C-5 hydroxyethyl and hydroxypropyl acyclonucleosides as substrates for thymidine kinase of herpes simplex virus type 1 (HSV-1 TK): Syntheses and biological evaluation

Article abstract of DOI:10.3390/molecules18055104

The efficient syntheses of 5-(2-hydroxyethyl)- and 5-(3-hydroxypropyl)- substituted pyrimidine derivatives bearing 2,3-dihydroxypropyl, acyclovir-, ganciclovirand penciclovir-like side chains are reported. A synthetic approach that included the alkylation of an N-anionic-5-substituted pyrimidine intermediate (method A) provided the target acyclonucleosides in significantly higher overall yields in comparison to those obtained by method B using sylilation reaction. The phosphorylation assays of novel compounds as potential substrates for thymidine kinase of herpes simplex virus type 1 (HSV-1 TK) showed that solely pyrimidine 5-substituted acyclonucleosides with a penciclovir-like side chain acted as a fraudulent substrates of HSV-1 TK. Moreover, the uracil derivative with penciclovir-like side chain with less bulky 2-hydroxyethyl substituent at C-5 proved to be a better substrate than the corresponding one with a 3-hydroxypropyl substituent. Therefore, this acyclonucleoside was selected as a lead compound for the development of a positron emission tomography HSV-1 TK activity imaging agent.

Full text of DOI:10.3390/molecules18055104

Molecules 2013, 18, 5104-5124; doi:10.3390/molecules18055104
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
C-5 Hydroxyethyl and Hydroxypropyl Acyclonucleosides as
Substrates for Thymidine Kinase of Herpes Simplex Virus
Type 1 (HSV-1 TK): Syntheses and Biological Evaluation
Andrijana Meščić 1, Svjetlana Krištafor 1, Ivana Novaković 2, Amar Osmanović 3, Ursina Müller 4,
Davorka Završnik 3, Simon M. Ametamey 4, Leonardo Scapozza 2 and Silvana Raić-Malić 1,*
1
Department of Organic Chemistry, Faculty of Chemical Engineering and Technology,
University of Zagreb, Marulićev trg 20, HR-10000 Zagreb, Croatia;
E-Mails: amescic@fkit.hr (A.M.); prekupec@fkit.hr (S.K.)
2
Pharmaceutical Biochemistry, School of Pharmaceutical Sciences, University of Geneva,
Quai Ernest-Ansermet 30, CH-1211 Geneva 4, Switzerland;
E-Mails: Ivana.Novakovic@unige.ch (I.N.); Leonardo.Scapozza@unige.ch (L.S.)
3
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Sarajevo,
Zmaja od Bosne 8, BIH-71 000 Sarajevo, Bosnia and Herzegovina;
E-Mails: amkooo@hotmail.com (A.O.); dzavrsnik@yahoo.com (D.Z.)
4
Center for Radiopharmaceutical Sciences, ETH Zurich (Swiss Federal Institute of Technology),
Wolfgang-Pauli Strasse 10, CH-8093 Zurich, Switzerland;
E-Mails: ursinamuller@gmail.com (U.M.); simon.ametamey@pharma.ethz.ch (S.M.A.)
* Author to whom correspondence should be addressed; E-Mail: sraic@fkit.hr;
Tel.: +385-1-4597-213; Fax: +385-1-4597-224.
Received: 31 January 2013; in revised form: 16 April 2013 / Accepted: 25 April 2013 /
Published: 2 May 2013
Abstract: The efficient syntheses of 5-(2-hydroxyethyl)- and 5-(3-hydroxypropyl)-
substituted pyrimidine derivatives bearing 2,3-dihydroxypropyl, acyclovir-, ganciclovir-
and penciclovir-like side chains are reported. A synthetic approach that included the
alkylation of an N-anionic-5-substituted pyrimidine intermediate (method A) provided the
target acyclonucleosides in significantly higher overall yields in comparison to those
obtained by method B using sylilation reaction. The phosphorylation assays of novel
compounds as potential substrates for thymidine kinase of herpes simplex virus type 1
(HSV-1 TK) showed that solely pyrimidine 5-substituted acyclonucleosides with a
penciclovir-like side chain acted as a fraudulent substrates of HSV-1 TK. Moreover, the
uracil derivative with penciclovir-like side chain with less bulky 2-hydroxyethyl

Molecules 2013, 18
substituent at C-5 proved to be a better substrate than the corresponding one with a
5105
3-hydroxypropyl substituent. Therefore, this acyclonucleoside was selected as a lead
compound for the development of a positron emission tomography HSV-1 TK activity
imaging agent.
Keywords: acyclic nucleoside analogues; 5-substituted pyrimidines; herpes simplex virus
type 1 thymidine kinase (HSV-1 TK); positron emission tomography (PET)
1. Introduction
Nucleoside analogues have acquired an important role as therapeutic agents in the field of
chemotherapy on account of their extensive biological activities [1,2]. Thereby, modified nucleosides
and nucleobases are a pharmacologically diverse family, which includes anticancer compounds,
antiviral agents, and immunosuppressive molecules. Interest in acyclic nucleosides began in the mid-
1970s when acyclovir was first reported as a potent anti-herpes drug [3,4]. Many variations, both of the
acyclic glycone and of the heterocyclic base, have been described [5,6]. Antiviral nucleoside analogues
are known to localize selectively in herpes simplex virus (HSV)-infected cells because of
monophosphorylation catalyzed by virus-encoded thymidine kinase (TK) [7]. Among these, acyclovir,
ganciclovir and penciclovir are reported to be potent against HSV types 1 and 2 [8,9] (Figure 1).
Figure 1. Acyclic nucleoside analogues and 2'-deoxyuridines as chemotherapeutic agents.
O
O
O
R5
N
N
N
N
HN
H2N
NH
HN
H2N
R5
N
N
O
N
O
X
HO
OH
OH
OH
IdU
I
O
FdU
F
acyclovir
X = O, ganciclovir
X = CH2, penciclovir
CF3 TFdU
Br
BVdU
OH
In another area, a great number of 5-substituted uracil derivatives, especially 2'-deoxyuridines, have
been investigated for the experimental and clinical treatment of neoplastic and viral diseases (Figure 1).
The selective monophosphorylation of antiviral nucleoside analogues by virus-encoded thymidine
kinase has been exploited in gene therapy of cancer [10]. Gene therapy offers promising new treatment
modalities in oncology and other diseases [11]. The suicide gene, HSV-1 TK, has already been
successfully employed in a variety of tumour models, both in vitro and in vivo [12,13]. A prodrug with
low toxicity becomes toxic only in cells where the HSV-1 TK gene is expressed, thus resulting in the
selective killing of transfected cells through termination of DNA synthesis. Phosphorylation of
radioactive-labelled prodrugs, usually derivatives of acyclonucleosides which are selective anti-herpes
agents, leads to metabolites trapped within the infected tumour cells, and the resulting accumulation of
radioactivity allows monitoring of the enzyme activity with positron emission tomography (PET) [14–16].
A striking difference is that while all pyrimidine nucleoside analogues show low, if any, bystander
killing effects, purine derivatives such as ganciclovir are endowed with bystander killer effect [17].

Molecules 2013, 18
5106
As found earlier, pyrimidine nucleoside analogues in which the acyclic sugar moiety is attached at
the C-6 rather than at the N-1 position have the potential for the development as a new PET imaging
probe. Therefore, thymines with 6-(2,3-dihydroxypropyl), 6-(1,3-dihydroxyisobutyl) and 6-(1,3-
dihydroxyisobutenyl) side chains have been developed as tracer molecules for monitoring HSV-1 TK
expression by means of PET [18–20]. Although the N-methylated thymine derivative, N-Me-
[18F]FHBT, bearing a 1,3-dihydroxyisobutyl side-chain at the C-6 position was not superior to the
purine analogue [18F]FHBG, an established PET reporter probe for imaging HSV-1 TK expression,
N-Me-[18F]FHBT clearly showed the feasibility of its use as a PET probe to monitor HSV-1 TK gene
expression in vivo [21].
In an effort to further explore the pyrimidine scaffold as a putative starting point for the
development of new fraudulent substrates of HSV-1 TK for non-invasive imaging of HSV-1 TK gene
expression with improved pharmacodynamic and pharmacokinetic profile, we have synthesized a
series of novel 5-substituted acyclic nucleoside analogues. Here we report on the synthesis of target
5-(2-hydroxyalkyl)-substituted uracil derivatives bearing 2,3-dihydroxypropyl (19), acyclovir- (21),
ganciclovir- (23) and penciclovir-like (31 and 32) substituents at N-1 position of the pyrimidine ring
(Figure 2). In order to evaluate target nucleoside mimetics 19, 21, 23, 31 and 32 as substrates for
HSV-1 TK, molecular modelling and phosphorylation assays of these compounds were performed.
Figure 2. Novel 5-(2-hydroxyalkyl)-substituted uracil derivatives bearing 2,3-dihydroxy-
propyl (19), acyclovir- (21), ganciclovir- (23) and penciclovir-like (31 and 32) side chains.
O
n
n = 2, 3
HN
OH
R1 =
O
N
O
OH
,
OH
R1
OH
O
,
OH
OH
OH
OH
2. Results and Discussion
2.1. Chemistry
5-(2-Hydroxyalkyl)uracil derivatives with 2,3-dihydroxypropyl (19), acyclovir- (21), ganciclovir-
(23) and penciclovir-like (31 and 32) side chains were prepared from the corresponding uracil
derivatives 10 and 1416 by the methods outlined in Schemes 13. The synthesis of compounds 10
and 1416, as precursors for N-alkylation, was performed in excellent yields (8099%) (Scheme 1).
5-(2-Hydroxyethyl)- and 5-(3-hydroxypropyl)-substituted uracils 1 and 2 served as starting materials
and their syntheses were reported earlier [22–24]. The synthesis of 5-substituted 2,4-dimethoxypyrimidine
derivatives 10 and 11 was carried out by acetylation, chlorination at the positions C-2 and C-4 with
POCl3, methoxylation with sodium methoxide and repeated acetylation [25] (Scheme 1).

Molecules 2013, 18
Scheme 1. Synthesis of the precursors 10 and 1416 for N-alkylation.
5107
O
Cl
O
OH
HN
n
ii)
OAc
i)
n
N
HN
OAc
n
O
N
H
Cl
N
O
N
H
n
2
3
n
2
3
n
2
3
iii)
5
6
3
4
1
2
OMe
OMe
O
i)
OAc
n
N
OH
n
N
+
N
MeO
N
MeO
N
MeO
N
n
2
3
n
2
3
7
10 11
8
9
iv)
OMe
OMe
OMe
OH
OAc
n
v)
OAc
n
vi)
N
N
N
O
N
O
N
H
O
N
H
n
2
3
n
2
3
16
12 13
14 15
Ac
Reagents and conditions: (i) acetic anhydride, pyridine, rt, 1 h; (ii) POCl3, N,N-diethylaniline, pyridine,
reflux, 1 h; (iii) NaOMe/MeOH, rt, 20 h; (iv) AcCl, rt, 20 h; (v) silica gel column chromatography;
(vi) NaOMe/MeOH, rt, 1 h.
Selective 2-dealkylation of 5-(acetoxyalkyl)-2,4-dimethoxypyrimidines [26] 10 and 11 in
non-aqueous acid gave the corresponding 4-methoxypyrimidin-2-one derivatives 14 and 15. Treatment
of the acetyl protected 2,4-dimethoxypyrimidines 10 and 11 with acetyl chloride afforded
N-acetylated 4-methoxypyrimidin-2-one derivatives 12 and 13, which after N-deprotection and
purification using column chromatography on silica gel yielded 14 and 15 (Scheme 1). Final
deacetylation of 14 gave 4-methoxypyrimidin-2-one 16 with a 2-hydroxyethyl substituent at C-5.
Further synthetic steps in the preparation of target compounds 19, 21 and 23 involved N-alkylation
applied by two synthetic methods (A and B, Scheme 2, Table 1). In order to ensure the introduction of
the substituent into position N-1 of the pyrimidine ring, the Hilbert-Johnson reactions [27] were
applied in synthetic method A. These reactions involved the treatment of 5-(2-acetoxyethyl)-2,4-
dimethoxypyrimidine derivative 10 with alkyl halides. Allyl bromide and 1,3-dibenzyloxy-2-
chloromethoxypropane [28] were used as agents for the introduction of 2,3-dihydroxypropyl (in 19)
and ganciclovir-like side chain (in 23), respectively. Furthermore, the treatment of 1,3-dioxolane with
acetyl bromide gave (2-acetoxyethoxy)methyl bromide [29] as a readily distilled colourless oil, which
served as the alkylating reagent for the introduction of the acyclovir-like side chain (in 21). Unlike
alkylation with allyl bromide which gave N-1-substituted 4-methoxypyrimidine 17 in 73% yield,
alkylation using (2-acetoxyethoxy)methyl bromide and 1,3-dibenzyloxy-2-chloromethoxypropane was
accompanied by 4-demethylation and the corresponding N-1-substituted pyrimidin-2,4-dione
derivatives 20 and 22 were isolated in moderate yield (43% and 36%, respectively).

Molecules 2013, 18
Scheme 2. Synthesis of 5-(2-hydroxyethyl)uracil derivatives with 2,3-dihydroxypropyl
5108
(19), acyclovir- (21) and ganciclovir-like (23) side chain.
OMe
OAc
N
MeO
N
10
iv)
vi)
i)
Synthetic
method A
O
O
OMe
OAc
OBn
OAc
OAc
OAc
HN
HN
N
O
N
O
N
O
N
O
O
20
22
vii)
17
OBn
v)
ii)
OMe
N
O
O
OAc
OH
R
Cl
R
OH
OH
N
HN
HN
23 28
O
O
N
O
N
O
O
OH
18
OH
OH
iii)
OH
21
23
iii)
O
N
vii)
OH
HN
OMe
O
OH
OH
OH
R
Cl
R
O
N
HN
26 27
OH
O
N
O
O
N
O
OH
19
OBn
OH
25
OBn
Synthetic
method B
x)
iii)
ix)
16
O
OMe
N
OMe
OH
HN
N
OR
R
14
viii)
N
Ac
14 16
H
O
N
H
O
O
N
H
1
OH
24
OH
Reagents and conditions: (i) allyl bromide; (ii) NaClO3, OsO4; (iii) 1 M NaOH, rt, 20 h; (iv)
(2-acethoxyethoxy)methyl bromide, Na2CO3, rt, 24 h; (v) NaOMe/MeOH, rt, 20 h; (vi) 1,3-dibenzyloxy-2-
chloromethoxypropane, K2CO3, CH2Cl2, rt, 24 h; (vii) BCl3, CH2Cl2, 3 h; (viii) NaH, DMF, 1-chloro-2,3-
dihydroxypropane; (ix) BSA, CH3CN, dioxolane, KI, TMSCl, rt, 48 h; (x) 1. HMDS, TMSCl;
2. 1,3-dibenzyloxy-2-chloromethoxypropane, TBAI, CH3CN, rt, 24 h.

Molecules 2013, 18
5109
Table 1. An overview of the yields in synthetic method A and B for target compounds 19,
21 and 23.
19
21
Yields
98%
99%
95%
90%
43%
74%
26%
21
23
Synthetic method A
1.
2.
3.
4.
Acetylation
Chlorination
Methoxylation
Acetylation
98%
99%
95%
90%
98%
99%
95%
90%
36%
67%
20%
23
5. Alkylation/Hydroxylation 73%/50%
6.
Deprotection
Overall yield:
39%
12%
19
Synthetic method B
Yields
98%
99%
95%
90%
80%
93%
25%
26%
4%
1.
2.
3.
4.
5.
6.
7.
8.
Acetylation
Chlorination
98%
99%
95%
90%
80%
-
-
-
Methoxylation
Acetylation
-
-
Demethoxylation
Deacetylation
Silylation/Akylation
Deprotection
-
-
75%
37%
18%
8%
65%
5%
Overall yield:
cis-Dihydroxylation of N-1-allyl pyrimidine derivative 17 with sodium chlorate in the presence
of osmium tetroxide afforded N-1-(2,3-dihydroxypropyl)pyrimidine derivative 18 which after the
4-demethylation and deacetylation with sodium hydroxide yielded the desired 5-(2-
hydroxyethyl)pyrimidine 19 bearing a 2,3-dihydroxypropyl group at N-1. The overall yield of this
method was 12%. The deacetylation of the side chains at C-5 and N-1 of 20 under basic conditions
afforded the 5-(2-hydroxyethyl)pyrimidine 21 with the best overall yield of 26%. Similarly, both
deacetylation in C-5 and debenzylation in N-1 side chains of 22 using boron trichloride gave the 5-(2-
hydroxyethyl)-substituted pyrimidine 23 with a ganciclovir-like side chain at N-1.
In search for a more efficient synthesis of 19, an alternate method B was applied using sodium
hydride as deprotonating agent and thus the uracil salt obtained in situ reacted with 1-chloro-2,3-
dihydroxypropane to give trihydroxy 4-methoxypyrimidin-2-one 24. Deprotection of 24 yielded target
the trihydroxypyrimidin-2,4-dione 19. In comparison to synthetic method A this method for
preparation of 19 is more efficient and the use of the highly poisonous reagent osmium tetroxide for
cis-hydroxylation was avoided. Similarly, compounds 21 and 23 were also prepared by silylation
reactions (synthetic method B). Since silylation of 5-(2-hydroxyethyl)uracil (1) using N,O-bis-
(trimethysilyl)acetamide (BSA) was unsuccessful, pyrimidin-2-one 16 was used for N-alkylation.
Trimethylsilylation of 16 [26] using BSA and in situ coupling employing trimethylchlorosilane,
potassium iodide and 1,3-dioxolane in dry acetonitrile was performed, and the expected product 21
was obtained after hydrolysis of 25 with sodium hydroxide (Scheme 2). Furthermore, compound 23
was prepared directly from 5-(2-hydroxyethyl)uracil (1) by the silylation of 1 and in situ reaction of
O-persilylated 1 with 1,3-dibenzyloxy-2-chloromethoxypropane to give N-1-alkylated pyrimidine 26

Molecules 2013, 18
5110
in 8% yield (method B). The removal of the benzyl protecting groups in 26 proceeded smoothly and
led to target compound 23.
For the introduction of a penciclovir-like side chain at N-1 position of the pyrimidine ring,
4-acetoxy-(3-acetoxymethyl)butyl iodide [30,31], prepared from a mesylate and the related bromide,
was used. Since neither direct Hilbert-Johnson-type reaction of 2,4-dimethoxypyrimidine derivative 10
with both alkyl halides and mesylate did not yield the corresponding pyrimidine acyclonucleoside, an
alternative method using 4-methoxypyrimidin-2-one derivatives 14 and 15 was applied.
Thus, the reaction of potassium salts of 14 and 15 with 4-acetoxy-(3-acetoxymethyl)butyl iodide led
to the target 5-(2-hydroxyethyl)- 31 and 5-(3-hydroxypropyl)pyrimidine 32 bearing a penciclovir-like
side chain (Scheme 3).
Scheme 3. Synthesis of 5-(2-hydroxyalkyl)uracil derivatives 31 and 32 with
penciclovir-like side chains.
O
OMe
OMe
n
OH
HN
i)
N
n
OAc
ii)
OAc
n
N
O
N
O
N
H
O
N
OH
OAc
n
2
3
n
2
3
n
2
3
OH
29 30
14 15
OAc
31 32
Reagents and conditions: (i) 4-Acetoxy-(3-acetoxymethyl)butyl iodide, K2CO3, DMF, 60 °C, 20 min; (ii) 1 M
NaOH, rt, 20 h.
2.2. In Vitro Validation of Compounds 19, 21, 23, 31 and 32 as Substrates of HSV-1 TK and hTK
To determine whether compounds 19, 21, 23, 31 and 32 are substrates of HSV-1 TK or human TK,
phosphorylation assays were performed by incubation of deoxythymidine (dT) and target compounds
with recombinant HSV-1 TK and human TK (hTK) according to a previously published protocol [32].
The monophosphorylation was monitored qualitatively by the formation of new peaks corresponding
to adenosine diphosphate (ADP), monophosphate derivatives of tested compounds and thymidine
monophosphate (dTMP). Results of phosphorylation assays for 19, 21 and 23 indicated that these
compounds are not phosphorylated by either HSV-1 TK or human TK (see Supplementary Materials).
While acyclonucleosides containing dihydroxypropyl (19), acyclovir- (21) and ganciclovir-like side
chain (23) were not phosphorylated, both 5-substituted acyclic pyrimidines 31 and 32 with a
penciclovir-like side chain showed to be substrates of HSV-1 TK. Furthermore, small alteration in
structures of 23, such as replacement of oxygen in the (1,3-dihydroxy-2-propoxy)methyl side chain at
N-1 with a methylene group (compound 31), has an impact on phosphorylation reaction of these
compounds. We can assume that the electron donating oxygen has an influence on the interaction of 23
and HSV-1 TK disabling the enzyme to phosphorylate fraudulent substrate 23 with a ganciclovir-like
side chain. Therefore, for compounds 31 and 32 while using viral HSV-1 TK, the formation of
monophosphate derivatives 31-MP (Figure 3) and 32-MP (Figure 4) as well as ADP were detected,
confirming that 31 and 32 are substrates of viral HSV-1 TK. However, the size of the alkyl substituent
at C-5 exhibited some effect on phosphorylation. Thus, compound 31 with a less bulky 2-hydroxyethyl

Molecules 2013, 18
5111
substituent at C-5 had a higher ADP/ATP ratio compared to compound 32, thus proving to be a better
substrate than 32. Furthermore, formation of 31-MP and 32-MP was proven by extracting the max.
absorbance wavelength of the new appearing peaks which corresponds to the max. absorbance
wavelength of compounds 31 and 32 (Figures 3 and 4). No phosphorylation was observed for
compounds 31 and 32 when human TK was used, indicating that these two compounds are not
substrates for hTK.
Figure 3. HPLC and DAD/HPLC chromatograms of the reaction of 31 and HSV-1 TK.
Figure 4. HPLC and DAD/HPLC chromatograms of the reaction of 32 and HSV-1 TK.
The phosphorylation patterns of compounds 31 and 32 by HSV-1 TK and hTK are presented in
Figure 5a. No significant difference in ADP/ATP ratio was noticed in phosphorylation assays with hTK.
Phosphorylation reaction of compound 31 and its fluorinated structural analogue, in which hydroxyl
group in C-5 substituent is replaced with fluorine, was performed at 30, 60 and 90 min of incubation
(Figure 5b). It is evident that formation of monophosphated derivatives for both compounds was
increased over time. By comparison of calculated values for monophosphated derivatives which were
formed at different times (30, 60 and 90 min), we can estimate that approximately 40% of 31-MP was
formed in 30 min and majority of 31-MP (~80%) in 60 min of incubation. On the contrary, fluorinated
derivative gave majority of corresponding monophosphate (~70%) already in 30 min and was almost
completely monophosphorylated in 60 min. The synthesis of fluorinated derivative of 31 and in vivo

Molecules 2013, 18
5112
results on small animal PET studies of 18F labeled compound 31, HHB-5-[18F]FEP, will be
presented elsewhere.
Figure 5. (a) Phosphorylation pattern of 1 mM of compounds 31 and 32, by HSV-1 TK
and hTK, and of compound 23 and dT by HSV-1 TK. Blank refers to the control
experiment performed in presence of the enzyme and in absence of the compound; (b)
Time course for conversion of 31 and its fluorinated analogue to corresponding
monophosphated derivatives.
0.35
0.3
0.25
0.2
HSV1 TK
0.15
hTK
0.1
0.05
0
30min
Blank
30 min
comp 31
30 min
comp 32
30 min
comp 23
30min
dT
(a)
9.0E+5
8.0E+5
7.0E+5
6.0E+5
5.0E+5
4.0E+5
3.0E+5
2.0E+5
1.0E+5
0.0E+0
O
F
HN
O
N
P
OH
O
N
OH
HN
O
30 min
60 min
90 min
P
OH
31‐MP
fluorinated 31‐MP
(b)
2.3. Cellular Activity Evaluation
Compounds 1732 were evaluated for their cytotoxic activity against normal human fibroblasts
(WI38). The results indicated that the target compounds exhibited no cytotoxic effects against normal
human fibroblasts up to 100 µM.
3. Experimental
3.1. General
Melting points (uncorrected) were determined with a Kofler micro hot-stage (Reichert, Wien,
Austria). Precoated Merck (Darmstadt, Germany) silica gel 60F-254 plates were used for thin layer

Molecules 2013, 18
5113
chromatography and the spots were detected under UV light (254 nm). Column chromatography was
performed using Fluka (Buchs, Switzerland) silica gel (0.0630.2 mm); glass columns were
1
13
slurry-packed under gravity. H- and C-NMR spectra were acquired on a Bruker 300 MHz NMR
spectrometer (Bruker Biospin, Rheinstetten, Germany) All data were recorded in DMSO-d6 at 298 K.
1
Chemical shifts were referenced to the residual solvent signal of DMSO at δ 2.50 ppm for H and δ
13
39.50 ppm for C. Individual resonances were assigned on the basis of their chemical shifts, signal
intensities, multiplicity of resonances and HH coupling constants. Mass spectra were recorded on an
Agilent 6410 instrument (Agilent Technologies, Wilmington, DE, USA) equipped with an electrospray
interface and triple quadrupole analyzer (LC/MS/MS). High performance liquid chromatography was
performed on an Agilent 1100 series system with UV detection (photodiode array detector) using a
Zorbax C18 reverse-phase analytical column (2.1 × 30 mm, 3.5 µm). All compounds used for
biological evaluation showed >95% purity in this HPLC system.
3.2. Procedures for the Preparation of Compounds
5-(2-Hydroxyethyl)pyrimidin-2,4-dione (1), 5-(3-hydroxypropyl)pyrimidin-2,4-dione (2), 5-(2-
acetoxyethyl)pyrimidin-2,4-dione (3), 5-(2-acetoxyethyl)-2,4-dichloropyrimidine (5), 5-(2-hydroxy-
ethyl)-2,4-dimethoxypyrimidine (8), 5-(2-acetoxyethyl)-2,4-dimethoxypyrimidine (10), 5-(2-acetoxy-
ethyl)-4-methoxypyrimidin-2-one (14) and 5-(2-hydroxyethyl)-4-methoxypyrimidin-2-one (16) were
synthesized in accord with original procedures given in the literature [2226].
5-(3-Acetoxypropyl)pyrimidin-2,4-dione (4). The mixture of compound 2 (30 g, 0.18 mol) and acetic
anhydride (138 mL, 1.46 mol) in anhydrous pyridine (327 mL) was stirred at room temperature for 1 h.
The reaction was quenched with water and the mixture was evaporated to dryness. After column
chromatography (CH2Cl2–CH3OH = 8:1) white crystals of compound 4 were isolated (35.14 g, 94%,
m.p.: 224227 °C). 1H-NMR: () 11.00 (1H, s, NH), 10.64 (1H, s, NH), 7.23 (1H, s, H-6), 3.97 (2H, t,
H-3', J = 6.5 Hz), 2.21 (2H, t, H-1', J = 7.3 Hz), 2.00 (3H, s, COCH3), 1.771.64 (2H, m, H-2') ppm.
13C-NMR: () 170.9 (COCH3), 164.9 (C-4), 151.8 (C-2), 138.6 (C-6), 111.5 (C-5), 63.7 (C-3'), 27.5
(C-2'), 23.2 (C-1'), 21.2 (COCH3) ppm. MS (ESI): m/z = 213.1 ([M+H]+).
5-(3-Acetoxypropyl)-2,4-dichloropyrimidine (6). The mixture of compound 4 (34 g, 0.16 mol), POCl3
(320 mL) and N,N-diethylaniline (32 mL) was refluxed for 1 h. The solvent was evaporated to dryness,
the residue was diluted with CCl4 (300 mL), washed twice with iced water and dried over MgSO4.
1
After the removal of the solvent, compound 6 (34.87 g, 87%) was isolated as a yellow oil. H-NMR:
() 8.8 (1H, s, H-6), 4.11 (2H, t, H-3', J = 6.3 Hz), 2.81 (2H, t, H-1', J = 7.7 Hz), 1.97 (2H, m, H-2'),
2.01 (3H, s, COCH3) ppm. 13C-NMR: () 170.1 (COCH3), 161.2 (C-4), 161.1 (C-2), 156.5 (C-6), 132.4
(C-5), 62.8 (C-3'), 26.9 (C-2'), 25.6 (C-1'), 20.4 (COCH3) ppm. MS (ESI): m/z = 250.1 ([M+H]+).
2-Methoxy-6,7-dihydro-5H-pyrano[2,3-d]pyrimidine (7) and 2,4-dimethoxy-5-(3-hydroxypropyl)-
pyrimidine (9). To a solution of sodium (16.7 g, 0.72 mol) in anhydrous methanol (703 mL) compound
6 (34.87 g, 0.14 mmol) was added. The reaction mixture was stirred at room temperature for 20 h,
neutralized with ether saturated with gaseous HCl at 0 °C. The solvent was evaporated and CHCl3
(560 mL) was added. The mixture was stirred at room temperature for 20 h and filtered. The filtrate

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was dried over MgSO4 and evaporated to dryness. After column chromatography (CH2Cl2–CH3OH =
35:1) compounds 7 (9.66 g, 42%, m.p.: 6870 °C) and 9 (5.29 g, 19%) were isolated.
7: 1H-NMR: () 8.15 (1H, s, H-6), 4.32 (2H, t, H-3', J = 5.2 Hz), 3.79 (3H, s, OCH3), 2.65 (2H, t, H-1',
J = 6.4 Hz), 1.921.88 (2H, m, H-2') ppm. 13C-NMR: () 168.5 (C-4), 164.4 (C-2), 159.2 (C-6), 109.2
(C-5), 68.3 (C-3'), 54.7 (OCH3), 21.0 (C-2'), 21.4 (C-1') ppm. MS (ESI): m/z = 167.1 ([M+H]+).
1
9: H-NMR: () 8.1 (1H, s, H-6), 4.47 (1H, t, OH, J = 5.2 Hz), 3.92 (3H, s, OCH3), 3.87 (3H, s,
OCH3), 3.41 (2H, q, H-3', J = 6.0 Hz), 2.47 (2H, t, H-1', J = 7.7 Hz), 1.65 (2H, k, H-2', J = 7.0 Hz)
ppm. 13C-NMR: () 169.3 (C-4), 164.0 (C-2), 157.5 (C-6), 115.1 (C-5), 60.5 (C-3’), 54.7 (OCH3), 54.1
(OCH3), 32.1 (C-2'), 22.9 (C-1') ppm. MS (ESI): m/z = 199.1 ([M+H]+).
5-(3-Acetoxypropyl)-2,4-dimethoxypyrimidine (11). To a stirred solution of compound 9 (5.2 g, 26.23 mmol)
in dry pyridine (48 mL) acetic anhydride (20 mL, 26.4 mmol) was added and the stirring was continued
for 1 h at room temperature. After the completion of reaction 15 mL of water was added. The solvent
was evaporated and the residue was purified by column chromatography (CH2Cl2–CH3OH = 30:1) to
1
yield compound 11 as a yellow oil (6.1 g, 97%). H-NMR: () 8.12 (1H, s, H-6), 3.98 (2H, t, H-3',
J = 6.5 Hz), 3.91 (3H, s, OCH3), 3.86 (3H, s, OCH3), 2.49 (2H, t, H-1', J = 7.8 Hz), 2.0 (3H, s,
13
COCH3), 1.81 (2H, k, H-2', J = 7.0 Hz) ppm. C-NMR: () 170.85 (COCH3), 169.29 (C-4), 162.9
(C-2), 157.7 (C-6), 114.3 (C-5), 63.6 (C-3'), 54.7 (OCH3), 54.2 (OCH3), 27.8 (C-2'), 22.9 (C-1'), 21.1
(COCH3) ppm. MS (ESI): m/z = 241.1 ([M+H]+).
5-(3-Acetoxypropyl)-4-methoxypyrimidin-2-one (15). To a dried two-necked flask compound 11 (3.23 g,
13.44 mmol) and acetyl chloride (26 mL, 0.36 mol) were added under argon atmosphere. The reaction
mixture was stirred at room temperature for 20 h. After the reaction was complete, the solvent was
evaporated at reduced pressure at 35 °C and coevaporated twice by the addition of toluene (6 mL). The
residue was chromatographed (EtOAc–CH3OH = 20:1). During this step, the intermediate 13 was
deacetylated to form a white crystalline product 15 (1.79 g, 59%, m.p.: 146149 °C). Compound 4 was
1
also isolated (966 mg, 34%). H-NMR: () 11.14 (1H, s, NH), 7.50 (1H, s, H-6), 3.97 (2H, t, H-3',
J = 6.5 Hz), 3.83 (3H, s, OCH3), 2.32 (2H, t, H-1', J = 7.4 Hz), 1.99 (3H, s, COCH3), 1.75 (2H, k, H-2',
13
J = 6.7 Hz) ppm. C-NMR: () 170.9 (COCH3), 170.8 (C-4), 156.6 (C-2), 143.1 (C-6), 105.8 (C-5),
63.6 (C-3'), 54.1 (OCH3), 27.8 (C-2'), 22.8 (C-1'), 21.1 (COCH3) ppm. MS (ESI): m/z = 227.2 ([M+H]+).
5-(2-Acetoxyethyl)-N-1-allyl-4-methoxypyrimidin-2-one (17). Method A: A solution of compound 10
(375 mg, 1.66 mmol) and allyl bromide (0.15 mL, 1.73 mmol) was heated under reflux for 8 h. After
evaporation of the solvent and column chromatography (ethyl acetate:CH3OH = 15:1) compound 17
1
was obtained as a colourless oil (305.3 mg, 73%). H-NMR: () 7.78 (1H, s, H-6), 5.90 (1H, m,
H-2"), 5.16 (1H, dd, H-3", J1 = 1.3 Hz, J2 =10.3 Hz), 5.06 (1H, dd, H-3", J1 = 1.4 Hz, J2 =17.2 Hz),
4.08 (2H, t, H-2', J = 6.6 Hz), 4.36 (2H, d, H-1", J = 5.4 Hz), 3.83 (3H, s, OCH3), 2.57 (2H, t, H-1',
J = 6.5 Hz), 1.95 (3H, s, COCH3) ppm. 13C-NMR: () 170.7 (COCH3), 170.1 (C-4), 155.6 (C-2), 147.5
(C-6), 103.6 (C-5), 133.7 (C-2"), 117.8 (C-3"), 62.6 (C-2'), 54.5 (OCH3), 51.1 (C-1"), 26.0 (C-1'), 21.1
(COCH3) ppm. MS (ESI): m/z = 253.1 ([M+H]+).

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5-(2-Acetoxyethyl)-N-1-(2,3-dihydroxypropyl)-4-methoxypyrimidin-2-one (18). Method A: OsO4 (1 mg,
0.004 mmol) was added to a solution of compound 17 (203.5 mg, 0.81 mmol), followed by addition of
sodium chlorate (103 mg, 0.97 mmol) dissolved in water (2.5 mL). The reaction mixture was stirred at
room temperature for 3 days. The mixture was then filtered through Celite and applied to an Amberlite
IR 4 B column using water as eluens. The product was evaporated to dryness and applied on a Dowex
(100–200 mesh, H+-form) column and eluted with water. The eluate was evaporated under reduced
pressure and the residue was purified by column chromatography (CH2Cl2–CH3OH = 10:1) to yield
1
compound 18 as a yellow oil (115.5 mg, 50%). H-NMR: () 7.71 (1H, s, H-6), 4.95 (1H, d, OH,
J = 5.5 Hz), 4.95 (1H, t, OH, J = 5.4 Hz), 4.024.11 (3H, m, H-2', H-3"), 3.84 (3H, s, OCH3),
3.703.78 (1H, m, H-2"), 3.423.48 (1H, m, H-3"), covered with water (H-1"), 2.58 (2H, t, H-1',
J = 6.5 Hz), 1.98 (3H, s, COCH3) ppm. 13C-NMR: () 170.8 (COCH3), 170.0 (C-4), 156.0 (C-2), 149.2
(C-6), 102.4 (C-5), 69.2 (C-2"), 64.1 (C-3"), 62.6 (C-2'), 54.4 (OCH3), 53.0 (C-1"), 25.9 (C-1'), 21.1
(COCH3) ppm. MS (ESI): m/z = 287.1 ([M+H]+).
5-(2-Hydroxyethyl)-N-1-(2,3-dihydroxypropyl)pyrimidin-2,4-dione (19). Method A: Compound 18
(92.4 mg, 0.32 mmol) was dissolved in 1 M NaOH (8 mL) and the mixture was stirred at room
temperature for 20 h. Then, it was neutralized by the addition of acetic acid. After evaporation of the
solvents and column chromatography (CH2Cl2–CH3OH = 5:1) white crystals of compound 19 were
obtained (29 mg, 39%, m.p.: 9598 °C).
Method B: Compound 24 (164 mg, 0.67 mmol) was dissolved in 1 M NaOH (20 mL) and the
mixture was stirred at room temperature for 20 h and neutralized by the addition of HCl. After
evaporation of the solvent and column chromatography (CH2Cl2–CH3OH = 5:1), compound 19 was
1
obtained (57.7 mg, 37%). H-NMR: () 11.18 (1H, bs, NH), 7.36 (1H, s, H-6), 4.95 (1H, d, OH,
J = 5.5 Hz), 4.69 (1H, t, OH, J = 5.6 Hz), 4.53 (1H, t, OH, J = 5.6 Hz), 3.89 (1H, dd, H-3", J1 = 3.8 Hz,
J2 =13.3 Hz), 3.643.72 (1H, m, H-2"), 3.45 (2H, q, H-2', J = 6.7 Hz), 3.363.41 (3H, m, H-3", H-1"),
13
2.292.34 (2H, m, H-1') ppm. C-NMR: () 164.6 (C-4), 151.5 (C-2), 144.3 (C-6), 109.3 (C-5), 69.6
(C-2"), 64.1 (C-3"), 60.0 (C-2'), 51.4 (C-1"), 30.4 (C-1') ppm. MS (ESI): m/z = 231.1 ([M+H]+). Anal.
Calcd for C9H14N2O5: C, 46.95; H, 6.13; N, 12.17. Found: C, 46.90; H, 6.15; N, 12.22.
N-1-[(2-Acetoxyethoxy)methyl]-5-(2-acetoxyethyl)pyrimidin-2,4-dione (20). Method A: To a stirred
solution of compound 10 (47.2 mg, 0.21 mmol) and Na2CO3 (21.9 mg, 0.2 mmol) in anhydrous
CH2Cl2 (1 mL), (2-acethoxyethoxy)methyl bromide [prepared in situ by adding acetyl bromide (0.02 mL,
0.2 mmol) to 1,3-dioxolane (0.01 mL, 0.2 mmol)] was added. The reaction mixture was stirred overnight
at room temperature and evaporated to dryness. After column chromatography (CH2Cl2–CH3OH = 20:1)
compound 20 was isolated as a crude product (28.2 mg, 43%, m.p.: 5355 °C). 1H-NMR: () 11.41 (1H,
bs, NH), 7.64 (1H, s, H-6), 5.09 (2H, s, H-1"), 4.064.17 (4H, m, H-3", H-4"), 3.69 (2H, t, H-2',
J = 4.6 Hz), 2.70 (2H, t, H-1', J = 6.4 Hz), 1.99 (3H, s, COCH3), 1.98 (3H, s, COCH3) ppm. 13C-NMR:
() 170.2 (COCH3), 163.7 (C-4), 150.9 (C-2), 142.0 (C-6), 109.4 (C-5), 76.3 (C-1"), 66.7 (C-3"), 62.9
(C-4"), 61.9 (C-2’), 25.8 (C-1'), 20.6 (COCH3), 20.6 (COCH3) ppm. MS (ESI): m/z = 315.1 ([M+H]+).
N-1-[(2-Hydroxyethoxy)methyl]-5-(2-hydroxyethyl)pyrimidin-2,4-dione (21). Method A: To a solution
of sodium (2.1 mg, 0.09 mmol) in anhydrous methanol (0.5 mL) compound 20 (27 mg, 0.09 mmol)

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dissolved in methanol (1 mL) was added. The reaction mixture was stirred at room temperature for 1 h
and neutralized with 1 M HCl/ether. After evaporation to dryness and column chromatography
(CH2Cl2–CH3OH = 5:1) compound 21 was isolated (14.6 mg, 74%) as a colourless oil.
Method B: Compound 25 (37 mg, 0.15 mmol) was dissolved in 1 M NaOH (10 mL) and the mixture
was stirred at room temperature for 20 h. Then, it was neutralized by the addition of acetic acid. After
evaporation of the solvents and column chromatography (ethyl acetate–CH3OH = 5:1) compound 21
1
was obtained (9.1 mg, 26%). H-NMR: () 11.25 (1H, bs, NH), 7.52 (1H, s, H-6), 5.08 (2H, s, H-1"),
4.60 (2H, bs, OH), 3.453.50 (6H, m, H-2', H-3", H-4"), 2.35 (2H, t, H-1', J = 6.6 Hz) ppm. 13C-NMR:
() 164.5 (C-4), 151.5 (C-2), 142.0 (C-6), 111.0 (C-5), 77.0 (C-1"), 71.0 (C-3"), 60.5 (C-4"), 60.0 (C-2'),
30.3 (C-1') ppm. MS (ESI): m/z = 231.1 ([M+H]+). Anal. Calcd for C9H14N2O5: C, 46.95; H, 6.13; N,
12.17. Found: C, 46.99; H, 6.15; N, 12.12.
5-(2-Acetoxyethyl)-N-1-[(1,3-dibenzyloxy-2-propoxy)methyl]pyrimidin-2,4-dione (22). Method A: To a
stirred solution of compound 10 (114 mg, 0.50 mmol) and K2CO3 (186.4 mg, 1.35 mmol) in anhydrous
CH2Cl2 (3.7 mL), 1,3-dibenzyloxy-2-chloromethoxypropane (257.6 mg, 0.8 mmol) was added under
argon atmosphere. The reaction mixture was stirred overnight at room temperature and after filtration
evaporated to dryness. After column chromatography (CH2Cl2–CH3OH = 35:1) compound 22 was
isolated as a colourless oil (87 mg, 36%). 1H-NMR: () 11.38 (1H, s, NH), 7.63 (1H, s, H-6), 7.40–7.22
(10H, m, Ph), 5.2 (2H, s, H-1"), 4.46 (4H, s, H-5"), 4.03 (2H, t, H-2', J = 6.8 Hz), 3.993.94 (1H, m,
H-3"), 3.573.40 (4H, m, H-4"), 2.44 (2H, t, H-1', J = 6.8 Hz), 1.98 (3H, s, COCH3) ppm. 13C-NMR:
() 170. 7 (COCH3), 164.2 (C-4), 151.5 (C-2), 143.9 (C-6), 138.7 (Ph), 128.7 (Ph), 127.9 (Ph), 127.8
(Ph), 109.7 (C-5), 77.2 (C-3"), 76.5 (C-1"), 72.7 (C-5"), 70.3 (C-4"), 62.4 (C-2'), 26.2 (C-1'), 21.1
(COCH3) ppm. MS (ESI): m/z = 483.2 ([M+H]+).
5-(2-Hydroxyethyl)-N-1-[(1,3-dihydroxy-2-propoxy)methyl]pyrimidin-2,4-dione (23). Method A: A
solution of compound 22 (63 mg, 0.13 mmol) in anhydrous CH2Cl2 (5 mL) was cooled to −68 °C and
1 M solution of BCl3 in CH2Cl2 (0.45 mL, 0.45 mmol) was added under argon atmosphere. The
reaction mixture was stirred at −68 °C for 3 h, quenched by the addition of CH2Cl2–CH3OH (1:1)
solution (15 mL) and allowed to warm to room temperature. The solvent was then evaporated and the
residue chromatographed (CH2Cl2–CH3OH = 25:1). Compound 23 was isolated as a white solid (22.7 mg,
67%, m.p.: 118120 °C).
Method B: In the same way as described for 22, the deprotection of 26 was performed. The reagents
used were: compound 26 (106 mg, 0.24 mmol), anhydrous CH2Cl2 (5 mL) and BCl3 (0.7 mL, 0.7 mmol).
After column chromatography (CH2Cl2–CH3OH = 10:1) compound 23 (40.9 mg, 65%) was isolated.
1H-NMR: () 11.27 (1H, s, NH), 7.53 (1H, s, H-6), 5.16 (2H, s, H-1"), 4.644.59 (3H, m, 3×OH),
13
3.523.40 (7H, m, H-2', H-4", H-3") 2.35 (2H, t, H-1', J = 6.6 Hz) ppm. C-NMR: () 164.5 (C-4),
151.4 (C-2), 142.1 (C-6), 110.7 (C-5), 80.9 (C-3"), 76.3 (C-1"), 61.4 (C-4"), 59.9 (C-2'), 30.3 (C-1')
ppm. MS (ESI): m/z = 261.1 ([M+H]+). Anal. Calcd for C10H16N2O6: C, 46.15; H, 6.20; N, 10.76.
Found: C, 46.10; H, 6.22; N, 10.72.
5-(2-Hydroxyethyl)-N-1-(2,3-dihydroxypropyl)-4-methoxypyrimidin-2-one (24). Method B: A mixture
of compound 14 (194.4 mg, 0.91 mmol) and sodium hydride (90.9 mg, 2.27 mmol, 60% dispersion in

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oil) in DMF (6 mL) was stirred at 60 °C for 1 h. To the resulting suspension glycerol α-chlorohydrine
(0.6 mL, 6.82 mmol) was added dropwise and the mixture was stirred at 70 °C overnight. The solvent
was evaporated under reduced pressure and after column chromatography (CH2Cl2–CH3OH = 5:1)
1
compound 24 (166.9 mg, 75%) was obtained. H-NMR: () 7.63 (1H, s, H-6), 4.92 (1H, d, OH,
J = 5.8 Hz), 4.71 (1H, t, OH, J = 5.8 Hz), 4.53 (1H, t, OH, J = 5.5 Hz), 4.05 (1H, dd, H-3", J1 = 3.3 Hz,
J2 = 13.0 Hz), 3.83 (3H, s, OCH3), 3.693.71 (1H, m, H-2"), 3.53.4 (2H, m, H-2'), covered with water
(H-3", H-1"), 2.40 (2H, t, H-1', J = 6.4 Hz) ppm. 13C-NMR: () 170.2 (C-4), 156.9 (C-2), 148.8 (C-6),
103.6 (C-5), 69.3 (C-2"), 64.1 (C-3"), 60.1 (C-2'), 54.3 (OCH3), 53.0 (C-1"), 30.0 (C-1') ppm. MS
(ESI): m/z = 245.1 ([M+H]+).
N-1-[(2-Hydroxyethoxy)methyl]-5-(2-hydroxyethyl)-4-methoxypyrimidin-2-one (25). Method B: To a
stirred solution of compound 16 (110 mg, 0.65 mmol) in acetonitrile (6 mL) under exclusion of
moisture N,O-bis-trimethysilylacetamide (BSA: 0.8 mL, 3.2 mmol) was added. The mixture was
stirred until a clear solution appeared (about 10 min). Dioxolane (0.12 mL, 1.66 mmol), KI (139 mg,
0.83 mmol) and TMSCl (0.1 mL) were added sequentially. The stirring was continued for 48 h at room
temperature. The volatile materials were evaporated under reduced pressure and the residue was
purified using column chromatography (CH2Cl2–CH3OH = 5:1) to give compound 25 (39.1 mg, 25%).
1H-NMR: () 7.78 (1H, s, H-6), 5.17 (2H, s, H-1"), 4.62 (1H, t, OH, J = 5.4 Hz), 4.59 (1H, t, OH,
J = 5.4 Hz), 3.453.56 (4H, m, H-3", H-4"), 3.84 (3H, s, OCH3), 3.62 (2H, t, H-2', J = 6.4 Hz), 2.45
13
(2H, t, H-1', J = 6.6 Hz) ppm. C-NMR: () 171.7 (C-4), 155.8 (C-2), 146.8 (C-6), 105.1 (C-5), 78.3
(C-1"), 71.3 (C-3"), 60.5 (C-4"), 59.8 (C-2'), 54.7 (OCH3), 29.9 (C-1') ppm. MS (ESI): m/z = 245.1
([M+H]+).
5-(2-Hydroxyethyl)-N-1-[(1,3-dibenzyloxy-2-propoxy)methyl]pyrimidin-2,4-dione (26) and 5-(2-
chloroethyl)-N-1-[(1,3-dibenzyloxy-2-propoxy)methyl]pyrimidin-2,4-dione (27). Method B: A mixture
of compound 1 (1.3 g, 8.33 mmol), hexamethyldisilazane (30 mL), trimethylsilyl chloride (0.58 mL,
4.57 mmol) and (NH4)2SO4 (416 mg, 3.15 mmol) was refluxed for 24 h under argon atmosphere. The
resultant solution was evaporated in vacuum using oil pump. To the residual oil in acetonitrile (50 mL),
TBAI (73.7 mg, 0.2 mmol) and 1,3-dibenzyloxy-2-chloromethoxypropane (4 g, 12.45 mmol) were
added under argon atmosphere. The reaction mixture was stirred at reflux overnight. After the reaction
was complete the solvent was evaporated and the residue purified using column chromatography
(CH2Cl2–CH3OH = 35:1) to obtain compounds 26 (289.6 mg, 8%) and 27 (87.3 mg, 2%).
1
26: H-NMR: () 11.30 (1H, s, NH), 7.53 (1H, s, H-6), 7.347.28 (10H, m, Ph), 5.21 (2H, s, H-1"),
4.56 (1H, t, OH, J = 5.4 Hz), 4.46 (4H, s, H-5"), 4.03.9 (1H, m, H-3") 3.503.40 (6H, m, H-2', H-4"),
2.30 (2H, t, H-1', J = 6.7 Hz) ppm. 13C-NMR: () 164.4 (C-4), 151.6 (C-2), 142.1 (C-6), 138.7 (C-Ph),
128.7 (C-Ph), 127.8 (C-Ph), 127.8 (C-Ph), 111.0 (C-5), 77.1 (C-3"), 76.5 (C-1"), 72.7 (C-5"), 70.3
(C-4"), 60.0 (C-2'), 30.3 (C-1') ppm. MS (ESI): m/z = 441.3([M+H]+).
27: 1H-NMR: () 11.43 (1H, s, NH), 7.69 (1H, s, H-6), 7.387.2 (10H, m, Ph), 5.20 (2H, s, H-1"), 4.46
(4H, s, H-5"), 4.043.95 (1H, m, H-3"), 3.62 (2H, t, H-2', J = 6.9 Hz), 3.553.40 (4H, m, H-4"), 2.58
13
(2H, t, H-1', J = 7.0 Hz) ppm. C-NMR: () 164.1 (C-4), 151.5 (C-2), 143.0 (C-6), 138.7 (C-Ph),

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128.7 (C-Ph), 127.8 (C-Ph), 127.7 (C-Ph), 109.8 (C-5), 77.2 (C-3"), 76.6 (C-1"), 72.7 (C-5"), 70.3
(C-4"), 43.5 (C-2'), 30.2 (C-1') ppm. MS (ESI): m/z = 459. 2 ([M+H]+).
5-(2-Chloroethyl)-N-1-[(1,3-dihydroxy-2-propoxy)methyl]pyrimidin-2,4-dione (28). Method B: In the
similar way as described for compound 23, the synthesis of 28 was performed. The reagents used
were: compound 27 (43.7 mg, 0.1 mmol), anhydrous CH2Cl2 (8 mL) and BCl3 (0.7 mL, 0.7 mmol).
After column chromatography (CH2Cl2–CH3OH = 5:1) compound 28 (15 mg, 57%) was isolated.
1H-NMR: () 11.36 (1H, s, NH), 7.65 (1H, s, H-6), 5.13 (2H, s, H-1"), 4.58 (2H, t, 2×OH, J = 5.5 Hz),
3.66 (2H, t, H-2', J = 6.9 Hz), 3.513.32 (5H, m, H-3", H-4"), 2.62 (2H, t, H-1', J = 6.9 Hz) ppm.
13C-NMR: () 164.2 (C-4), 151.3 (C-2), 143.2 (C-6), 110.7 (C-5), 81.0 (C-3"), 76.4 (C-1"), 61.4
(C-4"), 43.6 (C-2'), 30.3 (C-1') ppm. MS (ESI): m/z = 279.2([M+H]+).
N-1-[4-Acetoxy-(3-acetoxymethyl)butyl]-5-(2-acetoxyethyl)-4-methoxypyrimidin-2-one (29). To a stirred
solution of compound 14 (76.3 mg, 0.36 mmol) and K2CO3 (74.5 mg, 0.54 mmol) in anhydrous DMF
(7.6 mL), 4-acetoxy-(3-acetoxymethyl)butyl iodide (192 mg, 0.61 mmol) was added under argon
atmosphere. The reaction mixture was stirred at 60 °C for 20 min and then evaporated to dryness.
After column chromatography (CH2Cl2–CH3OH = 30:1) compound 29 was isolated as a colourless oil
1
(87.1 mg, 60%). H-NMR: () 7.89 (1H, s, H-6), 4.12 (2H, t, H-2', J = 6.6 Hz), 4.03 (4H, d, H-4",
J = 5.8 Hz), 3.85 (3H, s, OCH3), 3.83 (2H, t, H-1", J = 7.7 Hz), 2.60 (2H, t, H-1', J = 6.6 Hz), 2.02
(6H, s, 2×COCH3), 1.99 (3H, s, COCH3), within COCH3 signal (H-3") 1.67 (2H, q, H-2", J = 7.1 Hz)
ppm.13C-NMR: () 170.8 (COCH3), 169.9 (C-4), 155.6 (C-2), 147.7 (C-6), 103.3 (C-5), 64.0 (C-4"),
62.6 (C-2'), 54.3 (OCH3), 47.2 (C-1"), 34.9 (C-3"), 27.9 (C-1') 26.0 (C-2"), 21.1 (COCH3) ppm. MS
(ESI): m/z = 399.2 ([M+H]+).
N-1-[4-Acetoxy-(3-acetoxymethyl)butyl]-5-(3-acetoxypropyl)-4-methoxypyrimidin-2-one (30). The
synthesis of 30 was performed as described for 29. The reagents used were: compound 15 (1.54 g,
6.81 mmol), K2CO3 (1.41 g, 10.2 mmol), anhydrous DMF (40 mL) and 4-acetoxy-(3-
acetoxymethyl)butyl iodide (3.63 g, 11.6 mmol). After column chromatography (CH2Cl2–CH3OH =
1
50:1) compound 30 was isolated as a yellow oil (1.15 g, 41%). H-NMR: () 7.83 (1H, s, H-6),
4.043.98 (6H, m, H-4", H-3'), 3.843.80 (5H, m, OCH3, H-1"), 2.34 (2H, t, H-1', J = 7.5 Hz), 2.02
(6H, s, 2×COCH3), 2.00 (3H, s, COCH3), within COCH3 signal (H-3"), 1.78 (2H, k, H-2', J = 7.0 Hz),
13
1.67 (2H, q, H-2", J = 7.2 Hz) ppm. C-NMR: () 170.9 (COCH3), 170.1 (C-4), 155.7 (C-2), 146.6
(C-6), 106.5 (C-5), 64.1 (C-4"), 63.6 (C-3'), 54.4 (OCH3), 47.2 (C-1"), 34.9 (C-3"), 27.9 (C-2'), 27.7
(C-2"), 22.9 (C-1'), 21.1 (COCH3), 21.1 (COCH3) ppm. MS (ESI): m/z = 413.2 ([M+H]+).
5-(2-Hydroxyethyl)-N-1-[4-hydroxy-(3-hydroxymethyl)butyl]pyrimidin-2,4-dione (31). The synthesis
of 31 was performed as described for 19. The reagents used were: compound 29 (62.4 mg, 0.16 mmol)
and 1 M NaOH (5 mL). After column chromatography (CH2Cl2–CH3OH = 5:1) compound 31 was
obtained as a white solid (28.2 mg, 70%, m.p.: 8384 °C). 1H-NMR: () 11.15 (1H, bs, NH), 7.46 (1H,
s, H-6), 4.54 (1H, t, OH, J = 5.5 Hz), 4.40 (2H, t, 2×OH, J = 5.2 Hz), 3.69 (2H, t, H-1", J = 7.6 Hz),
3.45 (2H, q, H-2', J = 6.3 Hz), 3.423.32 (4H, m, H-4"), 2.32 (2H, t, H-1', J = 6.7 Hz), 1.55 (2H, q,
H-2", J = 7.3 Hz), 1.47 (1 H, m, H-3", J = 5.9 Hz) ppm. 13C-NMR: () 164.5 (C-4), 151.2 (C-2), 142.9
(C-6), 109.3 (C-5), 61.9 (C-4"), 59.9 (C-2'), 46.2 (C-1"), 41.2 (C-3"), 30.4 (C-1'), 28.2 (C-2") ppm. MS

Molecules 2013, 18
5119
(ESI): m/z = 259.1 ([M+H]+). Anal. Calcd for C11H18N2O5: C, 51.15; H, 7.02; N, 10.85. Found: C,
51.10; H, 6.99; N, 10.89.
5-(3-Hydroxypropyl)-N-1-[4-hydroxy-(3-hydroxymethyl)butyl]pyrimidin-2,4-dione (32). The synthesis
of 32 was performed in the same way as described for 31. The reagents used were: compound 30 (204 mg,
0.49 mmol) and 1 M NaOH (5 mL). After column chromatography (CH2Cl2–CH3OH = 5:1) compound
32 was obtained as white crystals (110 mg, 82%, m.p.: 113114 °C). 1H-NMR: () 11.14 (1H, s, NH),
7.46 (1H, s, H-6), 4.434.38 (3H, m, OH), 3.70 (2H, t, H-1", J = 7.3 Hz), 3.463.30 (6H, m, H-4",
H-3'), 2.22 (2H, t, H-1', J = 7.4 Hz), 1.631.42 (5H, H-2', H-2", H-3") ppm. 13C-NMR: () 164.4 (C-4),
151.2 (C-2), 141.9 (C-6), 113.0 (C-5), 61.9 (C-4"), 60.6 (C-3'), 46.2 (C-1"), 41.2 (C-3"), 31.9 (C-2'),
28.3 (C-2"), 23.3 (C-1') ppm. MS (ESI): m/z = 273.2 ([M+H]+). Anal. Calcd for C12H20N2O5: C, 52.93;
H, 7.40; N, 10.29. Found: C, 52.97; H, 7.43; N, 10.33.
3.3. Phosphorylation Assay of 19, 21, 23, 31 and 32
3.3.1. Protein Expression
The HSV-1 TK was expressed in E. coli as a GST-TK fusion protein and further purified as HSV-1
TK-GST protein by using glutathione affinity chromatography following protocols that have been
reported previously [33]. The human thymidine kinase 1 (hTK) was expressed in E. coli as a
6xHisTagged protein and purified following the previously published protocol [34].
3.3.2. HPLC System and Conditions
The high liquid performance chromatography system used Merck HITACHI LaChrom (Gynkotek
HPLC, Münich, Germany) equipped with a pump (L-7100), autosampler (L-7200) and a UV detector
(L-7400). The analyses were performed on an LiChroCART® 250-4 cartridge column packed with a
stationary phase made of spherical partical of silica (LiCrospher® 100 RP-18 endcapped, (5 µm)) and
coupled with a guard column LiChroCART® 4-4 of the same type (Merck, KGaA, Darmstadt,
Germany). Mobile phase consisted of 200 mM NaH2PO4, 25 mM tetrabutylammonium hydrogen
sulfate (TBAHS) as the ion-pairing reagent and 0.5% methanol (HPLC grade). All aqueous solutions
were prepared using de-ionized water from Millipore. The column was kept at a constant temperature
of 20 °C. The separation was performed isocratically at a flow-rate of 1.1 mL/min and the UV
detection was done at 254 nm [32].
3.3.3. The Phosphorylation Reaction
Phosphorylation of dT and compounds 19, 21, 23, 31, fluorinated derivative of 31 and 32 was
monitored by HPLC. Reactions were carried out in a final volume of 70 µL containing 50 mM HEPES
pH 7.5, 5 mM ATP, 5 mM MgCl2, 1 mM solution of dT and tested compounds and either 11 µg of
purified HSV-1 TK-GST or 0.8 µg of hTK. As the solutions of dT and tested compounds were
prepared with 100% DMSO attention was paid for not adding more than 5% DMSO in the sample
reaction (the final concentration of DMSO was 1%). The reaction was started with the addition of
HSV-1 TK-GST. The reaction was done at 37 °C and was stopped after 30, 60 and 90 min by a 10-fold

Molecules 2013, 18
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dilution with 50 mM EDTA (final concentration 5 mM) used to chelate the cofactor Mg2+. Each
reaction was done in triplicate. Finally, a volume of 40 µL of the sample reaction was injected for
analysis. The formation of the nucleotide monophosphate was monitored qualitatively. Two different
blank reactions (no enzyme or no compound) were performed under the same experimental conditions
to account for the background ATP hydrolysis. Control reactions were stopped after 30, 60 and 90 min
(Supplementary Materials). The resulting ADP/ATP ratios were calculated and used as a measure for
protein activity. The mean of at least triplicate measurements are reported.
3.3.4. Cellular Activity Evaluations
The WI38 (normal diploid human fibroblasts) cell lines were cultured as monolayers and
maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine
serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin in a humidified
atmosphere with 5% CO2 at 37 C. The panel cell lines were inoculated onto a series of standard
96-well microtiter plates on day 0, at 3000 cells to 6000 cells per well according to the doubling times
of specific cell line. Test agents were then added in five, 10-fold dilutions (1 × 108 to 1 × 104 M) and
incubated for further 72 h. Working dilutions were freshly prepared on the day of testing in the growth
medium. The solvent (DMSO) was also tested for eventual inhibitory activity by adjusting its
concentration to be the same as in the working concentrations (DMSO concentration never exceeded
0.1%). After 72 h of incubation, the cell growth rate was evaluated by performing the MTT assay:
experimentally determined absorbance values were transformed into a cell percentage growth (PG)
using the formulas proposed by NIH and described previously [35].This method directly relies on
control cells behaving normally at the day of assay because it compares the growth of treated cells with
the growth of untreated cells in control wells on the same plate—the results are therefore a percentile
difference from the calculated expected value. The IC50 and LC50 values for each compound were
calculated from dose-response curves using linear regression analysis by fitting the mean test
concentrations that give PG values above and below the reference value. If, however, all of the tested
concentrations produce PGs exceeding the respective reference level of effect (e.g., PG value of 50)
for a given cell line, the highest tested concentration is assigned as the default value (in the screening
data report that default value is preceded by a “>” sign). Each test point was performed in
quadruplicate in three individual experiments. The results were statistically analyzed (ANOVA, Tukey
post-hoc test at p < 0.05). Finally, the effects of the tested substances were evaluated by plotting the
mean percentage growth for each cell type in comparison to control on dose-response graphs.
4. Conclusions
5-(2-Hydroxyethyl)- and 5-(3-hydroxypropyl)-substituted 4-methoxypyrimidin-2-ones (10 and
1416) were prepared by an efficient synthetic route and subjected to N-1 alkylation to obtain target
compounds bearing 2,3-dihydroxypropyl (19), acyclovir- (21), ganciclovir- (23), and penciclovir-like
side chains (compounds 31, 32). The two synthetic methods A and B that were applied for
N-alkylation indicated that method A using activated N-anionic 5-substituted pyrimidine derivatives
was more successful than the silylation reaction method.

Molecules 2013, 18
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The influence of the acyclic glycone in target compounds 19, 21, 23, 31 and 32 on their
phosphorylation ability was observed. Thereby, 5-substituted pyrimidine derivatives 31 and 32 bearing
N-1 penciclovir-like side chain revealed to be substrates of HSV-1 TK. The structural congener of 31
bearing an oxygen isosteric replacement of methylene group in side chain of 23 was not
phosphorylated by HSV-1 TK. Moreover, 5-(2-hydroxyethyl)pyrimidin-2,4-dione 31 showed to be a
better substrate when compared to compound 32 with a longer substituent bearing an additional
methylene group.
Besides, both compounds 31 and 32 exhibited no cytotoxic effect against normal human fibroblasts
up to 100 µM. Compound 31 has been selected as a lead compound for development as a new PET
probe for imaging HSV-1 TK expression. Therefore, the fluorine-18 radiolabeling of the corresponding
pyrimidine precursor of 31 and the subsequent in vivo PET studies are currently underway.
Supplementary Materials
Supplementary Materials containing HPLC chromatograms of phosphorylation reaction of
compounds 19, 21 and 23 with HSV-1 TK and hTK, and HPLC chromatograms for time conversion
(30, 60 and 90 min of incubation) in phosphorylation of dT, 31 and its fluorinated derivative, as well as
blank reactions (no enzyme or no substrate) can be accessed at: http://www.mdpi.com/1420-3049/
18/5/5104/s1.
Acknowledgments
This study was performed in the framework of the SCOPES 2009–2012 programme (Swiss
National Science Foundation). Support of this study by the Ministry of Science, Education and Sports
of the Republic of Croatia (Project #125-0982464-2925) is also gratefully acknowledged. We thank
Sandra Kraljević-Pavelić, Ivana Ratkaj and Mirela Sedić in performing the cellular activity evaluations
of 17–32.
Conflict of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 132 are available from the authors.
© 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).