Antitumor imidazo[5,1-d]-1,2,3,5-tetrazines: compounds modified at the 3-position overcome resistance in human glioblastoma cell lines

Article abstract of DOI:10.1039/c6md00384b

Synthetic routes to 3-substituted imidazo[5,1-d]-1,2,3,5-tetrazines structurally related to temozolomide were explored. Interaction of 4-diazoimidazole-5-carboxamide with an isocyanate afforded high product yields when the isocyanate was available in acceptable purity. Alternatively, alkylation of the nor-temozolomide anion afforded high yields of new imidazotetrazines. Several compounds, evaluated against a panel containing matched MGMT± glioma cell lines, showed equal inhibitory activity irrespective of MGMT status; the N3-propargyl-imidazotetrazine (10m) was prioritised as an alternative to temozolomide able to bypass drug-resistance mechanisms. In Taq polymerase assays 10m, like temozolomide and its ring-opened counterpart MTIC, alkylated DNA at clusters of three and five guanine residues; covalent modification of N-7 sites of guanine were detected in piperidine cleavage assays. Compound 10m did not cross-link DNA but induced double-strand breaks evidenced by γ-H2AX detection. Propargyl-substituted imidazotriazene (13g), showed comparable activity to 10m indicating that ring-opening of the bicyclic nucleus of novel imidazotetrazine is probably required for activity.

Full text of DOI:10.1039/c6md00384b

View Article Online
View Journal
MedChemComm
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: D. Cousin, J.
Zhang, M. Hummersone, C. Matthews, M. Frigerio, T. D. Bradshaw and M. F.G Stevens, Med. Chem.
Commun., 2016, DOI: 10.1039/C6MD00384B.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/medchemcomm

Page 1 of 36
MedChemComm
View Article Online
DOI: 10.1039/C6MD00384B
Antitumor Imidazo[5,1ꢀd]ꢀ1,2,3,5ꢀtetrazines: Compounds
Modified at the 3ꢀPosition overcome Resistance in Human
Glioblastoma Cell Linesǂ
David Cousin,† Jihong Zhang,§ Marc G. Hummersone,† Charles S. Matthews,§
Mark Frigerio†, Tracey D. Bradshaw,*,§ and Malcolm F. G. Stevens*,§
Pharminox Ltd., Biocity, Pennyfoot St., Nottingham NG1 1GF, U.K. and School of
Pharmacy, University of Nottingham, Nottingham, NG7 2RD, U.K.
_____________________________________________________________
ǂ
Part 43 in the series `Antitumour Imidazotetrazines`, Part 42 is ref. 18. MFGS and TDB
were consultants to, and MFGS was a shareholder of, Pharminox Ltd.
*To whom correspondence should be addressed: tracey.bradshaw@nottingham.ac.uk tel.
44(0)115 951 3419; or malcolm.stevens@nottingham.ac.uk tel. 44(0)115 951 3404.
†Pharminox Ltd. §University of Nottingham.
1

MedChemComm
Page 2 of 36
View Article Online
DOI: 10.1039/C6MD00384B
Abstract
Synthetic routes to 3ꢀsubstituted imidazo[5,1ꢀd]ꢀ1,2,3,5ꢀtetrazines structurally related to
temozolomide were explored. Interaction of 4ꢀdiazoimidazoleꢀ5ꢀcarboxamide with an
isocyanate afforded high product yields when the isocyanate was available in acceptable
purity. Alternatively, alkylation of the norꢀtemozolomide anion afforded high yields of new
imidazotetrazines.
Several compounds, evaluated against a panel containing matched MGMT+/− glioma cell
lines, showed equal inhibitory activity irrespective of MGMT status; the N3ꢀpropargylꢀ
imidazotetrazine (10m) was prioritised as an alternative to temozolomide able to bypass
drugꢀresistance mechanisms. In Taq polymerase assays 10m, like temozolomide and its ringꢀ
opened counterpart MTIC, alkylated DNA at clusters of three and five guanine residues;
covalent modification of Nꢀ7 sites of guanine were detected in piperidine cleavage assays.
Compound (10m) did not crossꢀlink DNA but induced doubleꢀstrand breaks evidenced by γꢀ
H2AX detection. Propargylꢀsubstituted imidazotriazene (13g), showed comparable activity to
10m indicating that ringꢀopening of the bicyclic nucleus of novel imidazotetrazine is
probably required for activity.
2

Page 3 of 36
MedChemComm
View Article Online
DOI: 10.1039/C6MD00384B
Introduction
Originally synthesized in 1980 the anticancer drug temozolomide (1: TMZ) is the first
successful antitumor agent based on the bicyclic imidazo[5,1ꢀd]ꢀ1,2,3,5ꢀtetrazine skeleton,
and its chemistry was first described in 1984.1 Although originally envisaged as a competitor
to the antiꢀmelanoma drug DTIC,2 it has proven to be an unlikely commercial success story:
at a molecular weight of only 194 Da the agent is one of the most lucrative anticancer agents
in the current clinical armamentarium on a dollar sales/Da basis. Its robust pharmaceutical
properties, oral bioavailability, ability to pass the bloodꢀbrain barrier and suitability for outꢀ
patient use have contributed to the drug being the standard of care, in combination with
radiotherapy, for the treatment of glioblastoma multiforme (GBM); these properties also
commend the drug as a suitable partner in clinical trials of drug combinations with novel
agents.3 Unlike the precursor 3ꢀ(2ꢀchloroethyl) analog mitozolomide (2: MTZ), which
induced profound myelosupression in clinical trials, 1 has an acceptable safety profile:3
arguably superior to that of many `molecularlyꢀtargeted` agents. TMZ deserves a higher
status than that commonly accorded to ‘cytotoxic agents’ which are routinely vilified by the
modern drug discovery community. Surprisingly, in the years since its original synthesis in
1980 and launch in 1999, no mechanistic ‘meꢀtoo’ has entered the market. This may be
attributed to the unique chemical structure of 1 where every atom contributes to the efficient
delivery and activity of the molecule.3 A recent review has highlighted the difficulties in
developing secondꢀgeneration analogs of TMZ.4 The closest approach has been revealed in
the recent work of Wheelhouse and his colleagues who recruited the imidazotetrazine
prodrug scaffold to carry a substituted 3ꢀ(βꢀaminoethyl) residue which acted as a precursor of
an aziridinium warhead. 5 TMZ is a chemicallyꢀactivated prodrug, being hydrolytically ringꢀ
opened to the monomethyltriazene (3: MTIC). Protolytic fragmentation of 3 then generates
the methyldiazonium cation (4) with liberation of 5ꢀaminoimidazoleꢀ4ꢀcarboxamide (5:
3

MedChemComm
Page 4 of 36
View Article Online
DOI: 10.1039/C6MD00384B
AIC).6 The reactive intermediate (4) methylates genomic DNA especially at guanine residues
(G) in runs of guanines;7 the significant antitumor event is associated with methylation at
guanine Oꢀ6 sites in DNA. 6ꢀMethoxyguanineꢀDNA lesions (6) are repaired by capture of the
methyl group by a cysteine thiolate residue of methylguanineꢀDNA methyltransferase
(MGMT) and, in the absence of repair, 6ꢀmethoxyguanine sites mispair (in the WatsonꢀCrick
sense) with thymine residues. These loci are processed by the mismatch repair (MMR)
system to trigger futile cycles of thymine insertion adfacent to the methylated guanine base;
this eventually leads to DNA strand breaks, engagement of the apoptotic or autophagic
mechanisms and cell death.8, 9 The recognition that epigenetic silencing of the MGMT gene
by hypermethylation at Cꢀ5 sites of cytosine residues in GCꢀrich gene promoter sequences
can be measured in tumor samples in clinical practice, has allowed patients to be selected
with tumors more likely to respond to the drug.10,11 Conversely, tumors with a
hypomethylated (‘switched on’) MGMT gene, or with deficiency in MMR, are constituitively
resistant to TMZ and patients with such tumor profiles have limited treatment options; in
addition, acquired resistance pathways have been identified which restrict the clinical utility
of the drug. 10,11,12
Figure 1. Structures of the antitumour imidazotetrazines temozolomide (1) and mitozolomide
(2), methylation of DNA at guanine residues and repair of methylated DNA by MGMT.
4

Page 5 of 36
MedChemComm
View Article Online
DOI: 10.1039/C6MD00384B
In a previous publication we have shown that conjugating the 8ꢀcarboxamide
substituent in imidazotetrazines with DNA major and minor grooveꢀbinding motifs was not
rewarded with enhanced biological activity in in vitro systems.13 Accordingly, we initiated a
new research program with an audacious biological endꢀpoint. In a nutshell we asked the
following question: is it possible to develop safe new imidazotetrazine derivatives which
intrude novel alkyl lesions into DNA at the guanine Oꢀ6 position which are nonꢀrepairable by
MGMT and unaffected by the MMR status of the tumors? Potentially, such moieties might be
5

MedChemComm
Page 6 of 36
View Article Online
DOI: 10.1039/C6MD00384B
more efficacious than TMZ against the brain tumors for which the drug is currently approved,
but also extend the spectrum of action to tumor types currently inherently resistant to agents
of this class by virtue of their relatively abundant MGMT levels or MMR deficiency.
In many respects TMZ is a model prodrug, most notably because of the clean
chemical activation process from 1→3 to active alkylating species (4).6 In our design
considerations we have chosen to retain the imidazotetrazine scaffold which brings the
desirable physicoꢀchemical and pharmaceutical attributes mentioned earlier: but this
imperative limits the structural modifications which can be considered to only three sites.
(For the numbering system in imidazo[5,1ꢀd]ꢀ1,2,3,5ꢀtetrazines see Figure 2A). Although the
corresponding pyrazolo[5,1ꢀd]ꢀ1,2,3,5ꢀtetrazine core (Figure 2B) would be predicted to have
a similar ringꢀopening chemistry, earlier research efforts showed that adopting this related
heterocyclic template did not bring any particular benefits.14,15
Figure 2. Potential sites of chemical modification of imidazoꢀ (A) and
pyrazolo[5,1,d]ꢀ1,2,3,5ꢀtetrazines (B).
In this report we focus on the synthesis and biological properties of compounds
retaining an H atom at Cꢀ6 and a carboxamide moiety at Cꢀ8, and differing from TMZ only in
variations at Nꢀ3 (Figure 2A). Several Nꢀ3 modified compounds prepared, but inadequately
tested, in the original work1, 2 have been reꢀsynthesized and evaluated with new compounds
in a primary in vitro cell panel containing matched MGMT+/– human glioma cell lines.
Certain compounds showing equal activity against cells irrespective of their MGMT status
6

Page 7 of 36
MedChemComm
View Article Online
DOI: 10.1039/C6MD00384B
were subjected to a broader in vitro screen and further pharmacological evaluation. Making
the assumption that the Oꢀ6 position of guanine residues in DNA is still the locus of required
covalent modification by novel agents, we decided to synthesize and evaluate a broad range
of novel structural types, but specifically excluding agents with DNA crossꢀlinking potential,
like the illꢀfated MTZ.16,17
Chemistry
Synthetic routes to three types of imidazo[5,1ꢀd]ꢀ1,2,3,5ꢀtetrazines (10ꢀ12) differing
in the nature of the 3ꢀsubstituent were explored. The most widely applicable chemistry to
access a broad range of compounds was the Stone synthesis1(Synthetic Method 1: see
Experimental Section) starting with AIC (5) which was converted to 4ꢀdiazoimidazoleꢀ5ꢀ
carboxamide (7) and reacted with the appropriate isocyanate in DMSO (Scheme 1). Yields of
imidazotetrazines were near quantitative with pure isocyanates from commercial sources,
when available. We have previously used this route to synthesize 1, 2, 10aꢀd: to access other
compounds, isocyanates were prepared by classical routes including treatment of primary
amines with phosgene (or a phosgene equivalent), or the Curtius rearrangement of acyl
azides, prepared either from acid chlorides and sodium azide or directly from carboxylic
acids and diphenylphosphoryl azide. Many isocyanates required for this project prepared by
the above routes were used in an impure state and, because of their instability, it was rarely
possible to improve their purity by vacuum distillation. Accordingly, the modest to low
yields of imidazotetrazines reported (Table 1) underestimate the intrinsic efficiency of the
Stone synthesis. We have recently reported the synthesis of the (formerly) elusive 3ꢀ
hydroxymethylꢀimidazotetrazinone (8) and 9 (‘norꢀtemozolomide’).18 Although the anion
generated from 9 can be alkylated with methyl iodide to generate 1 in 70% yield, this
alternative approach to substituted 3ꢀalkylimidazotetrazines (Synthetic Method 2) generally
7

MedChemComm
Page 8 of 36
View Article Online
DOI: 10.1039/C6MD00384B
affords modest yields and is restricted in its scope.18 Nevertheless, the utility of such routes to
efficiently synthesize 11C TMZ is noteworthy 19, 20
.
Scheme 1. Synthetic Method 1 (5 → 7 → Products) and Synthetic Method 2 (8 → 9→
Products) for the preparation of 3ꢀsubstituted imidazotetrazines (10ꢀ12).
a Reagents and conditions: a) NaNO2, 1M HCl, H2O, b) RNCO, DMSO, c) 3N HCl, d) i)
NaH, DMF, ii) RX, e) DBU, RX, MeCN.
(e)
[via 9]
Examples of new 3ꢀalkylimidazotetrazines and related congeners (10) prepared
mainly by Synthetic Method 1 were those from isopropyl, cyclopropyl, cyclopentyl,
cyclohexyl, (cyclopentyl)methyl and (cyclohexyl)methyl isocyanates (10eꢀj), respectively.
Alternatively, the 3ꢀ(cyclopropyl)methylꢀsubstituted imidazotetrazine (10k) could be
prepared from 8 and (cyclopropyl)methyl bromide in the presence of DBU in acetonitrile
8

Page 9 of 36
MedChemComm
View Article Online
DOI: 10.1039/C6MD00384B
(Synthetic Method 2). Efforts to synthesize compounds suitable for biological evaluation with
3ꢀ(fluoromethyl), 3ꢀ(bromomethyl), 3ꢀ(iodomethyl), 3ꢀ(2ꢀfluoroethyl), 3ꢀ(2,2ꢀdifluoroethyl),
or 3ꢀ(2ꢀiodoethyl) substituents, and several fluorinated 3ꢀcyclopropyl and cyclopropylmethyl
variants, foundered because we were unable to prepare the required isocyanates in adequate
purity for application in Synthetic Method 1. More rewarding were the syntheses of
imidazotetrazines with a 3ꢀallyl (10l) and 3ꢀpropargyl substituent (10m). Syntheses of the
substituted 3ꢀpropargylimidazotetrazines (10n) and (10o) were obtained from 3ꢀ
hydroxymethylꢀimidazotetrazinone (8)18 and 1ꢀbromoꢀ2ꢀbutyne or 3ꢀbromoꢀ1ꢀ
(trimethylsilyl)ꢀ1ꢀpropyne, respectively, in the presence of DBU in acetonitrile.
Table 1. Synthetic Methods and Yields of 3ꢀAlkylimidazotetrazines (10), Imidazotetrazine
Carboxylic Acids, Esters, and Amides (11), 3ꢀAlkylimidazotetrazines Bearing Additional
Heteroatoms (12), and Imidazotriazenes (13).
Synthetic
Compound
R
Yield (%)
Method a
CH2Cl
1b
59
10a
10b
10c
10d
10e
10f
10g
10h
10i
CD3
1 (2)c
20 (75)
60
CH2CH3
1d
1
CH2CF3
66
CH(CH3)2
CH(CH2)2
CH(CH2)4
CH(CH2)5
CH2CH(CH2)4
CH2CH(CH2)5
1
29
1 (2)
1
7 (12)
31
1
18
1
3
1
15
10j
9

MedChemComm
Page 10 of 36
View Article Online
DOI: 10.1039/C6MD00384B
CH2CH(CH2)2
CH2CH=CH2
CH2C≡CH
2
12
10k
10l
1d
100
3 (30)
28
1 (2)c
10m
10n
10o
11a
11b
11c
11d
11e
11f
CH2C≡CCH3
CH2C≡CSiCH3
CH2CO2CH3
e
e
11
2
36
CH2CO2Et
1b (2)c
82 (13)
71
CH2CO2C(CH3)3
(R)ꢀCH(CH3)CO2CH3
(S)ꢀC(CH3)(CH2OH)CO2CH3
(CH2)2CO2Et
CH2CO2H
1
1
1
1
b
e
30
34
75
80
11g
11h
11i
(CH2)2CO2H
47
CH2CONH2
3
3
3
3
3
1b
1
2
2
1
e
45
CH2CONHCH3
CH2CON(CH3)2
CH2CON(CH2CH2)2O
(CH2)2CONH2
CH2OCH3
31
11j
8
11k
11l
33
24
11m
12a
12b
12c
12d
12e
12f
31
CH2OCH2Ph
34
CH2OAc
12
CH2OCOC(CH3)3
CH2SCH3
14
2
CH2SOCH3
98
CH2SO2CH3
1
68
12g
10

Page 11 of 36
MedChemComm
View Article Online
DOI: 10.1039/C6MD00384B
(CH2)2OSi(CH3)2C(CH3)3
e
7
12h
12i
(CH2)2OH
(CH2)2SCH3
(CH2)2SO2CH3
CH(CH2)2
e
45
26
95
54
97
69
69
70
97
93
93
97
90
1
1
4
4
4
4
4
4
4
4
4
4
12j
12k
13a
13b
13c
13d
13e
13f
13g
13h
13i
CH2CH(CH2)2
CH2CF3
CH2CHF2
(CH2)2OH
(CH2)2OCH3
CH2C≡CH
CH2C≡N
CH2CO2CH3
CH2CO2Et
13j
a See Experimental Section for details of Synthetic Methods 1ꢀ4. b Ref.21 c Ref.18
d Ref.2 e See Experimental Section.
The series of acetic and propionic acid esters (11aꢀ11f) were prepared efficiently from
the diazoimidazole (7) and the appropriate commerciallyꢀavailable isocyanates by Synthetic
Method 1. Alternatively esters (11a, 11b) can be prepared from 9 and methyl bromoacetate or
ethyl iodoacetate, respectively, albeit in low yields by Synthetic Method 2.18 Hydrolysis of
o
the ethyl esters (11b, 11f) in 5 Mꢀhydrochloric acid at 40ꢀ45 C afforded the corresponding
carboxylic acids (11g, 11h), respectively. These acids were then converted into the series of
amides (11iꢀ11m) via their mixed anhydrides, formed with isopropyl chloroformate and Nꢀ
methylmorpholine, followed by treatment with the appropriate amine (Synthetic Method 3)
(Scheme 2).
11

MedChemComm
Page 12 of 36
View Article Online
DOI: 10.1039/C6MD00384B
Scheme 2. Synthetic Method 3 for the conversion of acetic and propionic acid derivatives of
imidazotetrazines to amides.
a Reagents and conditions: a) iꢀBuOCOCl, NMM, DMF, ii) R1R2NH2.
Imidazotetrazines with additional O, S and N hetero atoms in the 3ꢀalkyl group (12)
fell into two categories depending on the length of the carbon linker (1ꢀ2 atoms) between the
imidazotetrazine nucleus and the additional heteroatom; most of the compounds (Table 1)
were prepared by modifications of Synthetic Methods 1 or 2. For other agents requiring
different approaches, details are given in the Experimental Section. We have shown
previously that the 3ꢀhydroxymethylꢀimidazotetrazine (8) can be prepared from norꢀ
temozolomide (9) and formaldehyde (79%).18 The bicycle with a 3ꢀ(2ꢀmethoxymethyl)
(MOM) substituent (12a) has been prepared previously by Synthetic Method 121 and the
benzyloxymethyl analog (12b) was prepared in the present work by the same route. Two
examples of esters (12c, 12d) which are potential prodrug forms of the hydroxymethylꢀ
imidazotetrazine (8) were prepared for biological evaluation by Synthetic Method 2 utilizing
bromomethyl acetate or chloromethyl pivaloate as alkylating agent, respectively, although
12

Page 13 of 36
MedChemComm
View Article Online
DOI: 10.1039/C6MD00384B
yields were poor.18 The thioether variant (12e) of the MOMꢀsubstituted imidazotetrazine
(above) was prepared by Synthetic Method 1. Oxidation of this thioether with periodic acid in
acetonitrile gave the sufoxide (12f) (46%), but a quantitative yield was obtained utilizing
o
ozone as oxidant in a DCMꢀMeOH mixture at ꢀ78 C. The corresponding sulfone (12g) was
prepared by the isocyanate route (68%) (Table 1). The prototypic example of a compound
with a 2ꢀcarbon linker 3ꢀ(2ꢀhydroxyethyl)ꢀimidazotetrazine (12i) was recovered (45%) by
deprotection of the tꢀbutyldimethylsilylꢀprotected derivative (12h) with THF in acetic acid at
o
25 C. The silyl precursor (12h) was itself prepared in meagre yield by Synthetic Method 1.
The methylthioethylꢀimidazotetrazine (12j) and its related sulfone (12k) were prepared by
Method 1 in 26 and 95% yields, respectively.
Certain examples of 1ꢀalkylꢀ3ꢀ(4ꢀcarbamoylimidazolꢀ5ꢀyl)triazenes (13) related in
structure to MTIC (3), which were required for pharmacological comparisons with their ringꢀ
closed imidazotetrazine counterparts, were prepared by interaction of the diazoimidazole (7)
and commercially available amines (10 mol. equiv.) in EtOAc at 25 oC (Synthetic Method 4)
(Scheme 3). Unoptimized yields are recorded in Table 1 and these samples were used for
biological studies without further purification. Previous experience with this synthetic
method has shown that > 95% purity is achieved.2, 22 Because of the availability of 2,2ꢀ
difluoroethylamine and aminoacetonitrile it was possible to prepare samples of triazenes
(13d, 13h); the corresponding imidazotetrazines bearing a 3ꢀ(2,2ꢀdifluoroethyl) or 3ꢀ
cyanomethyl group, respectively, were not accessible by Synthetic Method 1 because of the
unavailability of the required isocyanates. The triazenes (13i, 13j) were prepared from 7 and
methyl or ethyl glycinate hydrochloride salts in the presence of triethylamine (10 mol.
equiv.).
Scheme 3. Synthetic Method 4 for the preparation of 1ꢀalkylꢀ3ꢀ(4ꢀcarbamoylimidazolꢀ5ꢀ
yl)triazenes (13).
13

MedChemComm
Page 14 of 36
View Article Online
DOI: 10.1039/C6MD00384B
a Reagents and conditions: a) RNH2, EtOAc, 25 °C.
Biological Results and Discussion
Growth inhibitory assays on glioma (GBM) cell lines. All compounds listed in
23
Table 1 were shown to have > 95% purity by LCMS before biological evaluation (see
Experimental Section). It should be pointed out that, despite its clinical utility, TMZ yielded
only low growth inhibitory potency against the NCI 60 human cell line panel; assays were
performed after 48 h exposure of cells to TMZ, GI50 values > 100 μM in all lines.
Compounds were evaluated in a primary screen consisting of four GBMꢀderived cell lines:
two sensitive lines (SNB19V and U373V) gave GI50 values of 45.6 and 72.9 μM,
respectively, for TMZ; and two MGMTꢀtransfected lines (SNB19M and U373M) which
demonstrated approximately 11ꢀ and 5ꢀfold resistance to TMZ compared to their vector only
counterparts with GI50 values of 526 and 395 μM, respectively. GI50 values of other
compounds (Table 2) are the mean of at least three separate determinations, whereas for the
marker compound TMZ, n > 50. MTZ (2) was more than 3ꢀfold more potent than TMZ
against the sensitive vectorꢀonly (V) pair of cell lines, and 10ꢀfold more potent against the
MGMTꢀtransfected (M) lines than TMZ, yet the same trend persists; MGMTꢀtransfected cells
were > 2ꢀfold more resistant to MTZ than vector control cells.
Consistent with one of our objectives – to discover novel compounds which might be
active against glioma cell lines irrespective of their MGMT proficiency – compounds
14

Page 15 of 36
MedChemComm
View Article Online
DOI: 10.1039/C6MD00384B
selected for further scrutiny were required to have two activity characteristics: (i) they were at
least equiactive with, or preferably more potent than TMZ against the V lines; and crucially
(ii) maintained similar potency against the M lines. GI50 values of compounds with these
desirable qualities are listed in Table 2, together with values for some reference agents:
compounds which failed to pass muster on these two criteria were included in Table 2 only if
results were valuable in assessing SAR in the series, or not listed and deemed to be
‘deprioritized’ at this stage.
The 3ꢀchloromethyl analog (10a) of TMZ showed the type of desirable ‘flatline’
profile which was sought: the agent had higher potency (lower GI50 values) against the V
lines than TMZ and activity was maintained against the M lines. Results of representative
derivatives with lipophilic 3ꢀsubstituents (Table 2) demonstrate the practicability of the
glioma panel to select/deprioritize compounds. Thus compounds with cyclopropyl (10f),
cyclopropylꢀmethyl (10k) and allyl groups (10l) at Nꢀ3 had low potency against all four lines;
similarly, other compounds with lipophilic 3ꢀalkyl groups listed in Table 1, revealed profiles
which excluded them for further consideration at this stage (data not shown). In contrast three
compounds with propargylic groups (10mꢀo) were more potent than TMZ and equiactive
against V and M cell lines.
Of the acetic acid derivatives (Table 2) only the methyl (11a) and ethyl esters (11b)
had profiles consistent with greater activity than TMZ in V and M cell lines. The pivaloyl
ester (11c) and two αꢀsubstituted methyl acetates (11d
effects (data not shown). Interestingly the homologous ethyl propionate (11f) was less active
than the corresponding ethyl acetate (11b). Notably also the acetic acid (11g lacked the
required activity profile for further consideration despite being more active than TMZ against
, 11e) had markedly less inhibitory
)
both U373 cell lines. The homologous propionic acid (11h) was of low interest showing GI50
15

MedChemComm
Page 16 of 36
View Article Online
DOI: 10.1039/C6MD00384B
values > 250 μM against all four cell lines (data not shown). The primary acetamide (11i) had
higher potency than the secondary and tertiary acetamides (11j l). Consistent with the results
ꢀ
in the ester series, in the corresponding amides extension of the methylene chain to a twoꢀ
carbon ethylene chain led to a major loss of in vitro potency in the propionamide (11m)
(Table 2).
The 3ꢀhydroxymethylꢀimidazotetrazine (8), although showing equivalent activity
against the SNB19 V and M cell lines, proved to be too unstable for reliable bioꢀevaluation.
However, a seam of potentially useful activity extends through its more stable ethers and
esters with the MOM ether (12a), benzyloxymethyl (12b) derivatives, and two potential
prodrug forms of 8, the acetate (12c) and pivaloate esters (12d), being noteworthy for potent
inhibitory activity against both SNB19 and U373 V and M cell lines (Table 2). Interestingly
the thioꢀMOM analog (12e) was less active than its MOM counterpart, especially against the
SNB19 lines. Potency was restored to both V and M lines when the sulfide was oxidized to
sulfoxide (12f) or sulfone (12g). 3ꢀ(2ꢀHydroxyethyl)ꢀimidazotetrazine (12i), a homolog of 8,
was only inhibitory against the four cell lines at concentrations in the 200ꢀ400 μM range
(Table 2); other examples where the linker between the heteroatom and the bicycle was a
twoꢀcarbon fragment gave derivatives (12jꢀk) which were deprioritized on the activity
characteristics (i) and (ii) defined earlier (data not shown).
Table 2. In Vitro Growth Inhibitory Activity of 3ꢀSubstituted Imidazotetrazines (10ꢀ12) and
Related Triazenes (13) Against Humanꢀderived Glioblastoma Cell Lines.a
Mean GI50 (µM)b
Compound
SNB19V
45.6
SNB19M
526
U373V
72.9
U373M
395
1 (TMZ)
2 (MTZ)
10.0
68.4
22.0
40.3
16

Page 17 of 36
MedChemComm
View Article Online
DOI: 10.1039/C6MD00384B
3 (MTIC)
8
48.5
46.2
34.6
428
466
42.1
93.7
398
ꢀ
ꢀ
31.0
>500
223
29.3
306
24.4
360
149
199
36.1
33.0
30.7
46.4
55.6
143
140
72.3
263
92.5
132
355
33.1
28.8
39.4
28.9
56.5
14.4
7.5
10a
10f
286
386
10k
10l
258
239
141
35.6
39.6
39.1
47.5
52.4
367
37.8
37.6
39.5
49.0
61.1
255
37.6
36.5
30.3
32.1
63.2
129
10m
10n
10o
11a
11b
11f
265
296
69.5
65.4
260
11g
11i
38.0
65.8
67.7
62.3
321
95.3
246
11j
73.4
102
85.1
243
11k
11l
398
138
11m
12a
12b
12c
12d
12e
12f
30.8
52.2
48.3
45.0
107
37.9
48.5
40.4
28.0
151
26.0
16.5
11.8
13.2
59.0
13.7
20.2
26.8
13.8
20.1
18.3
12g
17

MedChemComm
Page 18 of 36
View Article Online
DOI: 10.1039/C6MD00384B
204
417
288
322
12i
>500
>500
68.8
68.8
441
>500
>500
131
>500
>500
209
>500
>500
423
13a
13b
13c
13d
13e
13f
13g
13h
13i
70.3
>500
>500
33.9
271
205
248
383
336
>500
39.1
111
>500
32.0
106
>500
35.6
242
47.7
33.6
49.7
37.4
32.6
30.3
46.4
23.4
13j
a SNB19V, U373V vector controls; SNB19M, U373M MGMTꢀtransfected. b Results
are means of ≥ 3 experiments (n = 4 per experiment). Where mean values < 500 μM,
SEM values ranged between 3.08% and 21.9% of given mean values.
Despite the fact that the ringꢀopened 1ꢀalkylꢀ3ꢀ(4ꢀcarbamoylimidazolꢀ5ꢀyl)triazene series of
compounds (13) were less stable than their bicyclic imidazotetrazine counterparts, analysis of
the inhibitory activities of triazenes tentatively supported the hypothesis that the established
chemical activation pathway 1→3 to methyldiazonium ion (4), previously thoroughly
established for TMZ (Figure 1),6 is also operating for the new triazenes. Significantly, MTIC
(3) shows an almost identical profile across the V (sensitive) and M (resistant) cell lines to
that of TMZ (Table 2); moreover, where direct comparisons between compounds were
possible, potencies of new triazenes (13) usually paralleled those of the imidazotetrazines
thereby validating the potential of the screening panel to identify compounds with
discriminating activities depending on the MGMT repair status of the cell lines. Thus
triazenes (13a, 13b) with small lipophilic cyclopropyl and cyclopropylmethyl groups were of
18

Page 19 of 36
MedChemComm
View Article Online
DOI: 10.1039/C6MD00384B
low potency across the screening panel, reproducing the behaviour of the corresponding
cyclic forms (10f, 10k); and triazenes substituted with hydroxyethyl (13e) and methoxyethyl
(13f) groups had low potency across the cell panel. Significantly, the propargylꢀsubstituted
triazene (13g), like the corresponding imidazotetrazine (10m), had activity superior to TMZ
irrespective of the V or M status of the glioma cell lines; replacement of the alkyne group of
13g with a nitrile functionality in the acetonitrile (13h) had a profound dyschemotherapeutic
effect.
Of notable significance were the biological profiles of the two glycine esters (13i, 13j) in the
triazene series which exactly mirrored the activities of the imidazotetrazine glycine esters
(11a, 11b). This observation is fully consistent with the hypothesis that the new
imidazotetrazines require ringꢀopening before exerting their inhibitory activities. We propose
that hydrolytic ringꢀopening of 11b first generates the corresponding triazene (13k). Our
earlier predictions on tautomeric preferences in monoalkyltriazenes such as MTIC (3)22 and
more extensive NMR studies by Vaughan and his colleagues on aryltriazenes bearing Hꢀ
bonding substituents ortho to the triazene linker,24 predict that the preferred tautomer of 13k
is stabilized by an intramolecular Hꢀbond which then steers the subsequent decomposition
pathway (Scheme 4). Protolytic fragmentation of 13k generates AIC (5) and a reactive
cationic species (14) which (presumably) can alkylate DNA. Because of the strongly acidic
nature of protons flanked by two EWGs as in 14, an alternative scenario involves ionization
of a proton from 14 leading to the formation of the (relatively) stable ethyl diazoacetate (15)
which also has the potential to alkylate DNA by a neutral carbene mechanism. However the
latter compound, which is commercially available, showed no activity against the four glioma
cell lines (GI50 > 800 μM) and is unlikely to be involved in the bioactivity of the
imidazotetrazine (11b) and its ringꢀopened partner, the triazene (13k).The same argument
probably explains the inactivity of the cyanomethyltriazene (13h).
19

MedChemComm
Page 20 of 36
View Article Online
DOI: 10.1039/C6MD00384B
Scheme 4. Decomposition of triazenes bearing glycinate groups.
Although the sulfone (12g) was identified as one of the most potent agents overall in the
glioma cell panel (above), this agent was not selected for further biological evaluation
because of its instability. Instead, the propargylꢀsubstituted imidazotetrazine (10m) and the
corresponding ringꢀopened triazene (13g) were investigated to determine how they compared
with TMZ and MTIC in a broader spectrum of tumor cell lines and, crucially, the specificity
of their interaction with DNA.
In previous work we have shown that variant glioma cell lines SNB19VR and
U373VR, demonstrating acquired resistance to TMZ can be derived from SNB19V and
25
U373V respectively
and used as tools to explore cross resistance between analogs,
including 10m and TMZ. U373VR reꢀexpressed MGMT, but loss of MSH6 (a protein key to
functional MMR) contributed to TMZ tolerance (and resistance) in SNB19VR cells. Whereas
TMZ gave GI50 values of 280.2 ꢀM and 288.8 ꢀM in SNB19VR and U373VR cells
9
respectively, 10m revealed GI50 values < 50 ꢀM against these cells. Thus, resistance to
TMZ is not only conferred by direct repair of O6ꢀMeG by MGMT but, in the absence of
MGMT, tolerance to lesions arises when O6ꢀMeGꢀT mispairs cannot be processed, a
consequence of MMR deficiency. Inherited defects in MMR genes MSH6 and MLH1 are
20

Page 21 of 36
MedChemComm
View Article Online
DOI: 10.1039/C6MD00384B
common in colorectal carcinoma (CRC).26 CRC cell lines HCT116 (hMLH1 mut) and DLD1
(hMSH6 mut), insensitive to TMZ (GI50 values > 500 µM) were > 10ꢀfold more sensitive to
imidazotetrazine (10m). The ability of novel imidazotetrazines to retain activity in MMR
deficient cells may thwart emergence of a hypermutation phenotype conferred by the absence
of MMR and promoted by TMZ treatment of glioma clinically.27, 28 However, it is likely that
(as yet unknown) mechanisms of resistance to these agents might emerge.
In U373VR, HCT116 and DLD1 cells the primary O6ꢀMeG lesion can be repaired by
MGMT, any unrepaired O6ꢀMeG lesions will be tolerated in MMR deficient HCT116, DLD1
and SNB19VR cells. In addition, more efficient repair of Nꢀ7 or Nꢀ3 methylated purines may
occur in SNB19VR cells through enhanced transcription of the BER protein NTHL1. Indeed,
PARP inhibition sensitized SNB19VR cells to TMZ >3ꢀfold,8 whereas PARP inhibition
potentiated the activity of 10m < 2ꢀfold (unpublished).
Our hypothesis underpinning the search for novel biologicallyꢀactive imidazotetrazines
reasons that cytotoxicity is a consequence of DNA alkylation which cannot be repaired by
MGMT. To examine DNA sequence selective alkylation by imidazotetrazine 10m and the
corresponding triazene 13g, Taq polymerase stop assays were performed. Vehicleꢀtreated
DNA showed very few sites of early termination. Compounds 10m and 13g at 100 µM
heavily alkylated DNA at clusters of three and five guanines (Fig. 3A). Alkylation damage
intensity increased with increasing concentrations of agents (1ꢀ1000 µM). Triazene 13g
showed quantitatively greater binding at equimolar doses consistent with the assumption that
the triazene is an activated form of prodrug 10m. These dominant polymerase halt sites at
runs of guanines are also major binding sites for TMZ and the corresponding triazene MTIC.7
The same region of plasmid pBR322 was used in piperidine cleavage assays to confirm
covalent modifications at Nꢀ7 guanine positions (Fig. 3B). The gel reveals the same clusters
21

MedChemComm
Page 22 of 36
View Article Online
DOI: 10.1039/C6MD00384B
of guanines as predominant sites of agentꢀDNA reaction, with runs of contiguous guanines
being the most reactive to analogs (10m) and (13g); DNA alkylation data (pattern, intensity,
Nꢀ7 guanine modification) are comparable to those of TMZ and MTIC.7
Imidazotetrazine 10m was investigated for its ability to crosslink DNA in the linear
pBR322 plasmid. The control, native pBR322 plasmid, migrates as a DNA band
corresponding to double stranded (DS) DNA and the control denatured pBR322 plasmid
migrates as a higher mobility DNA band coresponding to single stranded (SS) DNA. The
DNA crossꢀlinking agent cisplatin served as a positive control. Neither TMZ nor 10m at ≤
1000 ꢀM formed interꢀstrand crossꢀlinks after incubation for 2 h (Fig. 3C), or 40 h (not
shown).
Figure 3.
DNA adducts resulting in formation of DSBs induce rapid phosphorylation of Ser 139
at the carboxy terminus of histone H2AX (γH2AX).29 Qualitative techniques including
Western blot and immunohistochemistry allowed detection of γH2AX following treatment of
GBM cells with TMZ, 10m and 13g. A method to quantify γH2AX by flow cytometry was
subsequently developed. Results following exposure of U373V and U373M cells to 10m and
13g are shown (Fig. 3D). Whereas TMZ and MTIC were able to evoke DNA double strand
breaks in U373V cells only, 10m and the ringꢀopened triazene (13g) enhanced γH2AX
expression in both vector control and MGMTꢀtransfected U373 cells, albeit to a lesser extent
in U373M cells. Nevertheless, compared to DMSO controls, ≥2ꢀfold enhanced γH2AX levels
were evident in U373M cells treated with both compounds for 72 h.
Conclusions
Our primary goal at the outset of this work was to identify novel imidazotetrazines
which, unlike the clinicallyꢀused agent TMZ, display growth inhibitory activities against
22

Page 23 of 36
MedChemComm
View Article Online
DOI: 10.1039/C6MD00384B
glioma cell lines irrespective of their MGMT status. In the case of 3ꢀsubstituted
modifications of TMZ described herein this objective has been realized and several
compounds with desirable attributes are listed in Table 2. The propargylꢀsubstituted
imidazotetrazine (10m), selected as an exemplar compound, shows a ‘flat’ inhibitory profile
with GI50 values at < 40 µM against SNB 19V and M, and U373V and M cell lines: in
contrast the profile of TMZ is ‘spiky’ with both the M cell lines proving to be resistant to the
drug. Significantly, the propargylꢀsubstituted imidazotriazene (13g) shows a strikingly
similar ‘flat’ profile (Table 2) to that of the related imidazotetrazine prodrug (10m) and in
other examples where direct comparisons between imidazotetrazines and their related
triazenes were examined, both species showed parallel inhibitory activities: this implies that
ringꢀopening, like that shown in the TMZ to MTIC transformation,7 is also a requirement for
bioactivity in this new series. We will report detailed stability studies on 10m and 13g in a
future publication.
We have shown previously that, whereas TMZ gave GI50 values of 280.2 ꢁM and
288.8 ꢀM in cells lines with acquired resistance to the drug (SNB19VR and U373VR),
compound 10m showed GI50 values < 50 ꢀM against these cells.25 In CRC cell lines HCT116
(hMLH1 mut) and DLD1 (hMSH6 mut), insensitive to TMZ (IC50 values > 500 µM), were >
10ꢀfold more sensitive to imidazotetrazine (10m). This result provides encouragement that
new imidazotetrazines could be developed which will impede evolution of a hypermutation
phenotype and may possess broader spectrum antitumor activity compared to TMZ.
Taq polymerase stop assays confirmed that 10m and 13g, like TMZ and MTIC,
alkylated plasmid pBR322 DNA at runs of three and five guanines (Figure 3A). Similarly,
piperidine cleavage assays confirmed that both these agents induced covalent modifications
at Nꢀ7 guanine positions of the plasmid DNA (Figure 3B) with runs of contiguous guanines
being the prime targets. As might be expected for a putative monofunctional alkylating agent,
23

MedChemComm
Page 24 of 36
View Article Online
DOI: 10.1039/C6MD00384B
imidazotetrazine 10m, like TMZ, did not crosslink DNA in a linear pBR322 plasmid. That
alkylation/modification of guanine led to DNA DSBs in cells following their exposure to
10m (and ring opened 13g) can be concluded by detection of γH2AX foci. In contrast to
TMZ, 10m was able to evoke DNA double strand breaks in GBM cells irrespective of
MGMT status, therefore overcoming MGMT repair.
Finally, although the screening cascade has identified compounds with intriguing in
vitro properties, this is only the start of the process to select a clinical candidate. Going
forward we are hoping to select a novel imidazotetrazine with desirable ‘temozolomideꢀlike’
physicochemical characteristics and acceptable PK properties, to allow for oral
administration against in vivo glioma xenograft models; these models will include TMZꢀ
sensitive tumors and tumors with intrinsic and acquired resistance to TMZ.
Experimental Section
Chemistry. All compounds were purified by crystallization or flash chromatography
on Merck silica gel 60. In general imidazotetrazines and imidazotriazenes (Table 1)
decomposed vigorously in their melting ranges, which were highly variable depending on the
rate of heating and were not a reliable means to characterise compounds.
Synthetic Method 1. A substituted isocyanate (4.2 mmol) was added dropwise to a
stirred suspension of 4ꢀdiazoimidazoleꢀ5ꢀcarboxamide (7) (0.5 g, 3.65 mmol) in dry DMSO
o
(5 mL) at 25 C under nitrogen. After 24 h the mixture was poured onto ice and the product
was extracted into DCM (3 x 25 mL). The organic fraction was washed with water and dried
(Na2SO4), and solvent removed by vacuum evaporation. The solid residue was triturated with
EtOAc and the imidazotetrazine was collected and dried in vacuo at 25 oC. Selected examples
are the following: 8ꢀCarbamoylꢀ3ꢀ(iꢀpropyl)imidazo[5,1ꢀd]ꢀ1,2,3,5ꢀtetrazinꢀ4(3H)ꢀone
(10e) prepared from 4ꢀdiazoimidazoleꢀ5ꢀcarboxamide (7) and isopropyl isocyanate was
24

Page 25 of 36
MedChemComm
View Article Online
DOI: 10.1039/C6MD00384B
1
isolated as a beige solid (29%); LCMS (ES+) m/z 223 (M+H)+; H NMR (400 MHz, δ6ꢀ
DMSO) d 8.77 (1H, s, Hꢀ6), 7.77 (2H, brs, 2 x NH), 5.03 (1H, m, CH), 1.49 (6H, d, 2 x CH3);
8ꢀCarbamoylꢀ3ꢀpropargylimidazo[5,1ꢀd]ꢀ1,2,3,5ꢀtetrazinꢀ4(3H)ꢀone (10m) from 7 and
1
propargyl isocyanate was isolated (3%) as a white solid; LCMS (ES+) m/z 219 (M+H)+; H
NMR (400 MHz, δ6ꢀDMSO) d 8.86 (1H, s, Hꢀ6), 7.82 (1H, brs, NH), 7.70 (1H, brs, NH),
5.14 (2H, d, CH2), 3.52 (1H, t, CH). The product was identical to the same imidazotetrazine
(30%) prepared from norꢀtemozolomide (9) and propargyl bromide.18 Other imidazotetrazines
prepared by this method are listed in Table 1 with full spectroscopic characteristics (LCMS
and 1H NMR) detailed in the patent literature.23
Synthetic Method 2. Details of the synthesis of selected 3ꢀalkyl imidazotetrazines
prepared either (a) from the direct alkylation of norꢀtemozolomide (9) (for compounds 10b,
10m, 12c), or (b) (for compounds 10m, 11a, 11b) following the in situ generation of 9 from
the precursor 3ꢀhydroxymethyl imidazotetrazine (8) have been described in recent work.18
Other compounds prepared from 8 by this method are listed in Table 1. Full spectroscopic
characteristics (LCMS and 1H NMR) of compounds are detailed in the patent literature.23
8ꢀCarbamoylꢀ3ꢀ(butꢀ2ꢀynyl)imidazo[5,1ꢀd]ꢀ1,2,3,5ꢀtetrazinꢀ4(3H)ꢀone (10n). To a
o
solution of 8 in acetonitrile (0.17 M concentration of substrate in solvent) at 0 C was added
1ꢀbromoꢀ2ꢀbutyne (4 mol. equiv.) and DBU (1.4 mol. equiv.) and the mixture was stirred for
0.5 h. The mixture was acidified (1 M HCl) and products extracted into EtOAc. Combined
organic extracts were dried (MgSO4), filtered and concentrated in vacuo. The oily residue
was triturated with Et2O to furnish 10n (28%); 1H NMR (400 MHz, δ6ꢀDMSO) d 8.84 (1H, s,
Hꢀ6), 7.82 (1H, brs, NH), 7.70 (1H, brs, NH), 5.08 (2H, q, J = 2.4 Hz, CH2), 1.84 (3H, t, J =
2.4 Hz, CH3).
25

MedChemComm
Page 26 of 36
View Article Online
DOI: 10.1039/C6MD00384B
8ꢀCarbamoylꢀ3ꢀ(trimethylsilylpropꢀ2ꢀynyl)imidazo[5,1ꢀd]ꢀ1,2,3,5ꢀtetrazinꢀ4(3H)ꢀ
one (10o). Similarly prepared as above (11%) from 8 and 3ꢀbromoꢀ1ꢀ(trimethylsilyl)ꢀ1ꢀ
1
propyne; H NMR (400 MHz, δ6ꢀDMSO) d 8.87 (1H, s, Hꢀ6), 7.86 (1H, brs, NH), 7.72 (1H,
brs, NH), 5.18 (2H, s, CH2), 0.16 (9H, s, 3 x CH3).
Synthetic Method 3. Typical conditions were the following: a solution of iꢀpropyl
chloroformate (1M in toluene; 1.55 mL, 1.155 mmol) and Nꢀmethylmorpholine (0.127 mL,
1.55 mmol) were added to a stirred solution of either the imidazotetrazine acetic acid (11g) or
o
propionic acid (11h) (1.05 mmol) in DMF (1.34 mL) under nitrogen at ꢀ10 to ꢀ15 C. To the
stirred mixture was added either ammonia (0.5 M solution in 1,4ꢀdioxane; 0.396 mmol) and
triethylamine (0.198 mmol) (for compounds 11i or 11m), or the appropriate amine (for
compounds 11jꢀ11l). The reaction mixture was stirred (1 h) and allowed to warm to 25 oC (24
h). Et2O (5 mL) was added and the resulting precipitate was washed with acetonitrile and
DCM and finally more Et2O. The white product was purified by flash chromatography using
acetonitrile/DCM (9:1) as eluting solvent. A specific example is the following: 8ꢀ
Carbamoylꢀ3ꢀ(carbamoyl)methylimidazo[5,1ꢀd]ꢀ1,2,3,5ꢀtetrazinꢀ4(3H)ꢀone (11i) prepared
from imidazotetrazine (11g) and ammonia was isolated as a white solid (45%); LCMS (ES+)
1
m/z 238 (M+H)+ ; H NMR (400 MHz, δ6ꢀDMSO) δ 8.88 (1H, s, Hꢀ6), 7.84 (1H, brs, NH),
7.72 (1H, brs, NH), 7.70 (1H, brs, NH), 7.44 (1H, brs, NH), 4.88 (2H, s, CH2). Other
imidazotetrazines (11jꢀ11m) prepared by this Method are listed in Table 1. Full spectroscopic
characteristics (LCMS and 1H NMR) of compounds are detailed in the patent literature.23
Synthetic Method 4. A mixture of 4ꢀdiazoimidazoꢀ5ꢀcarboxamide (7) and the
o
appropriate amine (1 mol. equiv.) were stirred in dry EtOAc at 25 C for 4 h. The
imidazotriazenes (13aꢀh) were collected and washed with dry EtOAc. Compounds prepared
by this method are listed in Table 1 (with yields). 13i and 13j were prepared from the
hydrochloride salts of methyl and ethyl glycinate, respectively, in the presence of
26

Page 27 of 36
MedChemComm
View Article Online
DOI: 10.1039/C6MD00384B
triethylamine (1 mol. equiv.). (Note: imidazotriazenes are unstable in polar solvents and were
o
stored at 0ꢀ4 C. Their melting points vary according to the rate of heating and are often
accompanied by vigorous decomposition).
3ꢀ(8ꢀCarbamoylꢀ3,4ꢀdihydroꢀ4ꢀoxoimidazo[5,1ꢀd]ꢀ1,2,3,5ꢀtetrazinꢀ3ꢀyl)propanoic
acid (11h). A suspension of the ester (11f) 0.5 g, 1.784 mmol) in 5 M hydrochloric acid (2.4
mL) was heated at 45 oC for 16 h. The product was collected, washed with water followed by
acetone, and crystallized from an acetonitrile/water mixture to furnish the propanoic acid
(47%) as an offꢀwhite solid; LCMS (ES+) m/z 281 (M+H)+ ; 1H NMR (400 MHz, δ6ꢀDMSO)
δ 12.5 (1H, s, OH), 8.80 (1H, s, Hꢀ6), 7.75 (1H, brs, NH), 7.65 (1H, brs, NH), 4.45 (2H, t,
CH2), 2.75 (2H, t, CH2).
8ꢀCarbamoylꢀ3ꢀ(methylsulfinylmethyl)imidazo[5,1ꢀd]ꢀ1,2,3,5ꢀtetrazinꢀ4(3H)ꢀone
(12f). (i) The 3ꢀ(methylthiomethyl)imidazotetrazine (12e) prepared according to Synthetic
Method 1 (0.05 g) in DCM/MeOH (1:1 mixture, 5 mL) was cooled to ꢀ78 oC and ozone was
gently bubbled through the solution until saturated. The mixture was gassed with nitrogen,
allowed to warm to 25 oC and stirred with excess dimethyl sulphide for 0.5 h. The precipitate
was collected, washed with Et2O and furnished the pure title compound (98%) as a white
1
solid; LCMS (ES+) m/z 257 (M+H)+; H NMR (400 MHz, δ6ꢀDMSO) δ 8.92 (1H, s, Hꢀ6),
7.89 (1H, brs, NH), 7.74 (1H, brs, NH), 5.59 (1H, d, J = 13.2 Hz), 5.47 (1H, d, J = 13.2 Hz),
2.77 (3H, s, CH3). (ii) Oxidation of 12e (0.17 mmol) in acetonitrile (1 mL) containing FeCl3
(0.005 mmol) with periodic acid (0.18 mmol) at 25 oC for 1.5 h gave the same product (45%).
3ꢀ{2ꢀ(tꢀButyldimethylsilyloxy)ethyl}ꢀ8ꢀcarbamoylimidazo[5,1ꢀd]ꢀ1,2,3,5ꢀtetrazinꢀ
4(3H)ꢀone (12h). Triphosgene (1.675 g, 5.65 mmol.) was added portionwise to a stirred
mixture of 2ꢀ(tꢀbutyldimethylsilyloxy)ethylamine (3.0 g, 17.11 mmol.) in DCM(25 mL) and
saturated sodium hydrogen carbonate solution (25 mL) at 0 oC. After 1.5 h the organic layer
27

MedChemComm
Page 28 of 36
View Article Online
DOI: 10.1039/C6MD00384B
was washed with brine, dried (MgSO4) and concentrated in vacuo to give an oil which was
purified by vacuum distillation. 2ꢀ(tꢀButyldimethylsilyloxy)ethyl isocyanate distilled at 124
oC as a colorless oil which was used without further purification. The crude isocyanate was
reacted with 4ꢀdiazoimidazoleꢀ5ꢀcarboxamide (7) according to the conditions of Synthetic
1
Method 1 to afford 12i as a white solid 7%; LCMS (ES+) m/z 339 (M+H)+; H NMR (400
MHz, δ6ꢀDMSO) δ 8.87 (1H, s, Hꢀ6), 7.83 (1H, brs, NH), 7.70 (1H, brs, NH), 4.41 (2H, t,
CH2), 3.98 (2H, t, CH2), 0.78 (9H, s, 3 x CH3), ꢀ0.03 (6H, s, 2 x CH3).
8ꢀCarbamoylꢀ3ꢀ(2ꢀhydroxyethyl)imidazo[5,1ꢀd]ꢀ1,2,3,5ꢀtetrazinꢀ4(3H)ꢀone (12i).
A solution of 12h (0.05 g, 0.148mmol.) in THF:AcOH:H2O (5 mL of a 1:1:1 mixture) was
o
stirred a 25 C. After 3 h the reaction mixture was concentrated in vacuo and the residue
purified by flash column chromatography (SiO2, gradient 0ꢀ100% acetonitrile in DCM) to
1
furnish the 12h as a white solid (45%); LCMS (ES+) m/z 225 (M+H)+; H NMR (400 MHz,
δ6ꢀDMSO) δ 8.84 (1H, s, Hꢀ6), 7.81 (1H, brs, NH), 7.68 (1H, brs, NH), 4.85 (1H, t, OH),
4.34 (2H, t, CH2), 3.79 (2H, q, CH2O).
Biology. Stock solutions of TMZ and analogs were prepared (100 mM in DMSO) and
aliquots stored at ꢀ20 oC, protected from light. SNB19V, SNB19M, U373V and U373M cells
were gifts from ScheringꢀPlough Research Institute; HCT116 carcinoma cell lines were
purchased from the European Cell Culture Collection (ECCC, UK); DLD1 cells were a gift
from Dr. Srinivasan Madhusudan, University of Nottingham, U.K. Cells were maintained in
RPMI 1640 nutrient medium supplemented with 10% fetal bovine serum (FBS),
o
subcultivated twice weekly and incubated at 37 C in an atmosphere containing 5% CO2.
SNB19VR and U373VR, variant cell lines derived from SNB19V and U373V respectively8,25
were routinely maintained in medium additionally supplemented with 100 µM TMZ. All cell
lines were verified as being mycoplasma free.
28

Page 29 of 36
MedChemComm
View Article Online
DOI: 10.1039/C6MD00384B
Antiꢀproliferative assays. 3ꢀ(4,5ꢀDimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenylꢀtetrazolium bromide
(MTT) assays were performed as reported previously.8,25 Compound concentrations (µM)
required to inhibit 50 % cell growth (GI50) were calculated using nonꢀlinear regression
analysis.
TAQ polymerase Stop Assay. The sequence specificity of covalent DNA
modification by 1 (TMZ), 10m, 13g on the top strand of pBR322 plasmid was identified by
Taq polymerase stop assays.13,30 Linearized plasmid pBR322 DNA (0.5ꢀg) was treated with
compounds at concentrations 1, 10, 100 and 1000 ꢀM for 2 h at 37 oC and amplified with the
primer 5′ꢀTATGCꢀGACTCCTGCATTAGGꢀ3′, which binds to the top strand pBR322
sequence region 622ꢀ641. The primer was 5′ꢀend labeled with [γ32P] ATP using T4
polynucleotide kinase. After primer extension, samples were precipitated with ethanol and
sodium acetate, washed (70% ethanol) and lyophilized. Samples were resuspended in 6 ꢀL
formamide loading buffer, separated by denaturing polyacrylamide gel electrophoresis and
visualized by autoradiography.
Piperidine Cleavage Assay. A modification of the Maxam and Gilbert sequencing
technique was used to probe Nꢀ7 alkylation on the same region of plasmid pBR322 as
examined in the Taq polymerase assay.31 The 209ꢀbp probe subjected to modification was
amplified by PCR and defined by the top primer used in the Taq polymerase stop assay and a
second primer, 5′ꢀGCGCꢀCTATATCGCCGACATCꢀ3′, which binds to the bottom strand of
the plasmid at positions 435ꢀ452. Experimental conditions employed were as described by
Hartley et al.30 After amplification, the resulting PCR product was detected by 1% TAE
agarose gel electrophoresis, isolated and purified using the Bio101 GENECLEAN П Kit
according to the manufacturer’s protocol. Following precipitation and lyophilization, dried
DNA pellets were treated with 10% piperidine (100 ꢀL) at 90 oC for 15 min to produce strand
29