Synthesis and evaluation of derrubone and select analogues

Article abstract of DOI:10.1021/jo702366g

(Chemical Equation Presented) Recently, we reported that the natural product derrubone exhibits Hsp90 inhibitory activity. Due to its unique architectural scaffold and proposed rapid assembly, the synthesis of this natural product was pursued with the aim of identifying structure-activity relationships. Synthesis of the natural product was accomplished in eight highly convergent steps, which led to a facile method for the construction of related compounds. Biological evaluation of derrubone and its analogues identified several compounds that exhibit low micromolar inhibitory activity against breast and colon cancer cell lines.

Full text of DOI:10.1021/jo702366g

Synthesis and Evaluation of Derrubone and Select Analogues
Jedidiah M. Hastings, M. Kyle Hadden, and Brian S. J. Blagg*
Department of Medicinal Chemistry, The UniVersity of Kansas, 1251 Wescoe Hall DriVe, Malott 4070,
Lawrence, Kansas 66045-7563
bblagg@ku.edu
ReceiVed NoVember 1, 2007
Recently, we reported that the natural product derrubone exhibits Hsp90 inhibitory activity. Due to its
unique architectural scaffold and proposed rapid assembly, the synthesis of this natural product was
pursued with the aim of identifying structure-activity relationships. Synthesis of the natural product
was accomplished in eight highly convergent steps, which led to a facile method for the construction of
related compounds. Biological evaluation of derrubone and its analogues identified several compounds
that exhibit low micromolar inhibitory activity against breast and colon cancer cell lines.
Introduction
The 90 kDa heat shock proteins (Hsp90) continue to emerge
as promising chemotherapeutic targets for the development of
anti-tumor agents.1 As a molecular chaperone, Hsp90 is
responsible for the maturation of nascent polypeptides into their
biologically active, three-dimensional structures.2,3 This protein
folding process is dependent upon the hydrolysis of ATP, which
provides the energy necessary for conformational reorientation
of the polypeptide substrate and client protein release.4 Studies
have revealed two nucleotide binding pockets in the Hsp90
protein, one at the N-terminus and the other in the C-terminal
domain.5,6 Only the N-terminal ATP binding pocket manifests
ATPase activity, while the C-terminus appears to exhibit
allosteric properties.7
FIGURE 1. Derrubone.
pendent upon the Hsp90 protein folding machinery. In col-
laboration with the Matts research laboratory, we recently
reported an assay that was capable of identifying inhibitors of
both the N- and C-terminal ATP binding pockets.8 This assay
was optimized to identify small molecules that prohibit the
maturation and activation of firefly luciferase in rabbit reticu-
locyte. High-throughput screening of a small library of natural
products with this assay resulted in the identification of
derrubone as an Hsp90 inhibitor. Subsequent studies in vitro
determined that derrubone exhibits a novel mechanism of action
for Hsp90 inhibition.9
Although the N-terminal ATP binding site is the only pocket
that manifests ATPase activity, inhibitors of both nucleotide
binding pockets induce the degradation of client proteins de-
(1) Blagg, B. S. J.; Kerr, T. D. Med. Res. ReV. 2006, 26, 310-338.
(2) Chiosis, G.; Vilenchik, M.; Kim, J.; Solit, D. Drug DiscoVer Today
2004, 9, 881-886.
Derrubone (Figure 1) is a prenylated isoflavone that was first
isolated from Derris robusta in 1972.10 To date, no biological
activity has been reported for this molecule, and only two
(3) Neckers, L.; Neckers, K. Expert Opin. Emerging Drugs 2005, 10,
137-149.
(4) Pratt, W. B.; Toft, D. O. Exp. Biol. Med. 2003, 228, 111-133.
(5) Marcu, M. G.; Schulte, T. W.; Neckers, L. J. Natl. Cancer Inst. 2000,
92, 242-248.
(8) Galam, L.; Hadden, M. K.; Ma, Z.; Ye, Q.-Z.; Yun, B.-G.; Blagg,
B. S. J.; Matts, R. L. Bioorg. Med. Chem. 2007, 15, 1939-1946.
(9) Hadden, M. K.; Galam, L.; Matts, R. L.; Blagg, B. S. J. J. Nat. Prod.
In Press.
(10) East, A. J.; Ollis, W. D.; Wheeler, R. E. J. Chem. Soc. C: Org.
1969, 3, 365-374.
(6) Marcu, M. G.; Chadli, A.; Bouhouche, I.; Catelli, M.; Neckers, L. J.
Biol. Chem. 2000, 276, 37181-37186.
(7) Allan, R. K.; Mok, D.; Ward, B. K.; Ratajczak, T. J. Biol. Chem.
2006, 281, 7161-7171.
10.1021/jo702366g CCC: $40.75 © 2008 American Chemical Society
Published on Web 12/22/2007
J. Org. Chem. 2008, 73, 369-373
369

Hastings et al.
SCHEME 2. Synthesis of the Isoflavone Core
synthetic routes have been disclosed, both of which required
14 linear steps.11,12 Therefore, our goal was to provide a succinct
synthetic protocol for this natural product from which the
preparation of analogues could be easily accomplished and
structure-activity relationships revealed.
Results and Discussion
Synthesis of Derrubone. Retrosynthetic analysis of derru-
bone presented three primary disconnections that provided ample
opportunity for diversification without a lengthy protection-
deprotection strategy (Scheme 1). Installation of the aryl ketone
moiety via electrophilic aromatic substitution followed by
cyclization allowed access to the isoflavone core in three steps
from commercially available starting materials. Literature
precedence has shown that a variety of nitriles can serve as
precursors to the aryl ketone, thus allowing for introduction of
structural diversity.13,14
we followed a procedure recently reported by our laboratory17
that allowed for C-prenylation of phenols. Following this
protocol, the 7-hydroxyl of 6 was selectively allylated with allyl
bromide in the presence of potassium carbonate in a solution
of DMF (Scheme 3). Subsequent Claisen rearrangement led to
a 3:1 mixture of regioisomers, 8 and 9, with the unnatural
regioisomer 8 predominating. The regioisomers were inseparable
at this stage via flash chromatography. Therefore, the hydroxyl
groups of the crude mixture were masked as acetates 10 and
11, and the prenyl group was introduced via cross-metathesis
with 2-methyl-2-butene using Grubbs’ second generation cata-
lyst.18 Hydrolysis of acetates 12 and 13 afforded the unnatural
8-prenyl isomer 14 of derrubone in good yield. Unfortunately,
the yield of derrubone was less than desirable (<5%) and
required investigation of alternate methods for regioselective
prenylation of the aromatic ring.
SCHEME 1. Retrosynthetic Analysis of Derrubone
To avoid formation of the undesired regioisomer, the 7-hy-
droxyl of 6 was protected as the methoxy methyl ether to afford
phenol 15 (Scheme 4). Subsequent allylation of the free phenol
followed by Claisen rearrangement at 160 °C gave the desired
6-allyl isoflavone 16 in good yield. As before, cross-metathesis
with 2-methyl-2-butene provided the prenyl moiety. Completion
of the natural product was ultimately accomplished upon
hydrolysis of the methoxymethyl ether to give derrubone in eight
linear steps and overall yield of 16%.
Synthesis of Derrubone Analogues. Upon completion of the
natural product, the synthetic route was utilized for the prepara-
tion of analogues with the purpose of revealing structure-
activity relationships (Figure 2). In choosing constituents of the
library, we aimed to probe both the size and nature of the
binding pocket into which the 3-aryl side chain projects.
From the isoflavone core, two routes were proposed for
attachment of the prenyl side chain: (1) nucleophilic addition
to 1-bromo-3-methyl-2-butene, or (2) Claisen rearrangement and
subsequent olefin cross-metathesis. Both routes were envisioned
to produce derrubone via a succinct method that is amenable
to a diversity-oriented protocol for the rapid assembly of
derrubone analogues and elucidation of the first structure-
activity relationships.
In practice, the isoflavone backbone was obtained in three
steps from (3,4-methylenedioxy)phenylacetonitrile via electro-
philic aromatic substitution enlisting a zinc chloride catalyst.
Subsequent hydrolysis of the resulting imine (4) afforded phenyl
ketone 5 in good yield (Scheme 2). Utilizing the cyclization
procedure reported by Pelter and Foot,15 the isoflavone scaffold
(6) was obtained in excellent yield upon exposure to DMF under
Bischler-Napieralski-type conditions16 without protection of the
free phenols.
Direct prenylation at the 6-position of the isoflavone backbone
proved problematic as the 7-O-prenyl derivative was the
predominant product obtained under various conditions. To
circumvent formation of the undesired O-alkylated products,
(11) Hossain, M. M.; Kawamura, Y.; Yamashita, K.; Tsukayama, M.
Tetrahedron 2006, 62, 8625-8635.
FIGURE 2. A Library of derrubone analogues.
(12) Jain, A. C.; Jain, S. M. Tetrahedron 1972, 28, 5063-5067.
(13) Hakala, U.; Waehaelae, K. Tetrahedron Lett. 2006, 47, 8375-8378.
(14) Bolek, D.; Guetschow, M. J. Heterocycl. Chem. 2005, 42, 1399-
1403.
(15) Pelter, A.; Foot, S. Synthesis 1976, 326.
(16) Bischler, A.; Napieralski, B. Ber. 1893, 26, 1891-1903.
(17) Burlison, J. A.; Neckers, L.; Smith, A. B.; Maxwell, A.; Blagg, B.
S. J. J. Am. Chem. Soc. 2006, 128, 15529-15536.
(18) Scholl, M.; Ding, S.; Lee, C. W.; Grubbs, R. H. Org. Lett. 1999, 1,
953-956.
370 J. Org. Chem., Vol. 73, No. 2, 2008

Synthesis and EValuation of Derrubone and Analogues
SCHEME 3. Synthesis of the 8-Prenyl Regioisomer
SCHEME 4. Completed Synthesis of Derrubone
SCHEME 5. Synthesis of C3 Analogues
To explore the necessity of having an aromatic substituent
at C3, we proposed replacement of the aryl group with chlorine.
In addition, it is unknown whether one or both of the oxygen
atoms of the dioxolane serve as hydrogen-bond acceptors with
the putative binding pocket. To elucidate the role of the 3′- and
4′-oxygen atoms, a series of analogues were sought beginning
with an unsubstituted phenyl side chain and extending to a 3′,4′-
dichloro analogue. Substitution of the prenyl side chain was
proposed to explore the effects of C6 modification. As proposed,
alteration of the synthetic route was not required to obtain the
desired analogues.
Beginning from commercially available nitriles, aryl ketones
18a-e were obtained in good yields (Scheme 5). Cyclization
under Bischler-Napieralski conditions as described earlier also
worked well for these substrates and gave isoflavones 19a-e,
which were subsequently protected before O-allylation. Claisen
rearrangement of the allylic ethers gave the C-allylated products,
which underwent olefin cross-metathesis in the presence of
Grubbs’ second generation catalyst and 2-methyl-2-butene to
afford the prenyl side chain. Removal of the methoxy methyl
ether afforded analogues 22a-e in overall yields comparable
to that of the natural product.
SCHEME 6. Synthesis of C6 Analogues
Replacement of the prenyl moiety was readily accomplished
via cross-metathesis between 16 and the requisite olefin (Scheme
6). Subsequent hydrolysis of the methoxy methyl ether as
described previously afforded analogues 23a,b (35-45%).
Growth Inhibitory Activity by Derrubone and Analogues.
Data shown in Table 1 present the anti-proliferative activity of
derrubone and analogues against MCF-7 (breast) and HCT-116
(colon) cancer cell lines. The structure-activity relationships
revealed that modification at C6 has a dramatic effect on
inhibitory activity. When the prenyl moiety is removed or
replaced by a more polar functionality such as 6 or 23a,
respectively, activity is significantly decreased. When a lipo-
philic side chain is present in this position as in compounds 9
and 23b, anti-proliferative activity is comparable to that of the
natural product. Interestingly, when the prenyl side chain is
transposed from the 6-position to the 8-position (14), a small
increase in activity was observed against both cell lines.
Replacement of the aromatic ring at C3 with a chlorine atom
(22e) resulted in almost complete loss of activity (IC50 ) 91.6
µM in MCF-7 and IC50 >100 µM in HCT-116 cells). Removal
J. Org. Chem, Vol. 73, No. 2, 2008 371

Hastings et al.
of the 3′,4′-methylenedioxy moiety (22d) did not result in
substantial effects in cell growth assays. However, replacement
of the aromatic side chain with the 3′,4′-dichloro analogue
resulted in increased anti-proliferative activity, suggesting that
electron-withdrawing substituents at these positions could
improve activity. Interestingly, substitution at either C3′ or C4′
of the aromatic side chain greatly influenced activity. The 4′-
methoxy analogue (22a) demonstrated a 2-fold increase in
inhibitory activity versus derrubone, whereas a single C3′-
methoxy (22b) completely abolished anti-proliferation activity
at concentrations below 100 µM. When considered in tandem
with the data obtained from the unsubstituted phenyl analogue
(entry 22d), these results clearly suggest that substitution at C3′
is detrimental to anti-proliferative activity, while C4′ substitution
is beneficial but not essential.
preparation of derrubone analogues in an effort to elucidate
structure-function relationships for derrubone via evaluation
against breast and colon cancer cell lines. Anti-proliferative data
from two distinct cancer cell lines were obtained, identifying
several compounds that exhibit increased activity over the
natural product. In addition, these two compounds induced the
degradation of Hsp90-dependent client proteins in human breast
cancer cells, supporting their Hsp90 inhibitory activity in vitro.
Continued synthesis of derrubone analogues is currently un-
derway to identify compounds that exhibit optimal inhibitory
activity.
Experimental Section
2-(2-(Benzo[d][1,3]dioxol-5-yl)-1-iminoethyl)benzene-1,3,5-
triol (4).12 A solution of phloroglucinol (5 g, 39.7 mmol) and (3,4-
methylenedioxy)phenylacetonitrile (7 g, 43.6 mmol) in anhydrous
Et2O was prepared under an Ar atmosphere before zinc(II) chloride
(1.08 g, 7.9 mmol) was added to the mixture. The solution was
cooled to 0 °C. HCl gas was bubbled through the reaction mixture
for 10 min. The reaction was allowed to slowly warm to rt and
was stirred for 14 h. The solvent was decanted from the orange
oil. The residue was rinsed with cold Et2O then dissolved in EtOAc.
The organic layer was washed with saturated aqueous NaHCO3,
dried (Na2SO4), filtered, and evaporated to give 4 (9.7 g, 85%) as
a yellow, amorphous solid: 1H NMR (DMSO-d6, 400 MHz) δ 11.64
(s, 1H), 11.40 (br s, 1H), 10.73 (s, 1H), 6.85 (d, 2H, J ) 7.96 Hz),
6.81 (s, 1H), 6.03 (s, 2H), 5.98 (s, 2H), 4.42 (s, 2H) ppm.
2-(Benzo[d][1,3]dioxol-5-yl)-1-(2,4,6-trihydroxyphenyl)etha-
none (5).12 Compound 4 (9.7 g, 33.7 mmol) was suspended in
aqueous HCl (50% v/v, 100 mL). The suspension was warmed to
reflux and stirred for 3 h. The resulting solid was filtered and
washed with water and cold Et2O to afford 5 (9.2 g, 95%) as a
white powder: 1H NMR (DMSO-d6, 400 MHz) δ 12.22 (s, 1H),
10.40 (s, 1H), 6.83 (s, 1H), 6.80 (d, 1H, J ) 5.32 Hz), 6.68 (d, 1H,
J ) 7.92 Hz), 5.97 (s, 2H), 5.82 (s, 2H), 4.25 (s, 2H) ppm; 13C
(DMSO-d6, 125 MHz) δ 202.6, 164.8, 164.5, 146.9 (2C), 145.7
(2C), 129.5, 122.6, 109.8, 107.9, 103.6, 100.6, 94.7, 48.5 ppm; IR
(film) νmax 3297, 3202, 3082, 3026, 2920, 2853, 1655, 1612, 1502,
1371, 1323, 1250, 1197, 1036 cm-1; HRMS (ESI+) m/z 289.0700
([M + H]+, C15H13O6, requires 289.0712).
3-(Benzo[d][1,3]dioxol-5-yl)-5,7-dihydroxy-4H-chromen-4-
one (6).12,19 Boron trifluoride etherate (1.7 mL, 2 g, 13.9 mmol)
was added dropwise to a solution of 5 (1 g, 3.5 mmol) in anhydrous
DMF (11 mL) under an Ar atmosphere at rt. The solution was
stirred for 10 min before the dropwise addition of CH3SO2Cl (0.8
mL, 1.18 g, 10.5 mmol). The resulting solution was warmed to 80
°C and stirred for 2 h. The reaction was cooled to rt before H2O
(25 mL) was added. The aqueous mixture was extracted three times
with EtOAc. The combined organic extracts were washed with
saturated aqueous NaCl, dried (Na2SO4), filtered, and evaporated
under reduced pressure. The residue was purified by column
chromatography (SiO2, 96:4 CH2Cl2/MeOH) to afford 6 (0.83 g,
80%) as a pale yellow, amorphous solid: 1H NMR (DMSO-d6,
400 MHz) δ 12.89 (s, 1H), 10.91 (s, 1H), 8.38 (s, 1H), 7.14 (d,
1H, J ) 1.64 Hz), 7.02 (m, 2H), 6.40 (d, 1H, J ) 2.16 Hz), 6.24
(d, 1H, J ) 2.08 Hz), 6.06 (s, 2H) ppm; 13C (DMSO-d6, 125 MHz)
δ 179.9, 164.3, 162.0, 157.5, 154.6, 147.1 (2C), 124.4, 122.6, 121.9,
109.4, 108.2, 104.4, 101.1, 99.0, 93.7 ppm; HRMS (ESI+) m/z
299.0564 ([M + H]+, C16H11O6, requires 299.0556).
TABLE 1. Anti-Proliferative Activities and Derrubone and
Analogues
entry
(IC50, µM)
MCF-7
>100a
14.1 ( 1.7
10.6 ( 0.7
5.5 ( 0.4
>100
7.3 ( 2.5
12.3 ( 1.2
91.6 ( 1.1
62.6 ( 4.1
12.6 ( 0.9
11.9 ( 0.6
HCT-116
6
9
14
>100
14.2 ( 1.8
10.2 ( 0.8
7.3 ( 1.8
>100
22a
22b
22c
22d
22e
23a
23b
derrubone
5.2 ( 0.4
13.9 ( 1.3
>100
55.5 ( 11.3
10.8 ( 0.9
13.7 ( 3.4
a Values represent mean ( standard error for at least two separate
experiments performed in triplicate.
On the basis of the results obtained from anti-proliferative
studies, two compounds were chosen that contained modifica-
tions at each location, 14 and 22c, and tested for their ability to
induce Hsp90 client protein degradation (Figure 3). Both
compounds caused a decrease in Her2 and Raf levels, two well-
characterized proteins that depend upon Hsp90. Since actin is
not a Hsp90 substrate, it was used as a negative control to verify
that all protein levels were not affected by these inhibitors.
FIGURE 3. Western blot analysis of 14 (top) and 22c (bottom) in
MCF-7 cells (µM). Geldanamycin (500 nM) was used as the positive
control.
3-(Benzo[d][1,3]dioxol-5-yl)-5-hydroxy-7-(methoxymethoxy)-
4H-chromen-4-one (15). Compound 6 (1 g, 3.4 mmol) and NaH
(160 mg, 4.8 mmol, 60% disp. w/w) were dissolved in DMF (10
mL) and stirred for 10 min at rt. Chloromethyl methoxy ether (0.65
mL, 4.8 mmol) was added dropwise, and the mixture was stirred
Conclusion
In conclusion, we have completed the total synthesis of
derrubone in eight steps and an overall yield of 16%. In addition,
we have successfully applied this synthetic strategy toward the
(19) Baker, W.; Chadderton, J.; Harborne, J. B.; Ollis, W. D. J. Chem.
Soc. 1953, 1852-1860.
372 J. Org. Chem., Vol. 73, No. 2, 2008

Synthesis and EValuation of Derrubone and Analogues
for 9 h at rt. The reaction was quenched by the addition of saturated
aqueous NaHCO3 (50 mL) to the mixture. The resulting slurry was
further diluted with H2O (50 mL) and extracted three times with
EtOAc. The combined organic layers were washed with saturated
aqueous NaCl, dried (Na2SO4), filtered, and evaporated. The residue
was purified by column chromatography (SiO2, 4:1 hexanes/EtOAc)
to give 15 (870 mg, 75%) as a pale yellow, amorphous solid: 1H
NMR (CDCl3, 400 MHz) δ 12.79 (s, 1H), 7.89 (s, 1H), 7.07 (d,
1H, J ) 1.64 Hz), 6.94 (m, 2H), 6.60 (d, 1H, J ) 2.24 Hz), 6.53
(d, 1H, J ) 2.20 Hz), 6.02 (s, 2H), 5.26 (s, 2H), 3.52 (s, 3H) ppm;
13C NMR (CDCl3, 125 MHz) δ 180.8, 163.1, 162.6, 157.8, 153.0,
147.9, 147.8, 124.3, 123.8, 122.5, 109.6, 108.6, 106.9, 101.3, 100.2,
94.3, 94.2, 56.5 ppm; IR (film) νmax 3078, 2904, 1655, 1612, 1574,
1504, 1489, 1435, 1366, 1300, 1248, 1146, 1078, 1036, 1001, 939,
920, 822 cm-1; HRMS (ESI+) m/z 343.0801 ([M + H]+, C18H15O7,
requires 343.0818).
6-Allyl-3-(benzo[d][1,3]dioxol-5-yl)-5-hydroxy-7-(methoxy-
methoxy)-4H-chromen-4-one (16). DMF (1.0 mL) was added to
a mixture of NaH (40 mg, 0.52 mmol) and 15 (89 mg, 0.26 mmol).
The resulting suspension was stirred for 10 min. Allyl bromide (63
mg, 0.52 mmol) was added, and the reaction was stirred for 10 h
at rt. The reaction was quenched by addition of H2O. The solvent
was evaporated under reduced pressure, and the oily residue was
dissolved in EtOAc. The organic layer was extracted twice with
H2O, washed with saturated aqueous NaCl, and dried (Na2SO4).
The solvent was decanted from the drying agent and evaporated
under reduced pressure. The yellow solid was dissolved in DMF
(1 mL) and warmed to 160 °C in a focused microwave reactor at
300 W for 45 min. The solution was then diluted with EtOAc. The
mixture was washed three times with H2O. The organic layer was
then washed with saturated aqueous NaCl, dried (Na2SO4), filtered,
and evaporated under reduced pressure. The residue was purified
by column chromatography (SiO2, 3:1 hexanes/acetone) to give 16
(51 mg, 51%) as a pale yellow, amorphous solid: 1H NMR (CDCl3,
400 MHz) δ 12.99 (s, 1H), 7.88 (s, 1H), 7.06 (d, 1H, J ) 1.60
Hz), 6.79 (m, 2H), 6.68 (s, 1H), 6.00 (m, 3H), 5.30 (s, 2H), 5.03
(m, 2H), 3.51 (m, 5H) ppm; 13C NMR (CDCl3, 125 MHz) δ 178.0,
157.9, 156.6, 153.5, 150.4, 147.6, 145.1, 133.2, 121.8, 121.0, 119.7,
111.9, 109.1, 107.2, 106.9, 105.4, 103.8, 98.5, 91.6, 89.2, 53.6,
23.8 ppm; IR (film) νmax 3078, 2997, 2959, 2908, 2829, 1651, 1612,
1580, 1502, 1487, 1439, 1408, 1367, 1323, 1294, 1275, 1252, 1223,
1155, 1130, 1072, 1045, 987, 943, 920, 829 cm-1; HRMS (ESI+)
m/z 383.1121 ([M + H]+, C21H19O7, requires 383.1131).
(Na2SO4), filtered, and evaporated under reduced pressure. The
resulting solid was purified by chromatography (SiO2, 6:1 hexanes/
EtOAc) to afford 1 (9 mg, 95%) as a colorless, amorphous solid:
1H NMR (acetone-d6, 500 MHz) δ 13.27 (s, 1H), 9.75 (br s, 1H),
8.20 (s, 1H), 7.16 (d, 1H, J ) 1.65 Hz), 7.08 (dd, 1H, J ) 8.03,
1.65 Hz), 6.92 (d, 1H, J ) 8.00 Hz), 6.51 (s, 1H), 6.06 (s, 2H),
5.29 (tt, 1H, J ) 7.10, 1.35 Hz), 3.38 (d, 2H, J ) 7.20 Hz), 1.79
(s, 3H), 1.66 (s, 3H) ppm; 13C (acetone-d6, 125 MHz) δ 180.6,
161.8, 159.7, 155.9, 153.6, 147.6, 147.5, 130.9, 125.1, 122.8, 122.5,
122.2, 111.6, 109.6, 108.0, 105.1, 101.3, 93.0, 25.0, 21.1, 17.0 ppm;
IR (film) νmax 3580, 3524, 3433, 3412, 3005, 2964, 2924, 1701,
1620, 1580, 1433, 1365, 1321, 1250, 1229, 1094, 1038, 824 cm-1
;
HRMS (ESI+) m/z 367.1174 ([M + H]+, C21H19O6, requires
367.1182).
Anti-Proliferation Assays. MCF-7 cells were maintained in a
1:1 mixture of Advanced DMEM/F12 (Gibco) supplemented with
non-essential amino acids, L-glutamine (2 mM), streptomycin (500
µg/mL), penicillin (100 units/mL), and 10% FBS. HCT-116 cells
were maintained in Iscove’s Modified Dulbecco’s Medium (Sigma)
supplemented with L-glutamine (2 mM), streptomycin (500 µg/mL),
penicillin (100 units/mL), and 10% FBS. Cells were grown to
confluence in a humidified atmosphere (37 °C, 5% CO2), seeded
(2000/well, 100 µL) in 96-well plates, and allowed to attach
overnight. Compound or GDA at varying concentrations in DMSO
(1% DMSO final concentration) was added, and cells were returned
to the incubator for 72 h. At 72 h, the number of viable cells was
determined using an MTS/PMS cell proliferation kit (Promega) per
the manufacturer’s instructions. Cells incubated in 1% DMSO were
used as 100% proliferation, and values were adjusted accordingly.
IC50 values were calculated from separate experiments performed
in triplicate using GraphPad Prism.
Western Blot Analysis of 14 and 22c. MCF-7 cells were
cultured as described above and treated with various concentrations
of drug or GDA in DMSO (1% DMSO final concentration) for 24
h. The cells were collected in cold PBS and lysed in RIPA lysis
buffer containing 1 mM PMSF, 2 mM sodium orthovanadate, and
protease inhibitors on ice for 1 h. Lysates were clarified at 14 000g
for 10 min at 4 °C. Protein concentrations were determined using
the Pierce BCA protein assay kit per the manufacturer’s instructions.
Equal amounts of protein (20 µg) were electrophoresed under
reducing conditions, transferred to a nitrocellulose membrane, and
immunoblotted with the corresponding specific antibodies. Mem-
branes were incubated with an appropriate horseradish peroxidase
labeled secondary antibody, developed with a chemiluminescent
substrate, and visualized.
Derrubone (1). Grubbs’ second generation catalyst (1 mg, 0.0012
mmol) was added to a solution of 16 (10 mg, 0.026 mmol) in
CH2Cl2 (0.1 mL) and 2-methyl-2-butene (0.3 mL) under an Ar
atmosphere. The solution was stirred for 24 h at rt. The mixture
was concentrated under reduced pressure to give a green solid. The
solid was dissolved in MeOH (0.5 mL) and transferred to a screw-
cap vial. Concentrated HCl (50 µL) was added to the solution. The
mixture was warmed to 70 °C and stirred for 1 h. The reaction
was quenched by the addition of a saturated aqueous NaHCO3
solution (5 mL). The aqueous solution was extracted with EtOAc.
The organic layer was washed with saturated aqueous NaCl, dried
Acknowledgment. The authors gratefully acknowledge
support of this project by NIH R01 CA114393 and R01
CA120458. M.K.H. is a KU Cancer Center Postdoctoral Fellow.
Supporting Information Available: Experimental procedures
and characterization for all compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
JO702366G
J. Org. Chem, Vol. 73, No. 2, 2008 373