Malassezin - A novel agonist of the Arylhydrocarbon receptor from the yeast Malassezia furfur

Article abstract of DOI:10.1016/S0968-0896(00)00319-9

The yeast Malassezia furfur converts tryptophan into several indole compounds. One of these, malassezin, was identified as (2- 1H-indol-3-ylmethyl)-1H-indole-3-carbaldehyde (1). It was synthesized from N-Boc-indole-3-carbaldehyde in five steps with 12% ov

Full text of DOI:10.1016/S0968-0896(00)00319-9

Bioorganic & Medicinal Chemistry 9 (2001) 955±960
MalassezinÐA Novel Agonist of the Arylhydrocarbon Receptor
from the Yeast Malassezia furfur
Gregor Wille,a Peter Mayser,b,* Wiebke Thoma,b Thomas Monsees,b Annette Baumgart,c
Hans-Joachim Schmitz,c Dieter Schrenk,c Kurt Polborna,y and Wolfgang Steglicha,*
aDepartment Chemie der Ludwig-Maximilians-Universitat, Butenandtstr. 5-13 (F), 81377 Munchen, Germany
È
È
bCenter for Dermatology and Andrology, Justus-Liebig-University, 35385 Gieûen, Germany
cLebensmittelchemie und Umwelttoxikologie der Universitat Kaiserslautern, 67663 Kaiserslautern, Germany
È
Received 26 October 2000; accepted 16 November 2000
AbstractÐThe yeast Malassezia furfur converts tryptophan into several indole compounds. One of these, malassezin, was identi®ed
as 2-(1H-indol-3-ylmethyl)-1H-indole-3-carbaldehyde (1). It was synthesized from N-Boc-indole-3-carbaldehyde in ®ve steps with
12% overall yield. The compound easily cyclizes to indolo[3,2-b]carbazole (7) which is known to interact with the arylhydrocarbon
receptor (AHR). Similarly, malassezin was found to induce cytochrome P450 as an agonist of AHR (EC50=1.57 mM) in rat hepa-
tocytes. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
variety of colour and ¯uorescence phenomena is
induced.11 In this publication we describe the structure
of malassezin (1), a novel arylhydrocarbon receptor
(ACR) agonist from cultures of M. furfur.
Lipophilic yeasts of the genus Malassezia are part of the
residential ¯ora of human skin and many warm blooded
animals.1,2 They are also responsible for skin diseases
such as pityriasis versicolor in human beings, but its
pathogenesis is not fully understood.3,4 In pityriasis
versicolor the a€ected skin area exhibits ¯uorescence
under UV light (366 nm) and in the ®nal stage depig-
mentation appears due to interrupted melanin synth-
esis.5 The latter e€ect may be ascribed to an inhibition
of human tyrosinase6À8 by a secondary metabolite
produced by the fungus.
Results and Discussion
Isolation and structural elucidation
Malassezin (1) (Fig. 1) was originally obtained by a
tyrosinase assay guided fractionation of the EtOAc
extract from cultures of M. furfur. The crude extract
was subjected to gel permeation chromatography on
Sephadex LH-20, followed by preparative TLC and
HPLC on reverse-phase C-8 silica gel to yield <1 mg of
malassezin as a colourless solid (tR=21.2 min on analy-
tical HPLC using CH3CN:H2O as the eluent with a
gradient of 15:85 to 60:40 in 25 min).
Recently, we reported a new minimal medium for the
cultivation of Malassezia furfur (CBS 7019) which
simulates phenomena characteristic for the clinical var-
iance of pityriasis versicolor.9,10 With tryptophan as the
single nitrogen source, the formation of a series of
alkaloids and simple degradation products exhibiting a
Malassezin (1) exhibits a molecular ion at m/z 274.1113
in the HRMS (EI) corresponding to the molecular for-
mula C18H14N2O. The NMR signals of 1 summarized in
Table 1 were best resolved in DMSO-d6:CCl4 (4:1). The
aromatic protons 4-H to 7-H (and their counterparts 40-
H to 70-H) form a AA0BB0-pattern in the 1H NMR
spectrum that is supported by COSY experiments. Five
singlets can be assigned to a formyl group (dH 10.20), an
indole 2-H (dH 7.25), two indole NH groups and a
Abbreviations: CYP, cytochrome P450; DMEM, Dulbecco's modi®ed
Eagle's medium; EGTA, ethylene glycol-bis(b-aminoethyl ether)
N,N,N0,N0-tetraacetic acid; EROD, 7-ethoxyresoru®n O-deethylase;
FCS, fetal calf serum; Tris, tris(hydroxymethyl)aminomethane.
*Corresponding authors. Tel.: +49-641-99-43220; fax: +49-641-99-
43209; e-mail peter.mayser@derma.med.uni-giessen.de (P. Mayser);
Tel.: +49-89-2180-7755; fax: +49-89-2180-7756; e-mail: wos@
cup.uni-muenchen.de (W. Steglich).
yX-ray crystal structure analysis.
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00319-9

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G. Wille et al. / Bioorg. Med. Chem. 9 (2001) 955±960
Table 1. 1H and 13C NMR data (600 MHz) of malassezin (1) in
CCl4:DMSO-d6 (1:4)
Pos.
1H NMRa
COSY
13C NMRb
1
11.98c (s)
2
3
3a
4
5
151.3
113.0
126.5
120.1
121.8
122.6
111.6
136.2
Figure 1. Malassezin (1).
8.01 (d, 6.7)
7.1 (m)
7.1 (m)
5
4,6
5,7
6
6
7
methylene group (dH 4.55) that appears in the 13C NMR
spectrum at unusually high ®eld (dC 21.92). These facts
and an IR (KBr) absorption at 1630 cmÀ1 suggest the
presence of both an indole and an indole±carbaldehyde
moiety connected by a methylene group. In order to
elucidate the position of the formyl group we compared
the UV/Vis spectrum of 1 with those of 1:1 mixtures of
indole and indole-2- and 3-carbaldehyde, respectively.
Since correspondence was found only in the latter case,
the formyl group can be located at C-3 of the indole
nucleus. This suggests structure 1 for malassezin, in
accord with the occurrence of NOE's between the
methylene protons and H-20 and H-40 of the indole
nucleus. Structure 1 was proven by a total synthesis that
provided the compound in quantities sucient for bio-
logical evaluation.
7.35 (d, 7.0)
7a
10
20
30
3a0
40
50
60
70
7a0
8
10.98c (s)
7.25 (s)
123.7
110.8
125.5
118.2
118.5
121.1
111.5
135.4
21.9
7.48 (d, 8.1)
6.91 (t, 7.5)
7.02 (t, 7.6)
7.31 (d, 7.9)
50
40,60
50,70
60
4.55 (s)
10.20 (s)
9
184.4
ad (Multiplicity, J in Hz) in ppm.
bd in ppm.
cExchanges with D2O.
52%. Best results were obtained by using only a small
excess of sodium. In the ®nal step, 1 was synthesized by
a Vilsmeier formylation.16 At room temperature the
reaction proceeds sluggishly, however, by careful heat-
ing to 40 ꢀC, the desired product 1 was obtained in 74%
yield. At higher temperatures, only the condensation
product 7 was isolated as a poorly soluble solid.
Malassezin shows a high tendency for crystallization,
and suitable crystals for an X-ray crystallographic ana-
lysis (Fig. 2) could be grown from the mother liquor at
À20 ꢀC. Synthetic malassezin (1) agreed in all its spec-
troscopic data with the natural product, however, it was
devoid of any in vitro tyrosinase activity. This may be
explained by the presence of small amounts of an
unknown tyrosinase inhibitor in the natural malassezin
fractions. E€orts to identify this elusive compound are
under way.
Total synthesis
Recently, we have shown that indole and indole-3-car-
baldehyde occur in cultures of M. furfur.9 Commencing
with these compounds, we synthesized 1 in ®ve steps
(Scheme 1). Coupling of the anion obtained by ortho-
metalation12,13 of N-tosyl-indole (2) with N-Boc-indole-
3-carbaldehyde (3) a€orded the alcohol 4 in 74% yield.
Experiments to remove the hydroxy group in 4 by
hydrogenolysis with H2/Pd-C were unsatisfactory.
However, treatment of the crude alcohol 4 with acetyl
chloride followed by reaction of the resulting acetate 5
with sodium in liquid ammonia led to reductive removal
of the acetoxy group with simultaneous cleavage of all
protective groups.14 Pure 2,30-methylenebisindole15 (6)
was thereby obtained in varying yields ranging from 40±
Scheme 1. Reagents and conditions: (a) 1. tert-BuLi, THF, À20 ꢀC, 2. N-Boc-indole-3-carbaldehyde (3) in THF, 3. CH3COCl, À20 ꢀC to rt, 57%;
ꢀ
(b) Na, liquid NH3, THF, À78 ꢀC, 40±52%; (c) POCl3, D MF, 40 C, 74%; (d) cat. HCl, THF, re¯ux, 78%.

G. Wille et al. / Bioorg. Med. Chem. 9 (2001) 955±960
957
Figure 3. Natural AHR agonists: indolo[3,2-b]carbazole (7), 6-formyl-
indolo[3,2-b]carbazole (8) and tryptanthrines (9±11).
catalysed ERODactivity (EC 50=1.57 mM) in a con-
centration range similar to methyltryptanthrine (10).28
Malassezin is a non-planar molecule (see X-ray crystal
structure, Fig. 2) and does not ®t into a 7Â14 A
rectangle. Thus, it does not ful®ll major structural
requirements for potent AHR agonists.22 Further
investigations are needed to elucidate if malassezin
represents a novel prototype of AHR agonists. Alter-
natively, it may be easily converted into a potent agonist
such as indolo[3,2-b]carbazole (7). In supernatants of
cell cultures no 7 could be detected by ¯uorescence/
HPLC analysis. However, the formation of minor
amounts of 7 sucient for AHR activation, e.g. by
intracellular metabolism, cannot be excluded. It remains
to be elucidated if the yeast bene®ts from the biosynth-
esis of an AHR agonist and/or if AHR activation by
malassezin contributes to the pathogenesis of pityriasis
versicolor.
Figure 2. X-ray crystallographic structure of malassezin (1) (cyrstal-
lographic numeration of atoms was used).17,18
As expected,19,20 heating of 1 in THF with a catalytic
amount of concd HCl a€orded the indolocarbazole 7 in
78% yield. The NMR data and MS fragmentation pattern
of 7 were in agreement with those of the literature.21
Biological activity
The arylhydrocarbon receptor (AHR) is involved in cell
growth, di€erentiation and regulation of gene expres-
sion.22 It was shown that the AHR plays a role in the
transcriptional regulation of certain genes encoding
drug-metabolizing enzymes. Likewise, agonists of the
AHR induce an increased transcription of cytochrome
P450 (CYP) 1A1. The agonistic potency can be descri-
bed by the EC50 values for the induction of CYP 1A1-
dependent 7-ethoxyresoru®n O-dealkylase (EROD)
activity.23
Conclusion
Malassezin (1) is the ®rst alkaloid described from cul-
tures of a lipophilic Malassezia yeast. Compared to the
non-active 2,30-methylenebisindole (6) the activity of 1
as AHR agonist indicates the crucial in¯uence of the
formyl group as previously described for 7 and 8.20
Alternatively, 1 may be converted intracellularly into
the potent AHR agonist 7.
Indolo[3,2-b]carbazole19,22,24 (7) is a strong agonist of
the AHR and an in vivo dimerization product of (1H-
indol-3-yl)-methanol, a breakdown product from vege-
tables of the Brassica genus.25,26 Noteworthy, 7 was also
detected in faeces, urine, the gastrointestinal tract and
livers of rats, and also in human faeces. 6-Formyl-
indolo[3,2-b]carbazole (8) was found to be the strongest
AHR agonist known so far (Fig. 3).20,27 The ®rst AHR
agonists with a tryptanthrine skeleton (9±11) were iso-
lated from cultures of Candida yeasts containing
anthranilic acid and 5-substituted tryptophans (Fig. 3).28
Experimental
Material and methods
All common reagents and components of the yeast
medium were obtained from Sigma-Aldrich-Fluka
(Deisenhofen, Germany) and used without further pur-
i®cation unless otherwise indicated. Yeast strains where
supplied from the Centraalbureau voor Schimmelcul-
tures, The Netherlands. Merck silica gel 60 plates
(0.5 mm) were used for preparative TLC. Analytic
HPLC was performed on a Merck-Hitachi L-6200A
instrument, preparative HPLC on a Gilson Model 302.
For separation LiChrospher reversed phase material
(RP-8, anal: 4Â250, prep: 10Â250 mm) was used with
In our experiments with rat hepatocytes malassezin (1)
was submitted to ERODbioassays because of its struc-
tural similarity to the known AHR-agonists (Fig. 3). 1
was found to act as an AHR agonist inducing CYP 1A-

958
G. Wille et al. / Bioorg. Med. Chem. 9 (2001) 955±960
isocratic acetonitrile/water mixtures. Flow rates were 1
and 6.25 mL/min, respectively. Compounds were mon-
itored at 220 nm and dried on a Lyovac GT2. The
extinction of the dopaquinone was measured with a
Beckman DU-68 spectrophotometer (lmax=475 nm).
trated over glass wool, evaporated to dryness and dis-
solved in methanol. The crude extract was fractionated
by chromatography on Sephadex LH-20 with methanol
as eluent and further separated by preparative TLC
with toluene:ethyl formate:formic acid (10:5:3). The
main zones were partitioned between H2O and EtOAc
and the organic phases subjected to the tyrosinase
bioassay. The active fractions still contained several
components which were separated by HPLC. Malasse-
zin (1) was isolated from a tyrosinase-active peak
with tR=21.2 min on analytical HPLC using CH3CN:
H2O as the eluent with a gradient of 15:85 to 60:40 in
25 min.
All chemical reactions were carried out under an argon
atmosphere in ¯ame-dried vessels with dry, freshly dis-
tilled solvents under anhydrous conditions unless
otherwise indicated. Dry and air sensitive liquids were
handled via syringes. Dry THF was distilled from
sodium/benzophenone, pyridine and DMF were pur-
chased as dry solvents. Solvents for work-up procedures
were distilled before use. Reactions were monitored by
TLC on 0.25 mm silica gel plates (Merck, 60 F254) unless
otherwise indicated, using UV light for visualizing and
ethanolic p-anisaldehyde (2.5%) or acidic KMnO4
solutions as developing agents. Silica gel (Merck 60,
particle size 0.043±0.063 mm) was used for ¯ash column
chromatography.
Synthesis
3-Formylindole-1-carboxylic acid tert-butyl ester (3). To
a solution of indole-3-carbaldehyde (7.26 g, 50 mmol) in
dry THF (100 mL) were added Boc2O (12.00 g,
55 mmol) and a catalytic amount of DMAP at 0 ꢀC
under argon. The TLC showed complete reaction after
30 min, and the solvent was removed in vacuo (bath
temperature <35 ꢀC). Crystallization from MeOH
a€orded 3 as yellowish needles (11.02 g, 90%), mp
NMR spectra were recorded on Bruker AMX-600 (1H:
600.19 MHz, 13C: 150.92 MHz) and ARX-300 (1H:
300.13 MHz, 13C: 75.47 MHz) instruments and cali-
brated with the residual undeuterated solvent as the
internal reference. The following abbreviations were
used to denote the multiplicities: s, singlet; d, doublet; t,
triplet; q, quartet; m, multiplet; br broad; app apparent.
IR spectra: Perkin-Elmer 1420 or Bruker IFS 45 FT-IR.
High-resolution mass spectra (HRMS): Finnigan MAT
90 or MAT 95 Q, electron ionization (EI). X-ray crystal
structure analysis: Enraf-Nonius CAD4. UV/VIS: Per-
kin-Elmer Lambda 16. Melting points (mp) are uncor-
rected and were recorded on a Reichert Thermovar.
110 ꢀC. TLC: Rf 0.55 (hexanes:EtOAc 5:1); H NMR
1
(CDCl3) d 1.70 (s, 9H), 7.34 (dt, 1H, J=1 Hz,
J=7.0 Hz), 7.39 (dt, 1H, J=1 Hz, J=7.0 Hz), 8.12 (dd,
1H, J=1 Hz, J=7.5 Hz), 8.18 (s, 1H, H-2), 8.25 (dd,
1H, J=1 Hz, J=7.5 Hz); 13C NMR (CDCl3) d 27.3,
85.0, 114.6, 120.9, 121.5, 123.9, 125.4, 125.5, 135.4,
135.9, 148.2, 185.1; IR (KBr) 1744, 1679, 1558 cmÀ1
;
MS (EI) m/z 245.1 (M+). Anal. (C14H15NO3); calcd: C,
68.56; H, 6.21; N, 5.71. Found: C, 68.80; H, 6.18; N,
5.72.
DMEM, penicillin and streptomycin were purchased
from Biochrom (Berlin, Germany), FCS and col-
lagenase from Gibco (Karlsruhe, Germany), and iso-
citrate dehydrogenase from Roche (Mannheim,
Germany). Solutions for treatment of cell cultures were
obtained by dissolving malassezin (1) in DMSO and
preparing successive dilutions resulting in ®nal DMSO
concentrations of 0.5% in the culture medium.
3-{Hydroxy-[1-(4-methylphenylsulfonyl)-1H-indol-2-yl]-
methyl}-indole-1-carboxylic acid tert-butyl ester (4).
3.24 mL (5.5 mmol) of tert-BuLi (1.7 M in pentane)
were added at À20 ꢀC to a vigorously stirred solution
of 1-[(4-methylphenyl)sulfonyl]-1H-indole (2)29 (1.36 g,
5.01 mmol) in dry THF (40 mL). The mixture was
brought to À5 ꢀC within 40 min and allowed to warm to
20 ꢀC for 5 min. The solution was treated at À20 ꢀC with
3 (1.23 g, 5.0 mmol) dissolved in dry THF (20 mL), stir-
red for 1 h and then warmed to 20 ꢀC. The reaction
mixture was carefully quenched with water (40 mL), and
the mixture was extracted with ether (3Â50 mL). The
combined organic layers were washed with brine and
dried over MgSO4. Chromatography with hex-
anes:EtOAc (20:1 to 5:1 gradient) a€orded 4 as a yel-
lowish solid (1.91 g, 74%), mp 151 ꢀC. TLC: Rf 0.31
Cultures of Malassezia furfur and isolation of
malassezin (1); Medium
The medium for the induction of alkaloid synthesis
consisted of Tween 80 ultra1 (30 mL), cycloheximide
(0.5 g), chloramphenicol (0.05 g) and agar (20 g). The
volume of the medium was adjusted to 1000 mL with
distilled water. After sterilization, sterile ®ltered l-tryp-
tophan at a concentration of 0.3 g% was added at 50 ꢀC
and 10 mL portions of the medium were poured into
sterile Petri dishes (10 cm in diameter). The pH was
adjusted to 5.5 using HCl (0.1 M).
1
(hexanes:EtOAc 5:1); H NMR (CDCl3) d 1.67 (s, 9H),
2.34 (s, 3H), 3.78 (d, 1H, J=5 Hz), 6.36 (s, 1H), 6.58 (d,
1H, J=5 Hz), 6.93 (d, 1H, J=8 Hz), 7.03 (dt, 1H,
J=8.5 Hz, J=1 Hz), 7.17 (d, 2H, J=8.5 Hz), 7.18 (s,
1H), 7.21±7.36 (m, 4H), 7.69 (d, 2H, J=8.5 Hz), 8.16 (d,
1H, J=8.5 Hz), 8.18 (d, 1H, J=8.5 Hz); 13C NMR
(CDCl3) d 21.5, 28.1, 63.4, 83.8, 111.9, 114.7, 115.6,
119.8, 120.7, 121.4, 122.5, 123.7, 124.1, 124.3, 125.2,
126.3, 128.5, 128.8, 129.9, 136.7, 135.8, 137.5, 142.0,
Cultivation and isolation
A suspension of CBS 1878 M. furfur was smeared on
the minimal medium by a swab. After incubation for 14
days at 30 ꢀC, the content of the Petri dishes was pureed
and extracted with EtOAc for 12 h. The extract was ®l-
145.1, 149.6; IR (KBr) 3443, 1734, 1372, 1173 cmÀ1
;
HRMS (EI) m/z 516.1923 [M+], calculated for
C29H28NO5S, 516.1717.

G. Wille et al. / Bioorg. Med. Chem. 9 (2001) 955±960
959
3-{Acetoxy-[1-(4-methylphenylsulfonyl)-1H-indol-2-yl]-
methyl}-indole-1-carboxylic acid tert-butyl ester (5).
6.47 mL (11 mmol) of tert-BuLi (1.7 M in pentane)
were added at À20 ꢀC to a vigorously stirred solution of
2 (2.71 g, 10 mmol) in dry THF (80 mL). The mixture
was heated to À5 ꢀC within 40 min and allowed to warm
up to rt for 5 min. The solution was treated at À20 ꢀC
with 3 (2.45 g, 10 mmol) dissolved in dry THF (35 mL),
stirred for 2 h, and then treated with acetyl chloride
(1.36 mL, 20 mmol) and warmed to rt overnight. The
reaction mixture was carefully quenched with water
(50 mL), and the mixture was extracted with ether
(3Â80 mL). The combined organic layers were washed
with NaHCO3 (5%, 3Â70 mL), brine and dried over
MgSO4. Chromatography with hexanes:EtOAc (10:1)
a€orded 5 as a yellowish solid (3.18 g, 57%), mp 172 ꢀC,
besides unreacted alcohol 4 (852 mg, 1.65 mmol). TLC:
argon and then warmed to 20 ꢀC for 15 min. The liquid
was treated at 0 ꢀC with 6 (180 mg, 0.73 mmol) dissolved
in dry DMF (3 mL), and the ice bath was removed after
20 min. Careful warming to 40 ꢀC completed the for-
mylation reaction as indicated by TLC. The dark brown
solution was poured into saturated aqueous NaHCO3
(15 mL) at 20 ꢀC and extracted with ether (3Â20 mL).
The combined organic layers were washed with NaOH
(1 M, 320 mL) and brine, dried over MgSO4 and con-
centrated in vacuo (5 mL). A ®rst batch of 1 (102 mg)
crystallized from ether at À20 ꢀC in form of pinkish
crystals. Chromatography of the mother liquor with
hexanes:EtOAc (5:1±3:1 gradient) a€orded a second
batch (46 mg) of 1 (total yield: 148 mg, 74%), mp
240 ꢀC. TLC: Rf 0.52 (hexanes:EtOAc 1:1); H NMR
1
(DMSO-d6) d 4.57 (s, 2H), 6.94 (t, 1H, J=7 Hz), 7.06 (t,
1H, J=8 Hz), 7.13 (t, 2H, J=3 Hz, J=4.5 Hz), 7.26 (d,
1H, J=2.5 Hz), 7.34 (d, 1H, J=8 Hz), 7.38 (d, 1H,
J=4 Hz), 7.49 (d, 1H, J=8 Hz), 8.05 (t, 1H, J=4 Hz),
10.28 (s, 1H), 10.98 (s, 1H), 11.90 (s, 1H); 13C NMR
(DMSO-d6) d 22.1, 110.9, 111.6, 111.8, 113.2, 118.3,
118.7, 120.3, 121.3, 122.0, 122.7, 123.8, 125.7, 126.7,
135.5, 136.3, 151.4, 184.6; IR (KBr) 3402, 3379,
1618 cmÀ1; UV/Vis (CH3CN) lmax (lgE) 219 (4.73), 243
(4.23), 268 (4.20), 282 (4.15), 290 (4.17); HRMS (EI) m/
z 274.1106 [M+], calculated for C18H14N2O, 274.1116.
1
Rf 0.42 (hexanes:EtOAc 5:1); H NMR (acetone-d6) d
1.65 (s, 9H), 2.15 (s, 3H), 2.32 (s, 3H), 6.95 (t, 1H,
J=1 Hz), 7.23 (t, 1H, J=1 Hz), 7.26 (d, 2H, J=8.5 Hz),
7.27 (t, 2H, J=7 Hz), 7.35 (2 ddd, 2 H, J=1.5, J=6 Hz,
J=6 Hz and J=1.5 Hz, J=6 Hz, J=10 Hz), 7.54 (d,
1H, J=6 Hz), 7.57 (d, 1H, J=6 Hz), 7.74 (d, 2H,
J=8 Hz), 7.90 (t, 1H, J=1 Hz), 8.13 (d, 1H, J=8 Hz),
8.16 (d, 1H, J=8 Hz); 13C NMR (acetone-d6): d=21.1,
21.6, 28.3, 65.6, 85.1, 112.5, 115.7, 116.2, 119.7, 120.8,
122.4, 123.8, 124.9, 125.7, 126.1, 126.6, 127.6, 129.5,
130.1, 130.8, 136.5, 136.5, 138.3, 139.8, 146.4, 150.2,
170.3; IR (KBr) 1736, 1371, 1175 cmÀ1; UV/VIS
(CH3OH) lmax (lgE) 220 (4.63), 254 (4.41), 284 (3.97),
292 (3.96); HRMS (EI) m/z 558.1825 [M+], calcd for
C31H30N2O6S, 558.1805.
Indolo[3,2-b]carbazole (7). A solution of 1 (13 mg,
0.045 mmol) in THF (15 mL) was treated with one drop
of concd HCl in an open ¯ask. The mixture was re¯uxed
overnight, cooled to room temperature and ®ltered to
yield 7 as a yellowish precipitate (9 mg, 78%), mp
>350 ꢀC. TLC (RP-18): Rf 0.60 (MeOH 100%); 1H
NMR (DMSO-d6) d 7.10 (t, 2H, J=7.5 Hz), 7.31 (t, 2H,
J=7.5 Hz), 7.42 (d, 2H, J=7 Hz), 8.09 (s, 2H), 8.17 (d,
2H, J=7 Hz), 10.98 (s, 2H); 13C NMR (DMSO-d6) d
101.3, 111.3, 118.5, 121.1, 123.4, 123.5, 126.3, 135.9,
142.0; IR (KBr) 3403 cmÀ1; UV/Vis (MeOH) lmax
(qual.) 213, 249, 270, 320, 334 nm; MS (EI) m/z 256
(M+). The data are in agreement those given in ref 21.
2,30-Methylenebisindole (6). 20±25 mL of ammonia were
condensed into a solution of 5 (370 mg, 0.66 mmol) in
dry THF (3 mL) at À78 ꢀC under mechanical stirring.
The yellow muddy liquid was treated portion-wise with
sodium (50±100 mg) until the blue colour remained per-
manent for 30 min. The reaction was quenched with
NH4OAc (2 g, 26 mmol) and warmed up to 20 ꢀC. The
mixture was diluted with saturated aqueous NH4OAc
(30 mL) and extracted with ether (3Â50 mL). The com-
bined organic layers were washed with brine and dried
over MgSO4. Chromatography with hexanes:EtOAc
(5:1) a€orded 6 as a colourless solid that turned pink on
exposure to air15 (85 mg, 52%), mp 130±134 ꢀC. TLC:
EROD Bioassay
Cell culture and treatment. Hepatocytes were prepared
from male Wistar rats (Charles River, Kisslegg, Ger-
many) as described earlier.30 In brief, after anesthesia
with sodium pentobarbital (100 mg/kg b.w., ip), livers
were perfused in a two-step procedure with a calcium-
free, EGTA-supplemented bu€er and with a calcium-
containing bu€er supplemented with collagenase
(500 mg/L), respectively, each step taking 10 min.
Hepatocytes were dispersed in a BSA-containing wash-
ing bu€er, ®ltered through silk gauze, and washed with
washing bu€er. Viability was measured with a trypan
blue assay, and preparations showing viability >90%
were seeded on collagenated Petri dishes (6 cm diameter)
at a density of 70,000 cells/cm2 in DMEM containing
20% FCS, dexamethasone (0.1 mM), penicillin (100 U/
mL), and streptomycin (100 mg/mL). After 3 h, medium
was replaced by fresh medium, malassezin (1) was
added in DMSO (0.5% ®nal concentration), and the
cells were incubated for additional 48 h. DMSO served
as a control.
1
Rf 0.43 (hexanes:EtOAc 3:1); H NMR (acetone-d6) d
4.25 (s, 2H), 6.28 (q, 1H, J=1 Hz), 6.94 (dt, 1H,
J=7.5 Hz, J=1.5 Hz), 6.96 (dt, 1H, J=7.5 Hz,
J=1.5 Hz), 6.99 (dt, 1H, J=7.5 Hz, J=1.5 Hz), 7.09
(dd, 1H, J=8 Hz, J=1 Hz), 7.22 (dt, 1H, J=1 Hz), 7.27
(d, 1H, J=8.5 Hz), 7.39 (d, 1H, J=8 Hz), 7.43 (d, 1H,
J=8 Hz), 7.52 (d, 1H, J=8 Hz), 9.89 (br. s, 1H), 10.01
(br. s, 1H); 13C NMR (acetone-d6) d 25.0, 100.2, 111.4,
112.1, 113.3, 119.4, 119.5, 119.6, 120.2, 121.1, 122.1,
124.0, 128.4, 129.9, 137.5, 137.8, 140.2; IR (KBr)
3396 cmÀ1; UV/VIS (CH3CN) lmax (lgE) 223 (4.76), 272
(4.13), 280 (4.13), 289 (4.04); MS (EI) m/z 246.1 (M+).
The data are in agreement with those given in ref 15.
2-(1H-Indol-3-ylmethyl)-1H-indol-3-carbaldehyde, malas-
sezin (1). A solution of POCl3 (0.072 mL, 0.8 mmol) in
dry DMF (2 mL) was stirred at 0 ꢀC for 20 min under

960
G. Wille et al. / Bioorg. Med. Chem. 9 (2001) 955±960
8. Klinman, J. P. Chem. Rev. 1996, 96, 2541.
Analysis of CYP 1A activity
9. Mayser, P.; Wille, G.; Imkampe, A.; Thoma, W.; Arnold,
N.; Monsees, T. Mycoses 1998, 1, 265.
10. Mayser, P.; Pape, B. Antonie van Leeuwenhoek 1998, 73,
315.
11. Mayser, P. Habilitation Thesis. University of Gieûen,
Germany, 1999.
12. Sundberg, R. J.; Russell, H. F. J. Org. Chem. 1973, 38,
3324.
13. Saulnier, M. G.; Gribble, G. W. J. Org. Chem. 1982, 47, 757.
14. An alternative method for the conversion of carbinol 4
into 2,30-methylenebisindole (6) has been described by Tho-
lander and Bergman (ref 20).
Cells were washed with ice-cold saline and scraped of
with a tris-bu€ered glucose solution (50 mM Tris,
200 mM glucose, pH 7.4). The cells were then cen-
trifuged at 1000 g for 5 min and homogenized by soni-
cation (50 W) on ice. Activity of the CYP 1A isozymes
in the homogenates were measured by determination of
their ERODactivity according to the method of Burke
and Mayer.31 Protein contents were analyzed according
to Lowry.32
15. Jackson, A. H.; Prasitpan, N.; Shannon, P. V. R.; Tinker,
A. C. J. Chem. Soc. Perkin Trans. I 1987, 2543.
Statistical analysis
16. Gribble, G. W.; Pelcman, B. J. Org. Chem. 1992, 57, 3636.
17. X-ray crystal structure data of 1. Formula: C22H24N2O2;
Formula weight: 348.43 g/mol; Temperature: 293(2) K; Wave
length: 0.71073 A; Crystal system: triclinic; Space group: P-1;
Unit cell dimensions: a=8.000(2) A, a=102.65(2)ꢀ, b=
10.564(3) A, b=97.72(2)ꢀ, c=12.193(3) A, g=95.67(2)ꢀ;
Volume: 987.6(4) A3; Z=2; Density (calculated): 1.172 g/cm3;
Crystal habit: slightly pink plate; Absorption coecient:
0.075 mmÀ1; F(000): 372; Crystal size: 0.53Â0.33Â0.17 mm;
Theta range for data collection: 2.59ꢀ to 23.97ꢀ; Index ranges:
À9=h=9, 0=k=12, À13=l=13; Re¯ections collected: 3276;
Independent re¯ections: 3083 (Rint=0.0148); re¯ections
observed: 2263 (I>2sI) Absorption correction: Semi-empiri-
cal from psi-scans; Max. and min. transmission: 0.9994 and
0.9821; Re®nement method: Full-matrix least-squares on F2;
Data/restraints/parameters: 3083/0/237; Goodness-of-®t on
Treatments of primary hepatocytes were carried out
three times in duplicate. Dose±response curves, EC50-
values, and 95%-con®dence intervals were calculated
using a computerized log-probit procedure (Origin 5.0,
Microcal, Northampton, USA).
Tyrosinase bioassay
335 mL of a l-DOPA stock solution (6.4 mM in 0.05 M
phosphate bu€er at pH 7.0) and 805 mL of the bu€er
were mixed. After addition of 107 mL tyrosinase (EC
1.14.18.1; 500 U/mL in phosphate bu€er) the extinction
was measured over 1 min indicating the formation of
dopaquinone. Fractions of the crude extract obtained
by chromatography on Sephadex LH 20 were dissolved
in DMSO (100 mL) and added to the reaction with pure
DMSO as control. Inhibitory activity of the single frac-
tions was measured as reduced increase in extinction in
comparison to the control.
F2: 1.084; Final
R
indices [I>2s(I)]: R1=0.0526,
wR2=0.1314; R indices (all data): R1=0.0767, wR2=0.1479;
Largest di€. peak and hole: 0.324 and À0.185 e/A3.
18. X-ray crystal structures were calculated with SHELXS86
(Sheldrick, G.M. University of Gottingen, Germany, 1986)
and re®ned with SHELXL93 (Sheldrick, G. M. University of
Gottingen, Germany, 1993).
19. Pindur, U.; Muller, J. Arch. Pharm. 1987, 320, 280.
20. Tholander, J.; Bergman, J. Tetrahedron Lett. 1998, 39,
1619.
21. Kistenmacher, A.; Mullen, K. J. Heterocyclic Chem. 1992,
29, 1237.
Acknowledgements
The authors thank Dr. B. Ste€an, LMU Munchen, for
NMR measurements.
22. Gillner, M.; Bergman, J.; Cambillau, C.; Fernstrom, B.;
Gustafsson, J.-A. Mol. Pharm. 1985, 28, 357.
23. Burke, M. D.; Murray, G. I.; Lees, G. M. Biochem. J.
1983, 212, 15.
References and Notes
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J. C.; Brad®eld, C. A. Proc. Natl. Acad. Sci. U.S.A. 1991, 88,
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1. Gueho, E.; Midgley, G.; Guillot, J. Antonie Van Leeu-
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2. Yarrow, D.; Ahearn, D. C. In The Yeasts: A Taxonomic
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5. Further information about the disease and pictures from
patients are available on the web, http://www.dermis.net/
bilddb/diagnose/englisch/i111000.htm.
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29. Illi, V. O. Synthesis 1979, 136. We prepared compound 2
in 85% yield following the procedure of ref 13.
30. Schmitz, H.-J.; Hagenmaier, A.; Hagenmeier, H.; Bock,
K. W.; Schrenk, D. Toxicology 1995, 99, 47.
31. Burke, M. D.; Mayer, R. T. Drug Metab. Dispos. 1974, 2,
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32. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall,
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