Conjugates of the fungal cytotoxin illudin M with improved tumour specificity

Article abstract of DOI:10.1016/j.bmc.2008.08.015

A simplified procedure for the isolation of gram quantities of illudin M from culture broths of basidiomycete Omphalotus olearius is described. Esters of illudin M with docosahexaenoic acid, chlorambucil, demethylcantharidinic acid (endothall) and 2,2′-bi

Full text of DOI:10.1016/j.bmc.2008.08.015

Bioorganic & Medicinal Chemistry 16 (2008) 8592–8597
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Conjugates of the fungal cytotoxin illudin M with improved tumour specificity
a
a
b
Rainer Schobert a, , Bernhard Biersack , Sebastian Knauer , Matthias Ocker
*
a Organic Chemistry Laboratory, University of Bayreuth, Universitätsstraße 30, 95440 Bayreuth, Germany
b Department of Medicine 1, University Hospital Erlangen, Friedrich-Alexander-University Erlangen-Nuernberg, 91054 Erlangen, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A simplified procedure for the isolation of gram quantities of illudin M from culture broths of basidiomy-
cete Omphalotus olearius is described. Esters of illudin M with docosahexaenoic acid, chlorambucil, dem-
ethylcantharidinic acid (endothall) and 2,20-bipyridyl-5,50-dicarboxylic acid were synthesised and tested
for cytotoxicity and induction of apoptosis in two clinically relevant tumour cell lines (Panc-1 pancreas
carcinoma and HT-29 colon carcinoma) and in non-malignant human foreskin fibroblasts. The demeth-
ylcantharidin and the bipyridine conjugates retained the cytotoxicity of the parent illudin M while dis-
playing an improved specificity for the tumour cells over the fibroblasts.
Received 24 April 2008
Revised 31 July 2008
Accepted 4 August 2008
Available online 7 August 2008
Keywords:
Illudin
Cytotoxicity
Apoptosis
Ó 2008 Elsevier Ltd. All rights reserved.
Tumour specificity
1. Introduction
these pharmacological shortcomings. McMorris et al. reported8 im-
proved therapeutic indices for some derivatives albeit at the ex-
A frequent drawback of cytotoxic agents that rules them out as
candidates for further clinical studies is their lack of specificity for
tumour cells over normal body tissue, that is, their small therapeu-
tic index. An often-applied strategy to improve this specificity is
the covalent attachment of cancer cell seeking shuttle groups. For
instance, the conjugation of clinically established drugs like taxol,
pense of antiproliferative efficacy. Kinder et al. found that an
intact indane skeleton is not a prerequisite for antitumoural activ-
ity and that some structurally simplified bicyclic analogues of illu-
dins S and M even displayed a better discrimination between
different tumour cell lines.9 Irofulven (3), another semisynthetic
derivative by the McMorris group,10 was assessed by the NCI and
underwent several phase II clinical trials in hormone refractory
prostate cancer, ovarian, pancreatic, renal, colorectal, lung and
breast cancer.11 However, it turned out to be rather ineffective in
most of the trials except for some prostate and pancreatic cancer
camptothecin and doxorubicin to
x-3-fatty acids such as docosa-
hexaenoic acid dramatically improved their efficacy in mice bear-
ing various solid tumours.1–3
Illudin M (1) is a cytotoxic fungal sesquiterpene of the illudane
class comprising strongly electrophilic spirocyclopropane and en-
one systems. It is amenable to alkylation of bionucleophiles such
as DNA, RNA and proteins via a two-stage process (Fig. 1). Together
with its congener, illudin S (2), it was isolated from the culture
medium of Omphalotus olearius (formerly known as Clitocybe illu-
dens or Omphalotus illudens) mushrooms.4–6 The illudins were
found to be highly cytotoxic in various drug resistant cancer cells
with increased nucleotide excision repair or ABC drug transporter
activity. In some cell lines, one-digit nanomolar IC50 concentrations
were measured by the established colorimetric cytotoxicity assays,
OH
CH2OH
HO
HO
R
O
O
Illudin M (1): R = H
Illudin S (2): R = OH
Irofulven (3)
Nu2
Nu2
employing
3(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium
bromide (MTT) or sulforhodamine B (SRB) as dyes. Yet, the unfa-
vourable toxicity profiles and narrow therapeutic windows of the
illudins had prevented clinical application.7 Limited sets of struc-
turally varied derivatives of 1 and 2 were prepared to improve
HO
Nu1
O
OH
(1) and illudin
Nu1
Figure 1. Natural cytotoxins illudin
M
S
(2), semisynthetic
* Corresponding author. Fax: +49 0 921 552671.
E-mail address: Rainer.Schobert@uni-bayreuth.de (R. Schobert).
anticancer drug irofulven (3) and their general two-stage reaction with bionucle-
ophiles Nu.
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.08.015

R. Schobert et al. / Bioorg. Med. Chem. 16 (2008) 8592–8597
8593
cases, while severe side effects such as retinotoxicity were fre-
quently observed.12–19 Specific DNA adducts of 3 were structurally
characterised, recently.20 The rather daunting clinical results were
an incentive for us to investigate other derivatives of illudin M, in
particular those with an unmodified illudin M core linked to unsat-
urated fatty acid shuttles, or other potentially synergetic effector
components such as alkylating agents or compounds targeting
the nucleotide excision repair (NER) machinery. Herein, we report
their syntheses and antiproliferative properties in two clinically
relevant tumour cell lines, Panc-1 pancreas carcinoma and HT-29
colon carcinoma, as well as in non-malignant human foreskin
fibroblasts. We also disclose a simplified procedure for the isola-
tion of gram quantities of illudin M from culture broths of Omp-
halotus olearius.
salt thereof was first treated with Et3N and 2,4,6-trichlorobenzoyl
chloride to give a mixed anhydride within ca. 20 min. This was
treated with 1 and 4-(N-dimethylamino)pyridine (DMAP) to afford
the corresponding ester 4 in yields ranging from 30% to 75%. Ester
4a was obtained from docosahexaenoic acid (DHA), ester 4b from
one equivalent of endothall disodium salt and two equivalents of
1, ester 4c likewise from 2,20-bipyridyl-5,50-dicarboxylic acid, and
ester 4d from chlorambucil. The products were purified by column
chromatography on silica gel.
These carboxylic acids were chosen because tumour-specific
activities were anticipated of their esters 4. Polyunsaturated fatty
acids such as DHA are known to play an important role in the delay
of the progression of cancers caused by reactive oxygen species
(ROS), presumably by modulating hormone receptors, Akt kinase
and the nuclear factor jB (NF-jB). n ꢀ 3 fatty acids, in particular,
were shown to inhibit the formation of the tumour growth pro-
moter 13-hydroxy-octadecadienoic acid (13-HODE) by lowering
the uptake of linoleic acid, which is mediated via Gi protein-cou-
pled signal transduction pathways.24–26 Endothall (1-oxabicyclo-
[2.2.1.]-heptane-2,3-dicarboxylic acid) is the diacid form of the
demethyl analogue of cantharidin, the active principle of Epicanta
gorhami or Mylabris (‘blister beetles’), used in traditional Chinese
medicine for the treatment of liver, lung, intestinal and digestive
tract tumours. In clinical trials, cantharidin had shown therapeutic
effects on patients with primary hepatoma, but also occasional se-
vere side effects.27 Endothall was found to be far less toxic while
still active against tumours. The mode of action of the cantharidin
family members is thought to be related to their inhibition of the
serine–threonine protein phosphatase 2A (PP2A)28 which plays a
role in the tumourigenic transformation,29–31 the control of the cell
cycle32 and in cell proliferation.33 Other literature findings suggest
that the PP2A activity might be essential for the nuclear excisson
repair of DNA lesions induced by alkylants.34 Kelner, McMorris, Jas-
pers et al. had reported that 90% of illudin’s lethal effects in human
fibroblasts can be prevented by an active nucleotide excision repair
(NER) system.35 In in vitro experiments, the levels of PP2A in var-
ious organs paralleled the cytotoxicity of cantharidin derivatives.
Intestinal organs are particularly rich in PP2A. It is also worthy of
note that endothall inhibited the growth of primary hepatocellular
carcinoma cell lines more than that of normal hepatocytes.36 Thus
conjugation of illudin M to endothall as in 4b should increase the
organ selectivity and tumour specificity. 2,20-Bipyridyl-5,50-dicar-
boxylic acid is a dye-sensitiser and intercalator to DNA. More re-
cently, it has been attached via ester bonds to sugar-type tumour
markers with the intention to amplify the bioactivity by forming
oligomeric cluster complexes with metal ions available under
physiological conditions, such as Cu(II) or Fe(III).37 Similar ancillary
effects were expected from the bipyridine moiety of illudin M con-
jugate 4c. Chlorambucil is a clinically used bifunctional cross-link-
ing N-mustard, preferentially alkylating the N-7 atoms of guanine
residues in DNA. The conjugate 4d was therefore hoped to lead to
DNA lesions of unusual topology and so to altered responses by
proteins involved in the identification and repair of DNA defects.
2. Results and discussion
2.1. Isolation of illudin M
Omphalotus olearius (DSM 3398) was grown at 20 °C in a sterile
20 L plastic flask containing HA-medium21 (72 g yeast extract,
180 g malt extract, 72 g glucose, 18 L water, pH 5.5) under a con-
tinuous flow of sterile air. After about 4 weeks, the culture liquid
was extracted with ethyl acetate (six to seven extractions, each
with 10% of the volume) and the combined and dried extracts were
concentrated and purified by column chromatography (silica gel
60; hexane/ethyl acetate 2:1; Rf 0.35). Fractions containing pure
illudin M were pooled and concentrated to afford ca. 800 mg of
crystalline illudin M (1) upon standing. Very little illudin S (2)
and other minor side products were detected by analytical HPLC22
in the ethyl acetate extracts, and were easily removed during the
chromatographical work-up.
2.2. Ester conjugates of illudin M
Illudin M (1) was esterified with various mono- and dicarbox-
ylic acids at its secondary OH-group, according to the protocol of
Yamaguchi (Scheme 1).23 The respective carboxylic acid or sodium
O
1) Et3N, DMF, r.t.,
Cl3(C6H2)COCl
O
R
RCO2H
2) + 1, DMAP
HO
PhMe, r.t., 16 h
O
CH2
4a: R =
4d: R =
6
(CH2)3
N[(CH2)2Cl]2
HO
O
O
O
2.3. Biological evaluation
O
O
O
N
N
To assess the therapeutic window of the new illudin M esters 4
relative to the parent compound 1, cells of Panc-1 pancreatic carci-
noma and HT-29 colon adenocarcinoma, as well as non-malignant
human foreskin fibroblasts (HFs) were incubated for up to 120 h
HO
OH
O
O
O
O
with 1 and 4 at concentrations ranging from 0.01 to 10 lM, and fi-
O
nally stained with Trypan blue. The number of unstained (vital)
cells was counted and expressed as relative cell numbers com-
pared to untreated controls (Figure 2 shows results with 0.1, 1
O
O
OH
4b
4c
and 10
lM concentrations of the test compounds.). Although
Scheme 1. Synthesis of illudin M esters 4.
affording seemingly lower activity values, the Trypan blue method

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R. Schobert et al. / Bioorg. Med. Chem. 16 (2008) 8592–8597
Figure 2. Cytotoxic effects of illudin M (1) and its conjugates 4a–d at various concentrations (d: 10 lM; j: 1 lM; 4: 0.1 lM) in cells of Panc-1 pancreatic carcinoma, HT-29
colon adenocarcinoma and non-malignant human foreskin fibroblasts (HFs) upon incubation for 24–120 h. Y-axis shows number of viable cells relative to untreated controls
(100%) as ascertained by the Trypan blue exclusion assay. Values represent means of three independent experiments.
is more accurate than MTT or SRB assays since only truly dead cells
get stained, and since it allows to directly determine cell prolifer-
ation numbers rather than surrogate parameters. The DHA ester
4a was only very weakly active in all three kinds of cells at any
concentration. In contrast, the bis(illudinyl M) demethylcanthari-
dinate 4b and the bipyridine 4c exhibited a cytotoxic effect in
the tumour cells at concentrations between 1 and 10 lM which
was more pronounced and which set on more rapidly than that
of 1. For instance, cell numbers were quickly reduced to 33%
(Panc-1) or 26% (HT-29), respectively, after incubation with 1 lM
had a smaller and comparable cytotoxic effect in all three cell lines.
With respect to potential clinical applicability, conjugates 4b and
4c are clearly the most interesting ones.
The ability of compounds 4 to induce apoptosis in tumour cells
was investigated next. Treated cells were stained with propidium
iodide, and the labelled nuclei were analysed using a fluores-
cence-activated cell sorter (FACS). The percentage of apoptotic cells
was determined by measuring the fraction of nuclei with a sub-
diploid DNA content (Fig. 3). While compounds 4a and 4d caused
apoptosis rates of less than 10% in Panc-1 and HT-29 cells upon
of 4b for 48 h and to 6% (Panc-1) or 3% (HT-29), respectively, after
incubation for up to 96 h at concentration of 1 lM, an increase in
120 h. The non-malignant fibroblasts showed a merely moderate
response to 4b at 1 lM, with a drop of the number of viable cells
sub-diploid events was observed in FACS analysis for the same
concentration of conjugates 4b and 4c reaching up to 74% after
120 h in the case of the demethylcantharidin conjugate 4b acting
on Panc-1 cells. In HT-29 cells, 1 lM of compound 4b gave rise
to 53% apoptosis after 120 h. Compound 4c was less efficacious,
to 57% of untreated controls after 48 h and to 49% after 120 h. Illu-
din M (1) under the same conditions caused cell number reduc-
tions to 42% (Panc-1) or 26% (HT-29) or 39% (HF), respectively,
after 48 h and to 12% (Panc-1) or 4% (HT-29) or 28% (HF), respec-
tively, after 120 h. Thus, conjugate 4b had a greater cytotoxic im-
pact on the tumour cells while affecting the non-malignant HF
cells less when compared with the parent illudin M. This is also a
strong indication of 4b reaching its targets in an intact form rather
than decomposing beforehand into two equivalents of illudin M.
The cytotoxicity profile of the bipyridyl conjugate 4c resembled
eliciting 62% apoptosis in Panc-1 cells and 21% apoptosis in HT-
29 cells upon incubation at 1
were more resistant to apoptosis induced by 4b (33%, 1
120 h) and 4c (14%, 1 M, 120 h). The magnitude of apoptosis
l
M for 120 h. Non-transformed HFs
l
M,
l
induction as well as the spread between induction in the tumour
versus the non-malignant cells was the greatest for conjugate 4b.
In comparison, illudin M (1) showed a comparable induction of
apoptosis in the tumour cells (86% in Panc-1; 48% in HT-29), but
that of 4b. At a concentration of 1 lM 4c reduced the numbers of
Panc-1/HT-29/HF cells to 38%/36%/62% after 48 h of exposure and
an even greater one in the HF (89%). Only at 0.1 lM, illudin M
to 7%/11%/48% after 120 h. The chlorambucil conjugate 4d when
caused a higher apoptosis rate in Panc-1 cells than in HF. Thus,
applied at medium concentrations, for example, at 0.1 to 1 lM,
in terms of both cytotoxicity and apoptosis induction, at a concen-

R. Schobert et al. / Bioorg. Med. Chem. 16 (2008) 8592–8597
8595
Figure 3. Induction (in%) of apoptosis in Panc-1 cells, HT-29 cells and HF after treatment with different concentrations (d: 10
M (1) and its conjugates 4b–d by measuring the fraction of nuclei with a sub-diploid DNA content in a FACS. Values represent means of three independent experiments.
l
M; j: 1
l
M; 4: 0.1
l
M; – –: control) of illudin
Table 1
Vital cells (in%)a and apoptotic cells (in%)b of Panc-1 pancreatic carcinoma, HT-29
colon adenocarcinoma and non-malignant human foreskin fibroblasts upon exposure
for 120 h to 1
l
M of compounds 1 and 4a–d
Panc-1
HT-29
HF
Vital cells (%)
1
12.3 9.6
102.8 1.4
6.4 7.4
7.3 5.0
20.0 3.4
3.8 11.4
110.6 4.1
3.0 0.0
10.8 3.9
42.7 1.2
28.1 14.6
80.0 10.2
49.3 10.3
47.8 16.5
47.5 3.6
4a
4b
4c
4d
Apoptosis (%)
1
86.0 1.5
2.6 0.9
74.0 1.5
62.1 15.6
36.8 2.9
47.9 0.1
1.9 0.2
52.9 1.4
21.3 1.9
6.0 0.9
88.9 0.8
4.4 1.9
33.1 12.8
14.3 0.5
9.5 1.9
4a
4b
4c
4d
Figure 4. Percentage of vital and apoptotic Panc-1 pancreatic carcinoma cells, HT-
29 colon adenocarcinoma cells and non-malignant human foreskin fibroblasts upon
a
Values are derived from concentration–response curves obtained by measuring
the percentage of vital cells relative to untreated controls (100%) after 120 h
exposure for 120 h to 1
1.
lM of compounds 1 and 4a–d. Conditions as stated in Table
exposure to test compounds (1 lM) in the Trypan blue exclusion assay. Values
represent means of three independent experiments standard deviation.
b
Values are derived from the numbers of apoptotic cells as determined by
tained by the MTT assay was much weaker (ca 70% viable cells
upon exposure to 1–10 l
M for 24–48 h).38
measuring the fraction of nuclei with a sub-diploid DNA content in a FACS relative
to untreated controls (100%); they represent means of three independent experi-
ments standard deviation.
2.4. Conclusions
The finding that the esters of illudin M with endothall (4b) and
2,20-bipyridyl dicarboxylic acid (4c) retained the cytotoxicity of the
parent illudin M while displaying a significantly improved specific-
ity for tumour cells over normal fibroblasts is encouraging. As
endothall is known to target the liver, conjugate 4b will shortly
be tested on various liver cancers in vitro and in vivo. The failure
tration of 1 lM, the ester 4b was as efficacious as illudin M itself,
but distinctly more tumour-specific (Table 1 and Fig. 4). Although
irofulven (3) showed a comparably good selectivity in apoptosis
induction in breast cancer cells over normal mammary epithelial
cells at 1 lM concentration, its antiproliferative effect as ascer-

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R. Schobert et al. / Bioorg. Med. Chem. 16 (2008) 8592–8597
of the DHA conjugate 4a did not come as a big surprise. DHA con-
jugates of other cytotoxic drugs such as taxol also exhibited little if
any activity in tumour cell studies but were quite efficacious in
corresponding animal models with tumour xenografts. With our
simplified procedure for the production of gram quantities of illu-
din M at hand, larger arrays of ester conjugates can be readily pre-
pared and screened now. This should allow us to pinpoint those
properties of the effector groups attached to illudin M which most
distinctly improve its therapeutic index and tumour specificity.
51.0, 51.2, 51.3, 51.8, 75.9, 76.0, 77.8, 78.9, 79.6, 79.7, 79.8, 80.5,
80.6, 133.4, 133.5, 134.9, 135.8, 136.0, 145.9, 146.1, 146.2, 170.8,
171.8, 199.7, 199.9; m/z (EI, 70 eV) 646 (M+, 3%), 629 (3%), 416
(7%), 248 (7%), 231 (100%), 215 (77%), 187 (80%), 43 (69%); accurate
mass (EIMS) for C38H46O9: calcd 646.31418, obsd 646.31400.
3.2.3. 2,20-Bipyridyl-5,50-dicarboxylic acid bis(illudinyl M) ester
(4c).
Analogously to the synthesis of ester 4a, compound 4c (184 mg,
51%) was obtained from 2,20-bipyridyl-5,50-dicarboxylic acid
(125 mg, 0.51 mmol), NEt3 (163
zoyl chloride (182 L, 1.11 mmol), illudin M (273 mg, 1.11 mmol)
and DMAP (251 mg, 2.04 mmol) as a yellow oil; Rf 0.24 (ethyl ace-
tate/n-hexane 1:4);
max(ATR)/cmꢀ1 3480, 2967, 2928, 2867, 1717,
lL, 1.11 mmol), 2,4,6-trichloroben-
3. Experimental
3.1. General
l
m
1699, 1592, 1362, 1264, 1104, 1023, 944, 762, 730; dH (300 MHz;
CDCl3) 0.92–0.40 (4H, m), 1.15 (3H, s), 1.24 (3H, s), 1.39 (3H, s),
1.52 (3H, s), 3.60 (1H, s), 5.91 (1H, s), 6.56 (1H, s), 8.39 (1H, d,
J = 8.3 Hz), 8.56 (1H, d, J = 8.3 Hz), 9.24 (1H, s); dC (75 MHz; CDCl3)
6.0, 8.9, 14.6, 20.8, 24.8, 26.7, 31.5, 49.2, 76.0, 80.0, 121.4, 126.0,
133.6, 134.7, 136.3, 138.2, 146.0, 150.5, 158.4, 164.6, 199.8; m/z
(EI, 70 eV) 704 (M+, 4%), 268 (8%), 244 (51%), 227 (57%), 187
(100%), 159 (39%), 128 (35%), 115 (36%), 77 (21); accurate mass
(EIMS) for C42H44N2O8: calcd 704.30977, obsd 704.30920.
IR-spectra were recorded on a Perkin-Elmer One FT-IR spectro-
photometer. Magnetic resonance (NMR) spectra were recorded un-
der conditions as indicated on a Bruker Avance 300 spectrometer.
Chemical shifts (d) are given in parts per million downfield from
TMS as internal standard. Mass spectra were recorded using a Var-
ian MAT 311A (EI). Elemental analyses were carried out with a Per-
kin-Elmer 2400 CHN elemental analyser. For column
chromatography, Merck silica gel 60 (230–400 mesh) was used.
Starting compounds and components of the culture medium for
Omphalotus olearius were purchased from the usual sources, and
were used without further purification.
3.2.4. (Illudinyl M) 4-[40-di(chloroethyl)aminophenyl]-
butanoate (4d)
Analogously to the synthesis of ester 4a, compound 4d (132 mg,
66%) was obtained from chlorambucil (114 mg, 0.38 mmol), NEt3
3.2. Chemistry
(60 lL, 0.44 mmol), 2,4,6-trichlorobenzoyl chloride (67 lL,
0.44 mmol), illudin M (93 mg, 0.38 mmol) and DMAP (93 mg,
3.2.1. Docosahexaenoyl illudin M (4a)
Docosahexaenoic acid (100 mg, 0.31 mmol) was dissolved in
dry DMF (2 mL) and treated with NEt3 (49
0.75 mmol) as a yellow oil; Rf 0.23 (ethyl acetate/n-hexane/NEt3
1:5:0.06); ½aꢁ2D5 –25.2 (c 0.5, CHCl3);
m
max(ATR)/cmꢀ1 3495, 2962,
lL, 0.36 mmol) and
2,4,6-trichlorobenzoyl chloride (55 L, 0.36 mmol). The resulting
l
2928, 2865, 1726, 1696, 1612, 1518, 1446, 1360, 1252, 1179,
1143, 1105, 945, 909, 821, 802, 730; dH (300 MHz; CDCl3) 0.3–0.5
(1H, m), 0.7–1.0 (2H, m), 1.0–1.1 (4H, m), 1.17 (3H, s), 1.35 (3H,
s), 1.50 (3H, s), 1.8–2.0 (2H, m), 2.34 (2H, t, J = 7.4 Hz), 2.55 (2H,
t, J = 7.6 Hz), 3.5–3.7 (8H, m), 5.66 (1H, s), 6.50 (1H, s), 6.62 (2H,
d, J = 6.7 Hz), 7.04 (2H, d, J = 6.7 Hz); dC (75 MHz; CDCl3) 6.0, 8.8,
14.5, 20.7, 24.7, 26.7, 26.9, 31.4, 33.5, 34.0, 40.4, 48.8, 53.6, 76.0,
78.7, 112.3, 129.6, 130.6, 133.5, 135.2, 135.3, 144.2, 146.2, 173.2,
200.0; m/z (EI, 70 eV) 533 (M+, 35%), 464 (13%), 302 (100%), 254
(68%), 231 (26%), 215 (22%), 118 (39%); accurate mass (EIMS) for
suspension was stirred at room temperature under an atmosphere
of argon for 20 min. A solution of illudin M (76 mg, 0.31 mmol) and
DMAP (75 mg, 0.62 mmol) in dry toluene (5 mL) was added and
the resulting mixture was stirred for 16 h. After dilution with ethyl
acetate and washing with water, the organic phase was dried over
Na2SO4 and concentrated in vacuum. The residue was purified by
column chromatography (silica gel 60; ethyl acetate/n-hexane
1:9; Rf 0.31) to leave the product as a colourless oil (109 mg,
0.20 mmol, 65%); m
max(ATR)/cmꢀ1 3491, 3013, 2964, 1733, 1699,
29H3735Cl2NO4: calcd 533.20996, obsd 533.20990.
1609, 1445, 1362, 1255, 1148, 1106, 945, 706; dH (300 MHz, CDCl3)
0.3–0.4 (1H, m), 0.7–1.0 (5H, m), 1.05 (3H, s), 1.0–1.1 (1H, m), 1.16
(3H, s), 1.33 (3H, s), 1.48 (3H, s), 2.0–2.1 (2H, m), 2.3–2.4 (4H, m),
2.7–2.9 (10H, m), 3.54 (1H, s), 5.2–5.4 (12H, m), 5.64 (1H, m), 6.49
(1H, s); dC (75 MHz; CDCl3) 5.9, 8.7, 14.2, 14.5, 20.5, 20.6, 22.8, 24.7,
25.5, 25.6, 26.7, 31.4, 34.1, 48.7, 76.0, 78.7, 126.9, 127.7, 127.8,
127.9, 128.0, 128.2, 128.3, 128.5, 129.4, 132.0, 133.5, 135.1,
135.3, 146.1, 172.7, 200.0; m/z (EI, 70 eV) 558 (M+, 2%), 265 (5%),
231 (24%), 203 (26%), 105 (28%), 79 (65%), 67 (53%), 43 (100%).
Anal. Calcd for C37H50O4: C, 79.5; H, 9.0. Found: C, 79.3; H, 8.9%.
C
3.3. Cytotoxicity assays: cell lines and culture conditions
Panc-1 pancreatic carcinoma cells and HT-29 colon carcinoma
cells were cultured on six-well tissue culture plates (Becton Dick-
inson, Mannheim, Germany) in RPMI-1640 medium (Biochrom,
Berlin, Germany), containing 10% foetal calf serum (FCS, Biochrom),
penicillin (107 U/L) and streptomycin (10 mg/L) at 37 °C and 5%
CO2. Human foreskin fibroblasts (HFs) served as non-malignant
controls and were cultured in Dulbecco’s modified Eagle’s medium
(DMEM, Biochrom) with the same supplements. All cell lines were
obtained from the German Collection of Microorganisms and Cell
Cultures (DSMZ, Braunschweig, Germany).
Determination of cell viability. Cells were harvested after the
incubation period and stained with Trypan blue (Biochrom, Ger-
many). The number of unstained (intact) cells was counted in a
Neubauer chamber and expressed as relative cell numbers com-
pared to untreated controls (= 100%).
3.2.2. Bis(illudinyl M) endothallate (4b)
Analogously to the synthesis of ester 4a, compound 4b (75 mg,
30%) was obtained from disodium endothallate (93 mg,
0.40 mmol), NEt3 (130
ride (144 L, 0.94 mmol), illudin M (200 mg, 0.81 mmol) and
DMAP (199 mg, 1.62 mmol) as a colourless oil; Rf 0.44 (ethyl ace-
tate/n-hexane 1:3);
max(ATR)/cmꢀ1 3489, 2961, 1727, 1689,
lL, 0.94 mmol), 2,4,6-trichlorobenzoyl chlo-
l
m
1607, 1467, 1447, 1362, 1254, 1238, 1202, 1172, 1106, 1052,
944, 928, 909, 819, 730; dH (300 MHz, CDCl3) 0.3–0.5 (2H, m),
0.7–1.8 (34H, m), 3.0–3.1 (2H, m), 3.4–3.6 (4H, m), 4.7–4.9 (2H,
m), 5.63 (1H, s), 5.65 (1H, s), 6.49 (1H, s), 6.51 (1H, s); dC
(75 MHz; CDCl3) 6.1, 8.9, 14.1, 14.5, 14.6, 14.9, 20.6, 20.8, 21.0,
24.6, 24.7, 24.8, 25.6, 26.6, 26.7, 29.3, 29.5, 31.4, 31.5, 48.7, 48.8,
3.4. Flow cytometric analysis of apoptosis
For quantification of apoptosis, culture supernatants were col-
lected and the cells were washed two times with PBS, trypsinised

R. Schobert et al. / Bioorg. Med. Chem. 16 (2008) 8592–8597
8597
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and lysed in a hypotonic solution containing 0.1% sodium citrate,
0.1% Triton X-100 and 50 g/mL propidium iodide (Sigma). Analy-
l
sis of labelled nuclei was performed on a Calibur fluorescence-acti-
vated cell sorter (FACS) using CELLQuest software (both from
Becton Dickinson). The percentage of apoptotic cells was deter-
mined by measuring the fraction of nuclei with a sub-diploid
DNA content.
16. Yeo, W.; Boyer, M.; Chung, H. C.; Ong, S. Y. K.; Kim, R.; Zee, B.; Ma, B.; Lam,
K. C.; Mo, F. K. F.; Ng, E. K. W.; Ho, R.; Clarke, S.; Roh, J. K.; Beale, P.; Rha, S.
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Acknowledgements
This work was supported by the Deutsche Forschungsgemeins-
chaft (Grant Scho 402/8) and the ELAN programme of the Univer-
sity Hospital Erlangen (Grant 07.03.30.1).
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