7-Step total synthesis of (+)-EBC-329: Photoisomerisation reveals new: Seco -casbane family member

Article abstract of DOI:10.1039/c7ob01400g

The first seco-casbane, EBC-329, isolated from the Australian rainforest, was synthesised from (+)-2-carene in seven steps. This endeavour not only established the absolute stereochemical assignment as (8R,9S)-EBC-329, but also identified, via photoisomer

Full text of DOI:10.1039/c7ob01400g

Organic &
Biomolecular Chemistry
View Article Online
View Journal
COMMUNICATION
7-Step total synthesis of (+)-EBC-329:
Photoisomerisation reveals new seco-casbane
family member†
Cite this: DOI: 10.1039/c7ob01400g
Received 9th June 2017,
Accepted 7th August 2017
Timothy J. Vanden Berg,
David M. Pinkerton and Craig M. Williams
*
DOI: 10.1039/c7ob01400g
rsc.li/obc
The first seco-casbane, EBC-329, isolated from the Australian rain-
forest, was synthesised from (+)-2-carene in seven steps. This
endeavour not only established the absolute stereochemical
assignment as (8R,9S)-EBC-329, but also identified, via photo-
isomerisation, a new seco-casbane family member.
Casbanes are considered a rare family of gem-dimethyl-
cyclopropyl diterpene macrocycles.1,2 The parent skeletal
hydrocarbon, casbene (1) (Fig. 1), was the first family member
to be reported,3 and since then casbanes have been found in
both terrestrial4 and marine5 organisms. Our investigation
into the Australian rainforest plant Croton insularis6 has
revealed various casbanes [e.g. EBC-181 (2),7 EBC-304 (3)8 and
EBC-324 (4)9], and the first seco-casbanes 328 (5),10 329 (6)9
and 363 (7)10 (Fig. 1).
Surprisingly, casbanes have received little attention in
terms of total synthesis, especially given that they display a
range of biological activities.1,4,5,7,8 Crombie and Pattenden
were the first to synthesise casbene (1),11 soon followed by
Takahashi,12 and later by McMurry.13 However, no further
total syntheses of casbanes have surfaced since these reports
in the 1980s. In comparison, the seco-casbane EBC-329 (6) suc-
cumbed to total synthesis in 2015,14 one year after we dis-
closed the structure.9 The Thombal and Jadhav synthesis of
racemic 6 was achieved in 13 steps with a reported overall yield
of 10%; however, the supplied spectroscopic evidence was
inconsistent with that of the natural product (see ESI†).
Inspired to unambiguously determine the absolute stereo-
chemistry of EBC-329, and to further pursue its anti-cancer
properties (e.g. IC50 of 0.6 µM against the K562 chronic myolo-
genous leukemia cell line15), we embarked on a synthetic
campaign.
Fig. 1 Casbanes 1–7, and retrosynthetic analysis of EBC-329 (6).
Herein is reported a 7-step total synthesis of EBC-329 start-
ing from the chiral pool.
As EBC-329 (6) contains a gem-dimethylcyclopropane with
two embedded stereocentres, it was difficult to surpass the
chiral pool in the retrosynthetic analysis (Fig. 1), especially
considering the chiral pool was successfully exploited for the
synthesis of casbene (1).11–13 In this regard, commercially
available (+)-2-carene (8) was selected, even though it was likely
to lead to the minor (or non-natural) enantiomer based on the
cyclopropane stereochemical assignments seen in 1, 3, 4, 5
and 7. That being said, there is evidence that the absolute con-
figuration of casbane cyclopropane units can vary within the
same organism.5b
School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane,
4072, Australia. E-mail: c.williams3@uq.edu.au
†Electronic supplementary information (ESI) available: Experimental pro-
cedures, compound characterisation, X-ray crystallography and copies of 1H and
13C NMR, UV-Vis and UV-Vis circular dichroism spectra. CCDC 1521107. For ESI
and crystallographic data in CIF or other electronic format see DOI: 10.1039/
c7ob01400g
This journal is © The Royal Society of Chemistry 2017
Org. Biomol. Chem.

View Article Online
Communication
Organic & Biomolecular Chemistry
It was envisaged that oxidative ring opening of (+)-2-carene prone to oxidation in the open atmosphere, as has been
(8) would provide differentiated carbonyl groups. Such an previously reported.19
approach would allow judicious choice in chemoselective
Initially, a Petersen olefination20 strategy was pursued to
methods for installing the required unsaturation. Butenolide access enyne 12. Although this was productive, both standard
functionality could be installed sequentially or deployed in a and modified conditions provided unfavourable E/Z-isomer
concerted fashion using a Sonogashira16/cyclisation17 strategy ratios of 12 (i.e. the unwanted 12Z-12 as the major isomer).
(Fig. 1).
Fortunately, resorting to the Horner–Wadworth–Emmons olefi-
Attempts to procure ketoaldehyde 9 via ozonolysis13 of 8 nation protocol, using the known diisopropyl phosphonate
were unsatisfactory, thus prompting the use of a two-step pro- 13,21 installed the enyne motif in a favourable 2.4 : 1.0 E : Z
cedure involving dihydroxylation followed by oxidative cleavage ratio (see below). TBAF-mediated protodesilylation was sub-
(Scheme 1).18 Ketoaldehyde 9 was then subjected to Wittig sequently performed in situ to furnish enyne 14, followed by
olefination with known phosphorane 10, generating ketoester 11 hydrolysis of the methyl ester to yield the corresponding acid
with exclusive E selectivity (determined through an NOE differ- 15 in 63% yield over three steps (Scheme 1).
ence experiment, see ESI†) (Scheme 1). The overall yield of 11
The ester protection was removed prior to the installation
from 8 over three steps was enhanced to 52% if ketoaldehyde 9 of the butenolide because prior work in our group revealed the
was not purified prior to Wittig olefination. Ketoaldehyde 9 was methyl ester of EBC-329 to be unstable. The status of the car-
boxyl group (i.e. ester or free acid) did not appear to have an
effect on the outcome of the butenolide installation.
The final step of the synthesis required the installation of
the γ-alkylidenebutenolide. This was accomplished in 31%
yield with exclusive Z-selectivity (i.e. both isomers are 14Z)
using iodoacrylic acid 16,22 following previously reported one
pot palladium-free conditions.23 An NOE difference experi-
ment supported the butenolide configuration assigned to 6
(see ESI†). Integration of the 1H NMR spectrum recorded on
the butenolide mixture (i.e., 6 and 17) confirmed that the 12E/
12Z ratio was unvaried in the reactions subsequent to the
Horner–Wadsworth–Emmons olefination. Unfortunately, NOE
experiments performed on 12 through to 15 were unproduc-
tive. The two butenolide diastereomers were then separated by
normal-phase HPLC to yield pure EBC-329 (6) and impure 12Z-
EBC-329 (17) (Scheme 1), which could not be further purified.
The specific rotation obtained for synthetic 8S,9R-EBC-329
(6) was opposite to that reported for the natural sample, con-
firming the absolute stereochemistry of natural (−)-EBC-329
(6) as 8R,9S. This assignment was supported by comparison
between the UV-Vis circular dichroism spectra of synthetic and
natural EBC-329 (see ESI†).
As photoisomerisations of alkylidenebutenolides are
precedented,24–26 we exposed a deuterochloroform solution of
synthetic EBC-329 to laboratory fluorescent lighting for 60 h.
A new compound was generated which seemed to be in steady-
state equilibrium with EBC-329. No conversion was observed
in the absence of light under identical conditions. Based on
1H NMR spectral comparisons to both EBC-329 and
12Z-EBC-329 (17), the photochemical product was assigned
as one of either 14E-EBC-329 (18) or 12Z,14E-EBC-329 (19)
(Scheme 1). Unambiguous assignment of the photoisomer’s
stereochemistry could not be made with the available NMR
1
data. The H NMR spectrum of natural EBC-329 9 was then re-
evaluated to reveal this same photoisomer as a minor co-
isolate (see ESI†). Interestingly, the ratio of isomers observed
in the natural and irradiated synthetic samples were
comparable.
On this basis, the photoisomer was identified as a new
Scheme 1 Total synthesis of (+)-EBC-329. Double-headed arrows
depict key NOE observations.
member of the seco-casbane class of natural products, and it
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2017

View Article Online
Organic & Biomolecular Chemistry
Communication
was tentatively assigned the same absolute configuration as co-
isolated EBC-329 (i.e. 8R,9S). Although artefacts of isolation
may arise through photoisomerisation, the typical exposure of
Northern Australian terrestrial plants to prolonged, intense
sunlight supports the natural product status of the
photoisomer.
In conclusion, the synthesis of (+)-(8S,9R)-EBC-329 (6) was
achieved in a concise and efficient manner, culminating in a
seven step sequence. The synthetic approach described herein
is a substantial improvement over that previously reported,
and confirms for the first time the absolute stereochemistry of
naturally-occurring (−)-EBC-329 (6) as 8R,9S. In addition,
photoisomerisation of the final product led to the identifi-
cation of either 8R,9S-14E-EBC-329 (18) or 8R,9S-12Z-14E-
EBC-329 (19) as a new seco-casbane natural product.
The authors thank EcoBiotics Ltd and the University of
Queensland (UQ) for financial support. We gratefully acknowl-
edge Dr S. Chow and Ms Y. P. Tan for assistance with HPLC,
Prof. P. V. Bernhardt for assistance with collecting X-ray crystal-
lographic data, and Dr A. Savchenko for helpful discussions on
structure elucidation by NMR. T. J. V. thanks the Australian
Government for a Research Training Program Scholarship. The
Australian Research Council for a Future Fellowship award
(grant number FT110100851) to C. M. W. is also gratefully
acknowledged.
M. Zhao, M. Ma, Y. Xu, Z. Xiang, Y. Cai, J. Dong, X. Lei,
K. Huang and P. Yan, Mar. Drugs, 2013, 11, 455–465;
(d) M.-E. F. Hegazy, T. A. Mohamed, A. I. Elshamy, M. A. Al-
Hammady, S. Ohta and P. W. Paré, Molecules, 2016, 21, 308.
6 For previous work on this plant system by our group see:
(a) L. A. Maslovskaya, A. I. Savchenko, V. A. Gordon,
P. W. Reddell, C. J. Pierce, P. G. Parsons and
C. M. Williams, Org. Lett., 2011, 13, 1032–1035;
(b) L. A. Maslovskaya, A. I. Savchenko, V. A. Gordon,
P. W. Reddell, C. J. Pierce, P. G. Parsons and
C. M. Williams, Aust. J. Chem., 2015, 68, 652–659;
(c) L. A. Maslovskaya, A. I. Savchenko, C. J. Pierce,
V. A. Gordon, P. W. Reddell, P. G. Parsons and
C. M. Williams, Chem. – Eur. J., 2014, 20, 14226–14230;
(d) L. A. Maslovskaya, A. I. Savchenko, V. A. Gordon,
P. W. Reddell, C. J. Pierce, P. G. Parsons and
C. M. Williams, RSC Adv., 2016, 6, 25110–25113.
7 L. A. Maslovskaya, A. I. Savchenko, E. H. Krenske,
C. J. Pierce, V. A. Gordon, P. W. Reddell, P. G. Parsons and
C. M. Williams, Angew. Chem., Int. Ed., 2014, 53, 7006–
7009.
8 L. A. Maslovskaya, A. I. Savchenko, V. A. Gordon,
P. W. Reddell, C. J. Pierce, P. G. Parsons and
C. M. Williams, Chem.
–
Eur. J., DOI: 10.1002/
chem.201604674.
9 L. A. Maslovskaya, A. I. Savchenko, E. H. Krenske,
V. A. Gordon, P. W. Reddell, C. J. Pierce, P. G. Parsons and
C. M. Williams, Chem. Commun., 2014, 50, 12315–12317.
10 L. A. Maslovskaya, A. I. Savchenko, V. A. Gordon,
P. W. Reddell, C. J. Pierce, P. G. Parsons and
C. M. Williams, Eur. J. Org. Chem., 2016, 1673–1677.
11 (a) L. Crombie, G. Kneen and G. Pattenden, J. Chem. Soc.,
Chem. Commun., 1976, 66–68; (b) L. Crombie, G. Kneen,
G. Pattenden and D. Whybrow, J. Chem. Soc., Perkin Trans.
1, 1980, 1711–1717.
Conflicts of interest
T. J. V. and D. M. P. declare no conflicts of interest. C. M. W. is
a consultant to EcoBiotics Ltd.
Notes and references
1 M. J. Durán-Peña, J. M. Botubol Ares, I. G. Collado and 12 K. Toma, E. Miyazaki, T. Murae and T. Takahashi, Chem.
R. Hernández-Galán, Nat. Prod. Rep., 2014, 31, 940–952. Lett., 1982, 863–864.
2 Y.-H. Choi, J. Kim, J. M. Pezzuto, A. D. Kinghorn, 13 J. E. McMurry and G. K. Bosch, J. Org. Chem., 1987, 52,
N. R. Farnsworth, H. Latter and H. Wagner, Tetrahedron
Lett., 1986, 27, 5795–5798.
3 D. R. Robinson and C. A. West, Biochemistry, 1970, 9, 70–79.
4885–4893.
14 R. S. Thombal and V. H. Jadhav, Org. Biomol. Chem., 2015,
13, 9485–9491.
4 See for example: (a) Q.-L. Liang, C.-C. Dai, J.-H. Jiang, 15 C. B. Lozzio and B. B. Lozzio, Blood, 1975, 45, 321–334.
Y.-P. Tang and J.-A. Duan, Fitoterapia, 2009, 80, 514–516; 16 R. Chinchilla and C. Nájera, Chem. Rev., 2007, 107, 874–
(b) F.-G. Shao, R. Bu, C. Zhang, C.-J. Chen, J. Huang and
922.
J.-H. Wang, J. Asian Nat. Prod. Res., 2011, 13, 805–810; 17 (a) T. M. Ugurchieva and V. V. Veselovsky, Russ. Chem. Rev.,
(c) W.-W. Tao, J.-A. Duan, Y.-P. Tang, N.-Y. Yang, J.-P. Li and
Y.-F. Qian, Phytochemistry, 2013, 94, 249–253;
2009, 78, 337–373; (b) D. W. Knight, Contemp. Org. Synth.,
1994, 1, 287–315.
(d) F. A. e Silva Filho, J. N. da Silva Jr., R. Braz-Filho, 18 K. C. Nicolaou, V. A. Adsool and C. R. H. Hale, Org. Lett.,
C. A. de Simone, E. R. Silveira and M. A. Sousa Lima, Helv.
Chim. Acta, 2013, 96, 1146–1154.
5 See for example: (a) C.-X. Zhang, S.-J. Yan, G.-W. Zhang,
2010, 12, 1552–1555.
19 G. B. Hammond, M. B. Cox and D. F. Wiemer, J. Org.
Chem., 1990, 55, 128–132.
W.-G. Lu, J.-Y. Su, L.-M. Zeng, L.-Q. Gu, X.-P. Yang and 20 (a) D. J. Peterson, J. Org. Chem., 1968, 33, 780–784;
Y.-J. Lian, J. Nat. Prod., 2005, 68, 1087–1089; (b) Y. Li,
M. Carbone, R. M. Vitale, P. Amodeo, F. Castelluccio,
G. Sicilia, E. Mollo, M. Nappo, G. Cimino, Y.-W. Guo and
M. Gavagnin, J. Nat. Prod., 2010, 73, 133–138; (c) J. Yin,
(b) L. F. van Staden, D. Gravestock and D. J. Ager, Chem.
Soc. Rev., 2002, 31, 195–200, and references therein;
(c) Y. Yamakado, M. Ishiguro, N. Ikeda and H. Yamamoto,
J. Am. Chem. Soc., 1981, 103, 5568–5570.
This journal is © The Royal Society of Chemistry 2017
Org. Biomol. Chem.

View Article Online
Communication
Organic & Biomolecular Chemistry
21 (a) For the synthesis of 13 see: G. Yao and K. Steliou, Org.
Lett., 2002, 4, 485–488. Conditions used for the HWE olefi-
nation see: K. M. Brummond, J. Lu and J. Petersen, J. Am.
Chem. Soc., 2000, 122, 4915–4920.
22 (a) B. Wang, C. Lu, S.-Y. Zhang, G. He, W. A. Nack and
G. Chen, Org. Lett., 2014, 16, 6260–6263; (b) The first X-ray
crystal structure of iodoacrylic acid 16 is reported herein,
see ESI. CCDC 1521107.†
15, 5096–5098; (b) K. Sastraruji, S. G. Pyne, A. T. Ung,
P. Mungkornasawakul, W. Lie and A. Jatisatienr, J. Nat.
Prod., 2009, 72, 316–318.
25 For combined γ,δ- and ε,ζ-photoisomerisation of
γ-alkylidenebutenolide, see: T. Kajikawa, S. Hasegawa,
T. Iwashita, T. Kusumoto, H. Hashimoto,
a
D. M. Niedzwiedzki, H. A. Frank and S. Katsumura, Org.
Lett., 2009, 11, 5006–5009.
23 S. Inack-Ngi, R. Rahmani, L. Commeiras, G. Chouraqui, 26 For photoisomerisation at even more remote alkenes in a
J. Thibonnet, A. Duchêne, M. Abarbri and J.-L. Parrain,
Adv. Synth. Catal., 2009, 351, 779–788.
24 For γ,δ-photoisomerisation of a γ-alkenylbutenolide, see:
(a) S. D. Townsend and G. A. Sulikowski, Org. Lett., 2013,
conjugated γ-alkylidenebutenolide, see: T. Kajikawa,
K. Aoki, R. S. Singh, T. Iwashita, T. Kusumoto, H. A. Frank,
H. Hashimoto and S. Katsumura, Org. Biomol. Chem., 2009,
7, 3723–3733.
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2017