A new synthesis of temozolomide

Article abstract of DOI:10.1039/b205614c

An efficient condensation reaction for the synthesis of phenyloxycarbonyl substituted triazenylimidazoles was described. The condensation reaction made use of nitrosoimidazoles and phenyl methylcarbazate as the reacting products. The exposure of the triazenes to diffuse daylight induced the isomerization of the triazene-nitrogen bonds, resulting in a high yield of temozolomide.

Full text of DOI:10.1039/b205614c

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A new synthesis of temozolomide
Martin J. Wanner and Gerrit-Jan Koomen*
Laboratory of Organic Chemistry, Institute of Molecular Chemistry, University of Amsterdam,
Nieuwe Achtergracht 129, NL-1018 WS, Amsterdam. E-mail: gjk@science.uva.nl;
Fax: ϩ31-20-5255670
1
Received (in Cambridge, UK) 11th June 2002, Accepted 20th June 2002
First published as an Advance Article on the web 11th July 2002
An efficient condensation reaction between nitrosoimidazoles and phenyl methylcarbazate forms the basis of a new
synthetic route to phenyloxycarbonyl substituted triazenylimidazoles. Exposure of these triazenes to diffuse daylight
is sufficient to induce (E) to (Z)-isomerization of the triazene –N᎐N– bond, resulting in spontaneous formation of
᎐
temozolomide in high yield.
Introduction
Temozolomide is one of the antitumour drugs that is capable of
entering the central nervous system and is increasingly used to
treat malignant brain tumours.1–3 The mechanism of action of
temozolomide has been extensively investigated by Stevens and
co-workers.4–6 In plasma the tetrazinone ring of temozolomide
1 spontaneously hydrolyses to methyltriazenylimidazole-4-carb-
oxamide 2 (MTIC, Scheme 1), which falls apart to amino-
Scheme 2 Literature synthesis.
safer synthetic routes to temozolomide and analogues is still
under investigation.9–12 We wish to report on a new, economical
route to temozolomide that is based on condensation reac-
tions between nitrosoimidazoles and hydrazides, applying the
methodology we developed for 2-nitrosoadenosine.13
Results and discussion
4-Nitroimidazole 6 was chosen as starting material, and
the carboxamide substituent present in temozolomide was
introduced in the form of a nitrile (Scheme 3).
Recently
a useful synthesis of the 5-nitroimidazole-4-
Scheme 1 Bioactivation of temozolomide.
carbonitrile 7 was described via a cine-substitution reaction of
1,4-dinitroimidazole with potassium cyanide.14 The required
1,4-dinitroimidazole is the product of further nitration of 4-
nitroimidazole 6 with HNO3 and acetic anhydride. To improve
the solubility of imidazole 7 the N–H was protected with
dihydropyran to give THP-derivative 8 as a single regioisomer.
The THP-substituent is most likely positioned on N-1, as was
deduced from its azoxydimer 14 (vide infra). From our synthetic
work on nitrosoadenosine13 we noticed that partial reduction
of this type of electron deficient heterocyclic nitro compound
to the corresponding hydroxylamines proceeds readily with
hydrogen and 10% Pd on carbon as a catalyst without the use
of any additives. With nitroimidazole 8 however, considerable
overreduction to the corresponding amine occurred. Changing
the catalyst from Pd to Pt (10% on carbon) with EtOAc as a
solvent was found to be an important improvement, giving
almost quantitative formation of 9 after 1 hour at 1 atm of
hydrogen. Oxidation of this hydroxylamine to the nitroso
compound 10 proceeded efficiently with sodium periodate
imidazole-4-carboxamide AICA (3) and the methyldiazonium
cation. This methylating agent, which is the protonated form
of diazomethane, reacts at several positions in DNA and in
particular methylation of the O-6 position of guanosine has a
strong inhibitory effect with respect to cell replication. One of
the earlier triazene antitumour drugs based on this mechanism
is dacarbazine (DTIC, 5, Scheme 2), a drug that has been used
for many years against melanoma. Dacarbazine however
requires hepatic activation by P450 oxidation before it enters
the DNA-alkylation pathway via MTIC 2. In contrast, the
orally active and well-tolerated temozolomide is rapidly
absorbed and shows linear plasma pharmacokinetics.1
The existing synthesis of temozolomide is straightforward
and requires only a few steps from AICA (3) which is diazotized
to 4 (Scheme 2).7,8 Drawbacks from this synthesis are the
instability of diazonium intermediate 4 and the requirement of
excess methyl isocyanate. The development of improved and
DOI: 10.1039/b205614c
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This journal is © The Royal Society of Chemistry 2002
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Scheme 3 Reagents and conditions: (i) HNO3, Ac2O, 85%; (ii) KCN, NaHCO3, MeOH, H2O; rt, 62%; (iii) dihydropyran, p-TsOH, EtOAc;
(iv) H2, 10% Pt/C, EtOAc; rt, 60 min; (v) 2 eq. NaIO4 in H2O, EtOAc; 0 ЊC, 30 min; (vi) DCM–HOAc 5 : 2; rt, 2 h; (vii) TFA–HOAc–MeOH
3 : 6 : 10; rt, 5 h; (viii) conc. HCl–HOAc 2 : 1; 60–65 ЊC, 25 min; (ix) acetone–MeOH 2 : 1, 366 nm; rt, 1 h.
in water–ethyl acetate mixtures without the requirement of
phase-transfer catalysts. Small amounts of unsymmetrical
azoxy dimer 14 were formed as a result of a condensation reac-
tion between the starting hydroxylamine 9 and the nitroso
product 10. Although compound 14 is of no synthetical use, it
is helpful in elucidating the position of the THP-substituent. In
the 1H NMR spectrum of 14 the THP α-protons are shifted to
5.45 and 5.80 ppm, indicating the proximity of the azoxy sub-
stituent. If the THP was on the nitrogen atom next to the cyano
substituent these chemical shifts would not be expected.
Nitrosoimidazole 10, which is present as its monomer accord-
ing to 1H NMR in CDCl3-solution, did not crystallise and was
used without purification in the next condensation reaction. In
an initial attempt 1,1-dimethylhydrazine was used as nucleo-
phile with the intention of preparing N,N-dimethyltriazene 5
(dacarbazine). However, extensive reduction of the nitroso
substituent in 10 to hydroxylamine 9 occurred, due to the high
reduction potential of alkyl- or aryl-substituted hydrazines.
Acylated hydrazines show a more favourable reactivity pattern:
the reducing properties are strongly decreased while their
nucleophilicity towards electrophiles such as aldehydes or
ketones (e.g. semicarbazone formation) is largely preserved.
With phenyl methylcarbazate 11 and acetic acid as a catalyst15
efficient triazene formation was observed. Upon addition of
TFA and MeOH the THP-group was removed, providing
unprotected imidazole 12 which directly precipitated from the
reaction mixture in high overall yield.
Hydrolysis of a cyano group to a carboxamide requires
rather drastic acidic conditions. Wang and Stevens have shown
that the electronpoor tetrazinone ring system is sufficiently
acid stable to allow hydrolysis of e.g. cyanotemozolomide to
temozolomide with concentrated hydrochloric acid.10 With
aqueous hydrochloric acid and acetic acid as a co-solvent
temozolomide precursor 13 could be prepared from the
corresponding nitrile without appreciable hydrolysis of the
carboxy-triazene.
Ring closure of 12 or 13 to the desired tetrazinone ring
system was unsuccessful in spite of a variety of conditions such
as base catalysis, mild or strong acid catalysis and thermal con-
ditions. During one of these experiments however, a NMR tube
containing cyanotriazene 12 in d6-DMSO (2 mg in 0.6 mL) was
kept in diffuse daylight (window-sill) to observe any (E) to
(Z) isomerization of the triazene, which was expected to facili-
tate cyclization. The intermediate (Z)-triazene could not be
detected with H NMR, instead a clean conversion to cyano-
temozolomide 16 (Scheme 4) took place with concomitant
1
Scheme 4
formation of phenol. After 2 days the conversion was complete.
In an experiment to find a more suitable solvent, CDCl3 and
CD2Cl2 samples of 12 were exposed to daylight but this resulted
in complete fragmentation of starting material and/or product
to give phenyl methylcarbamate 18 and the instable diazo-
imidazole 17. Probably the polarity of the DMSO stimulates
the ring closing aminolysis and moreover DMSO may act as
a filter for light of shorter wavelengths. The light-sensitivity
of the tetrazinone ring system, both in the solid state and in
aqueous solution has been noticed before by Stevens.1 The
photoisomerisation–ring closing process was scaled up with
carboxamide 13 in a mixture of acetone and methanol as a
suitable solvent system to replace DMSO, with irradiation at
366 nm in a Rayonet RS. This way temozolomide was obtained
in 79% yield after trituration with acetone.
Via this route temozolomide was prepared in a reason-
able overall yield of 44% starting from 5-nitroimidazole-4-
carbonitrile 7. Intermediate triazenes such as 13 are not only
valuable photochemical precursors for temozolomide but can
also be useful as hydrolysis sensitive prodrugs of MTIC (2) and
the methyl diazonium ion formed thereof.
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5-{3-Methyl-3-[(phenyloxy)carbonyl]triazen-1-yl}imidazole-
4-carboxamide 13
Experimental
General
A suspension of nitrile 12 (1.35 g, 5.0 mmol) in a mixture of
concentrated HCl (22 mL) and acetic acid (11 mL) was stirred
vigorously at 60–65 ЊC (bath temperature) until the solids
had dissolved (approx. 20 min). The solution was heated for an
additional 5 min, cooled to room temperature and diluted with
water (3 portions of 10 mL) which induced crystallisation of
the product. After 16 h at 4 ЊC the mixture was filtered, the
product was washed thoroughly with water and dried in vacuo
to give 1.07 g (3.72 mmol, 74%) of carboxamide 13, mp 170 ЊC
(dec) (Found: C, 49.84; H, 4.28; N, 28.94. C12H12N6O3 requires
C, 50.00; H, 4.20; N, 29.15%); νmax/cmϪ1 (KBr) 3432, 1749,
1683, 1598; δH (d6-DMSO) 13.4 (1H, br, NH), 8.00 and 7.34
(2 × 1H, br, NH2), 7.92 (1H, s, 2-H), 7.51 (2H, m, ArH), 7.35
(3H, m, ArH), 3.54 (3H, s, CH3); δC (d6-DMSO) 158.9, 151.9,
150.6, 141.5, 136.2, 129.6, 126.3, 121.7, 121.2, 31.37.
All reagents and solvents were used as commercially avail-
able, unless indicated otherwise. Flash chromatography refers
to purification using the indicated eluents and Janssen Chimica
silica gel 60 (0.030–0.075 mm). Melting points were measured
with a Leitz melting point microscope. Elemental analyses were
performed by Kolbe, Mülheim an der Ruhr, Germany. Infrared
(IR) spectra were obtained from CHCl3 solutions unless
indicated otherwise, using a Bruker IFS 28 FT-spectro-
photometer and wavelengths are reported in cmϪ1. Proton
nuclear magnetic resonance (1H NMR) spectra and carbon
nuclear magnetic resonance (13C NMR; APT) spectra were
determined in CDCl3 at 300 K using a Bruker ARX 400
spectrometer, unless indicated otherwise.
5-Nitro-1-(tetrahydropyran-2-yl)imidazole-4-carbonitrile 8
A mixture of 5-nitroimidazole-4-carbonitrile 714 (4.14 g,
30 mmol), 3,4-dihydro-2H-pyran (5.49 mL, 60 mmol) and
p-TsOH (0.15 g) in EtOAc (60 mL) was stirred at room
temperature during 2 h. Et3N (0.105 mL) and light petroleum
(90 mL) were added and the solution was slowly filtered over
silica (15 g). The silica was washed with EtOAc (150 mL) and
the solvents were removed in vacuo. The residue obtained after
further drying (1 mbar, 45 ЊC) was stirred with a few mL light
petroleum to induce crystallisation. Yield: 6.38 g (2.93 mmol,
97.5%), mp 87–88 ЊC (Found: C, 48.77; H, 4.50; N, 25.12.
C9H10N4O3 requires C, 48.65; H, 4.54; N, 25.21%); νmax/cmϪ1
(KBr) 3140, 2951, 2859, 2242, 1566, 1475; δH 7.82 (1H, s), 5.45
(1H, m), 4.22 (1H, m), 3.77 (1H, m), 1.7–2.3 (6H, m);
δC (d6-DMSO) 136.1, 107.9, 102.1, 86.0, 69.2, 32.1, 24.5, 22.3.
Temozolomide 1
N2 was bubbled through a suspension of 13 (0.100 g,
0.347 mmol) in a mixture of acetone (10 mL) and methanol
(5 mL) in a normal 20 mL glass test tube. The mixture was
irradiated at room temperature in a Rayonet RS at 366 nm
during 1 h. Evaporation of the solvents and trituration with
acetone produced temozolomide 1 (0.053 g, 0.273 mmol, 79%)
as a slightly coloured crystalline solid, mp 175 ЊC (dec), liter-
ature values 212 ЊC (dec)7 or 185 ЊC (dec);8 νmax/cmϪ1 (KBr)
3494, 3124, 1745, 1680; δH (d6-DMSO) 8.83 (1H, s, 2-H), 7.80
and 7.68 (2 × 1H, br, NH2), 3.89 (3H, s, CH3); δC (d6-DMSO)
161.5, 139.2, 134.6, 130.5, 128.4, 36.13.
Azoxy dimer 14
From an earlier synthesis of nitrosoimidazole 10 the azoxy
dimer 14 was obtained, δH (selected signals) 7.94 (1H, s, 2-H),
7.88 (1H, s, 2Ј-H), 5.80 (1H, dd, J 10.5 and 2.1, N–CH–O), 5.45
(1H, dd, J 10.5 and 2.4, N–CH–O).
5-{3-Methyl-3-[(phenyloxy)carbonyl]triazen-1-yl}imidazole-
4-carbonitrile 12
10% Pt on carbon (0.20 g) was added to a solution of 8 (2.18 g,
10.0 mmol) in EtOAc (50 mL) and the mixture was stirred
vigorously under 1 atm of H2 during 1 h. The reaction was
followed with TLC (silica, eluent: EtOAc). The crystallised
product was dissolved by adding some ethanol and the catalyst
was removed by filtration over Celite. The solids were washed
with EtOAc (50 mL), the filtrate was cooled in ice, and under
vigorous stirring an ice-cold solution of NaIO4 (4.28 g,
20.0 mmol) in water (80 mL) was added quickly. Vigorous
stirring was continued for 30 min, the layers were separated and
the green organic layer was washed once with water (25 mL)
and dried over Na2SO4. Evaporation of the solvents gave
nitrosoimidazole 10 as an oil which was used without puri-
fication in the next step, δH 7.87 (1H, s, 2-H), 5.52 (1H, dd,
J 10.5 and 2.5, N–CH–O), 4.21 (1H, m), 3.77 (1H, m), 2.25 (1H,
m) 2.13 (1H, m), 2.0 (1H, m), 1.7 (3H, m).
A solution of nitrosoimidazole 10 (prepared from 10.0 mmol
8) in a mixture of DCM (15 mL) and acetic acid (6 mL) was
cooled in ice, phenyl methylcarbazate 1116 (1.83 g, 11 mmol)
was added and the cooling bath was removed. After stirring the
solution at room temperature for 2 h the DCM was removed
in vacuo and the resulting acetic acid solution was diluted with
methanol (10 mL) and trifluoroacetic acid (3 mL). Crystalliz-
ation started after ca. 1 h, and after a reaction time of 5 h the
mixture was cooled in ice and filtered. The yellow triazene 12
(2.05 g, 7.59 mmol, 76% from 8) was washed 3 times with cold
methanol and dried in vacuo, mp 105–110 ЊC (Found: C, 53.24;
H, 3.85; N, 31.02. C12H10N6O2 requires C, 53.44; H, 3.73;
N, 31.10%); νmax/cmϪ1 (KBr) 3578, 3451, 3120, 2233, 1739,
1637; δH (d6-DMSO) 13.84 (1H, br, NH), 8.00 (1H, s, 2-H), 7.50
(2H, m, ArH), 7.34 (3H, m, ArH), 3.52 (3H, s, CH3); in
13C NMR only 7 signals were observed; increasing the relax-
ation times or the temperature did not give improvement,
δC (d6-DMSO) 151.8, 150.7, 138.5, 129.6, 126.2, 121.6, 31.01.
Irradiation of 12 in d6-DMSO
A solution of 12 (4.0 mg) in d6-DMSO (0.6 mL) was kept in
1
diffuse daylight (window-sill) during 0.5 h. H NMR showed
starting material (54%), cyanotemozolomide (16) and phenol
(each 46%). Cyanotemozolomide 16: δH (d6-DMSO) 9.07 (1H,
s, 2-H), 3.93 (3H, s, CH3); phenol: 9.33 (1H, s), 7.17 (2H, m),
6.76 (3H, m).
Irradiation of 12 in CDCl3
A solution of 12 (4.0 mg) in CDCl3 (0.6 mL) was kept in diffuse
1
daylight (window-sill) during 4 h. In the H NMR spectrum
starting material (2-H at 7.63 ppm), an unidentified imidazole
(2-H at 7.76 ppm) and phenyl methylcarbamate δH 7–7.5
(5H, m, ArH), 4.95 (1H, br, NH), 2.90 (3H, d, J 4.7, CH3) were
observed.
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