Synthesis of 2-amino-6-(2-[18F]fluoro-pyridine-4-ylmethoxy)-9-(octyl-β- D-glucosyl)-purine: A novel radioligand for positron emission tomography studies of the O6-methylguanine-DNA methyltransferase (MGMT) status of tumour tissue

Article abstract of DOI:10.1016/S0040-4039(02)01394-1

The synthesis of the novel glucose conjugated O⁶-methylguanine-DNA methyltransferase (MGMT) inhibitor 2-amino-6-(2-[¹⁸F]fluoro-pyridine-4-ylmethoxy)-9-(octyl- α-D-glucosyl)-purine is reported. This compound might serve as a radiotracer for the determination of the MGMT status of tumour tissue.

Full text of DOI:10.1016/S0040-4039(02)01394-1

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 6301–6304
Synthesis of 2-amino-6-(2-[18F]fluoro-pyridine-
4-ylmethoxy)-9-(octyl-b- -glucosyl)-purine: a novel radioligand
D
for positron emission tomography studies of the
O6-methylguanine-DNA methyltransferase (MGMT) status of
tumour tissue
Ralf Schirrmacher,a,* Ute Mu¨hlhausen,a Bjo¨rn Wa¨ngler,a Esther Schirrmacher,a Jost Reinhard,b
Gerd Nagel,c Bernd Kaina,c Markus Piel,a Manfred Wießlerb and Frank Ro¨scha
aInstitute of Nuclear Chemistry, Section Radiopharmaceutical Chemistry, University of Mainz, Fritz-Strassmann-Weg 2,
D-55128 Mainz, Germany
bGerman Cancer Research Center, Division of Molecular Toxicology, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany
cDivision of Applied Toxicology, Institute of Toxicology, University of Mainz, Obere Zahlbacher Str. 67, D-55099 Mainz,
Germany
Accepted 10 July 2002
Abstract—The synthesis of the novel glucose conjugated O6-methylguanine-DNA methyltransferase (MGMT) inhibitor 2-amino-
6-(2-[18F]fluoro-pyridine-4-ylmethoxy)-9-(octyl-a-
D-glucosyl)-purine is reported. This compound might serve as a radiotracer for
the determination of the MGMT status of tumour tissue. © 2002 Elsevier Science Ltd. All rights reserved.
A major disadvantage in cancer therapy is the pre-exist-
ing and acquired resistance of tumour cells against
chemotherapeutics.1 The human DNA repair protein
O6-methylguanine-DNA methyltransferase (MGMT)
plays a critical role in cancer therapy with alkylating
reagents such as alkylnitrosoureas and alkyltriazenes.
Tumour cells and neoplastic tissue with elevated
MGMT levels can be resistant to alkylating therapeu-
tics. To be able to quantify the MGMT status non-
invasively could be helpful in finding the appropriate
cancer therapy. Although potent inhibitors of MGMT
have been found and introduced into adjuvant therapy,
in all cases no selectivity for neoplastic tissue has been
achieved and all showed poor water solubility, which
limits their applications.3 Recently, two MGMT-
inhibitors were labelled with [18F]fluorine and
[
131I]iodine, however without glucosidation.4 Following
our first approach to determine the level of MGMT in
tumour tissue using 18F-labelled derivatives of O6-
methylguanine,2 the aim of the present study was to
Scheme 1.
Keywords: positron emission tomography; MGMT; 18F-fluorination.
* Corresponding author. Tel.: +49 6131 3925371; fax: +49 6131 3925253; e-mail: schirrmacher@mail.kernchemie.uni-mainz.de
0040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)01394-1

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R. Schirrmacher et al. / Tetrahedron Letters 43 (2002) 6301–6304
synthesise a new 18F-fluorinated glucose-conjugated
MGMT inhibitor for the in vivo determination of the
MGMT-status of tumour tissue with positron emission
tomography (PET). Glucosidation represents a general
strategy for improving both drug solubility and target-
ing. Thus, appropriate glucosyl derivates of MGMT
inhibitors might provide targeted depletion of MGMT
activity in neoplastic tissue using the overexpressed
glucose-transporters while the uptake in normal tissue
is minimised. The labelling procedure employs a novel
precursor, chloro-compound 5%, and dried 2.2.2. Kryp-
tofix®/[18F]fluoride complex in DMF as a solvent. As
indicated in Scheme 1, selective nucleophilic aromatic
substitution of chloride with [18F]fluoride and subse-
quent cleavage of the benzoyl-protecting groups gave
compound 7 in an overall radiochemical yield of 5%
with a specific activity of 80 GBq/mmol at the end of a
60-minute synthesis.
h (TLC control) gave only the N9-substituted product.
5 and 5% were purified by column chromatography
(aceton/n-hexane 1:1) to give chemical yields ranging
from 20 to 40%. The varying of the chemical yields
depend highly on the effectiveness of the column chro-
matography step. If the flow rate of the mobile phase is
too low, deprotection of the benzoyl groups occurs due
to the basic nature of the silica gel. Thus, a fast
separation of the coupling products via flash chro-
matography is strongly recommended. Characterisation
1
of 5 and 5% was accomplished by H, 13C, 19F NMR,
ESI MS and elemental analysis (Table 1) as well as by
gradient reverse-phase HPLC (gradient eluent water/
CH3CN, 100% CH3CN after 20 min) and normal-phase
TLC (aceton/petrolether 4:1). Additionally, the 13C
chemical shifts of C-4, C-5 and C-8 of the guanine
moieties of 5, 5% and 6 were compared with compounds
of clearly established structural identity. Chemical shifts
were expected to be in the range of 153–155 ppm for
C-4, 115–116 ppm for C-5 and 138–140 ppm for C-8.6
All glucose conjugated compounds displayed 13C chem-
ical shifts within this required range as indicated in
Table 1. Furthermore, it is well known that N7-substi-
tuted MGMT inhibitors do not show any inhibitory
activity at all6,10 whereas compound 6 which was syn-
thesised from 5 clearly showed MGMT inhibition. No
deprotection of the labelling precursor 5% was per-
formed because the benzoyl groups are a necessary
requirement for the radioactive labelling with
[18F]fluoride. Every acidic H-atom has to be protected
to prevent the formation of a hydrogen–fluorine bond
which decreases the nucleophilicity of the [18F]fluoride
atom, ultimately leading to lesser radiochemical yields.9
The final cleavage of the benzoyl protecting groups in
the case of 5 could easily be achieved quantitatively by
dissolving 5 in methanol and trituration with NaOMe
1N at room temperature for 2 h. Subsequent neutralisa-
tion with Dowex (H+ 50WX2) ion exchanger and final
The required precursor 5% as well as the non-radioactive
standard compound 6 were obtained as illustrated in
Scheme 2. The O6-substituted guanine derivatives 3 and
3% were synthesised from the trimethylammonium salt
111 and the halogeno-pyridine methanols 212 and 2%13 in
DMSO at 60°C using a new coupling method which
applied DMAP as a catalyst.5 Using this method, com-
pound 3 and 3% could be obtained in yields of 80–85%.
The 8-bromooctyltetra-O-benzoyl-b-D-glucopyranoside
4 was obtained as described by Reinhard et al., who
reported the synthesis of monosaccharide-linked
inhibitors of MGMT in general.6 Extensive molecular
modelling studies revealed that the spacer length of the
(CH2)n alkyl-linker has to be in the range n=8–12 to
obtain strongest protein–spacer and protein–glucose
interaction. The coupling of 3 or 3% and 4 to obtain 5 or
5% was achieved applying the method of Kjellberg and
Liljenberg.7 The use of an equimolar amount of LiH as
a base for the deprotonation of the N9-H-atom and
subsequent reaction with 4 in DMF at 80°C within 2–3
X' ,X
X' ,X
N
N+
O
N
OH
N
N
N
Cl-
2, 2'
N
N
H
NaH, DMAP
N
H
N
1
NH2
N
3, 3'
NH2
RO
O
Br
O
X = F
X'= Cl
R = benzoyl
LiH
RO
RO
OR
4
N
N
O
N
O
X' ,X
O
N
X' ,X
O
N
OH
OR
N
LiOH 1N
O
N
8
O
N
8
HO
HO
RO
RO
N
NH2
N
NH2
OH
OR
5, 5'
6, 6'
Scheme 2.

R. Schirrmacher et al. / Tetrahedron Letters 43 (2002) 6301–6304
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R. Schirrmacher et al. / Tetrahedron Letters 43 (2002) 6301–6304
studies of MGMT protein status in normal and disease
tissues.
workup via column chromatography (CHCl3/MeOH
4:1) gave the pure product 6 which was identified by
1H, 13C, 19F NMR, ESI MS and elemental analysis
(Table 1) as well as by gradient reverse-phase HPLC
(gradient eluent water/CH3CN, 100% CH3CN after 20
min) and normal-phase TLC (CHCl3/MeOH 4:1).
In addition, for proving the feasibility of the non-
radioactive standard compound, a MGMT assay of
compound 6 was performed to obtain the IC50 value
which provides important information about the qual-
ity of the correspondent 18F-labelled radiotracer. The
MGMT assay has been previously described in detail.8
For compound 6, an IC50 value of 1.2 mM was deter-
mined which seems to be high enough for PET.
For the synthesis of radiolabelled compound 7, a differ-
ent route was chosen to introduce the [18F]fluoride in
one step without performing subsequent synthetic pro-
cedures. No-carrier-added (NCA) aqueous [18F]fluoride
prepared by the 18O(p,n)18F nuclear reaction on an
enriched water target (95+% 18O) was added to a
solution of K2CO3 1N (15 ml)/Kryptofix® 2.2.2. (10 mg)
in a Pyrex vessel. The water was evaporated using a
stream of nitrogen at 80°C and co-evaporated to dry-
ness with CH3CN (2×1 ml). Approximately 90% of the
starting [18F]F-radioactivity could be recovered after
the drying procedure. Precursor 6% (10 mg, 0.01 mmol)
in 0.4 ml DMF was added to the dried K[18F]F/Kryp-
tofix® 2.2.2. complex and the solution was heated at
100°C for 15 min. The radiochemical yield for this
substitution of the chlorine atom by the [18F]fluorine
isotope is approximately 15%. This relatively low yield
could possibly be explained by assuming a sterical
hindrance of the chlorine-[18F]fluorine substitution via
the bulky glucose unit which is freely movable due to
the long chain alkyl linker. LiOH 1N (0.1 ml) was
added and heating was continued for 10 min leading to
a quantitative deprotection of the glucose unit. The
resulting mixture was cooled to rt, Dowex (H+ 50WX2)
ion exchanger (70 mg) was added and stirred for an
additional 3 min to ensure the complete protonation of
the glucose moiety. The solution was passed through a
PTFE-filter (1 mm) and transferred to an HPLC-system.
The radioactive product corresponding to 7 was iso-
lated by gradient reversed-phase HPLC (gradient eluent
water/CH3CN, 100% CH3CN after 20 min) in high
chemical and radiochemical purity (>99%). The synthe-
sis and purification of the radioligand for biological
investigations were accomplished within 60 min from
end of bombardment of the target and gave compound
7 in an average overall radiochemical yield of 5%. This
procedure is thus compatible with the short half-life
(110 min) of fluorine-18 and will facilitate tomographic
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