Synthesis and anti-inflammatory structure-activity relationships of thiazine-quinoline-quinones: Inhibitors of the neutrophil respiratory burst in a model of acute gouty arthritis

Article abstract of DOI:10.1016/j.bmc.2008.09.052

Sixteen new thiazine-quinoline-quinones have been synthesised, plus one bicyclic analogue. These compounds inhibited neutrophil superoxide production in vitro with IC₅₀s as low 60 nM. Compounds with high in vitro anti-inflammatory activity were also tested in a mouse model of acute inflammation. The most active compounds inhibited both neutrophil infiltration and superoxide production at doses 2.5 μmol/kg, highlighting their potential for development as novel NSAIDs.

Full text of DOI:10.1016/j.bmc.2008.09.052

Bioorganic & Medicinal Chemistry 16 (2008) 9432–9442
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and anti-inflammatory structure–activity relationships
of thiazine–quinoline–quinones: Inhibitors of the neutrophil
respiratory burst in a model of acute gouty arthritis
Elizabeth W. Chia a, A. Norrie Pearce b, Michael V. Berridge a, Lesley Larsen c, Nigel B. Perry c,
Catherine E. Sansom c, Colette A. Godfrey c, Lyall R. Hanton d, Guo-Liang (Leon) Lu e,
Michaela Walton a, William A. Denny e, Victoria L. Webb f, Brent R. Copp b, Jacquie L. Harper a,
*
a Malaghan Institute of Medical Research, PO Box 7060, Wellington South, New Zealand
b Department of Chemistry, The University of Auckland, Private Bag 92019, Auckland, New Zealand
c New Zealand Institute for Crop & Food Research Ltd, Chemistry Department, University of Otago, PO Box 56, Dunedin, New Zealand
d Chemistry Department, University of Otago, PO Box 56, Dunedin, New Zealand
e Auckland Cancer Society Research Centre, The University of Auckland, Private Bag 92019 Auckland, New Zealand
f National Institute of Water & Atmospheric Research (NIWA) Ltd, Private Bag 14-901, Kilbirnie, Wellington, New Zealand
a r t i c l e i n f o
a b s t r a c t
Article history:
Sixteen new thiazine–quinoline–quinones have been synthesised, plus one bicyclic analogue. These com-
pounds inhibited neutrophil superoxide production in vitro with IC50s as low 60 nM. Compounds with
high in vitro anti-inflammatory activity were also tested in a mouse model of acute inflammation. The
most active compounds inhibited both neutrophil infiltration and superoxide production at doses
Received 5 August 2008
Revised 16 September 2008
Accepted 17 September 2008
Available online 20 September 2008
2.5
l
mol/kg, highlighting their potential for development as novel NSAIDs.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Thiazine–quinoline–quinones
Inflammation
Superoxide
Arthritis
Neutrophils
1. Introduction
pounds also translated into suppression of superoxide production
by infiltrating neutrophils in vivo in a murine model of gouty
inflammation highlighting the potential for further development
of synthetic analogues of the natural products as novel anti-inflam-
matory agents. To this end, we now report a more comprehensive
study into the structure–activity relationship of these thiazine–
quinoline–quinones to determine the key structural features re-
quired for anti-inflammatory activity.
Neutrophil infiltration and activation are linked with inflamma-
tion in a variety of diseases including gouty and rheumatoid arthri-
tis, asthma, cardiovascular disease and intestinal disease.1–5 One of
the hallmark features of neutrophil activation is the ‘respiratory
burst’ resulting in the production of destructive reactive oxygen
species such as superoxide that, if left uncontrolled, can lead to tis-
sue damage.6–8 Therefore the ability to block overproduction or
chronic production of superoxide has broad clinical potential for
the improved management of inflammatory conditions.
In previous work involving screening for inhibitors of human
neutrophil superoxide production, we isolated and characterized
two thiazine-containing 2-quinolinequinone carboxylic acid deriv-
atives ascidiathiazones A (1) and B (23) from a New Zealand ascid-
ian Aplidium sp.9 These natural products suppressed superoxide
production with anti-inflammatory IC50 (AI50) values of 1.55 for 1
O
O
O O
S
H
N
9
9
2
3
2
3
1'
O
1'
OH
OR
S
O O
N
N
H
N
7
7
5
O
5
O
O
1 R = H
8 R = Me
23
2. Chemistry
and 0.44 lM for 23. The anti-inflammatory effect of these com-
In general, the thiazine–quinoline–quinones were prepared by
the addition reaction of hypotaurine to quinoline–quinones
* Corresponding author. Tel.: +64 4 4996914.
E-mail address: jharper@malaghan.org.nz (J.L. Harper).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.09.052

E. W. Chia et al. / Bioorg. Med. Chem. 16 (2008) 9432–9442
9433
(Scheme 1). The required quinoline–quinones were prepared either
from commercially availableor synthesised 8-hydroxyquinolines by
oxidation using potassium nitrosodisulfonate (Fremy’s salt). The
hypotaurine addition gave varying proportions of the two regioi-
somers of the dioxothiazine ring. Separation of the two regioisomers
formed was accomplished by column chromatography. Proof of the
regiochemistry of the various purified thiazine-quinoline-quinones
was based on X-ray crystal structures, of the natural product ascidia-
thiazone A (1) previously reported9 and of the synthetic methyl
derivative 3 reported here (Fig. 1). The 13C NMR chemical shift of
the quinone carbonyl C-10 in 3, assigned by an HMBC correlation
with H-9, was 174.3 ppm, whereas the C-10 in regioisomer 22 reso-
nated at 178.8 ppm (Table 1). This change in the 13C NMR signals
of C-10 in the different regioisomers was comparable to that previ-
ously reported for methyl ester 8 and its regiosomer9 and was used
as the basis for assignment of the regiochemistry of the other thia-
zines (Table 1).
Figure 1. The crystal structure of compound 3 showing crystallographic number-
ing. Selected bond lengths (Å) and angles (°):C1–C2 1.519(3), C1–S1 1.7725(19),
C2–N1 1.457(2), C3–N1 1.326(2), C3–C4 1.384(2), C3–C6 1.520(2), C4–C5 1.450(2),
C4–S1 1.7646(17), C5–O3 1.230(2), C5–C7 1.500(2), C6–O4 1.208(2), C6–C8
1.479(2), C7–C8 1.385(2); C2 C1 S1 110.34(13), N1 C2 C1 109.81(15), N1 C3 C4
126.60(17), N1 C3 C6 113.27(15), C4 C3 C6 120.13(16), C3 C4 C5 122.35(16), C3 C4
S1 119.21(14), C5 C4 S1 118.44(13).
Compounds that could not be formed by hypotaurine addition
were prepared by modification of other thiazine–quinoline–
quinones. Thus aldehyde 5 was prepared from a selenium dioxide
oxidation of 3 and the unsaturated thiazine 25 was formed by a
base catalysed oxidation, using aqueous potassium hydroxide, of
3 (previously reported during the preparation of 24 from 89).
Amide 11 was prepared from acid 1 by a modified coupling
reaction using N,N-dimethylethylenediamine and N,N-diisopropyl-
ethylamine (DIPEA) with benzotriazol-1-yl-oxytripyrrolidinophos-
phonium hexafluorophosphate (PyBOP). The ester 12 was formed
from the reaction of the alcohol 7 with DIPEA, dimethylaminopyr-
idine (DMAP) and dimethylaminopropionyl chloride. The surpris-
ingly rapid formation of stable hemiacetals when the aldehyde 5
was dissolved in alcohols was used to form the hemiacetal 13.
The opened thiazine ring analogue 15 was prepared by a sequential
addition of sodium methanesulfinate followed by methylamine to
the quinoline–quinone. Addition of methylamine first, or at the
same time as the sodium methanesulfinate, resulted in a single
addition of methylamine.
3. Biological assays
3.1. In vitro anti-inflammatory activity
The natural product ascidiathiazone A, 1, was reported previ-
ously to act as an inhibitor of neutrophil superoxide production
in vitro and in vivo.9 To investigate the structural requirements
for anti-inflammatory (AI) activity, analogues of the natural prod-
ucts were tested for their ability to suppress superoxide production
by human neutrophils. Compounds were also tested for anti-prolif-
erative (AP) activity to identify compounds exhibiting selectivity
for high AI activity and low AP activity.
Initial structural modifications focussed on the carboxylic acid
group at the 7-position, 2-7 (Table 2). The absence of the carbox-
ylic acid functionality 2 resulted in a significant decrease in AI
activity and an undesirable increase in AP activity and therefore
O
O
O
O O
S
H
N
2
i
ii
+
S
O O
N
R
N
R
N
R
N
H
N
R
O
OH
O
O
R = H
R = H
2 R = H
4 R = CN
R = CN
R = CN
R = CON(CH3)2
R = CO2C8H17
R = CH3
R = CON(CH3)2
R = CO2C8H17
R = CH3
10 R = CON(CH3)2
9 R = CO2C8H17
3 R = CH3
21 R = CON(CH3)2
22 R = CH3
iii
2
iv
25 R = CH3,
5 R = CHO
v
13 R = CHOH(OCH3)
R = CO2CH3
R = CH2OH
16 R = CO2CH3
8 R = CO2CH3
1 R = CO2H
vi
iii
vii
11 R = CONHCH2CH2N(CH3)2
2
24 R = CO2H,
R = CH2OH
7 R = CH2OH
20 R = CH2OH
viii
12 R = CH2OCOCH2CH2N(CH3)2
R = (E)-CHCHCOPh
R = (E)-CHCHCOPh
6 R = (E)-CHCHCOPh
19 R = (E)-CHCHCOPh
Scheme 1. Reagents and conditions: (i) Fremy’s salt, aq acetone; (ii) hypotaurine, EtOH, CH3CN, H2O; (iii) 1 M KOH; (iv) SeO2, dioxane; (v) MeOH; (vi) 3 M HCl; (vii)
dimethylethylenediamine, DIPEA, PyBOP, DMF (viii) 3-dimethylaninopropionyl chloride, DMAP, DIPEA, DMF.

9434
E. W. Chia et al. / Bioorg. Med. Chem. 16 (2008) 9432–9442
Table 1
13C NMR data for thiazine-quinoline-quinonesa
C
2
3
4b
5
6
7
9
10
48.3
11
49.3
12c
48.4
13
48.9
15d
45.6
19
40.0
22
39.7
25
2
3
4a
5
5a
7
48.3
39.9
48.3
39.6
50.3
48.3
39.5
48.3
39.5
48.3
39.1
48.1
39.0
111.8
131.5
140.5
175.8
145.8
163.5
128.5
134.2
127.3
178.1
114.8
24.4
41.5
148.7
177.8
148.5
137.8
134.8
137.9
133.7
174.2
114.0
117.9
39.6
147.6
176.6
145.4
157.3
127.6
135.3
129.8
173.6
110.6
166.9
40.6
148.8
178.2
146.2
153.4
128.2
137.5
132.9
175.1
112.3
165.8
39.8
148.6
177.5
145.8
160.5
128.5
136.8
129.9
176.2
109.4
66.9
39.6
148.1
177.5
146.0
164.1
126.0
135.6
130.3
174.6
111.1
98.1
34.4
151.5
181.1
145.9
164.3
129.1
134.7
127.5
177.1
109.1
25.0
48.4
146.8
172.8
148.0
158.1
129.1
135.6
127.0
178.3
112.5
141.4
127.5
189.5
137.1
128.7
129.1
133.8
129.1
128.7
48.4
112.1
173.3
146.5
165.4
126.7
134.6
125.3
178.8
147.3
24.9
147.5
177.0
146.4
153.3
134.0
129.1
130.0
174.1
110.6
147.2
177.1
145.7
162.7
128.8
134.2
127.9
174.3
110.3
24.4
147.9
176.3
146.9
154.0
125.9
136.0
132.4
173.3
111.0
192.6
146.6
176.6
147.6
156.2
128.6
135.2
130.0
173.7
110.8
141.4
128.0
189.5
137.1
128.4
129.1
133.7
129.1
128.4
147.3
177.0
145.5
166.6
125.3
134.7
128.6
174.3
110.4
64.1
147.8
176.1
146.4
150.1
129.1
135.8
131.7
173.2
110.7
163.6
8
9
9a
10
10a
10
20
30
65.8
31.2
28.6
28.6
28.0
25.3
22.0
13.9
38.2
34.9
35.8
58.2
43.8
43.8
172.0
29.5
53.4
43.5
43.5
54.6
40
50
60
70
80
90
100
a
b
c
In d6-DMSO unless otherwise noted, chemical shifts in ppm, all assignments based on 2D NMR experiments.
In d6-acetone.
In D2O.
d
In d4-CDCl3.
loss of selectivity. A methyl group at the 7-position (3) resulted
in a further decrease in AI activity again with increased AP activ-
ity compared to 1. The presence of electron-withdrawing groups
at C-7 (4, 5, 6) resulted in AI activity comparable to that of 1,
but selectivity was lost. A methyl alcohol (7) at the 7-position
also had diminished AI activity and selectivity. Methylation of
the carboxylic acid group to give the methyl ester 8 (previously
reported9) had little effect on AI activity whereas increasing the
alkyl chain length to give the octyl ester 9 resulted in loss of AI
activity and a significant increase in AP activity into the submi-
cromolar range.
To determine whether increased aqueous solubility would en-
hance AI activity, a series of polar analogues 10-13 were prepared
with dimethylaminoethylamide 11 exhibiting notable AI activity
and greater selectivity compared to 1. Reduction of the methyl es-
ter to give 13 resulted in lowered AI potency. Together these re-
sults indicate that the functional group in the 7-position plays a
key role in the efficacy and specificity of compounds with 11
exhibiting the lowest AI:AP ratio and therefore highest selectivity
for AI activity.
Nextwelooked atthecontributionof thedifferentringsystemsof
1 to AI activity (Table 2, compounds 14-18). The dioxothiazinenaph-
thoquinone analogue 14 was essentially devoid of AI activity com-
pared to 2 indicating that the presence of a ring nitrogen favours
AI activity. Opening the thiazine ring (15) resulted in a loss of both
AI and AP activity compared to 3. Interestingly, without the thiazine
ring, quinoline quinones 16 and 17 had similar AI activity compared
to8 and 5, respectivelybutexhibited modestincreases in AP activity.
It appears that the presence of the thiazine ring lowers AP activity
thereby improving the AI selectivity of the thiazine–quinoline–qui-
nones. The structural fragment pyridine-2-carboxylic acid 18 was
completely inactive identifying the quinoline-quinone as the mini-
mum structure required for bioactivity.
To better understand the influence of the dioxothiazine ring on
the pattern of bioactivity, compounds 19-22 were used to investi-
gate the effect of ring regiochemistry on AI activity and selectivity.
All four compounds exhibited less potent AI activity with a com-
paratively small loss of AP activity in our assays compared to their
regioisomers, 6, 7, 10 and 3, respectively (Table 2) indicating that
[2,3-g] regiochemistry favours higher AI activity and low AP activ-
ity. Taken together, these data illustrate key contributions by the
pyridine and dioxothiazine rings to the AI activity and selectivity
of the ascidiathiazone structural class.
In addition to ascidiathiazone A 1, our previous natural product
isolation study also yielded the oxidized regioisomer ascidiatiaz-
one B 23.9 This 4H-1,4-benzothiazine-1,1-dioxide exhibited sub-
micromolar activity in the AI assay but with less selectivity than
that observed for 1 (Table 2). Regioisomer 24 exhibited enhanced
AI potency and selectivity compared to 23. Compound 24 also
exhibited enhanced AI activity and selectivity compared to 1. These
findings indicate that both the saturation state and the regiochem-
istry of the thiazine ring strongly influence AI activity and selectiv-
ity. Replacement of the carboxylic acid group 24 with a methyl 25
significantly lowered AI activity and increased anti-proliferative
activity.
3.2. In vivo activity
Acute gouty arthritis is characterised by the recruitment and
activation of neutrophils, and the production of superoxide, in re-
sponse to the known inflammatory stimulus, MSU crystals. A per-
itoneal model of gouty arthritis was used to determine whether the
in vitro AI activity of the compounds translated into an inhibitory
effect in vivo. Compounds 1, 8, 11, 23 and 24 were selected for
in vivo testing on the basis of their high AI activity and low AP
activity in vitro.
While modest suppression of MSU crystal-induced neutrophil
superoxide production was observed for natural products 1 and
23 at the higher dose of 25
and 24 exhibited significantly greater in vivo activity at
2.5 mol/kg (Table 3). Despite potent in vitro AI activity, 11 failed
lmol/kg, the synthetic analogues 8
l
to exhibit any inhibitory effect in vivo. The lack of in vivo activity
by 11 could result from a variety of different factors: differences in
the uptake and bioavailability of the compounds, susceptibility to
first pass clearance associated with delivery via the oral route
and the half-life of the compounds in vivo. Interestingly, 8 and
24 also inhibited neutrophil infiltration indicating that these com-
pounds have the potential to target neutrophilic inflammation by

E. W. Chia et al. / Bioorg. Med. Chem. 16 (2008) 9432–9442
9435
Table 2
In vitro anti-inflammatory activity of ascidiathiazone analoguesa
Compound
AI50
(l
M)b
AP50 (l
M)c
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
S
1
2
3
4
5
6
7
8
1.55 0.8
73 16
OH
N
H
N
N
N
N
N
N
N
N
O
O
O
S
29 0.5
6.79 0.6
N
H
O
O
S
115 33
14
8
N
H
O
O
S
1.86 0.88
5.34 0.70
8.01 1.38
2.62 2.00
3.52 3.0
3.08 1.22
1.2 0.2
N
H
CN
O
O
S
H
N
H
O
O
O
S
N
H
O
O
O
S
35
8
N
H
CH2OH
O
O
S
2.26 0.61
12
3
OMe
N
H
O
O
(continued on next page)

9436
E. W. Chia et al. / Bioorg. Med. Chem. 16 (2008) 9432–9442
Table 2 (continued)
Compound
AI50
(l
M)b
AP50 (l
M)c
O
O
O
O
O
O
O
O
O
O
O
O
O
S
9
15
6
0.42 0.03
O
N
H
N
N
N
N
N
O
O
O
S
10
11
12
13
14
15
16
26
3
35
5
N
N
H
O
O
O
S
H
1.57 0.60
91 20
4.17 1.21
n.d
N
N
H
N
O
O
O
S
62
5
O
N
N
H
O
O
O
S
13 0.1
OH
N
H
O
OMe
O
S
>167
2.55 1.00
N
H
O
O
MeO2S
MeHN
O
>333
97 13
N
O
1.74 1.01
4.00 1.56
OMe
N
O
O
(continued on next page)

E. W. Chia et al. / Bioorg. Med. Chem. 16 (2008) 9432–9442
9437
Table 2 (continued)
Compound
AI50
(l
M)b
AP50 (l
M)c
O
17
7.29 2.53
1.70 0.59
H
N
O
N
O
OH
18
19
>406
n.d.
O
O
H
N
>127
5.73 1.42
S
N
O
O
O
O
H
N
O
20
64
8
3.25 0.86
S
N
CH2OH
O
O
O
O
H
N
21
22
23
>149
6.13 1.44
6.87 3.35
16 3.26
N
S
N
N
N
O
O
O
O
O
H
N
>180
S
O
O
O
H
N
O
0.44 0.18
OH
S
O
O
O
O
O
O
O
S
24
0.06 0.02
27
6
OH
N
H
N
O
O
O
O
O
S
25
2.56 0.49
0.59 0.20
N
H
N
O
a
Values are means of two dose–response experiments SEM (n = 3).
IC50 for inhibition of PMA-induced superoxide production by neutrophils.
IC50 for inhibition of proliferation of HL60 cells.
b
c

9438
E. W. Chia et al. / Bioorg. Med. Chem. 16 (2008) 9432–9442
Table 3
In vivo inhibition of neutrophil superoxide production and neutrophil infiltration by compounds 1, 8, 11, 23 and 24
#
Structure
AI (lM)
% Inhibitiona
Superoxide Production
Neutrophil Infiltration
O
O
O
O
O
O
O
S
1
1.55
2.26
1.57
0.44
0.06
23b (p < 0.05)
25b (p < 0.05)
OH
N
H
N
N
N
N
N
O
O
O
O
O
O
O
S
8
93 (p < 0.05)
49 (p < 0.05)
OMe
N
H
O
O
S
H
11
23
24
2 (n.s.)
5 (n.s.)
N
N
H
H
N
N
O
O
59b (p < 0.05)
0b (n.s.)
OH
S
O
O
O
O
O
O
S
60 (p < 0.05)
65 (p < 0.05)
OH
N
H
O
n.s., not significant
a
All compounds were tested in vivo at 2.5
l
mol/kg.
b
Compounds with no effect at 2.5
lmol/kg were retested at 25 lmol/kg and results reported.
reducing both neutrophil recruitment and neutrophil superoxide
production.
production and infiltration in an in vivo model of gouty arthritis
thus identifying this structural class of inhibitors as promising can-
didates for development as anti-inflammatory drugs for the treat-
ment neutrophil-driven disease.
None of the compounds tested induced acute liver or renal tox-
icity measured as the levels of the liver enzymes and creatinine in
the serum compared with untreated mice and mice treated with
the anti-inflammatory agent colchicine. In all cases serum levels
did not significantly exceed those observed in control mice (Sup-
plementary Data Figures S1–4) although colchicine-treated mice
showed significantly elevated enzyme and creatinine levels, con-
sistent with colchicine’s narrow therapeutic window and acute
toxicity.
5. Experimental
5.1. Chemistry: general methods
All chemicals were purchased from Sigma–Aldrich or Acros
Organics. Octadecyl functionalised silica gel (C18), used for re-
versed-phase (RP) chromatography; silica gel 60, 200–400 mesh,
40–63 lm, for column chromatography and TLC plates silica gel
4. Conclusions
60 F254 were obtained from Merck. All solvents were distilled be-
fore use and were removed by rotary evaporation at temperatures
up to 35 °C. High resolution mass spectrometry was recorded using
a VG70-250S double focussing magnetic sector mass spectrometer
or a Bruker MicroTOFQ mass spectrometer. UV spectra were re-
corded in methanol using a Jasco V-550. NMR spectra were re-
corded at 500 or 300 MHz for 1H and 125 MHz or 75 MHz for 13C
In conclusion, we have identified the 7-position substituent, the
oxidation state of the thiazine ring and the regiochemistry of the
thiazine ring as playing a key role in the anti-inflammatory activity
and selectivity of the thiazine–quinoline–quinones. Selected thia-
zine–quinoline–quinones inhibited both neutrophil superoxide

E. W. Chia et al. / Bioorg. Med. Chem. 16 (2008) 9432–9442
9439
on Varian INOVA-500 or VXR-300 spectrometers. HPLC analyses
were carried out using an Agilent HP1100 on a C18 column (Phe-
C, 78.53; H, 4.76; N, 5.09; 1H NMR (500 MHz, CDCl3) 8.23 (1H, br
s), 8.20 (1H, d, J = 8.5 Hz), 8.08 (2H, dd, J = 7.5, 1.5 Hz), 8.07 (1H, d,
J = 15.5 Hz), 7.91 (1H, d, J = 15.5 Hz), 7.69 (1H, d, J = 8.5 Hz), 7.61
(1H, dt, J = 7 Hz), 7.54 (2H, t, J = 7.5 Hz), 7.48 (1H, t, J = 7.5 Hz), 7.34
(1H, dd, J = 8.5, 1.5 Hz), 7.21 (1H, dd, J = 7.5, 1.5 Hz). 13C NMR
(125 MHz, CDCl3) 190.5, 152.4, 151.1, 142.8, 138.2, 137.8, 137.0,
133.2, 128.8 (2C), 128.7 (2C), 128.6, 126.9, 122.0, 117.8, 110.6. UV
nomenex Luna ODS(3) 5
lm 100 A 150 ꢀ 3 mm) at 20 °C with a
2 ꢀ 4 mm C18 guard column. Peaks were detected at 210 and
254 nm and UV spectra recorded from 190 to 600 nm. Mobile
phase A (water/0.1% formic acid) and B (acetonitrile/0.1% formic
acid): t0 = 10% B, t12.5 = 100% B, t15 = 100% B, t16 = 10% B, t20 = 10%
B. The flow rate was 0.5 mL/min, with an injection volume of
(MeOH) kmax (loge) 337 (4.14), 294 (4.50), 206 (4.40) nm; ESIMS
5
l
L of 1 mg/mL solutions in methanol or DMSO. Preparative HPLC
m/z 298.0842 (calcd for C18H13NO2Na, 298.0844).
were carried out using a Gilson Preparative HPLC System with a
C18 column (Synergi-Max RP, 4
at 275 nm and 300 nm.
lm, 21.20 ꢀ 250 mm), monitoring
5.1.2. General procedure for oxidation of 8-hydroxyquinolines
to quinoline-quinones with Fremy’s salt (potassium
Potassium nitrosodisulfonate (Fremy’s salt) was freshly pre-
pared as described by Teuber.10 Compounds 1, 8, 16, 23 and 24
were prepared as described by Pearce et al.9; 8-hydroxyquino-
line-2-carboxaldehyde as described by Hassani et al.11; and 8-
hydroxyquinoline-2-carbonitrile as described by Schrader et al.12
and 2-hydroxymethyl-8-hydroxyquinoline as described by Yoneda
et al.13 14 was prepared as described by Schmitz and Bloor14 and
our data on 14 (see Registry No. 873536-72-2)15 match those re-
ported by Schmitz and Bloor, but the structure was drawn incor-
rectly in the 1988 paper and consequently recorded incorrectly
in the Chemical Abstracts Registry file (see RN 113831-02-0).
nitrosodisulfonate)
Typically, a solution of Fremy’s salt (1 g, 4 mmol) and potassium
dihydrogen phosphate (400 mg, 3 mmol) in water (75 mL) was
stirred at RT for 10 min then the quinolinol (6 mmol) in acetone
(70 mL) was added. After stirring for 30 min, a second solution of
Fremy’s salt (1 g) and potassium dihydrogenphosphate (400 mg)
in water (30 mL) was added, then a third equal amount after an-
other 30 min. After stirring for a further 2 h the mixture was ex-
tracted into dichloromethane, dried and evaporated in vacuo to
give the product as an orange gum. Purification by column chroma-
tography over silica gel eluting with ethyl acetate (0–40%) in
dichloromethane gave the products as orange solids.
5.1.1. General procedure for the preparation of 8-
hydroxyquinolines
Due to the instability of the quinones, these were mostly used
directly in the subsequent hypotaurine addition reaction.
To
a suspension of 8-hydroxyquinoline-2-carboxylic acid
(190 mg, 1.0 mmol) in oxalyl chloride (3 mL) under nitrogen was
added 2 drops of DMF. The solution was left at RT for 3 h then
evaporated in vacuo to give the acid chloride. To a stirred solution
of the acid chloride in dry CH2Cl2 (4 mL/mmol acid) under nitrogen
was added excess nucleophile with triethylamine. The solution
was refluxed for 30 min and then stirred at room temperature for
24 h. The reaction mixture was poured into CH2Cl2, washed with
sat. NaHCO3 then water and dried in vacuo. Column chromatogra-
phy over silica gel gave the 8-hydroxyquinoline products.
5.1.2.1. 5,8-Dihydro-5,8-dioxoquinoline (RN 858471-89-3). 8-
Hydroxyquinoline (1 g, 6.9 mmol) to give quinone (0.48 g, 44%).17 1H
NMR (300 MHz, CDCl3) 9.06 (1H, dd, J = 4.2, 1.5 Hz), 8.42 (1H, dd,
J = 7.8, 1.5 Hz), 7.71 (1H, dd, J = 7.8, 4.8 Hz), 7.16 (1H, d, J = 10.5 Hz),
7.06 (1H, d, J = 10.5 Hz); EIMS m/z 160.0403 (calcd for C9H6NO2,
160.0399); EIMS m/z 182.0216 (calcd for C9H5NO2Na, 182.0218).
5.1.2.2. 2-Methyl-5,8-dihydro-5,8-dioxoquinoline (RN 90800-
33-2). 2-Methyl-8-hydroxyquinoline (1 g, 6.9 mmol) to give qui-
none (0.45 g, 41%).17 1H NMR (300 MHz, CDCl3) 8.22 (1H, d,
J = 8.1 Hz), 7.50 (1H, d, J = 8.1 Hz), 7.05 (1H, d, J = 10.5 Hz), 6.96
(1H, d, J = 10.5 Hz), 2.71 (3H, s); EIMS m/z 173.04739 (calcd for
C10H7NO2, 173.04768).
5.1.1.1. 8-Hydroxyquinoline-N,N-dimethyl-2-carboxamide (RN
138878-37-2). From acid (250 mg, 1.32 mmol) and dimethyl
amine (33% in ethanol, 10 mL, 222 mmol). Elution with CH2Cl2/
EtOAc gave the product as a yellow gum (160 mg, 56%).16 1H NMR
(CDCl3) 8.24 (1H, d, J = 8.4 Hz), 7.94 (1H, s), 7.71 (1H, d, J = 8.4 Hz),
7.50 (1H, td, J = 8, 1 Hz), 7.35 (1H, d, J = 8.1 Hz), 7.21 (1H, br t,
J = 7.5 Hz), 3.20 (3H, s), 3.12 (3H, s).
5.1.2.3. 2-Hydroxymethyl-5,8-dihydro-5,8-dioxoquinoline (RN
251652-78-5). 2-Hydroxymethyl-8-hydroxyquinoline (300 mg,
1.7 mmol) to give quinone (265 mg, 82%).17 1H NMR (300 MHz,
CD3CN) 8.37 (1H, d, J = 8.4 Hz), 7.86 (1H, dd, J = 8.1, 0.6 Hz), 7.07
(1H, dd, J = 10.5, 0.6 Hz), 7.01 (1H, dd, J = 10.5, 0.6 Hz), 4.80 (2H, s),
3.78 (1H, br s); ESIMS m/z 212.0323 (calcd for C10H7NO3Na,
212.0318).
5.1.1.2. Octyl 8-hydroxyquinoline-2-carboxylate(RN880137-68-
8). From acid (50 mg, 0.26 mmol), 1-octanol (200
lL, 1.30 mmol)
and triethylamine (109 L, 0.78 mmol). Purified by reversed-phase
l
phenyl flash column chromatography eluting with MeOH, gave the
product as a bright orange solid (71 mg, 91%). 1H NMR (CDCl3) 8.24
(1H, d, J = 8.5 Hz), 8.13 (1H, d, J = 8.5 Hz), 7.53 (1H, dd, J = 8.0,
7.7 Hz), 7.34 (1H, d, J = 8.0 Hz), 7.21 (1H, d, J = 7.7 Hz), 4.44 (2H, t,
J = 6.8 Hz), 1.83 (2H, m), 1.49-1.28 (10H, m), 0.88 (3H, t, J = 6.9 Hz).
13C NMR (75 MHz, CDCl3) 165.0, 153.5, 145.4, 137.8, 137.1, 130.1,
129.7, 121.4, 117.4, 111.1, 66.2, 31.7, 29.1, 29.0, 28.6, 25.9, 22.5,
13.9. EIMS m/z 301.1677 (calcd for C18H23NO3, 301.1678).
5.1.2.4. 5,8-Dihydro-5,8-dioxoquinoline-2-carboxaldehyde (RN
326801-24-5). 8-Hydroxyquinoline-2-carboxaldehyde (200 mg,
1.2 mmol) to give the quinone (160 mg, 74%).17 1H NMR (300 MHz,
CDCl3) 10.29 (1H, s), 8.61 (1H, d, J = 7.8 Hz), 8.31 (1H, d, J = 7.8, Hz),
7.26 (1H, d, J = 10.5 Hz), 7.15 (1H, d, J = 10.5 Hz), EIMS m/z
187.02693 (calcd for C10H5NO3, 187.02694).
5.1.2.5. 5,8-Dihydro-5,8-dioxoquinoline-2-carbonitrile (RN 326801-
23-4). 8-Hydroxyquinoline-2-carbonitrile (1 g, 6.9 mmol) to give the
quinone (0.25 g, 24%).17 1H NMR (300 MHz, CDCl3) 8.60 (1H, d,
J = 8.1 Hz), 8.07 (1H, d, J = 7.8, Hz), 7.27 (1H, d, J = 9.6 Hz), 7.17 (1H, d,
J = 10.5 Hz), EIMS m/z 184.0270 (calcd for C10H4N2O2, 184.0273).
5.1.1.3. (E)-3-(8-Hydroxyquinolin-2-yl)-1-phenylprop-2-en-1-
one (RN 880137-67-7). To a stirred solution of 8-hydroxyquino-
line-2-carboxaldehyde (470 mg, 2.71 mmol) and acetophenone
(326 mg, 2.71 mmol) in ethanol (95%, 20 mL) was added aq KOH
(1M, 5 mL). The mixture was left at RT for 2 days, then aq HCl
(3 M) added until neutral and the product extracted into CH2Cl2.
Purification by column chromatography over silica gel eluting with
ethyl acetate 0–20% in CH2Cl2 gave the product as a yellow oil
(0.33 g, 44%). Found: C, 78.52; H, 4.79; N, 5.09. C18H13NO2 requires
5.1.2.6. 5,8-Dihydro-5,8-dioxoquinoline-N,N-dimethyl-2-carbox-
amide (RN880137-63-3). 8-Hydroxyquinoline-N,N-dimethyl-2-
carboxamide (160 mg, 0.74 mmol) to give the quinone (140 mg,
1
82%). H NMR (300 MHz, CDCl3) 8.51 (1H, d, J = 8.1 Hz), 8.03 (1H, d,

9440
E. W. Chia et al. / Bioorg. Med. Chem. 16 (2008) 9432–9442
J = 8.1, Hz), 7.18 (1H, d, J = 10.5 Hz), 7.08 (1H, d, J = 10.5 Hz), 3.15 (3H,
s), 3.12 (3H, s). ESIMS m/z 253.0577 (calcd for C12H10N2O3Na,
253.0589).
line (200 mg, 1.2 mmol) with hypotaurine (130 mg) isolated by
method 1 gave 3 as an orange solid (50 mg, 16%). 1H NMR (d6-DMSO)
9.38 (1H, s, NH), 8.36 (1H, d, J= 8 Hz, H-9), 7.84 (1H, d, J = 8 Hz, H-8),
3.97 (2H, m, H2-3), 3.51 (2H, m, H2-2) and 2.75 (3H, s, H3-1’). 13C
5.1.2.7.
(E)-3-(5,8-Dihydro-5,8-dioxoquinolin-2-yl)-1-phenyl-
NMR in Table 1. UV (MeOH) kmax (loge) 423 (3.26), 291 (3.95), 266
prop-2-en-1-one (RN880137-65-5). (E)-3-(8-Hydroxyquinolin-
2-yl)-1-phenylprop-2-en-1-one (60 mg, 0.22 mmol) to give the qui-
none (50 mg, 58%). 1H NMR (CDCl3) 8.45 (1H, d, J = 8.1 Hz), 8.27 (1H,
d, J = 15.6 Hz), 8.08 (2H, dt, J = 8.1, 1.2 Hz), 7.85 (1H, d, J = 15.3 Hz),
7.85 (1H, d, J = 7.8 Hz), 7.61 (1H, m), 7.52 (2H, m), 7.17 (1H, d,
J = 10.2 Hz), 7.08 (1H, d, J = 10.5 Hz). ESIMS m/z 312.0657 (calcd for
C18H11NO3Na, 312.0631).
(4.13), 234 (4.23) nm. HPLC 4.9 min. EIMS M+ m/z 278.0352 (calcd
for C12H10N2O4S, 278.0361).
Compound 22 as a yellow solid (40 mg, 13%). 1H NMR (d6-
DMSO) 9.29 (1H, s, NH), 8.37 (1H, d, J = 8 Hz, H-9), 7.76 (1H, d,
J = 8 Hz, H-8), 3.98 (2H, m, H2-3), 3.52 (2H, m, H2-2) and 2.79
(3H, s, H3-1’). 13C NMR in Table 1. UV (MeOH) kmax (log
e)
421(3.32), 293(4.04), 270(4.09), 235(4.28) nm. HPLC 5.3 min. EIMS
MH+ m/z 279.0435 (calcd for C12H11N2O4S, 279.0440).
Crystallographic data (excluding structure factors) for the struc-
ture of compound 3 in this paper have been deposited with the
Cambridge Crystallographic Data Centre as Supplementary Publi-
cation No. CCDC 695838. Copies of the data can be obtained, free
of charge, on application to CCDC, 12 Union Road, Cambridge
CB2 1EZ, UK, (fax: +44-(0)1223-336033 or e-mail: deposit@ccdc.
cam.ac.uk).
5.1.2.8. Octyl-5,8-dihydro-5,8-dioxoquinoline-2-carboxylate (RN
880137-66-6). To a stirred solution of [bis(trifluoroacetoxy)iodo]-
benzene (PIFA) (258 mg, 0.6 mmol) in MeCN/water 2:1 (3 mL) at 0
°C was added octyl 8-hydroxyquinoline-2-carboxylate (71 mg,
0.24 mmol) in CH2Cl2 (1 mL). The solution was stirred for 20 min,
poured into CH2Cl2 (20 mL), washed with water and dried in vacuo
to give octyl 5,8-dioxo-5,8-dihydroquinoline-2-carboxylate (69 mg,
90%) as a brown solid. 1H NMR (300 MHz, CDCl3) 8.58 (1H, d,
J = 8.1 Hz), 8.45 (1H, d, J = 8.1 Hz), 7.24 (1H, d, J = 10.5 Hz), 7.14 (1H,
d, J = 10.5 Hz), 4.46 (2H, t, J = 7.0 Hz), 1.83 (2H, m), 1.45-1.28 (10H,
m), 0.88 (3H, t, J = 6.7 Hz). 13C NMR (CDCl3) 183.7, 181.9, 163.8,
152.6, 147.0, 139.5, 138.0, 136.1, 130.1, 128.4, 66.9, 31.6, 29.1, 29.0,
28.4, 25.7, 22.5, 14.0. FABMS m/z 316.1555 (calcd for C18H22NO4,
316.1549).
Crystal data. C12H10N2O4S, M 278.28, triclinic, space group P-1 a
6.4790(2), b 7.7900(2), c 11.4908(3)Å,
c
K
a
77.9930(10) b 85.9670(10)
84.4930(10)°, V 563.89(3) Å3, Z 2, F(000) 288, k 0.71073 Å,
(Mo
a
) 0.300 cmꢁ1, T 90(2) K, 1841 unique reflections. Refinement of
l
173 parameters converged at final R1 = 0.0355, wR2(all data) =
0.0949.
5.1.3.4. 2H-Pyrido[2,3-g][1,4]benzothiazine-5,10-dione, 3,4-
dihydro-1,1-dioxide (2) (RN 880137-51-9). 5,8-Dihydro-5,8-
dioxoquinoline (30 mg, 0.19 mmol) with hypotaurine (21 mg) iso-
lated by method 2 gave 2 as an orange solid (20 mg, 40%). 1H NMR
(d6-DMSO) 9.43 (1H, s, NH), 9.06 (1H, dd, J = 1, 3 Hz, H-9), 8.49 (1H,
dd, J = 1, 6 Hz, H-7), 7.97 (1H, dd, J = 3, 6 Hz, H-8), 3.99 (2H, m, H2-
3) and 3.50 (2H, m, H2-2). 13C NMR in Table 1. UV (MeOH) kmax
5.1.3. General procedure for addition of hypotaurine
Typically, a solution of hypotaurine (220 mg, 2 mmol) in water
(4 mL) was added to the quinone (2.9 mmol) in acetonitrile
(10 mL) and ethanol (10 mL). The reaction mixture was stirred at
room temperature for 18 h then the solvents were removed in va-
cuo to give an orange solid. The crude product was purified by one
of two methods: Isolation method 1: Column chromatography on
silica gel eluting with methanol/chloroform (0:1 to 1:1) gave the
product as an orange solid. Isolation method2: Methanol was added,
the mixture sonicated for 1 min then the orange solid isolated by
filtration, then washed with further methanol.
(loge) 421 (3.29), 265 (4.07), 232 (4.17) nm; HPLC 5.65 min. EIMS
m/z 264.0201 (calcd for C11H8N2O4S, 264.0205).
5.1.3.5. 2H-Pyrido[2,3-g][1,4]benzothiazine-5,10-dione, 3,4-dihy-
dro-N,N-dimethyl-1,1-dioxo-7-carboxamide (10) (RN 880137-52-0)
and 1H-Pyrido[3,2-g][1,4]benzothiazine-5,10-dione, 2,3-dihydro-N,
5.1.3.1.Octyl-2H-pyrido[2,3-g][1,4]benzothiazine-5,10-dione, 3,4-
dihydro-1,1-dioxo-7-carboxylate (9) (RN 880137-44-0). Octyl-5,8-
dihydro-5,8-dioxoquinoline-2-carboxylate (140 mg) with hypotau-
rine (96 mg) isolated by method 1 to give a bright yellow solid
(92 mg, 49%). 1H NMR (d6-DMSO) 9.44 (1H, s, NH), 8.53 (1H, d,
J = 8 Hz, H-9), 8.39 (1H, d, J = 8 Hz, H-8), 4.36 (2H, t, J = 6.7 Hz, H2-
3’), 3.89 (2H, m, H2-3), 3.41 (2H, t, J = 6.0 Hz, H2-2), 1.75 (2H, m,
H2-4’), 1.40-1.26 (10H, m, H2-5’-9’), 0.85 (3H, t, J = 6.6 Hz, H3-10’).
N-dimethyl-1,1-dioxo-7-carboxamide (21) (RN 880137-53-1). 5,8-
Dihydro-5,8-dioxoquinoline-N,N-dimethyl-2-carboxamide (140 mg,
0.61 mmol) with hypotaurine (90 mg) isolated by method 1 gave
one regioisomer as an orange solid (52 mg, 26%) and the later eluting
21 as an orange solid (15 mg, 8%). Compound 10: 1H NMR (d6-DMSO)
9.48 (1H, s, NH), 8.57 (1H, d, J = 8 Hz, H-9), 8.07 (1H, d, J = 8 Hz, H-8),
4.00 (2 H, m, H2-3), 3.51 (2H, m, H2-2), 3.17 (3H, s, N-Me) and 3.04
(3H, s, N-Me). 13C NMR in Table 1. UV (MeOH) kmax (log
e) 422
(3.02), 296 (3.65), 236 (3.96) nm. HPLC 4.48 min. EIMS m/z 335.0561
13C NMR in Table 1. UV (MeOH) kmax (log
e) 422 (3.19), 297 (3.70),
274 (3.76), 237 (4.10) nm. HPLC 12.2 min. FABMS m/z 421.1419
(calcd for C20H25N2O6S 421.1433).
(calcd for C14H13N3O5S, 335.0576).
Compound 21: 1H NMR (d6-DMSO) 9.38 (1H, s, NH), 8.57 (1H, d,
J= 8 Hz, H-9), 7.99 (1H, d, J = 8 Hz, H-8), 3.99 (2H, m, H2-3), 3.52
(2H, m, H2-2), 3.18 (3H, s, N–Me) and 3.04 (3H, s, N-Me). UV
5.1.3.2. 2H-Pyrido[2,3-g][1,4]benzothiazine-5,10-dione, 3,4-dihy-
dro-1,1-dioxo-7-carbonitrile (4) (RN 880137-45-1). 5,8-Dihydro-
5,8-dioxoquinoline-2-carbonitrile (200 mg, 1.1 mmol) with hypotau-
rine (80 mg) isolated by method 2 to give an orange solid (94 mg,
30%). 1H NMR (d6-acetone) 8.78 (1H, d, J = 8 Hz, H-9), 8.73 (1H, br s,
NH), 8.51 (1H, d, J = 8 Hz, H-8), 4.27 (2H, m, H-3) and 3.52 (2H, m,
(MeOH) kmax (loge) 422 (3.29), 297 (3.94), 268 (4.01), 236
(4.21) nm. HPLC 4.21 min. EIMS MNa+ m/z 358.0475 (calcd for
C14H13N3O5SNa, 358.0474).
5.1.3.6. (E)-1-Phenyl-3-(2H-pyrido[2,3-g][1,4]benzothiazine-5,10-
dione, 3,4-dihydro-1,1-dioxide)prop-2-en-1-one (6) (RN 880137-
57-5) and (E)-1-phenyl-3-(1H-pyrido[3,2-g][1,4]benzothiazine-5,10-
dione, 3,4-dihydro-1,1-dioxide)prop-2-en-1-one 3804 (19). (E)-3-
(5,8-Dihydro-5,8-dioxoquinolin-2-yl)-1-phenylprop-2-en-1-one (50 mg,
0.17 mmol) with hypotaurine (30 mg) isolated by method 1 gave 6 as
an orange solid (15 mg, 22%) and the later eluting 19 also as an orange
solid (15 mg, 22%). Compound 6: 1H NMR (d6-DMSO) 9.35 (1H, s, NH),
8.46 (1H, d, J = 8 Hz, H-9), 8.42 (1H, d, J = 8 Hz, H-8), 8.26 (1H, d,
H-2). 13C NMR in Table 1. UV (MeOH) kmax (log
e) 397 (3.59), 273
(4.22), 232 (4.21) nm. HPLC 4.96 min. EIMS m/z 289.0152 (calcd for
C12H7N3O4S, 289.0157).
5.1.3.3. 2H-Pyrido[2,3-g][1,4]benzothiazine-5,10-dione, 3,4-dihy-
dro-7-methyl-1,1-dioxide(3) (RN880137-62-2) and 1H-pyrido[3,2-
g][1,4]benzothiazine-5,10-dione, 2,3-dihydro-7-methyl-1,1-diox-
ide(22) (RN880137-50-8). 2-Methyl-5,8-dihydro-5,8-dioxoquino-

E. W. Chia et al. / Bioorg. Med. Chem. 16 (2008) 9432–9442
9441
J = 16 Hz, H-2’), 8.14 (2H, d, J = 7 Hz, H-5’), 7.80 (1H, d, J = 16 Hz, 1’), 7.73
(1H, t, J = 7 Hz, H-7’), 7.63 (2H, t, J = 7 Hz, H-6’), 3.91 (2H, m, H2-3) and
J = 9 Hz, H-3), 6.64 (1H, d, J = 9 Hz, H-2) and 2.78 (3H, s, H3-1’).
13C NMR in Table 1. UV (MeOH) kmax (log
) 421(3.09), 267(3.94),
238(3.97), 210(3.90) nm. HPLC 4.11 min. EIMS m/z 276.0197 (calcd
e
3.42 (2H, m, H2-2). 13C NMR in Table 1. UV (MeOH) kmax (log
e) 313
(4.36), 274 (4.36), 244 (4.47) nm. HPLC 10.6 min. EIMS m/z 394.0549
(calcd for C20H14N2O5S 394.0623).
for C12H8N2O4S, 276.0205).
Compound 19: 1H NMR (d6-DMSO) 9.38 (1H, s, NH), 8.57 (1H, d,
J = 8 Hz, H-9), 8.42 (1H, d, J = 8 Hz, H-8), 8.43, d, J = 15 Hz, H-2’),
8.24 (2H, dd, J = 7.5, 1 Hz, H-5’), 7.92 (1H, d, J = 15 Hz, 1’), 7.85
(1H, d, J = 8 Hz, H-7’), 7.75 (2H, t, J = 7.5 Hz, H-6’), 4.00 (2H, m,
H2-2) and 3.54 (2H, t J = 6 Hz, H2-3). 13C NMR in Table 1. UV
5.1.7. 2-Methyl-6-methylsulfonyl-7-methylamino-5,8-dihydro-
5,8-dioxoquinoline (15)
To a solution of 2-methyl-quinoline-quinone (400 mg, 2.3 mmol)
in acetonitrile (10 ml) and ethanol (5 ml) was added sodium
methanesulfinate (236 mg, 2.31 mmol) in water (5 ml). The mixture
was left at RT for 2 h, then the solvents removed in vacuo. Purifica-
tion by column chromatography over silica gel, eluting with EtOAc
in DCM gave the intermediate methyl-6-methylsulfonyl-5,8-dihy-
dro-5,8-dioxoquinoline as an off white solid (320 mg, 55%). 1H
NMR (CDCl3) 8.52 (1H, d, J = 8 Hz, H-9), 7.40 (1H, d, J = 8 Hz, H-8),
7.19 (1H, s, H-3), 3.16 (3H, s, S-Me) and 2.75 (3H, s, C-Me). To a solu-
tion of methyl-6-methylsulfonyl-5,8-dihydro-5,8-dioxoquinoline
(220 mg, 0.88 mmol) in acetonitrile (5 ml) and methanol (5 ml)
was added methylamine (40% in water, 1 ml). The resultant yellow
solution was evaporated in vacuo. Purification over silica gel eluting
with 5% MeOH in CHCl3 gave the crude product as an orange solid
(75 mg, 31%), crystallisation from methanol gave 15 as an orange
crystalline solid (25 mg, 10%).1H NMR (CDCl3) 9.72 (1H, br s, NH),
8.34 (1H, d, J = 8 Hz, H-9), 7.56 (1H, d, J = 8 Hz, H-8), 3.46 (3H, d,
J = 6 Hz, H-3), 3.42 (3H, s, H-2) and 2.75 (3H, s, Me). 13C NMR in Table
(MeOH) kmax (loge) 326 (4.43), 203 (4.29) nm. HPLC 8.71 min. EIMS
m/z 395.0731 (calcd for C20H15N2O5S 395.0702).
5.1.3.7. 2H-Pyrido[2,3-g][1,4]benzothiazine-5,10-dione, 3,4-dihy-
dro-7-hydroxymethyl- 1,1-dioxide (7) and 1H-pyrido[3,2-g][1,4]ben-
zothiazine-5,10-dione,3,4-dihydro-7-hydroxymethyl-1,1-dioxide
(20). 5,8-Dihydro-2-(hydroxymethyl)-5,8-dioxoquinoline (54 mg,
0.29 mmol) with hypotaurine (22 mg) isolated by method 1 gave
adduct 7 as an orange solid (19 mg, 23%) and the later eluting 20
as a orange solid (14 mg, 17%). 1H NMR (d6-DMSO) 9.28 (1H, s,
NH), 8.38 (1H, d, J = 8.5 Hz, H-9), 7.92 (1H, d, J = 8 Hz, H-8), 5.74
(1H, t, J = 6 Hz, OH), 4.70 (2H, d, J = 5.5 Hz, H-1’), 3.86 (2H, t,
J = 6 Hz H-2) and 3.38 (2H, t, J = 6 Hz H-3). 13C NMR in Table 1. UV
(MeOH) kmax (loge) 421 (3.01), 290 (3.71), 266 (3.88), 259 (3.87),
234 (4.01), 200 (3.91). HPLC 3.41 min. ESIMS m/z 317.0196 (calcd
for C12H10N2O5SNa, 317.0208).
1. UV (MeOH) kmax (loge) 421 (3.29), 292 (3.86), 268 (3.95), 259
(3.96), 237 (4.15), 199 (3.96) nm. HPLC 6.35 min. EIMS m/z
303.0408 (calcd for C12H12N2O4SNa, 303.0415).
Compound 20: 1H NMR (d6-DMSO) 9.20 (1H, s, NH), 8.39 (1H, d,
J = 8.5 Hz, H-9), 7.85 (1H, d, J = 8 Hz, H-8), 5.77 (1H, t, J = 6 Hz, OH),
4.70 (2H, d, J = 5.5 Hz, H-1’), 3.86 (2H, m, H-2) and 3.38 (2H, m, H-
3). UV (MeOH) kmax (log
(4.06), 192 (3.95). HPLC 4.27 min. ESIMS m/z 382.1073 (calcd for
C16H20N3O6S, 382.1211).
e
) 424 (3.11), 291 (3.72), 267 (3.83), 236
5.1.8. 2H-Pyrido[2,3-g][1,4]-benzothiazine-5,10-dioxo-3,4-dihy-
dro-N-(2-dimethylaminoethyl)-1,1-dioxo-7-carboxamide (11)
To a solution of acid 1 (62 mg 0.20 mmol) in DMF (10 mL) was
added dimethylethylenediamine (0.026 mL, 0.24 mmol) followed
by N,N-diisipropylethylamine (DIPEA, 0.105 mL, 0.60 mmol). The
mixture was cooled to 0 °C and benzotriazol-1-yl-oxytripyrrolidino-
phosphonium hexafluorophosphate (PyBOP, 135 mg, 0.26 mmol)
was added, then stirred at room temperature for 3 h. The solvents
were removed in vacuo. The product was purified by reverse phase
(C18) column, followed by crystallization from MeCN and EtOAc
twice then preparative HPLC Mobile phase A (TFA/H2O, pH 2.5)
and B (90% MeCN/H2O): t0 = 1% B, t10 = 1% B, t11 = 15% B, t19 = 15%
B, t20 = 1% B, t22 = 1% B. Flow rate 13 mL/min. tR = 15.25 min. The
product was freeze dried to give the TFA salt of 11 as a yellow solid
(30 mg, 30%). 1H NMR (d6-DMSO): d 9.35 (s, 1H, H-4), 9.29 (br, 1H,
H-15), 8.96 (br, t, 1H, H-15), 8.57 (d, J = 8.1 Hz, 1H, H-9), 8.43 (d,
J = 8.1 Hz, 1H, H-8), 3.91 (br, 2H, H-3), 3.70 (br, q, J = 5.8 Hz, 2H, H-
13), 3.42(t, J = 6.0 Hz, 2H, H-2), 3.30(2H, H-14, overlappedbywater),
2.80 (s, 6H, H-16) ppm. 1H NMR (d4-MeOH): d 8.55 (d, J = 8.0 Hz, 1H,
H-9), 8.47 (d, J = 8.0 Hz, 1H, H-8), 4.09 (br, t, J = 5.9 Hz, 2H, H-3), 3.86
(t, J = 5.8 Hz, 2H, H-13), 3.52 (t, J = 5.9 Hz, 2H, H-2), 3.43 (t, J = 5.8 Hz,
2H, H-14), 2.99 (s, 6H, H-16) ppm. 13C NMR in Table 1. FABMS m/z
379.1076 (calcd for C16H19N4O5S, 379.1076).
5.1.4. 2H-Pyrido[2,3-g][1,4]benzothiazine-5,10-dione, 3,4-dihy-
dro-1,1-dioxo-7-carbaldehyde (5) (RN 880137-59-7)
A mixture of compound 3 (50 mg, 0.18 mmol) and selenium diox-
ide (80 mg, 0.72 mmol) in dioxane (4 mL) and water (0.5 mL) was
stirred under nitrogen at 90°C for 14 h. Further selenium dioxide
(160 mg) was added in batches whilst heating over 12 h. Separation
by column chromatography over silica gel eluting with 10–100%
MeOH in CHCl3 gave the aldehyde 5 as an orange solid (18 mg,
34%). 1H NMR (d6-DMSO) 10.23 (1H, s, CHO), 9.62 (1H, s, NH), 8.70
(1H, d, J = 8 Hz, H-9), 8.40 (1H, d, J = 8 Hz, H-8), 4.02 (2H, m, H2-3)
and 3.53 (2H, m, H2-2). 13C NMR in Table 1. UV (MeOH) kmax (log
e)
421 (3.14), 289 (3.77), 267 (3.90), 234 (4.07) nm. HPLC 4.66 min.E-
IMS m/z 292.0147 (calcd for C12H8N2O5S, 292.0154).
5.1.5. 2H-Pyrido[2,3-g][1,4]-benzothiazine-5,10-dione, 3,4-
dihydro-7-(hydromethoxymethyl)-1,1-dioxide (13) (RN 880137-
60-0)
A sample of aldehyde 5 (5 mg, 0.02 mmol) was left in methanol
(1 mL) overnight then the solvents removed to give the hemi acetal
(5 mg, 95%) as a yellow solid. 1H NMR (CD3OD) 8.53 (1H, d, J = 8 Hz,
H-9), 8.02 (1H, d, J = 8 Hz, H-8), 5.64 (1H, s, H-1’), 4.04 (2H, m, H2-
3), 3.59 (3H, m, H4-3’) and 3.43 (2H, m, H2-2). UV (MeOH) kmax
5.1.9. (2-Dimethylaminoethyl)-2H-pyrido[2,3-g][1,4]-benzo-
thiazine-5,10-dioxo-3,4-dihydro-1,1-dioxo-7-carboxylate (12)
To a suspension of 3-dimethylaminopropionic acid hydrochlo-
ride (230 mg, 1.5 mmol) in MeCN (20 mL) was added one drop of
DMF followed by oxalyl chloride (1.29 mL, 15.0 mmol) slowly. The
mixture was stirred for 4 h before all solvents were removed. The
resultant red brown solid was dissolved in DMF (10 mL), then added
to a solution of 7 (147 mg, 0.50 mmol), DMAP (6 mg, 0.05 mmol) and
(loge) 416 (2.95), 267 (3.72), 233 (3.91) nm. HPLC 4.65 min. EIMS
m/z 325.0490 (calcd for C13H13N2O6S 325.0494).
5.1.6. 4H-Pyrido[2,3-g][1,4]benzothiazine-5,10-dione-7-
methyl-1,1-dioxide (25) (RN 880137-62-2)
Compound 3 (10 mg, 0.036 mmol) was stirred in 1M KOH
(2 mL) for 3 h. The resultant red orange solution was passed down
a short column of weak cationic ion exchange resin (Amberlite IRC
86, 2 g) to give a yellow solution which was freeze dried to give the
pure product 25 as a yellow solid (9 mg, 91%). 1H NMR (d6-DMSO)
8.41 (1H, d, J = 8 Hz, H-9), 7.87 (1H, d, J = 8 Hz, H-8), 7.26 (1H, d,
DIPEA (522 lL, 3.0 mmol) in DMF (10 mL). The mixture was stirred
for 16 h then the solvents were removed in vacuo. The product
was purified by C18 column chromatography, then washed with a
little MeCN and EtOAc, and then further purified by preparative
HPLC Mobile phase A (TFA/H2O, pH 2.5) and B (90% MeCN/H2O) iso-

9442
E. W. Chia et al. / Bioorg. Med. Chem. 16 (2008) 9432–9442
cratic 9% B, flow rate 13 mL/min, tR = 11.11 min. The product was
freeze-dried to give the TFA salt of 12 as a brown solid (10 mg, 4%).
1H NMR (D2O): d 8.47 (d, J = 8.2 Hz, 1H, H-9), 7.92 (d, J = 8.2 Hz, 1H,
H-8), 5.42 (s, 2H, H-11), 4.14 (br t, J = 6.0 Hz, 2H, H-3), 3.61 (br t,
J = 6.0 Hz, 2H, H-2) 3.53 (t, J = 6.7 Hz, 2H, H-15), 3.10 (t, J = 6.7 Hz,
2H, H-14) and 2.94 (s, 6H, H-17) ppm. 13C NMR in Table 1. FABMS
m/z 394.1073 (calcd for C17H20N3O6S, 394.1073).
urate (MSU) crystals (3 mg) suspended in PBS (500 lL). Test com-
pounds were administered by oral gavage immediately following
administration of MSU. Control mice received PBS. Four hours after
MSU crystal administration, mice were euthanased by CO2. The
peritoneal cavity was washed with PBS (3 mL) containing 25 U/
mL heparin. Total cell numbers were counted and the sample vol-
ume adjusted to 1 ꢀ 106 cells/mL for subsequent analysis of super-
oxide production.
5.2. Biological assays
5.2.6. Detection of superoxide production by peritoneal
neutrophils
5.2.1. Reagents and chemicals
Peritoneal neutrophils were tested for superoxide production as
described above. To determine the number of neutrophils in each
sample, a small aliquot (100 lL) of each cell sample was spun onto
a glass slide and stained with the Diff-Quick Staining Kit (Dade
Behring, Newark, USA). The percentage of neutrophils in the peri-
toneal wash was determined microscopically using standard histo-
logical criteria. Superoxide production was then corrected for the
number of neutrophils/mL in each sample.
Polymorphprep (Axis-Shield, Norway) was obtained through
Medica Pacifica Ltd. (New Zealand). WST-1 and mPMS (1-meth-
oxy-5-methylphenazinium methylsulfate) were from Dojindo Lab-
oratories (Kumamoto, Japan). Cell culture reagents were obtained
from Invitrogen (New Zealand). All other reagents were obtained
through Sigma–Aldrich (New Zealand) unless otherwise stated.
5.2.2. Preparation of monosodium urate crystals (MSU)
Uric acid (250 mg) was added to 45 mL of ddH2O containing
300
lL of 5 M NaOH and the solution boiled until the uric acid
5.2.7. Serum toxicity markers
was dissolved. The solution was passed through a 0.2
l
M filter
Blood was collected from the heart of euthanased mice by car-
diac puncture. Whole blood samples were shipped over ice for
analysis (Gribbles Analytical Laboratories, Hamilton, New Zealand)
to determine the levels of liver enzymes (aspartate aminotransfer-
ase, alanine aminotransferase, alkaline phosphatase, lactate dehy-
drogenase) and the renal marker creatinine in the serum.
and 1 mL of 5 M NaCl was added. The hot solution was allowed
to cool then left at 26 °C for 7 days to allow crystal formation.
The resulting MSU crystals were washed with ethanol and acetone.
The resulting MSU crystals were needle shaped, 5–25 lm in length,
and were birefringent to polarised light. All MSU crystals were
tested as endotoxin free by LAL assay (<0.01 EU/10 mg).
Acknowledgement
5.2.3. Neutrophil superoxide assay
Human neutrophils were isolated from anti-coagulated whole
human blood using Polymorphprep (density 1.113 g/mL, 500 g,
35 min). Neutrophils (resuspended in 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
(1% DMSO/HBSS) for 30 min prior to addition of the detection
dye WST-1 (final concentration 0.35 mM). The respiratory burst
This work was supported by TerraMarine Pharmaceuticals and
the New Zealand Foundation for Research Science and Technology,
Contract CO1X0205.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2008.09.052.
was triggered by addition of PMA (final concentration 0.295 lM),
and dye reduction was monitored (450 nm) for 20 min at 37 °C.
Superoxide production was calculated as the rate of dye reduction
over time compared to buffer-treated cell controls.
References and notes
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HL60 cells (suspended in RPMI1640/5%FCS) 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 (dis-
solved in 1% DMSO/RPMI1640/5% FCS) and cultured for 48 hours
(37 °C, 5% CO2). MTT was then added to each well (final concentra-
tion 1.10 mM) and the cells were incubated for 2 h. Lysing buffer
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5.2.5. In vivo assay
C57BL/J6 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 aged between 8 and 10
weeks. Experimental procedures were approved by the Victoria
University Animal Ethics Committee (New Zealand) and carried
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