Design, synthesis, and biological evaluation of alcyopterosin a and illudalane derivatives as anticancer agents

Article abstract of DOI:10.1016/j.bmc.2005.10.033

The synthesis of alcyopterosin A and a series of new derivatives possessing an illudalane skeleton is described. The DNA binding properties of these compounds have been examined and compared to those of reference drugs using a UV spectroscopy technique. T

Full text of DOI:10.1016/j.bmc.2005.10.033

Bioorganic & Medicinal Chemistry 14 (2006) 1863–1870
Design, synthesis, and biological evaluation of alcyopterosin A
and illudalane derivatives as anticancer agents
Liliana M. Finkielsztein, Ana M. Bruno,
Sergio G. Renou and Graciela Y. Moltrasio Iglesias*
Departamento de Quı´mica Orga´nica, Facultad de Farmacia y Bioquı´mica, Universidad de Buenos Aires,
Junı´n 956, 1113, Buenos Aires, Argentina
Received 28 September 2005; revised 18 October 2005; accepted 20 October 2005
Available online 29 November 2005
Abstract—The synthesis of alcyopterosin A and a series of new derivatives possessing an illudalane skeleton is described. The DNA
binding properties of these compounds have been examined and compared to those of reference drugs using a UV spectroscopy
technique. The antitumor activity of selected compounds against a panel of 60 human tumor cell lines was tested in the in vitro
anticancer screening of the National Cancer Institute. Redox properties were also evaluated. Tested compounds showed significant
DNA affinity, derivatives 6 and 15 exhibited remarkable antiproliferative activity and have been identified as new leads in the
antitumor strategies.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
Illudalane sesquiterpenoids (Fig. 1) are a class of com-
pounds rare in nature and were isolated from the fungal
subdivision of Basidiomycotina1 and from the fern fam-
Figure 1. Illudalane skeleton.
ily of Pteridaceae.2 Antimicrobial,3,4 cytotoxic,5,6 and
antispasmodic activities7 are some of the properties
exhibited by members of this class. The alcyopterosins
HO
comprise a new set of these compounds whose isolation
from sub-Antarctic deep seawater soft coral Alcyonium
paessleri has been quite recently reported.8 They repre-
sent the first illudalane sesquiterpenoids ever isolated
from marine sources.
Cl
7
1
Figure 2. Chemical structures of alcyopterosin A (1) and alcyopterosin
homologue (7).
In a preliminary in vitro test, alcyopterosin A (1) (Fig. 2)
showed cytotoxicity toward HT-29 (human colon carci-
noma) cell line.8 The unusual structure as well as the
potential biological activities of these new compounds
make them attractive synthetic targets. In contrast to
the preparation method already published,9 we present
a versatile route to synthesize alcyopterosin A starting
from simple materials and allowing the obtainment of
other members of the family of alcyopterosins. In order
to establish the appropriate reagents and the optimum
reaction conditions for the synthesis, we attempted to
prepare an alcyopterosin homologue 7 (Fig. 2).
In the course of the synthetic pathway, a series of com-
pounds possessing the illudalane skeleton was obtained.
In this paper, we report the total synthesis of a naturally
occurring alcyopterosin A as well as the preparation of
new non-natural derivatives as potential antitumor
agents. Compounds were evaluated in terms of their
non-covalent DNA binding properties. Moreover, we
focused on the redox characteristics through the study
of cyclic voltammetry. Compounds 6, 9, and 15 were
Keywords: Synthesis; Alcyopterosin A; Illudalane derivatives;
Anticancer.
*
Corresponding author. Tel./fax: +54 1149648252; e-mail: gmoltra@
ffyb.uba.ar
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.10.033

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L. M. Finkielsztein et al. / Bioorg. Med. Chem. 14 (2006) 1863–1870
selected and evaluated in vitro against three cancer cell
lines: MCF-7 (breast), NCI-H460 (lung), and SF-268
(CNS) by NCI (National Cancer Institute). Compounds
6 and 15 were also evaluated in the in vitro anticancer
screening against 60 human tumor cell lines. Tested
compounds are potential novel templates for anticancer
chemotherapy and can serve as new clinical leads.
O
R1
Cl
(i)
Br
Br
8-11
8: R1= CH2CH2I
5
(i) NaCNBH3, ZnI2,
ClCH2CH2Cl, reflux
28%
9: R1= COCH3 24%
10: R1= CHOHCH2Cl
11: R =
(i) CuCl, NaBH4, ethanol, reflux
(i) NaBH4, ethanol, reflux, 15 min
(i) NaBH4, ethanol, reflux, 4 h
2. Chemistry
43%
42%
HC
CH2
1
Scheme 1 shows the retrosynthetic strategy for the
alcyopterosin A preparation. We started our synthetic
approach with the synthesis of b-chloroketone 2, ob-
tained from acylation of commercially available 4-bro-
mo-m-xylene (Scheme 2). The presence of the bromine
atom serves as a positional protective group to gain reg-
iochemical control of the productsÕ substitution pattern.
Conversion of 2 into indanone 3 was accomplished by
cyclization with concentrated sulfuric acid. An efficient
deoxygenation of indanone 3 into the corresponding in-
dane 4 became possible by using sodium cyanoborohy-
dride in the presence of zinc iodide.10 Thereafter,
conversion of 4 to a-chloroketone 5 was reached by a
classical Friedel–Crafts acylation reaction.
O
Scheme 3. Synthesis of the 1-(7-bromo-4,6-dimethylindan-5-yl)-2-
chloroethan-1-one derivatives (8–11).
refluxing ethanol. After 15 min of reflux, compound 5
was reduced to the corresponding b-chlorohydrin 10.
Upon refluxing over an extended period of time (4 h),
epoxide 11 was isolated as the main product. After
16 h of reaction, alcohol 6 was cleanly obtained as a
product of epoxide 11 ring opening.
The results obtained encouraged us to carry out the
synthesis of 7 from 5 in two steps: first with sodium
borohydride to give 6 and finally, with lithium
aluminum hydride to afford 7.
Obtaining compound 7 from a-chloroketone 5 allowed
us to try a variety of reducing agents with different reac-
tivities to be able to perform the transformation in
one-pot reaction. As a consequence a series of deriva-
tives (8–11) was obtained, as summarized in Scheme 3.
The synthesis of alcyopterosin A was achieved through
methylation of indanone 3. The reaction was successful
in a sealed glass tube using both an excess of iodome-
thane and sodium hydride in toluene. Similarly, com-
pounds 13–16 were synthesized under the same
conditions as those used for obtaining 4–7. The alcyop-
terosin A preparation was completed by substituting a
hydroxyl group in compound 16 for a chlorine atom
(Scheme 4).
Compounds 6, 10, and 11 were prepared from 5 at
different reaction times using sodium borohydride in
O
Cl
Cl
Br
3. Results and discussion
O
3.1. DNA binding properties
Compounds 1, 4–11, and 13–16 were tested for their
ability to bind DNA. The binding capacity of these com-
pounds was evaluated by measuring the hypochromic
Br
Br
Scheme 1. Retrosynthesis of alcyopterosin A.
O
O
(iii)
(ii)
(i)
78%
67%
99%
Br
Cl
Br
Br
Br
3
4
2
O
HO
HO
Cl
(vi)
(v)
41%
(iv)
93%
67%
Br
Br
7
5
6
Scheme 2. Synthesis of alcyopterosin homologue 2-(4,6-dimethylindan-5-yl)ethan-1-ol (7). Reagents and conditions: (i) ClCOCH2CH2Cl, AlCl3,
CS2; (ii) concentrated H2SO4; (iii) NaCNBH3, Znl2, CH2Cl2; (iv) ClCOCH2Cl, AlCl3, CS2; (v) NaBH4, ethanol, 16 h; (vi) LiAlH4, THF.

L. M. Finkielsztein et al. / Bioorg. Med. Chem. 14 (2006) 1863–1870
1865
O
O
O
(i)
66%
(ii)
(iii)
Cl
88%
79%
Br
Br
Br
Br
3
12
13
14
Cl
HO
HO
(vi)
(iv)
42%
(v)
69%
78%
Br
alcyopterosin A
16
15
1
Scheme 4. Synthesis of alcyopterosin A. Reagents and conditions: (i) Mel, NaH, toluene; (ii) NaCNBH3, Znl2, CH2ClCH2Cl; (iii) ClCOCH2Cl,
AlCl3, CS2; (iv) NaBH4, ethanol, 16 h; (v) LiAlH4, THF; (vi) Cl2SO, Py, CHCl3.
and bathochromic effects of their absorbance in the UV
spectra.11 The typical experiment was enhanced by
means of a slow rotation of DNA–drug mixture stirring,
in a 5:1 ratio during 24 h. The procedure was validated
by repeating assays with well-known intercalating
agents (m-AMSA and mitoxantrone) and a compound
which binds closely in the minor groove (bis-benzimide,
Hoechst No. 33258). The degree of interaction was ex-
pressed by the ratio between the final absorbance area
after 24 h (a24) and that of the compound at the same
concentration (a0), centered at maximal absorbance.
Values of 1 or higher indicate a total lack of affinity
and value 0 shows that the whole compound was bound
to DNA. The coefficient a24/a0 values obtained are sum-
marized in Table 1.
evaluated in a three cell line one dose prescreen.12–14
The three lines comprise MCF-7 (breast), NCI-H460
(lung), and SF-268 (CNS). These have been in use
by DTP (Development Therapeutic Program) for sev-
eral years to evaluate combinatorial libraries and have
proven to be an effective test of agents which exhibit-
ed some capability level to inhibit the growth of hu-
man tumor cells in culture. Two drugs were used as
a standard and evaluated against each cell line, NSC
19893 (5-FU) and NSC 123127 (Adriamycin). Results
are expressed as percentage test cell growth compared
with untreated control cells (PTC). Compounds, which
reduce the growth of any of the three cell lines by
32% or less, are considered in vitro active. Table 2
shows the values of PTC of selected targets. Com-
pound 15 has shown 0% of growth inhibition against
three cell lines. Similarly, 6 presented the same per-
centage of inhibition, except for the SF-268 (CNS)
line. Meanwhile, compound 9 has not reduced the
growth of any cell line by 32% or less. These results
suggest that derivatives 6 and 15 produce an impor-
tant effect on the growth of cancer cell lines.
The DNA binding assay showed that compounds tested
are excellent DNA ligands with affinity higher than
those of m-AMSA and bis-benzimide, except for 7, 10,
and 16. The presence of two carbon funtionalized chain
is essential (compounds 4 and 13 presented poor affini-
ty). Substitution at C-2 produces no remarkable modifi-
cation to the affinity (in cases 6, 15 and 7, 16). For
hydroxylic compounds (6, 7, 15, and 16) the absence
of the bromine atom reduces dramatically the DNA
binding degree.
Compounds 6 and 15, which passed the first criterion
of activity, were further evaluated at five concentra-
tions in 10-fold dilutions (10À4 to 10À8 M) against 60
different human tumor cell lines organized in subpanels
representing melanoma, leukemia, and cancers of
breast, prostate, lung, colon, ovary, kidney, and brain.
The experimental procedures have been described in
detail.13,15–17Three dose–response parameters are calcu-
lated for each experimental agent: the compound con-
centration required to cause 50% of net cell growth
3.2. In vitro antitumor activity
In a preliminary test performed by The National Can-
cer Institute (NCI), compounds 6, 9, and 15 were
Table 1. DNA binding assay [reference drugs: Mitoxantrone (Mx);
m-AMSA (m-A); and Bis-benzimide (B-b)]
a
a
Compound
a24/a0
Compound
a24/a0
Table 2. Three cell line prescreen
4
5
0.90
0.12
0.59
0.89
0.16
0.26
0.69
0.47
13
14
0.87
0.41
0.40
0.71
0.38
0.00
0.54
0.57
Compound
PTCd
MCF-7a
NCI-H460b
SF-268c
6
15
16
7
6
9
0
100
0
0
97
0
49
116
0
8
1
9
Mx
m-A
B-b
15
10
11
a Breast cell line.
b Lung cell line.
c CNS cell line.
a a24 = final absorbance (DNA-compound) after 24 h; a0 = initial
absorbance (compound) at the same concentration.
d PTC = percent test cell growth compared with untreated control cells.

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L. M. Finkielsztein et al. / Bioorg. Med. Chem. 14 (2006) 1863–1870
Table 4. Antitumor activity of compounds 6 and 15 in most sensitive
human tumor cell lines
(GI50); the compound concentration resulting in total
growth inhibition (TGI); and the concentration of the
compound leading to 50% net cell death (LC50). The
meangraph midpoints (MG-MID) represent average
values for each of the mentioned parameters and indi-
cate the average sensitivity of all cell lines to each test-
ed compound. The selective activity of a compound
against cancer cell lines from a special organ is charac-
terized by a high deviation of the particular cell line
parameter compared to the corresponding MG-MID
value.
Compound 6
Compound 15
a
Subpanel/cell line Log10 GI50 Subpanel/cell line Log10 GI50
a
Leukemia
CCRF-CEM
HL-60 (TB)
MOLT-4
Leukemia
SR
À4.95
À4.81
À4.86
À5.02
À4.88
À4.75
MOLT-4
NSLCb
RMPI-8226
NSLCb
HOP-92
NCI-H460
NCI-H522
CNS cancer
SF-268
À4.81
À4.82
À4.83
EKVX
À4.98
Colon cancer
HCT-15
KM12
À4.86
À4.86
À4.78
À4.77
À4.76
Table 3 shows that evaluated agents demonstrated a
remarkable activity in the in vitro antitumor screen-
ing, expressed by MG-MID GI50 values of À4.77
(compound 6) and À4.71 (compound 15). Moreover,
these results are of special interest because there is a
substantial difference between the cytostatic and the
cytotoxic activity in both compounds (MG-MID
GI50 and MG-MID LC50). Compound 15 displayed
broad spectrum activity, almost all subpanels tested
showed sensitivity to this agent. Leukemia, cancers
of lung, colon, and breast proved to be the most sen-
sitive subpanels to compound 6. In agreement with the
DNA binding assay, the presence of dimethyl substitu-
ent in the 2-position does not modify the antiprolifer-
ative activity. Representative data for antitumor
activity in most sensitive tumor cell lines are given
in Table 4.
SF-295
SNB-75
CNS Cancer
SF-295
SF-539
À4.82
À4.83
Melanoma
SK-MEL-5
UACC-62
SK-MEL-2
M14
À4.83
À4.77
À4.77
À4.77
Melanoma
SK-MEL-5
UACC-62
Renal cancer
CAKI-1
À4.82
À4.92
Renal cancer
786-0
À4.92
À4.86
À4.78
À4.82
À4.85
SN12C
CAKI-1
UO-31
Breast cancer
MDA-MB-231
MDA-MB-435
À4.96
À4.82
Ovarian cancer
IGROV1
À4.77
a Log10 GI50 = log of molar concentration that inhibits 50% net cell
growth.
b NSLC = non-small cell lung cancer.
3.3. Electrochemical properties
(a)
0,0015
0,001
0,0005
0
In an attempt to know if these illudalane derivatives are
capable of generating highly reactive radicals, which can
damage DNA and be implicated in the probable anti-
neoplastic mechanism, we investigated the electrochem-
ical behavior of the target compounds by cyclic
voltammetry.18 This methodology allows the distinction
between reversible and irreversible processes and more-
over, when none of them occurs. For compound 9 the
result was in accordance with an irreversible process.
For the selected agents (compounds 6 and 15), the cyclo-
voltammograms exhibited no variations with respect to
the support electrolyte. As a consequence this set of
compounds is not capable of forming oxy radicals and
then, the effect observed is not due to this mechanism.
-2,0
-1,5
-1,0
-0,5
0,0
0,5
1,0
1,5
2,0
E (V)
-0,0005
-0,001
-0,0015
0,0015
0,001
0,0005
0
(b)
-2,0
-1,5
-1,0
-0,5
0,0
0,5
1,0
1,5
2,0
E (V)
-0,0005
-0,001
-0,0015
Table 3. Average values (MG-MID) for in vitro antitumor activity on
60 human cell lines
Figure 3. Cyclovoltammograms of compounds 6 (a) and 9 (b).
Compound
MG-MIDa
Log10 TGIc
b
d
Log10 GI50
Log10 LC50
Figure 3 shows the cyclic voltammetry patterns of com-
pounds 6 and 9.
6
15
À4.77
À4.71
À4.40
À4.41
À4.12
À4.14
a MG-MID = meangraph midpoint = arithmetical mean value for all
tested cell lines.
b Log10 GI50 = log of molar concentration that inhibits 50% net cell
growth.
4. Conclusion
c Log10 TGI = log of molar concentration that produces a total growth
inhibition.
A method for the synthesis of alcyopterosin A was
developed. In contrast to other more complicated meth-
ods, the compound was prepared in a simple manner
and with readily available reagents. Furthermore, we
d Log10 LC50 = log of molar concentration that leads to 50% net cell
death.

L. M. Finkielsztein et al. / Bioorg. Med. Chem. 14 (2006) 1863–1870
1867
synthesized a new class of derivatives possessing the ill-
udalane skeleton. These compounds exhibited relevant
DNA binding properties with an affinity higher than
that of standard drugs. This study revealed that dimeth-
yl substituent in the 2-position produces no change in
the DNA binding and the presence of the bromine atom
leads to an increase of DNA binding ability. Com-
pounds 6 and 15 showed significant growth inhibition
activity against several human cancer lines. The study
of cyclic voltammetry demonstrated that these deriva-
tives are not capable of forming oxy radicals. Therefore,
their antitumor activity is not due to the production of
reactive oxygen species. Finally, 6 and 15 constitute
new potential anticancer leads. The mechanism of anti-
tumor activity is the subject of further investigations.
3.01 (t, J = 7.6 Hz, 2H, CH2), 4.39 (s, 2H, CH2). 13C
NMR: d 16.9, 20.5, 24.1, 33.5, 36.5, 50.6, 121.4, 128.8,
131.4, 138.7, 143.5, 147.0, 200.9. Anal. Calcd for
C13H14BrClO C, 51.77; H, 4.68. Found: C, 51.73; H,
4.66.
5.1.3. 1-(7-Bromo-2,2,4,6-tetramethylindan-5-yl)-2-chlo-
roethan-1-one (14). Yield: 79%; colorless oil, purified
by pTLC using hexane–ethyl acetate (8:2) as eluent.
1H NMR: d 1.19 (s, 6H, 2· CH3), 2.06 (s, 3H, CH3),
2.26 (s, 3H, CH3), 2.76 (s, 2H, CH2), 2.82 (s, 2H,
CH2), 4.40 (s, 2H, CH2). 13C NMR: d 16.5, 20.2, 29.6,
39.1, 47.9, 50.4, 50.7, 121.3, 128.6, 131.1, 138.4, 142.7,
146.0. Anal. Calcd for C15H18BrClO: C, 54.65; H,
5.50. Found: C, 54.67; H, 5.50.
5.2. 4-Bromo-5,7-dimethylindan-1-one (3)
5. Experimental
Chloroketone 2 (2.94 g, 10.7 mmol) was added in
small portions with swirling to concentrated
H2SO4 (33 mL). The resulting solution was heated
on a silicon bath at 90 ꢁC. After 1 h, the reaction
mixture was cautiously poured onto ice and then
extracted with ethyl acetate (50 mL). The organic
layer was washed with a solution of 10% NaOH
(2· 20 mL), water (20 mL), dried over anhydrous
Na2SO4, and evaporated in vacuo. The residue
was purified by pTLC using hexane–ethyl acetate
(8:2) as eluent to yield 3 (1.71 g, 67%) as a white
solid, mp: 101–103 ꢁC. 1H NMR: d 2.47 (s, 3H,
CH3), 2.57 (s, 3H, CH3), 2.71 (t, J = 6.1 Hz, 2H,
CH2), 3.02 (t, J = 6.1 Hz, 2H, CH2), 7.03 (s, 1H,
ar). 13C NMR: d 18.3, 23.5, 27.8, 37.5, 121.6,
132.7, 134.8, 137.7, 144.8, 156.5, 207.3. Anal. Calcd
for C11H11BrO: C, 55.26; H, 4.64. Found: C, 55.22;
H, 4.66.
Melting points (uncorrected) were determined with a
Thomas Hoover apparatus. Preparative thin layer chro-
matography (pTLC) was performed on a 20 · 20 cm
1
glass coated with silica gel 60 F254 (0.50 mm). H and
13C NMR spectra were recorded with a Bruker
400 MHz spectrometer with CDCl3 as solvent, employ-
ing TMS as internal standard. Chemical shifts are
reported as d values (parts per million). The following
NMR abbreviations are used: br (broad), s (singlet), d
(doublet), t (triplet), m (multiplet), and ex (exchangeable
with D2O). UV spectra were measured with a Jasco V-
570 Spectrophotometer. The cyclic voltammetry experi-
ments were carried out with an EQMAT-S1 potentiostat
controlled by EQSOFT software. Elemental analysis
was carried out in our laboratories with a Colleman
Analyser.
5.1. General procedure for the preparation of 2, 5, and 14
5.3. 4-Bromo-2,2,5,7-tetramethylindan-1-one (12)
AlCl3 (35.0 mmol) was added to a magnetically stirred
solution of aryl bromide (12.4 mmol) and the correspon-
dent acyl chloride (16.8 mmol) in CS2 (12 mL) at 0 ꢁC
over a period of 30 min. The reaction mixture was heat-
ed under reflux for a further 30 min. The resulting dark-
brown solution was cooled at room temperature and
carefully poured onto ice and was extracted with
Cl2CH2 (25 mL), washed with water (2· 10 mL), dried
over anhydrous Na2SO4, and evaporated in vacuo to
yield the crude product.
A solution of indanone 3 (0.40 g, 1.7 mmol) in toluene
(5 mL) was added to a glass tube containing NaH (0.22
g, 9.2 mmol). The tube was stoppered and stirred at
room temperature for 2 h. Then, methyl iodide
(0.72 mL, 11.6 mmol) was added in one portion, the
tube was sealed, and the resulting mixture was heated
in a silicon bath at 110 ꢁC for 90 h. The reaction mix-
ture was poured onto ice, extracted with dichlorometh-
ane (20 mL), washed with a solution of 5% HCl
(2· 10 mL) and water (15 mL), dried over anhydrous
Na2SO4, and evaporated in vacuo. The residue was
purified by pTLC using hexane–ethyl acetate (8:2) as
eluent to render 12 (0.29 g, 66%) as a pale yellow solid,
5.1.1. 1-(5-Bromo-2,4-dimethylphenyl)-3-chloropropan-1-
one (2)19. Yield: 99%; white solid, mp: 39–41 ꢁC (etha-
1
nol). H NMR: d 2.42 (s, 3H, CH3), 2.47 (s, 3H, CH3),
1
3.37 (t, J = 6.8 Hz, 2H, CH2), 3.90 (t, J = 6.8 Hz, 2H,
CH2), 7.16 (s, 1H, ar), 7.84 (s, 1H, ar). 13C NMR: d
21.6, 23.5, 39.5, 44.1, 122.1, 133.3, 135.2, 136.5, 138.7,
142.8, 198.9. Anal. Calcd for C11H12BrClO: C, 47.94;
H, 4.39. Found: C, 48.01; H, 4.38.
mp: 85–87 ꢁC. H NMR: d 1.25 (s, 6H, 2· CH3), 2.47
(s, 3H, CH3), 2.58 (s, 3H, CH3), 2.90 (s, 2H, CH2),
7.05 (s, 1H, ar). 13C NMR: d 18.1, 23.4, 25.8, 44.5,
46.2, 121.5, 132.7, 138.3, 144.8, 153.5. Anal. Calcd
for C13H15BrO: C, 58.44; H, 5.66. Found: C, 58.38;
H, 5.66.
5.1.2. 1-(7-Bromo-4,6-dimethylindan-5-yl)-2-chloroethan-
1-one (5). Yield: 93%; white solid, mp: 103–105 ꢁC, puri-
fied by pTLC using hexane–ethyl acetate (8:2) as eluent.
1H NMR: d 2.10 (s, 3H, CH3), 2.13 (m, J = 7.6 Hz, 2H,
CH2), 2.27 (s, 3H, CH3), 2.96 (t, J = 7.6 Hz, 2H, CH2),
5.4. General procedure for the preparation of 4, 8, and 13
ZnI2 (3 mmol) and NaCNBH3 (15 mmol) were added to
a solution of the correspondent a-chloroketone or inda-

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L. M. Finkielsztein et al. / Bioorg. Med. Chem. 14 (2006) 1863–1870
none (2 mmol) in ethylene dichloride (10 mL) at room
temperature. The reaction mixture was stirred at room
temperature or refluxed and, after completion of the
reaction (monitored by TLC), the mixture was cooled
and poured onto a solution of 6 N HCl (40 mL). Lastly,
the mixture was extracted with Cl2CH2 (20 mL) and the
organic extract was washed with water (15 mL), dried
over anhydrous Na2SO4, and evaporated in vacuo to
yield the crude product.
5.5.2. 1-(7-Bromo-4,6-dimethylindan-5-yl)-2-chloroethan-
1-ol (10). Yield: 43%; colorless oil, purified by pTLC
using hexane–ethanol (9:1) as eluent. 1H NMR: d
2.05–2.11 (m, 2H, CH2), 2.32 (s, 3H, CH3), 2.65 (br s,
1H, OH, ex), 2.55 (s, 3H, CH3), 2.96–3.02 (m, 4H,
2· CH2), 3.65 (dd, J = 3.8 Hz, J = 11.4 Hz, 1H, HCH),
3.98 (dd, J = 10.3 Hz, J = 11.4 Hz, 1H, HCH), 5.34–
5.41 (m, 1H, CH). Anal. Calcd for C13H16BrClO: C,
51.43; H, 5.31. Found: C, 51.47; H, 5.33.
5.4.1. 4-Bromo-5,7-dimethylindane (4)20. Yield: 78%;
white solid, mp: 32–34 ꢁC, purified by pTLC using hex-
5.5.3. 2-(7-Bromo-4,6-dimethylindan-5-yl)oxirane (11).
Yield: 42%; white solid, mp: 81–82 ꢁC, purified by pTLC
1
1
ane–ethyl acetate (9:1) as eluent. H NMR: d 2.11 (m,
using hexane–ethanol (8:2) as eluent. H NMR: d 2.10
J = 7.6 Hz, 2H, CH2), 2.19 (s, 3H, CH3), 2.36 (s, 3H,
CH3), 2.93 (t, J = 7.6 Hz, 2H, CH2), 2.98 (t,
J = 7.6 Hz, 2H, CH2), 6.87 (s, 1H, ar). 13C NMR: d
19.0, 22.8, 24.3, 32.9, 35.8, 119.7, 130.1, 132.7, 135.6,
142.2, 144.8. Anal. Calcd for C11H13Br: C, 58.69; H,
5.82. Found: C, 58.75; H, 5.84.
(m, J = 7.5 Hz, 2H, CH2), 2.30 (s, 3H, CH3), 2.48 (s,
3H, CH3), 2.74 (dd, J = 3.0 Hz, J = 5.6 Hz, 1H, HCH),
2.95–3.01 (m, 4H, 2· CH2), 3.24 (dd, J = 4.0 Hz,
J = 5.6 Hz, 1H, HCH), 3.96 (m, 1H). Anal. Calcd for
C13H15BrO: C, 58.44; H, 5.66. Found: C, 58.39; H, 5.65.
5.5.4. 2-(7-Bromo-2,2,4,6-dimethylindan-5-yl)ethan-1-ol
(15). Yield: 42%; white solid, mp: 74–76 ꢁC, purified
by pTLC using hexane–ethyl acetate (7:3) as eluent.
1H NMR: d 1.15 (s, 6H, 2· CH3), 1.39 (br s, 1H, OH,
ex) 2.17 (s, 3H, CH3), 2.42 (s, 3H, CH3), 2.76 (s, 2H,
CH2), 2.78 (s, 2H, CH2), 3.00 (t, J = 7.5 Hz, 2H, CH2),
3.71 (m, 2H, CH2). 13C NMR: d 16.7, 20.1, 29.8, 34.3,
38.5, 48.9, 51.0, 62.3, 121.6, 132.4, 134.2, 134.5, 141.8,
142.3. Anal. Calcd for C15H21BrO: C, 60.61; H, 7.12.
Found: C, 60.57; H, 7.10.
5.4.2. 7-Bromo-5-(2-iodoethyl)-4,6-dimethylindane (8).
Yield: 28%; white solid, mp: 80–81 ꢁC, purified by pTLC
1
using hexane–ethyl acetate (9:1) as eluent. H NMR: d
2.05 (m, J = 7.6, 2H, CH2), 2.17 (s, 3H, CH3), 2.39 (s,
3H, CH3), 2.92–2.97 (m, 4H, 2· CH2), 3.07–3.11 (m,
2H, CH2), 3.25–3.29 (m, 2H, CH2). 13C NMR: d 1.9,
16.6, 19.9, 23.8, 33.9, 36.4, 36.6, 121.6, 131.2, 133.6,
137.9, 142.9, 143.6. Anal. Calcd for C13H16BrI: C,
41.19; H, 4.25. Found: C, 41.14; H, 4.25.
5.4.3. 4-Bromo-2,2,5,7-tetramethylindane (13). Yield:
88%; colorless oil, purified by pTLC using hexane–ethyl
acetate (9:1) as eluent. 1H NMR: d 1.20 (s, 6H, 2· CH3),
2.16 (s, 3H, CH3), 2.36 (s, 3H, CH3), 2.75 (s, 2H, CH2),
2.81 (s, 2H, CH2), 6.87 (s, 1H, ar). 13C NMR: d 18.9,
22.8, 29.8, 39.3, 47.8, 50.4, 119.9, 130.1, 133.0, 135.6,
141.4, 144.0. Anal. Calcd for C13H17Br: C, 61.67; H,
6.77. Found: C, 61.72; H, 6.79.
5.6. General procedure for the preparation of 7 and 16
The correspondent alcohol (0.6 mmol) in dry THF
(3 mL) was added to a suspension of LiAlH4 (5 mmol)
in dry THF (15 mL) at 0 ꢁC. The reaction mixture was
heated under reflux during 24 h. After being cooled at
room temperature, the mixture was carefully poured
onto ice and a solution of 10% NaOH (5 mL) was add-
ed. The aqueous phase was extracted with Cl2CH2
(2· 10 mL). The extract was washed with water
(10 mL) dried over anhydrous Na2SO4 and evaporated
in vacuo to yield the crude product.
5.5. General procedure for the preparation of 6, 10, 11,
and 15
NaBH4 (12 mmol) was added over a period of 5 min to a
refluxing solution of the correspondent a-chloroketone
(1 mmol) in ethanol (18 mL). The reaction mixture was
heated under reflux until TLC showed the presence of
the desired product. The mixture was cooled at room
temperature and the solvent was removed in vacuo.
The residue was partitioned between CHCl3 (20 mL)
and water (10 mL). The organic layer was dried over
anhydrous Na2SO4, and evaporated in vacuo to yield
the crude product.
5.6.1. 2-(4,6-Dimethylindan-5-yl)ethan-1-ol (7). Yield:
67%; white solid, mp: 53–55 ꢁC, purified by pTLC using
hexane–ethyl acetate (8:2) as eluent. 1H NMR: d 1.42 (br
s, 1H, OH, ex), 2.07 (m, J = 7.6 Hz, 2H, CH2), 2.29 (s,
3H, CH3), 2.35 (s, 3H, CH3), 2.85 (t, J = 7.6 Hz, 2H,
CH2), 2.90 (t, J = 7.6 Hz, 2H, CH2), 2.99 (t,
J = 7.6 Hz, 2H, CH2), 3.77 (t, J = 7.6 Hz, 2H, CH2),
6.95 (s, 1H, ar). 13C NMR: d 16.6, 20.8, 25.2, 32.5,
33.2, 33.4, 62.4, 124.3, 132.3, 133.1, 135.3, 141.8,
142.3. Anal. Calcd for C13H18O: C, 82.06; H, 9.54.
Found: C, 82.11; H, 9.52.
5.5.1. 2-(7-Bromo-4,6-dimethylindan-5-yl)ethan-1-ol (6).
Yield: 41%; white solid, mp: 100–101 ꢁC, purified by
pTLC using hexane–ethyl acetate (7:3) as eluent. 1H
NMR: d 1.46 (br s, 1H, OH, ex), 2.08 (m, J = 7.6 Hz,
2H, CH2), 2.24 (s, 3H, CH3), 2.46 (s, 3H, CH3), 2.98
(m, 4H, 2· CH2), 3.04 (t, J = 7.4 Hz, 2H, CH2), 3.77
(t, J = 7.4 Hz, 2H, CH2). 13C NMR: d 16.8, 20.1, 23.8,
34.0, 34.3, 36.4, 62.3, 121.4, 132.1, 134.3, 134.5, 142.6,
143.1. Anal. Calcd for C13H17BrO: C, 58.01; H, 6.37.
Found: C, 57.98; H, 6.39.
5.6.2. 2-(2,2,4,6-Tetramethylindan-5-yl)ethan-1-ol (16)7.
Yield: 69%; white solid, mp: 65–67 ꢁC, purified by pTLC
1
using hexane–ethyl acetate (8:2) as eluent. H NMR: d
1.14 (s, 6H, 2· CH3), 1.43 (br s, 1H, OH, ex) 2.21 (s,
3H, CH3), 2.32 (s, 3H, CH3), 2.65 (s, 2H, CH2), 2.69
(s, 2H, CH2), 2.94 (t, J = 7.4 Hz, 2H, CH2), 3.73 (m,
2H, CH2), 6.85 (s, 1H, ar). 13C NMR: d 16.5, 20.8,
29.7, 33.2, 39.6, 47.6, 48.3, 62.4, 124.6, 132.3, 133.3,

L. M. Finkielsztein et al. / Bioorg. Med. Chem. 14 (2006) 1863–1870
1869
135.2, 141.1, 141.6. Anal. Calcd for C15H22O: C, 82.52;
H, 10.16. Found: C, 82.58; H, 10.18.
trode, a Ag/AgCl reference electrode, and MeOH of
electrochemical purity. The potentials were scanned
from À2000 to +2000 mV employing scan rates of
0.2 mV/s. The glassy carbon electrode was polished
intensively with aluminum oxide on a smooth polish-
ing cloth and degreased in methanol prior to each
electrochemical measurement. All the solutions exam-
ined by electrochemical techniques were first deaerated
for at least 10 min with N2, after which a continuous
stream of N2 was passed over the solution during
measurements. Lithium perchlorate 0.1 M was used
as support electrolyte. Sample concentration was
0.5 mM.
5.7. 1-(7-Bromo-4,6-dimethylindan-5-yl)ethan-1-one (9)
NaBH4 (0.49 g, 10.2 mmol) was added, in small por-
tions over a period of 15 min at 0 ꢁC, to a solution
of a-chloroketone 5 (0.22 g, 0.6 mmol) and CuCl
(0.20 g, 2 mmol) in MeOH (26 mL). After being heated
under reflux for 30 min, the resulting black precipitate
was removed by filtration, and the filtrate was acidified
with 5% aqueous HCl (10 mL) and extracted with Et2O
(2· 10 mL). The organic phase was washed with water
(2· 5mL), dried over anhydrous Na2SO4, and evapo-
rated in vacuo. The residue was purified by pTLC
using hexane–ethanol (8:2) as eluent to render 9
Acknowledgments
1
(0.04 g, 24%) as white solid, mp: 63–66 ꢁC. H NMR:
d 2.07–2.15 (m, 2H, CH2), 2.11 (s, 3H, CH3), 2.28 (s,
3H, CH3), 2.47 (s, 3H, CH3), 2.95 (t, J = 7.6 Hz, 2H,
CH2), 3.00 (t, J = 7.6 Hz, 2H, CH2). Anal. Calcd for
C13H15BrO: C, 58.44; H, 5.66. Found: C, 58.47; H,
5.67.
The authors thank the Development Therapeutics Pro-
gram of the National Cancer Institute, Bethesda, MD,
for providing the in vitro antitumor screening data
and CONICET and SECyT-UBA for their financial
support.
5.8. Alcyopterosin A (1)
References and notes
A solution of thionyl chloride (0.1 mL, 1.2 mmol) in
CHCl3 (1 mL) was added dropwise to a stirred solution
of 16 (0.10 g, 0.5 mmol) and pyridine (0.1 mL,
1.1 mmol) in CHCl3 (1 mL) at 0 ꢁC. It was stirred for
10 min and heated under reflux for 45 min. The reac-
tion mixture was diluted with water (5 mL), extracted
with CHCl3 (2· 5mL), and washed with a solution of
10% HCl (2· 5mL) and water (5 mL). The organic
layer was dried over anhydrous Na2SO4 and evaporat-
ed in vacuo. The residue was purified by pTLC using
hexane as eluent to render 1 (0.09 g, 78%) as a color-
less oil. 1H NMR: d 1.14 (s, 6H, 2· CH3), 2.20 (s,
3H, CH3), 2.30 (s, 3H, CH3), 2.64 (s, 2H, CH2), 2.68
(s, 2H, CH2), 3.11 (t, J = 8.3 Hz, 2H, CH2), 3.52 (t,
J = 8.3 Hz, 2H, CH2), 6.85 (s, 1H, ar). Anal. Calcd
for C15H21Cl: C, 76.09; H, 8.94. Found: C, 76.15; H,
8.97.
1. Wolf-Rainer, A. Curr. Med. Chem. 2001, 8, 583, and
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2. Murakami, T.; Tanaka, N. Prog. Chem. Org. Nat. Prod.
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´
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5.9. DNA affinity assay
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DNA solution: Calf thymus DNA (12.5 mg) was slow-
ly magnetically stirred in Tris–HCl buffer 10 mM, pH
7.4 (5 mL), for 24 h at 4 ꢁC. 0.6 mL was taken from
this solution and diluted to 25 mL with the same
buffer.
´
´
11. Asıs, S. E.; Bruno, A. M.; Martınez, A. R.; Sevilla, M. V.;
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The test compound solution was prepared at a 10À4 M
concentration using a minimal volume of ethanol and
then diluted adding water to a concentration of
2 · 10À5 M. A 3 mL sample of this solution was mixed
with 3 mL of the DNA solution. The mixture was slowly
rotated during 24 h and, then, its UV spectra were
recorded at 20 ꢁC using a 1 cm cell.
5.10. Cyclic voltammetry
Studies were carried out using a glassy carbon work-
ing electrode (A = 3.03 cm2), a platinum auxiliary elec-

1870
L. M. Finkielsztein et al. / Bioorg. Med. Chem. 14 (2006) 1863–1870
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´
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17. Data concerning the NCI screening methods in detail are
accessible from the NCI via the Internet from the
following address: http://dtp.nci.nih.gov/branches/btb/
ivclsp.html.