Probing the Structure-Activity Relationship of the Natural Antifouling Agent Polygodial against both Micro- and Macrofoulers by Semisynthetic Modification

Article abstract of DOI:10.1021/acs.jnatprod.6b01056

The current study represents the first comprehensive investigation into the general antifouling activities of the natural drimane sesquiterpene polygodial. Previous studies have highlighted a high antifouling effect toward macrofoulers, such as ascidians, tubeworms, and mussels, but no reports about the general antifouling effect of polygodial have been communicated before. To probe the structural and chemical basis for antifouling activity, a library of 11 polygodial analogues was prepared by semisynthesis. The library was designed to yield derivatives with ranging polarities and the ability to engage in both covalent and noncovalent interactions, while still remaining within the drimane sesquiterpene scaffold. The prepared compounds were screened against 14 relevant marine micro- and macrofouling species. Several of the polygodial analogues displayed inhibitory activities at sub-microgram/mL concentrations. These antifouling effects were most pronounced against the macrofouling ascidian Ciona savignyi and the barnacle Balanus improvisus, with inhibitory activities observed for selected compounds comparable or superior to several commercial antifouling products. The inhibitory activity against the microfouling bacteria and microalgae was reversible and significantly less pronounced than for the macrofoulers. This study illustrates that the macro- and microfoulers are targeted by the compounds via different mechanisms.

Full text of DOI:10.1021/acs.jnatprod.6b01056

Article
pubs.acs.org/jnp
Probing the Structure−Activity Relationship of the Natural
Antifouling Agent Polygodial against both Micro- and Macrofoulers
by Semisynthetic Modification
†
,¶
‡
§
⊥
⊥
Lindon W. K. Moodie, Rozenn Trepos, Gunnar Cervin, Lesley Larsen, David S. Larsen,
§
‡
∥
,†,#
Henrik Pavia, Claire Hellio, Patrick Cahill, and Johan Svenson*
†
Department of Chemistry, UiT The Arctic University of Norway, Breivika, N-9037, Tromsø, Norway
‡
Biodimar LEMAR UMR 6539, Universite
́
de Bretagne Occidentale, 6 Avenue le Gorgeu, 29200 Brest, France
, University of Gothenburg, SE-452 96 Stromstad, Sweden
Department of Chemistry, University of Otago, P.O. Box 56, Dunedin, New Zealand
§
Department of Marine Sciences - Tjar
̈
no
̈
̈
⊥
∥
Cawthron Institute, 98 Halifax Street East, Nelson 7010, New Zealand
#
Department of Chemistry, Material and Surfaces, SP Technical Research Institute of Sweden, Box 857, SE-501 15 Boras
̊
, Sweden
*
S Supporting Information
ABSTRACT: The current study represents the first comprehensive
investigation into the general antifouling activities of the natural
drimane sesquiterpene polygodial. Previous studies have highlighted
a high antifouling effect toward macrofoulers, such as ascidians,
tubeworms, and mussels, but no reports about the general
antifouling effect of polygodial have been communicated before.
To probe the structural and chemical basis for antifouling activity, a
library of 11 polygodial analogues was prepared by semisynthesis.
The library was designed to yield derivatives with ranging polarities
and the ability to engage in both covalent and noncovalent interactions, while still remaining within the drimane sesquiterpene
scaffold. The prepared compounds were screened against 14 relevant marine micro- and macrofouling species. Several of the
polygodial analogues displayed inhibitory activities at sub-microgram/mL concentrations. These antifouling effects were most
pronounced against the macrofouling ascidian Ciona savignyi and the barnacle Balanus improvisus, with inhibitory activities
observed for selected compounds comparable or superior to several commercial antifouling products. The inhibitory activity
against the microfouling bacteria and microalgae was reversible and significantly less pronounced than for the macrofoulers. This
study illustrates that the macro- and microfoulers are targeted by the compounds via different mechanisms.
1
9−21
22
olygodial (1) is the bioactive constituent of several species of
terrestrial plants and has also been isolated from marine
(SAR) studies focusing on the cytotoxic,
antifeedant,
1
−3
23
24
25
P
larvicidal, antifungal, and odiferous properties of 1.
4
5
sponges and nudibranchs. This drimane-type sesquiterpene
6
7
8
dialdehyde has exhibited antiallergic, antifeedant, antifouling,
antihyperalgesic, anti-inflammatory, antimicrobial,
2
6
9,10
antino-
1
1
12
ciception, and vasorelaxation activities. Such a diverse range
of biological effects infers multiple modes of action for 1. It has
been demonstrated that polygodial acts as a nonionic surfactant
to disrupt the cell membrane of microorganisms, yet this
compound also appears to interact with intracellular components
Compound 1 has been shown recently to display powerful
antifouling properties against the marine macrofoulers Ciona
savignyi (Ascidiacea), Spirobranchus caraniferus (Polychaeta), and
Mytilus galloprovincialis (Bivalvia). The low nanomolar 50%
effective concentrations (EC ) against larval metamorphosis of
the marine macrofoulers imply that 1 is a potentially useful lead
in the search for new antifouling compounds, especially as this
substance also has been shown to display a low toxicity toward
13
14
15
16
8
such as glutathione, ATPase, vanilloid receptors, and L-
13
cysteine-containing materials. This plethora of biological
effects makes 1 potentially useful as a lead compound for a
range of applications.
50
The chemistry and biological properties of the drimane
26,27
sesquiterpenes, which include 1, have been the subject of two
higher organisms and is not known to bioaccumulate.
17,18
extensive reviews, with the most recent published in 2004.
Chemical modifications of the dialdehyde and alkene motifs of 1
have resulted in a number of structure−activity relationship
Received: November 16, 2016
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.6b01056
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Scheme 1. Generation of Polygodial (1) Analogues and Structure of Reference Compounds
Scheme 2. Synthetic Routes to Structural Analogues Based on Polygodial (1)
Biofouling is the unwanted colonization and growth of
organisms on materials and surfaces submerged in water.
marine biofouling organisms and appears to be contact active,
whereby it can inhibit settlement and metamorphosis when
bound to a surface. Traditionally, antifouling formulations
28−30
8
It involves both microfouling, by marine microorganisms such as
bacteria and microalgae, and macrofouling by larger organisms,
slowly release a bioactive agent, with high potential for collateral
31
36
as typified by mussels, barnacles, ascidians, and macroalgae. All
activities at sea, including shipping, aquaculture, and the ocean
energy sector, face reduced efficiencies and increased costs due to
environmental harm. Eliminating the need to release a biocide
into the sea makes polygodial a particularly attractive alternative
to conventional antifouling biocides.
3
2
the settlement and growth of marine organisms. The
management of fouling in the marine environment is an age-
However, the SAR of 1 in relation to marine antifouling has, to
the best of our knowledge, not been explored. Neither has the
antifouling effect against other common foulers such as
barnacles, marine bacteria, and microalgae been assessed.
Establishing a SAR for 1 against marine biofouling organisms
would provide useful insights into the mode of action of this
compound. Establishing the width of the marine bioactivity
spectrum of 1 will guide the future development of 1 or
potentially commercially viable analogues thereof for marine
antifouling applications.
3
3
old problem, with the demand for effective environmentally
friendly antifouling technologies having accelerated in recent
years due to traditional ecotoxic biocidal approaches facing heavy
2
8
regulatory scrutiny. Natural products have been heralded as
possessing particular potential in yielding new nontoxic
28,34
antifouling solutions.
Both stationary marine organisms
that have evolved mechanisms to limit their colonization by
fouling species and natural products elicited from terrestrial
31,35
sources have been studied for their antifouling properties.
Compound 1 potently inhibits settlement and metamorphosis of
In this study, semisynthetic modifications of 1 yielded a library
of analogues that was used to probe the drimane scaffold SAR
B
DOI: 10.1021/acs.jnatprod.6b01056
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Article
Figure 1. Metamorphosis inhibition of C. savignyi larvae exposed to 1, 3, and 5. Inhibition of metamorphosis is relative to corresponding blank controls,
and values are means (n = 3) ± standard error.
Figure 2. Effects of 1, 4, and 6 on the settlement of B. improvisus cyprid larvae presented as percentages of settled (dark gray columns) and dead cyprids
(
light gray columns) and given as means ± standard error (n = 4). Remaining larvae were free swimming. Filtered seawater (FSW) and DMSO (0.1%, v/
v) were used as the negative control.
3
8
against the macrofouling organisms C. savignyi and Balanus
improvisus (Maxillopoda). The prepared compounds were also
screened against a selection of microfouling organisms, including
both marine bacteria and marine microalgae, to establish the
general antifouling properties of the compounds. The biological
effects of the prepared compounds were compared to structural
and commercial antifouling reference compounds. The natural
compound drimendiol (3), a close analogue of 1, was also
included and evaluated as an antifoulant for the first time.
conditions yielded cinnamolide (5). The alkene of both 3 and 5
was reduced by hydrogenation, to probe the role of the
unsaturated motif in bioactivity. The alkene of 3 provided a
useful functional handle for the oxidation of the B ring, which
underwent dihydroxylation (9) or epoxidation (10). Treatment
of diacetate 8 with SeO induced a selective allylic oxidation to
2
afford 11. Deprotection under basic conditions yielded triol 12.
These transformations provided a series of drimane compounds
with differing polarities. Additionally, the epimerized version of
1
, epipolygodial (2), was also synthesized.
Macrofouling Activity. Compound 1 has an established
RESULTS AND DISCUSSION
■
bioactivity against the larvae of tunicates, mussels, and
tubeworms in the nanomolar range and has been investigated
as a protective agent to prevent unwanted growth in
Compound Design. Although 1 and structurally related
drimane natural products have been accessed by total syn-
1
7,18
thesis,
the high abundance of 1 in various plant species and
39
37
aquaculture. In addition, 1 has also been shown to work as a
its ease of isolation encouraged a semisynthetic approach to
developing analogues. Following extraction from Drimys winteri
8
,39
contact active antifouling biocide.
As such, the antifouling
effect of a coating incorporating 1 does not rely on this
compound being released into the surrounding environment, a
particularly attractive scenario for developing marine antifouling
coatings that release a minimum amount of bioactive compounds
(
Winteraceae) and a single chromatographic step, 1 was isolated
in a practical yield (of 2.5% of dried plant material). Compound
3
identified as a suitable starting point to access a diverse range of
analogues of 1. The hydroxy group functional handles of 3 could
be masked as either the corresponding methyl ethers (7) or
acetates (8). Furthermore, the reaction of 3 under oxidative
23
, obtained by the NaBH -mediated reduction of 1, was
4
40
to the surrounding water. While the SAR activities of 1 have
23,24
been investigated against fungi, Drosophila melanogaster,
and
other biological targets, no attempts have been reported to
C
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dissect the individual structural contributions toward biofouling
activity. The current library, prepared semisynthetically via 1
isolated from D. winteri, represents the first such study. The
prepared analogues were initially screened for their ability to
inhibit macrofouling, measured as inhibition of settlement and/
or metamorphosis of C. savignyi and B. improvisus larvae. Sclareol
and 1,5-decalindiol were included as reference compounds in the
C. savignyi bioassays to probe the drimane scaffold, whereas the
commercial antifouling compound Sea-nine was included for the
B. improvisus bioassays. Representative examples demonstrating
antifouling effects are shown in Figures 1 and 2, and the compiled
macrofouling inhibitory capacity of the full library is presented in
Table 1.
compounds produced share the bicyclic drimane framework,
with the differences lying mainly within the degree of substitution
and oxidation. While the compounds displayed a range of ClogP
values, no apparent link between polarity and inhibitory effect
was noticeable. Going away from the drimane scaffold appears to
be detrimental, as shown by the lack of activity for 1,5-decalindol
and the relatively high inhibitory activity of sclareol. Compound
1 is reported typically to have multiple modes of action for a
single biological activity. The unusually high potency of 1 against
C. savignyi could reflect that this compound interferes with
biochemical targets linked to tail resorption and confers generally
toxic effects from disruption of cell membranes by way of its
surfactant properties. Comparing the potency of 1 (25-fold
higher inhibitory activity in comparison to the most active
analogues) with epi-polygodial (2) and analogues with a similar
CLogP suggests that the superior bioactivity is reliant on both the
presence and orientation of the dialdehyde motif and is not solely
dictated by polarity. No prominent inhibitory activity was seen
for the natural product drimendiol (3) in comparison to the
other synthetic derivatives. A similar trend between 1 and 3 has
been reported for antifeedant activity against the cotton worm
Table 1. Potency of 1 and Analogues against the Ascidian
C. savignyi and the Barnacle B. improvisus
C. savignyi
B. improvisus
a
compound
Mw
ClogP
EC50 (μg/mL) EC50 (μg/mL)
1
2
3
4
5
6
7
8
9
1
1
1
234.34
234.34
238.37
240.39
234.34
236.36
266.43
322.45
272.39
254.37
338.44
254.37
308.51
170.25
282.22
3.685
3.685
3.123
3.407
3.754
3.618
4.555
5.015
2.251
1.993
4.349
2.545
5.318
0.612
4.899
0.004
0.1
0.25
>5
48
Spodoptera littoralis.
1.0
0.5
1.5
1.5
0.1
Activities observed against the barnacle B. improvisus ranged
between 0.1 and >5 μg/mL, and a closer link between structure
and activity was observed. Compound 1 was highly active, with
an IC50 of 0.25 μg/mL. This represents an inhibitory activity
higher than those reported for the large majority of previously
0.7
0.2
0.8
0.7
>5
0.7
>5
>5
5
34,49
0.4
investigated antifouling natural products.
The effect of
0
0.4
compound hydrophilicity on settlement inhibition is more
pronounced against the balanide cyprids. All of the active
compounds (<5 μg/mL) have ClogP values between 3.1 and 3.8,
suggesting that, if a nonionic surfactant mechanism is operating,
there is an optimal polarity range. When the hydroxy groups of 3
were derivatized with methyl or acetyl groups (7 and 8,
respectively), the hydrophobicity increases to the detriment of
activity. It also appears that reducing the CLogP with respect to 1
by incorporating an additional oxygen functionality into the
drimane scaffold also impairs cyprid settlement inhibition.
Compounds 9, 10, and 12, which all contain at least three
oxygen atoms and have CLogP values below 2.6, demonstrated
this.
1
2
0.1
>5
>5
0.6
b
sclareol
,5-decalindiol
Sea-nine
1.9
n.t.
n.t.
0.25
1
>200
c
0.2
a
b
c
Calculated using ChemBio3D Ultra 14.0. Not tested. Data from ref
1 against Ciona intestinalis.
41
4
All of the synthetic compounds examined were potent
inhibitors of C. savignyi larval settlement and metamorphosis,
with submicrogram/mL EC50 values (Table 1). The natural
compound 1 was the most effective inhibitor, with an EC of 4
50
ng/mL, corresponding well with values reported in previous
A general surfactant mechanism is, however, not the sole
contributor to observed bioactivity. Compound 1 has been
8
studies. Both 2 and 11 displayed an EC value of 0.1 μg/mL and
5
0
4
7
were the most potent synthetic inhibitors, while the remaining
compounds were effective between 0.1 and 1.0 μg/mL. The
diterpenoid reference compound sclareol was also active, while
shown to form pyrroles with endogenous amines, which is
dependent on the β orientation of the aldehyde attached to C-9.
The difference in activity between 1 and its inactive C-9 epimer 2
against balanide cyprid settlement is striking. Lactone 6 was the
most active analogue against the cyprids, with an IC50 of 0.1 μg/
mL. This represents a 15-fold increase in activity in comparison
with the active unsaturated analogue 5. It is possible these two
compounds also may engage in reactions with endogenous
nucleophiles but with ranging affinity.
1,5-decalindol did not display significant inhibition at the
concentrations tested. It has been shown previously that 1
halts the metamorphosis of C. savignyi at the tail-resorption
3
9
stage. The tail-resorption stage is completed within 20 min of
metamorphosis onset and is characterized by massive apoptosis
42,43
of cells in the tail and reordering of structures in the trunk.
A
number of studies have investigated the biochemical drivers of
The results from the macrofouling studies demonstrate that 1
and its analogues interfere with the settlement and metamor-
phosis of both C. savignyi and B. improvisus at low concentrations.
The inhibitory effect of 1 against the tubeworm S. caraniferus and
44
ascidian metamorphosis, providing potential links between the
known modes of action of 1 in other scenarios and inhibition of
tail resorption. The established nonionic surfactant properties of
45
8
1
can confer generally toxic effects on C. savignyi larvae, but
the mussel M. galloprovincialis was recently reported at a
specific biochemical targets are also apparent. In particular, 1 is
concentration range of 2−4 ng/mL. This is comparable with data
obtained in the present study against C. savignyi. A shared and
specific covalent interaction is likely against these organisms
given the similar low EC values. Against C. savignyi, some of the
activity is lost upon structural alteration, implying both a specific
target and a drimane-linked nonionic surfactant effect. It appears
known to form covalent adducts intracellularly with amine
2
2,46,47
groups.
From a structural perspective, it is interesting that all of the
compounds prepared displayed pronounced inhibitory activity
against larval metamorphosis of C. savignyi. All the synthetic
50
D
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that the general “drimane effect” is sufficient to generate an
antifouling effect similar to that recently reported for the
commercial antifouling booster biocides chlorothalonil (EC 0.1
Table 3. MIC (in μg/mL) of Polygodial (1) and Analogues
against Marine Bacteria
a
50
50
S. putrefaciens
V. carchariae
V. harveyi
μg/mL) and tolylfluanid (EC 0.3 μg/mL). These inhibitory
5
0
compound
A
G
A
G
A
G
values for the commercial biocides are almost identical to those
4
1
b
reported by Bellas against C. intestinalis, implying a comparable
1
10
−
10
−
−
10
n.t.
10
−
−
−
10
−
−
−
−
10
10
−
10
n.t.
10
−
−
−
10
−
0.01
sensitivity of these two related species despite diverging from a
2
−
−
−
n.t.
−
−
−
51
common ancestor more than 180 million years ago. Bellas also
3
−
reported an EC for Sea-nine at 0.15 μg/mL against
4
0.01
n.t.
0.01
−
−
0.1
5
0
4
1
c
C. intestinalis. This general effect does not appear to extend
to B. improvisus, and several of the investigated compounds were
inactive at 5 μg/mL. This suggests that the drimane scaffold
alone is insufficient for a general inhibitory effect against barnacle
larvae. Compounds 1, 3, and 6 were nevertheless as active as the
commercial control Sea-nine (EC50 0.25 μg/mL), illustrating a
high inhibitory effect for select chemistries within the drimane
scaffold.
5
n.t.
−
−
−
−
−
−
−
1
n.t.
6
−
−
10
−
−
−
−
−
0.1
7
8
9
−
10
10
11
−
−
−
1
0.01
−
10
<0.01
−
−
1
1
2
−
1
−
<0.01
Sea-nine
Microfouling Activity. The capacity of compounds to
interfere with microfouling was assessed by screening the
prepared library against a range of species of marine microalgae
and bacteria that are involved in the initial formation of the
a
V. proteolyticus, R. litoralis, V. natriegens, V. aestuarianus, and P. irgensii
displayed no sensitivity to the tested compounds and are not included
b
c
in the table. Not active at >10 μg/mL. Not tested.
29
fouling biofilm and biocorrosion. It is of interest that the effect
of 1 against marine microorganisms has not been reported
previously. In total, four microalgae and eight marine bacterial
strains were included. The effect on both adhesion and growth
days (data not shown). None of the tested compounds displayed
antialgal properties comparable to those observed for the
5
4
commercial antifouling control Sea-nine, which is a herbicide.
(A and G, respectively, in the tables) was studied, and the results
The antibacterial effects of the test compounds were generally
low. Of the eight marine bacterial test strains included, five did
not show any sensitivity against the compounds at concentration
up to 10 μg/mL. V. carchariae and V. harveyi were the two most
sensitive strains and displayed reduced adhesion and growth
against half of the tested compounds. Compound 1 displayed
modest activity, with growth inhibition at 10 μg/mL against the
three sensitive bacterial strains. No obvious coupling between the
structure of the studied compounds and their antibacterial
potency could be inferred. The inhibitory effects were
bacteriostatic, as revealed by subsequent growth of the inhibited
bacterial strains in fresh medium (data not shown). These
observations are interesting, as both 1 and 3 have been
investigated as quorum sensing (QS) inhibitors by Paza et al.
against the bacteria Chromobacterium violaceum and Pseudomonas
expressed as the minimal inhibitory concentration (MIC) are
summarized in Tables 2 and 3.
Table 2. MIC (in μg/mL) of Polygodial (1) and Analogues
a
against Microalgae
C. closterium
H. coffeaformis
P. roscoffensis
compound
A
G
A
G
A
G
b
1
2
3
4
5
6
7
8
9
10
−
−
−
−
10
−
−
−
−
n.t.
0.01
−
0.01
−
−
−
−
−
−
−
10
−
0.01
−
n.t.
1
−
n.t.
1
−
c
n.t.
n.t.
10
−
−
−
n.t.
1
−
−
−
−
55
−
10
−
−
−
−
1
syringae. In this study 3 was shown to effectively inhibit QS and
to decrease biofilm formation. The biological effects were,
however, investigated at concentrations too high to be regarded
−
−
−
−
5
5
1
1
1
0
1
2
1
−
−
10
10
−
−
0.01
10
−
−
−
<0.01
as meaningful in an antifouling setting (400 and 800 μg/mL).
−
10
−
0.01
<0.01
−
10
<0.01
In the present study on marine bacteria, no inhibitory
antibacterial effect of 3 was observed at concentrations up to
10 μg/mL, and there were no indications that the drimane
scaffold is a general antibacterial scaffold. The bacterial strains
included in the current study were all Gram-negative, analogous
to C. violaceum and P. syringae used by Paza et al. However, the
marine origin of the screened bacteria and the significantly lower
concentrations employed in the present study may explain the
low activity of the tested compounds against marine bacteria.
Compound 1 has also been reported to inhibit other human
Sea-nine
<0.01
<0.01
<0.01
a
None of the tested compounds were active against P. purpureum.
Not active at >10 μg/mL. Not tested.
b
c
Six of the compounds displayed inhibitory activity against the
adhesion or growth of microalgae. The inhibitory effects against
microalgae were apparent at concentrations less than 1 μg/mL in
some instances but were generally inferior to other naturally
derived antifouling products evaluated against the same array of
5
6
pathogenic bacterial strains at moderate concentration levels.
3
5,52,53
microalgae.
P. purpureum was insensitive to all the test
The low inhibitory effect toward the marine microfouling
species implies that the high-affinity molecular target responsible
for the high activity among macrofoulers is not present in lower
organisms. A general nonionic surfactant effect is not
pronounced against any of the microorganisms either, granting
some of the microbial strains included evidently are more
sensitive than others. Our recent antifouling studies have shown
that cationic amphiphilic micropeptides efficiently target marine
compounds at concentrations up to 10 μg/mL. The effects were
mainly seen on adhesion of the microalgae, and only 6 and 12
also affected the growth of microalgae. Both 6 and 12 can be
regarded as active against microalgae, whereas of the two natural
products, 1 and 3, only 1 displayed a weak antialgal effect. The
inhibitory effects were biostatic, as revealed by recovery of algal
cells that were transferred to fresh medium and incubated for 5
E
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Journal of Natural Products
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bacteria and microalgae, an interaction that is mediated via a
balance of cationic charge and hydrophobic bulk, which is not
present in the currently studied neutral drimane sesquiter-
type natural products suggests that the mode of action is dictated
by differing mechanisms depending on target species, rather than
a general biocidal effect. General surfactant properties appear
partly responsible for observed activity against macrofoulers, but
there is evidence that specific biochemical targets also exist;
elucidating these targets is a priority for future research in this
area. Collectively, it appears that 1 and its synthetic analogues
exert a stronger influence on the macrofouling species, operating
at concentrations that are comparable to commercial antifouling
products, which are of greater financial consequence in the
struggle against marine fouling in industrial, commercial, and
recreational settings. Overall, 1 is a promising lead compound for
further development as a commercially viable antifouling agent.
5
3,57,58
penes.
Commercial Implications. The use of natural products to
combat biofouling has been heralded as a promising strategy for
the development of environmentally friendly antifouling
2
8,34
products.
The natural products that may be studied are
not limited to those primarily found in the marine environment,
as compounds isolated from terrestrial sources have also proven
5
9
useful. Juglone from the walnut family (Juglandaceae) and the
60
avermectins from the soil microbe Streptomyces avermitilis are
representative examples of promising antifouling compounds
from nonmarine organisms. The observations made in the
current study highlight the ability of 1 to inhibit effectively the
settlement and metamorphosis of higher marine macroorgan-
isms. From a commercial viewpoint, targeting these larger
organisms is of greater importance than their bacterial and algal
counterparts due to their greater influence on weight gain and
EXPERIMENTAL SECTION
General Experimental Procedures. Melting points were
■
determined using a Mettler Toledo FP62 automatic melting point
apparatus and are uncorrected. Specific optical rotations ([α]TD) were
recorded on a JASCO DIP-1000 digital polarimeter using a 100 mm cell.
Values were measured using the sodium D line (589 nm) at ambient
temperature. Samples were prepared in g/100 mL, in the solvent
indicated. Infrared spectra were recorded on a Bruker Optics Alpha FT-
IR spectrometer (with a diamond Attenuated Total Reflectance top
plate). NMR spectra were recorded on either a Varian 400-MR 400
32
reduced hydrodynamic efficiency. Additionally, their settle-
ment can also induce corrosion and blockage of underwater
tubes, membranes, and pipes in desalination plants and heat
2
9,61
exchangers.
Although the synthetic analogues of 1 did not match or better
the impressive low nanogram/mL activity seen for the natural
product in the screens employed, this is not unusual in the field of
natural products research. Several drugs based on natural
products are, in fact, synthetic versions of the originally isolated
natural products despite significant attempts at truncation and
1
13
MHz spectrometer for H nuclei and 100 MHz for C nuclei at 25 °C or
on a Varian 500 MHz AR Premium Shielded spectrometer operating at
00 MHz for H nuclei and 125 MHz for C nuclei at 25 °C. H NMR
chemical shifts are reported in parts per million (ppm) relative to the
1
13
1
5
chloroform (CHCl , δ 7.26) or methanol (CD OD, δ 3.31) peaks, and
3
3
1
3
coupling constants (J, Hz) were taken directly from the spectra.
NMR chemical shifts are reported in ppm relative to the chloroform
CDCl , δ 77.0) or methanol (MeOH, δ 49.0) peaks. High-resolution
C
62
analogue synthesis aimed at improving efficacy. The potent
activity profile of 1 against all investigated macrofoulers warrants
additional studies toward potential antifouling applications. The
observation that this compound class is only weakly active against
the microfoulers at relevant concentrations implies that a
combination of strategies is required to provide a comprehensive
biofouling protection if it aims at relying on 1. It must
nevertheless be pointed out that selectivity against certain classes
of fouling species may well be sufficient for commercialization, as
exemplified by the recently approved Selektope (medetomi-
(
3
mass spectrometry (HRMS) of the compounds was recorded on a
Bruker microTOF Q mass spectrometer using electrospray ionization
(ESI). All chemical reactions were conducted using anhydrous solvents
and an argon atmosphere unless stated otherwise. Reagents obtained
from commercial suppliers were used without purification. Anhydrous
solvents were dried using a PURE SOLV MD-6 solvent purification
system. Reactions were monitored by thin-layer chromatography carried
out on 0.2 mm Kieselgel F254 silica gel plates using UV light as a
visualizing agent and then stained and developed with heat using vanillin
in ethanolic sulfuric acid. Flash chromatography was performed using
63,64
dine), which is active only against tubeworms and barnacles.
0
.063−0.1 mm silica gel with the indicated eluent.
Plant Material and Other Organisms. The plant material used for
Conclusions. The current study has explored the potency of
polygodial (1) against an extensive panel of both macro- and
microfouling marine organisms. Given the high abundance of 1
in the leaves of D. winteri and its efficient isolation, 11 analogues
were prepared by semisynthesis to investigate, for the first time,
the structural contributions of 1 toward biofouling inhibition. To
generate a comprehensive overview of the antifouling potential
of these compounds, 14 relevant macrofouling and microfouling
species were included. The synthesized compounds displayed
powerful antifouling activities against the ascidian C. savignyi and
the barnacle B. improvisus. The effects observed against C. savignyi
were general, with most compounds displaying very similar
inhibitory activities ranging from 0.1 to 1.0 μg/mL. Although the
potencies against C. savignyi are high compared to other natural
marine antifouling agents, these values represent at least a 25-fold
reduction in activity compared to the native 1. Several of the
analyzed compounds were also active as inhibitors of the
settlement of barnacle cyprids, with settlement inhibition down
to 0.1 μg/mL. Select compounds reversibly interfered with
marine microorganisms, but measured potencies were orders of
magnitude lower than against macrofouling organisms. The exact
mode of action of 1 is unknown, but several prospective targets
are apparent. The observed bioactivity profile of the drimane-
the isolation of polygodial (1) from New Zealand Drimys winteri J.R.
Forst. and G. Forst was obtained from Topseeds NZ Ltd. and collected
in December 2014 in Dunedin (45°84 S, 170°52 E). The material was
collected and identified by Alison Evans from Topseeds NZ Ltd., New
Zealand. The material (voucher number OTA-070248) is stored at the
University of Otago, Dunedin, New Zealand. Eight marine bacterial
strains and four pure, but nonaxenic, marine microalgae (obtained from
Algobank, Caen, France) were used in the current study (Table 4). The
selected strains were representative of fouling species in both estuarine
65,66
and marine environments.
The bacteria were grown at 26 °C in a
marine medium, composed of 0.5% peptone (neutralized bacteriological
peptone, Oxoid Ltd.) in filtered (Whatman 1001-270, pore size 11 μm)
natural seawater. Algae were grown at 22 °C in F/2 medium prior to use.
For assays, the compounds were dissolved in 100% methanol (general
purpose grade, Fisher) and coated onto the internal surfaces of clear
polystyrene 96-well plates (Fisher Scientific) to afford wells containing
0
.01, 0.1, 1, and 10 μg/mL of each compound, with each concentration
replicated six times. Solvent was removed under vacuum, and the plates
were sterilized with UV-illumination. Growth media and Sea-nine were
employed as negative and positive commercial antifouling controls,
respectively.
Ascidian Antifouling Bioassay. The compounds were screened in
50
a laboratory bioassay based on the model taxa C. savignyi. Adult
C. savignyi were collected from Nelson Marina, Nelson, New Zealand,
F
DOI: 10.1021/acs.jnatprod.6b01056
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
tration of the compounds was replicated four times (n = 4), and dishes
with 10 μL of DMSO served as controls. The commercial antifouling
agent Sea-nine served as a positive control.
Table 4. Biofouling Microorganisms Studied in the Present
Study
species
Microalgae
code
Antibacterial Assays. Adhesion and growth of the bacterial strains
6
9
were assessed according to Thabard et al. Aliquots (100 μL) of
Algobank code
AC 170
8
bacterial suspension (2 × 10 colony forming units/mL) were added to
Cylindrotheca closterium
Halamphora coffeaeformis
Pleurochrysis roscoffensis
Porphyridium purpureum
Marine bacteria
each well under aseptic conditions, and the plates were incubated at 26
AC 713
°
C for 48 h. The lowest concentration yielding a decrease in optical
AC 32
density at 630 nm was defined as the minimal inhibitory concentration
for bacterial growth. Bacterial adhesion was determined by emptying
and rinsing the incubated wells with 100 μL of FSW after 48 h, before
allowing the plates to air-dry. Bacterial biofilms remaining adhered to the
walls of the dried wells were stained with 100 μL of 0.3% aqueous crystal
AC 122
a
ATCC
Polaribacter irgensii
Roseobacter litoralis
Shewanella putrefaciens
Vibrio aestuarianus
Vibrio carchariae
700398
49566
8071
70
violet (v/v) and visualized at 595 nm according to Sonal and Bholse.
The MIC value for adhesion was defined as the lowest concentration
inducing a reduction in optical density at 595 nm.
35048
35084
700106
14058
53559
Antialgal Assays. Stock solutions of each of the microalgal strains
Vibrio harveyi
(
5
0.1 mg chlorophyll a/mL) were prepared via chlorophyll analysis after
Vibrio natriegens
65
days of growth according to Chambers et al. Aliquots (100 μL) of the
Vibrio proteolyticus
algal stock solutions were transferred to wells of the pretreated
a
American tissue culture code.
microplates, and they were grown at 20 °C for 5 days under constant
−2
−1
light exposure (140 μmol m s ). To determine the growth of the
microalgae, the microplates were centrifuged at 4100 rpm (Allegra 25R
centrifuge, Beckman Coulter) for 10 min at 4 °C, and then the wells
were emptied. Aliquots (100 μL) of 100% methanol were added to wells
to liberate chlorophyll a, which was quantified using a fluorometric
and were held in a 1000 L recirculating system at 18 ± 1 °C, 34 ± 1 psu,
and 300 ± 50 mV ORP; constant full-spectrum fluorescent light
prevented premature spawning. Daily feeds comprised 250 mL of an
6
−1
(
8−9) × 10 cells mL Isochrysis galbana Parke algal culture. Three
6
5
method described by Chambers et al. The extent of microalgal
adhesion was determined by gentle removal of nonattached cells after 5
days of incubation using a multichannel pipet and addition of 100 μL of
gravid individuals were spawned, with ventral incisions made to expose
the egg and sperm ducts. Eggs and sperm were harvested and transferred
to separate 50 mL glass Petri dishes (90 mm diameter) filled with 20 or
1
00% methanol prior to fluorometric analysis of released chlorophyll a.
5
0 mL of 0.3-μm-filtered and UV-sterilized seawater (FSW),
Minimum inhibitory concentrations for algal growth and adhesion were
determined as for the bacteria. When inhibition was recorded, a toxicity
test was performed by transferring the contents of the wells into a flask of
fresh media. The growth of the inhibited strains was measured after 5
respectively. Each dish of eggs received eight drops of sperm suspension
from each of the two other individuals. After incubating at 18 °C for 1 h,
fertilized eggs were strained through a 20 μm sieve, rinsed three times
with 25 mL of reconstituted seawater (RSW; 33 ± 0.5 psu; Red Sea Salt,
Red Sea Aquatics, Cheddar, UK), transferred to a glass Petri dish along
with 25 mL of RSW, and held at 18 °C for 18 h to hatch. Hatched larvae
were transferred to conical flasks and diluted with RSW to yield 10 larvae
6
5
days of incubation according to Chambers.
71
Synthesis. Polygodial (1). Extraction of 1 from Drimys winteri: A
mixture of leaf and twigs (30 g) was dried, ground, and then shaken in
chloroform (300 mL) for 24 h. The solid was removed by filtration and
washed with chloroform; then the filtrate was evaporated in vacuo to
give a green gum (1.5 g). Purification by column chromatography over
silica gel, eluting with chloroform, gave pure 1 (740 mg, 2.5% from dried
−1
mL .
The bioassay was run in 12-well tissue culture plates (Corning Costar,
Corning Inc., Corning, NY; 23 mm diameter), filled with 7.1 mL
aliquots of the larval suspension. Stock solutions of each compound
prepared in 20% (v/v) ethanol were added to wells to yield final
concentrations of 0.001, 0.0025, 0.005, 0.01, 0.025, 0.05, 0.1, 1, 2.5, 5, 10,
plant) as an amorphous solid: [α]25
ν
−100.9 (c 0.7, CHCl
); IR (neat)
max 2925, 1719, 1674, 1640, 1365, 1069, 512 cm ; H NMR (CDCl ,
3
D
3
−
1 1
−1
and 20 μg mL . Blank controls contained carrier solvent only; positive
controls contained 0.0001, 0.0005, 0.0025, 0.0125, and 0.0625 μg mL−1
of 1; and negative controls contained sclareol or 1,5-decalindol at 0.05,
400 MHz) δ 9.54 (1H, d, J = 4.4 Hz, H-11), 9.46 (1H, s, H-12), 7.13
(1H, dt, J = 5.6, 2.3 Hz, H-7), 2.83 (1H, tt, J = 4.2, 2.2 Hz, H-9), 2.51
(1H, dtd, J = 20.3, 5.1, 2.1 Hz, H-6), 2.32 (1H, dddd, J = 20.4, 12.1, 3.9,
2.4 Hz, H-6), 1.85 (1H, dq, J = 13.1, 2.5 Hz, H-1), 1.57−1.46 (3H, m, H-
2, H-2, H-3), 1.38 (1H, td, J = 12.9, 4.5 Hz, H-1), 1.27 (1H, dd, J = 12.0,
4.7 Hz, H-5), 1.27−1.17 (1H, m, H-3), 0.96 (3H, s, H-14), 0.95 (3H, s,
−1
0
.1, 0.5, 1, 5, 10, 100, and 200 μg mL . Three replicates were performed
in all cases (n = 3). Plates were held at 18 °C, 12 h:12 h subdued light to
dark for 5 days, after which the number of larvae that had settled and
completed metamorphosis was counted. Dose−response curves were
plotted for the reference compounds using four-parameter logistic curve
H-15), 0.92 (3H, s, H-13); 13C NMR (CDCl
, 101 MHz) δ 202.1 (C-
3
11), 193.4 (C-12), 154.4 (C-7), 138.5 (C-8), 60.5 (C-9), 49.1 (C-5),
67
fitting on SigmaPlot 11.0. The corresponding concentrations that
41.9 (C-3), 39.7 (C-1), 37.0 (C-10), 33.28 (C-4*), 33.27 (C-13*), 25.4
reduced the number of larvae completing metamorphosis by 50%
(C-6), 22.1 (C-14), 18.2 (C-2), 15.4 (C-15) (*overlapped signal);
+
(
EC ) were calculated relative to blank controls.
Balanide Settlement Inhibition. The settlement inhibition of
HRESIMS m/z 257.1493 [M + Na] (calcd for C15
H
22NaO
2
,
50
257.1512).
71
cyprid larvae of B. improvisus used methods initially described by
Epipolygodial (2). A solution of 1 (28 mg, 0.12 mmol) in THF (5
5
2,68
Berntsson et al.
Cyprids were reared at the Tjar
̈
no
̈
-Marine Biological
mL) was treated with Cs CO (3.9 mg, 0.01 mmol) and heated to 50 °C.
2
3
Laboratory, Sweden. Stock solutions of the compounds in DMSO were
After 4 h, the reaction mixture was cooled to room temperature and
diluted with ethyl acetate. After washing with water and brine, the
organic layer was dried with sodium sulfate and filtered, and the solvent
was removed under reduced pressure. The resulting residue was purified
by column chromatography (12:1−9:1 petroleum ether/ethyl acetate)
to afford 2 (9 mg, 32%): [α]25 −218.0 (c 1.55, CHCl ); H NMR
prepared and serially diluted to generate the desired final concentrations
(
10 μL added to each dish). The compounds were added to untreated
polystyrene Petri dishes containing 10 mL of filtered (0.2 μm) seawater,
and a total of 18−22 newly molted cyprids were added to each test dish.
Test dishes were incubated at ambient temperature (20−25 °C) for 5
days. The number of metamorphosed juvenile barnacles, as well as live
and dead cyprids, was assessed under a dissecting microscope at the end
of the experiment. The EC concentrations represent the concentration
1
D
3
(CDCl , 400 MHz) δ 9.85 (1H, d, J = 2.6 Hz, H-11), 9.40 (1H, s, H-12),
3
7.09 (1H, dd, J = 4.9, 2.7 Hz, H-7), 3.25 (1H, t, J = 2.1 Hz, H-9), 2.56
(1H, dt, J = 20.5, 5.0 Hz, H-6), 2.21 (1H, dddd, J = 20.6, 11.6, 2.6, 1.8 Hz,
H-6), 1.78 (1H, dq, J = 12.2, 2.7, 2.0 Hz, H-1), 1.66−1.60 (1H, m, H-2),
1.56 (1H, dd, J = 11.6, 5.1 Hz, H-5), 1.53−1.41 (3H, m, H-1, H-2, H-3),
1.22−1.10 (1H, m, H-3), 0.96 (3H, d, J = 0.6 Hz, H-15), 0.93 (3H, s, H-
50
where 50% of the cyprids have failed to settle on the Petri dish surface.
An initial screen was performed at 5 μg/mL for all the compounds, and a
full EC50 determination was performed only on those compounds
displaying >50% inhibition at that concentration. Each test concen-
14), 0.90 (3H, s, H-13); 13C NMR (CDCl , 101 MHz) δ 202.4 (C-11),
3
G
DOI: 10.1021/acs.jnatprod.6b01056
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
1
(
2
92.9 (C-12), 153.6 (C-7), 137.5 (C-8), 58.6 (C-9), 44.3 (C-5), 42.2
C-3), 37.8 (C-10), 37.3 (C-1), 33.0 (C-4), 32.8 (C-13), 25.7 (C-6),
2.0 (C-14), 21.6 (C-15), 18.5 (C-2); HRESIMS m/z 257.1505 [M +
added, and the reaction stirred overnight. The reaction mixture was
diluted with ethyl acetate and filtered through Celite. Evaporation of the
solvent afforded the crude product, which was recrystallized from
petroleum ether as colorless crystals (18 mg, 72%): mp 133 °C
(petroleum ether); [α]25 −4.4 (c 0.4, CHCl ); IR (neat) ν 2913,
+
Na] (calcd for C H NaO , 257.1512). The slower eluting starting
material was also recovered (8 mg, 29%).
15
22
2
D
3
max
71
−1 1
Drimendiol (3). A solution of 1 (344 mg, 1.47 mmol) in methanol
1766, 1367, 1148, 1147, 959 cm ; H NMR (CDCl , 400 MHz) δ 4.21
3
(
25 mL) was treated with NaBH (222 mg, 5.87 mmol). After 2 h, water
4 mL) was added, and the resulting mixture concentrated in vacuo. The
(1H, d, J = 9.7 Hz, H-11), 4.11 (1H, dd, J = 9.7, 5.4 Hz, H-11), 2.55 (1H,
t, J = 8.0 Hz, H-8), 2.32 (1H, dd, J = 14.0, 4.8 Hz, H-7), 2.08 (1H, dd, J =
7.9, 5.5 Hz, H-9), 1.74−1.38 (6H, m, H-1, H-2, H-2, H-3, H-6, H-7),
1.30 (1H, td, J = 13.0, 5.0 Hz, H-6), 1.18 (1H, td, J = 14.4, 13.7, 4.3 Hz,
H-3), 0.93 (1H, td, J = 13.3, 3.4 Hz, H-1), 0.89 (3H, s, H-13) 0.85 (3H, s,
H-15), 0.82 (3H, s H-14), 0.82 (1H, m, H-5)* (signal partially obscured
4
(
residue was dissolved in ethyl acetate and washed with 1 M HCl, water,
and brine before drying over MgSO . The solvent was removed under
4
reduced pressure, and the crude product purified by column
chromatography (9:1−1:1 petroleum ether/ethyl acetate) to afford
the diol 3 as a white solid (254 mg, 73%): mp 68 °C (petroleum ether/
ethyl acetate); [α]25 −6.9 (c 0.1, CHCl ); IR (neat) ν 3276, 2922,
2
5
13
by H-14 signal); C NMR (CDCl , 101 MHz) δ 179.3 (C-12), 67.8 (C-
3
11), 51.6 (C-5), 50.1 (C-9), 42.1 (C-3), 40.6 (C-1), 37.6 (C-8), 35.5 (C-
D
3
max
−1 1
843, 1458, 1386, 1364, 987, 685 cm ; H NMR (CDCl , 400 MHz) δ
10), 33.7 (C-13), 33.0 (C-4), 22.5 (C-7), 22.1 (C-14), 18.6 (C-6), 18.2
3
+
.80−5.76 (1H, m, H-7), 4.33 (1H, d, J = 12.1 Hz, H-12), 3.96 (1H, d, J
12.1 Hz, H-12), 3.88 (1H, dd, J = 10.8, 2.0 Hz, H-11), 3.65 (1H, dd, J =
(C-2), 14.6 (C-15); HRESIMS m/z 259.1644 [M + Na] (calcd for
=
C H NaO , 259.1669).
15
24
2
1
(
0.8, 8.3 Hz, H-11), 2.17−1.82 (4H, m, H-1, H-6, H-6, H-9), 1.61−1.38
3H, m, H-2, H-2, H-3), 1.24 (1H, dd, J = 12.1, 4.6 Hz, H-5), 1.17 (1H,
td, J = 13.4, 3.8 Hz, H-3), 1.13 (1H, td, J = 13.2, 4.3 Hz, H-1), 0.87 (3H,
11,12-Dimethoxy-7-drimene (7). Compound 3 (37 mg, 0.16 mmol)
was dissolved in dimethylformamide and stirred under an inert
atmosphere. NaH (60% dispersion in oil, 62 mg, 1.6 mmol) and
iodomethane (97 μL, 1.6 mmol) were added, and the resulting mixture
13
s, H-14), 0.86 (3H, s, H-13), 0.74 (3H, s, H-15); C NMR (CDCl , 101
3
MHz) δ 137.0 (C-8), 127. Five (C-7), 67.5 (C-12), 61.5 (C-11), 54.5
was stirred overnight. The reaction was quenched with saturated NH Cl,
diluted with ethyl acetate, washed with water and brine, and dried over
4
(
3
C-9), 49.5 (C-5), 42.1 (C-3), 39.4 (C-1), 35.7 (C-10), 33.3 (C-13),
3.1 (C-4), 23.7 (C-6), 22.0 (C-14), 18.9 (C-2), 14.6 (C-15);
MgSO . After removal of solvent under reduced pressure, the resulting
4
+
HRESIMS m/z 261.1817 [M + Na] (calcd for C H NaO ,
oil was purified by column chromatography (petroleum ether/ethyl
1
5
26
2
2
61.1825).
β,9β-Drimane-11,12-diol (4). A flask containing platinum dioxide
12 mg) and ethyl acetate (3 mL) was evacuated and backfilled with
acetate, 9:1) to afford the title compound as a colorless oil (27 mg, 67%):
7
2
25
8
[α] −14.2 (c 1.2, CHCl ); IR (neat) ν 2920, 2865, 2806, 1741,
D
3
max
−
1 1
(
1458, 1387, 1364, 1109, 1087 cm ; H NMR (CDCl , 400 MHz) δ
3
nitrogen. This procedure was repeated three times before refilling with
5.83−5.80 (1H, m, H-7), 3.97 (1H, ddd, J = 11.2, 2.2, 1.1 Hz, H-12),
3.76 (1H, d, J = 11.3 Hz, H-12), 3.54 (1H, dd, J = 9.8, 3.5 Hz, H-11), 3.41
(1H, dd, J = 9.8, 5.5 Hz, H-11), 3.31 (3H, s, OMe-12), 3.28 (3H, s, OMe-
11), 2.12−2.03 (2H, m, H-6, H-9), 1.99−1.87 (2H, m, H-1, H-6), 1.56
(1H, qt, J = 13.3, 3.0 Hz, H-2), 1.49−1.42 (1H, m, H-2), 1.45−1.37 (1H,
m, H-3), 1.22 (1H, dd, J = 12.1, 4.4 Hz, H-5), 1.17 (1H, td, J = 13.2, 3.5
H (balloon). A solution of 3 (40 mg, 0.17 mmol) in ethyl acetate was
2
added, and the reaction stirred overnight. The reaction mixture was
diluted with ethyl acetate and filtered through Celite. Evaporation of the
solvent afforded the crude product, which was recrystallized from
chloroform as colorless crystals (35 mg, 83%): mp 148 °C (chloroform);
2
5
[
1
α] 13.8.0 (c 1.1, CHCl ); IR (neat) ν 3305, 2929, 2895, 1479,
Hz, H-3), 1.06 (1H, td, J = 13.1, 3.6 Hz, H-1), 0.88 (3H, s, H-14), 0.85
D
3
max
−1
1
13
386, 1364, 1041, 1028, 1015 cm ; H NMR (CDCl , 400 MHz) δ 3.94
(3H, s, H-13), 0.82 (3H, s, H-15); C NMR (CDCl , 101 MHz) δ 134.4
3
3
(
3
1
1H, dd, J = 10.4, 6.4 Hz, H-12), 3.83 (1H, dd, J = 11.1, 5.3 Hz, H-11),
.75 (1H, dd, J = 11.1, 9.8 Hz, H-11), 3.60 (1H, dd, J = 10.4, 7.0 Hz, H-
2), 2.23−2.16 (1H, m, H-8), 1.94−1.89 (1H, m, H-7), 1.67−1.59 (1H,
(C-8), 127.2 (C-7), 75.9 (C-12), 70.9 (C-11), 58.5 (OMe-11), 57.9
(OMe-12), 52.2 (C-9), 49.8 (C-5), 42.3 (C-3), 39.7 (C-1), 35.9 (C-10),
33.4 (C-13), 33.1 (C-4), 23.6 (C-6), 22.2 (C-14), 18.9 (C-2), 14.8 (C-
+
m, H-1), 1.59−1.49 (4H, m, H-2, H-6, H-7, H-9), 1.45−1.32 (2H, m, H-
15); HRESIMS m/z 289.2138 [M + Na] (calcd for C H NaO ,
1
7
30
2
2
1
0
, H-3), 1.29−1.22 (1H, m, H-6), 1.16 (1H, td, J = 14.0, 4.3 Hz, H-3),
.02 (1H, td, J = 13.1, 4.0 Hz, H-1), 0.89 (1H, dd, J = 12.0, 1.8 Hz, H-5),
.87 (3H, s, H-15), 0.85 (3H, s, H-13), 0.80 (3H, s, H-14); 13C NMR
289.2138).
74
11,12-Diacetoxy-7-drimene (8). Compound 3 (23 mg, 0.096
mmol) was dissolved in pyridine (1 mL) and, after the addition of acetic
anhydride (1 mL), stirred for 14 h. The reaction mixture was diluted
with ethyl acetate and washed with 2 M HCl, saturated Na CO , water,
(
CDCl , 101 MHz) δ 64.1 (C-12), 60.7 (C-11), 56.5 (C-5), 54.6 (C-9),
3
4
4
2.0 (C-3), 39.5 (C-1), 38.0 (C-8), 37.5 (C-10), 33.7 (C-13), 33.3 (C-
2
3
), 30.2 (C-7), 21.8 (C-14), 18.6 (C-2), 18.5 (C-6), 16.6 (C-15);
and brine, and the resulting organic layer dried over MgSO . After
4
+
HRESIMS m/z 263.1961 [M + Na] (calcd for C H NaO ,
removal of solvent under reduced pressure, the resulting residue was
purified by column chromatography (petroleum ether/ethyl acetate,
12:1−9:1) to afford the desired product (18 mg, 58%) as a colorless oil:
[α]25 14.8 (c 0.55, CHCl ); IR (neat) ν 2923, 1734, 1365, 1223,
1
5
28
2
2
63.1982).
−)-Cinnamolide (5). A solution of 3 (27 mg, 0.11 mmol) in
dichloromethane (15 mL) was treated with manganese dioxide (295 mg,
.3 mmol) and stirred under an aerial atmosphere for 48 h. The reaction
7
3
(
D
3
max
−1 1
3
1021 cm ; H NMR (CDCl , 400 MHz) δ 5.89 (1H, dt, J = 5.1, 2.3 Hz,
3
mixture was filtered through Celite, and the filter cake washed with
dichloromethane. The solvent was removed under reduced pressure to
afford the title compound (23 mg, 88%) as a white solid: mp 109 °C
H-7), 4.53 (1H, dq, J = 12.2, 1.0 Hz, H-12), 4.47 (1H, d, J = 12.1 Hz, H-
12), 4.27 (1H, dd, J = 11.6, 3.1 Hz, H-11), 4.06 (1H, dd, J = 11.6, 7.4 Hz,
H-11), 2.22−2.17 (1H, m, H-9), 2.11 (1H, dtd, J = 18.3, 5.1, 2.0 Hz, H-
6), 2.03 (3H, s, OAc), 1.99 (3H, s, OAc), 1.98−1.88 (2H, m, H-1, H-6),
1.53 (1H, tt, J = 13.6, 3.2 Hz, H-2), 1.49−1.46 (1H, m, H-2), 1.42 (1H,
dtd, J = 13.1, 3.1, 1.7 Hz, H-3), 1.24 (1H, dd, J = 12.2, 4.5 Hz, H-5), 1.17
(1H, td, J = 13.1, 3.9 Hz, H-3), 1.11 (1H, td, J = 12.8, 3.8 Hz, H-1), 0.88
(
dichloromethane); [α]25 = −26.3 (c 0.4, CHCl ); IR (neat) ν 2919,
D
3
max
−1 1
2
863, 1750, 1689, 1219, 1206, 1008, 963, 743 cm ; H NMR (CDCl ,
3
4
00 MHz) δ 6.88 (1H, dt, J = 3.9, 3.4 Hz, H-7), 4.37 (1H, t, J = 9.1 Hz,
H-11), 4.03 (1H, t, J = 9.2 Hz, H-11), 2.82 (1H, tdt, J = 9.3, 5.3, 3.5 Hz,
1
3
H-9), 2.41 (1H, ddt, J = 20.1, 5.3, 3.9 Hz, H-6), 2.11 (1H, dddd, J = 20.1,
(3H, s, H-14), 0.86 (3H, s, H-13), 0.81 (3H, s, H-15); C NMR
1
1.7, 4.9, 3.2 Hz, H-6), 1.64−1.44 (4H, m, H-1, H-2, H-2, H-3), 1.39
(CDCl , 101 MHz) δ 171.2 (OAc), 171.0 (OAc), 131.7 (C-8), 130.6
3
(
1H, dd, J = 11.7, 5.3 Hz, H-5), 1.28−1.16 (2H, m, H-1, H-3), 0.94 (3H,
(C-7), 68.0 (C-12), 63.0 (C-11), 51.0 (C-9), 49.5 (C-5), 42.0 (C-3),
39.4 (C-1), 35.9 (C-10), 33.3 (C-13), 33.1 (C-4), 23.7 (C-6), 22.0 (C-
14), 21.2 (OAc), 21.2 (OAc), 18.8 (C-2), 14.5 (C-15); HRESIMS m/z
13
s, H-14), 0.92 (3H, s, H-13), 0.81 (3H, s, H-15); C NMR (CDCl , 101
3
MHz) δ 170.4 (C-12), 136.5 (C-7), 127.3 (C-8), 67.4 (C-11), 51.0 (C-
9
+
), 49.8 (C-5), 42.3 (C-3), 39.7 (C-1), 34.4 (C-10), 33.3 (C-13), 33.0
345.2018 [M + Na] (calcd for C H NaO , 345.2036).
19
30
4
(
C-4), 25.1 (C-6), 21.5 (C-14), 18.4 (C-2), 13.6 (C-15); HRESIMS m/
8β,9β-Drimane-7α,8α,11,12-tetraol (9). Compound 3 (61 mg, 0.26
mmol) was dissolved in a mixture of acetone and water (4:1, 10 mL) and
cooled to 0 °C. N-Methylmorpholine N-oxide monohydrate (35 mg,
+
z 257.1491 [M + Na] (calcd for C H NaO , 257.1512).
15
22
2
cis-(−)-Dihydrocinnamolide (6). A flask containing platinum dioxide
3 mg) and ethyl acetate (2 mL) was evacuated and backfilled with
(
0.26 mmol) was added, followed by OsO (5 drops, 1% solution), and
4
nitrogen. This procedure was repeated three times before refilling with
the reaction allowed to warm to room temperature. After 12 h the
H (balloon). A solution of 5 (25 mg, 0.11 mmol) in ethyl acetate was
2
reaction mixture was extracted into ethyl acetate (3 × 15 mL), and the
H
DOI: 10.1021/acs.jnatprod.6b01056
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
combined organic layers were washed with brine. After drying over
sodium sulfate, the solvent was removed under reduced pressure and the
residue purified by column chromatography (ethyl acetate) to afford the
title compound as a white solid (57 mg, 83%): mp 172 °C (ethyl
acetate); [α]25 −24.9 (c 0.5, MeOH); IR (neat) ν 2926, 2885, 2504,
over sodium sulfate, and the solvent was removed in vacuo. Purification
by column chromatography (petroleum ether/ethyl acetate, 1:1)
provided the target compound (14 mg, 78%): [α]25 −16.0 (c 0.7,
D
−1 1
CHCl ); IR (neat) ν 3398, 2916, 2848, 1460, 1000, 752 cm ; H
3
max
NMR (CDCl , 400 MHz) δ 5.89 (1H, dd, J = 5.1, 1.8 Hz, H-7), 4.31
D
max
3
−1 1
2
385, 1441, 1373, 1080, 1047, 858, 678 cm ; H NMR (CD OD, 500
(1H, d, J = 12.6 Hz, H-12), 4.13 (1H, d, J = 12.5 HzH-12), 3.77 (1H, d, J
= 11.7 Hz, H-11), 3.73 (1H, d, J = 12.0 Hz, H-11), 2.12 (1H, dt, J = 18.5,
5.0 Hz, H-6), 1.91 (1H, dd, J = 18.2, 12.4 Hz, H-6), 1.83−1.77 (1H, m,
H-1), 1.72 (1H, dd, J = 12.2, 4.7 Hz, H-5), 1.57−1.48 (2H, m, H-2, H-2),
1.43 (1H, d, J = 11.1 Hz, H-1), 1.40 (1H, d, J = 13.3 Hz, H-3), 1.28−1.20
(1H, m, H-3), 0.92 (3H, s, H-14), 0.90 (3H, s, H-13), 0.81 (3H, s, H-
3
MHz) δ 3.93 (1H, dd, J = 11.1, 7.3 Hz, H-11), 3.92 (1H, dd, J = 3.4, 2.0
Hz, H-7), 3.78 (1H, dd, J = 11.1, 4.5 Hz, H-11), 3.68 (1H, d, J = 11.6 Hz,
H-12), 3.60 (1H, d, J = 11.6 Hz, H-12), 1.82 (1H, dd, J = 12.8, 3.8 Hz, H-
6
), 1.80 (1H, dd, J = 7.1, 4.5 Hz, H-9), 1.74 (1H, dtd, J = 12.8, 3.4, 1.4 Hz,
H-1), 1.69−1.58 (1H, m, H-2), 1.59 (1H, d, J = 13.3 Hz, H-5), 1.57 (1H,
13
td, J = 12.9, 2.1 Hz, H-6), 1.51−1.43 (1H, m, H-2), 1.41 (1H, dtd, J =
15); C NMR (CDCl , 101 MHz) δ 138.1 (C-8), 132.0 (C-7), 75.8 (C-
3
1
3.2, 3.5, 1.4 Hz, H-3), 1.23 (1H, td, J = 13.4, 3.9 Hz, H-3), 1.15 (1H, td,
9), 67.0 (C-12), 62.7 (C-11), 42.4 (C-5), 41.7 (C-3), 40.6 (C-10), 33.5
J = 12.9, 3.5 Hz, H-1), 0.88 (3H, s, H-13), 0.84 (3H, s, H-15), 0.83 (3H,
s, H-14); C NMR (CD OD, 126 MHz) δ 77.4 (C-8), 69.5 (C-7), 64.3
(C-13), 33.1 (C-4), 31.2 C-1), 24.2 (C-6), 22.4 (C-14), 18.7 (C-2), 15.4
13
+
(C-15); HRESIMS m/z 277.1778 [M + Na] (calcd for C15
H
26NaO
,
3
3
(
C-12), 60.0 (C-11), 56.1 (C-9), 47.5 (C-5), 43.1 (C-3), 40.9 (C-1),
277.1774).
3
8.4 (C-10), 33.6 (C-4, C-13), 27.1 (C-6), 22.0 (C-14), 19.7 (C-2), 16.4
+
(
C-15); HRESIMS m/z 295.1870 [M + Na] (calcd for C H NaO ,
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.6b01056.
15
28
4
■
2
95.1880).
β,9β-Drimane-[7,8-α]-oxirene-11,12-diol (10). A solution of
compound 3 (58 mg, 0.24 mmol) in CH Cl was treated with m-
*
S
8
2
2
chloroperoxybenzoic acid (mCPBA) (46 mg, 0.48 mmol, estimated
purity 55%). After 4 h a further portion of mCPBA was added (23 mg,
0
.24 mmol). After a total reaction time of 6 h, the reaction mixture was
1
13
H NMR and C NMR data for all synthetic compounds
PDF)
diluted with CH Cl , washed with saturated sodium bicarbonate, water,
2
2
(
and brine, and dried over sodium sulfate. The solvent was removed, and
the residue purified by column chromatography (petroleum ether/ethyl
acetate, 9:1−1:1), affording a mixture of the product and a structurally
related minor product. Recrystallization from petroleum ether provided
AUTHOR INFORMATION
Corresponding Author
*Tel: +46 706 855075. Fax: +46 10 512 37 49. E-mail: Johan.
svenson@sp.se.
■
1
0 as colorless crystals (44 mg, 72%): mp 116 °C dec (petroleum ether);
25
[α] +2.8 (c 1.15, CHCl ); IR (neat) ν 3301, 2923, 1456, 1387,
D 3 max
−1 1
1
071, 1040, 993, 753 cm ; H NMR (CDCl , 400 MHz) δ 4.13 (1H, d,
3
J = 12.5 Hz, H-12), 3.91 (1H, dd, J = 12.0, 2.4 Hz, H-11), 3.79 (1H, dd, J
ORCID
=
12.0, 9.3 Hz, H-11), 3.36 (1H, d, J = 12.5 Hz, H-12), 3.21 (1H, t, J = 2.0
Johan Svenson: 0000-0002-4729-9359
Hz, H-7), 2.13 (1H, dd, J = 15.1, 3.6 Hz, H-6), 1.86 (1H, dq, J = 13.0, 2.8
Hz, H-1), 1.73 (1H, dd, J = 9.3, 2.2 Hz, H-9), 1.66 (1H, ddd, J = 15.2,
Present Address
¶
1
1
1
6
3
1
2.8, 2.4 Hz, H-6), 1.49−1.35 (3H, m, H-2, H-2, H-3), 1.11 (1H, dd, J =
Department of Chemistry, Umea
Sweden.
̊ ̊
University, SE-901 87, Umea,
2.9, 4.1 Hz, H-5), 1.22−1.01 (2H, m, H-1, H-3), 0.86 (6H, m, H-13, H-
13
4), 0.76 (3H, s, H-15); C NMR (CDCl , 101 MHz) δ 67.3 (C-12),
3
Notes
1.2 (C-8), 60.5 (C-11), 59.6 (C-7), 56.4 (C-9), 44.6 (C-5), 41.9 (C-3),
9.5 (C-1), 35.1 (C-10), 33.0 (C-4), 33.0 (C-13), 22.6 (C-6), 22.4 (C-
4), 18.8 (C-2), 15.6 (C-15); HRESIMS m/z 277.1771 [M + Na]+
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
(
calcd for C H NaO , 277.1774).
15 26 3
■
7
4
1
1,12-Diacetoxy-7-drimen-9α-ol (11). A solution of diacetate 8
This work was partly supported with grants from the Norwegian
Research Council (ES508288), and J.S. and L.W.K.M. are
grateful for stipend support. J.S. was supported further by a
VINNMER M.C. incoming grant from VINNOVA (Grant 2014-
(
50 mg, 0.16 mmol) in 1,4-dioxane (6 mL) was treated with SeO (11
2
mg, 0.1 mmol) and heated at 95 °C. After 1 h, a further portion of SeO2
11 mg, 0.1 mmol) was added and stirred for a further 7 h. The reaction
(
mixture was allowed to cool to room temperature and filtered through
0
1435). H.P. and G.C. were supported by the Centre for Marine
Celite (washed with CH Cl /MeOH, 19:1). After solvent removal, the
2
2
Chemical Ecology (http://www.cemace.science.gu.se) at the
University of Gothenburg. The authors are grateful to the New
Zealand Ministry of Business, Innovation and Employment
(MBIE) for funding the Biocide Tool Box research program
product was purified by column chromatography (petroleum ether/
ethyl acetate, 12:1−9:1) to yield the target molecule as a colorless oil (35
mg, 67%): [α]25 −46.3 (c 1.2, CHCl ); IR (neat) ν 2918, 1735,
D
3
max
−1 1
1
5
366, 1225, 1022 cm ; H NMR (CDCl , 500 MHz) δ 6.04 (1H, dd, J =
3
.5, 2.0 Hz, H-7), 4.62 (1H, d, J = 12.3 Hz, H-12), 4.57 (1H, d, J = 12.1
(
UOAX1410), which supported the involvement of P.C. in this
Hz, H-12), 4.28 (1H, d, J = 11.9 Hz, H-11), 4.20 (1H, d, J = 11.8 Hz, H-
1), 2.14 (1H, dt, J = 18.7, 4.8 Hz, H-6), 2.05 (3H, s, OAc-12), 2.04 (3H,
s, OAc-11), 1.93 (1H, ddt, J = 18.7, 12.4, 1.6 Hz, H-6), 1.81 (1H, dd, J =
research project.
1
REFERENCES
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.93 (3H, s, H-14), 0.90 (3H, s, H-13), 0.90 (3H, s, H-15); 13C NMR
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