Concise syntheses of the abyssinones and discovery of new inhibitors of prostate cancer and MMP-2 expression

Article abstract of DOI:10.1021/ml100110x

The first asymmetric syntheses of four members of the abyssinone class of natural products (I, II, III, and IV 4′-OMe) via quinine- or quinidine-derived thiourea-catalyzed intramolecular conjugate additions of β-keto ester alkylidenes are reported. This concise strategy delivers all four natural products and their corresponding antipodes. A preliminary evaluation of all of these small molecules against a metastatic human prostate cancer cell line has identified that these compounds selectively and differentially inhibit cell growth and downregulate the expression of matrix metalloproteinase-2 (MMP-2) at nontoxic concentrations.

Full text of DOI:10.1021/ml100110x

pubs.acs.org/acsmedchemlett
Concise Syntheses of the Abyssinones and Discovery of
New Inhibitors of Prostate Cancer and MMP-2 Expression
Rebecca L. Farmer,†,§ Margaret M. Biddle,† Antoinette E. Nibbs,† Xiaoke Huang,§
Raymond C. Bergan,§,‡ and Karl A. Scheidt*,†,‡
†Department of Chemistry, Center for Molecular Innovation and Drug Discovery, Chemistry of Life Processes Institute,
Northwestern University, Silverman Hall, Evanston, Illinois 60208, ‡The Robert H. Lurie Comprehensive Cancer Center, and
§Department of Medicine, 303 East Superior Street, Chicago, Illinois 60611
ABSTRACT The first asymmetric syntheses of four members of the abyssinone
class of natural products (I, II, III, and IV 40-OMe) via quinine- or quinidine-derived
thiourea-catalyzed intramolecular conjugate additions of β-keto ester alkylidenes
are reported. This concise strategy delivers all four natural products and their
corresponding antipodes. A preliminary evaluation of all of these small molecules
against a metastatic human prostate cancer cell line has identified that these
compounds selectively and differentially inhibit cell growth and downregulate the
expression of matrix metalloproteinase-2 (MMP-2) at nontoxic concentrations.
KEYWORDS Abyssinones, prostate cancer, MMP-2 expression, hydrogen-bonding
catalysis
he abyssinones are a family of chiral, enantiomerically
enriched flavanones that exhibit a diverse range of
promising biological activities (see Figure 1).1-3
maintaining the integrity of the newly formed C2 stereo-
center, which would depend heavily on the identification of
mild decarboxylation/deprotection conditions. We antici-
pated that we could maximize efficiency if the decarboxyla-
tion and unmasking of the C7 phenol were performed in a
single flask as the last step of the synthesis.
T
Although extracts containing these small molecules have
been used as traditional remedies, any investigation of their
specific anticancer properties necessitates a general syn-
thetic approach that can access them in enantiomerically
enriched form. Our program geared toward the synthesis of
pyran-containing natural products prompted our interest in
these compounds.4,5 Despite their therapeutic promise, the
abyssinones have not been (a) synthesized with control over
the absolute stereochemistry and (b) evaluated for their
ability to inhibit cancer progression.6-8 The unadorned
structure of flavanones belies the challenge in executing a
strategy that installs and maintains the configuration at the
C2 position.9 This stereocenter in the abyssinones is quite
sensitive;mildly basic conditions promote reversible ring-
opening to achiral 20-hydroxychalcones.10 In addition, flava-
nones containing electron-donating substituents in the C40
The first challenge encountered in the total synthesis of the
abyssinones involved accessing the alkylidene substrates for
the thiourea cyclization (see Figure 2 for a representative
example with abyssinone I). Using the requisite aldehyde 5,
prepared from 4-hydroxybenzaldehyde,17 we attempted the
condensation with the protected β-keto ester 7 under standard
Knoevenagel conditions (piperidinium acetate, Dean-Stark
apparatus). These reactions produced the desired E alkylidene
8 in >95:5 E:Z for all aldehydes employed in the Knoevenagel
condensation. (We were not able to generate and isolate in
quantity the Z alkylidene species with which to conduct
cyclization studies.) Unfortunately, the reaction conditions also
led to the formation of significant amounts of racemic cycliza-
tion adducts ((9). After we surveyed various conditions, we
discovered that bis-morpholine aminal 6, used directly without
purification, underwent smooth Knoevenagel condensation
with β-ketoester 7 (2 equiv of glacial acetic acid at 22 °C in
toluene) to deliver alkylidene 8 with minimal levels of racemic
cyclized compounds. As predicted, the resulting alkylidene 8
underwent the desired intramolecular conjugate addition
when exposed to 10 mol % quinine-derived C60 thiourea
catalyst I.18 Multiple iterations of the cyclization of alkylidene
position are particularly susceptible to racemization.9,11
A
limited number of approaches for the stereoselective synth-
esis of flavanones have been developed, but these ap-
proaches are not general and would not provide the abyssi-
none core in a concise manner.12-16
Our synthetic plan is outlined in Figure 1. The key step in
the strategy is the application of our asymmetric thiourea-
catalyzed cyclization, which can provide controlled access to
either stereoisomer of these natural products.9,11 A Knoeve-
nagel condensation between an appropriately protected
β-keto ester and different aldehydes corresponding to each
abyssinone would provide the requisite alkylidenes for our
thiourea-catalyzed cyclization. Our greatest concern was
Received Date: May 11, 2010
Accepted Date: July 6, 2010
Published on Web Date: August 04, 2010
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Figure 1. Retrosynthetic analysis of abyssinone natural products.
Figure 2. Initial route to abyssinone I (1).
8 consistently afforded carboxyflavanone 9 in good yield
(78-80%) and with excellent enantioselectivity (90-91% ee).
Importantly, the alkylidene precursors underwent smooth
cyclization with excellent control over the newly formed C2
stereocenter using our chiral thiourea catalysis conditions.
Initially, the decarboxylation and deprotection of 9 were
with comparable levels of stereoselectivity. These results are
especially satisfying since (1) flavanones containing alkoxy
or hydroxy substituents in the C40 position are susceptible to
epimerizationdue to stabilized benzylic cationformation and
(2) flavanones can undergo reversible ring opening to form
20-hydroxychalcones upon exposure to mild bases. The over-
all yields for this process (conjugate addition, allyl deprotec-
tion, and decarboxylation) provided an efficient method for
the construction of these flavanones and allowed for the
evaluation ofoursynthetic method in terms of more complex
synthetic efforts.
We next sought to determine if the enantiomericallyenriched
abyssinones and their corresponding enantiomers would elicit
important and stereodependent biological activity. Members of
the broad flavonoid family (over 5000 natural products) have
exhibited a wide variety of anticancer effects, acting as antioxi-
dants, angiogenesis inhibitors, and potent cytotoxic agents.22 In
addition, several flavonoids have been shown to inhibit the
activity and downregulate the expression of pro-metastatic
enzymes, such as the matrix metalloproteinases (MMPs), in a
variety of tumor cell lines.23-26 However, these studies have
focused primarily on achiral isoflavones, and none of these
investigations to date have been conducted on the abyssinone
family of flavanone natural products.
accomplished in a single flask utilizing MgBr2 OEt2, but the
3
low yields for this reaction sequence (less than 25%) led us
to investigate higher yielding reactions conditions. Unfortu-
nately, various O-aryl-protected t-butyl carboxyflavanones
could not be converted into 1-4 without significant epimer-
ization or functionalization of the abyssinones containing
prenyl side chains. We anticipated that exchanging the
t-butyl ester for an allyl ester and replacing the methoxy-
methyl group with an allyl group might allow for mild, single-
flask decarboxylation/deprotection employing palladium
catalysis.19 The requisite allyl-protected alkylidenes 10-13
were synthesized directly from the corresponding aldehydes
using our mild bis-aminal approach. The key asymmetric
cyclizations of 10-13 were catalyzed by exposure to 10
mol % of either the quinine- or quinidine-derived thiourea I
or II20 at -25 °C in toluene (Ta bl e 1). The use of morpholine in
the presence of 5 mol % Pd(PPh3)4 promoted the deprotec-
tion and decarboxylation at 22 °C to afford the natural
product abyssinones I, II, III, and IV 40-OMe ether cleanly
and in highyields.21 The levels of enantioenrichment foreach
compound from the asymmetric conjugate addition
(i.e., 10-13 to 14-17) were excellent. The quinidine-derived
thiourea catalyst II provided each of the abyssinones (after
deprotection/decarboxylation) with the naturally occurring
configuration at C2, while employing the pseudoenantio-
meric thiourea I generated the unnatural (R)-abyssinones
Our studies focused on prostate cancer (PCa), which is the
second most common cause of cancer-related death in U.S.
men. Mortality from PCa is caused primarily by the develop-
ment of metastatic disease, in which PCa cells move from the
prostate gland to distant sites in the body and continue their
unchecked growth.27 Proteases such as MMPs increase cell
invasion, and thus, their synthesis by cancer cells facilitates
movement and metastatic behavior.28 MMP-2 has been shown
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Table 1. Catalytic Asymmetric Synthesis of Abyssinones
a Enantiomeric excess of abyssinones (1-4) after palladium(0) deprotection determined by HPLC analysis (Chiralcel OD-H or AD-H). b Isolated yield
from 10-13.
to be a particularly important target for human PCa, since its
increased expression in tissue portends metastasis.29 De-
creased MMP-2 expression also leads to decreased invasion of
human PCa cells in vitro and to decreased metastasis of human
PCa cells in a murine model of metastasis.25,30 Consequently,
the MMPs, and MMP-2 in particular, represent an important and
extensively studied therapeutic target. However, while some
first-generation MMP inhibitors have been effective in preclini-
cal models, there has been little success in subsequent clinical
trials, predominantly due to severe systemic toxicity.31,32
An alternative promising strategy for counteracting the
pro-invasive effect of MMP-2 is controlling the amount of this
enzyme produced by tumor cells (i.e., upregulation and
downregulation).30 We have demonstrated that PCa cells
treated with the isoflavone genistein exhibit decreased levels
of both MMP-2 gene transcript and protein, which leads to an
overall reduction in invasive potential.25 Furthermore, stud-
ies performed on additional cancer cell lines have demon-
strated that siRNA knockdown of MMP-2 leads to a reduction
of both invasion and tumor-induced angiogenesis.33 Devel-
oping a method to downregulate MMPexpression levels also
has the advantage of being potentially less toxic than tradi-
tional MMP inhibitors. Thus, we sought to evaluate the ability
of the abyssinones generated in our laboratory to inhibit PCa
cell growth and downregulate the expression of MMP-2,
since these types of interventions could both attenuate/
prevent metastasis and increase survival rates for PCa. We
were also eager to evaluate the effect of stereochemistry on
the ability of the abyssinones to inhibit cell proliferation and
downregulate MMP-2 expression.
Figure 3. CC50 values for each enantiomer and the racemic
mixtures of abyssinones 1-4 against proliferation of PC3-M cells.
For experimental details, see the Supporting Information.
interested to determine if the enantiomers of each compound
demonstrated differential cytotoxicity when compared to each
other and also to the racemic mixture.34 Furthermore, these
cytotoxicity studies were instrumental for defining nontoxic
levels of 1-4, which guided the MMP-2 transcript expression
evaluations (vide infra). Metastatic variant human PC3-M cells
were treated for 3 days with 0-50 μM racemic, (R)- or (S)-1-4
(12 compounds total) under conditions of exponential cell
growth, and then, MTT assays were performed.35 The CC50
values were determined for each compound and are shown in
Figure 3. As indicated by Figure 3, (S)-abyssinones III (3) and IV
40-OMe (4) both demonstrated statistically significant levels
of inhibition of PC3-M cell proliferation when compared to
the (R)-enantiomers and the racemic mixtures. In fact, both
Our studies began by evaluating the impact of enantioen-
riched and racemic abyssinones (I, II, III, and IV 40-OMe) on
metastatic PCa (PC3-M) cell growth. Given that enantioenriched
abyssinones have never been synthesized, we were particularly
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(S)-abyssinones III and IV 40-OMe caused an approximate 2-fold
decrease in the proliferation of PC3-M cells, as they possessed
CC50 values of 17 and 12 μM, respectively, as compared to the
CC50 values of the (R)-enantiomers and racemates at 42 [(R)-3],
57 [(()-3], 46 [(R)-4], and 33 μM [(()-4]. These results clearly
indicate the key role of stereochemistry in the biological activity
of these compounds and validate our enantioselective synthetic
approach for these particular natural products. Further evidence
for the importance of the enantioselective synthesis of the
abyssinones is also provided by the growth inhibition data for
the racemic compounds (Figure 3). Racemic abyssinone II was
actually significantly less active than both of the enantioen-
riched compounds. This particularly interesting result indicates
that there may be some functional antagonism between the
enantiomers of abyssinone II that lead to a significant reduction
of activity in the racemic mixture. Finally, we also performed
some preliminary experiments to try to determine if the abyssi-
nones were inhibiting cell growth via a cytotoxic (i.e., cell death)
or cytostatic (i.e., cell cycle arrest) mechanism. Interestingly,
when the absorbance values at 540 nm for the treated cells at
50 μM (t = 72 h) were compared with those for untreated cells
at the start of the experiment (t = 0 h), (S)-abyssinones III and
IV 40-OMe both appeared to reduce the number of cells to a level
at or below that of the start of the experiment. This result sugges-
ted that these compounds were acting through a cytotoxic
mechanism, which was verified by cell imaging that indicated
gross cell death at 72 h (see the Supporting Information).
However, all of the other compounds, including (R)-abyssinones
III and IV 40-OMe, demonstrated absorbance values that were
greater than that of the t = 0 measurement, indicating that they
were most likely acting through a cytostatic or combination
(cytostatic and cytotoxic) mechanism. Therefore, all of these
combined findings highlight the importance of our hydrogen-
bonding catalysis approach, since this information could not
have been obtained without sufficient amounts of the enantio-
enriched compounds for analysis.
We next chose to evaluate the impact of (R)- and (S)-1-4
(eight compounds total) on the levels of MMP-2 transcript, given
that several members of the flavonoid family of natural products
have been shown to downregulate the expression of this impor-
tant pro-metastatic enzyme. Our aim was not only to determine
the cytotoxicity of these compounds (i.e., their chemotherapeu-
tic potential) but also to evaluate their ability to target MMP-2
synthesis and to act as chemopreventative agents capable of
blocking progression to a more aggressive disease state. Cells
were treated with each compound at 3 μM for 3 days, since this
dose was not associated with cytotoxicity by the MTT assay.
Importantly, we chose this nontoxic dose for our analysis to eli-
minate any confounding nonspecific effects due to cell toxicity.
After abyssinone exposure, MMP-2 transcript levels were mea-
sured by isolating RNA and performing reverse transcription
and quantitative real time polymerase chain reactions (qRT-
PCR). MMP-2 transcript levels were measured instead of MMP-2
protein levels because MMP-2 expression is tightly regulated at
the transcriptional level36 and because flavonoids have been
shown to inhibit MMP-2 transcript expression. Furthermore,
decreased levels of MMP-2 mRNA expression have also been
correlated with a lower pathological stage of PCa and, by exten-
sion, an improved clinical outcome.29,37 The measurement of
Figure 4. MMP-2 expression levels of abyssinone-treated PC3-M
cells at 3 μM. For experimental details, see the Supporting Infor-
mation.
transcript levels provides a direct measure of the ability of the
abyssinones to target cell-based processes responsible for reg-
ulating this clinically relevant pro-metastatic enzyme. Once the
MMP-2 transcript levels were measured by qRT-PCR, they were
normalized to that of the internal control gene GAPDH
(glyceraldehyde-3-phosphate dehydrogenase), which was mea-
sured in both the treated and the untreated cells.38 GAPDH is
expressed at constant levels across a wide array of biological
systems and is widely used for control purposes. We found that
the GAPDH expression levels were the same for both the treated
and the untreated cells, which indicated that the effects on
MMP-2 expression were not a result of nonspecific downregula-
tion of transcription.
With both the MMP-2 studies and the cell growth studies, the
abyssinone enantiomers exhibited statistically significant differ-
ential biological activity (Figure 4). In particular, for both abys-
sinones III and IV 40-OMe, the (R)-enantiomers suppressed
MMP-2 expression to 65-78% of untreated control cells, res-
pectively, at a nontoxic concentration (3 μM). Interestingly, the
activity against MMP-2 expression for the unnatural (R)-enan-
tiomers was significantly greater than that for the corresponding
natural (S)-enantiomers. While there are limited examples of
natural products whose unnatural enantiomers demonstrate
comparable biological potency (e.g., fredericamycin A39), we
are not aware of compounds other than ent-roseophillin40 and
(R)-abyssinones III and IV 40-OMe that are more active than
their natural enantiomers. These examples clearly demonstrate
that the construction of unnatural antipodes by stereoselec-
tive synthesis provides new avenues for potential therapeutic
development. The in vitro assays described above interrogate
distinctly different biological functions and highlight the impor-
tance of successfully installing the stereochemical elements
during the syntheses of the abyssinones. The cytotoxicity and
MMP-2 studies conducted using each enantiomer show differ-
ent response profiles across the abyssinones (1-4), with pro-
mising abilities to differentially target cell growth and metastatic
potential.
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In summary, the first asymmetric syntheses of abyssinones
I-III and IV 40-OMe have been accomplished using a chiral
thiourea-catalyzed intramolecular conjugate addition. Atandem
allyl deprotection/allyl ester decarboxylation generates the final
products in a single flask while maintaining the stereochemical
integrity at the C2 center. Our synthetic route delivers the natural
products and the corresponding antipodes with excellent levels
of enantioselectivity, thereby facilitating the evaluation of in-
dividual stereoisomers and fueling the discovery of their differ-
ential bioactivity. The key observations that the abyssinones
specifically and differentially curtail cell growth and suppress
MMP-2 expression in whole cells at nontoxic concentrations
underscore the utility of our successful catalytic asymmetric syn-
thesis of these natural products. These combined experiments
fully integrate asymmetric catalysis, target synthesis application,
and chemical biology discovery with medicinal relevance.
Continued investigations of the abyssinones and related com-
pounds to understand their biological activity against various
cancer models are ongoing in our laboratories.
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SUPPORTING INFORMATION AVAILABLE General infor-
mation, general procedures, comparative analysis, selected NMR
spectra, HPLC traces, and general biological assay procedures and
additional data. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author: *To whom correspondence should be
addressed. Tel: 847-491-6659. Fax: 847-467-2184. E-mail:
scheidt@northwestern.edu.
(12) Brown, M. K.; Degrado, S. J.; Hoveyda, A. H. Highly Enantio-
selective Cu-Catalyzed Conjugate Additions of Dialkylzinc
Reagents to Unsaturated Furanones and Pyranones: Prepara-
tion of Air-Stable and Catalytically Active Cu-Peptide Com-
plexes. Angew. Chem., Int. Ed. 2005, 44, 5306–5310.
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R. C. R. Conjugate Addition to 3-Arylsulfinylchromones as a
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Funding Sources: Support for this work has been provided to K.
A.S. by the American Cancer Society (Scholar Grant 09-016-01
CDD), AstraZeneca, Amgen, GlaxoSmithKline, the H Foundation,
the Northwestern Medical Foundation (Dixon Translational Award),
and the Robert H. Lurie Cancer Center (NU) and to R.C.B. by a
Veterans Administration Merit Award. This work was funded in part
by the Chicago Biomedical Consortium with support from The
Searle Funds at The Chicago Community Trust. M.M.B. acknowl-
edges the NIH for a postdoctoral training fellowship (T32 AG00260)
directed by the Center for Molecular Innovation and Drug Discovery
(NU). A.E.N. and R.L.F. thank the NIH for predoctoral fellowships
(F31CA132617 and F31CA138097, respectively) and the Malkin
Scholars Program for cancer research grants.
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(17) See the Supporting Information for synthetic details.
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2010 American Chemical Society
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DOI: 10.1021/ml100110x ACS Med. Chem. Lett. 2010, 1, 400–405
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