Strategy for imidazotetrazine prodrugs with anticancer activity independent of MGMT and MMR

Article abstract of DOI:10.1021/ml300132t

The imidazotetrazine ring is an acid-stable precursor and prodrug of highly reactive alkyl diazonium ions. We have shown that this reactivity can be managed productively in an aqueous system for the generation of aziridinium ions with 96% efficiency. The

Full text of DOI:10.1021/ml300132t

Letter
pubs.acs.org/acsmedchemlett
Strategy for Imidazotetrazine Prodrugs with Anticancer Activity
Independent of MGMT and MMR
Elrashied A. E. Garelnabi,†,⊥ Dimitrios Pletsas,† Li Li,‡ Konstantinos Kiakos,∥ Nazira Karodia,§
John A. Hartley,∥ Roger M. Phillips,‡ and Richard T. Wheelhouse*,†
†School of Pharmacy, ‡Institute of Cancer Therapeutics, and §Chemical and Forensic Sciences, University of Bradford, Bradford BD7
1DP, U.K.
∥UCL Cancer Institute, Paul O'Gorman Building, 72 Huntley Street, London WC1E 6BT, U.K.
S
* Supporting Information
ABSTRACT: The imidazotetrazine ring is an acid-stable
precursor and prodrug of highly reactive alkyl diazonium
ions. We have shown that this reactivity can be managed
productively in an aqueous system for the generation of
aziridinium ions with 96% efficiency. The new compounds are
potent DNA alkylators and have antitumor activity independ-
ent of the O6-methylguanine-DNA methyltransferase and
DNA mismatch repair constraints that limit the use of
Temozolomide.
KEYWORDS: Imidazotetrazine, aziridinium, Temozolomide, cancer, 13C NMR, DNA
midazotetrazine 1 is a fascinating heterocycle, on account of
Iboth its chemistry and the therapeutic applications of its
derivatives. It forms the core of the anticancer prodrug
Temozolomide1 (1, R = CH3; TMZ). Although TMZ has
achieved “blockbuster” status,2 it remains the lone member of
its drug class because of the constraints placed on tumor
response to the prodrug by a requirement for DNA mismatch
repair (MMR) and resistance mediated through O6-alkylgua-
nine-DNA alkyltransferase (MGMT). Herein we report a
strategy that achieves remarkably effective control of the
reactive intermediates generated by the imidazotetrazine ring in
an aqueous environment, in this example, to generate
aziridinium ions. This design achieves compounds that react
with DNA but exhibit chemosensitivity independently of MMR
and MGMT.
parameters that determine the effectiveness of imidazotetrazine
prodrugs. The first is the rate of addition of water (or hydroxide
ion) to initiate the ring-opening reaction. This is slow at low
pH and, thus, confers acid stability and the convenience of oral
dosing.4 The pH-dependence of this reaction also influences
distribution of the prodrug around the body: hydrolysis kinetics
at pH 7.4 match closely the uptake rate (peak plasma
concentration after 30 min) and metabolic half-life (t1/2
=
1.29 h) in patients.1,5 The other significant kinetic parameter is
the reactivity of the latent electrophile. Hydrolysis of
methyldiazonium in a purely chemical system has t1/2 = 0.39
s.6 Again, a suboptimal value detracts from clinical effectiveness:
ethyldiazonium eliminates or reacts with water before it is able
to locate a reactive nucleic acid target site,7 while longer-lived
intermediary electrophiles such as chloronium achieve clinical
efficacy.8 For TMZ, these clinically useful, if not formally
optimized, pharmacokinetic properties were achieved serendip-
itously.
At neutral or alkaline pH, imidazotetrazines 1 (e.g., TMZ)
act as a source of diazonium ions 2, with few exceptions, ring-
opening as shown in Scheme 1.3 Methyldiazonium is released
from TMZ and methylates DNA; lethal interaction of this
adduct with MMR causes cell death. There are two kinetic
In the design of TMZ analogues with altered spectra of
activity, we reasoned that a neighboring group participation
(NGP)-based mechanism could be employed to control the
incipient alkyldiazonium ions. This would serve the dual
functions of directing reactivity and delivering an alternative
form of damage to DNA. Since the response of tumors to TMZ
is determined by the interaction of covalently modified DNA
with DNA repair systems,9 altering the electrophile would
necessarily alter the tumor response. In these respects, the
Scheme 1. Release of Electrophiles on Hydrolysis of the
a
Imidazotetrazine Ring
Received: May 28, 2012
a
Accepted: September 18, 2012
For TMZ, R = CH3; MTZ, R = CH2CH2Cl.
© XXXX American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
potential of the imidazotetrazine as an acid-stable precursor of
aziridines or aziridinium ions was explored. These are reactive
intermediates with proven clinical utility, being found widely in,
or generated by, synthetic and natural product antitumor drugs
and prodrugs. Furthermore, O6-aminoethylguanine is known to
be refractory to cleavage by MGMT.10
The synthesis of an aziridinium-precursor imidazotetrazine 3
is presented in Scheme 2. Imidazotetrazine ring closure is
a
Scheme 2
Figure 1. Gated decoupled 13C NMR spectrum showing identification
and quantitation of the final products of the 13C-labeled imidazote-
trazine 3 reaction in phosphate buffer, pD 7.8. Resonances were
assigned on the basis of JC,P coupling constants13 and spiking with
n
authentic, unlabeled alcohol 9.14
a
Scheme 3. Fate of the 13C Label
a
Reagents and conditions: (i) CuCl, AcOH, Δ, 12 h, 41%; (ii)
NH2NH2·H2O, EtOH, rt, 12 h 89%; (iii) NaNO2, AcOH, CH2Cl2/
H2O, 0−2 °C; (iv) CH2Cl2, rt, 48 h; (v) DMSO, rt, 48 h, 14% (over
steps iii−v).
achieved by reaction of diazoazole 4 with an isocyanate 5.11,12
This ring closure was placed toward the end of the synthesis to
avoid complications arising from reactivity of the imidazote-
trazine itself. Preparation of these derivatives posed a further
synthetic challenge, as β-aminoisocyanates were required. The
reactivity of the amine group was controlled by use of anilines,
rather than a protecting group strategy: β-anilinoisocyanates 5
were stable and isolable. For mechanistic studies, a 13C-labeled
version was prepared using enriched 13C(2)-acrylate 6 (the site
of the label is marked * in the figure and schemes); the label
was estimated as 75 atom % 13CH2 by 1H NMR. The analogous
bisimidazotetrazine 7 was similarly prepared from p-toluidine.
The prodrug activation mechanism was investigated by
reaction of 13C-labeled 3 in phosphate buffer. Final products
were identified and quantitated using gated decoupled 13C
NMR spectroscopy (Figure 1). Products arose from reaction of
the latent electrophiles with both nucleophiles in the system:
the D2O solvent and the phosphate of the buffer. In the
phosphate products 8a,b, the label was detected equally in the
two methylene positions; the alcohol products 9a,b showed a
small excess of label retained in the starting position. A
mechanism that accounts for these observations is presented in
Scheme 3. It is proposed that diazonium ion 10 is formed as in
Scheme 1. Subsequent reaction is either via intramolecular
displacement of N2 by the aniline or via direct hydrolysis. The
evidence of the NMR spectrum is that only ca. 4% direct
hydrolysis occurs, with the majority of material forming
aziridinium ion 11. This renders the two methylene positions
equivalent, and thus, equally susceptible to attack by
nucleophiles, as a consequence of which the label is scrambled
in the products. The cleanness of this result is gratifying, as
previous reports on 2-aminoethyldiazonium ions derived from
triazolines and alkylaminotriazenes showed only about 50%
ring closure to aziridines, with pathways of diazonium
a
Approximately 96% conversion to the aziridinium ion 11 and 4%
direct hydrolysis of the diazonium ion occur. Final products show
complete scrambling (1:1 mixtures) of the label, confirming the
intermediacy of a discrete aziridinium ion 11.
elimination and direct hydrolysis contributing significantly to
the detected products.15−17 The phosphate ester/alcohol
product ratio reflects the relative nucleophilicities of phosphate
and D2O.
The hydrolysis kinetics (pseudo-first-order disappearance of
imidazotetrazine, λmax = 330 nm) were measured for
compounds 3 and 7 and TMZ (Table 1). For all compounds,
the half-life measured under acidic conditions was much longer
than that determined at neutral or basic pH, although
compounds 3 and 7 showed increased reactivity under acidic
conditions as compared with TMZ.1,5 Even so, the t1/2 values
measured at pH 4.0 remain much longer than the uptake and
elimination rates of orally administered TMZ.1,5 It is interesting
to note that the site of structural variation in imidazotetrazines
3 and 7, compared with TMZ, is distant from and not
conjugated with the hydrolytic reaction center, C-4. This
change in acid reactivity demonstrates a curious complexity of
the imidazotetrazine system.
The reaction of compounds 3 and 7 and melphalan with
DNA, and the sequence specificity of covalent DNA
modification were assessed by a combination of cleavage-
based and polymerase stop assays. The piperidine cleavage
assay indicated that the new compounds alkylate identical GN7
sites at guanine-rich sequences to melphalan, with the
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Table 1. pH Dependence of Imidazotetrazine Hydrolysis Kinetics at 37 °C
pH = 4.0
pH = 7.4
pH = 8.0
a
b
a
b
a
b
k′
t1/2
k′
t1/2
k′
t1/2
TMZ
0.100 0.001
0.500 0.003
0.400 0.006
253.0
41.5
48.5
14.0 0.0
15.2 0.3
14.4 0.7
1.38
1.27
1.33
46.2 0.2
49.5 0.4
45.1 1.5
0.42
0.39
0.43
3
7
a
b
k′ pseudo-first-order rate constant, ×10−5 s−1; data are mean SD of three independent determinations. t1/2 half-life, h. Citrate-phosphate buffers.
bifunctional agent 7 being more reactive than the monofunc-
tional 3 (Figure 2). The same sites of reaction were identified
Figure 3. In vitro chemosensitivity (IC50/μM) of selected compounds
against A2780 (MMR+) and A2780-cp70 (MMR−) cell lines in the
absence (MGMT+) and presence (MGMT−) of the MGMT
inactivator PaTrin2 (10 μM). All data are the mean of ≥3
determinations; error bars are the SD; *IC50 >250 μM.
comparing the shaded and gray bars. For TMZ the IC50 was
>27-fold lower in the MMR-proficient cell line. This ratio was
reduced to 5.8-fold for new bifunctional agent 7 and 2.8-fold for
monofunctional 3. Moreover, in the absence of MMR (white
and gray bars), all compounds showed activity considerably
greater than that of TMZ, irrespective of the MGMT status of
the cells, showing that MMR-dependent activity and MGMT-
mediated resistance are now only minor determinants of the
antitumor effect.
Figure 2. DNA reaction by the piperidine cleavage method showing
GN7 alkylation on the upper strand of pBR322 DNA modified by
compounds 3 and 7 and melphalan (MEL). Arrows indicate the
position and sequence context of the alkylated guanines.
The activity of the new compounds was further investigated
in a panel of cell lines representing major tumor types (colon,
NSCL, pancreatic, and TMZ-resistant glioma); see Table S2 of
the Supporting Information. All cell lines were unresponsive
toward TMZ (IC50 >250 μM); the positive control MEL
elicited a range of responses (IC50 11−3 μM). Both new
compounds showed similar patterns of chemosensitivity, with
the bifunctional agent 7 having potency and selectivity similar
to that of MEL; the monofunctional agent 3 was 7−30-fold less
active.
Overall, remarkable control of the reactive intermediates
generated from imidazotetrazines has been achieved, with 96%
of an alkyldiazonium ion being directed to aziridinium ring
closure under aqueous conditions. In reaction with DNA,
compound 7 has sequence selectivity and reactivity similar to
that of melphalan. Moreover, the pH-dependence of the
hydrolysis kinetics mirrors that of the parent TMZ, so the new
compounds have the potential for oral bioavailability. The new
compounds disclosed herein have potent anticancer activity
that is independent of the two principal constraints on TMZ
using the polymerase stop assay, which detects all covalent
adducts (see Figure S8; Supporting Information). The reaction
of TMZ with the same DNA sequence has previously been
investigated by these methods19,20 and also shows a similar
pattern of sequence selectivity (see Figure S9).
The in vitro anticancer activity of compounds 3 and 7 was
assessed in a pair of MMR proficient and deficient cell lines:
A2780 ovarian carcinoma and its MMR-deficient variant
A2780-cp7018 (Figure 3). MGMT was inactivated using the
benzylguanine analog PaTrin2. Compound 7 was found to be
more active than TMZ, MTZ, and MEL; compound 3 was
approximately equipotent with MTZ. The relative potencies
correlate with their different DNA reactivities (Figure 2).
The extent of MGMT-mediated resistance can be assessed by
comparing the shaded and the black bars (i.e., MGMT−/
MGMT+ with MMR proficient). TMZ was >30-fold more
potent in the absence of MGMT; in contrast, both of the new
agents 3 and 7 showed equivalent potency in the absence and
presence MGMT. MMR dependence can be assessed by
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ASSOCIATED CONTENT
* Supporting Information
Full experimental details and example data are provided in the
Supporting Information. This material is available free of charge
via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Tel: 00 1 (0)1274 234710. E-mail r.t.wheelhouse@brad.ac.uk.
■
Present Address
⊥Department of Pharmaceutical Chemistry, Faculty of
Pharmacy, University of Khartoum, Sudan.
Funding
This work was supported, in part, by Grants C14492/A4884
(to R.T.W. and N.K.) and C2259/A9994 (to J.A.H.) from
Cancer Research U.K.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
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Decomposition of 1-Alkyltriazolinesa Mechanistic Study. J. Org.
Chem. 1993, 58, 2097−2103.
TMZ, Temozolomide; MTZ, mitozolomide; MGMT, O6-
methylguanine-DNA methyltransferase; MMR, DNA mismatch
repair
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Chem. Soc., Perkin Trans. 1 2000, 4432−4438.
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