Synthesis of radiolabeled O6-benzylguanine derivatives as new potential PET tumor imaging agents for the DNA repair protein O6-alkylguanine-DNA alkyltransferase

Article abstract of DOI:10.1002/jlcr.636

Novel radiolabeled O⁶-benzylguanine derivatives, 2-amino-6-O-[¹¹C]-[(methoxymethyl)benzyloxy]-9-benzyl purines ([¹¹C]p-O⁶AMBP, 1a; [¹¹C]m-O⁶-AMBP, 1b; [¹¹C]o-O⁶-AMBP, 1c), have been synthesized for evaluation as new potential positron emission tomography (PET) tumor imaging agents for the DNA repair protein, O⁶-alkylguanine-DNA alkyltransferase (AGT). The appropriate precursors for radiolabeling were obtained in three steps from starting material 2-amino-6-chloropurine with moderate to excellent chemical yields. Tracers were prepared by O-[¹¹C]methylation of hydroxymethyl precursors using [¹¹C]methyl triflate. Pure target compounds were isolated by solid-phase extraction (SPE) purification procedure in 45-60% radiochemical yields (decay corrected to the end of bombardment), and a synthesis time of 20-25 min. Copyright

Full text of DOI:10.1002/jlcr.636

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2002; 45: 1239–1252.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.636
Research Article
Synthesis of radiolabeled O6-benzylguanine
derivatives as new potential PET tumor
imaging agents for the DNA repair protein
O6-alkylguanine-DNA alkyltransferase
Qi-Huang Zheng1,*, Xuan Liu1, Xiangshu Fei1, Ji-Quan Wang1,
David W. Ohannesian3, Leonard C. Erickson3, K. Lee Stone1,
Tanya D. Martinez1, Kathy D. Miller2 and Gary D. Hutchins1
1 Department of Radiology, Indiana University School of Medicine, Indianapolis,
IN 46202, USA
2 Department of Medicine, Indiana University School of Medicine, Indianapolis,
IN 46202, USA
3 Cancer Center, Indiana University School of Medicine, Indianapolis, IN 46202,
USA
Summary
Novel radiolabeled O6-benzylguanine derivatives, 2-amino-6-O-[11C]-[(meth-
oxymethyl)benzyloxy]-9-benzyl purines ([11C]p-O6-AMBP, 1a; [11C]m-O6-
AMBP, 1b; [11C]o-O6-AMBP, 1c), have been synthesized for evaluation as
new potential positron emission tomography (PET) tumor imaging agents for
the DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGT). The
appropriate precursors for radiolabeling were obtained in three steps from
*Correspondence to: Qi-Huang Zheng, Department of Radiology, Indiana University School of
Medicine, 975 West Walnut Street, Room 028C, Indianapolis, IN 46202-5121, USA.
E-mail: qzheng@iupui.edu
Contract/grant sponsor: The Susan G. Komen Breast Cancer Foundation; contract/grant number:
IMG 2000 837
Contract/grant sponsor: Indiana University American Cancer Society, Institutional Grant
Committee; contract/grant number: IRG-84-002-17
Contract/grant sponsor: Indiana University Cores Centers of Excellence in Molecular Hematology.
Contract/grant sponsor: National Institutes of Health/National Cancer Institute; contract/grant
number: P20CA86350
Contract/grant sponsor: Indiana 21st Century Research and Technology Fund
Copyright # 2002 John Wiley & Sons, Ltd.
Received 19 April 2002
Revised 14 June 2002
Accepted 1 July 2002

1240
Q.-H. ZHENG ET AL.
starting material 2-amino-6-chloropurine with moderate to excellent chemical
yields. Tracers were prepared by O-[11C]methylation of hydroxymethyl
precursors using [11C]methyl triflate. Pure target compounds were isolated by
solid-phase extraction (SPE) purification procedure in 45–60% radiochemical
yields (decay corrected to the end of bombardment), and a synthesis time of
20–25 min. Copyright # 2002 John Wiley & Sons, Ltd.
Key Words: radiolabeled O6-benzylguanine derivatives; 2-amino-6-O-[11C]-
[(methoxymethyl)benzyloxy]-9-benzyl purines; carbon-11; positron emission
tomography; DNA repair protein; O6-alkylguanine-DNA alkyltransferase
Introduction
DNA repair enzyme O6-alkylguanine-DNA alkyltransferase (AGT) is
a suicide protein as it reverts alkylator cytotoxicity by transferring the
O6-alkyl group from the modified DNA guanine to cysteine-145 in its
active site in an irreversible, stoichiometric process, thereby restoring a
normal guanine at the site of the modified base.1 However, the resultant
alkylated AGT is inactivated for subsequent dealkylations. Therefore,
the ability of tumor cells to be resistant to toxic effects of alkylating
agents is dependent on cellular AGT levels; the higher the AGT content,
the less effective will be tumor killing by alkylator therapy.2 Tumor cells
frequently express high levels of AGT. Human AGT is encoded by the
O6-methylguanine-DNA methyltransferase (MGMT) gene.1 Elevated
levels of AGT are known to result in resistance of human gliomas to
nitrosoureas.3 AGT plays a critical role in protecting cancer cells from
the cytotoxic effects of chemotherapeutic drugs that alkylate the O6
position of guanine.4 The inactivation of AGT by administration of
substrates such as O6-benzylguanine (O6-BG) has been shown to
increase the cytotoxicity of alkylators in human tumor cell lines5,6 and
xenografts.7 O6-BG provides a means to effectively inactivate the AGT
protein and increase the chemotherapeutic effectiveness of alkylating
agents.8
The overexpression of AGT in tumors indicates that AGT is a
suitable target for tumor imaging agent development. Positron emission
tomography (PET) is a functional medical imaging modality that can
probe tumor cell physiology and gene expression. Using PET and a
positron-labeled O6-BG analog, which binds selectively to AGT, may
prove to be a useful tool for monitoring AGT levels in tumor tissues and
for evaluating the effectiveness of drug and genetic strategies to
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 1239–1252

SYNTHESIS OF RADIOLABELED O6-BENZYLGUANINE DERIVATIVES 1241
CH2O11CH3
O
p-CH2O11CH3, [11C] p-O6-MMBG
m-CH2O11CH3, [11C] m-O6-MMBG
N
N
o-CH2O11CH3, [11C] o-O6-MMBG
NH
H2N
N
F18
O
p-18F, [18F] p-O6-FBG
m-18F, [18F] m-O6-FBG
o-18F, [18F] o-O6-FBG
N
N
NH
H2N
N
O
7
6
5
4
N
1
N
8
O6-BG
NH
2
H2N
N
9
3
Figure 1. Structure of O6-BG, [11C]O6-MMBG and [18F]O6-FBG
down-regulate AGT in tumors and/or up-regulate its expression in
chemotherapy sensitive tissues, such as bone marrow.9
A number of carbon-11 and fluorine-18 labeled O6-BG analogs have
been developed in this laboratory.10 6-O-([11C]methoxymethylbenzyl)-
guanine ([11C]O6-MMBG) and 6-O-([18F]Fluorobenzyl)guanine ([18F]O6-
FBG) (Figure 1) are our initial target radiotracers; however, the low
radiochemical yields, long reaction time and complicated purification
methods in their radiochemical syntheses have prevented the reliable
routine production and automation of these tracers. In order to explore
novel radiolabeled O6-BG analogs as alternative candidate agents, we
have synthesized p-, m-, and o-derivatives of 2-amino-6-O-[11C]-
[(methoxymethyl)benzyloxy]-9-benzyl purines ([11C]p-O6-AMBP, 1a;
[11C]m-O6-AMBP, 1b; [11C]o-O6-AMBP, 1c).
Results and discussion
O6-BG is a well-known low-molecular-weight inhibitor of mammalian
AGT.11 The application of O6-BG and a number of alkylating agents in
combination cancer chemotherapy is currently being employed in
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 1239–1252

1242
Q.-H. ZHENG ET AL.
clinical trials. Numerous O6-BG derivatives and their related com-
pounds have been synthesized and tested for their ability to inhibit AGT
activity, which include those derivatives modified at the 2-amino group,
7-nitrogen, 9-nitrogen, and the para position of the O6-benzyl group.12–14
Our objective was to develop radiolabeled O6-BG analogs as PET
tumor imaging agents for AGT. The radiochemistry strategy was to
synthesize guanine derivatives modified at the 9-nitrogen position and
labeled at the O6-benzyl group. Here we report the synthesis of
radiolabeled O6-BG derivatives 1a–c.
The affinity of unlabeled standard samples of 1a–c was evaluated via
an in vitro AGT oligonucleotide assay.15–17 The three compounds 1a–c
proved to be potent AGT inhibitors albeit not as effective as the model
compound O6-BG, which was synthesized in our laboratory.10 The
concentration giving ꢀ 90% inhibition of AGT activity in HeLa cell
extracts were as follows: O6-BG=10 mM, p-O6-AMBP (1a)=ꢁ150 mM,
m-O6-AMBP (1b)=>150 mM, o-O6-AMBP (1c)=4150 mM (Table 1).
The ranking is therefore O6-BG >1a ꢀ 1b>1c. These results were not
unexpected, because none of the O6-BG derivatives have been reported
to be more potent AGT inhibitors than O6-BG, with the exception of
three classes of inhibitors that may have more activity than O6-BG
Table 1. AGT-inhibitory activity of O6-BG, p-O6-AMBP (1a), m-O6-AMBP
(1b), and o-O6-AMBP (1c), in fmol O6-methylguanine (O6-MeG) removed/mg of
protein
fmol O6-MeG removed/mg
of protein ꢂ S.D. (n)
HeLa diluent control
HeLa MR (methylation repair deficient)
O6-BG 0.1 mM
2020 ꢂ 496 (3)
48 ꢂ 44 (3)
2272 ꢂ 748 (3)
480 ꢂ 792 (4)
29 ꢂ 50.8 (3)
2044 ꢂ 395 (4)
1508 ꢂ 1132 (4)
840 ꢂ 728 (3)
68 ꢂ 112 (3)
2096 ꢂ 320 (4)
2020 ꢂ 748 (4)
816 ꢂ 608 (3)
333 ꢂ 416 (3)
1940 ꢂ 856 (4)
1612 ꢂ 920 (4)
1308 ꢂ 1024 (3)
888 ꢂ 319 (3)
O6-BG 1 mM
O6-BG 10 mM
p-O6-AMBP 1 mM
p-O6-AMBP 10 mM
p-O6-AMBP 50 mM
p-O6-AMBP 150 mM
m-O6-AMBP 1 mM
m-O6-AMBP 10 mM
m-O6-AMBP 50 mM
m-O6-AMBP 150 mM
o-O6-AMBP 1 mM
o-O6-AMBP 10 mM
o-O6-AMBP 50 mM
o-O6-AMBP 150 mM
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 1239–1252

SYNTHESIS OF RADIOLABELED O6-BENZYLGUANINE DERIVATIVES 1243
in vitro including: (1) those O6-BG derivatives bearing electron-
withdrawing groups (electronic effect) at the 8-position;18 (2) some
O6-(hetarylmethyl)guanines;19 and (3) 2,4-diamino-6-benzylpyrimidines
(BP) bearing electron-withdrawing groups at the 5-position.20 However,
these three classes of inhibitors are not ready for in vivo studies, since it
is difficult to make BG derivatives labeled at the 8-position; the toxicity
of O6-(hetarylmethyl)guanines remains to be investigated; and BP
derivatives are not as effective as O6-BG in mouse liver or spleen due to
their short half-life in vivo.21 The potentiation effect order of the
substituted O6-BG analogs is para > meta > ortho,22,23 most likely
because the steric effect at the ortho-position plays a more important
role than the electronic effect. Preliminary findings from the biological
assay of unlabeled 1a, 1b, and 1c warrant further evaluation of
radiolabeled 1a, 1b, and 1c as new potential PET tumor imaging agents
for the AGT in vivo.
The synthetic approaches for radiolabeled 1a–c and unlabeled 1a–c
were shown in Schemes 1 and 2.
The commercially available starting material 2-amino-6-chloropurine
(ACP, 2) was reacted with benzyl bromide to give the desired 9-isomer,
2-amino-9-benzyl-6-chloropurine (ACP-Bn, 3), and the byproduct
7-isomer. 3 was reacted with 1,4-diazabicyclo[2.2.2]octane (DABCO)
N
Cl
Cl
+
-
N
Cl
CH2Br
K2CO3
N
N
N
DABCO
N
N
N
NH
N
H2N
N
2
H2N
N
3
N
H2N
N
Bn
4
Bn
CH2OH
5a-c
NaH, DMSO
O
CH2OH
CH2O11CH3
HOH2C
O
N
11CH3OTfl/DMF
N
N
N
N
H2N
N
6a-c
N
H2N
N
1a-c
Bn
Bn
1a, [11C] p-O6-AMBP
1b, [11C] m-O6-AMBP
1c, [11C] o-O6-AMBP
Scheme 1. Synthetic approach for [11C]p-O6-AMBP, [11C]m-O6-AMBP, and
[11C]o-O6-AMBP
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 1239–1252

1244
Q.-H. ZHENG ET AL.
CH2OCH3
CH2OH
CH3I, NaH
O
CH2OCH3
4, NaH
N
N
CH2OH
N
CH2OH
H2N
N
7a-c
5a-c
Bn
1a-c
Scheme 2. Synthetic approach for unlabeled p-O6-AMBP, m-O6-AMBP, and
o-O6-AMBP
to convert into its quaternary ammonium salt, 1-(2-amino-9-benzyl-9H-
purin-6-yl)-4-aza-1-azoniabicyclo[2.2.2]octane chloride (4).24 The crude
product 4, without further purification, was reacted with the sodium
alkoxide of benzene 1,4-, 1,3-, or 1,2-dimethanols (5a, 5b, or 5c) to
provide the corresponding precursors, p-, m-, or o- substituted 2-amino-
6-O-[(hydroxymethyl)benzyloxy]-9-benzyl purine (p-O6-AHBP, 6a;
m-O6-AHBP, 6b; o-O6-AHBP, 6c). The overall chemical yields of 6a–c
from ACP were moderate to excellent.
To prepare the standard samples, unlabeled 1a, 1b, and 1c, 5a–c were
methylated by methyl iodide under strong basic condition using sodium
hydride to give 1,4-, 1,3-, or 1,2-methoxymethyl-benzyl alcohol (7a, 7b,
or 7c). To obtain the mono-methylated products of benzenedimethanol,
the suitable ratio of benzenedimethanol to sodium hydride was 1:5. 7a–c
were then coupled with quaternary ammonium salt 4 under basic
condition using sodium hydride to give the standard samples 1a–c.
The precursors 6a–c were labeled by [11C]methyl triflate25 through O-
[11C]methylation of hydroxymethyl position under basic conditions.26–29
The tracers were isolated by solid-phase extraction (SPE) purification26
to produce pure target compounds with 45–60% radiochemical yields
(based on 11CO2, decay corrected to end of bombardment), and 20–
25 min synthesis time. The large polarity difference between the
precursor and the labeled methylated product permitted the use of
SPE technique for purification of radioligands from radiolabeling
reaction mixture. The reaction mixture was diluted with NaHCO3 and
loaded onto C-18 cartridge by gas pressure. The cartridge column was
washed with water to remove unreacted precursor and reaction
solvent, and then the final labeled product was eluted with ethanol.
Chemical purity, radiochemical purity, and specific radioactivity were
determined by analytical HPLC methods. The chemical purity of
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 1239–1252

SYNTHESIS OF RADIOLABELED O6-BENZYLGUANINE DERIVATIVES 1245
precursors 6a, 6b, and 6c, and standard samples 1a, 1b, and 1c was
>97%. The radiochemical purity of target radiotracers 1a–c was
>99%, and the chemical purities of target radiotracers 1a–c were
ꢁ98% (1a), ꢁ95% (1b), ꢁ93% (1c). The average (n=5–10) specific
activity of target radiotracers 1a–c was 0.6–0.8 Ci/mmol at end-of-
synthesis (EOS).
Conclusion
We have developed synthetic procedures that provide radiolabeled
O6-BG derivatives 1a, 1b, and 1c. Preliminary findings from biological
assay indicate the synthesized analogs were less effective but have
similar inhibitory effectiveness on AGT in comparison with O6-BG. The
results warrant further evaluation of these radiotracers as new potential
PET tumor imaging agents for the DNA repair protein AGT in vivo.
Experimental
All commercial reagents and solvents were used without further
purification unless otherwise specified. The [11C]methyl triflate was
made according to a literature procedure25 by the metathetical reaction
of [11C]methyl bromide over a hot column of silver triflate supported on
porous graphite beads. One silver triflate column was used for 20–40
[11C]methyl triflate runs before replacement. Melting points were
determined on an MEL-TEMP II capillary tube apparatus and were
uncorrected. 1H NMR spectra were recorded on a Bruker QE 300 NMR
spectrometer using tetramethylsilane (TMS) as an internal standard.
Chemical shift data for the proton resonances were reported in parts per
million (d) relative to internal standard TMS (d 0.0). The low resolution
mass spectra were obtained using a Bruker Biflex III MALDI-Tof mass
spectrometer, and the high-resolution mass measurements were
obtained using a Kratos MS80 mass spectrometer, in the Department
of Chemistry at Indiana University. Chromatographic solvent propor-
tions are expressed on a volume: volume basis. Thin layer chromato-
graphy was run using Analtech silica gel GF uniplates (5 ꢃ 10 cm2).
Plates were visualized by UV light. Normal phase flash chromatography
was carried out on EM Science silica gel 60 (230–400 mesh) with a
forced flow of the indicated solvent system in the proportions described
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 1239–1252

1246
Q.-H. ZHENG ET AL.
below. All moisture-sensitive reactions were performed under a positive
pressure of nitrogen maintained by a direct line from a nitrogen source.
Analytical HPLC was performed using a Prodigy (Phenomenex) 5 mm
C-18 column, 4.6 ꢃ 250 mm2; 3:1:3 CH3CN: MeOH: 20 mM, pH
6.7 KHPOꢄ4 (buffer solution) mobile phase, flow rate 1.5 ml/min, and
UV (240 nm) and g-ray (NaI) flow detectors. Semi-prep C-18 silica
guard cartridge column 1 ꢃ 1 cm2 was obtained from E. S. Industries,
Berlin, NJ, and part number 300121-C18-BD 10 mm. Sterile vented
Millex-GS 0.22 mm vented filter unit was obtained from Millipore
Corporation, Bedford, MA.
2-Amino-9-benzyl-6-chloropurine (ACP-Bn, 3)
Benzyl bromide (2.25 ml, 18.7 mmol) was added to a mixture of 2-
amino-6-chloropurine (2, 2.13 g, 12.5 mmol), potassium carbonate
(5.2 g, 37.5 mmol), and anhydrous DMF (60 ml). The mixture was
stirred at room temperature overnight with exclusion of moisture. The
suspension was filtered and the filtrate was taken down to dryness under
vacuum. The residue chromatographed on a silica gel column by 4/1
CH2Cl2/EtOAc. Elution and evaporation afforded the title compound 3
1
(2.27 g, 70%) as a white solid, mp 202–2048C. H NMR (300 MHz,
DMSO-d6): d 5.31 (s, 2H, CH2N5), 6.96 (s, br, 2H, NH2, exchange
with D2O), 7.25–7.38 (m, 5H, ArH), 8.24 (s, 1H, 8-H).
1-(2-Amino-9-benzyl-9H-purin-6-yl)-4-aza-1-azoniabicyclo[2.2.2]octane
chloride (4)
ACP-Bn (3) (519.4 mg, 2 mmol) was reacted with 1,4-diazabicyclo
[2.2.2]octane (DABCO) (1.23 g, 11 mmol) in anhydrous DMF (4 ml) at
room temperature overnight. The mixture was filtered, and the resulting
solid was washed with acetone and dried under vacuum to afford a
quaternary ammonium salt 4 as a light yellow solid. The crude product
was used for the next step reaction without further purification.
2-Amino-6-O-[(40-hydroxymethyl)benzyloxy]-9-benzyl purine
(p-O6-AHBP, 6a)
Sodium hydride (80 mg, 2 mmol, 60% in oil) was added into a solution
of 1,4-benzenedimethanol (5a) (1.38 g, 10 mmol) in anhydrous DMSO
(1.0 ml). The mixture was stirred for 1 h at room temperature.
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 1239–1252

SYNTHESIS OF RADIOLABELED O6-BENZYLGUANINE DERIVATIVES 1247
The quaternary ammonium salt 4 (372 mg, 1 mmol) was added into the
mixture, which was stirred overnight. After addition of acetic acid
(185 ml), the mixture was adsorbed onto a silica gel column and eluted
with 8/1 CH2Cl2/MeOH to give a white solid 6a (260 mg, 72%), mp
179–1818C. 1H NMR (300 MHz, DMSO-d6): d 4.52 (d, 2H, J=5.89 Hz,
CH2OH, when exchanged with D2O, d changed to s), 5.24 (m, 1H, OH,
exchange with D2O), 5.26 (s, 2H, CH2N5), 5.49 (s, 2H, O6-CH2), 6.51
(s, 2H, NH2, exchange with D2O), 7.21–7.31 (m, 5H, N9-ArH),
7.33–7.36 (d, 2H, J=7.4 Hz, O6-ArH), 7.45–7.48 (d, 2H, J=7.4 Hz,
O6-ArH), 7.97 (s, 1H, 8-H). HRMS (CI, CH4): calcd for C20H19O2N5
361.1539, found 361.1521.
2-Amino-6-O-[(30-hydroxymethyl)benzyloxy]-9-benzyl purine
(m-O6-AHBP, 6b)
Prepared as 6a starting with 1,3-benzenedimethanol (5b) and quaternary
ammonium salt 4 on the same scale to give a white solid 6b (253 mg,
1
70%), mp 134–1368C. H NMR (300 MHz, DMSO-d6): d 4.55 (d, 2H,
J=5.89 Hz, CH2OH, when exchanged with D2O, d changed to s), 5.17
(t, 1H, J=5.1 Hz, OH, exchange with D2O), 5.26 (s, 2H, CH2N5), 5.51
(s, 2H, O6-CH2), 6.24 (s, 2H, NH2, exchange with D2O), 7.21–7.47 (m,
7H, ArH), 7.47 (s, 1H, O6-ArH), 7.84 (s, 1H, 8-H). HRMS (CI, CH4):
calcd for C20H19O2N5 361.1539, found 361.1526.
2-Amino-6-O-[(20-hydroxymethyl)benzyloxy]-9-benzyl purine
(o-O6-AHBP, 6c)
Prepared as 6a starting with 1,2-benzenedimethanol (5c) and quaternary
ammonium salt 4 on the same scale to give a white solid 6c (199 mg,
1
55%), mp 156–1588C. H NMR (300 MHz, DMSO-d6): d 4.68 (s, 2H,
CH2OH), 5.22 (m, 1H, OH, exchange with D2O), 5.27 (s, 2H, CH2N5),
5.58 (s, 2H, O6-CH2), 6.40 (s, 2H, NH2, exchange with D2O), 7.24–7.36
(m, 7H, ArH), 7.47–7.52 (m, 2H, ArH), 7.93 (s, 1H, 8-H). HRMS (CI,
CH4): calcd for C20H19O2N5 361.1539, found 361.1528.
The general method to prepare 1,4-, 1,3-, and 1,2-methyoxymethyl-
benzyl alcohol (7a, 7b, 7c)
Sodium hydride (80 mg, 2 mmol, 60% in oil) was added in the solution
5a, 5b, or 5c (1.38 g, 10 mmol) in anhydrous THF (15 ml). The mixture
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 1239–1252

1248
Q.-H. ZHENG ET AL.
was stirred at room temperature for 1 h. To this mixture was added neat
methyl iodide (123 ml, 2 mmol), and the resulting mixture was stirred
overnight. The mixture was evaporated and purified by flash column
chromatography to give the desired product 7a, 7b, or 7c as colorless oil
at a yield of 60–80%. 7a, 1H NMR (300 MHz, CDCl3): d 2.85 (s, br, 1H,
ArCH2OH, exchange with D2O), 3.34 (s, 3H, OCH3), 4.41 (s, 2H,
ArCH2O), 4.57 (s, br, 2H, ArCH2OH), 7.28 (s, 4H, ArH). 7b, 1H NMR
(300 MHz, CDCl3): d 3.22 (s, br, 1H, ArCH2OH, exchange with D2O),
3.35 (s, 3H, OCH3), 4.42 (s, 2H, ArCH2O), 4.58 (s, br, 2H, ArCH2OH),
7.23–7.31 (m, 4H, ArH). 7c, 1H NMR (300 MHz, CDCl3): d 3.39 (s, 3H,
OCH3), 3.64 (s, br, 1H, ArCH2OH, exchange with D2O), 4.53 (s, 2H,
ArCH2O), 4.62 (s, br, 2H, ArCH2OH), 7.28–7.37 (m, 4H, ArH).
2-Amino-6-O-[(40-methoxymethyl)benzyloxy]-9-benzyl purine
(p-O6-AMBP, 1a)
Sodium hydride (40 mg, 1 mmol, 60% in oil) was added to a solution of
7a (152 mg, 1 mmol) in anhydrous DMSO (0.5 ml), and the mixture was
stirred at room temperature for 1 h. To this mixture was added the
quaternary ammonium salt 4 (186 mg, 0.5 mmol), and the mixture was
stirred at room temperature for 3 h. The acetic acid (80 ml) was used to
neutralize the mixture. The mixture was evaporated and purified by
flash chromatography to give the unlabeled standard sample 1a (152 mg,
1
81%) as a white solid, mp 129–1308C. H NMR (300 MHz, CDCl3): d
3.35 (s, 3H, OCH3), 4.43 (s, 2H, CH2O), 5.07 (s, 2H, H2N, exchange
with D2O), 5.19 (s, 2H, CH2N5), 5.55 (s, 2H, O6-CH2), 7.19–7.21
(m, 2H, ArH), 7.29–7.32 (m, 5H, ArH), 7.46–7.49 (d, 2H, J=8.1 Hz,
ArH), 7.55 (s, 1H, 8-H). HRMS (CI, CH4): calcd for C21H21O2N5
375.1695, found 375.1695.
2-Amino-6-O-[(30-methoxymethyl)benzyloxy]-9-benzyl purine
(m-O6-AMBP, 1b)
Prepared as 1a starting with 7b and the quaternary ammonium salt 4 on
the same scale to give the unlabeled standard sample 1b (156 mg, 83%)
1
as a white solid, mp 98–998C. H NMR (300 MHz, CDCl3): d 3.38
(s, 3H, OCH3), 4.56 (s, 2H, CH2O), 4.92 (s, 2H, H2N, exchange with
D2O), 5.23 (s, 2H, CH2N5), 5.57 (s, 2H, O6-CH2), 7.22–7.26 (m, 2H,
ArH), 7.30–7.36 (m, 5H, ArH), 7.42–7.48 (d, 2H, J=8.1 Hz, ArH), 7.56
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 1239–1252

SYNTHESIS OF RADIOLABELED O6-BENZYLGUANINE DERIVATIVES 1249
(s, 1H, 8-H). HRMS (CI, CH4): calcd for C21H21O2N5 375.1695, found
375.1684.
2-Amino-6-O-[(20-methoxymethyl)benzyloxy]-9-benzyl purine
(o-O6-AMBP, 1c)
Prepared as 1a starting with 7c and the quaternary ammonium salt 4 on
the same scale to give the unlabeled standard sample 1c (107 mg, 57%)
1
as a light yellow solid, mp 132–1338C. H NMR (300 MHz, CDCl3): d
3.36 (s, 3H, OCH3), 4.63 (s, 2H, CH2O), 5.06 (s, br, 2H, NH2, exchange
with D2O), 5.20 (s, 2H, CH2N5), 5.63 (s, 2H, O6-CH2), 7.20–7.39 (m,
9H, ArH), 7.59 (s, 1H, 8-H). HRMS (CI, CH4): calcd for C21H21O2N5
375.1695, found 375.1685.
Typical experimental procedure for the radiosynthesis of 2-amino-
6-O-[11C]-[(methoxymethyl)benzyloxy]-9-benzyl purines ([11C]
p-O6-AMBP, 1a; [11C]m-O6-AMBP, 1b; [11C]o-O6-AMBP, 1c)
The precursor (p-O6-AHBP, 6a; m-O6-AHBP, 6b; or o-O6-AHBP, 1c)
(0.6–1.0 mg) was dissolved in CH3CN (300 ml). To this solution was
added tetrabutylammonium hydroxide (TBAH) (2–3 ml, 1 M solution in
methanol). The mixture was transferred to a small volume, three-neck
reaction tube. [11C]methyl triflate was passed into the air-cooled
reaction tube at ꢄ15 to ꢄ208C, which was generated by a Venturi
cooling device powered with 100 psi compressed air, until activity
reached a maximum (ꢁ3 min), then the reaction tube was heated at
70–808C for 3 min.The contents of the reaction tube were diluted with
0.1 M NaHCO3 (1 ml). This solution was passed onto a C-18 cartridge
by gas pressure. The cartridge was washed with 2 ꢃ 3 ml H2O, and the
aqueous washing was discarded. The product was eluted from the
column with 2 ꢃ 3 ml EtOH, and then passed onto a rotatory
evaporator. The solvent was removed by evaporation under high
vacuum. The labeled product 1a-c was formulated with 50 mM
NaH2PO4, whose volume was dependent upon the use of the labeled
product 1a-c in tissue biodistribution studies (ꢁ6 ml, 3 ꢃ 2 ml) or in
micro-PET imaging studies (1–3 ml) of the tumor bearing mice, sterile-
filtered through a sterile vented Millex-GS 0.22 mm cellulose acetate
membrane and collected into a sterile vial. Total radioactivity was
assayed and total volume was noted. The overall synthesis time was
ꢁ20 min. The decay corrected yield, from 11CO2, was 45–60%, and the
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 1239–1252

1250
Q.-H. ZHENG ET AL.
radiochemical purity was >99% by analytical HPLC. Retention times
in the analytical HPLC system were: RT6a=3.58 min, RT6b=3.84 min,
RT6c=3.98 min; RT1a=5.70 min, RT1b=5.90 min, RT1c=6.18 min.
In vitro AGT (MGMT) oligonucleotide assay
HeLa cells were cultured in Dulbecco’s modified Eagle’s medium
(DME) supplemented with 10% bovine calf serum. Measurement of
cellular AGT (MGMT) activity was performed by a modification of the
assay described by Wu et al.15 Control and drug treated cells were
washed in cold PBS, pH 7.4 and resuspended in cold assay buffer (Tris-
HCl, pH 8, 5% glycerol, 1 mM DTT, 1 mM EDTA). The cells were
sonicated in 400 ml of assay buffer for 5 s on ice. The lysate was pelleted
by centrifugation for 5 min, at 12,000 g, at 48C. The protein concentra-
tions were determined by the Pierce Coomassie plus protein assay
(Pierce, Rockford, IL). 50 mg of total cell protein were then reacted with
0.2 pmol of 50-Hex-labeled 18mer oligo in assay buffer for 2 h, at 378C.
The reaction was terminated by two phenol:chloroform extractions, one
chloroform:isoamyl alcohol extraction, and then ethanol-precipitated in
the presence of 1 mg carrier glycogen. Following precipitation, the
precipitate was washed with 70% ethanol, dried under vacuum, and
reacted with three units of PvuII (Promega, Madison, WI) in a total
volume of 20 ml for 2 h at 378C. The reaction was terminated by the
addition of 10 ml of gel loading buffer (96% formamide, 1 mM EDTA,
pH 8) and heating at 908C for 2 min. Samples (1a–c, and O6-BG) were
chilled on ice and loaded directly on to the gel. The samples were
electrophoresed through a 1.5 mm, 20% acrylamide, 7 M urea gel, at
300 V, for approximately 30 min.The gels were then placed on an
FMBIO II fluorescent scanner (MiraiBio, South San Francisco, CA)
and quantitated using the calculated fluorescent absorbance. AGT
specific activity (fmol of O6-methylguanine removed/mg of protein) was
calculated according to the following equation:
fluorescent units of 8-bp fragment/fluorescent units of 18-bp fragment
x 200 fmol/mg protein.
Acknowledgements
This work was partially supported by the Susan G. Komen Breast
Cancer Foundation grant IMG 2000 837 (to Q.H.Z.), the Indiana
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 1239–1252

SYNTHESIS OF RADIOLABELED O6-BENZYLGUANINE DERIVATIVES 1251
University American Cancer Society (ACS) Institutional Grant
Committee grant IRG-84-002-17 (to Q.H.Z.), the Indiana University
Cores Centers of Excellence in Molecular Hematology (CCEMH) pilot
and feasibility (P/F) grant (to Q.H.Z.), the NIH/NCI grant
P20CA86350 (to G.D.H.), the Indiana 21st Century Research and
Technology Fund (to G.D.H.). We thank Dr Bruce Mock and Barbara
Glick-Wilson for the efforts in producing carbon-11 precursors. The
reviewer and editor’s comments for the revision of the manuscript are
greatly appreciated.
References
1. Pegg AE. Cancer Res 1990; 50: 6119–6129.
2. Vaidyanathan G, Affleck DJ, Cavazos CM, et al. Bioconjugate Chem 2000;
11: 868–875.
3. Felker GM, Friedman HS, Dolan ME, et al. Cancer Chemother Pharmacol
1993; 32: 471–476.
4. Wani G, D’Ambrosio SM. Anticancer Res 1997; 17: 4311–4316.
5. Dolan ME, Moschel RC, Pegg AE. Proc Natl Acad Sci USA 1990; 87:
5368–5372.
6. Friedman HS, Dolan ME, Moschel RC, et al. J Natl Cancer Inst 1992; 84:
1926–1931.
7. Mitchell RB, Moschel RC, Dolan ME. Cancer Res 1992; 52: 1171–1175.
8. Dolan ME, Pegg AE. Clin Cancer Res 1997; 3: 837–847.
9. Williams DA, Maze R, Kurpad C, et al. Bone Marrow Transplantation
2000; 25: 105–109.
10. Liu X, Zheng Q-H, Fei XS, et al. Syn Commun 2003; 33(6): in press.
11. Pegg AE. Mutation Res 2000; 462: 83–100.
12. Dolan ME, Mitchell RB, Mummert C, et al. Cancer Res 1991; 51:
3367–3372.
13. Moschel RC, McDougall MG, Dolan ME, et al. J Med Chem 1992; 35:
4486–4491.
14. Nesseler E, Schirrmacher R, Hamkens W, et al. J Labeled Compd
Radiopharm 1999; 42 (Suppl. 1): S198–S200.
15. Wu R, Hurst-Calderone S, Kohn K. Cancer Res 1987; 47: 6229–6235.
16. Tano K, Shiota S, Collier J, et al. Proc Natl Acad Sci 1990; 87: 686–690.
17. Kreklau EL, Liu N, Li Z, et al. J Pharmacol Exp Ther 2001; 297: 524–530.
18. Chae MY, McDougall MG, Dolan ME, et al. J Med Chem 1995; 38:
359–365.
19. McElhinney RS, Donnelly DJ, McCormick JE, et al. J Med Chem 1998;
41: 5265–5271.
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 1239–1252

1252
Q.-H. ZHENG ET AL.
20. Terashima I, Kohda K. J Med Chem 1998; 41: 503–508.
21. Roy SK, Moschel RC, Dolan ME. Drug Metab Disposition 1996; 24:
1205–1211.
22. Kohda K, Terashima I, Koyama K, et al. Biol Pharm Bull 1995; 18:
424–430.
23. Mineura K, Fukuchi M, Kowada M, et al. Int J Cancer 1995; 63: 148–151.
24. Linn JA, McLean EW, Kelley TL. J Chem Soc Chem Commun 1994;
913–914.
25. Mock BH, Mulholland GK, Vavrek MT. Nucl Med Biol 1999; 26(4):
467–471.
26. Zheng Q-H, Mulholland GK. Nucl Med Biol 1996; 23(8): 981–986.
27. Zheng Q-H, Mulholland GK. J Label Compd Radiopharm 1997; 40
(Suppl. 1): 551–553.
28. Zheng Q-H, Winkle W, Carlson K, Mulholland GK. J Label Compd
Radiopharm 1997; 40 (Suppl. 1): 548–550.
29. Fei X, Zheng Q-H, Hutchins, GD, et al. J Label Compd Radiopharm 2002;
45: 449–470.
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 1239–1252