Spiralisones A-D: Acylphloroglucinol hemiketals from an Australian marine brown alga, Zonaria spiralis

Article abstract of DOI:10.1039/c2ob26988k

An intertidal sample of the Australian marine brown alga, Zonaria spiralis, exhibited promising kinase inhibitory and antibacterial activity. Chemical analysis returned six phloroglucinol-derived lipids, the new hemiketal spiralisones A-D (1-4) and the known chromones 5-6, and the known norsesquiterpenoid apo-9′-fucoxanthinone (7). Structures 1-7 were assigned on the basis of detailed spectroscopic analysis, biosynthetic considerations and total synthesis. Spiralisones undergo facile acid-mediated dehydration to yield the corresponding chromones, revealing for the first time that brown algal chromones may be handling artifacts rather than natural products. Hemiketals 1 and 2, and chromone 6, displayed inhibitory activity against the neurodegenerative disease kinase targets CDK5/p25, CK1δ and GSK3β, while hemiketals 1, 3 and 4, and chromone 6, displayed growth inhibitory activity against the Gram-positive bacteria Bacillus subtilis (ATCC 6051 and 6633). The promising kinase inhibitory and antibacterial properties of the Z. spiralis extract were attributed to the cumulative effect of many moderately potent phloroglucinol-derived lipid co-metabolites. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2ob26988k

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Organic &
Biomolecular
Chemistry
PAPER
Spiralisones A–D: acylphloroglucinol hemiketals from
an Australian marine brown alga, Zonaria spiralis†
Cite this: Org. Biomol. Chem., 2012, 10,
9671
Hua Zhang,‡§ Xue Xiao,‡ Melissa M. Conte, Zeinab Khalil and Robert J. Capon*
An intertidal sample of the Australian marine brown alga, Zonaria spiralis, exhibited promising kinase
inhibitory and antibacterial activity. Chemical analysis returned six phloroglucinol-derived lipids, the new
hemiketal spiralisones A–D (1–4) and the known chromones 5–6, and the known norsesquiterpenoid
apo-9’-fucoxanthinone (7). Structures 1–7 were assigned on the basis of detailed spectroscopic analysis,
biosynthetic considerations and total synthesis. Spiralisones undergo facile acid-mediated dehydration to
yield the corresponding chromones, revealing for the first time that brown algal chromones may be
handling artifacts rather than natural products. Hemiketals 1 and 2, and chromone 6, displayed inhibitory
activity against the neurodegenerative disease kinase targets CDK5/p25, CK1δ and GSK3β, while hemi-
ketals 1, 3 and 4, and chromone 6, displayed growth inhibitory activity against the Gram-positive bacteria
Bacillus subtilis (ATCC 6051 and 6633). The promising kinase inhibitory and antibacterial properties of the
Z. spiralis extract were attributed to the cumulative effect of many moderately potent phloroglucinol-
derived lipid co-metabolites.
Received 12th October 2012,
Accepted 1st November 2012
DOI: 10.1039/c2ob26988k
www.rsc.org/obc
efforts to develop clinically useful drugs to treat Alzheimer’s
diseases have included the search for inhibitors of BACE,
Introduction
Neurodegenerative diseases represent a very significant chal- CK1δ, CDK5 and GSK3β. Another major challenge to modern
lenge to modern healthcare, none more so than Alzheimer’s healthcare is that of infectious diseases, where clinically
disease where patients are confronted with progressive and important antibiotics are increasingly compromised and ren-
irreversible deterioration of cognitive ability, and a pathology dered less effective by drug resistance.
characterized by the aggregation and deposition of misfolded
In an effort to address the dual healthcare challenges
proteins.1 More specifically, on autopsy the brains of associated with neurodegenerative and infectious diseases,
Alzheimer’s patients exhibit amyloid pathology, mediated by we implemented a marine biodiscovery program in which
β-secretase (BACE) processing of the essential neuronal trans- ∼2600 southern Australian and Antarctic marine invertebrates
membrane amyloid precursor protein (APP) to generate and algae were screened for inhibitory activity against BACE
amyloid plaques,2 and tau pathology mediated by kinases (27, 1%), CK1δ (44, 1.7%), CDK5 (26, 1%) and GSK3β
such as casein kinase 1δ (CK1δ), cyclin-dependent kinase (69, 2.6%), as well as for antibiotic activity. This approach has
5 (CDK5) and glycogen synthase kinase 3β (GSK3β), that hyper- proved successful in the past, as evidenced by our recent
phosphorylate highly soluble microtubule-associated tau reports on biologically active alkaloids spanning the ianthelli-
protein to generate neurofibrillary tangles.3 Not surprisingly, done,4 dictyodendrin,5 lamellarin,6 ningalin,7 and massadine8
structure classes. In this report we describe our investigations
into an intertidal collection of the southern Australian brown
alga, Zonaria spiralis, which exhibited promising inhibitory
activities against BACE, CDK5 and GSK3β, as well as Gram-
Division of Chemistry and Structural Biology, Institute for Molecular Bioscience,
The University of Queensland, St. Lucia, Queensland 4072, Australia.
E-mail: r.capon@uq.edu.au; Fax: +61 7 3346 2090; Tel: +61 7 3346 2979
positive antibacterial activity. Comprehensive chemical
analysis yielded four new phloroglucinol-derived hemiketal
lipids, spiralisones A–D (1–4), as well as the known chromone
co-metabolites 5,7-dihydroxy-2-tridecanyl chromone (5)9
and 5,7-dihydroxy-2-(4Z,7Z,10Z,13Z,16Z-nonadecapentaenyl)-
chromone (6),10 and the degraded norterpene apo-9′-
†Electronic supplementary information (ESI) available: Details of synthetic pro-
cedures and bioassays, full NMR data for both natural and synthetic compounds,
along with 1H and/or 13C NMR spectra for all compounds, as well as representa-
tive figures of bioassay results. See DOI: 10.1039/c2ob26988k
‡These authors contribute equally to this paper.
§Current address: State Key Laboratory of Drug Research, Shanghai Institute of
Materia Medica, Chinese Academy of Sciences, Shanghai 201023, P. R. China.
This journal is © The Royal Society of Chemistry 2012
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fucoxanthinone (7).11
correlations (Fig. 1) allowed assembly of the structure elements
listed above, revealing spiralisone A (1) as an acylated phloro-
glucinol further cyclized to a hemiketal bearing a pendant
nonadeca-4,7,10,13-tetraenyl sidechain. The methylene inter-
rupted regiochemistry and Z geometry of the sidechain double
bonds were inferred from diagnostic overlapping resonances
for H2-6′, H2-9′ and H2-12′ (δH 2.77–2.83); shielded signals for
C-6′, C-9′ and C-12′ (δC 25.92, 25.89, and 25.89);12 HMBC corre-
lations (Fig. 1); and spectroscopic comparisons with authentic
unsaturated fatty acids such as arachidonic acid. With a zero
optical rotation spiralisone A (1) was assigned the racemic
structure as shown.
High resolution ESI(+)MS analysis of spiralisone B (2)
revealed an adduct ion ([M + Na]+) consistent with a molecular
formula (C28H36O5, Δmmu −0.6) indicative of a dehydro ana-
logue of 1. Comparison of the NMR data for 2 and 1 (Table 1)
confirmed this hypothesis with the only significant differences
being attributed to an additional methylene interrupted Δ16′
double bond (δH 5.30 and 5.38; δC 127.2 and 132.3) in 2. These
observations, together with a zero optical rotation, permitted
structure assignment of spiralisone B (2) as indicated (Fig. 2).
High resolution ESI(+)MS analysis of spiralisones C (3) and
D (4) revealed adduct ions ([M + Na]+) consistent with molecu-
lar formulae (C24H38O5 and C22H34O5; Δmmu 0.5 and −0.7,
respectively) indicative of lower saturated homologues of 1.
Comparison of the NMR data for 3 and 4 with 1 (Table 1) con-
firmed this hypothesis, with the differences being attributable
to pentadecanyl and tridecanyl sidechains as indicated. These
considerations, together with zero optical rotations, permitted
assignment of the racemic structures to spiralisones C (3) and
Results and discussion
A portion of the aqueous EtOH extract derived from Zonaria D (4) as indicated.
spiralis was decanted, concentrated in vacuo and subjected to
High resolution ESI(+)MS analysis of 5 and 6 revealed
sequential solvent trituration and reversed phase chromato- adduct ions ([M + Na]+) consistent with molecular formulae
graphy (SPE and HPLC) to yield the new spiralisones A–D (C22H32O4 and C28H34O4; Δmmu 0.8 and 0.6, respectively)
(1–4), along with the known co-metabolites 5–7. Structures indicative of dehydration analogues of 4 and 2, respectively.
were assigned to 1–7 on the basis of detailed spectroscopic Comparison of the NMR data for 5 with 4, and 6 with 2
analysis supported by chemical synthesis. An account of the (Table 1), revealed resonances diagnostic for chromone moi-
structure elucidation of spiralisones A–D (1–4), 5,7-dihydroxy- eties, permitting identification of 5 as 5,7-dihydroxy-2-trideca-
2-tridecanyl chromone (5)9 and 5,7-dihydroxy-2-(4Z,7Z,10Z,- nyl chromone, and 6 as 5,7-dihydroxy-2-(4Z,7Z,10Z,13Z,16Z-
13Z,16Z-nonadecapentaenyl) chromone (6),10 inclusive of syn- nonadecapentaenyl) chromone. First reported in 1987 from a
thetic studies, is outlined below.
mass spectrometric analysis of a rhododendron lace bug,9 our
High resolution ESI(+)MS analysis of spiralisone A (1) re-isolation of 5 provided the first comprehensive spectro-
revealed an adduct ion ([M + Na]+) consistent with a molecular scopic analysis and synthesis (see below). First reported in
formula (C28H38O5, Δmmu −0.6) requiring ten double bond 1982 from the Pacific brown alga Zonaria tournefortii,10a 6 was
equivalents (DBE). Analysis of the NMR (CDCl3) data for the subject of a 2009 total synthesis from 2,4,6-trihydroxy-
1 (Table 1) revealed resonances attributable to a ketone benzaldehyde and the ethyl ester of eicosapentaenoic acid.10b
(δC 195.0), a tetrasubstituted benzene bearing three oxy substi-
Discovery of chromones 5 and 6, as co-metabolites with
tuents (δC 164.7, 164.2, 160.2, 102.9, 97.0 and 96.2) and four hemiketals 4 and 2, raised the possibility that the former may
1,2-disubstituted double bonds (δC 130.8, 129.2 (x2), 128.9, be handling artifacts (dehydration products) of the latter.
128.5, 128.3, 128.1 and 127.7), accounting for nine double Indeed, during our studies we noted that hemiketals 4 and 2
bond equivalents (DBE) and requiring an additional ring underwent ready dehydration to return chromones 5 and 6
system. A quaternary hemiketal carbon (δC 102.5) accounted when exposed to mildly acidic conditions, such as acidic chro-
for the final oxygen atom and permitted assembly of the matographic media (e.g. silica) and spectroscopic solvents (e.g.
1
required ring system, while exchangeable H NMR resonances CDCl3). This observation prompted us to propose a plausible
were attributed to non-hydrogen bonded (δH 5.70) and hydro- biosynthesis (Fig. 3), and develop a short biomimetic synthesis
gen bonded (δH 11.92) phenol protons, respectively. Key HMBC (Scheme 1).
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Table 1 NMR data (CDCl3, 600 MHz) for spiralisones A–D (1–4)
1
2
3
4
No.
δC
δH (m., J in Hz)
δC
δH (m., J in Hz)
δC
δH (m., J in Hz)
δC
δH (m., J in Hz)
2
3
102.5
45.1
102.5
45.1
102.6
45.1
102.6
45.1
a 2.88 (d, 16.9)
b 2.74 (d, 16.9)
a 2.88 (d, 17.2)
b 2.74 (d, 17.2)
a 2.88 (d, 16.9)
b 2.75 (d, 16.9)
a 2.88 (d, 16.9)
b 2.74 (d, 16.9)
4
5
6
7
195.0
164.2
97.0
164.7
96.2
195.1
164.1
97.0
164.9
96.3
195.1
164.2
96.9
164.7
96.1
195.1
164.2
96.9
164.6
96.2
5.96 (br s)
5.89 (br s)
5.95 (d, 1.7)
5.89 (d, 1.7)
5.96 (d, 1.6)
5.89 (d, 1.6)
5.96 (br s)
5.89 (br s)
8
9
10
1′
102.9
160.2
40.8
102.8
160.2
40.8
103.0
160.2
41.4
102.9
160.3
41.4
1.89 (br t, 8.1)
1.58 (m)
1.89 (br t, 8.2)
1.58 (m)
1.87 (br t, 7.6)
1.48 (m)
1.87 (br t, 7.6)
1.48 (m)
2′
23.4
23.4
23.4
23.4
3′
4′
5′
6′
7′
8′
9′
10′
11′
12′
13′
14′
15′
16′
17′
18′
19′
5-OH
7-OH
27.0
2.15 (m)
27.0
2.14 (m)
29.73a
29.92a
29.91a
29.90a
29.88a
29.87a
29.8a
29.69a
29.68a
29.6a
32.2
1.34 (m)
29.73a
29.90a
29.87a
29.87a
29.8a
29.69a
29.68a
29.6a
32.1
1.34 (m)
129.2
129.2a
25.92a
128.9a
128.5a
25.89a
128.3a
128.1a
25.89a
127.7
130.8
27.5
5.30–5.43 (m)
5.30–5.43 (m)
2.77–2.85 (m)
5.30–5.43 (m)
5.30–5.43 (m)
2.77–2.85 (m)
5.30–5.43 (m)
5.30–5.43 (m)
2.77–2.85 (m)
5.31 (m)
5.38 (m)
2.03 (m)
1.34 (m)
1.26 (m)
129.17
129.23a
25.91a
128.8a
128.5a
25.88a
128.4a
128.34a
25.86a
128.29a
128.1a
25.8a
127.2
132.3
20.8
5.32–5.44 (m)
5.32–5.44 (m)
2.77–2.85 (m)
5.32–5.44 (m)
5.32–5.44 (m)
2.77–2.85 (m)
5.32–5.44 (m)
5.32–5.44 (m)
2.77–2.85 (m)
5.32–5.44 (m)
5.32–5.44 (m)
2.77–2.85 (m)
5.30 (m)
1.20–1.30 (m)
1.20–1.30 (m)
1.20–1.30 (m)
1.20–1.30 (m)
1.20–1.30 (m)
1.20–1.30 (m)
1.20–1.30 (m)
1.20–1.30 (m)
1.20–1.30 (m)
1.24 (m)
1.21–1.30 (m)
1.21–1.30 (m)
1.21–1.30 (m)
1.21–1.30 (m)
1.21–1.30 (m)
1.21–1.30 (m)
1.21–1.30 (m)
1.24 (m)
22.9
14.3
1.28 (m)
0.86 (t, 7.0)
22.9
14.3
1.27 (m)
0.86 (t, 7.0)
29.5
31.7
22.8
14.3
5.38 (m)
2.05 (m)
0.95 (t, 7.5)
11.91 (s)
5.99 (br s)
1.29 (m)
0.87 (t, 7.0)
11.92 (s)
14.5
11.93 (s)
5.52 (br s)
11.93 (s)
5.63 (br s)
5.70 (br s)
Fig. 1 Key 2D NMR correlations for spiralisone A (1).
Fig. 3 Plausible mixed polyketide/fatty acid biosynthesis of the hemiketal spir-
alisones A–D (1–4) and the chromones 5–6.
Fig. 2 Key 2D NMR correlations for spiralisone B (2).
example where X = coenzyme A [SCoA] or acyl carrier protein
[ACP], to yield acylphloroglucinol β-diketone intermediates.
The latter undergo rapid non-enzyme mediated (iii) intra-
molecular cyclization to yield hemiketals (spiralisones), and
(iv) dehydration to deliver chromones. The racemic nature of
spiralisone hemiketals supports a non-enzyme mediated cycli-
zation pathway.
The biosynthetic pathway outlined in Fig. 3 proceeds from
the common algal polyketide precursor phloroglucinol, en-
gaging with fatty acid biosynthesis. We hypothesize that (i)
condensation of acetyl-CoA and phloroglucinol can generate
an acylphloroglucinol intermediate, which in turn undergoes
(ii) sidechain elongation by a Claisen-type condensation
with an array of activated unsaturated/saturated fatty acids, for
Our biomimetic synthesis of spiralisone D (4) and the chro-
mone 5 (Scheme 1) draws on our proposed biosynthesis, and
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Zonaria spiralis extract as arising from the cumulative effect of
multiple moderately potent spiralisone inhibitors.
The co-metabolites 1–7 were assessed for antibacterial and
antifungal activities (ESI Table S8†). The hemiketals 1, 3, and
4, and the chromone 6, all exhibited moderate growth inhibi-
tory activity against Bacillus subtilis (ATCC 6051 and 6633) (IC50
2.5–10 μM), while none of the Zonaria spiralis metabolites 1–7
inhibited the Gram-positive bacteria Staphylococcus aureus
(ATCC 9144 and 25923), the Gram-negative bacteria Escherichia
coli (ATCC 11775) and Pseudomonas aeruginosa (ATCC 10145),
or the fungus Candida albicans (ATCC 90028). As with the
kinase inhibitory activity, the antibacterial properties of the
crude Zonaria spiralis extract was attributed to the “cumulative
effect” of multiple weakly antibacterial co-metabolites.
Scheme 1 Biomimetic synthesis of spiralisone D (4) and the chromone 5. (i)
tetradecanal, LDA, THF, −78 °C; (ii) DMP, CH2Cl2, 0 °C; (iii) BBr3, CH2Cl2, −78 °C ∼
rt; (iv) nPrSLi, HMPA, 80 °C.
Conclusions
In summary, our bioassay guided investigation into the chem-
istry of an Australian marine brown alga Zonaria spiralis led to
the discovery of an unprecedented class of acylphloroglucinol
hemiketals, the spiralisones A–D (1–4) which undergo facile
dehydration to chromones under mildly acidic conditions. Iso-
lation and characterization of these metabolites necessitated
great care, and highlighted the possibility that previously
reported algal chromones might be handling (dehydration)
artifacts, rather than natural products as previously supposed.
We proposed a plausible mixed polyketide/fatty acid biosyn-
thesis, and used this to develop successful biomimetic synth-
eses of the hemiketals 1 and 4, and their respective chromones
5 and 9. The spiralisones were established as the dominant
kinase inhibitory and antibacterial agents in the crude Zonaria
spiralis extract, with the potency displayed by the crude extract
being attributed to the cumulative concentration effect of
multiple mildly bioactive members of this class of mixed bio-
synthesis co-metabolites.
Scheme 2 Biomimetic synthesis of spiralisone A (1) and its corresponding
chromone (9). (i) arachidonal, LDA, THF, −78 °C; (ii) DMP, CH2Cl2, 0 °C; (iii) HCl/
MeOH, rt; (iv) TFA, CH2Cl2.
involves (i) aldol condensation of tridecanal and permethy-
lated acetylphloroglucinol, followed by (ii) oxidation of the
resulting alcohol to deliver the β-diketone product as an equili-
brium mixture of keto–enol tautomers. Deprotection with (iii)
BBr3 in CH2Cl2 initiated spontaneous cyclization to deliver the
target hemiketal 4 (30%), along with a methyl hemiketal side-
product (40%) and the chromone 5 (10%), whereas deprotec-
tion with (iv) lithium propane-1-thiolate in HMPA returned a
high yield of the chromone 5 (75%). Synthetic samples of 4
and 5 were identical in all respects with those isolated from
Zonaria spiralis.
Experimental section
General experimental procedures
Optical rotations were measured on a JASCO P-1010 polari-
meter with a 10 cm length cell. UV spectra were obtained on a
Cary 50 UV-visible spectrophotometer with 1 cm pathway cell.
While (i) and (ii) aldol coupling (Scheme 2) of arachidonal
with permethylated acetylphloroglucinol was successful, the
arachidonyl side chain proved unstable to the harsh methyl NMR experiments were performed on a Bruker Avance DRX600
spectrometer and referenced to residual 1H signals in the deut-
ether deprotection protocols. Fortunately, the corresponding
methoxymethyl (MOM) protected acetylphloroglucinol did
undergo successful deprotection with (iii) HCl/MeOH, to Agilent 1100 series LC/MSD instrument. HR-ESIMS data were
erated solvents. ESIMS experiments were carried out on an
return low yields of 1 (16%) and its corresponding chromone 9
(33%), and (iv) TFA in CH2Cl2, to return a high yield of 9 (85%).
acquired on a Bruker micrOTOF mass spectrometer by direct
infusion in MeCN at 3 μL min−1 using sodium formate clusters
Whereas the two new hemiketals 1, 2 and the chromone 6 as an internal calibrant. All HPLC analyses and purifications
showed inhibitory activity against CDK5/p25 (IC50 10, 3.0 and
10.0 μM, respectively), CK1δ (all IC50 < 10 μM) and GSK3β (all
IC50 < 10 μM), 3–5 and 7 failed to exhibit inhibitory activity up
to 30 μM (ESI Table S8†). None of the Zonaria spiralis meta-
bolites 1–7 inhibited BACE up to 10 μM. We attribute the
were performed on Agilent 1100 series LC instruments with
corresponding detectors, collectors and software inclusively.
All chemicals were purchased from Merck, Sigma-Aldrich or
Fluka. Solvents used for general purposes were of at least
analytical grade, and solvents used for HPLC were of HPLC
promising kinase inhibitory potency detected in the crude grade. Agilent Zorbax SB-C8 (5 μm, 4.6 × 150 mm (analytical)
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and 9.4 × 250 mm (semi-preparative)) columns were used for 13C NMR (CDCl3, 600 MHz) Table 1 and ESI Table S3;† ESI(+)
HPLC analyses and separations. A normal phase Biotage SNAP MS m/z 407 [M + H]+, ESI(−)MS m/z 405 [M − H]−; HR-ESI(+)
50 g cartridge and an Alltech SPE 5 g C18 cartridge were also MS m/z 429.2606 [M + Na]+ (calcd for C24H38O5Na, 429.2611).
used for crude fractionation. Flash column chromatography
was performed on silica gel LC60A (40–63 mm, Davisil).
SPIRALISONE D (4). Off-white solid; [α]D22 +0.8 (c 0.2, CHCl3); UV
1
(MeOH) λmax (log ε) 225 (4.22) and 288 (4.23) nm; H and 13C
NMR (CDCl3, 600 MHz) Table 1 and ESI Table S4;† ESI(+)MS
m/z 379 [M + H]+, ESI(−)MS m/z 377 [M − H]−; HR-ESI(+)MS
Collection and taxonomy
The alga material identified as Zonaria Spiralis (Order: Dictyo- m/z 401.2305 [M + Na]+ (calcd for C22H34O5Na, 401.2298).
tales; Family: Dictyotaceae) was collected in January 1993 by
5,7-DIHYDROXY-2-TRIDECANYL CHROMONE (5).9 Off-white solid; UV
the author (Capon) from intertidal rock platforms, near North (MeOH) λmax (log ε) 227 (4.14), 248 (4.14), 255 (4.12) and 292
1
Walkerville, Victoria, Australia.
(3.79) nm; H and 13C NMR (CDCl3, 600 MHz) ESI Table S5;†
ESI(+)MS m/z 361 [M + H]+, ESI(−)MS m/z 359 [M − H]−;
HR-ESI(+)MS m/z 383.2185 [M + Na]+ (calcd for C22H32O4Na,
Extraction and isolation
A portion (ca. 200 mL) of the EtOH extract of Zonaria spiralis 383.2193).
was decanted and concentrated in vacuo to return a dark
5,7-DIHYDROXY-2-(4Z,7Z,10Z,13Z,16Z-NONADECAPENTAENYL)
CHRO-
brown solid (1.7 g), which was subsequently partitioned MONE (6).10 Off-white solid; UV (MeOH) λmax (log ε) 221 (4.12),
between n-BuOH (2 × 40 mL) and H2O (50 mL). Bioassay analy- 238 (4.12), 245 (4.10) and 279 (3.76) nm; 1H and 13C NMR
sis localized the active agents in the n-BuOH solubles (1.3 g), (CDCl3, 600 MHz) ESI Table S6;† HR-ESI(+)MS m/z 435.2527
which was sequentially triturated with n-hexane (6 × 5 mL), [M + H]+ (calcd for C28H35O4, 435.2530).
CH2Cl2 (4 × 5 mL) and MeOH (2 × 5 mL) and concentrated
in vacuo to yield 93 mg, 211 mg and 873 mg solvent tritura- (c 0.04, MeOH); UV (MeOH) λmax (log ε) 206 (3.40), 218 (3.47)
tions respectively.
nm; 1H and 13C NMR (CD3OD, 600 MHz) ESI Table S7;†
APO-9′-FUCOXANTHINONE (7).11 Off-white solid; [α]2D2 −23.0
The CH2Cl2 solubles were eluted through a normal phase HR-ESI(+)MS m/z 289.1402 [M + Na]+ (calcd for C15H22O4Na,
Biotage SNAP cartridge using gradient elution from 92% 289.1410).
n-hexane/EtOAc to 34% n-hexane/EtOAc to return fractions I
(6.4 mg), II (143 mg) and III (62 mg). A portion (80 mg) of frac-
tion II was subjected to HPLC fractionation (Zorbax SB-C8
semi-preparative column, 4.0 mL min−1 gradient elution from
25% H2O/MeCN to 15% H2O/MeCN over 15 min) to yield,
in order of elution, spiralisone B (2, 38.8 mg), spiralisone D
(4, 2.0 mg), spiralisone A (1, 2.3 mg), chromone 6 (11.7 mg),
spiralisone C (3, 1.1 mg), and chromone 5 (1.2 mg). Fraction
III (62 mg) was subjected to SPE fractionation (Alltech C18 SPE
cartridge using a 10 mL stepwise gradient elution from 50%
H2O/MeOH to 100% MeOH) to return fractions IIIA–G.
Fraction IIIB (5.1 mg) was further fractionated by HPLC
(Zorbax SB-C8 semi-preparative column, 4.2 mL min−1 gradient
elution from 70% H2O/MeCN to 60% H2O/MeCN over 15 min)
Synthetic studies
to yield apo-9′-fucoxanthinone (7, 0.5 mg).
(Note – metabolite % yields were calculated as a weight to
weight estimate against the total n-BuOH solubles (1.3 g); 1
GENERAL PROCEDURES OF THE SYNTHESIS OF KETO–ENOL TAUTOMERS
Keto–enol (12a, 12a′). LDA (0.5 mL, 1.0 mmol, 2.0 M in THF)
(0.32%), 2 (5.3%), 3 (0.15%), 4 (0.28%), 5 (0.17%), 6 (1.6%) was added to a solution of 2-acetyl-1,3,5-trimethoxybenzene
and 7 (0.04%).)
10a (100 mg, 0.5 mmol) in THF (15 mL) at −78 °C, and the
SPIRALISONE A (1). Off-white solid; [α]D22 +0.9 (c 0.23, CHCl3); reaction mixture was stirred at this temperature for 30 min.
1
UV (MeOH) λmax (log ε) 227 (4.28) and 288 (4.20) nm; H and Tetradecanal (303 mg, 1.4 mmol in THF) was then added drop-
13C NMR (CDCl3, 600 MHz) Table 1 and ESI Table S1;† ESI(+) wise and the resulting reaction mixture was stirred for a
MS m/z 455 [M + H]+, ESI(−)MS m/z 453 [M − H]−; HR-ESI(+) further 3 h at −78 °C before quenching by addition of a satu-
MS m/z 477.2617 [M + Na]+ (calcd for C28H38O5Na, 477.2611).
rated aqueous solution of NH4Cl (5 mL). 11a was subsequently
SPIRALISONE B (2). Off-white solid; [α]D22 +0.3 (c 0.5, CHCl3); UV obtained by extraction with EtOAc (3 × 5 mL), drying over anhy-
(MeOH) λmax (log ε) 227 (4.13) and 288 (4.20) nm; H and 13C drous MgSO4 and concentration in vacuo. The crude sample of
1
NMR (CDCl3, 600 MHz) Table 1 and ESI Table S2;† ESI(+)MS 11a was dissolved in CH2Cl2 (10 mL) at 0 °C without further
m/z 453 [M + H]+ and 475 [M + Na]+, ESI(−)MS m/z 451 purification, and following the addition of DMP (212 mg,
[M − H]−; HR-ESI(+)MS m/z 475.2461 [M + Na]+ (calcd for 0.5 mmol) the reaction mixture was stirred at 0 °C for 3 h
C28H36O5Na, 475.2455).
before quenching by addition of saturated aqueous solutions
SPIRALISONE C (3). Off-white solid; [α]2D2 −4.4 (c 0.11, CHCl3); of Na2SO3 (5 mL) and NaHCO3 (5 mL). The quenched reaction
1
UV (MeOH) λmax (log ε) 226 (4.02) and 288 (4.04) nm; H and mixture was extracted with CH2Cl2 (3 × 5 mL), and the
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combined organic phases was dried over anhydrous MgSO4 1.25 M in MeOH) was added to a solution of keto–enol (12c,
and concentrated in vacuo to yield, after silica gel chromato- 12c′, 8.0 mg, 0.013 mmol) in MeOH (1 mL), and the reaction
graphy (EtOAc/hexane = 1 : 6), the keto–enol equilibrium mixture was stirred overnight. After removal of MeOH in vacuo,
mixture (12a, 12a′) (150 mg, 75% for 2 steps) as a white solid.
the residue was fractionated by HPLC (Zorbax SB-C8 semi-
preparative column, gradient elution 3.5 mL min−1 over
15 min from 30% H2O/MeCN to 100% MeCN) to yield 1
(1.0 mg, 16.1%) whose spectral data were in accord with those
of the natural product, and the corresponding chromone 9
(2.0 mg, 33.3%). (2) TFA (5.2 μL, 0.68 mmol) was added to a
solution of keto–enol (12c, 12c′, 8.0 mg, 0.013 mmol) in
CH2Cl2 (1 mL), and then the reaction mixture was stirred for
2 h. After removal of CH2Cl2 in vacuo, the residue was fractio-
nated by HPLC (Zorbax SB-C8 semi-preparative column, gradi-
ent elution 3.5 mL min−1 over 15 min from 30% H2O/MeCN to
100% MeCN) to yield 9 (5.0 mg, 85.0%) as colorless oil.
SPIRALISONE D (4) AND CHROMONE (5). Two different deprotec-
tion/cyclization methods were employed. (1) BBr3 (1.4 mL,
1.4 mmol, 1 M in CH2Cl2) was added dropwise to a solution of
keto–enol (12a, 12a′, 12 mg, 0.03 mmol) in dry CH2Cl2 (5 mL)
at −78 °C, and the reaction mixture was allowed to reach
ambient temperature overnight before quenching with water at
0 °C. The quenched reaction mixture was extracted with
CH2Cl2 (3 × 5 mL), and the combined organic phases was
dried over anhydrous MgSO4 and concentrated in vacuo. The
resulting residue was fractionated by HPLC (Zorbax SB-C8
semi-preparative column, gradient elution at 3.5 mL min−1
over 15 min from 30% H2O/MeCN to 100% MeCN) to yield 4
(4.3 mg, 40%) and 5 (1.0 mg, 10%) whose spectral data were in
accord with those of the natural products, and 8 (3.4 mg,
30%). (2) Lithium propane-1-thiolate (0.48 mL, 0.24 mmol,
0.5 M in HMPA) was added to a solution of keto–enol (12a,
12a′, 10 mg, 0.024 mmol) in dry HMPA (0.1 mL), and the reac-
tion mixture was maintained at 100 °C overnight before
quenching with HCl (4 ml, 0.4 mmol, 0.1 M). The resulting
mixture was extracted with CH2Cl2 (3 × 5 mL), and the com-
bined organic phases was dried over anhydrous MgSO4 and
concentrated in vacuo. The resulting residue was fractionated
by HPLC (Zorbax SB-C8 semi-preparative column, gradient
elution 3.5 mL min−1 over 15 min, from 30% H2O/MeCN to
100% MeCN) to yield 5 (6.4 mg, 75%).
Acknowledgements
We thank Noscira (Madrid, Spain) for preliminary kinase
inhibitor screening and A. Piggott for acquisition of HR-ESIMS
data. Z. Khalil acknowledges provision of an IMB Postgraduate
Award and a University of Queensland International Postgradu-
ate Scholarship. This work was funded in part by the Institute
for Molecular Bioscience, The University of Queensland, the
Australian Research Council (ARC LP0775547, LP0989954) and
Noscira (Madrid, Spain).
Notes and references
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9676 | Org. Biomol. Chem., 2012, 10, 9671–9676
This journal is © The Royal Society of Chemistry 2012