Fluorosubstitution and 7-alkylation as prospective modifications of biologically active 6-aryl derivatives of tricyclic acyclovir and ganciclovir analogues

Article abstract of DOI:10.1016/j.bmc.2005.01.004

A series of fluorine containing tricyclic analogues of acyclovir (ACV, 1) and ganciclovir (GCV, 2) were synthesized and evaluated for their activity against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) and cytostatic activity against HSV-1 thymi

Full text of DOI:10.1016/j.bmc.2005.01.004

Bioorganic & Medicinal Chemistry 13 (2005) 2089–2096
Fluorosubstitution and 7-alkylation as prospective
modifications of biologically active 6-aryl derivatives of
tricyclic acyclovir and ganciclovir analogues
Tomasz Ostrowski,a Bozenna Golankiewicz,a,* Erik De Clercqb and Jan Balzarinib
aInstitute of Bioorganic Chemistry, Polish Academy of Sciences, ul.Noskowskiego 12/14, 61-704 Poznan, Poland
bRega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
Received 30 July 2004; accepted 7 January 2005
Available online 2 February 2005
Abstract—A series of fluorine containing tricyclic analogues of acyclovir (ACV, 1) and ganciclovir (GCV, 2) were synthesized and
evaluated for their activity against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) and cytostatic activity against HSV-1
thymidine kinase (TK) gene-transduced human osteosarcoma tumour cells. It was found that fluorine substitution reduced the anti-
viral activity, but most of the new compounds were pronounced cytostatic agents with potency and selectivity similar to those of
parental ACV and GCV. Compounds 12, 13 and 16 seem to be promising as labeled substrates for 19F NMR studies of the
HSV TK–ligand interaction and/or monitoring of their metabolites in cells expressing HSV TK.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
and HSV-2 and subjected to a structure–antiviral activ-
ity analysis, 6-(4-MeOPh)-TACV (7) and 7-Me-6-Ph-
TACV (8) and their ganciclovir congeners (9 and 10,
respectively) showed similar potency and selectivity as
the parent compounds. Additionally, they are fluores-
cent and more lipophilic.3 Further studies using 6-
(4-MeOPh)-TACV and 6-(4-MeOPh)-TGCV as lead
compounds, recently yielded 6-(4-acyloxyphenyl)-TACV
and TGCV analogues, which exhibit stronger fluores-
cence and antiviral activity similar to that of parent
ACV and GCV.4 Moreover, they have been identified
as potent and highly selective cytostatic agents against
HSV-1-thymidine kinase (TK) gene-transduced human
osteosarcoma and murine mammary carcinoma tumour
cells.5
Our previous studies on modification of the guanine
moiety of the antiherpetic drugs acyclovir (ACV, 1)
and ganciclovir (GCV, 2) in order to modulate their
physical and biological properties have shown that intro-
ducing of 1,N2-(ethene-1,2-diyl) bridge to transform 1
and 2 into their tricyclic (T) analogues (TACV, 3 and
TGCV, 4), derivatives of 3,9-dihydro-9-oxo-5H-
imidazo[1,2-a]purine, together with an appropriate sub-
stituent at positions 6 and/or 7 of the appended ring may
lead to this aim.1,2 At the early stage of this search fluo-
rescent 6-phenyl-TACV (5) and 6-phenyl-TGCV (6)
appeared to be the most promising. The activity of
compound 6 against herpes simplex virus type 1 (HSV-
1) and type 2 (HSV-2) was very similar to that of the
parent ganciclovir, compound 5 being approximately
one order of magnitude less active than the parent
acyclovir.2
In the present work we prepared a further series of
TACV and TGCV derivatives using the aforementioned
lead compounds 7–10-derivatives bearing fluorine (com-
pounds 11–14) or trifluoromethyl group (15–17). We
added to this series two nonfluorinated congeners: 18,
an analogue of compound 14, and 19, thus combining
the structural features of both lead compounds 7 and
8 (Chart 1).
Of the next series of over 20 new tricyclic analogues,
which have been evaluated for activity against HSV-1
Keywords: Cytostatic activity; HSV-1 TK gene therapy; Tricyclic
acyclovir analogues; Fluorosubstitution; Anti-HSV-1, HSV-2 activity.
Selective fluorination has been a very effective tool for
modifying the reactivity of diverse organic compounds.
It has been known that it can have profound and
*
Corresponding author. Tel.: +48 61 8528503; fax: +48 61 8520532;
e-mail: bogolan@ibch.poznan.pl
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.01.004

2090
T. Ostrowski et al. / Bioorg. Med. Chem. 13 (2005) 2089–2096
cancer gene therapy, 19F NMR can also be considered
for imaging of the level and distribution of fluorinated
tricyclic nucleoside prodrug metabolites.
R3
O
O
1
9
6
7
8
N
7N
9N
N
1
NH
5
4
6
R2
8
2
2
3 N
R1
N 5
H
NH2
N3
N4
Introducing the benzyl or 4-fluorobenzyl group into the
7 position of the tricyclic system seemed of interest in
view of the potent in vitro antiviral activity found earlier
by others14,15 and by us16 for benzyl substituted purines
and their analogues.
R1
R1 R2
R3
H
H
H
H
H
CH3
H
CH3
H
H
CH3
4-FPhCH2
H
H
CH3
PhCH2
R1
A
B
A
B
A
B
A
A
B
B
A
B
A
A
A
B
A
A
A
H
H
Ph
Ph
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
1
2
2. Chemistry
4-CH3OPh
Ph
4-CH3OPh
Ph
All 6-mono and the majority of the 6,7-disubstituted tri-
cyclic analogues being presently studied were synthe-
sized by an alkylation–condensation reaction of ACV
or GCV with the appropriate brominated acetophe-
nones or propiophenones (Scheme 1), according to a
previously described method for other tricyclic deriva-
tives.2–4 In the case of the reaction of 1 with 2-bromo-
40-substituted propiophenones the yields of 6,7-disubsti-
tuted derivatives 13, 17 and 19 (28%, 19% and 25%,
respectively) were much lower in comparison with that
of corresponding 6-monosubstituted derivatives 11, 15
and 73 (88%, 71% and 81%, respectively). This was
due to the formation of the side products, the most
important of which being O6-alkylation products 130
and 170. This site of alkylation was unambiguously as-
cribed on the basis of diagnostic 1H and 13C NMR spec-
tra acquired from our recent work on dimers carrying
simultaneously O- and N-substituted guanine moie-
ties.17 For one of the above dimeric structures, for exam-
ple, the most conspicuous differences in NMR chemical
shifts (d in ppm) for O versus N regioisomers were,
respectively: 2-NH2, 6.45/7.08; C-5, 113.46/115.62; C-4,
154.28/149.41; C-2, C-6, 159.97, 160.43/153.73, 156.41.
The NMR data obtained now for compound 170 differ
from the above O-substitution only within the range of
4-FPh
4-FPh
4-FPh
Ph
HO
HO
O
A
B
O
4-CF3Ph
4-CF3Ph
4-CF3Ph
Ph
OH
4-CH3OPh CH3
Chart 1.
unexpected results. The literature on this topic is extre-
mely abundant. In the field of nucleosides there are hun-
dreds of analogues described that contain a fluorinated
glycone moiety, or are fluorinated at the nucleobase.6,7
As to the antiherpetic activity of acyclic analogues
examples have been reported in the literature of antivi-
rally efficient nucleobase-fluorinated derivatives6 and
of inefficient pseudosugar-fluorinated ones.8–10
19F NMR spectroscopy is a convenient tool for in vitro
and noninvasively in vivo identifying and monitoring
of fluorine-containing metabolites of drugs, for example,
fluoropyrimidine drugs.11 This technique also enables to
investigate the mode of binding of fluorinated substrates
to enzymes as well as conformational properties of com-
plexes formed.12 The main aim of our present synthetic
search was the exploration of new fluorine-substituted
tricyclic analogues of ACV and GCV as selective cyto-
static agents under conditions of HSV-1 TK gene ther-
apy. Since tricyclic analogues are so far the only
compounds for which the interaction with the HSV-1
thymidine kinase in solution has been measured by the
1H NMR spectrum,13 19F NMR can be used for studying
of the interaction between fluorinated nucleoside ana-
logues and HSV thymidine kinase. The advantage of
novel tricyclic acyclovir/ganciclovir analogues as tools
R3
O
N
i or ii
N
N
R2
N
R1
N
O
N
H
N
NH
NH2
11, 13-15, 17-19 R1 = A;
N
R1
12, 16 R1 = B; R2, R3 see Chart 1
CH3
R2
O
O
ii
N
N
N
R1
NH2
N
1
for H NMR enzyme–ligand studies over the existing
HO
O
A
B
ones is expected upon the following earlier results.13 1H
NMR spectra of the bound structure of the tricyclic
ligand with the HSV-1 thymidine kinase in solution have
been determined by transferred NOE technique. This
technique has not been successful neither in the case of
thymidine itself nor of acyclovir. Moreover, in view of
the progress on HSV thymidine kinase-mediated anti-
13', 17', 19'
R1 = A; R2 see Chart 1
HO
O
OH
Scheme 1. Reagents: (i) (1) NaH, DMF, R2COCH2Br, (2) NH4OH;
(ii) (1) NaH, DMF, R2COCH(Br)CH3, (2) NH4OH.

T. Ostrowski et al. / Bioorg. Med. Chem. 13 (2005) 2089–2096
2091
0.2–1.1 ppm. The aforementioned diagnostic data al-
lowed to correct the N-1 alkylated structure we assigned
formerly to the products of the reaction of 1 and 2 with
2-bromopropiophenone.3
variety of viruses including HSV-1 (strains KOS, F
and McIntyre), HSV-2 (strains G, 196 and Lyons), vac-
cinia virus (VV), vesicular stomatitis virus (VSV), HSV-
1 TKꢀ ACVr (TK-deficient acyclovir-resistant HSV-1)
strain, varicella-zoster virus (VZV) (strain Oka), TKꢀ
VZV mutants (strain 07-01) and cytomegalovirus
(CMV) (strains AD-169 and Davis) in human embry-
onic lung (HEL) cell cultures.
6,7-Disubstituted compounds 13, 17 and 19 were pre-
pared analytically pure in spite of their relative instabil-
ity under standard conditions of chromatography and
crystallization. We also tried to obtain their GCV ana-
logues but these products underwent decomposition in
the course of purification.
In general, introduction of a fluorine (11) or trifluorom-
ethyl (15) group in the 4 position of the 6-phenyl substi-
tuent of 5 decreased or abrogated antiherpetic activity.
The compounds were found inactive against VZV,
CMV, VV and VSV. Their activity against human her-
pes simplex virus types 1 and 2 and cytotoxicity are
shown in Table 1. For 6-(4-fluorophenyl)-TACV, 11,
there was a substantial loss of activity against HSV-1
and -2 in comparison with the activity of 4-unsubsti-
tuted 5 (EC50: 0.2–0.7 lg/mL for HSV-1 and 0.2–
1.3 lg/mL for HSV-2). Similarly, compound 12 (EC50
for HSV-1 and HSV-2: 0.384–1.92 lg/mL) and com-
pound 16 (EC50: 0.38–9.6 lg/mL) were less potent than
6 (EC50: 0.005–0.3 lg/mL) by two orders of magnitude.
Both TACV analogues (11 and 15) and TGCV ana-
logues (12 and 16) were relatively cytotoxic.
7-Arylalkyl-6-phenyl derivatives 14 and 18 were ob-
tained by arylalkylation of 6-phenyl-TACV (5). We have
reported previously that aryl substituent in the 6 posi-
tion of TACV partly directs arylalkylation reactions
into unusual positions.18 Benzylation of 5 with benzyl
bromide/K2CO3/DMF led to N-5 substituted dominant
product (18a) in 41% yield, whereas its N-4 isomer (18b,
6%) and C-7,N-5 disubstituted derivative (18c, 9%), but
not C-7 one, were minor products. Literature on aryl-
alkylation of protected or unprotected guanosine or
20-deoxyguanosine with benzyl bromide and related bro-
mides under alkaline conditions reports on using a vari-
ety of bases and solvents. Distribution of products has
been shown to depend on solvent and on 4-substituent
on the benzylating reagent.19 In our case, replacement
of DMF with methanol in benzylation of 5 resulted in
the formation of C-7 benzyl (18, 12% yield) and C-
7,N-5 dibenzyl (18c, 15%) products accompanied by
smaller amounts of their N-5 (18a, 7%) and N-4 (18b,
5%) congeners. Using 4-fluorobenzyl bromide as a re-
agent, we obtained the products in similar proportions.
Benzylation of 5 in 2,2,2-trifluoroethanol (TFE) was
regioselectively directed towards the C-7 position (25%
yield).
In the case of the 6,7-disubstituted derivatives 13 and 17,
the decrease of activity was similar to that of the 6-mon-
osubstituted ones. 6-(4-Fluorophenyl)-7-methyl-TACV,
13, seemed to be the best of the present series, since
the decrease of potency noted for this compound was
only 3–30-fold compared with that for 8 (EC50 HSV-1:
0.04–0.11 lg/mL; EC50 HSV-2: 0.07–0.16 lg/mL), with-
out loss of selectivity. 7-Methyl-6-(4-trifluoromethylphe-
nyl)-TACV, 17, was 5–40-fold less active and 5-fold less
selective than 13. 6-(4-Methoxyphenyl)-7-methyl-TACV
(19) turned out to be effective against particular strains
at the same concentrations as 13, but it was more toxic.
7-Arylalkylated derivatives 14 and 18, unlike their
7-methyl analogues, were devoid of anti-herpetic
activity.
3. Biological results
3.1. Antiviral activity
The newly synthesized compounds 11–19 were examined
for their inhibitory effects on the replication of a wide
Compounds 11–19 were shown to be inactive against a
HSV-1 TKꢀ ACVr strain. This observation seems to
Table 1. Activity against human herpes simplex virus type 1 (HSV-1) and 2 (HSV-2) and cytotoxicity of fluoro substituted tricyclic analogues of
acyclovir and ganciclovir
Compd
EC50 (lg/mL)a
MCCb(lg/mL)
HSV-1 (KOS) HSV-1 (F) HSV-1 (McIntyre) HSV-2 (G) HSV-2 (196) HSV-2 (Lyons) HSV-1/TKꢀ
11
12
>16
0.64
0.384
>16
48
1.92
9.6
>16
>16
0.384
0.384
>16
16
0.384
>16
0.384
0.384
>16
>16
1.92
>16
1.92
1.92
>16
>16
9.6
9.6
>16
>16
1.92
1.92
>16
>16
9.6
9.6
>16
>16
1.92
1.92
>16
>16
9.6
9.6
>16
>16
>80
>80
>16
>16
>80
>16
>16
>16
48
P80
400
13
14
400
P80
P80
400
15
16
17
18
1.92
9.6
16
>16
80
P80
P80
>400
>400
19
ACV
GCV
0.384
0.128
0.006
0.384
0.077
0.001
1.92
0.384
0.019
1.92
0.384
0.019
1.92
0.077
0.019
1.92
0.077
0.006
0.48
a 50% Effective concentration or compound concentration required to reduce virus-induced cytopathogenicity by 50%.
b Minimal cytotoxic concentration or compound concentration required to cause a microscopically detectable alteration of normal cell morphology.

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T. Ostrowski et al. / Bioorg. Med. Chem. 13 (2005) 2089–2096
Table 2. Inhibitory effects of fluoro substituted tricyclic analogues of
acyclovir and ganciclovir on the proliferation of osteosarcoma cells
[thymidine kinase-deficient OST cells (OST TKꢀ) and OST TKꢀ cells
transfected with the HSV-1 thymidine kinase gene (OST TKꢀ/HSV-1
TK+)]
in potential interaction sites of the compound pharma-
cophors with the enzyme.
5. Experimental
Compd
IC50 (lMa)
SIb
OST TKꢀ
OST TKꢀ/HSV-1 TK+
5.1. General methods
11
12
92 10
278 64
122 12
240 118
368 161
>500
0.27 0.08
0.0032 0.0011
0.043 0.030
0.73 0.34
341
Melting points were determined on MEL-TEMP II cap-
illary melting point apparatus and are uncorrected.
Mass spectra (LR) were measured on a AMD-604 mass
spectrometer by LSIMS method with m-nitrobenzyl
alcohol as a matrix. Elemental analyses were performed
by Microanalytical Laboratories of the Institute of Or-
ganic Chemistry Polish Academy of Sciences in Warsaw.
1H, 13C and 19F NMR spectra were recorded on a Unity
300 Varian spectrometer operating at 299.95 MHz,
75.43 MHz and 282.25 MHz, respectively. Tetramethyl-
silane was used as the internal standard in 1H and
13C NMR, while 10% trifluoroacetic acid in D2O was
used as the external standard in 19F NMR; the chemical
shifts are reported in ppm (d scale), coupling constants
(J) in Hz. Thin-layer chromatography (TLC) was
performed on Merck precoated 60 F254 gel plates. Short
column chromatography was carried out on Merck
silica gel 60H (40–63 lm).
86,875
2837
329
13
14
15
16
0.20 0.01
1840
58,140
1429
869
0.0086 0.0030
0.35 0.22
0.29 0.08
17
18
P500
252 42
62 16
19
ACV
GCV
0.012 0.003
0.095 0.049
0.00037 0.00004
5167
653
62 29
109 71
294,595
a 50% Inhibitory concentration or compound concentration required
to inhibit tumour cell proliferation by 50%.
b Selectivity index or ratio of IC50OST TKꢀ to IC50OST TKꢀ/HSV-1
TK+.
suggest that they require phosphorylation by the HSV-1
encoded TK in order to exert their anti-HSV activity in
cell culture.
5.2. Bromoketones
3.2. Cytostatic activity
2-Bromo-40-fluoroacetophenone was purchased from
Aldrich. All other bromoketones were obtained previ-
ously by diverse methods.20a–20d We found, as described
earlier,4 that the approach using bromine in tetra-
hydrofurane solution in the presence of aluminium chlo-
ride, was most convenient.21
The substitution of the tricyclic analogues of ACV and
GCV with fluorine had a much smaller influence on their
cytostatic activity against human osteosarcoma OST
TKꢀ cells transduced with the HSV-1 TK gene (OST
TKꢀ/HSV-1 TK+) than on the aforementioned antiviral
activity against HSV-1 and HSV-2 (Table 2). The selec-
tivity indices of all the new compounds 11–19 (including
the 7-arylalkyl substituted compounds) differed from
that of parental acyclovir and ganciclovir within less
than one order of magnitude. The ganciclovir analogues
were 4- or 5-fold lower but those of acyclovir analogues
ranged from 2-fold lower (11, 14) to 8-fold higher (19).
It should be the subject of further research to define
the most suitable fluorinated tricyclic nucleoside ana-
logue for the purpose of imaging. In this respect, it is
of crucial importance that the 18F-labeling of the hydro-
xy-precursor can be performed in a high yield and in a
short time period.
5.2.1. 2-Bromo-40-(trifluoromethyl)acetophenone20a. Iso-
lated by column chromatography using silica gel and
hexane–EtOAc (95:5) for elution to give pure product,
which was crystallized on evaporation, 82% yield. MS
(LR) 267 (M+), 269 (M++2).1H NMR (CDCl3) d 8.11,
7.78 (2d, 4H, C6H4), 4.45 (s, 2H, CH2).
5.2.2. 2-Bromo-40-fluoropropiophenone20b
. Chromato-
graphed on silica gel column using hexane–EtOAc
(95:5) for elution to obtain colorless oil in 78% yield.
1
MS (LR) 230 (M+ꢀ1), 232 (M++1). H NMR (CDCl3)
d 8.03–8.10, 7.12–7.20 (2m, 4H, C6H4), 5.24 (q, 1H,
CH), 1.90 (d, 3H, CH3).
2-Bromo-40-(trifluoromethyl)propiophenone20c
.
4. Conclusions
5.2.3.
Chromatographed on silica gel column using hexane–
EtOAc (95:5) as eluent to obtain colorless oil in 90%
yield. MS (LR) 280 (M+ꢀ1), 282 (M++1). 1H NMR
(CDCl3) d 8.15, 7.78 (2d, 4H, C6H4), 5.28 (q, 1H,
CH), 1.94 (d, 3H, CH3).
Compounds 12, 13 and 16 appear to be the best candi-
dates of the present series for the 19F NMR studies of
the HSV TK–ligand interactions and/or monitoring of
their metabolites in cells expressing HSV TK. 6,7-Disub-
stitution pattern leads to highly potent antiherpetic
agents only when the 7-position is substituted by methyl
(Table 1, compounds 13, 17, 19). Replacement of the 7-
methyl group with the 7-arylalkyl one results in a total
loss of antiherpetic activity (Table 1, compounds 14
and 18). The most interesting compounds should be
co-crystallized with HSV-1 TK to gain more insights
5.2.4. 2-Bromo-40-methoxypropiophenone20d. Purified by
silica gel column using hexane–EtOAc (98:2 ! 95:5) to
quantitatively afford product as an oil. MS (LR) 242
(M+ꢀ1), 243 (M+), 244 (M++1), 245 (M++2). 1H
NMR (CDCl3) d 8.02, 6.96 (2d, 4H, C6H4), 5.27 (q,
1H, CH), 3.89 (s, 3H, OCH3), 1.89 (d, 3H, CH3).

T. Ostrowski et al. / Bioorg. Med. Chem. 13 (2005) 2089–2096
2093
5.3. General procedure for the alkylation–condensation
reactions
MeOH to afford colorless crystals: mp >300 ꢁC (dec at
ca 250 ꢁC). 1H NMR (DMSO-d6) d 13.31 (br s, 1H,
NH), 8.46 (s, 1H, 7-H), 8.08 (s, 1H, 2-H), 8.15, 7.86
(2d, 4H, C6H4), 5.53 (s, 2H, NCH2O), 4.69 (t, 1H,
OH), 3.46–3.57 (m, 4H, CH2CH2). 13C NMR (DMSO-
d6) d 151.21 (C-9), 150.55 (C-3a), 146.72 (C-4a), 139.44
To an anhydrous suspension of acyclovir or ganciclovir
(1 mmol) in DMF (12 mL or 24 mL, respectively) was
added sodium hydride as 60% suspension in oil
(1.3 mmol). After being stirred with exclusion of mois-
ture for 1–2 h at room temperature, the resulting solu-
tion was treated with bromoketone (1.1 mmol; a
solution in 2 mL of DMF). The reaction mixture was
stirred at room temperature for the next 2–5 h (for acy-
clovir) or 5–20 h (for ganciclovir), made alkaline by
addition of concentrated aqueous ammonia and left
overnight. Volatile materials were evaporated, the resid-
ual oil was dissolved in CH2Cl2–MeOH (7.5:1) (in the
case of reactions with bromoacetophenones) or (9:1)
(in the case of reactions with bromopropiophenones),
applied onto a silica gel column and chromatographed
in CH2Cl2–MeOH gradient (7.5:1 ! 6:1) or using
CH2Cl2–MeOH (9:1), respectively. Fractions containing
the main product were evaporated to dryness and
recrystallized.
2
(C-2), 132.01 (C-10), 128.60 (C-40; J = 32.4 Hz), 127.69
3
(C-6), 125.91 (C-30, C-50; J = 3.5 Hz), 125.58 (C-20, C-
1
60), 124.04 (CF3; J = 272.3 Hz), 115.44 (C-9a), 105.33
(C-7), 72.39 (NCH2O), 70.59 (HOCH2CH2), 59.92
(HOCH2). 19F NMR (DMSO-d6) d ꢀ46.31. Anal. Calcd
for C17H14F3N5O3: C, 51.91; H, 3.59; N, 17.81. Found:
C, 51.72; H, 3.52; N, 17.69.
5.3.4. 3,9-Dihydro-3-[(1,3-dihydroxy-2-propoxy)methyl]-
9-oxo-6-(4-trifluoromethyl-phenyl)-5H-imidazo[1,2-a]pur-
ine (16). Crude isolated material (194 mg, 46% yield) was
stirred in H2O (25 mL) at room temperature for 3 h and
filtered. The solid was dried by co-evaporation with tol-
uene (twice), then dissolved in CH2Cl2–MeOH (3:1),
concentrated and kept at +5 ꢁC to afford white crystal-
line precipitate: mp > 300 ꢁC (soft. at 184–186 ꢁC, dec
at ca 230 ꢁC).1H NMR (DMSO-d6) d 13.31 (br s, 1H,
NH), 8.46 (s, 1H, 7-H), 8.06 (s, 1H, 2-H), 8.16, 7.86
(2d, 4H, C6H4), 5.62 (s, 2H, NCH2O), 4.63 (t, 2H,
OH, OH), 3.59–3.67 (p, 1H, CH), 3.27–3.48 (m, 4H,
CH2, CH2). 13C NMR (DMSO-d6) d 151.35 (C-9),
150.54 (C-3a), 146.99 (C-4a), 139.40 (C-2), 132.46 (C-
10), 129.38 (C-6), 128.49 (C-40; 2J = 31.7 Hz), 125.90
5.3.1. 3,9-Dihydro-3-[(2-hydroxyethoxy)methyl]-6-(4- flu-
orophenyl)-9-oxo-5H-imidazo-[1,2-a]purine (11). Crude
product (302 mg, 88% yield) was subjected to crystalliza-
tion from MeOH to give yellowish spangles: mp 270–
272 ꢁC (dec; soft at ca 250 ꢁC).1H NMR (DMSO-d6) d
13.14 (br s, 1H, NH), 8.24 (s, 1H, 7-H), 8.08 (s, 1H, 2-
H), 7.95–8.00, 7.32–7.38 (2m, 4H, C6H4), 5.53 (s, 2H,
NCH2O), 4.70 (t, 1H, OH), 3.48–3.57 (m, 4H,
CH2CH2). 13C NMR (DMSO-d6) d 162.12 (C-40;
1J = 246.2 Hz), 151.17 (C-9), 150.33 (C-3a), 146.45 (C-
4a), 139.29 (C-2), 128.17 (C-6), 127.20 (C-20, C-60;
3J = 8.6 Hz), 124.49 (C-10), 115.99 (C-30, C-50;
2J = 22.2 Hz), 115.38 (C-9a), 103.22 (C-7), 72.31
(NCH2O), 70.49 (HOCH2CH2), 59.85 (HOCH2). 19F
3
(C-30, C-50; J = 3.5 Hz), 125.65 (C-20, C-60), 124.10
(CF3; 1J = 272.0 Hz), 115.26 (C-9a), 105.15 (C-7),
80.41 (CH), 71.81 (NCH2O), 60.88 (HOCH2,
HOCH2).19F NMR (DMSO-d6) d ꢀ46.29. Anal. Calcd
for C18H16F3N5O4Æ0.5H2O: C, 50.00; H, 3.96; N,
16.20. Found: C, 50.05; H, 3.90; N, 15.74.
5.3.5.
O6-[1-(4-Fluorobenzoyl)ethyl]-9-[(2-hydroxyeth-
NMR (DMSO-d6)
C16H14FN5O3: C, 55.98; H, 4.11; N, 20.40. Found: C,
55.84; H, 3.96; N, 20.33.
d
ꢀ36.27. Anal. Calcd for
oxy)methyl]guanine (130). Crude solid (21 mg, 6%) was
dissolved in iPrOH and concentrated to give white pow-
der: mp 194–195 ꢁC (soft. at ca 180 ꢁC). 1H NMR
(DMSO-d6) d 8.15, 7.41 (q and t, 4H, C6H4), 8.05 (s,
1H, 8-H), 6.45 (q, 1H, CH), 6.28 (br s, 2H, NH2), 5.43
(s, 2H, NCH2O), 4.69 (t, 1H, OH), 3.44–3.50 (m, 4H,
CH2CH2), 1.57 (d, 3H, CH3).
5.3.2. 3,9-Dihydro-3-[(1,3-dihydroxy-2-propoxy)methyl]-
6-(4-fluorophenyl)-9-oxo-5H-imidazo[1,2-a]purine (12).
Crude isolated product (188 mg, 50% yield) was crystal-
lized from 50% aq EtOH to give white needles:
mp > 300 ꢁC (dec at ca 230 ꢁC).1H NMR (DMSO-d6)
d 13.14 (br s, 1H, NH), 8.23 (s, 1H, 7-H), 8.08 (s, 1H,
2-H), 7.95–8.01, 7.32–7.40 (2m, 4H, C6H4), 5.62 (s,
2H, NCH2O), 4.65 (t, 2H, OH, OH), 3.61–3.68 (p, 1H,
CH), 3.28–3.49 (m, 4H, CH2, CH2). 13C NMR
5.3.6. 3,9-Dihydro-3-[(2-hydroxyethoxy)methyl]-6-(4-flu-
orophenyl)-7-methyl-9-oxo-5H-imidazo[1,2-a]purine (13).
Chromatographically purified product (101 mg, 28%
yield) was dissolved in iPrOH and the resulting solution
was concentrated to obtain white needles: mp 241–
243 ꢁC (dec). 1H NMR (DMSO-d6) 12.50 (br s, 1H,
NH), 7.99 (s, 1H, 2-H), 7.60–7.67, 7.35–7.44 (2m, 4H,
C6H4), 5.47 (s, 2H, NCH2O), 4.69 (br s, 1H, OH),
1
(DMSO-d6) d 161.67 (C-40; J = 246.2 Hz), 150.77 (C-
9), 149.80 (C-3a), 146.02 (C-4a), 138.85 (C-2), 127.86
3
(C-6), 126.81 (C-20, C-60; J = 8.6 Hz), 124.12 (C-10),
2
115.56 (C-30, C-50; J = 21.6 Hz), 114.87 (C-9a), 102.67
3.42–3.54 (m, 4H, CH2CH2), 2.78 (s, 3H, 7-CH3). 13C
1
(C-7), 79.65 (CH), 71.31 (NCH2O), 60.37 (HOCH2,
HOCH2). 19F NMR (DMSO-d6) d ꢀ36.26. Anal. Calcd
for C17H16FN5O4: C, 54.69; H, 4.32; N, 18.76. Found:
C, 54.49; H, 4.32; N, 18.67.
NMR (DMSO-d6) d 161.92 (C-40;
154.32 (C-9), 150.06 (C-3a), 146.61 (C-4a), 138.89 (C-
J = 246.3 Hz),
3
2), 130.35 (C-20, C-60; J = 8.5 Hz), 124.77 (C-10),
123.93 (C-6), 116.22 (C-7), 115.80 (C-30, C-50;
2J = 21.7 Hz), 115.81 (C-9a), 72.13 (NCH2O), 70.40
(HOCH2CH2), 59.82 (HOCH2), 11.74 (7-CH3).19F
5.3.3. 3,9-Dihydro-3-[(2-hydroxyethoxy)methyl]-9-oxo-6-
(4-trifluoromethylphenyl)-5H-imidazo[1,2-a]purine (15).
Product after chromatography: 279 mg (71% yield).
Analytical sample was prepared by crystallization from
NMR (DMSO-d6)
C17H16FN5O3: C, 57.14; H, 4.51; N, 19.60. Found: C,
57.02; H, 4.48; N, 19.39.
d
ꢀ36.54. Anal. Calcd for

2094
T. Ostrowski et al. / Bioorg. Med. Chem. 13 (2005) 2089–2096
5.3.7. O6-[1-(4-Trifluoromethylbenzoyl)ethyl]-9-[(2-hydr-
oxyethoxy)methyl]guanine (170). Amorphous solid after
chromatography (102 mg, 24%) was purified by precipi-
tation from iPrOH: mp 166–169 ꢁC (soft. at ca 150 ꢁC).
1H NMR (DMSO-d6) d 8.24, 7.94 (2d, 4H, C6H4), 8.04
(s, 1H, 8-H), 6.43 (q, 1H, CH), 6.29 (br s, 2H, NH2),
5.41 (s, 2H, NCH2O), 4.67 (t, 1H, OH), 3.44–3.48 (m,
4H, CH2H2), 1.57 (d, 3H, CH3).13C NMR (DMSO-d6)
d 197.32 (C@O), 159.45, 158.86 (C-2, C-6), 154.82 (C-
4), 140.44 (C-8), 137.88 (C-10), 132.67 (C-40;
2J = 32.2 Hz), 129.18 (C-20, C-60), 125.89 (C-30, C-50;
3J = 4.0 Hz), 123.73 (CH3; 1J = 273.1 Hz), 113.26 (C-
5), 72.84 (CH-CH3), 72.06 (NCH2O), 70.48
(HOCH2CH2), 59.88 (HOCH2), 17.10 (CH3).
The mixture was evaporated at 35 ꢁC and the residue
was subjected to column chromatography, which was
performed in CH2Cl2–MeOH (97:3 ! 93:7). Evapora-
tion of fractions gave in the order of elution: the mixture
of 18c and 18b (162 mg; 15% and 5% yield, respectively,
1
on the basis of H NMR spectrum), homogeneous 18a
(49 mg, 7%) and homogeneous 18 (84 mg, 12%).
Compounds 18a–c were found to be identical by 1H
NMR as described previously.18
5.4.2.1. 7-Benzyl-3,9-dihydro-3-[(2-hydroxyethoxy)-
methyl]-9-oxo-6-phenyl-5H-imidazo-[1,2-a]purine
(18).
White crystals of 18 precipitated from concentrated
EtOH solution: mp 224–226 ꢁC (dec). 1H NMR
(DMSO-d6) d 12.93 (br s, 1H, NH), 7.99 (s, 1H, 2-H),
7.47–7.56, 7.10–7.29 (2m, 4H, C6H4), 5.49 (s, 2H,
NCH2O), 4.69 (t, 1H, OH), 4.68 (s, 2H, 7-CH2), 3.47–
3.55 (m, 4H, CH2CH2).13C NMR (DMSO-d6) d 153.67
(C-9), 149.92 (C-3a), 146.77 (C-4a), 138.99 (C-2),
140.12, 128.92, 128.81, 128.30, 128.06, 127.74, 127.50,
126.61, 125.79 (Ph, C-6), 117.85 (C-7), 115.88 (C-9a),
72.17 (NCH2O), 70.40 (HOCH2CH2), 59.83 (HOCH2),
29.88 (7-CH2). Anal. Calcd for C23H21 N5O3: C,
66.49; H, 5.10; N, 16.86. Found: C, 66.39; H, 5.15; N,
16.75.
5.3.8. 3,9-Dihydro-3-[(2-hydroxyethoxy)methyl]-7-meth-
yl-9-oxo-6-(4-trifluoromethyl-phenyl)-5H-imidazo[1,2-a]-
purine (17). Solid material after column chromatogra-
phy: 77 mg (19%). Crystalline white material precipi-
tated on evaporation of the fractions: mp > 300 ꢁC
(dec at 250–253 ꢁC).1H NMR (DMSO-d6) 12.87 (br s,
1H, NH), 8.01 (s, 1H, 2-H), 7.91, 7.81 (2d, 4H, C6H4),
5.48 (s, 2H, NCH2O), 4.69 (t, 1H, OH), 3.47–3.53 (m,
4H, CH2CH2), 2.84 (s, 3H, 7-CH3). 13C NMR
(DMSO-d6) d 154.46 (C-9), 150.26 (C-3a), 146.98 (C-
4a), 139.10 (C-2), 132.63 (C-10), 128.77 (C-20, C-60),
128.52 (C-40; 2J = 32.2 Hz), 125.74 (C-30, C-50;
1
3J = 3.5 Hz), 123.68 (C-6), 124.10 (CF3; J = 272.0 Hz),
5.4.3. Benzylation of 5 in 2,2,2-trifluoroethanol (TFE).
The same procedure as for reaction in MeOH; reagents:
7 (80 mg, 0.246 mmol), K2CO3 (64 mg, 0.463 mmol),
benzyl bromide (72 mg, 0.421 mmol), TFE (40 mL).
After 1 day of stirring, TLC in CH2Cl2–MeOH (9:1)
indicated the presence of 18, product of decomposition
and traces of 18a–c and no substrate in the reaction mix-
ture. Compound 18 was isolated by chromatography as
above to yield 26 mg (25%).
118.01 (C-7), 115.91 (C-9a), 72.26 (NCH2O), 70.53
(HOCH2CH2), 59.94 (HOCH2), 11.95 (7-CH3). 19F
NMR (DMSO-d6)
d
15.05. Anal. Calcd for
C18H16F3N5O3: C, 53.07; H, 3.96; N, 17.19. Found: C,
52.96; H, 3.87; N, 16.92.
5.3.9. 3,9-Dihydro-3-[(2-hydroxyethoxy)methyl]-6-(4-meth-
oxyphenyl)-7-methyl-9-oxo-5H-imidazo[1,2-a]purine (19).
Evaporation of appropriate fractions after chromato-
graphy gave 94 mg of the compound (25% yield). Ana-
lytical sample was obtained by crystallization from
5.4.4. 4-Fluorobenzylation of 5 in MeOH at room
temperature. To a suspension of 5 (325 mg, 1.0 mmol)
in anhydrous MeOH (100 mL) was added powdered
K2CO3 (262 mg, 1.9 mmol). After stirring at room tem-
perature for 1 h, to the resulting solution 4-fluorobenzyl
bromide (322 mg, 1.7 mmol) was added and the mixture
was stirred at room temperature for 4 days. The volatiles
were evaporated at 35 ꢁC and the residue was applied as
a suspension in CH2Cl2–MeOH (97:3) onto a silica gel
column. Separation was carried out using CH2Cl2–
MeOH (97:3 ! 95:5) as the eluent and resulted in isola-
tion of compounds 14b (15 mg, 3% yield), 14a (18 mg,
4%), 14 (55 mg, 13%) and 5 (94 mg, 29%), in the order
of elution.
1
MeOH: mp 211–214 ꢁC (dec). H NMR (DMSO-d6) d
12.59 (br s, 1H, NH), 7.98 (s, 1H, 2-H), 7.51, 7.09 (2d,
4H, C6H4), 5.47 (s, 2H, NCH2O), 4.70 (t, 1H, OH),
3.82 (s, 3H, OCH3), 3.42–3.55 (m, 4H, CH2CH2), 2.77
13
(s, 3H, 7-CH3). C NMR (DMSO-d6) d 159.36 (C-40),
154.37 (C-9), 150.03 (C-3a), 146.53 (C-4a), 138.87 (C-
2), 129.52 (C-20, C-60), 124.65 (C-10), 120.50 (C-7),
115.87 (C-9a), 114.31 (C-30, C-50), 72.19 (NCH2O),
70.44 (HOCH2CH2), 59.89 (HOCH2), 55.26 (OCH3),
11.82 (7-CH3). Anal. Calcd for C18H19N5O4Æ0.75H2O:
C, 56.46; H, 5.40; N, 18.29. Found: C, 56.44; H, 5.08;
N, 17.92.
5.4. Alkylation reactions
5.4.5. 4-Fluorobenzylation of 5 in MeOH at 50 ꢁC. Re-
agents were added in the same manner as described
5.4.1. Benzylation of 5 with benzyl bromide, K2CO3
and DMF as solvent was described in an earlier report.18
above;
5
(325 mg, 1.0 mmol), K2CO3 (552 mg,
4.0 mmol), 4-fluorobenzyl bromide (643 mg, 3.4 mmol),
anhydrous MeOH (50 mL). The solution was stirred at
50 ꢁC for 3 days, and then evaporated. Components of
the mixture were separated by column chromatography
in CH2Cl2–MeOH (97:3 ! 9:1) to afford products 14b
(133 mg, 25% yield), 14a (43 mg, 10%) and 14 (48 mg,
11%), in order of elution.
5.4.2. Benzylation of 5 in MeOH. To a suspension of 5
(540 mg, 1.66 mmol) in anhydrous MeOH (200 mL)
was added powdered K2CO3 (436 mg, 3.15 mmol). After
stirring at room temperature for 1 h, to the resulting
solution benzyl bromide (483 mg, 2.82 mmol) was added
in one portion and stirring was continued for 4 days.

T. Ostrowski et al. / Bioorg. Med. Chem. 13 (2005) 2089–2096
2095
5.4.5.1. 5,7-Di-(4-fluorobenzyl)-3,9-dihydro-3-[(2-hydr-
oxyethoxy)methyl]-9-oxo-6-phenylimidazo[1,2-a]purine (14b).
1H NMR (DMSO-d6) d 8.03 (s, 1H, 2-H), 7.48–7.52,
7.40–7.43, 7.00–7.11 (3m, 13H, 2 · C6H4, C6H5), 5.50
(s, 2H, NCH2O), 5.21 (s, 2H, N-5-CH2), 4.67 (t, 1H,
OH), 4.40 (s, 2H, 7-CH2), 3.41–3.51 (m, 4H, CH2H2).
19F NMR (DMSO-d6) d ꢀ38.65, ꢀ41.27.
cally on mock-infected HEL cell cultures at 3 days
after addition of the compound dilutions.
5.7. Cytostatic activity assays
To evaluate the cytostatic activity of the test compounds
against OST TKꢀ and OST TKꢀ/HSV-1 TK+, 104OST
cells/well were seeded in 96-well microtiter plates (Fal-
con) in 200 lL cell culture medium at 37 ꢁC, in the pres-
ence of five-fold dilutions (in normal growth medium) of
the test compounds. After 3 days the OST cells (after
being detached with trypsin solution (Gibco)) were
counted in a Coulter counter (Coulter Electronics,
Harpenden, UK). The IC50 was defined as the drug
concentration required to inhibit OST cell proliferation
by 50%.
5.4.5.2. 3,9-Dihydro-5-(4-fluorobenzyl)-3-[(2-hydroxyeth-
oxy)methyl]-9-oxo-6-phenyl-imidazo[1,2-a]purine (14a).
1H NMR (DMSO-d6) d 8.11 (s, 1H, 2-H), 7.91 (s, 1H,
7-H), 7.49–7.55, 7.02–7.10 (2m, 9H, C6H4, C6H5), 5.53
(s, 2H, NCH2O), 5.38 (s, 2H, N-5-CH2), 4.66 (t, 1H,
OH), 3.40–3.52 (m, 4H, CH2CH2). 19F NMR (DMSO-
d6) d ꢀ38.67.
5.4.5.3. 3,9-Dihydro-7-(4-fluorobenzyl)-3-[(2-hydroxyeth-
oxy)methyl]-9-oxo-6-phenyl-5H-imidazo[1,2-a]purine (14).
Keeping at +5 ꢁC of the concentrated MeOH solution of
14 gave a white crystalline precipitate: mp 243–245 ꢁC
Acknowledgements
This work was supported by the Polish State Committee
for Scientific Research (KBN) Grant no PBZ-KBN-059/
T09/18, an IDO Grant no 02/012 (to J.B.) from the
Katholieke Universiteit Leuven and a Grant from the
European Commission (QLRT-2001-01004).
1
(dec). H NMR (DMSO-d6) d 12.93 (br s, 1H, NH),
7.99 (s, 1H, 2-H), 7.45–7.56, 7.04–7.17 (2m, 9H,
C6H4C6H5), 5.48 (s, 2H, NCH2O), 4.69 (t, 1H, OH),
4.65 (s, 2H, 7-CH2), 3.45–3.55 (m, 4H, CH2CH2). 13C
NMR (DMSO-d6) d 160.50 (C-40; 1J = 242.5 Hz),
153.67 (C-9), 149.94 (C-3a), 146.77 (C-4a), 139.05 (C-
2), 136.23 (Ph), 129.28 (C-20, C-60; 3J = 8.1 Hz),
128.95, 127.98, 127.80 126.77 (C-6, Ph), 117.72 (C-7),
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