Synthesis and biological evaluation of acyclic nucleotide analogues with a furo[2,3-d]pyrimidin-2(3H)-one base

Article abstract of DOI:10.1139/V10-054

As a part of a broader structure-activity relationship (SAR) study of bicyclic nucleoside analogues (BCNAs) [anti-varicella-zoster virus (anti-VZV) and anti-human cytomegalovirus (anti-HCMV) agents], a novel series of 2-(phosphonomethoxy) ethyl (PME) subs

Full text of DOI:10.1139/V10-054

628
Synthesis and biological evaluation of acyclic
nucleotide analogues with a furo[2,3-d]pyrimidin-
2(3H)-one base
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Zlatko Janeba, Antonın Holy, Radek Pohl, Robert Snoeck, Graciela Andrei,
Erik De Clercq, and Jan Balzarini
Abstract: As a part of a broader structure–activity relationship (SAR) study of bicyclic nucleoside analogues (BCNAs)
[anti-varicella-zoster virus (anti-VZV) and anti-human cytomegalovirus (anti-HCMV) agents], a novel series of 2-(phosphono-
methoxy)ethyl (PME) substituted furo[2,3-d]pyrimidin-2(3H)-ones was synthesized. The target acyclic nucleotide analogues
were prepared by Sonogashira coupling of protected 5-iodo-1-[2-(phosphonomethoxy)ethyl]uracil with various 1-alkynes,
followed by in situ Cu(I)-promoted intramolecular cyclization and standard removal of the isopropyl ester groups. None
of the prepared PME analogues were active at subtoxic concentrations against VZV thymidine kinase competent (TK+),
VZV thymidine kinase deficient (TK–), HCMV, or any other viruses tested.
Key words: Sonogashira reaction, intramolecular cyclization, acyclic nucleotide analogues, phosphonates, bicyclic nucleo-
side analogues (BCNAs).
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Resume : Dans le cadre d’une large etude des analogues bicycliques de nucleotides (ABCN) [des agents actifs contre le virus
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de varicelle-zoster (anti-VVZ) et contre le cytomegalovirus humain (anti-CMVH)], on a realise la synthese d’une nouvelle se-
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rie de furo[2,3-d]pyrimidin-2(3H)-ones portant un substituant 2-(phosphonomethoxy)ethyle (PME). On a prepare les analogues
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acycliques de nucleotides voulus par un couplage de Sonogashira du 5-iodo-1-[2-(phosphonomethoxy)ethyl]uracile avec divers
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1-alcynes, suivi d’une cyclisation in situ intramoleculaire, catalysee par du Cu(I), et finalement d’une elimination standard
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des groupes esters isopropyles. Aucun des analogues PME n’est actif, a des concentrations sous-toxiques, contre le contre le
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virus de varicelle-zoster (anti-VVZ), contre le cytomegalovirus humain (anti-CMVH) ou contre tout autre virus teste.
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Mots-cles : reaction de Sonogashira, cyclysation intramoleculaire, analogues acycliques de nucleotide (ABCN), phosphonate.
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[Traduit par la Redaction]
in 92% yield by treatment of 5-hexynyl-1-methyluracil with
CuI in refluxing triethylamine/methanol mixture.1 Two years
later, the same authors described the preparation of the
6-n-butylfuro[2,3-d]pyrimidin-2(3H)-one 2’-deoxynucleoside
analogue.2
Introduction
Antiviral drugs currently belong to the most widely used
medications. The majority of them now available are de-
signed to help deal with infections caused by herpes simplex
virus (HSV), human immunodeficiency virus (HIV), hepatitis
B virus (HBV), hepatitis C virus (HCV), and cytomegalovirus
(CMV). Prominent among the antiviral drugs are nucleoside
and nucleotide analogues, mainly for their ability to inhibit
viral DNA polymerases and reverse transcriptases. Nowadays,
research of nucleic acid components steadily continues to pro-
vide new compounds with interesting biological activities and
high potential in the management of viral infections.
While studying 5-substituted pyrimidines as potential
inhibitors of thymidylate synthase and thus prospective anti-
viral agents, 6-n-butyl-3-methylfuro[2,3-d]pyrimidin-2(3H)-
one was reported by Robins and Barr1 in 1981 as a byproduct
(9% yield) of Sonogashira cross-coupling of 5-iodo-1-
methyluracil with 1-hexyne. The same compound was formed
Two decades later, the remarkably potent and selective ac-
tivity against varicella-zoster virus (VZV) was discovered
with furo[2,3-d]pyrimidin-2(3H)-one 2’-deoxynucleoside ana-
logues with longer alkyl chains at the C-6 position (e.g., com-
pound 1, Fig. 1).3 Subsequent extensive structure–activity
relationship (SAR) studies of these bicyclic nucleoside ana-
logues (BCNAs) followed4 and identified compound Cf 1743
(compound 3, Fig. 1) as one of the most potent antiviral
agents that have ever been reported.5 Its promising prodrug
FV100 (5’-valine ester of Cf 1743) is currently in phase II of
clinical development against herpes zoster (shingles).6
While SAR studies4 of BCNAs showed that some struc-
tural modifications of the furo[2,3-d]pyrimidin-2(3H)-one
Received 13 January 2010. Accepted 22 March 2010. Published on the NRC Research Press Web site at canjchem.nrc.ca on 10 June
2010.
1
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Z. Janeba, A. Holy, and R. Pohl. Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic,
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Flemingovo nam. 2, CZ-166 10 Prague 6, Czech Republic.
R. Snoeck, G. Andrei, E. De Clercq, and J. Balzarini. Rega Institute for Medical Research, Minderbroedersstraat 10, Leuven B-3000,
Belgium.
1Corresponding author (e-mail: janeba@uochb.cas.cz).
Can. J. Chem. 88: 628–638 (2010)
doi:10.1139/V10-054
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Janeba et al.
629
Fig. 1. Bicyclic nucleoside analogues.
base or the substituent at the C-6 position are tolerated,
sugar modifications of BCNAs are not acceptable for main-
taining potent anti-VZV activity. Thus, while 2’-deoxyribo-
sides are potent and selective anti-VZV agents, their ribo-
and arabino-analogues are significantly less active.4 Further-
more, 2’,3’-dideoxy analogues (e.g., compound 2, Fig. 1)7
and N-3 alkyl derivatives8 showed poor activity against
VZV but they surprisingly exhibited activity against human
cytomegalovirus (HCMV). Replacement of the sugar at the
N-3 position by the (2-hydroxyethoxy)methyl group (present
in the antiherpes drug acyclovir) afforded compounds (e.g.,
compound 4, Fig. 1) with weak activity against both VZV
and HCMV.9
Although not sufficient as a requirement, phosphorylation of
the furo[2,3-d]pyrimidin-2(3H)-one nucleoside analogues by
the VZV thymidine kinase (TK) is a prerequisite for their
eventual anti-VZV activity.4,10 This fact is supported by the
complete absence of antiviral activity of BCNAs in the thymi-
dine kinase deficient VZV TK– assays. Thus, the anti-VZV ac-
tivity of 2’-deoxyriboside BCNAs follows the activation
pathway characteristic for thymidine nucleosides (nucleoside
mechanism mode of action). On the other hand, BCNAs with
modified sugar moiety that inhibit HCMV replication do so by
a non-nucleoside mechanism of action, at an early stage of the
viral life cycle, possibly affecting viral entry into the cells.10
In 1986, De Clercq et al.11 described broad spectrum antivi-
ral activity of (S)-9-(3-hydroxy-2-phosphonomethoxy)propyla-
denine (HPMPA) and 9-(2-phosphonomethoxy)ethyladenine
(PMEA), compounds belonging to a group of acyclic nu-
cleoside phosphonates (ANPs). ANPs possess a phosphonate
group attached to the acyclic moiety of the nucleoside
through a stable P–C bond so they are resistant towards deg-
radation by cellular enzymes. Due to ‘‘by-passing’’ of the
first phosphorylating step, a number of ANPs proved to be
active against a broad range of DNA viruses and retrovi-
ruses, and during the course of some 30 years, they have be-
come a key class of antiviral agents.12
Results and discussion
Chemistry
The furo[2,3-d]pyrimidin-2(3H)-one nucleoside analogues
are usually prepared by the Sonogashira coupling of various
alkynes with 5-iodouracils, followed by Cu(I)-promoted in-
tramolecular cyclization.1–3 This procedure, with some mod-
ifications and optimization, was applied in the present work.
Recently, 5-bromouracil derivative was used to modify 1-
[2-(phosphonomethoxy)ethyl]uracil (PMEU) analogues in the
C-5 position of the uracil moiety via Pd-catalysed Suzuki–
Miyaura coupling.14 No record of the protected 5-iodo-1-[2-
(phosphonomethoxy)ethyl]uracil intermediate was found in
the literature even though the 5-iodouracil compounds could
be expected to be more reactive toward cross-coupling reac-
tions compared to their 5-bromouracil counterparts. Thus, we
decided to prepare the novel 5-iodouracil analogue as a suit-
able starting material for subsequent syntheses.
Starting PMEU compound 5 was prepared by a previously
described procedure:15 reaction of 4-methoxypyrimidin-2(1H)-
one16 with diisopropyl [(2-chloroethoxy)methyl]phosphonate17
in the presence of sodium hydride followed by hydrolysis in
aqueous methanol using Dowex 50 (H+ form). A much im-
proved yield was obtained during the alkylation step (79%)
compared to the original procedure (49%).15 The major differ-
ence in our procedure seems to be neutralization of the reac-
tion mixture before the work-up (evaporation of solvents) to
avoid possible product decomposition, since a slight excess of
NaH is used for the alkylation reaction.
The desired 5-iodouracil derivative (6) was prepared by
the reaction of compound 5 with iodine and ammonium cer-
ium(IV)nitrate (CAN) in refluxing acetonitrile (Scheme 1).18
This reactive intermediate can be conveniently prepared as a
white product by recrystallization in 90% yield. Treatment
of compound 6 with bromo(trimethyl)silane in a mixture of
acetonitrile/2,6-lutidine (*5:1) afforded the free phosphonic
acid (7). The presence of the sterically hindered mild base
2,6-lutidine is required to neutralize forming HBr and thus
prevent exchange of iodine atom in the C-5 position for bro-
mine or proton during the reaction and (or) its work-up.
Furo[2,3-d]pyrimidin-2(3H)-ones (10) were prepared in
moderate to high yields (50%–90% isolated yields for two
in situ steps) by the Sonogashira coupling of various terminal
alkynes (8) with 5-iodouracil derivative (6), followed by in
situ Cu(I)-promoted intramolecular cyclization (Scheme 1).
To further elucidate the mechanism of antiviral action of
BCNAs, which still remains unclear, we decided to synthe-
size a novel series of 2-(phosphonomethoxy)ethyl (PME)
furo[2,3-d]pyrimidin-2(3H)-ones,13 stable analogues of nucleo-
tides that by-pass the first phosphorylation step. It is the first
time that a combination of the furo[2,3-d]pyrimidin-2(3H)-one
base and the acyclic nucleoside phosphonate moiety is re-
ported.
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Can. J. Chem. Vol. 88, 2010
Scheme 1. Reagents: (i) I2, CAN, CH3CN, 80 8C; (ii) (CH3)3SiBr, 2,6-lutidine, CH3CN, RT; (iii) Pd(Ph3P)4, CuI, Et3N, DMF, 50 8C, then
CuI and 90 8C; (iv) Pd(Ph3P)4, CuI, Et3N, DMF (or THF), 50 8C; (v) CuI, Et3N, MeOH, reflux; (vi) (CH3)3SiBr, CH3CN, RT.
The lower yields can be attributed to difficult crystallization/
precipitation of highly lipophilic compounds (10). Neverthe-
less, the purity of diesters (10) proved to be very important
to ultimately obtain the pure final products (11). Also, ex-
traction of the reaction mixture with aqueous EDTA solution
during the work-up is necessary to get pure diesters (10)
with matching elemental analyses since phosphonates
strongly bind metal ions. Finally, the free phosphonic acids
(11) were prepared by standard treatment of compounds 10
with bromo(trimethyl)silane in acetonitrile followed by hy-
drolysis.
Fig. 2. Structure of compound 12 with atom numbering of the sub-
stituted furo[2,3-d]pyrimidin-2(3H)-ones used for assignment of
NMR signals.
Similarly, as during the BCNAs synthesis,1–3 5-alkynyl in-
termediates (9) were not isolated. Formation of compounds
9 and their conversion to fluorescent bicyclic products (10)
was monitored by thin-layer chromatography (TLC). There
were often fluorescent and nonfluorescent byproducts ob-
served, so we tried to optimize the reaction conditions. The
best yields of compounds 10 were obtained by the addition
of fresh CuI (0.5 equiv.) and by increasing the reaction tem-
perature from 50–90 8C after the starting compound 6 was
completely consumed (TLC, *1 h).
In spite of the fact that Sonogashira coupling and in situ
cyclization is a very practical procedure, which gives high
overall yields of furo[2,3-d]pyrimidin-2(3H)-ones (10), we
decided to prepare, isolate, and fully characterize one of the
intermediate 5-alkynyluracils and use it as pure starting com-
pound in the next cyclization step. Thus, coupling of the 5-
iodouracil derivative (6) with 1.3 equiv. of 1-hexyne (8b)
under standard Sonogashira coupling conditions (Pd(Ph3P)4,
CuI, Et3N, DMF, 50 8C, 2 h) afforded the 5-hexynyluracil
derivative (9b) in 82% yield (Scheme 1). Fluorescent by-
product was clearly visible on the TLC plate and the corre-
sponding 6-butylfuro[2,3-d]pyrimidin-2(3H)-one (10b) was
isolated from the reaction mixture in 2% yield.
compound 10b (78%) prepared by the standard in situ syn-
thesis in the DMF/Et3N mixture. Although, not all reaction
steps were optimized, the comparison of the results of the
in situ procedure with the two-pot-reaction sequence clearly
shows that the in situ approach for general preparation of
furo[2,3-d]pyrimidin-2(3H)-ones is preferable and more eco-
nomic.
For comparison, the Sonogashira coupling of the 5-
iodouracil derivative (6) with 1-hexyne (8b) was carried out
using THF as a solvent instead of DMF. In this case, the 5-
hexynyluracil derivative (9b) was isolated only in 59% yield
(compared to 82% in DMF) together with 5,6-disubstituted
furo[2,3-d]pyrimidin-2(3H)-one (12) as a byproduct (Fig. 2).
Formation of a similar type of byproduct during synthesis of
bicyclic nucleoside analogues was previously reported by
the group of McGuigan.19
To prepare the parent furo[2,3-d]pyrimidin-2(3H)-one de-
rivative unsubstituted at the C-6 position, ethynyl(trimethyl)-
silane (13) was coupled with the starting 5-iodouracil
compound (6) followed by deprotection of the trimethylsilyl
group with TBAF/THF (Scheme 2). The desired 5-ethyny-
luracil derivative (14) was obtained in 63% yield, but its
subsequent cyclization with CuI did not proceed well and
the reactions usually afforded complex mixtures. The best
Treatment of the 5-hexynyluracil derivative (9b) with CuI
(1 equiv.) in a mixture of MeOH/Et3N (3:2) at reflux gave
the cyclized product 10b in 90% yield. The calculated over-
all yield of compound 10b starting from 5-iodouracil deriva-
tive (6) was 74% and is comparable to the yield of
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Janeba et al.
631
Scheme 2. Reagents: (i) Pd(Ph3P)4, CuI, Et3N, DMF, 70 8C; (ii) TBAF, THF, RT; (iii) CuI, Et3N, EtOAc, reflux; (iv) (CH3)3SiBr, CH3CN,
RT; (v) CuI, Et3N, MeOH, reflux.
yield (38%) of the furo[2,3-d]pyrimidin-2(3H)-one (15) was
achieved by the Cu(I)-promoted intramolecular cyclization
in an EtOAc/Et3N mixture. Similar reaction of compound
14 with 1 equiv. of CuI in the MeOH/Et3N mixture gave a
complex mixture from which only a dimeric product of
Eglinton coupling,20 compound 17, was isolated in 10%
yield (Scheme 2). Finally, free phosphonic acid (16) was
prepared from compound 15 by the standard procedure in
74% yield (Scheme 2).
starting compound. An example of Sonogashira reaction prod-
uct, the 5-hexynyluracil derivative (9b) was isolated and fully
characterized, as well as the product 12, which was formed
during the coupling procedure in THF. Bearing in mind the
same overall yields of 6-butylfuro[2,3-d]pyrimidin-2(3H)-one
derivative (10b), the in situ coupling-intramolecular cycliza-
tion is clearly preferable to two-pot synthesis with isolation
of the 5-hexynyluracil intermediate (9b). Coupling of the 5-
iodouracil derivative (6) with ethynyl(trimethyl)silane (13)
under the Sonogashira reaction conditions, followed by
removal of the TMS group, afforded the 5-ethynyluracil
derivative (14). Intramolecular cyclization of compound 14
in EtOAc gave the desired furo[2,3-d]pyrimidin-2(3H)-one
(15), while a similar reaction in MeOH only afforded a
dimer (17) in very low yield. Free phosphonic acids (11 and
16) were obtained by the standard deprotection of the corre-
sponding diesters (10 and 15). None of the compounds tested
were active in any of the antiviral assays.
Biological activity
All the final 3-[2-(phosphonomethoxy)ethyl]furo[2,3-
d]pyrimidin-2(3H)-ones (11) and 16 were evaluated for their
inhibitory activity against a variety of DNA viruses includ-
ing herpes simplex virus type 1 (KOS) (HSV-1), HSV-2
(G), varicella-zoster virus (VZV) (thymidine kinase (TK)
competent (TK+) and TK deficient (TK–)), vaccinia virus
(Lederle), human cytomegalovirus (HCMV) (Davis) and the
lentiviruses, and human immunodeficiency virus type 1
(HIV-1) (IIIB) and HIV-2 (ROD). Interestingly, none of the
compounds tested were active at subtoxic concentrations, in-
cluding 11f and 11h, which represent the corresponding ana-
logues of the BCNA prototypes Cf 1368 and Cf 1743.3,5
These data revealed that the acyclic nucleotide analogues of
furo[2,3-d]pyrimidin-2(3H)-ones (11) and 16, designed to
by-pass the first phosphorylation step necessary for the anti-
VZV activity of BCNAs, may not be further metabolized to
their active antiviral metabolites and (or) the compounds
may not be recognized as an inhibitor at their eventual anti-
viral target.
Experimental section
Solvents were dried by standard procedures. Tetrahydro-
furan (THF) was freshly distilled from sodium/benzophe-
none under argon. All 1-alkynes, Pd(Ph3P)4, and CuI are
commercially available from Sigma-Aldrich. TLC was per-
formed on plates of Kieselgel 60 F254 (Merck). If not stated
otherwise, NMR spectra were recorded on Bruker Avance
400 (1H at 400.0 MHz, 13C at 100.6 MHz) in CDCl3,
DMSO-d6, or D2O. NMR spectra of several compounds
were recorded on Bruker Avance 600 spectrometer (1H at
600.0 MHz, 13C at 150.9 MHz) or Bruker Avance 500 (1H
at 500.0 MHz, 13C at 125.8 MHz). Chemical shifts (in ppm,
d scale) were referenced to the solvent signal (CDCl3 d:
7.26 ppm for 1H NMR and d: 77.0 ppm for 13C NMR,
DMSO-d6 for 1H NMR d: 2.5 ppm and for 13C d: 39.7
ppm). Chemical shifts in D2O were referenced to 1,4-diox-
Conclusions
In conclusion, novel series of the 2-(phosphonomethoxy)ethyl
derivatives of 6-alkylfuro[2,3-d]pyrimidin-2(3H)-ones was
effectively synthesized by a convenient one-pot procedure
consisting of Sonogashira coupling of the 5-iodouracil com-
pound (6) with 1-alkynes (8) and subsequent Cu(I)-promoted
cyclization. For this purpose, protected 5-iodo-1-[2-(phos-
phonomethoxy)ethyl]uracil (6) was prepared as a suitable
1
ane for H NMR d: 3.75 ppm and for 13C NMR d: 67.19
ppm. Melting points were determined on a Bu¨chi melting
point B-545 apparatus and are uncorrected. UV spectra
were measured on a DU spectrophotometer with solutions
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632
Can. J. Chem. Vol. 88, 2010
in MeOH. Mass spectra were measured on a LCQ classic
spectrometer using electrospray ionization (ESI). All new
compounds were fully characterized by mass spectrometry,
NMR spectroscopy, and elemental analysis (or HR mass
spectrometry in the case of compounds 6, 10h, 12, and 17).
Complete assignment of all NMR signals using a combina-
tion of 1H-1H COSY, 1H-13C HSQC, and 1H-13C HMBC
methods is provided.
(8b, 0.22 mL, 160 mg, 1.95 mmol) in dry and deoxygenated
DMF/Et3N (10:1, 20 mL) was heated under argon at 50 8C
for 2 h. Volatiles were flash evaporated, the residue was ex-
tracted with hot MeOH (3 Â 30 mL), and the solid was fil-
tered off. Volatiles were evaporated, the residue was
dissolved in CHCl3 (50 mL) and washed (2 Â 30 mL of sa-
turated EDTA (aq.), 1 Â 20 mL H2O). Organics were dried
(MgSO4) and filtered. Flash chromatography (4% MeOH in
CHCl3) afforded compounds 9b (512 mg, 82%) as brownish
oil and 10b (11 mg, 2%) as yellow foam.
1-{2-[(Diisopropoxyphosphoryl)methoxy]ethyl}-5-
iodouracil (6)
1-{2-[(Diisopropoxyphosphoryl)methoxy]ethyl}-5-(1-
hexynyl)uracil (9b)
A solution of 5 (8.28 g, 24.78 mmol), CAN (6.79 g,
12.39 mmol), and iodine (8.18 g, 32.22 mmol) in dry aceto-
nitrile (240 mL) was stirred at 80 8C under an inert atmos-
phere until the starting material was completely consumed
after 3 h as monitored by TLC analysis (5% MeOH/CHCl3).
Volatiles were evaporated, and the residue was dissolved in
EtOAc (250 mL) and washed with ice cold 5% Na2S2O3
(aq., 130 mL) and brine (50 mL). Organics were dried
(MgSO4), filtered, and solvents evaporated. The crude prod-
uct was crystallized (EtOAc) to give 6 (10.40 g, 87%) as
1H NMR (CDCl3) d: 9.00 (bs, 1H, NH), 7.48 (s, 1H, H-6),
4.73 (dh, 2H, JH,P = 7.7 Hz, Jvic = 6.2 Hz, POCH), 3.93 (m,
2H, H-1’), 3.79 (m, 2H, H-2’), 3.73 (d, 2H, JH,P = 8.4 Hz, H-
4’), 2.36 (t, 2H, Jvic = 7.1 Hz, H-3’’), 1.57–1.50 (m, 2H, H-
4’’), 1.46–1.36 (m, 2H, H-5’’), 1.35–1.28 (m, 12H, CH3),
0.90 (t, 3H, Jvic = 7.3 Hz, H-6’’). 13C NMR (CDCl3) d:
162.1 (C-4), 149.8 (C-2), 147.4 (C-6), 100.2 (C-5), 95.2 (C-
2’’), 71.2 (d, JC,P = 6.7 Hz, POC), 70.8 (C-1’’), 70.7 (d, JC,P
= 10.5 Hz, C-2’), 66.2 (d, JC,P = 167.8 Hz, C-4’), 48.8 (C-1’),
1
white crystals; mp 114 8C. H NMR (DMSO-d6) d: 11.66
30.6 (C-3’’), 24.02 (d, JC,P = 3.8 Hz, CH3), 23.96 (d, JC,P
=
(bs, 1H, NH), 8.07 (s, 1H, H-6), 4.56 (dh, 2H, JH,P
=
4.5 Hz, CH3), 22.0 (C-4’’), 19.3 (C-5’’), 13.6 (C-6’’). MS
(ESI) m/z: 415 [M + H]+. HR-MS (ESI) m/z: C19H31N2O6P-
Na [M + Na]+. Anal. calcd.: 437.1810; found: 437.1812.
7.7 Hz, Jvic = 6.2 Hz, POCH), 3.87 (m, 2H, H-1’), 3.77 (d,
2H, JH,P = 8.3 Hz, H-4’), 3.69 (m, 2H, H-2’), 1.23 and 1.21
(2 Â d, 2 Â 6H, Jvic = 6.2 Hz, CH3). 13C NMR (DMSO-d6)
d: 161.1 (C-4), 150.7 (C-2), 150.5 (C-6), 70.3 (d, JC,P
=
6-Butyl-3-{2-[(diisopropoxyphosphoryl)methoxy]
ethyl}furo[2,3-d]pyrimidin-2(3H)-one (10b)
1H and 13C NMR data were identical with compound 10b
prepared from compound 6 by the General Procedure A.
6.4 Hz, POC), 69.9 (d, JC,P = 11.6 Hz, C-2’), 67.7 (C-5),
64.9 (d, JC,P = 163.8 Hz, C-4’), 47.4 (C-1’), 23.9 (d, JC,P
=
3.8 Hz, CH3), 23.8 (d, JC,P = 4.5 Hz, CH3). MS (ESI) m/z:
459 [M – H]–. HR-MS (ESI) m/z: C13H21N2IO6P [M – H]–.
Anal. calcd.: 459.0176; found: 459.0181.
Method B
A stirred suspension of 6 (230 mg, 0.50 mmol), Pd(Ph3P)4
(29 mg, 25 mmol), CuI (20 mg, 0.11 mmol), 1-hexyne
(0.17 mL, 122 mg, 1.49 mmol), and Et3N (0.21 mL) in dry
and deoxygenated THF (10 mL) was stirred under argon at
50 8C for 3 h. Volatiles were flash evaporated, the residue
was extracted with hot MeOH (3 Â 30 mL), and the solid
was filtered off. Volatiles were evaporated, the residue was
dissolved in EtOAc (30 mL) and washed (2 Â 10 mL of sa-
turated EDTA (aq.), 1 Â 10 mL H2O). Organics were dried
(MgSO4) and filtered. Flash chromatography (5% MeOH/
CHCl3) afforded compounds 9b (246 mg, 59%) as a yellow
oil and 12 as a yellowish oil (60 mg, 12%).
5-Iodo-1-[2-(phosphonomethoxy)ethyl]uracil (7)
A mixture of compound 6 (230 mg, 0.50 mmol), bromo(-
trimethyl)silane (0.90 mL, 1.04 g, 6.82 mmol), and 2,6-luti-
dine (0.70 mL, 648 mg, 6.00 mmol) in acetonitrile (5 mL)
was stirred overnight at room temperature. The mixture was
concentrated in vacuo and then codistilled with acetonitrile
(3 Â 5 mL). The residue was dissolved in 50% aqueous
methanol (20 mL) and concd HCl was added until the pH
was between 2 and 3. After 1 h at room temperature, the
solvents were evaporated to dryness and the residue was pu-
rified using HPLC (20%–100% aq. MeOH) and crystallized
(H2O) to give 7 (70 mg, 37%) as white crystals; mp 194 8C.
UV–vis lmax (nm) (3 ((mol/L)–1 cm–1): 286 (16 300). H
NMR (DMSO-d6) d: 11.64 (bs, 1H, NH), 8.10 (s, 1H, H-6),
3.85 (m, 2H, H-1’), 3.68 (m, 2H, H-2’), 3.57 (d, 2H, JH,P
1
1-{2-[(Diisopropoxyphosphoryl)methoxy]ethyl}-5-(1-
hexynyl)uracil (9b)
1H and 13C NMR data were identical with compound 9b
prepared by Method A.
=
8.4 Hz, H-4’). 13C NMR (DMSO-d6) d: 161.1 (C-4), 150.8
(C-2), 150.5 (C-6), 69.7 (d, JC,P = 9.8 Hz, C-2’), 67.8 (C-5),
66.5 (d, JC,P = 159.7 Hz, C-4’), 47.1 (C-1’). MS (ESI) m/z:
374.9 [M – H]–. Anal. calcd. for C7H10N2IO6P.2/3H2O
(388.05): C 21.67, H 2.94, N 7.22, I 32.70, P 7.98; found:
C 21.67, H 3.13, N 6.92, I 32.96, P 7.77.
Diisopropyl {(2-[6-butyl-2-oxo-5-(hexyn-1-yl)furo[2,3-
d]pyrimidin-3(2H)-yl]ethoxy)methyl}phosphonate (12)
1H NMR (500.0 MHz, CDCl3) d: 7.89 (s, 1H, H-4), 4.68
(dh, 2H, JH,P = 7.7 Hz, Jvic = 6.2 Hz, POCH), 4.21 (m, 2H,
H-1’), 3.91 (m, 2H, H-2’), 3.69 (d, 2H, JH,P = 8.6 Hz, H-4’),
2.73 (t, 2H, Jvic = 7.4 Hz, H-1’’), 2.41 (t, 2H, Jvic = 7.1 Hz,
H-3’’’), 1.68 (m, 2H, H-2’’), 1.57 and 1.46 (2 Â m, 2 Â 2H,
H-4’’’ and H-5’’’), 1.36 (m, 2H, H-3’’), 1.28 and 1.24 (2 Â d,
2 Â 6H, Jvic = 6.2 Hz, CH3), 0.94 (t, 3H, Jvic = 7.4 Hz, H-
6’’’), 0.93 (t, 3H, Jvic = 7.3, H-4’’). 13C NMR (125.8 MHz,
CDCl3) d: 170.7 (C-7a), 162.0 (C-6), 155.3 (C-2), 140.8 (C-
Sonogashira coupling of 5-iodouracil derivative (6) with
1-hexyne (8b)
Method A
A stirred suspension of 6 (690 mg, 1.50 mmol), Pd(Ph3P)4
(87 mg, 75 mmol), CuI (87 mg, 0.46 mmol), and 1-hexyne
Published by NRC Research Press

Janeba et al.
633
4), 107.7 (C-4a), 97.8 (C-5), 97.2 (C-2’’’), 71.1 (d, JC,P
=
(125.8 MHz, CDCl3) d: 172.1 (C-7a), 159.6 (C-6), 155.4
6.6 Hz, POCH), 70.3 (d, JC,P = 11.6 Hz, C-2’), 68.2 (C-1’’’),
66.1 (d, JC,P = 168.2 Hz, C-4’), 52.1 (C-1’), 30.6 (C-4’’’),
29.0 (C-2’’), 26.8 (C-1’’), 24.0 (d, JC,P = 3.8 Hz, CH3), 23.9
(d, JC,P = 4.6 Hz, CH3), 22.0 (C-5’’’ and C-3’’), 19.2 (C-3’’’),
13.6 (C-6’’’ and C-4’’). MS (ESI) m/z: 495 [M + H]+. HR-
MS (ESI) m/z: C25H40N2O6P [M + H]+. Anal. calcd.:
495.2618; found: 495.2616.
(C-2), 140.5 (C-4), 107.4 (C-4a), 98.5 (C-5), 71.1 (d, JC,P
6.7 Hz, POC), 70.6 (d, JC,P = 11.8 Hz, C-2’), 66.0 (d, JC,P
168.8 Hz, C-4’), 51.5 (C-1’), 30.1 (C-1’’), 24.0 (d, JC,P
=
=
=
3.9 Hz, CH3), 23.9 (d, JC,P = 4.6 Hz, CH3), 20.2 (C-2’’),
13.5 (C-3’’). MS (ESI) m/z: 399 [M – H]–. Anal. calcd. for
C18H29N2O6P (400.41): C 53.99, H 7.30, N 7.00, P 7.74;
found: C 53.94, H 7.35, N 6.86, P 8.02.
6-Butyl-3-{2-[(diisopropoxyphosphoryl)methoxy]
ethyl}furo[2,3-d]pyrimidin-2(3H)-one (10b)
6-Butyl-3-{2-
[(diisopropoxyphosphoryl)methoxy]ethyl}furo[2,3-
d]pyrimidin-2(3H)-one (10b)
Treatment of 6 (690 mg, 1.50 mmol) with 1-hexyne (8b;
0.22 mL, 160 mg, 1.95 mmol) by General procedure A gave
10b (485 mg, 78%) as white crystals (EtOAc/Et2O); mp
A stirred suspension of 9b (438 mg, 1.06 mmol) and CuI
(200 mg, 1.05 mmol) in a mixture of MeOH (30 mL) and
Et3N (20 mL) was refluxed for 2 h. Volatiles were flash
evaporated, the residue was extracted with hot MeOH (3 Â
30 mL), and the solid was filtered off. Volatiles were evapo-
rated, the residue was dissolved in CHCl3 (50 mL) and
washed (2 Â 30 mL of saturated EDTA (aq.), 1 Â 20 mL
H2O). Organics were dried (MgSO4), filtered, and the product
was isolated by flash chromatography (4% MeOH/CHCl3)
and crystallized (EtOAc/Et2O) to give 10b (375 mg, 90%) as
1
139 8C. H NMR (CDCl3) d: 7.91 (s, 1H, H-4), 6.06 (s, 1H,
H-5), 4.66 (dh, 2H, JH,P = 7.7 Hz, Jvic = 6.2 Hz, POCH),
4.20 (m, 2H, H-1’), 3.88 (m, 2H, H-2’), 3.68 (d, 2H, JH,P
8.6 Hz, H-4’), 2.63 (t, 2H, Jvic = 7.5 Hz, H-1’’), 1.68–1.61
(m, 2H, H-2’’), 1.42–1.31 (m, 2H, H-3’’), 1.27 and 1.23
=
(2 Â d, 2 Â 6H, Jvic = 6.2 Hz, CH3), 0.92 (t, 3H, Jvic
=
7.4 Hz, H-4’’). 13C NMR (CDCl3) d: 172.1 (C-7a), 159.7
(C-6), 155.3 (C-2), 140.4 (C-4), 107.3 (C-4a), 98.4 (C-5),
71.0 (d, JC,P = 6.7 Hz, POC), 70.6 (d, JC,P = 11.8 Hz, C-2’),
66.1 (d, JC,P = 168.8 Hz, C-4’), 51.5 (C-1’), 28.8 (C-1’’), 27.9
(C-2’’), 24.0 (d, JC,P = 3.9 Hz, CH3), 23.9 (d, JC,P = 4.6 Hz,
CH3), 22.0 (C-3’’), 13.6 (C-4’’). MS (ESI) m/z: 415 [M + H]+.
Anal. calcd. for C19H31N2O6P (414.43): C 55.06, H 7.54, N
6.76, P 7.47; found: C 55.12, H 7.63, N 6.70, P 7.69.
white crystals; mp 142 8C. H and 13C NMR data were iden-
tical with compound 10b prepared from compound 6 by
General procedure A.
1
Synthesis of furo[2,3-d]pyrimidin-2(3H)-ones (10) by
Sonogashira coupling of 5-iodouracil derivative (6) with
1-alkynes (8) followed by in situ intramolecular cyclization
3-{2-[(Diisopropoxyphosphoryl)methoxy]ethyl}-6-
pentylfuro[2,3-d]pyrimidin-2(3H)-one (10c)
General procedure A
Treatment of 6 (464 mg, 1.01 mmol) with 1-heptyne (8c;
0.17 mL, 126 mg, 1.31 mmol) by General procedure A gave
10c (343 mg, 79%) as white crystals (EtOAc/EtOH); mp
120 8C. H NMR (CDCl3) d: 7.91 (s, 1H, H-4), 6.06 (s, 1H,
H-5), 4.65 (dh, 2H, JH,P = 7.7 Hz, Jvic = 6.2 Hz, POCH),
A stirred suspension of 6, Pd(Ph3P)4 (0.05 equiv.), CuI
(0.5 equiv.), and 1-alkyne (8; 1.3 equiv.) in dry and deoxy-
genated DMF/Et3N was stirred under argon at 50 8C for 2–
3 h. TLC (5% MeOH/CHCl3) showed complete conversion
to a faster-migrating product. CuI (0.5 equiv.) was added
and the reaction mixture was stirred at 90 8C for 2–4 h.
TLC (5% MeOH/CHCl3) showed complete conversion of
the intermediate 6-alkynyl derivative to a fluorescent product
(10). Volatiles were flash evaporated, the residue was dis-
solved in hot MeOH, and the solid filtered off. Volatiles
were evaporated and the residue was dissolved in CHCl3
(80 mL). Organics were washed (2 Â 10 mL of saturated
EDTA (aq.), 1 Â 10 mL H2O) and dried (MgSO4). After fil-
tration and evaporation, the product was isolated by flash
chromatography (5% MeOH/CHCl3) and crystallized from a
mixture of EtOAc with other organic solvents (hexanes,
EtOH, Et2O).
1
4.20 (m, 2H, H-1’), 3.89 (m, 2H, H-2’), 3.68 (d, 2H, JH,P
=
8.6 Hz, H-4’), 2.62 (t, 2H, Jvic = 7.2 Hz, H-1’’), 1.70–1.63
(m, 2H, H-2’’), 1.35–1.29 (m, 4H, H-3’’ and H-4’’), 1.25 and
1.21 (2 Â d, 2 Â 6H, Jvic = 6.2 Hz, CH3), 0.88 (t, 3H, Jvic
=
7.0 Hz, H-5’’). 13C NMR (CDCl3) d: 172.1 (C-7a), 159.8 (C-
6), 155.3 (C-2), 140.4 (C-4), 107.4 (C-4a), 98.4 (C-5), 71.0
(d, JC,P = 6.8 Hz, POC), 70.6 (d, JC,P = 11.8 Hz, C-2’), 66.1
(d, JC,P = 168.8 Hz, C-4’), 51.5 (C-1’), 31.1 (C-1’’), 28.2 (C-
2’’), 26.4 (C-3’’), 24.0 (d, JC,P = 3.9 Hz, CH3), 23.9 (d, JC,P
=
4.6 Hz, CH3), 22.2 (C-4’’), 13.9 (C-5’’). MS (ESI) m/z: 429
[M + H]+. Anal. calcd. for C20H33N2O6P (428.21): C 56.06,
H 7.76, N 6.54, P 7.23; found: C 56.11, H 7.89, N 6.47, P
7.49.
3-{2-[(Diisopropoxyphosphoryl)methoxy]ethyl}-6-
propylfuro[2,3-d]pyrimidin-2(3H)-one (10a)
3-{2-[(Diisopropoxyphosphoryl)methoxy]ethyl}-6-
hexylfuro[2,3-d]pyrimidin-2(3H)-one (10d)
Treatment of 6 (690 mg, 1.50 mmol) with 1-pentyne (8a;
0.19 mL, 133 mg, 1.95 mmol) by General procedure A gave
10a (402 mg, 67%) as white crystals (EtOAc/hexanes 1:1);
Treatment of 6 (920 mg, 2.00 mmol) with 1-octyne (8d;
0.38 mL, 287 mg, 2.60 mmol) by General procedure A gave
10d (586 mg, 64%) as white crystals (EtOAc/hexanes); mp
1
mp 128 8C. H NMR (500.0 MHz, CDCl3) d: 7.94 (s, 1H,
1
118 8C. H NMR (CDCl3) d: 7.92 (s, 1H, H-4), 6.06 (s, 1H,
H-4), 6.09 (t, 1H, J5,1’’ = 1.1 Hz, H-5), 4.68 (dh, 2H, JH,P
=
H-5), 4.65 (dh, 2H, JH,P = 7.7 Hz, Jvic = 6.2 Hz, POCH), 4.20
(m, 2H, H-1’), 3.89 (m, 2H, H-2’), 3.68 (d, 2H, JH,P = 8.6 Hz,
H-4’), 2.62 (t, 2H, Jvic = 7.2 Hz, H-1’’), 1.70–1.62 (m, 2H, H-
2’’), 1.40–1.19 (m, 18H, H-3’’ and H-4’’ and H-5’’ and CH3),
0.87 (t, 3H, Jvic = 6.9 Hz, H-6’’). 13C NMR (CDCl3) d: 172.1
7.7 Hz, Jvic = 6.2 Hz, POCH), 4.22 (m, 2H, H-1’), 3.91 (m,
2H, H-2’), 3.70 (d, 2H, JH,P = 8.6 Hz, H-4’), 2.63 (td, 2H,
Jvic = 7.4 Hz, J1’’,5 = 1.1 Hz, H-1’’), 1.72 (h, 2H, Jvic
=
7.4 Hz, H-2’’), 1.29 and 1.25 (2 Â d, 2 Â 6H, Jvic = 6.2 Hz,
CH3), 0.99 (t, 3H, Jvic = 7.4 Hz, H-3’’). 13C NMR
Published by NRC Research Press

634
Can. J. Chem. Vol. 88, 2010
(C-7a), 159.8 (C-6), 155.3 (C-2), 140.4 (C-4), 107.4 (C-4a),
(m, 2H, H-2’’), 1.33–1.19 (m, 26H, H-3’’ and H-4’’ and H-
5’’ and H-6’’ and H-7’’ and H-8’’ and H-9’’ and CH3), 0.86
(t, 3H, Jvic = 6.9 Hz, H-10’’). 13C NMR (CDCl3) d: 172.1
(C-7a), 159.8 (C-6), 155.3 (C-2), 140.4 (C-4), 107.4 (C-4a),
98.4 (C-5), 71.0 (d, JC,P = 6.8 Hz, POC), 70.6 (d, JC,P
=
11.8 Hz, C-2’), 66.1 (d, JC,P = 168.8 Hz, C-4’), 51.5 (C-1’),
31.4 (C-1’’), 28.6 (C-2’’), 28.2 (C-3’’), 26.7 (C-4’’), 24.0 (d,
JC,P = 3.9 Hz, CH3), 23.9 (d, JC,P = 4.6 Hz, CH3), 22.5 (C-
5’’), 14.0 (C-6’’). MS (ESI) m/z: 441 [M – H]–. Anal. calcd.
for C21H35N2O6P (442.49): C 57.00, H 7.97, N 6.33, P 7.00;
found: C 56.84, H 8.09, N 6.24, P 7.17.
98.4 (C-5), 71.0 (d, JC,P = 7.0 Hz, POC), 70.6 (d, JC,P
=
11.8 Hz, C-2’), 66.1 (d, JC,P = 168.8 Hz, C-4’), 51.5 (C-1’),
31.8 (C-1’’), 29.5 (C-2’’), 29.4 (C-3’’), 29.24 (C-4’’), 29.20
(C-5’’), 29.0 (C-6’’), 28.2 (C-7’’), 26.8 (C-8’’), 24.0 (d, JC,P
=
3.9 Hz, CH3), 23.9 (d, JC,P = 4.6 Hz, CH3), 22.6 (C-9’’), 14.1
(C-10’’). MS (ESI) m/z: 521 499 [M + H]+. Anal. calcd. for
C25H43N2O6P (498.59): C 60.22, H 8.69, N 5.62, P 6.21;
found: C 59.93, H 8.75, N 5.49, P 6.46.
3-{2-[(Diisopropoxyphosphoryl)methoxy]ethyl}-6-
heptylfuro[2,3-d]pyrimidin-2(3H)-one (10e)
Treatment of 6 (460 mg, 1.00 mmol) with 1-nonyne (8e;
0.21 mL, 162 mg, 1.30 mmol) by General procedure A gave
10e (226 mg, 50%) as white crystals (EtOAc/Et2O); mp
3-{2-[(Diisopropoxyphosphoryl)methoxy]ethyl}-6-(4-
pentylphenyl)furo[2,3-d]pyrimidin-2(3H)-one (10h)
1
116 8C. H NMR (CDCl3) d: 7.92 (s, 1H, H-4), 6.06 (s, 1H,
Treatment of 6 (460 mg, 1.00 mmol) with (4-pentylphe-
nyl)acetylene (8h; 0.25 mL, 224 mg, 1.30 mmol) by General
procedure A gave 10h (232 mg, 46%) as white crystals
(EtOAc/hexanes); mp 172 8C. 1H NMR (500.0 MHz,
CDCl3) d: 8.08 (s, 1H, H-4), 7.69–7.67 (m, 2H, o-Ph),
7.28–7.26 (m, 2H, m-Ph), 6.65 (s, 1H, H-5), 4.70 (dh, 2H,
JH,P = 7.7 Hz, Jvic = 6.2 Hz, POCH), 4.27– 4.25 (m, 2H, H-
1’), 3.95–3.94 (m, 2H, H-2’), 3.72 (d, 2H, JH,P = 8.6 Hz, H-
4’), 2.67–2.64 (m, 2H, H-1’’), 1.66–1.63 (m, 2H, H-2’’),
1.37–1.34 (m, 4H, H-3’’ and H-4’’), 1.30 and 1.27 (2 Â d,
2 Â 6H, Jvic = 6.2 Hz, CH3), 0.92–0.89 (m, 3H, H-5’’). 13C
NMR (125.8 MHz, CDCl3) d: 171.9 (C-7a), 155.9 (C-6),
155.3 (C-2), 145.1 (C-p-Ph), 141.2 (C-4), 129.0 (C-m-Ph),
125.8 (C-i-Ph), 124.9 (C-o-Ph), 108.0 (C-4a), 96.3 (C-5),
71.1 (d, JC,P = 6.7 Hz, POC), 70.5 (d, JC,P = 11.6 Hz, C-2’),
66.1 (d, JC,P = 168.9 Hz, C-4’), 51.6 (C-1’), 35.8 (C-1’’), 31.4
and 30.9 (C-2’’ and C-3’’), 24.00 (d, JC,P = 3.9 Hz, CH3),
23.95 (d, JC,P = 4.6 Hz, CH3), 22.5 (C-4’’), 14.0 (C-5’’). MS
(ESI) m/z: 505 [M + H]+. HR-MS (ESI) m/z: C26H38N2O6P
[M + H]+. Anal. calcd.: 505.2462; found: 505.2463.
H-5), 4.67 (dh, 2H, JH,P = 7.7 Hz, Jvic = 6.2 Hz, POCH),
4.21 (m, 2H, H-1’), 3.90 (m, 2H, H-2’), 3.69 (d, 2H, JH,P
=
8.6 Hz, H-4’), 2.63 (t, 2H, Jvic = 7.3 Hz, H-1’’), 1.71–1.63
(m, 2H, H-2’’), 1.35–1.23 (m, 20H, H-3’’ and H-4’’ and H-
5’’ and H-6’’ and CH3), 0.87 (t, 3H, Jvic = 6.9 Hz, H-7’’).
13C NMR (CDCl3) d: 172.2 (C-7a), 159.9 (C-6), 155.4 (C-
2), 140.3 (C-4), 107.4 (C-4a), 98.4 (C-5), 71.1 (d, JC,P
6.9 Hz, POC), 70.6 (d, JC,P = 11.8 Hz, C-2’), 66.2 (d, JC,P
=
=
168.8 Hz, C-4’), 51.5 (C-1’), 31.7 (C-1’’), 29.0 (C-2’’), 28.9
(C-3’’), 28.2 (C-4’’), 26.8 (C-5’’), 24.01 (d, JC,P = 3.9 Hz,
CH3), 23.96 (d, JC,P = 4.6 Hz, CH3), 22.6 (C-6’’), 14.0 (C-
7’’). MS (ESI) m/z: 457 [M + H]+. Anal. calcd. for
C22H37N2O6P (456.51): C 57.88, H 8.17, N 6.14, P 6.78;
found: C 57.67, H 8.14, N 5.99, P 7.05.
3-{2-[(Diisopropoxyphosphoryl)methoxy]ethyl}-6-
octylfuro[2,3-d]pyrimidin-2(3H)-one (10f)
Treatment of 6 (992 mg, 2.16 mmol) with 1-decyne (8f;
0.51 mL, 390 mg, 2.82 mmol) by General procedure A gave
10f (491 mg, 49%) as white crystals (EtOAc/Et2O); mp 114–
1
115 8C. H NMR (CDCl3) d: 7.92 (s, 1H, H-4), 6.06 (s, 1H,
H-5), 4.68 (dh, 2H, JH,P = 7.7 Hz, Jvic = 6.2 Hz, POCH), 4.21
(m, 2H, H-1’), 3.90 (m, 2H, H-2’), 3.69 (d, 2H, JH,P = 8.6 Hz,
H-4’), 2.63 (t, 2H, Jvic = 7.5 Hz, H-1’’), 1.72–1.62 (m, 2H, H-
2’’), 1.39–1.21 (m, 22H, H-3’’ and H-4’’ and H-5’’ and H-6’’
and H-7’’ and CH3), 0.87 (t, 3H, Jvic = 6.8 Hz, H-8’’). 13C
NMR (CDCl3) d: 172.1 (C-7a), 159.9 (C-6), 155.3 (C-2),
140.4 (C-4), 107.4 (C-4a), 98.4 (C-5), 71.1 (d, JC,P = 6.9 Hz,
Transformation of esters (10) to free phosphonic acids (11)
General procedure B
The dried starting esters (10; 1 mmol), bromo(trimethyl)-
silane (1.75 mL, 2.03 g, 13.26 mmol), and acetonitrile
(10 mL) were stirred at room temperature overnight. The
mixture was concentrated in vacuo and then codistilled with
a mixture of water/EtOH (9:1, 2 Â 5 mL). The residue was
crystallized (2 crops) to give free phosphonic acids (11).
POC), 70.6 (d, JC,P = 11.8 Hz, C-2’), 66.1 (d, JC,P
168.8 Hz, C-4’), 51.5 (C-1’), 31.8 (C-1’’), 29.2 (C-2’’), 29.1
(C-3’’), 29.0 (C-4’’), 28.2 (C-5’’), 26.8 (C-6’’), 24.2 (d, JC,P
=
=
3-[2-(Phosphonomethoxy)ethyl]-6-propylfuro[2,3-
d]pyrimidin-2(3H)-one (11a)
Treatment of 10a (250 mg, 0.62 mmol) gave 11a (167 mg,
85%) as white crystals (EtOAc/EtOH 3:1); mp 165 8C.
UV–vis lmax (nm) (3 ((mol/L)–1 cm–1): 329 (5400), 242 (8900).
1H NMR (DMSO-d6) d: 8.36 (s, 1H, H-4), 6.41 (s, 1H, H-5),
3.9 Hz, CH3), 24.0 (d, JC,P = 4.6 Hz, CH3), 22.6 (C-7’’), 14.1
(C-8’’). MS (ESI) m/z: 471 [M + H]+. Anal. calcd. for
C23H39N2O6P (470.54): C 58.71, H 8.35, N 5.95, P 6.58;
found: C 58.59, H 8.43, N 5.88, P 6.77.
6-Decyl-3-{2-
[(diisopropoxyphosphoryl)methoxy]ethyl}furo[2,3-
d]pyrimidin-2(3H)-one (10g)
4.11 (m, 2H, H-1’), 3.78 (m, 2H, H-2’), 3.57 (d, 2H, JH,P
=
8.5 Hz, H-4’), 2.61 (t, 2H, Jvic = 7.4 Hz, H-1’’), 1.71–1.58 (m,
2H, H-2’’), 0.94 (t, 3H, J = 7.4 Hz, H-3’’). 13C NMR (DMSO-
d6) d: 171.5 (C-7a), 157.9 (C-6), 154.6 (C-2), 142.8 (C-4),
106.0 (C-4a), 99.7 (C-5), 69.4 (d, JC,P = 10.5 Hz, C-2’),
66.5 (d, JC,P = 159.9 Hz, C-4’), 50.2 (C-1’), 29.4 and 20.0 and
13.5 (6-propyl). MS (ESI) m/z: 315 [M – H]–. Anal. calcd. for
C12H17N2O6PÁ1/5H2O (319.85): C 45.06, H 5.48, N 8.76,
P 9.68; found: C 45.21, H 5.45, N 8.64, P 9.90.
Treatment of 6 (800 mg, 1.74 mmol) with 1-dodecyne
(8g; 0.48 mL, 376 mg, 2.26 mmol) by General procedure A
gave 10g (700 mg, 81%) as white crystals (EtOAc/hexanes);
1
mp 112 8C. H NMR (CDCl3) d: 7.91 (s, 1H, H-4), 6.05 (s,
1H, H-5), 4.65 (dh, 2H, JH,P = 7.7 Hz, Jvic = 6.2 Hz, POCH),
4.20 (m, 2H, H-1’), 3.88 (m, 2H, H-2’), 3.68 (d, 2H, JH,P
=
8.6 Hz, H-4’), 2.61 (t, 2H, Jvic = 7.4 Hz, H-1’’), 1.72–1.60
Published by NRC Research Press

Janeba et al.
635
6-Butyl-3-[2-(phosphonomethoxy)ethyl]furo[2,3-
d]pyrimidin-2(3H)-one (11b)
H-2’’), 1.36–1.20 (m, 8H, H-3’’ and H-4’’ and H-5’’ and
H-6’’), 0.85 (t, 3H, J = 6.9 Hz, H-7’’). 13C NMR
(125.8 MHz, DMSO-d6) d: 171.6 (C-7a), 158.2 (C-6), 154.7
Treatment of 10b (200 mg, 0.48 mmol) gave 11b
(110 mg, 69%) as white crystals (H2O); mp 144 8C. UV–vis
(C-2), 143.0 (C-4), 106.2 (C-4a), 99.7 (C-5), 69.5 (d, JC,P
=
lmax (nm) (3 ((mol/L)–1 cm–1): 329 (7000), 243 (13 500). H
1
10.5 Hz, C-2’), 66.5 (d, JC,P = 159.9 Hz, C-4’), 50.3 (C-1’),
31.4 and 28.59 and 28.55 and 27.6 and 26.6 and 22.3 and
14.2 (6-heptyl). MS (ESI) m/z: 371 [M – H]–. Anal. calcd.
for C16H25N2O6PÁ1/4 H2O (376.86): C 50.99, H 6.82, N
7.43, P 8.22; found: C 51.12, H 6.77, N 7.41, P 8.40.
NMR (DMSO-d6) d: 8.36 (s, 1H, H-4), 6.40 (s, 1H, H-5),
4.11 (m, 2H, H-1’), 3.77 (m, 2H, H-2’), 3.58 (d, 2H, JH,P
=
8.6 Hz, H-4’), 2.64 (t, 2H, Jvic = 7.4 Hz, H-1’’), 1.68–1.50
(m, 2H, H-2’’), 1.42–1.27 (m, 2H, H-3’’), 0.90 (t, 3H, J =
7.4 Hz, H-4’’). 13C NMR (DMSO-d6) d: 171.5 (C-7a), 158.1
(C-6), 154.6 (C-2), 142.9 (C-4), 106.1 (C-4a), 99.6 (C-5),
69.4 (d, JC,P = 10.5 Hz, C-2’), 66.5 (d, JC,P = 159.9 Hz, C-
4’), 50.3 (C-1’), 28.7 and 27.2 and 21.7 and 13.8 (6-butyl).
MS (ESI) m/z: 329 [M – H]–. Anal. calcd. for C13H19N2O6P
(330.27): C 47.28, H 5.80, N 8.48, P 9.38; found: C 47.14,
H 5.76, N 8.33, P 9.59.
6-Octyl-3-[2-(phosphonomethoxy)ethyl]furo[2,3-
d]pyrimidin-2(3H)-one (11f)
Treatment of 10f (166 mg, 0.35 mmol) gave 11f (126 mg,
90%) as white crystals (H2O/EtOH); mp 164 8C. UV–vis
1
lmax (nm) (3 ((mol/L)–1 cm–1): 329 (5900), 243 (11 000). H
NMR (DMSO-d6) d: 8.36 (s, 1H, H-4), 6.40 (s, 1H, H-5),
4.11 (m, 2H, H-1’), 3.77 (m, 2H, H-2’), 3.57 (d, 2H, JH,P
=
3-[2-(Phosphonomethoxy)ethyl]-6-pentylfuro[2,3-
d]pyrimidin-2(3H)-one (11c)
8.6 Hz, H-4’), 2.63 (t, 2H, Jvic = 7.3 Hz, H-1’’), 1.68–1.52
(m, 2H, H-2’’), 1.38–1.15 (m, 10H, H-3’’ and H-4’’ and H-
5’’ and H-6’’ and H-7’’), 0.85 (t, 3H, J = 6.8 Hz, H-8’’). 13C
NMR (DMSO-d6) d: 171.5 (C-7a), 158.1 (C-6), 154.6 (C-2),
Treatment of 10c (250 mg, 0.58 mmol) gave 11c
(160 mg, 80%) as white crystals (H2O); mp 156 8C. UV–vis
lmax (nm) (3 ((mol/L)–1 cm–1): 329 (6700), 243 (13 600). H
1
142.8 (C-4), 106.1 (C-4a), 99.6 (C-5), 69.4 (d, JC,P
=
10.5 Hz, C-2’), 66.5 (d, JC,P = 159.9 Hz, C-4’), 50.2 (C-1’),
31.4 and 28.73 and 28.67 and 28.5 and 27.5 and 26.5 and
22.2 and 14.1 (6-octyl). MS (ESI) m/z: 385 [M – H]–. Anal.
calcd. for C17H27N2O6PÁ1/2 H2O (395.39): C 51.64, H 7.14,
N 7.09, P 7.83; found: C 51.58, H 7.18, N 7.02, P 8.11.
NMR (DMSO-d6) d: 8.36 (s, 1H, H-4), 6.40 (s, 1H, H-5),
4.11 (m, 2H, H-1’), 3.77 (m, 2H, H-2’), 3.58 (d, 2H, JH,P
=
8.6 Hz, H-4’), 2.63 (t, 2H, Jvic = 7.3 Hz, H-1’’), 1.61 (m, 2H,
H-2’’), 1.37–1.26 (m, 4H, H-3’’ and H-4’’), 0.87 (t, 3H, J =
6.9 Hz, H-5’’). 13C NMR (DMSO-d6) d: 171.5 (C-7a), 158.1
(C-6), 154.6 (C-2), 142.9 (C-4), 106.1 (C-4a), 99.6 (C-5),
69.4 (d, JC,P = 10.5 Hz, C-2’), 66.5 (d, JC,P = 159.9 Hz, C-
4’), 50.3 (C-1’), 30.8 and 27.5 and 26.2 and 21.9 and 14.1
(6-pentyl). MS (ESI) m/z: 343 [M – H]–. Anal. calcd. for
C14H21N2O6P (344.30): C 48.84, H 6.15, N 8.14, P 9.00;
found: C 48.53, H 5.97, N 7.99, P 9.06.
6-Decyl-3-[2-(phosphonomethoxy)ethyl]furo[2,3-
d]pyrimidin-2(3H)-one (11g)
Treatment of 10g (250 mg, 0.50 mmol) gave 11g
(187 mg, 90%) as white crystals (H2O/EtOH); mp 166–
167 8C. UV–vis lmax (nm) (3 ((mol/L)–1 cm–1): 329 (6900),
1
243 (13 000). H NMR (DMSO-d6) d: 8.36 (s, 1H, H-4),
6-Hexyl-3-[2-(phosphonomethoxy)ethyl]furo[2,3-
d]pyrimidin-2(3H)-one (11d)
6.40 (s, 1H, H-5), 4.11 (m, 2H, H-1’), 3.77 (m, 2H, H-2’),
3.58 (d, 2H, JH,P = 8.5 Hz, H-4’), 2.63 (t, 2H, Jvic = 7.1 Hz,
H-1’’), 1.66–1.54 (m, 2H, H-2’’), 1.37–1.15 (m, 14H, H-3’’
and H-4’’ and H-5’’ and H-6’’ and H-7’’ and H-8’’ and H-9’’),
0.85 (t, 3H, J = 6.7 Hz, H-10’’). 13C NMR (DMSO-d6) d:
171.5 (C-7a), 158.1 (C-6), 154.6 (C-2), 142.8 (C-4), 106.1
(C-4a), 99.6 (C-5), 69.4 (d, JC,P = 10.5 Hz, C-2’), 66.5 (d,
JC,P = 159.9 Hz, C-4’), 50.2 (C-1’), 31.4 and 29.1 and 29.0
and 28.8 and 28.5 and 27.5 and 26.5 and 22.2 and 14.1 (6-
decyl). MS (ESI) m/z: 413 [M – H]–. Anal. calcd. for
C19H31N2O6PÁ1/4H2O (418.94): C 54.47, H 7.58, N 6.69, P
7.39; found: C 54.52, H 7.53, N 6.61, P 7.70.
Treatment of 10d (490 mg, 1.11 mmol) gave 11d
(283 mg, 71%) as white crystals (H2O); mp 159–160 8C.
UV–vis lmax (nm) (3 ((mol/L)–1 cm–1): 329 (5900), 243 (11
1
100). H NMR (DMSO-d6) d: 8.36 (s, 1H, H-4), 6.39 (s, 1H,
H-5), 4.11 (m, 2H, H-1’), 3.78 (m, 2H, H-2’), 3.58 (d, 2H,
JH,P = 8.5 Hz, H-4’), 2.63 (t, 2H, Jvic = 7.4 Hz, H-1’’), 1.69–
1.54 (m, 2H, H-2’’), 1.39–1.18 (m, 6H, H-3’’ and H-4’’ and
H-5’’), 0.85 (t, 3H, J = 6.7 Hz, H-6’’). 13C NMR (DMSO-
d6) d: 171.5 (C-7a), 158.1 (C-6), 154.7 (C-2), 142.9 (C-4),
106.1 (C-4a), 99.6 (C-5), 69.4 (d, JC,P = 10.5 Hz, C-2’),
66.5 (d, JC,P = 159.9 Hz, C-4’), 50.3 (C-1’), 31.1 and 28.3
and 27.6 and 26.5 and 22.2 and 14.1 (6-hexyl). MS (ESI)
m/z: 357 [M – H]–. Anal. calcd. for C15H23N2O6P (358.33):
C 50.28, H 6.47, N 7.82, P 8.64; found: C 49.97, H 6.52, N
7.70, P 8.85.
6-(4-Pentylphenyl)-3-[2-(phosphonomethoxy)ethyl]furo[2,3-
d]pyrimidin-2(3H)-one (11h)
Treatment of 10h (130 mg, 0.26 mmol) gave 11h (85 mg,
78%) as white crystals (H2O/EtOH); mp 243–245 8C (dec).
UV–vis lmax (nm) (3 ((mol/L)–1 cm–1): 351 (16 400), 279
1
6-heptyl-3-[2-(phosphonomethoxy)ethyl]furo[2,3-
d]pyrimidin-2(3H)-one (11e)
(20 500). H NMR (DMSO-d6) d: 8.54 (s, 1H, H-4), 7.73
(m, 2H, o-Ph), 7.32 (m, 2H, m-Ph), 7.19 (s, 1H, H-5), 4.15
Treatment of 10e (270 mg, 0.59 mmol) gave 11e (187 mg,
84%) as white crystals (H2O/EtOH): mp 162–163 8C;
UV–vis lmax (nm) (3 ((mol/L)–1 cm–1): 328 (7900), 243 (14 700).
1H NMR (500.0 MHz, DMSO-d6) d: 8.35 (s, 1H, H-4),
6.40 (t, 1H, J5,1’’ = 1.1 Hz, H-5), 4.11 (m, 2H, H-1’), 3.77
(m, 2H, H-2’), 3.58 (d, 2H, JH,P = 8.5 Hz, H-4’), 2.63 (td,
2H, Jvic = 7.4 Hz, J1’’,5 = 1.1 Hz, H-1’’), 1.68–1.52 (m, 2H,
(m, 2H, H-1’), 3.81 (m, 2H, H-2’), 3.60 (d, 2H, JH,P =
8.6 Hz, H-4’), 2.61 (t, 2H, Jvic = 7.6 Hz, H-1’’), 1.64–1.53
(m, 2H, H-2’’), 1.36–1.20 (m, 4H, H-3’’ and H-4’’), 0.86 (t,
3H, J = 7.0 Hz, H-5’’). 13C NMR (DMSO-d6) d: 171.3 (C-
7a), 154.6 (C-6), 153.7 (C-2), 144.1 (C-p-Ph), 144.0 (C-4),
129.1 (C-m-Ph), 126.1 (C-i-Ph), 124.7 (C-o-Ph), 106.6 (C-
4a), 98.6 (C-5), 69.3 (d, JC,P = 10.5 Hz, C-2’), 66.5 (d, JC,P
=
Published by NRC Research Press

636
Can. J. Chem. Vol. 88, 2010
160 Hz, C-4’), 50.4 (C-1’), 35.1 and 31.0 and 30.5 and 22.1
and 14.0 (p-pentyl). MS (ESI) m/z: 419 [M – H]–. Anal.
calcd. for C20H25N2O6P (420.40): C 57.14, H 5.99, N 6.66,
P 7.37; found: C 56.96, H 6.01, N 6.53, P 7.61.
Anal. calcd. for C15H23N2O6P (358.13): C 50.28, H 6.47, N
7.82, P 8.64; found: C 50.22, H 6.53, N 7.69, P 8.98.
3-[2-(Phosphonomethoxy)ethyl]furo[2,3-d]pyrimidin-
2(3H)-one (16)
1-{2-[(Diisopropoxyphosphoryl)methoxy]ethyl}-5-
ethynyluracil (14)
A mixture of compound 15 (150 mg, 0.42 mmol), bro-
mo(trimethyl)silane (1.00 mL, 1.16 g, 7.58 mmol), and ace-
tonitrile (5 mL) was stirred at room temperature overnight.
The mixture was concentrated in vacuo and then codistilled
with a mixture of water/ethanol (9:1, 2 Â 5 mL). The resi-
due was crystallized to give 16 (85 mg, 74%) as yellowish
crystals (EtOAc/EtOH 3:1), dec. over 200 8C. UV–vis lmax
TMSA (13; 0.9 mL, 640 mg, 6.51 mmol) and then Et3N
(5.5 mL) were added to a suspension of 6 (2 g, 4.34 mmol),
Pd(Ph3P)4 (251 mg, 0.22 mmol), and CuI (83 mg,
0.44 mmol) in deoxygenated DMF (20 mL). The reaction
was sealed with a septum and stirred at 45 8C for 1 h. Vola-
tiles were evaporated in vacuo, and toluene was added to the
residue and then evaporated (2 Â 5 mL). The residue was
flash chromatographed (4% MeOH/CHCl3) to give a yellow-
ish solid upon drying. The TMS intermediate was dissolved
in dry THF (40 mL), TBAF (1 mol/L THF solution, 5 mL)
was added, and the reaction mixture was stirred at ambient
temperature for 0.5 h. Volatiles were evaporated, the residue
was dissolved in CHCl3(50 mL), and the solution was
washed (saturated EDTA (aq.), and H2O) and dried
(MgSO4). Volatiles were evaporated, and the residue was
chromatographed (4% MeOH/CHCl3) and crystallized to
give 14 (1.04 g, 63%) as white crystals (EtOAc/hexanes);
1
(nm) (3 ((mol/L)–1 cm–1): 325 (5200), 238 (8700). H NMR
(500.0 MHz, D2O) d: 8.55 (s, 1H, H-4), 7.58 (d, 1H, J6,5
=
2.7 Hz, H-6), 6.80 (d, 1H, J5,6 = 2.7 Hz, H-5), 4.31 (m, 2H,
H-1’), 3.94 (m, 2H, H-2’), 3.71 (d, 2H, JH,P = 8.7 Hz, H-4’).
13C NMR (125.8 MHz, D2O) d: 174.3 (C-7a), 159.6 (C-2),
148.3 (C-4 and C-6), 110.4 (C-4a), 107.8 (C-5), 72.5 (d,
JC,P = 11.7 Hz, C-2’), 69.0 (d, JC,P = 158.7 Hz, C-4’), 54.4
(C-1’). MS (ESI) m/z: 273 [M – H]–. Anal. calcd. for
C9H11N2O6PÁ1/3H2O (280.17): C 38.58, H 4.20, N 10.00, P
11.06; found: C 38.80, H 4.33, N 9.66, P 10.82.
5,5’-Buta-1,3-diyne-1,4-diylbis{(1-{2-
1
[(diisopropoxyphosphoryl)methoxy]ethyl}-uracil)} (17)
A stirred suspension of 14 (400 mg, 1.11 mmol), CuI
(213 mg, 1.11 mmol), Et3N (6 mL), and MeOH (14 mL)
was refluxed for 5 h. Reddish gel was formed. DMF (2 mL)
was added and the reaction stirred at room temperature
overnight. TLC (20% MeOH/CHCl3) showed complex mix-
ture. Volatiles were flash evaporated and the residue codis-
tilled with toluene (2 Â 5 mL). Flash chromatography (15%
MeOH/CHCl3) and crystallization gave compound 17
(82 mg, 10%) as yellow crystals (EtOAc/EtOH), dec. over
mp 147–148 8C. H NMR (600 MHz, DMSO-d6) d: 11.64
(bs, 1H, NH), 7.97 (s, 1H, H-6), 4.55 (dh, 2H, JH,P
=
7.7 Hz, Jvic = 6.2 Hz, POCH), 4.08 (s, 1H, HC:C), 3.90–
3.87 (m, 2H, H-1’), 3.77 (d, 2H, JH,P = 8.3 Hz, H-4’), 3.72–
3.69 (m, 2H, H-2’), 1.22 and 1.20 (2 Â d, 2 Â 6H, Jvic
=
6.2 Hz, CH3). 13C NMR (150.9 MHz, DMSO-d6) d: 162.4
(C-4), 150.5 (C-6), 150.1 (C-2), 96.6 (C-5), 83.7 (HC:C),
76.5 (C:CH), 70.4 (d, JC,P = 6.4 Hz, POC), 69.9 (d, JC,P
11.5 Hz, C-2’), 64.9 (d, JC,P = 164 Hz, C-4’), 47.7 (C-1’),
=
24.0 (d, JC,P = 3.7 Hz, CH3), 23.9 (d, JC,P = 4.4 Hz, CH3).
1
240 8C. H NMR (500.0 MHz, DMSO-d6) d: 11.77 (bs, 2H,
MS (ESI) m/z: 381 [M
+
Na]+. Anal. calcd. for
NH), 8.15 (s, 2H, H-6), 4.56 (dh, 4H, JH,P = 7.7 Hz, Jvic
=
C15H23N2O6PÁ1/4 EtOAc (380.35): C 50.52, H 6.63, N 7.37,
6.2 Hz, POCH), 3.90 (t, 4H, J1’,2’ = 4.8 Hz, H-1’), 3.78 (d,
4H, JH,P = 8.3 Hz, H-4’), 3.71 (t, 4H, J2’,1’ = 4.8 Hz, H-2’),
1.23 and 1.21 (2 Â d, 2 Â 12H, Jvic = 6.2 Hz, CH3). 13C
NMR (125.8 MHz, DMSO-d6) d: 162.3 (C-4), 152.0 (C-6),
149.9 (C-2), 95.8 (C-5), 76.7 (C:C-C:C), 75.4 (C:C-
P 8.14; found: C 50.28, H 6.68, N 7.20, P 8.43.
3-{2-[(Diisopropoxyphosphoryl)methoxy]ethyl}furo[2,3-
d]pyrimidin-2(3H)-one (15)
A stirred suspension of 14 (620 mg, 1.63 mmol), CuI
(310 mg, 1.63 mmol), Et3N (60 mL), and EtOAc (200 mL)
was refluxed under argon for 12 h. Volatiles were evapo-
rated, the residue was dissolved in hot MeOH (50 mL), and
the solid filtered off. Volatiles were evaporated and the resi-
due dissolved in CHCl3 (80 mL). Organics were washed
(2 Â 30 mL of saturated EDTA (aq.), 1 Â 20 mL H2O) and
dried (MgSO4). Isolation by flash chromatography (8%
MeOH/CHCl3) and crystallization afforded compound 15
C:C), 70.4 (d, JC,P = 6.4 Hz, POC), 69.8 (d, JC,P
=
11.6 Hz, C-2’), 64.9 (d, JC,P = 163.7 Hz, C-4’), 48.0 (C-1’),
24.0 (d, JC,P = 3.7 Hz, CH3), 23.9 (d, JC,P = 4.5 Hz, CH3).
MS (ESI) m/z: 737 [M + Na]+. HR-MS (ESI) m/z:
C30H45N4O12P2 [M + H]+. Anal. calcd.: 715.2504; found:
715.2504.
Antiviral activity assays
1
(236 mg, 38%) as white crystals (EtOAc); mp 168 8C. H
Varicella-zoster virus (VZV) drug susceptibility tests were
performed on confluent hen egg lysozyme (HEL) cells in
96-well microtiter plates by the plaque reduction assay.
Monolayers were infected with 20 plaque forming units
(PFU) of cell-associated virus per well. For each assay, virus
controls (infected–untreated cells) were included. After a 2 h
incubation period, the virus inoculum was removed and the
media replaced by the different dilutions (in duplicate) of
the tested molecules. Serial dilutions of test compounds
were incubated with the infected monolayers for 5 d. After
a 5 d incubation period, the cells were fixed and stained
NMR (500.0 MHz, DMSO-d6) d: 8.54 (s, 1H, H-4), 7.73 (d,
1H, J6,5 = 2.7 Hz, H-6), 6.81 (d, 1H, J5,6 = 2.7 Hz, H-5), 4.49
(dh, 2H, JH,P = 7.7 Hz, Jvic = 6.2 Hz, POCH), 4.17 (m, 2H,
H-1’), 3.80 (m, 2H, H-2’), 3.77 (d, 2H, JH,P = 8.2 Hz, H-4’),
1.16 and 1.12 (2 Â d, 2 Â 6H, Jvic = 6.2 Hz, CH3). 13C NMR
(125.8 MHz, DMSO-d6) d: 171.7 (C-7a), 154.6 (C-2), 145.2
(C-4), 144.8 (C-6), 105.1 (C-5), 104.5 (C-4a), 70.3 (d, JC,P
6.3 Hz, POC), 69.4 (d, JC,P = 11.4 Hz, C-2’), 64.8 (d, JC,P
=
=
163.8 Hz, C-4’), 50.9 (C-1’), 23.9 (d, JC,P = 3.7 Hz, CH3),
23.8 (d, JC,P = 4.6 Hz, CH3). MS (ESI) m/z: 359 [M + H]+.
Published by NRC Research Press

Janeba et al.
637
´
R.; Yarnold, C. J.; Carangio, A.; Velazquez, S.; Andrei, G.;
Snoeck, R.; De Clercq, E.; Balzarini, J. Drugs Future 2000,
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with Giemsa, and the level of virus-induced cytopathic ef-
fect was determined by counting the number of plaques for
each dilution. Activity was expressed as EC50 (effective
compound concentration required to reduce virus plaque for-
mation by 50%) compared to the untreated control.
For the HCMV assays, confluent HEL fibroblasts were
grown in 96-well microtiter plates and infected with the hu-
man cytomegalovirus strains Davis and AD-169 at 100 PFU
per well. After a 2 h incubation period, residual virus was re-
moved and the infected cells were further incubated with me-
dium containing different concentrations of the test
compounds (in duplicate). After incubation for 7 d at 37 8C,
virus-induced cytopathogenicity was monitored microscopi-
cally after ethanol fixation and staining with Giemsa. Antiviral
activity was expressed as the EC50 or compound concentration
required to reduce virus-induced cytopathogenicity by 50%.
To examine the inhibitory effect of the compounds against
virus-induced cytopathicity in HEL cells (herpes simplex vi-
rus type 1 (HSV-1), HSV-2 (G), vaccinia virus, and vesicular
stomatitis virus), confluent cell cultures in microtiter 96-well
plates were inoculated with 100 CCID50 of virus (1 CCID50
being the virus dose to infect 50% of the cell cultures). After
a 1 h virus adsorption period, residual virus was removed,
and the cell cultures were incubated in the presence of vary-
ing concentrations (200, 40, 8, . . . mmol/L) of the test com-
pounds. Viral cytopathicity was recorded as soon as it
reached completion in the control virus-infected cell cultures
that were not treated with the test compounds.
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The methodology of the anti-HIV assays was as follows:
human T-lymphocyte CEM (*3 Â 105 cells/cm3) cells
were infected with 100 CCID50 of HIV-1(IIIB) or HIV-
2(ROD)/mL and seeded in 200 mL wells of a microtiter
plate containing appropriate dilutions of the test compounds.
After 4 d of incubation at 37 8C, HIV-induced CEM giant
cell formation was examined microscopically.
Acknowledgments
This study was performed as a part of research project
(OZ40550506) of the Institute of Organic Chemistry and Bi-
ochemistry, v.v.i. This study was supported by the Center
for New Antivirals and Antineoplastics (1M0508) by the
Ministry of Education, Youth and Sports of the Czech Re-
public, and by Gilead Sciences and the Institute of Organic
Chemistry and Biochemistry (IOCB) Research Centre. The
research of JB was supported by the ‘‘Geconcerteerde On-
derzoeksacties Vlaanderen’’ (GOA no. 05/19). We thank
Leentje Persoons, Vicky Broeckx, Frieda De Meyer, Anita
Camps, Lies Van den Heurck, Steven Carmans, and Leen
Ingels for excellent technical assistance.
´
(11) De Clercq, E.; Holy, A.; Rosenberg, I.; Sakuma, T.; Balzar-
ini, J.; Maudgal, P. C. Nature 1986, 323 (6087), 464. doi:10.
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