Synthesis and antiviral activity of 2,4-diamino-5-cyano-6-[2- (phosphonomethoxy)ethoxy]pyrimidine and related compounds

Article abstract of DOI:10.1016/j.bmc.2004.04.002

Synthesis of 2,4-diamino-5-cyano-6-{[(diisopropoxyphosphoryl)methoxy] ethoxy}pyrimidine was based on the formation of the pyrimidine ring by cyclization followed by modification of the side chain by alkylation. The 5-cyano group was also transformed to a 5-formyl and 5-hydroxymethyl group by reduction. As a side product an unexpected dimer was formed. Resulting compounds were converted to the free phosphonic acids by treatment with bromotrimethylsilane followed by hydrolysis. The 5-cyano and 5-formyl derivatives showed pronounced antiretroviral activity, comparable to that of the reference drugs adefovir and tenofovir.

Full text of DOI:10.1016/j.bmc.2004.04.002

Bioorganic & Medicinal Chemistry 12 (2004) 3197–3202
Synthesis and antiviral activity of 2,4-diamino-5-cyano-6-
[2-(phosphonomethoxy)ethoxy]pyrimidine and related compounds
b
ꢀ a,*
ꢀ
ꢀ a
ꢀ
ꢀ a
Dana Hockova, Antonın Holy, Milena Masojıdkova, Graciela Andrei,
Robert Snoeck,b Erik De Clercqb and Jan Balzarinib
aInstitute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo nꢀam. 2,
CZ-166 10 Prague 6, Czech Republic
bRega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
Received 22 December 2003; accepted 2 April 2004
Available online 8 May 2004
Abstract—Synthesis of 2,4-diamino-5-cyano-6-{[(diisopropoxyphosphoryl)methoxy]ethoxy}pyrimidine was based on the formation
of the pyrimidine ring by cyclization followed by modification of the side chain by alkylation. The 5-cyano group was also
transformed to a 5-formyl and 5-hydroxymethyl group by reduction. As a side product an unexpected dimer was formed. Resulting
compounds were converted to the free phosphonic acids by treatment with bromotrimethylsilane followed by hydrolysis. The
5-cyano and 5-formyl derivatives showed pronounced antiretroviral activity, comparable to that of the reference drugs adefovir
and tenofovir.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
NH2
N
NH2
N
N
N
N
2-(Phosphonomethoxy)alkyl derivatives of purine and
pyrimidine bases––acyclic nucleoside phosphonates
(ANPs)––possess significant antiviral and cytostatic
activity.1 These nucleotide analogues contain an iso-
polar phosphonomethyl ether moiety instead of the
nucleotide phosphate ester group, which excludes their
enzymatic degradation and/or eliminates problems with
intracellular phosphorylation necessary for nucleoside
activation. Among the ANPs, particularly 9-[2-(phos-
phonomethoxy)ethyl]adenine (PMEA, adefovir, Fig. 1)
and [(R)-2-phosphonomethoxypropyl]adenine ((R)-
PMPA, tenofovir, Fig. 1) are active against DNA viru-
ses and retroviruses.2 In this paper, these commercially
available drugs are included as reference compounds for
the antiviral evaluation assays.
O
N
O
OH
R
O
P
O
P
OH
OH
OH
OH
O
PMEA: R = H
(R)-PMPA: R = (R)-CH3
(S)-HPMPC
NH2
R
N
H2N
N
O
1a, R = H
1b, R =CH3
1c, R = Cl
1d, R = Br
O
P
OH
OH
O
Figure 1.
The SAR studies demonstrated that the margins of
structural alteration are very narrow.3 Except for the
antiviral activity of the cytosine derivative (S)-HPMPC
(cidofovir, Fig. 1),4 the choice of the base is limited
mostly to adenine, guanine, or 2,6-diaminopurine, and
to their 8-aza and 3-deaza congeners.5 The pharmaco-
phore of purine acyclic nucleoside phosphonates is
characterized by the presence of amino group(s) at the
pyrimidine part of the purine system.
Keywords: Nucleotide analogues; Acyclic nucleoside phosphonates;
Antiviral; Pyrimidines.
Recently we discovered a new type of antiviral ANPs orig-
inating from 2-substituted 4-amino-6-hydroxypyrimidines.6
* Corresponding author. Tel.: +420-220183262; fax: +420-220183560;
e-mail: lasice@uochb.cas.cz
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.04.002

3198
D. Hockovꢀa et al. / Bioorg. Med. Chem. 12 (2004) 3197–3202
Alkylation of these bases by the appropriate phospho-
nate-protected synthon afforded a mixture of O6- and
N1-regioisomers, which were converted to the free
phosphonates. While none of the isomeric 1-[2-(phos-
phonomethoxy)ethyl]pyrimidin-6-one derivatives was
antivirally active, among 6-[2-(phosphonomethoxy)eth-
oxy]pyrimidine derivatives, compounds derived from
2,4-diaminopyrimidine significantly inhibited replication
of retroviruses and herpesviruses in cell culture. These
novel subclass of pyrimidine ANPs can be considered as
analogues of 2,6-diaminopurine with an open imidazole
ring in the purine moiety.
cell culture, we decided to further enlarge the diversity of
the functional groups at the 5-position of pyrimidine
moiety.
2. Results and discussion
As a suitable parent compound 2,4-diamino-5-cyano-6-
[2-(diisopropoxyphosphorylmethoxy)ethoxy]pyrimidine
(3) was selected. For the creation of its intermediate 2,4-
diamino-5-cyano-6-(2-hydroxyethoxy)pyrimidine (2) a
known two-step approach starting from tetracyanoeth-
ylene was applied.8 The second step is based on the
formation of pyrimidine ring by the cyclization of
dicyanoketene ethylene acetal with guanidine.9 The hy-
droxy group in the side chain of compound 2 was
alkylated by (diisopropoxyphosphoryl)methyl tosylate7
(Scheme 1) to form the desired 2,4-diamino-5-cyano-6-
[2-(diisopropoxyphosphorylmethoxy)ethoxy]pyrimidine
(3). This alkylation reaction is aggravated by the insol-
ubility of starting material in THF and so the standard
conditions had to be optimized.
To further investigate the structure–activity relationship
in this new group of antiviral compounds, recently we
reported7 the synthesis and antiviral activity of 2,4-di-
amino-6-[2-(phosphonomethoxy)ethoxy]pyrimidine deriv-
atives (1, Fig. 1) bearing various substituents at the
C5-position. In this case, the imidazole ring of the
purine base is even more closely mimicked. Among these
compounds, the 5-methyl derivative 1b was markedly
active against human immunodeficiency virus [at least
10-fold more than the 5-unsubstituted compound (1a)]
and Moloney murine sarcoma virus-induced cytopath-
icity in cell culture (EC50: ꢀ0.00018 lmol/mL) but also
cytostatic to CEM cell cultures. In contrast, the 5-hal-
ogen-substituted derivatives 1c and 1d showed
pronounced antiretroviral activity (EC50: 0.0023–
0.0110 lmol/mL), comparable to that of the reference
drugs adefovir and tenofovir, but were devoid of mea-
surable toxicity at 0.3 lmol/mL.
The cyano group at the 5-position was further modified
by reduction. The synthesis of 5-formyl derivative 5 was
achieved by hydrogenation of compound 3 in a mixture
of water and sulfuric acid over palladium on charcoal.
The formyl group underwent consecutive reduction
(Scheme 2) by sodium borohydride in methanol to af-
ford 5-(hydroxymethyl)pyrimidine 7. This compound
was rather unstable and when the reaction time of the
reduction was prolonged, only an unexpected product 8
was isolated instead of the compound 7. Traces of the
same product were isolated also from the hydrogenation
Since several 5-substituted 2,4-diaminopyrimidine
derivatives markedly inhibited retrovirus replication in
NH2
NH2
1. HOCH2CH2OH
NC
NC
CN
CN
CN
O
CN
H2NC(O)NH2
NaH/Et3N//DMF
N
N
O
P
OiPr
OiPr
OH
O
2. H2NC(NH)NH2 HCNS
NaOMe/MeOH
TsO
P(O)(OiPr)2
H2N
N
H2N
N
O
2
3
BrSiMe3/CH3CN
Pd/C/H2
aq. H2SO4
NH2
O
C
NH2
O
C
NH2
BrSiMe3
CH3CN
CN
O
N
H
O
P
N
H
O
P
N
O
P
OH
OH
OiPr
OiPr
OH
OH
O
O
O
H2N
N
O
H2N
N
O
H2N
N
6
5
4
(+ traces of 8)
Scheme 1.
NH2
NH2
N
NH2
O
C
NH2
CH2OH
O
N
N
H
O
P
N
O
P
NaBH4/MeOH
OiPr
OiPr
OiPr
OiPr
+
O
O
H2N
N
O
O
N
NH2
H2N
N
O
H2N
N
7
5
O
O
O
RO P
O P OR
OR
8, R = iPr
9, R = H
BrSiMe3/CH3CN
OR
Scheme 2.

D. Hockovꢀa et al. / Bioorg. Med. Chem. 12 (2004) 3197–3202
3199
Table 1. Antiviral activity of test compounds in cell culture
of 5-cyano derivative 3 when the reaction mixture was
allowed to react for three days.
a
b
Example
EC50 (lmol/mL)
CC50
(lmol/mL)
(CEM)
HIV-1
(IIIB)
HIV-2
(ROD)
MSV
2,4-Diamino-6-[2-(diisopropoxyphosphorylmethoxy)eth-
oxy]pyrimidines bearing 5-cyano and 5-formyl groups
(compounds 3 and 5) smoothly afforded, on treatment
with bromotrimethylsilane followed by hydrolysis, the
free phosphonic acids 4 and 6, that were purified by ion-
exchange column chromatography on Dowex 1 · 2
(OAcꢁ). In contrast, the 5-hydroxymethyl compound 7
afforded no desired product under the above reaction
conditions or under the modified conditions in the
presence of lutidine.11
4 (5-CN)
0.011
0.0045
0.0027
0.050
0.0095
0.021
––
P 0.3
P 0.3
P 0.2
6 (5-CHO) 0.0045
0.080
9
Reference compounds
1a (5-H) 0.0031
1b (5-CH3) 0.00023
PMEA 0.0033
(R)-PMPA 0.0012
0.0016
0.00023
0.0066
0.0014
0.00015
0.00016
0.0022
0.0046
0.042
0.0047
0.056
0.41
a 50% effective concentration.
b 50% cytostatic concentration.
The same standard procedure was used for deprotection
of both the phosphono diester groups in dimer 8 to form
1
bisphosphonic acid 9. In H NMR spectrum of com-
activity against several DNA and retroviruses. In con-
trast to the parent 5-unsubstituted compound 1a, none
of the derivatives 4, 6, and 9 were active against herpes
simplex virus type 1 or type 2, cytomegalovirus and
vaccinia virus. Compounds 4 and 6 were inhibitory
against HIV at 0.0027–0.011 lmol/mL, and against
MSV at 0.0095–0.021 lmol/mL (Table 1). These antivi-
ral potencies were comparable with those found for the
5-unsubstituted 2,4-diamino-6-[2-(phosphonomethoxy)-
ethoxy]pyrimidine against HIV but inferior the values
found for MSV. These values were also comparable to
those recorded for adefovir and tenofovir. Compared
with the 5-methyl derivative 1b, the antiretroviral
activities were lower. Compound 9 was 10- to 20-fold
less inhibitory to HIV than 4 and 6. The compounds
showed no appreciable cytotoxic activity against E6SM,
HEL, or CEM cell growth and did not affect micro-
scopically visible cell morphology.
pound 9 a two-proton singlet (d 3.40) was found to-
gether with two four-proton multiplets of H-10 and H-20
and four-proton doublet of P-CH2 (see Experimental).
In 13C NMR spectrum (APT), except for signals
assigned to carbons of the pyrimidine base and a side
chain, also a signal of the methylene group was observed
(d 15.28). These results together with the mass spectrum
of derivative 9 are in good agreement with suggested
structure. Formation of the dimer during the reduction
is caused probably by the reaction of 5-unsubstituted
side product with 5-hydroxymethyl derivative 7. Similar
type of dimers linked by methylene group has been
observed previously.10
Our attempts to modify the cyano group at the 5-posi-
tion by hydrolysis failed. Acidic hydrolysis of 3 in re-
fluxing aqueous H2SO4 gave a very complex reaction
mixture. At room temperature and lower acid concen-
tration only starting compound was isolated. Under
basic conditions the side chain attached via an ether
bond at the 6-position was cleaved. When sodium
methoxide in methanol was used, only 2,4-diamino-5-
cyano-6-methoxypyrimidine (10) was isolated. Hydro-
lysis in aqueous NaOH afforded compound 11 (Scheme 3).
3. Experimental
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 Buchi
melting point apparatus. NMR spectra were measured
on an FT NMR spectrometer Varian UNITY 500 (1H at
500 MHz and 13C at 125.7 MHz 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). Dimethylformamide and acetonitrile were dis-
2.1. Biological activity
The 5-cyano and 5-formyl 2,4-diamino-6-[2-(phosphono-
methoxy)ethoxy]pyrimidines (4 and 6) and the bis-
phosphonic acid 9 were investigated for their inhibitory
ꢁ
tilled from P2O5 and stored over molecular sieves (4 A).
NH2
NH2
CN
NaOMe/MeOH
CN
N
N
3.1. 2,4-Diamino-5-cyano-6-[2-(diisopropoxyphosphoryl-
methoxy)ethoxy]pyrimidine (3)
H2N
N
O
H2N
N
10
OMe
3
O
P
OiPr
OiPr
O
Starting 2,4-diamino-5-cyano-6-(2-hydroxyethoxy)pyr-
imidine (2) was prepared in two steps from tetracyano-
ethylene according to the literature, yield 73%.8;9 For
C7H9N5O2 (195.2) calcd: C, 43.08; H, 4.65; N, 35.88.
Found: C, 42.84; H, 4.71; N, 35.55. FABMS: 196 [MHþ]
(20). 1H NMR (DMSO-d6): 6.94 (br s, 2H) and 6.88 (br
s, 2H) (NH2); 4.83 (t, 1H, J(OH,20) ¼ 5.2, OH); 4.28 (t,
2H, J(10,20) ¼ 5.4, H-10); 3.65 (q, 2H, J(10,20) ¼
NH2
CONH2
OH
N
aq. NaOH
H2N
N
11
Scheme 3.

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D. Hockovꢀa et al. / Bioorg. Med. Chem. 12 (2004) 3197–3202
J(OH,20) ¼ 5.3, H-20). 13C NMR: 171.14 (C-6); 165.99
(C-4); 163.06 (C-2); 116.27 (CN); 67.86 (C-5); 63.09 (C-
10); 59.37 (C-20). To the suspension of this compound
(2.4 g, 12 mmol) in triethylamine (8 mL) (diisopropoxy-
phosphoryl)methyl tosylate (4.8 g, 13.7 mmol) was ad-
ded followed by dimethylformamide (20 mL) and NaH
(60% disp. in mineral oil, 1.3 g). The reaction mixture
was stirred at room temperature for 1 h, evaporated in
vacuo and codistilled successively with toluene and
ethanol. The residue was chromatographed on silica gel.
The fractions eluted with 4% MeOH in CHCl3 gave 2 g
(45%) of the product, that was without further purifi-
cation used in the next step. FABMS: 374 [MHþ] (10),
pad of Celite and the catalyst was washed with hot water
and hot methanol (100 mL each). The filtrate was neu-
tralized with aqueous NaOH and solvents evaporated.
The residue was mixed with hot methanol, salts were
filtered off, and the mixture was taken down. Product
was purified by column chromatography on silica gel
(elution with 3% MeOH in CHCl3) to give 0.63 g (42%)
of the desired product (also 0.11 g (4%) of compound 8
was isolated as a side product). FABMS: 377 (MHþ)
(10). 1H NMR (DMSO-d6): 9.82 (s, 1H, CHO); 8.30 (br
d, 1H), 7.44 (br d, 1H, J ¼ 4.0), and 7.09 (br s, 2H)
(NH2); 4.58 (m, 2H, P-OCH); 4.43 (m, 2H, H-10); 3.83
(m, 2H, H-20); 3.81 (d, 2H, J(P,CH) ¼ 8.3, P-CH2); 1.22
(d, 6H) and 1.21 (d, 6H, J ¼ 6.2) (CH3). 13C NMR:
184.77 (CHO); 171.50 (C-6); 164.62 (C-4); 163.91 (C-2);
91.84 (C-5); 70.69 (d, J(P,C) ¼ 12.2, C-20); 70.32
(d, J(P,C) ¼ 6.3, P-OC); 65.04 (d, J(P,C) ¼ 164.6, P-C);
64.83 (C-10); 23.95 (d, 2C, J(P,C) ¼ 3.4) and 23.82
(d, 2C, J(P,C) ¼ 4.4) (CH3).
1
152 [baseHþ] (100). H NMR (DMSO-d6): 6.95 (br s,
2H) and 6.90 (br s, 2H) (NH2); 4.59 (m, 2H, P-OCH);
4.40 (m, 2H, H-10); 3.81 (d, 2H, J(P,CH) ¼ 8.3, P-CH2);
3.80 (m, 2H, H-20); 1.24 (d, 6H) and 1.23 (d, 6H) (CH3).
13C NMR: 170.86 (C-6); 165.95 (C-4); 162.99 (C-2);
116.05 (CN); 70.61 (d, J(P,C) ¼ 11.7, C-20); 70.41 (d,
J(P,C) ¼ 6.3, P-OC); 65.15 (C-5); 65.12 (d, J(P,C) ¼
164.1, P-C); 63.03 (C-10); 24.01 (d, 2C, J(P,C) ¼ 3.9) and
23.88 (d, 2C, J(P,C) ¼ 4.4) (CH3).
3.4. 2,4-Diamino-5-formyl-6-[2-(phosphonomethoxy)-
ethoxy]pyrimidine (6)
3.2. 2,4-Diamino-5-cyano-6-[2-(phosphonomethoxy)-
ethoxy]pyrimidine (4)
5-Formyl-2,4-diamino-6-[2-(diisopropoxyphosphorylmeth-
oxy)ethoxy]pyrimidine (5, 0.36 g, 0.95 mmol), aceto-
nitrile (10 mL), and BrSiMe3 (1 mL) was stirred for 2.5 h
at room temperature. After evaporation in vacuo and
codistillation with acetonitrile, the residue was treated
with water and triethylamine was added to alkaline
reaction. The mixture was evaporated to dryness and the
residue was applied onto column of Dowex 1 · 2 (ace-
tate, 25 mL) column, which was then washed with water
followed by gradient of acetic acid (0–2 M, 1 L each).
The main UV absorbing fraction was evaporated, the
residue was three-times codistilled with water and crys-
tallized from water–methanol to afford the product as a
white solid: (230 mg, 82%), mp 225–227 ꢁC. For
C8H13N4O6PÆ5/4H2O (314.7) calcd: C, 30.53; H, 4.96; N,
17.80. Found: C, 30.87; H, 5.01; N, 17.65. FABMS: 293
(MHþ) (10). 1H NMR (D2O+NaOD): 9.73 (s, 1H,
CH@O); 4.46 (m, 2H, H-10); 3.95 (m, 2H, H-20); 3.59 (d,
2H, J(P,CH) ¼ 8.4, P-CH2). 13C NMR: 187.89 (CH@O);
172.46 (C-6); 164.285 (C-4); 163.85 (C-2); 92.44 (C-5);
70.25 (d, J(P,C) ¼ 9.3, C-20); 69.18 (d, J(P,C) ¼ 148.4, P-
C); 66.11 (C-10).
2,4-Diamino-5-cyano-6-[2-(diisopropoxyphosphorylmeth-
oxy)ethoxy]pyrimidine (3, 0.19 g, 0.5 mmol), acetonitrile
(10 mL), and BrSiMe3 (1 mL) was stirred overnight at
room temperature. After evaporation in vacuo and
codistillation with acetonitrile, the residue was treated
with water and concd aqueous ammonia was added to
alkaline reaction. The mixture was evaporated to dry-
ness and the residue was applied onto column of Dowex
50 · 8 (Hþ-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 minimum volume of water was applied on Dowex
1 · 2 (acetate, 25 mL) column, which was then washed
with water followed by gradient of acetic acid (0–3 M).
The main UV absorbing fraction was evaporated,
the residue was three-times codistilled with water
and crystallized from water–ethanol to afford product as
a white solid: (120 mg, 86%), mp 294–295 ꢁC. For
C8H12N5O5PÆH2O (307.2) calcd: C, 31.28; H, 4.59; N,
22.80; P, 10.08. Found: C, 31.27; H, 4.67; N, 22.54;
P, 9.94. FABMS: 290 (MHþ) (10). 1H NMR
(D2O+NaOD): 4.48 (m, 2H, H-10); 3.95 (m, 2H, H-20);
3.74 (d, 2H, J(P,CH) ¼ 8.4, P-CH2). 13C NMR: 171.87
(C-6); 166.15 (C-4); 163.30 (C-2); 116.85 (CN); 70.88 (d,
J(P,C) ¼ 10.7, C-20); 67.69 (d, J(P,C) ¼ 156.2, P-C);
66.72 (C-5); 64.52 (C-10).
3.5. 2,4-Diamino-5-hydroxymethyl-6-[2-(diisopropoxy-
phosphorylmethoxy)ethoxy]pyrimidine (7)
To the solution of compound 5 (0.27 g, 0.7 mmol) in
methanol (5 mL) NaBH4 (32 mg, 0.9 mmol) was added
in portions. The reaction mixture was stirred at room
temperature for 30 min, applied on short column of
silica gel and washed with 40% MeOH in CHCl3. Sol-
vents were evaporated to obtain crude product as a
sticky colorless foam (0.24, 89%). The product appeared
to be rather unstable (in further attempts to purify
compound 7 dimer 8 was isolated). FABMS: 379 [MHþ]
(15). 1H NMR (DMSO-d6): 5.88 (br s, 2H) and 5.85 (br
s, 2H) (NH2); 4.59 (m, 2H, P-OCH); 4.45 (br s, 1H,
OH); 4.27 (br s, 2H, CH2OH); 4.26 (m, 2H, H-10); 3.80
3.3. 2,4-Diamino-5-formyl-6-[2-(diisopropoxyphosphoryl-
methoxy)ethoxy]pyrimidine (5)
5-Cyano-2,4-diamino-6-[2-(diisopropoxyphosphorylmeth-
oxy)ethoxy]pyrimidine (3, 1.5 g, 4 mmol) was hydro-
genated in water (36 mL) and sulfuric acid (6 mL) over
5% palladium on charcoal (0.25 g) under stirring for 20 h
at room temperature. The mixture was filtered through a

D. Hockovꢀa et al. / Bioorg. Med. Chem. 12 (2004) 3197–3202
3201
(d, 2H, J(P,CH) ¼ 8.3, P-CH2); 3.77 (m, 2H, H-20); 1.24
(d, 6H) and 1.23 (d, 6H, J ¼ 6.2) (CH3). 13C NMR:
166.55 (C-6); 164.80 (C-4); 161.59 (C-2); 89.13 (C-5);
71.18 (d, J(P,C) ¼ 11.7, C-20); 70.35 (d, J(P,C) ¼ 5.8, P-
OC); 65.15 (d, J(P,C) ¼ 164.1, P-C); 64.11 (C-10); 52.88
(CH2OH); 24.00 (d, 2C, J(P,C) ¼ 3.4) and 23.87 (d, 2C,
J(P,C) ¼ 4.4) (CH3).
(br s, 4H, NH2); 3.83 (s, 3H, OCH3). 13C NMR: 171.42
(C-6); 163.26 (C-2); 165.86 (C-4); 116.24 (CN); 62.89
(C-5); 53.76 (OCH3).
3.9. 2,4-Diamino-6-hydroxypyrimidine-5-carboxamide
(11)
Compound 3 (0.9 g, 2.4 mmol) was added to the aqueous
10% solution of NaOH (20 mL) and reaction mixture
was refluxed for 3 h. Acetic acid was added to adjust pH
to 6–7. After cooling to rt, white solid was filtered off
and washed with methanol. This product was identified
as compound 11 (0.30 g, 74%). FABMS: 152
(MHþ)H2O) (100), 170 (MHþ) (10). For C5H7N5O2
(169.1) calcd: C, 35.51; H, 4.17; N, 41.41. Found: C,
3.6. 5,50-Methylenebis{2,4-diamino-6-[2-(diisopropoxy-
phosphorylmethoxy)ethoxy]pyrimidine} (8)
The same reaction procedure as described for compound
7 was applied, but the reaction time was longer (3 h), a
small amount of water was added and the reaction
mixture was evaporated to dryness. Preparative TLC
followed by preparative HPLC afforded compound 8 as
a white solid (90 mg, 36%). FABMS: 709.5 [MHþ] (100).
1H NMR (DMSO-d6): 5.97 (br s, 4H) and 5.81 (br s, 4H)
(NH2); 4.33 (m, 4H, H-10); 3.83 (m, 4H, H-20); 3.82
(d, 4H, J(P,CH) ¼ 8.6, P-CH2); 4.62 (m, 4H, P-OCH);
3.42 (s, 2H, CH2); 1.25 (d, 12H) and 1.24 (d, 12H,
J ¼ 6.2) (CH3).
1
35.81; H, 4.00; N, 41.79. H NMR (DMSO-d6): 10.46
(br s, 1H, OH), 7.05 (br s, 2H), and 6.86 (br, 2H) (NH2).
13C NMR (D2O+NaOD): 175.66 (CO); 166.74 (C-4);
163.87 (C-2); 161.59 (C-6); 120.07 (C-5).
3.10. Antiviral activity assays
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.
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, . . ., lg/mL) of the test compounds. Viral
cytopathicity was recorded as soon as it reached com-
pletion in the control virus-infected cell cultures that
were not treated with the test compounds.
3.7. 5,50-Methylenebis{2,4-diamino-6-[2-(phosphonomethoxy)-
ethoxy]pyrimidine} (9)
Compound 8 (0.28 g, 0.4 mmol), acetonitrile (10 mL),
and BrSiMe3 (1 mL) were stirred at room temperature
for 20 h. After evaporation in vacuo and codistillation
with acetonitrile, the residue was treated with water and
triethylamine was added to alkaline reaction. The mix-
ture was evaporated to dryness and the residue was
applied onto column of Dowex 1 · 2 (acetate, 25 mL)
column, which was then washed with water followed by
gradient of acetic acid (0–2 M, 1 L each) and hot formic
acid (2 M, the product was very unsoluble). Combined
fractions containing product were evaporated, the resi-
due was three-times codistilled with water and crystal-
lized from water–methanol to afford product as a white
solid: (110 mg, 51%). FABMS: 541 (MHþ) (40). For
C15H26N8O10P2 (540) calcd: C, 33.34; H, 4.85; N, 20.74.
Found: C, 33.48; H, 4.98; N, 20.54. 1H NMR
(D2O+NaOD): 4.45 (m, 4H, H-10); 3.95 (m, 4H, H-20);
3.59 (d, 4H, J(P,CH) ¼ 8.6, P-CH2); 3.40 (s, 2H, CH2).
13C NMR: 166.52 (C-6); 164.09 (C-4); 160.65 (C-2);
88.25 (C-5); 70.52 (d, J(P,C) ¼ 10.7, C-20); 69.14 (d,
J(P,C) ¼ 150.4, P-C); 65.53 (C-10); 15.28 (CH2).
3.11. Inhibition of HIV-1-induced cytopathicity in CEM
cells
The methodology of the anti-HIV assays has been
described
previously.12
Briefly,
human
CEM
(ꢀ3 · 105 cells/mL) cells were infected with 100 CCID50
HIV-1 ((IIIB) or HIV-2 (ROD) per mL and seeded in
200 lL-wells of 96-well microtiter plates, containing
appropriate dilutions of the test compounds. After four
days of incubation at 37 ꢁC, CEM giant cell formation
was examined microscopically.
3.8. 2,4-Diamino-5-cyano-6-methoxypyrimidine (10)
To the solution of compound 3 (0.3 g, 0.8 mmol) in
methanol (8 mL) sodium methoxide (1 mL, 1 M solution
in methanol) was added. The reaction mixture was
stirred at room temperature for 20 h but only starting
material was detected by TLC. Then the solution was
refluxed for 45 h and a white solid was filtered off after
cooling. This product was identified as compound 10
(0.1 g, 77%). FABMS: 166 (MHþ) (100). For C6H7N5O
(165.2) calcd: C, 43.64; H, 4.27; N, 42.41. Found:
3.12. Inhibition of MSV-induced transformation of
murine C3H/3T3 embryo fibroblasts
The anti-MSV assay was performed as described previ-
ously.12 Murine C3H/3T3 embryo fibroblast cells were
seeded at 5 · 105 cells/mL into 1-cm2 wells of 48-well
microplates. Twenty four hours 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
1
C, 43.44; H, 4.26; N, 42.08. H NMR (DMSO-d6): 6.96

3202
D. Hockovꢀa et al. / Bioorg. Med. Chem. 12 (2004) 3197–3202
described previously)13 for 90–120 min at 37 ꢁC. The
medium was then replaced by 1 mL of fresh medium
containing various concentrations of the test com-
pounds. After six days, transformation of the cell cul-
ture was examined microscopically.
4. For review, see: (a) De Clercq, E. Collect. Czech. Chem.
Commun. 1998, 63, 480; (b) Safrin, S.; Cherrington, J.;
Jaffe, H. S. Adv. Exp. Med. Biol. 1999, 458, 111; (c) De
Clercq, E.; Andrei, G.; Balzarini, J.; Hatse, S.; Liekens, S.;
Naesens, L.; Neyts, J.; Snoeck, R. Nucleos. Nucleot. 1999,
18, 759; (d) Snoeck, R.; Noel, J. C.; Muller, C.; De Clercq,
E.; Bossens, M. J. Med. Virol. 2000, 60, 205.
ꢀ
ꢂ
5. (a) Balzarini, J.; Holy, A.; Jindrich, J.; Naesens, L.;
Snoeck, R.; Schols, D.; De Clercq, E. Antimicrob. Agents
Chemother. 1993, 37, 332; (b) Balzarini, J.; Aquaro, S.;
Acknowledgements
ꢀ
Perno, C. F.; Witvrouw, M.; Holy, A.; De Clercq, E.
Biochem. Biophys. Res. Commun. 1996, 219, 337;
This study was performed as a part of research project #
Z4055905 of the Institute of Organic Chemistry and
Biochemistry. It was supported by the Programme of
Targeted Projects of the Academy of Sciences of the
Czech Republic (# S4055109), by Gilead Sciences
(Foster City, CA, USA), by ISEP/FORTIS, the ‘Ge-
concerteerde Onderzoeksacties’ (GOA-00/12), and the
ꢀ
(c) Naesens, L.; Neyts, J.; Balzarini, J.; Holy, A.;
Rosenberg, I.; De Clercq, E. J. Med. Virol. 1993, 39,
167; (d) Kreider, J. W.; Balogh, K.; Olson, R. O.; Martin,
J. C. Antiviral Res. 1990, 14, 51; (e) Yu, K. L.; Bronson, J.
J.; Yang, H.; Patick, A.; Alam, M.; Brankovan, V.;
Datema, R.; Hitchcock, M. J.; Martin, J. C. J. Med.
ꢀ
Chem. 1992, 35, 2958; (f) Otova, B.; Zidek, Z.; Holy, A.;
Votruba, I.; Sladka, M.; Marinov, I.; Leskova, V. In Vivo
ꢀ
ꢀ
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.
ꢀ
ꢀ
1997, 11, 163; (g) Otova, B.; Francova, K.; Franek, F.;
Koutnık, P.; Votruba, I.; Holy, A.; Sladka, M.; Schram-
ꢂ
ꢀ
ꢀ
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lova, J. Anticancer Res. 1999, 19, 3173; (h) Rose, W. C.;
Crosswell, A. R.; Bronson, J. J.; Martin, J. C. J. Natl.
Cancer Inst. 1990, 82, 510.
ꢀ
ꢀ
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6. (a) Holy, A.; Votruba, I.; Masojıdkova, M.; Andrei, G.;
Snoeck, R.; Naesens, L.; De Clercq, E.; Balzarini, J. J.
Med. Chem. 2002, 45, 1918; (b) Balzarini, J.; Pannecou-
que, C.; De Clercq, E.; Aquaro, S.; Perno, C. F.; Egberink,
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