Synthesis and initial biological evaluation of myxocoumarin B

Article abstract of DOI:10.1039/c8ob02273a

The myxocoumarins A and B from Stigmatella aurantiaca MYX-030 are natural products featuring unusual nitro- and long-chain alkyl substitution. While myxocoumarin A was shown to exhibit strong antifungal properties, the antifungal potential of myxocoumarin

Full text of DOI:10.1039/c8ob02273a

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Synthesis and initial biological evaluation of
myxocoumarin B†
Cite this: DOI: 10.1039/c8ob02273a
a
a
a
b
Jonas I. Müller,‡ Kalina Kusserow,‡ Gesa Hertrampf, Aleksandar Pavic,
b
a,c
Jasmina Nikodinovic-Runic and Tobias A. M. Gulder
*
The myxocoumarins A and B from Stigmatella aurantiaca MYX-030 are natural products featuring unusual
nitro- and long-chain alkyl substitution. While myxocoumarin A was shown to exhibit strong antifungal
properties, the antifungal potential of myxocoumarin B was not yet assessed due to low production titers
during initial isolation. We therefore developed a total synthesis of myxocoumarin B that involves a late-
stage Pd-catalyzed nitration of the coumarin core. The availability of synthetic material facilitated the
initial evaluation of the bioactivity of myxocoumarin B, which revealed a lack of activity against medically
relevant Candida sp. and low cytotoxicity in vitro against human fibroblasts (MRC-5) and in vivo
(zebrafish).
Received 13th September 2018,
Accepted 18th October 2018
DOI: 10.1039/c8ob02273a
rsc.li/obc
exhibit promising inhibitory activities against a wide range of
fungal pathogens, including Botrytis cinerea, Magnaporthe
Introduction
Natural products have a huge impact as leads and drugs in grisea, Phaeospaeria nodorum, Blumeria graminis, and Fusarium
medicine and agrochemical applications. Owing to the contin- culmorum. Due to the initially low production titers of myxo-
ued emergence of new and untreatable maladies and the evol- coumarin B (7), combined with the unfortunate inability to
ution of pathogens exhibiting resistance against existing treat- regrow the producing strain from all existing stock cultures,
ment options, the continued discovery of novel scaffolds with the antifungal potential of 7 could not be evaluated during
potent biological activities is of utmost importance. A rich this study. Given the significant activity of 6, we thus decided
source of such natural products with truly novel molecular to develop a short synthetic access towards 7 to facilitate its
architectures and activity profiles are myxobacteria. These biological evaluation.
mostly soil-dwelling δ-proteobacteria are particularly talented
producers of unusual ribosomal and non-ribosomal peptides
and polyketides with often unprecedented modes of
Results and discussion
1
–5
actions.
It is thus not surprising that many myxobacterial
natural products have intensely been studied in recent years. A broad range of synthetic approaches for the construction of
Intriguing examples include the antineoplastic pretubulusin coumarin core structures can be found in the literature,
6
7,8
14
15
16
17
(
1), the antifilarial corallopyronin A (2),
the antimalarial including Perkin, Reformatsky, Wittig and Knoevenagel
9
,10
chlorotonil A (3),
the cystobactamids, e.g. 4, as potent anti- reactions. The most widely used method is the Pechmann con-
1
1
18
biotics against Gram-negative bacteria, and the antifungal densation by which phenolic substrates are fused to β-keto
soraphen A1α (5) (Fig. 1). In 2013, we reported the discovery esters catalysed by a (Lewis) acid. Disconnecting the respective
of the myxocoumarins A (6) and B (7) from liquid cultures of building blocks accordingly in myxocoumarin B (7) leads to
Stigmatella aurantiaca MYX-030. Compound 6 was shown to resorcinol 8 with substitution at C-5 and β-keto ester 9, which
in turn can readily be prepared from 10 by regio-selective alkyl-
1
2
1
3
ation with iodo-octane (Scheme 1A). Owing to the activation of
the ortho/para aromatic positions by the phenolic functions in
a
Biosystems Chemistry, Department of Chemistry and Center for Integrated Protein
Science Munich (CIPSM), Technical University of Munich, Germany.
8
, the introduction of suitable substituents at C-5 within this
E-mail: tobias.gulder@ch.tum.de
b
Institute of Molecular Genetics and Genetic Engineering, University of Belgrade,
substrate is not directly possible. An elegant access to the
corresponding nitro-substituted resorcinol 13 is provided by
Pd-catalyzed nitration of aryl chlorides as developed by the
Vojvode Stepe 444a, Belgrade, Serbia
c
Technische Universität Dresden, Chair of Technical Biochemistry, Bergstraße 66,
01062 Dresden, Germany
1
9
Buchwald laboratory. Following this approach, the commer-
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/
cially available substrate 11 was smoothly converted to 12 in
c8ob02273a
These authors contributed equally to this work.
‡
75% yield (Scheme 1B). BCl -promoted O-demethylation gave
3
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Fig. 1 Structures of myxobacterial compounds 1–7.
1
3 in 71% yield. Despite literature precedence for the utiliz-
ation of nitro-substituted phenols in Pechmann reactions to
2
0
yield the corresponding nitro coumarins, our attempts to
convert 13 to 7 under various condensation conditions failed
to provide the desired product (data not shown). As this likely
Scheme 1 A. Retro-synthetic analysis (A), unsuccessful synthetic routes
(B, C) and first generation synthesis (D) of myxocoumarin B (7). Reagents
can be rationalized by the deactivation of resorcinol 13 due to and conditions: (a) 1. nBuLi (2.5 eq.), DIPA (2.5 eq.), THF, −78 °C,
1
Pd
h. 2. 10 (1.0 eq.), 0 °C, 1 h. 3. R–I (1.2 eq.), −78 °C to rt, 16 h. (b)
the nitro substitution, we decided to use an alternative strategy
that involves a later-stage nitration at the fully elaborated cou-
marin core (Scheme 1C). Following a protocol by Thorn et al.
one of the hydroxy functions in phloroglucinol (14) was
2
(dba)
3
(0.5 mol%), tBuBrettPhos (1.2 mol%), TDA (5.0 mol%), NaNO
(2.0 eq.), toluene, 130 °C, 40 h. (d)
SO (3.6 eq.), NaNO (2.8 eq.), H O,
2
(2 eq.), tBuOH, 130 °C, 24 h. (c) BCl
3
1. NH
4
OH, RT, 18 h. 2. HCl (e) 1. H
2
4
2
2
0
2
°C, 15 min. 2. KI (3.6 eq.), H O, rt, 5.5 h (49% over two steps d, e). (f) 9
replaced by an amino group by stirring of this substrate in (1.0 eq.), SSA (9.6 eq.), CHCl , 65 °C, 20 h. (g) CuI (0.2 eq.), KNO2
3
2
1
(
1.1 eq.), 18-crown-6 (1.0 eq.), N,N’-diethylethylenediamine (0.4 eq.),
DMF, 100 °C, 26 h. (h) 9 (1.0 eq.), TFA (6.2 eq.), MW, 100 °C, 30 min. (i)
BCl (2.1 eq.), toluene, 110 °C, 18 h. ( j) Tf O (1.3 eq.), pyridine (2.0 eq.),
DCM, 0 °C to rt, 30 min. (k) Pd (dba) (0.5 mol%), tBuBrettPhos
1.2 mol%), TDA (5.0 mol%), NaNO (2 eq.), tBuOH, 130 °C, 24 h. (l) LiCl
ammonium hydroxide. The resulting crude hydrochloride 15
was directly used in a Sandmeyer reaction to generate 5-iodore-
sorcinol (16) in 49% yield over two steps. The latter was uti-
lized in a Pechmann condensation reaction with 9 to deliver
3
2
2
3
(
2
iodo coumarin 17, although only in unsatisfactory 5% yield. 17 (6.0 eq.), DMF, 155 °C, 18 h. All yields are un-optimized.
was subjected to an Ullmann-type copper-catalyzed nitration
2
2
utilizing a protocol by Saito et al. However, only traces of
myxocoumarin B (7) were detectable in the reaction mixture by
MS analysis.
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Given the power of the Pd-catalyzed nitration utilized in Table 1 MIC values, IC50 and LC50 for myxocoumarin B (7)
route B for the synthesis of 12, we decided to evaluate this
C. albicans
C. krusei
MRC-5
Zebrafish
reaction for the introduction of the desired nitro functionality
later in the synthetic route. Coumarin 19 was constructed by a
Pechmann condensation of 3,5-dimethoxyphenol (18) with 9
in 26% yield. Likely owing to a shielding effect of the long-
alkyl chain R located at the coumarin core, a regioselective
O-demethylation of the desired methoxy functionality using
a
a
b
c
MIC
MIC
IC50
LC50
[µg mL−1]
[µg mL−1]
[µg mL−1]
[µg mL−1]
Test
Compound 7
µM
>500
>500
35
100
120
344
a
b
Minimum inhibitory concentration. Calculated IC50 value corres-
ponds to the concentration required to inhibit 50% of cell growth.
BCl
3
was possible in 47% yield. The resulting phenol 20 was
c
Calculated LC50 value corresponds to the concentration required to
converted into 21 by triflation in 98% yield. Triflate 21 served kill 50% of the embryos.
as the substrate in a Pd-catalyzed nitration again following
1
9
the Buchwald protocol. This indeed delivered the fully func-
tionalized coumarin core 22 in 48% yield. The latter was
O-deprotected in 48% yield to conclude the first total synthesis
of 7. Taken together, this sequence gave access to myxocou-
marin B (7) in five steps from 18, with an overall yield of
approx. 3%.
To improve the unsatisfyingly low yield of the overall
sequence, we set out to further streamline the synthetic route.
As the yields of Pechmann condensation generally improve
with the electron-richness of the employed phenols, we
decided to first construct the corresponding dihydroxy deriva-
tive of 19, coumarin 24. This compound is available by con-
densation of phloroglucinol 23 with 9 in 42% yield. Given the
apparent protective effect of the long-chain n-octyl group as
observed in the selective O-demethylation of 19 to 20 shown
above, we hypothesized that the desired activation by regio-
selective mono O-triflation of 24 should be possible, as well as
the following Pd-catalyzed nitration reaction in the presence
of the free phenol. Indeed, selective triflation proceeded
smoothly to deliver 25 in 51% yield, along with >5% of the bis-
triflated analog and 18% of re-isolated starting material 24,
thus leading to 63% of 25 brsm (Scheme 2). Compound 25 was
subjected to Pd-catalyzed nitration with slightly modified
conditions (5-fold increase of Pd-catalyst and ligand), furnish-
ing 39% of the natural product 7. Overall, this route thus
reduced the number of required steps from five (Scheme 1D)
to only three, accompanied by a more than 3-fold increase in
overall yield.
Fig. 2 Toxicity assessment of myxocoumarin B (7) in zebrafish embryos
exposed to different concentrations and representative images of
zebrafish embryos at 114 hpf upon treatment with 250 µM of myxocou-
marin B (7) compared to a DMSO control.
Having sufficient amounts of synthetic myxocoumarin B (7)
in hands we next aimed at the evaluation of its antifungal
effects against fungal pathogens, i.e., Candida albicans and
C. krusei (see ESI† for details). Unfortunately, no effect on the
planktonic growth of these strains was observed up to concen-
trations of 500 µg mL− (Table 1).
1
Cytotoxicity of 7 in vitro was assessed against healthy
human lung fibroblasts cell line MRC-5 and was found to be
moderate (IC50 at 100 µM). Toxicity of 7 in vivo was determined
using zebrafish embryos and found to be at least 3-fold lower
(
IC50 at 344 µM). Importantly, myxocoumarin B (7) elicited no
toxic response (lethal nor teratogenic) in zebrafish embryos
upon treatment with doses up to 250 µM, with all embryos
dying upon exposure to 500 µM (Fig. 2).
Conclusions
In conclusion we developed a short total synthetic access to 7
with a late-stage Pd-catalyzed nitration reaction as the key step.
Our work nicely showcases that the Pd-catalyzed nitration reac-
1
9
tion developed by Buchwald et al. can readily be applied to
elaborate substrates such as the employed triflated coumarin
derivatives 21 and 25 and therefore constitutes a valuable
transformation for late-stage natural product functionali-
zation. The obtained synthetic material facilitated initial
assessment of the biological properties of 7, which, in contrast
to myxocoumarin A (6), despite only small structural differ-
ences, revealed a complete lack of activity against fungal patho-
gens and low toxicity both in vitro and in vivo. Access to 7 now
permits investigations into the likely ecological functions of
Scheme 2 Alternative synthetic route to myxocoumarin B (7) via triflate
2
7
(
5. Reagents and conditions: (a) 9 (1.0 eq.), TFA (6.2 eq.), MW, 110 °C,
5 min. (b) Tf O (1.2 eq.), pyridine (3.0 eq.), DCM, 0 °C, 4 h. (c) Pd (dba)
2.5 mol%), tBuBrettPhos (6.0 mol%), TDA (5.0 mol%), NaNO (2 eq.),
2
2
3
2
tBuOH, 130 °C, 24 h.
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the myxocoumarin class of natural products in their natural
producer. This work is currently ongoing in our laboratory.
6 A. Ullrich, Y. Chai, D. Pistorius, Y. A. Elnakady,
J. E. Herrmann, K. J. Weissmann, U. Kazmaier and
R. Müller, Angew. Chem., Int. Ed., 2009, 48, 4422.
7
H. Irschik, R. Jansen, G. Höfle, K. Gerth and
H. Reichenbach, J. Antibiot., 1985, 38, 145.
Conflicts of interest
8
T. F. Schäberle, A. Schiefer, A. Schmitz, G. M. König,
A. Hoerauf and K. Pfarr, Int. J. Med. Microbiol., 2014, 304,
There are no conflicts to declare.
72.
9
K. Gerth, H. Steinmetz, G. Höfle and R. Jansen, Angew.
Chem., Int. Ed., 2007, 47, 600.
Acknowledgements
1
0 J. Held, T. Gebru, M. Kalesse, R. Jansen, K. Gerth,
R. Müller and B. Mordmüller, Antimicrob. Agents
Chemother., 2014, 58, 6378.
Funding of this work by the Ministry of Education, Science
and Technological Development of the Republic of Serbia,
Project No. 173048 (to AP and JNR), the DAAD (Deutscher 11 S. Baumann, J. Herrmann, R. Raju, H. Steinmetz,
Akademischer Austauschdienst, Bilateral Project of Germany
K. I. Mohr, S. Hüttel, K. Harmrolfs, M. Stadler and
with the Republic of Serbia to JNR and TAMG – 2016/2017)
R. Müller, Angew. Chem., Int. Ed., 2014, 53, 14605.
and by the DFG (to TAMG, Emmy-Noether program GU 1233/1- 12 K. Gerth, N. Bedorf, H. Irschik, G. Höfle and
1
and Center for Integrated Protein Science Munich CIPSM) is
H. Reichenbach, J. Antibiot., 1994, 47, 23.
gratefully acknowledged.
All experiments involving zebrafish embryos were performed
13 T. A. M. Gulder, S. Neff, T. Schüz, T. Winkler, R. Gees and
B. Böhlendorf, Beilstein J. Org. Chem., 2013, 9, 2579.
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that ethical approval is not needed for zebrafish embryos pro- 15 R. L. Shriner, Org. React., 1942, 1, 1.
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Lett., 1998, 39, 2391.
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