5-Substituted-2,4-diamino-6-[2-(phosphonomethoxy) ethoxy]pyrimidines-Acyclic Nucleoside Phosphonate Analogues with Antiviral Activity

Article abstract of DOI:10.1021/jm030932o

2,4-Diamino-6-hydroxypyrimidines substituted in position 5 by an allyl, benzyl, cyanomethyl, ethoxycarbonylmethyl, phenyl, cyclopropyl, or methyl group were prepared either by C5-alkylation or by formation of the pyrimidine ring by cyclization. Their alky

Full text of DOI:10.1021/jm030932o

5064
J . Med. Chem. 2003, 46, 5064-5073
5-Su bstitu ted -2,4-d ia m in o-6-[2-(p h osp h on om eth oxy)eth oxy]p yr im id in essAcyclic
Nu cleosid e P h osp h on a te An a logu es w ith An tivir a l Activity
Dana Hockova´,*,† Anton´ın Holy´,*,† Milena Masoj´ıdkova´,† Graciela Andrei,‡ Robert Snoeck,‡ Erik De Clercq,‡ and
J an Balzarini‡
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo na´m. 2, CZ-166 10,
Prague 6, Czech Republic, and Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10,
B-3000 Leuven, Belgium
Received J une 18, 2003
2,4-Diamino-6-hydroxypyrimidines substituted in position 5 by an allyl, benzyl, cyanomethyl,
ethoxycarbonylmethyl, phenyl, cyclopropyl, or methyl group were prepared either by C5-
alkylation or by formation of the pyrimidine ring by cyclization. Their alkylation with
2-[(diisopropoxyphosphoryl)methoxy]ethyl tosylate afforded N1- and O6-regioisomers that were
separated and converted to the free phosphonic acids by treatment with bromotrimethylsilane
followed by hydrolysis. Reaction of 2,4-diamino-6-{[(diisopropoxyphosphoryl)methoxy]ethoxy}-
pyrimidine with elemental bromine, N-chloro-, or N-iodosuccinimide gave the corresponding
phosphorus-protected 5-bromo-, 5-chloro-, and 5-iodo derivatives, respectively. Their depro-
tection afforded 2,4-diamino-5-bromo- and -5-chloro-6-[2-(phosphonomethoxy)ethoxy]pyrimi-
dines. 2,4-Diamino-5-methyl-6-[2-(phosphonomethoxy)ethoxy]pyrimidine was synthesized also
by cross-coupling of the 5-bromo compound with AlMe3, followed by deprotection. The compounds
showed poor, if any, inhibitory activity against DNA viruses such as herpes simplex virus type
1 and type 2, cytomegalovirus, varicella-zoster virus, and vaccinia virus. In contrast, several
5-substituted 2,4-diaminopyrimidine derivatives markedly inhibited retrovirus replication in
cell culture. The 5-methyl derivative was exquisitely inhibitory to human immunodeficiency
virus and Moloney murine sarcoma virus-induced cytopathicity in cell culture (EC50 ∼ 0.00018
µmol/mL) but also cytostatic to CEM cell cultures. In contrast, the 5-halogen-substituted
derivatives showed pronounced antiretroviral activity (EC50 ) 0.0023-0.0110 µmol/mL),
comparable to that of the reference drugs adefovir and tenofovir, but were devoid of measurable
toxicity at 0.3 µmol/mL.
In tr od u ction
2-(Phosphonomethoxy)alkyl derivatives of purine and
pyrimidine basessacyclic nucleoside phosphonates
(ANPs)spossess significant antiviral and cytostatic
activity.1 These nucleotide analogues contain an isopolar
phosphonomethyl ether moiety instead of the nucleotide
phosphate ester group that excludes their enzymatic
degradation and/or eliminates problems with intracel-
lular phosphorylation necessary for nucleoside activa-
tion. Among ANPs, particularly 9-[2-(phosphonometh-
oxy)ethyl]adenine (PMEA, adefovir, 1a , Figure 1) is
active against DNA viruses and retroviruses;2 its pro-
drug, adefovir dipivoxil,3 has been recently approved for
hepatitis B therapy (Hepsera).4 Structure-activity re-
lationship studies in the series of PMEA congeners
included modification of the parent molecule both in the
side chain and in the heterocyclic moiety. Substitution
of the 2-(phosphonomethoxy)ethyl chain in position 2
by a hydroxymethyl group leads to another structural
type of active compound, (S)-N-[3-hydroxy-2-(phospho-
nomethoxy)propyl] (HPMP) derivatives, of which the
adenine derivative HPMPA (1e) exhibits potent anti-
DNA-viral activity.5 Substitution of PMEA side chain
F igu r e 1.
* Corresponding authors. D.H.: phone, +420 220183262; fax, +420
220183560; e-mail, lasice@uochb.cas.cz. A.H.: phone, +420 220183384;
fax, +420 220183560; e-mail, holy@uochb.cas.cz.
† Academy of Sciences of the Czech Republic.
‡ Katholieke Universiteit Leuven.
in position 2 by the methyl group results in [(R)-2-
phosphonomethoxypropyl]adenine (PMPA, tenofovir,
1c) with very high potency and selectivity against HIV-1
10.1021/jm030932o CCC: $25.00 © 2003 American Chemical Society
Published on Web 10/15/2003

Acyclic Nucleoside Phosphonate Analogues
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23 5065
and HIV-2;6 its oral prodrug, tenofovir disoproxil fuma-
rate, has been approved for treatment of HIV infection
(Viread).
Sch em e 1
The SAR studies demonstrated that the margins of
structural alteration are very narrow.7 The specifity of
antiviral action is determined by the structure of the
side chain. Except for the antiviral activity of the
cytosine derivative (S)-HPMPC (cidofovir, Vistide, 2,
Figure 1),8 the choice of the base is limited mostly to
adenine, guanine, and 2,6-diaminopurine and to their
8-aza and 3-deaza congeners. The 2,6-diaminopurine
and guanine ANP derivatives are often very potent
antivirals9 and/or exhibit antitumor properties.10 The
pharmacophore of purine acyclic nucleoside phospho-
nates is characterized by the presence of amino groups
at the pyrimidine part of the purine system. While N6-
substitution in adenine and 2,6-diaminopurine deriva-
tives still preserves the (antiviral and/or cytostatic)
activity in the PME and PMP series,11 other alterations
of amino groups generally result in complete loss of
activity.12
Recently, we discovered a new type of antiviral acyclic
nucleoside phosphonate13 originating from 2-substituted
4-amino-6-hydroxypyrimidines. Alkylation of these bases
by the appropriate phosphonate-protected synthon af-
forded a mixture of O6- and N1-regioisomers that were
converted to the free phosphonates. While none of the
isomeric 1-[2-(phosphonomethoxy)ethyl]pyrimidin-6-one
derivatives 4 was antivirally active, among 6-[2-
(phosphonomethoxy)ethoxy]pyrimidine derivatives, com-
pounds derived from 2,4-diaminopyrimidine (3a , 3c, 3d )
and 2-amino-4-hydroxypyrimidine (3b) (Figure 1) sig-
nificantly inhibited replication of retroviruses and her-
pesviruses in cell culture. Compounds 3 can be consid-
ered as analogues of 2,6-diaminopurine and guanine
PME derivatives [PMEDAP (1b), PMEG] with an open
imidazole ring in the purine moiety. This structural
relation is strongly supported by the finding that the
corresponding analogue of PMPDAP, i.e., 2,4-diamino-
6-[(R)-2-(phosphonomethoxy)propyl]oxypyrimidine (3c),
not only has the same selective antiretroviral activity
as its 2,4-diaminopurine congener (R)-PMPDAP (1d ) but
this activity is also limited solely to the (R)-enantiomer;
the (S)-enantiomer of 3c is similarly devoid of antiviral
activity as is (S)-PMPDAP.
the C5-position.14 This was the method of choice for
synthesis of 5-allyl, benzyl, cyanomethyl, and ethoxy-
carbonylmethyl pyrimidines 5a -d , using NaHCO3 as
a base and dimethylformamide as a solvent. The known
procedure was applied for the C5-methylation of 2,4,-
diamino-6-hydroxypyrimidine (6) (yield 22% of 5e).15
5-Phenyl pyrimidine derivative 5f was prepared by
condensation16 of commercially available ethyl phenyl-
cyanoacetate (7f) with guanidine in the presence of
sodium ethoxide. Analogously, 2,4-diamino-5-cyclopro-
pyl-6-hydroxypyrimidine (5g) was obtained from ethyl
cyclopropylcyanoacetate (7g); the latter compound was
synthesized according to the literature17 by condensa-
tion of diethyl carbonate with cyclopropaneacetonitrile
using NaH as a base.
The ultimate aim of our study was the synthesis of
5-substituted 6-[2-(phosphonomethoxy)ethoxy]pyrimi-
dines derived from the above bases. As expected, alky-
lation of 5a -g with 2-(diisopropoxyphosphorylmethoxy)-
ethyl tosylate 10 under standard conditions18 (Cs2CO3,
dimethylformamide, heating) afforded a mixture of N1-
alkyl and O6-alkyl regioisomers in the ratio approxi-
mately from 1:1 to 1:2 (Scheme 2). Resulting 5-substi-
tuted 2,4-diamino-1-{2-[(diisopropoxyphosphoryl)meth-
oxy]ethyl}pyrimidin-6(1H)-ones 8a -g and 5-substituted
2,4-diamino-6-{2-[(diisopropoxyphosphoryl)methoxy]-
ethoxy}pyrimidines 9a -g were separated by prepara-
tive HPLC. The isomeric diesters gave, on treatment
with bromotrimethylsilane followed by hydrolysis, the
free phosphonic acids 11a -g and 12a -g. 5-Ethoxycar-
bonylmethyl derivatives 8d and 9d afforded by the
above procedure two products: in addition to the esters
11d and 12d , the free carboxylic acids 11h and 12h
were isolated as a result of the ethyl ester hydrolysis.
All compounds 11 and 12 were purified by ion-exchange
column chromatography on Dowex 1 × 2 (OAc-).
To extend this study, 5-halogeno derivatives (Br, Cl,
I) were also prepared13a from the preformed diester-
protected molecule of the O6-isomer 13. Bromination
of 13 with elemental bromine in DMF/CCl4 gave smoothly
the 5-bromo derivative 14a ; similarly, 5-chloro deriva-
tive 15a and 5-iodo derivative 16a were obtained from
compound 13 on reaction with N-chlorosuccinimide or
N-iodosuccinimide, respectively. Deprotection by bro-
motrimethylsilane in acetonitrile followed by hydrolysis
gave the corresponding free phosphonates: 5-bromo
To further investigate the structure-activity relation-
ship of this new group of antiviral compounds, in this
paper we report on the synthesis and antiviral activity
of 2,4-diamino-6-[2-(phosphonomethoxy)ethoxy]pyrimi-
dine derivatives bearing various substituents at the C5-
position of the parent compound 3a and its N1-isomer
4. The substituents were selected with regard to their
spatial requirements and electronegativity: i.e., an allyl,
benzyl, cyanomethyl, ethoxycarbonylmethyl, phenyl,
chloro, bromo, cyclopropyl, and methyl group.
Ch em istr y
Two synthetic methods were used to prepare the
starting 5-substituted-2,4-diaminopyrimidine bases 5a -
g: (a) direct C5-alkylation of pyrimidine base or (b)
formation of pyrimidine ring by cyclization (Scheme 1).
Strong electrophiles, such as methyl bromides bearing
an electron-withdrawing group, can regioselectively
monoalkylate 2,4-diamino-6-hydroxypyrimidine (6) on

5066 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23
Hockova´ et al.
Sch em e 2
of the appropriate hydrogens and carbons due to spin-
spin interactions J (H,P) and J (C,P), respectively.
An tivir a l Activity. Contrary to the parent compound
3a , none of the 5-substituted compounds 12, 14b, or 15b
were active against either HSV-1, HSV-2, a thymidine
kinase (TK)-deficient HSV-1 strain, cytomegalovirus
(CMV), or vaccinia virus (Table 1). 5-Methyl derivative
12e was active against wild-type VZV and TK-deficient
VZV strain. Among the newly synthesized congeners of
3a and/or 4, only three 5-substituted 6-[2-(phospho-
nomethoxy)ethoxy] (PMEO) pyrimidine derivatives (12e,
14b, and 15b) showed a pronounced antiviral activity
in cell culture against retroviruses. Both the 5-methyl
derivative 12e and the 5-bromo and 5-chloro derivatives
14b and 15b were exquisitely inhibitory to Moloney
murine sarcoma virus (MSV) in C3H/3T3 cell cultures
[50% effective concentration (EC50) ranging from 0.00016
to 0.007 µmol/mL, respectively]. Compound 12e was also
very effective against HIV-1 and HIV-2 in CEM cell
cultures (EC50 ) 0.00023-0.00043 µmol/mL). The 5-bro-
mo and 5-chloro derivatives 14b and 15b were also
active, though less efficient, against these viruses at
EC50 values of 0.0031-0.0095 µmol/mL. These values
were comparable with those recorded for adefovir and
tenofovir.
To exclude the possibility that the antiviral effect of
12e (Table 1, 12e entry 1) could be due to contamination
by traces of palladium originating from the catalyst, we
have synthesized this compound also by alkylation of
the 2,4-diamino-6-hydroxy-5-methylpyrimidine (5e); the
thus obtained preparation of compound 12e exhibited
antiviral effects (Table 1, 12e entry 2) comparable with
the compound isolated from cross-coupling alkylation
of the diester 13.
A uniform interpretation of the above experimental
data is difficult: the highly active methyl derivative 12e
contrasts with inactive analogue 12g, bearing the
slightly more sterically demanding cyclopropyl group.
Nor is electronegativity the reason for the lack of
antiviral activity of the cyanomethyl (12b), ethoxycar-
bonylmethyl (12d ), or phenyl derivative (12f). Substitu-
tion at the position C-5 by the large electronegative
bromine atom in 14b and chloro atom in 15b is
consistent with preserving the antiviral activity. How-
ever, as evidenced by the limited stability of the
analogous 5-iodo derivative, which is easily transformed
to compound 3a , it cannot be excluded that compounds
14b and 15b underwent a similar transformation inside
the cell.20
derivative 14b and its 5-chloro congener 15b. However,
the 5-iodo derivative 16a gave under the conditions of
this reaction and/or ion-exchange chromatography pu-
rification the 5-unsubstituted 2,4-pyrimidine derivative
3a as the main product. Direct iodination of 3a by
elemental iodine in a dioxane-water mixture under the
conditions described for pyrimidine nucleosides also
failed.
As an alternative approach to the 5-C-substituted
derivatives of compound 3a we have also considered the
cross-coupling of compound 14a with organometallic
reagents. This method has been widely applied to 5-halo
pyrimidines19 but not yet to 2,4-diamino-6-hydroxypy-
rimidine derivatives. In our study, trimethylaluminum
was used for cross-coupling with bromo derivative 14a
as an alternative method for preparation of the 5-methyl
derivative 9e (Scheme 3). However, the main isolated
product was monoester 17 (31%), diester 9e was ob-
tained in 8% yield only. Deprotection of the combined
products 9e and 17 gave phosphonic acid 12e in 27%
overall yield. Nevertheless, attempts to extend this
method to the synthesis of the 5-ethyl derivative using
triethylaluminum under the same conditions failed; only
traces of the desired product were detected.
All N1- (11) and O6-isomers (12, 14b) were inactive
in vitro against all RNA viruses tested [i.e. vesicular
stomatitis virus (VSV), parainfluenza-3 virus, reovirus-
1, Sindbis virus, Coxsackie B4 virus].
By analogy to the 5-unsubstituted compounds,13a the
structure assignment of the N1- and O6-isomers of
ANPs synthesized in this study is based on their 13C
1
and H NMR spectra. The N1-isomers were character-
The majority of the compounds showed no appreciable
cytotoxic activity against E6SM, HEL, or CEM cell
growth. The antivirally active compounds 12e and 14b
did not affect microscopically visible cell morphology at
0.18 and 0.15 µmol/mL (HEL) or 1.4 and 1.2 µmol/mL
(E6SM), nor were they inhibitory to HEL cell prolifera-
tion at 0.07-0.18 µmol/mL. However, in sharp contrast
with the 5-bromo- and 5-chloro-substituted compounds
(IC50 g 0.3 µmol/mL), the 5-methyl derivative 12e
showed a pronounced cytostatic activity against prolif-
erating CEM cells [50% inhibitory concentration (IC50)
ized by the chemical shift of carbon atom C-1′ (δ ∼ 40
ppm) and C-6 (δ ∼ 154 ppm), while in the 13C NMR
spectra of O6-isomers there is a low-field shift of C-1′
carbon (δ ∼ 64 ppm), which is due to linkage to the
oxygen atom, and a less significant low-field shift of the
C-6 carbon (δ ∼ 166 ppm). We have also observed
characteristic differences in 1H NMR spectra in the
chemical shifts of H-1′ (N1-isomers, δ ∼ 4.0 ppm; O6-
isomers, δ ∼ 4.3 ppm). The presence of the phosphonate
function at the side chain was manifested by splitting

Acyclic Nucleoside Phosphonate Analogues
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23 5067
Sch em e 3
Ta ble 1. Antiviral Activity of Test Compounds
EC50a (µmol/mL)
VZV
b
CC50
CMV
HIV-1 (IIIB) HIV-2 (ROD) AD169 Davis
HSV-1 HSV-2
(µmol/mL)
(CEM)
formula
OKA
07/1
(KOS )
(G)
0.77
0.79
VV
MSV
11a
12a
11b
12b
11c
12c
11d
12d
11e
12e
12e
11f
12f
11g
12g
11h
12h
14b
15b
>∼0.3
>∼0.3
>∼0.3
>∼0.3
>∼0.3
>∼0.3
>∼0.3
>∼0.3
>∼0.3
0.00043
0.00023
>∼0.3
>∼0.3
>∼0.3
>∼0.3
>∼0.3
>∼0.3
0.0073
0.0031
>∼0.3
>∼0.3
>∼0.3
>∼0.3
>∼0.3
>∼0.3
0.11
>∼0.15 >∼0.15 >∼0.15 >∼0.15
>∼0.15 >∼0.15 >∼0.15 >∼0.15
>∼0.15 >∼0.15 >∼0.15 >∼0.15
>∼0.15 >∼0.15 >∼0.15 >∼0.15
>∼0.15 >∼0.15 >∼0.15 0.084
>∼1.2
>∼1.2
>∼1.2
>∼1.2
>∼1.2
>∼1.2
>∼1.2
>∼1.2
>∼1.2
>∼1.2
>∼1.2
>∼1.2
>∼1.2
>∼1.2
>∼1.2
>∼1.2
>∼1.2
>∼1.2 >∼0.12
>∼1.2 >∼0.12
>∼0.3
>∼0.3
>∼0.3
>∼0.3
>∼0.3
>∼0.3
>∼0.3
>∼0.3
>∼0.3
0.015
0.0047
>∼0.3
>∼0.3
>∼0.3
>∼0.3
>∼0.3
>∼0.3
>∼0.3
0.3
>∼1.2 >∼1.2 >∼0.12
0.66
>∼1.2 >∼0.12
>∼1.2 >∼1.2 0.3
>∼0.15 >∼0.15 0.14
0.11
>∼1.2 >∼1.2 0.3
>∼0.15 >∼0.15 >∼0.15 >∼0.15
>∼0.15 >∼0.15 >∼0.15 >∼0.15
>∼0.12 >∼0.12
>∼1.2 >∼1.2 0.011
>∼1.2 >∼1.2 0.035
>∼1.2 >∼1.2 >∼0.12
>∼1.2 >∼1.2 0.00022
0.078
>∼0.3
0.00031
0.00023
>∼0.3
>∼0.3
>∼0.3
>∼0.3
>∼0.3
>∼0.3
0.0095
0.0044
>∼0.15 >∼0.15 0.018
>∼0.12 0.72
0.042
>∼1.2 0.86
0.00016
>∼0.15 >∼0.15 >∼0.15 >∼0.15
>∼0.15 >∼0.15 >∼0.15 >∼0.15
>∼0.15 >∼0.15
>∼1.2 >∼1.2 >∼0.12
>∼1.2 >∼1.2 >∼0.12
>∼1.2 >∼1.2 >∼0.12
>∼1.2 >∼1.2 >∼0.12
>∼1.2 >∼1.2 0.082
>∼1.2 >∼1.2 0.046
>∼0.15 >∼0.15 >∼0.15 >∼0.15
>∼0.15 >∼0.15 >∼0.15 >∼0.15
>∼0.15
0.15
> ∼0.15 0.7
0.70
0.7
0.007
>∼0.12 >0.6
>∼1.2
>∼1.2 >∼1.2 0.0028
Reference Compounds
0.0045 0.0095
3a
3d
0.003
0.0016
0.014
0.0066
0.0014
>0.19
0.13
0.1
>0.19
0.025
0.033 0.18 0.00015
0.042
0.023
0.056
0.41
>0.014
0.0033
0.0012
0.12
0.00017 0.00024 0.0048c 0.017c
0.0065 >0.002 7
>0.17
>0.17
PMEA
(R)PMPA
0.27
>0.17
0.03
0.17
0.035
0.17
0.024
0.024
0.0022
0.004 6
>0.17
>0.17
>0.17
a
b
50% effective concentration. 50% cytostatic concentration. c EC50 values are the average for three HSV-1 strains (KOS, F, McIntyre)
and three HSV-2 strains (G, 196, Lyons).
caster (UK); and 2,4-diamino-6-hydroxypyrimidine, bromo
derivatives used for its alkylations, trimethylaluminum, and
ethyl phenylcyanoacetate were obtained from Sigma-Aldrich
) 0.012-0.015 µmol/mL]. The molecular basis of this
phenomenon is currently unclear.
(Praha, Czech Republic). Dimethylformamide and acetonitrile
were distilled from P2O5 and stored over molecular sieves (4
Exp er im en ta l Section
Å).
Unless otherwise stated, solvents were evaporated at 40 °C/2
kPa, and compounds were dried at 2 kPa over P2O5. Melting
points were determined on a Bu¨chi melting point apparatus.
TLC was performed on Silufol UV254 plates (Kavalier Votice,
Czech Republic) in chloroform-methanol. NMR spectra were
measured on an FT NMR spectrometer Varian UNITY 500
(1H at 500 M and 13C at 125.7 M frequency) in dimethyl
sulfoxide-d6 or D2O. Mass spectra were measured on a ZAB-
EQ (VG Analytical) spectrometer using FAB (ionization by Xe,
accelerating voltage 8 kV, glycerol matrix).
2-[(Diisop r op oxyp h osp h or yl)m eth oxy]eth yl Tosyla te
(10). Freshly prepared (2-acetoxyethoxy)methyl chloride21 (153
g, 1 mol) was added dropwise under stirring at 140 °C to
triisopropyl phosphite (212 g, 1.02 mol) at such a rate that
the evolving isopropyl chloride still condensed. After the
vigorous reaction subsided, the mixture was stirred at 140 °C
for two more hours, and the volatiles were distilled off at 80
°C/15 Torr. Distillation of the residue in vacuo gave fraction
140-155 °C/1 Torr (242.8 g, 86%). This compound (211.7 g,
0.75 mol) was taken up in anhydrous methanol (2 L), and
sodium methoxide (50 mL, 1 M solution in methanol) was
added. After 2 h standing at the ambient temperature, the
Ma ter ia ls. Bromotrimethylsilane, N-chlorosuccinimide, N-
iodosuccinimide, and cesium carbonate were purchased from
Fluka (Switzerland); cyclopropylacetonitrile was from Lan-

5068 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23
Hockova´ et al.
reaction was complete. The solution was neutralized by adding
Dowex 50 × 8 (H+-form) prewashed with methanol. The
suspension was filtered, made slightly alkaline by adding
several drops of triethylamine, and the volatiles were taken
down in vacuo. The oily residue was taken in ether (500 mL),
washed with saturated NaCl solution, and dried, and the
solvent was evaporated. The residue was dried at 50 °C/0.1
Torr to yield 130 g (0.54 mol, 72%) of diisopropyl (2-hydroxy-
ethoxy)methanephosphonate.22
Syn th esis of 5-Su bstitu ted -2,4-d ia m in o-6-h yd r oxyp y-
r im id in es by Cycliza tion (Gen er a l P r oced u r e). To a
solution of EtONa (1.85 g, 27 mmol) in absolute ethanol (25
mL) were added guanidine hydrochloride (1.25 g, 12.5 mmol,
95%) and ethyl phenylcyanoacetate (2.3 mL, 12.5 mmol) or
ethyl cyclopropylcyanoacetate (1.7 g, 12.5 mmol. The mixture
was refluxed for 5 h under Ar atmosphere, stirred at room
temperature overnight, and then evaporated in vacuo. Water
was added and the mixture was neutralized by acetic acid.
Crude product was filtered off and recrystalllized from water-
ethanol.
2,4-Dia m in o-6-h yd r oxy-5-p h en ylp yr im id in e (5f). Yield:
0.96 g, 38%), mp: 262-263 °C. Anal. (C10H10N4O) C, H, N.
FABMS: 203 (MH+) (100). 1H NMR (DMSO-d6): 10.20 brs,
1H (NH or OH); 7.30 m, 4 H and 7.15 t, 1 H (arom H); 6.22
brs, 2 H, and 5.55 brs, 2 H (NH2).
This product (60 g, 0.25 mol) in pyridine (500 mL) was
treated portionwise with tosyl chloride (72 g, 0.3 mol) under
cooling by ice. 4-(Dimethylamino)pyridine (1 g) was added, and
the mixture was stirred for 3 h in an ice-bath and left to stand
overnight at 5 °C. Methanol (20 mL) was added and the
mixture was concentrated in vacuo at 30 °C (maximum bath
temperature) to half of the original volume. The residue was
diluted with ethyl acetate (700 mL) and the solution was
washed successively with 100-mL portions of water (2×), 5%
HCl (to acid reaction), water, saturated KHCO3, and water.
It was dried and taken down in vacuo. Chromatography of the
residue in two portions on silica gel columns (1 L) in toluene
gave the UV-absorbing product, which was collected and dried
over P2O5 in vacuo to yield 69 g (70%) of compound 10 as a
thick colorless oil that was used for further syntheses. 1H NMR
(DMSO-d6): 7.78 d, 2H and 7.48 d, 2H, J ) 8.3 (ar H); 4.56 m,
2H (P-OCH); 4.12 m, 2H and 3.70 m, 2H (O-CH2); 3.70 d,
2H, J (P,CH) ) 8.2 (P-CH2); 2.42 s, 3H (CH3); 1.23 d, 6H and
1.21 d, 6H, J (CH3,CH) ) 6.2 (CH3). 13C NMR (DMSO-d6):
145.06, 132.51, 130.30 2C and 127.76, 2C (arom C); 70.36 d,
2C, J (P,C) ) 6.4 (P-OC); 69.87, J (P,C) ) 11.7 and 69.61 (O-
CH2); 64.87 d, J (P,C) ) 164.1 (P-C); 23.94 d, 2C, J (P,C) ) 3.9
and 23,81 d, 2C, J (P,C) ) 4.9 (CH3); 21.23 (CH3).
C-5-Alk yla tion of 2,4-Dia m in o-6-h yd r oxyp yr im id in e
(Gen er a l P r oced u r e). To a suspension of 2,4-diamino-6-
hydroxypyrimidine (2.0 g, 16 mmol) and sodium hydrogen
carbonate (1.24 g, 9.6 mmol, 1.1 equiv) in dimethylformamide
(20 mL) was added bromo derivative (17.6 mmol, 1.1 equiv).
The mixture was stirred at room temperature for 2 days.
Solvent was evaporated in vacuo and the residue was codis-
tilled with toluene and ethanol. Water (50 mL) was added and
pH adjusted with acetic acid to 6-7. Crude product was filtered
off and recrystallized from water-ethanol.
5-Allyl-2,4-d ia m in o-6-h yd r oxyp yr im id in e (5a ). Yield:
0.91 g, 31%. Mp: 255-256 °C. Anal. (C7H10N4O‚H2O) C, H,
N. FABMS: 167 (MH+) (30). 1H NMR (DMSO-d6): 9.88 brs,
1H (NH or OH); 6.00 brs, 2 H, and 5.63 brs, 2 H (NH2); 5.68
ddt, 1 H, J (2′,1′) ) 6.0, J (2′,3′) ) 10.0 and 17.1, (H-2′); 4.99
brdt, 1 H, J (3t′,2′) ) 17.1, J (3t′,1′) ) J gem ) 1.5 (H-3′trans); 4.84
brdt, 1 H, J (3c′,2′) ) 10.0, J (3t′,1′) ) J gem ) 1.5 (H-3′cis); 2.91
d, 2 H, J (1′,2′) ) 6.0 (H-1′). 13C NMR: 162.45 (C-4), 161.90
(C-2), 53.44 (C-6); 137.07 (C-2′); 113.65 (C-3′); 85.09 (C-5); 26.82
(C-1′).
2,4-Dia m in o-5-ben zyl-6-h yd r oxyp yr im id in e (5b). Yield:
1.6 g, 46%. Mp: 253-254 °C. Anal. (C11H12N4O) C, H, N.
FABMS: 217 (MH+) (100). 1H NMR (DMSO-d6): 9.96 brs, 1H
(NH or OH); 7.24 d, 2 H, 7.19 t, 2H and 7.09 t, 1 H (arom H);
6.08 brs, 2 H, and 5.74 brs, 2 H (NH2); 3.52 s, 2 H (CH2Ph).
2,4-Dia m in o-5-cya n om eth yl-6-h yd r oxyp yr im id in e (5c).
Yield: 2.0 g, 72%. Mp: dec. Anal. (C6H7N5O‚1/2H2O) C, H, N.
FABMS: 166 (MH+) (55). 1H NMR (DMSO-d6): 10.06 brs, 1H
(NH or OH); 6.24 brs, 2 H, and 6.19 brs, 2 H (NH2); 3.36 s, 2
H (H-1′).
2,4-Dia m in o-5-cyclop r op yl-6-h yd r oxyp yr im id in e (5g).
Yield: 1.62 g, 78%. Mp: dec. Anal. (C7H10N4O) C, H, N.
1
EIMS: 166 (M) (65). H NMR (DMSO-d6): 9.79 brs, 1H (NH
or OH); 6.08 brs, 2 H and 5.79 brs, 2 H (NH2); 1.01 m, 1 H,
0.66, 2, and 0.37 m, 2 H (cyclopropyl).
Alk yla tion of 5-u bstitu ted -2,4-d ia m in o-6-h yd r oxyp y-
r im id in es (Gen er a l P r oced u r e). 5-Substituted-2,4-diamino-
6-hydroxypyrimidine (4 mmol) was codistilled with toluene and
the residue was sonicated with dimethylformamide (7 mL) and
cesium carbonate (0.65 g, 2 mmol) and heated to 80 °C.
Tosylate 10 (1.7 g, 4.4 mmol, 1.1 equiv) was added, and the
reaction mixture was heated at 100 °C for 20-30 h, evaporated
in vacuo, and codistilled with toluene and ethanol. The residue
was treated with hot chloroform and filtered and the filtrate
taken down in vacuo. Preparative HPLC afforded N- and
O-isomers as colorless or yellow foams that were used in the
next step without further purification.
5-Allyl-2,4-d ia m in o-1-{2-[(d iisop r op oxyp h osp h or yl)-
m eth oxy]eth yl}p yr im id in -6(1H)-on e (8a ). Yield: 0.25 g,
16%. FABMS: 389 (MH+) (35). 1H NMR (DMSO-d6): 6.37 brs,
2 H, and 5.55 brs, 2 H (NH2); 5.69 ddt, 1 H, J (2′′,1′′) ) 6.1,
J (2′′,3′′) ) 10.0 and 17.1, (H-2′′); 5.00 ddt, 1 H, J (3t′′,2′′) ) 17.1,
J (3t′′,1′′) ) 1.5, J gem ) 2.3 (H-3′′trans); 4.83 ddt, 1 H, J (3c′′,2′′)
) 10.0, J (3t′′,1′′) ) 1.5, J gem ) 2.3 (H-3′′cis); 4.57 m, 2 H (P-
OCH); 3.98 t, 2 H, J (1′,2′) ) 5.8 (H-1′); 3.78 d, 2 H, J (P,CH) )
8.3 (P-CH2); 3.64 t, 2 H, J (2′,1′) ) 5.8 (H-2′); 2.94 dt, 2 H,
J (1′′,3′′) ) 1.5, J (1′′,2′′) ) 6.1 (H-1′′); 1.23, 6, and 1.21 d, 6 H,
J (CH3,CH2) ) 6.1 (CH3).13C NMR: 161.62 (C-4); 159.93 (C-2);
153.83 (C-6); 136.99 (C-2′′); 113.64 (C-3′′); 85.06 (C-5); 70.39
d, 2 C, J (P,C) ) 6.3 (P-OC); 70.26 d, J (P,C) ) 11.2 (C-2′); 65.02
d, J (P,C) ) 163.6 (P-C); 40.21 (C-1′); 27.81 (C-1′′); 23.98 d, 2
C, J (P,C) ) 3.9 and 23.86 d, 2 C, J (P,C) ) 4.4 (CH3).
5-Allyl-2,4-d ia m in o-6-[2-(d iisop r op oxyp h osp h or yl-
m et h oxy)et h oxy]p yr im id in e (9a ). Yield: 0.25 g, 16%.
FABMS: 389 (MH+) (20). 1H NMR (DMSO-d6): 5.79 brs, 2 H,
and 5.70 brs, 2 H (NH2); 5.72 ddt, 1 H, J (2′′,1′′) ) 6.0, J (2′′,3′′)
) 10.0 and 17.0, (H-2′′); 5.00 ddt, 1 H, J (3t′′,2′′) ) 17.0, J (3t′′,1′′)
) 1.5, J gem ) 2.0 (H-3′′trans); 4.87 ddt, 1 H, J (3c′′,2′′) ) 10.0,
J (3t′′,1′′) ) 1.5, J gem ) 2.0 (H-3′′cis); 4.59 m, 2 H (P-OCH); 4.26
m, 2 H (H-1′); 3.79 d, 2 H, J (P,CH) ) 8.3 (P-CH2); 3.75 m, 2
H, (H-2′); 3.00 dt, 2 H, J (1′′,3′′) ) 1.5, J (1′′,2′′) ) 6.0 (H-1′′);
1.24, 6, and 1.22 d, 6 H, J (CH3,CH2) ) 6.2 (CH3). 13C NMR:
166.79 (C-6); 163.64 (C-4); 160.92 (C-2); 136.43 (C-2′′); 114.16
(C-3′′); 85.92 (C-5); 71.23 d, J (P,C) ) 11.7 (C-2′); 70.28 d, 2 C,
J (P,C) ) 6.3 (P-OC); 65.23 d, J (P,C) ) 164.1 (P-C); 64.23
(C-1′); 26.37 (C-1′′); 23.98 d, 2 C, J (P,C) ) 3.9 and 23.86 d, 2
C, J (P,C) ) 4.4 (CH3).
2,4-Dia m in o-5-ben zyl-1-{2-[(d iisop r op oxyp h osp h or yl)-
m eth oxy]eth yl}p yr im id in -6(1H)-on e (8b). Yield: 0.25 g,
14%. FABMS: 439 (MH+) (100). 1H NMR (DMSO-d6): 7.23 d,
2 H, 7.18, 2, and 7.10 t, 1 H (arom H); 6.42 brs, 2 H, and 5.66
brs, 2 H (NH2); 4.57 m, 2 H (P-OCH); 4.01 t, 2 H, J (1′,2′) )
5.8 (H-1′); 3.79 d, 2 H, J (P,CH) ) 8.2 (P-CH2); 3.66 t, 2 H,
J (2′,1′) ) 5.8 (H-2′); 3.54 s, 2 H (H-1′′); 1.24, 6, and 1.21 d, 6
H, J (CH3,CH2) ) 6.1 (CH3).13C NMR: 162.12 (C-4); 160.11 (C-
2); 153.87 (C-6); 142.33, 128.27, 2 C, 127.90, 2 C, and 125.31
(arom C); 87.32 (C-5); 70.38 d, 2 C, J (P,C) ) 6.3 (P-OC); 70.24
d, J (P,C) ) 11.2 (C-2′); 65.03 d, J (P,C) ) 164.1 (P-C); 40.30
2,4-Dia m in o-5-eth oxyca r bon ylm eth yl-6-h yd r oxyp yr i-
m id in e (5d ).14 Yield: 1.97 g, 59%. Mp: dec. Anal. (C8H12N4O3)
C, H, N. FABMS: 213 (MH+) (100). 1H NMR (DMSO-d6): 9.89
brs, 1H (NH or OH); 6.05 brs, 2 H, and 5.82 brs, 2 H (NH2);
4.01 q, 2 H, J (CH2,CH3) ) 7.1 (OCH2); 3.17 s, 2 H (H-1′); 1.16
t, 3 H (CH3).
2,4-Dia m in o-6-h yd r oxy-5-m eth ylp yr im id in e (5e) was
prepared by monomethylation accoording to the literature
and15 crystallized from water-ethanol. Yield: 42%. Mp: 275-
277 °C. Anal. (C5H8N4O‚1/2H2O) C, H, N. FABMS: 140 (MH+)
(60). 1H NMR (DMSO-d6): 10.00 brs, 1H (NH or OH); 5.98
brs, 2 H and 5.65 brs, 2 H (NH2); 1.61 s, 3 H (CH3).

Acyclic Nucleoside Phosphonate Analogues
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23 5069
(C-1′); 29.04 (C-1′′); 23.98 d, 2 C, J (P,C) ) 3.9 and 23.86 d, 2
C, J (P,C) ) 4.4 (CH3).
(NH2); 4.52 m, 2 H (P-OCH); 4.26 m, 2 H (H-1′); 3.68 m, 2 H,
(H-2′); 3.67 d, 2 H, J (P,CH) ) 8.3 (P-CH2); 1.21, 6, and 1.18
d, 6 H, J (CH3,CH2) ) 6.1 (CH3). 13C NMR: 166.09 (C-6); 163.11
(C-4); 161.51 (C-2); 134.07, 130.98, 2 C, 128.61, 2 C, and 126.52
(arom C); 91.31 (C-5); 71.18 d, J (P,C) ) 12.2 (C-2′); 70.38 d, 2
C, J (P,C) ) 6.4 (P-OC); 65.36 d, J (P,C) ) 164.1 (P-C); 64.46
(C-1′); 24.11 d, J (P,C) ) 3.9, 23.98 d, 2 C, J (P,C) ) 4.9 (CH3).
2,4-Dia m in o-5-b en zyl-6-[2-(d iisop r op oxyp h osp h or yl-
m et h oxy)et h oxy]p yr im id in e (9b ). Yield: 0.40 g, 23%.
FABMS: 439 (MH+) (35). 1H NMR (DMSO-d6): 7.30-7.10 m,
5 H (arom H); 5.88 brs, 2 H, and 5.74 brs, 2 H (NH2); 4.58 m,
2 H (P-OCH); 4.28 m, 2 H (H-1′); 3.77 d, 2 H, J (P,CH) ) 8.3
(P-CH2); 3.76 m, 2 H, (H-2′); 3.61 s, 2 H, (H-1′′); 1.22, 6, and
1.20 d, 6 H, J (CH3,CH2) ) 6.1 (CH3).
2,4-Dia m in o-5-cyclop r op yl-1-{2-[(d iisop r op oxyp h os-
p h or yl)m et h oxy]et h yl}p yr im id in -6(1H)-on e (8g). Yield:
1
0.29 g, 19%. FABMS: 389 (MH+) (100). H NMR (DMSO-d6):
2,4-Dia m in o-5-cya n om eth yl-1-{2-[(d iisop r op oxyp h os-
p h or yl)m eth oxy]eth yl}p yr im id in -6(1H)-on e (8c). Yield:
6.36 brs, 2 H, and 5.62 brs, 2 H (NH2); 4.56 m, 2 H (P-OCH);
3.94 t, 2 H, J (1′,2′) ) 4.8 (H-1′); 3.77 d, 2 H, J (P,CH) ) 8.3
(P-CH2); 3.61 t, 2 H, J (2′,1′) ) 4.8 (H-2′); 1.22, 6, and 1.21 d,
6 H, J (CH3,CH2) ) 6.2 (CH3); 1.07 m, 1 H, 0.67, 2, and 0.38 m
2 H (cyclopropyl).13C NMR: 161.82 (C-4), 161.46 (C-2); 154.12
(C-6); 87.29 (C-5); 70.35 d, 2 C, J (P,C) ) 6.4 (P-OC); 70.24 d,
J (P,C) ) 11.2 (C-2′); 64.97 d, J (P,C) ) 163.6 (P-C); 39.85 (C-
1′); 23.97 d, 2 C, J (P,C) ) 3.9 and 23.85 d, 2 C, J (P,C) ) 4.9
(CH3); 6.79, 2 C and 5.91 (cyclopropyl).
1
0.25 g, 16%. FABMS: 388 (MH+) (100). H NMR (DMSO-d6):
6.66 brs, 2 H, and 6.17 brs, 2 H (NH2); 4.56 m, 2 H (P-OCH);
4.00 t, 2 H, J (1′,2′) ) 5.6 (H-1′); 3.78 d, 2 H, J (P,CH) ) 8.1
(P-CH2); 3.65 t, 2 H, J (2′,1′) ) 5.6 (H-2′); 3.38 s, 2 H (H-1′′);
1.22, 6, and 1.20 d, 6 H, J (CH3,CH2) ) 6.2 (CH3). 13C NMR:
161.39 (C-4); 160.28 (C-2); 154.50 (C-6); 119.68 (CN); 77.53 (C-
5); 70.40 d, 2 C, J (P,C) ) 6.4 (P-OC); 70.00 d, J (P,C) ) 11.2
(C-2′); 64.99 d, J (P,C) ) 163.6 (P-C); 40.26 (C-1′); 23.98 d, 2
C, J (P,C) ) 3.9 and 23.85 d, 2 C, J (P,C) ) 4.9 (CH3).
2,4-Dia m in o-5-cya n om et h yl-6-[2-(d iisop r op oxyp h os-
p h or ylm eth oxy)eth oxy]p yr im id in e (9c). Yield: 0.26 g,
17%. FABMS: 388 (MH+) (75). 1H NMR (DMSO-d6): 6.29 brs,
2 H, and 6.02 brs, 2 H (NH2); 4.59 m, 2 H (P-OCH); 4.33 m,
2 H (H-1′); 3.81 d, 2 H, J (P,CH) ) 8.5 (P-CH2); 3.79 m, 2 H,
(H-2′); 3.48 s, 2 H, (H-1′′); 1.24, 6, and 1.23 d, 6 H, J (CH3,-
CH2) ) 6.1 (CH3).13C NMR: 166.93 (C-6); 163.38 (C-4); 161.84
(C-2); 119.00 (CN); 78.12 (C-5); 71.09 d, J (P,C) ) 11.7 (C-2′);
70.32 d, 2 C, J (P,C) ) 6.4 (P-OC); 65.17 d, J (P,C) ) 163.6
(P-C); 64.52 (C-1′); 23.98 d, 2 C, J (P,C) ) 3.9 and 23.85 d, 2
C, J (P,C) ) 4.9 (CH3).
2,4-Dia m in o-1-{2-[(d iisop r op oxyp h osp h or yl)m eth oxy]-
et h yl}-5-(et h oxyca r b on ylm et h yl)-p yr im id in -6(1H )-on e
(8d ). Yield: 0.25 g, 15%. FABMS: 435 (MH+) (80), no further
characterization, unstable.
2,4-Dia m in o-6-[2-(d iisop r op oxyp h osp h or ylm eth oxy)-
eth oxy]-5-(eth oxycar bon ylm eth yl)-pyr im idin e (9d). Yield:
0.22 g, 13%. FABMS: 435 (MH+) (20), no further characteriza-
tion, unstable.
2,4-Dia m in o-1-{2-[(d iisop r op oxyp h osp h or yl)m eth oxy]-
et h yl}-5-m et h ylp yr im id in -6(1H)-on e (8e). Yield: 0.39 g,
27%. FABMS: 363 (MH+) (20). 1H NMR (DMSO-d6): 6.29 brs,
2 H, and 5.58 brs, 2 H (NH2); 4.57 m, 2 H (P-OCH); 3.97 t, 2
H, J (1′,2′) ) 5.8 (H-1′); 3.77 d, 2 H, J (P,CH) ) 8.3 (P-CH2);
3.62 t, 2 H, J (2′,1′) ) 5.8 (H-2′); 1.64 s, 3 H, (CH3); 1.22, 6,
and 1.20 d, 6 H, J (CH3,CH2) ) 6.2 (CH3).13C NMR: 161.85
(C-4); 160.09 (C-2), 153.50 (C-6); 82.27 (C-5); 74.46 d, J (P,C)
) 11.2 (C-2′); 70.37 d, 2 C, J (P,C) ) 6.3 (P-OC); 64.98 d, J (P,C)
) 164.1 (P-C); 40.17 (C-1′); 23.89 d, 2 C, J (P,C) ) 4.9 and
23.85 d, 2 C, J (P,C) ) 4.9 (CH3); 9.39 (CH3).
2,4-Dia m in o-6-[2-(d iisop r op oxyp h osp h or ylm eth oxy)-
et h oxy]-5-m et h ylp yr im id in e (9e). Yield: 0.61 g, 42%.
FABMS: 363 (MH+) (35). 1H NMR (DMSO-d6): 6.41 brs, 2 H,
and 6.33 brs, 2 H (NH2); 4.59 m, 2 H (P-OCH); 4.31 m, 2 H
(H-1′); 3.80 d, 2 H, J (P,CH) ) 8.3 (P-CH2); 3.78 m, 2 H (H-
2′); 1.74 s, 3 H (CH3); 1.24, 6, and 1.22 d, 6 H, J (CH3,CH2) )
6.2 (CH3).
2,4-Diam in o-5-cyclopr opyl-6-[2-(diisopr opoxyph osph o-
r ylm eth oxy)eth oxy]p yr im id in e (9g). Yield: 0.55 g, 35%.
FABMS: 389 (MH+) (95). 1H NMR (DMSO-d6): 5.84 brs, 2 H,
and 5.70 brs, 2 H (NH2); 4.59 m, 2 H (P-OCH); 4.26 m, 2 H
(H-1′); 3.81 d, 2 H, J (P,CH) ) 8.3 (P-CH2); 3.76 m, 2 H (H-
2′); 1.24, 6, and 1.22 d, 6 H, J (CH3,CH2) ) 6.2 (CH3); 1.11 m,
1 H, 0.75, 2, and 0.38 m 2 H (cyclopropyl). 13C NMR: 167.34
(C-6), 165.30 (C-4); 160.73 (C-2); 88.69 (C-5); 71.27 d, J (P,C)
) 12.2 (C-2′); 70.27 d, 2 C, J (P,C) ) 6.4 (P-OC); 65.24 d, J (P,C)
) 164.5 (P-C); 63.95 (C-1′); 23.99 d, 2 C, J (P,C) ) 3.9 and
23.865 d, 2 C, J (P,C) ) 4.9 (CH3); 7.02, 2 C and 4.38
(cyclopropyl).
5-Su bstitu ted -2,4-d ia m in o-1-[2-(p h osp h on om eth oxy)-
eth yl]p yr im id in -6(1H)-on es a n d 5-Su bstitu ted -2,4-d ia m i-
n o-6-[2-(p h osp h on om eth oxy)eth oxy]p yr im id in es (Gen -
er a l P r oced u r e). A mixture of diisopropyl ester (0.5 mmol),
acetonitrile (10 mL), and BrSiMe3 (1 mL) was stirred overnight
at room temperature. After evaporation in vacuo and co-
distillation with acetonitrile, the residue was treated with
water and concentrated aqueous ammonia was added to the
alkaline reaction. The mixture was evaporated to dryness and
the residue was applied onto a column of Dowex 50 × 8 (H+-
form, 20 mL) and washed with water. Elution with 2.5%
aqueous ammonia and evaporation in vacuo afforded crude
product as ammonium salt. This residue in a minimum volume
of water was applied on a Dowex 1 × 2 (acetate) (25 mL)
column, which was then washed with water followed by
gradient of acetic acid (0-0.75 M for O-isomers, 0.5-2 M for
N-isomers). The main UV-absorbing fraction was evaporated,
the residue was codistilled three times with water and crystal-
lized from water-ethanol to afford the product as a white solid.
5-Allyl-2,4-d ia m in o-1-[2-(p h osp h on om eth oxy)eth yl]p y-
r im id in -6(1H)-on e (11a ). Yield: 114 mg, 76%. Mp: 254-255
°C. Anal. (C10H17N4O5P‚1/2H2O) C, H, N, P. FAB-MS: 303 (M-
1
H-) (60). H NMR (DMSO-d6): 6.60 brs, 2 H, and 5.69 brs, 2
H (NH2); 5.70 ddt, 1 H, J (2′′,1′′) ) 6.2, J (2′′,3′′) ) 10.0 and
17.1, (H-2′′); 5.01 ddt, 1 H, J (3t′′,2′′) ) 17.1, J (3t′′,1′′) ) 1.5,
J gem ) 2.3 (H-3′′trans); 4.84 ddt, 1 H, J (3c′′,2′′) ) 10.0, J (3t′′,1′′)
) 1.5, J gem ) 2.3 (H-3′′cis); 3.99 t, 2 H, J (1′,2′) ) 6.0 (H-1′);
3.62 t, 2 H, J (2′,1′) ) 6.0 (H-2′); 3.60 d, 2 H, J (P,CH) ) 8.3
(P-CH2); 2.95 brd, 2 H, J (1′′,2′′) ) 6.2 (H-1′′).
5-Allyl-2,4-d ia m in o-6-[2-(p h osp h on om eth oxy)eth oxy]-
p yr im id in e (12a ). Yield: 113 mg, 75%. Mp: 240-241 °C.
Anal. (C10H17N4O5P) C, H, N, P. FAB-MS: 303 (M-H-) (25).
1H NMR (DMSO-d6): 5.98 brs, 2 H, and 5.92 brs, 2 H (NH2);
5.73 ddt, 1 H, J (2′′,1′′) ) 6.1, J (2′′,3′′) ) 10.0 and 17.1, (H-2′′);
5.00 brd, 1 H, J (3t′′,2′′) ) 17.1 (H-3′′trans); 4.88 brd, 1 H, J (3c′′,2′′)
) 10.0 (H-3′′cis); 4.27 m, 2 H (H-1′); 3.73 m, 2 H, (H-2′); 3.57 d,
2 H, J (P,CH) ) 8.3 (P-CH2); 3.00 brd, 2 H, J (1′′,2′′) ) 6.1 (H-
1′′).
2,4-Dia m in o-1-{2-[(d iisop r op oxyp h osp h or yl)m eth oxy]-
eth yl}-5-p h en ylp yr im id in -6(1H)-on e (8f). Yield: 0.18 g,
1
10%. FABMS: 425 (MH+) (100). H NMR (DMSO-d6): 7.31 t,
2 H, 7.26 d, 2 H and 7.17 t, 1 H (arom H); 6.65 brs, 2 H, and
5.42 brs, 2 H (NH2); 4.58 m, 2 H (P-OCH); 4.04 t, 2 H, J (1′,2′)
) 5.8 (H-1′); 3.80 d, 2 H, J (P,CH) ) 8.3 (P-CH2); 3.68 t, 2 H,
J (2′,1′) ) 4.6 (H-2′); 1.23, 6, and 1.22 d, 6 H, J (CH3,CH2) )
6.1 (CH3).13C NMR: 160.80 (C-4); 159.52 (C-2); 154.35 (C-6);
135.88, 131.03, 2 C, 128.12, 2 C, and 125.64 (arom C); 90.30
(C-5); 70.39 d, 2 C, J (P,C) ) 6.4 (P-OC); 70.11 d, J (P,C) )
11.7 (C-2′); 65.02 d, J (P,C) ) 164.1 (P-C); 40.15 (C-1′); 24.01
d, J (P,C) ) 3.4, 23.98 d, J (P,C) ) 3.4 and 23.86 d, 2 C, J (P,C)
) 4.4 (CH3).
2,4-Dia m in o-5-ben zyl-1-[2-(p h osp h on om eth oxy)eth yl]-
p yr im id in -6(1H)-on e (11b). Yield: 125 mg, 70%. Mp: 252-
253 °C. Anal. (C14H19N4O5P‚2H2O) C, H, N. FABMS: 355
(MH+) (50).
2,4-Dia m in o-6-[2-(d iisop r op oxyp h osp h or ylm eth oxy)-
et h oxy]-5-p h en ylp yr im id in e (9f). Yield: 0.36 g, 21%.
1
FABMS: 425 (MH+) (100). H NMR (DMSO-d6): 7.35 m, 2H
and 7.25 m, 3 H (arom H); 6.00 brs, 2 H, and 5.55 brs, 2 H

5070 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23
Hockova´ et al.
1H NMR (DMSO-d6): 7.24 d, 2 H, 7.19, 2, and 7.09 t, 1 H
(arom H); 6.57 brs, 2 H, and 5.75 brs, 2 H (NH2); 4.00 t, 2 H,
J (1′,2′) ) 6.0 (H-1′); 3.63 t, 2 H, J (2′,1′) ) 6.0 (H-2′); 3.60 d, 2
H, J (P,CH) ) 8.3 (P-CH2); 3.54 s, 2 H (H-1′′). 13C NMR
(D2O): 164.10 (C-4); 161.18 (C-2); 155.56 (C-6); 140.10, 128.68,
2 C, 127.73, 2 C, and 126.19 (arom C); 89.33 (C-5); 70.95 d,
J (P,C) ) 10.7 (C-2′); 69.61 d, J (P,C) ) 149.9 (P-C); 42.94 (C-
1′); 28.66 (C-1′′).
2,4-Diam in o-5-ben zyl-6-[2-(ph osph on om eth oxy)eth oxy]-
p yr im id in e (12b). Yield: 121 mg, 68%. Mp: 241-242 °C.
Anal. (C14H19N4O5P‚2/3H2O) C, H, N. FABMS: 355 (MH+) (50).
1H NMR (DMSO-d6): 7.23, 4, and 7.10 m, 1 H (arom H); 6.15
brs, 4 H (NH2); 4.30 m, 2 H (H-1′); 3.74 m, 2 H (H-2′); 3.62 s,
2 H (H-1′′); 3.58 d, 2 H, J (P,CH) ) 8.6 (P-CH2). 13C NMR
(D2O): 168.05 (C-6); 163.70 (C-4); 160.70 (C-2); 139.95, 128.74,
2 C, 127.89, 2 C, and 126.32 (arom C); 90.38 (C-5); 70.66 d,
J (P,C) ) 8.8 (C-2′); 67.75 d, J (P,C) ) 149.4 (P-C); 65.66 (C-
1′); 27.50 (C-1′′).
(P-CH2); 1.79 s, 3 H (CH3). 13C NMR: 163.51 (C-4); 160.81
(C-2); 154.69 (C-6); 85.56 (C-5); 70.70 d, J (P,C) ) 10.7 (C-2′);
69.33 d, J (P,C) ) 150.4 (P-C); 42.27 (C-1′); 11.40 (CH3).
2,4-Diam in o-5-m eth yl-6-[2-(ph osph on om eth oxy)eth oxy]-
p yr im id in e (12e). Yield: 81 mg, 57%. Mp: 253-254 °C. Anal.
1
(C8H15N4O5P) C, H, N, P. FABMS: 279 (MH+) (70). H NMR
(D2O + NaOD): 4.37 m, 2 H (H-1′); 3.91 m, 2 H (H-2′); 3.58 d,
2 H, J (P,CH) ) 8.4 (P-CH2); 1.84 s, 3 H (CH3). 13C NMR:
167.06 (C-6); 163.58 (C-4); 160.01 (C-2); 85.75 (C-5); 70.47 d,
J (P,C) ) 10.3 (C-2′); 68.96 d, J (P,C) ) 148.9 (P-C); 65.42 (C-
1′); 10.54 (CH3).
2,4-Dia m in o-5-p h en yl-1-[2-(p h osp h on om eth oxy)eth yl]-
p yr im id in -6(1H)-on e (11f). Yield: 140 mg, 78%. Mp: 235-
236 °C. Anal. (C13H17N4O5P‚1/2H2O) C, H, N, P. FABMS: 341
1
(MH+) (30). H NMR (DMSO-d6): 7.32 t, 2 H, 7.26 d, 2 H and
7.18 t, 1 H (arom H); 6.81 brs, 2 H, and 5.49 brs, 2 H (NH2);
4.03 t, 2 H, J (1′,2′) ) 6.0 (H-1′); 3.66 t, 2 H, J (2′,1′) ) 6.0 (H-
2′); 3.62 d, 2 H, J (P,CH) ) 8.3 (P-CH2).
2,4-Dia m in o-5-cya n om eth yl-1-[2-(ph osp h on om eth oxy)-
eth yl]p yr im id in -6(1H)-on e (11c). Yield: 120 mg, 77%. Mp:
250-251 °C. Anal. (C9H14N5O5P‚1/2H2O) C, H, N. FABMS: 304
2,4-Diam in o-5-ph en yl-6-[2-(ph osph on om eth oxy)eth oxy]-
p yr im id in e (12f). Yield: 133 mg, 75%. Mp: 230-231 °C.
Anal. (C13H17N4O5P‚3/2H2O) C, H, N, P. FABMS: 341 (MH+)
1
(MH+) (50). H NMR (DMSO-d6): 6.71 brs, 2 H, and 6.20 brs,
1
(40). H NMR (DMSO-d6): 7.36, 2, and 7.25 m, 3 H (arom H);
2 H (NH2); 3.99 t, 2 H, J (1′,2′) ) 6.1 (H-1′); 3.62 t, 2 H, J (2′,1′)
) 6.1 (H-2′); 3.60 d, 2 H, J (P,CH) ) 8.3 (P-CH2); 3.39 s, 2 H
(H-1′′). 13C NMR (D2O + NaOD): 161.66 (C-2); 159.15 (C-4);
153.64 (C-6); 117.40 (CN); 81.09 (C-5); 68.10 d, J (P,C) ) 11.3
(C-2′); 67.06 d, J (P,C) ) 149.4 (P-C); 40.13 (C-1′).
2,4-Dia m in o-5-cya n om eth yl-6-[2-(ph osp h on om eth oxy)-
eth oxy]p yr im id in e (12c). Yield: 123 mg, 81%. Mp: 258-
259 °C, dec. Anal. (C9H14N5O5P) C, H, N. FABMS: 304 (MH+)
(60). 1H NMR (DMSO-d6): 6.28 brs, 2 H, and 6.02 brs, 2 H
(NH2); 4.31 m, 2 H (H-1′); 3.75 m, 2 H (H-2′); 3.53 d, 2 H, J (P,-
CH) ) 8.2 (P-CH2); 3.49 s, 2 H (H-1′′).
2,4-Dia m in o-5-(eth oxyca r bon ylm eth yl)-1-[2-(p h osp h o-
n om eth oxy)eth yl]p yr im id in -6(1H)-on e (11d ) a n d 2,4-Di-
am in o-5-(car boxym eth yl)-1-[2-(ph osph on om eth oxy)eth yl]-
p yr im id in -6(1H)-on e (11h ). Starting from a 2-fold amount
(1 mmol) of diisopropyl ester 8d , the Dowex 1 × 2 column
chromatography gave two products:
11d : eluted by 1 M AcOH. Yield: 97 mg, 28%. Mp: dec.
Anal. (C11H19N4O7P‚H2O) C, H, N, P. FABMS: 351 (MH+) (20).
1H NMR (DMSO-d6): 6.63 brs, 2 H and 5.88 brs, 2 H (NH2);
4.00 q, 2 H, J (CH2,CH3) ) 7.1 (O-CH2); 3.96 t, 2 H, J (1′,2′) )
6.1 (H-1′); 3.60 t, 2 H, J (2′,1′) ) 6.1 (H-2′); 3.59 d, 2 H, J (P,-
CH) ) 8.2 (P-CH2); 3.21 s, 2 H (H-1′′); 1.17 t, 3 H, J (CH3,-
CH2) ) 7.1 (CH3).
6.35 brs, 2 H and 5.79 brs, 2 H (NH2); 4.27 m, 2 H (H-1′); 3.65
m, 2 H (H-2′); 3.48 d, 2 H, J (P,CH) ) 8.4 (P-CH2).
2,4-Dia m in o-5-cyclop r op yl-1-[2-(p h osp h on om eth oxy)-
eth yl]p yr im id in -6(1H)-on e (11g). Yield: 120 mg, 79%.
Mp: 282-283 °C. Anal. (C10H17N4O5P) C, H, N, P. FABMS:
1
305 (MH+) (95). H NMR (D2O + NaOD): 4.10 t, 2 H, J (1′,2′)
) 5.3 (H-1′); 3.78 t, 2 H, J (2′,1′) ) 5.3 (H-2′); 3.51 d, 2 H, J (P,-
CH) ) 8.6 (P-CH2); 1.23 m, 1 H, 0.89, 2, and 0.40 m, 2 H
(cyclopropyl).
2,4-Dia m in o-5-cyclop r op yl-6-[2-(p h osp h on om eth oxy)-
eth oxy]p yr im id in e (12g). Yield: 125 mg, 82%. Mp: 255-
256 °C. Anal. (C10H17N4O5P) C, H, N, P. FABMS: 305 (MH+)
(95). 1H NMR (D2O + NaOD): 4.38 m, 2 H (H-1′); 3.93 m, 2 H
(H-2′); 3.62 d, 2 H, J (P,CH) ) 8.4 (P-CH2); 1.24 m, 1 H, 0.93,
2, and 0.45 m, 2 H (cyclopropyl).
P r ep a r a tion of 2,4-Dia m in o-5-m eth yl-6-[2-(p h osp h o-
n om eth oxy)eth oxy]p yr im id in e (12e) by Cr oss-Cou p lin g
Rea ction . To a solution of bromo derivative 14a (0.85 g, 2
mmol) and Pd(PPh3)4 (0.23 g, 10 mol %) in dry tetrahydrofuran
(35 mL) was added a 2 M solution of AlMe3 in toluene (5 mL,
5 equiv) under Ar atmosphere. The resulting mixture was
refluxed for 20 h and taken down in vacuo. The residue was
treated with hot chloroform and filtered, and the filtrate was
evaporated in vacuo. Chromatography on silica gel afforded
unreacted starting material (14a , 0.12 g, 14%) and diisopropyl
ester 9e (0.06 g, 8%) FABMS: 363 (MH+) (100). 1H NMR
(DMSO-d6): 6.41 brs, 2 H, and 6.33 brs, 2 H (NH2); 4.59 m, 2
H (P-OCH); 4.31 m, 2 H (H-1′); 3.80 d, 2 H, J (P,CH) ) 8.3
(P-CH2); 3.78 m, 2 H (H-2′); 1.74 s, 3 H (CH3); 1.24, 6, and
1.22 d, 6 H, J (CH3,CH2) ) 6.2 (CH3).
The solid that was filtered off from hot chloroform was
triturated with a mixture of water, methanol, and aqueous
NH3 and filtered. The filtrate was taken down in vacuo and
deionized on a column of Dowex 50 × 8. Further purification
by Dowex 1 × 2 column chromatography (elution with gradient
of acetic acid) afforded monoisopropyl ester 17. Yield: 0.2 g,
31%. FABMS: 321 (MH+) (100). 1H NMR (DMSO-d6): 6.98
brs, 4 H (NH2); 4.41 m, 2 H (P-OCH); 4.26 m, 2 H (H-1′); 3.75
m, 2 H (H-2′); 3.55 d, 2 H, J (P,CH) ) 8.4 (P-CH2); 1.71 s, 3 H
(CH3); 1.15 d, 6 H, J (CH3,CH2) ) 6.1 (CH3).
Both intermediates 9e and 17 were combined, dry acetoni-
trile (15 mL) and BrSiMe3 (1.5 mL) were added, and the
resulting mixture was stirred overnight at room temperature.
Solvent was evaporated in vacuo and the residue was codis-
tilled with acetonitrile. Water and aqueous NH3 was added
and the solution was evaporated to dryness. The residue was
deionized on a Dowex 50 × 8 column and further purified by
Dowex 1 × 2 column chromatography (elution with a gradient
of acetic acid). Crystallization (water-ethanol) gave compound
12e. Yield: 0.15 g, overall yield 27%. Mp: 253-255 °C. Anal.
(C8H15N4O5P) C, H, N, P. FABMS: 279 (MH+) (100). 1H NMR
11h : eluted by 2 M AcOH. Yield: 90 mg, 27%. Mp: 235-
236 °C. Anal. (C9H15N4O7P‚H2O) C, H, N, P. FABMS: 323
1
(MH+) (10). H NMR (DMSO-d6): 6.73 brs, 2 H and 5.91 brs,
2 H (NH2); 3.98 t, 2 H, J (1′,2′) ) 6.0 (H-1′); 3.62 t, 2 H, J (2′,1′)
) 6.0 (H-2′); 3.60 d, 2 H, J (P,CH) ) 8.3 (P-CH2); 3.15 s, 2 H
(H-1′′).
2,4-Dia m in o-5-(eth oxyca r bon ylm eth yl)-6-[2-(p h osp h o-
n om eth oxy)eth oxy]p yr im id in e (12d ) a n d 2,4-Dia m in o-
5-(ca r boxym eth yl)-6-[2-(p h osp h on om eth oxy)eth oxy]p y-
r im id in (12h ). Starting from 2-fold amount (1 mmol) of
diisopropyl ester 9d , the Dowex 1 × 2 column chromatography
gave two products:
12d : eluted by 0.25 M AcOH. Yield: 130 mg, 34%. Mp: 199-
200 °C. Anal. (C11H19N4O7P‚2H2O) C, H, N, P. FABMS: 351
1
(MH+) (20). H NMR (DMSO-d6): 6.21 brs, 2 H and 6.12 brs,
2 H (NH2); 4.24 m, 2 H (H-1′); 4.04 q, 2 H, J (CH2,CH3) ) 7.1
(O-CH2); 3.70 m, 2 H (H-2′); 3.54 d, 2 H, J (P,CH) ) 8.5 (P-
CH2); 3.30 s, 2 H (H-1′′); 1.17 t, 3 H, J (CH3,CH2) ) 7.1 (CH3).
12h : eluted by 0.5-1.0 M AcOH. Yield: 72 mg, 22%. Mp:
228-229 °C. Anal. (C9H15N4O7P‚H2O) C, H, N, P. FABMS: 323
1
(MH+) (20). H NMR (DMSO-d6): 6.21 brs, 2 H and 6.14 brs,
2 H (NH2); 4.26 m, 2 H (H-1′); 3.70 m, 2 H (H-2′); 3.55 d, 2 H,
J (P,CH) ) 8.5 (P-CH2); 3.21 s, 2 H (H-1′′).
2,4-Dia m in o-5-m eth yl-1-[2-(p h osp h on om eth oxy)eth yl]-
p yr im id in -6(1H)-on e (11e). Yield: 82 mg, 59%. Mp: 284-
285 °C. Anal. (C8H15N4O5P) C, H, N, P. FABMS: 279 (MH+)
1
(20). H NMR (D2O + NaOD): 4.13 t, 2 H, J (1′,2′) ) 5.2 (H-
1′); 3.97 t, 2 H, J (2′,1′) ) 5.2 (H-2′); 3.50 d, 2 H, J (P,CH) ) 8.5

Acyclic Nucleoside Phosphonate Analogues
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23 5071
(D2O + NaOD): 4.37 m, 2 H (H-1′); 3.91 m, 2 H (H-2′); 3.58 d,
2 H, J (P,CH) ) 8.4 (P-CH2); 1.84 s, 3 H (CH3).
with a linear gradient of acetic acid (0-1 M, 1 L each); the
UV-absorbing fraction was taken down in vacuo, the residue
was codistilled with water, and the crystalline residue was
filtered from the water supension. The crystalline product was
washed with ethanol and ether and dried in vacuo. Yield: 1.7
g, 58%. Mp: 218 °C. Anal. (C7H12BrN4O5P): C, H, Br, N, P.
2,4-Dia m in o-5-b r om o-6-[2-(d iisop r op oxyp h osp h or yl-
m et h oxy)et h oxy]p yr im id in e (14a ). Compound 13 (4.3 g,
12.9 mmol) in DMF (40 mL) was treated with 0.7 M bromine
solution in CCl4 (20 mL). After 3 h of stirring at room
temperature, the reaction mixture was made alkaline by
addition of triethylamine and evaporated, and the residue
(14a ) was crystallized from ethanol. Yield: 3.9 g, 72%. Mp:
165 °C. Anal. (C12H22BrN4O5P): C, H, Br, N, P. FABMS: 427
(M+, 59), 428 (57). 1H NMR (DMSO-d6): 6.33 br, 2H, and 6.13
s, 2H (NH2); 4.59 m, 2H (P-OCH); 4.33 m, 2H (H-1′); 3.82 d,
2H, J (P,CH) ) 8.4 (P-CH2); 3.79 m, 2H (H-2′); 1.23 d, 6H,
and 1.22 d, 6H J (CH3,CH) ) 6.1 (CH3). 13C NMR (DMSO-d6):
164.65 (C-6); 162.04 (C-4); 161.10 (C-2)]; 71.64 (C-5); 70.84 d,
J (P,C) ) 12.2 (C-2′); 70.37 d, 2C, J (P,C) ) 6.4 (P-O-C); 65.27
(C-1′); 65.23 d, J (P,C) ) 164.9 (P-C); 24.03 d, 2C, J (P,C) )
3.9 and 23.91 d, 2C, J (P,C) ) 4.4 (CH3).
1
FABMS: 343 (MH+, 90), 349 (M - I, 55), 253 (base, 100), H
NMR (D2O + NaOD): 4.14 m, 2H (H-1′); 3.93 m, 2H (H-2′);
3.61 d, 2H, J (P,CH) ) 8.4 (P-CH2); 13C NMR (D2O + NaOD):
165.32 (C-6); 161.84 (C-4); 160.79 (C-2); 73.64 (C-5); 70.47 d,
J (P,C) ) 10.3 (C-2′); 69.29 d, J (P,C) ) 149.4 (P-C); 66.50 (C-
1′).
2,4-Diam in o-5-ch lor o-6-[2-(ph osph on om eth oxy)eth oxy]-
p yr im id in e (15b) was prepared from the diester 15a (2.1 g,
5.7 mmol) analogously as described for compound 14b. Yield:
1.6 g, 63%. Mp: 233 °C. Anal. (C7H12ClN4O5P): C, H, Cl, N,
1
P. FABMS: 293 (MH+, 90), H NMR (D2O + NaOD): 4.43 m,
2H (H-1′); 3.92 m, 2H (H-2′); 3.58 d, 2H, J (P,CH) ) 8.5 (P-
CH2); 13C NMR (D2O + NaOD): 164.33 (C-6); 161.03 (C-4);
160.05 (C-2); 85.51 (C-5); 70.48 d, J (P,C) ) 10.3 (C-2′); 69.29
d, J (P,C) ) 149.4 (P-C); 66.40 (C-1′).
2,4-Dia m in o-5-ch lor o-6-[2-(d iisop r op oxyp h osp h or yl-
m eth oxy)eth oxy]p yr im id in e (15a ). N-Chlorosuccinimide
(1.18 g, 1.5 equiv) was added with stirring to the suspension
of compound 13 (2.5 g, 7.5 mmol) in acetonitrile (70 mL). After
90 min at ambient temperature, the homogeneous reaction
mixture was diluted with toluene (300 mL) and the solution
was extracted successively (50 mL each) with 5% NaHSO3,
(3×), sat. KHCO3, and water, dried, and evaporated. The
crystalline residue was triturated with acetone (50 mL),
filtered, and dried in vacuo. Yield: 2.60 g, 94.4%. Mp: 165
°C. Anal. (C12H22ClN4O5P): C, H, Cl, N, P. FABMS: 383 (M,
Dep r otection of 2,4-Dia m in o-6-[2-(d iisop r op oxyp h os-
p h or ylm eth oxy)eth oxy]-5-iod op yr im id in e (16a ) was per-
formed from 2.25 g (6.1 mmol) of compound 16a in essentially
the same manner as described for compound 14b. The UV-
absorbing fraction from Dowex 1 chromatography was codis-
tilled twice with water and the residue boiled briefly with
water (100 mL). The supension was left to stand at ambient
temperature overnight, filtered, washed with acetone and
ether, and dried. Yield: 1.57 g, 74%. Mp: 263 °C. Anal.
(C7H13N4O5P): C, H, N, P. FABMS: 265 (MH+), 1H NMR (D2O
+ NaOD): 5.39 s, 1H (H-5); 4.25 m, 2H (H-1′); 3.90 m, 2H
(H-2′); 3.58 d, 2H, J (P,CH) ) 8.4 (P-CH2); 13C NMR (D2O +
NaOD): 170.77 (C-6); 166.61 (C-4); 162.71 (C-2); 76.82 (C-5);
70.44 d, J (P,C) ) 10.2 (C-2′); 69.17 d, J (P,C) ) 150.4 (P-C);
66.18 (C-1′).
An tivir a l Activity Assa ys. The antiviral, other than anti-
HIV-1, assays were based on inhibition of virus-induced
cytopathicity in either E6SM (HSV-1, HSV-2, VV) or HEL
(VZV, CMV) cell cultures, following previously established
procedures.21 Briefly, confluent cell cultures in microtiter 96-
well plates were inoculated with 100 CCID50 of virus, 1 CCID50
being the virus dose required to infect 50% of the cell cultures.
After a 1- to 2-h virus adsorption period, residual virus was
removed, and the cell cultures were incubated in the presence
of varying concentrations (400, 200, 100, ... µg/mL) of the test
compounds. 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.
1
80). H NMR (DMSO-d6): 6.48 br, 2H, and 6.19 s, 2H (NH2);
4.58 m, 2H (P-OCH); 4.34 m, 2H (H-1′); 3.81 d, 2H, J (P,CH)
) 8.4 (P-CH2); 3.79 m, 2H (H-2′); 1.23 d, 6H, and 1.21 d, 6H
J (CH3,CH) ) 6.1 (CH3). 13C NMR (DMSO-d6): 163.74 (C-6);
160.75 (C-4); 159.89 (C-2); 83.00 (C-5); 70.82 d, J (P,C) ) 11.7
(C-2′); 70.39 d, 2C, J (P,C) ) 6.4 (P-O-C); 65.17 (C-1′); 65.16
d, J (P,C) ) 164.1 (P-C); 24.02 d, 2C, J (P,C) ) 3.9 and 23.89
d, 2C, J (P,C) ) 4.4 (CH3).
2,4-Dia m in o-6-[2-(d iisop r op oxyp h osp h or ylm eth oxy)-
eth oxy]-5-iod op yr im id in e (16a ). N-Iodosuccinimide (2.22 g,
1.5 equiv) was added under stirring to the solution of com-
pound 13 (2.5 g, 7.5 mmol) in acetonitrile (70 mL). After 90
min at ambient temperature, the reaction mixture was diluted
with toluene (300 mL) and the solution was extracted succes-
sively (50 mL each) with 5% NaHSO3 (3×), sat. aqueous
KHCO3, and water, dried, and evaporated. The crystalline
residue was triturated with ether (50 mL), filtered, and dried
in vacuo. Yield: 3.0 g, 87.3%. Mp: 151 °C. Anal. (C12H22
-
IN4O5P): C,H,I,N,P. FABMS: 475 (MH+, 90), 349 (M-I, 55),
253 (base, 100), 1H NMR (DMSO-d6): 6.20 br, 2H, and 6.13 s,
2H (NH2); 4.54 m, 2H (P-OCH); 4.31 m, 2H (H-1′); 3.84 d,
2H, J (P,CH) ) 8.3 (P-CH2); 3.78 m, 2H (H-2′); 1.23 d, 6H,
and 1.33 d, 6H J (CH3,CH) ) 6.3 (CH3). 13C NMR (DMSO-d6):
167.31 (C-6); 164.56 (C-4); 162.45 (C-2)] 70.88 d, J (P,C) ) 12.2
(C-2′); 70.35 d, 2C, J (P,C) ) 6.3 (P-O-C); 65.53 (C-1′); 65.36
d, J (P,C) ) 164.1 (P-C); 43.06 (C-5); 24.03 d, 2C, J (P,C) )
3.9 and 23.96 d, 2C, J (P,C) ) 3.9 (CH3).
In h ib it ion of H IV-1-In d u ced Cyt op a t h icit y in CE M
Cells. The methodology of the anti-HIV assays has been
described previously.22 Briefly, human CEM (∼3 × 105
cells.mL-1) cells were infected with 100 CCID50 HIV-1 ((IIIB)
or HIV-2 (ROD) per mL and seeded in 200 µL-wells of 96-well
microtiter plates, containing appropriate dilutions of the test
compounds. After 4 days of incubation at 37 °C, CEM giant
cell formation was examined microscopically.
2,4-Diam in o-5-br om o-6-[2-(ph osph on om eth oxy)eth oxy]-
p yr im id in e (14b). The diester 14a (3.5 g, 8.5 mmol) in
acetonitrile (50 mL) was treated with bromotrimethylsilane
(7 mL) overnight, the volatiles were evaporated in vacuo, and
the residue codistilled with acetonitrile (2 × 25 mL). Water
(50 mL) was added and the solution was set to pH 10 by
addition of concentrated aqueous NH3. After 10 min at this
pH, the solution was taken down and the residue was applied
on a Dowex 50 × 8 column (H+-form) (100 mL). The column
was then washed with water to the drop of acidity of the eluate.
The resin was then suspended in water (150 mL) and concen-
trated aqueous NH3 was added to pH 10.0 with stirring. After
10 min at this pH the suspension was filtered, the resin
washed repeatedly with water (total, 400 mL), and the filtrate
was evaporated in vacuo. The residue in water (20 mL) was
alkalified by concentrated aqueous NH3 to pH 10 and applied
onto a column (100 mL) of Dowex 1 × 2. The column was
washed with water, until the UV-absorption dropped, and then
In h ibition of MSV-In d u ced Tr a n sfor m a tion of Mu r in e
C3H/3T3 Em br yo F ibr obla sts. The anti-MSV assay was
performed as described previously.22 Murine C3H/3T3 embryo
fibroblast cells were seeded at 5 × 105 cells mL-1 into 1-cm2
wells of 48-well microplates. At 24 h later, the cell cultures
were infected with 80 focus-forming units of MSV (prepared
from tumors induced following intramuscular inoculation of
3-day-old NMRI mice with MSV, as described previously23) for
90-120 min at 37 °C. The medium was then replaced by 1
mL of fresh medium containing various concentrations of the
test compounds. After 6 days, transformation of the cell culture
was examined microscopically.
Ack n ow led gm en t. This study was performed as a
part of research project # Z4055905 of the Institute of
Organic Chemistry and Biochemistry. It was supported
by the Program of Targeted Projects of the Academy of

5072 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23
Hockova´ et al.
J .; J affe, H. S. Cidofovir. Review of current and potential clinical
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E.; Andrei, G.; Balzarini, J .; Hatse, S.; Liekens, S.; Naesens, L.;
Neyts, J .; Snoeck, R. Antitumor potential of acyclic nucleoside
phosphonates. Nucleosides Nucleotides 1999, 18, 759-771. (d)
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Cidofovir, a new approach for the treatment of cervix intraepi-
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205-209.
Sciences of the Czech Republic (# S4055109), by Gilead
Sciences (Foster City, CA) by ISEP/FORTIS, the “Ge-
concerteerde Onderzoeksacties” (GOA-00/12), and the
Rene´ Descartes Prize-2001 of the European Commission
(HPAW-2002-90001). The authors’ thanks are also due
to the staff of the mass spectrometry and analytical
departments (Head, K. Ubik) of the Institute of Organic
Chemistry and Biochemistry and J .Gu¨nter for perform-
ing preparative HPLC.
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