Noduliprevenone: A novel heterodimeric chromanone with cancer chemopreventhe potential

Article abstract of DOI:10.1002/chem.200801574

A study was conducted to report a novel heterodimeric chromanone with can chemopreventive potential. It was demonstrate that the aim of chemopreventive strategy can include the inhibition of phase 1 enzymes accompanied with an increased phase II metabolis

Full text of DOI:10.1002/chem.200801574

DOI: 10.1002/chem.200801574
Noduliprevenone: A Novel Heterodimeric Chromanone with Cancer
Chemopreventive Potential
[a]
Alexander Pontius,[a] AnjaKrick, Stefan Kehraus,[a] Silke E. Foegen,[b]
Michael Müller,[b] Karin Klimo,[c] Clarissa Gerhäuser,[c] and Gabriele M. Kçnig*[a]
Chemoprevention is considered a new strategy in fighting
diverse types of cancer. To influence cancer development
and progression, signaling pathways,[1] for example, enzymes
like cyclooxygenase-2,[2] transcription factors like NF-kB,[3]
and several metabolic processes leading to activation or de-
activation of carcinogenic agents, are envisaged as promising
approaches.[4,5]
The fungus Nodulisporium sp. is an endophyte of a Medi-
terranean alga, and produces the novel polyketide noduli-
prevenone (1), an unprecedented structure consisting of two
Cytochrome P450 (CYP) enzymes activate xenobiotics
and thus contribute to the generation of potent carcinogens.
Accordingly, inhibition of cytochrome P450 isoforms, such
as CYP1A enzymes, which play an important role in poly-
cyclic aromatic hydrocarbon (PAH) induced cancer forms, is
advantageous for chemoprevention.[6] In contrast, the induc-
tion of phase II enzymes inactivates carcinogens and acceler-
ates their renal or biliar elimination, forming conjugates
with polar ligands, such as glutathione, glucuronic acid, and
acetic or sulfuric acid.[7] NAD(P)H:quinone reductase (QR)
contributes in detoxification by catalysing the two-electron-
accepted reduction of quinones to their corresponding, low-
mutagenic hydroquinones and is therefore often used as bio-
marker.[8] Accordingly, the aim of cancer chemopreventive
strategy can include the inhibition of phase I enzymes ac-
companied with an increased phase II metabolism.
uniquely modified xanthone-derived units. The absolute
configuration of compound 1, including four chiral centres
and a chiral axis, was assigned using CD-spectroscopy,
Mosherꢀs method and selective NOE gradient measure-
ments. Compound 1 was identified as a competitive inhibitor
of cytochrome P450 1A activity with an IC50 value 6.5Æ
1.6 mm and induces at the same time twofold NAD(P)H:qui-
none reductase (QR) activity in Hepa 1c1c7 mouse culture
cells with a concentration of 5.3Æ1.1 mm.
[a] A. Pontius, Dr. A. Krick, Dr. S. Kehraus, Prof. Dr. G. M. Kçnig
Institute for Pharmaceutical Biology
University of Bonn
Nussallee 6, 53115 Bonn (Germany)
Fax : (+49)228-73-3250
E-mail: g.koenig@uni-bonn.de
The molecular formula of compound 1 was deduced by
accurate mass measurement (HRESIMS) to be C33H34O15,
implying 17 degrees of unsaturation. UV absorption maxima
at 279 and 353 nm gave proof for an extended aromatic
system. The 13C NMR spectrum depicted 33 resonances, 32
of which could be recognised as pairs with close to identical
chemical shifts, indicating a heterodimeric structure with
almost the same substitution pattern of the subunits
(Table S1 in the Supporting Information). Each subunit in-
cludes three carbonyl groups and six sp2 carbons, the latter
forming the aromatic nucleus. Two of the aromatic carbon
[b] Dr. S. E. Foegen, Prof. Dr. M. Müller
Institute of Pharmaceutical Sciences
Albert-Ludwigs-University Freiburg
Albertstrasse 25, 79104 Freiburg (Germany)
[c] K. Klimo, Dr. C. Gerhäuser
Department of Toxicology and Cancer Risk Factors
DKFZ-German Cancer Research Center
Im Neuenheimer Feld 280, 69120 Heidelberg (Germany)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200801574.
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COMMUNICATION
resonances were characterised by a downfield shift due to
substitution with oxygen (dC =161.7, C-1’/159.5 ppm, C-1;
dC =157.8, C-4a’/159.8 ppm, C-4a). The 1H NMR spectrum
contained two typical signals for chelated hydroxyl groups
(dH =11.47 ppm, OH-1’; dH =11.94 ppm, OH-1) characteris-
tic for the presence of phenolic moieties adjacent to carbon-
yl groups (dC =195.4 ppm, C-9’; dC =197.7 ppm, C-9). Since
only one uncoupled aromatic proton resonance (dH =
6.49 ppm, CH-2’; dH =6.43 ppm, CH-4) was present on each
aromatic system, the phenyl rings had to be fivefold substi-
substitution pattern for both molecules is comparable.[10,11]
The nearly congruent CD curves with the same sign for the
1La and 1Lb band CD suggested the M absolute configuration
for compound 1 (Figure S2in the Supporting Information).
NOE enhancements observed for the H NMR resonance
of H-8aa’ upon irradiation of the H-5’ and CH3-13’ resonan-
ces indicated that the methylene proton H-8aa’, the
1
carboxymethyl group and H-5’ were located on the same
G
side of the molecule with proton 8aa’ and the carboxymeth-
yl group in pseudo-axial positions (Figure 1, and Figure S3
1
tuted. From the data of H-13C HMBC experiments a hetero-
dimeric chromanone structure was deduced (Figure S1 in
the Supporting Information). The architecture of subunit I
was clarified by HMBC correlations of H2-8a’ to C-9a’, H-2’
to C-9’ and OH-1’ to C-9’. The substitution pattern of the ar-
omatic moiety was deduced on the basis of HMBC cou-
plings and supported by the NOESY cross-peaks from both
OH-1’ and CH3-11’ to CH-2’ proving their meta position to
each other. The chromanone heterocycle of subunit II was
assigned by HMBC correlations of H2-8a to C-9a, H-4 to
C-9, CH3-11 to C-4a and OH-1 to C-9. In contrast to sub-
unit I, here NOESY cross-peaks were only observed between
the vicinal residues CH3-11 and CH-4, proving that both
benzene moieties are not identically substituted. Taking all
information of spectroscopic analyses into account, the sub-
Figure 1. Selective gradient NOEs (black arrows) of both monomeric
subunits depicted as Newman projections (along the 5’-10a’/5-10a axis, re-
spectively).
A
quaternary carbons C-4’ of subunit I and C-2of subunit II.
According to the degree of unsaturation one more ring had
1
to be present in 1. The H-1H COSY experiment revealed
two aliphatic spin systems due to couplings between H-5’,
H2-6’ and H2-7’, as well as from OH-5 through to H2-7.
Long-range HMBC correlations between H2-6’, H2-7’ and
the carbonyl group C-8’ (dC =176.0 ppm), but also between
H-5’ and C-8’ indicated a g-lactone ring in subunit I. HMBC
correlations of H2-6 and H2-7 to the carbonyl group C-8
(dC =173.8 ppm) in addition to the correlation of the me-
thoxyl group CH3-14 (dH =3.63 ppm) to C-8 suggested a 4-
hydroxy-butyric acid methyl ester moiety for subunit II. The
connection of the lactone ring to C-10a’ followed from the
HMBC cross-peaks between H-5’ and C-4a’, C-8a’ as well as
C-10a’, while the ester chain was found to be bonded to C-
10a because of cross-peaks between H-5 to both C-8a and
C-10a. Further HMBC correlations between the methoxyl
groups CH3-13’ (dH =3.70 ppm), CH3-13 (dH =3.77 ppm) and
the corresponding carbonyl groups C-12’ (dC =169.8 ppm),
C-12( dC =170.9 ppm) evidenced two carboxymethyl func-
tionalities. Correlations of H2-8a’ to C-12’ and H2-8a to C-12
positioned these ester groups at the quaternary carbons 10a’
and 10a, respectively.
in the Supporting Information). Accordingly, as expected
for bulkier moieties the lactone ring must have pseudo-
equatorial orientation. This was confirmed by a further
NOE correlation between the lactone methylene group H2-
6’ and H-8ab’. For subunit II irradiation of the resonance for
H-5 caused enhancements of H-8aa and CH3-13, suggesting
the same orientation of the carboxymethyl group, H-5 and
H-8aa, with the ester chain in a pseudo-equatorial position
(Figure S3 in the Supporting Information). Consequently,
the relative configuration of the chiral centres was deduced
as 5’R*, 10a’S* and 5R*, 10aS*, respectively. Furthermore,
two important NOESY correlations between CH3-13’ and
CH3-11, as well as between H-7’a and 1-OH allowed us to
deduce, in conjunction with the M-configuration of the
chiral axis, the absolute configuration of both chiral carbons
of subunit I as 5’S and 10a’R (Figure 2and Figure S4 in the
Supporting Information). By using a modified Mosherꢀs
method, the absolute configuration of the secondary alcohol
group at the chiral centre C-5 was assigned as 5R (Figure S5
in the Supporting Information).[12] Taking into account the
relative configuration deduced from NOE measurements
the absolute configuration of subunit II was assigned as 5R
and 10aS. We propose the trivial name noduliprevenone for
compound 1.
Compound 1 has a chiral axis between C-4’and C-2as
well as four chiral centres. The absolute configuration of the
chiral axis was determined by using CD spectroscopy. The
CD spectrum of 1 was compared with that of the reference
molecule (M)-orsellinic acid camphanate, the absolute con-
figuration of which was established by X-ray crystallograph-
Biosynthetically, noduliprevenone (1) is an octaketide
presumably derived from anthraquinone and xanthone pre-
cursor molecules.[13] The chromanone basic structures of 1
are supposedly formed through a ring cleavage of the xan-
[9]
ic studies (Figure S2in the Supporting Information).
Sector rule lines depicted for the 1Lb band show that the
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9861

G. M. Kçnig et al.
Figure 2. Selective gradient NOEs (white arrows) between the two sub-
units of 1. The preferred conformation was calculated using Cerius24.0
(MSI) molecular modeling software package.
thone nucleus, finally creating the lactone and acyclic
methyl ester chain of subunits I and II as shown in Fig-
ure S6 in the Supporting Information.[14] Chromanone-type
structures substituted with a butanolide ring (called ergo-
chrome F unit) are extremely rare and have only been ob-
served in a few fungal derived compounds, that is, xantho-
quinodin A3 and B3,[14] ergoxanthin,[15] chaetomanone,[16]
and the lachnones 3, 4 and 5[17] (Figure S7 in the Supporting
Information). Substitution with an acyclic hydroxybutanoic
methyl ester is unprecedented. Also, noduliprevenone is
unique in being the first heterodimeric compound that incor-
porates two such unusually modified chromanone units. The
connection of the subunits probably resulted from oxidative
phenol coupling, involving two different activated sites of
the aromatic rings. Moreover, the linkage of the subunits
must be stereoselectively controlled, thus leading to the M
conformer of 1.
The chemopreventive potential of compound 1 was inves-
tigated applying two phase I and II metabolic enzymes.
First, the influence on the initiation of cancer development
was analysed by performing a CYP1A inhibition assay.
Noduliprevenone (1) was identified as an inhibitor of
CYP1A activity in vitro, with an IC50 value of 6.5Æ1.6 mm
(n=3; Figure 3A). The compound demonstrated competi-
tive inhibition with respect to the substrate 3-cyano-7-
Figure 3. A) Dose-dependent inhibition of CYP1A enzymatic activity by
1 (black bars, mm scale) in comparison with a-naphthoflavone (white
bars, nm scale), respectively. Means significantly different from control
(C) (*P<0.01, **P<0.001). B) Competitive inhibition of CYP1A activi-
ty by 1. Dixon (left) and Cornish Bowden plot (right) of the results of ki-
!
&
*
netic experiments using 2.5 mm ( ), 5 mm ( ), and 10 mm ( ) CEC, respec-
*
tively, as a substrate. C) Induction of QR activity by 1 ( ) in comparison
*
with sulforaphane used as a positive control ( ).
Experimental Section
The algal sample originates from Corfu (Greece). The sample was pro-
cessed immediately after collection. The isolation of the fungus was car-
ried out using an indirect isolation method. Algal samples were rinsed
three times with sterile water. After surface sterilisation with 70% EtOH
for 15 s the alga was rinsed in sterile artificial sea water (ASW). Subse-
quently the alga was aseptically cut into small pieces and placed on agar
plates containing isolation medium: agar (15 g/L), ASW (800 mLLÀ1),
glucose (1 gLÀ1), peptone from soymeal (0.5 gLÀ1), yeast extract
(0.1 gLÀ1), benzyl penicillin (250 mgLÀ1
) and streptomycin sulfate
(250 mgLÀ1). Fungi growing out of the algal tissue were separated on bio-
malt medium (biomalt 20 gLÀ1, agar 10 gLÀ1, ASW 800 mLLÀ1) until the
culture was pure. The strain 707/GrKo3 was assigned as Nodulisporium
sp. and was identified by Dr. R. A. Samson, Centralbureau voor Schim-
mel Cultures, Utrecht (The Netherlands).
The fungal strain was cultivated for ten weeks on 10 L solid Czapek-Dox
medium (Becton Dickinson, France) with agar (15 gLÀ1) at room temper-
ature in Fernbach flasks. Fungal biomass and media were homogenised
by using an Ultra-Turrax apparatus and extracted with ethyl acetate. The
ethyl acetate extract (16.35 g) of the Nodulisporium fermentation was
separated by liquid–liquid extraction (hexane–MeOH). The MeOH ex-
tract was fractionated by using column chromatography over silica gel
(Merck, 63–200 mm, 7”23 cm) with a CH2Cl2/acetone/MeOH gradient
yielding 12fractions. Fraction 4 (1.53 g) was further fractionated by RP
column chromatography (Macherey–Nagel Polygoprep 60–50 C18, 4”
23 cm) with a MeOH/H2O gradient. Subfraction 4 was then purified by
RP HPLC (Knauer C18 Eurosphere 100 250”8 mm, 5 mm) with a mobile
phase (2mLmin À1) consisting of 55/45 MeOH/H2O resulting in 18 frac-
ethoxycoumarin, determined by Dixon– and Cornish–
A
Bowden plots (Figure 3B). Competitive inhibition was also
reflected by a decrease in IC50 values obtained with decreas-
ing substrate concentrations (data not shown). Otherwise,
after a 48 h treatment period, noduliprevenone (1) was
found to induce the twofold specific activity of QR in a
dose-dependent manner with a concentration of 5.3Æ1.1 mm
(n=3) (Figure 3C) without evident cytotoxicity at concen-
trations below 25 mm.
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Chem. Eur. J. 2008, 14, 9860 – 9863

Medicinal Chemistry
COMMUNICATION
tions. Fraction 17 (18.2mg) was finally separated (same RP18 HPLC
column) with mixture MeOH/H2O 55/45 (2.5 mLminÀ1) affording the
pure compound 1 (12.0 mg).
Compound 1 was tested noneffectively in agar diffusion assays[18] against
the bacteria Bacillus megaterium, Escherichia coli, the fungi Microbo-
tryum violaceum, Eurotium rubrum and Mycotypha microspora, and the
green micro-alga Chlorella fusca. Cytotoxicity of the compound was in-
vestigated by using 36 cancer cell lines.[19] The pure compound showed no
activity in the assay at concentrations of 1 mgmLÀ1 and 10 mgmLÀ1, re-
spectively.
Data for 1: White solid; [a]D24 =+89.44 (c=0.15 in CHCl3); 1D and 2D
NMR data: see Table S1 in the Supporting Information; IR (ATR): n˜ =
2922, 2359, 1732, 1633, 1365, 1155 cmÀ1; UV/Vis (MeOH): lmax (loge)=
279 (4.13), 353 nm (3.60 molÀ1 dm3 cmÀ1); HR ESIMS: m/z calcd for
[C33H34O15Na]+: 693.1795; found: 693.1807.
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Keywords: cancer
chemoprevention
·
heterodimeric
271.
chromanone · medicinal chemistry · natural products ·
structure elucidation
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Received: August 1, 2008
Published online: October 1, 2008
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9863