Design, synthesis and evaluation of 2-aryl benzoxazoles as promising hit for the A2A receptor

Article abstract of DOI:10.1080/14756366.2017.1334648

The development of adenosine A_2A receptor antagonists has received much interest in recent years for the treatment of neurodegenerative diseases. Based on docking studies, a new series of 2-arylbenzoxazoles has been identified as potential A_2AR antagonists. Structure-affinity relationship was investigated in position 2, 5 and 6 of the benzoxazole heterocycle leading to compounds with a micromolar affinity towards the A_2A receptor. Compound F1, with an affinity of 1 μm, presented good absorption, distribution, metabolism and excretion properties with an excellent aqueous solubility (184 μm) without being cytotoxic at 100 μm. This compound, along with low-molecular weight compound D1 (K_i = 10 μm), can be easily modulated and thus considered as relevant starting points for further hit-to-lead optimisation.

Full text of DOI:10.1080/14756366.2017.1334648

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Design, synthesis and evaluation of 2-aryl
benzoxazoles as promising hit for the A2A receptor
Romain Duroux, Nicolas Renault, Joana Esteves Cuelho, Laurence Agouridas,
David Blum, Luisa V. Lopes, Patricia Melnyk & Saïd Yous
To cite this article: Romain Duroux, Nicolas Renault, Joana Esteves Cuelho, Laurence Agouridas,
David Blum, Luisa V. Lopes, Patricia Melnyk & Saïd Yous (2017) Design, synthesis and evaluation
of 2-aryl benzoxazoles as promising hit for the A2A receptor, Journal of Enzyme Inhibition and
Medicinal Chemistry, 32:1, 850-864, DOI: 10.1080/14756366.2017.1334648
To link to this article: http://dx.doi.org/10.1080/14756366.2017.1334648
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Date: 01 July 2017, At: 06:58

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2017
VOL. 32, NO. 1, 850–864
https://doi.org/10.1080/14756366.2017.1334648
RESEARCH PAPER
Design, synthesis and evaluation of 2-aryl benzoxazoles as promising hit for the
A2A receptor
Romain Durouxa, Nicolas Renaultb, Joana Esteves Cuelhoc, Laurence Agouridasa, David Bluma, Luisa V. Lopesc,
Patricia Melnyka and Saïd Yousa
aINSERM, CHU Lille, UMR-S 1172 – JPArc – Centre de Recherche Jean-Pierre AUBERT Neurosciences et Cancer, Universite de Lille, Lille, France;
c
bINSERM, CHU Lille, U995 – LIRIC – Lille Inflammation Research International Center, Universite de Lille, Lille, France; Instituto de Medecina
Molecular, Lisbon, Portugal
ABSTRACT
ARTICLE HISTORY
Received 24 March 2017
Revised 10 May 2017
Accepted 18 May 2017
The development of adenosine A2A receptor antagonists has received much interest in recent years for the
treatment of neurodegenerative diseases. Based on docking studies, a new series of 2-arylbenzoxazoles
has been identified as potential A2AR antagonists. Structure-affinity relationship was investigated in pos-
ition 2, 5 and 6 of the benzoxazole heterocycle leading to compounds with a micromolar affinity towards
the A2A receptor. Compound F1, with an affinity of 1 lm, presented good absorption, distribution, metab-
olism and excretion properties with an excellent aqueous solubility (184 lm) without being cytotoxic at
100 lm. This compound, along with low-molecular weight compound D1 (Ki ¼ 10 lm), can be easily modu-
lated and thus considered as relevant starting points for further hit-to-lead optimisation.
KEYWORDS
Benzoxazole; A2A receptor;
solubility; neurodegenera-
tive disease
Introduction
constant drawbacks remain such as high toxicity and poor solubil-
ity7,8,13,14. These important limitations have obstructed the devel-
opment of drugs targeting this receptor. Therefore, one of the
main challenges regarding A2A antagonist development is to
improve solubility and lower toxicity while keeping good affinity
at A2A receptor. Selectivity parameters are now more debated
since studies have highlighted the therapeutic potential of dual
A1/A2A antagonists15 as well as a non-selective ligand proven by
caffeine, for neurodegenerative disease.
Alzheimer’s (AD) and Parkinson’s diseases (PD), currently the most
important neurodegenerative pathologies, are characterised by a
progressive neuronal death1. Current therapies are restricted to
symptomatic interventions and do not prevent progressive neur-
onal loss. Therefore, novel therapeutic solutions are needed and
one of the promising strategies consists of targeting the adeno-
sine A2A receptor.
Epidemiological studies have shown that people consuming
regularly over a lifetime caffeine-based beverages are substantially
less likely to develop PD and AD2,3. Caffeine exerts its biological
effects primarily by antagonising adenosine receptors (GPCRs). The
adenosine A2A receptor subtype, one of the four adenosine recep-
tors, was shown in multiple studies to be responsible for the neu-
roprotective effects of caffeine in experimental models of AD and
PD4–6. Besides, many A2A antagonists have been synthesised over
the past few years and some showed promising results in manag-
ing cognitive dysfunction in both diseases7. For example, antago-
nists (Figure 1) such as Istradefylline (KW-6002), Preladenant (SCH
42088) and Tozadenant (SYN 115)9 have been investigated clinic-
ally in PD with promising results, especially Istradefylline which
With the aim of developing novel A2A antagonists with good
water solubility, we designed a series of benzoxazoles bearing a
protonable amine function (Figure 2(A)). We first focused on a dif-
fuse hydrophobic zone located at the top of the active site that is
generally occupied by an aryl group in well-known antagonists
(Figure 2(B)). Because of the presence of an acidic cluster (Glu169,
Asp170) in this pocket, a tertiary amine which can allow for an
ionic interaction could be a good alternative to an aryl group
(Figure 2(C)). The present work describes the medicinal chemistry
approach leading to a series of 2-arylbenzoxazoles which best
compounds display micromolar affinity towards the A2A receptor
and good water solubility.
has been approved in Japan as an adjunct to L-DOPA therapy10,11
.
Less research work has been undertaken regarding AD but it is
now well established that A2A antagonists lead to the improve-
ment of spatial memory accompanied by the decrease of Ab amyl-
Methods
Chemistry
oid burden, Tau hyperphosphorylation and neurotoxicity6,12
.
All reagents and solvents were purchased and used without fur-
Therefore, developing A2A antagonists constitutes a promising
ther purification. Reactions were monitored by TLC performed on
R
therapeutic strategy for the treatment of both AD and PD. MachereyeNagel AlugramV Sil 60/UV254 sheets (thickness 0.2 mm).
However, although many antagonists have been developed so far, Some purification of products was carried out by column
ꢀ ꢀ
Jean-Pierre Aubert Research Center, UMR-S1172, Faculte de Pharmacie (Universite Lille), 3 rue du Pr Laguesse, BP
CONTACT Saïd Yous
83, 59006 Lille Cedex, France
said.yous@univ-lille2.fr
Supplemental data for this article can be accessed here.
ß 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unre-
stricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
851
O
Me
NH2
N
OMe
OMe
N
N
O
N
N
O
O
N
N
O
N
N
N
N
Istradefylline (KW6002)
N
Preladenant
Ki (A2A) = 12 nM
Solubility= 1.5 µM
Ki (A2A) = 0.5 nM
Solubility= 20 nM
Cl
NH2
O
HO
O
HO
N
N
N
OH
S
N
N
N
N
NH
N
O
N
H
N
N
NH2
N
O
Tozadenant (SYN115)
ZM-241385
Ki (A2A) = 1.6 nM
1,2,4 triazine
Ki (A2A) = 5 nM
Ki (A2A) = 4 nM
Solubility = 45 µM
Figure 1. Selective adenosine A2A receptor antagonists.
Figure 2. Molecular modelling-guided design. (A) Representation of various modulations around the benzoxazole scaffold. (B) Predicted binding mode of ZM-241385 in
the apoA2AR-T4E pocket (dark) compared with the X-ray binding mode (gray). (C) Putative binding mode of compound F1 in the apoA2AR-T4E pocket.

852
R. DUROUX ET AL.
chromatography using MachereyeNagel silica gel (230e400 mesh). 1H NMR (300 MHz, CDCl3): 8.85 (br s, 1H, OH), 8.34 (br s, 1H, NH),
€
Melting points were determined on a BUCHI B-540 apparatus and
7.50 (m, 1H, H5'), 7.28 (m, 1H, H3'), 7.10 (d, 1H, H6, J ¼ 8.1 Hz), 6.87
are uncorrected. NMR spectra were recorded on a Bruker Avance
(m, 1H, H3), 6.72 (m, 1H, H5), 6.58 (dd, 1H, H4', J ¼ 1.7 Hz and
300 spectrometer operating at 300 MHz (1H) or 75 MHz (13 C).
J ¼ 3.5 Hz), 2.30 (s, 3H, CH3). IR (ꢀ, cmꢂ1): 3388 (NH), 3182–2854
Chemical shifts are in parts per million (ppm) and were referenced
(OH), 1649 (C ¼ O).
to the residual proton peaks in deuterated solvents. Mass spectra
were recorded with an LCMS (Waters Alliance Micromass ZQ
2000). LCMS analysis was performed using a Waters XBridge C18
column (5 lm particle size column, dimensions 50 ꢀ 4.6 mm). A
gradient starting from 98% H2O/formate buffer 5 mm (pH 3.8) and
reaching 100% CH3CN/formate buffer 5 mm (pH 3.8) within 4 min
at a flow rate of 2 ml/min was used followed by a return to the
starting conditions within 1 min. FT-IR spectra were recorded on a
Thermo Nicolet Avatar 320 FT-IR spectrometer. The purity of final
compounds was verified by high-pressure liquid chromatography
(HPLC) columns: C4 Interchrom UPTISPHERE. Analytical HPLC was
performed on a Shimadzu LC-2010AHT system equipped with a
UV detector set at 254 and 215 nm. Compounds were dissolved in
50 ml acetonitrile and 950 ml buffer A and injected into the sys-
tem. The following eluent systems were used: buffer A (H2O/TFA,
100:0.1) and buffer B (CH3CN/H2O/TFA, 80:20:0.1). HPLC retention
times (HPLC tR) were obtained at a flow rate of 0.2 ml/min for
35 min using the following conditions: a gradient run from 100%
of buffer A over 1 min, then to 100% of buffer B over the next
30 min.
N-(2-Hydroxy-5-nitro-phenyl) furan-2-carboxamide (2d): The title
compound was prepared from furoic acid and 4-nitro-2-aminophe-
1
nol to afford 2d as a yellow solid (84%): mp 165 ꢁC (methanol). H
NMR (300 MHz, CDCl3): 9.77 (br s, 1H, OH), 9.13 (br s, 1H, NH), 8.79
(d, 1H, H6, J ¼ 1.9 Hz), 8.25 (dd, 1H, H4, J ¼ 1.9 Hz and J ¼ 8.6 Hz),
8.07 (m, 1H, H5'), 7.81 (d, 1H, H3', J ¼ 3.5 Hz), 7.32 (d, 1H, H3,
J ¼ 8.6 Hz), 6.78 (dd, 1H, H4', J ¼ 1.7 Hz and J ¼ 3.5 Hz). IR (ꢀ, cmꢂ1):
3376 (NH), 3000–2500 (OH), 1640 (C ¼ O).
General procedure for the synthesis of benzoxazole (3a–3d)
A suspension of amide 2 (2.21 mmol) and TsOH (5.53 mmol) was
refluxed in toluene (150 ml) equipped with a Dean–Stark apparatus
until complete dissolution for 17 h. The solution was then cooled
to room temperature, hydrolysed with water and basified with a
6 M solution of NaOH up to basic pH (10–12). The organic layer
was separated, dried over K2CO3 and concentrated in vacuo. Solid
was suspended from the appropriate solvent and filtered.
2-(Furan-2-yl)-5-methyl-1,3-benzoxazole (3a): The title com-
pound was prepared from amide 2a to afford 3a as a beige solid
1
(82%): mp 64 ꢁC (petroleum ether). H NMR (300 MHz, CDCl3): 7.67
(m, 1H, H5'), 7.54 (m, 1H, H3'), 7.44 (d, 1H, H7, J ¼ 8.3 Hz), 7.25 (m,
1H, H4), 7.17 (m, 1H, H6), 6.70 (dd, 1H, H4', J ¼ 1.7 Hz and
J ¼ 3.5 Hz), 2.49 (s, 3H, CH3). LC-MS (ESI) m/z found: 200 [M þ H]þ.
2-(3,4-Dimethoxyphenyl)-5-methyl-1,3-benzoxazole (3b): The
title compound was prepared from amide 2b to afford 3b as a
beige solid (80%): mp 136 ꢁC (diethyl ether). 1H NMR (300 MHz,
CDCl3): 7.83 (dd, 1H, H6', J ¼ 1.9 Hz and J ¼ 8.4 Hz), 7.75 (d, 1H, H2',
J ¼ 1.9 Hz), 7.53 (m, 1H, H6), 7.43 (d, 1H, H5', J ¼ 8.4 Hz), 7.13 (m,
1H, H4), 6.98 (d, 1H, H7, J ¼ 8.4 Hz), 4.02 (s, 3H, OMe), 3.97 (s, 3H,
OMe), 2.48 (s, 3H, CH3). LC-MS (ESI) m/z found: 270 [M þ H]þ.
2-(Furan-2-yl)-6-methyl-1,3-benzoxazole (3c): The title com-
pound was prepared from amide 2c to afford 3c as a beige solid
General procedure for the synthesis of amide (2a–2d)
To a solution of acid (17.8 mmol) in DCM (100 ml) at 0 ꢁC were
added thionyl chloride (71.4 mmol) and 5 drops of DMF. This solu-
tion was stirred for 3 h at reflux, cooled to room temperature and
concentrated in vacuo. The residue was diluted in 50 ml of EtOAc
and added to a solution of aminophenol (16.6 mmol) and Et3N
(35.6 mmol) in 35 ml of EtOAc at 0 ꢁC. The reaction mixture was
stirred at room temperature overnight, hydrolysed with water and
extracted twice with EtOAc. An acid–base workup with saturated
NaHCO3 and 1 M HCl solution was performed and the organic
layer was concentrated in vacuo. The solid was then suspended in
a mixture of EtOH/H2O (250 ml/10 ml) and NaOH was added
(54 mmol). The reaction mixture was stirred at reflux for 4 h,
cooled to room temperature, acidified slowly with 6 M HCl up to
acidic pH. Resulting solid was filtered, washed with water and
recrystallised in an appropriate solvent.
1
(68%): mp 54 ꢁC (diethyl ether). H NMR (300 MHz, CDCl3): 8.04 (m,
1H, H5'), 7.61 (d, 1H, H4, J ¼ 8.1 Hz), 7.40 (d, 1H, H3', J ¼ 3.5 Hz), 7.38
(m, 1H, H7), 7.27–7.18 (m, 1H, H5), 6.79 (dd, 1H, H4', J ¼ 1.7 Hz and
J ¼ 3.5 Hz), 2.44 (s, 3H, CH3). LC-MS (ESI) m/z found: 200 [M þ H]þ.
2-(Furan-2-yl)-5-nitro-1,3-benzoxazole (3d): The title compound
was prepared from amide 2b to afford 3d as a yellow solid (82%):
mp 182 ꢁC (diethyl ether). 1H NMR (300 MHz, CDCl3): 8.62 (d, 1H,
H4, J ¼ 2.1 Hz), 8.34 (dd, 1H, H6, J ¼ 2.1 Hz and J ¼ 8.4 Hz), 7.75 (d,
1H, H7, J ¼ 8.4 Hz), 7.68 (m, 1H, H5'), 7.39 (d, 1H, H3', J ¼ 3.5 Hz),
6.68 (dd, 1H, H4', J ¼ 1.7 Hz and J ¼ 3.5 Hz). IR (ꢀ, cmꢂ1): 1519 (Ar-
NO2). LC-MS (ESI) m/z found: 231 [M þ H]þ.
N-(2-Hydroxy-5-methylphenyl)furan-2-carboxamide (2a): The title
compound was prepared from 2-furoic acid and 4-methyl-2-amino-
phenol to afford 2a as a beige solid (80%): mp 186 ꢁC (acetonitrile).
1H NMR (300 MHz, [D6]DMSO): 9.76 (br s, 1H, OH), 9.09 (br s, 1H,
NH), 7.92 (m, 1H, H5'), 7.72 (m, 1H, H6), 7.28 (dd, 1H, H4, J ¼ 2.2 Hz
and J ¼ 8.4 Hz), 6.79 (m, 2H, H3 and H3'), 6.70 (dd, 1H, H4', J ¼ 1.7 Hz
and J ¼ 3.5 Hz), 2.21 (s, 3H, CH3). IR (ꢀ, cmꢂ1): 3380 (NH), 2750–3100
(OH), 1640 (C¼ O). LC-MS (ESI) m/z found: 218 [Mþ H]þ.
Methyl 2-[2-(3,4-dimethoxyphenyl)-1,3-benzoxazol-5-yl] acetate
(3e): To a suspension of methyl 2-(3-amino-4-hydroxyphenyl) acet-
ate (1.6 g, 8.83 mmol) in T3P (solution in EtOAc) (4.2 g, 13.3 mmol),
were added 3,4-dimethoxybenzoic acid (1.61 g, 8.83 mmol) and
DIPEA (1.46 ml, 8.83 mmol). The reaction mixture was heated over-
night at 120 ꢁC, cooled to room temperature, suspended in water
and extracted three times with EtOAc. Combined organic layers
were washed 1 M NaOH solution, dried over MgSO4 and concen-
trated in vacuo. Solid was recrystallised from EtOH to afford com-
pound (3e) as a beige solid (1.59 g, 55%): mp 110 ꢁC. 1H NMR
(300 MHz, CDCl3): 7.82 (dd, 1H, H6', J ¼ 8.4 Hz and J ¼ 1.8 Hz), 7.74
(d, 1H, H4, J ¼ 1.8 Hz), 7.64 (m, 1H, H2'), 7.49 (d, 1H, H7, J ¼ 8.4 Hz),
7.24 (dd, 1H, H6, J ¼ 8.4 Hz and J ¼ 1.8 Hz), 6.98 (d, 1H, H5',
N-(2-Hydroxy-5-methylphenyl)-3,4-dimethoxybenzamide
(2b):
The title compound was prepared from 3,4-dimethoxybenzoic acid
and 4-methyl-2-aminophenol to afford 2b as a white solid (82%):
mp 164 ꢁC (acetonitrile). 1H NMR (300 MHz, CDCl3): 9.47 (br s, 1H,
OH), 9.41 (br s, 1H, NH), 7.61 (dd, 1H, H4, J ¼ 2.0 Hz and J ¼ 8.4 Hz),
7.55 (d, 1H, H6, J ¼ 2.0 Hz), 7.42 (m, 1H, H2'), 7.07 (d, 1H, H3,
J ¼ 8.4 Hz), 6.83 (dd, 1H, H6', J ¼ 1.7 Hz and J ¼ 8.3 Hz), 6.79 (d, 1H,
H5', J ¼ 8.3 Hz), 3.84 (s, 3H, OMe), 3.82 (s, 3H, OMe), 2.22 (s, 3H,
CH3). IR (ꢀ, cmꢂ1): 3368 (NH), 3182–2854 (OH), 1653 (C ¼ O). LC-MS
(ESI) m/z found: 288 [M þ H]þ.
N-(2-Hydroxy-4-methylphenyl)furan-2-carboxamide (2c): The title
compound was prepared from furoic acid and 5-methyl-2-amino-
phenol to afford 2c as a brown solid (72%): mp 170ꢁC (acetonitrile). J ¼ 8.4 Hz), 4.01 (s, 3H, OMe), 3.95 (s, 3H, OMe), 3.75 (s, 2H, CH2),

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1
853
3.70 (s, 3H, CO2Me). IR (ꢀ, cmꢂ1): 1730 (ester C ¼ O). LC-MS (ESI) m/ >300 ꢁC. H NMR (300 MHz, [D6]DMSO): 13.12 (br s, 1H, NHþ), 8.04
z found: 328 [M þ H]þ.
(m, 1H, H5'), 7.83 (m, 1H, H6), 7.68 (m, 2H), 7.33 (d, 1H, H3',
J ¼ 3.5 Hz), 7.30–7.25 (m, 2H), 6.98–6.89 (m, 3H), 6.66 (dd, 1H, H4',
J ¼ 1.7 Hz and J ¼ 3.5 Hz), 4.37 (s, 2H, CH2), 3.75–3.52 (m, 6H,
General procedure for the synthesis of compound (4a–4c)
To a solution of compound (3a–3c) (20.1 mmol) in CCl4 (150 ml)
was added N-bromosuccinimide (NBS) (24.1 mmol) and benzoyl
peroxide (1.41 mmol) and the reaction mixture was refluxed under
a halogen lamp (230 W). After 3 h and 30 min stirring, the mixture
was cooled to room temperature and the succinimide was filtered
off. Then, the solution was concentrated in vacuo, and the solid
was suspended in diethyl ether and filtered.
H
piperazine), 3.06 (m, 2H, Hpiperazine). 13C NMR (75 MHz, [D6]DMSO):
156.0 (C), 150.6 (C), 150.0 (C), 148.0 (CH), 141.9 (C), 141.7 (C), 129.6
(2 CH), 129.4 (CH), 127.5 (C), 123.5 (CH), 120.4 (CH), 116.4 (2 CH),
116.2 (CH), 113.4 (CH), 111.6 (CH), 58.7 (CH2), 50.6 (2 CH2), 45.7 (2
CH2). LC-MS (ESI) m/z Found: 360 [M þ H]þ. HPLC: C4 column:
tR ¼ 15.5 min, purity >99%.tert-Butyl4-{[2-(Furan-2-yl)-1,3-benzoxa-
zol-5-yl]methyl}piperazine-1-carboxylate (A3): The title compound
was prepared from compound 4a and boc-piperazine. Solid was
recrystallised from methanol to afford A3 as a white solid (83%):
5-(Bromomethyl)-2-(furan-2-yl)-1,3-benzoxazole (4a): The title
compound was prepared from benzoxazole 3a to afford 4a as a
beige solid (70%): mp 130 ꢁC. 1H NMR (300 MHz, CDCl3): 7.77 (m,
1H, H5'), 7.67 (m, 1H, H3'), 7.54 (d, 1H, H7, J ¼ 8.4 Hz), 7.42 (dd, 1H,
H6, J ¼ 1.8 Hz and J ¼ 8.4 Hz), 7.30 (m, 1H, H4), 6.64 (dd, 1H, H4',
J ¼ 1.7 Hz and J ¼ 3.5 Hz), 4.64 (s, 2H, CH2). LC-MS (ESI) m/z found:
278 [M þ H]þ, 280 [M þ H]þ.
1
mp 128 ꢁC (methanol). H NMR (300 MHz, CDCl3): 7.69 (m, 1H, H5'),
7.67 (m, 1H, H4), 7.49 (d, 1H, H7, J ¼ 8.3 Hz), 7.34 (dd, 1H, H6,
J ¼ 1.5 Hz and J ¼ 8.3 Hz), 7.26 (m, 1H, H3'), 6.62 (dd, 1H, H4',
J ¼ 1.7 Hz and J ¼ 3.5 Hz), 3.63 (s, 2H, CH2), 3.44 (t, 4H, Hpiperazine
,
J ¼ 4.9 Hz), 2.42 (t, 4H, Hpiperazine, J ¼ 3.5 Hz), 1.45 (s, 9H, (3 CH3).
NMR 13C (CDCl3, d ppm): 163.0 (C), 155.6 (C), 154.0 (C), 149.8 (C),
147.7 (C), 141.9 (C), 141.8 (CH), 132.2 (CH), 127.9 (C), 121.5 (CH),
115.8 (CH), 113.3 (CH), 111.2 (CH), 79.6 (2 CH2), 60.8 (CH2), 51.9 (2
CH2), 26.5 (3 CH3). IR (ꢀ, cmꢂ1): 1679 (C ¼ O). LC-MS (ESI) m/z
found: 328 [M-tBu þ H]þ, 284 [M þ H]þ. HPLC: C4 column:
tR ¼ 14.7 min, purity >99%.
6-(Bromomethyl)-2-(furan-2-yl)-1,3-benzoxazole (4b): The title
compound was prepared from benzoxazole 3b to afford 4b as a
1
brown solid (65%): mp 122 ꢁC, H NMR (300 MHz, CDCl3): 7.72–7.68
(m, 2H, H5' and H4), 7.60 (m, 1H, H7'), 7.40 (dd, 1H, H5, J ¼ 8.2 Hz
and J ¼ 1.6 Hz), 7.30 (dd, 1H, H3', J ¼ 0.7 Hz and J ¼ 3.5 Hz), 6.63
(dd, 1H, H4', J ¼ 1.7 Hz and J ¼ 3.5 Hz), 4.64 (s, 2H, CH2). IR (ꢀ,
cmꢂ1): 750 (C-Br).
5-(Bromomethyl)-2-(3,4-dimethoxyphenyl)-1,3-benzoxazole (4c):
The title compound was prepared from benzoxazole 3c to afford
4c as a beige solid (75%): mp 140 ꢁC. 1H NMR (300 MHz, CDCl3):
7.86 (dd, 1H, H6', J ¼ 1.9 Hz and J ¼ 8.4 Hz), 7.76 (m, 2H, H4 and
H2'), 7.53 (d, 1H, H7 or H5', J ¼ 8.5 Hz), 7.39 (dd, 1H, H6, J ¼ 1.7 Hz
and J ¼ 8.4 Hz), 7.00 (d, 1H, H7 or H5', J ¼ 8.4 Hz), 4.65 (s, 2H, CH2),
4.02 (s, 3H, OMe), 3.99 (s, 3H, OMe). IR (ꢀ, cmꢂ1): 750 (C-Br).
4-(4-{[2-(Furan-2-yl)-1,3-benzoxazol-5-yl]methyl}piperazin-1-
yl)phenol (A5): The title compound was prepared from compound
4a and 4-piperazinephenol. Solid was purified by flash chromatog-
raphy using DCM/EtOAc (9/1) to afford A5 as a white solid (83%):
1
mp 212 ꢁC. H NMR (300 MHz, [D6]DMSO): 8.79 (br s, 1H, OH), 8.07
(m, 1H, H5'), 7.71 (m, 2H, H7 and H4), 7.46 (d, 1H, H3', J ¼ 3.5 Hz),
7.41 (dd, 1H, H6, J ¼ 1.2 Hz and J ¼ 8.4 Hz), 6.82 (dd, 1H, H4',
J ¼ 1.8 Hz and J ¼ 3.5 Hz), 6.78 (d, 2H, Hphenyl, J ¼ 8.7 Hz), 6.63 (d,
2H, Hphenyl, J ¼ 8.7 Hz), 3.64 (s, 2H, CH2), 2.96 (m, 4H, Hpiperazine),
2.55 (m, 4H, Hpiperazine). 13C NMR (75 MHz, [D6]DMSO): 155.4 (C),
151.3 (C), 149.2 (C), 147.6 (CH), 144.7 (C), 142.1 (C), 141.7 (C), 135.9
(C), 126.9 (CH), 120.2 (CH), 118.2 (2 CH), 115.9 (2 CH), 115.5 (CH),
113.3 (CH), 110.8 (CH), 62.2 (CH2), 53.2 (2 CH2), 50.5 (2 CH2). IR (ꢀ,
cmꢂ1): 3195–2780 (OH). LC-MS (ESI) m/z found: 376 [M þ H]þ.
HPLC: C4 column: tR ¼ 10.2 min, purity 99% C18 column:
tR ¼ 14.3 min, purity >99%.
General procedure for the synthesis of compound (A1–A3 and
A5–A8)
To a solution of amine (2.05 mmol) in acetone (15 ml) were added
compound (4a–4c) (1.87 mmol) and Et3N (2.05 mmol). The reaction
mixture was refluxed for 1 h, cooled to room temperature and
concentrated in vacuo. Solid was suspended in water and
extracted three times with EtOAc. Combined organic layers were
dried over MgSO4 and concentrated in vacuo to give a solid which
was then purified.
2-(Furan-2-yl)-5-({4-[4-(2-methoxyethoxy)phenyl]piperazin-1-
yl}methyl)-1,3-benzoxazole (A6): The title compound was prepared
from compound 4a and 1-[4-(2-methoxyethoxy)phenyl]piperazine.
Solid was recrystallised from ethanol to afford A6 as a white solid
1
(33%): mp 123 ꢁC. RMN H (300 MHz, [D6]DMSO): 8.08 (m, 1H, H5'),
2-(Furan-2-yl)-5-(piperidin-1-ylmethyl)-1,3-benzoxazole
hydro-
chloride (A1): The title compound was prepared from compound
4a and piperidine. Solid was suspended in diethyl ether with HCl
gas, concentrated in vacuo and recrystallised from acetonitrile to
afford A1 as a white solid (74%): mp >300 ꢁC. 1H NMR (300 MHz,
[D6]DMSO): 12.43 (br s, 1H, NHþ), 8.02 (m, 1H, H6), 7.75 (m, 1H,
H5'), 7.70 (m, 1H, H4), 7.66 (d, 1H, H7, J ¼ 8.1 Hz), 7.32 (d, 1H, H3',
J ¼ 3.5 Hz), 6.65 (dd, 1H, H4', J ¼ 1.7 Hz and J ¼ 3.5 Hz), 4.26 (d, 2H,
CH2, J ¼ 5.0 Hz), 3.47 (m, 2H, Hpiperidine), 2.61 (m, 2H, Hpiperidine),
2.33 (m, 2H, Hpiperidine), 1.92–1.80 (m, 3H, Hpiperidine), 1.35 (m, 1H,
7.72 (m, 2H, H7 and H4), 7.46 (dd, 1H, H3', J ¼ 0.7 Hz and J ¼ 3.5 Hz),
7.40 (dd, 1H, H6, J ¼ 1.4 Hz and J ¼ 8.4 Hz), 6.88–6.79 (m, 5H, H4',
Hphenyl), 3.99 (m, 2H, OCH2), 3.64 (s, 2H, CH2), 3.61 (m, 2H, CH2O),
3.29 (s, 3H, OCH3), 3.01 (m, 4H, Hpiperazine), 2.54 (m, 4H, Hpiperazine).
RMN 13C (75 MHz, [D6]DMSO): 155.4 (C), 152.5 (C), 149.2 (CH),
147.5 (C), 145.9 (C), 142.1 (C), 141.7 (C), 135.9 (C), 126.1 (C), 119.9
(CH), 118.1 (2 CH), 115.4 (2 CH), 114.1 (CH), 112.3 (CH), 110.2 (CH),
71.2 (CH2), 67.7 (CH2), 60.8 (CH2), 53.4 (2 CH2), 50.6 (2 CH2), 33.7
(CH3). LC-MS (ESI) m/z found: 434 [M þ H]þ. HPLC: C4 column:
tR ¼ 16.1 min, purity >99%.
H
piperidine). 13C NMR (75 MHz, [D6]DMSO): 156.5 (C), 151.0 (C), 146.3
(CH), 142.1 (C), 142.0 (C), 129.1 (CH), 125.1 (C), 122.9 (CH), 115.3
(CH), 112.5 (CH), 111.8 (CH), 60.9 (CH2), 52.3 (2 CH2), 22.5 (2 CH2),
22.1 (CH2). LC-MS (ESI) m/z found: 283 [M þ H]þ. HPLC: C4 column:
tR ¼ 16.2 min, purity 98%.
2-(Furan-2-yl)-6-(piperidin-1-ylmethyl)-1,3-benzoxazole
hydro-
chloride (A7): The title compound was prepared from compound
4c and piperidine. Solid was suspended in diethyl ether with HCl
gas, concentrated in vacuo and recrystallised from acetonitrile to
afford A7 (276 mg, 68%): mp >300 ꢁC. 1H NMR (300 MHz,
2-(Furan-2-yl)-5-[(4-phenylepiperazin-1-yl)methyl]-1,3-benzoxa-
zole hydrochloride (A2): The title compound was prepared from [D6]DMSO): 11.08 (br s, 1H, NHþ), 8.16–8.10 (m, 2H, H7 and H5'),
compound 4a and phenylpiperazine. Solid was suspended in 7.82 (d, 1H, H4, J ¼ 8.1 Hz), 7.63 (m, 1H, H5), 7.51 (d, 1H, H3',
diethyl ether with HCl gas, concentrated in vacuo and recrystal- J ¼ 3.2 Hz), 6.83 (m, 1H, H4'), 4.38 (m, 2H, CH2), 3.28 (m, 2H,
lised from ethanol to afford A2 as a white solid (65%): mp
Hpiperidine), 2.83 (m, 2H, Hpiperidine), 1.76–1.66 (m, 5H, Hpiperidine),

854
R. DUROUX ET AL.
1.35 (m, 1H, Hpiperidine). 13C NMR (75 MHz, [D6]DMSO): 156.1 (C), (dd, 1H, H6, J ¼ 1.8 Hz and J ¼ 8.4 Hz), 6.80 (dd, 1H, H4', J ¼ 1.7 Hz
149.9 (C), 148.0 (CH), 142.4 (C), 141.7 (C), 129.0 (C), 127.8 (CH), and J ¼ 3.5 Hz), 3.72 (s, 2H, CH2). IR (ꢀ, cmꢂ1): 1710 (acid C ¼ O).
120.1 (CH), 116.3 (CH), 114.4 (CH), 113.4 (CH), 59.1 (CH2), 52.0 (2 LC-MS (ESI) m/z found: 244 [M þ H]þ.
CH2), 22.6 (2 CH2), 21.9 (CH2). LC-MS (ESI) m/z found: 283 [M þ H]þ.
HPLC: C4 column: tR ¼ 15.7 min, purity 96%.
General procedure for the synthesis of amide (7a–7d)
2-(3,4-Dimethoxyphenyl)-5-(piperidin-1-ylmethyl)-1,3-benzoxa-
zole hydrochloride (A8): The title compound was prepared from
compound 4b and piperidine. Solid was suspended in diethyl
ether with HCl gas, concentrated in vacuo and recrystallised from
To a solution of acid (6) (1.5 mmol) in toluene (4 ml) was added at
0 ꢁC SOCl2 (5.97 mmol). The mixture was refluxed during 1 h,
cooled to room temperature and concentrated in vacuo. The oil
was then diluted with EtOAc (26 ml) and added dropwise to a
solution of amine (1.6 mmol) and Et3N (2.25 mmol) in EtOAc
(30 ml) while being stirred and cooled in an ice bath. After 1 h and
30 min stirring at room temperature, the mixture was hydrolysed
with water, extracted twice with EtOAc and combined organic
layers were washed with a saturated NaHCO3 solution, brine, dried
over MgSO4 and concentrated in vacuo to give a solid which was
suspended in diethyl ether and filtered.
acetonitrile to afford A8 as
a white solid (200 mg, 60%):
mp >300 ꢁC, 1H NMR (300 MHz, [D6]DMSO): 12.31 (br s, 1H, NHþ),
7.90 (dd, 1H, H6', J ¼ 1.6 Hz and J ¼ 8.4 Hz), 7.82 (dd, 1H, H6,
J ¼ 1.9 Hz and J ¼ 8.4 Hz), 7.78 (d, 1H, H2', J ¼ 1.6 Hz), 7.71 (d, 1H,
H4, J ¼ 1.9 Hz), 7.62 (d, 1H, H5', J ¼ 8.4 Hz), 6.97 (d, 1H, H7,
J ¼ 8.4 Hz), 4.27 (d, 2H, CH2, J ¼ 5.1 Hz), 3.99 (s, 3H, OMe), 3.96 (s,
3H, OMe), 3.45 (m, 2H, Hpiperidine), 2.64 (m, 2H, Hpiperidine), 2.34 (m,
2H, Hpiperidine), 1.86 (m, 3H, Hpiperidine), 1.35 (m, 1H, Hpiperidine). 13C
NMR (75 MHz, [D6]DMSO): d 164.4 (C), 152.4 (C), 151.5 (C), 149.3
(C), 142.6 (C), 128.4 (CH), 124.7 (C), 122.5 (CH), 121.5 (C), 119.0
(CH), 111.5 (CH), 111.1 (CH), 110.1 (CH), 60.9 (CH2), 56.1 (CH3), 56.1
(CH3), 52.6 (2 CH2), 22.5 (2 CH2), 22.0 (CH2). LC-MS (ESI) m/z found:
353 [M þ H]þ. HPLC: C4 column: tR ¼ 14.3 min, purity 99%.
2-[2-(Furan-2-yl)-1,3-benzoxazol-5-yl]-1-(piperidin-1-yl)ethan-1-
one (7a): The title compound was prepared from compound 6
and piperidine to afford 7a as a white solid (76%): mp 112 ꢁC. 1H
NMR (300 MHz, CDCl3): 8.16 (d, 1H, H5', J ¼ 1.7 Hz), 7.71 (m, 2H, H4
and H7), 7.68 (m, 1H, H6), 7.34 (d, 1H, H3', J ¼ 3.5 Hz), 6.81 (dd, 1H,
H4', J ¼ 1.7 Hz and J ¼ 3.5 Hz), 3.78 (s, 2H, CH2), 3.39 (m, 4H,
Synthesis of 2-(Furan-2-yl)-5-(piperazin-1-ylmethyl)-1,3-benzoxa-
zole (A4): A solution of compound (A3) (100 mg, 0.35 mmol)
diluted in 5 ml of DCM with TFA (2 ml, 26 mmol) was stirred for 3 h
at room temperature, hydrolysed with water and basified with 1 M
solution of NaOH up to basic pH and extracted three times with
DCM. Combined organic layers were dried over MgSO4 and con-
centrated in vacuo. Solid was suspended in diethyl ether with a
H
piperidine), 1.68 (m, 4H, Hpiperidine), 1.47 (m, 2H, Hpiperidine). IR (ꢀ,
cmꢂ1): 1640 (C ¼ O).
2-[2-(Furan-2-yl)-1,3-benzoxazol-5-yl]-1-(4-phenylpiperazin-1-yl)e-
than-1-one (7b): The title compound was prepared from com-
pound 6 and phenylpiperazine to afford 7b as a white solid (78%):
mp 128 ꢁC. 1H NMR (300 MHz, CDCl3): 7.68 (m, 1H, H5'), 7.63 (m,
drop of ethanol and filtered to afford compound (A4) as a beige 1H, H3'), 7.52 (d, 1H, H7, J ¼ 8.4 Hz), 7.32–7.23 (m, 4H, Hphenyl),
1
solid (60%): mp 192 ꢁC. H NMR (300 MHz, [D6]DMSO): 8.08 (m, 1H, 6.89–6.87 (m, 3H, H4, H6 and Hphenyl), 6.63 (dd, 1H, H4', J ¼ 1.7 Hz
H5'), 7.74–7.71 (m, 2H, H4 and H7), 7.46 (dd, 1H, H3', J ¼ 0.7 Hz and and J ¼ 3.5 Hz), 3.91 (s, 2H, CH2), 3.83 (t, 2H, Hpiperazine, J ¼ 4.6 Hz),
J ¼ 3.5 Hz), 7.38 (dd, 1H, H6, J ¼ 1.6 Hz and J ¼ 8.3 Hz), 6.83 (dd, 1H, 3.66 (t, 2H, Hpiperazine, J ¼ 4.6 Hz), 3.16 (t, 2H, Hpiperazine, J ¼ 4.6 Hz),
H4', J ¼ 1.7 Hz and J ¼ 3.5 Hz), 3.65 (s, 2H, CH2), 3.45–3.39 (m, 4H, 3.02 (t, 2H, Hpiperazine, J ¼ 4.6 Hz). IR (ꢀ, cmꢂ1): 1640 (C ¼ O).
H
piperazine), 2.43 (m, 4H, Hpiperazine). 13C NMR (75 MHz, [D6]DMSO):
2-[2-(Furan-2-yl)-1,3-benzoxazole-5-yl]-1-(morpholine-4-yl)
ethan-1-one (7c): The title compound was prepared from com-
pound 6 and morpholine to afford 7c as a white solid (57%): mp
118 ꢁC. 1H NMR (300 MHz, CDCl3): 7.67 (m, 1H, H5'), 7.59 (m, 1H,
H3'), 7.52 (d, 1H, H7, J ¼ 8.4 Hz), 7.27 (m, 2H, H4 and H6), 6.63 (dd,
1H, H4', J ¼ 1.7 Hz and J ¼ 3.5 Hz), 3.85 (s, 2H, CH2), 3.66 (m, 4H,
155.4 (C), 149.3 (C), 147.6 (CH), 142.0 (C), 141.7 (C), 135.2 (C), 127.0
(CH), 120.3 (CH), 115.6 (CH), 113.3 (CH), 110.9 (CH), 61.9 (CH2), 50.6
(2 CH2), 44.1 (2 CH2). LC-MS (ESI) m/z found: 284 [M þ H]þ. HPLC:
C4 column: tR ¼ 15.8 min, purity 99%.
2-[2-(Furan-2-yl)-1,3-benzoxazol-5-yl]acetonitrile (5): To a solu-
tion of 5-(bromomethyl)-2-(3,4-dimethoxyphenyl)-1,3-benzoxazole
(4a) (1.23 g, 3.53 mmol) in a mixture of EtOH (56 ml) and H2O
(15 ml) was added KCN (1.15 g, 17.7 mmol). After one night stirring
at reflux, the reaction mixture was cooled to room temperature
and concentrated in vacuo. Solid was suspended in water and
extracted three times with EtOAc. Combined organic layer were
dried over MgSO4 and concentrated in vacuo. Solid was then
recrystallised in methanol to afford (5) as a beige solid (582 mg,
56%): mp 140 ꢁC. 1H NMR (300 MHz, CDCl3): 7.71–7.70 (m, 2H, H5'
and H3'), 7.58 (d, 1H, H7, J ¼ 8.3 Hz), 7.36–7.31 (m, 2H, H4 and H6),
6.64 (dd, 1H, H4', J ¼ 1.7 Hz and J ¼ 3.5 Hz), 3.90 (s, 2H, CH2). IR (ꢀ,
cmꢂ1): 2240 (CN). LC-MS (ESI) m/z found: 225 [M þ H]þ.
H
morpholine), 3.50 (m, 4H, Hmorpholine). IR (ꢀ, cmꢂ1): 1630 (C ¼ O).
2-[2-(Furan-2-yl)-1,3-benzoxazol-5-yl]-1-{4-[4-(2-methoxyethoxy)-
phenyl]piperazin-1-yl}ethan-1-one (7d): The title compound was
prepared from compound 6 and 1-[4-(2-methoxyethoxy)phenyl]pi-
perazine to afford 7d as a beige solid (73%): mp 114 ꢁC. 1H NMR
(300 MHz, [D6]DMSO): 7.67 (m, 1H, H5'), 7.62 (m, 1H, H3'), 7.51 (d,
1H, H7, J ¼ 8.4 Hz), 7.29 (m, 2H, H6 and H4), 6.84 (m, 4H, Hphenyl
and Hphenyl), 6.63 (dd, 1H, H4', J ¼ 1.7 Hz and J ¼ 3.5 Hz), 4.05 (t, 2H,
CH2O, J ¼ 4.6 Hz), 3.89 (s, 2H, CH2), 3.81 (t, 2H, Hpiperazine
,
,
J ¼ 4.7 Hz), 3.71 (t, 2H, CH2O, J ¼ 4.6 Hz), 3.63 (t, 2H, Hpiperazine
J ¼ 4.7 Hz), 3.43 (s, 3H, OCH3), 3.02 (t, 2H, Hpiperazine, J ¼ 4.7 Hz),
2.88 (t, 2H, Hpiperazine, J ¼ 4.7 Hz). IR (ꢀ, cmꢂ1): 1630 (C ¼ O).
2-[2-(Furan-2-yl)-1,3-benzoxazol-5-yl] acetic acid (6): A solution
of compound (5) (1.1 g, 4.91 mmol) in a mixture of AcOH (5.5 ml),
H2SO4 (5.5 ml) and H2O (5.5 ml) was refluxed for 17 h. After cooling
to room temperature, cold water was added and the solution was
extracted with EtOAc. The organic layer was extracted twice with
a saturated NaHCO3 solution, and then the aqueous layer was
acidified with 1 M HCl solution and extracted twice with EtOAc.
Combined organic layer were dried over MgSO4 and concentrated
General procedure for the synthesis of compound (B1–B4)
To a solution of amide (1.06 mmol) (7a–7d) in THF (5 ml) was
added LiAlH4 (2.65 mmol). After 1 h stirring at room temperature,
water (0.1 ml), 1 M NaOH solution (0.1 ml) and water (0.3 ml) were
added successively to get a white mineral solid which was filtered
off and washed with EtOAc (50 ml). Organic layer was then washed
under vacuum. Solid was suspended in diethyl ether and filtered with water, brine solution, dried over MgSO4 and concentrated in
to afford compound (6) as a white solid (702 mg, 60%): mp 203 ꢁC. vacuo to afford a solid which was then purified.
1H NMR (300 MHz, CDCl3): 12.38 (br s, 1H, OH), 8.07 (m, 1H, H5'),
2-(Furan-2-yl)-5-[2-(piperidine-1-yl) ethyl]-1,3-benzoxazole hydro-
7.68 (d, 1H, H7, J ¼ 8.4 Hz), 7.66 (m, 1H, H3'), 7.45 (m, 1H, H4), 7.32 chloride (B1): The title compound was prepared from amide 7a.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
855
Solid was suspended in diethyl ether with HCl gas, concentrated The aqueous layer was acidified with 6 M HCl solution up to acid
in vacuo and recrystallised from acetonitrile to afford B1 as a pH (1–3) and the formed solid was filtered. Crude was heated in
white solid (10%): mp >300 ꢁC. 1H NMR (300 MHz, [D6]DMSO): DMF (5 ml) at 80 ꢁC for 3 h, cooled to room temperature, hydro-
10.53 (br s, 1H, NHþ), 8.0 (m, 1H, H5'), 7.74 (d, 1H, H7, J ¼ 8.4 Hz), lysed with water and acidified with 1 M HCl solution and extracted
7.69 (m, 1H, H4), 7.46 (d, 1H, H3', J ¼ 3.5 Hz), 7.33 (dd, 1H, H6, three times with EtOAc. Combined organic layers were washed
J ¼ 3.4 and J ¼ 8.4 Hz), 6.82 (dd, 1H, H4', J ¼ 1.7 Hz and J ¼ 3.5 Hz),
with brine, dried over MgSO4 and concentrated in vacuo. Solid
3.52–3.48 (m, 2H, CH2CH2), 3.33–3.19 (m, 4H, Hpiperidine), 2.93–2.90
(m, 2H, CH2CH2), 1.80–1.70 (m, 5H, Hpiperidine), 1.43–1.39 (m, 1H,
H
(CH), 142.0 (C), 141.9 (C), 134.9 (C), 126.8 (CH), 120.1 (CH), 115.7
(CH), 113.3 (CH), 111.3 (CH), 57.2 (CH2), 52.5 (2 CH2), 29.7 (CH2),
22.8 (2 CH2), 21.9 (CH2). LC-MS (ESI) m/z found: 297 [M þ H]þ.
HPLC: C4 column: tR ¼ 16.3 min, purity >99%.
was suspended in diethyl ether and filtered.
3-[2-(Furan-2-yl)-1,3-benzoxazol-5-yl] propanoic acid (8a): The
title compound was prepared from compound 4a to afford 8a as
a white solid (43%): mp 207 ꢁC. 1H NMR (300 MHz, CDCl3): 12.19
(br s, 1H, OH), 8.05 (m, 1H, H5'), 7.66–7.62 (m, 2H, H6 and H4), 7.42
(d, 1H, H3', J ¼ 3.5 Hz), 7.28 (d, 1H, H7, J ¼ 8.2 Hz), 6.79 (m, 1H, H4'),
2.94 (t, 2H, CH2CH2, J ¼ 7.5 Hz), 2.59 (t, 2H, CH2CH2, J ¼ 7.5 Hz). IR
(ꢀ, cmꢂ1): 1686 (C ¼ O). LC-MS (ESI) m/z found: 258 [M þ H]þ.
3-[2-(3,4-Dimethoxyphenyl)-1,3-benzoxazol-5-yl] propanoic acid
(8b): The title compound was prepared from compound 4c to
afford 8b as a white solid (47%): mp 182 ꢁC. 1H NMR (300 MHz,
CDCl3): 12.14 (br s, 1H, OH), 7.77 (dd, 1H, H6', J ¼ 2.0 Hz and
J ¼ 8.4 Hz), 7.67–7.60 (m, 3H, H4, H2' and H5'), 7.25 (dd, 1H, H6,
J ¼ 1.6 Hz and J ¼ 8.3 Hz), 7.17 (d, 1H, H7, J ¼ 8.3 Hz), 3.88 (s, 3H,
OMe), 3.86 (s, 3H, OMe), 2.94 (t, 2H, CH2CH2, J ¼ 7.7 Hz), 2.59 (t, 2H,
CH2CH2, J ¼ 7.7 Hz). IR (ꢀ, cmꢂ1): 1710 (C ¼ O).
piperidine). 13C NMR (75 MHz, [D6]DMSO): 155.5 (C), 149.0 (C), 147.7
2-(Furan-2-yl)-5-[2-(4-phenylpiperazin-1-yl) ethyl]-1,3-benzoxa-
zole (B2): The title compound was prepared from amide 7b. Solid
was recrystallised from ethanol to afford B2 as a white solid
(12%): mp 160 ꢁC. 1H NMR (300 MHz, [D6]DMSO): 8.06 (m, 1H, H5'),
7.65 (m, 1H, H4), 7.56 (1H, d, H7, J ¼ 8.2 Hz), 7.43 (d, 1H, H3',
J ¼ 3.5 Hz), 7.31 (dd, 1H, H6, J ¼ 1.3 Hz and J ¼ 8.2 Hz), 7.22–7.17 (m,
2H, Hphenyl), 6.92 (d, 2H, Hphenyl, J ¼ 7.9 Hz), 6.80 (dd, 1H, H4',
J ¼ 1.7 Hz and J ¼ 3.5 Hz), 6.78–6.73 (m, 1H, Hphenyl), 3.12 (m, 4H,
H
piperazine), 2.88 (t, 2H, CH2CH2, J ¼ 7.3 Hz), 2.64–2.57 (m, 6H,
CH2CH2 and Hpiperazine). 13C NMR (75 MHz, [D6]DMSO): 155.2 (C),
151.5 (C), 148.5 (C), 147.5 (CH), 141.7 (C), 142.1 (C), 138.2 (C), 129.4
(CH), 126.9 (CH), 120.0 (CH), 119.2 (CH), 115.8 (CH), 115.4 (CH),
113.2 (CH), 110.8 (CH), 60.4 (CH2), 53.1 (2 CH2), 48.7 (2 CH2), 33.0
(CH2). LC-MS (ESI) m/z found: 374 [M þ H]þ. HPLC: C4 column:
tR ¼ 15.8 min, purity >99%.
Synthesis of compounds (9a–9c)
Same procedure as described for compounds (7a–7d) has been
used.
3-(2-(Furan-2-yl)-1,3-benzoxazol-5-yl)-1-(4-phenylpiperazin-1-
yl)propan-1-one (9a): The title compound was prepared from com-
pound 8a and phenylpiperazine to afford 9a as a white solid
2-(Furan-2-yl)-5-[2-(morpholin-4-yl)
ethyl]-1,3-benzoxazole
hydrochloride (B3): The title compound was prepared from amide
7c. Solid was suspended in diethyl ether with HCl gas, concen-
trated in vacuo and recrystallised from acetonitrile to afford B3 as
a white solid (80%): mp >300 ꢁC. 1H NMR (300 MHz, [D6]DMSO):
11.36 (br s, 1H, NHþ), 8.06 (m, 1H, H5'), 7.73–7.67 (m, 2H, H7 and
H4), 7.43 (m, 1H, H3'), 7.31 (m, 1H, H6), 7.80 (m, 1H, H4'), 3.92–3.82
(m, 4H, CH2CH2), 3.31–3.18 (m, 8H, Hmorpholine). LC-MS (ESI) m/z
found: 299 [M þ H]þ. HPLC: C4 column: tR ¼ 14.8 min, purity 99%.
2-(Furan-2-yl)-5-(2-{4-[4-(2-methoxyethoxy)phenyl]piperazin-1-
yl}ethyl)-1,3-benzoxazole (B4): The title compound was prepared
from amide 7d. Solid was suspended recrystallised from aceto-
nitrile to afford B4 as a beige solid (73%): mp >300 ꢁC. 1H NMR
(300 MHz, CDCl3): 7.67 (m, 1H, H5'), 7.62 (m, 1H, H3'), 7.48 (d, 1H,
H7, J ¼ 8.3 Hz), 7.27 (m, 2H, H6 and H4), 7.22 (m, 1H, Hphenyl), 6.90
(m, 4H, Hphenyl), 6.63 (dd, 1H, H4', J ¼ 1.7 Hz and J ¼ 3.5 Hz), 4.09 (t,
2H, OCH2, J ¼ 4.6 Hz), 3.73 (t, 2H, MeOCH2, J ¼ 4.6 Hz), 3.45 (s, 3H,
OMe), 3.15 (m, 4H, Hpiperazine), 2.98 (m, 2H, CH2CH2), 2.73 (m, 6H,
Hpiperazine and CH2CH2). 13C NMR (75 MHz, CDCl3): 155.5 (C), 152.9
(C), 148.8 (C), 145.9 (C), 145.7 (CH), 142.7 (C), 141.9 (C), 137.3 (C),
126.1 (CH), 119.9 (CH), 118.1 (2 CH), 115.4 (2 CH), 114.1 (CH), 112.3
(CH), 110.2 (CH), 71.2 (CH2), 67.7 (CH2), 60.8 (CH2), 59.2 (CH2), 53.4
(2 CH2), 50.6 (2 CH2), 33.7 (CH3). LC-MS (ESI) m/z found: 448
[M þ H]þ. HPLC: C4 column: tR ¼ 15.2 min, purity 99%.
1
(55%): mp 164 ꢁC. H NMR (300 MHz, CDCl3): 7.66 (m, 1H, H5'), 7.58
(m, 1H, H3'), 7.48 (d, 1H, H7, J ¼ 8.3 Hz), 7.29–7.23 (m, 4H, Hphenyl),
6.91–6.87 (m, 3H, H4, H6 and Hphenyl), 6.62 (dd, 1H, H4', J ¼ 1.7 Hz
and J ¼ 3.5 Hz), 3.57 (m, 2H, Hpiperazine, J ¼ 5.7 Hz), 3.55 (m, 2H,
Hpiperazine), 3.17–3.11 (m, 4H, CH2CH2 and Hpiperazine), 3.04 (m, 2H,
H
piperazine), 2.73 (t, 2H, CH2CH2, J ¼ 7.7 Hz). IR (ꢀ, cmꢂ1): 1658
(C ¼ O).
3-[2-(3,4-Dimethoxyphenyl)-1,3-benzoxazole-5-yl]-1-(piperidin-1-
yl)propan-1-one (9b): The title compound was prepared from com-
pound 8b and piperidine to afford 9b as a white solid (66%): mp
130 ꢁC. 1H NMR (300 MHz, CDCl3): 7.85 (dd, 1H, H6', J ¼ 8.4 Hz and
J ¼ 1.9 Hz), 7.75 (d, 1H, H2', J ¼ 1.9 Hz), 7.56 (m, 1H, H4), 7.58 (d, 1H,
H7', J ¼ 8.4 Hz), 7.21 (dd, 1H, H6, J ¼ 1.5 Hz and J ¼ 8.1 Hz), 7.17 (d,
1H, H7, J ¼ 8.4 Hz), 4.02 (s, 3H, OMe), 3.97 (s, 3H, OMe), 3.57 (t, 1H,
CH2CH2, J ¼ 5.6 Hz), 3.35 (t, 2H, Hpiperidine, J ¼ 5.6 Hz), 3.10 (t, 2H,
CH2CH2, J ¼ 7.7 Hz), 2.67 (t, 2H, CH2CH2, J ¼ 7.7 Hz), 1.68–1.47 (m,
6H, Hpiperidine). IR (ꢀ, cmꢂ1): 1625 (C ¼ O).
3-(2-(3,4-Dimethoxyphenyl)-1,3-benzoxazol-5-yl)-1-(4-phenylpiper-
azin-1-yl)propan-1-one (9c): The title compound was prepared from
compound 8b and phenylpiperazine to afford 9c as a white solid
(54%): mp 160ꢁC. 1H NMR (300 MHz, CDCl3): 7.85 (dd, 1H, H6',
J ¼ 2.0 Hz and J ¼ 8.5 Hz), 7.75 (d, 1H, H2', J ¼ 2.0 Hz), 7.60 (d, 1H, H4,
J ¼ 1.2 Hz), 7.47 (d, 1H, H7, J ¼ 8.3Hz), 7.28–7.20 (m, 3H, H6 and
Hphenyl), 6.99 (d, 1H, H5', J ¼ 8.5 Hz), 6.87–6.82 (m, 3H, Hphenyl), 4.02
(s, 3H, OMe), 3.98 (s, 3H, OMe), 3.79 (m, 2H, Hpiperazine), 3.55 (m, 2H,
Hpiperazine), 3.16–3.11 (m, 4H, CH2CH2 and Hpiperazine), 3.03 (m, 2H,
General procedure for the synthesis of acid (8a–8b)
A solution of dimethyl malonate (9.06 mmol) in acetone (33 ml)
with K2CO3 (13.6 mmol) was stirred for 20 min at room tempera-
ture. Then compound (4a, 4c) (4.53 mmol) was added and the
mixture was stirred at reflux for 2 h, cooled to room temperature
and concentrated in vacuo. Solid was suspended in water and
H
piperazine), 2.74 (t, 2H, CH2CH2, J ¼ 7.4 Hz). IR (ꢀ, cmꢂ1): 1635 (C¼ O).
Synthesis of compounds (C1–C3)
extracted twice with EtOAc. Combined organic layer were dried Same procedure as described for compounds (B1–B4) has been
over MgSO4 and then concentrated in vacuo. The solid was sus- used.
pended in H2O (7 ml) and NaOH (18.1 mmol) was added. The mix-
2-(Furan-2-yl)-5-(3-(4-phenylpiperazin-1-yl)propyl)-1,3-benzoxa-
ture was stirred overnight at 40 ꢁC and then washed with EtOAc. zole hydrochloride (C1): The title compound was prepared from

856
R. DUROUX ET AL.
compound 9a. Solid was suspended in diethyl ether with HCl gas, CH2CH2), 3.64 (t, 2H, CH2CH2, J ¼ 6.6 Hz). IR (ꢀ, cmꢂ1): 3400 (OH).
LC-MS (ESI) m/z found: 300 [M þ H]þ.
concentrated in vacuo and recrystallised from acetonitrile to afford
1
C1 as a white solid (12%): mp >300 ꢁC. H NMR (300 MHz, CDCl3):
11.00 (br s, 1H, NHþ), 8.07 (m, 1H, H5'), 7.71–7.67 (m, 2H, H4 and
H3'), 7.44 (m, 1H, H6), 7.32–7.24 (m, 3H, H7 and Hphenyl), 6.98 (m,
2-(2-(3,4-Dimethoxyphenyl)-1,3-benzoxazol-5-yl)ethyl methanesul-
fonate (11). To a solution of compound (10) (800 mg, 2.37 mmol)
in DCM (20 ml) with Et3N (0.49 ml, 3.55 mmol) at 0 ꢁC, was added
dropwise mesyl chloride (0.28 ml, 3.55 mmol). After 4 h stirring at
room temperature, mixture was hydrolysed with water and
extracted twice with DCM. Combined organic layers were dried
over MgSO4 and concentrated in vacuo. Solid was suspended in
diethyl ether and filtered to afford compound (11) as a beige solid
(895 mg, 100%): mp 112 ꢁC. 1H NMR (300 MHz, CDCl3): 7.85 (dd,
1H, H6', J ¼ 8.4 Hz and J ¼ 2.0 Hz), 7.76 (d, 1H, H4', J ¼ 2.0 Hz), 7.61
(d, 1H, H4, J ¼ 1.6 Hz), 7.51 (d, 1H, H7, J ¼ 8.3 Hz), 7.21 (dd, 1H, H6,
J ¼ 8.3 Hz and J ¼ 1.6 Hz), 7.00 (d, 1H, H5', J ¼ 8.4 Hz), 4.47 (t, 2H,
CH2CH2, J ¼ 6.8 Hz), 4.02 (s, 3H, OMe), 3.97 (s, 3H, OMe), 3.18 (t, 2H,
CH2CH2, J ¼ 6.9 Hz), 2.88 (s, 3H, CH3). LC-MS (ESI) m/z found: 378
2H, Hphenyl), 6.81 (m, 2H, Hphenyl and H4'), 3.76 (m, 2H), 3.57 (m,
2H), 3.12 (m, 6H), 2.80 (m, 2H, CH2CH2CH2), 2.14 (m, 2H,
CH2CH2CH2). 13C NMR (75 MHz, [D6]DMSO): 155.3 (C), 150.1 (C),
148.7 (C), 147.6 (CH), 142.0 (C), 141.9 (C), 138.3 (C), 129.6 (2 CH),
126.9 (CH), 120.4 (CH), 119.6 (CH), 116.4 (2 CH), 115.5 (CH), 113.3
(CH), 111.0 (CH), 55.5 (CH2), 51.1 (2 CH2), 45.9 (2 CH2), 32.4 (CH2),
25.6 (CH2). LC-MS (ESI) m/z found: 388 [M þ H]þ. HPLC: C4 column:
tR ¼ 15.8 min, purity 98%.
2-(3,4-Dimethoxyphenyl)-5-[3-(piperidin-1-yl)propyl]-1,3-benzox-
azole hydrochloride (C2): The title compound was prepared from
compound 9b. Solid was suspended in diethyl ether with HCl gas,
concentrated in vacuo and recrystallised from ethyl acetate to
afford C2 as a white solid (72%): mp 214 ꢁC. 1H NMR (300 MHz, [M þ H]þ.
[D6]DMSO): 10.20 (br s, 1H, NHþ), 7.77 (dd, 1H, H6', J ¼ 2.0 Hz and
J ¼ 8.4 Hz), 7.68 (d, 1H, H5', J ¼ 8.4 Hz), 7.66–7.63 (m, 2H, H2' and
General procedure for the synthesis of compound (B5–B6)
H4), 7.21 (dd, 1H, H6, J ¼ 1.5 Hz and J ¼ 8.3 Hz), 7.17 (d, 1H, H7,
J ¼ 8.3 Hz), 3.88 (s, 3H, OMe), 3.86 (s, 3H, OMe), 3.38 (m, 2H,
CH2CH2CH2), 2.99 (m, 2H, CH2CH2CH2), 2.76 (m, 2H, CH2CH2CH2),
2.08 (m, 2H, Hpiperidine), 1.75–1.65 (m, 5H, Hpiperidine), 1.35 (m, 1H,
Hpiperidine). 13C NMR (75 MHz, [D6]DMSO): 163.2 (C), 152.5 (C), 149.5
(C), 149.3 (C), 142.4 (C), 137.8 (C), 125.9 (C), 121.3 (CH), 119.2 (CH),
119.2 (CH), 112.4 (CH), 110.9 (CH), 110.2 (CH), 56.2 (CH3), 56.1
(CH3), 55.9 (CH2), 52.4 (2 CH2), 32.5 (CH2), 25.6 (CH2), 22.9 (2 CH2),
21.9 (CH2). LC-MS (ESI) m/z found: 381 [M þ H]þ. HPLC: C4 column:
tR ¼ 17.3 min, purity 98%.
To a solution of compound 11 (0.79 mmol) in DMF (8 ml), were
added K2CO3 (1.46 mmol) and amine (1.03 mmol). After overnight
stirring at 80 ꢁC, the reaction mixture was cooled, hydrolysed with
water and extracted three times with EtOAc. Combined organic
layers were dried over MgSO4 and concentrated in vacuo to afford
a solid which was then purified.
2-(3,4-Dimethoxyphenyl)-5-(2-(piperidin-1-yl)ethyl)-1,3-benzoxa-
zole hydrochloride (B5): The title compound was prepared from
compound 11 and piperidine. Solid was suspended in diethyl
ether with HCl gas, concentrated in vacuo and recrystallised from
ethanol to afford B5 as a white solid (25%): mp 260 ꢁC. 1H NMR
(300 MHz, [D6]DMSO): 10.63 (br m, 1H, NHþ), 7.79 (dd, 1H, H6',
J ¼ 8.4 Hz and J ¼ 2.0 Hz), 7.73 (d, 1H, H7, J ¼ 8.3 Hz), 7.68 (d, 1H,
H2', J ¼ 1.3 Hz), 7.66 (d, 1H, H4, J ¼ 2.0 Hz), 7.31 (dd, 1H, H6,
J ¼ 8.4 Hz and J ¼ 1.7 Hz), 7.18 (d, 1H, H5', J ¼ 8.6 Hz), 3.89 (s, 3H,
OMe), 3.87 (s, 3H, OMe), 3.50 (m, 2H, CH2CH2), 3.27–3.20 (m, 4H,
CH2CH2 and Hpiperidine), 2.92 (m, 2H, Hpiperidine), 1.82–1.70 (m, 5H,
Hpiperidine), 1.42 (m, 1H, Hpiperidine). 13C NMR (75 MHz, [D6]DMSO):
163.4 (C), 152.5 (C), 149.5 (C), 149.3 (C), 142.4 (C), 137.8 (C), 125.9
(C), 121.3 (CH), 119.2 (CH), 119.2 (CH), 112.4 (CH), 110.9 (CH), 110.2
(CH), 57.3 (CH2), 56.2 (CH3), 56.1 (CH3), 52.4 (2 CH2), 29.7 (CH2),
22.8 (2 CH2), 21.9 (CH2). LC-MS (ESI) m/z found: 367 [M þ H]þ.
HPLC: C4 column: tR ¼ 16.1 min, purity 99%.
2-(3,4-Dimethoxyphenyl)-5-[3-(4-phenylpiperazin-1-yl)propyl]-
1,3-benzoxazole hydrochloride (C3): The title compound was pre-
pared from compound 9c. Solid was suspended in diethyl ether
with HCl gas, concentrated in vacuo and recrystallised from etha-
nol to afford C3 as a white solid (73%): mp 194 ꢁC. 1H NMR
(300 MHz, [D6]DMSO): 10.81 (br s, 1H, NHþ), 7.77 (dd, 1H, H6',
J ¼ 1.8 Hz and J ¼ 8.4 Hz), 7.70–7.65 (m, 3H, H4, H6 and H7),
7.29–7.22 (m, 3H, H2' and Hphenyl), 7.17 (d, 1H, H5', J ¼ 8.4 Hz), 6.98
(m, 2H, Hphenyl), 6.87–6.82 (m, 1H, Hphenyl), 3.88 (s, 3H, OMe), 3.86
(s, 3H, OMe), 3.77 (m, 2H, CH2CH2CH2), 3.56 (m, 2H, Hpiperazine),
3.11 (m, 6H, Hpiperazine), 2.79 (t, 2H, CH2CH2CH2, J ¼ 7.4 Hz), 2.13 (m,
2H, CH2CH2CH2). 13C NMR (75 MHz, [D6]DMSO): 163.2 (C), 152.5 (C),
150.1 (C), 149.5 (C), 149.3 (C), 142.4 (C), 137.8 (C), 129.6 (2 CH),
125.9 (CH), 121.3 (CH), 120.4 (C), 119.3 (CH), 119.2 (CH), 116.4 (2
CH), 112.4 (CH), 110.9 (CH), 110.2 (CH), 56.2 (CH2), 56.1 (CH3), 55.5
(CH3), 51.1 (2 CH2), 45.9 (2 CH2), 32.5 (CH2), 25.7 (CH2). LC-MS (ESI)
m/z found: 458 [M þ H]þ. HPLC: C4 column: tR ¼ 16.6 min,
purity >99%.
2-(3,4-Dimethoxyphenyl)-5-[2-(4-phenylpiperazin-1-yl)ethyl]-1,3-
benzoxazole (B6): The title compound was prepared from com-
pound 11 and phenylpiperazine. Solid was recrystallised from
ethanol to afford B6 as a white solid (28%): mp 140 ꢁC. 1H NMR
(300 MHz, CDCl3): 7.85 (dd, 1H, H6', J ¼ 2.0 Hz and J ¼ 8.4 Hz), 7.76
(d, 1H, H2', J ¼ 2.0 Hz), 7.61 (m, 1H, H4), 7.48 (d, 1H, H5', J ¼ 8.4 Hz),
7.31–7.26 (m, 2H, H7 and Hphenyl), 7.20 (dd, 1H, H6, J ¼ 1.5 Hz and
2-(2-(3,4-dimethoxyphenyl)-1,3-benzoxazol-5-yl)ethanol (10). To a
solution of compound (3e) (1 g, 3.06 mmol) in THF (10 ml) was J ¼ 8.3 Hz), 7.01–6.85 (m, 4H, Hphenyl), 4.03 (s, 3H, OMe), 3.98 (s, 3H,
OMe), 3.27–3.24 (t, 4H, Hpiperazine, J ¼ 4.8 Hz), 3.01–2.96 (m, 2H,
added LiAlH4 (340 mg, 9.2 mmol). After 1 h stirring at room tem-
CH2CH2), 2.75–2.71 (m, 6H, CH2CH2 and Hpiperazine). 13C NMR
perature, water (0.34 ml), 1 M NaOH solution (0.34 ml) and water
(300 MHz, CDCl3): 163.4 (C), 152.0 (C), 151.3 (C), 149.4 (C), 149.2 (C),
142.5 (C), 136.9 (C), 129.1 (2 CH), 125.5 (CH), 121.1 (CH), 119.9 (C),
119.7 (CH), 119.4 (CH), 116.1 (2 CH), 111.0 (CH), 110.0 (2 CH), 60.9
(CH2), 56.1 (CH3), 56.0 (CH3), 53.3 (2 CH2), 49.2 (2 CH2), 33.7 (CH2).
LC-MS (ESI) m/z found: 444 [M þ H]þ. HPLC: C4 column:
tR ¼ 15.7 min, purity 99%.
(1.02 ml) were added successively until get a white solid which
was filtered off and washed with EtOAc (60 ml). Organic layer was
washed with water, dried over MgSO4 and concentrated in vacuo.
Solid was recrystallised in acetonitrile to afford compound (10) as
1
a beige solid (603 mg, 86%): mp 210 ꢁC. H NMR (300 MHz, CDCl3):
9.13 (br s, 1H, OH) 7.86 (dd, 1H, H6', J ¼ 1.8 Hz and J ¼ 8.4 Hz), 7.76
(d, 1H, H2', J ¼ 1.8 Hz), 7.61 (m, 1H, H4), 7.50 (d, 1H, H7, J ¼ 8.4 Hz),
7.24 (dd, 1H, H6, J ¼ 1.2 Hz and J ¼ 8.4 Hz), 7.00 (d, 1H, H5',
2-(Furan-2-yl)-1,3-benzoxazol-5-amine (D1). To a solution of 2-
J ¼ 8.4 Hz), 4.03 (s, 3H, OMe), 3.98 (s, 3H, OMe), 3.92 (m, 2H, (furan-2-yl)-5-nitro-1,3-benzoxazole (3.07 g, 13.3 mmol) (3d) in

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
857
EtOH (130 ml) were added Pd/C (10%, 100 mg) and hydrazine The obtained yellow oil was suspended in diethyl ether with HCl
monohydrate (0.78 ml, 16 mmol). The mixture was heated at 70 ꢁC gas to formed a solid which was filtered to afford compound E1
for 3 h, cooled to room temperature, the Pd/C was filtered off and as a beige solid (11 mg, 8%): mp 169 ꢁC. 1H NMR (300 MHz,
the filtrate was concentrated in vacuo. Crude was suspended in [D6]DMSO): 10.69 (br m, 1H, NHþ), 8.04 (m, 1H, H5'), 7.55 (d, 1H,
water and extracted three times with EtOAc. Combined organic H7, J ¼ 8.8 Hz), 7.38 (m, 1H, H3'), 7.09 (m, 1H, H4), 6.88 (dd, 1H, H6,
layer were washed with brine, dried over MgSO4 and concentrated J ¼ 1.9 Hz and J ¼ 8.8 Hz), 6.79 (dd, 1H, H4', J ¼ 1.7 Hz and
in vacuo. Solid was recrystallised from hexane to afford compound J ¼ 3.5 Hz), 5.37 (br m, 1H, NH), 3.57 (t, 2H, CH2CH2, J ¼ 6.3 Hz),
(D1) as grey solid (2.16 g, 81%): mp 112 ꢁC. 1H NMR (300 MHz, 3.49–3.46 (m, 2H, Hpiperidine), 3.24 (t, 2H, CH2CH2, J ¼ 6.2 Hz), 2.90
[D6]DMSO): 8.01 (m, 1H, H5'), 7.40 (d, 1H, H7, J ¼ 8.7 Hz), 7.34 (d, (m, 2H, Hpiperidine), 1.79 (m, 5H, Hpiperidine), 1.39 (m, 1H, Hpiperidine).
13C NMR (75 MHz, [D6]DMSO): 155.3 (C), 147.2 (CH), 145.0 (C),
1H, H3', J ¼ 3.5 Hz), 6.84 (d, 1H, H4, J ¼ 2.1 Hz), 6.77 (dd, 1H, H4',
J ¼ 1.7 Hz and J ¼ 3.5 Hz), 6.66 (dd, 1H, H6, J ¼ 2.1 Hz and 143.6 (C), 142.7 (C), 124.3 (C), 114.9 (CH), 114.1 (CH), 113.2 (CH),
J ¼ 8.7 Hz), 5.16 (br s, 2H, NH2). 13C NMR (75 MHz, [D6]DMSO):
111.5 (CH), 102.6 (CH), 54.5 (CH2), 52.7 (2 CH2), 39.2 (CH2), 22.8 (2
CH2), 21.8 (CH2). LC-MS (ESI) m/z found: 312 [M þ H]þ. HPLC: C4
154.9 (C), 147.3 (C), 147.0 (CH), 142.6 (C), 142.5 (C), 142.4 (C), 114.4
(CH), 113.6 (CH), 113.1 (CH), 110.9 (CH), 103.0 (CH). IR (ꢀ, cmꢂ1):
column: tR ¼ 15.9 min, purity 99%.
3424 (NH2). LC-MS (ESI) m/z found: 201 [M þ H]þ. HPLC: C4 column:
2-Bromo-N-(2-(furan-2-yl)-1,3-benzoxazol-5-yl)acetamide (13): To
tR ¼ 14.2 min, purity 98%.
a solution of compound D1 (210 mg, 1.05 mmol) and Et3N (0.18 ml,
1.26 mmol) in DCM (10 ml) at 0 ꢁC was added dropwise a solution
of bromoacetyl bromide (0.11 ml, 1.26 mmol) diluted in DCM
(5 ml). The reaction mixture was stirred at room temperature for
N-(4-(Benzyloxy)phenethyl)-2-(furan-2-yl)-1,3benzoxazol-5-amine
(12). To a solution of compound D1 (800 mg, 4 mmol) in DMF
2 h, hydrolysed with water and extracted twice with DCM.
(15 ml) were added 1-(benzyloxy)-4-(2-bromoethyl)benzene (1.4 g,
4.8 mmol)16 and K2CO3 (828 mg, 5.99 mmol). The reaction mixture
Combined organic layers were washed with brine, dried over
MgSO4 and concentrated in vacuo. The solid was suspended in
was stirred at 80 ꢁC overnight, cooled to room temperature, hydro-
lysed with water, acidified with 1 M HCl solution, and extracted
with EtOAc three times. Combined organic layers were washed
diethyl ether and filtered to afford compound 13 as a white solid
(249 mg, 74%): mp 203 ꢁC. 1H NMR (300 MHz, CDCl3): 8.27 (br s,
1H, NH), 7.96 (m,1H, H5'), 7.70–7.69 (m, 1H, H4), 7.56–7.50 (m, 2H,
with brine, dried over MgSO4 and concentrated in vacuo to give a
H7 and H6), 7.30 (dd, 1H, H3', J ¼ 0.7 Hz and J ¼ 3.5 Hz), 6.39 (dd,
yellow oil. Purification by flash chromatography was realised with
EtOAc/Cyclohexane as solvent (1/9 up to 3/7) to give the product
as a yellow oil which was suspended in diethyl ether and filtered
1H, H4', J ¼ 1.7 Hz and J ¼ 3.5 Hz), 4.08 (s, 2H, CH2). IR (ꢀ, cmꢂ1):
3246 (NH), 1643 (C ¼ O).
to afford compound 12 as a pale brown solid (430 mg, 26%): mp
184 ꢁC. 1H NMR (75 MHz, [D6]DMSO, J Hz): 8.10 (m, 1H, H5'), 7.75
General procedure for the synthesis of compounds (F1–F3)
(m, 1H), 7.58 (m, 1H), 7.46–7.26 (m, 7H), 7.21 (d, 2H, Hphenyl
,
To a solution of compound 13 (0.59 mmol) in acetone (5 ml) were
added K2CO3 (0.88 mmol) and the amine (0.65 mmol). The reaction
mixture was refluxed for 2 h, cooled to room temperature and
concentrated in vacuo. The crude was suspended in water and
extracted three times with EtOAc. Combined organic layer were
washed with brine, dried over MgSO4 and concentrated in vacuo
to afford a solid which was then purified.
J ¼ 8.7 Hz), 6.96 (d, 2H, Hphenyl, J ¼ 8.7 Hz), 6.83 (dd, 1H, H4',
J ¼ 1.7 Hz and J ¼ 3.5 Hz), 5.06 (s, 2H, CH2), 3.52 (t, 2H, CH2CH2,
J ¼ 7.5 Hz), 2.99 (t, 2H, CH2CH2, J ¼ 7.5 Hz). LC-MS (ESI) m/z found:
411 [M þ H]þ.
4-(2-{[2-(Furan-2-yl)-1,3-benzoxazol-5-yl]amino}ethyl)phenol (E2). A
solution of compound 12 (740 mg, 1.66 mmol) in MeOH (15 ml)
with Pd/C (50 mg) under H2 atmosphere was stirred at 25 ꢁC for
overnight. Pd/C was filtered off and the filtrate was concentrated
in vacuo. Solid was recrystallised in CH3CN to afford compound E2
as a yellow crystal (104 mg, 18%): mp 174 ꢁC. 1H NMR (300 MHz,
[D6]DMSO): 9.16 (br s, 1H, OH), 8.01 (m, 1H, H5'), 7.43 (d, 1H, H7,
N-[2-(Furan-2-yl)-1,3-benzoxazol-5-yl]-2-(piperidin-1-yl)acetamide
hydrochloride (F1): The title compound was prepared from com-
pound 13 and piperidine. Solid was suspended in diethyl ether
with HCl gas, concentrated in vacuo and recrystallised from etha-
nol to afford F1 as a white solid (57%): mp >300 ꢁC. 1H NMR
(300 MHz, [D6]DMSO): 11.26 (br s, 1H, NHþ), 9.99 (br s, 1H, NH),
8.17 (m, 1H, H5'), 8.08 (m, 1H, H4), 7.76 (d, 1H, H7, J ¼ 8.8 Hz), 7.58
(dd, 1H, H6, J ¼ 1.9 Hz and J ¼ 8.8 Hz), 7.46 (d, 1H, H3', J ¼ 3.5 Hz),
6.82 (dd, 1H, H4', J ¼ 1.7 Hz and J ¼ 3.5 Hz), 4.18 (s, 2H, CH2),
3.50–3.47 (m, 2H, Hpiperidine), 3.10 (m, 2H, Hpiperidine), 1.80–1.67 (m,
J ¼ 8.7 Hz), 7.33 (d, 1H, H3', J ¼ 3.5 Hz), 7.07 (d, 2H, Hphenyl
,
J ¼ 8.4 Hz), 6.80 (m, 1H, H4), 6.77 (dd, 1H, H4', J ¼ 1.7 Hz and
J ¼ 3.5 Hz), 6.69 (d, 2H, Hphenyl, J ¼ 8.4 Hz), 6.71 (m, 1H, H6), 5.73 (br
t, 1H, J ¼ 5.6 Hz, NH), 3.24–3.17 (m, 2H, J ¼ 6.9 Hz, CH2CH2), 2.75 (t,
2H, J ¼ 7.6 Hz, CH2CH2). 13C NMR (75 MHz, [D6]DMSO): 156.1 (C),
154.9 (C), 147.7 (C), 147.0 (CH), 142.8 (C), 142.5 (C), 142.2 (C); 130.3
(C), 130.0 (2 CH), 115.5 (2 CH), 114.4 (CH), 113.1 (CH), 112.8 (CH),
111.1 (CH), 100.2 (CH); 46.1 (CH2), 34.4 (CH2). IR (ꢀ, cmꢂ1): 3340
(OH). LC-MS (ESI) m/z found: 321 [M þ H]þ. HPLC: C4 column:
tR ¼ 14.8 min, purity 97%.
5H,
H
piperidine), 1.40 (m, 1H, Hpiperidine). 13C NMR (75 MHz,
[D6]DMSO): 163.4 (C), 156.0 (C), 147.7 (CH), 146.6 (C), 141.9 (C),
141.8 (C), 135.9 (C), 118.2 (CH), 115.8 (CH), 113.3 (CH), 111.4 (CH),
110.8 (CH), 57.6 (CH2), 53.4 (2 CH2), 22.7 (2 CH2), 21.6 (CH2). LC-MS
(ESI) m/z found: 326 [M þ H]þ. HPLC: C4 column: tR ¼ 15.1 min, pur-
ity 98%.
N-[2-(Furan-2-yl)-1,3-benzoxazol-5-yl]-2-(4-phenylpiperazin-1-
yl)acetamide (F2): The title compound was prepared from com-
2-(Furan-2-yl)-N-(2-(piperidin-1-yl)ethyl)-1,3
benzoxazol-5-amine
hydrochloride (E1). To a solution of compound D1 (800 mg, pound 13 and phenylpiperazine. Solid was recrystallised from
4 mmol) in DMF (16 ml) were added K2CO3 (1.7 g, 12 mmol) and N- ethanol to afford F2 as a white solid (48%): mp 174 ꢁC. 1H NMR
2-chloroethyl piperidine hydrochloride (1471 mg, 7.99 mmol). The (300 MHz, CDCl3): 9.26 (br s, 1H, NHþ), 8.02 (m, 1H, H4), 7.68–7.67
reaction mixture was stirred at 70 ꢁC overnight, cooled to room (m, 1H, H5'), 7.56 (dd, 1H, H6, J ¼ 2.0 Hz and J ¼ 8.8 Hz), 7.51 (d, 1H,
temperature, hydrolysed with water and extracted three times H7, J ¼ 8.6 Hz), 7.3–7.29 (m, 3H, Hphenyl and H3'), 6.98–6.88 (m, 3H,
with EtOAc. Combined organic layer were dried over MgSO4 Hphenyl and Hphenyl), 6.63 (dd, 1H, H4', J ¼ 1.7 Hz and J ¼ 3.5 Hz),
and concentrated in vacuo. Purification by flash chromatography 3.30 (t, 4H, Hpiperazine, J ¼ 4.9 Hz), 3.26 (s, 2H, CH2), 2.83 (t, 4H,
was performed with DCM/EtOH/NH3 (90/10/1) as
a
solvent. Hpiperazine, J ¼ 4.9 Hz). 13C NMR (75 MHz, CDCl3): 168.2 (C), 156.2 (C),

858
R. DUROUX ET AL.
152.9 (C), 146.9 (C), 145.8 (CH), 142.5 (C), 142.2 (C), 134.9 (C), 129.2 in the presence of 1 U/ml of adenosine deaminase (Roche, Basel,
(2 CH), 120.2 (CH), 117.8 (CH), 116.3 (2 CH), 114.5 (CH), 112.3 (CH), Switzerland). All samples were assayed in duplicate. Non-specific
111.2 (CH), 110.5 (CH), 62.0 (CH2), 50.6 (2 CH2), 49.5 (2 CH2). LC-MS
(ESI) m/z found: 403 [M þ H]þ. HPLC: C4 column: tR ¼ 14.7 min,
purity >99%.tert-Butyl-4-({[2-(furan-2-yl)-1,3-benzoxazol-5-yl]carba-
moyl}methyl)piperazine-1-carboxylate (F3): The title compound
was prepared from compound 13 and tert-butyl piperazine-1-carb-
oxylate. Solid was recrystallised from ethanol to afford F3 as a
binding was determined for each assay in the presence of the
antagonist ZM-24135 (8.3 nm). Microplates were incubated for 1 h
at room temperature and the reaction was stopped by vacuum fil-
tration with a Skatron semi-automatic cell harvester with chilled
incubation solution (pH 7.4, Tris 50 mm MgCl 10 mm) to filter mats
1.5 mm (Molecular Devices, Sunnyvale, CA). Three millilitres of scin-
tillation cocktail (OptiPhase “HiSafe” 2, PerkinElmer) were added
and radioactivity bound to the filters was determined after 12 h.
Molecules inhibited binding by ꢃ30% at 10 lm were submitted to
Ki evaluation. This percentage was calculated using Excel 2013 as
a ratio of data with ligands to data without ligands. Data were
analysed using Graph Pad Prism, version 5.01 (San Diego, CA).
Inhibition constants (Ki) were calculated from the IC50 values by
non-linear regression analysis, the Cheng and Prusoff equation
and KD value of 1.0 nm were used. Displacement reference curves
were performed with ZM-24135 (0–6 nm in 6%, 40% or 60% of
DMSO). No difference was observed between each concentration.
Affinity towards A1R (human recombinant CHO cells, [3H]-
DPCPX (1 nm), Cerep catalogue reference 0002, as described by
Townsend-Nicholson19), A2BR (human recombinant HEK-293 cells,
[3H]-CPX (5 nm), Cerep catalogue reference 0005, as described by
Stehle20) and A3R (human recombinant HEK-293 cells, [125I]-AB-
MECA (0.15 nm), Cerep catalogue reference 0006, as described by
Salvatore21) was determined by CEREP laboratories. KD values used
were: 1.7 nm for [3H]-DPCPX, 65 nm for [3H]-CPX and 0.15 nm for
[125I]-AB-MECA. For these three receptors, the stock solution of
compounds was prepared in DMSO. The final concentration of
DMSO was no more than 1%. Data were analysed using
1
white solid (90%): mp 186 ꢁC. H NMR (300 MHz, CDCl3): 9.15 (br s,
1H, NH), 7.98 (d, 1H, H4, J ¼ 2.0 Hz), 7.68–7.67 (m, 1H, H5'), 7.55 (dd,
1H, H6, J ¼ 2.0 Hz and J ¼ 8.6 Hz), 7.50 (d, 1H, H7, J ¼ 8.6 Hz), 7.28
(m, 1H, H3'), 6.62 (dd, 1H, H4', J ¼ 1.7 Hz and J ¼ 3.5 Hz), 3.54 (t, 4H,
Hpiperazine, J ¼ 4.9 Hz), 3.19 (s, 2H, CH2), 2.60 (t, 4H, Hpiperazine
,
J ¼ 4.9 Hz), 1.48 (s, 9H, 3 CH3). 13C NMR (75 MHz, CDCl3): 167.9 (C),
156.2 (C), 154.6 (C), 147.0 (C), 145.9 (CH), 142.5 (C), 142.2 (C), 134.8
(C), 117.8 (CH), 114.5 (CH), 112.3 (CH), 111.2 (CH), 110.5 (CH), 80.1
(CH2), 62.1 (2 CH2), 53.3 (2 CH2), 28.4 (3 CH3). IR (l, cmꢂ1): 1684
(C ¼ O), 1673 (C ¼ O). LC-MS (ESI) m/z found: 371 [M-tBu þ H]þ,
427 [M þ H]þ. HPLC: C4 column: tR ¼ 15.1 min, purity 99%.
N-(2-(Furan-2-yl)-1,3-benzoxazol-5-yl)-2-(piperazin-1-yl)acetamide
dihydrochloride (F4): To a solution of (F3) (180 mg, 0.422 mmol) in
MeOH (10 ml) was added 6 M HCl (1.41 ml, 8.44 mmol) and the
reaction mixture was stirred at 40 ꢁC for 15 h. The precipitated
product was filtered and washed with diethyl ether to afford com-
pound (F4) as a white solid (121 mg, 72%): mp 192 ꢁC. 1H NMR
(300 MHz, [D6]DMSO): 11.16 (br s, 1H, NH), 9.88 (br s, 2H, NH2þ),
8.16 (d, 1H, H4, J ¼ 1.8 Hz), 8.07 (m, 1H, H5'), 7.75 (d, 1H, H7,
J ¼ 8.8 Hz), 7.59 (dd, 1H, H6, J ¼ 1.8 Hz and J ¼ 8.8 Hz), 7.45–7.44 (m,
1H, H3'), 6.81 (dd, 1H, H4', J ¼ 1.7 Hz and J ¼ 3.5 Hz), 4.22 (s, 2H,
CH2), 3.57 (s, 4H, Hpiperazine, J ¼ 4.9 Hz), 3.43 (s, 4H, Hpiperazine
,
J ¼ 4.9 Hz). 13C NMR (75 MHz, [D6]DMSO): 163.7 (C), 155.9 (C), 147.7
(CH), 146.5 (C), 141.9 (C), 141.8 (C), 135.9 (C), 118.2 (CH), 115.7
(CH), 113.3 (CH), 111.4 (CH), 110.8 (CH), 57.6 (CH2), 49.1 (4 CH2).
LC-MS (ESI) m/z found: 327 [M þ H]þ. HPLC: C4 column:
tR ¼ 14.9 min, purity 99%.
R
R
SigmaPlotV version 4.0 for WindowsV (# 1997 by SPSS Inc.,
Chicago, IL).
Cell culture and cytotoxicity assay
The human neuroblastoma cell line (SY5Y) was cultured in
Dulbecco’s modified Eagle medium (DMEM) (Gibco, Waltham, MA)
supplemented with 2 mm L-glutamine, 100 mg/ml streptomycin,
100 IU/ml penicillin, 1 mm non-essential amino acids and 10% (v/v)
heat-inactivated foetal bovine serum (Sigma-Aldrich, Saint-Louis,
MO), and grown at 37 ꢁC in a humidified incubator with 5% CO2.
Cells were seeded at 2000 cells per well onto 96-well plates in
DMEM medium. Cells were starved for 24 h to obtain synchronous
cultures and were then incubated in a culture medium that con-
tained various concentrations of test compounds, each dissolved
in less than 0.1% DMSO. After 72 h of incubation, cell growth was
estimated by the colorimetric MTT (thiazolyl blue tetrazolium
bromide) assay.
Molecular docking
Molecular docking was performed using Gold suite v5.217 within
the Hermes v1.6 GUI (CCDC#). Thus after adding hydrogens,
water molecules were deleted and docking was performed in a
10 Å around co-crystallised ligands then ligands deleted. Early ter-
mination of three docking solutions within 1.5 Å was set up in
order to highlight ligands converging towards a few binding
modes.
Pharmacological assays
Displacement binding assays
Radioligands were obtained from the following source: [3H]-
ZM241385 from IsoBio (10–50 Ci/ml), [3H]-DPCPX from PerkinElmer
(Waltham, MA) (120 Ci/mmol), [3H]-CPX from Perkin Elmer (120 Ci/
mmol), [125I]-AB-MECA from Chelatec (2200 Ci/mmol) (Saint-
Herblain, France).
For A2A receptor binding evaluation, the stock solution of com-
pounds was prepared in DMSO. The final concentration of DMSO
was no more than 3% for radioligand binding assay.
Absorption, distribution, metabolism and excretion (ADME)
assessment
Aqueous solubility (in phosphate-buffered saline, PBS, pH 7.4; incu-
bation room temperature for 24 h as described by Lipinski22
Eurofins Cerep SA catalogue reference G235), partition coefficient
(log D, n-octanol/PBS, pH 7.4, room temperature for 60 min as
described by Sangster23; Eurofins Cerep SA catalogue reference
0417), human plasma protein binding evaluated at 10 lm concen-
tration for 4 h at 37 ꢁC as described by Banker24 (Eurofins Cerep
SA catalogue reference 2194), A-B and B-A permeability coefficient
evaluated at 10 lm for 40 min as described by Hidalgo25 (Papp,
Competition binding curves of the A2A receptor antagonist
[3H]-ZM241385 by the designed A2A antagonists described above,
were performed as before18 in Human HEK293 A2AR membranes
(PerkinElmer). 0.5 ml of membranes (0.5 U of A2AR) were incubated
with [3H]-ZM241385 (2 nm) and increasing concentrations of the Caco-2 cells, pH 6.5/7.4; Eurofins Cerep SA catalogue reference
designed A2AR antagonists (0–600 nm) in a final volume of 300 ll G228), metabolic stability in human liver microsomes evaluated at

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
859
0.1 lm concentration for 0, 15, 30, 45, 60 min at 37 ꢁC as described Indeed, interaction with this key amino acid is well known to lock
by Obach26 (Eurofins Cerep SA catalogue reference 0607) were the A2A receptor33 and more generally class-A GPCR34 in their
determined in standard assays by Eurofins Cerep SA, France www. inactive conformation. The 3,4 dimethoxyphenyl, found on
cerep.fr).
Istradefylline, was also used to create this interaction. Furthermore,
different nature and size of linkers was used to bring selected
amine function. Indeed, these basic functions in designed ligands
(Figure 2(C)) occupy the same pocket as the phenol part of the
ZM-241385 and allow not only to interact with Glu169 through an
ionic interaction but also to improve solubility.
Results and discussion
Structure-based insights
Our design was guided by molecular modelling studies which
took into consideration the two structures of adenosine A2A recep-
tors co-crystallised with high-affinity A2A antagonists ZM-241385
and T4E (1,2,4-triazines) in respective 3EML27,28 and 3UZC29 PDB
entries. Although several molecular dynamics studies have shown
Chemistry
Synthetic routes used to prepare benzoxazole derivatives are
depicted in Schemes 1–3. The first set of benzoxazoles (3a–d) was
prepared from commercially available aminophenols using two dif-
ferent synthetic routes (Scheme 1). The first one led to molecules
3a–d, in two steps. Indeed, aminophenol derivatives 1a–e reacted
with appropriate acyl chlorides to give an equimolar mixture of
mono and diacyl compounds which, when treated under basic
conditions, gave amides 2a–d. Subsequent cyclisation under acidic
conditions afforded benzoxazoles 3a–d in good yield35. The
second synthetic route allowed to transform aminophenol 1e into
the importance of water molecules for ligand recognition30,31
,
these molecules represent a bias in molecular docking with many
options like their rigid pre-docking displacement, a tolerance for
moving them or simply delete them. Since T4E-bound structures
show that the key water molecules in ZM-241385-bound crystal
structures could be displaced by aryl substituents, we decided to
avoid this experimental bias in order to benefit with the whole
cavity.
In contrast with ZM-241385 bound structure, the T4E-bound
one was identified as the most suitable target to predict correct
docking poses of both T4E (no data show) and ZM-241385 within
a 2.0 Å structural deviation in comparison with experimental co-
crystallised poses (Figure 2(B)). This is due to the greater volume
of T4E-bound pocket that allows to accommodate bulky di-aryl
substituted triazine as well as linear ZM-241385 ligands.
Consequently, apoA2AR-T4E pocket appears to be more relevant
to accommodate a wide diversity of chemical structures and was
therefore used to perform our docking calculations.
A set of benzoxazole-based molecules have been docked, using
Gold suite v5.2 within the Hermes v1.6 GUI (CCDC#), into the
apoA2AR-T4E pocket and the ones that satisfied interactions with
essential amino acids32 Phe168, Glu169, Trp246 and Asn253 were
selected. ZM-241385 was shown as an example (Figure 2(B)) rather
than the triazine because it is structurally closer to our benzoxa-
zole ligands (Figure 2(C)). As illustrated in Figure 2(B), the central
heterocycle of ZM-241385 interacts through a hydrogen bond
with Asn253 and p-stacking with Phe168. Benzoxazole ring seems
to recapitulate these interactions and could, therefore, constitute a
good alternative as a central core. The furan, found in many A2A
antagonists, was selected to interact with Trp246 by aromatic
interaction and thus made the antagonist character of our ligands.
R
compound 3e in one step using T3PV
.
36
A radical bromination of compounds 3a–c was performed to
generate the key brominated intermediates 4a–c (Scheme 2)
which allowed the introduction of the tertiary amine at different
distances from the central heterocycle. Molecules A1–8 displaying
a one-methylene linker were obtained by reacting 4a–c with vari-
ous amines. Compound A4 was synthesised from A3 deprotection
using TFA. Based on binding results (see Table 1), we focused the
further synthetic effort on C-5 substituted compounds. Treatment
of 4a with potassium cyanide followed by an acidic hydrolysis
gave carboxylic acid 6 which was subjected to amidification and
reduction to afford target compounds B1–4 (two-methylene
linker).
To get molecules C1–3 (three-methylene linker), the same pro-
cedure was used starting from carboxylic acids 8a–b, obtained by
malonic substitution performed on compounds 4a–b followed by
a basic hydrolysis and then a decarboxylation reaction by heating
in DMF.
To obtain molecules B5–6 (Scheme 3), ester function of com-
pound 3e was first reduced to alcohol 10 using LiAlH4. The latter
was then activated by the action of mesyl chloride to afford mol-
ecule 11. The classical nucleophilic substitution was then per-
formed to give compounds B5–6.
OH
NH
Ar
5
OH
NH
N
a
b
R
R
Ar
R
O
6
2
2a-d
1a-e
3a-d
O
N°
Position
5
R
Ar
c
O
3a
Me
CO CH
OMe
OMe
2
3
OCH
3
3b
3c
3d
5
Me
N
OCH
3
O
O
6
5
Me
3e
O
NO2
Scheme 1. Reagents and conditions: (a) i) ArCO2H, SOCl2, DMF, DCM, ii) Et3N, EtOAc, aminophenol (1a–d); iii) NaOH, EtOH/H2O, then 6 M HCl, 60–80% over 2 steps; (b)
R
APTS, toluene, reflux, 70–80%, (c) T3PV (50% in EtOAc), DIPEA, 3,4-dimethoxybenzoic acid, 35%.

860
R. DUROUX ET AL.
N
N
O
O
X
5
6
N
O
N
O
N
N
c
Ar
HN
O
A3
for
7a-d, X = CO
g
A4
A1-3, A5-8
B1-4,
X = CH2
b
f
5
N
Ar
O
5
Br
N
O
O
d
N
O
a
R
Ar
H3C
6
6
4a-c
5 (R= CN)
3a-c
e
6 (R= CO2H)
h
O
N
i
X
g
HO
N
O
N
Ar
Ar
O
8a-b
9a-c,
X = CO
C1-3, X = CH2
Scheme 2. Reagents and conditions: (a) NBS, benzoyl peroxide, CCl4, reflux/ht (230 W), 60–75%; (b) R2R1NH, Et3N, acetone, 50–85%; (c) TFA, DCM, 60%; (d) KCN, EtOH/
H2O, 50–60%; (e) H2O/H2SO4/AcOH, reflux, 60–70%; (f) i) SOCl2, toluene; ii) secondary amine, Et3N, EtOAc, 50–60%; (g) LiAlH4, THF, 60–75%; (h) i) dimethyl malonate,
K2CO3, acetone; ii) NaOH, H2O then 6 M HCl; iii) DMF, reflux, 40–43%.
OMs
N
OH
CO CH
2
3
c
b
a
O
N
N
O
N
O
O
N
OCH
OCH
OCH
OCH
3
3
3
3
OCH
OCH
OCH
OCH
3
3
3
3
B5-6
11
3e
10
Scheme 3. Reagents and conditions: (a) LiAlH4, THF, 86%; (b) MsCl, Et3N, DCM, quant.; (c) secondary amine, K2CO3, DMF, 60 ꢁC, 25–28%.
To get target molecules with an amide (F1–4) or ethylamine determined for those exhibiting a specific displacement superior
to 35%.
The first set of derivatives A1–8, allowed drawing some early
linker (E1–2), 5-nitro benzoxazole derivative (3d) was reduced with
hydrazine hydrate in the presence of Pd/C to afford compound D1
(Scheme 4). Nucleophilic substitution gave compounds E1 and 12.
The latter was deprotected under H2 atmosphere afforded E2. To
get molecules F1–3, treatment of D1 with bromoacetyl bromide
gave compound 13 that was substituted with various amines.
Compound F4 was obtained from F3 deprotection using 6 M HCl
in MeOH.
SARs (Table 1). First, comparing molecules A1–6, piperidine
emerged as the preferred amine since a dramatic decrease in
affinity was observed with all other selected ones. Surprisingly, the
phenylpiperazine group featured in many A2A antagonists was not
tolerated in our series.
Regarding the position of the protonable amine function, com-
pounds with a chain at the C-5 position appeared to exhibit a
higher affinity than a compound with a chain at the C-6 position
(molecules A1 versus A7). Therefore, the protonable amine was
placed in the C-5 position for following molecules. Concerning the
aryl in position 2 of the benzoxazole, replacing the furan of A1 by
a 3,4-dimethoxyphenyl (A8) totally abolished affinity. This result is
Structure-affinity relationship and early ADME studies
Affinities of benzoxazole derivatives for the human adenosine
receptor were determined by a competitive radioligand displace- in agreement with literature since the preference of the adenosine
ment assay using [3H]-ZM241385 as radioligand18. All compounds
were first screened at 10 lm concentration, and Ki values were
A2A receptor for furan substituent is well documented even if the
3,4-dimethoxyphenyl is found on the Istradefylline molecule7,13
.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
861
Table 1. A2A receptor affinity and cytotoxicity data of compounds A1–8, B1–6 and C1–3.
5
N
O
O
N
2
N
2
n
Ar
N
O
6
A1-A6, A8, B1-B6, C1-C3
A7
a
c
Cpd.
Ar
Ki
SEM (µM) /
CC
/
50
N
b
d
% inhib
% inhib
ZM -
0.0013
0.002
241385
N
A1
A2
A3
A4
A5
A6
1%
33%
30%
0%
19
3.1
N
N
10%
Boc
N
N
15%
15%
11%
14%
O
HN
N
N
n = 1
N
HO
27%
37%
N
N
O
H CO
3
A7
A8
-
5%
0%
0%
N
18%
OCH
3
OCH
3
N
B1
1%
7
1.4
3%
N
N
B2
B3
B4
0%
11%
-
O
N
O
11 0.25
10%
n = 2
N
N
O
H CO
3
N
B5
B6
10%
5%
40%
19%
OCH
3
OCH
O
3
N
N
N
N
C1
28%
-
n = 3
N
C2
C3
10%
11%
73 µM
OCH
3
OCH
3
N
N
aDisplacement of specific [3H]-ZM 241385 binding to hA2A receptors stably expressed in HEK293 cells. bDisplacement percentage of specific [3H]-ZM 241385 at 10 lm.
cCompound concentration causing 50% of SY5Y cell death after 24 h treatment. dPercentage of dead SY5Y cells after 24 h treatment at 100 lm.

862
R. DUROUX ET AL.
O
N
O
N
N
H
E1
Br
d
RO
c
O
N
O
O
N
O
OBn
b
a
3d
H N
2
N
H
D
1
12 (R = Bn)
E2 (R = H)
e
O
N
O
O
N
O
O
O
f or g
N
Br
N
H
N
H
F1-4
13
Scheme 4. Reagents and conditions: (a) hydrazine hydrate, Pd/C (10%), EtOH, 81%; (b) 1-(benzyloxy)-4-(2-bromoethyl)benzene, K2CO3, DMF, 70 ꢁC, 15%; (c) H2, Pd/C
(10%), MeOH, 48%; (d) N-chloroethylpiperidine, K2CO3, DMF, 70 ꢁC, 8%; (e) bromoacetylbromide, NEt3, DCM, 75%; (f) for F1–3, secondary amine, K2CO3, acetone,
48–90%; (g) for F4, i) boc-piperazine, K2CO3, acetone, ii) 6 M HCl, MeOH, 72%.
Table 2. A2A receptor affinity and cytotoxicity data of compounds D1, E1–2 and F1–4.
O
N
O
O
O
R
R
N
N
H
N
H
F1-F4
D1, E1-E2
c
c
a
a
CC
/
CC
/
50
Cpd.
R
Cpd.
R
Ki (mM)
/
Ki (mM) /
50
b
d
b
d
% inhib
% inhib
% inhib
% inhib
N
4%
1%
D1
H
10 0.5
F1
1
0.08
O
N
N
O
3%
12%
1%
12%
0%
E1
E2
F2
F3
F4
11 0.02
N
O
HO
N
Boc
HN
N
15%
25%
8%
55 µM
N
O
aDisplacement of specific [3H]-ZM 241385 (2 nm) binding to hA2A receptors stably expressed in HEK293 cells. bDisplacement percentage of specific [3H]-ZM 241385 at
c
10 lm. Compound concentration causing 50% of SY5Y cell death after 24 h treatment. dPercentage of dead SY5Y cells after 24 h treatment at 100 lm.
Table 3. Preliminary ADME studies of F1.
Permeability Caco-2-
(10ꢂ6 cm/s)c
f
Solubility (lm)
HLMe
t1/2 (min)
Clint
d
Cpd.
F1
PBS, pH 7.4a
PPB (%)b
83
A/B
50
B/A
24
LogD7.4
(ll/min/mg)
184
2.33
c
11
630
aEvaluated after 24 h stirring. bPPB ¼ plasma protein binding. Compound was tested at 10 lm concentration. Permeability ¼ Compound was tested at 10 lm concen-
tration at pH 6.5/7.4. dDetermined between a mixture PBS7.4/octanol. HLM ¼ human liver microsome. Clint ¼ Compound was tested at 0.1 lm concentration.
e
f

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
863
Throughout Table 1, a similar tendency was observed for the active compound (F1) toward the hA2AR (Ki ¼ 1 lm). Furthermore,
other analogues suggesting that the piperidine chain at C-5 and
the furan at C-2 of the benzoxazole ring are the best moieties for
A2A receptor affinity.
the latter presented good preliminary ADME properties with a
very interesting solubility (184 lm) as well as good log D7.4 (2.33)
without cytotoxicity at 100 lm. Thus, both F1 and D1, which can
be easily modulated in position 7, appear to be promising starting
points for further optimisation.
Concerning the linker, the comparison between B1 and A1
revealed that increasing the length to two carbons is beneficial
for affinity. Moreover, as can be seen from affinity data of E1
(11 lm) and F1 (1lm) (Table 2), a three-atom linker is also well tol-
erated. When comparing the latter two compounds, rigidifying the
linker by incorporating an amide in place of an amine allows an
increase in affinity. Besides, F1 was found to be the most active
compound in this series with a Ki value of 1 lm. As can be seen
from Figure 2(C) the three-atom linker seems necessary to allow the
interaction between the piperidine and Glu169. This ligand also
exhibited a slight selectivity (see Table 4 of the Supplementary
Material) versus A1 receptor (2.5-fold). Concerning the two others
adenosine receptors, preliminary studies showed a probably good
interaction with hA2B (73% inhibition at 10lm) but a highly select-
ivity over adenosine A3 receptor (11% inhibition at 10lm). These
informations do not constitute a brake for the development of F1
as a potential drug candidate at this “hit” identification stage.
Interestingly, replacing the protonable amine of E1 by the ami-
noethyl phenol chain present in ZM-241385 (E2) led to a drastic
drop in affinity, highlighting the importance of the tertiary amine
in this series.
Acknowledgements
We express our thanks to the 300 MHz NMR facilities funded by
the Region Nord-Pas de Calais (France), the Ministere de la
Jeunesse, de l’Education Nationale et de la Recherche (MJENR) and
the Fonds Europeens de Developpement Regional (FEDER). This
work was supported by Lille 2 University, ANR “Adoratau”, Comue
Univ Lille Nord de France. Romain Duroux is the recipient of a fel-
lowship from Lille 2 University.
Disclosure statement
No potential conflict of interest was reported by the authors.
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