FULL PAPER

DOI: 10.1002/chem.201103278

Marilines A–C: Novel Phthalimidines from the Sponge-Derived Fungus

Stachylidium sp.

Celso Almeida,[a, e] Yasmin Hemberger,[b] Sven Marcel Schmitt,[a] Sarah Bouhired,[a]

Lavanya Natesan,[a] Stefan Kehraus,[a] Konstantinos Dimas,[c] Michael Gtschow,[d]
Gerhard Bringmann,*[b] and Gabriele M. Kçnig*[a]
Dedicated to Professor Joachim Thiem on the occasion of his 70th birthday

Abstract: A marine-derived fungus of
the genus Stachylidium was isolated
from the sponge Callyspongia cf. C.
flammea. Chemical investigation of the
bioactive fungal extract led to the iso-
lation of the novel phthalimidine deriv-
atives marilines A1 (1 a), A2 (1 b), B
(2), and C (3). The absolute configura-
tions of the enantiomeric compounds
1 a and 1 b were assigned by a combina-
tion of experimental circular dichroism
(CD) investigations and quantum
Keywords: CD spectroscopy · hu-
man leukocyte elastase · marine
fungus · phthalimidine · Stachylidi-
um
chemical CD calculations. The skeleton
of marilines is most unusual, and its
biosynthesis is suggested to require un-
common biochemical reactions in
fungal secondary metabolism. Both
enantiomers, marilines A1 (1 a) and A2
(1 b), inhibited human leukocyte elas-
tase (HLE) with an IC50 value of
0.86 mm.

Introduction plants and bacteria. Fungal-derived phthalimidines are de-

Inspired by the impressive level of biodiversity in the
marine environment, the pharmacological potential of natu-
ral products from marine organisms has been intensely in-
vestigated.[1] Phthalimidines are secondary metabolites
mainly isolated from fungi, but also reported to be found in
[a] Dr. C. Almeida, S. M. Schmitt, S. Bouhired, L. Natesan,
Dr. S. Kehraus, Prof. Dr. G. M. Kçnig
Institute for Pharmaceutical Biology, University of Bonn
Nussallee 6, 53115 Bonn (Germany)
Fax: (+ 49) 228-733250
E-mail: g.koenig@uni-bonn.de
[b] Y. Hemberger, Prof. Dr. G. Bringmann
Institute of Organic Chemistry, University of Wrzburg
Am Hubland, 97074 Wrzburg (Germany)
Fax: (+ 49) 931-3184755
E-mail: bringman@chemie.uni-wuerzburg.de
[c] Dr. K. Dimas
Laboratory of Pharmacology
Faculty of Medicine, University of Thessaly, Biopolis
41110 Larissa (Greece)
[d] Prof. Dr. M. Gtschow
Pharmaceutical Institute, Pharmaceutical Chemistry I
University of Bonn
An der Immenburg 4, 53121 Bonn (Germany)
[e] Dr. C. Almeida
Current address:
Laboratory of Tropical Bioorganic Chemistry
Faculty of Natural Exact Sciences and Technology
University of Panama (Panama)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201103278.
scribed as having a wide spectrum of bioactivities, for exam-
ple, the phytotoxic activity of cichorine, a metabolite isolat-
ed from Alternaria cichorii and Aspergillus silvaticus, which
causes necrotic lesions in Russian knapweed.[2, 3] Porritoxin,
from Alternaria porri, is known to be a non-specific toxin in-
hibiting the growth of lettuce and stone-leek roots, two im-
portant commercial plants.[4–8] Stachybocins and spirodihy-
drobenzo-furanlactams were isolated from Stachybotrys sp.
and are reported to possess endothelin receptor antagonistic
effects. The latter also has HIV-1 protease inhibitory proper-
ties.[9, 10] The antifungal pestalachlorides isolated from Pesta-
lotopsis adusta,[11] the aldose reductase inhibitors salfredins
from Crucibulum sp.,[12] and the cytotoxic hericenone B iso-
lated from Hericium erinaceum[13] additionally demonstrate
the broad spectrum of bioactivity found for fungal-derived
phthalimidine-like structures.
During our search for new natural products produced
from the marine-derived fungus Stachylidium sp. four novel
phthalimidine derivatives, marilines A1/2–C (Figure 1) were
isolated from a culture grown on agar-biomalt medium sup-
plemented with artificial sea salt. The fungus is not able to
grow without the sea salt supplement. Although phthalimi-
dine-like structures are not rare, the structural skeleton of
marilines A1/2–C is most unusual, and its biosynthesis is sug-
gested to require unique reactions in fungal secondary me-
tabolism. Marilines A1/2–C were tested in a broad range of
assays to determine their biological activity. Both enantio-
mers, marilines A1 (1 a) and A2 (1 b), inhibited human leuko-
cyte elastase (HLE) with the same potency (IC50 = 0.86 mm).

Chem. Eur. J. 2012, 18, 8827 – 8834  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 8827
 Results and Discussion

Structural elucidation of marilines A–C (1–3): The molecu-

lar formula of compound 1 was deduced by accurate mass
measurement (HREIMS) to be C33H43NO5, requiring 13 de-
grees of unsaturation. The 13C NMR and DEPT135 spectra
contained 33 carbon resonances, including nine resulting
from methyl groups, seven from sp2 methines, one from
a sp3 methine, and four signals from methylene groups,
while the remaining 12 resonances were assigned to quater-
nary carbon atoms. 1H NMR and 1H,1H COSY spectra
showed four resonance signals in the aromatic region (dH =
6.95 (s), 6.67 (d), 6.56 (dd), 7.19 (d) ppm; Table 1) indicating
together with 13C NMR and 1H,13C HMBC data the pres-
ence of two phenyl moieties, one of them 1,2,4- and the
other pentasubstituted. Furthermore, the molecule was
Figure 1. Structural formulae of compounds 1–3.
found to contain three trisubstituted double bonds (dC =
120.6, 141.5, 124.6, 132.0, 120.8, 137.9 ppm), which were part
of a mono- and a hemiterpene moiety. The C1’ to C10’ part
of the molecule was deduced from two proton coupling spin

Table 1. 1H (300 MHz) and 13C (75 MHz) NMR spectroscopic data for marilines A–C (1–3).
Position 1 ([D6]acetone) 2 ([D6]acetone) 3 ([D4]MeOH)
dH[a] (J in Hz) dN, dC, mult.[a,b] dH[a] (J in Hz) dC, mult.[a,b] dH[a] (J in Hz) dC, mult.[a,b]
132.4, N
1 166.2, qC 167.3, qC 171.7, qC
2 116.4, qC 116.3, qC 114.5, qC
3 157.2, qC 156.9, qC 158.4, qC
4 119.4, qC 119.3, qC 118.9, qC
5 161.9, qC 161.7, qC 162.3, qC
6 6.95, s 101.8, CH 6.92, s 101.8, CH 6.68, s 105.0, CH
7 150.0, qC 150.0, qC 152.1, qC
8 4.97, q (6.8) 57.5, CH 4.62, q (6.8) 56.9, CH 4.53, q (6.8) 53.2, CH
9 1.27, d (6.8) 19.4, CH3 1.44, d (6.8) 18.9, CH3 1.42, d (6.8) 20.8, CH3
10 3.99, s 62.1, CH3 3.98, s 62.1, CH3 3.95, s 62.5, CH3
11 2.12, s 8.8, CH3 2.09, s 8.8, CH3 2.15, s 8.6, CH3
12 120.1, qC a: 3.33, dt (14.0, 5.5) 43.6, CH2
b: 3.85, dt (14.0, 5.5)
13 156.8, qC 3.71, m 61.7, CH2
14 6.67, d (2.6) 101.2, CH
15 160.8, qC
16 6.56, dd (2.6, 8.6) 105.5, CH
17 7.19, d (8.6) 131.9, CH
18 3.81, br s 55.7, CH3
1’ a: 4.68, dd (6.6, 12.3) 66.2, CH2 a: 4.65, dd (6.6, 12.3) 66.2, CH2
b: 4.72, dd (6.6, 12.3) b: 4.71, dd (6.6, 12.3)
2’ 5.53, t (6.6) 120.6, CH 5.51, t (6.6) 120.6, CH
3’ 141.5, qC 141.5, qC
4’ 2.12, m 40.1, CH2 2.12, m 40.1, CH2
5’ 2.14, m 27.0, CH2 2.14, m 27.0, CH2
6’ 5.10, t (6.6) 124.6, CH 5.10, t (6.6) 124.6, CH
7’ 132.0, qC 132.1, qC
8’ 1.63, br s 25.8, CH3 1.63, br s 25.8, CH3
9’ 1.77, br s 16.7, CH3 1.76, br s 16.7, CH3
10’ 1.59, br s 17.7, CH3 1.58, br s 17.7, CH3
1’’ a: 4.54, dd (6.6, 12.0) 66.1, CH2
b: 4.58, dd (6.6, 12.0)
2’’ 5.35, t (6.6) 120.8, CH
3’’ 137.9, qC
4’’ 1.68, br s 18.2, CH3
5’’ 1.67, br s 25.7, CH3
[a] Assignments are based on extensive 1D and 2D NMR experiments (1H,13C, 1H,15N HMBC, HSQC, COSY). [b] Implied multiplicities determined by
DEPT.

8828 www.chemeurj.org  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Chem. Eur. J. 2012, 18, 8827 – 8834
Marilines A–C
systems, the first from 2’-H to 1’-H2 and 9’-H3, and the
second ranged from 4’-H through to 10’-H3 and 8’-H3. 1H,13C
HMBC data correlated 9’-H3 to C2’, C3’ and C4’, which dis-
closed the structure of this terpenoid fragment. Based on lit-
erature comparisons we established the configuration of the
D2’/3’ double bond as E.[14] 1H NMR resonance signals that
arose from the hemiterpene unit C1’’ to C5’’ included two
singlet methyl resonances, that is, CH3-4’’ (dH = 1.68 (br s)
ppm) and CH3-5’’ (dH = 1.67 (br s) ppm), both with 1H,13C
HMBC correlations to C3’’ and the sp2 methine CH-2’’
(dH = 5.35 (t) ppm). 2’’-H coupled to the oxygenated methyl-
ene protons 1’’-H2, thus completing this partial structure.
A 13C NMR signal at dC = 57.5 ppm (C8) was found to be
characteristic for a carbon atom neighboring a nitrogen
atom, which according to its chemical shift in the 15N NMR
spectrum (dN = 132.4 ppm) was part of an amide functionali-
ty. The 1H,1H COSY showed correlations from 8-H to 9-H3.
Furthermore, 1H,13C HMBC spectra showed correlations be-
tween 8-H and the carbonyl carbon C1, as well as to C6, C7
and C2 of the pentasubstituted aromatic ring. 1H,13C HMBC
correlations between 9-H3 and C7 as well as 1H,15N HMBC
correlations between 9-H3 and the amide nitrogen (Table
S1; Supporting Information) gave evidence for a phthalimi-
dine skeleton, that is, the C1 to C9 part of the structure.
One of the substituents of the aromatic ring of the phthal-
ACHTUNGREimidine moiety was established to be a methyl group (CH3-
11, dH = 2.12 (s) ppm), which was positioned at C4 as de-
duced from HMBC correlations. The quaternary carbons C3
and C5 had 13C NMR shifts typical of aromatic carbons
bound to oxygen. The methoxy group OCH3-10 was estab-
lished to be located at C3, as evident from 1H,13C HMBC
correlations. CH2-1’ of the monoterpene moiety was at-
tached to oxygen as concluded from the chemical shifts of
C1’ (dC = 66.2 ppm) and 1’-H2 (dH = 4.68 (dd), 4.72 (dd)
ppm). 1’-H2 showed a long-range heteronuclear coupling
with C5 of the aromatic ring via an oxygen bridge, thus plac-
ing the terpene moiety at C5. The latter deduction was also
supported by 1H,1H NOESY correlations from 1’-H2 to 6-H.
Three further resonance signals in the 1H NMR spectrum
resulted from a trisubstituted benzene ring. 16-H and 17-H
(dd, J = 8.6 Hz) were placed in ortho position to each other,
while 14-H and 16-H (d, J = 2.6 Hz) were meta-positioned.
1
H,13C HMBC correlations from 1’’-H2 to C13 and from
OCH3-18 to C15 showed that the oxygenated quaternary ar-
omatic carbons C13 (dC = 156.8 ppm) and C15 (dC =
160.8 ppm) were connected to a hemiterpene (C1’’ to C5’’)
and a methoxy moiety (OCH3-18), respectively. 1H,1H
NOESY correlations of 18-H3 to 14-H and 16-H, and of 1’’-
H2 to 14-H confirmed the position of these substituents.
1
H,15N HMBC measurements showed two distinct correla-
tions from 9-H3 and 17-H to the nitrogen atom (dN =
132.4 ppm), evidencing that the tri-substituted benzene ring
is connected to the phthalimidine nucleus via C12. 1H,1H
NOESY correlations from 17-H to 9-H3 and to 8-H con-
firmed the structure. We propose the trivial name mariline
A (1) for this new compound (Figure 1).
Chem. Eur. J. 2012, 18, 8827 – 8834  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
FULL PAPER
The molecular formula of 2 was determined by accurate-
mass measurements (HRESIMS) to be C23H33NO4, requiring
eight degrees of unsaturation. The C1 to C9 and C1’ to C10’
parts of the molecule were established to be identical to
those of compound 1 based on 13C and 1H NMR, as well as
1
H,13C HMBC and 1H,1H COSY spectra (Table 1, and Sup-
porting Information). The structure of 2, however, was lack-
ing the second aromatic ring and an ethyl-1-ol moiety was
linked to the nitrogen atom instead. This was evidenced by
heteronuclear long-range correlations from 12-H2 to C8 and
C1 through the nitrogen atom. Furthermore, the methylene
CH2-13 showed proton coupling with CH2-12, whereby the
13
C NMR shift of C13 indicated that it was connected to
a hydroxy group (dC = 61.7 ppm). This completed the planar
structure of compound 2, for which we propose the name
mariline B.
The molecular formula of 3 was deduced by accurate-
mass measurements (HRESIMS) to be C11H13NO3, yielding
six degrees of unsaturation. NMR data suggested the pres-
ence of a phthalimidine nucleus with most substituents on
the aromatic ring as present in 1 and 2. Compound 3, how-
ever, possessed a free hydroxy group, which was confirmed
by an IR absorption band at 3346 cm 1. This OH group was
placed at C5, which, from its chemical shift (dC = 162.3 ppm)
was clearly an oxygenated aromatic carbon atom. The spec-
troscopic data for compound 3 did not show any signals for
a substituent on the nitrogen atom, which is thus present as
an NH group. We propose the name mariline C for 3.
Stereochemical issues: Although compounds 1–3 each pos-
sess a chiral carbon, C8, the specific optical rotations were,
in all cases, close to zero, and CD measurements produced
only marginal CD effects. The structures of the molecules
suggested a CD effect (expected at around 260 nm) due to
the proximity of the stereogenic center to the chromophore.
It was thus assumed that these chiral compounds had been
obtained as racemic mixtures. Optical resolution of com-
pound 1 by analytical HPLC on a chiral stationary phase
(Lux cellulose-1; Phenomenex; 25 cm  0.46 cm) resulted in
two base-line separated peaks in ratios of approximately 1:1,
3:2, and 3:7 in three independent cultures. To exclude the
existence of a rotationally hindered N,C axis as a second
possible stereogenic element and, therefore, the possible oc-
currence of diastereomers, quantum-chemical calculations
were carried out. The molecular geometries of both, the
ground state and the transition states of the rotation around
the N,C axis of the global minimum of mariline A were opti-
mized by DFT using B3LYP/6-31G*.[15–17] These structures
were subjected to RI-SCS-MP2/TZVP[18, 19] single-point cal-
culations to provide more reliable values for the enthalpy of
formation leading to a rotational barrier of DE°1 = 11.6 kcal
mol 1 for the atropisomerization from (M)- to (P)-1 and of
DE°2 = 14.4 kcal mol 1 for the one from (P)- to (M)-1. The
calculated values evidenced a free rotation around the N,C-
axis and therefore in the following stereochemical character-
ization only the configuration at the stereogenic center at
C8 had to be elucidated. Furthermore, the values of the ro-
www.chemeurj.org 8829

tational barrier showed that the M-atropo-diastereomer was
energetically much more favored as compared to the P-
isomer, so that, although 1 is configurationally unstable at
the N,C axis, axial chirality does play a role. The reason is
probably the larger steric repulsion between the methyl
group at C8 and the alkoxyl side chain at the benzyl part of
mariline A in the P conformation (Figure 2).
Figure 2. Molecular structures of the main conformers of (M,S)-1 and
(P,S)-1.
For the two enantiomeric forms we propose the names
marilines A1 (1 a) and A2 (1 b). After chromatographic reso-
lution of 1 a and 1 b by HPLC on a chiral stationary phase,
their CD spectra were measured, showing opposite CD ef-
fects, confirming the existence of enantiomers (Figures 3,
S15 and S16; Supporting Information). The absolute config-
urations of 1 a and 1 b were determined by the combination
of electronic circular dichroism (CD) spectroscopy and
quantum-chemical CD calculations, an efficient and reliable
method for the assignment of absolute stereostructures.[20]
Due to the very high molecular flexibility of mariline A
(1), a detailed investigation of the conformational space of
Figure 3. Assignment of the absolute configurations of the two enantiomers of mariline A (1) by comparison of experimental and quantum-chemically
calculated CD spectra.
8830 www.chemeurj.org  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
G. Bringmann, G. M. Kçnig et al.
compound 1 was carried out using the B3LYP/SV(P)[21]
method, yielding more than 550 minimum structures. For all
conformers that contributed to the overall CD spectrum by
more than 0.5 % TDB3LYP CD calculations were per-
formed, in combination with the COSMO model[22, 23] to ac-
count for solvent effects. After Boltzmann weighting accord-
ing to the relative energies from the DFT optimizations, the
comparison of the calculated CD spectrum with the experi-
mental one did not permit an unambiguous assignment of
the absolute configuration. This problem was most likely as-
sociated with dispersion effects caused by the highly flexible
alkoxy side chains. For an improvement one possibility
would be to perform the complete conformational analysis
with, for example, the dispersion-corrected B97-D functional
in combination with a larger basis set like TZVP. To prevent
the very time-consuming renewed DFT optimization of all
550 conformers, another option is the calculation of single-
point energies for all previously DFT-optimized structures
with RI-SCS-MP2 in combination with the TZVP basis set.
Especially in this case, with such a high number of conform-
ers, a very accurate valuation of the energies with regard to
the Boltzmann weighting was indispensable. These single-
point calculations led to 86 relevant conformers, which
nearly all possessed the M conformation (within a range of
3.5 kcal mol 1 above the global minimum). Boltzmann
weighting with these more reliable values for the enthalpy
of formation now yielded a significantly improved overall
CD spectrum. The curve calculated for (R)-1 almost exactly
matched the experimental one of Peak A (Figure 3, left),
while the CD curve predicted for (S)-1 was in good agree-
ment with the CD spectrum measured for Peak B (Figure 3,
right). These results led to the clear attribution of the abso-
lute configurations of the two enantiomers of mariline A
Chem. Eur. J. 2012, 18, 8827 – 8834
Marilines A–C
(1), showing that the more rapidly eluting stereoisomer,
mariline A1 (1 a), has the 8R configuration and mariline A2
(1 b), the more slowly eluting enantiomer, is S-configured at
C8.
Racemization and Biosynthesis: Several cases of racemiza-
tion reactions on natural products with a chiral center in
lactam, lactone or dihydrofuran rings have previously been
reported,[11, 24–26] which suggested a post-biosynthetic racemi-
zation also for 1 a or 1 b. A similar case is the dihydroisoben-
zofuran derivative pestacin, with the chiral carbon C1 in a-
position to the oxygen in the dihydrofuran ring.[25] Racemi-
zation in the case of pestalachloride A, isopestacin, and pes-
tacin was proposed to occur after their biosynthetic forma-
tion, via resonance-stabilized cationic intermediates. Based
on structural considerations, a similar mechanism may be
feasible for 1 a and 1 b (Figure S31a; Supporting Informa-
tion), but less favored since the substituent at C8 is a methyl
group whereas in pestalachloride A (and in a similar fashion
in isopestacin and pestacin) an aromatic moiety is located at
the respective position, so that the intermediate is a benzyl
cation. Recently, Schmalz and co-workers showed that pest-
alone can be converted to a racemic mixture of pestalachlor-
ide A under mild conditions with NH3 (Figure S31b; Sup-
porting Information).[27] In a similar way compounds 1 a and
1 b might also be formed from the respective precursors
(Figure S31c; Supporting Information). Noteworthy is also
a previous literature report on the easy racemization of the
phthalimidine-like compound pagoclone.[28] It was reported
that this isoindolinone already racemized under weakly
basic or acidic conditions and at moderately elevated tem-
peratures. Racemization of pagoclone was found to be rapid
with 0.005 n KOH at 50 8C for 2 h, and suggested to occur
via a retro-Michael/Michael reaction. This type of reaction
is, however, unlikely to occur in the case of 1 a and 1 b. Nev-
ertheless, to evaluate whether racemization had occurred
during the extraction and isolation process, enantiomerically
pure marilines A1 and A2 were subjected to increasing con-
centrations of KOH and HCl as outlined in the experimen-
tal section. But even under harsh conditions no racemization
was observed as evidenced by HPLC analysis on a chiral
phase (Figures S18 and S19; Supporting Information). Since
racemization of marilines A1 or A2 does not occur under
various reaction conditions, it may be concluded that both
enantiomers are genuine products of the biosynthetic pro-
cess and, thus, not the result of post-biosynthetic racemiza-
tion reactions.
Compounds 2 and 3 were collected as racemic mixtures,
too, since again no CD effects and no optical rotation values
were obtained. Exhaustive attempts to separate the enantio-
mers with three different chiral stationary HPLC columns
were unsuccessful.
Compounds with a related phthalimidine (isoindolinone)
nucleus as found for 1–3 are known from fungal metabolism,
however, they all lack the methyl group at C8. The structur-
ally most simple phthalimidines possess an unsubstituted ni-
trogen atom as found in mariline C (3), namely 6-hydroxy-
Chem. Eur. J. 2012, 18, 8827 – 8834  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
FULL PAPER
4-methoxy-5-methylphthalimidine (cichorine),[2, 3] zinnimi-
dine,[4] duricaulic acid,[29] memnobotrin A and the related
stachybotrylactam[30] and the more complex xylactam.[31] Re-
ported N-substituted phthalimidines include compounds
with a phenylethyl,[13, 32] ethyl-1-ol,[6, 7] or a carbonic acid[9, 12]
moiety attached. Of these, examples of fungal phthalimi-
dines structurally related to marilines A1 and A2 (1 a and
1 b) include hericenone B and stachybotrin C,[13, 32] both with
a phenylethyl moiety connected to the nitrogen atom. In
compounds 1 a and 1 b, however, the nitrogen is substituted
with a derivatized phenyl ring, hitherto, only present in
a similar way in the phthalimidine structure clitocybin A.[33]
Porritoxin, porritoxin sulfonic acid,[6, 7] memnobotrin B,[30]
and stachybotramide[34] contain an N-ethyl-1-ol moiety and
are structurally related to mariline B (2) (all related struc-
tures are depicted in Figure S32; Supporting Information).
Compounds 1–3 are related to their oxygen-containing
counterparts, phthalides, which are more common in
nature.[35] Biosynthetic studies focusing on phthalide struc-
tures, for example, for mycophenolic acid[36] or for phtha-
lide-like metabolites isolated from Talaromyces flavus,[37]
evidenced the tetraketide nature of the phthalide nucleus.
Compounds 1–3 also possess the basic skeleton of the tetra-
ketide 3-methyl-orsellinic acid, yet with the acetate-derived
methyl group in orsellinic acid replaced by an ethyl group in
the precursor molecules of 1–3.[38] To our knowledge no bio-
synthetic studies have so far been performed for phthalimi-
dine-like molecules. The biosynthesis of phthalimidine, how-
ever, most probably involves similar polyketide-type reac-
tions as known for phthalides.
The most unusual structural characteristic of the phthal-
ACHTUNGREimidine derivatives 1–3 is the methyl substituent at C8. Its
biosynthetic origin seems to require either a propionate
starter unit or a C-methylation (e.g., via an SAM-dependent
methyltransferase) at C8. A third possibility would be the
loss of a carbon atom from a pentaketide intermediate. To
our knowledge, only early publications in the 1960s on the
fungal metabolite barnol showed C-methylation of an ace-
tate starter unit (incorporation of CH3 from SAM) to finally
give an ethyl side chain (Figure S33; Supporting Informa-
tion). In these studies no incorporation of propionate was
observed.[39] In 1989 Kawahara et al. published the structure
of fungal metabolites where such a biosynthetic scenario can
also be envisaged, for example, the dihydrocoumarine stella-
tin (Figure S34; Supporting Information).[40] Studies in 1981
and 1988 on the biosynthesis of the fungal polyketides
pseurotin A, austrocorticinic acid,[41, 42] and aurovertin B[43]
report incorporation of a propionate starter unit, and in the
case of aurovertin B additionally the C-methylation of an
acetate starter unit evidenced by feeding studies. Generally,
however, it is believed that fungi do not incorporate propio-
nate in their metabolites.[44] In the light of these early litera-
ture reports and the compounds reported here, this assump-
tion has to be questioned.
Biological activity: Marilines A1 and A2 inhibited the serine
protease HLE with an IC50 value of 0.86 mm for both enan-
www.chemeurj.org 8831

tiomers (Figure S22 and Table S10; Supporting Informa-
tion). Cholesterol esterase from porcine pancreas was par-
tially inhibited (Figure S23 and Table S10; Supporting Infor-
mation). In both cases a large “offset”, that is, the remaining
cholesterol esterase activity in the presence of infinite inhib-
itor concentration, was observed. The IC50 values obtained
were 0.18 mm (offset = 49  3 %) for mariline A1 and 0.63 mm
(offset = 54  3 %) for mariline A2. Thus, both compounds
are only capable to reduce the activity of cholesterol ester-
ase by approximately 50 %. Such a kinetic behavior might
reflect binding of the compounds to a site of cholesterol es-
terase different from the active site.[45] Mariline B (2)
showed no inhibitory activity towards HLE and cholesterol
esterase (IC50 > 10 mm, Table S10; Supporting Information).
Compound 3 was not tested in these assays, due to the small
amounts isolated. Marilines A1 and A2 were further assayed
for their activities against the proteases bovine chymotryp-
sin, bovine trypsin, papain from Carica papaya, and the es-
terase acetylcholinesterase from Electrophorus electricus
and found to be inactive (IC50 > 10 mm, S30; Supporting In-
formation).
Mariline A1 was tested against five cancer cell lines and
exhibited a mean GI50 of 24.4 mm, while mariline A2 was
tested against 19 cancer cell lines and showed a GI50 of
11.02 mm. The observed antiproliferative pattern of mariline
A2 did not correlate with that of any of the standard anti-
proliferative compounds with known mechanism of action
as deduced by COMPARE analysis. The first match on the
standard agents database (correlation 0.427) was rhizoxin,
a metabolite produced by a bacterium associated with
a fungus (Table S8; Supporting Information).
Marilines A1, A2, and B were tested for antiplasmodial ac-
tivity and exhibited IC50 values of 6.68, 11.61, and 13.84 mm,
respectively, against the liver stage of Plasmodium berghei.
Marilines B and C showed also antagonistic activity on the
cannabinoid receptor CB2 with Ki values of 5.97 and
5.94 mm, respectively. Mariline A2 further exhibited antago-
nistic activity against the histamine receptor H2 with a Ki
value of 5.92 mm, the dopamine receptor DAT with a Ki
value of 5.63 mm, and the adrenergic receptor Beta3 with
a Ki value of 5.63 mm (Figures S26–28; Supporting Informa-
tion).
Marilines A1/2–C were further evaluated for inhibition of
protein kinases and the growth of rifampicin-resistant Myco-
bacterium tuberculosis but no activity was found (detailed
description in Supporting Information; S30). Furthermore,
mariline A1 was tested against the pathogens of the tropical
infectious diseases African sleeping sickness and leishmania-
sis. The compound showed only very moderate anti-parasitic
activity against Trypanosoma brucei brucei with an IC50 of
17.7 mm, and was not active against Leishmania major
(IC50 > 100 mm). The other compounds were not tested for
reasons of lacking availability.
The most interesting activity obtained from the multitude
of bioactivity tests was found towards HLE. This serine pro-
tease is considered to be a primary source of tissue damage
associated with inflammatory diseases such as chronic ob-
8832 www.chemeurj.org  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
G. Bringmann, G. M. Kçnig et al.
structive pulmonary disease, cystic fibrosis, and adult respi-
ratory distress syndrome.[46, 47] Inhibition of HLE is thera-
peutically useful in the treatment of such diseases and there
has been considerable interest in developing small molecular
weight inhibitors of HLE.[46–49]
Conclusion
Marilines A–C, isolated from the marine-derived fungus Sta-
chylidium sp., represent novel phthalimidine derivatives
with most unusual structures. Their biosynthesis is suggested
to require reactions unique in fungal secondary metabolism.
Among a multitude of bioactivities found, the most interest-
ing activity was towards human leukocyte elastase (HLE),
an enzyme which is considered to be a primary source of
tissue damage associated with inflammatory diseases such as
chronic obstructive pulmonary disease, cystic fibrosis, and
adult respiratory distress syndrome. This study demonstrates
the great potential of marine-derived fungi to produce com-
pounds with unprecedented structures and promising biolog-
ical activities.
Experimental Section
General procedures: Optical rotations were measured on a Jasco DIP
140 polarimeter. UV and IR spectra were obtained using a Perkin–Elmer
Spectrum BX instrument. CD spectra were recorded in MeOH at room
temperature using a JASCO J-810-150S. All NMR spectra were recorded
in [D4]MeOH or [D6]acetone employing a Bruker Avance 300 DPX spec-
trometer. Spectra were referenced to residual solvent signals with reso-
nances at dH/C 3.35/49.0 ppm for [D4]MeOH and dH/C 2.04/29.8 for
[D6]acetone ppm. The 1H-15N HMBC spectrum was referenced externally
to urea (the 15N chemical shift is reported relative to liquid ammonia).
HREIMS were recorded on a Finnigan MAT 95 spectrometer. HRE-
SIMS were measured on a Bruker Daltonik micrOTOF-Q Time-of-Flight
mass spectrometer with ESI source. HPLC was carried out using
a system composed of a Waters 515 pump together with a Knauer K-
2300 differential refractometer. HPLC columns were from Knauer (250 
8 mm, 5 mm, Eurospher-100 Si and C18, flow rate 2 mL min 1), and from
Macherey–Nagel (250  4.6 mm, 5 mm, Nucleodur Isis, C18, flow rate:
1 mL min 1). For resolution of enantiomeric mixtures the HPLC column
Lux Cellulose-1 from Phenomenex (250  4.5 mm, 5 mm) was used with
a flow rate of 1 mL min 1 (n-hexane/isopropanol 9:1). Merck silica gel 60
(0.040–0.063 mm, 70–230 mesh) was used for vacuum liquid chromatogra-
phy (VLC). Columns were wet-packed under vacuum using petroleum
ether (PE). Before applying the sample solution, the columns were equi-
librated with the first designated eluent. Standard columns for crude ex-
tract fractionation had dimensions of 13  4 cm.
Fungal material: The marine-derived fungus Stachylidium sp. was isolat-
ed from the sponge Callyspongia sp. cf. C. flammea and identified by Dr.
P. Massart and Dr. C. Decock from Belgian Coordinated Collections of
Microorganisms, Mycothque de lUniversit Catholique de Louvain
(BCCM/MUCL). A specimen is deposited at the Institute for Pharma-
ceutical Biology, University of Bonn, isolation number “293 K04”, run-
ning number “220”.
Culture, extraction and isolation: Compounds 1–3 were isolated from
two different cultures of Stachylidium sp., a first one with 12 L and
a second one with 10 L of media volume. Both were performed on an
agar-biomalt medium (biomalt 20 g L 1, agar 15 g L 1 agar) supplemented
with sea salt during 2 months (12 L culture) and 40 d (10 L culture). An
extraction with EtOAc (5 L) yielded 5.9 g and 2.1 g of extract, respective-
Chem. Eur. J. 2012, 18, 8827 – 8834
Marilines A–C
ly. EtOAc extracts were subjected to VLC fractionation in a silica open
column using a gradient solvent system with petroleum ether/acetone at
10:1, 5:1, 2:1, 1:1, 100 % acetone and 100 % MeOH, resulting thus 6
VLC fractions for each culture. Compounds 1 a and 1 b were isolated
from both cultures, whereas 3 was only isolated from the first culture
(12 L), and 2 from the second culture (10 L).
Compounds 1 a and 1 b were isolated from VLC 2, followed by NP-
HPLC fractionation using petroleum ether/acetone 11:1 (fraction 2 of 7)
and RP-HPLC fractionation using 90 % MeOH (fraction 3 of 4; 12.6 mg,
tR = 31 min). The racemic mixture was then separated using a Phenomen-
ex Lux Cellulose-1 HPLC column using n-hexane/isopropanol 9:1 (mari-
line A1; 7.2 mg, tR = 21 min; mariline A2 ; 4.8 mg, tR = 29 min). Compound
2 was isolated from VLC 4, followed by NP-HPLC fractionation using
petroleum ether/acetone 9:2 (fraction 5 of 7; 2.6 mg, tR = 48 min). Com-
pound 3 was isolated from VLC 4, followed by NP-HPLC fractionation
using petroleum ether/acetone 2:1 (fraction 4 of 7), followed by RP-
HPLC with a Macherey–Nagel Isis column using 30 % MeOH (fraction 2
of 3; 1.4 mg, tR = 7 min).
Mariline A1 ACHTUNGRE(1 a): Colorless oil (600 mg L 1, 0.12 %); [a]23
D = + 14 (c =
0.225 in acetone); 1H and 13C NMR data see Table 1; IR (ATR): n = 3330
(br), 2925, 1668, 1605 cm 1; UV/VIS (MeOH): lmax (loge) = 219 (4.11),
268 nm (3.73 mol 1 dm3 cm 1); CD (MeOH; c = 2.8  10 4 mol L 1):
De 202 = 9.0, De 218 = + 12.4, De 248 = + 6.1, De298 = 3.2 cm2 mol 1;
LREIMS: m/z: 533.3 [M] + ; HREIMS: m/z: calcd for C33H43NO5 :
533.3141 [M] + ; found: 533.3139.
Mariline A2 ACHTUNGRE(1 b): Colorless oil (400 mg L 1, 0.08 %); [a]23
D = 14 (c = 0.175
in acetone); 1H and 13C NMR data see Table 1; IR (ATR): n = 3330 (br),
2925, 1668, 1605 cm 1; UV/VIS (MeOH): lmax (loge) = 219 (4.11), 268 nm
(3.73 mol 1 dm3 cm 1); CD (MeOH; c = 3.1  10 4 mol L 1): De 202 = + 15.8,
De 218 = 12.2, De 248 = 5.2, De 293 = + 4.3 cm2 mol 1; LREIMS: m/z: 533.3
[M] + ; HREIMS: m/z: calcd for C33H43NO5 : 533.3141 [M] + ; found:
533.3139.
Mariline B (2): White amorphous solid (260 mg L 1, 0.124 %); 1H and
13
C NMR data see Table 1; IR (ATR): n = 3391 (br), 2926, 1664,
1607 cm 1; UV/VIS (MeOH): lmax (loge) = 207 (4.46), 262 nm
(3.95 mol 1 dm3 cm 1); LRESIMS: m/z: 388.5 [M+H] + ; HRESIMS: m/z:
calcd for C23H33NO4Na: 410.2302 [M+Na] + ; found: 410.2290.
Mariline C (3): Amorphous solid (108 mg L 1, 0.022 %); 1H and 13C NMR
data see Table 1; IR (ATR): n = 3346 (br), 2926, 1670, 1610 cm 1; UV/
VIS (MeOH): lmax (loge) = 216 (3.90), 259 nm (3.71 mol 1 dm3 cm 1);
LRESIMS: m/z: 206.0 [M H] , 207.9 [M+H] + ; HRESIMS: m/z: calcd
for C11H13NO3Na: 230.0788 [M+Na] + ; found: 230.0786.
Racemization experiments: Racemization experiments were based on lit-
erature data,[28] where racemization at a chiral-substituted phthalimidine
nucleus was achieved under very low acidic/alkaline conditions. Each ex-
periment contained 200 mg of mariline A1 dried in a stream of N2 in
a glass vial which was suspended in 400 mL solution with the following
conditions: a) water, b) 0.0005 m KOH, c) 0.0025 m KOH, d) 0.5 m KOH,
e) 0.0005 n HCl, d) 0.5 n HCl. 200 mg mariline A2 were also tested at the
range of 0.5 m KOH. All samples were then incubated in a water bath at
55 8C for 2 h, after which they were neutralized with the same molar pro-
portion of acid/alkaline solution. After drying in gaseous nitrogen the
samples were injected in an HPLC chiral-phase column with the same
solvent system as used to separate the enantiomers marilines A1 and A2
for detection of racemization (see General Experimental Procedures).
Bioassays: Marilines A1 and A2 (and, where possible, mariline B) were
tested towards a panel of proteases and esterases, that is, bovine chymo-
trypsin, bovine trypsin, HLE, papain from Carica papaya, porcine choles-
terol esterase, and acetylcholinesterase from Electrophorus electricus as
described earlier.[45, 50–52] Protein kinases DYRK1A and CDK5 were as-
sayed according to the procedure described by Bettayeb et al.[53] Cytotox-
ic activity against a panel of cancer cell lines was performed according to
Saroglou et al. and Monks et al.[54, 55] Antiplasmodial activity against Plas-
modium berghei liver stages was assayed as described by Ploemen
et al.[56] Compounds were further tested against two antibiotic resistant
strains of Mycobacterium tuberculosis according to the procedure de-
scribed by Bauer et al.[57] Binding assays were performed against a panel
of 46 psychoactive receptors (activity considered with at least 50 % inhib-
Chem. Eur. J. 2012, 18, 8827 – 8834  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
FULL PAPER
ition at the 10 mm level), namely 5HT1A, 5HT1B, 5HT1D, 5HT1E,
5HT2A, 5HT2B, 5HT2C, 5HT3, 5HT5 A, 5HT6, 5HT7, Alpha1A,
Alpha1B, Alpha1D, Alpha2A, Alpha2B, Alpha2C, Beta1, Beta2, Beta3,
BZP Rat Brain Site, D1, D2, D3, D4, D5, DAT, DOR, Gaba A, H1, H2,
H3, H4, KOR, M1, M2, M3, M4, M5, MOR, NET, SERT, Sigma1,
Sigma2, CB1, CB2. Experiments are fully described by the bioassay pro-
viders.[58] Anti-parasitic activities against Leishmania major and Trypan-
soma brucei brucei were assessed as described earlier.[59, 60]
Computational details: Conformational analyses, the TDDFT excited-
states calculations, and the calculations of the single-point energies for
1 were carried out using the ORCA software package.[61] Optimizations
were performed using the B3LYP functional in combination with the
Ahlrich’s split-valence double-zeta basis set. Based on the DFT optimized
structures single UV and CD spectra were simulated with B3LYP/SV(P)
(nstates = 50) in combination with COSMO, with corresponding oscillator
and rotational strength values from the length formalism.[62] For Boltz-
mann statistical weighting, RI-SCS-MP2/TZVP single-point energies
were used. SpecDis 1.51[63] was used to sum up single UV and CD spec-
tra, for the generation of Gaussian curves (using a sigma value of
0.16 eV), UV shifting (11 nm),[64, 65] and for comparison with the experi-
mental CD data.
Acknowledgements
We thank the kind help of Dr. Laurent Meijer (Protein Phosphorylation
& Disease, CNRS, Roscoff, France) for performing the protein kinases
assays and the effort of Dr. Miguel PrudÞncio (Instituto de Medicina Mo-
lecular, Malaria Unit, Faculdade de Medicina, Universidade de Lisboa,
Portugal) for performing the antiplasmodial activity assays; the Ki deter-
minations and antagonist functional data were generously provided by
the National Institute of Mental Healths Psychoactive Drug Screening
Program, Contract # HHSN-271-2008-00025-C (NIMH PDSP). The
NIMH PDSP is directed by Bryan L. Roth MD, PhD at the University of
North Carolina at Chapel Hill and Project Officer Jamie Driscol at
NIMH, Bethesda MD, USA. The authors wish to thank Stephanie Haut-
mann for technical assistance. We also kindly thank the financial support
from FCT (Science and Technology Foundation, Portugal). We are also
grateful to the Deutsche Forschungsgemeinschaft (DFG, SFB 630 “Rec-
ognition, Preparation, and Functional Analysis of Agents against Infec-
tious Diseases” and FOR 845 “Post-Genomic Strategies for New Antibi-
otic Drugs and Targets”) for funding and to Prof. Dr. Heidrun Moll and
PD Dr. August Stich for the anti-infective tests.
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Received: October 18, 2011
Revised: March 20, 2012
Published online: June 18, 2012
Chem. Eur. J. 2012, 18, 8827 – 8834