Synthesis and antimycobacterial evaluation of benzofurobenzopyran analogues

Article abstract of DOI:10.1016/j.bmc.2006.12.009

We recently reported that 3,3-dimethyl-3H-benzofuro[3,2,f][1]-benzopyran and its hydrogenated analogue are selective in vitro inhibitors of mycobacterial growth. However, their lack of in vivo activity on a murine model of Mycobacterium tuberculosis infec

Full text of DOI:10.1016/j.bmc.2006.12.009

Bioorganic & Medicinal Chemistry 15 (2007) 2177–2186
Synthesis and antimycobacterial evaluation of
benzofurobenzopyran analogues
Soizic Prado,a,b Yves L. Janin,a,* Brigitte Saint-Joanis,b Priscille Brodin,b Sylvie Michel,c
Michel Koch,c Stewart T. Cole,b Franc¸ois Tillequinc and Pierre-Etienne Bosta
aLaboratoire de Chimie Organique, URA 2128 CNRS-Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France
bUnite´ de Ge´ne´tique Mole´culaire Bacte´rienne, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France
cLaboratoire de Pharmacognosie de l’Universite´ Rene´ Descartes, UMR/CNRS 8638,
Faculte´ des Sciences Pharmaceutiques et Biologiques, 4 avenue de l’Observatoire, 75006 Paris, France
Received 25 October 2006; revised 25 November 2006; accepted 8 December 2006
Available online 12 December 2006
Abstract—We recently reported that 3,3-dimethyl-3H-benzofuro[3,2,f][1]-benzopyran and its hydrogenated analogue are selective
in vitro inhibitors of mycobacterial growth. However, their lack of in vivo activity on a murine model of Mycobacterium tuberculosis
infection due to their poor bioavailability led to a structure–activity relationship investigation. We wish to report here the prepa-
ration of some structural analogues along with their biological effect on the growth of Mycobacterium smegmatis, M. tuberculosis,
as well as on VERO cells for the most active compound.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
specific for Mycobacteria. We also started a structure–
activity relationship study. This synthetic work not
only attempts to obtain analogues with an in vivo
antimycobacterial effect but also to generate active
compounds suitable for a biochemical target identifi-
cation using affinity chromatography. This paper
describes the synthesis of various compounds
structurally related to 1 and the first elements of the
structure–activity relationship obtained.
The antimycobacterial activity of the 3,3-dimethyl-3H-
benzofuro[3,2,f][1]-benzopyran (1), or its dihydro ana-
logue 2, was measured1 in view of the antibacterial
activity reported for the chromene-containing struc-
tures of various compounds2–5 and that of the less
structurally related antimycobacterial agent;6–8 the
cytotoxic9 and hepatotoxic10 usnic acid (3) (Fig. 1).
From our results, a survey of other antimycobacterials
related to compound 1 pointed out the series of diben-
zofurans or dibenzothiophenes reported in the 50’s for
their in vitro antimycobacterial activity.11–14 Among
them, the sulfate 4 was reported to be the most active
in vitro but, similarly to 1 or 2, these compounds were
found inactive in vivo in the mouse model. On the
other hand, compounds 1 and 2 display a most
remarkable selectivity of action on the Mycobacterium
genus.1 For this reason, biological studies have been
undertaken in order to elucidate their mechanism
of action and identify a potential biological target
Keywords: Antimycobacterials; Benzofuro[3,2,f][1]-benzopyran; Struc-
ture–activity relationship study; Mycobacterium tuberculosis.
*
Corresponding author. Tel.: +33 6 85 42 49 47; fax: +33 1 45 68 84
04; e-mail: yljanin@pasteur.fr
Figure 1.
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.12.009

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S. Prado et al. / Bioorg. Med. Chem. 15 (2007) 2177–2186
2. Chemistry
and, in our case, 4-hydroxybenzophenone (6) provided
the chromane analogue 23. Following a preparation of
the previously described 7-hydroxychromane 24,22 we
synthesized analogue 25, bearing an internal hydrogen
bond between the carbonyl function and the chromene
C–D ring system via a Friedel-Craft reaction with ben-
zoyl chloride. This internal hydrogen bond was intro-
duced in an attempt to reduce the single bond rotation
existing in structures 16, 17, and 23. A possible occur-
rence of an equilibrium between a putative, biologically
active, flat conformation (the one depicted) and an inac-
tive one (the opposite) was the reason behind this part of
our work. Moreover, we further demonstrated the po-
tential for chemical diversity in this series of analogues
and prepared the nitrogen-bearing derivative 27. This
was achieved via a condensation reaction between the
aldehyde-bearing chromene 26 (made from 4-hydroxy-
benzaldehyde using the two-step preparation depicted
in Scheme 1) and 3-pyridylmagnesiumchloride.23
(Scheme 2).
In many instances the synthesis of the analogues de-
scribed here started from commercially available phe-
nols. As depicted on Scheme 1, from the hydroxy-
bearing compounds 5–9, the corresponding propargylic
ethers 10–14 were made using the remarkable copper
chloride-catalysed method.15,16 Note: for the structures
of some of the numbered synthetic intermediates not
depicted on the schemes, see the Experimental part. This
was followed by a thermal treatment17–19 in boiling
dichlorobenzene which provided, via a Claisen cycliza-
tion, the chromene-containing compounds 15–19 as
depicted in Figure 2.
From the unsymmetrical 3-phenylphenol (9), the Claisen
cyclization of the corresponding propargylic ether 14 led
to a mixture of the corresponding mixture of chromenes
19a and 19b which could not be separated in our hands
(data not shown). As described below, this two-compo-
nent mixture was evaluated anyway.
Further hydroxy-bearing compounds were prepared pri-
or to the construction of the chromene ring according to
the synthetic path depicted in Scheme 1. From benzo-
quinone (28), using modified procedures,24–27 involving
the Michael addition of enamines 29 followed by a cycli-
zation under acidic conditions, we prepared the phenol
30. Attempts to use more functionalized enamines were
not very successful and further work is still ongoing. It is
noteworthy that such reactions are related to the
Nenitzescu hydroxyindole preparation from benzoqui-
Since analogue 16 turned out to be biologically interest-
ing, we prepared from it alcohol 20 or the further re-
duced compound 21 by treatment with either sodium
borohydride or ammonium formate in the presence of
palladium over charcoal. The use of a montmorillonite
K10-catalysed reaction20,21 between allylbromide (22)
Scheme 1. Reagents and condition: (i) ClC(Me)2CCH, DBU, MeCN,
CuCl2; (ii) 1,3-dichlorobenzene, reflux.
Scheme 2. Reagents: (i) NaBH4, EtOH; (ii) NH3, HCO2H/Pd/C,
EtOH; (iii) K10, CH2Cl2; (iv) C6H5COCl, AlCl3, CH2Cl2; (v)
3-pyridylMgCl, THF.
Figure 2.

S. Prado et al. / Bioorg. Med. Chem. 15 (2007) 2177–2186
2179
none and enamines28 which sometimes proceeds in mod-
est yield and often requires extensive optimisation of the
reaction conditions.29 From this phenol, analogue 32
was obtained using the usual route (see Scheme 1). Trac-
es of the isomeric chromene were observed in the course
of its preparations and could be removed during the
purification steps (Scheme 3).
The preparation of compound 1, via the cyclization of
the corresponding propargylic ether, always takes place
with the occurrence of traces of the linear analogues 37
and we could remove these traces to below detectable
levels by recrystallization.1 However, in order to remove
any ambiguity concerning its improbably powerful anti-
mycobacterial property, we devised an unequivocal syn-
thetic access to compound 37. The bromination of 2-
hydroxydibenzofuran (33) enabled the preparation of
pure 5-bromo derivative 34 from which, through the
usual route depicted in Scheme 1, the brominated linear
analogue 36 was obtained. Hydrogenation of 36 using
palladium over charcoal and one or two equivalents of
ammonium formate allowed the preparation of the line-
ar analogue 37 or the further reduced chromane-bearing
compound 38 (Scheme 4).
Scheme 5. Reagents: (i) 3-ClPhCO3H, CF3CO2H, CH2Cl2; (ii)
MeONa, MeOH; (iii) see Scheme 1.
From the previously described30 2,8-diacyldibenzofuran
(39), a limited Baeyer–Villinger reaction led to the 2-ac-
etyl-8-acyl derivative (40) along with a proportion of the
corresponding 2,8-diacetyl derivative (41). The follow-
ing hydrolysis of the mixture of these esters provided a
mixture of 2-hydroxydibenzofurans 42 and 43 which
Scheme 6. Reagents: (i) NBS, EtOH; (ii) CH3COCl, AlCl3, CH2Cl2.
could be separated. However, if the propargylic ether
44 could be obtained from 42 (and transformed into
analogues
45)
alkylation
trials
with
2,8-
dihydroxydibenzofuran (43) only led to extensive
decomposition (Scheme 5).
As the chromane analogue 2 is as effective on Mycobac-
terium tuberculosis growth in vitro as compound 1, we
undertook some chemical transformation of 2 aiming
at introducing substituents on its ring C. Such chemistry
is not possible on compound 1 because of the reactive
chromene double bond.1 Bromination of 2 gave the
5-bromo derivative 46 in good yield. On the other hand,
the aluminium chloride-catalysed acylation of 2 gave the
11-acyl analogue 47 along with the bisfuran derivative
Scheme 3. Reagents: (i) a—toluene, b—H2SO4; (ii) see Scheme 1.
48 resulting from
a Lewis acid-catalysed pyran
rearrangement into a furan ring.31,32 The separation of
compounds 47 and 48 turned out to be difficult and
could only be achieved in low yield (Scheme 6).
3. Biological results and discussion
The antimycobacterial activity was screened on the fast
growing saprophyte Mycobacterium smegmatis mc2155
and on the virulent strain M. tuberculosis H37Rv for
all synthesized compounds, using the new Microdilution
Resazurin Assay (MRA).33 The minimal inhibitory
concentration (MIC95) is defined as the amount of
compound required for >95% inhibition of bacterial
growth. The compounds found to be the most active
Scheme 4. Reagents and condition: (i) NBS, EtOH; (ii) see Scheme 1;
(iii) or (iv) NH3, HCO2H (1 or 2 equiv), Pd/C, EtOH, reflux.

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S. Prado et al. / Bioorg. Med. Chem. 15 (2007) 2177–2186
Table 1. In vitro biological assays
6 days would result in loss of biological effect. The
replacement of the furan B ring was attempted by an
ether linkage (compound 15), or a single carbon–carbon
bond (compound 18 and the mixture 19a and 19b). This
was not successful from a biological point of view. In a
different approach, the furan ring was replaced by a car-
bonyl group (compounds 16, 17, and 23), a hydroxym-
ethylene (compounds 20 and 27) or a methylene
(compound 21). This met with a certain degree of suc-
cess. However, as shown in Table 1, the most active
compounds were evaluated for their cytotoxicity on
VERO cells and turned out to be cytotoxic at the same
concentration range, contrary to the pyranodibenzofu-
rans 1 and 2. For this reason, further work on this type
of tricyclic analogues was stopped. The recent report on
the activity of various acetophenones35,36 was of interest
in the light of the antimycobacterial activity we mea-
sured for 4,4’-dihydroxybenzophenone (7) or com-
pounds 16, 23, and 25. The linear tetracyclic analogues
36–38, which had been prepared in order to lift any
doubt concerning trace amounts of the linear isomer
37 at the origin of the measured antimycobacterial activ-
ities for compound 1, turned out to be somewhat less ac-
tive. An important difference of activity on M.
tuberculosis was observed between compounds 37 and
38 but this may just reflect a slow precipitation of com-
pound 38 out of the culture media of M. tuberculosis as
mentioned above. The biological results for the few
angular tetracyclic analogues 45–47 made so far are dis-
appointing as they illustrate a very narrow structure–ac-
tivity relationship for this series. However, the recurrent
problem of water solubility is acute for all these com-
pounds; the estimated CLogP of compound 1 is actually
5.5. The substituents so far introduced (bromine or ace-
tyl) on the benzofuro[3,2,f][1]-benzopyran structure do
not improve this physical characteristic. These results
lead to the conclusion that there is a need to devise ori-
ginal synthetic access to analogues of 1 and 2 bearing
more diverse substituents thereby providing improved
water solubility. From the chemistry point of view,
two other syntheses of the core structure of 1 or 2 were
reported.37,38 On the other hand, the design of prepara-
tions of polyfunctionalized hydroxydibenzofurans along
with methods to circumvent the recurrent problem of
the unequivocal cyclization of asymmetric propargylic
ethers into chromenes (as depicted in Fig. 1) would still
be quite useful. A recent report of dibenzofuran synthe-
sis39 should be mentioned here as well as our recent
work on the synthesis of 2,2-dimethylchromenes from
various salicylaldehydes.40
Compound MIC95 (lg/mL) MIC95 (lg/mL) IC50
M. smegmatis
M. tuberculosis (VERO Cells)
Isoniazid
1
12
10
0.1
10
>500
80
2
10
62
10
100
ND
ND
32
7
125
>500
40
15
16
17
18
19a,b
20
21
23
25
27
30
32
33
36
37
38
39
42
43
45
46
47
48
70
20
32
62
ND
ND
ND
16
>500
>500
20
>500
>500
20
>500
50
30
30
16
75
4
62
62
62
ND
ND
ND
ND
ND
10
31
500
120
25
>500
ND
120
20
30
35
ND
ND
16
40
5
>500
10
>500
15
8
15
16
16
>500
>500
>500
>500
>500
>500
>500
>500
ND
ND
ND
ND
ND, not determined.
against M. tuberculosis were further evaluated for their
cytotoxicity on mammalian VERO cell line growth
using the dimethylthiazolyldiphenyl tetrazolium
(MTT)-based assay.
Many propargylic ether intermediates not shown in Ta-
ble 1 (compounds 10, 13, 14, 31, and 44) were tested on
M. smegmatis and M. tuberculosis but their antimyco-
bacterial activity turned out to be above 100 lg/mL.
Other tricyclic intermediates were found to be almost
as active as the pyranodibenzofuran 1. For instance,
2-hydroxydibenzofuran (33) and the dibenzofurans 39,
42, and 43 displayed antimycobacterial activities. This
is in accordance with the results previously reported
for the mono-substituted series of dibenzofurans such
as the sulfate derivative 411–14 or more recently reported
carbazoles.34 However and also in accordance with these
previously reported results, compounds 39, 42, and 43
turned out to be also cytotoxic for VERO cells. Of note
is the drastic difference of effect on M. tuberculosis or M.
smegmatis observed for some analogues (see compounds
21, 30, 38, and 42). These contrasting results, which were
confirmed at least by two experiments, are of interest for
the design of biological assays on Mycobacterium in gen-
eral and deserve further investigations. Aside from the
fact that the two bacteria have different biochemistry
and physiology, it is probably important to mention that
an assay on M. smegmatis takes 2 days, whereas an as-
say on M. tuberculosis requires at least 6 days. We sug-
gest that this time factor explains some of the differences
observed. In the case of the most lipophilic compounds,
a slow crystallization out of the growth medium over
4. Experimental
1H NMR and 13C NMR spectra were recorded on a
Bruker spectrometer at 400 and 100 MHz, respectively.
Most often, two-dimensional experiments were per-
formed in order to further demonstrate structural
assignments. Shifts (d) are given in ppm with respect
to the TMS signal and coupling constants (J) are given
in Hertz. Column chromatography was performed over
Merck silica gel 60 (0.035–0.070 mm), using a solvent
pump operating at pressure between 2 and 7 bar

S. Prado et al. / Bioorg. Med. Chem. 15 (2007) 2177–2186
2181
(25–50 mL/mn) and an automated collecting system
driven by a UV detector set to 254 nm unless stated
otherwise. Mass spectra were obtained on an Agilent
1100 series LC/MSD system using an atmospheric elec-
trospray ionization system, a C18 column, a gradient of
water and either acetonitrile containing 0.07% of either
formic acid or water and methanol containing 0.07%
ammonium formate.
(s, 6H). 13C (CDCl3): 195.2; 160.2; 159.7; 133.0; 132.2;
131.9; 130.8; 119.8; 115.5; 85.7; 75.0; 72.7; 29.9.
3-(2-Methylbutyn-2-yloxy)biphenyl (13). Using the gen-
eral procedure described above and after a chromatog-
raphy over silica gel (cyclohexane/dichloromethane,
95:5), compound 13 was obtained as a pale oil (1.1 g,
68%) from 4-phenylphenol (8). LC/MS (ES) m/z 237
1
[M+H]+. H (CDCl3): 7.80 (m, 2H); 7.66 (m, 2H); 7.54
General procedure for synthesis of propargylic ethers
(m, 3H); 7.43 (m, 2H); 2.70 (s, 1H); 1.85 (s, 6H). 13C
(CDCl3): 155.5; 141.4; 141.3; 136.0; 127.8; 127.3;
122.1; 86.7; 75.4; 73.7; 30.1.
To a solution of the phenol (1 equiv) in anhydrous ace-
tonitrile (50 mL for 5.5 mmol; dried over 4A molecular
sieve) under argon and cooled using an ice bath were
added DBU (1.3 equiv) and copper chloride dihydrate
(0.01 equiv). The solution was maintained at 0 ꢁC and
chlorobutyne (1.3 equiv) was added. After stirring for
5 h, the mixture was concentrated at reduced pressure
and the residue was partitioned between water and tolu-
ene. The organic fraction was washed with hydrochloric
acid 1 N, sodium hydroxide 1 N, sodium hydrogenocar-
bonate 1 N and brine and dried over anhydrous sodium
sulfate. After concentration to dryness, the residue was
purified as described below.
3-(2-Methylbut-3-yn-2-yloxy)biphenyl (14). Using the
general procedure described above and after a chroma-
tography over silica gel (cyclohexane/dichloromethane,
99.5:0.5), compound 14 was obtained from 3-phenylphe-
nol (9) as a pale oil (1.1 g, 69%). LC/MS (ES) m/z 237
1
[M+H]+. H (CDCl3): 7.62 (m, 2H); 7.46 (m, 3H); 7.54
(m, 3H); 7.35 (m, 2H); 7.23 (m,1H); 2.62 (s, 1H); 1.71
(s, 6H). 13C (CDCl3): 156.3; 142.5; 141.3; 129.5; 127.9;
127.5; 127.2 (three signals); 122.1; 121.9; 91.0; 74.4;
75.8; 30.1.
2,2-Dimethyl-6-phenoxy-2H-chromene (15). From com-
pound 10, using the general procedure described above,
compound 15 was obtained after chromatography over
silica gel (cyclohexane/dichloromethane, 95:5) as an oil
0.19 g, (75%). LC/MS (ES) m/z 253 [M+H]+. 1H
(CDCl3): 7.33 (m, 2H); 7.07 (dd, 1H, J = 7 and 1);
6.98 (m, 2H); 6.81 (d, 2H, J = 3); 6.78 (s, 1H); 6.71 (d,
2H, J = 3); 6.30 (d, 1H, J = 9); 5.67 (d, 1H, J = 9);
1.47 (s, 6H). 13C (CDCl3): 158.8; 150.4; 149.4; 132.0;
129.9; 122.8; 122.6; 122.4; 120.6; 118.0; 117.8; 117.5;
75.5; 30.0.
General procedure for the cyclization of the propargylic
ethers
Chromene was obtained from propargylic ethers by
heating them in 1,2-dichlorobenzene (100 mL or
7 mmol) for 12 h. Purification of the residue, obtained
after concentration to dryness, was made by a chroma-
tography over silica gel and led to the chromene-con-
taining compounds described below.
1-(2-Methylbut-3-yn-2-yloxy)-4-phenoxybenzene
(10).
Using the general procedure described above and after
a chromatography over silica gel (cyclohexane/dichloro-
methane, 98:2), compound 10 was obtained as an oil
(0.4 g, 60%) from 4-phenoxyphenol (5). LC/MS (ES)
(2,2-Dimethyl-2H-chromen-6-yl)(phenyl)methanone (16).
From compound 11, using the procedure described
above, chromene 16 was obtained after chromatography
over silica gel (cyclohexane/dichloromethane, 50:50) as
an oil (88%). LC/MS (ES) m/z 265 [M+H]+. 1H (CDCl3):
7.75 (m, 1H); 7.49–7.60 (m, 6H); 6.83 (dd, 1H, J = 1.2
and 9.8); 6.37 (d, 1H, J = 9.8); 5.69 (dd, 1H, J = 1.4
and 9.8); 1.5 (s, 6H). 13C (CDCl3): 195.9; 157.6; 138.7;
132.7; 132.2; 131.6; 130.6; 130.1; 129.2; 128.6; 122.1;
121.0; 116.3; 77.9; 28.8.
1
m/z 253 [M+H]+. H (CDCl3): 7.35 (m, 2H); 7.21 (m,
2H); 7.09 (dd, 1H, J = 7 and 1); 7.02 (d, 2H, J = 8);
6.96 (d, 2H, J = 9); 2.57 (s, 1H); 1.66 (s, 6H). 13C
(CDCl3): 158.3; 152.9; 151.6; 130.0; 123.8; 123.2;
119.9; 118.5; 86.6; 74.1; 73.1; 29.9.
(4-(2-Methylbut-3-yn-2-yloxy)phenyl)(phenyl)methanone
(11). Using the procedure described above, propargylic
ether 11 was obtained from 4-hydroxybenzophenone
(6) after a chromatography over silica gel (cyclohex-
ane/dichloromethane, 6:4) as an oil (75%). LC/MS
(2,2-Dimethyl-2H-chromen-6-yl)(phenyl)methanone (17).
From compound 12, using the procedure described
above, chromene 17 was obtained after chromatography
over silica gel (dichloromethane/ethanol, 98:02) as a yel-
low amorphous solid (58%). LC/MS (ES) m/z 279
[MꢀH]ꢀ. 1H (CDCl3): 7.75 (m, 2H); 7.62 (dd, 1H,
J = 9 and 2); 7.50 (d,1H, J = 2); 7.18 (s, 1H, OH); 6.94
(m, 2H); 6.84 (d, 1H, J = 9); 6.35 (d, 1H, J = 9); 5.70
(d, 1H, J = 9); 1.5 (s, 6H). 13C (CDCl3): 195.8; 160.6;
157.4; 133.0; 132.6; 131.6; 130.9; 130.6; 129.2; 122.0;
121.0; 116.4; 115.6; 77.9; 28.7.
1
(ES) m/z 279 [MꢀH]ꢀ. H (CDCl3): 7.78 (m, 4H); 7.58
(m, 1H); 7.49 (m, 2H); 7.31 (m, 2H); 2.66 (s, 1H); 1.74
(s, 6H). 13C (CDCl3): 196.1; 160.1; 138.5; 132.3; 132.2;
131.6; 130.2; 128.6; 119.5; 85.6; 75.2; 72.2; 30.1; 30.0.
(4-Hydroxyphenyl)(4-(2-methylbut-3-yn-2-yloxy)phenyl)-
methanone (12). Using the procedure described above,
propargylic ether 12 was obtained from 4,40-dihydroxy-
benzophenone (7) after a chromatography over silica gel
(cyclohexane/dichloromethane, 6:4) as an oil (75%). LC/
2,2-Dimethyl-6-phenyl-2H-chromene (18). Cyclization of
compound 13 under conditions described above gave
compound 18 as an oil (0.35g, 37%) after chromatogra-
phy over silica gel (cyclohexane/dichloromethane, 98:2).
1
MS (ES) m/z 263 [MꢀH]ꢀ weak. H (CDCl3): 7.78 (m,
4H); 7.29 (m, 2H); 6.91 (m, 2H); 2.69 (s, 1H, s); 1.74

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S. Prado et al. / Bioorg. Med. Chem. 15 (2007) 2177–2186
1
LC/MS (ES) m/z 235 [M+H]+. H (CDCl3): 7.45 (m,
2H); 7.41 (m, 2H); 7.37 (dd, 1H, J = 8 and 2); 7.32
(dd, 1H, J = 7 and 1); 7.23 (d, 1H, J = 2); 6.87 (d, 1H,
J = 8); 6.41 (d, 1H, J = 9); 5.68 (d, 1H, J = 9); 1.48 (s,
6H). 13C (CDCl3): 152.9; 141.3; 134.3; 131.5; 129.0;
128.1; 127.0 (three signals); 125.4; 121.8; 122.7; 121.8;
116.9; 75.5; 28.4.
dryness. The residue was purified by chromatography
over silica gel (cyclohexane/dichloromethane, 3:7 to
pure dichloromethane) to give, in order of elution, the
following not quite expected22 four compounds. Com-
pound 2,2,8,8-tetramethyl-3,4,9,10-tetrahydro-2H,8H-
1
pyrano[2,3-f]chromene: 3% yield, oil. H (CDCl3): 6.81
(d, 1H, J = 8.3); 6.35 (d, 1H, J = 8.3); 2.71 (t, 2H,
J = 6.7); 2.62 (t, 2H, J = 6.8); 1.77 (m, 4H); 1.33 (m,
12H). 13C (CDCl3): 153.2; 152.2; 111.7; 109.9; 108.9;
74.3; 73.9; 33.3; 32.8; 27.5; 27.1; 22.4; 17.5. Compound
2,2,8,8-tetramethyl-2,3,4,6,7,8-hexahydropyrano[3,2-g]chro-
(2,2-Dimethyl-2H-chromen-6-yl)(phenyl)methanol (20).
Compound 16 (1.07 g, 4.05 mmol) was dissolved in eth-
anol (80 mL) and sodium borohydride (0.23g,
6.07 mmol) was added. This was stirred at room temper-
ature overnight and concentrated to dryness. The resi-
due was dissolved in dichloromethane, the organic
phase was washed with water, dried over sodium sulfate
and concentrated to dryness to give a quantitative yield
of 20. Solid, LC/MS (ES) m/z 249 [MꢀOH]ꢀ, 266
[MꢀOH + NH3]+. 1H (CDCl3): 7.49–7.60 (m, 4H);
7.10 (dd, 1H, J = 2.2 and 8.2); 7.00 (d, 1H, J = 2.2);
6.75 (d, 1H, J = 8.2); 6.30 (d, 1H, J = 9.8); 5.77 (s,
1H); 5.61 (d, 1H, J = 9.8); 2.18 (s br, 1H); 1.43 (s,
6H). 13C (CDCl3): 152.8; 144.3; 136.7; 131.4; 128.8;
127.9; 127.8; 126.9; 125.1; 122.7; 121.6; 116.6; 76.7;
76.2; 28.5; 28.4.
1
mene: 3.2% yield, solid. H (CDCl3): 1.32 (s, 12H); 6.74
(s, 1H); 6.24 (d, 1H, J = 8.3); 2.69 (t, 4H, J = 6.7); 1.77
(t, 4H, J = 6.7). 13C (CDCl3): 153.3; 129.7; 113.0;
105.1; 74.2; 33.5; 27.2; 22.2. Compound 2,2-dimethylch-
roman-5-ol: 4.6% yield, solid. 1H (CDCl3): 6.97 (m, 1H);
6.43 (dd, 1H, J = 1.0 and 8.2); 6.34 (dd, 1H, J = 1.0 and
7.9); 4.70 (s, 1H); 2.68 (t, 2H, J = 6.8); 1.83 (t, 2H,
J = 6.8); 1.35 (s, 6H). 13C (CDCl3): 155.5; 154.2; 127.5;
110.3; 108.8; 106.3; 74.3; 32.5; 27.0; 17.2. Compound
1
24: LC/MS (ES) m/z 179 [M+H]+ 23% yield, wax. H
(CDCl3): 6.92 (d, 1H, J = 8.1); 6.36 (dd, 1H, J = 2.5
and 8.1); 6.29 (d, 1H, J = 2.5); 4.59 (m, 1H); 2.71 (t,
2H, J = 6.7); 1.79 (t, 2H, J = 6.7); 1.34 (s, 6H). 13C
(CDCl3): 155.2; 130.5; 113.7; 107.7; 104.2; 74.8; 33.3;
27.3; 22.1.
6-Benzyl-2,2-dimethylchroman (21). Compound 16
(0.15 g, 0.56 mmol), ammonium formate (0.5 g, 8.5
mmol) and 10% palladium over charcoal (0.03 g,
0.03 mmol) were refluxed in ethanol (40 mL) for 1 h.
The suspension was filtered and concentrated to dry-
ness. The residue was purified by chromatography over
silica gel (cyclohexane/dichloromethane, 7:3) to give
compound 21 (0.13 g, 85%) as a solid. LC/MS (ES) m/
z 267 [M+H]+. 1H (CDCl3): 7.60 (m, 2H); 7.23 (m,
3H); 6.96 (m, 2H); 6.75 (m, 1H); 3.93 (s, 2H); 2.71 (t,
2H, J = 6.7); 1.82 (t, 2H, J = 6.7); 1.30 (s, 6H). 13C
(CDCl3): 152.7; 142.2; 132.5; 130.1; 129.3; 128.8;
128.2; 126.3; 121.1; 117.5; 74.4; 41.5; 33.3; 27.3; 22.9.
7-Hydroxy-2,2-dimethylchroman-6-yl)(phenyl)methanone
(25). Compound 24 (0.26 g, 1.4 mmol) and benzoyl chlo-
ride (0.2 mL, 0.0017 mol) were dissolved in dichloro-
methane (50 mL, distilled over P2O5). Dry aluminium
chloride (0.23 g, 1.7 mmol) was added and the reaction
mixture was protected from humidity by a calcium
guard and stirred for an hour. Water was added and this
was extracted with dichloromethane. The organic layer
were dried over sodium sulfate and concentrated to dry-
ness. A first chromatography over silica gel eluting with
a mixture of dichloromethane and cyclohexane, 7:3, pro-
vided a fraction (0.21 g) containing a mixture of isomer-
ic compounds. This was recrystallized in methanol
(20 mL) to yield pure compound 25 (0.088 g, 24%).
(2,2-Dimethyl-2H-chroman-6-yl)(phenyl)methanone (23).
4-Hydroxybenzophenone (6) (0.5 g, 2.52 mmol), 1-bro-
mo-3-methylbut-2-ene (0.64 mL, 5.04 mol) and mont-
morillonite K10 (10 g) were stirred in dichloromethane
(50 mL) overnight. The suspension was filtered and
concentrated to dryness. The residue was purified by
chromatography over silica gel (cyclohexane/dichloro-
methane, 4:6 to 1:1) to give a small fraction of pure com-
pound 23 which had to be further sublimated (0.04 g,
1
LC/MS (ES) m/z 283 [M+H]+. H (CDCl3): 12.27 (s,
1H); 7.58 (m, 5H); 7.30 (s, 2H); 6.43 (s, 1H); 2.67 (t,
2H, J = 6.8); 1.82 (t, 2H, J = 6.8); 1.38 (s, 6H). 13C
(CDCl3): 200.3; 164.2; 161.9; 139.1; 135.5; 131.6;
129.2; 128.7; 113.6; 113.0; 105.2; 76.4; 33.1; 27.4; 22.1.
2,2-Dimethyl-6-formyl-2H-chromene (26). Using the
above-mentioned
7%). LC/MS (ES) m/z 267 [M+H]+. H (CDCl3): 7.78
protocol
4-(2-methylbut-3-yn-2-
1
(m, 1H); 7.60 (m, 2H); 7.50 (m, 2H); 6.84 (d, 1H,
J = 8.5); 2.84 (t, 2H, J = 6.7); 1.87 (t, 2H, J = 6.7);
1.39 (s, 6H). 13C (CDCl3): 196.2; 158.8; 138.9; 132.9;
132.0; 131.8; 130.1; 129.5; 128.5; 121.2; 117.4; 75.9;
32.9; 27.3; 22.7.
yloxy)benzaldehyde, precursor to compound 26, was ob-
tained from 4-formylphenol (5.0 g, 41 mmol) after puri-
fication by chromatography over silica gel (cyclohexane/
dichloromethane, 50:50, v/v) as a pale oil (3.70 g,
1
44.79%). LC/MS (ES) m/z 189 [M+H]+. H (CDCl3):
9.92 (s, 1H); 7.84 (m, 2H); 7.34 (m, 2H); 2.67 (s, 1H);
1.73 (s, 6H). 13C (CDCl3): 191.3; 161.6; 131.2 (two sig-
nals); 131.0; 119.7 (two signals); 85.3; 75.3; 72.8; 29.8.
This propargylic ether was cyclized using the above-
mentioned protocol to give, after purification by chro-
matography over silica gel (cyclohexane/dichlorometh-
ane, 70:30 v/v), compound 26 as a pale oil (1.59 g,
51.4%). LC/MS (ES) m/z 268 [M+H]+. 1H (CDCl3):
8.65 (d, 1H, J = 1); 8.53 (dd, 1H, J = 4 and 1); 7.73
2,2-Dimethylchroman-7-ol (24). Resorcinol (10.08 g,
(4.76 mL,
91 mmol)
and
2-methylbut-3-en-2-ol
91.0 mmol) were refluxed in 80% formic acid (50 mL)
for 4 h as previously described.22 The solution was
concentrated, diluted in water, made basic with solid
sodium hydrogen carbonate and extracted with
dichloromethane. The organic layer were washed with
water, dried over sodium sulfate, and concentrated to

S. Prado et al. / Bioorg. Med. Chem. 15 (2007) 2177–2186
2183
(m, 1H); 7.28 (m, 1H); 7.10(dd, 1H, J = 8 and 2); 6.98 (d,
1H, J = 2); 6.77 (d, 1H, J = 8); 6.30 (d, 1H, J = 9); 5.81
(s; 1H); 5.65 (d, 1H, J = 9); 1.49 (s, 6H). 13C (CDCl3):
153.2; 149.1; 148.6; 139.6; 135.7; 134.4; 131.6; 127.8;
125.0; 123.7; 122.4; 121.8; 116.8; 74.2; 76.8; 28.4.
1.97 mmol) using the general procedure for the cycliza-
tion of the propargylic ethers described above and was
purified by chromatography over silica gel (cyclohex-
ane/dichloromethane, 98:2). LC/MS (ES) m/z 255
1
[M+H]+. H (CDCl3): 7.12 (d, 1H, J = 8); 6.77 (d, 1H,
J = 9); 6.68 (d, 1H, J = 9); 5.65 (d, 1H, J = 8); 2.81 (m,
1H); 2.73 (m, 1H); 1.92 (m, 2H,); 1.49 (s, 6H). 13C
(CDCl3):155.5; 149.8; 148.5; 131.1; 130.7; 119.9; 114.6;
114.1; 113.8; 113.0; 111.4; 75.8; 27.5; 24.4; 23.9.
(2,2-Dimethyl-2H-chromen-6-yl)-pyridin-3-yl-methanol
(27). A 2 M isopropyl magnesium chloride solution in
THF (1.58 mL, 3.16 mmol) was added under argon to
a solution of 3-bromopyridine (0.5 g, 3.16 mmol) in
anhydrous THF (16 mL, distilled over sodium). The
reaction mixture was stirred at room temperature for
1 h. The chromene 26 was then added and the reaction
mixture was stirred for 2 h. The solution was evaporated
to dryness and the residue was dissolved in CH2Cl2
(50 mL) and washed with H2O (50 mL). The organic
layer was dried over anhydrous Na2SO4, filtered, and
evaporated. Purification by chromatography of the res-
idue over silica gel (cyclohexane/dichloromethane, 5:5)
gave compound 27 as an amorphous solid (30%). LC/
1-Bromo-dibenzofuran-2-ol (34). To a solution of 2-
hydroxydibenzofuran (33) (5 g, 27.00 mmol) in ethanol
(50 mL), N-bromosuccinimide (4.8g, 40.7 mmol) was
added and this was stirred overnight. The solvent was
then evaporated to dryness and the residue purified by
chromatography over silica gel (cyclohexane/dichloro-
methane, 5:5). A recrystallization of the chromatograph-
ic fraction containing 34 and a dibrominated side-
product in methanol gave compound 34 as white crys-
tals (4.5 g, 63%). LC/MS (ES) m/z 261, 263 [MꢀH]ꢀ.
1H (CDCl3): 8.45 (d, 1H, J = 8); 7.61 (d, 1H, J = 8);
7.53 (dd, 1H, J = 8 and 2); 7.51 (d, 1H, J = 9); 7.43
(dd, 1H, J = 8 and 2); 7.19 (d, 1H, J = 9); 5.5 (s, 1H,
OH). 13C (CDCl3):157.2; 151.0; 148.8; 128.1; 124.4;
124.1; 122.8; 122.4; 115.1; 112.0; 111.9; 102.7; 75.8.
1
MS (ES) m/z 268 [M+H]+. H (CDCl3): 8.65 (d, 1H,
J = 1); 8.53 (dd, 1H, J = 5 and 1); 7.73 (m, 1H); 7.28
(m, 1H); 7.10 (dd, 1H, J = 8 and 2); 6.98 (d, 1H,
J = 2); 6.77 (d, 1H, J = 8); 6.30 (d, 1H, J = 9); 5.81 (s,
1H); 5.65 (d, 1H, J = 9); 1.45 (s, 6H). 13C (CDCl3):
153.2; 149.07; 148.6; 139.6; 135.7; 134.4; 131.6; 127.8;
125.0; 123.7; 122.4; 121.8; 116.8; 76.8; 74.2; 28.4.
1-Bromo-2-(2-methylbut-3-yn-2-yloxy)dibenzo[b,d]furan
(35). Following the general procedure described above a
quantitative yield of compound 35 was obtained from
compound 34 as an oil. LC/MS (ES) m/z 329,331
6,7,8,9-Tetrahydrodibenzo[b,d]furan-2-ol (30). To a solu-
tion of benzoquinone (28) (1 g, 9.25 mmol) in toluene
(50 mL) at 0 ꢁC was added drop-wise the commercially
available enamine 29 (2 g, 12 mmol). The reaction mix-
ture was stirred at 0 ꢁC for 4 h. The solid precipitate
was filtered off and washed with toluene and this was
dried in a desiccator (3.1 g). It was then dispersed in
H2SO4 70% (16 mL) and the suspension stirred at room
temperature for 3 h. The mixture was diluted with water
and extracted with ethyl acetate. The organic layer was
dried over anhydrous sodium sulfate, filtered, and evap-
orated to dryness. A purification of the residue by chro-
1
[M+H]+. H (CDCl3): 8.60 (dd, 1H, J = 8 and 2); 7.75
(d, 1H, J = 8); 7.57 (d, 1H, J = 8); 7.53 (dd, 1H, J = 8
and 2); 7.49 (d, 1H, J = 8); 7.42 (dd, 1H, J = 8 and 2);
2.61 (s, 1H); 1.71 (s, 6H). 13C (CDCl3): 157.2; 152.8;
151.3; 125.1; 124.8; 123.1; 122.2; 121.5; 111.9; 111.8;
110.5; 86.7; 73.3; 30.0.
1-Bromo-2,2-dimethyl-2H-benzofuro[2,3-g][1]benzopyran
(36). Cyclization according to the general procedure,
after chromatography over silica gel (cyclohexane), gave
a quantitative yield of compound 36 as an oil. LC/MS
matography
over
silica
gel
(cyclohexane/
dichloromethane, 5:5) gave compound 30 as an amor-
phous solid (0.73 g, 42%). LC/MS (ES) m/z 189
1
(ES) m/z 327; 330 [MꢀH]ꢀ. H (CDCl3): 8.50 (d, 1H,
1
[M+H]+. H (CDCl3): 7.25 (d, 1H, J = 8); 6.85 (d, 1H,
J = 8); 7.52 (d, 1H, J = 8); 7.48 (dd, 1H, J = 8 and 2);
7.37 (dd, 1H, J = 8 and 2); 6.45 (d, 1H, J = 9); 5.80 (d,
1H, J = 9); 1.40 (s, 6H). 13C (CDCl3): 157.2; 151.3;
146.3; 133.1; 127.8; 125.0; 124.1; 122.9; 122.1; 122.8;
111.7; 107.9; 104.0; 75.8; 27.7.
J = 3); 6.73 (dd, 1H, J = 8 and 3); 4.73 (s, 1H); 2.75
(m, 1H); 2.58 (m, 1H); 1.95 (m, 1H); 1.93 (m, 1H). 13C
(CDCl3):155.7; 151.5; 149.7; 130.1; 113.2; 111.4; 104.3;
27.3; 23.9; 23.3; 20.8.
2,2-Dimethyl-2H- benzofuro[2,3-g][1]benzopyran (37). To
a solution of 36 (0.15 g, 0.46 mmol) in ethanol (50 mL)
were added 10% Pd/C (0.02 g, 0.02 mmol) and ammoni-
um formate (0.03 g, 0.46 mmol). The reaction mixture
was heated at reflux during 5 h. After chromatography
over silica gel (cyclohexane/dichloromethane, 98:2),
compound 37 (0.045 g, 40%) was obtained as a white
amorphous solid. LC/MS (ES) m/z 251 [M+H]+. 1H
(CDCl3): 7.88 (d, 1H, J = 8); 7.55 (d, 1H, J = 8); 7.45
(dd, 1H, J = 8 and 2); 7.36 (s, 1H); 7.33 (dd, 1H, J = 8
and 2); 7.19 (s, 1H); 6.49 (d, 1H, J = 8); 5.78 (d, 1H,
J = 8); 1.51 (s, 6H). 13C (CDCl3): 157.3; 151.5; 149.3;
132.5; 217.2; 127.1; 127.7; 122.8; 122.5; 212.8; 120.9;
112.1; 111.5; 108.2; 74.5; 28.0.
8-(2-Methylbut-3-yn-2-yloxy)-1,2,3,4-tetrahydrodiben-
zo[b,d]furan (31). Using the general procedure for syn-
thesis of propargylic ethers described above compound
31 was obtained from 30 (1 g, 5.32 mmol) as an oil in
1
37% yield. LC/MS (ES) m/z 255 [M+H]+. H (CDCl3):
7.31 (d, 1H, J = 8); 7.26 (d, 1H, J = 2); 7.10 (dd, 1H,
J = 8 and 2); 2.75 (m, 1H); 1.97 (m, 1H); 1.87 (m, 1H);
1.97 (m, 1H); 1.77 (s, 1H); 1.69 (s, 3H). 13C
(CDCl3):155.2; 151.4; 151.3; 129.4; 119.1; 113.5; 112.6;
110.7; 91.7; 87.0; 74.4; 73.3; 30.0; 23.9; 23.3; 23.0; 20.3.
3,3-Dimethyl-8,9,10,11-tetrahydro-benzofuro[3,2-f][1]benzo-
pyran (32). Compound 32 was obtained as a solid (0.2 g,
40%) from ether propargylic ether 31 (0.59 g,

2184
S. Prado et al. / Bioorg. Med. Chem. 15 (2007) 2177–2186
2,2-Dimethyl-3,4-dihydro-2H-benzofuro[2,3-g][1]benzo-
pyran (38). To a solution of 36 (0.40 g, 1.22 mmol) in
ethanol (50 mL) were added 10% Pd/C (0.06 g,
(0.006 g, 38%). LC/MS (ES) m/z 293 [M+H]+. 1H
(CDCl3): 8.64 (1H, J = 2); 8.11 (dd, 1H, J = 2 and 8);
7.60 (d, 1H, J = 8); 7.34 (d, 1H, J = 8); 7.15 (1H,
J = 9); 6.98 (1H, J = 8); 5.91 (d, 1H, J = 9); 2.73 (s,
1H); 1.70 (s, 6H). 13C (CDCl3): 197.6; 160.1; 152.1;
149.3; 133.1; 132.7; 128.1; 125.3; 123.3; 119.5; 117.3;
116.5; 111.9; 111.7; 76.3; 27.7; 27.2.
0.06 mmol)
and
ammonium
formate
(0.15 g,
2.44 mmol). The reaction mixture was heated at reflux
during 5 h. After filtration, the filtrate was concentrated
to dryness and purified by chromatography over silica
gel (cyclohexane/dichloromethane, 98:2) yielding com-
pound 38 (0.11 g, 35%) as a white amorphous solid.
LC/MS (ES) m/z 253 [M+H]+. 1H (CDCl3): 7.90 (d,
1H, J = 8); 7.52 (d, 1H, J = 8); 7.42 (dd, 1H, J = 8 and
2); 7.35 (s, 1H); 7.29 (dd, 1H, J = 8 and 2); 7.29 (s,
1H); 3.00 (t, 2H, J = 6); 1.91 (t, 2H, J = 6); 1.45 (s,
3H); 1.40 (s, 3H). 13C (CDCl3): 157.0 (C6a); 150.9;
150.4; 127.1; 124.9; 123.7; 122.6; 121.6; 120.8; 111.8;
111.4; 108.2; 74.5; 33.2; 27.3; 23.8.
5-Bromo-3,3-dimethyl-2,3-dihydro-1H-benzofuro[3,2-f]chro-
mene (46). Chromane 2 (0.32 g, 1.3 mmol) was dissolved
in ethanol (40 mL). N-bromosuccinimide (0.27 g,
1.57 mmol) was added and the solution was stirred for
an hour. This was concentrated to dryness and the resi-
due was purified by chromatography over silica gel
(cyclohexane/dichloromethane, 97:3) to yield compound
46 (0.38 g, 88%). LC/MS (ES) m/z 331, 333 [M+H]+. 1H
(CDCl3): 7.96 (d, 1H, J = 7.7); 7.66 (s, 1H); 7.56 (d, 1H,
J = 8.1); 7.47 (m, 1H); 7.35 (m, 1H); 3.28 (t, 2H,
J = 6.8); 2.01 (t, 2H, J = 6.8); 1.45 (s, 6H). 13C (CDCl3):
157.1; 150.0; 146.6; 127.2; 125.1; 123.0; 122.5; 122.3;
114.3; 112.1; 111.3; 75.5; 32.6; 26.9; 21.8.
1-(8-Hydroxydibenzo[b,d]furan-2-yl)ethanone (42). The
2,8-diacyldibenzofuran (39)30 (1 g, 3.96 mmol) and tri-
fluoroacetic acid (0.89 mL, 11.9 mmol) were dissolved
in dichloromethane (100 ml). Compound 3-chloro-
benzoperoxoic acid (0.89 g, 3.96 mmol) was added and
the solution was stirred for 3 days. The resulting solu-
tion was diluted in dichloromethane, washed with 1 N
sodium hydroxide, brine and dried over sodium sulfate.
The residue, containing a mixture of unreacted 39,
monoacetate 40, and bisacetate 41, was heated to reflux
for 15 min in a 1 N methanolate solution (50 mL). This
was concentrated to dryness and dispersed in water. The
aqueous phase was washed with dichloromethane (to re-
move compound 39) and made acidic using 2 N hydro-
chloric acid. This was then extracted with ethyl acetate.
The organic layer was washed with brine to neutrality,
dried over sodium sulfate and concentrated to dryness.
The residue was purified by chromatography over silica
gel (dichloromethane/ethanol, 99.5:0.5) yielding com-
pounds 42 (0.22 g, 24%) and 2,8-dihydroxydibenzofuran
(43) (0.14 g, 19%; previously described30). Compound
42: LC/MS (ES) m/z 225 [MꢀH]ꢀ. 1H (DMSO-d6):
9.57 (s, 1H); 8.74 (d, 1H, J = 1.6); 8.09 (dd, 1H,
J = 1.1 and 8.6); 7.72 (d, 1H, J = 8.6); 7.55 (m, 2H);
7.00 (dd, 1H, J = 2.6 and 8.8); 2.7 (s, 3H). 13C
(DMSO-d6): 198.0; 159.6; 154.8; 150.8; 133.0; 128.5;
125.0; 124.8; 123.2; 117.3; 115.0; 112.5; 107.3; 27.7.
Acylation of compound, preparation of 1-(3,3-dimethyl-
2,3-dihydro-1H-benzofuro[3,2-f]chromen-5-yl)ethanone
(47) and 1-(2-isopropyl-1,2-dihydro-benzo[d]benzo[1,2-b;
4,3-b0]difuran-4-yl)-ethanone (48). In a flask protected
from moisture by a calcium chloride guard, acetic anhy-
dride (0.11 mL, 1.2 mmol) was dissolved in dichloro-
methane (30 mL) and dry aluminium chloride (0.16 g,
1.2 mmol) was added. This was stirred for 10 min and
compound 2 (0.2 g, 0.79 mmol) was added. The result-
ing red solution was stirred for 24 h and concentrated
to dryness. The residue was purified by chromatography
over silica gel eluting with dichloromethane-cyclohex-
ane, 3:7, to yield a fraction containing a mixture of 47
and 48 (0.12 g). This was recrystallized in methanol
twice to provide pure compound 48 (0.01 g, 4.3% yield).
LC/MS (ES) m/z 295 [M+H]+. 1H (CDCl3): 7.92 (s, 1H);
7.88 (d, 1H, J = 7.5); 7.53 (m, 2H); 7.38 (m, 1H); 3.64
(dd, 1H, J = 9.2 and 16.0); 3.33 (dd, 1H, J = 8.9 and
16.0); 2.74 (s, 3H); 2.12 (sept, 1H, J = 6.7); 1.16 (d,
3H, J = 6.7); 1.11 (d, 3H, J = 6.7). 13C (CDCl3): 197.1;
158.7; 157.2; 151.3; 129.1; 126.2; 123.5; 123.1; 122.6;
121.1; 119.4; 112.4; 109.6; 90.5; 33.9; 32.1; 31.6; 18.6;
18.4.
1-(8-(2-methylbut-3-yn-2-yloxy)dibenzo[b,d]furan-2-yl)-
ethanone (44). Using the above-mentioned protocol, the
propargylic ether44 was obtained from compound 42
after a purification by chromatography over silica gel
(cyclohexane/dichloromethane, 95:5) as a pale oil
(0.04 g, 14.3%). (LC/MS): not ionized by electrospray.
1H (CDCl3): 8.57 (1H, J = 2); 8.11 (dd, 1H, J = 2 and
8); 7.86 (d, 1H, J = 2); 7.60 (d, 1H, J = 8); 7.50 (1H,
J = 8); 7.49 (1H, J = 2 and 8); 2.73 (s, 1H); 2.64 (s,
1H); 1.70 (s, 6H). 13C (400 MHz, CDCl3): 196.3; 158.6;
152.2; 150.5; 131.3; 127.0; 123.8; 122.9; 122.2; 120.6;
113.1; 110.7; 110.5; 85.0; 73.3; 72.4; 28.6; 25.7.
The mother liquors were concentrated to dryness and
the residue subjected to a second chromatography over
silica gel using a finer type (20–45 lm). This allowed
the isolation of a small portion of quasi-pure compound
47 (0.01 g; 4.3% yield; 95% pure). LC/MS (ES) m/z 295
[M+H]+. 1H (CDCl3): 7.99 (d, 1H, J = 7.6); 7.83 (s,
1H); 7.53 (m, 1H); 7.51 (m, 1H); 7.36 (m, 1H); 3.29 (t,
2H, J = 6.9); 2.72 (s, 3H); 2.04 (t, 2H, J = 6.9); 1.48 (s,
6H). 13C (CDCl3): 200.3; 158.2; 149.9; 149.8; 128.2;
128.0; 126.8; 124.7; 122.3; 123.0; 116.5; 112.3; 110.9;
75.2; 32.7; 32.2; 27.1; 21.5.
1-(3,3-Dimethylbut-3H-benzofuro[3,2-f]chromen-10-yl)-
ethanone (45). Compound 45 was obtained from ether
44 using the general procedure described above after
purification by chromatography over silica gel (cyclo-
hexane/dichloromethane, 50:50) as white crystals
4.1. MIC determinations
MICs were determined using the new Microdilution
Resazurin Assay (MRA).33 Resazurin salt powder (Sig-
ma) was prepared at 0.01% (wt/vol) in distilled water,

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Acknowledgments
We thank Sanofi-Aventis as well as Pfizer for very gen-
erous donations of scientific equipment. This work re-
ceived the financial support of the Institut Pasteur
(GPH-5, DVPI) and the European Community
(LHSP-CT-2005-018923).
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