Design of a hypoxia-activated prodrug inhibitor of O6- alkylguanine-DNA alkyltransferase

Article abstract of DOI:10.1016/j.bmcl.2012.08.008

The efficacy of agents that alkylate the O-6 position of guanine is inhibited by O⁶-alkylguanine-DNA alkyltransferase (AGT) which removes these lesions from the tumor DNA. To increase differential toxicity, inhibitors must selectively deplete AGT in tumors, while sparing normal tissues where this protein serves a protective function. A newly synthesized prodrug of the AGT inhibitor O⁶-benzylguanine (O⁶-BG) with an α,α-dimethyl-4-nitrobenzyloxycarbonyl moiety masking the essential 2-amino group has demonstrated the feasibility of targeting hypoxic regions that are unique to solid tumors, for drug delivery. However, these modifications resulted in greatly decreased solubility. Recently, new potent global AGT inhibitors with improved formulatability such as O⁶-[(3-aminomethyl) benzylguanine (1) have been developed. However, acetylamino (N-(3-(((2-amino-9H- purin-6-yl)oxy)methyl)benzyl)acetamide) (2) exhibits a pronounced decrease in activity. Thus, 1 would be inactivated by N-acetylation and probably N-glucuronidation. To combat potential conjugational inactivation while retaining favorable solubility, we synthesized 6-((3-((dimethylamino)methyl) benzyl)oxy)-9H-purin-2-amine (3) in which the 3-aminomethyl moiety is protected by methylation; and to impart tumor selectivity we synthesized 2-(4-nitrophenyl)propan-2-yl(6-((3-((dimethylamino)methyl)benzyl)oxy) -9H-purin-2-yl)carbamate (7), a hypoxia targeted prodrug of 3 utilizing an α,α-dimethyl-4-nitrobenzyloxycarbonyl moiety. Consistent with this design, 7 demonstrates both hypoxia selective conversion by EMT6 cells of 7 to 3 and hypoxic sensitization of AGT containing DU145 cells to the cytotoxic actions of laromustine, while exhibiting improved solubility.

Full text of DOI:10.1016/j.bmcl.2012.08.008

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6242–6247
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design of a hypoxia-activated prodrug inhibitor of O6-alkylguanine-DNA
alkyltransferase
⇑
Rui Zhu , Helen A. Seow , Raymond P. Baumann, Kimiko Ishiguro, Philip G. Penketh ,
Krishnamurthy Shyam, Alan C. Sartorelli
Department of Pharmacology and Developmental Therapeutics Program, Cancer Center, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520-8066,
United States
a r t i c l e i n f o
a b s t r a c t
The efficacy of agents that alkylate the O-6 position of guanine is inhibited by O6-alkylguanine-DNA alkyl-
transferase (AGT) which removes these lesions from the tumor DNA. To increase differential toxicity,
inhibitors must selectively deplete AGT in tumors, while sparing normal tissues where this protein serves
a protective function. A newly synthesized prodrug of the AGT inhibitor O6-benzylguanine (O6-BG) with
Article history:
Received 30 April 2012
Revised 24 July 2012
Accepted 1 August 2012
Available online 10 August 2012
an
a,a-dimethyl-4-nitrobenzyloxycarbonyl moiety masking the essential 2-amino group has demon-
strated the feasibility of targeting hypoxic regions that are unique to solid tumors, for drug delivery.
However, these modifications resulted in greatly decreased solubility. Recently, new potent global AGT
inhibitors with improved formulatability such as O6-[(3-aminomethyl)benzylguanine (1) have been
developed. However, acetylamino (N-(3-(((2-amino-9H-purin-6-yl)oxy)methyl)benzyl)acetamide) (2)
exhibits a pronounced decrease in activity. Thus, 1 would be inactivated by N-acetylation and probably
N-glucuronidation. To combat potential conjugational inactivation while retaining favorable solubility,
we synthesized 6-((3-((dimethylamino)methyl)benzyl)oxy)-9H-purin-2-amine (3) in which the 3-ami-
nomethyl moiety is protected by methylation; and to impart tumor selectivity we synthesized 2-(4-nitro-
phenyl)propan-2-yl(6-((3-((dimethylamino)methyl)benzyl)oxy)-9H-purin-2-yl)carbamate (7), a hypoxia
Keywords:
Hypoxia
Prodrug
O6-Alkylguanine-DNA alkyltransferase
AGT
MGMT
Inhibitor
Targeted
targeted prodrug of 3 utilizing an
a,a-dimethyl-4-nitrobenzyloxycarbonyl moiety. Consistent with this
design, 7 demonstrates both hypoxia selective conversion by EMT6 cells of 7 to 3 and hypoxic sensitiza-
tion of AGT containing DU145 cells to the cytotoxic actions of laromustine, while exhibiting improved
solubility.
Ó 2012 Elsevier Ltd. All rights reserved.
A number of antitumor agents alkylate the O-6 position of
guanine residues in DNA.1 These include the nitrosoureas
carmustine (BCNU) and lomustine (CCNU) and the methylating
agents temozolomide (TMZ), procarbazine, dacarbazine (DTIC)
and streptozocin,1 as well as the 1,2-bis(sulfonyl)hydrazine pro-
drug class of antitumor chloroethylating agents exemplified by
laromustine and KS119 developed in this laboratory.2,3 The repair
protein O6-alkylguanine-DNA alkyltransferase (AGT), stoichiomet-
rically transfers alkyl and chloroethyl groups from the O-6 position
of guanine in DNA to cysteine 145 in its active site.4,5 The O-6
position of guanine in DNA is restored to its native state and the
alkylated form of AGT is then degraded. Thus, one AGT molecule
repairs only a single guanine O-6 position lesion. This action repre-
sents the primary mechanism of tumor and host cell resistance to
agents that alkylate the O-6 position of guanine in DNA.1
The AGT inhibitor O6-benzylguanine (O6-BG) reacts with the
active site of AGT to form S-benzylcysteine, inactivating the
protein and increasing the sensitivity of cells to agents that alkyl-
ate the O-6 position of guanine.6,7 However, global inactivation of
AGT, in most cases, does not result in a therapeutic advantage as
this sensitizes both normal and tumor tissues.8,9 In instances
where the therapeutic effect of the alkylator is largely due to a low-
er tumor AGT activity, global AGT ablation would be expected to be
therapeutically deleterious, as it would collapse the very differen-
tial responsible for efficacy. Thus, non-toxic doses of O6-BG have
been shown in patients to deplete the AGT content of tumors,
enhancing their sensitivity to BCNU, but also sensitizing host
tissues, necessitating a reduction in BCNU dosage to ineffective
levels.8,9 Selectively targeting the release of AGT inhibitors to
tumor tissue would result in differential sensitization and circum-
vent this weakness.10 We have attempted to exploit hypoxic
Abbreviations: AGT, O6-alkylguanine-DNA alkyltransferase; O6-BG, O6-benzyl-
guanine; BCNU, carmustine; CCNU, lomustine; DTIC, dacarbazine; TMZ,
temozolomide.
⇑
Corresponding author. Tel.: +1 203 785 4524; fax: +1 203 737 2045.
E-mail addresses: philip.penketh@gmail.com, philip.penketh@yale.edu (P.G.
Penketh).
Both authors have contributed equally to this manuscript.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.008

R. Zhu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6242–6247
6243
regions, which are unique to solid tumors, to target the release of
O6-BG by masking its essential 2-amino group.10 This should sensi-
tize not only the hypoxic regions themselves but also adjacent
normoxic regions of the tumor by a bystander effect resulting from
the diffusion of the released inhibitor into the surrounding tumor
tissue. This targeting is based on the observation that nitro aro-
matic groups are reduced in mammalian tissues in a one electron
manner to generate a nitro radical anion as the initial product.11
Nitro radical anions are rapidly reoxidized by molecular oxygen
to regenerate the parental compound, so reduction beyond this
stage, to hydroxylamino or amino functionalities, only occurs in
very low oxygen environments.11 When the nitro group is reduced
to a hydroxylamino or amino functionality the masking group
fragments and liberates O6-BG.10 Using a similar strategy, nitrohet-
erocycles have been successfully used diagnostically to selectively
label hypoxic regions in human tumors using agents such as
[F-18]fluoromisonidazole.12 We initially synthesized three nitrob-
enzyloxycarbonyl prodrug derivatives of O6-BG (4–6)10 (Fig. 1)
and evaluated their ability to undergo reductive fragmentation to
generate O6-BG under normoxic and oxygen deficient conditions
using both enzyme systems and intact cells.10 Of these, the gem-di-
methyl analogue (6) emerged as the most promising agent, giving
the highest yield of O6-BG under oxygen deficient conditions, with
little or no activation under normoxia. Compound 6 produced a
significant enhancement of the cytotoxicity of laromustine selec-
tively under hypoxic conditions to DU145 human prostate
carcinoma cells, which express very high levels (42,000 mole-
cules/cell) of AGT.10 However, the utility of 6 was strongly
advantages gained by the aminomethyl substitution. Such conju-
gation reactions can be prevented by replacing the protons on
the amino group in compound 1 with protective methyl groups
to give the N,N-dimethylaminomethyl analogue 3 (Fig. 1) while
retaining the ability to be converted to a relatively water-soluble
salt to permit formulation for clinical usage. We have, therefore,
synthesized 2 and 3, to test the effects of acetylation and methyl-
ation of the 3-aminomethyl moiety of 1 on its AGT inhibitory
activity; and have synthesized 7, as a a,a-dimethyl-4-nitrobenzyl-
oxycarbonyl based hypoxia selective prodrug of 3, as shown in
Figure 2. Compounds 1, 4–6 were synthesized as described
earlier.10,13 Compound 7 is a hypoxia selective AGT inhibitor
prodrug that preferentially liberates 3 under hypoxic conditions,
while exhibiting improved solubility, and resistance to potential
conjugational inactivation. We also prepared 7W the isethionic
acid salt of 7 to further increase water-solubility.
Initially, compounds 1–3 were assessed for AGT inhibitory
activity using two methods; O6-benzylguanine (O6-BG) was also
included to serve as a positive control in these studies. In the first
protocol, the sensitivity of purified cloned human recombinant
AGT to direct inactivation by the inhibitors in a simple model
system was determined (Fig. 3). In the second protocol, the ability
of the inhibitors to inactivate AGT in intact cells (HL-60 promyelo-
cytic leukemia which naturally expresses ꢀ17,000 AGT molecules/
cell), where it resides in the nucleus largely bound to DNA, was
assayed (Fig. 4). Both of these protocols gave very similar results,
suggesting that access to AGT was approximately equivalent for
compounds 1–3. While all four compounds were found to possess
AGT inhibitory activity, compound 2 was considerably less active
than the other three agents, demonstrating that N-acetylation of
the aminomethyl group markedly reduces efficacy. In addition,
compound 3 was comparable in activity to the aminomethyl ana-
logue 1. In agreement with Pauly et al.,13 compound 1, the most
potent of the four analogues, was found to be more effective than
O6-BG. In the intact HL-60 cell experiments, 2 h exposure to O6-BG
and compounds 1, 2 and 3 gave IC50 values of 0.039, 0.009, 0.086,
compromised by the fact that the addition of the lipophilic a,a-di-
methyl-4-nitrobenzyloxycarbonyl moiety to O6-BG reduced its
aqueous solubility by >70-fold (Table 1), hampering formulation
and therapeutic utility. Therefore, it is essential for an efficacious
prodrug to contain a hydrophilic (solubilizing) moiety to counter
the hydrophobicity of this hypoxic region targeting moiety.
Recently Pauly et al.13 reported that O6-[(3-aminomethyl)
benzyl]guanine (1) (Fig. 1) was superior to O6-BG as an inactivator
of human AGT. This finding is particularly important since this
modification not only results in an increase in potency but it also
permits salt formation, thereby aiding in formulation. Thus, it
would be anticipated that prodrugs of compound 1, containing
and 0.025 lM, respectively. Thus, compound 3 retains reasonable
activity as an inhibitor of AGT, and the solubility advantage of com-
pound 1 (Table 1), i.e., its ability to form a water-soluble salt, while
avoiding the possibility of being largely inactivated by metabolic
N-acetylation. The methylation of the amino function also permits
a significant decrease in the cost of goods by eliminating a blocking
and unblocking step in the synthesis of compound 3. O6-BG, 1, 2
and 3 were also evaluated for their ability to enhance the cytotoxic
effects of laromustine against an EMT6 cell line (murine mammary
carcinoma) that had been transfected with the human AGT gene
(Fig. 5). This cell line (EMT6hAGT18) expresses ꢀ18,000 AGT
the
a,a-dimethyl-4-nitrobenzyloxycarbonyl moiety, could be
formulated as a consequence of improved aqueous solubility. How-
ever, the primary 3-aminomethyl group on the benzyl moiety may
be susceptible to conjugation reactions (i.e., N-acetylation and
N-glucuronidation;14) in patients, potentially resulting in a de-
crease in activity (N-acetylation to form compound 2) (Fig. 1)
and/or rapid excretion (N-glucuronidation), decreasing the
Figure 1. Chemical structures and names of key compounds.

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R. Zhu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6242–6247
Table 1
Comparison of the aqueous solubilities of key compounds
Agent
Compound description
Aqueous solubility
22 °C
Solubility ratio
w.r.t. O6-BG
O6-BG
1
3
6
7
7W
O6-Benzylguanine standard AGT inhibitor
(O6-[(3-Aminomethyl)benzylguanine)
718 25
1.77 0.11 mM
6.49 0.07 mM
9.8 0.7
326 10
>10 mM
l
M
1.00
2.47
9.04
0.014
0.454
>13.9a
(O6-[(3-N,N-dimethylaminomethyl)benzylguanine)
O6-BG with
3 With -dimethyl-4-nitrobenzyloxycarbonyl moiety attached to the 2-amino group of guanine
Hydroxyethylsulfonate salt of 7
a
,a
-dimethyl-4-nitrobenzyloxycarbonyl moiety attached to the 2-amino group of guanine
lM
a,a
lM
a
The aqueous solubility at room temperature was determined by adding 1% by volume of a saturated solution of agent to 0.99 mL of distilled water at 22 °C with very rapid
mixing. To this mixture ꢀ200 mg of white quartz (50 + 70 mesh) was added and the mixture shaken periodically for 10 min to stimulate precipitation from super saturated
solutions. The mixture was centrifuged for 2 min at 10,000 g then an aliquot of the supernatant was diluted 10-fold with 30 mM potassium phosphate pH 5.4 buffer
containing 30% CH3CN and the sample analyzed by HPLC.10 The concentrations were then calculated by comparisons with standards of known concentration.
Values represent the mean of 3 determinations S.E.M.
a
Aqueous solubility limit not reached.
Figure 2. The synthesis of key compounds. Compound 2 was prepared by reacting the sodium salt of 3-(acetylamino)methylbenzyl alcohol with 1-(2-amino-9H-purin-6-yl)-
1-methylpyrrolidin-1-ium chloride (8) in N,N-dimethylformamide in the presence of 4-(N,N-dimethylamino)pyridine. Compound 3 was synthesized using an analogous
procedure in which 3-(N,N-dimethylamino)methylbenzyl alcohol (9) was used in lieu of 3-(acetylamino)methylbenzyl alcohol.16 Compound 7 was prepared from compound
3 using a procedure analogous to the one employed for the synthesis of the 3-nitrophenoxycarbonyl derivative of O6-BG.10 The free amine was then converted to the water-
soluble hydroxyethylsulfonate salt (7W) by reaction with isethionic acid.
molecules/cell. As expected, all four AGT inhibitors produced insig-
nificant cytotoxicity on their own, but pretreatment of cells with
these agents resulted in a significant potentiation of the cytotoxic
effects of laromustine (Fig. 5). In this intact cell system, compound
3 was significantly more potent than both O6-BG and 1, which gave
essentially equivalent enhancements of cytotoxicity. The greater
potency of the dimethylamino analogue in this assay may reflect
differences in the permeability, export, metabolism or other factors
not relating directly to their relative potencies as inactivators of
human AGT in this cell line. Compound 2 was by far the least effec-
tive, supporting the contention that conjugation of the primary
amino group on the benzyl moiety of compound 1 in vivo would
reduce its potency. These findings suggest that compound 3 offers
the greatest potential of these O6-BG analogues.

R. Zhu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6242–6247
6245
Figure 3. Comparative inhibition of purified recombinant human AGT activity by O6-BG and the O6-BG analogs 1, 2, 3. The ability of O6-BG, 1, 2, and 3 to inhibit AGT was
assessed using an assay based upon the repair of DNA containing cross-link precursor lesions (O6-(2-chloroethyl)guanine and N1,O6-ethanoguanine).17 This DNA was prepared
by reacting 1,2-bis(sulfonyl)-1-(2-chloroethyl)hydrazine, synthesized as previously described15 with L1210 DNA.10 Varying concentrations (0.01, 0.1, and 1.0 M) of O6-BG, 1,
l
2, and 3 were incubated with 40 fm of AGT (37 °C for 10 min) and the treated AGT assayed for its ability to repair the cross-link precursor lesions relative to AGT incubated in
the absence of inhibitors.10 Repair of the cross-link precursor lesions results in decreased DNA cross-linking. Therefore, inhibition of AGT repair results in an increase in the
measured cross-linking. Values represent the means of 3 independent determinations ( S.E.M.)
Figure 4. AGT inhibition in HL60 promyelocytic leukemia cells in vitro. Cells from the human HL60 leukemia cell line, were treated with graded concentrations of O6-BG (Â),
1 (D
), 2 (h), or 3 (s) for 2 h and AGT levels were assessed using a [3H]AGT based binding assay as described previously.18 Dose response curves were generated and IC50
values were determined.
While compound 3 has an impressive ability to sensitize AGT
expressing cells to laromustine in vitro, it retains the inherent flaw
of global AGT inhibition, that untargeted inhibitors not only ablate
AGT in the tumors, where the repair protein is a hindrance to
treatment, but also in normal tissues where it serves a protective
function. Therefore, for an AGT inhibitor to have a meaningful im-
pact in cancer therapy as a modulator of guanine O-6 alkylating
agents it is necessary for the inhibitor to be delivered preferentially
to the tumor target. This ensures that normal tissues are largely
spared sensitization and that the dosage of the guanine O-6 alkyl-
ating drug is maintained without substantially increased host tox-
icity. To accomplish this we synthesized compound 7 which
assay. Cells received a 4 h pre-exposure to various concentrations
of compound 7 under either normoxic or hypoxic conditions. The
cells were then challenged with laromustine to ascertain the de-
gree of sensitization. The high AGT activity (42,000 AGT molecules
per cell) in the DU145 cell line results in substantial resistance to
laromustine and other agents that rely on the alkylation of O-6 po-
sition of guanine residues in DNA for the bulk of their activity10,20–
22
so the loss of AGT activity should be readily apparent. The 4 h
pretreatment resulted in only a moderate sensitization to the cyto-
toxic actions of laromustine under normoxic conditions, but a
much more pronounced sensitization under hypoxic conditions.
Thus, a 5 lM pretreatment with compound 7 resulted in normoxic
utilizes the same
a
,
a
-dimethyl-4-nitrobenzyloxycarbonyl hypoxic
and hypoxic survival fractions of 0.43 and 0.027, respectively
(Fig. 6). This is consistent with the prodrug 7 liberating the AGT
inhibitor 3 preferentially under hypoxic conditions. However,
despite this ꢀ16-fold hypoxic/oxic surviving fraction differential,
corresponding to a >4-fold difference in LC90 concentration, com-
pound 7 showed a significantly greater degree of normoxic sensiti-
zation than previously observed with 6,10 indicating the possible
presence of more significant non-reductive routes of activation
with compound 7 than 6. The conversion of 7 to 3 by EMT6 cell
suspensions under normoxic and hypoxic conditions was studied
and a >60-fold differential in the formation of 3 was observed
under hypoxic conditions (Table 2). The low IC50 value of 3 for
region targeting moiety used in 6 to mask the essential 2-amino
group of the more water soluble compound 3. In support of this,
compounds 7 and 7W were found to have >30 and >1000 times
the aqueous solubility of compound 6, respectively (Table 1). Some
of the advantage gained by the enhanced solubility of 7 and 7W
could be offset by the greater basicity of the tertiary amine as
the extracellular pH is lower in hypoxic tumors than in normal
tissue, while their intracellular pH is relatively unchanged and this
can impair the uptake of basic chemotherapeutic agents.19
A comparison was made between the ability of compound 7 to
potentiate the cytotoxic effects of laromustine under hypoxic and
normoxic conditions in DU145 cells using a clonogenic cell survival
AGT of 0.025 lM may account for these observations as very little

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R. Zhu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6242–6247
Figure 5. The effects of O6-BG and compounds 1, 2, and 3 in the presence or absence of laromustine. EMT6hAGT18 cells, which express 18,000 molecules of AGT per cell, were
treated with a 2 M) for 18 h. In instances where cells were treated with two compounds,
M concentration of O6-BG, 1, 2, or 3 in the presence or absence of laromustine (100
the AGT inhibitor was administered two hours before laromustine treatment (100
M, 18 h). Cell survival was measured using a clonogenic assay described previously.7 The
l
l
l
maximum DMSO concentrations to which cells were exposed were 60.05% and non-toxic. Points represent mean S.E.M of 3 to 5 individual experiments.
Table 2
Liberation of
conditions
3 from 7 by incubation with EMT6 cells under oxic and hypoxic
EMT6 activation
Oxic
Hypoxic
[7]
[3]
[7]
[3]
lM T = 0 h
lM T = 0 h
lM T = 1 h
lM T = 1 h
197.57 3.8
0.0a
195.5 2.9
0.2 0.04
199.78 5.8
0.0a
149.6 9.3
12.3 1.9
Production of 3 from 7 (ꢀ200
l
M) by EMT6 cells (2 Â 107/ml) under oxic and
hypoxic conditions over a one hour incubation period at 37 °C. The concentrations
were determined by HPLC as previously described10 except the elution solvent’s
aqueous constituent buffer was 0.05 M Glycine, 1.0 mM NaN3, pH 10.0. Compounds
3 and 7 elute at ꢀ9.3 and ꢀ29.7 min, respectively. Values represent the mean of 6
determinations S.E.M.
a
Below the limit of detection (<0.1 lM).
activation would be required under oxic conditions to result in an
enhancement of laromustine cytotoxicity. If the levels of non-
reductive routes of activation are not too excessive in patients,
the hypoxia-activated AGT inhibitor prodrug 7W, in conjunction
with laromustine, or other agents that rely on alkylation of the
O-6 position of guanine residues in DNA for their activity, may
be useful in a clinical setting.
Acknowledgments
This work was supported in part by U.S. Public Health Service
Grants CA090671, CA122112, and CA129186 from the National
Cancer Institute and a grant from the National Foundation for
Cancer Research.
Figure 6. The effects of various concentrations of compound 7 on the cytotoxicity
of laromustine under oxic and hypoxic conditions in DU145 cells. DU145 cells were
pretreated with graded concentrations of 7 (5, 10, 20, 40
lM) for 4 h followed by
100 M laromustine for a total exposure time of 24 h under normoxic (closed
l
symbols) or hypoxic (open symbols) conditions and cell survival assessed using a
clonogenic assay.7 Oxygen deficiency was established using a nitrogen flush and
seal combined with a dual enzyme oxygen scavenging system consisting of 2 U/ml
of glucose oxidase, 120 U/ml of catalase, and 10 mM of added glucose to maintain
hypoxia.23 The maximum DMSO concentrations to which cells were exposed were
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