Diastereomeric quinolinone alkaloids from the marine-derived fungus Penicillium janczewskii

Article abstract of DOI:10.1021/np058018g

From Penicillium janczewskii, obtained from a marine sample, two new diastereomeric quinolinones, 3S*,4R*-dihydroxy-4-(4′- methoxyphenyl)-3,4-dihydro-2(1H)-quinolinone (1) and 3R*,4R*- dihydroxy-4-(4′-methoxyphenyl)-3,4-dihydro-2(Lif)-quinolinone (2), wer

Full text of DOI:10.1021/np058018g

J. Nat. Prod. 2005, 68, 1397-1399
1397
Diastereomeric Quinolinone Alkaloids from the Marine-Derived Fungus
Penicillium janczewskii|
Jian He,† Ulrich Lion,† Isabel Sattler,*,† Friedrich A. Gollmick,† Susanne Grabley,† Jingmin Cai,‡
Marinus Meiners,‡ Henning Schu¨nke,§ Karsten Schaumann,§ Ute Dechert, and Michael Krohn
Leibniz-Institute for Natural Products Research and Infection Biology, Hans-Kno¨ll-Institute, Beutenbergstrasse 11a,
D-07745 Jena, Germany, Fachhochschule Ostfriesland (FHO), Constantiaplatz 4, D-26723 Emden, Germany, BRAIN
Biotechnology Research and Information Network AG, Darmsta¨dter Strasse 34, D-64673 Zwingenberg, Germany, and
Alfred-Wegener-Institut fu¨r Polar- und Meeresforschung (AWI), Am Handelshafen 12, D-27570 Bremerhaven, Germany
Received February 11, 2005
From Penicillium janczewskii, obtained from a marine sample, two new diastereomeric quinolinones,
3S*,4R*-dihydroxy-4-(4′-methoxyphenyl)-3,4-dihydro-2(1H)-quinolinone (1) and 3R*,4R*-dihydroxy-4-(4′-
methoxyphenyl)-3,4-dihydro-2(1H)-quinolinone (2), were identified, along with two known alkaloids,
peniprequinolone (3) and 3-methoxy-4-hydroxy-4-(4′-methoxyphenyl)-3,4-dihydro-2(1H)-quinolinone (4).
Cytotoxicity testing on eight tumor cell lines revealed a moderate specificity of 2 on SKOV-3 cells.
The exploitation of the marine environment has been
intriguingly successful in recent years in the search for
structurally unusual and biologically highly active natural
products.1 To avoid depletion of marine resources and to
enable access to large quantities of interesting compounds,
there is a particular interest in those marine organisms
that are culturable. Thus, we are studying microbial fungi
isolated from marine sources for their potential of providing
new natural products.2 In a combined approach of biological
and chemical screening3 we are gaining a thorough under-
standing of the secondary metabolite pattern of these fungi.
Penicillium janczewskii Zalessky4,5 (strain H-TW5/869)
was selected from our screening program for further
studies, because of a rich metabolite pattern as detected
by thin-layer chromatography on silica gel with various
staining reagents, and cytotoxic activity of its methanolic
extracts. The extracts from mycelia and culture filtrate
showed significant cytotoxicity against CHO-K1 cells. In
addition to two known fungal metabolites (3 and 4), our
studies yielded new diastereomeric dihydroquinoline al-
kaloids 1 and 2. We here report the isolation and structure
elucidation of 1 and 2, as well as the biological activity of
these compounds.
869. Because of experimental feasibility and the predomi-
nant occurrence of metabolites, the culture filtrate was
chosen for compound isolation. It was extracted through
adsorption on Amberchrom CG161c resin and subsequent
elution with MeOH. Initial separation of the natural
products from the concentrated and lyophilized elute (14
g) was performed by flash reversed-phase chromatography
(RP18, MeOH/H2O gradient). The nonpolar fraction was
repeatedly submitted to column chromatography (CHCl3/
MeOH) and finally purified by preparative HPLC (RP18,
MeOH/H2O gradient) to yield compounds 1 (2 mg) and 2
(3 mg).
Compound 1 was obtained as a white solid, and its
molecular formula was established as C16H15NO4 by HRE-
IMS (m/z 285.0994 [M]+, ∆0.7 mmu, 228.1040 [M - C2-
HO2]+, ∆ -1.5 mmu) and supported by ESIMS, which
revealed the quasi-molecular weight through peaks at m/z
308.1 [M + Na]+ and 593.2 [2M + Na]+. The molecular
formula indicated 10 degrees of unsaturation within the
molecule. The IR bands at 1705, 1684, 1604, and 1511 cm-1
suggested the presence of a carbonyl group and an aromatic
ring. 13C NMR and DEPT data revealed one methoxy group
(δ 54.9), two oxygenated sp3-hybridized carbons [one me-
thine (δ 74.8) and one quaternary carbon (δ 75.8)], 12
aromatic carbons, eight of which were methine groups, and
1
one amide carbonyl carbon (δ 170.4). The H NMR spec-
trum indicated that the aromatic signals represented two
sets of protons: one was a pair of ortho-coupled doublets
attributed to four symmetrical protons at δ 7.18, 6.78, and
another was an ABCD system at δ 7.38, 7.22, 6.99, and
6.89, respectively. Furthermore, one methoxy group at δ
3.68 and one methine group at δ 4.29 were confirmed. Thus,
it was determined that the basic skeleton of 1 consisted of
two aromatic rings, one of them substituted with a cyclic
amide. Cross-peaks in the HMBC spectrum of 1 between
H-3 and C-2, C-4, C-4a and between NH-1 and C-2, C-3,
C-4a, C-8, C-8a suggested the presence of a cyclic amide
(Figure 1). HMBC correlations between OCH3 (δ 3.68) and
C-4′ (δ 158.2) revealed that the methoxy group was located
at C-4′ of the benzyl ring, and those between H-3 and C-1′
and H-2′/6′ and C-4 revealed the location of that aromatic
system. Finally, comparison of the 1H and 13C NMR
spectral data of 1 with analogous data for the known
peniprequinolone (3) and 4,6-8 which were also obtained
The isolation of the metabolites was performed from a
crude product of a 50 L cultivation of P. janczewskii H-TW5/
|
Dedicated to Professor Dr. Axel Zeeck on the occasion of his 65th
birthday.
* To whom correspondence should be addressed. Tel: 0049-3641-656920.
Fax: 0049-3641/656679. E-mail: isabel.sattler@hki-jena.de.
† Hans-Kno¨ll-Institute.
‡ Fachhochschule Ostfriesland.
§ Alfred-Wegener-Institute.
BRAIN AG.
10.1021/np058018g CCC: $30.25
© 2005 American Chemical Society and American Society of Pharmacognosy
Published on Web 08/19/2005

1398 Journal of Natural Products, 2005, Vol. 68, No. 9
Notes
Table 2. Cytotoxicity Dataa of Compounds 1-3
cell line
1
2
3
MDA-MB 231
DU-145
SKOV-3
HT-29
52.9
50.6
44.6
58.8
56.3
80.3
55.2
52.4
31.8
48.4
8.1
32.8
56.8
65.1
39.0
36.6
8.4
30.8
20.2
4.0
nd
A549
CAKI-1
SK-MEL 2
K562
nd
nd
nd
a % viability at 10 µg/mL compound concentration; nd not
determined.
Figure 1. Key HMBC correlations of 1.
Table 1. 13C and 1H NMR Dataa for 1 and 2
than in the anti-facial situation of compound 1. The
observed NOE correlations between H-2′/6′ and H-3 were
quantified by peak volumes and calibrated against the
vicinal aromatic NOE between H-6 and H-7 (Figure 1).
These calculations yielded a 13-fold higher peak volume
of the NOE between H-3 and H-2′/6′ in compound 2
compared to that in compound 1. Therefore, the structure
of compound 1 was determined as 3S*,4R*-dihydroxy-4-
(4′-methoxyphenyl)-3,4-dihydro-2(1H)-quinolinone, and 2
was 3R*,4R*-dihydroxy-4-(4′-methoxyphenyl)-3,4-dihydro-
2(1H)-quinolinone. The diastereomers are very likely bio-
synthetic hydration products of an intermediate related to
compound 5.
1
2
b
c
b
c
position δC
δH
δC
δH
3
74.8 4.29 (s)
170.4
74.4 4.46 (s)
170.4
2
4
75.8
76.5
4a
131.1
129.5
5
128.4 7.38 (dd, 1.0, 7.0)
128.7 6.74 (brd, 7.0)
6
122.5 6.99 (ddd, 1.0, 7.0, 8.0) 122.0 6.87 (m)
7
126.5 7.22 (dt, 1.0, 8.0)
115.2 6.89 (dd, 1.0, 8.0)
135.7
128.0 7.21 (dt, 1.0, 7.0)
8
115.3 6.90 (m)
137.1
134.4
8a
1′
133.4
2′, 6′
3′, 5′
4′
1-NH
3-OH
4-OH
OCH3
128.5 7.18 (d, 9.0)
112.6 6.78 (d, 9.0)
158.2
128.1 7.28 (d, 9.0)
113.0 6.89 (d, 9.0)
158.2
Peniprequinolone (3)6 and 3-methoxy-4-hydroxy-4-(4′-
methoxyphenyl)-3,4-dihydro-2(1H)-quinolinone (4)7,8 are
two known quinolinone type alkaloids that in this study
were for the first time isolated from a marine-derived
fungus. These compounds were originally isolated by
bioassay-guided methods from the fungus P. cf. simplicis-
simum with nematicidal activity toward Pratylenchus
penetrans of 3 and toxicity against brine shrimp of 4,
respectively. Compounds 1-4 obtained in this study were
evaluated for their cytotoxic potential against human
tumor cell lines (Table 2) by determining cell viability at a
standard concentration of 10 µg/mL. The two new com-
pounds 1 and 2 showed a low to moderate general toxicity
against MDA-MB 231 (human breast adenocarcinoma),
DU-145 (human prostate carcinoma), HT-29 (human colon
carcinoma), A549 (human non small cell lung carcinoma),
CAKI-1 (human kidney carcinoma), SK-MEL 2 (human
melanoma), and K562 (human myeloid leukemia) cells,
with 2 being slightly more potent. In addition, a signifi-
cantly stronger cytotoxicity against SKOV-3 cells (human
ovary adenocarcinoma) was found for 2. As 1 and 2 differ
only in their relative stereochemistry, these significant
differences in efficacy as well as selectivity are very
surprising. Finally, in accordance with its reported anti-
fungal activity,6 compound 3 acts as a strong cytotoxic
agent, however, with no selectivity; 4 did not show signifi-
cant cytotoxicity.
10.19 (brs)
10.46 (brs)
5.30 (brs)
5.90 (brs)
54.9 3.68 (s)
5.12 (brs)
5.48 (brs)
55.0 3.74 (s)
a In DMSO-d6; chemical shifts in ppm. b 75 MHz. c 300 MHz;
multiplicities and J values (Hz) are shown in parentheses.
Figure 2. Acidic dehydration of compounds 1 and 2.
in the present investigation, indicated a family of structur-
ally related compounds with differences in their substitu-
tion patterns. Thus, 1 was determined to be 3,4-dihydroxy-
4-(4′-methoxyphenyl)-3,4-dihydro-2(1H)-quinolinone.
Compound 2 was isolated as a white amorphous solid.
The molecular formula was also determined as C16H15NO4
by HRESIMS of [M + Na]+. The compound was recognized
as a diastereomer of 1 with different relative stereochem-
istry at positions 3 and 4 on the basis of its very similar
spectroscopic data (Table 1, Experimental Section). This
assumption was proven through acidic dehydration by
TsOH in acetone. A mixture of 1 and 2 (1:1) yielded
3-hydroxy-4-(4-methoxyphenyl)-2(1H)-quinolinone (5) as
the sole product (Figure 2). The structure of 5 was unam-
Experimental Section
General Experimental Procedures. Optical rotations
were recorded on a Perkin-Elmer 241 polarimeter. IR spectra
were recorded with a Perkin-Elmer model 298 spectrometer
(KBr, disks). 1D NMR (1H, 13C) spectra were recorded on a
Bruker DPX-300 spectrometer; 2D NMR (COSY, HMBC,
HMQC, NOESY) spectra, on a Bruker DPX-500 spectrometer.
HREIMS were recorded with the AMD-402 instrument of Be
geometry equipped with direct inlet system (AMD Intectra).
ESIMS were recorded by use of a Quattro triple quadrupole
mass spectrometer (VG Biotech, Altrincham, England) and
Finnigan MAT 311A (EI: 70 eV, direct inlet, high resolution
with perfluorokerosine as a standard). TLC was performed on
silica gel plates (Macherey-Nagel, Sil G/UV254, 0.20 mm); spots
were detected under a UV lamp and after staining with
1
bigiously assigned through H NMR and ESIMS data.9,10
The relative stereochemistry of 1 and 2 was determined
through conformational analysis and quantitative NOE
data obtained from NOESY spectra. Basic conformational
analysis by different standard calculation methods (AM2,
MMDO) clearly indicated an almost perpendicular orienta-
tion of the two aromatic rings with a significant rotational
barrier around the C-4-C-1′ bond. A fixed conformational
arrangement of the amide ring is rather unlikely. It can
be assumed, though, that such conformational states in
which H-3 and H-2′/6′ are situated in close proximity are
more likely in the syn-facial arrangement of compound 2

Notes
Journal of Natural Products, 2005, Vol. 68, No. 9 1399
anisaldehyde/H2SO4. Solid-phase extraction was done on Am-
berchrom CG-161C (TOSOHAAS); TLC was performed on DC
Alugram SIL G/UV 254 (0,25 mm, Macherey-Nagel GmbH&Co,
Germany), and staining reagents were prepared according to
standard laboratory procedures. Staining was achieved through
spraying onto the plate and subsequent heating. Chromatog-
raphy was done on LiChroprep RP-18 (25-40 µm, Merck
KGaA, Germany), Sephadex LH-20 (Pharmacia Biotech AB,
Sweden), and silica gel 60 (0.040-0.063 mm, Merck KgaA,
Germany). All solvents were of gas-distilled grade.
Microorganism and Fermentation. The strain of Peni-
cillium janczewskii Zalessky (strain KMPB H-TW5/869) was
isolated from surface water collected during a research cruise
in 1995 from the German Bight (North Sea) northeast of the
island Helgoland (54°22′0′′ N, 8°16′0′′ E). The fungus was
maintained on agar slants before the identification according
to the protocol of Pitt.4,5 The isolate was deposited as DSM17433
at the Deutsche Sammlung von Mikroorganismen und Zellkul-
turen GmbH (DSMZ, Braunschweig, Germany). Inocculum
was prepared from agar slants. After 7 days of cultivation at
25 °C spores and mycelium were cut by a cork borer and used
for inocculating the culture broth. The large-scale fermentation
was performed in resting cultures in Fernbach flasks in a total
volume of 50 L in glucose/potato extract broth.4 Screening
cultures were run in 200 mL of culture media in Erlenmeyer
flasks. The cultures were incubated at 25 °C for 29 days.
Screening Extracts. Cultures were separated by filtration.
The lyophilized mycelium was extracted with 50 mL of
methanol. The suspension was sonicated for 6 min, and
extraction was performed for 3 h. The solvent of the separated
extract was removed in vacuo, and the residue was dissolved
in 1 mL of methanol and stored at -20 °C in microplates.
Samples of the culture filtrate were prepared by solid-phase
extraction on Amberchrom 161c (Supelco).11
Downstream Processing and Purification. After up-
scale cultivation, biomass and culture liquid were separated
by filtration through cheesecloths. Biomass was lyophilized to
give 312 g of dried mycelia. The culture broth was adsorbed
on a Amberchrom CG 161c column, washed with distilled
water, and eluted with methanol. The methanolic extract was
evaporated in vacuo to give 14 g of crude extract. The
lyophilized culture filtrate was separated by flash chromatog-
raphy (RP18, MeOH/H2O, gradient) to obtain fractions 1 to 4.
Fraction 4 was purified using Sephadex LH-20 (3 × 80 cm,
MeOH) combined with repeated column chromatography
(CHCl3/MeOH, gradient) on silica gel to yield compound 3 (15
mg), compound 4 (20 mg), and a mixture of 1 and 2 The latter
was further purified by preparative HPLC (RP18, MeOH/H2O
gradient from 25/75 to 45/55 in 110 min) to obtain 1 (2 mg,
tR,1 ) 45 min) and 2 (3 mg, tR,2 ) 55 min).
Cytotoxicity Testing. The cytotoxic activities of methan-
olic extracts and pure compounds were determined in a
sulforhodamine B (SRB) staining assay (Sigma, Schnelldorf,
Germany). For a typical experiment, cells were inoculated in
96-well microtiter plates in amounts of 90 µL at plating
densities ranging from 5000 to 10 000 cells/well depending on
the doubling time of individual cell lines. After cell inoculation,
the microtiter plates were incubated for 24 h prior to addition
of the screening extracts or compounds. Extracts (50% MeOH)
or compounds solubilized in 100% MeOH or dimethyl sulfoxide
were stored frozen prior to use. The final test concentration
for pure compounds of 1 µg/test well (100 µL) with complete
medium corresponds to a micromolar test range. Following
extract or compound addition, the plates were incubated for
an additional 48 h, then washed with PBS. The assay was
terminated by fixing of the cells with cold trichloroacetic acid
solution (TCA) for 30 min on ice (final concentration: adherent
cells 10% TCA; suspension cells 16% TCA). The supernatant
was discarded, and the plates were washed five times with
tap water and air-dried. SRB solution (100 µL) at 0.4% (w/v)
in 1% acetic acid was added to each well, and plates were
incubated for 10 min at room temperature. After staining,
unbound dye was removed by washing five times with 1%
acetic acid and the plates were air-dried. Bound stain was
subsequently solubilized in 10 mM Tris/KOH (pH 10.5), and
the absorbance was read at 550 nm.
3S*,4R*-Dihydroxy-4-(4′-methoxyphenyl)-3,4-dihydro-
2(1H)-quinolinone (1): white solid; [R]15 -12.9° (c 0.7,
D
1
MeOH); IR (KBr) νmax 3300, 1705, 1684, 1604, 1511 cm-1; H
and 13C NMR data, see Table 1; HMBC correlations (DMSO-
d6) NH-1/C-2, NH-1/C-3, NH-1/C-4a, NH-1/C-8, NH-1/C-8a,
H-3/C-2, H-3/C-4, H-3/C-4a, H-3/C-1′, OH-3/C-1′, OH-3/C-3,
OH-3/C-4, OH-4/C-3, OH-4/C-4, OH-4/C-4a, OH-4/C-1′, H-5/
C-4, H-5/C-8a, H-6/C-5, H-6/C-7, H-6/C-8, H-6/C-4a, H-6/C-8a,
H-7/C-6, H-7/C-8a, H-7/C-8, H-8/C-4, H-8/C-6, H-8/C-4a, H-8/
C-8a, H2′(6′)/C-4, H2′(6′)/C-3′(5′), H2′(6′)/C-4′, H3′(5′)/C-1′, H3′-
(5′)/C-4′, OCH3/C-4′; ESIMS m/z 308.1 [M + Na]+, 285.9 [M +
H]+, 593.2 [2M + Na]+.
3R*,4R*-Dihydroxy-4-(4′-methoxyphenyl)-3,4-dihydro-
2(1H)-quinolinone (2): white solid; [R]15 -4.2° (c 0.5,
D
1
MeOH); IR (KBr) νmax 3200, 1710, 1678, 1612, 1511 cm-1; H
and 13C NMR data, see Table 1; ESIMS m/z 308.1 (M + Na)+,
593.5 [2M + Na]+; HRESIMS m/z 308.0911 [M + Na]+ (calcd
for C16H15NO4Na, 308.0899).
Acidic Dehydration of 1 and 2. To a solution of 1 and 2
(1:1, 3 mg) in acetone (2 mL) TsOH was added (0.5 mg), and
the mixture was stirred at room temperature for 2 h. After
solvent evaporation, the residue was purified by silica gel
column chromatography (P.E./EtOAc, 4:1) to give compound
5 (1 mg).
3-Hydroxy-4-(4′-methoxyphenyl)-2(1H)-quinolinone, 5:
white solid; 1H NMR (CDCl3, 400 MHz) δ 8.31 (1H, brs, NH),
7.33 (1H, d, J ) 7.6 Hz), 7.25 (1H, m), 7.11 (2H, d, J ) 9.0
Hz), 7.07 (1H, t, J ) 7.4 Hz), 6.85 (1H, d, J ) 7.9 Hz), 6.80
(2H, d, J ) 9.0 Hz), 4.35 (1H, s, OH), 3.75 (3H, s, OCH3);
ESIMS m/z 268.1 [M + H].
Acknowledgment. We thank B. Lehmann, I. Perner, F.
Rhein, B. Ritzka, K. Scheer, S. Grimm, and S. Hauck for
excellent technical assistance, and Drs. X. Huang and E.
Roemer for valuable suggestions. This collaboration project
was funded by grants from the German Federal Ministry for
Education and Research (03F0227 and CHN 98/306).
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NP058018G