The anti-cancer, anti-inflammatory and tuberculostatic activities of a series of 6,7-substituted-5,8-quinolinequinones

Article abstract of DOI:10.1016/j.bmc.2010.03.021

A variety of 6,7-substituted-5,8-quinolinequinones were synthesised and assessed for their anti-tumour and anti-inflammatory activities, and their ability to inhibit the growth of Mycobacterium bovis BCG. In particular, the introduction of a sulfur group

Full text of DOI:10.1016/j.bmc.2010.03.021

Bioorganic & Medicinal Chemistry 18 (2010) 3238–3251
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
The anti-cancer, anti-inflammatory and tuberculostatic activities of a series
of 6,7-substituted-5,8-quinolinequinones
Benjamin J. Mulchin a, Christopher G. Newton a,b, James W. Baty a, Carole H. Grasso a, William John Martin a,
Michaela C. Walton a, Emma M. Dangerfield a,b, Catherine H. Plunkett a,b, Michael V. Berridge a,
a,
Jacquie L. Harper a, Mattie S. M. Timmer b, , Bridget L. Stocker
*
*
a Malaghan Institute of Medical Research, PO Box 7060, Wellington, New Zealand
b School of Chemical and Physical Sciences, Victoria University of Wellington, PO Box 600, Wellington, New Zealand
a r t i c l e i n f o
a b s t r a c t
Article history:
A variety of 6,7-substituted-5,8-quinolinequinones were synthesised and assessed for their anti-tumour
and anti-inflammatory activities, and their ability to inhibit the growth of Mycobacterium bovis BCG. In
particular, the introduction of a sulfur group at the 7-position of the quinolinequinone led to the discov-
ery of two compounds, 6-methylamino-7-methylsulfanyl-5,8-quinolinequinone (10a) and 6-amino-7-
methylsulfonyl-5,8-quinolinequinone (12), that exhibited selectivity for leukemic cells over T-cells, a
highly desirable property for an anti-cancer drug. A number of anti-inflammatory (AI) compounds were
also identified, with 6,7-bis-methylsulfanyl-5,8-quinolinequinone (18a) exhibiting the highest AI activity
Received 15 January 2010
Revised 10 March 2010
Accepted 11 March 2010
Available online 15 March 2010
Keywords:
Quinolinequinone
Cancer
Tuberculosis
Inflammation
Synthesis
(0.11 lM), while 6,7-dichloro-5,8-quinolinequinone (7a), 6,7-dichloro-2-methyl-5,8-quinolinequinone
(7b), and 6,7-bis-phenylsulfanyl-quinoline-5,8-diol (19) also exhibited good AI activity and specificity.
Several quinolinequinone TB-drug candidates were identified. Of these, 6-amino-7-chloro-5,8-quinolin-
equinone (11) and 6-amino-7-methanesulfinyl-5,8-quinolinequinone (14), exhibited low MICs (1.56–
3.13 lg/mL) for the 100% growth inhibition of M. Bovis BCG. Some general trends pertaining to the func-
tional group substitution of the quinolinequinone core and biological activity were also identified.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Other biologically active 5,8-quinolinequinones include the potent
anti-inflammatory agents Ascidiathiazones A (2) and B (3), recently
Derivatives of 5,8-quinolinequinones have a wide spectrum of
biological properties that include anti-tumour, anti-inflammatory
and anti-bacterial activities and accordingly, much effort is spent
in developing new and more effective quinolinequinone-based
therapeutics.1–6 Notable 5,8-quinolinequinones include the anti-
tumour agent streptonigrin (1) (Fig. 1), isolated from the bacterium
Streptomyces flocculus in 1959.7,8 Streptonigrin was one of the first
compounds to be systematically modified in an attempt to corre-
late specific structural features with anti-cancer properties.9 Ear-
lier structure–activity studies concluded that the 7-amino-
quinolinequinone-moiety of streptonigrin was crucial for anti-tu-
mour activity,10 however later work revealed that the addition of
electron-withdrawing groups at the 6- and/or 7-positions of the
quinolinequinone resulted in enhanced rates of DNA degrada-
tion,11,12 a measure of anti-tumour activity. Unfortunately, the
high toxicity of streptonigrin has limited its therapeutic value.13,14
isolated from the New Zealand ascidian Aplidium sp.,15 the anti-
tuberculostatic thiazole-containing quinolinequinones, such as
4,16,17 and 7-heptadecylsulfanyl-6-hydroxy-5,8-quinolinequinone
(5), which exhibited promising anti-malarial activity.18,19
Given the potential of quinolinequinones in the treatment of a
variety of diseases and, in particular, the potential of less complex
quinolinequinones (e.g., 5) to have interesting biological activities,
we synthesised and tested a number of 6,7-substituted-5,8-qui-
nolinequinones for their anti-proliferative, anti-inflammatory, and
tuberculostatic activities. Few 6,7-substituted-5,8-quinolinequinon-
es have been screened in multiple assays and given the broad biolog-
ical profiles of quinolinequinones, this makes it difficult to determine
the drug’s specificity. Moreover, relatively few bicyclic thiol-substi-
tuted quinolinequinones have been synthesised12,16–18,20–28 and the
subsequent biological studies performed on these compounds have
primarilyfocussedontheroleofmono-ordi-thio-quinolinequinones
as anti-malarial18,19,29 and anti-fungal24–26 agents. A comparison of
the activities of a variety of 6,7-substituted-5,8-quinolinequinones
in a number of different assays provides valuable information on
how the substitution pattern affects biological activity and will high-
light key factors important for the development of better and more
* Corresponding authors. Tel.: +64 4 463 6529; fax: +64 4 463 5241 (M.S.M.T.);
tel.: +64 4 499 6914; fax: +64 4 499 6915 (B.L.S.).
E-mail addresses: mattie.timmer@vuw.ac.nz (M.S.M. Timmer), bstocker@mala
ghan.org.nz (B.L. Stocker).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.03.021

B. J. Mulchin et al. / Bioorg. Med. Chem. 18 (2010) 3238–3251
3239
O
O
O
O
O
S
OMe
N
S
N
HO2C
N
N
NH2 HO2C
N
N
H
N
O
O
O
Me
HO
NH2
Ascidiathiazone A (2)
4
O
O
O
O
O
HO2C
N
S
MeO
OH
SC17H35
OMe
N
H
N
Streptonigrin (1)
O
Ascidiathiazone B (3)
Figure 1. Representative 5,8-quinolinequinones.
5
specific quinolinequinone-based therapeutics. In this study, we fo-
cussed on the biological effects of chloro, amino, andthio-substituted
quinolinequinones.
a to the aliphatic amine and the nearest carbonyl could be used.
Unfortunately, such HMBC was only observed for the 6-substituted
methylamine isomer 8a (R2 = Me). The tendency of quinolinequi-
nones to tautomerise nevertheless allowed for the assignment of
each regioisomer. As a consequence of tautomerisation (Scheme
1), C6, C7 and C8 are broader and less visible in the 13C NMR spec-
tra for the 6-substituted isomer, whilst C5, C6 and C7 are less vis-
ible for the 7-subsituted isomer. The HMBC between H4 to C5 can
thus be used to determine the regioselectivity of addition.
2. Chemistry
The synthesis of the 6,7-substituted-5,8-quinolinediones com-
menced with the formation of known dichloro-quinones 7a and
7b via the sodium chlorate oxidation12 of 8-hydroxyquinoline 6a
or 8-hydroxy-2-methyl-quinoline 6b, respectively (Table 1). The
dichloro-quinones were then treated with a variety of amine
nucleophiles to give the 6-substituted-products 8a–e and/or the
7-substituted products 9a, 9c–e in good (65–96%) yield. Here, the
presence or absence of a Lewis acid and the choice of solvent af-
fected the regioselectivity of the reaction. The addition of
NiCl2ꢀ6H2O favoured formation of the 6-isomer (Table 1, entries
1, 3, 4). This is thought to occur via the chelation of the Lewis acid
to the nitrogen atom and C8 carbonyl of the quinolinequinone,
thus increasing the electron-withdrawing power of the carbonyl
and the reactivity at the conjugated 6-position.30–33 The use of a
more polar solvent also increased the yield of the 6-isomer, though
the rationale for this is not well understood. In the absence of the
Lewis acid, a mixture of the 6- and 7-substituted isomers was ob-
tained (entries 2, 5–7). This ratio was improved in favour of the 7-
isomer when a less polar solvent was used31,32 (e.g., dioxane, entry
2). In all instances, the regio-isomers were separable by flash col-
umn chromatography.
It is also worthwhile to note that the 1H NMR chemical shifts
and polarity of the amino-5,8-quinolinequinone regioisomers can
be used to determine the site of addition.31 The chemical shift of
H2 for the 7-isomer has been shown to be slightly upfield when
compared to the 6-isomer, while that of H4 is slightly downfield,
thus making the value of
D(H2–H4) larger for the 6-isomer than
the 7-isomer. The 6-isomer has also been reported to be more polar
than the 7-isomer, as determined by Rf values (silica gel TLC).
These correlations held true for the assignment of all of our com-
pounds [e.g., for the 6-isomer 8a:
D(H2–H4) = 0.67 ppm, Rf = 0.23
(EtOAc) cf. 7-isomer 9a: (H2–H4) = 0.45 ppm, Rf = 0.32 (EtOAc)].
D
With the required amino-quinones in hand, the sulfur function-
ality was then introduced. Treatment of 7-chloro-quinolinequi-
none 8a with sodium thiomethylate (NaSMe) in EtOH at rt gave
the desired thio-quinone 10a in good (88%) yield (Scheme 2),
whilst the phenyl-substituted thiol 10b was prepared via the reac-
tion of dichloro-quinolinequinones 8a in pyridine with thiophenol
(PhSH). The sulfides 10a and 10b were then oxidised to the sulfox-
ides 13a and 13b, respectively, using one equivalent of KMnO4 or
m-CPBA, with the m-CPBA oxidation giving improved yields. Inter-
To confirm the regioselectivity of the substitution reaction, it
was initially hoped that the long range HMBC between the protons
Table 1
Formation of 6- and 7-substituted quinolinequinones
O
O
O
Cl
Cl
NHR2
+
Cl R1
Cl
NaClO3
HCl
R2-NH2
R1
N
conditions
R1
N
R1
N
N
NHR2
OH
O
O
O
6a R1 = H
7a R1 = H
8a-e
9a-e
6b R1 = Me
7b R1 = Me
Entry
Starting material
Conditionsa
R1
R2
Yieldb 6-isomer
Yieldb 7-isomer
1
2
3
4
5
6
7
7a
7a
7a
7a
7a
7a
7b
A
B
A
C
D
D
D
H
H
H
H
H
H
Me
Me
Me
CHPh2
CH2CH2Cl
CH2CH2Cl
CH2CH2Br
CH2CH2Br
8a, 95%
8a, 20%
8b, 96%
8c, 82%
8c, 46%
8d, 53%
8e, 44%
9a, —
9a, 67%
9b, —
9c, —
9c, 48%
9d, 25%
9e, 21%
a
Conditions: (A) NiCl2ꢀ6H2O (1.1 equiv), EtOH; (B) 1,4-dioxane, pyr. (2.1 equiv); (C) EtOH/H2O (2:1), NiCl2ꢀ6H2O (1.1 equiv), 2 M NaOH (2.1 equiv); (D) EtOH/H2O (2:1), 2 M
NaOH (2.1 equiv).
b
Isolated yield.

3240
B. J. Mulchin et al. / Bioorg. Med. Chem. 18 (2010) 3238–3251
6-substitution
O
O
O
O
O
O
S
Cl
SR3
O
4
O
R3
a
b
4
5
5
4a
NHR
Cl
4a
8a
NR
Cl
3
2
3
2
6
6
N
NHMe
N
NHMe
N
NHMe
6
O
9a
N
1
8a
7
7
N
1
8
O
8
OH
16a R3 = Me
17a R3 = Me
16b R3 = Ph
7-substitution
Scheme 3. Formation of 6-sulfur-substituted quinones. Regents and conditions: (a)
NaSMe, EtOH, 16a: 94% or HSPh, pyr., 16b: 90%; (b) m-CPBA (1.0 equiv), CH2Cl2,
0 °C, 10 min, 79%.
O
4
OH
5
4
5
4a
Cl
4a
8a
Cl
3
2
3
2
6
N
1
8a
7
NHR
7
N
1
NR
8
O
8
O
(e.g., 7?8), this is disfavoured for quinolinequinones containing
a 6- or 7-substituted amine (e.g., 8)—a consequence of enamine
formation (Scheme 4) and electron delocalisation over the quino-
line quinone-ring leading to unfavourable orbital interactions with
hard amine nucleophiles.
In the 7-amino-substituted series (Scheme 3), 7-methylamine
quinolinequinone 9a was treated with either NaSMe or PhSH to
give sulfides 16a and 16b in 94% and 90% yield, respectively. Oxi-
dation of sulfide 16a with one equivalent of m-CPBA then gave
sulfoxide 17a in good yield.
To prepare additional sulfur-substituted quinolinequinones for
biological evaluation, dichloro-quinolinequinone 7a was treated
with 2.1 equiv of sodium thiomethoxide in MeCN to give the corre-
sponding disulfide 18a in excellent (90%) yield (Scheme 5). Alter-
natively, treatment of 7a with 2.1 equiv of HSPh in pyridine gave
the bis(phenylsulfanyl) analogue 18b, also in excellent yield. When
excess HSPh (3 equiv) was added to a solution of 7a in pyridine,
diol 19 was isolated in 42% yield—a consequence of the reduction
of the quinone moiety by the excess HSPh, which itself was
Scheme 1. Tautomerisation of amino-substituted quinolinequinones.
estingly, during column chromatography of the amine-substituted
quinolinequinones using ammonia in the eluent, aminolysis prod-
ucts were observed. This reaction was subsequently used to our
advantage to produce an additional series of compounds. Thus,
by subjecting quinolinequinone 8b to a solution of ammonia in
water/methanol and subsequent workup, amine 11 was isolated
in 84% yield. In an analogous manner, methylthio substituted qui-
none 15 was obtained from 10a in good yield, while oxidation of
sulfide 10a to the corresponding sulfone, using two equivalents
of m-CPBA, followed by aminolysis resulted in the formation of
the free amine 12 in 93% yield. Aminolysis of sulfoxide 13a also
proceeded uneventfully to give amine 14 in 88% yield.
To explain the tendency of the N-alkylated amino-quinolinequi-
nones to undergo aminolysis, it needs to be recognised that Mi-
chael addition to the 6-position of the quinolinequinone is
favoured (Scheme 4). Here, enamine formation and delocalisation
of the negative charge through both the quinone-ring and sulfox-
ide/sulfone substituent favours nucleophilic attack at the imine.
Subsequent protonation of the alkylamino-substituent, followed
by b-elimination results in reestablishment of the quinone system
and net nucleophilic substitution at the 6-position. It is also con-
ceivable that this type of substitution may occur in vitro/in vivo,
rendering these compounds efficient electrophilic traps for irre-
versible/covalent inhibition of enzymatic targets.
:Nu
O
O
H
N
O
H
N+
:
Nu
R
R
N
S
O
N
N
S
O
O-
+H+
The specificity of the aminolysis reaction, in that Michael addi-
tion takes place when ammonia is used as the nucleophile but not
the thiolate, follows literature precedent34 whereby hard nucleo-
philes, such as amines and alkoxides, predominantly result in dis-
placement of the amine substituent. The large excess of ammonia
also ensures complete aminolysis of the alkylamine. Similarly, dis-
placement of the 7-chloride in chloro-quinolinequinone 8b is not
observed. Softer nucleophiles (e.g., thiols) favour substitution of
chlorides,35 and though amine substitution of chlorides is known
O
O
O
Nu H
N+
Nu
R
H
S
O
-RNH2
N
S
O
O-
Scheme 4. Nucleophilic substitution via a Michael/Retro Michael mechanism.
O
O
O
O
NHR2
Cl
NHR2
SR3
NHR2
NH2
a
c
b
R3
Me
N
N
N
S
N
S
O
O
O
O
O
14
O
2
3
2 = Me
10a R2 = Me, R3 = Me
R = Me, R = Me
8a
8b
13a
13b
R
R
2 = CHPh2
10b
R
2 = Me, R3 = Ph
R
2 = Me, R3 = Ph
O
b
b
d
O
11 R2 = H
NH2
NH2
Me
S
O O
N
SMe
N
O
O
12
15
Scheme 2. Formation of 7-sulfur-substituted quinones. Reagents and conditions: (a) NaSMe, EtOH, 10a: 88%; or HSPh, pyr., 10b: 95%; (b) aq NH3, 11: 84%, 15: 76%, 14: 88%;
(c) m-CPBA (1.0 equiv), CH2Cl2, 0 °C, 35 min, 13a: 74%; or KMnO4, AcOH, 13b: 30%; (d) (i) m-CPBA (2.0 equiv), CH2Cl2, 0 °C, 30 min; (ii) aq NH3, 93%.

B. J. Mulchin et al. / Bioorg. Med. Chem. 18 (2010) 3238–3251
3241
OH
lostatic activities. To investigate the potential of the compounds to
act as anti-cancer drugs, their anti-proliferative activity was mea-
sured against HL60 cells (IC50) and human T-cells (IC50) from
healthy volunteers using the MTT assay.36,37 To determine the
anti-inflammatory activity (AI) of the quinolinequinones, their
ability to block the production of superoxide by activated human
neutrophils,37,38 a key readout linked to a number of inflammatory
diseases including gout and cardiovascular disease,39–41 was mea-
sured. The potential of the quinolinequinones in treating tubercu-
losis was determined via the Alamar Blue tuberculostatic assay42,43
using Mycobacterium bovis BCG as the bacterial model. Here, the
MIC was determined as the minimal amount of compound leading
to 100% growth inhibition.
A summary of these assay results is presented (Table 2). The
compounds have been broadly grouped into those containing
two halogen substituents (entries 1 and 2), an amine and halogen
functionality (entries 3–12), those with a mono-sulfur functional-
ity (entries 13–24), and the di-sulfur containing derivatives (en-
tries 25–28).
O
O
SR3
SR3
SPh
SPh
Cl
Cl
a
b
N
N
N
OH
19
O
O
7a
3
18a
18b
R
= Me
R
3 = Ph
c
O
O
O
O
O
O
S
S
S
Tol
Tol
O
Tol
+
N
N
Cl
O
O
O
20
21
Scheme 5. Formation of disulfides and sulfones. Reagents and conditions: (a)
NaSMe (2.1 equiv), MeCN, 18a: 90%; or HSPh (2.1 equiv), THF, pyr., 18b: 92%; (b)
HSPh (3 equiv), THF, pyr., 42%; (c) p-toluenesulfinic acid (2.1 equiv), H2O, MeCN, 20:
44%, 21: 18%.
oxidised to the disulfide (PhSSPh). Somewhat unsurprisingly, sub-
jection of dichloro-quinolinequinone 7a to 1 equiv of HSPh under a
variety of conditions led to an inseparable mixture of the 6- and 7-
sulfur substituted products in a 1:1 ratio, even when NiCl2ꢀ6H2O
was added as a Lewis acid. This lack of regioselectivity occurred
as a consequence of the favourable reactivity of the soft sulfur
3.2. Anti-proliferative activity
Since the early work on streptonigrin, it is well known that qui-
nolinequinones show promise as anti-cancer agents; however, in
general, their toxicity has limited therapeutic application. A similar
trend was observed for our 6,7-amino and/or halogen functional-
ised quinolinequinones with most exhibiting potent anti-prolifera-
nucleophile towards the
a,b-unsaturated ketone—such nucleo-
philic addition is so facile, that the slight enhancement of electro-
philicity at the C6 position brought about via the addition of the
Lewis acid has no effect on chemoselectivity. To prepare further
sulfone derivatives, dichloro-quinolinequinone 7a was then trea-
ted with 2.1 equiv of p-toluenesulfinic acid. This resulted in the
formation of the disulfone 20 in 44% yield, and the 6-isomer 21
(18% yield) following recrystallisation.
Finally, to follow-up from our previous studies whereby we
synthesised and tested a variety of Ascidathiazone analogues,5
the more simple tricyclic quinolinequinones 22 and 23 were pre-
pared (Scheme 6). Here, ethanolic solutions of the b-chloro-ethyl-
amine precursors 8c and 9c were subjected to a slight excess of
sodium sulfide in H2O,16 which, following substitution of the pri-
mary chloride by the thiol and subsequent intra molecular cyclisa-
tion, then gave the simple ascidiathiazone quinolinequinone
analogues 22 and 23 in 59% and 76% yield, respectively.
tive activity against HL60 cells [IC50 values of ca. 1–4
3–12)] and T-cells [IC50 values of ca. 1.5–5 M (entries 3–12)].
lM (entries
l
There was little distinction between the anti-proliferative activities
for the regioisomers (e.g., compare entries 5 and 6, 7 and 8, 9 and
10, 11 and 12), or with the type of amino substituent (cf. 3, 4, and
5). The dichloro-quinolinequinones (entries 1 and 2), exhibited
notably lower anti-proliferative activity against T-cells and HL60
cells. The introduction of a sulfur atom, at either the 6- or 7-posi-
tion however, provided more promising results. Most notable were
the activities of thiol 10a (entry 14), and the sulfoxide 12 (entry
23). With an HL60 IC50 of 3.4 lM and a T-cell IC50 of 19.5 lM, thiol
10a showed modest selectivity towards tumour cells and is a wor-
thy candidate for further evaluation. This somewhat selective
activity appears to be specific to this particular thiol, as its
amine-analogue 15 (entry 13), its regio-isomer 16a (entry 15),
and its thiophenyl analogue 10b (entry 16) exhibited comparable
anti-proliferative activity across the two assays. The sulfoxides
(entries 20–22) did not exhibit very promising anti-proliferative
activity; however the sulfone 12 (entry 23) exhibited good selec-
tivity with the HL60 anti-proliferative activity (IC50 = 4.53
being almost fivefold higher than the corresponding T-cell anti-
proliferative activity (IC50 = 19.3 M). The di-thiols 18a, 18b, and
19 (entries 25–27) exhibited little anti-proliferative specificity,
though it is interesting to note that the reduction of the diphenyl
quinolinequinone 18b to the diol 19 led to a slight reduction in
the anti-proliferative activities (cf. entry 26 and entry 27). Finally,
of the quinolinequinones tested, the chloro-sulfone analogue 21
(entry 24) and the bis-tolyl-analogue 20 (entry 28) showed com-
paratively modest anti-proliferative activity.
Taken as a whole, these results indicate that 6,7-substituted-
5,8-quinolinequinones containing an amine and a halogen at the
6- or 7-position or two sulfur-containing groups show little speci-
ficity towards HL60 cells compared to human T-cells, thus making
them less desirable anti-cancer drugs. Of these two classes of com-
pounds, the amine-functionalised quinolinequinones show greater
anti-proliferative activity. Quinolinequinones with an amine- and
thiol-functionality at the 6- or 7-position however, hold promise
as selective anti-cancer drugs with two lead compounds, thiol
10a and sulfone 12, having been identified.
3. Biological results and discussion
3.1. Overview
lM)
Having synthesised a variety of quinolinequinones, we then
tested them for anti-proliferative, anti-inflammatory and tubercu-
l
O
O
H
N
H
N
a
a
Cl
Cl
N
Cl
N
S
O
O
O
22
8c
9c
O
Cl
S
N
N
H
N
N
H
O
O
23
Scheme 6. Formation of tricyclic structures. Regents and conditions: (a) Na2Sꢀ9H2O,
EtOH, 22: 59%, 23: 76%.

3242
B. J. Mulchin et al. / Bioorg. Med. Chem. 18 (2010) 3238–3251
Table 2
Quinolinequinones and their biological activities
Entry
Compound
HL60 IC50
31.8 3.8
(
lM)
T-cell IC50
(lM)
AI50
(
l
M)
Tuberculostatic activity; MIC (lg/mL)
O
Cl
Cl
1
32.9 15.3
1.3 0.9
3.4 1.9
>200
6.3–12.5
N
O
7a
O
Cl
Cl
2
3
18.7 3.4
44.9 4.5
6.3–12.5
1.56–3.13
6.3–12.5
12.5–25
12.5–25
3.13–6.3
6.3–12.5
6.3–12.5
12.5–25
N
O
7b
O
NH2
Cl
3.26 0.12
2.45 0.11
2.45 0.46
4.30 0.09
1.65 0.25
1.20 0.03
1.62 0.17
1.94 0.34
2.04 0.06
2.59 0.01
3.56 0.08
5.21 0.34
2.56 0.56
2.50 0.69
2.29 0.05
2.27 0.09
N
N
O
11
O
H
N
Ph
Ph
4
33.9 0.61
78.9 0.36
41.1 2.1
20.0 4.2
20.7 6.4
37.0 2.3
31.0 4.7
Cl
O
8b
O
H
N
Me
5
N
N
Cl
O
8a
O
Cl
Me
6
N
H
O
O
9a
H
N
7
N
N
N
N
Cl
Br
O
8d
9d
O
Cl
Br
8
N
H
O
O
H
N
9
Cl Cl
O
O
8c
9c
Cl
Cl
10
N
H
O

B. J. Mulchin et al. / Bioorg. Med. Chem. 18 (2010) 3238–3251
3243
Table 2 (continued)
Entry
Compound
HL60 IC50
(
lM)
T-cell IC50
(l
M)
AI50
(
l
M)
Tuberculostatic activity; MIC (
12.5–25
lg/mL)
O
H
N
11
12
1.30 0.07
2.23 0.10
34.2 0.7
N
O
Cl
Br
8e
O
Cl Br
1.21 0.02
1.59 0.08
73.2 1.5
12.5–25
6.3–12.5
N
N
H
O
9e
O
NH
2
Me
13
14
15
16
17
18
19
20
7.64 0.49
3.44 0.07
7.92 0.23
8.03 0.30
5.88 0.04
4.24 0.15
3.52 0.13
1.30 0.01
8.32 0.56
19.5 1.0
7.57 0.23
12.2 1.4
6.91 0.42
5.88 1.40
6.02 0.40
1.93 0.07
>150
N
N
N
N
N
N
N
N
S
O
15
O
H
N
Me
Me
>200
12.5–25
S
O
10a
O
S
Me
Me
>200
>50
N
H
O
16a
O
H
N
Me
Ph
>150
12.5–25
12.5–25
6.3–12.5
6.3–12.5
1.56–3.13
S
O
10b
O
S
Ph
Me
92.8 1.1
N
H
O
16b
O
H
N
>200
S
O
22
O
S
>200
N
H
O
23
O
NH
2
Me
>200
S
O
O
14
(continued on next page)

3244
B. J. Mulchin et al. / Bioorg. Med. Chem. 18 (2010) 3238–3251
Table 2 (continued)
Entry
Compound
HL60 IC50
12.1 0.2
(
lM)
T-cell IC50
11.8 2.0
(lM)
AI50
(
l
M)
Tuberculostatic activity; MIC (lg/mL)
O
H
N
Me
Me
21
22
23
24
25
26
27
28
>200
>200
>200
>200
25–50
N
O
S
O
13a
O
O
S
Me
Me
33.2 0.4
13.4 1.5
19.3 1.4
35.5 2.8
1.69 0.07
7.77 1.5
10.0 1.6
40.5 0.9
>50
N
N
N
N
N
N
N
N
H
O
17a
O
NH2
Me
4.53 0.25
38.1 2.7
1.23 0.02
3.68 0.15
6.95 1.36
61.6 3.2
25–50
S
O
O
O
12
O
O O
S
Tol
>50
Cl
O
21
O
S
S
Me
Me
0.11 0.01
24.8 0.6
2.3 0.1
>200
6.3–12.5
O
18a
O
S
S
Ph
Ph
>50
O
18b
OH
S
S
Ph
Ph
25–50
OH
19
O
O O
S
Tol
Tol
6.3–12.5
S
O
O
O
20
3.3. Anti-inflammatory activity
chloro-functionality typically showed modest anti-inflammatory
activity (entries 4–12), while the amine- and sulfur-containing qui-
nolinequinones showed poor activity (entries 13–23). Here, it
should be noted that the tricyclic quinolinequinones 22 and 23
(entries 18 and 19), the stripped-back analogues of ascidiathiaz-
ones A and B, respectively, showed poor anti-inflammatory activ-
ity. Previous AI studies on other ascidiathiazone analogues
revealed that the addition of a polar-group to the pyridine ring
greatly increases AI activity, and it was also suggested that the oxi-
dation state and regiochemistry of the thiazine ring played a criti-
cal role in AI activity.5 Our results support this statement.
When considering the anti-inflammatory activities of the 6,7-
substituted-5,8-quinolinequinones, it is interesting to note that
there was an inverse correlation between anti-inflammatory activ-
ity and anti-proliferative activity. Of the quinolinequinones tested,
two of the more promising anti-inflammatory candidates were the
dichloro-derivatives 7a and 7b, which, with AI activities of 1.3
lM
and 3.4 M, respectively, exhibited greater than 10-fold selectivity
l
for inhibition of superoxide production when compared to their
anti-proliferative T-cell activities (entries 1 and 2). With the
exception of the poorly inhibitory chloro-amine quinolinequinone
11 (entry 3), the quinolinequinones containing an amine- and
As a class of compounds, the di-thiol containing quinolinequi-
nones showed better AI activity. The di-thiomethyl derivative 18a

B. J. Mulchin et al. / Bioorg. Med. Chem. 18 (2010) 3238–3251
3245
(AI = 0.11 lM), exhibited 10-fold greater activity in the AI assay
4. Conclusion
compared to its anti-proliferative activity (entry 25). Interestingly,
the di-thiophenyl derivative 18b exhibited modest AI activity (ca.
A variety of 6,7-substituted 5,8-quinolinequinolines have been
synthesised and several lead candidates identified as potential
anti-cancer, anti-inflammatory or tuberculostatic agents. In partic-
ular, the introduction of a sulfur group at the 7-position of the qui-
nolinequinone led to the discovery of a number of biologically
active compounds that exhibit selectivity for leukemic cells over
T-cells, a highly desirable property for an anti-cancer drug. Several
lead anti-inflammatory and tuberculostatic quinolinequinones
have also been identified. When determining the biological speci-
ficity of the quinolinequinones, some broad functional group pat-
terns appear to be important. Typically, a thiol and amine-
functionalised quinolinequinone leads to better anti-proliferative
activity, while a di-chloro or di-thiol-substituted quinolinequinone
gives better anti-inflammatory activity. In the case of tuberculo-
static activity, the functional-group distinctions are less clear and
the observed activities may better reflect the differences in the qui-
nolinequinones bioavailability and the inherent difficulties of
drugs penetrating the thick mycobacterial cell wall. We are cur-
rently further investigating the biological profiles and modes of ac-
tion of our lead candidates.
25
l
M, entry 26), however in its reduced form, diol 19, showed
M, entry 27). Finally,
remarkably enhanced AI activity (ca. 2.3
l
the disulfone 20 (entry 28) exhibited particularly poor AI activity.
Taken as a whole, these results illustrate that quinolinequinones
containing an amine-functionality at the 6- or 7-position show
poor AI activity compared to di-chloro or di-thio quinolinequinon-
es. Based on our results dichloro 7a, methyl-dichloro 7b, di-thio-
methyl 18a and diol 19 have been identified as lead AI drug
candidates.
3.4. Tuberculostatic activity
On the whole, most of the quinolinequinones tested exhib-
ited good to modest tuberculostatic activity with typical MIC’s
ranging from approximately
3 to 50 lg/mL. Ethambutol, a
front-line drug used for the treatment of tuberculosis, served
as a positive control and exhibited an MIC = 3.1–6.3
our assay.
lg/mL in
When considering the tuberculostatic activity of the quinolin-
equinones, it was more difficult to correlate specific structural fea-
tures with biological activity. Generally, the quinolinequinones
containing a halogen at the 6- and/or 7-position exhibited slightly
better tuberculostatic activity than derivatives containing a thiol
group at the 6- or 7-position (compare entries 1–12 with 13–19);
however the precise levels of activity depended on the substitution
pattern. Previous studies have revealed that chloro-ethylamino-
quinolinequinones, such as 8c (entry 9), and the subsequent tricy-
clic compound 22 (entry 18) exhibited promise as TB-drugs.44
Though our halo-ethylamines 8d–e (entries 7, 11) and 9c–e (en-
tries 8, 10, 12) showed good activities, as did the tricyclic deriva-
tives 22 and 23 (entries 18, 19), these were not the most
promising compounds identified in our assay. Of particular note
was the tuberculostatic activity of the primary amine 11 (entry
5. Experimental
5.1. Chemistry
5.1.1. General methods
Unless otherwise stated all reactions were performed under
atmospheric air. THF (Lab-Scan) was distilled from LiAlH4. MeCN
(Panreac) was distilled from calcium hydride, and EtOAc (Pure Sci-
ence) was also distilled. EtOH (absolute, Pure Science), hexanes
(Pure Science), MeOH (Pure Science), AcOH (Aiax Finechem),
CH2Cl2 (LabServ), 30% aqueous NH3 (J. T. Baker Chemical Co.),
Et2O (Merk), TFA (Aldrich), sodium chlorate (BDH), Sodium chlo-
rate (BDH), 8-hydroxyquinoline (BDH), concd HCl (Univar), 2-
methyl-8-quinolinol (Aldrich), NiCl2ꢀ6H2O (Riedel de Häen),
methylamine (Riedel de Häen), NaHCO3 (Pure Science), MgSO4
(Pure Science), pyridine (Roth), b-chloro-ethylamino hydrochloride
(Aldrich), b-bromo-ethylamino hydrochloride (BDH), sodium thio-
methoxide (Acros), Toluene (Fischer Scientific), thiophenol (Jans-
sen Chemicals), m-CPBA (Janssen Chemicals), KMnO4 (AnalaR), p-
toluenesulfinic acid sodium salt (Aldrich), sodium sulfide (Univar),
diphenylmethylamine (Aldrich), AcCl (B&M), NaOH (Pure Science),
and NH4OAc (AnalaR) were used as received. All solvents were re-
moved by evaporation under reduced pressure. Reactions were
monitored by TLC-analysis on ALUGRAM SIL G/UV254 TLC plates
with either visual detection, or detection by UV-absorption
(254 nm). Column chromatography was performed using Pure Sci-
3). With an MIC = 1.56–3.13 lg/mL, amine 11 was one of the more
promising TB drug candidates identified in our assay. The quinolin-
equinones containing two thiol functionalities also exhibited
mixed tuberculostatic activity with the di-thiomethyl derivative
18a showing better activity than the di-thiophenyl derivative
18b (entry 27 cf. entry 28).
The tuberculostatic activities of the quinolinequinones contain-
ing a sulfoxide or sulfone functionality were again mixed. A quinol-
inequinone containing
exhibited good tuberculostatic activity (MIC = 1.56–3.13
however its free amine sulfone counterpart 12 exhibited poor
activity (MIC = 25–50 M, entry 23), thus preventing the correla-
a
free amine, sulfone 14 (entry 20)
lg/mL),
l
tion between the presence of a free amine and tuberculostatic
ence silica gel (40–63 lm) as the stationary phase and the solvent
activity to be made. [This lack of correlation is further illustrated
systems indicated. High-resolution mass spectra were recorded on
a Waters Q-TOF Premier™ Tandem Mass Spectrometer using posi-
tive electro-spray ionisation. Infrared spectra were recorded as
thin films using a Bruker Tensor 27 FTIR spectrometer equipped
with an Attenuated Total Reflectance (ATR) sampling accessory,
and are reported in wave numbers (cmꢁ1). Melting points (Mp)
were obtained on a Gallenkamp Melting Point Apparatus and UV
data obtained on an Agilent 8453 spectrometer. Nuclear magnetic
resonance (NMR) spectra were acquired at 20 °C in CDCl3, D2O or
DMSO-d6 as indicated using a Varian Unity-INOVA 500 MHz spec-
trometer, where 1H and 13C were measured at 500 and 125 MHz,
respectively. 1H NMR spectral data are presented as illustrated:
chemical shift (d) in ppm, multiplicity [s (singlet), d (doublet), t
(triplet), m (multiplet over the range specified)], number of pro-
tons, (nH), coupling constants (J) in hertz, assignment of protons).
when the activity of free amine 11 (MIC = 1.56–3.13 lg/mL, entry
3) is compared with the activity of the free amine thiol analogue
15 (MIC = 6.3–12.5, entry 13)]. Of special note, however, is the
tuberculostatic activity of the di-sulfone 20 (entry 28). Though
the mono-sulfones tested exhibited only poor tuberculostatic
activity (entries 23 and 24), di-sulfone 20 showed markedly better
activity and, more importantly, had tuberculostatic activity that
was approximately fivefold greater than its anti-proliferative
activity.
In summary, three lead quinolinequinone TB-drug candidates,
amine 11, sulfoxide 14, and di-sulfone 20 were identified. Though
the di-sulfone 20 had a lower MIC when compared to the other two
quinolinequinones (6.3–12 lg/mL vs 1.56–3.13 lg/mL), it exhib-
ited only modest anti-proliferative activity and therefore may also
be a suitable TB-drug.

3246
B. J. Mulchin et al. / Bioorg. Med. Chem. 18 (2010) 3238–3251
1H NMR spectra are referenced to the CHCl3 (d 7.26 ppm), H2O (d
4.79 ppm) or DMSO (d 2.50 ppm) residual solvent peaks. 13C
NMR spectra were proton decoupled and referenced to TMS (d
0 ppm). NMR peak assignments were made using COSY, HSQC,
and HMBC experiments.
J2,4 = 1.7 Hz, H-4), 7.58 (dd, 1H, J3,4 = 7.8, J2,3 = 4.6 Hz, H-3) 6.09 (s,
1H, N–H), 3.48 (d, 3H, JNH,N-Me = 5.6 Hz, N-Me); 13C NMR
(125 MHz, CDCl3): d 180.1 (C-5), 175.3 (C-8), 155.3 (C-2), 148.5
(C-8a), 144.4 (C-6), 134.6 (C4), 126.7 (C-4a), 126.5 (C-3), 112.2
(C-7), 32.7 (CH3); HRMS(ESI) m/z calcd for [C10H7N2O2Cl+H]+:
223.0274, obsd.: 223.0274.
5.1.2. Procedures for the synthesis of the quinolinequinones
5.1.2.1. 6,7-Dichloro-5,8-quinolinequinone (7a)
.
Sodium chlo-
5.1.2.4. 6-(Benzhydryl-amino)-7-chloro-5,8-quinolinequinone
(8b). A solution of 6,7-dichloro-5,8-quinolinequinone (7a) (0.20 g,
0.87 mmol) and NiCl2ꢀ6H2O (0.21 g, 0.95 mmol, 1.1 equiv) in EtOH
(10 mL) was stirred for 45 min at rt. Diphenylmethylamine
(0.18 mL, 0.95 mmol, 1.1 equiv) was then added and after stirring
at rt for 1 h, the EtOH was removed under reduced pressure, the
reaction mixture was dissolved in CH2Cl2, washed with satd aq
NaHCO3, followed by brine, then dried (MgSO4). Filtration, concen-
tration in vacuo and purification by column chromatography
(CH2Cl2/EtOAc, 1/0?5/1, v/v) yielded a bright orange solid that
was crystallised from CH2Cl2/hexanes to give quinolinequinone
8b as a bright orange micro-crystalline powder (0.31 g, 0.83 mmol,
96%). Rf = 0.48 (EtOAc); Mp 172.2–173.5 °C; IR (thin film): 3335,
3061, 1676, 1598, 1568, 1508, 1454, 1309, 1266, 1203, 1142,
rate (53 g, 0.50 mol, 5 equiv) was added over a period of 1 h to a
solution of 8-hydroxyquinoline (14.5 g, 0.10 mol) in concd HCl
(600 mL) at 40 °C and the reaction mixture stirred for 2 h before
being diluted with water to a total volume of 2 L. The white precip-
itate that formed was removed by filtration and discarded. The fil-
trate was then extracted with CH2Cl2 (6 ꢂ 250 mL), the organic
phases were combined, washed with water and concentrated in
vacuo to give a yellow solid. The solid was then recrystallised in
MeOH to yield 7a as bright yellow crystals (6.67 g, 29 mmol,
29%). Rf = 0.52 (EtOAc); Mp 219.4–221.0 °C (Lit.12 221–223 °C); IR
(thin film): 2989, 2915, 2831, 1691, 1675, 1572, 1557, 1273,
1194, 1136, 1100, 1020, 949, 895, 828, 724, 694 cmꢁ1; UV–vis
(MeOH) kmax (log e
): 201 (4.23), 240 (4.33), 272 (4.24) nm; 1H
NMR (500 MHz, CDCl3): d 9.11 (dd, 1H, J2,3 = 4.1, J2,4 = 0.9 Hz, H-
2), 8.54 (dd, 1H, J3,4 = 8.0, J2,4 = 1.2 Hz, H-4), 7.77 (dd, 1H,
J3,4 = 8.1, J2,3 = 4.9 Hz, H-3); 13C NMR (125 MHz, CDCl3): d 175.6
(C-5), 174.3 (C-8), 155.4 (C-2), 146.8 (C-8a), 144.4 (C-6 or C-7),
143.1 (C-6 or C-7), 135.6 (C-4), 128.3 (C-4a), 128.3 (C-3); HRMS(E-
SI) m/z calcd for [C9H3NO2Cl2+Na]+: 249.9439, obsd.: 249.9445.
1070, 701 cmꢁ1; UV–vis (MeOH) kmax (log
e): 201 (6.47), 231
(6.31), 271 (6.19) nm; 1H NMR (500 MHz, CDCl3): d 9.00 (dd, 1H,
J2,3 = 4.7, J2,4 = 1.7 Hz, H-2), 8.32 (dd, 1H, J3,4 = 7.9, J2,4 = 1.7 Hz, H-
4), 7.56 (dd, 1H, J3,4 = 7.9, J2,3 = 4.7 Hz, H-3) 7. 24–7.42 (m, 10H,
CH arom.), 6.92 (d, 1H, JNH,CHPh2 = 8.5 Hz, CH), 6.45 (s, 1H, NH);
13C NMR (125 MHz, CDCl3): d 179.9 (C-5), 175.3 (C-8), 155.2 (C-
2), 148.2 (C-8a), 143.0 (C-4a), 141.6 (C-6), 134.8 (C-4) 129.1 (CH-
o arom.), 128.1 (CH-p arom.), 127.2 (CH-m arom.), 126.9 (C-ipso
arom.), 126.7 (C-3), 114.3 (C-7), 61.8 (CHPh2); HRMS(ESI) m/z calcd
for [C22H15N2O2Cl+Na]+: 397.0720, obsd.: 397.0644.
5.1.2.2. 6,7-Dichloro-2-methyl-5,8-quinolinequinone (7b). So-
dium chlorate (16.5 g, 155 mmol, 5 equiv) was added over a period
of 1 h to a solution of 8-hydroxy-2-methyl-quinoline (4.93 g,
31.0 mmol) in concd HCl (200 mL) at 40 °C and the reaction mix-
ture stirred for 2 h before being diluted to 1 L with water. The
white precipitate that formed was removed by filtration and dis-
carded and the filtrate extracted with CH2Cl2 (6 ꢂ 100 mL). The or-
ganic phases were combined and washed with water, then
concentrated in vacuo to give a yellow solid that was set to crystal-
lise in the minimum volume of boiling MeOH to yield dichloride 7b
as bright yellow crystals (2.05 g, 8.4 mmol, 27%). Rf = 0.20 (EtOAc);
Mp 179.1–181.3 °C (Lit.12 Mp 180–181 °C); IR (film): 1699, 1691,
1676, 1593, 1569, 1466, 1290, 1260, 1208, 1146, 1084, 930, 864,
5.1.2.5. 7-Chloro-6-(2-chloro-ethylamino)-5,8-quinolinequinone
(8c). To
a solution of 6,7-dichloro-5,8-quinolinequinone (7a)
(0.11 g, 0.48 mmol, 1 equiv) in EtOH/H2O (7.5 mL, 2:1, v/v) was
added NiCl2ꢀ6H2O (0.13 g, 0.55 mmol, 1.15 equiv) and the solution
stirred for 45 min at rt. 2-Chloro-ethylamine hydrochloride
(0.075 g, 0.65 mmol, 1.35 equiv) and 2 m NaOH (0.5 mL, 1.0 mmol,
2.1 equiv) were then added and the solution refluxed for 15 min.
The solvents were removed under reduced pressure and the resi-
due taken up in CH2Cl2, washed with satd aq NaHCO3 and brine,
dried (MgSO4), filtered and concentrated in vacuo. Purification of
the residue by column chromatography (CH2Cl2/EtOAc, 35/1?1/
2, v/v) gave quinolinequinone 8c (0.12 g, 0.39 mmol, 82%) as dark
red crystals. Rf = 0.23 (EtOAc); Mp 165.7–166.6 °C (Lit. Mp 151 °C,
Ref. 29); IR (film): 1687, 1657, 1595, 1570, 1514, 1452, 1440,
828, 727, 640 cmꢁ1; UV–vis (MeOH) kmax (log
e): 280 (4.10), 245
(4.17), 195 (3.82) nm; 1H NMR (500 MHz, CDCl3): d 8.37 (d,
J3,4 = 8.0 Hz, 1H, H-4), 7.59 (d, J3,4 = 8.0 Hz, 1H, H-3), 2.78 (s, 3H,
CH3); 13C NMR (125 MHz, CDCl3): 175.6 (C5), 174.5 (C8), 166.1
(C2), 146.3 (C8a), 143.9 (C7), 142.8 (C6), 135.6 (C4), 128.3 (C3),
126.1 (C4a), 25.3 (CH3); HRMS(ESI) m/z calcd for [C10H5NO2Cl2+-
Na]+: 263.9595, obsd.: 263.9600.
1360, 1329, 1304, 1257, 1215, 1191, 1111, 1075, 822, 679 cmꢁ1
UV–vis (MeOH) kmax (log ): 470 (3.53), 271 (4.11), 232 (4.24)
nm; 1H NMR (500 MHz, CDCl3)
8.99 (dd, J2,4 = 1.7 Hz,
;
e
d
5.1.2.3. 7-Chloro-6-methylamino-5,8-quinolinequinone (8a).
A
J2,3 = 4.7 Hz, 1H, H-2), 8.35 (dd, J2,4 = 1.7 Hz, J3,4 = 7.9 Hz, 1H, H-4),
solution of 6,7-dichloro-5,8-quinolinequinone (7a) (1.37 g,
5.99 mmol) and NiCl2ꢀ6H2O (1.57 g, 6.59 mmol, 1.1 equiv) in EtOH
(25 mL) was stirred for 45 min at rt. Methylamine (40%, 0.62 mL,
7.19 mmol, 1.2 equiv) was then added and after stirring at rt for
1 h, the EtOH was removed under reduced pressure, the residue
was dissolved in CH2Cl2, washed with satd aq NaHCO3, followed
by brine, then dried (MgSO4). Filtration, concentration in vacuo
and purification by column chromatography (CH2Cl2/EtOAc, 3/
1?1/1, v/v) yielded a red solid which was crystallised from a min-
imal amount of boiling CH2Cl2/hexanes (1/4, v/v) to give quinone
8a as deep brick-red crystals (1.26 g, 5.7 mmol, 95%). Rf = 0.23
(EtOAc); Mp 219.0–219.2 °C (Lit.45 Mp 194–195 °C); IR (thin film):
3347, 2926, 2856, 1678, 1596, 1561, 1507, 1424, 1308, 1217, 1131,
7.59 (dd, J2,3 = 4.7 Hz, J3,4 = 7.9 Hz, 1H, H-3), 6.34 (br s, 1H, N–H),
4.21 (m, 2H, H-20), 3.78 (t, JH-2 ,H-3 = 5.8 Hz, 2H, H-30); 13C NMR
(125 MHz, CDCl3) 179.7 (C5), 175.5 (C8), 155.3 (C2), 148.1 (C8a),
143.4 (C6), 134.8 (C4), 126.8 (C4a), 126.8 (C3), 113.4 (C7), 46.0
(C20), 44.1 (C30); HRMS(ESI) m/z calcd for [C11H8N2O2Cl2+Na]+:
292.9861, obsd.: 292.9865.
0
0
5.1.2.6. 6-(2-Bromo-ethylamino)-7-chloro-5,8-quinolinequinone
(8d) and 7-(2-bromo-ethylamino)-6-chloro-5,8-quinolinequi-
none (9d). 2-Bromo-ethylamine hydrochloride (0.33 g, 2.9 mmol,
1.5 equiv) and 2 m NaOH (1.7 mL, 3.5 mmol, 1.8 equiv) were added
to a solution of the 6,7-dichloro-5,8-quinolinequinone (7a) (0.43 g,
1.9 mmol, 1 equiv) in EtOH (35 mL) and H2O (15 mL) and the solu-
tion refluxed for 15 min. The solvents were then removed under
reduced pressure and the residue dissolved in CH2Cl2, washed with
satd aq NaHCO3 and brine, dried (MgSO4), filtered and concen-
912, 737, 682 cmꢁ1; UV–vis (MeOH) kmax (log
e): 200 (4.08), 232
(4.16), 270 (4.01), 470 (3.42) nm; 1H NMR (500 MHz, CDCl3): d
9.02 (dd, 1H, J2,3 = 4.6, J2,4 = 1.7 Hz, H-2), 8.35 (dd, 1H, J3,4 = 7.8,

B. J. Mulchin et al. / Bioorg. Med. Chem. 18 (2010) 3238–3251
3247
trated. Purification of the crude product by column chromatogra-
phy (CH2Cl2/EtOAc, 3/1?0/1, v/v) yielded the 6-substituted isomer
8d as dark red crystals (0.36 g, 1.03 mmol, 53%) and the 7-substi-
tuted isomer 9d as orange crystals (0.17 g, 0.48 mmol, 25%). Data
for 6-substituted isomer 8d: Rf = 0.24 (EtOAc); Mp 166.0–
167.3 °C; IR (film): 1679, 1592, 1548, 1445, 1324, 1264, 1219,
(10 mL). The reaction mixture was left to stir for 4 h, until TLC-anal-
ysis showed the complete consumption of starting material and the
presence of two products. The solvent was removed under reduced
pressure and the residue taken up in CH2Cl2, washed with satd aq
NaHCO3 and brine, dried (MgSO4), filtered and concentrated in va-
cuo. Purification of the crude product by column chromatography
(CH2Cl2/EtOAc, 1/0?4/1, v/v) gave the title compound 9a (0.30 g,
1.27 mmol, 67%) as a mauve solid, together with small amounts of
its structural isomer 8a (0.090 g, 0.38 mmol, 20%) as a red solid. Both
were crystallised from a minimum amount of boiling solvent
(CH2Cl2/hexanes, 1/4, v/v). Data for 6-substituted isomer 9a:
Rf = 0.32 (EtOAc); Mp 220.3–221.9 °C; IR (thin film): 3306, 1690,
1147, 1082, 854, 681, 645 cmꢁ1; UV–vis (MeOH) kmax (log
e): 464
(3.69), 271 (4.22), 232 (4.34) nm; 1H NMR (500 MHz, CDCl3) d
9.03 (dd, J2,4 = 1.7 Hz, J2,3 = 4.7 Hz, 1H, H-2), 8.38 (dd, J2,4 = 1.7 Hz,
J3,4 = 7.9 Hz, 1H, H-4), 7.62 (dd, J2,3 = 4.7 Hz, J3,4 = 7.9 Hz, 1H, H-3),
6.35 (br s, 1H, N–H), 4.28 (m, 2H, H-20), 3.64 (m, 2H, H-30); 13C
NMR (125 MHz, CDCl3) 179.7 (C5), 175.2 (C8), 155.4 (C2), 148.1
(C8a), 143.3 (C6), 134.8 (C4), 126.8 (C4a), 126.8 (C3), 113.5 (C7),
45.8 (C20), 32.7 (C30); HRMS(ESI) m/z calcd for [C11H8N2O2ClBr+-
Na]+: 336.9355, obsd.: 336.9356. Data for 7-substituted isomer
9d: Rf = 0.36 (EtOAc); Mp 143.0 °C (decomp); IR (film): 1687,
1649, 1604, 1565, 1516, 1437, 1355, 1301, 1245, 1197, 1138,
1594, 1562, 1508, 1416, 1327, 1305, 1206, 1097, 907, 724 cmꢁ1
UV–vis (MeOH) kmax (log ): 201 (3.78), 236 (3.84), 273 (3.78) 480
;
e
(3.17) nm; 1H NMR (500 MHz, CDCl3): d 8.92 (dd, 1H, J2,3 = 4.6,
J2,4 = 1.7 Hz, H-2), 8.47 (dd, 1H, J3,4 = 8.0, J2,4 = 0.8 Hz, H-4), 7.66
(dd, 1H, J3,4 = 8.0, J2,3 = 4.6 Hz, H-3), 6.27 (s, 1H, N–H), 3.40 (d, 3H,
JNH,N-Me = 5.8, N–CH3); 13C NMR (125 MHz, CDCl3): d 178.9 (C-5),
175.7 (C-8), 153.4 (C-2), 145.9 (C-8a), 145.3 (C-7), 134.7 (C-4),
130.0 (C-4a), 128.5 (C-3), 107.6 (C-6), 32.7 (CH3); HRMS(ESI) m/z
calcd for [C10H7ClN2O2+Na]+: 245.0094, obsd.: 245.0094.
1096, 850, 724, 544 cmꢁ1; UV–vis (MeOH) kmax (log
e): 465
(3.15), 288 (3.70) nm; 1H NMR (500 MHz, CDCl3) d 8.94 (dd,
J2,4 = 1.7 Hz, J2,3 = 4.7 Hz, 1H, H-2), 8.47 (dd, J2,4 = 1.7 Hz,
J3,4 = 7.9 Hz, 1H, H-4), 7.67 (dd, J2,3 = 4.7 Hz, 3,4 = 7.9 Hz, 1H, H-3),
6.46 (br s, 1H, N–H), 4.26 (m, 2H, H-20), 3.63 (t, JH-2 ,H-3 = 5.8 Hz,
2H, H-30); 13C NMR (125 MHz, CDCl3) 178.5 (C8), 175.7 (C5),
153.7 (C2), 146.0 (C8a), 144.2 (C7), 134.8 (C4), 129.6 (C4a), 128.5
(C3), 111.8 (C6), 45.9 (C20), 32.0 (C30); HRMS(ESI) m/z calcd for
[C11H8N2O2ClBr+Na]+: 336.9355, obsd.: 336.9359.
0
0
5.1.2.9. 6-Chloro-7-(2-chloro-ethylamino)-5,8-quinolinequinone
(9c). 2-Chloro-ethylamine hydrochloride (0.82 g, 7.1 mmol,
1.5 equiv) and 2 m NaOH (4.3 mL, 8.5 mmol, 1.8 equiv) were added
to a solution of the 6,7-dichloro-5,8-quinolinequinone (7a) (1.06 g,
4.7 mmol, 1 equiv) in EtOH (45 mL) and H2O (22.5 mL) and the
solution refluxed for 15 min. The solvents were then removed un-
der reduced pressure and the residue dissolved in CH2Cl2, washed
with sat aq NaHCO3 and brine, dried (MgSO4), filtered and concen-
trated. Purification of the crude product by column chromatography
(CH2Cl2/EtOAc, 3/1?0/1, v/v) yielded the 7-substituted isomer 9c as
orange crystals (0.68 g, 2.2 mmol, 48%) and the 6-substituted isomer
8cas dark redcrystals(0.65 g, 2.1 mmol, 46%). Datafor 7-substituted
isomer 9c: Rf = 0.36 (EtOAc); Mp 148.6–150.0 °C; IR (film): 1687,
1649, 1605, 1566, 1518, 1438, 1302, 1251, 1205, 1145, 1097, 859,
5.1.2.7. 6-(2-Bromo-ethylamino)-7-chloro-2-methyl-5,8-quinol-
inequinone (8e) and 7-(2-Bromo-ethylamino)-6-chloro-2-me-
thyl-5,8-quinolinequinone (9e). 2-Bromo-ethylamine hydro-
chloride (3.6 g, 33 mmol, 1.5 equiv) and 2 m NaOH (20 mL,
40 mmol, 1.8 equiv) were added to a solution of the 6,7-dichloro-
2-methyl-5,8-quinolinequinone (7b) (4.7 g, 22 mmol, 1 equiv) in
EtOH (400 mL) and H2O (200 mL) and the solution refluxed for
15 min. The solvents were then removed under reduced pressure
and the residue dissolved in CH2Cl2, washed with satd aq NaHCO3
and brine, dried (MgSO4), filtered and concentrated. Purification of
the crude product by column chromatography (CH2Cl2/EtOAc, 3/
1?0/1, v/v) yielded the 6-substituted isomer 8e as dark red crys-
tals (3.6 g, 9.7 mmol, 44%) and the 7-substituted isomer 9e as or-
ange crystals (1.73 g, 4.71 mmol, 21%). Data for 6-substituted
isomer 8e: Rf = 0.31 (EtOAc); Mp 168.4–169.3 °C; IR (film): 1676,
1604, 1571, 1515, 1449, 1404, 1357, 1311, 1230, 1143, 1116,
813, 723, 547 cmꢁ1; UV–vis (MeOH) kmax (log
e): 462 (3.32), 273
(3.97), 235 (4.00) nm; 1H NMR (500 MHz, CDCl3) d 8.93 (dd,
J2,4 = 1.7 Hz, J2,3 = 4.7 Hz, 1H, H-2), 8.46 (dd, J2,4 = 1.7 Hz,
J3,4 = 7.9 Hz, 1H, H-4), 7.67 (dd, J2,3 = 4.7 Hz, J3,4 = 7.9 Hz, 1H, H-3),
6.44 (br s, 1H, N–H), 4.23 (m, 2H, H-20), 3.79 (t, JH-2 ,H-3 = 5.8 Hz,
2H, H-30); 13C NMR (125 MHz, CDCl3) 178.6 (C8), 175.7 (C5), 153.7
(C2), 146.0 (C8a), 144.3 (C7), 134.7 (C4), 129.6 (C4a), 128.5 (C3),
111.8 (C6), 46.1 (C20), 44.0 (C30); HRMS(ESI) m/z calcd for
[C11H8N2O2Cl2+Na]+: 292.9861, obsd.: 292.9866.
0
0
930, 859, 731 cmꢁ1; UV–vis (MeOH) kmax (log
e): 486 (3.50), 280
(4.26), 238 (4.40) nm; 1H NMR (500 MHz, CDCl3) d 8.21 (d,
J3,4 = 8.0 Hz, 1H, H-4), 7.42 (d, J3,4 = 8.0 Hz, 1H, H-3), 6.32 (br s,
1H, N–H), 4.24 (m, 2H, H-20), 3.61 (m, 2H, H-30), 2.73 (s, 3H, H-
Me); 13C NMR (125 MHz, CDCl3) 179.7 (C5), 175.5 (C8), 166.0
(C2), 147.7 (C8a), 143.1 (C6), 134.9 (C4), 126.7 (C3), 124.6 (C4a),
112.9 (C7), 45.8 (C2’), 32.2 (C30), 25.4 (Me); HRMS(ESI) m/z calcd
for [C12H10N2O2ClBr+Na]+: 350.9512, obsd.: 350.9510. Data for 7-
substituted isomer 9e: Rf = 0.44 (EtOAc); Mp 211.3–211.7 °C; IR
(film): 3313, 1693, 1605, 1583, 1555, 1513, 1445, 1330, 1300,
5.1.2.10. 6-Methylamino-7-methylsulfanyl-quinolinequinone
(10a). To a solution of the 7-chloro-6-methylamino-quinoline
(8a) (0.353 g, 1.59 mmol, 1 equiv) in EtOH (15 mL) was added so-
dium thiomethoxide (0.177 g, 2.54 mmol, 1.6 equiv) and the reac-
tion stirred for 90 min at rt. The solvent was then removed under
reduced pressure and the mixture dissolved in CH2Cl2, washed
with satd aq NaHCO3 followed by brine, then dried over MgSO4 be-
fore being filtered and concentrated in vacuo. Purification by flash
chromatography (CH2Cl2/ EtOAc, 10/1?0/1, v/v) gave the desired
quinone 10a (0.33 g, 2.24 mmol, 88%) as a red-brown solid, which
yielded reddish-purple crystals after recrystallisation from toluene.
Rf = 0.14 (EtOAc); IR (film): 3688, 3167, 2921, 2852, 1684, 1563,
1262, 1147, 729 cmꢁ1; UV–vis (MeOH) kmax (log
e): 485 (3.53),
301 (4.00), 274 (4.07), 236 (4.16) nm; 1H NMR (500 MHz, CDCl3)
d 8.34 (d, J3,4 = 8.0 Hz, 1H, H-4), 7.51 (d, J3,4 = 8.0 Hz, 1H, H-3),
6.39 (br s, 1H, N–H), 4.25 (m, 2H, H-20), 3.62 (m, 2H, H-30); 13C
NMR (125 MHz, CDCl3) 178.8 (C8), 176.0 (C5), 163.9 (C2), 145.4
(C8a), 144.0 (C7), 134.9 (C4), 128.5 (C3), 127.4 (C4a), 111.6 (C6),
45.9 (C20), 32.0 (C30), 25.0 (Me); HRMS(ESI) m/z calcd for
[C12H10N2O2ClBr+Na]+: 350.9512, obsd.: 350.9516.
1512, 1413, 1299, 1211, 1116 cmꢁ1 1H NMR (500 MHz, CDCl3) d
;
9.00 (dd, J2,4 = 1.8 Hz, J2,3 = 4.6 Hz, 1H, H-2), 8.33 (dd, J2,4 = 1.8 Hz,
J3,4 = 7.8 Hz, 1H, H-4), 7.56 (dd, J2,3 = 4.6 Hz, J3,4 = 7.8 Hz, 1H, H-3),
6.52 (br s, 1H, N–H), 3.49 (d, J9,N–H = 5.9 Hz, 3H, H-9), 2.37 (s, 3H,
H-10); 13C NMR (125 MHz, CDCl3) 181.5 (C5), 178.5 (C8), 155.4
(C2), 150.2 (C6), 149.6 (C8a), 134.7 (C4), 127.6 (C4a), 126.6 (C3),
112.4 (C7), 34.0 (NHMe), 19.0 (SMe); HRMS(ESI) m/z calcd for
[C11H10N2O2S+Na]+: 257.0361, obsd.: 257.0364.
5.1.2.8.
(9a)46
6-Chloro-7-methylamino-5,8-quinolinequinone
Methylamine (40% aq, 0.072 mL, 2.33 mmol, 1.2 equiv)
.
and pyridine(0.33 mL, 4.0 mmol, 2.1 equiv)were added toa solution
of dichloro-quinolinequinone 7a (0.44 g, 1.9 mmol) in 1,4-dioxane

3248
B. J. Mulchin et al. / Bioorg. Med. Chem. 18 (2010) 3238–3251
5.1.2.11. 6-Methylamino-7-phenylsulfanyl-5,8-quinolinequinone
(10b). Methylamino-quinolinequinone (8a) (0.030 g, 0.13 mmol)
was dissolved in pyridine (12 mL) and thiophenol (0.016 mL,
0.16 mmol, 1.2 equiv) was added. After the reaction mixture was stir-
red for 16 h, the pyridine was removed under reduced pressure and
the residue dissolved in CH2Cl2, washed with satd aq NaHCO3 and
brine, dried (MgSO4), filtered and concentrated in vacuo. Purification
bycolumnchromatography(hexanes/CH2Cl2, 10/1?0/1,v/vfollowed
by CH2Cl2/EtOAc, 10/1?0/1) yielded a purple-red solid which was
crystallised in the minimum volume of boiling solvent (CH2Cl2/hex-
anes, 1/3, v/v) to give 10b as purple-red crystals (0.037 g, 0.12 mmol,
95%). Rf = 0.19(EtOAc);Mp176.4–177.5 °C;IR(thin film):3263, 1740,
1682, 1558, 1306, 1273, 1212, 733, 688 cmꢁ1; UV–vis (MeOH) kmax
H-4), 7.73 (dd, 1H, J3,4 = 7.9, J2,3 = 4.7 Hz, H-3) 7.65 (m, 2H, NH2);
13C NMR (125 MHz, DMSO-d6): d 179.6 (C-5), 174.3 (C-8), 154.8
(C-2), 148.7 (C-8a), 147.1 (C-6), 134.6 (C-4), 127.6 (C-4a), 127.3
(C-3), 110.8 (C-7); HRMS(ESI) m/z calcd for [C9H5N2O2Cl+Na]+:
230.9932, obsd.: 230.9937.
5.1.2.15. 6-Amino-7-methylsulfonyl-5,8-quinolinequinone
(12). To a solution of 6-methylamino-7-methylsulfanyl-5,8-qui-
nolinequinone (10a) (18 mg, 0.077 mmol) in CH2Cl2 (1 mL) was
slowly added a solution of m-CPBA (40 mg, 0.16 mmol, 2.1 equiv)
in CH2Cl2 (1 mL). After 30 min, the reaction mixture was quenched
with satd aq NaHCO3 and extracted with CH2Cl2. The combined or-
ganic layers were dried (MgSO4), filtered, and concentrated. The
residue was dissolved in MeOH (1 mL), and 30% aq NH3 (0.2 mL)
was added. The resulting solution was stirred for 1 h, concentrated
and purified via silica gel column chromatography (CH2Cl2?1%
MeOH in CH2Cl2) to give amine 12 as an orange solid (0.018 g,
0.071 mmol, 93%). Rf = 0.28 (CH2Cl2/MeOH, 9/1, v/v), IR (film):
3301, 1697, 1602, 1575, 1419, 1330, 1288, 1219, 1170, 1064,
(log e
): 202 (4.40), 237 (4.18), 253 (4.19) 454 (3.16) nm; 1H NMR
(500 MHz, CDCl3): d 9.05 (dd, 1H, J2,3 = 4.6, J2,4 = 1.7 Hz, H-2), 8.40
(dd, 1H, J3,4 = 7.8, J2,4 = 1.7 Hz, H-4), 7.60 (dd, 1H, J3,4 = 7.8,
J2,3 = 4.6 Hz, H-3) 7.23 (m, 4H, CH-o and CH-m arom.), 7.11 (m, 1H,
CH-p arom.), 6.69 (s, 1H, NH), 3.44 (d, 3H, JNH,CH3 = 4.4 Hz, CH3); 13
C
NMR (125 MHz, CDCl3): d 181.4 (C-8), 178.4 (C-5), 155.5 (C-2),
150.5 (C-6), 149.1 (C-8a), 134.7 (C-4), 129.1 (CH-o or CH-m arom.),
127.0 (CH-o or CH-m arom.), 126.4 (C-3), 125.7 (CH-p arom.), 33.4
(CH3); HRMS(ESI) m/z calcd for [C16H12N2O2S2+Na]+: 319.0517, obsd.:
319.0517.
; d 9.03 (dd,
1008, 843 cmꢁ1 1H NMR (500 MHz, DMSO-d6)
J2,4 = 1.7 Hz, J2,3 = 4.6 Hz, 1H, H-2), 8.54 (br s, 2H, NH2), 8.39 (dd,
J2,4 = 1.7 Hz, J3,4 = 7.9 Hz, 1H, H-4), 7.79 (dd, J2,3 = 4.6 Hz,
J3,4 = 7.9 Hz, 1H, H-3), 3.31 (s, 3H, SO2Me); 13C NMR (125 MHz,
DMSO-d6) 180.1 (C5), 176.7 (C8), 155.8 (C2), 150.2 (C6), 148.2
(C8a), 134.9 (C4), 127.9 (C4a), 127.7 (C3), 109.8 (C7), 45.0 (SO2Me);
HRMS(ESI) m/z calcd for [C10H9N2O4S]+: 253.0278, obsd.: 253.0283.
5.1.2.12. 7-Methanesulfinyl-6-methylamino-5,8-quinolinequi-
none (13a). To a solution of 6-methylamino-7-methylsulfanyl-
5,8-quinolinedione (10a) (12 mg, 0.054 mmol) in CH2Cl2 (2 mL) at
0 °C was added m-CPBA (13 mg, 0.052 mmol, 1.0 equiv). After stir-
ring at rt for 30 min, the mixture was concentrated in vacuo and
purified by flash chromatography (CH2Cl2/EtOH/MeOH/30% aq
NH3, 300/2/2/1?35/2/2/1, v/v) to give the desired sulfoxide 13a as
orange crystals (9 mg, 0.040 mmol, 74%). 1H NMR (500 MHz, CDCl3)
d 10.16 (br s, 1H, N–H), 9.01 (dd, J2,4 = 1.7 Hz, J2,3 = 4.8 Hz, 1H, H-2),
8.35 (dd, J2,4 = 1.7 Hz, J3,4 = 7.8 Hz, 1H, H-4), 7.61 (dd, J2,3 = 4.8 Hz,
J3,4 = 7.8 Hz, 1H, H-3), 3.39 (d, JMe,N–H = 5.9 Hz, 3H, NMe), 3.02 (s,
3H, SMe); 13C NMR (125 MHz, CDCl3) 181.0 (C8), 176.4 (C5), 155.6
(C2), 152.2 (C7), 148.0 (C8a), 135.2 (C4), 129.4 (C4a), 126.2 (C3),
109.5 (C6), 39.8 (SOMe), 33.6 (NMe); HRMS(ESI) m/z calcd for
[C11H10N2O3S+H]+: 251.0485, obsd.: 251.0488.
5.1.2.16. 6-Amino-7-methanesulfinyl-5,8-quinolinequinone
(14). To a solution of 7-methanesulfinyl-6-methylamino-5,8-qui-
nolinequinone (13a) (12 mg, 0.048 mmol) in MeOH (1 mL) was
added 30% aq NH3 (0.2 mL). The resulting solution was stirred for
1 h, concentrated and purified via silica gel column chromatogra-
phy (CH2Cl2?1% MeOH in CH2Cl2) to give amine 14 as orange crys-
tals (10 mg, 0.042 mmol, 88%). Rf = 0.44 (CH2Cl2/EtOH 13/1, v/v);
Mp 239.1 °C (decomp.); IR (film): 3301, 1697, 1602, 1575, 1419,
1330, 1288, 1219, 1170, 1064, 1008, 843 cmꢁ1; UV–vis (MeOH)
kmax (log e
): 441 (3.15), 267 (3.76), 231 (3.95) nm; 1H NMR
(500 MHz, CDCl3) d 9.13 (br s, 1H, N–H), 9.05 (dd, J2,4 = 1.7 Hz,
J2,3 = 4.7 Hz, 1H, H-2), 8.41 (dd, J2,4 = 1.7 Hz, J3,4 = 7.9 Hz, 1H, H-4),
7.64 (dd, J2,3 = 4.7 Hz, J3,4 = 7.9 Hz, 1H, H-3), 6.17 (br s, 1H, N–H),
3.03 (s, 3H, SMe); HRMS(ESI) m/z calcd for [C10H8N2O3S+Na]+:
259.0153, obsd.: 259.0146.
5.1.2.13. 7-Benzenesulfinyl-6-methylamino-5,8-quinolinequi-
none (13b). To a solution of 6-methylamino-7-phenylsulfanyl-
5,8-quinolinequinone (10b) (0.45 g, 1.66 mmol) in AcOH (10 mL)
was added KMnO4 (0.29 g, 1.83 mmol, 1.1 equiv) and the reaction
stirred at rt for 1.5 h. The reaction was then neutralised with satd
aq NaHCO3, washed with brine, dried with MgSO4 and filtered be-
fore being concentrated in vacuo. Purification by column chroma-
tography (CH2Cl2/EtOAc, 1/0?0/1, v/v) lead to the isolation of
sulfoxide 13b as a bright orange film (0.16 g, 30%). Rf = 0.25
(EtOAc); IR (thin film): 2923, 1688, 1600, 1567, 1443, 1416,
5.1.2.17. 6-Amino-7-methylsulfanyl-5,8-quinolinequinone
(15). To a solution of 6-methylamino-7-methylsulfanyl-5,8-qui-
nolinequinone (10a) (14 mg, 0.060 mmol) in MeOH (1 mL) was
added 30% aq NH3 (0.2 mL). The resulting solution was stirred for
1 h, concentrated and purified via silica gel column chromatogra-
phy (CH2Cl2?1% MeOH in CH2Cl2) to give amine 15 as brown crys-
tals (10 mg, 0.045 mmol, 76%). Rf = 0.32 (CH2Cl2/MeOH, 8/1, v/v);
Mp 222.6–223.6 °C (decomp.); Lit.16 Mp 222 °C); IR (film): 3235,
1686, 1591, 1572, 1531, 1396, 1279, 1263, 1208, 1003, 750,
1393, 1331, 1306, 1274, 1106, 1075, 994, 748, 686 cmꢁ1 1H
;
NMR (500 MHz, CDCl3): d 9.96 (s, 1H, NH), 9.01 (s, 1H, H-2), 8.30
(s, 1H, H-3), 7.86 (s, 1H, CH-p), 7.55 (m, 5H, H3, CH-o and CH-m),
3.38, (d, JNH,CH3 = 5.0 Hz, CH3); HRMS(ESI) m/z calcd for
[C16H12N2O3S+Na]+: 335.0466, obsd.: 335.0463.
685 cmꢁ1; UV–vis (MeOH) kmax (log
e): 486 (3.31), 257 (3.89),
230 (3.99) nm; 1H NMR (500 MHz, CDCl3) d 9.01 (dd, J2,4 = 1.6 Hz,
J2,3 = 4.6 Hz, 1H, H-2), 8.36 (dd, J2,4 = 1.6 Hz, J3,4 = 7.8 Hz, 1H, H-4),
7.58 (dd, J2,3 = 4.6 Hz, J3,4 = 7.8 Hz, 1H, H-3), 5.99 (br s, 2H, N–H),
2.44 (s, 3H, H-9); 13C NMR (125 MHz, DMSO-d6) 180.4 (C5),
177.6 (C8), 154.8 (C2), 151.4 (C6), 149.4 (C8a), 134.4 (C4), 127.8
(C4a), 127.1 (C3), 110.0 (C7), 17.0 (C9); HRMS(ESI) m/z calcd for
[C10H8N2O2S+H]+: 221.0385, obsd.: 221.0390.
5.1.2.14. 6-Amino-7-chloro-5,8-quinolinequinone (11)32
. To a
solution of 6-(benzhydryl-amino)-7-chloro-5,8-quinolinequinone
(8b) (15 mg, 0.040 mmol) in MeOH (1 mL) was added 30% aq
NH3 (0.2 mL). The resulting solution was stirred for 1 h, concen-
trated and purified via silica gel column chromatography
(CH2Cl2?1% MeOH in CH2Cl2) to give amine 11 as a bright orange
solid (7 mg, 0.033 mmol, 84%). Rf = 0.33 (EtOAc); IR (thin film):
3522, 3005, 1707, 1584, 1539, 1421, 1360, 1222, 1093, 1050,
5.1.2.18. 6-Methylsulfanyl-7-methylamino-5,8-quinolinequinone
(16a). To a solution of the 7-chloro-6-methylamino-quinoline
(9a) (1.1 g, 4.8 mmol, 1 equiv) in EtOH (50 mL) was added sodium
thiomethoxide (0.53 g, 7.6 mmol, 1.6 equiv) and the reaction stir-
;
905, 713 cmꢁ1 1H NMR (500 MHz, DMSO-d6): d 8.94 (dd, 1H,
J2,3 = 4.7, J2,4 = 1.5 Hz, H-2), 8.32 (dd, 1H, J3,4 = 7.9, J2,4 = 1.5 Hz,

B. J. Mulchin et al. / Bioorg. Med. Chem. 18 (2010) 3238–3251
3249
red for 90 min at rt. The solvent was then removed under reduced
pressure and the mixture dissolved in CH2Cl2, washed with satd aq
NaHCO3 followed by brine, then dried over MgSO4 before being fil-
tered and concentrated in vacuo. Purification by flash chromatog-
raphy (CH2Cl2/ EtOAc, 10/1?0/1, v/v) gave the desired sulfide
16a (1.05 g, 4.5 mmol, 94%) as a mauve solid, which yielded red-
dish-purple crystals after recrystallisation from toluene. Rf = 0.17
(EtOAc); Mp 170.1–173.8 °C; IR (film): 3674, 3302, 3087, 2923,
2851, 1688, 1591, 1552, 1509, 1413, 1303, 1257, 1096 cmꢁ1; UV–
5.1.2.21. 6,7-Bis-methylsulfanyl-5,8-quinolinequinone (18a). 6,7-
Dichloro-5,8-quinolinequinone (7a) (0.326 g, 1.42 mmol) was dis-
solved in MeCN (5 mL) at 40 °C, and a saturated solution of sodium
thiomethoxide (0.209 g, 2.98 mmol, 2.1 equiv) in water was added
drop-wise. The solution was then refluxed overnight, cooled to rt,
then extracted twice with CH2Cl2, washed with satd aq NaHCO3
then brine and dried over MgSO4 before and being filtered and con-
centrated in vacuo. Purification by column chromatography
(CH2Cl2/EtOAc, 1/0?0/1, v/v) yielded a brown-red solid that was
recrystallised (CH2Cl2/hexanes, 1/3, v/v) to give disulfide 18a as
dark red crystals (0.32 g, 1.28 mmol, 90%). Rf = 0.40 (EtOAc); Mp
(decomp.) 93.0–95 °C; IR (thin film): 2929, 1659, 1582, 1476,
1429, 1303, 1284, 1203, 1140, 809, 692 cmꢁ1; UV–vis (MeOH) kmax
vis (MeOH) kmax (log e): 488 (3.64), 260 (4.31), 232 (4.34), 197
(4.25) nm; 1H NMR (500 MHz, CDCl3) d 8.90 (dd, J2,4 = 1.1 Hz,
J2,3 = 3.4 Hz, 1H, H-2), 8.46 (dd, J2,4 = 1.1 Hz, J3,4 = 7.8 Hz, 1H, H-4),
7.64 (dd, J2,3 = 3.4 Hz, J3,4 = 7.8 Hz, 1H, H-3), 6.68 (br s, 1H, N–H),
3.51 (d, J10,N–H = 5.8 Hz, 3H, H-10), 2.34 (s, 3H, H-9); 13C NMR
(125 MHz, CDCl3) 180.3 (C8), 179.2 (C5), 153.5 (C2), 151.2 (C7),
146.8 (C8a), 135.0 (C4), 131.3 (C4a), 128.6 (C3), 110.1 (C6), 34.0
(NHMe), 19.2 (SMe); HRMS(ESI) m/z calcd for [C11H10N2O2S+Na]+:
257.0361, obsd.: 257.0368.
(log e
): 202 (4.53), 231 (4.53), 285 (4.32) nm; 1H NMR (500 MHz,
CDCl3): d 8.97 (dd, 1H, J2,3 = 4.6, J2,4 = 1.5 Hz, H-2), 8.36 (dd, 1H,
J3,4 = 7.9, J2,4 = 1.5 Hz, H-4), 7.63 (dd, 1H, J3,4 = 7.9, J2,3 = 4.6 Hz, H-
3) 2.78 (s, 1H, S-CH3) 2.73 (s, 1H, S-CH3); 13C NMR (125 MHz,
CDCl3): d 178.0 (C-5), 177.2 (C-8), 154.2 (C-2), 148.8 (C-6 or C-7),
148.5 (C-4a), 145.8 (C-6 or C-7), 134.7 (C-4), 129.8 (C-8a), 127.4
(C-3), 18.5 (CH3), 18.4 (CH3); HRMS(ESI) m/z calcd for
[C11H9NO2S2+Na]+: 273.9972, obsd.: 273.9972.
5.1.2.19. 7-Methylamino-6-phenylsulfanyl-5,8-quinolinequinone
(16b). 7-Chloro,6-methylamino-5,8-quinolinequinone (9a) (0.17 g,
0.77 mmol) was dissolved in pyridine (25 mL) and thiophenol
(0.094 mL, 0.91 mmol, 1.2 equiv) was added. The reaction mixture
was refluxed for 24 h by which 1H NMR analysis showed complete
consumption of the starting material. The pyridine was removed un-
derreducedpressureandthe residuewasdissolvedinCH2Cl2, washed
with satd aq NaHCO3 and brine, dried (MgSO4) and filtered before
being concentrated in vacuo. Purification by column chromatography
(hexanes/CH2Cl2, 10/1?0/1, v/v followed by CH2Cl2/EtOAc, 10/1?1/
3) yielded a purple-red solid which was crystallised in a minimum
amount of boiling solvent (CH2Cl2/hexanes, 1/3, v/v) to give thioether
16b as purple-red crystals (0.203 g, 0.69 mmol, 90%). Rf = 0.52
(EtOAc); Mp 198.0–199.8 °C; IR (thin film): 3290, 1700, 1591, 1555,
1518, 1413, 1307, 1268, 1202, 1140, 1098, 731 cmꢁ1; UV–vis (MeOH)
5.1.2.22. 6,7-Bis-phenylsulfanyl-5,8-quinolinequinone (18b). To
a solution of 6,7-dichloro-5,8-quinolinequinone (7a) (0.307 g,
1.35 mmol) in THF (5 mL) were added thiophenol (0.291 mL,
2.84 mmol, 2.1 equiv) and pyridine (0.326 mL, 4.05 mmol, 3 equiv)
and the reaction mixture stirred for 30 min at rt. The reaction mix-
ture was then extracted with CH2Cl2, washed with satd aq NaHCO3
then brine, dried over MgSO4 and filtered before being concen-
trated in vacuo. Purification by column chromatography (CH2Cl2/
EtOAc, 3/1?1/1, v/v) yielded a red solid that was recrystallised
(CH2Cl2/hexanes) to give disulfide 18b as dark red crystals
(0.465 g, 1.24 mmol, 92%). Rf = 0.62 (EtOAc); Mp 180.7–182.0 °C;
Lit. 177–178 °C;24 IR (thin film): 3075, 2241, 1668, 1581, 1494,
1476, 1440, 1268, 1199, 1138, 744, 726, 689 cmꢁ1; UV–vis (MeOH)
kmax (log e
): 201 (3.01), 248 (2.82) nm; 1H NMR (500 MHz, CDCl3): d
kmax (log e
): 462 (2.67), 240 (3.86), 201 (4.29) nm; 1H NMR
8.95 (dd, 1H, J2,3 = 4.4, J2,4 = 1.6 Hz, H-2), 8.50 (dd, 1H, J3,4 = 7.9,
J2,4 = 1.6 Hz, H-4), 7.67 (dd, 1H, J3,4 = 7.9, J2,3 = 4.4 Hz, H-3), 7.23 (m,
4H, CH-o and CH-m arom.), 7.13 (m, 1H, CH-p arom.), 6.88 (s, 1H,
NH), 3.48 (d, 3H, JNH,CH3 = 5.9 Hz, CH3); 13C NMR (125 MHz, CDCl3):
d 180.0 (C-8), 179.1 (C-5), 153.4 (C-2), 151.4 (C-7), 146.3 (C-8a),
138.2 (Ci arom), 135.2(C-4), 131.0 (C-4a) 129.2(CH-o orCH-m arom.),
128.6(C-3),126.4(CH-oorCH-marom.), 125.6(CH-parom.),104.0(C-
6), 33.4 (CH3); HRMS(ESI) m/z calcd for [C16H12N2O2S2+Na]+:
319.0517, obsd.: 319.0517.
(500 MHz, CDCl3): d 8.91 (dd, 1H, J2,3 = 4.7, J2,4 = 1.5 Hz, H-2), 8.25
(dd, 1H, J3,4 = 7.9, J2,4 = 1.5 Hz, H-4), 7.57 (dd, 1H, J3,4 = 7.9,
J2,3 = 4.7 Hz, H-3), 7.31 (m, 10H, CH arom.); 13C NMR (125 MHz,
CDCl3): d 178.1 (C-5 or C-8), 177.2 (C-5 or C-8), 154.6 (C-2),
149.7 (C-6 or C-7), 148.3 (C-8a), 146.6 (C-6 or C-7), 135.1 (C-4),
133.2 (CH arom.), 132.7 (CH arom), 132.0 (CH arom.), 131.4 (CH
arom.) 129.7 (C-4a), 129.2 (CH arom.), 128.3 (CH arom.), 128.1
(CH arom.) 127.6 (C-3.); HRMS(ESI) m/z calcd for [C21H13NO2S2+-
Na]+: 398.0285, obsd.: 398.0289.
5.1.2.20. 6-Methanesulfinyl-7-methylamino-5,8-quinolinequi-
none (17a). To a solution of 7-methylamino-6-methylsulfane-
5,8-quinolinedione (16a) (0.025 g, 0.107 mmol) in CH2Cl2 (3 mL)
at 0 °C was added m-CPBA (0.027 g, 0.107 mmol, 1.0 equiv). After
stirring at rt for 30 min, the mixture was concentrated in vacuo
and purified by flash chromatography (CH2Cl2/EtOH/MeOH/30%
aq NH3, 300/2/2/1?35/2/2/1, v/v) to give the desired sulfoxide
17a as orange crystals (0.021 g, 0.084 mmol, 79%). Rf = 0.48
(CH2Cl2/EtOH 13/1, v/v); Mp 151.4–152.7 °C; IR (film): 3076,
1703, 1596, 1560, 1470, 1415, 1393, 1341, 1306, 1283, 1142,
5.1.2.23. 6,7-Bis-methylsulfanyl-quinoline-5,8-diol (19). To a
solution of 6,7-dichloro-5,8-quinolinequinone (7a) (0.272 g,
1.19 mmol) in THF (5 mL) was added thiophenol (0.550 mL,
5.00 mmol, 4.2 equiv) and pyridine (0.331 mL, 4.19 mmol, 3 equiv)
and the solution stirred for 3 h at rt. The reaction mixture was then
extracted with CH2Cl2, washed with satd aq NaHCO3 then brine,
dried over MgSO4 and filtered before being concentrated in vacuo.
Purification by column chromatography (CH2Cl2/EtOAc, 1/0?5/1,
v/v) yielded a pale yellow solid that was recrystallised (CH2Cl2)
to give quinolinediol 19 as long yellow needles (0.188 g,
0.50 mmol, 42%). Rf = 0.88 (EtOAc); Mp 160.4–161.6 °C, Lit.24
160.5–161 °C; IR (thin film): 1578, 1476, 1398, 1372, 1228, 1146,
1096, 1081, 1003, 962, 726 cmꢁ1; UV–vis (MeOH) kmax (log
430 (3.06), 289 (3.58), 258 (3.69), 232 (3.79), 210 (3.61) nm; 1H
NMR (500 MHz, CDCl3) 10.13 (br s, 1H, N–H), 8.93 (dd,
e):
1082, 740, 688 cmꢁ1; UV–vis (MeOH) kmax (log
e): 205 (4.56), 260
d
(4.31), 353 (3.56) nm; 1H NMR (500 MHz, CDCl3): d 8.92 (dd, 1H,
J2,3 = 4.4, J2,4 = 1.7 Hz, H-2), 8.63 (dd, 1H, J3,4 = 8.5, J2,4 = 1.7 Hz, H-
4), 7.57 (dd, 1H, J3,4 = 8.5, J2,3 = 4.4 Hz, H-3) 7.15 (m, 10H, CH
arom.); 13C NMR (125 MHz, CDCl3): d 149.9 (C-2), 149.1 (C-8),
148.1 (C-5), 138.8 (C-8a), 136.5 (Ci), 135.1 (Ci), 133.3 (C-4), 129.2
(CH arom.), 128.8 (CH arom.), 127.7 (CH arom.), 127.2 (CH arom.),
126.4 (CH arom.), 125.8 (CH arom.), 122.4 (C-3), 119.9 (C-4a),
J2,4 = 1.7 Hz, J2,3 = 4.7 Hz, 1H, H-2), 8.35 (dd, J2,4 = 1.7 Hz,
J3,4 = 7.9 Hz, 1H, H-4), 7.66 (dd, J2,3 = 4.7 Hz, J3,4 = 7.9 Hz, 1H, H-3),
3.42 (d, JMe,N–H = 5.6 Hz, 3H, NMe), 2.99 (s, 3H, SMe); 13C NMR
(125 MHz, CDCl3) 180.6 (C8), 176.8 (C5), 153.4 (C2), 152.7 (C7),
147.0 (C8a), 133.7 (C4), 129.3 (C4a), 128.4 (C3), 109.5 (C6), 39.8
(SMe), 33.6 (NMe); HRMS(ESI) m/z calcd for [C11H10N2O3S+Na]+:
273.0310, obsd.: 273.0303.

3250
B. J. Mulchin et al. / Bioorg. Med. Chem. 18 (2010) 3238–3251
117.5, (C-6 or C-7) 115.3 (C-6 or C-7); HRMS(ESI) m/z calcd for
J3,4 = 7.9 Hz, 1H, H-3), 6.20 (br s, 1H, N–H), 3.82 (m, 2H, H-20),
[C21H15NO2S2+H]+: 378.0623, obsd.: 378.0622.
3.04 (t, JH-2 ,H-3 = 4.7 Hz, 2H, H-3’); 13C NMR (125 MHz, CDCl3)
178.2 (C5), 176.2 (C8), 153.0 (C2), 146.7 (C8a), 141.4 (C7), 133.9
(C4), 130.5 (C4a), 127.8 (C3), 111.6 (C6), 41.8 (C20), 23.8 (C30);
HRMS(ESI) m/z calcd for [C11H8N2O2S+H]+: 233.0385, obsd.:
233.0391.
0
0
5.1.2.24. 6,7-Bis-(toluene-4-sulfonyl)-5,8-quinolinequinone
(20) and 7-Chloro-6-(toluene-4-sulfonyl)-5,8-quinolinequinone
(21). A solution of p-toluenesulfinic acid, sodium salt (0.198 g,
1.11 mmol, 2.1 equiv) in H2O (3 mL) was added to a warm solution
of 6,7-dichloro-5,8-quinolinequinone (7a) (0.121 g, 0.53 mmol) in
MeCN (10 mL). The reaction was refluxed for 5 h, cooled, and neu-
tralised with NaOH. The reaction mixture was diluted with water
and extracted with CH2Cl2. The organic layer was washed with
brine, dried (MgSO4), filtered and concentrated. Purification of
the residue by column chromatography (CH2Cl2/EtOAc/MeOH, 1/
1/0?0/1/0.01, v/v) yielded sulfone 21 (0.070 g, 0.10 mmol, 18%)
as a yellow solid, which was crystallised in the minimum volume
of boiling solvent (H2O, and EtOH) to give yellow powder-like crys-
tals, and bis-sulfone 20 (0.229 g, 0.23 mmol, 44%) as an orange
film. Data for 21: Rf = 0.58 (CH2Cl2/MeOH, 9:1); Mp (decomp.)
240.5–243.5 °C; IR (thin film): 3374, 2361, 2136, 1698, 1641,
1597, 1556, 1527, 1368, 1277, 1238, 1084 cmꢁ1; UV–vis (MeOH)
5.2. Biological assays
5.2.1. Reagents and chemicals
Unless otherwise stated, cell culture reagents were obtained
from Invitrogen (New Zealand) and all other reagents were pur-
chased from Sigma–Aldrich (New Zealand). HL60 cells, originally
from ATCC, were obtained from Dr. Graeme Findlay (University
of Auckland, NZ). RPMI-1640 media was obtained from GIBCO-
BRL, Grand Island, NY, USA. The Vacutainer cell preparation tubes
were obtained from BD, Franklin Lakes, NJ, USA. 3-(4,5-Dimethyl-
thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was from
Sigma–Aldrich and 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-
disulfophenyl)-2H-tetrazolium, monosodium salt (WST-1) from
Dojindo (Kumamoto, Japan). Polymorphprep density gradient
(Axis-Shield, Norway) was obtained through Media Pacifica Ltd
(New Zealand). MACS T cell activation/expansion kit was obtained
from Miltenyi Biotec, Gladbach, Germany. M. bovis BCG Pasteur
strain 1173P was gifted by AgResearch Wallaceville Animal Re-
search Centre, Upper Hutt, New Zealand. Dubos broth base and
OADC (Oleic acid-albumin-dextrose-catalase) were supplied by
Fort Richard, Auckland, New Zealand. Alamar Blue solution
(BUF012B) was obtained from AbD serotec, UK.
kmax (log e
): 200 (3.56), 226 (3.42) nm; 1H NMR (500 MHz, D2O):
d 8.79 (s, 1H, H-2), 8.28 (s, 1H, H-4), 7.84 (s, 2H, CH-o or CH-m),
7.62 (s, 1H, H-3) 7.36 (s, 2H, CH-o or CH-m), 2.35 (s, 3H, CH3);
13C NMR (125 MHz, CDCl3): d 183.3.1 (C-5), 177.5 (C-8), 171.2 (C-
8a), 154.4 (C-2), 149.1 (C-8a), 144.5 (C-6 or C-7), 139.4 (Ci arom.),
135.3 (C-4), 129.4 (CH-m arom.), 127.5 (C-4a), 127.2 (C-3) 126.1
(CH-o arom.), 114.6 (C6-or C-7), 20.6 (CH3); Data for 20: Rf = 0.40
(EtOAc); IR (thin film): 1708, 1644, 1595, 1360, 1273, 1166,
1116, 1066, 1021, 813, 738, 704, 667 cmꢁ1 1H NMR (500 MHz,
;
CDCl3): d 8.77 (s, 1H, H-2), 8.60 (s, 1H, H-4), 7.91 (s, 4H, CH-o or
CH-m), 7.34 (s, 1H, H-3) 7.27 (s, 4H, CH-o or CH-m), 2.43 (s, 6H,
CH3); HRMS(ESI) m/z calcd for [C23H19NO7S2+Na]+: 508.0495,
obsd.: 508.0503.
5.2.2. HL60 cell culture
HL60 cells were maintained in RPMI-1640 supplemented with
5% (v/v) fetal calf serum (FCS), 2 mM glutamate, 100 U/mL penicil-
lin, and 100
lg/mL streptomycin, and were kept at 37 °C in a
5.1.2.25. 2,3-Dihydro-1H-4-thia-1,5-diaza-anthraquinone (22).
A
humidified incubator maintained at 5% CO2.
solution of sodium sulfide (0.28 g, 1.2 mmol, 1.5 equiv) in H2O
(25 mL) was added to quinolinequinone (8c) (0.24 g, 0.72 mmol)
in EtOH (50 mL) and the solution was stirred at rt for 50 min and
refluxed for an additional 10 min. The solvents were removed un-
der reduced pressure and the residue was purified by column chro-
matography (CH2Cl2/EtOAc, 50/1?1/2, v/v) to yield the title
compound 22 as dark purple crystals (0.11 g, 0.46 mmol, 59%).
Rf = 0.07 (EtOAc), Rf = 0.27 (CH2Cl2/MeOH, 11:1); Mp 262.5–
263.3 °C (Lit.16 Mp 263 °C); IR (film): 1672, 1593, 1558, 1510,
5.2.3. In vitro activation of T cells
Blood samples were obtained from healthy volunteers, and
peripheral blood mononuclear cells (PBMC) were immediately iso-
lated by the FICOLL™-hypaque™ method using BD Vacutainer cell
preparation tubes containing sodium heparin. PBMC were washed
in PBS and resuspended in RPMI-1640 medium with 10% FCS,
100 U/mL penicillin, and 100 lg/mL streptomycin. T cells were
activated using a MACS T cell activation/expansion kit following
the manufacturer’s protocol. Briefly, PBMC (5 ꢂ 106) were incu-
bated for two days at 37 °C with 2.5 ꢂ 106 anti-biotin MACS iBead
particles that had been pre-loaded with CD2-biotin, CD3-biotin
and CD28-biotin. Cells were then split into two equal parts with
culture medium supplemented with 20 U/mL IL-2 and incubated
for another two–four days. All assays were performed in triplicate
and the mean result recorded.
1399, 1336, 1320, 1270, 1150, 1119, 1086, 910, 734, 685 cmꢁ1
;
UV–vis (MeOH) kmax (log e): 335 (3.26), 268 (3.51), 230 (3.71),
209 (3.72) nm; 1H NMR (500 MHz, CDCl3) d 8.93 (dd, J2,4 = 1.7 Hz,
J2,3 = 4.8 Hz, 1H, H-2), 8.30 (dd, J2,4 = 1.7 Hz, J3,4 = 7.8 Hz, 1H, H-4),
7.53 (dd, J2,3 = 4.8 Hz, J3,4 = 7.8 Hz, 1H, H-3), 6.01 (br s, 1H, N–H),
3.80 (m, 2H, H-20), 3.05 (m, 2H, H-30); HRMS(ESI) m/z calcd for
[C11H8N2O2S+Na]+: 255.0204, obsd.: 255.0206.
5.1.2.26. 3,4-Dihydro-2H-1-thia-4,5-diaza-anthraquinone (23).
A
5.2.4. Anti-proliferative MTT assay
solution of sodium sulfide (0.26 g, 1.1 mmol, 1.5 equiv) in H2O
(25 mL) was added to quinolinequinone (9c) (0.22 g, 0.72 mmol)
in EtOH (50 mL) and the solution was stirred at rt for 50 min and
refluxed for an additional 10 min. The solvents were removed un-
der reduced pressure and the residue was purified by column chro-
matography (CH2Cl2/EtOAc, 50/1?1/2, v/v) to yield the title
compound 23 as dark purple crystals (0.13 g, 0.54 mmol, 76%).
Rf = 0.17 (EtOAc), Rf = 0.33 (CH2Cl2/MeOH, 11:1); Mp 264.2 °C (de-
comp); IR (film): 1666, 1629, 1590, 1556, 1497, 1337, 1314, 1200,
Anti-proliferative activity was measured using the 2-day MTT
assay.36,37 Phenoxodiol was used as
a
positive control
(IC50 = 3.2
l
M).47 HL60 cells and T cells were plated into 96-well
plates in the presence of different concentrations of quinolinequi-
nones (final cell concentration = 0.2 ꢂ 106 cells/mL in complete
RPMI) and incubated for 48 h (37 °C, 5% CO2). 10 lL of MTT
(5 mg/mL in HBSS) was then added to each well and the cells incu-
bated for 2 h before adding 100 L of SDS lysing buffer (10% w/v
l
SDS, 45% v/v dimethylformamide/H2O, pH 4.7) and further over-
night incubation. The absorbance at 570 nm was measured in a
FLUOstar OPTIMA plate reader (BMG Labtechnologies Pty. Ger-
many). All assays were performed in triplicate and the mean result
recorded.
1153, 1113, 910, 720 cmꢁ1; UV–vis (MeOH) kmax (log
e): 579 (2.27),
330 (3.43), 269.0 (3.66), 230.0 (3.73), 201 (3.67) nm; 1H NMR
(500 MHz, CDCl3) d 8.87 (dd, J2,4 = 1.7 Hz, J2,3 = 4.7 Hz, 1H, H-2),
8.37 (dd, J2,4 = 1.7 Hz, J3,4 = 7.9 Hz, 1H, H-4), 7.58 (dd, J2,3 = 4.7 Hz,

B. J. Mulchin et al. / Bioorg. Med. Chem. 18 (2010) 3238–3251
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5.2.5. Superoxide assay
Measurement of superoxide production was performed using the
tetrazolium salt, WST-1 as previously described and reviewed.37,38
Blood was obtained from healthy volunteers by venous puncture
and purified using Polymorphprep density gradient according to
manufacturer’s instructions. Purified neutrophils were cultured in
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l
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g/ml WST-1
g/ml phorbol
pound or DMSO control added. After 30 min, 250
l
l
was added and neutrophils stimulated with 0.2
12-myristate 13-acetate (PMA). Immediately following PMA addi-
tion, the plate was loaded into a Versamax spectrophotometer and
the absorbance measured at 450 nm over 20 min at 37 °C. The Vmax
was then calculated for each sample and normalised against the
positive PMA-stimulated, DMSO cell control. All assays were per-
formed in triplicate and the mean result recorded. Superoxide dis-
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mutase served as a positive control (0.8 0.05 lM).
5.2.6. Alamar blue tuberculostatic assay
Antimicrobial susceptibility testing was performed in black,
clear-bottomed, 96-well optical bottom microplates using the Ala-
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The authors would like to thank the Cancer Society of New Zea-
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Supplementary data
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Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.03.021.
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