Substitution of aminomethyl at the meta-position enhances the inactivation of O6-alkylguanine-DNA alkyltransferase by O6- benzylguanine

Article abstract of DOI:10.1021/jm800675p

O⁶-Benzylguanine is an irreversible inactivator of O ⁶-alkylguanine-DNA alkyltransferase currently in clinical trials to overcome alkyltransferase-mediated resistance to certain cancer chemotherapeutic alkylating agents. In order to produce more soluble alkyltransferase inhibitors, we have synthesized three aminomethyl-substituted O ⁶-benzylguanines and the three methyl analogs and found that the substitution of aminomethyl at the meta-position greatly enhances inactivation of alkyltransferase, whereas para-substitution has little effect and ortho-substitution virtually eliminates activity. Molecular modeling of their interactions with alkyltransferase provided a molecular explanation for these results. The square of the correlation coefficient (R²) obtained between E-model scores (obtained from GLIDE XP/QPLD docking calculations) vs log(ED₅₀) values via a linear regression analysis was 0.96. The models indicate that the ortho-substitution causes a steric clash interfering with binding, whereas the meta-aminomethyl substitution allows an interaction of the amino group to generate an additional hydrogen bond with the protein.

Full text of DOI:10.1021/jm800675p

7144
J. Med. Chem. 2008, 51, 7144–7153
Substitution of Aminomethyl at the Meta-Position Enhances the Inactivation of
O6-Alkylguanine-DNA Alkyltransferase by O6-Benzylguanine
Gary T. Pauly,† Natalia A. Loktionova,‡ Qingming Fang,‡ Sai Lakshmana Vankayala,§ Wayne C. Guida,§,| and
Anthony E. Pegg*,‡
Laboratory of ComparatiVe Carcinogenesis, National Cancer Institute at Frederick, P.O. Box B, Building 538, Frederick, Maryland 21702,
Departments of Cellular and Molecular Physiology and Pharmacology, The PennsylVania State UniVersity College of Medicine,
P.O. Box 850, Hershey, PennsylVania 17033, Department of Chemistry and Center for Molecular DiVersity in Drug Design, DiscoVery, and
DeliVery, UniVersity of South Florida, Tampa, Florida 33620, and Drug DiscoVery Program, H. Lee Moffitt Cancer Center & Research
Institute, Tampa, Florida 33612
ReceiVed June 4, 2008
O6-Benzylguanine is an irreversible inactivator of O6-alkylguanine-DNA alkyltransferase currently in clinical
trials to overcome alkyltransferase-mediated resistance to certain cancer chemotherapeutic alkylating agents.
In order to produce more soluble alkyltransferase inhibitors, we have synthesized three aminomethyl-
substituted O6-benzylguanines and the three methyl analogs and found that the substitution of aminomethyl
at the meta-position greatly enhances inactivation of alkyltransferase, whereas para-substitution has little
effect and ortho-substitution virtually eliminates activity. Molecular modeling of their interactions with
alkyltransferase provided a molecular explanation for these results. The square of the correlation coefficient
(R2) obtained between E-model scores (obtained from GLIDE XP/QPLD docking calculations) vs log(ED50)
values via a linear regression analysis was 0.96. The models indicate that the ortho-substitution causes a
steric clash interfering with binding, whereas the meta-aminomethyl substitution allows an interaction of
the amino group to generate an additional hydrogen bond with the protein.
Introduction
structure that rotates the alkylated guanine deoxynucleoside from
the base stack into an active site pocket that contains the Cys145
acceptor residue.3,19 This places the O6-alkylguanine in the
correct position for repair in a hydrophobic cleft where the
Cys145 and Val148 carbonyls accept hydrogen bonds from
the exocyclic amine of the guanine, the N of Ser159 donates a
hydrogen bond to the substrate guanine-O6, and the hydroxyl
of Tyr114 donates a hydrogen bond to guanine-N3. These
interactions exactly position the alkyl group for attack by
Cys145. This residue is highly reactive by virtue of its activation
to a thiolate anion by a Glu172/His146/water/Cys145 hydrogen
bond network.3,11,20 BG binds much more weakly at the active
site pocket, since all of the interactions with the alkyltransferase
DNA-binding domain are lost, but it is held in a position that
allows attack by Cys145 via the interactions with the guanine
moiety described above and the interaction of the benzyl group
with the side chain of Pro140.3,11 Mutation of this Pro residue
profoundly reduces the ability of BG to inactivate human
alkyltransferase.21,22 Despite the weak binding, the reactivity
of benzyl in bimolecular displacement reactions such as that
occurring in the alkyltransferase active site facilitates the
inactivation reaction. However, the rate constant for the reaction
O6-Alkylguanine-DNA alkyltransferase (alkyltransferasea) is
a unique DNA repair protein that acts in a single step to restore
DNA with O6-alkylguanine adducts by transferring the alkyl
group to an acceptor site, which in human alkyltransferase is
located at Cys145.1-3 Alkyltransferase activity in tumors is an
important source of resistance to therapeutic alkylating agents
such as dacarbazine, temozolomide, 1,3-bis(2-chloroethyl)-1-
nitrosourea (BCNU) or 1-(2-chloroethyl)-3-(trans-4-methyl-
cyclohexyl)-1-nitrosourea.1,2,4-8 O6-Benzylguanine (BG) is a
potent inhibitor of human alkyltransferase, which acts as a
pseudosubstrate. After binding in the active site, it leads to the
formation of S-benzylcysteine at the Cys145 acceptor site,
irreversibly inactivating the protein.9-11 A related compound
O6-(4-bromothenyl)guanine was found to be a slightly more
potent inactivator of alkyltransferase12 and is thought to act in
the same way. Both compounds are undergoing clinical
trials.4,7,8,13-18 Although there have been some responses
in these trials providing proof of concept for the target, it is
clear that improved inhibitors will be needed.
Since the original development of BG, structural and bio-
chemical studies have provided a much greater understanding
of the repair reaction catalyzed by alkyltransferase and the
binding and reaction of this inhibitor. After binding the substrate
DNA via the minor groove using a helix-turn-helix motif,
the alkyltransferase protein brings about a change in the DNA
with BG free base is only about 600 M-1 ·s-1 10
which is
,
>10000 times less than that for the repair of O6-methylguanine
in DNA or of oligodeoxyribonucleotides containing BG, which
are much more potent inactivators.23,24 Such oligodeoxyribo-
nucleotides are not ideal for clinical use, and other attempts to
improve the binding of low molecular weight pseudosubstrates
are needed.
* To whom correspondence should be addressed. Phone: 717-531-8152.
Fax: 717-531-5157. E-mail: aep1@psu.edu.
†
Another serious problem with BG is its poor solubility. In
an attempt to make more soluble derivatives, we have now tested
O6-[(aminomethyl)benzyl]guanines (compounds 1-3 shown in
Figure 1) for the ability to inactivate human alkyltransferase. It
was found that the nature and position of the substitution
profoundly affected inhibition and that compound 2, the
National Cancer Institute at Frederick.
The Pennsylvania State University College of Medicine.
University of South Florida.
H. Lee Moffitt Cancer Center & Research Institute.
‡
§
|
a Abbreviations: alkyltransferase, O6-alkylguanine-DNA alkyltransferase;
BG, O6- benzylguanine; BCNU, 1,3-bis(2-chloroethyl)-1-nitrosourea; DMSO,
dimethyl sulfoxide; DMF, dimethylformamide.
10.1021/jm800675p CCC: $40.75
2008 American Chemical Society
Published on Web 10/31/2008

InactiVation by O6-Benzylguanine
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22 7145
Figure 1. Substituted BGs and an N6-[(hydroxymethyl)benzyl]-2-aminoadenine (7) resulting from a rearrangement of 3.
Scheme 1. Synthesis of O6-[(Aminomethyl)benzyl]guanines and O6-(Methylbenzyl)guanines
substitution with an aminomethyl group at the meta- position,
provides a more potent and much more soluble inhibitor than
BG. Comparison with O6-(methylbenzyl)guanines (compounds
4-6) was used to determine if the improved activity was due
to stabilization of the transition state by a positional effect of
substitution on the benzyl ring or to the ability of the meta-
aminomethyl group to bind more tightly to the active site.
Compounds 4-6 did not show improvement over BG, suggest-
ing that the latter was the case. Molecular modeling studies using
one of the crystal structures available for the protein that
involved its interaction with compounds 1-6 and BG were
carried out and provide a plausible explanation for these results
and show that the meta-substituent on 2 provides additional
interactions with the active site pocket that increase the affinity
for binding to the alkyltransferase protein.
Results and Discussion
Synthesis of Methyl- and Aminomethyl-Substituted BGs.
Preparation of the three isomeric O6-[(aminomethyl)benzyl]gua-
nines (1-3) was adapted from the method reported by Keppler
et al.25 to make compound 1 as shown in Scheme 1. 2-Amino-
6-chloropurine was converted to 1-(2-amino-9H-purin-6-yl)-1-
methylpyrrolidinium chloride (8). The amine groups of the three
isomers of (aminomethyl)benzyl alcohol were protected as the
corresponding trifluoroacetamides (9, 10, and 11).Treatment of
compound 8 in DMF with one of the three protected (aminom-

7146 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22
Pauly et al.
Table 1. Inactivation of Purified Human Alkyltransferase in Vitro
ED50 (µM)a
substituted compound 6 was similar to 3 in leading to a large
loss of inhibitory potency (ED50 values of 90 µM and 185 µM
in the presence and absence of DNA) (Table 1). These results
suggest that the improved activity of 2 was not caused by
stabilization of the transition state due simply to a substitution
on the benzyl ring at the meta-position.
In contrast to studies with folate ester derivatives of BG where
the ED50 values were affected by the presence of a (His)6 tag
on the alkyltransferase protein,26 there was no difference
between assays with untagged alkyltransferase protein and
assays using either a C-terminal or N-terminal (His)6-tagged
alkyltransferase with any of the compounds listed in Table 1.
Some of these folate derivatives are less potent inactivators of
a polymorphic form of human alkyltransferase in which Ile143
is changed to a Val and Lys178 to an Arg (I143V/K178R).26
This variant was only slightly less susceptible than wild type
to compound 2 (results not shown).
alkyltransferase tested
inhibitor
no DNA
with DNA
wild type
wild type
wild type
wild type
BG
2
1
0.30
0.017
0.23
80
0.10
0.004
0.053
45
3
wild type
wild type
wild type
5
4
6
0.25
0.32
185
0.16
0.20
90
wild type
S159A mutant
S159A mutant
7
2
1
>1000
0.013
0.16
>1000
0.003
0.028
a Results shown are the mean values for three to five assays with different
preparations of recombinant alkyltransferase, which agreed within (20%.
ethyl)benzyl alcohols in the presence of potassium tert-butoxide
gave the corresponding trifluoroacetyl protected O6-(aminom-
ethyl)benzylguanines (12, 13, and 14). Removal of the trifluo-
roacetate protecting groups with potassium carbonate in
methanol-water then gave the three O6-(aminomethyl)ben-
zylguanines (1, 2, and 3). Similarly, the treatment of 8 with
one of the three isomers of methylbenzyl alcohol in the presence
of potassium tert-butoxide gave the three methylbenzylguanines
4, 5, and 6 (Scheme 1).
Molecular Docking of Inhibitors to Human Alkyltrans-
ferase. Computational docking studies were performed using
the GLIDE program (version 4.5, Schro¨dinger, LLC, New York,
2007). The docked structures were chosen for comparison with
experimentally determined ED50 values using either the Glide
Score or E-model scoring function. For enhanced docking
accuracy, the best docked structures using GLIDE extra preci-
sion (XP) mode were used to calculate ligand partial charges
in the protein environment and then redocked with XP using
Schro¨dinger’s QPLD (quantum polarized ligand docking)
method.27 The Maestro user interface (version 8.0, Schro¨dinger,
LLC, New York, 2007) was employed to set up the GLIDE
docking studies and for visualization of the results. The
alkyltransferase X-ray crystal structure chosen for our modeling
studies was human alkyltransferase bound to DNA containing
O6-methylguanine and Cys145 mutated to Ser to prevent the
alkylation reaction from taking place.19 In our modeling studies,
Ser145 was mutated back to Cys. To validate the docking
approach, self-docking was performed with XP/QPLD using the
partial native ligand O6-methylguanine. The rms of the docked
pose when compared to the crystallographically observed
position of the O6-methylguanine moiety was 0.14 Å, and thus,
GLIDE produced a docking mode that closely resembled the
X-ray crystal structure. Thus, our hypothesis was that GLIDE
would be capable of producing docking poses for the compounds
studied that are similar to the position, orientation, and
conformation adopted by the ligands prior to nucleophilic attack
by Cys145 and that the docking scores obtained would correlate
well with the experimentally observed ED50 values for enzyme
inactivation. Although there is a crystal structure available for
human alkyltransferase benzylated at Cys145,11 this structure
was not employed to model the BG analogues described in this
study, since the GLIDE program is unsuitable for modeling
covalent bonds formed between the ligand and protein.
During the attempt to recrystallize a sample of O6-[2-
(aminomethyl)benzyl]guanine (3) from boiling water, an in-
soluble precipitate formed. This precipitate was recovered by
filtration. The proton NMR spectrum of the precipitated material
was not consistent with the original sample of compound 3,
even though MS analysis of the original sample and the
precipitate gave similar molecular ions with m/z ) 271.1 [M +
H]+. A sample of compound 3 was dissolved in water and
heated at 65 °C for 1 week. The precipitate was then recovered
for further analysis by NMR. The NMR spectrum of the
precipitated material is consistent with N6-[2-(hydroxymethyl)-
benzyl]-2-aminoadenine (7 in Figure 1), which we propose is
formed by intramolecular attack of the benzylic amine at the
6-position of the purine, displacing the oxygen. This rearrange-
ment was not observed with 1 and 2.
Inactivation of Purified Human Alkyltransferase in Vitro.
Compound 1, the para-substituted aminomethyl derivative of
BG, was only slightly more active than BG itself, but the meta-
substituted derivative 2 was considerably more active with an
ED50 of approximately 4 nM compared to 100 nM for BG when
assayed in the presence of DNA and 17 nM compared to 300
nM when assayed in the absence of DNA (Table 1). The
inactivation of the S159A mutant alkyltransferase by compounds
1 and 2 differed only slightly from the inactivation of the wild
type (Table 1). This rules out the possibility that there is an
interaction between the side chain of this serine residue in
alkyltransferase and the aminomethyl group of these inhibitors.
The ortho-substituted derivative 3 was much less active than
BG or the other substituted BGs with an ED50 of 45 µM when
assayed in the presence of DNA and 80 µM when assayed in
the absence of DNA (Table 1). This derivative was assayed
immediately after a solution was made up, since as described
above it is unstable in aqueous solution, rearranging to form 7
with a half-life of about 50 h at 37 °C. This compound was
also tested and was inactive as an alkyltransferase inhibitor (less
than 10% inhibition at 1 mM).
In addition to O6-methylguanine, compounds 1-6 and BG
were docked to human alkyltransferase using the GLIDE XP.
Each pose of these compounds was redocked with XP using
the QPLD method. The coefficient of determination (i.e., the
square of the correlation coefficient, R2) was calculated between
QPLD Glide Scores vs log(ED50) values, determined in the
presence of DNA, as part of a linear regression analysis. An
acceptable R2 value of 0.86 between log(ED50) values and XP/
QPLD Glide Scores was obtained (Figure 2).
E-model scores were also used for correlation studies. It had
been previously observed by Bytheway and Cochran28 that
E-model scores correlated better with log(ED50) values than
Glide Scores in their particular study. In our study, the
The improved activity of an aminomethyl meta-substituent
is not mimicked by a simple methyl group. There was no
difference between BG itself and compound 4 or 5, which had
ED50 values of about 0.3 µM in the absence of DNA and about
0.15 µM in the presence of DNA (Table 1). The ortho-methyl

InactiVation by O6-Benzylguanine
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22 7147
Table 2. Binding Interactions Derived from Docking Compounds 1-6,
BG, and O6-Methylguanine to Human Alkyltransferase
E-model Glide
good vdwa bad vdwa
compd
score
score H-bonds
contacts
contacts
2
1
4
BG
-88.1
-82.0
-81.5
-78.2
-77.5
-56.6
-52.2
-52.0
-9.42
-9.46
-9.54
-8.79
-9.30
-7.16
-6.75
-6.12
4
3
3
3
3
4
2
1
297
312
306
287
289
177
260
180
8
8
7
4
8
3
5
1
5
O6-methylguanine
3
6
a vdw, Van der Waals.
except for the interaction with Tyr114 involve interaction with
protein backbone rather than the amino acid side chains.
Figure 5 shows an overlay of all of the inhibitor poses in the
active site with O6-methylguanine. It is clearly apparent that
compounds 3 and 6 are not oriented in the catalytic site of
human alkyltransferase in same way as the other inhibitors.
Inactivation of Alkyltransferase in HT29 Cells in Cul-
ture. The striking difference between 1 and 2 in the inactivation
of alkyltransferase was also seen when the compounds were
added to the culture medium of HT29 cells (Figure 6). When
cell extracts were prepared from these cells and residual
alkyltransferase activity measured, it was found that within 30
min or less of exposure to 0.5-2.5 µM compound 2, the
alkyltransferase was almost completely reduced. Compound 1
was clearly less effective; the 1 µM dose only produced 20%
loss of activity in 30 min (Figure 6A). This difference was also
seen when the ability of 1 and 2 to sensitize HT29 cells to
BCNU was examined (Figure 6B). Alkyltransferase activity
provides resistance to BCNU, and alkyltransferase inhibitors
overcome this resistance.9,12,29-31 Compound 2 was much more
effective than compound 1 in sensitizing HT29 cells to the
chloroethylating agent (Figure 6B). Previously published studies
have shown that BG passes very readily through cell membranes
and very rapidly inactivates alkyltransferase in HT29 cells.9,29,32
Even though 2 produces a comparable degree of alkyltransferase
inactivation, the rate of inactivation by 2 seen in Figure 6A is
significantly slower than with BG. This is likely to be due to a
reduced uptake of 2 due to its positive charge.
Figure 2. Plot of log(ED50) values versus QPLD_Glide_scores with
DNA. Numbering corresponds to compounds shown in Figure 1. The
ED50 values determined in the presence of DNA from Table 1 were
used.
Figure 3. Plot of log(ED50) values versus QPLD_E-model scores with
DNA. Numbering corresponds to compounds shown in Figure 1. The
ED50 values determined in the presence of DNA from Table 1 were
used.
coefficient of determination obtained between E-model scores
(obtained from XP/QPLD calculations) vs log(ED50) values via
a linear regression analysis gave an exceptional R2 of 0.96,
shown in Figure 3. It is noteworthy that the E-model is a
composite scoring function, derived from a combination of the
Glide Score itself coupled with Coulombic energy, van der
Waals energy, and ligand strain energy terms, and is used by
GLIDE to select the best docking pose for each individual
inhibitor regardless of the scoring function subsequently used
and can be used to rank order the inhibitors as well.
Conclusions
The addition of a meta-aminomethyl group to BG forming
compound 2 not only greatly increases the solubility but results
in an approximately 20-fold improvement in the ability to
inactivate purified human alkyltransferase. This improvement
is seen in assays conducted with or without added DNA. It is
noteworthy that the molecular modeling studies showed a similar
trend and an R2 of 0.90 (data not shown) for a linear regression
analysis of log(ED50), determined in the absence of DNA, vs
XP/QPLD Glide Scores, and an R2 of 0.98 (data not shown)
for log(ED50), determined in the absence of DNA, vs E-model
scores were obtained. As previously noted, inactivation of
alkyltransferase by BG33 is enhanced 3- to 4-fold by the presence
of DNA, which stimulates the rate of alkyl transfer.34 In contrast,
some BG derivatives with bulky substituents are much less
potent inhibitors in the presence of DNA, since they cannot be
accommodated in the active site when DNA is bound there.34,35
The inactivation of alkyltransferase by compounds 1 and 2 was
increased by the presence of DNA, indicating that these
compounds, like BG, do not compete with DNA for access to
the active site. However compound 1, the para-aminomethyl
derivative, did not show any improvement in inhibitory potency
over BG itself. These results are very well explained by the
From examination of the QPLD based docking modes, one
can conclude that the binding affinities correlate quite well with
the number of hydrogen bonds and good van der Waals contacts
formed between the inhibitor and the alkyltransferase binding
site. There is insufficient space to adequately accommodate the
ortho-substituted benzylguanines. Thus, because of fewer favor-
able van der Waals contacts and because of their fewer hydrogen
bonds, compounds 3 and 6 are the least potent of the inhibitors
we tested in the modeling studies. On the other hand, compounds
1 and 2 exhibit three and four hydrogen bonds, respectively,
and both form good van der Waals contacts with the alkyl-
transferase protein. All the values of these docking based binding
interactions are summarized in Table 2.
Figure 4 shows the key hydrogen bonding interactions of
compound 2 with human alkyltransferase. Interactions with
residues Tyr114, Cys145, Ser159, and Asn137 are depicted. All

7148 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22
Pauly et al.
Figure 4. Key hydrogen bonding interactions of compound 2 (O6-[3-(aminomethyl)benzyl]guanine) with the protein residues represented in stick
model. The inhibitor is represented in green for carbon, blue for nitrogen, and red for oxygen. The rest of the protein is shown as pale green ribbon
cartoon.
Figure 5. Overlay of the position of all of the docked compounds in the human alkyltransferase active site with the native ligand O6-methylguanine
and parent inhibitor BG. All of the potent inhibitor poses in green (compounds 1, 2, 4, 5, and BG) are oriented in the same way as the native ligand
O6-methylguanine. The ineffective inhibitors O6-[2-(aminomethyl)benzyl]guanine (3) in red and O6-[2-(methyl)benzyl]guanine (6) in yellow are
not oriented in the same way as O6-methylguanine. The rest of the color code is the same as in Figure 4.
molecular modeling studies, which indicate the formation of
an additional hydrogen bond when compound 2 is bound in
the active site. The aminomethyl group from 2 but not 1 is able
to interact with Asn137. This also explains why a simple methyl
substitution (compound 5) was ineffective, since it cannot form
this hydrogen bond. The modeling studies also show clearly
why the ortho-substituted BG derivatives (3 and 6) are much
less effective (>200-fold) than BG since, because of steric
clashes, they cannot be positioned in the same way as the parent
compound.
actual crystal structures of BG bound to wild type alkyltrans-
ferase or its C145S mutant, the remarkable R2 values obtained
using XP/QPLD with E-model scoring for ranking the com-
pounds tested here show that the structure of human alkyltrans-
ferase bound to DNA containing O6-methylguanine and Cys145
mutated to Ser can be used as a starting structure for this process.
The mutation of Ser145 back to Cys, as is appropriate for the
wild type enzyme, and refinement of the structure obtained with
MacroModel were essential in order to obtain the R2 values we
report. The model we have generated should be highly useful
in the design of more potent inhibitors by performing additional
modeling studies on BG analogues and by virtually screening
databases of commercially available compounds to identify
potential new lead compounds for further elaboration.
Our results not only demonstrate that 2 may be a valuable
alkyltransferase inhibitor but also show clearly the value of
molecular modeling for the design of improved alkyltransferase
inhibitors. Even though it has not yet been possible to obtain

InactiVation by O6-Benzylguanine
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22 7149
Figure 6. Inactivation of alkyltransferase in HT29 cells. Panel A shows the loss of alkyltransferase (hAGT) activity as a function of time after
addition of the compound indicated to cultures of HT29 cells. Panel B shows the effect of exposing HT29 cells to the compound indicated for 2 h
prior to the addition of 40 µM BCNU. Cell survival was then measured using a colony forming assay as described in the Experimental Section.
were collected on a Thermo Finnigan TSQ Quantum spectrometer
in positive ion electrospray mode scanning from m/z ) 100 to m/z
) 1500 in 1 s. The electrospray voltage was 3.5 kV, and the transfer
tube was at 350 °C. Elemental analysis was performed by Atlantic
Microlab, Inc. (Norcross, GA). All silica gel chromatography was
carried out using Davisil, grade 633, 200-425 mesh, 60 Å.
4-(Aminomethyl)benzyl alcohol was made according to the pro-
cedure reported by Keppler et al.25 The NMR spectrum of this
material was in agreement with the published data. 2-(Aminom-
ethyl)benzyl alcohol was made as described by McCalmont et al.,39
and the NMR spectrum agreed with the reported data.
3-(Aminomethyl)benzyl Alcohol. A sample of 3-cyanobenzoic
acid (20 g, 136 mmol) was suspended in 100 mL of dry
tetrahydrofuran (THF). Borane-THF complex (400 mL of 1.0 M
solution in THF) was added dropwise over 1 h, and the mixture
was stirred for an additional 3 h. The reaction was quenched with
220 mL of concentrated HCl/water (1:1 v/v) to cleave the borate
ester complex. The THF was then removed under vacuum. Solid
NaOH pellets, 70 g total, were added slowly to neutralize the acid
and to bring the pH of the solution to 12. The aqueous phase was
then extracted 4 times with 500 mL portions of ether. The ether
extract was dried over MgSO4 and concentrated under vacuum to
give 18 g (131 mmol, 96%) of 3-(aminomethyl)benzyl alcohol as
a light-colored oil. 1H NMR (CDCl3/TMS) δ ) 1.95 (broad s, 3H,
-OH and sNH2, exchangeable with D2O), 3.86 (s, 2H,
-CH2-NH2), 4.66 (s, 2H, -CH2-OH), 7.18-7.35 (m, 4H, Ar)
ppm, in agreement with data published by Lee et al.40 who made
this compound by a different route.
A major advantage of compound 2 is that it is much more
soluble (approximately 1000 times at physiological pH) than
BG. The limited solubility of BG requires formulation in a
diluent containing polyethylene glycol. Although adequate blood
levels for alkyltransferase inactivation in a variety of tumors
can be achieved with this route of administration,4,13-17 the
potentiation of the myelotoxicity of the alkylnitrosourea-based
and methylating chemotherapies due to inactivation of alkyl-
transferase in the bone marrow limits the effectiveness of BG.
Regional delivery is an obvious way to attempt to circumvent
this limitation, but in many cases, only a small volume can be
administered, and the poor solubility of BG prevents its use in
this way. This could be avoided by using 2.
In particular, 2 is suitable for testing for protocols involving
administration by convection-enhanced therapy to treat brain
tumors. Convection-enhanced therapy uses a hydrostatic pressure
gradient-dependent direct intracerebral infusion to establish a
bulk flow interstitial current that produces a relatively uniform
spatial distribution of drugs at concentrations nearer the infused
concentration at significant distances from the point of delivery.
It is currently under development for use with a variety of agents
directed against brain tumors.36-38 The hydrophilicity of the
protonated 2 is also likely to prevent back-loss across the
blood-brain barrier. The slower uptake of 2 compared to BG
is not likely to prove a significant obstacle to its use, since total
alkyltransferase inactivation is easily obtained within 30 min
of exposure to 1 µM levels. Obviously, the success of the
compound in clinical use will depend on its biological stability
and bioavailability.
1-(2-Amino-9H-purin-6-yl)-1-methylpyrrolidinium Chloride (8).
This compound was prepared from 2-amino-6-chloropurine as
described.25 The 1H NMR spectrum of this product was in
agreement with the data in that publication.
General Synthesis of 2,2,2-Triflouro-N-[(hydroxymethyl)ben-
zyl]acetamides. Protection of the amine groups of the three
aminomethylbenzyl alcohols as trifluoroacetamides was performed
according to the procedure used by Keppler et al. to make 2,2,2-
trifluoro-N-[(4-(hydroxymethyl)benzyl]acetamide (9).25
2,2,2-Trifluoro-N-[(4-(hydroxymethyl)benzyl]acetamide (9). The
yield of this reaction was 89%, giving a white solid with a melting
point of 91-92 °C. 1H NMR (CDCl3/TMS) δ ) 1.77 (s, 1H, -OH,
exchanges with D2O), 4.52 (d, J ) 6.0, 2H, -CH2-NH-), 4.70
(s, 2H, -CH2-OH), 6.63 (broad s, 1H, -NH-CO-, exchanges
with D2O), 7.26-7.39 (m, 4H, Ar) ppm, in agreement with the
published data.25
Experimental Section
Chemistry. Chemicals were obtained from Aldrich (Milwaukee,
WI) and were used without further purification. UV spectra were
determined on a Beckman Coulter DU 7400 spectrophotometer.
Molar extinction coefficients for compounds 1-3 were determined
by simply dissolving approximately 10 mg of the material in 1.00
L of 0.01 M phosphate buffer, pH 7.0, and recording the UV
absorbance spectrum. Compounds 4-7 did not dissolve completely,
even at this low concentration. As a result, these four compounds
were predissolved in 1 mL of DMSO and then diluted to 1.00 L
with phosphate buffer prior to recording their UV absorbance
spectra. 1H NMR spectra were recorded with a Varian INOVA 400
MHz spectrometer. Chemical shifts are reported as δ values in parts
per million relative to TMS as an internal standard. Splitting pattern
abbreviations are as follows: s ) singlet, d ) doublet, t ) triplet,
and m ) multiplet. Coupling constants are in hertz. Mass spectra
2,2,2-Trifluoro - N- [(3- (hydroxymethyl) benzyl] acetamide (10).
The yield of this reaction was 91%, giving a white solid with a
1
melting point of 73-74 °C. H NMR (CDCl3/TMS) δ ) 1.84 (s,
1H, -OH, exchanges with D2O), 4.52 (d, J)5.6, 2H,

7150 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22
Pauly et al.
-CH2-NH-), 4.70 (s, 2H, -CH2-OH), 6.65 (broad s, 1H,
-NH-CO-, exchanges with D2O), 7.20-7.4 (m, 4H, Ar) ppm.
2,2,2-Trifluoro-N-[2-(hydroxymethyl)benzyl]acetamide (11). The
yield of this reaction was 76%, giving a white solid with a melting
point of 75-76 °C. 1H NMR (CDCl3/TMS) δ ) 2.20 (s, 1H, -OH,
exchanges with D2O), 4.61 (d, J)5.6, 2H, -CH2-NH-), 4.78 (s,
2H, -CH2-OH), 7.30-7.44 (m, 4H, aromatic), 7.76 (broad s, 1H,
-NH-CO-, exchanges with D2O) ppm.
General Synthesis of the N-[(2-amino-9H-purin-6-yloxymeth-
yl)benzyl]-2,2,2-trifluoroacetamides (12-14). The procedure used
by Keppler et al.25 to make N-[4-(2-amino-9H-purin-6-yloxymeth-
yl)benzyl]-2,2,2-trifluoroacetamide (12) was used. Compound 8 was
suspended in 35-40 mL/g DMF. To this was added between 1.4
and 2.0 equiv of one of the three protected aminomethylbenzyl
alcohols (9-11) together with 2 equiv, with respect to the benzyl
alcohol, of potassium tert-butoxide. After 3-5 h the reactions were
quenched with acetic acid in water and the solvents were evaporated.
The products were then purified by silica gel chromatography,
eluting with methanol/dichloromethane (1:50) followed by the same
solvents (1:10).
N-[4-(2-Amino-9H-purin-6-yloxymethyl)benzyl]-2,2,2-trifluoro-
acetamide (12). This reaction gave a white solid with a melting
range of 213-215 °C in 34% yield based on the purine. 1H NMR
(DMSO-d6/TMS) δ ) 4.40 (s, 2H, -CH2-NH-), 5.47 (s, 2H,
-O-CH2-), 6.27 (s, 2H, guanine-NH2, exchanges with D2O),
7.27-7.52 (m, 4H, Ar), 7.83 (s, 1H, guanine H8), 10.02 (broad s,
1H, -NH-CO- exchanges with D2O), 12.44 (broad s, 1H, guanine
H9, exchanges with D2O) ppm. Keppler et al.25 report the signal
at 4.40 ppm as a doublet.
N-[3-(2-Amino-9H-purin-6-yloxymethyl)benzyl]-2,2,2-trifluoro-
acetamide (13). This reaction gave a white solid which decomposed
above 210 °C in 35% yield based on the purine. 1H NMR (DMSO-
d6/TMS) δ ) 4.42 (s, 2H, -CH2-NH-), 5.48 (s, 2H, -O-CH2-),
6.27 (s, 2H, guanine-NH2, exchanges with D2O), 7.23-7.45 (m,
4H, Ar), 7.83 (s, 1H, guanine H8), 10.03 (broad s, 1H, -NH-CO-
exchanges with D2O), 12.45 (broad s, 1H, guanine H9, exchanges
with D2O) ppm.
H9 proton nor the benzylic amine protons are observed. A sample
of this material was recrystallized from water for elemental analysis.
Analysis calculated for C13H14N6O1 (C, H, N). The material was
converted to the hydrochloride salt by suspending it in water and
adding 1 equiv of HCl. The material was then filtered and
lyophilized. Analysis of the hydrochloride salt was calculated for
C13 H15N6O1Cl1 as the monohydrate (C, H, N, Cl).
O6-[2-(Aminomethyl)benzyl]guanine (3). 3 was recovered as a
white solid which decomposed above 170 °C in 74% yield. UV
(0.01 M sodium phosphate pH 7.0), λmax ) 241 nm (ε ) 7400),
1
λmax ) 282 nm (ε ) 8400). H NMR (DMSO-d6/TMS) δ ) 3.82
(s, 2H, -CH2-NH2), 5.55 (s, 2H, -O-CH2-) 6.27 (s, 2H,
guanine-NH2, exchanges with D2O), 7.22-7.50 (m, 4H, Ar), 7.83
(s, 1H, guanine H8) ppm. As with compound 1, neither the guanine
H9 proton nor the benzylic amine protons are observed. Elemental
analysis calculated for C13H14N6O1 (C, H, N). This material was
not converted to a hydrochloride salt because of the rearrangement
observed below.
N6-[2-(Hydroxymethyl)benzyl]-2-aminoadenine (7). Upon at-
tempting to recrystallize O6-[2-(aminomethyl)benzyl)]guanine (3)
from water, it was observed that the material came out of solution
upon heating. The NMR spectrum (1H) of the recovered solid
material indicated decomposition or rearrangement. The mass
spectrum of the original sample of compound 3 and the precipitate
contained similar molecular ions at m/z ) 271.1 [M + H]+.
A sample of O6-[2-(aminomethyl)benzyl]guanine (3) was dis-
solved in water and heated to 65 °C for 1 week. The insoluble
precipitate was then filtered, washed with water, and dried under
vacuum. This material with a melting range of 219-221 °C was
identified as N6-[(2-hydroxymethyl)benzyl]-2-aminoadenine (7)
based on the NMR spectrum. UV (0.01 M sodium phosphate with
0.1% v/v DMSO, pH 7.0), λshoulder ) 250 nm (ε ) 8000), λmax
284 nm (ε ) 12 800). 1H NMR (DMSO-d6/TMS) δ ) 4.64 (singlet
with broad shoulder downfield, 4H, -CH2-NH- and
)
a
-CH2-OH), 5.30 (s, 1H, -CH2-OH exchanges with D2O), 5.67
(s, 2H, purine-NH2, exchanges with D2O), 7.15-7.40 (m, 4H, Ar),
7.47 (broad d, J)7.2, 1H, benzylic NH, exchanges with D2O), 7.65
(s, 1H, purine H8), 12.06 (broad s, 1H, purine H9, exchanges with
D2O) ppm. Elemental analysis calculated for C13H14N6O1 ·0.5H2O
(C, H, N). The conversion of a very dilute solution of compound
3 to compound 7 was followed by UV spectroscopy in 0.1 M
sodium phosphate buffer at pH 7.4 and 37 °C and was found to
proceed with a half-life of 51 h with first order kinetics.
N-[2-(2-Amino-9H-purin-6-yloxymethyl)benzyl]-2,2,2-trifluoro-
acetamide (14). This reaction gave a white solid which decomposed
1
slowly above 180 °C in 25% yield based on the purine. H NMR
(DMSO-d6/TMS) δ ) 4.58 (d, J ) 5.6, 2H, -CH2-NH-), 5.57
(s, 2H, -O-CH2-), 6.29 (s, 2H, guanine-NH2, exchanges with
D2O), 7.26-7.60 (m, 4H, Ar), 7.81 (s, 1H, guanine H8), 10.00 (t,
J ) 5.6, 1H, -NH-CO- exchanges with D2O), 12.43 (broad s,
1H, guanine H9, exchanges with D2O) ppm.
General Synthesis of O6-[(Methyl)benzyl]guanines (4-6). To a
solution of one of the three isomers of methylbenzyl alcohol (2.5
g, 20 mmol) in 100 mL of dry DMF, potassium tert-butoxide (4.5
g, 80 mmol) was added. After 30 min, compound 8 (2.54 g, 10
mmol) was added. The mixture was stirred for 18 h, and then the
solvent was evaporated under vacuum. The resulting material was
purified over silica gel, loading with dichloromethane/methanol
(25:1) and eluting with the same solvents (10:1). The three
compounds produced in this way were each recrystallized from
ethanol/water (1:1).
General Method for the Removal of 2,2,2-Trifluoroacetyl
Protecting Groups. Trifluoroacetyl protecting groups were removed
from compounds (9-11) using the procedure described by Keppler
et al.25 to make O6-[4-(aminomethyl)benzyl]guanine (1).
O6-[4-(Aminomethyl)benzyl]guanine (1). 1 was recovered as a
white solid which decomposed above 185 °C in 76% yield. UV
(0.01 M sodium phosphate pH 7.0), λmax ) 241 nm (ε ) 7400),
1
λmax ) 282 nm (ε ) 8500). H NMR (DMSO-d6/TMS) δ ) 3.72
(s, 2H, -CH2-NH2), 5.45 (s, 2H, -O-CH2-) 6.26 (s, 2H,
guanine-NH2, exchanges with D2O), 7.31-7.47 (m, 4H, Ar), 7.82
(s, 1H, guanine H8) ppm, in agreement with the data published by
Keppler et al.25 Neither the guanine H9 proton nor the benzylic
amine protons are observed.25 A sample of this material was
recrystallized from water for elemental analysis. Analysis calculated
for C13H14N6O1 ·0.2H2O (C, H, N). The material was converted to
the hydrochloride salt by suspending it in water and adding 1 equiv
of HCl. The material was then filtered and lyophilized. Analysis
of the hydrochloride salt was calculated for C13H15N6O1Cl1 ·0.5H2O
(C, H, N, Cl).
O6-[4-(Methyl)benzyl]guanine (4). The above procedure gave
2.2 g (8.6 mmol, 43%) of 4 as a white solid with a melting point
of 205-207 °C. UV (0.01 M sodium phosphate with 0.1% v/v
DMSO, pH 7.0), λmax ) 241 nm (ε ) 7600), λmax ) 282 nm (ε )
1
8700). H NMR (DMSO-d6/TMS) δ ) 2.31 (s, 3H, -CH3), 5.45
(s, 2H, -O-CH2-), 6.27 (s, 2H, guanine-NH2, exchanges with
D2O), 7.16-7.44 (m, 4H, Ar), 7.81 (s, 1H, guanine H8), 12.41
(broad s, 1H, guanine H9, exchanges with D2O), in agreement with
the data published by Dolan et al.9 who made this compound by a
different route. Elemental analysis for C13H13N5O1 (C, H, N).
O6-[3-(Methyl)benzyl]guanine (5). The above procedure gave
2.2 g (8.6 mmol, 43%) of 5 as a white solid with a melting point
of 201-203 °C. UV (0.01 M sodium phosphate with 0.1% v/v
DMSO, pH 7.0), λmax ) 241 nm (ε ) 7500), λmax ) 282 nm (ε )
O6-[3-(Aminomethyl)benzyl]guanine (2). 2 was recovered as a
white solid with a melting range of 158-160 °C in 68% yield. UV
(0.01 M sodium phosphate, pH 7.0), λmax ) 241 nm (ε ) 7500),
1
λmax ) 282 nm (ε ) 8600). H NMR (DMSO-d6/TMS) δ ) 4.02
1
8700). H NMR (DMSO-d6/TMS) δ ) 2.32 (s, 3H, -CH3), 5.44
(s, 2H, -CH2-NH2), 5.48 (s, 2H, -O-CH2-) 6.30 (s, 2H,
guanine-NH2, exchanges with D2O), 7.29-7.48 (m, 4H, Ar), 7.83
(s, 1H, guanine H8) ppm. As with compound 1, neither the guanine
(s, 2H, -O-CH2-), 6.27 (s, 2H, guanine-NH2, exchanges with
D2O), 7.13-7.37 (m, 4H, Ar), 7.81 (s, 1H, guanine H8), 12.41

InactiVation by O6-Benzylguanine
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22 7151
(broad s, 1H, guanine H9, exchanges with D2O). Elemental analysis
for C13H13N5O1 (C, H, N).
O6-[2-(Methyl)benzyl]guanine (6). The above procedure gave
1.8 g (7.0 mmol, 35%) of 6 as a white solid with a melting point
of 211-213 °C. UV (0.01 M sodium phosphate with 0.1% v/v
DMSO, pH 7.0), λmax 241 nm (ε ) 7600), λmax ) 282 nm (ε )
1
8600). H NMR (DMSO-d6/TMS) δ ) 2.35 (s, 3H, -CH3), 5.48
(s, 2H, -O-CH2-), 6.28 (s, 2H, guanine-NH2, exchanges with
D2O), 7.18-7.49 (m, 4H, Ar), 7.82 (s, 1H, guanine H8), 12.43
(broad s, 1H, guanine H9, exchanges with D2O). Elemental analysis
for C13H13N5O1 ·0.25H2O (C, H, N).
Inactivation of Purified Recombinant Human Alkyltrans-
ferase. Human alkyltransferase proteins with and without (His)6
tags were expressed in E. coli and purified to homogeneity as
previously described.26 ED50 values for the inactivation of purified
human alkyltransferase in vitro were obtained essentially as
previously described.34,35 Briefly, purified recombinant human
alkyltransferase was incubated with different concentrations of
potential inhibitors in 0.5 mL of reaction buffer (50 mM Tris-HCl,
pH 7.6, 0.1 mM EDTA, 5.0 mM dithiothreitol) containing 50 µg
of hemocyanin for 30 min at 37 °C. For assay in the presence of
DNA, 10 µg of calf thymus DNA was added to the 0.5 mL of
reaction buffer. The remaining alkyltransferase activity was then
determined after incubation with [3H]methylated calf thymus DNA
substrate for 30 min at 37 °C by measuring the [3H]methylated
protein formed, which was collected on nitrocellulose filters. A
graph of the percentage of the alkyltransferase activity remaining
against inhibitor concentration was then plotted, and the ED50 values
representing the amount of inhibitor needed to produce a 50% loss
of activity were calculated from the equation for the best fit
exponential curve (KaleidaGraph, Synergy Software, Reading, PA
19606).
Cell Culture and Inactivation of Cellular Alkyltransferase.
HT29 cells were grown in RPMI 1640 medium supplemented with
10% fetal bovine serum and maintained by seeding at 1 × 105
cells/25-cm2 flask at weekly intervals. Inactivation of cellular
alkyltransferase by potential inhibitors was determined in cells that
reached about 80% confluence. After different times of exposure
to the compounds, cells were harvested by trypsinization and cell
pellets were washed with PBS. Cell extracts were prepared as
previously described by sonication on ice and centrifugation at
15000g, and supernatant extracts were stored at -70 °C until
assayed. Crude cell protein was determined using Bio-Rad protein
assay dye (Bio-Rad, Hercules, CA).
Figure 7. Prepared protein of the human alkyltransferase without DNA.
O6-Methylguanine and a bound water (WAT1) are also shown.
alkyltransferase bound to a DNA oligonucleotide and containing
O6-methylguanine determined at 3.2 Å resolution (PDB ID:
1T38).19,45 For our studies, DNA was removed except for O6-
methylguanine (see Figure 7). Solvent molecules in the protein
crystal structure were deleted, except for a water molecule in the
active site (WAT 1), Ser145 was mutated to Cys145, and the protein
was then prepared for the docking studies by processing it using
Schro¨dinger’s protein preparation facility. This procedure minimizes
the protein to 0.30 Å rmsd using the OPLS-2001 force field.
Subsequent refinement of the protein/ligand complex was performed
using the mixed torsional/low-mode conformational sampling
method within Schro¨dinger’s MacroModel program. The OPLS2001
force field was used, and a distance dependent dielectric “constant”
further attenuated by a factor of 2 was employed. A 5 Å shell of
residues surrounding the partial native ligand (O6-methylguanine),
the ligand itself, and a nearby water molecule (WAT 1) were fully
flexible during the conformational sampling/energy minimization
phase of the conformational search procedure. The rest of protein
was frozen at its crystallographically determined position including
residues Asn157 and Arg135, which were within the 5 Å shell of
mobile residues. The lowest energy conformer that retained the
native ligand pose in the X-ray crystal structure of PDB 1T38 was
used for subsequent studies. Structures of O6-methylguanine, BG,
and compounds 1-6 were prepared using Schro¨dinger’s LigPrep
facility.
The remaining alkyltransferase activity was measured as removal
of O6-[3H]methylguanine from [3H]methylated calf thymus DNA.9
Cell extracts were incubated with [3H]methylated DNA substrate
for 30 min at 37 °C in 1 mL of reaction buffer (50 mM Tris-HCl,
pH 7.6, 0.1 mM EDTA, 5.0 mM dithiothreitol). The DNA was
precipitated and hydrolyzed, and modified bases were separated
by reverse-phase HPLC. The results were expressed as the
percentage of the alkyltransferase activity present in cultures to
which no compound was added.
The initial docking studies were done with GLIDE (version 4.5,
Schro¨dinger, LLC, New York, 2007) operating in either SP or XP
mode.42-44 Maestro (version 8.0, Schro¨dinger Suite 2007, Schro¨-
dinger, LLC New York, 2007) was employed as the graphical user
interface and for generation of the graphics used in the figures.
The best docked structures from XP were used to calculate the
ligand partial charges in the environment of the protein and then
redocked using Schro¨dinger’s QPLD (quantum polarized ligand
docking) method.27 In order to validate the docking approach, self-
docking was performed using the partial native ligand O6-
methylguanine using XP/QPLD. The rms of the docked pose when
compared to the crystallographically observed position was 0.14
Å, and thus, GLIDE produced a docking mode that closely
resembled the X-ray crystal structure.
Sensitization of Cells to Killing by BCNU. The effect of
alkyltransferase inactivators on the sensitivity of cells to BCNU
was determined using a colony-forming assay. Cells were plated
at a density of 106 in 25 cm2 flasks and 24 h later were exposed to
different concentrations of potential inhibitors for 2 h as indicated
before exposure to 40 µM BCNU for 2 h as previously described.35,41
After 2 h, the medium was replaced with fresh medium containing
the alkyltransferase inhibitor but no BCNU and the cells were left
to grow for an additional 16-18 h. The cells were then replated at
densities of 250-1000 cells per 25 cm2 flask and grown for 8 days
until discrete colonies had formed. The colonies were washed with
0.9% saline solution, stained with 0.5% crystal violet in ethanol,
and counted.
Molecular Docking Studies. The computational modeling
studies relied upon the GLIDE (Grid-based Ligand Docking from
Energetics) program (Glide, version 4.5, Schro¨dinger, LLC New
York, 2007)42-44 for the docking simulations. These simulations
were performed using the X-ray crystal structure of the human
Acknowledgment. This research was supported in part by
the Intramural Research Program of the National Institutes of
Health, National Cancer Institute, Center for Cancer Research.

7152 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22
Pauly et al.
Lomeguatrib, a potent inhibitor of O6-alkylguanine-DNA alkyltrans-
ferase: phase I safety, pharmacodynamic, and pharmacokinetic trial
and evaluation in combination with temozolomide in patients with
advanced solid tumors. Clin. Cancer Res. 2006, 12, 1577–1584.
(19) Daniels, D. S.; Woo, T. T.; Luu, K. X.; Noll, D. M.; Clarke, N. D.;
Pegg, A. E.; Tainer, J. A. Novel modes of DNA binding and nucleotide
flipping by the human DNA repair protein AGT. Nat. Struct. Mol.
Biol. 2004, 11, 714–720.
Work in A.E.P.’s laboratory was supported by Grants CA-
018137, CA-097209, and CA-071976 from the National Cancer
Institute, National Institutes of Health, U.S.A.
Supporting Information Available: Elemental analysis results
for compounds 1-7. This material is available free of charge via
the Internet at http://pubs.acs.org.
(20) Guengerich, F. P.; Fang, Q.; Liu, L.; Hachey, D. L.; Pegg, A. E. O6-
Alkylguanine-DNA alkyltransferase: low pKa and reactivity of cysteine
145. Biochemistry 2003, 42, 10965–10970.
References
(1) Pegg, A. E. Repair of O6-alkylguanine by alkyltransferases. Mutat.
Res. 2000, 462, 83–100.
(2) Margison, G.; Povey, A. C.; Kaina, B.; Santiba´n˜ez-Koref, M. F.
Variability and regulation of O6-alkylguanine-DNA alkyltransferase.
Carcinogenesis 2003, 24, 625–635.
(3) Tubbs, J. L.; Pegg, A. E.; Tainer, J. A. DNA binding, nucleotide
flipping, and the helix-turn-helix motif in base repair by O6-
alkylguanine-DNA alkyltransferase and its implications for cancer
chemotherapy. DNA Repair (Amsterdam) 2007, 6, 1100–1115.
(4) Gerson, S. L. MGMT: its role in cancer aetiology and cancer
therapeutics. Nat. ReV. Cancer 2004, 4, 296–307.
(21) Xu-Welliver, M.; Pegg, A. E. Point mutations at multiple sites
including highly conserved amino acids maintain activity but render
O6-alkylguanine-DNA alkyltransferase insensitive to O6-benzylgua-
nine. Biochem. J. 2000, 347, 519–526.
(22) Pegg, A. E.; Xu-Welliver, M.; Loktionova, N. A. The DNA Repair
Protein O6-Alkylguanine-DNA Alkyltransferase as a Target for Cancer
Chemotherapy In DNA Alterations in Cancer: Genetic and Epigenetic
Changes; M. Ehrlich, E., Ed.; Eaton Publishing: Natick, MA, 2000;
pp 471-488.
(23) Pegg, A. E.; Goodtzova, K.; Loktionova, N. A.; Kanugula, S.; Pauly,
G. T.; Moschel, R. C. Inactivation of human O6-alkylguanine-DNA
alkyltransferase by modified oligodeoxyribonucleotides containing O6-
benzylguanine. J. Pharmacol. Exp. Ther. 2001, 296, 958–965.
(24) Luu, K. X.; Kanugula, S.; Pegg, A. E.; Pauly, G. T.; Moschel, R. C.
Repair of oligodeoxyribonucleotides by O6-alkylguanine-DNA alky-
ltransferase. Biochemistry 2002, 41, 8689–8697.
(25) Keppler, A.; Gendreizig, S.; Gronemeyer, T.; Pick, H.; Vogel, H.;
Johnsson, K. A general method for the covalent labeling of fusion
proteins with small molecules in vivo. Nat. Biotechnol. 2003, 21, 86–
89.
(26) Fang, Q.; Loktionova, N. A.; Moschel, R. C.; Javanmard, S.; Pauly,
G. T.; Pegg, A. E. Differential inactivation of polymorphic variants
of human O6-alkylguanine-DNA alkyltransferase. Biochem. Pharma-
col. 2008, 75, 618–626.
(27) Cho, A. E.; Guallar, V.; Berne, B.; Friesner, R. A. Importance of
accurate charges in molecular docking: quantum mechanical/molecular
mechanical (QM/MM) approach. J. Comput. Chem. 2005, 26, 915–
931.
(28) Bytheway, I.; Cochran, S. Validation of molecular docking calculations
involving FGF-1 and FGF-2. J. Med. Chem. 2004, 47, 1683–1693.
(29) 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.
(5) Pegg, A. E.; Dolan, M. E. Overcoming Resistance to Alkylating Agents
by Inhibitors of O6-Alkylguanine-DNA Alkyltransferase In DNA
Repair in Cancer Therapy; Panasci, L. C., Alaoui-Jamali, M., Eds.;
Humana Press: Totowa, NJ, 2004; pp 143-177.
(6) Dolan, M. E.; Pegg, A. E. O6-Benzylguanine and its role in
chemotherapy. Clin. Cancer Res. 1997, 3, 837–847.
(7) Middleton, M. R.; Margison, G. P. Improvement of chemotherapy
efficacy by inactivation of a DNA-repair pathway. Lancet Oncol. 2003,
4, 37–44.
(8) Khan, O.; Middleton, M. R. The therapeutic potential of O6-
alkylguanine-DNA alkyltransferase inhibitors. Expert Opin. InVest.
Drugs 2007, 16, 1573–84.
(9) Dolan, M. E.; Moschel, R. C.; Pegg, A. E. Depletion of mammalian
O6-alkylguanine-DNA alkyltransferase activity by O6-benzylguanine
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.
(10) Pegg, A. E.; Boosalis, M.; Samson, L.; Moschel, R. C.; Byers, T. L.;
Swenn, K.; Dolan, M. E. Mechanism of inactivation of human O6-
alkylguanine-DNA alkyltransferase by O6-benzylguanine. Biochemistry
1993, 32, 11998–12006.
(11) Daniels, D. S.; Mol, C. D.; Arvai, A. S.; Kanugula, S.; Pegg, A. E.;
Tainer, J. A. Active and alkylated human AGT structures: a novel
zinc site, inhibitor and extrahelical binding. EMBO J. 2000, 19, 1719–
1730.
(12) McElhinney, R. S.; Donnelly, D. J.; McCormick, J. E.; Kelly, J.;
Watson, A. J.; Rafferty, J. A.; Elder, R. H.; Middleton, M. R.;
Willington, M. A.; McMurry, T. B. H.; Margison, G. P. Inactivation
of O6-alkylguanine-DNA alkyltransferase 1. Novel O6-(hetarylmeth-
yl)guanines having basic rings in the side chain. J. Med. Chem. 1998,
41, 5265–5271.
(13) Quinn, J. A.; Desjardins, A.; Weingart, J.; Brem, H.; Dolan, M. E.;
Delaney, S. M.; Vredenburgh, J.; Rich, J.; Friedman, A. H.; Reardon,
D. A.; Sampson, J. H.; Pegg, A. E.; Moschel, R. C.; Birch, R.;
McLendon, R. E.; Provenzale, J. M.; Gururangan, S.; Dancey, J. E.;
Maxwell, J.; Tourt-Uhlig, S.; Herndon, J. E., 2nd.; Bigner, D. D.;
Friedman, H. S. Phase I trial of temozolomide plus O6-benzylguanine
for patients with recurrent or progressive malignant glioma. J. Clin.
Oncol. 2005, 23, 7178–7187.
(14) Warren, K. E.; Aikin, A. A.; Libucha, M.; Widemann, B. C.; Fox, E.;
Packer, R. J.; Balis, F. M. Phase I study of O6-benzylguanine and
temozolomide administered daily for 5 days to pediatric patients with
solid tumors. J. Clin. Oncol. 2005, 23, 7646–7653.
(15) Broniscer, A.; Gururangan, S.; MacDonald, T. J.; Goldman, S.; Packer,
R. J.; Stewart, C. F.; Wallace, D.; Danks, M. K.; Friedman, H. S.;
Poussaint, T. Y.; Kun, L. E.; Boyett, J. M.; Gajjar, A. Phase I trial of
single-dose temozolomide and continuous administration of O6-
benzylguanine in children with brain tumors: a pediatric brain tumor
consortium report. Clin. Cancer Res. 2007, 13, 6712–6718.
(16) Batts, E. D.; Maisel, C.; Kane, D.; Liu, L.; Fu, P.; O’Brien, T.; Remick,
S.; Bahlis, N.; Gerson, S. L. O6-Benzylguanine and BCNU in multiple
myeloma: a phase II trial. Cancer Chemother. Pharmacol. 2007, 60,
415–421.
(17) Gajewski, T. F.; Sosman, J.; Gerson, S. L.; Liu, L.; Dolan, E.; Lin,
S.; Vokes, E. E. Phase II trial of the O6-alkylguanine-DNA alkyl-
transferase inhibitor O6-benzylguanine and 1,3-bis(2-chloroethyl)-1-
nitrosourea in advanced melanoma. Clin. Cancer Res. 2005, 11, 7861–
7865.
(30) Kreklau, E. L.; Kurpad, C.; Williams, D. A.; Erickson, L. C. Prolonged
inhibition of O6
-methylguanine-DNA methyltransferase in human
-benzylguanine in vitro and in vivo. J. Pharmacol.
tumor cells by O6
Exper. Ther. 1999, 291, 1269–1275.
(31) Griffin, R. J.; Arris, C. E.; Bleasdale, C.; Boyle, F. T.; Calvert, A. H.;
Curtin, N. J.; Dalby, C.; Kanugula, S.; Lembicz, N. K.; Newell, D. R.;
Pegg, A. E.; Golding, B. J. Resistance-modifying agents. 7. Inhibition
of O6-alkylguanine-DNA alkyltransferase by O6-alkenyl-, O6-cycloalk-
enyl- and O6-(2-oxoalkyl)-guanines, and potentiation of temozolomide
cytotoxicity in vitro by O6-(1-cyclopentenylmethyl)guanine. J. Med.
Chem. 2000, 43, 4071–4083.
(32) Moschel, R. C.; McDougall, M. G.; Dolan, M. E.; Stine, L.; Pegg,
A. E. Structural features of substituted purine derivatives compatible
with depletion of human O6-alkylguanine-DNA alkyltransferase.
J. Med. Chem. 1992, 35, 4486–4491.
(33) Goodtzova, K.; Crone, T.; Pegg, A. E. Activation of human O6-
alkylguanine-DNA alkyltransferase by DNA. Biochemistry 1994, 33,
8385–8390.
(34) Pegg, A. E.; Chung, L.; Moschel, R. C. Effect of DNA on the
inactivation of O6-alkylguanine-DNA alkyltransferase by 9-substituted
O6-benzylguanine derivatives. Biochem. Pharmacol. 1997, 53, 1559–
1564.
(35) Nelson, M. E.; Loktionova, N. A.; Pegg, A. E.; Moschel, R. C.
2-Amino-O4-benzylpteridine derivatives: potent inactivators of O6-
alkylguanine-DNA alkyltransferase. J. Med. Chem. 2004, 47, 3887–
3891.
(36) Sampson, J. H.; Akabani, G.; Archer, G. E.; Berger, M. S.; Coleman,
R. E.; Friedman, A. H.; Friedman, H. S.; Greer, K.; Herndon, J. E.,
2nd.; Kunwar, S.; McLendon, R. E.; Paolino, A.; Petry, N. A.;
Provenzale, J. M.; Reardon, D. A.; Wong, T. Z.; Zalutsky, M. R.;
Pastan, I.; Bigner, D. D. Intracerebral infusion of an EGFR-targeted
toxin in recurrent malignant brain tumors. Neuro-Oncology 2008, 10,
320–329.
(18) Ranson, M.; Middleton, M. R.; Bridgewater, J.; Lee, S. M.; Dawson,
M.; Jowle, D.; Halbert, G.; Waller, S.; McGrath, H.; Gumbrell, L.;
McElhinney, R. S.; Donnelly, D.; McMurry, T. B.; Margison, G. P.
(37) Ferguson, S.; Lesniak, M. S. Convection enhanced drug delivery of
novel therapeutic agents to malignant brain tumors. Curr. Drug
DeliVery 2007, 4, 169–180.

InactiVation by O6-Benzylguanine
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22 7153
(38) Lopez, K. A.; Waziri, A. E.; Canoll, P. D.; Bruce, J. N. Convection-
enhanced delivery in the treatment of malignant glioma. Neurol. Res.
2006, 28, 542–548.
J. K.; Shaw, D. E.; Francis, P.; Shenkin, P. S. Glide: a new approach
for rapid, accurate docking and scoring. 1. Method and assessment of
docking accuracy. J. Med. Chem. 2004, 47, 1739–1749.
(39) McCalmont, W. F.; Patterson, J. R.; Lindenmuth, M. A.; Heady, T. N.;
Haverstick, D. M.; Gray, L. S.; Macdonald, T. L. Investigation into
the structure-activity relationship of novel concentration dependent,
dual action T-type calcium channel agonists/antagonists. Bioorg. Med.
Chem. 2005, 13, 3821–3839.
(40) Lee, T. R.; Niu, J.; Lawrence, D. S. The extraordinary active site
substrate specifity of pp60c-src a multiple specificity protein kinase.
J. Biol. Chem. 1995, 270, 5375–5380.
(41) Javanmard, S.; Loktionova, N. A.; Fang, Q.; Pauly, G. T.; Pegg, A. E.;
Moschel, R. C. Inactivation of O6-alkylguanine-DNA alkyltransferase
by folate esters of O6-benzyl-2′-deoxyguanosine and of O6-[4-
(hydroxymethyl)benzyl]guanine. J. Med. Chem. 2007, 50, 5193–5201.
(42) Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic,
J. J.; Mainz, D. T.; Repasky, M. P.; Knoll, E. H.; Shelley, M.; Perry,
(43) Halgren, T. A.; Murphy, R. B.; Friesner, R. A.; Beard, H. S.; Frye,
L. L.; Pollard, W. T.; Banks, J. L. Glide: a new approach for rapid,
accurate docking and scoring. 2. Enrichment factors in database
screening. J. Med. Chem. 2004, 47, 1750–1759.
(44) Friesner, R. A.; Murphy, R. B.; Repasky, M. P.; Frye, L. L.;
Greenwood, J. R.; Halgren, T. A.; Sanschagrin, P. C.; Mainz, D. T.
Extra precision glide: docking and scoring incorporating a model of
hydrophobic enclosure for protein-ligand complexes. J. Med. Chem.
2006, 49, 6177–6196.
(45) Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T. N.;
Weissig, H.; Shindyalov, I. N.; Bourne, P. E. The Protein Data Bank.
Nucleic Acids Res. 2000, 28, 235–242.
JM800675P