Benzofuran derivatives as a novel class of inhibitors of mTOR signaling

Article abstract of DOI:10.1016/j.ejmech.2013.12.020

High-throughput screening (HTS) hit 1 was previously identified as an inhibitor of the Akt/mTOR (Akt/mammalian target of rapamycin) signaling, which is a major target in oncology. The cytotoxicity of 1 was determined on a panel of human cancer cells lines

Full text of DOI:10.1016/j.ejmech.2013.12.020

European Journal of Medicinal Chemistry 74 (2014) 41e49
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Benzofuran derivatives as a novel class of inhibitors of mTOR signaling
Christophe Salomé a, Vanessa Narbonne a, Nigel Ribeiro a, Frédéric Thuaud a,
Maria Serova b, Armand de Gramont b, Sandrine Faivre b, Eric Raymond b,
,
Laurent Désaubry a
*
a Therapeutic Innovation Laboratory, UMR7200, CNRS/Université de Strasbourg, Illkirch, France
b Department of Medical Oncology, Beaujon University Hospital, INSERM U728/AP-HP, Clichy, France
a r t i c l e i n f o
a b s t r a c t
Article history:
High-throughput screening (HTS) hit 1 was previously identified as an inhibitor of the Akt/mTOR (Akt/
mammalian target of rapamycin) signaling, which is a major target in oncology. The cytotoxicity of 1 was
determined on a panel of human cancer cells lines with an IC50 comprised between 30 and 140 mM.
Subsequent structureeactivity relationship (SAR) studies led us to the identification of compounds that
displayed an enhanced cytotoxicity. We demonstrated also that these molecules directly bind to mTOR
complex 1 (mTORC1) and inhibit its kinase activity.
Received 26 November 2013
Received in revised form
20 December 2013
Accepted 21 December 2013
Available online 2 January 2014
Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords:
Benzofuran
Cytotoxicity
mTOR
mTORC1
1. Introduction
of resistance in cancer cells combined to a lack of mTORC2 complex
inhibition.
The AKT/mammalian target of rapamycin (mTOR) pathway is
considered as one of the most commonly activated and deregulated
signaling pathways in human cancer [1e3]. Rapamycin (sirolimus),
a naturally occurring macrolide, was the first identified potent in-
hibitor of mTOR. This kinase is associated with other proteins in
two molecular complexes: mTORC1 and mTORC2. Rapamycin binds
to the cytosolic immunophilin protein FKBP12 (FK-binding protein
12), which selectively inhibits mTORC1, without interacting with
mTORC2. Several rapamycin derivatives (rapalogues) such as tem-
sirolimus, everolimus and deforolimus, are currently commercial-
ized to treat cancers. Temsirolimus and everolimus have been
approved for the first- and second-line treatment, respectively, of
patients with clear renal cell carcinomas. Moreover, temsirolimus
was also approved for the treatment of mantle cell lymphoma.
Current clinical development of rapalogues was impaired by
toxicity when combined with chemotherapy and several targeted
therapies [1e3]. Furthermore, the antiproliferative activity of
rapalogues appears to be limited by various molecular mechanisms
Novel anticancer drugs designed to overcome potential mech-
anisms of resistance to rapalogues are currently in development
[1]. Several companies have identified drugs that inhibit mTOR by
competing with adenosine triphosphate (ATP) into the ATP-binding
sites. These compounds do not require FKBP12 to bind with mTOR
and, thereby, inhibit both mTORC1 and mTORC2 complexes. Some
of these molecules do not only inhibit mTOR, but also phosphati-
dylinositide 3-kinases (PI3K). Several specific mTOR inhibitors and
dual PI3K/mTOR inhibitors are currently investigated in phase III
trials.
Compound 1 (also known as ChemBridge 5219657, Fig. 1) was
identified in a high-throughput screen as an inhibitor of Akt/mTOR
signaling [4,5]. In 786-O renal carcinoma cells, 1 blocked Akt/
mTOR-induced nuclear export of the transcription factor FOXO1a
(IC50 ¼ 20
mM) and cell proliferation (IC50 ¼ 10
mM) by an un-
identified mechanism downstream of Akt. The privileged structure
of benzofurans, which is uncommon among the known inhibitors
of the Akt/mTOR cascade, prompted us to define the structural
requirements of 1 for its antiproliferative effects and to examine the
pharmacological potential of this new class of cytostatic agents.
Herein, we report the first SAR study on 1 and our efforts to identify
its mechanism of action.
* Corresponding author. Tel.: þ33 368 854 141; fax: þ33 368 854 310.
E-mail address: desaubry@unistra.fr (L. Désaubry).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.12.020

42
C. Salomé et al. / European Journal of Medicinal Chemistry 74 (2014) 41e49
2. Results and discussions
Compound 7r was then selected as the starting point for the
design of an affinity ligand to identify the molecular target of this
class of compounds by pull-down assays. En route to this objective,
we synthesized 7t and demonstrated that the introduction of a
linker on the terminal piperidine moiety was well tolerated for the
cytotoxicity. In the meanwhile, we examined the influence of the
benzyl moiety. Functionalization of the benzylic position as a ke-
tone (10) or an alcohol (11) maintained the cytotoxicity almost
unaltered. Gratifyingly, the deletion of this benzylic methylene
(13a) conserved the cytotoxicity. We combined these data to design
the affinity probe 13e (Scheme 2) that entails a 2-phenylbenzofuran
substituted by a linker based on 7t.
Western blot analysis of pull-down assays gave positive results
with antibodies targeting mTOR but not other proteins that belong
to the same signaling pathway, such as Akt, Rictor, Raptor, PI3K and
PDK1 (pyruvate dehydrogenase lipoamide kinase isozyme 1)
(Fig. 3a). No unspecific binding was observed with Affi-Gel 10 beads
containing only the linker as a negative control. Our lead com-
pounds 7s and 13e were shown to inhibit in a dose dependent
manner the phosphorylation of S6 ribosomal protein, which is a
target of the mTOR complex 1 (mTORC1) (Fig. 3b and c). In addition,
the phosphorylation of AKT (Ser473) was increased after exposure
to high doses of 13e, suggesting a specific inhibition of mTORC1 [9].
In comparison with clinical mTOR inhibitor, 7r and 13a dis-
played a cytotoxicity in the same range of concentration than
everolimus and OZI027 [10] on a panel of human cancer cell lines,
but with a different profile of selectivity (Table 2), suggesting that
this new class of anticancer agents may find clinical applications
that are complementary to those of everolimus and OZI027.
2.1. Chemical synthesis
The synthesis of 2-benzyl-benzofuran derivatives started from
the condensation of phenol 2 with chloroacetophenone 3 to afford
the 2-benzoyl-5-methoxybenzofurane 4 [6]. Subsequent reduction
of 4 by NaBH3CN and TMSCl [7] and demethylation by BBr3 afforded
the key intermediate 6 (Scheme 1). A Mannich reaction on phenol 6
with various secondary amines afforded the expected adducts 7. A
Mannich condensation was also performed on phenol 8 obtained
by demethylation of 4, to generate 10, which upon a reduction with
NaBH4 afforded the alcohol 11.
The synthesis of 2-phenyl-benzofurane 13a was performed, in
one step, from benzoquinone as described by Lyubchanskaya [8]
(Scheme 2). Next, a Mannich reaction with amines 9aeb afforded,
as previously, the expected adducts 13aeb. To detect, by pull-down
experiments, the molecular target of these compounds, we
saponified 13b and conjugated the cognate carboxylic acid with an
amino linker using ECDI/HOBT in CH2Cl2 at 0 ꢀC to afford the azide
13c that was hydrogenated to the amine 13d and conjugated to Affi-
Gel 10.
2.2. Biological results
2.2.1. In vitro cytotoxicity
First, we examined the antiproliferative effects of 1 on a panel of
human colon (HT29, HCT116, Colo205, HCC2998), ovarian
(OVCAR3, IGROV1), lung (HOP62, HOP92), breast (MCG7), mela-
noma (MDA-MB-231), prostate (DU145) and head and neck
(SQ20B) cancer cell lines to verify its cytotoxicity and to identify a
cell line suitable to assay its derivatives and analogs (Fig. 2).
HCC2998, SQ20B and HOP92 cell lines were found to be particularly
3. Conclusion
In conclusion, we performed the first hit to lead optimization of
1 and demonstrated that the replacement of the dimethylamine
and benzyl moieties by a 4-piperidino-piperidine and a phenyl
significantly improves cytotoxicity. We also demonstrated that this
new class of compounds interacts with the complex mTORC1 to
inhibit its activity. The optimization of 13a is currently under
investigation to increase its antiproliferative potency.
sensitive to 1 cytotoxicity (IC50 z 40 mM).
Based on these data, the head and neck cancer cell line SQ20B
was chosen to test the cytotoxicity of synthesized compounds.
These cells have a constitutively active mutation in epidermal
growth factor receptor (EGFR) resulting in a constitutive activation
of Akt. They are used as a cellular model of radioresistant cancers
that are weakly sensitive to cisplatine.
4. Experimental protocol
Our SAR study began by investigating the effects of substitutions
on the amine moiety (Table 1). Replacement of the dimethylamino
moiety of 1 by a bulkier and more hydrophobic diethylamino 7a, N-
methyl-cyclohexylamino (7b), tetrahydroisoquinolino (7c) or N-
methyl-benzylamino (7d) slightly increased cytotoxicity. Substitu-
tion by a N,O-dimethylhydroxylamino group (7e) was well toler-
ated, but introduction of a hydroxyl (7feh) led to a significant loss
of potency. Inclusion of the amine in a morpholine ring (7i) slightly
increased the activity, and the insertion in N-substituted basic
piperazine rings was well allowed (7jem). Interestingly, hydroxyl-
piperidino (7n, o) derivatives were as active as diethylamine 7a.
Introduction of a carboxamide (7p) or a phenyl group (7q) in the
position 4 of the piperidino moiety was detrimental. Gratifyingly, a
major improvement in potency resulted from the substitution by a
4-piperidinopiperidine (7r) or a 4-pyrrolidinopiperidine moiety
(7s).
4.1. Biology
4.1.1. Cell lines
Hepatocarcinoma SK-HEP1, HEPG2, prostate DU145, renal CAKI1
and ovarian OVCAR3 cell lines were obtained from the ATCC
(Rockville, MD). Colon Colo205, HCT116, HT29, and HSCLC HOP62
cell lines were purchased from NCI cell line bank. SQ20B was kindly
provided by Prof. E. Deutsch (Institute Gustave Roussy, France).
ColoR was developed in our laboratory. Cells were grown as
monolayers in RPMI medium supplemented with 10% fetal calf
serum (Invitrogen, Cergy-Pontoise, France), 2 mM glutamine,
100 units/ml penicillin and 100 m
g/ml streptomycin at 37 ꢀC in a
humidified 5% CO2 atmosphere, and regularly checked for the
absence of Mycoplasma.
4.1.2. Cell cytotoxicity assay
Cell survival was determined using the MTT assay (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma,
Saint-Quentin Fallavier, France). The conversion of yellow water-
soluble tetrazolium MTT into purple insoluble formazan is cata-
lyzed by mitochondrial dehydrogenases and used to estimate the
number of viable cells. In brief, cells were seeded in 96-well tissue
culture plates at a density of 2 ꢁ 103 cells/well. After drug exposure,
Fig. 1. Structure of HTS hit 1 [4].

C. Salomé et al. / European Journal of Medicinal Chemistry 74 (2014) 41e49
43
Scheme 1. Synthesis of 1 and its derivatives 7aet, 10 and 11.
Scheme 2. Synthesis of 2-phenylbenzofuran derivatives 13aee.
cells were incubated with 0.4 mg/ml MTT for 4 h at 37 ꢀC. After
incubation, the supernatant was discarded, insoluble formazan
precipitates were dissolved in 0.1 mL of DMSO and the absorbance
was measured at 560 nm by use of a microplate reader (Thermo,
France). Wells with untreated cells or with drug-containing me-
dium without cells were used as positive and negative controls
respectively. Everolimus and OZI027 were purchased from Selleck
Chemicals (Munich, Germany).
4.1.3. Western blot analysis
Cells were lysed in buffer containing 50 mM HEPES (pH 7.6),
150 mM NaCl, 1% Triton X-100, 2 mM sodium vanadate, 100 mM
NaF, and 0.4 mg/ml phenylmethylsulfonyl fluoride. Equal amounts
of protein (30 mg/lane) were subjected to SDS-PAGE and transferred
to nitrocellulose membranes. Membranes were blocked with 5%
milk in 0.05% Tween 20/phosphate-buffered saline and then incu-
bated with the primary antibody overnight. Membranes were then
washed and incubated with the secondary antibody conjugated to
horseradish peroxidase. Bands were visualized by using the
enhanced chemiluminescence Western blotting detection system.
Densitometric analysis was performed under conditions that yiel-
ded a linear response.
Fig. 2. Inhibition of cell proliferation by 1 on various cancer cell lines (IC50, mM). IC50
values were determined from doseeresponse curves of viability assessed by MTT assay
after a 72 h treatment according to the method described under Experimental section.

44
C. Salomé et al. / European Journal of Medicinal Chemistry 74 (2014) 41e49
Table 1 (continued)
Table 1
Cytotoxicity of benzofuran derivatives against SQ20B human cancer cell line (IC50
m
,
Cpd
NR1R2
R3
IC50 (mM)
M).a
10
COPh
17 ꢂ 1.6
13 ꢂ 3.4
11
CH(OH)Ph
13a
Ph
2.8 ꢂ 0.7
Cpd
NR1R2
R3
IC50 (mM)
a
Data are the average of two independent IC50 value determinations.
1
7a
NMe2
NEt2
CH2Ph
CH2Ph
33 ꢂ 2.8
12 ꢂ 4.2
4.1.4. Pull-down assay
7b
7c
CH2Ph
CH2Ph
12 ꢂ 2.2
16 ꢂ 0.6
Sub-confluent SQ20B cells were collected and washed in
phosphate-buffered saline 1ꢁ (PBS). Lysis was performed by
freezing the cells in liquid N2 in 500 mL of lysis buffer (50 mM Trise
HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, protease
inhibitor cocktail 1ꢁ). Cellular debris was removed by centrifuga-
tion at 10,000 ꢁ g for 10 min. Five hundred microgram of total
7d
7e
7f
CH2Ph
CH2Ph
CH2Ph
12 ꢂ 5.0
40 ꢂ 7.4
30 ꢂ 5.7
protein extract was incubated for 10e12 h at 4 ꢀC with 40
mL of
NMeOMe
agarose conjugate 13e. The beads were washed extensively with
lysis buffer and bound-proteins were eluted by SDS sample buffer
(50 mM TriseHCl pH 6.8, 2% SDS, 10% glycerol, 1.4 M b-mercap-
toethanol, and bromophenol blue). Eluted proteins were recovered
from the beads by centrifugation. Total proteins, unbound proteins,
or eluted proteins were resolved by 12%e8% SDS-PAGE and electro-
transferred to nitrocellulose sheets (Schleicher and Schuell, Dassel,
Germany). The membrane was blocked in PBS containing 5% nonfat
dry milk and 0.1% Tween 20 and then incubated with the primary
antibody overnight. After washing, the blots were incubated with
appropriate secondary antibodies. Horseradish peroxidase-
conjugated (HRP) AffiniPure donkey anti-rabbit at 1/10000 (Jack-
son ImmunoResearch) and HRP donkey anti-goat at 1/1000 (Santa
Cruz biotechnology, Santa Cruz, CA) were used. Finally, protein-
antibody complexes were visualized by an enhanced chem-
iluminescence detection system (SuperSignal West Pico, Pierce).
7g
7h
7i
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
27 ꢂ 6.2
>200
N(CH2CH2OH)2
6.5 ꢂ 2.3
18 ꢂ 4.7
36 ꢂ 5.1
21 ꢂ 6.7
7j
7k
7l
4.2. Chemistry
All reagents and solvents for synthesis were purchased from
SigmaeAldrich, Fluka, or Acros and used without further purifica-
tion. Column chromatography was carried out on silica gel 60
(Merck, 70e230 mesh). 1H NMR spectra at 300 MHz or 400 MHz
and 13C NMR spectra at 75 MHz or 100 MHz were recorded with
DPX 300 SY Brucker spectrometers with the deuterated solvent as
the lock and residual solvent as the internal reference. All chemical
shift values and coupling constants J are quoted in ppm and in Hz,
respectively. Infrared spectra were recorded using a PerkineElmer
881. Analytical RP-HPLC-MS was performed using a C18 column
7m
7n
CH2Ph
CH2Ph
49 ꢂ 6.4
11 ꢂ 2.5
7o
7p
CH2Ph
CH2Ph
13 ꢂ 2.9
85 ꢂ 11
(30 mm ꢁ 1 mm; 1.9
mm) using the following parameters: (1) the
solvent system A (acetonitrile) and B (0.05% TFA in H2O); (2) the
linear gradient t ¼ 0 min with 98% B, t ¼ 5 min with 5% B, t ¼ 6 min
with 5% B, t ¼ 7 min with 98% B, and t ¼ 9 min with 98% B; (3) flow
rate of 0.3 mL/min; (4) column temperature 50 ꢀC; (5) ratio of
products determined by integration of spectra recorded at 210 or
254 nm; (6) ionization mode ESI. Melting points (mp [ꢀC]) were
taken on samples in open capillary tubes.
7q
7r
CH2Ph
CH2Ph
>200
3.0 ꢂ 0.9
7s
7t
CH2Ph
CH2Ph
3.0 ꢂ 1.1
14 ꢂ 0.9
4.2.1. General procedure for the Mannich reaction (method A)
The secondary amine (2 eq) was added to an EtOH (0.1e0.2 M)
solution of 5-hydroxy-benzofuran derivative (1 eq) and formalde-
hyde (37% in water, 2 eq). The solution was stirred at reflux (3 he
16 h) and concentrated under vacuum. The residue was purified by

C. Salomé et al. / European Journal of Medicinal Chemistry 74 (2014) 41e49
45
Fig. 3. Binding and inhibition of mTORC1. (a) Pull-down assay results demonstrating an interaction with mTOR or with a protein that interacts with mTOR. Western blot analysis of
proteins pulled down from the negative control-coupled Affi-Gel beads (NC), 13d-coupled Affi-Gel beads (13e) and the crude protein extract (ꢃ). (b) Inhibition of the phosphor-
ylation of pS6 (mTORC1 substrate) and activation of p-AKT (Ser473) in SQ20B cells, exposed to 7s or 13a (0e10
Right panel: quantification of pS6 and pAKT expression relative to actin levels (c) Akt/mTOR signaling.
mM) determined by immunoblotting of whole-cell lysates (left panel).
flash column chromatography on silica gel or by recrystallization in
5H), 7.09 (s, 2H), 3.84 (s, 3H); 13C NMR (100 MHz, CDCl3)
d 156.7,
EtOH to obtain a white solid.
153.0, 137.3, 132.8, 129.5, 128.5, 127.5, 118.5, 116.4, 113.2, 103.9, 55.9;
IR (neat): 3119, 3058 (CeH ar), 2960, 2929, 2833 (CeH), 1644, 1598,
1576, 1547, 1470, 1445, 1224 (CeO methoxy), 1161, 970, 898, 831,
4.2.2. 2-Benzyl-4-((dimethylamino)methyl)benzofuran-5-ol (1)
729, 697 cme1
.
Using Method A with formaldehyde (68
mL, 0.90 mmol),
benzofuran 6 (100 mg, 0.45 mmol) and dimethylamine (40 mg,
0.90 mmol), 1 (92 mg, 72%) was obtained after recrystallization in
4.2.4. 2-Benzyl-5-methoxybenzofuran (5)
EtOH as a white solid; 1H NMR (300 MHz, CDCl3)
d
7.36e7.27 (m,
5H), 7.21 (d, J ¼ 9.1 Hz, 1H), 6.75 (d, J ¼ 9.1 Hz, 1H), 6.24 (s, 1H), 4.08
(s, 2H), 3.76 (s, 2H), 2.35 (s, 6H); 13C NMR (75 MHz, CDCl3)
158.4,
A solution of 4 (7.63 g, 30.3 mmol) and TMSCl (23 mL,
181.5 mmol) in anhydrous CH3CN (100 mL) was cooled to 0 ꢀC and
NaBH3CN (11.41 g, 181.5 mmol) was added in one time. The mixture
was stirred at room temperature (60 h). The solution was filtered
through a pad of celite and the pad was washed with CH2Cl2
(200 mL). The organic layer was washed with a 2 N aqueous HCl
solution (50 mL), brine (50 mL), dried (MgSO4) and concentrated
under vacuum. The residue was purified by flash chromatography
on silica gel using heptane/CH2Cl2 (1/1) as a solvent. Benzofuran 5
was obtained as yellow oil (6.49 g, 90%): 1H NMR (200 MHz, CDCl3)
d
153.8, 149.0, 137.4, 129.1, 128.8, 128.1, 126.9, 112.7, 111.7, 110.4, 101.2,
59.5, 44.8, 35.3; IR (neat): 3029, 2995, 2953, 2915, 2850, 1611, 1494,
1450, 1423, 1228, 997, 972, 823, 737, 694; LCeMS (M þ Hþ)(ESIþ)
282.10 [M þ Hþ] (calcd for C18H19NO2Hþ 282.10).
4.2.3. (5-Methoxybenzofuran-2-yl)(phenyl)methanone (4)
2-Hydroxy-5-methoxybenzaldehyde (5) (10 g, 65.7 mmol) was
d
7.22e7.16 (m, 6H), 6.87e6.70 (m, 2H), 6.23 (s, 1H), 4.0 (s, 2H), 3.73
added to a 2-butanone solution (400 mL) of a-chloroacetophenone
(s, 3H).
6 (10.16 g, 65.7 mmol) and K2CO3 (10.9 g, 78.8 mmol). The mixture
was stirred at reflux (5 h). Then, the volatiles were evaporated and
the residue was dissolved in EtOAc. The organic layer was washed
with a 2 N aqueous NaOH solution (2 ꢁ 50 mL), brine (2 ꢁ 50 mL),
dried (MgSO4) and concentrated under vacuum. The brown solid
was recrystallized in EtOH to obtain brown crystals (16.20 g, 98%):
4.2.5. 2-Benzylbenzofuran-5-ol (6)
A 1 M CH2Cl2 solution of BBr3 (35.9 mL, 35.9 mmol) was added,
at ꢃ78 ꢀC, to an anhydrous CH2Cl2 (100 mL) mixture of 5 (3.43 g,
14.4 mmol). The medium was stirred at room temperature (16 h).
H2O (20 mL) was added and the layers were separated. The organic
1H NMR (400 MHz, CDCl3)
d
8.03 (d, J ¼ 8.4 Hz, 2H), 7.61e7.45 (m,

46
C. Salomé et al. / European Journal of Medicinal Chemistry 74 (2014) 41e49
Table 2
(120 mg, 0.90 mmol), 7c (92 mg, 55%) was obtained after recrys-
tallization in EtOH/EtOAc as white crystals: Rf (pentane/Et2O; 8/2)
Cytotoxicity of 7r, 13a, everolimus and OZI027 against human cancer cell lines (IC50
,
m
M).a
0.36; 1H NMR (400 MHz, CDCl3)
d
¼ 7.39e7.11 (m, 10H), 7.02 (d,
Cell lines
7r
13a
Everolimus
OZI027
J ¼ 7.8 Hz, 1H), 6.75 (d, J ¼ 8.8 Hz, 1H), 6.28 (s, 1H), 4.09 (s, 2H), 4.0
(s, 2H), 3.79 (s, 2H), 2.98 (t, J ¼ 5.3 Hz, 2H), 2.89 (m, 2H); 13C NMR
Colo205 (colon)
ColoR (colon)
HCT116 (colon)
HT29 (colon)
CAKI1 (kidney)
SK-HEP1 (liver)
DU145 (prostate)
OVCAR3 (ovary)
HOP62 (lung)
3.1 ꢂ 0.5
3.7 ꢂ 0.5
12 ꢂ 1.9
5.1 ꢂ 1.0
6.9 ꢂ 1.7
11 ꢂ 2.1
20 ꢂ 1.6
19 ꢂ 3.3
11 ꢂ 3.4
3.0 ꢂ 0.9
6.3 ꢂ 2.2
18 ꢂ 1.5
57 ꢂ 4.2
45 ꢂ 5.9
26 ꢂ 2.0
42 ꢂ 7.7
13 ꢂ 1.8
22 ꢂ 3.4
54 ꢂ 6.1
2.8 ꢂ 0.7
20 ꢂ 4.7
8.7 ꢂ 1.9
12 ꢂ 5.1
15 ꢂ 2.8
14 ꢂ 2.5
12 ꢂ 1.8
8.0 ꢂ 2.1
16 ꢂ 2.6
19 ꢂ 3.3
5.5 ꢂ 1.6
5.8 ꢂ 0.6
5.8 ꢂ 1.4
5.6 ꢂ 1.0
55 ꢂ 4.2
1.9 ꢂ 0.3
3.2 ꢂ 1.3
4.1 ꢂ 0.8
8.1 ꢂ 3.3
41 ꢂ 7.2
1.3 ꢂ 0.4
(100 MHz, CDCl3)
d 158.4, 153.5, 149.0, 137.2, 133.6, 133.4, 129.0,
128.7, 128.6,128.4,128.3,126.8,126.6,125.9,112.7,110.8,110.4,101.1,
57.6, 55.6, 50.2, 35.2, 28.7; IR (neat): 3027 (CeH ar), 2799 (CeH);
1601, 1583, 1494 (C]C ar), 1249 (CeO phenol), 1226, 793; 740, 701;
LCeMS (M þ Hþ)(ESIþ) 370.1 [M þ Hþ] (calcd for C25H23NO2Hþ
370.2).
SQ20B (head and neck)
4.2.9. 2-Benzyl-4-((benzyl(methyl)amino)methyl)benzofuran-5-ol
(7d)
a
Data are the average of two independent IC50 value determinations.
Using Method A with formaldehyde (68
mL, 0.90 mmol),
benzofuran 6 (100 mg, 0.45 mmol) and N-methyl-benzylamine
(109 mg, 0.90 mmol), 7d (140 mg, 44%) was obtained as an oil after
purification by column chromatography on silica gel using pentane/
t-BuOMe (9/1) as solvent: Rf (pentane/Et2O; 1/1) 0.77; 1H NMR
layer was washed with H2O (20 mL). The aqueous layers were
combined and washed with CH2Cl2 (2 ꢁ 20 mL). The organic layers
were combined, dried (MgSO4) and concentrated under vacuum.
The residue was purified by flash chromatography on silica gel
using heptane/EtOAc (8/2) as a solvent to obtain 6 [11] as a yellow
oil (2.70 g, 84%): Rf 0.22 (heptane/EtOAc; 8/2); 1H NMR (300 MHz,
(400 MHz, CDCl3)
(d, J ¼ 8.0 Hz, 1H), 6.15 (s, 1H), 3.99 (s, 2H), 3.76 (s, 2H), 3.54 (s, 2H),
2.19 (s, 3H); 13C NMR (100 MHz, CDCl3)
158.3, 153.4, 148.9, 137.2,
d
7.28e7.15 (m, 10H), 7.12 (d, J ¼ 10.7 Hz, 1H), 6.67
d
CDCl3)
d
7.29e7.16 (m, 6H), 6.81 (d, J ¼ 2.6 Hz, 1H), 6.64 (d,
136.8,129.4,128.9,128.6,128.5,128.1,127.6,126.8,112.6,111.5,110.3,
101.1, 61.5, 57.2, 41.5, 35.2; IR (neat): 3027 (CeH ar), 2838 (CeH),
1611, 1493 (C]C ar), 1445 (CeH CH3), 1229 (CeO phenol), 1029,
994, 957, 853, 792, 731, 698; LCeMS (M þ Hþ)(ESIþ) 358.1 [M þ Hþ]
(calcd for C24H23NO2Hþ 358.2).
J ¼ 8.6 Hz, 1H), 6.20 (s, 1H), 4.58 (s, 1H), 4.0 (s, 2H); 13C NMR
(100 MHz, CDCl3)
d 158.9, 151.3, 150.1, 137.2, 129.7, 128.9, 128.6,
126.8, 111.8, 111.2, 105.6, 103.3, 35.1; IR (neat): 3166 (OH phenol),
2920, 2853, 1621, 1599, 1494, 1469, 1194, 1170, 950, 899, 837, 741,
707 cme1; LCeMS (M þ Hþ)(ESIþ) 225.10 [M þ Hþ] (calcd for
C
15H12O2Hþ 225.10).
4.2.10. 2-Benzyl-4-((methoxy(methyl)amino)methyl)benzofuran-5-
ol (7e)
4.2.6. 2-Benzyl-4-((diethylamino)methyl)benzofuran-5-ol (7a)
Using Method A with formaldehyde (68 L, 0.90 mmol),
benzofuran 6 (100 mg, 0.45 mmol) and diethylamine (66 mg,
0.90 mmol), 7a (90 mg, 65%) was obtained as an oil after purifica-
tion by column chromatography on silica gel using pentane/Et2O
(8/2) as solvent: Rf (pentane/Et2O; 1/1) 0.43; 1H NMR (400 MHz,
Using Method A with formaldehyde (68
mL, 0.90 mmol),
m
benzofuran 6 (100 mg, 0.45 mmol) and N,O-dimethylhydroxyl-
amine (55 mg, 0.90 mmol), 7e (68 mg, 50%) was obtained as an oil
after purification by column chromatography on silica gel using
Pentane/EtOAc (9/1) as solvent: Rf (pentane/EtOAc; 9/1) 0.33; 1H
NMR (400 MHz, CDCl3)
J ¼ 8.8 Hz, 1H), 6.31 (s, 1H), 4.2e3.8 (br s, 3H), 4.10 (s, 2H), 3.59 (s,
2H), 2.71 (s, 3H); 13C NMR (100 MHz, CDCl3)
158.5, 152.3, 149.2,
d 8.85 (s, 1H), 7.37e7.25 (m, 6H), 6.81 (d,
CDCl3)
d
7.27e7.17 (m, 5H), 7.09 (d, J ¼ 8.6 Hz, 1H), 6.62 (d,
J ¼ 8.6 Hz, 1H), 6.13 (s, 1H), 3.98 (s, 2H), 3.78 (s, 2H), 2.55 (q,
d
J ¼ 7.1 Hz, 4H), 1.03 (t, J ¼ 7.1 Hz, 6H); 13C NMR (100 MHz, CDCl3)
137.2, 128.9, 128.6, 126.8, 112.8, 111.5, 110.7, 101.3, 60.2, 59.4, 44.2,
35.2; IR (neat): 3028 (CeH ar), 2892 (CeH); 1603, 1494, 1439 (CeH
CH3), 1230 (CeO phenol), 1074, 865, 789; 735, 702; LCeMS (Me
NH(CH3)(OCH3))(ESIþ) 237.1 [M ꢃ NH(CH3)(OCH3)]þ (calcd for
d
158.2, 153.8, 148.8, 137.3, 128.9, 128.6, 126.7, 112.6, 111.7, 110.0,
101.0, 53.5, 46.7, 35.2, 11.4; IR (neat): 2967, 2925, 2847 (CeH), 1612,
1494 (C]C ar), 1443 (CeH CH3), 1229 (CeO phenol), 1195, 1135,
958, 792, 771; 738, 702; LCeMS (M þ Hþ)(ESIþ) 310.1 [M þ Hþ]
(calcd for C20H23NO2Hþ 310.2).
C
16H13Oþ2 237.1).
4.2.11. 2-Benzyl-4-(((2-hydroxyethyl)(methyl)amino)methyl)
benzofuran-5-ol (7f)
4.2.7. 2-Benzyl-4-((cyclohexyl(methyl)amino)methyl)benzofuran-
5-ol (7b)
Using Method A with formaldehyde (68
mL, 0.90 mmol),
Using Method A with formaldehyde (68
mL, 0.90 mmol),
benzofuran 6 (100 mg, 0.45 mmol) and N-methyl-2-aminoethanol
(68 mg, 0.90 mmol), 7f (36 mg, 27%) was obtained as an oil after
purification by column chromatography on silica gel using EtOAc as
benzofuran 6 (100 mg, 0.45 mmol) and N-methyl-cyclohexylamine
(102 mg, 0.90 mmol), 7b (38 mg, 24%) was obtained as an oil after
purification by column chromatography on silica gel using pentane/
EtOAc (9/1) as eluant: Rf (Pentane/EtOAc; 8/2) ¼ 0.43; 1H NMR
solvent: 1H NMR (400 MHz, CDCl3)
d 7.37e7.25 (m, 5H), 7.21 (d,
J ¼ 8.7 Hz, 1H), 6.74 (d, J ¼ 8.7 Hz, 1H), 6.23 (s, 1H), 5.52e5.01 (br m,
(300 MHz, CDCl3)
d
7.39e7.27 (m, 5H), 7.19 (d, J ¼ 8.9 Hz, 1H), 6.72
1H), 4.07 (s, 2H), 3.87 (s, 2H), 3.8 (t, J ¼ 5.5 Hz, 2H), 2.7 (t, J ¼ 5.5 Hz,
2H), 2.38 (s, 3H); 13C NMR (100 MHz, CDCl3)
d 158.5, 153.4, 149.1,
(d, J ¼ 8.9 Hz, 1H), 6.23 (s, 1H), 4.08 (s, 2H), 3.93 (s, 2H), 2.6 (m, 1H),
2.3 (s, 3H), 1.93e0.91 (m, 10H); 13C NMR (75 MHz, CDCl3)
d
157.9,
137.3, 129.1, 128.7, 128.2, 126.9, 112.8, 111.7, 110.5, 101.2, 59.9, 59.1,
58.0, 42.2, 35.3; IR (neat): 3380 (OH); 2980 (CeH); 1610, 1494 (C]C
ar); 1436 (CeH), 1420; 1228 (CeO phenol); 792; 734, 702; LCeMS
(M þ Hþ)(ESIþ) 312.2 [M þ Hþ] (calcd for C19H21NO3Hþ 312.1).
153.7, 148.5, 137.0, 128.7, 128.4, 127.7, 126.5, 112.3, 111.4, 109.7, 100.8,
62.1, 53.7, 36.4, 34.9, 27.9, 25.8, 25.5; IR (neat): 2926 (CeH); 2852
(CeH CH3); 1612, 1494 (C]C ar), 1449 (CeH CH2), 1230 (CeO
phenol), 796; 734; 702 (CeH); LCeMS (M þ Hþ)(ESIþ) 350.2
[M þ Hþ] (calcd for C23H27NO2Hþ 350.2).
4.2.12. 2-Benzyl-4-((cyclohexyl(2-hydroxyethyl)amino)methyl)
benzofuran-5-ol (7g)
4.2.8. 2-Benzyl-4-((3,4-dihydroisoquinolin-2(1H)-yl)methyl)
In a sealed tube, 6 (200 mg, 0.90 mmol), 2-(cyclohexylamino)
benzofuran-5-ol (7c)
ethanol (390 mL, 4.5 mmol) and formaldehyde (37% in water, 363 mL,
Using Method A with formaldehyde (68
benzofuran 6 (100 mg, 0.45 mmol) and tetrahydroisoquinoline
m
L, 0.90 mmol),
4.5 mmol) were stirred at room temperature (6 d) in dioxane
(2 mL). The volatiles were evaporated and the crude residue was

C. Salomé et al. / European Journal of Medicinal Chemistry 74 (2014) 41e49
47
purified by flash chromatography on silica gel using pentane/Et2O
4.2.16. 2-Benzyl-4-((4-benzylpiperazin-1-yl)methyl)benzofuran-5-
ol (7k)
Using Method A with formaldehyde (68
(1/1) as solvent. Concentrated HCl (2 mL, 23.5 mmol) was added to
a BuOH/H2O (10/1) solution of the purified residue. The solution
was stirred at reflux (4 h). The mixture was concentrated under
vacuum and the aqueous layer was basified with a saturated
Na2CO3 aqueous solution and washed with Et2O (3 ꢁ 30 mL). The
organic layers were combined dried (MgSO4), and concentrated
under vacuum. The crude residue was purified by flash chroma-
tography on silica gel using CH2Cl2/MeOH/Et3N (9/1/0.1) as solvent
mL, 0.90 mmol),
benzofuran 6 (100 mg, 0.45 mmol) and N-benzyl-piperazine
(158 mg, 0.90 mmol), 7k (72 mg, 39%) was obtained after recrys-
tallization in EtOH as white crystals: Rf (EtOAc/heptane; 1/1) 0.38;
1H NMR (400 MHz, CDCl3)
d
7.38e7.21 (m, 10H), 7.20 (d, J ¼ 8.8 Hz,
1H), 6.73 (d, J ¼ 8.8 Hz, 1H), 6.24 (s, 1H), 4.08 (s, 2H), 3.83 (s, 2H),
3.56 (s, 2H), 2.61e2.58 (br m, 8H); 13C NMR (100 MHz, CDCl3)
to obtain 56 mg of 7g (32%); 1H NMR (400 Hz, CDCl3)
d
7.36e7.14
d 158.3, 153.3, 148.9, 137.2, 129.1, 128.9, 128.6, 128.3, 128.2, 127.2,
(m, 5H), 7.17 (d, J ¼ 8.7 Hz, 2H), 6.73 (d, J ¼ 8.7 Hz, 1H), 6.23 (s, 1H),
4.08 (s, 2H), 4.00 (s, 2H), 3.74e3.69 (m, 3H), 2.78e2.70 (m, 3H),
1.94e1.80 (m, 2H), 1.36e1.15 (m, 7H); 13C NMR (75 MHz, CDCl3)
127.2, 126.7, 112.5, 110.7, 110.3, 101.3, 62.8, 57.8, 52.8, 52.7, 35.2; IR
(neat): 3029 (CeH ar); 2810 (CeH); 1600, 1494, 1443 (C]C ar);
1227 (CeO phenol); 1130, 952, 795, 783; 735, 698 (CeH); LCeMS
(M þ Hþ)(ESIþ) 413.2 [M þ Hþ] (calcd for C27H28N2O2Hþ 413.2).
d
158.7, 153.9, 149.4, 137.6, 129.4, 129.0, 128.3, 127.2, 113.2, 112.4,
110.5, 101.4, 61.1, 60.7, 53.2, 51.6, 35.6, 28.4, 26.2, 26.0; IR (neat):
3028 (CeH ar), 2921 (CH2), 1603, 1494 (C]C ar), 1434 (CeH CH3),
1373 (NeH amine), 1232 (CeO phenol), 1174, 953, 791,732, 699;
LCeMS (M þ Hþ)(ESIþ) 268.1 [M þ Hþ] (calcd for C17H17NO2Hþ
268.1).
4.2.17. 2eBenzyl-4-((4-(2-(2-hydroxyethoxy)ethyl)piperazin-1-yl)
methyl)benzofuran-5-ol (7l)
Using Method A with formaldehyde (68
mL, 0.90 mmol),
benzofuran 6 (100 mg, 0.45 mmol) and 1-[2-(2-hydroxyethoxy)
ethyl]piperazine (157 mg, 0.90 mmol), 7l (90 mg, 65%) was ob-
tained as an oil after purification by column chromatography on
silica gel using EtOAc/MeOH (8/2) as solvent; 1H NMR (400 MHz,
4.2.13. 2,20-(((2-Benzyl-5-hydroxybenzofuran-4-yl)methyl)
azanediyl)diethanol (7h)
CDCl3)
(s, 2H), 3.84 (s, 2H), 3.74e3.62 (m, 6H), 2.89e2.06 (br m, 10H), 13
NMR (100 MHz, CDCl3) 158.6, 153.3, 149.1, 137.3, 129.1, 128.8,
d
7.38e7.19 (m, 6H), 6.72 (d, J ¼ 8.8 Hz, 1H), 6.23 (s, 1H), 4.08
Using Method A with formaldehyde (68
mL, 0.90 mmol),
C
benzofuran 6 (100 mg, 0.45 mmol) and diethanolamine (95 mg,
0.90 mmol), 7h (32 mg, 21%) was obtained as a colorless oil after
purification by column chromatography on silica gel using EtOAc/
MeOH (9/1): Rf (EtOAc/MeOH; 9/1) 0.3; 1H NMR (300 MHz, CDCl3)
d
128.5, 127.0, 112.7, 110.9, 110.5, 101.3, 72.6, 67.6, 62.2, 57.9, 5, 52.53,
35.3.
d
7.37e7.25 (m, 5H), 7.18 (d, J ¼ 8.7 Hz, 1H), 6.73 (d, J ¼ 8.7 Hz, 1H),
4.2.18. 4-((2-Benzyl-5-hydroxybenzofuran-4-yl)methyl)piperazine-
1-carbaldehyde (7m)
6.24 (s, 1H), 5.55e5.15 (br m, 3H), 4.14 (s, 2H), 3.96 (s, 2H), 3.75 (t,
J ¼ 5.1 Hz, 4H), 2.77 (t, J ¼ 5.1 Hz, 4H); 13C NMR (75 MHz, CDCl3)
Using Method A with formaldehyde (68
mL, 0.90 mmol),
d
158.9, 153.2, 149.4, 137.5, 129.3, 129.0, 128.7, 127.2, 113.2, 112.5,
benzofuran 6 (100 mg, 0.45 mmol) and N-formyl-piperazine
(103 mg, 0.90 mmol), 7m (135 mg, 50%) was obtained as a white
solid after purification by column chromatography on silica gel
using pentane/EtOAc (1/1) as solvent: Rf (pentane/EtOAc; 1/1) 0.17;
110.8, 101.4, 60.5, 56.7, 56.0, 35.5; IR (neat): 3270 (OH), 2917, 2849
(CeH), 1602, 1574, 1567, 1556 (C]C ar), 1434 (CeH CH2), 1227 (CeO
phenol), 1070 (OH), 956, 792; 702; LCeMS (M þ Hþ)(ESIþ) 342.1
[M þ Hþ] (calcd for C20H23NO4Hþ 342.2).
1H NMR (400 MHz, CDCl3)
d 7.96 (s, 1H), 7.28e7.13 (m, 6H), 6.65 (d,
J ¼ 8.8 Hz, 1H), 6.13 (s, 1H), 3.98 (s, 2H), 3.76 (s, 2H), 3.55 (m, 2H),
3.36 (t, J ¼ 5.0 Hz, 2H), 2.49 (s, 4H); 13C NMR (100 MHz, CDCl3)
4.2.14. 2-Benzyl-4-(morpholinomethyl)benzofuran-5-ol (7i)
Using Method A with formaldehyde (68
mL, 0.90 mmol),
d
160.6, 158.7, 152.9, 149.9, 137.1, 128.9, 128.6, 128.4, 126.8, 112.6,
benzofuran 6 (100 mg, 0.45 mmol) and morpholine (91 mg,
0.90 mmol), 7i (74 mg, 51%) was obtained after recrystallization in
EtOH as a white solid: Rf (pentane/EtOAc; 8/2) ¼ 0.73; 1H NMR
110.7, 110.1, 100.9, 57.8, 53.0, 45.3, 39.7, 35.2; IR (neat): 2960, 2845
(CeH), 1698, 1682 (C]O amide), 1651 (NeH amide), 1600, 1494
(C]C ar), 1434 (CeH CH2), 1269, 1227 (CeO phenol), 1133, 1022,
793, 782; 734, 700; LCeMS (M þ Hþ)(ESIþ) 351.1 [M þ Hþ] (calcd
for C21H22N2O3Hþ 351.2).
(400 MHz, CDCl3)
d 10.5 (br s, 1H), 7.39e7.28 (m, 5H), 7.23 (d,
J ¼ 8.8 Hz, 1H), 6.75 (d, J ¼ 8.8 Hz, 1H), 6.26 (s, 1H), 4.09 (s, 2H), 3.84
(s, 2H), 3.78 (t, J ¼ 4.0 Hz, 4H), 2.61 (br s, 4H); 13C NMR (100 MHz,
CDCl3)
d 158.5, 153.1, 149.0, 137.1, 128.9, 128.6, 128.3, 126.8, 112.6,
4.2.19. 1-((2-Benzyl-5-hydroxybenzofuran-4-yl)methyl)piperidin-
3-ol (7n)
110.5,110.3,101.0, 66.8, 58.3, 53.1, 35.2; IR (neat): 2970, 2852 (CeH),
1600, 1494 (C]C ar), 1454 (CeH CH2), 1227 (CeO phenol), 1113,
858, 794, 783; 735, 700; LCeMS (M þ Hþ)(ESIþ) 324.10 [M þ Hþ]
(calcd for C20H21NO3Hþ 324.10).
Using Method A with formaldehyde (68
mL, 0.90 mmol),
benzofuran 6 (100 mg, 0.45 mmol) and 3-hydroxypiperidine
(91 mg, 0.90 mmol), 7n (43 mg, 28%) was obtained as an oil after
purification by column chromatography on silica gel using EtOAc as
4.2.15. 2-Benzyl-4-((4-methylpiperazin-1-yl)methyl)benzofuran-5-
ol (7j)
solvent: Rf (EtOAc) 0.51; 1H NMR (400 MHz, CDCl3)
d
7.39e7.20 (m,
6H), 6.74 (d, J ¼ 8.8 Hz, 1H), 6.24 (s, 1H), 4.09 (s, 2H), 3.828e3.80 (s,
3H), 2.29e1.27 (m, 9H); RMN 13C (100 MHz, CDCl3)
158.4, 153.2,
Using Method A with formaldehyde (68
mL, 0.90 mmol),
d
benzofuran 6 (100 mg, 0.45 mmol) and N-methylpiperazine (90 mg,
0.90 mmol), 7j (145 mg, 95%) was obtained as a yellow solid after
purification by column chromatography on silica gel using EtOAc/
MeOH (9/1) as solvent: Rf (EtOAc/MeOH; 9/1) 0.32; 1H NMR
149.0, 137.2, 128.9, 128.6, 128.2, 126.8, 112.6, 110.9, 110.3, 101.1, 66.8,
60.1, 58.0, 53.1, 35.2, 32.4, 22.7; IR (neat): 3386 (OH), 3027 (CeH ar),
2938, 2808 (CeH), 1610, 1494 (C]C ar), 1441 (CeH CH2), 1347 (OH),
1228 (CeO phenol), 1153, 1107 (OH), 959, 791; 730, 702; LCeMS
(M þ Hþ)(ESIþ) 338.1 [M þ Hþ] (calcd for C21H23NO3Hþ 338.2).
(300 MHz, CDCl3)
(s, 1H), 4.07 (s, 2H), 3.83 (s, 2H), 2.69e2.38 (br m, 8H), 2.31 (s, 3H);
13C NMR (75 MHz, CDCl3)
158.7, 153.6, 149.3, 138.9, 129.3, 129.0,
d
7.38e7.18 (m, 6H), 6.72 (d, J ¼ 8.7 Hz, 1H), 6.25
d
4.2.20. 1-((2-Benzyl-5-hydroxybenzofuran-4-yl)methyl)piperidin-
128.6, 127.1, 112.9, 111.1, 110.7, 101.5, 58.2, 55.3, 53.0, 46.2, 35.5; IR
(neat): 2921, 2849 (CeH), 2801; 1607, 1488 (C]C ar), 1440 (CeH
CH3), 1223 (CeO phenol), 1144, 1132, 806; 738, 700; LCeMS
(M þ Hþ)(ESIþ) 337.2 [M þ Hþ] (calcd for C21H24N2O2Hþ 337.2).
4-ol (7o)
Using Method A with formaldehyde (68
benzofuran 6 (100 mg, 0.45 mmol) and 4-hydroxypiperidine
(91 mg, 0.90 mmol), 7o (28 mg, 18%) was obtained after
mL, 0.90 mmol),

48
C. Salomé et al. / European Journal of Medicinal Chemistry 74 (2014) 41e49
recrystallization in EtOH as white crystals: Rf (pentane/Et2O, 1/1)
0.09; 1H NMR (400 Hz, CDCl3)
7.38e7.20 (m, 6H), 6.74 (d,
J ¼ 8.8 Hz,1H), 6.23 (s,1H), 4.08 (s, 2H), 3.82 (s, 2H), 2.95e2.80 (br s,
2H), 2.31e2.45 (br s, 2H), 1.99e1.94 (m, 2H), 1.72e1.64 (m, 2H); 13
NMR (100 MHz, CDCl3) 160.1, 158.3, 153.4, 148.9, 137.2, 128.9,
128.6,128.1,126.7,112.6,111.0,110.2,101.0, 57.8, 35.2, 34.1; IR (neat):
3306 (OH secondary alcohol), 2944, 2825 (CeH), 1610, 1504, 1494
(C]C ar), 1447 (CeH CH2), 1054; 1228 (CeO phenol), 1054, 1016,
799, 793, 779; 735, 699; LCeMS (M þ Hþ)(ESIþ) 338.1 [M þ Hþ]
(calcd for C21H23NO3Hþ 338.2).
2.03 (m, 3H), 1.91 (d, J ¼ 12.8 Hz, 2H), 1.78e1.60 (m, 6H); 13C NMR
d
(100 MHz, CDCl3) d 158.3, 153.5, 148.9, 137.3, 129.0, 128.6, 128.1,
126.8, 112.7, 111.1, 110.1, 101.1, 61.3, 57.9, 52.0, 51.4, 35.2, 31.3, 23.3.
C
d
4.2.25. 10-((2-Benzyl-5-hydroxybenzofuran-4-yl)methyl)-N-(2-(2-
hydroxyethoxy)ethyl)-[1,40-bipiperidine]-4-carboxamide (7t)
Using Method A with formaldehyde (68
mL, 0.90 mmol),
benzofuran 6 (100 mg, 0.45 mmol) and N-(2-(2-hydroxyethoxy)
ethyl)-[1,40-bipiperidine]-4-carboxamide (269 mg, 0.90 mol), 7t
(90 mg, 65%) was obtained as an oil after purification by column
chromatography on silica gel using acetone as solvent; 1H NMR
4.2.21. 1-((2-Benzyl-5-hydroxybenzofuran-4-yl)methyl)piperidine-
4-carboxamide (7p)
(400 MHz, CDCl3)
d
7.72 (d, J ¼ 7.6 Hz, 2H), 7.34e7.15 (m, 5H), 6.22
(s, 1H), 5.59 (br t, 1H), 4.08 (s, 2H), 3.81e3.76 (m, 5H), 3.59 (q,
J ¼ 4.8 Hz, 4H), 3.52e3.43 (m, 2H), 3.10 (d, J ¼ 12.0 Hz, 2H), 2.99 (d,
J ¼ 11.6 Hz, 2H), 2.42e2.37 (m,1H), 2.18e2.10 (m, 5H),1.99e1.58 (m,
8H).
Using Method A with formaldehyde (68
mL, 0.90 mmol),
benzofuran 6 (100 mg, 0.45 mmol) and 4-carboxamidepiperidine
(115 mg, 0.90 mmol), 7p (28 mg, 17%) was obtained after recrys-
tallization in EtOH as white crystals: 1H NMR (400 MHz, CDCl3)
d
7.29e7.11 (m, 6H), 6.64 (d, J ¼ 8.8 Hz, 1H), 6.15 (s, 1H), 4.0 (s, 2H),
3.74 (s, 2H), 3.01 (d, J ¼ 11.6 Hz, 2H), 2.20e2.10 (br m, 4H), 1.95e1.68
(m, 5H); 13C NMR (100 MHz, CDCl3)
158.4, 152.9, 148.9, 137.1,
128.9, 128.6, 128.2, 126.7, 112.4, 110.8, 110.2, 101.0, 57.9, 52.6, 39.1,
35.1, 28.6; IR (neat): 3331, 3165 (NeH amide), 2928, 2812 (CeH),
1614 (C]O amide), 1494 (C]C ar), 1443, 1434 (CeH CH2), 1416,
1229 (CeO phenol), 1142, 800, 792, 779; 734, 699; LCeMS
(M þ Hþ)(ESIþ) 365.1 [M þ Hþ] (calcd for C22H24N2O3Hþ 364.2).
4.2.26. 2-Benzoylbenzofuran-5-ol (8)
d
A 1 M CH2Cl2 solution of BBr3 (35.9 mL, 35.9 mmol) was added,
at ꢃ78 ꢀC, to an anhydrous CH2Cl2 (100 mL) mixture of 4 (3.43 g,
14.4 mmol). The medium was stirred at room temperature (16 h).
H2O (20 mL) was added and the layers were separated. The
organic layer was washed with H2O (20 mL). The aqueous layers
were combined and washed with CH2Cl2 (2 ꢁ 20 mL). The organic
layers were combined, dried (MgSO4) and concentrated under
vacuum. The residue was purified by flash chromatography on
silica gel using heptane/EtOAc (8/2) as a solvent to obtain 8 as a
yellow oil (2.70 g, 84%): Rf 0.22 (heptane/EtOAc; 8/2); 1H NMR
4.2.22. 2-Benzyl-4-((4-phenylpiperidin-1-yl)methyl)benzofuran-5-
ol (7q)
Using Method A with formaldehyde (68
mL, 0.90 mmol),
benzofuran (100 mg, 0.45 mmol) and 4-phenylpiperidine
6
(300 MHz, CDCl3)
d
7.89 (d, J ¼ 8.0 Hz, 2H), 7.54e7.32 (m, 5H),
(145 mg, 0.90 mmol), 7q (108 mg, 61%) was obtained after recrys-
tallization in EtOH as white crystals: Rf (pentane/EtOAc; 9/1) 0.32;
6.93e6.98 (m, 2H), 3.66 (s, 1H); 13C NMR (100 MHz, CDCl3)
d
185.0,
153.7, 152.6, 151.0, 137.2, 133.0, 129.4, 128.6, 127.8, 118.6, 117.1,
112.9, 106.8.
1H NMR (400 MHz, CDCl3)
d
7.41e7.23 (m, 11H), 6.79 (d, J ¼ 8.8 Hz,
1H), 6.29 (s, 1H), 4.12 (s, 2H), 3.89 (s, 2H), 3.20 (d, J ¼ 11.6 Hz, 2H),
2.64 (m, 1H), 2.36e2.25 (td, J ¼ 3.2, 11.6 Hz, 2H), 1.96e1.86 (m, 4H);
13C NMR (100 MHz, CDCl3)
d 158.4, 153.6, 148.9, 145.6, 137.3, 129.0,
4.2.27. (4-((1,40-Bipiperidin)-10-ylmethyl)-5-hydroxybenzofuran-2-
128.7, 128.5, 128.2, 126.9, 126.8, 126.4, 112.6, 111.2, 110.2, 101.1, 58.3,
53.9, 42.4, 35.2, 33.4; IR (neat): 3028 (CeH ar), 2928, 2808 (CeH),
1603, 1493 (C]C ar), 1451 (CeH CH2), 1230 (CeO phenol), 1142;
731, 700; LCeMS (M þ Hþ)(ESIþ) 398.2 [M þ Hþ] (calcd for
yl)(phenyl)methanone (10)
Using Method A with formaldehyde (224 mL, 2.52 mmol), 8 [12]
(300 mg, 1.26 mmol) and 4-piperidinopiperidine (424 mg,
2.52 mmol), 10 (500 mg, 95%) was obtained as a white solid after
purification by column chromatography on silica gel using EtOAc/
MeOH (7/3) as solvent; Rf ¼ 0.19 (EtOAc/MeOH 7/3); 1H NMR
C
27H27NO2Hþ 398.2).
4.2.23. 4-((1,40-Bipiperidin)-10-ylmethyl)-2-benzylbenzofuran-5-ol
(7r)
(400 MHz, CDCl3) d 8.07e8.04 (m, 2H), 7.66e7.62 (m,1H), 7.57e7.52
(m, 2H), 7.44 (s, 1H), 7.42 (d, J ¼ 8.8 Hz, 1H), 7.03 (d, J ¼ 8.8 Hz, 1H),
3.91 (s, 2H), 3.03e2.98 (m, 2H), 2.53e2.50 (m, 4H), 2.38e2.31 (m,
1H), 2.20e2.14 (m, 2H), 1.88 (d, J ¼ 12.8 Hz, 2H), 1.71e1.54 (m, 6H),
Using Method A with formaldehyde (68
mL, 0.90 mmol),
benzofuran 6 (100 mg, 0.45 mmol) and 4-piperidopiperidine
(151 mg, 0.90 mmol), 7r (74 mg, 41%) was obtained after recrys-
tallization in MeOH as a white solid: 1H NMR (400 MHz, CDCl3)
1.48e1.43 (m, 2H); 13C NMR (100 MHz CDCl3)
d 184.1, 154.5, 152.8,
150.4, 137.3, 132.8, 129.5, 128.5, 126.5, 118.9, 113.9, 112.7, 111.9, 62.1,
57.7, 53.2, 50.3, 27.9, 26.4, 24.7.
d
7.12e7.04 (m, 5H), 6.96 (d, J ¼ 8.8 Hz, 1H), 6.49 (d, J ¼ 8.8 Hz, 1H),
5.99 (s, 1H), 3.84 (s, 2H), 3.56 (s, 2H), 2.86 (d, J ¼ 10.4 Hz, 2H), 2.27e
1.21 (m, 17H); 13C NMR (100 MHz, CDCl3)
158.3, 153.4, 148.9, 137.2,
d
128.9, 128.6, 128.1, 126.7, 112.6, 111.2, 110.1, 101.1, 62.3, 57.9, 53.1,
50.2, 35.2, 27.9, 26.4, 24.8; IR (neat): 2958, 2933, 2848 (CeH), 2793,
2756; 1613, 1598, 1493 (C]C ar); 1446 (CeH CH2), 1228 (CeO
phenol),1101, 953, 792, 782; 735, 699; LCeMS (M þ Hþ)(ESIþ) 405.3
[M þ Hþ] (calcd for C26H32N2O2Hþ 405.2).
4.2.28. 4-((1,40-Bipiperidin)-10-ylmethyl)-2-(hydroxy(phenyl)
methyl)benzofuran-5-ol (11)
NaBH4 (27 mg, 0.72 mmol) was added at 0 ꢀC to a MeOH (8 mL)
solution of the benzofuran 10 (100 mg, 0.24 mmol). The mixture
was stirred at room temperature (3 h). Water was added cautiously.
The white precipitate was filtered and recrystalized in MeOH to
obtain 92 mg (90%) of 11 as white crystals; 1H NMR (400 MHz,
4.2.24. 2-Benzyl-4-((4-(pyrrolidin-1-yl)piperidin-1-yl)methyl)
benzofuran-5-ol (7s)
CDCl3)
d
7.48e7.45 (m, 2H), 7.39e7.28 (m, 3H), 7.17 (d, J ¼ 8.8 Hz,
Using Method A with formaldehyde (68
m
L, 0.90 mmol),
1H), 6.72 (d, J ¼ 8.8 Hz, 1H), 6.34 (s, 1H), 5.85 (s, 1H), 3.72 (s, 2H),
3.03e2.98 (m, 2H), 2.47e2.42 (m, 4H), 2.27e2.20 (m,1H), 2.05e1.97
(m, 2H), 1.80e1.75 (m, 2H), 1.62e1.54 (m, 6H), 1.42e1.40 (m, 2H);
benzofuran 6 (100 mg, 0.45 mmol) and 4-(pyrrolidin-1-yl)piperi-
dine (139 mg, 0.90 mmol), 7s (116 mg, 66%) was obtained as a white
solid after filtration; 1H NMR (400 MHz, CDCl3)
d
7.34e7.44 (m, 5H),
13C NMR (100 MHz, CDCl3)
d 159.4, 153.6, 149.1, 140.7, 128.6, 128.2,
7.15 (d, J ¼ 8.6 Hz, 1H), 6.69 (d, J ¼ 8.6 Hz, 1H), 6.19 (s, 1H), 4.04 (s,
127.4, 126.8, 113.6, 111.7, 110.6, 101.6, 70.5, 62.2, 57.8, 53.1, 50.2, 27.7,
26.2, 24.7.
2H), 3.76 (s, 2H), 2.99 (d, J ¼ 11.6 Hz, 2H), 2.55e2.50 (m, 4H), 2.16e

C. Salomé et al. / European Journal of Medicinal Chemistry 74 (2014) 41e49
49
4.2.29. 4-((1,40-Bipiperidin)-10-ylmethyl)-2-phenylbenzofuran-5-ol
(13a)
conjugated to Affigel-10 without any further purification: 1H NMR
(400 MHz, CDCl3)
7.30 (m, 2H), 6.93 (s, 1H), 6.80 (d, J ¼ 8.8 Hz, 1H), 3.92 (s, 2H), 3.71e
3.58 (m, 24H), 3.50e3.43 (app q, J ¼ 5.0 Hz, 2H), 3.15 (d, J ¼ 11.5 Hz,
2H), 3.08e2.95 (m, 4H), 2.26e2.14 (m, 6H), 1.93e1.70 (m, 8H); LCe
MS (M þ Hþ)(ESIþ) 740.4 [M þ Hþ] (calcd for C40H60N4O9Hþ 741.4).
d
7.84 (d, J ¼ 7.2 Hz, 2H), 7.48e7.42 (m, 2H), 7.38e
Using Method A with formaldehyde (68
mL, 0.90 mmol),
benzofuran 6 (100 mg, 0.45 mmol) and 4-piperidinopiperidine
(151 mg, 0.90 mmol), 13a (90 mg, 65%) was obtained as an oil af-
ter purification by column chromatography on silica gel using
acetone as solvent; 1H NMR (400 MHz, CDCl3)
d
7.72 (d, J ¼ 7.6 Hz,
2H), 7.34e7.30 (m, 2H), 7.25e7.14 (m, 2H), 6.80 (s, 1H), 6.68 (d,
J ¼ 8.7 Hz, 1H), 3.78 (s, 2H), 3.03 (d, J ¼ 12.0 Hz, 2H), 2.42e2.39 (m,
4H), 2.27e2.21 (m, 1H), 2.08e2.018 (m, 2H),1.76 (d, J ¼ 12.8 Hz, 2H),
1.61e1.46 (m, 6H), 1.36e1.34 (m, 2H); 13C NMR (100 MHz, CDCl3)
Acknowledgment
We are grateful to region Alsace and MNESR for fellowships to
Christophe Salomé and Frédéric Thuaud. We thank also the SATT
Ile-de-France Innov for financial support.
d
156.3, 153.8, 149.0, 130.6, 128.8, 128.6, 128.4, 124.8, 113.7, 111.4,
110.5, 99.1, 62.3, 57.9, 53.2, 50.3, 27.9, 26.4, 24.8.
4.2.30. N-(20-Azido-3,6,9,12,15,18-hexaoxaicosyl)-10-((5-hydroxy-
2-phenylbenzofuran-4-yl)methyl)-(1,40-bipiperidine)-4-
carboxamide (13c)
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.12.020.
Using Method A with formaldehyde (70
mL, 0.840 mmol),
benzofuran 12 (94 mg, 0.42 mmol) and ethyl (1,40-bipiperidine)-4-
carboxylate [13] (151 mg, 0.90 mol), 13b (161 mg, 80%) was ob-
tained as an oil after purification by column chromatography on
silica gel using acetone as solvent. EDCI (25 mg, 0.129 mmol), HOBT
(17 mg, 0.129 mmol) were added to a CH2Cl2 (4 mL) solution of 13b
(54 mg, 0.108 mmol). The solution was stirred at 0 ꢀC (15 min). A
CH2Cl2 (2 mL) solution of 20-azido-3,6,9,12,15,18-hexaoxaicosan-1-
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(400 MHz, CDCl3)
d
7.84 (d, J ¼ 7.3 Hz, 2H), 7.49e7.40 (m, 2H), 7.38e
7.30 (m, 2H), 6.92 (s,1H), 6.80 (d, J ¼ 8.8 Hz,1H), 6.28 (s, 1H), 3.92 (s,
2H), 3.72e3.61 (m, 22H), 3.56 (t, J ¼ 5.0 Hz, 2H), 3.48 (app q,
J ¼ 5.0 Hz, 2H), 3.40 (t, J ¼ 5.3 Hz, 2H), 3.15 (d, J ¼ 11.2 Hz, 2H), 3.02
(d, J ¼ 11.2 Hz, 2H), 2.52e2.09 (m, 6H), 1.94e1.68 (m, 8H); 13C NMR
(100 MHz, CDCl3)
d 174.9, 156.3, 153.7, 149.0, 130.6, 128.8, 128.6,
128.5, 124.8, 113.3, 111.3, 110.5, 99.1, 70.7, 70.7, 70.6, 70.6, 70.5, 70.5,
70.3, 70.2, 70.1, 70.0, 61.8, 57.8, 52.9, 50.7, 48.8, 39.1, 29.7, 28.9, 27.8;
LCeMS (M þ Hþ)(ESIþ) 767.4 [M þ Hþ] (calcd for C40H58N6O9Hþ
767.4).
4.2.31. N-(20-Amino-3,6,9,12,15,18-hexaoxaicosyl)-10-((5-hydroxy-
2-phenylbenzofuran-4-yl)methyl)-(1,40-bipiperidine)-4-
carboxamide (13d)
Pd/C (5%, 1 mg) was added to an EtOH (5 mL) solution of 13c
(5 mg, 0.01 mmol). The mixture was stirred at room temperature and
under 40 psi of hydrogen (16 h). The mixture was filtered on a pad of
celite. The pad was washed with EtOH and the filtrate was evapo-
rated to obtain 5 mg (quant.) of 13d as a colorless oil which was