Benzofuran-, benzothiophene-, indazole- and benzisoxazole-quinones: Excellent substrates for NAD(P)H:quinone oxidoreductase 1

Article abstract of DOI:10.1016/j.bmc.2013.03.071

A series of heterocyclic quinones based on benzofuran, benzothiophene, indazole and benzisoxazole has been synthesized, and evaluated for their ability to function as substrates for recombinant human NAD(P)H:quinone oxidoreductase (NQO1), a two-electron reductase upregulated in tumor cells. Overall, the quinones are excellent substrates for NQO1, approaching the reduction rates observed for menadione.

Full text of DOI:10.1016/j.bmc.2013.03.071

Bioorganic & Medicinal Chemistry 21 (2013) 2999–3009
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Benzofuran-, benzothiophene-, indazole- and benzisoxazole-
quinones: Excellent substrates for NAD(P)H:quinone
oxidoreductase 1
a
b
a
a
b
Jeffery J. Newsome , Mary Hassani , Elizabeth Swann , Jane M. Bibby , Howard D. Beall ,
a,c,
⇑
Christopher J. Moody
a
Department of Chemistry, University of Exeter, Stocker Road, Exeter EX4 4QD, UK
Department of Biomedical and Pharmaceutical Sciences, The University of Montana, 32 Campus Drive #1552, Missoula, MT 59812-1552, USA
School of Chemistry, University of Nottingham, University Park, Nottingham NG7 2RD, UK
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of heterocyclic quinones based on benzofuran, benzothiophene, indazole and benzisoxazole has
been synthesized, and evaluated for their ability to function as substrates for recombinant human
NAD(P)H:quinone oxidoreductase (NQO1), a two-electron reductase upregulated in tumor cells. Overall,
the quinones are excellent substrates for NQO1, approaching the reduction rates observed for menadione.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 31 December 2012
Revised 14 March 2013
Accepted 22 March 2013
Available online 6 April 2013
Keywords:
Quinone
Benzofuran
Benzothiophene
Indazole
Quinone reductase
NQO1
1
. Introduction
Quinonesare widespread in nature, and participate in important
whilst the benzimidazole quinones have been widely investigated
as analogues of MMC.
2
1–27
1
Despite the aforementioned studies, we sought to extend the range
of heterocyclic quinones studied as substrates for NQO1, focusing on
benzofurans, benzothiophenes, and indazoles. Furoquinones are quite
well described, and although most of their naturally occurring com-
biological redox processes. For example, the ubiquinones act as elec-
tron-transfer agents in the respiratory chain, and the heterocyclic
pyrroloquinolinequinone (coenzyme PQQ) is a redox co-factor. In-
deed heterocyclic derivatives form an important subset of qui-
1
pounds are naphthofuranquinones, relatively simple derivatives such
2
28
nones, often possessing potent biological activity, for example as
as acamelin 5 are known. Benzothiophene quinones are less com-
phosphatase inhibitors.3 The best known heterocyclic quinone is
the clinically used cancer therapeutic agent, the indolequinone nat-
mon, although caldariellaquinone 6 appears to fulfil an important re-
dox role in thermophilic and acidophlic archaea of the Sulfolobus
4–7
29,30
ural product mitomycin C (MMC) 1,
although other heterocyclic
genus that lack ubiquinones as electron-transfer agents.
On the
8
quinones such as streptonigrin 2 have also been widely studied.
Given the biological importance of quinone reduction, our own
studies have focused on the two-electron reduction of quinones by
other hand, indazolequinones are somewhat rarer, although com-
31
pounds such as 7 have been prepared as MMC analogues, whilst oth-
ers have been investigated as substrates for carbonyl reductase.32
Therefore in an attempt to widen the group of NQO1 substrates/
inhibitors, and to probe further the active site of the enzyme, we have
explored a new series of heterocyclic quinones based on benzofuran,
benzothiophene, indazole and benzisoxazole.
the enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1, DT-diaph-
orase, QR1, EC 1.6.99.2).9
–12
In particular, we have investigated a
1
3–17
broad range of indolequinone based substrates and inhibitors,
the quinoline-5,8-dione system found in streptonigrin,
1
8
and
both benzimidazole-4,7-diones 3 and benzothiazole-4,7-diones 4
1
9
(
Fig. 1). Others have also studied 5-undecyl-6-hydroxybenzo-
2. Results and discussion
2.1. Chemistry
thiazole-4,7-dione (UHDBT), an analogue of ubiquinone, as an
inhibitor of electron transport by binding to cytochrome bc ,
1
2
0
⇑
Corresponding author. Fax: +44 115 951 3564.
E-mail address: c.j.moody@nottingham.ac.uk (C.J. Moody).
In order to make meaningful comparisons with the more widely
studied indolequinones, we initially elected to investigate
0
968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.03.071

3
000
J. J. Newsome et al. / Bioorg. Med. Chem. 21 (2013) 2999–3009
O
O
Me
O
O
Me
MeO
MeO
O
a-d
MeO
O
O
OCONH
OMe
NH
N
CO H
2
2
O
OMe
O
OH
H N
H
2
N
N
2
H N
Me
2
13
Me
N
12
HO
MeO
Scheme 2. Reagents and conditions: (a) HNO
3
, AcOH, rt; (b) Sn, HCl, EtOH, rt; (c)
OMe
LiAlH
, THF, 0 °C; (d) Fremy’s salt, NaH
2
PO
4
, aq acetone, rt (53% over four steps).
1
2
4
O
O
O
MeO
N
R
X
Me
H
O
MeO
O
O
a
MeO
MeO
Me
S
Me
S
3
4
X = NMe
X = S
5
1
4
15
O
O
OCONH
+
2
MeS
H
2
N
b
N
S
Me
N
6
O
O
H
6
7
O
OH
Me
d
e
R
O
MeO
MeO
Me
Figure 1. Some heterocyclic quinones.
S
S
O
18
16 R = NO2
c
relatively simple 5-methoxy-heterocyclic quinones in the benzofu-
ran and benzothiophene series. The synthesis of quinone 11 started
1
7 R = NH2
with the known benzofuran 8, readily prepared from benzoqui-
Scheme 3. Reagents and conditions: (a) POCl
3 2 2
, DMF, CH Cl , 0–50 °C (20%, plus 40%
none in a Nenitzescu type reaction.33 Methylation gave the known
of 4-formyl isomer); (b) HNO , AcOH, rt (61%); (c) Sn, HCl, EtOH, reflux (71%); (d)
3
NaBH
4
2 4
, MeOH, rt; (e) Fremy’s salt, NaH PO , aq acetone, rt (41% over two steps).
5
-methoxy derivative 9, nitration of which gave a mixture of the
desired 4-nitro compound 10 along with its 6-nitro isomer in
excellent overall yield, but in a 1:2 ratio. Although nitrobenzofuran
R
Me
1
0 could be isolated, it was more convenient to reduce the mixture
Me
MeO
MeO
O
a
CO Et
of nitro compounds to the corresponding amines, reduce the ke-
tone with sodium borohydride and then oxidize the aniline with
Fremy’s salt and purify the desired quinone 11 at the final stage
2
S
S
OEt
2
0 R = NO2
1
9
b
2
1 R = NH2
(Scheme 1). The intermediate iminoquinone was not observed
and was presumably readily hydrolyzed under the reaction
conditions.
c
The isomeric benzofuranquinone 13, with the alcohol group at
the 2-position, was synthesized from the known benzofuran 12,
readily available from 4-methoxyphenol by carbene O–H insertion,
and cyclization. The sequence of nitration, reduction and oxida-
tion to the quinone was carried out without purification of the
intermediate compounds and delivered the pure benzofuranqui-
none 13 in 53% over the four steps (Scheme 2).
O
O
Me
NH2
Me
MeO
d
MeO
3
4
S
OH
S
OH
2
3
22
Scheme 4. Reagents and conditions: (a) HNO
reflux (86%); (c) LiAlH , THF, rt (80%); (d) Fremy’s salt, NaH
aq HCl (2 M), acetone. (67%).
3
, AcOH, rt (74%); (b) Sn, HCl, EtOH,
In the benzothiophene series, we started with the known
4
2
4
PO , aq acetone, rt, then
5
5
-methoxy-2-methylbenzothiophene 14 (Scheme 3).3 Formyla-
tion under Vilsmeier conditions gave the desired 3-aldehyde 15,
but in poor yield (20%), the major product being the unwanted
quinone, provided the benzothiophenequinone 18 in reasonable
overall yield (Scheme 3).
4
-formyl compound. Nevertheless, subjecting aldehyde 15 to the
usual sequence of nitration, reduction, and final oxidation to the
By analogy with the conversion of benzofuran 12 into
benzofuranquinone 13, the known starting material,36 ethyl
5
-methoxy-3-methylbenzothiophene-2-carboxylate 19, was con-
Me
Me
O
O
verted into benzothiophenequinone 23 by the now familiar se-
quence of nitration, reduction and final oxidation to the quinone
HO
a
MeO
Me
Me
O
O
(Scheme 4).
8
9
A range of 3-unsubstituted benzothiophenequinones was also
prepared as shown in Scheme 5. Reaction of 3-methoxycinnamic
acid with thionyl chloride and methanol readily provided the
known benzothiophene-2-carboxylate starting material 24 in 37%
yield. Nitration, followed by hydrogenation over Pd/C resulted
in reduction of the nitro group with concomitant hydrogenolysis
of the chlorine, and gave the 4-aminobenzothiophene 26, which
was either directly oxidized to quinone 27, or converted into
quinone 29 by initial reduction of the ester (Scheme 5). Benzothio-
phenequinones 31–33 were also prepared by standard transforma-
tions as shown in Scheme 5.
b
3
7
Me
Me
NO
O
O
OH
Me
c-e
2
MeO
MeO
Me
O
O
O
1
1
10
Scheme 1. Reagents and conditions: (a) KH, MeI, DMF, rt (42%); (b) fuming HNO
AcOH, rt (81% as 1:2 mixture of 4- and 6-nitro isomers); (c) Sn, HCl, EtOH, reflux; (d)
NaBH , MeOH, rt; (e) Fremy’s salt, NaH PO , aq acetone, rt (20% over three steps).
3
,
4
2
4

J. J. Newsome et al. / Bioorg. Med. Chem. 21 (2013) 2999–3009
3001
R
Me
O
OH
Me
NOH
OH
Cl
X
MeO
a
MeO
MeO
MeO
MeO
O
a
CO Me
2
S
OMe
S
2
5 X = Cl, R = NO2
2
4
41
4
2
b
2
6 X = H, R = NH2
b
c
NO2
Me
N
Me
d
c
MeO
MeO
N
NH2
O
O
O
MeO
MeO
MeO
CO Me
44
2
43
S
OH
S
O
d, e
28
27
NH2
f
O
e
Me
N
MeO
MeO
H
O
O
S
O
O
30
45
S
R
O
g
Scheme 7. Reagents and conditions: (a) NH
DIAD, Ph P, THF, rt (100%); (c) HNO , AcOH, rt (91%); (d) Sn, HCl, EtOH, reflux; (e)
Fremy’s salt, NaH PO , aq acetone, rt (20% over two steps).
2
OHÁHCl, EtOH, pyridine, rt (98%); (b)
3
3
2
9 R = OH
O
O
2
4
h
CHO
3
2 R = OAc
3 R = H
i, j
S
O
O
O
O
OH
Me
Me
MeO
MeO
3
31
X
OH
S
Scheme 5. Reagents and conditions: (a) HNO
MeOH–THF (83%); (c) Fremy’s salt, NaH PO , aq acetone, rt (75%); (d) LiAlH
, aq acetone, rt (88%); (f) MnO , CH Cl
O, pyridine, rt (69%);
3
, AcOH, reflux (87%); (b) H
, THF, rt
, reflux
2
, Pd/C,
2
4
4
1
redox
2
3 (X = O)
18
(
(
(
78%); (e) Fremy’s salt, NaH
38%); (g) Fremy’s salt, NaH PO
2
PO
4
2
2
2
E
E
=
1.12 V
E
redox
=
1.01 V
2
4
, aq acetone, rt (79%); (h) Ac
2
3 (X = S)
i) MsCl, Et
3
N, CH
2
Cl
2
4
, rt; (j) LiAlH , THF, rt (5% over two steps).
=
1.16 V
redox
Me
O
O
Me
N
O
O
OH
Me
N
MeO
MeO
MeO
MeO
a, b
N
N
N
N
H
O
Me
Me
OMe
MeO
35
40
=
3
4
39
=
Eredox
MeO
1.26 V
E
redox
1.09 V
c
O
OH
Me
O
Me
NH2
NO2
Me
N
MeO
Me
N
MeO
d
MeO
N
N
OH
N
N
O
Me
O
Me
Me
H
MeO
MeO
36
4
=
6
47
=
3
7 X = NO2
8 X = NH2
Eredox
1.28 V
Eredox
1.44 V
e
3
Figure 2. Eredox values (v. Fc) for benzofuran-, benzothiophene- and indazole-
quinones 13, 18, 23, 39 and 40 compared to related indolequinones 46 and 47.1
3,14
f
delivered the required indazolequinone 39 (Scheme 6). The methyl
group was further functionalized by radical bromination and con-
version into the 3-hydroxymethylindazolequinone 40 (Scheme 6).
Finally an example of a benzisoxazolequinone was prepared. 2-
Hydroxy-5-methoxyacetophenone 41 was converted into its oxime
O
O
O
O
OH
Me
N
MeO
MeO
g, h
N
N
N
Me
Me
3
9
40
4
2 that underwent facile intramolecular Mitsunobu reaction to
provide the benzisoxazole 43 in quantitative yield. Thereafter,
nitration, reduction and oxidation as before gave the required ben-
zisoxazolequinone 45 (Scheme 7)
Scheme 6. Reagents and conditions: (a) Cl
CH Cl , rt; (b) Zn, AcOH, rt (35% over two steps); (c) HNO
MeI, KOH, DMSO, rt (49%); (e) Sn, HCl, EtOH, reflux (96%); (f) Fremy’s salt, NaH
3
CCH
2
O
2
CN@NCO
, AcOH, rt (81%); (d)
PO
, aq acetone (36% over two
2
CH
2
Cl
3
,
BF
3
ÁOEt
2
,
2
2
3
2
4
,
4
aq acetone, rt (78%); (g) NBS, AIBN, CCl ; (h) AgNO
steps).
3
Reduction potentials were measured for benzofuran-, benzo-
thiophene- and indazole-quinones 13, 18, 23, 39 and 40 using cyc-
lic voltammetry in DMF as solvent with tetra-n-butylammonium
tetrafluoroborate as supporting electrolyte as previously de-
The synthesis of indazolequinones started from the dim-
ethoxyindazole 35, itself prepared by the literature method from
1
3
scribed. The Eredox values, with reference to ferrocene (Fc), are
shown in Figure 2; values for the related indolequinones 46 and
47 are also shown. The data show that whilst the indazole quinone
39 has a similar redox potential to the indolequinones (Eredox v. Fc
3
,5-dimethoxyacetophenone 34 by electrophilic amination with
bis(trichloroethyl) azodicarboxylate followed by zinc reduction
3
8
and cyclization. Nitration, methylation, reduction and oxidation

3
002
J. J. Newsome et al. / Bioorg. Med. Chem. 21 (2013) 2999–3009
Table 1
Metabolism of heterocyclic quinones by recombinant human NQO1
O
O
R3
O
O
R3
MeO
MeO
R2
N
X
X
A
B
Entry
Ring
Compd
X
R2
Me
R3
CH
Me
CH(OH)Me
Me
NQO1a (ave) (
lmol/min/mg)
NQO1a (init) (
lmol/min/mg)
1.25 ± 0.03b
2.49 ± 1.27d
87.9 ± 25.6
80.8 ± 6.7
nd
c
1
2
3
4
5
6
7
8
9
A
A
A
A
A
A
A
A
A
A
A
B
B
B
46
47
11
13
18
23
27
29
31
32
33
39
40
45
NMe
NMe
O
O
S
S
S
S
2
OH
c
CH
Me
2
OH
nd
585 ± 32
1025 ± 54
236 ± 35
468 ± 68
555 ± 71
927 ± 89
897 ± 64
900 ± 86
553 ± 66
55.3 ± 5.1
247 ± 14
290 ± 28
CH
Me
2
OH
c
CH
2
OH
nd
nd
c
CH
2
OH
Me
OH
Me
H
H
H
H
CO
CH
2
53.9 ± 3.8
63.8 ± 11.7
27.4 ± 5.7
24.5 ± 7.9
5.51 ± 0.87
19.6 ± 5.3
38.1 ± 7.1
27.6 ± 3.2
2
S
S
S
CHO
CH OAc
10
11
12
13
14
2
Me
—
—
H
Me
NMe
NMe
O
CH
Me
2
OH
—
a
Ave and init refer to average and initial rates of metabolism as measured by the HPLC and spectrophotometric assays, respectively; average rates are determined from
irreversible NADH oxidation whereas initial rates are determined from cytochrome c reduction. For reference, initial rate for menadione reduction was 1225 ± 15
mg.
lmol/min/
b
Ref. 13.
nd = not determined.
c
d
Ref. 14.
À1.20 to À1.40 V), the other heterocyclic quinones are consider-
ably easier to reduce. Consistent with this finding, quinone 39
had the lowest reduction rate by NQO1 (Table 1).
reduction rate observed for menadione (1225 ± 15 lmol/min/
1
9
mg), a simple naphthoquinone that has been used to measure
activity of the enzyme, making these compounds some of the best
NQO1 substrates seen to date.
2
.2. Enzyme studies
The results presented here complement our previous work on bior-
eductive activation of indolequinone antitumor agents by NQO1. Novel
heterocyclic quinones have been synthesized, characterized and stud-
ied biologically as substrates for recombinant human NQO1. These
data add to our understanding of the structural requirements for effi-
cient metabolism by the quinone reductase enzyme.
The new heterocyclic quinones were evaluated for their ability to
act as substrates for NQO1. We used two assays for studying quinone
metabolism by recombinant human NQO1 based on HPLC and spec-
trophotometry. The former HPLC system is capable of quantifying
both NADH oxidation and quinone reduction, and gives average
rates of reduction over a 30–40 min period.39,13 Quinone reduction
is reversible due to redox cycling of the hydroquinone, so results (Ta-
3
3
. Experimental section
À1
À1
ble 1) are reported as lmol NADH oxidized min mg NQO1. The
.1. General chemistry experimental details
Commercially available reagents were used throughout without
alternative spectrophotometric method uses cytochrome c as the
terminal electron acceptor and gives initial rates of reduction that
are generally higher than the HPLC method.18 Nevertheless the rel-
ative order of metabolism is essentially the same with the two meth-
ods, and with the exception of entries 1, 2, 5 and 6, both methods
were used to enable reliable comparison between the new heterocy-
clic quinones. Interestingly, quinones 27 and 33 had similar initial
reduction rates, but the average rate for 27 was 10-fold higher than
for 33. This suggests that the 27 hydroquinone redox cycles more
efficiently than the 33 hydroquinone, most likely due to the elec-
purification unless otherwise stated. Light petroleum refers to the
fraction with bp 40–60 °C and was distilled before use. Ether refers
to diethyl ether. Reactions were routinely carried out under a
nitrogen or argon atmosphere. Analytical thin layer chromatogra-
phy was carried out on aluminum-backed plates coated with silica
gel, and visualized under UV light at 254 and/or 360 nm. Flash
chromatography was carried out on silica gel. Fully characterized
compounds were chromatographically homogeneous.
4
0
À1
tron-withdrawing methyl carboxylate group present on 27. In
Infrared spectra were recorded in the range 4000–600 cm
contrast to our previous studies on indolequinones,1
3,39
electron-
using FT spectrometers. NMR spectra were carried out at 300 and
1
13
withdrawing groups did not appear to increase rates of reduction
for the benzothiophene series.
4
00 MHz ( H frequencies, corresponding C frequencies are 75
and 100 MHz). Chemical shifts are quoted in ppm with TMS as inter-
1
3
The new quinones are all excellent substrates for rhNQO1. In
the benzofuran and benzothiophene series, reduction rates were
higher when the hydroxyalkyl substituent was at the C-2 position
rather than C-3 (Table 1), possibly due to stabilizing hydrogen-
bonding interactions with key amino acid residues in the NQO1
active site. As with the benzimidazole- and benzothiazole-qui-
nones, all of the new quinones were much better substrates for
NQO1 than the widely studied indolequinones,13,39,14 as seen by
comparison with indolequinones 46 and 47 included in Table 1
for comparison. In fact, the reduction rates for benzofuran- and
benzothiophene-quinones 13, 29, 31 and 32 approach the initial
nal standard. J values are recorded in Hz. In the C spectra, signals
corresponding to CH, CH or CH groups, as assigned from DEPT,
2
3
are noted; all others are C. High and low resolution mass spectra
were recorded on a Micromass GCT TDF High Resolution mass spec-
trometer, or at the EPSRC Mass Spectrometry Service (Swansea).
1
9
3.2. Synthesis of benzofuranquinones
3.2.1. 3-Acetyl-5-methoxy-2-methylbenzofuran 9
3
3
3-Acetyl-5-hydroxy-2-methylbenzofuran 8 (1.62 g, 8.5 mmol)
in DMF (30 mL) was added to a stirring suspension of potassium

J. J. Newsome et al. / Bioorg. Med. Chem. 21 (2013) 2999–3009
3003
hydride (0.61 g, 15.2 mmol) in DMF (50 mL) at 0 °C. The mixture
was stirred at room temperature for 45 min. Iodomethane
1.80 g, 12.7 mmol) was added dropwise at 0 °C and the mixture
allowed to warm to room temperature. After 2 h saturated
ammonium chloride solution was added and the mixture ex-
tracted with ethyl acetate. The ethyl acetate layer was washed
3.2.4. 2-Hydroxymethyl-5-methoxy-3-methylbenzofuran-4,7-
dione 13
To a solution of methyl 5-methoxy-3-methylbenzofuran-2-car-
(
3
4
boxylate 12 (0.20 g, 0.91 mmol) in acetic acid (6 mL), cooled to
À10 °C was added a mixture of concentrated nitric acid (1 mL)
and acetic acid (4 mL). The mixture was stirred at room tempera-
ture for 1 h. The reaction mixture was poured in an ice/water mix-
ture and the resulting precipitate filtered off and dried. The crude
material was obtained as a 2.8:1 ratio of 4- and 6-nitro products
and used directly in the next step. To a suspension of the above
mixture in ethanol (30 mL) were added tin powder (0.48 g,
4.0 mmol) and hydrochloric acid (3 M; 7 mL). The mixture was stir-
red at room temperature for 2 h. The solution was decanted from
the excess tin and neutralized with saturated aqueous sodium
hydrogen carbonate. The suspension obtained was added to an
equal volume of water. The mixture was extracted with ethyl ace-
4
thoroughly with hydrochloric acid (1 M), dried (MgSO ) and con-
centrated. The crude product was purified by chromatography
eluting with dichloromethane to yield the title compound as a col-
41
orless solid (0.74 g, 42%), mp 69–70 °C (lit.,
300 MHz; CDCl ) 7.48 (1H, d, J 2.6, 4-H), 7.33 (1H, d, J 8.9,
-H), 6.89 (1H, dd, J 8.9, J 2.6, 6-H), 3.88 (3H, s, OMe), 2.76 (3H,
s, Me), 2.62 (3H, s, Me).
mp 72 °C); d
H
(
7
3
3
.2.2. 3-Acetyl-5-methoxy-2-methyl-4-nitrobenzofuran 10
To a solution of 3-acetyl-5-methoxy-2-methylbenzofuran 9
(
0.217 g, 1.1 mmol) in acetic acid (4 mL) cooled to 0 °C was added
4
tate. The organic layer was dried (MgSO ) and concentrated. The
a mixture of fuming nitric acid (0.5 mL) and acetic acid (2 mL). The
mixture was stirred at room temperature for 2 h and then poured
on to an ice/water mixture and the precipitate obtained was filtered
off and dried. NMR analysis of the mixture showed a 2:1 ratio of
crude material was used directly in the next step without
purification.
To
a suspension of lithium aluminum hydride (0.091 g,
2.4 mmol) in THF (10 mL) at 0 °C was added a solution of the
above mixture of benzofurans in THF (10 mL) and the reaction
was stirred for 15 min. The reaction mixture was quenched by
the addition of water (0.2 mL), sodium hydroxide (1 M; 0.2 mL)
and silica gel (2 g). The granular precipitate was filtered off
6
-nitro and 4-nitro products (0.21 g, 81%), used without further
purification. Small quantities of each isomer were obtained by pre-
1
parative TLC (dichloromethane elution) and were identified by H
NMR spectroscopy: 3-acetyl-5-methoxy-2-methyl-4-nitrobenzofu-
ran 10, d
ArH), 3.93 (3H, s, OMe), 2.74 (3H, s, Me), 2.49 (3H, s, Me); 3-acetyl-5-
methoxy-2-methyl-6-nitrobenzofuran, d (300 MHz; CDCl ) 7.97
1H, s, ArH), 7.76 (1H, s ArH), 4.02 (3H, s, OMe), 2.83 (3H, s, Me),
H
(300 MHz; CDCl
3
) 7.50 (1H, d, J 9.0, ArH), 7.03 (1H, d, J 9.0
4
through a pad of Celite. The filtrate was dried (MgSO ) and con-
centrated in vacuo to give the alcohol that was used directly in
the next step without purification, or characterization. To a solu-
tion of the benzofuran-2-methanol in acetone (50 mL) was added
a solution of potassium nitrosodisulfonate (0.64 g, 2.4 mmol) in
sodium dihydrogen phosphate buffer (0.3 M; 50 mL). The mixture
was stirred at room temperature for 1 h. The excess acetone was
removed in vacuo. The resulting residue was extracted with
dichloromethane and washed with water. The organic layer was
H
3
(
2
.62 (3H, s, Me).
3
.2.3. 3-(1-Hydroxyethyl)-5-methoxy-2-methylbenzofuran-4,7-
dione 11
To a mixture of 3-acetyl-5-methoxy-2-methyl-4-nitrobenzofu-
ran 10 and its 6-nitro isomer (1:2 ratio) (0.80 g, 3.4 mmol) in
ethanol (100 mL) were added tin powder (4.00 g, 34.0 mmol) and
hydrochloric acid (3 M; 40 mL). The mixture was heated under re-
flux for 30 min. Upon cooling the solution was decanted from the
tin and neutralized with saturated aqueous sodium hydrogen car-
bonate. The mixture was extracted with ethyl acetate and the or-
2 4
dried (Na SO ) and concentrated. The crude product was purified
by chromatography eluting with ethyl acetate/dichloromethane
(1:4) and recrystallized (ethyl acetate) to yield the title compound
as an orange solid (0.108 g, 53%, over four steps); mp 205–206 °C;
(found: C, 58.4; H, 4.5. C11
4.6); (found: M , 222.0528. C11
H
10
O
H
5
+ 0.2H
2
O requires C, 58.5; H,
+
10
O
5
requires 222.0528); kmax
À1
ganic layer dried (MgSO
4
) and concentrated, to yield a mixture of
(KBr)/cm
3452, 3057, 2939, 1691, 1660, 1610, 1587;
) 5.80 (1H, s, 6-H), 4.69 (2H, d, J 4.0 Hz, CH OH),
3.86 (3H, s, OMe), 2.31 (3H, s, Me), 1.98 (1H, br s, OH); d
d
H
4
- and 6-amino derivatives (0.56 g) that was used directly. Sodium
(300 MHz; CDCl
3
2
borohydride (0.10 g, 2.6 mmol) was added to a stirred solution of
the above mixture (0.56 g, 2.6 mmol) in methanol (50 mL). After
C
(100 MHz; acetone) 177.8 (C), 175.3 (C), 160.3 (C), 156.6 (C),
2
0 min, acetone (10 mL) was added. The solvent was removed
and the residue dissolved in ethyl acetate and washed with water.
The organic layer was dried (MgSO ) and concentrated. To a solu-
149.2 (C), 125.4 (C), 116.7 (C), 105.7 (CH), 56.4 (Me), 54.0
+
2
(CH ), 7.6 (Me); m/z (EI) 222 (M , 35%), 193 (13), 151 (23), 109
4
(25), 69 (100).
tion of the crude product in acetone (40 mL) was added a solution
of potassium nitrosodisulfonate (1.00 g, 3.7 mmol) in sodium dihy-
drogen phosphate buffer (0.3 M; 40 mL). The mixture was stirred
at room temperature for 1 h. The acetone was removed in vacuo,
and the resulting residue was extracted with dichloromethane
The intermediate nitro compound methyl 5-methoxy-3-
methyl-4-nitrobenzofuran-2-carboxylate, can be isolated and
characterized. To a solution of methyl 5-methoxy-3-methylben-
zofuran-2-carboxylate 12 (0.130 g, 0.6 mmol) in acetic acid
(5 mL), cooled to À10 °C was added a mixture of concentrated ni-
tric acid (0.5 mL) and acetic acid (3 mL). The mixture was stirred
at room temperature for 1 h. The reaction mixture was poured in
an ice/water mixture and the resulting precipitate filtered off and
dried. The crude material was obtained as a 3:1 ratio of 4- and
6-nitro products (0.12 g, 80%). Purification by chromatography
eluting with dichloromethane gave methyl 5-methoxy-3-methyl-
2 4
and washed with water. The organic layer was dried (Na SO )
and concentrated. The crude material was purified by chromatog-
raphy eluting with dichloromethane/ethyl acetate (19:1) and
recrystallized (dichloromethane/ether) to yield the title compound
(
0.052 g, 20%, over three steps based on the 4-nitro starting mate-
rial), as an orange crystalline solid, mp 157–158 °C; kmax (MeOH)/
À1
nm 436 (log
e
3.18), 320 (3.66), 260 (3.98); kmax (KBr)/cm 3414,
4-nitrobenzofuran-2-carboxylate, mp 146–149 °C;
kmax (KBr)/
À1
3
060, 2990, 2915, 1683, 1657, 1607, 1587; d
H
(300 MHz; CDCl
3
)
cm 3103, 2996, 2945, 1706, 1629, 1588; d
H
(300 MHz; CDCl
3
)
5
.76 (1H, s, 6-H), 4.81 (1H, m, CHMe), 3.85 (3H, s, OMe), 3.78
7.64 (1H, d, J 9.1 Hz, ArH), 7.20 (1H, d, J 9.1 Hz, ArH), 3.99 (3H,
s, OMe), 3.96 (3H, s, OMe), 2.47 (3H, s, Me); d (75 MHz;
CDCl ) 160.0 (C), 148.8 (C), 147.6 (C), 143.3 (C), 133.6 (C), 123.0
(C), 121.5 (C), 115.1 (CH), 113.7 (CH), 57.7 (Me), 52.4 (Me), 8.9
(Me).
(
1H, d, J 10.7 Hz, OH), 2.38 (3H, s, Me), 1.45 (3H, d, J 6.7 Hz, CHMe);
(100 MHz; CDCl ) 179.2 (C), 175.4 (C), 159.8 (C), 153.4 (C), 150.6
C), 125.0 (C), 124.8 (C), 105.7 (CH), 62.4 (CH), 56.9 (Me), 24.4 (Me),
2.2 (Me).
C
d
C
3
3
(
1

3
004
J. J. Newsome et al. / Bioorg. Med. Chem. 21 (2013) 2999–3009
3
.3. Synthesis of benzothiophenequinones
4
layer was dried over MgSO and evaporated under reduced pres-
sure to yield the title compound (175 mg, 71%) as a yellow solid;
+
3
1
.3.1. 5-Methoxy-2-methylbenzothiophene-3-carboxaldehyde
5
mp 88–90 °C; (found: MH , 222.0587. C11
H
11NO
2
S + H requires
À1
222.0589);
m
max (KBr)/cm 3437, 2960, 2921, 2852, 1655, 1602,
To a mixture of dry DMF (19.6 mL, 169.40 mmol) and dry
1559, 1459, 1421, 1340, 1224, 1201; d
(1H, s, CHO), 6.96 (1H, d, J 8.5, ArH), 6.91 (1H, d, J 8.5, ArH), 6.02
(2H, br s, NH ), 3.89 (3H, s, OMe), 2.85 (3H, s, Me); d (100 MHz;
CDCl ) 184.7 (CH), 160.8 (C), 144.2 (C), 134.3 (C), 132.1 (C), 130.9
H 3
(300 MHz; CDCl ) 10.08
dichloromethane (9.1 mL) was added dropwise phosphorus oxy-
chloride (16.4 mL, 169.40 mmol) at 0 °C. The ice bath was removed.
The reaction mixture was stirred for an additional 30 min and cooled
in an ice bath. 5-Methoxy-2-methylbenzothiophene 1435 (3.02 g,
6.94 mmol) was added portionwise to the solution over 5 min at
°C. The reaction mixture was stirred and heated to 50 °C overnight,
2
C
3
(C), 123.1 (C), 110.5 (CH), 108.6 (CH), 56.5 (Me), 14.9 (Me); m/z
+
1
0
(CI) 222 (MH , 100%), 206 (5).
poured into an ice-cold aqueous sodium hydroxide (1 M). The mix-
ture was extracted with dichloromethane, dried over MgSO , fil-
3.3.4. 3-Hydroxymethyl-5-methoxy-2-methylbenzothiophene-
4,7-dione 18
4
tered, and concentrated. The residue obtained was purified by
Sodium borohydride (44 mg, 2.31 mmol) was added in one por-
tion to a solution of 4-amino-5-methoxy-2-methylbenzothioph-
ene-3-carboxaldehyde 17 (170 mg, 0.77 mmol) in dry methanol
(21 mL) cooled at 0 °C. The reaction mixture was stirred at room
temperature for 1 h. The reaction mixture was evaporated off
and the crude product obtained was purified by chromatography,
eluting with ethyl acetate–dichloromethane 1:1, to yield the
unstable amino alcohol intermediate as a yellow solid that was used
in the next step with no further purification.
To a solution of the amino alcohol intermediate dissolved in ace-
tone (50 mL) was added a solution of potassium nitrosodisulfonate
(907 mg, 3.32 mmol) in sodium dihydrogen phosphate buffer
(0.3 M; 40 mL). The reaction was stirred at room temperature for
1 h. The excess acetone was removed in vacuo. The resulting residue
was extracted with dichloromethane and the combined organic
layer evaporated off. The residue obtained was purified by chroma-
tography, eluting with dichloromethane, to yield the title compound
chromatography, eluting with dichloromethane, to yield the title
+
compound (390 mg, 20%) as a yellow oil; (found: MH , 207.0477.
À1
C
2
1
11
H
10
O
2
S + H requires 207.0480);
m
max (film)/cm 2957, 2930,
834, 1735, 1670, 1597, 1559, 1455, 1413, 1351, 1270, 1247, 1224,
151; d (300 MHz; CDCl ) 10.31 (1H, s, CHO), 8.14 (1H, d, J 2.5, 4-
H
3
H), 7.60 (1H, d, J 9.0, 7-H), 7.00 (1H, dd, J 9.0, 2.5, 6-H), 3.88 (3H, s,
OMe), 2.89 (3H, s, Me); d (75 MHz; CDCl ) 184.0 (CH), 159.6 (C),
59.3 (C), 138.9 (C), 130.5 (C), 129.3 (C), 122.6 (CH), 116.0 (CH),
C
3
1
1
+
06.4 (CH), 56.1 (Me), 14.8 (Me); m/z (CI) 207 (MH , 100%), 179
(
10); and the 4-formyl derivative (809 mg; 40%) as a beige solid;
+
mp 119–120 °C; (found: MH , 207.0477. C11
2
H
10SO
2
+ H requires
À1
07.0480);
m
max (KBr)/cm 3421, 2910, 2846, 1661, 1566, 1454,
1
H 3
430, 1314, 1243, 1183, 1131, 1071; d (300 MHz; CDCl ) 10.68
(
1H, s, CHO), 8.09 (1H, br s, 3-H), 7.86 (1H, d, J 8.8, ArH), 6.96 (1H,
d, J 8.8, ArH), 3.97 (3H, s, OMe), 2.63 (3H, s, Me); d (100 MHz; CDCl
90.7 (CH), 162.3 (C), 148.0 (C), 139.9 (C), 134.1 (C), 129.7 (CH),
C
3
)
1
1
2
22.7 (CH), 118.1 (C), 108.6 (CH), 55.8 (Me), 17.0 (Me); m/z (CI)
(75 mg, 41%) as a yellow crystalline solid; mp 196–198 °C; (found:
+
+
07 (MH , 100%), 179 (10).
MH , 239.0372. C11
H
10
O
4
S + H requires 239.0378); kmax (acetoni-
trile)/nm 200 (log
(3.21);
e
4.14), 232 (4.12), 288 (4.18), 348 (3.47), 416
À1
3
.3.2. 5-Methoxy-2-methyl-4-nitrobenzothiophene-3-
m
max (KBr)/cm 3425, 2925, 2852, 1663, 1636, 1601, 1447,
carboxaldehyde 16
To a solution of 5-methoxy-2-methylbenzothiophene-3-car-
boxaldehyde 15 (388 mg, 1.88 mmol) in acetic acid (3.2 mL),
1344, 1324, 1255, 1220, 1120, 1086, 1070; d
5.96 (1H, s, 6-H), 4.68 (2H, s, CH OH), 3.88 (3H, s, OMe), 2.53 (3H,
s, Me); d (100 MHz; CDCl ) 179.5 (C), 178.1 (C), 160.1 (C), 144.4
(C), 142.9 (C), 138.4 (C), 137.0 (C), 108.1 (CH), 56.9 (Me), 56.5
H 3
(300 MHz; CDCl )
2
C
3
cooled to 0–5 °C was added a mixture of nitric acid (638
ll,
+
9
.42 mmol) in acetic acid (4.7 mL). The mixture was stirred at room
2
(CH ), 13.7 (Me); m/z (CI) 239 (MH , 85%), 223 (50), 221 (100).
temperature overnight. The reaction mixture was poured into ice/
water, neutralized with a saturated aqueous sodium hydrogen car-
bonate, extracted with dichloromethane, dried over MgSO , fil-
4
3.3.5. Ethyl 5-methoxy-3-methyl-4-nitrobenzothiophene-2-
carboxylate 20
tered, and the filtrate evaporated under reduced pressure and
azeotroped with toluene to remove the remaining acetic acid.
The crude product obtained was purified by chromatography, elut-
To a solution of ethyl 5-methoxy-3-methylbenzothiophene-2-
3
6
carboxylate 19 (480 mg, 1.92 mmol) in acetic acid (3.2 mL),
cooled to 0–5 °C was added a mixture of nitric acid (1.3 mL,
19.20 mmol) in acetic acid (4.8 mL). The mixture was stirred at
room temperature overnight. The reaction mixture was dropped
ing with dichloromethane, to yield the title compound (286 mg;
+
6
C
2
1
1%) as a yellow solid; mp 205–207 °C; (found: MH , 252.0333.
À1
11
H NO
9 4
S + H requires 252.0331);
m
max (KBr)/cm 2975, 2941,
845, 2771, 1679, 1598, 1536, 1459, 1425, 1375, 1271, 1117,
082; d (300 MHz; CDCl ) 10.05 (1H, s, CHO), 7.80 (1H, d, J 8.9,
4
in ice/water, extracted with dichloromethane, dried over MgSO ,
filtered, evaporated and azeotroped with toluene to remove the
acetic acid. The crude product obtained was purified by chroma-
tography, eluting with dichloromethane, to yield the title com-
H
3
ArH), 7.66 (1H, d, J 8.9, ArH), 3.97 (3H, s, OMe), 2.90 (3H, s, Me);
(75 MHz; CDCl ) 183.7 (CHO), 160.4 (C), 150.4 (C), 131.0 (C),
29.4 (C), 128.8 (C), 125.1 (CH), 111.4 (CH), 57.7 (Me), 16.4 (Me),
d
C
3
pound (417 mg, 74%) as a light yellow solid; mp 158–160 °C;
+
1
(found: MH , 296.0589. C13
(KBr)/cm
H
13NO
5
S + H requires 296.0592);
m
max
+
À1
one C unobserved; m/z (CI) 252 (MH , 100%), 222 (65), 206 (30),
3422, 2988, 2939, 2850, 1711, 1608, 1527, 1446,
(300 MHz; CDCl
.85 (1H, d, J 8.9, ArH), 7.26 (1H, d, J 8.9, ArH), 4.40 (2H, q, J 7.1,
1
94 (10).
1370, 1297, 1247, 1193, 1174, 1151, 1051; d
H
3
)
7
3
.3.3. 4-Amino-5-methoxy-2-methylbenzothiophene-
OCH
2
Me), 3.98 (3H, s, OMe), 2.64 (3H, s, Me), 1.41 (3H, t, J 7.1,
(100 MHz; CDCl ) 162.6 (C), 148.6 (C), 137.1 (C),
136.7 (C), 133.8 (C), 131.5 (C), 130.6 (C), 125.1 (CH), 113.3 (CH),
carboxaldehyde 17
To suspension of 5-methoxy-2-methyl-4-nitrobenzo-
thiophenecarboxaldehyde 16 (280 mg, 1.12 mmol) in ethanol
23 mL) was added tin powder (601 mg, 5.02 mmol) and hydro-
2
CH Me); d
C
3
a
+
2
61.7 (CH ), 57.3 (Me), 14.2 (Me), 12.1 (Me); m/z (CI) 296 (MH ,
100%), 279 (8), 266 (15).
(
chloric acid (3 M; 8.1 mL). The mixture was stirred and heated un-
der reflux for 1 h. Upon cooling, the reaction mixture was decanted
from the excess of tin and neutralized with a saturated aqueous
3.3.6. Ethyl 4-amino-5-methoxy-3-methylbenzothiophene-2-
carboxylate 21
solution of NaHCO
Celite and extracted with ethyl acetate. The combined organic
3
. The suspension obtained was filtered through
To a suspension of ethyl 5-methoxy-3-methyl-4-nitrobenzothi-
ophene-2-carboxylate 20 (370 mg, 1.25 mmol) in ethanol (27 mL)

J. J. Newsome et al. / Bioorg. Med. Chem. 21 (2013) 2999–3009
3005
was added tin powder (676 mg, 5.64 mmol) and hydrochloric acid
3 M; 9 mL). The mixture was stirred and heated under reflux for
h. Upon cooling, the reaction mixture was decanted from the ex-
cess of tin and neutralized with a saturated aqueous solution of
NaHCO . The suspension obtained was filtered through Celite and
extracted with ethyl acetate. The combined organic layer was dried
over MgSO , filtered and evaporated under reduced pressure to
yield the title compound (285 mg, 86%) as a yellow solid; mp
3.3.9. Methyl 3-chloro-5-methoxy-4-nitrobenzothiophene-2-
carboxylate 25
(
1
Methyl
3-chloro-5-methoxybenzothiophene-2-carboxylate
2437 (1.00 g, 3.90 mmol) was dissolved in acetic acid (20 mL). Fum-
ing nitric acid (0.25 mL) was added carefully, and the mixture was
stirred at room temperature before being heated under reflux over-
night. After cooling, the mixture was poured into water (125 mL),
and the precipitate collected, washed with water and dried to give
the title compound (1.03 g, 87%) used without further purification.
A sample was recrystallized from methanol to give yellow crystals,
3
4
+
1
2
1
1
8
20–123 °C; (found: MH , 266.0844. C13
H
15NO
3
S + H requires
À1
66.0851);
mmax (KBr)/cm 3460, 3368, 2968, 2925, 1705, 1621,
+
35
536, 1471, 1440, 1332, 1259, 1209, 1178, 1144, 1094, 1055,
009; d (300 MHz; CDCl ) 7.11 (1H, d, J 8.6, ArH), 7.03 (1H, d, J
.6, ArH), 4.35 (2H, q, J 7.0, OCH Me), 3.89 (3H, s, OMe), 3.06
Me); NH not observed; d
) 165.7 (C), 145.5 (C), 143.3 (C), 136.7 (C), 136.5
C), 130.5 (C), 127.3 (C), 115.3 (CH), 113.4 (CH), 63.1 (CH ), 59.0
Me), 18.1 (Me), 16.4 (Me); m/z (CI) 266 (MH , 100%), 265 (40),
mp 170–173 °C; (found: M , 300.9821. C11
H
8
ClNO
5
S requires
À1
H
3
300.9812); kmax (KBr)/cm 1721, 1607, 1534, 1514, 1438, 1374,
2
1293, 1241; d
(1H, d, J 9.0, ArH), 3.99 (3H, s, OMe), 3.97 (3H, s, OMe); d
(100 MHz; CDCl ) 160.8 (C), 149.4 (C), 132.1 (C), 130.1 (C), 127.4
H 3
(300 MHz; CDCl ) 7.86 (1H, d, J 9.0, ArH), 7.33
(
(
(
(
3H, s, Me), 1.40 (3H, t, J 7.0, CH
2
2
C
C
100 MHz; CDCl
3
3
2
(C), 125.4 (CH), 122.8 (C), 114.6 (CH), 57.4 (Me), 52.9 (Me); 1 C
+
+
unobserved; m/z (EI) 303/301 (MH , 100%).
2
20 (10).
3
.3.10. Methyl 4-amino-5-methoxybenzothiophene-2-
3
.3.7. 4-Amino-5-methoxy-3-methylbenzothiophene-2-
carboxylate 26
methanol 22
To suspension of lithium aluminum hydride (162 mg,
.23 mmol) in dry THF (6 mL) at 0 °C was added a solution of
ethyl 4-amino-5-methoxy-3-methylbenzothiophene-2-carboxyl-
ate 21 (280 mg, 1.06 mmol) in dry THF (3 mL). The mixture was
allowed to warm up to room temperature and stirred for 2 h.
The mixture was cooled to 0 °C and quenched by the addition
of water (0.5 mL), aqueous sodium hydroxide (1 M; 0.5 mL) and
silica gel. The granular precipitate was filtered off through a
The 3-chloro-4-nitrobenzothiophene 25 (1.00 g, 3.3 mmol) and
sodium acetate (0.60 g, 7.3 mmol) were dissolved in methanol
(200 mL) and THF (55 mL). Palladium-on-carbon (5%; 0.5 g) was
added, and the mixture was shaken under a hydrogen atmosphere
for 5 h. The mixture was filtered through Celite, the filtrate evapo-
rated, and the residue purified by chromatography eluting with
light petroleum / ethyl acetate (4:1) to give the title compound
(0.69 g, 88%) as a pale yellow solid, mp 149–151 °C; (found: C,
a
4
55.6; H, 4.7; N, 5.7. C11
H11NO
3
S requires C, 55.7; H, 4.7; N, 5.9%);
À1
pad of Celite. The filtrate was dried over MgSO
4
, filtered and con-
k
max (KBr)/cm 3445, 3359, 1702, 1625, 1528, 1470, 1206; d
H
centrated in vacuo to yield the title compound (188 mg, 80%)
as an orange solid; mp 114–115 °C; (found: MH , 224.0753.
(300 MHz; CDCl
ArH), 7.09 (1H, d, J 8.6, ArH), 4.27 (2H, br s, NH
OMe), 3.91 (3H, s, OMe); d (100 MHz; CDCl
(C), 136.0 (C), 132.4 (C), 131.8 (C), 127.8 (C), 126.5 (CH), 113.7
(CH), 111.4 (CH), 56.8 (Me), 52.3 (Me); m/z (EI) 237 (M , 70%),
3
) 8.01 (1H, d, J 0.8, 3-H), 7.20 (1H, dd, J 8.6, 0.8,
), 3.93 (3H, s,
) 163.3 (C), 142.9
+
2
À1
C
3
1
11
H
11NO
2
S + H requires 224.0745);
m
max (KBr)/cm 3436, 3352,
C
3
273, 2923, 2851, 1599, 1462, 1265, 1203, 1157, 1136, 1044,
019; d (300 MHz; CDCl ) 7.12 (1H, d, J 8.6, ArH), 6.90 (1H, d,
OH), 3.88 (3H, s, OMe), 2.54 (3H, s,
not observed; d (100 MHz; CDCl ) 142.3 (C),
34.8 (C), 131.7 (C), 131.1 (C), 127.5 (C), 126.8 (C), 110.6 (CH),
+
H
3
J 8.6, ArH), 4.75 (2H, s, CH
Me); OH, NH
2
222 (100), 194 (35), 83 (68).
2
C
3
1
1
6
3.3.11. Methyl 5-methoxy-4,7-dioxobenzothiophene-2-
carboxylate 27
+
2
09.1 (CH), 56.9 (CH ), 55.5 (Me), 13.4 (Me); m/z (CI) 224 (MH ,
+
0%), 223 (M , 80), 206 (100), 194 (15).
A mixture of 4-aminobenzothiophene 26 (50 mg, 0.21 mmol)
and potassium nitrosodisulfonate (230 mg, 0.84 mmol) in acetone
(7.5 mL) and sodium dihydrogen phosphate buffer (0.3 M;
7.5 mL) was stirred at room temperature overnight. The acetone
was evaporated, water (70 mL)was added, and the mixture was ex-
tracted with dichloromethane (3 Â 25 mL). The combined extracts
3
4
.3.8. 2-Hydroxymethyl-5-methoxy-3-methylbenzothiophene-
,7-dione 23
To
a solution of 4-amino-5-methoxy-3-methylbenzo-thio-
phene-2-methanol 22 (180 mg, 0.81 mmol) in acetone (48 mL)
was added a solution of potassium nitrosodisulfonate (889 mg,
2 4
were dried (Na SO ), evaporated and the residue purified by chro-
3
3
.26 mmol) in sodium dihydrogen phosphate buffer (0.3 M;
9 mL). The reaction was stirred at room temperature for 1 h.
matography eluting with dichloromethane to give the title com-
pound (40 mg, 75%) as a dark yellow solid, mp 234 °C; (found: C,
The excess acetone was removed in vacuo. The resulting residue
was extracted with dichloromethane and the combined organic
layer concentrated. The residue obtained was stirred at room tem-
perature in a 1:1 mixture of hydrochloric acid (2 M)-acetone for
52.8; H, 3.3. C11
2960, 1720, 1689, 1633, 1597, 1534, 1439, 1244, 1268, 1085; d
(300 MHz; CDCl ) 8.15 (1H, s, 3-H), 6.08 (1H, s, 6-H), 3.94 (3H, s,
OMe), 3.90 (3H, s, OMe); d (75 MHz; CDCl ) 179.6 (C), 175.1 (C),
8 5
H O S requires C, 52.4; H, 3.2%); kmax (KBr) 3088,
H
3
C
3
1
h. The acetone was removed in vacuo. The resulting residue
was extracted with dichloromethane. The organic layer was
washed with water, dried over MgSO , filtered and evaporated un-
161.4 (C), 160.8 (C), 148.0 (C), 139.3 (C), 138.5 (C), 130.7 (CH),
109.0 (CH), 57.0 (Me), 53.1 (Me); m/z (EI) 252 (M , 100%), 237 (34).
+
4
der reduced pressure to yield the title compound (130 mg, 67%) as
an orange crystalline solid; mp 215–216 °C; (found: MH ,
3.3.12. 4-Amino-5-methoxybenzothiophene-2-methanol 28
A solution of the benzothiophene 26 (2.00 g, 8.43 mmol) in dry
THF (28 mL) was added dropwise to lithium aluminum hydride
(1.07 g, 28.1 mmol) in THF (55 mL). The mixture was stirred at
room temperature for 16 h, and then water was carefully added.
The mixture was filtered through Celite, and extracted with ethyl
acetate (3 Â 30 mL). The combined extracts were washed with
+
2
39.0379. C11
10 4
H O S + H requires 239.0378); kmax (acetonitrile)/
nm 200 (log
e
3.86), 232 (3.88), 284 (3.90), 352 (3.24), 412 (3.09);
À1
m
1
5
max (KBr)/cm 3385, 3054, 2920, 2843, 1677, 1638, 1600, 1538,
461, 1442, 1342, 1327, 1253, 1227, 1134; d
.96 (1H, s, 6-H), 4.87 (2H, s, CH OH), 3.87 (3H, s, OMe), 2.47
(100 MHz; CDCl ) 180.0 (C),
76.9 (C), 160.4 (C), 146.6 (C), 143.2 (C), 136.5 (C), 135.7 (C),
H 3
(300 MHz; CDCl )
2
(
3H, s, Me), 1.71 (1H, br s, OH); d
C
3
water (30 mL), dried (MgSO
recrystallized from methanol to give the title compound (1.38 g,
78%), mp 130–132 °C; (found: C, 57.2; H, 5.3; N, 6.6. C10 re-
quires C, 57.4; H, 5.3; N, 6.7%); kmax (KBr)/cm- 3401, 3310, 3016,
4
), and evaporated to a solid that was
1
1
1
+
07.7 (CH), 58.2 (CH
00%), 221 (30).
2
), 56.7 (Me), 13.2 (Me); m/z (CI) 239 (MH ,
H11NO
2
1

3
006
J. J. Newsome et al. / Bioorg. Med. Chem. 21 (2013) 2999–3009
À1
1
(
602, 1480, 1463; d
1H, s, 3-H), 6.95 (1H, d, J 8.6, ArH), 4.87 (2H, s, CH
OMe); OH, NH not observed; d (75 MHz; CDCl
43.3 (C), 133.7 (C), 130.4 (C), 128.6 (C), 117.1 (CH), 111.9 (CH),
H
(300 MHz; CDCl
3
) 7.19 (1H, d, J 8.6, ArH), 7.09
), 3.90 (3H, s,
) 144.0 (C),
C
12
H
10
O
5
S requires C, 54.1; H, 3.8%); kmax (KBr)/cm 3091, 2983,
2963, 1725, 1677, 1642, 1602, 1534, 1467, 1438, 1333, 1232,
1141, 1085, 1030, 962, 854, 798; d (300 MHz; CDCl ) 7.49 (1H,
s, 3-H), 5.99 (1H, s, 6-H), 5.26 (2H, s, CH ), 3.88 (3H, s, OMe),
2.13 (3H, s, Me); d (75 MHz; CDCl ) 179.7 (C), 175.6 (C), 170.3
(C), 160.3 (C), 146.0 (C), 144.6 (C), 138.7 (C), 125.5 (CH), 108.5
2
2
C
3
H
3
1
1
2
+
10.7 (CH), 61.0 (CH
2
), 56.8 (Me); m/z (EI) 209 (M , 62%), 194
C
3
(
100), 166 (25).
+
(
2
CH), 60.2 (CH ), 56.8 (Me), 20.7 (Me); m/z (EI) 266 (M , 32%),
3
2
.3.13. 2-Hydroxymethyl-5-methoxybenzothiophene-4,7-dione
9
251 (8), 224 (100), 207 (36), 178 (20).
A mixture of the 4-aminobenzothiophene 28 (0.32 g, 1.5 mmol)
and potassium nitrosodisulfonate (1.61 g, 6.0 mmol) in acetone
47 mL) and sodium dihydrogen phosphate buffer (0.3 M; 47 mL)
was stirred at room temperature for 1 h. Work-up as described
above gave the title compound (0.29 g, 88%) as a dark orange solid,
3.3.17. 5-Methoxy-2-methylbenzothiophene-4,7-dione 33
(a) A stirred solution of the alcohol 29 (200 mg, 0.89 mmol) in
dichloromethane (11 mL) at À10 °C was treated with triethylamine
(140 mg, 0.19 mL, 1.34 mmol). After 10 min, methanesulfonyl
chloride (110 mg, 0.08 mL, 0.98 mmol) was added, and the mixture
allowed to stir at room temperature overnight. Water (11 mL) and
dichloromethane (30 mL) were added, the organic layer separated,
(
mp 213 °C (from methanol); (found: C, 53.3; H, 3.3. C10
H
8
O
4
S re-
À1
quires C, 53.6; H. 3.60%); kmax (KBr)/cm
3413, 3062, 2986,
2
8
947, 1680, 1628, 1598, 1572, 1326, 1246, 1140, 1084, 1043,
64, 792; d (300 MHz; CDCl ) 7.43 (1H, s, 3-H), 5.99 (1H, s, 6-
OH), 3.88 (3H, s, OMe), 2.05 (1H, t, J 6.0,
(100 MHz; CDCl ) 180.2 (C), 175.9 (C), 160.6 (C),
56.3 (C), 142.3 (C), 139.2 (C), 121.5 (CH), 108.7 (CH), 58.8 (CH ),
4
washed with water (50 mL), brine (50 mL), dried (MgSO ) and
evaporated to give the mesylate as a yellow solid, used without
any purification.
(b) The above product was dissolved in THF (11 mL) and added
to lithium aluminum hydride (330 mg, 8.63 mmol) in THF (9 mL).
The mixture was stirred overnight at room temperature, before
the careful addition of water. The mixture was filtered through Cel-
ite, and the filtrate extracted with ethyl acetate (3 Â 50 mL). The
combined extracts were washed with saturated aqueous sodium
H
3
H), 4.91 (2H, d, J 6.0, CH
CH OH); d
1
5
2
2
C
3
2
+
7.3 (Me); m/z (EI) 224 (M , 100%) 209 (28), 194 (35), 125 (26).
3
3
.3.14. 4-Amino-5-methoxybenzothiophene-2-carboxaldehyde
0
A
solution of the benzothiophene-2-methanol 28 (0.90 g,
4
hydrogen carbonate (10 mL), water (10 mL), dried (MgSO ) and
4
.30 mmol) in dichloromethane (100 mL) was stirred with manga-
evaporated. The residue was purified by chromatography to give
nese(IV) oxide (3.78 g, 43 mmol) under reflux for 24 h. The solution
was filtered through Celite, washed through with dichlorometh-
ane, and the combined filtrate and washings concentrated under
vacuum. The residue was purified by chromatography eluting with
light petroleum/ethyl acetate (4:1) to give the title compound
the title compound (9 mg, 5%) as a yellow solid, mp 217–220 °C;
+
(found: M , 208.0194. C10
H
8
O
3
S requires 208.0194); kmax (KBr)/
À1
cm
3066, 2923, 1680, 1640, 1596, 1535, 1468, 1329, 1246,
1083, 968, 858, 792; d
(1H, s, 6-H), 3.86 (3H, s, OMe), 2.56 (3H, s, Me); d
CDC1
H
(300 MHz; CDCl
3
) 7.22 (1H, s, 3-H), 5.94
(75 MHz;
3
) 179.9 (C), 175.9 (C), 160.0 (C), 148.5 (C), 142.4 (C), 139.4
C
(
0.29 g, 33%) as a pale yellow solid, mp 128–130 °C; (found: C,
5
7.9; H, 4.3; N, 6.5. C10
H
9
NO
2
S requires C, 57.9; H, 4.4; N, 6.8%);
(C), 124.1 (CH), 108.3 (CH), 56.7 (Me), 15.9 (Me); m/z (EI) 208
(M , 64%), 193 (21), 178 (30), 149 (47), 71 (44), 69 (100).
À1
+
k
max (KBr)/cm
3474, 3373, 2826, 1667, 1520, 1483;
) 10.00 (1H, s, CHO), 7.97 (1H, s, H-3), 7.19 (1H,
d, J 8.6, ArH), 7.09 (1H, d, J 8.6, ArH), 4.42 (2H, br s, NH ), 3.90
) 184.4 (CH), 143.0 (C), 142.2 (C),
d
H
(
300 MHz; CDCl
3
2
3.4. Synthesis of indazolequinones
(
C 3
3H, s, OMe); d (75 MHz; CDCl
1
5
36.2 (C), 133.2 (C), 130.8 (CH), 127.7 (C), 114.6 (CH), 111.9 (CH),
6.7 (Me); m/z (EI) 207 (M , 70%), 192 (100), 164 (40), 77 (35).
3.4.1. 5,7-Dimethoxy-3-methylindazole 35
+
To
.81 mmol) in dry dichloromethane (44 mL) were added bis(tri-
chloroethyl) azodicarboxylate (3.35 g, 8.81 mmol) and BF
ÁEtO
(539 l, 4.40 mmol). The mixture was stirred overnight at room
a solution of 3,5-dimethoxyacetophenone 34 (1.58 g,
8
3
.3.15. 2-Formyl-5-methoxybenzothiophene-4,7-dione 31
3
A
mixture of the 4-aminobenzothiophene 30 (50 mg,
l
0
.24 mmol) and potassium nitrosodisulfonate (260 mg, 0.96 mmol)
temperature, quenched with aqueous ammonium acetate solution
in acetone (7.5 mL) and sodium dihydrogen phosphate buffer
0.3 M; 7.5 mL) was stirred at room temperature overnight.
Work-up as described above gave the title compound (42 mg,
(25%; 70 mL) and extracted with ethyl acetate (4 Â 70 mL). The or-
(
2 4
ganic layer was dried over Na SO , filtered and evaporated under
reduced pressure. The crude product obtained was purified by flash
chromatography. A mixture of hydrazine intermediate and starting
material (20:1) was obtained. To a solution of the above product in
glacial acetic acid (49 mL) was added zinc dust (4.94 g,
75.55 mmol). The mixture was stirred at room temperature for
1 h and water (50 mL) followed by aqueous sodium hydroxide
(1 M) were added until pH = 10. The mixture was extracted with
7
C
1
9%) as a dark orange solid, mp 218 °C; (found: C, 53.9; H, 2.8.
À1
10
6
H O
4
S requires C, 54.0; H, 2.7%); kmax (KBr)/cm 3070, 1688,
649, 1600, 1522, 1325, 1249, 1148, 1079, 867; d
) 10.03 (1H, s, CHO), 8.16 (1H, s, 3-H), 6.12 (1H, s, 6-H),
.92 (3H, s, OMe); d (75 MHz; CDCl ) 183.0 (CH), 179.4 (C),
H
(300 MHz;
CDCl
3
3
1
1
C
3
74.9 (C), 161.0 (C), 149.6 (C), 147.6 (C), 138.7 (C), 132.6 (CH)
+
09.2 (CH), 57.1 (Me); m/z (EI) 222 (M , 100%), 207 (32), 192 (37).
2 4
ethyl acetate. The organic layer was dried over Na SO , filtered
and evaporated under reduced pressure. The crude product was
purified by flash chromatography, eluting with ethyl acetate–light
petroleum 1:1 to yield the title compound (590 mg; 35%) as a beige
solid; mp 154–156 °C (lit., mp 155–156 °C); d (300 MHz; CDCl )
H 3
11.72 (1H, br s, NH), 6.64 (1H, d, J 2.3, ArH), 6.44 (1H, t, J 2.3, ArH),
3
3
.3.16. 2-Acetoxymethyl-5-methoxybenzothiophene-4,7-dione
2
3
8
Acetic anhydride (2.7 mL) was added to a solution of the alcohol
9 (75 mg, 0.33 mmol) in pyridine (16 mL), and the mixture was
2
stirred overnight at room temperature. The mixture was diluted
with water (27 mL), and extracted with dichloromethane
3.95 (3H, s, OMe), 3.80 (3H, s, OMe), 2.45 (3H, s, Me).
(
3 Â 50 mL). The combined extracts were washed with water
3.4.2. 5,7-Dimethoxy-3-methyl-4-nitroindazole 36
To a solution of 5,7-dimethoxy-3-methylindazole 35 (200 mg,
1.04 mmol) in acetic acid (8 mL), cooled to 0–5 °C, was added a
(
50 mL) and brine (50 mL), dried (MgSO ) and evaporated. The res-
4
idue was purified by chromatography eluting with dichlorometh-
ane/ethyl acetate (19:1) to give the title compound as a yellow
solid (58 mg, 66%), mp 153–155 °C; (found: C, 54.0; H, 3.7.
mixture of nitric acid (69
ll, 1.04 mmol) in acetic acid (1 mL).
The mixture was stirred at room temperature for 1 h. The reaction

J. J. Newsome et al. / Bioorg. Med. Chem. 21 (2013) 2999–3009
3007
was quenched by addition of brine, extracted with ethyl acetate,
dried over MgSO , filtered, evaporated and azeotroped with tolu-
ene to remove the acetic acid. The crude product obtained was
purified by flash chromatography, eluting with ethyl acetate/light
petroleum (1:1), to yield the title compound (201 mg, 81%) as a
bright yellow crystalline solid, recrystallized from dichlorometh-
3.4.5. 5-Methoxy-1,3-dimethylindazole-4,7-dione 39
4
To a solution of 4-amino-5,7-dimethoxy-1,3-dimethylindazole
38 (0.250 g, 1.15 mmol) in acetone (70 mL) was added a solution
of potassium nitrosodisulfonate (1.250 g, 4.59 mmol) in sodium
dihydrogen phosphate buffer (0.3 M, 58 mL). The reaction was stir-
red at room temperature for 1 h. The excess acetone was removed
in vacuo. The resulting residue was extracted with dichlorometh-
ane–pentane; mp 219–220 °C; (found: C, 50.7; H, 4.5; N, 17.6.
+
C
2
3
10
H
11
N
3
O
4
requires C, 50.6; H, 4.7; N, 17.7%); (found: MH ,
4
ane. The organic layer was washed with water, dried over MgSO ,
À1
38.0829. C10
H
11
N
3
O
4
+ H requires 238.0828);
m
max (KBr)/cm
filtered, evaporated under reduced pressure to yield the title com-
pound (208 mg, 78%) as a bright yellow crystalline solid, recrystal-
lized from dichloromethane–pentane; mp 185–186 °C; (found: C,
398, 3149, 2917, 1597, 1517, 1308, 1223, 1115, 1059; d
) 13.43 (1H, s, NH), 6.53 (1H, s, ArH), 4.05 (3H, s,
OMe), 4.00 (3H, s, OMe), 2.50 (3H, s, Me); d (100 MHz; d -DMSO)
48.9 (C), 148.2 (C), 138.6 (C), 129.0 (C), 124.3 (C), 114.7 (C), 94.8
H
(
300 MHz; CDCl
3
C
6
10 2 3
58.2; H, 4.8; N, 13.6. C10H N O requires C, 58.2; H, 4.9; N,
+
1
(
13.6%); (found: M , 206.0683. C10
(acetonitrile)/nm 272 (log
1680, 1657, 1590, 1529, 1510, 1340, 1216, 1018; d
CDCl ) 5.79 (1H, s, 6-H), 4.16 (3H, s, NMe), 3.85 (3H, s, OMe),
.49 (3H, s, Me); d (75 MHz; CDCl ) 177.8 (C), 176.1 (C), 162.0
H
10
N
2
O
3
requires 206.0691); kmax
+
À1
CH), 58.3 (Me), 56.6 (Me), 13.2 (Me); m/z (CI) 238 (MH , 95%), 221
e 3.91), 320 (3.84); mmax (KBr)/cm
(
55), 207 (100), 192 (80).
H
(300 MHz;
3
2
C
3
3
.4.3. 5,7-Dimethoxy-1,3-dimethyl-4-nitroindazole 37
(
(
(
C), 149.0 (C), 137.4 (C), 117.1 (C), 107.3 (CH), 57.0 (Me), 38.4
Me), 12.9 (Me); m/z (EI) 206 (M , 100%), 191 (85), 177 (62), 123
30).
To a stirred solution of 5,7-dimethoxy-3-methyl-4-nitroindaz-
ole 36 (116 mg, 0.60 mmol) in DMSO (2 mL) was added potas-
sium hydroxide (135 mg, 2.42 mmol). The mixture was stirred
+
at room temperature for 30 min and iodomethane (150
ll,
3
.4.6. 3-Hydroxymethyl-5-methoxy-1-methylindazole-4,7-
dione 40
A solution of 5-methoxy-1,3-dimethylindazole-4,7-dione 39
51 mg, 0.25 mmol), AIBN (12 mg, 0.07 mmol) and N-bromosuccin-
imide (86 mg, 0.49 mmol) in CCl (3 mL) was purged five times
2
.42 mmol) was added dropwise to the solution. The reaction
mixture was then stirred at room temperature for a further 4 h.
The crude mixture was diluted with ethyl acetate, washed thor-
oughly with hydrochloric acid (2 M), dried over MgSO , filtered
4
and evaporated. The crude product obtained was purified by flash
chromatography, eluting with ethyl acetate/light petroleum (1:1),
to yield the title compound (71 mg, 49%) as a bright yellow crys-
talline solid, recrystallized from dichloromethane–pentane; mp
(
4
with vacuum followed with nitrogen. The reaction mixture was
heated at reflux under a nitrogen atmosphere overnight. The mix-
ture was concentrated under reduced pressure and purified by
flash chromatography, eluting with dichloromethane, to yield the
bromide intermediate, which was used in the next step with no fur-
ther purification. The bromide intermediate dissolved in acetone
1
68–169 °C; (found: C, 52.8; H, 5.1; N, 16.9. C11
H
13
N
3
O
4
requires
re-
+
C, 52.6; H, 5.2; N, 16.7%); (found: M , 251.0910. C11
H
13
N
3
O
4
À1
quires 251.0906);
522, 1324, 1280, 1220, 1050; d
ArH), 4.19 (3H, s, NMe), 4.02 (3H, s, OMe), 3.97 (3H, s, OMe),
.40 (3H, s, Me); d (75 MHz; CDCl ) 149.0 (C), 148.6 (C), 138.9
m
max (KBr)/cm
3435, 2929, 2841, 1577,
(
(
9 mL) and water (4 mL) was added to a suspension of silver nitrate
92 mg, 0.54 mmol) in an aqueous solution of acetone (50%;
1
H
(300 MHz; CDCl
3
) 6.45 (1H, s,
1
3 mL). The reaction mixture was heated under reflux overnight
and extracted with ethyl acetate. The combined organic layer
was dried over MgSO , filtered and evaporated under reduced pres-
2
C
3
(
(
C), 128.9 (C), 126.3 (C), 117.1 (C), 94.6 (CH), 58.9 (Me), 56.4
Me), 39.0 (Me), 13.4 (Me); m/z (EI) 251 (M , 100%), 234 (28),
+
4
sure. The residue obtained was purified by flash chromatography,
eluting with light petroleum–ethyl acetate 1:2, to yield the title
compound (29 mg; 36%) as a yellow solid; mp 177–179 °C; (found:
MH , 223.0721. C10
2
21 (29), 206 (72), 175 (40). No nOe enhancement observed after
pre-irradiation of the Me group at 2.39 ppm. 4.9% nOe enhance-
ment observed on the aromatic proton at 6.45 ppm after pre-irra-
diation at 4.02 ppm (5-OMe). 2.3% nOe enhancement observed at
+
H
10
N
2
O
4
+ H requires 223.0719); kmax (acetoni-
À1
trile)/nm 276 (log
e
4.23), 308 (3.74);
m
max (KBr)/cm 3368, 2928,
3
.97 ppm (7-OMe) after pre-irradiation of NMe at 4.19 ppm. 3.5%
1
689, 1653, 1593, 1525, 1505, 1240, 1204, 1015; d
CDCl ) 5.87 (1H, s, 6-H), 4.86 (2H, s, CH OH), 4.17 (3H, s, NMe),
.90 (3H, s, OMe); d (75 MHz; CDCl ) 177.5 (C), 177.0 (C), 161.8
C), 152.6 (C), 137.8 (C), 117.4 (C), 107.7 (CH), 58.2 (CH ), 57.2
Me), 38.7 (Me); m/z (ES) 223 (MH , 95%), 164 (35), 149 (30), 90
H
(300 MHz;
nOe enhancements observed on the aromatic proton at 6.45 ppm
and 1.3% nOe enhancement observed at 4.02 ppm (5-OMe) after
pre-irradiation at 3.97 ppm (7-OMe).
3
2
3
(
(
C
3
2
+
3
.4.4. 4-Amino-5,7-dimethoxy-1,3-dimethylindazole 38
(100).
To a suspension of 5,7-dimethoxy-1,3-dimethyl-4-nitroindaz-
ole 37 (55.3 mg, 0.22 mmol) in ethanol (3.7 mL) were added tin
3.5. Synthesis of benzisoxazolequinones
powder (120 mg, 0.99 mmol) and hydrochloric acid (3 M;
1
.5 mL). The mixture was heated under reflux for 1 h. Upon cool-
3.5.1. (E)-2-Hydroxy-5-methoxyacetophenone oxime 42
ing, the solution was decanted from the excess tin and neutral-
ized with a saturated aqueous solution of sodium hydrogen
carbonate. The precipitate obtained was extracted with ethyl ace-
tate. The organic layer was filtered through a pad of Celite, dried
To a solution of 2-hydroxy-5-methoxyacetophenone 41 (2.50 g,
15.1 mmol) and hydroxylamine hydrochloride (2.10 g, 30.1 mmol)
in ethanol (50 mL) was added dropwise dry pyridine (2.68 mL,
30.1 mmol). The reaction mixture was stirred at room temperature
overnight and evaporated under reduced pressure. The crude prod-
uct obtained was purified by flash chromatography, eluting with
4
over MgSO , filtered and evaporated under reduced pressure to
yield the title compound (47 mg; 96%) as a colorless solid; mp
+
7
9–80 °C; (found: M , 221.1163. C11
H
15
N
3
O
2
requires 221.1164);
ethyl acetate–light petroleum 1:2, to yield the title compound
À1
42
m
max (KBr)/cm
3434, 3343, 2927, 2836, 1590, 1527, 1453,
(300 MHz; CDCl ) 6.48 (1H, s, 6-H),
.11 (3H, s, NMe), 3.84 (3H, s, OMe), 3.82 (3H, s, OMe), 2.66
(75 MHz; CDCl ) 139.9 (C), 138.4 (C), 138.3 (C),
(1.68 g, 98%) as a colorless solid; mp 112–113 °C (lit.,
mp
+
1
4
351, 1271, 1197, 1111; d
H
3
121 °C); (found: MH , 182.0813. C9H11NO + H requires
182.0817); mmax (KBr)/cm 3348, 2964, 2920, 2831, 1641, 1497,
3
À1
(
3H, s, Me); d
C
3
1404, 1368, 1284, 1232, 1208, 1176, 1052, 1011; dH (300 MHz;
1
3
30.5 (C), 123.2 (C), 116.3 (C), 99.3 (CH), 59.2 (Me), 56.4 (Me),
8.3 (Me), 14.5 (Me); m/z (EI) 221 (M , 27%), 206 (100), 191
CDCl ) 10.70 (1H, br s, OH), 7.53 (1H, br s, OH), 7.17 (1H, d, J 3.0,
6-H), 7.11 (1H, dd, J 9.0, 3.0, 4-H), 6.93 (1H, d, J 9.0, 3-H), 3.80
3
+
(
14), 163 (8).
(3H, s, OMe), 2.62 (3H, s, Me); dC (100 MHz; CDCl ) 159.3 (C),
3

3
008
J. J. Newsome et al. / Bioorg. Med. Chem. 21 (2013) 2999–3009
1
5
52.3 (C), 151.6 (C), 118.7 (C), 117.7 (CH), 116.5 (CH), 113.1 (CH),
6.0 (Me), 10.9 (Me); m/z (CI) 182 (MH , 63%), 166 (100), 151
3.6. Biology
+
(
15), 125 (5).
3.6.1. HPLC analysis
Reduction of the quinones was followed by HPLC using an All-
3
.5.2. 5-Methoxy-3-methylbenzisoxazole 43
Diisopropyl azodicarboxylate (8.5 mL, 43.5 mmol) was added
tech C18 (5
l
m, 250 mm  4.6 mm) column with a Waters HPLC
system (2487 Dual k Absorbance detector, two 515 HPLC pumps,
717plus Autosampler, Millennium32 Chromatography Manager).
The solvent program used a linear gradient of 5–80% B over
10 min, 80% B for 5 min, then 80% B to 5% B over 5 min (solution
A, 10 mM potassium phosphate buffer, pH 6.0; solution B, metha-
nol). Reactions were run in 25 mM Tris–HCl (pH 7.4) containing
dropwise to a stirred solution of the oxime 42 (5.24 g, 29.0 mmol)
and triphenylphosphine (11.35 g, 43.5 mmol) in dry THF (192 mL)
under nitrogen. The reaction mixture was stirred at room temper-
ature overnight, and evaporated under reduced pressure. The crude
product was purified by chromatography, eluting with ethyl ace-
tate/light petroleum (1:3), to yield the title compound (4.72 g,
200 lM NADH (Sigma), 50 lM quinone, and recombinant human
43
1
00%) as a yellow solid; mp 28–30 °C (lit., mp not given); (found:
NQO1 (gift from David Ross, University of Colorado, Denver, CO).
NADH oxidation was quantified at 340 nm following 30–40 min
incubations at 22 °C.
+
À1
MH , 164.0708. C
H
9 9
NO
972, 2935, 1730, 1528, 1484, 1458, 1439, 1252, 1219, 1127,
079, 1028; d (300 MHz; CDCl ) 7.44 (1H, d, J 9.0, 7-H), 7.16
2
+ H requires 164.0712); mmax (KBr)/cm
2
1
H
3
(
2
(
(
1H, dd, J 9.0, 2.3, 6-H), 6.96 (1H, d, J 2.3, 4-H), 3.87 (3H, s, OMe),
.56 (3H, s, Me); d (75 MHz; CDCl ) 158.4 (C), 156.2 (C), 154.9
3.6.2. Spectrophotometric method
C
3
Quinone reduction by recombinant human NQO1 was also
quantified using a modification of an assay that uses cytochrome
C), 122.5 (C), 120.2 (CH), 110.5 (CH), 101.3 (CH), 55.9 (Me), 10.1
+
44
Me); m/z (CI) 164 (MH , 100%), 148 (5).
c as the terminal electron acceptor. Reaction mixtures contained
1
mM NADH (Sigma), 25
lM quinone, 70 lM cytochrome c (Sigma)
3
.5.3. 5-Methoxy-3-methyl-4-nitrobenzisoxazole 44
and 0.1–3.0 g/mL rhNQO1 in 25 mM Tris–HCl (pH 7.4) with 0.07%
l
To a solution of nitric acid/sulfuric acid 9:1 (22 mL), cooled in a
BSA and 0.1% Tween-20. Reactions were run at least in triplicate at
salt and ice bath, was added 5-methoxy-3-methylbenzisoxazole 43
2.22 g, 13.6 mmol) portionwise. The mixture was stirred at room
22 °C in a Beckman DU 7500 spectrophotometer at 550 nm (molar
absorptivity 21.1 mM- cm for cytochrome c). Initial reduction
1
À1
(
temperature overnight. The mixture was poured into ice/water,
basified with a saturated aqueous sodium hydrogen carbonate, ex-
rates (lmol cytochrome c reduced/min/mg NQO1) were calculated
from the linear portion (0–30 s) of the reaction curves.
4
tracted with dichloromethane, dried over MgSO , and evaporated
under reduced pressure to yield the title compound (2.58 g, 91%)
Acknowledgments
+
as
a
yellow solid; mp 103–105 °C; (found: MH , 209.0551.
À1
C
9
H
8
N
2
O
4
+ H requires 209.0562);
m
max (KBr)/cm
3092, 2954,
(300 MHz;
) 7.69 (1H, d, J 9.2, ArH), 7.36 (1H, d, J 9.2, ArH), 4.00 (3H, s,
OMe), 2.50 (3H, s, Me); d (75 MHz; CDCl ) 158.2 (C), 153.0 (C),
49.1 (C), 131.7 (C), 116.9 (CH), 115.3 (C), 113.6 (CH), 58.1 (Me),
This work was supported by the FORCE Cancer Charity, the
Cancer Research Campaign (as was) [CRUK], and NIH Grant
P20RR017670 (H.D.B.). We also thank the EPSRC Mass Spectrom-
etry Centre at Swansea for mass spectra, Dr Stephen Green for
help with the electrochemical experiments, and Professor David
Ross (University of Colorado, Denver) for generous gifts of
enzyme.
2
850, 1525, 1513, 1475, 1371, 1325, 1267, 1060; d
H
CDCl
3
C
3
1
1
+
0.9 (Me); m/z (CI) 209 (MH , 100%), 192 (5).
3
4
.5.4. 5-Methoxy-3-methylbenzisoxazole-4,7-dione 45
To a suspension of 5-methoxy-3-methyl-4-nitrobenzisoxazole
4 (2.5 g, 12.0 mmol) in ethanol (200 mL) was added tin powder
References and notes
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2
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2 4
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4
3.2 mmol) in sodium dihydrogen phosphate buffer (0.3 M;
00 mL). The reaction was stirred at room temperature for 1 h,
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4
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C
3
1
1
4755.
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+
9
H
7
NO
4
+ H requires 194.0453); kmax (acetonitrile)/nm 220 (log
e
À1
.92), 256 (3.89), 380 (3.30);
mmax (KBr)/cm 3437, 3060, 2917,
698, 1667, 1578, 1486, 1467, 1440, 1355, 1336, 1251, 1205,
186, 1032, 1013; d (300 MHz; CDCl ) 5.98 (1H, s, 6-H), 3.93
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74.5 (C), 165.2 (C), 161.8 (C), 158.1 (C), 117.7 (C), 107.1 (CH),
H
3
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1
5
+
7.9 (Me), 11.0 (Me); m/z (CI) 194 (MH , 100%), 166 (5).

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