Synthesis, biological activity, and evaluation of the mode of action of novel antitubercular benzofurobenzopyrans substituted on A ring

Article abstract of DOI:10.1016/j.ejmech.2010.09.048

The 8-, 9-, 10-, and 11-halo, hydroxy, and methoxy derivatives of the antimycobacterial 3,3-dimethyl-3H-benzofuro[3,2-f][1]benzopyran were synthesized by condensation of the diazonium salts of 2-chloroanilines (13-17) with 1,4-benzoquinone (18), reduction of the intermediate phenylbenzoquinones 19-22 to dihydroxybiphenyls, cyclisation to halo-2-hydroxydibenzofurans 24-27, and construction of the pyran ring by thermal rearrangement of the corresponding dimethylpropargyl ethers 35-38. Palladium catalyzed nucleophilic aromatic substitution permitted conversion of the halo to the corresponding hydroxy derivatives which were methylated to methoxy-3,3-dimethyl-3H-benzofuro[3,2-f][1] benzopyran. All compounds substituted on the A ring were found more potent than the reference compound 1 against Mycobacterium bovis BCG and the virulent strain Mycobacterium tuberculosis H37Rv. The effect of the most active derivatives on mycolate synthesis was explored in order to confirm the preliminary hypothesis of an effect on mycobacterial cell wall biosynthesis. The linear 9-methoxy-2,2-dimethyl-2H-benzofuro[2,3-g][1]benzopyran (46) exhibiting a good antimycobacterial activity and devoid of cytotoxicity appeared to be the most promising compound.

Full text of DOI:10.1016/j.ejmech.2010.09.048

European Journal of Medicinal Chemistry 45 (2010) 5833e5847
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, biological activity, and evaluation of the mode of action of novel
antitubercular benzofurobenzopyrans substituted on A ring
a
a
a
b
c
Aikaterini Termentzi , Inana Khouri , Thomas Gaslonde , Soizic Prado , Brigitte Saint-Joanis ,
Fabienne Bardou , Elsa P. Amanatiadou , Ioannis S. Vizirianakis , Jana Kordulakova , Mary Jackson ,g,
Roland Brosch , Yves L. Janin , Mamadou Daffé , François Tillequin , Sylvie Michel
Laboratoire de Pharmacognosie, Université Paris Descartes, UMR/CNRS 8638, Faculté des Sciences Pharmaceutiques et Biologiques, 4 avenue de l’Observatoire, 75006 Paris, France
Molécules de Communication et Adaptation des Micro-organismes, FRE 3206 CNRS/MNHN Muséum National d’Histoire Naturelle, 57 rue Cuvier (CP54), 75005 Paris, France
Unité de Génétique Moléculaire Bactérienne, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France
d
e
e
f
c
c
h
d
a
a,
*
a
b
c
d
CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), Département “Mécanismes Moléculaires des Infections Mycobactériennes”, 205 Route de Narbonne,
F-31077 Toulouse & Université de Toulouse, UPS, IPBS, F-31077 Toulouse, France
e
Laboratory of Pharmacology, Department of Pharmaceutical Sciences, Aristotle University of Thessaloniki, GR-54124 Greece
Department of Biochemistry, Faculty of Natural Sciences, Comenius University, Mlynska dolina, 84215 Bratislava, Slovakia
Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523-1682, USA
Institut Pasteur, Unité de Chimie Organique, URA 2128-CNRS, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France
f
g
h
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 July 2010
Received in revised form
The 8-, 9-, 10-, and 11-halo, hydroxy, and methoxy derivatives of the antimycobacterial 3,3-dimethyl-3H-
benzofuro[3,2-f][1]benzopyran were synthesized by condensation of the diazonium salts of 2-chloroani-
lines (13e17) with 1,4-benzoquinone (18), reduction of the intermediate phenylbenzoquinones 19e22 to
dihydroxybiphenyls, cyclisation to halo-2-hydroxydibenzofurans 24e27, and construction of the pyran
ring by thermal rearrangement of the corresponding dimethylpropargyl ethers 35e38. Palladium cata-
lyzed nucleophilic aromatic substitution permitted conversion of the halo to the corresponding hydroxy
derivatives which were methylated to methoxy-3,3-dimethyl-3H-benzofuro[3,2-f][1]benzopyran. All
compounds substituted on the A ring were found more potent than the reference compound 1 against
Mycobacterium bovis BCG and the virulent strain Mycobacterium tuberculosis H37Rv. The effect of the most
active derivatives on mycolate synthesis was explored in order to confirm the preliminary hypothesis of an
effect on mycobacterial cell wall biosynthesis. The linear 9-methoxy-2,2-dimethyl-2H-benzofuro[2,3-g][1]
benzopyran (46) exhibiting a good antimycobacterial activity and devoid of cytotoxicity appeared to be the
most promising compound.
16 September 2010
Accepted 17 September 2010
Available online 1 October 2010
Keywords:
Antimycobacterials
3,3-Dimethyl-3H-benzofuro[3,2-f][1]
benzopyran
Structure-activity relationship
Mycolates
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
interesting as they displayed little cytotoxicity in mammalian VERO
cells, stimulating further pharmacomodulation studies in this
Tuberculosis remains a serious and highly contagious infectious
disease, which currently represents the second leading cause of
death worldwide. Although a renewal of research in this field is
currently taking place [1e3], there is still an urgent need for the
discovery of novel antitubercular agents. In our continuous search
for such compounds, previous work in our group associated two
functional “natural like” antimicrobial structures, dibenzofuran and
series. However, as compounds 1 and 2 had no activity in vivo [5],
further investigation in the benzofurobenzopyran series was made
in attempts to improve the activity and/or bioavailability. To this
goal, the exploration of the putative mechanism of action of this
series at both cellular and molecular levels also appeared as an
important issue. In this respect, the specific activity of compounds 1
and 2 against Mycobacterium and Nocardia asteroids [4], which share
in common related cell wall components, suggested that their
activity should lie in the perturbation of the biosynthesis of mycolic
acids present in the cell walls of both microorganisms.
2
,2-dimethylpyran subunits, to successfully create a hybrid
compound 1 and its reduced analogue 2, that showed promising
antitubercular activity in vitro [4] (Fig.1). Both compounds appeared
Previous attempts towards the establishment of structure-activity
relationships in the benzofurobenzopyran series led the synthesis of
several related compounds (Fig. 2). Replacementof the furanB ring by
an ether linkage (3), a single carbonecarbon bond, a carbonyl group,
*
Corresponding author. Tel.: þ33 1 53 73 98 03; fax: þ33 1 40 46 96 58.
E-mail address: sylvie.michel@parisdescartes.fr (S. Michel).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.09.048

5834
A. Termentzi et al. / European Journal of Medicinal Chemistry 45 (2010) 5833e5847
rearrangement of a propargylic ether, since this method had previ-
ously given satisfactory results in several related series [9e11].
2
2
.1.1. Synthesis of the biphenyls
.1.1.1. Synthesis of biphenyls through palladium catalyzed reac-
tions. Several experiments were carried out to get the required
biphenyls through Pd catalyzed SuzukieMiyaura reaction between
2
-bromohydroquinone (9) and various 2-chlorophenylboronic acid
Fig. 1. Chemical structure of 3,3-dimethyl-3H-benzofuro[3,2-f][1]benzopyran (1) and
its reduced analogue (2).
(Scheme 1) [12,13]. The use of Pd/C with different base and solvent
systems did not give the expected products. In contrast the use of
Pd(PPh
3
)
4
in toluene:H
2
O (5:1), in the presence of Na
2
CO
3
, with the
a hydroxymethylene group or a methylene gave either inactive or
cytotoxic products [5]. Also, several related “seco” compounds
including a pyridine ring (4), although displaying interesting anti-
mycobacterial activities, were found to be also cytotoxic thus
discouraging further research in this direction [6,7]. These data
strongly suggested that the tetracyclic benzofurobenzopyran core
was an indispensable structural component for the retention of the
biological activity.
Modifications, such as bromination (5) or acylation on position 5
of C ring of the benzofurobenzopyran skeleton resulted in inactive
derivatives [5]. Concerning the substitution on the A ring, acylation
at position 10 and nitration at position 8 also gave inactive
compounds, against both Mycobacterium smegmatis and Mycobac-
terium tuberculosis (6 & 7) [5,8]. In contrast, the heterocyclic
analogue with a nitrogen atom at position 10 (8) was a relatively
0
0
0
phenylboronic acid 10 gave the desired 2 -chloro-4 -methoxy[1,1 -
biphenyl]-2,5-diol (11), (6% yield) and an oxidized product 2-(2-
chloro-4-methoxyphenyl)-1,4-benzoquinone (12) (19%).
Consequently, the need for large quantities of biphenyls as
starting materials led to envisage an alternate synthesis involving
a more versatile, efficient and cheaper method (Scheme 2).
2
.1.1.2. Synthesis of biphenyls 24e28 through intermediate aryl-
benzoquinones. Following a reaction scheme previously developed
by Schimmelschmidt [14], the formation of 2-hydroxydibenzofur-
ans conveniently substituted on the C ring was envisioned through
a three steps process involving (i) formation of an aryl-1,4-benzo-
quinone through aromatic nucleophilic substitution of a diazonium
salt with p-benzoquinone, (ii) reduction to the corresponding p-
diphenol, and (iii) cyclisation into 2-hydroxybenzofuran in alkaline
medium.
potent antitubercular agent with MICs95 of 8
mg/ml and 16 mg/ml
against M. smegmatis and M. tuberculosis, respectively [8].
The required diazonium derivatives were conveniently obtained
from commercially available 2-chloro-halogeno-anilins (13e17) and
immediately reacted with 1,4-benzoquinone (18) in excess (1.2 eq) to
give the corresponding 2-phenyl-1,4-benzoquinones 19e22 in satis-
factory yields (84e98%), with the exception of 2-(2,6-dichlor-
ophenyl)-1,4-benzoquinone (22) (12%), due to the known difficulties
in the formation of the diazonium salt of 2,6-dichloroanilin [15].
Similarly, the condensation of p-benzoquinone with the diazonium
salt obtained from 2-chloro-5-methoxyanilin (17) successfully affor-
ded 2-(2-chloro-5-methoxyphenyl)-1,4-benzoquinone (23) in 84%
yield. The arylbenzoquinones 19e23 were then reduced to the
desired corresponding biphenyls 24e28 in 55e91% yield, by treat-
These results suggested to perform a systematic pharmacomo-
dulation on the basic core of 1 and explore various substitutions on
each position of the A ring. Consequently, we describe here the
synthesis and antimycobacterial activity of a series of 8, 9, 10 and
11-halo, methoxy and hydroxy 3,3-dimethyl-3H-benzofuro[3,2-f]
[1]benzopyran. Moreover, their ability to inhibit the mycolate
synthesis was investigated, in order to confirm the hypothesis of an
effect on mycobacteria cell wall biosynthesis.
2
. Results and discussion
2.1. Chemistry
ment with saturated aqueous Na
2 2 4
S O (Scheme 2) [14].
The synthetic methods previouslyapplied for the synthesis of 3,3-
2
.1.2. Synthesis of dibenzofurans
The use of sodium hydride in different conditions did not permit
dimethyl-3H-benzofuro[3,2-f][1]benzopyran derivatives do not
allow facile modulations of substitutions in the A ring [3e8].
Consequently, anovelaccessallowingsubstitutionsonallpositionsof
the A ringwasenvisioned, throughthe creationofasuitablebiphenyl,
followed by closure into 2-hydroxydibenzofuran. Construction of the
dimethylpyran ring was anticipated at the last step, through thermal
to observe cyclisation of biphenyls 24e28 into the desired diben-
zofurans. Thus, the creation of the furan ring was envisioned and
successfully achieved through intramolecular aromatic nucleophilic
substitution with KOH and NaOAc at high temperature in a sealed
tube (Scheme 2) [14]. In some cases significant improvements of the
yields were obtained under microwave irradiation. In all cases, the
addition of a small amount (w0.2eq) of sodium dithionite permitted
to avoid biphenyl oxidation in the course of the reaction. Under
optimized conditions, the use of acetonitrile as solvent permitted
the formation of 6-chloro-2-hydroxydibenzofuran (29), 7-bromo-2-
hydroxydibenzofuran (30) and 8-chloro-2-hydroxydibenzofuran
(31) in 92, 76 and 90% yields, respectively. In the case of 9-chloro-2-
Fig. 2. Previous pharmacomodulations on A, B and C rings.
Scheme 1. Biphenyl synthesis with palladium catalyst.

A. Termentzi et al. / European Journal of Medicinal Chemistry 45 (2010) 5833e5847
5835
Scheme 2. Biphenyl synthesis through the p-benzoquinone pathway.
hydroxydibenzofuran (32), the best yield (44%) was obtained in
2.1.4. Formation of hydroxy- and methoxy-3,3-dimethyl-3H-
benzofuro[3,2-f]benzopyrans
The halides 41e44 were converted into the corresponding
phenols by nucleophilic aromatic substitution with KOH, catalyzed
water solution. The best results (35% yield) for 2-hydroxy-8-
methoxydibenzofuran (33) were achieved at solid stage with
microwaves. It is interesting to note that, when reaction was carried
0
0
0
out on 2 ,3 -dichloro[1,1 -biphenyl]-2,5-diol (24) at solid stage
under microwave irradiation, the formation of the expected
compound 29 (12%) was accompanied by that of larger amounts of
by a recently described system based on Pd
2 3
dba in the presence of
a suitable ligand [17]. (Scheme 5). In the cases of 41 and 43, the use of
0
0
0
2-di-tert-butylphosphino-3,4,5,6-tetramethyl-2 ,4 ,6 -triisopropyl-
0
2
,7-dihydroxydibenzofuran (34) (15%) probably resulting from the
1,1 -biphenyl, (ligand L1) permitted to obtain phenols 48 and 50 in
occurrence of a benzyne intermediate. (Scheme 3).
satisfactory 86e92% yields. The same reaction applied to bromo-
compound 42 afforded the corresponding phenol 49 in a moderate
6
2% yield, together with the dimerization product 52 isolated in 11%
2
.1.3. Formation of the pyran ring
The 3,3-dimethyl-3H-benzofuro[3,2-f]benzopyrans derivatives
yield (Fig. 4). In contrast, the more sterically hindered compound 44
could only be converted into the corresponding phenol when using
2
41-45 were prepared by thermal rearrangement of the corre-
0 0 0
-di-tert-butylphosphino-2 ,4 ,6 -triisopropylbiphenyl (ligand L2).
sponding propargyl ethers 35e39, obtained by alkylation of the
phenolic group of 2-hydroxydibenzofurans 29e33 with 3-chloro-3-
methylbut-1-yne (Scheme 2) [16]. Purification of the final rear-
rangement mixtures by column chromatography only permitted to
isolate the angular derivatives, except in the case of 39 for which
the angular compound 45, obtained in 75% yield, was accompanied
by its linear isomer 46 (16%). (Fig. 3). In the case of compound 34,
which bears two hydroxyl groups, reaction with an excess of 3-
chloro-3-methylbut-1-yne led successively to the formation of the
dipropargyl ether 40 and the dipyranodibenzofuran 47 (Scheme 4).
Under optimized conditions, the desired phenol 51 was only
obtained in a low 23% yield and was accompanied by 11% of 3,3-
dimethyl-3H-benzofuro[3,2-f][1]benzopyran 1, resulting from
reductive elimination.
Methylation of phenols 48, 49 and 51 using dimethylsulfate in
the presence of sodium hydride at room temperature, gave the
corresponding methoxy-3,3-dimethyl-3H-benzofuro[3,2-f]benzo-
pyrans 53, 54 and 55 (Scheme 5).
Also, 10-hydroxy-3,3-dimethyl-1,2-dihydro-3H-benzofuro[3,2-
f][1]benzopyran (58), the dihydro derivative of compound 50, was
Scheme 3. Formation of 2,7-dihydroxydibenzofuran (34).

5836
A. Termentzi et al. / European Journal of Medicinal Chemistry 45 (2010) 5833e5847
increased potency, in comparison with the hit compound 1. In
contrast, the 10-chloroderivative 43 was surprisingly totally devoid
of activity against M. tuberculosis, but the activity against M. bovis
was conserved. The most interesting compound in this series was
the 9-bromocompound 42, with a MIC95 [ 37
mM against M.
tuberculosis. Furthermore, this compound failed to show any toxic
activity on VERO cells, with an IC50 ¼ 3500
mM and a selectivity
Fig. 3. Chemical structure of compound 46.
index of 93.
Methoxylated compounds (53, 54, 45, 55) were also found more
active than the hit compound 1 against M. tuberculosis with MIC95
obtained in three steps from the previously described 10-acetyl-3,3-
dimethyl-3H-benzofuro[3,2-f][1]benzopyran (6), (Scheme 6) [5].
Hydrogenation of 6 using Pd/C as catalyst first afforded 10-acetyl-
from 45 to 89
active (MIC95 ¼ 89
substituted on the 8, 10, 11 positions (53, 45, 55), exhibited similar
anti-TB activity, with MICs95 values of 45 M. Compound 55, with
an IC50 of 280 M on VERO cells, was the most interesting
compound of this series. The anti-TB activity of the 9-methoxylated
m
M. The 9-methoxylated derivative 54 was the less
mM). The others benzofurobenzopyrans,
3
,3-dimethyl-1,2-dihydro-3H-benzofuro[3,2-f][1]benzopyran (56),
m
which was subsequently converted to ester 57 through Baeyere
Villiger oxidation with meta-chloroperbenzoic acid. Final saponifi-
cation of 57 gave the expected hydroxy-compound 58.
m
linear isomer 46 was similar to that of the angular one 45 (MIC95
M), with a total lack of toxicity against mammalian cells (IC50
7400 M), and a selectivity index of 83.
¼
89 m
¼
2
2
.2. Biology
m
From a structure activity viewpoint, it appeared that the elec-
tronic effects of the substituents in the A ring played a dramatic role
on the antimycobacterial activity, as exemplified by compounds
substituted at C-10: 6, 56, 43, 50, 58, and 45. Indeed, the potency
was significantly decreased when the A ring was substituted by an
.2.1. Antimycobacterial activity and cytotoxicity
The antimycobacterial activities of the novel benzofur-
obenzopyrans were screened on Mycobacterium bovis BCG and on
the virulent strain M. tuberculosis H37Rv using the Microdilution
Resazurin Assay (MRA) [18]. Results are expressed as MIC95
electron-withdrawing group (R ¼ CH
ucts 6 (MIC95 > 1742 M) and 56 (MIC95 > 340
substitution by an electron-donating group, such as a hydroxy (i.e.,
50: MIC95 ¼ 47 M, 58: MIC95 ¼ 46.5 M) or methoxy group (i.e., 45:
MIC95 ¼ 45 M) resulted in a significantly increased potency.
3
COe) as illustrated by prod-
(
Minimal Inhibitory Concentration), which is the amount of
m
mM). In contrast,
compound required for >95% inhibition of bacterial growth.
Isoniazid (INH) was used as positive control. The cytotoxicity of all
final products was also evaluated on mammalian VERO cells, using
doxorubicin (DOX) as control. The results are presented in Table 1.
As a general rule all compounds substituted on the A ring were
found more potent than the reference compound 1, and more active
against M. tuberculosis than against M. bovis, indicating that
substitution of benzofurobenzopyran core by hydroxy, methoxy or
halogen groups enhanced the antimycobacterial activity.
m
m
m
Finally, the dimer 52 and the complex compound 47, bearing
two pyran rings fused onto the dibenzofuran basic core, displayed
no significant activity against both M. tuberculosis and M. bovis.
2.2.2. Inhibition of mycolates biosynthesis
Preliminary results permitted to establish that the lead
compound 1 did not act on nucleic acids or protein biosynthesis. In
addition, the activity on Nocardia asteroides, as well as preliminary
results (unpublished results), suggested that compound 1 should
affect the biosynthesis of mycolates and lipids present in Myco-
bacterium cell wall [19].
Mycolic acids (MAs) are major components of the lipid-rich
envelope of the Mycobacterium genus, playing a key role in both the
architecture and permeability of the mycobacterial envelope
[20,21]. Furthermore, the biosynthesis pathway leading to mycolic
acids is the target of the most effective antituberculous drug
isoniazid (INH) [22e25].
The good activity (MIC95 from 23.5 to 95
compounds (48e51), was, unfortunately, accompanied by a strong
in vitro cytotoxicity against VERO cells (from 3 to 9 M) suggesting
mM) of hydroxylated
m
a possible contribution to the antitubercular (anti-TB) activity.
The present study confirmed the results previously observed for
compound 1 and 2 indicating that the 1-2-double bond is not
indispensable for anti-TB activity [1]. Indeed, dihydro compound 58
(
m
MIC95 ¼ 46
M), with a 1,2-double bond.
Halogenated (41, 42, 44) compounds bearing chloro or bromo
substituents in positions 8, 9, and 11, respectively, exhibited an
m
M) was as active as its counterpart 50 (MIC95 ¼ 45
Scheme 4. Synthesis of compound 47.

A. Termentzi et al. / European Journal of Medicinal Chemistry 45 (2010) 5833e5847
5837
Scheme 5. Functionalization of the halogenobenzofurobenzopyrans to hydroxyl- and methoxyl- benzofurobenzopyrans.
Extensive characterization of fatty acid synthesis in Mycobacteria
has demonstrated the role of two fatty acid synthases (FAS), namely
FAS-I and FAS-II systems [26,27] : FAS-I (a multifunctional poly-
peptide type-I FAS) performs de novo synthesis of small fatty acids
from acetate producing acyl chains (C16, C18, and C22e26) [26,28,29]
and FAS-II (type-II FAS), consisting of discrete monofunctional
proteins, unable to perform de novo synthesis, preferentially extend
the FAS-I products to a mixture of longer homologous acids [26].
FAS-II elongates the FAS-I products to give meromycolate precur-
sors, which are further modified and condensed with FAS-I prod-
treated with the radioactive [1-14C]_acetate and the inhibition of
each molecule was evaluated on the basis of reduced incorporation
of radioactivity in comparison to the control. Thiolactomycine
(TLM), a thiolactone antibiotic inhibiting both the FAS-II system and
the elongation steps leading to the synthesis of
a-mycolate and
epoxymycolate, was used as a comparison standard at two different
concentrations, active in cell-free extracts [37]. The percentage of
inhibition was determined for the different classes of mycolates:
epoxy-,
control.
a-prim, a-mycolates and fatty acids, compared with the
ucts to form mycolic acids, very long chain (C60eC90
-hydroxylated fatty acids [14,27,30e33].
The most widespread class of mycolic acids, found both in M.
tuberculosis and M. smegmatis, is the so-called -mycolates one.
M. tuberculosis is further characterized by the presence of keto-
mycolates and methoxymycolates, the latest, generally typifying
slow-growing pathogenic species. In the case of M. smegmatis,
a rapidly-grower and non-pathogen of the genus Mycobacterium,
)
a
-alkylated
Results are presented in Fig. 5.
b
Treatment with TLM inhibited the synthesis of all types of
mycolates and accumulated fatty acids. Among the different
samples tested a similar inhibition profile was observed with the
linear methoxylated molecule 46.
All other angular molecules tested strongly inhibited the synthesis
of epoxy-mycolates (>60%). In the case of methoxylated molecules
(53, 54, 45, and 55) the inhibition approximately reached 90%.
Leading unsubstituted compound 1 strongly inhibited the
a
cell wall contains, in addition to
a-mycolate, epoxymycolic acids
and the shorter chain ’-mycolates [20,34]. M. smegmatis is often
a
synthesis of the epoxy-mycolates and induced an accumulation
0
used as a model for the study of M. tuberculosis mycolic acid
biosynthesis pathway due to the similarity of FAS-I and FAS-II
systems in the different mycobacterial organisms [35].
a
-mycolates and fatty acids at its MIC. Treatment with this mole-
-mycolates.
cule did not affect the synthesis of
a
Compounds methoxylated at positions 8 and 10 (53 and 45)
The effect of the benzofurobenzopyran derivatives in the
mycolate biosynthesis was performed on the M. smegmatis mycolic
acid synthesis, according to the protocol of Vaubourgeix et al [36].
Compound 1, halogenated compounds 41, 42 and 44 and all
methoxylated molecules (45, 46, and 53e55) were chosen among
different samples for in vitro evaluation of the mycolate synthesis.
The selection of the molecules was made on the basis of better
activity and relatively low cytotoxicity toward mammalian cell
lines. The concentrations applied were chosen equal to the MIC95
for all compounds. For this purpose, the cell-free system, which
contains all the enzyme machinery producing mycolic acid, was
showed the same inhibition profile, strongly accumulating fatty
0
acids and slightly accumulating
a
-mycolates. The synthesis of
a-mycolates was practically unaffected by these two compounds.
Scheme 6. Synthesis of 10-hydroxy-3,3-dimethyl-1,2-dihydro-3H-benzofuro[3,2-f][1]
benzopyran (58).
Fig. 4. Chemical structure of compound 52.

5838
A. Termentzi et al. / European Journal of Medicinal Chemistry 45 (2010) 5833e5847
0
a -mycolates, a-
Table 1
induced a moderate inhibition of the syntheses of
mycolates, and fatty acids.
In vitro activity of all final products against M. tuberculosis and M. bovis and cyto-
toxicity on VERO cells.
As a general rule, angular benzofurobenzopyrans seem to inhibit
the biosynthesis of the epoxy-mycolates of the mycobacteria cell
wall and their antimycobacterial effect should be attributed to
this property. The mechanism of action of 9-methoxy-3,3-
MIC95a
M. bovis
MIC95a
M. tuberculosis
IC50a
(VERO cells)
Select
Indexb
INH
DOX
1
4.4
e
1.8
e
>100
3.6
320
e
e
e
dimethyl-3H-benzofuro[3,2-f][1]benzopyran (54) is different, since
100
e
100
>1712
>340
3.2
e
0
this compound induces a strong accumulation of
a
-mycolates.
6
56
>340
e
e
Interestingly the inhibition profile of the linear methoxy isomer 46
is similar with that of thiolactomycine, causing accumulation of
fatty acids, with parallel inhibition of all types of mycolates. These
results strongly suggest that the mechanisms of action of linear and
angular benzofurobenzopyran are different.
Halogenated
41
42
43
44
>350
150
100
87.5
37.5
353
100
3500
290
100
1.2
93.3
0.8
>350
43.8
2.3
Hydroxylated
4
4
5
5
5
8
9
0
1
8
94
94
79
94
140
23.5
23.5
47
95
46.5
3.3
9.2
5
9.1
18
0.1
0.4
0.1
0.1
0.4
3. Conclusion
Benzofurobenzopyrans, bearing halogen, hydroxy, and methoxy
substituents at positions 8, 9, 10, and 11 on the A ring, were
prepared in order to determine the structural requirements indis-
pensable to observe antimycobacterial activity in this series. Target
compounds were synthesized with satisfactory yields through
a novel access allowing substitutions on all positions of the A ring.
The biological results suggest that the substitution on the A ring
by an electron-withdrawing group lead to inactive compounds
(e.g.: 6, 7, 56). On the contrary, substitution by electron-donating
hydroxy or methoxy groups significantly enhances the anti-
mycobacterial activity. It should be noted that in the case of
hydroxylation, the enhancement of the antimycobacterial activity is
accompanied by a significant increase of the cytotoxicity against
mammalian cells. Among halogenated compounds, 9-bromo-3,3-
dimethyl-3H-benzofuro[3,2-f][1]benzopyran (42) appears as the
most interesting derivative, molecule with increased antitubercular
potency and significantly decreased cytotoxicity (selectivity index
93 for VERO cells).
Methoxylated
53
54
45
55
46
47
52
89
>360
89
89
89
45
89
45
45
89
>300
>200
100
100
100
280
7400
5000
8600
2.2
1.1
2.2
6.2
83.2
e
>300
>200
e
All tests were performed two times in duplicate.
a
All the values provided are in
The selective index was calculated as the ration of IC50 in VERO cells to MIC95 in
m
M.
b
Mycobacterium tuberculosis.
The 11-methoxylated derivative 55 induced a weaker accumulation
of fatty acids. In contrast, 9-methoxy-3,3-dimethyl-3H-benzofuro
3,2-f][1]benzopyran (54) had a different inhibition profile: while it
practically did not affect the synthesis of fatty acids and
lates, it significantly enhanced the synthesis of
[
a
-myco-
-mycolates (90%).
0
a
Finally, 9-methoxy-2,2-dimethyl-2H-benzofuro[2,3-g][1]ben-
zopyran (46) might be considered of special interest. Indeed, this
linear compound associates a good antitubercular potency with
a reduced toxicity towards mammalian cells, suggesting to further
explore structure-activity relationships in the benzofuro[2,3-g][1]
benzopyran in a near future.
8
-Chloro-3,3-dimethyl-3H-benzofuro[3,2-f][1]benzopyran (41)
and 9-bromo-3,3-dimethyl-3H-benzofuro[3,2-f][1]benzopyran (42)
inhibited the synthesis of epoxy-mycolates, approximately by 65%
and induced a strong accumulation of fatty acids. The 9-bromo
derivative also inhibited thesynthesisof a-mycolates by25%. The 11-
chlorinated compound 44 had a different inhibition profile: it
All compounds were shown to interact with the mycobacterial
cell wall lipid biosynthesis. However, whereas angular 3,3-dimethyl-
strongly inhibited the synthesis of epoxy-mycolates and only
Fig. 5. The mycolic and fatty acid inhibition profile of the synthesized benzofurobenzopyrans. TLM was used as a positive control in two concentrations. Experiment was performed
twice in duplicates. The data presented above indicate the mean values obtained by two independent measurements.

A. Termentzi et al. / European Journal of Medicinal Chemistry 45 (2010) 5833e5847
5839
ꢀ
3
H-benzofuro[3,2-f][1]benzopyran inhibit the synthesis of the
and the mixture was stirred at 0e5 C for 1.5 h to ensure the
formation of the diazonium salt. The diazonium salt solution was
slowly added to a water solution (54 mL) of 1,4-benzoquinone (18)
(0.80 g, 7.4 mmol) and sodium acetate (1.2 g,14.6 mmol). Isolation of
the precipitate by filtration, followed by thorough washing of with
water (3 ꢁ 10 mL) gave 2-(2,3-dichlorophenyl)-1,4-benzoquinone
epoxy-mycolates, the linear analogue 46 was shown to act through
a different mechanism of action close to that of thiolactomycine,
causing accumulation of fatty acids and inhibition of all types of
mycolates.
4
. Experimental section
(19) (1.40 g, 91%) as a yellow amorphous powder. TLC Silica gel 60
ꢂ1
F254, CH
2
Cl
2
/C
6
H
12 80:20, Rf ¼ 0.62. IR (KBr)
n
max (cm ): 3070, 3065,
4.1. Chemistry
2960, 2918, 1657, 1599, 1450, 1410, 1334, 1291, 1283, 1100, 909, 863,
8
39, 777, 711. UV
l
max (nm) (log
3
) (MeOH): 210 (4.45), 246(4.16), 299
1
0
Column chromatographies were performed on Merck silica gel
(3.48). H NMR (400 MHz, CDCl
3
)
d
7.60 (d, J ¼ 8.1 Hz, 1H, H-4 ), 7.34
0
0
(
20e45
mm or 35e70
mm). Thin layer chromatographies were
(t, J ¼ 8.1 Hz,1H, H-5 ), 7.17 (d, J ¼ 8.1 Hz,1H, H-6 ), 6.96 (d, J ¼ 10.2 Hz,
performed using Merck TLC Silica gel 60 F254. IR spectra were
obtained from potassium bromide pellets on a Nicolet-510 instru-
ment. UV spectra were recorded in spectroscopic grade MeOH on
1H, H-6), 6.92 (dd, J ¼ 1.6,10.24 Hz,1H, H-5), 6.82 (d, J ¼ 1.6 Hz,1H, H-
13
3
3). C NMR (75 MHz, CDCl ) d 187.1 (C-4), 184.7 (C-1), 146.0 (C-2),
0
0
136.8 (C-6), 136.5 (C-5), 134.9 (C-3), 134.6 (C-3 ), 133.8 (C-2 ), 131.6
1
0
0
0
0
a Jenway 6705 spectrometer. H NMR spectra were recorded on a
(C-1 ), 131.5 (C-4 ), 128.6 (C-6 ), 127.5 (C-5 ). MS (ESI) m/z: 251 (M ꢂ
Bruker Avance 400 (400 MHz) spectrometer and 13C NMR on
a Bruker Avance 300 (75 MHz) spectrometer. When necessary, the
structures of the novel compounds were insured and the signals
H)
ꢂ
(
35
Cl, Cl), 253 (M ꢂ H)ꢂ
35
(
35
Cl, Cl), 255 (M ꢂ H)ꢂ
37
( Cl, Cl).
37
37
4.1.3. 2-(4-Bromo-2-chlorophenyl)-1,4-benzoquinone (20)
1
1
unambiguously assigned by 2D NMR techniques: He H COSY,
The procedure described for the preparation of 19, applied to 4-
bromo-2-chloroanilin (14) (1.00 g, 4.9 mmol) and 1,4-benzoqui-
none (18) (0.63 g, 5.8 mmol), gave 20 (1.30 g, 90%) as a yellow
1
1
13
1
13
1
He H NOESY, Ce H HMQC, and Ce H HMBC. These experi-
ments were performed using standard Bruker microprograms on
a Bruker Avance 400 spectrometer. Mass spectra were recorded
with a ZQ 2000 spectrometer using electron spray ionization.
Microwave experiments were realized on a Biotage apparatus Ini-
amorphous solid. TLC Silica gel 60 F254, CH
2
Cl
2
, Rf ¼ 0.89. IR (KBr)
ꢂ1
n
max (cm ): 3081, 3065, 2918, 1648, 1590, 1466, 1346, 1287, 1089,
1062, 976, 929, 836, 762. UV
l
max (nm) (log
3) (MeOH): 207 (4.65),
ꢀ
1
tiatorÔ 2.0. The temperature was set at 185 C and the level of
217sh (4.53), 298 (3.71). H NMR (400 MHz, CDCl
3
)
d
7.70 (d, J ¼ 1.7
0
0
absorbance at “Very High” in the case of solid phase reactions.
Hz, 1H, H-3 ), 7.54 (dd, J ¼ 1.7, 8.2 Hz, 1H, H-5 ), 7.14 (d, J ¼ 8.2 Hz,
0
1
H, H-6 ), 6.96 (d, J ¼ 10.1 Hz, 1H, H-6), 6.92 (dd, J ¼ 2.3, 10.1 Hz, 1H,
0
0
0
13
4
.1.1. 2 -Chloro-4 -methoxy[1,1 -biphenyl]-2,5-diol (11) and 2-(2-
H-5), 6.84 (d, J ¼ 2.3 Hz, 1H, H-3). C NMR (75 MHz, CDCl
3
) d 187.0
chloro-4-methoxyphenyl)-1,4-benzoquinone (12)
(C-4), 184.7 (C-1), 145.1 (C-2), 136.8 (C-6), 136.5 (C-5), 135.2 (C-3),
0 0 0 0 0
134.1 (C-2 ), 132.6 (C-3 ), 131.7 (C-6 ), 131.3 (C-1 ), 130.2 (C-5 ), 124.1
A biphasic mixture of 2-chloro-4-methoxyphenylboronic acid
0
-
ꢂ
35
Cl, Br), 297 [M ꢂ H]ꢂ
79
35
(10) (100 mg, 0.53 mmol), 2-bromohydroquinone (9) (91.5 mg,
(C-4 ). MS (ESI ) m/z: 295 [M ꢂ H]
(
( Cl,
8
1
Br or Cl, Br), 299 [M ꢂ H]ꢂ
37
79
37
81
0
2
8
.48 mmol), Pd(PPh
3
)
4
(17 mg, 14.7 nmol), and Na
2
CO
3
(308 mg,
( Cl, Br).
.9 mmol) in toluene:H
2
O (5:1) (10 mL) was stirred under argon at
ꢀ
0
C for 20 h. The reaction mixture was extracted with
4.1.4. 2-(2,5-Dichlorophenyl)-1,4-benzoquinone (21)
dichloromethane (3 ꢁ 20 mL). The combined organic layers were
dried over Na SO , filtered and evaporated under reduced pressure.
Column chromatography on silica gel (CH Cl ) gave successively 2-
2-chloro-4-methoxyphenyl)-1,4-benzoquinone (12) (22 mg, 19%)
The procedure described for the preparation of 19, applied to
2,5-dichloroanilin (15) (2.00 g, 12.3 mmol) and 1,4-benzoquinone
(18) (1.60 g, 14.8 mmol), gave 21 (2.80 g, 93%) as a yellow amor-
2
4
2
2
(
phous solid. TLC Silica gel 60 F254, CH
2
Cl
2
, Rf ¼ 0.74. IR (KBr)
n
max
0
0
0
ꢂ1
and 2 -chloro-4 -methoxy[1,1 -biphenyl]-2,5-diol (11) (7 mg, 6%).
(cm ): 3085, 3050, 1653, 1466, 1330, 1275, 1096, 993, 925, 839,
8
16, 426. UV
lmax (nm) (log 3) (MeOH): 208 (4.46), 215 (4.39), 229sh
1
4
.1.1.1. 2-(2-Chloro-4-methoxyphenyl)-1,4-benzoquinone (12). TLC
(3.96), 253sh (4.03), 292 (3.45). H NMR (400 MHz, CDCl ) d 7.45 (d,
3
1
0
0
Silica gel 60 F254, Solv. CH
d
2
Cl
2
, Rf ¼ 0.80. H NMR (400 MHz, CDCl
3
)
J ¼ 8.6 Hz, 1H, H-3 ), 7.41 (dd, J ¼ 2.28, 8.6 Hz, 1H, H-4 ), 7.28 (d, J ¼
0
0
0
7.14 (d, J ¼ 8.5 Hz, 1H, H-6 ), 7.02 (d, J ¼ 2.5 Hz, 1H, H-3 ), 6.90 (d, J
2.2 Hz, 1H, H-6 ), 6.96 (d, J ¼ 10.1 Hz, 1H, H-6), 6.92 (dd, J ¼ 2.2, 10.1
0
13
¼
10.0 Hz, 1H, H-6), 6.87 (dd, J ¼ 2.5, 8.4 Hz, 1H, H-5 ), 6.84 (dd, J ¼
Hz, 1H, H-5), 6.85 (d, J ¼ 2.2 Hz, 1H, H-3). C NMR (75 MHz, CDCl
3
)
2
.5, 10.0 Hz, 1H, H-5), 6.79 (d, J ¼ 2.5 Hz, 1H, H-3), 3.85 (s, 1H,
d 186.9 (C-4), 184.6 (C-1), 144.9 (C-2), 136.8 (C-6), 136.5 (C-5), 135.3
13
0
0
0
þ
0
0
eOCH
3
). C NMR (75 MHz, CDCl
3
)
d
187.4 (C-4), 185.5 (C-1), 161.1
(C-3), 133.7 (C-1 ), 132.8 (C-5 ), 131.5 (C-2 ), 131.0 (C-3 ), 130.7 (C-4 ),
0
0
0
35
Cl, Cl), 255 (M þ H)þ
35
(
C-4 ), 145.7 (C-2), 136.9 (C-6), 136.4 (C-5), 134.9 (C-3), 134.0 (C-2 ),
130.5 (C-6 ). MS (ESI) m/z: 253 (M þ H)
(
0
0
0
0
35
Cl, Cl), 257 (M þ H)þ
37
37
37
1
(
31.6 (C-6 ), 124.4 (C-1 ), 115.4 (C-3 ), 112.9 (C-5 ), 55.7 (eOCH
3
). MS
(
( Cl, Cl).
ESI) m/z: 249 (M þ H)þ
(
35
Cl), 251 (M þ H)þ
( Cl).
37
4
.1.5. 2-(2-Chloro-5-methoxyphenyl)-1,4-benzoquinone (23)
The procedure described for the preparation of 19, applied to 2-
0
0
0
4
.1.1.2. 2 -Chloro-4 -methoxy[1,1 -biphenyl]-2,5-diol (11). TLC Silica
1
gel 60 F254, Solv. CH
2
Cl
2
/EtOAc, 9:1, Rf ¼ 0.68. H NMR (400 MHz,
chloro-5-methoxyanilin (17) (1.00 g, 6.3 mmol) and 1,4-benzoqui-
none (18) (0.82 g, 7.6 mmol), gave 23 (1.40 g, 84%) as a yellow
amorphous solid. TLC Silica gel 60 F254, CH
0
0
CD
6
(
eOCH
(
1
(
3
OD)
d
7.24 (d, J ¼ 8.5 Hz, 1H, H-6 ), 7.03 (d, J ¼ 2.5 Hz, 1H, H-3 ),
0
.89 (dd, J ¼ 2.5, 8.5 Hz, 1H, H-5 ), 6.76 (d, J ¼ 8.5 Hz, 1H, H-3), 6.69
2
Cl
2
, Rf ¼ 0.51. IR (KBr)
ꢂ1
dd, J ¼ 3.0, 8.5 Hz, 1H, H-4), 6.60 (d, J ¼ 3.0 Hz, 1H, H-6), 3.85 (s, 1H,
n
max (cm ): 3069, 3059, 2976, 2945, 1653, 1594, 1567, 1474, 1275,
1
3
0
3
). C NMR (75 MHz, CD
3
OD)
d
159.5 (C-4 ), 149.4 (C-5), 146.8
1224, 1170, 1100, 1030, 921, 824, 644. UV
207 (4.51), 288 (3.71), 234sh (4.20). H NMR (400 MHz, CDCl
d
lmax (nm) (log
3
) (MeOH):
0
0
0
1
C-2), 134.2 (C-2 ), 132.3 (C-6 ), 129.1 (C-1 ), 126.7 (C-1), 117.5 (C-6),
3
)
0
0
0
0
16.2 (C-3), 115.7 (C-4), 114.8 (C-3 ), 112.9 (C-5 ), 55.5 (eOCH
3
). MS
7.37 (d, J ¼ 8.9 Hz, 1H, H-3 ), 6.94 (dd, J ¼ 3.00, 8.9 Hz, 1H, H-4 ),
ESI) m/z: 251 (M þ H)þ
(
35
Cl), 253 (M þ H)þ
( Cl).
37
6.92 (d, J ¼ 10.1 Hz, 1H, H-6), 6.87 (dd, J ¼ 2.1, 10.1 Hz, 1H, H-5), 6.80
0
(
d, J ¼ 2.1 Hz, 1H, H-3), 6.76 (d, J ¼ 3.0 Hz, 1H, H-6 ), 3.82 (s, 3H,
13
4
.1.2. 2-(2,3-Dichlorophenyl)-1,4-benzoquinone (19)
In a typical experiment, a solution of NaNO (0.45 g, 6.5 mmol) in
water (1.6 mL) was added to a solution of 2,3-dichloroanilin (13)
1.00 g, 6.1 mmol) in 9% aqueous hydrochloric acid solution (6.5 mL),
eOCH
3
). C NMR (75 MHz, CDCl
3
)
d
187.3 (C-4), 185.0 (C-1), 158.2
0
0
2
(C-5 ), 146.1 (C-2), 136.8 (C-6), 136.4 (C-5), 135.0 (C-3), 133.1 (C-1 ),
0
0
0
0
3
130.6 (C-3 ), 124.3 (C-2 ), 116.3 (C-4 ), 116.2 (C-6 ), 55.7 (CeOCH ).
35
Cl), 249 (M ꢂ H)ꢂ
37
(
MS (ESI) m/z: 247 (M ꢂ H)ꢂ
(
( Cl).

5840
A. Termentzi et al. / European Journal of Medicinal Chemistry 45 (2010) 5833e5847
0
0
0
4
.1.6. 2 ,3 -Dichloro[1,1 -biphenyl]-2,5-diol (24)
A saturated aqueous solution of Na (200 mL) was added to
a solution of 2-(2,3-dichlorophenyl)-1,4-benzoquinone (19) (1.00 g,
.0 mmol) in Et O (400 mL). The mixture was vigorously stirred for
.5 h. The organic layer was dried over Na SO , filtered and evap-
orated under reduced pressure. Crystallization from toluene gave
crude material was dissolved in Et
with a saturated aqueous solution of Na
layer was dried over Na SO , filtered and evaporated under reduced
2
O (400 mL) and stirred for 2.5 h
2
S
2
O
4
2 2 4
S O
(200 mL). The organic
2
4
0
0
0
4
1
2
pressure. Crystallization from toluene gave 2 ,6 -dichloro[1,1 -
2
4
biphenyl]-2,5-diol (27) (0.56 g, 9% overall yield), as white prisms,
ꢀ
m.p. 146e147 C. TLC Silica gel 60 F254, CH
n
2
Cl
2
, Rf ¼ 0.13. IR (KBr)
0
0
0
ꢂ1
2
,3 -dichloro[1,1 -biphenyl]-2,5-diol (24) (0.92 g, 91%) as white
prisms, m.p. 151e152 C. TLC Silica gel 60 F254, CH
max (cm ): 3307, 3054, 3027, 1509, 1457, 1407, 1353, 1310,
244, 1189, 1034, 814, 777, 757. UV
max (cm ): 3490, 3346, 3031, 1559, 1497, 1450, 1427, 1189, 808,
ꢀ
2
Cl
2
, Rf ¼ 0.17. IR
793, 777, 730, 718. UV
l
max (nm) (log
3
) (MeOH): 209 (4.53), 303
ꢂ1
1
0
(
KBr)
n
(3.69). H NMR (400 MHz, CD OD)
3
d
7.43 (d, J ¼ 8.0 Hz, 2H, H-5 , H-
0
0
1
(
l
max (nm) (log
3
) (MeOH): 217
3 ), 7.29 (t, J ¼ 8.0 Hz,1H, H-4 ), 6.77 (d, J ¼ 8.7 Hz,1H, H-3), 6.72 (dd,
1
13
4.49), 305 (3.67). H NMR (400 MHz, CD OD)
3
d
7.50 (dd, J ¼ 1.8, 7.7
J ¼ 2.8, 8.7 Hz, 1H, H-4), 6.48 (d, J ¼ 2.8 Hz, 1H, H-6). C NMR (75
0
0
0
0
Hz,1H, H-4 ), 7.30 (t, J ¼ 7.7 Hz,1H, H-5 ), 7.24 (dd, J ¼ 1.8, 7.7 Hz,1H,
MHz, CD
3
OD)
d
149.7 (C-5), 147.1 (C-2), 136.9 (C-1 ), 135.4 (C-2 , C-
0
0
0
0
0
H-6 ), 6.75 (d, J ¼ 8.7 Hz, 1H, H-3), 6.70 (dd, J ¼ 2.8, 8.7 Hz, 1H, H-4),
6 ), 128.9 (C-4 ), 127.5 (C-3 , C-5 ), 124.8 (C-1), 116.5 (C-6), 116.1 (C-
13
ꢂ
35
Cl, Cl), 255 (M ꢂ H)ꢂ
35
6
1
(
.55 (d, J ¼ 2.8 Hz,1H, H-6). C NMR (75 MHz, CD
3
OD)
d
149.6 (C-5),
4),115.9 (C-3). MS (ESI) m/z: 253 (M ꢂ H)
(
0
0
0
0
35
Cl, Cl), 257 (M ꢂ H)ꢂ
37
37
37
46.9 (C-2), 140.5 (C-1 ), 132.5 (C-3 ), 132.1 (C-2 ), 129.9 (C-4 ), 128.9
(
(
Cl, Cl).
0
0
C-6 ), 127.1 (C-1), 126.8 (C-5 ), 116.5 (C-6), 116.1 (C-4), 115.7 (C-3).
ꢂ
35
35
ꢂ
35
37
0
0
0
MS (ESI) m/z: 253 (M ꢂ H)
(
Cl, Cl), 255 (M ꢂ H)
(
Cl, Cl),
4.1.10. 2 -Chloro-5 -methoxy[1,1 -biphenyl]-2,5-diol (28)
ꢂ
37
37
ꢂ
2
57 (M ꢂ H)
(
Cl, Cl), 507 (2MeH) .
Sodium dithionite reduction of 2-(2-chloro-5-methoxyphenyl)-
1
,4-benzoquinone (23) (1.00 g, 4.0 mmol), under the conditions
0
0
0
0
0
4
.1.7. 4 -Bromo-2 -chloro[1,1 -biphenyl]-2,5-diol (25)
described for the reduction of 19 to 24, gave 2 -chloro-5 -methoxy
[1,1 -biphenyl]-2,5-diol (28) (0.55 g, 55%), as white needles, m.p.
129e130 C (toluene). TLC Silica gel 60 F254, CH
0
Sodium dithionite reduction of 2-(4-bromo-2-chlorophenyl)-
,4-benzoquinone (20) (0.90 g, 3.0 mmol), under the conditions
described for the reduction of 19 to 24, gave 4 -bromo-2 -chloro
1,1 -biphenyl]-2,5-diol (25) (0.90 g, 88%), as whitish prisms, m.p.
72e173 C (toluene). TLC Silica gel 60 F254, CH
max (cm ): 3303, 3073, 3032, 1583, 1508, 1477, 1462, 1370,
306, 1186, 1131, 1084, 1069, 808, 788, 768. UV
MeOH): 212 (4.50), 217 (4.55), 236sh (4.14), 306 (3.73). H NMR
400 MHz, CD
ꢀ
1
2
Cl
2
/AcOEt 90:10, Rf
0
0
ꢂ1
¼ 0.55. IR (KBr)
n
max (cm ): 3408, 3003, 2968, 2944, 2840, 1594,
0
[
1
1575, 1511, 1466, 1450, 1314, 1221, 1170, 1021, 1170, 1022, 802, 783.
ꢀ
1
2
Cl
2
, Rf ¼ 0.15. IR
UV
lmax (nm) (log
3
) (MeOH): 211 (4.45), 216 (4.46), 294 (3.73). H
ꢂ1
0
(
KBr)
n
NMR (400 MHz, CD
3
OD)
d
7.34 (d, J ¼ 8.6 Hz, 1H, H-3 ), 6.89 (dd, J ¼
0
0
1
(
(
l
max (nm) (log
3)
2.9, 8.6 Hz, 1H, H-4 ), 6.86 (d, J ¼ 2.9 Hz,1H, H-6 ), 6.74 (d, J ¼ 8.7 Hz,
1
1H, H-3), 6.68 (dd, J ¼ 2.8, 8.7 Hz,1H, H-4), 6.57 (d, J ¼ 2.8 Hz,1H, H-
0
13
0
3
OD)
d
7.66 (d, J ¼ 1.9 Hz, 1H, H-3 ), 7.49 (dd, J ¼ 1.9,
6), 3.80 (s, 3H, eOCH
3
). C NMR (75 MHz, CD
3
OD)
d
158.1 (C-5 ),
0
0
0
8
.2 Hz, 1H, H-5 ), 7.23 (d, J ¼ 8.2 Hz, 1H, H-6’), 6.75 (d, J ¼ 8.7 Hz, 1H,
149.5 (C-5), 146.9 (C-2), 138.6 (C-1 ), 129.6 (C-3 ), 127.4 (C-1), 124.8
(C-2 ), 116.9 (C-6), 116.8 (C-3), 116.1 (C-4), 115.3 (C-6 ), 113.9 (C-4 ),
0
0
0
H-3), 6.70 (dd, J ¼ 2.8, 8.7 Hz, 1H, H-4), 6.56 (d, J ¼ 2.8 Hz, 1H, H-6).
13
0
). MS (ESI) m/z: 249 (M ꢂ H)ꢂ
35Cl), 251 (M ꢂ H)ꢂ
C NMR (75 MHz, CD
3
0
OD)
d
149.6 (C-5), 147.0 (C-2), 137.2 (C-1 ),
54.6 (CeOCH
3
(
0
0
0
37
1
(
34.6 (C-2 ), 133.0 (C-6 ), 131.4 (C-3 ), 129.4 (C-5 ), 126.1 (C-1), 120.6
( Cl).
0
C-4 ), 116.7 (C-6), 116.1 (C-3), 115.7 (C-4). MS (ESI) m/z: 297 (M ꢂ
ꢂ
7
35
Cl, Br), 299 (M ꢂ H)ꢂ
79
35
Cl, Br or Cl, Br), 301 (M ꢂ H)ꢂ
81
37
79
H)
(
(
4.1.11. 6-Chloro-2-hydroxydibenzofuran (29)
3
81
(
Cl, Br).
Potassium hydroxyde (66 mg,1.2 mmol), sodium acetate (83 mg,
mmol), and sodium dithionite (10 mg, 0.07 mmol) were added to
a solution of 2 ,3 -dichloro[1,1 -biphenyl]-2,5-diol (24) (100 mg,
1
0
0
0
0
0
0
4
.1.8. 2 ,5 -Dichloro[1,1 -biphenyl]-2,5-diol (26)
Sodium dithionite reduction of 2-(2,5-dichlorophenyl)-1,4-
0.40 mmol) in acetonitrile (4 mL) in a 35 mL tube, which was sealed
under vacuum and heated for 30 min at 175e180 C under stirring.
ꢀ
benzoquinone (21) (2.50 g, 9.9 mmol), under the conditions
0
0
0
ꢀ
described for the reduction of 19 to 24, gave 2 ,5 -dichloro[1,1 -
The reaction mixture was extracted with dichloromethane (3 ꢁ 20
biphenyl]-2,5-diol (26) (2.20 g, 88%), as white prisms, m.p. 163 C
2 4
mL). The combined organic layers were dried over Na SO , filtered
(
(
toluene). TLC Silica gel 60 F254, CH
cm ): 3529, 3389, 3062, 3034, 1516, 1474, 1446, 1353, 1298, 1201,
2
Cl
2
, Rf ¼ 0.14. IR (KBr)
n
max
and evaporated under reduced pressure. Column chromatography
on silica gel (CH Cl /cyclohexane, 9:1) gave 6-chloro-2-hydrox-
ꢂ1
2
2
1
092, 1026, 827, 807, 797, 772. UV
l
max (nm) (log
3
) (MeOH): 210
ydibenzofuran (29) (79 mg, 91%) as an amorphous white solid. TLC
1
ꢂ1
(
4.45), 216 (4.45), 307 (3.63). H NMR (400 MHz, CD
3
OD) 7.45 (d, J
d
Silica gel 60 F254, Solv. CH
2
Cl
2
, Rf ¼ 0.46. IR (KBr)
n
max (cm ): 3276,
0
0
¼
8.9 Hz, 1H, H-3 ), 7.32 (dd, J ¼ 2.5, 8.9 Hz, 1H, H-4 ), 7.32 (d, J ¼ 2.5
3080, 2933, 1477, 1450, 1420, 1224, 1193, 1170, 1142, 848, 812, 792,
743. UV max (nm) (log ) (MeOH): 219sh (4.55), 212 (4.47), 247sh
(4.19), 258 (4.12), 282sh (3.99), 291 (4.15), 324 (3.71). H NMR (400
MHz, DMSO-d6)
0
Hz, 1H, H-6 ), 6.76 (d, J ¼ 8.7 Hz, 1H, H-3), 6.71 (dd, J ¼ 2.8, 8.7 Hz,
l
3
13
1
1
H, H-4), 6.57 (d, J ¼ 2.8 Hz, 1H, H-6). C NMR (75 MHz, CD
3
OD)
0
0
0
d
149.6 (C-5), 147.0 (C-2), 139.6 (C-1 ), 132.1 (C-5 ), 131.7 (C-2 ), 131.3
d
9.59 (s, 1H, -OH), 7.85 (d, J ¼ 7.9 Hz, 1H, H-9), 7.46
0
0
0
(
C-6 ), 130.3 (C-3 ), 128.1 (C-4 ), 126.0 (C-1), 116.6 (C-6), 116.2 (C-3),
(d, J ¼ 8.9 Hz, 1H, H-4), 7.45 (d, J ¼ 7.9 Hz, 1H, H-7), 7.36 (d, J ¼ 2.6
ꢂ
35
Cl, Cl), 255 (M ꢂ H)ꢂ
35
1
(
15.9 (C-4). MS (ESI) m/z: 253 (M ꢂ H)
(
Hz, 1H, H-1), 7.28 (t, J ¼ 7.8 Hz, 1H, H-8), 7.00 (dd, J ¼ 2.6, 8.9 Hz, 1H,
3
5
37
ꢂ
37
37
13
Cl, Cl), 257 (M ꢂ H)
(
Cl, Cl).
H-3). C NMR (75 MHz, DMSO-d6)
d
153.7 (C-2), 152.3 (C-5a), 150.2
(C-4a), 126.6 (C-7), 126.2 (C-6), 124.5 (C-9b), 123.1 (C-8), 118.9 (C-9),
0
0
0
4.1.9. 2 ,6 -Dichloro[1,1 -biphenyl]-2,5-diol (27)
116.5 (C-9a), 116.0 (C-3), 111.7 (C-4), 105.6 (C-1). MS (ESI) m/z: 217
(M ꢂ H)ꢂ
(
35
Cl), 219 (M ꢂ H)ꢂ
( Cl).
37
A solution of NaNO (1.90 g, 27 mmol) in water (3.2 mL) was
2
added to a solution of 2,6-dichloroanilin (16) (4.00 g, 24.6 mmol) in
water (88 mL) and concentrated aqueous solution of hydrochloric
acid (12 N, 45 mL). The mixture was stirred at 0e5 C for 3 h to
ensure the formation of the diazonium salt. The diazonium salt
solution was then slowly added to a solution of 1,4-benzoquinone
4.1.12. 2,7-Dihydroxydibenzofuran (34)
ꢀ
Potassium hydroxide (200 mg, 3.6 mmol), sodium acetate (83
0
0
mg, 1 mmol), sodium dithionite (10 mg, 0.07 mmol), and 2 ,3 -
0
dichloro[1,1 -biphenyl]-2,5-diol (24) (100 mg, 0.4 mmol) were
(
18) (3.20 g, 29.6 mmol) in water (108 mL) containing sodium
thoroughly mixed in a 10 mL microwave vial. The vial was sealed
and left for 15 min at 185 C in the microwave apparatus, with the
ꢀ
acetate (4.8 g, 59 mmol). After 24 h stirring at room temperature,
the solid precipitate was isolated by filtration to obtain crude
absorption level set at “Very High”. The reaction mixture was
2
-(2,6-dichlorophenyl)-1,4-benzoquinone (22) (0.74 g, w12%). The
diluted with water (20 mL) and extracted with dichloromethane

A. Termentzi et al. / European Journal of Medicinal Chemistry 45 (2010) 5833e5847
5841
(
3 ꢁ 20 mL). The combined organic layers were dried over Na
filtered and evaporated under reduced pressure. Column chroma-
tography on silica gel (CH Cl ) successively afforded 6-chloro-2-
2
SO
4
,
(d, J ¼ 9.0 Hz,1H, H-4), 7.38 (t, J ¼ 8.2 Hz,1H, H-7), 7.28 (d, J ¼ 7.8 Hz,
13
1H, H-8), 7.01 (dd, J ¼ 2.5, 8.8 Hz, 1H, H-3). C NMR (75 MHz,
CD OD) 157.3 (C-5a), 153.2 (C-2), 150.1 (C-4a), 127.6 (C-9), 127.2
(C-7), 123.2 (C-9a), 122.6 (C-8), 122.1 (C-9b), 115.8 (C-3), 111.3 (C-4),
2
2
3
d
hydroxydibenzofuran (29) (10 mg, 12%) and 2,7-dihydroxydi
benzofuran (34) (11.5 mg, 15%), as an amorphous whitish solid. TLC
Silica gel 60 F254, Solv. CH
3
ꢂ
35
109.7 (C-6), 107.4 (C-1). MS (ESI) m/z: 217 (M ꢂ H)
( Cl), 219
ꢂ1
(M ꢂ H)ꢂ
37
2
Cl
2
, Rf ¼ 0.04. IR (KBr)
n
max (cm ): 3202,
( Cl).
105, 3031, 2930, 1645, 1595, 1470, 1443, 1326, 1210, 1186, 1143,
1
1
104, 812. UV
NMR (400 MHz, CD
.8 Hz, 1H, H-4), 7.24 (d, J ¼ 2.6 Hz, 1H, H-1), 6.91 (d, J ¼ 2.1 Hz, 1H,
H-6), 6.81 (dd, J ¼ 2.6, 8.8 Hz, 1H, H-3), 6.80 (dd, J ¼ 2.1, 8.4 Hz, 1H,
H-8). 13C NMR (75 MHz, CD
OD) 158.3 (C-7),157.6 (C-5a),153.0 (C-
a), 150.3 (C-2), 125.1 (C-9b), 120.5 (C-9), 116.4 (C-9a), 112.9 (C-8),
l
max (nm) (log
3
) (MeOH): 207 (4.21), 307 (3.54). H
4.1.16. 8-Methoxy-2-hydroxydibenzofuran (33)
The procedure described for the preparation of 29 and 34 from 24
3
OD)
d
7.70 (d, J ¼ 8.4 Hz, 1H, H-9), 7.29 (d, J ¼
0
0
8
by microwave irradiation, applied to 2 -chloro-5 methoxybiphenyl-
2,5-diol (28) (100 mg, 0.40 mmol) gave, after chromatography on
silica gel (CH Cl ), 8-methoxy-2-hydroxybenzofuran (33) (30 mg,
2 2
3
d
4
1
35%) as a white amorphous solid. TLC Silica gel 60 F254, Solv. CH
2
Cl
2
,
ꢂ1
10.8 (C-4), 110.8 (C-3), 104.6 (C-1), 97.6 (C-6). MS (ESI) m/z: 199
Rf ¼ 0.15. IR (KBr)
n
max (cm ): 3381, 3097, 3062, 3000, 2930, 1599,
ꢂ
(
M ꢂ H) .
1485,1459,1435,1186,1155,1034, 839, 816, 793. UV
MeOH): 213 (4.41), 217 (4.49), 256 (3.89), 304 (4.13), 328 (3.62). H
NMR (400 MHz, CD OD)
lmax (nm) (log 3)
1
(
4
.1.13. 7-Bromo-2-hydroxydibenzofuran (30)
3
d
7.41 (d, J ¼ 2.6 Hz,1H, H-9), 7.38 (d, J ¼ 8.9
The procedure described for the preparation of 29 from 24 in
Hz,1H, H-6), 7.34 (d, J ¼ 8.9 Hz,1H, H-4), 7.34 (d, J ¼ 2.8 Hz,1H, H-1),
0
0
0
a sealed tube, applied to 4 -bromo-2 -chloro[1,1 -biphenyl]-2,5-diol
7.0 (dd, J ¼ 2.6, 8.9 Hz,1H, H-7), 6.93 (dd, J ¼ 2.5, 8.9 Hz,1H, H-3), 3.86
13
(25) (100 mg, 0.34 mmol) gave 7-bromo-2-hydroxydibenzofuran
3 3
(s, 3H, eOCH ). C NMR (75 MHz, CD OD) d 155.8 (C-8),152.8 (C-4a),
(
30) (60 mg, 75%) as a white amorphous solid. TLC Silica gel 60 F254
,
151.5 (C-5a), 151.0 (C-2), 124.9 (C-9b), 124.7 (C-9a), 115.1 (C-3), 114.8
ꢂ1
Solv. CH
2
3
2
Cl
2
, Rf ¼ 0.24. IR (KBr)
n
max (cm ): 3256, 3100, 2956,
(C-7),111.5 (C-6),111.3 (C-4),105.3 (C-1),103.2 (C-9), 55.0 (CeOCH
MS (ESI) m/z: 213 (M ꢂ H) .
3
).
ꢂ
925, 1477, 1438, 1415, 1219, 1184, 1170, 905, 804. UV
lmax (nm) (log
) (MeOH): 207 (4.56), 220sh (4.45), 248 (4.25), 257 (4.26), 295
1
(
4.19), 320 (3.74). H NMR (400 MHz, CD
3
OD)
d
7.85 (d, J ¼ 8.3 Hz,
4.1.17. 6-Chloro-2-(1,1-dimethylpropyn-1-oxy)dibenzofuran (35)
Potassium iodide (119 mg, 0.72 mmol) and DBU (164 mL, 1.1
mmol) wereadded to a solution of 6-chloro-2-hydroxydibenzofuran
1
8
¼
H, H-9), 7.74 (d, J ¼ 1.7 Hz, 1H, H-6), 7.46 (dd, J ¼ 1.7, 8.3 Hz, 1H, H-
) 7.40 (d, J ¼ 8.9 Hz,1H, H-4), 7.36 (d, J ¼ 2.6 Hz,1H, H-1), 6.98 (dd, J
13
2.6, 8.9 Hz, 1H, H-3). C NMR (75 MHz, CD
3
OD)
d
157.1 (C-5a),
(29) (120 mg, 0.55 mmol) in anhydrous toluene (4 mL) under argon.
1
53.6 (C-2), 150.4 (C-4a), 125.4 (C-8), 123.9 (C-9a), 123.6 (C-9b),
3-Chloro-3-methylbut-1-yne (166
m
L, 1.1 mmol) was added and the
ꢀ
1
1
H)
21.4 (C-9), 119.6 (C-7), 115.7 (C-3), 114.5 (C-6), 111.5 (C-4), 105.3 (C-
reaction mixture was stirred for 2 h at 65 C under argon. The
reaction was quenched with water (10 mL) and extracted with
dichloromethane (3 ꢁ 20 mL). The combined organic layers were
ꢂ
79
Br), 263 (M ꢂ H)ꢂ
81
). MS (ESI) m/z: 261 (M ꢂ H)
(
( Br), 523 (2M-
ꢂ
79
ꢂ 81
( Br), 525 (2M-H) ( Br).
2 4
dried over Na SO , filtered and evaporated under reduced pressure.
4.1.14. 8-Chloro-2-hydroxydibenzofuran (31)
Column chromatography over silica gel (cyclohexane/CH
2 2
Cl , 9:1)
The procedure described for the preparation of 29 from 24 in
gave 6-chloro-2-(1,1-dimethylpropyn-1-oxy)dibenzofuran (35)
a sealed tube, applied to 26 (100 mg, 0.40 mmol) gave 8-Chloro-2-
hydroxydibenzofuran (31) (70 mg, 81%) as a white amorphous
(134 mg, 86%) as an amorphous white solid. TLC Silica gel 60 F254
,
ꢂ1
Solv. CH
2
Cl
2
/C
6
H
12 50:50, Rf ¼ 0.52. IR (KBr)
n
max (cm ): 3300, 2984,
solid. TLC Silica gel 60 F254, Solv. CH
2
Cl
2
, Rf ¼ 0.37. IR (KBr)
n
max
2926, 2852, 1474, 1443, 1190, 1171, 1140, 960, 890, 867, 781, 735. UV
max (nm) (log ) (MeOH): 210 (4.65), 218 (4.78), 255 (4.27), 285
(4.34), 310 (3.80). H NMR (400 MHz, CDCl
ꢂ1
(
cm ): 3233, 2925, 2855, 1598, 1470, 1442, 1419, 1166, 1065, 866,
l
3
1
8
2
d
04, 750, 695. UV
54 (4.10), 298 (4.12), 329sh (3.57). H NMR (400 MHz, CD
7.94 (d, J ¼ 2.1 Hz, 1H, H-9), 7.50 (d, J ¼ 8.8 Hz, 1H, H-6), 7.42 (dd, J
2.1, 8.8 Hz,1H, H-7), 7.41 (d, J ¼ 8.8 Hz,1H, H-4), 7.36 (d, J ¼ 2.6 Hz,
l
max (nm) (log
3
) (MeOH): 209 (4.49), 244 (4.15),
3
)
d
7.85 (d, J ¼ 7.7 Hz, 1H,
1
3
OD)
H-9), 7.82 (d, J ¼ 2.4 Hz,1H, H-1), 7.60 (d, J ¼ 8.9 Hz,1H, H-4), 7.49 (d, J
¼ 7.7 Hz,1H, H-7), 7.41 (dd, J ¼ 2.4, 8.9 Hz,1H, H-3), 7.31 (t, J ¼ 7.8 Hz,
0
0
0
13
¼
1H, H-8), 2.66 (s, 1H, H-3 ), 1.74 (s, 6H, H-1 a, 1 b). C NMR (75 MHz,
13
1
H, H-1), 6.99 (dd, J ¼ 2.5, 8.8 Hz, 1H, H-3). C NMR (75 MHz,
CD OD) 155.2 (C-5a), 153.5 (C-2), 150.9 (C-4a), 127.5 (C-8), 126.6
C-7), 125.9 (C-9a), 123.7 (C-9b), 120.0 (C-9), 116.1 (C-3), 112.3 (C-6),
3
CDCl ) d 152.6 (C-5a), 152.0 (C-4a), 151.5 (C-2), 127.2 (C-7), 126.5 (C-
3
d
6), 124.3 (C-9b), 123.4 (C-8), 123.2 (C-3), 119.1 (C-9), 117.1 (C-9a),
0
0
0
0
(
114.3 (C-1),111.9 (C-4), 86.1 (C-2 ), 74.2 (C-3 ), 73.2 (C-1 ), 29.6 (C-1 a,
ꢂ
35
0
þ
35
Cl), 287 (M þ H)þ
37
1
H)
11.6 (C-4), 105.4 (C-1). MS (ESI) m/z: 217 (M ꢂ H)
(
Cl), 219 (M ꢂ
1 b). MS (ESI) m/z: 285 (M þ H)
(
( Cl), 591 (2M
ꢂ
37
þ
35
Cl), 593 (2M þ Na)þ
37
(
Cl).
þ Na)
(
( Cl).
4
.1.15. 9-Chloro-2-hydroxydibenzofuran (32)
Potassium hydroxyde (300 mg, 5.4 mmol), sodium acetate (83
mg, 1 mmol), sodium dithionite (28 mg, 0.19 mmol), and water (150
4.1.18. 7-Bromo-2-(1,1-dimethylpropyn-1-oxy)dibenzofuran (36)
The procedure described for the preparation of 35 from 29, in
a procedure involving 7-bromo-2-hydroxydibenzofuran (30) (236
0
0
m
L) were added to a solution of 2 ,6 -dichlorobiphenyl-2,5-diol (27)
mg, 0.89 mmol), DBU (269
mL, 1.77 mmol), potassium iodide (192
(
100 mg, 0.40 mmol) in acetonitrile (4 mL) in a 35 mL tube, which
mg, 1.16 mmol), and 3-chloro-3-methylbut-1-yne (273 m
mmol) in anhydrous toluene (6 mL) gave 7-bromo-2-(1,1-dime-
L, 1.78
ꢀ
was sealed under vacuum and heated for 2.5 h at 175e180 C under
stirring. The reaction mixture was extracted with dichloromethane
thylpropyn-1-oxy)dibenzofuran (36) (205 mg, 70%) as a white
(
3 ꢁ 20 mL). The combined organic layers were dried over Na
2
SO
4
,
amorphous solid. TLC Silica gel 60 F254, Solv. CH
2
Cl
2
/C
6
H
12 50:50, Rf
ꢂ1
filtered and evaporated under reduced pressure to give, by crys-
tallization from cyclohexane, 9-chloro-2-hydroxydibenzofuran
¼ 0.56. IR (KBr)
n
max (cm ): 3296, 3089, 3062, 2984, 2930, 2850,
1632, 1595, 1474, 1464, 1418, 1186, 1169, 1139, 1124, 1049, 949, 918,
883, 858, 820. UV max (nm) (log ) (MeOH): 218 (4.43), 246 (4.10),
256 (4.16), 294 (4.18), 303sh (4.08). H NMR (400 MHz, CDCl
ꢀ
(32) (37 mg, 44%), as white needles, m.p. 113e115 C. TLC Silica gel
l
3
ꢂ1
1
6
2
(
2
d
0 F254, Solv. CH
2
Cl
2
, Rf ¼ 0.47. IR (KBr)
n
max (cm ): 3292, 2957,
3
) d 7.80
925, 2851, 1477, 1420, 1227, 1193, 1162, 933, 808, 777, 744. UV
) (MeOH): 209 (4.50), 217 (4.45), 240 (4.24), 257 (4.08),
82sh (3.95), 290 (4.09), 325 (3.72). H NMR (400 MHz, CD
l
max
(d, J ¼ 8.1 Hz,1H, H-9), 7.80 (d, J ¼ 2.3 Hz,1H, H-1), 7.76 (d, J ¼ 1.1 Hz,
nm) (log
3
1H, H-6), 7.51 (d, J ¼ 8.8 Hz, 1H, H-4), 7.49 (dd, J ¼ 1.4, 8.2 Hz, 1H,
1
0
3
OD)
H-8), 7.39 (dd, J ¼ 2.3, 8.8 Hz, 1H, H-3), 2.64 (s, 1H, H-3 ), 1.77 (s, 1H,
0
0
13
7.71 (d, J ¼ 2.3 Hz, 1H, H-1), 7.45 (d, J ¼ 8.2 Hz, 1H, H-6), 7.41
3
H-1 a, 1 b). C NMR (75 MHz, CDCl ) d 157.1 (C-5a), 152.9 (C-4a),

5842
A. Termentzi et al. / European Journal of Medicinal Chemistry 45 (2010) 5833e5847
0
0
0
0
0
1
51.5 (C-2), 125.9 (C-8), 123.8 (C-9a), 122.9 (C-3), 121.6 (C-9), 120.3
9), 86.3 (C-2 ), 74.0 (C-3 ), 73.4 (C-1 ), 29.6 (C-1 a, 1 b). MS (ESI) m/z:
0
þ
(
C-7), 115.3 (C-6), 115.1 (C-9b), 114.0 (C-1), 111.6 (C-4), 86.3 (C-2 ),
281 (M þ H) .
0
0
þ
0
0
þ
7
(
4.1 (C-3 ), 73.4 (C-1 ), 29.6 (C-1 a, 1 b). MS (ESI) m/z: 329 (M þ H)
7
9 81
Br), 331 (M þ H)
(
Br).
4.1.22. 2,7-Di(1,1-dimethylpropyn-1-oxy)dibenzofuran (40)
The procedure described for the preparation of 35 from 29, in
a procedure applied to 2,7-dihydroxydibenzofurane (34) (44 mg,
4.1.19. 8-Chloro-2-(1,1-dimethylpropyn-1-oxy)dibenzofuran (37)
The procedure described for the preparation of 35 from 29, in
a procedure involving 8-chloro-2-hydroxydibenzofuran (31) (250
mg, 1.15 mmol), DBU (342 L, 2.30 mmol), potassium iodide (248
mg, 1.50 mmol), and 3-chloro-3-methylbut-1-yne (346 L, 2.30
0.22 mmol), DBU (100
0.57 mmol), and 3-chloro-3-methylbut-1-yne (100
in anhydrous toluene (2.5 mL), with stirring for 6 h at 65 C under
argon gave 2,7-di(1,1-dimethylpropyn-1-oxy)dibenzofuran (40)
mL, 0.66 mmol), potassium iodide (95 mg,
m
L, 0.65 mmol)
ꢀ
m
m
mmol) in anhydrous toluene (4 mL) gave 8-chloro-2-(1,1-dime-
thylpropyn-1-oxy)dibenzofuran (37) (160 mg, 49%) as a white
(50 mg, 69%) as a pale yellow oil. TLC Silica gel 60 F254, Solv. CH
2
Cl
2
/
ꢂ1
C
6
H
12 50:50, Rf ¼ 0.50. IR (KBr)
n
max (cm ): UV
lmax (nm) (log 3)
1
amorphous solid. TLC Silica gel 60 F254, Solv. CH
2
Cl
2
/C
6
H
12 50:50, Rf
(MeOH): 217 (4.45), 255 (4.04), 295 (4.05). H NMR (400 MHz,
ꢂ1
¼
0.69. IR (KBr)
435, 1166, 1135, 1108, 886, 820, 808, 691. UV
n
max (cm ): 3270, 3241, 2984, 2925, 2852, 1477,
CD
3
OD)
d
7.83 (d, J ¼ 8.5 Hz, 1H, H-9), 7.76 (d, J ¼ 2.2 Hz, 1H, H-1),
1
l
max (nm) (log
3)
7.55 (d, J ¼ 1.9 Hz, 1H, H-6), 7.47 (d, J ¼ 8.8 Hz, 1H, H-4), 7.30 (dd, J ¼
2.2, 8.8 Hz, 1H, H-3), 7.21 (dd, J ¼ 1.9, 8.5 Hz, 1H, H-8). 2.67 (s, 1H, H-
1
(
MeOH): 209sh (4.63), 218 (4.80), 253 (4.22), 292 (4.28). H NMR
0 00 00 00 0 0 00
3 or H-3 ), 2.63 (s,1H, H-3 or H-3 ),1.75 (s, 6H, H-1 a,1 b or H-1 a,
(
1
7
2
d
400 MHz, CDCl
3
)
d
7.92 (d, J ¼ 1.7 Hz, 1H, H-9), 7.81 (d, J ¼ 2.1 Hz,
0
0
13
H, H-1), 7.51 (d, J ¼ 8.9 Hz, 1H, H-4), 7.50 (d, J ¼ 8.7 Hz, 1H, H-6),
1 b). C NMR (75 MHz, CD
3
OD) d 157.2 (C-5a), 155.7 (C-7), 152.9 (C-
.43 (dd, J ¼ 1.9, 8.7 Hz, 1H, H-7), 7.39 (dd, J ¼ 2.3, 8.9 Hz, 1H, H-3),
4a), 151.1 (C-2), 124.5 (C-9b), 121.5 (C-3), 120.3 (C-9), 119.7 (C-9a),
0
0
0
13
0 00
117.3 (C-8), 113.8 (C-1), 111.2 (C-4), 105.0 (C-6), 86.4 (C-2 or C-2 ),
.67 (s, 1H, H-3 ), 1.74 (s, 6H, H-1 a, 1 b). C NMR (75 MHz, CDCl
155.2 (C-5a), 153.2 (C-4a), 151.2 (C-2), 128.0 (C-8), 127.2 (C-7),
25.9 (C-9a), 123.5 (C-9b), 123.3 (C-3), 120.5 (C-9), 114.1 (C-1), 112.7
3
)
0
00
0
00
0
00
0
85.8 (C-2 or C-2 ), 74.3 (C-3 or C-3 ), 73.9 (C-3 or C-3 ), 73.4 (C-1
00 0 00 0 0 00 00
1
or C-1 ), 73.1 (C-1 or C-1 ). 29.6 (C-1 a, 1 b or C-1 a, 1 b). MS (ESI)
0
0
0
0
þ
þ
(
C-4), 111.7 (C-6), 86.1 (C-2 ), 74.3 (C-3 ), 73.5 (C-1 ), 29.6 (C-1 a, 1.b).
m/z: 333 (M þ H) , 355 (M þ Na) .
þ
35
Cl), 287 (M þ H)þ
37
MS (ESI) m/z: 285 (M þ H)
(
( Cl).
4.1.23. 8-Chloro-3,3-dimethyl-3H-benzofuro[3,2-f][1]benzopyran (41)
4
.1.20. 9-Chloro-2-(1,1-dimethylpropyn-1-oxy)dibenzofuran (38)
The procedure described for the preparation of 35 from 29, in
a procedure involving 9-chloro-2-hydroxydibenzofuran (32) (180
mg, 0.82 mmol), DBU (246 L, 1.50 mmol), potassium iodide (178
mg, 1.03 mmol), and 3-chloro-3-methylbut-1-yne (250 L, 1.50
A solution of 6-chloro-2-(1,1-dimethylpropyn-1-oxy)dibenzo-
ꢀ
furan (35) (130 mg, 0.46 mmol) in DMF (5 mL) was heated at 150 C
for 20 h. After removal of the solvent under reduced pressure,
purification by column chromatography over silica gel (solvent
cyclohexane) gave 8-chloro-3,3-dimethyl-3H-benzofuro[3,2-f][1]
benzopyran (41) (130 mg, 100%) as a white amorphous solid. TLC
m
m
mmol) in anhydrous toluene (8 mL) gave 9-chloro-2-(1,1-dime-
thylpropyn-1-oxy)dibenzofuran were obtained (38) (226 mg, 98%)
Silica gel 60 F254, Solv. CH
(cm ): 3093, 3038, 2968, 2922, 2855, 1621, 1574, 1474, 1431, 1415,
1361, 1259, 1236, 1217, 1154, 1143, 1119, 1094, 975, 894, 816, 788,
2
Cl
2
/C
6
H
12 50:50, Rf ¼ 0.66. IR (KBr)
n
max
ꢂ1
as a white amorphous solid. TLC Silica gel 60 F254, Solv. CH
2
Cl
2
/
ꢂ1
C
2
UV
6
H
12 50:50, Rf ¼ 0.72. IR (KBr)
n
max (cm ): 3303, 3081, 2992,
929, 2855, 1645, 1583, 1469, 1442, 1182, 1162, 1135, 929, 782, 753.
max (nm) (log ) (MeOH): 210sh (4.60), 222 (4.72), 256 (4.22),
86 (4.28), 312 (3.78). H NMR (400 MHz, CDCl
Hz,1H, H-1), 7.53 (d, J ¼ 8.9 Hz,1H, H-4), 7.51 (d, J ¼ 7.8 Hz,1H, H-6),
.42 (t, J ¼ 7.8 Hz, 1H, H-7), 7.41 (dd, J ¼ 2.4, 8.9 Hz, 1H, H-3), 7.36 (d,
729. UV
lmax (nm) (log 3) (MeOH): 210 (4.44), 233 (4.36), 283 (4.06),
1
l
3
295 (4.24), 306 (4.32), 349 (3.68). H NMR (400 MHz, CDCl ) d 7.92
3
1
2
3
)
d
8.31 (d, J ¼ 2.4
(d, J ¼ 7.9 Hz, 1H, H-11), 7.48 (d, J ¼ 7.9 Hz, 1H, H-9), 7.43 (d, J ¼ 8.8
Hz, 1H, H-6), 7.30 (t, J ¼ 7.8 Hz, 1H, H-10), 7.08 (d, J ¼ 9.8 Hz, 1H, H-
7
1), 7.01 (d, J ¼ 8.8 Hz, 1H, H-5), 5.90 (d, J ¼ 9.8 Hz, 1H, H-2), 1.55 (s,
0
0
0
13
13
J ¼ 7.5 Hz, 1H, H-8), 2.66 (s, 1H, H-3 ), 1.74 (s, 6H, H-1 a, 1 b).
NMR (75 MHz, CDCl 157.3 (C-5a), 152.5 (C-4a), 151.1 (C-2), 128.3
C-9), 127.4 (C-7), 123.3 (C-8), 123.0 (C-3), 122.5 (C-9a), 116.4 (C-9b),
C
6H, H-3a, 3b). C NMR (75 MHz, CDCl
3
)
d
152.6 (C-7a), 151.0 (C-6a),
3
)
d
148.9 (C-4a), 132.5 (C-2), 126.8 (C-9), 126.1 (C-8), 123.3 (C-10), 120.5
(C-11), 119.5 (C-11b), 118.9 (C-1), 117.1 (C-11a), 116.8 (C-5), 115.9 (C-
11c), 111.5 (C-9), 75.8 (C-3), 27.4 (C-3a, 3b). MS (ESI) m/z: 285 (M þ
(
0
0
1
1
16.4 (C-1), 111.2 (C-4), 110.1 (C-6), 86.1 (C-2 ), 74.2 (C-3 ), 73.5 (C-
0
0
0
þ
35
þ
35
þ
þ
þ
37
), 29.6 (C-1 a, 1 b). MS (ESI) m/z: 285 (M þ H)
(
Cl), 287 (M þ
H)
(
(
(
Cl), 287 (M þ H)
(
Cl). HRESIMS calcd. for C17
H
H
13ClO
13ClO
2
2
þ
37
þ
35
þ
37
35
H)
(
Cl), 307 (M þ Na)
(
Cl), 309 (M þ Na)
(
Cl), 591 (2M þ
Cl), 285.0679 (M þ H) , found 285.0685; calcd. for C17
þ
35
þ
37
37
Na)
(
Cl), 593 (2M þ Na)
(
Cl).
Cl), 287.0835 (M þ H) , found 287.0664.
4.1.21. 8-Methoxy-2-(1,1-dimethylpropyn-1-oxy)dibenzofuran (39)
4.1.24. 9-Bromo-3,3-dimethyl-3H-benzofuro[3,2-f][1]benzopyran (42)
The procedure described for the preparation of 35 from 29, in
A solution of 7-bromo-2-(1,1-dimethylpropyn-1-oxy)dibenzo-
furan (36) (200 mg, 0.61 mmol) in DMF (6 mL) was heated at 150 C
for 9 h. The solvent was removed under reduced pressure. Purifi-
cation by column chromatography over silica gel (solvent cyclo-
hexane) gave 9-bromo-3,3-dimethyl-3H-benzofuro[3,2-f][1]benzo
ꢀ
a procedure involving 8-methoxy-2-hydroxydibenzofuran (33) (70
mg, 0.33 mmol), DBU (100 L, 0.66 mmol), potassium iodide (71
mg, 0.43 mmol), and 3-chloro-3-methylbut-1-yne (100
m
m
L, 0.65
ꢀ
mmol) in anhydrous toluene (3 mL), with stirring for 16 h at 65 C
under argon gave 8-methoxy-2-(1,1-dimethylpropyn-1-oxy)
dibenzofuran (39) (60 mg, 66%) as a white amorphous solid. TLC
pyran (42) (160 mg, 80%) as a white amorphous solid. TLC Silica gel
ꢂ1
60 F254, Solv. CH
2
Cl
2
/C
6
H
12 50:50, Rf ¼ 0.48. IR (KBr)
n
max (cm ):
Silica gel 60 F254, Solv. CH
cm ): 3097, 2918, 2848, 1645, 1594, 1181, 1460, 1176, 1131, 1030,
2
Cl
2
/C
6
H
12 80:20, Rf ¼ 0.72. IR (KBr)
n
max
3078, 3043, 2979, 2926, 2852, 1478, 1412, 1365, 1274, 1217, 1194,
1155, 963, 887, 805, 741, 716. UV max (nm) (log ) (MeOH): 207
(4.47), 234 (4.33), 279 (4.04), 303 (4.33), 313 (4.41), 349 (3.65). H
NMR (400 MHz, CDCl
ꢂ1
(
l
3
1
8
2
2
08. UV
97 (3.89), 326sh (3.43). H NMR (400 MHz, CD
.4 Hz, 1H, H-1), 7.45 (d, J ¼ 8.9 Hz, 1H, H-6), 7.44 (d, J ¼ 8.8 Hz, 1H,
lmax (nm) (log 3) (MeOH): 212 (4.60), 217 (4.40), 254 (3.85),
1
3
OD)
d
7.75 (d, J ¼
3
)
d
7.83 (d, J ¼ 8.4 Hz,1H, H-11), 7.71 (d, J ¼ 1.7
Hz, 1H, H-8), 7.45 (dd, J ¼ 1.7, 8.3 Hz, 1H, H-10), 7.30 (d, J ¼ 8.8 Hz,
H-4), 7.38 (d, J ¼ 2.6 Hz, 1H, H-9), 7.31 (dd, J ¼ 2.4, 8.8 Hz, 1H, H-3),
1H, H-6), 7.01 (d, J ¼ 9.8 Hz, 1H, H-1), 6.95 (d, J ¼ 8.7 Hz, 1H, H-5),
13
7
3
.05 (dd, J ¼ 2.7, 8.9 Hz, 1H, H-7), 3.92 (s, 3H, eOCH
3
), 2.61 (s, 1H, H-
5.86 (d, J ¼ 9.8 Hz, 1H, H-2), 1.51 (s, 6H, H-3a, 3b). C NMR (75 MHz,
0
0
0
13
), 1.69 (s, 6H, H-1 a, 1 b). C NMR (75 MHz, CD
3
OD) 155.7 (C-8),
d
3
CDCl ) d 157.3 (C-7a), 151.2 (C-6a), 148.8 (C-4a), 132.5 (C-2), 125.8
1
53.4 (C-4a), 151.6 (C-5a), 150.7 (C-2), 124.9 (C-9b), 124.6 (C-9a),
(C-10), 123.6 (C-11a), 122.9 (C-11), 119.9 (C-9), 119.0 (C-11b), 119.0
(C-1), 116.5 (C-5), 115.7 (C-11c), 115.2 (C-8), 111.2 (C-6), 75.8 (C-3),
122.6 (C-3), 115.2 (C-7), 114.1 (C-1), 112.2 (C-6), 111.5 (C-4), 103.7 (C-

A. Termentzi et al. / European Journal of Medicinal Chemistry 45 (2010) 5833e5847
5843
ꢂ
ꢂ1
2
(
7.4 (C-3a, 3b). MS (ESI) m/z: 327 (M ꢂ H) (79Br), 329 (M ꢂ H)ꢂ
0.68. IR (KBr)
1209, 1193, 1143, 968, 824, 796, 737. UV
17 (4.57), 259 (4.12), 268 (4.11), 287 (3.99), 312sh (4.19), 321 (4.24),
n
max (cm ): 3051, 3004, 2956, 2923, 1481, 1446, 1259,
8
1
Br).
l
max (nm) (log ) (MeOH):
3
2
1
4
.1.25. 10-Chloro-3,3-dimethyl-3H-benzofuro[3,2-f][1]benzopyran
352sh (3.77), 369sh (3.61). H NMR (400 MHz, CDCl
3
)
d
7.50 (d, J ¼
(43)
2.5 Hz, 1H, H-11), 7.48 (d, J ¼ 9.0 Hz, 1H, H-8), 7.33 (d, J ¼ 8.6 Hz, 1H,
H-6), 7.10 (d, J ¼ 9.9 Hz, 1H, H-1), 7.08 (dd, J ¼ 2.5, 9.0 Hz, 1H, H-9),
A solution of 8-chloro-2-(1,1-dimethylpropyn-1-oxy)dibenzo-
furan (37) (150 mg, 0.53 mmol) in DMF (5 mL) was heated at
30 C for 20 h. The solvent was removed under reduced pressure.
6.96 (d, J ¼ 8.6 Hz, 1H, H-5), 5.89 (d, J ¼ 9.9 Hz, 1H, H-2), 3.97 (s, 3H,
ꢀ
13
1
eOCH
3
), 1.55 (s, 6H, H-3a, 3b). C NMR (75 MHz, CDCl
3
)
d
155.6 (C-
Purification by column chromatography over silica gel (solvent
cyclohexane) gave 10-chloro-3,3-dimethyl-3H-benzofuro[3,2-f][1]
benzopyran (43) (125 mg, 83%) as a white amorphous solid. TLC
10), 151.9 (C-7a), 151.8 (C-6a), 148.3 (C-4a), 132.1 (C-2), 124.8 (C-
11a), 119.8 (C-11b), 119.2 (C-1), 116.1 (C-5), 115.7 (C-11c), 114.4 (C-9),
112.0 (C-8), 111.2 (C-6), 105.8 (C-11), 75.6 (C-3), 56.2 (CeOCH
3
), 27.3
ꢂ
Silica gel 60 F254, Solv. CH
2
Cl
2
/C
6
H
12 50:50, Rf ¼ 0.73. IR (KBr)
n
max
(C-3a, 3b). MS (ESI) m/z: 279 (M ꢂ H) . HRESIMS calcd. for
ꢂ1
þ
(
cm ): 2980, 2921, 2848, 1482, 1459, 1443, 1416, 1260, 1159, 1089,
C
18
H
16
O
3
, 281.1173 (M þ H) , found 281.1170.
9
(
64, 801, 781, 708. UV
4.50), 281 (3.99), 303 (4.20), 313 (4.36), 352 (3.66). H NMR (400
MHz, CDCl
7.96 (d, J ¼ 2.1 Hz, 1H, H-11), 7.47 (d, J ¼ 8.7 Hz, 1H,
H-8), 7.40 (dd, J ¼ 2.1, 8.7 Hz, 1H, H-9), 7.31 (d, J ¼ 8.8 Hz, 1H, H-6),
.01 (d, J ¼ 9.8 Hz, 1H, H-1), 6.96 (d, J ¼ 8.8 Hz, 1H, H-5), 5.87 (d, J
9.8 Hz, 1H, H-2). 13C NMR (75 MHz, CDCl
155.3 (C-7a), 151.7
C-6a), 148.7 (C-4a), 132.6 (C-2), 127.9 (C-10), 126.7 (C-9), 125.8 (C-
1a), 121.8 (C-11), 118.9 (C-1), 118.8 (C-11b), 116.9 (C-5), 115.8 (C-
lmax (nm) (log 3) (MeOH): 211 (4.50), 215
1
4.1.27.2. 9-Methoxy-2,2-dimethyl-2H-benzofuro[2,3-g][1]benzopyran
(46). TLC Silica gel 60 F254, Solv. CH Cl /C
12 70:30, Rf ¼ 0.63. UV
max (nm) (log ) (MeOH): 209sh (4.29), 216 (4.37), 230sh (4.18), 310
(3.92), 331sh (3.78). H NMR (400 MHz, CDCl
3
)
d
2
2
6
H
l
3
1
7
3
)
d
7.45 (d, J ¼ 8.9 Hz,
¼
3
)
d
1H, H-7), 7.35 (d, J ¼ 2.6 Hz, 1H, H-10), 7.34 (s, 1H, H-11), 7.19 (s, 1H,
H-5), 7.05 (dd, J ¼ 2.6, 8.9 Hz, 1H, H-8), 6.50 (d, J ¼ 9.8 Hz, 1H, H-4),
(
1
5.80 (d, J ¼ 9.8 Hz,1H, H-3), 3.95 (s, 3H, eOCH
3
),1.52 (s, 6H, H-2a, 2b).
C NMR (75 MHz, CDCl ) d 155.5 (C-9), 152.0 (C-5a), 151.8 (C-6a),
3
13
1
1c), 112.7 (C-8), 111.2 (C-6), 75.8 (C-3), 27.3 (C-3a, 3b). MS (ESI) m/
þ
35
þ
37
þ
z: 285 (M þ H)
(
Cl), 287 (M þ H)
(
Cl), 307 (M þ Na) .
148.8 (C-11a), 132.8 (C-3), 124.9 (C-10a), 124.3 (C-10b), 122.9 (C-4),
121.5 (C-4a), 115.0 (C-8), 112.0 (C-7), 108.6 (C-5), 107.3 (C-11), 103.6
3
5
þ
HRESIMS calcd. for C17
H13ClO
2
(
Cl), 285.0679 (M þ H) , found
7
þ
2
2
85.0646; calcd. for C17
H13ClO
2
(3 Cl), 287.0835 (M þ H) , found
(C-10), 76.0 (C-2), 56.1 (CeOCH
3
), 27.6 (C-2a, 2b). MS (ESI) m/z: 279
ꢂ
þ
87.0547.
(M ꢂ H) . HRESIMS calcd. for C18
16 3
H O , 281.1173 (M þ H) , found
281.1171.
4
.1.26. 11-Chloro-3,3-dimethyl-3H-benzofuro[3,2-f][1]benzopyran
0
0
(44)
4.1.28. 3,3,10,10-Tetramethyl-3H,10H-furo[3,2-f:5,4-f ]dichromene
(47)
A solution of 9-chloro-2-(1,1-dimethylpropyn-1-oxy)dibenzo-
ꢀ
furan (38) (200 mg, 0.7 mmol) in DMF (7 mL) was heated at 150 C
for 4 h. The solvent was removed under reduced pressure. Purifi-
cation by column chromatography over silica gel (solvent cyclo-
hexane) gave 11-chloro-3,3-dimethyl-3H-benzofuro[3,2-f][1]benzo
pyran (44) (176 mg, 85%) as a white amorphous solid. TLC Silica gel
6
A solution of 2,7-di(1,1-dimethylpropyn-1-oxy)dibenzofuran
(40) (50 mg, 0.15 mmol) in DMF (2.5 mL) was heated at 150 C for
32 h. The solvent was removed under reduced pressure. Purifica-
tion by column chromatography over silica gel (solvent cyclo-
3,3,10,10-tetramethyl-3H,10H-furo[3,2-f:5,4-f ]
dichromene (47) (13 mg, 26%) as a white amorphous solid. TLC
ꢀ
0
hexane)
gave
ꢂ1
0 F254, Solv. CH
2
Cl
2
/C
6
H12 50:50, Rf ¼ 0.75. IR (KBr)
n
max (cm ):
3
1
(
059, 3027, 2969, 2922, 2868, 1460, 1421, 1408, 1368, 1360, 1252,
213, 1139, 1115, 958, 931, 809, 765, 708. UV max (nm) (log
MeOH): 212 (4.42), 237 (4.28), 285 (4.07), 295 (4.20), 306 (4.30),
Silica gel 60 F254, Solv. CH
2
Cl
2
/C
6
H
12 50:50, Rf ¼ 0.53. IR (KBr)
n
max
ꢂ1
l
3
)
(cm ): 3054, 2970, 2924, 2850, 1642, 1431, 1280, 1213, 1202, 1159,
1108, 1050, 805, 727. UV
(4.44), 246 (4.42), 284 (4.41), 292 (4.43), 333 (4.04). H NMR (400
MHz, CD OD)
lmax (nm) (log 3) (MeOH): 205 (4.39), 232
1
1
3
1
52 (3.65). H NMR (400 MHz, CD
), 7.53 (d, J ¼ 8.0 Hz, 1H, H-8), 7.44 (t, J ¼ 7.9 Hz, 1H, H-9), 7.38 (d, J
7.8 Hz, 1H, H-10), 7.38 (d, J ¼ 8.6 Hz, 1H, H-6), 7.03 (d, J ¼ 8.8 Hz,
3
OD)
d
7.90 (d, J ¼ 10.0 Hz, 1H, H-
3
d
7.72 (d, J ¼ 8.4 Hz, 1H, H-13) 7.30 (d, J ¼ 8.7 Hz, 1H,
¼
H-6), 7.05 (d, J ¼ 9.8 Hz, 1H, H-1), 6.91 (d, J ¼ 9.8 Hz, 1H, H-8), 6.87
(d, J ¼ 8.7 Hz, 1H, H-5), 6.84 (d, J ¼ 8.4 Hz, 1H, H-12), 5.85 (d, J ¼ 9.8
Hz, 1H, H-2), 5.79 (d, J ¼ 9.8 Hz, 1H, H-9), 1.53 (s, 12H, H-3a, 3b, 10a,
1
3
1
H, H-5), 5.85 (d, J ¼ 10.0 Hz, 1H, H-2), 1.48 (s, 6H, H-3a, 3b).
C
3
NMR (75 MHz, CD OD) d 157.9 (C-7a), 151.3 (C-6a), 149.5 (C-4a),
13
129.8 (C-2), 127.3 (C-9), 126.8 (C-11), 124.5 (C-10), 123.3 (C-11a),
3
10b). C NMR (75 MHz, CD OD) d 153.2 (C-7a), 152.3 (C-11a), 151.3
1
23.1 (C-1), 119.0 (C-11b),117.7 (C-5), 116.2 (C-11c), 111.3 (C-6), 110.4
(C-6a), 148.5 (C-4a), 131.8 (C-2), 130.5 (C-9), 121.5 (C-13), 120.2 (C-
13b), 119.4 (C-1), 117.7 (C-13a), 116.0 (C-8), 115.1 (C-13c), 114.4 (C-5),
112.0 (C-12), 110.7 (C-6), 106.7 (C-7b), 76.7 (C-10), 75.6 (C-3), 27.8
þ
35
(
2
(
(
C-8), 74.9 (C-3), 27.0 (C-3a, 3b). MS (ESI) m/z: 285 (M þ H)
(
Cl),
Cl), 285.0679
(37Cl), 287.0835
þ
37
35
87 (M þ H)
(
Cl). HRESIMS calcd. for C17
H
13ClO
13ClO
2
(
þ
þ
þ
M þ H) ; found 285.0670 & calcd. for C17
H
2
(C-10a, 10b), 27.4 (C-3a, 3b). MS (ESI) m/z: 333 (M þ H) . HRESIMS
þ
M þ H) ; found 287.0646.
calcd. for C22H
20
O
3
, 333.1485 (M þ H) , found 333.1499.
4.1.27. 10-Methoxy-3,3-dimethyl-3H-benzofuro[3,2-f][1]
4.1.29. 8-Hydroxy-3,3-dimethyl-3H-benzofuro[3,2-f][1]benzopyran
benzopyran (45) and 9-methoxy-2,2-dimethyl-2H-benzofuro[2,3-g]
(48)
[
1]benzopyran (46)
A solution of 8-methoxy-2-(1,1-dimethylpropyn-1-oxy)diben-
A
solution of 8-chloro-3,3-dimethyl-3H-benzofuro[3,2-f][1]
ꢂ3
benzopyran (41) (50 mg, 0.18 mmol), Pd
2
dba
3
(3.3 mg, 3.6 ꢁ 10
0 0 0
mmol), 2-di-tert-butylphosphino-3,4,5,6-tetramethyl-2 ,4 ,6 -trii-
zofuran (39) (50 mg, 0.18 mmol) in DMF (2 mL) was heated at
1
Purification by column chromatography over silica gel (solvent
cyclohexane) successively afforded 10-methoxy-3,3-dimethyl-3H-
benzofuro[3,2-f][1]benzopyran (45) (37.5 mg, 75%) and 9-methoxy-
ꢀ
0
ꢂ3
40 C for 20 h. The solvent was removed under reduced pressure.
sopropyl-1,1 -biphenyl (6.9 mg, 14.4 ꢁ 10 mmol), and KOH (29.5
mg, 0.53 mmol) in 1,4-dioxane/water 1:1 (1.5 mL) was heated
ꢀ
at 100 C under stirring for 8 h in a Schlenk tube previously evac-
uated and back-filled with argon. The reaction mixture was
extracted with dichloromethane (3 ꢁ 5 mL). The combined organic
2
,2-dimethyl-2H-benzofuro[2,3-g][1]benzopyran (46) (8 mg, 16%)
as white amorphous solids.
2 4
layers were dried over Na SO , filtered and evaporated under
reduced pressure. Column chromatography over silica gel (solvent:
cyclohexane/dichloromethane 1:0 to 1:1) gave 8-hydroxy-3,3-
dimethyl-3H-benzofuro[3,2-f][1]benzopyran (48) (42 mg, 92%) as
4
.1.27.1. 10-Methoxy-3,3-dimethyl-3H-benzofuro[3,2-f][1]benzo-
pyran (45). TLC Silica gel 60 F254, Solv. CH Cl /C
2
2
6
H12 70:30, Rf ¼

5844
A. Termentzi et al. / European Journal of Medicinal Chemistry 45 (2010) 5833e5847
a white amorphous solid. TLC Silica gel 60 F254, Solv. CH
2
Cl
2
/C
6
H
12
4.1.31. 10-Hydroxy-3,3-dimethyl-3H-benzofuro[3,2-f][1]
benzopyran (50)
The procedure described for the preparation of 48 from 41,
applied to 10-chloro-3,3-dimethyl-3H-benzofuro[3,2-f][1]benzo-
ꢂ1
5
2
7
2
0:50, Rf ¼ 0.1. IR (KBr)
n
max (cm ): 3373, 3066, 3034, 2978, 2971,
921, 1594, 1485, 1431, 1252, 1197, 1158, 1115, 987, 909, 808, 793,
34. UV max (nm) (log ) (MeOH): 207 (4.29), 216 (4.32), 241 (4.38),
69 (3.85), 293 (4.06), 305 (4.07), 346 (3.60), 360sh (3.53). H NMR
7.60 (d, J ¼ 8.5 Hz, 1H, H-11), 7.35 (d, J ¼ 8.4 Hz,
H, H-6), 7.25 (t, J ¼ 8.5 Hz, 1H, H-10), 7.11 (d, J ¼ 10.0 Hz, 1H, H-1),
l
3
1
ꢂ3
pyran (43) (100 mg, 0.36 mmol), Pd
2
dba
3
(6.6 mg, 7.2 ꢁ 10
0 0 0
mmol), 2-di-tert-butylphosphino-3,4,5,6-tetramethyl-2 ,4 ,6 -trii-
(
400 MHz, CDCl
3
)
d
0
ꢂ3
1
sopropyl-1,1 -biphenyl (13.8 mg, 29 ꢁ 10 mmol), and KOH (59
7
.05 (d, J ¼ 8.5 Hz, 1H, H-9), 6.98 (d, J ¼ 8.4 Hz, 1H, H-5), 5.89 (d, J ¼
mg, 1.06 mmol) in 1,4-dioxane/water 1:1 (3.0 mL), with heating for
13
ꢀ
1
0.0 Hz, 1H, H-2), 5.50 (s, 1H, -OH), 1.55 (s, 6H, H-3a, 3b). C NMR
75 MHz, CDCl 151.0 (C-6a), 148.6 (C-4a), 144.9 (C-7a), 141.2 (C-
),132.2 (C-2),125.8 (C-11a),123.5 (C-10),120.2 (C-11b),119.2 (C-1),
24 h at 100 C, gave 10-hydroxy-3,3-dimethyl-3H-benzofuro[3,2-f]
(
8
3
)
d
[1]benzopyran (50) (80 mg, 86%) as a white amorphous solid. TLC
Silica gel 60 F254, Solv. CH
(cm ): 3401, 3097, 3058, 2976, 2926, 2871, 1474, 1424, 1194, 1140,
2
Cl
2
/C
6
H
12 50:50, Rf ¼ 0.26. IR (KBr)
n
max
ꢂ1
1
16.1 (C-5), 115.9 (C-11c), 114.2 (C-11), 113.2 (C-9), 111.2 (C-6), 75.8
ꢂ
(
C-3), 27.4 (C-3a, 3b). MS (ESI) m/z: 265 (M ꢂ H) . HRESIMS calcd.
1112, 967, 852, 809, 743. UV
259 (4.29), 304 (3.87), 325sh (3.63), 335sh (3.52). H NMR (400
MHz, CDCl
7.45 (d, J ¼ 2.6 Hz,1H, H-11), 7.41 (d, J ¼ 8.8 Hz,1H, H-
), 7.28 (d, J ¼ 8.8 Hz, 1H, H-6), 7.01 (d, J ¼ 9.8 Hz, 1H, H-1), 6.95 (dd,
J ¼ 2.6, 8.8 Hz, 1H, H-9), 6.93 (d, J ¼ 8.8 Hz, 1H, H-5), 5.84 (d, J ¼ 9.8
lmax (nm) (log 3) (MeOH): 209 (4.34),
þ
1
for C17
H
14
O
3
, 267.1017 (M þ H) , found 267.1017.
3
) d
8
4
.1.30. 9-Hydroxy-3,3-dimethyl-3H-benzofuro[3,2-f][1]benzopyran
0
0
0
(49) and 3,3,3 ,3 -tetramethyl-9,9 -bi(3H-benzofuro[3,2-f]
13
Hz, 1H, H-2), 1.51 (s, 6H, H-3a, 3b). C NMR (75 MHz, CDCl
C-10), 151.9 (C-6a), 151.1 (C-7a), 148.2 (C-4a), 132.2 (C-2), 125.0 (C-
1a),120.9 (C-11b),119.1 (C-1), 116.3 (C-5), 115.8 (C-11c), 114.9 (C-9),
12.1 (C-8), 111.1 (C-6), 107.8 (C-11), 75.7 (C-3), 27.3 (C-3a, 3b). MS
3
) d 151.9
chromene) (52)
(
1
The procedure described for the preparation of 48 from 41,
applied to 9-bromo-3,3-dimethyl-3H-benzofuro[3,2-f]benzopyran
1
ꢂ3
(
42) (120 mg, 0.36 mmol), Pd
2
dba
3
(6.6 mg, 7.2 ꢁ 10 mmol), 2-di-
ꢂ
ꢂ
(ESI) m/z: 265 (M ꢂ H) , 531 (2M-H) . HRESIMS calcd. for C17
14 3
H O ,
0
0
0
0
tert-butylphosphino-3,4,5,6-tetramethyl-2 ,4 ,6 -triisopropyl-1,1 -
ꢂ
265.0861 (M ꢂ H) , found 265.0875.
ꢂ3
biphenyl (13.8 mg, 29 ꢁ 10 mmol), and KOH (59 mg, 1.06 mmol)
ꢀ
in 1,4-dioxane/water 1:1 (3.4 mL), with heating for 3 h at 100 C,
4.1.32. 11-Hydroxy-3,3-dimethyl-3H-benzofuro[3,2-f][1]
successively afforded after extraction and column chromatography
over silica gel (solvent: cyclohexane/dichloromethane 1:0 to 1:1) 9-
hydroxy-3,3-dimethyl-3H-benzofuro[3,2-f][1]benzopyran (49) (60
benzopyran (51)
A solution of 11-chloro-3,3-dimethyl-3H-benzofuro[3,2-f][1]
benzopyran (44) (160 mg, 0.56 mmol), Pd
2
dba
3
(10.6 mg, 11.5 ꢁ
0
0
0
mg, 62%) and 3,3,3 ,3 -tetramethyl-9,9 -bi(3H-benzofuro[3,2-f]
chromene) (52) (20 mg, 11%) as white amorphous solids.
ꢂ3
0
0
0
1
0
mmol), 2-di-tert-butylphosphino-2 ,4 ,6 -triisopropylbiphenyl
ꢂ3
(t-BuXPhos) (19.2 mg, 45 ꢁ10 mmol), and KOH (95 mg,1.7 mmol)
ꢀ
in 1,4-dioxane/water 1:1 (5 mL) was heated at 100 C under stirring
for 22 h in a Schlenk tube previously evacuated and back-filled with
argon. The reaction mixture was extracted with dichloromethane
(3 ꢁ 5 mL). The combined organic layers were dried over Na SO ,
4
(
.1.30.1. 9-Hydroxy-3,3-dimethyl-3H-benzofuro[3,2-f][1]benzopyran
49). TLC Silica gel 60 F254, Solv. CH Cl /C
12 50:50, Rf ¼ 0.11. IR
max (cm ): 3373, 3076, 2964, 2921, 1637, 1630, 1442, 1422,
255, 1142, 1113, 1080, 961, 817, 807, 718. UV max (nm) (log
MeOH): 208 (4.38), 215sh (4.25), 233 (4.27), 253 (4.20), 271 (3.94),
2
2
6
H
ꢂ1
(
KBr)
n
2
4
filtered and evaporated under reduced pressure. Column chroma-
tography over silica gel (solvent: cyclohexane/dichloromethane 1:0
to 1:1) gave 11-hydroxy-3,3-dimethyl-3H-benzofuro[3,2-f][1]ben-
1
(
2
d
l
3)
1
82 (3.99), 313sh (4.22), 321 (4.25). H NMR (400 MHz, CDCl
7.82 (d, J ¼ 8.5 Hz, 1H, H-11), 7.26 (d, J ¼ 8.6 Hz, 1H, H-6), 7.03 (d, J
9.8 Hz, 1H, H-1), 7.02 (d, J ¼ 2.3 Hz, 1H, H-8), 6.85 (d, J ¼ 8.6 Hz,
H, H-5), 6.85 (dd, J ¼ 2.3, 8.5 Hz, 1H, H-10), 5.84 (d, J ¼ 9.8 Hz, 1H,
3
)
zopyran (51) (36 mg, 23%) as a white amorphous solid. TLC Silica gel
ꢂ1
6
3
0 F254, Solv. CH
2
Cl
2
/C
6
H
12 50:50, Rf ¼ 0.29. IR (KBr)
n
max (cm ):
¼
318, 2968, 2917, 2851, 1598, 1472, 1423, 1290, 1259, 1143, 1115,
(nm) (log ) (MeOH): 217 (4.36),
1
13
1033, 960, 936, 773, 718. UV
l
max
3
H-2), 5.11 (s, 1H, -OH), 1.45 (s, 6H, H-3a, 3b). C NMR (75 MHz,
CDCl 158.9 (C-7a), 155.3 (C-9), 151.9 (C-4a), 148.9 (C-6a), 132.0
C-2), 122.6 (C-11), 120.0 (C-11b), 119.3 (C-1), 119.2 (C-11a), 115.3 (C-
1c), 114.6 (C-5), 111.2 (C-10), 110.8 (C-6), 98.8 (C-8), 75.9 (C-3), 27.4
1
2
(
1
42 (4.39), 292 (4.26), 305 (4.20), 346 (3.69), 361 (3.63). H NMR
400 MHz, CDCl
7.93 (d, J ¼ 10.0 Hz, 1H, H-1), 7.33 (d, J ¼ 8.6 Hz,
H, H-6), 7.31 (t, J ¼ 8.0 Hz, 1H, H-9), 7.18 (d, J ¼ 8.2 Hz, 1H, H-10),
3
) d
3
) d
(
1
ꢂ
6.98 (d, J ¼ 8.6 Hz, 1H, H-5), 6.70 (d, J ¼ 7.8 Hz, 1H, H-8), 5.81 (d, J ¼
(
C
C-3a, 3b). MS (ESI) m/z: 265 (M ꢂ H) . HRESIMS calcd. for
13
ꢂ
10.0 Hz, 1H, H-2), 5.64 (s, 1H, -OH), 1.53 (s, 6H, H-3a, 3b). C NMR
75 MHz, CDCl 158.9 (C-11), 150.7 (C-6a), 150.0 (C-7a), 148.9 (C-
a), 130.7 (C-2), 127.7 (C-9), 122.5 (C-1), 119.9 (C-11b), 116.1 (C-5),
17
H
14
O
3
, 265.0861 (M ꢂ H) , found 265.0864.
(
4
3
) d
0
0
0
4
(
.1.30.2. 3,3,3 ,3 -Tetramethyl-9,9 -bi(3H-benzofuro[3,2-f]chromene)
52). TLC Silica gel 60 F254, Solv. CH Cl /C
max (cm ): 3042, 2976, 2925, 2855, 1485, 1435, 1414,
max (nm) (log 3)
115.9 (C-11c), 112.5 (C-11a), 110.6 (C-6), 108.6 (C-8), 104.6 (C-10),
ꢂ
2
2
6
H
12 70:30, Rf ¼ 0.40.
75.2 (C-3), 27.4 (C-3a, 3b). MS (ESI) m/z: 265 (M ꢂ H) . HRESIMS
ꢂ1
ꢂ
IR (KBr)
1
(
CD
n
calcd. for C
7 14 3
H
O , 265.0861 (M ꢂ H) , found 265.0861.
1
271, 1254, 1188, 1158, 1112, 964, 807. UV
MeOH): 207 (4.72), 237sh (4.49), 334 (4.52). H NMR (400 MHz,
l
1
4.1.33. 10-Acetyl-3,3-dimethyl-1,2-dihydro-3H-benzofuro[3,2-f][1]
benzopyran (56)
0
3
OD)
d
8.08 (d, J ¼ 8.2 Hz, 2H, H-11, H-11 ), 7.86 (d, J ¼ 1.6 Hz,
0
0
2
H, H-8, H-8 ), 7.68 (dd, J ¼ 1.6, 8.2 Hz, 2H, H-10, H-10 ), 7.35 (d, J
Palladium on charcoal (5% Pd) (4 mg) was added to a solution of
10-acetyl-3,3-dimethyl-3H-benzofuro[3,2-f][1]benzopyran (6) (45
mg, 0.15 mmol) in EtOH (10 ml). The reaction mixture was stirred
under hydrogen pressure (4 bar) for 24 h and filtered. After evap-
oration of the solvent, column chromatography over silica gel
(solvent: dichloromethane/cyclohexane 95:5) gave 10-acetyl-3,3-
dimethyl-1,2-dihydro-3H-benzofuro[3,2-f][1]benzopyran (56) (40
mg, 88%) as a brown solid. TLC Silica gel 60 F , Solv. CH Cl /MeOH
0
0
¼
8.7 Hz, 2H, H-6, H-6 ), 7.14 (d, J ¼ 9.8 Hz, 2H, H-1, H-1 ), 6.96
0
0
0
(
d, J ¼ 8.7 Hz, 2H, H-5, H-5 ), 5.89 (d, J ¼ 9.8 Hz, 2H, H-2, H-2 ),
0
13
1
d
.54 (s, 12H, H-3a, 3b, H-3 a, 3 b). C NMR (75 MHz, CD
3
OD)
0
0
157.7 (C-7a, C-7’a), 151.6 (C-6a, C-6 a), 148.6 (C-4a, C-4 a), 139.9
0
0
0
(
1
1
C-9, C-9 ), 132.3 (C-2, C-2 ), 123.8 (C-11a, C-11 a), 122.3 (C-11, C-
0 0 0 0
1 ), 122.2 (C-10, C-10 ), 119.5 (C-11b, C-11 b), 119.3 (C-1, C-1 ),
0 0 0
16.1 (C-5, C-5 ), 115.7 (C-11c, C-11 c), 111.1 (C-6, C-6 ), 110.4 (C-8,
2
54
2
2
0
0
0
0
ꢂ1
C-8 ), 75.8 (C-3, C-3 ), 27.4 (C-3a, 3b, C-3 a, 3 b). MS (ESI) m/z:
(9.5/0.5), Rf ¼ 0.55, IR (KBr) nmax (cm ): 2969, 2922, 1673, 1488,
1447, 1416, 1354, 1248, 1162, 1120, 953, 824, 799. UV (nm) (log
ꢂ
þ
4
97 (M ꢂ H) . HRESIMS calcd. for C34
H
26
O
4
, 499.1902 (M þ H) ,
l
max
1
found 499.1881.
3) (MeOH): 220 (4.26), 244 (4.36), 267 (4.34). H NMR (300 MHz,

A. Termentzi et al. / European Journal of Medicinal Chemistry 45 (2010) 5833e5847
5845
CDCl
3
)
d
8.62 (d, J ¼ 2.0 Hz,1H, H-11), 8.09 (dd, J ¼ 2.0, 9.0 Hz,1H, H-
), 7.55 (d, J ¼ 9.0 Hz,1H, H-8), 7.37 (d, J ¼ 9.0 Hz,1H, H-6), 6.9 (d, J ¼
.0 Hz, 1H, H-5), 3.33 (t, J ¼ 7.0 Hz, 2H, H-1), 2.71 (s, 3H, CH CO),
.12 (t, J ¼ 7.0 Hz, 2H, H-2), 1.41 (s, 6H, H-3a, 3b). C NMR (75 MHz,
197.5 (CH CO), 159.6 (C-7a), 150.9 (C-6a), 150.0 (C-4a),
32.1 (C-10), 127.5 (C-9), 125.9 (C-11a), 122.9 (C-11), 121.9 (C-11b),
17.9 (C-5), 115.8 (C-11c), 111.4 (C-8), 110.3 (C-6), 74.0 (C-3), 32.2
was poured into a flask containing NaH (13.5 mg, 0.56 mmol).
Dimethylsulfate (21 L, 0.22 mmol) was slowly added and the
9
9
2
m
3
reaction was stirred at room temperature for 1 h. The reaction
mixture was quenched with water (10 mL) and extracted
with dichloromethane (3 ꢁ 10 mL). The organic layers were
concentrated under reduced pressure. Chromatography over silica
gel (solvent: cyclohexane/dichloromethane 1:0 to 1:1) gave
8-methoxy-3,3-dimethyl-3H-benzofuro[3,2-f][1]benzopyran (53)
13
CDCl
3
)
d
3
1
1
(
(
C-2), 26.9 (CH
M þ Na) .
3
CO), 26.6 (C-3a,3b), 21.1 (C-1). MS (ESI): m/z 317
þ
(22 mg, 70%) as a white amorphous solid. TLC Silica gel 60 F254, Solv.
ꢂ1
CH
2
Cl
2
/C
6
H
12 80:20, Rf ¼ 0.57. IR (KBr)
n
max (cm ): 3058, 2976,
4.1.34. 10-Acetoxy-3,3-dimethyl-1,2-dihydro-3H-benzofuro[3,2-f]
2933, 2836, 1626, 1582, 1497, 1483, 1463, 1423, 1271, 1208, 1185,
1158, 1115, 1065, 980, 901, 851, 808, 793, 734. UV max (nm) (log
(MeOH): 219 (4.47), 240 (4.49), 267 (3.98), 293 (4.16), 305 (4.15),
[1]benzopyran (57)
l
3)
A solution of 10-acetyl-3,3-dimethyl-1,2-dihydro-3H-benzofuro
1
[3,2-f][1]benzopyran (56) (50 mg, 0.17 mmol) and 3-chloroper-
3
331 (3.63), 345 (3.65), 361sh (3.54). H NMR (400 MHz, CDCl )
oxybenzoic acid (625 mg, 3.6 mmol) in CHCl (50 mL) was stirred
at room temperature for 21 h. The reaction mixture was washed
3
d
7.64 (d, J ¼ 7.9 Hz,1H, H-11), 7.40 (d, J ¼ 8.7 Hz,1H, H-6), 7.30 (t, J ¼
7.9 Hz, 1H, H-10), 7.12 (d, J ¼ 9.8 Hz, 1H, H-1), 7.03 (d, J ¼ 8.0 Hz, 1H,
H-9), 6.97 (d, J ¼ 8.7 Hz,1H, H-5), 5.88 (d, J ¼ 9.8 Hz,1H, H-2), 4.11 (s,
with aqueous sodium hydroxide solution (2 ꢁ 25 mL) and water (2
13
ꢁ
25 mL). The organic layer was dried over magnesium sulfate,
filtered, and evaporated under reduced pressure. Column chro-
3 3
3H, eOCH ), 1.54 (s, 6H, H-3a, 3b). C NMR (75 MHz, CDCl ) d 150.9
(C-7a), 148.6 (C-4a), 146.0 (C-6a), 145.7 (C-8), 132.1 (C-2), 125.8 (C-
11a), 123.2 (C-10), 119.7 (C-11b), 119.2 (C-1), 116.1 (C-5), 115.7 (C-
matography over silica gel (solvent: dichloromethane/cyclohexane
9
:1) gave 10-Acetoxy-3,3-dimethyl-1,2-dihydro-3H-benzofuro[3,2-
f][1]benzopyran (57) as a yellow amorphous solid (22 mg, 40%).
TLC Silica gel 60 F254, Solv. CH Cl /C
) (MeOH): 245 (4.22), 256 (4.20), 293(4.23). H NMR
300 MHz, CDCl
): 7.69 (dd, J ¼ 0.5, 2.5 Hz, 1H, H-11), 7.55 (dd, J ¼
.5, 8.5 Hz, 1H, H-8), 7.34 (d, J ¼ 9.0 Hz, 1H, H-6), 7.15 (dd, J ¼ 2.5,
.5 Hz, 1H, H-9), 6.95 (d, J ¼ 9.0 Hz, 1H, H-5), 3.21 (t, J ¼ 7.0 Hz, 2H,
CO), 1.97 (t, J ¼ 7.0 Hz, 2H, H-2), 1.38 (s, 6H,
H-3a, 3b). 13C NMR (75 MHz, CDCl
) 170.1 (CH CO), 154.2 (C-7a),
51.0 (C-6a), 149.5 (C-4a), 145.7 (C-10), 125.7 (C-11a), 122.2 (C-
1b), 119.8 (C-9), 117.7 (C-5), 115.3 (C-11c), 115.0 (C-11), 111.9 (C-8),
11c), 114.3 (C-11), 111.4 (C-6), 108.9 (C-9), 75.7 (C-3), 56.2 (CeOCH
3
),
þ
27.4 (C-3a, 3b). MS (ESI) m/z: 303 (M þ Na) . HRESIMS calcd. for
þ
2
2
6
H
12 (9/1), Rf ¼ 0.25. UV
l
max
C
18
H
16
O
3
, 281.1173 (M þ H) , found 281.1174.
1
(
(
nm) (log 3
3
4.1.37. 9-Methoxy-3,3-dimethyl-3H-benzofuro[3,2-f][1]benzopyran
(54)
The procedure described for the preparation of 53 from 48,
applied to 9-hydroxy-3,3-dimethyl-3H-benzofuro[3,2-f][1]benzo-
pyran (49) (40 mg, 0.15 mmol), NaH (18 mg, 0.75 mmol), and
0
8
H-1), 2,37 (s, 3H, CH
3
3
3
1
1
dimethylsulfate (28
mL, 0.29 mmol), gave 9-methoxy-3,3-dimethyl-
3H-benzofuro[3,2-f][1]benzopyran (54) (31.5 mg, 75%) as a white
1
10.2 (C-6), 73.8 (C-3), 32.2 (C-2), 26.5 (C-3a, 3b), 21.2 (CH
3
CO),
amorphous solid. TLC Silica gel 60 F254, Solv. CH
2
Cl
2
/C
6
H
12 80:20, Rf
þ
ꢂ1
2
0.8 (C-1). MS (ESI): m/z 333 (M þ Na) .
¼ 0.69. IR (KBr)
nmax (cm ): 3053, 2966, 2922, 2838, 1637, 1501,
1419, 1275, 1150, 1112, 1079, 964, 827, 807, 715. UV
lmax (nm) (log 3)
4
.1.35. 10-Hydroxy-3,3-dimethyl-1,2-dihydro-3H-benzofuro[3,2-f]
(MeOH): 207 (4.46), 235 (4.30), 253 (4.25), 271 (4.00), 281 (4.04),
1
[1]benzopyran (58)
321 (4.33), 350sh (4.02). H NMR (400 MHz, CDCl
3
)
d
7.85 (d, J ¼ 8.7
Sodium hydroxide pellets (55 mg, 1.37 mmol) were added to
Hz, 1H, H-11), 7.27 (d, J ¼ 8.7 Hz, 1H, H-6), 7.07 (d, J ¼ 2.3, 1H, H-8),
a solution of 10-acetoxy-3,3-dimethyl-1,2-dihydro-3H-benzofuro
7.04 (d, J ¼ 9.8 Hz, 1H, H-1), 6.93 (dd, J ¼ 2.3, 8.8 Hz, 1H, H-10), 6.85
[3,2-f][1]benzopyran (57) (22 mg, 0.07 mmol) in ethanol (20 mL).
(d, J ¼ 8.7 Hz, 1H, H-5), 5.83 (d, J ¼ 9.8 Hz, 1H, H-2), 3.92 (s, 3H,
ꢀ
13
The reaction mixture was heated at 50 C for 2.5 h. After evapo-
ration of the solvent under reduced pressure, the solid residue was
partitioned between dichloromethane (20 mL) and water (20 mL).
The aqueous layer was acidified by addition of concentrated
aqueous HCl solution (12 N, 10 mL) and extracted with dichloro-
methane (20 mL). The organic layer dried over magnesium sulfate,
filtered, and evaporated under reduced pressure gave 10-hydroxy-
eOCH
3
), 1.51 (s, 6H, H-3a, 3b). C NMR (75 MHz, CDCl
3
)
d
159.6 (C-
9), 158.4 (C-7a), 151.2 (C-6a), 148.5 (C-4a), 131.9 (C-2), 122.4 (C-11),
120.0 (C-11b), 119.4 (C-1), 117.5 (C-11a), 115.03 (C-11c), 114.4 (C-11),
110.8 (C-6), 96.4 (C-8), 75.6 (C-3), 55.7 (eOCH
3
), 27.4 (C-3a, 3b). MS
þ
þ
þ
(ESI) m/z: 281 (M þ H) , 303 (M þ Na) , 583 (2M þ Na) . HRESIMS
þ
calcd. for C18H
16
O
3
, 281.1173 (M þ H) , found 281.1174.
3
,3-dimethyl-1,2-dihydro-3H-benzofuro[3,2-f][1]benzopyran (58)
4.1.38. 11-Methoxy-3,3-dimethyl-3H-benzofuro[3,2-f][1]
benzopyran (55)
The procedure described for the preparation of 53 from 48,
applied to 11-hydroxy-3,3-dimethyl-3H-benzofuro[3,2-f][1]benzo-
pyran (51) (20 mg, 0.08 mmol), NaH (9.5 mg, 0.38 mmol), and
as a yellow amorphous solid (17 mg, 95%). TLC Silica gel 60 F254
Solv. CH
,
ꢂ1
2
Cl
2
/C
6
H12 (9/1), Rf ¼ 0.25. IR (KBr)
n
max (cm ): 3362;
3
229; 2973; 2922; 1595; 1474; 1451; 1424; 1381; 1369; 1193; 1159;
1120; 801. UV
l
max (nm) (log
3
) (MeOH): 259 (4.05), 303 (4.23), 321
1
(
3.73), 335 (3.64). H NMR (300 MHz, CDCl
3
):
d
7.35 (d, J ¼ 2.3 Hz,
dimethylsulfate (15.5
mL, 0.16 mmol), gave 11-methoxy-3,3-
1
6
H, H-11), 7.41 (d, J ¼ 8.7 Hz, 1H, H-8), 7.29 (d, J ¼ 8.9 Hz, 1H, H-6),
dimethyl-3H-benzofuro[3,2-f][1]benzopyran (55) (13 mg, 62%) as
a white amorphous solid. TLC Silica gel 60 F254, Solv. CH
.95 (dd, J ¼ 2.3, 8.7 Hz, 1H, H-9), 6.92 (d, J ¼ 8.9 Hz, 1H, H-5), 3.39
2
Cl
2
/C
6
H
12
ꢂ1
(
3
t, J ¼ 6.8 Hz, 2H, H-1), 1.9 (t, J ¼ 6.8 Hz, 2H, H-2), 1.3 (s, 6H, H-3a,
70:30, Rf ¼ 0.45. IR (KBr)
n
max (cm ): 3104, 2976, 2925, 2836, 1594,
13
b). C NMR (75 MHz, CDCl
3
)
d
154.5, 151.1, 151 (C-6a, C-7a, C-10),
1496, 1470, 1463, 1423, 1375, 1279, 1256, 1212, 1154, 1119, 1088, 967,
804, 773, 718. UV max (nm) (log ) (MeOH): 214 (4.42), 238 (4.40),
282sh (4.13), 293 (4.31), 305 (4.28), 345 (3.73), 360 (3.64). H NMR
(400 MHz, CDCl
7.85 (d, J ¼ 10.0 Hz, 1H, H-1), 7.43 (t, J ¼ 8.2 Hz,
125.8 (C-11a), 122 (C-11b), 117 (C-5), 115.2 (C-11c), 114.4 (C-9), 111.8
l
3
1
(
C-8), 110.1 (C-6), 108.0 (C-11), 73.8 (C-3), 32.3 (C-2), 26.5 (C-3a, 3b),
þ
2
C
0.8 (C-1). MS (ESI): m/z 267 (M þ H) . HRESIMS m/z calcd for
3
) d
þ
17
H
16
O
3
, 267.1095 (M þ H) , found 267.1020.
1H, H-9), 7.33 (d, J ¼ 8.7 Hz, 1H, H-6), 7.22 (d, J ¼ 8.2 Hz, 1H, H-8),
6
.97 (d, J ¼ 8.7 Hz,1H, H-5), 6.83 (d, J ¼ 8.2 Hz, 1H, H-10), 5.80 (d, J ¼
13
4
.1.36. 8-Methoxy-3,3-dimethyl-3H-benzofuro[3,2-f][1]benzopyran
53)
A solution of 8-hydroxy-3,3-dimethyl-3H-benzofuro[3,2-f][1]
benzopyran (48) (30 mg, 0.11 mmol) in anhydrous DMF (1.5 mL)
10.0 Hz, 1H, H-2), 4.09 (s, 3H, eOCH
3
), 1.53 (s, 6H, H-3a, 3b).
C
(
3
NMR (75 MHz, CDCl ) d 158.3 (C-7a), 154.4 (C-11), 150.7 (C-6a),
148.9 (C-4a), 130.4 (C-2), 127.9 (C-9), 122.8 (C-1), 121.5 (C-11b),
115.9 (C-11c), 115.9 (C-5), 113.5 (C-11a), 110.6 (C-6), 104.8 (C-8),

5846
A. Termentzi et al. / European Journal of Medicinal Chemistry 45 (2010) 5833e5847
1
03.7 (C-10), 75.0 (C-3), 55.5 (CeOCH
3
), 27.4 (C-3a, 3b). MS (ESI) m/
4.2.3.1. Preparation of cell wall extracts. Freshly grown bacteria
were washed with 10 mM potassium phosphate buffer, pH 7.0, and
resuspended in 50 mM -mercaptoethanol (buffer A). Bacteria were
þ
þ
z: 281 (M þ H) , 303 (M þ Na) . HRESIMS calcd. for C18
16 3
H O ,
þ
2
4
4
81.1173 (M þ H) , found for 281.1170.
b
lysed by cell distruption, by two passages through a One-shot Cell
Disrupter (Constant Systems Ltd.) at 0.8 kbar. The crude extract was
.2. Biology
ꢀ
centrifuged for 15 min at 3000 ꢁ g at 4 C. The very dense layer at
.2.1. MIC determinations
the surface of the supernatant, consisting of cell wall and
membrane fragments [39], was removed, resuspended in buffer A
and homogenized with a syringe (needle size 0.7 ꢁ 30 mm). The
protein concentration of the extracts was determined with the DC
method (Bio-Rad) for insoluble proteins by boiling in 0.5 N NaOH at
MICs were determined using the Microdilution Resazurin
Assay (MRA) [18]. Resazurin salt powder (Sigma) was prepared
at 0.01% (wt/vol) in distilled water, sterilized by filtration
through a 0.22
stock solutions were prepared in dimethylsulfoxide (DMSO) at
concentration of 10 mg/mL and frozen until used. The inocula
were prepared from M. bovis BCG or M. tuberculosis H37Rv strains
grown in 7H9, supplemented with 10% ADC enrichment (Difco).
Two microliters (one in case of M. tuberculosis) of two-fold serial
ꢀ
mm membrane and stored at 4 C until used. Drug
ꢀ
100 C for 10 min.
4.2.3.2. Evaluation of the activity of all final products to inhibit MA
biosynthesis in vitro. MA biosynthesis was assayed in vitro in
a reaction medium containing 100 mM potassium phosphate
dilutions of each drug were prepared in 200
100 L for M. tuberculosis) directly in 96-well plates at concen-
trations from 100 g/mL to 0.1 g/mL (500 g/mL to 0.9 g/mL
for M. tuberculosis). Growth controls containing DMSO and
m
L of 7H9 medium
buffer, pH 7.0, 3 mM MgCl
CoASH and 500 g of cell wall extract proteins in a total volume of
500 L. Mixtures were first incubated in the presence or absence of
2 3
, 7 mM KHCO , 7 mM ATP, 0.7 mM
(
m
m
m
m
m
m
m
the compounds (10 mM solutions in DMSO, such as the final solvent
isoniazid (from 1
plates were covered, sealed and incubated at 37 C. After 8 days
m
g/mL to 1 ng/mL were also included. The
concentration in the reaction medium is 5% v/v). Reaction mixtures
were incubated at 37 C for 15 min and then 50 mM of [1- C]
ꢀ
ꢀ
14
for M. bovis or 6 days for M. tuberculosis, 30
m
L of resazurin
_acetate (PerkinElmer Life Sciences) were added for the MA and
fatty acid biosynthesis. Reaction mixtures were incubated at 37 C
for 60 min. The reactions were stopped by saponification and the
MA profiles were analyzed by TLC.
ꢀ
solution was added to each well and plates were allowed to
ꢀ
incubate at 37 C for an additional 24 h. A change from blue to
pink indicates the reduction of resazurin and therefore bacterial
growth. The MIC was defined as the lowest drug concentration
that prevented this color change.
Results were expressed as % inhibition of mycolate and fatty acid
synthesis in comparison to the control. The concentrations of the
compounds were equal to their antituberculosis MICs determined
for M. tuberculosis.
4.2.2. Cytotoxicity evaluation
14
A cell line, which grows attached to tissue culture plates, was
[1- C]_acetate (specific activity, 56.60 mCi/mmol) was
used for the determination of the antiproliferative activity of the
synthesized benzofuranobenzopyrans. Monkey kidney epitheliar
VERO cell cultures were maintained in Dulbecco’s Modified
Engle’s medium (DMEM) supplemented with 10% v/v fetal bovine
purchased from PerkinElmer Life Sciences.
Acknowledgements
serum (FBS) and antibiotics (penicillin and streptomycin 100
ml). Cells were incubated at 37 C in a humidified atmosphere
containing 5% CO and maintained at densities that permitted
2
m
g/
One of us (A. Termentzi) wishes to acknowledge “Ville de Paris”
for postdoctoral financial support.
ꢀ
logarithmic growth. For the evaluation of cytotoxicity of the
synthesized compounds, cells at their logarithmic phase of
growth were removed from culture and seeded in 24-well plate
Appendix. Supplementary data
ꢂ4
Supplementary data associated with this article can be found in
the online version at doi:10.1016/j.ejmech.2010.09.048.
cultures at cell density 2 ꢁ 10 cells/ml. Cells then were allowed
to attach in plates for 3 h before the addition of any compound.
All compounds were diluted in dimethylsulfoxide (DMSO),
whereas the final concentration of the solvent in cell culture did
not exceed the level of 0.4% v/v. That solvent concentration
caused no measurable growth inhibition for both cell lines.
Chemical molecules were added in cultures at different concen-
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