Synthesis of potential prodrug systems for reductive activation. Prodrugs for anti-angiogenic isoflavones and VEGF receptor tyrosine kinase inhibitory oxindoles

Article abstract of DOI:10.1016/j.tet.2009.04.014

A number of potential prodrug systems for reductive activation have been investigated. The prodrug systems chosen for the study were the 2-nitrophenylacetyl, 3-methyl-3-(3,6-dimethyl-1,4-benzoquinon-2-yl)butanoyl and 4-nitrobenzyl groups, readily attached

Full text of DOI:10.1016/j.tet.2009.04.014

Tetrahedron 65 (2009) 4894–4903
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of potential prodrug systems for reductive activation. Prodrugs
for anti-angiogenic isoflavones and VEGF receptor tyrosine
kinase inhibitory oxindoles
,
Emilie A. Blanche a, Lesley Maskell b, Marie A. Colucci c, Jacqueline L. Whatmore b
,
*
,c,
Christopher J. Moodya
*
a Department of Chemistry, University of Exeter, Exeter EX4 4QD, UK
b Peninsula Medical School, St. Luke’s Campus, Exeter EX1 2LU, UK
c School of Chemistry, University of Nottingham, University Park, Nottingham NG7 2RD, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
A number of potential prodrug systems for reductive activation have been investigated. The prodrug
systems chosen for the study were the 2-nitrophenylacetyl, 3-methyl-3-(3,6-dimethyl-1,4-benzoquinon-
2-yl)butanoyl and 4-nitrobenzyl groups, readily attached to acidic OH or NH groups in drug molecules, and
released upon bioreductive activation. The drug molecules studied were the naturally occurring isoflavone
biochanin A, an inhibitor of VEGF-induced angiogenesis, and the pyrrolylmethylidenyl oxindole SU5416
(semaxanib) and its 6-hydroxy derivative, inhibitors of VEGF receptor tyrosine kinase. Following coupling
the prodrug system to the drug, the compounds were evaluated chemically and biologically. Under
chemical reducing conditions, the 3-methyl-3-(3,6-dimethyl-1,4-benzoquinon-2-yl)butanoic acid based
prodrugs appear to fragment the most efficiently, followed by the 2-nitrophenylacetate esters with the 4-
nitrobenzyl ethers being the least efficient. The potentially pro-anti-angiogenic compounds were also
assayed for their ability to block VEGF-induced angiogenesis in HUVECS in comparison to the free agents.
Control compounds that cannot be activated under bioreductive conditions are less potent than the free
drug, whereas many of the potential prodrugs not only exhibit a dose response, but appear at least
equipotent with the free drug.
Received 23 January 2009
Received in revised form 16 March 2009
Accepted 2 April 2009
Available online 10 April 2009
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
with the most common exogenous reductase being a bacterial
nitroreductase.
The design of prodrug systems for use in cancer therapy is
a topic of current interest.1 Such prodrugs have the potential to
improve tumour selectivity of chemotherapeutic agents and hence
reduce unwanted toxic side effects. One prodrug strategy that has
been widely investigated uses bioreduction of readily reducible
compounds such as quinones, nitro aromatics or N-oxides, to re-
lease the active drug from its prodrug.2–6 This strategy takes two
forms. Firstly, one can rely on the combination of hypoxia and the
upregulation of reductase enzymes such as NQO1 (2-electron re-
ductase) and cytochrome c reductase (1-electron) to effect a tu-
mour selective reduction that releases the active species. In the
second approach, an exogenous activating enzyme is delivered to
tumour cells using antibody or gene therapy (ADEPT or GDEPT),
Some examples of bioreductive prodrug strategies are shown for
nitroarenes and quinones in Scheme 1. For illustrative purposes we
only show reduction of the nitro groups to hydroxylamino, followed
by lone-pair initiated fragmentation. The product of further re-
duction, the aniline, would behave similarly. Likewise we only show
reduction of quinones to hydroquinones by a 2-electron process,
although it is recognized that for both nitroarenes and quinones,
bioreductive activation can also proceed by 1-electron pathways
involving radical anion intermediates. In the first two examples the
reduction process results in release of the active drug DH, after
protonation of the leaving group Dꢀ in water, with the formation of
a relatively innocuous byproduct. Thus reduction of the 2-nitro-
phenylacetyl derivative 1 (R¼H or Me) would lead to the corre-
sponding hydroxylamine 2 followed by spontaneous cyclization to
the N-hydroxyoxindole 3 with concomitantreleaseof DH.7 Likewise,
in the second example, reduction of the quinone 4 to the corre-
sponding hydroquinone 5 results in rapid lactonization to give 6
with expulsion of the leaving group;8,9 the method has been used as
* Corresponding authors. Tel.: þ44 115 846 8500.
E-mail addresses: jackie.whatmore@pms.ac.uk (J.L. Whatmore), c.j.moody@
nottingham.ac.uk (C.J. Moody).
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.04.014

E.A. Blanche et al. / Tetrahedron 65 (2009) 4894–4903
4895
R
R
R
R
R
R
D
D
DH
+
O
..
O
O
N
NO2
NHOH
OH
3
1
2
..
HO
Me
O
O
O
O
Me
Me
Me
Me
O
O
Me
Me
+ 2 e–
O2
D
D
DH
+
HO
Me
Me
Me
Me
Me
OH
5
6
4
D
D
..
HOHN
O2N
: Nuc
7
Nuc
O
DH
+
CO2
HOHN
HON
OCOD
O
D
8
9
..
HOHN
O2N
10
R
R
R
R
O –
: Nuc
O –
O
O
O
O
D
Nuc
Me
D
MeO
MeO
MeO
MeO
[O]
+ 2 e–
O2
Me
Me
DH
+
Me
+
..
N
N
N
N
Me
Me
Me
Me
O(H)
O(H)
12
11
13
14
Scheme 1. Proposed mechanisms of bioreductively initiated release of drugs DH from nitroarene and quinone prodrugs.
a bioreductive drug delivery system for melphalan.10,11 In the other
examples shown in Scheme 1 release of the drug DH results in the
generation of an electrophilic species, which could also exert its own
cytotoxic effect. The most common systems of this type involve the
nitrobenzyl group as shown for the 4-nitrobenzyl compound 7,
bioreduction of which would lead to the release of the drug DH si-
multaneous with formation of a reactive electrophile 8. Interception
of 8 by a biological nucleophile (DNA or protein) then leads to irre-
versible modification of the biomolecule and hence toxicity. The
corresponding nitrobenzyloxycarbonyl compound 10 behaves sim-
ilarly. The 4-nitrobenzyl derivatives 7 have found use as prodrugs for
phosphoramide mustards,12,13 whereas the nitrobenzyloxycarbonyl
compounds 10 have been widely applied in GDEPT,6 as prodrugs for,
for example, minor groove alkylating agents,14 doxorubicin15 and
cytotoxic acridines.16 Although all the nitro compounds shown in
Scheme 1 are nitrobenzene derivatives, bioreductiveprodrugs based
on nitro-imidazoles, -furans and -thiophenes have also been de-
veloped.17–23 Our own contributions in this area have involved the
indolequinone 11 prodrug system, reduction of which gives a hydro-
quinone 12 capable of releasing drug DH whilst simultaneously
generating the iminium electrophile 13, reaction of which with
biological nucleophiles gives, after reoxidation, the adduct 14.24–28
We have studied this quinone system using various model drugs,
whilst others have used PARP inhibitors29 and camptothecin.22
In continuation of our interest in bioreductively activated
drugs,24–28,30 and in anti-angiogenic agents,31 we initiated a study
of potential prodrugs for anti-angiogenic isoflavones and VEGF
receptor tyrosine kinase inhibitory oxindoles, and we now report
the full details of this work.32
2. Results and discussion
The development of the microvasculature is a critical aspect of
tumour growth as recognized by Folkman some 35 years ago.33,34
This process of angiogenesis is a prerequisite for tumours to grow
beyond the minimum volume, and although it is stimulated by
a number of factors, there is considerable evidence that vascular
endothelial growth factor (VEGF) is a major contributor to solid
tumour growth by the promotion of both angiogenesis and vascular
permeability.35,36 Hence the inhibition of VEGF expression, in-
duction or function represents an attractive target for the de-
velopment of novel therapeutic agents. To date a number of small
molecule inhibitors of VEGF receptor tyrosine kinases have been
developed and are in clinical trial,37 with AvastinÒ (bevacizumab),
a monoclonal antibody against VEGF and SutentÒ (sunitinib) al-
ready receiving FDA approval.
The compounds chosen for study were the isoflavone biochanin
A 15, the oxindole SU5416 16 and its 6-hydroxy derivative 17
(Fig. 1). Biochanin A 15, and other soya-derived isoflavones such as
genistein, are known to block the effects of VEGF,31,38,39 and are
readily functionalized through the 7-hydroxy group. The oxindole
SU5416 16, also known as semaxanib, is a potent inhibitor of the
VEGF receptor tyrosine kinase Flk-1/KDR, and a proven anti-an-
giogenic agent,40–43 although its clinical development has now
been discontinued. A related oxindole (SU11248) is now marketed
as SutentÒ (sunitinib). Oxindoles are readily functionalized through
their NH group, although we have also prepared the 6-hydroxy
derivative 17 to provide an alternative point of attachment of the
prodrug moiety.

4896
E.A. Blanche et al. / Tetrahedron 65 (2009) 4894–4903
Me
20 are prepared as controls since they cannot participate in the
OMe
reductively activated fragmentations shown in Scheme 1. This is
particularly important in the case of the nitrophenylacetate esters
since they could also release the free drug by a hydrolytic rather
than a reductive process.
OH
O
Me
N
H
O
HO
O
15
N
R
An analogous set of derivatives 24–29 was prepared from
SU5416 16, obtained from condensation of oxindole with 3,5-
dimethylpyrrole-2-caboxaldehyde,45 using similar protocols, as
outlined in Scheme 3. No attempt was made to optimize the rather
poor yields observed in some of these coupling reactions.
Finally, the 6-hydroxy derivative 17 of SU5416 was investigated.
The starting material, 6-hydroxyoxindole,46 was converted into the
corresponding benzyl ether, reaction of which with 3,5-dimethyl
pyrrole-2-aldehyde gave the SU5416 derivative 30. Alternatively,
reaction of 6-hydroxyoxindole with the pyrrole aldehyde gave 6-
hydroxy SU5416 17 in good yield. Subsequent reactions of 17 with
4-nitrobenzyl bromide, 2-nitrophenylacetic acid or the aforemen-
tioned benzoquinonylbutanoic acid gave the novel derivatives 31–
33 (Scheme 4).
H
biochanin A
16 R=H; SU5416
(semaxanib)
17 R=OH
Figure 1. Structures of anti-angiogenic agents studied.
Initially, commercially available biochanin A 15 was converted
into the benzyl and 4-nitrobenzyl ethers 18 and 19 by alkylation
reactions. As expected, the two hydroxyl groups in biochanin A
exhibit different reactivity due to the involvement of the 5-OH in
intramolecular hydrogen bonding, and therefore alkylation oc-
curred predominantly at the 7-OH group, as confirmed by NOE
NMR spectroscopy. Nitro-substituted phenylacetic acids were
readily attached to biochanin A using Mitsunobu or carbodiimide
coupling protocols to give compounds 20–22, the dimethyl
substituted precursor to 22 being prepared by a literature method.7
Finally the quinone derivative 23 was obtained by coupling to 3-
In order to assess the ability of the various prodrug systems to
release their phenolate or oxindole leaving group upon reductive
activation, selected compounds were subjected to a simple chem-
ical reduction. Thus 7-(4-nitrobenzyloxy)biochanin A 19 was
treated with indium powder in ethanolic ammonium chloride so-
lution, an efficient method for the reduction of nitroarenes to
methyl-3-(3,6-dimethyl-1,4-benzoquinon-2-yl)butanoic
(Scheme 2). The benzyl and 3-nitrophenylacetyl derivatives 18 and
acid44
OMe
Me
OH
O
O
X
Me
N
H
O
XC6H4CH2Br, DMF
XC6H4CH2Br, DMF
O
16
15
(X= H) NaH
(X= H) K2CO3
(X = NO2) KH
N
(X = NO2) KH
18 X = H (51%)
X
19 X = NO2 (75%)
24 X = H (93%)
25 X = NO2 (62%)
OMe
OH
O
O
Me
ArCR2CO2H
Me
Ph3P, DIAD, toluene
or
O
N
H
15
R
ArCR2CO2H
R
DCC, DMAP, CH2Cl2
16
O
X
O
N
Ph3P, DIAD, toluene
O
Y
Y
R
R
X
20 R = X = H, Y = NO2 (4%)
21 R = Y = H, X = NO2 (27%)
26 R = X = H, Y = NO2 (6%)
27 R = Y = H, X = NO2 (27%)
22 R = Me, Y = H, X = NO2 (76%)
28 R = Me, Y = H, X = NO2 (5%)
OMe
Me
OH
O
O
Me
N
H
O
O
Quinone-CMe2CH2CO2H
DCC, DMAP, CH2Cl2
N
O
15
Quinone-CMe2CH2CO2H
DCC, DMAP, CH2Cl2
O
O
O
Me
Me
16
Me
Me
Me
O
Me
Me
Me
O
23 (87%)
29 (52%)
Scheme 2.
Scheme 3.

E.A. Blanche et al. / Tetrahedron 65 (2009) 4894–4903
4897
Me
O
BnBr, K2CO3
O
O
MeOH
50%
Me
N
H
N
H
HO
BnO
O2NC6H4CH2Br
O
O
KH, DMF
95%
Me
HO
O
O
EtOH
heat
88%
EtOH
heat
OHC
Me
89% of 30
34
N
H
NO2
35
Me
Me
Me
Me
Me
O
N
N
H
O
H
ArCH2Br
K2CO3
O
ArCR2CO2H
N
H
N
H
HO
O
Ph3P, DIAD, toluene
DMF
O
R
O
34
60% of 31
or
30 X = H
R
17
DCC, DMAP, CH2Cl2
O
X
31 X = NO2
X
Quinone-CMe2CH2CO2H
EDCI, DMAP
CH2Cl2
ArCH2CO2H
DCC, DMAP
CH2Cl2
Y
42%
36 R = X = H, Y = NO2 (72%)
37 R = Y = H, X = NO2 (32%)
22%
Me
Me
38 R = Me, Y = H, X = NO2 (61%)
Me
Me
Me
N
H
N
H
O
O
N
H
N
O
O
H
O
O
O
Quinone-CMe2CH2CO2H
O
O
Me
O
O
34
O
Me
O
O
DCC, DMAP, CH2Cl2
34%
O2N
Me
33
Me
Me
Me
Me
Me
32
39
Scheme 4. Yields in scheme clarified.
Scheme 5.
anilines47 and the reaction monitored by NMR spectroscopy. The
signal for the benzylic CH2 group at ca. 5.20 disappeared after
release of coumarin from the 4-nitrobenzyl ether 35 was very slow,
whereas the 2-nitrophenylacetic acid derivative 37, and the ben-
zoquinone 39 underwent fragmentationwith release of coumarin as
shown in Figure 2. In order to check that this was due to reductive
activation rather than simple hydrolysis of the phenylacetate esters,
the 3-nitro compound 36, in which there is no possibility for
mechanism based reductive fragmentation (Scheme 1), was studied.
Under conditions that led to rapid increase in fluorescence for
compound 37, no fluorescence was observed for 36.
d
about 40 min, concomitant with the appearance of signals due to
free biochanin A. Likewise reduction of the 2-nitrophenylacetyl
derivative 21 also triggers the release of biochanin A. In the oxin-
dole series, reduction of the 4-nitrobenzyl compound 25 proceeded
cleanly to give the corresponding 4-aminobenzyl derivative that
did not fragment further. In contrast, indium reduction of the 2-
nitrophenylacetyl derivative 27 did result in release of free SU5416
16.
1
For a more complete study of the ability of the prodrug systems
to fragment upon reductive activation, a model system was also
investigated. Thus the strongly fluorescent compound, 7-hydroxy-
4-methylcoumarin 34, was functionalized as described previously
for biochanin A 15 to give the coumarin derivatives 35–39 (Scheme
5). These compounds in which the coumarin 7-OH is blocked are
essentially non-fluorescent and hence can act as pro-fluorescent
indicators for efficient reductive activation leading to release of the
fluorophore itself. A similar approach has been used with 7-ami-
nocoumarin derivatives.48
Compound 35
Compound 37
0.8
Compound 39
0.6
0.4
0.2
0
The pro-fluorescent compounds (10 mmol) were dissolved in
a chloroform–ethanol–water mixture in a cuvette and treated with
an excess of sodium dithionite as reducing agent. The progress of the
reaction was monitored by following the fluorescence at 445 nm
(excitation wavelength 360 nm) in a fluorescence spectrometer pre-
0
20
40
60
Time (min)
80
100
120
calibrated so as to reach 70% fluorescence for 10
marin. It was immediately apparent that under these conditions
mmol of free cou-
Figure 2. Increase in fluorescence upon release of 4-methyl-7-hydroxycoumarin 34
from compounds 35, 37 and 39 following reduction with sodium dithionite.

4898
E.A. Blanche et al. / Tetrahedron 65 (2009) 4894–4903
A selection of the reductively activated prodrugs was assayed for
angiogenesis observed at 10 mM for all compounds, and with sim-
their ability to inhibit VEGF-induced angiogenesis in human um-
bilical vein endothelial cells (HUVECs) in comparison to the free
agents 15–17. This assay examines the ability of human endothelial
cells grown on a fibrin matrix to form three-dimensional structures
when stimulated with the angiogenic promoter VEGF. The potency
of each drug to inhibit this angiogenesis at a range of concentra-
tions was then examined. HUVECs have been reported to express
a range of bioreductive enzymes including NQO1,49,50 and cyto-
chrome c reductase (unpublished data from this laboratory) in-
dicating that the cellular systems for bioreduction of the prodrugs
are present.
ilar dose response profiles. A small number of 6-substituted de-
rivatives of pyrrolylmethylidenyl oxindoles have been investigated
previously; in the SU5416 series, the 6-fluoro-analogue is reported
to be slightly more potent than its 6-unsubstituted counterpart,40
whereas in the closely related SU6668 series of compounds that
contain an additional 2-carboxyethyl group at the pyrrole 4-posi-
tion, introduction of a large aryl substituent at the oxindole 6-po-
sition appears to increase potency.41 Such an effect may be
responsible for the activity of compounds 30 and 31.
3. Conclusions
Firstly, to control for the possible biological effects of com-
pounds deriving from the fragmentation of the prodrug systems,
such compounds were tested alone for their ability to inhibit VEGF-
stimulated angiogenesis. Thus 4-aminobenzyl alcohol (a possible
product from prodrugs 19, 25 and 31), oxindole (a possible product
from prodrug 27) and 3,3-dimethyloxindole (a possible product
from prodrug 28) were tested, but none of these compounds sig-
nificantly inhibited angiogenesis at the concentrations tested.
Secondly, to avoid the complication of hydrolytic cleavage of the
ester bonds competing with the planned reductive fragmentation,
the aryl esters 20–23 and 32 and 33 were omitted from this study.
The results are summarized in Figure 3. In the biochanin A series,
compound 18 that cannot be cleaved under bioreductive conditions
is less potent than the free drugdit did not inhibit VEGF-stimulated
angiogenesis to the same degree of significance as the free drug.
The potential prodrug 19 appeared to exhibit a dose response, but is
at least equipotent with free biochanin A. Although the ability of
biochanin A to inhibit angiogenesis has also been noted elsewhere
in the literature,38 the molecular mechanism is not known.
The SU5416 (semaxanib) series of compounds are generally
more potent than the isoflavones, with free SU5416 16 itself causing
We have synthesized and evaluated a range of novel derivatives
of the anti-angiogenic compounds biochanin A and SU5416 (sem-
axanib) as potential prodrug systems that can be bioreductively
activated. Under chemical reducing conditions, the 3-methyl-3-
(3,6-dimethyl-1,4-benzoquinon-2-yl)butanoic acid based prodrugs
appear to fragment the most efficiently, followed by the 2-nitro-
phenylacetate esters with the 4-nitrobenzyl ethers being the least
efficient. In cellular systems all of the compounds that fragment
rapidly by bioreduction significantly inhibited VEGF-stimulated
angiogenesis at concentrations comparable to their parent com-
pound, suggesting that the active drug is being released in a bi-
ological system. Several of the compounds, particularly the
derivatives of SU5416 have potent anti-angiogenic activity, and
form the basis of further study.
4. Experimental section
4.1. General procedures
Commercially available reagents were used throughout without
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 ni-
trogen or argon atmosphere. Analytical thin layer chromatography
was carried out on aluminium-backed plates coated with Merck
Kieselgel 60 GF254, and visualized under UV light at 254 and/or
360 nm. Chromatography was carried out using Merck Kieselgel
60H silica or Matrex silica 60. Fully characterized compounds were
chromatographically homogeneous. Infrared spectra were recorded
in the range 4000–600 cmꢀ1 using Nicolet Magna FT-550 or Perkin
Elmer FT-1600 spectrometers. NMR spectra were carried out on
Bruker 300, 400 and 500 MHz instruments (1H frequencies, cor-
responding 13C frequencies are 75, 100 and 125 MHz). Chemical
94% inhibition of VEGF-stimulated angiogenesis at 1
mM and 100%
inhibition at 10 M (Fig. 3). Again the benzyl derivative 24 that
m
cannot undergo bioreductive fragmentation is considerably less
potent than the free drug and does not significantly inhibit VEGF-
induced angiogenesis at either 1 mM or 10 mM. However, its 4-
nitrobenzyl analogue 25, a potential prodrug, is more potent than
24, inducing significant inhibition of VEGF-stimulated angiogenesis
at 1 mM, suggesting that bioreductive release does occur. The sim-
ilarity between the 3-and 2-nitrophenylacetyl compounds 26 and
27 suggests that the compounds may be undergoing hydrolytic
rather than reductive fragmentation.
The 6-substituted derivatives of SU5416 17 and 30–31 all appear
equipotent (Fig. 3) with significant inhibition of VEGF-stimulated
120
100
80
60
40
20
0
15
18
19
16
24
25
Compounds tested (mM)
Figure 3. Inhibition of VEGF-stimulated angiogenesis in HUVECs by biochanin A 15, SU5416 16 and 6-hydroxy SU5416 17 and various derivatives. HUVECs were seeded onto pre-
26
27
28
17
30
31
formed fibrin matrices and treated with 100 ng/mL VEGFꢂtest compounds at 1 (yellow), 10 (blue), or 100 (red)
mM. After 5 days the formation of tubular structures was quantified.
Results are expressed as percentage inhibition of a VEGF control and show meanꢂSD.

E.A. Blanche et al. / Tetrahedron 65 (2009) 4894–4903
4899
shifts are quoted in parts per million with TMS as internal standard.
J values are recorded in hertz. In the 13C spectra, signals corre-
sponding to CH, CH2 or CH3 groups, as assigned from DEPT, 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 Centre (Swansea).
d, J 2.1, ArH), 6.56 (1H, d, J 2.1, ArH), 4.03 (2H, s, CH2), 3.85 (3H, s,
OMe); m/z (CI) 313 (18%), 285 (100), 164 (21).
4.1.4. 5-Hydroxy-3-(4-methoxyphenyl)-7-(2-nitrophenylacetoxy)-
chromen-4-one 21
To a solution of biochanin A 15 (375 mg, 1.26 mmol) in toluene
(15 mL) was added 2-nitrophenylacetic acid (456 mg, 2.52 mmol),
triphenylphosphine (663 mg, 2.52 mmol) and diisopropyl azodi-
carboxylate (0.5 mL, 2.52 mmol) dropwise. The reaction mixture
was stirred at room temperature for 18 h. Ethyl acetate was added
and the organic phase was washed with sodium hydroxide solution
(2 M), water and brine, dried (MgSO4), filtered and concentrated.
Purification by chromatography eluting with ethyl acetate–
dichloromethane (1:19) gave the title compound (155 mg, 27%) as
a colourless solid, mp 155–156 ꢁC (ethanol). (Found: C, 64.4; H, 3.5;
N, 2.9. C24H17NO8 requires: C, 64.4; H, 3.8; N, 3.1%.) nmax (KBr)/cmꢀ1
3431, 1772,1649,1608,1577,1511; dH (300 MHz; CDCl3) 12.82 (1H, s,
OH), 8.23 (1H, dd, J 8.2, 1.2, ArH), 7.95 (1H, s, 2-H), 7.68 (1H, m, ArH),
7.58 (1H, m, ArH), 7.49 (1H, d, J 6.7), 7.47 (2H, dd, J 2.1, 6.8, ArH), 7.00
(2H, dd, J 2.1, 6.8, ArH), 6.80 (1H, d, J 2.1, CH), 6.62 (1H, d, J 2.1, CH),
4.29 (2H, s, CH2), 3.86 (3H, s, OMe); dC (75 MHz; CDCl3) 181.7 (C),
168.1 (C), 162.8 (C), 160.3 (C), 157.3 (C), 156.2 (C), 153.7 (CH), 148.8
(C), 134.5 (CH), 134.0 (CH), 130.5 (CH), 129.6 (CH), 129.4 (C), 126.0
(CH),124.5 (C),122.9 (C),114.5 (CH),110.0 (C), 105.7 (CH),101.3 (CH),
55.8 (Me), 40.6 (CH2); m/z (CI) 404 (21%), 313 (51), 286 (26), 284
(100).
4.1.1. 7-Benzyloxy 5-hydroxy-3-(4-methoxyphenyl)chromen-4-
one 18
To biochanin A 15 (300 mg, 1.0 mmol) in DMF (9 mL) was added
potassium carbonate (159 mg, 1.1 mmol) and the mixture was
stirred at room temperature for 1.5 h. Benzyl bromide (0.14 mL,
1.1 mmol) was added and the mixture was stirred at room tem-
perature for 4 h before adding water (5 mL). The product was
extracted using ethyl acetate. The combined organic layers were
washed with brine (2ꢃ10 mL) and were evaporated. The residue
was purified by chromatography eluting with dichloromethane to
give the title compound (200 mg, 51%) as a colourless solid; mp 197–
199 ꢁC (ethanol) (lit.,51 mp 190 ꢁC (acetone–methanol)). (Found: C,
74.0; H, 4.7. C23H18O5 requires: C, 73.8; H, 4.8%.) nmax (KBr)/cmꢀ1
3436, 1665, 1607, 1571, 1253, 1179, 1163; dH (300 MHz; CDCl3) 12.87
(1H, br s, OH), 7.86 (1H, s, 2-H), 7.42 (7H, m, ArH), 6.98 (2H, dd, J 2.1,
6.8, ArH), 6.47 (2H, m, ArH), 5.13 (2H, s, CH2), 3.85 (3H, s, OMe); dC
(100 MHz; CDCl3) 180.8 (C), 164.6 (C), 162.7 (C), 159.8 (C), 157.9 (C),
152.7 (CH), 135.7 (C), 130.1 (CH), 128.7 (CH), 128.4 (CH), 127.5 (CH),
123.7 (C), 122.9 (C), 114.1 (CH), 98.9 (CH), 93.3 (CH), 70.5 (CH2), 55.4
(Me); one C unobserved; m/z (EI) 374 (Mþ, 46%), 91 (100).
4.1.5. 5-Hydroxy-3-(4-methoxyphenyl)-7-[2-(2-nitrophenyl)-2,2-
dimethylacetoxy]chromen-4-one 22
4.1.2. 5-Hydroxy-3-(4-methoxyphenyl)-7-(4-nitrobenzyloxy)-
chromen-4-one 19
To biochanin A 15 (163 mg, 0.57 mmol), 2-methyl-2-(2-nitro-
phenyl)propanoic acid7 (100 mg, 0.48 mmol) and 4-dimethylami-
nopyridine (11 mg, 0.01 mmol) in dichloromethane (4 mL) was
added dicyclohexylcarbodiimide (1 M in dichloromethane;
0.57 mL, 0.57 mmol) dropwise. The mixture was stirred at room
temperature overnight. The solvent was evaporated and the resi-
due was purified by chromatography, eluting with dichloro-
methane to give the title compound (173 mg, 76%) as a pale yellow
solid; mp 172–173 ꢁC (ethanol). (Found: C, 65.8; H, 4.4; N, 3.0.
C26H21NO8 requires: C, 65.7; H, 4.5; N, 2.9%.) (Found: MHþ,
476.1343. C26H21NO8þH requires: 476.1345.) nmax (KBr)/cmꢀ1 3436,
1762, 1637, 1607, 1582, 1515, 1247, 1128; dH (300 MHz; CDCl3) 12.83
(1H, s, OH), 8.05 (1H, d, J 7.7, ArH), 7.94 (1H, s, 2-H), 7.70 (2H, m,
ArH), 7.51 (1H, m, ArH), 7.47 (2H, dd, J 1.9, 6.9, ArH), 6.99 (2H, dd, J
1.9, 6.9, ArH), 6.80 (1H, d, J 2.0, ArH), 6.54 (1H, d, J 2.0, ArH), 3.85
(3H, s, OMe), 1.84 (6H, s, Me); dC (100 MHz; CDCl3) 181.3 (C), 172.9
(C), 162.3 (C), 159.9 (C), 156.9 (C), 156.1 (C), 153.3 (CH), 148.3 (C),
138.4 (C), 133.7 (CH), 130.1 (CH), 128.3 (CH), 128.1 (CH), 126.1 (CH),
124.0 (C), 122.6 (C), 114.1 (CH), 109.5 (C), 105.3 (CH), 101.1 (CH), 55.4
(Me), 46.6 (C), 27.2 (Me); m/z (CI) 476 (MHþ, 3%), 313 (18), 285
(100), 162 (14), 134 (18).
To a solution of biochanin A 15 (100 mg, 0.35 mmol) in DMF
(3 mL) was added potassium hydride (21 mg, 0.52 mmol) portion-
wise and the mixture was stirred at room temperature for 1 h.
4-Nitrobenzyl bromide (91 mg, 0.42 mmol) was added and the
solution was stirred at room temperature overnight. Water was
added and the reaction mixture was extracted with ethyl acetate
and toluene. The combined organic layers were dried (MgSO4),
filtered and evaporated in vacuo. Purification by chromatography
eluting with ethyl acetate–toluene (1:4) gave the title compound
(110 mg, 75%) as a colourless solid; mp 220–223 ꢁC (ethanol).
(Found: Mþ, 419.1006. C23H17NO7 requires: 419.1005.) nmax (KBr)/
cmꢀ1 3416, 3073, 2924, 2847, 1736, 1644, 1618; dH (400 MHz; CDCl3)
12.90 (1H, s, OH), 8.28 (2H, d, J 8.8, ArH), 7.89 (1H, s, 2-H), 7.62 (2H,
d, J 8.8, ArH), 7.46 (2H, dd, J 2.2, 6.7, ArH), 6.99 (2H, dd, J 2.2, 6.7,
ArH), 6.47 (1H, d, J 2.3, ArH), 6.45 (1H, d, J 2.3, ArH), 5.24 (2H, s,
CH2), 3.85 (3H, s, OMe); dC (100 MHz; CDCl3) 180.9 (C), 163.7 (C),
162.9 (C), 159.8 (C), 157.9 (C), 152.8 (C), 143.0 (C), 130.1 (CH), 127.7
(CH), 124.0 (CH), 123.8 (C), 122.7 (C), 114.1 (CH), 106.7 (C), 98.7 (CH),
93.3 (CH), 77.2 (CH), 68.9 (CH2), 55.4 (Me); m/z (EI) 419 (Mþ, 100),
284 (39), 255 (30), 132 (15), 105 (18), 78 (31).
4.1.3. 5-Hydroxy-3-(4-methoxyphenyl)-7-(3-nitrophenylacetoxy)-
chromen-4-one 20
4.1.6. 5-Hydroxy-3-(4-methoxyphenyl)-7-[3-methyl-3-(3,6-
dimethyl-1,4-benzoquinon-2-yl)butan-1-oyloxy]chromen-4-one 23
To 3-methyl-3-(3,6-dimethyl-1,4-benzoquinon-2-yl)butanoic
acid (200 mg, 0.45 mmol), biochanin A 15 (356 mg, 1.35 mmol) and
4-dimethylaminopyridine (10 mg, 0.10 mmol) in dichloromethane
(5 mL) was added dicyclohexylcarbodiimide (1 M in dichloro-
methane; 0.9 mL, 0.90 mmol). The reaction mixture was stirred at
room temperature overnight. The solvent was evaporated and the
residue purified by chromatography, eluting with ethyl acetate–
toluene (1:9) to give the title compound (197 mg, 87%) as a colour-
less solid; mp 110 ꢁC (ethanol). (Found: MHþ, 503.1698.
C29H26O8þH requires: 503.1706.) nmax (KBr)/cmꢀ1 3088, 2965,
2939, 1751, 1644, 1613, 1516, 1255, 1122; dH (300 MHz; CDCl3) 12.80
(1H, s, OH), 7.92 (1H, s, 2-H), 7.44 (2H, dd, J 2.1, 6.7, ArH), 6.98 (2H,
dd, J 2.1, 6.7, ArH), 6.63 (1H, d, J 2.1, ArH), 6.48 (1H, d, J 2.1, ArH), 6.46
To biochanin A 15 (300 mg,1.06 mmol), 3-nitrophenylacetic acid
(147 mg, 0.81 mmol) and 4-dimethylaminopyridine (19 mg,
0.16 mmol) in dichloromethane (5 mL) was added dicyclohex-
ylcarbodiimide (1 M in dichloromethane; 1.1 mL, 1.06 mmol)
dropwise. The mixture was stirred at room temperature for 48 h.
The solvent was evaporated and the residue was purified by chro-
matography, eluting with dichloromethane to give the title com-
pound (142 mg, 40%) as a colourless solid; mp 149–151 ꢁC (ethanol).
(Found: C, 64.4; H, 3.8; N, 3.1. C24H17NO8 requires: C, 64.4; H, 3.8; N,
3.1%.) nmax (KBr)/cmꢀ1 3467, 3080, 1763, 1608, 1529, 1515, 1350,
1251, 1144; dH (300 MHz; CDCl3) 12.85 (1H, s, OH), 8.28 (1H, s, ArH),
8.21 (1H, d, J 8.2, ArH), 7.94 (1H, s, 2-H), 7.73 (1H, d, J 7.7, ArH), 7.58
(1H, m, ArH), 7.45 (2H, d, J 8.7, ArH), 6.98 (2H, d, J 8.7, ArH), 6.75 (1H,

4900
E.A. Blanche et al. / Tetrahedron 65 (2009) 4894–4903
(1H, s, ArH quinone), 3.84 (3H, s, OMe), 3.27 (2H, s, CH2), 2.18 (3H, s,
Me), 1.99 (3H, s, Me), 1.54 (6H, s, Me), irradiation of OH resulted in
a 3.2% enhancement of H-6 signal and no enhancement of the H-8
signal; dC (75 MHz; CDCl3) 191.0 (C), 187.1 (C), 181.2 (C), 170.3 (C),
162.5 (C), 160.0 (C), 156.9 (C), 155.6 (C), 155.6 (C), 153.2 (CH), 151.7
(C), 148.0 (C), 139.9 (C), 131.6 (CH), 130.1 (CH), 124.1 (C), 122.4 (C),
114.2 (CH), 109.5 (C), 105.3 (CH), 100.7 (CH), 55.3 (Me), 47.6 (CH2),
38.7 (C), 15.9 (Me), 14.2 (Me), 28.9 (Me); m/z (CI) 313 (21%), 285
(100), 284 (13), 221 (14), 207 (10).
4.1.11. 3-[(3,5-Dimethylpyrrol-2-yl)methylidenyl]-1-(2-
nitrophenylacetyl)indolin-2-one 27
To a solution of SU5416 16 (100 mg, 0.42 mmol) in toluene
(5 mL) were added 2-nitrophenylacetic acid (152 mg, 0.84 mmol),
triphenylphosphine (221 mg, 0.84 mmol) and diisopropyl azodi-
carboxylate (0.33 mL, 1.68 mmol) dropwise. The reaction mixture
was stirred at room temperature for 18 h. Ethyl acetate was added
and the organic phase was washed with sodium hydroxide solution
(2 M), water and brine, dried (MgSO4), filtered and concentrated in
vacuo. Purification by chromatography eluting with ethyl acetate–
light petroleum (1:4) gave the title compound (46 mg, 27%) as an
orange solid; mp 203 ꢁC (dichloromethane–ethanol). (Found: MHþ,
402.1476. C23H19N3O4þH requires: 402.1454.) nmax (KBr)/cmꢀ1
3426, 3354, 2919, 1685, 1547, 1342; dH (300 MHz; CDCl3) 12.58 (1H,
br s, NH), 8.20 (1H, d, J 6.8, ArH), 8.16 (1H, d, J 9.4, ArH), 7.63 (1H, td,
J 7.3, 1.0, ArH), 7.49 (3H, m, ArH), 7.42 (1H, s, ]CH), 7.19 (2H, m,
ArH), 6.06 (1H, s, pyrrole-H), 4.92 (2H, s, CH2), 2.45 (3H, s, Me), 2.37
(3H, s, Me); dC (75 MHz; CDCl3) 170.5 (C), 160.0 (C), 149.3 (C), 138.6
(C), 136.5 (C), 134.9 (C), 133.4 (CH), 133.1 (CH), 130.5 (C), 128.3 (CH),
127.4 (CH), 126.5 (C), 126.1 (CH), 125.1 (CH), 124.5 (CH), 124.2 (CH),
121.0 (C),116.3 (CH),113.6 (CH), 44.0 (CH2),14.0 (Me),11.7 (Me); one
C unobserved; m/z (CI) 402 (MHþ, 5%), 267 (17), 253 (10), 240 (18),
239 (100), 238 (48), 237 (10).
4.1.7. 3-[(3,5-Dimethylpyrrol-2-yl)methylidenyl]indolin-2-one
(SU5416) 16
Prepared in 77% yield from oxindole and 3,5-dimethylpyrrole-2-
carboxaldehyde45 as described in the literature;40 mp 226–227 ꢁC
(ethanol) (lit.,40 mp not given).
4.1.8. 1-Benzyl-3-[(3,5-dimethylpyrrol-2-yl)methylidenyl]indolin-
2-one 24
Prepared (93%) by benzylation of 16 as described in previously.31
4.1.9. 3-[(3,5-Dimethylpyrrol-2-yl)methylidenyl]-1-(4-
nitrobenzyl)indolin-2-one 25
To a solution of SU5416 15 (100 mg, 0.42 mmol) in DMF (3 mL)
was added portionwise potassium hydride (washed from 30% in oil;
26 mg, 0.63 mmol) under a nitrogen atmosphere. The solution was
stirred at room temperature for 1 h before adding 4-nitrobenzyl
bromide (100 mg, 0.46 mmol) portionwise. The mixture was stirred
at room temperature overnight and quenched with water. Brine
was added to the reaction mixture, which was extracted with ether.
The combined organic layers were dried (MgSO4), filtered and
evaporated in vacuo. The crude material was purified by chroma-
tography, eluting with ethyl acetate–light petroleum (3:7) to give
the title compound (97 mg, 62þ%) as a yellow orange solid; mp 195–
198 ꢁC (ethanol). (Found: MH , 374.1506. C22H19N3O3þH requires:
374.1504.) nmax (KBr)/cmꢀ1 3047, 2919, 2852, 1675, 1567, 1347; dH
(300 MHz; CDCl3) 13.14 (1H, br s, NH), 8.18 (2H, d, J 8.8, ArH), 7.54
(1H, m, ArH), 7.47 (1H, s, ]CH), 7.45 (2H, d, J 8.8, ArH), 7.10 (2H, m,
ArH), 6.70 (1H, m, ArH), 6.01 (1H, d, J 2.5, pyrrole-H), 5.19 (2H, s,
CH2), 2.39 (3H, s, Me), 2.36 (3H, s, Me); dC (75 MHz; CDCl3) 168.2
(C), 144.1 (C), 138.4 (C), 138.1 (C), 133.7 (C), 127.9 (CH), 127.2 (C),
125.7 (CH), 125.5 (C), 124.1 (CH), 123.8 (CH), 122.1 (CH), 117.3 (CH),
113.0 (CH), 110.7 (C), 108.2 (CH), 103.0 (C), 43.1 (CH2), 14.0 (Me), 11.7
(Me).
4.1.12. 3-[(3,5-Dimethylpyrrol-2-yl)methylidenyl]-1-[2-(2-
nitrophenyl)-2,2-dimethylacetyl]indolin-2-one 28
To SU5416 16 (300 mg, 1.26 mmol), 2-methyl-2-(2-nitro-
phenyl)propanoic acid7 (272 mg, 1.30 mmol) and triphenylphos-
phine (397 mg, 1.51 mmol) in toluene (15 mL) was added
diisopropyl azodicarboxylate (0.37 mL, 1.89 mmol) dropwise. The
mixture was stirred at 70 ꢁC overnight. The solvent was evaporated
and the residue was purified twice by chromatography, eluting first
with ethyl acetate–light petroleum (1:4) and then with dichloro-
methane–light petroleum (1:1) to give the title compound (27 mg,
5%) as an orange solid; mp 147–150 ꢁC (ethanol). (Found: C, 69.8; H,
5.6; N, 9.7. C25H23N3O4 requires: C, 69.9; H, 5.4; N, 9.8%.) (Found:
Mþ, 429.1674. C25H23N3O4 requires: 429.1688.) nmax (KBr)/cmꢀ1
3436, 2927, 1700, 1686, 1565, 1523, 1343, 1289, 1260, 1148; dH
(300 MHz; CDCl3) 12.02 (1H, br s, NH), 8.10 (1H, d, J 9.2, ArH), 7.68
(2H, m, ArH), 7.54 (1H, m, ArH), 7.42 (1H, dd, J 1.3, 7.6, ArH), 7.25
(2H, m, ArH), 7.22 (1H, s, ]CH), 7.17 (1H, dt, J 1.2, 7.6, ArH), 5.93 (1H,
s, pyrrole-H), 2.28 (3H, s, Me), 2.25 (3H, s, Me), 1.93 (6H, s, Me); dC
(100 MHz; CDCl3) 176.5 (C), 166.5 (C), 148.2 (C), 139.7 (C), 137.2 (C),
136.1 (C), 133.8 (C), 132.0 (CH), 129.3 (CH), 127.0 (C), 126.9 (C), 126.8
(CH), 126.2 (CH), 125.0 (CH), 124.2 (CH), 123.5 (CH), 116.3 (CH), 116.1
(CH), 113.0 (CH), 109.8 (C), 50.4 (C), 27.6 (Me), 13.8 (Me), 11.6 (Me);
m/z (EI) 429 (Mþ, 18), 237 (100), 209 (42).
4.1.10. 3-[(3,5-Dimethylpyrrol-2-yl)methylidenyl]-1-(3-
nitrophenylacetyl)indolin-2-one 26
To SU5416 16 (200 mg, 0.84 mmol), 3-nitrophenylacetic acid
(228 mg, 1.26 mmol) and triphenylphosphine (331 mg, 1.26 mmol)
in toluene (10 mL) was added diisopropyl azodicarboxylate
(0.25 mL, 1.26 mmol) dropwise. The mixture was stirred at room
temperature overnight, and then at 70 ꢁC for 24 h. The solvent was
evaporated and the residue was purified by chromatography,
eluting with dichloromethane to give the title compound (19 mg,
6%) as an orange solid; mp 220–222 ꢁC (ethanol). (Found: C, 68.55;
H, 4.8; N, 10.2. C23H19N3O4 requires: C, 68.8; H, 4.8; N, 10.5%.)
(Found: Mþ, 401.1395. C23H19N3O4 requires: 401.1375.) nmax (KBr)/
cmꢀ1 3423, 1698, 1687, 1599, 1580, 1531, 1348, 1318, 1148; dH
(300 MHz; CDCl3) 12.60 (1H, br s, NH), 8.30 (1H, s, ArH), 8.23 (1H,
m, ArH), 8.18 (1H, m, ArH), 7.72 (1H, m, ArH), 7.55 (1H, d, J 7.9, ArH),
7.51 (1H, m, ArH), 7.43 (1H, s, ]CH), 7.21 (2H, m, ArH), 6.08 (1H, s,
pyrrole-H), 4.70 (2H, s, CH2), 2.46 (3H, s, Me), 2.36 (3H, s, Me); dC
(100 MHz; CDCl3) 171.0 (C), 168.6 (C), 148.3 (C), 138.9 (C), 136.4 (C),
136.2 (CH), 135.8 (C), 135.3 (C), 129.3 (CH), 127.5 (C), 126.7 (C), 126.1
(CH), 125.3 (CH), 124.7 (CH), 124.4 (CH), 122.2 (CH), 116.4 (CH), 116.3
(CH), 113.9 (CH), 109.3 (C), 44.3 (CH2), 14.1 (Me), 11.8 (Me); m/z (EI)
401 (Mþ, 50%), 238 (100), 221 (47), 209 (18), 136 (16), 89 (21).
4.1.13. 3-[(3,5-Dimethylpyrrol-2-yl)methylidenyl]-1-[3-methyl-3-
(3,6-dimethyl-1,4-benzoquinon-2-yl)butan-1-oyl]indolin-2-one 29
To 3-methyl-3-(3,6-dimethyl-1,4-benzoquinon-2-yl)butanoic
acid44 (245 mg, 1.04 mmol), SU5416 16 (295 mg, 1.24 mmol) and 4-
dimethylaminopyridine (24 mg, 0.02 mmol) in dichloromethane
(5 mL) was added dicyclohexylcarbodiimide (1 M in dichloro-
methane; 1.24 mL,1.24 mmol) dropwise. The mixture was stirred at
room temperature overnight. The solvent was evaporated and the
residue was purified by chromatography, eluting with dichloro-
methane to give the title compound (215þmg, 52%) as an orange
solid; mp 125–127 ꢁC (ethanol). (Found: M , 456.2029. C28H28N2O4
requires: 456.2049.) nmax (KBr)/cmꢀ1 3423, 2924, 1638, 1650, 1636,
1540, 1355, 1315, 1262, 1149, 1081; dH (300 MHz; CDCl3) 12.54 (1H,
br s, NH), 8.11 (1H, m, ArH), 7.50 (1H, m, ArH), 7.41 (1H, s, ]CH), 7.18
(2H, m, ArH), 6.49 (1H, d, J 1.5, CH), 6.07 (1H, d, J 2.3, pyrrole-H),
3.97 (2H, s, CH2), 2.47 (3H, s, Me), 2.37 (3H, s, Me), 2.21 (3H, s, Me),
1.93 (3H, s, Me), 1.58 (6H, s, Me); dC (100 MHz; CDCl3) 191.3 (C),
187.6 (C), 174.0 (C), 168.5 (C), 154.5 (C), 148.3 (C), 138.3 (C), 137.5 (C),

E.A. Blanche et al. / Tetrahedron 65 (2009) 4894–4903
4901
136.0 (C), 134.6 (C), 131.4 (CH), 127.3 (C), 126.6 (C), 125.9 (CH), 124.3
(CH), 124.0 (CH), 116.3 (CH), 116.0 (CH), 113.6 (CH), 109.9 (C), 52.8
(CH2), 37.9 (C), 28.7 (Me), 15.9 (Me), 14.2 (Me), 14.1 (Me), 11.7 (Me);
m/z (CI) 457 (MHþ, 43%), 456 (Mþ, 21), 240 (13), 239 (100), 238 (77),
221 (65), 220 (10).
0.12 mmol) was added and the reaction mixture stirred at rt for 3 h.
The solution was then heated to 60 ꢁC for 1 h. On cooling the re-
action mixture was diluted with ethyl acetate (20 mL) and washed
with water (2ꢃ20 mL) and dried (MgSO4). The solvent was re-
moved in vacuo and the crude product was recrystallized from
ethyl acetate–methanol to give the title compound (27.5 mg, 60%) as
an yellow crystalline solid; mp 253–255 ꢁC (decomp.) (from ethyl
acetate–methanol). (Found: MHþ, 390.1438. C22H19N3O4þH re-
quires: 390.1454.) nmax (KBr)/cmꢀ1 3449, 1674, 1631, 1568, 1348,
1312, 1150, 1102; dH (500 MHz; DMSO-d) 13.15 (1H, br s, NH), 10.76
(1H, s, NH), 8.27 (2H, d, J 8.5, ArH), 7.73 (2H, d, J 8.5, ArH), 7.63 (1H,
d, J 8.5, ArH), 7.42 (1H, s, ]CH), 6.67 (1H, dd, J 8.5, 2.5, ArH), 6.53
(1H, d, J 2.5, ArH), 5.96 (1H, d, J 2.0, pyrrole-H), 5.29 (2H, s, CH2),
2.32 (3H, s, Me), 2.27 (3H, s, Me); dC (100 MHz; DMSO-d) 169.8 (C),
157.0 (C), 146.9 (C), 145.2 (C), 139.3 (C), 134.6 (C), 130.4 (C), 128.1
(CH), 126.4 (C), 123.5 (CH), 121.8 (CH), 119.2 (C), 119.1 (CH), 112.8
(CH), 112.1 (C), 107.6 (CH), 96.8 (CH), 68.2 (CH2), 13.4 (Me), 11.2
(Me); m/z (ES) 390 (MHþ, 100%), 263 (95), 149 (80).
4.1.14. 6-Hydroxy-3-[(3,5-dimethylpyrrol-2-yl)methylidenyl]-
indolin-2-one 17
A solution of 6-hydroxyoxindole46 (150 mg, 1.01 mmol), pyrro-
lidine (0.13 mL, 1.51 mmol) and 3,5-dimethylpyrrole-2-carbox-
aldehyde45 (149 mg, 1.21 mmol) in ethanol (10 mL) was heated to
reflux at 90 ꢁC for 4 h. The ethanol was removed in vacuo and the
crude product was purified by chromatography eluting with ethyl
acetate–light petroleum (2:3) to give the title compound (225 mg,
88%) as an orange crystalline solid; mp >300 ꢁC (from ethyl ace-
tate–light petroleum). (Found: Mþ, 254.1055. C15H14N2O2 requires:
254.1055.) nmax (KBr)/cmꢀ1 3697, 3606, 1602, 1266, 1105; dH
(400 MHz; DMSO-d) 13.07 (1H, s, NH), 10.57 (1H, s, OH), 9.35 (1H, s,
NH), 7.44 (1H, d, J 8.2, ArH), 7.28 (1H, s, ]CH), 6.36 (1H, dd, J 8.2, 2.2,
ArH), 6.31 (1H, d, J 2.2, ArH), 5.91 (1H, d, J 2.1, pyrrole-H), 2.26 (3H, s,
Me), 2.23 (3H, s, Me); dC (100 MHz; DMSO-d) 170.4 (C), 157.1 (C),
140.4 (C), 134.2 (C), 129.8 (C), 126.8 (C), 121.1 (CH), 119.7 (CH), 117.5
(C), 114.2 (C), 112.3 (CH), 108.6 (CH), 97.7 (CH), 13.9 (Me), 11.7 (Me);
m/z (EI) 254 (Mþ, 100%).
4.1.17. 3-[(3,5-Dimethylpyrrol-2-yl)methylidenyl]-6-(2-
nitrophenylacetoxy)indolin-2-one 32
To 6-hydroxy-3-[(3,5-dimethylpyrrol-2-yl)methylidenyl]indo-
lin-2-one 17 (30.0 mg, 0.118 mmol), 2-nitrophenylacetic acid
(21.4 mg, 0.128 mmol) and 4-dimethylaminopyridine (2.5 mg,
24 mmol) in dichloromethane (2 mL) was added dicyclohexyl-
4.1.15. 6-Benzyloxy-3-[(3,5-dimethylpyrrol-2-yl)methylidenyl]-
indolin-2-one 30
carbodiimide (1 M in dichloromethane; 0.118 mL) dropwise. The
mixture was stirred at room temperature for 18 h. Dichloro-
methane was added (10 mL) and the organic layer was washed with
water (10 mL), brine (10 mL), dried (MgSO4) and solvent removed
in vacuo. The crude product was purified by chromatography
eluting with dichloromethane–ethyl acetate (9:1) and re-
crystallization from ethanol and then ethyl acetate–hexane to give
the title compound (11 mg, 22%) as a yellow crystalline solid; mp
201–203 ꢁC (from ethyl acetate–hexane). (Found: MHþ, 418.1413.
C23H19N3O5þH requires: 418.1403.) nmax (KBr)/cmꢀ1 3697, 1674,
1601, 1568, 1349, 1140; dH (500 MHz; CDCl3) 13.02 (1H, br s, NH),
8.20 (1H, d, J 8.0, ArH), 7.67–7.64 (2H, m, NH and ArH), 7.53 (1H, t, J
8.0, ArH), 7.46 (1H, d, J 8.0, ArH), 7.41 (1H, d, J 8.5, ArH), 7.32 (1H, s,
]CH), 6.78 (1H, dd, J 8.5, 2.0, ArH), 6.70 (1H, d, J 2.0, ArH), 5.97 (1H,
d, J 0.5, pyrrole-H), 4.26 (2H, s, CH2), 2.37 (3H, s, Me), 2.31 (3H, s,
Me); dC (100 MHz; DMSO-d) 169.5 (C), 169.1 (C), 148.5 (C), 148.1 (C),
138.4 (C), 135.9 (C), 134.2 (CH), 133.9 (CH), 131.9 (C), 129.6 (C), 129.1
(CH), 126.6 (C), 125.0 (CH), 123.6 (CH), 123.2 (C), 118.6 (CH), 113.6
(CH), 112.6 (CH), 111.7 (C), 103.2 (CH), 40.0 (CH2), 13.5 (Me), 11.2
(Me); m/z (ES) 418 (MHþ, 100%), 371 (17), 223 (17).
(a) To 6-hydroxyoxindole46 (50 mg, 0.336 mmol) in methanol
(5 mL) was added potassium carbonate (48.6 mg, 0.35 mmol) and
benzyl bromide (42 mL, 0.35 mmol). The solution was heated to
reflux for 2.5 h. On cooling the reaction mixture was diluted with
ethyl acetate (20 mL) and washed with sodium hydroxide (2 M;
10 mL) and water (10 mL). The solvent was removed in vacuo and
the crude product was purified by chromatography eluting with
ethyl acetate–light petroleum (1:4 to 2:3) to give 6-benzyloxyox-
indole (40 mg, 50%) as an pale pink sticky solid used without fur-
ther purification; dH (300 MHz; CDCl3) 7.67 (1H, s, NH), 7.46–7.34
(5H, m, ArH), 7.13 (1H, d, J 8.1, ArH), 6.64 (1H, dd, J 8.1, 2.1, ArH), 6.55
(1H, d, J 2.1, ArH), 5.10 (2H, s, CH2), 3.49 (2H, s, CH2); dC (100 MHz;
CDCl3) 179.4 (C), 159.5 (C), 144.1 (C), 137.2 (C), 128.9 (CH), 128.3
(CH), 127.8 (CH), 125.5 (CH), 117.8 (C), 108.7 (C), 98.4 (CH), 70.6
(CH2), 36.2 (CH2).
(b) A solution of 6-benzyloxyoxindole (35 mg, 0.15 mmol), pyr-
rolidine (0.03 mL, 0.35 mmol) and 3,5-dimethylpyrrole-2-carbox-
aldehyde45 (34.7, 0.28 mmol) in ethanol (4 mL) was heated to reflux
at 90 ꢁC for 4 h. The ethanol was removed in vacuo and the crude
product was purified by chromatography eluting with ethyl ace-
tate–light petroleum (2:3) to give the title compound (45 mg, 89%)
as an orange crystalline solid; mp >241–243 ꢁC (from ethyl ace-
tate–light petroleum). (Found: Mþ, 344.1519. C22H20N2O2 requires:
344.1525.) nmax (KBr)/cmꢀ1 3697, 3450, 1674, 1631, 1597, 1570, 1455,
1366, 1312, 1279; dH (400 MHz; CDCl3) 12.90 (1H, br s, NH), 7.62
(1H, br s, NH), 7.45–7.25 (7H, m, 6ꢃArH and ]CH), 6.69 (1H, dd, J
8.4, 2.3, ArH), 6.55 (1H, d, J 2.3, ArH), 5.95 (1H, d, J 2.6, pyrrole-H),
5.10 (2H, s, CH2), 2.36 (3H, s, Me), 2.31 (3H, s, Me); dC (100 MHz;
CDCl3) 170.1 (C), 157.9 (C), 138.0 (C), 137.0 (C), 135.9 (C), 131.4 (C),
128.6 (CH), 128.0 (CH), 127.4 (CH), 127.0 (C), 122.1 (CH), 119.8 (C),
118.3 (CH), 112.3 (CH), 111.9 (C), 108.2 (CH), 97.2 (CH), 70.4 (CH2),
13.9 (Me), 11.6 (Me); m/z (ES) 344 (Mþ, 3%), 253 (100%), 91 (2%).
4.1.18. 6-[3-Methyl-3-(3,6-dimethyl-1,4-benzoquinon-2-
yl)butanoyloxy]-3-[(3,5-dimethylpyrrol-2-yl)methylene]-
indolin-2-one 33
To 6-hydroxy-3-[(3,5-dimethylpyrrol-2-yl)methylidenyl]indo-
lin-2-one 17 (32 mg, 0.126 mmol), 3-methyl-3-(3,6-dimethyl-1,4-
benzoquinon-2-yl)butanoic acid44 (29.7 mg, 0.126 mmol), 4-dime-
thylaminopyridine (2.5 mg, 24 mmol) and triethylamine (17.6 mL,
0.126 mmol) in dichloromethane (3 mL) was added 1-(3-dimethy-
laminopropyl)-3-ethyl carbodiimide hydrochloride (24.2 mg,
0.126 mmol) portionwise. The mixture was stirred at room tem-
perature for 18 h. Dichloromethane was added (10 mL) and the
organic layer was washed with water (10 mL), brine (10 mL), dried
(MgSO4) and solvent removed in vacuo. The crude product was
purified by chromatography eluting with ethyl acetate–light pe-
troleum (1:1) and recrystallized from ethyl acetate–hexane to give
the title compound (25 mg, 42%) as an yellow crystalline solid; mp
177–179 ꢁC (from ethyl acetate–hexane). (Found: MHþ, 473.2074.
C28H28N2O5þH requires: 473.2077.) nmax (KBr)/cmꢀ1 3449, 2986,
1710, 1673, 1568, 1363, 1133; dH (500 MHz; CDCl3) 13.00 (1H, br s,
NH), 7.74 (1H, s, NH), 7.39 (1H, d, J 8.5, ArH), 7.32 (1H, s, ArH), 6.64
4.1.16. 3-[(3,5-Dimethylpyrrol-2-yl)methylidenyl]-6-(4-
nitrobenzyloxy)indolin-2-one 31
To 6-hydroxy-3-[(3,5-dimethylpyrrol-2-yl)methylidenyl]indo-
lin-2-one 17 (30 mg, 0.118 mmol) in DMF (2 mL) was added po-
tassium carbonate (17 mg, 0.12 mmol). The reaction mixture was
stirred for 10 min and then 4-nitrobenzyl bromide (27 mg,

4902
E.A. Blanche et al. / Tetrahedron 65 (2009) 4894–4903
(1H, dd, J 8.5, 2.0, ArH), 6.56 (1H, d, J 2.0, ArH), 6.43 (1H, d, J 1.5,
ArH), 5.98 (1H, br s, pyrrole-H), 3.24 (2H, s, CH2), 2.37 (3H, s, Me),
2.31 (3H, s, Me), 2.17 (3H, s, Me), 1.97 (3H, d, J 1.5, Me), 1.54 (6H, s,
Me); dC (100 MHz; CDCl3) 191.1 (C), 187.2 (C), 171.6 (C), 169.8 (C),
152.3 (C), 148.4 (C), 148.2 (C), 139.5 (CH), 137.3 (C), 137.2 (C), 133.1
(C), 131.5 (CH), 127.2 (C), 124.4 (C), 123.7 (CH), 117.8 (CH), 114.4 (CH),
112.9 (CH), 110.7 (C), 103.3 (CH), 47.6 (CH2), 38.8 (C), 29.0 (Me), 16.0
(Me), 14.2 (Me), 14.0 (Me), 11.7 (Me); m/z (ES) 473 (MHþ, 100%), 413
(22), 371 (20), 363 (22), 358 (20), 282 (24), 223 (23).
(1H, dd, J 1.0, 7.5, ArH), 7.13 (2H, dd, J 2.2, 6.5, ArH), 6.28 (1H, d, J 1.0,
3-H), 4.00 (2H, s, CH2), 2.44 (3H, s, Me); dC (75 MHz; CDCl3) 168.5
(C), 160.9 (C), 154.5 (C), 153.4 (C), 152.3 (C), 134.5 (CH), 134.0 (CH),
129.6 (CH), 129.4 (C), 126.0 (CH), 125.8 (CH), 118.5 (CH), 118.4 (C),
115.0 (CH), 110.8 (CH), 90.5 (C), 40.6 (CH2), 19.6 (Me); m/z (CI) 340
(MHþ, 9%), 177 (100).
4.1.22. 4-Methyl-7-[2,2-dimethyl-2-(2-nitrophenyl)acetoxy]-
coumarin 38
To 2-methyl-2-(2-nitrophenyl)propanoic acid7 (217 mg,
1.04 mmol), 4-methyl-7-hydroxycoumarin 34 (219 mg, 1.24 mmol)
and 4-dimethylaminopyridine (24 mg, 0.02 mmol) in dichloro-
methane (5 mL) was added dicyclohexylcarbodiimide (1 M in
dichloromethane; 1.24 mL, 1.24 mmol) dropwise. The mixture was
stirred at room temperature overnight. The solvent was evaporated
and the residue was purified by chromatography, eluting with ethyl
acetate–light petroleum (1:1) to give the title compound (234 mg,
61%) as a colourless solid; mp 139–140 ꢁC (ethanol). (Found: C,
65.0; H, 4.4; N, 3.7. C20H17NO6 requires: C, 65.4; H, 4.7; N, 3.8%.)
(Found: MHþ, 368.1128. C20H17NO6þH requires: 368.1134.) nmax
(KBr)/cmꢀ11750, 1728, 1523, 1353; dH (300 MHz; CDCl3) 8.05 (1H, d,
J 7.6, ArH), 7.71 (2H, m, ArH), 7.60 (1H, d, J 8.8, ArH), 7.52 (1H, m,
ArH), 7.13 (1H, dd, J 2.2, 8.6, ArH), 7.07 (1H, m, ArH), 6.27 (1H, s, 3-
H), 2.43 (3H, s, Me), 1.85 (6H, s, Me); dC (75 MHz; CDCl3) 173.8 (C),
160.6 (C), 154.1 (C), 153.2 (C), 152.0 (C), 148.3 (C), 138.5 (C), 133.9
(CH), 128.3 (CH), 128.1 (CH), 126.1 (CH), 125.4 (CH), 118.3 (CH), 118.0
(C), 114.6 (CH), 110.4 (CH), 46.6 (C), 27.3 (Me), 18.8 (Me); m/z (CI)
368 (MHþ, 100%), 192 (45), 177 (100), 164 (24), 148 (17), 134 (21).
4.1.19. 4-Methyl-7-(4-nitrobenzyloxy)coumarin 35
To 4-methyl-7-hydroxycoumarin 34 (400 mg, 2.2 mmol) in DMF
(20 mL) was added potassium hydride (136 mg, 3.4 mmol) por-
tionwise. The mixture was stirred at room temperature for 1 h
before adding 4-nitrobenzyl bromide (732 mg, 3.4 mmol). The
mixture was then stirred at room temperature overnight. Water
was then added and the mixture was extracted with ethyl acetate.
The combined organic layers were washed with brine, dried
(MgSO4), filtered and concentrated in vacuo. The solid obtained was
stirred in light petroleum, filtered and rinsed with light petroleum
and a small amount of ethyl acetate to give the title compound
(670 mg, 95%) as a colourless solid; mp 203–204 ꢁC (ethanol).
(Found: MHþ, 312.0880. C17H13NO5þH requires: 312.0872.) nmax
(KBr)/cmꢀ1 3067, 1721, 1623, 1603, 1516, 1388, 1342, 1285; dH
(300 MHz; CDCl3) 8.27 (2H, d, J 8.7, ArH), 7.63 (2H, d, J 8.7, ArH), 7.54
(1H, d, J 8.8, ArH), 6.95 (1H, dd, J 2.5, 8.8, ArH), 6.87 (1H, d, J 2.5,
ArH), 6.18 (1H, s, 3-H), 5.34 (2H, s, CH2), 2.41 (3H, s, Me); dC
(75 MHz; CDCl3) 161.4 (C), 161.2 (C), 155.5 (C), 152.8 (C), 148.0 (C),
143.5 (C), 128.1 (CH), 126.2 (CH), 124.4 (CH), 114.6 (C), 113.1 (CH),
112.9 (CH), 102.3 (CH), 69.4 (CH2), 19.1 (Me); m/z (CI) 312 (MHþ,
100%), 177 (81), 106 (18).
4.1.23. 4-Methyl-7-[3-methyl-3-(3,6-dimethyl-1,4-benzoquinon-2-
yl)butan-1-oyloxy]coumarin 39
To 3-methyl-3-(3,6-dimethyl-1,4-benzoquinon-2-yl)butanoic
acid (250 mg, 1.1 mmol), 4-methyl-7-hydroxycoumarin 34
(400 mg, 2.2 mmol) and dimethylaminopyridine (13 mg, 0.1 mmol)
in dichloromethane (6 mL) was added dicyclohexylcarbodiimide
(1 M in dichloromethane; 1.1 mL, 1.1 mmol) dropwise and the re-
action mixture was stirred at room temperature overnight. The
solvent was evaporated and the residue was purified by chroma-
tography eluting with ethyl acetate–light petroleum (2:3) to give
the title compound (151 mg, 34%) as a colourless solid; mp 117 ꢁC
(ethanol). (Found: MHþ, 395.1477. C23H22O6þH requires: 395.1494.)
nmax (KBr)/cmꢀ1 3431, 2950, 2914, 1751, 1726, 1654, 1613, 1127, 1106;
dH (300 MHz; CDCl3) 7.58 (1H, d, J 8.5, ArH), 7.02 (1H, d, J 2.2, ArH),
6.96 (1H, dd, J 2.2, 8.5, ArH), 6.46 (1H, d, J 1.5, ArH quinone), 6.27
(1H, d, J 1.1, ArH), 3.29 (2H, s, CH2), 2.42 (3H, d, J 1.3, Me), 2.18 (3H, s,
Me),1.99 (3H, d, J 1.5, Me),1.55 (6H, s, Me); dC (75 MHz; CDCl3) 214.8
(C), 207.1 (C), 191.4 (C), 187.5 (C), 171.2 (C), 157.7 (C), 153.1 (C), 152.2
(C), 148.0 (C), 140.2 (C), 132.0 (CH), 125.8 (CH), 118.5 (C), 118.3 (CH),
115.0 (CH), 110.8 (CH), 47.9 (CH2), 29.3 (Me), 19.1 (Me), 16.4 (Me),
14.6 (Me); one C unobserved; m/z (CI) 395 (MHþ, 8%), 221 (48), 177
(100), 148 (5).
4.1.20. 4-Methyl-7-(3-nitrophenylacetoxy)coumarin 36
To 3-nitrophenylacetic acid (188 mg, 1.04 mmol), 4-methyl-7-
hydroxycoumarin 34 (219 mg, 1.24 mmol) and 4-dimethylamino-
pyridine (24.3 mg, 0.025 mmol) in dichloromethane (5 mL) was
added dicyclohexylcarbodiimide (1 M in dichloromethane;
1.24 mL, 1.24 mmol) dropwise. The mixture was stirred at room
temperature overnight. The solvent was evaporated and the resi-
due was purified by chromatography, eluting with dichloro-
methane to give the title compound (255 mg, 72%) as a colourless
solid; mp 188–190 ꢁC (ethanol). (Found: C, 63.7; H, 3.9; N, 4.1.
C18H13NO6 requires: C, 63.7; H, 3.9; N, 4.1%.) (Found: MHþ,
340.0831. C18H13NO6þH requires: 340.0821.) nmax (KBr)/cmꢀ1 3093,
3069, 2927, 1751, 1615, 1533, 1522, 1388, 1351, 1262, 1135; dH
(300 MHz; CDCl3) 8.29 (1H, s, ArH), 8.23 (1H, d, J 8.2, ArH), 7.75 (1H,
d, J 7.7, ArH), 7.61 (2H, m, ArH), 7.13 (1H, d, J 2.3, ArH), 7.08 (1H, dd, J
2.3, 8.6, ArH), 6.29 (1H, s, 3-H), 4.05 (2H, s, CH2), 2.45 (3H, s, Me); dC
(100 MHz; CDCl3) 168.3 (C), 160.4 (C), 154.1 (C), 152.7 (C), 151.8 (C),
135.6 (CH), 134.6 (C), 129.8 (CH), 125.5 (CH), 124.5 (CH), 122.8 (CH),
118.1 (C), 117.8 (CH), 114.8 (CH), 110.3 (CH), 40.6 (CH2), 18.8 (Me);
one C unobserved; m/z (CI) 340 (MHþ, 15%), 177 (100), 164 (18).
4.1.24. Reduction of 3-[(3,5-dimethylpyrrol-2-yl)methylene]-1-(4-
nitrobenzyl)indolinone 25
4.1.21. 4-Methyl-7-(2-nitrophenylacetoxy)coumarin 37
To 4-methyl-7-hydroxycoumarin 34 (400 mg, 2.2 mmol) in tol-
uene (24 mL) was added 2-nitrophenylacetic acid (614 mg,
3.4 mmol), triphenylphosphine (460 mg, 3.4 mmol) and diiso-
propyl azodicarboxylate (0.66 mL, 3.4 mmol) dropwise. The solu-
tion was stirred at room temperature for 48 h. The mixture was
then filtered and the solid was rinsed with light petroleum and
a small amount of ethyl acetate to give the title compound (248 mg,
32%) as a colourless solid; mp 157–158 ꢁC (ethanol). (Found: C,
63.9; H, 3.7; N, 3.9. C18H13NO6 requires: C, 63.7; H, 3.9; N, 4.1%.)
(Found: MHþ, 340.0826. C18H13NO6þH requires: 340.0821.) nmax
(KBr)/cmꢀ1 3078, 1762, 1731, 1710, 1603, 1513; dH (300 MHz; CDCl3)
8.22 (1H, dd, J 1.1, 8.1, ArH), 7.66 (1H, m, ArH), 7.57 (2H, m, ArH), 7.48
To 3-[(3,5-dimethylpyrrol-2-yl)methylene]-1-(4-nitrobenzyl)-
indolinone 25 (18 mg, 0.05 mmol) in ethanol (0.5 mL) was added
ammonium chloride saturated solution (0.1 mL) and indium pow-
der (40 mg, 0.35 mmol). The mixture was heated under reflux for
4 h and then cooled to room temperature, water (1 mL) was added
and the pH was adjusted to 9 with sodium hydroxide solution
(2 M). The product was extracted with ethyl acetate, dried (MgSO4),
filtered and the solvent evaporated in vacuo. Purification by chro-
matography eluting with ethyl acetate–light petroleum (3:7) gave
1-(4-aminobenzyl)-3-[(3,5-dimethylpyrrol-2-yl)methylidenyl]indo-
lin-2-one (16 mg, 90%) as a yellow solid; mp 212–215 ꢁC (ethanol).
(Found: MHþ, 344.1776. C22H21N3OþH requires: 344.1763.) nmax

E.A. Blanche et al. / Tetrahedron 65 (2009) 4894–4903
4903
(KBr)/cmꢀ1 3447, 3360, 2924, 2847, 1659, 1567, 1460, 1347, 1265,
1178; dH (300 MHz; CDCl3) 13.28 (1H, br s, NH), 7.60 (1H, d, J 7.3,
ArH), 7.43 (1H, s, ]CH), 7.13 (2H, d, J 8.1, ArH), 7.06 (2H, m, ArH),
6.81 (1H, d, J 7.3, ArH), 6.63 (2H, d, J 8.3, ArH), 5.98 (1H, s, pyrrole-
H), 4.98 (2H, s, CH2), 3.57 (2H, br s, NH2), 2.39 (3H, s, Me), 2.34 (3H,
s, Me); dC (75 MHz; CDCl3) 165.5 (C), 146.0 (C), 139.4 (C), 137.0 (C),
132.6 (C), 128.8 (CH), 127.4 (C), 126.8 (C), 126.0 (C), 125.9 (CH), 123.5
(CH), 121.9 (CH), 117.4 (CH), 115.7 (CH), 112.9 (CH), 112.2 (C), 109.2
(CH), 30.1 (CH2), 14.3 (Me), 12.0 (Me); m/z (CI) 344 (MHþ, 85%), 343
(Mþ, 100), 251 (96).
11. Killian, D. M.; Chikhale, P. J. J. Neurochem. 2001, 76, 966–974.
12. Li, Z. R.; Han, J. Y.; Jiang, Y. Y.; Browne, P.; Knox, R. J.; Hu, L. Q. Bioorg. Med. Chem.
2003, 11, 4171–4178.
13. Jiang, Y. Y.; Han, J. Y.; Yu, C. Z.; Vass, S. O.; Searle, P. F.; Browne, P.; Knox, R. J.; Hu,
L. Q. J. Med. Chem. 2006, 49, 4333–4343.
14. Hay, M. P.; Atwell, G. J.; Wilson, W. R.; Pullen, S. M.; Denny, W. A. J. Med. Chem.
2003, 46, 2456–2466.
15. Hay, M. P.; Wilson, W. R.; Denny, W. A. Bioorg. Med. Chem. 2005, 13, 4043–4055.
16. Zaarur, N.; Gabai, V. L.; Porco, J. A.; Calderwood, S.; Sherman, N. Y. Cancer Res.
2006, 66, 1783–1791.
17. Lee, H. H.; Palmer, B. D.; Wilson, W. R.; Denny, W. A. Bioorg. Med. Chem. Lett.
1998, 8, 1741–1744.
18. Parveen, I.; Naughton, D. P.; Whish, W. J. D.; Threadgill, M. D. Bioorg. Med. Chem.
Lett. 1999, 9, 2031–2036.
19. Reigan, P.; Edwards, P. N.; Gbaj, A.; Cole, C.; Barry, S. T.; Page, K. M.; Ashton, S. E.;
Luke, R. W. A.; Douglas, K. T.; Stratford, I. J.; Jaffar, M.; Bryce, R. A.; Freeman, S.
J. Med. Chem. 2005, 48, 392–402.
20. Ferrer, S.; Naughton, D. P.; Parveen, I.; Threadgill, M. D. J. Chem. Soc., Perkin
Trans. 1 2002, 335–340.
21. Ferrer, S.; Naughton, D. P.; Threadgill, M. D. Tetrahedron 2003, 59, 3437–3444.
22. Zhang, Z.; Tanabe, K.; Hatta, H.; Nishimoto, S. Org. Biomol. Chem. 2005, 3,
1905–1910.
23. Thomson, P.; Naylor, M. A.; Everett, S. A.; Stratford, M. R. L.; Lewis, G.; Hill, S.;
Patel, K. B.; Wardman, P.; Davis, P. D. Mol. Cancer Ther. 2006, 5, 2886–2894.
24. Moody, C. J.; Swann, E. Farmaco 1997, 52, 271–279.
25. Naylor, M. A.; Swann, E.; Everett, S. A.; Jaffar, M.; Nolan, J.; Robertson, N.;
Lockyer, S. D.; Patel, K. B.; Dennis, M. F.; Stratford, M. R. L.; Wardman, P.; Adams,
G. E.; Moody, C. J.; Stratford, I. J. J. Med. Chem. 1998, 41, 2720–2731.
26. Everett, S. A.; Naylor, M. A.; Barraja, P.; Swann, E.; Patel, K. B.; Stratford, M. R. L.;
Hudnott, A. R.; Vojnovic, B.; Locke, R. J.; Wardman, P.; Moody, C. J. J. Chem. Soc.,
Perkin Trans. 2 2001, 843–860.
27. Swann, E.; Moody, C. J.; Stratford, M. R. L.; Patel, K. B.; Naylor, M. A.; Vojnovic, B.;
Wardman, P.; Everett, S. A. J. Chem. Soc., Perkin Trans. 2 2001, 1340–1345.
28. Everett, S. A.; Swann, E.; Stratford, M. R. L.; Patel, K. B.; Naylor, M. A.; Tian, N.;
Newman, R. G.; Vojnovic, B.; Moody, C. J.; Wardman, P. Biochem. Pharmacol.
2002, 63, 1629–1639.
29. Ferrer, S.; Naughton, D. P.; Threadgill, M. D. Tetrahedron 2003, 59, 3445–3454.
30. Couch, G. D.; Burke, P. J.; Knox, R. J.; Moody, C. J. Tetrahedron 2008, 64,
2816–2823.
31. Whatmore, J. L.; Swann, E.; Barraja, P.; Newsome, J. J.; Bunderson, M.; Beall, H.
D.; Tooke, J. E.; Moody, C. J. Angiogenesis 2002, 5, 45–51.
4.1.25. In vitro angiogenesis model
Angiogenesis assays were carried out using a method based on
that of Koolwijk et al.52 Human fibrinogen (5 mg/mL) was dissolved
in serum free Hams F12 and filtered through a 0.2 mm filter. Fibrin
matrices were prepared by polymerizing the solution of fibrinogen
with 1.5 U/mL of human thrombin. Six hundred microlitre of this
mixture was added to each well of a 24 well plate. After polymeri-
zation, matrices were soaked in Hams F12 containing 20% FCS for
120 min at 37 ꢁC to inactivate the thrombin. Highly confluent
HUVECs were plated onto the surface of the matrix and cultured for
24 h. After this time the medium was replaced with medium con-
taining anti-angiogenic agents ꢂ100 ng/mL of VEGF. Drugs were
tested at concentrations shown in Figure 3. Controls received VEGF
and carrier alone. After a maximum of 5 days the formation of tu-
bular structures of endothelial cells in the three-dimensional matrix
was analyzed. Two randomly chosen microscopic fields per well
were examined and the number of times a tubular structure crossed
a 1 cm grid line were scored to provide an index of the degree of
angiogenesis in each well. Each condition was carried out in tripli-
cate wells, and each drug was tested using 2–4 different cell pop-
ulations. Since assays were performed on primary cells isolated from
different individuals a certain degree of variability was observed in
cell responses and this was reflected in the standard deviations
observed. Statistical analyses were carried out on the raw data using
the using the Mann–Whitney U Test and P<0.05 was considered
statistically significant. Values are expressed as meanꢂSD.
32. Maskell, L.; Blanche, E. A.; Colucci, M. A.; Whatmore, J. L.; Moody, C. J. Bioorg.
Med. Chem. Lett. 2007, 17, 1575–1578.
33. Folkman, J. N. Engl. J. Med. 1971, 285, 1182–1186.
34. Folkman, J. Ann. Surg. 1972, 175, 409–416.
35. Claffey, K. P.; Robinson, G. S. Cancer Metastasis Rev. 1996, 15, 165–176.
36. Ferrara, N.; DavisSmyth, T. Endocr. Rev. 1997, 18, 4–25.
37. Fujita, Y.; Abe, R.; Shimizu, H. Curr. Pharm. Des. 2008, 14, 3820–3834.
38. Zhou, J. R.; Mukherjee, P.; Gugger, E. T.; Tanaka, T.; Blackburn, G. L.; Clinton, S. K.
Cancer Res. 1998, 58, 5231–5238.
Acknowledgements
39. He, H.; Venema, V. J.; Guo, X. L.; Venema, R. C.; Marrero, M. B.; Caldwell, R. B.
J. Biol. Chem. 1999, 274, 25130–25135.
40. Sun, L.; Tran, N.; Tang, F.; App, H.; Hirth, P.; McMahon, G.; Tang, C. J. Med. Chem.
1998, 41, 2588–2603.
We thank the Association for International Cancer Research
(AICR), and the FORCE Cancer Charity (Exeter) for financial support
of this research, and the EPSRC Mass Spectrometry Centre at
Swansea for mass spectra.
41. Sun, L.; Tran, N.; Liang, C. X.; Tang, F.; Rice, A.; Schreck, R.; Waltz, K.; Shawver, L.
K.; McMahon, G.; Tang, C. J. Med. Chem. 1999, 42, 5120–5130.
42. Fong, T. A. T.; Shawver, L. K.; Sun, L.; Tang, C.; App, H.; Powell, T. J.; Kim, Y. H.;
Schreck, R.; Wang, X. Y.; Risau, W.; Ullrich, A.; Hirth, K. P.; McMahon, G. Cancer
Res. 1999, 59, 99–106.
43. Mendel, D. B.; Laird, A. D.; Smolich, B. D.; Blake, R. A.; Liang, C. X.; Hannah, A. L.;
Shaheen, R. M.; Ellis, L. M.; Weitman, S.; Shawver, L. K.; Cherrington, J. M. Anti-
Cancer Drug Des. 2000, 15, 29–41.
44. Zheng, A.; Shan, D.; Wang, B. J. Org. Chem. 1999, 64, 156–161.
45. Corwin, A. H.; Krieble, R. H. J. Am. Chem. Soc. 1941, 63, 1829–1834.
46. Beckett, A. H.; Daisley, R. W.; Walker, J. Tetrahedron 1968, 24, 6093–6109.
47. Pitts, M. R.; Harrison, J. R.; Moody, C. J. J. Chem. Soc., Perkin Trans. 1 2001,
955–977.
48. James, A. L.; Perry, J. D.; Stanforth, S. P. J. Heterocycl. Chem. 2006, 43, 515–517.
49. Siegel, D.; Ross, D. Free Radical Biol. Med. 2000, 29, 246–253.
50. Gustafson, D. L.; Siegel, D.; Rastatter, J. C.; Merz, A. L.; Parpal, J. C.; Kepa, J. K.;
Ross, D.; Long, M. E. J. Pharmacol. Exp. Ther. 2003, 305, 1079–1086.
51. King, F. E.; Jurd, L. J. Chem. Soc. 1952, 3211–3215.
References and notes
1. Melton, R. G.; Knox, R. J. Enzyme Prodrug Strategies for Cancer Therapy; Kluwer
Academic/Plenum: New York, NY, 1999.
2. Jaffar, M.; Williams, K. J.; Stratford, I. J. Adv. Drug Delivery Rev. 2001, 53, 217–228.
3. Naylor, M. A.; Thomson, P. Mini-Rev. Med. Chem. 2001, 1, 17–29.
4. Skelly, J. V.; Knox, R. J.; Jenkins, T. C. Mini Rev. Med. Chem. 2001, 1, 293–306.
5. Wardman, P. Curr. Med. Chem. 2001, 8, 739–761.
6. Denny, W. A. Curr. Pharm. Des. 2002, 8, 1349–1361.
7. Atwell, G. J.; Sykes, B. M.; O’Connor, C. J.; Denny, W. A. J. Med. Chem. 1994, 37,
371–380.
8. Carpino, L. A.; Triolo, S. A.; Berglund, R. A. J. Org. Chem. 1989, 54, 3303–3310.
9. Amsberry, K. L.; Borchardt, R. T. Pharm. Res. 1991, 8, 323–330.
10. Weerapreeyakul, N.; Hollenbeck, R. G.; Chikhale, P. J. Bioorg. Med. Chem. Lett.
2000, 10, 2391–2395.
52. Koolwijk, P.; vanErck, M. G. M.; deVree, W. J. A.; Vermeer, M. A.; Weich, H. A.;
Hanemaaijer, R.; vanHinsbergh, V. W. M. J. Cell Biol. 1996, 132, 1177–1188.