Benzofuranquinones as inhibitors of indoleamine 2,3-dioxygenase (IDO). Synthesis and biological evaluation

Article abstract of DOI:10.1039/c3ob42258e

A series of benzofuranquinones, analogues of the marine metabolite annulin A, has been synthesized and evaluated as inhibitors of human indoleamine 2,3-dioxygenase (IDO). The synthesis was carried out by copper(ii)-mediated reaction of bromobenzoquinones with 1,3-dicarbonyl compounds followed by functional group interconversions. The most potent compounds were 5-methoxy-2-methylbenzofuranquinones containing a CH₂OR group at position-3 with IC₅₀ ～ 0.2 mM. The corresponding hydroquinones were inactive. Compounds based on the benzimidazolequinone framework are also active IDO inhibitors. The quinones do not generate significant levels of oxidative stress at concentrations that inhibit IDO. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c3ob42258e

Organic &
Biomolecular Chemistry
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Benzofuranquinones as inhibitors of indoleamine
2,3-dioxygenase (IDO). Synthesis and biological
evaluation†
Cite this: Org. Biomol. Chem., 2014,
12, 2663
Catarina Carvalho,a David Siegel,b Martyn Inman,a Rui Xiong,b David Rossb and
Christopher J. Moody*a
A series of benzofuranquinones, analogues of the marine metabolite annulin A, has been synthesized and
evaluated as inhibitors of human indoleamine 2,3-dioxygenase (IDO). The synthesis was carried out by
copper(II)-mediated reaction of bromobenzoquinones with 1,3-dicarbonyl compounds followed by func-
tional group interconversions. The most potent compounds were 5-methoxy-2-methylbenzofuranqui-
nones containing a CH2OR group at position-3 with IC50 ∼ 0.2 mM. The corresponding hydroquinones
were inactive. Compounds based on the benzimidazolequinone framework are also active IDO inhibitors.
The quinones do not generate significant levels of oxidative stress at concentrations that inhibit IDO.
Received 13th November 2013,
Accepted 20th February 2014
DOI: 10.1039/c3ob42258e
www.rsc.org/obc
large number of human cancers.11 It is a monomeric catalytic
haem-containing enzyme with a known structure wherein the
Introduction
One very attractive, but different, approach to cancer therapy is active site is composed of two subsites with the haem pocket
to recruit the body’s own immune system to reject solid accommodating the indole ring of the natural substrate trypto-
tumours, and therefore some effort has gone into trying to phan,12 and whose mechanism has been widely studied.13–16
understand how tumours escape the host immune system.1–3
There are a number of known inhibitors of IDO, encom-
The essential amino acid tryptophan (Trp) plays a key role in passing many structural types as outlined in recent
“immune escape” – T-lymphocytes are extremely sensitive to reviews.17,18 The most widely studied are based on 1-methyl-
Trp shortage – and its degradation in tumours inhibits T-cell tryptophan (1-MT), a competitive inhibitor with the natural
proliferation and, as a result, prevents immunological rejec- substrate, but generally only poorly active (Ki = 62 µM).19 Never-
tion of the tumour.4,5 The first step in Trp catabolism is the theless even such a relatively poor inhibitor of IDO can
oxidative cleavage of the indole 2,3-bond, catalysed by the increase the efficacy of chemotherapeutic agents such as pacli-
haem-containing enzyme indoleamine 2,3-dioxygenase (IDO), taxel in the inhibition of tumour growth in in vivo models.7,8
and is the first and rate-limiting step in the kynurenine Other examples of IDO inhibitors shown in Fig. 1 include aryli-
pathway in mammalian cells. IDO (and its relative IDO26) midazoles such as 4-phenylimidazole (IC50 = 48 μM),20 a com-
therefore plays a major role, its function suppressing the pound that binds to the haem iron of the enzyme, some simple
immune response.1,4,7–9 Hence there is increasing evidence to analogues of brassinin (Ki = 98 μM), a natural product obtained
support the hypothesis that inhibition of IDO produces signifi- from cruciferous vegetables,21,22 S-benzylisothioureas (IC50
=
cant anticancer effects. For example, studies using siRNA to 61 μM),23 and the naturally occurring annulins (A, Ki = 0.69 μM;
silence IDO have resulted in recovery of T-cell responses and B, Ki = 0.12 μM; C, Ki = 0.14 μM).24 The latter structures inspired
reinstallation of antitumour immunity.10
a range of studies on naphthoquinone derivatives such as
IDO is an attractive target for a drug development pro- dehydro-α-lapachone (IC50 = 0.21 μM),25 and a series of pyrano-
gramme since it is known to be constitutively activated in a naphthoquinones.26 More structurally complex natural products
such as the marine metabolite exiguamine A (Ki = 0.21 µM),27
and the marine fungal natural product plectosphaeroic acid
aSchool of Chemistry, University of Nottingham, University Park, Nottingham
NG7 2RD, UK. E-mail: c.j.moody@nottingham.ac.uk; Fax: (+44) (0)115 951 3564
bDepartment of Pharmaceutical Sciences, Skaggs School of Pharmacy, University of
(IC50 = ∼2 μM) have also been identified as inhibitors of
IDO.28 In the latter case, the IDO inhibitory activity resides
in the aminophenoxazinone moiety rather than the more
structurally complex pyrroloindole fragment.28
However, it was the structures of the naturally occurring
isobenzofuranquinones annulins A and C that captured our
Colorado, Anschutz Medical Campus, 12850 East Montview Blvd., Aurora, Colorado
80045, USA
†Electronic supplementary information (ESI) available: Copies of NMR spectra.
See DOI: 10.1039/c3ob42258e
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Scheme 1
Table 1 Preparation of benzofuranquinones from bromoquinones and
1,3-dicarbonyl compoundsa
Entry
1
R
2
R2
R3
3
Yield/%
1
2
3
4
5
6
7
a
b
b
b
c
b
b
5-OMe
6-OMe
6-OMe
6-OMe
5,6-Benzo
6-OMe
6-OMe
a
a
b
c
a
d
e
Me
Me
Me
Me
Me
i-Pr
Ph
CO2Me
CO2Me
CO2But
COMorph
CO2Me
CO2Me
CO2Et
a
b
c
d
e
f
34
49
21
32
29
46
35
g
a Morph = 4-morpholino.
1,3-dicarbonyl compounds.39,40 Use of the parent 1,3-dicarbo-
nyl compound 2 in place of the enamine allowed the extension
of this method to the synthesis of benzofuranquinones 3
bearing electron-withdrawing groups at the 3-position
(Scheme 1, Table 1). In most cases, in contrast to the enamine
annulation, copper(II) bromide was found to be a superior
reagent to copper(II) acetate.
The products could be further functionalised by transform-
ations at the 3-position; thus, the tert-butyl ester 3c could be
cleaved by treatment with trifluoroacetic acid to give the
corresponding carboxylic acid 4, and the methyl esters 3a, 3b,
3e and 3f could be reduced to their corresponding alcohols 5.
In some cases, the latter transformation required a two-step
procedure involving reduction of the quinone using sodium
dithionite, followed by ester reduction with lithium aluminium
hydride, with the benzofuranquinone product 5 being obtained
after re-oxidation upon stirring the quenched reaction mixture
under air. Methylation of the alcohol 5a with iodomethane and
silver(I) oxide gave the corresponding methyl ether 6. To investi-
gate the effect of incorporating a leaving group at the 3-position,
alcohols 5a and 5c could be converted to 2,4,6-trifluoroaryl
ethers 7 by treatment with thionyl chloride, followed by reac-
tion with 2,4,6-trifluorophenol and potassium carbonate
(Scheme 2). The presence of such potential leaving groups has
been found to be beneficial in the inhibition of other enzymes
such as NQO1 and NQO2 (human quinone reductases QR1
and 2), and thioredoxin reductase.34,41–45
Fig. 1 Structures of known inhibitors of IDO.
attention in view of our longstanding interest in the synthesis
and biologically properties of heterocyclic quinones,29–38 and
therefore we embarked on a study of the synthesis and biologi-
cal evaluation of related benzofuranquinones as inhibitors
of IDO.
Results and discussion
Chemistry
In a previous paper,38 we described the synthesis of benzofur-
anquinones from benzofurans by way of a classical nitration,
reduction sequence followed by oxidation of the 4-aminoben-
zofuran to the quinone. Although successful, the method was
lengthy, particularly taking into account the need to synthesize
the starting benzofurans, and therefore we sought a more
efficient method. We have previously reported the synthesis of
indolequinones via a regioselective copper-mediated oxidative
annulation of bromoquinones 1 with enamines derived from
Further modification of the 5-methoxybenzofuranquinones
5a and 7a could be effected by substitution with primary
amines, which readily undergo addition-elimination reactions
to displace the methoxy group in a regioselective manner to
give the 5-amino substituted benzofuranquinones 9a–c
(Scheme 3). The benzofuranquinone 8,38 an isomer of 5a in
which the 2-methyl and 3-hydroxymethyl groups have been
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Biology
With a range of benzofuranquinones in hand, we turned our
attention to their evaluation as IDO inhibitors. Human IDO,
with histidine tagged N-terminus, was expressed in E. coli and
purified using Ni2+ affinity chromatography as previously
described.46 Compounds were evaluated for their ability to
inhibit IDO-catalyzed oxidative degradation of L-tryptophan to
N-formylkynurenine. This was converted into kynurenine, by
trichloroacetic acid cleavage of the N-formyl group, which was
assayed by reaction with Ehrlich’s reagent to produce a
product with strong absorbance at 480 nm. The data that show
the amount of enzyme activity remaining at four different
inhibitor concentrations are shown in Table 2. For selected
benzofuranquinones more detailed IC50 values were also deter-
mined. The known IDO inhibitor 4-phenylimidazole was used
as a control.
Scheme 2 [Ar = 2,4,6-F3-C6H2] modification of benzofuranquinone
esters.
The most active inhibitors of IDO in the benzofuranqui-
none series are the 5- or 6-methoxy substituted quinones
bearing a CH2OH, CH2OMe or CH2OAr group at position-3
that show 90–97% enzyme inhibition at 10 μM (Table 2,
entries 8, 9, 11–13). Incorporation of electron withdrawing
ester or acid substituents at C-3 is detrimental to biological
activity (entries 1, 2, 4–7), although the morpholino amide
(entry 3) retains some activity. The fusion of an additional
benzene ring at C-5/C-6 (entries 4, 10, 14) results in less potent
compounds, as does the substitution of the 5-methoxy group
by amino groups (entries 15–18). For comparison, the hydro-
quinones 10a and 10b corresponding to 3a and 3b were
assayed, and found to be ineffective IDO inhibitors with
80–90% enzyme activity remaining at 10 μM inhibitor concen-
trations. These data demonstrate that the benzofuranquinones
(entries 8, 9, 11–13), particularly compounds 5a and 5b (IC50
=
0.24 and 0.50 μM respectively), are potent inhibitors of IDO
and are comparable with many of the known IDO inhibitors
shown in Fig. 1, including the naturally occurring isobenzo-
furanquinones annulin A and annulin C,24 and that much
simpler benzofuranquinones are as efficient at inhibiting
IDO as the larger highly substituted isobenzofuranquinone
natural products.
Scheme 3 [Ar = 2,4,6-F3-C6H2] conversion of 5-methoxybenzofuran-
quinones into 5-amino derivatives.
We also examined a number of other heterocyclic quinones
as IDO inhibitors, and the results are shown in Table 3, again
using 4-phenylimidazole as a control.
Although the range of heterocyclic quinones shown
in Table 3 is limited, some conclusions can be drawn.
The benzothiophenequinone 11 (Table 3, entry 1) has
similar activity to its benzofuran counterpart 5a (30%
activity remaining vs. 36% at 1 μM concentration of inhibitor).
Indolequinones (entries 2–4) are poor inhibitors of IDO;
the parent indolequinone 14 has previously been reported
with a Ki of 190 20 nmol.19 Whilst the indazole derivative 15
has modest activity, it is the three benzimidazolequinones
16–18 (entries 6–8) that are the most potent IDO
inhibitors (IC50 ∼ 0.2 μM). Overall the data demonstrate that
benzimidazolequinones are also potent compounds, and are
comparable with the benzofuranquinones, and some of the
Scheme 4 Reduction of benzofuranquinones to corresponding
hydroquinones.
exchanged, was also subjected to the same reaction, and gave
the corresponding amino derivative 9d.
For comparison, the hydroquinones 10a and 10b, corres-
ponding to benzofuranquinones 3a and 3b were synthesised
by reduction with sodium dithionite (Scheme 4), and a range
of previously prepared heterocyclic quinones (see Table 3) were
also studied.33,36,38
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Table 2 Inhibition of rhIDO by benzofuranquinonesa
Percent activity remaining of IDO at
Entry
R2
R3
R5
R6
IC50 (μM)
0.1 µM
1 µM
10 µM
100 µM
1
2
3
4
5
6
7
8
3a
3b
3d
3e
3f
3g
4
5a
5b
5c
5d
6
7a
7b
8a
8b
8c
8d
—
Me
Me
Me
Me
i-Pr
Ph
Me
Me
Me
Me
i-Pr
Me
Me
Me
Me
Me
CO2Me
CO2Me
COMorph
CO2Me
CO2Me
CO2Et
OMe
H
H
Benzo
H
H
H
OMe
H
Benzo
H
OMe
OMe
Benzo
NH(CH2)4Me
NH(CH2)3NMe2
NH(CH2)3NMe2
NH(CH2)2NMe2
H
OMe
OMe
7.2
34
96
100
80
95
100
90
95
83
95
95
85
84
100
85
82
84
86
90
81
98
65
95
100
85
95
36
45
95
40
26
41
85
72
57
56
50
40
72
10
85
70
40
90
3
5
6
5
50
12
12
55
2
2
25
5
5
7
OMe
OMe
OMe
H
CO2H
CH2OH
CH2OH
CH2OH
CH2OH
CH2OMe
CH2OAr
CH2OAr
CH2OH
CH2OH
CH2OAr
Me
0.24
0.50
9
OMe
4
10
11
12
13
14
15
16
17
18
19
70
10
4
OMe
H
H
≈1.0
8
95
35
22
22
12
90
16
12
14
10
H
H
H
H
Me
CH2OH
4-Phenylimidazole
(control)
70
a Morph = 4-morpholino; Ar = 2,4,6-F3-C6H2.
Table 3 Inhibition of rhIDO by heterocyclic quinones
Percent activity remaining of IDO
at
Entry
X
Y
Z
S
R2
R3
R5
R6
IC50 (μM) 0.1 µM 1 µM 10 µM 100 µM Ref.
1
2
3
4
5
6
7
8
9
11
12
13
14
15
16
17
18
—
C
C
C
C
C
N
N
N
C
C
C
C
N
C
C
C
Me
CH2OH
CH2OH
CHMeOH OMe
OMe
OMe
H
H
H
H
1.0
∼10
∼10
26a
∼1.0
0.25
74
94
99
99
82
67
69
66
32
86
87
43
53
24
32
30
5
48
46
13
28
1.1
13
11
4
27
33
6
—
—
9
38
33
33
NMe Me
NMe Me
NH
H
—
H
H
OMe
H
H
H
NMe
CH2OH
H
38
36
36
36
NMe CH2OH
NMe CH2OAc
NMe CH2OArb
—
—
—
OMe
OMe
OMe
0.20
0.18
8
4-Phenylimidazole
(control)
70
a Ki reported as 190 20 nmol.19 b Ar = 4-nitrophenyl.
known inhibitors shown in Fig. 1 in their ability to inhibit BxPc-3 cells were chosen since they have moderate levels of
IDO. quinone reductase (NQO1) activity and have a large cyto-
To determine if these quinones were themselves cytotoxic plasmic to nuclear ratio which makes them ideal for examin-
to cancer cells we treated BxPc-3 human pancreatic cancer ing damage to microtubules and actin cytoskeleton. Quinones
cells with benzofuranquinone 5a and heterocyclic quinone 16. 5a (Table 2) and 16 (Table 3) were selected because they
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Fig. 3 Quinone-induced cytotoxicity in BxPc-3 cells. Quinones 5a and
16 were examined for their ability to induce cytotoxicity in BxPc-
3 human pancreatic cancer cells. For these studies, cells were treated
quinones (10 µM) for 4 h after which cytotoxicity was assayed using
trypan blue exclusion. β-Lapachone (2.5 µM × 4 h) was included as a
positive control. Results are expressed as mean standard deviation of
three determinations.
1,3-dicarbonyl compounds. The compounds were evaluated
for their ability to inhibit IDO, and the study identified two
compounds (5a and 5b with IC50 = 0.24 and 0.50 μM respect-
ively), that are potent inhibitors of IDO comparable to the
naturally occurring annulins. In contrast, hydroquinones were
much less active, and whereas indolequinones were essentially
inactive, benzimidazolequinones showed comparable activity
to the benzofurans. Importantly, the quinones do not do not
generate significant levels of oxidative stress at concentrations
that inhibit IDO.
Fig. 2 Immunostaining of microtubules and actin cytoskeleton follow-
ing treatment with quinones. BxPc-3 human pancreatic cancer cells
were treated with quinones 5a and 16 (10 µM × 4 h) and then analysed
by immunostaining and confocal microscopy for quinone-induced
damage to microtubules and actin cytoskeleton. Cells were also exam-
ined for intracellular oxidative stress (CellROX) following treatment with
quinones (10 µM × 4 h). Treatment with β-lapachone (2.5 µM × 4 h) was
included as a positive control for quinone induced oxidative stress.
Experimental section
Chemistry: general experimental details
Commercially available reagents were used throughout
without purification unless otherwise stated. All anhydrous
solvents were used as supplied, except tetrahydrofuran and
dichloromethane that were freshly distilled according to stan-
dard procedures. Reactions were routinely carried out under
an argon atmosphere unless otherwise stated, and all glass-
ware was flame-dried before use. Light petroleum refers to the
fraction with bp 40–60 °C. Ether refers to diethyl ether.
Analytical thin layer chromatography was carried out on
aluminium backed plates coated with silica gel, and visualised
under UV light at 254 and/or 360 nm and/or by chemical stain-
ing. Flash chromatography was carried out using silica gel,
with the eluent specified.
Infrared spectra were recorded using an FT-IR spectrometer
over the range 4000–600 cm−1. NMR spectra were recorded at
400 or 500 MHz (1H frequency, 100 or 125 MHz 13C frequency).
Chemical shifts are quoted in parts per million (ppm), and are
referenced to residual H in the deuterated solvent as the
internal standard. Coupling constants, J, are quoted in Hz.
In the 13C NMR spectra, signals corresponding to CH, CH2,
represent two different classes of potent quinone IDO inhibi-
tors. Exposure of BxPc-3 cells to 5a or 16 did not result in
visible damage to microtubules or actin cytoskeleton nor
did they generate detectable levels of oxidative stress as
determined by confocal microscopy (Fig. 2). Treatment,
however, with β-lapachone a quinone which undergoes NQO1-
dependent redox cycling, did generate high levels of oxidative
stress and significant damage to microtubules and the actin
cytoskeleton as shown in Fig. 2. In agreement with these data
quinones 5a and 16 did not induce significant levels of cyto-
toxicity while treatment with β-lapachone did result in pro-
nounced cell death (Fig. 3). These data confirm that at
concentrations that inhibit IDO these quinones do not gene-
rate significant levels of oxidative stress.
Conclusions
In summary, we have prepared a range of benzofuranquinones or CH3 groups are assigned from the DEPT spectra. Mass
by copper(II)-mediated reaction of bromobenzoquinones with spectra were recorded on a time-of-flight mass spectrometer
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using electrospray ionisation (ESI), or an EI magnetic sector (acetonitrile)/nm 232 (log ε 4.11), 259 (log ε 3.84), 303 (log ε
instrument. 4.12), 402 (log ε 2.90); δH (400 MHz; CDCl3) 5.85 (1 H, s, H-5),
The synthesis of compounds 11–13 and 15–18 has been 3.94 (3 H, s, OMe), 3.86 (3 H, s, OMe), 2.68 (3 H, s, 2-Me); δC
described previously.33,36,38
(100 MHz; CDCl3) 179.9, 169.4, 165.2, 162.3, 158.1, 148.6,
127.0, 113.1, 108.2 (CH), 56.9 (Me), 52.3 (Me), 14.3 (Me); m/z
(ESI) 273 (M + Na+, 100%).
General procedure for the synthesis of benzofuran-4,7-diones
6-Methoxy-2-methyl-3-(morpholine-4-carbonyl)benzofuran-
4,7-dione 3d.
A solution of 1,3-dicarbonyl compound (1.0 equiv.) in aceto-
nitrile (5–10 mL mmol−1) was added to a mixture of bromoqui-
none (1.0 equiv.), copper(II) bromide or copper(II) acetate
monohydrate (1.5–2.0 equiv.) and potassium carbonate
(3.0 equiv.). The resulting mixture was stirred at reflux for the
indicated time, cooled to room temperature, diluted with
dichloromethane (20 mL mmol−1), filtered through Celite and
concentrated in vacuo. Column chromatography of the residue
gave the benzofuran-4,7-dione.
Prepared by the general procedure from 2-bromo-6-methoxy-
benzoquinone (0.217 g, 1.0 mmol), acetoacetylmorpholine
(0.171 g, 1.0 mmol), copper(II) bromide (0.448 g, 2.0 mmol)
and potassium carbonate (0.414 g, 3.0 mmol) stirred at reflux
in acetonitrile (10 mL) for 5 h. Column chromatography
eluting with ethyl acetate and light petroleum (1 : 1) gave the
title compound as an orange solid (0.098 g, 32%), mp
168–170 °C; (found: M + Na+, 328.0785. C15H15NO6Na requires:
328.0792); νmax (CHCl3)/cm−1 3011, 1689, 1657, 1638; δH
(400 MHz; CDCl3) 5.83 (1 H, s, 5-H), 3.89 (3 H, s, OMe),
3.88–3.72 (5 H, m, CH2), 3.58–3.52 (1 H, m, CH2), 3.42–3.36 (1
H, m, CH2), 3.32–3.26 (1 H, s, CH2), 2.51 (3 H, s, 2-Me); δC
(75 MHz; CDCl3) 181.2, 168.8, 161.3, 159.7, 159.4, 147.8, 127.1,
115.3, 106.9 (CH), 66.6 (CH2), 66.5 (CH2), 57.0 (Me), 47.3
(CH2), 42.4 (CH2), 13.1 (Me); m/z (EI) 328 (M + Na+, 100%).
Methyl 4,9-dihydro-2-methyl-4,9-dioxonaphtho[2,3-b]furan-
3-carboxylate 3e.
Methyl
4,7-dihydro-5-methoxy-2-methyl-4,7-dioxobenzo-
furan-3-carboxylate 3a.
Prepared by the general procedure from 2-bromo-5-methoxy-
benzoquinone39 (0.200 g, 0.92 mmol), methyl acetoacetate
(0.099 mL, 0.92 mmol), copper(II) acetate monohydrate
(0.299 g, 1.38 mmol) and potassium carbonate (0.381 g,
2.76 mmol) stirred at reflux in acetonitrile (10 mL) for 1.5 h.
Column chromatography eluting with ethyl acetate and light
petroleum (1 : 2) gave the title compound as a yellow solid
(78.5 mg, 34%); mp 203–205 °C; (found: M + Na+, 273.0365.
C12H10O6 + Na+ requires: 273.0375); νmax (CHCl3)/cm−1 1718,
1701, 1661, 1610, 1551, 1456, 1248, 1187, 1026, 992; λmax
(acetonitrile)/nm 232 (log ε 4.17), 267 (log ε 3.87), 302 (log ε
3.92), 404 (log ε 3.08); δH (400 MHz; CDCl3) 5.80 (1 H, s, H-6),
3.92 (3 H, s, OMe), 3.87 (3 H, s, OMe), 2.68 (3 H, s, 2-Me); δC
(100 MHz; CDCl3) 175.4, 174.7, 164.0, 162.4, 160.6, 150.2,
123.6, 112.5, 104.6 (CH), 57.0 (Me), 52.2 (Me), 14.0 (Me); m/z
(ESI) 523 (2M + Na+, 100%), 273 (M + Na+, 65%).
Prepared by the general procedure from 2-bromo-1,4-naphtho-
quinone (0.474 g, 2.0 mmol), methyl acetoacetate (0.232 g,
2.0 mmol), copper(II) bromide (0.670 g, 3.0 mmol) and potass-
ium carbonate (0.828 g, 6.0 mmol) stirred at reflux in aceto-
nitrile (20 mL) for 16 h. Column chromatography eluting with
ethyl acetate and dichloromethane (1 : 49) gave the title com-
pound as a yellow solid (0.158 g, 29%), mp 163–165 °C; (found:
M + Na+, 293.0409. C15H10O5Na requires: 293.0420); νmax
(CHCl3)/cm−1 3606, 3011, 1602; δH (400 MHz; CDCl3) 8.23–8.21
(2 H, m, ArH), 7.80–7.77 (2 H, m, ArH), 4.01 (3 H, s, OMe), 2.75
(3 H, s, 2-Me); δC (75 MHz; CDCl3) 178.8, 173.5, 164.8, 162.6,
151.4, 134.2 (CH), 133.7 (CH), 133.6, 131.5, 128.1, 127.4 (CH),
126.5 (CH), 113.3, 52.4 (Me), 14.2 (Me); m/z (EI) 293 (M + Na+,
82%), 563 (2M + Na+, 100).
Methyl
4,7-dihydro-6-methoxy-2-methyl-4,7-dioxobenzo-
furan-3-carboxylate 3b.
Prepared by the general procedure from 2-bromo-6-methoxy-
benzoquinone39 (0.200 g, 0.92 mmol), methyl acetoacetate
(0.099 mL, 0.92 mmol), copper(II) acetate monohydrate
(0.299 g, 1.38 mmol) and potassium carbonate (0.381 g,
2.76 mmol) stirred at reflux in acetonitrile (10 mL) for 1.5 h.
Column chromatography eluting with ethyl acetate and light
petroleum (1 : 2) gave the title compound as a yellow solid
(114 mg, 49%); mp 191–193 °C; (found: M + Na+, 273.0369.
C12H10O6 + Na+ requires: 273.0375); νmax (CHCl3)/cm−1 3011,
1690, 1662, 1611, 1458, 1313, 1248, 1094, 848; λmax
Methyl 4,7-dihydro-2-isopropyl-6-methoxy-4,7-dioxobenzo-
furan-3-carboxylate 3f.
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Prepared by the general procedure from 2-bromo-6-methoxy- (Me), 28.3 (Me), 14.2 (Me); m/z (EI) 607 (2M + Na+, 68%), 315
benzoquinone (0.434 g, 2.0 mmol), methyl 4-methyl-3-oxopen- (100).
tanoate (0.288 g, 2.0 mmol), copper(II) bromide (0.670 g,
4,7-Dihydro-6-methoxy-2-methyl-4,7-dioxobenzofuran-3-car-
3.0 mmol) and potassium carbonate (0.828 g, 6.0 mmol) boxylic acid 4.
stirred at reflux in acetonitrile (20 mL) for 4 h. Column chrom-
atography eluting with ethyl acetate and dichloromethane
(1 : 49) gave the title compound as a bright yellow solid (0.256 g,
46%), mp 163–165 °C; (found: M + Na+, 301.0693. C14H14O6Na
requires: 301.0683); νmax (CHCl3)/cm−1 3606, 3048, 1690, 1661,
1603; δH (400 MHz; CDCl3) 5.86 (1 H, s, 5-H), 3.96 (3 H, s,
Trifluoroacetic acid (0.4 mL) was added to a stirred solution of
OMe), 3.88 (3 H, s, OMe), 3.66 (1 H, sept, J 7.0, CH), 1.38 (6 H,
d, J 7.0, Me); δC (75 MHz; CDCl3) 180.2, 172.1, 169.3, 162.5,
158.4, 148.4, 127.1, 111.4, 108.0 (CH), 56.9 (Me), 52.4 (Me),
27.9 (CH), 20.5 (Me); m/z (EI) 301 (100%).
Ethyl 4,7-dihydro-6-methoxy-4,7-dioxo-2-phenylbenzofuran-
3-carboxylate 3g.
3c (0.100 g, 0.34 mmol) in dichloromethane (4 mL), and the
resulting mixture was stirred at room temperature for 18 h,
washed with water (3 × 5 mL), dried (Na2SO4), filtered and con-
centrated. Flash column chromatography eluting with ethyl
acetate gave the title compound as an orange solid (0.059 g,
73%), mp 165–167 °C; (found: M + Na+, 259.0218. C11H8O6Na
requires: 259.0213); νmax (CHCl3)/cm−1 3044, 2699, 1738, 1698,
1636, 1574, 1324; δH (400 MHz; CDCl3) 6.00 (1 H, s, 5-H), 3.98
(3 H, s, OMe), 2.86 (3 H, s, 2-Me); δC (75 MHz; CDCl3) 185.8,
169.5, 168.0, 160.7, 147.3, 125.3, 112.9, 105.9 (CH), 57.7 (Me),
14.5 (Me); one carbon unobserved; m/z (EI) 259 (M + Na+,
100%).
Prepared by the general procedure from 2-bromo-6-methoxy-
benzoquinone (0.217 g, 1.0 mmol), ethyl benzoylacetate
(0.192 g, 1.0 mmol), copper(II) bromide (0.448 g, 2.0 mmol)
and potassium carbonate (0.414 g, 3.0 mmol) stirred at reflux
in acetonitrile (10 mL) for 4 h. Column chromatography
eluting with ethyl acetate and light petroleum (1 : 3) gave the
title compound as an orange solid (0.114 g, 35%), mp
154–156 °C; (found: M + Na+, 349.0658. C18H14O6Na requires:
349.0688); νmax (CHCl3)/cm−1 3012, 2985, 1730, 1689, 1662,
1606; δH (400 MHz; CDCl3) 7.89 (2 H, m, ArH), 7.51 (3 H, m,
ArH), 5.90 (1 H, s, 5-H), 4.49 (2 H, q, J 7.2, CH2), 3.92 (3 H, s,
OMe), 1.41 (3 H, t, J 7.2, Me); δC (75 MHz; CDCl3) 180.3, 169.0,
162.8, 159.3, 158.7, 147.8, 131.1 (CH), 128.9 (CH), 128.6, 127.5
(CH), 113.0, 107.4 (CH), 62.4 (CH2), 57.0 (Me), 13.9 (Me); one
carbon unobserved; m/z (EI) 349 (M + Na+, 100%).
4,7-Dihydro-3-hydroxymethyl-5-methoxy-2-methylbenzofuran-
4,7-dione 5a.
A solution of 3a (75.0 mg, 0.30 mmol) in THF (3 mL) was
added at 0 °C to a suspension of lithium aluminium hydride
(57.0 mg, 1.50 mmol) in THF (6 mL) under a nitrogen atmos-
phere. The reaction mixture was stirred at room temperature
for 1.5 h, and then acetone (0.72 mL) and a solution of ferric
chloride (1 M; 0.36 mL) in hydrochloric acid (1 M; 0.36 mL)
were added. The mixture was stirred at room temperature for
5 min and then extracted with dichloromethane (3 × 5 mL).
The combined organic extracts were washed with water
(10 mL), brine (10 mL), dried (MgSO4), filtered and the solvent
removed in vacuo. The residue was purified by column chrom-
atography using light petroleum–EtOAc (2 : 1) to give the title
compound (19.8 mg, 30%) as an orange solid; mp 183 °C;
tert-Butyl 4,7-dihydro-6-methoxy-2-methyl-4,7-dioxobenzo-
furan-3-carboxylate 3c.
(found:
M + +
Na+, 245.0424. C11H10O5 Na+ requires:
245.0420); νmax (CHCl3)/cm−1 2360, 1666, 1602, 1554, 1187; δH
(400 MHz; CDCl3) 5.79 (1 H, s, H-6), 4.62 (2 H, s, CH2), 3.87 (3
H, s, OMe), 2.42 (3 H, s, 2-Me); δC (100 MHz; CDCl3) 179.2,
175.3, 159.7, 154.9, 150.3, 125.8, 119.7, 105.9 (CH), 56.9 (Me),
54.8 (CH2), 12.0 (Me); m/z (ESI) 245 (M + Na+, 42%).
Methyl 4,7-dihydroxy-6-methoxy-2-methylbenzofuran-3-car-
boxylate 10b.
Prepared by the general procedure from 2-bromo-6-methoxy-
benzoquinone (0.217 g, 1.0 mmol), tert-butyl acetoacetate
(0.158 g, 1.0 mmol), copper(II) bromide (0.448 g, 2.0 mmol)
and potassium carbonate (0.414 g, 3.0 mmol) stirred at reflux
in acetonitrile (10 mL) for 5 h. Column chromatography
eluting with ethyl acetate and light petroleum (1 : 4) gave the
title compound as a yellow solid (0.060 g, 21%), mp 152–154 °C;
(found: M + Na+, 315.0832. C15H15O6Na requires: 315.0839);
νmax (CHCl3)/cm−1 3011, 1721, 1688, 1663, 1610; δH (400 MHz;
CDCl3) 5.85 (1 H, s, 5-H), 3.88 (3 H, s, OMe), 2.67 (3 H, s,
2-Me), 1.64 (9 H, s, tBu); δC (75 MHz; CDCl3) 179.9, 169.5,
164.4, 160.9, 158.1, 148.4, 127.2, 115.1, 108.2 (CH), 82.7, 56.8
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To a solution of 3b (439 mg, 1.75 mmol) in chloroform br s, OH), 2.51 (3 H, s, 2-Me); δC (75 MHz; CDCl3) 183.3, 173.0,
(3.7 mL) was added a solution of sodium dithionite (1.84 g, 156.0, 151.6, 134.4 (CH), 133.8 (CH), 132.8, 132.5, 130.2, 127.1
10.5 mmol) in water (7.3 mL). After being stirred at room (CH), 127.0 (CH), 120.7, 55.0 (CH2), 12.3 (Me); m/z (EI) 265
temperature for 3 h, the precipitate formed was filtered and (100%).
dried in vacuo to yield the title compound as a colourless solid
4,7-Dihydro-3-hydroxymethyl-2-isopropyl-6-methoxybenzofuran-
(351 mg, 79%); mp 183–184 °C; (found: M + Na+, 275.0525. 4,7-dione 5d.
C12H12O6 + Na+ requires: 275.0526); δH (400 MHz; DMSO-d6)
9.78 (1 H, s, OH), 8.70 (1 H, s, OH), 6.41 (1 H, s, H-5), 3.94
(3 H, s, OMe), 3.78 (3 H, s, OMe), 2.66 (3 H, s, 2-Me); δC
(100 MHz; DMSO-d6) 167.5, 162.0, 146.5, 143.7, 141.4, 124.3,
108.1, 107.1, 96.7 (CH), 56.5 (Me), 53.0 (Me), 14.6 (Me); m/z
(ESI) 275 (M + Na+, 47%), 253 (M + H+, 46%).
To a solution of 3f (0.244 g, 0.88 mmol) in chloroform (5 mL)
was added solution of sodium dithionite (0.764 g,
4,7-Dihydro-3-hydroxymethyl-6-methoxy-2-methylbenzofuran-
4,7-dione 5b.
a
4.39 mmol) in water (5 mL), and the mixture was stirred vigor-
ously for 1 h. The aqueous phase was extracted with dichloro-
methane (3 × 5 mL), and the combined organic phases were
dried (MgSO4), filtered and concentrated. The residue was dis-
solved in THF (10 mL) and lithium aluminium hydride
(0.167 g, 4.39 mmol) was added. The resulting mixture was
A solution of 10b (96.8 mg, 0.38 mmol) in THF (3 mL) was stirred at room temperature for 1.5 h, then quenched by
added at 0 °C to a suspension of lithium aluminium hydride addition of ethyl acetate (1 mL), water (1 mL) and silica gel
(75.0 mg, 1.98 mmol) in THF (6 mL). The reaction mixture was
(1 g). The mixture was stirred under air for 30 min, filtered
stirred at room temperature for 1.5 h, cooled to 0 °C and and concentrated. Column chromatography of the residue
quenched by addition of water (0.15 mL), sodium hydroxide eluting with ethyl acetate and dichloromethane (1 : 19) gave
(1 M; 0.15 mL) and silica gel (150 mg). The granular precipitate
the title compound as an orange solid (0.096 g, 44%), mp
was filtered off through a pad of Celite. The filtrate was dried 128–130 °C; (found: M + Na+, 273.0711. C13H14O5Na requires:
(Na2SO4) and concentrated in vacuo to give the title compound 273.0739); νmax (CHCl3)/cm−1 3691, 3606, 1687, 1646, 1603; δH
as an orange solid (44.7 mg, 52%); mp 142–144 °C; (found:
(400 MHz; CDCl3) 5.85 (1 H, s 5-H), 4.64 (2 H, br s, CH2), 3.95
M + Na+, 245.0424. C11H10O5 + Na+ requires: 245.0420); νmax (1 H, br s, OH), 3.91 (3 H, s, OMe), 3.20 (1 H, sept, J 7.0, CH),
(CHCl3)/cm−1 3479, 3011, 1686, 1647, 1588, 1321, 1095; λmax 1.38 (6 H, d, J 7.0, Me); δC (75 MHz; CDCl3) 184.8, 168.4, 164.8,
(acetonitrile)/nm 222 (log ε 4.30), 267 (log ε 4.06), 318 (log ε
160.2, 148.2, 129.5, 119.3, 106.3 (CH), 57.1 (Me), 54.7 (CH2),
3.99), 433 (log ε 3.05); δH (400 MHz; CDCl3) 5.83 (1 H, s, H-5), 27.0 (CH), 21.0 (Me); m/z (EI) 273 (M + Na+, 98%), 413 (100).
4.60 (2 H, s, CH2), 3.88 (3 H, s, OMe), 2.43 (3 H, s, 2-Me);
δC (100 MHz; CDCl3) 184.6, 168.5, 160.1, 156.7, 148.2, 129.4,
121.0, 106.3 (CH), 57.1 (Me), 55.0 (CH2), 12.3 (Me); m/z (ESI)
245 (M + Na+, 42%).
4,7-Dihydro-5-methoxy-3-methoxymethyl-2-methylbenzofuran-
4,7-dione 6.
4,9-Dihydro-3-hydroxymethyl-2-methylnaphtho[2,3-b]furan-
4,9-dione 5c.
A mixture of 5a (15.0 mg, 0.067 mmol), silver(I) oxide (23.4 mg,
0.10 mmol) and iodomethane (1 mL) was heated to reflux for
22 h and then filtered through Celite. The filter cake was
washed with dichloromethane (5 mL) and the filtrate and
To a solution of 3e (0.143 g, 0.53 mmol) in THF (10 mL) was washings were combined and concentrated in vacuo. The
added lithium aluminium hydride (0.101 g, 2.65 mmol). The residue was purified by column chromatography, eluting with
resulting mixture was stirred at room temperature for 1.5 h, light petroleum–ethyl acetate (1 : 1) to give the title compound
then quenched by addition of ethyl acetate (1 mL), water as an orange solid (8.2 mg, 51%); mp 164–165 °C; (found: M +
(1 mL) and silica gel (1 g). The mixture was stirred under air Na+, 259.0549. C12H12O5 + Na+ requires: 259.0582); νmax
for 30 min, filtered and concentrated. Column chromato- (CHCl3)/cm−1 1691, 1658, 1533, 1390, 1121, 1020, 842; λmax
graphy of the residue eluting with ethyl acetate and dichloro- (acetonitrile)/nm 225 (log ε 4.07), 259 (log ε 3.92), 310 (log ε
methane (1 : 49) gave the title compound as a yellow solid 3.65), 426 (log ε 3.02); δH (400 MHz; CDCl3) 5.75 (1 H, s, H-6),
(0.048 g, 38%), mp 166–168 °C; (found: M + Na+, 265.0481. 4.54 (2 H, s, CH2), 3.84 (3 H, s, OMe), 3.41 (3 H, s, OMe), 2.46
C14H10O4Na requires: 265.0471); νmax (CHCl3)/cm−1 3691, (3 H, s, 2-Me); δC (100 MHz; CDCl3) 177.7, 175.5, 159.9, 157.6,
3606, 3011, 1661, 1601; δH (400 MHz; CDCl3) 8.25–8.19 (2 H, 149.9, 125.2, 116.5, 105.4 (CH), 63.3 (CH2), 58.5 (Me), 56.8
m, ArH), 7.81–7.77 (2 H, m, ArH), 4.71 (2 H, s, CH2), 3.95 (1 H, (Me), 12.3 (Me); m/z (ESI) 259 (M + Na+, 100%).
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4,7-Dihydro-3-(2,4,6-trifluorophenoxy)methyl-5-methoxy- were washed with water (10 mL), dried (MgSO4), filtered and
2-methylbenzofuran-4,7-dione 7a.
concentrated. Column chromatography of the residue eluting
with dichloromethane and light petroleum (2 : 1) gave the title
compound as a yellow solid (0.038 g, 70%), mp 174–176 °C;
(found: M + Na+, 395.0513. C20H11F3O4Na requires: 395.0502);
νmax (CHCl3)/cm−1 3045, 3007, 1674, 1601, 1551, 1509; δH
(400 MHz; CDCl3) 8.24–8.15 (2 H, m, ArH), 7.78–7.75 (2 H, m,
ArH), 6.70 (2 H, t, J 8.4, ArH), 5.36 (2 H, s, CH2), 2.56 (3 H, s,
Me); δC (75 MHz; CDCl3) 181.4, 173.3, 160.4, 157.6 (dt, J 247,
15, CF), 156.4 (ddd, J 250, 15, 7, CF), 151.1, 133.9 (CH), 133.8
(CH), 133.2, 132.3, 131.5 (td, J 15, 5, C), 128.9, 126.9 (CH),
126.8 (CH), 115.7, 100.8 (td, J 27, 7, CH), 65.0 (CH2), 12.4 (Me);
m/z (EI) 395 (M + Na+, 58%), 413 (100).
Thionyl chloride (0.64 mL, 8.87 mmol) was added at 0 °C to a
solution of 5a (37.5 mg, 0.17 mmol) in dichloromethane
(2 mL). After being stirred at room temperature for 2 h, the
solvent was removed in vacuo and the resulting residue was
purified by column chromatography, eluting with light pet-
roleum–ethyl acetate (2 : 1) to give 3-chloromethyl-5-methoxy-
2-methylbenzofuran-4,7-dione as a yellow solid (23.5 mg,
58%). To the chloride (23.5 mg, 0.097 mmol) in anhydrous
DMF (2 mL) was added sodium hydride (60% dispersion in
mineral oils; 4.68 mg, 0.195 mmol) followed by 2,4,6-trifluoro-
phenol (31.0 mg, 0.211 mmol) and the reaction mixture was
stirred at room temperature for 5 h. Water (3 mL) was then
added and the reaction mixture was extracted with ether
(3 × 5 mL) and dried (MgSO4). The solvent was removed under
reduced pressure and the residue was purified by column
chromatography, eluting with light petroleum–ethyl acetate
(2 : 1) to give the title compound as a yellow solid (20.1 mg,
58%); mp 124–125 °C; (found: M + Na+, 375.0459. C17H11F3O5
+ Na+ requires: 375.0451); νmax (CHCl3)/cm−1 2927, 2855, 1692,
1660, 1509, 1454, 1390, 1120, 1042, 842; δH (400 MHz; CDCl3)
6.66 (2 H, t, J 8.0, ArH), 5.76 (1 H, s, H-6), 5.20 (2 H, s, CH2),
3.84 (3 H, s, OMe), 2.46 (3 H, s, Me); δC (100 MHz; CDCl3)
177.3, 175.4, 159.9, 159.0, 157.6 (dt, J 246, 15, CF), 156.3 (ddd,
J 250, 14, 7, CF), 149.9, 147.3, 124.7, 114.8, 105.5 (CH), 100.8
(ddd, JCF 27, 19, 8, CH), 64.7 (CH2), 56.8 (Me), 12.0 (Me); m/z
(ESI) 375 (M + Na+, 100%).
4,7-Dihydro-3-hydroxymethyl-2-methyl-5-(pentylamino)ben-
zofuran-4,7-dione 8a.
Pentylamine (0.15 mL, 1.31 mmol) was added to a solution of
5a (29.1 mg, 0.13 mmol) in anhydrous acetonitrile (1.5 mL)
and the resulting mixture was stirred at room temperature for
45 min. The solvent was removed in vacuo and the crude
product was purified by column chromatography on silica gel,
previously inactivated with triethylamine, eluting with light
petroleum–ethyl acetate (1 : 1) to give the title compound as a
purple solid (23.4 mg, 64%); mp 102 °C (dichloromethane–
light petroleum); (found: M + Na+, 300.1199. C15H19NO4 + Na+
requires: 300.1212); νmax (CHCl3)/cm−1 3491, 3007, 2961, 2863,
1671, 1633, 1591, 1467, 1390, 1190; λmax (acetonitrile)/nm 214
(log ε 4.76), 245 (log ε 4.56), 341 (log ε 4.17), 518 (log ε 3.82);
δH (400 MHz; CDCl3) 5.84 (1 H, s br, NH), 5.28 (1 H, s, H-6),
4.59 (2 H, s, CH2), 3.14 (2 H, q, J 6.9, CH2), 2.38 (3 H, s, Me),
1.72–1.65 (2 H, quin, J 6.9, CH2), 1.39–1.35 (4 H, m, 2 × CH2),
0.93 (3 H, t, J 6.9, Me); δC (100 MHz; CDCl3) 180.7, 175.1,
153.2, 152.9, 147.9, 123.5, 118.9, 95.8 (CH), 55.0 (CH2), 43.0
(CH2), 29.1 (CH2), 27.8 (CH2), 22.3 (CH2), 13.9 (Me), 11.8 (Me);
m/z (ESI) 300 (M + Na+, 100%).
4,9-Dihydro-2-methyl-3-(2,4,6-trifluorophenoxy)methylnaphtho-
[2,3-b]furan-4,9-dione 7b.
4,7-Dihydro-5-(3-(dimethylamino)propylamino)-3-(hydroxy-
methyl)-2-methylbenzofuran-4,7-dione 8b.
Thionyl chloride (0.105 mL, 1.45 mmol) was added to a stirred
solution of 5c (0.035 g, 0.14 mmol) in dichloromethane N,N-Dimethylpropane-1,3-diamine (0.16 mL, 1.28 mmol) was
(2 mL), and the mixture was stirred at room temperature for added to a solution of dione 5a (28.5 mg, 0.13 mmol) in anhy-
6 h, concentrated and the excess thionyl chloride was azeo- drous acetonitrile (1.5 mL) and the resulting mixture was
troped with dichloromethane (2 mL). The residue was dis- stirred at room temperature for 1 h. The solvent was removed
solved in DMF (2 mL), and 2,4,6-trifluorophenol (0.107 g, in vacuo and the residue purified by column chromatography,
0.72 mmol) and potassium carbonate (0.100 g, 0.72 mmol) eluting with methanol–dichloromethane (0–20%) to give the
were added sequentially. The mixture was stirred at room title compound as
a purple solid (20.5 mg, 55%); mp
temperature for 16 h, diluted with water (20 mL) and extracted 127–128 °C (dichloromethane–light petroleum); (found: M +
with ethyl acetate (3 × 10 mL). The combined organic phases H+, 293.1495. C15H20N2O4 + H+ requires: 293.1496); νmax
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(CHCl3)/cm−1 3053, 2360, 2341, 1733, 1716, 1698, 1192, 1007; (acetonitrile)/nm 214 (log ε 4.44), 246 (log ε 4.22), 341 (log ε
λmax (acetonitrile)/nm 215 (log ε 4.29), 246 (log ε 4.02), 343 3.78), 507.2 (log ε 3.54); δH (400 MHz; CDCl3) 6.48 (1 H, s br,
(log ε 3.64), 315 (log ε 3.31); δH (400 MHz; CDCl3) 7.83 (1 H, NH), 5.24 (1 H, s, H-6), 4.63 (2 H, s, CH2), 3.15 (2 H, q, J 5.7,
s br, NH), 5.23 (1 H, s, H-6), 4.59 (2 H, s, CH2), 3.26 (2 H, q, CH2), 2.61 (2 H, t, J 5.7, CH2), 2.30 (6 H, s, Me), 2.26 (3 H, s,
J 6.0, CH2), 2.53 (2 H, t, J 6.0, CH2), 2.37 (3 H, s, Me), 2.34 (6 H, Me); δC (100 MHz; CDCl3) 179.1, 175.3, 153.9, 153.2, 148.5,
s, Me), 1.86 (2 H, quin, J 6.0, CH2); δC (100 MHz; CDCl3) 180.8, 123.3, 117.3, 96.1 (CH), 56.4 (CH2), 54.8 (CH2), 45.1 (CH3), 39.9
175.1, 153.4, 152.6, 148.8, 123.5, 118.9, 95.2 (CH), 57.9 (CH2), (CH2), 8.5 (Me); m/z (ESI) 279 (M + H+, 100%).
55.0 (CH2), 45.0 (Me), 42.9 (CH2), 24.2 (CH2), 11.8 (Me); m/z
(ESI) 293 (M + H+, 100%).
Methyl 4,7-dihydroxy-6-methoxy-2-methylbenzofuran-3-car-
boxylate 10a.
4,7-Dihydro-3-(2,4,6-trifluorophenoxy)methyl-5-(3-(dimethyl-
amino)propylamino)-2-methylbenzofuran-4,7-dione 8c.
To a solution of 3a (200 mg, 0.80 mmol) in chloroform (4 mL)
was added
a solution of sodium dithionite (835 mg,
4.80 mmol) in water (8 mL) and the reaction mixture was
stirred at room temperature for 3 h. The precipitate formed
was filtered and dried in vacuo to give the title compound as a
colourless solid (161 mg, 80%); mp 238–239 °C; (found: M +
Na+, 275.0527. C12H12O6Na+ requires: 275.0526); δH (400 MHz;
DMSO-d6) 9.64 (1 H, s, OH), 9.52 (1 H, s, OH), 6.52 (1 H, s,
H-6), 3.94 (3 H, s, OMe), 3.72 (3 H, s, OMe), 2.67 (3 H, s, Me);
δC (100 MHz, DMSO-d6) 167.5, 163.3, 142.6, 136.9, 133.8,
131.1, 114.8, 108.5, 100.8 (CH), 57.0 (Me), 53.0 (Me), 14.9 (Me);
m/z (ESI) 527 (2M + Na+, 100%), 275 (M + Na+, 40).
To a solution of 7a (20.1 mg, 0.057 mmol) in anhydrous aceto-
nitrile (1 mL) was added N,N-dimethylpropane-1,3-diamine
(0.071 mL, 0.57 mmol) and the resulting mixture was stirred at
room temperature for 1 h. The solvent was removed in vacuo
and the crude product was purified by column chromato-
graphy, eluting with methanol–dichloromethane (0–10%) to
give the title compound as a purple solid (8.4 mg, 35%); mp
60–62 °C; (found: M + H+, 423.1546. C21H21F3N2O4 + H+
requires: 423.1526); νmax (CHCl3)/cm−1 3693, 3639, 2360, 2338,
1658, 1631, 1601, 1120; λmax (acetonitrile)/nm 212 (log ε 4.05),
246 (log ε 3.74), 336 (log ε 3.39), 507 (log ε 3.03); δH (400 MHz;
CDCl3) 7.50 (1 H, s br, N-H), 6.65 (2 H, t, J 8.4, ArH), 5.23 (1 H,
s, H-6), 5.17 (2 H, s, CH2), 3.22 (2 H, q, J 6.0, CH2), 2.45 (2 H, t,
J 6.0, CH2), 2.38 (3 H, s, Me), 2.28 (6 H, s, Me), 1.82 (2 H, quin,
J 6.0, CH2); δC (125 MHz; CDCl3) 178.9, 175.1, 157.5 (dt, JCF
245, 14, CF), 156.4 (ddd, JCF 249, 15, 8, CF), 156.9, 152.9, 149.0,
131.3 (dt, JCF 15, 5, C), 122.4, 113.8, 100.7 (ddd, JCF 26, 20, 7,
CH), 94.9 (CH), 65.0 (CH2), 57.9 (CH2), 45.2 (2Me), 42.8 (CH2),
24.6 (CH2), 11.7 (Me); m/z (ESI) 423 (M + H+, 100%).
Biology
IDO assay. IDO inhibitors were assayed as previously
described.47 Briefly, the oxidative cleavage of L-tryptophan cata-
lyzed by rhIDO, expressed in E. coli and purified using Ni2+
affinity chromatography as described,46 was measured as
described previously by Takikawa et al.,48 and modified by
Austin et al.46 Reactions (0.25 mL) were performed in 50 mM
potassium phosphate buffer pH 7.4, containing 20 mM
ascorbic acid, 10 μM methylene blue, 0.4 mg mL−1 catalase
and 4 μg mL−1 rhIDO. DMSO or inhibitor dissolved in DMSO
was added for 10 min then reactions were initiated by the
addition of 400 μM L-tryptophan. After 30 min at 37 °C, reac-
tions were terminated by the addition of 100 μL of 30% (w/v)
trichloroacetic acid. Samples were heated to 65 °C for 15 min
and then centrifuged at 13k rpm for 5 min. Supernatant
(100 μL) was transferred to a 96-well plate and 100 μL of
4-dimethylaminobenzaldehyde (Ehrlich’s reagent, 2% in acetic
acid) was added to each well. After 2 min, the absorbance was
determined using a microplate reader at 490 nm. Results were
quantified against a standard curve generated using authentic
kynurenine. Final results were calculated as percent of DMSO
treated controls. IC50 values were calculated using GraphPad
Prism 4.0 using 6–8 inhibitor concentrations (0.03–100 µM)
from three determinations.
4,7-Dihydro-5-(2-(dimethylamino)ethylamino)-2-(hydroxy-
methyl)-3-methylbenzofuran-4,7-dione 8d.
To a solution of 4,7-dihydro-2-(hydroxymethyl)-5-methoxy-3-
methylbenzofuran-4,7-dione38 (50.0 mg, 0.225 mmol) in anhy-
drous acetonitrile (3 mL) was added N,N-dimethylethane-1,2-
diamine (0.24 mL, 2.25 mmol). After being stirred for 3 h at
room temperature, the solvent was removed in vacuo and the
crude product purified by column chromatography, eluting
with dichloromethane to dichloromethane–methanol (9 : 1) to
give the title compound as a purple solid (62.5 mg, quantitative
yield); mp 146–147 °C (dichloromethane–n-hexane); (found:
Analysis of quinone-induced toxicity
M + H+, 279.1341. C14H18N2O4 + H+ requires: 279.1339); νmax BxPc-3 human pancreatic cancer cells were obtained from
(CHCl3)/cm−1 3352, 1687, 1632, 1589, 1381, 1006; λmax ATCC and grown on glass coverslips in RPMI1640
2672 | Org. Biomol. Chem., 2014, 12, 2663–2674
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Paper
supplemented with 10% (v/v) fetal bovine serum, 100 units
mL−1 penicillin and 100 µg mL−1 streptomycin. To determine
if quinones induce toxicity by damaging microtubules or the
actin cytoskeleton, BxPx-3 cells were treated with quinones 5a
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as a positive control. Immediately following treatment with
quinones cells were fixed in 3.5% (v/v) formaldehyde in phos-
X. B. Ren, X. S. Hao, H. T. Sun, C. Vladau, J. A. Franek,
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were then blocked in RPMI1640 supplemented with 10% (v/v)
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fetal bovine serum for 1 h at room temperature. Immunostain- 12 H. Sugimoto, S. I. Oda, T. Otsuki, T. Hino, T. Yoshida and
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DNA and then can be observed in the nucleus following exci- 17 T. Di Pucchio, S. Danese, R. De Cristofaro and S. Rutella,
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100 mm plates in complete medium and allowed to attach for 19 G. Carr, M. K. W. Chung, A. G. Mauk and R. J. Andersen,
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Acknowledgements
We thank the Association for International Cancer Research
and the University of Nottingham for support.
24 A. Pereira, E. Vottero, M. Roberge, A. G. Mauk and
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