Towards more specific O6-methylguanine-DNA methyltransferase (MGMT) inactivators

Article abstract of DOI:10.1016/j.bmc.2011.01.038

Searching for a novel family of inactivators of the human DNA repair protein O⁶-methylguanine-DNA methyltransferase (MGMT) which is known to bind to the DNA minor groove, we have computationally modelled and synthesised two series of 2-amino-6-

Full text of DOI:10.1016/j.bmc.2011.01.038

Bioorganic & Medicinal Chemistry 19 (2011) 1658–1665
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Towards more specific O6-methylguanine-DNA methyltransferase (MGMT)
inactivators
Sergio Lopez a, Geoffrey P. Margison b, R. Stanley McElhinney a, , Alessandra Cordeiro a,
T. Brian H. McMurry a, Isabel Rozas a,
⇑
a School of Chemistry, University of Dublin, Trinity College, Dublin 2, Ireland
b Cancer Research UK Carcinogenesis Group, Paterson Institute for Cancer Research, University of Manchester, Manchester M20 4BX, UK
a r t i c l e i n f o
a b s t r a c t
Searching for a novel family of inactivators of the human DNA repair protein O6-methylguanine-DNA
methyltransferase (MGMT) which is known to bind to the DNA minor groove, we have computationally
modelled and synthesised two series of 2-amino-6-aryloxy-5-nitropyrimidines with morpholino or
aminodiaryl substituents (potential minor groove binders) at the 4-position. Synthesis of these com-
pounds was achieved by successive substitution of each of the two Cl atoms of 2-amino-4,6-dichloro-
5-nitropyrimidine by the corresponding amino and aryloxy derivatives. Biochemical evaluation of these
compounds as MGMT inactivators showed poor activities, but in general the 4-bromothenyloxy deriva-
tives showed better inactivation than the benzyloxy versions. DNA binding assessment was not possible
due to insolubility problems.
Article history:
Received 23 November 2010
Revised 14 January 2011
Accepted 19 January 2011
Available online 27 January 2011
Keywords:
DNA repair
MGMT
PaTrin2
5-Nitropyrimidine
Docking
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
tion.8–10 MGMT inactivators [such as O6-(4-bromothenyl)guanine,
(originally named PaTrin-2 and now Lomeguatrib, Fig. 1) and
It is well known that resistance to the cytotoxic effects of
antitumor alkylating agents such as the nitrosoureas (e.g., bis-
chloroethylnitrosourea, BCNU) or methylating agents (e.g., tem-
ozolomide, TMZ) can be mediated by the human DNA repair
protein O6-methylguanine-DNA methyltransferase (MGMT).
MGMT removes alkyl groups from guanine residues that have been
alkylated at the O6-position by these drugs. The alkyl group is
transferred to a cysteine residue in its active site in an auto-inacti-
vating stoichiometric suicidal process.1,2 MGMT can be expressed
at high levels in certain tumour cells,2,3 and hence it has become
an important target for the development of drugs designed to im-
prove the effectiveness of the treatment of tumours with this type
of alkylating agent.3–5
MGMT binds to the minor groove of DNA in a rapid on–off pro-
cess and alkyl transfer occurs when MGMT encounters a recogni-
sed substrate O6-alkylguanine lesion.6 More recently it has been
shown that at least in vitro, minor groove binding can be coopera-
tive.7 The minor groove of DNA is also the interaction site for many
enzymes and transcription control proteins. Consequently, com-
pounds that target the minor groove have been investigated for
their potential for treating cancer via inhibition of the action of
DNA-dependent enzymes or by direct inhibition of transcrip-
O6-benzylguanine] that have been in clinical trials, have so far been
shown not to improve patient outcomes.11 Hence, new approaches
need to be considered to enhance the inactivation of this protein in
order to overcome the resistance to alkylating agents which are
still some of the preferred drugs in cancer chemotherapy.
Current inactivators are based on free guanine and thus do not
directly exploit the DNA minor groove binding characteristics of
MGMT. Therefore, a novel approach to MGMT inactivation was
considered: by attaching a minor groove binding moiety to an
MGMT inactivator, we reasoned that it might better present the
inactivating moiety to the active site of the protein. The possibility
that the minor groove binding characteristics of the compound
might result in an attenuation of the cooperative binding of
MGMT7 and reduce the overall rate of repair of cytotoxic DNA
damage, was also considered.
Here we describe the modelling, synthesis and biochemical
assessment of 4-nitropyrimidine derivatives that were intended
to act as MGMT inactivators coupled to di-aromatic amino deriva-
tives which could interact with the DNA minor groove (Fig. 1). The
results obtained for the corresponding morpholine derivatives
(1 and 2 in Fig. 1) are also presented and it has to be noted that
the ring numbering of these compounds is different that the rest
of the pyrimidines here presented. Henceforth, and for the sake
of clarity, we will use the ring numbers shown for compounds 3
and 4 in Figure 1 when referring to ring substitution for all
compounds.
⇑
Corresponding author. Tel.: +353 18963731; fax: +353 16712826.
E-mail address: rozasi@tcd.ie (I. Rozas).
In memoriam.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.01.038

S. Lopez et al. / Bioorg. Med. Chem. 19 (2011) 1658–1665
1659
Br
OR1
4
S
R1
NO2
3
N
5
4-Br-thenyl benzyl
morpholine
1
2
O
6
N
X= CH2
X= NH
X= O
3a
3b
3c
3d
4a
4b
4c
4d
H2N
N
2
1
N
O
N
1, 2
X= CO
N
H
H2N
N
OR1
6
NO2
1
X
N
PaTrin-2
5
2
H2N
N
N
H
NH2
4
3
3a-d, 4a-d
Figure 1. Structure of PaTrin-2 and the nitro derivatives 1–4 proposed in this study.
LIGPLOT software (v. 4.4.2) was used to analyse the docking re-
sults, helping to determine the interactions formed between ligand
and residues in the active site. This program takes into account
hydrogen bonding (by means of distance cut-offs) and hydrophobic
interactions and, despite its simplicity, provides a good insight of
the interactions established within the complexes: the result
obtained for the MGMT:PaTrin-2 complex is shown in Figure 3a.
Several hydrophobic contacts are established between the
4-bromothenyl or the purine group and various MGMT residues
(Met134, Val148, Asn157, Gly160 Tyr114 and Gly156). In general,
the PaTrin-2 molecule is totally surrounded by hydrogen bond con-
tacts, which indicates very good packing in the binding site of the
protein, conferring substantial stability to the complex.
Details of the interactions observed in the docking of all the
compounds studied and the active site of MGMT are presented in
the Supplementary data section and here only the general trends
will be discussed. In Figure 3, the plots obtained for compounds
1, 2, 3b and 4b are shown, as examples of the docking experiments
performed and the interactions established.
2. Results
2.1. Molecular modelling
To predict the affinity of the proposed compounds for MGMT
and to gain insight into the forces operating in the MGMT–ligand
complexes,
a molecular modelling study was undertaken.
PaTrin-2 was included to allow a better understanding of the cor-
responding MGMT activity. Thus, docking studies were carried out
based on the crystal structure of MGMT12 as a template and using
the AUTODOCK4.2 package.13 Fully optimized structures of the ligands
(using B3LYP/6-31G14) were used for the docking experiments
with the rigid structure of MGMT in a ligand-flexible approach. A
detailed interaction analysis for each MGMT–ligand complex was
then performed, and the binding energies were evaluated using a
scoring function, based on the AMBER force-field that considers
van der Waals’ and electrostatic interactions.15
The repair mechanism of MGMT involves the transfer of an alkyl
residue from guanine to the Cys145, and Pegg and co-workers16
proposed that this Cys145 is highly reactive and exists as a thiolate
anion at neutral pH. Thus, the interaction of the S atom in the
Cys145 with the ether linkage of PaTrin-2 is crucial for the inacti-
vation process to occur. Figure 2 shows the results obtained for the
docking experiment of PaTrin-2 into the MGMT protein indicating
how the ether O atom and the CH2 group of the thenyl moiety are
close to this Cys145.
Docking studies also provide information about the role of the
amino acid residues in the active site of the binding process and
the type of interactions involved. We found that some amino acids
in the MGMT binding pocket are always involved in the interac-
tions established with the ligands to form the complexes. For
example, Pro140 was found to form hydrophobic interactions with
all of the compounds except PaTrin-2. In all of the complexes,
Figure 2. 3D-Representation using Rasmol (v.2.6) of the MGMT:PaTrin-2 complex obtained from molecular docking. The ether O atom of PaTrin-2 (red) can be found near the
Cys145 (yellow).

1660
S. Lopez et al. / Bioorg. Med. Chem. 19 (2011) 1658–1665
Figure 3. LIGPLOT representation. of the docking of (a) PaTrin-2, morpholine derivatives (b) 1 and (c) 2, and diaromatic derivatives with NH linker (d) 3b and (e) 4b, into the
MGMT binding pocket.
residues Arg135 and Ser159 were found to form hydrophobic or
hydrogen bond interactions depending on the ligand. Interestingly,
Tyr158 is involved in hydrophobic interactions only in complexes
with 4-bromothenyl derivatives. The key residue, Cys145 appears
to be involved in the formation of all the complexes studied but
not always establishing interactions near the 4-bromothenyl or
benzyl groups. We can consider the interaction of these residues
ligand-specific because the involvement of the rest of the amino
acid residues cannot be rationalised.
The binding energies obtained with the scoring function, which
should be considered only as an indication of the potential of these
compounds as MGMT inactivators, are shown in Table 1. An impor-
tant conclusion is the strong interaction observed for the lead com-
pound, PaTrin-2, followed by compounds 1 and 2 (morpholine
substituted nitropirymidines) which show half of this binding
strength. Thus, a better MGMT inhibitory activity would be ex-
pected for those compounds in comparison with the rest, which
demonstrated much weaker binding. Additionally, larger interac-
tion energy values are obtained for the 4-bromothenyl derivatives
compare to the corresponding benzyl derivatives, in agreement
with the best inactivating activity previously observed experimen-
tally with PaTrin-2 and its 6-benzyloxy analogue.17 The di-
aromatic compounds (3a–d and 4a–d) are significantly poorer
ligands, in agreement with the poor fit of the aromatic rings into
the MGMT binding pocket due to steric hindrance. The significant
improvement in interaction energy of the morpholino derivatives
compared to the amino di-aromatic compounds could be explained
because the attached group (morpholine) at the 4 position of the
5-nitropyrimidine is less bulky, more flexible and fits better into
the active site of the protein.
In general, positive results were obtained from the docking
experiments with PaTrin-2 and all the compounds proposed,

S. Lopez et al. / Bioorg. Med. Chem. 19 (2011) 1658–1665
1661
Table 1
The morpholino pyrimidine 6 was prepared using excess of
morpholine in acetone. We found that this compound was ob-
tained in higher yield at reflux temperature, whereby the disubsti-
tuted derivative was not detected in any of the experiments.
Nevertheless, this was expected due to the lower nucleophilicity
of secondary amines over alkoxyde groups. Further incorporation
of 4-bromothenyloxy and benzyloxy groups into the morpholino
derivative 6 was achieved using an improved version of the meth-
od previously reported by Pegg et al.10 These authors reported the
displacement of chlorine in 2-amino-4-chloro-5-nitropyrimidine
to afford 2-amino-4-benzyloxy-5-nitropyrimidine at high temper-
atures (160 °C) with sodium in excess benzyl alcohol, also used as
solvent. However, this approach would not be desirable for the
expensive 4-bromothenyl alcohol and, hence, we used an equimo-
lar amount of benzyl or 4-bromothenyl alcohol and sodium hy-
dride in DMSO as solvent. Applying this protocol, compounds 1
and 2 were finally prepared (Scheme 1).
Having optimised the introduction of both the substituent in
the 4-position and, then, the alkoxy moieties in the 6-position,
we focused on the incorporation of aryl diamines, such as 7a–d
(commercially available), to the 4 position of the 5-nitropyrimidine
5 (Scheme 2). These amines could be positively charged at
physiological pH and possess a concave shape, two important
factors involved in DNA minor-groove recognition,21 as has been
investigated in our laboratory.22 However, when compound 5
was treated with one equivalent of bis-(4-aminophenyl)methylene
(7a) and triethylamine, a yellow precipitate was obtained that
proved by NMR analysis to be the symmetric di-pyrimidine 8
(Scheme 2). Lower reaction temperatures or higher dilution of
the reagents also led to the formation of this insoluble precipitate,
and the desired monopyrimidine derivative was not detected.
Thus, the selective introduction of one alkoxy group into 5 prior
to the coupling reaction with these aryl amines was investigated.
The mono alkoxylation of 5 was performed using an equivalent
amount of the corresponding alcohol and sodium hydride under
the reactions conditions herein reported for the synthesis of 1
and 2. However, the reaction was carried out in THF, due to the
problems encountered in the purification of the products when
DMSO was used. Optimization of the displacement of only one
chlorine atom was required as substantial amounts of the di-
substituted product were also obtained as previously reported.
The reaction temperature was lowered to 0 °C to control the reac-
tivity of the alkoxide and subsequent isolation of the compounds 9
and 10 (Scheme 2) was accomplished by successive column chro-
matographies on silica gel as the products and unreacted starting
material have similar Rf values.
IC50 values for inactivation of MGMT by compounds 1, 2, 3a–d and 4c–d, the 4-
unsubtitued compound (11) and PaTrin-2 and docking binding energies calculated
with AUTODOCK 4.2 for compounds 1, 2, 3a–d, 4a–d and PaTrin-2
OR1
NO2
N
H2N
N
N(H)R2
M) Binding E (kcal molꢀ1
)
a
Compound R1
R2
BrTn Morpholine–
Bn Morpholine–
IC50
(l
1
2
3a
3b
3c
3d
4a
4b
4c
4d
11
PaTrin-2
1.0
19.0
ꢀ4.44
ꢀ4.49
ꢀ4.16
ꢀ3.50
ꢀ1.52
ꢀ1.98
ꢀ2.67
ꢀ1.80
ꢀ0.77
ꢀ2.83
—
BrTn H2N–Ph–CH2–Ph– 20.0
BrTn H2N–Ph–NH–Ph–
BrTn H2N–Ph–O–Ph–
BrTn H2N–Ph–CO–Ph–
4.7
13.0
5.7
b
Bn
Bn
Bn
Bn
H2N–Ph–CH2–Ph–
H2N–Ph–NH–Ph–
H2N–Ph–O–Ph–
H2N–Ph–CO–Ph–
—
—
b
>600c
>600c
0.32
BrTn H–
0.003
ꢀ8.95
a
Bn = benzyl; BrTn = 4-bromothenyl.
These compounds could not be tested because lack of material due to their
b
difficult preparation.
c
These compounds showed no inactivation at the highest concentration used in
the assay (600 lM).
indicating stabilising interactions with the MGMT active site and
for that reason we proceeded to prepare the new nitropyrimidines
1–4.
2.2. Chemistry
The preparation of nitropyrimidines as analogues of O6-benzyl-
guanine has previously been reported.18,19 In this work we present
the preparation of the corresponding O6-4-bromothenyl and
O6-benzyl nitropyrimidines introducing moieties at the amino
group at the 4-position that we considered would interact with
the DNA minor groove putatively presenting the MGMT inactivator
moiety more effectively to the active site of the protein. To prepare
these new derivatives, the synthesis of compound 5 is absolutely
essential and we have already reported an efficient pathway for
its synthesis.20
Starting from compound 5, two possible routes could be consid-
ered to obtain the final nitropyrimidines that are conveniently
functionalised at the 4-position. That is the subsequent substitu-
tion of each of the Cl atoms by amines and alkoxyde groups, or
inversely.
For the first route, compound 5 was treated with sodium meth-
oxide, as a model alkoxide for the more expensive 4-bromothenyl
alcohol. Even at room temperature, the displacement of both of the
chlorines of pyrimidine 5 by methoxide was observed. Since the
selectivity of the reaction could not be controlled and the desired
product was obtained in a very poor yield, the alternative route
was investigated (Scheme 1).
The final target compounds 3a–d and 4a–d (Scheme 2) were ob-
tained in moderate to high yield (41–90%) from the addition reac-
tion of compounds 9 and 10, respectively to the corresponding
commercial diaryl amines, 7a–d.
2.3. Biochemistry
The 5-nitropyrimidines 1, 2, 3a–d, and 4c–d were tested for
their ability to inactivate purified recombinant MGMT in vitro.
Cl
OR1
Cl
NO2
NO2
N
NO2
Cl
N
N
N
(i)
(ii)
H2N
N
N
H2N
N
H2N
N
O
O
1 (42%), R1 = 4-Bromothenyl
2 (46%), R1 = Benzyl
5
6
(88%)
Scheme 1. Reagents and conditions: (i) morpholine in acetone at reflux, 1.5 h; (ii) R1OH, NaH in DMSO, 80 °C, 2 h.

1662
S. Lopez et al. / Bioorg. Med. Chem. 19 (2011) 1658–1665
Cl
Cl
Cl
N
NO2
Cl
NO2
O2N
(i, 7a)
N
N
N
H2N
N
H2N
N
N
N
H
NH2
H
5
8
(ii)
OR1
OR1
X
NO2
Cl
NO2
NH
N
N
(i)
NH2
H2N
N
H2N
N
9 (24%), R1= 4-bromothenyl
10 (42%), R1= benzyl
3a-d (41, 56, 90, 68%), R1 = 4-bromothenyl
4a-d (26, 30, 85, 82%), R1 = benzyl
7a, X= CH2
7b, X= NH
7c, X= O
X
H2N
NH2
7d, X= CO
Scheme 2. Reagents and conditions: (i) 7a–d, TEA in refluxing THF; (ii) R1OH, NaH in THF, 0 °C.
PaTrin-2 was used as a positive control in all assays and the result
obtained for the 4-unsubstituted O6-(4-bromothenyl) derivative
(11) was considered for comparison.19 The resulting IC50 values
are presented in Table 1. Compounds 4c and 4d were unable to
inactivate MGMT at the highest concentrations used and only
DNA, we tried to perform thermal denaturation experiments in
salmon sperm DNA (68% Adenine–Thymine base pair content).
Unfortunately, these families of molecules were not soluble in
the buffers tested (phosphate, MES, Tris) or in mixtures of DMSO
or ethanol and the buffers. Hence, it was not possible to measure
the ability of these compounds to bind to DNA.
can be concluded that the IC50 for these compounds is >600
All other compounds showed much greater inactivation with
IC50 values ranging from 1 to 20 M (Table 1). The 2-amino-4-
lM.
l
3. Conclusions
(4-bromothenyloxy)-6-morpholino-5-nitropyrimidine 1 was the
most active compound synthesised, but substitution by polyaro-
matic amines in the benzyloxy derivatives (4c and 4d), as just
mentioned, clearly obliterated activity. This demonstrates that
bulky groups at the 4-position of 5-nitropyrimidines negatively af-
fect MGMT-inactivation capacity. The difficult preparation of the
less accessible benzyloxy analogues 4a and 4b (X = CH2, NH) did
not provide enough material for their biochemical evaluation;
however, it seems likely that they would follow the same pattern.
In the case of the morpholino derivatives, the benzyloxy com-
pound 2 exhibited an activity approximately 20 times lower than
the corresponding 4-bromothenyloxy derivative 1. This trend
was previously observed in our laboratory, since PaTrin-2 was
found to be ten times more potent an inactivator than the corre-
Searching for novel MGMT inactivators that can also bind to the
DNA minor groove, we have performed docking experiments with
PaTrin-2 and two series of 2-amino-6-aryloxy-5-nitropyrimidines
with (a) morpholino or (b) aminodiaryl (known minor-groove
binder skeletons) substituents at the 4-position. Based on the com-
puted stabilising interactions of these compounds with the MGMT
active site, we synthesised both series of nitropyrimidines.
Thus, starting from 2-amino-4,6-dichloro-5-nitropyrimidine it
was possible to prepare the morpholino derivatives by first substi-
tuting one of the Cl groups by morpholine and then introducing the
aryloxy group by substitution of the second Cl group. In the case of
the diaryloxy derivatives, the prerequisite was the introduction of
the aryloxy group followed by the substitution of the second Cl
group by the corresponding diaryldiamino compound.
sponding O6-benzylguanine (IC50 0.04 M).16,23
l
These results also indicate that, in general, the substitution at
the 4-position of the pyrimidine ring is not well tolerated, since
the introduction of morpholine resulted in a poorer biological
activity. The 4-bromothenyloxy derivative unsubstituted in 4-posi-
tion (11) previously prepared by our laboratory,17,24 was more po-
tent than any of the substituted versions (Table 1), suggesting as
well that the 4-position is not best suited to derivatisation.
Thus far, no clear pattern has emerged by the comparison of the
IC50 values of the amino diaryl compounds (3). However, it seems
that those compounds with diaryl moieties containing groups that
can form strong hydrogen bonds (X = NH, CO) were better MGMT
inactivators and this may be a consequence of interactions with
the amino acid residues involved in the MGMT binding process.
However, it must be noted that compared to the IC50 value for
Biochemical evaluation of these compounds as MGMT inhibi-
tors demonstrated poor activities. The IC50 for the benzyloxy diaro-
matic compounds, 4c and 4d, is greater than 600 lM. All of the
other derivatives, morpholino (1 and 2) and 4-bromothenyloxy
amino diaryl (3a–d), showed better inactivation (IC50s between 1
and 20 lM).
All of the compounds prepared had substantially higher IC50s
than PaTrin-2 and while the lack of the guanine core skeleton in
these dual agents might explain this, previous nitro derivatives
(both benzyloxy- and 4-bromothenyloxy-) have shown reasonably
potent MGMT inactivation.18,24 This might suggest that, even if
binding of our compounds to DNA was weak, it could have the
effect of reducing the compound’s ability to inactivate MGMT:
for alkyl transfer from alkyguanines in DNA, MGMT rotates the
damaged base around the phosphodiester linkages and into
the MGMT active site pocket, while an arginine residue occupies
the vacated site in the helix. This may not be possible if the base
is part of a structure that is bound to DNA, possibly in an
PaTrin-2 (0.003
poor activity.
lM), all of the present compounds showed a very
To assess the ability of the conjugated MGMT-inactivator/min-
or-groove binder derivatives (compounds 3 and 4) to bind to

S. Lopez et al. / Bioorg. Med. Chem. 19 (2011) 1658–1665
1663
inappropriate topolgy, rather than being an integral part of the
DNA molecule. On the other hand, if DNA binding is involved, for
the present compounds, this may be too weak and hence much
stronger anchoring to DNA might be necessary to facilitate MGMT
inactivation.
ysis Laboratory, School of Chemistry and Chemical Biology, Univer-
sity College Dublin and at the Instituto de Química Médica (CSIC) in
Madrid, Spain.
4.2.1. 2-Amino-4-chloro-6-morpholino-5-nitropyrimidine (6)
In this context the actual nature of the minor-groove binding
moieties may be important since these structures have been
shown to better interact with the minor groove when cationic ami-
dine-like groups are incorporated at both sides of the diaromatic
system.21,25 Finally, poor activity could be due to a non-optimal
distance between the two functionalities (minor-groove binding
and MGMT inactivation), incorporated in these molecules: linkers
between these functions might therefore be incorporated in future
dual agents.
A solution of morpholine (479 mg, 5.5 mmol) in acetone
(10 mL) was added dropwise to a stirred solution of 2-amino-4,
6-dichloro-5-nitropyrimidine (418 mg, 2 mmol) in acetone
(10 mL), and the reaction mixture was refluxed for 1.5 h. The sol-
vent was removed in vacuum and the residue was treated with
water (10 mL). The suspension was filtered off and the yellow solid
obtained was washed with water, suction-dried and collected
(459 mg, 88%). mp 210–212 °C. 1H NMR (DMSO-d6) d 3.37 (t, 4H,
J 4.9, NCH2CH2O); 3.63 (t, 4H, J 4.9, NCH2CH2O); 7.49 (s, 1H, NH);
7.60 (s, 1H, NH). 13C NMR (DMSO-d6) d 46.6 (NCH2CH2O); 65.8
(NCH2CH2O); 122.0 (C5); 153.5, 156.8, 159.9 (C2, C4, C6). IR mmax
3470, 3326, 3211 (NH2); 1549 (NO2) cmꢀ1. HRMS (ES) [M+H]+ calcd
for C8H11ClN5O3 260.0550; found 260.0548. Anal. Calcd for
C8H10ClN5O3: C, 37.0; H, 3.9; N, 27.0. Found: C, 37.0; H, 4.0; N, 26.7.
4. Experimental
4.1. Modelling
The crystal structure of MGMT (PDB entry: 1QNT, resolution
1.90 Å) was used to derive a model for ligand binding in the active
site. To reduce the required computational time for docking, we
described the active site around the Cys145 residue, which is lo-
cated within the residues 83–176 of the C-terminal domain.
Hydrogen atoms were added to the crystal structure assuming a
pH of 7, using the SYBYL program (v.7.2). Point charges were as-
signed to the MGMT protein according to the TRIPOS force-field.
SYBYL (v.7.2) was used for the representation of the ligands, using
the Gasteiger-Hückel algorithm for the calculation of the charges.
The AUTODOCK program (v. 4.2)12 was then used to perform the dock-
ing study. The residues Tyr114, Gly131, Met134, Arg135, Asn137,
Pro140, Cys145, Val148, Tyr158, Ser159 and Gly160 were consid-
ered essential for the binding process, as previously reported by
Wiessler and co-workers26 we selected the centre of these residues
as the position of the active site and centre of this grid (coordinates
XYZ: 1.440, 41.180, 37.690). The grid point spacing was defined as
0.2 Å. Finally, the LIGPLOT program (v.4.4.2) was used for determin-
4.2.2. General procedure for the preparation of 4-alkoxy-6-
morpholino derivatives
Sodium hydride (25 mg, 1 mmol) was added to a stirred solu-
tion of the corresponding alcohol (1.2 mmol) in dry DMSO (1 mL)
under an argon atmosphere. Effervescence was observed due to
the formation of hydrogen gas. After 30 min, 6 (260 mg, 1 mmol)
was added in small portions and the reaction mixture was heated
to 80 °C for 2–3 h. DMSO was removed in high vacuum and the res-
idue was treated with water (4 mL), giving a viscous product.
4.2.2.1. 2-Amino-4-(4-bromothenyloxy)-6-morpholino-5-nitro-
pyrimidine (1).
The product was recrystallised from a mix-
ture methanol/water to afford a yellow solid (191 mg, 46%). mp
152–154 °C. 1H NMR (DMSO-d6) d 3.35–3.45 (m, 4H, NCH2CH2O);
3.55–3.75 (m, 4H, NCH2CH2O); 5.52 (s, 2H, OCH2ThBr); 7.29 (br s,
3H, H30, NH2); 7.70 (s, 1H, H50). 13C NMR (DMSO-d6) d 46.9
(NCH2CH2O); 62.2, 66.0 (OCH2Th, NCH2CH2O); 108.2, 111.5 (C40,
C5); 125.6 (C50); 131.0 (C30); 140.2 (C20); 158.34, 160.0, 162.9
(C2, C4, C6). IR mmax 3475, 3337, 3225 (NH2); 1545 (NO2) cmꢀ1
.
ing and plotting
interaction.
a 2D representation of the ligand–protein
HRMS (ES) [M+Na]+ calcd for C13H14BrN5O4SNa 437.9848; found
437.9848. Anal. Calcd for C13H14BrN5O4S: C, 37.5; H, 3.4; N, 16.8.
Found: C, 37.3; H, 3.4; N, 16.4.
4.2. Chemistry
All the commercial chemicals were obtained from Sigma–
Aldrich or Fluka and were used without further purification.
Deuterated solvents for NMR use were purchased from Apollo.
Dry solvents were prepared using standard procedures, according
to Vogel,27 with distillation prior to use. Chromatographic columns
used Silica Gel 60 (230–400 mesh ASTM). Solvents for synthesis
purposes were used at GPR grade. Analytical TLC was performed
using Merck Kieselgel 60 F254 silica gel plates. Visualisation was
by UV light (254 nm). NMR spectra were recorded in a Bruker
DPX-400 Avance spectrometer, operating at 400.13 MHz and
600.1 MHz for 1H NMR; and at 100.6 MHz and 150.9 MHz for 13C
NMR. Shifts are referenced to the internal solvent signals and J val-
ues recorded in Hertz. NMR data were processed using Bruker
Win-NMR 5.0 software. HRMS spectra were measured on a Micro-
mass LCT electrospray TOF instrument with a WATERS 2690 auto-
sampler with methanol, water or ethanol as carrier solvents.
Melting points were determined using a Stuart Scientific Melting
Point SMP1 apparatus and are uncorrected. Infrared spectra were
recorded on a Mattson Genesis II FTIR spectrometer equipped with
a Gateway 2000 4DX2-66 workstation and on a Perkin Elmer Spec-
trum One FT-IR Spectrometer equipped with Universal ATR sam-
pling accessory. Samples analysis was carried out in Nujol using
NaCl plates. Elemental analyses were carried out at the Microanal-
4.2.2.2. 2-Amino-4-benzyloxy-6-morpholino-5-nitropyrimidine
(2).
The product was purified by column chromatography on
bright yellow solid
silica gel (3:1 EtOAc/hexane) to give
a
(137 mg, 42%). Mp 170–172 °C. 1H NMR (DMSO-d6) d 3.36 (t, 4H,
J 4.4, NCH2CH2O); 3.62 (t, 4H, J 4.4, NCH2CH2O); 5.39 (s, 2H,
OCH2C6H5); 7.23 (br s, 2H, NH2); 7.32–7.42 (m, 5H, OCH2C6H5).
13C NMR (DMSO-d6) d 48.9 (NCH2CH2O); 66.0, 68.0 (OCH2C6H5,
NCH2CH2O); 111.7 (C5); 127.9, 128.1, 128.6, 136.4 (OCH2C6H5);
158.3, 160.3, 163.5 (C2, C4, C6). IR mmax 3469, 3336, 3223 (NH2);
1548 (NO2) cmꢀ1. HRMS (ES) [M+Na]+ calcd for C15H17N5O4Na
354.1178; found 354.1178. Anal. Calcd for C15H17N5O4: C, 54.4;
H, 5.2; N, 21.1. Found: C, 54.5; H, 5.2; N, 20.7.
4.2.3. 2-Amino-4-(4-bromothenyloxy)-6-chloro-5-nitro-
pyrimidine (9)
Sodium hydride (96 mg, 4 mmol) was added to a stirred solu-
tion of 4-bromothenyl alcohol (772 mg, 4 mmol) in dry THF
(30 mL) under an argon atmosphere. Effervescence was observed.
After 30 min, the mixture was cooled to 0 °C, followed by the addi-
tion of 5 (1.24 g, 6 mmol). The mixture was stirred overnight (the
temperature rose from 0 °C to 13 °C during that period). Aqueous
THF (10 mL) was added dropwise and the solvent was removed
leaving a sticky oily residue. Hexane (30 mL) was added to form

1664
S. Lopez et al. / Bioorg. Med. Chem. 19 (2011) 1658–1665
a yellow solid, which was filtered and washed with hexane. The so-
lid formed was purified by column chromatography on silica gel
(2:3 EtOAc/hexane) to give a bright yellow solid (342 mg, 24%).
mp 146–148 °C. 1H NMR (DMSO-d6) d 5.61 (s, 2H, OCH2ThBr);
7.34 (s, 1H, H30); 7.75 (s, 1H, H50); 8.12 (s, 1H, NH); 8.21 (s, 1H,
6.54 (d, 2H, J 8.5, H200); 6.76 (d, 2H, J 8.5, H30); 6.83 (d, 2H, J 8.5,
H300); 7.34 (s, 1H, H3000); 7.37 (d, 2H, H20); 7.54 (s, 1H, C6H4NHC6H4);
7.58 (br s, 2H, NH2); 7.72 (s, 1H, H5000); 10.4 (s, 1H, pyrimidine-
NHC6H4). 13C NMR (DMSO-d6) d 62.5 (ThBrCH2O); 108.2, 109.6
(C5–NO2), 113.9, 115.1, 122.6, 124.9, 125.6, 128.0, 131.0, 132.0,
140.3, 144.2, 156.5, 158.8, 160.5, 164.4. IR mmax 3451, 3411
(NH2); 3331, 3218 (NH2); 1552 (NO2) cmꢀ1. HRMS (ES) [M+H]+
calcd for C21H19BrN7O3S 528.0375; found 528.0384. Anal. Calcd
for C21H18BrN7O3Sꢂ0.5EtOAc: C, 48.3; H, 3.9; N, 17.1. Found: C,
48.25; H, 3.7; N, 16.9.
13
NH). C NMR (DMSO-d6) d 63.3 (OCH2ThBr); 108.4 (C40); 123.0,
126.0, 131.7, 139.0 (C5–NO2, C30, C20, C50); 152.8, 160.5, 161.7
(C2, C4, C6). IR mmax 3466, 3422, 3305 (NH2); 1543 (NO2) cmꢀ1
.
HRMS (ES) [M+H]+ calcd for C9H7BrClN4O3S 364.9111; found
364.9120.
4.2.4. 2-Amino-4-benzyloxy-6-chloro-5-nitropyrimidine (10)
Sodium hydride (96 mg, 4 mmol) was added to a stirred solu-
tion of benzyl alcohol (432 mg, 4 mmol) in dry THF (30 mL) under
an argon atmosphere. Effervescence was observed. After 1 h, the
mixture was cooled to 0 °C, followed by the addition of 5 (1.09 g,
5.2 mmol). The mixture was stirred overnight (the temperature
rose from 0 °C to 13 °C during that period). Water (5 mL) was
added dropwise to quench the excess of base. THF was removed
in vacuum and the residue was treated with EtOAc, washed with
water and dried over Na2SO4. Filtration and evaporation of the sol-
vent left a brown residue which was purified by column chroma-
tography on silica gel (1:2 EtOAc/hexane) to afford a bright
yellow solid (476 mg, 42%). Mp 168–170 °C. 1H NMR (DMSO-d6)
d 5.46 (s, 2H, OCH2C6H5); 7.27–7.50 (m, 5H, OCH2C6H5); 8.06 (s,
1H, NH); 8.13 (s, 1H, NH). 13C NMR (DMSO-d6) d 69.3 (OCH2C6H5);
123.1 (C5), 128.1, 128.4, 128.5, 135.4 (OCH2C6H5); 152.6 160.7,
162.2 (C2, C4, C6). IR mmax 3478, 3318, 3204 (NH2); 1544
(NO2) cmꢀ1. HRMS (ES) [M+H]+ calcd for C11H10ClN4O3 281.0441;
found 281.0440.
4.2.5.3. 2-Amino-4-(4-bromothenyloxy)-6-[N-(4-(4-aminophen-
oxy)anilino)]-5-nitropyrimidine (3c).
The residue was puri-
fied by column chromatography on silica gel (2:1 EtOAc/hexane)
to afford a bright yellow solid (95 mg, 90%). mp 202–204 °C. 1H
NMR (DMSO-d6) d 5.00 (br s, 2H, C6H4NH2); 5.60 (s, 2H, ThBrCH2O);
6.59 (d, 2H, J 8.5, H200); 6.78 (d, 2H, J 8.5, H300); 6.83 (d, 2H, J 9.0,
H20); 7.33 (s, 1H, H3000); 7.57 (d, 2H, J 9.0, H20); 7.64 (s, 2H, NH2);
7.71 (s, 1H, H5000); 10.4 (s, 1H, NH). 13C NMR (DMSO-d6) d 62.4
(ThBrCH2O); 108.2, 109.7 (C5–NO2), 115.0, 116.6, 121.1, 125.0,
125.6, 131.0, 131.1, 140.1, 145.7, 145.8, 156.1, 156.5, 160.4,
164.2. IR mmax 3430, 3416, 3337 (NH2); 1559 (NO2) cmꢀ1. HRMS
(ES) [M+H]+ calcd for C21H18BrN6O4S 529.0215; found 529.0224
Anal. Calcd for C21H17BrN6O4Sꢂ0.2EtOAc: C, 47.9; H, 3.4; N, 15.4.
Found: C, 47.9; H, 3.45; N, 15.0.
4.2.5.4. 2-Amino-4-(4-bromothenyloxy)-6-[N-(4,40-aminoben-
zophenone)]-5-nitropyrimidine (3d).
Filtration and evapo-
ration of the solvent gave a yellow residue, which was purified
by column chromatography on silica gel (2:1 EtOAc/hexane) to af-
ford a bright yellow solid (74 mg, 68%). mp 196–198 °C. 1H NMR
(DMSO-d6) d 5.61 (s, 2H, ThBrCH2O); 6.13 (br s, 2H, C6H4NH2);
6.61 (d, 2H, J 8.5, H300); 7.34 (s, 1H, H3000); 7.54 (d, 2H, J 8.5, H200);
7.61 (d, 2H, J 8.5, H30); 7.72 (s, 1H, H5000); 7.79 (s(br), 1H, NH);
7.82 (br s, 1H, NH); 7.89 (d, 2H, J 8.5, H20); 10.6 (s, 1H, C6H4NH).
13C NMR (DMSO-d6) d 62.6 (ThBrCH2O); 108.2, 110.3 (C5–NO2),
112.7, 121.8, 124.1, 125.6, 130.0, 131.1, 132.6, 134.5, 140.0,
141.0, 153.8, 156.2, 160.4, 164.1; 192.6 (CO). IR mmax 3483, 3415
(NH2); 3367, 3321 (NH2); 1653 (CO); 1531 (NO2) cmꢀ1. HRMS
(ES) [M+H]+ calcd for C22H18BrN6O4S 541.0215; found 541.0224.
Anal. Calcd for C22H17BrN6O4Sꢂ0.2EtOAc: C, 49.0; H, 3.35; N, 15.0.
Found: C, 49.0; H, 3.25; N, 15.0.
4.2.5. General procedure for the preparation of 4-(4-
bromothenyloxy)-6-diaromatic derivatives, 3
A solution of the corresponding diamine (0.8 mmol) in THF
(6 mL) was added to a stirred solution of 9 (73 mg, 0.2 mmol)
and triethylamine (112 lL, 0.8 mmol) in THF (6 mL). The mixture
was refluxed for 2.5–48 h, depending of the starting diamine.
THF was removed, water (20 mL) was added and the product
was extracted with EtOAc (2 ꢁ 15 mL), washed with water and
dried over Na2SO4.
4.2.5.1. 2-Amino-4-(4-bromothenyloxy)-6-[N-(4-(4-aminoben-
zyl)anilino)]-5-nitropyrimidine (3a).
Filtration and evapora-
4.2.6. General procedure for the preparation of 4-benzyloxy-6-
diaromatic derivatives, 4
tion of the solvent left a yellow residue, which was purified by
column chromatography on silica gel (2:1 EtOAc/hexane) to obtain
a yellow solid (43 mg, 41%). Mp 172–174 °C. 1H NMR (DMSO-d6) d
3.73 (s, 2H, C6H4CH2C6H4); 4.93 (br s, 2H, C6H4NH2); 5.60 (s, 2H,
ThBrCH2O); 6.50 (d, 2H, J 8.5, H300); 6.89 (d, 2H, J 8.5, H200); 7.15
(d, 2H, J 8.5, H30); 7.34 (s, 1H, H3000); 7.53 (d, 2H, J 8.5, H20); 7.65
(s, 1H, NH); 7.67 (s, 1H, NH); 7.71 (s, 1H, H5000); 10.4 (s, 1H,
A solution of the corresponding diamine (1 mmol) in THF
(15 mL) or THF (10 mL)/DMF (2 mL) was added to a stirred solution
of 10 (56 mg, 0.2 mmol) and triethylamine (139 lL, 1 mmol) in
THF (5 mL) and the mixture was refluxed overnight or 48 h. THF,
along with the excess of triethylamine, were removed on the ro-
tary-evaporator and DMF was removed in high vacuum.
NHC6H4). 13C NMR (DMSO-d6)
d 40.0 (C6H4CH2C6H4); 62.44
(ThBrCH2O); 108.2, 109.8 (C5–NO2), 114.2, 123.4, 125.6, 128.5,
128.7, 129.4, 131.0, 135.7, 139.1, 140.1, 146.8 (C5–NO2, ThBr,
C6H4); 156.5, 160.4, 164.2 (C2, C4, C6). IR mmax 3450, 3409 (NH2);
3336, 3296 (NH2); 1554 (NO2) cmꢀ1. HRMS (ES) [M+H]+ calcd for
4.2.6.1.
2-Amino-4-benzyloxy-6-[N-(4-(4-aminobenzyl)anili-
The residue was treated with
no)]-5-nitropyrimidine (4a).
water and extracted with EtOAc (4 ꢁ 25 mL). The organic layer
was washed with water, brine and dried over Na2SO4. Filtration
and evaporation of the solvent gave a residue, which was purified
by column chromatography on silica gel (2:1 EtOAc/hexane) to af-
ford a yellow solid (23 mg, 26%). mp 210–212 °C. 1H NMR (DMSO-
d6) d 3.74 (s, 2H, C6H5CH2C6H5); 4.86 (br s, 2H, C6H4NH2); 5.47 (s,
2H, C6H5CH2O); 6.48 (d, 2H, J 8.2, H300); 6.87 (d, 2H, J 8.2, H200);
7.16 (d, 2H, J 8.2, H30); 7.33 (m, 1H, H4000); 7.41 (t, 2H, J 7.0, H3000);
7.50 (d, 2H, J 7.0, H2000); 7.54 (d, 2H, J 8.2, H20); 10.46 (s, 1H, NH).
13C NMR (DMSO-d6) d 40.3 (C6H5CH2C6H5); 68.6 (C6H5CH2O);
110.2 (C5–NO2); 114.4, 114.5, 123.6, 128.2, 128.4, 128.7, 128.9,
C22H20BrN6O3S 527.0423; found 527.0432. Anal. Calcd for
C
22H19BrN6O3Sꢂ0.2EtOAc: C, 50.3; H, 3.8; N, 15.4. Found: C, 50.4;
H, 3.7; N, 15.45.
4.2.5.2. 2-Amino-4-(4-bromothenyloxy)-6-[N-(4-(4-aminophe-
nylamino)anilino)]-5-nitropyrimidine (3b).
Filtration and
evaporation of the solvent gave a brown residue, which was puri-
fied by column chromatography on silica gel (2:1 EtOAc/hexane) to
afford a brown solid (59 mg, 56%). Mp 200–202 °C. 1H NMR
(DMSO-d6) d 4.79 (br s, 2H, C6H4NH2); 5.59 (s, 2H, ThBrCH2O);

S. Lopez et al. / Bioorg. Med. Chem. 19 (2011) 1658–1665
1665
129.0, 129.6, 136.0, 136.5, 139.3, 147.2, 156.8, 161.0, 165.0. IR mmax
3398, 3352 (NH2); 3158 (NH2); 1555 (NO2) cmꢀ1. HRMS (ES)
[M+H]+ calcd for C24H23N6O3 444.1753; found 444.1757. Anal.
Calcd for C24H22N6O3ꢂ0.7EtOAc: C, 63.9; H, 5.5; N, 16.7. Found: C,
63.6; H, 5.9; N, 16.9.
continued at 37 °C until the reaction was complete (1 h). For each
assay, serial dilutions of PaTrin-2 were used as a positive control.
Following the incubations, DNA was hydrolysed to acid solubility
using 1 M perchloric acid and removed and washed once by centri-
fugation. The acid-insoluble [3H]-methylated MGMT protein was
quantitated by liquid scintillation counting. IC50 values were deter-
mined from the slope of the concentration-dependence plot and
essentially represent the mean of 5 determinations.
4.2.6.2.
2-Amino-4-benzyloxy-6-[N-(4-(4-aminophenylami-
The residue was puri-
no)anilino)]-5-nitropyrimidine (4b).
fied by column chromatography on silica gel (2:1 EtOAc/hexane)
to afford a brown solid (27 mg, 30%). mp 198–200 °C. 1H NMR
(DMSO-d6) d 4.30 (br s, 2H, C6H4NH2); 5.59 (s, 2H, C6H5CH2O);
6.50 (d, 2H, J 8.4, H200); 6.75 (d, 2H, J 8.4, H30); 6.80 (d, 2H, J 8.4,
H300); 7.48 (m, 5H, H4000, H3000, H20); 7.51 (m, 5H, H2000, C6H5NHC6H5,
NH2); 10.49 (s, 1H, NH). 13C NMR (DMSO-d6) d 68.5 (C6H5CH2O);
110.0 (C5–NO2); 114.0, 115.2, 122.7, 124.9, 128.2, 128.4, 128.9,
132.1, 136.6, 144.3, 144.5, 156.6, 160.9, 165.1. IR mmax 3451
(NH2); 3312, 3215 (NH2); 1555 (NO2) cmꢀ1. HRMS (ES) [M+H]+
calcd for C23H22N7O3 445.1706; found 445.1710. Anal. Calcd for
C23H21N7O3ꢂ1.1EtOAc: C, 60.9; H, 5.6; N, 18.1. Found: C, 61.4; H,
6.1; N, 18.5.
Acknowledgements
The authors are grateful to the Irish Cancer Society, Cancer
Research UK and CHEMORES for financial support. The authors
are indebted to Dr. John O’Brien for the NMR studies.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.01.038.
References and notes
4.2.6.3. 2-Amino-4-benzyloxy-6-[N-(4-(4-aminophenoxy)anili-
1. Pegg, A. E. Mutat. Res. 1990, 233, 165.
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no)]-5-nitropyrimidine (4c).
The residue was purified by col-
umn chromatography on silica gel (2:1 EtOAc/hexane) to afford a
bright yellow solid (76 mg, 85%). Mp 198–200 °C. 1H NMR (DMSO-
d6) d 4.91 (br s, 2H, C6H4NH2); 5.37 (s, 2H, C6H5CH2O); 6.51 (d, 2H,
J 8.5, H200); 6.79 (d, 2H, J 8.5, H300); 6.74 (d, 2H, J 8.5, H20); 6.74 (d,
2H, J 8.5, H30); 7.26 (t, 1H, J 7.5, H4000); 7.32 (t, 2H, J 7.5, H3000); 7.38–
7.44 (m, 2H, H2000); 7.49 (m, 4H, H20, NH2); 10.3 (s, 1H, NH). 13C
NMR (DMSO-d6) d 68.3 (C6H5CH2O); 109.9 (C5–NO2); 115.0, 116.6,
121.1, 125.0, 127.9, 128.1, 128.6, 132.2, 136.3, 145.7, 145.8, 156.0,
156.5, 160.7, 164.7. IR mmax 3450, 3409 (NH2); 3326, 3218 (NH2);
7. Adams, C. A.; Melikishvili, M.; Rodgers, D. W.; Rasimas, J. J.; Pegg, A. E.; Fried, M.
G. J. Mol. Biol. 2009, 389, 248.
8. Chemotherapeutic Agents, 6th ed. In Burger’s Medicinal Chemistry and Drug
Discovery; Abraham, D. J., Ed.; John Wiley and Sons, Inc., 2003; Vol. 5,.
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1113.
1555 (NO2) cmꢀ1
.
HRMS (ES) [M+H]+ calcd for C23H21N6O4
11. (a) Friedman, H. S.; Pluda, J.; Quinn, J. A.; Ewesuedo, R. B.; Long, L.; Friedman, A.
H.; Cokgor, I.; Colvin, O. M.; Haglund, M. M.; Ashley, D. M.; Rich, J. N.; Sampson,
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Colvin, O. M.; Haglund, M. M.; Pegg, A. E.; Moschel, R. C.; McLendon, R. E.;
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D.; Friedman, H. S. J. Clin. Oncol. 2002, 20, 2277; (c) Ranson, M.; Middleton, M.
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445.1624; found 445.1635. Anal. Calcd for C23H20N6O4ꢂ0.4EtOAc:
C, 61.6; H, 4.9; N, 17.5. Found: C, 61.5; H, 4.7; N, 17.8.
4.2.6.4.
none)]-5-nitropyrimidine (4d).
2-Amino-4-benzyloxy-6-[N-(4,40-diaminobenzophe-
The residue was treated with
water and extracted with EtOAc (4 ꢁ 25 mL). The organic layer was
washed with water, brine and dried over Na2SO4. Filtration and
evaporation of the solvent gave a yellow residue, which was purified
by column chromatography on silica gel (2:1 EtOAc/hexane) to af-
ford a bright yellow solid (78 mg, 82%). mp 222–224 °C. 1H NMR
(DMSO-d6) d 5.48 (s, 2H, C6H5CH2O); 6.15 (br s, 2H, C6H4NH2); 6.61
(d, 2H, J 8.5, H300); 7.36 (t, 1H, J 7.5, H4000); 7.42 (t, 2H, J 7.5, H3000);
7.49–7.58 (m, 4H, H200, H2000); 7.62 (d, 2H, J 8.5, H30); 7.76 (s(br),
1H, NH); 7.78 (br s, 1H, NH); 7.91 (d, 2H, J 8.5, H20); 10.7 (s, 1H,
C6H4NH). 13C NMR (DMSO-d6) d 68.4 (C6H5CH2O); 110.5 (C5–NO2),
112.7, 121.7, 124.1, 127.9, 128.2, 128.6, 130.0, 132.6, 134.4, 136.2,
141.1, 153.8, 156.2, 160.7, 164.7; 192.6 (CO). IR mmax 3484, 3414
(NH2); 3376, 3316 (NH2); 1632 (CO); 1538 (NO2) cmꢀ1. HRMS (ES)
[M+Na]+ calcd for C24H20N6O4Na 479.1444; found 479.1436. Anal.
Calcd for C24H20N6O4ꢂ0.9EtOAc: C, 61.9; H, 5.1; N, 15.7. Found: C,
61.9; H, 5.05; N, 15.9.
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Determination of the IC50 values for inactivation of MGMT by
the study compounds was by a method previously described.28 In
brief, the compounds were dissolved at 10 mM in dry DMSO and
serial dilutions prepared in DMSO. In each assay, fixed amounts
of MGMT (50–60 fmol) were incubated with aliquots of the dilu-
tion series of test compound in a total volume of 200
containing 10 g of calf thymus DNA at 37 °C for 1 h. Excess
[3H]-methylated DNA substrate (100
L containing 4 g of DNA
and 100 fmol of O6-[3H]methylguanine) was added and incubation
lL of buffer
l
l
l
28. Watson, A. J.; Margison, G. P. Methods Mol. Biol. 2000, 152, 49.