Synthesis and preliminary biological evaluation of O6-[4-(2- [18F]fluoroethoxymethyl)benzyl]guanine as a novel potential PET probe for the DNA repair protein O6-alkylguanine-DNA alkyltransferase in cancer chemotherapy

Article abstract of DOI:10.1016/j.bmc.2005.05.061

A novel fluorine-18-labeled O⁶-benzylguanine (O⁶-BG) derivative, O⁶-[4-(2-[¹⁸F]fluoroethoxymethyl)benzyl] guanine (O⁶-[¹⁸F]FEMBG, [¹⁸F]1), has been synthesized for evaluation as

Full text of DOI:10.1016/j.bmc.2005.05.061

Bioorganic & Medicinal Chemistry 13 (2005) 5779–5786
Synthesis and preliminary biological evaluation
of O6-[4-(2-[18F]fluoroethoxymethyl)benzyl]guanine
as a novel potential PET probe for the DNA repair protein
O6-alkylguanine-DNA alkyltransferase in cancer chemotherapy
Ji-Quan Wang,a Emiko L. Kreklau,b Barbara J. Bailey,b
Leonard C. Ericksonb and Qi-Huang Zhenga,*
aDepartment of Radiology, Indiana University School of Medicine, Indianapolis, IN 46202, USA
bIndiana University Cancer Center and Department of Pharmacology and Toxicology,
Indiana University School of Medicine, Indianapolis, IN 46202, USA
Received 26 May 2005; accepted 26 May 2005
Available online 1 July 2005
Abstract—A novel fluorine-18-labeled O6-benzylguanine (O6-BG) derivative, O6-[4-(2-[18F]fluoroethoxymethyl)benzyl]guanine (O6-
[18F]FEMBG, [18F]1), has been synthesized for evaluation as a potential positron emission tomography (PET) probe for the DNA
repair protein O6-alkylguanine-DNA alkyltransferase (AGT) in cancer chemotherapy. The appropriate radiolabeling precursor
N
2,9-bis(p-anisyldiphenylmethyl)-O6-[4-(hydroxymethyl)benzyl]guanine (6) and reference standard O6-[4-(2-fluoroethoxymeth-
yl)benzyl]guanine (O6-FEMBG, 1) were synthesized from 1,4-benzenedimethanol and 2-amino-6-chloropurine in four or six steps,
respectively, with moderate to excellent chemical yields. The target tracer O6-[18F]FEMBG was prepared in 20–35% radiochemical
yields by reaction of MTr-protected precursor 6 with [18F]fluoroethyl bromide followed by quick deprotection reaction and purifi-
cation with a simplified Silica Sep-Pak method. Total synthesis time was 60–70 min from the end of bombardment. Radiochemical
purity of the formulated product was >95%, with a specific radioactivity of >1.0 Ci/lmol at the end of synthesis. The activity of
unlabeled O6-FEMBG was evaluated via an in vitro AGT oligonucleotide assay. Preliminary findings from biological assay indicate
that the synthesized analogue has similarly strong inhibiting effect on AGT in comparison with O6-BG and O6-4-fluorobenzylgua-
nine (O6-FBG). The results warrant further in vivo evaluation of O6-[18F]FEMBG as a new potential PET probe for AGT.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
cysteine-145 residue in its active site in a suicidal man-
ner, whereby the AGT molecule is irreversibly inactivat-
ed, the alkylated protein is then rapidly degraded, and
its recovery requires de novo protein synthesis.1–3 The
ability of tumor cells to be resistant to the toxic effects
of alkylating agents is thus dependent on cellular AGT
levels. Inactivation of AGT by administration of direct
substrates such as O6-benzylguanine (O6-BG) has been
shown to increase the cytotoxicity of alkylators and
the chemotherapeutic efficacy of alkylating agents.
Therefore, it is highly imperative to detect the AGT lev-
els in tumor cells. The overexpression of AGT in cancers
provides a target for the development of medical probes.
Alkylating agents are extensively used in the chemother-
apy of various cancers. The cytotoxicity of these agents
stems from their ability to alkylate DNA guanine resi-
dues at their O6-position. The effectiveness of the alkyl-
ating agents is limited by tumor overexpression of the
DNA repair protein O6-alkylguanine-DNA alkyltrans-
ferase (AGT), also commonly referred to as O6-methyl-
guanine-DNA methyltransferase (MGMT), which
removes cytotoxic O6-alkylguanine adducts. AGT trans-
fers alkyl groups such as methyl, ethyl, or benzyl from
the O6-position of the guanine residues in DNA to the
Positron emission tomography (PET) is a non-invasive
biomedical imaging technique. O6-BG derivatives with
suitable positron emitting radionuclides attached to
the benzyl ring are potentially useful in the non-invasive
imaging of the DNA repair protein AGT. The efforts of
Keywords: O6-[4-(2-[18F]fluoroethoxymethyl)benzyl]guanine; Positron
emission tomography; Probe; O6-alkylguanine-DNA alkyltransferase;
Cancer.
*
Corresponding author. Tel.: +1 317 278 4671; fax: +1 317 278
9711; e-mail: qzheng@iupui.edu
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.05.061

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our group have been to develop novel positron labeled
O6-BG derivatives for PET to monitor the AGT in can-
cers and its response to chemotherapy. A series of car-
bon-11 labeled O6-BG derivatives as shown in Figure
1 have been synthesized and evaluated in this laborato-
ry.4–7 However, the short half-life (t1/2) of the radionu-
clide carbon-11, 20.4 min, limits the imaging protocol
to a maximum of about 90 min post-injection. If imag-
ing were to extend past 90 min, more complete informa-
tion could be obtained regarding the concentration and
localization of AGT levels in cancer tissue. It is attrac-
tive, therefore, to pursue analogues of O6-BG, which
can be labeled with the longer-lived radionuclide fluo-
rine-18 (t1/2 110 min). It is hoped that fluorine-18 labeled
O6-BG analogues, which permit imaging of up to 5 h
post-injection, will result in a better match between the
pharmacokinetics of binding and the physical decay of
the label. To this end, we turn our efforts toward the
development of fluorine-18 labeled O6-BG derivatives.
A potentially useful fluorine-18 labeled O6-BG deriva-
tive, O6-(4-[18F]fluorobenzyl)guanine (O6-[18F]FBG) as
shown in Figure 1, has been described in the literature.1
It is expected that the methodology with [18F]fluoride
([18F]Fꢀ) for the radiolabeling of O6-[18F]FBG will not
be applicable to all O6-BG derivatives, as specific com-
pound may require specific labeling method. Therefore,
we designed and synthesized a novel fluorine-18 labeled
O6-BG derivative, O6-[4-(2-[18F]fluoroethoxymeth-
yl)benzyl]guanine (O6-[18F]FEMBG, [18F]1), and per-
formed the radiolabeling of the precursor using
2-[18F]fluoroethyl bromide ([18F]FEBr)8,9 within
a
multipurpose fluorine-18 radiosynthesis system. The
biological activity of new unlabeled O6-BG derivative
O6-[4-(2-fluoroethoxymethyl)benzyl]guanine (O6-FEM-
BG, 1) was evaluated via an in vitro AGT oligonucleo-
tide assay.
2. Results and discussion
2.1. Chemistry and radiochemistry
The synthetic approach for the monomethoxytrityl
(MTr-) protected precursor N2,9-bis(p-anisyldiphenylm-
ethyl)-O6-[4-(hydroxymethyl)benzyl]guanine (6), and
reference standard O6-[4-(2-fluoroethoxymethyl)ben-
zyl]guanine (O6-FEMBG, 1) is shown in Scheme 1. 1,4-
Benzenedimethanol was converted to its monosodium
alkoxide, which was reacted with 2-amino-6-chloropu-
rine (2) to give O6-[4-(hydroxymethyl)benzyl]guanine
(3) in 59% yield.10 Oxidation of compound 3 with
pyridinium chlorochromate (PCC) provided O6-(4-form-
ylbenzyl)guanine (4) in 35% yield.10 Protection of N2
and N9 positions of the guanine moiety in compound 4
with monomethoxytrityl group using monomethoxytri-
tyl chloride (MTrCl)11–14 afforded N2,9-bis(p-anisyldi-
phenylmethyl)-O6-(4- formylbenzyl)guanine (5) in
43% yield. Reduction of compound 5 with sodium
Figure 1. Chemical structures of O6-BG, carbon-11 labeled O6-BG derivatives, and O6-[18F]FBG.

J.-Q. Wang et al. / Bioorg. Med. Chem. 13 (2005) 5779–5786
5781
Scheme 1. Synthetic approach for O6-FEMBG (1) and O6-[18F]FEMBG ([18F]1).
borohydride (NaBH4) gave the MTr-protected precursor
6 in 96% yield. Compound 6 was alkylated with
1-bromo-2-fluoroethane under basic condition to pro-
duce N2,9-bis(p-anisyldiphenylmethyl)-O6-[4-(2- fluoro-
ethoxymethyl)benzyl]guanine (7) in 66% yield.
Deprotection of compound 7 with 1 N HCl furnished
the reference standard O6-FEMBG (1) in 66% yield.
The overall chemical yield for the synthesis of precursor
6 was 8.5% in four steps. The overall chemical yield
for the reference standard 1 was 5.1% in six steps. The
chemical purity of precursor 6 and standard sample 1
was >95% measured by HPLC method.
tillation of [18F]FEBr was completed, the reaction
mixture of precursor 6 with [18F]FEBr was heated at
120 ꢁC for 10–15 min to form a radiolabeled intermedi-
ate N2,9-bis(p-anisyldiphenylmethyl)-O6-[4-(2- [18F]flu-
oroethoxymethyl)benzyl]guanine
([18F]7).
The
radiolabeling reaction was monitored by analytical
radio-HPLC method using a Prodigy (Phenomenex)
5 lm C-18 column, 4.6 · 250 mm; 3:1:1 CH3CN/
ꢀ
MeOH/20 mM, pH 6.7 KHPO4
mobile phase,
1.5 mL/min flow rate, and UV (240 nm) and c-ray
(NaI) flow detectors. Retention times (tRs) in the analyt-
ical HPLC system for [18F]FEBr, 6 and [18F]7 are 2.31,
10.15, and 13.38 min, respectively. The radiolabeling
mixture was passed through a Silica Sep-Pak column
to remove unreacted [18F]FEBr. The large polarity dif-
ference between 6 and [18F]7, and unreacted [18F]FEBr
permitted the use of a simple solid-phase extraction
(SPE) technique11–13,16 for fast isolation of 6 and [18F]7
from the radiolabeling reaction mixture. The Sep-Pak
was first eluted with 15% MeOH/CH2Cl2 and the solvent
was evaporated under high vacuum (0.1–1.0 mmHg) to
give a mixture of 6 and [18F]7. As the remaining unre-
acted [18F]FEBr may affect the deprotection reaction
The synthetic approach for the target tracer O6-[4-(2-
[18F]fluoroethoxymethyl)benzyl]guanine (O6-[18F]FEM-
BG, [18F]1) is also shown in Scheme 1. 2-Bromoethanol
was reacted with trifluoromethane sulfonic anhydride to
give 2-bromoethyl triflate in 47% yield.15 Nucleophilic
substitution of 2-bromoethyl triflate with [18F]KF/
Kryptofix 2.2.2 in CH3CN provided the radiolabeled re-
agent [18F]FEBr, which was distilled under a nitrogen
flow at 130 ꢁC for 5 min and bubbled into a reaction ves-
sel containing the precursor 6 in CH3CN. After the dis-

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J.-Q. Wang et al. / Bioorg. Med. Chem. 13 (2005) 5779–5786
of [18F]7 through the interaction with N2 and N9 posi-
tions of the guanine moiety in compound [18F]7, they
had to be removed prior to deprotection of [18F]7. The
existence of the [18F]FEBr would also affect the purifica-
tion of labeled product from its mixture with precursor
and the quality control (QC) of the tracer production.17
Less than 10% of [18F]FEBr was usually left unreacted,
and the unreacted [18F]FEBr was stayed in the Sep-Pak
column. Then, the [18F]FEBr remainder was removed by
washing the column with some additional more polar
solvent, AcOH/EtOH/H2O combination (2:8:90). Com-
pound [18F]7 was treated with 1 N HCl at 80 ꢁC for 5–
10 min and the reaction mixture was neutralized with
6 N NaOH to provide the target tracer [18F]1. To simpli-
fy the synthetic procedure, the final reaction mixture
was purified with SPE method instead of HPLC, which
makes it amenable to automation. The crude product
was passed through another Silica Sep-Pak column to
remove the precursor 6 and radioactive byproducts with
ethanol. The large polarity difference between [18F]1 and
the precursor 6 and radioactive byproducts permitted
the use of SPE technique for fast purification of radio-
tracer [18F]1. The radiochemically pure compound
[18F]1 was isolated from the Sep-Pak using a 2:8:90
HOAc/EtOH/H2O eluant and the combined product
containing fraction was treated with 2 M NaOH and
150 mM NaH2PO4 mixed solution to adjust pH to
5.5–7.0. The overall radiochemical yield of [18F]1 was
20–35% (n = 5), and the synthesis time was 60–70 min
(n = 5) from the end of bombardment (EOB). Chemical
purity, radiochemical purity, and specific radioactivity
were determined by analytical HPLC. Retention times
in the analytical HPLC system were: tR6 = 10.15 min,
The specific radioactivity of radiotracer [18F]1 was 1.0–
1.3 Ci/lmol at the end of synthesis (EOS).
Overall, the three-step radiolabeling procedure includ-
ing the production of radiolabeled precursor
[18F]FEBr, the radiolabeling of the precursor 6 with
[18F]FEBr to form the radiolabeled intermediate
[18F]7, and the deprotection of [18F]7 to give the target
tracer [18F]1 were combined into a multipurpose fluo-
rine-18 radiosynthesis system. This prototype device
can perform three-step 18F-labeling reactions in three
separate reaction vials. Purification capabilities include
a novel chromatography methodology with a simplified
dual Silica Sep-Pak method11–13,16 and direct distilla-
tion. Organic solvents can be removed when necessary
by conventional roto-evaporation, and the product
recovered for formulation and direct aseptic delivery
to the final product vial. Automated cleaning routines
have also been developed to prepare the device for sub-
sequent syntheses and to minimize radiation exposure
to production personnel. In comparison with the fluo-
rine-18 radiolabeling method reported in the litera-
ture,1 our improved method has several advantages
such as higher radiochemical yield and specific radioac-
tivity, shorter purification time, and more automatic
operating processes.
2.2. In vitro O6-methylguanine-DNA methyltransferase
oligonucleotide assay
Human AGT is encoded by the MGMT gene.18 The cel-
lular MGMT specific activity of O6-FEMBG was evalu-
ated using breast cancer MCF-7 cells in comparison
with the parent compound O6-BG and compound O6-
4-fluorobenzylguanine (O6-FBG),5–7 via a modified
technique of an in vitro MGMT oligonucleotide assay
tR7 = 13.38 min,
2.66 min. The radiochemical purity of target radiotracer
tR1 = 2.66 min,
and
tR[18F]1 =
[18F]1 was >96%, and the chemical purity was ꢁ93%.
Figure 2. Percent MGMT activity of O6-FEMBG (FEMBG) in comparison with O6-BG (BG) and O6-FBG (FBG) in different concentrations. *FGB
was slightly but significantly more effective than BG at 0.5 lM, and FEMBG was slightly but significantly less effective that BG at 1.0 lM.

J.-Q. Wang et al. / Bioorg. Med. Chem. 13 (2005) 5779–5786
5783
Table 1. MGMT specific activity of O6-FEMBG in comparison with
O6-BG and O6-FBG in fmol O6-methylguanine (O6-MeG) removed/
mg of protein
points were determined on a MEL-TEMP II capillary
tube apparatus and were uncorrected. H NMR spectra
1
were recorded on a Bruker QE 400 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 the internal
standard TMS (d 0.0). Low-resolution mass spectra
(LRMS) were obtained using a Bruker Biflex III MAL-
DI-TOF mass spectrometer, and high-resolution mass
spectra (HRMS) 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 chromatography was run using Analtech silica
gel GF uniplates (5 · 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 below. All moisture-sensitive
reactions were performed under a positive pressure of
nitrogen maintained by a direct line from a nitrogen
source.
Dose (lM)
O6-BGa
O6-FBGa
O6-FEMBGa
0.1
0.5
1
4500 575
1868 328
166 65
4500 575
918 118*
76 23
4500 575
1071 225
432 33*
10
50
100
<50
<50
<50
5
5
5
<50
<50
<50
5
5
5
<50
<50
<50
5
5
5
a MGMT specific activity (fmol O6-MeG removed/mg of protein).
* p < 0.01 compared to respective O6-BG treatment.
developed in our laboratory.19 The MGMT assay was
originally described by Wu et al.20 Both reference com-
pounds O6-BG and O6-FBG were previously prepared
in this laboratory.4 The new compound O6-FEMBG
proved to be a potent AGT inhibitor similar to the
two reference compounds. The results are summarized
in Figure 2 and Table 1. The data are reported as IC90
values because therapeutic efficacy of O6-BG and O6-
BG related compounds require >90% inhibition of
MGMT. In this case, the IC90 value of each compound
is also more precisely represented by the data than the
IC50 value. The relative differences among the com-
pounds, however, appear to be similar based on either
IC90 or IC50 values. The respective IC90 values for O6-
BG (BG) and O6-FBG (FBG) are between 0.5 and
1.0 lM, whereas the IC90 value for O6-FEMBG (FEM-
BG) is ꢁ1.0 lM. As seen in the bar graph and the table,
O6-FBG was slightly but significantly more effective
than O6-BG at 0.5 lM, and O6-FEMBG was slightly
but significantly less effective than O6-BG at 1.0 lM.
In summary, O6-FBG and O6-FEMBG appear to be
similar in efficacy compared to O6-BG at inhibiting cel-
lular MGMT activity in MCF-7 cells.
Analytical HPLC was performed using a Prodigy (Phe-
nomenex) 5 lm C-18 column, 4.6 · 250 mm; 3:1:1
CH3CN/MeOH/20 mM, pH 6.7 KHPO4 (buffer solu-
ꢀ
tion) mobile phase; flow rate 1.5 mL/min; and UV
(240 nm) and c-ray (NaI) flow detectors. Semi-prep
SiO2 Sep-Pak type cartridge was obtained from Waters
Corporation, Milford, MA. Sterile Millex-GS 0.22 lm
vented filter unit was obtained from Millipore Corpora-
tion, Bedford, MA.
4.2. O6-[4-(Hydroxymethyl)benzyl]guanine (3)
To a 250 mL two-necked flask, 1,4-benzenedimethanol
(20 g, 144.75 mmol) was added, and then heated to
130 ꢁC until it was completely melted. Under nitrogen,
sodium (0.60 g, 26.10 mmol) was added in two portions.
When gas evolution ceased and all sodium disappeared,
the temperature was lowered to 115 ꢁC, and 2-amino-6-
chloropurine, 2 (2.171 g, 12.80 mmol) was added. The
mixture was stirred at 115 ꢁC for 24 h. While it was still
hot, the mixture was poured into a beaker containing
600 mL water with constant stirring. It was stirred until
the solution was cooled to room temperature. Undis-
solved solid was filtered off, and the filtrate was neutral-
ized with glacial acetic acid. The resultant precipitate
was collected by filtration and crystallized from 1:1
MeOH/H2O to give compound 3 (2.063 g, 59%) as an
off-white solid, mp 226–229 ꢁC (lit.8 229–231 ꢁC dec).
1H NMR (DMSO-d6, ppm) d 12.42 (s, 1H, NH, D2O
exchangeable), 7.80 (s, 1H, 8-CH), 7.44 (d, 2H,
J = 6.62 Hz, phenyl), 7.32 (d, 2H, J = 6.62 Hz, phenyl),
6.29 (s, 2H, NH2, D2O exchangeable), 5.45 (s, 2H,
CH2), 5.20 (s, 1H, OH, D2O exchangeable), 4.49 (s,
2H, CH2).
3. Conclusion
The synthetic procedures that provide the MTr-protect-
ed precursor, reference standard O6-FEMBG, and fluo-
rine-18 labeled O6-BG derivative O6-[18F]FEMBG have
been well developed. A novel fluorine-18-radiolabeling
methodology within a multiuse 18F-radiosynthesis mod-
ule for the preparation of fluorine-18 tracer and a novel
chromatography methodology with a simplified dual Sil-
ica Sep-Pak method for the purification of fluorine-18
tracer have also been developed. Preliminary findings
from in vitro biological assay indicate that the synthe-
sized analogue O6-FEMBG has an inhibitory effect on
AGT (MGMT) similar to that of O6-BG or O6-FBG.
The chemistry and in vitro biological results warrant
further in vivo evaluation of O6-[18F]FEMBG as a
new potential PET probe for AGT in cancers.
4. Experimental
4.1. General
4.3. O6-(4-Formylbenzyl)guanine (4)
To a 250 mL two-necked flask, compound 3 (1.00 g,
3.69 mmol), sodium acetate (0.11 g, 1.34 mmol), pyridi-
nium chlorochromate (PCC, 1.21 g, 5.61 mmol), and
All commercial reagents and solvents were used without
further purification unless otherwise specified. Melting

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J.-Q. Wang et al. / Bioorg. Med. Chem. 13 (2005) 5779–5786
dry pyridine (15 mL), were added. The mixture was stir-
red overnight under nitrogen. To the dark brown mix-
ture methanol (15 mL) was added. After stirring for
2 h, the solvents were removed. Methanol (60 mL) was
added to the solid residue to extract the product, then
silica gel was added to absorb the mixture and dried un-
der vacuum. The dry silica gel bearing the reaction mix-
ture was transferred to the top of a silica gel column
eluted with 30:1 MeCN/H2O to give compound 4
(0.35 g, 35%) as an off-white solid, mp > 247 ꢁC dec,
trile (10 mL), KOH (0.60 g, 10.69 mmol) was added.
The mixture was stirred at 80 ꢁC overnight. To the
cooled reaction, saturated NH4Cl solution was then
added. The reaction mixture was extracted with EtOAc.
The organic phase was washed with brine, dried over
MgSO4, and evaporated to dryness. The residue was dis-
solved in 2:1 hexane/EtOAc and subjected to column
chromatography eluted with 2:1 hexane/EtOAc to give
compound 7 (0.14 g, 66%) as a white solid, mp 90 ꢁC
1
(dec). H NMR (CDCl3, ppm) d 7.49 (s, 1H, 8-CH),
1
(lit.8 >247 ꢁC dec). H NMR (DMSO-d6, ppm) d 12.58
6.95–7.35 (m, 28H, MTr-phenyls), 6.78 (d, 2H,
J = 8.82 Hz, O6-phenyl), 6.66 (d, 2H, J = 8.82 Hz, O6-
phenyl), 5.69 (s, 1H, NH), 4.74 (s, 2H, benzyl CH2),
4.57 (d of t, 2H, J1 = 47.80 Hz, J2 = 3.68 Hz, CH2
CH2F), 4.57 (s, 2H, benzyl CH2), 3.79 (s, 3H, OCH3),
3.75 (s, 3H, OCH3), 3.68 (d of t, 2H, J1 = 29.41 Hz,
J2 = 3.68 Hz, CH2CH2F). LRMS (ESI, m/e): 273
(100%), 862 [(M+H)+, 19%]. HRMS (ESI, m/e): calcd
for C55H49FN5O4, 862.3769; found, 862.3798.
(br s, 1H, NH, D2O exchangeable), 10.00 (s, 1H,
CHO), 7.91 (d, 2H, J = 8.09 Hz, phenyl), 7.82 (s, 1H,
8-CH), 7.71 (d, 2H, J = 8.08 Hz, phenyl), 6.32 (s, 2H,
NH2, D2O exchangeable), 5.60 (s, 2H, CH2).
4.4. N2,9-Bis(p-anisyldiphenylmethyl)-O6-(4-formylben-
zyl)guanine (5)
To a 100 mL two-necked flask equipped with a condens-
er, compound 4 (0.29 g, 1.08 mmol), MTrCl (1.00 g,
4.7. O6-[4-(2-Fluoroethoxymethyl)benzyl]guanine (1)
3.23 mmol),
4-(dimethylamino)pyridine
(DMAP,
0.029 g, 0.24 mmol), DMF (25 mL), and triethylamine
(1.2 mL) were added. The solution was stirred at 50–
60 ꢁC for 4 h. The solution was transferred to a separa-
tion funnel by the aid of EtOAc, and washed twice with
brine. The organic layers were combined, dried over
MgSO4, and evaporated to dryness. The residue was dis-
solved in CH2Cl2, transferred to the top of a silica gel
column, and eluted with 3:1 hexane/EtOAc to give com-
pound 5 (0.38 g, 43%) as a white solid, mp 80 ꢁC (dec).
1H NMR (DMSO-d6, ppm) d 10.00 (s, 1H, CHO), 7.85
(d, 2H, J = 7.36 Hz, O6-phenyl), 7.57 (s, 1H, 8-CH),
6.62-7.40 (m, 30H, MTr-phenyls), 6.11 (s, 1H, NH),
4.98 (s, 2H, CH2), 3.72 (s, 3H, OCH3), 3.67 (s, 3H,
OCH3). LRMS (EI, m/e): 273 (100%), 813 (M+, 0.4%).
HRMS (EI, m/e): calcd for C53H43N5O4, 813.3315;
found 813.3314.
To a solution of compound 7 (0.14 g, 0.16 mmol) in
methanol (9 mL), 1 N HCl (2 mL) was added. The reac-
tion solution was stirred at room temperature for 20 min
and then neutralized with 1 N NaOH. Silica gel was
added to absorb the solution, dried under vacuum,
and transferred to the top of a silica gel column eluted
with 2:1 EtOAc/hexane, then 30:1 MeCN/H2O to give
compound 1 (0.047 g, 91%) as a white solid, mp 144–
1
146 ꢁC. H NMR (DMSO-d6, ppm) d 12.48 9 (s, 1H,
NH, D2O exchangeable), 7.79 (s, 1H, 8-CH), 7.48 (d,
2H, J = 5.88 Hz, phenyl), 7.35 (d, 2H, J = 5.88 Hz,
phenyl), 6.30 (s, 2H, NH2, D2O exchangeable), 5.46 (s,
2H, benzyl CH2), 4.55 (d, 2H, J = 47.06 Hz, CH2
CH2F), 4.52 (s, 2H, benzyl CH2), 3.66 (d, 2H,
J = 31.62 Hz, CH2CH2F). LRMS (CI, m/e): 318
[(M+H)+, 100%]. HRMS (CI, m/e): calcd for
C15H17FN5O2 318.1366; found 318.1359.
4.5. N2,9-Bis(p-anisyldiphenylmethyl)-O6-[4-(hydroxy-
methyl)benzyl]guanine (6)
4.8. 2-Bromoethyl triflate
To a solution of compound 5 (0.38 g, 0.46 mmol) in eth-
anol (15 mL) NaBH4 (0.045 g, 1.19 mmol) was added
over three portions at 0 ꢁC. The mixture was warmed
to room temperature in a period of 3 h. The solution
was absorbed by silica gel, which was dried in vacuum
and transferred to the top of a silica gel column. The col-
umn was eluted with 1:1 hexane/EtOAc to give com-
pound 6 (0.36 g, 96%) as a white solid, mp 110 ꢁC
(dec). 1H NMR (DMSO-d6, ppm) d 7.52 (s, 1H, 8-
CH), 6.65–7.32 (m, 32H, phenyls), 6.08 (s, 1H, NH),
5.19 (t, 1H, J = 5.88 Hz, OH), 4.82 (s, 2H, CH2), 4.47
(d, 2H, J = 5.15 Hz, CH2), 3.72 (s, 3H, OCH3), 3.68 (s,
3H, OCH3). LRMS (CI, m/e): 274 (100%), 815 (M+,
0.2%). HRMS (CI, m/e): calcd for C53H45N5O4,
815.3472; found 815.3448.
To a 100 mL two-necked flask, pyridine (1.7 mL,
21.02 mmol) and dry CH2Cl2 (20 mL) were added. The
flask was cooled in an ice–salt bath. Then trifluorome-
thane sulfonic anhydride (3.4 mL, 20.21 mmol) was add-
ed. After 5 min, 2-bromoethanol (1.42 mL, 20.03 mmol)
was added. The reaction mixture was stirred for 1.5 h,
during which time it was gradually warmed to room
temperature. The reaction mixture was then filtered,
and the solid was washed with 1:1 CH2Cl2/hexane.
The filtrate was passed through a short silica gel column
eluted with 1:1 CH2Cl2/hexane. The solvent was re-
moved, and the residue was distilled under vacuum to
give 2-bromoethyl triflate (2.44 g, 47%) as a colorless
1
oil. H NMR (CDCl3, ppm) d 4.75 (t, 2H, J = 6.62 Hz,
CH2), 3.62 (t, 2H, J = 6.62 Hz, CH2).
4.6. N2,9-Bis(p-anisyldiphenylmethyl)-O6-[4-(2-fluoroeth-
oxymethyl)benzyl]guanine (7)
4.9. O6-[4-(2-[18F]Fluoroethoxymethyl)benzyl]guanine
(O6-[18F]FEMBG, [18F]1)
To a solution of compound 6 (0.20 g, 0.25 mmol) and 1-
bromo-2-fluoroethane (0.4 mL, 5.37 mmol) in acetoni-
No-carrier-added (NCA) aqueous H18F (0.5 mL) pre-
pared by 18O(p,n)18F nuclear reaction in a RDS-112

J.-Q. Wang et al. / Bioorg. Med. Chem. 13 (2005) 5779–5786
5785
cyclotron on an enriched H218O water (95+%) target
was added to a Pyrex vessel, which contains K2CO3
(4 mg, in 0.2 mL H2O) and Kryptofix 2.2.2 (10 mg, in
0.5 mL CH3CN). Azeotropic distillation at 120 ꢁC for
15 min with HPLC grade CH3CN (3 · 1 mL) under a
nitrogen stream efficiently removed water to form anhy-
drous [18F]KF/Kryptofix 2.2.2 complex. 2-Bromoethyl
triflate (10–20 mg, dissolved in 0.5 mL CH3CN) was
introduced to this complex. The reaction mixture was
sealed and heated at 120 ꢁC for 10–15 min to produce
2-[18F]fluoroethyl bromide ([18F]FEBr) and was subse-
quently allowed to cool down and connect to another
reaction vessel containing the precursor 6 (3–5 mg, dis-
solved in 0.5 mL CH3CN), which was air-cooled at
ꢀ15 to ꢀ20 ꢁC with a Venturi cooling device powered
with 100 psi compressed air. Then, the reaction vessel
containing [18F]FEBr was heated to 130 ꢁC to distill
[18F]FEBr, under a nitrogen stream, into the solution
of precursor 6. When radioactivity reached a maximum,
the reaction mixture was heated at 120 ꢁC for 10–
15 min, and subsequently allowed to cool down. The
radiolabeling mixture was passed through a Silica Sep-
Pak cartridge. The Sep-Pak column was first eluted with
15% MeOH/CH2Cl2 (3.5 mL), and the fractions were
passed onto a rotatory evaporator. Then, the Sep-Pak
column was eluted with 2:8:90 AcOH/EtOH/H2O to re-
move the unreacted [18F]FEBr. The organic solvent in
15% MeOH/CH2Cl2 fractions was removed by evapora-
tion under high vacuum (0.1–1.0 mmHg). The residue
was acidified with 1 N HCl (0.6 mL) and heated for
10 min at 80 ꢁC to give the target tracer O6-[18F]FEM-
BG ([18F]1). The content was neutralized with 6 N
NaOH (0.1 mL), diluted with ethanol (3 mL), and evap-
orated under vacuum. The crude product was passed
through another Silica Sep-Pak cartridge with the aid
of ethanol. The Sep-Pak was eluted with EtOH to re-
move the radioactive byproducts and unreacted precur-
sor 6. The radiochemically pure product [18F]1 was
eluted from the Sep-Pak with 2 : 8 : 90 AcOH/EtOH/
H2O and adjusted pH to 5.5–7.0 by addition of a 2 M
NaOH and 150 mM NaH2PO4 mixed solution. The
solution was sterile-filtered through a 0.22 lm cellulose
acetate membrane and collected into a sterile vial. The
overall radiochemical yield of [18F]1 was 20–35%, and
the synthesis time was 60–70 min from EOB. Retention
times in the analytical HPLC system were:
tR6 = 10.15 min, tR[18F]KF = 1.88 min, tR[18F]FEBr =
2.31 min, tR[18F]7 = 13.38 min, and tR[18F]1 = 2.66 min.
Rockford, IL). Fifty micrograms of total cell protein
was then reacted with 0.2 pmol of 50-Hex-labeled
18mer oligo in the assay buffer for 2 h, at 37 ꢁC. The
reaction was terminated by two phenol:chloroform
extractions, one chloroform:isoamyl alcohol extraction,
and then ethanol-precipitated in the presence of 1 lg
carrier glycogen. Following precipitation, the precipitate
was washed with 70% ethanol, dried under vacuum, and
reacted with 3 units of PvuII (Promega, Madison, WI)
in a total volume of 20 lL for 2 h at 37 ꢁC. The reaction
was terminated by the addition of 10 lL of gel loading
buffer (96% formamide, 1 mM EDTA, pH 8) and heat-
ing at 90 ꢁC for 2 min. Samples (O6-BG, O6-FBG and
O6-FEMBG) 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 a
FMBIO II fluorescent scanner (MiraiBio, South San
Francisco, CA) and quantitated using the calculated
fluorescent absorbance. MGMT specific activity (fmol
of O6-methylguanine removed/mg of protein) was calcu-
lated according to the following equation:
ðfluorescent units of 10 base pair fragment
=fluorescent units of 18 base pair fragmentÞ
ꢂ ð200 fmol=mg of proteinÞ
4.11. Statistical analysis
O6-BG, O6-FBG, and O6-FEMBG treatments were sep-
arately compared with control treatment by StudentÕs t-
test to determine the statistical significance of differenc-
es. Differences were considered significant at p < 0.01
unless otherwise stated. All data are presented as the
mean standard deviation (SD) from at least three
independent measurements.
Acknowledgments
This work was partially supported by the Susan
G. Komen Breast Cancer Foundation grants IMG
02-1550 and BCTR 0504022, and the Indiana Genom-
ics Initiative (INGEN) of Indiana University, which is
supported in part by Lilly Endowment Inc. The refer-
eesÕ criticisms and editorÕs comments for the revision
of the manuscript are greatly appreciated.
4.10. In vitro MGMT oligonucleotide assay
References and notes
MCF-7 cells were cultured in DulbeccoÕs modified Ea-
gleÕs medium (DMEM) supplemented with 10% bovine
calf serum. Measurement of cellular MGMT specific
activity was performed by a modification19 of the assay
described by Wu et al.20 Control and drug-treated cells
were washed in cold PBS, pH 7.4 and resuspended in
the cold assay buffer (Tris–HCl, pH 8, 5% glycerol,
1 mM DTT, 1 mM EDTA). The cells were sonicated
in 400 lL of the assay buffer for 5 s on ice. The lysate
was pelleted by centrifugation for 5 min, at 12,000 · g,
at 4 ꢁC. The protein concentrations were determined
by the Pierce Coomassie plus protein assay (Pierce,
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