Synthesis and growth-inhibitory activities of imidazo?5,1-d]-1,2,3,5-tetrazine-8-carboxamides related to the anti-tumour drug temozolomide, with appended silicon, benzyl and heteromethyl groups at the 3-position

Article abstract of DOI:10.1039/c7md00554g

A series of 3-(benzyl-substituted)-imidazo?5,1-d]-1,2,3,5-tetrazines (13) and related derivatives with 3-heteromethyl groups has been synthesised and screened for growth-inhibitory activity in vitro against two pairs of glioma cell lines with temozolomide-sensitive and -resistant phenotypes dependent on the absence/presence of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). In general the compounds had low inhibitory activity with GI50 values >50 μM against both sets of cell lines. Two silicon-containing derivatives, the TMS-methylimidazotetrazine (9) and the SEM-analogue (10), showed interesting differences: compound (9) had a profile very similar to that of temozolomide with the MGMT+ cell lines being 5 to 10-fold more resistant than MGMT- isogenic partners; the SEM-substituted compound (10) showed potency across all cell lines irrespective of their MGMT status.

Full text of DOI:10.1039/c7md00554g

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Hummersone, T. D. Bradshaw, J. Zhang, C. J. Moody, M. Foreiter, H. Summers, W. Lewis, R. Wheelhouse
and M. F.G Stevens, Med. Chem. Commun., 2018, DOI: 10.1039/C7MD00554G.
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RESEARCH ARTICLE
Synthesis and growth-inhibitory activities of imidazo[5,1-d]-1,2,3,5-tetrazine-
8-carboxamides related to the anti-tumour drug temozolomide, with
appended silicon, benzyl and heteromethyl groups at the 3-position†‡
David Cousin,a Marc G. Hummersone,a Tracey D. Bradshaw,* b Jihong Zhang,b Christopher J.
Moody,c Magdalena B. Foreiter,c Helen S. Summers,c William Lewis,c Richard T. Wheelhouse
d and Malcolm F.G. Stevens*b
A series of 3-(benzyl-substituted)-imidazo[5,1-d]-1,2,3,5-tetrazines (13) and related derivatives with 3-
heteromethyl groups has been synthesised and screened for growth-inhibitory activity in vitro against two
pairs of glioma cell lines with temozolomide-sensitive and -resistant phenotypes dependent on the
absence/presence of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). In general
the compounds had low inhibitory activity with GI50 values > 50 µM against both sets of cell lines. Two silicon-
containing derivatives, the TMS-methylimidazotetrazine (9) and the SEM-analogue (10), showed interesting
differences: compound (9) had a profile very similar to that of temozolomide with the MGMT+ cell lines being
5 to 10-fold more resistant than MGMT– isogenic partners; the SEM-substituted compound (10) showed
potency across all cell lines irrespective of their MGMT status.
_________________________________________________________________________
a Pharminox Ltd, Biocity, Pennyfoot St., Nottingham NG1 1GF, UK.
b School of Pharmacy, University of Nottingham, NG7 2RD, UK. *E-mail: tracey.bradshaw@nottingham.ac.uk,
malcolm.stevens@nottingham.ac.uk
c School of Chemistry, University of Nottingham, NG7 2RD, UK. Email: c.j.moody@nottingham.ac.uk
d Institute of Cancer Therapeutics, School of Pharmacy and Medical Sciences, University of Bradford,
Bradford, BD7 1DP, UK.
† Part 44 in the series ‘Antitumour Imidazotetrazines’. Part 43 is ref.12.
‡ Electronic supplementary information (ESI) available: experimental details, crystallographic data;
NMR spectroscopic studies. See xxxxxxxxxxxx

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Introduction
The anticancer drug temozolomide (1, TMZ) was originally synthesised in 1980 and its
properties were first described in 1984.1 The chemical and biological properties of the
extensive family of anticancer imidazotetrazines have been extensively reviewed over the
years, most recently in 2014.2,3
TMZ is a small molecule of MW only 194 Da but has a constellation of desirable
pharmaceutical properties which led to the drug being approved, in combination with
radiotherapy, for the treatment of glioblastoma multiforme (GBM). Contributing to its
commercial and clinical success is the benefit of the drug being administered orally in an outꢀ
patients setting. Surprisingly, in the nearly two decades since its marketing launch, no
mechanistic ‘meꢀtoos’ have entered the market in this niche clinical application. It is the
ambitious objective of our present research to develop a new agent with the pharmaceutical
virtues of TMZ, but which overcomes known resistance mechanisms inhibiting its broader
use.
It is well established that TMZ is a prodrug that is converted chemically into the triazene 5ꢀ
(3ꢀmethyltriazenꢀ1ꢀyl)imidazoleꢀ4ꢀcarboxamide (2: MTIC), which is a precursor of a fugitive
methylating species, the methyldiazonium ion (3) and 5ꢀaminoimidazoleꢀ4ꢀcarboxamide (4:
AIC) (Scheme 1).4,5 Methylation of DNA (5) at runs of guanine residues generates inter alia
6ꢀmethoxyguanineꢀDNA lesions (6) that can be repaired, restoring the integrity of DNA by
removal of the methyl group by the DNA repair protein O6ꢀmethylguanineꢀDNA
methyltransferase (MGMT): tumours where the MGMT gene is epigenetically silenced
respond better to the drug than those where the MGMT gene is switched on.6,7 Although
repair of methylated DNA by MGMT represents the major pathway by which tumours derive
resistance to TMZ, other mechanisms, both constitutive and acquired, such as mismatch
repair (MMR) deficiency, further compromise the clinical utility of the drug,8ꢀ10 and long
term survival from this most malevolent of diseases is rare. Surprisingly, a case has been
made recently to propose a wider role for TMZ in the era of precision medicine.11

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Scheme 1. Structures of temozolomide (1) and MTIC (2), the formation of O6ꢀmethylguanine residues (6) in DNA at runs of
guanines (G) and repair of methylated DNA by O6ꢀmethylꢀDNA methyltransferase (MGMT).
The versatile Stone synthesis of imidazotetrazines originally employed to synthesise TMZ
involves the reaction of 5ꢀdiazoimidazoleꢀ4ꢀcarboxamide (7), prepared by nitrosation of AIC
(4), and an isocyanate and can be exploited to furnish a range of related compounds (Scheme
2).1 We have recently reported on our efforts to identify new imidazotetrazines,12 mainly
prepared by this method, which can introduce novel alkyl lesions into the Oꢀ6 position of
guanine residues in DNA which cannot be repaired by MGMT and are unaffected by the
MMR status of tumour cells. Of the 50 compounds described, the imidazotetrazine with a 3ꢀ
propargyl substituent (8), for example, was equiactive against two sensitive GBMꢀderived
cell lines (SNB19V and U373V) and two MGMTꢀtransfected lines (SNB19M and U373M).
A closer examination of the properties of 8 in a Taq polymerase assay showed that this
propargylꢀimidazotetrazine alkylated plasmid pBR322 DNA at runs of three to five guanines.
In addition, a piperidine cleavage assay confirmed that 8 covalently modified Nꢀ7 sites in
plasmid DNA in runs of contiguous guanines. These preferential locations are also targeted
by TMZ.13
Herein we report new investigations on compounds that can be considered as lipophilic
modifications of the propargyl substituent. These include two siliconꢀcontaining
imidazotetrazines that have had important roles in past synthetic work and are now shown to
have interesting differences in their activities on GBM cells in vitro, as well as a series of 3ꢀ
benzylimidazotetrazines that, in general, have low growth inhibitory activity.
Chemistry
The TMSꢀmethylimidazotetrazine (9), formed from 7 and TMSꢀmethyl isocyanate has been
converted into 1 with TBAF and acetic acid in acetonitrile at room temperature in 78% yield
(Scheme 2),14 providing the first practicable route to the prodrug 1 avoiding use of the

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volatile methyl isocyanate. In an alternative approach the imidazotetrazine (10) formed from
7 and TMSꢀethyloxymethyl isocyanate (SEM isocyanate) could be deprotected by use of
excess boron trifluoride in chloroform to provide the 3ꢀhydroxymethylimidazotetrazine (11)
in 95% yield. In turn 11 could be transformed to TMZ (1) with DBU and methyl iodide in
acetonitrile in a modest 22% yield via an intermediate norꢀtemozolomide (12) generated in
situ.15 The method has been adopted recently, by others, to synthesise the [11C]ꢀmethyl
variant of temozolomide.16 The benzylꢀimidazotetrazine (13a) has been synthesised
previously (36%) from 7 and benzyl isocyanate in DMSO14 or from norꢀtemozolomide (12)
and benzyl alcohol under Mitsunobu conditions (25%).15 The syntheses of three other
imidazotetrazines (13e,14 13i,17 and 13t14) have been reported previously, but the compounds
were not subjected to pharmacological investigation against the discriminating four cell line
panel.
In the present work, reaction of 7 and phenethyl isocyanate afforded the homologue
(13b) (Scheme 2). Methoxyꢀsubstituted benzyl isocyanates were converted into
imidazotetrazines (13cꢀ13h) in yields that reflect the variable purities of the different
isocyanates: commerciallyꢀavailable isocyanates of high purity, gave yields of
imidazotetrazines ranging from 80ꢀ90% in the Stone synthesis, whereas less pure isocyanates
afforded yields of products in the 30ꢀ50% range. Attempts to improve the quality of
isocyanates by vacuum distillation were thwarted by their decomposition under these
conditions. Isocyanates derived from 5ꢀmembered heterocycles, generally prepared by
Curtius rearrangement of the corresponding acyl azides,17,18 were cyclised with 7 to afford the
series (13iꢀ13m) where the heteromethyl moieties could be considered as benzylic surrogates,
with the heterocycle offering additional nonꢀcovalent bonding opportunities. Synthesis of the
(1,2,3ꢀtriazolꢀ4ꢀyl)methylimidazotetrazine (13n) required a different approach: this was
prepared (42%) from 8 and TMSꢀazide in an aqueous tꢀbutanol medium containing sodium
ascorbate and copper(II) sulfate. Commercially available αꢀsubstituted benzyl isocyanates
were cyclised with 7 to afford the imidazotetrazines (13oꢀ13t) in moderate to good yields.

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Scheme 2. Synthetic routes to 3ꢀsubstituted imidazotetrazines. Reagents and Conditions: a) HC≡CCH2NCO, DMSO, 25 °C;
b) TMSCH2NCO, DMSO, 25 °C; c) TBAF, AcOH, MeCN, 25 °C; d) SEMꢀNCO, DMSO, 25 °C; e) BF3, CHCl3, 0 °C; f)
DBU, MeI, MeCN, 25 °C; g) R1R2CHNCO, DMSO, 25 °C) BnOH, PSꢀPPh3, DIAD, DMF; i) TMSꢀN3, Na ascorbate,
Cu(II)SO4, tꢀBuOH, H2O, 25 °C.
On the assumption that the new imidazotetrazines would, like TMZ and 8, act as
prodrugs and undergo ringꢀopening to their respective imidazotriazenes 21,4 and 14a,12
respectively, a small series of new triazenes (14bꢀ14g) was prepared by coupling 7 and the
appropriate amines (Scheme 3) for biological evaluation in comparison with their ringꢀclosed
counterparts. Yields of triazenes were in the range 75ꢀ98%.
Scheme 3. Synthesis of 1ꢀsubstitutedꢀ3ꢀ(4ꢀcarbamoylimidazolꢀ5ꢀyl)triazenes (14). Reagents and Conditions: a) RCH2NH2,
EtOAc, 25 °C.
Crystallography

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The influence of coplanarity of the 8ꢀcarboxamide group with the bicyclic nucleus of
imidazotetrazines in ordering crystal packing has previously been reported for
mitozolomide19,20 The ability of the carboxamide group to make multiple Hꢀbonded
structures has led to the characterisation of several polymorphs and coꢀcrystals of
temozolomide.21 Similar influences dominate crystal packing in the TMSꢀ
methylimidazotetrazine (9) and its SEM analogue (10) (Figures 1 and 2). In contrast to the
TMSꢀderivative (9), the SEM derivative (10) shows intermolecular Hꢀbonding between the
planes of stacked imidazotetrazines. These Hꢀbonds arise from an outꢀofꢀplane torsion in the
carboxamide that allows it to access the sideꢀchain SEM oxygen of imidazotetrazines in the
plane below. These may account for increased stability in the solid phase, evident as lesser
water solubility observed qualitatively when handling.
(A)
(B)
Figure 1 (A) XꢀRay crystal structure of 3ꢀtrimethylsilylmethylimidazotetrazine (9); (B) crystal packing through hydrogen
bonding.
(A)

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(B)
Figure 2 (A) XꢀRay crystal structure of 3ꢀ(2ꢀtrimethylsilylethoxy)methylimidazotetrazine (10); (B) crystal packing through
hydrogen bonding.
1H NMR spectroscopic studies
Kinetics experiments observed by 1H NMR spectroscopy proved invaluable in identifying
methyldiazonium ion as an intermediate in the prodrug activation chemistry of TMZ.4,5 The
aqueous chemistry of the new silylꢀimidazotetrazines (9) and (10) was investigated by similar
methods using a neutral phosphate buffer in D2O, pD = 7.0 (equivalent to pH = 7.4).4 Data
for compound 9 are shown in Figure 3 and exhibit significant parallels to TMZ. In the
aromatic region, exponential loss of the 6ꢀH signal (δH = 8.67) is accompanied by
concomitant evolution of the final heterocyclic product of the reaction, AIC (4) 2ꢀH at δH =
7.34 (for confirmation, see Figure S3, ESI). The silane region showed a similarly clean
conversion of the TMS substituent of 9 δH = 0.22) to final product (δH = 0.16) presumed to be
trimethylsilanol.

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Figure 3. Reaction of TMSꢀmethylimidazotetrazine (9) in deuterated phosphate buffer.
The signal for the methylene resonance of 9 (δH = 3.99) disappeared exponentially with time
and the products derived from this part of the starting molecule were very informative. Two
products were detected, methanol (δH = 3.36) and methyl phosphate (δH = 3.45), each in
predominantly deuterated methyl forms (Figure 3, inset); there was no evidence of
formaldehyde production. The product ratios and deuterium incorporation data were closely
analogous to those obtained from TMZ (Figure S4, ESI).4 Unlike TMZ, however, which
ultimately transfers an intact methyl group to a nucleophile, any methylation products from 9
necessarily derive a proton or deuteron from solvent.

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Scheme 4 Reaction pathways of TMSꢀmethylimidazotetrazine (9) in deuterated phosphate buffer (pD = 7.00). Solid arrows
indicate a reaction pathway consistent with the NMR spectroscopic data. Dotted arrows show theoretical alternative routes
for which no evidence was obtained.
A reaction pathway consistent with these observations is summarised in Scheme 4, route A.
The imidazotetrazine undergoes hydrolytic ringꢀopening to the triazene which fragments to
the diazonium ion. The diazonium ion loses the TMS group to water to liberate diazomethane
that is deuterated by solvent to methyl diazonium and methylates a proximal nucleophile.
There is no evidence to support alternative pathways (B–D) that would involve premature
hydrolysis of the TMS group as these would necessarily lead to formaldehyde (in the hydrate
form) as product (ESI). Whilst the silane group of compound 9 renders it more lipophilic
than TMZ, the current study implies that in vitro biological responses are likely to be similar
as they are elicited through similar chemical routes.
The aqueous behaviour of the SEMꢀsubstituted imidazotetrazine (10) was similarly
investigated (see Figure 4). The signalꢀtoꢀnoise ratio was determined by the poor water
solubility of the imidazotetrazine and the need to identify the multiplet structures in the
aliphatic products. All of the signals from 10 were clearly visible and clean conversion to the
products AIC, formaldehyde hydrate and trimethylsilylethanol was evident, (see also Figure
S5, ESI) without any accumulation of intermediates, Scheme 5, route A, is consistent with
this result.
Figure 4 Reaction of SEMꢀimidazotetrazine (10) in deuterated phosphate buffer (pD = 7.00).

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It was notable that product peaks were already evident in the first increment of the kinetic
run, just a few minutes after initiation of the reaction. Decay of 10 below the detection limit
of the experiment was complete within 3 h, in contrast to persistence beyond 10 h for the
TMS derivative (9) (Figure 3) or TMZ.4,5 Assignment of the silyl product as
trimethylsilyethanol was based on the small chemical shift changes in the two multiplets of
the ethylene unit during reaction. The second order multiplet structure of the starting
material was preserved in the products and the methylene adjacent to the silane shifted from
0.98 to 0.89 ppm. This result shows that reaction of 10 is by hydrolysis of the
imidazotetrazine and then loss of formaldehyde rather than fragmentation starting at the
silane terminus of the molecule. There was no evidence of earlyꢀstage loss of the SEM group
(e.g. Scheme 5 routes B, C). This result suggests that 10 would be unlikely to have activity as
a biological alkylating agent per se.
Scheme 5 Proposed reaction pathway for decomposition of the SEMꢀimidazotetrazine (10).
Biological results and discussion
Growth inhibitory assays on glioma (GBM) cell lines
Prior to biological evaluation, all compounds were assessed to have >95% purity by LCMS.18
Compounds were evaluated against four GBMꢀderived cell lines: two lines sensitive to TMZ
(SNB19V and U373V) gave GI50 values of 35.7 and 68.0 µM, respectively; two lines
transfected with MGMT (SNB19M and U373M) showed approximately 13ꢀfold and 5ꢀfold
resistance to TMZ with GI50 values of 470 and 369 µM, respectively (Table 1). The pattern of
growth inhibition values for the corresponding ringꢀopened triazene MTIC (2) is consistent
with the known prodrug character of TMZ.4 and these results are in accord with previously
published work on these two compounds.12 Also we reported12 that the 3ꢀpropargylꢀ
imidazotetrazine (8) and its ringꢀopened triazene derivative (14a) showed growthꢀinhibitory
potency superior to TMZ across all four cell lines, confirming that these variants overcome
the principal resistance mechanism involving expression of MGMT: these results are
included in Table 1 for comparison purposes.

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In interpreting the results of tumour cell in vitro growth inhibition assays of the new
compounds revealed in the present work, it is important to bear in mind that, despite its
clinical success, TMZ has a very low order of potency against the NCI60 cell panel; typically
IC50 values are > 100 µM across all cell lines. In keeping with our objective to discover new
imidazotetrazines active against GBM tumours irrespective of their MGMT status, to warrant
further interest compounds were required to be approximately equiactive with, or more potent
than, TMZ against the “V” lines and maintain potency against the “M” lines – i.e. to have a
‘flat’ activity profile, as exemplified by the marker compound (8). Consequently, new
compounds with a GI50 value > 50 µM against the temozolomideꢀsensitive SNB19V cell line
were, in the absence of other unusual selectivities against the cell panel, deꢀprioritised and
their inhibitory activities recorded at the default value (Table 1). As predicted by the NMR
study, the profile of the TMSꢀmethylimidazotetrazine (9) against the four cell lines is
strikingly similar in its ‘spikey’ nature to that of TMZ itself (Table 1) with sub 50 µM
potency only against the SNB19V cell line, and those lines with transfected MGMT (M)
being approximately 10ꢀ and 5ꢀfold resistant to the compound, compared with their vector
(V) only counterparts. On the other hand the 3ꢀSEM compound (10) showed surprising
potency against all four cell lines, notably against the more resistant U373 V and M lines.
The NMR study suggests that this nonꢀselective growthꢀinhibitory activity may be due to
liberation of cytotoxic formaldehyde within cells.
Disappointingly all the 3ꢀbenzylimidazotetrazines (13aꢀ13h) showed GI50 values greater than
the cutꢀoff value and in most cases were only inhibitory in the range 100ꢀ200 µM (data not
shown). Of the heteromethyl compounds (13iꢀ13n) the (furanꢀ2ꢀyl)methyl derivative (13i)
was the most inhibitory, but did not achieve the threshold GI50 value, and gave and gave a
disappointingly featureless profile in the NCI60 screen, reminiscent of TMZ (Figure S6,
ESI). Bucking the trend, both enantiomers of the α–methylbenzylimidazotetrazines (13o and
13p) showed good and equivalent potency against both SNB19V and M lines although they
were less active against the U373 pair. Other αꢀsubstituted benzylimidazotetrazines (13qꢀt)
were uninteresting with GI50 values > 50 µM. With the exception of propargyltriazene (14a),
already mentioned, the series of related triazenes (14bꢀ14g) were deprioritised with GI50
values in the 100ꢀ200 µM range in most cases. Again, the (furanꢀ2ꢀyl)methyltriazene (14d),
the ringꢀopened counterpart of imidazotetrazine (13i), was the most active albeit exceeding
the required threshold GI50 value of < 50 µM.

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Table 1 In vitro growth inhibitory activities of 3ꢀsubstituted imidazotetrazines and related triazenes against humanꢀderived
glioblastoma cell lines a
Mean GI50 (µM) b
Compound (s) SNB19V
SNB19M
U373V
U373M
1, TMZ
2
8
35.7 ± 12
470 ± 88.7 68.0 ± 32
369 ± 86.1
48.5 ± 17.9 472 ± 88.7 93.7 ± 39.7 398 ± 33.7
35.6 ± 14.2 37.8 ± 11.7 37.6 ± 11.4 36.1 ± 9.6
9
10
13aꢀ13n
13o
13p
13qꢀ13t
14a
28.4 ± 5.9
29.9 ± 5.3
>50
32.7 ± 1.3
34.7 ± 0.8
>50
302 ± 69.5 45.6 ± 14.5 249.5 ± 72.1
27.5 ± 1.4
>50
49.7 ± 5.2
45.3 ± 6.0
>50
25.3 ± 3.0
>50
>50
>50
>50
24.2 ± 2.7
>50
>50
>50
>50
39.1 ± 5.3
33.9 ± 1.5
32.0 ± 6.9
35.6 ± 1.2
14bꢀ14g
>50
>50
>50
>50
a Glioma lines: SNB19V and U373V (vector transfection only, MGMT activity 4 and 56 fmol/mg MGMT, respectively);
SNB19M and U373M (stable MGMT transfection 649 and 648 fmol/mg, respectively). b GI50 values were determined by a
7ꢀday MTT assay and where GI50 values are < 50 µM are shown as the mean ±SD of ≥ 3 independent experiments (n=4ꢀ8
per experiment). All compounds were dissolved in DMSO at stock concentration 100 mM, stored at –20 °C and diluted in
culture medium immediately prior to use.
Conclusions
The 3ꢀbenzylimidazotetrazine series does not reveal useful inhibitory activity against the
glioblastoma cell panel or a characteristic profile like the methyl analogue TMZ notable for
enhanced activity against the V cell lines and resistance towards the MGMTꢀexpressing M
lines.12 A possible reason for this observation could be a chemical inability of the compounds
(or their ringꢀopened forms) to transfer a benzyl fragment to DNA or, alternatively, that an
O6ꢀbenzylguanine intrusion, if indeed formed, is not perceived to be a cytotoxic lesion by the
cell. NMR Spectroscopic investigation (Figures S7, 8, ESI) implied that there may be more
than one pathway for reaction of derivative 13a in phosphate buffer and showed that benzyl
group transfer to solvent (benzyl alcohol) was not the major reaction product. Moreover,
investigation of tautomerism in the triazene showed that the tautomer present in DMSO
solution was not that from which a diazonium ion could be released (Figure S9, ESI). The
failure to generate O6ꢀbenzylguanineꢀDNA seems the likely reason for inactivity as it is
known that O6ꢀbenzylguanine is a substrate for MGMT, and had DNA benzylation occurred,
it should have been reflected in activity against MGMT– cell lines.22 Despite its overall
potency against the four cell line panel, the SEMꢀsubstituted imidazotetrazine (10), which
outperforms that of the lead propargylimidazotetrazine (8) as reported in the present work,
has pharmaceutical liabilities in terms of stability and potential vulnerability to metabolic
degradation. The NMR study confirms that this compound does not fragment by a TMZꢀlike
alkylation mechanism. Tentatively we suggest that its overall inhibitory properties may be
explained by its liberating cytotoxic formaldehyde within cells.
In future publications we will report on the extensive series of compounds we have
synthesised and evaluated with a propargyl substituent at Nꢀ3 and modifications of the 8ꢀ

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carboxamide group in an effort to select a candidate which overcomes known mechanisms by
which GBM tumours mount resistance towards the prototype compound TMZ. Additionally,
by fineꢀtuning the pharmaceutical properties of future drug candidates by varying the nature
of the substituent at Cꢀ8, we are seeking to identify a new agent for a proofꢀofꢀprinciple in
vivo evaluation in a mouse xenograft model implanted with GBM tumours sensitive and
resistant to TMZ.
Experimental section
Chemistry
Most imidazotetrazines and all imidazotriazenes decomposed in their melting ranges which
varied according to the rate of heating. Melting points were not a reliable means to assess
purity. Compounds were purified by crystallisation or flash chromatography on Merck silica
gel 60. Compounds 9,14 10,14 13a,14 13e,1 13i 17 and 13t14 have been reported previously.
General method for the synthesis of 3ꢀsubstituted imidazotetrazines (13)
To a stirred suspension of 4ꢀdiazoimidazoleꢀ5ꢀcarboxamide (7) (0.5 g, 3.65 mmol) in dry
DMSO (5 mL) was added dropwise a substituted isocyanate (4.2 mmol) at 25 °C under
nitrogen. After 24 h the mixture was poured into ice and the imidazotetrazine extracted in
DCM (3 × 25 mL). The organic fraction was washed with water, dried (Na2SO4) and solvent
removed by vacuum evaporation. The product was triturated with EtOAc, collected by
filtration and dried in vacuo at 25 °C. A selected example was the 3ꢀbenzylimidazotetrazine
13a, formed from 7 and benzyl isocyanate, isolated as a grey solid (48%), m.p. 188 °C
(decomp.) with physical characteristics (IR, 1H and 13C NMR) identical to an authentic
sample.14
General method for the synthesis of imidazotriazenes (14)
A mixture of 4ꢀdiazoimidazoꢀ5ꢀcarboxamide (7) and an amine (1 mol. equiv.) was stirred at
25 °C for 4 h in dry EtOAc. Products 14 aꢀg were collected and washed with dry EtOAc.
(Note: imidazotriazenes are unstable in polar solvents and were stored at 0–4 °C. Their
melting points vary according to the rate of heating and are often accompanied by vigorous
effervescence.) If pure when analysed by highfield NMR, compounds were used in
biological evaluations without further purification.
Experimental details and characterization data for imidazotetrazines 13 and imidazotriazenes
14 are given in the ESI.
Crystallography
Details of Xꢀray crystallography are given in the ESI.

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14
NMR Kinetics
1H NMR kinetic experiments were performed on a Jeol ECA600 instrument observing 1H at
600.1723 MHz. Data were processed using Delta NMR software (5.0.5, Jeol Resonance Ltd).
Phosphate buffer (200 mM in D2O) was prepared by mixing solutions of NaH2PO4 (0.138 g
in 5 mL D2O) and Na2HPO4 (0.178 g in 5 mL D2O) with monitoring by a recently calibrated
pH meter until a reading of pD=7.000 was obtained. Imidazotetrazines were dissolved in d6ꢀ
DMSO (10 mM).
In a typical NMR experiment, buffer (0.7 mL) was placed in an NMR tube, equilibrated at 37
°C in the probe for 10 min and the magnet shimmed. The tube was ejected, imidazotetrazine
stock (200 µL) added, mixed thoroughly and returned to the spectrometer. Shims were
adjusted and spectra recorded every 15 min for 12–18 h. Spectra were acquired with 64
transients, sweep width 15 ppm and digitised to 65 K data points. The residual H2O/HOD
peak was presaturated during the relaxation delay (5 s). Data were zeroꢀfilled once and an
exponential window function (0.3–1.0 Hz) was applied during Fourier transformation.
Artefacts due to water suppression were removed digitally. At the end of reaction, a further
high resolution spectrum was acquired and peaks identified by spiking with small samples of
authentic materials (AIC, HCHO, Figures S3, S5, ESI).
Antiꢀproliferative assays
HCT 116 cells were purchased from the American Type Tissue collection
Stock solutions of TMZ and other compounds listed in Table 1 were prepared in DMSO (100
mM) and aliquots stored at –20 °C, protected from light. SNB19V, SNB19M, U373V and
U373M were originally obtained from ScheringꢀPlough Research Institute, USA and were
verified as being mycoplasma free. Cells were maintained in RPMI 1640 nutrient medium
supplemented with 10% foetal bovine serum (FBS), subꢀcultivated twice weekly and
incubated at 37 °C in an atmosphere containing 5% CO2.
3ꢀ(4,5ꢀDimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide (MTT) assays were performed
as reported previously.8,9 Compound concentration required to inhibit 50% growth (GI50 /
µM) were calculated using nonꢀlinear regression analysis.
Conflicts of interest
DC, MGH, JZ and HSS were former employees of Pharminox Ltd; TDB and MFGS were consultants
to, and MFGS a shareholder of, Pharminox Ltd.

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The synthesis and biological evaluation of imidazotetrazines substituted at N-3 is described.