Microwave-assisted synthesis of derivatives of khellinone under phase-transfer catalytic conditions

Article abstract of DOI:10.1007/s00706-008-0931-3

The microwave-assisted synthesis of four new 5-acetyl-4,7-dimethoxy-6- hydroxybenzofuran (khellinone) analogs is described. The structures of the obtained derivatives in the solid state are evaluated on the basis of ¹³C CP/MAS NMR spectra and theoretical calculations at DFT level. A single crystal X-ray diffraction structure is presented for 8-acetyl-7-hydroxy- 4-methylcoumarin. 1,4-Bis(5-acetyl-4,7-dimethoxybenzofuran-6-yloxy)butane was evaluated for potential anticancer activity in an in vitro screening panel of 60 human tumor cell lines. Selected leukemia, non-small cell lung cancer, CNS, melanoma, ovarian, and breast cancer cell lines were sensitive to this compound.

Full text of DOI:10.1007/s00706-008-0931-3

Monatsh Chem 139, 1337–1348 (2008)
DOI 10.1007/s00706-008-0931-3
Printed in The Netherlands
Microwave-assisted synthesis of derivatives of khellinone
under phase-transfer catalytic conditions
1
1
2
_
Elzbieta Hejchman , Dorota Maciejewska , Irena Wolska
1
Department of Organic Chemistry, Faculty of Pharmacy, Medical University of Warsaw, Warsaw, Poland
´
Department of Crystallography, Faculty of Chemistry, Adam Mickiewicz University, Poznan, Poland
2
Received 23 January 2008; Accepted 15 March 2008; Published online 9 June 2008
# Springer-Verlag 2008
Abstract The microwave-assisted synthesis of four
new 5-acetyl-4,7-dimethoxy-6-hydroxybenzofuran
(khellinone) analogs is described. The structures of
the obtained derivatives in the solid state are evaluated
on the basis of 13C CP/MAS NMR spectra and theo-
retical calculations at DFT level. A single crystal
X-ray diffraction structure is presented for 8-acetyl-
7-hydroxy-4-methylcoumarin. 1,4-Bis(5-acetyl-4,7-
dimethoxybenzofuran-6-yloxy)butane was evaluated
for potential anticancer activity in an in vitro screen-
ing panel of 60 human tumor cell lines. Selected leu-
kemia, non-small cell lung cancer, CNS, melanoma,
ovarian, and breast cancer cell lines were sensitive to
this compound.
induce apoptosis in several human cancer cell lines
[2]. Dihydrobenzofurans of the neolignan type (from
Persea or Croton species) exhibit potent and broad
cytotoxicity against different human cancer cell lines
[3, 4]. Benzofurans isolated from Brazilian propolis
are mildly cytotoxic toward highly metastatic murine
colon carcinoma and human fibrosarcoma cells [5].
It has been reported that a Chilean rancher success-
fully applied an herbal tea from the leaves of Ammi
visnaga (Apiaceae) for his slow paralysis [6]. Inspired
¨
by this case, Hansel et al. isolated khellinone 1 from
this plant (Fig. 1), a compound with weak but un-
usually selective activity as a potassium-channel
blocker [7]. Next, symmetrical dimers linked via the
alkylation of the 6-hydroxy groups of two molecules
of khellinone, were discovered as the blockers of the
voltage-gated potassium channel Kv1.3 [8].
Keywords Khellinone; Coumarin; Microwave-assisted syn-
thesis; X-ray diffraction; Anticancer activity.
Coumarins are physiologically active benzo-2-
pyron derivatives, used by us previously as inter-
mediates in syntheses of benzofurancarboxylic acids
[9]. Their cytotoxic effects, highly dependent on the
nature and positions of the functional groups, were
most extensively examined in recent years [10]. 7-
Hydroxycoumarin (Fig. 1) has been reported to inhib-
it the proliferation of a number of human malignant
cells in vitro as well as to demonstrate activity against
prostate cancer, malignant melanoma, and metastatic
renal cell carcinoma [11–16]. Lopez-Gonzalez et al.
determined the cytostatic and apoptotic action of
7-hydroxycoumarin on human lung carcinoma cell
Introduction
Benzo[b]furan derivatives of plant origin have been
the subject of increased interest in recent years due
to their diverse biological activities such as anti-
cancer, antioxidant, antimicrobial, and antifungal
properties [1]. Cyclopenta[b]benzofuran derivatives
isolated from Aglaia inhibit protein synthesis and
Correspondence: Elz_bieta Hejchman, Department of Organic
Chemistry, Faculty of Pharmacy, Medical University of
Warsaw, Banacha 1, 02-097 Warsaw, Poland. E-mail:
e_hejchman@windowslive.com

1338
E. Hejchman et al.
Fig. 1 The structures of khellinone and 7-hydroxycoumarin
Scheme 1
lines assuming that the coumarins may become new
options in treatment of non-small cell lung cancer
[17]. Geiparvarin, containing coumarin and furan moi-
eties, isolated from Geiera parviflora (Fig. 1) is an
antiproliferative compound and may represent a new
drug which targets tubulin [10].
Therefore we have adopted the substituted ben-
zo[b]furan and 7-hydroxycoumarin systems as phar-
macophores and introduced the linker to increase the
flexibility of the target compounds.
In this paper we present microwave-assisted syn-
thesis, structural data and 60 cell lines anticancer
assay of the khellinone and coumarin derivatives
(Schemes 1 and 2). These compounds, except the
symmetrical dimer 3 have not been synthesized
yet. For the compound 3 we have elaborated the
new efficient method of synthesis under microwave-
assisted conditions.
Scheme 2
The beneficial effects of microwave irradiation are
finding an increased role in the preparation of novel,
biologically active compounds. This technique has
shown an advantage where processes require forcing
conditions or prolonged reaction times, also when
sensitive reagents are involved in the synthesis. It

Microwave-assisted synthesis of derivatives of khellinone under phase-transfer catalytic conditions
1339
usually allows simplifying the operations, resulting
in cleaner reaction and increased yield. Using the
phase transfer catalysis (PTC) technique under mi-
crowave irradiation to carry out the preparation of
aromatic ethers by Williamson method was found to
be highly useful in terms of mildness of conditions,
yield, and convenience [18–21].
We have applied a modified method for the O-
alkylation of phenols called S-L(!)-L PTC [22]. It
retains the convenience of the solid–liquid PTC
technique but is faster and allows optimizing the re-
action temperature. The rate of S-L PTC is increased
by using a trace amount of water (so called !-phase)
which forms a thin film around the solid particle of a
phenolate. It becomes the third, middle phase rich
in the catalyst, localized between the solid reactant
and organic phase, containing the alkylating agent.
Microwave heating under low energy input (ca.
100 W) allows the use of the thermally unstable (at
temperatures beyond 120–130ꢀC) phase-transfer cat-
alyst tetrabutylammonium bromide.
Systematic examinations of khellinone and cou-
marin derivatives in solid state are not easy, because
of a number of unrelated phenomena: difficulties to
obtain single crystals suitable to X-ray diffraction
analysis and co-crystallization with solvent. Some-
times they are oils at room temperature. Thus, we
decided to use the 13C CP=MAS NMR method and
theoretical calculations of shielding constants at
DFT level of theory to obtain information about
the structure of the solid dimers and relevant com-
ponents of these dimers.
All attempts to obtain suitable single crystals
of the reported new khellinone derivatives failed.
Consequently, the X-ray structure of the building
block in the synthesis of 1-(5-acetyl-4,7-dimethoxy-
benzofuran-6-yloxy)-4-(8-acetyl-4-methylcoumarin-
7-yloxy)butane (7), namely 8-acetyl-7-hydroxy-4-
methylcoumarin (5) has been solved.
oven. Then, the alkylating agent 1,4-dibromobutane,
tetrabutylammonium bromide, and water were added
to the sodium salt. The mixture was heated in the
microwave oven for 10 min to yield 1-(5-acetyl-4,7-
dimethoxybenzofuran-6-yloxy)-4-bromobutane (2).
The bromoalkyl derivative of khellinone 2 was then
used as an alkylating agent in the reactions with
khellinone sodium salt (1a), 7-hydroxy-4-methyl-
coumarin sodium salt (4a), and 8-acetyl-7-hydroxy-
4-methylcoumarin sodium salt (5a) under the same
conditions. The products 1,4-bis(5-acetyl-4,7-dimeth-
oxybenzofuran-6-yloxy)butane (3), 1-(5-acetyl-4,7-
dimethoxybenzofuran-6-yloxy)-4-(4-methylcoumarin-
7-yloxy)butane (6), and 1-(5-acetyl-4,7-dimeth-
oxybenzofuran-6-yloxy)-4-(8-acetyl-4-methylcou-
marin-7-yloxy) butane (7) were isolated by column
chromatography on silica gel.
7-Hydroxy-4-methylcoumarin (4) was obtained
by heating under reflux a mixture of resorcinol, ethyl
acetoacetate, and catalytic amounts of p-toluene-
sulfonic acid with azeotropic removal of water and
ethanol, according to Furuta [23]. Using 2,6-dihy-
droxyacetophenone instead of resorcinol yielded
8-acetyl-7-hydroxy-4-methylcoumarin (5) under the
same conditions. Sodium salts 4a and 5a of the cou-
marins 4 and 5 were obtained in the same way as
described for khellinone. The khellinone-derived un-
symmetrical dimer 10 was prepared in two steps
using the S-L(!)-L PTC technique (Scheme 2). In
the first step 2,3-dihydro-2,2-dimethyl-7-benzofura-
nol (8) was subjected to the microwave-assisted
alkylation with 1,4-dibromobutane in the presence
of anhydrous potassium carbonate, powdered potas-
sium hydroxide, and tetrabutylammonium bromide.
The resulting 1-(2,2-dimethyl-2,3-dihydrobenzofu-
ran-7-yloxy)-4-bromobutane (9) was then used as
an alkylating agent in the reaction with khellinone
sodium salt 1a, under the conditions shown in
Scheme 1. The product 1-(5-acetyl-4,7-dimethoxy-
benzofuran-6-yloxy)-4-(2,3-dihydro-2,2-dimethyl-
benzofuran-7-yloxy)butane (10) was isolated by
column chromatography on silica gel.
Results and discussion
Syntheses
X-Ray structure of 8-acetyl-7-hydroxy-4-methyl-
coumarin (5)
The khellinone-derived dimers 3–5 were synthesized
in two steps based on S-L(!)-L PTC technique
(Scheme 1). First, the sodium salt of khellinone 1a
was prepared by heating equimolar amounts of khel-
linone (1) and powdered sodium hydroxide, sus-
pended in toluene, in the monomode microwave
The molecular and crystal structure of 8-acetyl-7-
hydroxy-4-methylcoumarin (5) in solid state was an-
alyzed by single crystal X-ray diffraction. All details
of the measurements, crystal data, and structure re-

1340
E. Hejchman et al.
Table 2 Hydrogen bonding geometry for 5
D-H ꢁꢁ ꢁ ꢁA dðDꢂHÞ dðHꢁꢁꢁꢁAÞ dðDꢁꢁꢁꢁAÞ <ðDHAÞ
Table 1 Crystal data, data collection, and structure refine-
ment for 5
Compound
5
˚
A
˚
A
˚
A
deg
O70-H7 ꢁ ꢁ ꢁ ꢁO100 1.07(3) 1.50(3)
2.481(2) 149(2)
3.182(2) 135(1)
3.696(2) 168(1)
Empirical formula
Formula weight
T=K
Wavelength=A
Crystal system, space group
C12H10O4
218.20
293(2)
0.71073
triclinic, P ꢂ1
i
C50–H5 ꢁ ꢁ ꢁ ꢁO20 0.97(2) 2.42(2)
ii
C90–H9A ꢁ ꢁ ꢁ ꢁ70 0.98(2) 2.73(2)
˚
i
ii
Symmetry codes: x, 1 þ y, z; 1 þ x, y, ꢂ1 þ z
Unit cell dimensions
˚
finement are given in Table 1. A view of the molec-
ular structure together with the atomic numbering
scheme is shown in Fig. 2 (the drawings were per-
formed with the Mercury program [24]). The results
indicate that compound 5 crystallizes in the triclinic
space group P-1 with one ordered molecule in the
asymmetric unit. Table 2 presents selected bond
lengths, valence, and torsion angles. The interatomic
distances and bond angles of the coumarin group are,
in the majority, in good agreement with published
values for other 4-methylcoumarin derivatives [25–
29]. But the C70–C80 and C80–C8a0 bond lengths in
the benzene ring are rather longer due to the acetyl
substituent at C80 indicating a ꢀ-electron delocaliza-
tion within this fragment of the molecule. Similar
structural changes have also been found for o-hydro-
xyacetophenone derivatives [30].
a=A
˚
7.534(3)
8.372(3)
9.108(3)
69.03(3)
72.21(3)
89.08(3)
b=A
˚
c=A
ꢁ=ꢀ
ꢂ=ꢀ
ꢃ=ꢀ
3
˚
Volume=A
507.9(3)
2, 1.427
0.108
228
2.53–27.97
ꢂ9 ꢃ h ꢃ 9
ꢂ11 ꢃ k ꢃ 10
ꢂ11 ꢃ l ꢃ 11
Z, Dx=Mg ꢁ mꢂ3
ꢄ=mmꢂ1
F(000)
ꢅ range for data collection=ꢀ
hkl range
Reflections:
collected
unique (Rint)
observed (I>2ꢆ(I))
6405
2219(0.013)
1577
Data=restraints=parameters
Absorption correction
Goodness-of-fit on F2
R(F) (I>2ꢆ(I))
2219=0=186
multi-scan
1.036
0.0371
0.1213
The coumarin moiety is nearly planar with a max-
0
˚
imum deviation of 0.029(1) A for C7 . The dihedral
angle between the least-squares planes of the 2-pyr-
one and benzene rings is only 1.71(7)ꢀ. The methyl
C90 and the hydroxy O70 atoms are nearly coplanar
with the coumarin ring system (the appropriate tor-
sion angles are given in Table 2). The disposition of
the acetyl group with respect to the coumarin moiety
can be described by the torsion angles C70–C80–
C100–C110 of 174.2(1)ꢀ and C70–C80–C100–O100
wR(F2) (all data)
Max=min. ꢀꢇ=e ꢁ A
ꢂ3
˚
0.212=ꢂ0.146
˚
Table 3 Selected bond lengths=A, angles=deg, and selected
torsional angles=deg for 5
C20–O20
C30–C40
C70–O70
C70–C80
C80–C80a
1.203(2)
1.345(2)
1.347(2)
1.416(2)
1.412(2)
C20–O10–C80a
C60–C70–O70
O70–C70–C80
122.8(1)
117.4(1)
121.2(1)
C20–C30–C40–C90
O70–C70–C80–C80a
C70–C80–C100C110
178.9(1)
ꢂ177.8(1)
174.2(1)
Fig. 2 A view of the molecule of 5

Microwave-assisted synthesis of derivatives of khellinone under phase-transfer catalytic conditions
˚
1341
0
of ꢂ4.7(2)ꢀ. In consequence the O100, C100, and
C110 atoms of this group are found to be only mar-
ginally out of the plane of the two-ring framework
(max deviation of 0.152(2) A for C11 ). Strong in-
tramolecular hydrogen bonding is present between
O70 and O100 atoms (see Fig. 2 and Table 3) which
Fig. 4 Projection of the crystal structure of 5 along the b axis
showing parallel sheets
Fig. 3 The interconnections within a sheet for 5
Fig. 5 The distances between stacked benzene rings (A) and between stacked 2-pyrone rings (B) for 5

1342
E. Hejchman et al.
is characterized by the disposition of the hydroxyl
and acetyl groups in the plane of molecule. The an-
gle between the best planes of the coumarin moiety
and the C70=O70=H7=O100=C100 ring is only 3.4(4)ꢀ.
In the crystal of 5, the packing of the molecules is
stabilized by intermolecular C–Hꢁ ꢁ ꢁO hydrogen
bonds and stacking forces. The molecules are linked
by C6–H6ꢁ ꢁ ꢁO2 and C11–H11Aꢁ ꢁ ꢁO8 interactions
forming sheets parallel to the (101) plane (Figs. 3
and 4). Cohesion between sheets results in ꢀꢁ ꢁ ꢁꢀ
stacking interactions by partly overlapping mole-
cules from neighboring parallel sheets. The benzene
and 2-pyrone rings overlap alternately; for example
benzene rings between plane 1 and 2 and 2-pyrone
rings between plane 2 and 3, according to Fig. 4. The
perpendicular distance between stacked benzene rings
˚
from neighboring parallel sheets is 3.35 A and be-
˚
tween stacked 2-pyrone rings is 3.39 A, as shown in
Fig. 5. The geometric parameters of all intra- and
intermolecular hydrogen bonds are given in Table 3.
Fig. 6 The 13C CP=MAS NMR spectra of 5-acetyl-6-hydroxy-4,7-dimethoxybenzofuran (1), 1,4-bis(5-acetyl-4,7-dimethoxy-
benzofuran-6-yloxy)butane (3), 7-hydroxy-4-methylcoumarin (4), 8-acetyl-7-hydroxy-4-methylcoumarin (5), 1-(2,3-dihydro-
2,2-dimethylbenzofuran-7-yloxy)-4-bromobutane (9), and 1-(5-acetyl-4,7-dimethoxybenzofuran-6-yloxy)-4-(2,3-dihydro-2,2-
dimethylbenzofuran-7-yloxy)butane (10) in solid state. Sidebands are marked with an asterisk

Microwave-assisted synthesis of derivatives of khellinone under phase-transfer catalytic conditions
1343
NMR spectra and molecular modeling
of their components 5-acetyl-6-hydroxy-4,7-dimeth-
oxybenzofuran (1), 7-hydroxy-4-methylcoumarin
(4), 8-acetyl-7-hydroxy-4-methylcoumarin (5), as
well as 1-(2,3-dihydro-2,2-dimethylbenzofuran-7-
yloxy)-4-bromobutane (9). The notation used in the
discussion of NMR results is shown in Schemes 1 and
2. The most probably assignment of signals is also
given in Fig. 6 and Table 4.
The methoxy groups at aromatic rings affected
the conformational variability in the solid state. We
could even determine by X-ray crystallography non-
averaged two or more structures in the asymmetric
unit or multiple resonances in NMR solid state spec-
tra for such compounds [31, 32]. These differences
are predominantly caused by different dihedral an-
gles between the planes of methoxy groups and the
planes of the aromatic rings. An analogical pheno-
menon could take place for acetyl groups [33].
Surprisingly, the solid state 13C NMR spectra of 1,
3, 4, 5, 9, and 10 are simple and resemble the solution
ones. Small differences between the values of the
chemical shifts in solution and in solid state could
be interpreted by intermolecular and intramolecu-
lar interactions. We calculated the theoretical shield-
ing constants ꢆ=ppm for various conformations of
Theoretical computations provided two types of in-
1
formation: the shielding constants of H and 13C
atoms of the molecules, and structural data of those
conformations, which correspond to the NMR ex-
perimental resonances. In the case of NMR solu-
tion spectra, the calculation helped us to assign
properly resonances of those 13C atoms for which
1D and 2D dimensional NMR experiments gave un-
clear information, while the theoretical analysis of
13C NMR resonances for the solid state allowed us
to propose chemical shifts derived stable conforma-
tions. Theoretical shielding constants were comput-
ed employing the DFT B3 LYP=6-31G
using CHF-GIAO approach for geometries opti-
mized at PM3 level of theory for all analyzed com-
pounds, and additionally for X-ray derived atomic
coordinates of 5.
In Fig. 6 we have shown 13C CP=MAS NMR
spectra of those compounds, which are solid at
room temperature: 1,4-bis(5-acetyl-4,7-dimethoxy-
benzofuran-6-yloxy)butane (3), 1-(5-acetyl-4,7-
dimethoxybenzofuran-6-yloxy)-4-(2,3-dihydro-2,2-
dimethylbenzofuran-7-yloxy)butane (10), and three
ꢄꢄ
method
Table 4 13C CP=MAS chemical shift in solid state ppm
No.
13C chemical shifts in solid state for components=ppm
ꢄ
C2,C20
C3,C30
C4,C40
C4a/C3a
C5,C50
C6,C60
C7,C70
ꢄ
1
4
5
9
142.3
163.9
153.0
86.3
105.9
105.9
105.4
42.4
151.6
157.0
153.0
118.5
105.8
105.9
131.0
133.7
115.3
151.6
115.5
113.8
109.0
124.5
168.2
165.7
142.3
115.5
110.9
127.5
ꢄ
C8,C80
C8a/C7a
C9
C10
C11
Linker
ꢄ
1
4
5
9
58.1
114.2
108.8
–
153.5
202.9
20.5
17.9
29.7
32.1
–
203.1
58.1
–
33.0
158.8
157.0
145.9
–
–
29.1, 30.2
ꢄ
13C chemical shifts in solid state for dimers=ppm
C2,C20
C3,C30
C4,C40
C3a, 3a0
C5,C50
C6,C60
C7,C70
3
10
142.6
148.1
87.4
102.5
105.9
41.5
142.6
143.6
120.5
113.2
114.5
127.6
120.2
123.4
115.9
145.1
143.6
109.3
132.1
132.8
143.0
C8,C80
C7a,C70a
C9
C10
C11
Linker
3
10
57.5
59.4
–
147.1
145.9
148.1
200.2
200.1
28.6
30.1
59.3
60.4
73.0, 26.4
70.9, 65.9
26.0, 25.5
ꢄ

1344
E. Hejchman et al.
Fig. 7 Proposed and analyzed conformations in solid state of 1, 3, 3a, 4, 9, and 10
analyzed compounds of 1, 3, 4, 5, 9, and 10 and
compared them with the experimental chemical shifts
ꢈ=ppm. If we found the linear correlation between
theoretical ꢆ and experimental ꢈ data, we proposed
the probable structure of the most stable conformer
in solid state. In the spectrum of 5-acetyl-6-hy-
droxy-4,7-dimethoxybenzofuran (khellinone) (1) we
observed less resonances than carbon atoms in the
molecule. The values of shielding constants are
strongly dependent on locations of methoxy groups
in different examined conformations. The best agree-
ment with experimental chemical shifts we obtained
for the structure shown in Fig. 7, with the intramo-
lecular hydrogen bond between OHꢁ ꢁ ꢁO¼C groups,
and almost perpendicular orientation of both meth-
oxy group planes to the benzene ring plane. The
solid state spectrum of 7-hydroxy-4-methylcoumarin
(4) represents a more classic example. All reso-
nances are shifted to higher frequencies, what is in
good agreement with a single pattern of shielding
constants calculated for a dimer stabilized by the
intermolecular interactions OHꢁ ꢁ ꢁO type also shown
in Fig. 7. In the analysis of 8-acetyl-7-hydroxy-4-
methylcoumarin (5) we used the X-ray derived
atomic coordinates and optimized at the PM3 level
of theory. For both types of coordinates we have
obtained very good linear correlations between the
theoretical and experimental values (r2 ¼ 0.998 and
0.996). It should mean that our conclusions con-
cerning the solid state structures of the compounds

Microwave-assisted synthesis of derivatives of khellinone under phase-transfer catalytic conditions
1345
Table 5 Cytotoxicity of 3 (GI50=ꢄM)a
Leukemia
Non-small cell
lung cancer
CNS
cancer
Melanoma
Ovarian
cancer
Prostate
cancer
Breast
cancer
CCRF-CEM 2.10
MOLT-4 1.86
RPMI-8226 2.06
EKVX 2.72
NCI-H23 5.66
SF-295
2.74
UACC-257
2.90
OVCAR-4
3.99
PC-3
2.14
NCI=ADR-RES
2.24 T47D 2.75
MGMb 4.59
a
The cytotoxicity GI50 values are the concentrations corresponding to 50% growth inhibition; b Mean graph midpoint for all
human cancer cell lines (ca. 60) tested. The panels and cells employed included the following: leukemia CCRF-CEM, HL-60
(TB), K-562, MOLT-4, RPMI-8226, SR; non-small cell lung cancer: A549=ATCC, EKVX, HOP-62, HOP-92, NCI-H226, NCI-
H23, NCI-H322M, NCI-H460; colon cancer: COLO 205, HCC-2998, HCT-116, HCT-15, HT-29, KM12, SW-620; CNS cancer:
SF-268, SF-295, SF-539, SNB-19, SNB-75, U251; Melanoma: LOX IMVI, MALME-3M, M14, SK-MEL-2, SK-MEL-5, SK-
MEL-28, UACC-257, UACC-62; Ovarian cancer: OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, SK-OV-3; Renal cancer: 786-
0, A498, ACHN, CAKI-1, SN12C, TK-10, UO-31; Prostate cancer: PC-3, DU-145; Breast cancer: MCF-7, NCI=ADR-RES,
MDA-MB-231=ATCC, HS 578T, MDA-MB-435, BT-549, I-47D
obtained by us based on molecular modeling and
the experimental 13C CP=MAS NMR chemical
shifts are justified. We considered the solid 1-(2,3-
dihydro-2,2-dimethylbenzofuran-7-yloxy)-4-bromo-
butane (9), because the unsubstituted 2,3-dihydro-
2,2-dimethyl-7-benzofuranol is liquid at room
temperature. Satisfying agreement of the theoretical
versus the experimental values of resonances was
obtained for the conformation shown in Fig. 7, in
which the butyl linker exhibits an extended zig-
zag conformation, and the butyloxy group is not
placed within the benzene ring plane. Atom C400 is
pointed at the C60 atom, what is reflected by low
frequency shift of C60 atom resonance by 4.1 ppm
as compared to the solution spectrum. Separate,
sharp resonances of gem-methyl groups at C20 atom
(29.6 and 29.1 ppm) show that there are no dynamics
at C-20 in the solid state. The discussion presented
above helped us to carry out the conformational
analysis of dimers 3 and 10. We considered two
orientations of the heterocyclic systems: the first
one with O1 and O10 atoms at the same side of the
aliphatic linker with a folded conformation of the
linker, and the second with O1 and O10 atoms turned
in opposite direction and a zig-zag conformation of
the aliphatic linker (see Fig. 7). The latter conforma-
tions are more stable, by average 15 kJ=mol. Such
conformations are compatible with the X-ray struc-
ture of another khellinone dimer [34]. For both types
of conformations of 3 and 10 we calculated the
theoretical shielding constants. We found better
linear correlations between the experimental chemi-
cal shifts and those theoretical values which corre-
spond to lower energy conformations (r2 ꢅ 0.98). On
this basis we were encouraged to propose extended
conformations for both compounds 3 and 10 (see
Fig. 7). The separations between the aromatic parts
0
0
˚
(C6ꢁ ꢁ ꢁC6 and C6ꢁ ꢁ ꢁC7 ) are close to 7.60 A, and
in each molecule both aromatic systems are almost
parallel to each other. The methoxy groups 8 and
80 in 3 show smaller deviations from the benzofuran
ring plane than the other two methoxy groups
11 and 110, and both acetyl groups which are almost
perpendicular to them. The benzofuran system in
compound 10 demonstrates the same features.
Both methyl groups at C20 atom in 1,2-dihydro-
benzofuran system show only one signal of double
intensity.
Anticancer assay
Compound 3 was accepted for evaluation by the Di-
vision of Cancer Treatment and Diagnosis National
Cancer Institute (Bethesda, USA) in the full panel of
60 different cell lines, representing human leukemia,
non-small cell lung cancer, colon cancer, central ner-
vous system (CNS) cancer, melanoma, ovarian can-
cer, renal cancer, prostate cancer, and breast cancer
cell lines. The details are available at the web site
http:==dtp.nci.nih.gov. Results from representative
cell lines are listed in Table 5, along with mean
graph midpoint (MGM) values for all 60 lines.
Conclusions
An efficient method of microwave-assisted synthesis
was worked out for a series of benzofuran and
coumarin derivatives. 1,4-Bis(5-acetyl-4,7-dimeth-

1346
E. Hejchman et al.
O-Alkylation of sodium salts of phenols 1a, 4a, 5a, and 8a
The alkylating agent (1,4-dibromobutane or bromoalkyl
component 2 or 9, 3 mmol), 193 mg tetrabutylammonium
bromide (0.6mmol), and 0.15cm3 water were added to the
appropriate sodium salt. The mixture was heated under reflux
in the microwave oven for 10–15min (150W). The products
were purified by column chromatography on silica gel (eluent
CHCl3-AcOEt or cyclohexane-AcOEt).
oxybenzofuran-6-yloxy)butane (3) at micromolar
concentrations reduces the growth of human leuke-
mia, non-small cell lung cancer, CNS, melanoma,
ovarian, and breast cancer cells.
The stable solid state conformations of 1,4-bis(5-
acetyl-4,7-dimethoxybenzofuran-6-yloxy)butane (3),
1-(5-acetyl-4,7-dimethoxybenzofuran-6-yloxy)-4-
(2,3-dihydro-2,2-dimethylbenzofuran-7-yloxy)butane
(10), and their three components: 5-acetyl-6-hydroxy-
4,7-dimethoxybenzofuran (1), 7-hydroxy-4-methyl-
coumarin (4), 8-acetyl-7-hydroxy-4-methylcoumarin
(5) as well as 1-(2,2-dimethyl[2,3]dihydrobenzo-
furan-7-yloxy)-4-bromobutane (9) were proposed
on the basis of 13C CP=MAS NMR solid state spec-
tra and molecular modeling at DFT level of theory.
The conformation of the molecule of 8-acetyl-
7-hydroxy-4-methylcoumarin (5) was confirmed by
X-ray analysis. The crystal structure of 5 is stabi-
lized by intermolecular C–Hꢁ ꢁ ꢁO hydrogen bonds
and stacking forces between parallel sheets.
1-(5-Acetyl-4,7-dimethoxybenzofuran-6-yloxy)-4-
bromobutane (2, C16H19O5Br)
This compound was prepared according to the methods de-
scribed above using 0.709 g khellinone (3mmol) and 0.12 g
sodium hydroxide (3mmol). The resulting sodium salt of
khellinone, 0.36 cm3 1,4-dibromobutane (3 mmol, 0.65 g),
0.194 g tetrabutylammonium bromide (0.6 mmol), and
0.15 cm3 water were combined and heated under reflux in
the microwave oven. The reaction was monitored on TLC
on silica gel plates (eluent CHCl3:AcOEt, 10:1, Rf ¼ 0.68).
The reaction mixture was dissolved in 10cm3 chloroform
and the inorganic salts were filtered off. Solvent was evapo-
rated and the residue purified by column chromatography on
silica gel (eluent CHCl3:AcOEt, 20:1). Yield 0.476 g, 43%, oil;
1H NMR (200MHz, CDCl3): ꢈ ¼ 1.84 (m, 2 H, 300-H2), 1.98
(m, 2 H, 200-H2), 2.47 (s, 3 H, 10-H3), 3.44 (t, J ¼ 6.6 Hz, 2 H,
400-H2), 3.93 (s, 3 H, 8-H3), 4.01 (m, 5 H, 11-H3, 100-H2), 6.82
(d, J ¼ 2.2 Hz, 1 H, 3-H), 7.52 (d, J ¼ 2.2 Hz, 1 H, 2-H) ppm;
13C NMR (50 MHz, CDCl3): ꢈ ¼ 28.69 (300-C), 29.30 (200-C),
32.86 (10-C), 33.60 (400-C), 61.13 (8-C), 61.18 (11-C), 74.02
(100-C), 105.04 (3-C), 116.43 (3a-C), 124.39 (5-C), 134.38 (7-
C), 143.98 (6-C), 144.47 (4-C), 144.61 (2-C), 148.62 (7a-C),
202.06 (9-C) ppm; IR (neat): ꢉꢁ¼ 2939, 1703, 1604, 1477,
1346, 1265, 1136, 1068, 746 cmꢂ1; TOF MS ESþ: m=z ¼
393.0314 [C16H19O579Br þ Na].
Experimental
All chemicals were purchased from the major chemical sup-
pliers as high or highest purity grade and used without any
further purification. Microwave oven Plazmatronika 1000W
(http:==www.plazmatronika.pl) equipped with a single mode
cavity suitable for microscale synthesis and microwave choked
outlet connected to external condenser, was used at its 50,
30, and 15% power. Melting points were determined with
1
a Digital Melting Point Apparatus 9001. H and 13C NMR
1,4-Bis(5-acetyl-4,7-dimethoxybenzofuran-6-yloxy)butane
(3) [8]
spectra in solution were recorded at 25ꢀC with a Varian Unity
plus-200, and standard Varian software was employed. Several
(as indicated) 1H and 13C NMR spectra in solution were
recorded with a Bruker Avance DMX 400. The solid state
13C CP=MAS NMR spectra were measured using a Bruker
Avance DMX 400. The powdered samples were spun at 8–
10kHz. Contact time of 6 ms, repetition time of 10 s, and
spectral width of 24 kHz were used for accumulation of
4000 scans. Chemical shifts ꢈ [ppm] were referenced to TMS.
IR spectra were recorded on a FT IR Perkin Elmer instrument.
High resolution mass spectra were recorded on Quattro LCT
(TOF). Theoretical shielding constants were computed at
Yield 1.34 g, 85%, mp 119–120ꢀC (Ref. [8] 119–120ꢀC); TLC:
eluent CHCl3:AcOEt, 10:1, Rf ¼ 0.51; 1H NMR (400MHz,
CDCl3): ꢈ ¼ 1.89 (m, 4 H, 200, 300-H2), 2.54 (s, 6 H, 10, 100-
H3), 3.99 (s, 6 H, 8,80-H3), 4.07 (s, 6 H, 11, 110-H3), 4.12 (m, 4
H, 100, 400-H2), 6.87 (d, J ¼ 1.8 Hz, 2 H, 3, 30-H), 7.58 (d,
J ¼ 1.8 Hz, 2 H, 2, 20-H) ppm; 13C NMR (100MHz, CDCl3):
ꢈ ¼ 26.85 (200, 300-C), 33.01 (10, 100-C), 61.29 (8, 80-C) 61.40
(11, 110-C), 74.96 (100, 400-C), 105.17 (3, 30-C), 116.53 (3a,
30a-C), 124.68 (5, 50-C), 134.59 (7, 70-C), 144.10 (6, 60-C),
144.70 (2, 20), 144.88 (4, 40-C), 148.53 (7a, 70a -C), 202.24
(9, 90-C) ppm; IR (KBr): ꢉꢁ¼ 3157, 3127, 3000–2841 (mul-
tiple bands), 1702, 1593, 1478, 1440, 1378, 1348, 1270,
1194, 1171, 1140, 1072, 1005, 988, 956, 921, 879, 843,
ꢄꢄ
DFT level of theory (B3LYP=6-31G hybrid functional and
CHF-GIAO approach) for geometries optimized at PM3 level
of theory for all analyzed compounds, and additionally for
X-ray derived atomic coordinates of 5.
755 cmꢂ1
.
1-(5-Acetyl-4,7-dimethoxybenzofuran-6-yloxy)-4-(4-methyl-
coumarin-7-yloxy)butane (6, C26H26O8)
Preparation of sodium salts of phenols 1, 4, 5, and 8
Phenolates 1a, 4a, 5a, and 8a were prepared by heating equi-
molar amounts of the appropriate phenol and powdered sodi-
um hydroxide (3mmol) in 2 cm3 toluene under reflux in the
monomode microwave oven (300W, 10 min). The products
were used without further purification.
Yield 0.84g, 60%, oil; TLC: eluent CHCl3:AcOEt, 10:1, Rf ¼
1
0.54; H NMR (200MHz, CDCl3): ꢈ ¼ 1.96 (m, 4 H, 200,300-
H2), 2.40 (d, J ¼ 1.2 Hz, 3 H, 90-H3), 2.54 (s, 3 H, 10-H3), 4.00
(s, 3H, 11-H3), 4.08 (s, 3 H, 8-H3,), 4.07 (m, 4 H, 100-H2, 400-

Microwave-assisted synthesis of derivatives of khellinone under phase-transfer catalytic conditions
1347
H2), 6.12 (d, J ¼ 1.2 Hz, 1 H, 30-H), 6.83 (d, J ¼ 2.2 Hz, 3-H),
1-(5-Acetyl-4,7-dimethoxybenzofuran-6-yloxy)-4-(2,3-dihydro-
2,2-dimethylbenzofuran-7-yloxy)butane (10, C26H30O7)
Yield 0.54g, 40%, mp 64–66ꢀC; TLC: eluent CHCl3:AcOEt,
6.86 (d, J ¼ 8.7 Hz, 1 H, 60-H), 6.89 (m, 1 H, 80-H), 7.50 (d,
J ¼ 8.7 Hz, 1 H, 50-H), 7.59 (d, J ¼ 2.2 Hz, 1 H, 2-H) ppm; 13C
NMR (50 MHz, CDCl3): ꢈ ¼ 18.68 (90-C), 25.67 (200-C),
26.68 (300-C), 32.89 (10-C), 61.13 (8-C), 61.19 (11-C), 68.13
(400-C), 74.56 (100-C), 101.44 (60-C), 105.06 (3-C), 111.84
(80-C), 112.60 (30-C), 113.47 (40a -C), 116.40 (3a-C), 124.41
(5-C), 125.51 (50-C), 134.40 (7-C), 144.00 (6-C), 144.60 (2-C,
4-C), 148.65 (7a-C), 152.61 (40-C), 155.28 (20-C), 161.38 (80a-
C), 162.16 (70-C), 202.12 (9-C); IR (neat): ꢉꢁ¼ 2943, 1715,
1614, 1477, 1431, 1386, 1347, 1265, 1200, 1140, 1070,
989, 878, 846, 748 cmꢂ1; TOF MS ESþ: m=z ¼ 489.1525
[C26H26O8 þ Na].
1
10:1, Rf ¼ 0.78); H NMR (300MHz, CDCl3): ꢈ ¼ 1.50 (s, 6
H, 80,90-H3), 1.94 (m, 4 H, 200, 300-H2), 2.52 (s, 3 H, 10-H3),
3.01 (s, 1 H, 30-H), 3.98 (s, 3 H, 11-H3), 4.06 (s, 3 H, 8-
H3), 4.10 (m, 2 H, 100-H2), 4.12 (m, 2 H, 400-H2), 6.76
(s, 3 H, 40,50,60-H), 6.87 (s,1 H, 3-H), 7.57 (s, 1 H, 2-H)
13
ppm; C NMR (75 MHz, CDCl3): ꢈ ¼ 26.02 (300-C), 26.94
(200-C), 28.50 (80, 90-C), 33.14 (10-C), 43.59 (30-C), 61.40
(8-C), 61.51 (11-C), 68.91 (400-C), 75.06 (100-C), 87.40
(20-C), 105.29 (3-C), 113.21 (60-C), 116.61 (3a-C), 117.74
(50-C), 120.52 (40-C), 124.75 (5-C), 128.59 (30a-C), 134.65
(7-C), 144.05 (70-C), 144.20 (4-C) 144.79 (2-C), 144.98
(6-C), 148.03 (7a-C), 148.94 (70a-C), 202.43 (9-C) ppm;
IR (KBr): ꢉꢁ¼ 3153, 3129, 2964–2848 (multiple bands),
1704, 1614, 1592, 1542, 1478, 1491, 1374, 1346, 1303,
1283, 1269, 1198, 1170, 1139, 1075, 954, 871, 776,
759, 723, 632 cmꢂ1; TOF MS ESþ: m=z ¼ 477.1889
[C26H30O7 þ Na].
1-(5-Acetyl-4,7-dimethoxybenzofuran-6-yloxy)-4-(8-acetyl-
4-methylcoumarin-7-yloxy)butane (7, C28H28O9)
Yield 0.82 g, 54%, oil; TLC: eluent CHCl3:AcOEt, 10:1, Rf ¼
1
0.36; H NMR (400MHz, CDCl3): ꢈ ¼ 1.84 (m, 2 H, 200-H2),
1.97 (m, 2 H, 300-H2), 2.37 (s, 3 H, 90-H3), 2.50 (s, 3 H, 110-
H3), 2.56 (s, 3 H, 9-H3), 3.97, 4.04 (2 s, 6 H, 8, 11-H3), 4.08
(t, J ¼ 6.0 Hz, 2 H, 400-H2), 4.13 (t, J ¼ 6.0 Hz, 2 H, 100-H2),
6.10 (s, 1 H, 30-H), 6.85 (d, J ¼ 3.0 Hz, 1 H, 3-H), 6.89 (d,
J ¼ 9.2 Hz, 1 H, 60-H), 7.54 (d, J ¼ 9.2 Hz, 1 H, 50-H), 7.59 (d,
J ¼ 3.0 Hz, 2-H) ppm; 13C NMR (50 MHz, CDCl3): ꢈ ¼ 18.92
(90-C), 25.81 (200-C), 26.66 (300-C), 32.56 (110-C), 33.06 (10-
C), 61.28 (8,11-C), 68.95 (100, 400-C), 105.22 (3-C), 108.58 (60-
C), 112.63 (30-C), 114.05 (40a-C), 116.51 (3a-C), 119.73
(80-C), 124.42 (5-C), 126.57 (50-C), 134.49 (7-C), 144.14 (6-
C), 144.58 (4-C), 144.80 (2-C), 148.81 (7a-C), 150.76 (40-C),
152.38 (80a-C), 158.13 (20-C), 160.23 (70-C), 199.50 (9-C),
202.34 (100-C) ppm; TOF MS ESþ: m=z ¼ 531.1631
[C28H28O9 þ Na].
7-Hydroxy-4-methylcoumarin (4)
A mixture of 2.2 g resorcinol (20 mmol), 4.0cm3 ethyl ace-
toacetate (30 mmol) and 0.417g p-toluenesulfonic acid
(2.2mmol) in 35cm3 toluene was heated at reflux for 8 h with
azeotropic removal of water and ethanol by use of a Dean-
Stark trap. The reaction mixture was left overnight. Then
50cm3 ice–water were added and the mixture was stirred
for 45 min. The precipitate was filtered off and dried. Crystal-
lization (MeOH) yielded a colorless solid, 3.17 g (90%), mp
187–188ꢀC (Ref. [35] 188ꢀC (MeOH, H2O)).
8-Acetyl-7-hydroxy-4-methylcoumarin (5)
Using of 2,6-dihydroxyacetophenone (Aldrich) instead of res-
orcinol resulted in yellow solid, yield 80%, mp 174–175ꢀC
(EtOH) (Ref. [36] 172–175ꢀC (EtOH)).
1-(2,2-Dimethyl[2,3]dihydrobenzofuran-7-yloxy)-4-
bromobutane (9, C14H19BrO2)
2,3-Dihydro-2,2-dimethyl-7-benzofuranol (Aldrich) (3mmol,
0.493 g), 0.12g sodium hydroxide (3mmol), 2.073 g anhy-
drous potassium carbonate (15 mmol), 1.8 cm3 1,4-dibromo-
butane (15 mmol, 3.25 g), 0.1 g tetrabutylammonium bromide
(0.3mmol) were combined and heated under reflux in the
microwave oven (500 W). The reaction was monitored on
TLC on silica gel plates (eluent cyclohexane:AcOEt, 5:1,
Rf ¼ 0.78). The reaction mixture was dissolved in CHCl3
(10 cm3), the inorganic salts were filtered off. Solvent was
evaporated and the residue purified by column chromatogra-
phy on silica gel (eluent cyclohexane:AcOEt, 10:1). Yield
0.74g, 82%, mp 40–41ꢀC. 1H NMR (400MHz, CDCl3):
ꢈ ¼ 1.51 (s, 6 H, 80, 90-H3), 1.96 (q, J ¼ 6.8 Hz, 2 H, 200-H2),
2.05 (q, J ¼ 6.8 Hz, 2 H, 300-H2), 3.02 (s, 2 H, 30-H), 3.49 (t,
J ¼ 6.4 Hz, 2 H, 100-H2), 4.094 (t, J ¼ 6.4 Hz, 2 H, 400-H2), 6.76
(m, 3 H, 40, 50, 60-H) ppm; 13C NMR (100 MHz, CDCl3):
ꢈ ¼ 28.20 (300-C), 28.40 (80,90-C), 29.58 (200-C), 33.72 (100-
C), 43.46 (30-C), 68.30 (400-C), 87.36 (20-C), 113.44 (60-C),
117.94 (50-C), 120.40 (40-C), 128.48 (30a-C), 143.73 (70-C),
148.04 (70a-C) ppm; IR (KBr): ꢉꢁ¼ 3070, 3049, 2969–2872
(multiple bands), 1614, 1592, 1471, 1492, 1436, 1369,1303,
1281, 1243,1199, 1129, 1075, 955, 877, 846, 775, 755, 720,
X-Ray diffraction studies
Crystals of 5 suitable for X-ray analysis were grown by slow
evaporation from ethanol solution. The data were collected
on an Oxford Diffraction KM4CCD diffractometer [37] at
293 K, using graphite-monochromated MoKꢁ radiation. The
unit cell parameters were determined by least-squares treat-
ment of setting angles of highest-intensity reflections chosen
from the whole experiment. Intensity data were corrected
for the Lorentz and polarization effects [37]. The structures
were solved by direct methods by use of the SIR2004
program [38] and refined by the full-matrix least-squares
method with the SHELXL97 program [39]. The function
2
2
2
2
ꢂwðjFoj ꢂ jFcj Þ was minimized with wꢂ1 ¼ ½ꢆ2ðFoÞ þ
2
2
2
ð0:0655PÞ þ 0:0654Pꢆ, where P ¼ ðFo þ 2Fc Þ=3. All non-
hydrogen atoms were refined anisotropically. The hydro-
gen atoms were placed in the calculated positions and then
their positional and thermal parameters were refined. An
empirical extinction correction was also applied accord-
ing to the formula Fc ¼ kFc½1 þ ð0:001ꢊFc l =sin2ꢅÞꢆ
[39], and the extinction coefficient ꢊ was equal to
0.071(9).
ꢂ1=4
3
0
2
627 cmꢂ1
.

1348
The deposition number CCDC 678518 for 5 contains the
supplementary crystallographic data for this paper. These data
can be obtained free of charge via www.ccdc.cam.ac.uk=data_
request=cif, or by emailing data_request@ccdc.cam. ac.uk, or
by contacting The Cambridge Crystallographic Data Centre,
12, Union Road, Cambridge CB2 1EZ, UK; fax: þ44 1223
336033.
E. Hejchman et al.
17. Lopez-Gonzalez JS, Prado-Garcia H, Aguilar-Cazares D,
Molina-Guarneros JA, Morales-Fuentes J, Mandoki JJ
(2004) Lung Cancer 43:275
18. Thierney JP, Lidstom P (2005) Microwave Assisted
Organic Synthesis (ed.) Blackwell Publishing Ltd, 296p
19. Kappe CO, Dallinger D (2006) In: Van der Eycken E,
Kappe CO (eds) Nature Reviews Drug Discovery 5:51,
Microwave-Assisted Synthesis of Heterocycles, Series:
Topics in Heterocyclic Chemistry, Vol. 1. XIV, 309p and
references therein
Acknowledgements
The authors thank the Interdisciplinary Center for Mathemati-
cal and Computational Modeling (ICM) of Warsaw University
for the computational grant.
20. Bogdal D, Pielichowski J, Jaskot K (1998) Org Prep Proc
Int 30:427
21. Shieh WC, Dell S, Repic O (2001) Org Lett 3(26):4279
22. Yadav GD, Desai NM (2006) Catal Commu 7:325
23. Furuta T, Takeuchi H, Isozaki M, Takahashi Y, Kanehara
M, Suigimoto M, Watanabe T, Noguchi K, Dore TM,
Kurahashi T, Iwamura M, Tsien RY (2004) Chem Bio
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24. Macrae CF, Edgington PR, McCabe P, Pidcock E, Shields
GP, Taylor R, Towler M, van de Streek J (2006) Mercury:
visualization and analysis of crystal structures. J Appl
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