2-Amino-O4-benzylpteridine derivatives: Potent inactivators of O6-alkylguanine-DNA alkyltransferase

Article abstract of DOI:10.1021/jm049758+

2-Amino-O⁴-benzylpteridine (1), 2-amino-O⁴-benzyl-6, 7-dimethylpteridine (2), 2-amino-O⁴-benzyl-6-hydroxymethylpteridine (4), 2-amino-O⁴-benzylpteridine-6-carboxylic acid (5), 2-amino-O ⁴-benzyl-6-for

Full text of DOI:10.1021/jm049758+

J . Med. Chem. 2004, 47, 3887-3891
3887
2-Am in o-O4-ben zylp ter id in e Der iva tives: P oten t In a ctiva tor s of
O6-Alk ylgu a n in e-DNA Alk yltr a n sfer a se
Michael E. Nelson,† Natalia A. Loktionova,‡ Anthony E. Pegg,‡ and Robert C. Moschel*,†
Laboratory of Comparative Carcinogenesis, National Cancer Institute at Frederick, P.O. Box B, Building 538,
Frederick, Maryland 21702, and Departments of Cellular and Molecular Physiology and Pharmacology,
The Pennsylvania State University College of Medicine, P.O. Box 850, Hershey, Pennsylvania 17033
Received March 29, 2004
2-Amino-O4-benzylpteridine (1), 2-amino-O4-benzyl-6,7-dimethylpteridine (2), 2-amino-O4-
benzyl-6-hydroxymethylpteridine (4), 2-amino-O4-benzylpteridine-6-carboxylic acid (5), 2-amino-
O4-benzyl-6-formylpteridine (6), and O4-benzylfolic acid (7) are shown to be as potent or more
potent inactivators of the human DNA repair protein O6-alkylguanine-DNA alkyltransferase
(alkyltransferase) in vitro than O6-benzylguanine, the prototype alkyltransferase inactivator
currently in clinical trials. Additionally, the negatively charged (at physiological pH) inactivators
2-amino-O4-benzylpteridine-6-carboxylic acid (5) and O4-benzylfolate (7) are far more water
soluble than O6-benzylguanine. The activity of O4-benzylfolic acid (7) is particularly noteworthy
because it is roughly 30 times more active than O6-benzylguanine against the wild-type
alkyltransferase and is even capable of inactivating the P140K mutant alkyltransferase that
is resistant to inactivation by O6-benzylguanine. All the pteridine derivatives except 2-amino-
O4-benzylpteridine-6-carboxylic acid are effective in enhancing cell killing by 1,3-bis(2-
chloroethyl)-1-nitrosourea (BCNU). However, the effectiveness of O4-benzylfolate as an adjuvant
for cell killing by BCNU appears to be a function of a cell’s R-folate receptor expression. Thus,
O4-benzylfolate is least effective as an adjuvant in A549 cells (which express little if any
receptor), is moderately effective in HT29 cells (which express low levels of the receptor), but
is very effective in KB cells (which are known to express high levels of the R-folate receptor).
Therefore, O4-benzylfolic acid shows promise as an agent for possible tumor-selective alkyl-
transferase inactivation, which suggests it may prove to be superior to O6-benzylguanine as a
chemotherapy adjuvant.
In tr od u ction
formulated, more water-soluble, or more tumor-specific
than O6-benzylguanine.10 Our ongoing studies have led
to the discovery of a new class of potent alkyltransferase
inactivators, i.e., 2-amino-O4-benzylpteridine derivatives
(1, 2, and 4-7) that readily sensitize cells to killing by
BCNU. Furthermore, some members of this class offer
significant advantages over O6-benzylguanine with
respect to water solubility and tumor cell selectivity. In
particular, O4-benzylfolic acid (7), a very water-soluble
inactivator, shows promise for selectively inactivating
alkyltransferase in tumor cells that overexpress folic
acid receptors. Therefore, 2-amino-O4-benzylpteridine
derivatives represent a promising new class of alkyl-
transferase inactivator with representatives that may
be superior to O6-benzylguanine as a chemotherapy
adjuvant.
O6-Benzylguanine derivatives,1,2 some O6-benzylpy-
rimidines,3 and related compounds4,5 are known to be
inactivators of the human DNA repair protein O6-
alkylguanine-DNA alkyltransferase (alkyltransferase).6
This repair protein is the primary source of resistance
many tumor cells exhibit to chemotherapeutic agents
that modify the O6-position of DNA guanine residues.6
Therefore, inactivation of this protein can bring about
a significant improvement in the therapeutic effective-
ness of these chemotherapy drugs. The prototype inac-
tivator O6-benzylguanine is currently in clinical trials
in the U.S. as an adjuvant in combination with the
chloroethylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea
(BCNU) and the methylating agent temozolomide.7,8
A
similar alkyltransferase inactivator, O6-(4-bromo-
thenyl)guanine is in clinical trials in the UK.9
Resu lts a n d Discu ssion
Although O6-benzylguanine is a promising chemo-
therapy adjuvant, it is not an ideal drug because its
inactivating potency is modest, it exhibits only limited
water solubility. and it is not a specific inactivator for
alkyltransferase in tumor cells vs normal cells of a host.
Therefore, we have sought to prepare additional alkyl-
transferase inactivators that are more potent, better
The 2-amino-O4-benzylpteridine derivatives studied
are 2-amino-O4-benzylpteridine (1), 2-amino-O4-benzyl-
6,7-dimethylpteridine (2), 2-amino-O4-benzyl-6-hydroxy-
methylpteridine (4), 2-amino-O4-benzylpteridine-6-car-
boxylic acid (5), 2-amino-O4-benzyl-6-formylpteridine
(6), and O4-benzylfolic acid (7). Pteridines 1 and 2 were
prepared by the method of Pfleiderer and Lohrmann
from 2,4,5-triamino-O6-benzylpyrimidine (3) (structure
shown in Scheme 1) and glyoxal or diacetyl, respec-
tively.11 Pteridines 4-7 were prepared as illustrated in
Scheme 1. Thus, treatment of the triaminopyrimidine
* To whom correspondence should be addressed. Phone: 301-846-
5852. Fax: 301-846-6146. E-mail: moschel@mail.ncifcrf.gov.
† National Cancer Institute at Frederick.
‡ The Pennsylvania State University College of Medicine.
10.1021/jm049758+ CCC: $27.50 © 2004 American Chemical Society
Published on Web 06/18/2004

3888 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 15
Nelson et al.
access of these 2-amino-O4-benzylpteridines to the
protein’s active site. This contrasts with the situation
for O6-benzylguanine (Table 1), which exhibits an
enhanced ED50 in the presence of calf thymus DNA.12
However, previous studies with 9-substituted O6-ben-
zylguanine derivatives, which are also sterically hin-
dered from entering the alkyltransferase active site by
DNA, show that these are effective inactivators of
alkyltransferase in vivo, suggesting that the amount of
cellular alkyltransferase bound to DNA must be small
or readily exchangeable with the free form.12
It is not totally clear yet why compounds 1, 5, and 7
are so significantly better than O6-benzylguanine against
the alkyltransferase in the absence of calf thymus DNA.
For these compounds it may be that the respective
pteridine derivative “leaving groups” are easier to
displace by the active site cysteine residue of the
alkyltransferase than is guanine from O6-benzylgua-
nine. Compound 1 is known to be sensitive to acid
hydrolysis,11 indicating that reaction of the benzyl group
with water to displace the protonated pteridine leaving
group is also facile. Interestingly, 1 is more acid-labile
than 2,11 which may reflect why 2 exhibits a higher ED50
than 1 (Table 1). Clearly, there is not enough data
available at present to conclude that ease of leaving
group displacement as reflected by relative rates of acid
hydrolysis is responsible for the very low ED50 values
exhibited by 1, 5, and 7. To draw such conclusions,
extensive comparisons of the rates of acid hydrolysis of
1, 2, and 4-7 with hydrolysis rates for O6-benzylgua-
nine under identical conditions will be required. With
respect to O4-benzylfolic acid (7), a combination of ease
of displacement and favorable steric interactions with
the protein probably contributes to its remarkable
effectiveness as an alkyltransferase inactivator. It is
possible that the glutamyl residue of 7 (particularly the
carboxyl groups) provides an additional binding site on
the alkyltransferase protein surface. This would be
consistent with the inactivation of mutant P140K
because the resistance of this mutant to O6-benzylgua-
nine results from its prevention of the binding of the
benzyl group to the protein via stacking with Pro140.
At present we do not know the alkyltransferase site(s)
that interact with the folate but have ruled out Arg128
because mutant R128L is inactivated with an ED50 of
0.02, very similar to the behavior of the wild type.
(3) with dihydroxyacetone in dimethylacetamide (DMA)/
H2O in the presence of sodium ascorbate and air
afforded the hydroxymethylpteridine (4), which was
oxidized to the 6-carboxyl derivative (5) with perman-
ganate in acetone water solution. Alternatively, 4 was
oxidized to the 6-formylpteridine derivative 6 by treat-
ment with iodoxybenzoic acid (IBX) in dimethyl sulfox-
ide. Treatment of 6 with p-aminobenzoylglutamate
(pAB-glu) followed by reduction with sodium cyanoboro-
hydride in dimethylformamide led to formation of O4-
benzylfolic acid (7).
The ability of these various compounds to inactivate
the human alkyltransferase protein in the presence and
absence of calf thymus DNA is summarized Table 1.
Data for O6-benzylguanine are included for comparison.
The data are expressed as concentration of inactivator
required to reduce the activity of the alkyltransferase
protein by 50% (i.e., ED50). As indicated, in the absence
of calf thymus DNA, pteridine derivatives 2, 4, and 6
exhibit activity similar to that of O6-benzylguanine
whereas derivatives 1, 5, and 7 are superior to O6-
benzylguanine as alkyltransferase inactivators. In par-
ticular, O4-benzylfolic acid (7) is roughly 30 times more
effective than O6-benzylguanine against the wild-type
human alkyltransferase and displays an ED50 in the
nanomolar range. Interestingly, this same compound is
also effective against the P140K mutant alkyltrans-
ferase (although at significantly higher concentrations).
This protein is essentially resistant to inactivation by
O6-benzylguanine and related derivatives.10 Previously,
only oligodeoxyribonucleotides containing O6-benzylgua-
nine residues were known to inactivate the P140K and
other mutant alkyltransferase proteins.10
We have confirmed that benzylation of the active site
cysteine is indeed involved in the mechanism of inac-
tivation of the alkyltransferase by 7 as it is with O6-
benzylguanine. To demonstrate this, peptide fragments
In the presence of calf thymus DNA, the ED50 values
for alkyltransferase inactivation increase significantly,
suggesting that DNA binding of the protein hinders
Sch em e 1

2-Amino-O4-benzylpteridine Derivatives
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 15 3889
Ta ble 1. Inactivation of Human O6-Alkylguanine-DNA Alkyltransferase in Vitro in the Absence and Presence of Calf Thymus (ct)
DNA
- ctDNAa
ED50 (µM)
+ctDNAa
ED50 (µM)
inactivator
n
n
O6-benzylguanine
0.32 ( 0.08
0.045 ( 0.01
0.4
0.2
0.09
0.19
0.01 ( 0.001
4
4
1
1
2
2
3
0.12 ( 0.02
0.45 ( 0.05
0.5
0.4
1.83 ( 0.62
1.05
3
6
1
1
3
2
3
2-amino-O4-benzylpteridine (1)
2-amino-O4-benzyl-6,7-dimethylpteridine (2)
2-amino-O4-benzyl-6-hydroxymethylpteridine (4)
2-amino-O4-benzyl-6-carboxypteridine (5)
2-amino-O4-benzyl-6-formylpteridine (6)
O4-benzylfolic acid (7)b
0.47 ( 0.05
a
The ED50 value was calculated from a graph of residual alkyltransferase activity over a range of 8-10 concentrations, and the
experiment was repeated 1-4 times as indicated by n. Results are shown as the mean for two separate estimations and the mean ( SD
b
for three more separate estimations. ED50 against the P140K mutant alkyltransferase in the absence of ctDNA is 12 µM. In the presence
of ctDNA, the compound is inactive at concentrations of e1 mM.
Ta ble 2. Concentrations of 2-Amino-O4-benzylpteridine
Derivatives Required To Kill 90% of HT29 Cells (ED90) with
BCNU (40 µM)a
inactivator
ED90 (µM)
O6-benzylguanine
0.4,b 0.7c
2-amino-O4-benzylpteridine (1)
0.2b
0.6b
2-amino-O4-benzyl-6,7-dimethylpteridine (2)
2-amino-O4-benzyl-6-hydroxymethylpteridine (4) 0.7b
2-amino-O4-benzyl-6-carboxypteridine (5)
2-amino-O4-benzyl-6-formylpteridine (6)
O4-benzylfolic acid (7)
inactive at 30 µMb
0.7c
15,d 24c
a
Cells were completely resistant to 40 µM BCNU treatment in
b
the absence of alkyltransferase inactivator. Studies carried out
in Dulbecco’s medium (which contains 9.1 µM folic acid). The
alkyltransferase inactivator was present before, during, and after
treatment with BCNU for 16-18 h before replating (see Experi-
mental Section). c Studies carried out in RPMI medium (which
contains 2.3 µM folic acid). The alkyltransferase inactivator was
F igu r e 1. Cell killing by O4-benzylfolic acid and BCNU. The
plating efficiency was 606 ( 33 colonies per 1000 cells for
HT29, 700 ( 98 colonies per 1000 cells for KB, and 689 ( 8
colonies per 1000 cells for A549 cells and was not affected by
the doses of BCNU used.
d
present before and during BCNU treatment only. Studies carried
out in folate-free RPMI medium. The alkyltransferase inactivator
was present before and during BCNU treatment only.
obtained by digestion of the alkyltransferase protein
with trypsin were separated and analyzed by MALDI-
TOF mass spectroscopy on an Applied Biosystems 4700
Proteomics analyzer, using R-cyanohydroxycinnamic
acid as the sample matrix. A peak of m/z 1315.75,
corresponding to the theoretical mass of the unmodified
tryptic fragment GNPVPILIPCHR, was observed in the
spectrum from the unmodified protein. This peak was
not observed in the tryptic digest of either the O6-
benzylguanine or O4-benzylfolic acid treated protein, but
a new peak at m/z 1405.78 or 1405.75, respectively,
appeared instead, corresponding to the theoretical mass
of GNPVPILIP(benzyl-C)HR. This 1405.75 peak was
then subjected to collision-induced fragmentation in a
tandem MS/MS analysis on the same instrument, and
the dominant peaks observed in the resulting spectra
corresponded to the theoretical immonium ions, y-ions,
and internal fragment ions that would be expected from
fragmentation of the modified peptide containing S-
benzylcysteine.
some active process but that uptake is limited. This
would be consistent with uptake occurring via the
R-form of the folic acid receptor, and 7 was more
effective when tested in a folate-free medium (Table 2).
Further support for this concept is shown in Figure 1,
which shows A549 lung tumor, HT29 colon tumor, and
KB nasopharyngeal tumor cell killing by 40 µM BCNU
following a 2 h exposure to 7 in folate-free growth
medium. KB cell killing by 80 µM BCNU in combination
with 7 is also illustrated. The A549 and HT29 cells are
completely resistant to killing by 40 µM BCNU in the
absence of modulator because of their alkyltransferase
activity. The KB cells, which have a higher alkyltrans-
ferase level, are completely resistant to even 80 µM
BCNU. As shown, the effectiveness of 7 as an adjuvant
was lowest in A549 cells, was somewhat greater in HT29
cells, and was greatest in KB cells. A549 cells express
little, if any, R-folate receptor, HT29 cells express low
levels of the receptor, and KB cells express high levels
of the receptor.14-17 Thus, O4-benzylfolic acid may be a
useful agent for selectively inactivating alkyltransferase
in tumors that overexpress the R-folate receptor. These
tumors are numerous and include adenocarcinomas,
ovarian, endometrial, and bronchioloalveolar carcino-
mas, some non-small-cell lung carcinomas, small-cell
lung carcinomas, squamous cell carcinomas, colorectal
carcinomas, gastric carcinomas, and kidney tumors.17
Such tumor selectivity would be very advantageous
because the side effects associated with systemic alkyl-
Not surprisingly, the 2-amino-O4-benzylpteridine de-
rivatives 1, 2, 4, 6, and 7 are all capable of enhancing
HT29 cell killing by BCNU (Table 2), which is a common
property of alkyltransferase inactivators.1-6,13 2-Amino-
O4-benzylpteridine-6-carboxylic acid (5) is not effective
probably because the negative charge on the molecule
at physiological pH prevents its easy entry into cells.
Even though O4-benzylfolic acid is also anionic at
physiological pH, it does enhance cell killing by BCNU
but it was much less effective than compounds 1, 2, 4,
and 6, suggesting that its uptake is brought about by

3890 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 15
Nelson et al.
transferase inactivation7,8 could be significantly re-
duced.
158.1, 151.3, 137.4, 135.5, 129.0, 128.5, 128.45, 122.5, 68.75.
Anal. (C14H11N5O3‚H2O) C, H, N.
2-Am in o-O4-ben zyl-6-for m ylp ter id in e (6). Iodoxybenzoic
acid (IBX) (1.7 g, 6.1 mmol) was stirred in DMSO (16 mL) until
it dissolved. 2-Amino-O4-benzyl-6-hydroxymethylpteridine (4)
(1.16 g, 4.1 mmol) was added with constant stirring at room
temperature to produce a dark-orange solution. The reaction
was complete in 2 h as monitored by TLC (CH2Cl2/MeOH, 10:
1). The reaction mixture was poured into H2O (150 mL) to
produce a pale-yellow precipitate, which was collected by
filtration. This solid was stirred at 40 °C in CH2Cl2/acetone
(1:1, 250 mL) for approximately 30 min and was filtered to
remove the iodosobenzoic acid byproduct. This process was
repeated twice. The dissolved product was evaporated onto
silica (50 mL) and was eluted from a silica gel column with
CH2Cl2/CH3CN (7:3). Solvent was evaporated to give 2-amino-
O4-benzyl-6-formylpteridine (6) (0.26 g, 0.92 mmol, 22.4%). UV
(MeOH/0.05 M phosphate, pH 6.8, 5:95) λmax 236 nm (ꢀ )
13 600), 261 (sh) (ꢀ ) 9600) 309 nm (ꢀ ) 5200), 370 (ꢀ ) 9300);
1H NMR δ 9.96 (1H, s, 6-CHO), 9.19 (1H, s, H-7), 8.03 (1H, s,
N2Ha, exchanges with D2O), 7.89 (1H, s, N2Hb, exchanges with
D2O), 7.60 (2H, m, ArH), 7.42 (3H, m, ArH), 5.62 (2H, s,
ArCH2); 13C NMR (100 MHz) δ 191.2, 166.9, 163.1, 159.0,
149.2, 141.0, 135.4, 129.1, 128.51, 128.49, 122.8, 69.0. Anal.
(C14H11N5O2) C, H, N.
O4-Ben zylfolic Acid (7). 2-Amino-O4-benzyl-6-formylpte-
ridine (6) (0.26 g, 0.92 mmol) and p-aminobenzoylglutamate
(pAB-glu) (0.29 g, 1.1 mmol) were stirred in DMF (4.4 mL)
until they were completely dissolved. NaBH3CN (0.08 g, 1.3
mmol) was added with stirring. After approximately 5 min,
the reaction color changed from yellow-orange to red. TLC
(CH2Cl2/MeOH/AcOH, 90:5:5) showed complete loss of 6. The
reaction mixture was poured into vigorously stirred water (50
mL), producing a yellow precipitate that dissolved when the
pH of the suspension was adjusted to 7.2 by the addition of 2
M NaOH. Activated charcoal (20 mg) was added and was then
filtered out. The solution pH was then adjusted to 3.0 by the
addition of 2 M HCl, producing a yellow precipitate that was
collected by filtration. The solid was dissolved in CH2Cl2/MeOH
(3:1) and evaporated onto silica (30 mL). The product was
eluted from a silica gel column with CH2Cl2/MeOH/AcOH (90:
5:5). The solvent was evaporated and the product was sus-
pended in vigorously stirred H2O to produce a fine solid (7),
which was filtered and dried under vacuum (87 mg, 0.16 mmol,
17.7%). UV (0.05 M phosphate, pH 6.8) λmax 277 nm (ꢀ )
19 000), 289 nm (sh) (ꢀ ) 18 100), 368 nm (ꢀ ) 8200); 1H NMR
δ 12.31 (2H, br s, 2CO2H), 8.79 (1H, s, 7-H), 8.12 (1H, d, J )
7.7 Hz, glu-NH, exchanges with D2O), 7.64 (2H, d, J ) 8.7
Hz, pABArH), 7.57 (2H, m, BnArH), 7.41 (3H, m, BnArH), 7.29
(2H, br-s, N2H2, exchange with D2O), 6.95 (1H, t, J ) 6.1 Hz,
6-CH2NH, exchanges with D2O), 6.64 (2H, d, J ) 8.8 Hz,
pABArH), 5.58 (2H, s, BnCH2), 4.50 (2H, d, J ) 5.7 Hz, 6-CH2-
NH, singlet in D2O), 4.33 (1H, m, gluR-CH, dd in D2O), 2.32
(2H, t, J ) 7.5 Hz gluγ-CH2), 2.04 (1H, m, gluâ-CH2a), 1.90
(1H, m, gluâ-CH2b); 13C NMR (100 MHz) δ 173.9, 173.7, 166.4,
166.3, 161.3, 156.5, 150.8, 150.3, 149.1, 135.9, 129.0, 128.8,
128.5, 128.3, 121.6, 121.3, 111.2, 68.4, 51.7, 46.1, 30.4, 24.0.
Anal. (C26H25N7O6‚0.5H2O) C, H, N. Alternatively, the crude
product before silica gel chromatography (see above) was
dissolved in H2O, the pH of which was adjusted to 7.0 by the
addition of 2 M NaOH, and the solution was evaporated to
give the sodium salt. This product was purified on a Sephadex
LH-20 column (3 cm × 80 cm) and was eluted with aqueous
0.1 M sodium acetate solution at 1 mL/min. UV absorption
was monitored continuously at 280 nm. Fractions (10 mL) 54-
90 containing the product were combined, and the pH was
adjusted to 2.5 with HCl to precipitate the product, which was
collected by filtration and dried under vacuum.
Exp er im en ta l Section
Ma ter ia ls a n d Meth od s. Unless otherwise stated, chemi-
cals were obtained from Aldrich (Milwaukee, WI) or Sigma (St.
Louis, MO) and were used without further purification. UV
spectra were determined on a Beckman Coulter DU 7400
spectrophotometer. 1H and 13C NMR spectra were recorded in
DMSO-d6 with a Varian INOVA 400 MHz spectrometer.
Chemical shifts are reported as δ values in parts per million
relative to TMS as internal standard. The splitting pattern
abbreviations are as follows: s ) singlet, d) doublet, dd )
double doublet, t ) triplet, m ) multiplet. Elemental analyses,
performed by Atlantic Microlab, Inc. (Norcross, GA) were
within 0.4% of the theoretical values calculated for C, H, and
N. Thin-layer chromatographic analyses were performed using
precoated, aluminum-backed silica gel plates, and the spots
were visualized with UV light. All silica gel column chroma-
tography was carried out using Davisil grade 633, 200-425
mesh, 60 Å. Compounds 1-3 were prepared by the method of
Pfleiderer and Lohrmann.11
2-Am in o-O4-ben zyl-6-h ydr oxym eth ylpter idin e (4). 2,4,5-
Triamino-O6-benzylpyrimidine (3) (3.26 g, 14.1 mmol) was
dissolved in DMA/H2O (1:1, 28 mL) and stirred at room
temperature. Sodium ascorbate (2.85 g, 14.4 mmol) was added
followed by dihydroxyacetone dimer (2.57 g, 14.3 mmol). The
reaction mixture was heated to 40 °C, and air was bubbled
into the reaction flask through a Pasteur pipet. The reaction
was monitored by TLC (CH2Cl2/MeOH, 10:1). After 4 h all the
starting material was consumed, and the reaction mixture was
poured into 250 mL of H2O to produce a yellow-orange solid.
This solid was collected by filtration, was dissolved in CH2-
Cl2/MeOH (3:1, 500 mL), and was dried over MgSO4. The
solution was filtered and evaporated onto silica gel (100 mL).
Product was eluted from a silica gel column with CH2Cl2/
MeOH (20:1) and fractions containing product were pooled and
evaporated to produce 2-amino-O4-benzyl-6-hydroxymethylp-
teridine (4) (1.12 g, 28.1%). UV (MeOH/0.05 M phosphate, pH
6.8, 5:95) λmax 234 nm (ꢀ ) 18 800), 264 nm (ꢀ ) 9200), 366 (ꢀ
) 6900); 1H NMR δ 8.88 (1H, s, H-7), 7.56 (2H, m, ArH), 7.40
(3H, m, ArH), 7.28 (2H, s, N2H2, exchange with D2O), 5.58 (1H,
t, J ) 5.9 Hz, 6-CH2OH, exchanges with D2O), 5.55 (2H, s,
ArCH2), 4.62 (2H, d, J ) 5.9 Hz, 6-CH2OH changes to a singlet
in D2O); 13C NMR (100 MHz) δ 166.4, 161.2, 156.5, 151.1,
150.0, 135.8, 128.9, 128.5, 128.3, 121.2, 68.4, 62.7. Anal.
(C14H13N5O2‚0.5H2O) C, H, N.
2-Am in o-O4-b en zylp t er id in e-6-ca r b oxylic Acid (5).
2-Amino-O4-benzyl-6-hydroxymethylpteridine (4) (0.24 g, 0.84
mmol) was suspended in acetone/0.5 M phosphate buffer, pH
7 (1:1, 14 mL), and stirred at room temperature. Potassium
permanganate (0.34 g, 2.18 mmol) was added in four portions
at 30 min intervals. The resulting suspension was then stirred
at room temperature for an additional 3 h. The reaction
mixture was diluted with H2O (50 mL). Sodium sulfite was
added until all of the permanganate was consumed, producing
a brown-black precipitate, which was removed by filtration,
leaving a clear, yellow solution. The pH was adjusted to 2.5
by the addition of 2 M HCl, producing a yellow solid, which
was collected by filtration. The solid was dissolved in H2O (50
mL) by adjusting the pH to 7.0 through the addition of 0.1 M
NaOH until the pH remained constant for 30 min. Any
suspended solid material was filtered and the solution was
evaporated to give the sodium salt. This product was purified
on a 3 cm × 80 cm Sephadex LH-20 column and was eluted
with H2O (1 mL/min). UV absorption was monitored continu-
ously at 280 nm. Fractions (10 mL) 34-44 containing the
product were combined, and the pH was adjusted to 2.5 with
HCl to precipitate the product, which was collected by filtration
In Vitr o Alk yltr a n sfer a se Activity Assa y. Purified re-
combinant human alkyltransferase was incubated with dif-
ferent concentrations of inactivator in 0.5 mL of reaction buffer
(50 mM Tris-HCL, pH 7.6, 0.1 mM EDTA, 5.0 mM dithiothrei-
tol) containing 50 µg of hemocyanin for 30 min at 37 °C. The
remaining alkyltransferase activity was determined after
1
and dried under vacuum to afford 5 (0.17 g, 67%). H NMR δ
13.52 (1H, s, CO2H, exchanges with D2O), 9.25 (1H, s, H-7),
7.83 (1H, N2Ha, exchanges with D2O), 7.70 (1H, N2Hb, ex-
changes with D2O), 7.58 (2H, m, ArH), 7.41 (3H, m, ArH), 5.60
(2H, s, ArCH2); 13C NMR (100 MHz) δ 166.9, 164.8, 162.6,

2-Amino-O4-benzylpteridine Derivatives
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 15 3891
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.18 The
results were expressed as the percentage of the alkyltrans-
ferase activity remaining. The concentration of inhibitor that
led to a 50% loss of alkyltransferase activity (ED50) was
calculated from graphs of the percentage of remaining alkyl-
transferase activity against inactivator concentration. For
assays in the presence of DNA, 10 µg of calf thymus DNA was
added before incubation with the inactivators.
(5) 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. T. Resistance-modifying
agents. 8. Inhibition of O6-alkylguanine-DNA alkyltransferase
by O6-alkenyl-, O6-chcloalkenyl-, and O6-(2-oxoalkyl)guanines
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inhibition of O6-alkylguanine-DNA alkyltransferase. Prog. Nu-
cleic Acid Res. Mol. Biol. 1995, 51, 167-223.
Cell Cu ltu r e a n d Cytotoxicity Assa ys. Cells were grown
either in Dulbecco’s modified Eagle’s medium supplemented
with 10% fetal bovine serum plus 1.5 mM glutamine and 50
µg/mL gentamycin (HT29) or in RPMI 1640 medium in the
presence of 10% fetal bovine serum (HT29, A549, and KB
cells). The effect of alkyltransferase inactivators on the
sensitivity of cells to BCNU was determined using a colony-
forming assay.13 Cells were plated at a density of 106 in 25
cm2 flasks and 24 h later were incubated with different
concentrations of alkyltransferase inactivator for 2 h before
exposure to 40 µM (HT29 cells and A549 cells) or 80 µM (KB
cells) of BCNU for 2 h. The BCNU was first dissolved in
absolute ethanol at a concentration of 8 mM, was diluted with
the same volume of ice-cold phosphate-buffered saline, and was
immediately used to treat the cells. The medium was replaced
with fresh medium containing alkyltransferase inactivator
(unless otherwise indicated), and the cells were left to grow
for an additional 16-18 h. The alkyltransferase inactivator
was added to the medium after the treatment with BCNU to
ensure that the inactivator was present during the entire
period that DNA adducts are formed by BCNU. The cells were
then replated at densities of 200-2000 cells per 25 cm2 flasks
and grown for 8 days until discrete colonies were formed. The
colonies were washed with 0.9% saline solution, were stained
with 0.5% crystal violet in ethanol, and counted. The plating
efficiency of cells not treated with drugs was about 50% for
HT29 and A549 cells and 80% for KB cells. In experiments to
assess the effect of folate present in the medium on the
sensitivity of cells to the alkyltransferase inactivators and
BCNU, the cells were incubated with inactivators for 2 h and
with BCNU for 2 h in RPMI 1640 folate-free medium. After
this period, the medium was replaced with fresh RPMI 1640
medium, which contains 2.3 µM folic acid.
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Cokgor, I.; Haglund, M. M.; Ashley, D. M.; Rich, J .; Dolan, M.
E.; Pegg, A. E.; Moschel, R. C.; McLendon, R. E.; Tracy, K.;
Herndon, J . E.; Bigner, D. D.; Schold, S. C. Phase I trial of O6-
benzylguanine for patients undergoing surgery for malignant
glioma. J . Clin. Oncol. 1998, 16, 3570-3575.
(8) Quinn, J . A.; Pluda, J .; Dolan, M. E.; Delaney, S.; Kaplan, R.;
Rich, J . N.; Friedman, A. H.; Reardon, D. A.; Sampson, J . H.;
Colvin, O. M.; Haglund, M. M.; Pegg, A. E.; Moschel, R. C.;
McLendon, R. E.; Provenzale, J . M.; Gururangan, S.; Tourt-
Uhlig, S.; Herndon, J . E.; Bigner, D. D.; Friedman, H. S. Phase
II trial of carmustine plus O6-benzylguanine for patients with
nitrosourea-resistant recurrent or progressive malignant glioma.
J . Clin. Oncol. 2002, 20, 2277-2283.
(9) Middleton, M. R.; Margison, G. P. Improvement of chemotherapy
efficacy by inactivation of a DNA-repair pathway. Lancet Oncol.
2003, 4, 37-44.
(10) Luu, K. X.; Kanugula, S.; Pegg, A. E.; Pauly, G. T.; Moschel, R.
C. Repair of oligodeoxyribonucleotides by O6-alkylguanine-DNA
alkyltransferase. Biochemistry 2002, 41, 8689-8697.
(11) Pfleiderer, W.; Lohrmann, R. Synthese von 2-amino-4-alkoxy-
pteridinen (Synthesis of 2-amino-4-alkoxypteridines). Chem. Ber.
1961, 94, 12-18.
(12) Pegg, A. E.; Chung, L.; Moschel, R. C. Effect of DNA on the
inactivation of O6-alkylguanine-DNA alkyltransferase by 9-sub-
stituted O6-benzylguanine derivatives. Biochem. Pharmacol.
1997, 53, 1559-1564.
(13) Dolan, M. E.; Moschel, R. C.; Pegg, A. E. Depletion of mammalian
O6-alkylguanine-DNA alkyltransferase activity by O6-benzylgua-
nine 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. 1991, 87, 5368-5372.
(14) Ross, J . F.; Chaudhuri, P. K.; Ratnam, M. Differential regulation
of folate receptor isoforms in normal and malignant tissues in
vivo and in established cell lines. Physiologic and clinical
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(15) Nonancourt-Didion, M. d.; Gueant, J .-L.; Adjalla, C.; Chery, C.;
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R is one of the mechanism explaining the adaptation of HT29
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