6-[2-(Phosphonomethoxy)alkoxy]pyrimidines with antiviral activity

Article abstract of DOI:10.1021/jm011095y

6-Hydroxypyrimidines substituted at positions 2 and 4 by hydrogen, methyl, amino, cyclopropylamino, dimethylamino, methylsulfanyl, or hydroxyl group afford by the reaction with diisopropyl 2-(chloroethoxy)methylphosphonate in the presence of NaH, CS₂CO₃, or DBU a mixture of N¹- and O⁶-[2-(diisopropylphosphorylmethoxy)ethyl] isomers which were converted to the free phosphonic acids by treatment with bromotrimethylsilane followed by hydrolysis. Analogously, 2,4-diamino-6-hydroxypyrimidine gave on reaction with [(R)- and (S)-2-(diisopropylphosphorylmethoxy)propyl] tosylate, followed by deprotection, the enantiomeric 6-[2-(phosphonomethoxy)propoxy]pyrimidines. 2,4-Diamino-6-sulfanylpyrimidine gave, on treatment with diisopropyl 2-(chloroethoxy)methylphosphonate in the presence of NaH and subsequent deprotection, 2,4-diamino-6-{[2-(phosphonomethoxy)ethyl]sulfanyl}pyrimidine. 2-Amino-4-hy-droxy-6-[2-(phosphonomethoxy)ethyl]pyrimidine was obtained from the appropriate 2-amino-4-chloropyrimidine derivative by alkaline hydrolysis and ester cleavage. Direct alkylation of 2-amino-4,6-dihydroxypyrimidine afforded a mixture of 2-amino-4,6-bis[2-(phosphonomethoxy)-ethyl]- and 2-amino-1,4-bis[2-(phosphonomethoxy)ethyl]pyrimidine. None of the N¹-[2-(phosphonomethoxy)ethyl] isomers exhibited any antiviral activity against DNA viruses or RNA viruses tested in vitro. On the contrary, the O⁶-isomers, namely the compounds derived from 2,4-diamino-, 2-amino-4-hydroxy-, or 2-amino-4-[2-(phosphonomethoxy)ethoxy]l-6-hydroxypyrimidine, inhibited the replication of herpes viruses [herpes simplex type 1 (HSV-1) and type 2 (HSV-2), varicella-zoster virus (VZV), and cytomegalovirus (CMV)] and retroviruses [Moloney sarcoma virus (MSV) and human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2)], their activity being most pronounced against the latter. The antiviral activity was lower if the oxygen at the position 6 was replaced by a sulfur atom, as in 2,4-diamino-6-[2-(phosphonomethoxy)ethylsulfanyl]pyrimidine. In analogy to N⁹-[2-(phosphonomethoxy)propyl]-2,6-diaminopurine (PMPDAP), solely the (R)-2,4-diamino-6-[2-(phosphonomethoxy)propoxy]-pyrimidine exerted antiviral activity, whereas its (S)-enantiomer was essentially inactive.

Full text of DOI:10.1021/jm011095y

1
918
J . Med. Chem. 2002, 45, 1918-1929
6
-[2-(P h osp h on om eth oxy)a lk oxy]p yr im id in es w ith An tivir a l Activity
,
†
†
†
‡
‡
‡
Anton ´ı n Hol y´ ,* Ivan Votruba, Milena Masoj ´ı dkov a´ , Graciela Andrei, Robert Snoeck, Lieve Naesens,
‡
‡
Erik De Clercq, and J an Balzarini
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo n a´ m. 2,
CZ-166 10 Praha 6, Czech Republic, and Rega Institute for Medical Research, Katholieke Universiteit Leuven,
Minderbroedersstraat 10, B-3000 Leuven, Belgium
Received November 7, 2001
6
-Hydroxypyrimidines substituted at positions 2 and 4 by hydrogen, methyl, amino, cycloprop-
ylamino, dimethylamino, methylsulfanyl, or hydroxyl group afford by the reaction with
diisopropyl 2-(chloroethoxy)methylphosphonate in the presence of NaH, Cs CO , or DBU a
2
3
1
6
mixture of N - and O -[2-(diisopropylphosphorylmethoxy)ethyl] isomers which were converted
to the free phosphonic acids by treatment with bromotrimethylsilane followed by hydrolysis.
Analogously, 2,4-diamino-6-hydroxypyrimidine gave on reaction with [(R)- and (S)-2-(diisoprop-
ylphosphorylmethoxy)propyl] tosylate, followed by deprotection, the enantiomeric 6-[2-(phospho-
nomethoxy)propoxy]pyrimidines. 2,4-Diamino-6-sulfanylpyrimidine gave, on treatment with
diisopropyl 2-(chloroethoxy)methylphosphonate in the presence of NaH and subsequent
deprotection, 2,4-diamino-6-{[2-(phosphonomethoxy)ethyl]sulfanyl}pyrimidine. 2-Amino-4-hy-
droxy-6-[2-(phosphonomethoxy)ethyl]pyrimidine was obtained from the appropriate 2-amino-
4
2
-chloropyrimidine derivative by alkaline hydrolysis and ester cleavage. Direct alkylation of
-amino-4,6-dihydroxypyrimidine afforded a mixture of 2-amino-4,6-bis[2-(phosphonomethoxy)-
1
ethyl]- and 2-amino-1,4-bis[2-(phosphonomethoxy)ethyl]pyrimidine. None of the N -[2-(phospho-
nomethoxy)ethyl] isomers exhibited any antiviral activity against DNA viruses or RNA viruses
6
tested in vitro. On the contrary, the O -isomers, namely the compounds derived from
2
,4-diamino-, 2-amino-4-hydroxy-, or 2-amino-4-[2-(phosphonomethoxy)ethoxy]-6-hydroxy-
pyrimidine, inhibited the replication of herpes viruses [herpes simplex type 1 (HSV-1) and
type 2 (HSV-2), varicella-zoster virus (VZV), and cytomegalovirus (CMV)] and retroviruses
[
(
Moloney sarcoma virus (MSV) and human immunodeficiency virus type 1 (HIV-1) and type 2
HIV-2)], their activity being most pronounced against the latter. The antiviral activity was
lower if the oxygen at the position 6 was replaced by a sulfur atom, as in 2,4-diamino-6-[2-
9
(
2
phosphonomethoxy)ethylsulfanyl]pyrimidine. In analogy to N -[2-(phosphonomethoxy)propyl]-
,6-diaminopurine (PMPDAP), solely the (R)-2,4-diamino-6-[2-(phosphonomethoxy)propoxy]-
pyrimidine exerted antiviral activity, whereas its (S)-enantiomer was essentially inactive.
In tr od u ction
-[2-(Phosphonomethoxy)alkyl]purines are attractive
also selective antitumor properties.9 In addition to
adenine and 2,6-diaminopurine derivatives, their gua-
nine counterparts PMEG (5) and (R)-PMPG (6) are
9
1
for their antiviral and cytostatic activity. Among them,
particularly 9-[2-(phosphonomethoxy)ethyl]adenine
PMEA, adefovir, 1, Chart 1) is active against DNA
1
0
potent antivirals and exhibit powerful antitumor
1
1
activity. However, their potential use is substantially
(
1
2
2
limited by their narrow safety margin. The acyclic
nucleoside phosphonates of the PME or PMP-type
derived from all the above purine bases induce
NO production in macrophages, stimulate in vitro
viruses and retroviruses; its bis(pivaloyloxymethyl)
ester (adefovir dipivoxil)3 is in the ultimate clinical
4
phase for use in hepatitis B therapy. Adefovir has
pronounced cytostatic activity. Another related adenine
5
1
3
secretion of cytokines, and display antiarthritic activ-
derivative, 9-(R)-[2-(phosphonomethoxy)propyl]adenine
1
4
ity.
(PMPA, tenofovir, 2), is a promising anti-HIV drug: its
prodrug Viread has been approved for treatment of
AIDS. Also the 2,6-diaminopurine congeners of these
two drugs demonstrated interesting biological proper-
ties: the (R)-9-[2-(phosphonomethoxy)propyl] derivative
Thorough SAR studies in the series of acyclic nucleo-
6
15
side phosphonates led to the conclusion that, except
1
6
for an N∼C interchange at positions 3 and 8, any
substitution or other alteration of the purine base at
the position 2 or 8 results in the loss of antiviral or
(
R)-PMPDAP (4) belongs among the most active com-
7
15
pounds against retroviruses in vitro and the corre-
cytostatic activity. The influence of substitution of the
sponding PME derivative PMEDAP (3) exhibits, in
addition to inhibiting DNA viruses and retroviruses,
amino group at position 6 (in adenine or 2,6-diamino-
purine) is different. Generally, this group can be re-
placed by numerous mono- or dialkyl, cycloalkyl, alk-
8
*
Corresponding author: Dr. Anton ´ı n Hol y´ , DSc., Institute of
17
enyl, or alkynyl functions while preserving antiviral
Organic Chemistry and Biochemistry, Academy of Sciences of the
Czech Republic, Flemingovo n a´ m. 2, CZ-166 10 Praha 6, Czech
Republic. Phone: (4202)-20183333. Fax: (4202)-24310090. E-mail:
holy@uochb.cas.cz.
and/or cytostatic activity, particularly in the PMEDAP
18
series. Presumably, at least in some cases, this activity
†
may be due to the intracellular deamination resulting
Academy of Sciences of the Czech Republic.
Katholieke Universiteit Leuven.
‡
19
in guanine derivatives.
1
0.1021/jm011095y CCC: $22.00 © 2002 American Chemical Society
Published on Web 03/27/2002

6
-[2-(Phosphonomethoxy)alkoxy]pyrimidines
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9 1919
Ch a r t 1
Sch em e 1a
a
(i) Ra-Ni/EtOH, reflux; (ii) 10/Cs2CO3/DMF, 100 °C; (iii) (a)
BrSiMe3/acetonitrile; (b) H2.
treatment of the appropriate 6-hydroxypyrimidine with
diisopropyl 2-(chloroethoxy)methylphosphonate (10) in
the presence of a suitable base [NaH, Cs2CO3, or DBU
The pharmacophore of purine acyclic nucleoside phos-
phonates is characterized by the presence of amino
groups at the pyrimidine part of the purine system.
Replacement of these groups by aminomethyl or ami-
dino functions, which are more strongly basic compared
to their amino counterparts and cannot participate in
the heteroaromatic π-electron system, resulted in com-
pletely inactive products both in the PME- and (R)-PMP-
(1,8-diazabicyclo[5.4.0]undec-7-ene)] in dimethylforma-
mide. The reaction course is illustrated by the reaction
of compound 10 with the parent compound of the series,
4
-amino-6-hydroxypyrimidine (12). The starting mate-
rial was obtained by desulfurization of 4-amino-6-
hydroxy-2-sulfanylpyrimidine (11). Treatment with the
synthon 10 in the presence of Cs2CO3 afforded a mixture
2
0
series.
We have also investigated acyclic nucleoside phos-
phonates derived from pyrimidines bearing amino group-
s). Only the cytosine derivative of the structurally
1
of two isomers: N -[2-(diisopropylphosphorylmethoxy)-
ethyl]-4-aminopyrimidin-6-one (13a ) and 4-amino-6-[2-
(
(diisopropylphosphorylmethoxy)ethoxy]pyrimidine (14a).
related HPMPs[3-hydroxy-2-(phosphonomethoxy)prop-
yl]sseries [(S)-HPMPC, cidofovir, Vistide, 7] showed
antiviral21 and antitumor activity, while its 1-[2-
The isomeric diesters gave, on treatment with bromo-
trimethylsilane followed by hydrolysis, the free phos-
phonic acids 13b and 14b, respectively (Scheme 1).
Their structure was unequivocally assigned on the basis
22
1
5
(
(
phosphonomethoxy)ethyl] (PMEC, 8) or (R)-1-[2-
phosphonomethoxy)propyl]23 congeners were essen-
13
of C NMR spectra. Similarly, 2,4-diamino-6-hydroxy-
1
tially inactive in both aspects. N -[2-(phosphono-
1
pyrimidine (15) gave N -[2-(diisopropylphosphoryl-
methoxy)ethyl] derivatives of 2-aminopyrimidine, 2,4-
methoxy)ethyl]-2,4-diaminopyrimidin-6-one (16a ) and
2
4
diaminopyrimidine (9), and their regioisomers were
devoid of antiviral or cytostatic activity. However, the
critical feature of these compounds which distinguishes
them from the above compounds might be their qua-
ternary character. In an attempt to overcome this
drawback, we synthesized and investigated the biologi-
cal activity of the structurally related nonquaternary
acyclic nucleoside phosphonates derived from pyrimi-
dine bases bearing an hydroxyl group at position 6.
2
,4-diamino-6-[2-(diisopropylphosphorylmethoxy)ethoxy]-
pyrimidine (17a ) and the free phosphonic acids 16b and
1
6
7b. In the same reaction sequence, 2-amino-4-hydroxy-
-methylpyrimidine (18) was transformed to the iso-
meric diesters 19a and 20a and free phosphonic acids
9b and 20b, respectively (Scheme 2).
With regard to the high biological activity of N -
1
6
substituted 2,6-diaminopurine derivatives (vide supra),
we have also examined the PME derivatives of 2-amino-
4
-cyclopropylamino-6-hydroxypyrimidine (22) and 2-
Ch em istr y
amino-4-(dimethylamino)-6-hydroxypyrimidine (25).
Starting pyrimidines were prepared by treatment of
2-amino-4-chloro-6-hydroxypyrimidine (21) with cyclo-
propylamine and dimethylamine, respectively. It should
The major effort in this study focused on the synthesis
of pyrimidine N -[2-(phosphonomethoxy)ethyl] (PME)
derivatives. These syntheses were accomplished by
1

1
920 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9
Hol y´ et al.
Sch em e 2
Sch em e 4a
Sch em e 3a
a
1
0/ base/DMF 100 °C; (ii) K2CO3/DABCO/H2O; (iii) (a) BrSiMe3,
(b) H2O.
Sch em e 5
a
i
In formulae 23, 24, 26, and 27, (a) R ) Pr, (b) R ) H.
be noted that this reaction should not be performed in
DMF: heating of compound 21 with cyclopropylamine
in DMF in an autoclave gave the dimethylamino deriva-
tive 25 as the sole product, evidently due to the reaction
with the solvent. Reaction of pyrimidines 22 and 25 with
the synthon 10 under standard conditions gave diesters
of N -isomers 23a and 26a and O -isomers 24a and 27a .
Their deprotection gave N-phosphonates 23b and 26b
and O-phosphonates 24b and 27b, respectively (Scheme
alkylation of sulfanylpyrimidines with the synthon 10
afforded the S-alkyl derivatives only. Thus, 2,4-diamino-
6-sulfanylpyrimidine (34) gave diester 35a and there-
from the free phosphonate 35b. The preference of
S-alkylation over the formation of N/O-isomers is clearly
manifested by the reaction of 4-amino-6-hydroxy-2-
sulfanylpyrimidine (11) with synthon 10 in the presence
of 1 equiv of sodium hydride. The 2-[2-(diisopropylphos-
phorylmethoxy)ethyl]sulfanyl derivative 36a , formed as
the only reaction product, gave by ester cleavage the
free phosphonate 36b (Scheme 6).
1
6
3
).
2
-Amino-4-chloro-6-hydroxypyrimidine (21) also gave,
with the synthon 10 in the presence of a base, a mixture
1
6
of diesters: N -isomer 30 and O -isomer 28. The latter
compound was transformed by alkaline hydrolysis in the
2
5
presence of DABCO (1,4-diazabicyclo[2.2.2]octane) to
-amino-4-hydroxy-6-[2-(diisopropylphosphorylmeth-
The alkylation of the 6-hydroxypyrimidines is not
limited to the ω-chloroalkyl derivatives (e.g., 10). Reac-
tions of 2,4-diamino-6-hydroxypyrimidine (15) with [(R)-
or (S)-2-(diisopropylphosphoryl)methoxy]propyl tosylate
[(R)-37, (S)-37] in the presence of cesium carbonate
gave the enantiomeric 2,4-diamino-6-[2-(diisopropyl-
phosphorylmethoxy)prop-1-oxy]pyrimidines [(R)-38a ,
(S)-38a ] and the corresponding free phosphonates (ana-
logues of PMP-compounds) [(R)-38b, (S)-38b] (Scheme
2
oxy)ethoxy]pyrimidine (29a ), which gave, after ester
cleavage, the free phosphonate 29b (Scheme 4). Com-
pound 29a is not formed by direct alkylation of 2-amino-
4
,6-dihydroxypyrimidine (31) with the synthon 10.
Disregarding the character of the base used, this reac-
tion gave isomeric disubstituted products only: 2-
amino-4,6-bis[2-(diisopropylphosphorylmethoxy)ethoxy]-
pyrimidine (32a ) and 2-amino-1-[2-(diisopropylphos-
phorylmethoxy)ethyl]-6-[2-(diisopropylphosphorylmeth-
oxy)ethoxy]pyrimidine (33a ). Bromotrimethylsilane-
mediated cleavage of ester groups afforded the free
phosphonic acids 32b and 33b (Scheme 5).
1
7). The formation of N -isomers was in these cases
nearly nondetectable.
Another unequivocal reaction alternative which leads
6
exclusively to the O -isomers makes use of the reactivity
of the 6-chlorine atom at the pyrimidine ring (Scheme
8): reaction of 4-amino-6-chloro-2-methylsulfanylpyri-
midine (39) with sodium alkoxide of diethyl 2-(hydroxy-
ethoxy)methylphosphonate 40 generated in situ gave
compound 41a as the only product. Its deprotection
In contrast to 6-hydroxypyrimidines which give, due
to -NdC-(OH)- T -NH-C(dO)- tautomerism, by
alkylation with the synthon 10 in the presence of a
1
6
strong base always a mixture of N - and O -isomers, the

6
-[2-(Phosphonomethoxy)alkoxy]pyrimidines
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9 1921
Sch em e 6a
pyrimidine ring [i.e., 17b and 29b] emerged as the most
active compounds. They were inhibitory to herpes
simplex virus type 1 (HSV-1), HSV-2, and the thymidine
-
kinase (TK)-deficient TK /HSV-1 strain at EC50 values
ranking between 6.5 and 24 µg/mL (Table 1). The
compounds were even more potent against two wild-
type VZV and two TK-deficient VZV strains (EC50: 0.6-
2
.5 µg/mL), but they were not active against cyto-
megalovirus (CMV) at subtoxic concentrations (data not
shown). Compound 17b was not active against adeno-
virus type 2 and type 3 infections in HEL cells.
Both 17b and 29b were exquisitely inhibitory to
Moloney murine sarcoma virus (MSV) in C3H/3T3 cell
cultures (EC50: 0.04-0.08 µg/mL). Compound 17b was
also very effective (EC50: 0.4-0.8 µg/mL) against HIV-1
and HIV-2 in CEM cell cultures. In contrast, 29b was
not inhibitory at 0.8 µg/mL, that is, at a compound
concentration close to its toxicity threshold.
a
(i) 10, 1 equiv of NaH/DMF; (ii) (a) BrSiMe3/CH3CN; (b) H2O.
PMEO derivatives bearing an amino group solely in
the C-4, but not in the C-2, position of the pyrimidine
ring (i.e., compound 14b) or that have the 4-NH2 group
replaced by dimethylamino (27b), cyclopropylamino
Sch em e 7
(24b), or by methyl group (20b) or, wherein the 2-amino
group is replaced by a 2-methylsulfanyl group (i.e., 41b),
were devoid of significant antiviral activity (Table 1).
Replacement of the ether oxygen of compounds 17b or
2
9b by sulfur in 6-[2-(phosphonomethoxy)ethylsulfanyl]
derivatives resulted in a marked (5- to 10-fold) decrease
(35b) or complete loss (36b) of the antiviral activity. In
contrast, the sulfanyl derivative 35b gained some anti-
adenovirus activity (EC50: 74-78 µg/mL), comparable
with PMEDAP (38-47 µg/mL) but inferior to (S)-
HPMPA (EC50: 0.21-0.24 µg/mL). Esterification of the
phosphonate residue in 35b by isopropyl groups (as in
compound 35a ) annihilated the antiviral activity of the
parent compound.
Sch em e 8
afforded the free phosphonate 41b. This alternative
could be a method of choice for large-scale syntheses.
Limiting factor will be the availability of the starting
We have also synthesized the (R)- and (S)-6-[2-
(phosphonomethoxy)propoxy] (PMPO) homologues of
the most active PMEO derivative 17b. Interestingly, the
(R)-enantiomer of 38b showed pronounced anti-herpes
and anti-retroviral activity that was comparable to the
activity of 17b, whereas the (S)-derivative of 38b was
virtually devoid of antiviral activity. The residual activi-
ties of the (S)-enantiomer of 38b noted for MSV and
HIV-1 and HIV-2 may be due to contamination by traces
(e1%) of the (R)-enantiomer of 38b. It should also be
noted that the free pyrimidine bases of 14b, 24b, and
27b (i.e., compounds 13b, 22, and 25, respectively) were
devoid of antiviral activity, except for a marginal activity
against MSV.
6
(4)-halogenopyrimidine derivatives.
The structure of N - and O -isomers of ANPs synthe-
1
6
1
3
sized in this study could be derived from their C NMR
spectra. The N -isomers were characterized by the
1
chemical shift of carbon atom C-1′ (δ ∼ 40 ppm) and by
3
triplets of carbons C-2 [ J (C-2′,H-1′) ) 2.9] and C-6
3
[
J (C-6,H-1′) ) 3.9], and by doublets of carbons C-4
2
3
1
[
J (C-4,H-5) ) 4.9] and C-5 [ J (C,H) ) 164.2]. In the
1
6
C NMR spectra of O -isomers there is a low-field shift
of C-1′ carbon (δ ∼ 65 ppm), which is due to the linkage
to oxygen atom. We have also observed characteristic
alkylation low-field shifts of pyrimidine base carbons
C-6 and C-2 (ca. 15 and 3 ppm, respectively) and an
absence of vicinal interaction of R-hydrogen atoms of
the side chain with carbon C-6, which is typical for the
None of the isomeric 1-[2-(phosphonomethoxy)ethyl]
(PME) pyrimidin-6-one derivatives substituted at the
C-2 and/or C-4 positions of the pyrimidine ring with
4-methyl (19b), 2,4-diamino (16b), 2-amino-4-dimeth-
ylamino (26b), or even 2-amino-4-[2-(phosphonomethoxy)-
ethoxy] (33b) group showed appreciable antiviral activ-
ity with the exception of a poor but notable anti-MSV
activity.
1
N -isomers. The presence of the phosphonate function
at the side chain is manifested by splitting of the
appropriate hydrogens and carbons due to spin-spin
interactions J (H,P) and J (C,P), respectively.
1
6
The N and O -isomers were inactive in vitro against
all RNA viruses tested [vesicular stomatitis virus (VSV),
parainfluenza-3 virus, reovirus-1, Sindbis virus,
Coxsackie B-4 virus, or Punta Toro virus].
Biology
Among the 6-[2-(phosphonomethoxy)ethoxy] (PMEO)
pyrimidine derivatives, several analogues showed a
pronounced antiviral activity in cell culture. PMEO
derivatives that carry an amino group at C-2 of the
The majority of the compounds showed no appreciable
cytostatic activity against E6SM, HEL, and CEM cell

1
922 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9
Hol y´ et al.
Ta ble 1. Antiviral Activity of PMEO and PMPO Pyrimidine Derivatives in Cell Culture
EC50 (µg/mL)
HSV-1
(KOS)
HSV-2
(G)
HSV-1 TK-
(VMW 1837)
VZV
(OKA)
VZV
(YS)
VZV TK-
(07/1)
VZV TK-
(YS/R)
HIV-1
(IIIB)
HIV-2
(ROD)
formula
MSV
138 ( 3
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
4
2
3b
4a
>400
>400
>400
>16
240
>400
>400
>400
>16
>400
24
>400
>400
>400
>16
>80
>400
>80
>400
9.6
>400
>400
>400
g80
>400
>400
>400
>16
>400
9.6
>400
>400
>400
>16
240
>50
>50
>50
>50
32
>50
>50
>50
>50
>50
1.1
>50
>50
>50
>50
>50
>50
>50
>50
0.9
>50
>50
>50
>50
>50
2.5
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
1.6
>50
>50
>50
>50
>50
>50
>50
>50
0.6
>100
>100
>100
>100
>20
>20
4b
6b
7b
9b
0b
2
3b
4b
5
6b
7b
9b
2b
3b
5a
5b
6b
8b(R)
8b(S)
1b
13 ( 7.6
>100
>100
2.92 ( 2.48
0.04 ( 0.002
122 ( 3
80.0 ( 28.3
0.80 ( 0.17
>100
25.0 ( 7.1
0.43 ( 0.32
>100
6.5
1.2
>400
>400
>400
>16
240
>400
>80
>400
9.6
>50
>50
>50
>50
>20
>50
>50
>50
1.1
89 ( 37.6
133 ( 19
>100
>100
>100
>100
>100
>100
>40
>100
>100
>400
>80
>400
9.6
132 ( 5
105 ( 30
133 ( 11
0.08
>100
>100
>100
>100
>100
>100
>0.8
>0.8
240
240
50
>50
>50
>50
7
>50
>50
>50
20
>50
>50
>50
15
43 ( 0.75
107 ( 10
57 ( 38
>100
80 ( 35
>100
>400
>400
29
240
16
>400
>400
48
>50
>50
7.5
47 ( 18
5.5 ( 2.1
>100
52 ( 14
3.0 ( 1.4
>100
1.7
>400
>400
>50
>50
>50
>50
72 ( 17
0.046 ( 0.012
6.1 ( 1.4
>40d
48
9.6
3.8
5.9
6.3
5.7
1.9 ( 0.5
1.3 ( 0.4
>80
>80
>80
>80
>50
>50
10
>50
>50
10
>50
>50
10
>50
>50
10
51
33
>80
>80
>100
>100
b
b
b
b,c
b,c
b,c
b,c
d
d
PMEA
7
7
7
0.25
1.8
2.5
a
b
Fifty percent effective concentration, or compound concentration required to inhibit virus-induced cytopathicity by 50%. Data taken
c
-
from ref 33. Data taken from ref 30; data are the average values for two wild-type (YS, OKA) and two TK (07/1, YS/R) VZV strains.
Data taken from ref 27.
d
Ta ble 2. In Vitro Cytotoxicity of Free Phosphonates
cantly (40-80×) higher compared to their EC50 values,
e.g., against retroviruses (Table 1).
%
control
In conclusion, we report on a novel subclass of acyclic
pyrimidine) nucleoside phosphonates that are endowed
formula
L-1210
L-929
HeLaS3
CCRF-CEM
(
1
1
1
1
1
2
2
2
2
2
3
3
3
3b
4b
6b
7b
9b
0b
4b
6b
7b
9b
2b
3b
5b
98
81
76
62
95
99
71
90
88
50
77
80
83
80
96
90
84
96
61
81
85
93
99
104
94
86
93
89
83
87
88
92
89
96
72
72
98
92
79
86
95
95
63
100
95
with inhibitory activity against both DNA and retro-
viruses. From our studies, it could be concluded that
the 6-[2-(phosphonomethoxy)ethoxy]pyrimidines must
bear an (unsubstituted) amino group concomitantly on
both C-2 and C-4, or an amino on C-2 and an OH group
on C-4, to display antiviral activity. Alkyl ether deriva-
tives are preferred over alkyl thioethers. The most active
compounds of the 6-[2-(phosphonomethoxy)ethoxy] and
87
98
104
61
109
101
89
56
77
88
94
92
6
-[2-(phosphonomethoxy)propoxy]pyrimidine series have
an in vitro antiviral activity that is comparable to PMEA
and (R)-PMPA and should be further investigated for
their utility as novel antiviral agents.
(
4
S)-38b
1b
89
87
growth. The antivirally active compounds 17b, 35b, 38b
R), and 29b did not affect microscopically visible cell
morphology at 50 (HEL) or 400 (E6SM) µg/mL, and 17b,
5b, and 29b were inhibitory to HEL cell proliferation
Exp er im en ta l Section
(
Unless otherwise stated, solvents were evaporated at 40 °C/2
kPa, and compounds were dried at 2 kPa over P O . Melting
2 5
3
points were determined on a B u¨ chi melting point apparatus.
TLC was performed on Silufol UV254 plates (Kavalier Votice,
Czech Republic) in chloroform-ethanol (4:1). Paper electro-
phoresis (20 V/cm, 1 h) was made on Whatman 3 MM paper
in 0.05 M triethylammonium hydrogen carbonate pH 7.5.
at 25-50 µg/mL.
Detailed evaluation of cytostatic activity in vitro was
performed in two mouse cell lines (L1210, L929) and
two cell lines of human origin (HeLaS3, CCRF-CEM).
The cells were grown in the presence of the tested
compounds at a constant drug concentration (10 µM)
and, after a 72 h incubation period, the cells were
counted and the inhibition expressed relative to cell
count in the control culture grown in the culture
medium without compound (Table 2). The IC50 values
of 17b and 29b were 12.4 µM and 11.2 µM (3.3 µg/mL
and 3.0 µg/mL, respectively) for CCRF-CEM cells, 25
and 7.5 µM (6.5 µg/mL and 2.0 µg/mL, respectively) for
L-1210 cells, and 25 and 2 µM (6.5 µg/mL and 5.3 µg/
mL, respectively) for L929 cells. However, the IC50
values of 17b and 29b in CCRF-CEM cells are signifi-
NMR spectra were measured on an FT NMR spectrometer
1
13
Varian UNITY 500 ( H at 500 M and C at 125.7 M frequency)
in CDCl , dimethyl sulfoxide-d , or D O. Mass spectra were
3
6
2
measured on a ZAB-EQ (VG Analytical) spectrometer using
FAB (ionization by Xe, accelerating voltage 8 kV, glycerol
matrix). UV spectra were measured on a Shimadzu type UV
1
240 Mini spectrophotometer in aqueous solutions.
Ma ter ia ls. Bromotrimethylsilane, cesium carbonate, so-
dium hydride, and dimethylamine (30% solution in ethanol)
were purchased from Fluka (Switzerland); DBU, DABCO,
cyclopropylamine, and all pyrimidine derivatives were ob-
tained from Sigma-Aldrich (Praha, Czech Republic). Dimeth-
ylformamide and acetonitrile were distilled from P O and
2
5
stored over molecular sieves (4 Å).

6
-[2-(Phosphonomethoxy)alkoxy]pyrimidines
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9 1923
4-amino-6-hydroxypyrimidine (12), mp 272 °C. Yield, 10.0 g
Meth od s. Deion iza tion of th e Rea ction Mixtu r es. The
+
1
solution of reaction products in water (20-25 mL) was applied
(64.4%). Anal. (C
(CD SOCD ): 4.97 s, 1H (H-5); 6.42 brs, 2H (NH
(H-2); 11.41 brs, 1H (OH).
-Am in o-6-[2-(p h osp h on om e t h oxy)e t h oxy]p yr im i-
4
H
5
N
3
O) C, H, N. MS: 112 (MH ). H NMR
+
onto a column of Dowex 50 × 8 (H -form) (100 mL, if not stated
3
3
2
); 7.77 s, 1H
otherwise), and the column was washed with water (20%
aqueous methanol for phosphonate diesters) till the drop of
the UV absorption (254 nm) and acid reaction of the eluate.
Standard elution rate: 3 mL/min. Elution was continued with
4
d in e (14b) a n d 4-Am in o-1-[2-(p h osp h on om eth oxy)eth yl]-
p yr im id in -6(1H)-on e (13b). Compound 12 (3.6 g, 33.6 mmol)
in DMF (70 mL) was treated with NaH (1.36 g, 34 mmol, 60%
dispersion in paraffin oil) for 0.5 h under stirring, and
diisopropyl 2-(chloroethoxy)methylphosphonate (10) (9.4 mL,
40.5 mmol) was added. The mixture was stirred for 8 h at 80
°C, filtered through Celite pad, and evaporated in vacuo. The
residue in chloroform was purified on silica gel; elution with
chloroform-ethanol (97.5:2.5) afforded 4-amino-6-[2-(diiso-
propylphosphorylmethoxy)ethoxy]pyrimidine (14a ), which was
crystallized from ethyl acetate-petroleum ether. Yield, 3.0 g
2
.5% ammonia (in water or 20% aqueous methanol, respec-
tively), and the UV-absorbing eluate was collected and evapo-
rated in vacuo.
P u r ifica tion of th e P h osp h on a tes by Colu m n Ch r o-
m a togr a p h y on Dow ex 1 × 2. Unless stated otherwise, 100
mL columns of Dowex 1 × 2 (100-200 mesh, acetate form,
prewashed with water) were used. The sample was dissolved
in water (20-25 mL), alkalified with concentrated aqueous
ammonia to pH 9-9.5, and applied onto the column. Elution
with water (3 mL/min) was continued till the drop of the initial
UV absorption (254 nm) of the eluate. The column was then
eluted with the linear gradient of acetic acid (0-0.4 M acetic
acid, 1 L each; 3 mL/min, fractions 30 mL).
An tivir a l Activity Assa ys. The antiviral assays, other
than HIV-1, were based on inhibition of virus-induced cyto-
pathicity in either E
CMV, adenovirus type 2 and 3) cell cultures, following previ-
ously established procedure. 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 1-2 h virus adsorption period,
residual virus was removed, and the cell cultures were
incubated in the presence of varying concentrations (400, 200,
1
(26.8%); mp 112 °C. Anal. (C H N O P) C, H, N, P. H NMR
1
3
24
3
5
(CD SOCD ): 1.22 d, 6 H and 1.23 d, 6 H, J (CH ,CH) ) 6.1 (4
3
3
3
× CH ); 3.78 brt, 2 H, J (2′,1′) ) 4.5 (H-2′); 3.78 d, J (CH -P)
3
2
) 8.4 (CH -P); 4.31 brt, 2H, J (1′,2′) ) 4.5 (H-1′); 4.59 m, 2 H
2
(P-OCH); 5.67 s, 1 H (H-5); 6.62 bs, 2H (NH
2). 13C NMR (CD
SOCD ): 23.84 d, 2C, J (CH
CH ); 23.99 d, 2C, J (CH ); 64.42 (C-1′); 64.99
,P) ) 2.9 (2 × CH
d, J (CH ,P) ) 165.0 (CH
2
); 8.07 s, 1H (H-
3
3
3
,P) ) 3.9 (2 ×
6
SM (HSV-1, HSV-2, VV) or HEL (VZV,
3
3
3
2
2
-P); 70.35 d, 2C, J (CH,P) ) 5.9
2
6
(2CHO); 70.88 d, J (2′,P) ) 11.7 (C-2′); 85.80 (C-5); 157.85
(C-2), 165.64 and 168.89 (C-4 and C-6).
This compound was treated with BrSiMe
3
(10 mL) in
acetonitrile (70 mL) overnight at room temperature. After
evaporation in vacuo, water (100 mL) was added to the residue,
followed by concentrated aqueous ammonia to give an alkaline
reaction, and the mixture was evaporated. The residue was
deionized on a Dowex 50 × 8 column (100 mL) further purified
by Dowex 1 × 2 (acetate form) column (150 mL) chromatog-
raphy [elution with water followed by linear gradient of acetic
acid (0-1 M, 1L each)]. The main UV-absorbing fraction was
evaporated, the residue codistilled with water (3 × 50 mL) and
crystallized from water. Yield, 1.8 g (80%) of 4-amino-6-[2-
(phosphonomethoxy)ethoxy]pyrimidine (14b), mp 254 °C. Anal.
1
00, ... 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.
An ti-Ad en ovir u s Activity in HEL Cells (cf. r ef 27). On
day 0, confluent cultures of human embryonic lung (HEL)
fibroblasts were infected with 100 CCID50 of human adenovirus
type 2 or type 3. Virus was removed after 2 h adsorption at
1
3
7 °C and replaced by serial dilutions of the compounds. On
(C H N O P) C, H, N, P. H NMR (D O + NaOD): 3.71 d,
7
12
3
5
2
day 7-10 p.i., microscopy was performed to determine the
cytopathic effect for calculation of the EC50 and the minimum
cytotoxic concentration (MCC).
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
2H, J (CH ,P) ) 8.5 (CH P); 3.94 m, 2H (H-2′); 4.37m, 2H (H-
2
2
1′); 5.95 d, 1H, J (5,2) ) 0.95 (H-5); 8.10 d, 1H, J (2,5) ) 0.95
(H-2). 13C NMR (D O + NaOD): 69.11 (C-1′), 70.10 d, J (CH ,P)
2
2
) 155.7 (CH -P); 73.38 d, J (2′,P) ) 11.0 (C-2′); 89.29 (C-5);
2
160.16 (C-2); 167.99 (C-6); 172.02 (C-4).
2
8
5
described previously. Briefly, human CEM (∼3 × 10 cells
Further elution of the crude reaction mixture on silica gel
column with chloroform-ethanol (95:5) gave the oily 4-amino-
1-[2-(diisopropylphosphorylmethoxy)ethyl]pyrimidin-6(1H)-
one (13a ), which was dried in vacuo [yield, 4.6 g (41.1%)]. This
3
-
1
B
mL ) cells were infected with 100 CCID50 HIV-1 ((III ) or
HIV-2 (ROD)/mL and seeded in 200 µL wells of a microtiter
plate, containing appropriate dilutions of the test compounds.
After 4 days of incubation at 37 °C, CEM giant cell formation
was examined microscopically.
compound was treated with BrSiMe (10 mL) in acetonitrile
(70 mL) overnight at room temperature. After evaporation in
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
vacuo, the residue was treated with water (100 mL). After 10
min, concentrated aqueous ammonia was added to give an
alkaline reaction, and the mixture was evaporated. The residue
was applied onto a column (100 mL) of Dowex 50 × 8 and
eluted with water. The main UV-absorbing fraction was
evaporated, and the residue was crystallized from 70% aqueous
ethanol (ether added to turbidity). Yield, 2.8 g (91%) of
2
8
performed as described previously. Murine C3H/3T3 embryo
fibroblast cells were seeded at 5 × 10 cells mL into 1 cm2
wells of a 48-well microplate. At 24 h later, the cell cultures
were infected with 80 focus-forming units of MSV (prepared
from tumors induced following intramuscular inoculation of
5
-1
2
9
3
9
-day-old NMRI mice with MSV, as described previously ) for
4-amino-1-[2-(phosphonomethoxy)ethyl]pyrimidin-6(1H)-one
1
0-120 min at 37 °C. The medium was then replaced by 1
(13b), mp 233 °C. Anal. (C
(CD SOCD ): 3.56 d, 2H, J (CH
J (2′,1′) ) 4.9 (H-2′); 3.90t, 2H, J (1′,2′) ) 4.9 (H-1′); 5.06 s, 1 H
(H-5); 6.45 brs, 2H (NH ); 6.90 brs, 2H (P-OH); 7.98 s, 1H
(H-2). 13C NMR (CD
SOCD ): 44.44 (C-1′), 66.54 d, J (CH ,P)
) 160.2 (CH
7
H
12
N
3
O
5
P) C, H, N, P. H NMR
mL of fresh medium containing various concentrations of the
test compounds. After 6 days, transformation of the cell culture
was examined microscopically.
Cytosta tic Activity Assa ys. Inhibition of the cell growth
was estimated in mouse leukemia L1210 cells (ATCC CCL
3
3
2
2
,P) ) 8.8 (CH P); 3.64 t, 2H,
2
3
3
2
2
-P); 70.13 d, J (2′,P) ) 11.7 (C-2′); 84.78 (C-5);
2
19), CCRF-CEM T lymphoblastoid cells (ATCC CCL 119),
152.35 (C-2), 161.05 and 163.74 (C-4 and C-6).
murine L929 cells (ATCC CCL 1), and human cervix carcinoma
2,4-Dia m in o-6-[2-(p h osp h on om eth oxy)eth oxy]p yr im i-
d in e (17b ) a n d 2,4-Dia m in o-1-[2-(p h osp h on om et h oxy)-
eth yl]p yr im id in -6(1H)-on e (16b). A mixture of 2,4-diamino-
6-hydroxypyrimidine (15) (2.52 g, 20 mmol), cesium carbonate
(3.25 g, 10 mmol) in dimethylformamide (40 mL) was stirred
for 30 min at 80 °C, and diisopropyl 2-chloroethoxymeth-
ylphosphonate (10) (3.5 mL, 23.4 mmol) was added. The
mixture was stirred for 16 h at 100 °C and filtered from salts.
The filtrate was taken down in vacuo, and the residue
3
0
HeLa S3 cells (ATCC CCL 2.2) as described.
-Am in o-6-h yd r oxyp yr im id in e (12). 4-Amino-6-hydroxy-
-sulfanylpyrimidine (11) (20 g, 0.14 mol) in boiling ethanol
300 mL) was treated under stirring with Raney-Ni until the
4
2
(
starting material disappeared. The supension was filtered
while hot, the precipitate washed with hot ethanol (300 mL),
and the filtrate evaporated to dryness. The residue afforded
on crystallization from ethanol (ether added to turbidity)

1
924 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9
Hol y´ et al.
chromatographed on silica gel column (300 mL) with chloro-
form. The eluate gave a product which was crystallized from
ethyl acetate-petroleum ether to afford 1.2 g (17.2%) of 2,4-
dia m in o-6-[2-(diisopr opylph osph or ylm et h oxy)et h oxy]-
H (H-5). 13C NMR (D
154.7 (CH P); 74.09 d, J (2′,P) ) 11.8 (C-2′); 81.05 (C-5); 159.73,
166.79 and 168.04 (C-2, C-4 and C-6). The upfield position of
C-1′ (δ 45.15) indicates N-substitution. Two signals of NH
2 2
O): 45.15 (C-1′); 70.41 d, J (CH -P) )
2
2
1
pyrimidine (17a ), mp 159 °C. Anal. (C13
H
25
N
4
i
O
5
P) C, H, N, P.
groups (δ 6.61 and 5.88) which are observed in H NMR
+
+
MS: 349.3 (MH ) (100), 265.1 (MH - 2 × Pr) (6); 139 (26);
spectrum in DMSO exclude the substitution at the exo-
+
1
1
1
3
27.1 (BaseH ) (37). H NMR (CD
3
SOCD
); 3.74 m, 2 H (H-2′);
-P); 4.22 m, 2H (H-1′); 4.59
) ) 6.2 (2xCH); 5.02 s, 1 H
3
): 1.24 d, 6H and
positions 2-NH
expected substitution at N .
2 2
and/or 4-NH and are consistent with the
1
.24 d, 6 H, J (CH
.78 d, 2H, J (CH
dh, 2 H, J (CH,P) ) 8.2, J (CH,CH
3
,CH) ) 6.2 (4 × CH
3
2
-P) ) 8.2 (CH
2
2-Am in o-4-m et h yl-6-[2-(p h osp h on om et h oxy)et h oxy]-
p yr im id in e (20b) a n d 2-Am in o-4-m eth yl-1-[2-(p h osp h o-
n om eth oxy)eth yl]p yr im id in -6(1H)-on e (19b). Diisopropyl
2-(chloroethoxy)methylphosphonate (10) (15 mL, 62.5 mmol)
was added to a mixture of 2-amino-6-hydroxy-4-methylpyri-
midine (18) (6.25 g, 50 mmol) and cesium carbonate (11.3 g,
25 mmol) in DMF (70 mL) which had been stirred at 100 °C
for 1 h prior to addition. The reaction mixture was then stirred
at 100 °C for 14 h, filtered while hot, and evaporated in vacuo.
The residue gave, on extraction with chloroform and subse-
quent purification on silica gel column (250 mL), compound
3
1
3
(
H-6); 5.85 bs, 2 H and 6.00 bs, 2H (2 × NH
SOCD ): 23.85 d, 2C, J (CH
,P) ) 4.6 (2 × CH
J (CH ); 63.65 (C-1′); 65.02 d, J (CH
,P) ) 4.1 (2 × CH
64.4 (CH
P); 70.32 d, 2C, J (CH,P) ) 6.0 (2 × CH-O); 71.05
2
). C NMR (CD
); 23.99 d, 2C,
-P) )
3
-
3
3
3
3
3
2
1
2
d, J (2′,P) ) 11.9 (C-2′); 76.35 (C-5); 163.01, 166.15 and 169.92
(
C-2, C-4 and C-6).
This compound (1.0 g, 2.9 mmol) was treated with BrSiMe
3
(4 mL) in acetonitrile (40 mL) overnight. The solvents were
stripped down in vacuo, the residue was codistilled with
acetonitrile (2 × 25 mL), and water (50 mL) was added to the
residue. The solution was alkalized with concentrated aqueous
ammonia and evaporated in vacuo. The residue was deionized
on a Dowex 50 × 8 column (100 mL) and the UV-absorbing
fraction of the ammonia eluate was collected. It was taken
down in vacuo, redissolved in water (20 mL), brought to pH
2
0a , which was crystallized from ethyl acetate-petroleum
ether. Yield, 5.75 g (33,1%), mp 72-73 °C. Anal. (C14 P)
): 1.33 d, 6H, J ) 6.2 and 1.34 d,
); 3.82 d, 2H,
26 3 5
H N O
1
C, H, N, P. H NMR (CDCl
H, J ) 6.2 (4 × CH ); 2.26 d, 3H, J ) 0.6 (CH
J (H,P) ) 8.2 (P-CH
3
6
3
3
2
); 3.90 m, 2H (2 × H-2′); 4.76 dh, 2H,
i
J (H,P) ) 7.6 and J (H,H) ) 6.2 (2 × OCH ( Pr)); 4.42 m, 2H (2
1
3
×
H-1′); 4.90 b, 2H (NH
CDCl ): 23.61 (CH
); 23.90 d, 2C, J (C,P) ) 4.9 (2 × CH
4.04 d, 2C, J (C,P) ) 3.9 (2 × CH ); 64.60 (C-1′); 65.99 d, J (C,P)
167.6 (P-CH ); 71.06 d, J (C,P) ) 6.8 (OCH ( Pr)); 71.17 d,
2
); 5.95 q, 1H, J ) 0.6 (H-5); C NMR
9
-10 by concentrated aqueous ammonia, and applied onto a
(
3
3
3
);
column (70 mL) Dowex 1 × 2 (acetate form) thoroughly
prewashed with water. Elution with water gave (with reten-
tion) product which was crystallized from water to afford 2,4-
diamino-6-[2-(phosphonomethoxy)ethoxy]pyrimidine (17b) (0.60
2
3
i
)
2
J (C,P) ) 10.8 (C-2′); 97.03 (C-5); 162.48, 168.27 and 170.36
(C-2, C-4 and C-6).
7 13 4 5
g, 78.3%), mp 279 °C (water). EUp 0.80. Anal. (C H N O P) C,
This compound (5.55 g, 16 mmol) was treated with BrSiMe
3
H, N, P. UV spectrum [λmax (ꢀmax)] (pH 2): 276 (9100), (pH 7):
65 (7500). These data agree with the UV spectra published31
(5 mL) in acetonitrile (50 mL) at room temperature overnight,
and the volatiles were evaporated in vacuo. The residue was
dissolved in water (100 mL), and concentrated aqueous am-
monia was added to an alkaline reaction. The mixture was
deionized on a Dowex 50 × 8 column (100 mL) and further
purified by Dowex 1 × 2 column (150 mL) chromatography
2
for 2,4-diamino-6-methoxypyrimidine ((λmax 263 and 275,
1
respectively). H NMR (CD
J (CH ,P) ) 8.7 (CH P); 3.74 t, 2 H, J (2′,1′) ) 4.9 (H-2′); 4.23
t, 2 H, J (1′,2′) ) 4.9 (H-1′); 5.07 s, 1 H (H-5); 5.86 bs, 2 H and
3 3
SOCD , 40 °C): 3.58 d, 2 H,
2
2
1
6
.01 bs, 2 H (2 × NH
2 2 2
). H NMR (D O): 3.69 d, 2H, J (CH ,P)
[elution with water followed by linear gradient of acetic acid
)
8.7 (CH P); 3.91 m, 2H (H-2′); 4.30 m, 2 H (H-1′); 5.45 s, 1
2
3
1
(0-0.4 M, 1 L each)]. The main UV-absorbing fraction was
H (H-5). C NMR (D
2
O): 69.30 (C-1′); 70.28 d, J (CH
P); 73.35 d, J (2′,P) ) 10.3 (C-2′); 79.63 (C-5); 165.46,
69.35 and 171.08 (C-2, C-4 and C-6). Low-field position of
2
-P) )
evaporated, and the residue was codistilled with water (3 ×
1
1
51.3 (CH
2
5
2
0 mL) and crystallized from water. Yield, 3.67 g (87%) of
-amino-4-methyl-6-[2-(phosphonomethoxy)ethoxy]pyrimi-
C-1′-carbon signal in the O-isomers (diester 17a and free
phosphonate 17b) (δ 63.65 and 69.30, respectively) indicates
that the PME group is linked to the oxygen atom at C-6.
1
dine (20b), mp 245 °C. Anal. (C
8
H
14
N
3
O
5
P) C, H, N, P. H NMR
(
D
2
O + NaOD): 2.26 s, 3H (CH ); 3.57 d, 2H, J (H,P) ) 8.5
3
(P-CH
2
); 3.92 m, 2H (2 × H-2′); 4.39 m, 2H (2 × H-1′); 6.13 s,
Further elution of the silica gel column with chloroform gave
13
1
7
2
H (H-5). C NMR (D
2.06 d, J (C,P) ) 149.4 (P-CH
′); 98.75 (C-5); 165.50, 172.80 and 173.63 (C-2, C-4 and
2
3
O + NaOD): 25.42 (CH ); 68.85 (C-1′);
1
.8 g (26%) of amorphous 2,4-diamino-1-[2-(diisopropylphos-
phorylmethoxy)ethyl]pyrimidin-6(1H)-one (16a ) (R 0.35, S1)
. mp 196-197 °C. MS:
2
); 73.09 d, J (C,P) ) 10.2 (C-
F
which was dried in vacuo over P
2
O
5
i
C-6).
Further elution of the silica gel column followed by crystal-
lization from ethyl acetate-petroleum ether gave 2-amino-4-
methyl-1-[2-(diisopropylphosphorylmethoxy)ethyl]pyrimidin-
+
+
+
3
49.2 (MH ) (100); 265.1(MH - 2 × Pr) (35); 126 (BH ) (26).
1
H NMR (CD
3
SOCD
); 3.62 t, 2 H, J (2′,1′) ) 4.9 (H-2′); 3.78 d,
-P) ) 8.3 (CH -P); 3.96 t, 2H, J (1′,2′) ) 4.9 (H-1′);
3 3
): 1.23 d, 6 H and 1.21 d, 6 H, J (CH ,-
CH) ) 6.3 (4 × CH
3
2
4
H, J (CH
2
2
6
(1H)-one (19a ) (4.2 g, 24%), mp 88 °C. Anal. (C14
H
26
N
3
O
5
P)
): 1.29 d, 6H, J ) 6.2 and 1.32 d,
); 2.12 d, 3H, J ) 0.8 (CH ); 3.74 d, 2H,
.57 m, 2 H (2 × CH); 4.59 s, 1 H (H-5); 6.55 bs, 2 H and 5.84
1
C, H, N, P. H NMR (CDCl
H, J ) 6.2 (4 × CH
J (H,P) ) 8.6 (P-CH
3
1
3
bs, 2H (2 × NH
2
). C NMR (CD
); 24.00 d, 2C, J (CH
-P) ) 163.1 (CH
3
SOCD
,P) ) 3.9 (2 × CH
P); 70.22 d, J (2′,P) )
3
): 23.94 d, 2C, J (CH
3
,P)
6
3
3
)
4.9 (2 × CH
3
3
3
); 39.39
2
); 3.90 m, 2H (2 × H-2′); 4.20 m, 2H (2 ×
(
C-1′); 65.00 d, J (CH
2
2
H-1′); 4.72 dh, 2H, J (H,P) ) 7.6 and J (H,H) ) 6.2 (2 × OCH);
1
5
1.7 (C-2′); 70.40 d, 2C, J (CH,P) ) 6.9 (2 × CH-O); 76.66 (C-
13
5
2
.62 b, 2H (NH
2
3
); 5.79 q, 1H, J ) 0.8 (H-5). C NMR (CDCl ):
); 155.71 (C-6); 162.22 (C-2); 163.38 (C-4).
3.64 (CH ); 23.97 d, 2C, J (C,P) ) 3.9 and 23.91 d, 2C, J (C,P)
3
The residue (5.17 mmol) was treated with BrSiMe
3
(6 mL)
) 4.9 (2 × CH ); 43.06 (C-1′); 66.17 d, J (C,P) ) 168.1 (P-CH );
3
2
in acetonitrile (60 mL) overnight and worked up as described
for the O-isomer. Purification of the desalted mixture on Dowex
71.27 d, J (C,P) ) 6.8 (OCH); 72.88 d, J (C,P) ) 11.7 (C-2′);
102.22 (C-5); 156.37, 162.93 and 164.33 (C-2, C-4 and C-6).
This product (4.0 g, 11.5 mmol) was treated analogously
with BrSiMe3 (5 mL) in acetonitrile (50 mL) to afford, after
chromatography of the deionized reaction mixture on Dowex
1 × 2 and crystallization from water, 2-amino-4-methyl-1-[2-
(phosphonomethoxy)ethyl]pyrimidin-6(1H)-one (19b). Yield,
1
column (elution with water) gave a product which was
crystallized from water to afford 0.95 g (65%) of 2,4-diamino-
1
2
-[2-(phosphonomethoxy)ethyl]pyrimidin-(1H)-one (16b), mp
28 °C (water). EUp 0.90. Anal. (C P‚H O) C, H, N, P.
7
H
13
N
4
O
5
2
UV spectrum [λmax (ꢀmax)] (pH 2): 264 (18000), (pH 7): 267
3
2
1
(
12200). The λmax value coincides with the value published
for 2,4-diamino-1-methylpyrimidin-6(1H)-one (268 nm). 1H
NMR (CD SOCD ): 3.59 d, 2 H, J (CH ,P) ) 8.4 (CH P); 3.60
t, 2 H, J (2′,1′) ) 6.1 (H-2′); 3.96 t, 2 H, J (1′,2′) ) 6.1 (H-1′);
2.3 g (76%), mp 283 °C. Anal. (C H N O P) C, H, N, P. H
8
14
3
5
NMR (D O + NaOD): 2.12 s, 3H (CH ); 3.50 d, 2H, J (H,P) )
2
3
3
3
2
2
8.7 (P-CH ); 3.82 t, 2H, J ) 5.3 (2 × H-2′); 4.18 t, 2H, J ) 5.3
2
1
3
(2 × H-1′); 5.77 s, 1H (H-5). C NMR (D O + NaOD): 25.43
2
1
4
.61 s, 1 H (H-5); 5.88 bs, 2 H and 6.61 bs, 2 H (2 × NH
NMR (D O): 3.48 d, 2 H, J (CH ,P) ) 8.4 (CH P); 3.76 t, 2 H,
J (1′,2′) ) 5.2 (H-1′); 4.09 t, 2H, J (2′,1′) ) 5.2 (H-2′); 5.06 s, 1
2
). H
(CH ); 45.60 (C-1′); 72.55 d, J (C,P) ) 142.0 (P-CH ); 73.17
3
2
2
2
2
(C-2′); 102.40 (C-5); 160.25, 168.32 and 169.48 (C-2, C-4 and
C-6).

6
-[2-(Phosphonomethoxy)alkoxy]pyrimidines
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9 1925
2
-Am in o-4-cyclopr opylam in o-6-h ydr oxypyr im idin e (22).
mL) was added. The mixture was stirred at 100 °C for 24 h,
filtered while hot, and evaporated in vacuo. The residue was
extracted with hot chloroform (100 mL), filtered, and purified
by chromatography on silica gel column (200 mL). Elution with
chloroform gave 2-amino-4-(dimethylamino)-6-[2-(diisopropyl-
phosphorylmethoxy)ethoxy]pyrimidine (27a ) as a thick oil (4.6
2
-Amino-4-chloro-6-hydroxypyrimidine monohydrate (21, 5.0
g, 30.5 mmol) was refluxed in ethanol (150 mL) with cyclo-
propylamine (15 mL) for 12 h. The mixture was evaporated in
vacuo, codistilled with ethanol (3 × 50 mL), adsorbed from
methanol on silica gel and applied onto a column of silica gel
(
200 mL) in chloroform. Elution with chloroform-ethanol
3
g, 47%). It was treated with BrSiMe (5 mL) and acetonitrile
gradient afforded a crystalline product which was filtered from
ether and dried in vacuo to afford 2-amino-4-cyclopropylamino-
6
(50 mL) overnight at room temperature. After evaporation in
vacuo, the residue was treated with water (100 mL), alkalized
with concentrated aqueous ammonia, and the mixture was
evaporated. The residue was deionized on Dowex 50 × 8
column (100 mL) and purified by Dowex 1 × 2 column (150
mL) chromatography [elution with water followed by linear
gradient of acetic acid (0-0.4 M, 1 L each)]. The main UV-
absorbing fraction was evaporated, and the residue was
codistilled with water (3 × 50 mL) and crystallized from water.
Yield, 1.1 g (31%) of 2-amino-4-(dimethylamino)-6-[2-(phospho-
-hydroxypyrimidine (22), mp 229 °C. Yield, 3.0 g (59%). Anal.
1
(
C
7
H
10
N
4
O) C, H, N. H NMR (DMSO): 0.40m, 2H and 0.62,
m, 2H (C-CH , N-CH); 2.30 m, 1H; 4.66 s, 1H (H-5); 6.08
brs, 2H (NH ); 6.55 bs, 2H (NH); 9.73 brs, 1H (OH).
-Am in o-4-cyclopr opylam in o-6-[2-(ph osph on om eth oxy)-
2
2
2
eth oxy]p yr im id in e (24b) a n d 2-Am in o-4-cyclop r op yla m i-
n o-1-[2-(p h osp h on om e t h oxy)e t h yl]p yr im id in -6(1H )-
on e (23b). The mixture of compound 22 (3.0 g, 18 mmol) and
cesium carbonate (2.92 g, 9 mmol) in DMF (50 mL) was stirred
at 100 °C for 1 h, and compound 10 (6 mL) was added. The
reaction mixture was stirred at 100 °C for 24 h, filtered while
hot, and evaporated in vacuo. The residue was extracted with
hot chloroform (100 mL), filtered, concentrated in vacuo, and
purified by chromatography on silica gel column (2 × 200 mL).
Elution with chloroform gave 2-amino-4-cyclopropylamino-6-
9 17
nomethoxy)ethoxy]pyrimidine (27b), mp 168 °C. Anal. (C H -
1
N
4
O
5
P) C, H, N, P. H NMR (DMSO): 2.93 s, 6H (N(CH
3.58 d, 2H, J (H,P) ) 8.8 (P-CH ); 3.74 m, 2H (H-2′); 4.26 m,
2H (2 × H-1′); 5.20 s, 1H (H-5); 6.03 bs, 2H (NH
(DMSO): 36.93, 2C (N(CH ); 64.28 (C-1′); 66.96 d, J (C,P) )
3 2
) );
2
1
3
2
). C NMR
3 2
)
160.7 (P-CH ); 70.89 d, J (C,P) ) 11.4 (C-2′); 75.02 (C-5);
161.92, 164.91 and 169.93 (C-2, C-4 and C-6).
2
[
2-(diisopropylphosphorylmethoxy)ethoxy]pyrimidine (24a ) as
Further elution of the silica gel column with chloroform-
ethanol gradient afforded 2-amino-4-(dimethylamino)-1-[2-
(diisopropylphosphorylmethoxy)ethyl]pyrimidin-6(1H)-one (26a)
as a thick oil (2.0 g, 20.5%), which was treated with BrSiMe3
(5 mL) and acetonitrile (50 mL) overnight and worked up
similarly. After Dowex 1 chromatography, the product gave,
on crystallization from water, 1.17 g (75.5%) of 2-amino-4-
(dimethylamino)-1-[2-(phosphonomethoxy)ethyl]pyrimidin-6(1H)-
a thick oil (1.8 g, 25.8%). It was treated with BrSiMe (5 mL)
3
and acetonitrile (50 mL) overnight at room temperature. After
evaporation in vacuo, the residue was treated with water (100
mL), alkalized with concentrated aqueous ammonia, and
evaporated. The residue was deionized on a column (100 mL)
of Dowex 50 × 8 and further purified by Dowex 1 × 2 column
(
150 mL) chromatography [elution with water followed by
1
linear gradient of acetic acid (0-0.4 M, 1 L each)]. The main
UV-absorbing fraction was evaporated, and the residue was
codistilled with water (3 × 50 mL) and crystallized from water.
Yield, 1.0 g (70.7%) of 2-amino-4-cyclopropylamino-6-[2-
one (26b), mp 235 °C. Anal. (C H N O P) C, H, N, P. H NMR
9
17
4
5
(DMSO): 2.89 s, 6H (N(CH ) ); 3.59 d, 2H, J (H,P) ) 8.3 (P-
3
2
CH ); 3.61 t, 2H, J (2′,1′) ) 6.0 (2 × H-2′); 3.98 t, J (1′,2′) ) 6.0
2
1
3
(H-1′); 4.69 s, 1H (H-5); 6.69 bs, 2H (NH ). C NMR (DMSO):
2
(phosphonomethoxy)ethoxy]pyrimidine (24b), mp 244 °C. Anal.
36.93, 2C (N(CH ) ); 40.0 (C-1′); 66.76 d, J (C,P) ) 158.7 (P-
3
2
1
(
C
10
H
17
N
4
O
5
P) C, H, N, P. H NMR (D
H and 0.80 m, 2H (C-CH ); 2.53 m, 1H (N-CH); 3.57 d, 2H,
J (CH ,P) ) 8.4 (CH P); 3.91 m, 2H (H-2′); 4.32 m, 2H (H-1′);
.61 s, 1 H (H-5).
Further elution of the silica gel column with chloroform-
2
O + NaOD): 0.54 m,
CH ); 69.82 d, J (2′,P) ) 10.3 (C-2′); 75.79 (C-5); 154.67, 161.94
2
2
2
and 162.37 (C-2, C-4 and C-6).
2
2
2
-Am in o-4-ch lor o-6-[2-(d iisop r op ylp h osp h or ylm et h -
5
oxy)eth oxy]p yr im id in e (28) a n d 2-Am in o-4-ch lor o-1-[2-
(d iisop r op ylp h osp h or ylm eth oxy)eth yl]p yr im id in -6(1H)-
on e (30). 2-Amino-4-chloro-6-hydroxypyrimidine monohydrate
(21) (25 mmol) was codistilled with toluene (3 × 50 mL) in
vacuo, and the residue was treated with DMF (50 mL), DBU
(3.8 mL), and diisopropyl 2-(chloroethoxy)methylphosphonate
(10) (7 mL). The mixture was stirred for 16 h at 100 °C, and
the volatiles were evaporated at 50 °C/2 kPa. The residue was
taken in chloroform (200 mL), filtered, and washed with
saturated NaCl solution (100 mL). The aqueous wash was
extracted with chloroform (5 × 50 mL), and the combined
extracts were dried with magnesium sulfate and evaporated.
Silica gel column (150 mL) chromatography by chloroform-
ethanol gradient gave 2-amino-4-chloro-6-[2-(diisopropylphos-
ethanol gradient afforded (after crystallization from ethanol-
ether) yellow 2-amino-4-cyclopropylamino-1-[2-(diisopropyl-
phosphorylmethoxy)ethyl]pyrimidin-6(1H)-one (23a ) (1.6 g,
2
3%), which was treated with BrSiMe
3
(5 mL) and acetonitrile
(50 mL) overnight and worked up similarly. The product eluted
with water during Dowex 50 chromatography. It was evapo-
rated in vacuo and crystallized from water to yield 2-amino-
4
2
Anal. (C10
m, 2H and 0.81 m, 2H (C-CH
-cyclopropylamino-1-[2-(phosphonomethoxy)ethyl]pyrimidin-
-one (23b), not melting under 290 °C. Yield, 0.90 g (71.8%).
1
H
17
N
4
O
5
P) C, H, N, P. H NMR (D
2
O + NaOD): 0.54
2
); 2.50 m,1H (N-CH); 3.59 d,
-P); 3.95 t, 2H, J (2′,1′) ) 4.9 (H-2′);
2
4
+
(
(
H, J (CH
.30 t, 2H, J (1′,2′) ) 4.9 (H-1′); 5.54 s, 1H (H-5). C NMR (D
NaOD): 9.43 (2 × CH , cyclopropyl); 25.96 (N-CH); 69.14
C-1′); 72.19 d, J (C,P) ) 149.4 (P-CH ); 73.31 d, J (C,P) ) 10.3
C-2′); 78.75 (C-5); 165.53, 170.07 and 173.71 (C-2, C-4 and
2
,P) ) 8.5 (CH
2
1
3
2
O
phorylmethoxy)ethoxy]pyrimidine (28) (2.5 g, 27.2%), mp 89
+
2
°
(
C. Anal. (C13
H
23ClN
O
SOCD
); 3.78 d, J (CH
P) C, H, Cl, N, P. MS: 368.3 (MH )
3
5
100). 1H NMR (CD
2
): 1.22 d, 6 H and 1.23 d, 6 H,
-P) ) 8.3 (CH -P); 3.80
3
3
J (CH ,CH) ) 6.1 (CH
3
3
2
2
C-6).
m, 2H (H-2′); 4.37 m, 2H (H-1′); 4.58 m, 2 H (P-OCH); 6.06 s,
1
3
2
-Am in o-4-(dim eth ylam in o)-6-h ydr oxypyr im idin e (25).
1 H (H-5); 7.05 bs, 2 H (NH
d, 2C, J (CH ,P) ) 4.9 and 23.97 d, 2C, J (P,C) ) 3.9 (CH
2
). C NMR (CD
3
SOCD
3
): 23.83
);
2
-Amino-4-chloro-6-hydroxypyrimidine monohydrate (5.0 g)
3
3
was stirred with 30% dimethylamine in ethanol (180 mL) at
00 °C in an autoclave for 16 h. The crystalline product was
filtered, washed with water, acetone, and ether, and dried in
vacuo to afford 2-amino-4-(dimethylamino)-6-hydroxypyrimi-
65.00 d, J (P,C) ) 164.1 (P-C); 65.15 (C-1′); 70.35 d, 2C, J (P,C)
) 6.7 (P-OC); 70.52 d, 2C, J (P,C) ) 11.7 (C-2′); 94.46 (C-5);
160.12 (C-2); 162.97 (C-4); 170.56 (C-6).
Further elution and crystallization from ethyl acetate-ether
gave 2-amino-4-chloro-1-[2-(diisopropylphosphorylmethoxy)-
ethyl]pyrimidin-6(1H)-one (30) (4.8 g, 52.4%), mp 95 °C. Anal.
1
dine (25), not melting under 300 °C. Yield, 4.3 g (81%). Anal.
1
(
C
6
H
3
10
N
4
O) C, H, N. H NMR (CD
3
SOCD
); 9.75 brs, 1H (OH).
-Am in o-4-(d im e t h yla m in o)-6-[2-(p h osp h on om e t h -
3
): 2.89 s, 6H (N-
CH
); 4.51 s, 1H (H-5); 6.18 brs, 2H (NH
2
+
1
(
C
13
H
23ClN
3
O
5
P) C, H, Cl, N, P. MS: 368.4 (MH ) (100). H
SOCD ): 1.20 d, 6 H and 1.22 d, 6 H, J (CH ,CH) )
); 3.69 t, 2 H, J (2′,1′) ) 5.5 (H-2′); 3.76 d, J (CH -P) )
-P); 4.06 t, 2H, J (1′,2′) ) 5.5 (H-1′); 4.55 m, 2 H (P-
2
NMR (CD
6.1 (CH
8.1 (CH
3
3
3
oxy)eth oxy]p yr im id in e (27b) a n d 2-Am in o-4-(d im eth yl-
amino)-1-[2-(phosphonomethoxy)ethyl]pyrimidin-6(1H)-one
3
2
2
1
3
(
26b). Compound 25 (4.0 g, 26 mmol) and cesium carbonate
OCH); 5.67 s, 1 H (H-5); 7.60 bs, 2 H (NH
SOCD ): 23.84 d, 2C, J (CH ,P) ) 3.9 and 23.97 d, 2C, J (P,C)
) 3.9 (CH ); 40.29 (C-1′); 65.03 d, J (P,C) ) 164.1 (P-C); 69.01
2 3
). C NMR (CD -
(4.22 g, 13 mmol) in DMF (60 mL) was stirred at 100 °C for 1
3
3
h, and diisopropyl 2-(chloroethoxy)methylphosphonate (10) (8
3

1
926 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9
Hol y´ et al.
d, J (P,C) ) 11.7 (C-2′); 70.41 d, 2C, J (P,C) ) 5.9 (P-OC); 98.28
C-5); 155.73 (C-6); 157.87 (C-4); 161.48 (C-2).
-Am in o-4-h yd r oxy-6-[2-(p h osp h on om eth oxy)eth oxy]-
p yr im id in e (29b). A mixture of compound 28 (5.7 g, 15.5
mmol), DABCO (3.6 g), and K CO (9.0 g) in water (100 mL)
(phosphonomethoxy)ethoxy]-1-[2-(phosphonomethoxy)ethyl]py-
(
rimidin-6(1H)-one (33b) as a yellowish amorphous product, mp
19 3 10 2 3
H N O P -
) C, H, N, P. 1H NMR (CD
1
08 °C. Anal. (C10
SOCD ): 3.73 d, 2 H, J (CH
9.0 (2 × CH -P); 3.85 bt, 2H, J (2′,1′) ) 5.0 (H-2′); 3.95 m,
2
3
2
2
,P) ) 9.0 and 3.77 d, 2H, J (CH ,P)
)
2
2
2
3
H (H-2′′); 4.17 bt, 2 H, J (1′,2′) ) 5.0 (H-1′); 4.33 m, 2 H, 2H
was refluxed for 150 min under stirring, cooled, and acidified
+
(H-1′′); 5.90 s, 1H (H-5).
by addition of Dowex 50 × 8 (H -form). The suspension was
alkalified with concentrated aqueous ammonia and, after 5
min stirring, filtered, and the resin was washed with 50%
aqueous methanol (200 mL). The filtrate was evaporated to
dryness, ethanol (50 mL) was added, and the mixture was
evaporated to dryness. The residue gave by purification on
silica gel column (150 mL) with chloroform-ethanol gradient
crystalline 2-amino-6-[2-(diisopropylphosphorylmethoxy)ethoxy]-
2,4-Diam in o-6-{[2-(ph osph on om eth oxy)eth yl]su lfan yl}-
p yr im id in e (35b ). Suspension of 2,4-diamino-6-sulfanyl-
pyrimidine hemisulfate (34) (2.616 g, 13.7 mmol) in DMF (40
mL) was stirred with sodium hydride (1.0855 g, 27 mmol, 60%
dispersion in paraffin oil) for 1 h, and compound 10 (9 mL,
17.2 mmol) was added. The mixture was stirred for 8 h at 80
°C, filtered through Celite pad, and evaporated in vacuo. The
residue was purified on silica gel column (150 mL) in chloro-
form-ethanol (49:1) to afford 2,4-diamino-6-{[2-(diisoprop-
4
-hydroxypyrimidine (29a ) (4.2 g, 78%), mp 154 °C. Anal.
1
(
C
13
H
24
N
3
O
6
P) C, H, N, P. H NMR (CD
and 1.23 d, 6 H, J (CH
,CH) ) 6.2 (4 × CH
′); 3.76 d, J (CH -P) ) 8.3 (CH -P); 4.19 m, 2H (H-1′); 4.59
m, 2 H (P-OCH); 4.75 s, 1 H (H-5); 6.65 bs, 2 H, 2H (NH );
,P)
); 65.03 d, J (P,C) )
3
SOCD
3
): 1.24 d, 6H
3
3
); 3.74 m, 2 H (H-
ylphosphorylmethoxy)ethyl]sulfanyl}pyrimidine (35a ) (4.0 g,
1
2
2
2
80%), mp 109 °C. Anal. (C13
H
25
N
4
O
4
PS) C, H, N, P, S. H NMR
2
(CD
(CH
3
SOCD
); 3.17 t, 2H, J (1′,2′) ) 6.6 (H-1′); 3.68 t, 2H, J (2′,1′) ) 6.6
-P) ) 8.3 (CH -P); 4.60 m, 2 H (P-
OCH); 5.60 s, 1 H (H-5); 5.95 brs, 2 H and 6.17 brs, 2H (NH ).
This compound in acetonitrile (50 mL) was treated with
BrSiMe (5 mL) overnight, and the volatiles were evaporated
3 3
): 1.24 d, 6H and 1.25 d, 6 H, J (CH ,CH) ) 6.1
1
3
1
0.45 s, 1H (OH). C NMR (CD
3 3 3
SOCD ): 23.87 d, 2C, J (CH
3
)
1
7
4.9 and 24.01 d, 2C, J (P,C) ) 3.9 (CH
3
(H-2′); 3.77 d, 2H, J (CH
2
2
64.6 (P-C); 65.04 (C-1′); 70.37 d, 2C, J (P,C) ) 6.3) (P-OC);
0.87 d, J (P,C) ) 11.7 (C-2′); 79.95 (C-5); 155.68 (C-4); 164.25
2
(C-2); 171.01 (C-6).
3
This diester (4.0 g, 11.5 mmol) was treated with BrSiMe
10 mL) in acetonitrile (80 mL) overnight and evaporated in
3
in vacuo. The residue was treated with water (100 mL),
alkalified with concentrated aqueous ammonia, and evapo-
rated. The deionized residue was purified by Dowex 1 × 2
column (100 mL) chromatography [elution with water followed
by linear gradient of acetic acid (0-0.5 M, 1.5 L each)]. The
main UV-absorbing fraction was evaporated, and the residue
was codistilled with water (3 × 50 mL) and crystallized from
water. Yield, 2.8 g (91%) 2,4-diamino-6-{[2-(phosphonomethoxy)-
(
vacuo, the residue was treated with water (50 mL) and
alkalified with concentrated aqueous ammonia, and the mix-
ture was evaporated in vacuo. The residue was deionized on a
Dowex 50 × 8 column (100 mL), and the UV-absorbing
ammonia eluate was evaporated to dryness. It was dissolved
in minimum hot water by addition of concentrated aqueous
ammonia and acidified with concentrated HCl to pH 3-3.5.
The precipitate of compound 29b was collected, washed with
7 13 4 4
ethyl]sulfanyl}pyrimidine (35b), mp 246 °C. Anal. (C H N O -
1
PS) C, H, N, P, S. H NMR (D
) 6.8 (H-1′); 3.56 d, 2H, J (CH
J (2′,1′) ) 6.8 (H-2′); 5.70 s, 1 H (H-5); 6.32 brs, 2H (2-NH
6.48 brs, 2H (4-NH ).
-Am in o-6-h yd r oxy-2-{[2-(p h osp h on om eth oxy)eth yl]-
su lfa n yl}p yr im id in e (36b). Compound 11 (monohydrate)
4.85 g, 30 mmol) in DMF (50 mL) was stirred with NaH (30
2
O + NaOD): 3.18t, 2H, J (1′,2′)
,P) ) 8.7; (CH P); 3.68 t, 2H,
);
water and ethanol, and dried in vacuo. Yield, 0.7 g (23%), mp
2
2
1
2
3
2
27 °C. Anal. (C
.58 d, J (CH -P) ) 8.6 (CH
H (H-1′); 5.36 s, 1 H (H-5); 6.55 bs, 2 H, 2H (NH
SOCD ): 64.81(C-1′); 66.86 d, J (CH
7
H
12
N
3
O
6
P) C, H, N, P. H NMR (CD
3
SOCD
3
):
2
2
2
-P); 3.76 m, 2 H (H-2′); 4.29 m,
2
1
3
2
). C NMR
4
(
CD
3
3
2
,P) ) 160.2 (CH -
2
P); 70.70 d, J (2′,P) ) 11.7 (C-2′); 78.60 (C-5); 162.76 (C-2);
(
1
71.30, 2C (C-4 and C-6).
mmol) at room temperature till dissolution and synthon 10 (8
mL) was added in one portion. The mixture was stirred at 80
°C for 6 h and evaporated at 50 °C in vacuo. The residue was
extracted with hot chloroform (total 300 mL) and filtered
through Celite pad, and the filtrate was purified on a silica
gel column (300 mL) by chloroform-ethanol mixture. Yield,
after crystallization from ethyl acetate-ether, 5.3 g (48.3%)
Additional, somewhat impure compound 29b (1.0 g) was
obtained from the deionized filtrate by elution from Dowex 1
2 column with 1 M formic acid.
-Am in o-4,6-bis[2-(p h osp h on om eth oxy)eth oxy]p yr im i-
din e (32b) an d 2-Am in o-4-[2-(ph osph on om eth oxy)eth oxy]-
-[2-(ph osph on om eth oxy)eth yl]pyr im idin -6(1H)-on e (33b).
×
2
1
of compound 36a , mp 163-164 °C. Anal. (C13
H
24
N
3
O
5
PS) C,
,CH) )
3
); 3.25 t, 2H,
A mixture of 2-amino-4,6-dihydroxypyrimidine (31) (12.7 g, 0.1
mol) and cesium carbonate (27.8 g (85 mmol) in DMF (200 mL)
was stirred 1 h at 100 °C, and compound 10 (30 mL) was
added. The mixture was stirred 16 h at 100 °C, filtered while
hot, and evaporated in vacuo. Purification of the residue by
silica gel column chromatography (200 mL) afforded 2.8 g of
the oily residue of compound 32a which was then treated with
1
H, N, P, S. H NMR (CD
3
SOCD
3 3
): 1.23 d, 6 H, J (CH
6
.2 and 1.24 d, 6 H, J (CH
3
,CH) ) 6.2 (4 × CH
J (1′,2′) ) 6.3 (H-1′); 3.72 t, 2H, J (2′,1′) ) 6.3 (H-2′); 3.77 d,
2
H, J (CH
H (H-5); 6.48 s, 2 H (NH
): 23.94 d, 2C, J (P,C) ) 4.4 and 24.04 d, 2C, J (P,C) )
.9 (4 × CH ); 28.85 (C-1′); 64.86 d, J (P,C) ) 164.6 (P-C); 70.45
d, 2C, J (P,C) ) 6.3 (2 × CHO); 71.20 d, J (2′,P) ) 11.7 (C-2′);
1.54 (C-5); 162.48, 163.90 and 164.91 (C-2, C-4, and C-6).
This compound was treated with BrSiMe (10 mL) in
2
-P) ) 8.3 (CH
2
-P); 4.59 m, 2 H (2 × OCH); 4.40 s,
); 11.55 bs, 1H (OH). 13C NMR (CD
-
3
1
2
SOCD
3
3
3
BrSiMe (7 mL) in acetonitrile (50 mL) overnight. The residue
3
was dissolved in water (100 mL), alkalified with concentrated
aqueous ammonia and evaporated. The deionized residue was
codistilled with ethanol and filtered from ethanol. Yield, 1.4 g
8
3
(
3.5%) 2-amino-4,6-bis[2-(phosphonomethoxy)ethoxy]pyrimi-
acetonitrile (70 mL) at ambient temperature overnight and
worked up under standard conditions. The deionized crude
reaction mixture was chromatographed on Dowex 1 × 2
column (150 mL) (elution with water followed by a linear
gradient of formic acid (0-1 M, 1.5 L each). The product
fraction was evaporated and codistilled with water, and the
residue was crystallized from water. Yield, 3.1 g (76%) of
1
dine (32b), mp 127 °C. Anal. (C10
NMR (CD SOCD
H (2 × H-2′); 4.29 m, 2H (2 × H-1′); 5.36 s, 1H (H-5); 6.55 bs,
H
19
N
3
O
10
P
2
) C, H, N, P. H
3
3
): 3.58 d, 2H, J (H,P) ) 8.6 (P-CH ); 3.76 m,
2
2
2
)
5
1
3
H (NH
2
). C NMR (DMSO): 64.81, 2C (C-1′); 66.86 d, J (C,P)
160.2 (P-CH ); 70.70 d, 2C, J (C,P) ) 11.7 (C-2′); 78.60 (C-
2
); 162.76 (C-2); 171.30 (C-4, C-6).
compound 36b, not melting <280 °C. Anal. (C
7
H
12
N
3
O
5
PS) C,
O + NaOD): 3.29 t, 2H, J (2′,1′) ) 6.5
-P) ) 8.7 (CH -P); 3.80 t, 2H, J (1′,2′)
6.5 (H-1′); 4.92 s, 1 H (H-5).
2,4-Dia m in o-6-(S)-[2-(p h osp h on om et h oxy)p r op oxy]-
Further elution of the silica gel column gave a thick oil of
1
H, N, P, S. H NMR (D
2
diester 33a (4.8 g), which was similarly treated with BrSiMe
10 mL) in acetonitrile (70 mL) overnight and evaporated in
3
(H-2′); 3.54 d, 2H, J (CH
2
2
(
)
vacuo. The residue was passed through Dowex 50 × 8 column
+
(
H -form) (150 mL), and the column was eluted with water.
After washing out the inorganic acids, the UV-absorbing
product fraction was eluted with water. It was evaporated in
vacuo, and the residue was stirred with ethanol-acetone
mixture (1:1, 100 mL) to give 1.8 g (4.5%) of 2-amino-4-[2-
p yr im id in e [(S)-38b ]. (S)-[2-(Diisopropylphosphorylmeth-
oxy)propyl] tosylate [(S)-37] (25.7 g, 63 mmol) in DMF (40 mL)
was added at 90 °C to a stirred mixture of 2,4-diamino-6-
hydroxypyrimidine (15) (60 mmol), DMF (40 mL), and DBU

6
-[2-(Phosphonomethoxy)alkoxy]pyrimidines
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9 1927
(
°
10.6 mL, 60 mmol). The reaction mixture was stirred at 100
C 24 h and evaporated in vacuo. The residue was taken in
chloroform (200 mL), filtered, and washed with saturated NaCl
cellent technical assistance of Mrs. B eˇ la Nov a´ kov a´ , Mrs.
Anita Van Lierde, Mrs. Frieda De Meyer, Miss L.Vanden-
heurck, Mrs. Ann Absillis, and Mrs. Lizette van Ber-
ckelaer is gratefully acknowledged. The authors’ thanks
are also due to the staff of the mass spectrometry and
analytical departments of the Institute of Organic
Chemistry and Biochemistry.
(100 mL). The aqueous wash was extracted with chloroform
(
5 × 50 mL), and the combined chloroform solutions were dried
with magnesium sulfate and evaporated. Chromatography on
silica gel column (150 mL) by chloroform-ethanol gradient
gave the O -isomer 38a as an oily residue which was dried in
vacuo over phosphorus pentoxide overnight. Acetonitrile (80
6
3
mL) and BrSiMe (20 mL) were added to the residue, and the
Refer en ces
solution was left to stand overnight in a stoppered flask. The
volatiles were evaporated in vacuo, the residue was treated
with water (100 mL), alkalified with concentrated aqueous
ammonia, and evaporated. The deionized residue was purified
by Dowex 1 × 2 column (200 mL) chromatography [elution
with water followed by linear gradient of acetic acid (0-0.5
M, 1.5 L each). The main UV-absorbing fraction was evapo-
rated, and the residue was codistilled with water (3 × 50 mL)
(
1) Reviews: (a) Hol y´ , A. Phosphonylmethyl analogues of nucleo-
tides and their derivatives: chemistry and biology. Nucleosides
Nucleotides 1987, 6, 147-155. (b) Hol y´ A. Isopolar Phosphorus-
Modified Nucleotide Analogues. In Advances in Antiviral Drug
Design; De Clercq, E., Ed.; J AI Press: Greenwich, CT, 1994; pp
179-232. (c) Freeman, S.; Gardiner, J .M. Acyclic nucleosides
as antiviral compounds. Mol. Biotechnol. 1996, 5, 125-137.
2) (a) Naesens, L.; Snoeck, R.; Andrei, G.; Balzarini, J .; Neyts, J .;
De Clercq, E. HPMPC (cidofovir), PMEA (adefovir) and related
acyclic nucleoside phosphonate analogues: A review of their
pharmacology and clinical potential in the treatment of viral
infections. Antivir. Chem. Chemother. 1997, 8, 1-23. (b) Ying,
C.; De Clercq, E.; Neyts, J . Lamivudine, adefovir and tenofovir
exhibit long-lasting anti-hepatitis B virus activity in cell culture.
J . Viral Hepat. 2000, 7, 79-83.
(
and crystallized from water. Yield, 3.5 g (19.7%) of compound
1
(
S)-38b, mp 281 °C. Anal. (C
NMR (D
dd, 1H, J (CH
J (CH ,P) ) 9.4, J (gem) ) 12.2 (CH
dd, 1H, J (1′b,2′) ) 5.5, J (gem)) )10.5 (H-1′b); 4.14 dd, 1H,
8
H
15
N
4
O
5
P‚H
2
O) C, H, N, P. H
2
O + NaOD): 1.26 d, 3H, J (3′,2′) ) 6.4 (H-3′); 3.51
,P) ) 9.4, J (gem) ) 12.2 (CH P); 3.60 dd, 1H,
P); 3.92 m, 2H (H-2′); 4.06
b
b
a
a
(
3) (a) Starrett, J . E., J r.; Tortolani, D. R.; Hitchcock, M. J .; Martin,
J . C.; Mansuri, M. M. Synthesis and in vitro evaluation of a
phosphonate prodrug: bis(pivaloyloxymethyl) 9-(2-phosphonyl-
methoxyethyl)adenine. Antiviral Res. 1992, 19, 267-273. (b)
Adefovir dipivoxil - Anti-HIV - Anti-HBV. Drugs Future 2000,
1
3
J (1′a,2′) ) 3.7, J (gem) ) 10.5 (H-1′a); 5.41 s, 1 H (H-5).
NMR (D O): 15.84 (C-3′); 66.99 d, J (CH -P) ) 149.9 (P-C);
9.68 (C-1′); 75.14 d, J (2′,P) ) 11.2 (C-2′); 76.84 (C-5); 166.74
C-2); 162.83 (C-4); 171.05 (C-6).
,4-Dia m in o-6-(R)-[2-(p h osp h on om et h oxy)p r op oxy]-
C
2
2
6
(
2
5, 844-847.
2
(
4) Perrillo, R.; Schiff, E.; Yoshida, E.; Statler, A.; Hirsch, K.; Wright,
T.; Gutfreund, K.; Lamy, P.; Murray, A. Adefovir dipivoxil for
the treatment of lamivudine-resistant hepatitis B mutants.
Hepatology 2000, 32, 129-134. (b) Gilson, R. J .; Chopra, K. B.;
Newell, A. M.; Murray-Lyon, I. M.; Nelson, M. R.; Rice, S. J .;
Tedder, R. S.; Toole, J .; J affe, H. S.; Weller, I. V. A placebo-
controlled phase I/II study of adefovir dipivoxil in patients with
chronic hepatitis B virus infection. J . Viral. Hepat. 1999, 6, 387-
p yr im id in e [(R)-38b]. This compound was prepared similarly
from (R)-[2-(diisopropylphosphorylmethoxy)propyl] tosylate
[(R)-37] (50 mmol), 2,4-diamino-6-hydroxypyrimidine (15) (60
mmol), and DBU (60 mmol) in DMF (70 mL). The reaction
mixture was stirred at 100 °C for 24 h. Further workup
followed the procedure described for the (S)-enantiomer. Yield,
3
.55 g (24%) compound (R)-38b, not melting under 290 °C.
3
95. (c) Torresi, J .; Locarnini, S. Antiviral chemotherapy for the
1
13
Anal. (C
8
H
15
N
4
O
5
P‚H
2
O) C, H, N, P. H NMR and C NMR
treatment of hepatitis B virus infections. Gastroenterology. 2000,
118, S83-S103.
spectra are identical with those of the (S)-enantiomer.
-Am in o-2-m eth ylsu lfa n yl-6-[2-(p h osp h on om eth oxy)-
(
5) (a) Otov a´ , B.; Sladk a´ , M.; Votruba, I.; Hol y´ , A.; K rˇ en, V.
Cytostatic effect of 9-(2-phosphonomethoxyethyl)adenine (PMEA).
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Prague 1993, 39, 136-141. (b) Otov a´ , B.; Sladk a´ , M.; Bla zˇ ek,
K.; Schramlov a´ , J .; Votruba, I.; Hol y´ , A. Cytostatic effect of 9-(2-
phosphonomethoxyethyl)adenine (PMEA). II. Lymphoblastic
leukemia in Sprague-Dawley rats. Folia Biol. Prague 1993, 39,
4
eth oxy]p yr im id in e (41b). Diethyl 2-hydroxyethylphospho-
nate (40) (5.3 g, 25 mmol) in DMF (40 mL) was treated at 0
°
C with NaH (1.0 g, 60% dispersion in paraffin oil, 25 mmol)
and, after 1 h stirring at 0 °C, 4-amino-6-chloro-2-methylsul-
fanylpyrimidine (39) (3.5 g, 20 mmol) was added in one portion.
The mixture was stirred 16 h at 100 °C and evaporated to
dryness in vacuo, and the residue was extracted with hot
chloroform (total, 300 mL). The chloroform extract was evapo-
rated in vacuo, and the residue containing compound 41a was
1
42-149. (c) Otov a´ , B.; K rˇ enov a´ , D.; Zidek, Z.; Hol y´ , A.; Votruba,
I.; K rˇ en, V. Cytostatic effect of 9-(2-phosphonomethoxyethyl)
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Folia Biol. Prague 1993, 39, 311-314. (d) Hatse, S.; Balzarini,
J .; De Clercq, E. Induction of erythroid differentiation of human
leukemia K562 cells by the acyclic nucleoside phosphonate 9-(2-
phosphonylmethoxyethyl)adenine (PMEA). Nucleos. Nucleot.
3
treated with BrSiMe (10 mL) in acetonitrile (50 mL) at room
temperature overnight. The mixture was evaporated to dry-
ness in vacuo, and the residue was deionized on a column (100
mL) of Dowex 50 × 8. This product in water (20 mL) was
dissolved by addition of concentrated aqueous ammonia and
acidified by HCl to pH 3-3.5. The precipitate was collected,
washed with water and ethanol, and dried in vacuo. Yield, 0.8
g (13.5%) of 4-amino-2-methylsulfanyl-6-[2-(phosphonomethoxy)-
1
995, 14, 649-652. (e) Hatse, S.; De Clercq, E.; Balzarini, J .
Evidence for distinction of the differentiation-inducing activities
and cytostatic properties of 9-(2-phosphonylmethoxyethyl)ad-
enine and a variety of differentiation-inducing agents in human
erythroleukemia K562 cells. Mol. Pharmacol. 1996, 50, 1231-
1
242. (f) Otov a´ , B.; Zidek, Z.; Hol y´ , A.; Votruba, I.; Sladk a´ , M.;
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8 14 3 5
ethoxy]pyrimidine (41b), mp 210-211 °C. Anal. (C H N O -
1
1, 163-167. (g) Hatse, S.; Naesens, L.; Degreve, B.; Segers, C.;
1
PS) C, H, N, P, S. H NMR (D
2
O): 2.48 s, 3H (SCH
3
); 3.72 d,
Vandeputte, M.; Waer, M.; De Clercq, E.; Balzarini, J . Potent
antitumor activity of the acyclic nucleoside phosphonate 9-(2-
phosphonylmethoxyethyl)adenine in choriocarcinoma-bearing
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2
H, J (H,P) ) 8.5 (P-CH
2
); 3.94 m, 2H (2 × H-2′); 4.36 m, 2H
1
3
(
6
2 × H-1′); 5.68 s, 1H (H-5); C NMR (D
2
O): 16.02 (SCH
9.20 (C-1′); 70.01 d, J (C,P) ) 156.3 (P-CH ); 73.53 d, J (C,P)
10.7 (C-2′); 84.82 (C-5); 167.93, 172.13 and 173.70 (C-2, C-4
3
);
2
)
and C-6).
(
6) (a) Tsai, C. C.; Follis, K. E.; Sabo, A.; Beck, T. W.; Grant, R. F.;
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(
#203/94/K001), by the program of targeted projects
of the Academy of Sciences of the Czech Republic
#S4055109), by Gilead Sciences (Foster City, CA), by
(
the Fifth Framework Programme of the European
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(
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