4-nitrobenzyloxycarbonyl derivatives of O 6-benzylguanine as hypoxia-activated prodrug inhibitors of O 6-alkylguanine-DNA alkyltransferase (AGT), which produces resistance to agents targeting the o -6 position of DNA guanine

Article abstract of DOI:10.1021/jm201115f

A series of 4-nitrobenzyloxycarbonyl prodrug derivatives of O ⁶-benzylguanine (O⁶-BG), conceived as prodrugs of O ⁶-BG, an inhibitor of the resistance protein O⁶- alkylguanine-DNA alkyltransferase (AGT), were synthesized and evaluated for their ability to undergo bioreductive activation by reductase enzymes under oxygen deficiency. Three agents of this class, 4-nitrobenzyl (6-(benzyloxy)-9H-purin-2-yl)carbamate (1) and its monomethyl (2) and gem-dimethyl analogues (3), were tested for activation by reductase enzyme systems under oxygen deficient conditions. Compound 3, the most water-soluble of these agents, gave the highest yield of O⁶-BG following reduction of the nitro group trigger. Compound 3 was also evaluated for its ability to sensitize 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl] hydrazine (laromustine)-resistant DU145 human prostate carcinoma cells, which express high levels of AGT, to the cytotoxic effects of this agent under normoxic and oxygen deficient conditions. While 3 had little or no effect on laromustine cytotoxicity under aerobic conditions, significant enhancement occurred under oxygen deficiency, providing evidence for the preferential release of the AGT inhibitor O⁶-BG under hypoxia.

Full text of DOI:10.1021/jm201115f

ARTICLE
pubs.acs.org/jmc
4-Nitrobenzyloxycarbonyl Derivatives of O6-Benzylguanine as
Hypoxia-Activated Prodrug Inhibitors of O6-Alkylguanine-DNA
Alkyltransferase (AGT), Which Produces Resistance to Agents
Targeting the O-6 Position of DNA Guanine
Rui Zhu, Mao-Chin Liu, Mei-Zhen Luo, Philip G. Penketh,* Raymond P. Baumann, Krishnamurthy Shyam,
and Alan C. Sartorelli
Department of Pharmacology and Developmental Therapeutics Program, Cancer Center, Yale University School of Medicine,
333 Cedar Street, New Haven, Connecticut 06520-8066, United States
ABSTRACT: A series of 4-nitrobenzyloxycarbonyl prodrug
derivatives of O6-benzylguanine (O6-BG), conceived as pro-
drugs of O6-BG, an inhibitor of the resistance protein
O6-alkylguanine-DNA alkyltransferase (AGT), were synthesized
and evaluated for their ability to undergo bioreductive activation
by reductase enzymes under oxygen deficiency. Three agents of
this class, 4-nitrobenzyl (6-(benzyloxy)-9H-purin-2-yl)carba-
mate (1) and its monomethyl (2) and gem-dimethyl analogues
(3), were tested for activation by reductase enzyme systems
under oxygen deficient conditions. Compound 3, the most water-soluble of these agents, gave the highest yield of O6-BG following
reduction of the nitro group trigger. Compound 3 was also evaluated for its ability to sensitize 1,2-bis(methylsulfonyl)-1-
(2-chloroethyl)-2-[(methylamino)carbonyl]hydrazine (laromustine)-resistant DU145 human prostate carcinoma cells, which
express high levels of AGT, to the cytotoxic effects of this agent under normoxic and oxygen deficient conditions. While 3 had little
or no effect on laromustine cytotoxicity under aerobic conditions, significant enhancement occurred under oxygen deficiency,
providing evidence for the preferential release of the AGT inhibitor O6-BG under hypoxia.
’ INTRODUCTION
S-benzylcysteine in the active site of the protein by acting as a
substrate, depleting AGT and increasing the sensitivity of both
tumor and host cells to agents that chloroethylate and methylate
the O-6 position of guanine in DNA.15ꢀ17 Relatively nontoxic
levels of O6-BG have been shown experimentally in both cell and
animal tumor systems, as well as in patients, to deplete the AGT
content of tumors. This action sensitizes cell systems in vitro and
experimental tumors in vivo to BCNU; however, since AGT
levels are also depleted by O6-BG in normal cells, a concomitant
sensitization of host tissues occurs, requiring a considerable
reduction in the dosage of BCNU. Thus, in cancer patients
pretreated with O6-BG, the resulting myelosuppression requires
an 80% decrease in BCNU dosage, resulting in an ineffective
therapeutic level of the nitrosourea.18,19 These findings imply
that methodology producing a selective or preferential depletion
of AGT in tumor tissue relative to normal tissue is required to
circumvent AGT induced tumor resistance to guanine O-6
alkylating agents.
Alkylating agents are among the most potent and widely used
antineoplastic agents. A particularly important class of alkylating
agents consists of compounds that methylate and chloroethylate
the O-6 position of DNA guanine. This class includes as chloro-
ethylating agents, the chloroethylnitrosoureas (e.g., carmustine
or BCNU and lomustine or CCNU) and the sulfonylhydrazine
prodrugs [e.g., 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methyl-
amino)carbonyl]hydrazine (laromustine, onrigin, cloretazine,
VNP40101M, 101M)1ꢀ6 and 1,2-bis(methylsulfonyl)-1-(2-
chloroethyl)-2[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine
(KS119)]7,8 synthesized in our laboratory, and the methylating
agents temozolomide, procarbazine, dacarbazine, and streptozo-
tocin. O6-Alkylation of guanine residues in DNA is repaired by
the protein O6-alkylguanine-DNA alkyltransferase (AGT), which
restores the O-6 position of guanine in DNA to its native state by
transferring the O6-methyl or O6-(2-chloroethyl) group to a
cysteine residue in the active site of the protein.9,10 There is no
known biological acceptor that removes the alkyl moiety from
this cysteine residue, so one AGT molecule can only repair a
single alkyl lesion. The presence of increasing levels of AGT
appears to correlate relatively well with elevated tumor and host
tissue resistance to guanine O6-methylating and chloroethylating
agents.11ꢀ13
The oxygen deficient tumor cell fractions present in solid
tumors, which we believe are major sites of tumor vulnerability,
can be used to selectively activate prodrugs susceptible to re-
ductive fragmentation to generate a potent inhibitor of AGT
(Figure 1).20,21 To this end, we have synthesized a series of
O6-Benzylguanine (O6-BG) is among the most effective
known inhibitors of AGT;14 this agent reacts with AGT to form
Received: August 19, 2011
Published: September 28, 2011
r
2011 American Chemical Society
7720
dx.doi.org/10.1021/jm201115f J. Med. Chem. 2011, 54, 7720–7728
|

Journal of Medicinal Chemistry
ARTICLE
Figure 1. Influence of oxygen concentration on nitro reduction. Shown is the chemical mechanism underlying the inverse oxygen concentration
dependency of net prodrug reduction that leads to molecular fragmentation and the release of the AGT inhibitor O6-BG.
Scheme 1
agents containing a 4-nitrobenzyloxycarbonyl trigger/linker and
have presented evidence to show selective/preferential activation
under oxygen deficiency to generate the AGT inhibitor O6-BG.
These agents contain the nitrobenzyloxycarbonyl moiety in place
of one of the protons in the 2-amino group of O6-BG. Since a free
amino group in O6-BG is essential for potent AGT inhibitory
activity, the proposed prodrug should be much less active than
the parent compound (i.e., O6-BG) until unmasking of the
2-amino group occurs following initial one electron nitro reduc-
tion in the oxygen deficient cellular fractions.22,23 In the past, we
have used the α-methyl-4-nitrobenzyloxycarbonyl moiety as a
masking group in KS119, a highly selective sulfonylhydrazine
prodrug that displayed excellent activity against oxygen deficient
EMT6 murine mammary carcinoma cells with little or no activity
against their normoxic counterparts.7,8 The proposed activation
mechanism is depicted in Scheme 1.
’ CHEMISTRY
We have synthesized three 4-nitrobenzyloxycarbonyl deriva-
tives of O6-BG, i.e., the desmethyl (1), the monomethyl (2), and
the gem-dimethyl (3) analogues, as shown in Scheme 2. These
compounds differ structurally only in the degree of methylation
of the benzylic carbon in the linker region. Thus, treatment of
(2-amino-6-(benzyloxy)-9H-purin-9-yl)methyl pivalate (4)22 with
phosgene in the presence of pyridine in anhydrous dichloro-
methane gave the corresponding 2-isocyanatopurine (5) which,
when reacted with the appropriate benzyl alcohol, gave the
corresponding carbamate (6, 7, or 8).24 The reaction time and
yield of the carbamate depended greatly upon the structure of the
benzyl alcohol. Thus, intermediate 5, when reacted with 4-ni-
trobenzyl alcohol, α-methyl-4-nitrobenzyl alcohol, or α,α-di-
methyl-4-nitrobenzyl alcohol for 2 h, gave carbamate yields of
89% (6), 63% (7), and 14% (8), respectively. However, with
prolonged reaction times, compounds 7 and 8 were produced in
yields of 81% (6 h) and 68% (40 h), respectively. It is obvious that
steric hindrance to nucleophilic addition produced by the α-
methyl and the α,α-dimethyl groups on the benzylic carbon is
responsible for the observed decrease in reaction rates, with the
more hindered α,α-dimethyl alcohol being the least reactive.
Two methods were employed for the deprotection of the
pivaloyloxymethyl group in compounds 6, 7, and 8. Treatment
of 7 and 8 with ethanolic sodium hydroxide, followed by
neutralization with 10% acetic acid, gave 2 and 3 in 60% and
63% yields, respectively; however, this procedure resulted in the
cleavage of the carbamate linkage in 6 to give the original starting
O6-BG, once released, would be expected to enhance the kill of
oxygen deficient tumor cells treated with agents such as laromus-
tine, carmustine, and KS119; in addition, it is probable that the
activated compounds will diffuse from the oxygen deficient
fractions into adjacent oxygenated tumor cells. These phenom-
ena should permit selective depletion of solid tumor AGT,
thereby allowing a nearly full dosage of guanine O6-methylating
and chloroethylating agents to be used in the treatment of cancer
patients, resulting in increases in the therapeutic efficacy of
prodrugs containing agents targeting the O-6 position of DNA
guanine in tumors that exhibit resistance to these compounds by
virtue of the presence of relatively high levels of AGT.
7721
dx.doi.org/10.1021/jm201115f |J. Med. Chem. 2011, 54, 7720–7728

Journal of Medicinal Chemistry
ARTICLE
Scheme 2a
a Reagents and conditions: (i) phosgene CH2Cl2/pyridine, 0 °C to room temperature, 20 h; (ii) room temperature, 2ꢀ40 h; (iii) EtOH/1 M NaOH
(6:1), 0 °C, 30 min, followed by neutralization with 10% acetic acid, or 0.1ꢀ1 M NH3 in CH3OH, 20ꢀ40 h.
Table 1. Yields of O6-BG after Prodrug Reduction by
The ability of the chemical and enzyme systems to reduce the
nitro group was also evaluated using nitrobenzene as a model
Zn/EDTAa
nitroaromatic. N-Phenylhydroxylamine and aniline, which are
commercially available, were used as standards for quantifying
the product yields by HPLC under the various reducing condi-
tions employed. The chemical reduction of nitrobenzene
(Table 5), as well as that of the three O6-BG prodrugs, was
carried out using zinc/EDTA. This methodology, which afforded
the complete reduction of the nitro group in nitrobenzene to give
aniline, resulted in the formation of O6-BG from each of the three
synthesized prodrugs, the rank order being 3 g 2 > 1 (Table 1).
Enzymatic reduction of nitrobenzene with xanthine/xanthine
oxidase, as well as with NADPH:cytochrome P450 reductase,
gave a mixture of partially reduced (N-phenylhydroxylamine)
and fully reduced (aniline) products. Xanthine/xanthine oxidase
gave a much higher yield of aniline on a molar basis than
NADPH:cytochrome P450 reductase, which gave N-phenylhy-
droxylamine as the main product of nitroreduction (Table 5).
The relative yields of reduction products had a bearing on the
yield of O6-BG from each prodrug. Thus, enzymatic reduction
with xanthine/xanthine oxidase resulted in the formation of
O6-BG in every case when the three O6-BG prodrugs were employed
as substrates. The rank order of O6-BG formation was unchanged
from the chemical reduction, i.e., 3 g 2 > 1 (Table 2). Enzymatic
reduction by NADPH:cytochrome P450 reductase gave slightly
different results. While all three parental prodrugs (∼20 μM)
were reduced by this enzyme, only 3 gave a significant yield of O6-
BG (Table 3). In addition, a greater proportion of 3 was also
reduced, implying that it was a better substrate than either 1 or 2
for NADPH:cytochrome P450 reductase (Table 3). As observed
earlier, nitroreduction by this enzyme gave the hydroxylamine as
the principal product, as evidenced by the observation that
approximately 8 times as much N-phenylhydroxylamine as ani-
line was formed when nitrobenzene was employed as the
substrate (Table 5). Compound 3 also generated very small
quantities of O6-BG under aerobic conditions. From these results
we can infer that (a) the amino derivatives of 1, 2, and 3 readily
fragment to form O6-BG, with 3 giving the best yield, (b) the
hydroxylamino derivative of 3 generates O6-BG, while the
hydroxylamino derivatives of 1 and 2 do not readily fragment
to give O6-BG, and (c) 3 has a greater tendency to form O6-BG
under aerobic conditions than 1 and 2, possibly because of a low
prodrug
% molar O6-BG yield
1
2
3
4.0 ( 1.1
35.4 ( 3.6
54.8 ( 3.2
a A solution of each agent in 50 mM Tris-HCl, 10 mM EDTA, pH 7.0
buffer was shaken with ∼4 mg/mL Zn dust and allowed to settle. It was
then centrifuged and the supernatant analyzed by HPLC for O6-BG. The
results represent the mean of three independent experiments ( SEM.
material O6-BG. The greater stability of the carbamate linkage in
7 and 8 relative to 6 may be not only due to the steric protection
afforded by the C-methyl groups in compounds 7 and 8 but also
because the electron-releasing inductive effect of the C-methyl
groups rendered the carbonyl carbon less prone to attack by the
hydroxide ion. Therefore, a milder method was devised for the
selective removal of the pivaloyloxymethyl group in compound 6.
Thus, not only did 0.1 M ammonia in methanol afford an
excellent yield of 1 from compound 6 but a similar deprotection
procedure employing 1 M ammonia in methanol gave better
yields of 2 and 3 from compounds 7 and 8, respectively.
’ RESULTS
The synthesized agents were evaluated for their ability to
generate O6-BG by chemical (Table 1) and enzymatic activation
(Tables 2 and 3), as well as by their ability to inhibit AGT
(Table 4). The poor inhibition of AGT by the intact prodrugs 1,
2, and 3 compared to that by O6-BG was essential for the success
of this strategy. Therefore, we compared their relative abilities to
inhibit AGT using a modification of previously described
methodology.25 This assay is based upon the ability of AGT to
repair O6-(2-chloroethyl)guanine and N1,O6-ethanoguanine ad-
ducts in DNA and thereby prevent the formation of 1-(N3-
deoxycytidinyl)-2-(N1-deoxyguanosinyl)ethane cross-links. No
significant inhibition of AGT was observed over a 30 min test
period at 37 °C with 1, 2, and 3 up to the maximum concentra-
tion tested (50 μM), while O6-BG produced about a 50%
1
inhibition at /500 of this concentration under the same condi-
tions (Table 4).
7722
dx.doi.org/10.1021/jm201115f |J. Med. Chem. 2011, 54, 7720–7728

Journal of Medicinal Chemistry
ARTICLE
Table 2. Production of O6-BG from 1, 2, and 3 by Xanthine (X)/Xanthine Oxidase (XO) under Oxygen Deficient and Normoxic
Conditionsa
X/XO activation
1, normoxic
1, oxygen deficient
2, normoxic
2, oxygen deficient
3, normoxic
3, oxygen deficient
[prodrug] μM, T = 0
[O6-BG] μM, T = 0
[prodrug] μM, T = 1
[O6-BG] μM, T = 1
15.4 ( 0.4
BLD
18.8 ( 0.7
BLD
19.0 ( 0.2
BLD
21.4 ( 1.7
BLD
17.7 ( 0.6
BLD
20.8 ( 0.9
BLD
11.7 ( 2.1
BLD
13.4 ( 0.3
2.8 ( 0.5
12.3 ( 2.7
BLD
8.0 ( 1.5
11.6 ( 0.7
11.4 ( 2.7
BLD
4.0 ( 1.1
14.8 ( 0.3
a Production of O6-BG from ∼20 μM 1, 2, and 3 by 1 mM xanthine (X) and 0.16 units/mL xanthine oxidase (XO) was determined under oxygen
deficient and normoxic conditions over a 1 h incubation period at 37 °C. The concentrations are given with (SEM (N = 3) and were determined by
HPLC. BLD = below the limit of detection (<0.05 μM).
Table 3. Production of O6-BG from 1, 2, and 3 by NADPH:Cytochrome P450 Reductase under Oxygen Deficient and Normoxic
Conditionsa
P450 reductase activation
1, normoxic
1, oxygen deficient
2, normoxic
2, oxygen deficient
3, normoxic
3, oxygen deficient
[prodrug] μM, T = 0
[O6-BG] μM, T = 0
[prodrug] μM, T = 1
[O6-BG] μM, T = 1
18.2 ( 0.9
BLD
17.6 ( 0.5
BLD
19.8 ( 2.1
BLD
18.0 ( 1.1
BLD
20.3 ( 1.2
BLD
19.3 ( 0.9
BLD
15.5 ( 2.8
BLD
13.8 ( 2.6
BLD
18.8 ( 2.0
BLD
17.3 ( 1.6
0.1 ( 0.1
18.2 ( 2.8
0.1 ( 0.1
4.2 ( 1.0
13.9 ( 0.2
a Production of O6-BG from ∼20 μM 1, 2, and 3 by 1 mM NADPH and 1.04 units/mL NADPH:cytochrome P450 reductase was determined under
oxygen deficient and normoxic conditions over a 1 h incubation period at 37 °C. The concentrations are given with (SEM (N = 3) and were determined
by HPLC. BLD = below the limit of detection (<0.05 μM).
Table 4. AGT Inactivation by Various Agentsa
Reductive activation of the synthesized prodrugs was also
studied using two tumor cell lines, the murine EMT6 mammary
agent
percent AGT inactivation
carcinoma and the human DU145 prostate carcinoma (Figure 2).
While only 3 generated measurable quantities of O6-BG in the
case of the DU145 under conditions of oxygen deficiency, all
three compounds generated O6-BG in the rank order 3 > 2 > 1 in
the case of the EMT6 carcinoma. Little or no aerobic activation
was seen with these agents in both the enzymatic and cellular
studies. As with the enzymatic studies, some loss of parental
agent was observed with both of these cell lines under normoxic
conditions. This normoxic loss was most marked with 1; how-
ever, this loss did not result in the formation of O6-BG. These
findings collectively indicate that the linker used in 3 is superior to
those employed in 1 and 2. Furthermore, clonogenic assays using
3 at 40 μM sensitized DU145 prostate carcinoma cells to various
concentrations of laromustine (50ꢀ200 μM) under conditions of
oxygen deficiency but not under aerobic conditions (Figure 3).
O6-BG
47 ( 3
<1
1
2
3
<1
<1
a Comparison of the levels of purified recombinant human AGT
inactivation by 50 μM 1, 2, or 3 and 0.1 μM O6-BG after a 30 min
incubation at 37 °C by assaying the ability of each agent to repair
substrate DNA containing O6-(2-chloroethyl)guanine and N1,O6-etha-
noguanine lesions relative to AGT incubated in the absence of agent.
Values are the mean of three determinations ((SEM where applicable).
Table 5. Nitrobenzene Reducing Agent Dependent Aniline:
Phenylhydroxylamine Reduction Product Ratiosa
Zn/EDTA
xanthine/xanthine oxidase
NADPH:P450 reductase
1:0.0
1:0.7 ( 0.2
1:7.8 ( 1.9
’ DISCUSSION AND CONCLUSIONS
a The influence of different reducing agents on the nitrobenzene
reduction product ratio of aniline:N-phenylhydroxylamine. Nitroben-
zene was reduced with either Zn dust/EDTA, xanthine/xanthine
oxidase, or NADPH:cytochrome P450 reductase as described in the
Experimental Section, and the reduction products were analyzed by
HPLC. The concentration ratios are the mean of three deteriminations
( SEM.
The three factors that would be expected to have an impact on
the activation of the synthesized O6-BG prodrugs are (a) the ease
and extent of reduction of the nitro group, (b) the relative
position of the nitro group with respect to the side chain, and (c)
the ease of fragmentation of the CꢀO bond, where C is the
benzylic carbon, once the nitro group is converted to the
hydroxylamino or amino function. Although all three prodrugs
have similar half-wave reduction potentials, as measured by
differential pulse polarography (Table 6), 3 is a much better
substrate for NADPH:cytochrome P450 reductase than 2 and 1.
The relative position of the nitro group with respect to the side
chain is identical for all three prodrugs and therefore cannot
explain the differences in the yields of O6-BG generated from
these agents. However, any factor that stabilizes the partial
positive charge on the benzylic carbon in the transition state of
level of reduction despite the presence of oxygen or because of
the relief of steric strain or because of other possible routes of
activation. However, this background rate was always relatively
small when observed. Loss of initial material was observed with
all three prodrugs under normoxic conditions with both enzyme
systems, but this loss of parental agent was not associated with
any significant O6-BG production.
7723
dx.doi.org/10.1021/jm201115f |J. Med. Chem. 2011, 54, 7720–7728

Journal of Medicinal Chemistry
ARTICLE
Figure 2. Loss of parental prodrugs (1, upper row; 2, middle row; 3, lower row) and the subsequent generation of O6-BG over a 3 h 37 °C incubation
period in medium in the presence of either 107/mL DU145 cells (left-hand column) or 107/mL EMT6 cells (right-hand column). In all panels,
9 represents the concentration of parental prodrug under normoxic conditions, 1 represents the concentration of parental prodrug under oxygen
deficient conditions, 2 represents the concentration of generated O6-BG under normoxic conditions, and [ represents the concentration of generated
O6-BG under oxygen deficient conditions. The plotted values represent the mean of three separate experiments ( SEM.
the fragmentation step, following reduction of the nitro group to
a hydroxylamino or amino function, will facilitate the release of
O6-BG. In the reduction product of 3 the charge is stabilized not
only by the electron-releasing mesomeric (resonance) effect of
the hydroxylamino or amino group in the para position but also
by the electron-releasing inductive effect of not one but two
methyl groups. However, in the absence of the benzylic methyl
group(s), a complete reduction of the nitro group to the highly
electron-releasing amino function, relative to the more weakly
electron releasing hydroxylamino group, appears to be needed
for the release of O6-BG from the compounds under study. Thus,
3 is not only more easily reduced by a key nitroreductase, i.e.,
NADPH:cytochrome P450 reductase, than 2 and 1 but also has
the structural features necessary for a more facile fragmentation
once the nitro group is reduced.
analogue. It is conceivable that factors similar to those described
for the 6-thioguanine prodrugs are operative in this case.
In the enzymatic and cellular studies, loss of parental prodrug
was also seen to various degrees under normoxic conditions; this
occurred without the concomitant generation of O6-BG. This
loss could be due to the reaction of the prodrugs with reactive
oxygen species generated as a consequence of prodrug redox
cycling under normoxic conditions and in the case of xanthine
oxidase directly from the oxidation of xanthine with oxygen. In
the presence of cells, additional metabolic routes not resulting in
O6-BG formation may contribute to this normoxic loss. The
cellular sensitization studies which indicated pronounced poten-
tiation of the toxicity of laromustine only under oxygen deficient
conditions strongly support the lack of formation of AGT
inhibitory agents of any type under normoxic conditions.
Surprisingly, the rank order of water solubility for the three
prodrugs is 3 > 2 > 1. The absence of a chiral center also makes 3
a more attractive candidate for further possible development as a
pharmaceutical than 2. Thus, 3, conceived as a hypoxia-activated
prodrug of O6-BG, appears to be a promising new agent with the
following desirable properties:
Thomson et al.26 recently reported the evaluation of 4-nitro-
benzyl and gem-dimethyl substituted 4-nitrobenzyl derivatives of
6-thioguanine as hypoxia-selective agents. While reduction of the
nitro group did not result in the release of 6-thioguanine in the
case of the desmethyl analogue, hypoxia-selective release of
6-thioguanine was indeed observed with the gem-disubstituted
7724
dx.doi.org/10.1021/jm201115f |J. Med. Chem. 2011, 54, 7720–7728

Journal of Medicinal Chemistry
ARTICLE
with a guanine O-6 alkylating agent following nonselective
(global) ablation of AGT.
’ EXPERIMENTAL SECTION
All chemicals, solvents, reagents, and enzymes were purchased from
Sigma-Aldrich (St. Louis, MO) and used without further purification
with the exceptions of laromustine which was provided by Vion
Pharmaceuticals Inc. (New Haven, CT), the fluorescent dye Hoechst
33258 which was purchased from Molecular Probes, Inc. (Eugene, OR.),
and purified recombinant human AGT which was a kind gift from
Dr. Joann Sweasy (Yale Medical School, New Haven, CT).
Melting points were determined on a Thomas-Hoover Unimelt
melting point apparatus and are uncorrected. NMR spectra were
recorded on a Varian EM-390 (90 MHz) or Bruker Avance DRX-400
(400 MHz) NMR spectrometer with tetramethylsilane as an internal
standard. Mass spectra were recorded on a VG-ZAB-SE mass spectro-
meter in the fast atom bombardment mode (glycerol matrix). Column
chromatography was conducted with Merck silica gel 60, 230ꢀ400
mesh. Thin layer chromatography was performed on EM precoated
silica gel sheets containing a fluorescent indicator. High resolution mass
spectra were obtained at the W. M. Keck Foundation Biotechnology
Resource Laboratory at Yale University. The purity of the tested
compounds was determined by analytical HPLC, using a Beckman
System Gold HPLC system, comprising a 168 UV/vis detector, a 502
autosampler, and a 127P solvent module monitoring at 280 nm with a
40 nm bandwidth. The purity of the tested compounds was >95% in
each and every case.
Figure 3. Sensitization of DU145 human prostate carcinoma cells to
laromustine by 3 induced inhibition of AGT in clonogenic experiments.
DU145 AGT containing cells in confluent monolayers were exposed to
40 μM 3 for 6 h and then treated with graded concentrations of
laromustine for a total of 24 h under conditions of normoxia (9) or
oxygen deficiency (2). The cytotoxicity of laromustine alone under
conditions of normoxia (1) or oxygen deficiency ([) is also displayed.
After treatment, cells were detached by trypsinization and survival was
quantified using clonogenicity. The Y axis indicatesthe percent survivaland
the X axis the concentration of laromustine employed. Oxygen was
removed from test samples enzymatically in the presence of 2 units/mL
glucose oxidase, 120 units/mL catalase, and 25 mM glucose during incuba-
tion and plating of cells to measure cloning efficiency. All points are the
result of at least three independent experimental determinations ( SEM.
(6-(Benzyloxy)-2-((((4-nitrobenzyl)oxy)carbonyl)amino)-
9H-purin-9-yl)methyl Pivalate (6). To a stirred solution of (2-amino-
6-(benzyloxy)-9H-purin-9-yl)methyl pivalate (4) (1.06 g, 3 mmol) and
dry pyridine (3 mL) in 70 mL of anhydrous dichloromethane was added
a 20% solution of phosgene in toluene (1.5 mL, 3.4 mmol) dropwise at
0ꢀ5 °C. The mixture was stirred overnight, while the ice bath was
allowed to warm to room temperature. A solution of 4-nitrobenzyl
alcohol (0.46 g, 3 mmol) in anhydrous dichloromethane (10 mL) was
added, and the reaction mixture was stirred at room temperature for 2 h.
It was then evaporated with 5 g of silica gel to dryness in vacuo and the
residue was purified by column chromatography (silica gel, dichloro-
methaneꢀethanol, 30:1) to give 1.42 g (89%) of the title compound as a
Table 6. Polarographic Half-Wave Reduction Potentials of
Synthesized Prodrugs
prodrug
1
2
3
polarographic half-wave
ꢀ463 mVa
ꢀ447 mVa
ꢀ449 mVa
reduction potential, pH 7.0
a The supporting electrolyte contained 10% DMSO in all cases for
consistency. The DMSO was required to aid in the dissolution of 1. The
presence of 10% DMSO resulted in the measured polarographic half-
wave reduction potentials being shifted approximately 50 mV more
negative than in its absence.
1
white solid: mp 146ꢀ148 °C; H NMR (CDCl3, 90 MHz) δ 10.20
(a) The intact 3 prodrug is >500-fold less effective as an AGT
inhibitor than O6-BG.
(b) Reduction of 3 by common nitroreductases such as
NADPH:cytochrome P450 reductase and xanthine/
xanthine oxidase under oxygen-deficient conditions re-
sults in the generation of O6-BG.
(c) The activation rate for 3 under normoxic conditions is low
or below the limit of detection under the conditions and
incubation times examined to date.
(s, D2O exchangeable,1H), 8.24 (d, J = 8.5 Hz, 2H), 8.03 (s, 1H),
7.33ꢀ7.61 (m, 7H), 6.08 (s, 2H), 5.62 (s, 2H), 5.37 (s, 2H), 1.16
(s, 9H); MS m/z 535 [M + H]+.
Compounds 7 and 8 were synthesized using procedures analogous to
the one described above except that for compound 7, after the addition
of 1-(4-nitrophenyl)ethanol, the reaction mixture was stirred at room
temperature for 6 h, and for compound 8, the reaction mixture was stirred
at room temperature for 40 h after the addition of 2-(4-nitrophenyl)-
propan-2-ol.
(6-(Benzyloxy)-2-(((1-(4-nitrophenyl)ethoxy)carbonyl)-
amino)-9H-purin-9-yl)methyl Pivalate (7). Compound 7 was
obtained in an 81% yield as a white solid: mp 140ꢀ142 °C; 1H NMR
(CDCl3, 90 MHz) δ 10.34 (s, D2O exchangeable, 1H), 8.26 (d, J = 9 Hz,
2H), 8.04 (s, 1H), 7.54 (d, J = 9 Hz, 2H), 7.34ꢀ7.41 (m, 5H), 6.07
(s, 2 H), 6.06 (q, J = 6.6 Hz, 1H), 5.61 (s, 2H), 1.65 (d, J = 6.6 Hz, 3H),
1.15 (s, 9H); MS m/z 549 [M + H]+.
(6-(Benzyloxy)-2-((((2-(4-nitrophenyl)propan-2-yl)oxy)-
carbonyl)amino)-9H-purin-9-yl)methyl Pivalate (8). Compound
8 was obtained in a 68% yield as a white solid: mp 92ꢀ94 °C; 1H NMR
(CDCl3, 90 MHz) δ 10.20 (s, D2O exchangeable, 1H), 8.19 (d, J = 9 Hz,
2H), 7.97 (s, 1H), 7.45 (d, J = 9 Hz, 2H), 7.29ꢀ7.45 (m, 5H), 6.02
(s, 2 H), 5.58 (s, 2H), 1.87 (s, 6H), 1.13 (s, 9H); MS m/z 563 [M + H]+.
(d) Once O6-BG is released in the oxygen deficient compart-
ment, it is conceivable that the inhibitor will diffuse into the
surrounding oxygenated tumor cells and sensitize them to
the cytodestructive effects of guanine O-6 alkylating agents
(i.e., will exert a bystander effect). However, very exten-
sive release and equilibration around host tissues could
be problematic, since it would attenuate the targeting
effect.
(e) The trigger/linker employed (i.e., the gem-dimethyl
4-nitrobenzyloxycarbonyl moiety) ensures that release
of O6-BG in normal tissues will be relatively low relative
to oxygen deficient areas of solid tumors. This should
minimize the undesirable effects that result from treatment
7725
dx.doi.org/10.1021/jm201115f |J. Med. Chem. 2011, 54, 7720–7728

Journal of Medicinal Chemistry
ARTICLE
4-Nitrobenzyl (6-(Benzyloxy)-9H-purin-2-yl)carbamate (1).
A suspension of 6 (0.53 g, 1 mmol) in 0.1 M ammonia in methanol
(40 mL) was stirred at room temperature until TLC showed a complete
disappearance of the starting material (approximately 40 h). The white
solid that was formed was collected by filtration after cooling the reaction
mixture inaniceꢀwater bathfor 1 h, washed withmethanolandether, and
dried to give 0.36 g (85%) of the target molecule: mp 233ꢀ235 °C; 1H
NMR (400 MHz, DMSO-d6) δ 13.24 (s, 1H), 10.51 (s, 1H), 8.29ꢀ8.21
(m, 3H), 7.74 (d, J = 8.7 Hz, 2H), 7.61ꢀ7.49 (m, 2H), 7.46ꢀ7.26 (m,
3H), 5.59 (s, 2H), 5.34 (s, 2H); 13C NMR (101 MHz, DMSO-d6)
δ 151.8, 151.7, 147.0, 144.7, 136.3, 128.9, 128.5, 128.3, 123.6, 67.5, 64.5.
HRMS, calculated for C20H16N6O5, m/z, 421.1255 [(M + H)+]; found,
421.1257.
1-(4-Nitrophenyl)ethyl (6-(Benzyloxy)-9H-purin-2-yl)carba-
mate (2). Method 1. To an ice-cooled solution of 7 (1.2 g, 2.2 mmol) in
ethanol (60 mL) was added an ice-cooled solution of 1 M sodium
hydroxide (10 mL), and the mixture was stirred at 0 °C for 30 min. The
reaction mixture was neutralized with 10% acetic acid and was evapo-
rated with 5.8 g of silica gel to dryness in vacuo. The residue was
chromatographed on a silica gel column (60 Å, 70ꢀ230 mesh) and
eluted with CH2Cl2/EtOH, 20:1 (v/v), to give 0.57 g (60%) of the title
compound as a white solid.
mixture was incubated for a further 30 min at 37 °C to allow the residual
AGT activity to repair the DNA. This mixture was then diluted 10-fold
with 5 mM Tris-HCl, 1 mM EDTA, and 1 mM NaN3, pH 8.0 buffer,
then incubated at 50 °C for 2ꢀ3 h to allow the unrepaired cross-link
precursors to progress to cross-links. The level of cross-linking was then
measured using a previously described fluorescence methodology.6 This
involved dilution of the sample to a volume of 1.5 mL with 5 mM Tris-
HCl, 1.0 mM EDTA, and 1.0 mM NaN3 buffer, pH 8.0, containing
0.1 μg/mL of Hoechst H33258 fluorescent dye, and measuring the
fluorescence using a Hoefer Scientific Instruments TKO 100 fluorom-
eter. The mixture was then heated in a 100 °C hot-block for 5 min and
then plunged into a water bath at room temperature for 5 min, and the
fluorescence measured again. The percentage of DNA molecules that
were cross-linked (i.e., containing at least one cross-link per molecule)
was then calculated from the change in fluorescence.
Polarographic Determination of Half-Wave Reduction
Potentials. Differential pulse polarography (DPP) voltagrams of the
synthesized agents were obtained using a pH 7.0 buffer composed of
100 mM potassium chloride and 50 mM potassium phosphate as the
supporting electrolyte containing 10% DMSO by volume. Compounds
were added as 0.5% of a 10 mM solution in DMSO to give final
concentrations of 50 μM. The samples were purged with nitrogen to
remove dissolved oxygen, and DPP voltammograms were obtained
using a Princeton Applied Research electrochemical trace analyzer
model 394, linked to a model 303A static mercury drop electrode
(Princeton Applied Research, Oak Ridge, TN, U.S.). Scans were
performed from 0 to ꢀ900 mV (2 mV/s) using a platinum counter
electrode versus an Ag/AgCl reference electrode (saturated KCl/AgCl
electolyte). A pulse amplitude of 50 mV was used, and the polarographic
half-wave reduction potential E1/2 was calculated from the peak current
potential (EP) according to the following equation: E1/2 = EP ꢀ pulse
amplitude/2.27
Method 2. Compound 2 was also synthesized using a procedure
analogous to the one described for 1 except that 1 M ammonia in
methanol was used as a base in lieu of 0.1 M ammonia and was obtained
1
as a white solid: yield, 79%; mp 134ꢀ135 °C (dec); H NMR (400
MHz, DMSO-d6) δ 13.22 (s, 1H), 10.44 (s, 1H), 8.42ꢀ8.12 (m, 3H),
7.76 (d, J = 8.7 Hz, 2H), 7.56 (dd, J = 7.9, 1.5 Hz, 2H), 7.46ꢀ7.26 (m,
3H), 5.96 (q, J = 6.6 Hz, 1H), 5.59 (s, 2H), 1.56 (d, J = 6.6 Hz, 3H); 13C
NMR (101 MHz, DMSO-d6) δ 151.7, 151.3, 146.9, 136.3, 128.9, 128.5,
128.3, 126.9, 123.7, 71.2, 67.5, 22.5. HRMS, calculated for C21H18N6O5,
m/z, 435.1411 [(M + H)+]; found, 435.1414.
2-(4-Nitrophenyl)propan-2-yl (6-(Benzyloxy)-9H-purin-2-
yl)carbamate (3). Compound 3 was synthesized using procedures
analogous to the ones described for 2 and was obtained as a white solid:
yield, 63% (method 1), 82% (method 2); mp 246 °C (dec); 1H NMR
(400 MHz, DMSO-d6) δ 13.21 (s, 1H), 10.36 (s, 1H), 8.34ꢀ8.19
(m, 3H), 7.79 (dd, J = 9.2, 2.1 Hz, 2H), 7.57 (dd, J = 7.8, 1.4 Hz, 2H),
7.46ꢀ7.27 (m, 3H), 5.61 (s, 2H), 1.84 (s, 6H); 13C NMR (101 MHz,
DMSO-d6) δ 154.1, 151.8, 150.5, 146.3, 136.3, 128.9, 128.4, 128.3,
125.8, 123.5, 80.2, 67.5, 28.4. HRMS, calculated for C22H20N6O5, m/z,
449.1568 [(M + H)+]; found, 449.1571.
AGT Inactivation Assay. AGT substrate DNA was prepared by
treating L1210 DNA (175 μg/mL) in 10 mM Tris-HCl buffer (pH
∼7.4) with 0.2 mM 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine
for 3 min at 37 °C. This substrate DNA was then stored at 0 °C until
used. A 10 μL aliquot of substrate DNA containing O6-(2-chlor-
oethyl)guanine and N1,O6-ethanoguanine lesions (∼50 fmol) was
incubated for 30 min at 37 °C with 40 fmol of purified recombinant
human AGT which was pretreated (or not) with various inhibitors for
30 min at 37 °C. This assay gives a moderately linear decrease in these
DNA lesions in proportion to the level of AGT activity until the lesions
have been largely titrated. The level of remaining cross-link precursors in
this mixture was then determined and the level of inhibition of AGT
calculated from this value. The procedure is as follows. About 40 fmol of
AGT in AGT stabilization buffer of the following composition was used:
20 mM Tris-HCl, 1 mM EDTA, 1 mg/mL of bovine serum albumin,
0.1 mM dithiothreitol, and 2 μg/mL of L1210 DNA, pH 7.4 (the L1210
DNA greatly contributes to the AGT stability but does not add
significantly to the total DNA in the final assay). This was mixed with
an equal volume of 2ꢁ concentration of inhibitor in an equivalent
amount of buffer to give a mixture of the desired inhibitor and AGT
concentration. This mixture was then incubated for 30 min at 37 °C.
Then an amount of 10 μL was added to 10 μL of substrate DNA, and the
Determination by HPLC. HPLC measurements of the concentra-
tions of nitrobenzene, N-phenylhydroxylamine, aniline, 1, 2, and 3 were
performed using a Beckman 127P solvent module and a Beckman 168
UV/vis detector (Beckman, Fullerton, CA, U.S.). A standard curve for
estimation of total agent was established, and a linear relationship
between concentration and the area under the curve was found in all
cases. Samples in 50% v/v acetonitrile were separated on a 5 μm,
220 mm ꢁ 4.6 mm Applied Biosystems RP-18 C-18 reverse phase
column (Applied Biosystems, Carlsbad, CA, U.S.) by elution with 34%
acetonitrile in buffer (0.03 M KH2PO4, 1.0 mM NaN3, pH 5.4) for 5 min
followed by a 34ꢀ70% acetonitrile linear gradient in buffer, at a flow rate
of 0.6 mL/min from 5 to 35 min. After this point the concentration of
acetonitrile was maintained at this level for 5 min and then returned to
the starting concentration over an additional 5 min. Absorbance was
monitored at 280 nm using a Beckman 168 UV/vis detector.
Reduction of Nitrobenzene with Zn/EDTA. A solution of
nitrobenzene at 1 mM in 50 mM Tris-HCl, 10 mM EDTA, pH 7.0, was
quickly shaken with approximately 4 mg/mL Zn dust and allowed to
settle at room temperature, then centrifuged at 10000g for 4 min. The
supernatant was then diluted 10-fold with 30 mM potassium phosphate
buffer, pH 5.4, and analyzed by HPLC.
Reduction of Nitrobenzene with Xanthine/Xanthine Oxi-
dase. A solution of nitrobenzene at 100 μM in 100 mM potassium
phosphate buffer, pH 7.4, containing 2.5 mM xanthine added as 1% of a
250 mM solution in 1 M NaOH was purged with nitrogen and 0.16
units/mL xanthine oxidase (Sigma, bovine milk, X4500-5UN) added
and the mixture incubated at 37 °C. Samples were taken at 2 h and
analyzed by HPLC for aniline, nitrobenzene, and N-phenylhydroxyla-
mine. Nitrobenzene, N-phenylhydroxylamine, and aniline eluted at
approximately 20, 10, and 8 min, respectively.
Reduction of Nitrobenzene with NADPH:Cytochrome
P450 Reductase. A solution of nitrobenzene at 100 μM in 100 mM
7726
dx.doi.org/10.1021/jm201115f |J. Med. Chem. 2011, 54, 7720–7728

Journal of Medicinal Chemistry
ARTICLE
potassium phosphate buffer, pH 7.85, the pH optimum for NADPH:
cytochrome P450 reductase (Sigma, human recombinant C8113),
containing 10 mM NADPH, 10 mM glucose, 2 units/mL glucose oxidase,
120 units/mL catalase, and 4 units/mL of NADPH:cytochrome P450
reductase, added after 5 min to allow the oxygen to be depleted, was
incubated for 2 h in a sealed tube at 37 °C. Then the mixture was analyzed
by HPLC for aniline, nitrobenzene, and N-phenylhydroxylamine.
Reduction of 1, 2, and 3 with Zn/EDTA. A solution of each
agent at 50 μM in 50 mM Tris-HCl, 10 mM EDTA, pH 7.0, was quickly
shaken with approximately 4 mg/mL Zn dust and allowed to settle at
room temperature, then centrifuged at 10000g for 4 min. The super-
natant was then analyzed by HPLC.
Reduction of 1, 2, and 3 by NADPH:Cytochrome P450
Reductase and Xanthine Oxidase. In these experiments, oxygen
deficiency was enzymatically generated as previously described.28
Briefly, glucose/glucose oxidase was used to rapidly consume the
available free oxygen and catalase was employed to remove the
generated hydrogen peroxide. In the absence of other components, this
system itself does not measurably reduce 1, 2, and 3 in the time frames
employed in these experiments. The reaction mixtures in a total volume
of 0.5 mL were as follows: 100 mM potassium phosphate buffer, pH 7.4,
containing 10 mM glucose, 5 μL of a solution of 200 units/mL glucose
oxidase and 12 000 units/mL catalase, 20 μM test agent added as a 100ꢁ
solution in DMSO plus either 5 μL of 104 units/mL NADPH:
cytochrome P450 reductase plus 1 mM NADPH or 0.16 units/mL
xanthine oxidase plus 1 mM xanthine. The aerobic reaction was identical
except that the glucose needed for the oxygen deficiency system to
operate was omitted. Furthermore, the aerobic reaction volumes were
scaled up 10-fold and the mixture was incubated as a shallow layer with
shaking in sealed 25 cm2 plastic culture flasks to ensure that full air
saturation was maintained and evaporation prevented. The reaction
mixtures were incubated at 37 °C, and samples were removed at 0 and
1 h after initiation, mixed with an equal volume of CH3CN, and cen-
trifuged at 10000g for 10 min to sediment any precipitated protein. The
supernatant was then analyzed as described above for the remaining
agent and reduction products using HPLC.
caps screwed on tightly. This action facilitated oxygen depletion of the
medium by glucose oxidase through removal of residual oxygen contain-
ing air and denial of the entry of additional air. After treatment,
monolayers were rinsed with phosphate buffered saline, and cells were
detached by trypsinization, suspended in culture medium, and counted
and sequential cell dilutions were plated in duplicate into six-well plates
at a density of 1 ꢁ 102, 1 ꢁ 103, and 1 ꢁ 104 cells per well. At 10ꢀ14
days later, colonies were fixed, stained with 0.25% crystal violet in 80%
methanol, and quantified. DMSO concentrations were e0.05% and
nontoxic. Cells under aerobic conditions were treated under similar
conditions and cytotoxic agent concentrations but in unsealed flasks
without glucose oxidase and catalase. Cells were then washed, harvested
by trypsinization, and assayed for survival using a clonogenic assay
described previously.3,30,31 In the absence of cells, no measurable direct
metabolism of 3 or of laromustine was detected in the presence of the
glucose oxidase and catalase enzyme components of the oxygen
deficiency generating system.28
’ AUTHOR INFORMATION
Corresponding Author
*Telephone: (203) 785-4524. Fax: (203) 737-2045. E-mail:
philip.penketh@gmail.com.
’ ACKNOWLEDGMENT
This work was supported in part by U.S. Public Health Service
Grants CA090671, CA122112, and CA129186 from the Na-
tional Cancer Institute and a grant from the National Foundation
for Cancer Research.
’ ABBREVIATIONS USED
O6-BG, O6-benzylguanine; AGT, O6-alkylguanine-DNA alkyl-
transferase
Cellular Activation of 1, 2, and 3 under Normoxic and
Oxygen Deficient Conditions. Experiments, following the time
course of prodrug loss and O6-BG generation when ∼20 μM 1, 2, or 3
was incubated with either EMT6 or DU145 cells, were conducted as
follows. Cells at 107/mL in Dulbecco’s minimal essential medium
(DMEM) supplemented with 10% fetal bovine serum (EMT6) or in
α-minimum essential medium with 10% fetal bovine serum (DU145)
containing 10 mM glucose were shaken as cell suspensions as shallow
layers (5 mL) in 25 cm2 plastic culture flasks for the normoxic
incubations. For the oxygen deficient condition incubations, the cells
were incubated in sealed microfuge tubes with an additional 10 mM
glucose and 2 units/mL glucose oxidase plus 120 units/mL catalase as
additional components. Samples were removed at 0, 1, 2, and 3 h after the
start of the incubations and mixed with an equal volume of acetonitrile to
lyze the cells, precipitate the macromolecules, and extract the remaining
prodrug and generated O6-BG. The mixture was allowed to stand for
15 min at room temperature, centrifuged at 10000g for 10 min and the
supernatant analyzed by HPLC for parental prodrug and O6-BG.
Cytotoxicity Studies. Cell survival (clonogenic) assays were
performed using a previously described method.3 The 25 cm2 plastic
tissue culture flasks were seeded with 2.5 ꢁ 105 cells each and 3 days later
cells were pretreated for 6 h in the presence of 3 prior to the addition of
laromustine dissolved in 10 mL of medium for 24 h at 37 °C. All agents
were initially dissolved in DMSO and then diluted to the required concentra-
tion. For oxygen-deficient conditions, cells were incubated with laro-
mustine in the presence of 2 units/mL glucose oxidase (Sigma G6641),
120 units/mL catalase (Sigma, C1345) in high glucose DMEM
(Invitrogen).28,29 Flasks were flushed with nitrogen for 10 s and the
’ REFERENCES
(1) Shyam, K.; Penketh, P. G.; Loomis, R. H.; Rose, W. C.; Sartorelli,
A. C. Antitumor 2-(aminocarbonyl)-1,2-bis(methylsulfonyl)-1-(2-chloro-
ethyl)hydrazines. J. Med. Chem. 1996, 39, 796–801.
(2) Finch, R. A.; Shyam, K.; Penketh, P. G.; Sartorelli, A. C. 1,
2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-(methylamino)carbonylhydra-
zine (101M): a novel sulfonylhydrazine prodrug with broad-spectrum
antineoplastic activity. Cancer Res. 2001, 61, 3033–3038.
(3) Baumann, R. P.; Seow, H. A.; Shyam, K.; Penketh, P. G.;
Sartorelli, A. C. The antineoplastic efficacy of the prodrug cloretazine
is produced by the synergistic interaction of carbamoylating and
alkylating products of its activation. Oncol. Res. 2005, 15, 313–325.
(4) Ishiguro, K.; Shyam, K.; Penketh, P. G.; Sartorelli, A. C. Role of
O6-alkylguanine-DNA alkyltransferase in the cytotoxic activity of clor-
etazine. Mol. Cancer Ther. 2005, 4, 1755–1763.
(5) Ishiguro, K.; Seow, H. A.; Penketh, P. G.; Sartorelli, A. C. Mode of
action of the chloroethylating and carbamoylating moiety of the prodrug
cloretazine. Mol. Cancer Ther. 2006, 5, 969–976. PMCID: PMC2680221.
(6) Penketh, P. G.; Baumann, R. P.; Ishiguro, K.; Shyam, K.; Seow,
H. A.; Sartorelli, A. C. Lethality to leukemia cell lines of DNA interstrand
cross-links generated by cloretazine derived alkylating species. Leuk. Res.
2008, 32, 1546–1553. PMCID: PMC2888535.
(7) Shyam, K.; Penketh, P. G.; Shapiro, M.; Belcourt, M. F.; Loomis,
R. H.; Rockwell, S.; Sartorelli, A. C. Hypoxia-selective nitrobenzyloxy-
carbonyl derivatives of 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydra-
zine. J. Med. Chem. 1999, 42, 941–946.
(8) Seow, H. A.; Penketh, P. G.; Shyam, K.; Rockwell, S.; Sartorelli,
A. C. 1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2[[1-(4-nitrophenyl)-
ethoxy]carbonyl]hydrazine (KS119): an anticancer agents targeting
7727
dx.doi.org/10.1021/jm201115f |J. Med. Chem. 2011, 54, 7720–7728

Journal of Medicinal Chemistry
ARTICLE
hypoxic cells. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 9282–9287.
PMCID: PMC1166587.
species generated by decomposition. Cancer Chemother. Pharmacol.
2004, 53, 279–287.
(26) Thomson, P.; Naylor, M. A.; Stratford, M. R. L.; Lewis, G.; Hill,
S.; Patel, K. B.; Wardman, P.; Davis, P. D. Hypoxia-driven elimination of
thiopurines from their nitrobenzyl prodrugs. Bioorg. Med. Chem. Lett.
2007, 17, 4320–4322.
(27) Bianco, P.; Haladjian, J. Study of two forms of ferredoxin from
Desulfovibrio gigas by differential pulse polarography. Biochem. Biophys.
Res. Commun. 1977, 78, 323–327.
(9) Pegg, A. E. Mammalian O6-alkylguanine-DNA alkyltransferase:
regulation and importance in response to alkylating carcinogenic and
therapeutic agents. Cancer Res. 1990, 50, 6119–6129.
(10) Brent, T. P.; Remack, J. S. Formation of covalent complexes
between human O6-alkylguanine-DNA alkyltransferase and BCNU-
treated defined length oligodeoxynucleotides. Nucleic Acids Res. 1988,
16, 6779–6788.
(11) Baumann, R. P.; Shyam, K.; Penketh, P. G.; Remack, J.; Brent,
T. P.; Sartorelli, A. C. 1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2
mac_hyphen;[(methylamino)carbonyl]hydrazine (VNP40101M): II.
Role of O6-alkylguanine DNA alkyltransferase in cytotoxicity. Cancer
Chemother. Pharmacol. 2004, 53, 288–295.
(28) Baumann, R.; Penketh, P.; Seow, H.; Shyam, K.; Sartorelli, A.
Generation of oxygen deficiency in cell culture using a two-enzyme
system to evaluate agents targeting hypoxic tumor cells. Radiat. Res.
2008, 170, 651–660. PMCID: PMC2667281.
(29) Baumann, R. P.; Penketh, P. G.; Ishiguro, K.; Shyam, K.; Zhu,
Y. L.; Sartorelli, A. C. Reductive activation of the prodrug 1,2-bis-
(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophe-
nyl)ethoxy]carbonyl]hydrazine (KS119) selectively occurs in oxygen-
deficient cells and overcomes O6-alkylguanine-DNA alkyltransferase
mediated KS119 tumor cell resistance. Biochem. Pharmacol. 2010,
79, 1553–1561. PMCID: PMC2885764.
(30) Rockwell, S. In vivoꢀin vitro tumor systems: new models for
studying the response of tumors to therapy. Lab. Animal Sci. 1977,
27, 831–851.
(12) Gerson, S. L.; Willson, J. K. O6-Alkylguanine-DNA alkyltrans-
ferase. A target for the modulation of drug resistance. Hematol. Oncol.
Clin. North Am. 1995, 9, 431–450.
(13) Schold, S., Jr.; Brent, T. P.; von Hofe, E.; Friedman, H. S.; Mitra,
S.; Bigner, D. D.; Swenberg, J. A.; Kleihues, P. O6-Alkylguanine-DNA
alkyltransferase and sensitivity to procarbazine in human brain-tumor
xenografts. J. Neurosurg. 1989, 70, 573–577.
(14) Dolan, M. E.; Moschel, R. C.; Pegg, A. E. Depletion of
mammalian O6-alkylguanine-DNA alkyltransferase activity by O6-ben-
zylguanine provides a means to evaluate the role of this protein in
protection against carcinogenic and therapeutic alkylating agents. Proc.
Natl. Acad. Sci. U.S.A. 1990, 87, 5368–5372.
(15) Dolan, M. E.; Mitchell, R. B.; Mummert, C.; Moschel, R. C.;
Pegg, A. E. Effect of O6-benzylguanine analogues on sensitivity of human
tumor cells to the cytotoxic effects of alkylating agents. Cancer Res. 1991,
51, 3367–3372.
(31) Belcourt, M. F.; Hodnick, W. F.; Rockwell, S.; Sartorelli, A. C.
The intracellular location of NADH:cytochrome b5 reductase modu-
lates the cytotoxicity of the mitomycins to Chinese hamster ovary cells.
J. Biol. Chem. 1998, 273, 8875–8881. PMCID: PMC40257.
(16) Baer, J. C.; Freeman, A. A.; Newlands, E. S.; Watson, A. J.;
Rafferty, J. A.; Margison, G. P. Depletion of O6-alkylguanine-DNA
alkyltransferase correlates with potentiation of temozolomide and
CCNU toxicity in human tumor cells. Br. J. Cancer 1993, 67, 1299–1302.
(17) Magull-Seltenreich, A.; Zeller, W. J. Sensitization of human
colon tumor cell lines to carmustine by depletion of O6-alkylguanine-
DNA alkyltransferase. J. Cancer Res. Clin. Oncol. 1995, 121, 225–229.
(18) Quinn, J. A.; Pluda, J.; Dolan, M. E.; Delaney, S.; Kaplan, R.;
Rich, J. N.; Friedman, A. H.; Reardon, D. A.; Sampson, J. H.; Colvin,
O. M.; Haglund, M. M.; Pegg, A. E.; Moschel, R. C.; McClendon, R. E.;
Provenzale, J. M.; Guruangan, S.; Tourt-Uhlig, S.; Herndon, J. E., II;
Bigner, D. D.; Freidman, H. S. Phase II trial of carmustine plus
O6-benzylguanine for patients with nitrosourea-resistant recurrent or
progressive malignant glioma. J. Clin. Oncol. 2002, 20, 2277–2283.
(19) Schilsky, R. L.; Dolan, M. E.; Bertucci, D.; Ewesuedo, R. B.;
Vogelzang, N. J.; Mani, S.; Wilson, L. R.; Ratain, M. J. Phase I clinical and
pharmacological study of O6-benzylguanine followed by carmustine in
patients with advanced cancer. Clin. Cancer Res. 2000, 6, 3025–3031.
(20) Kennedy, K. A.; Teicher, B. A.; Rockwell, S.; Sartorelli, A. C.
The hypoxic tumor cell: a target for selective cancer chemotherapy.
Biochem. Pharmacol. 1980, 29, 1–8.
(21) Sartorelli, A. C. Therapeutic attack of hypoxic cells of solid
tumors: presidential address. Cancer Res. 1988, 48, 775–778.
(22) Chae, M.-Y.; McDougall, M. G.; Dolan, M. E.; Swenn, K.; Pegg,
A. E.; Moschel, R. C. Substituted O6-benzylguanine derivatives and their
inactivation of human O6-alkylguanine DNA alkyltransferase. J. Med.
Chem. 1994, 37, 342–347.
(23) Chae, M.-Y.; Swenn, K.; Kanugula, S.; Dolan, M. E.; Pegg, A. E.;
Moschel, R. C. 8-Substituted O6-benzylguanine, substituted 6(4)-(benzy-
loxy)pyrimidine derivatives, and related compounds as inhibitors of human
O6-alkylguanine DNA alkyltransferase. J. Med. Chem. 1995, 38, 359–365.
(24) Wei, G.; Loktionova, N. A.; Pegg, A. E.; Moschel, R. C.
β-Glucuronidase-cleavable prodrugs of O6-benzylguanine and O6-ben-
zyl-20-deoxyguanine. J. Med. Chem. 2005, 48, 256–261.
(25) Penketh, P. G; Shyam, K; Baumann, R. P; Remack, J. S; Brent,
T. P; Sartorelli, A. C. 1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-
[(methylamino)carbonyl]hydrazine (VNP40101M): I. Direct inhibi-
tion of O6-alkylguanine-DNA alkyltransferase (AGT) by electrophilic
7728
dx.doi.org/10.1021/jm201115f |J. Med. Chem. 2011, 54, 7720–7728