Synthesis and Preliminary Biological Evaluation of 6-O-[11C]-[(methoxymethyl)benzyl]guanines, New Potential PET Breast Cancer Imaging Agents for the DNA Pepair Protein AGT

Article abstract of DOI:10.1016/S0960-894X(02)01048-X

Novel radiolabeled O⁶-benzylguanine derivatives, 6-O-[11C]-[(methoxymethyl)benzyl]guanines ([11C]p-O⁶-MMBG, 1a; [11C]m-O⁶-MMBG, 1b; ([11C]o-O⁶-MMBG, 1c), have been synthesized for evaluation as new potential positron emission tomography (PET) breast cancer imaging agents for DNA repair protein, O⁶-alkylguanine-DNA alkyltransferase (AGT).

Full text of DOI:10.1016/S0960-894X(02)01048-X

Bioorganic & Medicinal Chemistry Letters 13 (2003) 641–644
Synthesis and Preliminary Biological Evaluation of
6-O-[11C]-[(methoxymethyl)benzyl]guanines, New Potential PET
Breast Cancer Imaging Agents for the DNA Repair Protein AGT
Xuan Liu,a Qi-Huang Zheng,a,* Xiangshu Fei,a Ji-Quan Wang,a David W. Ohannesian,b
Leonard C. Erickson,b K. Lee Stonea and Gary D. Hutchinsa
aDepartment of Radiology, Indiana University School of Medicine, Indianapolis, IN 46202, USA
bDepartment of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN 46202, USA
Received 21 August 2002; accepted 23 November 2002
Abstract—Novel radiolabeled O6-benzylguanine derivatives, 6-O-[11C]-[(methoxymethyl)benzyl]guanines ([11C]p-O6-MMBG, 1a;
[11C]m-O6-MMBG, 1b; ([11C]o-O6-MMBG, 1c), have been synthesized for evaluation as new potential positron emission tomo-
graphy (PET) breast cancer imaging agents for DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGT).
# 2003 Elsevier Science Ltd. All rights reserved.
Breast cancer is the most common cancer among
women and the second leading cause of cancer death in
women. Breast cancer is commonly treated by various
combinations of surgery, radiation therapy, chemo-
therapy, hormone therapy and gene therapy.1 In the
diagnosis and treatment of breast cancer, positron
emission tomography (PET) has become a clinically
valuable and accepted new medical imaging tool.2 PET
coupled with radiopharmaceutical 2-[18F]fluoro-2-
deoxy-d-glucose (FDG) is widely used in clinical set-
tings to diagnose breast cancer.3 FDG is the only PET
breast cancer imaging agent used clinically at this point
in time. However, FDG is not in all cases satisfactory.
Only a limited number of PET studies using other
radiotracers have been conducted to image breast can-
cer and monitor its response to treatment, due to the
limited accessibility of radiotracers. Therefore, novel
PET tracers for breast cancer imaging are needed.
Radiotracer development is a key area for advancement
of research and clinical applications of PET in cancer
detection and treatment.4
In breast tumors, resistance to chemotherapeutic drugs
that alkylate the O6 position of guanine correlates with
the levels of the AGT. 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, stoichio-
metric process, thereby restoring a normal guanine at
the site of the modified base.6 However, the resultant
alkylated AGT is inactivated for subsequent deal-
kylations. Therefore, the ability of tumor cells to be
resistant to toxic effects of alkylating agents is depen-
dent on cellular AGT levels; the higher the AGT con-
tent, the less effective will be tumor killing by alkylator
therapy.7 Breast tumor cells frequently express high
levels of AGT. AGT plays a critical role in protecting
breast cancer cells from the cytotoxic effects of chemo-
therapeutic drugs that alkylate the O6 position of gua-
nine.8 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 lines and xenografts.9,10 O6-BG pro-
vides a means to effectively inactivate the AGT protein
and increase the chemotherapeutic effectiveness of
alkylating agents.11
Elevated levels of the DNA repair protein, O6-alkyl-
guanine-DNA alkyltransferase (AGT), are known to
result in resistance of human gliomas to nitrosoureas.5
The overexpression of AGT in breast tumors12 indicates
that AGT is a suitable target for the development of PET
breast tumor imaging agent to detect, image and monitor
breast cancer and its response to chemotherapy. Using
*Corresponding author. Tel.: +1-317-278-4671; fax: +1-317-278-
1072; e-mail: qzheng@iupui.edu
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(02)01048-X

642
X. Liu et al. / Bioorg. Med. Chem. Lett. 13 (2003) 641–644
PET with a positron emitting radionuclide carbon-11 or
fluorine-18 labeled O6-BG analogue, in which the O6-
benzyl moiety binds selectively to AGT, may prove to
be a useful tool for monitoring AGT levels in breast
tumor tissues and for evaluating the effectiveness of
drug and genetic strategies to down-regulate AGT in
breast tumors and/or up-regulate its expression in
chemotherapy sensitive tissues, such as bone marrow.
(DABCO-purine, 3) at a yield of 90%.17 The crude
product 3, without further purification, was reacted
with the sodium alkoxide of 4-, 3-, or 2-methoxymethyl-
benzyl alcohol (5a–c), which was prepared from benzene
1,4-, 1,3-, or 1,2-dimethanol (4a–c) with 60–80% yields,
to give the corresponding standard samples unlabeled
1a–c with 59–62% yields.
Compound 3 was reacted with the sodium alkoxide of
4a–c to provide the corresponding precursors, p-, m-,
or o- substituted 6-O-[(hydroxymethyl)benzyl]guanine
(p-O6-HMBG, 6a;18 m-O6-HMBG, 6b; o-O6-HMBG,
6c). The overall chemical yields of 6a–c from ACP were
moderate (47–52%).
O6-BG is a well-known low-molecular-weight inhibitor
of mammalian AGT.13 The application of O6-BG and a
number of alkylating agents in combination cancer
chemotherapy is currently being employed in clinical
trials. Numerous O6-BG derivatives and their related
compounds 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.14À16 Our objective was to develop radiolabeled
O6-BG analogues as PET imaging agents for the AGT
in breast cancer as well as in other cancers. The radio-
chemistry strategy was to synthesize guanine derivatives
amendable to labeling at the O6-benzyl group. Here we
report the synthesis, radiolabeling and preliminary bio-
logical evaluation of completely novel O6-BG derivatives,
6-O-[11C]-[(methoxymethyl)benzyl]guanines ([11C]p-O6-
MMBG, 1a; [11C]m-O6-MMBG, 1b; [11C]o-O6-MMBG,
1c).
Compounds 5a–c, 6a–c and 1a–c have analytical data
1
such as mp, HNMR and MS in agreement with the
indicated structures.19
The precursors 6a–c were labeled by the [11C]methyl
triflate20 through O-[11C]methylation of the hydroxy-
methyl position under basic conditions.21,22 The tracers
were isolated by the semi-preparative reversed phase
high performance liquid chromatography (HPLC) pur-
ification procedure, which employed a Prodigy (Phe-
nomenex) 5 mm C-18 column, 10 Â 250 mm; 3:1:3
CH3CN:MeOH:20 mM, pH 6.7 KHPO-4 mobile phase,
5.0 mL/min flow rate, and UV (240 nm) and g-ray (NaI)
flow detectors, to produce radiochemically pure target
compounds radiolabeled 1a–c (35–70 mCi) with 10–20%
radiochemical yields, based on 11CO2, decay corrected
to end of bombardment (EOB). There are three posi-
tions in precursors 6a–c including hydroxymethyl posi-
tion at O6 benzyl group, N7 and N9 positions at purine
ring that could be labeled by the [11C]methyl triflate to
produce desired labeled products 1a–c and undesired
N9-[11C]methylated products 7a–c and N7-[11C]methy-
lated products 8a–c. The radiochemical yields of desired
products 1a–c were low due to the undesired products
7a–c and 8a–c. The O-[11C]methylation radiolabeling
reaction was carried out under basic conditions which
employed organic base tetrabutylammonium hydroxide
(TBAH, 1 M solution in methanol), the ratio of labeled
products 1a–c and 7a–c, 8a–c was affected by the
amount of base TBAH, more base TBAH was used,
more products 7a–c and 8a–c were produced, the sui-
table amount of base TBAH for the reaction was
2–4 mL. The ranking of radiochemical yields was 1a–c
>7a–c >8a–c. Chemical purity, radiochemical purity,
and specific radioactivity were determined by the ana-
lytical HPLC methods, which employed a Prodigy
(Phenomenex) 5 mm C-18 column, 4.6 Â 250 mm; 3:1:3
CH3CN:MeOH:20 mM, pH 6.7 KHPO-4 mobile phase,
1.5 mL/min flow rate, and UV (240 nm) and g-ray (NaI)
flow detectors. Retention times in the analytical HPLC
system were: RT6a=2.95 min, RT6b=2.82 min, RT6c=
2.87 min; RT1a=3.22 min, RT1b=3.16 min, RT1c=
3.10 min. Retention times in the semi-preparative HPLC
system were: RT6a=3.24 min, RT6b=3.04 min, RT6c=
3.10 min; RT1a=3.58 min, RT1b=3.67 min, RT1c=
3.64 min. The chemical purities of the precursors 6a, 6b,
and 6c, and the standard samples 1a, 1b, and 1c were
>95%. The radiochemical purities of the target radio-
The synthetic approach for the radiolabeled 1a–c and
unlabeled 1a–c is shown in Scheme 1.
The commercially available starting material 2-amino-
6-chloropurine (ACP, 2) was reacted with 1,4-diaza-
bicyclo[2.2.2]octane (DABCO) to provide the impor-
tant intermediate quaternary amine 1-(2-amino-9H-
purin-6-yl)-4-aza-1-azoniabicyclo[2.2.2]octane chloride
Scheme 1. Synthesis of 1a–c.

X. Liu et al. / Bioorg. Med. Chem. Lett. 13 (2003) 641–644
643
tracers 1a–c were >99%, and the chemical purities of
Acknowledgements
the target radiotracers 1a–c were >90%. The average
(n=5–8) specific radioactivity of the target radiotracers
1a–c was 0.6–0.8 Ci/mmol at end-of-synthesis (EOS).
This work was partially supported by the Susan G.
Komen Breast Cancer Foundation grant IMG 2000 837
(to Q.H.Z.), the Indiana 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 National Institutes of Health/National Cancer
Institute grant P20CA86350 (to G.D.H.), the Indiana
21st Century Research and Technology Fund (to
G.D.H.), and the Lilly Endowment Inc. (to Indiana
Genomics Initiative (INGEN) of Indiana University).
We thank Dr. Bruce Mock and Barbara Glick-Wilson
for the efforts in producing carbon-11 precursors. The
reviewer’s criticisms and editor’s comments for the
revision of the manuscript are greatly appreciated.
The affinities of the unlabeled standard samples 1a–c
were evaluated via an in vitro AGT oligonucleotide
assay using breast cancer MCF-7 and MDA-MB-435
cells.23,24 The three compounds 1a–c proved to be fairly
potent AGT inhibitors in comparison with the parent
compound O6-BG, which was synthesized in our
laboratory.25 The concentration giving ꢀ90% inhibi-
tion of AGT activity in breast cancer MCF-7 cell
extracts were as follows: O6-BG=1 mM, 1a=ꢀ1 mM,
1b=ꢁ10 mM, 1c=>50 mM (Table 1). The ranking is
therefore O6-BG ꢀ1a >1b >1c. The results show that
the modified compound 1a exhibits strong inhibitory
effectiveness on AGT greater than or equal to the par-
ent compound O6-BG; and the modified compounds 1b
and 1c are fairly potent AGT inhibitors too albeit not as
effective as the parent compound O6-BG. The potentia-
tion effect order of the substituted O6-BG analogues is
para >meta >ortho,26,27 most likely because the steric
effect at the ortho-position plays a more important role
than the electronic effect. Preliminary findings from in
vitro biological assay of the unlabeled 1a, 1b, and 1c
warrant further in vivo evaluation of the radiolabeled
1a, 1b, and 1c.
References and Notes
1. Boxhorn, H. K. E.; Eck, S. L. Hematol. Oncol. Clin. North
Am. 1998, 12, 665.
2. Varagnolo, L.; Stokkel, M. P.; Mazzi, U.; Pauwels, E. K.
Nucl. Med. Biol. 2000, 27, 103.
3. Vranjesevic, D.; Filmont, J. E.; Meta, J.; Silverman, D. H.;
Phelps, M. E.; Rao, J.; Valk, P. E.; Czernin, J. J. Nucl. Med.
2002, 43, 325.
4. Fowler, J. S.; Volkow, N. D.; Wang, G. J.; Ding, S. L.;
Dewey, S. L. J. Nucl. Med. 1999, 40, 1154.
5. Felker, G. M.; Friedman, H. S.; Dolan, M. E.; Moschel,
R. C.; Schold, C. Cancer Chemother. Pharmacol. 1993, 32,
471.
In summary, the synthetic procedures that provide
novel radiolabeled O6-BG derivatives 1a, 1b, and 1c
have been developed. Preliminary findings from bio-
logical assay indicate the synthesized analogues have
similar strong inhibitory effectiveness on AGT in com-
parison with the O6-BG. These results warrant further
evaluation of these radiotracers as new potential PET
breast cancer imaging agents for the DNA repair
protein AGT in vivo.
6. Pegg, A. E. Cancer Res. 1990, 50, 6119.
7. Vaidyanathan, G.; Affleck, D. J.; Cavazos, C. M.; Johnson,
S. P.; Shankar, S.; Friedman, H. S.; Colvin, M. O.; Zalutsky,
M. R. Bioconjug. Chem. 2000, 11, 868.
8. Wani, G.; D’Ambrosio, S. M. Anticancer Res. 1997, 17,
4311.
9. Dolan, M. E.; Moschel, R. C.; Pegg, A. E. Proc. Natl.
Acad. Sci. U.S.A. 1990, 87, 5368.
10. Mitchell, R. B.; Moschel, R. C.; Dolan, M. E. Cancer Res.
1992, 52, 1171.
11. Dolan, M. E.; Pegg, A. E. Clin. Cancer Res. 1997, 3, 837.
12. Eberhagen, I.; Bankmann, M.; Kaina, B. Anticancer Res.
1995, 15, 761.
13. Pegg, A. E. Mutation Res. 2000, 462, 83.
14. Dolan, M. E.; Mitchell, R. B.; Mummert, C.; Moschel,
R. C.; Pegg, A. E. Cancer Res. 1991, 51, 3367.
15. Moschel, R. C.; McDougall, M. G.; Dolan, M. E.; Stine,
L.; Pegg, A. E. J. Med. Chem. 1992, 35, 4486.
16. Nesseler, E.; Schirrmacher, R.; Hamkens, W.; Rosch, F. J.
Label. Compd. Radiopharm. 1999, 42 (Suppl. 1), S198.
17. Linn, J. A.; McLean, E. W.; Kelley, T. L. J. Chem. Soc.
Chem. Commun. 1994, 913.
Table 1. AGT-inhibitory activity of O6-BG, p-O6-MMBG (1a),
m-O6-MMBG (1b), and o-O6-MMBG (1c), in fmol O6-methylguanine
(O6-MeG) removed/mg of protein
Fmol O6-MeG
removed/mg of
proteinÆSD (n=3)
MCF-7 control
2599Æ167
33Æ28
MDA-MB-435 (methylation repair deficient)
MCF-7 O6-BG 0.1 mM
MCF-7 O6-BG 1 mM
MCF-7 O6-BG 10 mM
MCF-7 O6-BG 50 mM
MCF-7 1a 0.1 mM
MCF-7 1a 1 mM
MCF-7 1a 10 mM
MCF-7 1a 50 mM
MCF-7 1b 0.1 mM
MCF-7 1b 1 mM
MCF-7 1b 10 mM
MCF-7 1b 50 mM
MCF-7 1c 0.1 mM
MCF-7 1c 1 mM
MCF-7 1c 10 mM
MCF-7 1c 50 mM
2389Æ28
164Æ93
50Æ16
19Æ26
457Æ293
292Æ286
35Æ41
18. Chae, M. Y.; Swenn, K.; Kanugula, S.; Dolan, M. E.;
Pegg, A. E.; Moschel, R. C. J. Med. Chem. 1995, 38, 359.
19. 5a–c, colorless oil. 5a, 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),
58Æ76
1644Æ738
1137Æ427
21Æ18
1
7.28 (s, 4H, ArH). 5b, H NMR (300 MHz, CDCl3): d 3.22 (s,
15Æ18
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). 5c, 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,
2345Æ100
2299Æ210
2489Æ261
660Æ54

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X. Liu et al. / Bioorg. Med. Chem. Lett. 13 (2003) 641–644
4H, ArH). 6a, a white solid, mp 226–229 ꢂC (lit.,18 229–231 ꢂC
dec.). 1H NMR (DMSO-d6): d 4.50–4.53 (d, J=9.5 Hz, 2H,
CH2OH), 5.24 (br, s, 1H, OH, exchanges with D2O), 5.50 (s,
2H, ArCH2), 6.32 (s, 2H, NH2, exchanges with D2O), 7.29–
7.51 (m, 4H, ArH), 7.85 (s, 1H, 8-H), 12.65 (br, s, 1H, >NH,
exchanges with D2O). 6b, a white solid, mp 192–194 ꢂC. 1H
NMR (DMSO-d6): d 4.53–4.56 (d, J=9.5 Hz, 2H, CH2OH),
5.24 (br, s, 1H, OH, exchanges with D2O), 5.49 (s, 2H,
ArCH2), 6.31 (s, 2H, NH2, exchanges with D2O), 7.30–7.34
(m, 1H, Ar-5-H), 7.36–7.38 (m, 2H, Ar-4-H and Ar-6-H), 7.47
(s, 1H, Ar-2-H), 7.85 (s, 1H, 8-H), 12.48 (br, s, 1H, >NH,
exchanges with D2O). HRMS (CI, CH4) calcd for
C13H13N5O2 271.1069, found 271.1065. 6c, a white solid, mp
198–202 ꢂC dec. 1H NMR (DMSO-d6): d 4.66–4.69 (d,
J=9.3 Hz, 2H, CH2OH), 5.24 (br, s, 1H, OH, exchanges with
D2O), 5.54 (s, 2H, ArCH2), 6.32 (s, 2H, NH2, exchanges with
D2O), 7.27–7.39 (m, 2H, Ar-4-H, Ar-5-H), 7.49–7.51 (d,
J=7.4 Hz, 2H, Ar-3-H and Ar-6-H), 7.84 (s, 1H, 8-H), 12.48
(br, s, 1H, >NH, exchanges with D2O). HRMS (CI, CH4)
calcd for C13H13N5O2 271.1069, found 271.1060. 1a, a white
solid, mp 163–165 ꢂC. 1H NMR (300 MHz, CD3OD): d 3.36 (s,
3H, OCH3), 4.44 (s, 2H, CH2O), 4.90 (s, 2H, NH2, exchange
with D2O), 5.53 (s, 2H, O6–CH2), 7.32–7.34 (d, 2H, J=8.1 Hz,
ArH), 7.48–7.51 (d, 2H, J=8.1 Hz, ArH), 7.84 (s, 1H, 8-H).
HRMS (CI, CH4): calcd for C14H15N5O2 285.1226, found
285.1222. 1b, a white solid, mp 190–192 ꢂC (dec.); 100–110 ꢂC
21. Zheng, Q.-H.; Mulholland, G. K. Nucl. Med. Biol. 1996,
23, 981.
22. Typical experimental procedure for the radiosynthesis of
[11C]p-O6-MMBG, 1a; [11C]m-O6-MMBG, 1b; [11C]o-O6-
MMBG, 1c. The precursor (p-O6-HMBG, 6a; m-O6-HMBG,
6b; or o-O6-HMBG, 6c) (0.6–1.0 mg) was dissolved in CH3CN
(300 mL). To this solution was added TBAH (2–4 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 À15 to À20 ꢂC,
which was generated by a Venturi cooling device powered with
100 psi compressed air, until radioactivity reached a maximum
(ꢁ3 min), then the reaction tube was heated at 70–80 ꢂC for
3 min. The contents of the reaction tube were diluted with
0.1 M NaHCO3 (1 mL) and 1:1 CH3CN:H2O (0.6 mL), and
injected onto the preparative HPLC column. The product
fraction was collected, the solvent was removed by rotatory
evaporation, and the final product 1a, 1b, or 1c was for-
mulated in saline containing 5% ethanol (1–3 mL), sterile-fil-
tered 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 and formulation time was 30–40 min EOB.
The decay corrected yields of the target radiotracers 1a–c,
from 11CO2, were 10–20%.
23. Wu, R.; Hurst-Calderone, S.; Kohn, K. Cancer Res. 1987,
47, 6229.
1
(sinter). H NMR (300 MHz, CD3OD): d 3.37 (s, 3H, OCH3),
4.64 (s, 2H, CH2O), 4.86 (s, br, 2H, NH2, exchange with D2O),
5.63 (s, 2H, O6–CH2), 7.31–7.40 (m, 3H, ArH), 7.56–7.59 (m,
1H, ArH), 7.83 (s, 1H, 8-H). HRMS (CI, CH4): calcd for
C14H15N5O2 285.1226, found 285.1234. 1c, a white solid, mp
204–205 ꢂC (dec.). 1H NMR (300 MHz, CD3OD): d 3.36 (s,
3H, OCH3), 4.46 (s, 2H, CH2O), 4.90 (s, br, 2H, NH2,
exchange with D2O), 5.55 (s, 2H, O6–CH2), 7.28–7.49 (m, 4H,
ArH), 7.84 (s, 1H, 8-H). HRMS (CI, CH4): calcd for
C14H15N5O2 285.1226, found 285.1221.
24. Zheng, Q.-H.; Liu, X.; Fei, X.; Wang, J.-Q.; Ohannesian,
D. W.;Erickson,L.C.;Stone,K. L.;Martinez,T. D.;Miller, K.D.;
Hutchins, G. D. J. Label. Compd. Radiopharm. 2002, 45, 1239.
25. Liu, X.; Zheng, Q.-H.; Hutchins, G. D.; Fei, X.; Erickson,
L. C.; Miller, K. D.; Mock, B. H.; Glick-Wilson, B. E.; Win-
kle, W. L.; Stone, K. L.; Carlson, K. A. Syn. Commun. 2003,
in press.
26. Kohda, K.; Terashima, I.; Koyama, K.; Watanabe, K.;
Mineura, K. Biol. Pharm. Bull. 1995, 18, 424.
20. Mock, B. H.; Mulholland, G. K.; Vavrek, M. T. Nucl.
Med. Biol. 1999, 26, 467.
27. Mineura, K.; Fukuchi, M.; Kowada, M.; Terashima, I.;
Kohda, K. Int. J. Cancer 1995, 63, 148.