Investigating the role of stereochemistry in the activity of anticancer acylfulvenes: Synthesis, reductase-mediated bioactivation, and cellular toxicity

Article abstract of DOI:10.1021/jm051104t

Acylfulvenes comprise a family of semisynthetic natural product derivatives with potent antitumor activities. Previous studies indicated that acylfulvenes are bioactivated by NADPH-dependent alkenal/one reductase (AOR), presumably generating intermediates

Full text of DOI:10.1021/jm051104t

J. Med. Chem. 2006, 49, 2593-2599
2593
Investigating the Role of Stereochemistry in the Activity of Anticancer Acylfulvenes: Synthesis,
Reductase-Mediated Bioactivation, and Cellular Toxicity
Jiachang Gong,†,‡ James F. Neels,†,§ Xiang Yu,| Thomas W. Kensler,| Lisa A. Peterson,‡, and Shana J. Sturla*,†,‡
Department of Medicinal Chemistry, College of Pharmacy, UniVersity of Minnesota, Minneapolis, Minnesota 55455, The Cancer Center,
UniVersity of Minnesota, Minneapolis, Minnesota 55455, Department of Chemistry, UniVersity of Minnesota, Minneapolis, Minnesota 55455,
Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland 21205, and School of Public Health, UniVersity of Minnesota,
Minneapolis, Minnesota 55455
ReceiVed NoVember 1, 2005
Acylfulvenes comprise a family of semisynthetic natural product derivatives with potent antitumor activities.
Previous studies indicated that acylfulvenes are bioactivated by NADPH-dependent alkenal/one reductase
(AOR), presumably generating intermediates with the capacity to alkylate cellular targets, such as DNA,
proteins, and glutathione. This process is thought to induce apoptosis, and the chemical and biochemical
pathways involved are topics of current investigation. In this study, four acylfulvene analogues were
synthesized: (-)-acylfulvene, (+)-acylfulvene, (-)-(hydroxymethyl)acylfulvene, and (+)-(hydroxymethyl)-
acylfulvene. These compounds were synthesized by a chiral-resolution method, described for the first time
in this report, and by asymmetric synthesis using a method formally demonstrated previously. Cell toxicity
studies indicate a positive correlation between AOR level and acylfulvene sensitivity. The absolute
configuration of acylfulvene analogues has a significant influence on cytotoxicity. (-)-(Hydroxymethyl)-
acylfulvene is 25 times more potent than (+)-(hydroxymethyl)acylfulvene in cells transfected with an AOR
overexpression vector. Based on kinetic parameters, the rates of AOR-mediated activation are more strongly
dependent on acylfulvene substitution than on absolute stereochemistry. These data support the role of AOR-
mediated metabolism and indicate the involvement of other stereochemically dictated pathways, such as
transport and biomolecule binding, in contributing to the cytotoxicity of acylfulvenes.
Chart 1. Structures of Natural Illudins and Semisynthetic
Derivatives, Acylfulvenes
Introduction
DNA-alkylating agents are among the most effective classes
of drugs for the treatment of cancer.1 Due to their low selectivity
and high toxicity, extensive research has focused on the
development of antitumor agents with increased selectivity and
decreased toxicity to normal cells. Illudins are mushroom-
derived sesquiterpene natural products. Illudin S (1) and M (2)
(Chart 1) are toxic to a number of tumor cells, but have low
therapeutic indices as antitumor compounds.2-4 Acylfulvenes,
semisynthetic derivatives of illudin S, are selectively cytotoxic
to certain tumor cells and thus are promising chemotherapeutic
agents.5-10 One member of this family, (-)-(hydroxymethyl)-
acyfulvene (4, (-)-HMAF; Chart 1), is currently in phase II
clinical trials for a number of cancers.3;6;10-12 Acylfulvenes are
alkylating agents that covalently bind to biological macromol-
ecules, including DNA and protein.13;14 It was observed that
60-70% of HMAF that was taken up by cells binds to
macromolecules.8;15 The selective toxicity profile of acylfulvenes
may originate from energy-dependent selective uptake mech-
anisms, from differences in metabolic bioactivation to reactive
species, and from differences in DNA and protein alkylation.13;16
Illudins and acylfulvenes can alkylate cellular targets by
different chemical pathways (Scheme 1): Direct nucleophilic
addition of cellular nucleophiles by conjugate addition to the
8-position, or enzymatic reduction of the carbon-carbon double
bond of the R,â-unsaturated ketone, leading to reactive inter-
mediate 5. Both pathways may lead to the formation of adducts
of cellular macromolecules and could be involved in the
induction of apoptosis.13;14;17
(-)-HMAF undergoes NADPH-dependent metabolism to
generate 6, and this process is catalyzed by rat liver cytosol as
well as recombinant rat alkenal/one oxidoreductase (AOR).18
AOR reduces a variety of R,â-unsaturated aldehydes and ketones
including cytotoxic and carcinogenic byproducts of lipid
peroxidation.18-20 Its cellular localization, cofactor preference
and inhibition profile are identical to the acylfulvene-reducing
enzyme that was partially characterized from rat liver cyto-
sol.13;14 In addition, a positive correlation was observed between
(-)-HMAF sensitivity and levels of AOR expression in tumor
cells.18
Acylfulvenes are obtained by semisynthesis from isolated
natural products and have (R)-configurations at the 2-position.
(-)-Acylfulvene (3, (-)-AF; Chart 1) and (-)-HMAF are
readily obtained by treatment of illudin S with dilute H2SO4, in
the absence or presence of formaldehyde, respectively.6 Semi-
synthesis methods limit investigations of structure-activity
relationships including study of the influence of absolute
stereochemistry on biological effects. A method for the asym-
metric synthesis of acylfulvenes using a Sharpless asymmetric
dihydroxylation reaction and the allenic Pauson-Khand type
* To whom correspondence should be addressed. E-mail: sturl002@
umn.edu. Phone: 612-626-0496. Fax: 612-624-0139.
† Department of Medicinal Chemistry, University of Minnesota.
‡ The Cancer Center, University of Minnesota.
§ Department of Chemistry, University of Minnesota.
| Johns Hopkins Bloomberg School of Public Health.
School of Public Health, University of Minnesota.
10.1021/jm051104t CCC: $33.50 © 2006 American Chemical Society
Published on Web 03/23/2006

2594 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8
Gong et al.
Scheme 1. Chemical Routes for Acylfulvene Metabolism and Adduct Formation
Scheme 2. Brummond Method for the Racemic Synthesis of
Chart 2. Structures of Chiral Derivatizing Agents Used for the
Resolution of Acylfulvene Synthetic Intermediates
Acylfulvenesa 21
Scheme 3. Synthesis of Diastereomeric Esters Used for the
Chiral Resolution of Acylfulvene Synthetic Intermediatesa
a Reagents and conditions: (a) t-BuLi, TBS ether of 3-trimethylsilyl-
propyn-1-ol, THF, -78 °C, 60%; (b) CeCl3, ethynylmagnesium bromide,
THF, 0 °C, 95%; (c) Ac2O, DMAP, Et3N; (d) [CuH(PPh3)]6, toluene, H2O,
55% for two steps; (e) K2CO3, MeOH, H2O, 96%; (f) Mo(CO)6, DMSO,
toluene, 72%.
reaction was reported by Brummond and co-workers.21 Ad-
ditionally, the total synthesis of either enantiomer of acylfulvenes
was accomplished recently by McMorris and co-workers
utilizing the Padwa carbonyl ylide 1,3-dipolar cycloaddition
methodology and enantiomerically pure starting materal.22 In
MV 522 adenocarcinoma cells, the synthetic (-)-acylfulvenes
were 5-6 times more potent than the corresponding (+)-
isomers.22 The detailed mechanism of how stereochemistry
influences toxicity is not known.
a Reagents and conditions: (a) TBAF, THF, 95%; (b) respective chiral
acid, HBTU, DMAP, THF, 86%; (c) camphanic acid chloride, DMAP, THF,
88%.
In our investigations of the mechanism of action of acylful-
venes, we aim to understand the chemical origins of selective
cytotoxicity, including the relationship of absolute stereochem-
istry to biological activity, and the role of bioactivation. We
report here two synthetic approaches to obtaining both enanti-
omers of acylfulvenes. Using compounds obtained by total
synthesis, we have examined the influence of stereochemistry
on the cytotoxicity of acylfulvenes to human embryonic kidney
cells engineered to overexpress AOR. Furthermore, using
purified AOR, we disclose the first report of configuration-based
differences in the kinetics of AF and HMAF reductive activation.
derivatized with an enantiomerically pure chiral acid or acid
chloride. Three chiral reagents, (+)-methoxyphenylacetic acid
11, (-)-O-acetylmandelic acid 12, and (-)-camphanic acid
chloride 13 (Chart 2), were chosen as derivatizing agents and
their resolving efficiencies were compared. Thus, cis- and trans-
10 were treated with TBAF and independently afforded free
racemic diols cis- and trans-14. The diols were coupled with
11, 12 or 13 to afford diastereomeric esters (15-20) in 70-
90% yields (Scheme 3). The resulting diastereomeric esters 15,
16, 17, and 18 were separated by reversed phase HPLC (Figure
1, isocratic elution, 1:1 MeOH:H2O). Balancing various factors
including degree of separation, total time needed for collection,
long-term chemical stability of the compounds and cost of the
chiral derivative, 11 was the derivatizing agent of choice for
obtaining diastereomerically pure material.
Results and Discussion
Chemistry. To obtain both enantiomers of acylfulvene, we
explored two preparative approaches: chiral resolution and
asymmetric synthesis. Brummond and co-workers have reported
a concise synthesis of (()-acylfulvenes (Scheme 2) in which
the TBS-protected diol 10 is an intermediate.23 We chose to
investigate the chiral resolution of this substrate (10) because
of its chemical stability and convenient functionality. Racemic
10 was synthesized in eight steps starting from 1,1-diacetylcy-
clopropane 7.23 The secondary alcohol was deprotected and
Diastereomeric esters 15 and 18 were resolved by preparative
HPLC. 1H NMR analysis indicated high optical purities of the
resulting esters (ee > 98%). The chiral esters were hydrolyzed
in the presence of one equivalent of NaOH and the resulting
free secondary hydroxyl was reacted with TBS-Cl to give the
corresponding enantiomerically pure 10. A method for direct

Stereochemistry and AcylfulVene ActiVity
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8 2595
Scheme 5. Asymmetric Synthesis of Acylfulvenes Using the
Method Reported by Brummond and Co-workersa
a AD-R represents an asymmetric dihydroxylation reaction utilizing the
following reagents: (DHQ)2PYR, K2OsO2(OH)4, K3Fe(CN)6, K2CO3,
CH3SO2NH2. AD-â utilizes analogous conditions, replacing the DHQ ligand
with DHQD. DHQ, dihydroquinine; DHQD, dihydroquinidine.
Figure 1. Overlay of HPLC chromatograms of the diastereomeric ester
derivatives. The top trace displays cis-ester derivatives 15-17 and the
bottom trace displays trans-ester derivatives 18-20. Absolute stereo-
chemistry assignments for (R)-15 and (S)-15 were determined after
preparative separation of the compounds, hydrolysis of the ester, and
measurement of optical rotations. The (R)- and (S)-assignments for 16,
17, 18, and 19 were not determined and are indicated as a convention
based on the results from compound 15.
laboratory, the asymmetric synthesis pathway is a more viable
route and substrates for the biological studies described here
were prepared in this manner.
Biochemistry
Scheme 4. Representative Synthesis of an AF from Direct
McMorris and co-workers reported that the sensitivity of MV
522 adenocarcinoma cells to acylfulvenes was dependent on
the compound’s absolute stereochemistry.22 It was observed that
the IC50 values of (-)-acylfulvenes are 5-6 times lower than
those of the corresponding (+)-isomers after 2 or 48 h.22 These
differences in toxicities may originate from differences in
cellular uptake, enzyme-mediated metabolic bioactivation, or
subsequent interaction with biological systems. To investigate
the potential role of metabolism, we measured the sensitivity
of cells engineered to express either low or high AOR levels
and determined kinetic parameters for AOR-catalyzed NADPH-
mediated reductive bioactivation of the analogues prepared using
the synthetic routes described above.
Human embryonic kidney cells (293 cell line) were transiently
transfected either with an episomal rAOR overexpression vector
(pCEP4-rAOR) or control vector (pCEP4).18 Following hygro-
mycin selection, AOR transfected cells produced a 7-8-fold
higher expression of AOR than control cells. AOR levels were
determined spectrophotometrically.18 The cytotoxicity of each
compound to the AOR-transfected cells was assessed by
measuring cell viability via MTT assay 24 h posttreatment. Our
observations echoed the previous report that enhanced AOR
activity greatly sensitized cells to HMAF toxicity.18 The toxicity
of each compound tested in this study was 15-20-fold higher
in AOR transfected cells compared to control cells. The IC50
values of control cells toward (-)-AF and (-)-HMAF were
1.4 and 1.2 µM, respectively. These cells were significantly more
resistant to the (+)-enantiomers, such that IC50 values were not
determined because of the exceedingly high levels of compound
required. Relative sensitivities of the cells to the four agents
tested are indicated in Figure 2. Consistent with the findings of
McMorris and co-workers,22 (-)-configuration agents demon-
strated significantly increased toxicity compared to the (+)-
counterparts. (-) -HMAF and (-)-AF are the two most potent
compounds, with relative IC50 values of 55 and 95 nM,
respectively. More than 25-fold and 50-fold higher concentra-
tions of (+)-HMAF and (+)-AF, or 1420 and 5000 nM,
respectively, were required to achieve the same cytotoxicity as
the corresponding (-)-enantiomers in AOR-transfected 293
cells.
Reaction of MeLi with a Diastereomerically Pure Estera
a Reagents and conditions: (a) CeCl3, MeLi, THF, 90%; (b) IBX, DMSO,
88%.
conversion of the resolved esters 15 and 18 to the corresponding
optically pure diol 21 was developed (Scheme 4) to circumvent
the alternative inefficient sequence of hydrolysis and protection.
Thus, esters 15 or 18 were treated with 10 equiv MeLi, providing
21 in 90% yield. To assign the absolute stereochemistry of the
tertiary alcohol in the resolved ester, 21 was oxidized with IBX
and the optical rotation of the corresponding acylfulvene was
measured. Initially, cis-and trans-isomers of intermediates were
handled separately because we observed large differences in
the reactivity of cis- and trans-21 toward Dess-Martin oxida-
tion. However, both diols were converted to acylfulvene by IBX,
24;25
the Dess-Martin precursor, in DMSO in 90% yield.
To determine the optimal method of obtaining enantiomeri-
cally pure acylfulvenes, we completed the asymmetric synthesis
formally demonstrated by Brummond and co-workers (Scheme
5). Following published procedures, E-enyne 22 was obtained
in three steps from 7. Enyne 22 was subjected to Sharpless
asymmetric dihydroxylation conditions to generate (4S,5S) and
(4R,5R)-23 using (DHQ)PYR or (DHQD)PYR as chiral cata-
lysts, respectively. Each enantiomer of 23 was advanced to
HMAF utilizing the same conditions as for the racemic
synthesis. To determine the optical purity of the TBS-protected
diol 10 produced by this method, it was treated with TBAF,
and the resulting free alcohol was coupled with chiral acid 11.
NMR and HPLC analysis of the resulting diastereomeric esters
were consistent with the presence of a single isomer (ee% >
95%). The completion of the synthesis validated the effective-
ness of Brummond’s method for the asymmetric synthesis of
either enantiomer of HMAF. Considering the time-consuming
nature of the HPLC separation, we currently find that in our
We measured kinetic parameters for AOR-catalyzed metabo-
lism of each compound to determine if stereochemistry at the

2596 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8
Gong et al.
cells vs 8392 B-cells (insensitive).9 Additionally, our observa-
tions may be related to downstream effects such as the binding
to cellular macromolecules including DNA and proteins, which
is thought to be an important mode of toxicity for acylfulvenes.9
Acylfulvene-DNA lesions inhibit DNA synthesis,15 and the
influences of protein adducts are unknown. Both cellular
transport and adduct formation are likely to be sensitive to
substrate stereochemistry and may be important contributing
factors.
Conclusions
We have synthesized both enantiomers of acylfulvene and
HMAF by two preparative routes: chiral resolution and asym-
metric synthesis. Both pathways are effective for obtaining
enantiomerically pure acylfulvenes. The relative toxicities of
this series of compounds were compared in 293 cells transfected
to overexpress the metabolic enzyme AOR. Additionally, kinetic
parameters for AOR-catalyzed metabolism were determined
independently with isolated enzyme. (-)-HMAF is the best
AOR substrate, and (+)-isomers are metabolized with lower
overall efficiency compared to the (-)-isomers; these data are
consistent with cytotoxicity data. However, the natural stereo-
chemistry of the tertiary alcohol only contributed to an ap-
proximate 2-fold enhancement of catalytic efficiency, whereas
toxicities were over an order of magnitude higher. Furthermore,
AOR-mediated metabolism is influenced more strongly by the
presence or absence of the hydroxylmethyl substituent than by
absolute stereochemistry. This assertion is illustrated by an
approximate 2-fold increase in Vmax/Km for (+)-HMAF over
(-)-AF, but a 10-fold decrease in cytotoxicity. Overall, these
data support that metabolic activation contributes to the relative
toxicities of acylfulvene analogues but indicate that other
biochemical interactions sensitive to stereochemistry are im-
portant. These may include differences in cellular transport as
well as subsequent binding to DNA and protein targets. Current
studies are focused on the use of synthetic analogues of
acylfulvenes to probe further mechanisms of selective toxicity.
Figure 2. AOR transfected 293 cells were challenged for 24 h with
the indicated compounds. Cell viability was measured by MTT assay
in 96-well plates. Each point represents the mean of eight determina-
tions, and bars indicate standard deviation.
Table 1. Kinetic Parameters for Acylfulvene Metabolism by AORa
substrate
Km (µM)
Vmax (µM/min)
Vmax/Km (min-1
)
(+)-AF
465
538
243
213
1.7
5.0
4.5
8.3
3.7
9.3
18.5
39.0
(-)-AF
(+)-HMAF
(-)-HMAF
a Values represent averages determined from three runs with errors within
10%.
2-position affects the rate of this process. Enzymatic activation
of acylfulvenes results in loss of conjugation in the five-
membered ring, cyclopropyl ring-opening, and aromatization.
In the UV-vis spectra, the absorptions at 330 and 420 nm
disappear. Due to the interference of NADPH at 330 nm, the
change of the absorbance at 420 nm was monitored spectro-
photometrically to determine kinetic parameters for AOR-
catalyzed reduction of the R,â-unsaturated ketones. HPLC
analysis was also performed on these incubation mixtures to
confirm that 6 (Nu ) OH, R′ ) H, Scheme 1) was the major
metabolite. The UV change and metabolite formation required
both AOR and NADPH. The identity of 6 was confirmed by
coelution with a synthetic standard,26 as well as by LC-MS
analysis.
Experimental Section
General. All chemicals were purchased from Sigma-Aldrich and
used without further purification. Solvents were dried on an M.
Braun (Stratham, NH) solvent purification system prior to use. 1H
NMR spectra were acquired on a 300 MHz Varian Mercury NMR
spectrometer, and chemical shifts were reported relative to the
residual nondeuterated solvent signals. HPLC analysis was carried
out on an Agilent 1100 series instrument with diode array detector.
HRMS were recorded on a Bruker BioTOF II mass spectrometer
with an electrospray ionization source. Polypropylene glycol (PPG)
was used as matrix in all HRMS experiments. Flash chromatography
(silica gel, 200-400 mesh) was used for product purification.
Normal phase TLC was used for product checking and the spots
were detected by exposure to UV-lamp or I2.
Kinetic parameters for the metabolism of synthetic acylful-
venes by AOR are shown in Table 1. Both HMAF enantiomers
display higher Km values than AF. These results are consistent
with earlier AOR studies in which a Km value of 145 µM for
(-)-HMAF was determined by monitoring the appearance of
the hydroxyl metabolite 6.18
Despite the strong correlation between cell sensitivity and
AOR expression levels, cytotoxicities do not correlate directly
with bioactivation parameters. For example, the AOR activation
parameters positively correlate with (-)-HMAF as the most
active analogue and (+)-AF as the least active, but there is only
a 10-fold difference in Vmax/Km, while there is a 100-fold
enhancement in cytotoxicity for (-)-HMAF vs (+)-AF. Inter-
estingly, (+)-HMAF is activated about twice as efficiently as
(-)-AF, but is about 40-fold less potent in the cell-based assay.
The nonlinear relationship between kinetic parameters and cell
sensitivity suggests that additional biochemical factors are
important in dictating cell sensitivity to these drugs. Cellular
accumulation, i.e., selective uptake or efflux of a drug, is an
important mechanism that accounts for differences in cytotox-
icity, is implicated in drug resistance, and has been the subject
of extensive investigation.27-29 In the case of acylfulvenes,
relative cellular accumulations have been compared in relatively
sensitive and insensitive cells. In that study, a 263-fold increase
was observed in accumulation of the drug in MV522 (sensitive)
IBX was prepared by a published procedure.24;25 Racemic 10
was prepared as previously reported23 and was used as precursor
for chiral resolution. (4S,5S)-23 and (4R,5R)-23 were prepared using
a published method.21 (-)- and (+)-3, and (-)- and (+)-4 were
prepared by chiral resolution of diastereomeric intermediates using
the procedures described here, or using (4S,5S)-23 and (4R,5R)-23
as precursors, by published methods.21;22 The enantiomeric excesses
of compounds obtained by asymmetric synthesis were determined
by derivatization of the intermediates (1R,2S)-trans-10 and (1S,2R)-
trans-10 with camphanic acid, as described below, and analysis
1
by H NMR and HPLC.
Chemical Synthesis. (1R*,2S*)-1,2-Dihydroxy-2,4-dimethyl-
3-spiro-cyclopropyl-bicyclo[4.3.0]nona-5,9-dien-7-one (rac cis-
14). TBAF (3 mL, 1.0 M solution in THF) was added to an ice-
cold solution of 10 (50 mg, 0.15 mmol) in 2 mL of THF. The

Stereochemistry and AcylfulVene ActiVity
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8 2597
solution was stirred at 0 °C for 10 min and was then warmed slowly
to 25 °C and stirred for additional 30 min. The solvent was
evaporated under reduced pressure, and the residue was purified
by flash chromatography eluting with 50% EtOAc/CH2Cl2 to yield
the title compound as a white solid. 97% yield. mp 75-77 °C; 1H
NMR (300 MHz, CDCl3): δ 0.96 (m, 4H), 1.09 (s, 3H), 1.27 (s,
3H), 2.19 (s, 1H), 2.29 (d, J ) 8.4 Hz, 1H), 3.02 (s, 2H), 4.43 (d,
J ) 7.5 Hz, 1H), 6.26 (s, 1H); HRMS calcd for C13H16O3 [M +
Na]+: 243.0997. Found: 243.1025.
(s, 1H), 2.55 (s, 1H), 5.67 (s, 1H), 5.69 (s, 1H), 6.25 (s, 2H); HRMS
calcd for C23H28O6 [M + Na]+ ) 423.1784, found 423.1843.
(1S*,2S*)-2-Dihydroxy-2,4-dimethyl-3-spiro-cyclopropyl-1-
[(2S)-2-methoxyphenylacetyloxy]bicyclo[4.3.0]nona-5,9-dien-7-
one (18). The title compound was prepared as a white solid by the
same procedure as ester-15, with rac trans-14 (50 mg, 0.23 mmol),
(+)-methoxyphenylacetic acid 11 (115 mg, 0.69 mmol), HBTU
(259 mg, 0.69 mmol), DMAP (27.7 mg, 0.23 mmol), and Et3N
1
(0.143 mL, 1.15 mmol) in 2 mL of THF. 74% yield. H NMR
(C6D6) δ 0.05-0.16 (m, 2H), 0.53-0.66 (m, 2H), 0.68-0.75 (m,
1H), 0.84 (s, 3H), 0.88-0.93 (m, 1H), 0.98 (s, 3H), 1.16-1.22
(m, 4H), 1.24-1.40 (m, 4H), 1.65 (s, 3H), 1.70 (s, 3H), 2.44 (m,
2H), 2.47 (s, 1H), 2.49 (s, 1H), 2.71 (m, 1H), 5.57 (d, J ) 1.7 Hz,
1H), 5.91 (s, 1H), 5.94 (s, 1H), 5.99 (s, 1H), 6.04 (s, 1H), 6.33 (d,
J ) 1.7 Hz, 1H), 7.01-7.09 (m, 6H), 7.42-7.47 (m, 4H); HRMS
calcd for C23H24O6 [M + Na]+ ) 419.1482. Found 419.1528.
(1S*,2S*)-2-Dihydroxy-2,4-dimethyl-3-spiro-cyclopropyl-1-
[(2S)-2-acetyloxy-phenylacetyloxy]bicyclo[4.3.0]nona-5,9-dien-
7-one (19). The title compound was prepared as a white solid by
the same procedure as ester-15, with rac trans-14 (50 mg, 0.23
mmol), (-)-O-acetylmandelic acid 12 (134 mg, 0.69 mmol), HBTU
(259 mg, 0.69 mmol), DMAP (27.7 mg, 0.23 mmol), Et3N (0.143
mL, 1.15 mmol) in 2 mL THF. 75% yield. 1H NMR (C6D6) δ 0.52-
0.60 (m, 1H), 0.81-0.97 (m, 2H), 0.98-1.08 (m, 2H), 1.14 (s,
3H), 1.21 (s, 3H), 1.29-1.42 (m, 3H), 1.57 (s, 3H), 1.58 (s, 3H),
2.93 (s, 2H), 3.39 (m, 3H), 3.41 (s, 3H), 3.50 (s, 2H), 3.70 (m,
1H), 4.28 (m, 1H), 4.83 (s, 1H), 4.92 (s, 1H), 5.03 (s, 1H), 5.07 (s,
1H), 5.60 (s, 1H), 5.84 (s, 1H), 7.35-7.46 (m, 7H), 7.51-7.55
(m, 3H); HRMS calcd for C22H24O5 [M + Na]+ ) 391.1521. Found
391.1372.
(1S*,2S*)-1,2-Dihydroxy-2,4-dimethyl-3-spiro-cyclopropyl-
bicyclo[4.3.0]nona-5,9-dien-7-one (rac trans-14). The title com-
pound was prepared by the same procedure as rac cis-14, with
TBAF (3 mL, 1.0 M solution in THF) and 10 (50 mg, 0.15 mmol)
in 2 mL of THF. In this case, the solution was stirred at 0 °C for
10 min and was then warmed slowly to 25 °C and stirred for
additional 2 h. The solvent was evaporated under reduced pressure,
and the residue was purified by flash chromatography eluting with
50% EtOAc/CH2Cl2 to yield the title compound as a white solid.
1
95% yield. mp 72-74 °C; H NMR (300 MHz, CDCl3): δ 0.53
(M, 1H), 0.80 (M, 1H), 1.05 (M, 1H), 1.16 (s, 3H), 1.34 (M, 1H),
1.56 (s, 3H), 2.42 (d, J ) 8.4 Hz, 1H), 1.94 (s, 2H), 4.64 (d, J )
7.5 Hz, 1H), 6.20 (s, 1H). HRMS calcd for C13H16O3 [M + Na]+:
243.0997. Found: 243.1005.
(1R*,2S*)-2-Dihydroxy-2,4-dimethyl-3-spiro-cyclopropyl-1-
[(2S)-2-methoxy-phenylacetyloxy]bicyclo[4.3.0]nona-5,9-dien-7-
one (15). To an oven-dried 10 mL round-bottomed flask was added
rac cis-14 (50 mg, 0.23 mmol), (+)-methoxyphenylacetic acid 11
(115 mg, 0.69 mmol), O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyl-
uronium hexafluorophosphate (HBTU) (259 mg, 0.69 mmol),
DMAP (27.7 mg, 0.23 mmol), Et3N (0.143 mL, 1.15 mmol), and
2 mL of THF. The mixture was stirred at 25 °C for 1 day. After
this time, the reaction mixture was poured into 20 mL of H2O and
extracted with EtOAc. The organic solution was washed with a
5% aqueous K2CO3 solution and then dried over anhydrous Na2-
SO4. Solvent was evaporated under reduced pressure, and the
resulting residue was purified by flash chromatography eluting first
with 20% then 35% EtOAc/CH2Cl2 to yield the corresponding ester
(1S*,2S*)-2-Dihydroxy-2,4-dimethyl-3-spiro-cyclopropyl-1-
{4,7,7-trimethyl-3-oxo-2-oxabicyclo[2,2,1]heptane-1-carboxyl}-
bicyclo[4.3.0]nona-5,9-dien-7-one (20). The title compound was
prepared as a white solid by the same procedure as ester-17, with
rac trans-14 (50 mg, 0.23 mmol), (-)-camphanic acid chloride 13
(149 mg, 0.69 mmol), DMAP (27.7 mg, 0.23 mmol), and Et3N
1
(0.143 mL, 1.15 mmol) in 2 mL of THF. 85% yield; H NMR
(C6D6) δ 0.12-0.21 (m, 2H); 0.58-0.68 (m, 2H), 0.70 (s, 3H),
0.72 (s, 3H), 0.89 (s, 3H), 0.91 (s, 3H), 1.08 (s, 3H), 1.10 (s, 3H),
1.12-1.37 (m, 7H), 1.65-1.77 (m, 2H), 1.88-1.92 (m, 1H), 1.97-
2.11 (m, 2H), 2.53-2.58 (m, 4H), 5.97 (s, 1H), 5.99 (s, 1H), 6.07
(d, J ) 1.7 Hz, 1H,), 6.13 (d,, J ) 1.7 Hz, 1H); HRMS calcd for
C23H28O6 [M + Na]+ ) 423.1784, found 423.1812.
1
as a white solid. 70% yield. H NMR (CDCl3) δ 0.85 (m, 4H);
1.20 (m, 10H), 1.51 (s, 3H), 1.53 (s, 3H), 2.22 (s, 6H), 2.85 (m,
2H), 2.97 (s, 1H), 2.99 (s, 1H), 5.68 (s, 1H), 5.81 (s, 1H), 5.88 (s,
1H), 5.98 (s, 1H), 6.19 (s, 1H), 7.32-7.58 (m, 10H); HRMS calcd
for C23H24O6 [M + Na]+ ) 419.1471. Found 419.1482.
(1R*,2S*)-2-Dihydroxy-2,4-dimethyl-3-spiro-cyclopropyl-1-
[(2S)-2-acetyloxy-phenylacetyloxy]bicyclo[4.3.0]nona-5,9-dien-
7-one (16). The title compound was prepared as a white solid by
the same procedure as ester-15, with rac cis-14 (50 mg, 0.23 mmol),
(-)-O-acetylmandelic acid 12 (134 mg, 0.69 mmol), HBTU (259
mg, 0.69 mmol), DMAP (27.7 mg, 0.23 mmol), and Et3N (0.143
mL, 1.15 mmol) in 2 mL of THF. 80% yield. 1H NMR (CDCl3) δ
0.63-0.95 (m, 8H), 1.07 (s, 3H), 1.52 (s, 3H), 1.54 (s, 3H), 1.58
(s, 3H), 2.86 (s, 2H), 2.93 (s, 2H), 3.42 (s, 3H), 3.44 (s, 3H), 4.80
(s, 1H), 4.90 (s, 1H), 5.65 (s, 1H), 5.74 (s, 1H), 5.78 (s, 1H), 6.10
(s, 1H), 7.38-7.45 (m, 10H); HRMS calcd for C22H24O5 [M +
Na]+ ) 391.1521, found 391.1517.
(1S,2R)-8,9-Dihydroxy-3,6,8-trimethyl-7-spiro-cyclopropyl-
bicyclo[4.3.0]nona-1,3,5-triene (21). To a 25 mL round-bottomed
flask was added CeCl3‚7H2O (232 mg, 0.62 mmol). It was dried at
140 °C under vacuum with stirring for 4 h and was then cooled on
ice and suspended in THF. The suspension was stirred at 25 °C
for 2 h and then cooled to -78 °C. MeLi (0.4 mL of a 1.6 M
solution in ether, 0.62 mmol) was added dropwise, and the resulting
brown suspension was stirred at -78 °C for an additional 30 min.
A solution of resolved ester (1S,2R)-15 (25 mg, 0.063 mmol) in
0.5 mL of THF was added dropwise and the mixture was stirred
for 1 h. The mixture was poured into an ice-cold solution of dilute
HCl and warmed to 25 °C, resulting in a yellow solution. The
mixture was extracted twice with EtOAc, and the combined organic
extracts were washed with 5% K2CO3 solution, dried over
anhydrous Na2SO4, and filtered. The solvent was evaporated under
reduced pressure, and the residue was purified by flash chroma-
tography eluting with 20% EtOAc/hexanes to yield 21 (11 mg, 0.05
mmol, 90% yield) as a yellow gum. 1H NMR (CDCl3) δ 0.80 (m,
1H), 0.90 (m, 1H), 0.97 (m, 1H), 1.15 (s, 1H), 1.20 (m, 1H), 1.58
(d, J ) 7.8 Hz, 1H), 1.84 (s, 1H), 2.05 (s, 3H), 2.83 (s, 1H), 4.30
(1R*,2S*)-2-Dihydroxy-2,4-dimethyl-3-spiro-cyclopropyl-1-
{4,7,7-trimethyl-3-oxo-2-oxabicyclo[2,2,1]heptane-1-carboxyl}-
bicyclo[4.3.0]nona-5,9-dien-7-one (17). To an oven-dried 10 mL
round-bottomed flask were added rac cis-14 (50 mg, 0.23 mmol),
(-)-camphanic acid chloride 13 (149 mg, 0.69 mmol), DMAP (27.7
mg, 0.23 mmol), Et3N (0.143 mL, 1.15 mmol), and 2 mL of THF.
The mixture was stirred at 25 °C for 1 day. After this time, the
reaction mixture was poured into 20 mL of H2O and extracted with
EtOAc. The organic solution was washed with a 5% aqueous K2-
CO3 solution and then dried over anhydrous Na2SO4. Solvent was
evaporated under reduced pressure, and the resulting residue was
purified by flash chromatography eluting first with 20% then 35%
EtOAc/CH2Cl2 to yield the corresponding ester as a white solid.
(d, J ) 7.8 Hz, 1H), 6.05 (s, H), 6.32 (s, 1H). [R]25 ) -15.7 (c
D
2.1 mg/mL, CHCl3).
(-)-Acylfulvene ((-)-3) (by IBX-mediated oxidation). The title
compound was prepared according to the published procedure22
with diol (8R,9S)-21 (30 mg, 0.14 mmol) and IBX (110 mg, 0.28
1
1
85% yield; H NMR (C6D6) δ 0.29 (s, 4H) 0.60 (s, 3H), 0.63 (s,
mmol) in 4 mL of DMSO. 80% yield. H NMR (CDCl3) δ 0.70
3H), 0.71 (s, 4H), 0.78 (s, 3H), 0.80 (s, 3H), 0.88 (m, 12H), 1.35
(m, 1H), 1.10 (m, 1H), 1.30 (m, 1H), 1.38 (s, 3H), 1.50 (m, 1H),
2.00 (s, 3H), 2.15 (s, 3H), 3.92 (s, 1H), 6.43 (s, 1H), 7.18 (s, 1H).
(s, 6H), 1.56-1.68 (m, 4H), 1.90-2.14 (m, 4H), 2.51 (m, 2H),2.54

2598 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8
Gong et al.
[R]25 ) -487.2 (c 3.5 mg/mL, EtOH). (+)-Acylfulvene ((+)-3)
Acknowledgment. We acknowledge Professor Kay Brum-
mond for helpful advice regarding the synthesis of acylfulvenes
and for generously providing detailed experimental protocols
and chemical standards. We acknowledge support from the
Office of the Dean of the Graduate School of the University of
Minnesota. J.G. was supported by a Postdoctoral Fellowship
from the Carcinogenesis and Chemoprevention Program of The
Cancer Center, University of Minnesota. S.J.S. was supported
in part by Career Transition Award CA-108604 from the
National Cancer Institute.
D
was prepared in the same manner: [R]25 ) +493.2 (c 4.1 mg/
D
mL, EtOH).
(-)-HMAF ((-)-4). The title compound was prepared according
to the published procedure6 with (-)-3 (20 mg, 0.09 mmol) and
formaldehyde (55 mg, 1.85 mmol) in 2 mL of acetone and 2 mL
1
of 5% H2SO4 solution. 60% yield. H NMR (CDCl3) δ 0.71 (m,
1H), 1.09 (m, 1H), 1.36 (m, 1H), 1.38 (s, 3H), 1.49 (m, 1H), 2.15
(s, 3H), 2.19 (s, 3H), 3.90 (s, 1H), 4.68 (s, 1H), 7.10 (s, 1H). [R]25
D
) -609.8 (c 2.5 mg/mL, EtOH). (+)-4 was prepared in the same
manner: [R]25 ) +618.4 (c 1.4 mg/mL, EtOH).
D
Expression of AOR in Escherichia coli and Purification.
Recombinant rAOR was purified mainly following what was
previously described.20 Briefly, plasmid pTrcHisA containing rAOR
cDNA with a 6-His tag at N-terminus was transformed into
chemically competent E. coli strain BL21. AOR was expressed in
Luria-Bertani medium containing 100 µg/mL carbenicillin as
described previously.30 Recombinant AOR was purified using
nickel-nitrilotriacetic acid superflow resin (Qiagen Inc.) according
to manufacturer’s recommendations. Elutions were analyzed by
SDS-polyacrylamide gel electrphoresis using Coomassie Blue
staining. Fractions with >95% purity were pooled and dialyzed
overnight against 1 L of dialysis buffer (10 mM potassium
phosphate, pH 7.3, 1 mM dithiothreitol, 5 mg/mL complete protease
inhibitor cocktail (Roche Inc.), 0.1% Tween-20) at 4 °C overnight.
Protein concentration was determined by Bio-Rad protein assay.
Aliquots were stored at -80 °C for up to 12 months without
significant loss of enzymatic activity.
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20
Plasmid pCEP4-rAOR was cloned as described previously and
transfected into 293 cells with Lipofectamine 2000 reagents
(Invitrogen) according to the manufacturer’s recommendations.
Determination of AOR Enzymatic Activities in Cultured
Cells. Cultured cells were collected and lysed in reaction buffer
(0.5X PBS (phosphate buffer saline, pH ) 7.2), 0.01% Triton
X-100) by freezing and thawing in liquid nitrogen and 37 °C water
bath three times. Cell lysate was then centrifuged at 12 000g for
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Beckman DU800 spectrophotometer.
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velocities were measured by monitoring the decay of the absorbance
at 420 nm (ꢀ ) 2194 cm-1‚M-1 for AF, 2210 cm-1‚M-1 for HMAF)
at 37 °C. Stock solutions of analytes were prepared in DMSO (50
mM) and were diluted to a final incubation concentration between
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analyte, 5 mM NADP+, 10 mM glucose-6-phosphate, and 10 units
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recorded. A 10 and 20 min incubation was performed for HMAF
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