An improved synthesis of 5,6-dimethylxanthenone-4-acetic acid (DMXAA)

Article abstract of DOI:10.1016/S0223-5234(02)01406-X

5,6-Dimethylxanthenone-4-acetic acid (DMXAA) is a novel anticancer agent with a number of unique activities, and is in clinical trial. The current synthesis of DMXAA involves six steps, beginning with a heterogeneous reaction to form an isonitrosoacetanilide, and gives an overall yield of 11% from 2,3-dimethylaniline. We report an alternative synthesis of the key intermediate 3,4-dimethylanthranilic acid via nitration of 3,4-dimethylbenzoic acid and separation of the key desired isomer by ready crystallisation. This, together with improvements in the rest of the synthesis, provide a shorter and higher-yielding route to DMXAA (22% overall from 3,4-dimethylbenzoic acid).

Full text of DOI:10.1016/S0223-5234(02)01406-X

European Journal of Medicinal Chemistry 37 (2002) 825ꢀ828
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Laboratory note
An improved synthesis of 5,6-dimethylxanthenone-4-acetic acid
(DMXAA)
Graham J. Atwell, Shangjin Yang, William A. Denny *
Auckland Cancer Society Research Centre, School of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland 1000,
New Zealand
Received 24 May 2002; received in revised form 30 July 2002; accepted 30 July 2002
Abstract
5,6-Dimethylxanthenone-4-acetic acid (DMXAA) is a novel anticancer agent with a number of unique activities, and is in clinical
trial. The current synthesis of DMXAA involves six steps, beginning with a heterogeneous reaction to form an isonitrosoacetanilide,
and gives an overall yield of 11% from 2,3-dimethylaniline. We report an alternative synthesis of the key intermediate 3,4-
dimethylanthranilic acid via nitration of 3,4-dimethylbenzoic acid and separation of the key desired isomer by ready crystallisation.
This, together with improvements in the rest of the synthesis, provide a shorter and higher-yielding route to DMXAA (22% overall
from 3,4-dimethylbenzoic acid).
´
# 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: DMXAA; Synthesis; Anti-vascular agent; Selective nitration
1. Introduction
toxic reagents hydroxylamine and chloral hydrate. The
first step of this synthesis is a heterogeneous reaction,
requiring a careful time-temperature profile, and produ-
cing a crude product (2) of low purity that is difficult to
clean up. In this paper we report an alternative synthesis
of 4 from a readily-accessible precursor, together with
improvements in the subsequent synthesis of DMXAA.
5,6-Dimethylxanthenone-4-acetic acid (DMXAA, 8)
[1] is a novel anticancer agent currently in clinical trial,
with a number of unique activities, including the ability
to shut down blood flow in tumors [2,3], induce
production of tumor necrosis factor [4,5], and inhibit
tumor angiogenesis [6]. It shows curative antitumor
activity in colon 38 tumors in mice, activity that is
potentiated by a number of other agents, including
radiation [7], the serotonin receptor antagonist cypro-
2. Results and discussion
The best alternative route to 4 appeared to be via
direct nitration of 3,4-dimethylbenzoic acid (9), followed
by reduction. The nitration of 3,4-dimethylbenzoic acid
heptadine [8], immunotherapy [9], and enzymeꢀ
therapy [10].
/
prodrug
The reported synthesis of DMXAA (Fig. 1) [1]
involves the key intermediate 3,4-dimethylbenzoic acid
(4). This is prepared from 2,3-dimethylaniline (1) by
condensation with hydroxylamine and chloral hydrate
to give the isonitrosoacetanilide (2), followed by acid-
catalysed ring closure to the isatin (3) and oxidative ring
opening of this to give 4 in 37% overall yield for the
three steps. However, this method requires use of the
(9) with KNO3ꢀ/H2SO4 fuming nitric acid was reported
[11] to give a mixture of the three nitro isomers (10aꢀ
/
10c) in a ratio of 1:2:1 (Fig. 2). We repeated this work
using fuming HNO3, and obtained the same mixture of
isomers in 94% overall yield, with a more desirable
product ratio, determined by 1H-NMR as 1:8:2. Crystal-
lisation of this mixture by neutralisation of a hot
aqueous solution of the sodium salts with acetic acid
removed 10a, giving a mixture of 10b and 10c only, in
the ratio 3.4:1. Hydrogenation of this mixture, followed
by crystallisation of the resulting mixture of amino
* Correspondence reprints
E-mail address: b.denny@auckland.ac.nz (W.A. Denny).
´
0223-5234/02/$ - see front matter # 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
PII: S 0 2 2 3 - 5 2 3 4 ( 0 2 ) 0 1 4 0 6 - X

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63 to 71%. Traces of the isomeric meta-iodoacid (from
11) in the preparation of 5 did not couple, ensuring
isomerically pure 7. Finally, careful control of the water
content to avoid sulphonated byproducts raised the
yield of DMXAA from 7 in the sulphuric acid ring
closure reaction from 70 to 93%. Overall, the yield of
DMXAA by this new route from 3,4-dimethylbenzoic
acid (9) was 22%, double that (11%) reported previously
from 2,3-dimethylaniline (1) [1].
3. Experimental
Analyses were performed by the Microchemical
Laboratory, University of Otago, Dunedin, NZ. M.p.s
were determined on a digital melting point apparatus,
and are as read. NMR spectra were measured at 400 or
200 (1H) and 100 MHz (13C), and are referenced to
Me4Si. Mass spectra were recorded at nominal 5000
resolution. HPLC of mixed nitroacids (10) was con-
Fig. 1. Original synthesis of DMXAA
ducted on a Bondclone C18 10 mm column of 3.9ꢁ
mm, at a flow rate of 1 ml minꢂ1, using a gradient
elution profile of A; 10ꢀ100%: B; 90ꢀ0% over 25 min.
Solution A: 80% MeCNꢀH2O. Solution B: 0.045 M
ammonium formate, pH 3.5.
/
300
/
/
/
3.1. Nitration of 3,4-dimethylbenzoic acid (9)
3,4-Dimethylbenzoic acid (9) (5.0 g, 33 mmol) was
1.52, 17
added portionwise to stirred fuming HNO3 (dꢃ
/
mL) maintained below ꢂ5 8C. The mixture was stirred
/
at 0 8C for a further 10 min, then poured into water
(250 mL). After cooling to 0 8C the precipitated solid
was collected, washed with ice-cold water and dried to
give a mixture of 4,5-dimethyl-2-nitrobenzoic acid (10a),
3,4-dimethyl-2-nitrobenzoic acid (10b) and 3,4-di-
methyl-5-nitrobenzoic acid (10c) (6.11 g, 94%) in the
Fig. 2. Alternative route to anthranilic acid 4
1
approximate ratio of 1:8:2 as determined by H-NMR.
compounds 4 and 11 by neutralisation of a hot aqueous
solution of the sodium salts with acetic acid gave 4 (96%
pure, 42% overall yield from 9).
The mixture was dissolved in water (55 mL) containing
NaOH (1.33 g) and the hot solution was treated with
AcOH (3.0 mL) and then cooled slowly to 0 8C. After 2
h at 0 8C the precipitated solid was collected and
washed with ice-cold water and dried to give a mixture
of 10b and 10c (4.52 g, 70% yield based on 9), in the
Together with this alternative synthesis of 4, we also
report on improvements to the reported [1] synthesis of
DMXAA from 4. These experiments are on a larger
scale, using 4 prepared as both above and also as
previously [1]. Diazotisation of 4 as previously [1], but
using sulphamic acid to remove excess NaNO2 before
treatment with KI, improved the yield of 3,4-dimethyl-2-
iodobenzoic acid (5) from 69 to 78%. This gives an
overall yield of 5 from starting material (over three
steps) of 33%, compared to 25% (over four steps) by the
isonitrosoacetanilide route. Coupling of 5 obtained by
this method with 2-hydroxyphenylacetic acid to give 8
was improved by rigorous drying of the DMSO and the
tris[2-(2-methoxyethoxy)ethyl]amine catalyst over mi-
crowave-heated molecular sieves, raising the yield from
1
approximate ratio of 3.4:1, as determined by H-NMR.
1
Compound 10b: H-NMR [(CD3)2SO]: d 7.78 (d, Jꢃ
/
7.9 Hz, 1H,), 7.50 (d, Jꢃ8.0 Hz, 1H), 2.38 (s, 3H), 2.12
/
1
(s, 3H). Compound 10c: H-NMR [(CD3)2SO]: d 8.12
(d, Jꢃ1.2 Hz, 1H), 8.03 (s, 1H), 2.41 (s, 3H), 2.36 (s,
3H).
/
3.2. Reduction of mixed nitroacids (10)
A solution of the mixed isomers 10b and 10c (4.52 g,
23.2 mmol) in MeOH (30 mL) was hydrogenated over
5% PdꢀC at 55 psi for 10 h, and the catalyst and solvent
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G.J. Atwell et al. / European Journal of Medicinal Chemistry 37 (2002) 825ꢀ
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828
827
were removed to provide a mixture of 3,4-dimethylan-
thranilic acid (4) and 3-amino-4,5-dimethylbenzoic acid
(11) as a solid (3.75 g, 98% yield). This mixture was
dissolved in 1.0 N aq. NaOH (23 mL, 23 mmol) and
water (27 mL), and the hot solution was clarified by
filtration and then treated with AcOH (1.40 mL, 24
mmol). Following cooling at 0 8C for 2 h, the product
was collected, washed thoroughly with ice-cold water
and dried to give 4 (2.34 g, 42% overall yield from 9)
molecular sieve) and tris[2-(2-methoxyethoxy)ethyl]a-
mine (TDA-1; 95%, 12 mL, dried over molecular sieves)
was added. The mixture was stirred at 20 8C with the
exclusion of moisture until homogeneous. Copper(I)
chloride (7 g) and the powdered 6 (72 g, 0.229 mol) were
then added, and the reaction mixture was stirred at ca.
40 8C until solidification occurred. The mixture was
then heated in an oil bath at 85 8C for 4 h, until
homogeneous. After cooling to r.t., the mixture was
diluted with a solution of NaOH (4 g) in water (2.7 L),
filtered through a Celite pad, and then acidified with
glacial AcOH (200 mL). The precipitated solid was
collected, washed well with water, and dried under
vacuum at 100 8C to give a crude product (60 g,
87%). This was suspended in boiling MeOH (400 mL),
hot EtOAc (400 mL) was added, and the resulting
homogeneous solution was boiled down to 350 mL,
during which time the product separated. After cooling
1
(96% pure by HPLC; see conditions above). H-NMR
[(CD3)2SO]: d 7.51 (d, Jꢃ
/
8.2 Hz, 1H), 6.40 (d, Jꢃ8.2
/
Hz, 1H), 2.21 (s, 3H), 2.00 (s, 3H).
3.3. 3,4-Dimethyl-2-iodobenzoic acid (5)
Finely powdered 4 (100 g, 0.605 mol) was added to a
mixture of concd. H2SO4 (215 mL) and water (1270
mL). The mixture was stirred at 90 8C until homo-
geneous, then cooled to 0 8C. The stirred mixture was
treated dropwise, below the surface, with a solution of
at ꢂ5 8C overnight, the solid was collected, washed
/
with cold EtOAc and dried to give 2-[2-(carboxyl-
methyl)phenoxy]-3,4-dimethylbenzoic acid (7) (49 g,
71%), which was used directly. A sample recrystallised
NaNO2 (46.8 g, 0.678 mol) in water (100 mL) at 0ꢀ
over a 1 h period. The reaction mixture was stirred for a
further 15 min at 0ꢀ2 8C, then treated with sulphamic
/
2 8C
/
from EtOAc had m.p. 240ꢀ
[(CD3)2SO]: d 12.40 (s, 2H, CO2H), 7.63 (d, Jꢃ
Hz, H-6), 7.27 (dd, Jꢃ7.4, 1.2 Hz, H-6?), 7.20 (d, Jꢃ
7.9 Hz, 1H, H-5), 7.06 (m, 1H, H-4?), 6.90 (dd, Jꢃ7.40,
7.40 Hz, 1H, H-5?), 6.09 (d, Jꢃ8.13 Hz, 1H, H-3?), 3.70
/
242 8C. 1H-NMR
7.9
acid (7 g) and stirred for 15 min. A cold solution of KI
(340 g) in water (200 mL) was added in one portion to
the cold (2 8C) diazotised solution. After the addition
the stirred mixture was warmed up to room temperature
(r.t.) over a period of 1 h, then gradually heated to
75 8C over a period of 2 h, and kept at 75 8C for a
further 30 min. After addition of Na2S2O5 (5 g), the
reaction mixture was cooled to r.t. and the solid was
collected, washed well with cold water, and dried to give
a crude product (155 g) which contained (HPLC) 78% of
5 and 13% of 3,4-dimethyl-2-hydroxybenzoic acid. The
crude product was used directly. Extraction of the
mixture with hot diisopropyl ether gave pure 5: m.p.
/
/
/
/
/
(m, 2H, CH2), 2.37 (s, 3H, CH3), 2.00 (s, 3H, CH3).
HPLC: 99.5%. Anal. Calc. for C17H16O5: C, 68.0; H,
5.4. Found: C, 68.1; H, 5.3%.
3.5. 5,6-Dimethyl-9-oxo-9H-xanthen-4-yl)acetic acid
(DMXAA) (8)
Powdered crude 7 (100 g, 0.333 mol) was added to a
stirred mixture of concd. H2SO4 (1300 mL) and water
(478 mL). The mixture was heated to 90 8C over a 30
min period, and maintained this temperature until
homogeneous and for a further 25 min. The mixture
was then cooled to 10 8C, stirred well, and diluted
slowly with water (6 L). The precipitated solid was
collected, washed with water (8 L) and suspended in a
mixture of MeOH (1.5 L) and water (1 L), with the
exclusion of light. Addition of concd. aq. NH3 (ca. 20
mL) gave a homogeneous solution, that was immedi-
ately acidified with glacial AcOH (1 L), the precipitated
product was collected, washed with water (10 L), Milli-
Q water (6 L), cold MeOH (500 mL), and dried under
139ꢀ
140 8C. 1H-NMR [(CD3)2SO]: d 13.14 (s, 1H,
/
CO2H), 7.22 (q, 2H, H-5,6), 2.43 (s, 3H, CH3), 2.35 (s,
3H, CH3). HPLC: 97%. Anal. Calc. for C9H9IO2: C,
39.2; H, 3.3; I, 46.0. Found: C, 39.6; H, 3.3; I, 45.6%.
3.4. 2-[2-(Carboxylmethyl)phenoxy]-3,4-
dimethylbenzoic acid (7)
The above crude acid 5 (100 g, 0.362 mol) was
dissolved in a solution of KOH (23.4 g, 0.417 mol) in
water (450 mL). The solution was concentrated under
reduced pressure at 100 8C, and the resulting solid was
heated under vacuum at 100 8C for 12 h to give the dry
potassium salt (6). Sodium metal (13.6 g, 0.591 mol) was
dissolved in MeOH (400 mL), then 2-hydroxyphenyla-
cetic acid (45 g, 0.295 mol) was added, and the solution
was evaporated to dryness under reduced pressure, then
heated under vacuum at 100 8C for 24 h to give the
anhydrous disodium salt. This was dissolved in anhy-
drous DMSO (350 mL, dried over microwave-dried
vacuum at 100 8C to give 8 (87 g, 93%): m.p. 259ꢀ
260 8C. 1H-NMR [(CD3)2SO]: d 12.63 (brs, 1H,
CO2H), 8.09 (dd, Jꢃ7.9, 1.6 Hz, 1H, H-1), 7.90 (d,
Jꢃ8.1 Hz, 1H, H-8), 7.78 (dd, Jꢃ7.2, 1.5 Hz, 1H, H-
3), 7.40 (dd, Jꢃ7.6, 7.6 Hz, 1H, H-2), 7.27 (d, Jꢃ8.1
/
/
/
/
/
/
Hz, 1H, H-7), 3.97 (s, 2H, CH2), 2.40 (s, 3H, CH3), 2.35
(s, 3H, CH3). HPLC: 98.0%. Anal. Calc. for C17H14O4:
C, 72.3; H, 5.0. Found: C, 72.5; H, 5.3%.

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