Synthesis, 18F-radiolabelling and biological evaluations of C-6 alkylated pyrimidine nucleoside analogues

Article abstract of DOI:10.1081/NCN-200033914

Synthesis of pyrimidine derivatives with a side-chain attached to the C-6 of pyrimidine ring (6-14) is reported. Target compounds 8 and 12 were subjected to in vitro phosphorylation tests, determination of their binding affinities to herpes simplex virus

Full text of DOI:10.1081/NCN-200033914

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Synthesis, 18F‐Radiolabelling and Biological Evaluations
of C‐6 Alkylated Pyrimidine Nucleoside Analogues
a
Dr. Silvana Raić‐Malić , Anass Johayem b , Simon M. Ametamey b , Sanja Batinac a , Erik De
Clercq c , Gerd Folkers d & Leonardo Scapozza d
a Department of Organic Chemistry, Faculty of Chemical Engineering and Technology ,
University of Zagreb , Zagreb, HR‐10000, Croatia
b Center for Radiopharmaceutical Science of ETH, PSI and USZ , Villigen PSI, 5232,
Switzerland
c Rega Institute for Medical Research , Katholieke Universiteit Leuven , Leuven,
Minderbroedersstraat 10, Leuven, B‐3000, Belgium
d Department of Chemistry and Applied Biosciences ETH , Institute of Pharmaceutical
Sciences , Winterthurerstr. 190, Zürich, CH‐8057, Switzerland
Published online: 01 Jun 2007.
To cite this article: Dr. Silvana Raić‐Malić , Anass Johayem , Simon M. Ametamey , Sanja Batinac , Erik De Clercq , Gerd
Folkers & Leonardo Scapozza (2004) Synthesis, 18F‐Radiolabelling and Biological Evaluations of C‐6 Alkylated Pyrimidine
Nucleoside Analogues, Nucleosides, Nucleotides and Nucleic Acids, 23:11, 1707-1721, DOI: 10.1081/NCN-200033914
To link to this article: http://dx.doi.org/10.1081/NCN-200033914
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 23, No. 11, pp. 1707–1721, 2004
18
Synthesis, F-Radiolabelling and Biological Evaluations of
C-6 Alkylated Pyrimidine Nucleoside Analogues
1,
2
2
*
´
´
Silvana Raic-Malic, Anass Johayem, Simon M. Ametamey,
Sanja Batinac,1 Erik De Clercq,3 Gerd Folkers,4
and Leonardo Scapozza4
1Department of Organic Chemistry, Faculty of Chemical Engineering
and Technology, University of Zagreb, Zagreb, Croatia
2Center for Radiopharmaceutical Science of ETH, PSI and
USZ, Villigen PSI, Switzerland
3Rega Institute for Medical Research, Katholieke Universiteit Leuven,
Leuven, Belgium
4Department of Chemistry and Applied Biosciences ETH, Institute of
Pharmaceutical Sciences, Zu¨rich, Switzerland
ABSTRACT
Synthesis of pyrimidine derivatives with a side-chain attached to the C-6 of
pyrimidine ring (6–14) is reported. Target compounds 8 and 12 were subjected to in
vitro phosphorylation tests, determination of their binding affinities to herpes simplex
virus (HSV-1) thymidine kinase (TK) and catalytic turnover constants. Fluorinated
pyrimidine derivative 12 (40 mM) exhibited better binding affinity for HSV-1 TK than
acyclovir (ACV, 170 mM) and ganciclovir (GCV, 48 mM). Catalytic turnover constant
(kcat) of 12 (0.08 sÀ1) was close to the kcat values of ACV (0.10 sÀ1) and GCV
(0.10 sÀ1). Furthermore, compounds 8 and 12 showed no cytotoxic effects in HSV-1
TK-transduced and non-transduced cell lines. Besides, compounds 8 and 12 did not
exhibit antiviral or cytostatic activities against several viruses and malignant tumor
*
´ ´
Correspondence: Dr. Silvana Raic-Malic, Department of Organic Chemistry, Faculty of Chemical
Engineering and Technology, University of Zagreb, Zagreb HR-10000, Croatia; Fax: +385-1-
4597-250; E-mail: silvana.raic@fkit.hr.
1707
DOI: 10.1081/NCN-200033914
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com
Copyright D 2004 by Marcel Dekker, Inc.

´ ´
Raic-Malic et al.
1708
cell lines that were evaluated. The new fluorinated pyrimidine derivative 16 that is
phosphorylated by HSV-1 TK could be developed as non-toxic PET-tracer molecule.
Thus, 18F labelling of the precursor 14 was performed by nucleophilic substitution
using [18F] tetrabutylammonium fluoride as the fluorinating reagent.
Key Words: Pyrimidine nucleoside analogues; 18F-radiolabelling; HSV-1 TK;
Phosphorylation; Binding affinity.
INTRODUCTION
Acyclic nucleoside analogues are known to be potent anti-herpesvirus agents.[1]
Among these agents, acyclovir and ganciclovir were reported to be efficient antiviral
agents with low host toxicity,[2,3] ganciclovir being the more active against herpes
simplex virus type 1 (HSV-1) and 2 (HSV-2).[4,5] Acyclovir and ganciclovir are quite
selective as compared to the other nucleoside antiviral agents.[2] The selectivity of these
acyclic nucleoside analogues is due, in part, to the fact that they are phosphorylated
only in virus-infected cells, where a virus specific thymidine kinase of low substrate
specificity converts the nucleoside analogues to their monophosphate derivatives.[5] The
monophosphates are converted to diphosphates and then to the corresponding
triphosphates by cellular enzymes.[4] The triphosphates prevent viral replication by
inhibition of the viral DNA polymerase. Fluoro derivatives of ganciclovir and
penciclovir, i.e. 9-[(3-fluoro-1-hydroxy-2-propoxy)methyl]guanine and 9-(4-fluoro-3-
hydroxymethylbutyl)guanine were evaluated as tracers for non-invasive positron
emission tomography (PET) imaging of herpes simplex virus type 1 thymidine kinase
(HSV-1 TK) gene expression.[6–9]
Previous studies have shown that C-6 alkylated pyrimidine derivatives, i.e.
6-hydroxypropylthymine[10] and 6-(3-hydroxy-2-hydroxymethylpropyl)thymine, have
good binding affinities for HSV-1 TK and no cytotoxic effects. Furthermore, the crystal
˚
structures of these two compounds in complex with HSV-1 TK (refined to 2.2 A) were
solved and confirmed to their excellent substrate-binding affinities.[11] These findings
led us to synthesize new C-6 alkylated pyrimidine derivatives which might find
application as new non-toxic PET-tracer molecules that are specifically and efficiently
phosphorylated by the HSV-1 TK.
In this paper, we describe the synthesis of novel pyrimidine derivatives containing
2,3-dihydroxypropyl (8) and 2-fluoro-3-hydroxypropyl (12) side-chains attached to C-6
of the pyrimidine moiety, the evaluation of their cytotoxic properties, binding affinities
to HSV-1 TK and kinetic turnover constants. Furthermore, we report the labelling of
the nucleoside precursors with protected functional groups in the side-chain with 18F.
RESULTS AND DISCUSSION
Chemistry
The pyrimidine derivatives containing an acyclic side-chain attached to the C-6 of
the pyrimidine ring (6–14) were synthesized (Schemes 1 and 2).

C-6 Alkylated Pyrimidine Nucleoside Analogues
1709
Scheme 1. Reagents and conditions: (i) POCl3, reflux; (ii) NaOCH3, MeOH, r.t.; (iii) n-BuLi,
THF; (iv) –50°C, acetic acid; (v) AcCl, H2O; (vi) 1. BCl3, CH2Cl2, 2. NaHCO3; (vii) BCl3, CH2Cl2.
Chlorination of 2,4-dihydroxy-5,6-dimethylpyrimidine (1) with phosphoryl chloride
afforded 2,4-dichloro-5,6-dimethylpyrimidine (2). Compound 2 was then treated with
sodium methoxide in methanol to give 2,4-dimethoxy-5,6-dimethylpyrimidine (3)
according to the procedure described in the literature.[12]
The key intermediate, 6-(3-benzyloxy-2-hydroxypropyl)-2,4-dimethoxy-5-methyl-
pyrimidine (6) was prepared via an addition reaction of the lithiated 2,4-dimethoxy-5,

´ ´
Raic-Malic et al.
1710
Scheme 2. Reagents and conditions: (i) DAST, CH2Cl2; (ii) AcCl, H2O; (iii) BCl3, CH2Cl2.
6-dimethylpyrimidine (4) with benzyloxyacetaldehyde (5),[13] according to an analogous
procedure described in the literature.[14] Attempts to hydrolyze the 2- and 4-methoxy
groups of 6 in methanolic hydrogen chloride or a mixture of aqueous sodium hydroxide
were unsuccessful. However, treatment of 6 with acetyl chloride, containing several drops
of water, effected a conversion to the 2,4-dihydroxypyrimidine derivative 7. Debenzy-
lation of 7 was achieved by using boron trichloride in dichloromethane, while
deacetylation was accomplished with sodium hydrogen carbonate to produce 6-(2,
3-dihydroxypropyl)pyrimidine-2,4-dione (8, Scheme 1).
Fluorination: Compound 10 was obtained by the fluorination of 6 using
diethylaminosulfur trifluoride (DAST) as the fluorinating reagent (Scheme 2).[15]
Deprotection of the methoxy groups in 10 and, in the next step, the benzyloxy
group in 11 were carried out with acetyl chloride and boron trichloride, respectively, to
give the target compound 12 (Scheme 2).
Scheme 3. Reagents and conditions: (i) AcCl/CH2Cl2; (ii) CH3SO2Cl/pyridine; (iii) [18F]Bu4NF/
DMSO; (iv) HCl, H2O.

C-6 Alkylated Pyrimidine Nucleoside Analogues
Radiochemistry
1711
Preparation of the precursor 14 for 18F labelling is outlined in Scheme 3. To avoid
intramolecular cyclization and formation of conformationally constrained carbon-bridged
pyrimidine nucleosides,[16] a precursor with protected carbonyl and hydroxyl groups
was synthesized.
The primary hydroxyl group in 9 was protected with an acetyl group (13), while the
secondary hydroxyl group was converted to the mesylate 14. The radiosynthesis of the
18F-labelled compound, 18F-HHT was performed via a two-step reaction sequence. The
first step, a classical nucleophilic substitution on a mesyl leaving, was accomplished
using [18F]-tetrabutylammonium fluoride as the fluorinating agent in DMSO. The second
step involving the removal of the protecting groups was achieved byhydrolysis with
aqueous HCl. The radiochemical yield was rather low and did not exceed 1%. A
probable reason might be the instability of the precursor under basic conditions.
The structures of the compounds 2, 3, and 6–14 were confirmed by analyses of
1
1
their H and 13C NMR spectra. The H NMR spectroscopic data of the 2, 3, and 6–14
are displayed in Table 1.
The chemical shifts and coupling constants are consistent with the proposed
structures. The 13C NMR data are given in the Experimental Section. Presence of the
fluorine atom in 10–12 was confirmed in 13C NMR spectra which show characteristic
fluorine-carbon interactions with one-bond (at C-2’) and two-bonds (at C-1’ and C-3’)
coupling constants of ca. 175 Hz and ca. 22 Hz, respectively.[17] The chemical shift
assignment of novel compounds agrees with these for the related acyclic pyrimidine
nucleoside analogues.[15,16]
Biological Evaluations of Compound 8 and Its
Fluorinated Derivative 12
Phosphorylation Pattern
To explore the substrate acceptance of HSV-1 TK, new compounds with a 2,
3-dihydroxypropyl (8) or 2-fluoro-3-hydroxypropyl side-chain (12) were subjected to
in vitro phosphorylation tests and determination of binding affinity with HSV-1 TK.
Phosphorylation of compounds 8 and 12 was monitored by the formation of
their monophosphorylated derivatives using DAD (Diode Array Detector) HPLC.
Comparison of the phosphorylation patterns of compound 8 with HSV-1 TK and
without HSV-1 TK showed, besides ATP [retention time (RT)=16.9 min] and 8
(RT=4.8 min), the formation of two new compounds, the ADP and monophosphate
derivative of 8 (Fig. 1). The decrease of the peak intensity of compound 8, while
phosphorylation occurred, was also observed. Similar to that, monitoring of phos-
phorylation of 12 revealed the formation of ADP (RT=5.4 min) and a decrease of
the peak intensity for compound 12 (RT=7.4 min, Fig. 1). In the chromatogram, peak
of monophosphorylated compound 12 together with ATP peak had retention time of
12.7 min.
The results of the analyses clearly showed the formation of new peaks cor-
responding to the monophosphate derivatives of 8 (RT=7.1 min) and 12 (RT=12.9 min).
Thus, this suggests that compounds 8 and 12 are indeed substrates for HSV-1 TK.

´ ´
Raic-Malic et al.
1712

C-6 Alkylated Pyrimidine Nucleoside Analogues
1713
Figure 1. Chromatogram analyses of 8 (a) and 12 (b) with retention times (RT).
Binding Affinity Assays
To evaluate the binding affinities of compounds 8 and 12, competition studies were
performed using radioactively labelled thymidine and the compounds 8 and 12 as
competitive ligands. Initial velocity (Vi) was determined by monitoring the increase of
radioactively labelled dTMP over time in the presence of various concentrations of the
compounds. The affinities of 8 and 12 were calculated as Ki values (Table 2).
Comparison of the binding affinities for 8 and 12 showed that the 2-fluorinated
pyrimidine derivative 12 had a better affinity (Ki=39.54 mM) for the HSV-1 TK than its
dihydroxy compound 8 (Ki=62.35 mM). It is interesting to note that 12 exhibited better
affinity for HSV-1 TK than acyclovir (ACV) and ganciclovir (GCV) (Table 2).
A Spectrophotometric Assay for Quantitative
Determination of kcat of HSV-1 TK
The transfer of the g-phosphate of ATP to the substrate, as catalysed by HSV-1 TK
represents the first and limiting step of coupled reaction. The last step of the cascade,

´ ´
Raic-Malic et al.
1714
Table 2. Binding affinities (Ki) and catalytic turnover constants (kcat) of the compounds 8 and 12
with HSV-1 TK.
Compd.
Ki [mM]
kcat [sÀ1
]
Specificitya
8
62.35 5.6
39.54 3.5
0.04 0.01
0.08 0.01
0.35
0.00036
0.00115
1
12
dT[18,19]
ACV[18,19]
GCV[18,20]
PCV[21,22]
0.2
170
48
0.10
0.10
0.00034
0.0012
0.025
1.5
0.045
aThe specificity is defined as [kcat(ligand)/Ki(ligand)/ kcat(dT)/KM(dT)].
namely the consumption of the cofactor NADH by L-lactate dehydrogenase and formation
of NAD+, corresponds in an equimolar way to the initial phosphorylation of the HSV-1
TK substrate and leads to a decrease of absorbance at 340 nm. This method has been
applied and further developed in a previous study[22] to characterize the catalytic
turnover constant (kcat) of several substrates of HSV-1 TK. This simple in vitro method
is based on the correlation between the kcat and the decrease of absorbance at 340 nm per
mg of HSV-1 TK (Dabs340nmminÀ1 mgÀ1). Deoxythymidine (dT), acyclovir (ACV),
ganciclovir (GCV) and penciclovir (PCV) were chosen as reference compounds because
their kcat values have been reported in the literature. The following kcat values were used
for dT, ACV, GCV and PCV: 0.35 sÀ1, 0.10 sÀ1, 0.10 sÀ1 and 0.045 sÀ1, respectively.
The Dabs340nmminÀ1 mgÀ1 values obtained for 8 and 12 were introduced in the
calibration curve equation and their kcat were calculated as described in the literature
(Table 2).[21] The presence of compounds 8 and 12 induced a clear decrease in
absorption, indicating that both compounds are substrates for HSV-1 TK.
The calculated kcat value for 8 was 0.04 sÀ1 (Table 2). For the 2-fluorinated
compound 12, kcat value was 0.08 sÀ1, which was close to that of ACV and GCV
(0.10 sÀ1) and higher than that for PCV (0.045 sÀ1). The specificity of HSV-1 TK for
the fluorinated derivative 12 was three-fold higher than that for 8. Furthermore, 12 and
GCV had almost the same specificity to HSV-1 TK, while comparison of 12 with ACV
showed that HSV-1 TK was three times more specific for 12 than for ACV.
Antiviral and Antitumor Evaluations
The compounds 6, 8, 9, 11 and 12 were evaluated against HSV-1 (strain KOS),
HSV-1 (TK-deficient strain KOS), HSV-2 (strain G), vaccinia virus and vesicular
stomatitis virus. The compounds were also evaluated for their cytostatic activity
against malignant tumor cell lines: murine leukemia (L1210) and human T-lymphocyte
(Molt4/C8 and CEM) cells. No specific antiviral or antitumor activities were observed
against any of the evaluated viruses or tumor cell lines. Target compounds 8 and 12 did
not exhibit any cytotoxic effects (minimum cytotoxic concentration > 400 mg/ml).
Functional Assay
Cytotoxicity assays in HSV-1 TK-transduced and non-transduced osteosarcoma
(MG63) and non-small lung cancer cells (H122 NSCLC) showed that both compounds
8 and 12 were not cytotoxic at the tested concentration of 200 mg/ml.

C-6 Alkylated Pyrimidine Nucleoside Analogues
CONCLUSIONS
1715
Novel pyrimidine derivatives with a 2,3-dihydroxypropyl (8) or 2-fluoro-3-
hydroxypropyl (12) side-chain were synthesized. The key intermediate 6 for the preparation
of 8 and 12 was obtained by introduction of the C-6 side-chain of pyrimidine ring via an
addition reaction of the lithiated pyrimidine derivative (4) with benzyoxyacetaldehyde (5).
To explore the substrate acceptance of HSV-1 TK, compounds 8 and 12 were
subjected to in vitro kinetic evaluations and determination of catalytic turnover
constants. Comparison of binding affinities of fluorinated pyrimidine derivative 12 and
ACV and GCV showed that 12 had an affinity of 40 mM which is better than that
of ACV (170 mM) and GCV (48 mM). The catalytic turnover constant (kcat) of 12
(0.08 sÀ1) was close to those of ACV (0.10 sÀ1) and GCV (0.10 sÀ1). Compounds 8 and
12 did not exhibit any cytotoxic effects (minimum cytotoxic concentration > 400 mg/
ml). No specific antiviral or antitumor activities were observed against any of the
evaluated viruses or tumor cell lines.
The non-toxic (unlabelled) fluorinated compound 12 can be efficiently phosphor-
ylated by HSV-1 TK and thus fulfils the necessary requirements for application as a
tracer molecule for non-invasive TK imaging with PET. 18F labelling of the precursor
14 was performed, although the radiochemical yield of 18F incorporation into 14 was
very low.
EXPERIMENTAL SECTION
General Methods
Melting points were determined with Kofler micro hot-stage (Reichert, Wien) and
are uncorrected. Precoated Merck silica gel 60F-254 plates were used for thin layer
chromatography (TLC) and the spots were detected under UV light (254 nm). Column
chromatography (CLC) was performed using silica gel (0.063–0.2 mm, Merck); glass
column was slurry-packed under gravity. The electron impact mass spectra were
recorded with a Fisous Trio-2000 spectrometer with ionising energy 70 eV at the Paul
Scherrer Institute. Elemental analyses were performed in the Central Analytic Service,
1
Ruðer Bosˇkovi Institute, Zagreb. H and 13C NMR spectra were recorded on a Bruker
AMX-300, operating at 75.46 MHz for the 13C resonance. The samples were dissolved in
1
DMSO-d6 and measured in 5 mm NMR tubes. The H and 13C NMR chemical shift
values (d) are expressed in ppm referred to TMS and coupling constants (J) in Hz.
2,4-Dichloro-5,6-dimethylpyrimidine (2).[12] The mixture of 2,4-dihydroxy-5,
6-dimethylpyrimidine (1) (5 g, 3.57 mmol) and POCl3 (10 ml, 109.24 mmol) was heated
under reflux for 1 h. Excess of POCl3 was then removed under reduced pressure and
the residue was added to ice, washed with ether and dried over sodium sulphate. The
crude product was recrystallized from ethanol to give 2 (3.883 g, 60.2 %, mp 70–71°C);
MS m/z 177 (M+); 13C NMR (DMSO) d 155.24 (C-2), 171.48 (C-3), 127.74 (C-5), 160.54
(C-6), 22.83 (CH3), 14.40 (CH3).
2,4-Dimethoxy-5,6-dimethylpyrimidine (3).[12] To a solution of sodium (571 mg,
24.83 mmol) in methanol (14 ml) was added 2 (2 g, 11.17 mmol). The reaction mixture
was refluxed for 3 h. The solvent was evaporated and water was added to dissolve NaCl.

´ ´
Raic-Malic et al.
1716
The oily layer was extracted with dichloromethane, dried over sodium sulphate and
concentrated under reduced pressure. The residue was kept in refrigerator and gave
colourless crystals of 3 (1.748 g, 93.6 %, mp 39–40°C); MS m/z 169 (M+); 13C NMR
(DMSO) d 162.10 (C-2), 168.67 (C-3), 107.25 (C-5), 165.67 (C-6), 53.96 (OCH3), 53.74
(OCH3), 21.46 (CH3), 9.67 (CH3).
6-(3-Benzyloxy-2-hydroxypropyl)-2,4-dimethoxy-5-methylpyrimidine (6). N-
Butyllithium (1.6 M, 2.2 ml, 3.5 mmol) was added dropwise to a solution of 3
(500 mg, 2.94 mmol) in anhydrous tetrahydrofuran (7 ml) at À70°C under argon
atmosphere. The temperature was warmed to À55°C and the solution was stirred for
30 min. Benzyloxyacetaldehyde (5)[13] (530 mg, 3.53 mmol) dissolved in tetrahydro-
furan was added dropwise to the solution and the stirring was continued for 2 h. The
solution was neutralized by the addition of acetic acid and the temperature was raised
to room temperature. The solvent was removed under reduced pressure and the residue
was partitioned between dichloromethane and water. Organic layer was dried and
the solvent was removed. The crude product was purified by column chromatography
(n-hexane:ethyl acetate - 4:1) providing oily compound 6 (593 mg, 63 %); MS m/z 318
(M+); 13C NMR (DMSO) d 162.10 (C-2), 169.02 (C-4), 108.39 (C-5), 166.58 (C-6),
138.51–127.36 (C6H5), 53.97 (OCH3), 53.82 (OCH3), 38.54 (C-1’), 68.83 (C-2’), 74.08
(C-3’), 72.21 (CH2Ph), 9.79 (CH3). Anal. Calcd for C17H22N2O4: C, 64.13; H 6.97; N,
8.80. Found: C, 63.96; H 6.98; N, 8.77.
6-(2-Acetoxy-3-benzyloxypropyl)-5-methyl-(1H,3H)-pyrimidine-2,4-dione (7).
A solution of 6 (354 mg) in acetyl chloride (5 ml) was refluxed for 10 h. Few
drops of water were added and the mixture was stirred at room temperature overnight.
The solution was evaporated under reduced pressure. The residue was applied to
column chromatography (CH2Cl2:MeOH - 30:1) to give 7 (193 mg, 52 %); MS m/z
332 (M+); 13C NMR (DMSO) d 169.66 (C=O), 150.75 (C-2), 164.80 (C-4), 106.18 (C-5),
146.51 (C-6), 138.1–127.49 (C6H5), 31.57 (C-1’), 70.25 (C-2’), 70.51 (C-3’), 72.06
(CH2Ph), 20.67 (CH3), 9.75 (CH3).
6-(2,3-Dihydroxypropyl)-5-methyl-(1H,3H)-pyrimidine-2,4-dione (8). A solu-
tion of 7 (110 mg, 0.329 mmol) in dichloromethane (8 ml) was cooled to À75°C. Boron
trichloride (1M, 1.65 ml, 1.65 mmol) was added via syringe under argon atmosphere.
The mixture was stirred at À75°C for 2 h and then the temperature was raised to À40°C.
A solution of MeOH/CH2Cl2 (1:1, 10 ml) was added and the cooling bath was removed.
The solution was neutralized with NaHCO3 and stirred for an additional 30 min. After
filtration, the solvent was removed under reduced pressure. Water (2 ml) was added to
the residue and the solvent was evaporated. The crude product was applied to column
chromatography (CH2Cl2:MeOH - 7:1) to afford 8 (35 mg, 53 %, mp 186–187°C); MS
m/z 200 (M+); 13C NMR (DMSO) d 150.81 (C-2), 164.73 (C-4), 105.24 (C-5), 149.08
(C-6), 34.58 (C-1’), 70.08 (C-2’), 65.59 (C-3’), 9.82 (CH3). Anal. Calcd for C8H12N2O4:
C, 48.0; H 6.04; N, 13.99. Found: C, 48.12; H 5.98; N, 13.95.
6-(2,3-Dihydroxypropyl)-2,4-dimethoxy-5-methylpyrimidine (9). The com-
pound 6 (500 mg, 1.57 mmol) was treated according to the procedure analogous to
that for the preparation of 8. The crude product was submitted to column chromatography
(CH2Cl2:MeOH - 10:1) to give oily product 9 (240 mg, 67 %); MS m/z 228 (M+); 13C

C-6 Alkylated Pyrimidine Nucleoside Analogues
1717
NMR (DMSO) d 162.12 (C-2), 169.02 (C-4), 108.39 (C-5), 167.17 (C-6), 54.0 (OCH3),
53.83 (OCH3), 38.34 (C-1’), 71.09 (C-2’), 65.64 (C-3’), 9.84 (CH3). Anal. Calcd for
C10H16N2O4: C, 52.62; H 7.07; N, 12.27. Found: C, 52.50; H 7.09; N, 12.23.
6-(3-Benzyloxy-2-fluoropropyl)-2,4-dimethoxy-5-methylpyrimidine (10). Com-
pound 6 (369 mg, 1.159 mmol) was dissolved in anhydrous dichloromethane (4 ml).
Diethylaminosulfur trifluoride (DAST, 0.22 ml, 1.679 mmol) was then added dropwise
with stirring at room temperature under argon atmosphere. After 50 min the reaction
mixture was partitioned between dichloromethane and aqueous sodium bicarbonate.
The organic layer was concentrated and purified by silica column chromatography
(n-hexane:ethyl acetate - 4:1) to give oily 10 (126 mg, 34 %); MS m/z 320 (M+); 13C NMR
(DMSO) d 164.51 (C-2), 169.63 (C-4), 108.96 (C-5), 169.63 (C-6), 135.66–127.99
(C6H5), 54.52 (OCH3), 54.42 (OCH3), 35.35 (C-1’, J=23 Hz), 91.93 (C-2’, J=172 Hz),
71.55 (C-3’, J=20 Hz), 72.75 (CH2Ph), 9.55 (CH3). Anal. Calcd for C17H21FN2O3: C,
63.74; H 6.61; N, 8.74. Found: C, 63.81; H 6.59; N, 8.72.
6-(3-Benzyloxy-2-fluoropropyl)-5-methyl-(1H,3H)-pyrimidine-2,4-dione (11).
The compound 11 (113 mg, 0.353 mmol) was treated according to the procedure
analogous to that for the preparation of 7. Crude product 11 was purified by column
chromatography (CH2Cl2:MeOH - 30:1) afforded white crystals of 11 (43 mg, 42 %,
mp 132–132.5°C); MS m/z 292 (M+); 13C NMR (DMSO) d 151.21 (C-2), 165.17
(C-4), 106.73 (C-5), 146.54 (C-6), 135.66–128.02 (C6H5), 32.45 (C-1’, J=22 Hz),
91.15 (C-2’, J=175 Hz), 71.32 (C-3’, J=20 Hz), 72.75 (CH2Ph), 10.15 (CH3).
Anal. Calcd for C15H17FN2O3: C, 61.63; H 5.86; N, 9.58. Found: C, 61.51; H 5.87;
N, 9.56.
6-(2-Fluoro-3-hydroxypropyl)-5-methyl-(1H,3H)-pyrimidine-2,4-dione (12).
The compound 11 (71 mg, 0.243 mmol) was treated according to a procedure that
was analogous to that for the preparation of 8. The residue was submitted to column
chromatography (CH2Cl2:MeOH - 10:1) to afford crystals of 12 (26 mg, 53 %, mp
151–152°C); MS m/z 202 (M+); 13C NMR (DMSO) d 150.78 (C-2), 164.71 (C-4),
106.10 (C-5), 146.54 (C-6), 31.9 (C-1’, J=22 Hz), 91.49 (C-2’, J=174 Hz), 62.67 (C-3’,
J=21 Hz), 9.70 (CH3). Anal. Calcd for C8H11FN2O3: C, 47.52; H 5.48; N, 13.86.
Found: C, 47.40; H 5.49; N, 13.82.
6-(3-Acetoxy-2-hydroxypropyl)-2,4-dimethoxy-5-methylpyrimidine (13). A so-
lution of 9 (202 mg, 0.885 mmol) and acetyl chloride (0.063 ml, 0.887 mmol) in
dichloromethane (2ml) was stirred at room temperature for 30 min. The solvent was
evaporated under reduced pressure. Column chromatography (CH2Cl2:MeOH - 20:1) of
the residue gave 13 (109 mg, 46 %). MS m/z 270 (M+).
6-(3-Acetoxy-2-mesylpropyl)-2,4-dimethoxy-5-methylpyrimidine (14). To
compound 13 (86 mg, 0.318 mmol) dissolved in anhydrous pyridine (1.5 ml),
methanesulfonyl chloride (0.074 ml, 0.954 mmol) was added under argon atmosphere.
The solution was kept on ice for 1 h. Ice water was added to the mixture. After
extraction with dihloromethane, column chromatography was performed to give oily 14
(86 mg, 81 %). MS m/z 348 (M+). Anal. Calcd for C13H20N2O7S: C, 44.82; H 5.79; N,
8.04. Found: C, 44.75; H 5.80; N, 8.01.

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Raic-Malic et al.
1718
6-(2-[18F]Fluoro-3-hydroxypropyl)-5-methyl-(1H,3H)-pyrimidine-2,4-dione (16)
(18F-HHT). Aqueous H18F (37 GBq) from the cyclotron target was added to 10 ml
of tetrabutylammoniumhydroxide (1M solution in MeOH). The solution was heated at
110°C and evaporated to dryness with the aid of a N2 flow. This process was repeated
three times by adding acetonitrile (1 ml). To the residue was added a solution of 8–
10 mg of 14 in dry DMSO (1ml), and the reaction mixture was heated at 120°C for
30 min. The reaction was quenched by adding water (7 ml), and the resulting mixture
was passed through a Sep-Pak Plus C18 cartridge (Waters). The Sep-Pak cartridge was
washed with water (3 ml) and the fluorine-18 labelled intermediate retained on the C18
was eluted with MeCN (5 ml). The MeCN elute was evaporated to dryness under
reduced pressure and a flow of N2. The residue was acidified with 37% HCl (1 ml) and
heated for 45 min at 120°C. Upon cooling, the contents were neutralized with 6N NaOH
(1 ml). Purification of the crude product was achieved by high-performance liquid
chromatography (HPLC) (Column: Bondclone C-18, 300 Â 7.8 mm). The mobile phase
was water and the flow rate was 6 ml/min. The retention time of 18F-HHT was 11 min.
HPLC Assays for Monitoring the Phosphorylation of
Substrates 8 and 12
DAD HPLC (L-7455 Merck) system was based on reverse-phase ion-pair
chromatography using a previously published protocol[19] (Column, RP-18; solvent,
0.2 M NaH2PO4, 25 mM tetrabutylammonium hydrogen sulphate, 3% methanol; flow
1.1 ml/min; detection, UV 254 nm and diode array). Reactions were carried out in a
final volume of 75 ml containing 50 mM Tris, pH 7.2, 5 mM ATP, 5 mM MgCl2, 2 mM
substrate and 1–5 mg of HSV-1 thymidine kinase (TK). The reaction was stopped after
1 h at 37°C by a 10-fold dilution in water EDTA-solution (2.5 mM) and freezing at
À20°C prior to injection. The formation of the pyrimidine monophosphate was
monitored qualitatively. Two different blank reactions (no enzyme or no substrate)
were run concurrently to account for the (minimal) ATP independent hydrolysis.
Determination of Kcat by Spectrophotometric Assays
Kcat values were determined as described in the literature.[22] Reaction mixture
with a final volume of 75 ml containing 50 mM Tris buffer pH 7.2, 1 mM 1,4-dithio-
DL-threitol, 0.21 mM phosphoenolpyruvate, 2.5 mM MgCl2, 5 mM ATP, 0.18 mM
NaDH, 0.8 mg pyruvate kinase, 0.5 mg L-lactate dehydrogenase, and three different
concentrations of the substrate (0.2 mM, 0.6 mM, and 1.0 mM) were incubated at
37°C. Two minutes later, 1 to 6 mg of HSV-1 TK was added in order to initiate the
reaction. The changes in absorbance at 340 nm corresponding to ADP formation of the
TK-dependent reaction at 37°C were monitored during 10 minutes. The data presented
are the results of three independent series of measurements performed in triplicate.
Control experiments were performed in order to take into account the spontaneous
hydrolysis of ATP under the experimental conditions.
Binding Affinity Determination
Kinetic studies measuring the conversion of 3H-labeled dT to dTMP in the
presence of various concentrations (0, 50, 200 mM) of 8 and 12 were performed using

C-6 Alkylated Pyrimidine Nucleoside Analogues
1719
the DEAE-cellulose method.[20,23] Reactions were carried out in 30 ml of 50 mM Tris,
pH 7.2, 5 mM ATP, 5 mM MgCl2, 1.5 mg/ml BSA. The appropriate amount of enzyme
and concentartions of [3H]thymidine were chosen for Michaelis–Menten conditions for
initial velocity measurements. The Ki values were determined by a nonlinear fit of the
raw data to the equation: V=Vmax [dT]/{Km(1 + [compd.]/Ki) + [dT]} for competitive
inhibition[24] using the Origin 6.0 software. The calculated values were based on at
least three independent experiments.
Functionality Assay
HSV-1 TK-transduced and non-transduced osteosarcoma (MG63) and non-small
lung cancer (H125 NSCLC) cell lines were split 4 days before the cytotoxicity assay.
Therefore, cells reached maximum speed of growth when the assay was started. The
cells were trypsinized in 10 ml of fresh RPMI 1640 medium with 10% foetal calf
serum and 1% antibiotic PSF solution. The cell concentration of a 1:1 dilution with
trypan blue was determined using a Neubauer’s counting chamber. The suspension was
diluted with medium to a final concentration of 20000 cells per ml. The cell suspension
(2000 cells in 100 ml) were seeded in each well of a 96 well NunclonTM Surface plate
from NuncTM. After 1.5 h incubation at 37°C the cells were attached to the bottom of
the wells. Sterile solutions (100 ml) of GCV, 8 and 12 in RPMI 1640 were added to the
cells leading to final substrate concentrations of 0, 50, 200, 500, 1000, and 2000 mM.
All samples were prepared in triplicate for each cell line. GCV was used as reference
control for a cytotoxic drug to HSV-1 TK expressing cells. The wells with no
thymidine analogue allow calculating the standard value of 100% of cell growth. The
plates were incubated for 4.5 days at 37°C in the presence of 5% CO2. Viability of the
cells was measured using the XTT reagent. The XTT reagent (50 ml) was added to each
well. While the plates were incubated at 37°C in the presence of 5% CO2 the living
cells converted the XTT (tetrazolium derivative) by biodegradation to a water-soluble
reddish formazan derivative.[25,26] After 9 h incubation the number of cells in each vial
was determined by colorimetric UV absorption measurement of the converted XTT
with an ELISA reader at a wavelength of 450 nm.
Antitumor Activity Assays
Antitumor activity against L1210 (murine leukemia), Molt4/C8 and CEM (human
T-lymphocytes) cell lines were measured essentially as originally described for the
mouse leukemia (L1210) cell line.[27]
Antiviral Activity Assays
Antiviral activity against HSV-1, HSV-2, vaccinia virus and vesicular stomatitis
virus was determined as described previously.[28]
ACKNOWLEDGMENTS
The authors thank Dr. Oliver Zerbe for his contribution in the interpretation of the
1H and 13C NMR spectra and to Patrick Ballmer for help in performing cytotoxicity

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Raic-Malic et al.
1720
assays. We also thank Lizette van Berckelaer for excellent technical assistance in
performing the antitumor cell activity assays, as well as Ann Absillis, Anita Van
Lierde, Frieda De Meyer, Anita Camps and Lies Vandenheurck for excellent technical
assistance in performing the antiviral activity assays.
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Received March 22, 2004
Accepted June 8, 2004