Syntheses of cytosporones A, C, J, K, and N, metabolites from medicinal fungi

Article abstract of DOI:10.1071/CH15144

The syntheses of the fungal metabolites cytosporones A, (±)-C, and N are reported. And the syntheses of cytosporones J and K are described for the first time. The preparation of racemic cytosporone J and racemic cytosporone K, natural products containing

Full text of DOI:10.1071/CH15144

CSIRO PUBLISHING
Aust. J. Chem. 2015, 68, 1583–1592
Full Paper
http://dx.doi.org/10.1071/CH15144
Syntheses of Cytosporones A, C, J, K, and N, Metabolites
from Medicinal Fungi
A
A B
,
Andrew M. Beekman and Russell A. Barrow
A
Research School of Chemistry, Australian National University, Acton,
ACT, 2601, Australia.
B
Corresponding author. Email: rab@anu.edu.au
The syntheses of the fungal metabolites cytosporones A, (ꢀ)-C, and N are reported. And the syntheses of cytosporones J
and K are described for the first time. The preparation of racemic cytosporone J and racemic cytosporone K, natural
products containing the rare 3-isochromanone substructure, was achieved in 8 linear steps with an overall yield of 45 % and
7 linear steps in 46 % yield, respectively, resulting in the complete characterization of these compounds for the first time.
The key steps included a recently described homologation of benzoic acid to the analogous phenyl acetate using Birch
reductive alkylation conditions, acylation of the appropriate phenyl acetate derivative, and a selective reduction and
spontaneous biomimetic lactonization to yield the 3-isochromanone skeleton. The synthesized natural products were
evaluated for their biological activity against several clinical strains of human pathogens with all compounds displaying
weak antimicrobial activity.
Manuscript received: 23 March 2015.
Manuscript accepted: 24 April 2015.
Published online: 5 June 2015.
Introduction
precursors to the 3-isochromanones via a reduction of the
carbonyl at carbon 100 and subsequent lactonization at carbon
1 (Fig. 1).
We describe here the previously unreported syntheses of
cytosporones A, (ꢀ)-C, and N and the first syntheses of
cytosporone J and cytosporone K. The natural product structures
are confirmed through complete characterization and an extend-
ed biological profile is presented.
The on-going success of natural products in the pharmaceutical
industry highlights why natural products investigations remain a
staple of chemical research.[1] However, the chemical diversity
that newly discovered compounds occupy presents a substantial
challenge for identifying pharmaceutical leads.
Fungi represent a rich source of bio-diverse natural products
with a track record of pharmaceutical potential,[1a,2] and with
,99000 species described from a conservative estimate of
3.5 million, the exploration of fungal chemistry still shows
great potential.[3] The knowledge base presented by ethnophar-
macology is a valuable source for the accelerated discovery of
pharmaceutical leads as the compounds identified from tradi-
tional medicines frequently possess inherently low toxicity, a
consequence of generations of human testing.
In 2000, Clardy and coworkers revealed cytosporone A (1)
and (ꢀ)-cytosporone C (2) (Fig. 1) from an endophytic Cytos-
pora sp. of fungus isolated from the host plant Conocarpus
erecta from Costa Rica.[4] In 2009, Proksch and coworkers
identified the single enantiomer (þ)-cytosporone C as well as
newly discovered metabolites cytosporones J (3), K (4), M (5),
and N (6) from an endophytic Pestalotiopsis sp. of fungi isolated
from the mangrove Rhizophora mucronata, a plant used in
Chinese traditional medicine.[5] Due to the small quantity of
3–5 isolated, complete characterization of these metabolites was
not achieved. Metabolites 1–6 have been tested for their antimi-
crobial properties and cytotoxic activity.[5,6]
Results and Discussion
When work began on the syntheses of these natural fungal
metabolites, few methods existed in the literature for the prep-
aration of the 3-isochromanone skeleton, and only a small
number of these enable substitution at position 1.[8] Conse-
quently, we hoped to exploit a biomimetic route to the final
heterocyclic core.
We envisaged that (ꢀ)-cytosporone C (2) could be obtained
by a reduction of the benzylic ketone in cytosporone N (6)
resulting in spontaneous lactonization (Scheme 1). Subsequent
hydrolysis of the methyl ester in 6 would return the carboxylic
acid present in cytosporone A (1). Formation of 6 could be
achieved by acylation of an appropriately protected 3,5-
dihydroxyphenyl acetate derivative, which could be formed
by the homologation and protecting group interconversions of
3,5-dimethoxybenzoic acid.
We recently reported a two-step homologation of 3,5-
dimethoxybenzoic acid to methyl 3,5-dimethoxyphenyl acetate
via a Birch reductive alkylation and subsequent oxidative–
decarboxylation using lead tetraacetate.[9] With 7 in hand,
investigation into an appropriate protecting group for the phenol
Cytosporones C, J, and K (2–4) possess the 3-isochromanone
skeleton, rarely found in nature with less than twenty previously
reported natural products containing the substructure.[4,5,7]
Cytosporones A (1), M (5), and N (6) appear to be biosynthetic
Journal compilation Ó CSIRO 2015
www.publish.csiro.au/journals/ajc

1584
A. M. Beekman and R. A. Barrow
moieties was performed. Liu and coworkers reported that
demethylation of ethyl 2-(3,5-dimethoxyphenyl)-3-oxodecanoate,
towards the synthesis of cytosporone B, could not be
achieved.[10] After considering several different phenol protect-
ing groups, we used the isopropyl protecting group owing to its
simplicity to incorporation, labile nature in the presence of
aluminium chloride,[11] and stability to perchloric acid, which
we found to be particularly effective in the acylation of 1-alkyl-
3,5-dimethoxy-phenyl rings.[12]
give 8 in good yield (87 %, Scheme 2). Regioselective acylation
was achieved using perchloric acid and freshly prepared octa-
noic anhydride to give a near quantitative return (98 %) of the
2-acylated product 9, as indicated by the desymmetrization of
the aromatic resonances in the 1H NMR spectrum. The isopropyl
protecting groups were then removed under mild conditions to
furnish cytosporone N (6) in good yield. The obtained data
matched those reported by Proksch and coworkers for the
natural product.[5] The syntheses of 6 and cytosporone M (5)
were reported in 2012 by Dong and coworkers to exemplify the
synthetic utility of their new rhodium phosphoramidite-
catalyzed alkene hydroacylation.[13] The authors determined
that natural cytosporone M (5) possessed the R configuration
([a]2D0 ꢁ4.0 (c 0.02 in methanol (MeOH)).
Following this route, demethylation of 7 was achieved with
boron tribromide in excellent yield (99 %) and subsequent
isopropylation was achieved under Williamson conditions to
OH
HO
Saponification of the lactone moiety in 6 was achieved in
good yield to provide cytosporone A (1), with the analytical data
matching that reported in the literature (Scheme 3).[4] Despite
the propensity for d-keto acids to tautomerize,[14] none of the
tautomeric hydroxylactones were observed by NMR using
[D6]acetone as the solvent. Our attention then turned to the
stereoselective reduction of carbon 100 to give enantiopure
cytosporone C. The use of both Brown’s DIP-chloride (Diiso-
pinocampheylchloroborane)[15] and Corey’s CBS (Corey-
Bakshi-Shibata) reagent[16] both proved ineffective at reducing
6, returning the starting material only. It is hypothesized that
steric hindrance around the desired site of reaction is too great
for these bulky reagents to react as desired. Consequently,
reduction was performed using sodium borohydride in methanol
to return (ꢀ)-cytosporone C (2) in excellent yield (Scheme 3),
with the analytical data matching that reported by Clardy and
coworkers.[4] The syntheses of 1 and 2 were also recently
reported by Beatriz and coworkers.[17] Interestingly, the authors
reported the demethylation of 2-(3,5-dimethoxyphenyl)-3-
oxodecanoic acid with aluminium chloride, a transformation
that could not be achieved by Liu and coworkers in 2010[10] or in
our laboratories.
R1
1ꢀ
1
R2O
O
O
R2 ꢁ H
A (1): R1
M (5): R1
ꢁ
ꢁ
R2 ꢁ Me
R2 ꢁ Me
OH
N (6): R1
ꢁ
HO
OH
1
R1
O
O
C (2): R1
J (3): R1
K (4): R1
ꢁ
ꢁ
OH
Having developed the desired chemistry for the preparation
of the 3-isochromanone skeleton, our attention turned to the
preparation of cytosporones J (3) and K (4). Preparation of the
requisite anhydride followed a procedure described by us
previously,[18] which began with the preparation of alcohol
ꢁ
OAc
Fig. 1. Structures of cytosporones A, C, J, K, M, and N.
Deprotection
OH
O
HO
HO
OH
C7H15
O
O
O
Rꢂ
Rꢂ
C7H15
C7H15
O
O
O
R
Acylation
O
O
O
Reduction and
lactonization
Hydrolysis
Cytosporone A (R ꢁ Me)
Cytosporone N (R ꢁ H)
(ꢃ)-cytosporone C
Protecting group
interconversion
O
O
O
O
O
O
O
Rꢂ
Rꢂ
O
O
O
OH
Homologation
O
3,5-dimethoxybenzoic acid
Scheme 1. Retrosynthetic analysis of cytosporones A, N, and (ꢀ)-C.

First Syntheses of Cytosporones J and K
1585
(R)-10 from enantiopure (R)-propylene oxide and 6-bromohexene,
followed by protection to give acetoxynonene (R)-11 and
ozonolysis to provide aldehyde (R)-12. Lindgren oxidation
yielded the desired carboxylic acid (R)-13 in excellent yield
(92 %) and high purity (Scheme 4). Carboxylic acid (R)-13 was
then treated with trifluoroacetic anhydride, and the resulting
mixed anhydride was immediately used in Friedel–Crafts acyl-
ation with 8 to selectively provide (R)-14 (Scheme 5). The
isopropyl ethers were cleaved under the mild conditions
described by Banwell et al.[11] to give the keto-ester (R)-15,
which when treated with sodium borohydride yielded (2R,10R)
and (2R,10S)-cytosporone K ((2R)-4) in excellent yield. Despite
can be applied with reasonable confidence.[19] We assume that
the stereocentres present in cytosporone C and cytosporone M
are reproduced in cytosporones J and K, as they are proposed to
emerge from the same biosynthetic pathway, which is probable
given their concurrent isolation.[20] Based on the study by Dong
et al.,[13] we can assume that the stereocentres at 60 (in 3) and 2
(in 4) are R, and those stereocentres alone account for an [a]D20
ꢁ8.5 (in 3) and an [a]2D0 ꢁ5.7 (in 4) due to the racemic centres at
1 ((60R)-3) and 10 in ((2R)-4) equating to an [a]2D0 0. Applying
this information to the specific rotation of natural cytosporone J
(3; [a]2D0 þ11 (c 0.1 in MeOH)) allows us to propose that
stereocentre 60 contributes an [a]2D0 E þ19.5, the same sign
and similar magnitude to that measured for cytosporone C (2;
[a]2D0 þ11 (c 0.1 in MeOH)).[21] Likewise, the optical rotation of
natural cytosporone K (4; [a]2D0 þ7 (c 0.02 in MeOH)) allows us
to propose that stereocentre 1 contributes an [a]2D0 Eþ12.7. At
this stage, the chirality that these specific rotations correspond to
is unclear.
1
(2R)-4 representing a diastereomeric mixture, both the H and
13C NMR data obtained (800 MHz and 200 MHz respectively,
[D6]acetone) for these two compounds presented identically.
The characteristic data matched that provided by Proksch and
coworkers.[5] Additionally, for the first time, full spectroscopic
data of cytosporone K (4) were obtained.
(1S,60R) and (1R,60R)-cytosporone J ((60R)-3) was sub-
sequently prepared by deacetylation of (2R)-4 using ammonium
hydroxide in methanol. Again, despite (60R)-3 representing a
diastereomeric mixture, both the 1H and 13C NMR data obtained
(800 MHz and 200 MHz respectively, [D6]acetone) for these
two compounds presented identically. The data matched that
reported in the literature,[5] and full characteristic data were
obtained and reported for the first time. The synthetic sequence
was repeated with (S)-propylene oxide, instead of (R)-propylene
oxide, to give (2S)-4 and (60S)-3.
In 2000, when Clardy and coworkers reported the elucidation
of the first cytosporones, they made the observation that the
cytosporones could be biosynthetically related to the common
and very well studied curvularins.[4] They noted that the curvu-
larins (exemplified by curvularin (17)) could be biosynthetically
OH
HO
OH
C7H15
HO
HO
C7H15
NaOH, MeOH
65ꢅC, 16 h, 99 %
O
O
O
The specific rotations of the diastereomeric mix of (1S,60R)
and (1R,60R)-cytosporone J ((60R)-3; [a]2D0 ꢁ8.5 (c 0.83 in
MeOH)) and (10R,2R) and (10S,2R)-cytosporone K ((2R)-4;
[a]2D0 ꢁ5.7 (c 1.60 in MeOH)) were determined. This informa-
tion, coupled with the specific rotation of natural cytosporone
C (2; [a]2D0 þ11 (c 0.1 in MeOH))[5] and the specific rotation of
cytosporone M (5; [a]D20 ꢁ4.0 (c 0.02 in MeOH),[13] allows us to
apply van’t Hoff’s principle of optical superposition to propose
the sign and magnitude of the stereocentre at 1 and 10 in the
natural cytosporones J and K, respectively. As the stereocentres
in cytosporones J and K are more than two methylene units
apart, we can assume that they are not interacting. Hence, van’t
Hoff’s supposition that individual asymmetric centres in a chiral
molecule make independent contribution to the optical rotation
1
6
O
O
NaBH4, MeOH
r.t., 15 min, 89 %
OH
C7H15
O
HO
2
O
Scheme 3. Synthesis of cytosporone A (1) and (ꢀ)-cytosporone C (2).
O
O
O
O
O
1a) Li/NH3, THF, ꢄ33ꢅC, 1 h,
b) BrCH2CO2Me, THF, ꢄ33ꢅC to r.t., 2 h, 93 %
2) Pb(OAc)4, CH2Cl2, r.t., 1 h, 91 %
O
OH
7
O
O
O
O
iPr
iPr
Octanoic anhydride,
HClO4
1) BBr3, CH2Cl2, ꢄ78ꢅC to r.t., 16 h, 99 %
2) K2CO3, iPrI, acetone, 56ꢅC, 16 h, 87 %
Toluene, r.t., 24 h, 98 %
OH
HO
8
O
O
O
O
O
iPr
iPr
C7H15
C7H15
AlCl3
CH2Cl2, r.t., 2 h, 69 %
O
O
6
9
O
O
Scheme 2. Synthesis of cytosporone N (6) from 3,5-dimethoxybenzoic acid.

1586
A. M. Beekman and R. A. Barrow
OH
1) Mg, THF, 66ꢅC, 15 min
Br
2) CuI, THF, ꢄ40ꢅC, 4 h, 89 %
(R)-10
O
OAc
1) O3, CH2Cl2, ꢄ78ꢅC, 15 min
Ac2O, pyridine, r.t., 3 h, 98 %
2) PPh3, CH2Cl2, ꢄ78ꢅC, 1 h, 78 %
(R)-11
OAc
O
OAc
O
NaClO2, NaH2PO4
HO
t-BuOH, r.t., 45 min, 92 %
(R)-12
(R)-13
Scheme 4. Preparation of carboxylic acid (R)-13 from 6-bromohexene and (R)-propylene oxide.
OH
O
O
HO
O
iPr
iPr
1) TFAA
CH2Cl2, r.t. 2 h
1ꢀ
7ꢀ
(R)-13
2) 8, HClO4
toluene, r.t., 24 h, 96 %
ACP-S
O
O
O
OAc
(R)-14
O
O
HO
OH
16
AlCl3, CH2Cl2
r.t., 2 h, 71 %
NaBH4, MeOH
r.t., 2 h, 92 %
O
OH
O
OAc
HO
O
(R)-15
O
O
6
7ꢂ
10
OH
HO
1ꢂ
2
NH4OH, MeOH
r.t., 16 h, 99 %
5ꢂ
4ꢂ
O
4
O
4
O
OAc
Curvularin (17)
(2R)-
4
7
OH
HO
OH
HO
1
6ꢂ
OH
5
1
6ꢂ
2ꢂ
4ꢂ
O
4
O
OH
(6ꢂR)-
3
O
O
Cytosporone K (4)
Scheme 5. Synthesis of cytosporone K and cytosporone J.
Scheme 6. Reduction of precursor 16 at C-700 or C-100 leading to the
curvularins or cytosporones, respectively.
derived from a cytosporone A-type precursor (16) (Scheme 6),
which undergoes a macrolactonization to give a 12-membered
ring instead of the d-lactone seen in cytosporone C (2). At this
time, cytosporone J (3) and cytosporone M (5) were yet to be
reported, but their discovery in 2009 by Proksch et al.[5] sup-
ported the biosynthesis proposed by Clardy et al. A survey of the
literature indicated that the absolute configuration of the
hydroxyl group on the side chain of the cytosporones is R,[13]
and the majority of the curvularin-type compounds isolated
possess the S stereochemistry about C-4. There are only two
reports of the R stereochemistry of the curvularins about C4 in
nature[22] unlike the numerous reports (.27) on the S stereo-
chemistry.[23] Dihydrocurvularin ((10-OH)-17) has been
reported as a natural product once in the literature,[24] but was
present as an apparent mixture of epimers about C10, suggesting
that it was an artefact. These factors led us to hypothesize that
the biosynthetic pathway, which forms the curvularins and
cytosporones, diverges based on the occurrence of a reduction
of 16 at either C-100 or at C-700. If the reduction first occurs at
C-700, then macrolactonization results in a curvularin. In con-
trast, if reduction occurs first at C-100, then a cytosporone is
produced due to spontaneous formation of the d-lactone
(Scheme 6). Subsequently, the reduction at C-60 of the cytos-
porone (4) occurs to produce the R hydroxy unit.
Compounds 1, 2, 6, (R)-14, and (S)-14 as well as (2R)-4, (2S)-
4, (60R)-3, and (60S)-3 were subject to biological assay to
evaluate their antibacterial activity against clinical strains of
Staphylococcus epidermidis, Escherichia coli, Pseudomonas
aeruginosa, and Mycobacterium smegmatis, a selection of
Gram-positive and Gram-negative human pathogenic bacteria.
Kanamycin was employed in the assay as a positive control and
for comparison. The IC50 (half-maximal inhibitory concentra-
tion) values for the R enantiomers and their precursors are
displayed in Table 1. Both enantiomers were tested, but there
was not a significant difference in activity between the enantio-
mers. Thus, only the IC50 values for the natural side chain
stereochemistry are presented. The MIC (minimum inhibitory
concentration) of all compounds was greater than 600 mg mLꢁ1
.
Previous studies have shown that the cytosporones have no
significant cytotoxic activity and mild antifungal activity.[4–6]
These results may suggest another reason for the use of the host
plant as a Chinese traditional medicinal.

First Syntheses of Cytosporones J and K
1587
Table 1. Antimicrobial activity of 1, 2, 6, (2R)-4, (69R)-3, and (R)-14 showing 50 % inhibition of selected strains
IC50 [mg mLꢁ1
]
StrainA
Kanamycin
1
2
6
(2R)-4
(60R)-3
(R)-14
Staphylococcus epidermidis
Escherichia coli
90
,1
209
,1
.600
592
593
.600
486
568
.600
508
589
538
452
465
555
501
473
413
582
.600
497
Pseudomonas aeruginosa
Mycobacterium smegmatis
519
436
408
449
441
AClinical strains obtained from The Canberra Hospital, Australia.
Conclusions
membrane filter) methanol at the concentration and flow rate
stated with detection at 254 nm and 300 nm. Analytical enan-
tioselective HPLC was performed using a Phenomenex LUX
cellulose-4 5 m 250 mm ꢂ 4.6 mm column. Elution was carried
out using pre-filtered (0.45 mm nylon membrane filter) ethanol
and n-hexane at the concentration and flow rate stated with
detection at 254 nm and 300 nm. Intermediates were purified to
allow for sufficient characterization and effective subsequent
reactivity, as exemplified in the Supplementary Material. In
general, reagents were purchased from commercial suppliers
and used without further purification unless otherwise stated.
Purification techniques were directed by typical procedures. All
solvents were dried and distilled either immediately before use
or stored as appropriate. Ethereal solvents diethyl ether (Et2O)
and THF were refluxed over sodium and benzophenone until
completely dried and free of moisture. Toluene was refluxed
over sodium. Ethanol (EtOH) and tert-butanol (t-BuOH) were
refluxed with and distilled from magnesium. CH2Cl2 was
refluxed with and distilled from CaH2. Acetone was refluxed
with and distilled from Na2SO4. The distillation fraction of
petroleum spirit used was 60–808C.
The synthesis of five fungal metabolites (1–4 and 6) was
achieved, including the preparation of two natural products,
cytosporones J and K, for the first time. Racemic cytosporone J
(3) was prepared in 8 linear steps with an overall yield of 45 %,
and racemic cytosporone K (4) was prepared in 7 linear steps in
46 % yield. Complete characterization of these compounds was
achieved for the first time. The route employed displayed the
power of biomimetic synthesis in the preparation of natural
products, and is a viable route for the synthesis of several other
cytosporones. The synthetic products showed identical charac-
teristic data to those reported for the natural products. Selected
compounds subjected to biological tests exhibited weak anti-
microbial activity against clinical strains of human pathogens.
Experimental
General Experimental Procedures
1H and 13C NMR spectra were recorded at 298 K, and at 300,
400, 500, or 800 MHz, and 75, 100, 125, or 200 MHz, respec-
tively, on an Inova 300, Varian Mercury 300, Varian MR-400,
Inova 500, or Avance 800 instrument. Chemical shifts are
1
Methyl 2-(3,5-Dimethoxyphenyl)acetate (7)
reported in ppm (d). H NMR spectra are referenced to the
resonance from residual CHCl3 at 7.26 ppm and the central peak
in the resonance from residual CHD2COCD3 at 2.05 ppm.
13C NMR spectra are referenced to the central peak in the signal
from CDCl3 at 77.0 ppm and the central peak in the resonance
from (CD3)2CO at 29.8 ppm. When ambiguous, assignments
were made based on prior literature and 2D NMR analysis
including HSQC, HMBC, and NOESY data. Electrospray mass
spectrometry (EI–MS) and high-resolution electrospray mass
spectrometry (HREI–MS) data were recorded on a VG autospec
mass spectrometer or Waters autospec premier mass spec-
trometer, operating at 70 eV. Electrospray ionization mass
spectrometry (ESI–MS) and high-resolution electrospray ioni-
zation mass spectrometry (HRESI–MS) data were recorded on a
Bruker Apex 3 spectrometer. Infrared (IR) spectra were recor-
ded on a Bruker Alpha-P spectrophotometer as neat solid or
film. UV spectra were measured on a Cary 4G UV-Visible
Spectrophotometer using a 1 cm solution cell in the solvent
indicated. Optical data were measured on a PerkinElmer 341
polarimeter. Concentrations are expressed as g per 100 mL,
measured at the sodium D-line (589 nm) at 208C in the solvent
indicated. Flash chromatography was performed under pressure
using normal phase silica gel (230–400 mesh Scharlau 60,
40–60 mm) as solid support and HPLC-graded solvents as
eluents. Semi-preparative HPLC was performed using an
Agilent 1100 system, using a Phenomenex LUNA 5 m C18
250 ꢂ 10.00 mm column and an Agilent 1100 series diode array
detector for eluate detection. Elution was carried out using 0.1 %
v/v TFA in milli-Q water and pre-filtered (0.45 mm nylon
To a solution of 3,5-dimethoxybenzoic acid (1.82 g, 10.0 mmol)
in THF (20 mL) was condensed NH3 (50 mL). Lithium (153 mg,
21.9 mmol) was added in portions at ꢁ338C whereon a deep blue
colour persisted. Methyl bromoacetate (1.14 mL, 1.84 g,
12.0 mmol) was then added dropwise, causing the solution to
become pale yellow. NH3 was evaporated under a stream of
nitrogen and the resulting solid residue was dissolved in water
and Et2O. The layers were separated and the aqueous layer was
cooled to 08C and acidified to pH 4 with careful addition of 1 M
HCl(aq). The acidic solution was extracted with ethyl acetate
(EtOAc), dried with MgSO4, filtered, and concentrated under
vacuum to give 3,5-dimethoxy-1-(2-methoxy-2-oxoethyl)
cyclohexa-2,5-diene-1-carboxylic acid as a colourless solid
(2.38 g, 93 %; 9.26 mmol). nmax (neat)/cmꢁ1 2982, 2651, 1703,
1649, 1695. dH (CDCl3, 300 MHz) 4.77 (2H, s, 2,6-H), 3.67 (3H,
s, 20-OCH3), 3.58 (6H, s, 3,5-OCH3), 2.7–2.9 (4H, m, 4-HA,
4-HB, 10-H). dC (CDCl3, 75 MHz) 180.9 (COOH), 171.4 (20-C),
154.2 (3,5-C), 93.9 (2,6-C), 54.5 (3,5-OCH3), 51.6 (20-OCH3),
47.8 (1-C), 45.6 (10-C), 31.0 (4-C). m/z (ESI) 279 (100 %, [M þ
Na]þ), 257 (40, [M þ H]þ). HRESI m/z 257.1024; calcd for
C12H17O6 257.1025.
To a solution of 3,5-dimethoxy-1-(2-methoxy-2-oxoethyl)
cyclohexa-2,5-dienecarboxylic acid (1.85 g, 7.20 mmol) in
CH2Cl2 (150 mL) was added lead tetraacetate (4.15 g,
9.40 mmol). The reaction was monitored by TLC and after
40 min, water (40 mL) was added. The mixture was filtered
through a pad of silica. The organic phase was separated, washed
with saturated NaHCO3(aq), dried with MgSO4, filtered, and

1588
A. M. Beekman and R. A. Barrow
concentrated under vacuum to give an orange oil. Purification
was achieved by flash column chromatography on silica with
33 % EtOAc in petroleum spirits as the eluent (RF ¼ 0.6) to yield
the title compound as a yellow oil (1.38 g, 91 %; 6.57 mmol).
t, J 7.5, 200-H), 1.70–1.50 (2H, m, 300-H), 1.31 (12H, d, J 6.1,
OCH(CH3)2), 1.36–1.22 (8H, m, 400-700-H), 0.86 (3H, t, J 6.8,
800-H). dC (CDCl3, 75 MHz) 207.3 (100-C), 171.8 (1-C), 159.4
(50-C), 157.1 (30-C), 134.3 (10-C), 109.2 (60-C), 107.9 (20-C),
100.5 (40-C), 70.4 (OCH(CH3)2), 69.9 (OCH(CH3)2), 51.9
(1-OCH3), 44.5 (200-C), 38.8 (2-C), 31.7, 29.4, 29.2, 24.6, 22.6
(700-C), 21.9 (OCH(CH3)2), 14.0 (800-C). m/z (EI) 392 (10 %,
[M]þꢃ), 293 (55), 127 (100), 57 (55). HREI m/z 392.2573; calcd
for C23H36O5 392.2563.
n
max (neat)/cmꢁ1 1723, 1602. dH (CDCl3, 300 MHz) 6.44 (2H,
m, 20,60-H), 6.37 (1H, m, 40-H), 3.78 (6H, s, 30,50-OCH3), 3.69
(3H, s, 1-OCH3), 3.56 (2H, s, 2-H). dC (CDCl3, 75 MHz) 171.8
(1-C), 160.8 (30,50-C), 136.0 (10-C), 107.3 (20,60-C), 99.2 (40-C),
55.3 (30,50-OCH3), 52.1 (1-OCH3), 41.4 (2-C). m/z (EI) 210
(100 %, [M]þꢃ), 151 (95), 121 (30), 91 (20). HREI m/z 210.0890;
calcd for C11H14O4 210.0892.
Cytosporone N: Methyl 2-(3,5-Dihydroxy-2-
octanoylphenyl)acetate (6)
Methyl 2-(3,5-Diisopropoxyphenyl)acetate (8)
To a solution of methyl 2-(3,5-diisopropoxy-2-octanoylphenyl)
acetate (9; 100 mg, 0.255 mmol) in CH2Cl2 (5 mL) was added
aluminium chloride (102 mg, 0.765 mmol), and the solution was
allowed to stir at room temperature for 2 h, after which time
NH4Cl(aq) was added and the solution was extracted with
EtOAc. The organic layer was washed with water and brine,
dried with MgSO4, filtered, and concentrated to give a yellow
amorphous solid. Purification was achieved via flash column
chromatography on silica using 50 % EtOAc in petroleum spirits
as eluent (RF ¼ 0.3), yielding a colourless amorphous solid
(54.2 mg, 69 %; 0.176 mmol). Purity was confirmed via HPLC
on a Phenomenex LUNA 5 m C18 column using 75 % methanol
in water (0.1 % TFA) as solvent with a flow rate of 1.5 mL minꢁ1
resulting in a retention time of 10.7 min. nmax (neat)/cmꢁ1 3376,
To a solution of methyl 2-(3,5-dimethoxyphenyl)acetate (7;
1.05 g, 5.00 mmol) in CH2Cl2 (50 mL) at ꢁ788C was added
boron tribromide (1.0 M in hexane, 11.0 mL, 11.0 mmol)
dropwise. The solution was allowed to stir and slowly warm
to room temperature overnight at which time ice was added.
The layers were separated and the organic layer was washed
with NaHCO3(aq), water and brine, dried with MgSO4, filtered,
and concentrated under vacuum to yield methyl 2-(3,5-
dihydroxyphenyl)acetate as a colourless solid (1.01 g, 99 %;
4.90 mmol). nmax (neat)/cmꢁ1 3117, 1716, 1601. dH ([D6]acetone,
300 MHz) 8.22 (2H, s, 30,50-OH), 6.32–6.24 (3H, m, 20,40,60-H),
3.62 (3H, s, 1-OCH3), 3.46 (2H, s, 2-H). dC ([D6]acetone,
75 MHz) 172.1 (1-C), 159.4 (30,50-C), 137.4 (10-C), 108.6 (20,60-C),
102.0 (40-C), 51.9 (1-OCH3), 41.4 (2-C). m/z (ESI) 205 (100 %,
[M þ Na]þ), 193 (35, [M þ H]þ), 169 (38). HRESI m/z
205.0467; calcd for C9H10O4Na23 205.0477.
To a solution of methyl 2-(3,5-dihydroxyphenyl)acetate
(1.01 g, 4.90 mmol) in acetone (50 mL) was added potassium
carbonate (2.71 g, 19.6 mmol) and iodomethane (0.763 mL,
1.74 g, 12.3 mmol), and the reaction was stirred at reflux for
16 h. After cooling, the reaction mixture was filtered and the
filtrate was concentrated to give a brown solid. Purification was
achieved via flash column chromatography on silica using 33 %
EtOAc in petroleum spirits as eluent (RF ¼ 0.4) to give the title
compound as an off-white solid (1.13 g, 87 %; 4.26 mmol). nmax
(neat)/cmꢁ1 2976, 1737, 1590. dH (CDCl3, 300 MHz) 6.39 (2H,
m, 20,60-H), 6.33 (1H, m, 40-H), 4.05 (2H, septet, J 6.0, OCH
(CH3)2), 3.68 (3H, s, 1-OCH3), 3.52 (2H, s, 2-H), 1.31 (12H, d,
J 6.0, OCH(CH3)2). dC (CDCl3, 75 MHz) 171.8 (1-C), 159.0
(30,50-C), 135.7 (10-C), 108.8 (20,60-C), 102.3 (40-C), 69.7 (OCH
(CH3)2) 51.9 (1-OCH3), 41.3 (2-H), 21.9 (OCH(CH3)2). m/z (EI)
266 (40 %, [M]þꢃ), 182 (100), 123 (50). HREI m/z 266.1518;
calcd for C15H22O4 266.1518.
3220, 2925, 2848, 1702, 1683. lmax (CH3OH)/nm (e/Mꢁ1 cmꢁ1
)
202 (log e 3.96), 219 (3.72), 269 (3.34). dH (CDCl3, 400 MHz)
11.63 (1H, s, 30-OH), 6.27 (1H, d, J 2.5, 60-H), 6.25 (1H, d, J 2.5,
40-H), 3.81 (2H, s, 2-H), 3.74 (3H, s, 1-OCH3), 2.82 (2H, t, J 7.2,
200-H), 1.70–1.60 (2H, m, 300-H), 1.40–1.25 (8H, m, 400-700-H),
0.87 (3H, t, J 7.0, 800-H). dC (CDCl3, 75 MHz) 206.8 (100-C),
172.3 (1-C), 163.7 (50-C), 160.4 (30-C), 136.3 (10-C), 116.7 (20-
C), 112.7 (60-C), 103.2 (40-C), 52.6 (1-OCH3), 43.5 (200-C), 41.4
(2-C), 31.7, 29.2, 29.1, 24.9, 22.6 (700-C) 14.1 (800-C). dH ([D6]
acetone, 300 MHz) 9.72 (1H, s, 30-OH), 6.37 (1H, d, J 2.5, 60-H),
6.32 (1H, d, J 2.5, 40-H), 3.67 (2H, s, 2-H), 3.61 (3H, s, 1-OCH3),
2.90 (2H, t, J 7.2, 200-H), 1.65–1.55 (2H, m, 300-H), 1.35–1.20
(8H, m, 400-700-H), 0.87 (3H, t, J 7.0, 800-H). dC ([D6]acetone,
75 MHz) 205.9 (100-C), 171.7 (1-C), 160.3 (50-C), 159.3 (30-C),
136.6 (10-C), 120.5 (20-C), 111.5 (60-C), 102.3 (40-C), 51.6
(1-OCH3), 44.1 (200-C), 39.5 (2-C), 32.2 (60-C), 29.7, 29.6, 24.7,
23.0 (500-C), 14.0 (800-C). m/z (ESI) 331 (100 %, [M þ Na]þ), 102
(35). m/z (HRESI) 309.1705; [M þ H]þ requires 309.1702.
Cytosporone A: 2-(3,5-Dihydroxy-2-octanoylphenyl)
acetic acid (1)
Methyl 2-(3,5-Diisopropoxy-2-octanoylphenyl)acetate (9)
To a solution of cytosporone N (6; 10.0 mg, 32.3 mmol) in
methanol (2 mL) was added 1 M NaOH(aq) solution until the
solution became cloudy. Methanol was then added dropwise
until the solution became clear again and the reaction was heated
at 508C for 16 h. After cooling, water was added and the solution
was extracted with EtOAc. The organic layer was then dried
with MgSO4, filtered, and concentrated to give the title com-
pound as an amorphous solid (9.4 mg, 99 %; 32 mmol). Purity
was confirmed via HPLC on a Phenomenex LUNA 5 m C18
column using 75 % methanol in water (0.1 % TFA) as solvent
with a flow rate of 1.5 mL minꢁ1 resulting in a retention time of
6.1 min. nmax (neat)/cmꢁ1 3371, 3224, 2929, 2841, 1706, 1687.
lmax (CH3OH)/nm (e/Mꢁ1 cmꢁ1) 202 (log e 3.99), 219 (3.78),
270 (3.29). dH ([D6]acetone, 300 MHz) 6.34 (1H, d, J 2.1, 60-H),
6.28 (1H, d, J 2.1, 40-H), 3.64 (2H, s, 2-H), 2.87 (2H, m, J 7.2,
To a solution of methyl 2-(3,5-diisopropoxyphenyl)acetate (8;
0.319 g, 1.20 mmol) in toluene (10 mL) was added HClO4 (2
drops), and the solution was stirred at room temperature for 8 h
at which time a further 2 drops of HClO4 was added and the
solution was stirred for a further 16 h. NaHCO3(aq) was added
and the solution was extracted with EtOAc. The organic layer
was washed with brine, dried with MgSO4, filtered, and con-
centrated to give a brown oil. Purification by flash column
chromatography on silica using 20 % EtOAc in petroleum spirit
as eluent (RF ¼ 0.3) yielded the title compound as a colourless
amorphous solid (0.461 g, 98 %; 1.18 mmol). nmax (neat)/cmꢁ1
2916, 2836, 1705, 1680, 1617. dH (CDCl3, 300 MHz) 6.33 (1H,
d, J 2.0, 60-H), 6.31 (1H, d, J 2.0, 40-H), 4.51 (2H, septet, J 6.1,
OCH(CH3)2), 3.65 (3H, s, 1-OCH3), 3.59 (2H, s, 2-H), 2.81 (2H,

First Syntheses of Cytosporones J and K
1589
200-H), 1.65–1.55 (2H, m, 300-H), 1.35–1.25 (8H, m, 400-700-H),
0.87 (3H, t, J 7.0, 800-H). dC ([D6]acetone, 75 MHz) 206.1
(100-C), 171.9 (1-C), 160.4 (50-C), 159.4 (30-C), 136.7 (10-C),
120.7 (20-C), 111.6 (60-C), 102.4 (40-C), 44.3 (200-C), 39.6 (2-C),
32.3, 29.8, 29.7, 24.8, 23.1 (700-C), 14.2 (80-C). m/z (ESI) 295
(100 %, [M þ H]þ). m/z (HRESI) 295.1554; [M þ H]þ requires
295.1546.
(3.00 mL, 3.25 g, 31.8 mmol), and the solution was stirred at
room temperature for 3 h. NH4Cl(aq) was added and the solution
was extracted with Et2O. The organic layer was then washed
with NH4Cl(aq) and saturated NaHCO3(aq), dried with MgSO4,
filtered, and concentrated to give a colourless oil (3.07 g, 98 %,
16.7 mmol). [a]D20 þ8.3 (c 10.6 in MeOH). nmax (neat)/cmꢁ1
1742, 1235. dH (CDCl3, 400 MHz) 5.80 (1H, ddt, J 17.0, 10.2,
6.7, 8-H), 5.1–4.8 (3H, m, 2,9-H), 2.1–2.0 (2H, m, 7-H), 2.02
(3H, s, C(O)CH3), 1.8–1.2 (8H, m, 3–6-H), 1.20 (3H, d, J 6.3,
1-H). dC (CDCl3, 100 MHz) 170.8 (C(O)CH3), 140.1 (8-C),
114.3 (9-C), 71.0 (2-C), 35.9 (3-C), 33.7 (7-C), 28.9, 28.8, 25.2,
21.4 (C(O)CH3), 20.0 (1-C). m/z (EI) 184 (40 %, [M]þꢃ), 137
(100). HREI m/z 184.1466; calcd for C11H20O2 184.1463.
Cytosporone C: (ꢀ)-1-Heptyl-6,8-dihydroxyisochroman-
3-one (2)
To a solution of cytosporone N (6; 20.0 mg, 64.6 mmol) in
methanol (5 mL) was added sodium borohydride (2.4 mg,
65 mmol), and the solution was stirred at room temperature for
2 h. The solution was diluted with water and extracted with
EtOAc, and the organic layer was dried with MgSO4, filtered,
and concentrated to give an amorphous colourless solid. Puri-
fication was achieved via flash column chromatography on
silica using 66 % EtOAc in petroleum spirits as eluent (RF ¼ 0.3)
to give the title compound as a colourless amorphous solid
(16.0 mg, 89 %; 57.5 mmol). Purity was confirmed via HPLC on
a Phenomenex LUNA 5 m C18 column using 75 % methanol in
water (0.1 % TFA) as solvent with a flow rate of 1.5 mL minꢁ1
resulting in a retention time of 16.7 min. nmax (neat)/cmꢁ1 3372,
2923, 2852, 1710, 1651. lmax (CH3OH)/nm (e/Mꢁ1 cmꢁ1) 208
(log e 3.90), 282 (3.38). dH ([D6]acetone, 400 MHz) 6.35 (1H, d,
J 2.2, 5-H), 6.24 (1H, d, J 2.2, 7-H), 5.56 (1H, dd, J 8.9, 5.1, 1-H),
3.80 (1H, d, J 19.7, 4-HA), 3.46 (1H, d, J 19.7, 4-HB), 2.00–1.70
(2H, m, 10-H), 1.60–1.40 (2H, m, 20-H), 1.40–1.20 (8H, m, 30-60-
H), 0.87 (3H, t, J 7.0, 70-H). dC ([D6]acetone, 100 MHz) 170.6
(3-C), 159.0 (8-C), 154.4 (6-C), 133.4 (4a-C), 114.0 (8a-C),
106.2 (5-C), 101.8 (7-C), 78.3 (1-C), 36.4 (10-C), 35.4 (4-C),
32.5, 29.9, þ2ꢃ9.9, 26.4, 23.2 (60-C), 14.3 (70-C). m/z (EI) 278
(55 %, [M] ), 207 (55), 179 (100), 151 (55). m/z (HREI)
278.1518; [M]þꢃ requires 278.1518.
(R)-Non-8-en-2-yl Acetate ((R)-11)
The title compound was synthesized in the same manner as
(S)-non-8-en-2-yl acetate from (R)-non-8-en-2-ol. [a]2D0 ꢁ7.8
(c 10.2 in MeOH). All remaining spectroscopic data were
identical to those of (S)-non-8-en-2-yl acetate.
(S)-8-Oxooctan-2-yl Acetate ((S)-12)
(S)-Non-8-en-2-yl acetate (2.76 g, 15.0 mmol) was dissolved in
CH2Cl2 (40 mL) and cooled to ꢁ788C. Ozone was then bubbled
through the solution until a blue colour persisted at which time
oxygen was bubbled through the solution for 15 min. Triphe-
nylphosphine (7.87 g, 30.0 mmol) was then added to the solution
and the reaction mixture was allowed to slowly warm to room
temperature. The solvent was removed under vacuum and the
crude product was purified via flash column chromatography on
silica using 10 % EtOAc in petroleum spirits as eluent (RF ¼ 0.2)
yielding a colourless oil (2.18 g, 78 %; 11.7 mmol). [a]2D0 þ2.0
(c 10.3 in MeOH). nmax (neat)/cmꢁ1 2981, 2733, 1750, 1728. dH
(CDCl3, 300 MHz) 9.76 (1H, t, J 1.8, 8-H), 5.00–4.75 (1H, m,
2-H), 2.42 (2H, dt, J 7.3, 1.8, 7-H), 2.02 (3H, s, C(O)CH3), 1.7–
1.2 (8H, m, 3–6-H), 1.19 (3H, d, J 6.2, 1-H). dC (CDCl3,
100 MHz) 202.4 (8-C), 170.6 (C(O)CH3), 70.7 (2-C), 43.6
(7-C), 35.5 (3-C), 28.7, 25.0, 21.7, 21.2 (C(O)CH3), 19.8 (1-C).
m/z (ESI) 209 (100 %, [M þ Na]þ). HRESI m/z 209.1155; calcd
for C10H18O233Na 209.1154.
(S)-Non-8-en-2-ol ((S)-10)
To a solution of copper iodide (286 mg, 1.5 mmol) in THF
(10 mL) at ꢁ408C was added freshly prepared hex-5-en-1-
ylmagnesium bromide (prepared from 0.530 g, 22.0 mmol
magnesium and 2.68 mL, 3.26 g, 20.0 mmol 6-bromohex-1-ene
in 20 mL THF), and the mixture was stirred for 15 min. (S)-
Propylene oxide (1.40 mL, 1.16 g, 20.0 mmol) was then added
dropwise and the solution was allowed to stir at ꢁ408C for 4 h.
NH4Cl(aq) was then added and the solution extracted with Et2O.
The organic layer was separated, dried with MgSO4, filtered,
and concentrated to give the title compound as a colourless oil
(2.53 g, 89 %; 17.8 mmol). [a]2D0 þ8.2 (c 1.23 in MeOH). nmax
(neat)/cmꢁ1 3398, 3081, 1639. dH (CDCl3, 300 MHz) 5.81 (1H,
ddt, J 16.9, 10.3, 6.6, 8-H), 5.0–4.9 (2H, m, 9-H), 3.9–3.7 (1H,
m, 2-H), 2.1–2.0 (2H, m, 7-H), 1.2–1.5 (8H, m, 3–6-H), 1.18
(3H, d, J 6.2, 1-H). dC (CDCl3, 75 MHz) 139.0 (8-C), 114.2
(9-C), 68.1 (2-C), 39.3 (3-C), 33.7 (7-C), 29.1, 28.9, 25.6, 23.5
(1-C). m/z (EI) 142 (60 %, [M]þꢃ), 127 (100). HREI m/z
142.1356; calcd for C9H18O 142.1358.
(R)-8-Oxooctan-2-yl Acetate ((R)-12)
The title compound was synthesized in the same manner as
(S)-8-oxooctan-2-yl acetate from (R)-non-8-en-2-yl acetate.
[a]2D0 ꢁ3.1 (c 10.6 in MeOH). All remaining spectroscopic data
were identical to those of (S)-8-oxooctan-2-yl acetate.
(S)-7-Acetoxyoctanoic Acid ((S)-13)
Sodium chlorite (488 mg, 5.40 mmol) and sodium dihydrogen
phosphate (344 mg, 2.87 mmol) were dissolved in water (8 mL),
and this solution was added to a solution of (S)-8-oxooctan-2-yl
acetate (585 mg, 2.80 mmol), 2-methyl-2-butene (2.0 M in THF,
3.00 mL, 421 mg, 6.00 mmol) in t-BuOH (4 mL). The mixture
was allowed to stir at room temperature for 45 min before being
quenched with water and extracted with EtOAc. The organic
layer was dried with MgSO4, filtered, and concentrated to give
the title compound as a colourless oil (520 mg, 92 %;
2.58 mmol). [a]D20 þ2.9 (c 1.07 in MeOH). nmax (neat)/cmꢁ1
3107, 2985, 2739, 1736, 1713. dH (CDCl3, 300 MHz) 5.00–4.80
(1H, m, 7-H), 2.34 (2H, t, J 7.5, 2-H), 2.02 (3H, s, C(O)CH3),
1.75–1.25 (8H, m, 3–6-H), 1.20 (3H, d, J 6.3, 8-H). dC (CDCl3,
100 MHz) 178.9 (1-C), 170.7 (C(O)CH3), 70.8 (7-C), 37.8
(2-C), 35.6 (6-C), 28.9, 25.1, 21.9, 21.3(C(O)CH3), 19.9 (8-C).
(R)-Non-8-en-2-ol ((R)-10)
The title compound was synthesized in the same manner as
(S)-non-8-en-2-ol from (R)-propylene oxide. [a]2D0 ꢁ8.9 (c 1.16
in MeOH). All remaining spectroscopic data were identical to
those of (S)-non-8-en-2-ol.
(S)-Non-8-en-2-yl Acetate ((S)-11)
To a solution of (S)-non-8-en-2-ol (2.42 g, 17.0 mmol) in pyri-
dine (3.00 mL, 2.95 g, 37.2 mmol) was added acetic anhydride

1590
A. M. Beekman and R. A. Barrow
m/z (ESI) 201 (100 %, [M – H]–). HRESI m/z 201.1135; calcd for
C10H17O4 201.1132.
chromatography on silica using 66 % EtOAc in petroleum spirits
as eluent (RF ¼ 0.2), yielding a colourless amorphous solid
(250 mg, 71 %; 0.682 mmol). [a]2D0 þ7.0 (c 5.30 in MeOH). nmax
(neat)/cmꢁ1 3180, 2910, 2838, 1725, 1701, 1645. dH (CDCl3,
300 MHz) 6.38 (1H, d, J 2.3, 60-H), 6.31 (1H, d, J 2.3, 40-H), 4.83
(1H, tq, J 6.3, 6.3, 700-H), 3.67 (2H, s, 2-H), 3.62 (3H, s,
1-OCH3), 2.90 (2H, apparent t, 200-H), 1.96 (3H, s, C(O)CH3),
1.75–1.25 (8H, m, 300-600-H), 1.17 (3H, d, J 6.3, 800-H). dC
(CDCl3, 75 MHz) 206.2 (100-C), 172.06 (1-C), 170.6 (C(O)CH3),
160.7 (50-C), 159.7 (30-C), 137.0 (10-C), 120.9 (20-C), 111.9
(60-C), 102.7 (40-C), 71.1 (700-C), 51.9 (1-OCH3), 44.3 (200-C),
39.9 (2-C), 36.5 (600-C), 30.5, 30.5, 24.9, 21.2 (C(O)CH3), 20.2
(800-C). m/z (ESI) 367 (55 %, [M þ H]þ), 102 (100). HRESI m/z
367.1758; calcd for C19H27O7 367.1757.
(R)-7-Acetoxyoctanoic Acid ((R)-13)
The title compound was synthesized in the same manner as
(S)-7-acetoxyoctanoic acid from (R)-8-oxooctan-2-yl acetate.
[a]2D0 ꢁ2.6 (c 1.01 in MeOH). All remaining spectroscopic data
were identical to those of (S)-7-acetoxyoctanoic acid.
Methyl (S)-2-(2-(7-Acetoxyoctanoyl)-3,5-
diisopropoxyphenyl)acetate ((S)-14)
To a solution of (S)-7-acetoxyoctanoic acid (520 mg, 2.58 mmol)
in anhydrous CH2Cl2 (5 mL) was added trifluoroacetic anhy-
dride (0.364 mL, 542 mg, 2.58 mmol), and the solution was
stirred at room temperature for 2 h. The solvent was removed
under vacuum and the resulting mixed anhydride was added to a
solution of methyl 2-(3,5-diisopropoxyphenyl)acetate (266 mg,
1.00 mmol) in toluene (10 mL), and HClO4 (2 drops) was added.
The resulting solution was stirred at room temperature for 8 h at
which time a further 2 drops of HClO4 was added and the
solution was stirred for a further 16 h. NaHCO3(aq) was added
and the solution was extracted with EtOAc. The organic layer
was washed with brine, dried with MgSO4, filtered, and con-
centrated to give a brown oil. Purification by flash column
chromatography on silica using 33 % EtOAc in petroleum spirit
as eluent (RF ¼ 0.2) yielded the title compound as a colourless
amorphous solid (432 mg, 96 %; 0.960 mmol). [a]2D0 þ5.9
(c 4.00 in MeOH). nmax (neat)/cmꢁ1 2919, 2831, 1738, 1711,
1690, 1637. dH (CDCl3, 300 MHz) 6.35 (1H, d, J 2.1, 60-H), 6.33
(1H, d, J 2.1, 40-H), 4.88 (1H, tq, J 6.4, 6.4, 700-H), 4.55 (1H,
septet, J 6.1, 30-OCH(CH3)2), 4.53 (1H, septet, J 6.1, 50-OCH
(CH3)2), 3.67 (3H, s, 1-OCH3), 3.60 (2H, s, 2-H), 2.83 (2H, dd,
J 7.8, 7.8, 200-H), 2.02 (3H, s, C(O)CH3), 1.7–1.3 (8H, m, 300-600-
H), 1.34 (6H, d, J 6.1, 30-OCH(CH3)2), 1.32 (6H, d, J 6.1,
50-OCH(CH3)2), 1.20 (3H, d, J 6.4, 800-H). dC (CDCl3, 100 MHz)
206.9 (100-C), 171.7 (1-C), 170.7 (C(O)CH3), 159.5 (50-C), 157.1
(30-C), 134.3 (10-C), 124.5 (20-C), 109.3 (60-C), 100.5 (40-C),
70.9 (700-C), 70.5, 69.9, 51.9 (1-OCH3), 44.3 (200-C), 38.7 (2-C),
35.7 (600-C), 29.2, 25.3, 24.0, 21.9 (OCH(CH3)2), 21.3 (C(O)
CH3), 19.9 (800-C). m/z (EI) 450 (20 %, [M]þꢃ), 293 (100), 181
(55). HREI m/z 450.2620; calcd. for C25H38O7 450.2618.
Methyl (R)-2-(2-(7-Acetoxyoctanoyl)-3,5-
dihydroxyphenyl)acetate ((R)-15)
The title compound was synthesized in the same manner as methyl
(S)-2-(2-(7-acetoxyoctanoyl)-3,5-dihydroxyphenyl)acetate from
methyl (R)-2-(2-(7-acetoxyoctanoyl)-3,5-diisopropoxyphenyl)
acetate. [a]2D0 ꢁ6.3 (c 4.90 in MeOH). All remaining spectro-
scopic data were identical to those of methyl ((S)-2-(2-(7-
acetoxyoctanoyl)-3,5-dihydroxyphenyl)acetate.
(2S,10R) And (2S,10S)-7-(6,8-Dihydroxy-3-oxoisochroman-
1-yl)heptan-2-yl Acetate ((2S)-4)
To a solution of methyl (S)-2-(2-(7-acetoxyoctanoyl)-3,5-
dihydroxyphenyl)acetate (100.0 mg, 0.272 mmol) in methanol
(10 mL) was added sodium borohydride (11.3 mg, 0.300 mmol),
and the solution was stirred at room temperature for 2 h. The
solution was diluted with water and extracted with EtOAc, and
the organic layer was dried with MgSO4, filtered, and concen-
trated to give a colourless solid. Purification was achieved via
flash column chromatography on silica using 80 % EtOAc in
petroleum spirits as eluent (RF ¼ 0.4) to give the title compounds
as a colourless amorphous solid (92.1 mg, 92 %; 0.273 mmol).
Purity was confirmed via HPLC analysis on a Phenomenex LUX
cellulose-4 5 m column using 35 % ethanol in n-hexane as sol-
vent with a flow rate of 0.5 mL minꢁ1 resulting in a single
retention time for the diastereomeric mixture of 8.6 min. [a]D20
þ6.5 (c 1.02 in MeOH). nmax (neat)/cmꢁ1 3168, 2917, 2820,
1736, 1711, 1631. lmax (CH3OH)/nm (e/Mꢁ1 cmꢁ1) 203 (log e
3.81), 280 (3.41). dH ([D6]acetone, 800 MHz) 6.37 (d, J 2.0, 1H,
50-H), 6.22 (1H, d, J 2.0, 70-H), 5.55 (1H, dd, J 8.9, 5.1, 10-H),
4.82 (1H, m, 2-H), 3.78 (1H, d, J 19.2, 40-HA), 3.45 (1H, d,
J 19.2, 40-HB), 1.94 (3H, s, C(O)CH3), 1.90–1.75 (2H, m, 7-H),
1.60–1.25 (8H, m, 3–6-H), 1.15 (3H, d, J 6.2, 1-H). dC ([D6]
acetone, 200 MHz) 169.7 (C(O)CH3), 169.6 (30-C), 158.2
(80-C), 153.6 (60-C), 132.6 (40a-C), 113.0 (80a-C), 105.3 (50-C),
100.9 (70-C), 77.4 (10-C), 70.2 (2-C), 35.6 (7-C), 35.5 (40-C),
34.5 (3-C), 29.5, 25.4, 25.1, 20.3, 19.3 (1-C). m/z (ESI) 337
(65 %, [M þ H]þ), 181 (100). HRESI m/z 337.1649; calcd for
C18H25O6 337.1651.
Methyl (R)-2-(2-(7-Acetoxyoctanoyl)-3,5-
diisopropoxyphenyl)acetate ((R)-14)
The title compound was synthesized in the same manner as
methyl (S)-2-(2-(7-acetoxyoctanoyl)-3,5-diisopropoxyphenyl)
acetate from (R)-7-acetoxyoctanoic acid. [a]2D0 ꢁ6.7 (c 4.70 in
MeOH). All remaining spectroscopic data were identical to
those of (S)-2-(2-(7-acetoxyoctanoyl)-3,5-diisopropoxyphenyl)
acetate.
Methyl (S)-2-(2-(7-Acetoxyoctanoyl)-3,5-
dihydroxyphenyl)acetate ((S)-15)
To a solution of methyl (S)-2-(2-(7-acetoxyoctanoyl)-3,5-
diisopropoxyphenyl)acetate (432 mg, 0.960 mmol) in CH2Cl2
(50 mL) was added aluminium chloride (306 mg, 2.30 mmol),
and the solution was allowed to stir at room temperature for 2 h.
After all of the starting material was consumed, as monitored by
TLC, NH4Cl(aq) was added and the solution was extracted with
EtOAc. The organic layer was washed with water and brine,
dried with MgSO4, filtered, and concentrated to give a yellow
amorphous solid. Purification was achieved via flash column
(2R,10R) And (2R,10S)-7-(6,8-Dihydroxy-3-oxoisochroman-
1-yl)heptan-2-yl acetate ((2R)-4). Includes Cytosporone K
The title compounds were synthesized in the same manner as
(2S,10R) and (2S,10S)-7-(6,8-dihydroxy-3-oxoisochroman-1-yl)
heptan-2-yl acetate from methyl (R)-2-(2-(7-acetoxyoctanoyl)-
3,5-dihydroxyphenyl)acetate. Purity was confirmed via HPLC
analysis on a Phenomenex LUX cellulose-4 5 m column using
35 % ethanol in n-hexane as solvent with a flow rate of

First Syntheses of Cytosporones J and K
1591
0.5 mL minꢁ1 resulting in a single retention time for the dia-
stereomeric mixture of 13.4 min. [a]2D0 ꢁ5.7 (c 1.60 in MeOH).
All remaining spectroscopic data were identical to those of
(2S,10R) and (2S,10S)-7-(6,8-dihydroxy-3-oxoisochroman-1-yl)
heptan-2-yl acetate.
18 h on a shaker incubator. After incubation, the plate was read
at A600 to assess the relative turbidity (i.e. growth) of the treated
cultures. An inhibition level of 50 % (IC50) was determined
based on comparison with the average turbidity readings of the
untreated control. Inhibition was calculated as:
½logðA=BÞꢂð50ꢁCÞ=ðDꢁCÞþlogðBÞꢄ
(1R,60S) And (1S,60S)-6,8-Dihydroxy-1-(6-hydroxyheptyl)
isochroman-3-one ((60S)-3)
IC50 ¼ 10
To a solution of (2S,10R) and (2S,10S)-7-(6,8-dihydroxy-3-
oxoisochroman-1-yl)heptan-2-yl acetate (10.0 mg, 29.7 mmol)
in methanol (2 mL) was added ammonium hydroxide (2 mL),
and the solution was stirred at room temperature for 16 h. The
reaction was then cooled, and water was added and the solution
was extractedwith EtOAc. The organiclayer was then dried with
MgSO4, filtered, and concentrated to give the title compounds as
a colourless amorphous solid (8.6 mg, 99 %; 29 mmol). Purity
was confirmed via HPLC analysis on a Phenomenex LUX cel-
lulose-4 5 m column using 35 % ethanol in n-hexane as solvent
with a flow rate of 0.5 mL minꢁ1 resulting in a retention time of
8.2 min. [a]2D0 þ7.9 (c 0.96 in MeOH). nmax (neat)/cmꢁ1 3168,
where A represents the higher concentration of the test com-
pound of the two data points that bracket 50 % inhibition;
B represents the lower concentration of the test compound of
the two data points that bracket 50 % inhibition; C is the
inhibitory activity (%) at concentration B; and D is the inhibitory
activity (%) at concentration A. The MIC refers to the concen-
tration at which no growth was observed.
Supplementary Material
1H NMR and 13C NMR for all new compounds as well analytical
data for the synthesized natural products are available on the
Journal’s website.
3102, 2935, 2802, 1718, 1653. lmax (CH3OH)/nm (e/Mꢁ1 cmꢁ1
)
203 (log e 4.02), 281 (3.32). dH ([D6]acetone, 800 MHz) 6.37
(1H, d, J 2.1, 5-H), 6.22 (1H, d, J 2.1, 7-H), 5.55 (1H, dd, J 8.9,
5.1, 1-H), 3.78 (1H, d, J 19.2, 4-HA), 3.68 (1H, m, 60-H), 3.45
(1H, d, J 19.2, 4-HB), 1.60–1.25 (2H, m, 10-H), 1.5–1.10 (8H, m,
20-50-H), 1.08 (3H, d, J 6.2, 70-H). dC ([D6]acetone, 200 MHz)
169.6 (3-C), 158.1 (8-C), 153.5 (6-C), 132.5 (4a-C), 113.0 (8a-
C), 105.3 (5-C), 100.8 (7-C), 77.4 (1-C), 69.1 (20-C), 35.6 (70-C),
35.5 (4-C), 34.5 (30-C), 29.5, 25.4, 25.1, 20.2, 19.3 (10-C). m/z
(ESI) 295 (45 %, [M þ H]þ), 181 (100). HRESI m/z 295.1542;
calcd for C16H23O5 295.1540.
Acknowledgements
This study was undertaken with the support of the ANU and an NHMRC
project grant (1028092). AMB acknowledges the ANU for an Australian
Postgraduate Award.
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