Antiviral activity of triazine analogues of 1-(S)-[3-hydroxy-2- (phosphonomethoxy)propyl]cytosine (cidofovir) and related compounds

Article abstract of DOI:10.1021/jm061281+

Treatment of 5-azacytosine sodium salt with diisopropyl [(2-chloroethoxy)methyl]phosphonate followed by removal of ester groups with BrSi(CH₃)₃ afforded 1-[2-(phosphonomethoxy)ethyl]-5- azacytosine (3). Reaction of 5-azacytosine with

Full text of DOI:10.1021/jm061281+

J. Med. Chem. 2007, 50, 1069-1077
1069
Antiviral Activity of Triazine Analogues of
1-(S)-[3-Hydroxy-2-(phosphonomethoxy)propyl]cytosine (Cidofovir) and Related Compounds
Marcela Krecˇmerova´,*,† Anton´ın Holy´,*,† Alois P´ıskala,† Milena Masoj´ıdkova´,† Graciela Andrei,‡ Lieve Naesens,‡ Johan Neyts,‡
Jan Balzarini,‡ Erik De Clercq,‡ and Robert Snoeck‡
Gilead Sciences & IOCB Research Centre, Centre for New AntiVirals and Antineoplastics (IOCB), Institute of Organic Chemistry and
Biochemistry, Academy of Sciences of the Czech Republic, FlemingoVo na´m. 2, CZ-166 10, Prague 6, Czech Republic, and Rega Institute for
Medical Research (REGA), Katholieke UniVersiteit LeuVen, Minderbroedersstraat 10, B-3000 LeuVen, Belgium
ReceiVed NoVember 3, 2006
Treatment of 5-azacytosine sodium salt with diisopropyl [(2-chloroethoxy)methyl]phosphonate followed by
removal of ester groups with BrSi(CH3)3 afforded 1-[2-(phosphonomethoxy)ethyl]-5-azacytosine (3). Reaction
of 5-azacytosine with [(trityloxy)methyl]-(2S)-oxirane followed by etherification with diisopropyl (bro-
momethyl)phosphonate and removal of ester groups gave 1-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-
5-azacytosine (1). The synthesis of 6-azacytosine congener 2 was analogous using N4-benzoylated
intermediates. Compound 1 was shown to exert strong activity against a broad spectrum of DNA viruses
including adenoviruses, poxviruses, and herpesviruses (i.e., herpes simplex viruses, varicella zoster virus,
and human cytomegalovirus). Decomposition of 1 in alkaline solutions resulted in products 17 and 18.
While the N-formylguanidine derivative 17 proved active, the carbamyolguanidine derivative 18 was devoid
of antiviral activity.
Introduction
substitutions in the cytosine base; other data published on the
modification of the cytosine moiety in the literature are rather
scarce.12
Cidofovir {HPMPC, CDV,a 1-(S)-[3-hydroxy-2-(phospho-
nomethoxy)propyl]cytosine} is a potent and selective anti-DNA
virus agent that was developed and discovered in our labora-
tories.1 Cidofovir suppresses the in vitro growth of all human
and animal DNA viruses thus far examined.2 It is officially
approved for the treatment of cytomegalovirus retinitis,3 but it
is also used off label, either systemically or topically, for the
treatment of papillomatosous (anogenital or laryngeal)4 infec-
tions, progressive multifocal leukoencephalopathy (caused by
JC polyomavirus),5 adenovirus6 infections, and some severe
infections caused by poxviruses7 (e.g., vaccinia, orf, and
molluscum contagiosum). The potential use of cidofovir as an
antipoxvirus agent is supported by its potent activity against
smallpox virus8 and monkeypox virus.9 Both are highly infec-
tious viruses for human beings and could be purposely used in
a bioterrorist attack.
Aza analogues of cytosine and its nucleosides have been
investigated in our institute for several decades. The clinically
most important compounds of this group are the antileukemic
agents13-15 5-azacytidine16,17 (azacitidine) and 5-aza-2′-deoxy-
cytidine18-20 (decitabine; Figure 1). Also the related 1-â-D-
arabinofuranosyl-5-azacytosine21,22 (fazarabine) exhibits remark-
able antileukemic activity and was investigated in clinical
trials.23,24 The most promising drug candidate from this group
is decitabine, which has progressed to phase III clinical studies
for myelodisplastic syndrome (MDS). 5-Azacytidine and to a
higher extent its 2′-deoxy congener have shown considerable
anti-HIV activity even at 1 µM concentrations.25 2′,3′-Dideoxy-
5-azacytidine26 also exhibited a potent anti-HIV activity, yet
with higher cytotoxicity than 2′,3′-dideoxycytidine. A selective
antiviral effect against hepatitis B virus (HBV) was observed
with 2′,3′-dideoxy-L-5-azacytidine27 (EC50 ) 1.5 µM), which
was active as 2′,3′-dideoxy-D-cytidine (EC50 ) 1 µM) and was
not cytotoxic. The acyclic 5-azapyrimidine nucleoside 1-[(1,3-
dihydroxy-2-propoxy)methyl]-5-azacytosine28 was shown to be
strongly inhibitory against human cytomegalovirus (HCMV) and
Epstein-Barr virus (EBV).29
In the ongoing search for new cidofovir analogues and
derivatives, accruing attention is given to the development of
neutral ester prodrugs to enhance oral absorption and improve
pharmacological parameters.10 So far, our effort in this area has
mainly been focused at the modification of the phosphonate-
bearing side chain (ref 11 and references therein) and on
In this paper we describe the synthesis of 5-aza- and
6-azacytosine congeners of acyclic nucleotide analogues. Focus
is given to their (S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]
derivatives (S)-HPMP-5-azaC (1) and (S)-HPMP-6-azaC (2)
(Figure 1), which are analogous to cidofovir.30 In the series of
2-(phosphonomethoxy)ethyl derivatives, we have previously
reported on the synthesis of the 6-azacytosine derivative,31 and
we describe here the synthesis of N-1-[2-(phosphonomethoxy)-
ethyl]-5-azacytosine (3) that we used as a model compound to
examine its chemical stability and biological properties.
* To whom correspondence should be addressed. (M.K.) Phone: +420
220183475. Fax: +420 220183560. E-mail: marcela@uochb.cas.cz. (A.H.)
Phone: +420 220183384. Fax: +420 220183560. E-mail: holy@
uochb.cas.cz.
† Academy of Sciences of the Czech Republic.
‡ Katholieke Universiteit Leuven.
a Abbreviations: Ac, acetyl; ANP, acyclic nucleoside phosphonate;
tBuONa, sodium tert-butoxide; CDV, cidofovir; DMF, N,N-dimethylfor-
mamide; DMSO, dimethyl sulfoxide; HCMV, human cytomegalovirus;
HHV-6, human herpesvirus 6; HPMP, 1-[3-hydroxy-2-(phosphonomethoxy)-
propyl]; HPMP-5-azaC, 1-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-
5-azacytosine; HPMP-6-azaC, 1-(S)-[3-hydroxy-2-(phosphonomethoxy)-
propyl]-6-azacytosine; HSV-1, herpes simplex virus type 1; HSV-2, herpes
simplex virus type 2; iPr, 2-propyl; PME, 2-(phosphonomethoxy)ethyl;
PME-azaC, 1-[2-(phosphonomethoxy)ethyl]-5-azacytosine; PMEC, 1-[2-
(phosphonomethoxy)ethyl]cytosine; PMP, (R)-2-(phosphonomethoxy)pro-
pyl; Tr, triphenylmethyl; VZV, varicella zoster virus.
Chemistry
The regioselectivity of alkylation follows the pattern of
regiospecificity in similar reactions of 5-azacytosine. In neutral
10.1021/jm061281+ CCC: $37.00 © 2007 American Chemical Society
Published on Web 02/14/2007

1070 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5
KrecˇmeroVa´ et al.
Figure 1.
Scheme 1a
fully with the free base instead of its sodium salt. The structure
of 7 was confirmed on the basis of the proton-coupled 13C NMR
spectra. Whereas carbon atom C-4 (δ 166.70) has a coupling
constant only with H-6, J(C-4,H-6) ) 12.3 Hz, in the case of
carbon atoms C-2 (δ 154.26) and C-6 (δ 159.89) we found, in
addition to interaction with proton H-6, J(C-2,H-6) ) 5.8 Hz
and J(C-6,H-6) ) 203.5 Hz, also long-range coupling constants
with proton H-1′, J(C-2,H-1′) ) 2.9 Hz and J(C-6,H-1′) ) 3.9
Hz. Another structural proof is the IR spectrum, where the
characteristic vibrations of 7 correspond to those of other
1-substituted 5-azacytosines, e.g., 1-methyl-5-azacytosine,33 but
differ from those of N-3-substituted derivatives. Still another
difference distinguishing N-1- and N-3-alkylated 5-azacytosines
consists in their UV spectra: while λmax of N-1-alkyl derivatives
in neutral solutions is about 247 nm and shifts at pH 2 to a
value of about 254 nm, λmax values of N-3-substituted derivatives
in neutral solution are much higher (about 265 nm).34 Compound
7 gave after deprotection the final phosphonate 1 (which showed
247.0 nm in H2O at pH 7 and 254.4 nm at pH 2). The structure
of the N-1 regioisomer thereby was definitely confirmed.
a Conditions: (a) DMF, 110 °C, 3 days; (b) (CH3)3SiBr, CH3CN, rt, 24 h.
For the synthesis of phosphonomethyl ethers, 8 was benzoy-
lated at the amino group to N4-benzoyl-1-[(2S)-2-hydroxy-3-
(triphenylmethoxy)propyl]-6-azacytosine (9) by the action of
benzoic anhydride in N,N-dimethylformamide. Compound 9
gave on treatment with diisopropyl [(tosyloxy)methyl]phospho-
nate in the presence of excess sodium hydride in N,N-
dimethylformamide fully protected phosphonate 10. Reaction
of 10 with bromotrimethylsilane followed by hydrolysis led
simultaneously to removal of the trityl group and phosphonate-
protecting diisopropyl ester groups. The final removal of the
benzoyl group from the N4 position was achieved by sodium
methoxide-catalyzed methanolysis.
Figure 2.
conditions alkylation of the N-3 position is dominant; in most
cases mixtures of N-1- and N-3-monosubstituted and N-1,N-
3-disubstituted compounds are formed. Selective alkylation of
the N-1 position was described only with the sodium salt of
5-azacytosine.32 Reaction of a sodium salt of 5-azacytosine with
diisopropyl [(2-chloroethoxy)methyl]phosphonate (4) in dim-
ethylformamide afforded regioselectively the desired diisopropyl
1-[2-(phosphonomethoxy)ethyl]-5-azacytosine (5), which was
subsequently deprotected to the free phosphonic acid 3 by the
action of bromotrimethylsilane in acetonitrile followed by
hydrolysis (Scheme 1).
The preparation of the 5-azacytosine phosphonate 11 was
more complicated due to the alkali-labile character of the 1,3,5-
triazine ring in protic solvents,34 which excludes the use of
benzoyl or another alkali-labile group for amino group protec-
tion. Reaction of 7 with diisopropyl [((p-tolylsulfonyl)oxy)-
methyl]phosphonate under standard conditions (excess of
sodium hydride, N,N-dimethylformamide, room temperature)
was not successful, leading to a complicated mixture of products
mostly substituted at the N-3 position as witnessed by UV
spectroscopy after deprotection (λmax ) 265 nm in H2O). The
transformation of 7 to 11 was performed by replacement of
Synthesis of both (S)-HPMP derivatives, i.e., (S)-1-[3-
hydroxy-2-(phosphonomethoxy)propyl] derivatives 1 and 2,
started with nucleophilic opening of an oxirane ring in (2S)-2-
[(trityloxy)methyl]oxirane (6) (Scheme 2). Its reaction with
5-aza- or 6-azacytosine performed in dry dimethyl sulfoxide in
the presence of a catalytic amount of sodium hydroxide gave
regiospecifically in both cases N-1-substituted compounds
1-[(2S)-2-hydroxy-3-(triphenylmethoxy)propyl]-5-azacytosine (7)
and 1-[(2S)-2-hydroxy-3-(triphenylmethoxy)propyl]-6-azacy-
tosine (8) in excellent yields. Thus, it was proven that alkylation
of 5-azacytosine at the N-1 position can be performed success-

Triazine Analogues of CidofoVir
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5 1071
Scheme 2a
a Conditions: (a) 5-azacytosine, NaOH, DMSO, 120 °C, 10 h; (b) 6-azacytosine, NaOH, DMSO, 120 °C, 3 h; (c) benzoic anhydride, DMF, rt, 4 days;
(d) TsOCH2P(O)(OiPr)2, NaH, DMF, rt; (e) (CH3)3SiBr, CH3CN, rt; (f) CH3ONa/CH3OH, rt; (g) BrCH2P(O)(OiPr)2, dioxane, NaH or tBuONa, 80-100 °C,
5 h; (h) 80% acetic acid, 80 °C, 2 h.
Scheme 3a
Scheme 4a
a Conditions: (a) 0.25 M triethylammonium hydrogen carbonate, 37 °C,
72 h, followed by 80 °C, 12 h; (b) 1 M NH4OH, 48 h.
a Conditions: (a) DMF, 120 °C, 3 h; (b) (CH3)3SiBr, CH3CN, rt.
Besides synthesis of 1, we also utilized the tritylated
intermediate 7 for preparation of acyclic nucleoside analogue
15, (2S)-(2,3-dihydroxypropyl)-5-azacytosine. Detritylation of
7 was performed by a standard procedure with acetic acid
(Scheme 2). Another compound of our interest was 5,6-dihydro
derivative 16 (Figure 2), i.e., (S)-1-[3-hydroxy-2-(phospho-
nomethoxy)propyl]-5,6-dihydro-5-azacytosine, a compound pre-
pared from 1 by catalytic hydrogenation.
Figure 3.
Similarly to other N-1-substituted 5-azacytosine derivatives
(riboside, 2′-deoxyriboside, arabinoside), also the 5-aza analogue
of HPMPC decomposes according to Scheme 3. The first step
is a reversible ring opening of the sym-triazine to the N-
formylguanidine derivative 17, which can close back to the
cyclic structure. This hydrolytic reaction is slow and reaches
equilibrium within several days. However, the reversible ring-
opening hydrolysis is accompanied by irreversible deformylation
reaction of the intermediary formyl derivative that gives rise to
antivirally inactive 2-{[(2S)-3-hydroxy-2-(phosphonomethoxy)-
propyl]carbamoyl}guanidine (18).
In the HPMP series, the antiviral activity is usually connected
with (S)-enantiomers; therefore, we prepared the triazine HPMP
analogues as the (S)-enantiomers. However, in certain cases,
an activity against DNA viruses was also observed with the
(R)-isomers.36 Considering the remarkable antiviral activity of
compound 1, we considered it necessary to prepare also the
(R)-enantiomer of this compound, i.e., 1-(R)-[3-hydroxy-2-
(phosphonomethoxy)propyl]-5-azacytosine (19; Figure 3). The
compound was made not only to study its biological activity,
but also as a standard for determination of the enantiomeric
purity of 1. The synthesis was performed following the
diisopropyl [((p-tolylsulfonyl)oxy)methyl]phosphonate30 with
diisopropyl (bromomethyl)phosphonate.35 The conversion de-
gree depends on the reaction conditions. Thus, reaction of 7
with diisopropyl (bromomethyl)phosphonate and sodium hydride
in dimethylformamide gave a low yield (approximately 15%)
of 11 contaminated by several byproducts; among them we have
characterized the N-3 isomer 12 (further characterized as acetate
13, Figure 2). However, the same reaction performed in dioxane
in the presence of NaH or sodium tert-butoxide was much more
advantageous: the yield of 11 increased (up to 30-40%), while
the formation of undesired N-3-substituted byproducts was
suppressed, and the residual 7 could be regenerated. Deprotec-
tion of 11 to free phosphonic acid 1 was carried out by treatment
with bromotrimethylsilane in acetonitrile; to strictly avoid
alkaline conditions, the final purification of the product was
achieved by anion-exchange chromatography on Dowex 1 in
acetate form using 1.5 M acetic acid as an eluent. The thus
obtained 1 was partially contaminated with its 3-O-acetyl
derivative 14 (up to 10%). On recrystallization of the product
from water, this impurity was removed.

1072 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5
KrecˇmeroVa´ et al.
procedure described for 1 with the use of commercially available
optically pure (2R)-2-[(trityloxy)methyl]oxirane as a starting
material. The enantiomeric purity of both enantiomers 1 and
19 was determined by the method of capillary electrophoresis.
In the series of so-called “PMP“ derivatives we have prepared
1-[(R)-2-(phosphonomethoxy)propyl]-5-azacytosine (20). The
synthesis followed standard conditions leading to PMP deriva-
tives, i.e., condensation of the appropriate base component (5-
azacytosine) with (R)-{2-[(diisopropoxyphosphoryl)methoxy]-
propyl} p-toluenesulfonate (21) under basic conditions37 followed
by treatment of the intermediary ester 22 with bromotrimeth-
ylsilane and hydrolysis (Scheme 4).
Biological Activity
The antiviral activity spectrum of the different derivatives
was evaluated against a variety of DNA viruses (Table 1), RNA
viruses, and retroviruses. Among the 5-azacytosine congeners
of acyclic nucleoside phosphonate analogues, compound 1
showed potent and selective activity against several DNA
viruses, including different herpesviruses (HSV-1, HSV-2, VZV,
HCMV, and HHV-6), adenovirus (Ad2), and poxvirus (vaccinia
virus) with 50% effective concentration (EC50) values of 0.71
µg/mL for Ad2, 2.56 µg/mL for vaccinia virus, and 0.02-0.6
µg/mL for herpesviruses. The antiviral activity of 1 was
comparable to that of the reference drug (S)-HPMPC against
HSV-1, HSV-2, and vaccinia virus, or 2-7-fold more active
against VZV, HCMV, HHV-6, and Ad2. Compound 1 proved
to be 2-fold less cytotoxic for HEL cells than (S)-HPMPC [50%
cytostatic concentration (CC50) 140 µg/mL for 1 compared to
61 µg/mL for (S)-HPMPC], but 2-fold more toxic for human
T-lymphoblast HSB-2 cells [CC50 ) 16 µg/mL for compound
1 compared to 37 µg/mL for (S)-HPMPC]. For all these DNA
viruses, compound 1 showed a 2-16-fold higher antiviral
selectivity index (ratio of CC50 to EC50) than (S)-HPMPC.
Among the potential decomposition products 17 and 18, the
N-formylguanidine derivative 17, which can close back to the
cyclic structure, showed activity with EC50 values equivalent
to those obtained for the original compound 1 (VZV and
HCMV) or at 3-25-fold higher EC50 values for HSV-1, HSV-
2, HHV-6, Ad2, and vaccinia virus. In contrast, the carbam-
oylguanidine derivative 18, obtained by irreversible deformy-
lation of the intermediate derivative 17, was devoid of antiviral
activity. The fact that product 17 can irreversibly decompose
into product 18 may explain the lost of activity observed with
the first decomposition product as a function of time (data not
shown). Whether the antiviral activity observed with the
derivative 17 is due to its conversion to the cyclic structure,
giving the original compound 1, needs further investigations.
The 5-azacytosine congener in the so-called “PME” series
(3) showed weak activity against the different herpesviruses
tested (EC50 values ranging from 10 to 50 µg/mL) and lacked
activity against vaccinia virus, while in the “PMP” series the
5-azacytosine derivative 20 was inactive against the DNA
viruses (herpes-, adeno-, and poxviruses) evaluated here,
similarly to other PMP derivatives already described.
The (R)-enantiomer 19 proved to be significantly less active
than the (S)-enantiomer 1, with 30-165-fold higher EC50 values.
Also, the isomeric 6-azacytosine analogue 2 was considerably
less active than 1.
The acyclic nucleoside analogue (2S)-(2,3-dihydroxypropyl)-
5-azacytosine (15) proved to be inactive against the different
DNA viruses tested, while the 5,6-dihydro derivative 16
inhibited the replication of herpesviruses with EC50 values
ranging from 3 to 12 µg/mL and proved also to be inhibitory
to vaccinia virus (EC50 ) 38 µg/mL).

Triazine Analogues of CidofoVir
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5 1073
(C12H23N4O5P) C, H, N, P. FABMS: m/z 335 (MH+) (45), 251
(M - (2 × isopropyl) + 3H) (100).
None of the 5-azacytosine congeners of acyclic nucleoside
phosphonate analogues showed activity against RNA viruses
or HIV, except for 16, which was able to inhibit HCV
subgenomic replicon replication in Huh-5-2 cells with an EC50
value of 24 µg/mL. Interestingly, both the HPMP and PME
5-azacytosine derivatives (i.e., compounds 1 and 3) were
inhibitory to Moloney murine sarcoma virus (MSV)-induced
transformation of C3H/3T3 cell cultures with EC50 values of
2.2 and 7.5 µg/mL, respectively. The isomeric 6-azacytosine
analogue 2 was also able to inhibit MSV with an EC50 value of
17 µg/mL, as well as the potential decomposition product 17
(EC50 ) 13 µg/mL).
1-[2-(Phosphonomethoxy)ethyl]-5-azacytosine (3). A solution
of 5 (2.5 g, 7.5 mmol) in acetonitrile (60 mL) was stirred with
bromotrimethylsilane (7 mL, 51.6 mmol) in the dark for 24 h. The
mixture was evaporated, the residue was codistilled with acetonitrile
(3 × 50 mL), and 0.2 M triethylammonium hydrogen carbonate
(50 mL) was added. After 10 min, Dowex 50 (pyridinium form)
was added to reach a neutral reaction, the solid was filtered off
and washed with water, and the pH of the filtrate was adjusted to
8.0 with aqueous ammonia solution. The solution was concentrated
to approximately 20 mL, alkalized to pH 9, and applied to a column
of Dowex 1X2 (100 mL, acetate form). Elution was performed first
with water, followed by a gradient of acetic acid (0-0.4 M, 2 L);
finally the product was eluted with 1 M acetic acid. The product-
containing fractions were evaporated, and the residue was coevapo-
rated with water and three times with ethanol. The solid was
warmed with ethanol to crystallization, then left to stand at 4 °C
overnight, collected by suction, washed with ethanol and ether, and
dried in vacuo. Yield: 1.2 g, 64%, of a white solid. Mp: 228 °C.
Anal. Calcd for C6H11N4O5P: C, 28.81; H, 4.43; N, 22.40; P, 12.38.
Found: C, 28.52; H, 4.53; N, 21.51; P, 11.68. FABMS: m/z 251
(MH+) (100). HRMS (QTOF): m/z, C6H12N4O5P (MH+), calcd
251.0545, found 251.0544.
Conclusion
In conclusion, we have prepared the isomeric aza analogues
of HPMPC and PMEC, i.e., the sym-triazine (5-azacytosine)
and 1,2,4-triazine (6-azacytosine) derivatives. While the activity
of the latter compound was marginal, the 5-aza analogue of
HPMPC demonstrated antiviral activity specifically against
DNA viruses, comparable or even better than that of the parent
compound. These findings warrant further investigations on this
new class of antiviral compounds.
Reaction of (2S)-2-[(Trityloxy)methyl]oxirane with 5-Aza-
and 6-Azacytosine (General Procedure). A suspension of 5-aza-
cytosine or 6-azacytosine (2 g, 17.8 mmol) and 6 (5.63 g, 17.8
mmol) in dry dimethyl sulfoxide (20 mL) was heated to 120 °C.
One pellet of sodium hydroxide (60 mg, 1.5 mmol) was added,
and heating under stirring was continued till dissolution and then
for an additional 10 h (in the case of 7) or 3 h (for the preparation
of 8). The reaction mixture was cooled to room temperature and
poured onto a column of neutral alumina (150 mL) equilibrated in
toluene. Elution was performed first with a mixture of toluene-
ethyl acetate (1:1) till a drop in the UV absorption, followed by
ethyl acetate (200 mL) and the system ethyl acetate-acetone-
ethanol-water (18:3:1:1). The purity of the products was controlled
by TLC in the system ethyl acetate-acetone-ethanol-water (18:
3:1:1). All product-containing fractions (still containing dimethyl
sulfoxide) were taken down, and the residue was codistilled with
dimethylformamide (2 × 100 mL) and then with toluene (100 mL).
The semisolid residue was crystallized from a toluene-acetone (2:
1) mixture, and the crystalline material was collected by suction,
washed with diethyl ether, and dried in the air.
1-[(2S)-2-Hydroxy-3-(triphenylmethoxy)propyl]-5-azacy-
tosine (7). Yield: 6.5 g, 83%, of white crystals. Mp: 130-132
°C. Anal. Calcd for C25H24N4O3‚0.5H2O: C, 68.63; H, 5.76; N,
12.81. Found: C, 68.82; H, 5.87; N, 12.25. HRMS (FAB): m/z,
C25H25N4O3 (MH+), calcd 429.1927, found 429.1931. FABMS: m/z
429 (MH+) (2), 243 (trityl) (100), 113 (5-azacytosine + H) (25).
IR (CHCl3, cm-1): 3540 [νas(NH2)], 3485 [νas(NH2) bonded], 3422
[νs(NH2)], 3346, 3193 [νs(NH2) bonded + ν(OH) intramolecularly
bonded], 1685 [ν(CdO)], 1633 [âs(NH2)], 1507 [ν(ring) triazine],
1491, 1463 [ν(ring) triazine + phenyl], 1120 [âas(NH2)], 1090,
1084, 1055 [νas(C-O-C) + ν(C-OH)], 1598, 1491, 1450 [ν(ring)
triazine], 708.
Experimental Section
Unless stated otherwise, the solvents were evaporated at 40 °C/2
kPa and the compounds were dried at 13 Pa. Melting points were
determined on a Kofler block and are uncorrected. Analytical TLC
was performed on silica gel 60 F254 plates (Merck KGaA,
Darmstadt, Germany); chromatographic systems are described in
the text. Column chromatography was performed on silica gel 60
µm (Fluka) or aluminum oxide (50-150 µm, pH 7.0 ( 0.5, Fluka).
Reversed-phase HPLC separations were performed on a Waters
Delta 600 instrument with a Waters 2487 dual λ absorbance detector
using columns XTerra RP18 (3.9 × 150 mm, analytical column)
1
and XTerra RP18 (10 × 150 mm, preparative column). H NMR
spectra were measured on a Varian Unity 500 instrument (at 500
MHz) in DMSO-d6 solutions (referenced to the solvent signal at δ
2.50) or in D2O solutions with internal standard sodium 3-(trim-
ethylsilyl)propane-1-sulfonate (DSS). 1H NMR chemical shifts (δ,
ppm) and coupling constants (J, Hz) were obtained by first-order
analysis of the spectra. 13C NMR spectra were recorded on the same
instrument (at 125.7 MHz) using the APT pulse sequence in DMSO-
d6 (referenced to the solvent signal δ 39.70). Mass spectra were
measured on a ZAB-EQ (VG Analytical) spectrometer using FAB
(ionization with xenon, accelerating voltage 8 kV, glycerol matrix).
Optical rotations were measured on an AUTOPOL IV polarimeter
(Rudolph Research Analytical) at 20 °C; [R]D values are given in
10-1 deg cm2 g-1
.
Materials and Solvents. Most of the chemicals and ion-exchange
resins (Dowex 50WX8-200 and Dowex 1X2-400) were purchased
from Sigma-Aldrich (Czech Republic). Dimethylformamide and
acetonitrile were dried by distillation from CaH2 (DMF in vacuo)
and stored over molecular sieves (4 Å). Diisopropyl [(tosyloxy)-
methyl]phosphonate,30 diisopropyl (bromomethyl)phosphonate,35
and diisopropyl [(2-chloroethoxy)methyl]phosphonate31 were pre-
pared by previously described procedures. (2S)- and (2R)-2-
[(Trityloxy)methyl]oxiranes were purchased from DAISO Co. Ltd.
(Japan).
1-[(2S)-2-Hydroxy-3-(triphenylmethoxy)propyl]-6-azacy-
tosine (8). Yield: 6.0 g, 73%, of white crystals. Mp: 199-200
°C. Anal. (C25H24N4O3‚2H2O) C, H, N. FABMS: m/z 429 (MH+)
(0.6), 243 (trityl) (100).
N4-Benzoyl-1-[(2S)-2-hydroxy-3-(triphenylmethoxy)propyl]-
6-azacytosine (9). Benzoic anhydride (814 mg, 3.27 mmol) was
added to a solution of 8 (1.4 g, 3.27 mmol) in dimethylformamide
(15 mL) and the mixture stirred for 4 days at room temperature.
Ethanol (5 mL) was added, the solution taken down, and the residue
codistilled with toluene (100 mL) and then chromatographed on a
column of silica gel (300 mL) in the system toluene-ethyl acetate
(1:1). Yield: 1.12 g, 64%, of a solid foam. Anal. Calcd for
C32H28N4O4‚0.25H2O: C, 71.56; H, 5.35; N, 10.43. Found: C,
71.49; H, 5.44; N, 9.95. HRMS (FAB): m/z, C32H29N4O43 (MH+),
1-{[(Diisopropoxyphosphoryl)methoxy]ethyl}-5-azacytosine
(5). A mixture of the sodium salt of 5-azacytosine (4.69 g, 35 mmol)
and 4 (11.5 mL, 46.5 mmol) in DMF (65 mL) was stirred at 110
°C for 3 days. The solid was filtered off and washed with DMF.
The combined filtrates were evaporated, the product was extrac-
ted with chloroform, and the chloroform solution was evaporated
to dryness. The crude product 5 was purified by chromatography
on silica gel using a gradient of methanol in chloroform (0-10%)
and subsequently crystallized from a mixture of ethyl acetate-
ether. Yield: 4.17 g, 36%, of white crystals. Mp: 140 °C. Anal.

1074 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5
KrecˇmeroVa´ et al.
calcd 533.2189, found 533.2204. FABMS: m/z 533 (MH+) (1.3),
243 (trityl) (100), 105 (benzoyl) (46), 77 (phenyl) (10).
mino)pyridine (40 mg, 0.33 mmol) and acetic anhydride (240 mg,
2.4 mmol) were added to a solution of 12 (800 mg, 1.3 mmol)
in acetonitrile (20 mL), and the mixture was set aside for
24 h at ambient temperature. Methanol (20 mL) was added and
the solution evaporated. The residue was chromatographed on a
column of silica gel (200 mL) in the system ethyl acetate-acetone-
ethanol-water (36:6:1:1). Yield: 450 mg, 53%, of a colorless
syrup.
N4-Benzoyl-1-{(2S)-2-[(diisopropoxyphosphoryl)methoxy]-3-
(triphenylmethoxy)propyl}-6-azacytosine (10). A solution of 9
(1.04 g, 1.96 mmol) and diisopropyl [((p-tolylsulfonyl)oxy)methyl]-
phosphonate (1.05 g, 3.0 mmol) in DMF (10 mL) was stirred at
-20 °C, and a 60% dispersion of sodium hydride in mineral oil
(235 mg, 5.9 mmol) was added. The reaction mixture was then
slowly warmed to room temperature, stirred for an additional 2 h,
and filtered through Celite. The filtrate was taken down in vacuo
and the residue chromatographed on a column of silica gel (300
mL) in ethyl acetate. Yield: 1.02 g, 73%, of white foam. Anal.
(C39H43N4O7P) C, H, N, P. FABMS: m/z 711 (MH+) (0.6), 469
(M - trityl + 2H) (4), 243 (trityl) (100), 105 (benzoyl) (38).
(S)-1-[3-Hydroxy-2-(phosphonomethoxy)propyl]-6-azacy-
tosine (2). Bromotrimethylsilane (1.9 mL, 14 mmol) was added to
a solution of 10 (1.02 g, 1.44 mmol) in acetonitrile (10 mL) and
the reaction mixture kept in the dark for 2 days. The yellow solution
was evaporated and the residue codistilled with acetonitrile (20 mL)
and with methanol (20 mL) and then treated with 0.1 M sodium
methoxide in methanol (30 mL) for 24 h at ambient temperature.
Dowex 50 (H+ form) was added till the reaction was weakly acidic
(pH 5) and the resulting suspension applied to a column of Dowex
50 (H+, 50 mL). Elution with water (500 mL) removed salts,
followed by 1% aqueous ammonia (elution of the product). The
content of UV-absorbing fractions was analyzed by TLC in the
system 2-propanol-ammonia-water (7:1:2) (Rf ) 0.1), and the
product-containing fractions were evaporated. The residue was
applied to a column of Dowex 50 (Na+, 20 mL) and the column
eluted with water. The product-containing fractions were evaporated
to dryness, and the resulting solid was stirred with a mixture of
ethanol-hexane (1:1) (10 mL), filtered off, and dried in vacuo.
Yield: 250 mg, 54%, of the sodium salt of 3, white solid. Mp:
254 °C. [R]D: -19.4 (c 0.508, H2O). HRMS (FAB): m/z, C7H12N4-
Na2O6P (MH+), calcd 325.0290, found 325.0302. FABMS: m/z
325 (MH+) (22).
(S)-1-[3-Hydroxy-2-(phosphonomethoxy)propyl]-5-azacy-
tosine (1). Bromotrimethylsilane (4.7 mL, 35 mmol) was added to
a solution of 11 (1.34 g, 3.13 mmol) in acetonitrile (30 mL), and
the mixture was set aside at ambient temperature for 20 h. The
mixture was then evaporated at 30 °C, the residue was coevaporated
with acetonitrile (2 × 30 mL), and 90% aqueous methanol (50 mL)
was added. The solution was neutralized with 1 M triethylammo-
nium hydrogen carbonate to pH 7 and evaporated. The residue was
partitioned between water (100 mL) and ether (100 mL) and the
aqueous layer evaporated to a volume of 5 mL and applied to a
column of Dowex 1 (AcO- form, 100 mL). Elution was performed
with water (500 mL) and then with a linear gradient of acetic acid
(0.1-1 M, 1.5 L), and last, the product was eluted with 1.5 M
acetic acid. UV-absorbing fractions were taken down in vacuo, and
the residue was coevaporated several times with water (20 mL) till
complete removal of acetic acid. The thus obtained product 1 was
contaminated with 14 (11.5% according to HPLC). Pure 1 was
obtained after double recrystallization from water. Yield: 350 mg,
40%, of white crystals. Mp: 175-178 °C. [R]D: -43.7 (c 0.308,
H2O). Enantiomeric purity: 95% (determined by capillary electro-
phoresis). UV: λmax ) 247 nm (pH 7), 254 nm (pH 2). Anal.
(C7H13N4O6P·H2O) C, H, N, P. FABMS: m/z 281.1 (MH+) (4).
HRMS (FAB): m/z, C7H14N4O6P (MH+), calcd 281.0651, found
281.0657.
(R)-1-[3-Hydroxy-2-(phosphonomethoxy)propyl]-5-azacy-
tosine (19). 19 was prepared analogously to 1, starting from (2R)-
2-[(trityloxy)methyl]oxirane. The spectral data are identical with
those of compound 1. [R]D: +47.8 (c 0.189, H2O). Enantiomeric
purity: 100% (determined by capillary electrophoresis).
Reaction of Compound 7 with Diisopropyl (Bromomethyl)-
phosphonate (Method A). A suspension of 7 (785 mg, 1.8 mmol)
in dry dioxane (4 mL) was stirred with sodium tert-butoxide (220
mg, 2.3 mmol). After a complete dissolution of starting material
(approximately 15 min) diisopropyl (bromomethyl)phosphonate
(700 mg, 2.7 mmol) was added and the mixture heated at 80 °C
for 6 h, then cooled to ambient temperature, neutralized dropwise
with acetic acid to pH 7, and taken down. The residue was
chromatographed on silica gel (300 mL) in the system chloroform-
methanol-triethylamine (100:5:1). Three fractions were obtained
in the following elution sequence: products 12 and 11 and
regenerated starting compound 7 (215 mg, 27%).
N-3-[(Diisopropoxyphosphoryl)methyl]-1-[(2S)-2-hydroxy-3-
(triphenylmethoxy)propyl]-5-azacytosine (12). Yield: 200 mg,
18%, of colorless syrup (80% purity), for characterization trans-
formed to 13.
1-{(2S)-2-[(Diisopropoxyphosphoryl)methoxy-3-(triphenyl-
methoxy)]propyl}-5-azacytosine (11). Yield: 400 mg, 36%, of a
white foam. [R]D: -36.3 (c 0.715, CHCl3). Anal. (C32H39N4O6P)
C, H, N, P. FABMS: m/z 629 (M + Na) (0.6), 365 (M - trityl +
2H) (0.2), 243 (trityl) (100).
Reaction of Compound 7 with Diisopropyl (Bromomethyl)-
phosphonate (Method B). Sodium hydride (60% suspension in
mineral oil, 100 mg, 2.5 mmol) was added to a suspension of
compound 7 (830 mg, 2.0 mmol) in dioxane (5 mL). The mixture
was stirred for 30 min at room temperature, then diisopropyl
(bromomethyl)phosphonate (674 mg, 2.6 mmol) was added, and
the reaction mixture was stirred at 100 °C for 5 h. After being
cooled to room temperature, the mixture was applied to a column
of silica gel (150 mL) and chromatographed in the system
chloroform-methanol-triethylamine (100:5:1). The following
products were obtained: 12 (260 mg, 20%), 11 (500 mg, 41%),
and regenerated 7 (250 mg, 30%).
(S)-1-[3-Acetoxy-2-(phosphonomethoxy)propyl]-5-azacy-
tosine (14). This compound formed as an impurity in 1 during its
elution from Dowex 1 with 1.5 M acetic acid (method A). To
confirm its structure, a sample of a mother liquor after crystallization
of 1 (5 mL, mass content approximately 50 mg) was separated by
preparative HPLC using 0.02 M triethylammonium acetate buffer
(isocratic elution, flow rate 5 mL/min); two products were
obtained: free phosphonic acid 1 (retention time 1.9 min) and
acetate 14 (retention time 3.8 min). Fractions containing 14 were
evaporated to dryness, and the residue was coevaporated with water
(5 × 50 mL) and dried in vacuo at 60 °C for 8 h to give the
triethylammonium salt of 14 as a colorless oil. FABMS: m/z 345.1
(M + Na) (6), 323.2 (MH+) (4).
(2S)-(2,3-Dihydroxypropyl)-5-azacytosine (15). A mixture of
7 (1.16 g, 2.7 mmol) in 80% acetic acid (100 mL) was heated at
80 °C for 2 h. The solution was evaporated in vacuo and the residue
codistilled with water (4 × 30 mL). The crude product was adsorbed
onto silica gel (20 mL) from methanol, applied to a silica gel column
(100 mL) equilibrated in the system ethyl acetate-acetone-
ethanol-water (15:3:4:3), and chromatographed in this system. The
residue of 15 was finally crystallized from aqueous ethanol. Yield:
211 mg, 40%. [R]D: -73.9 (c 0.187, H2O). Anal. (C6H10N4O3‚
1/2H2O) C, H, N. FABMS: m/z 187 (MH+) (100).
(S)-1-[3-Hydroxy-2-(phosphonomethoxy)propyl]-5,6-dihydro-
5-azacytosine (16). A mixture of 1 (110 mg, 0.39 mmol) in
methanol (15 mL) and acetic acid (0.5 mL) was stirred till complete
dissolution (1 h, approximately) and then hydrogenated on 10%
palladium on charcoal (20 mg) under atmospheric pressure at room
temperature till UV absorption disappeared (48 h). The catalyst
was filtered off through a Celite pad, the filtrate passed through a
Nylon membrane filter (Whatman, 0.2 µm) and evaporated in vacuo,
and the product lyophilized. Yield: 80 mg, 73%. [R]D: -2.9 (c
0.259, H2O). FABMS: m/z 283 (MH+) (16). HRMS (FAB): m/z,
C7H16N4O6P (MH+), calcd 283.0807, found 283.0807.
N-3-[(Diisopropoxyphosphoryl)methyl]-1-[(2S)-2-acetoxy-3-
(triphenylmethoxy)propyl]-5-azacytosine (13). 4-(Dimethyla-

Triazine Analogues of CidofoVir
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5 1075
3-Formyl-2-{[(2S)-3-hydroxy-2-(phosphonomethoxy)propyl]-
carbamoyl}guanidine (17). A solution of 1 (100 mg, 0.34 mmol)
in 0.25 M triethylammonium hydrogen carbonate (5 mL) was
incubated at 37 °C for 72 h and then for 12 h at 80 °C. The reaction
course was monitored by measurement of the absorption maximum
decrease in the UV spectrum at λmax ) 245 nm. For each
measurement, a 10 µL sample of the reaction mixture was diluted
with water to an overall volume of 3 mL and the absorbance value
at 245 nm determined. The reaction reached equilibrium when no
further decrease of absorbance occurred. The reaction mixture was
evaporated to dryness and the residue coevaporated with water (5
× 3 mL) and then with methanol (2 × 3 mL). The residue in water
(3 mL) was applied to a column of Dowex 50 (Na+ form, 20 mL)
and the column eluted with water. The UV-absorbing eluate was
concentrated in vacuo and lyophilized to give 105 mg (92%) of a
sodium salt of 17 as a colorless amorphous material.
2-{[(2S)-3-Hydroxy-2-(phosphonomethoxy)propyl]carbamoyl}-
guanidine (18). A solution of 1 (200 mg, 0.67 mmol) in 1 M
aqueous ammonia (2.5 mL) was stirred at room temperature for
48 h and then evaporated and the residue coevaporated with water
(5 mL). The crude product was purified by reversed-phase HPLC,
isocratic elution with water. The desired product in fractions (not
absorbing in UV) was detected by TLC on silica gel plates in the
system 2-propanol-25% NH4OH-water (7:1:2) followed by spray-
ing of the plate with a mixture of 5% NaOH-5% K3[Fe(CN)6]-
5% Na[Fe(CN)5NO] (1:1:1) (which gave orange spots of 18). The
product-containing fractions were evaporated and dried in vacuo.
Yield: 150 mg, 83%, as a white foam. [R]D: +31.2 (c 0.226, H2O).
FABMS: m/z 271(MH+) (100). HRMS (FAB): m/z, C6H16N4O6P
(MH+), calcd 271.0807, found 271.0808.
1-{(R)-2-[(Diisopropoxyphosphoryl)methoxy]propyl}-5-aza-
cytosine (22). A suspension of a sodium salt of 5-azacytosine (280
mg, 2.1 mmol), 21 (830 mg, 2.03 mmol), and a catalytic amount
of cesium carbonate (approximately 10 mg) in DMF (6 mL) was
heated at 120 °C for 3 h. After being cooled to room temperature,
the mixture was filtered through a Celite pad, the filtrate was
evaporated, and the residue was coevaporated with toluene and
chromatographed on a column of silica gel (300 mL) in the system
chloroform-methanol (9:1). Yield: 310 mg, 44%, of a white solid.
[R]D: -78.4 (c 0.257, CHCl3). Anal. (C13H25N4O5P) C, H, N, P.
FABMS: m/z 349 (MH+) (64), 265 (M - (2 × isopropyl) + 3H)
(100), 113 (BH+) (42).
1-[(R)-2-(Phosphonomethoxy)propyl]-5-azacytosine (20). A
solution of 22 (270 mg, 0.8 mmol) in acetonitrile (6 mL) was treated
with bromotrimethylsilane (0.6 mL, 4.4 mmol) by the same
procedure as described for 1. Yield: 205 mg, 97%, of a white foam.
[R]D: -67.4 (c 0.270, H2O). FABMS: m/z 365 (MH+) (100).
HRMS (FAB): m/z, C7H14N4O5P (MH+), calcd 265.0702, found
265.0698.
Antiviral Activity Assays. The compounds were evaluated
against the following viruses: herpes simplex virus type 1 (HSV-
1) strain Kos, thymidine kinase-deficient (TK-) HSV-1 Kos strain
resistant to ACV (ACVr), herpes simplex virus type 2 (HSV-2)
strains Lyons and G, varicella zoster virus (VZV) strain Oka, TK-
VZV strain 07-1, human cytomegalovirus (HCMV) strains AD-
169 and Davis, a clinical isolate of adenovirus type 2 (Ad2), human
herpes virus 6 subtype A (HHV-6A) strain GS, vaccinia virus
Lederle strain, respiratory syncitial virus (RSV) strain Long,
vesicular stomatitis virus (VSV), Coxsackie B4, parainfluenza 3,
reovirus-1, Sindbis, Punta Toro, yellow fever virus (YFV), human
immunodeficiency virus type 1 strain IIIB, human immunodefi-
ciency virus type 2 strain ROD, and hepatitis C virus (HCV). The
antiviral, other than anti-HIV and anti-HCV, assays were based on
inhibition of virus-induced cytopathicity or plaque formation in
human embryonic lung (HEL) fibroblasts, African green monkey
cells (Vero), human epithelial cells (HeLa), or human T-lympho-
blasts (HSB-2), according to previously established procedures.38
Confluent cell cultures in microtiter 96-well plates were inoculated
with 100 CCID50 of virus (1 CCID50 being the virus dose to infect
50% of the cell cultures) or with 20 plaque-forming units (PFUs).
After a 1-2 h adsorption period, residual virus was removed, and
the cell cultures were incubated in the presence of varying
concentrations of the test compounds. Viral cytopathicity or plaque
formation (VZV) was recorded as soon as it reached completion
in the control virus-infected cell cultures that were not treated with
the test compounds. Antiviral activity was expressed as the EC50,
or the concentration required to reduce virus-induced cytopatho-
genicity or viral plaque formation by 50%.
Inhibition of HIV-Induced Cytopathicity in CEM Cells. The
methodology for the anti-HIV assays has been described previ-
ously.39 Briefly, human CEM cells (∼3 × 105 cells/mL) were
infected with 100 CCID50 HIV-1 or HIV-2/mL and seeded in 200
µL well microtiter plates, containing appropriate dilutions of the
test compounds. After 4 days of incubation at 37 °C, CEM giant
cell formation was examined microscopically.
Inhibition of HCV Subgenomic Replicon Replication in Huh-
5-2 Cells. Huh-5-2 cells (a cell line with a persistent HCV replicon,
I389luc-ubi-neo/NS3-3′/5.1, a replicon with firefly luciferase-
ubiquitin-neomycin phosphotransferase fusion protein and an
EMCV-IRES-driven NS3-5B HCV polyprotein) were cultured in
RPMI medium (Gibco) supplemented with 10% fetal calf serum,
2 mM L-glutamine (Life Technologies), 1× nonessential amino
acids (Life Technologies), 100 IU/mL penicillin, 100 µg/mL
streptomycin, and 250 µg/mL G418 (Geneticin, Life Technologies).
The cells were seeded at a density of 7000 cells/well in a 96-well
View Plate (Packard) in medium containing the same components
as described above, except for G418. The cells were allowed to
adhere and proliferate for 24 h. At that time, the culture medium
was removed and five serial dilutions (5-fold dilutions starting at
100 µg/mL or 100 µM) of the test compounds were added in culture
medium lacking G418. Interferon R 2a (500 IU) was included as a
positive control in each experiment for internal validation, and an
HCV polymerase (2′C-methylcytidine) and/or an HCV protease
inhibitor (VX-950), can be included as well as a reference. The
plates were further incubated at 37 °C and 5% CO2 for 72 h.
Replication of the HCV replicon in Huh-5-2 cells results in
luciferase activity in the cells. Luciferase activity was measured
by adding 50 µL of 1× Glo-lysis buffer (Promega) for 15 min
followed by 50 µL of the Steady-Glo luciferase assay reagent
(Promega). Luciferase activity was measured with a luminometer,
and the signal in each individual well was expressed as a percentage
of the untreated cultures. The 50% effective concentrations (EC50)
were calculated from these data sets. Parallel cultures of Huh-5-2
cells, seeded at a density of 7000 cells/well on classical 96-well
cel culture plates (Becton-Dickinson), were treated in a similar
fashion except that no Glo-lysis buffer or Steady-Glo luciferase
reagent was added. The effect of the compounds on the proliferation
of the cells was measured 3 days after addition of the various
compounds by means of the CellTiter 96 AQueous nonradioactive
cell proliferation assay (MTS, Promega). In this assay 3-(4,5-
dimethylthiazol-2-yl)-5-[3-(carboxymethoxy)phenyl]-2-(4-sulfophe-
nyl)-2H-tetrazolium (MTS) was bioreduced by cells into a formazan
that is soluble in tissue. The number of cells correlates directly
with the production of the formazan. The MTS-stained cultures
were quantified in a plate reader at 498 nm. The 50% cytostatic
concentrations (EC50) were calculated from these data sets.
Inhibition of MSV-Induced Transformation of Murine C3H/
3T3 Embryo Fibroblasts. The anti-MSV assay was performed as
described previously.39 Murine C3H/3T3 embryo fibroblast cells
were seeded at 5 × 105 cells/mL into 1-cm2 wells of 48-well
microplates. After 24 h, the cell cultures were infected with 80
focus-forming units of MSV (prepared from tumors induced
following intramuscular inoculation of 3 day old NMRI mice with
MSV, as described previously40) for 90-120 min at 37 °C. The
medium was then replaced by 1 mL of fresh medium containing
various concentrations of the test compounds. After 6 days,
transformation of the cell culture was examined microscopically.
Cytotoxicity Assays. Cytotoxicity measurements were based on
the inhibition of cell growth. HEL cells were seeded at a rate of 5
× 103 cells/well into 96-well microtiter plates and allowed to
proliferate for 24 h. Then, medium containing different concentra-
tions of the test compounds was added. After 3 days of incubation

1076 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5
KrecˇmeroVa´ et al.
(5) (a) Segarra-Newnham, M.; Vodolo, K. M. Use of cidofovir in
progressive multifocal leukoencephalopathy. Ann. Pharmacother.
2001, 35, 741-744. (b) Gasnault, J.; Kousignian, P.; Kahraman, M.;
Rahoiljaon, J.; Matheron, S.; Delfraissy, J. F.; Taoufik, Y. Cidofovir
in AIDS-associated progressive multifocal leukoencephalopathy: A
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at 37 °C, the cell number was determined with a Coulter counter.
The cytostatic concentration was calculated as the CC50, or the
compound concentration required to reduce cell proliferation by
50% relative to the number of cells in the untreated controls. CC50
values were estimated from graphic plots of the number of cells
(percentage of control) as a function of the concentration of the
test compounds. Alternatively, cytotoxicity for cell morphology was
expressed as the minimum cytotoxic concentration (MCC), or the
compound concentration that caused a microscopically detectable
alteration of cell morphology.
(7) Bray, M.; Wright, M. E. Progressive vaccinia. Clin. Infect. Dis. 2003,
36, 766-774.
Acknowledgment. This study was performed as a research
project of the Institute of Organic Chemistry and Biochemistry,
Academy of Sciences of the Czech Republic, Grant Z4 055
0506, and the Centre for New Antivirals and Antineoplastics,
Grant 1M0508. We gratefully acknowledge the financial support
of the NIH (Grant 1UC1 AI062540-01), the European Com-
mission (Rene´ Descartes Prize-2001, Grant HPAV-2002-
100096), the Grant Agency of the Academy of Sciences (Grant
1QS 400550501), and Gilead Sciences (Foster City, CA). Our
thanks are also due to Dr. P. Fiedler from the Institute of Organic
Chemistry and Biochemistry for measurement of the IR spectra,
the staff of the Mass Spectrometry (Dr. J. Cvacˇka, Head) and
Analytical (Dr. L. Holasova´, Head) Departments of the Institute
of Organic Chemistry and Biochemistry, and Dr. V. Kasˇicˇka
for electrophoretic determination of the enantiomeric purity of
the compounds. We thank Ann Absilis, Anita Camps, Steven
Carmans, Frieda De Meyer, Katrien Geerts, Leentje Persoons,
Wim Van Dam, and Lies Van den Heurck for excellent technical
assistance with the antiviral assays and Christiane Callebaut for
invaluable editorial assistance.
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Supporting Information Available: Elemental analysis data,
1H and 13C NMR data of all new compounds, NMR spectra of target
compounds, HRMS (compounds 3, 7, and 9), and activities of
compounds against RNA viruses and retroviruses. This material is
available free of charge via the Internet at http://pubs.acs.org.
(14) Kantarjian, H. M.; O’Brien, S. M.; Keating, M.; Beran, M.; Estey,
E.; Girald, S.; Kornblau, S.; Rios, M. B.; deVos, D.; Talpay, M.
Results of decitabine therapy in the accelerated and blastic phases
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