

(C ),122.29 (C ),121.74 (C ), 121.45 (C ), 121.18 (C ), 118.37 (C ),

Ar

eq., 0.85 mmol) in ACN (4 mL). After puriﬁcation by silica gel col-

umn chromatography, eluting DCM/MeOH 95:5, the clean com-

pound 15b was obtained as an amorphous creamy solid. (28 mg,

115.30 (C ), 70.50 (CH

2

eO), 62.30 (d, J ¼ 6.9 Hz, CH

2

eOeP), 62.30

2

6

3

(CH

C

d

2

eN), 49.20 (C , C pip), 31.96 (d, J ¼ 137.2 Hz, CH

2

eP), 29.49 (C ,

5

31

pip), 15.24 (d,J ¼ 6.0 Hz, CH

3

). P NMR (162 MHz, MeOD)

1

Ar

19

3

2%) H NMR (400 MHz, MeOD)

d

8.42 (bs, 1H, H ), 8.33 (d,

27.43. F NMR (376 MHz, MeOD)

d

ꢂ59.34. HRMS-ESI (m/z)

Ar

þ

J ¼ 8.0 Hz, 1H, H ), 7.70 (m, 5H, H ), 7.38 (d, J ¼ 8.0 Hz, 2H, H ),

[MþH] calcd for C39

42 3 3 6

H F N O P 724.2760, found 724.2757.

Ar

7

.28 (m, 3H, H ), 6.98 (m, 2H, H ), 6.88 (m, 2H, H ), 5.01 (s, 2H,

4

CH

2

eO), 4.37 (d, J ¼ 7.4 Hz,1H, H pip), 3.98 (m, 4H, CH

2

eOeP), 3.75

4.1.14.5. Diethyl {[4-(2-{[1-({2-[3-(triﬂuoromethoxy)phenyl]-1H-1,3-

benzodiazol-6-yl}methyl)piperidin-4-yl]oxy}phen-oxymethyl)phenyl]

methyl}phosphonate (15e). The title compound was obtained

following the general procedure 5 from benzimidazole 14e (36 mg,

2

6

(

s, 2H, CH

2

eN), 3.20 (d, J ¼ 21.8 Hz, 2H, CH

2

eP), 2.89 (bs, 2H, H , H

2

6

3

5

pip), 2.51 (bs, 2H, H , H pip), 1.89 (m, 4H, H , H pip), 1.20 (t,

1

3

quat

J ¼ 7.1 Hz, 6H, CH

3

). C NMR (101 MHz, MeOD)

d

150.89 (C

),

quat

1

50.05 (C

), 147.19 (C

), 136.29 (d, J ¼ 3.0 Hz, C

), 131.36

1 eq., 0.12 mmol), SOCl

compound 11a (50 mg, 1 eq., 0.12 mmol), diisopropylethylamine

(104 L, 5.3 eq., 0.61 mmol) in ACN (2 mL). After puriﬁcation by

2

(244 mL, 29 eq., 3.35 mmol) and then

quat

(

C

), 131.04 (C

), 130.94 (C

), 130.72 (C

), 130.63 (C

),

Ar

1

29.83 (C ), 129.78 (C ), 129.67 (d, J ¼ 6.6 Hz, C ), 127.50 (d,

m

Ar

J ¼ 3.3 Hz, C ), 126.38 (C ), 125.36 (C ), 125.14 (C ), 123.08 (C ),

silica gel column chromatography, eluting DCM/MeOH 95:5, the

clean compound 15e was obtained as an amorphous creamy solid.

Ar

122.66 (C ), 122.19 (C ), 121.45 (C ), 118.29 (C ), 115.33 (C ),

1

7

4

0.50 (CH eO), 62.48 (CH eOeP),

2

eN), 62.30 (d, J ¼ 7.0 Hz, CH

2

(18 mg, 25%) H NMR (400 MHz, MeOD)

d

8.08 (d, J ¼ 8.0 Hz, 1H,

2

6

3

5

Ar

9.37 (C , C pip), 31.96 (d, J ¼ 138.1 Hz, CH

2

eP), 29.70 (C , C pip),

H ), 8.03 (s, 1H, H ), 7.62 (m, 3H, H ), 7.41 (m, 3H, H ), 7.31 (m,

3

1

19

Ar

1

5.25 (d, J ¼ 5.9 Hz, CH

3

). P NMR (162 MHz, MeOD)

d

27.42. F

3H, H ), 6.99 (m, 2H, H ), 6.88 (m, 2H, H ), 5.02 (s, 2H, CH

2

eO),

þ

4

NMR (376 MHz, MeOD)

d

ꢂ64.25. HRMS-ESI (m/z) [MþH] calcd for

4.39 (s, 1H, H pip), 3.99 (dqd, J ¼ 8.0, 7.0, 1.6 Hz, 4H, CH

2

eOeP),

2

C

39

H F

42 3

N

3

O

5

P 708.2811, found 708.2812.

3.77 (s, 2H, CH eP), 2.91 (s, 2H, H ,

2

eN), 3.21 (d, J ¼ 21.7 Hz, 2H, CH

2

6

2

6

3

5

H pip), 2.54 (s, 2H, H , H pip), 1.91 (s, 4H, H , H pip), 1.21 (dt,

13

quat

4

.1.14.3. Diethyl {[4-(2-{[1-({2-[3-(triﬂuoromethyl)phenyl]-1H-1,3-

J ¼ 7.0, 0.4 Hz, 6H, CH

3

). C NMR (101 MHz, MeOD)

d

150.88 (C

),

quat

Ar

quat

benzodiazol-6-yl}methyl)piperidin-4-yl]oxy}phen-oxymethyl)phenyl]

methyl}phosphonate (15c). The title compound was obtained

following the general procedure 5 from benzimidazole 14c (47 mg,

150.06 (C

), 149.71 (d, J ¼ 2.0 Hz, 1H, C ), 147.15 (C

), 136.29 (d,

quat

Ar

quat

Ar

J ¼ 3.9 Hz, C

), 131.78 (C ), 131.00 (d, J ¼ 9.5 Hz, C

), 130.69

quat

Ar

(C

), 129.66 (d, J ¼ 6.7 Hz, C ), 127.51 (d, J ¼ 3.2 Hz, C ), 125.01

Ar

1

eq., 0.16 mmol), SOCl

compound 11a (70 mg, 1 eq., 0.16 mmol), diisopropylethylamine

144 L, 5.3 eq., 0.85 mmol) in ACN (4 mL). After puriﬁcation by

2

(340

mL, 29 eq., 4.68 mmol) and then

(C ), 122.29 (C ), 122.21 (C ), 121.82 (C ), 121.45 (C ), 119.27

Ar

2

(C ), 118.92 (C ), 118.31 (C ), 115.33 (C ), 70.50 (CH eO), 62.43

2

6

(

m

(CH

2

eN), 62.31 (d, J ¼ 7.0 Hz, CH

2

eOeP), 49.34 (C , C pip), 32.02

3

5

silica gel column chromatography, eluting DCM/MeOH 95:5, the

(d, J ¼ 138.0 Hz, CH

2

eP), 29.67 (C , C pip), 15.24 (d, J ¼ 6.0 Hz, CH

3

).

31

19

clean compound 15c was obtained as an amorphous creamy solid.

P NMR (162 MHz, MeOD)

d

27.43. F NMR (376 MHz, MeOD)

1

Ar

þ

(

24 mg, 21%) H NMR (400 MHz, MeOD)

d

8.41 (bs, 1H, H ), 8.33 (d,

d

ꢂ59.35. HRMS-ESI (m/z) [MþH] calcd for

39 42 3 3 6

C H F N O P

Ar

J ¼ 8.0 Hz, 1H, H ), 7.71 (m, 5H, H ), 7.32 (m, 4H, H ), 6.94 (m, 4H,

724.2760, found 724.2759.

Ar

4

H ), 5.01 (s, 2H, CH

2

eO), 4.40 (s, 1H, H pip), 3.96 (m, 4H,

CH eOeP), 3.82 (s, 2H, CH

2

eN), 3.19 (d, J ¼ 23.3 Hz, 2H, CH

2

eP),

4.1.14.6. Diethyl ({3-[2-({1-[(2-phenyl-1H-1,3-benzodiazol-6-yl)

2

6

2

6

3

5

2

.97 (bs, 2H, H , H pip), 2.68 (bs, 2H, H , H pip), 1.95 (m, 4H, H , H

methyl]piperidin-4-yl}oxy)phenoxymethyl]-phenyl}methyl)phospho-

nate (16a). The title compound was obtained following the general

procedure 5 from benzimidazole 14a (22 mg, 1 eq., 0.099 mmol),

13

pip), 1.18 (t, J ¼ 6.9 Hz, 6H, CH

3

). C NMR (101 MHz, MeOD)

d

150.10

Ar

(

C

C ), 137.46 (d, J ¼ 1.7 Hz, C ), 136.93 (C ), 136.27 (d, J ¼ 3.7 Hz,

quat

Ar

), 129.87 (C ), 129.82 (C ), 129.68 (d, J ¼ 6.7 Hz, C ), 127.54 (d,

SOCl

2

(210

mL, 29 eq., 2.87 mmol) and then compound 11b (43 mg, 1

Ar

J ¼ 3.0 Hz, C ), 123.85 (C ), 122.34 (C ), 121.45 (C ), 118.42 (C ),

eq., 0.099 mmol), diisopropylethylamine (90

mL, 5.3 eq., 0.53 mmol)

Ar

1

(

C

d

[

15.29 (C ), 70.51 (CH

2

eO), 62.30 (d, J ¼ 6.8 Hz, CH

2

eOeP), 62.25

in ACN (3 mL). After puriﬁcation by silica gel column chromatog-

raphy, eluting DCM/MeOH 95:5, the clean compound 16a was ob-

2

6

3

CH

2

eN), 49.20 (C , C pip), 31.91 (d, J ¼ 137.5 Hz, CH

2

eP), 29.34 (C ,

). P NMR (162 MHz, MeOD)

5

31

1

pip), 15.21 (d, J ¼ 5.9 Hz, CH

3

tained as an amorphous creamy solid. (24 mg, 38%) H NMR

19

Ar

27.44. F NMR (376 MHz, MeOD)

d

ꢂ64.33. HRMS-ESI (m/z)

(400 MHz, MeOD)

d

8.09 (d, J ¼ 7.2 Hz, 2H, H ), 7.57 (m, 5H, H ),

þ

Ar

Ar Ar

MþH] calcd for C39

H F

42 3

N

3

O

5

P 708.2811, found 708.2809.

7.33 (m, 5H, H ), 7.03 (m, 2H, H ), 6.91 (m, 2H, H ), 5.06 (s, 2H,

4

CH

2

eO), 4.43 (s, 1H, H pip), 3.98 (ddq, J ¼ 8.0, 7.0, 1.0 Hz, 4H,

4.1.14.4. Diethyl {[4-(2-{[1-({2-[4-(triﬂuoromethoxy)phenyl]-1H-1,3-

2

eOeP), 3.84 (s, 2H, CH eP),

2

eN), 3.23 (d, J ¼ 21.7 Hz, 2H, CH

2

6

2

6

3

5

benzodiazol-6-yl}methyl)piperidin-4-yl]oxy}phen-oxymethyl)phenyl]

2.99 (bs, 2H, H , H pip), 2.62 (bs, 2H, H , H pip), 1.97 (m, 4H, H , H

12

M. Bessi ꢀe res, E. Plebanek, P. Chatterjee et al.

European Journal of Medicinal Chemistry 214 (2021) 113211

pip), 1.20 (td, J ¼ 7.0, 1.0 Hz, 6H, CH

3

). ¹³C NMR (101 MHz, MeOD)

4.1.14.9. Diethyl {[3-(2-{[1-({2-[4-(triﬂuoromethoxy)phenyl]-1H-1,3-

benzodiazol-6-yl}methyl)-piperidin-4-yl]oxy}-phenoxymethyl)

phenyl]methyl}phosphonate (16d). The title compound was ob-

tained following the general procedure 5 from benzimidazole 14d

quat

d

152.28 (C

), 150.16 (C

), 147.08 (C

), 137.88 (d, J ¼ 3.3 Hz,

quat

Ar quat

C

(

), 131.61 (d, J ¼ 9.4 Hz, C

), 130.13 (C ), 129.47 (C

), 129.13

Ar

d, J ¼ 6.6 Hz, C ), 128.78 (C ), 128.73 (d, J ¼ 7.0 Hz, C ), 128.32 (d,

Ar Ar Ar Ar

J ¼ 3.4 Hz, C ), 126.44 (C ), 125.92 (d, J ¼ 3.7 Hz, C ), 122.36 (C ),

(37 mg, 1 eq., 0.12 mmol), SOCl

then compound 11b (50 mg, 1 eq., 0.12 mmol), diisopropylethyl-

amine (104 L, 5.3 eq., 0.64 mmol) in ACN (3 mL). After puriﬁcation

2

(246 mL, 29 eq., 3.34 mmol) and

Ar

1

2

21.44 (C ), 118.51 (C ), 115.19 (C ), 70.54 (CH eO), 62.35 (d,

2

6

J ¼ 6.9 Hz, CH

2

eOeP), 62.21 (CH

2

eN), 49.29 (C , C pip), 32.32 (d,

m

3

5

31

J ¼ 137.7Hz, CH

2

eP), 29.41(C , C pip), 15.25 (d, J ¼ 6.0 Hz, CH

3

).

P

by silica gel column chromatography, eluting DCM/MeOH 95:5, the

clean compound 16d was obtained as an amorphous creamy solid.

þ

NMR (162 MHz, MeOD)

d

27.36 HRMS-ESI (m/z) [MþH] calcd for

1

C

H

37 43

N

3

O

5

P 640.2941, found 640.2938.

(19 mg, 31%) H NMR (400 MHz, MeOD)

d

8.20 (d, J ¼ 8.8 Hz, 2H,

Ar

H ), 7.63 (s, 2H, H ), 7.39 (m, 7H, H ), 7.03 (m, 2H, H ), 6.92 (m,

Ar

4

4.1.14.7. Diethyl {[3-(2-{[1-({2-[4-(triﬂuoromethyl)phenyl]-1H-1,3-

2H, H ), 5.07 (s, 2H, CH

6.8, 3.0 Hz, 4H, CH

2H, CH

2.06e1.84 (m, 4H, H , H pip), 1.22 (t, J ¼ 6.8 Hz, 6H, CH

2

eO), 4.44 (s, 1H, H pip), 3.99 (dqd, J ¼ 7.8,

benzodiazol-6-yl}methyl)-piperidin-4-yl]oxy}-phenoxymethyl)

phenyl]methyl}phosphonate (16b). The title compound was ob-

tained following the general procedure 5 from benzimidazole 14b

2

eOeP), 3.83 (s, 2H, CH

2

eN), 3.25 (d, J ¼ 21.6 Hz,

2

6

2 6

2

eP), 2.98 (bs, 2H, H , H pip), 2.60 (bs, 2H, H , H pip),

3

5

13

3

). C NMR

quat

(

34 mg, 1 eq., 0.115 mmol), SOCl

then compound 11b (50 mg, 1 eq., 0.115 mmol), diisopropylethyl-

amine (100 L, 5.3 eq., 0.59 mmol) in ACN (3 mL). After puriﬁcation

2

(243

m

L, 29 eq., 3.35 mmol) and

(101 MHz, MeOD)

d

151.43 (C

), 151.25 (C

), 150.14 (C

),

quat

Ar

147.14 (C

), 137.91 (d, J ¼ 3.3 Hz, C

), 131.60 (d, J ¼ 9.1 Hz, C

Ar

m

129.10 (d, J ¼ 6.4 Hz, C ), 128.72 (d, J ¼ 6.5 Hz, C ), 128.60 (C ),

Ar

by silica gel column chromatography, eluting DCM/MeOH 95:5, the

clean compound 16b was obtained as an amorphous creamy solid.

128.32 (C ), 128.30 (d, J ¼ 2.7 Hz, C ), 125.90 (d, J ¼ 3.7 Hz, C ),

Ar Ar Ar Ar Ar

122.31 (C ), 121.45 (C ), 121.20 (C ), 118.46 (C ), 115.24 (C ),

70.55 (CH eOeP), 62.32 (CH eN),

eO), 62.35 (d, J ¼ 6.9 Hz, CH

1

Ar

(

19 mg, 22%) H NMR (400 MHz, MeOD)

d

8.45 (bs, 1H, H ), 8.36 (d,

2

Ar

2

6

3

5

J ¼ 7.8 Hz, 1H, H ), 7.83 (m, 1H, H ), 7.78 (m, 1H, H ), 7.63 (m, 2H,

49.34 (C , C pip), 32.32 (d, J ¼ 137.2 Hz, CH

2

eP), 29.57 (C , C pip),

Ar

31

19

H ), 7.42 (bs, 1H, H ), 7.32 (m, 4H, H ), 7.02 (m, 2H, H ), 6.91 (m,

15.25 (d, J ¼ 6.1 Hz, CH

NMR (376 MHz, MeOD)

P 724.2760, found 724.2755.

3

). P NMR (162 MHz, MeOD)

d

27.36

F

Ar

4

þ

2

H, H ), 5.06 (s, 2H, CH

eOeP), 3.80 (s, 2H, CH

eP), 2.94 (bs, 2H, H , H pip), 2.55 (bs, 2H, H , H pip), 1.96 (m,

2

eO), 4.42 (d, J ¼ 7.4 Hz, 1H, H pip), 3.99

d

ꢂ57.38 HRMS-ESI (m/z) [MþH] calcd for

(

m, 4H, CH

2

eN), 3.25 (d, J ¼ 21.7 Hz, 2H,

39 42 3 3 6

C H F N O

2

6

2

6

CH

4

MeOD)

2

3

5

13

H, H , H pip), 1.22 (t, J ¼ 7.1 Hz, 6H, CH

3

). C NMR (101 MHz,

quat

d

150.87 (C

), 150.11 (C

), 147.19 (C

), 137.94 (d,

4.1.14.10. Diethyl

{[3-(2-{[1-({2-[3-(triﬂuoromethoxy)phenyl]-1H-

quat

J ¼ 3.0 Hz, C

), 131.58 (d, J ¼ 9.5 Hz, C

), 131.39 (C

), 131.06

1,3-benzodiazol-6-yl}methyl)piperidin-4-yl]oxy}phenoxymethyl)

phenyl]methyl}phosphonate (16e). The title compound was ob-

tained following the general procedure 5 from benzimidazole 14e

quat

Ar

(

C

), 130.63 (C

), 129.83 (C ), 129.82 (C ), 129.77 (C ), 129.09

Ar

(

d, J ¼ 6.8 Hz, C ), 128.71 (d, J ¼ 6.9 Hz, C ), 128.30 (d, J ¼ 2.8 Hz,

Ar

C ), 126.40 (C ), 125.89 (d, J ¼ 3.7 Hz, C ), 125.36 (C ), 123.06

(27 mg, 1 eq., 0.088 mmol), SOCl

then compound 11b (38 mg, 1 eq., 0.088 mmol), diisopropylethyl-

amine (79 L, 5.3 eq., 0.47 mmol) in ACN (2 mL). After puriﬁcation

2

(186 mL, 29 eq., 2.56 mmol) and

Ar

(

C ), 122.23 (C ), 121.45 (C ), 118.38 (C ), 115.28 (C ), 70.56

2

CH

2

eO), 62.38 (CH

2

eN), 62.34 (d, J ¼ 6.9 Hz, CH

2

eOeP), 49.43 (C ,

m

6

3

5

C pip), 32.28 (d, J ¼ 138.1Hz, CH

2

eP), 29.75 (C , C pip), 15.26 (d,

by silica gel column chromatography, eluting DCM/MeOH 95:5, the

clean compound 16e was obtained as an amorphous creamy solid.

3

1

19

J ¼ 6.0 Hz, CH

376 MHz, MeOD)

P 708.2811, found 708.2808.

3

). P NMR (162 MHz, MeOD)

d

27.36 F NMR

þ

1

(

C

d

ꢂ64.31. HRMS-ESI (m/z) [MþH] calcd for

(22 mg, 35%) H NMR (400 MHz, MeOD)

d

8.06 (ddd, J ¼ 7.90, 1.6,

Ar

39

H F

42 3

N

3

O

5

1.0 Hz,1H, H ), 8.01 (s,1H, H ), 7.61 (q, J ¼ 8.2 Hz, 3H, H ), 7.33 (m,

Ar Ar Ar

6

H, H ), 6.98 (m, 2H, H ), 6.88 (m, 2H, H ), 5.01 (s, 2H, CH

2

eO),

4

4.1.14.8. Diethyl {[3-(2-{[1-({2-[3-(triﬂuoromethyl)phenyl]-1H-1,3-

4.39 (s, 1H, H pip), 3.94 (dqd, J ¼ 8.2, 7.0, 1.1 Hz, 4H, CH

2

eOeP),

2

benzodiazol-6-yl}methyl)piperidin-4-yl]-oxy}phenoxymethyl)phenyl]

methyl}phosphonate (16c). The title compound was obtained

following the general procedure 5 from benzimidazole 14c (34 mg,

3.80 (s, 2H, CH eP), 2.95 (s, 2H, H ,

2

eN), 3.20 (d, J ¼ 21.7 Hz, 2H, CH

2

6

2

6

3

5

H pip), 2.57 (s, 2H, H , H pip), 1.91 (s, 4H, H , H pip), 1.21e1.10 (dt,

1

3

quat

J ¼ 7.0, 0.4 Hz, 6H, CH

3

). C NMR (101 MHz, MeOD)

d

150.93 (C

),

quat

1

eq., 0.115 mmol), SOCl

compound 11b (50 mg, 1 eq., 0.115 mmol), diisopropylethylamine

100 L, 5.3 eq., 0.59 mmol) in ACN (3 mL). After puriﬁcation by

2

(243

m

L, 29 eq., 3.35 mmol) and then

150.15 (C

C

), 149.73 (C

), 147.09 (C

), 137.88 (d, J ¼ 3.2 Hz,

quat

Ar

quat

Quat

), 131.73 (C ), 131.61 (d, J ¼ 9.3 Hz, C

), 130.71 (C

), 129.10

Ar

(

m

(d, J ¼ 6.5 Hz, C ), 128.72 (d, J ¼ 6.7 Hz, C ), 128.31 (d, J ¼ 3.2 Hz,

Ar

silica gel column chromatography, eluting DCM/MeOH 95:5, the

clean compound 16c was obtained as an amorphous creamy solid.

C ), 125.90 (d, J ¼ 3.6 Hz, C ), 125.01 (C ), 122.34 (C ), 121.44

Ar

4

(C ), 118.93 (C ), 118.48 (C ), 115.20 (C ), 73.12 (C pip), 70.53

1

Ar

2

(

20 mg, 24%) H NMR (400 MHz, MeOD)

d

8.40 (bs, 1H, H ), 8.31 (d,

(CH

2

eO), 62.34 (d, J ¼ 6.9 Hz, CH

2 2

eOeP), 62.17 (CH eN), 49.24 (C ,

Ar

6

3

5

J ¼ 7.9 Hz, 1H, H ), 7.67 (m, 4H, H ), 7.28 (m, 5H, H ), 6.90 (m, 4H,

C pip), 32.33 (d, J ¼ 137.5 Hz, CH

2

eP), 29.45 (C , C pip), 15.25 (d,

Ar

4

31

19

H ), 5.01 (s, 2H, CH

.0 Hz, 4H, CH

CH

2

eO), 4.36 (s, 1H, H pip), 3.94 (ddq, J ¼ 8.0, 7.1,

J ¼ 6.0 Hz, CH

(376 MHz, MeOD)

39 42 3 3 6

C H F N O P 724.2760, found 724.2757.

3

). P NMR (162 MHz, MeOD)

d

27.36. F NMR

þ

1

2

eOeP), 3.71 (s, 2H, CH

2

eN), 3.19 (d, J ¼ 21.7 Hz, 2H,

d

ꢂ59.38. HRMS-ESI (m/z) [MþH] calcd for

2

6

2

6

2

eP), 2.86 (s, 2H, H , H pip), 2.44 (d, J ¼ 8.7 Hz, 2H, H , H pip),

3

5

1

CH

1

.91 (dd, J ¼ 12.9, 8.4 Hz, 4H, H , H pip), 1.16 (dt, J ¼ 7.1, 1.0 Hz, 6H,

1

3

quat

3

). C NMR (101 MHz, MeOD)

d

150.79 (C

), 150.07 (C

),

quat

47.25 (C

31.38 (C

),137.94 (d, J ¼ 3.2 Hz, C

),131.57 (d, J ¼ 9.4 Hz, C

4.1.15. 2-(2-Bromophenoxy)tetrahydropyran (18)

quat

Ar

), 131.06 (C

), 130.65 (C

), 129.81 (C ), 129.79

Under inert atmosphere, dihydropyran (3.96 mL, 1.5 eq.,

43.35 mmol) was added to a solution of 2-bromophenol 17

(3.35 mL, 1 eq., 28.90 mmol) in dichloromethane (35 mL). A cata-

lytic amount of pyridinium p-toluenesulfonate (726 mg, 0.1 eq.,

2.89 mmol) was then inserted in the ﬂask, and the mixture was

stirred at room temperature for 12 h. The volatiles were then

removed under reduced pressure, and the residue was puriﬁed by

silica gel column chromatography, eluting pure PE, to afford com-

pound 18 as a colorless oil. (7.43 g, quant.), [47]. CAS: 57999-46-9.

Ar

(

C ), 129.07 (d, J ¼ 6.7 Hz, C ), 128.70 (d, J ¼ 6.7 Hz, C ), 128.29 (d,

Ar Ar Ar Ar

J ¼ 3.1 Hz, C ), 126.37 (C ), 125.88 (d, J ¼ 3.6 Hz, C ), 123.08 (C ),

Ar

1

22.15 (C ), 121.45 (C ), 118.31 (C ), 115.31 (C ), 70.57 (CH

2

eO),

2

6

2.53(CH

2

eN), 62.34 (d, J ¼ 7.0 Hz, CH

2

eOeP), 49.56(C , C pip),

3

5

3

2.29 (d, J ¼ 137.6Hz, CH

2

eP), 29.89 (C , C pip), 15.29, 15.26 (d,

31

19

J ¼ 6.0 Hz, CH

3

). P NMR (162 MHz, MeOD)

d

27.36. F NMR

þ

(

376 MHz, MeOD)

d

ꢂ64.31. HRMS-ESI (m/z) [MþH] calcd for

39 42 3 3 5

C H F N O P 708.2811, found 708.2807.

13

M. Bessi ꢀe res, E. Plebanek, P. Chatterjee et al.

European Journal of Medicinal Chemistry 214 (2021) 113211

4

.1.16. tert-Butyl 4-[hydroxy-(2-tetrahydropyran-2-yloxyphenyl)

4.1.19. tert-Butyl 4-{[2-(benzyloxy)phenyl](hydroxy)methyl}

piperidine-1-carboxylate (22)

methyl] piperidine-1-carboxylate (19)

Under Ar atmosphere, compound 18 (1 g, 1 eq., 3.89 mmol) was

Method A: In an autoclave, piperidine derivative 21 (1 g, 1 eq.,

2.55 mmol) was dissolved in ethyl acetate (43 mL). The solution and

the atmosphere in the apparatus were degassed with argon before

the addition of 10% Pd/C (500 mg). The solution and the atmo-

sphere were again saturated with argon, and then the autoclave

ꢀ

dissolved in THF (8 mL) and cooled at ꢂ78 C. After stirring for

5

min, n-BuLi (2.5 M in hexane, 1.56 mL, 1 eq., 3.89 mmol) was

ꢀ

added dropwise to the mixture and stirred 30 min. at ꢂ78 C. A

solution of 1-(tert-Butoxycarbonyl)-4-piperidinecarboxaldehyde

(

830 mg, 1 eq., 3.89 mmol) in THF (8 mL) was then slowly intro-

2

was sealed and ﬁlled with H until a pressure of 7 bars. The reaction

ꢀ

duced by cannulation into the ﬂask. After 10 min at ꢂ78 C, the

solution was then allowed to stir at room temperature for 3 h and

quenched by a slow addition of water (10 mL). The aqueous phase

was subsequently extracted three times with ethyl acetate (3 x

mixture was stirred for 24 h at room temperature, ﬁltrated onto

Celite®.The ﬁltrate was evaporated, giving pure product 22 as

colorless crystals. (733 mg, quant.) Method B: Under inert atmo-

sphere, compound 19 (200 mg, 1 eq., 0.51 mmol) was dissolved in

3

0 mL) and the resulted organic layers dried over MgSO

4

and

dichloromethane (4 mL). Triethylsilane (408

and TFA (196 L, 5 eq., 2.55 mmol) were successively added to this

solution, and stirred at room temperature for 1 h. The reaction was

quenched with a saturated aqueous solution of NaHCO (5 mL),

extracted with EtOAc (3 x 10 mL), washed with brine (10 mL), dried

over MgSO and concentrated under reduced pressure. The product

mL, 5 eq., 2.55 mmol)

concentrated under reduced pressure. The resulting product was

then loaded onto a silica gel column and puriﬁed eluting Petroleum

ether/Ethyl Acetate 8:2 to afford desired piperidine derivative 19 as

white solid. (914 mg, 60%) H NMR (250 MHz, CDCl

m

3

1

3

)

d

7.18 (m, 3H,

Ar

H ), 6.97 (dt, J ¼ 7.3, 1.3 Hz, 1H, H ), 5.40 (m, 1H, OeCHeO), 4.62

4

2

6

(

CH

dt, J ¼ 12.2, 6.8 Hz, 1H, CHeOH), 4.07 (m, 2H, H , H ), 3.81 (m, 1H,

was then puriﬁed by a ﬂash column chromatography (PE/EtOAc

95:5), affording desired product 22 as colorless crystals. (30 mg,

2

6

2

eO), 3.59 (m, 1H, CH eO), 2.54 (m, 3H, OH, H , H ), 1.79 (m, 9H,

3

4

5

3

5

1

Ar

, H , H , H ) 1.42 (s, 9H, CH

3

Boc), 1.21 (m, 2H, H , H ). HRMS

392.2438 found 392.2433.

21%) H NMR (400 MHz, CDCl

3

)

d

7.03 (m, 2H, H ), 6.78 (m, 2H,

þ

Ar

2

6

(

ESI): m/z [MþH] calcd for C22

H

34NO

5

H ), 5.71 (s, 1H, OH), 4.01 (bd,J ¼ 8.2 Hz, 2H, H , H pip), 4.01

2

6

(

bt,J ¼ 11.9 Hz, 2H, H , H pip), 2.53 (d,J ¼ 7.1 Hz, 2H, CH

2

),1.69 (m,

4

3

5

1

d

1

4

2

H, H pip),1.59 (bd,J ¼ 14.4 Hz, 2H, H , H pip),1.43 (s, 9H, CH Boc),

3

4.1.17. 1-(Benzyloxy)-2-bromobenzene (20)

3

5

13

.15 (bd,J ¼ 11.9, 4.1 Hz, 2H, H , H pip). C NMR (101 MHz, CDCl

3

)

To a solution of 2-bromophenol (17) (1.34 mL, 1 eq., 11.6 mmol)

quat

Ar

155.04 (C]O), 154.06 (C

2

eCH ), 131.23 (C ), 127.21 (C ),

in DMF (20 mL), benzyl chloride (1.46 mL, 1.1 eq., 12.7 mmol) and

potassium carbonate (3.19 g, 2 eq., 23.1 mmol) were added. The

resulting mixture was stirred for 12 h at 70 C. After evaporation of

all volatiles, the residue was dissolved in a mixture of EtOAc (80 mL)

and water (40 mL). The aqueous phase was extracted thrice with

EtOAc (3 x 80 mL), the organic phases were washed three times

with water (3 x 40 mL), three times with brine (3 x 40 mL), dried

over MgSO4 and concentrated under reduced pressure. After a

ﬁltration on silica gel, eluting petroleum ether/ethyl acetate 8:2, the

desired product 20 was obtained as a colorless oil. (3.1 g, quant.),

quat

Ar

quat

26.51 (C

eOH), 120.22 (C ), 115.26 (C ), 79.45 (C

Boc),

4

2

6

3

5

2

4.10 (C pip), 36.88 (C , C pip), 36.80 (CH ), 31.98 (C , C pip),

8.50 (CH

92.1913, found 292.1906.

ꢀ

þ

3

Boc). HRMS-ESI (m/z) [MþH] calcd for C17

H26NO

3

4.1.20. tert-Butyl 4-{[2-({4-[(diethoxyphosphoryl)methyl]phenyl}

methoxy)phenyl] methyl}piperidine-1-carboxylate (23a)

In a 10e20 mL microwave vial, phosphonate derivative 2a

(1.32 mg, 1.5 eq., 4.12 mmol) was dissolved in DMA (20 mL). To this

mixture were sequentially added potassium carbonate (760 mg, 2

eq., 5.49 mol), phenol 22 (800 mg, 1 eq., 2.45 mmol) and ﬁnally a

catalytic amount of sodium iodide (few crystals). This reaction was

[

48]. CAS: 31575-75-4.

ꢀ

4.1.18. tert-butyl 4-{[2-(benzyloxy)phenyl](hydroxy)methyl}

stirred for 30 min at 140 C under microwave irradiation, and the

piperidine-1-carboxylate (21)

obtained solution was dissolved in water (20 mL) and diethyl ether

(100 mL). The aqueous phase was then extracted twice ethyl ace-

tate (2 x 50 mL), and the organic phases washed with water (5 x

Under Ar atmosphere, bromobenzene 20 (3.36 g, 1.1 eq.,

ꢀ

1

2.7 mmol) was dissolved in THF (19 mL) and cooled at ꢂ78 C.

After stirring for 10 min, n-BuLi (2.5 M in hexane, 5.03 mL, 1.1

4

40 mL), once with brine (40 mL), dried over MgSO and evaporated.

eq.12.7 mmol) was added dropwise to the solution and stirred

The residue was consequently puriﬁed by column chromatography

(DCM/MeOH 99:1) to afford the intermediate 23a as a white solid,

which was directly engaged in the next step.

ꢀ

3

0 min. at ꢂ78 C. A mixture of 1-(tert-Butoxycarbonyl)-4-

piperidinecarboxaldehyde (2.47 g, 1 eq., 11.6 mmol) in THF

(

19 mL) was then slowly introduced by cannulation into the ﬂask.

ꢀ

After 10 min at ꢂ78 C, the solution was then allowed to stir at

room temperature for 3 h and quenched by the slow addition of

water (20 mL). The aqueous phase was subsequently extracted

three times with ethyl acetate (3 x 80 mL) and the resulted organic

4.1.21. tert-Butyl 4-{[2-({3-[(diethoxyphosphoryl)methyl]phenyl}

methoxy)phenyl] methyl}piperidine-1-carboxylate (23b)

In a 2e5 mL microwave vial, phosphonate derivative 2b

(331 mg, 1.5 eq., 1.03 mmol) was dissolved in DMA (5 mL). To this

mixture were sequentially added potassium carbonate (190 mg, 2

eq., 1.37 mol), phenol 22 (200 mg, 1 eq., 0.69 mmol) and ﬁnally a

catalytic amount of sodium iodide (few crystals). This reaction was

4

layers dried over MgSO and concentrated under reduced pressure.

The resulting product was then loaded onto a silica gel column and

puriﬁed eluting Petroleum ether/Ethyl Acetate 8:2 to afford desired

1

ꢀ

piperidine derivative 21 as colorless crystals. (2.94 g, 65%). H NMR

stirred for 30 min at 140 C under microwave irradiation, and the

Ar

(

250 MHz, CDCl

3

)

d

7.36 (m, 5H, H ), 7.22 (m, 2H, H ), 6.94 (m, 2H,

obtained solution was dissolved in water (10 mL) and ethyl acetate

(50 mL). The aqueous phase was then extracted with ethyl acetate

(3 x 30 mL), and the organic phases washed with water (5 x 10 mL),

Ar

2

H ), 5.08 (s, 2H, CH2benzylic), 4.61 (bs, 1H, CHeOH), 4.01 (m, 2H, H ,

4

6

2

4

6

3

5

H , H pip), 2.53 (m, 3H, H , H , H pip),1.59 (m, 2H, H , H pip), 1.43

3

5

13

(

s, 9H, CH

3

Boc), 1.15 (m, 2H, H , H pip). C NMR (101 MHz, CDCl

3

)

4

once with brine (10 mL), dried over MgSO and evaporated. The

quat

Ar

d

(

155.77 (C]O), 154.86 (C

), 136.66 (C

), 131.05 (C

), 128.71

residue was consequently puriﬁed by column chromatography

(DCM/MeOH 99:1) to afford 23b compound as a colorless oil.

Ar

C ), 128.46 (C ), 128.29 (C ), 128.15 (C ), 127.32 (C ), 121.02

Ar

quat

1

Ar

(

C ), 112.01 (C ), 79.24 (C

Boc), 75.05 (CHeOH), 70.24

(330 mg, 91%) H NMR (400 MHz, CDCl

3

)

d

7.24 (m, 4H, H ), 7.07

(m, 2H, H ), 6.82 (m, 2H, H ), 5.01 (s, 2H, CH eO), 3.96 (m, 6H,

eOeP, H , H pip), 3.10 (d, J ¼ 21.6 Hz, 2H, CH eP), 2.55 (d,

2

6

3

5

Ar

(

2

CH2benzylic) 43.93 (C⁴pip), 42.56 (C , C pip), 28.74 (C , C pip),

2

3

5

þ

2

6

8.47 (CH

3

Boc), 28.25 (C , C pip). HRMS-ESI (m/z) [MþH] calcd

398.2332, found 398.2326.

CH

J ¼ 7.1 Hz, 4H, CH

2

6

4

for C24

H32NO

4

2

eCH,H , H pip), 1.73 (m, 1H, H pip), 1.56

14

M. Bessi ꢀe res, E. Plebanek, P. Chatterjee et al.

European Journal of Medicinal Chemistry 214 (2021) 113211

3

5

(

CH

eCH

bd,J ¼ 14.4 Hz, 2H, H , H pip), 1.39 (s, 9H, CH

3

Boc), 1.17 (m, 8H,

eq., 0.61 mmol) in ACN (3 mL). After puriﬁcation by silica gel col-

umn chromatography, eluting DCM/MeOH 95:5, the clean com-

pound 25a was obtained as an amorphous creamy solid. (13 mg,

3

5

13

quat

3

Phosph, H , H pip). C NMR (101 MHz, CDCl

3

)

d

156.58 (C

quat

2

), 154.86 (C]O), 137.79 (d, J ¼ 3.0 Hz, C

), 131.90 (d,

quat

Ar

1

Ar

J ¼ 9.0 Hz, C

), 131.01 (C ), 129.17 (d, J ¼ 6.7 Hz, C ), 129.04

18%) H NMR (400 MHz, MeOD)

d

8.11 (dd, J ¼ 8.0, 2.0 Hz, 2H, H ),

quat

Ar

(

C

), 128.73 (d, J ¼ 3.4 Hz, C ), 128.35 (d, J ¼ 6.6 Hz, C ), 127.18

7.65 (m, 2H, H ), 7.57 (m, 3H, H ), 7.42 (d, J ¼ 7.8 Hz, 2H, H ), 7.33

Ar

quat

6

Ar

C ), 125.51 (d, J ¼ 3.6 Hz, C ), 120.48 (C ), 111.74 (C ), 79.11 (C

(m, 3H, H ), 7.16 (dt, J ¼ 8.4, 1.6 Hz, 1H), 7.10 (dd, J ¼ 7.3, 1.6 Hz, 1H,

2

Ar

Boc), 69.58 (CH

pip), 37.34 (CH

(

C

2

eO), 62.15 (d, J ¼ 6.9 Hz, CH

2

eOeP), 44.28(C , C

H ), 6.98 (d, J ¼ 8.4 Hz, 1H, H ), 6.86 (t, J ¼ 7.4 Hz, 1H, H ), 5.08 (s,

4

2

), 36.66 (C pip), 33.82 (d, J ¼ 138.0Hz, CH

2

eP), 32.15

2H, CH

2

eO), 4.02 (m, 6H, CH

2

eOeP, CH

2

eN), 3.25 (d, J ¼ 21.2 Hz,

3

5

31

2 6

C , C pip), 28.46 (CH

162 MHz, MeOD)

P 532.2828, found 532.2819.

3

Boc), 16.37 (d, J ¼ 6.7 Hz, CH

3

)

P NMR

2

eP), 3.18 (bd, J ¼ 12.1 Hz, 2H, H , H pip), 2.64 (d, J ¼ 6.7 Hz,

þ

2

6

4

d

26.22. HRMS-ESI (m/z) [MþH] calcd for

2

), 2.47 (bs, 2H, H , H pip), 1.82 (m, 1H, H pip), 1.74 (bd,

3

5

3

5

29

H43NO

6

J ¼ 14.5 Hz, 2H, H , H pip), 1.45 (dq, J ¼ 11.2, 2.7 Hz, 2H, H , H pip),

13

1

.24 (t, J ¼ 7.1 Hz, 6H, CH

3

). C NMR (101 MHz, MeOD)

d

156.62

quat

4

.1.21.1. Diethyl

({4-[2-(piperidin-4-ylmethyl)phenoxymethyl]

(C

eCH

2

), 153.11 (C

), 136.42 (d, J ¼ 4.3 Hz, C

), 130.93 (d,

quat

Ar Ar

Ar

phenyl} methyl)phosphonate (24a). Following general procedure 2,

triﬂuoroacetic acid (18.9 mL, 100 eq., 245 mmol) was added to a

solution of 23a in DCM (15 mL). Pure compound 24a was obtained

after ﬂash column chromatography (DCM/MeOH 95:5), affording

J ¼ 9.2 Hz, C

),130.57 (C ),130.24 (C ), 129.72 (d, J ¼ 6.4 Hz, C ),

Ar Ar Ar

Ar

129.38 (C ), 128.81 (C ), 128.30 (C ), 127.26 (d, J ¼ 3.2 Hz, C ),

Ar

128.29 (C ), 127.13 (C ), 126.49 (C ), 120.25 (C ), 111.74 (C ),

69.26 (CH eOeP), 61.89 (CH eN),

eO), 62.30 (d, J ¼ 7.3 Hz, CH

), 35.25 (C pip), 31.88 (d, J ¼ 138.1 Hz,

2

1

2

6

4

desired product as a light brown solid. (356 mg, 88%) H NMR

52.69 (C , C pip), 36.24 (CH

2

Ar

3

5

31

(

400 MHz, MeOD)

d

7.43 (d, J ¼ 8.3 Hz, 2H, H ), 7.34 (dd, J ¼ 8.0,

CH

(162 MHz, MeOD)

38 45 3 4

C H N O P 638.3146, found 638.3144.

2

eP), 30.13 (C , C pip), 15.24 (d, J ¼ 6.0 Hz, CH

3

)

P NMR

Ar

þ

2

(

4

(

.4 Hz, 2H, H ), 7.15 (m, 2H, H ), 7.01 (dd, J ¼ 8.0 Hz, 1H, H ), 6.88

d

27.27. HRMS-ESI (m/z) [MþH] calcd for

Ar

d, J ¼ 7.2 Hz, 1H, H ), 5.10 (s, 2H, CH

H, CH

under MeOD peak, 2H, CH

pip), 2.66 (d, J ¼ 7.0 Hz, 2H, CH

2

eO), 4.04 (dq, J ¼ 7.3, 7.2 Hz,

2

6

2

eOeP), 3.35 (under MeOD peak, m, 2H, H , H pip), 3.26

2

6

2

eP), 2.86 (dt, J ¼ 12.5, 2.7 Hz, 2H, H , H

4.1.21.4. Diethyl

({3-[2-({1-[(2-phenyl-1H-1,3-benzodiazol-6-yl)

4

2

), 1.93 (m, 1H, H pip), 1.82 (bd,

methyl]piperidin-4-yl}methyl)phenoxymethyl]-phenyl}methyl)phos-

phonate (26a). The title compound 26a was obtained following the

general procedure 5 from benzimidazole 14a (26 mg, 1 eq.,

3

5

3

5

J ¼ 15.4 Hz, 2H, H , H pip), 1.42 (dq, J ¼ 12.4, 3.4 Hz, 2H, H , H pip),

13

1

d

.26 (t, J ¼ 7.2 Hz, 6H, CH

3

). C NMR (101 MHz, MeOD)

quat

156.65(C

eCH

2

), 136.36 (d, J ¼ 3.9 Hz, C

), 131.03 (d,

0.12 mmol), SOCl

2

(246

mL, 29 eq., 3.37 mmol) and then compound

quat

Ar

J ¼ 9.4 Hz, C

), 130.60(C ), 129.76(d, J ¼ 6.8 Hz, C ), 127.62(C ),

24b (50 mg, 1 eq., 0.12 mmol), diisopropylethylamine (105 m

L, 5.3

Ar

1

6

3

27.38(C ), 127.32 (C ), 120.44(C ), 120.35(C ), 111.87(C ),

eq., 0.61 mmol) in ACN (3 mL). After puriﬁcation by silica gel col-

umn chromatography, eluting DCM/MeOH 95:5, the clean com-

pound 26a was obtained as an amorphous creamy solid. (20 mg,

2

6

9.36(CH

2

eO), 63.33 (d, J ¼ 7.1 Hz, CH

2

eOeP), 43.88 (C , C pip),

4

3

6.09(CH

2

), 34.35(C pip), 31.96(d, J ¼ 138.2 Hz, CH

2

eP), 28.56 (C ,

). P NMR (162 MHz, MeOD) 27.49.

P 432.2304, found

5

31

1

Ar

C pip), 15.24(d, J ¼ 6.2 Hz, CH

3

d

24%) H NMR (400 MHz, MeOD)

d

8.10 (dd, J ¼ 8.1, 2.7 Hz, 2H, H ),

þ

Ar

HRMS-ESI (m/z) [MþH] calcd for C24

H35NO

4

7.67 (s, 1H, H ), 7.62 (d, J ¼ 8.6 Hz, 1H, H ), 7.54 (m, 3H, H ), 7.42

Ar

4

32.2306.

(s, 1H, H ), 7.32 (m, 3H, H ), 7.24 (m, 1H, H ), 7.15 (dt, J ¼ 8.1,

Ar

1

.6 Hz, 1H), 7.10 (dd, J ¼ 7.4, 1.6 Hz, 1H, H ), 6.98 (d, J ¼ 7.8 Hz, 1H,

Ar Ar

4

.1.21.2. Diethyl

({3-[2-(piperidin-4-ylmethyl)phenoxymethyl]

H ), 6.86 (td, J ¼ 7.4, 0.8 Hz, 1H, H ), 5.07 (s, 2H, CH

6H, CH eOeP, CH

eN), 3.24 (d, J ¼ 21.6 Hz, 2H, CH

J ¼ 11.6 Hz, 2H, H , H pip), 2.64 (d, J ¼ 6.7 Hz, 2H, CH

2

eO), 3.96 (m,

eP), 3.18 (bd,

), 2.47 (s, 2H,

phenyl} methyl)phosphonate (24b). Following general procedure 2,

triﬂuoroacetic acid (4.5 mL, 100 eq., 58.3 mmol) was added to a

solution of compound 23b (310 mg, 1 eq., 0.58 mmol) in DCM

9 mL). Pure compound 24b was obtained after ﬂash column

chromatography (DCM/MeOH 95:5), affording desired product

2

6

2

6

4

3

5

H , H pip), 1.80 (m, 1H, H pip), 1.74 (bd, J ¼ 14.9 Hz, 2H, H , H pip),

3

5

13

(

1.45 (bq, J ¼ 14.4 Hz, 2H, H , H pip), 1.20 (t, J ¼ 7.1 Hz, 6H, CH

3

).

), 149.72

), 131.64 (d, J ¼ 9.2 Hz,

C

quat

NMR (101 MHz, MeOD)

d

156.59 (C

eCH

2

), 153.14 (C

1

quat

Ar

colorless oil. (235 mg, 94%) H NMR (400 MHz, MeOD)

d

7.39 (s, 1H,

(d, J ¼ 2.0 Hz, C

quat Ar

), 138.00 (d, J ¼ 3.4 Hz, C

Ar

H ), 7.26 (m, 3H, H ), 7.11 (dt, J ¼ 7.6, 1.6 Hz, 1H, H ), 7.06 (dd,

C

), 130.59 (C ), 130.25 (C ), 129.37 (C ), 129.04 (d, J ¼ 6.7 Hz,

Ar

J ¼ 7.6, 1.6 Hz, 1H, H ), 6.93 (d, J ¼ 8.1 Hz, 1H, H ), 6.83 (dt, J ¼ 7.5,

C ), 128.82 (C ), 128.43 (d, J ¼ 6.5 Hz, C ), 128.34 (d, J ¼ 3.2 Hz,

Ar Ar Ar Ar Ar

Ar

0

(

.7 Hz, 1H, H ), 5.01 (s, 2H, CH

2

eO), 3.96 (m, 4H, CH

m, 2H, H , H pip), 3.21 (d, J ¼ 21.6 Hz, 2H, CH eP), 2.80 (dt,

J ¼ 12.9, 2.3 Hz, 2H, H , H pip), 2.61 (d, J ¼ 7.1 Hz, 2H, CH ), 1.86 (m,

2

eOeP), 3.27

C ), 128.29 (C ), 127.17 (C ), 126.50 (C ), 125.72 (C ), 125.62 (d,

2

6

Ar

2

J ¼ 3.6 Hz, C ), 120.31 (C ), 111.65 (C ), 69.23 (CH

2

eO), 62.38 (d,

eOeP), 61.80 (CH eN), 52.70 (C , C pip), 36.13

2 2

2

6

2

6

2

J ¼ 6.9 Hz, CH

4

3

5

4

3

5

1

H, H pip), 1.75 (bd, J ¼ 14.1 Hz, 2H, H , H pip), 1.45 (dq, J ¼ 13.0,

(CH

2

), 35.38 (C pip), 32.30 (d, J ¼ 137.8 Hz, CH

2

eP), 30.07 (C , C

P NMR (162 MHz, MeOD) 27.27.

P 638.3146, found

3

5

13

31

0.9 Hz, 2H, H , H pip), 1.17 (t, J ¼ 7.1 Hz, 6H, CH

3

)$ C NMR

pip), 15.26 (d, J ¼ 6.0 Hz, CH

3

)

d

quat

þ

(

101 MHz, MeOD)

d

157.91 (C

eCH

2

), 139.20 (d, J ¼ 3.2 Hz, C

),

HRMS-ESI (m/z) [MþH] calcd for C38

45 3 4

H N O

quat

Ar

1

7

3

33.95 (d, J ¼ 9.3 Hz, C

), 131.97(C ), 130.47 (d, J ¼ 6.7 Hz, C ),

638.3141.

Ar

quat

Ar

29.82 (d, J ¼ 6.6 Hz, C ), 129.74 (d, J ¼ 3.1 Hz, C ), 129.02(C

),

Ar

28.72(C ), 127.05 (d, J ¼ 3.7 Hz, C ), 121.76(C ), 113.04(C ),

4.1.21.5. Diethyl {[3-(2-{[1-({2-[4-(triﬂuoromethyl)phenyl]-1H-1,3-

benzodiazol-6-yl}methyl)piperidin-4-yl]methyl}-phenoxymethyl)

phenyl]methyl}phosphonate (26b). The title compound 26b was

obtained following the general procedure 5 from benzimidazole

2

6

0.61(CH

7.31 (CH

2

eO), 63.79 (d, J ¼ 6.9 Hz, CH

2

eOeP), 45.11(C , C pip),

4

3

2

), 35.84 (C pip), 33.54 (d, J ¼ 138.0Hz, CH

2

eP), 29.73 (C ,

P NMR (162 MHz, MeOD) 27.01.

P 432.2304, found

5

31

C pip), 16.62 (d, J ¼ 6.1 Hz, CH

3

)

d

þ

HRMS-ESI (m/z) [MþH] calcd for C24

H35NO

4

14b (60 mg,1 eq., 0.20 mmol), SOCl

then compound 24b (80 mg, 1 eq., 0.20 mmol), diisopropylethyl-

amine (168 L, 5.3 eq., 0.99 mmol) in ACN (4 mL). After puriﬁcation

2

(452 mL, 29 eq., 6.20 mmol) and

4

32.2299.

m

4.1.21.3. Diethyl

({4-[2-({1-[(2-phenyl-1H-1,3-benzodiazol-6-yl)

by silica gel column chromatography, eluting DCM/MeOH 95:5, the

methyl]piperidin-4-yl}methyl)phenoxy-methyl]phenyl}-methyl)phos-

phonate (25a). The title compound 25a was obtained following the

general procedure 5 from benzimidazole 14a (26 mg, 1 eq.,

clean compound 26b was obtained as an amorphous creamy solid.

1

Ar

(24 mg, 19%) H NMR (400 MHz, MeOD)

d

8.46 (s, 1H, H ), 8.37 (d,

Ar

J ¼ 7.6 Hz, 1H, H ), 7.75 (m, 4H, H ), 7.33 (m, 5H, H ), 7.14 (dd,

Ar

0

.12 mmol), SOCl

2

(246

mL, 29 eq., 3.37 mmol) and then compound

J ¼ 7.8, 1.8 Hz, 2H), 7.01 (dd, J ¼ 8.3, 1.1 Hz, 1H, H ), 6.88 (td, J ¼ 7.4,

Ar

2

4a (50 mg, 1 eq., 0.12 mmol), diisopropylethylamine (105

mL, 5.3

1.2 Hz, 1H, H ), 5.08 (s, 2H, CH

2

eO), 4.26 (s, 2H, CH

2

eN), 3.99 (dqd,

15

M. Bessi ꢀe res, E. Plebanek, P. Chatterjee et al.

European Journal of Medicinal Chemistry 214 (2021) 113211

J ¼ 9.5, 7.1, 1.5 Hz, 4H, CH

2

eOeP), 3.38 (under MeOD peak, m, 2H,

H , H pip), 3.22 (under MeOD peak, m, 2H, CH eP), 2.81 (t,

), 1.90 (m, 1H,

H pip),1.82 (bd, J ¼ 13.5 Hz, 2H, H , H pip), 1.55 (bt, J ¼ 12.0 Hz, 2H,

pip),1.22 (t, J ¼ 7.1 Hz, 6H, CH

3

). ¹³C NMR (101 MHz, MeOD)

d 156.60

2

6

quat

2

(C

eCH

2

), 151.64 (C

), 150.52 (C

), 138.00 (d, J ¼ 3.3 Hz,

2

6

quat

Ar

quat

Ar

J ¼ 12.0 Hz, 2H, H , H pip), 2.68 (d, J ¼ 6.8 Hz, 2H, CH

2

C

), 131.64 (d, J ¼ 9.2 Hz, C

), 130.59 (C ), 129.03 (d, J ¼ 6.9 Hz,

4

3

5

Ar

C ), 128.50 (C ), 128.43 (d, J ¼ 6.6 Hz, C ), 128.38 (C ), 128.33 (d,

3

5

13

Ar

H , H pip), 1.21 (td, J ¼ 7.1, 0.6 Hz, 6H, CH

3

). C NMR (101 MHz,

J ¼ 3.1 Hz, C ), 127.15 (C ), 125.62 (d, J ¼ 3.7 Hz, C ), 125.34 (C ),

Ar Ar Ar Ar

quat

MeOD)

d

156.60 (C

eCH

2

), 151.84 (C

), 137.93 (d, J ¼ 3.3 Hz,

123.12 (d, J ¼ 4.1 Hz, C ), 121.74 (C ), 121.25 (C ), 120.29 (C ),

Ar Ar

quat

Ar

C

(

), 131.69 (d, J ¼ 9.3 Hz, C

), 131.56 (C

), 130.57 (C ), 130.36

119.20 (C ), 111.65 (C ), 69.23 (CH

2

eO), 62.38 (d, J ¼ 6.9 Hz,

Ar

2

6

4

C ), 129.94 (C ), 129.90 (C

), 129.10 (d, J ¼ 6.6 Hz, C ), 128.45

CH

2

eOeP), 61.90 (CH

2

eN), 52.87 (C , C pip), 36.20 (CH

2

), 35.45 (C

Ar

3

5

(

d, J ¼ 6.5 Hz, C ), 128.34 (d, J ¼ 3.4 Hz, C ), 127.88 (C ), 127.35

pip), 32.26 (d, J ¼ 137.8 Hz, CH

2

eP), 30.19 (C , C pip), 15.25 (d,

Ar

31

19

C ), 125.68 (d, J ¼ 3.9 Hz, C ), 123.25 (C ), 123.18 (C ), 120.40

J ¼ 6.0 Hz, CH

(376 MHz, MeOD)

39 44 3 3 5

C H F N O P 722.2971, found 722.2965.

3

)

P NMR (162 MHz, MeOD)

ꢂ59.39. HRMS-ESI (m/z) [MþH] calcd for

d

27.27. F NMR

Ar

þ

C ), 111.68 (C ), 69.24 (CH

2

eO), 62.41 (d, J ¼ 6.9 Hz, CH

2

eOeP),

), 34.86 (C pip),32.16

eP), 29.22 (C , C pip), 15.24 (d, J ¼ 6.0 Hz, CH

d

2

6

4

6

0.93 (CH

2

eN), 52.34 (C , C pip), 35.70 (CH

2

3

5

(

d, J ¼ 138.0Hz, CH

2

3

)

3

1

19

P NMR (162 MHz, MeOD)

d

27.22. F NMR (376 MHz, MeOD)

4.1.21.8. Diethyl {[3-(2-{[1-({2-[3-(triﬂuoromethoxy)phenyl]-1H-1,3-

benzodiazol-6-yl}methyl)piperidin-4-yl]methyl}-phenoxymethyl)

phenyl]methyl}phosphonate (26e). The title compound 26e was

obtained following the general procedure 5 from benzimidazole

þ

d

7

ꢂ64.36. HRMS-ESI (m/z) [MþH] calcd for

39 44 3 3 4

C H F N O P

06.3022, found 706.3013.

4

.1.21.6. Diethyl {[3-(2-{[1-({2-[3-(triﬂuoromethyl)phenyl]-1H-1,3-

14e (60 mg,1 eq., 0.20 mmol), SOCl

then compound 24b (80 mg, 1 eq., 0.20 mmol), diisopropylethyl-

amine (168 L, 5.3 eq., 0.99 mmol) in ACN (4 mL). After puriﬁcation

2

(452 mL, 29 eq., 6.20 mmol) and

benzodiazol-6-yl}methyl)piperidin-4-yl]methyl-}phenoxymethyl)

phenyl]methyl}phosphonate (26c). The title compound 26c was

obtained following the general procedure 5 from benzimidazole

m

by silica gel column chromatography, eluting DCM/MeOH 95:5, the

1

4c (60 mg, 1 eq. 0.20 mmol), SOCl

then compound 24b (80 mg, 1 eq., 0.20 mmol), diisopropylethyl-

amine (168 L, 5.3 eq., 0.99 mmol) in ACN (4 mL). After puriﬁcation

2

(452

m

L, 29 eq., 6.20 mmol) and

clean compound 26e was obtained as an amorphous creamy solid.

1

(26 mg, 20%) H NMR (400 MHz, MeOD)

d

8.10 (ddd, J ¼ 7.8, 1.3,

Ar

Ar Ar

m

0.6 Hz, 1H, H ), 8.05 (s, 1H, H ), 7.66 (m, 3H, H ), 7.45 (m, 2H, H ),

Ar

by silica gel column chromatography, eluting DCM/MeOH 95:5, the

clean compound 26c was obtained as an amorphous creamy solid.

7.28 (m, 4H, H ), 7.14 (dt, J ¼ 7.4, 1.7 Hz, 1H), 7.10 (dd, J ¼ 7.7, 1.7 Hz,

Ar

1H, H ), 6.98 (d, J ¼ 8.0 Hz,1H, H ), 6.86 (td, J ¼ 7.4,1.0 Hz,1H, H ),

1

Ar

(

26 mg, 20%). H NMR (400 MHz, MeOD)

d

8.46 (s, 1H, H ), 8.37 (d,

5.07 (s, 2H, CH

J ¼ 21.7 Hz, 2H, CH

J ¼ 6.7 Hz, 2H, CH ), 2.47 (bt, J ¼ 11.8 Hz, 2H, H , H pip), 1.81 (m, 1H,

3 5

2

eO), 3.98 (m, 6H, CH

eP), 3.17 (bd, J ¼ 10.2 Hz, 2H, H , H pip), 2.64 (d,

2 2

eOeP, CH eN), 3.25 (d,

Ar

2

6

J ¼ 7.8 Hz, 1H, H ), 7.85 (d, J ¼ 7.8 Hz, 1H, H ), 7.79 (t, J ¼ 7.8 Hz, 1H,

2

Ar

2

6

H ), 7.68 (m, 2H, H ), 7.44 (s, 1H, H ), 7.35 (m, 3H, H ), 7.27 (m,

2

Ar

4

1

H, H ), 7.16 (dt, J ¼ 8.3, 1.8 Hz, 1H, H ), 7.11 (dd, J ¼ 7.4, 1.5 Hz, 1H,

H pip), 1.74 (bd, J ¼ 13.0 Hz, 2H, H , H pip), 1.45 (bq, J ¼ 13.3 Hz,

Ar

3 5 13

H ), 6.99 (d, J ¼ 7.8 Hz, 1H, H ), 6.87 (td, J ¼ 7.5, 1.0 Hz, 1H, H ),

2H, H , H pip), 1.20 (t, J ¼ 6.8 Hz, 6H, CH

3

). C NMR (101 MHz,

quat

5

.09 (s, 2H, CH eO), 3.97 (m, 6H, CH eOeP, CH eN), 3.26 (d,

2

MeOD)

d

156.58 (C

eCH

2

), 151.29 (C

), 131.63 (C

), 149.72 (d, J ¼ 2.0 Hz,

2

6

quat

Ar

quat

J ¼ 21.7 Hz, 2H, CH

2

eP), 3.13 (bd, J ¼ 11.3 Hz, 2H, H , H pip), 2.65 (d,

C

), 137.99 (d, J ¼ 3.3 Hz, C

), 131.61 (d, J ¼ 9.9 Hz,

Ar

2

6

4

quat

Ar

J ¼ 6.8 Hz, 2H, CH

), 2.38 (bs, 2H, H , H pip), 1.78 (m, 1H, H pip),

), 130.75 (C ), 130.58 (C ), 129.02 (d, J ¼ 7.1 Hz, C ), 128.41 (d,

3

5

3

5

Ar

1

.73 (bd, J ¼ 13.4 Hz, 2H, H , H pip), 1.44 (bq, J ¼ 13.4 Hz, 2H, H , H

J ¼ 6.9 Hz, C ), 128.36 (C ), 128.34 (d, J ¼ 3.7 Hz, C ), 127.15 (C ),

Ar Ar Ar Ar

1

3

pip), 1.22 (t, J ¼ 7.1 Hz, 6H, CH

3

). C NMR (101 MHz, MeOD)

d

156.59

125.61 (d, J ¼ 3.7 Hz, C ), 125.43 (C ), 125.01 (C ), 122.57 (C ),

Ar Ar Ar Ar Ar

quat

(

C

eCH

2

), 151.20 (C

), 138.03 (d, J ¼ 3.4 Hz, C

), 131.63 (d,

122.46 (C ), 120.30 (C ), 120.29 (C ), 118.97 (C ), 118.50 (C ),

quat

Ar

quat

Ar

J ¼ 9.1 Hz, C

), 131.42 (C

), 131.10 (C

), 130.59 (C ), 130.55

111.64 (C ), 69.22 (CH

2

eO), 62.36 (d, J ¼ 6.9 Hz, CH

2

eOeP), 61.80

Ar

2

6

4

(

C ), 129.87 (C ), 129.83 (C ), 129.02 (d, J ¼ 6.4 Hz, C ), 128.46

(CH

J ¼ 137.7 Hz, CH

NMR (162 MHz, MeOD)

2 2

eN), 52.70 (C , C pip), 36.14 (CH ), 35.41 (C pip), 32.23 (d,

Ar

3

5

31

C ), 128.37 (d, J ¼ 4.4 Hz, C ), 128.33 (d, J ¼ 3.2 Hz, C ), 127.09

2

eP), 30.12 (C , C pip), 15.25 (d, J ¼ 6.0 Hz, CH

3

).

ꢂ59.39.

44 3 3 5

H F N O P 722.2971, found

P

Ar

19

C ), 126.50 (C ), 125.60 (d, J ¼ 3.8 Hz, C ), 125.34 (C ), 123.12 (d,

d

27.27. F NMR (376 MHz, MeOD)

d

Ar

þ

J ¼ 4.1 Hz, C ), 122.64 (C ), 120.26 (C ), 111.64 (C ), 69.22

HRMS-ESI (m/z) [MþH] calcd for C39

2

(

CH

2

eO), 62.36 (d, J ¼ 7.0 Hz, CH

2

eOeP), 62.12 (CH

2

eN), 52.87 (C ,

eP),

). P NMR (162 MHz,

ꢂ64.34 HRMS-ESI (m/z)

P 706.3022, found 706.3013.

722.2964.

6

4

C pip), 36.33 (CH

2

), 35.64 (C pip), 32.30 (d, J ¼ 137.2 Hz, CH

2

3

5

31

3

0.46 (C , C pip), 15.25 (d, J ¼ 5.9 Hz, CH

3

4.2. Assays for anti-EBOV activity and cytotoxicity

19

MeOD) d 27.29. F NMR (376 MHz, MeOD) d

þ

[MþH] calcd for C39

H F

44 3

N

3

O

4

All work with infectious virus was conducted in a BSL-4 labo-

ratory at the Centers for Disease Control and Prevention. All labo-

ratorians adhered to international practices appropriate for this

biosafety level. Anti-EBOV activities were determined in Huh7 cells

(Apath LLC), using recombinant virus expressing the ﬂuorescent

reporter protein ZsGreen (EBOV-ZsG), as described previously

[49,50]. Brieﬂy, 3000 Huh7 cells were seeded in each well of a 384-

well plate. The following day, the cells were treated with varying

concentrations of compound for 2 h, before infection with EBOV-

ZsG virus at a multiplicity of infection of 0.3. The ZsGreen ﬂuores-

cence was determined 3 days post-infection using a Synergy H1MD

plate reader (BioTek). Cytotoxicity was determined on cells that

received compound, but no virus, using CellTiter-Glo (Promega)

according to the manufacturer’s instructions. The 50% effective

(EC50) and 50% cytotoxic concentrations (CC50) were determined

using GraphPad Prism 7.0 (GraphPad Software) to ﬁt a 4-parameter

equation to semi-log plots of the concentration-response data. The

4.1.21.7. Diethyl {[3-(2-{[1-({2-[4-(triﬂuoromethoxy)phenyl]-1H-1,3-

benzodiazol-6-yl}methyl)piperidin-4-yl]methyl}-phenoxymethyl)

phenyl]methyl}phosphonate (26d). The title compound 26d was

obtained following the general procedure 5 from benzimidazole

1

4d (60 mg,1 eq., 0.20 mmol), SOCl

then compound 24b (80 mg, 1 eq., 0.20 mmol), diisopropylethyl-

amine (168 L, 5.3 eq., 0.99 mmol) in ACN (4 mL). After puriﬁcation

2

(452 mL, 29 eq., 6.20 mmol) and

m

by silica gel column chromatography, eluting DCM/MeOH 95:5, the

clean compound 26d was obtained as an amorphous creamy solid.

1

(

31 mg, 24%) H NMR (400 MHz, MeOD)

d

8.21 (d, J ¼ 8.9 Hz, 2H,

Ar

Ar Ar

H ), 7.67 (m, 2H, H ), 7.49 (d, J ¼ 9.0 Hz, 2H, H ), 7.44 (s, 1H, H ),

Ar

7.31 (m, 4H, H ), 7.16 (dt, J ¼ 8.3, 1.5 Hz, 1H), 7.12 (dd, J ¼ 7.6, 1.6 Hz,

1

5

Ar

H, H ), 7.00 (d, J ¼ 8.3 Hz,1H, H ), 6.88 (td, J ¼ 7.5,1.0 Hz,1H, H ),

.10 (s, 2H, CH eO), 4.00 (m, 6H, CH eOeP, CH eN), 3.27 (d,

2

6

J ¼ 21.8 Hz, 2H, CH

2

eP), 3.18 (bd, J ¼ 11.8 Hz, 2H, H , H pip), 2.67 (d,

2 6 4

J ¼ 6.8 Hz, 2H, CH

2

), 2.47 (bs, 2H, H , H pip), 1.81 (m, 1H, H pip),

Selectivity Index (SI) was calculated as the CC50/EC50

.

3

5

3

5

1.75 (bd, J ¼ 15.4 Hz, 2H, H , H pip), 1.46 (bq, J ¼ 10.4 Hz, 2H, H , H

Assays to test the inhibition of EBOV-GP-mediated entry were

16

M. Bessi ꢀe res, E. Plebanek, P. Chatterjee et al.

European Journal of Medicinal Chemistry 214 (2021) 113211

performed with pseudotyped HIV particles as described [49].

Brieﬂy, particles were generated by co-transfection of Lenti-X 293T

cells with plasmids expressing EBOV-GP and a HIV luciferase re-

porter vector. Huh7 cells in 384-well plates were treated with

EBOV

FDA

HF

Ebola virus

US food and drug administration

Hemorrhagic fever

Glycoprotein

Microwaves

Niemann Pick C1 protein

Pyridinium p-toluenesulfonate

Ribonucleic acid

room temperature

Nucleophilic substitution

Triﬂuoroacetic acid

GP

ꢀ

varying concentrations of compound for 1 h at 37 C, then the

MW

NPC1

PPTS

RNA

r.t.

pseudotyped particles were added. Three days later, ﬁreﬂy lucif-

erase activity was determined using the Bright-Glo luciferase assay

system (Promega).

For ﬁlipin staining, HeLa cells were treated with the indicated

concentrations of compounds, or the vehicle control at a ﬁnal

concentration of 0.1% (v/v). Test compounds were dissolved in

DMSO, the positive control compound U18666A (Sigma-Aldrich)

was dissolved in water. Twenty-four hours later, the cells were

washed with Phosphate Buffered Saline (PBS) three times, then

ﬁxed with 3% paraformaldehyde for 1 h at room temperature. The

cells were again washed three times with PBS, then incubated with

SN

TFA

THF

TLC

Tetrahydrofuran

thin layer chromatography

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18

Article Doi

Design, synthesis and biological evaluation of 2-substituted-6-[(4-substituted-1-piperidyl)methyl]-1H-benzimidazoles as inhibitors of ebola virus infection

DOI: 10.1016/j.ejmech.2021.113211

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Full text of DOI:10.1016/j.ejmech.2021.113211

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