Anti-inflammatory thiazine alkaloids isolated from the New Zealand ascidian Aplidium sp.: Inhibitors of the neutrophil respiratory burst in a model of gouty arthritis

Article abstract of DOI:10.1021/np060626o

Ascidiathiazones A (3) and B (4), two new tricyclic thiazine-containing quinolinequinone alkaloids, were isolated from the New Zealand ascidian Aplidium species. Both compounds inhibited the in vitro production of superoxide by PMA-stimulated human neutrophils in a dose-dependent manner with IC₅₀ 1.55 ± 0.32 and 0.44 ± 0.09 μM, respectively. In vivo inhibition of superoxide production by peritoneal neutrophils in a murine model of gout was observed for both compounds with oral doses of 25.6 μmol/kg. Ascidiathiazone A (3) was synthesized in four steps from 8-hydroxyquinoline-2- carboxylic acid.

Full text of DOI:10.1021/np060626o

936
J. Nat. Prod. 2007, 70, 936-940
Anti-inflammatory Thiazine Alkaloids Isolated from the New Zealand Ascidian Aplidium sp.:
Inhibitors of the Neutrophil Respiratory Burst in a Model of Gouty Arthritis
A. Norrie Pearce,† Elizabeth W. Chia,‡ Michael V. Berridge,‡ George R. Clark,† Jacquie L. Harper,‡ Lesley Larsen,§
Elizabeth W. Maas, Michael J. Page,| Nigel B. Perry,§ Victoria L. Webb, and Brent R. Copp*,†
TerraMarine Pharmaceuticals, Department of Chemistry, The UniVersity of Auckland, PriVate Bag 92019, Auckland, New Zealand, Malaghan
Institute of Medical Research, P.O. Box 7060, Wellington South, New Zealand, New Zealand Institute for Crop & Food Research Ltd.,
Chemistry Department, UniVersity of Otago, P.O. Box 56, Dunedin, New Zealand, National Institute of Water & Atmospheric Research (NIWA)
Ltd., PriVate Bag 14-901, Kilbirnie, Wellington, New Zealand, and NIWA, P.O. Box 893, Nelson, New Zealand
ReceiVed December 17, 2006
Ascidiathiazones A (3) and B (4), two new tricyclic thiazine-containing quinolinequinone alkaloids, were isolated from
the New Zealand ascidian Aplidium species. Both compounds inhibited the in Vitro production of superoxide by PMA-
stimulated human neutrophils in a dose-dependent manner with IC50 1.55 ( 0.32 and 0.44 ( 0.09 µM, respectively. In
ViVo inhibition of superoxide production by peritoneal neutrophils in a murine model of gout was observed for both
compounds with oral doses of 25.6 µmol/kg. Ascidiathiazone A (3) was synthesized in four steps from 8-hydroxyquinoline-
2-carboxylic acid.
Inflammation is a common characteristic of many debilitating
human diseases including gout, rheumatoid arthritis, asthma, and
chronic obstructive pulmonary disease. Many of the current
treatments involve the use of nonsteroidal anti-inflammatory drugs
(NSAIDs) that target the cyclooxygenase and phospholipase
enzymes in the inflammatory cascade. Although these therapies can
be effective, some individuals fail to respond to treatment or
experience cardiovascular and gastrointestinal side effects.1-3
Refractory disease and poor tolerance to side effects highlight a
need for alternative anti-inflammatory treatments. Many inflam-
matory diseases, including gouty arthritis and non-atopic asthma,
are associated with the infiltration of neutrophils and the subsequent
production of damage-causing superoxide. Therefore inhibition of
superoxide production by human neutrophils represents an alternate
target of the acute inflammatory response.4
During screening for anti-inflammatory natural products from
gave the activity concentrated in the water fraction. Repeated C18
chromatography followed by size exclusion chromatography on
Sephadex LH-20 yielded two related biologically active compounds,
3, a yellow powder (0.02% dry wt), and 4, a pink powder (0.005%
dry wt), which were characterized as the free acids.
The major compound, ascidiathiazone A (3), gave bright yellow
solutions in neutral and acidic aqueous media, which turned pink
upon the addition of base. The UV-visible spectrum revealed
absorption maxima at 205, 237, 274, 290, and 420 nm, which
exhibited a bathochromic shift under basic conditions to 211, 236,
329, and 467 nm, suggesting the presence of an extended aromatic
chromophore and acidic/basic groups. The IR spectrum showed
strong broad absorptions at 1660 and 1651 cm-1, indicating the
presence of a carboxylic acid and/or a 1,4-quinone. A molecular
formula of C12H8N2O6S for 3 was established by negative ion
ESIFTMS.
New Zealand biota, an extract of the ascidian Aplidium sp. exhibited
strong inhibition of superoxide production by human neutrophils
stimulated with PMA.5 The genus Aplidium (family Polyclinidae)
is one of the largest genera of ascidians and is found worldwide.6
A wide variety of biologically active compounds have been reported
from Aplidium species including simple prenylated quinones and
hydroquinones (e.g., 1), which exhibit antiproliferative activities.7
Recently, several prenylated quinones bearing a fused 1,1-dioxo-
1,4-thiazine ring, e.g., conicaquinone A (2), have been reported
from the Mediterranean ascidian Aplidium conicum.8-10 We now
report that bioassay-guided fractionation of the extract of the New
Zealand ascidian Aplidium sp. resulted in the discovery of two
thiazine-containing 2-quinolinequinone carboxylic acid derivatives,
which we have named ascidiathiazones A (3) and B (4). The
structures of 3 and 4 were determined by spectroscopic methods,
with confirmation of 3 also being achieved by X-ray crystal-
lography. The major natural product 3 was synthesized, and in Vitro
and in ViVo anti-inflammatory activities are reported.
The 1H NMR spectrum of 3 (DMSO-d6) was simple, comprised
of two exchangeable singlets (δ 13.80 and 9.43), two ortho-coupled
aromatic doublets at δ 8.51 and 8.38, and two coupled methylene
resonances (δ 3.89 and 3.41, Table 1). The 13C NMR spectrum of
3 (Table 1) contained 12 signals, consistent with the molecular
formula, and was comprised of two methylene carbons (δ 48.1 and
39.0) and 10 sp2 carbons (δ 110.7-176.2).
Results and Discussion
Bioassay-guided fractionation of a MeOH-CH2Cl2 extract of
Aplidium sp. using reversed-phase C18 flash CC (H2O/MeOH/TFA)
* To whom correspondence should be addressed. Fax: 64-9-373-7422.
E-mail: b.copp@auckland.ac.nz.
1
1
Extensive interpretation of COSY, H-13C gHSQC, and H-
13C and 1H-15N gHMBC NMR data (Table 1) allowed construction
of two fragments, comprised of a 6,7-disubstituted quinoline-
quinone-2-carboxylic acid ring system and a second fragment of a
1,2-disubstituted ethane. The presence of a quinolinequinone
† The University of Auckland.
‡ Malaghan Institute of Medical Research.
§ New Zealand Institute for Crop & Food Research Ltd.
NIWA, Kilbirnie.
| NIWA, Nelson.
10.1021/np060626o CCC: $37.00
© 2007 American Chemical Society and American Society of Pharmacognosy
Published on Web 05/11/2007

Anti-inflammatory Thiazine Alkaloids from Aplidium sp.
Journal of Natural Products, 2007, Vol. 70, No. 6 937
Table 1. 1H and 13C NMR Data for Ascidiathiazones A (3) and B (4)
ascidiathiazone A (3)
ascidiathiazone B (4)
a
d
pos
δC/δN
δ
H (mult, J in Hz)b
HMBCc
δC
δ
H (mult, J, Hz)f
HMBCc
2
3
4
4a
5
5a
6
7
8
9
9a
10
10a
11
48.1
39.0
3.41 (t, 6)
3.89 (m)
9.43 (br s)
N-4, 10*, 10a*
4a
114.1
141.8
nde
150.8
181.1
127.8
137.0
126.0
158.2
nd
147.3
176.5
113.9
165.8
nog
6.29 (d, 7.9)
7.34 (d, 7.9)
10*, 10a
4a
103.0
147.7
176.2
146.2
300.0
151.3
128.9
135.6
131.5
173.2
110.7
165.1
8.45 (d, 7.9)
8.18 (d, 7.9)
5, 8, 9a, 10*
5a, 11
8.38 (d, 8.1)
8.51 (d, 8.1)
9a, 11
5*, 5a, 7, 10
COOH
13.80 (br s)
a 13C NMR spectrum recorded in DMSO-d6 at 100 MHz. 15N chemical shifts deduced indirectly from 1H-15N HMBC NMR experiment (optimized
b
for 6 Hz and referenced to liquid NH3 using urea as an external standard). Spectrum recorded in DMSO-d6 at 400 MHz. c1H-13C HMBC NMR
x
x
experiment optimized for JCH ) 8.3 Hz. Correlations observed for experiment optimized for JCH ) 2 Hz are indicated by asterisks. d13C NMR
e
f
g
spectrum recorded in DMSO-d6 at 75 MHz. nd: not determined. Spectrum recorded in DMSO-d6 at 300 MHz. no: not observed.
fragment was suggested by the observation of 1H-13C HMBC
x
correlations (experiment optimized for JCH of 8.3 Hz) from H-9
(δ 8.51) to δ 151.3 (C-7) and 146.2 (C-5a) and a quinonoid
resonance at δ 173.2 (C-10), while a carboxylic acid group could
be placed at C-7 by virtue of an observed correlation from H-8 (δ
8.38) to a resonance at δ 165.1 (C-11). The presence of the
carboxylic acid was supported by conversion of 3 to the methyl
ester 5 by treatment with thionyl chloride in methanol (see
1
Figure 1. Selected H-13C HMBC correlations observed for 4
Experimental Section). Placement of a nitrogen atom was achieved
and 6.
1
by detection of a strong H-15N HMBC correlation from H-8 (δ
8.38) to a pyridine-type 15N resonance at 300.0 ppm.11 Observation
was prepared by total synthesis (Vide infra). Significant chemical
shift differences were observed for H-2/H-3/H-6/9 and H-7/8 (∆δ
0.12-0.33) and C-3/C-4a/C-5/C-5a and C-9a (∆δ 5.6-18.7),
indicating that compound 6 was not the structure of the isolated
of a long-range correlation from H-9 to a second quinonoid carbon
resonance at δ 176.2 (C-5) in a 1H-13C HMBC NMR experiment
optimized for 2 Hz completed the quinolinequinone fragment.
The assignment of the two remaining quinolinequinone carbon
minor metabolite. Further evidence for the regiochemistry of the
1
thiazine ring was contained in correlations observed in H-13C
HMBC NMR experiments on 4 and 6. In the case of 6, correlations
were observed between both the relatively shielded â-enamine
proton H-2 (δ 6.62) and pyridine ring proton H-9 (δ 8.57) and the
same quinonoid resonance C-10 (δ 177.6), while for 4 a correlation
from the â-enamine proton H-2 (δ 6.29) was observed to one
quinonoid resonance C-10 (δ 176.5) and from the pyridine ring
proton H-6 (δ 8.45) to the other quinonoid resonance C-5 (δ 181.1)
(Figure 1). Thus the structure of the minor natural product was
proposed to be 4. The 4H-1,4-benzothiazine-1,1-dioxide ring system
contained in 4 is rare in nature, with the only prior report being
the euthyroidenone family of alkaloids isolated from a New Zealand
bryozoan.14
Ascidiathiazone A (3) was synthesized via the route shown in
Scheme 1. Commercially available 8-hydroxyquinoline-2-carboxylic
acid was treated with SOCl2 in MeOH at reflux to yield methyl
ester 7, which was oxidized to quinone 8 with Fremy’s salt. Stirring
quinone 8 in EtOH with cerium chloride heptahydrate and hypo-
taurine for 3 days yielded the chromatographically separable 1,1-
dioxothiazine regioisomers 5 and 9 in a 10:1 ratio. Spectroscopic
data observed for synthetic 5 were identical to those observed for
semisynthetically prepared 5. Hydrolysis of the ester group in 5
by heating in concentrated HCl yielded 3, which was identical with
the natural product in all respects. In contrast, saponification of 5
yielded oxidized acid 6.
resonances (δ 147.7 (C-4a) and 110.7 (C-10a)) and a 1,1-dioxo-
1,4-thiazine ring was achieved in the following manner. Inspection
of the 1H NMR spectrum, combined with COSY, HSQC (1H-13C
and 1H-15N), and HMBC (1H-13C and 1H-15N) NMR data
analysis (Table 1), indicated the presence of a -NHCH2CH2- spin
system, while the chemical shifts of the two associated methylene
13C resonances (δ 48.1 and 39.0) were similar to those previously
reported for a 1,1-dioxo-1,4-thiazine moiety (-NHCH2CH2SO2-
).8-10,12 Fusion of this moiety to positions 4a and 10a was deduced
by the observation of an 1H-13C HMBC correlation from H2-3 (δ
3.89) to C-4a (δ 147.7) in an experiment optimized for 8.3 Hz,
while weaker correlations were observed from H2-2 (δ 3.41) in a
2 Hz optimized 1H-13C HMBC experiment to carbons at δ 110.7
(C-10a) and crucially to the signal at δ 173.2, already assigned to
C-10. This completed the structural assignment of compound 3,
including the proposed assignment of the thiazine ring regiochem-
istry. Confirmation of the proposed structure 3 was achieved with
the aid of a single-crystal X-ray diffraction study (see Supporting
Information).13
The minor compound, ascidiathiazone B (4), was characterized
as an oxidized derivative of 3 by (-)-ESIFTMS, showing two fewer
hydrogens than 3, and NMR data, which indicated replacement of
the H2-2-H2-3 spin system with two ortho-coupled vinylic protons
1
at δ 7.34 and 6.29 (Table 1). As observed for 3, long-range H-
13C HMBC NMR correlations established the presence of a
quinolinequinone-carboxylic acid fragment (Table 1). This left the
possible structure of the minor natural product as being either 4 or
the regioisomer 6.
Ascidiathiazones A (3) and B (4) and compounds 5 and 8
strongly inhibited in Vitro superoxide production by PMA-
stimulated human neutrophils in a dose-dependent manner (Table
2).4 Whereas the anti-inflammatory effect of synthetic intermediate
8 was associated with cell stasis or toxicity (AP50), natural products
Compelling proof of the identity of 4 was achieved by direct
comparison of NMR data observed for 4 and 6, the latter of which

938 Journal of Natural Products, 2007, Vol. 70, No. 6
Pearce et al.
Experimental Section
Scheme 1. Synthesis of Ascidiathiazone A (3) and the
Regioisomer of Ascidiathiazone B (6)
General Experimental Procedures. UV-vis spectra were run as
methanol solutions on a UV-2102 PC Shimadzu UV-vis scanning
spectrophotometer. IR spectra were acquired as either dry films or Nujol
mulls on a Spectrum One FTIR spectrometer with the 1603 cm-1
absorption band of polystyrene being used as reference. NMR spectra
were recorded on either a Bruker Avance DRX-600 spectrometer at
1
600 MHz for H and 150 MHz for 13C, a Bruker Avance DRX-400
1
spectrometer at 400 MHz for H and 100 MHz for 13C, or a Bruker
1
Avance 300 spectrometer at 300 MHz for H and 75 MHz for 13C.
Residual solvent signals were used as reference (DMSO-d6: δH, 2.50;
δC, 39.4; CD3OD: δH, 3.30; δC, 49.0; CDCl3: δH, 7.25; δC, 77.0 ppm).
MS were recorded on either a VG-7070 or a Thermo LTQ-FT mass
spectrometer. Compound purity was determined by reversed-phase
HPLC (Waters 600 HPLC photodiode array system, Alltech Econosil
C18, 5 µm, 4.6 × 125 mm, H2O (0.05% TFA) to MeCN over 25 min
at 1.0 mL/min and monitoring at 254 nm).
Biological Material. The ascidian was collected (950 g) by scuba
at a depth of 28 m from Tom Bowling Bay, North Cape, North Island,
New Zealand, and kept frozen until used. A voucher specimen of the
orange-brown colonial ascidian Aplidium sp. is held at the National
Institute for Water and Atmospheric Research, Private Bag 14-901,
Kilbirnie, Wellington, New Zealand, as MNP7215 and MNP7347. A
color in situ photograph is included in the Supporting Information.
Aplidium sp. (TerraMarine accession codes: MNP7215, MNP7347)
forms erect lamellate colonies to 10 cm in maximum height and 1 cm
thick. Colonies are occasionally attached to the substratum by a short,
wide stalk. Common clocal apertures of 1-2 mm in diameter are evenly
distributed throughout the test. Zooids are arranged in regular circular
systems around slightly raised common clocal apertures. Living
specimens have an opaque brown-colored test, but zooids are nonpig-
mented. Aplidium sp. most closely resembles Aplidium gilVum.21
However, the morphology of the colony (sandy stalk), transparent test,
pointed branchial lobes, and structure of the atrial lappet set the two
species apart. Apidium sp. has not been described before in New Zealand
and does not resemble any Australian Aplidium species described by
Kott;6 it is therefore considered to be a new species endemic to New
Zealand waters.
Isolation and Purification. The frozen animals were freeze-dried
(25.4 g) and extracted with MeOH (6 × 100 mL) followed by CH2Cl2
(2 × 100 mL). The combined extracts were filtered and dried to produce
9.96 g of crude extract, which was subjected to C18 reversed-phase
flash CC (water through to MeOH/TFA). The anti-inflammatory assay
showed the activity to be concentrated in the water fraction. Repeated
C18 CC (water through to 100% MeOH) followed by size exclusion
CC on Sephadex LH-20 yielded 3 (5.1 mg, 0.02% dry wt) and the
later eluting compound 4 (1.3 mg, 0.005% dry wt). The compounds
were characterized as the free acids.
3 and 4 selectively suppressed superoxide production. To rule out
nonspecific antioxidant effects, 3-5 were also tested in a xanthine/
xanthine oxidase enzyme-based superoxide scavenging assay. As
shown in Table 2, all three compounds were considerably less
effective as superoxide scavengers than as suppressors of superoxide
production by neutrophils.
The in Vitro results observed for 3-5, with their indications of
potent and selective inhibition of neutrophil superoxide production
and low antiproliferative activity, prompted us to evaluate the
compounds in an animal model for gout. In this model, monosodium
urate crystals were injected intraperitoneally (ip) into mice, and
the recruitment of neutrophils and their ability to produce superoxide
were measured.15
Peritoneal neutrophils collected from mice treated orally with 5
(25.6 µmol/kg) exhibited marked inhibition of superoxide produc-
tion (84 ( 3% inhibition) followed by 4 (59 ( 9% inhibition) and
3 (23 ( 11% inhibition) (Figure 2). Interestingly 5 showed the
greatest inhibitory effect in ViVo, whereas it was the weakest
inhibitor in Vitro (Table 2). Compound 5 also inhibited urate-
induced neutrophil recruitment into the peritoneal cavity by 60%
at 25 µmol/kg, whereas 3 and 4 were inactive (data not shown).
Oral bioavailability and pharmacokinetics of the ester compared
with the acid may play a role in these contrasting patterns of activity.
Renal and heptatic toxicity is a common problem with many
existing NSAIDs.16 Colchicine is one such drug that is still used
therapeutically to treat gout despite the known hepatic side effects.17
None of the test compounds caused significant elevation of liver
and renal toxicity markers in sera from treated mice compared to
the na¨ıve and disease controls in ViVo (Supporting Information
Figure S3), and the serum markers were consistently lower than
colchicine. These results provided evidence that the liver and renal
side effects of these new anti-inflammatory compounds may be
low or nonexistent in the context of the acute inflammation model.
Ascidiathiazone A (3): yellow powder; mp 155 °C (dec); UV
(MeOH) λmax (log ꢀ) 416 (3.40), 268 (4.21), 236 (4.46), 216 (4.37);
(MeOH/TFA) 421 (3.56), 274 (4.18), 237 (4.55), 205 (4.43); (MeOH/
KOH) 467 (3.59), 329 (3.97), 236 (4.52), 211 (5.07) nm; fluorescence
(MeOH/TFA), Ex 250 nm, Em 508 nm; (MeOH/KOH) Ex 250 nm,
Em 507 nm; IR (film) νmax 3034, 1660, 1651, 1585, 1417, 1193, 1129
cm-1; NMR data are reported in Table 1; HRESI(-)FTMS m/z
307.0030 [M - H]- (calcd for C12H7N2O6S, 307.0025); purity 96%;
tR ) 15.41 min.
Ascidiathiazone B (4): pink powder; mp 280 °C (dec); UV (MeOH/
TFA) λmax (log ꢀ) 417 (3.11), 269 (3.91), 237 (4.02), 214 (4.50);
(MeOH/KOH) 472 (3.29), 311 (3.62), 279 (3.81), 237 (4.04), 207 (4.95)
nm; fluorescence (MeOH/TFA) Ex 250 nm, Em 505 nm; (MeOH/KOH)
Ex 250 nm, Em 506 nm; IR (film) νmax 3414, 1681, 1638, 1524, 1279,
1127 cm-1; NMR data are reported in Table 1; HRESI(-)FTMS m/z
304.9874 [M - H]- (calcd for C12H5N2O6S, 304.9868); purity 99%;
tR ) 15.54 min.
In summary, the screening of New Zealand biota for bioactive
molecules capable of inhibiting the respiratory burst of human
neutrophils has led to the discovery of two new natural products,
3 and 4, containing rare 1,1-dioxo-1,4-thiazine rings. We presume
that these compounds are biosynthesized by a similar route to our
synthesis, i.e., by addition of hypotaurine, which has been reported
in another ascidian,18 to quinoline-5,8-quinone-2-carboxylic acid.
While this compound has not been reported as a natural product, a
possible precursor, 5-hydroxyquinoline-2-carboxylic acid, is known.19
The combination of in ViVo anti-inflammatory activity by oral
dosage, specificity for inhibition of superoxide production by
neutrophils, lack of toxicity markers, and ease of synthesis makes
this new compound class highly attractive for further study as
potential anti-inflammatory pharmaceuticals.20
Semisynthesis of Ascidiathiazone A Methyl Ester (5). A sample
of 3 (8 mg, 0.026 mmol) was stirred in dry MeOH (1.5 mL) under
nitrogen and cooled to -5 °C in a salted ice-bath. Excess SOCl2 (40
µL) was added, and the mixture was stirred 15 min before being allowed
to come to room temperature. It was then stirred at 40 °C for 2.5 h and
then dried under vacuum. The crude mixture was separated on a C18
Sep-pak, the product eluting with 10% MeOH/water, yielding 5 (3 mg,
36% yield) as a yellow gum: IR (film) νmax 1728 cm-1 1H NMR
;

Anti-inflammatory Thiazine Alkaloids from Aplidium sp.
Journal of Natural Products, 2007, Vol. 70, No. 6 939
Table 2. In Vitro Activities of Compounds 3, 4, 5, 7, and 8a
b
c
compound
AI50
AP50
X/XO d
3
4
5
7
8
1.55 ( 0.32 (n ) 7)
0.44 ( 0.09 (n ) 4)
2.26 ( 0.31 (n ) 4)
276.7 ( 56.95 (n ) 2)
1.75 ( 0.15 (n ) 2)
0.65 ( 0.06
73.08 ( 7.33 (n ) 5)
16.73 ( 2.02 (n ) 2)
11.78 ( 2.28 (n ) 2)
96.93 ( 96.52 (n ) 2)
4.11 ( 0.27 (n ) 2)
55.90 ( 7.90 (n ) 2)
17.89 ( 1.14 (n ) 2)
18.23 ( 5.66 (n ) 2)
NDe
ND
SOD
methotrexate
allopurinol
6.92 nM ( 0.84 nM
42.24 ( 5.86
b
a Values are IC50 values (units of µM unless stated otherwise) representing the mean of at least two determinations ( standard error. AI50:
anti-inflammatory activity. Concentration of compound required to inhibit in Vitro superoxide production by PMA-stimulated human neutrophils by
c
d
50%. AP50: antiproliferative activity. Concentration of compound required to inhibit HL60 cell proliferation by 50%. X/XO: xanthine/xanthine
e
oxidase inhibitory activity. Concentration of compound required to inhibit superoxide production in xanthine/xanthine oxidase assay by 50%. ND:
not determined.
mmol) was stirred in dry MeOH (2 mL) at 0 °C. SOCl2 (60 µL, 0.82
mmol) was added dropwise, and the suspension was stirred at 0 °C for
10 min under N2 and then at 65 °C for 2 h, during which time the
bright yellow suspension dissolved into solution and became light brown
in color. Water (10 drops) was added to quench the reaction mixture,
which was evaporated to dryness under vacuum to yield 7 (103 mg
1
(96%)) as a bright orange solid: mp 93-94 °C; H NMR (CD3OD,
400 MHz) δ 8.83 (1H, d, J ) 8.6 Hz), 8.30 (1H, d, J ) 8.6 Hz), 7.73
(1H, dd, J ) 7.9, 7.9 Hz), 7.62 (1H, d, J ) 8.0 Hz), 7.35 (1H, d, J )
7.8 Hz), 4.13 (3H, s); 13C NMR (CD3OD, 100 MHz) δ 164.1, 152.6,
143.8, 143.5, 134.9, 132.5, 131.9, 122.1, 119.3, 115.7, 54.6; EIMS m/z
203 [M]+ (60), 171 925), 143 (100); HREIMS m/z 203.0581 (calcd
for C11H9NO3, 203.0582); purity 100%; tR ) 21.62 min.
Methyl Quinoline-5,8-dione-2-carboxylate (8). To a solution of
Fremy’s salt (370 mg, 1.38 mmol) in water (10 mL) and aqueous KH2-
PO4 (0.167 M, 3 mL) at 0 °C was added 7 (60 mg, 0.30 mmol) in
MeOH (4 mL). The purple suspension was stirred for 10 min at 0 °C,
after which time it was allowed to come to room temperature and stirred
for 1 h. A second portion of Fremy’s salt, water, and aqueous KH2PO4
(0.167 M) were added. The reaction mixture was stirred for a further
2 h and extracted with 3 × 20 mL portions of CH2Cl2. The organic
extracts were dried under reduced pressure, affording crude 8 as an
orange solid (58 mg (90%)). This crude material was used in the
subsequent reaction without further purification. 1H NMR (CDCl3, 300
MHz) δ 8.59 (1H, d, J ) 8.1 Hz), 8.48 (1H, d, J ) 8.1 Hz), 7.25 (1H,
d, J ) 10.5 Hz), 7.14 (1H, d, J ) 10.5 Hz), 4.07 (3H, s); 13C NMR
(CDCl3, 75 MHz) δ 183.6, 182.0, 164.5, 152.3, 147.0, 139.6, 138.1,
136.2, 130.6, 128.5, 53.5.
Figure 2. Inhibition of superoxide production by peritoneal
neutrophils from gouty mice treated with 3-5 relative to mono-
sodium urate (MSU) alone treated mice. Mice were injected
intraperitoneally with MSU crystals (3 mg) in the presence or
absence of compounds 3-5 (oral gavage, 25.6 µmol/kg) or
colchicine (sc, 5 µmol/kg, control). The neutrophils were isolated
from the peritoneum at 4 h and tested for superoxide production
as described in the Experimental Section.
Ascidiathiazone A Methyl Ester 5 and Regioisomer 9. To a
solution of quinone 8 (58 mg, 0.27 mmol) and cerium chloride
heptahydrate (101 mg, 0.27 mmol) in absolute EtOH (25 mL) was added
hypotaurine (29 mg, 0.27 mmol) in water (2 mL). The reaction mixture
was stirred at room temperature for 3 days, then dried under reduced
pressure. The residue was triturated with MeOH, affording a MeOH-
insoluble red-brown solid (5) (47 mg (55%)). The filtrate was subjected
to C18 reversed-phase flash CC, yielding the thiazine isomer 9 (6.0
mg (7% yield)) as a bright yellow solid. Alternatively, to quinone 8
(220 mg, 1 mmol) in MeCN (7.5 mL) and EtOH (7.5 mL) was added
hypotaurine (120 mg, 1.1 mmol) in water (3 mL). The reaction mixture
was stirred at room temperature overnight and then solvents were
removed in Vacuo. MeOH was added and an insoluble orange product
filtered off and washed with MeOH to yield pure 5 (122 mg, 37%).
(DMSO-d6, 400 MHz) δ 8.54 (1H, d, J ) 8.1 Hz), 8.40 (1H, d, J )
8.1 Hz), 3.95 (3H, s), 3.89 (2H, t, J ) 5.8 Hz), 3.40 (2H, t, J ) 5.8
Hz); HMBC correlation from methyl protons δ 3.95 to a carbonyl
carbon at δ 163.0; FABMS m/z 323 [M + H]+; HRFABMS m/z
323.0336 (calcd for C13H11N2O6S, 323.0338).
Compound 6. Methyl ester 5 (87 mg, 0.27 mmol) was stirred in 2
N KOH (15 mL) at room temperature for 2 h. Concentrated HCl was
added dropwise until the reaction mixture turned acidic, after which
the solvents were removed under reduced pressure. The residue was
taken up in water and subjected to C8 reversed-phase flash CC, yielding
6 (13 mg (16%)) as a bright yellow solid: mp 240 °C (dec); IR (Nujol
mull) νmax 3399, 1642, 1519, 1299, 1270, 1161, 1118 cm-1; UV
(MeOH) λmax (log ꢀ) 414 (3.55), 330 (3.77), 266 (4.26), 240 (4.35),
212 (4.42); (MeOH/TFA) 424 (3.55), 268 (4.23), 213 (4.73); (MeOH/
KOH) 483 (3.78), 316 (3.99), 278 (4.19), 239 (4.45), 210 (5.04) nm;
1H NMR (DMSO-d6, 600 MHz) δ 13.90 (1H, br s, COOH), 11.51 (1H,
d, J ) 5.6 Hz, NH), 8.57 (1H, d, J ) 8.1 Hz, H-9), 8.42 (1H, d, J )
8.1 Hz, H-8), 7.17 (1H, dd, J ) 8.9, 5.6 Hz, H-3), 6.62 (1H, d, J ) 8.9
Hz, H-2); 13C NMR (DMSO-d6, 150 MHz) δ 177.6 (C-10), 175.5
(C-5), 165.1 (C-11), 151.8 (C-7), 146.5 (C-5a), 141.5 (C-4a), 135.8
(C-9), 130.8 (C-9a), 130.4 (C-3), 128.8 (C-8), 115.2 (C-10a), 112.0
(C-2); HRESI(-)FTMS m/z 304.9872 [M - H]- (calcd for C12H5N2O6S,
304.9868).
1
Methyl ester 5: mp 254 °C (dec); IR (film) νmax 1721 cm-1; H
NMR (DMSO-d6, 300 MHz) δ 9.46 (1H, s, NH), 8.54 (1H, d, J ) 8.1
Hz, H-9), 8.40 (1H, d, J ) 8.1 Hz, H-8), 3.95 (3H, s, H3-12), 3.89
(2H, br m, H2-3), 3.38 (H2-2, obscured by water peak); 13C NMR
(DMSO-d6, 100 MHz) δ 175.7 (C-5), 172.7 (C-10), 163.7 (C-11), 149.4
(C-7), 147.3 (C-4a), 145.9 (C-5a), 135.4 (C-9), 131.4 (C-9a), 128.7
(C-8), 110.3 (C-10a), 52.5 (C-12), 47.7 (C-2), 39.0 (C-3); FABMS m/z
323 [M + H]+; HRFABMS m/z 323.0336 (calcd for C13H11N2O6S,
323.0338); purity 98%; tR ) 15.46 min.
1
Methyl ester 9: IR (film) νmax 1727 cm-1; H NMR (DMSO-d6,
300 MHz) δ 9.67 (1H, s, NH), 8.51 (1H, d, J ) 8.0 Hz, H-6), 8.35
(1H, d, J ) 8.0 Hz, H-7), 4.09 (2H, m, H2-3), 3.98 (3H, s, H3-12),
3.49 (2H, t, J ) 5.8 Hz, H2-2); 13C NMR (DMSO-d6, 75 MHz) δ 177.7
(C-5), 168.2 (C-10), 163.7 (C-11), 150.8 (C-4a), 149.8 (C-8), 145.2
(C-9a), 136.9 (C-6), 129.8 (C-5a), 127.8 (C-7), 111.9 (C-10a), 53.1
Synthesis of Ascidiathiazone A. Methyl 8-Hydroxyquinoline-2-
carboxylate (7). 8-Hydroxyquinoline-2-carboxylic acid (100 mg, 0.53

940 Journal of Natural Products, 2007, Vol. 70, No. 6
Pearce et al.
(C-12), 48.4 (C-2), 40.3 (C-3); HRESI(-)FTMS m/z 321.0186 [M -
H]- (calcd for C13H9N2O6S, 321.0176).
Detection of Superoxide Production by Peritoneal Neutrophils.
Four hours after MSU administration, mice were euthanized by CO2
exposure. The peritoneal cavity was washed with PBS (3 mL, pH 7.4)
containing 25 U/mL heparin. Total cell numbers were counted and the
sample volume was adjusted to 1 × 106 neutrophils/mL. The cells were
then tested for superoxide production as described above. To determine
the number of neutrophils in each sample, a small aliquot (100 µL) of
each cell sample was fixed onto slides using a cytocentrifuge and stained
with the Diff-Quick staining kit (Dade Behring, Newark, NJ). The
percentage of neutrophils in the peritoneal wash was determined
microscopically using standard histological criteria. Superoxide produc-
tion is represented as superoxide produced per million neutrophils.
Ascidiathiazone A. Methyl ester 5 (17 mg, 0.05 mmol) was
dissolved in concentrated HCl (2 mL) and stirred 5 h at room
temperature, then at 100 °C for 1 h. The solution was dried under
reduced pressure and subjected to C18 reversed-phase flash CC, yielding
3 as a bright yellow solid (11 mg, 68%) that was identical to the natural
product in all respects. HRESI(-)FTMS m/z 307.0030 [M - H]- (calcd
for C12H7N2O6S, 307.0025).
Neutrophil Superoxide Assay.4 Human neutrophils were isolated
from anticoagulated whole human blood using Polymorphprep (density
1.113 g/mL, centrifuge 500g, 35 min). Neutrophils, resuspended in
Hank’s balanced salt solution (HBSS), were plated out in 96-well flat-
bottomed plates at 1 × 106 cells/well. Neutrophils were treated with
different concentrations of the test compound (dissolved in HBSS, 1%
v/v DMSO) for 30 min prior to addition of the detection dye WST-1
(final concentration of 0.35 mM). At this concentration, no effect of
DMSO on the ability of neutrophils to generate a respiratory burst in
response to PMA was observed. The respiratory burst was initiated by
addition of phorbol 12-myristate 13-acetate (PMA, final concentration
of 0.295 µM), and dye reduction was monitored in real time (450 nm)
for 20 min at 37 °C. Respiratory burst activity was calculated as the
rate of dye reduction over time compared to buffer-treated cells.
Cell Culture Conditions. HL60 cells were cultured in RPMI-1640
medium supplemented with 10% v/v heat-inactivated fetal bovine serum
(FBS), 2 mM GlutaMAX-1, 100 U/mL penicillin, and 100 µg/mL
streptomycin sulfate, at 37 °C in a humidified atmosphere maintained
at 5% CO2. Cell densities were maintained at less than 0.5 × 106 cells/
mL. Cell viability was confirmed by trypan blue exclusion.
Acknowledgment. This work was supported by the New Zealand
Foundation for Research Science and Technology, contract CO1X0205.
Supporting Information Available: Color photograph of Aplidium
sp., X-ray crystallographic data for 3, Figure S3 showing toxicity
markers, and 1H and 13C NMR spectroscopic data for compounds 3-9.
This material is available free of charge via the Internet at http//
pubs.acs.org.
References and Notes
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on application to the Director, CCDC, 12 Union Road, Cambridge
CB2 1EZ, UK (fax: +44-(0)1223-336033 or e-mail: deposit@
ccdc.cam.ac.uk).
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Antiproliferative Assay.22 HL60 cells (suspended in RPMI1640,
5% v/v FBS) were plated out in 96-well flat-bottomed plates at 2 ×
104 cells/well. The cells were treated with different concentrations of
the test compound (dissolved in RPMI1640, 5% v/v FBS, 1% v/v
DMSO) and incubated for 48 h (37 °C in a humidified atmosphere
maintained at 5% CO2). 3-(4,5-Dimethylthiozol-2-yl)-2,5-diphenyl-
tetrazolium bromide (MTT) was then added to each well (final
concentration of 1.10 mM), and the cells were incubated for 2 h. An
equal volume of lysing buffer (10% w/v SDS, 45% v/v DMF in H2O,
pH 4) was added to each well, and the plates were incubated overnight
to ensure complete lysis. Proliferative activity was determined by
comparing the absorbance of the samples at 570 nm to the buffer-
treated cells.
Xanthine Oxidase Assay.4 Varying dilutions of test compound
(dissolved in 0.1M Tris (pH 7,4), 0.2 mM EDTA, 1% v/v DMSO)
were plated out in 96-well flat-bottomed plates. WST-1 and xanthine
were added to a final concentration of 0.35 and 2.38 mM, respectively.
Xanthine oxidase was then added to a final concentration of 15 mU/
mL. The dye reduction was monitored in real time (450 nm) for 20
min at 37 °C. Enzyme activity was calculated as the rate of dye
reduction over time compared to buffer-treated controls.
In ViWo Murine Gout Model (MSU-Induced Inflammation)
Assay. C57BL/6 male mice were bred and housed in a conventional
animal facility at the Malaghan Institute of Medical Research. All
animals used for the experiments were between 8 and 12 weeks old.
Experimental procedures were approved by the animal ethics committee
in accordance with Victoria University of Wellington (New Zealand)
guidelines for the care of animals. Mice were injected intraperitoneally
(ip) with monosodium urate (MSU) crystals (3 mg) suspended in 500
µL of phosphate-buffered saline (PBS, pH 7.4). Test compounds were
administered by oral gavage (25.6 µmol/kg) at the same time as
administration of MSU. The positive control, colchicine (5 µmol/kg),
was administered subcutaneously (sc), immediately after ip injection
of MSU crystals. Colchicine was dissolved in PBS (pH 7.4). Control
mice received an equivalent volume of PBS (pH 7.4) by oral gavage.
NP060626O