Total synthesis and anticancer activities of (-)- and (+)-thespesone

Article abstract of DOI:10.1021/jo1012493

Figure presented. The natural p-naphthoquinone (-)-thespesone 1 and its (+)-enantiomer were synthesized for the first time by bisacylation of a 5-lithiodihydrobenzofuran 2′ with 4-methyl-3-tert-butoxycyclobut-3-ene-1, 2-dione 3. The racemate of the required 2-arylpropan-1-ol precursor 10 was kinetically resolved by an enzyme-catalyzed acetylation exclusively of the (S)-enantiomer. Saponification of this acetate mediated by the same enzyme, porcine pancreas lipase (PPL), afforded the (S)-2-arylpropan-1-ol in 96% ee. Its unreacted (R)-enantiomer could be obtained with 77% ee. (-)-(S)-Thespesone was far more efficacious against a panel of six cancer cell lines including multiresistant ones than its (+)-enantiomer and also when compared to thymoquinone, an established natural antitumoral p-quinone from Nigella sativa. Unlike the latter, (-)-thespesone was well tolerated by nonmalignant fibroblasts.

Full text of DOI:10.1021/jo1012493

pubs.acs.org/joc
Total Synthesis and Anticancer Activities of (-)- and (þ)-Thespesone
Sandra Breyer, Katharina Effenberger-Neidnicht, and Rainer Schobert*
Organic Chemistry Laboratory, University of Bayreuth, 95440 Bayreuth, Germany
rainer.schobert@uni-bayreuth.de
Received June 25, 2010
The natural p-naphthoquinone (-)-thespesone 1 and its (þ)-enantiomer were synthesized for the first
time by bisacylation of a 5-lithiodihydrobenzofuran 20 with 4-methyl-3-tert-butoxycyclobut-3-ene-
1,2-dione 3. The racemate of the required 2-arylpropan-1-ol precursor 10 was kinetically resolved by
an enzyme-catalyzed acetylation exclusively of the (S)-enantiomer. Saponification of this acetate
mediated by the same enzyme, porcine pancreas lipase (PPL), afforded the (S)-2-arylpropan-1-ol in
96% ee. Its unreacted (R)-enantiomer could be obtained with 77% ee. (-)-(S)-Thespesone was far more
efficacious against a panel of six cancer cell lines including multiresistant ones than its (þ)-enantiomer
and also when compared to thymoquinone, an established natural antitumoral p-quinone from
Nigella sativa. Unlike the latter, (-)-thespesone was well tolerated by nonmalignant fibroblasts.
Introduction
and the cytotoxicities of the two enantiomers and the race-
mate of thespesone against a panel of six cancer cell lines and
nonmalignant foreskin fibroblasts. Scheme 1 outlines the
retrosynthesis, which was based on a disconnection of the aro-
matic and the quinoid rings. p-Naphthoquinones had been
previously obtained from halo benzenes by regioselective
bisacylation of their lithiated derivatives with cyclobut-3-
ene-1,2-diones such as 3.10-13 The required 5-bromo-dihydro-
benzofuran 2 was to be prepared from 2-hydroxy-4-methyl-
acetophenone 5 via functional group transformations14
comprising the establisment of the S-configured stereocenter
in the benzylic position of bromide 4 by an enzymatic reso-
lution with porcine pancreas lipase.
The heartwood of portia or milo trees (Thespesia populnea;
malvaceae) that grow in Asia, Africa, Hawaii, Florida, California,
and the Caribbean Islands1 has been traditionally used as a
cardiac stimulant. It was also reported to have antifungal, anti-
biotic, antifertility, antibacterial, anti-inflammatory, antioxi-
dant, purgative, hepatoprotective, and antitumor properties.2-5
The constituents believed responsible for the majority of these
effects are so-called mansonones,6 highly oxo-functionalized
sesquiterpenes of the cadinane type. They were isolated from
extracts of the reddish brown heartwood.7 Some of them have
cytotoxic, antifungal, or antioxidant properties.5-8 (-)-
Thespesone 1 was first isolated in 19839 but was not fully
characterized until 2004.8 Herein we describe its total synthesis
Results and Discussion
€
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Syntheses of Racemic and Enantiopure Thespesones 1.
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Published on Web 08/16/2010
DOI: 10.1021/jo1012493
r
2010 American Chemical Society

Breyer et al.
JOCArticle
SCHEME 1. Retrosynthetic Analysis of (-)-Thespesone 1
SCHEME 2. Synthesis of Chiral Precursors 4a
5 was benzylated to give ether 6, which was treated with
methyl magnesium iodide to afford the tertiary alcohol 7.
Elimination of water using KHSO415 left the styrene deriva-
tive 8 in 90% overall yield (Scheme 2). Initially, we intended
to convert the latter to the enantiopure alcohol (S)-10, an
immediate precursor to the required dibromide (S)-4, by
means of an asymmetric dihydroxylation reaction followed
by a selective reduction of the tertiary hydroxy group of diol
(R)-9 with Raney-Ni.16-18 However, Sharpless dihydroxyla-
tion of 8 with AD-Mix β proceeded with low enantioselec-
tivity. Product alcohol 10 was eventually isolated with 7% ee
in favor of the S-enantiomer. This finding is in line with
earlier reports on the low selectivity of AD-reactions of
sterically encumbered styrenes.19,20 Replacement of the ben-
zoxy group of 8 by hydroxy or acetate residues gave insuffi-
cient conversion due to formation of a water-soluble
phenolate, while an ortho-methoxy analogue did not react
at all. An alternative attempt to prepare alcohol (S)-10 by
enantioselective hydroboration of 8 using Ipc-borane also
failed.21-25 More successful was an enzymatic resolution of
racemic alcohol (()-10 as obtained by hydroboration of 8
aReagents and conditions: (i) BnBr, K2CO3, THF/DMF, reflux, 20 h,
99%; (ii) CH3MgI, Et2O, 0 °C to reflux (4 h), then sat. NH4Cl; (iii) KHSO4,
DMF, 80 °C, 1 h. *ee after three runs.
with BH3 in THF. Its acetylation mediated by porcine
pancreas lipase (PPL)26-30 in the presence of vinyl acetate
and methyl tert-butyl ether as the solvent led to acetate (S)-11
with 85% ee in 45% chemical yield. Residual 10 could be
recovered and rereacted. After three runs, aside the acetate
(S)-11, the unreacted (R)-10 was isolated in an optical purity
of 77% ee. The hydrolysis of the enriched (S)-11 in aqueous
phosphate buffer (pH 7) was also amenable to catalysis by
PPL and afforded the target alcohol (S)-10 with 96% ee,
again with the option to repeat this procedure with recovered
starting acetate until maximum conversion. Bromination of
the enantiomers of alcohol 10 with NBS/PPh3 afforded the
respective dibromide 4.31 The enantiomeric purity of com-
pounds 10 and 11 was in each case determined by gas
chromatography on a Lipodex E column after deprotection
of the hydroxy groups to give the respective stereoisomers of
2-(2-hydroxy-4-methylphenyl)propan-1-ol 100.
The benzyl ether of the enantiopure or racemic dibromide
4 had to be cleaved using AlCl3/PhNEt232 since the custom-
ary hydrogenation at Pd/C had failed. As some 5-bromo-3,6-
dimethyldihydrobenzofuran 2 was inevitably formed as a
side product of this deprotection, the crude dibromophenol
was treated with K2CO3 in acetone right away to obtain pure
2 in over 80% yield. Scheme 3 shows this sequence for the
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TABLE 1. Inhibitory Concentrations (μM) IC50(72 h)a of (()-1, (-)-1, (þ)-1, and 15-17 When Applied to Cancer and Nonmalignant Cells
cell line/compound
518A2
HL-60
HT-29
HT-29/Colc
KB-V1/Vbl
MCF-7/Topo
HF
(()-1
(-)-1
(þ)-1
15
16
17
26 ( 4
20 ( 5
>100
71 ( 12
21 ( 13
93 ( 10
28 ( 6b
82 ( 25
67 ( 29
84 ( 23
77 ( 1
17 ( 6
16 ( 6
21 ( 25
32 ( 6b
12c
70 ( 12
11 ( 8
>100
27 ( 6b
3.3c
>100
>100
>100
33 ( 20b
>100
>100
47 ( 19
>20c
0.7c
28 ( 9b
2.3c
0.2c
aValues are derived from concentration-response curves obtained by measuring the percentual absorbance of viable cells relative to untreated
controls (100%) after 72 h of exposure of 518A2 melanoma, HL-60 leukemia, HT-29, and HT-29/Colc colon carcinoma, MCF-7/Topo breast
carcinoma, and KB-V1/Vbl cervix carcinoma cells as well as human foreskin fibroblasts (HF) to the test compounds in the MTT assay. Values represent
means of four independent experiments ( standard deviation. bValues taken from literature.38,39 cSRB-assays (48 h) with sensitive wild-type cells; values
taken from literature.5
SCHEME 3. Synthesis of (-)-(S)-Thespesone 1 from (S)-4
a p-benzoquinone occurring in the seed oil of Nigella sativa
that showed antitumor effects in various in vitro and ani-
mal studies,34-37 and of Karalai’s best perfomers, the
o-quinoid mansonones D 16 and E 17. IC50 values after
24 and 48 h of exposure to the test compounds are listed in
the Supporting Information. Table 1 summarizes only the
IC50(72 h) values.
The natural (-)-1 was generally far more active against the
cancer cells than its (þ)-enantiomer and even surpassed the
efficacy of thymoquinone 15. Unlike the latter, it was tole-
rated very well by the nonmalignant fibroblasts, which is
interesting from a pharmacological viewpoint. While less
active than mansonone E 17 in the colon, cervix, and breast
carcinoma cell lines, (-)-1 was comparable in activity to its
close furanoid analogue 16.
dibromide (S)-4 that leads up to the natural (-)-(S)-thespe-
sone. Li-halogen exchange of 2 with BuLi and subsequent
trapping of the intermediate aryllithium compound with
3-tert-butoxy-4-methylcyclobut-3-ene-1,2-dione 3 left the
acyloine 12. This was not isolated but heated in toluene to
initiate an electrocyclic opening--closure sequence afford-
ing the hydroquinone 13, which underwent an auto-oxida-
tion to the quinone 14.10-13 Its tert-butyl group was finally
removed with TFA in CH2Cl2.12 Racemic and (þ)-thespe-
sone were synthesized analogously starting from either (()-4
or (R)-4.
In Vitro Cytotoxicities of the Thespesone Stereoisomers.
Karalai et al.5 reported the cytotoxicities of nine compounds
isolated from T. populnea against four cancer cell lines
(MCF-7, HeLa, HT-29, and KB carcinomas). For unknown
reasons they left out thespesone 1. Later on, Murugesan et al.
reported an LD50(7 d) > 10 μM for it when applied to MCF-
7 breast carcinoma cells.33 We now tested (-)-1, (þ)-1, and
(()-1 for growth inhibition of cells of human HL-60 leuke-
mia, 518A2 melanoma, HT-29 colon carcinoma, and the
multidrug-resistant variants of HT-29/Colc, MCF-7/Topo
(breast), and KB-V1/Vbl (cervix; a HeLa-derivative), as
well as of nonmalignant human foreskin fibroblasts (HF).
We compared the results with those of thymoquinone 15,
Conclusions
The first synthesis of natural (-)-thespesone 1 and its (þ)-
enantiomer was based upon an enzyme-mediated kinetic
resolution of a chiral precursor alcohol and a known bisa-
cylation of a 5-lithiodihydrobenzofuran with cyclobut-3-en-
1,2-dione to establish the p-naphthoquinone. The resolution
of racemic alcohol (()-10 via acetylation and rehydrolysis of
the so-formed acetate, both mediated by porcine pancreas
lipase (PPL), afforded the alcohol (S)-10 with 96% ee in high
chemical yield. The unreacted enantiomeric alcohol (R)-10
could also be enriched to 77% ee by several more cycles of
PPL-mediated acetylations. Unlike many other constituents
of T. populnea, thespesone had not yet been screened for
anticancer activity. We closed this gap and found that the
natural (-)-enantiomer of thespesone was far more active
against various cancer cell lines including multiresistant ones
than its (þ)-enantiomer and also when compared to the
(34) Badary, O. A.; Gamal El-Din, A. M. Cancer Detect. Prev. 2001, 25,
362–368.
(35) Badary, O. A.; Al-Shabanah, O. A.; Nagi, M. N.; Al-Rikabi, A. C.;
Elmazar, M. M. Eur. J. Cancer Prev. 1999, 8, 225–260.
(36) Gali-Muhtasib, H.; Roessner, A.; Schneider-Stock, R. Int. J. Bio-
chem. Cell Biol. 2006, 38, 1249–1253.
(37) Gali-Muhtasib, H.; Ocker, M.; Kuester, D.; Krueger, Z.; El-Hajj, S.;
Diestel, A.; Evert, M.; El-Najjar, N.; Peters, B.; Jurjus, A.; Roessner, A.;
Schneider-Stock, R. J. Cell. Mol. Med. 2008, 12, 330–342.
(33) Inbaraj, J. J.; Gandhidasan, R.; Murugesan, R. Free Radical Biol.
Med. 1999, 26, 1072–1078.
6216 J. Org. Chem. Vol. 75, No. 18, 2010

Breyer et al.
JOCArticle
established antitumoral thymoquinone 15. The large spread
between efficacy against cancer cells and tolerance by non-
malignant fibroblasts makes (-)-thespesone 1 a potential
new candidate for anticancer therapy.
(S)-2-(2-(Benzyloxy)-4-methylphenyl)propan-1-ol(S)-10. Acetate
(S)-11 (1.5 g, 5.0 mmol, 85% ee) was dissolved in methanol (42 mL)
and phosphate buffer (pH 7, 126 mL) and treated with PPL (1.5 g)
at 35 °C for 24 h to allow for ca. 50% conversion. The mixture was
extracted with ethyl acetate (6 ꢀ 100 mL), and the extracts were
washed with brine (100 mL), dried over anhydrous Na2SO4, and
evaporated to dryness. The crude product was purified by column
chromatography (cyclohexane/ethyl acetate 6:1, silica gel 60) to
Experimental Section
General Remarks. Melting points are uncorrected. IR spectra
were recorded on an FT-IR spectrophotometer equipped with
an ATR sampling unit. Nuclear magnetic resonance (NMR)
spectra were recorded under conditions as indicated. Chemical
shifts are given in parts per million (δ) downfield from tetra-
methylsilane as internal standard for 1H and 13C. Gas chroma-
tograpy was conducted on a Lipodex E column (80-200 °C, rate
5 °C/min). For chromatography silica gel 60 (230-400 mesh)
was used. All starting compounds were purchased from com-
mercial sources and used without purification.
yield 356 mg (1.39 mmol, 30%) of (S)-10 as a colorless oil. [R]24
-
D
3.3 (c 1, CHCl3); 96% ee; 2.68 mmol of starting acetate (S)-11 was
recovered and resubmitted to this protocol.
(S)-1-(Benzyloxy)-4-bromo-2-(1-bromopropane-2-yl)-5-methyl-
benzene (S)-4. A solution of alcohol (S)-10 (220 mg, 0.86 mmol,
96% ee) in CH2Cl2 (20 mL) was cooled to 0 °C, treated with
N-bromosuccinimide (489 mg, 2.75 mmol) and triphenylphos-
phane (270 mg, 1.03 mmol), and allowed to warm to room tem-
perature overnight. The solvent was evaporated and the residue
was taken up in ethyl acetate (2 mL) and purified by column
chromatography (cyclohexane/ethyl acetate 4:1, silica gel 60).
Yield: 325 mg (0.82 mmol, 95%); colorless oil; Rf 0.8 (cyclo-
(()-2-(2-(Benzyloxy)-4-methylphenyl)propan-1-ol (()-10. A
solution of compound 8 (6.7 g, 28.1 mmol) in dry THF (100 mL)
was cooled to 0 °C and treated dropwise with BH3 (1 M in THF,
28.7 mL). The resulting mixture was stirred at this temperature
for 2 h, then treated with NaOH (10%, 36.8 mL) and H2O2
(30%, 19.2 mL), and finally stirred at room temperature for
a further 1.5 h. Brine (80 mL) was added, and the mixture
was extracted with ethyl acetate (3 ꢀ 100 mL). The combined
organic layers were washed with brine (100 mL), dried over
anhydrous Na2SO4, and evaporated to dryness. The result-
ing crude product was purified by column chromatography
(ethyl acetate/cyclohexane 1:4, silica gel 60) to yield 6.27 g
(24.46 mmol, 87%) of (()-10 as a colorless oil; Rf 0.5
(cyclohexane/ethyl acetate 1:1); IR (ATR) νmax 3389, 3031,
2922, 2870, 1610, 1578, 1505, 1453, 1412, 1381, 1288, 1255,
hexane/ethyl acetate 1:1); [R]24 -3.8 (c 1, CHCl3). IR (ATR)
D
ν
max 2924, 2954, 1726, 1602, 1492, 1454, 1385, 1249, 1174, 1091,
1024, 799, 733, 696 cm-1; 1H NMR (CDCl3, 300 MHz) δ 1.39 (d,
J=6.7 Hz, 3 H), 2.38 (s, 3 H), 3.49 (dd, J=15.8 Hz, J=8.8 Hz,
1 H), 3.57 (ddq, J=6.7 Hz, J=8.8 Hz, J=8.9 Hz, 1 H), 3.66 (dd,
J=15.8 Hz, J=8.9 Hz, 1 H), 5.07 (s, 2 H), 6.84 (s, 1 H), 7.36 (s,
1 H), 7.4-7.5 (m, 5 H); 13C NMR (CDCl3, 75 MHz) δ 18.5, 23.1,
35.3, 39.1, 70.4, 114.6, 115.8, 127.2, 128.1, 128.7, 131.1, 131.5,
136.7, 137.2, 155.3; m/z (EI) 397 (3) [Mþ-1], 395 (4), 227 (8), 225
(8), 147 (4), 132 (3), 91 (100), 65 (5). Anal. Calcd for C17H18-
Br2O: C, 51.28; H, 4.56. Found: C, 51.31; H, 4.63.
(S)-5-Bromo-3,6-dimethyl-2,3-dihydrobenzofurane (S)-2. Ben-
zyl ether (S)-4 (190 mg, 0.48 mmol) was dissolved in CH2Cl2
(15 mL), and PhNEt2 (0.77 mL, 712 mg, 4.77 mmol) and AlCl3
(191 mg, 1.43 mmol) were added. The mixture was stirred at
room temperature for 1 h and then quenched with 1 M aqueous
HCl (20 mL). It was extracted with diethyl ether (4ꢀ50 mL), and
the extracts were washed with 1 M aqueous HCl and brine
(100 mL each). The organic layer was dried over anhydrous
Na2SO4, and the solvent was removed in vacuum. The residue
thus obtained was treated with K2CO3 (144 mg, 1.04 mmol) and
acetone (50 mL), and the resulting mixture was heated under
reflux overnight, then cooled to room temperature, filtered, and
evaporated. The crude product was purified by column chromato-
graphy (cyclohexane/ethyl acetate 10:1, silica gel 60) to leave
(S)-2 (90 mg, 0.39 mmol, 83%) as a colorless oil; Rf 0.7 (cyclo-
hexane/ethyl acetate 1:1); [R]24D -12.2 (c 1, CHCl3). IR (ATR)
1
1162, 1134, 1016, 809, 732, 695 cm-1; H NMR (CDCl3, 300
MHz) δ 1.44 (d, J=7.0 Hz, 3 H), 2.51 (s, 3 H), 2.76 (br, 1H, OH),
3.47 (ddq, J=6.4 Hz, J=6.6 Hz, J=7.0 Hz, 1 H), 3.79 (dd, J=
17.0 Hz, J=6.6 Hz, 1 H), 3.91 (dd, J=17.0 Hz, J=6.4 Hz, 1 H),
5.18 (s, 2 H), 6.96 (s, 1 H), 6.98 (d, J=7.5 Hz, 1 H), 7.29 (d, J=
7.5 Hz, 1 H), 7.4-7.6 (m, 5 H); 13C NMR (CDCl3, 75 MHz) δ
16.5, 21.0, 34.7, 67.0, 69.7, 112.5, 121.3, 126.9, 127.4, 128.2,
129.0, 136.6, 136.9, 155.9; m/z (EI) 256 (13) [Mþ], 225 (11), 148
(14), 135 (26), 91 (100), 65 (9). Anal. Calcd for C17H20O2: C,
79.65; H, 7.86. Found: C, 79.59, H, 8.06.
(S)-2-(2-(Benzyloxy)-4-methylphenyl)propyl Acetate (S)-11.
A solution of alcohol (()-10 (5.75 g, 22.43 mmol) in methyl
tert-butyl ether (100 mL) and vinyl acetate (3.05 mL, 2.84 g, 33.0
mmol) was treated with PPL (1.23 g), and the resulting mixture
was stirred at room temperature for 6 days. It was filtered, and
the filtrate was concentrated in a vacuum before being purified
and separated from unreacted alcohol 10 by column chromato-
graphy (cyclohexane/ethyl acetate 8:1, silica gel 60). Yield: 1.5 g
(5.0 mmol, 45%) of (S)-11 as a colorless oil of Rf 0.8 (cyclo-
hexane/ethyl acetate 1:1); [R]24D 13.7 (c 1, CHCl3); 85% ee. IR
(ATR) νmax 3032, 2968, 1735, 1612, 1579, 1506, 1454, 1370,
1226, 1164, 1023, 810, 735 cm-1; 1H NMR (CDCl3, 300 MHz) δ
1.40 (d, J=7.1 Hz, 3 H), 2.06 (s, 3 H), 2.43 (s, 3 H), 3.72 (ddq, J=
6.3 Hz, J=7.1 Hz, J=7.5 Hz, 1 H), 4.30 (dd, J=17.8 Hz, J=
7.5 Hz, 1 H), 4.38 (dd, J=17.8 Hz, J=6.3 Hz, 1 H), 5.14 (s, 2 H),
6.87 (s, 1 H), 6.88 (d, J=7.9 Hz, 1 H), 7.21 (d, J=7.9 Hz, 1 H),
7.4-7.6 (m, 5 H); 13C NMR (CDCl3, 75 MHz) δ 16.9, 20.6, 21.1,
31.7, 68.3, 69.7, 112.6, 121.3, 126.9, 127.0, 127.5, 128.3, 137.1,
155.9, 170.7; m/z (EI) 298 (3) [Mþ], 238 (11), 133 (21), 92 (12), 91
(100), 43 (12). Anal. Calcd for C19H22O3: C, 76.48; H, 7.43.
Found: C, 76.28; H, 7.51.
ν
max 2921, 2852, 1737, 1659, 1633, 1463, 1377, 1260, 1092, 804,
721 cm-1; 1H NMR (CDCl3, 300 MHz) δ 1.28 (d, J = 6.8 Hz,
3 H), 2.31 (s, 3 H), 3.49 (ddq, J=6.8 Hz, J=8.6 Hz, J=8.8 Hz,
1 H), 4.05 (dd, J=16.0 Hz, J=8.6 Hz, 1 H), 4.65 (dd, J=16.0
Hz, J = 8.8 Hz, 1 H), 6.66 (s, 1 H), 7.24 (s, 1 H); 13C NMR
(CDCl3, 75 MHz) δ 19.3, 23.1, 36.3, 79.0, 111.7, 114.6, 127.3,
132.0, 137.2, 159.3; m/z (EI) 228 (44) [Mþþ1], 226 (42), 147 (15),
132 (100), 115 (8), 91 (16), 63 (8), 51 (16); HRMS (EI) calcd for
C10H11BrO [M]þ 225.9993, found 225.9980.
(S)-7-tert-Butoxy-1,5,8-trimethyl-1,2-dihydronaphtho[2,1-b]furan-
6,9-dione (S)-14. A solution of (S)-2 (63 mg, 0.28 mmol) in dry
THF (15 mL) was cooled to -78 °C, treated dropwise with
n-BuLi (2.1 M in hexane, 0.14 mL, 0.29 mmol) and stirred at this
temperature for 1 h. A solution of dione 3 (69 mg, 0.42 mmol) in
THF (10 mL) was added, stirring was continued for 90 min at
-78 °C, and the mixture was finally quenched with saturated
aqueous NH4Cl (10 mL) and diluted with diethyl ether (25 mL).
The phases were separated, and the aqueous one was extracted
with diethyl ether (3ꢀ50 mL). The combined organic layers were
washed with brine (50 mL), dried over anhydrous Na2SO4, and
evaporated to dryness. The remainder was dissolved in toluene
(S)-2-(2-(Benzyloxy)-4-methylphenyl)propan-1-ol (R)-10. The
unreacted alcohol 10 was enantiomerically enriched to 77% ee
by repeating the above reaction two more times to afford 1.99 g
(7.76 mmol, 35%) of alcohol (R)-10 as a colorless oil. [R]24D þ
1.6 (c 1, CHCl3).
J. Org. Chem. Vol. 75, No. 18, 2010 6217

Breyer et al.
JOCArticle
(50 mL) and heated under reflux for 2 h. The reaction mixture
was concentrated in vacuum, and the residue thus obtained was
left overnight under ambient atmosphere at room temperature
to give quinone 14, which was purified by column chromato-
graphy (cyclohexane/ethyl acetate 4:1, silica gel 60). Yield: 50 mg
(0.16 mmol, 57%); yellow oil of Rf 0.7 (cyclohexane/ethyl ace-
tate 1:1); [R]24D -128.4 (c 1, CHCl3). IR (ATR) νmax 2962, 2929,
RPMI-1640 medium supplemented with 10% fetal calf serum
(FCS), 100 IU/mL penicillin G, 100 μg/mL streptomycin sul-
fate, 0.25 μg/mL amphotericin B and 250 μg/mL gentamycine.
The 518A2, the KB-V1/Vbl and the HF cells were cultured in
Dulbecco’s Modified Eagle Medium containing 10% FCS, 100
IU/mL penicillin G, 100 μg/mL streptomycin sulfate, 0.25 μg/
mL amphotericin B and 250 μg/mL gentamycine. The MCF-7/
Topo cells were grown in E-MEM medium supplemented with
2.2 g/L NaHCO3, 110 mg/L sodium pyruvate and 5% FCS. The
cells were maintained in a moisture-saturated atmosphere (5%
CO2) at 37 °C in 75-mL culture flasks. They were serially pas-
saged following trypsinisation by 0.05% trypsin/0.02% EDTA.
Mycoplasma contamination was routinely monitored, and only
mycoplasma-free cultures were used.
Inhibition of Cell Growth and Metabolic Activity (MTT Assay).
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide] was used to identify viable cells which reduce it to a
violet formazan. HL-60 cells (5ꢀ105/mL), and cells (5ꢀ104/mL)
of 518A2, HT-29, KB-V1/Vbl, MCF-7/Topo carcinomas and
foreskin fibroblasts (HF) were seeded in 96-well tissue culture
plates and cultured for 24 h.40 Incubation (5% CO2, 95%
humidity, 37 °C) of the cells following treatment with the test
compounds was continued for 24, 48, or 72 h. Blank and solvent
controls were treated identically. MTT in phosphate buffered
saline (5 mg/mL) was added to a final concentration of 0.05%
(HL-60, 518A2, HF) or 0.1% (KB-V1/Vbl, MCF-7/Topo, HT-
29, HT-29/Colc). After 2 h the formazan precipitate was dis-
solved in 10% sodium dodecylsulfate in DMSO containing
0.6% acetic acid in the case of HL-60 cells. For the adherent
518A2, HT-29, HT-29/Colc, KB-V1/Vbl, MCF-7/Topo and HF
cells the microplates were swiftly turned to discard the medium
before adding the solvent mixture. The microplates were gently
shaken in the dark for 30 min and absorbance at 570 and 630 nm
(background) was measured with an ELISA plate reader. All
experiments were carried out in quadruplicate; the percentage
of viable cells was calculated as mean ( SD with controls set to
100%.
1
1659, 1600, 1566, 1457, 1365, 1259, 1105, 1016, 799 cm-1; H
NMR (CDCl3, 300 MHz) δ 1.28 (d, J = 6.8 Hz, 3 H), 1.42 (s,
9 H), 2.04 (s, 3 H), 2.66 (s, 3 H), 4.06 (ddq, J=6.8 Hz, J=8.3 Hz,
J=8.7 Hz, 1 H), 4.37 (dd, J=16.5 Hz, J=8.7 Hz, 1 H), 4.57 (dd,
J=16.5 Hz, J=8.3 Hz, 1 H), 6.82 (s, 1 H); 13C NMR (CDCl3, 75
MHz) δ 10.6, 19.8, 23.6, 29.6, 37.1, 62.3, 80.2, 116.9, 123.5,
130.2, 132.9, 134.4, 157.9, 163.9, 183.6, 187.6; m/z (EI) 258 (56)
[Mþ - C4H8], 216 (37), 159 (8), 111 (20), 91 (59), 57 (100). Anal.
Calcd for C19H22O4: C, 72.59; H, 7.05. Found: C, 72.61; H, 7.06.
(-)-7-Hydroxy-1,5,8-trimethyl-1,2-dihydronaphtho[2,1-b]furan-
6,9-dione (-)-1. A solution of ether (S)-14 (40 mg, 0.13 mmol) in
CH2Cl2 (10 mL) was cooled to 0 °C, treated with TFA (0.5 mL),
and stirred at this temperature for 1 h. The volatiles were re-
moved in vacuum, and the crude product was purified by
column chromatography (cyclohexane/ethyl acetate 4:1, silica
gel 60) to yield (-)-1 (26 mg, 0.1 mmol, 77%) as a yellow solid of
mp 155-156 °C (lit.9 156-157 °C); Rf 0.6 (cyclohexane/ethyl
acetate 1:1); [R]24 -151.5 (c 0.1, CHCl3) [lit.9 [R]29 -336
D
D
(c 0.01, CHCl3)]; IR (ATR) νmax 3318, 2956, 2923, 2840, 1640,
1
1598, 1560, 1464, 1385, 1351, 1259, 1094, 1020, 796 cm-1; H
NMR (CDCl3, 300 MHz) δ 1.27 (d, J = 6.9 Hz, 3 H), 2.01 (s,
3 H), 2.69 (s, 3 H), 4.12 (ddq, J=6.9 Hz, J=8.1 Hz, J=8.7 Hz,
1 H), 4.39 (dd, J=16.5 Hz, J=8.7 Hz, 1 H), 4.59 (dd, J=16.5
Hz, J=8.1 Hz, 1 H), 6.79 (s, 1 H), 7.71 (br, 1 H, OH); 13C NMR
(CDCl3, 75 MHz) δ 8.3, 19.7, 23.8, 37.1, 80.4, 116.2, 117.7,
120.7, 128.8, 131.2, 134.2, 153.8, 165.6, 180.5, 186.3; m/z (EI)
258 (67) [Mþ], 216 (42), 159 (12), 111 (16), 91 (43), 57 (100);
HRMS (EI) calcd for C15H15O4 [M þ 1]þ 259.0970, found
259.0965.
Cell Lines and Culture Conditions. The HL-60 cells were
obtained from the German Collection of Biological Material
(DSMZ), Braunschweig, the 518A2 cells from the Department
of Oncology and Hematology of the Martin-Luther University
Halle, Germany, the KB-V1/Vbl and the MCF-7/Topo cells
from the Institute of Pharmacy of the University Regensburg,
Germany, and the human foreskin (HF) fibroblasts as well as
the HT-29 cells from the University Hospital Erlangen, Germany.
The multidrug resistant cells were bred by repeated treatment
with vinblastine sulfate (KB-V1/Vbl: 340 nM), topotecan hy-
drochloride (MCF-7/Topo: 550 nM), or colchicine (HT-29/
Colc: 62.5 nM). The HL-60 and HT-29 cells were grown in
Acknowledgment. This work was supported by a grant
from the Deutsche Forschungsgemeinschaft (Scho 402/8-3).
Supporting Information Available: Syntheses and properties
of compounds 6 - 10, optical rotations of compounds (R)-1,
(R)-2, (R)-4, (R)-14, 1H and 13C NMR spectra of compounds 1
- 14; chiral GC of 10, inhibitory concentrations IC50(24 h/48 h)
of (()-/(-)-/(þ)-1 and 15. This material is free of charge via the
Internet at http://pubs.acs.org.
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6218 J. Org. Chem. Vol. 75, No. 18, 2010