Total synthesis and antibacterial activity of dysidavarone A

Article abstract of DOI:10.1021/ol400156u

A concise total synthesis of dysidavarone A possessing the new "dysidavarane" carbon skeleton has been accomplished by a convergent strategy, involving a stereoselective reductive alkylation of a Wieland-Miescher type ketone under Birch conditions and an

Full text of DOI:10.1021/ol400156u

ORGANIC
LETTERS
2013
Vol. 15, No. 4
964–967
Total Synthesis and Antibacterial Activity
of Dysidavarone A
†
Bjorn Schmalzbauer, Jennifer Herrmann, Rolf Muller,* and Dirk Menche*
‡
,‡
,†
€
€
ꢀ
€
Kekule-Institut fur Organische Chemie und Biochemie, University of Bonn,
Gerhard-Domagk-Str. 1, 53121 Bonn, Germany, and Helmholtz-Institut fu€r
Pharmazeutische Forschung Saarland (HIPS) and Institut fu€r Pharmazeutische
€
Biotechnologie, Universitat des Saarlandes, Gebaude C 2.3, 66123 Saarbrucken,
€
€
Germany
rom@mx.uni-saarland.de; dirk.menche@uni-bonn.de
Received January 18, 2013
ABSTRACT
A concise total synthesis of dysidavarone A possessing the new “dysidavarane” carbon skeleton has been accomplished by a convergent
strategy, involving a stereoselective reductive alkylation of a Wieland-Miescher type ketone under Birch conditions and an advantageous
intramolecular palladium-catalyzed R-arylation of a sterically hindered ketone. Dysidavarone A showed potent antimicrobial and antiproliferative
activities based on characteristic morphological changes of treated cells.
The dysidavarones AꢀD present structurally unique
marine natural products that were recently isolated from
the sponge Dysidea avara.1 As shown in Scheme 1 for
dysidavarone A 1, the 3D architectures of these quinone
sesquiterpenes are characterized by an unprecedented,
challenging tetracyclic core that is presumably derived
from a rearranged drimane skeleton.1 Preliminary biolo-
gical evaluations have demonstrated that they possess
antiproliferative activities against various human cancer
cell lines as well as inhibitory activities against protein
tyrosine phosphatase 1B (PTP1B).1 The postulated tricyc-
lic biosyntheticprecursors, avaronand avarol, show a wide
spectrum of biological activity and low toxicity,2 ranging
from antibacterial,3 cytostatic,4 anti-inflammatory,5 anti-
platelet,6 antioxidant,7 and anti-HIV8 activity. Moreover,
avarol is a potent inhibitor of the cytokine TNF-R, making
it attractive for the treatment of inflammatory diseases
such as psoriasis.9 A more detailed biological evaluation of
the dysidavarones themselves, however, has been ham-
pered by the scarcity of these natural products. The intri-
guing new skeleton together with the sparse natural supply
attracted our interest in developing a total synthesis of this
unique class of natural products, not only for unambig-
uous structural assignment, but also to support further
biological evaluations. Herein, we report the first total
synthesis of dysidavarone A by a highly concise strategy
involving an advantageous intramolecular R-arylation
of an elaborate ketone. Notably, this presents the first
† University of Bonn.
‡
€
Universitat des Saarlandes.
ꢀ
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(5) Ferrandiz, M. L.; Sanz, M. J.; Bustos, G.; Paya, M.; Alcaraz,
(1) Jiao, W. H.; Huang, X. J.; Yang, J. S.; Yang, F.; Piao, S. J.; Gao,
H.; Li, J.; Ye, W. C.; Yao, X. S.; Chen, W. S.; Lin, H. W. Org. Lett. 2012,
14, 202.
M. J.; De Rosa, S. Eur. J. Pharmacol. 1994, 253, 75–82.
(6) Belisario, M. A.; Maturo, M.; Avagnale, G.; De Rosa, S.;
Scopacasa, F.; De Caterina, M. Pharmacol. Toxicol. 1996, 79, 300–304.
(7) (a) Pejin, B.; Iodice, C.; Tommonaro, G.; De Rosa, S. J. Nat.
Prod. 2008, 71, 1850–1853. (b) Belisario, M. A.; Maturo, M.; Pecce, R.;
De Rosa, S.; Villani, G. R. Toxicology 1992, 72, 221–233.
(2) Atta-ur-Rahman Studies in Natural Products Chemistry; Elsevier:
Oxford, UK, 2012; Vol. 36, pp 189ꢀ218.
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Muller, W. E. Zbl. Bakt. Hyg. 1985, 260A, 379–386.
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Gasic, M. J.; Heidemann, D.; Bernd, A.; Kurulec, B.; Eich, E.; Seibert,
Cancer Inst. 1987, 78, 663–666. (b) Loya, S.; Hizi, A. FEBS Lett. 1990,
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r
10.1021/ol400156u
Published on Web 02/07/2013
2013 American Chemical Society

total synthesis of a dysidavarone-type natural product
and unequivocally confirms the full 3D architecture of
this class of compounds. Furthermore, the convergent
sequence was readily scalable and enabled more detailed
biological evaluation of dysidavarone A revealing potent
antibacterial and antiproliferative activities as well as
specific morphological changes of treated cells.
Scheme 2. Synthesis of Benzyl Bromide 3
Scheme 1. Retrosynthesis Analysis of Dysidavarone A
original work. Recrystallization from n-pentane raised the
ee to excellent 99.9%. As shown in Scheme 3, the stereo-
selective coupling of the two building blocks 3 and 4 by
a reductive alkylation under Birch conditions proceeded
with good yields (72%) and furnished the desired diaster-
eomer 2 with high selectivities (dr >20:1).15,16
The results of the pivotal intramolecular R-arylation
of ketone 2 are summarized in Table 1. No conversion
was observed in initial studies with catalytic amounts
of PdCl2(PPh3)2 and (DtBPF)PdCl2,18 while Pd(OAc)2
17
As shown in Scheme 1, our retrosynthetic plan relied
on protected phenol 2 as a late-stage intermediate,
which was planned to be converted to the tetracyclic core
of 1 by a challenging intramolecular palladium-catalyzed
R-arylation.10 The quinone functionality in turn was ex-
pected to arise at a late step of the route after liberation
of the labile phenol by oxidation. Intermediate 2 in turn
should be generated through a stereoselective reductive
alkylation under Birch type conditions from known
Wieland-Miescher type ketone 411 with orcinol 5 derived
benzyl bromide 3.
As shown in Scheme 2, the synthesis of aromatic building
block 3 started from orcinol 5, which was monoethylated
with diethylsulfate12 followed by selective bromination of
the aromatic core with NBS.13 After protection of the
remaining phenol as a tert-butyl ether,14 the benzylic position
was brominated with NBS/AIBN.
in the absence of ligands only led to degradation.19 Also,
a combination of Pd(OAc)2 with P(t-Bu)320 was tried but
again no conversion was observed. It was assumed that the
sterical hindrance of the two bulky residues in the ortho
position of the bromine might prevent the reaction. There-
fore, we attempted to cleave the tert-butyl ether to evaluate
the unprotected phenol. However, these attempts likewise
failed due to the high instability of the liberated phenol.
Finally, a combination of Pd(OAc)2with ligand 9, which
was developed by the group of Buchwald, led to a success-
ful ring-closure in good yields (66%) considering the
complexity and high steric hindrance of the substrate.19
Catalyst-loadings of 10 mol % were required to achieve
complete conversion. Use of XPhos-precatalyst21 also
resulted in product formation, but in lower yields and
(15) The stereochemistry of 2 was confirmed by NMR methods:
The 1,2-dioxolane protected Wieland-Miescher type
ketone 4 was prepared in three steps in enantiopure form
according to a previously reported procedure involving
an organo-catalyzed Hajos-Parish-Eder-Sauer-Wiechert
type aldol condensation.11 The ee of the crude product of
the condensation as determined by chiral HPLC analysis
was very good (95%) despite the use of (L)-R-Phenylalanin
in contrast to (L)-β-Phenylalanin, which was used in the
(16) This type of reductive alkylation often served as key-step in the
total synthesis of sesquiterpenoidal quinones and hydroquinones albeit
with simpler aromates. For some examples, see: (a) Sarma, A. S.;
Chattopadhyay, P. J. Org. Chem. 1982, 47, 1727–1731. (b) An, J.;
Wiemer, D. F. J. Org. Chem. 1996, 61, 8775–8779. (c) Stahl, P.; Kissau,
L.; Mazitschek, R.; Huwe, A.; Furet, P.; Giannis, A.; Waldmann, H.
J. Am. Chem. Soc. 2001, 123, 11586–11593. (d) Sakurai, J.; Oguchi, T.;
Watanabe, K.; Abe, H.; Kanno, S.; Ishikawa, M.; Katoh, T. Chem.;
Eur. J. 2008, 14, 829–837.
(17) Muratake, H.; Natsume, M. Tetrahedron Lett. 1997, 38, 7581–
7582.
(18) Grasa, G. A.; Colacot, T. J. Org. Lett. 2007, 9, 5489–5492.
(19) Fox, J. M.; Huang, X.; Chieffi, A.; Buchwald, S. L. J. Am. Chem.
Soc. 2000, 122, 1360–1370.
(10) For a review on metal-catalyzed R-arylation of carbonyl see:
(a) Johansson, C. C. C.; Colacot, T. J. Angew. Chem., Int. Ed. 2010, 49,
676–707. (b) Bellina, F.; Rossi, R. Chem. Rev. 2010, 110, 1082–1146.
(11) Hagiwara, H.; Uda, H. J. Org. Chem. 1988, 53, 2308–2311.
(12) Bredereck, H.; Hennig, I.; Rau, W. Chem. Ber. 1953, 86, 1085–
1095.
(13) Tatsuta, K.; Furuyama, A.; Yano, T.; Suzuki, Y.; Ogura, T.;
Hosokawa, S. Tetrahedron Lett. 2008, 49, 4036–4039.
(14) Holcombe, J. L.; Livinghouse, T. J. Org. Chem. 1986, 51, 111–
113.
(20) Kawatsura, M.; Hartwig, J. F. J. Am. Chem. Soc. 1999, 121,
1473–1478.
Org. Lett., Vol. 15, No. 4, 2013
965

Table 1. Results of the R-Arylation of 2
Scheme 3. Completion of the Total Synthesis of Dysidavarone A
entry catalysta/ligand
base
solvent
temp
yield
b
1
2
3
4
5
6
7
PdCl2(PPh3)2/ꢀ
Cs2CO3
THF
75ꢀ100 °C
rtꢀ75 °C
80 °C
(DtBPF)PdCl2a/ꢀ t-BuONac THF
(DtBPF)PdCl2/ꢀ t-BuONad THF
Pd(OAc)2/ꢀ
t-BuONad toluene 80 °C
Pd(OAc)2/P(tBu)3 t-BuONad toluene 80 °C
Pd(OAc)2/9
t-BuONad toluene 70 °C
t-BuONad toluene 80 °C
66%
43%
XPhos-Precat/ꢀ
a Catalyst loading: 10 mol %, except for entry 2, where 2 mol %
catalyst were used. b 3 equiv base was used. c 1.1 equiv base. d 1.3 equiv
base was used.
Finally, we discovered that 5N HCl in THF26 not only
achieved deprotection, but also resulted in the required
rearrangement of the exocyclic double bond to the more
stable endocyclic olefin after prolonged reaction times. The
free phenol 13 proved fairly stable in solution but decom-
position was observed upon concentration. Therefore,
it was used directly for the final oxidation with Salcomine
[N,N0-bis(salicylidene)ethylene-diamino-cobalt(II)] under
an O2-atmosphere16d to yield dysidavarone A with a
yield of 60% over both steps (Scheme 3). The identity
of the synthesized material with natural dysidavarone A
was confirmed by NMR-, mass-, and CD-spectral data,1,27
which also proves the full stereostructure of this class of
compounds in general.
With synthetic dysidavarone A in hand the biological
potency of this scarce natural product could be studied in
more detail. As shown in Table 2, it was evaluated against
various microorganisms and cell lines demonstrating
potent inhibitory effects against Gram-positive bacteria
(Table 2, entries 8ꢀ15), in particular against various
Staphylococci with activities in low or even below μg/mL
concentrations (Table 2, entries 11ꢀ15). Furthermore,
it showed promising antiproliferative potencies against
cancer cell lines (Table 2, entries 17, 18), while no inhibi-
tory activity against various fungi/yeast (Table 2, entries
1ꢀ3) and Gram-negative bacteria (Table 2, entries 4ꢀ7)
was observed. Likewise, dysidavarone A showed no
anti-inflammatory activities (see Supporting Information
section).
To attain further hints about the mode of action, dysi-
davarone A treated cells were screened on several apoptosis
markers using U-2 OS cells. The cultured cells were
stained by labeling mitochondria, endoplasmic reticulum,
lysosomes, and nuclei and inspected by fluorescence micro-
scopy (see Supporting Information section). As shown in
Figure 1, treated cells showed striking alterations compared
to the control population. In the course of treatment a
required significantly longer reaction times. Notably, this
advanced application22 of the method in complex target
synthesis proceeds in an unusual 6-endo mode.
After successful formation of the bridged eight-membered
ring, ketal 10 was deprotected with 3N hydrochloric acid.16d
Afterward, both carbonyl-functions of compound 11 were
methenylated by Ph3PþCH3Brꢀ and tBuOK.23
Subsequent attempts to rearrange the terminal double
bond 12 to 13 with RhCl316d or iodine24 failed, resulting in
the cleavage of the tert-butoxy group and formation of
a variety of byproducts. Deprotection was also evaluated
with TFA in DCM25 and TFMS in trifluoroethanol,14 but
both reagents likewise resulted in various side products.
(21) (a) Biscoe, M. R.; Buchwald, S. L. Org. Lett. 2009, 11, 1773–
1775. (b) Hellal, M.; Singh, S.; Cuny, G. D. J. Org. Chem. 2012, 77,
4123–4130.
(22) (a) MacKay, J. A.; Bishop, R. L.; Rawal, V. H. Org. Lett. 2005,
7, 3421–3424. (b) Liao, X.; Stanley, L. M.; Hartwig, J. F. J. Am. Chem. Soc.
2011, 133, 2088–2091. (c) Bhat, V.; Allan, K. M.; Rawal, V. H. J. Am.
ꢀ
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Chem. Soc. 2011, 133, 5798–5801. (d) Sole, D.; Vallverdu, L.; Solans, X.;
Font-Bardıa, M.; Bonjoch, J. J. Am. Chem. Soc. 2003, 125, 1587–1594. (e)
´
ꢀ
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Sole, D.; Peidro, E.; Bonjoch, J. Org. Lett. 2000, 2, 2225–2228.
(23) Fitjer, L.; Quabeck, U. Synth. Commun. 1985, 15, 855–864.
(24) Ling, T.; Xiang, A. X.; Theodorakis, E. A. Angew. Chem., Int.
Ed. 1999, 38, 3089–3091.
(27) Synthetic dysidavarone A was a crystalline compound (mp
98ꢀ103) and had an optical rotation of þ125, while natural dysidavar-
one A was reported as an oil with an optical rotation of þ30. This
discrepancy may possibly be caused by a higher purity of the synthetic
material as compared to the natural material, as evidenced from the
NMR spectra supplied in the isolation paper.
(25) Tae, H. S.; Hines, J.; Schneekloth, A. R.; Crews, C. M. Org. Let.
2010, 12, 4308–4311.
(26) Eder, U.; Haffer, G.; Neef, G.; Sauer, G.; Seeger, A.; Wiechert,
R. Chem. Ber. 1977, 110, 3161–3167.
966
Org. Lett., Vol. 15, No. 4, 2013

Table 2. MIC50 and GI50 Values of Dysidavarone A 1
MIC50
entry
test organism
[μg/mL]
1
Mucor hiemalis
∼64
2
Pichia anomala
∼64
3
Candida albicans
>64
4
Escherichia coli TolC
∼64
5
Escherichia coli DH5R
>64
6
Chromobacterium violaceum
Pseudomonas aeruginosa PA14
Bacillus subtilis
>64
7
>64
Figure 1. Changes of cellular morphology and lysosomal pH of
cultivated U-2 OS cells upon 3 h treatment with 20 μg/mL
dysidavarone A (a) in comparison to control cells (b). Cells were
incubated and stained with acridine orange (red: acidic lyso-
somes; green: neutral compartments) and Hoechst33342 (blue:
nuclei).
8
>64
9
Enterococcus faecalis DSM20478
Enterococcus faecium DSM20477
Micrococcus luteus
>64
10
11
12
13
∼64
5.5 ( 0.2
7.1 ( 2.1
9.9 ( 0.8
Staphylococcus aureus DSM346
Staphylococcus aureus DSM11822
(multidrug resistant)
14
Staphylococcus aureus N315
(methicillin resistant)
4.0 ( 0.1
unique 3Dstructureofthisclass of quinonesesquiterpenes.
Key transformations of the convergent synthesis include a
stereoselective reductive alkylation of a Wieland-Miescher
type ketone and an advantageous R-arylation of a highly
hindered elaborate ketone using the Buchwald ligand. The
concise route enabled access to sufficient material for
further biological evaluation of this scarce metabolite,
revealing potent antibacterial and antiproliferative activ-
ities by specifically changing the morphology of treated
cells. Present studies are now directed to further analyze
and potentially use this promising biological potency.
15
16
Staphylococcus aureus Newman
Staphylococcus carnosus DSM20501
6.5 ( 0.6
0.2 ( 0.1
GI50 [μg/mL]
17
18
human HL-60 myeloid leukemia cell line
(ACC-3)
1.65
human HepG2 hepatocellular carcinoma
cell line (ACC-180)
4.26
significant disruption of lysosomal integrity (deacidifi-
cation) was observed. In addition, unusual pore formation
took place which was accompanied by specific morpholo-
gical alterations of the cells, that is, membrane blebbing
and chromatin condensation. The macroscopic effects of 1
partially resemble those of bacterial pore-forming toxins28
and may suggest that these compounds trigger a lysosomal
dysfunction that culminates in the release of cathepsins
from lysosomes in the cytosol, which in turn causes pro-
teolytic cell damage and may activate the mitochondrial
apoptosis pathway.29
Acknowledgment. This work was generously supported
by the D.F.G. (SFB 623). The authors thank Sebastian
Essig, Michael Dieckmann, Thomas Debnar (University
of Heidelberg), Dr. Manuel Kretschmer (Columbia Uni-
versity New York) and Dr. Aubry Miller (German Cancer
Research Center, Heidelberg) for helpful suggestions in
devising the synthetic route as well as Andreas J. Schneider
(University of Bonn) for HPLC-support and Friederike
Eberhagen (University of Bonn) for technical support.
In summary, the first total synthesis of dysidavarone A
was achieved in a highly concise route in 10 steps (longest
linear sequence) with a total yield of 11% and confirms the
Supporting Information Available. Experimental pro-
1
cedures, spectral data and copies of H- and 13C NMR
spectra for all new compounds and details of biological
experiments. This material is available free of charge via
the Internet at http://pubs.acs.org.
(28) Kennedy, C. L.; Smith, D. J.; Lyras, D.; Chakravorty, A.; Rood,
J. I. PLoS Pathogens 2009, 5, e1000516.
ꢁ
(29) Cesena, M. H.; Pegana, K.; Spesa, A.; Turka, B. Exp. Cell. Res.
ꢁ
2012, 313, 1245–1251.
The authors declare no competing financial interest.
Org. Lett., Vol. 15, No. 4, 2013
967