Synthesis and biological activity of azido analogues of 5,6-dimethylxanthenone-4-acetic acid for use in photoaffinity labeling

Article abstract of DOI:10.1021/jm0702175

5,6-Dimethylxanthenone-4-acetic acid (1) is scheduled for phase III clinical trials as a vascular disrupting agent. However, its biochemical receptor(s) have yet to be identified. In this report, the synthesis of azido analogues of 1 that could be used fo

Full text of DOI:10.1021/jm0702175

© Copyright 2007 by the American Chemical Society
Volume 50, Number 16
August 9, 2007
Articles
Synthesis and Biological Activity of Azido Analogues of 5,6-Dimethylxanthenone-4-acetic Acid
for Use in Photoaffinity Labeling
Brian D. Palmer,† Kimiora Henare,† See-Tarn Woon,† Rachel Sutherland,† Charu Reddy,† Liang-Chuan S. Wang,†
Claudine Kieda,‡ and Lai-Ming Ching*,†
Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, UniVersity of Auckland, Auckland, New Zealand, and
Centre de Biophysique Moleculaire, UPR 4301 CNRS, Orleans Cedex, France
ReceiVed February 24, 2007
5,6-Dimethylxanthenone-4-acetic acid (1) is scheduled for phase III clinical trials as a vascular disrupting
agent. However, its biochemical receptor(s) have yet to be identified. In this report, the synthesis of azido
analogues of 1 that could be used for photoaffinity labeling of proteins as an approach toward identifying
its molecular targets is described. While 5-azidoxanthenone-4-acetic acid (2) and 5-azido-6-methylxantheone-
4-acetic acid (3) were found to have biological activities similar to that of 1, 6-azido-5-methylxanthenone-
4-acetic acid (4) was unstable and could not be evaluated. Both azido compounds 2 and 3 activated NF-κB,
induced the production of tumor necrosis factor in cultured mouse splenocytes, and induced hemorrhagic
necrosis of colon 38 tumors in mice. Photoreaction of lysates from spleen cells with tritiated 2 resulted in
two radiolabeled protein bands at 50 and 14 kDa that could be competitively inhibited with cold 1 and cold
2. The azido compounds 2 and 3 exhibit all the requirements for use in photoaffinity labeling of potential
receptor(s) for 1.
Introduction
A number of vascular disrupting agents are in the clinic for
the treatment of cancer, most notably bevacizumab, a mono-
clonal antibody against the vascular endothelial growth factor,
which has shown activity for colorectal cancers.1-4 Combret-
astatin, the taxanes, and vinca alkaloids all cause antivascular
effects through a disruption of normal polymerization of
tubulin.5-7 5,6-Dimethylxanthenone-4-acetic acid (1) (DMXAA,
AS1404), a synthetic small molecule that is scheduled to enter
phase III clinical trial as a vascular disrupting agent, has a
distinct mode of action that does not appear to involve tubulin
as the main target (Figure 1). The molecular mode of action of
1 is not yet fully elucidated and may involve multiple pathways,
depending on the cell type and context. In preclinical studies,
Figure 1. Chemical structures of 5,6-dimethylxantheone-4-acetic acid
(1) and its azido analogues.
* To whom correspondence should be addressed. Phone:
1 at its maximum tolerated dose (MTD) caused rapid and
+64-9-3737-599, extension 86140. Fax: +64-9-3737-502. E-mail:
irreversible inhibition of tumor blood flow,8,9 and the resultant
l.ching@auckland.ac.nz.
ischemia and subsequent necrosis could account for the killing
of nearly 90% of the tumor cells in established, vascularized
† University of Auckland.
‡ Centre de Biophysique Moleculaire.
10.1021/jm0702175 CCC: $37.00 © 2007 American Chemical Society
Published on Web 07/06/2007

3758 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 16
Palmer et al.
Scheme 1a
implants.8 Selective apoptosis of vascular endothelial cells in
colon 38 tumors was observed within 30 min of 1 administra-
tion,10,11 and a significant correlation between the induction of
vascular endothelial cell apoptosis and the reduction of tumor
blood flow was obtained.11 Maintenance of the tumor vascular
collapse such that widespread necrosis develops involves the
induction of a cascade of vasoreactive mediators such as
serotonin,12 nitric oxide,13 and tumor necrosis factor (TNF) and
other cytokines.14-16
a Reagents and conditions: (i) NaNO2, aqueous H2SO4, 0-5 °C, 10 min,
then aqueous NaN3, 0-5 °C, 1 h.
The addition of inhibitors of ΙκB, parthenolide and salicylate,
results in loss of production of TNF and IFN-γ in primary
splenocyte cultures, indicating a role for NF-κB.17 On the other
hand, induction of apoptosis of human umbilical cord vein
endothelial cells by 1 can occur in the absence of NF-κΒ
activation, indicating alternative signaling pathways may pre-
dominate for 1’s effects on endothelial cells.18 One implication
of the multiplicity of pathways that are involved in its action is
that 1 may possess more than one cellular target, and approaches
toward identification of its receptor(s) must take this complexity
into account. The method of photoaffinity labeling would allow
cellular proteins to be screened for binding to a photoreactive
analogue of 1, a technique that has been used successfully to
identify protein targets for a number of other anticancer agents
including verapamil,19 paclitaxel,20-22 and thalidomide.23 We
have previously shown that tritiated 1 associates only weakly
to cell lysates, suggesting that it has a low affinity of binding
to its receptor(s).24 With this in mind, the use of a photoaffinity-
labeling approach to the identification of its receptor was
considered appropriate, since the probe becomes covalently
attached to the target protein(s)25 by use of this technique. Of
course, low affinity binding of 1 could also lead to nonspecific
labeling of many proteins and the formation of false positives,
and so all identified proteins would have to be subsequently
evaluated for their receptor status.
In this paper we describe the synthesis and biological activity
of azido analogues (2-4) of 1 that could be used for photoaf-
finity labeling and subsequent identification of potential recep-
tor(s) for 1. We chose to use an azide group as the photoreactive
substituent because it is readily introduced via diazotization of
an aromatic amine, it is not as sterically demanding as alternative
photoreactive groups, and it undergoes efficient conversion to
a reactive nitrene species upon photolysis. Others have used
aryl diazirine, aryl ketone, and diazoester groups for this
purpose.26 Tritiation of the selected photoprobe was used to
introduce a reporter tag suitable for detection of probe-protein
adducts.
These were obtained by reduction of the nitroxanthenones 12
and 20, which were prepared using chemistry similar to that
developed in the original studies for construction of the
xanthenone-4-acetic acid ring system.28 Thus, the methylnitroa-
nilines 6 and 14 were converted into isatins 8 and 16 using a
recent modification of the isonitrosoacetanilide Sandmeyer
procedure.29 Oxidation to the anthranilic acids 9 and 17 followed
by diazotization and reaction with KI gave iodides 10 and 18.
The potassium salt of these acids reacted with the disodium
salt of 2-hydroxyphenylacetic acid under TDA-1 catalysis to
give the diphenyl ethers 11 and 19, which were cyclized to give
the xanthenones 12 and 20 using 85% H2SO4. While the 5-azido
compounds 2 and 3 were stable at room temperature, the 6-azido
compound 4, which has the azido group in resonance with the
carbonyl group of the xanthenone ring, was moderately unstable
under the same conditions, with decolorization occurring on
standing (particularly in solution) and the formation of polar,
baseline products by TLC analysis. For this reason, the azide 4
was considered to be unsuitable for tritiation and for further
use as a probe.
Biological Activity
Induction of Hemorrhagic Necrosis. A hallmark of the
antivascular effects of 1 is the induction of hemorrhagic necrosis
of subcutaneously growing solid tumors in mice within 24 h of
a single injection at the MTD.30-32 The azido analogues were
examined for their ability to induce hemorrhagic necrosis. Mice
with colon 38 tumors were injected intraperitoneally with 1, 2,
or 3 at respective MTDs of 25, 200, and 190 mg/kg. After 24
h, mice were killed and the tumor was excised. The histologies
of all tumors were compared with those of untreated tumors,
and a representative field of the untreated and treated tumors is
shown in Figure 2. Untreated tumors had less than 5% necrosis,
and tumors treated with all drugs exhibited greater than 95%
hemorrhagic necrosis. Thus, all three agents had the ability to
cause widespread necrosis, although the azido analogues were
8-fold lower in potency than 1.
Cytotoxicity of the Azido Analogues Compared with 1.
The direct cytotoxicity of the analogues compared with 1 on
HECPP murine endothelial cells was measured using the MTT
assay. Varying concentrations of each compound were added
to cultures of HECPP murine endothelial cells, and the percent
of metabolically active cells in culture was assessed after 24 h
(Figure 3). Increasing concentrations of each drug led to a
decrease in cell viability. The azido analogues were more
cytotoxic than 1 in vitro, and the concentration required to cause
50% reduction in cell viability was 600 and 300 µg/mL,
respectively, for 2 (Figure 3B) and 3 (Figure 3C), compared
with 1000 µg/mL for 1 (Figure 3A).
Chemistry
Previous structure-activity relationship (SAR) studies of the
activity of substituted xanthenone-4-acetic acids indicated a clear
preference for substitution at the 5 and 6 positions of the ring
system, with 5,6-disubstitution providing particularly potent
compounds.27 5,6-Dimethylxanthenone-4-acetic acid (1) was
chosen from among these for clinical evaluation. In these studies
the 5-amino compound (5) displayed some activity in inducing
hemorrhagic necrosis in colon 38 tumors, albeit at a significantly
higher optimum dose than 1.28 These earlier SAR studies
suggested that the 5 and 6 positions of the xanthenone-4-acetic
acid ring system would be best for incorporation of azide
functionality in order to retain a spectrum of biological activity
similar to that of 1. The 5-azido analogue (2) of 5 was prepared
in good yield from 5 by conversion to the diazonium salt,
followed by reaction with aqueous sodium azide (Scheme 1).
The methylated azides 3 and 4 were similarly prepared from
the corresponding amino compounds 13 and 21 (Scheme 2).
Activation of NF-KB. 1 has been shown to activate NF-κB
in a number of cell lines in culture24,33 as well as in vivo in
tumors,34 although the precise role of the activated NF-κΒ in
the action of 1 is not clear. The ability of 2 and 3 to also activate
NF-κB in HECPP cells was investigated as an indication that
the azido analogues share a spectrum of biological activity

Analogues of 5,6-Dimethylxanthenone-4-acetic Acid
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 16 3759
Scheme 2a
a Reagents and conditions: (i) (AcO)2CHCOCl, KHCO3, DCM, 20 °C, 1 h; (ii) H2NOH‚HCl, EtOH-H2O, reflux, 90 min; (iii) concentrated H2SO4,
80-93 °C, 30-150 min; (iv) H2O2, aqueous KOH, KCl, 20 °C, 2 h; (v) NaNO2, concentrated H2SO4-85% H3PO4, 0-10 °C, 30 min, then aqueous KI, CuI,
10-80 °C, 10 min; (vi) 1 equiv of aqueous KOH, 20 °C, 5 min, dry well, then 2-hydroxyphenylacetic acid disodium salt, TDA-1, CuCl, p-dioxane, reflux,
16 h; (vii) 80% H2SO4, 80 °C, 15 min; (viii) H2, 5% Pd-C, MeOH-EtOAc, 60 psig, 2 h; (ix) NaNO2, aqueous H2SO4, 0-5 °C, 5 min, then aqueous NaN3,
5-20 °C, 1.5 h.
pg/mL TNF at 100 µg/mL. TNF production was obtained with
3 at 30 and 100 µg/mL only, and less than 50 pg/mL TNF was
induced.
Photoaffinity Labeling and Specificity of Binding. Both
azido compounds had similar activity and potency to each other.
The less toxic 5-azidoxanthenone-4-acetic acid (2) was chosen
for custom tritiation and evaluated for its utility in photoaffinity
labeling. Lysates from murine splenocyte were incubated with
tritiated 2 and then UV-irradiated. Other aliquots of cell lysates
were preincubated with cold 1 or cold 2 before the addition of
tritiated 2 and photoreaction. Parts A and B of Figure 6 show
the Coomassie Blue stained gel and the autoradiograph,
respectively. Two bands (50 and 14 kDa) were visible in the
autoradiograph. More intense banding pattern was observed in
lane 2 (UV-treated) compared to lane 1 (no UV treatment),
indicating that photoactivation had induced binding between
[3H]-2 and the proteins. Cold 1 reduced the radioactive intensity
Figure 2. Histology of colon 38 tumor section (A) untreated or 24 h
after treatment with (B) 1 (25 mg/kg), (C) 2 (200 mg/kg), or (D) 3
(190 mg/kg).
of the 14 kDa band (Figure 6B, lanes 3 and 4) but not that of
the 50 kDa band. Excess cold 2, however, was shown to
competitively inhibit [3H]-2 from reacting with proteins in both
the 50 and the 14 kDa bands (lane 6, Figure 6B).
similiar to that of 1. NF-κB was activated with 1 at 10 µg/mL
and above (Figure 4A). With 2, activated NF-κB was observed
at 300 and 1000 µg/mL (Figure 4B), indicating a 30-fold lower
potency than 1 in this activity. Activated NF-κB was observed
with 3 at 30 and 100 µg/mL (Figure 4C), but no determinations
could be obtained at higher doses because it was found to be
toxic and sufficient amounts of nuclear extracts could not be
obtained for testing.
Discussion and Conclusion
As shown in Figure 2, 5-azidoxanthenone-4-acetic acid (2)
and 5-azido-6-methylxanthenone-4-acetic acid (3) retained the
ability to cause profound necrosis of subcutaneously growing
colon 38 in mice, a hallmark of the in vivo antitumor response
to 1. The two stable azido analogues were both less potent than
1. Their MTDs in mice were approximately 8-fold higher than
that for 1, but at their respective MTDs, the degree of necrosis
induced with the azido analogues was equivalent to that induced
with 1 (Figure 2). The in vivo antitumor activity of this class
of agents does not correlate with their in vitro cytotoxicity.36
Excellent correlations are observed, however, between in vivo
induction of tumor necrosis and in vitro activation of NF-κB37
and enhancement of immune effector cell activity.36,38 The
biological evaluations here were consistent with previous
observations. Although their MTDs in vivo were 8-fold higher,
Cytokine Induction in Murine Splenocyte Cultures. A key
component of the action of 1 is the induction of cytokines, and
we have previously used primary splenocyte cultures as an in
vitro model for investigating this action of 1.35 The ability of
the azido analogues to induce TNF in murine splenocyte cultures
was compared with that of 1 (Figure 5). Both azido analogues
induced TNF secretion into the culture supernatants, but clearly
1 was more active. Maximal TNF production (180 pg/mL) was
obtained at 10 µg/mL of 1, whereas 2 maximally induced 110

3760 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 16
Palmer et al.
Figure 3. Percent survival of HECPP cells in culture 24 h after the addition of varying concentrations of (A) 1, (B) 2, or (C) 3.
Figure 4. EMSA showing activated NF-κB in nuclear extracts from HECPP cells cultured for 2 h with indicated concentrations of (A) 1, (B) 2,
or (C) 3.
Figure 5. TNF activity in murine splenocyte culture supernatants 4 h after treatment with indicated concentrations of (A) 1, (B) 2, or (C) 3.
the azido analogues were more toxic than 1 to cultured
endothelial cells but were approximately 10-fold less potent than
1 in activating NF-κB (Figure 4) and in stimulating TNF
production in cultured splenocytes (Figure 5). Despite differ-
ences in potencies, the results indicated that the azido com-
pounds share a similar spectrum of biological effects as 1 and
are therefore most likely to be acting on the same molecular
targets as 1. Moreover, compound 2 was shown to undergo
photoreaction to specifically bind to cellular proteins extracted
from murine splenocytes (Figure 6). We conclude that the two
stable azidoxanthenone analogues 2 and 3 described in this
report would be suitable for use in a photoaffinity approach to
identify the receptor(s) for 1.
Experimental Section
Chemistry. Combustion analyses were performed by the Mi-
crochemical Laboratory, University of Otago, Dunedin, NZ. Melting
points were determined using an Electrothermal model 9200 digital
melting point apparatus and are as read. NMR spectra were obtained
on a Bruker Avance-400 spectrometer and are referenced to
Me4Si. APCI mass spectrometry was performed on a Surveyor MSQ
single quadrupole spectrometer (ThermoFinnigan), using methanol

Analogues of 5,6-Dimethylxanthenone-4-acetic Acid
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 16 3761
resulting solution was cooled and poured onto crushed ice. The
resulting brown solid was filtered off, washed with water, and
dissolved in 1 N NaOH (200 mL). The pH was adjusted to 6 by
the addition of acetic acid, and the resulting brown precipitate was
removed by filtration. The filtrate was acidified by the addition of
concentrated HCl and the precipitate was filtered off and washed
well with water to give the isatin 8 as a yellow solid (3.33 g,
1
36%): mp (EtOH) 238 °C; H NMR ((CD3)2SO) δ 1.39 (br s, 1
H), 7.69 (d, J ) 7.6 Hz, 1 H), 7.13 (d, J ) 7.6 Hz, 1 H), (2.50,
s, 3H). Anal. (C9H6N2O4‚0.25H2O) C, H, N.
7-Methyl-6-nitro-1H-indole-2,3-dione (16). Reaction of the
isonitrosoacetanilide 1529 with concentrated H2SO4 as described
above for the preparation of 8, except that the reaction time was
2.5 h at a temperature of 93 °C, gave the isatin 16 (54%) as an
1
orange powder: mp 148-151 °C; H NMR ((CD3)2SO) δ 1.45
(br s, 1 H), 7.55 (d, J ) 8.0 Hz, 1 H), 7.53 (d, J ) 8.0 Hz, 1 H),
2.25 (s, 3 H). Anal. (C9H6N2O4‚0.125H2O) C, H, N.
2-Amino-4-methyl-3-nitrobenzoic Acid (9). The isatin 8 (3.30
g, 0.016 mol) was dissolved in a solution of KOH (0.99 g, 0.018
mol) and KCl (2.5 g, 0.034 mol) in water (30 mL), and the solution
was cooled to 10 °C. Hydrogen peroxide (3.3 g of a 27% aqueous
solution, 0.026 mol) was added dropwise, and stirring was continued
at this temperature for 5 min, then at room temperature for 2 h.
The resulting orange suspension was acidified with acetic acid
and the precipitate was filtered off and washed well with water to
give 9 as a bright-yellow powder (2.71 g, 86%): mp (ethyl acetate/
petroleum ether) 221-223 °C. 1H NMR ((CD3)2SO) δ 7.87
(d, J ) 8.1 Hz, 1 H), Anal. (C8H8N2O4) C, H, N.
Figure 6. (A) SDS-PAGE gel of murine splenocyte proteins incubated
with tritiated 2 without UV treatment (lane 1) and with UV treatment
(lane 2), preincubated with 10- and 1000-fold excess of cold 1 (lanes
3 and 4, respectively), and preincubated with 10- and 1000-fold excess
of cold 2 prior to UV treatment (lanes 5 and 6, respectively). (B)
Autoradiograph of the gel showing radioactive protein bands at 50 and
14 kDa.
2-Amino-3-methyl-4-nitrobenzoic Acid (17). Reaction of the
isatin 16 with H2O2 exactly as described above for the isomer 8
gave the anthranilic acid 17 (68%) as a yellow solid: mp (ethyl
solutions and simultaneous positive and negative ion acquisition.
Flash column chromatography was performed on silica gel 60
support (Scharlau, 230-400 mesh ASTM), using the indicated
eluants.
1
acetate/petroleum ether) 208-211 °C; H NMR (CDCl3) δ 7.94
(d, J ) 8.8 Hz, 1 H), 6.94 (d, J ) 8.8 Hz, 1 H), 6.22 (br, 2 H),
2.22 (s, 3 H). APCI found: [M - H]- ) 195. Anal. (C8H8N2O4)
C, H, N.
(5-Azido-9-oxo-9H-xanthen-4-yl)acetic Acid (2). Concentrated
H2SO4 (8 mL) was added to powdered amino compound 528 (0.20
g, 0.74 mmol), which had been cooled to 5 °C in an ice bath.
Stirring was continued until the mixture became homogeneous, and
then an ice-water slurry (∼50 g) was added in one portion. The
clear, colorless solution was recooled to 5 °C, and a solution of
NaNO2 (73 mg, 1.06 mmol) in water (5 mL) was added dropwise
over 5 min. After the mixture was stirred for an additional 10 min
at 0-5 °C, a solution of NaN3 (0.28 g, 4.30 mmol) in cold water
(10 mL) was added in one portion, and the mixture was stirred
with cooling for 1 h. The white precipitate was filtered off, washed
well with water, and air-dried to give the azide 2 as a white solid
2-Iodo-4-methyl-3-nitrobenzoic Acid (10). The amine 9 (7.00
g, 0.036 mol) was dissolved in concentrated H2SO4 (130 mL), and
the solution was cooled to 5 °C. A chilled solution of NaNO2 (2.69
g, 0.039 mol) in concentrated H2SO4 (130 mL) was added dropwise
with stirring, keeping the temperature below 10 °C. Chilled 85%
H3PO4 (255 mL) was then added at such a rate that the temperature
remained below 10 °C (this requires an acetone-dry ice bath), and
stirring was continued for a further 30 min. The resulting tan
suspension was poured onto an ice-water mixture, and a solution
of KI (29.6 g, 0.18 mol) in water (150 mL) was added in one
portion. CuI (25.6 g, 0.020 mol) was added, and the mixture was
warmed to 80 °C. After 10 min at this temperature, ethyl acetate
was added and the mixture was stirred well, then filtered through
Celite. The ethyl acetate layer was removed from the filtrate, washed
with aqueous sodium sulfite solution, then water, and worked up
to give an orange solid, which was slurried with hot benzene to
leave the iodide 10 as a tan powder (6.53 g, 59.5%). A portion
crystallized from aqueous methanol as a cream solid: mp 244 °C;
1H NMR ((CD3)2SO) δ 13.8 (br, 1 H), 7.72 (d, J ) 7.9 Hz, 1 H),
7.54 (d, J ) 7.9 Hz, 1 H), 2.31 (s, 3 H) (COOH resonance not
visible). APCI found: [M - H]- ) 306. Anal. (C8H6INO4‚
0.25MeOH) C, H, N.
2-Iodo-3-methyl-4-nitrobenzoic Acid (18). Reaction of the
anthranilic acid 17 exactly as described above for the isomer 9 gave
the iodide 18 (84%) as a tan powder: mp (benzene/petroleum ether)
168-172 °C; 1H NMR ((CD3)2SO) δ 13.80 (br, 1 H), 7.91 (d, J )
8.3 Hz, 1 H), 7.52 (d, J ) 8.3 Hz, 1 H), 2.52 (s, 3 H). APCI found:
[M - H]- ) 306. Anal. (C8H6INO4‚0.25C6H6) C, H, N.
2-[2-(Carboxymethyl)phenoxy]-4-methyl-3-nitrobenzoic Acid
(11). To a solution of the acid 10 (6.53 g, 0.021 mol) in methanol
(300 mL) was added 1 N KOH (21 mL, 0.02 mol). After 5 min the
solution was concentrated to dryness and dried well in a vacuum
oven to leave the potassium salt of the acid as a colorless powder.
p-Dioxane (500 mL) was added, followed by the disodium salt of
2-hydroxyphenylacetic acid (4.94 g, 0.025 mol), TDA-1 (0.81 g,
2.52 mmol), and CuCl (0.25 g, 2.52 mmol), and the mixture was
1
(0.21 g, 96%): mp 169 °C (dec); H NMR ((CD3)2SO) δ 12.60
(br, 1 H), 8.12 (dd, J ) 8.0, 1.7 Hz, 1 H), 7.98 (dd, J ) 8.0, 1.6
Hz, 1 H), 7.84 (dd, J ) 7.3, 1.7 Hz, 1 H), 7.71 (dd, J ) 7.8,
1.6 Hz, 1 H), 7.50-7.45 (m, 2 H), 3.99 (s, 2 H). APCI found:
[M + H]+ ) 296. Anal. (C15H19N3O4) C, H, N. The azide 2 was
randomly tritiated in the xanthenone ring by SibTech, Inc., using
a custom proprietary procedure.39 The specific activity thus obtained
was 0.1 mCi/mmol.
6-Methyl-7-nitro-1H-indole-2,3-dione (8). A solution of diac-
etoxyacetyl chloride (21.9 g, 0.113 mol) in dichloromethane
(50 mL) was added dropwise to a vigorously stirred mixture of
2-nitro-3-methylaniline (6) (13.21 g, 0.086 mol) and KHCO3 (43.5
g, 0.434 mol) in dichloromethane (200 mL) at -10 °C. After 5
min, the cooling bath was removed and the mixture was stirred at
room temperature for 1 h. The solid material was removed by
filtration, which was washed with dichloromethane. The combined
dichloromethane solution was concentrated to dryness, and the
residue was dissolved in ethanol (120 mL). A solution of hydroxy-
lamine hydrochloride (30.2 g, 0.434 mol) in water (60 mL) was
added, and the solution was refluxed for 90 min. The solvent was
removed in vacuo and the residue was extracted with ethyl acetate,
washed with water, then worked up to give the crude isonitrosoac-
etanilide 7 as an orange syrup (63%), which was used directly.
Concentrated H2SO4 (80 mL) was added to crude 7 (10.0 g, 0.045
mol), and the mixture was warmed at 80 °C for 30 min. The

3762 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 16
Palmer et al.
refluxed under nitrogen for 16 h. The solvent was removed in vacuo,
and the residue was dissolved in 1 N NaOH (500 mL) and filtered.
The filtrate was acidified with concentrated HCl, and the solution
was extracted with ethyl acetate. The extract was dried and
concentrated to ∼50 mL. Addition of petroleum ether precipitated
the ether 11 as a brown powder sufficiently pure for the next
step (3.25 g, 46%). A small portion was recrystallized from
aqueous acetone to give 11 as a fine, tan powder: mp 220-221
2.41 (s, 3 H). APCI found: [M + H]+ ) 310 (60%), 282 (100%).
Anal. (C16H11N3O4‚0.5H2O) C, H, N.
(6-Azido-5-methyl-9-oxo-9H-xanthen-4-yl)acetic Acid (4). Di-
azotization of the amino compound 21, followed by reaction with
NaN3 exactly as described above for the isomeric amine 13, gave
the azide 4 as a tan solid (85%): mp, slowly sintered upon heating.
This material was unstable at room temperature, especially
when in solution, gradually discoloring and forming polar products
on standing over several hours. The compound could be stored at
1
°C; H NMR ((CD3)2SO) δ 13.8-11.0 (br, 2 H), 7.97 (d, J ) 8.1
1
Hz, 1 H), 7.49 (d, J ) 8.1 Hz, 1 H), 7.30 (dd, J ) 7.5, 1.5 Hz,
1 H), 7.12 (ddd, J ) 7.73, 7.4, 1.5 Hz, 1 H), 7.0 (ddd, J ) 7.5,
7.4, 1.5 Hz, 1 H), 6.28 (d, J ) 7.7 Hz, 1 H), 3.65 (br s, 2 H),
2.36 (s, 3 H). APCI found: [M - H]- ) 330. Anal. (C16H13NO7)
C, H, N.
2-[2-(Carboxymethyl)phenoxy]-3-methyl-4-nitrobenzoic Acid
(19). Reaction of the potassium salt of the iodide 18 with the
disodium salt of 2-hydroxyphenyl acetic acid, exactly as described
above for the isomeric iodide 10, gave the diphenyl ether 19 (51%)
as tan cubes: mp (ethyl acetate/petroleum ether) 228-232 °C; 1H
NMR ((CD3)2SO) δ 12.15 (br, 2 H), 7.97 (d, J ) 8.2 Hz, 1 H),
7.49 (d, J ) 8.2 Hz, 1 H), 7.29 (dd, J ) 7.5, 1.5 Hz, 1 H), 7.13
(ddd, J ) 8.02, 7.5, 1.5 Hz, 1 H), 6.99 (ddd, J ) 8.0, 7.5, 0.9 Hz,
1 H), 6.38 (dd, J ) 7.5, 0.9 Hz, 1 H), 3.65 (br s, 2 H), 2.37
(s, 3 H). APCI found: [M - H]- ) 330. Anal. (C16H13NO7)
C, H, N.
(5-Amino-6-methyl-9-oxo-9H-xanthen-4-yl)acetic Acid (13).
A solution of the diacid 11 (3.20 g, 9.66 mmol) in 80% H2SO4
(v/v, 30 mL) was warmed at 80 °C for 15 min. The cooled solution
was poured onto crushed ice, which was diluted further with water.
The brown precipitate was filtered off, adsorbed onto silica from
an ethyl acetate solution, and chromatographed on silica. Elution
with ethyl acetate-petroleum ether (1:1) gave foreruns, while ethyl
acetate eluted the nitroxanthenone 12 as a tan powder (0.75 g, 25%).
The material was immediately dissolved in ethyl acetate-methanol
(1:1) (60 mL) and hydrogenated over 5% Pd-C (30 mg) at 60 psi
for 2 h. The catalyst was filtered off and the filtrate concentrated
to give a yellow solid, which was slurried in methanol to leave 13
as a pale-yellow solid (0.57 g, 21% overall from 11): mp 242-
246 °C; 1H NMR ((CD3)2SO) δ 12.50 (br, 1 H), 8.90 (dd, J ) 8.0,
1.7 Hz, 1 H), 7.78 (dd, J ) 7.2, 1.7 Hz, 1 H), 7.40 (dd, J ) 8.0,
7.2 Hz, 1 H), 7.34 (d, J ) 8.0 Hz, 1 H), 7.12 (d, J ) 8.0 Hz, 1 H),
5.25 (br s, 2 H), 4.02 (s, 2 H), 2.29 (s, 3 H). APCI found: [M +
H]+ ) 284. Anal. (C16H13NO4) C, H, N.
-30 °C for at least 6 months. H NMR ((CD3)2SO) δ 8.13-8.07
(m, 2 H), 7.80 (d, J ) 7.2 Hz, 1 H), 7.47-7.40 (m, 2 H), 3.90
(s, 2 H), 2.34 (s, 3 H). APCI found: [M + H]+ ) 310.
Biology. Mice and Reagents. C57Bl/6 mice were bred at the
Animal Research Unit, Auckland University, and were housed under
conditions of constant temperature, lighting, and humidity. All
experiments used male mice, 8-12 weeks old, and conformed to
local institutional guidelines. The sodium salt of 1, synthesized at
the Auckland Cancer Society Research Centre,27 was dissolved in
culture medium for in vitro experiments and saline for injections
into mice.
Determination of Hemorrhagic Necrosis. Colon 38 adenocar-
cinoma fragments (∼1 mm3) were subcutaneously implanted into
the left flank of anesthetized (sodium pentobarbital, 82 mg/kg) mice
and allowed to grow to a diameter of approximately 5 × 6 mm,
which generally required 14 days. Groups of colon 38 tumor-bearing
mice were administered with 1 (25 mg/kg), 2 (200 mg/kg), or 3
(190 mg/kg) by intraperitoneal injection, while another group was
left untreated. Twenty-four hours after treatment, tumors were
excised from the mice immediately after cervical dislocation, fixed
in formalin, sectioned, and stained with hematoxylin and eosin. A
section across the major axis of the tumor was examined on a grid
marked at 0.4 mm intervals and was scored for percentage of
necrosis as previously described.12 At least three mice were used
for each treatment group.
Cell Culture. All cultures were maintained at 37 °C under
humidified atmosphere of 5% CO2 in air. HECPP murine endot-
helial cells40 were cultured in M199 medium (Gibco BRL Life
Technologies, Gaithersburg, MD) supplemented with 10% fetal calf
serum (FCS), 100 U/mL ampicillin, and 100 µg/mL streptomycin.
Splenocytes were obtained from mice following cervical disloca-
tion. Spleens were removed and the cells squeezed out from the
capsule into 10 mL of culture medium (Alpha-MEM supplemented
with 10% FCS, 100 U/mL ampicillin, and 100 µg/mL streptomycin).
Spleen cells were aspirated to form a single cell suspension, and
red blood cells were removed by osmotic lysis. Nucleated cells
were counted using a hemocytometer, and the cell concentration
was adjusted to 3 × 107 cells/mL. Murine splenocytes were cultured
in flat-bottomed 96-well plates at 3 × 106 cells/well, with drugs in
a total volume of 200 µL of culture medium. After the appropriate
incubation period, which was 4 h for the majority of the experi-
ments, 150 µL of the supernatant from each well was removed
and stored at -20 °C until analyzed for cytokines.
Cytotoxicity Assay. The MTT (3-(4,5-cimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) colorimetric assay was used to assess
loss of cell viability in culture.41 HECPP cells were seeded in flat-
bottomed 96 well plates at a density of 105 cells/well in 100 µL
and incubated overnight. Drugs were added the following day, and
after a further 24 h of incubation, MTT (10 µL of a solution of 5
mg/mL in PBS) was added to each well. The cells were incubated
for a further 2 h to allow living cells to cleave the pale-yellow
MTT substrate and form dark-blue formazan crystals. The formazan
crystals in the cells were dissolved by the addition of 100 µL of
0.04 N HCl/isopropanol, and the absorbance in each well was
measured with a microplate reader at 550 nm with a 630 nm
reference filter.
(6-Amino-5-methyl-9-oxo-9H-xanthen-4-yl)acetic Acid (21).
Cyclization of the diacid 19 with 80% H2SO4 followed by
immediate reduction of the resulting nitroxanethenone 20, exactly
as described above for the isomeric diacid 11, gave crude product
21 which was purified by chromatography on silica. Elution
with 5% methanol in ethyl acetate gave the amino compound 21
as a pale-yellow powder (32% overall yield from 19): mp 290-
1
293 °C; H NMR ((CD3)2SO) δ 7.95 (dd, J ) 8.0, 1.7 Hz, 1 H),
7.74 (d, J ) 8.7 Hz, 1 H), 7.61 (dd, J ) 7.3, 1.7 Hz, 1 H), 7.27
(dd, J ) 8.7, 7.3 Hz, 1 H), 66.71 (d, J ) 8.7 Hz, 1 H), 6.19 (br,
2 H), 3.77 (br s, 2 H), 2.19 (s, 3 H). APCI found: [M + H]+
284. Anal. (C16H13NO4‚0.125H2O) C, H, N.
)
(5-Azido-6-methyl-9-oxo-9H-xanthen-4-yl)acetic Acid (3). Con-
centrated H2SO4 (10 mL) was added to the powdered amine 13
(0.23 g, 0.832 mmol), and the resulting solution was immediately
cooled in an ice bath. After 2 min, ice-water (60 g) was added in
one portion and the mixture was stirred until the internal temperature
was 3 °C. A small amount of insoluble material was removed by
filtration, and the solution was recooled to 3 °C. A solution of NaN3
(63 mg, 0.91 mmol) in water (0.5 mL) was added dropwise. After
the mixture was stirred for 5 min, a solution of NaN3 (0.30 g, 4.61
mmol) in water (2 mL) was added, and the mixture was stirred at
3 °C for 30 min, then at room temperature for 1 h. The resulting
solid was filtered off, washed well with water, and dried in vacuo
to give the azide 3 as a cream powder (0.23 g, 92%): mp 160-
163 °C (dec); 1H NMR ((CD3)2SO) δ 8.14 (dd, J ) 7.9, 1.7 Hz, 1
H), 7.91 (d, J ) 8.1 Hz, 1 H), 7.83 (dd, J ) 7.3, 1.7 Hz, 1 H), 7.48
(dd, J ) 7.9, 7.3 Hz, 1 H), 7.36 (d, J ) 8.1 Hz, 1 H), 4.03 (s, 2 H),
Measurement of TNF. Supernatants from splenocytes cultures
were collected 4 h after drug treatment and assayed for the presence
of TNF using commercially available ELISA kits (OptEIA Mouse
TNF-R Kit, Pharmingen, San Diego, CA), following the manufac-
turer’s instructions. Each sample was assayed in duplicate, and

Analogues of 5,6-Dimethylxanthenone-4-acetic Acid
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 16 3763
References
triplicate wells were set up for each test condition. The mean (
SEM values were calculated.
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Preparation of Nuclear Extracts and Assay for Activated NF-
KB. HECPP cells (2.5 × 107) were incubated with 1 or azido
analogues and harvested after 2 h. Nuclear proteins were prepared
as previously described.35 Cells were lysed in cell lysis buffer
(15 mM KCl, 10 mM HEPES (pH 7.6), 2 mM MgCl2, 0.1 mM
EDTA, 25 µM dithiothreitrol (DTT), 25 µM phenylmethylsulphonyl
fluoride (PNPGB), and 0.5% Nonidet P-40) for 30 min. The pellet
of nuclei was then incubated in 10 µL of nuclei lysis buffer (0.5 M
KCl, 25 mM HEPES (pH 7.6), 0.1 mM EDTA, 1 mM DTT, 25
µM PNPGB) on ice for 30 min, and then 100 µL of dialysis buffer
(25 mM HEPES, pH 7.6, 0.1 mM EDTA, 1 mM DTT, 10%
glycerol, 25 µM PNPGB) was added. After centrifugation at 20000g
for 15 min, the supernatant was collected and the protein concentra-
tion was determined with Bradford reagent at 596 nm.42 NF-κB
complexes in the nuclear extracts were determined using the
electrophoretic mobility shift assay (EMSA) as previously de-
scribed.33 Briefly, the oligonucleotide (5′-AGCTTACAAGG-
GACTTTC-3′) containing the NF-κB consensus binding site from
the κ immunoglobulin enhancer gene,43 annealed to its comple-
mentary strand, was radiolabeled using the Klenow fragment of
DNA polymerase I (Klenow Fill-In Kit, Stratagene, La Jolla, CA)
and [R-32P]dCTP (370 mBq/mL, 10 mCi/mL, Redivue, Amersham)
in a fill-in reaction for 5′-protruding ends. DNA binding reactions
were carried out in total volume of 15 µL containing 5 µg of nuclear
protein, 4 µL of binding buffer (20 mM KCl, 12 mM HEPES, pH
7.6, 2.5 mM MgCl2, 0.4 mM EDTA, 0.5 mM DTT, 25 µM PNPGB,
12% glycerol), and 1.5 µg of poly(dI/dC). Samples were incubated
on ice for 10 min before adding the 32P-labeled probe (20 000 cpm).
Reactions were terminated by the addition of a loading dye (250
mM Tris (pH 7.5), 0.2% bromophenol blue, 0.2% xylene cyanol,
and 4% glycerol). Samples were loaded onto a 4% polyacrylamide
gel and subjected to electrophoresis in 0.25× TBE buffer (22.3
mM Tris, 22.2 mM borate, 0.5 mM EDTA) at 150 V for 2 h. The
dried gels were exposed to autoradiography (Kodak Scientific
Imaging Film) at -80 °C overnight.
Preparation of Cell Lysate for Photoaffinity Labeling and
1D Gel Electrophoresis. Murine splenocytes (2 × 107) were lysed
with Milli-Q water for 5 min and centrifuged (1100g, 10 min) to
remove cell debris. Lysate (25 µg) was incubated with 1.8 µg of
[3H]-2 (0.1 mCi/mmol, Sibtech, Inc., Newington, CT) in a volume
of 10 µL. To test for specificity of binding, other aliquots were
preincubated with 18 or 1800 µg of unlabeled 1 or 2 dissolved in
Milli-Q water for 15 min on ice before the addition of [3H]-2. After
5 min, the samples were UV-irradiated (254 nm, Stratlinker 1800,
Stratagene, La Jolla, CA) for 2 min and then the proteins were
separated by SDS gel electrophoresis. Each sample (15 µL) was
mixed with sample buffer (15 µL (Laemmli sample buffer plus
5% of 2-mercaptoethanol), incubated at 98 °C for 4 min, and left
at room temperature till loading. The sample mixture (20 µL) and
prestained molecular weight protein standards (5 µL, Bio-Rad) were
loaded onto the gel. Electrophoresis was carried out at 120 V until
the dye front reached the bottom of the glass plates (90 min). The
gel was then stained with Coomassie Blue (1 h) and destained with
40% methanol/5% acetic acid (1 h). The gel was then treated for
1 h with Amplify fluorographic solution (Amersham Pharmacia
Biotech Inc, Piscataway, NJ). The gel was then transferred to 3MM
filter paper, vacuum-dried for 1 h at 80 °C (model 583, Bio-Rad,
Hercules, CA), and exposed at -80 °C for 1 month to detect protein
bands with radioactivity. The autoradiograph was overlaid on the
Coomassie Blue stained gel to identify the protein bands that were
radioactive.
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