Synthesis and antiproliferative activity of benzofuran-based analogs of cercosporamide against non-small cell lung cancer cell lines

Article abstract of DOI:10.1016/j.ejmech.2013.09.013

A novel series of 3-methyl-1-benzofuran derivatives were synthesized and screened in vitro for their antiproliferative activity against two human NSCLC cell lines (NSCLC-N6 mutant p53 and A549 wild type p53). Most promising compounds presented a structural analogy with the west part of cercosporamide, a natural product of biological interest. In particular, compounds 10, 12 and 31 showed cytotoxic activities at micromolar concentrations (IC₅₀ < 9.3 μM) and compounds 13, 18 and 32 displayed moderate IC₅₀ values (25-40 μM).

Full text of DOI:10.1016/j.ejmech.2013.09.013

European Journal of Medicinal Chemistry 69 (2013) 823e832
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antiproliferative activity of benzofuran-based analogs of
cercosporamide against non-small cell lung cancer cell lines
Marc-Antoine Bazin a, Lizeth Bodero a, Christophe Tomasoni b, Bénédicte Rousseau b,
,
Christos Roussakis b, Pascal Marchand a
*
a Université de Nantes, Nantes Atlantique Universités, Laboratoire de Chimie Thérapeutique, Cibles et Médicaments des Infections et du Cancer, IICiMed EA
1155, UFR des Sciences Pharmaceutiques et Biologiques, 1 rue Gaston Veil, 44035 Nantes, France
b Université de Nantes, Nantes Atlantique Universités, Département Cancer Pulmonaire et Cibles Moléculaires, Cibles et Médicaments des Infections et du
Cancer, IICiMed EA 1155, UFR des Sciences Pharmaceutiques et Biologiques, 1 rue Gaston Veil, 44035 Nantes, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of 3-methyl-1-benzofuran derivatives were synthesized and screened in vitro for their
antiproliferative activity against two human NSCLC cell lines (NSCLC-N6 mutant p53 and A549 wild type
p53). Most promising compounds presented a structural analogy with the west part of cercosporamide, a
natural product of biological interest. In particular, compounds 10, 12 and 31 showed cytotoxic activities
Received 16 July 2013
Received in revised form
3 September 2013
Accepted 5 September 2013
Available online 18 September 2013
at micromolar concentrations (IC50 < 9.3
values (25e40 M).
m
M) and compounds 13, 18 and 32 displayed moderate IC50
m
Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords:
Benzofuran
Cercosporamide
Antiproliferative activity
NSCLC
1. Introduction
derivatives represent an important source of antiproliferative
products. Sugano et al. reported that 4-hydroxy-3-methyl-6-
phenylbenzofuran-2-carboxylic acid ethyl ester derivatives
showed selective and potent cytotoxicity against a tumorigenic cell
line [7]. Other previous studies by Hranjec et al. have shown the
antiproliferative effects of benzofuran-2-carboxamides on tumor
cell lines [8]. Thus, as a part of our research program on bioactive
compounds on NSCLC [9,10], we thought that benzofuran-based
analogs of cercosporamide could display antiproliferative activity
on two lung cancer cell lines, NSCLC-N6 and A549.
From a structural point of view, cercosporamide is a usnic acid
analog that contains a dihydroxybenzamide moiety. In this way, 3-
methylbenzofuran derivatives functionalized in the position 2 were
synthesized while keeping a structural analogy with the west part
of cercosporamide (Fig. 1). The in vitro antiproliferative activity of
these compounds was determined by measurement of their IC50
values towards two cancer cell lines.
Lung cancer remains the leading cause of cancer-related deaths
among both men and women worldwide. Non-small cell lung
cancer (NSCLC) accounts for approximately 85% of all cases of lung
cancer [1]. Antiproliferative agents-based chemotherapy (eg
vinorelbine, paclitaxel, carboplatin) improves survival in advanced
NSCLC whereas surgery remains the treatment of choice of early
NSCLC [2]. The development of chemotherapeutic agents display-
ing an original mechanism of action leading to a high activity and a
low toxicity is still needed. Among them, natural products repre-
sent a source of novel antiproliferative agents.
(ꢀ)-Cercosporamide (Fig. 1) is an antifungal natural agent iso-
lated from the phytopathogen fungus Cercosporidium henningsii [3].
Initially this phytotoxin was found to inhibit selectively CaPkc1
kinase [4]. Recent studies showed that cercosporamide had anti-
tumor activity through MAPK-interacting kinase 1 (Mnk1) inhibi-
tion and suppressed both growth of lung metastases [5] and acute
myeloid leukemia precursors [6]. Due to these relevant biological
properties, we were interested in synthesizing cercosporamide
analogs based on benzofuran scaffold. Indeed, benzofuran
2. Results and discussion
2.1. Chemistry
Benzofuran scaffold was built from 3,5-dimethoxyphenol to
achieve a suitable substitution on the benzene ring (Scheme 1).
* Corresponding author. Tel.: þ33 240 412 874; fax: þ33 240 412 876.
E-mail address: pascal.marchand@univ-nantes.fr (P. Marchand).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.09.013

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M.-A. Bazin et al. / European Journal of Medicinal Chemistry 69 (2013) 823e832
step sequence from 3: brominationecyanationehydration
(Scheme 1).
A modification of the ethyl ester moiety of 6 into amides was
O
OH
OH
O
R
HO
HO
H2N
performed via the preparation of the carboxylic acid precursor 7
and activation in the presence of coupling reagents such as 2-
chloro-1-methylpyridinium iodide (CNMPI) and O-(benzotriazol-
OH
O
O
O
H2N
O
O
1-yl)-N,N,N0,N0-tetramethyluronium
tetrafluoroborate
(TBTU)
(Table 1).
(-)-cercosporamide
esters, aldehydes, amides
TBTU [18] was used instead of CNMPI for the synthesis of amides
with less nucleophilic amines and the yields remained acceptable
(55e90%).
Fig. 1. Structure of (ꢀ)-cercosporamide and target compounds derived from 7-
carbamoyl-4,6-dihydroxy-3-methylbenzofuran.
Unsubstituted dicarboxamide derivative 13 was obtained by
aminolysis of ester 6 in a methanolic solution of ammonia in 67%
yield.
Phenol derivative was first acetylated by a Friedel-Crafts-type re-
action using acetyl chloride and boron trichloride, at low temper-
ature in dichloromethane [11]. Applying this reaction conditions, 2-
hydroxy-4,6-dimethoxyacetophenone 1 was obtained in very good
yield (97%) without isolating the O-acetylated by-product. Ketone 1
was then O-alkylated, with ethyl bromoacetate and potassium
carbonate as a base in N,N-dimethylformamide (DMF) to afford
ester 2 which was next cyclized with cesium carbonate in DMF
under microwave radiation [12,13]. Interestingly, ethyl 4,6-
dimethoxy-3-methyl-1-benzofuran-2-carboxylate 3 was obtained
by a one-pot reaction from ketone 1 using the previously described
procedure to synthesize compound 2. In this methodology, ester 2
was not isolated and the benzofuran derivative 3 was provided by
rising the temperature from 70 ꢁC to 100 ꢁC. Finally, the yield was
enhanced from 43% (two steps) to 84% (one step).
Bromine atom was then readily introduced at the position 7 of
the benzofuran ring [14,15], by reaction of compound 3 with
bromine in acetic acid at room temperature for 30 min, to afford
compound 4 in excellent yield. Subsequent cyanation with copper
cyanide in N-methylpyrrolidinone, under microwave radiation at
200 ꢁC, gave nitrile derivative 5 in good yield [16]. Finally, hydration
of nitrile function in sulfuric acid medium provided carboxamide 6.
Interestingly, as recently described in indole series [17], a direct
aminocarbonylation at C-7 of benzofuran derivative 3, using
chlorosulfonyl isocyanate (CSI) as electrophilic reagent, led to
compound 6 in good yield (74%) after acidic hydrolysis of the cor-
responding N-chlorosulfonylcarboxamide intermediate. In com-
parison, compound 6 was obtained in 71% yield using the three-
To prepare the aldehyde analog of ester 6, the strategy was to
carry out a reduction-oxidation sequence starting from ethyl 4,6-
dimethoxy-3-methyl-1-benzofuran-2-carboxylate 3 without car-
boxamide substitution at C-7 to avoid the reduction of the amide
function in the presence of lithium aluminium hydride (LAH) in the
first step (Scheme 2). It’s to be noticed that the reduction of ester 4
failed due to the presence of the bromine atom on the benzofuran
ring; degradation of the reaction mixture was observed.
The ethyl ester 3 was easily reduced, with LAH, to alcohol 14
which was converted to aldehyde 15 with manganese dioxide,
quantitatively [7]. Unfortunately, attempt to directly access the
benzofuran-7-carboxamide analog of 15, by treatment with CSI,
failed. Thus, the previously described strategy (bromination-cyan-
ation-hydration) was applied to introduce a carboxamide function
at the position 7 of benzofuran-2-carbaldehyde 15 (Scheme 2). 2-
Formylbenzofuran-7-carboxamide 18 was obtained in three steps,
in 17% overall yield.
The last step of the synthetic work was to prepare 4,6-
dihydroxybenzofuran-7-carboxamide derivatives by demethyla-
tion of 4,6-dimethoxybenzofuran-7-carboxamide precursors. The
first attempts were unsuccessful using boron tribromide [19] and
compounds 6 and 8 as starting material (Scheme 3). At low tem-
perature (ꢀ10 ꢁC) in the presence of 2 equivalents of Lewis acid,
starting material was recovered but with an excess of reagent (5e
10 equiv.) and higher temperatures (room temperature to reflux),
monodemethylation (UPLC-MS monitoring) and then degradation
of the reaction mixture were observed. In the latter experimental
(d)
OMe
O
OMe
O
O
OMe
O
OMe
(a)
(b)
(c)
MeO
CO2Et
MeO
CO2Et
MeO
OH
MeO
OH
R
1
2
3 R = H
(e)
(f)
4 R = Br
5 R = CN
6 R = CONH2
(h)
OMe
OMe
(g)
(i)
(j for 8-9) or (k for 10-12)
(l for 13)
MeO
MeO
CO2H
CONHR'
O
O
R' = H
CONH2
CONH2
7
8-13
Scheme 1. Reagents and conditions: (a) AcCl, BCl3, CH2Cl2, ꢀ10 ꢁC then reflux, 3 h, 97%; (b) BrCH2CO2Et, K2CO3, DMF, 70 ꢁC, 2 h, 99%; (c) Cs2CO3, CH3CN, reflux, 17 h, 55%; (d)
BrCH2CO2Et, K2CO3, DMF, 70 ꢁC, 1 h then 100 ꢁC, 18 h, 84%; (e) Br2, AcOH, rt, 30 min, 93%; (f) CuCN, NMP, 200 ꢁC, MW, 10 min, 84%; (g) H2SO4, 60 ꢁC, 6 h, 91%; (h) CSI, CH3CN, 0 ꢁC to
rt, 2 h then HCl 1M, rt, 74%; (i) NaOH, EtOH, reflux, 4.5 h, 79%; (j) CNMPI, Et3N, CH2Cl2, reflux, 10 h, 64e69%; (k) TBTU, i-Pr2NEt, DMF, rt, 24 h, 55e90%; (l) NH3, MeOH, sealed tube,
reflux, 60 h, 67%.

M.-A. Bazin et al. / European Journal of Medicinal Chemistry 69 (2013) 823e832
825
Table 1
carbaldehyde 28, even by rising the temperature, due to prob-
lems of solubility in acetonitrile. Finally, ester 26 reacted with CSI to
provide 7-carboxamidebenzofuran derivative 31 in good yield
(87%) and its benzyl groups were cleaved by catalytic hydro-
genolysis to give 7-carboxamide-4,6-dihydroxybenzofuran deriva-
tive 32.
Structures of N2-(het)aryl-4,6-dimethoxy-3-methylbenzofuran-2,7-dicarboxamide
8e12.
Compound R0
Coupling Yield
reagent
(%)a
OMe
O
2.2. Biological evaluation
8
9
10
11
12
n-C3H7
C6H5eCH2 CNMPI
C6H5
2-Pyridyl
4-Pyridyl
CNMPI
64
69
90
55
58
MeO
CONHR'
TBTU
TBTU
TBTU
Two cell lines were used in this study, A549 and NSCLC-N6,
originating from an adenocarcinoma and an epidermoid lung
cancer, respectively. The NSCLC-N6 is a cell line derived from an
NSCLC of a previously untreated patient (moderately differentiated
classified as T2N0M0) [26]. The A549 line was obtained from the
ATCC (reference CCL-185) [27] and is known to have a wild type p53
gene, while NSCLC-N6 has a mutant p53 gene, similar to tumors in
situ.
CONH2
a
Isolated yields.
conditions,
2-formyl-4,6-dihydroxy-3-methyl-1-benzofuran-7-
carboxamide 21 was produced in low yield (21%).
To circumvent this problem, we decided to introduce the benzyl
moiety as protecting group of 4,6-dihydroxybenzofuran derivatives
in order to perform the cleavage in milder conditions.
In this second route, 2,4-bis(benzyloxy)-6-hydroxyacetophenone
25 was initially prepared to be cyclized into benzofuran ring as
described before for the dimethoxy series (Scheme 4).
As indicated in the literature [20,21], 6-hydroxy-2,4-
dibenzyloxyacetophenone 25 was furnished by an initial tri-
benzylation of triacetoxyphloroglucinol 22 to avoid additive C-
benzylation by direct O-benzylation of phloroglucinol [22,23].
Subsequent acylation of 23, through the formation of a mixed an-
It is known that the chemoresistance observed for NSCLC is
largely due to a very high proportion of cells in the G0/G1 phase of
the cell cycle [28e30]. Therefore, as many anticancer drugs have a
cycle-dependent activity, their effectiveness on such slowly
developing tumors is limited. Consequently, we focus our research
on cytostatic molecules that stop cell growth in G1-phase of the cell
cycle, before initiating cells to apoptotic death. In addition, this
prevents excessive toxicity in vivo. This approach was successfully
carried out to discover the NSCLC antitumor activity of triazine
A190 [9], used as reference compound (Table 2).
Compounds 3e18, 21, 26, 28e32 were tested for their in vitro
antiproliferative activity against NSCLC-N6 and A549 human lung
cancer cell lines using MTT assay. The results expressed as IC50 were
summarized in Table 2.
hydride, afforded acetophenone 24 followed by
a mono-
deprotection in the presence of titanium tetrachloride to give the
desired compound 25 (Scheme 4).
Ethyl 4,6-dibenzyloxy-3-methyl-1-benzofuran-2-carboxylate
26 was then obtained using the method previously reported
which included O-alkylation with ethyl bromoacetate and base-
catalyzed cyclization of the alkylated product at 100 ꢁC in DMF. In
these experimental conditions, removal of ester group at the po-
sition 2 of compound 26 was observed and benzofuran by-product
27 was isolated in 20% yield after purification by column
chromatography.
Interestingly, benzofuran-2-carbaldehyde 28 was prepared in
good yield (83%) by the VilsmeiereHaack formylation of 27 without
C7 electrophilic attack [24]. This approach was more direct than the
two-step route of reduction and oxidation from the ester 26.
The benzyl protective groups of 26 and 28 were removed under
transfer hydrogenation conditions using Pd/C and cyclohexene, in a
mixture of methanol/tetrahydrofuran at reflux, to afford dihy-
droxybenzofuran derivatives 29 and 30 in 92% and 61% yield,
respectively [25]. Efforts to install a carboxamide function at the
position 7 of the analogs 29 and 30, by the use of CSI, remained
unsuccessful since a complex mixture was observed in the medium.
The same reaction failed from 4,6-dibenzyloxybenzofuran-2-
Three series (ester, aldehyde and amide), based on 3-methyl-1-
benzofuran scaffold, were developed considering the substitution
at the position 2 of the ring.
Among the esters, 4,6-dimethoxybenzofuran derivatives 3e6,
substituted or not at the position 7 were found to be inactive.
Benzyl analog 26 of compound 3 remained inactive and the cor-
responding debenzylated derivative 29 too. Interestingly, the
introduction of a carboxamide function at the position 7 of com-
pounds 26 and 29 resulted in obtaining an antiproliferative effect of
compounds 31 and 32. Indeed, benzyl derivative 31 exhibited a
cytotoxic activity against both cell lines with IC50 values inferior to
7.2
mM and IC50 values ranged from 24.0 to 32.9
mM for compound
32.
In the second series, the same trends were observed since
benzofuran-2-carboxaldehydes 15e17, 28 and 30 were inactive. The
only difference concerned compounds 18 and 21. 4,6-
Dimethoxybenzofuran derivatives 18 displayed IC50 values of
44.4 mM and 63.8 mM against NSCLC-N6 and A549, respectively,
whereas ester analog 6 was totally inactive. In the contrary, 4,6-
dihydroxybenzofuran derivative 21 (IC50 values NSCLC-N6 of
OMe
OMe
OMe
(a)
(b)
MeO
MeO
MeO
CHO
CO2Et
CH2OH
O
O
O
R
3
14
15 R = H
16 R = Br
17 R = CN
(c)
(d)
(e)
(f) X
18 R = CONH2
Scheme 2. Reagents and conditions: (a) LiAlH4, THF, rt, 15 min, 95%; (b) MnO2, CH2Cl2, reflux, 2 h, 98%; (c) Br2, AcOH, rt, 2 h, 79%; (d) CuCN, NMP, 200 ꢁC, MW, 40 min, 50%; (e)
H2SO4, 60 ꢁC, 6 h, 44%; (f) CSI, CH3CN, 0 ꢁC to rt, 15 h then HCl 1 M, rt.

826
M.-A. Bazin et al. / European Journal of Medicinal Chemistry 69 (2013) 823e832
OMe
O
OH
Structural analogy with the west part of the cercosporamide,
namely 4,6-dihydroxy-3-methyl-1-benzofuran-7-carboxamide,
(a)
was found on compounds 21 and 32, displaying low anti-
proliferative activity against tested NSCLC cell lines, for the first
one, and possessing promising activity for the second one. Gener-
ally, based on this scaffold, substitutions of the phenols by a methyl
or a benzyl were preferred (21 vs 18, 32 vs 31) in terms of antitumor
activity. In addition, 4,6-dimethoxybenzofuran derivatives fur-
nished also the most interesting products in dicarboxamide series
(compounds 10, 12 and 13).
The results suggested that the design of new compounds,
inspired from cercosporamide structure, was a valuable starting
point to obtain in vitro antiproliferative agents against NSCLC cell
lines.
MeO
HO
COR
COR
O
CONH2
CONH2
19 (R = OC2H5, failure)
20 (R = NHC3H7, failure)
21 (R = H, 21%)
6
8
18
Scheme 3. Reagents and conditions: (a) BBr3, CH2Cl2, reflux, 10 h.
60.8
m
M and IC50 values A549 of 108.4
ester counterpart 32 (IC50 values NSCLC-N6 of 32.9
values A549 of 24.0 M).
m
M) was less active than
mM and IC50
m
In the last series, six 4,6-dimethoxybenzofuran-2-carboxamide
3. Conclusion
derivatives 8e13 were tested. Among them, N-propylcarboxamide
8
and N-benzylcarboxamide
substituted carboxamides 10 (phenyl) and 12 (4-pyridyl) exhibited
good cytotoxic activity (IC50 values inferior to 9.2 M). Surprisingly,
9
were inactive. N-(Hetero)aryl
In summary, a series of 3-methyl-1-benzofuran derivatives were
synthesized and screened for their antiproliferative activity against
two human NSCLC cell lines (NSCLC-N6 and A549). Among them,
three compounds (10, 12 and 31) displayed cytotoxic activity and
could be possible candidates for further evaluation. However, we
retained for additional study compounds with IC50 values between
m
the design of 2-pyridyl isomer 11 of compound 12 led to a dramatic
decrease in antiproliferative activity.
Finally, 2,7-dicarboxamidebenzofuran derivative 13 showed
promising antitumor activity against NSCLC-N6 and A549 cell lines
10
cytostatic molecules in vivo and in this strategy a molecule whose
IC50 value is less than 5 M generally proves to be too toxic in an-
imals and when its IC50 is greater than 60 M, dose adjustment
in vivo is difficult to succeed. As we proceed for reference com-
pound A190 (IC50 values NSCLC-N6 of 39.4 M and IC50 values A549
of 52.6 M), a cytostatic compound which showed a very inter-
mM and 50 mM. Indeed, the goal of the project was to find
with IC50 values of 22.6 mM and 39.4 mM, respectively.
The present study investigated the biological effect of several
substituents on 3-methyl-1-benzofuran ring at the position 2
(ester, aldehyde, amide), the position 7 (H, Br, CN, CONH2) and the
positions 4 and 6 (OMe, OBn, OH). In most cases, compounds with
methoxy groups at the 4 and 6-positions of benzofuran remained
inactive, except amides 10, 12, 13 and aldehyde 18, more active or as
active as the reference compound A190 (Table 2). It was obvious
that the antiproliferative activity was improved by the introduction
of a carboxamide (CONH2) group at the 7-positon (15 vs 18, 26 vs 31
and 29 vs 32).
m
m
m
m
esting in vivo profile with original mechanism of action [9], com-
pounds 13, 18 and 32 will be selected for further pharmacological
investigations. In this context, the compounds will be studied as
follows: the cell responses by the observation of cell proliferation
arrest to choose cytostatic agents, the induction of apoptosis by cell
OH
OAc
OBn
OBn
O
(a)
(b)
(c)
HO
OH
AcO
OAc
BnO
OBn
BnO
OBn
22
23
24
OBn
OBn
OBn
O
(d)
(e)
(f from 27)
BnO
26 R = CO2C2H5
BnO
R
CHO
BnO
OH
O
O
25
28
27 R = H (20%, by-product)
X
(h)
(g)
(h from 26)
OH
OBn
O
OH
(g)
(h)
X
(g)
HO
BnO
HO
COR'
O
COR'
COR'
O
CONH2
CONH2
31 R' = OC2H5
33 R' = H
29 R' = OC2H5
30 R' = H
32 R' = OC2H5
21 R' = H
Scheme 4. Reagents and conditions: (a) Ac2O, pyridine, 120 ꢁC, 4 h, 98%; (b) BnCl, NaH, H2O, DMF, 0 ꢁC to rt, 17 h, 78%; (c) AcOH, TFAA, CH2Cl2, 0 ꢁC, 1.5 h, 75%; (d) TiCl4,
CH2Cl2, ꢀ5 ꢁC, 1 h, 84%; (e) BrCH2CO2Et, K2CO3, DMF, 70 ꢁC, 2 h then 100 ꢁC, 20 h, 52%; (f) POCl3, DMF, rt, 20 h then NaOH 8N, 83%; (g) Cyclohexene, Pd/C 10%, MeOH, THF, reflux, 5 h,
92% (for 29) and 2 h, 61% (for 30 and 32); (h) CSI, CH3CN, 0 ꢁC to rt, 5 h then HCl 1 M, rt, 87%.

M.-A. Bazin et al. / European Journal of Medicinal Chemistry 69 (2013) 823e832
827
Table 2
Inhibition effects of compounds 3-6, 8e13, 15e18, 21, 26, 28e32 on the growth of NSCLC-N6 and A549 tumor cell lines in vitro.
OR3
R1
R3O
O
R2
Compound
R1
R2
R3
NSCLC-N6a IC50 ꢂ SEM (
m
M)b
A549a IC50 ꢂ SEM (
m
M)b
A190c
3
4
5
6
39.4 ꢂ 2.1
52.6 ꢂ 2.8
CO2CH2CH3
CO2CH2CH3
CO2CH2CH3
CO2CH2CH3
CONH(n-C3H7)
CONHCH2C6H5
CONHC6H5
CONH(2-Pyridyl)
CONH(4-Pyridyl)
CONH2
CHO
CHO
CHO
CHO
CHO
CO2CH2CH3
CHO
CO2CH2CH3
CHO
CO2CH2CH3
CO2CH2CH3
H
Br
CN
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
n.a.d
n.a.
>87.4
n.a.
n.a.
>93.6
>81.4
<9.3
>84.4
<9.3
>87.4
n.a.
n.a.
>93.6
CONH2
CONH2
CONH2
CONH2
CONH2
CONH2
CONH2
H
Br
CN
CONH2
CONH2
H
8
9
54.8 ꢂ 2.6
10
11
12
13
15
16
17
18
21
26
28
29
30
31
32
<9.3
>84.4
<9.3
22.6 ꢂ 1.3
39.4 ꢂ 2.1
n.a.
n.a.
n.a.
n.a.
49.5 ꢂ 8.0
n.a.
63.8 ꢂ 2.3
108.4 ꢂ 13.6
>72.0
44.4 ꢂ 4.2
60.8 ꢂ 3.4
>72.0
>80.6
65.6 ꢂ 6.8
n.a.
Bn
Bn
H
H
Bn
H
H
H
H
>80.6
68.6 ꢂ 21.6
n.a.
CONH2
CONH2
<7.2
32.9 ꢂ 1.4
<7.2
24.0 ꢂ 5.7
a
Non-small cell lung cancer (NSCLC) cell lines.
b
c
Mean from at least eight determinations. SEM: Standard Error of the Mean.
A190 the 4,6-diamino-1,2-dihydro-1-(400-chlorophenyl)-2-(1-tricyclo[3.3.1.13,4] decyl-1,3,5-triazine hydrochloride, is used as reference compound.
d
n.a.: not active.
cycle arrest, the molecular mechanisms involved. Finally, to
confirm the in vitro results on the cell lines, the antitumoral effect
would be also studied with an in vivo experiment on NSCLC-N6
xenografted nude mice. Unfortunately, no significant difference of
IC50 values between the two cell lines (wild type p53 or mutant
p53) was observed for the tested compounds. These data would
indicate that p53 is not the target involved in the observed anti-
tumor activity.
4.1.1. 1-(2-Hydroxy-4,6-dimethoxyphenyl)acetophenone
(xanthoxylin) (1)
3,5-Dimethoxyphenol (15.0 g, 97.3 mmol) was dissolved in
100 mL of anhydrous dichloromethane. Boron trichloride (1 M so-
lution in dichloromethane, 102.2 mL, 102.2 mmol) was slowly
added under nitrogen at ꢀ10 ꢁC. After 5 min, acetyl chloride
(7.3 mL, 102.2 mmol) was added dropwise. The mixture was heated
and kept under reflux for 3 h and was stirred overnight at room
temperature. The reaction mixture was cooled at 0 ꢁC and was
slowly hydrolyzed with 1 M HCl. The organic phase was separated
and the aqueous layer was extracted twice with dichloromethane.
The organic layers were dried over sodium sulfate and evaporated
under reduced pressure. Recrystallization from methanol afforded
1 (18.5 g, 97%) as a white powder.
4. Experimental protocols
4.1. Chemistry
All reactions were monitored by TLC analysis using Merck sili-
cagel 60F-254 thin-layer plates. Column chromatography was car-
ried out on silicagel Merck 60 (70e230 mesh ASTM). Melting points
were determined on an Electrothermal IA 9000 melting point
apparatus and are uncorrected. Infrared spectra (IR) were recorded
on a Shimadzu IRAffinity-1 IR-FT spectrophotometer equipped with
a MIRacle 10 accessory ATR. 1H and 13C NMR spectra were per-
formed in DMSO-d6 or CDCl3 using a Bruker AVANCE 400 MHz
Rf 0.28 (CH2Cl2); Mp: 80.5 ꢁC (lit [31]. 78.5e79.5 ꢁC); IR (KBr)
3102, 3021, 2945, 1612, 1595, 1572, 1422, 1366, 1269, 1219, 1207,
1155, 1111, 1082, 893, 835 cmꢀ1 1H NMR (400 MHz, CDCl3):
14.04 (s, 1H, eOH), 6.07 (d, 1H, J ¼ 2.0 Hz, H5), 5.93 (d, 1H,
J ¼ 2.0 Hz, H3), 3.86 (s, 3H, eOCH3), 3.83 (s, 3H, eOCH3), 2.62 (s,
3H, eCOCH3); 13C NMR (100 MHz, DMSO-d6):
202.8, 166.2, 165.9,
n
;
d
d
162.8, 105.5, 93.6, 90.8, 56.0, 55.7, 32.7; MS (ESI) m/z (%): 197.1
(100) [M þ H]þ.
spectrometer. Chemical shifts are reported as
d values in parts per
million (ppm) relative to tetramethylsilane as internal standard and
coupling constants (J) are given in Hertz (Hz). The following ab-
breviations are used to describe peak patterns when appropriate: s
(singlet), d (doublet), t (triplet), q (quartet), m (multiplet). Mass
spectra were recorded using an Electrospray Ionization Method
with Waters ZQ 2000 spectrometer. Microwave-promoted re-
actions were performed on a CEM Discover SP monomode appa-
ratus. Elemental analyses were performed on a Thermo Scientific
Elemental Analyser Flash EA 1112 and were found within ꢂ0.4% of
the theoretical values.
4.1.2. Ethyl (2-acetyl-3,5-dimethoxyphenoxy)acetate (2)
To a stirred solution of compound 1 (3.95 g, 20.1 mmol) in DMF
(50 mL) were successively added ethyl bromoacetate (2.46 mL,
22.2 mmol) and potassium carbonate (8.35 g, 60.4 mmol). The
reaction mixture was heated to 60 ꢁC for 20 h. After cooling, the
reaction mixture was diluted with ethyl acetate and water was
added. The organic layer was separated, washed twice with water
and brine, dried over sodium sulfate and evaporated under
reduced pressure. The crude product was purified by silica gel

828
M.-A. Bazin et al. / European Journal of Medicinal Chemistry 69 (2013) 823e832
chromatography using dichloromethane as eluent to afford 2
(3.86 g, 68%) as a white powder.
(5 mL) in a 10 mL glass vial equipped with a small magnetic stir bar.
The vial was sealed and microwave irradiated (100 W) at 200 ꢁC for
10 min. After cooling, the mixture was filtered and the solid was
washed with methanol and dried to give a gray solid. The crude
product was purified by silica gel chromatography using dichloro-
methane as eluent to afford 5 (354 mg, 84%) as a white powder.
Rf 0.13 (CH2Cl2); Mp: 62.0 ꢁC; IR (KBr)
n
2974, 1732, 1694, 1452,
1416, 1287, 1231, 1200, 1135, 1020, 820 cmꢀ1 1H NMR (400 MHz,
CDCl3):
;
d
6.15 (d,1H, J ¼ 2.0 Hz, Har), 5.99(d,1H, J ¼ 2.0 Hz, Har), 4.62(s,
2H, eOCH2e), 4.25 (q, 2H, J ¼ 7.2 Hz, eCH2CH3), 3.80 (s, 6H, 2 ꢃ e
OCH3), 2.54 (s, 3H, eCOCH3), 1.29 (t, 3H, J ¼ 7.2 Hz, eCH2CH3); 13C
Rf 0.44 (CH2Cl2); Mp: 219.7 ꢁC; IR (KBr)
1595, 1506, 1331, 1282, 1221, 1160, 1126, 1016, 976, 825, 767 cmꢀ1
1H NMR (400 MHz, CDCl3):
n 2982, 2227, 1718, 1620,
NMR (100 MHz, CDCl3):
d
201.5, 168.5, 162.3, 158.6, 156.7, 114.7, 91.9,
;
91.7, 66.2, 61.5, 56.0, 55.6, 32.7,14.3; MS (ESI) m/z (%): 283.1 [M þ H]þ.
d
6.27 (s, 1H, Har), 4.40 (q, 2H, J ¼ 7.1 Hz,
Anal. Calcd for C14H18O6: C 59.57; H 6.43. Found: C 59.82; H 6.80%.
eCH2CH3), 4.01 (s, 3H, eOCH3), 4.00 (s, 3H, eOCH3), 2.65 (s, 3H, 3e
CH3), 1.41 (t, 3H, J ¼ 7.1 Hz, eCH2CH3); 13C NMR (100 MHz, CDCl3):
4.1.3. Ethyl 4,6-dimethoxy-3-methyl-1-benzofuran-2-carboxylate
(3)
d 164.4, 160.4, 159.8, 155.9, 140.4, 126.8, 113.1, 112.9, 89.8, 77.2, 61.0,
56.9, 56.0, 14.4, 11.0; MS (ESI) m/z (%): 290.0 [M þ H]þ. Anal. Calcd
for C15H15NO5: C 62.28; H 5.23; N 4.84. Found: C 62.13; H 5.02; N
4.70%.
4.1.3.1. Method a. Compound 2 (2.00 g, 7.1 mmol) and cesium
carbonate (9.23 g, 28.3 mmol) were suspended in DMF (20 mL) in a
100 mL round bottom flask equipped with a magnetic stir bar. The
opened flask was heated under microwave irradiation (30 W) at
80 ꢁC for 90 min. After cooling, water (50 mL) and ethyl acetate
(50 mL) were added. The organic phase was separated, dried over
sodium sulfate and evaporated under reduced pressure. Compound
3 was obtained without further purification as a white powder
(1.18 g, 63%).
4.1.6. Ethyl 7-carbamoyl-4,6-dimethoxy-3-methyl-1-benzofuran-2-
carboxylate (6)
4.1.6.1. Method a. Concentrated sulfuric acid (10.5 mL) was added
to compound 5 (3.00 g, 10.3 mmol). The mixture was heated to
60 ꢁC for 6 h. After cooling, ethyl acetate (100 mL) was added and
the solution was neutralized with a saturated sodium bicarbonate
solution. The organic layer was then separated, dried over sodium
sulfate and evaporated under reduced pressure to afford 6 (2.90,
91%) as a yellow powder.
4.1.3.2. Method b. To a stirred solution of compound 1 (15.50 g,
79.0 mmol) in DMF (150 mL) were added potassium carbonate
(32.76 g, 237.0mmol) and ethyl bromoacetate (9.64 mL, 86. 91 mmol).
The mixture was heated at 70 ꢁC for 1 h and then at 100 ꢁC overnight.
The reaction mixture was cooled to room temperature and filtered on
CeliteÒ. The solution was diluted with ethyl acetate (200 mL) and the
organic layer was washed with brine and water, dried over sodium
sulfate and evaporated under reduced pressure. The crude product
was purified by silica gel chromatography using dichloromethane as
eluent to afford 3 (17.64 g, 84%) as a white powder.
4.1.6.2. Method b. To a stirred suspension of compound 5 (200 mg,
0.76 mmol) in acetonitrile (10 mL) at 0 ꢁC under argon was added
chlorosulfonyl isocyanate (72 mL, 0.83 mmol). The mixture was then
warmed to room temperature for 2 h and the reaction mixture was
quenched with 1 M HCl (3 mL). After 1 h at room temperature, the
precipitate was filtered and washed with water to afford 6 (172 mg,
74%) as a white powder.
Rf 0.56 (CH2Cl2); Mp: 130.7 ꢁC; IR (KBr)
1631, 1606, 1576, 1508, 1450, 1373, 1279, 1220, 1195, 1150, 1028, 807,
765 cmꢀ1; 1H NMR (400 MHz, CDCl3):
n
3031, 2986, 2935, 1721,
Rf 0.61 (CH2Cl2); Mp: 202.5 ꢁC; IR (KBr)
n
3430, 3387, 2982, 2939,
1699, 1644, 1601, 1457, 1380, 1319, 1282, 1215, 1141, 804, 767 cmꢀ1
1H NMR (400 MHz, CDCl3):
7.11 (bs, 1H, eNH2), 6.34 (s, 1H, Har),
;
d
6.60 (d, 1H, J ¼ 1.8 Hz, Har),
d
6.26 (d, 1H, J ¼ 1.8 Hz, Har), 4.41 (q, 2H, J ¼ 7.1 Hz, eCH2CH3), 3.88 (s,
5.70 (bs, 1H, eNH2), 4.39 (q, 2H, J ¼ 7.1 Hz, eCH2CH3), 4.01 (s, 3H, e
3H, eOCH3), 3.83 (s, 3H, eOCH3), 2.68 (s, 3H, 3eCH3), 1.42 (t, 3H,
OCH3), 3.98 (s, 3H, eOCH3), 2.68 (s, 3H, 3eCH3), 1.41 (t, 3H,
J ¼ 7.1 Hz, eCH2CH3); 13C NMR (100 MHz, CDCl3):
d
161.7, 160.5,
J ¼ 7.1 Hz, eCH2CH3); 13C NMR (100 MHz, CDCl3):
d 164.9, 160.4,
156.6, 156.5, 139.0, 127.3, 112.8, 94.6, 87.8, 60.7, 55.7, 55.5, 14.5, 11.2;
MS (ESI) m/z (%): 265.0 [M þ H]þ. Anal. Calcd for C14H16O5: C 63.63;
H 6.10. Found: C 64.01; H 6.19%.
160.2, 158.5, 155.2, 140.1, 126.2, 113.8, 101.2, 90.5, 60.9, 57.1, 55.7,
14.4, 11.0; MS (ESI) m/z (%): 308.0 [M þ H]þ. Anal. Calcd for
C15H17NO6: C 58.63; H 5.58; N 4.56. Found: C 59.02; H 5.72; N 4.60%.
4.1.4. Ethyl 7-bromo-4,6-dimethoxy-3-methyl-1-benzofuran-2-
carboxylate (4)
4.1.7. 7-Carbamoyl-4,6-dimethoxy-3-methyl-1-benzofuran-2-
carboxylic acid (7)
To a stirred solution of compound 3 (1.30 g, 4.92 mmol) in glacial
To a stirred solution of compound 6 (2.10 g, 6.83 mmol) in
ethanol (15 mL) was added aqueous 1 M NaOH (6.8 mL, 6.83 mmol).
The reaction mixture was refluxed for 4 h. After cooling, ethanol
was removed under reduced pressure and the residue was
precipitated with 1 M HCl. The suspension was then filtered to
afford 7 (1.50 g, 79%) as a yellow powder.
acetic acid (50 mL) was added dropwise bromine (265
mL,
5.16 mmol) at room temperature. After 30 min, the reaction
mixture was quenched with water (30 mL) and the precipitate was
then filtered and washed with water. The crude product was then
recrystallized in a mixture methanol/water (19:1) to afford 4
(1.57 g, 93%) as a white powder.
Rf 0.61 (CH2Cl2/EtOH 9:1); Mp: 255.3 ꢁC; IR (KBr)
2951, 1847, 1675, 1598, 1568, 1319, 1282, 1221, 1163, 1147, 976, 792,
712, 605 cmꢀ1 1H NMR (400 MHz, DMSO-d6):
13.21 (bs, 1H, e
n 3461, 3314,
Rf 0.22(CH2Cl2);Mp: 168e170 ꢁC;IR(KBr)
1327, 1271, 1217, 1144, 1128, 978, 814, 766 cmꢀ1; 1H NMR (400 MHz,
CDCl3):
6.34(s,1H, Har), 4.42 (q, 2H, J ¼ 7.2 Hz, eCH2CH3), 3.98 (s, 3H,
eOCH3), 3.94 (s, 3H, eOCH3), 2.67 (s, 3H, eCH3),1.43 (t, 3H, J ¼ 7.2 Hz,
eCH2CH3); 13C NMR (100 MHz, CDCl3):
200.8, 168.3, 158.4, 157.2,
n 2990,1703,1612,1589,
;
d
d
CO2H), 7.63 (bs, 1H, eNH2), 7.48 (bs, 1H, eNH2), 6.63 (s, 1H, Har),
4.00 (s, 3H, eOCH3), 3.94 (s, 3H, eOCH3), 2.64 (s, 3H, 3eCH3); 13C
d
NMR (100 MHz, DMSO-d6): d 164.3, 160.8, 157.8, 156.4, 152.7, 139.6,
153.6, 120.3, 98.5, 93.0, 71.4, 61.3, 56.7, 56.2, 32.6, 14.3; MS (ESI) m/z
124.9, 111.9, 104.4, 91.4, 56.7, 56.0, 11.0; MS (ESI) m/z (%): 280.0
[M þ H]þ. Anal. Calcd for C13H13NO6: C 55.92; H 4.69; N 5.02.
Found: C 55.84; H 4.68; N 4.87%.
(%): 343.0 (100) [M þ H]þ, 345.1 [M þ Hþ2]þ. Anal. Calcd for
C14H15BrO5: C 49.00; H 4.41. Found: C 49.15; H 4.43%.
4.1.5. Ethyl 7-cyano-4,6-dimethoxy-3-methyl-1-benzofuran-2-
carboxylate (5)
Compound 4 (500 mg, 1.46 mmol) and copper(I) cyanide
(261 mg, 2.91 mmol) were suspended in N-methylpyrrolidone
4.1.8. Synthesis of amides 8e9 using CNMPI procedure
4.1.8.1. 4,6-Dimethoxy-3-methyl-N2-propyl-1-benzofuran-2,7-
dicarboxamide (8). Compound 7 (200.0 mg, 0.72 mmol) was sus-
pended in dichloromethane (30 mL). 2-Chloro-1-methylpyridinium

M.-A. Bazin et al. / European Journal of Medicinal Chemistry 69 (2013) 823e832
829
iodide (183.0 mg, 0.72 mmol), amine (0.72 mmol) and triethyl-
amine (251.6 L, 1.79 mmol) were successively added and the
powder. Yield 55% (70 mg). Rf 0.66 (CH2Cl2/EtOH, 9:1); Mp:
259.7 ꢁC; IR (KBr)
3401, 3319, 2916, 1649, 1634, 1562, 1470, 1429,
1306, 1250, 1069, 841, 787 cmꢀ1 1H NMR (400 MHz, DMSO-d6):
9.66 (s,1H, eNHe), 8.37 (dd,1H, J ¼ 4.6 and 1.4 Hz, H3-pyridine), 8.18
(d, 1H, J ¼ 8.2 Hz, H6-pyridine), 7.86 (ddd, 1H, J ¼ 8.2, 7.4 and 1.4 Hz,
m
n
mixture was refluxed for 10 h. The reaction mixture was diluted
with dichloromethane (50 mL) and the organic layer was washed
with water (3 ꢃ 30 mL), dried over sodium sulfate and evaporated
under reduced pressure. The crude product was purified by silica
gel chromatography using CH2Cl2/EtOH 9:1 as eluent to give the
desired product.
;
d
H
5-pyridine), 7.75 (s,1H, eNH2), 7.50 (s,1H, eNH2), 7.18 (dd,1H, J ¼ 7.4
and 4.6 Hz, H4-pyridine), 6.64 (s, 1H, Har), 3.99 (s, 3H, eOCH3), 3.93 (s,
3H, eOCH3), 2.66 (s, 3H, 3eCH3); 13C NMR (100 MHz, DMSO-d6):
White powder. Yield 64% (147 mg). Rf 0.43 (CH2Cl2/EtOH, 9:1);
d 163.8, 158.9, 157.7, 156.8, 152.3, 151.0, 148.2, 140.5, 138.5, 123.6,
Mp: 219 ꢁC; IR (KBr)
n
3375, 3185, 2958, 2933, 1653, 1644, 1601,
1522, 1506, 1463, 1380, 1322, 1218, 1144, 1120, 801 cm-1 1H NMR
(400 MHz, DMSO-d6):
120.0, 113.6, 112.2, 103.5, 92.1, 56.9, 56.2, 10.7; MS (ESI) m/z (%):
356.2 [M þ H]þ. Anal. Calcd for C18H17N3O5: C 60.84; H 4.82; N
11.82. Found: C 60.67; H 4.80; N 11.89%.
;
d
7.96 (t, 1H, J ¼ 6.0 Hz, eNHe), 7.62 (s, 1H, e
NH2), 7.42 (s, 1H, eNH2), 6.62 (s, 1H, Har), 3.99 (s, 3H, eOCH3), 3.93
(s, 3H, eOCH3), 3.20e3.25 (m, 2H, eCH2CH2CH3), 2.62 (s, 3H, 3e
CH3), 1.52e1.58 (m, 2H, eCH2CH2CH3), 0.90 (t, 3H, J ¼ 7.4 Hz, e
4.1.9.3. 4,6-Dimethoxy-3-methyl-N2-pyridin-4-yl-1-benzofuran-2,7-
dicarboxamide (12). Compound 12was obtained following the
representative procedure described for compound 10. Yellow
powder. Yield 58% (74 mg). Rf 0.48 (CH2Cl2/EtOH, 9:1); Mp:
CH2CH2CH3); 13C NMR (100 MHz, DMSO-d6):
d 164.1, 159.2, 157.6,
156.2, 151.8, 141.7, 120.4, 112.2, 104.1, 91.6, 56.8, 56.0, 40.1, 22.5, 11.4,
10.5; MS (ESI) m/z (%): 321.1 [M þ H]þ. Anal. Calcd for C16H20N2O5: C
59.99; H 6.29; N 8.74. Found: C 60.08; H 6.34; N 9.06%.
235.8 ꢁC; IR (KBr)
n
3395, 3358, 2922, 1682, 1587, 1504, 1319, 1283,
10.91
1221, 1136, 1119, 1055 cm-1; 1H NMR (400 MHz, DMSO-d6):
d
(s, 1H, eNHe), 8.57 (d, 2H, J ¼ 5.5 Hz, H3 and 5-pyridine), 7.95 (d, 2H,
J ¼ 5.5 Hz, H2 and 6-pyridine), 7.69 (s, 1H, eNH2), 7.50 (s, 1H, eNH2),
6.63 (s, 1H, Har), 3.99 (s, 3H, eOCH3), 3.91 (s, 3H, eOCH3), 2.65 (s,
4.1.8.2. N2-Benzyl-4,6-dimethoxy-3-methylbenzofuran-2,7-
dicarboxamide (9). Compound
representative procedure described for compound 8. White pow-
der. Yield 69% (183 mg). Rf 0.77 (CH2Cl2); Mp: 278.8 ꢁC; IR (KBr)
3369, 3172, 2933, 1650, 1604, 1509, 1466, 1430, 1384, 1325, 1261,
1218, 1163, 1144, 1123, 810, 693 cmꢀ1 1H NMR (400 MHz, DMSO-
d6):
9 was obtained following the
3H, 3eCH3); 13C NMR (100 MHz, DMSO-d6):
d 164.0, 158.7, 158.6,
n
156.6, 152.4, 148.2, 147.1 (2 C), 140.5, 124.8, 114.5 (2 C), 112.1, 103.8,
91.8, 56.8, 56.1, 10.8; MS (ESI) m/z (%): 356.1 [M þ H]þ. Anal. Calcd
for C18H17N3O5: C 60.84; H 4.82; N 11.82. Found: C 60.80; H 4.67; N
12.02%.
;
d
8.54 (t,1H, J ¼ 6.2 Hz, eNHe), 7.59 (s, 1H, eNH2), 7.37 (s, 1H, e
NH2), 7.32e7.33 (m, 4H, Hbenzyl), 7.22e7.26 (m, 1H, Hbenzyl), 6.59 (s,
1H, Har), 4.44 (d, 2H, J ¼ 6.2 Hz, eCH2e), 3.96 (s, 3H, eOCH3), 3.90
(s, 3H, eOCH3), 2.60 (s, 3H, 3eCH3); 13C NMR (100 MHz, DMSO-d6):
4.1.10. 4,6-Dimethoxy-3-methyl-1-benzofuran-2,7-dicarboxamide
(13)
d
164.1, 159.3, 157.7, 156.2, 151.8, 141.5, 139.6, 128.2 (2 C), 127.4 (2 C),
A solution of ammonia (7 N in methanol) was added to com-
pound 6 (400 mg, 1.3 mmol) in a sealed tube and kept to reflux
condition for 60 h. Solvent was then evaporated and the powder
obtained was triturated with methanol and filtered. The crude
product was purified by silica gel chromatography using CH2Cl2/
EtOH 99:1 as eluent to give 13 (244 mg, 67%) as a beige powder.
126.7, 121.1, 112.1, 104.1, 91.6, 56.8, 56.0, 41.9, 10.6; MS (ESI) m/z (%):
369.1 [M þ H]þ. Anal. Calcd for C20H20N2O5: C 65.21; H 5.47; N 7.60.
Found: C 65.47; H 5.55; N 7.81%.
4.1.9. Synthesis of amides 10e12 using TBTU procedure
4.1.9.1. 4,6-Dimethoxy-3-methyl-N2-phenyl-1-benzofuran-2,7-
dicarboxamide (10). To a stirred solution of compound 7 (100.0 mg,
0.36 mmol) in DMF (5 mL) were successively added O-(benzo-
Rf 0.49 (CH2Cl2/EtOH, 9:1); Mp: 271.1 ꢁC; IR (KBr)
2922, 1659, 1607, 1566, 1391, 1310, 1219, 1171, 1142, 1113 cmꢀ1
NMR (400 MHz, DMSO-d6): 7.60 (bs, 1H, eNH2), 7.55 (bs, 1H, e
n
3420, 3345,
;
1H
d
triazol-1-yl)-N,N,N0,N0-tetramethyluronium
tetrafluoroborate
(149.5 mg, 0.47 mmol), N,N-diisopropylethylamine (124.8 L,
NH2), 7.42 (bs, 1H, eNH2), 7.32 (bs, 1H, eNH2), 6.62 (s, 1H, Har), 3.99
m
(s, 3H, eOCH3), 3.93 (s, 3H, eOCH3), 2.62 (s, 3H, 3eCH3); 13C NMR
0.72 mmol) and amine (0.47 mmol). The reaction mixture was
stirred at room temperature for 24 h. Water (30 mL) and
dichloromethane (30 mL) were then added and the aqueous layer
was extracted with dichloromethane (2 X 30 mL). The combined
organic layers were dried over sodium sulfate and evaporated un-
der reduced pressure. The crude product was purified by silica gel
chromatography using CH2Cl2/EtOH 95:5 as eluent to give the
desired product.
(100 MHz, DMSO-d6): d 164.1, 161.1, 157.8, 156.3, 151.8, 141.7, 121.0,
112.2, 103.9, 91.6, 56.8, 56.0, 10.6; MS (ESI) m/z (%): 279.1 [M þ H]þ.
Anal. Calcd for C13H14N2O5: C 56.11; H 5.07; N 10.07. Found: C 55.93;
H 5.12; N 9.81%.
4.1.11. (4,6-Dimethoxy-3-methylbenzofuran-2-yl)methanol (14)
To a stirred suspension of lithium aluminum hydride (0.86 g,
22.7 mmol) in anhydrous THF (200 mL) was added dropwise a
solution of compound 3 (5.00 g, 18.9 mmol) in anhydrous THF
(100 mL) at room temperature under nitrogen atmosphere. The
mixture was stirred at room temperature for 15 min and quenched
with aqueous saturated NaHCO3 solution (300 mL). The aqueous
layer was extracted with ethyl acetate (2 ꢃ 100 mL). The combined
organic layers were dried over sodium sulfate and evaporated un-
der reduced pressure to afford 14 (3.99 g, 95%) as a white powder.
White powder. Yield 90% (115 mg). Rf 0.77 (CH2Cl2/EtOH, 9:1);
Mp: 237.7 ꢁC; IR (KBr)
n
3369, 3191, 2921, 1684, 1635, 1540, 1442,
1316, 1255, 1215, 1144, 1120, 979, 807, 749, 687 cmꢀ1 1H NMR
(400 MHz, DMSO-d6):
9.96 (s, 1H, eNHe), 7.71 (d, 2H, J ¼ 7.6 Hz,
2 and 6-phenyl), 7.63 (s, 1H, eNH2), 7.45 (s, 1H, eNH2), 7.35 (dd, 2H,
;
d
H
J ¼ 7.4 and J ¼ 7.6 Hz, H3 and 5-phenyl), 7.11 (t, 1H, J ¼ 7.4 Hz, H4-phenyl),
6.63 (s, 1H, Har), 3.98 (s, 3 H, eOCH3), 3.92 (s, 3H, eOCH3), 2.63 (s,
3H, 3eCH3); 13C NMR (100 MHz, DMSO-d6):
d
164.0, 158.2, 157.8,
Rf 0.16 (CH2Cl2); Mp: 109.2 ꢁC; IR (KBr)
1623, 1601, 1507, 1454, 1417, 1389, 1319, 1262, 1216, 1198, 1155, 1112,
996, 812, 792 cm-1 1H NMR (400 MHz, CDCl3):
6.55 (d, 1H,
n 3337, 3240, 2992, 2933,
156.4, 152.1, 141.5, 138.2, 128.6 (2 C), 123.8, 122.1, 120.5 (2 C), 112.2,
103.9, 91.8, 56.8, 56.1, 10.7; MS (ESI) m/z (%): 355.1 [M þ H]þ. Anal.
Calcd for C19H18N2O5: C 64.40; H 5.12; N 7.91. Found: C 64.48; H
5.21; N 7.84%.
;
d
J ¼ 1.9 Hz, Har), 6.26 (d, 1H, J ¼ 1.9 Hz, Har), 4.67 (d, 2H, J ¼ 5.5 Hz, e
CH2OH), 3.85 (s, 3H, eOCH3), 3.83 (s, 3H, eOCH3), 2.33 (s, 3H, 3e
CH3), 1.69 (t, 1H, J ¼ 5.5 Hz, eOH); 13C NMR (100 MHz, CDCl3):
4.1.9.2. 4,6-Dimethoxy-3-methyl-N2-pyridin-2-yl-1-benzofuran-2,7-
dicarboxamide (11). Compound 11was obtained following the
representative procedure described for compound 10. Beige
d 159.2, 156.2, 155.3, 148.8, 113.4, 112.7, 93.9, 88.0, 55.7, 55.5, 55.4,
9.8; MS (ESI) m/z (%): 205.0 [M þ HeH2O]þ. Anal. Calcd for
C12H14O4: C 64.85; H 6.35. Found: C 64.86; H 6.01%.

830
M.-A. Bazin et al. / European Journal of Medicinal Chemistry 69 (2013) 823e832
4.1.12. 4,6-Dimethoxy-3-methyl-1-benzofuran-2-carbaldehyde
(15)
dropwise boron tribromide (1 M in CH2Cl2, 3.0 mL, 3.0 mmol). The
mixture was then refluxed for 10 h. The reaction mixture was
diluted with water and EtOAc. The organic phase was separated,
dried over sodium sulfate and evaporated under reduced pressure.
The crude product was purified by silica gel chromatography using
dichloromethane/EtOH 19:1 as eluent to afford 21 (15 mg, 21%) as a
beige powder.
To a stirred solution of compound 14 (3.50 g, 15.7 mmol) in
distilled dichloromethane (100 mL) was added activated man-
ganese(IV) oxide (8.22 g, 94.5 mmol). The mixture was refluxed for
2 h. The reaction mixture was filtered on CeliteÒ and washed with
dichloromethane. The organic filtrate was evaporated in vacuo to
give 15 (3.40 g, 98%) as a yellow powder.
Rf 0.24 (CH2Cl2/EtOH, 19:1); Mp > 360 ꢁC; IR (KBr)
1659, 1612, 1393, 1327, 1238, 1190, 827 cmꢀ1 1H NMR (400 MHz,
DMSO-d6): 14.14 (bs, 1H, eOH), 11.54 (bs, 1H, eOH), 9.92 (s, 1H, e
CHO), 8.36 (bs, 1H, eNH2), 7.44 (bs, 1H, eNH2), 6.25 (s, 1H, Har), 2.68
(s, 3H, 3eCH3); 13C NMR (100 MHz, DMSO-d6):
179.9, 171.0, 168.0,
n 3454, 3177,
Rf 0.37 (CH2Cl2); Mp: 154.3 ꢁC; IR (KBr)
1623, 1601, 1500, 1451, 1319, 1261, 1221, 1147, 1117, 1098, 868, 813,
779 cmꢀ1; 1H NMR (400 MHz, CDCl3):
9.81 (s, 1H, eCHO), 6.55 (d,
n
2964, 2927, 2841, 1672,
;
d
d
1H, J ¼ 1.6 Hz, Har), 6.25 (d, 1H, J ¼ 1.6 Hz, Har), 3.88 (s, 3H, eOCH3),
d
3.84 (s, 3H, eOCH3), 2.66 (s, 3H, 3eCH3); 13C NMR (100 MHz,
160.7, 156.0, 147.9, 131.7, 111.7, 99.8, 93.3, 10.8; MS (ESI) m/z (%):
236.0 [M þ H]þ. Anal. Calcd for C11H9NO5: C 56.18; H 3.86; N 5.96.
Found: C 55.89; H 3.70; N 6.22%.
CDCl3):
d 177.4, 163.3, 158.1, 157.3, 147.2, 112.8, 95.1, 87.7, 77.2, 55.8,
55.6, 10.1; MS (ESI) m/z (%): 221.0 [M þ H]þ. Anal. Calcd for
C
12H12O4: C 65.45; H 5.49. Found: C 65.30; H 5.72%.
4.1.17. Phloroglucinol triacetate (22)
4.1.13. 7-Bromo-4,6-dimethoxy-3-methyl-1-benzofuran-2-
carbaldehyde (16)
Compound 16 was obtained following the same procedure as
described for compound 4. The crude product was purified by silica
gel chromatography using dichloromethane as eluent to afford 16
(1.26 g, 79%) as a yellow powder.
To a solution of phloroglucinol (10.0 g, 79.2 mmol) in pyridine
(50 mL) was added acetic anhydride (30 mL, 317.2 mmol). The
mixture was stirred and refluxed for 4 h. Pyridine was then
removed under reduced pressure and the residue was washed with
diisopropyl ether to give 22 (19.6 g, 98%) as a white powder.
Rf 0.23 (cyclohexane/EtOAc 5:1); Mp: 106.6 ꢁC (litt [32]. 106e
Rf 0.41 (CH2Cl2); Mp: 180e181 ꢁC;IR(KBr)
n
3075, 2926,1661,1612,
1587,1319,1258,1217,1126,1094, 826, 793 cmꢀ1; 1H NMR (400 MHz,
DMSO-d6): 9.88 (s, 1H, eCHO), 6.71 (s, 1H, Har), 4.02 (s, 3H, eOCH3),
4.01 (s, 3H, eOCH3), 2.65 (s, 3H, 3eCH3); 13C NMR (100 MHz, DMSO-
107 ꢁC); IR (KBr)
n
3105, 1757, 1601, 1366, 1182, 1121, 1018, 914 cmꢀ1
6.84 (s, 3H, Har), 2.28 (s, 9H, eCH3); 13
;
C
1H NMR (400 MHz, CDCl3):
d
d
NMR (100 MHz, CDCl3): d 168.7 (C]O),151.2 (CareO), 112.9 (CareH),
21.2 (eCH3); MS (ESI) m/z (%): 251.1 [M ꢀ H]ꢀ.
d6): d 178.5,158.5,156.7,153.8,147.2,131.0,112.5, 93.0, 83.2, 57.4, 56.6,
10.0; MS (ESI) m/z (%): 299.0 [M þ H]þ, 301.0 [M þ Hþ2]þ. Anal. Calcd
4.1.18. Phloroglucinol tribenzyl ether (23)
for C12H11BrO4: C 48.18; H 3.71. Found: C 48.07; H 3.93%.
A mixture of phloroglucinol triacetate 22 (3.0 g, 11.9 mmol),
benzyl chloride (4.93 mL, 42.8 mmol) and sodium hydride (60% in
mineral oil) (3.43 g, 85.7 mmol) in DMF (60 mL) was cooled to 0 ꢁC
and water (0.64 mL, 35.7 mmol) was added dropwise over a period
of 10 min. The reaction mixture was stirred overnight at room
temperature poured into water. The aqueous layer was extracted
with ethyl acetate and organic layer was washed with brine, dried
over Na2SO4 and filtered. The solvent was evaporated in vacuo and
the residue was recrystallized from methanol to give 23 (3.12 g,
78%) as a white powder.
4.1.14. 2-Formyl-4,6-dimethoxy-3-methyl-1-benzofuran-7-
carbonitrile (17)
Compound 17 was obtained following the same procedure as
described for compound 5. The crude product was purified by silica
gel chromatography using dichloromethane as eluent to afford 17
(732 mg, 50%) as an orange powder.
Rf 0.34 (cyclohexane/ethyl acetate 1:1); Mp: 263.9 ꢁC; IR (KBr)
n
2227, 1653, 1617, 1595, 1506, 1436, 1322, 1261, 1224, 1172, 1123, 970,
810 cm-1; 1H NMR (400 MHz, DMSO-d6):
d
9.88 (s, 1H, eCHO), 6.74
Rf 0.66 (cyclohexane/EtOAc 4:1); Mp: 88.5 ꢁC (lit [33]. 93.0e
(s, 1H, Har), 4.07 (2 ꢃ s, 2 ꢃ 3H, 2 ꢃ eOCH3), 2.63 (s, 3H, 3eCH3); 13
C
94.0 ꢁC); IR (KBr)
n
2922, 1593, 1449, 1377, 1155, 1063, 1026, 804,
1H NMR (400 MHz, CDCl3):
7.33e7.43 (m, 15H,
Ar), 6.28 (s, 3H, H2,4,6), 5.01 (s, 6H, eCH2); 13C NMR (100 MHz,
NMR (100 MHz, DMSO-d6):
d
178.4, 165.4, 161.4, 156.3, 147.5, 112.8,
727, 692 cmꢀ1
;
d
112.0, 103.2, 92.1, 77.2, 57.6, 57.1, 9.6; MS (ESI) m/z (%): 246.0
[M þ H]þ. Anal. Calcd for C13H11NO4: C 63.67; H 4.52; N 5.71. Found:
C 63.98; H 4.60; N 5.79%.
H
CDCl3): d 160.6 (3 C), 136.8 (3 C), 128.6 (6 CH), 128.0 (3 CH), 127.6 (6
CH), 94.9 (3 CH), 70.1 (3-CH2); MS (ESI) m/z (%): 397.2 [M þ H]þ.
4.1.15. 2-Formyl-4,6-dimethoxy-3-methyl-1-benzofuran-7-
carboxamide (18)
4.1.19. 2,4,6-Tribenzyloxyacetophenone (24)
Acetic acid (2.2 mL, 38.4 mmol) and TFAA (6.4 mL, 45.3 mmol)
were stirred for 5 min at room temperature. Phloroglucinol tri-
benzyl ether 23 (5.0 g, 12.6 mmol) was dissolved in CH2Cl2
(200 mL) and then added at 0 ꢁC. After 1.5 h, the deep purple
solution was diluted with AcOEt and poured into saturated aq.
NaHCO3 solution. The yellow organic layer was washed with brine,
dried with Na2SO4 and concentrated under reduced pressure. The
crude product was purified by silica gel chromatography using
dichloromethane/cyclohexane 6:4 as eluent to afford 24 (4.1 g,
75%) as a yellowish oil.
Compound 18 was obtained following the same procedure as
described for compound 6. The crude product was purified by silica
gel chromatography using dichloromethane/EtOH 9:1 as eluent to
afford 18 (296 mg, 44%) as a yellowish powder.
Rf 0.49 (CH2Cl2/EtOH 9:1); Mp: 260.1 ꢁC; IR (KBr)
n
3381, 3185,
2927, 1727, 1653, 1607, 1571, 1316, 1261, 1218, 1144, 1120, 816 cmꢀ1
1H NMR (400 MHz, DMSO-d6):
9.84 (s, 1H, eCHO), 7.63 (s, 1H, e
;
d
NH2), 7.50 (s,1H, eNH2), 6.63 (s,1H, Har), 3.99 (s, 3H, eOCH3), 3.93 (s,
3H, eOCH3), 2.64 (s, 3H, 3eCH3); 13C NMR (100 MHz, DMSO-d6):
d
178.2, 164.1, 159.3, 157.4, 154.0, 147.1, 127.5, 111.7, 104.3, 91.7, 56.8,
Rf 0.43 (cyclohexane/EtOAc 4:1); IR (KBr)
1599, 1584, 1431, 1153, 1111, 735, 694 cmꢀ1
CDCl3): 7.26e7.39 (m, 15H, HAr), 6.24 (s, 2H, H3,5), 5.04 (s, 4H, 2-
CH2), 4.99 (s, 2H, eCH2), 2.47 (s, 3H, eCOCH3); 13C NMR
(100 MHz, CDCl3): 201.6 (C]O), 161.1, 157.2 (2 C), 136.5 (2 C),
136.4, 128.7 (2 C), 128.6 (4 C), 128.2, 128.0 (2 C), 127.5 (2 C), 127.1
(4 C), 115.1, 93.5 (2 C), 70.6 (2 C), 70.3, 32.7 (eCOCH3); MS (ESI) m/z
(%): 439.1 [M þ H]þ.
n
3032, 2870, 1692,
56.3, 9.8; MS (ESI) m/z (%): 264.0 [M þ H]þ. Anal. Calcd for
;
1H NMR (400 MHz,
C
13H13NO5: C 59.31; H 4.98; N 5.32. Found: C 59.34; H 4.86; N 5.44%.
d
4.1.16. 2-Formyl-4,6-dihydroxy-3-methyl-1-benzofuran-7-
carboxamide (21)
d
To a stirred solution of compound 18 (80 mg, 0.3 mmol) in dry
dichloromethane (6 mL) stirring under argon at ꢀ10 ꢁC was added

M.-A. Bazin et al. / European Journal of Medicinal Chemistry 69 (2013) 823e832
831
4.1.20. 2-Hydroxy-4,6-dibenzyloxyacetophenone (25)
and evaporated under reduced pressure. The crude product was
purified by silica gel chromatography using dichloromethane as
eluent to afford 28 (506 mg, 83%) as a white powder.
TiCl4 (1 M in CH2Cl2, 6.3 mL, 6.3 mmol) was added dropwise
at ꢀ5 ꢁC to a stirred solution of 24 (4.6 g, 10.5 mmol) in CH2Cl2
(70 mL). After 1.5 h of stirring, the mixture was slowly poured into
aqueous saturated NaHCO3, washed with water and brine, dried
over Na2SO4 and concentrated under reduced pressure. The crude
product was purified by silica gel chromatography using dichloro-
methane/cyclohexane 1:3 as eluent to afford 25 (3.1 g, 84%) as a
white powder after trituration in methanol.
Rf 0.25 (CH2Cl2); Mp: 106e108 ꢁC; IR (KBr)
n
2920, 1665, 1612,
1591, 1319, 1254, 1233, 1163, 1092, 733, 696, 660 cmꢀ1 1H NMR
(400 MHz, CDCl3): 9.84 (s, 1H, eCHO), 7.37e7.45 (m, 10H, Hbenzyl),
;
d
6.66 (d, 1H, J ¼ 1.8 Hz, H7), 6.45 (d, 1H, J ¼ 1.8 Hz, H5), 5.14 (s, 2H, e
CH2), 5.09 (s, 2H, eCH2), 2.69 (s, 3H, 3eCH3); 13C NMR (100 MHz,
CDCl3):
d 177.4, 162.2, 158.0, 156.2, 147.3, 136.1, 128.7 (4 C), 128.3,
Rf 0.28 (cyclohexane/CH2Cl2 1:1); Mp: 102e104 ꢁC (lit [21].
128.2, 127.6 (2 C), 127.3 (2 C), 113.1, 96.7, 89.3, 70.7, 70.4, 10.3; MS
(ESI) m/z (%): 373.1 [M þ H]þ. Anal. Calcd for C24H20O4: C 77.40; H
5.41. Found: C 77.28; H 5.76%.
104 ꢁC); IR (KBr)
n
1614, 1587, 1265, 1171, 1103, 1080, 826, 727,
692 cmꢀ1
;
1H NMR (400 MHz, CDCl3):
d
14.03 (s, 1H, eOH), 7.26e
7.41 (m, 10H, HAr), 6.16 (d, J ¼ 2.3 Hz, 1H, Har), 6.10 (d, J ¼ 2.3 Hz, 1H,
Har), 5.06 (sapp, 4H, 2-CH2), 2.55 (s, 3H, eCOCH3); 13C NMR
4.1.23. General procedure of debenzylation e synthesis of
compounds 29, 30 and 32
(100 MHz, CDCl3):
d 203.2 (C]O), 167.6, 165.1, 162.0, 135.9, 135.6,
128.8 (2 C), 128.7 (2 C), 128.5, 128.4, 128.0 (2 C), 127.7 (2 C), 106.3,
94.7, 92.4, 71.1, 70.3, 33.3 (eCOCH3); MS (ESI) m/z (%): 349.2
[M þ H]þ.
To
a
solution of 4,6-dibenzyloxybenzofurane derivative
(0.24 mmol) in a mixed solution of MeOH (2.5 mL), THF (2.5 mL)
and cyclohexene (1 mL) was added Pd/C 10% (102 mg, 0.10 mmol).
The suspension was refluxed for 5 h (compound 29) or for 2 h
(compounds 30 and 32). Pd/C was filtered on CeliteÒ, and the
filtrate was concentrated in vacuo. The crude product was purified
by silica gel chromatography using dichloromethane as eluent to
afford 29, and was triturated in dichloromethane to obtain com-
pounds 30 and 32.
4.1.21. Ethyl 4,6-dibenzyloxy-3-methyl-1-benzofuran-2-
carboxylate (26) and 4,6-dibenzyloxy-3-methyl-1-benzofurane (27)
To a stirred solution of compound 25 (3.34 g, 9.6 mmol) in DMF
(20 mL) were successively added ethyl bromoacetate (1.17 mL,
10.5 mmol) and potassium carbonate (3.98 g, 28.8 mmol). The re-
action mixture was heated to 70 ꢁC for 2 h, then to 100 ꢁC for 20 h.
The reaction mixture was cooled to room temperature and filtered
on CeliteÒ. The solution was diluted with ethyl acetate (200 mL)
and the organic layer was washed with brine and water, dried over
sodium sulfate and evaporated under reduced pressure. The crude
product was purified by silica gel chromatography using cyclo-
hexane/dichloromethane 4:1 as eluent to afford 26 (2.08 g, 52%) as
a white powder and 27 (0.65 g, 20%) as a white powder.
4.1.23.1. Ethyl 4,6-dihydroxy-3-methyl-1-benzofuran-2-carboxylate
(29). Brown solid. Yield 92% (52 mg). Rf 0.16 (CH2Cl2/EtOH, 19:1);
Mp: 220e223 ꢁC; IR (KBr)
n
3329, 1680, 1620, 1329, 1169, 1152, 1082,
835, 627 cm-1; 1H NMR (400 MHz, DMSO-d6):
d
10.28 (s, 1H, eOH),
9.82 (s, 1H, eOH), 6.39 (s, 1H, Har), 6.25 (s, 1H, Har), 4.31 (q, 2H,
J ¼ 7.1 Hz, eCH2CH3), 2.63 (s, 3H, 3eCH3), 1.34 (t, 3H, J ¼ 7.1 Hz, e
CH2CH3); 13C NMR (100 MHz, DMSO-d6):
d 159.6 (2 C), 156.7, 154.5,
137.3, 126.8, 110.3, 98.1, 88.9, 60.1, 14.2, 10.9; MS (ESI) m/z (%): 237.1
[M þ H]þ. Anal. Calcd for C12H12O5: C 61.02; H 5.12. Found: C 60.74;
H 5.15%.
4.1.21.1. Ethyl 4,6-dibenzyloxy-3-methyl-1-benzofuran-2-carboxylate
(26). Rf 0.27 (cyclohexane/CH2Cl2, 4:1); Mp: 103e105 ꢁC; IR (KBr)
n
1707, 1616, 1595, 1273, 1150, 1096, 1063, 1024, 806, 739, 698 cmꢀ1
;
1H NMR (400 MHz, CDCl3):
d
7.34e7.44 (m,10H, Hbenzyl), 6.68 (d,1H,
4.1.23.2. 4,6-Dihydroxy-3-methyl-1-benzofuran-2-carbaldehyde
J ¼ 1.4 Hz, H7), 6.45 (d, 1H, J ¼ 1.4 Hz, H5), 5.13 (s, 2H, eCH2), 5.07 (s,
2H, eCH2), 4.41 (q, 2H, J ¼ 7.1 Hz, eCH2CH3), 2.70 (s, 3H, 3eCH3),
1.41 (t, 3H, J ¼ 7.1 Hz, eCH2CH3); 13C NMR (100 MHz, CDCl3):
(30). Brown powder. Yield 61% (26 mg). Rf 0.45 (CH2Cl2/EtOH,
19:1); Mp > 360 ꢁC; IR (KBr)
n
3464, 1593, 1302, 1140, 1072, 818,
789 cmꢀ1
;
1H NMR (400 MHz, DMSO-d6):
d
10.52 (bs, 1H, eOH),
d
160.6, 160.5, 156.6, 155.5, 139.2, 136.4 (2 C), 128.7 (2 C), 128.6 (2 C),
10.10 (bs, 1H, eOH), 9.78 (s, 1H, eCHO), 6.38 (d, 1H, J ¼ 1.7 Hz, Har),
128.2, 128.1, 127.6 (2 C), 127.2 (3 C), 113.2, 96.4, 89.4, 70.6, 70.2, 60.7,
6.26 (d, 1H, J ¼ 1.7 Hz, Har), 2.65 (s, 3 H, 3eCH3); 13C NMR (100 MHz,
14.5, 11.4; MS (ESI) m/z (%): 417.2 [M þ H]þ. Anal. Calcd for
DMSO-d6): d 177.2, 161.2, 157.9, 155.6, 146.1, 131.6, 110.4, 98.3, 88.9,
C
26H24O5: C 74.98; H 5.81. Found: C 75.09; H 5.66%.
9.6; MS (ESI) m/z (%): 193.1 [M þ H]þ. Anal. Calcd for C10H8O4: C
62.50; H 4.20. Found: C 62.80; H 4.16%.
4.1.21.2. 4,6-Dibenzyloxy-3-methyl-1-benzofurane (27). Rf 0.92
(CH2Cl2); Mp: 132e134 ꢁC; IR (KBr)
2924, 1616, 1497, 1452, 1377,
1314, 1148, 1117, 1094, 1065, 1024, 808, 737, 696 cmꢀ1 1H NMR
(400 MHz, CDCl3): 7.32e7.40 (m, 10H, Hbenzyl), 7.19 (d, 1H,
n
4.1.23.3. Ethyl 7-carbamoyl-4,6-dihydroxy-3-methyl-1-benzofuran-
;
2-carboxylate (32). Brown powder. Yield 61% (55 mg). Rf 0.61
d
(CH2Cl2/EtOH, 19:1); Mp: 296e297 ꢁC; IR (KBr)
n
3458, 3140, 1659,
1614, 1331, 1244, 1169, 1009, 829, 766, 739 cmꢀ1 1H NMR
(400 MHz, DMSO-d6): 13.80 (s, 1H, eOH),11.39 (bs, 1H, eOH), 8.30
J ¼ 1.0 Hz, H2), 6.68 (d, 1H, J ¼ 1.8 Hz, H7), 6.46 (d, 1H, J ¼ 1.8 Hz, H5),
;
5.12 (s, 2H, eCH2), 5.07 (s, 2H, eCH2), 2.34 (d, 3H, J ¼ 1.0 Hz, 3e
d
CH3); 13C NMR (100 MHz, CDCl3):
d
157.9, 157.3, 154.1, 139.2, 137.0,
(bs, 1H, eNH), 7.14 (bs, 1H, eNH), 6.19 (s, 1H, H5), 4.32 (q, 2H,
J ¼ 7.1 Hz, eCH2CH3), 2.61 (s, 3H, 3eCH3), 1.32 (t, 3H, J ¼ 7.1 Hz, e
136.9, 128.6 (2 C), 128.5 (2 C), 128.0, 127.8, 127.6 (2 C), 127.1 (2 C),
115.8, 112.6, 95.6, 89.8, 70.6, 70.0, 10.0; MS (ESI) m/z (%): 345.1
[M þ H]þ. Anal. Calcd for C23H20O3: C 80.21; H 5.85. Found: C 80.60;
H 5.98%.
CH2CH3); 13C NMR (100 MHz, DMSO-d6):
d 169.9, 165.5, 159.0 (2 C),
153.6,138.1,126.8,110.3, 98.4, 92.0, 60.5,14.2,10.7; MS (ESI) m/z (%):
280.2 [M þ H]þ. Anal. Calcd for C13H13NO6: C 55.92; H 4.69; N 5.02.
Found: C 56.13; H 4.61; N 5.04%.
4.1.22. 4,6-Dibenzyloxy-3-methyl-1-benzofuran-2-carbaldehyde
(28)
4.1.24. Ethyl 4,6-dibenzyloxy-7-carbamoyl-3-methyl-1-
benzofuran-2-carboxylate (31)
Phosphorus oxychloride (145 mL, 1.55 mmol) and DMF (2.1 mL)
were stirred at room temperature for 1 h under argon atmosphere.
Compound 27 (465 mg, 1.35 mmol) was then added portionwise
and the resulting mixture was stirred overnight, quenched with 8 N
aqueous NaOH. The reaction mixture was extracted with ethyl ac-
etate (50 mL) and the organic layer was dried over sodium sulfate
To a stirred suspension of compound 26 (1.50 g, 3.60 mmol) in
acetonitrile (60 mL) at 0 ꢁC under argon was added chlorosulfonyl
isocyanate (495 mL, 5.68 mmol). The mixture was then warmed to
room temperature for 5 h and the reaction mixture was quenched
with 1 M HCl. After 1 h at room temperature, the precipitate was

832
M.-A. Bazin et al. / European Journal of Medicinal Chemistry 69 (2013) 823e832
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filtered and washed with water to afford 31 (1.44 g, 87%) as a white
powder.
Rf 0.26 (CH2Cl2/EtOH, 19:1); Mp: 177e179 ꢁC; IR (KBr)
n
3385,
3211, 1701, 1632, 1611, 1275, 1138, 1113, 1067, 723, 692, 617 cmꢀ1; 1H
NMR (400 MHz, DMSO-d6): 7.73 (bs, 1H, eNH), 7.36e7.56 (m, 11H,
benzyl and eNH), 6.92 (s, 1H, H5), 5.36 (s, 2H, eCH2), 5.30 (s, 2H, e
d
H
CH2), 4.36 (q, 2H, J ¼ 7.1 Hz, eCH2CH3), 2.68 (s, 3H, 3eCH3), 1.35 (t,
3H, J ¼ 7.1 Hz, eCH2CH3); 13C NMR (100 MHz, DMSO-d6):
d 164.3,
159.3, 156.8, 155.1, 152.7, 138.9, 136.7, 136.3, 128.6 (2 C), 128.4 (2 C),
128.0, 127.8, 127.5 (4 C), 125.8, 112.3, 105.5, 94.4, 70.8, 70.0, 60.5, 14.2,
11.0; MS (ESI) m/z (%): 460.1 [M þ H]þ. Anal. Calcd for C27H25NO6: C
70.58; H 5.48; N 3.05. Found: C 70.69; H 5.55; N 2.92%.
4.2. Biological evaluation
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4.2.1. Cytotoxicity activity assays (NSCLC-N6 and A549)
The antiproliferative activity of compounds 3-6, 8e13, 15e18, 21,
26, 28e32 was evaluated. The NSCLC-N6 cell line [26], derived from a
human non-small-cell bronchopulmonary carcinoma (moderately
differentiated, rarely keratinized, classified as T2N0M0), and A549
obtained from ATCC collection reference CCL-185 [27] were used for
all experiments. Both cell lines were cultured in RPMI 1640 medium
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4 from 2-hydroxy-3-bromo-4,6-
dimethoxyacetophenone [15] using the procedure described for compound
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with 5% fetal calf serum, to which were added 100 IU penicillin.mLꢀ1
100
g streptomycin.mLꢀ1 and 2 mM glutamine, at 37 ꢁC in an air/
,
m
carbon dioxide atmosphere (95:5, v/v). Cytotoxicity was determined
by continuous drug exposure. Experiments were performed in 96
wells microtiter plates (105 cells mLꢀ1 for NSCLC-N6 and 2 ꢃ10ꢀ4 cells
mLꢀ1 for A549). Cell growth was estimated by a colorimetric assay
based on the conservation of tetrazolium dye (MTT) to a blue for-
mazan product by live mitochondria [34]. Eight repeats were per-
formed for each concentration. Control growth was estimated from 8
determinations. Optical density at 570 nm corresponding to solubi-
lized formazan was read for each well on a Titertek Multiskan MKII.
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Appendix A. Supplementary data
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