Total Synthesis of Mycenarubin A, Sanguinolentaquinone and Mycenaflavin B and their Cytotoxic Activities

Article abstract of DOI:10.1002/ejoc.201800417

Here we report the first total synthesis of the fungal alkaloids mycenarubin A, sanguinolentaquinone and mycenaflavin B. The pyrroloquinoline alkaloid mycenarubin A was obtained in 10 steps (21 % total yield, 92 % ee) from the known key precursor 6,7-bis(benzyloxy)indole by an asymmetric alkylation and a biomimetic ring closure as the key steps. The indolo-6,7-quinone sanguinolentaquinone was obtained in eight steps (28 % total yield). Mycenaflavin B was also obtained in eight steps starting from the same key precursor (total yield 15 %) by a biomimetic ring closure and an acid-catalysed decarboxylation reaction as the key steps. The cytotoxic activities of mycenarubin A and mycenaflavin B were evaluated against mouse fibroblasts (L929) and human malignant melanoma cells (RPMI-7951).

Full text of DOI:10.1002/ejoc.201800417

A Journal of
Accepted Article
Title: Total Synthesis of Mycenarubin A, Sanguinolentaquinone and
Mycenaflavin B and Their Cytotoxic Activities
Authors: Jana Backenköhler, Bernhard Reck, Markus Plaumann, and
Peter Spiteller
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10.1002/ejoc.201800417
European Journal of Organic Chemistry
FULL PAPER
Total Synthesis of MycenarubinA, Sanguinolentaquinone and
Mycenaflavin B and TheirCytotoxic Activities
Jana Backenköhler,[a] Bernhard Reck,[a] Markus Plaumann[b] and Peter Spiteller*[a]
Dedicated to Professor Wolfgang Steglich on the occasion of his 85th birthday.
Abstract: Here we report the first total synthesis of the fungal
alkaloids mycenarubin A, sanguinolentaquinone and mycenaflavin B.
The pyrroloquinoline alkaloid mycenarubin A was obtained in ten
steps (21 % total yield, 92 % ee) from the known key precursor 6,7-
dibenzyloxyindole via an asymmetric alkylation and a biomimetic ring
closure as the key steps. The indolo-6,7-quinone sanguinolenta-
quinone was obtained in eight steps (28 % total yield). Mycenaflavin
B was obtained in eight steps starting from the same key precursor
(total yield 15 %) with a biomimetic ring closure and an acid-
catalysed decarboxylation reaction as the key steps. The cytotoxic
activities of mycenarubin A and mycenaflavin B were evaluated
against mouse fibroblasts (L929) and human malignant melanoma
cells (RPMI-7951).
Introduction
Figure 1. Pyrroloquinoline andindoloquinonealkaloids from mushrooms.
Pyrroloquinoline alkaloids isolated from marine organisms like
isobatzellines, damirones and makaluvamines are known for
their numerous biological activities, for instance their potent anti-
tumour activity.[1−5] In 1993 Steglich et al. isolated the first fungal
Results and Discussion
pyrroloquinoline alkaloid haematopodin (1) from the fruiting
bodies of Mycena haematopus.[6] Other pyrroloquinoline
alkaloids were isolated from Mycena rosea (mycenarubin A
(2)),[7] Mycena sanguinolenta (sanguinone A and B along with
the indolo-6,7-quinone alkaloid sanguinolentaquinone (3))[8] and
Mycena pelianthina (pelianthinarubin A and B).[9] Recently, the
previously unknown pyrroloquinoline alkaloids mycenaflavin A
(4a) and mycenaflavin B (4b) have been isolated from the
fruiting bodies of Mycena haematopus (Figure 1).[10]
Two synthetic routes are known for the synthesis of
pyrroloquinoline alkaloids.[11] In the first route the quinoline ring
is constructed first and then the pyrrole ring is closed. This
strategy was successfully used for the syntheses of the marine
pyrroloquinoline alkaloids damirone A, B and C.[12,13] The second
and most popular route starts with the formation of an indole
scaffold followed by the closure of the piperidine ring. This
synthetic strategy is similar to the proposed biosynthesis of
pyrroloquinoline alkaloids.[11] Different methods for closing the
piperidine ring have been described.[6,14−18] For instance, the
piperidine ring can be constructed via a biomimetic Michael
addition and reoxidation of the hydroquinone. This is an elegant
cyclisation method reported by Steglich et al.,[6] Lown et al.,[16]
So far, the biological activities and the ecological role of the
fungal alkaloids have not been studied in detail. But
mycenarubin A (2) (1.7 μmol) and the pelianthinarubins A and B
(1.1 μmol) did not show antimicrobial activities against selected
bacteria (Escherichia coli, Bacillus brevis and B. subtilis) or fungi
(Cladosporium cucumerinum).[7,9] However, the anti-tumour
activities of these compounds and the biological activities of
mycenaflavin B (4b) have not been examined so far on account
of the limited quantities of mushroom material available.
Hence, we developed total syntheses of mycenarubin A (2) and
mycenaflavin B (4b) and investigated the cytotoxic activities of
the synthesised compounds.
[17]
Cava et al. and our group.[18] The advantage of this method is
that no functionalisation at the C-4 position of the indole is
necessary. Thus, we intended to apply this strategy to
synthesise mycenarubin A (2) (Scheme 1) and mycenaflavin B
(4b).
In contrast to many marine pyrroloquinoline alkaloids[19]
mycenarubin A (2) and mycenaflavin B (4b) possess side chains
at N-5. These side chains should be introduced via reductive
amination. The challenge of the synthesis of mycenarubin A (2)
was the introduction of the stereocentre at the C-4 position.
We were able to synthesise an enantiopure tryptophan
derivative 5 by using an enantioselective alkylation of N-
(diphenylmethylene)glycine ethyl ester (14) in the presence of a
cinchona alkaloid (Scheme 1). The required alkylation agent 6
could be obtained from the known aldehyde 7.
[a]
[b]
J. Backenköhler,Dr.B.Reck, Prof.Dr. P. Spiteller
Institutfür Organischeund Analytische Chemie,UniversitätBremen,
Leobener Str.7, 28359 Bremen (Germany)
E-mail:psp@uni-bremen.de
Dr. M. Plaumann
Institutfür Biometrie und MedizinischeInformatik, Otto von Guericke
UniversitätMagdeburg, Leipziger Straße 44 (Haus 2), 39120
Magdeburg (Germany)
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10.1002/ejoc.201800417
European Journal of Organic Chemistry
FULL PAPER
the side chain via reductive amination. It was important to
choose a protecting group for the amino group on the side chain
which is selectively removable at the end of the total synthesis.
Thus, we chose the Teoc protecting group which can be easily
removed by fluoride ions. The corresponding aldehyde 15 was
synthesised via two steps from 3-aminopropanol. First, 3-
aminopropanol was N-Teoc protected using 4-nitrophenyl 2-
(trimethylsilyl)ethyl carbonate and then the alcohol was oxidised
to the aldehyde 15 by TEMPO oxidation (69 % yield over two
steps).[23,24] The side chain 15 was introduced via reductive
amination (NaBH(OAc)3, AcOH, CH2Cl2, 91 %). Usage of
NaCNBH3 in MeOH afforded 11 in lower yield (54 %) and purity.
During our attempts to synthesise mycenarubin A (2), we
observed an acid-catalysed decarboxylation reaction of the
protected mycenarubin A 27. Thus, we synthesised a racemic,
OH-protected mycenarubin
F
derivative 33 which was
decarboxylated and deprotected in one step to obtain
mycenaflavin B (4b). Additionally, we synthesised the indolo-6,7-
quinone alkaloid sanguinolentaquinone (3). Both syntheses will
be discussed later.
In the next step, the benzylic groups were deprotected under
hydrogen atmosphere. Surprisingly, after removal of the catalyst
and hydrogen atmosphere the resulting hydroquinone was not
oxidised to the quinone by oxygen in the air atmosphere.
Scheme 1.Retrosynthetic analysis for the synthesis ofmycenarubin A(2).
At first, the alkylating agent 6 for the enantioselective alkylation
was prepared via three steps from the known 6,7-
[18]
dibenzyloxyindole-3-carbaldehyde (7),
as outlined in Scheme
2. The Boc protection (Boc2O, DMAP) of the carbaldehyde 7
gave the protected indole 8 (97 %). Reduction of the aldehyde
with NaBH4, followed by benzylic bromination with PBr3 afforded
the alkylation agent 6 in 94 % yield over two steps. The
alkylation agent
6 is moisture- and heat-sensitive, thus
purification and mass spectrometric analysis are not possible.
Recently, Ku et al. developed an asymmetric phase-transfer
alkylation of N-(diphenylmethylene)glycine tert-butyl ester in the
presence of a cinchona alkaloid and successfully applied this
strategy to the enantioselective synthesis of an (S)-tryptophan
derivative (99 % ee).[20] Hence, we applied the above mentioned
strategy to synthesise the (S)-tryptophan derivative 5. In contrast
to Ku et al. we used the known N-(diphenylmethylene)glycine
ethyl ester (14)[21] for alkylation and Corey’s cinchona catalyst
13.[22] Phase-transfer catalytic conditions as described by Ku et
al. (50 % aq. KOH in toluene/chloroform (7:3) at 0 °C) gave the
tryptophan derivative 5 in 52 % yield.[20] When the alkylation was
performed at −78 °C in dichloromethane and with CsOH×H2O, 5
was obtained in higher yields (91 %, 97% ee). The enantiopurity
was determined by deprotection of the Schiff base under acidic
conditions (2 M aq. HCl, THF), followed by derivatisation with
(R)-Mosher′s acid chloride (DMAP, Et3N, CH2Cl2) and GC-MS
analysis (see the SI).
Scheme 2. Synthesis of the protected mycenarubin A 12. Reagents and
conditions: a) Boc2O, DMAP, THF/CH3CN (1:1), 12 h, rt, 97 %. b) NaBH4,
THF/EtOH (3:1), 3 h, rt, 98 %. c) PBr3, Et2O, 10 min, rt, 96 %. d) Corey’s
cinchona catalyst 13, N-(diphenylmethylene)glycine ethyl ester (14),
CsOH×H2O, CH2Cl2, 4 h, −78 °C, 91 % (97 % ee). e) 2 M aq. HCl, THF, 4 h, rt,
93 %. f) 15, NaBH(OAc)3, AcOH, CH2Cl2, 12 h, rt, 91 % g) H2, Pd/C, MeOH,
1 h, rt then Et3N, MnO2, air,1 h, rt, 49 %.
The coupling product was converted into the free amine 10 by
hydrolytic cleavage of the imine with aqueous HCl in THF (93 %).
The Boc protective group was not removed under these
conditions. Surprisingly, purification of the amine 10 by using
acid-base extraction was not possible. Therefore, we purified the
amine 10 by column chromatography (cyclohexane/EtOAc 5:1 to
EtOAc/MeOH 19:1). In the next step, we intended to introduce
Presumably the 2,3-double bond of the pyrrole ring was also
reduced during the hydrogenation due to the electron-
withdrawing effect of the Boc protecting group.
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10.1002/ejoc.201800417
European Journal of Organic Chemistry
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After addition of MnO2 the reaction mixture turned red
immediately, indicating the formation of the pyrroloquinoline 12.
The pyrroloquinoline 12 was obtained in 49 % yield after column
chromatography. LiOH-induced ester hydrolysis at 55 °C gave
the N-Teoc protected mycenarubin A 16 in 89 % yield (Scheme
3). Finally, we intended to cleave the N-Teoc protective group
with TBAF. First, we treated the N-Teoc protected mycenarubin
A 16 with an excess of TBAF in DMF. These conditions led to
Teoc deprotection but unfortunately the formed mycenarubin A
(2) was mostly N-formyl protected. Suchý et al. have already
described the formylation of amines in presence of DMF and a
base.[25] To avoid the undesired formylation reaction, we treated
the N-Teoc protected mycenarubin A 16 with an excess of TBAF
in DMSO but we could not observe any conversion. Addition of
CsF to the TBAF containing reaction mixture immediately led to
the formation of mycenarubin A (2). Treatment of the N-Teoc
protected mycenarubin A 16 with CsF alone did not result in
deprotection. Thus, a mixture of CsF and TBAF is necessary for
the Teoc deprotection. Purification of mycenarubin A (2) was
achieved by ion exchange chromatography (DOWEX 50WX8,
eluent: 25 % aq. NH3; 76 % yield, 92 % ee). The enantiomeric
excess of the synthesised mycenarubin A (2) was determined by
N-Boc protection, derivatisation of the carboxylic acid with a
proline derivative and separation of the resulting diastereomers
by LC-MS (see the SI). The specific rotation of the synthesised
the guideline for our synthesis.[26] The benzylic groups of the
protected alcohol 21 were removed by hydrogenation in the
presence of Pd/C in EtOAc (98 %). Unfortunately, the 2,3 double
bond of the pyrrole ring was also reduced due to electron-
withdrawing effect of the Boc protective group. Moreover, the
indoline 22 was not oxidised to the corresponding quinone by
aerial oxygen. Using DDQ the double bond was reestablished
and the catechol was oxidised to afford the quinone 23 in one
step with 91 % yield. Next, we intended to introduce a side chain
at the C-4 position using a Michael addition. We used the DMB-
protected 3-aminopropanol 25[26] as the nucleophile because
primary amines do not react selectively with quinones. The
resulting hydroquinone was oxidised to the quinone by oxygen in
an air atmosphere. Additionally, a transamination reaction led to
the removal of the Boc group. Treatment of the protected
sanguinolentaquinone 24 with trifluoroacetic acid in dichloro-
methane gave sanguinolentaquinone (3) in 72 % yield after
purification by column and size exclusion chromatography.
20
mycenarubin A (2) ([α]D = +647 (c = 0.0034, H2O)) was lower
than the specific rotation of the natural product reported in the
25
literature ([α]D = +1190 (c = 0.00807, H2O)).[7] Thus, we
isolated mycenarubin A (2) from an extract of Mycena rosea by
the known procedure[7] and found a lower specific rotation for the
20
natural product ([α]D = +669 (c = 0.0055, H2O)) which is in
agreement with the specific rotation of the synthesised
mycenarubin A (2). Mycenarubin A (2) slowly decomposes even
at −40 °C, hence the optical rotation decreases gradually.
Scheme 3. Synthesis ofmycenarubin A(2). Reagents and conditions: a) 0.1 M
aq. LiOH, THF, 48 h, 55 °C, 89 %. b) CsF, TBAF (1 M in THF), DMSO, 5 min,
rt, 76 % (92 % ee).
Scheme 4. Total synthesis of sanguinolentaquinone (3). Reagents and
conditions: a) (COCl)2, diethyl ether, 30 min, 0 °C then MeOH, 30 min, rt, 86 %.
b) LiAlH4, THF, 3 h, reflux, 92 %. c) TBDMSCl, imidazole, DMF, 12 h, rt, 97 %.
d) Boc2O, DMAP, THF, 12 h, rt, 92 %. e) H2 (1 atm), Pd/C, EtOAc, 4 h, rt,
98 %. f) DDQ, toluene, 5 min, rt, 91 %. g) 3-[N-(3,4-dimethoxybenzyl)amino]-
propan-1-ol (25), air, CH3CN, 72 h, rt, 59 %. h) CF3CO2H, CH2Cl2, 1 h, rt, 72 %.
Different strategies for the synthesis of mycenaflavin B (4b)
were applied (see the SI). The first generation synthesis
(Scheme 4) started with the protected methyl indolyl-3-glyoxylic
acid 18 which was prepared by the reaction of the indole 17 with
oxalyl chloride followed by the addition of MeOH (yield 86 %).
Reduction of 18 with an excess of lithium aluminium hydride at
reflux in THF (92 %) yielded the alcohol 19, which was protected
as a TBDMS ether (yield 97 %). N-protection of 20 with Boc2O
and DMAP in THF gave the fully protected indole 21 in 93 %
yield. Henceforward, we used the procedure of Steglich et al. as
However, attempts to oxidatively cyclise sanguinolentaquinone
(3) to obtain the mycenaflavin B derivative 26 by using Dess-
Martin periodinane have not been successful (Scheme 5).
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10.1002/ejoc.201800417
European Journal of Organic Chemistry
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Scheme 5. Attempts to oxidise sanguinolentaquinone (3) to form the
mycenaflavin B derivative 26. Reagents and conditions. a) Dess-Martin
periodinane, CH2Cl2,2 h, 0 °C to rt.
During our attempts to synthesise mycenarubin A (2) we
observed an unexpected side reaction (Scheme 6). When the N-
Boc protected mycenarubin A 27 was treated with a Lewis or
Brønsted acid,
a
decarboxylation reaction took place.
Fortunately, we were able to use this strategy for the total
synthesis of mycenaflavin B (4b).
Scheme 6. Attempted deprotection of the N-Boc protected mycenarubin A
derivative 27 with a Lewis or Brønsted acid. Reagents and conditions: a) SnCl4,
DMF or AcOH, THF, H2O.
Scheme 7. Total synthesis of mycenaflavin B (4b). Reagents and conditions:
a) Eschenmoser’s salt, CH2Cl2, 2 h, rt, 96 %. b) N-(Diphenylmethylene)glycine
ethyl ester (14), PBu3, CH3CN, 12 h, reflux, 92 %. c) 2 M HCl, THF, 4 h, 0 °C to
rt, 99 %. d) 3-(Tetrahydro-2H-pyran-2-yloxy)propan-1-al (34), NaCNBH3,
MeOH, 12 h, rt, 88 %. e) H2, Pd/C, MeOH, 1 h, rt, then Et3N, air, 1 h, 39 %. f)
LiOH, THF/H2O (1:1), 48 h, 55 °C. g) AcOH, MeOH/H2O (2:1), 40 °C, 2 h. h)
AcOH, rt, 72 h, 48 % over three steps.
Since the carboxylic acid should be removed in the last step of
the synthesis of mycenaflavin B (4b), the synthesis of an
enantiopure tryptophan derivative 5 was not necessary. Thus,
we developed a shorter synthesis of a racemic tryptophan
derivative 30 (Scheme 7). The synthesis started with Mannich
reaction of the indole 17 with Eschenmoser’s salt (96 %).[26]
Somei-Kametani coupling of the known gramine 29 and N-
(diphenylmethylene)glycine ethyl ester (14) in the presence of
0.5 eq. tributylphosphine gave the protected tryptophan
derivative 30 in 92 % yield. Although tributylphosphine has only
a catalytic effect on the reaction, 0.5 eq. tributylphosphine are
advisable because residues of benzophenone (impurity from
benzophenone imine used for the synthesis of 14) deactivate the
phosphine catalyst. The coupling product can be converted into
the free amine 31 upon hydrolysis of the imine with aqueous
hydrochloric acid (93 %). In the case of 31 purification by a
simple acid-base extraction was possible (presumably, due to
the absence of the N-Boc protecting group). Again the side
chain was introduced via reductive amination (3-(tetrahydro-2H-
pyran-2-yl)oxypropanal (34),[27] NaCNBH3, 88 %). We used a
THP-protected propanal derivative 34 because the THP group
can be removed during the final acidic decarboxylation due to
the acid labiality of the THP protecting group (Scheme 7).
The benzylic groups were removed by catalytic hydrogenation,
subsequently the hydroquinone was oxidised to the correspond-
ing quinone 33 under air atmosphere (39 % yield).
In this case the addition of MnO2 was not necessary,
presumably again due to the absence of the N-Boc protecting
group. A one pot synthesis was finally used to synthesise
mycenaflavin B (4b) from the pyrroloquinoline 33. First, the ethyl
ester was converted into the carboxylic acid using aq. LiOH in
THF at 55 °C. After acidification with AcOH complete
decarboxylation took place at 40 °C within 2 h. Finally, further
addition of AcOH and stirring at rt for 3 days led to THP
deprotection and to the formation of mycenaflavin B (4b) (48%
over three steps). Purification of mycenaflavin B (4b) was
achieved by HPLC. The 13C chemical shifts and the width of 1H
and 13C signals of mycenaflavin B (4b) strongly depend on the
pH value. Only small changes of the pH value lead to shifting
and broadening of the signals. Particularly, the signals of C-2
and C-8a are affected by a change of the pH value.
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The resulting synthetic mycenarubin A (2) and mycenaflavin B
(4b) were evaluated for their antimicrobial activity against three
soil bacteria. Mycenarubin A (2) did not display antimicrobial
activity against Azospirillum brasilense, Azoarcus tolulyticus and
Azovibrio restrictus (1 μmol). Mycenaflavin B (4b) did not exhibit
antimicrobial activity against Azospirillum brasilense and
Azoarcus tolulyticus but revealed a moderate activity against
Azovibrio restrictus (inhibition zone: Ø = 0.8 cm, 1 μmol).[10]
Unfortunately, no anti-tumour activities were found for
mycenaflavin B (4b). The cytotoxicity of mycenaflavin B (4b)
could result from its planarity and therefore the possibility of
DNA-intercalation.
Conclusions
The enantioselective total synthesis of mycenarubin A (2) and
total syntheses of sanguinolentaquinone (3) and mycenaflavin B
(4b) have been achieved in 10, 8 and 8 steps, respectively,
starting from the key precursor 6,7-dibenzyloxyindole (17). Key
steps to obtain mycenarubin A (2) were an enantioselective
alkylation reaction with N-(diphenylmethylene)glycine ethyl ester
(14) in the presence of a cinchona alkaloid derived catalyst and
a biomimetic ring closure. The key step for the synthesis of
mycenaflavin B (4b) was an acid catalysed decarboxylation
reaction forming the enamine. The total yields are ranging from
Additionally, we tested the cytotoxic activities of mycenarubin A
(2) and mycenaflavin B (4b). Two fast growing cell lines, mouse
fibroblast L929 and human malignant melanoma RPMI-7951,
were incubated for up to 52 h using three different
concentrations of each compound (up to 870 μM for
mycenarubin A (2) and up to 1170 μM for mycenaflavin B (4b)).
(a)
100
21 % (92 % ee) for mycenarubin
A
(2), 28 % for
80
60
40
20
0
sanguinolentaquinone (3) to 15 % for mycenaflavin B (4b).
These efficient synthetic routes allowed the examination of the
antimicrobial and anti-tumour activity of the synthesised
compounds. Non-antimicrobial and non-cytotoxic properties
were observed for mycenarubin A (2). Mycenaflavin B (4b) has
shown a moderate activity against the soil bacterium Azovibrio
restrictus (inhibition zone: Ø = 0.8 cm, 1 μmol) and a moderate
cytotoxicity with a CC50 of 400 μM for fibroblasts and 500 μM for
melanoma cells, respectively.
0
200
400
c [μmol/L]
600
800
100
80
60
40
20
0
(b)
ExperimentalSection
General: Syntheses which are sensitive to oxygen or air humidity were
performed in glassware dried by heating in vacuo and under an argon
atmosphere. Dry CH2Cl2, CH3CN, THF and toluene were directly taken
from the solvent purification system SPS-800 constructed by M. Braun.
DMF and DMSO were distilled over phosphorus pentoxide and stored
over molecular sieve (3 Å). Methanol and ethanol were distilled over
calcium oxide and also stored over molecular sieve (3 Å). Et2O was
stored over sodium. Purification of reaction products was carried out by
column chromatography using silica gel 60 (40−63 µm) as stationary
phase. Analytical thin-layer chromatography was performed with TLC
aluminum foils coated with silica gel 60 F254 purchased from Merck.
Visualisation was accomplished by UV light (254 nm and 366 nm) or
staining with KMnO4 solution. 1H NMR spectra were recorded on an
Avance WB-360, an Avance NB-360 (both 360 MHz) at 293 K or an
Avance DRX-600 spectrometer (Bruker Daltonics, 600 MHz) at 300 K
and are reported in ppm using the solvent as an internal standard (CDCl3
at 7.26 ppm, CD3OD at 3.31 ppm, D2O at 4.75 ppm[28] and d6-DMSO at
2.50 ppm). Data are reported as s (singlet), d (doublet), t (triplet), m
(multiplet) and br (broad). Coupling constants are quoted in Hz and
signals were integrated. Proton-decoupled 13C NMR spectra were
recorded on an Avance WB-360 (91 MHz) at 293 K or an Avance DRX-
600 (151 MHz) spectrometer at 300 K and are reported in ppm using
solvent as an internal standard (CDCl3 at 77.16 ppm, CD3OD at
49.00 ppm and d6-DMSO at 39.52 ppm). The 13C chemical shifts
measured in D2O were calibrated by the IUPAC recommendations[29]
using the conversion factor of 0.25144953 for calculating the absolute
frequency of 13C (0.0 ppm). EIMS and HR-EIMS spectra were obtained
on a MAT 95 spectrometer (Finnigan MAT). ESIMS spectra were
0
200
400
600
c [μmol/L]
800
1000
1200
Figure 2. Concentration-dependent effects of mycenarubin
A (2) or
mycenaflavin B (4b) on the vitality of different cell lines. The mouse fibroblasts
L929 (diamonds) and cells of the human malignant melanoma cell line RPMI-
7951 (triangles) were incubated for up to 52 h with mycenarubin A(2) (a) or for
up to 44 h with mycenaflavin B (4b) (b) with the concentrations indicated. The
dotted and the dashed regression lines refer to the data points obtained for
L929 cells andRPMI-7951 cells,respectively.
Incubation times of more than 50 h and concentrations of
mycenarubin A (2) up to 870 μM had no adverse effects on the
vitality of the fibroblasts and melanoma cells (Figure 2 (a)). A
complete different behaviour is observable for mycenaflavin B
(4b). The alkaloid 4b revealed a moderate cytotoxicity with a
50 % cytotoxic concentration (CC50) of 400 μM for fibroblasts and
500 μM for melanoma cells, respectively (Figure
2 (b)).
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10.1002/ejoc.201800417
European Journal of Organic Chemistry
FULL PAPER
recorded on an Esquire LC (Bruker). HR-ESIMS spectra were obtained
on a QExactive Plus Orbitrap (Thermo Fisher Scientific). Infrared spectra
were recorded on a Nicolet iS10 FT-IR spectrometer (Thermo Fisher
Scientific). Melting points were measured with a melting point apparatus
530 from Büchi, UV-Vis spectra were recorded on a Genesys 10S UV-
VIS spectrophotometer (Thermo Fisher Scientific). Optical rotation values
were measured on a Perkin-Elmer 243 polarimeter and CD spectra were
solvent was removed in vacuo. The residue was purified by flash column
chromatography (SiO2, CHCl3/MeOH 19:1) to afford 9 (1.22 g, 2.65 mmol,
1
98 %) as a yellow solid. Rf = 0.45 (CHCl3/MeOH 19:1); m.p. 122 °C; H
NMR (360 MHz, CDCl3, 293 K): δ = 1.60 (s, 9H, Boc), 1.83 (sbr, 1H, OH),
3
4.76 (s, 2H, H-1′), 5.18 (s, 4H, CH2Ph), 7.05 (d, J(H-5,H-4) = 8.3 Hz, 1H,
H-5), 7.27 – 7.48 ppm (m, 12H, CH2Ph, H-4, H-2); 13C NMR (90.5 MHz,
CDCl3, 293 K): δ = 28.1 (Boc), 57.2 (C-1′), 73.1 (CH2Ph), 75.3 (CH2Ph),
83.3 (Boc), 113.6 (C-5), 114.5 (C-4), 119.8 (C-3), 125.9 (C-2), 127.0 (C-
3a), 127.8 (CH2Ph), 127.9 (CH2Ph), 128.2 (CH2Ph), 128.5 (CH2Ph),
129.0 (CH2Ph), 130.1 (C-7a), 137.5 (CH2Ph), 138.0 (CH2Ph, C-7), 148.6
(C-6), 150.6 ppm (Boc); IR (ATR): ν = 695, 717, 736, 771, 797, 836, 845,
853, 905, 925, 989, 1001, 1027, 1045, 1079, 1146, 1159, 1223, 1237,
1262, 1347, 1371, 1388, 1421, 1456, 1475, 1500, 1557, 1614, 1676,
1747, 2857, 2936, 2979, 3028, 3059, 3413 cm−1; MS (EI, 70 eV): m/z
(%): 459 (3) [M]+∙, 268 (14) [M – Boc – Bn]+, 250 (7) [M – Boc – Bn –
H2O]+, 222 (7), 91 (100) [C7H7]+; HRMS (EI): calcd. for C28H29NO5 [M]+
459.20402; found: 459.20385.
recorded on
a
Chirascan spectrometer (Applied Photophysics).
Preparative HPLC separations were performed on Waters 510 pumps
equipped with an automated gradient controller 680 and a Waters 486
UV/vis detector. The samples were separated on a Nucleodur C-18ec
column (5 μm, 16 × 250 mm) by the use of the following gradient
programm: 10 min at 100% H2O (+ 0.1% AcOH) then within 20 min to
MeOH-H2O (50:50), then within 5 min linear to 100% MeOH (flow rate:
6 mL/min; UV/vis detection at 440 nm).
Cytotoxicity Tests: A Dulbecos Modified Eagle Medium (500 mL DMEM,
50 mL FCS and 1 g Ciprofloxacin) was used as the incubation medium.
The cells were incubated for up to 52 h. After the incubation time, the
cells were carefully washed and trypan blue solution was added to
100 μL cell suspension. The dead cells take up the dye and the rate
between the healthy cells and the dead cells was determined by counting
two loaded chambers (each with 9 squares) of a hemocytometer under
the microscope (further details see the SI).
6,7-Dibenzyloxy-3-(bromomethyl)-1-(tert-butyloxycarbonyl)indole (6).
Alcohol 9 (0.57 g, 1.25 mmol) was suspended in diethyl ether (20 mL)
and treated with PBr3 (44 μL, 0.45 mmol) at 0 °C. The mixture was stirred
for 10 minutes at ambient temperature. Sat. aq. NaHCO3 solution
(20 mL) was added and the phases were separated. The organic phase
was dried over MgSO4 and the solvent was removed in vacuo to afford 6
(0.63 g, 1.20 mmol, 96 %) as a yellow solid which was used immediately
without further purification. 1H NMR (360 MHz, CDCl3, 293 K): δ = 1.59 (s,
9H, Boc), 4.64 (s, 2H, H-1′), 5.17 (s, 2H, CH2Ph), 5.19 (s, 2H, CH2Ph),
Antimicrobial Assay: For plate diffusion assays, 2 (0.29 mg, 1 μmol) or
4b (0.24 mg, 1 μmol) was dissolved in H2O and was dropped onto paper
discs (Ø 6 mm, thickness 0.5 mm). These paper discs were dried under
sterile conditions and then placed on agar plates inoculated with the
respective test organism (Azospirillum brasilense (sp7), Azoarcus
tolulyticus (Td-1) and Azovibrio restrictus (DSM 23866, DSMZ)). The
plates were incubated at 37 °C for 24 h.
3
7.10 (d, J(H-5,H-4) = 7.7 Hz, 1H, H-5), 7.28 – 7.48 (m, 11H, CH2Ph, H-
4), 7.53 ppm (s, 1H, H-2); 13C NMR (90.5 MHz, CDCl3, 293K): δ = 24.9
(C-1′), 28.1 (Boc), 73.1 (CH2Ph), 75.4 (CH2Ph), 83.7 (Boc), 113.7 (C-5),
114.5 (C-4), 116.5 (C-3), 126.4 (C-3a), 127.2 (C-2), 127.9 (CH2Ph),
128.0 (CH2Ph), 128.2 (CH2Ph), 128.6 (CH2Ph), 129.0 (CH2Ph), 130.2 (C-
7a), 137.5 (CH2Ph), 138.0 (CH2Ph, C-7), 148.3 (C-6), 151.0 ppm (Boc);
IR (ATR): ν = 674, 695, 718, 740, 759, 767, 796, 821, 839, 855, 906, 928,
991, 1001, 1009, 1027, 1042, 1080, 1152, 1198, 1211, 1226, 1240, 1260,
1348, 1378, 1419, 1439, 1456, 1477, 1497, 1572, 1596, 1615, 1638,
1676, 1754, 2862, 2946, 2978, 3023, 3059, 3399 cm−1.
6,7-Dibenzyloxy-1-(tert-butyloxycarbonyl)indole-3-carbaldehyde (8).
To a solution of aldehyde 7[18] (0.40 g, 1.10mmol) in a mixture of THF
(10 mL) and CH3CN (10 mL) Boc2O (0.36 g, 1.65 mmol) and DMAP
(0.13 g, 1.10mmol) were added. The mixture was stirred at ambient
temperature for 12 h. The solvent was removed under reduced pressure
and the crude product was purified by flash column chromatography
(SiO2, CHCl3/MeOH 19:1) to afford 8 (0.49 g, 1.07mmol, 97 %) as a
yellow solid. Rf = 0.83 (CHCl3/MeOH 19:1); m.p. 107 °C; 1H NMR
(360 MHz, CDCl3, 293 K): δ = 1.61 (s, 9H, Boc), 5.20 (s, 4H, CH2Ph),
(S)-6,7-Dibenzyloxy-1-(tert-butyloxycarbonyl)-N-(diphenyl-
methylene)tryptophan ethyl ester (5). A solution of bromide 6 (0.71 g,
1.36 mmol) in dry CH2Cl2 (20 mL) was cooled to −78 °C. Subsequently,
N-(diphenylmethylene)glycine ethyl ester (14)[21] (0.37 g, 1.36 mmol), O-
allyl-N-9-anthracenylmethylcinchonidium
bromide (13)[22]
(84 mg,
3
7.16 (d, J(H-5,H-4) = 8.5 Hz, 1H, H-5), 7.27 − 7.49 (m, 10H, CH2Ph),
3
0.0136 mmol) and CsOH×H2O (0.78 g, 5.17 mmol) were added and the
reaction mixture was stirred at −78 °C for four hours. Water (20 mL) and
EtOAc (20 mL) were added and the phases were separated. The
aqueous phase was extracted with EtOAc (2×20 mL). The combined
organic phases were dried (MgSO4) and the solvent was removed under
reduced pressure. The crude product was purified by flash column
chromatography (SiO2, cyclohexane/EtOAc 5:1) to afford 5 (0.88 g,
1.24 mmol, 91 %) as a yellow viscous oil. The enantioselectivity was
7.98 (d, J(H-4,H-5) = 8.5 Hz, 1H, H-4), 8.03 (s, 1H, H-2), 10.03 ppm (s,
1H, CHO); 13C NMR (90.5 MHz, CDCl3, 293 K): δ = 28.0 (Boc), 72.6
(CH2Ph), 75.2 (CH2Ph), 85.2 (Boc), 114.5 (C-5), 117.4 (C-4), 121.1 (C-3),
123.0 (C-3a), 127.9 (CH2Ph), 128.0 (CH2Ph), 128.1 (CH2Ph), 128.3
(CH2Ph), 128.6 (CH2Ph), 128.9 (CH2Ph), 130.4 (C-7a), 137.0 (C-7),
137.1 (CH2Ph), 137.7 (CH2Ph), 138.8 (C-2), 147.7 (C-6), 151.4 (Boc),
185.6 ppm (CHO); IR (ATR): ν = 695, 714, 735, 745, 772, 804, 817, 851,
905, 922, 1012, 1028, 1034, 1048, 1080, 1147, 1181, 1224, 1236, 1263,
1284, 1332, 1372, 1385, 1394, 1418, 1457, 1475, 1503, 1557, 1579,
1618, 1676, 1755, 2833, 2862, 2909, 2938, 2979 cm−1; MS (EI, 70 eV):
m/z (%): 457 (0.5) [M]+∙, 401 (0.5) [M – C4H9]+, 357 (0.5) [M – Boc]+, 266
(8) [M – Boc − Bn]+, 91 (100) [C7H7]+; HRMS (EI): calcd. for C28H27NO5
[M]+ 457.18837; found: 457.18885.
determined by GC-MS (97 % ee, see the SI). Rf
=
0.37
(cyclohexane/EtOAc 5:1); [α]D = −85.5 (c = 1.446, CHCl3); 1H NMR
(360 MHz, CDCl3, 293K): δ = 1.30 (t, 3J(CH3,CH2) = 7.1 Hz, 3H,
CO2CH2CH3), 1.55 (s, 9H, Boc), 3.24 (dd, 2J(H-1′,H-1′) = 14.2 Hz,
20
2
3
3J(H1′,H-2′) = 9.1 Hz, 1H, H-1′), 3.35 (dd, J(H-1′,H-1′) = 14.2 Hz, J(H-
3
1′,H-2′) = 4.4 Hz, 1H, H-1′), 4.17 (m, 2H, CO2CH2CH3), 4.39 (dd, J(H-
3
2′,H-1′) = 9.1 Hz, J(H-2′,H-1′) = 4.4 Hz, 1H, H-2′), 5.17 (s, 2H, CH2Ph),
6,7-Dibenzyloxy-1-(tert-butyloxycarbonyl)-3-(hydroxymethyl)indole
(9). The protected aldehyde 8 (1.25 g, 2.70 mmol) was dissolved in a
mixture of THF (45 mL) and ethanol (15 mL). NaBH4 (0.21 g, 5.46 mmol)
was added and the mixture was stirred at ambient temperature for two
hours. The solvent was removed under reduced pressure. The residue
was dissolved in EtOAc (50 mL) and was washed with sat. aq. NaHCO3
solution (100 mL). The organic phase was dried over MgSO4 and the
5.23 (s, 2H, CH2Ph), 6.69 (d, 3J(H,H) = 7.2 Hz, 2H, Ph), 6.90 (m, 1H, H-4,
H-5), 7.17 − 7.65 ppm (m, 18H, CH2Ph, Ph); 13C NMR (90.5 MHz, CDCl3,
293 K):
δ = 14.2 (CO2CH2CH3), 27.9 (Boc), 28.6 (C-1′), 61.0
(CO2CH2CH3), 65.2 (C-2′), 72.9 (CH2Ph), 75.0 (CH2Ph), 82.6 (Boc),
113.1 (C-5), 114.0 (C-4), 115.9 (C-3), 126.4 (C-2), 127.5 (Ph), 127.6 (Ph),
127.7 (Ph), 127.8 (Ph), 127.9 (Ph), 128.0 (Ph), 128.2 (Ph), 128.3 (C-3a),
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201800417
European Journal of Organic Chemistry
FULL PAPER
128.4 (Ph), 128.7 (Ph), 128.8 (Ph), 129.3 (C-7a), 130.2 (Ph), 135.7 (Ph),
137.4 (Ph), 137.6 (C-6), 137.9 (Ph), 139.2 (Ph), 148.4 (Boc), 150.1 (C-5),
170.9 (C-4′), 171.7 ppm (CO2Et); IR (ATR): ν = 694, 742, 781, 796, 853,
907, 1023, 1066, 1105, 1151, 1233, 1255, 1351, 1368, 1419, 1446, 1496,
1575, 1597, 1615, 1733, 2850, 2925, 2977 cm−1; MS (EI, 70 eV): m/z
(%): 708 (2) [M]+, 608 (8) [M – Boc]+, 517 (19) [M – Boc – Bn]+, 489 (9)
[M – Boc – Bn – CO]+, 342 (14), 307 (14), 266 (10), 193 (17), 91 (100)
[C7H7]+; HRMS (EI): calcd. for C45H44N2O6 [M]+ 708.31939; found:
708.32019.
7.40 ppm (m, 11H, CH2Ph, H-2); 13C NMR (151 MHz, CDCl3, 300 K): δ =
−1.4 (Si(CH3)3), 14.1 (CO2CH2CH3), 17.8 (CH2Si(CH3)3), 28.1 (Boc, C-1′),
28.6 (C-5′), 38.7 (C-6′), 45.4 (C-4′), 61.1 (C-2′), 61.5 (CH2CH2Si(CH3)3),
63.1 (CO2CH2CH3), 72.9 (CH2Ph), 75.2 (CH2Ph), 83.2 (Boc), 113.3 (C-5),
114.0 (C-4), 114.3 (C-3), 126.4 (C-2), 127.7 (CH2Ph), 127.8 (CH2Ph),
127.9 (C-3a), 128.1 (CH2Ph), 128.5 (CH2Ph), 128.9 (CH2Ph), 129.7 (C-
7a), 137.4 (CH2Ph), 137.8 (CH2Ph), 138.0 (C-7), 148.5 (C-6), 150.5 (Boc),
157.2 (SiCH2CH2OC=O), 172.2 ppm (CO2Et); IR (ATR): ν = 695, 734,
801, 836, 934, 1023, 1152, 1232, 1350, 1369, 1421, 1454, 1498, 1608,
1716, 1751, 2951, 3321cm−1; MS (EI, 70 eV): m/z (%): 745 (5) [M]+∙, 645
(10) [M –Boc]+, 554 (4) [M – Boc − Bn]+, 527 (7), 436 (6), 408 (7), 342
(100) [M – Boc – CH(CO2Et)NHCH2CH2NHC(O)OCH2CH2Si(CH3)3]+, 303
(18), 275 (16), 185 (23), 174 (8), 116 (12) [HOCH2CH2Si(CH3)3], 91 (81)
[C7H7]+, 73 (23) [Si(CH3)3/CO2Et]+; HRMS (EI): calcd. for C41H55N3O8Si
[M]+∙ 745.37529; found: 745.37499.
(S)-6,7-Dibenzyloxy-1-(tert-butyloxycarbonyl)tryptophan ethyl ester
(10). To a solution of the protected tryptophan 5 (0.53 g, 0.75 mmol) in
THF (14 mL) 1 M HCl (14 mL) was added and the mixture was stirred at
ambient temperature for four hours. THF was removed under reduced
pressure. The residue was neutralised with sat. aq. NaHCO3 solution and
the aqueous phase was extracted with EtOAc (3×50 mL). The combined
organic phases were dried over MgSO4. The solvent was removed in
vacuo and the residue was purified by flash column chromatography
(SiO2, first cyclohexane/EtOAc 5:1 to remove impurities like
benzophenone then EtOAc/MeOH 19:1) to afford 10 (0.38 g, 0.68mmol,
1-(tert-Butyloxycarbonyl)-N-[2-(trimethylsilyl)ethoxycarbonyl]-
mycenarubin A ethyl ester (12). An argon-purged suspension of the
tryptophan derivative 11 (0.13 g, 0.174 mmol) and 10 % Pd/C (40 mg) in
MeOH (90 mL) was stirred under a hydrogen atmosphere (1 atm,
balloon) at ambient temperature. After one hour, the catalyst was filtered
off. Subsequently, triethylamine (100 μL) and MnO2 (1.0 g) were added
to the filtrate which was stirred for an additional hour under air
atmosphere. The reaction mixture was filtered and the solvent was
removed under reduced pressure. The crude product was purified by
flash column chromatography (SiO2, CHCl3/MeOH 49:1) to afford 12
(48 mg, 85 μmol, 49%) as a red solid. Rf = 0.19 (CHCl3/MeOH 49:1);
20
93 %) as a brown oil which slowly solidifies. [α]D = +2.5 (c = 0.264,
3
CHCl3); 1H NMR (360 MHz, CDCl3, 293 K): δ = 1.24 (t, J(CH3,CH2) =
7.1 Hz, 3H, CO2CH2CH3), 1.60 (s, 9H, Boc), 1.66 (sbr, 2H, NH2), 2.94 (dd,
3
2J(H-1′,H-1′) = 14.4 Hz, J(H-1′,H-2′) = 7.5 Hz, 1H, H-1′), 3.14 (dd, 2J(H-
3
1′,H-1′) = 14.4 Hz, J(H-1′,H-2′) = 5.3 Hz, 1H, H-1′), 3.80 (dd, 3J(H-2′,H-1′)
3
= 7.5 Hz, 3J(H-2′,H-1′) = 5.3 Hz, 1H, H-2′), 4.16 (q, J(CH2,CH3) = 7.1 Hz,
2H, CO2CH2CH3), 5.17 (s, 2H, CH2Ph), 5.20 (s, 2H, CH2Ph), 7.04 (d,
3
20
1
3J(H-5,H-4) = 8.5 Hz, 1H, H-5), 7.22 (d, J(H-4,H-5) = 8.5 Hz, 1H, H-4),
[α]D = +511 (c = 0.009, MeOH); H NMR (600 MHz, CDCl3, 300 K): δ =
7.27 – 7.49 ppm (m, 11H, CH2Ph, H-2); 13C NMR (91 MHz, CDCl3,
293 K): δ = 14.1 (CO2CH2CH3), 27.9 (Boc), 30.4 (C-1′), 54.3 (C-2′), 60.8
(CO2CH2CH3), 72.7 (CH2Ph), 75.0 (CH2Ph), 82.8 (Boc), 113.1 (C-5),
113.9 (C-4), 115.3 (C-3), 125.9 (C-2), 127.5 (CH2Ph), 127.6 (CH2Ph),
127.7 (CH2Ph), 127.9 (CH2Ph), 128.1 (C-3a), 128.3 (CH2Ph), 128.7
(CH2Ph), 129.6 (CH2Ph), 137.3 (CH2Ph), 137.7 (C-7a), 137.8 (C-7),
148.3 (C-6), 150.3 (Boc), 174.8 ppm (CO2Et); IR (ATR): ν = 695, 718,
737, 756, 770, 795, 837, 846, 854, 905, 925, 1010, 1027, 1079, 1149,
1188, 1224, 1238, 1262, 1329, 1350, 1369, 1392, 1419, 1456, 1475,
1499, 1570, 1600, 1614, 1677, 1746, 2856, 2904, 2935, 2978, 3025,
3058, 3387cm−1; MS (EI, 70 eV): m/z (%): 544 (2) [M]+, 444 (4) [M –
Boc]+, 353 (9) [M –Boc – Bn]+, 342 (23), 252 (12), 189 (4), 161 (4), 132
(4), 91 (100) [C7H7]+; HRMS (EI): calcd. for C32H36N2O6 [M]+∙: 544.25679;
found: 544.25726.
0.01 (s, 9H, Si(CH3)3), 0.95 (t, J(CH2Si,CH2)= 8.6 Hz, 2H, CH2Si(CH3)3),
1.20 (t, J(CH3,CH2) = 7.1 Hz, 3H, CO2CH2CH3), 1.61 (Boc), 1.73 − 1.78
3
3
(m, 1H, H-2′), 1.92 − 1.96 (m, 2H, H-2′), 3.14− 3.25 (m, 4H, H-3, H-1′, H-
3′), 3.29 − 3.34 (m, 1H, H-3′), 3.65 − 3.67 (m, 1H, H-1′), 4.10 − 4.13 (m,
3
4H, CO2CH2CH3, CH2CH2Si), 4.37 − 4.41 (m, 1H, H-4), 5.13 (t, J(NH,H-
3′) = 6.2 Hz, 1H, NH), 5.46 (s, 1H, H-6), 7.32 ppm (s, 1H, H-2); 13C NMR
(151 MHz, CDCl3, 300 K): δ = −1.4 (Si(CH3)3), 14.2 (CO2CH2CH3), 17.9
(CH2Si(CH3)3), 24.0 (C-3), 27.8 (Boc), 27.9 (C-2′), 38.4 (C-3′), 49.6 (C-1′),
62.1 (C-4), 62.4 (CO2CH2CH3), 63.3 (CH2CH2Si), 86.6 (Boc), 96.3 (C-6),
114.3 (C-2a), 125.1 (C-8b), 125.8 (C-2), 129.6 (C-8a), 147.8 (Boc), 151.7
(C-5a), 157.3 (SiCH2CH2OC=O), 169.0 (C-8), 170.4 (CO2Et), 179.2 ppm
(C-7); IR (ATR): ν = 695, 719, 737, 769, 796, 837, 906, 921, 1027, 1080,
1150, 1186, 1238, 1261, 1311, 1348, 1370, 1392, 1420, 1455, 1475,
1500, 1528, 1558, 1603, 1683, 1746, 2935, 2978, 3327 cm−1; UV/vis
(MeOH): λmax (ε) = 507 (2416), 336 (17265), 254 (20580), 202 nm
(25993 mol−1 dm3 cm−1); MS (ESI+): m/z: 562 [M + H]+, 584 [M + Na]+;
HRMS (ESI+): calcd. for C27H39N3O8SiNa+ [M + Na]+ 584.23986; found:
584.24023.
(S)-N-[(N-(2-(Trimethylsilyl)ethyloxycarbonyl)-3-aminopropyl]-6,7-
dibenzyloxy-1-(tert-butyloxycarbonyl)tryptophan ethyl ester (11). To
a solution of the tryptophan derivative 10 (0.20 g, 0.37 mmol) in CH2Cl2
(5 mL) sodium triacetoxyborohydride (0.11 g, 0.51 mmol) and acetic acid
(24 μL) were added. After five minutes
a
solution of N-(2-
N-[2-(Trimethylsilyl)ethoxycarbonyl]mycenarubin
A
(16).
The
(trimethylsilyl)ethyloxycarbonyl)-3-aminopropan-1-al (15)[23,24] (0.092 g,
0.42 mmol) in CH2Cl2 (1 mL) was added and the reaction mixture was
stirred at ambient temperature overnight. Sat. aq. NaHCO3 solution
(10 mL) was added and the phases were separated. The aqueous phase
was extracted with CH2Cl2 (3×10 mL). The combined organic phases
were dried (MgSO4), the solvent was removed in vacuo and the crude
product was purified by flash column chromatography (SiO2,
CHCl3/MeOH 49:1) to afford 11 (0.25 g, 0.34 mmol, 91 %) as a yellow oil.
pyrroloquinoline 12 (52 mg, 93 μmol) was dissolved in THF (5 mL) and
0.1 M aq. LiOH solution (5 mL) was added. The reaction mixture was
stirred for 16 h at 55 °C. Subsequently, AcOH (31 μL) was added and the
solvent was removed in vacuo. The crude product was purified by flash
column chromatography (SiO2, CH2Cl2/MeOH 3:2) to afford 16 (36 mg,
20
83 μmol, 89%) as a purple solid. Rf = 0.75 (CH2Cl2/MeOH 3:2); [α]D
=
1
+249 (c = 0.007, MeOH); H NMR (600 MHz, CD3OD, 300 K): δ = 0.04 (s,
9H, Si(CH3)3), 0.97 (t, J(CH2Si,CH2) = 8.3 Hz, 2H, CH2Si(CH3)3). 1.86 −
3
1
Rf = 0.13 (CHCl3/MeOH 49:1); [α]D20 = +1.85 (c = 1.328, CHCl3); H NMR
1.98 (m, 2H, H-2’), 3.14 − 3.41 (m, 6H, H-3, H-1’, H-3’), 3.79 − 3.83 (m,
3
3
(600 MHz, CDCl3, 300K): δ = −0.02 (s, 9H, Si(CH3)3), 0.91 (t,
1H, H-3’), 4.12 (t, J(CH2,CH2Si) = 8.3 Hz, CH2CH2Si), 4.23 (d, J(H-4,H-
3) = 7.2 Hz, 1H, H-4), 5.42 (s, 1H, H-6), 6.95 ppm (s, 1H, H-2). 13C NMR
(151 MHz, CD3OD, 300 K): δ = −1.4 (Si(CH3)3), 18.6 (CH2Si(CH3)3), 25.5
(C-3), 29.2 (C-2’), 39.3 (C-1’), 50.4 (C-3’), 63.9 (CH2CH2Si), 66.9 (C-4),
93.4 (C-6), 117.9 (C-2a), 125.9 (C-8b), 126.1 (C-2), 127.0 (C-8a), 157.4
(SiCH2CH2OC=O), 159.3 (C-5a), 173.1 (C-8), 176.5 (CO2H), 180.5 ppm
(C-6). IR (ATR): ν = 668, 694, 719, 738, 755, 769, 795, 857, 905, 926,
3
3J(CH2Si,CH2) = 8.5 Hz, 2H, CH2Si(CH3)3), 1.05 (t, J(CH3,CH2) = 7.1 Hz,
3H, CO2CH2CH3), 1.53 (s, 9H, Boc), 1.76 − 1.80 (m, 2H, H-5′), 2.68 –
2.80 (m, 2H, H-4′), 3.10 − 3.24 (m, 4H, H-1′, H-6′), 3.71 (t, 3J(H-2’,H-1’) =
7.1 Hz, 1H, H-2′), 3.99 − 4.09 (m, 4H, CO2CH2CH3, OCH2CH2Si), 5.10 (s,
3
2H, CH2Ph), 5.11 (s, 2H, CH2Ph), 5.42 (sbr, 1H, NH), 6.97 (d, J(H-5,H-4)
= 8.4 Hz, 1H, H-5), 7.20 (d, 3J(H-4,H-5) = 8.4 Hz, 1H, H-4), 7.21 −
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201800417
European Journal of Organic Chemistry
FULL PAPER
3
3
941, 1027, 1034, 1044, 1066, 1078, 1106, 1151, 1187, 1225, 1240, 1261,
1328, 1352, 1368, 1393, 1418, 1442, 1455, 1476, 1498, 1574, 1599,
1725, 1745, 2901, 2976, 3057, 3388, 3675 cm−1. UV/vis (MeOH): λmax (ε)
= 529 (1590), 357 (16965), 245 (24612), 216 nm (12536 mol−1dm3cm−1);
MS (ESI−): m/z = 432 [M − H]−, 866 [2M − H]−. HRMS (ESI−): calcd. for
C20H26N3O6Si− [M − H]− 432.15964; found: 432.15988.
(sbr, 1H, OH), 2.97 (t, J(H-1’,H-2’) = 6.3 Hz, 2H, H-1’), 3.87 (t, J(H-2′,H-
1′) = 6.3 Hz, 2H, H-2′), 5.21 (s, 2H, CH2Ph), 5.22 (s, 2H, CH2Ph), 6.92 (s,
3
3
1H, H-2), 6.93 (d, J(H-5,H-4) = 8.7 Hz, 1H, H-5), 7.24 (d, J(H-4,H-5) =
8.7 Hz, 1H, H-4), 7.34 − 7.51 (m, 10H, CH2Ph), 7.93 ppm (sbr, 1H, NH);
13C NMR (90.5 MHz, CDCl3, 293 K): δ = 28.9 (C-1‘), 62.7 (C-2‘), 73.1
(CH2Ph), 75.6 (CH2Ph), 110.6 (C-5), 112.5 (C-3), 114.0 (C-4), 122.3 (C-
2), 124.6 (C-3a), 127.8 (CH2Ph), 128.0 (CH2Ph), 128.3 (CH2Ph), 128.5
(CH2Ph), 128.7 (CH2Ph), 131.6 (C-7a), 134.6 (C-7), 137.7 (CH2Ph),
138.1 (CH2Ph), 146.5 ppm (C-6); IR (ATR): ν = 666, 695, 751, 910, 929,
955, 991, 1020, 1042, 1065, 1184, 1217, 1257, 1305, 1335, 1347, 1369,
1440, 1453, 1467, 1505, 1628, 2864, 2924, 3033, 3061, 3242,
3399 cm−1; MS (EI, 70 eV): m/z (%) = 373 (18) [M]+∙, 282 (25)
[M − Bn]+,255 (5) [M –Bn – H2O]+, 254 (30) [M –Bn – CO]+, 236 (5) [M –
Bn – CO – H2O]+, 91 (100) [C7H7]+; HRMS (EI): calcd. for C24H23NO3
[M]+∙: 373.16725; found: 373.16622.
Mycenarubin A (2). To a solution of the protected pyrroloquinoline 16
(10.0 mg, 21 μmol) in DMSO (2 mL) CsF (14.0 mg, 92 μmol) and 1 M
TBAF in THF (0.1 mL, 100 μmol) were added. The reaction mixture was
stirred for 5 minutes. The solvent was removed in vacuo. The crude
product was purified by ion-exchange chromatography (DOWEX 50WX8,
eluent: 25% aq. NH3) to afford 2 (5.1 mg, 17.6 μmol, 76%, 92 % ee) as a
20
20
red solid. [α]D = +647 (c = 0.0034, H2O) (natural product: [α]D = +669
(c = 0.0055, H2O)); CD (H2O): λ (Δε) = 243 (−3.2), 263 (+1.0), 280 (+1.0),
310 (+1.4), 363 (−3.5), 514 nm (+1.3 L mol−1 cm−1); H NMR (600 MHz,
1
D2O, 300 K): δ = 2.01 – 2.12 (m, 2H, H-2’), 3.08 − 3.22 (m, 4H, H-3, H-3’),
6,7-Dibenzyloxy-3-(2-(tert-butyldimethylsilyloxy)ethyl)indole (20). To
a solution of the alcohol 19 (0.84 g, 2.25 mmol) in DMF (20 mL)
imidazole (0.34 g, 5.00 mmol) and TBDMSCl (0.51 g, 3.37 mmol) were
added. The reaction mixture was stirred for 12 h at ambient temperature.
Water (50 mL) was added and the aqueous layer was extracted with
ethyl acetate (3×50 mL). The combined organic phases were washed
with water (3×50mL) and dried over MgSO4. The solvent was removed
under reduced pressure and the crude product was purified by flash
column chromatography (SiO2, CHCl3) to afford 20 (1.07 g, 2.20mmol,
2
3
3
3.34 (ddd, J(H-1’,H-1’) = 14.1 Hz, J(H-1’,H-2’) = 7.1 Hz, J(H-1’,H-2’) =
3
7.1 Hz, 1H, H-1’), 3.75 (ddd, 2J(H-1’,H-1’) = 14.1 Hz, J(H-1’,H-2’) =
3
3
7.1 Hz, J(H-1’,H-2’) = 7.1 Hz, 1H, H-1’), 4.26 (d, J(H-4,H-3) = 6.8 Hz,
1H, H-4), 5.36 (s, 1H, H-6), 6.96 ppm (s, 1H, H-2); 13C NMR (151 MHz,
D2O, 300 K): δ = 26.9 (C-3), 28.0 (C-2’), 39.8 (C-3’), 51.2 (C-1’), 68.2 (C-
4), 95.3 (C-6), 118.9 (C-2a), 127.0 (C-8b), 127.8 (C-8a), 128.8 (C-2),
159.3 (C-5a), 174.3 (C-8), 179.3 (CO2H), 182.3 ppm (C-7); (ATR): ν =
666, 694, 738, 766, 808, 998, 1024, 1106, 1152, 1235, 1246, 1326, 1370,
1417, 1442, 1455, 1498, 1519, 1548, 1579, 1606, 1651, 1672, 1716,
1751, 2951, 3032, 3065, 3324 cm−1; UV/vis (MeOH): λmax (ε) = 527 (377),
355 (4000), 246 (7088), 200 nm (4211mol−1dm3cm−1); MS (ESI+): m/z =
290 [M + H]+, 312 [M + Na]+. HRMS (ESI+): calcd. for C14H15N3O4Na+
[M + Na]+ 312.09548; found: 312.09541.
1
97 %) as a colorless oil. Rf = 0.57 (CHCl3); H NMR (360 MHz, CDCl3,
3
293 K): δ = 0.19 (s, 6H, SiCH3), 1.07 (s, 9H, SiC(CH3)3), 3.08 (t, J(H-
3
1’,H-2’) = 7.3 Hz, 2H, H-1’), 4.01 (t, J(H-2’,H-1’) = 7.3 Hz, 2H, H-2’), 5.31
3
(s, 2H, CH2Ph), 5.33 (s, 2H, CH2Ph), 6.94 (d, J(H-2,NH) = 2.2 Hz, 1H,
H-2), 7.05 (d, 3J(H-5,H-4) = 8.6 Hz, 1H, H-5), 7.38 (d, 3J(H-4,H-5) =
8.6 Hz, 1H, H-4), 7.44 −7.63 (m, 10H, CH2Ph), 7.99 ppm (sbr, 1H, NH);
13C NMR (90.5 MHz, CDCl3, 293 K): δ = ‒5.2 (SiCH3), 18.5 (SiC(CH3)3),
26.1 (SiC(CH3)3), 29.2 (C-1’), 63.9 (C-2’), 73.0 (CH2Ph), 75.4 (CH2Ph),
110.3 (C-5), 113.3 (C-3), 114.0 (C-4), 121.9 (C-2), 125.0 (C-3a), 127.7
(CH2Ph), 127.9 (CH2Ph), 128.1 (CH2Ph), 128.4 (CH2Ph), 128.5 (CH2Ph),
131.2 (C-7a), 134.4 (C-7), 137.8 (CH2Ph), 138.2 (CH2Ph), 146.2 ppm (C-
6); IR (ATR): ν = 662, 696, 714, 729, 750, 756, 771, 789, 809, 834, 853,
913, 938, 1005, 1014, 1030, 1067, 1083, 1098, 1131, 1185, 1225, 1254,
1276, 1303, 1348, 1372, 1390, 1439, 1451, 1466, 1477, 1498, 1509,
1576, 1631, 2856, 2884, 2928, 2951, 3426 cm−1; MS (EI, 70 eV): m/z (%)
= 487 (33) [M]+∙, 396 (10) [M – Bn]+, 368 (21) [M –Bn – CO]+, 264 (18)
[M – Bn – OTBDMS]+, 236 (32) [M – Bn – CO – OTBDMS]+, 208 (14), 91
(100) [C7H7]+; HRMS (EI): calcd. for C30H37O3NSi [M]+∙: 487.25372,
found: 487.25392.
Methyl 6,7-dibenzyloxyindole-3-glyoxylate (18). To a solution of the
key precursor 17 (3.28 g, 10.0 mmol) in diethyl ether (50 mL) oxalyl
chloride (1.5 mL, 17.2 mmol) was added dropwise at 0 °C. The reaction
mixture was stirred at 0 °C for 30 min and then quenched with MeOH
(2 mL, 50mmol). The mixture was allowed to warm to ambient
temperature over 30 min. The resulting yellow precipitate was collected
by filtration, washed with cold diethyl ether and dried in vacuo to afford
18 (3.58 g, 8.6 mmol, 86 %) as a yellow solid. m.p. 146 °C; 1H NMR
(360 MHz, d6-DMSO, 293K): δ = 3.88 (s, 3H, CO2CH3), 5.18 (s, 2H,
CH2Ph), 5.20 (s, 2H, CH2Ph), 7.20 (d, 3J(H-5,H-4) = 8.7 Hz, 1H, H-5),
7.29 − 7.52 (m, 10H, CH2Ph), 7.80 (d, 3J(H-4,H-5) = 8.7 Hz, 1H, H-4),
8.28 (d, 3J(H-2,NH) = 3.3 Hz, 1H, H-2), 12.43 ppm (sbr, 1H, NH); 13C
NMR (90.5 MHz, d6-DMSO, 293 K): δ = 52.6 (CO2CH3), 71.3 (CH2Ph),
74.4 (CH2Ph), 112.5 (C-5), 112.8 (C-3), 116.3 (C-4), 121.7 (C-3a), 127.8
(CH2Ph), 127.9 (CH2Ph), 128.0 (CH2Ph), 128.1 (CH2Ph), 128.4 (CH2Ph),
128.5 (CH2Ph), 131.4 (C-7a), 133.7 (C-7), 137.2 (CH2Ph), 137.3 (C-2),
147.5 (C-6), 163.9 (C-2‘), 178.5 ppm (C-1‘); IR (ATR): ν = 682, 695, 717,
743, 773, 840, 879, 906, 959, 974, 1026, 1062, 1104, 1121, 1143, 1222,
1237, 1254, 1278, 1296, 1330, 1366, 1383, 1416, 1453, 1470, 1507,
1610, 1640, 1733, 2862, 2950, 3031, 3150, 3313 cm−1; MS (EI, 70 eV):
m/z (%) = 415 (17) [M]+∙, 356 (3) [M ‒ CO2CH3]+, 324 (15) [M ‒ Bn]+, 296
(6) [M ‒ Bn ‒ CO]+, 264 (4), 236 (4), 91 (100) [C7H7]+; HRMS (EI): calcd.
for C25H21NO5 [M]+∙: 415.14142; found: 415.14088.
6,7-Dibenzyloxy-1-(tert-butyloxycarbonyl)-3-(2-(tert-butyldimethyl-
silyloxy)ethyl)indole (21). To a solution of the protected alcohol 20
(1.13 g, 2.32 mmol) in THF (100mL) Boc2O (0.76 g, 3.48 mmol) and a
catalytic amount of DMAP were added. The reaction mixture was stirred
at ambient temperature for 12 h. The solvent was removed under
reduced pressure and the crude product was purified by flash column
chromatography (SiO2, cyclohexane/EtOAc 3:1) to afford 21 (1.26 g,
2.15 mmol, 92 %) as a colorless solid. Rf = 0.59 (cyclohexane/EtOAc
1
3:1); m.p. 106 °C; H NMR (360 MHz, CDCl3, 293K): δ = 0.12 (s, 6H,
3
SiCH3), 1.01 (s, 9H, SiC(CH3)3), 1.68 (s, 9H, Boc), 2.94 (t, J(H-1’,H-2’) =
3
6.7 Hz, 2H, H-1’), 3.97 (t, J(H-2’,H-1’) = 6.7 Hz, 2H, H-2’), 5.24 (s, 2H,
6,7-Dibenzyloxy-3-(2-hydroxyethyl)indole (19). A suspension of lithium
aluminum hydride (2.6 g, 68 mmol) in THF (100 mL) was heated to reflux.
To this suspension a solution of the ester 18 (2.0 g, 4.8 mmol) in THF
(50 mL) was added dropwise. The mixture was stirred for three hours
under reflux and afterwards allowed to cool to ambient temperature.
Excess LiAlH4 was quenched with water. The reaction mixture was
filtered and the residue was washed with hot THF. The filtrate was dried
over MgSO4 and evaporated to afford 19 (1.64 g, 4.4 mmol, 92 %) as
CH2Ph), 5.27 (s, 2H, CH2Ph), 7.10 (d, 3J(H-5,H-4) = 8.5 Hz, 1H, H-5),
7.25 (d, 3J(H-4,H-5) = 8.5 Hz, 1H, H-4), 7.36 – 7.58 ppm (m, 11H, CH2Ph,
H-2); 13C NMR (90.5 MHz, CDCl3, 293 K): δ = −5.3 (SiCH3), 18.4
(SiC(CH3)3), 26.0 (SiC(CH3)3), 28.1 (Boc), 28.5 (C-1’), 62.7 (C-2’), 73.1
(CH2Ph), 75.2 (CH2Ph), 82.7 (Boc), 113.3 (C-5), 114.0 (C-4), 117.2 (C-3),
125.5 (C-2), 127.7 (CH2Ph), 127.8 (CH2Ph), 128.1 (CH2Ph), 128.4
(CH2Ph), 128.8 (C-3a), 128.9 (CH2Ph), 129.6 (C-7a), 137.6 (C-7), 138.1
(CH2Ph), 148.7 (C-6), 150.3 ppm (Boc); IR (ATR): ν = 663, 695, 716, 730,
1
violet crystals. m.p. 86 °C; H NMR (360 MHz, CDCl3, 293 K): δ = 1.55
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201800417
European Journal of Organic Chemistry
FULL PAPER
739, 749, 757, 770, 781, 797, 811, 837, 912, 925, 935, 975, 1010, 1025,
1042, 1061, 1097, 1134, 1160, 1224, 1239, 1255, 1316, 1349, 1372,
1388, 1424, 1439, 1455, 1465, 1495, 1509, 1746, 2856, 2884, 2908,
2927, 2950, 3426 cm−1; MS (EI, 70 eV): m/z (%) = 587 (2) [M]+∙, 487 (3)
[M – Boc]+, 396 (13), 368 (6), 264 (10), 236 (15), 208 (7), 91 (100)
[C7H7]+, 73 (9) [Si(CH3)3]+, 57 (15) [C4H9]+; HRMS (EI): calcd. for [M]+
C35H45NO5Si: 587.30615, found: 587.30695.
temperature under air atmosphere. Subsequently, water (100 mL) was
added and the aqueous phase was extracted with chloroform (6×20 mL).
The combined organic phases were dried over MgSO4 and the solvent
was removed under reduced pressure. The crude product was purified by
flash column chromatography (SiO2, CHCl3/MeOH 9:1) to afford 24
1
(0.23 g, 0.44 mmol, 59 %) as a red oil. Rf = 0.37 (CHCl3/MeOH 9:1); H
NMR (360 MHz, CDCl3, 293 K): δ = −0.03 (s, 6H, SiCH3), 0.82 (s, 9H,
3
SiC(CH3)3), 1.85 – 1.88 (m, 2H, H-2’’), 2.81 (t, J(H-1’,H-2’) = 6.8 Hz, 2H,
H-1’), 3.38 (sbr, 2H, H-1’’), 3.72 – 3.87 (m, 10H, DMB, H-3’’, H-2’), 4.46 (s,
2H, DMB), 5.57 (s, 1H, H-5), 6.75 – 6.79 (m, 3H, DMB), 7.14 (s, 1H, H-2),
11.90 ppm (sbr, 1H, NH); 13C NMR (90.5 MHz, CDCl3, 293K): δ = −5.3
(SiCH3), 18.3 (SiC(CH3)3), 25.9 (SiC(CH3)3), 29.5 (C-2’’), 30.4 (C-1’), 47.5
(C-1’’), 55.9 (DMB), 59.2 (C-3’’), 63.3 (C-2’), 104.5 (C-5), 111.0 (DMB),
111.1 (DMB), 120.8 (C-3), 121.2 (DMB), 126.0 (C-3a), 128.1 (DMB),
128.9 (C-2), 130.0 (C-7a), 148.7 (DMB), 149.2 (DMB), 163.1 (C-4), 170.0
(C-7), 179.6 ppm (C-6); IR (ATR): ν = 660, 749, 774, 809, 831, 918, 1025,
1058, 1090, 1139, 1154, 1223, 1257, 1359, 1402, 1461, 1506, 1557,
1593, 1658, 2855, 2927, 3150 cm−1; UV/vis (MeOH): λmax (ε) = 541 (478),
380 (5274), 252 (5691), 231 (7622), 204 nm (17886 mol−1dm3cm−1); MS
(ESI+): m/z = 529 [M + H]+, 551 [M + Na]+; HRMS (ESI+): calcd. for
C28H41N2O6Si+ [M+ H]+ 529.2728; found: 529.2728.
1-(tert-Butyloxycarbonyl)-3-(2-(tert-butyldimethylsilyloxy)ethyl)-6,7-
dihydroxyindoline (22). An argon-purged suspension of compound 21
(1.02 g, 1.74 mmol) and 10% Pd/C (0.20 g) in EtOAc (200 mL) was
stirred under a hydrogen atmosphere (1 atm, balloon) at ambient
temperature. After four hours, the catalyst was filtered off and the solvent
was removed under reduced pressure. The crude product was purified by
flash column chromatography (SiO2, cyclohexane/EtOAc 9:1) to afford 22
(0.70 g, 1.7 mmol, 98 %) as
a
colorless solid. Rf
=
0.32
1
(cyclohexane/EtOAc 9:1); m.p. 65 °C; H NMR (360MHz, CDCl3, 293K):
δ = 0.07 (s, 3H, SiCH3), 0.08 (s, 3H, SiCH3), 0.92 (s, 9H, SiC(CH3)3),
1.55 (s, 9H, Boc), 1.55 – 1.63 (m, 1H, H-1’), 1.96 (dddd, 2J(H-1’,H-1’) =
3
14.3 Hz, J(H-1’,H-2’) = 5.1 Hz, 3J(H-1’,H-2’) = 5.1 Hz, 3J(H-1’,H-3) =
5.1 Hz, 1H, H-1’), 3.28 – 3.35 (m, 1H, H-3), 3.67 – 3.79 (m, 3H, H-2, H-
2
3
2’), 4.13 (dd, J(H-2,H-2) = 11.5 Hz, J(H-2,H-3) = 9.3 Hz, 1H, H-2) 5.80
3
3
(sbr, 1H, OH), 6.53 (d, J(H-5,H-4) = 8.0 Hz, 1H, H-5), 6.64 (d, J(H-4,H-
5) = 8.0 Hz, H-4), 11.46 ppm (s, 1H, OH); 13C NMR (91 MHz, CDCl3,
293 K): δ = −5.3 (SiCH3), 18.3 (SiC(CH3)3), 26.0 (SiC(CH3)3), 28.4 (Boc),
37.6 (C-3), 37.9 (C-1’), 56.1 (C-2), 61.6 (C-2’), 82.9 (Boc), 110.3 (C-5),
114.5 (C-4), 127.2 (C-3a), 128.2 (C-7a), 132.2 (C-7), 145.9 (C-6),
154.9 ppm (Boc); IR (ATR): ν = 660, 721, 776, 807, 825, 883, 907, 938,
965, 986, 1005, 1024, 1080, 1097, 1155, 1202, 1219, 1250, 1294, 1309,
1339, 1366, 1376, 1417, 1472, 1613, 1655, 2855, 2884, 2928, 2952,
3476 cm−1; MS (EI, 70 eV): m/z (%) = 409 (17) [M]+∙, 353 (28) [M –
C(CH3)3]+, 309 (100) [M – Boc]+, 296 (94), 252 (87) [M – Boc – C(CH3)3]+,
Sanguinolentaquinone (3). To a stirred solution of the compound 24
(50 mg, 94 μmol) in dichloromethane (3 mL) trifluoroacetic acid (300 μL)
was added. The reaction mixture was stirred for one hour at ambient
temperature. The solvent was removed under reduced pressure, the
residue was dissolved in toluene and the solution was concentrated in
vacuo. The residue was purified by flash column chromatography (SiO2,
CHCl3/MeOH 9:1 to MeOH) and size exclusion chromatography
(sephadex LH-20, MeOH) to afford 3 (18 mg, 68 μmol, 72 %) as a red
3
solid. 1H NMR (600 MHz, CD3OD, 300K): δ = 1.96 (tt, J(H-2’’,H-1’’) =
3
3
6.4 Hz, J(H-2’’,H-3’’) = 6.4 Hz, 2H, H-2’’), 2.88 (t, J(H-1’,H-2’) = 5.5 Hz,
∙
3
3
176 (73), 57 (43) [C(CH3)3]+; HRMS (EI): calcd. for C21H35NO5Si [M]+∙
2H, H-1’), 3.52 (t, J(H-1’’,H-2’’) = 6.4 Hz, 2H, H-1’’), 3.73 (t, J(H-3’’,H-
3
409.22845; found: 409.22984.
1’’) = 6.4 Hz, 2H, H-3’’), 3.83 (t, J(H-2’,H-1’) = 5.5 Hz, 2H, H-2’), 5.40 (s,
1H, H-5), 7.04 ppm (s, 1H, H-2); 13C NMR (151 MHz, CD3OD, 300 K): δ =
30.2 (C-1’), 31.9 (C-2’’), 42.5 (C-1’’), 60.7 (C-3’’), 64.4 (C-2’), 93.6 (C-5),
122.9 (C-3), 124.1 (C-3a), 129.1 (C-2), 130.4 (C-7a), 160.4 (C-4), 174.1
(C-7), 178.6 ppm (C-6); IR (ATR): ν = 668, 702, 798, 863, 1019, 1089,
1260, 1410, 1559, 1653, 2962 cm−1; UV/vis (MeOH): λmax (ε) = 522 (222),
333 (3220), 241 (5806), 203 nm (3982mol−1dm3cm−1); MS (ESI+): m/z =
265 [M + H]+, 287 [M + Na]+, 303 [M + K]+, 551 [2M + Na]+; HRMS (ESI+):
calcd. for C13H17N2O4+ [M + H]+ 265.11841; found: 265.11828.
1-(tert-Butoxycarbonyl)-3-(2-(tert-butyldimethylsilyloxy)ethyl)-1H-
indolo-6,7-quinone (23). To a solution of the indoline 22 (0.39 g,
0.95 mmol) in toluene (40 mL) DDQ (0.48 g, 2.14 mmol) was added. The
reaction mixture was stirred at ambient temperature for five minutes. The
suspension was filtered and the solvent was removed under reduced
pressure. The crude product was purified by flash column
chromatography (SiO2, cyclohexane/EtOAc 1:1) to afford 23 (0.35 g,
0.86 mmol, 91 %) as a red solid. Rf = 0.76 (cyclohexane/EtOAc 1:1); m.p.
110 °C; 1H NMR (360 MHz, CDCl3, 293 K): δ = −0.04 (s, 6H, SiCH3),
rac-6,7-Dibenzyloxy-N-(diphenylmethylene)tryptophan ethyl ester
(30). To a solution of the gramine 29[26] (0.20 g, 0.52 mmol) and
N-(diphenylmethylene)glycine ethyl ester (14)[21] (0.18 g, 0.64 mmol) in
acetonitrile (6 mL) tributylphosphine (0.64 mL, 0.26 mmol) was added.
The mixture was stirred at reflux for 12 h. Subsequently, the solvent was
removed under reduced pressure. The crude product was purified by
flash column chromatography (SiO2, cyclohexane/EtOAc 5:1) to afford 30
3
0.81 (s, 9H, SiC(CH3)3), 1.57 (s, 9H, Boc), 2.63 (t, J(H-1’,H-2’) = 6.1 Hz,
3
3
2H, H-1’), 3.70 (t, J(H-2’,H-1’) = 6.1 Hz, 2H, H-2’), 6.09 (d, J(H-5,H-4) =
3
9.9 Hz, 1H, H-5), 7.30 (d, J(H-4,H-5) = 9.9 Hz, 1H, H-4), 7.37 ppm (s,
1H, H-2); 13C NMR (91 MHz, CDCl3, 293 K): δ = −5.4 (SiCH3), 18.3
(SiC(CH3)3), 25.9 (SiC(CH3)3), 27.4 (Boc), 27.6 (C-1’), 63.1 (C-2’), 86.4
(Boc), 122.7 (C-3), 125.7 (C-5), 127.1 (C-7a), 130.8 (C-2), 134.8 (C-3a),
137.0 (C-4), 147.6 (Boc), 167.0 (C-7), 183.0 ppm (C-6); IR (ATR): ν =
660, 679, 702, 765, 774, 791, 836, 860, 920, 941, 1005, 1038, 1079,
1101, 1127, 1153, 1256, 1273, 1291, 1325, 1370, 1391, 1418, 1445,
1460, 1473, 1490, 1612, 1664, 1738, 2854, 2883, 2927, 2957,
3136 cm−1; UV/vis (MeOH): λmax (ε) = 380 (5538), 218 (21420), 204 nm
(18803 mol−1dm3cm−1); MS (ESI+): m/z = 428 [M + Na]+, 444 [M + K]+,
833 [2M + Na]+; HRMS (ESI+): calcd. for C21H31NO5SiNa+ [M+ Na]+
428.1864; found: 428.1865.
1
(0.30 g, 0.48 mmol, 92 %) as a yellow viscous oil. Rf = 0.17; H NMR
(360 MHz, CDCl3, 293K): δ = 1.22 (t, 3J(CH3,CH2) = 7.1 Hz, 3H,
CO2CH2CH3), 3.29 (dd, 2J(H-1’,H-1’) = 14.2 Hz, 3J(H-1’,H-2’) = 8.9 Hz,
3
1H, H-1’), 3.47 (dd, 2J(H-1’,H-1’) = 14.2 Hz, J(H-1’,H-2’) = 4.6 Hz, 1H, H-
3
1’), 4.11 – 4.25 (m, 2H, CO2CH2CH3), 4.43 (dd, J(H-2’,H-1’) = 4.6 Hz,
3J(H-2’,H-1’) = 8.9 Hz, 1H, H-2’), 5.17 – 5.26 (m, 4H, CH2Ph), 6.57 – 6.58
3
(m, 2H, Ph), 6.76 – 6.79 (m, 2H, H-2, H-5), 6.91 (d, J(H-4,H-5) = 8.5 Hz,
3
1H, H-4), 7.11 (t, J(H,H) = 7.6 Hz, 2H, Ph), 7.21 – 7.66 (m, 16H, Ph),
8.06 ppm (sbr, 1H, NH); 13C NMR (91 MHz, CDCl3, 293 K): δ = 14.0
(CO2CH2CH3), 29.2 (C-1’), 60.7 (CO2CH2CH3), 65.9 (C-2’), 72.6 (CH2Ph),
75.0 (CH2Ph), 109.9 (C-5), 111.7 (C-3), 113.8 (C-4), 122.9 (C-2), 124.7
(C-3a), 127.4 (CH2Ph), 127.5 CH2Ph), 127.8 (CH2Ph), 127.9 (CH2Ph),
128.1 (CH2Ph), 128.2 (CH2Ph), 128.3 (CH2Ph), 128.6 (CH2Ph), 130.0
(CH2Ph), 130.8 (C-7a), 133.8 (C-7), 135.7 (CH2Ph), 137.6 (CH2Ph),
4-(N-(3-Hydroxypropyl)-N-(3,4-dimethoxybenzyl)amino)-3-(2-(tert-
butyldimethylsilyloxy)ethyl)indolo-6,7-quinone (24). To a solution of
the quinone 23 (0.30 g, 0.74 mmol) in acetonitrile (30 mL) 3-[N-(3,4-
dimethoxybenzyl)amino]propan-1-ol[26] (25) (0.33 g, 1.48 mmol) was
added. The reaction mixture was stirred three days at ambient
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10.1002/ejoc.201800417
European Journal of Organic Chemistry
FULL PAPER
137.8 (CH2Ph), 139.3 (CH2Ph), 145.8 (C-6), 170.3 (C-3’), 172.0 ppm
(CO2Et); IR (ATR): ν = 693, 735, 782, 846, 908, 936, 1027, 1067, 1137,
1177, 1212, 1257, 1298, 1314, 1340, 1372, 1442, 1508, 1575, 1596,
1623, 1728, 2924, 2978, 3029, 3060, 3433 cm−1; MS (EI, 70 eV): m/z (%)
= 608 (21) [M]+∙, 517 (38) [M ‒ Bn]+, 489 (22) [M – Bn − CO]+, 342 (37),
307 (31), 91 (100) [C7H7]+; HRMS (EI): calcd. for C40H36O4N2 [M]+∙
608.26696; found: 608.26750.
70 eV): m/z
(%)
=
586 (10) [M]+∙, 342 (100) [M –
CH(CO2Et)NHCH2CH2CH2OTHP]+, 160 (58) [THPOCH2CH2CH2NH2]+,
91 (100) [C7H7]+; HRMS (EI): calcd. for C35H42O6N2 [M]+∙ 586.30429;
found: 586.30318.
rac-O-(Tetrahydro-2H-pyran-2-yl)mycenarubin E ethyl ester (33). An
argon-purged suspension of the tryptophan derivative 32 (0.19 g,
0.72 mmol) and 10 % Pd/C (65 mg) in MeOH (90 mL) was stirred under a
hydrogen atmosphere (1 atm, balloon) at ambient temperature. After one
hour, the catalyst was filtered off. Subsequently, triethylamine (100 μL)
was added to the filtrate which was stirred for an additional hour under air
atmosphere. The solvent was removed under reduced pressure and the
crude product was purified by flash column chromatography (SiO2,
CHCl3/MeOH 49:1) to afford 33 (0.11 g, 0.27 mmol, 39 %) as a red solid.
Rf = 0.02 (CHCl3/MeOH 49:1); 1H NMR (360 MHz, CDCl3, 293 K): δ =
rac-6,7-Dibenzyloxytryptophan ethyl ester (31). To a solution of the
protected tryptophan 30 (0.76 g, 1.25 mmol) in THF (5 mL) 1 M HCl
(20 mL) was added dropwise at 0 °C. The reaction mixture was stirred for
four hours at ambient temperature. Subsequently, THF was removed
under reduced pressure and the aqueous residue was washed with
diethyl ether (2×20 mL). The pH was adjusted to 9 with saturated
NaHCO3 solution and the aqueous phase was extracted with
dichloromethane (3×20mL). The combined dichloromethane phases
were dried over MgSO4 and the solvent was removed under reduced
pressure to afford 31 (0.55 g, 1.23 mmol, 99%) as a brownish viscous oil.
3
1.24 (t, J(CH3CH2)= 7.1 Hz, 3H, CO2CH2CH3), 1.57 – 2.08 (m, 8H, H-2’,
THP), 3.18 – 3.36 (m, 3H, H-3, H-1’), 3.47 – 3.56 (m, 2H, H-3’, THP),
3.77 – 3.95 (m, 3H, H-1’, H-3’, THP), 4.16 (q, J(CH2CH3) = 7.1 Hz, 2H,
CO2CH2CH3), 4.35* (dd, J(H-4,H-3) = 6.7 Hz, J(H-4,H-3) = 1.8 Hz, 0.5H,
3
3
3
3
1H NMR (360 MHz, CDCl3, 293 K): δ = 1.22 (t, J(CH3,CH2) = 7.1 Hz, 3H,
2
3
3
CO2CH2CH3), 2.99 (dd, J(H-1’,H-1’) = 14.5 Hz, 3J(H-1’,H-2’) = 7.5 Hz,
H-4), 4.46* (dd, J(H-4,H-3) = 6.5 Hz, J(H-4,H-3) = 1.5 Hz, 0.5H, H-4),
4.53 – 4.55* (m, 0.5H, THP), 4.58 – 4.60* (m, 0.5H, THP), 5.48 (s, 0.5H,
H-6), 5.49 (s, 0.5H, H-6), 7.08 (m, 1H, H-2), 11.45 ppm (sbr, 1H, NH); 13C
NMR (91 MHz, CDCl3, 293K): δ = 14.3 (CO2CH2CH3), 20.0+20.5* (THP),
24.4+24.6* (C-3), 25.5 (THP), 27.8+27.9* (C-2’), 30.9+31.0* (THP),
48.7+49.1* (C-1’), 62.4+62.4* (CO2CH2CH3), 62.8+63.0* (C-4),
63.0+63.7* (THP), 64.2+64.6* (C-3’), 94.6+94.6* (C-6), 99.6+99.9* (THP),
114.2+114.4* (C-2a), 125.2 (C-2), 125.3 (C-8a), 126.1 (C-8b), 153.2 (C-
5a), 170.7+170.9 (C-8), 170.9+171.0 (CO2Et), 180.5 ppm (C-7) (*two
diastereomeric pairs of enantiomers); IR (ATR): ν = 668, 693, 716, 734,
765, 866, 880, 901, 924, 940, 1027, 1058, 1066, 1184, 1241, 1250, 1320,
1378, 1394, 1410, 1442, 1522, 1559, 1593, 1617, 1670, 1734, 2901,
2972, 2988, 3675 cm−1; UV/vis (MeOH): λmax (ε) = 520 (323), 350 (3909),
242 (5828), 200 nm (4323 mol−1dm3cm−1); MS (ESI+): m/z = 403 [M + H]+,
425 [M + Na]+, 441 [M + K]+, 827 [2M + Na]+; MS (ESI−): m/z = 401 [M –
H]−; HRMS (ESI+): calcd. for C21H27N2O6+ [M + H]+ 403.18636; found:
403.18628.
3
1H, H-1’), 3.21 (dd, 2J(H-1’,H-1’) = 14.5 Hz, J(H-1’,H-2’) = 4.7 Hz, 1H, H-
3
1’), 3.79 (m, 1H, H-2’), 4.13 (q, J(CH2,CH3) = 7.1 Hz, 2H, CO2CH2CH3),
3
5.18 (s, 2H, CH2Ph), 5.20 (s, 2H, CH2Ph), 6.91 (d, J(H-5,H-4) = 8.5 Hz,
3
1H, H-5), 6.92 (s, 1H, H-2), 7.23 (d, J(H-4,H-5) = 8.5 Hz, 1H, H-4), 7.32
– 7.60 (m, 10H, CH2Ph), 7.97 (sbr, 1H, NH); 13C NMR (91 MHz, CDCl3,
293 K):
δ = 14.3 (CO2CH2CH3), 30.8 (C-1’), 55.1 (C-2’), 61.1
(CO2CH2CH3), 73.1 (CH2Ph), 75.5 (CH2Ph), 110.6 (C-5), 111.5 (C-3),
114.0 (C-4), 122.9 (C-2), 124.7 (C-3a), 127.8 (CH2Ph), 128.0 (CH2Ph),
128.3 (CH2Ph), 128.5 (CH2Ph), 128.6 (CH2Ph), 131.5 (C-7a), 134.5 (C-7),
137.8 (CH2Ph), 138.1 (CH2Ph), 146.5 (C-6), 175.1 ppm (CO2Et); IR
(ATR): ν = 696, 736, 791, 844, 967, 903, 1021, 1067, 1119, 1136, 1181,
1200, 1217, 1259, 1301, 1351, 1441, 1453, 1509, 1576, 1630, 1728,
2868, 2939, 3309 cm−1; MS (ESI+): m/z = 445 [M + H]+, 467 [M + Na]+,
483 [M + K]+; HRMS (ESI+): calcd. for C27H29N2O4 [M + H]+ 445.21218;
found: 445.21221.
rac-6,7-Dibenzyloxy-N-(3-(tetrahydro-2H-pyran-2-yloxy)propyl)-
tryptophan ethyl ester (32). To a solution of the tryptophan 31 (0.23 g,
0.52 mmol) in MeOH (5 mL) 3-(tetrahydro-2H-pyran-2-yloxy)propan-1-al
(34)[27] (0.13 g, 0.79mmol) and sodium cyanoborohydride (0.10 g,
1.56 mmol) were added. The reaction mixture was stirred at ambient
temperature for 12 h. The solvent was removed under reduced pressure
and the crude product was purified by flash column chromatography
(SiO2, CHCl3/MeOH 49:1) to afford 32 (0.27 g, 0.46 mmol, 88%) as a
brownish oil. Rf = 0.2 (CHCl3/MeOH 49:1); 1H NMR (360 MHz, CDCl3,
Mycenaflavin B (4b). The pyrroloquinoline 33 (31 mg, 77 μmol) was
dissolved in THF (4 mL) and 0.1 M aqueous LiOH solution (4 mL) was
added. The solution was stirred for 48 hours at 55 °C (ester hydrolysis).
Subsequently, the solution was neutralised with AcOH and the solvents
were removed under reduced pressure. The residue was dissolved in a
mixture of MeOH (2 mL), H2O (1 mL) and AcOH (5 mL). The solution was
stirred for two hours at 40 °C (decarboxylation step). After cooling to
ambient temperature the reaction mixture was stirred 72 h at ambient
temperature (THP deprotection). The solvents were removed under
reduced pressure and the crude product was purified by HPLC to afford
4b (9 mg, 37 μmol, 48 % over three steps) as a brown solid. HPLCprep: Rt
3
293 K): δ = 1.11 (t, J(CH3,CH2) = 7.1 Hz, 3H, CO2CH2CH3), 1.49 – 1.80
3
(m, 8H, THP), 2.58 – 2.78 (m, 2H, H-3’), 3.08 (d, J(H-1’,H-2’) = 6.7 Hz,
2H, H-1’), 3.37 – 3.44 (m, 1H, H-5’), 3.46 – 3.50 (m, 1H, THP), 3.57 –
3
3
3.62 (m, 1H, H-2’), 3.74 – 3.86 (m, 2H, H-5’, THP), 4.07 (q, J(CH2,CH3)
= 30.2 min; 1H NMR (600 MHz, d6-DMSO, 300 K): δ = 1.88 (tt, J(H-2’,H-
3
3
= 7.1 Hz, 2H, CO2CH2CH3), 4.52* (t, J(H-1’,H-2’) = 3.5 Hz, 0.5H, H-1’),
3’) = 5.8 Hz, J(H-2’,H-1’) = 6.2 Hz, 2H, H-2’), 3.47 (t, 3J(H-3’,H-2’) =
3
3
4.55* (t, J(H-1’,H-2’) = 3.5 Hz, 0.5H, H-1’), 5.19 (s, 2H, CH2Ph), 5.21 (s,
5.8 Hz, 2H, H-3’), 4.09 (t, J(H-1’,H-2’) = 6.2 Hz, 2H, H-1’), 4.72 (s, 1H,
3
3
3
2H, CH2Ph), 6.89 (d, J(H-2,NH) = 2.1 Hz, 1H, H-2), 6.92 (d, J(H-5,H-4)
NH); 5.58 (s, 1H, H-6), 6.81 (d, J(H-3,H-4) = 6.8 Hz, 1H, H-3), 7.28 (d,
3
= 8.6 Hz, 1H, H-5), 7.25 (d, J(H-4,H-5) = 8.6 Hz, 1H, H-4), 7.31 – 7.51
3J(H-4,H-3) = 6.8 Hz, 1H, H-4), 7.71 ppm (s, 1H, H-2); 13C NMR
(151 MHz, d6-DMSO, 300 K): δ = 31.5 (C-2’), 49.1 (C-1’), 57.5 (C-3’),
91.1 (C-6), 104.5 (C-3), 119.8 (C-8b), 120.4 (C-2a), 125.4 (C-8a), 126.5
(C-2), 131.5 (C-4), 145.8 (C-5a), 170.6 (C-8), 179.0 ppm (C-7); IR (ATR):
ν = 668, 757, 811, 911, 984, 1051, 1148, 1196, 1264, 1310, 1374, 1394,
1456, 1559, 1577, 1653, 2901, 2988cm−1; UV/vis (MeOH): λmax (ε) = 522
(3896), 435 (5202), 418 (4883). 241 (11457), 225 (14395), 201 nm
(10085 mol−1dm3cm−1); MS (ESI+): m/z = 245 [M + H]+, 267 [M + Na]+;
MS (ESI−): m/z = 243 [M – H]−; HRMS (ESI+): calcd. for C13H13N2O3+
[M + H]+ 245.09207; found: 245.09190.
(m, 10H, CH2Ph), 8.16 ppm (sbr, 1H, NH); 13C NMR (91 MHz, CDCl3,
293 K): δ = 14.1 (CO2CH2CH3), 19.5+19.5* (THP), 25.4 (THP), 29.4 (C-
1’), 30.1+30.1* (C-4’), 30.6 (THP), 45.5+45.6* (C-3’), 60.5 (CO2CH2CH3),
62.1+62.2* (THP), 62.3 (C-2’), 65.7+65.8* (C-5’); 72.9 (CH2Ph), 75.3
(CH2Ph), 98.7+98.8* (THP), 110.3 (C-5), 111.5 (C-3), 113.8 (C-4), 122.6
(C-2), 124.7 (C-3a), 127.6 (CH2Ph), 127.8 (CH2Ph), 128.0 (CH2Ph),
128.3 (CH2Ph), 128.4 (CH2Ph), 131.2 (C-7a), 134.3 (C-7), 137.6 (CH2Ph),
138.0 (CH2Ph), 146.2 (C-6), 174.9 ppm (CO2Et) (*two diastereomeric
pairs of enantiomers); IR (ATR): ν = 695, 735, 790, 845, 906, 941, 1026,
1067, 1084, 1183, 1216, 1260, 1301, 1350, 1377, 1443, 1497, 1509,
1575, 1629, 1657, 1728, 2869, 2930, 3030, 3062, 3362 cm−1; MS (EI,
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natural products • total synthesis
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FULL PAPER
Text for Table of Contents
Total Synthesis*
J. Backenköhler, Dr. B. Reck, Dr. M.
Plaumann, Prof. Dr. P. Spiteller*
Page No. – Page No.
Total Synthesis of Mycenarubin A,
Sanguinolentaquinone and
Mycenaflavin B and their Cytotoxic
Activities
The first total synthesis of the fungal pyrroloquinoline alkaloids mycenarubin A, sanguinolentaquinone and mycenaflavin B are
described. Key steps for the synthesis of mycenarubin A are an enantioselective alkylation and a biomimetic Michael addition
leading to the pyrrolo[4,3,2-de]quinoline core. Mycenaflavin B has been obtained via a biomimetic ring closure and an acidic
decarboxylation reaction. Moreover, the cytotoxic activities of mycenarubin A and mycenaflavin B have been evaluated
against mouse fibroblasts (L929) and human malignant melanoma cells (RPMI-7951).
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