Arbutin Derivatives Isolated from Ancient Proteaceae: Potential Phytochemical Markers Present in Bellendena, Cenarrhenes, and Persoonia Genera

Article abstract of DOI:10.1021/acs.jnatprod.7b01038

Extensive phytochemical studies of the paleoendemic Tasmanian Proteaceae species Bellendena montana, Cenarrhenes nitida, and Persoonia gunnii were conducted employing pressurized hot water extraction. As part of these studies, six novel glycosides were is

Full text of DOI:10.1021/acs.jnatprod.7b01038

Article
pubs.acs.org/jnp
Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
Arbutin Derivatives Isolated from Ancient Proteaceae: Potential
Phytochemical Markers Present in Bellendena, Cenarrhenes, and
Persoonia Genera
Bianca J. Deans,† Nathan L. Kilah,† Gregory J. Jordan,‡ Alex C. Bissember,*,† and Jason A. Smith*,†
†School of Physical Sciences−Chemistry, University of Tasmania, Hobart, Tasmania 7001, Australia
‡School of Biological Sciences, University of Tasmania, Hobart, Tasmania 7001, Australia
S
* Supporting Information
ABSTRACT: Extensive phytochemical studies of the paleoendemic Tasmanian
Proteaceae species Bellendena montana, Cenarrhenes nitida, and Persoonia gunnii were
conducted employing pressurized hot water extraction. As part of these studies, six novel
glycosides were isolated, including rare examples of glycoside-containing natural products
featuring tiglic acid esters. These polar molecules may represent potential phytochemical
markers in ancient Proteaceae.
species, excluding Persoonia gunnii, employed peak-matching
against polyol standards to identify chemical components.
Interestingly, the authors noted that the sugar-containing
extracts obtained from B. montana, C. nitida, and all nine
Persoonia species contained numerous unidentified compounds.
Significantly, Bieleski and Briggs state, “These additional peaks
were almost always characteristic of a genus, and they clearly
indicate the presence in the leaf extracts of compounds other
than sugars or polyols that could have taxonomic value.”8 These
intriguing results suggest that both Bellendena and Cenarrhenes
species and Persoonia species contain polar components that
have not been isolated and identified and, more importantly,
that polar phytochemicals present in these species may
potentially serve as novel chemical markers.
This prompted us to undertake a proof of concept study to
explore the aforementioned hypothesis. By design, the
monotypic Bellendena and Cenarrhenes genera and a plant
species from the Persoonia genera were selected. In this work,
the first natural product isolation studies of the Proteaceae
species C. nitida and P. gunnii and a more extensive isolation
study of B. montana are reported.9 This research was facilitated
by pressurized hot water extraction (PHWE), employing an
efficient and practical method recently developed in our
laboratory.10 In total, 11 different glycosides were isolated, 6
of which are new compounds. These polar molecules could
represent novel phytochemical markers in ancient Proteaceae.
he Proteaceae family represents one of the most diverse
Tand ancient lineages of eudicot vascular plants, with over
1700 species and ∼80 genera.1 Studies on this family have
significantly contributed to unraveling the evolution of vascular
plants.2 Furthermore, Proteaceae play an important role in
contributing to understanding the biogeography of the
southern hemisphere.3 This family spans this region, including
Southeast Asia, and is centered in Australia. Proteaceae are
found in Tasmania, the southeastern island state of Australia.
This island contains strong endemism and is regarded as a “hot
spot” of global importance for paleoendemic plants.4
Significantly, many ancient clades, which have succumbed to
selection pressures in other regions, are still found in this
isolated, geographically restricted territory.5
Tasmania is home to 64 species of Proteaceae from 14 extant
genera; 20 of these plant species are endemic, and these
originate from nine genera.6 Three of these genera are endemic
(Agastachys, Bellendena, and Cenarrhenes). Some of these
species are strongly paleoendemic, notably Bellendena montana
and Cenarrhenes nitida, which are restricted to Tasmania and
are the only living members of groups ∼85 and ∼30 million
years old, respectively (Figure 1).2d This places them among
the most relictual members of this ancient family. Bellendena,
Cenarrhenes, and Persoonia genera have been the focus of
various taxonomic investigations.7
In 2005, Bieleski and Briggs disclosed a broad phytochemical
survey of Proteaceae species that employed quantitative gas−
liquid chromatography to analyze polyol sugars present in the
leaves of over 120 Proteaceae members.8 This study, which
specifically featured B. montana, C. nitida, and nine Persoonia
Received: December 14, 2017
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.7b01038
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Figure 1. Simplified representation of the phylogeny of Proteaceae
estimated by Sauquet and co-workers.2d This DNA-based phylogeny
was estimated using Bayesian methods, with age estimates from a
relaxed molecular clock calibrated with fossil data. Genera are color-
coded according to their “age index”. The age index estimates the
mean time between speciation events (assuming no extinction) and is
equal to the age of each group divided by log2(n + 1), where n is the
number of species. Thus, the age index essentially indicates the
antiquity of the genus. Asterisks denote genera containing Tasmanian
endemic species. s.l. = sensu lato.
Figure 2. Novel arbutin derivatives 1, 5, and 6 and known arbutin (4)
and compounds 2 and polifolioside (3), isolated from B. montana.
Compounds 1−4 were isolated from B. montana leaves and
compounds 1, 2, 5, and 6 from B. montana flowers.
RESULTS AND DISCUSSION
■
B. montana. Previous chemical investigations of B. montana
have focused on the isolation of alkaloids from aerial parts
(leaves, stems, and flowers). A total of 15 different alkaloids
have been reported from these species to date. The majority of
these natural products were tropane alkaloids, including
bellendine, isobellendine, and darlingine.9,11 Methyl (p-
hydroxybenzoyl)acetate was also isolated from B. montana.9
The aforementioned compounds were extracted in a non-
discriminative method, with leaves, stems, and flowers extracted
together en mass, which prevented the discrete location of
these phytochemicals within the morphology of the plant to be
identified. It was later shown that upon separate examination of
the flowers and the other aerial parts of B. montana the alkaloid
profiles differed, with bellendine concentrated in flowers and
isobellendine in other parts of the plant.11a
Arbutin (4) has been previously isolated from members of the
Proteaceae family.14 In addition, various arbutin derivatives
have also been isolated from Proteaceae and other families.15
The presence of arbutin as a decomposition artifact resulting
from the isolation process is unlikely in our study, as the free
acid chain portions of both compounds 1 and 2 were not
1
observed by H NMR spectroscopy or TLC analysis at any
stage. Hence, arbutin is not an artifact of isolation and instead
represents a true component of B. montana.
1
On the basis of the H and 13C NMR and IR spectroscopic
data, the molecular formula of C21H22O10 was proposed for
compound 1, which was supported by HRESIMS data (m/z
calculated [M + Na]+, 457.1106; found, 457.1105). The IR
spectrum featured bands at 1722 and 1668 cm−1 that were
consistent with keto carbonyl and ester carbonyl stretches,
1
PHWE was performed on B. montana leaf material. The
aqueous PHWE mixture was extracted with EtOAc to provide a
crude extract. The remaining aqueous phase was also
evaporated to afford a crude extract. These respective crude
samples were purified by flash column chromatography. In this
way, the new natural product 1 (2.0% yield w/w) and known
compounds 2 (3.4% yield w/w), polifolioside (3) (0.67% yield
w/w), and arbutin (4) (0.71% yield w/w) were isolated (Figure
respectively. The H NMR spectrum contained resonances at
δH 6.95−6.97 (m, 2H, H-3, H-5) and 6.76 (d, J = 9 Hz, 2H, H-
2, H-6), which were consistent with a hydroquinone moiety, as
well as resonances that were consistent with a β-D-glucose.
Notably, the anomeric proton at δH 4.76−4.79 (m, 1H, H-1′)
and the 6′-methylene protons at δH 4.55 (d, J = 10.9 Hz, 1H,
H-6a′) and 4.27 (dd, J = 11.6, 7.4 Hz, 1H, H-6b′) were
observed downfield due to deshielding from the adjacent ester
moiety. These data were consistent with the presence of an
arbutin core structure associated with a related derivative
1
2). The H, 13C, and 2D NMR spectroscopic data (HSQC,
HMBC, COSY) for polifolioside (3), as well as HRESIMS data
(m/z [M + Na]+ calcd for C25H26O15Na, 589.1164; found,
589.1162), were consistent with reported data for heterocycle 3
and a similar compound.12 The 1H and 13C NMR spectroscopic
data for arbutin (4) were consistent with reported data.13
isolated from Persoonia linearis x pinifolia.16 The H NMR
1
spectrum also featured signals at δH 7.91 (d, J = 8.4 Hz, 2H, H-
2″, H-6″) and δH 6.93−6.96 (m, 2H, H-3″, H-5″), consistent
with a p-hydroxyphenyl moiety.17 The H NMR and HSQC
1
B
DOI: 10.1021/acs.jnatprod.7b01038
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
spectra revealed a resonance at δH 4.05 (s, 1.4H, H-8″; partial
deuterium exchange observed) corresponding to methylene
protons, which was consistent with a resonance reported for
methylene protons of benzoylacetic acid,17 as well as those
reported for methyl (p-hydroxybenzoyl)acetate.9 Furthermore,
the H-8″ methylene protons engaged in deuterium exchange
(D2O, methanol-d4; partial exchange was also observed in
acetone-d6). This underscores the acidity of the protons flanked
by both carbonyl groups. The 13C NMR spectrum displayed
resonances at δC 190.7 (C-7″, CO) and 167.7 (C-9″, C
O), consistent with keto and ester moieties in the benzoyl
acetate fragment.18
HMBC correlations between the ester carbonyl at δC 167.7
(C-9″, CO) and the 6′-methylene protons at δH 4.55 (d, J =
10.9 Hz, 1H, H-6a′) and 4.25 (dd, J = 11.8, 6.7 Hz, H-6b′)
were consistent with an ester linkage through C-6′ of the β-D-
glucose moiety (Figure 3).16 Both keto and ester 13C NMR
acetate was not isolated despite the extraction solvent (35%
EtOH/65% H2O v/v). Furthermore, 3-(4-hydroxyphenyl)-3-
oxopropanoic acid and its expected decarboxylation product, p-
hydroxyacetophenone, were not observed by NMR spectros-
copy at any stage of the extraction/isolation process. This
reinforces the mildness of the PHWE method that we have
developed.10
Perry and Brennan previously isolated compound 2 from the
native New Zealand Proteaceae species Toronia toru.19 This is
the first and only report of this compound in the literature. The
1H, 13C, and 2D NMR spectroscopic data of compound 2 were
consistent with the data reported by these authors, as well as
HRESIMS data (m/z calculated [M + Na]+, 393.1156; found,
393.1160). However, the specific rotation ([α]22 −41, c 1,
D
MeOH) contrasted with the reported data ([α]19 +18, c 2.5,
D
MeOH).19 For this reason, the structure of compound 2 was
secured by X-ray crystallography (Figure 4).
PHWE was performed on B. montana flowers. The aqueous
PHWE mixture was extracted with EtOAc to provide a crude
extract. The remaining aqueous phase was also evaporated to
afford a crude extract. These respective crude samples were
purified by flash column chromatography. In this way,
compounds 1 and 2 (2.4% combined yield w/w), 5 (0.27%
yield w/w), and 6 (0.03% yield w/w) were isolated (Figure 2).
On the basis of the H and 13C NMR and IR spectroscopic
1
data the molecular formula of C31H34O14 was proposed for
compound 5, which was supported by HRESIMS data (m/z
calculated [M + Na]+, 653.1841; found, 653.1848). Key IR
stretches present at 1711 and 1601 cm−1 were consistent with
the presence of keto carbonyl and ester groups, respectively.
Figure 3. Selected key HMBC correlations for compound 1.
resonances at δC 190.7 and 167.7 correlated with the methylene
protons at δH 4.05 (s, 1.4H) in the HMBC spectrum, which
further supported the presence of a benzoylacetyl moiety in
compound 1. Moreover, HMBC correlations between the δH
7.91 (d, J = 8.4 Hz, 2H, H-2″, H-6″) methine protons and δC
190.7 (C-7″, CO) carbonyl resonance confirmed this. The
structure of the novel arbutin derivative 1 was also secured by
single-crystal X-ray crystallography (Figure 4).
1
The H and 13C NMR spectroscopic data suggested structural
similarities with compound 2. Specifically, two sets of doublets
at δH 6.94−6.96 (m, 2H, H-3, H-5) and 6.73 (d, J = 9 Hz, 2H,
H-2, H-6) were consistent with the hydroquinone portion. In
addition, resonances at δH 4.83 (d, J = 8.3 Hz, 1H, H-1′) were
assigned to the anomeric proton, and signals at δH 4.61 (dd,
1H, H-6a′) and 4.26−4.27 (m, 1H, H-6b′) were consistent with
a β-D-glucose moiety.16 Moreover, the H NMR spectrum
1
featured signals at δH 6.29 (s, 1H, H-5a″), 6.16 (s, 1H, H-5b″),
4.31 (t, J = 6.4 Hz, 2H, H-4″), and 2.72 (t, J = 6.4 Hz, 2H, H-
3″) assigned to methylene resonances, which were consistent
with those reported within aforementioned arbutin derivatives,
including compound 2.19 The presence of methylene
resonances at δH 5.79 (d, J = 0.8 Hz, 1H, H-10a″) and 5.66
(d, J = 0.9 Hz, 1H, H-10b″), as well as δH 4.24−4.27 (m, 2H,
H-9″) and 2.61 (t, J = 6.4 Hz, 2H, H-8″), suggested the
presence of a repeated portion of the side chain. COSY
correlations were observed between the methylene protons at
δH 4.31 (t, J = 6.4 Hz, 2H, H-4″) and 2.72 (t, J = 6.4 Hz, 2H,
H-3″) and the methylene protons at δH 4.24−4.27 (m, 2H, H-
9″) and 2.61 (t, J = 6.4 Hz, 2H, H-8″) (Figure 5a). These data
supported the presence of two separate chain portions. The 1H,
13C, COSY, and HSQC spectroscopic data were consistent with
the presence of a 2-methylene-4-hydroxybutanoyl moiety by
analogy with compound 2.17
Figure 4. ORTEP representations of glucoside 1 and natural product
2. Ellipsoids are drawn at the 50% probability level. In the case of
compound 2, two independent molecules were present in the
asymmetric unit, one of which was partially disordered over two
sites of uneven occupancy. The disordered molecule in the asymmetric
unit has been omitted for clarity (an image of both molecules is
available in Figure S1 in the Supporting Information).
The 13C NMR spectrum associated with compound 5
featured a key resonance at δC 190.7 that was consistent with
the presence of a keto carbonyl, as observed for compound 1,
and signals at δC 167.6 (CO, C-9‴), 165.93 (C-1″), and
165.90 (C-6″) that were consistent with three ester resonances.
HMBC spectroscopic analysis provided important information
regarding the connections in compound 5 (Figure 5b).
Specifically, a correlation between the methylene protons in
Interestingly, in 1971, Bick and co-workers reported the
isolation of methyl (p-hydroxybenzoyl)acetate (the methyl
ester analogue of glucoside 1) from B. montana.9 The extraction
solvent mixture they employed contained MeOH, and we
suggest that transesterification may have occurred during the
isolation process. During our study, ethyl (p-hydroxybenzoyl)-
C
DOI: 10.1021/acs.jnatprod.7b01038
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(apparent d, 1.7H, H-8‴; partial deuterium exchange
observed). These data were consistent with the proposed
benzoyl acetate group (Figure 6). In addition, respective
Figure 5. Selected key (a) COSY and (b) HMBC correlations for
compound 5.
the β-glucose at δH 4.61 (dd, J = 11.8, 2 Hz, 1H, H-6′) and the
ester moiety at δC 165.93 (C-1″) supported the ester
connection to the arbutin portion. The ester resonance at δC
165.90 (C-6″) featured HMBC correlations with the methylene
protons at δH 4.31 (t, J = 6.4 Hz, 2H, H-4″), 2.72 (t, J = 6.4 Hz,
2H, H-3″), and 2.61 (t, J = 6.4 Hz, 2H, H-8″), which
pinpointed the location of the ester group between both 2-
methylene-4-hydroxybutanoic acid chain portions. Further-
more, HMBC correlations were observed between the
methylene signals at δH 6.29 (s, 1H, H-5a″) and 6.16 (s, 1H,
H-5b″) and the ester resonance at δC 165.90 (C-6″). In
addition, HMBC correlations were also evident between the
methylene signals at δH 5.79 (d, J = 0.78, 1H, H-10a″) and 5.66
(d, J = 0.9 Hz, 1H, H-10b″) and the ester resonance at δC
165.93 (C-1″). These data provided support for the proposed
connections in compound 5. The HMBC spectroscopic data
also indicated correlations between the ester at δC 167.6 (C
O, C-9‴) and both the methylene resonances at δH 4.24−4.27
(m, 2H, H-9″) and the methylene signal at δH 4.00 (s, 1.8H, H-
8‴). This was consistent with an ester linkage between the
benzoyl acetate and the 2-methylene-4-hydroxybutanoic acid
portion. In addition, the HMBC spectrum contained strong
cross-peaks between the keto carbonyl at δC 190.7 (C-7‴) and
methine protons at δH 7.90 (d, J = 7 Hz, 2H, H-2‴, H-6‴) and
methylene protons at δH 4.00 (s, 1.8H, H-8‴), which was
consistent with the presence of the benzoyl acetate moiety.
This was also observed in compound 1.
Figure 6. Selected key (a) COSY and (b) HMBC correlations for
compound 6.
HMBC correlations between the ester carbonyl signal at δC
165.9 (C-9‴) and the methylene resonance at δH 3.99
(apparent d, 1.7H, H-8‴) and the methylene peaks at 4.23−
4.27 (m, 2H, H-4″) and 2.65 (t, J = 6.4 Hz, 2H, H-3″)
supported the connection between the benzoyl acetate and 2-
methylene-4-hydroxybutanoyl portions. A linkage between the
arbutin and 2-methylene-4-hydroxybutanoyl portions was
supported by HMBC correlations between the methylene
resonances at δH 6.22 (d, J = 0.6 Hz, 1H, H-5a″) and 5.71 (d, J
= 1.2 Hz, 1H, H-5b″) and the ester carbonyl signal δC 167.6
(C-1″). Furthermore, HMBC correlations between the
anomeric proton δH 4.83 (d, J = 8.3 Hz, 1H, H-1′) and the
oxygenated tertiary carbon resonance at δC 151.1 (C-1)
indicated the connection between the β-glucose and hydro-
quinone moieties.
Notably, arbutin derivatives 5 and 6, which feature both
benzoyl acetate and 2-methylene-4-hydroxybutanoyl portions,
represent hybrid ester analogues of compounds 1 and 2. The
phytochemical composition of the B. montana flowers was
considerably different from that of the aforementioned leaf
material. This was apparent when the 1H NMR spectra of their
respective crude aqueous extracts were compared. Although
compound 1 was present in the leaf extract, it was not isolated
from the flowers. We suggest that compounds 5 and 6
represent authentic components of the plant, rather than
artifacts of extraction/isolation. This is because no ethyl ester
containing compounds were isolated as part of this study
despite the extraction solvent used (35% EtOH/H2O v/v).
Furthermore, 2-methylene-4-hydroxybutanoic acid was not
observed by NMR spectroscopy at any stage of the
extraction/isolation process.
1
On the basis of the H and 13C NMR and IR spectroscopic
data the molecular formula of C26H28O12 was proposed for
compound 6, which was supported by HRESIMS data (m/z
calculated [M + Na]+, 555.1473; found, 555.1469). The IR
spectrum featured bands at 1711 and 1601 cm−1 that were
consistent with keto carbonyl and ester carbonyl stretches,
respectively. The NMR spectroscopic data featured signals that
were consistent with the presence of an arbutin core, as
observed with various arbutin derivatives, including compounds
2 and 5.16,19 Moreover, the 1H, 13C, COSY, and HSQC
spectroscopic data were consistent with the presence of a 2-
methylene-4-hydroxybutanoyl moiety by analogy with com-
pounds 2 and 5.17
C. nitida. Phytochemical isolation studies of C. nitida have
yet to be reported. PHWE was performed on C. nitida leaves.
The aqueous PHWE mixture was extracted with EtOAc to
provide a crude extract. The remaining aqueous phase was also
evaporated to afford a crude extract. These respective crude
samples were purified separately by flash column chromatog-
raphy. In this way, new compound 7 (9.14% combined yield w/
w) was isolated from both fractions (Figure 7). On the basis of
The HMBC spectroscopic data revealed key correlations
between the keto carbonyl signal at δC 190.8 (C-7‴) and the
benzoyl acetate aromatic protons at δH 7.89 (d, J = 9 Hz, 2H,
H-2‴, H-6‴) and also the methylene resonance at δH 3.99
1
the H and 13C NMR and IR spectroscopic data the molecular
D
DOI: 10.1021/acs.jnatprod.7b01038
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Figure 7. Novel arbutin derivative 7, isolated from C. nitida leaves
(left). Selected key HMBC correlations for compound 7 (right).
formula of C21H22O11 was proposed for compound 7, which
was supported by HRESIMS data (m/z calculated [M + Na]+,
473.1054; found, 473.1060). The IR spectrum featured bands
at 1722 and 1668 cm−1 that were consistent with keto and ester
carbonyl stretches, respectively.
The 1H NMR spectrum revealed several similarities to
compound 1. Specifically, resonances at δH 6.95 (d, J = 8.9 Hz,
2H, H-3, H-5) and 6.76 (d, J = 8.6 Hz, 2H, H-2, H-6) were
consistent with the presence of an arbutin fragment and key β-
glucose resonances, including the anomeric proton at δH 5.05
(d, J = 7.6 Hz, 1H, H-1′) and methylene protons at δH 4.51
(dd, J = 11.8, 2.0 Hz, 1H, H-6a′) and 4.24 (dd, J = 12.4, 6.5 Hz,
1H, H-6b′). The presence of a second aromatic system was
supported by signals at δH 7.51 (d, J = 2.0 Hz, 1H, H-2″), 7.48
(dd, J = 8.4, 2.0 Hz, 1H, H-6″), and 6.91 (d, J = 8.4 Hz, 1H, H-
5″), which were consistent with a 3,4-dihydroxyphenyl
system.20 Both 1H NMR and HSQC spectroscopic data
revealed the presence of methylene protons at δH 4.00−4.02
(m, 1.6H, H-8″; partial deuterium exchange), which were
consistent with the reported resonances for benzoyl acetate
derivatives17,18 and compound 1. The 13C NMR spectroscopic
data featured resonances at δC 190.7 (CO, C-7″) and 167.7
(CO, C-9″), which were consistent with both keto and ester
groups in a benzoyl acetate portion.18 Key HMBC correlations
were observed between δC 190.7 (CO, C-7″) and aromatic
protons at δH 7.51 (d, J = 2.0 Hz, 1H, H-2″) and 7.48 (dd, J =
8.4, 2.0 Hz, 1H, H-6″) and the methylene resonances at δH
4.00−4.02 (m, 1.6H, H-8″). These data supported the presence
of a 3,4-dihydroxybenzoyl acetate portion (Figure 7). The
NMR spectroscopic data featured signals that were consistent
with the presence of an arbutin core, as observed with various
arbutin derivatives, including compounds 2, 5, and 6.16,19
P. gunnii. Few phytochemical studies of P. gunnii have been
reported, and these previous investigations utilized only
qualitative analytical methods (i.e., TLC and Mayer’s test).21
Other members of the Persoonia genus have been examined in
more detail. This includes the mainland Australian species
Persoonia elliptica, which contained phenolic compounds,22
anthocyanins,23 and arbutin and its derivatives,16 and Persoonia
salicina, which featured anthocyanins.24
Figure 8. Arbutin derivatives 8, 9, pyroside (10), and compound 11,
isolated from P. gunnii leaves (in addition to arbutin (4)).
Figure 9. ORTEP representations of tiglic acid esters 8 and 9.
Ellipsoids are drawn at the 50% probability level.
spectroscopic data for arbutin (4), pyroside (10),13,25 and
glucoside 1119 were consistent with reported data. In addition,
the X-ray crystal structure of arbutin (4) as the monohydrate
was obtained (Figure S3 in the Supporting Information); the
structure of the hemihydrate has previously been reported.26
Compound 11 has been previously isolated from the fruits of
the Australian endemic Persoonia linearis x pinifolia.16 It displays
potent antimicrobial activity against the bacteria Bacillus subtilis
(Gram positive) and Escherichia coli (Gram negative) and the
fungus Phytophthora cinnamomi.16 This type of biological
activity, particularly against Phytophthora cinnamomi, may
suggest a survival adaptation by Persoonia against the extremely
damaging fungal root rot.
1
On the basis of the H and 13C NMR and IR spectroscopic
data the molecular formula of C22H28O11 was proposed for
compound 8, which was supported by HRESIMS data (m/z
calculated [M + Na]+, 491.1524; found, 491.1531). The IR
spectrum featured bands at 1701 and 1658 cm−1, which were
consistent with two ester stretches. The NMR spectroscopic
data featured signals that were consistent with the presence of
an arbutin core as observed with various arbutin derivatives,
including compounds 2 and 5−7. The presence of two olefinic
protons at δH 6.85 (t, J = 5.6 Hz, 1H, H-3″) and 6.80 (t, J = 6.1
Hz, 1H, H-8″), two sets of methylene protons at δH 4.29 (d, J =
5.9 Hz, 2H, H-4″) and 4.88 (d, J = 6.1 Hz, 2H, H-9″), and two
methyl groups at δH 1.93 (s, 3H, H-10″) and 1.85 (s, 3H, H-5″)
was consistent with the presence of two (E)-4-hydroxy-2-
methyl-2-butenoic acid fragments.27
PHWE was performed on P. gunnii leaves. The aqueous
PHWE mixture was extracted with EtOAc to provide a crude
extract. The remaining aqueous phase was also evaporated to
afford a crude extract. These respective crude samples were
purified by flash column chromatography. In this way, new
tiglic acid esters 8 (0.45% yield w/w) and 9 (2.9% yield w/w)
and the known pyroside (10) (1.5% yield w/w), arbutin (4)
(6.2% yield w/w), and glucoside 11 (1.6% yield w/w) were
isolated (Figure 8). The respective structures of novel arbutin
derivatives 8 and 9 were secured by single-crystal X-ray
1
crystallography (Figure 9). In each case, the H and 13C NMR
The 13C NMR spectroscopic data revealed key resonances at
δC 167.4 (C-1″) and 167.1 (C-6″), which were consistent with
the presence of two ester functional groups in compound 8.
spectroscopic evidence and HRMS data were consistent with
1
the structures of compounds 8 and 9. The H and 13C NMR
E
DOI: 10.1021/acs.jnatprod.7b01038
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
The duplication of resonances at δC 127.3 (C-2″) and 130.3
(C-7″) and the presence of olefinic carbons at δC 141.7 (C-3″)
and 135.4 (C-8″), methylene signals at δC 58.4 (C-4″) and 61.0
(C-9″), and methyl resonances at δC 11.7 (C-5″) and 11.4 (C-
10″) were consistent with the presence of two (E)-4-hydroxy-2-
methyl-2-butenoic acid parts. Interestingly, the presence of an
ester linkage between the two (E)-4-hydroxy-2-methyl-2-
butenoic acid portions has not been reported. The COSY
spectroscopic data revealed correlations from the olefinic
proton at δH 6.85 (t, J = 5.6 Hz, H-3″) to both the methyl
group at δH 1.93 (H-10″) and the methylene signal at δH 4.29
(m, 2H, H-4″) in the first portion (Figure 10a). COSY
HMBC correlations between the olefinic signal at δC 135.4 (C-
8″) and both the methyl group at δH 1.93 (H-10″) and
methylene resonance at δH 4.88 (d, 2H, H-9″) were consistent
with a second (E)-4-hydroxy-2-methyl-2-butenoic acid frag-
ment.
1
On the basis of the spectroscopic H and 13C NMR and IR
data the molecular formula of C17H22O9 was proposed for
compound 9, which was supported by HRESIMS data (m/z
calculated [M + Na]+, 393.1156; found, 393.1163). An IR band
was present at 1697 cm−1 that was consistent with a hydrogen-
bonded ester stretch. The NMR spectroscopic data featured
signals that were consistent with the presence of an arbutin core
as observed with various arbutin derivatives, including
compounds 2 and 5−8. The NMR spectroscopic data were
consistent with the presence of an (E)-4-hydroxy-2-methyl-2-
butenoic acid moiety (by analogy with compound 8).27 The
13C NMR spectroscopic data featured an ester carbonyl signal
at δC 167.6 (CO, C-1″), quaternary carbon resonances at δC
127.4 (C-2″), 141.4 (C-3″), and 58.4 (C-4″), and a methyl
resonance at 11.3 (C-5″), which were all consistent with an
(E)-4-hydroxy-2-methyl-2-butenoic acid system.27 The COSY
spectrum revealed correlations between the olefinic proton
signal at δH 6.87 (td, J = 6, 1.3 Hz, 1H, H-3″) and both the
methylene resonances at δH 4.30−4.32 (m, 2H, H-4″) and the
methyl protons at δH 1.86 (d, J = 1 Hz, 3H, H-5″) (Figure 11a).
Figure 10. Selected key (a) COSY and (b) HMBC correlations for
compound 8.
correlations were also observed between the olefinic proton at
δH 6.80 (t, J = 6.1 Hz, 1H, H-8″) and both the methyl group at
δH 1.93 (H-10″) and methylene signal at δH 4.88 (d, J = 6.1 Hz,
H-9″). These data are consistent with the presence of two (E)-
4-hydroxy-2-methyl-2-butenoic acid components.
Figure 11. Selected key (a) COSY and (b) HMBC correlations for
compound 9.
The HMBC correlations revealed the key linkages in the
structure of compound 8 (Figure 10b). Specifically, HMBC
correlations between the β-D-glucose methylene proton
resonances at δH 4.56 (dd, J = 11.8, 2 Hz, 1H, H-6a′) and
4.26−4.30 (m, 1H, H-6b′) and the ester carbonyl peak at δC
167.4 (C-1″) supported the ester connection to the arbutin
core. HMBC correlations between the methyl signal at δH 1.85
and the ester carbonyl at δC 167.4 (C-1″) supported the ester
linkage between the arbutin and the first tigloyl chain (C-1″−
C-6″). HMBC correlations in the (E)-4-hydroxy-2-methyl-2-
butenoic acid portion included correlations between the
quaternary carbon at δC 127.3 (C-2″) and the methylene
signal at δH 4.27−4.30 (m, 2H, H-4″) and methyl resonances at
δH 1.85 (C-5″). HMBC correlations were evident between the
methyl group at δH 1.85 (C-5″) and the olefinic carbon at δC
141.7 (C-3″). In addition, HMBC correlations were observed
between the methylene carbon resonance at δC 58.4 (C-4″) and
the olefinic proton at δH 6.85 (H-3″). Observed HMBC
correlations in the second (E)-4-hydroxy-2-methyl-2-butenoic
acid portion (C-6″ to C-10″) included those between the ester
carbonyl at δC 167.1 (C-6″) and the methyl group at δH 1.93
(H-10″), the olefinic proton at δH 6.80 (H-8″), and the
methylene signal at δH 4.88 (d, H-9″). In addition, HMBC
correlations between the quaternary carbon resonance at 130.3
(C-7″) and the methyl group at δH 1.93 (H-10″) and
methylene protons at δH 4.88 (d, 2H, H-9″) were obsesrved.
These data further supported the presence of an (E)-4-hydroxy-
2-methyl-2-butenoic acid portion. Key HMBC correlations
were found between the ester carbonyl resonance at δC 167.6
(CO, C-1″) and the β-D-glucose methylene protons at δH
4.56 (dd, J = 11.8, 2 Hz, 1H, H-6a′) and 4.25−4.30 (d, 1H, H-
6b′), as well as with both the methyl protons at δH 1.86 (d, J =
1 Hz, 3H, H-5″) and the olefinic proton at δH 6.87 (td, J = 6,
1.3 Hz, 1H, H-3″) (Figure 11b). These data provided support
for the presence of the ester linkage between the arbutin and
(E)-4-hydroxy-2-methyl-2-butene systems (Figure 11b). In
addition, the quaternary carbon resonance at δC 127.4 (C-2″)
displayed HMBC correlations with methylene protons at δH
4.30−4.32 (m, 2H, H-4″) and methyl protons at δH 1.86 (d, J =
1 Hz, 3H, H-5″).
Notably, both compounds 8 and 9 represent the first
examples of glucoside-containing natural products featuring
(E)-4-hydroxy-2-methyl-2-butenoic acid ester moieties that
have been isolated. Previous reports of (E)-4-hydroxy-2-
methyl-2-butenoate ester natural products are limited and
include a variety of sesquiterpenoids isolated from the
Chloranthus genus.28
Finally, we sought to provide further evidence in support of
the isolated novel arbutin derivatives 1 and 7 that contained
benzoyl acetate moieties. Compounds 1 and 7 were both
independently reacted with NaBH4. Reduction of glucoside 1
with NaBH4 in EtOH afforded diol 12 (Figure 12). This was
F
DOI: 10.1021/acs.jnatprod.7b01038
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Chemical shifts were recorded in ppm. Spectra were calibrated by
assignment of the residual solvent peak to δH 4.79 for D2O, δH 3.31
and δC 49.00 for methanol-d4, and δH 2.05 and δC 29.84 for acetone-d6.
Coupling constants (J) were recorded in Hz. HRESIMS analyses were
conducted on a Thermo-Scientific LTQ-Orbitrap operating in positive
ionization mode. MS samples were prepared in MeOH.
Figure 12. Compounds 12 and 13, formed from the respective
reductions of glucosides 1 and 7.
X-ray crystallographic data for the structure determination of
compounds 1, 2, and 8 were recorded on either the MX1 or MX2
beamlines at the Australian Synchrotron (Mo Kα radiation),30 while
the data for compounds 4 and 9 were recorded on a Bruker AXS D8
Quest instrument (Cu Kα radiation). All data sets were collected on
single crystals mounted on nylon loops with viscous immersion oil and
placed into a chilled nitrogen stream. The structure of compound 2
was solved by intrinsic phasing methods with SHELXT,31 and the
structures of compounds 4, 8, and 9 were solved by charge-flipping
methods with SUPERFLIP.32 All structures were refined using full-
matrix least squares on F2 with SHELXL31 within the OLEX2 suite.33
Non-hydrogen atoms were refined with anisotropic displacement
parameters. Hydrogen atoms were visible in the diffraction map but
were included at calculated positions riding on the atoms to which
they are attached. Refinement of the Flack parameter34 and
comparisons with the known configuration of the glucose component
derivatives were used to assign absolute configuration of the
stereogenic carbon atoms. All structures were examined by the
likelihood method,35 with analysis of the Bayesian statistics of the
Bijvoet pairs performed in PLATON,36 which agreed with the
assignment of the absolute structure. Molecular graphics were
produced with OLEX2.33 Crystallographic data have been deposited
with the Cambridge Crystallographic Data Centre (file numbers
1581874−1581877 and 1587038).
TLC was performed using Merck silica gel 60-F254 plates.
Developed chromatograms were visualized by UV absorbance (254
nm) or through application of heat to a plate stained with cerium
molybdate (Ce(NH4)2(NO3)6, (NH4)6Mo7O24·4H2O, H2SO4, H2O).
Manual flash column chromatography was performed with flash grade
silica gel (60 μm) and the indicated eluent in accordance with standard
techniques.37 Automated flash column chromatography was performed
with a Grace Reveleris X2 flash column chromatography system with
40 μm silica gel cartridges and the indicated eluent gradient. Plant
material was ground using a Sunbeam spice/coffee bean grinder.
PHWE was undertaken employing a Breville espresso machine, Model
800ES.
Plant Material. Fresh leaves were collected from a number of
healthy mature specimens of Bellendena montana at the summit of Mt.
Wellington in December 2016 (42.8963° S, 147.2342° E). Fresh
flowers were collected from healthy mature specimens of B. montana at
the summit of Mt. Wellington in January 2017 (42.8963° S, 147.2367°
E). Fresh leaves were collected from a number of healthy mature
specimens of Cenarrhenes nitida at Mt. Wedge, South-West National
Park, Tasmania, in November 2015 (42.8417° S, 146.2883° E). Fresh
leaves were collected from a number of healthy mature specimens of
Persoonia gunnii at Mt. Wedge, South-West National Park, Tasmania,
in November 2015 (42.8424° S, 146.2898° E). Voucher specimens of
B. montana (voucher number HO589254), C. nitida (voucher number
HO589253), and P. gunnii (voucher number HO589257) were
provided to the Tasmanian Herbarium, Tasmanian Museum and Art
Gallery, and verified by Dr. Miguel de Salas. All leaf material and
flowers, obtained as described above, were dried in an oven maintained
at 40 °C for 24 h prior to undertaking extraction studies.
PHWE of B. montana Leaf Material. B. montana leaves (9.4 g)
were coarsely ground in a spice grinder, mixed with sand (2 g), placed
into the portafilter (sample compartment) of an unmodified espresso
machine, and extracted using 35% v/v EtOH/H2O (200 mL of a hot
solution). This was repeated a further two times with equal portions of
ground leaf material (28.2 g in total). The ensuing extracts were
combined and concentrated under reduced pressure on a rotary
evaporator to remove EtOH (45 °C bath temperature). NaCl (15 mL
of a saturated aqueous solution) was added to this aqueous extract, and
the mixture was extracted with EtOAc (3 × 60 mL). The combined
organic extracts were dried (Na2SO4), filtered, and concentrated under
supported by 1H and 13C NMR spectroscopy and MS (m/z [M
+ H]+ found, 168), which was consistent with the reduction of
both keto and ester carbonyl groups. The reduction of a
benzoyl acetate has been reported under similar conditions.29
Traces of arbutin (4) were also isolated from this reaction.
Reduction of compound 7 with NaBH4 in EtOH afforded the
β-hydroxy ester 13, as supported by 1H and 13C NMR
spectroscopy and HRESIMS (m/z calculated [M + Na]+,
249.0747; found, 249.0752). Hence, the formation of reduction
products 12 and 13 supported the presence of benzoyl acetate
moieties in novel glucosides 1 and 7.
In conclusion, extensive phytochemical studies of the
paleoendemic Tasmanian Proteaceae species B. montana, C.
nitida, and P. gunnii were conducted employing PHWE. As part
of these studies, six novel glucosides were isolated, including
rare examples of glycoside-containing natural products
featuring tiglic acid ester moieties. More specifically, com-
pounds 1, 2, and 4−6 were isolated from B. montana (∼85
million year old genus), the related glucoside 7 was isolated
from C. nitida (∼30 million year old genus), and germane
compounds 4 and 8−11 were isolated from P. gunnii (∼20
million year old genus). Notably, glucoside 2 has been isolated
from Toronia toru (Proteaceae native to New Zealand)19 and
compound 11 has been isolated from Persoonia linearis x
pinifolia (Proteaceae native to southeastern Australia).16 We
suggest that these polar natural products may represent novel
phytochemical markers in ancient Proteaceae. However, greater
sampling of these and other Proteaceae species is required
before any firm conclusions regarding the implications of the
findings can be drawn. For example, it is possible that (i) all
Proteaceae may feature this phytochemical profile in various
forms, (ii) convergent evolution is responsible (i.e., phyto-
chemistry has evolved independently in the Persoonioid/
Bellendena clade containing B. montana and P. gunnii and in the
Proteoideae/Symphionematoideae/Grevilleoideae clade con-
taining C. nitida), and (iii) the phytochemistry of these plants
is ancestral and has been lost in some, but not all, Proteaceae.
Thus, our future research will focus on greater sampling of
these and other ancient Proteaceae.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Unless otherwise specified,
reactions were performed under an air atmosphere. Solvents were
analytical grade and were purified by standard laboratory procedures.
Reagents were purchased from Sigma-Aldrich (Sydney, Australia), AK
Scientific, Combi-Blocks, and Oakwood and were used without
purification.
Polarimetry was performed with a Rudolph Research Analytical
Autopol III automatic polarimeter with a 0.5 dm cell. Infrared
spectrometry was performed on a Shimadzu FTIR 8400s spectrom-
eter, with samples prepared either as thin films on NaCl plates or using
an ATR attachment. NMR experiments were performed either on a
Bruker Avance III NMR spectrometer operating at 400 MHz (1H) or
100 MHz (13C) or on a Bruker Avance III NMR spectrometer
operating at 600 MHz (1H) or 150 MHz (13C). The deuterated
solvents used were D2O, methanol-d4, and acetone-d6 as specified.
G
DOI: 10.1021/acs.jnatprod.7b01038
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
reduced pressure to provide a crude brown solid (1.25 g; extract A).
The remaining aqueous phase was also concentrated under reduced
pressure (45 °C bath temperature) to provide a crude brown solid
(15.8 g; extract B).
Extract A. Acetone was added to crude extract A (1.25 g), obtained
as described immediately above. The mixture was adsorbed onto
silica/Celite (∼4/1 ratio) and subjected to automated flash
chromatography (24 g silica cartridge, 0 → 20% MeOH/CH2Cl2; 16
min) to provide new compound 1 (77 mg), compound 2 as a yellow
powder (190 mg),19 and an impure sample of compound 1 (243 mg).
This impure sample was adsorbed onto silica and subjected to flash
column chromatography (20% MeOH/CH2Cl2) to afford compound
1 as a colorless powder (162 mg).
98.8, 93.8, 88.9, 87.5, 76.6, 76.2, 73.4, 69.8, 65.7, 61.7 ppm; HRESIMS
m/z [M + Na]+ calcd for C25H26O15Na 589.1164, found 589.1162.
Arbutin (4): [α]22 −57 (c 1, MeOH), lit.38 [α]22 −55.8 (c 0.56,
D
D
MeOH); 1H NMR (600 MHz, methanol-d4) δ 6.98 (d, J = 9 Hz, 2H),
6.71 (d, J = 9 Hz, 2H), 4.74 (d, J = 7.4 Hz, 1H), 3.90 (d, J = 12 Hz,
1H), 3.71 (dd, J = 12.2, 4.3 Hz, 1H), 3.41−3.45 (m, 2H), 3.39−3.40
(m, 2H) ppm; 13C NMR (150 MHz, methanol-d4) δ 152.4, 151.0,
118.0, 115.2, 102.3, 76.7, 76.6, 73.6, 70.1, 61.2 ppm.
PHWE of B. montana Flowers. B. montana flowers (entire flower
inflorescence spikes; 10 g) were finely ground in a spice grinder, mixed
with sand (10 g), placed into the portafilter (sample compartment) of
an unmodified espresso machine, and extracted using 35% EtOH/65%
H2O v/v (200 mL of a hot solution). This was repeated a further two
times with equal portions of ground flower material (30 g in total).
The ensuing extracts were then combined and concentrated under
reduced pressure on a rotary evaporator to remove EtOH (45 °C bath
temperature). This aqueous mixture was extracted with EtOAc (3 × 60
mL). The combined organic extracts were dried (Na2SO4), filtered,
and concentrated under reduced pressure to provide a light yellow
solid (0.76 g; extract A). The remaining aqueous phase was also
concentrated under reduced pressure (45 °C bath temperature) to
provide a crude brown solid (14.2 g; extract B).
Extract A. Acetone was added to crude extract A (0.76 g), obtained
as described immediately above, and the mixture was adsorbed onto
silica/Celite (∼3/1 ratio) and then subjected to automated flash
chromatography (24 g silica cartridge, 0 → 25% MeOH/CH2Cl2; 15
min) to provide previously unreported compound 5 as a colorless
powder (82 mg, 0.27% yield w/w), compound 1 (95 mg, 0.32% yield
w/w), a fraction containing mixtures of compounds 1 and 2 (210 mg
in total; ∼1/1.3 ratio of 1 and 2), and an impure fraction (31 mg).
This fraction was further purified via a pipet column (20% MeOH/
80% CH2Cl2), to provide previously unreported compound 6 as a pale
yellow oil (11 mg, 0.03% yield w/w).
Extract B. MeOH/MeCN/H2O (20/79/1 v/v/v) was added to
extract B (15.8 g), obtained as described above. The mixture was
adsorbed onto silica/Celite (4:1 ratio) and fractionated through a plug
of silica with EtOAc (4 × 50 mL), 10% MeOH/90% EtOAc (2 × 50
mL), and 20% MeOH/80% EtOAc (1 × 100 mL) as eluents. The
fractions containing the major compounds (as judged by TLC analysis
and 1H NMR spectroscopic analysis) were combined and concen-
trated under reduced pressure to provide an orange-brown solid (4.08
g). The ensuing residue was adsorbed onto silica/Celite (∼1/3 ratio)
and subjected to automated flash column chromatography (40 g silica
cartridge, 0 → 25% MeOH/CH2Cl2; 22 min). This provided
compound 1 (229 mg), compound 2 (227 mg), compound 3 as a
pale yellow film (190 mg, 0.67% yield w/w), and impure fractions A
(750 mg) and B (406 mg). Fraction A was adsorbed onto silica/Celite
(∼3/1 ratio) and subjected to automated flash column chromatog-
raphy (24 g silica cartridge, 0 → 30% MeOH/CH2Cl2; 10 min) to
provide compound 1 (105 mg; total combined mass 573 mg, 2.0%
yield w/w) and compound 2 (483 mg). Fraction B was adsorbed onto
silica/Celite (∼3/1 ratio) and subjected to automated flash column
chromatography (24 g silica cartridge, 0 → 30% MeOH/CH2Cl2; 15
min) to provide compound 2 (72 mg; total combined mass 972 mg,
3.4% yield w/w) and arbutin (4) as a pale yellow oil (202 mg, 0.71%
yield w/w).13 Compound 1 was recrystallized from EtOH/H2O to
provide crystals suitable for X-ray crystallographic analysis. Compound
2 was recrystallized from MeCN/H2O to provide crystals suitable for
X-ray crystallographic analysis.
Extract B. MeOH/MeCN/H2O (20/79/1 v/v/v) was added to
crude extract B (14.2 g), obtained as described above. The mixture was
adsorbed onto Celite and fractionated through a plug of silica with
EtOAc (100 mL), 10% MeOH/90% EtOAc (100 mL), and 20%
MeOH/80% EtOAc (6 × 50 mL) as eluents. The fractions containing
the major compounds (as judged by TLC analysis and 1H NMR
spectroscopic analysis) were combined and concentrated under
reduced pressure to provide crude sample A (3.68 g). The plug itself
was then soaked in 30% MeOH/70% EtOAc (100 mL) for 48 h and
filtered, and the filtrate was concentrated under reduced pressure.
MeOH (30 mL) was added to the ensuing solids and the mixture
magnetically stirred. After 1 h the mixture was filtered and the filtrate
concentrated under reduced pressure. The combined organic fractions
were concentrated under reduced pressure to provide crude sample B
(3.93 g). Crude samples A and B were combined (7.61 g in total). The
ensuing solid was adsorbed onto silica/Celite (∼3/1 ratio) and
subjected to automated flash column chromatography (40 g silica
cartridge, 0 → 100% (1/40/59 H2O/MeOH/CH2Cl2)/CH2Cl2; 25
min) to provide a mixture of compounds 1 and 2 (422 mg in total;
∼1/1.3 ratio of 1 and 2) and sucrose as a beige powder (374 mg,
1.25% yield w/w).
Compound 1: [α]22 −49 (c 1, MeOH); IR (NaCl) 1722, 1668,
D
1607, 1584, 1513, 1400, 1349, 1331, 1319, 1300, 1269, 1220, 1202,
1
1163, 1154, 1072, 1061, 1046, 851, 829, 774 cm−1; H NMR (400
MHz, acetone-d6) δ 7.91 (d, J = 8.4 Hz, 2H), 6.93−6.97 (m, 4H), 6.76
(d, J = 9 Hz, 2H), 4.76−4.79 (m, 1H), 4.55 (d, J = 10.9 Hz, 1H), 4.25
(dd, J = 11.8, 6.7 Hz, 1H), 4.05 (s, 1.4H; partial deuterium exchange),
3.65−3.71 (m, 1H), 3.47−3.53 (m, 1H), 3.40−3.45 (m, 2H) ppm; 13C
NMR (150 MHz, acetone-d6) δ 190.7, 167.7, 162.3, 152.6, 151.2,
131.1, 128.5, 118.2, 115.6, 115.4, 102.3, 76.9, 74.0, 73.8, 70.4, 64.2,
45.1 ppm; HRESIMS m/z [M + Na]+ calcd for C21H22O10Na
457.1105, found 457.1112.
Compound 2: mp 148−149 °C; [α]22 −41 (c 1, MeOH), lit.19
D
[α]19D +18 (c 2.5, MeOH); IR (NaCl) 3282, 1700, 1628, 1510, 1448,
1213, 1164, 1072, 1018, 832, 776 cm−1; 1H NMR (600 MHz, D2O) δ
6.94 (d, J = 8.9 Hz, 2H), 6.78 (d, J = 8.9 Hz, 2H), 6.22 (s, 1H), 5.74
(s, 1H), 4.91 (d, J = 7.8 Hz, 1H), 4.50 (dd, J = 11.8, 2.2 Hz, 1H), 4.30
(dd, J = 12.4, 7.4 Hz, 1H), 3.75−3.78 (m, 1H), 3.62 (t, J = 6.3 Hz,
2H), 3.57−3.60 (m, 1H), 3.52−3.55 (m, 1H), 3.49 (t, J = 9.2 Hz, 1H),
2.47 (t, J = 6.3 Hz, 2H) ppm; 13C NMR (150 MHz, D2O) δ 168.4,
151.2, 150.2, 136.1, 128.9, 118.3, 116.1, 101.0, 75.5, 73.6, 72.9, 70.0,
63.7, 60.0, 34.0 ppm; HRESIMS m/z [M + Na]+ calcd for
C17H22O9Na 393.1156, found 393.1160.
Compound 5: [α]22 −38 (c 1.7, MeOH); IR (NaCl) 2359, 1711,
D
1601, 1510, 1324, 1282, 1214, 1169, 1072 cm−1; 1H NMR (600 MHz,
acetone-d6) δ 7.90 (d, J = 8.7 Hz, 2H), 6.94−6.97 (m, 4H), 6.74 (d, J
= 8.8 Hz, 2H), 6.29 (s, 1H), 6.16 (s, 1H), 5.79 (d, J = 0.8 Hz, 1H),
5.66 (d, J = 0.9 Hz, 1H), 4.83 (d, J = 8.3 Hz, 1H), 4.61 (m, 3H), 4.31
(t, J = 6.4 Hz, 2H), 4.24−4.27 (m, 3H), 4.00 (s, 1.8H, deuterium
exchange observed), 3.77 (m, 1H), 3.55 (t, J = 8.9 Hz, 1H), 3.46−3.48
(m, 2H), 2.72 (t, J = 6.4 Hz, 2H), 2.61 (t, J = 6.4 Hz, 2H) ppm; 13C
NMR (150 MHz, acetone-d6) δ 190.7, 167.6, 165.93 165.90, 162.4,
152.6, 151.1, 137.0, 136.6, 131.1, 128.5, 126.92, 126.90, 118.1, 115.5,
115.3, 102.1, 77.0, 74.0, 73.8, 70.6, 64.2, 62.90, 62.85, 45.1, 31.2, 31.1
ppm; HRESIMS m/z [M + Na]+ calcd for C31H34O14Na 653.1841,
found 653.1848.
Compound 3: [α]20D −105 (c 0.9, MeOH), lit.12 [α]23D −82 (c 1.0,
MeOH); 1H NMR (600 MHz, acetone-d6) δ 12.54 (s, 1H), 7.71 (d, J
= 2 Hz, 1H), 7.56 (dd, J = 8.4, 2 Hz, 1H), 7.01 (d, J = 8 Hz, 1H), 6.51
(d, J = 2 Hz, 1H), 6.29 (d, J = 2 Hz, 1H), 5.77 (s, 1H), 4.53 (d, J = 7.8
Hz, 1H), 4.48 (d, J = 2 Hz, 1H), 4.15 (overlapped dd, J = 4.5, 2 Hz,
1H), 4.00 (q, J = 9.6, 4.7 Hz, 1H), 3.84 (dd, J = 11.3, 5 Hz, 1H), 3.50−
3.58 (m, 3.6 H), 3.40 (t, J = 8.6 Hz, 1H), 3.21−3.28 (m, 2.4 H) ppm;
13C NMR (150 MHz, acetone-d6) δ 178.7, 164.3, 162.1, 157.3, 157.1,
148.4, 145.0, 133.6, 121.9, 121.7, 115.8, 115.5, 106.5, 104.6, 103.2,
Compound 6: [α]22 −46 (c 0.4, MeOH); IR (NaCl) 1711, 1654,
D
1601, 1583, 1509, 1282, 1214, 1170, 1073 cm−1; 1H NMR (600 MHz,
acetone-d6) δ 7.89 (d, J = 9 Hz, 2H), 6.92−6.95 (m, 4H), 6.72 (d, J =
H
DOI: 10.1021/acs.jnatprod.7b01038
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
9 Hz, 2H), 6.22 (d, J = 0.6 Hz, 1H), 5.71 (d, J = 1.2 Hz, 1H), 4.80 (d, J
= 7.7 Hz, 1H), 4.58 (dd, J = 11.6, 2 Hz, 1H), 4.23−4.27 (m, 3H), 3.99
(apparent d, 1.7H, deuterium exchange observed), 3.75 (m, 1H) 3.53
(t, J = 8.8 Hz, 1H), 3.42−3.47 (m, 2H), 2.65 (t, J = 6.4 Hz, 2H) ppm;
13C NMR (150 MHz, acetone-d6) δ 190.8, 167.6, 165.9, 162.4, 152.6,
151.1, 136.7, 131.1, 128.5, 126.9, 118.1, 115.5, 115.3, 102.2, 77.0, 74.0,
73.8, 70.6, 64.2, 62.9, 45.2, 31.1 ppm; HRESIMS m/z [M + Na]+ calcd
for C26H28O12Na 555.1473, found 555.1469.
PHWE of Cenarrhenes nitida. C. nitida leaves (15 g) were finely
ground in a spice grinder, mixed with sand (2 g), placed into the
portafilter (sample compartment) of an unmodified espresso machine,
and extracted using 35% EtOH/65% H2O v/v (200 mL of a hot
solution). This was repeated one more time with equal portions of
ground leaf material (30 g in total). The ensuing extracts were then
combined and concentrated under reduced pressure on a rotary
evaporator to remove EtOH (45 °C bath temperature). NaCl (40 mL
of a saturated aqueous solution) was added to this aqueous extract, and
the ensuing mixture was extracted with EtOAc (3 × 60 mL). The
combined organic extracts were dried (Na2SO4), filtered, and
concentrated under reduced pressure to provide a crude pale green
solid (1.63 g; extract A). The remaining aqueous phase was also
concentrated under reduced pressure (45 °C bath temperature) to
provide a crude pale brown solid (19.0 g; extract B).
Extract A. MeOH was added to crude extract A (1.63 g), obtained
as described immediately above. The mixture was adsorbed onto
silica/Celite (∼4/1 ratio) and then subjected to automated flash
chromatography (12 g silica cartridge, 70 → 100% EtOAc/hexanes
and then 0 → 10% MeOH/EtOAc; 17 min) to provide previously
unreported compound 7 (922 mg) and an impure sample of
compound 7 (241 mg). The latter was adsorbed onto silica and
purified by flash column chromatography (20% MeOH/80% CH2Cl2)
to provide previously unreported compound 7 as a colorless powder
(151 mg).
reduced pressure to provide a crude pale green solid (1.31 g; extract
A). The remaining aqueous phase was also concentrated under
reduced pressure (45 °C bath temperature) to provide a crude pale
brown solid (11.8 g; extract B).
Extract A. Extract A, obtained as described above, was washed with
CH2Cl2 (15 mL). The ensuing green residue (1.09 g) was adsorbed
onto silica and purified by automated flash column chromatography
(12 g silica cartridge, 0 → 10% MeOH/CH2Cl2; 10 min) to afford
fractions A (322 mg) and B (102 mg). Fraction A was adsorbed onto
silica and purified by flash column chromatography (15% MeOH/85%
CH2Cl2) to provide new compound 8 (99 mg). Fraction B was
subjected to flash column chromatography (90 → 100% EtOAc/
hexanes then 5% MeOH/95% CH2Cl2) to provide compound 8 as a
white powder (36 mg; total combined mass 135 mg, 0.45% yield w/w)
and previously unreported compound 9 (27 mg).
Extract B. A sample of extract B (2.36 g), obtained as described
above, was adsorbed onto silica and fractionated through a plug of
silica with EtOAc (5 × 25 mL), 5% MeOH/95% EtOAc (5 × 25 mL),
and 10% MeOH/90% EtOAc (3 × 25 mL) as eluents. The fractions
1
containing the major compound (as judged by TLC analysis and H
NMR spectroscopic analysis) were combined and concentrated under
reduced pressure to provide a pale brown solid (1.27 g). The ensuing
crude solid was then adsorbed onto silica and subjected to automated
flash column chromatography (12 g silica cartridge, 0 → 10% MeOH/
EtOAc; 12 min) to afford pyroside (10) (92 mg; extrapolated 1.5%
yield w/w) as a colorless powder, compound 9 (179 mg; extrapolated
2.9% yield w/w) as colorless needles, arbutin (4) (372 mg;
extrapolated 6.2% yield w/w) as a pale yellow powder, and compound
11 (95 mg; extrapolated 1.6% yield w/w) as a pale yellow powder.19
Compounds 8 and 9 were both recrystallized from methanol-d4 to
provide crystals suitable for X-ray crystallographic analysis. Compound
4 was recrystallized from MeOH to provide crystals suitable for X-ray
crystallographic analysis.
Extract B. MeOH/H2O (95/5 v/v) was added to extract B (19.0 g),
obtained as described above, and the mixture was adsorbed onto silica/
Celite (4/1 ratio) and fractionated through a plug of silica with EtOAc
(2 × 50 mL), 5% MeOH/95% EtOAc (3 × 50 mL), 10% MeOH/90%
EtOAc (4 × 50 mL), and 20% MeOH/80% EtOAc (2 × 50 mL) as
eluents. The fractions containing the major compound (as judged by
Compound 8: mp 177−178 °C; [α]22 −30 (c 1.3, MeOH); IR
D
(NaCl) 1701, 1658, 1508, 1460, 1452, 1377, 1354, 1321, 1282, 1267,
1
1205, 1136, 1095, 1064, 1051, 1022, 1004, 962, 827, 773 cm−1; H
NMR (600 MHz, methanol-d4) δ 6.94 (d, J = 8.6 Hz, 2H), 6.85 (t, J =
5.6 Hz, 1H), 6.80 (t, J = 6.1 Hz, 1H), 6.69 (d, J = 8.6 Hz, 2H), 4.88 (d,
J = 6.1 Hz, 3H, overlapped with H2O signal), 4.74 (d, J = 7 Hz, 1H),
4.56 (d, J = 11.8 Hz, 1H), 4.26−4.31 (m, 3H), 3.66 (t, J = 8.7 Hz, 1H),
3.46 (t, J = 8.2 Hz, 1H), 3.38−3.40 (m, 2H), 1.93 (s, 3H), 1.85 (s, 3H)
ppm; 13C NMR (150 MHz, methanol-d4) δ 167.4, 167.1, 152.5, 150.8,
141.7, 135.4, 130.3, 127.3, 118.1, 115.3, 102.1, 76.5, 73.9, 73.5, 70.6,
64.1, 61.0, 58.4, 11.7, 11.4 ppm; HRESIMS m/z [M + Na]+ calcd for
C22H28O11Na 491.1524, found 491.1531.
1
TLC analysis and H NMR spectroscopic analysis) were combined
and concentrated under reduced pressure to provide a yellow solid
(4.86 g). The ensuing residue was adsorbed onto silica and subjected
to automated flash column chromatography (24 g silica cartridge, 0 →
20% MeOH/EtOAc, 12 min) to provide a yellow solid (2.20 g), which
was subjected to automated flash column chromatography (24 g silica
cartridge, 0 → 20% MeOH/EtOAc; 9 min) to provide compound 7
(1.67 g; total combined mass 2.74 g, 9.14% yield w/w).
Compound 9: mp 196−197 °C; [α]22 −23 (c 1, MeOH); IR
D
Compound 7: [α]22 −78 (c 0.6, MeOH); IR (ATR) 1728, 1697,
(NaCl) 1697, 1508, 1444, 1375, 1336, 1325, 1211, 1128, 1070, 1041,
1014, 829, 777 cm−1; 1H NMR (600 MHz, methanol-d4) δ 6.94 (d, J =
9 Hz, 2H), 6.87 (td, J = 6, 1.3 Hz, 1H), 6.70 (d, J = 9 Hz, 2H), 4.72 (d,
J = 7.4 Hz, 1H), 4.53 (dd, J = 11.8, 2 Hz, 1H), 4.25−4.32 (m, 3H),
3.62−3.67 (m, 1H), 3.43−3.49 (m, 2H), 3.36−3.41 (m, 1H), 1.86 (d, J
= 1 Hz, 3H) ppm; 13C NMR (150 MHz, methanol-d4) δ 167.6, 152.5,
150.9, 141.4, 127.4, 118.1, 115.2, 102.2, 76.5, 74.0, 73.5, 70.5, 63.8,
58.4, 11.3 ppm; HRESIMS m/z [M + Na]+ calcd for C17H22O9Na
393.1156, found 393.1163.
D
1662, 1593, 1508, 1440, 1292, 1207, 1172, 1153, 1124, 1085, 1024,
831, 785 cm−1; 1H NMR (600 MHz, acetone-d6) δ 7.51 (d, J = 2.0 Hz,
1H), 7.48 (dd, J = 8.4, 2.0 Hz, 1H), 6.95 (d, J = 8.9 Hz, 2H), 6.91 (d, J
= 8.4 Hz, 1H), 6.76 (d, J = 8.6 Hz, 2H), 5.05 (d, J = 7.6 Hz, 1H), 4.51
(dd, J = 11.8, 2.0 Hz, 1H), 4.24 (dd, J = 12.4, 6.5 Hz, 1H), 4.18 (t, J =
3.0 Hz, 1H), 4.00−4.02 (m, 1.6H, partial deuterium exchange), 3.99−
4.00 (m, 1H), 3.56−3.59 (m, 2H) ppm; 13C NMR (150 MHz,
acetone-d6) δ 190.7, 167.7, 152.5, 151.3, 150.8, 145.0, 129.0, 122.5,
118.1, 115.6, 115.0, 114.9, 100.3, 72.0, 71.4, 70.7, 67.7, 64.6, 45.2 ppm;
HRESIMS m/z [M + Na]+ calcd for C21H22O11Na 473.1054, found
473.1060.
Pyroside (10): [α]22 −46 (c 2.6, MeOH); IR (NaCl) 3370, 1713,
D
1511, 1447, 1368, 1252, 1215, 1074, 1042, 1021 cm−1; 1H NMR (600
MHz, methanol-d4) δ 6.95 (d, J = 9 Hz, 2H), 6.71 (d, J = 9 Hz, 2H),
4.72 (d, J = 7.3 Hz, 1H), 4.40 (dd, J = 11.8, 4.4 Hz, 1H), 4.26 (dd, J =
11.8, 6.3 Hz, 1H), 3.56−3.60 (m, 1H), 3.41−3.45 (m, 1H), 3.35−3.39
(m, 1H), 2.07 (s, 3H) δ ppm; 13C NMR (150 MHz, methanol-d4) δ
171.3, 152.6, 150.8, 118.2, 115.2, 102.2, 76.5, 73.9, 73.5, 70.3, 63.3,
19.3 ppm.
PHWE of Persoonia gunnii. P. gunnii leaves (10 g) were finely
ground in a spice grinder, mixed with sand (5 g), placed into the
portafilter (sample compartment) of an unmodified espresso machine,
and extracted using 35% EtOH/65% H2O v/v (200 mL of a hot
solution). This was repeated a further two times with equal portions of
ground leaf material (30 g in total). The ensuing extracts were then
combined and concentrated under reduced pressure on a rotary
evaporator to remove EtOH (45 °C bath temperature). NaCl (20 mL
of a saturated aqueous solution) was added to this aqueous extract, and
this mixture was extracted with EtOAc (3 × 60 mL). The combined
organic extracts were dried (Na2SO4), filtered, and concentrated under
Compound 11: [α]22 −44 (c 2.6, MeOH), lit.19 [α]D −48 (c 2.5,
D
MeOH); IR (NaCl) 3344, 2359, 1707, 1510, 1457, 1363, 1213, 1072,
1043, 1021, 777 cm−1; 1H NMR (methanol-d4, 600 MHz) δ 6.94 (d, J
= 9.6 Hz, 2H), 6.69 (d, J = 9.6 Hz, 2H), 6.38 (br s, 1H), 6.04 (br s,
1H), 4.74 (d, J = 7.0 Hz, 1H), 4.54−4.60 (m, 2H), 4.28 (dd, J = 12.7,
7.0 Hz, 1H), 3.82−3.95 (m, 1H), 3.63−3.72 (m, 2H), 3.36−3.47 (m,
I
DOI: 10.1021/acs.jnatprod.7b01038
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
5H) ppm; 13C NMR (150 MHz, D2O) δ 168.3, 151.3, 150.4, 139.1,
127.8, 118.4, 116.2, 101.3, 76.1, 75.6, 73.0, 70.4, 69.5, 64.3, 60.6 ppm.
1-(4-Hydroxyphenyl)-1,3-propanediol (12). NaBH4 (8 mg, 0.21
mmol) was added to a magnetically stirred solution of compound 1
(16 mg, 37 μmol) in EtOH (5 mL) maintained at 0 °C. After 10 min,
the reaction mixture was warmed to room temperature. After 7 h, the
mixture was concentrated under reduced pressure to provide a yellow
solid (39 mg), which was adsorbed onto silica and purified by flash
column chromatography (10% MeOH/90% CH2Cl2) to provide title
compound 12 (2 mg) as a clear, colorless oil and arbutin (1 mg) as a
for preparation of voucher specimens. The authors also thank
the Tasmanian Government Department of Primary Industries,
Parks, Water and Environment (DPIPWE) as well as the
Wellington Park Management Trust for the provision of
collection flora permits. B.J.D. thanks the Australian Govern-
ment for a Research Training Program Scholarship. N.L.K.’s
contribution to this research was supported under the
Australian Research Council’s Discovery Early Career Research
Award funding scheme (project number DE150100263). Part
of this research was undertaken on the MX1 and MX2
beamlines at the Australian Synchrotron, Victoria, Australia.
1
clear, colorless oil. H NMR (600 MHz, methanol-d4): δ 7.19 (d, J =
8.1 Hz, 2H), 6.76 (d, J = 8.1 Hz, 2H), 4.71−4.73 (m, 1H), 3.64−3.69
(m, 1H), 3.57−3.61 (m, 1H), 1.96−2.02 (m, 1H), 1.83−1.88 (m, 1H)
ppm. 13C NMR (150 MHz, methanol-d4): δ 156.4, 135.7, 128.6, 114.6,
70.8, 58.8, 41.2 ppm. ESIMS: m/z 168 {[M + H]+}.
REFERENCES
■
(1) Weston, P. H. In The Families and Genera of Vascular Plants;
Kubitski, K., Ed.; Springer-Verlag: Berlin, 1990; Vol. 9, pp 364−404.
(2) (a) Jordan, G. J.; Dillon, R. A.; Weston, P. H. Am. J. Bot. 2005, 92,
789−796. (b) Jordan, G. J.; Weston, P. H.; Carpenter, R. J.; Dillon, R.
A.; Brodribb, T. J. Am. J. Bot. 2008, 95, 521−530. (c) Mast, A. R.;
Willis, C. L.; Jones, E. H.; Downs, K. M.; Weston, P. H. Am. J. Bot.
2008, 95, 843−870. (d) Sauquet, H.; Weston, P. H.; Anderson, C. L.;
Barker, N. P.; Cantrill, D. J.; Mast, A. R.; Savolainen, V. Proc. Natl.
Acad. Sci. U. S. A. 2009, 106, 221−225. (e) Brodribb, T. J.; Jordan, G.
J.; Carpenter, R. J. New Phytol. 2013, 199, 559−570. (f) Carpenter, R.
J.; Tarran, M.; Hill, R. S. Aust. Syst. Bot. 2017, 30, 148−158.
(3) Weston, P. H. In Molecular Plant Taxonomy: Methods and
Protocols; Besse, P., Ed.; Springer: New York, 2014; Vol. 1115.
(4) Kooyman, R. M.; Rossetto, M.; Sauquet, H.; Laffan, S. W. PLoS
One 2013, 8, e80685.
Ethyl 3-Hydroxy-3-(3,4-dihydroxyphenyl)propanoate (13).
NaBH4 (47 mg, 1.24 mmol) was added to a magnetically stirred
solution of compound 7 (102 mg, 23 μmol) in EtOH (5 mL)
maintained at 0 °C. After 10 min, the reaction mixture was warmed to
room temperature. After 7 h, the mixture was concentrated under
reduced pressure to provide a yellow solid (190 mg), which was
adsorbed onto silica and purified by flash column chromatography
(25% MeOH/75% CH2Cl2) to provide title compound 13 (7.1 mg) as
a pale yellow film: IR (NaCl) 3245, 1710, 1607, 1509, 1445, 1371,
1
1282, 1205, 1114, 1033 cm−1; H NMR (600 MHz, methanol-d4) δ
6.83 (d, J = 2 Hz, 1H), 6.73 (d, J = 8 Hz, 1H), 6.70 (dd, J = 8, 2 Hz,
1H), 4.12 (q, J = 7.2 Hz, 2H), 2.72 (dd, J = 14.8, 8.6 Hz, 2H), 2.62
(dd, J = 11.8, 4.8 Hz, 1H), 1.23 (t, J = 7.1 Hz, 3H) ppm; 13C NMR
(150 MHz, methanol-d4) δ 171.7, 144.9, 144.5, 135.2, 117.1, 114.7,
112.8, 70.2, 60.2, 43.9, 13.0 ppm; HRESIMS m/z [M + Na]+ calcd for
C11H14O5 249.0747, found 249.0752.
(5) Jordan, G. J.; Harrison, P. A.; Worth, J. R. P.; Williamson, G. J.;
Kirkpatrick, J. B. Global Ecol. Biogeogr. 2016, 25, 127−140.
(6) De Salas, M. F.; Baker, M. L. 2017, A Census of the Vascular Plants
of Tasmania, Including Macquarie Island; Tasmanian Herbarium,
Tasmanian Museum and Art Gallery: Hobart, Tasmania, Australia,
2017; www.tmag.tas.gov.au..
(7) (a) Venkata Rao, C. Proc. Linn. Soc. N.S.W 1957, 257−271.
(b) Wiltshire, R. J. E.; Stace, H. M. Telopea 1997, 7 (3), 181−185.
(c) Barker, N. P.; Weston, P. H.; Rutschmann, F.; Sauquet, H. J.
Biogeogr. 2007, 34, 2012−2027.
(8) Bieleski, R. L.; Briggs, B. G. Aust. J. Bot. 2005, 53, 205−217.
(9) Bick, I. R. C.; Bremner, J. B.; Gillard, J. W. Phytochemistry 1971,
10, 475−477.
(10) (a) Just, J.; Deans, B. J.; Olivier, W. J.; Paull, B.; Bissember, A.
C.; Smith, J. A. Org. Lett. 2015, 17, 2428−2430. (b) Just, J.; Jordan, T.
B.; Paull, B.; Bissember, A. C.; Smith, J. A. Org. Biomol. Chem. 2015,
13, 11200−11207. (c) Just, J.; Bunton, G. L.; Deans, B. J.; Murray, N.
L.; Bissember, A. C.; Smith, J. A. J. Chem. Educ. 2016, 93, 213−216.
(d) Deans, B. J.; Bissember, A. C.; Smith, J. A. Aust. J. Chem. 2016, 69,
1219−1222. (e) Deans, B. J.; Just, J.; Chhetri, J.; Burt, L. K.; Smith, J.
N.; Kilah, N. L.; de Salas, M.; Gueven, N.; Bissember, A. C.; Smith, J.
A. ChemistrySelect. 2017, 2, 2439−2443. (f) Deans, B. J.; Olivier, W. J.;
Girbino, D.; Bissember, A. C.; Smith, J. A. Fitoterapia 2018, 123, 65.
(11) (a) Bick, I. R. C.; Gillard, J. W.; Leow, H.-M. Aust. J. Chem.
1979, 32, 1827−1840. (b) Ralph, I.; Bick, I. R. C.; Gillard, J. W.; Leow,
H.-M. Phytochemistry 1986, 25, 972−973. (c) Motherwell, W. D. S.;
Isaacs, N. W.; Kennard, O.; Bick, I. R. C.; Bremner, J. B.; Gillard, J. W.
J. Chem. Soc. D 1971, 133−134.
(12) (a) Pachaly, P.; Klein, M. Planta Med. 1987, 53, 442−444.
(b) Lee, M. H.; Son, Y. K.; Han, Y. N. Arch. Pharmacal Res. 2002, 25,
842−850.
(13) Boonkla, W.; Pitchuanchom, S.; Meepowpan, P.; Thaisuchat,
H.; Nuntasaen, N.; Punyanitya, S.; Pompimon, W. Am. J. Appl. Sci.
2011, 8, 1093−1097.
(14) Gibbs, R. D. Chemotaxonomy of Flowering Plants; McGill-
Queen’s University Press: Montreal, 1974; Vol. III.
(15) Xu, W.-H.; Liang, Q.; Zhang, Y.-J.; Zhao, P. Chem. Biodiversity
2015, 12, 54−81.
(16) Macleod, J. K.; Rasmussen, H. B.; Willis, A. C. J. Nat. Prod.
1997, 60, 620−622.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.7b01038.
1H, 13C, and 2D NMR spectra and crystallographic data
(PDF)
Crystallographic data for compound 1 (CIF)
Crystallographic data for compound 2 (CIF)
Crystallographic data for compound 4 (CIF)
Crystallographic data for compound 8 (CIF)
Crystallographic data for compound 9 (CIF)
AUTHOR INFORMATION
Corresponding Authors
*E-mail for A.C.B.: Alex.Bissember@utas.edu.au.
*E-mail for J.A.S.: Jason.Smith@utas.edu.au.
■
ORCID
Nathan L. Kilah: 0000-0002-0615-3791
Alex C. Bissember: 0000-0001-5515-2878
Jason A. Smith: 0000-0001-6313-3298
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors acknowledge the University of Tasmania School of
Physical Sciences−Chemistry and the Commonwealth Scien-
tific and Industrial Research Organisation (CSIRO) for
financial support, the University of Tasmania Central Science
Laboratory for access to NMR spectroscopy and mass
spectrometry services, and Dr. Miguel de Salas at the
Tasmanian Herbarium (Tasmanian Museum and Art Gallery)
J
DOI: 10.1021/acs.jnatprod.7b01038
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(17) Tirpak, R. E.; Olsen, R. S.; Rathke, M. W. J. Org. Chem. 1985,
50, 4877−4879.
(18) Jung, J.-C.; Park, O.-S. Synth. Commun. 2007, 37, 1665−1673.
(19) Perry, N. B.; Brennan, N. J. J. Nat. Prod. 1997, 60, 623−626.
(20) Saito, S.; Kawabata, J. Tetrahedron 2005, 61, 8101−8108.
(21) Bick, I. R. C.; Bremner, J. B.; Paano, A. M. C.; Preston, N. W. A
Survey of Tasmanian Plants for Alkaloids; University of Tasmania:
Hobart, Tasmania, Australia, 1991.
(22) Cannon, J. R.; Metcalf, B. W. Aust. J. Chem. 1971, 24, 1925−
1931.
(23) Gascoigne, R. M.; Ritchie, E.; White, D. E. J. Proc. R. Soc. N.S.W.
1948, 82, 44−70.
(24) Cornforth, J. W. J. Proc. Soc. N. S. W. 1938, 72, 255−256.
(25) Perry, N. B.; Benn, M. H.; Foster, L. N.; Routledge, A.; Weavers,
R. T. Phytochemistry 1996, 42, 453−459.
(26) Nycz, J. E.; Malecki, G.; Morag, M.; Nowak, G.; Ponikiewski, L.;
Kusz, J.; Switlicka, A. J. Mol. Struct. 2010, 980, 13−17.
(27) Wolff, M.; Seemann, M.; Grosdemange-Billiard, C.; Tritsch, D.;
Campos, N.; Rodriguez-Concepcion, M.; Boronat, A.; Rohmer, M.
Tetrahedron Lett. 2002, 43, 2555−2559.
(28) (a) Kim, S.-Y.; Kashiwada, Y.; Kawazoe, K.; Murakami, K.; Sun,
H.-D.; Li, S.-L.; Takaishi, Y. Phytochem. Lett. 2009, 2, 110−113.
(b) Ran, X.-H.; Teng, F.; Chen, C.-X.; Wei, G.; Hao, X.-J.; Liu, H.-Y. J.
Nat. Prod. 2010, 73, 972−975. (c) Ni, G.; Zhang, H.; Liu, H.-C.; Yang,
S.-P.; Geng, M.-T.; Yue, J.-M. Tetrahedron 2013, 69, 564−569.
(d) Wang, L.-J.; Xiong, J.; Liu, S.-T.; Liu, X.-H.; Hu, J.-F. Chem.
Biodiversity 2014, 11, 919−928.
(29) De Castro, K. A.; Ko, J.; Park, D.; Park, S.; Rhee, H. Org. Process
Res. Dev. 2007, 11, 918−921.
(30) Cowieson, N. P.; Aragao, D.; Clift, M.; Ericsson, D. J.; Gee, C.;
Harrop, S. J.; Mudie, N.; Panjikar, S.; Price, J. R.; Riboldi-Tunnicliffe,
A.; Williamson, R.; Caradoc-Davies, T. J. Synchrotron Radiat. 2015, 22,
187−190.
(31) Sheldrick, G. M. Acta Crystallogr., Sect. A: Found. Adv. 2015, 71,
3−8.
(32) Palatinus, L.; Chapuis, G. J. Appl. Crystallogr. 2007, 40, 786−
790.
(33) Dolomanov, O. V.; Bourhis, L. J.; Gildea, R. J.; Howard, J. A. K.;
Puschmann, H. J. Appl. Crystallogr. 2009, 42, 339−341.
(34) Flack, H. D. Acta Crystallogr., Sect. A: Found. Crystallogr. 1983,
39, 876−881.
(35) Hooft, R. W. W.; Straver, L. H.; Spek, A. L. J. Appl. Crystallogr.
2008, 41, 96−103.
(36) Spek, A. L. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2009, 65,
148−155.
(37) Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43, 2923−
2925.
(38) Morikawa, H.; Kasai, R.; Otsuka, H.; Hirata, E.; Shinzato, T.;
Aramoto, M.; Takeda, Y. Chem. Pharm. Bull. 2004, 52, 1086−1090.
K
DOI: 10.1021/acs.jnatprod.7b01038
J. Nat. Prod. XXXX, XXX, XXX−XXX