Synthesis and in vitro evaluation of 5-fluoro-6-[(2-iminopyrrolidin-1-YL) methyl]Uracil, TPI(F): An inhibitor of human thymidine phosphorylase (TP)

Article abstract of DOI:10.1080/15257770903451603

An investigation was conducted to determine if the 5-fluoro analog of TPI (5-chloro-6-[(2-iminopyrrolidin-1-yl)methyl]uracil), a potent inhibitor of human thymidine phosphorylase (TP), has an IC50 in a range that might allow to use it labeled for imaging of TP expression in vivo. The previously unreported fluoro analog, TPI(F), was prepared and tested against TPI and TPI(Br) using an inhibition assay of [H-3]thymidine cleavage. An assay, performed in the presence of 0.4 mg/ml of human TP, yielded IC50 values of 2.5 nM, 2.7 nM, and 9.0 nM for TPI, TPI(Br), and TPI(F), respectively. The results indicate that further studies to develop 18F-labeled TPI(F) as a potential radiopharmaceutical for PET imaging of TP expression in vivo are warranted.

Full text of DOI:10.1080/15257770903451603

Nucleosides, Nucleotides and Nucleic Acids, 29:49–54, 2010
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770903451603
SYNTHESIS AND IN VITRO EVALUATION OF
5-FLUORO-6-[(2-IMINOPYRROLIDIN-1-YL)METHYL]URACIL, TPI(F):
AN INHIBITOR OF HUMAN THYMIDINE PHOSPHORYLASE (TP)
John R. Grierson, J. Scott Brockenbrough, Janet S. Rasey, Linda Wiens, and
Hubert Vesselle
Department of Radiology, Division of Nuclear Medicine, University of Washington, Seattle,
Washington, USA
An investigation was conducted to determine if the 5-fluoro analog of TPI (5-chloro-6-[(2-
2
iminopyrrolidin-1-yl)methyl]uracil), a potent inhibitor of human thymidine phosphorylase (TP),
has an IC50 in a range that might allow to use it labeled for imaging of TP expression in vivo. The
previously unreported fluoro analog, TPI(F), was prepared and tested against TPI and TPI(Br)
using an inhibition assay of [H−3]thymidine cleavage. An assay, performed in the presence of 0.4
mg/ml of human TP, yielded IC50 values of 2.5 nM, 2.7 nM, and 9.0 nM for TPI, TPI(Br),
and TPI(F), respectively. The results indicate that further studies to develop 18F-labeled TPI(F) as a
potential radiopharmaceutical for PET imaging of TP expression in vivo are warranted.
Keywords Human thymidine phosphorylase; TP; transition state inhibitor analog; TPI;
TPI(F)
INTRODUCTION
Thymidine phosphorylase (TP) is an essential enzyme involved in
endogenous nucleotide salvage.[1,2] It specifically cleaves the glycosidic
bond in thymidine to produce thymine and 2-deoxyribose-1α-phosphate
(2dR−1P; Scheme 1). The reaction is non-energy dependent, reversible
under physiologic conditions, and strongly coupled to thymine degradation.
As a result, TP generally functions to clear thymidine from blood, which
keeps plasma concentrations low. However, there is growing interest in the
potential role TP plays in tumor biology since it was discovered that 2dR-1P
Received 9 February 2009; accepted 29 October 2009.
This work was supported by NIH grant ROI CA 115559.
Address correspondence to John R. Grierson, Department of Radiology, University of Washington,
UW Medical Center Room RR215-Box 357115, 1959 NE Pacific Street, Seattle, WA, 98195, USA. E-mail:
grierson@u.washington.edu
49

50
J. R. Grierson et al.
O
N
Me
NH
O
HO
HO
TP
Me
O
O
NH
O
P
O-
+
Pi
+
O
O
O-
N
H
O
OH
OH
Thymine
SCHEME 1 Equation of reversible TP catalysis with thymidine as the substrate.
2dR-1P
Thymidine
(or 2dR) promotes neovacularization, in vitro and in experimental tumor
models.[3–8]
Our research group’s goal is to investigate tumor biology, particularly
lung cancer, using PET imaging and to actively develop novel agents for
that purpose. Recently, we reported a radiosynthesis and in vitro charac-
terization of 5ꢁ-deoxy-5ꢁ-[18F]fluorothymidine (DFT; Figure 1) as a targeted
radiopharmaceutical for imaging TP in vivo.[9] This fluorinated thymidine
analog is a TP substrate and was actively metabolized to 2,5-dideoxy-5-
[18F]fluororibose-1α-phosphate (ddFR-1P) in cells. We hypothesized that
labeled ddFR-1P would be trapped in cells due to its phosphate charge and,
consequently, accumulate in proportion to intracellular TP activity. Unfortu-
nately, ddFR-1P proved too unstable to be useful as a PET imaging agent.
The instability of ddFR-1P prompted us to consider an alternative
strategy that directly targeted TP with a potent 18F-labeled enzyme inhibitor.
That goal required a 18F-labeled compound with exceptional binding
potency, since the imaging agent and target would have a 1:1 stoichiometry
and there would be no metabolic turnover to feed a labeled metabolite
pool. Fortunately, a TP inhibitor, TPI (5-chloro-6-[(2-iminopyrrolidin-1-
yl)methyl]uracil; Figure 1), has been previously shown to have an IC50 of
35 nM.[10] Moreover, TPI is a weak inhibitor of the related enzyme uridine
phosphorylase (IC50 > 1000 nM).[10]
O
N
O
O
Me
X
N
F
NH
NH
NH
18F
O
N
H
O
N
H
O
O
N
HN
OH
N
TPI:
TPI(Br): X = Br
X = F
TPI(F)-(IM)
DFT
X = Cl
TPI(F):
FIGURE 1 Structures of 5ꢁ-deoxy-5ꢁ-[18F]fluorothymidine (DFT) and TP inhibitors designated as TPI,
TPI(Br), TPI(F), and TPI(F)-(IM).

Synthesis and TP Inhibition of TPI(F)
51
The high TPI inhibitory binding with TP is similar to that found in other
receptor-binding 18F-labeled molecules that have been successfully imaged
in vivo,[11,12] so a fluorinated analog was of interest. Replacement of the
chlorine atom in TPI with a fluorine atom was obvious, but the critical
question was whether that analog retained the high inhibitory potency of
TPI. Reported herein is a preliminary investigation that focused on the
synthesis of 5-fluoro-6-[(2-iminopyrrolidin-1-yl)methyl]uracil (TFI(F)) and
determination of its IC50 value with TP. The goal was to determine if TPI(F)
had adequate potency to justify its fluorine-18 labeling and evaluation as an
imaging agent of TP expression in vivo.
RESULTS AND DISCUSSION
TPI(F) was prepared by alkylating 2−iminopyrrolidine with
6-chloromethyl-5-fluorouracil[13,14] in methanol, promoted by 1,8-
diazabicyclo[5.4.0]undec-7-ene.[15] TPI and TPI(Br) were also prepared
to directly compare the relative 50% inhibitory values (IC50) with
human TP. TPI(F) was an obvious choice for a fluorinated TPI analog,
given that a fluorine atom represents minimal structural modification
of the parent drug. A recent synthesis and evaluation of 5-fluoro-6-
[(1H-imidazol-1-yl)methyl]uracil (TPI(F)-(IM) (Figure 1) supports that
view.[13,14] However, in that case, the additional impact of the imidazole
ring, as the pendent amine moiety, was unclear.
The relative potencies (IC50 values) for TPI and TPI(Br) and TPI(F)
were determined to be 2.5 nM and 2.7 nM, and 9.0 nM, respectively. The
inhibition curves are shown in Figure 2. However, our values for TPI and
TPI(Br) are lower than previously reported.[10] This may be attributed to
the specific enzyme concentrations used in the respective enzyme assays.
Regardless, the trend for TPI, TPI(Br), and TPI(F) is clear. Fluorination,
alone, does not improve potency. Nevertheless, the potency of TPI(F) still
falls within the range that is typical of many PET radiotracer ligands. Thus,
radiolabeling of TPI(F) with high specific activity [18F]fluoride ion (1–2
Ci/µmol) and evaluation of its potential for imaging regional TP expression
in vivo is warranted.
EXPERIMENTAL
All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA)
1
and used as received. NMR spectra: H (300 MHz, δ, TMS); 13C (75 MHz,
δ, TMS); 19F (282 MHz, δ, CCl3F), were recorded using a Bruker AV301
multinuclear instrument. Mass spectrometry (MS) was performed using
a Micromass Quattro Premier XE instrument and electrospray ionization

52
J. R. Grierson et al.
FIGURE 2 Inhibition of TP activity in the presence of various concentrations of TPI(F), TPI(Br), and
TPI. The IC50 value for each compound was extrapolated from its corresponding concentration curve
yielding values of 2.5 nM, 2.7 nM, and 9.0 nM for TPI, TPI(Br), and TPI(F), respectively. Values represent
the means with standard deviations of three separate experiments.
(ES). Characteristic data is given for selected synthetic intermediates and
products, which were not available.
(A) Chemical Syntheses
i. 5-Fluoro-6-chloromethyluracil was prepared according to methods re-
ported by Lai and Kalman et al.[13,14] and used without purification
(MS-ES− (M-H) m/z 177, 179) for the synthesis of TPI(F). Its synthesis
involves the preparation of several key intermediates: (a) 2,4-Difluoro-3-
oxo-butyric acid ethyl ester was prepared according to McBee et al.[16] 1H
NMR (CDCl3) 1.35 (t, J = 7.5 Hz, 3H), 4.34 (q, J = 7.05 Hz, 2H), 5.23
(d, JHF = 44 Hz, 2H), 5.49 (d, JHF = 47 Hz, 1H); 13C NMR (CDCl3)
13.9, 63.2, 83.2 (dd, JCF = 184 Hz, JCF = 3 Hz), 89.6 (dd, JCF = 196
ꢁ
ꢁ
Hz, J CF = 1.5 Hz), 162.2 (d, JCF = 23 Hz), 195.3 (dd, JCF = 18 Hz,
JꢁCF = 22 Hz); 19F NMR (CDCl3) −204.4 (JFH = 47.4 Hz), -236.7 (JFH
=
44.3 Hz); (b) 5-Fluoro-6-(fluoromethyl)-2-(methylthio)pyrimidin-4(1H)-
one was prepared according to Duschinsky et al.[17]: m.p. 222–223◦C;
MS-ES− (M-H) m/z 191; 1H NMR (d6-DMSO) 2.50 (s, 3H), 5.31 (dd, JCF
= 48 Hz, JꢁCF = 3 Hz, 1H), <10.0 (amide not observed); 19F NMR (D2O)
−157.4, −222.4 (td, JFH = 48 Hz, JFF = 8.5 Hz).
ii. 2-Iminopyrrolidine hydrochloride was prepared by reported
methods[18,19] and recrystallized from anhydrous EtOH to afford
a hygroscopic, granular, white solid: m.p. 172.4–172.8◦C; mass
spectrometry (ESI+ (M+H), m/z 85).

Synthesis and TP Inhibition of TPI(F)
53
iii. TPI(F)-hydrochloride was prepared by adaptation of the TPI synthesis
of Yano et al.[15] to obtain a white crystalline hydrochloride salt that
1
exhibited: m.p. 240◦C–243◦C (dec); MS-ES− (M-H), m/z 225; H-NMR
(D2O) 2.11 (pentuplet, J = 7.7 Hz, 2H), 2.93 (t, J = 8.0 Hz, 2H), 3.68 (t,
J = 7.3 Hz, 2H), 4.55 (s, 2H), 4.7 (HOD); 19F-NMR (D2O) -169.42 (t, JFH
= 2.5 Hz);
iv. TPI-hydrochloride and TPI(Br)-hydrochloride were prepared, as de-
scribed by Yano[15] and were characterized by mass spectrometry and 1H-
NMR. The 1H NMR spectra of TPI, TPI(Br) and TPI(F) were essentially
identical.
(B) Determination of IC50 for TPI(F) with [H-3]Thymidine and
Recombinant Human TP
Thymidine phosphorylase activity was assayed in vitro using a modifica-
tion of previously described methods.[20] An amount of 100 µl aliquots of
0.4 mg/ml human recombinant thymidine phosphorylase (Sigma-Aldrich
Catalog No. T9319) in 0.5% bovine serum albumin were placed on ice.
At time zero minutes the reaction was initiated by addition of 300 µl of
assay buffer (4◦C) containing 76 mM sodium phosphate, pH 7.4 and 1.6
mM [3H-5ꢁ]-thymidine (Moravek Radiochemicals Catalog No. MT-846W;
Moravek, Brea, CA, USA) plus or minus the appropriate concentrations
of TPI compounds, serially diluted in double distilled water. The mixture
was vortexed and placed in a 37◦C gently shaking water bath. Time zero
minute controls were terminated immediately without being removed from
ice. After 30 minutes at 37◦C, reactions were terminated by the addition of
400 µl of a 4◦C slurry of 5% charcoal (Sigma-Aldrich Catalog No. 242276,
“Darco G-60”) in 10% trichloroacetic acid. The terminated mixture was
centrifuged (10,000 × g/10 minutes/room temperature) and 0.4 ml of
the supernate was counted in 5 ml of EcoScint A (National Diagnostics,
Atlanta, GA, USA) in a Beckman LS5000C liquid scintillation counter. For
an individual experiment, values for each condition were determined from
the mean of two separate determinations after subtracting the time zero
control values. IC50 values were extrapolated from the averaged data from
three separate experiments and represent the concentration of inhibitor
that inhibited TP activity by 50%.
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