Antitumor imidazotetrazines. 40.1 Radiosyntheses of [4-11c-carbonyl]- and [3-n-11c-methyl]-8-carbamoyl-3-methylimidazo[5,1-d]-1,2, 3,5-tetrazin-4(3h)-one (temozolomide) for positron emission tomography (PET) studies

Article abstract of DOI:10.1021/jm020921f

8-Carbamoyl-3-methylimidazo[5,1-d]-1,2,3,5-tetrazin-4(3H)-one (temozolomide, 1) is an anti-cancer prodrug. As part of investigations to probe its postulated mode of action using PET we have developed two rapid radiosynthetic routes for the preparation of temozolomide labeled with the short-lived positron emitter, carbon-11 (t_1/2 = 20.4 min). Reaction of 5-diazoimidazole-4-carboxamide (7) with the novel labeling agent [¹¹C-methyl] methyl isocyanate (8) gave [3-N- ¹¹C-methyl]temozolomide (9) in 14-20% radiochemical yield from [¹¹C-methyl] methyl isocyanate (8) (decay corrected). The position of radiolabeling in the 3-N-methyl group was confirmed by [^11/13C]colabeling and subsequent carbon-13 NMR spectroscopy. Similarly, the reaction of 5-diazoimidazole-4-carboxamide (7) with [¹¹C-carbonyl]methyl isocyanate (10) gave [4-¹¹C-carbonyl]temozolomide (11) in 10-15% radiochemical yield from [¹¹C-carbonyl]methyl isocyanate (10) (decay corrected). Apyrogenic samples of [3-N-¹¹C-methyl]temozolomide (9) and [4-¹¹C-carbonyl]temozolomide (11), with good chemical and radiochemical purities, have been prepared and used in human PET studies.

Full text of DOI:10.1021/jm020921f

5448
J . Med. Chem. 2002, 45, 5448-5457
An titu m or Im id a zotetr a zin es. 40.1 Ra d iosyn th eses of [4-11C-Ca r bon yl]- a n d
[3-N-11C-Meth yl]-8-ca r ba m oyl-3-m eth ylim id a zo[5,1-d ]-1,2,3,5-tetr a zin -4(3H)-on e
(Tem ozolom id e) for P ositr on Em ission Tom ogr a p h y (P ET) Stu d ies
Gavin D. Brown,*,† Sajinder K. Luthra,‡ Cathryn S. Brock,† Malcolm F. G. Stevens,§ Patricia M. Price,† and
Frank Brady‡
MRC Clinical Sciences Centre, Imperial College of Science, Technology and Medicine, Hammersmith Hospital, Du Cane Road,
London W12 0NN, UK, Imaging Research Solutions Ltd, Cyclotron Building, Hammersmith Hospital, Du Cane Road,
London W12 0NN, UK, and Cancer Research Laboratories, School of Pharmaceutical Sciences, University of Nottingham,
Nottingham NG7 2RD
Received April 25, 2002
8-Carbamoyl-3-methylimidazo[5,1-d]-1,2,3,5-tetrazin-4(3H)-one (temozolomide, 1) is an anti-
cancer prodrug. As part of investigations to probe its postulated mode of action using PET we
have developed two rapid radiosynthetic routes for the preparation of temozolomide labeled
with the short-lived positron emitter, carbon-11 (t1/2 ) 20.4 min). Reaction of 5-diazoimidazole-
4-carboxamide (7) with the novel labeling agent [11C-methyl]methyl isocyanate (8) gave [3-N-
11C-methyl]temozolomide (9) in 14-20% radiochemical yield from [11C-methyl]methyl isocyanate
(8) (decay corrected). The position of radiolabeling in the 3-N-methyl group was confirmed by
[
11/13C]colabeling and subsequent carbon-13 NMR spectroscopy. Similarly, the reaction of
5-diazoimidazole-4-carboxamide (7) with [11C-carbonyl]methyl isocyanate (10) gave [4-11C-
carbonyl]temozolomide (11) in 10-15% radiochemical yield from [11C-carbonyl]methyl isocyanate
(10) (decay corrected). Apyrogenic samples of [3-N-11C-methyl]temozolomide (9) and [4-11C-
carbonyl]temozolomide (11), with good chemical and radiochemical purities, have been prepared
and used in human PET studies.
Sch em e 1
In tr od u ction
Brain tumors rank among the most aggressive of
tumors. Malignant brain tumors such as anaplastic
astrocytoma and glioblastoma multiforme are largely
incurable despite the attempts of surgical and radio-
therapy treatments. Alternative cytotoxic chemotherapy
regimens currently used include nitrosoureas such as
carmustine and lomustine; the hydrazine derivative,
procarbazine and the triazene, dacarbazine; or a com-
bination of procarbazine, lomustine, and vincristine.2
However, these drugs have several severe side effects
and have had little impact on overall survival rates,3
consequently the prognosis for malignant brain tumors
remains poor.
vanced malignant melanoma,9 and paediatric solid
tumors.10,11 In Europe, temozolomide (1) is widely
regarded as the preferred drug for the treatment of
malignant brain tumors, while in the USA the first
comparative studies12 have shown it gives better clinical
results than the drug treatment of choice, procarbazine.
In 1998 temozolomide (1) was licensed by the European
Medicines Evaluation Agency (EMEA) for clinical use
in the treatment of glioblastoma multiforme. Then in
1999 it was licensed by both the EMEA and the Food
and Drug Administration in the USA for clinical use in
the treatment of anaplastic astrocytoma.
The mechanism of action of temozolomide (1) has been
investigated and inferred through a number of scientific
studies of its crystalline structure,13 chemical decom-
position,14 and its interaction with DNA using molecular
modeling techniques.15 From these studies it is postu-
lated that temozolomide (1) behaves as a prodrug
(Scheme 1). Given orally, it is rapidly absorbed systemi-
In light of the high mortality rates associated with
these cancers, the role of cytotoxic chemotherapeutic
drugs continues to be investigated. One such drug which
in recent years has emerged as arguably the most
effective anticancer agent for the treatment of malig-
nant brain tumors is 8-carbamoyl-3-methylimidazo[5,1-
d]-1,2,3,5-tetrazin-4(3H)-one (temozolomide, 1). Temo-
zolomide (1) initially demonstrated broad-spectrum
antitumor activity against murine tumors.4 In subse-
quent clinical trials conducted under the auspices of the
Cancer Research Campaign, UK, it showed notable
antitumor activity against recurrent glioblastoma
multiforme,5-7 recurrent anaplastic astrocytoma,8 ad-
* To whom correspondence should be addressed. Tel: +44-208-
3833162. Fax: +44-208-3832029. E-mail: gavin.brown@ic.ac.uk.
† MRC Clinical Sciences Centre.
‡ Imaging Research Solutions Ltd.
§ Cancer Research Laboratories.
10.1021/jm020921f CCC: $22.00 © 2002 American Chemical Society
Published on Web 11/05/2002

Antitumor Imidazotetrazines. 40
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 25 5449
Sch em e 2
Sch em e 3
cally and at physiological pH undergoes nucleophilic
attack by water at the 4-carbonyl position of the
tetrazinone ring to produce 5-(3-methyltriazen-1-yl)-
imidazole-4-carboxamide (MTIC, 2). Under the same
conditions, MTIC (2) further reacts with water to
liberate 5-aminoimidazole-4-carboxamide (3) with the
generation of the highly reactive methyldiazonium
cation 4.14 Finally, as the active species, the methyl-
diazonium cation 4 preferentially methylates DNA at
the O6 position of guanine in guanine-rich regions.14 All
nucleophilic sites on DNA have the potential to become
methylated. However, because the sensitivity of tumor
cells to temozolomide correlates with levels of the DNA
repair protein O6-alkylguanine DNA alkyltransferase
activity, it is clear that methylation at the O6 position
of guanine is the significant cytotoxic lesion.16,17
For any given drug a precise knowledge of its mode
of action is an important prerequisite in drug develop-
ment and the further development of treatment strate-
gies. With this aim, positron emission tomography has
been used with [11C]temozolomide to investigate the
proposed prodrug activation of temozolomide (1) in vivo.
To achieve this, we have separately labeled temozolo-
mide (1) in two different positions using carbon-11. The
positions of labeling were specifically chosen in order
to monitor the retention or loss of the radiolabel
following activation of the prodrug in accordance with
the proposed mechanism (Scheme 1). It was anticipated
that retention of the label in tumor would occur if
temozolomide (1) was labeled in the 3-N-methyl position
since the N-methyl group is believed to eventually
alkylate DNA. On the other hand the label in the
4-carbonyl position should be lost as [11C]carbon dioxide.
Here we report suitable radiochemistry strategies de-
veloped to label temozolomide (1) in each of these
positions.
Sch em e 4
However, attempts to achieve this by reaction of temo-
zolomide (1) with 1-chloroethyl chloroformate, a known
N-dealkylating agent19 proved unsuccessful.1,20
A second direct approach was the cyclization of MTIC
(2) with [11C]phosgene (6) (Scheme 3), this time with a
view to introducing carbon-11 in the 4-carbonyl position.
This seemed a more favorable route since structurally
related 3-methylpyrazolotetrazinones are known to
undergo the corresponding cyclization reaction with
phosgene.21 MTIC (2) was prepared in good chemical
yield (50-55%) by the reaction of 5-diazoimidazole-4-
carboxamide (7) with methylamine.22 Trial reactions
using varied concentrations of MTIC (2) and phosgene
were carried out at low temperature and gave a mixture
of undetermined products but failed to produce any
temozolomide (1). Similarly, there have been other
unsuccessful attempts to react MTIC (2) with phosgene
or phosgene equivalents such as 1,1′-carbonyldiimida-
zole, 4-nitrophenyl chloroformate, and chloroformic acid
trichloromethyl ester.23
In the absence of suitable direct labeling routes, we
focused on the original cyclization reaction first used
to prepare temozolomide (1).4 This slow cyclization
reaction between 5-diazoimidazole-4-carboxamide (7)
and methyl isocyanate produces clinical grade temozo-
lomide (1) in high yield. An attractive feature of this
route was that it could be doubly employed for the
synthesis of temozolomide labeled with carbon-11 in
either the 3-N-methyl or 4-carbonyl group (Scheme 4).
Thus the reaction of 5-diazoimidazole-4-carboxamide (7)
with [11C-methyl]methyl isocyanate (8) would give [3-N-
11C-methyl]temozolomide (9) and similarly the reaction
of 5-diazoimidazole-4-carboxamide (7) with [11C-carbo-
nyl]methyl isocyanate (10) would give [4-11C-carbonyl]-
temozolomide (11). To utilize this labeling approach it
was therefore necessary to first develop radiosynthetic
Resu lts a n d Discu ssion
Ra d iola belin g Str a tegies. The structure of temo-
zolomide (1) severely restricts possible strategies for
direct radiolabeling with carbon-11 due to the short half-
life (t1/2 ) 20.4 min) of the radionuclide. Two direct
approaches were considered for introducing carbon-11
in the 3-N-methyl and 4-carbonyl positions, respectively.
The first approach was to [11C]methylate the N-desm-
ethyl analogue 518 of temozolomide (1) to give temozo-
lomide labeled with carbon-11 in the 3-N-methyl posi-
tion (Scheme 2). The N-desmethyl analogue 5 required
for this reaction might be obtained from temozolomide
(1) itself by removal of the 3-N-methyl group attached
to the tetrazinone ring using an N-dealkylating agent.

5450 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 25
Brown et al.
Sch em e 5
routes to [11C-methyl]methyl isocyanate (8) and [11C-
carbonyl]methyl isocyanate (10).
Ra d iosyn th eses of [11C-Ca r bon yl]m eth yl Isocy-
a n a t e (10) a n d
[
11C-Meth yl]m et h yl Isocya n a t e
(8). Probably the most commonly used route to the
synthesis of organo-isocyanates is the direct phosgena-
tion of an amine, and there are several reports using
this approach for the preparation of [11C-carbonyl]-
organo-isocyanates.24-26 However, this route is unsuit-
able for the preparation of [11C-carbonyl]methyl isocy-
anate (10). This is due to difficulties in dispensing
accurately the precise amount of gaseous methylamine
required for the radiosynthesis which is typically per-
formed at the microscale level. Hence, attempts at the
reaction between [11C]phosgene (6) and methylamine
have resulted in the formation of [11C-carbonyl]N,N′-
dimethylurea due to reaction between the initially
formed [11C-carbonyl]methyl isocyanate (10) and any
excess amine.25 To avoid this problem an alternative
route has been developed based on the [11C]phosgena-
tion of N,N-bis(trimethylsilyl)methylamine (Scheme
5a).25 Using this route we have been able to produce
[11C-carbonyl]methyl isocyanate (10) in sufficient ra-
diochemical yield {ca. 55% from [11C]phosgene (6) (decay
corrected)} for use in the radiosynthesis of [4-11C-
carbonyl]temozolomide (11). However, a novel route to
[11C-carbonyl]methyl isocyanate (10) via the [11C]phos-
genation of N-sulfinylmethylamine (Scheme 5b) has now
been developed which should also prove suitable.27
A novel route for the radiosynthesis of [11C-methyl]-
methyl isocyanate (8)28 was developed using the reaction
between iodomethane and silver cyanate.29 First, a
method for producing methyl isocyanate from io-
domethane was devised by passing gaseous iodomethane,
under a flow of nitrogen, across a heated silver cyanate
column contained in a tube furnace. The outflow of
products from the column were collected in toluene and
analyzed by combined gas chromatography-mass spec-
trometry (GC-MS). The percentage conversion of io-
domethane to methyl isocyanate was examined over the
100-180 °C temperature range. Reaction temperatures
above 160 °C gave methyl isocyanate in essentially
quantitative yields. However, the yield of [11C-methyl]-
methyl isocyanate (8) from [11C]iodomethane (12)
(Scheme 6) was only 70-75% under the same optimized
conditions giving 9.7-11.2 GBq (263-304 mCi) of [11C-
methyl]methyl isocyanate (8) at the end of the radio-
synthesis. Radio-GC analysis of the reaction mixture
(Figure 1) showed no unreacted [11C]iodomethane (12)
present (the retention time for authentic iodomethane
) 4.1 min). The main loss was due to radioactivity (ca.
25%) remaining on the silver cyanate column; however,
higher reaction temperatures failed to drive off this
bound radioactivity as [11C-methyl]methyl isocyanate
(8). To verify the radiosynthesis, [11C-methyl]methyl
isocyanate (8) was derivatized by reaction with 1-(2-
F igu r e 1. Radio-GC analysis of [11C-methyl]methyl isocyanate
(8).
Sch em e 6
methoxyphenyl)piperazine30 to form 4-(2-methoxyphe-
nyl)piperazine-1-[11C]methyl carboxamide (13) (Scheme
6).
Cycliza tion Step . The cycloaddition reaction be-
tween 5-diazoimidazole-4-carboxamide (7) and methyl
isocyanate is a very slow and light sensitive process.
On the gram scale, the reaction takes 20 days to reach
completion when carried out in dichloromethane or ethyl
acetate at 25 °C.4 Clearly, these conditions could not be
employed in radiosyntheses involving the short-lived
radioisotope carbon-11 (t1/2 ) 20.4 min) due to the long
reaction time. Similar long reaction times are observed
for the corresponding cyclization reactions to produce
other imidazotetrazinones such as mitozolomide [the
2-chloroethyl congener of temozolomide (1)]. However,
these reactions can be accelerated by employing the
highly polar solvents, hexamethylphosphoramide31 or
DMSO32 as the reaction medium. For the synthesis of
temozolomide (1), the most rapid reaction (1 day) was
achieved at 30 °C in DMSO using a >150-fold excess of
methyl isocyanate to the diazo precursor 7. Under these
conditions temozolomide (1) was prepared in 83-89%
yield. Using the same stoichiometry, reactions in hex-
amethylphosphoramide, N,N-dimethylformamide, ac-
etonitrile, and tetrahydrofuran all gave low yields of
temozolomide (1) (3-6%) after stirring for 1 day at
30 °C.
The reaction was then scaled down to the micromolar
level and the stoichiometry reversed in order to estab-

Antitumor Imidazotetrazines. 40
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 25 5451
F igu r e 2. Automated system for the radiosynthesis of [3-N-11C-methyl]temozolomide (9) from [11C]iodomethane (12) via [11C-
methyl]methyl isocyanate (8).
lish reaction conditions that could be translated to a no-
carrier-added radiosynthetic method. Using micromolar
amounts of methyl isocyanate reduced the risk of
polymerization to allow sealed reactions to be carried
out under thermal conditions. As expected, the yield of
temozolomide (1) (15-18%) was significantly reduced
from the yield (83-89%) obtained when using excess
methyl isocyanate. Several more polar N-substituted
and N,N-disubstituted carboxamides were also exam-
ined but only N,N-dimethylformamide produced a mea-
surable amount of temozolomide (1) (3-6%). These low
yields were not entirely unexpected since organo-isocy-
anates can sometimes react with N,N-disubstituted
carboxamides under thermal conditions to form N-
persubstituted amidines.33 Under further modified con-
ditions, reactions between 5-diazoimidazole-4-carboxa-
mide (7) and methyl isocyanate in the presence of boron
trifluoride etherate34 or triethyloxonium tetrafluorobo-
rate34 as catalysts failed to reduce the reaction time,
while a third catalytic approach using polyphosphoric
acid35 resulted in decomposition of the diazo precursor
7. Unable to further reduce the reaction time, the
thermal reaction conditions developed using DMSO
were translated to carbon-11 radiochemistry for the
radiosyntheses of both [3-N-11C-methyl]temozolomide (9)
and [4-11C-carbonyl]temozolomide (11).
5-diazoimidazole-4-carboxamide (7) in DMSO, and the
reaction mixture was stirred for 10 min at 100 °C. The
resultant mixture was then injected onto an HPLC
column and eluted with a phosphate buffer. The radio-
active product eluting with the same retention time as
authentic temozolomide (1) was collected. A typical
HPLC profile of the reaction is shown (Figure 3). The
product was Millipore filtered and transferred into a
sterile vial. The overall radiosynthesis time was ca. 47
min from the end of radionuclide production. Using this
method, [3-N-11C-methyl]temozolomide (9) as formu-
lated for clinical use was obtained from [11C-methyl]-
methyl isocyanate (8) in ca. 14-20% radiochemical yield
(decay corrected), giving on average 577 MBq (range
497-721 MBq) of [3-N-11C-methyl]temozolomide (9). The
average specific radioactivity was 64.0 GBq µmol-1
(range 59-69 GBq µmol-1) at the end of the radiosyn-
thesis, corresponding to 1.00-1.20 µg (5.2-6.2 nmol) of
stable temozolomide (1). The radiochemical and chemi-
cal purities of the formulated product as determined by
analytical HPLC were >97%. During and after radioac-
tive decay, mass spectrometry (CI +ve mode) of the
product gave spectra identical to that of authentic
temozolomide (1).
To confirm the position of labeling in the 3-N-methyl
position of temozolomide, a [11/13C]colabeling experiment
was performed. This involved modification to the auto-
mated synthesis system (Figure 2) by the insertion of a
small vial containing [13C]iodomethane between valve
5 and the heated silver cyanate salt. Initially, [11C]-
iodomethane (12) was prepared and then transferred
under a flow of nitrogen into the small vial containing
[13C]iodomethane. Under the same nitrogen flow, the
R a d iosyn t h esis of [3-N-11C-Meth yl]t em ozolo-
m id e (9) fr om [11C-Meth yl]m eth yl Isocya n a te (8).
A fully automated synthesis system (Figure 2) was
developed for the routine production of [3-N-11C-methyl]-
temozolomide (9) from [11C-methyl]methyl isocyanate
(8). Using this system [11C-methyl]methyl isocyanate (8),
prepared as described, was passed into a solution of

5452 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 25
Brown et al.
F igu r e 3. A typical HPLC profile from the radiosynthesis of
[3-N-11C-methyl]temozolomide (9).
resultant [11/13C]iodomethane mixture was transferred
out of the vial and across silver cyanate at 180 °C to
form [11/13C-methyl]methyl isocyanate. Reaction of [11/13C-
methyl]methyl isocyanate with 5-diazoimidazole-4-car-
boxamide (7) gave [3-N-11/13C-methyl]temozolomide which
was then isolated by semipreparative HPLC. After
radioactive decay, the isolated product was examined
by proton-decoupled carbon-13 NMR spectroscopy (62.9
MHz). A single peak at δ ) 36.0 (relative to tetrameth-
ylsilane) was observed (Figure 4b), having the same
chemical shift as the 3-N-methyl group of authentic
temozolomide (1) (Figure 4a) and identified as belonging
to a methyl group by DEPT editing, thus confirming the
position of labeling. Furthermore, mass spectrometry
(CI +ve mode) of the product gave a peak at m/z ) 196
[M + H]+ corresponding to [3-N-13C-methyl]temozolo-
mide.
F igu r e 4. Carbon-13 NMR spectra of temozolomide (1) (a +
b) and [3-N-13C-methyl]temozolomide (c) showing (a) proton
decoupled spectrum of temozolomide, (b) DEPT edited spec-
trum of temozolomide (1) [CH and CH3 are positive)] and (c)
proton decoupled spectrum of [3-N-13C-methyl]temozolomide
(the dotted arrows denote the 13C-signal for the 3-N-methyl
group).
methyl isocyanate (10) and [4-11C-carbonyl]temozolo-
mide (11) radiosyntheses. The radiosynthesis of [11C-
carbonyl]methyl isocyanate (10) required nonpolar
organic solvents such as toluene or diethyl ether. In
contrast, the cyclization reaction between 5-diazoimi-
dazole-4-carboxamide (7) and [11C-carbonyl]methyl iso-
cyanate (10) to give [4-11C-carbonyl]temozolomide (11)
was limited to DMSO. Consequently, [11C-carbonyl]-
methyl isocyanate (10) was prepared in o-xylene then
distilled into a solution of the diazo precursor 7 in
DMSO for the cyclization step. Ideally, it was preferred
to carry out the entire radiosynthesis as a “one-pot”
method to simplify the automation process required to
carry out the radiosynthesis. However, attempts to
prepare either [11C-carbonyl]methyl isocyanate (10) or
[4-11C-carbonyl]temozolomide (11) in solvent mixtures
containing a nonpolar organic solvent and DMSO had
limited success. For example, the yield of [11C-carbonyl]-
methyl isocyanate (10) from [11C]phosgene (6) in a
toluene:DMSO (50:50) mixture was only 5-12% while
the radiosynthesis of [4-11C-carbonyl]temozolomide (11)
proved even more sensitive and could only be produced
in neat DMSO.
Ra d iosyn th esis of [4-11C-Ca r bon yl]tem ozolom id e
(11) fr om [11C-Ca r bon yl]m eth yl Isocya n a te (10).
The radiosynthesis of [4-11C-carbonyl]temozolomide (11)
from [11C-carbonyl]methyl isocyanate (10) was based on
a “two-pot” method and performed using the automated
synthesis system shown in Figure 5. Essentially, [11C-
carbonyl]methyl isocyanate (10) was prepared in o-
xylene in vessel 2 by the [11C]phosgenation of N,N-
bis(trimethylsilyl)methylamine and distilled into vessel
3 containing 5-diazoimidazole-4-carboxamide (7) in
DMSO. The resultant mixture was stirred for 10 min
at 100 °C to produce [4-11C-carbonyl]temozolomide (11)
which was then isolated by semipreparative HPLC
using the same conditions used for the isolation of [3-N-
11C-methyl]temozolomide (9). The radiosynthesis time,
including isolation and Millipore filtration of the for-
mulated product, was ca. 50 min from the end of
radionuclide production.
Using this “two-pot” method, [4-11C-carbonyl]temo-
zolomide (11) as formulated for clinical use, was ob-
tained from [11C-carbonyl]methyl isocyanate (10) in ca.
10-15% radiochemical yield (decay corrected), giving on
average 373 MBq (range 300-481 MBq) of [4-11C-
The “two-pot” method was employed due to the
different solvent requirements of the [11C-carbonyl]-

Antitumor Imidazotetrazines. 40
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 25 5453
F igu r e 5. Automated system for the radiosynthesis of [4-11C-carbonyl]temozolomide (11) from [11C]phosgene (6) via [11C-carbonyl]-
methyl isocyanate (10).
carbonyl]temozolomide (11). The average specific radio-
activity was 52 GBq µmol-1 (range 46-61 GBq µmol-1
11 (t1/2 ) 20.4 min) for use in human PET studies. To
achieve this we explored the use of rapid chemical
routes, a feature essential for radiosyntheses using
)
at the end of the radiosynthesis, corresponding to 0.67-
0.89 µg (3.5-4.6 nmol) of stable temozolomide (1). The
radiochemical and chemical purities of the formulated
product as determined by analytical HPLC were >97%.
During and after radioactive decay, mass spectrometry
(CI +ve mode) of the product gave spectra identical to
that of authentic temozolomide (1).
short-lived positron emitters such as carbon-11 (t1/2
)
20.4 min). The best route proved to be the reaction of
5-diazoimidazole-4-carboxamide (7) with methyl isocy-
anate, the same chemistry used for large scale synthesis
of the clinical grade drug. However, the main disad-
vantage of this route under the original reaction condi-
tions4 was its slow nature, although the reaction has
since been accelerated by the use of DMSO as the
reaction solvent.32 We have attempted to further ac-
celerate the reaction with the use of catalysts and
alternative solvents and found that in both cases the
reaction times were either increased or resulted in
decomposition of the diazo precursor 7. However, we
were able to thermally accelerate the reaction suf-
ficiently to allow its practical application in the radio-
syntheses of temozolomide labeled with carbon-11 (t1/2
) 20.4 min) in the 3-N-methyl and 4-carbonyl positions.
Attempts were made to confirm the position of label-
ing by preparing [4-11/13C-carbonyl]temozolomide using
[13C]carbon tetrachloride in [11C/13C]colabeling experi-
ments with [11C]carbon tetrachloride and subsequent
carbon-13 NMR. This involved repeating the radiosyn-
thesis of [11C-carbonyl]methyl isocyanate (10) from [11C]-
phosgene (6) via [11C]carbon tetrachloride but with the
introduction of [13C]carbon tetrachloride between valves
6 and 7 of the automated synthesis system (Figure 5)
prior to the [11C]phosgene (6) radiosynthesis. However,
although [11C/13C]organo-isocyanates can be successfully
prepared in this way,27 attempts to produce [4-11/13C-
carbonyl]temozolomide were unsuccessful. This was
possibly due to contamination of the DMSO reaction
mixture by unreacted [13C]carbon tetrachloride during
the distillation step. The synthesis of temozolomide (1)
in chlorinated solvents such as carbon tetrachloride
requires extremely long reaction times (>20 days)4 so
the presence of any [13C]carbon tetrachloride can only
have impeded the final cyclization reaction step.
For labeling in the 3-N-methyl position, the novel
labeling agent [11C-methyl]methyl isocyanate (8) was
prepared in good radiochemical yield by the reaction of
[11C]iodomethane (12) with silver cyanate. Reaction of
5-diazoimidazole-4-carboxamide (7) with [11C-methyl]-
methyl isocyanate (8) gave [3-N-11C-methyl]temozolo-
mide (9). [4-11C-carbonyl]temozolomide (11) was simi-
larly prepared by the reaction of 5-diazoimidazole-4-
carboxamide (7) with [11C-carbonyl]methyl isocyanate
(10). In both cases automated synthesis systems were
developed and used to carry out the radiosyntheses,
giving apyrogenic samples of [3-N-11C-methyl]temozo-
lomide (9) and [4-11C-carbonyl]temozolomide (11) with
Con clu sion s
The anticancer prodrug temozolomide (1) has been
labeled with the short-lived positron emitter, carbon-

5454 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 25
Brown et al.
(perfluorotributylamine) and run in the electron impact (EI)
mode or tuned for positive ions in the chemical ionization (CI
+ve) mode using ammonia as the reactant gas and scanning
between 40 and 500 AMU. Spectral data were collected using
a PDP 11/23 processor (Digital Computers) and analyzed using
the Sidar software program (Nermag).
Com bin ed Ga s Ch r om a togr a p h y-Ma ss Sp ectr om etr y
(GC-MS). GC-MS experiments were carried out using a
Varian Vista 6000 gas chromatograph interfaced to the mass
spectrometer operating in EI mode. Gas chromatographic
separations were performed on a nonpolar dimethylsiloxane
bonded phase column (BP1 from SGE Ltd; thickness of bonded
phase ) 5 µm; column length ) 25 m; internal diameter )
0.503 mm) using helium (5 psi, 0.34 bar) as the carrier gas.
All samples (0.1-0.2 µL), neat liquids or solutions, were
injected directly onto the column via an on-column injector
(OCI3 from SGE Ltd). The column was temperature pro-
grammed from 35 °C using the following sequence: 35 °C (5
min) ramped to 180 °C at 25 °C min-1. From 180 °C (5 min)
ramped to 275 °C at 25 °C min-1. The column was conditioned
between injections by heating at 275 °C for 15 min.
NMR Sp ectr a . NMR spectra were obtained for solutions
in CD3OD using a Bruker AM250 spectrometer (1H, 250.13
MHz; 13C, 62.90 MHz) at ambient temperature. Chemical
shifts are reported in ppm (δ) relative to internal tetrameth-
ylsilane (TMS) (δ ) 0.00; downfield is positive).
Syn th esis of Meth yl Isocya n a te fr om Iod om eth a n e.
Iodomethane (1 µL, 16 µmol) was distilled under a stream of
nitrogen (10 mL min-1) and passed over silver cyanate (250
mg, 1.67 mmol) contained in a glass column (25 × 0.5 cm)
placed inside a Carbolite MTF 9/15 tube furnace (18 × 1.5 cm)
having variable temperature control. The silver cyanate
column was preconditioned by flushing with nitrogen at 100
°C for 20 min. The temperature was then adjusted to the
desired reaction temperature for 20 min before distilling the
iodomethane. The outflow from the column was collected in
toluene (250 µL) and the collected sample analyzed by GC-
MS.
Syn th esis of Tem ozolom id e (1). (a ) Ma cr osca le Syn -
th esis. Temozolomide (1) was prepared from 5-diazoimidazole-
4-carboxamide (7)22 in ca. 85% chemical yield using the
previously reported method.32 Mass spectrometry (CI +ve
mode) of the product gave a peak at m/z ) 195 [M + H]+.
Elemental C:H:N analysis gave 37.05% C, 3.07% H, and
43.34% N (expected values calculated from C6H6N6O2, formula
weight ) 194 were 37.11% C, 3.09% H, and 43.30% N).
Temozolomide (1): 13C NMR in d6-DMSO, δ(ppm) relative to
TMS: 161.4, 139.1 (carbonyl); 134.5, 130.4 (quaternary car-
bons); 128.3 (C6-CH); 36.0 (N-methyl). (b) Micr osca le Syn -
th esis. Methyl isocyanate (1 µL, 17 µmol) was added to a
reaction vial containing 5-diazoimidazole-4-carboxamide (7)22
(2.0 mg, 15 µmol) in anhydrous DMSO (250 µL). The vial was
sealed and placed in an oil bath, and the reaction mixture was
allowed to stir for 10 min at 100 °C. The reaction mixture was
then injected onto an HPLC column (µ-Bondapak C18, 30 ×
0.78 cm). The column was eluted with a mixture of water,
ethanol, and phosphate buffer (98:2:0.1) at a flow rate of 3 mL
min-1, and the eluent was continuously monitored for absor-
bance at 324 nm. The area of the HPLC peaks of the reaction
components eluting at 9-10 min and 14-15 min, having the
same retention times as authentic 5-diazoimidazole-4-carboxa-
mide (7) and temozolomide (1), respectively, were measured.
This procedure was repeated for a series of reactions in which
DMSO was replaced by a range of solvents (N-methylforma-
mide, N,N-dimethylformamide, N,N-dimethylacetamide, ac-
etonitrile, 1-methyl-2-pyrrolidinone, hexamethylphosphora-
mide, tetrahydrofuran, acetic acid, and ethyl acetate).
F igu r e 6. PET image of mean radioactivity concentration
during the first 90 min after injection of [3-N-11C-methyl]-
temozolomide (9) (the image represents a transverse section
of brain with a right parietal lesion). The concentration of
radioactivity present in brain tissue decreases in the order:
red > orange > yellow > green > light blue > dark blue.
good chemical and radiochemical purities. Human PET
studies using [3-N-11C-methyl]temozolomide (9) and
[4-11C-carbonyl]temozolomide (11) have been carried out
to determine the in vivo pharmacokinetics and to probe
postulated mode of action of this anticancer prodrug. A
transverse brain image from a [3-N-11C-methyl]temo-
zolomide (9) PET study is shown, the subject was a
patient with a recurrent right parietal grade IV astro-
cytoma, glioblastoma multiforme (Figure 6). Further
PET studies designed to demonstrate in vivo prodrug
activation of temozolomide (1) in accordance with its
proposed mechanism of action (Scheme 1) have now
been completed. These studies, in which exhaled [11C]-
carbon dioxide was also monitored, were performed on
patients after administration of [3-N-11C-methyl]temo-
zolomide (9) and [4-11C-carbonyl]temozolomide (11),
respectively. The results of this work are published
elsewhere.36,37
Exp er im en ta l Section
Ma ter ia ls. Methyl isocyanate was purchased from Ubichem
Ltd. Phosgene was purchased from Fluka Ltd. [13C]Carbon
tetrachloride (90 at. %) was purchased from CK Gas Products
Ltd. For reference purposes, authentic samples of temozolo-
mide (1) and 5-diazoimidazole-4-carboxamide (7) were donated
by the Cancer Research Laboratories, School of Pharmaceuti-
cal Sciences, University of Nottingham. All other reagents and
solvents were purchased from Sigma-Aldrich Company Ltd.
Meth od s. Ma ss Sp ectr om etr y. Mass spectra of reaction
products were obtained using a quadrupole mass spectrometer
(Nermag R10/10C). Samples were introduced into the ioniza-
tion source of the spectrometer using the probe facility and
vaporized by passing a current through the probe filament.
The spectrometer was calibrated conventionally using FC-43
Reactions between 5-diazoimidazole-4-carboxamide (7) (2.0
mg, 15 µmol) and methyl isocyanate (1 µL, 17 µmol) were also
carried out in ethyl acetate (500 µL) or dichloromethane (500
µL) containing either boron trifluoride etherate (1 µL, 8 µmol),
polyphosphoric acid (1 µL), or triethyloxonium tetrafluorobo-
rate (1.0 M in dichloromethane) (5 µL, 5 µmol). The reactions
were analyzed by HPLC using the conditions described above.

Antitumor Imidazotetrazines. 40
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 25 5455
Attem p ted Dem eth yla tion of Tem ozolom id e (1). Typi-
cally, temozolomide (1) (200 mg, 0.01 mol) was dissolved in a
mixture of toluene and acetonitrile (30 mL, 50:50). 1-Chloro-
ethyl chloroformate (1.0 mL, 0.01 mol) was added and the
reaction mixture stirred at room temperature or heated under
reflux for 1.5 h. Repeat experiments were carried out in which
the reagent concentrations and reaction times were varied.
Samples of the reaction mixture were analyzed by mass
spectrometry (CI +ve mode) and TLC. TLC analyses were
carried out using Silica 60F254 plates (0.25 mm thickness)
eluted with a mixture of acetone and glacial acetic acid (7:1,v/
v). The plates were monitored for absorbance at 254 nm.
Rea ction betw een MTIC (2) a n d P h osgen e. Typically,
MTIC (2)22 (100 mg, 0.6 mmol) was dissolved in tetrahydro-
furan (15 mL) and the solution cooled to -20 °C using a carbon
tetrachloride/liquid nitrogen cooling mixture. Phosgene in
toluene (1.93 M, 1.5 mL) was added and the reaction mixture
stirred under nitrogen for 1 h. Repeat experiments were
carried out in which the reagent concentrations and reaction
times were varied. Samples of the reaction mixture were
analyzed by mass spectrometry (CI +ve mode) and TLC using
the conditions described above. The Rf values of reaction
mixture components were compared to the Rf value (0.68) for
authentic temozolomide (1) under the same TLC conditions.
Ra d ioch em istr y. Ra d iosyn th esis of [11C]Iod om eth a n e
(12). [11C]Iodomethane (12) was prepared from cyclotron
produced [11C]carbon dioxide using the automated synthesis
system shown in Figure 2. A full account of the method has
been previously reported.38 To summarize, [11C]carbon dioxide
was transferred by a flow of nitrogen (10 mL min-1) into vessel
1 containing 0.1 M lithium aluminum hydride in THF (200
µL). The THF was evaporated by heating vessel 1 under a
nitrogen flow. Vessel 1 was then cooled by a flow of compressed
air. Hydriodic acid (200 µL) contained in a Teflon (PTFE) loop
was added to vessel 1 which was then sealed and briefly heated
to 160 °C. The [11C]iodomethane (12) produced was distilled
from vessel 1 via a sodium hydroxide trap for further reaction.
The preparation time was ca. 13 min from the end of radio-
nuclide production.
injected onto an HPLC column (µ-Bondapak C18, 25 × 0.78
cm) and eluted with a mixture of water: ethanol (70:30) at a
flow rate of 3 mL min-1. The eluent was monitored for
radioactivity and absorbance at 254 nm. A radioactive product
was observed at 14-16 min having the same retention time
as authentic 4-(2-methoxyphenyl)piperazine-1-methylcarboxa-
mide. The radioactive product was collected and after radioac-
tive decay the residue analyzed by mass spectrometry (CI +ve
mode). The mass spectrum obtained was identical to that of
authentic 4-(2-methoxyphenyl)piperazine-1-methylcarboxam-
ide. C13H19N3O2, formula weight ) 249. Mass spectrometry (CI
+ve mode), m/z ) 250 [M + H]+.
Ra d iosyn th esis of [11C-Ca r bon yl]m eth yl Isocya n a te
(10) fr om [11C]P h osgen e (6). [11C-Carbonyl]methyl isocyan-
ate (10) was prepared from [11C]phosgene (6) using a previ-
ously reported method.25 To summarize, vessel 2 (Figure 5)
containing N,N-bis(trimethylsilyl)methylamine (1 µL, 5 µmol)
in anhydrous o-xylene (250 µmol) was cooled to -10 °C using
a crushed ice/ethanol mixture. [11C]Phosgene (6) was passed
into vessel 2 for 4 min. Vessel 2 was then removed from the
cooling mixture to allow the reaction mixture to warm to room
temperature. Typically the conversion of [11C]phosgene (6) to
[11C-carbonyl]methyl isocyanate (10) was 55-60% (decay cor-
rected). The total radiosynthesis time {including [11C]phosgene
(6) production} was ca. 15 min from the end of radionuclide
production.
For analysis, 1-(2-methoxyphenyl)piperazine (2 mg, 10 µmol)
in anhydrous toluene (250 µL) was added to vessel 2, and the
resultant solution was stirred for 10 min at room temperature.
The solution was then analyzed by HPLC using the conditions
described above. A radioactive product was observed at 14-
16 min having the same retention time as authentic 4-(2-
methoxyphenyl)piperazine-1-methylcarboxamide. Mass spec-
trometry (CI +ve mode) analysis of the radioactive product
gave a spectrum identical to that of authentic 4-(2-methox-
yphenyl)piperazine-1-methylcarboxamide. C13H19N3O2, for-
mula weight ) 249. Mass spectrometry (CI +ve mode), m/z )
250 [M + H]+.
Ra d iosyn th esis of [3-N-11C-Meth yl]tem ozolom id e (9)
fr om [11C-Meth yl]m eth yl Isocya n a te (8). [11C-Methyl]m-
ethyl isocyanate (8) was passed for 1 min in a stream of
nitrogen (10 mL min-1) into vessel 2 (Figure 2) containing a
solution of 5-diazoimidazole-4-carboxamide (7)22 (2.0 mg,15
µmol) in anhydrous DMSO (250 µL). The vial was sealed and
lowered into an oil bath, and the reaction mixture was stirred
for 10 min at 100 °C. The reaction mixture was then injected
onto an HPLC column (µ-Bondapak C18, 30 × 0.78 cm). The
column was eluted with a mixture of water, ethanol, and
phosphate buffer (98:2:0.1) at a flow rate of 3 mL min-1, and
the eluent was continuously monitored for radioactivity and
UV absorbance. The radioactive product eluting between 14
and 16 min, having the same retention time as authentic
temozolomide (1) was collected and passed through a Millipore
filter (0.22 µm, Millex GS, Millipore) and transferred into a
sterile vial. The pH of the formulated solution was ca. 6.0. The
radiosynthesis time was ca. 47 min from the end of radionu-
clide production, and the average radiochemical yield of [3-N-
11C-methyl]temozolomide (9) was 577 MBq (range 497-721
MBq). The average specific radioactivity was 64 GBq µmol-1
(range 59-69 GBq µmol-1) at the end of the radiosynthesis,
corresponding to 1.00-1.20 µg (5.2-6.2 nmol) of stable temo-
zolomide (1).
Samples of [3-N-11C-methyl]temozolomide (9) as formulated
for human injection were analyzed by HPLC using a Nucleosil
5 C18 column (25 × 0.39 cm) eluted at 0.8 mL min-1 with a
mixture of 0.5% acetic acid and acetonitrile (90:10). The eluate
was monitored continuously for radioactivity and absorbance
at 324 nm. The retention time for 5-diazoimidazole-4-carboxa-
mide (7) was 4.6 min. The formulated product gave one
radioactive peak and one stable peak both with the same
retention time (7.0 min) as authentic temozolomide (1). The
radiochemical and chemical purities were >97%. A sample of
[3-N-11C-methyl]temozolomide (9) as formulated for human
injection, was analyzed during and after radioactive decay by
Ra d iosyn th esis of [11C]P h osgen e (6). [11C]Phosgene (6)
was prepared from cyclotron produced [11C]methane using the
automated synthesis system shown in Figure 5. A full account
of the method has been previously reported.39 To summarize,
[11C]methane was cryogenically trapped in succession on
Porapak traps 1 and 2 and transferred into vessel 1 containing
chlorine (30 mL). The contents of vessel 1 were then passed
under a stream of oxygen through furnace 1 containing heated
pumice stone (2.5 g) impregnated with copper(II) chloride at
400 °C. Under the same stream of oxygen, the resultant [11C]-
carbon tetrachloride produced in furnace 1 was converted to
[11C]phosgene (6) by passage through furnace 2 containing iron
granules (1.5 g) at 300 °C. The [11C]phosgene (6) was passed
through an antimony trap to remove excess chlorine, a plastic
sinter filter to remove antimony chloride and trapped for
further use in anhydrous o-xylene (250 µL) contained in vessel
2. The preparation time was ca. 11 min from the end of
radionuclide production.
Ra d iosyn th esis of [11C-Meth yl]m eth yl Isocya n a te (8)
fr om [11C]Iod om eth a n e (12). [11C]Iodomethane (12) was
distilled under nitrogen (10 mL min-1) across silver cyanate
(250 mg, 1.67 mmol) at 180 °C for 1 min, and the products
were trapped in vessel 2 (Figure 2) containing anhydrous
toluene (250 µL). Typically the conversion of [11C]iodomethane
(12) to [11C-methyl]methyl isocyanate (8) was 70-75% (decay
corrected). The total radiosynthesis time {including [11C]-
iodomethane (12) production} was ca. 13 min from the end of
radionuclide production.
A sample (1 µL) of the product was analyzed by combined
radio-gas chromatography. A radioactive peak was observed
at 2.5-3.5 min (Figure 1) having the same retention time as
authentic methyl isocyanate. For further analysis, 1-(2-meth-
oxyphenyl)piperazine (2 mg, 10 µmol) in anhydrous toluene
(250 µL) was added to vessel 2 and the resultant solution was
stirred for 10 min at room temperature. The solution was then

5456 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 25
Brown et al.
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M.; Liu-Mares, W.; Vezina, L. G.; Ettinger, A. G.; Reaman, G.
H. Phase I study of temozolomide in children and adolescents
with recurrent solid tumors a report from the children’s cancer
group. J . Clin. Oncol. 1998, 16, 3037-3043.
(11) Estlin, E. J .; Lashford, L.; Ablett, S.; Price, L.; Gowing, R.;
Gholkar, A.; Kohler, J .; Lewis, I. J .; Morland, B.; Pinkerton, C.
R. Phase I study of temozolomide in paediatric patients with
advanced cancer. Br. J . Cancer 1998, 78, 652-661.
(12) Yung, W. K.; Albright, R. E.; Olson, J .; Fredericks, R.; Fink, K.;
Prados, M. D.; Brada, M.; Spence, A.; Hohl, R. J .; Shapiro, W. A
phase II study of temozolomide vs procarbazine in patients with
glioblastoma multiforme at first relapse. Br. J . Cancer 2000, 83,
588-593.
(13) Lowe, P. R.; Sansom, C. E.; Schwalbe, C. H.; Stevens, M. F. G.;
Clark, A. S. Antitumour imidazotetrazines. 25. Crystal structure
of 8-carbamoyl-3- methylimidazo[5,1-d]-1,2,3,5-tetrazin-4(3H)-
one (temozolomide) and structural comparisons with the related
drugs mitozolomide and DTIC. J . Med. Chem. 1992, 35, 3377-
3382.
(14) Denny, B. J .; Wheelhouse, R. T.; Stevens, M. F. G.; Tsang, L. L.
H.; Slack, J . A. NMR and molecular modeling investigation of
the mechanism of activation of the antitumour drug temozolo-
mide and its interaction with DNA. Biochemistry 1994, 33,
9045-9051.
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molecular modelling studies on the mechanism of action of the
antitumour drug temozolomide. Contrib. Oncol. 1995, 49, 40-
49.
(16) McGarrity, J . F.; Smyth, T. J . Hydrolysis of diazomethane-
kinetics and mechanism. J . Am. Chem. Soc. 1980, 102, 7303-
7308.
(17) Wedge, S. R.; Porteous, J . K.; Newlands, E. S. 3-Aminobenzamide
and/or O6-benzylguanine evaluated as an adjuvant to temozo-
lomide or BCNU treatment in cell lines of variable mismatch
repair status and O6-alkylguanine-DNA alkyltransferase activ-
ity. Br. J . Cancer 1996, 74, 1030-1036.
mass spectrometry (CI +ve mode). The spectra obtained were
identical to that of authentic temozolomide (1). C6H6N6O2,
formula weight ) 194. Mass spectrometry (CI +ve mode), m/z
) 195 [M + H]+.
Ra d iosyn th esis of [4-11C-Ca r bon yl]tem ozolom id e (11)
fr om
[
11C-Ca r bon yl]m et h yl Isocya n a t e (10). Vessel 2
(Figure 5) containing [11C-carbonyl]methyl isocyanate (10) was
lowered into oil bath 1 at 150 °C. The [11C-carbonyl]methyl
isocyanate (10) was then distilled under a flow of nitrogen (10
mL min-1) for 2 min into vessel 3 containing 5-diazoimidazole-
4-carboxamide (7)22 (2.0 mg, 15 µmol) in anhydrous DMSO (250
µL). Vessel 3 was lowered into oil bath 2, and the reaction
mixture was stirred for 10 min at 100 °C. The [4-11C-carbonyl]-
temozolomide (11) produced was isolated from the reaction
mixture by semipreparative HPLC as described above. The
product was collected and passed through a Millipore filter
(0.22 µm, Millex GS, Millipore) and transferred into a sterile
vial. The pH of the formulated solution was ca. 6.0. The
radiosynthesis time was ca. 50 min from the end of radionu-
clide production, and the average radiochemical yield of [4-11C-
carbonyl]temozolomide (11) was 373 MBq (range 300-481
MBq). The average specific radioactivity was 52 GBq µmol-1
(range 46-61 GBq µmol-1) at the end of the radiosynthesis,
corresponding to 0.67-0.89 µg (3.5-4.6 nmol) of stable temo-
zolomide (1).
Samples of [4-11C-carbonyl]temozolomide (11) as formulated
for human injection were analyzed by HPLC as described
above. The formulated product gave one radioactive peak and
one stable peak both with the same retention time (7.0 min)
as authentic temozolomide (1). The radiochemical and chemical
purities were >97%. A sample of [4-11C-carbonyl]temozolomide
(11), as formulated for human injection, was analyzed during
and after radioactive decay by mass spectrometry (CI +ve
mode). The spectra obtained were identical to that of authentic
temozolomide (1). C6H6N6O2, formula weight ) 194. Mass
spectrometry (CI +ve mode), m/z ) 195 [M + H]+.
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