Antitumour imidazotetrazines. Part 39. Synthesis of bis(imidazotetrazine)s with saturated spacer groups

Article abstract of DOI:10.1039/b005652i

Bis(imidazotetrazine)s (16), related in structure to the antitumour agents mitozolomide (1a) and tempzolomide (1b), but linked through the N(3)-N(3′) atoms of the imidazo[5, 1-d][1,2,3,5]tetrazine ring-systems, are prepared by interaction of 5-diazoimidazole-4-carboxamide (8) and diisocyanates (15). The presence of the polymethylene linker with/without sulfur and oxygen heteroatoms does not substantially affect the acid stability, base-catalysed decomposition, antitumour activity or DNA base alkylation preference characteristic of the unlinked imidazotetrazines mitozolomide and temozolomide., The Royal Society of Chemistry 2000.

Full text of DOI:10.1039/b005652i

View Article Online / Journal Homepage / Table of Contents for this issue
1
Antitumour imidazotetrazines. Part 39. Synthesis of
bis(imidazotetrazine)s with saturated spacer groups
Jill Arrowsmith, Sharon A. Jennings, David A. F. Langnel, Richard T. Wheelhouse† and
Malcolm F. G. Stevens*
1
Cancer Research Laboratories, School of Pharmaceutical Sciences, University of Nottingham,
University Park, Nottingham, UK NG7 2RD. E-mail: malcolm.stevens@nottingham.ac.uk
Received (in Cambridge, UK) 13th July 2000, Accepted 25th September 2000
First published as an Advance Article on the web 9th November 2000
Bis(imidazotetrazine)s (16), related in structure to the antitumour agents mitozolomide (1a) and temozolomide (1b),
but linked through the N(3)–N(3Ј) atoms of the imidazo[5,1-d][1,2,3,5]tetrazine ring-systems, are prepared by inter-
action of 5-diazoimidazole-4-carboxamide (8) and diisocyanates (15). The presence of the polymethylene linker with/
without sulfur and oxygen heteroatoms does not substantially affect the acid stability, base-catalysed decomposition,
antitumour activity or DNA base alkylation preference characteristic of the unlinked imidazotetrazines
mitozolomide and temozolomide.
Introduction
In recent papers we have reported on the outcome of efforts
to target the imidazotetrazine moiety to specific DNA
sequences. Thus, mitozolomide and temozolomide have been
conjugated through the 8-carboxamide group to H-bonding
Of the N(3) substituted family of 8-carbamoylimidazo[5,1-d]-
[
1,2,3,5]tetrazin-4(3H)-ones only mitozolomide (1a) and
temozolomide (1b) have pronounced antitumour activities
7
motifs and spermidine and peptidic DNA minor and major
2,3
in vivo. Mitozolomide achieved chemotherapeutic notoriety
10
groove-recognition moieties. Sadly, these initiatives have not
in the 1980s. Although in a single dose it could elicit cures
in most established mouse tumours, sadly, in human trials it
produced profound, life-threatening, bone marrow suppression
led to any significant biological breakthroughs. For example,
although conjugation of mitozolomide to the A–T selective
DNA minor groove-binding octapeptide SPKKSPKK did
enhance the potency of DNA interaction compared with that
of the unconjugated drug, covalent alkylation still occurred
2
4
(
thrombocytopenia). In contrast, the second-generation agent
temozolomide, although less active in mouse tumours, can be
used safely on an out-patients basis to treat human malignant
10
in the major groove. We have been able to rationalise these
5
brain tumours for which use it is now approved and marketed
setbacks from our understanding of the molecular mechanism
throughout the world. The 3-ethyl analogue (1c) is inactive in
all mouse tumour models.
6
of action of temozolomide. By analogy, the chemistry of
3
breakdown of the temozolomide conjugates 2 probably
involves nucleophilic attack by water at C(4) of the bicycle with
ring-opening and loss of a molecule of carbon dioxide. The
acyclic methyltriazene 3 thus formed then undergoes proteo-
lytic fragmentation to afford the (conjugated) 5-amino-
imidazole-4-carboxamide 4 with detachment of the reactive
methanediazonium species 5 which methylates guanine residues
6
of DNA 6 to generate the cytotoxic O -methylguanine lesion 7
(
Scheme 1). Irrespective of the binding preference imposed
by the targeting moiety, the short-lived methanediazonium
The orally active temozolomide delivers a methylating
reagent, methanediazonium ion, to nucleophilic sites on DNA.
The cytotoxicity of temozolomide is principally associated
with methylation at the O(6) position of guanine residues in the
11
species 5 (pseudo-first order rate constant for hydrolysis = 1.8
Ϫ1
12
s
in unbuffered aqueous THF) has time to diffuse to, and
methylate, preferred nucleophilic sites within runs of guanine
bases in the major groove. This process thwarts efforts to
utilise the carboxamide moiety to target the imidazotetrazine
methylating component to specific gene sequences.
In an alternative strategy to broaden the clinical utility of
imidazotetrazines we have now synthesised bis(imidazotetra-
zine)s linked by saturated spacer groups. We anticipated that
these new agents might forge DNA crosslinks which might not
be reversed by known DNA repair mechanisms.
6,7
major groove of DNA and is dependent on the mismatch
repair pathway (MMR). This lesion is subjected to repair by
6
O -methylguanine-DNA alkyltransferase (ATase) and, there-
fore, the susceptibility of a tumour to temozolomide depends
8
on the relative expression of MMR and ATase in a cell. Further-
more, other products of promiscuous DNA methylation, which
give rise to strandbreaks, are identified and tagged for repair
9
by poly(ADP-ribose)polymerase (PARP). Modulation of these
clinically compromising repair functions by specific inhibitors is
being studied in an effort to extend the spectrum of susceptible
tumour types.
Results and discussion
The most practicable route to 3-substituted imidazotetrazines
is from the interaction of 5-diazoimidazole-4-carboxamide
2
(
8) and isocyanates (9) (Scheme 2). In the present work we
†
Present address: Cancer Research Group, School of Pharmacy,
University of Bradford, UK BD7 1DP.
have prepared some previously unreported N(3)-substituted
4
432
J. Chem. Soc., Perkin Trans. 1, 2000, 4432–4438
DOI: 10.1039/b005652i
This journal is © The Royal Society of Chemistry 2000

View Article Online
Scheme 2
Scheme 1
Difunctional acids or esters 12, depending on availability,
were used as starting materials for the synthesis of a range
of novel bis(imidazotetrazine)s. Esters were converted to acid
hydrazides 13 and thence, by nitrosation, to acid azides 14. The
imidazotetrazines (1d–g) bearing a one-carbon fragment at
N(3), which are potential reagents for coupling to an unsub-
stituted imidazotetrazinone (1h) to form the prototypic
bis(imidazotetrazine) 10. The chloromethyl isocyanate (9;
18
Curtius rearrangement of succinyl azide (14a) in a mixture of
refluxing chloroform–benzene was used safely to furnish the
diisocyanate 15a. The propensity of the diisocyanate to under-
go polymerisation dictated that crude material obtained from
the evaporated mixture was reacted without further purifi-
cation; reaction with diazoimidazolecarboxamide 8 in DMSO
at 25 ЊC afforded the ethylene-linked bis(imidazotetrazine) 16a
in poor yield (25%). Similarly prepared, from commercially
available diisocyanates (15b–e), were the polymethylene-linked
bis(imidazotetrazine)s (16b–e) in 60–97% yields (Scheme 3).
The contrast in yields between 16a and 16b–e probably reflects
the higher purity of the commercially available diisocyanates
(15b–e), their ease of reactivity with 8 or the facility with which
the products precipitated from the reaction mixture. For
example, formation of 16a took 28 days to reach completion,
whereas synthesis of 16b–e was complete in 12 hours. It is
our experience that long reaction times inevitably lead to by-
product formation [e.g. cyclisation of 8 to azahypoxanthine
R = CH Cl), cyclised with 8 in DMSO or HMPA to give the
2
imidazotetrazine 1d. Similarly, 8 and methoxymethyl isocyanate
(
9; R = CH OMe), ethoxymethyl isocyanate (9; R = CH OEt)
2
2
and isocyanatomethyl 4-chlorophenyl sulfide (9; R = CH SC -
2
6
H Cl-p) afforded the imidazotetrazines (1e–g), respectively. In
4
contrast, the sterically hindered tert-butyl and trichloromethyl
isocyanates failed to produce the respective imidazotetrazines.
An obvious route to the bis(imidazotetrazine) 10 would
involve interaction of the unsubstituted imidazotetrazin-4(3H)-
13
one (1h) with formaldehyde (Scheme 2). The reported
synthesis of 1h from diazoimidazolecarboxamide 8 and the
sterically challenged trimethylsilyl isocyanate (9; R = TMS) has
proven to be impossible to reproduce on a preparative scale and
under a range of conditions only recovered starting materials
or azahypoxanthine (11), the product of slow intramolecular
cyclisation of 8, were isolated. Other independent synthetic
strategies to obtain the elusive imidazotetrazinone 1h have not
14
19
been successful.
(11)].
15
Ege et al. have shown that N(3)-linked bis(pyrazolotetra-
zine)s can be prepared from diisocyanates and diazopyrazoles
and to adapt this method to synthesise the bis(imidazotetra-
The diisocyanates (15f–l) bearing additional hetero atoms
in the linker fragment were obtained as oils in yield of 50–77%
by Curtius rearrangements of the precursor acid azides (14f–l)
and were reacted directly with 8 in DMSO to furnish the
bis(imidazotetrazine)s (16f–l) in moderate to good yields
(Table 1).
zine) 10 would require diisocyanatomethane (9; R = CH NCO)
2
available from the potentially hazardous rearrangement of
malonyl diazide. We did not consider it prudent to attempt this
reaction even though it was apparently performed safely by the
The crude bis(imidazotetrazine)s (16a–l) always contained
16
Ϫ1
legendary Curtius himself. In an alternative approach, the
contaminants absorbing in the 1600–1700 cm region of the
known ester 1i, prepared from 8 and ethyl isocyanatoacetate
IR spectrum—possibly triazine-1,3,5-triones formed by tri-
merisation of the isocyanates. No suitable crystallisation
solvent was found to remove these contaminants but a brief
washing of the bis(imidazotetrazine) with petroleum–ether
followed by formic acid proved to be an effective process to
furnish pure compounds; these were characterised by FAB MS,
(
9; R = CH CO Et), was hydrolysed to the acetic acid deriva-
2 2
17
tive 1j. A logical sequence from 1j to the isocyanatomethyl-
imidazotetrazine 1l would be the following: conversion of 1j
to the acid azide 1k; Curtius rearrangement of the azide to 1l;
and finally, construction of the second imidazotetrazine ring by
interaction of 1l with a second tranche of diazoimidazolecarb-
oxamide 8. However this strategy foundered on our inability to
secure a reliable and safe synthesis of the acid azide 1k using
a range of conditions. We therefore abandoned our efforts to
synthesise the methylene-linked bis(imidazotetrazine) 10.
1
13
H and C NMR, and in some cases C, H, N microanalyses.
1
The H NMR spectrum of the hexamethylene-linked imidazo-
tetrazine 16c clearly shows that the structure is symmetrical
about the 3,4-hexamethylene bond.
The availability of the sulfide-linked imidazotetrazine 16f
J. Chem. Soc., Perkin Trans. 1, 2000, 4432–4438
4433

Table 1 Yields and physical characteristics of bis(imidazotetrazine)s 16
Microanalysis found (required)
Com-
pound
Yield
(%)
a
b
1
c
13
d
Mp/ЊC
Formula
MW
C
H
N
H NMR
C NMR
1
b
c
6a
e
165–167
155–158
167–169
C H N O
386
414
442
37.31 (37.30)
40.57 (40.58)
43.54 (43.44)
2.61 (2.59)
3.41 (3.80)
4.10 (4.07)
43.49 (43.52)
40.57 (40.58)
37.96 (38.00)
8.86 (2 H, s, 6-H, 6Ј-H), 7.88 (2 H, s, 2 × NH), 7.72 (2 H, s, 2 × NH),
.78 (4 H, s, 2 × CH2)
8.81 (2 H, s, 6-H, 6Ј-H), 7.86 (2 H, s, 2 × NH), 7.70 (2 H, s, 2 × NH),
.34 (4 H, br, 2 × CH ), 1.94 (4 H, br s, 2 × CH )
—
1
2
10 12
4
4
4
4
82
42
C H N O
162.4, 139.9, 135.3, 131.4,
129.5, 49.2, 26.0
161.7, 139.2, 134.7, 130.7,
128.8, 48.9, 28.2, 25.6
1
4
14 12
4
2
2
C H N O
8.81 (2 H, s, 6-H, 6Ј-H), 7.86 (2 H, s, 2 × NH), 7.70 (2 H, s, 2 × NH),
1
6
18 12
4
.28 (4 H, t, J 7.0, 2 × CH ), 1.18 (4 H, br, 2 × CH ), 1.43 (4 H, br,
2
2
2
× CH2)
d
e
44
60
160
165
C H N O
470
526
45.55 (45.96)
49.85 (50.19)
4.71 (4.68)
5.74 (5.70)
35.70 (35.74)
31.72 (31.94)
8.80 (2 H, s, 6-H, 6Ј-H), 7.85 (2 H, s, 2 × NH), 7.68 (2 H, s, 2 × NH),
—
1
8
22 12
4
4
4
.30 (4 H, t, J 7.5, 2 × CH ), 1.83 (4 H, br, 2 × CH ), 1.27 (8 H, br,
2
2
4
× CH2)
C H N O
8.67 (2 H, s, 6-H, 6Ј-H), 7.85 (2 H, s, 2 × NH), 7.65 (2 H, s, 2 × NH),
—
2
2
30 12
4
.15 (4 H, br, 2 × CH ), 1.62 (4 H, br, 2 × CH ), 1.17 (16 H, br,
2
2
8
× CH2)
f
79
33
63
24
50
47
130–132
168–170
160–162
144
C H N O S
418
402
446
34.05 (34.45)
35.66 (35.82)
37.57 (37.67)
—
2.64 (2.41)
2.21 (2.49)
3.19 (3.16)
—
—
—
—
—
—
—
8.89 (2 H, s, 6-H, 6Ј-H), 7.84 (2 H, br, 2 × NH), 7.27 (2 H, br,
× NH), 5.65 (4 H, s, 2 × CH2)
8.94 (2 H, s, 6-H, 6Ј-H), 7.89 (2 H, br, 2 × NH), 7.75 (2 H, s,
× NH), 5.88 (4 H, s, 2 × CH2)
8.87 (2 H, s, 6-H, 6Ј-H), 7.86 (2 H, br, 2 × NH), 7.73 (2 H, br,
× NH), 5.65 (4 H, s, 2 × CH ), 3.79 (4 H, s, 2 × CH )
161.3, 138.7, 134.2, 131.2,
129.2, 50.0
161.7, 139.7, 134.4, 131.9,
130.0, 76.8
161.9, 139.9, 134.7, 131.9,
130.0, 78.5, 68.9
162.3, 139.5, 135.1, 130.1, 50.8,
31.6
1
2
10 12
4
5
6
2
g
h
i
C H N O
1
2
10 12
2
C H N O
1
4
14 12
2
2
2
f
C H N O S
478
8.87 (2 H, s, 6-H, 6Ј-H), 7.88 (2 H, br, 2 × NH), 7.72 (2 H, br,
× NH), 5.51 (4 H, s, 2 × CH ), 3.03 (4 H, s, 2 × CH )
1
4
14 12
4
2
2
2
2
2
j
164–166
141
C H N O S
446
37.53 (37.67)
—
3.05 (3.16)
—
8.89 (2 H, s, 6-H, 6Ј-H), 7.89 (2 H, br, 2 × NH), 7.76 (2 H, br,
× NH), 4.57 (4 H, t, J 6.8, 2 × CH ), 3.12 (4 H, t, J 6.8, 2 × CH )
162.0, 139.5, 134.8, 131.4,
129.4, 48.4, 29.6
—
1
4
14 12
4
2
2
2
g
k
C H N O S
492
8.85 (2 H, s, 6-H, 6Ј-H), 7.83 (2 H, br, 2 × NH), 7.70 (2 H, br,
1
5
16 12
4
2
× NH), 4.53 (4 H, t, J 6.0, 2 × CH ), 4.00 (2 H, s, CH ), 3.07 (4 H,
2
2
t, J 6.0, 2 × CH2)
l
86
146–148
C H N O S
4 2
478
34.90 (35.15)
2.67 (2.93)
—
8.86 (2 H, s, 6-H, 6Ј-H), 7.86 (2 H, br, 2 × NH), 7.73 (2 H, br,
× NH), 4.62 (4 H, t, J 6.4, 2 × CH ), 3.24 (4 H, t, J 6.4, 2 × CH )
161.9, 139.6, 134.7, 131.4,
129.4, 48.2, 35.7
1
4
14
2
2
2
2
a
f
b
c
d
e
All compounds melted with vigorous decomposition. Determined by FAB MS. Spectra recorded at 250 MHz in (CD ) SO. Spectra recorded at 62.9 MHz in (CD ) SO. Purity of starting isocyanate not known.
3
2
3 2
ϩ
g
ϩ
FAB MS m/z 479.0781. C H N O S requires m/z 479.0777 (M ϩ H ). FAB MS m/z 493.0937. C H N O S requires m/z 493.0929 (M ϩ H ).
1
4
14 12
4
15 16 12
4 2

View Article Online
Table 2 Decomposition of imidazotetrazines and bis(imidazotetra-
zine)s at pH 7.4 in phosphate buffer
₁
t₂/h

Analytical
Compound
25 ЊC
37 ЊC
wavelength/nm
1
1
6c
6d
6l
a
b
4.41
—
—
—
5.07
1.03
1.30
2.77
3.27
1.28
328
328
331
331
331
1
1
1
Scheme 3
offered the prospect of developing a new reductive desulfuris-
ation route to temozolomide (1b). Firstly, the sulfide was heated
with ethanol-washed Raney nickel in refluxing trifluoroethanol,
but no temozolomide was detected, despite the fact that the
target molecule is stable under the reaction conditions. Back
20
and Yang have used nickel boride, prepared by the reduction
2ϩ
of Ni salts with sodium borohydride, to effect efficient desul-
furisation of sulfide substrates. Again, no temozolomide was
formed using this reagent and only tetrazinone ring-opening
was observed.
Fig. 1 Decomposition (absorbance values versus wavelength in nm)
of imidazotetrazines in phosphate buffer at pH 7.4: A, temozolomide
(
1b); B, the bis(imidazotetrazine) (16c). Spectra were recorded at
The acid stability of the imidazotetrazine nucleus of 1a–c is
intervals of 15 min for A; 30 min for B.
2,6
a characteristic property of this class of agent. The stabilities
of representative bis(imidazotetrazine)s in phosphate buffer
6
were compared by a UV spectrophotometric method. At pH
5
.0 there was no measurable decomposition of any compound
(
data not shown). The t₂ values for mitozolomide (1a) and
₁

temozolomide (1b) at pH 7.4 and 37 ЊC were 1.03 and 1.30 h,
respectively (Table 2). Lengthening the linker from a hexa- (16c)
to an octa-methylene unit (16d) had little effect on t₂ (2.77 and
₁

3
.27 h, respectively); compound 16l with a disulfide residue
attached to the ethylene group at N(3) was more unstable with a
t
₁
(1.28 h) comparable to that of mitozolomide.
₂

Scrutiny of the UV curves reveals underlying differences in
the decomposition mechanisms depending on the nature of the
N(3)-attached group. The overall UV curves for temozolomide
(
(
1b) and the hexamethylene-linked bis(imidazotetrazine) (16c)
Fig. 1A and B) are nearly identical, with the decay of the
bands at 328 and 331 nm, respectively, and the emergence of a
peak at 270 nm indicating that decomposition probably follows
6
the accepted pathway —via the alkyltriazenes 17 or 18, leading
to the formation of 5-aminoimidazole-4-carboxamide (AIC) 19
and, the generation of the rapidly solvolysed methanediazo-
nium 5 or a hexane-1,6-bis(diazonium) reactive species 20,
respectively (Scheme 4). This proposal is corroborated by the
presence of a sharp isosbestic point at 295 nm in both cases
indicating that the only UV-absorbing species in the decom-
position mixtures from 1b and 16c are the starting materials and
Scheme 4
19. Presumably, the electronic ‘insulation’ of the two imidazo-
tetrazine rings by the hexamethylene linkage in 16c ensures
that each bicycle undergoes independent ring-opening to the
unstable bis(triazene) 18 and thence further decomposition.
J. Chem. Soc., Perkin Trans. 1, 2000, 4432–4438
4435

View Article Online
Fig. 2 Decomposition (absorbance values versus wavelength in nm)
of bis(imidazotetrazine) 16l in phosphate buffer at pH 7.4. Spectra were
recorded at intervals of 5 min.
The decomposition curves of 16l are significantly different; no
isosbestic point is observed and no peak develops at 270 nm
(
Fig. 2). Presumably, the electronic influence of the β-disulfide
residue dictates that an alternative ring-opening mechanism
may apply in this case.
Bis(imidazotetrazine)s of a different type (21a–c) were
formed from the imidazotetrazines (1a–c) and formaldehyde
in concentrated sulfuric acid at 30 ЊC (Scheme 5). These com-
Fig. 3 Partial autoradiogram of 6% denaturing sequencing gel show-
ing the blocks to Taq DNA polymerase after treatment with agents:
lane 1, control of unmodified BamHI-SalI fragment of pBR322 DNA;
lane 2, cisplatin at 1 µM; lanes 3–6, mitozolomide (1a) at 1, 10, 100 µM
and 1 mM, respectively; lanes 7–10, temozolomide (1b) at 1, 10, 100 µM
and 1 mM, respectively; lanes 11–14, the bis(imidazotetrazine) 16l at 30,
1
00, 300 µM and 1 mM, respectively. Bands marked G3–G5 refer to
sequences of 3–5 consecutive guanine residues.
alkylation with the reference compounds cisplatin, mitozol-
omide (1a) and temozolomide (1b) (lanes 2–10).
The polymerase stop assay showed that the bis(imidazotetra-
zine) 16l has broadly the same sequence selectivity as the
chloroethylating and methylating agents 1a and 1b. This is
unsurprising since the sites of reaction of DNA with small
molecular weight electrophiles are principally determined by
the nucleophilicity of microenvironments within DNA. How-
ever there was a significant increase in the efficiency of DNA
modification by the bis(imidazotetrazine) 16l (compare lanes
12 and 14 with lanes 9 and 10 in Fig. 3). This can be related to
the stability of ultimate electrophilic species derived from
intermediates in the different decomposition pathways. Thus
the longer-lived electrophiles derived from the active drugs 1a
and 1b can locate and react with DNA whereas the ethane-
diazonium ion generated from inactive 1c can undergo a
Scheme 5
pounds were isolated as hydrates. The cyano analogue of
temozolomide (22) also formed the methylene-linked carbox-
1
amide 21b when reacted similarly. Presumably, the cyano group
undergoes hydrolysis in the reaction conditions prior to inter-
action with formaldehyde. Unfortunately, the scope of this
reaction was limited: no reaction occurred between 1a and 1b
with acetaldehyde, propionaldehyde, butyraldehyde, valeralde-
hyde, chloroacetaldehyde, tribromoacetaldehyde or phenylacet-
aldehyde.
22
The bis(imidazotetrazine) 16l was selected for further evalu-
ation in a Thermus aquaticus (Taq) DNA polymerase assay
competitive elimination reaction with evolution of ethene.
10,21
The particularly effective alkylation by 16l provides indirect
evidence that the decomposition pathway in this case may lead
to an episulphonium ion as the ultimate electrophile. Such
ions are considerably more stable than electrophiles derived
to determine the preferred base sequences within DNA modi-
fied by covalent alkylation. Sites of modification were deter-
mined following a 2 hour incubation of BamHI linearised
pBR322 DNA with the test agent. Following annealing
of a 5Ј end-labelled primer complementary to bases 621–640
of the BamHI-SalI fragment of pBR322 extension with the
thermostable Taq DNA polymerase produced a full length
fragment of 263 base pairs. Covalent modification of the DNA
by drug serves to block the progress of the polymerase causing
termination of chain elongation. The products of linear
amplification were run on a sequencing gel to reveal sites of
covalent modification. In the partial DNA sequence shown
23
from the prodrugs 1a–c and presumably more effective DNA
alkylating agents.
The new bis(imidazotetrazine)s also showed comparable
in vitro activities in the National Cancer Institute (USA) 60
24
human tumour cell panel with mitozolomide (1a). Mean GI50
values (mean concentrations to inhibit cell growth of all cell
lines in the panel by 50%) were in the 10–100 µM range with
evidence of some selectivity against cells of leukaemic, CNS
and melanoma origin (GI50 values 1–5 µM), which is typical of
the profile of this class of agent. However, no compelling case
(
Fig. 3), clearly guanine-rich sequences are the preferred sites of
4
436 J. Chem. Soc., Perkin Trans. 1, 2000, 4432–4438

View Article Online
can be made at the moment for considering these bifunctional
alkylating analogues as alternatives to the clinically useful
monofunctional temozolomide.
NH), 8.87 (1 H, s, 6-H); δ (DMSO-d ) 15.1 (CH ), 51.0 (CH ),
C 6 3 2
62.8 (CH ), 129.6 (C-6), 131.1 (C-8), 134.5 (C-8a), 140.3 (C-4),
2
162.9 (CONH ) (Found: C, 40.19; H, 4.26; N, 35.15. C H N O
2
8
10
6
3
requires: C, 40.34; H, 4.23; N, 35.28%).
Experimental
Melting points are uncorrected. H and C NMR spectra were
acquired on a Bruker ARX 250 instrument observing H at
8
-Carbamoyl-3-(4-chlorophenylthiomethyl)imidazo[5,1-d]-
1
13
[1,2,3,5]tetrazin-4(3H)-one 1g
1
13
1
13
Isocyanatomethyl 4-chlorophenyl sulfide (9; R = CH SC H Cl-
2 6 4
p) was obtained from chloromethyl 4-chlorophenyl sulfide and
silver cyanate according to the method of Jones and Powers.
The isocyanate had νmax (KBr)/cm 2255–2264 (N᎐C᎐O) and
was used in an ethereal solution without purification and
reacted with 8 in DMSO (see above). The cream thiomethyl-
imidazotetrazine (87%) had mp 145–147 ЊC (decomp.); ν
2
50.13 MHz and C at 62.9 MHz. H and C spectra were
referenced to tetramethylsilane and coupling constants are
recorded in Hz. Analytical TLC was performed using Merck
Kieselgel 60 F254 plates and flash chromatography was carried
out using silica columns, Kieselgel 60 mesh grade 24; com-
pounds were visualised with UV irradiation. IR spectra were
recorded on a Mattson Instruments Galaxy Series FTIR 2020
instrument. Mass spectra were recorded on either a Bio-ion 20
plasma desorption instrument or a VG Autospec. Fast atom
bombardment (FAB) mass spectra were recorded on a VG
Autospec.
Chemicals, solvents and diisocyanates 15b–e were purchased
from Sigma-Aldrich Chemical Company Ltd. or Lancaster
Synthesis Ltd. The materials for the linear Taq stop PCR were
provided by Professor J. A. Hartley, UCL Middlesex Hospital.
Mitozolomide and temozolomide were synthesised by the
2
6
Ϫ1
᎐ ᎐
max
Ϫ1
(
KBr)/cm 3090, 1736 (C᎐O), 1680 (C᎐O), 1609, 1479, 1366,
᎐ ᎐
1
269, 1096, 1044; δ (DMSO-d ) 5.80 (2 H, s, CH ), 7.51 (4 H,
H 6 2
dd, J 8.5, arom C–H), 7.71 (1 H, br s, NH), 7.85 (1 H, br s, NH),
.88 (1 H, s, 6-H); δC (DMSO-d ) 53.1 (CH ), 129.3 (arom
8
6
2
C–H), 129.5 (C-6), 131.5 (C-8), 131.8 (arom C), 133.1 (arom
C), 134.0 (arom CH), 134.2 (C-8a), 138.6 (C-4), 161.5 (CONH2)
(
Found: C, 42.64; H, 2.72; N, 24.86. C H ClN O S requires:
12 9 6 2
C, 42.80; H, 2.69; N, 24.96%).
2
1
13
Synthesis of isocyanates 15a and 15f–l: general method
method of Stevens et al. and had H, C NMR and IR spectra
identical to authentic samples. Chloromethyl isocyanate was
prepared from chloroacetyl chloride by the Curtius rearrange-
ment of the corresponding acid azide. Methoxymethyl and
ethoxymethyl isocyanates were prepared from chloromethyl
methyl ether or chloromethyl ethyl ether, respectively, and silver
The acid hydrazides 13a and l3f–l were prepared from the
corresponding methyl or ethyl esters 12a, 12f–l in 95% ethanol
containing an excess of hydrazine hydrate. The hydrazides
25
(
0.5 g) were introduced into a vigorously stirred emulsion of
carbon tetrachloride (5 mL), water (5 mL) and 10 M hydro-
chloric acid (1.0 mL) at 0–5 ЊC. Sodium nitrite (2.2 mol equiv.)
in water (5 mL) was added dropwise and the mixture was stirred
at 0 ЊC for 1 h. The organic layer was separated and the aqueous
layer was extracted with benzene (2 × 10 mL). The combined
26
isocyanate, according to the method of Jones and Powers.
8
4
-Carbamoyl-3-(chloromethyl)imidazo[5,1-d][1,2,3,5]tetrazin-
(3H)-one 1d
organic fractions containing the diazides were dried (CaCl2)
Chloromethyl isocyanate (9; R = CH Cl) (0.5 g) in diethyl ether
2
and heated under reflux (2–4 h) to complete conversion to
(
0
5 mL) was stirred with 5-diazoimidazole-4-carboxamide (8;
.20 g) in HMPA (1 mL) at 25 ЊC, in the dark, under nitrogen,
the isocyanates. The solvent was then removed by vacuum
evaporation to furnish the diisocyanates as oils which showed
for 12 h. The white product (0.2 g, 59%) which precipitated
Ϫ1
νmax (film) at 2240–2260 (N᎐C᎐O) cm . The diisocyanates were
from the reaction mixture had mp 130 ЊC (vigorous decomp.);
Ϫ1
used without further purification.
νmax (KBr)/cm 3428, 3092, 1750 (C᎐O), 1674 (C᎐O), 1366,
1
057, 743; δH (DMSO-d ) 6.28 (2 H, s, CH ), 7.77 (1 H, br s,
6
2
NH), 7.91 (1 H, br s, NH), 8.95 (1 H, s, 6-H); δ (DMSO-d )
General method for the synthesis of bis(imidazotetrazine)s 16
C
6
5
1
6.3 (CH ), 130.3 (C-6), 132.2 (C-8), 134.6 (C-8a), 139.9 (C-4),
linked through the N(3)–N(3Ј) positions
2
61.4 (CONH ) (Found: C, 31.58; H, 2.27; N, 36.46. C H -
2
6
5
To the crude diisocyanate 15 (3.0 mmol) in DMSO (5 mL) was
added 5-diazoimidazole-4-carboxamide 8 (6.0 mmol) and the
mixture was stirred (28 d to prepare 16a; 12 h to prepare 16b–l)
at 0 ЊC under a nitrogen atmosphere. The mixture was diluted
with ice–water and the beige solid bis(imidazotetrazine), which
precipitated from the reaction mixture, was collected and puri-
fied by sequential washing with petroleum–ether, formic acid
and water. Yields and physical characteristics of compounds
16a–l are recorded in Table 1.
ClN O requires: C, 31.52; H, 2.20; N, 36.76%).
6
2
8
4
-Carbamoyl-3-(methoxymethyl)imidazo[5,1-d][1,2,3,5]tetrazin-
(3H)-one 1e
Methoxymethyl isocyanate (9; R = CH OMe) (0.5 g) in diethyl
2
ether (5 mL), was cyclised with 8 (0.20 g) in DMSO (1 mL)
under similar conditions (see above). The white methoxy-
methylimidazotetrazine (0.1 g, 31%) had mp 143–145 ЊC
Ϫ1
(
(
(
(
(
(
3
vigorous decomp.); νmax (KBr)/cm 3091, 1744 (C᎐O), 1688
C᎐O), 1607, 1262, 910; δ (DMSO-d ) 3.41 (3 H, s, CH ) 5.61
2 H, s, CH ), 7.72 (1 H, br s, NH), 7.86 (1 H, br s, NH), 8.88
1 H, s, 6-H); δC (DMSO-d ) 57.0 (CH ), 79.4 (CH ), 129.6
C-6), 131.4 (C-8), 134.4 (C-8a), 139.7 (C-4), 161.6 (CONH2)
Found: C, 37.29; H, 3.64; N, 37.28. C H N O requires: C,
N,NЈ-Bis[3,4-dihydro-4-oxo-3-(2-chloroethyl)imidazo[5,1-d]-
[1,2,3,5]tetrazin-8-ylcarbonyl]diaminomethane (21a)
᎐
H
6
3
2
6
3
2
Mitozolomide (1a; 1.0 g, 4.12 mmol) and 37% formaldehyde
solution (0.18 mL, 2.47 mmol) were stirred in concentrated
sulfuric acid (20 mL) for 5 h at 25 ЊC. The white diamino-
methane (0.41 g, 40%), precipitated when the solution was
7
8
6
3
7.50; H, 3.60; N, 37.49%).
Ϫ1
poured into ice–water, had mp 108–112 ЊC; νmax (KBr)/cm
8
4
-Carbamoyl-3-(ethoxymethyl)imidazo[5,1-d][1,2,3,5]tetrazin-
(3H)-one 1f
3
331, 3092, 1738 (C᎐O), 1661, 1254; δ (DMSO-d ) 4.02 (4 H, t,
᎐
H 6
J 6.0, 2 × CH Cl), 4.64 (4 H, t, J 6.0, 2 × NCH ), 4.97 (2 H, t,
2
2
Similarly prepared, from ethoxymethyl isocyanate (9; R = CH2-
OEt) and 8 in DMSO, the cream ethoxymethylimidazotetra-
J 5.6, NCH N), 8.92 (2 H, t, J 5.6, 2 × NH), 8.93 (2 H, s, 2 ×
2
6-H); δC (DMSO-d ) 41.7 (CH Cl), 44.3 (NCH N), 50.3
6
2
2
zine (36%) had mp 245–248 ЊC (vigorous decomp.); ν (KBr)/
(NCH ), 129.6 (C-6, C-6Ј), 130.3 (C-8, C-8Ј), 134.5 (C-8a,
max
2
Ϫ1
cm
3090, 1740 (C᎐O), 1680 (C᎐O), 1458, 1258, 1096;
C-8aЈ), 139.2 (C-4, C-4Ј), 159.8 (C᎐O) (Found: C, 35.03;
δ (DMSO-d ) 1.13 (3 H, t, J 6.4, CH ), 3.66 (2 H, q, J 6.4, CH )
H, 3.03; N, 32.63. C H Cl N O .H O requires C, 34.86; H,
H
6
3
2
15 14
2
12
4
2
5
.64 (2 H, s, NCH O), 7.72 (1 H, br s, NH), 7.86 (1 H, br s,
3.13; N, 32.62%).
2
J. Chem. Soc., Perkin Trans. 1, 2000, 4432–4438
4437

View Article Online
N,NЈ-Bis(3,4-dihydro-4-oxo-3-methylimidazo[5,1-d][1,2,3,5]-
tetrazin-8-ylcarbonyl)diaminomethane (21b)
approximately 3 h. Gels were transferred to filter paper, dried
and visualised by autoradiography.
Similarly prepared, from temozolomide (1b) and formalde-
hyde in sulfuric acid, this diaminomethane (64%) had mp 134–
Acknowledgements
Ϫ1
1
1
36 ЊC; νmax (KBr)/cm 3387, 1761, 1736, 1655, 1578, 1458,
252; δ (DMSO-d ) 3.87 (6 H, s, 2 × CH ), 4.96 (2 H, t, J 5.9,
We are grateful to the Cancer Research Campaign, UK for
long-term support of the CRC Experimental Cancer Chemo-
therapy Research Group. For the award of studentships
we thank Schering-Plough Research Institute, Kenilworth,
New Jersey, USA (J. A. and D. A. F. L.) and the University of
Nottingham (S. A. J.).
H
6
3
CH ), 8.86 (2 H, s, 2 × 6-H), 8.87 (2 H, t, J 5.9, 2 × NH);
2
δ (DMSO-d ) 36.5 (CH ), 44.2 (CH ), 128.9 (C-6, C-6Ј), 129.7
C
6
3
2
(
C-8, C-8Ј), 135.0 (C-8a, C-8aЈ), 139.3 (C-4, C-4Ј), 159.9 (C᎐O)
᎐
(
Found: C, 34.84; H, 3.52; N, 37.23. C H N O ؒ2.5H O
13
12 12
4
2
requires C, 35.06; H, 3.85, N, 37.75%).
The same diaminomethane (62%) was obtained from1
8
-cyano-3-methylimidazo[5,1-d][1,2,3,5]tetrazin-4(3H)-one 22
References
with formaldehyde in concentrated sulfuric acid.
1
Part 38. D. A. F. Langnel, J. Arrowsmith and M. F. G. Stevens,
ARKIVOC, in press (WEB).
2 M. F. G. Stevens, J. A. Hickman, R. Stone, N. W. Gibson,
N,NЈ-Bis(3,4-dihydro-4-oxo-3-ethylimidazo[5,1-d][1,2,3,5]-
tetrazin-8-ylcarbonyl)diaminomethane (21c)
G. U. Baig, E. Lunt and C. G. Newton, J. Med. Chem., 1984, 27,
1
96.
Similarly prepared, from 1c and formaldehyde in sulfuric
3 M. F. G. Stevens, J. A. Hickman, S. P. Langdon, D. Chubb,
L. Vickers, R. Stone, G. U. Baig, C. Goddard, N. W. Gibson,
J. A. Slack, C. Newton, E. Lunt, C. Fizames and F. Lavelle, Cancer
Res., 1987, 47, 5846.
E. S. Newlands, G. R. P. Blackledge, J. A. Slack, C. Goddard,
C. J. Brindley, L. Holden and M. F. G. Stevens, Cancer Treat. Rep.,
1985, 69, 801.
5 E. S. Newlands, S. M. O’Reilly, M. G. Glaser, M. Bower, H. Evans,
C. Brock, M. H. Brampton, I. Colquhoun, P. Lewis, J. M. Rice-
Edwards, R. D. Illingworth and P. G. Richards, Eur. J. Cancer, 1996,
acid, this diaminomethane (40%) had mp 177–179 ЊC; ν
max
Ϫ1
(
KBr)/cm 3418, 4123, 1742, 1655, 1572, 1460, 1277, 1250;
δ (DMSO-d ) 1.39 (6 H, t, J 7.0, 2 × CH ), 4.33 (4 H, q, J 7.0,
H
6
3
4
2
6
4
1
× CH CH ), 4.96 (2 H, t, J 4.9, NCH N), 8.86 (2 H, s, 2 ×
2
3
2
-H), 8.87 (2 H, t, J 4.9, 2 × NH); δ (DMSO-d ) 14.0 (CH ),
C
6
3
4.2 (CH ), 44.6 (CH ), 129.1 (C-6, C-6Ј), 129.7 (C-8, C-8Ј),
2
2
35.0 (C-8a, C-8aЈ), 138.9 (C-4, C-4Ј), 159.98 (C᎐O) (Found:
᎐
C, 38.47; H, 4.19; N, 35.88. C H N O ؒ2.25H O requires
15
16 12
4
2
3
2A, 2236.
C, 38.42; H, 4.41; N, 35.84%).
6
B. J. Denny, R. T. Wheelhouse, M. F. G. Stevens, L. L. H. Tsang and
J. A. Slack, Biochemistry, 1994, 33, 9045.
7 A. S. Clark, B. Deans, M. F. G. Stevens, M. J. Tisdale,
Linear Taq polymerase stop PCR assay
R. T. Wheelhouse, B. J. Denny and J. A. Hartley, J. Med. Chem.,
The sequence specificity of covalent DNA modification by
mitozolomide (1a), temozolomide (1b) and the disulfide-linked
bis(imidazotetrazine) 16l was determined by a polymerase stop
1
995, 38, 1493.
8
H. S. Friedman, R. E. McLendon, T. Kerby, M. Dugan,
S. H. Bigner, A. J. Henry, D. M. Ashley, J. Krischer, S. Lovell,
K. Rasheed, F. Marchev, A. J. Seman, I. Cokgor, J. Rich, E. Stewart,
O. M. Colvin, J. M. Provezale, D. D. Bigner, M. M. Haglund,
A. H. Friedman and P. L. Modrich, J. Clin. Oncol., 1998, 16, 3851;
D. S. Middlemas, C. F. Stewart, M. N. Kirstein, C. Poquette,
H. S. Friedman, P. J. Houghton and T. P. Brent, Clin. Cancer Res.,
21
assay.
Ϫ1
pBR322 Plasmid DNA (125 µL, 160 µg mL ) was linearised
with BamHI restriction enzyme (3 µL), in 10 × Reaction 3
buffer (15 µL) and water (7 µL), and precipitated with NaOAc
(
(
3 M, 15 µL) and ethanol (95%, 495 µL). Linearised DNA
0.5 µg) was treated with drug at concentrations in the range
2
000, 6, 998.
9 S. Boulton, L. C. Pemberton, J. K. Porteous, N. J. Curtin, R. J.
1
–1000 µM as detailed in Fig. 3 for 2 hours at 37 ЊC (solutions
Griffin, B. T. Golding and B. W. Durkacz, Br. J. Cancer, 1995, 72,
8
2
49; S. Boulton, S. Kyle and B. W. Durkacz, Carcinogenesis, 1999,
0, 199.
were made up to a final volume of 50 µL with buffer: 25 mM
tris(2-hydroxyethyl)amine, 1 mM EDTA, pH 7.2). The DNA
was treated with NaOAc (3 M, 10 µL), water (10 µL) and
precipitated with ethanol (95%, 300 µL), washed and lyo-
philised. The BamHI-SalI fragment was used as a template
for extension of a 20-base oligonucleotide primer of sequence
1
0 J. Arrowsmith, S. Missailidis and M. F. G. Stevens, Anti-Cancer
Drug Des., 1999, 14, 205.
1 R. H. Smith, S. R. Koepke, Y. Tondeur, C. L. Denlinger and
C. J. Michejda, J. Chem. Soc., Chem. Commun., 1985, 936.
1
12 J. F. Mcgarrity and T. Smyth, J. Am. Chem. Soc., 1980, 102, 7303.
3 Y. Wang and M. F. G. Stevens, Bioorg. Med. Chem., 1996, 6, 185.
14 Y. Wang and M. F. G. Stevens, J. Chem. Soc., Perkin Trans. 1, 1995,
783.
1
5
Ј-TATGCGACTCCTGCATTAGG-3Ј. The primer was 5Ј-end
32
labelled with [γ- P] ATP using T4 polynucleotide kinase. The
linear amplification of DNA was performed in a total volume
of 100 µL containing 0.5 µg DNA, 10 µL 10 × buffer (670 mM
2
1
1
5 G. Ege, K. Gilbert and K. Maurer, Chem. Ber., 1987, 120, 1375.
6 T. Curtius, J. Prakt. Chem., 1895, 52, 210.
17 Y. Wang, M. F. G. Stevens and W. T. Thomson, J. Chem. Soc., Chem.
Commun., 1994, 1687.
8 C. King, J. Am. Chem. Soc., 1964, 86, 437.
9 J. K. Horton and M. F. G. Stevens, J. Chem. Soc., Perkin Trans. 1,
Tris pH 8.4, 20 mM MgCl ), 0.25 ng labelled primer, 250 µM
2
dNTP mix and 1 U Taq polymerase. The samples were mixed,
overlaid with 2 drops of mineral oil and then incubated in
a thermal cycler. The amplification procedure was carried out
for 30 cycles, each consisting of 1 min denaturation at 95 ЊC,
1
1
1
981, 1433.
0 T. G. Back and K. Yang, J. Chem. Soc., Chem. Commun., 1990,
19.
2
2
min annealing at 60 ЊC and 2 min chain elongation at 72 ЊC.
8
Following amplification, the samples were precipitated with
NaOAc (3 M, 1 µL) and ethanol (95%, 300 µL), washed and
lyophilised. The samples were taken up in formamide dye
21 M. Ponti, S. Forrow, R. L. Souhami, M. D’Incalci and J. A. Hartley,
Nucleic. Acids Res., 1991, 19, 2929.
2
2 Y. Wang, R. T. Wheelhouse, L. Zhao, D. A. F. Langnel and
M. F. G. Stevens. J. Chem. Soc., Perkin Trans. 1, 1998, 1669.
3 J. G. Henkel and G. S. Amata, J. Med. Chem., 1988, 31, 1282.
4 M. R. Boyd and K. D. Paull, Drug Dev. Res., 1995, 34, 91.
5 G. Schroeter, Ber. Dtsch. Chem. Ges., 1909, 42, 3356.
(
4 µL), denatured at 90 ЊC (2 min) and removed onto ice. The
2
2
2
DNA fragments were separated on 0.4 mm, 6% polyacrylamide
gels (80 mL sequagel 6, 20 mL sequagel complete) with a
Tris–boric acid–EDTA buffer system at 55 ЊC, 3000 V for
26 L. W. Jones and D. H. Powers, J. Am. Chem. Soc., 1924, 46, 2518.
4
438
J. Chem. Soc., Perkin Trans. 1, 2000, 4432–4438